A bacterial-type ABC transporter is involved in aluminum tolerance in rice.

PubMed

Huang, Chao Feng; Yamaji, Naoki; Mitani, Namiki; Yano, Masahiro; Nagamura, Yoshiaki; Ma, Jian Feng

2009-02-01

Aluminum (Al) toxicity is a major factor limiting crop production in acidic soil, but the molecular mechanisms of Al tolerance are poorly understood. Here, we report that two genes, STAR1 (for sensitive to Al rhizotoxicity1) and STAR2, are responsible for Al tolerance in rice. STAR1 encodes a nucleotide binding domain, while STAR2 encodes a transmembrane domain, of a bacterial-type ATP binding cassette (ABC) transporter. Disruption of either gene resulted in hypersensitivity to aluminum toxicity. Both STAR1 and STAR2 are expressed mainly in the roots and are specifically induced by Al exposure. Expression in onion epidermal cells, rice protoplasts, and yeast showed that STAR1 interacts with STAR2 to form a complex that localizes to the vesicle membranes of all root cells, except for those in the epidermal layer of the mature zone. When expressed together in Xenopus laevis oocytes, STAR1/2 shows efflux transport activity specific for UDP-glucose. Furthermore, addition of exogenous UDP-glucose rescued root growth in the star1 mutant exposed to Al. These results indicate that STAR1 and STAR2 form a complex that functions as an ABC transporter, which is required for detoxification of Al in rice. The ABC transporter transports UDP-glucose, which may be used to modify the cell wall.

Export of Extracellular Polysaccharides Modulates Adherence of the Cyanobacterium Synechocystis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fisher, ML; Allen, R; Luo, YQ

2013-09-10

The field of cyanobacterial biofuel production is advancing rapidly, yet we know little of the basic biology of these organisms outside of their photosynthetic pathways. We aimed to gain a greater understanding of how the cyanobacterium Synechocystis PCC 6803 (Synechocystis, hereafter) modulates its cell surface. Such understanding will allow for the creation of mutants that autoflocculate in a regulated way, thus avoiding energy intensive centrifugation in the creation of biofuels. We constructed mutant strains lacking genes predicted to function in carbohydrate transport or synthesis. Strains with gene deletions of slr0977 (predicted to encode a permease component of an ABC transporter),more » slr0982 (predicted to encode an ATP binding component of an ABC transporter) and slr1610 (predicted to encode a methyltransferase) demonstrated flocculent phenotypes and increased adherence to glass. Upon bioinformatic inspection, the gene products of slr0977, slr0982, and slr1610 appear to function in O-antigen (OAg) transport and synthesis. However, the analysis provided here demonstrated no differences between OAg purified from wild-type and mutants. However, exopolysaccharides (EPS) purified from mutants were altered in composition when compared to wild-type. Our data suggest that there are multiple means to modulate the cell surface of Synechocystis by disrupting different combinations of ABC transporters and/or glycosyl transferases. Further understanding of these mechanisms may allow for the development of industrially and ecologically useful strains of cyanobacteria. Additionally, these data imply that many cyanobacterial gene products may possess as-yet undiscovered functions, and are meritorious of further study.« less

Efflux pump-mediated benzalkonium chloride resistance in Listeria monocytogenes isolated from retail food.

PubMed

Jiang, Xiaobing; Yu, Tao; Liang, Yu; Ji, Shengdong; Guo, Xiaowei; Ma, Jianmin; Zhou, Lijun

2016-01-18

In this study, efflux pump-mediated benzalkonium chloride (BC) resistance, including plasmid-encoded (Qac protein family and BcrABC) and chromosome-borne efflux pumps, was investigated in Listeria monocytogenes from retail food in China. Among the 59 L. monocytogenes strains, 13 (22.0%) strains were resistant to BC. The PCR results showed that bcrABC was harbored by 2 of 13 BC resistant strains. However, none of the qac genes were detected among the 59 strains. The bcrABC was absent in both of the plasmid cured strains, indicating that this BC resistance determinant was plasmid-encoded in the two bcrABC-positive strains. In the presence of reserpine, most of the bcrABC-negative strains had decreases in the MICs of BC, suggesting the existence of other efflux pumps and their role in BC resistance. After exposed to reserpine, the reduction in BC MICs was observed in the two cured strains, indicating that efflux pumps located on chromosome was also involved in BC resistance. Our findings suggest that food products may act as reservoirs for BC resistant isolates of L. monocytogenes and plasmid- and chromosome-encoded efflux pumps could mediate the BC resistance of L. monocytogenes, which is especially relevant to the adaption of this organism in food-related environments with frequent BC use. Copyright © 2015 Elsevier B.V. All rights reserved.

An operon from Lactobacillus helveticus composed of a proline iminopeptidase gene (pepI) and two genes coding for putative members of the ABC transporter family of proteins.

PubMed

Varmanen, P; Rantanen, T; Palva, A

1996-12-01

A proline iminopeptidase gene (pepI) of an industrial Lactobacillus helveticus strain was cloned and found to be organized in an operon-like structure of three open reading frames (ORF1, ORF2 and ORF3). ORF1 was preceded by a typical prokaryotic promoter region, and a putative transcription terminator was found downstream of ORF3, identified as the pepI gene. Using primer-extension analyses, only one transcription start site, upstream of ORF1, was identifiable in the predicted operon. Although the size of mRNA could not be judged by Northern analysis either with ORF1-, ORF2- or pepI-specific probes, reverse transcription-PCR analyses further supported the operon structure of the three genes. ORF1, ORF2 and ORF3 had coding capacities for 50.7, 24.5 and 33.8 kDa proteins, respectively. The ORF3-encoded PepI protein showed 65% identity with the PepI proteins from Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus delbrueckii subsp. lactis. The ORF1-encoded protein had significant homology with several members of the ABC transporter family but, with two distinct putative ATP-binding sites, it would represent an unusual type among the bacterial ABC transporters. ORF2 encoded a putative integral membrane protein also characteristic of the ABC transporter family. The pepI gene was overexpressed in Escherichia coli. Purified PepI hydrolysed only di and tripeptides with proline in the first position. Optimum PepI activity was observed at pH 7.5 and 40 degrees C. A gel filtration analysis indicated that PepI is a dimer of M(r) 53,000. PepI was shown to be a metal-independent serine peptidase having thiol groups at or near the active site. Kinetic studies with proline-p-nitroanilide as substrate revealed Km and Vmax values of 0.8 mM and 350 mmol min-1 mg-1, respectively, and a very high turnover number of 135,000 s-1.

ALS3 encodes a phloem-localized ABC transporter-like protein that is required for aluminum tolerance in Arabidopsis.

PubMed

Larsen, Paul B; Geisler, Matt J B; Jones, Carol A; Williams, Kelly M; Cancel, Jesse D

2005-02-01

Aluminum (Al) toxicity in acid soils is a worldwide agricultural problem that severely limits crop productivity through inhibition of root growth. Previously, Arabidopsis mutants with increased Al sensitivity were isolated in order to identify genes important for Al tolerance in plants. One mutant, als3, exhibited extreme root growth inhibition in the presence of Al, suggesting that this mutation negatively impacts a gene required for Al tolerance. Map-based cloning of the als3-1 mutation resulted in the isolation of a novel gene that encodes a previously undescribed ABC transporter-like protein, which is highly homologous to a putative bacterial metal resistance protein, ybbM. Northern analysis for ALS3 expression revealed that it is found in all organs examined, which is consistent with the global nature of Al sensitivity displayed by als3, and that expression increases in roots following Al treatment. Based on GUS fusion and in situ hybridization analyses, ALS3 is primarily expressed in leaf hydathodes and the phloem throughout the plant, along with the root cortex following Al treatment. Immunolocalization indicates that ALS3 predominantly accumulates in the plasma membrane of cells that express ALS3. From our results, it appears that ALS3 encodes an ABC transporter-like protein that is required for Al resistance/tolerance and may function to redistribute accumulated Al away from sensitive tissues in order to protect the growing root from the toxic effects of Al.

Overexpression of Both ERG11 and ABC2 Genes Might Be Responsible for Itraconazole Resistance in Clinical Isolates of Candida krusei

PubMed Central

He, Xiaoyuan; Zhao, Mingfeng; Chen, Jinyan; Wu, Rimao; Zhang, Jianlei; Cui, Rui; Jiang, Yanyu; Chen, Jie; Cao, Xiaoli; Xing, Yi; Zhang, Yuchen; Meng, Juanxia; Deng, Qi; Sui, Tao

2015-01-01

Objective To study the main molecular mechanisms responsible for itraconazole resistance in clinical isolates of Candida krusei. Methods The 14α-demethylases encoded by ERG11 gene in the 16 C.krusei clinical isolates were amplified by polymerase chain reaction (PCR), and their nucleotide sequences were determined to detect point mutations. Meanwhile, ERG11 and efflux transporters (ABC1 and ABC2) genes were determined by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) for their expression in itraconazole-resistant (R), itraconazole-susceptible dose dependent (SDD) and itraconazole-susceptible (S) C.krusei at the mRNA level. Results We found 7-point mutations in ERG11 gene of all the C.krusei clinical isolates, including 6 synonymous mutations and 1 missense mutation (C44T). However, the missense mutation was found in the three groups. The mRNA levels of ERG11 gene in itraconazole-resistant isolates showed higher expression compared with itraconazole-susceptible dose dependent and itraconazole-susceptible ones (P = 0.015 and P = 0.002 respectively). ABC2 gene mRNA levels in itraconazole-resistant group was significantly higher than the other two groups, and the levels of their expression in the isolates appeared to increase with the decrease of susceptibility to itraconazole (P = 0.007 in SDD compared with S, P = 0.016 in SDD with R, and P<0.001 in S with R respectively). While ABC1 gene presented lower expression in itraconazole resistant strains. However, the mRNA levels of ERG11, ABC1 and ABC2 in a C.krusei (CK10) resistant to both itraconazole and voriconazole were expressed highest in all the itraconazole-resistant isolates. Conclusions There are ERG11 gene polymorphisms in clinical isolates of C.krusei. ERG11 gene mutations may not be involved in the development of itraconazole resistance in C.krusei. ERG11 and ABC2 overexpression might be responsible for the acquired itraconazole resistance of these clinical isolates. PMID:26308936

A Genetic Locus Necessary for Rhamnose Uptake and Catabolism in Rhizobium leguminosarum bv. trifolii

PubMed Central

Richardson, Jason S.; Hynes, Michael F.; Oresnik, Ivan J.

2004-01-01

Rhizobium leguminosarum bv. trifolii mutants unable to catabolize the methyl-pentose rhamnose are unable to compete effectively for nodule occupancy. In this work we show that the locus responsible for the transport and catabolism of rhamnose spans 10,959 bp. Mutations in this region were generated by transposon mutagenesis, and representative mutants were characterized. The locus contains genes coding for an ABC-type transporter, a putative dehydrogenase, a probable isomerase, and a sugar kinase necessary for the transport and subsequent catabolism of rhamnose. The regulation of these genes, which are inducible by rhamnose, is carried out in part by a DeoR-type negative regulator (RhaR) that is encoded within the same transcript as the ABC-type transporter but is separated from the structural genes encoding the transporter by a terminator-like sequence. RNA dot blot analysis demonstrated that this terminator-like sequence is correlated with transcript attenuation only under noninducing conditions. Transport assays utilizing tritiated rhamnose demonstrated that uptake of rhamnose was inducible and dependent upon the presence of the ABC transporter at this locus. Phenotypic analyses of representative mutants from this locus provide genetic evidence that the catabolism of rhamnose differs from previously described methyl-pentose catabolic pathways. PMID:15576793

Complete sequence of Enterococcus faecium pVEF3 and the detection of an omega-epsilon-zeta toxin-antitoxin module and an ABC transporter.

PubMed

Sletvold, H; Johnsen, P J; Hamre, I; Simonsen, G S; Sundsfjord, A; Nielsen, K M

2008-07-01

Glycopeptide resistant Enterococcus faecium (GREF) persists on Norwegian poultry farms despite the ban on the growth promoter avoparcin. The biological basis for long-term persistence of avoparcin resistance is not fully understood. This study presents the complete DNA sequence of the E. faecium R-plasmid pVEF3 and functional studies of some plasmid-encoded traits (a toxin-antitoxin (TA) system and an ABC transporter) that may be of importance for plasmid persistence. The pVEF3 (63.1 kbp), isolated from an E. faecium strain of poultry origin sampled in Norway in 1999, has 71 coding sequences including the vanA avoparcin/vancomycin resistance encoding gene cluster. pVEF3 encodes the TA system omega-epsilon-zeta, and plasmid stability tests and transcription analysis show that omega-epsilon-zeta is functional in Enterococcus faecalis OGIX, although with decreasing effect over time. The predicted ABC transporter was not found to confer reduced susceptibility to any of the 28 substances tested. The TA system identified in the pVEF-type plasmids may contribute to vanA plasmid persistence on Norwegian poultry farms. However, size and compositional heterogeneity among E. faecium vanA plasmids suggest that additional plasmid maintenance systems in combination with host specific factors and frequent horizontal gene transfer and rearrangement causes the observed plasmid composition and distribution patterns.

Whole genome re-sequencing identifies a mutation in an ABC transporter (mdr2) in a Plasmodium chabaudi clone with altered susceptibility to antifolate drugs☆

PubMed Central

Martinelli, Axel; Henriques, Gisela; Cravo, Pedro; Hunt, Paul

2011-01-01

In malaria parasites, mutations in two genes of folate biosynthesis encoding dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) modify responses to antifolate therapies which target these enzymes. However, the involvement of other genes which modify the availability of exogenous folate, for example, has been proposed. Here, we used short-read whole-genome re-sequencing to determine the mutations in a clone of the rodent malaria parasite, Plasmodium chabaudi, which has altered susceptibility to both sulphadoxine and pyrimethamine. This clone bears a previously identified S106N mutation in dhfr and no mutation in dhps. Instead, three additional point mutations in genes on chromosomes 2, 13 and 14 were identified. The mutated gene on chromosome 13 (mdr2 K392Q) encodes an ABC transporter. Because Quantitative Trait Locus analysis previously indicated an association of genetic markers on chromosome 13 with responses to individual and combined antifolates, MDR2 is proposed to modulate antifolate responses, possibly mediated by the transport of folate intermediates. PMID:20858498

LhnR and upstream operon LhnABC in Agrobacterium vitis regulate the induction of tobacco hypersensitive responses, grape necrosis and swarming motility.

PubMed

Zheng, Desen; Hao, Guixia; Cursino, Luciana; Zhang, Hongsheng; Burr, Thomas J

2012-09-01

The characterization of Tn5 transposon insertional mutants of Agrobacterium vitis strain F2/5 revealed a gene encoding a predicted LysR-type transcriptional regulator, lhnR (for 'LysR-type regulator associated with HR and necrosis'), and an immediate upstream operon consisting of three open reading frames (lhnABC) required for swarming motility, surfactant production and the induction of a hypersensitive response (HR) on tobacco and necrosis on grape. The operon lhnABC is unique to A. vitis among the sequenced members in Rhizobiaceae. Mutagenesis of lhnR and lhnABC by gene disruption and complementation of ΔlhnR and ΔlhnABC confirmed their roles in the expression of these phenotypes. Mutation of lhnR resulted in complete loss of HR, swarming motility, surfactant production and reduced necrosis, whereas mutation of lhnABC resulted in loss of swarming motility, delayed and reduced HR development and reduced surfactant production and necrosis. The data from promoter-green fluorescent protein (gfp) fusions showed that lhnR suppresses the expression of lhnABC and negatively autoregulates its own expression. It was also shown that lhnABC negatively affects its own expression and positively affects the transcription of lhnR. lhnR and lhnABC constitute a regulatory circuit that coordinates the transcription level of lhnR, resulting in the expression of swarming, surfactant, HR and necrosis phenotypes. © 2012 THE AUTHORS. MOLECULAR PLANT PATHOLOGY © 2012 BSPP AND BLACKWELL PUBLISHING LTD.

Grass flower development.

PubMed

Hirano, Hiro-Yuki; Tanaka, Wakana; Toriba, Taiyo

2014-01-01

Grasses bear unique flowers lacking obvious petals and sepals in special inflorescence units, the florets and the spikelet. Despite this, grass floral organs such as stamens and lodicules (petal homologs) are specified by ABC homeotic genes encoding MADS domain transcription factors, suggesting that the ABC model of eudicot flower development is largely applicable to grass flowers. However, some modifications need to be made for the model to fit grasses well: for example, a YABBY gene plays an important role in carpel specification. In addition, a number of genes are involved in the development of the lateral organs that constitute the spikelet. In this review, we discuss recent progress in elucidating the genes required for flower and spikelet development in grasses, together with those involved in fate determination of the spikelet and flower meristems.

Convergent Loss of ABC Transporter Genes From Clostridioides difficile Genomes Is Associated With Impaired Tyrosine Uptake and p-Cresol Production.

PubMed

Steglich, Matthias; Hofmann, Julia D; Helmecke, Julia; Sikorski, Johannes; Spröer, Cathrin; Riedel, Thomas; Bunk, Boyke; Overmann, Jörg; Neumann-Schaal, Meina; Nübel, Ulrich

2018-01-01

We report the frequent, convergent loss of two genes encoding the substrate-binding protein and the ATP-binding protein of an ATP-binding cassette (ABC) transporter from the genomes of unrelated Clostridioides difficile strains. This specific genomic deletion was strongly associated with the reduced uptake of tyrosine and phenylalanine and production of derived Stickland fermentation products, including p -cresol, suggesting that the affected ABC transporter had been responsible for the import of aromatic amino acids. In contrast, the transporter gene loss did not measurably affect bacterial growth or production of enterotoxins. Phylogenomic analysis of publically available genome sequences indicated that this transporter gene deletion had occurred multiple times in diverse clonal lineages of C. difficile , with a particularly high prevalence in ribotype 027 isolates, where 48 of 195 genomes (25%) were affected. The transporter gene deletion likely was facilitated by the repetitive structure of its genomic location. While at least some of the observed transporter gene deletions are likely to have occurred during the natural life cycle of C. difficile , we also provide evidence for the emergence of this mutation during long-term laboratory cultivation of reference strain R20291.

Convergent Loss of ABC Transporter Genes From Clostridioides difficile Genomes Is Associated With Impaired Tyrosine Uptake and p-Cresol Production

PubMed Central

Steglich, Matthias; Hofmann, Julia D.; Helmecke, Julia; Sikorski, Johannes; Spröer, Cathrin; Riedel, Thomas; Bunk, Boyke; Overmann, Jörg; Neumann-Schaal, Meina; Nübel, Ulrich

2018-01-01

We report the frequent, convergent loss of two genes encoding the substrate-binding protein and the ATP-binding protein of an ATP-binding cassette (ABC) transporter from the genomes of unrelated Clostridioides difficile strains. This specific genomic deletion was strongly associated with the reduced uptake of tyrosine and phenylalanine and production of derived Stickland fermentation products, including p-cresol, suggesting that the affected ABC transporter had been responsible for the import of aromatic amino acids. In contrast, the transporter gene loss did not measurably affect bacterial growth or production of enterotoxins. Phylogenomic analysis of publically available genome sequences indicated that this transporter gene deletion had occurred multiple times in diverse clonal lineages of C. difficile, with a particularly high prevalence in ribotype 027 isolates, where 48 of 195 genomes (25%) were affected. The transporter gene deletion likely was facilitated by the repetitive structure of its genomic location. While at least some of the observed transporter gene deletions are likely to have occurred during the natural life cycle of C. difficile, we also provide evidence for the emergence of this mutation during long-term laboratory cultivation of reference strain R20291. PMID:29867812

Complete genome sequencing and analysis of a Lancefield group G Streptococcus dysgalactiae subsp. equisimilis strain causing streptococcal toxic shock syndrome (STSS)

PubMed Central

2011-01-01

Background Streptococcus dysgalactiae subsp. equisimilis (SDSE) causes invasive streptococcal infections, including streptococcal toxic shock syndrome (STSS), as does Lancefield group A Streptococcus pyogenes (GAS). We sequenced the entire genome of SDSE strain GGS_124 isolated from a patient with STSS. Results We found that GGS_124 consisted of a circular genome of 2,106,340 bp. Comparative analyses among bacterial genomes indicated that GGS_124 was most closely related to GAS. GGS_124 and GAS, but not other streptococci, shared a number of virulence factor genes, including genes encoding streptolysin O, NADase, and streptokinase A, distantly related to SIC (DRS), suggesting the importance of these factors in the development of invasive disease. GGS_124 contained 3 prophages, with one containing a virulence factor gene for streptodornase. All 3 prophages were significantly similar to GAS prophages that carry virulence factor genes, indicating that these prophages had transferred these genes between pathogens. SDSE was found to contain a gene encoding a superantigen, streptococcal exotoxin type G, but lacked several genes present in GAS that encode virulence factors, such as other superantigens, cysteine protease speB, and hyaluronan synthase operon hasABC. Similar to GGS_124, the SDSE strains contained larger numbers of clustered, regularly interspaced, short palindromic repeats (CRISPR) spacers than did GAS, suggesting that horizontal gene transfer via streptococcal phages between SDSE and GAS is somewhat restricted, although they share phage species. Conclusion Genome wide comparisons of SDSE with GAS indicate that SDSE is closely and quantitatively related to GAS. SDSE, however, lacks several virulence factors of GAS, including superantigens, SPE-B and the hasABC operon. CRISPR spacers may limit the horizontal transfer of phage encoded GAS virulence genes into SDSE. These findings may provide clues for dissecting the pathological roles of the virulence factors in SDSE and GAS that cause STSS. PMID:21223537

Complete genome sequencing and analysis of a Lancefield group G Streptococcus dysgalactiae subsp. equisimilis strain causing streptococcal toxic shock syndrome (STSS).

PubMed

Shimomura, Yumi; Okumura, Kayo; Murayama, Somay Yamagata; Yagi, Junji; Ubukata, Kimiko; Kirikae, Teruo; Miyoshi-Akiyama, Tohru

2011-01-11

Streptococcus dysgalactiae subsp. equisimilis (SDSE) causes invasive streptococcal infections, including streptococcal toxic shock syndrome (STSS), as does Lancefield group A Streptococcus pyogenes (GAS). We sequenced the entire genome of SDSE strain GGS_124 isolated from a patient with STSS. We found that GGS_124 consisted of a circular genome of 2,106,340 bp. Comparative analyses among bacterial genomes indicated that GGS_124 was most closely related to GAS. GGS_124 and GAS, but not other streptococci, shared a number of virulence factor genes, including genes encoding streptolysin O, NADase, and streptokinase A, distantly related to SIC (DRS), suggesting the importance of these factors in the development of invasive disease. GGS_124 contained 3 prophages, with one containing a virulence factor gene for streptodornase. All 3 prophages were significantly similar to GAS prophages that carry virulence factor genes, indicating that these prophages had transferred these genes between pathogens. SDSE was found to contain a gene encoding a superantigen, streptococcal exotoxin type G, but lacked several genes present in GAS that encode virulence factors, such as other superantigens, cysteine protease speB, and hyaluronan synthase operon hasABC. Similar to GGS_124, the SDSE strains contained larger numbers of clustered, regularly interspaced, short palindromic repeats (CRISPR) spacers than did GAS, suggesting that horizontal gene transfer via streptococcal phages between SDSE and GAS is somewhat restricted, although they share phage species. Genome wide comparisons of SDSE with GAS indicate that SDSE is closely and quantitatively related to GAS. SDSE, however, lacks several virulence factors of GAS, including superantigens, SPE-B and the hasABC operon. CRISPR spacers may limit the horizontal transfer of phage encoded GAS virulence genes into SDSE. These findings may provide clues for dissecting the pathological roles of the virulence factors in SDSE and GAS that cause STSS.

Maltose Uptake by the Novel ABC Transport System MusEFGK2I Causes Increased Expression of ptsG in Corynebacterium glutamicum

PubMed Central

Henrich, Alexander; Kuhlmann, Nora; Eck, Alexander W.; Krämer, Reinhard

2013-01-01

The Gram-positive Corynebacterium glutamicum efficiently metabolizes maltose by a pathway involving maltodextrin and glucose formation by 4-α-glucanotransferase, glucose phosphorylation by glucose kinases, and maltodextrin degradation via maltodextrin phosphorylase and α-phosphoglucomutase. However, maltose uptake in C. glutamicum has not been investigated. Interestingly, the presence of maltose in the medium causes increased expression of ptsG in C. glutamicum by an unknown mechanism, although the ptsG-encoded glucose-specific EII permease of the phosphotransferase system itself is not required for maltose utilization. We identified the maltose uptake system as an ABC transporter encoded by musK (cg2708; ATPase subunit), musE (cg2705; substrate binding protein), musF (cg2704; permease), and musG (cg2703; permease) by combination of data obtained from characterization of maltose uptake and reanalyses of transcriptome data. Deletion of the mus gene cluster in C. glutamicum Δmus abolished maltose uptake and utilization. Northern blotting and reverse transcription-PCR experiments revealed that musK and musE are transcribed monocistronically, whereas musF and musG are part of an operon together with cg2701 (musI), which encodes a membrane protein of unknown function with no homologies to characterized proteins. Characterization of growth and [14C]maltose uptake in the musI insertion strain C. glutamicum IMcg2701 showed that musI encodes a novel essential component of the maltose ABC transporter of C. glutamicum. Finally, ptsG expression during cultivation on different carbon sources was analyzed in the maltose uptake-deficient strain C. glutamicum Δmus. Indeed, maltose uptake by the novel ABC transport system MusEFGK2I is required for the positive effect of maltose on ptsG expression in C. glutamicum. PMID:23543710

ArgR is an essential local transcriptional regulator of the arcABC operon in Streptococcus suis and is crucial for biological fitness in an acidic environment.

PubMed

Fulde, Marcus; Willenborg, Joerg; de Greeff, Astrid; Benga, Laurentiu; Smith, Hilde E; Valentin-Weigand, Peter; Goethe, Ralph

2011-02-01

Streptococcus suis is one of the most important pathogens in pigs and can also cause severe infections in humans. Despite its clinical relevance, very little is known about the factors that contribute to its virulence. Recently, we identified a new putative virulence factor in S. suis, the arginine deiminase system (ADS), an arginine catabolic enzyme system encoded by the arcABC operon, which enables S. suis to survive in an acidic environment. In this study, we focused on ArgR, an ADS-associated regulator belonging to the ArgR/AhrC arginine repressor family. Using an argR knockout strain we were able to show that ArgR is essential for arcABC operon expression and necessary for the biological fitness of S. suis. By cDNA expression microarray analyses and quantitative real-time RT-PCR we found that the arcABC operon is the only gene cluster regulated by ArgR, which is in contrast to the situation in many other bacteria. Reporter gene analysis with gfp under the control of the arcABC promoter demonstrated that ArgR is able to activate the arcABC promoter. Electrophoretic mobility shift assays with fragments of the arcABC promoter and recombinant ArgR, and chromatin immunoprecipitation with antibodies directed against ArgR, revealed that ArgR interacts with the arcABC promoter in vitro and in vivo by binding to a region from -147 to -72 bp upstream of the transcriptional start point. Overall, our results show that in S. suis, ArgR is an essential, system-specific transcriptional regulator of the ADS that interacts directly with the arcABC promoter in vivo.

Transcription of putative tonoplast transporters in response to glyphosate and paraquat stress in Conyza bonariensis and Conyza canadensis and selection of reference genes for qRT-PCR.

PubMed

Moretti, Marcelo L; Alárcon-Reverte, Rocio; Pearce, Stephen; Morran, Sarah; Hanson, Bradley D

2017-01-01

Herbicide resistance is a challenge for modern agriculture further complicated by cases of resistance to multiple herbicides. Conyza bonariensis and Conyza canadensis are invasive weeds of field crops, orchards, and non-cropped areas in many parts of the world. In California, USA, Conyza populations resistant to the herbicides glyphosate and paraquat have recently been described. Although the mechanism conferring resistance to glyphosate and paraquat in these species was not elucidated, reduced translocation of these herbicides was observed under experimental conditions in both species. Glyphosate and paraquat resistance associated with reduced translocation are hypothesized to be a result of sequestration of herbicides into the vacuole, with the possible involvement of over-expression of genes encoding tonoplast transporters of ABC-transporter families in cases of glyphosate resistance or cationic amino acid transporters (CAT) in cases of paraquat resistance. However, gene expression in response to herbicide treatment has not been studied in glyphosate and paraquat resistant populations. In the current study, we evaluated the transcript levels of genes possibly involved in resistance using real-time PCR. First, we evaluated eight candidate reference genes following herbicide treatment and selected three genes that exhibited stable expression profiles; ACTIN, HEAT-SHOCK-PROTEIN-70, and CYCLOPHILIN. The reference genes identified here can be used for further studies related to plant-herbicide interactions. We used these reference genes to assay the transcript levels of EPSPS, ABC transporters, and CAT in response to herbicide treatment in susceptible and resistant Conyza spp. lines. No transcription changes were observed in EPSPS or CAT genes after glyphosate or paraquat treatment, suggesting that these genes are not involved in the resistance mechanism. Transcription of the two ABC transporter genes increased following glyphosate treatment in all Conyza spp. lines. Transcription of ABC transporters also increased after paraquat treatment in all three lines of C. bonariensis. However, in C. canadensis, paraquat treatment increased transcription of only one ABC transporter gene in the susceptible line. The increase in transcription of ABC transporters after herbicide treatment is likely a stress response based on similar response observed across all Conyza lines regardless of resistance or sensitivity to glyphosate or paraquat, thus these genes do not appear to be directly involved in the mechanism of resistance in Conyza spp.

Transcription of putative tonoplast transporters in response to glyphosate and paraquat stress in Conyza bonariensis and Conyza canadensis and selection of reference genes for qRT-PCR

PubMed Central

Alárcon-Reverte, Rocio; Pearce, Stephen; Morran, Sarah; Hanson, Bradley D.

2017-01-01

Herbicide resistance is a challenge for modern agriculture further complicated by cases of resistance to multiple herbicides. Conyza bonariensis and Conyza canadensis are invasive weeds of field crops, orchards, and non-cropped areas in many parts of the world. In California, USA, Conyza populations resistant to the herbicides glyphosate and paraquat have recently been described. Although the mechanism conferring resistance to glyphosate and paraquat in these species was not elucidated, reduced translocation of these herbicides was observed under experimental conditions in both species. Glyphosate and paraquat resistance associated with reduced translocation are hypothesized to be a result of sequestration of herbicides into the vacuole, with the possible involvement of over-expression of genes encoding tonoplast transporters of ABC-transporter families in cases of glyphosate resistance or cationic amino acid transporters (CAT) in cases of paraquat resistance. However, gene expression in response to herbicide treatment has not been studied in glyphosate and paraquat resistant populations. In the current study, we evaluated the transcript levels of genes possibly involved in resistance using real-time PCR. First, we evaluated eight candidate reference genes following herbicide treatment and selected three genes that exhibited stable expression profiles; ACTIN, HEAT-SHOCK-PROTEIN-70, and CYCLOPHILIN. The reference genes identified here can be used for further studies related to plant-herbicide interactions. We used these reference genes to assay the transcript levels of EPSPS, ABC transporters, and CAT in response to herbicide treatment in susceptible and resistant Conyza spp. lines. No transcription changes were observed in EPSPS or CAT genes after glyphosate or paraquat treatment, suggesting that these genes are not involved in the resistance mechanism. Transcription of the two ABC transporter genes increased following glyphosate treatment in all Conyza spp. lines. Transcription of ABC transporters also increased after paraquat treatment in all three lines of C. bonariensis. However, in C. canadensis, paraquat treatment increased transcription of only one ABC transporter gene in the susceptible line. The increase in transcription of ABC transporters after herbicide treatment is likely a stress response based on similar response observed across all Conyza lines regardless of resistance or sensitivity to glyphosate or paraquat, thus these genes do not appear to be directly involved in the mechanism of resistance in Conyza spp. PMID:28700644

Living colors in the gray mold pathogen Botrytis cinerea: codon-optimized genes encoding green fluorescent protein and mCherry, which exhibit bright fluorescence.

PubMed

Leroch, Michaela; Mernke, Dennis; Koppenhoefer, Dieter; Schneider, Prisca; Mosbach, Andreas; Doehlemann, Gunther; Hahn, Matthias

2011-05-01

The green fluorescent protein (GFP) and its variants have been widely used in modern biology as reporters that allow a variety of live-cell imaging techniques. So far, GFP has rarely been used in the gray mold fungus Botrytis cinerea because of low fluorescence intensity. The codon usage of B. cinerea genes strongly deviates from that of commonly used GFP-encoding genes and reveals a lower GC content than other fungi. In this study, we report the development and use of a codon-optimized version of the B. cinerea enhanced GFP (eGFP)-encoding gene (Bcgfp) for improved expression in B. cinerea. Both the codon optimization and, to a smaller extent, the insertion of an intron resulted in higher mRNA levels and increased fluorescence. Bcgfp was used for localization of nuclei in germinating spores and for visualizing host penetration. We further demonstrate the use of promoter-Bcgfp fusions for quantitative evaluation of various toxic compounds as inducers of the atrB gene encoding an ABC-type drug efflux transporter of B. cinerea. In addition, a codon-optimized mCherry-encoding gene was constructed which yielded bright red fluorescence in B. cinerea.

Prediction and analysis of three gene families related to leaf rust (Puccinia triticina) resistance in wheat (Triticum aestivum L.).

PubMed

Peng, Fred Y; Yang, Rong-Cai

2017-06-20

The resistance to leaf rust (Lr) caused by Puccinia triticina in wheat (Triticum aestivum L.) has been well studied over the past decades with over 70 Lr genes being mapped on different chromosomes and numerous QTLs (quantitative trait loci) being detected or mapped using DNA markers. Such resistance is often divided into race-specific and race-nonspecific resistance. The race-nonspecific resistance can be further divided into resistance to most or all races of the same pathogen and resistance to multiple pathogens. At the molecular level, these three types of resistance may cover across the whole spectrum of pathogen specificities that are controlled by genes encoding different protein families in wheat. The objective of this study is to predict and analyze genes in three such families: NBS-LRR (nucleotide-binding sites and leucine-rich repeats or NLR), START (Steroidogenic Acute Regulatory protein [STaR] related lipid-transfer) and ABC (ATP-Binding Cassette) transporter. The focus of the analysis is on the patterns of relationships between these protein-coding genes within the gene families and QTLs detected for leaf rust resistance. We predicted 526 ABC, 1117 NLR and 144 START genes in the hexaploid wheat genome through a domain analysis of wheat proteome. Of the 1809 SNPs from leaf rust resistance QTLs in seedling and adult stages of wheat, 126 SNPs were found within coding regions of these genes or their neighborhood (5 Kb upstream from transcription start site [TSS] or downstream from transcription termination site [TTS] of the genes). Forty-three of these SNPs for adult resistance and 18 SNPs for seedling resistance reside within coding or neighboring regions of the ABC genes whereas 14 SNPs for adult resistance and 29 SNPs for seedling resistance reside within coding or neighboring regions of the NLR gene. Moreover, we found 17 nonsynonymous SNPs for adult resistance and five SNPs for seedling resistance in the ABC genes, and five nonsynonymous SNPs for adult resistance and six SNPs for seedling resistance in the NLR genes. Most of these coding SNPs were predicted to alter encoded amino acids and such information may serve as a starting point towards more thorough molecular and functional characterization of the designated Lr genes. Using the primer sequences of 99 known non-SNP markers from leaf rust resistance QTLs, we found candidate genes closely linked to these markers, including Lr34 with distances to its two gene-specific markers being 1212 bases (to cssfr1) and 2189 bases (to cssfr2). This study represents a comprehensive analysis of ABC, NLR and START genes in the hexaploid wheat genome and their physical relationships with QTLs for leaf rust resistance at seedling and adult stages. Our analysis suggests that the ABC (and START) genes are more likely to be co-located with QTLs for race-nonspecific, adult resistance whereas the NLR genes are more likely to be co-located with QTLs for race-specific resistance that would be often expressed at the seedling stage. Though our analysis was hampered by inaccurate or unknown physical positions of numerous QTLs due to the incomplete assembly of the complex hexaploid wheat genome that is currently available, the observed associations between (i) QTLs for race-specific resistance and NLR genes and (ii) QTLs for nonspecific resistance and ABC genes will help discover SNP variants for leaf rust resistance at seedling and adult stages. The genes containing nonsynonymous SNPs are promising candidates that can be investigated in future studies as potential new sources of leaf rust resistance in wheat breeding.

The ABC of Biofilm Drug Tolerance: the MerR-Like Regulator BrlR Is an Activator of ABC Transport Systems, with PA1874-77 Contributing to the Tolerance of Pseudomonas aeruginosa Biofilms to Tobramycin.

PubMed

Poudyal, Bandita; Sauer, Karin

2018-02-01

A hallmark of biofilms is their tolerance to killing by antimicrobial agents. In Pseudomonas aeruginosa , biofilm drug tolerance requires the c-di-GMP-responsive MerR transcriptional regulator BrlR. However, the mechanism by which BrlR mediates biofilm drug tolerance has not been elucidated. Here, we demonstrate that BrlR activates the expression of at least 7 ABC transport systems, including the PA1874-PA1875-PA1876-PA1877 (PA1874-77) operon, with chromatin immunoprecipitation and DNA binding assays confirming BrlR binding to the promoter region of PA1874-77. Insertional inactivation of the 7 ABC transport systems rendered P. aeruginosa PAO1 biofilms susceptible to tobramycin or norfloxacin. Susceptibility was linked to drug accumulation, with BrlR contributing to norfloxacin accumulation in a manner dependent on multidrug efflux pumps and the PA1874-77 ABC transport system. Inactivation of the respective ABC transport system, furthermore, eliminated the recalcitrance of biofilms to killing by tobramycin but not norfloxacin, indicating that drug accumulation is not linked to biofilm drug tolerance. Our findings indicate for the first time that BrlR, a MerR-type transcriptional activator, activates genes encoding several ABC transport systems, in addition to multiple multidrug efflux pump genes. Moreover, our data confirm a BrlR target contributing to drug tolerance, likely countering the prevailing dogma that biofilm tolerance arises from a multiplicity of factors. Copyright © 2018 American Society for Microbiology.

Novel 2,4-Dichlorophenoxyacetic Acid Degradation Genes from Oligotrophic Bradyrhizobium sp. Strain HW13 Isolated from a Pristine Environment

PubMed Central

Kitagawa, Wataru; Takami, Sachiko; Miyauchi, Keisuke; Masai, Eiji; Kamagata, Yoichi; Tiedje, James M.; Fukuda, Masao

2002-01-01

The tfd genes of Ralstonia eutropha JMP134 are the only well-characterized set of genes responsible for 2,4-dichlorophenoxyacetic acid (2,4-D) degradation among 2,4-D-degrading bacteria. A new family of 2,4-D degradation genes, cadRABKC, was cloned and characterized from Bradyrhizobium sp. strain HW13, a strain that was isolated from a buried Hawaiian soil that has never experienced anthropogenic chemicals. The cadR gene was inferred to encode an AraC/XylS type of transcriptional regulator from its deduced amino acid sequence. The cadABC genes were predicted to encode 2,4-D oxygenase subunits from their deduced amino acid sequences that showed 46, 44, and 37% identities with the TftA and TftB subunits of 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) oxygenase of Burkholderia cepacia AC1100 and with a putative ferredoxin, ThcC, of Rhodococcus erythropolis NI86/21, respectively. They are thoroughly different from the 2,4-D dioxygenase gene, tfdA, of R. eutropha JMP134. The cadK gene was presumed to encode a 2,4-D transport protein from its deduced amino acid sequence that showed 60% identity with the 2,4-D transporter, TfdK, of strain JMP134. Sinorhizobium meliloti Rm1021 cells containing cadRABKC transformed several phenoxyacetic acids, including 2,4-D and 2,4,5-T, to corresponding phenol derivatives. Frameshift mutations indicated that each of the cadRABC genes was essential for 2,4-D conversion in strain Rm1021 but that cadK was not. Five 2,4-D degraders, including Bradyrhizobium and Sphingomonas strains, were found to have cadA gene homologs, suggesting that these 2,4-D degraders share 2,4-D degradation genes similar to those of strain HW13 cadABC. PMID:11751829

A Cluster of Five Genes Essential for the Utilization of Dihydroxamate Xenosiderophores in Synechocystis sp. PCC 6803.

PubMed

Obando S, Tobias A; Babykin, Michael M; Zinchenko, Vladislav V

2018-05-21

The unicellular freshwater cyanobacterium Synechocystis sp. PCC 6803 is capable of using dihydroxamate xenosiderophores, either ferric schizokinen (FeSK) or a siderophore of the filamentous cyanobacterium Anabaena variabilis ATCC 29413 (SAV), as the sole source of iron in the TonB-dependent manner. The fecCDEB1-schT gene cluster encoding a siderophore transport system that is involved in the utilization of FeSK and SAV in Synechocystis sp. PCC 6803 was identified. The gene schT encodes TonB-dependent outer membrane transporter, whereas the remaining four genes encode the ABC-type transporter FecB1CDE formed by the periplasmic binding protein FecB1, the transmembrane permease proteins FecC and FecD, and the ATPase FecE. Inactivation of any of these genes resulted in the inability of cells to utilize FeSK and SAV. Our data strongly suggest that Synechocystis sp. PCC 6803 can readily internalize Fe-siderophores via the classic TonB-dependent transport system.

Role of the Fur Regulon in Iron Transport in Bacillus subtilis

PubMed Central

Ollinger, Juliane; Song, Kyung-Bok; Antelmann, Haike; Hecker, Michael; Helmann, John D.

2006-01-01

The Bacillus subtilis ferric uptake regulator (Fur) protein mediates the iron-dependent repression of at least 20 operons encoding ∼40 genes. We investigated the physiological roles of Fur-regulated genes by the construction of null mutations in 14 transcription units known or predicted to function in siderophore biosynthesis or iron uptake. We demonstrate that ywbLMN, encoding an elemental iron uptake system orthologous to the copper oxidase-dependent Fe(III) uptake system of Saccharomyces cerevisiae, is essential for growth in low iron minimal medium lacking citric acid. 2,3-Dihydroxybenzoyl-glycine (Itoic acid), the siderophore precursor produced by laboratory strains of B. subtilis, is of secondary importance. In the presence of citrate, the YfmCDEF ABC transporter is required for optimal growth. B. subtilis is unable to grow in minimal medium containing the iron chelator EDDHA unless the ability to synthesize the intact bacillibactin siderophore is restored (by the introduction of a functional sfp gene) or exogenous siderophores are provided. Utilization of the catecholate siderophores bacillibactin and enterobactin requires the FeuABC importer and the YusV ATPase. Utilization of hydroxamate siderophores requires the FhuBGC ABC transporter together with the FhuD (ferrichrome) or YxeB (ferrioxamine) substrate-binding proteins. Growth with schizokinen or arthrobactin is at least partially dependent on the YfhA YfiYZ importer and the YusV ATPase. We have also investigated the effects of a fur mutation on the proteome and documented the derepression of 11 Fur-regulated proteins, including a newly identified thioredoxin reductase homolog, YcgT. PMID:16672620

Role of the Fur regulon in iron transport in Bacillus subtilis.

PubMed

Ollinger, Juliane; Song, Kyung-Bok; Antelmann, Haike; Hecker, Michael; Helmann, John D

2006-05-01

The Bacillus subtilis ferric uptake regulator (Fur) protein mediates the iron-dependent repression of at least 20 operons encoding approximately 40 genes. We investigated the physiological roles of Fur-regulated genes by the construction of null mutations in 14 transcription units known or predicted to function in siderophore biosynthesis or iron uptake. We demonstrate that ywbLMN, encoding an elemental iron uptake system orthologous to the copper oxidase-dependent Fe(III) uptake system of Saccharomyces cerevisiae, is essential for growth in low iron minimal medium lacking citric acid. 2,3-Dihydroxybenzoyl-glycine (Itoic acid), the siderophore precursor produced by laboratory strains of B. subtilis, is of secondary importance. In the presence of citrate, the YfmCDEF ABC transporter is required for optimal growth. B. subtilis is unable to grow in minimal medium containing the iron chelator EDDHA unless the ability to synthesize the intact bacillibactin siderophore is restored (by the introduction of a functional sfp gene) or exogenous siderophores are provided. Utilization of the catecholate siderophores bacillibactin and enterobactin requires the FeuABC importer and the YusV ATPase. Utilization of hydroxamate siderophores requires the FhuBGC ABC transporter together with the FhuD (ferrichrome) or YxeB (ferrioxamine) substrate-binding proteins. Growth with schizokinen or arthrobactin is at least partially dependent on the YfhA YfiYZ importer and the YusV ATPase. We have also investigated the effects of a fur mutation on the proteome and documented the derepression of 11 Fur-regulated proteins, including a newly identified thioredoxin reductase homolog, YcgT.

Genes of the N-Methylglutamate Pathway Are Essential for Growth of Methylobacterium extorquens DM4 with Monomethylamine

PubMed Central

Gruffaz, Christelle; Muller, Emilie E. L.; Louhichi-Jelail, Yousra; Nelli, Yella R.; Guichard, Gilles

2014-01-01

Monomethylamine (MMA, CH3NH2) can be used as a carbon and nitrogen source by many methylotrophic bacteria. Methylobacterium extorquens DM4 lacks the MMA dehydrogenase encoded by mau genes, which in M. extorquens AM1 is essential for growth on MMA. Identification and characterization of minitransposon mutants with an MMA-dependent phenotype showed that strain DM4 grows with MMA as the sole source of carbon, energy, and nitrogen by the N-methylglutamate (NMG) pathway. Independent mutations were found in a chromosomal region containing the genes gmaS, mgsABC, and mgdABCD for the three enzymes of the pathway, γ-glutamylmethylamide (GMA) synthetase, NMG synthase, and NMG dehydrogenase, respectively. Reverse transcription-PCR confirmed the operonic structure of the two divergent gene clusters mgsABC-gmaS and mgdABCD and their induction during growth with MMA. The genes mgdABCD and mgsABC were found to be essential for utilization of MMA as a carbon and nitrogen source. The gene gmaS was essential for MMA utilization as a carbon source, but residual growth of mutant DM4gmaS growing with succinate and MMA as a nitrogen source was observed. Plasmid copies of gmaS and the gmaS homolog METDI4690, which encodes a protein 39% identical to GMA synthetase, fully restored the ability of mutants DM4gmaS and DM4gmaSΔmetdi4690 to use MMA as a carbon and nitrogen source. Similarly, chemically synthesized GMA, the product of GMA synthetase, could be used as a nitrogen source for growth in the wild-type strain, as well as in DM4gmaS and DM4gmaSΔmetdi4690 mutants. The NADH:ubiquinone oxidoreductase respiratory complex component NuoG was also found to be essential for growth with MMA as a carbon source. PMID:24682302

Trafficking and processing of bacterial proteins by mammalian cells: Insights from chondroitinase ABC.

PubMed

Muir, Elizabeth; Raza, Mansoor; Ellis, Clare; Burnside, Emily; Love, Fiona; Heller, Simon; Elliot, Matthew; Daniell, Esther; Dasgupta, Debayan; Alves, Nuno; Day, Priscilla; Fawcett, James; Keynes, Roger

2017-01-01

There is very little reported in the literature about the relationship between modifications of bacterial proteins and their secretion by mammalian cells that synthesize them. We previously reported that the secretion of the bacterial enzyme Chondroitinase ABC by mammalian cells requires the strategic removal of at least three N-glycosylation sites. The aim of this study was to determine if it is possible to enhance the efficacy of the enzyme as a treatment for spinal cord injury by increasing the quantity of enzyme secreted or by altering its cellular location. To determine if the efficiency of enzyme secretion could be further increased, cells were transfected with constructs encoding the gene for chondroitinase ABC modified for expression by mammalian cells; these contained additional modifications of strategic N-glycosylation sites or alternative signal sequences to direct secretion of the enzyme from the cells. We show that while removal of certain specific N-glycosylation sites enhances enzyme secretion, N-glycosylation of at least two other sites, N-856 and N-773, is essential for both production and secretion of active enzyme. Furthermore, we find that the signal sequence directing secretion also influences the quantity of enzyme secreted, and that this varies widely amongst the cell types tested. Last, we find that replacing the 3'UTR on the cDNA encoding Chondroitinase ABC with that of β-actin is sufficient to target the enzyme to the neuronal growth cone when transfected into neurons. This also enhances neurite outgrowth on an inhibitory substrate. Some intracellular trafficking pathways are adversely affected by cryptic signals present in the bacterial gene sequence, whilst unexpectedly others are required for efficient secretion of the enzyme. Furthermore, targeting chondroitinase to the neuronal growth cone promotes its ability to increase neurite outgrowth on an inhibitory substrate. These findings are timely in view of the renewed prospects for gene therapy, and of direct relevance to strategies aimed at expressing foreign proteins in mammalian cells, in particular bacterial proteins.

Biochemical characterization of an ABC transporter LptBFGC complex required for the outer membrane sorting of lipopolysaccharides.

PubMed

Narita, Shin-ichiro; Tokuda, Hajime

2009-07-07

Seven Lpt proteins (A through G) are thought to be involved in lipopolysaccharide transport from the inner to outer membrane of Escherichia coli. LptB belongs to the ATP-binding cassette transporter superfamily. Although the lptB gene lacks neighboring genes encoding membrane subunits, bioinformatic analyses recently indicated that two distantly located consecutive genes, lptF and lptG, could encode membrane subunits. To examine this possibility, LptB was expressed with LptF and LptG. We report here that both LptF and LptG formed a complex with LptB. Furthermore, an inner membrane protein, LptC, which had been implicated in lipopolysaccharide transport, was also included in this complex.

Genome-Wide Identification, Characterization and Phylogenetic Analysis of ATP-Binding Cassette (ABC) Transporter Genes in Common Carp (Cyprinus carpio).

PubMed

Liu, Xiang; Li, Shangqi; Peng, Wenzhu; Feng, Shuaisheng; Feng, Jianxin; Mahboob, Shahid; Al-Ghanim, Khalid A; Xu, Peng

2016-01-01

The ATP-binding cassette (ABC) gene family is considered to be one of the largest gene families in all forms of prokaryotic and eukaryotic life. Although the ABC transporter genes have been annotated in some species, detailed information about the ABC superfamily and the evolutionary characterization of ABC genes in common carp (Cyprinus carpio) are still unclear. In this research, we identified 61 ABC transporter genes in the common carp genome. Phylogenetic analysis revealed that they could be classified into seven subfamilies, namely 11 ABCAs, six ABCBs, 19 ABCCs, eight ABCDs, two ABCEs, four ABCFs, and 11 ABCGs. Comparative analysis of the ABC genes in seven vertebrate species including common carp, showed that at least 10 common carp genes were retained from the third round of whole genome duplication, while 12 duplicated ABC genes may have come from the fourth round of whole genome duplication. Gene losses were also observed for 14 ABC genes. Expression profiles of the 61 ABC genes in six common carp tissues (brain, heart, spleen, kidney, intestine, and gill) revealed extensive functional divergence among the ABC genes. Different copies of some genes had tissue-specific expression patterns, which may indicate some gene function specialization. This study provides essential genomic resources for future studies in common carp.

Genome-Wide Identification, Characterization and Phylogenetic Analysis of ATP-Binding Cassette (ABC) Transporter Genes in Common Carp (Cyprinus carpio)

PubMed Central

Peng, Wenzhu; Feng, Shuaisheng; Feng, Jianxin; Mahboob, Shahid; Al-Ghanim, Khalid A.

2016-01-01

The ATP-binding cassette (ABC) gene family is considered to be one of the largest gene families in all forms of prokaryotic and eukaryotic life. Although the ABC transporter genes have been annotated in some species, detailed information about the ABC superfamily and the evolutionary characterization of ABC genes in common carp (Cyprinus carpio) are still unclear. In this research, we identified 61 ABC transporter genes in the common carp genome. Phylogenetic analysis revealed that they could be classified into seven subfamilies, namely 11 ABCAs, six ABCBs, 19 ABCCs, eight ABCDs, two ABCEs, four ABCFs, and 11 ABCGs. Comparative analysis of the ABC genes in seven vertebrate species including common carp, showed that at least 10 common carp genes were retained from the third round of whole genome duplication, while 12 duplicated ABC genes may have come from the fourth round of whole genome duplication. Gene losses were also observed for 14 ABC genes. Expression profiles of the 61 ABC genes in six common carp tissues (brain, heart, spleen, kidney, intestine, and gill) revealed extensive functional divergence among the ABC genes. Different copies of some genes had tissue-specific expression patterns, which may indicate some gene function specialization. This study provides essential genomic resources for future studies in common carp. PMID:27058731

Multiple ABC glucoside transporters mediate sugar-stimulated growth in the heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120.

PubMed

Nieves-Morión, Mercedes; Flores, Enrique

2018-02-01

Cyanobacteria are generally capable of photoautotrophic growth and are widely distributed on Earth. The model filamentous, heterocyst-forming strain Anabaena sp. PCC 7120 has long been considered as a strict photoautotroph but is now known to be able to assimilate fructose. We have previously described two components of ABC glucoside uptake transporters from Anabaena that are involved in uptake of the sucrose analog esculin: GlsC [a nucleotide-binding domain subunit (NBD)] and GlsP [a transmembrane component (TMD)]. Here, we created Anabaena mutants of genes encoding three further ABC transporter components needed for esculin uptake: GlsD (NBD), GlsQ (TMD) and GlsR (periplasmic substrate-binding protein). Phototrophic growth of Anabaena was significantly stimulated by sucrose, fructose and glucose. Whereas the glsC and glsD mutants were drastically hampered in sucrose-stimulated growth, the different gls mutants were generally impaired in sugar-dependent growth. Our results suggest the participation of Gls and other ABC transporters encoded in the Anabaena genome in sugar-stimulated growth. Additionally, Gls transporter components influence the function of septal junctions in the Anabaena filament. We suggest that mixotrophic growth is important in cyanobacterial physiology and may be relevant for the wide success of these organisms in diverse environments. © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

Genetic Analysis of Benzothiophene Biodesulfurization Pathway of Gordonia terrae Strain C-6

PubMed Central

Lian, Kehui; Zhang, Yue; Tian, Huimei; Ji, Kaihua; Li, Guoqiang

2013-01-01

Sulfur can be removed from benzothiophene (BT) by some bacteria without breaking carbon-carbon bonds. However, a clear mechanism for BT desulfurization and its genetic components have not been reported in literatures so far. In this study, we used comparative transcriptomics to study differential expression of genes in Gordonia terrae C-6 cultured with BT or sodium sulfate as the sole source of sulfur. We found that 135 genes were up-regulated with BT relative to sodium sulfate as the sole sulfur source. Many of these genes encode flavin-dependent monooxygenases, alkane sulfonate monooxygenases and desulfinase, which perform similar functions to those involved in the 4S pathway of dibenzothiophene (DBT) biodesulfurization. Three of the genes were found to be located in the same operon, designated bdsABC. Cell extracts of pET28a-bdsABC transfected E. coli Rosetta (DE3) converted BT to a phenolic compound, identified as o-hydroxystyrene. These results advance our understanding of enzymes involved in the BT biodesulfurization pathway. PMID:24367657

Evolutionary Analysis of MIKCc-Type MADS-Box Genes in Gymnosperms and Angiosperms

PubMed Central

Chen, Fei; Zhang, Xingtan; Liu, Xing; Zhang, Liangsheng

2017-01-01

MIKCc-type MADS-box genes encode transcription factors that control floral organ morphogenesis and flowering time in flowering plants. Here, in order to determine when the subfamilies of MIKCc originated and their early evolutionary trajectory, we sampled and analyzed the genomes and large-scale transcriptomes representing all the orders of gymnosperms and basal angiosperms. Through phylogenetic inference, the MIKCc-type MADS-box genes were subdivided into 14 monophyletic clades. Among them, the gymnosperm orthologs of AGL6, SEP, AP1, GMADS, SOC1, AGL32, AP3/PI, SVP, AGL15, ANR1, and AG were identified. We identified and characterized the origin of a novel subfamily GMADS within gymnosperms but lost orthologs in monocots and Brassicaceae. ABCE model prototype genes were relatively conserved in terms of gene number in gymnosperms, but expanded in angiosperms, whereas SVP, SOC1, and GMADS had dramatic expansions in gymnosperms but conserved in angiosperms. Our results provided the most detailed evolutionary history of all MIKCc gene clades in gymnosperms and angiosperms. We proposed that although the near complete set of MIKCc genes had evolved in gymnosperms, the duplication and expressional transition of ABCE model MIKCc genes in the ancestor of angiosperms triggered the first flower. PMID:28611810

Positive regulation of Leptospira interrogans kdp expression by KdpE as Demonstrated with a novel β-galactosidase reporter in Leptospira biflexa.

PubMed

Matsunaga, James; Coutinho, Mariana L

2012-08-01

Leptospirosis is a potentially deadly zoonotic disease that afflicts humans and animals. Leptospira interrogans, the predominant agent of leptospirosis, encounters diverse conditions as it proceeds through its life cycle, which includes stages inside and outside the host. Unfortunately, the number of genetic tools available for examining the regulation of gene expression in L. interrogans is limited. Consequently, little is known about the genetic circuits that control gene expression in Leptospira. To better understand the regulation of leptospiral gene expression, the L. interrogans kdp locus, encoding homologs of the P-type ATPase KdpABC potassium transporter with their KdpD sensors and KdpE response regulators, was selected for analysis. We showed that a kdpE mutation in L. interrogans prevented the increase in kdpABC mRNA levels observed in the wild-type L. interrogans strain when external potassium levels were low. To confirm that KdpE was a positive regulator of kdpABC transcription, we developed a novel approach for constructing chromosomal genetic fusions to the endogenous bgaL (β-galactosidase) gene of the nonpathogen Leptospira biflexa. We demonstrated positive regulation of a kdpA'-bgaL fusion in L. biflexa by the L. interrogans KdpE response regulator. A control lipL32'-bgaL fusion was not regulated by KdpE. These results demonstrate the utility of genetic fusions to the bgaL gene of L. biflexa for examining leptospiral gene regulation.

SPIB and BATF provide alternate determinants of IRF4 occupancy in diffuse large B-cell lymphoma linked to disease heterogeneity

PubMed Central

Care, Matthew A.; Cocco, Mario; Laye, Jon P.; Barnes, Nicholas; Huang, Yuanxue; Wang, Ming; Barrans, Sharon; Du, Ming; Jack, Andrew; Westhead, David R.; Doody, Gina M.; Tooze, Reuben M.

2014-01-01

Interferon regulatory factor 4 (IRF4) is central to the transcriptional network of activated B-cell-like diffuse large B-cell lymphoma (ABC-DLBCL), an aggressive lymphoma subgroup defined by gene expression profiling. Since cofactor association modifies transcriptional regulatory input by IRF4, we assessed genome occupancy by IRF4 and endogenous cofactors in ABC-DLBCL cell lines. IRF4 partners with SPIB, PU.1 and BATF genome-wide, but SPIB provides the dominant IRF4 partner in this context. Upon SPIB knockdown IRF4 occupancy is depleted and neither PU.1 nor BATF acutely compensates. Integration with ENCODE data from lymphoblastoid cell line GM12878, demonstrates that IRF4 adopts either SPIB- or BATF-centric genome-wide distributions in related states of post-germinal centre B-cell transformation. In primary DLBCL high-SPIB and low-BATF or the reciprocal low-SPIB and high-BATF mRNA expression links to differential gene expression profiles across nine data sets, identifying distinct associations with SPIB occupancy, signatures of B-cell differentiation stage and potential pathogenetic mechanisms. In a population-based patient cohort, SPIBhigh/BATFlow-ABC-DLBCL is enriched for mutation of MYD88, and SPIBhigh/BATFlow-ABC-DLBCL with MYD88-L265P mutation identifies a small subgroup of patients among this otherwise aggressive disease subgroup with distinct favourable outcome. We conclude that differential expression of IRF4 cofactors SPIB and BATF identifies biologically and clinically significant heterogeneity among ABC-DLBCL. PMID:24875472

The ATP-binding cassette transporter Cbc (choline/betaine/carnitine) recruits multiple substrate-binding proteins with strong specificity for distinct quaternary ammonium compounds

PubMed Central

Chen, Chiliang; Malek, Adel A.; Wargo, Matthew J.; Hogan, Deborah A.; Beattie, Gwyn A.

2017-01-01

Summary We identified a choline, betaine and carnitine transporter, designated Cbc, from Pseudomonas syringae and Pseudomonas aeruginosa that is unusual among members of the ATP-binding cassette (ABC) transporter family in its use of multiple periplasmic substrate-binding proteins (SBPs) that are highly specific for their substrates. The SBP encoded by the cbcXWV operon, CbcX, binds choline with a high affinity (Km, 2.6 μM) and, although it also binds betaine (Km, 24.2 μM), CbcXWV-mediated betaine uptake did not occur in the presence of choline. The CbcX orthologue ChoX from Sinorhizobium meliloti was similar to CbcX in these binding properties. The core transporter CbcWV also interacts with the carnitine-specific SBP CaiX (Km, 24 μM) and the betaine-specific SBP BetX (Km, 0.6 μM). Unlike most ABC transporter loci, caiX, betX and cbcXWV are separated in the genome. CaiX-mediated carnitine uptake was reduced by CbcX and BetX only when they were bound by their individual ligands, providing the first in vivo evidence for a higher affinity for ligand-bound than ligand-free SBPs by an ABC transporter. These studies demonstrate not only that the Cbc transporter serves as a useful model for exploring ABC transporter component interactions, but also that the orphan SBP genes common to bacterial genomes can encode functional SBPs. PMID:19919675

The ATP-binding cassette transporter Cbc (choline/betaine/carnitine) recruits multiple substrate-binding proteins with strong specificity for distinct quaternary ammonium compounds.

PubMed

Chen, Chiliang; Malek, Adel A; Wargo, Matthew J; Hogan, Deborah A; Beattie, Gwyn A

2010-01-01

We identified a choline, betaine and carnitine transporter, designated Cbc, from Pseudomonas syringae and Pseudomonas aeruginosa that is unusual among members of the ATP-binding cassette (ABC) transporter family in its use of multiple periplasmic substrate-binding proteins (SBPs) that are highly specific for their substrates. The SBP encoded by the cbcXWV operon, CbcX, binds choline with a high affinity (K(m), 2.6 microM) and, although it also binds betaine (K(m), 24.2 microM), CbcXWV-mediated betaine uptake did not occur in the presence of choline. The CbcX orthologue ChoX from Sinorhizobium meliloti was similar to CbcX in these binding properties. The core transporter CbcWV also interacts with the carnitine-specific SBP CaiX (K(m), 24 microM) and the betaine-specific SBP BetX (K(m), 0.6 microM). Unlike most ABC transporter loci, caiX, betX and cbcXWV are separated in the genome. CaiX-mediated carnitine uptake was reduced by CbcX and BetX only when they were bound by their individual ligands, providing the first in vivo evidence for a higher affinity for ligand-bound than ligand-free SBPs by an ABC transporter. These studies demonstrate not only that the Cbc transporter serves as a useful model for exploring ABC transporter component interactions, but also that the orphan SBP genes common to bacterial genomes can encode functional SBPs.

Genome-Wide Identification of Differentially Expressed Genes Associated with the High Yielding of Oleoresin in Secondary Xylem of Masson Pine (Pinus massoniana Lamb) by Transcriptomic Analysis

PubMed Central

Liu, Qinghua; Zhou, Zhichun; Wei, Yongcheng; Shen, Danyu; Feng, Zhongping; Hong, Shanping

2015-01-01

Masson pine is an important timber and resource for oleoresin in South China. Increasing yield of oleoresin in stems can raise economic benefits and enhance the resistance to bark beetles. However, the genetic mechanisms for regulating the yield of oleoresin were still unknown. Here, high-throughput sequencing technology was used to investigate the transcriptome and compare the gene expression profiles of high and low oleoresin-yielding genotypes. A total of 40,690,540 reads were obtained and assembled into 137,499 transcripts from the secondary xylem tissues. We identified 84,842 candidate unigenes based on sequence annotation using various databases and 96 unigenes were candidates for terpenoid backbone biosynthesis in pine. By comparing the expression profiles of high and low oleoresin-yielding genotypes, 649 differentially expressed genes (DEGs) were identified. GO enrichment analysis of DEGs revealed that multiple pathways were related to high yield of oleoresin. Nine candidate genes were validated by QPCR analysis. Among them, the candidate genes encoding geranylgeranyl diphosphate synthase (GGPS) and (-)-alpha/beta-pinene synthase were up-regulated in the high oleoresin-yielding genotype, while tricyclene synthase revealed lower expression level, which was in good agreement with the GC/MS result. In addition, DEG encoding ABC transporters, pathogenesis-related proteins (PR5 and PR9), phosphomethylpyrimidine synthase, non-specific lipid-transfer protein-like protein and ethylene responsive transcription factors (ERFs) were also confirmed to be critical for the biosynthesis of oleoresin. The next-generation sequencing strategy used in this study has proven to be a powerful means for analyzing transcriptome variation related to the yield of oleoresin in masson pine. The candidate genes encoding GGPS, (-)-alpha/beta-pinene, tricyclene synthase, ABC transporters, non-specific lipid-transfer protein-like protein, phosphomethylpyrimidine synthase, ERFs and pathogen responses may play important roles in regulating the yield of oleoresin. These DEGs are worthy of special attention in future studies. PMID:26167875

Cloning and Characterization of Benzoate Catabolic Genes in the Gram-Positive Polychlorinated Biphenyl Degrader Rhodococcus sp. Strain RHA1

PubMed Central

Kitagawa, Wataru; Miyauchi, Keisuke; Masai, Eiji; Fukuda, Masao

2001-01-01

Benzoate catabolism is thought to play a key role in aerobic bacterial degradation of biphenyl and polychlorinated biphenyls (PCBs). Benzoate catabolic genes were cloned from a PCB degrader, Rhodococcus sp. strain RHA1, by using PCR amplification and temporal temperature gradient electrophoresis separation. A nucleotide sequence determination revealed that the deduced amino acid sequences encoded by the RHA1 benzoate catabolic genes, benABCDK, exhibit 33 to 65% identity with those of Acinetobacter sp. strain ADP1. The gene organization of the RHA1 benABCDK genes differs from that of ADP1. The RHA1 benABCDK region was localized on the chromosome, in contrast to the biphenyl catabolic genes, which are located on linear plasmids. Escherichia coli cells containing RHA1 benABCD transformed benzoate to catechol via 2-hydro-1,2-dihydroxybenzoate. They transformed neither 2- nor 4-chlorobenzoates but did transform 3-chlorobenzoate. The RHA1 benA gene was inactivated by insertion of a thiostrepton resistance gene. The resultant mutant strain, RBD169, neither grew on benzoate nor transformed benzoate, and it did not transform 3-chlorobenzoate. It did, however, exhibit diminished growth on biphenyl and growth repression in the presence of a high concentration of biphenyl (13 mM). These results indicate that the cloned benABCD genes could play an essential role not only in benzoate catabolism but also in biphenyl catabolism in RHA1. Six rhodococcal benzoate degraders were found to have homologs of RHA1 benABC. In contrast, two rhodococcal strains that cannot transform benzoate were found not to have RHA1 benABC homologs, suggesting that many Rhodococcus strains contain benzoate catabolic genes similar to RHA1 benABC. PMID:11673430

Genetic characterization of plasmid-associated triphenylmethane reductase in Listeria monocytogenes.

PubMed

Dutta, Vikrant; Elhanafi, Driss; Osborne, Jason; Martinez, Mira Rakic; Kathariou, Sophia

2014-09-01

The enzyme triphenylmethane reductase (TMR) reduces toxic triphenylmethane dyes into colorless, nontoxic derivatives, and TMR-producing microorganisms have been proposed as bioremediation tools. Analysis of the genome of Listeria monocytogenes H7858 (1998-1999 hot dog outbreak) revealed that the plasmid (pLM80) of this strain harboring a gene cassette (bcrABC) conferring resistance to benzalkonium chloride (BC) and other quaternary ammonium disinfectants also harbored a gene (tmr) highly homologous to TMR-encoding genes from diverse Gram-negative bacteria. The pLM80-associated tmr was located two genes downstream of bcrABC as part of a putative IS1216 composite transposon. To confirm the role of tmr in triphenylmethane dye detoxification, we introduced various tmr-harboring fragments of pLM80 in a pLM80-cured derivative of strain H7550, from the same outbreak as H7858, and assessed the resistance of the constructs to the triphenylmethane dyes crystal violet (CV) and malachite green. Transcriptional and subcloning data suggest that the regulation of TMR is complex. Constructs harboring fragments spanning bcrABC and tmr were CV resistant, and in such constructs tmr transcription was induced by sublethal levels of either BC or CV. However, constructs harboring only tmr and its upstream intergenic region could also confer resistance to CV, albeit at lower levels. Screening a panel of BC-resistant L. monocytogenes strains revealed that all those harboring bcrABC and adjacent pLM80 sequences, including tmr, were resistant to CV and decolorized this dye. The findings suggest a potential role of TMR as a previously unknown adaptive attribute for environmental persistence of L. monocytogenes. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Gene expression in Listeria monocytogenes exposed to sublethal concentration of benzalkonium chloride.

PubMed

Tamburro, Manuela; Ripabelli, Giancarlo; Vitullo, Monia; Dallman, Timothy James; Pontello, Mirella; Amar, Corinne Francoise Laurence; Sammarco, Michela Lucia

2015-06-01

In this study, tolerance at sublethal concentration of benzalkonium chloride and transcription levels of mdrL, ladR, lde, sigB and bcrABC genes in Listeria monocytogenes strains were evaluated. Viable cells reduction occurred in 45% of strains and clinical isolates showed lower sensitivity than isolates from foods. An increased transcription of an efflux system encoding gene was found in 60% of strains, and simultaneous mdrL overexpression and ladR underexpression occurred in 30% of isolates. A significant association between reduced benzalkonium chloride activity and both mdrL and sigB overexpression was observed; sigB expression also correlated with both mdrL and ladR genes. The bcrABC gene was only found in six strains, all isolated from foods and sensitive to benzalkonium chloride, and in four strains an underexpression was observed. Disinfection at sublethal concentration was less effective in clinical isolates, and mdrL and sigB expression was significantly affected by disinfection. Further insights are needed to understand the adaptation to benzalkonium chloride and to evaluate whether changes in gene expression could affect the L. monocytogenes virulence traits and persistence in the environment. Copyright © 2015 Elsevier Ltd. All rights reserved.

Growth of Aerobic Ripening Bacteria at the Cheese Surface Is Limited by the Availability of Iron

PubMed Central

Back, Alexandre; Irlinger, Françoise

2012-01-01

The microflora on the surface of smear-ripened cheeses is composed of various species of bacteria and yeasts that contribute to the production of the desired organoleptic properties. The objective of the present study was to show that iron availability is a limiting factor in the growth of typical aerobic ripening bacteria in cheese. For that purpose, we investigated the effect of iron or siderophore addition in model cheeses that were coinoculated with a yeast and a ripening bacterium. Both iron and the siderophore desferrioxamine B stimulated the growth of ripening bacteria belonging to the genera Arthrobacter, Corynebacterium, and Brevibacterium. The extent of stimulation was strain dependent, and generally, the effect of desferrioxamine B was greater than that of iron. Measurements of the expression of genes related to the metabolism of iron by Arthrobacter arilaitensis Re117 by real-time reverse transcription-PCR showed that these genes were transcribed during growth in cheese. The addition of desferrioxamine B increased the expression of two genes encoding iron-siderophore ABC transport binding proteins. The addition of iron decreased the expression of siderophore biosynthesis genes and of part of the genes encoding iron-siderophore ABC transport components. It was concluded that iron availability is a limiting factor in the growth of typical cheese surface bacteria. The selection of strains with efficient iron acquisition systems may be useful for the development of defined-strain surface cultures. Furthermore, the importance of iron metabolism in the microbial ecology of cheeses should be investigated since it may result in positive or negative microbial interactions. PMID:22367081

Structure, Regulation, and Putative Function of the Arginine Deiminase System of Streptococcus suis

PubMed Central

Gruening, Petra; Fulde, Marcus; Valentin-Weigand, Peter; Goethe, Ralph

2006-01-01

Streptococcus suis is an important cause of infectious diseases in young pigs. Little is known about the virulence factors or protective antigens of S. suis. Recently, we have identified two proteins of the arginine deiminase system (ADS) of S. suis, which were temperature induced and expressed on the streptococcal surface (N. Winterhoff, R. Goethe, P. Gruening, M. Rohde, H. Kalisz, H. E. Smith, and P. Valentin-Weigand, J. Bacteriol. 184:6768-6776, 2002). In the present study, we analyzed the complete ADS of S. suis. Due to their homologies to the recently published S. gordonii ADS genes, the genes for arginine deiminase, ornithine carbamoyl-transferase, and carbamate kinase, which were previously designated adiS, octS, and ckS, respectively, were renamed arcA, arcB, and arcC, respectively. Our data revealed that arcA, arcB, and arcC of the S. suis ADS are transcribed from an operon (arcABC operon). Additionally, putative ADS-associated genes were cloned and sequenced which, however, did not belong to the arcABC operon. These were the flpS gene upstream of the arcABC operon with homology to the flp transcription regulator of S. gordonii and the arcD, arcT, arcH, and argR genes downstream of the arcABC operon with high homologies to a putative arginine-ornithine antiporter, a putative dipeptidase of S. gordonii, a putative β-N-acetylhexosaminidase of S. pneumoniae, and a putative arginine repressor of S. gordonii, respectively. The transcriptional start point of the arcABC operon was determined, and promoter analysis provided evidence that multiple factors contribute to the regulation of the ADS. Thus, a putative binding site for a transcription regulator of the Crp/Fnr family, an ArgR-binding site, and two cis-acting catabolite response elements were identified in the promoter-operator region of the operon. Consistent with this, we could demonstrate that the ADS of S. suis is inducible by arginine and reduced O2 tension and subject to carbon catabolite repression. Furthermore, comparing an arcA knockout mutant in which expression of the three operon-encoded proteins was abolished with the parental wild-type strain showed that the arcABC operon of S. suis contributes to survival under acidic conditions. PMID:16385025

Structure, regulation, and putative function of the arginine deiminase system of Streptococcus suis.

PubMed

Gruening, Petra; Fulde, Marcus; Valentin-Weigand, Peter; Goethe, Ralph

2006-01-01

Streptococcus suis is an important cause of infectious diseases in young pigs. Little is known about the virulence factors or protective antigens of S. suis. Recently, we have identified two proteins of the arginine deiminase system (ADS) of S. suis, which were temperature induced and expressed on the streptococcal surface (N. Winterhoff, R. Goethe, P. Gruening, M. Rohde, H. Kalisz, H. E. Smith, and P. Valentin-Weigand, J. Bacteriol. 184:6768-6776, 2002). In the present study, we analyzed the complete ADS of S. suis. Due to their homologies to the recently published S. gordonii ADS genes, the genes for arginine deiminase, ornithine carbamoyl-transferase, and carbamate kinase, which were previously designated adiS, octS, and ckS, respectively, were renamed arcA, arcB, and arcC, respectively. Our data revealed that arcA, arcB, and arcC of the S. suis ADS are transcribed from an operon (arcABC operon). Additionally, putative ADS-associated genes were cloned and sequenced which, however, did not belong to the arcABC operon. These were the flpS gene upstream of the arcABC operon with homology to the flp transcription regulator of S. gordonii and the arcD, arcT, arcH, and argR genes downstream of the arcABC operon with high homologies to a putative arginine-ornithine antiporter, a putative dipeptidase of S. gordonii, a putative beta-N-acetylhexosaminidase of S. pneumoniae, and a putative arginine repressor of S. gordonii, respectively. The transcriptional start point of the arcABC operon was determined, and promoter analysis provided evidence that multiple factors contribute to the regulation of the ADS. Thus, a putative binding site for a transcription regulator of the Crp/Fnr family, an ArgR-binding site, and two cis-acting catabolite response elements were identified in the promoter-operator region of the operon. Consistent with this, we could demonstrate that the ADS of S. suis is inducible by arginine and reduced O2 tension and subject to carbon catabolite repression. Furthermore, comparing an arcA knockout mutant in which expression of the three operon-encoded proteins was abolished with the parental wild-type strain showed that the arcABC operon of S. suis contributes to survival under acidic conditions.

Rice Ferredoxin-Dependent Glutamate Synthase Regulates Nitrogen-Carbon Metabolomes and Is Genetically Differentiated between japonica and indica Subspecies.

PubMed

Yang, Xiaolu; Nian, Jinqiang; Xie, Qingjun; Feng, Jian; Zhang, Fengxia; Jing, Hongwei; Zhang, Jian; Dong, Guojun; Liang, Yan; Peng, Juli; Wang, Guodong; Qian, Qian; Zuo, Jianru

2016-11-07

Plants assimilate inorganic nitrogen absorbed from soil into organic forms as Gln and Glu through the glutamine synthetase/glutamine:2-oxoglutarate amidotransferase (GS/GOGAT) cycle. Whereas GS catalyzes the formation of Gln from Glu and ammonia, GOGAT catalyzes the transfer of an amide group from Gln to 2-oxoglutarate to produce two molecules of Glu. However, the regulatory role of the GS/GOGAT cycle in the carbon-nitrogen balance is not well understood. Here, we report the functional characterization of rice ABNORMAL CYTOKININ RESPONSE 1 (ABC1) gene that encodes a ferredoxin-dependent (Fd)-GOGAT. The weak mutant allele abc1-1 mutant shows a typical nitrogen-deficient syndrome, whereas the T-DNA insertional mutant abc1-2 is seedling lethal. Metabolomics analysis revealed the accumulation of an excessive amount of amino acids with high N/C ratio (Gln and Asn) and several intermediates in the tricarboxylic acid cycle in abc1-1, suggesting that ABC1 plays a critical role in nitrogen assimilation and carbon-nitrogen balance. Five non-synonymous single-nucleotide polymorphisms were identified in the ABC1 coding region and characterized as three distinct haplotypes, which have been highly and specifically differentiated between japonica and indica subspecies. Collectively, these results suggest that ABC1/OsFd-GOGAT is essential for plant growth and development by modulating nitrogen assimilation and the carbon-nitrogen balance. Copyright © 2016 The Author. Published by Elsevier Inc. All rights reserved.

Transcriptomic insights on the ABC transporter gene family in the salmon louse Caligus rogercresseyi.

PubMed

Valenzuela-Muñoz, Valentina; Sturm, Armin; Gallardo-Escárate, Cristian

2015-04-09

ATP-binding cassette (ABC) protein family encode for membrane proteins involved in the transport of various biomolecules through the cellular membrane. These proteins have been identified in all taxa and present important physiological functions, including the process of insecticide detoxification in arthropods. For that reason the ectoparasite Caligus rogercresseyi represents a model species for understanding the molecular underpinnings involved in insecticide drug resistance. llumina sequencing was performed using sea lice exposed to 2 and 3 ppb of deltamethrin and azamethiphos. Contigs obtained from de novo assembly were annotated by Blastx. RNA-Seq analysis was performed and validated by qPCR analysis. From the transcriptome database of C. rogercresseyi, 57 putative members of ABC protein sequences were identified and phylogenetically classified into the eight subfamilies described for ABC transporters in arthropods. Transcriptomic profiles for ABC proteins subfamilies were evaluated throughout C. rogercresseyi development. Moreover, RNA-Seq analysis was performed for adult male and female salmon lice exposed to the delousing drugs azamethiphos and deltamethrin. High transcript levels of the ABCB and ABCC subfamilies were evidenced. Furthermore, SNPs mining was carried out for the ABC proteins sequences, revealing pivotal genomic information. The present study gives a comprehensive transcriptome analysis of ABC proteins from C. rogercresseyi, providing relevant information about transporter roles during ontogeny and in relation to delousing drug responses in salmon lice. This genomic information represents a valuable tool for pest management in the Chilean salmon aquaculture industry.

The Yersinia pestis Siderophore, Yersiniabactin, and the ZnuABC system both contribute to Zinc acquisition and the development of lethal septicemic plague in mice

PubMed Central

Bobrov, Alexander G.; Kirillina, Olga; Fetherston, Jacqueline D.; Miller, M. Clarke; Burlison, Joseph A.; Perry, Robert D.

2014-01-01

Summary Bacterial pathogens must overcome host sequestration of zinc (Zn2+), an essential micronutrient, during the infectious disease process. While the mechanisms to acquire chelated Zn2+ by bacteria are largely undefined, many pathogens rely upon the ZnuABC family of ABC transporters. Here we show that in Yersinia pestis, irp2, a gene encoding the synthetase (HMWP2) for the siderophore yersiniabactin (Ybt) is required for growth under Zn2+-deficient conditions in a strain lacking ZnuABC. Moreover, growth stimulation with exogenous, purified apo-Ybt provides evidence that Ybt may serve as a zincophore for Zn2+ acquisition. Studies with the Zn2+-dependent transcriptional reporter znuA∷lacZ indicate that the ability to synthesize Ybt affects the levels of intracellular Zn2+. However, the outer membrane receptor Psn and TonB as well as the inner membrane (IM) ABC transporter YbtPQ, that are required for Fe3+ acquisition by Ybt, are not needed for Ybt-dependent Zn2+ uptake. In contrast, the predicted IM protein YbtX, a member of the Major Facilitator Superfamily, was essential for Ybt-dependent Zn2+ uptake. Finally, we show that the ZnuABC system and the Ybt synthetase HMWP2, presumably by Ybt synthesis, both contribute to the development of a lethal infection in a septicemic plague mouse model. PMID:24979062

Genome-wide identification, phylogenetic analysis, and expression profiles of ATP-binding cassette transporter genes in the oriental fruit fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae).

PubMed

Xiao, Lin-Fan; Zhang, Wei; Jing, Tian-Xing; Zhang, Meng-Yi; Miao, Ze-Qing; Wei, Dan-Dan; Yuan, Guo-Rui; Wang, Jin-Jun

2018-03-01

The ATP-binding cassette (ABC) is the largest transporter gene family and the genes play key roles in xenobiotic resistance, metabolism, and development of all phyla. However, the specific functions of ABC gene families in insects is unclear. We report a genome-wide identification, phylogenetic, and transcriptional analysis of the ABC genes in the oriental fruit fly, Bactrocera dorsalis (Hendel). We identified a total of 47 ABC genes (BdABCs) from the transcriptomic and genomic databases of B. dorsalis and classified these genes into eight subfamilies (A-H), including 7 ABCAs, 7 ABCBs, 9 ABCCs, 2 ABCDs, 1 ABCE, 3 ABCFs, 15 ABCGs, and 3 ABCHs. Comparative phylogenetic analysis of the ABCs suggests an orthologous relationship between B. dorsalis and other insect species in which these genes have been related to pesticide resistance and essential biological processes. Comparison of transcriptome and relative expression patterns of BdABCs indicated diverse multifunctions within different B. dorsalis tissues. The expression of 4, 10, and 14 BdABCs from 18 BdABCs was significantly upregulated after exposure to LD 50 s of malathion, avermectin, and beta-cypermethrin, respectively. The maximum expression level of most BdABCs (including BdABCFs, BdABCGs, and BdABCHs) occurred at 48h post exposures, whereas BdABCEs peaked at 24h after treatment. Furthermore, RNA interference-mediated suppression of BdABCB7 resulted in increased toxicity of malathion against B. dorsalis. These data suggest that ABC transporter genes might play key roles in xenobiotic metabolism and biosynthesis in B. dorsalis. Copyright © 2017 Elsevier Inc. All rights reserved.

Evolutionary Analysis of MIKCc-Type MADS-Box Genes in Gymnosperms and Angiosperms.

PubMed

Chen, Fei; Zhang, Xingtan; Liu, Xing; Zhang, Liangsheng

2017-01-01

MIKC c -type MADS-box genes encode transcription factors that control floral organ morphogenesis and flowering time in flowering plants. Here, in order to determine when the subfamilies of MIKC c originated and their early evolutionary trajectory, we sampled and analyzed the genomes and large-scale transcriptomes representing all the orders of gymnosperms and basal angiosperms. Through phylogenetic inference, the MIKC c -type MADS-box genes were subdivided into 14 monophyletic clades. Among them, the gymnosperm orthologs of AGL6, SEP , AP1 , GMADS , SOC1 , AGL32 , AP3 / PI , SVP , AGL15 , ANR1 , and AG were identified. We identified and characterized the origin of a novel subfamily GMADS within gymnosperms but lost orthologs in monocots and Brassicaceae. ABCE model prototype genes were relatively conserved in terms of gene number in gymnosperms, but expanded in angiosperms, whereas SVP , SOC1 , and GMADS had dramatic expansions in gymnosperms but conserved in angiosperms. Our results provided the most detailed evolutionary history of all MIKC c gene clades in gymnosperms and angiosperms. We proposed that although the near complete set of MIKC c genes had evolved in gymnosperms, the duplication and expressional transition of ABCE model MIKC c genes in the ancestor of angiosperms triggered the first flower.

An ABC Transporter System of Yersinia pestis Allows Utilization of Chelated Iron by Escherichia coli SAB11

PubMed Central

Bearden, Scott W.; Staggs, Teanna M.; Perry, Robert D.

1998-01-01

The acquisition of iron is an essential component in the pathogenesis of Yersinia pestis, the agent of bubonic and pneumonic plague. A cosmid library derived from the genomic DNA of Y. pestis KIM6+ was used for transduction of an Escherichia coli mutant (SAB11) defective in the biosynthesis of the siderophore enterobactin. Recombinant plasmids which had a common 13-kb BamHI fragment were isolated from SAB11 transductants in which growth but not enterobactin synthesis was restored on media containing the iron chelator EDDA [ethylenediamine-di(o-hydroxyphenyl acetic acid)]. Subcloning and transposon mutagenesis revealed a 5.6-kb region, designated yfe, essential for SAB11 growth stimulation. In vitro transcription-translation analysis identified polypeptides of 18, 29.5, 32, and 33 kDa encoded by the yfe locus. Sequence analysis shows this locus to be comprised of five genes in two separate operons which have potential Fur-binding sequences in both promoters. A putative polycistronic operon, yfeABCD, is Fur regulated and responds to iron and manganese. A functional Fur protein is required for the observed manganese repression of this operon. This operon encodes polypeptides which have strong similarity to the ATP-binding cassette (ABC) family of transporters and include a periplasmic binding protein (YfeA), an ATP-binding protein (YfeB), and two integral membrane proteins (YfeC and -D), which likely function in the acquisition of inorganic iron and possibly other ions. The ∼21-kDa protein encoded by the separately transcribed yfeE gene may be located in the cell envelope, since a yfeE::TnphoA fusion is PhoA+. Mutations in this gene abrogate growth of SAB11 on iron-chelated media. PMID:9495751

An ABC transporter system of Yersinia pestis allows utilization of chelated iron by Escherichia coli SAB11.

PubMed

Bearden, S W; Staggs, T M; Perry, R D

1998-03-01

The acquisition of iron is an essential component in the pathogenesis of Yersinia pestis, the agent of bubonic and pneumonic plague. A cosmid library derived from the genomic DNA of Y. pestis KIM6+ was used for transduction of an Escherichia coli mutant (SAB11) defective in the biosynthesis of the siderophore enterobactin. Recombinant plasmids which had a common 13-kb BamHI fragment were isolated from SAB11 transductants in which growth but not enterobactin synthesis was restored on media containing the iron chelator EDDA [ethylenediamine-di(o-hydroxyphenyl acetic acid)]. Subcloning and transposon mutagenesis revealed a 5.6-kb region, designated yfe, essential for SAB11 growth stimulation. In vitro transcription-translation analysis identified polypeptides of 18, 29.5, 32, and 33 kDa encoded by the yfe locus. Sequence analysis shows this locus to be comprised of five genes in two separate operons which have potential Fur-binding sequences in both promoters. A putative polycistronic operon, yfeABCD, is Fur regulated and responds to iron and manganese. A functional Fur protein is required for the observed manganese repression of this operon. This operon encodes polypeptides which have strong similarity to the ATP-binding cassette (ABC) family of transporters and include a periplasmic binding protein (YfeA), an ATP-binding protein (YfeB), and two integral membrane proteins (YfeC and -D), which likely function in the acquisition of inorganic iron and possibly other ions. The approximately 21-kDa protein encoded by the separately transcribed yfeE gene may be located in the cell envelope, since a yfeE::TnphoA fusion is PhoA+. Mutations in this gene abrogate growth of SAB11 on iron-chelated media.

Regulation of Sulfur Assimilation Pathways in Burkholderia cenocepacia through Control of Genes by the SsuR Transcription Factor▿

PubMed Central

Łochowska, Anna; Iwanicka-Nowicka, Roksana; Zielak, Agata; Modelewska, Anna; Thomas, Mark S.; Hryniewicz, Monika M.

2011-01-01

The genome of Burkholderia cenocepacia contains two genes encoding closely related LysR-type transcriptional regulators, CysB and SsuR, involved in control of sulfur assimilation processes. In this study we show that the function of SsuR is essential for the utilization of a number of organic sulfur sources of either environmental or human origin. Among the genes upregulated by SsuR identified here are the tauABC operon encoding a predicted taurine transporter, three tauD-type genes encoding putative taurine dioxygenases, and atsA encoding a putative arylsulfatase. The role of SsuR in expression of these genes/operons was characterized through (i) construction of transcriptional reporter fusions to candidate promoter regions and analysis of their expression in the presence/absence of SsuR and (ii) testing the ability of SsuR to bind SsuR-responsive promoter regions. We also demonstrate that expression of SsuR-activated genes is not repressed in the presence of inorganic sulfate. A more detailed analysis of four SsuR-responsive promoter regions indicated that ∼44 bp of the DNA sequence preceding and/or overlapping the predicted −35 element of such promoters is sufficient for SsuR binding. The DNA sequence homology among SsuR “recognition motifs” at different responsive promoters appears to be limited. PMID:21317335

BmeRABC5 is a multidrug efflux system that can confer metronidazole resistance in Bacteroides fragilis.

PubMed

Pumbwe, Lilian; Chang, Abraham; Smith, Rachel L; Wexler, Hannah M

2007-01-01

The RND-family efflux pump gene bmeB5 was previously shown to be overexpressed in metronidazole-resistant laboratory mutants of Bacteroides fragilis. In the present study, we characterized the bmeABC5 genes and an upstream putative TetR-family regulator gene (bmeR5). bmeR5 (645 bp) was located 51 bp upstream of bmeA5 and encoded a 24.9-kDa protein. Deletant strains lacking bmeB5 or bmeR5 were constructed from a wild-type B. fragilis strain ADB77. Strain antimicrobial susceptibility was determined and gene expression was quantified. bmeR5 was overexpressed in Escherichia coli using a 6x-His tag system; BmeR5-His6 was isolated from inclusion bodies and its binding to bmeABC5 promoter regions was determined. BmeR5-His6 bound specifically to the bmeR5-bmeC5 intergenic region (IT1). Deletion of bmeR5 (ADB77DeltabmeR5) resulted in a significant (p < 0.05) increase in expression of bmeA5, bmeB5, and bmeC5, and > two-fold increase in minimum inhibitory concentrations (MICs) of ampicillin, cefoxitin, cefoperazone, ciprofloxacin, imipenem, metronidazole, ethidium bromide, and sodium dodecyl sulfate (SDS). MICs were reduced by the efflux pump inhibitor carbonyl cyanide m-chlorophenyl hydrazone (CCCP). The MICs of ampicillin, cefoperazone, metronidazole, and SDS were reduced by approximately two-fold in ADB77DeltabmeB5. A multidrug (metronidazole)-resistant, nim-negative B. fragilis clinical isolate overexpressed bmeABC5 genes, had a G-->T point mutation in IT1, and significantly reduced binding to BmeR5-His6. These data demonstrate that BmeR5 is a local repressor of bmeABC5 expression and that mutations in IT1 can lead to a derepression and resistance to multiple antimicrobial agents, including metronidazole.

Increased expression of the yeast multidrug resistance ABC transporter Pdr18 leads to increased ethanol tolerance and ethanol production in high gravity alcoholic fermentation

PubMed Central

2012-01-01

Background The understanding of the molecular basis of yeast tolerance to ethanol may guide the design of rational strategies to increase process performance in industrial alcoholic fermentations. A set of 21 genes encoding multidrug transporters from the ATP-Binding Cassette (ABC) Superfamily and Major Facilitator Superfamily (MFS) in S. cerevisiae were scrutinized for a role in ethanol stress resistance. Results A yeast multidrug resistance ABC transporter encoded by the PDR18 gene, proposed to play a role in the incorporation of ergosterol in the yeast plasma membrane, was found to confer resistance to growth inhibitory concentrations of ethanol. PDR18 expression was seen to contribute to decreased 3 H-ethanol intracellular concentrations and decreased plasma membrane permeabilization of yeast cells challenged with inhibitory ethanol concentrations. Given the increased tolerance to ethanol of cells expressing PDR18, the final concentration of ethanol produced during high gravity alcoholic fermentation by yeast cells devoid of PDR18 was lower than the final ethanol concentration produced by the corresponding parental strain. Moreover, an engineered yeast strain in which the PDR18 promoter was replaced in the genome by the stronger PDR5 promoter, leading to increased PDR18 mRNA levels during alcoholic fermentation, was able to attain a 6 % higher ethanol concentration and a 17 % higher ethanol production yield than the parental strain. The improved fermentative performance of yeast cells over-expressing PDR18 was found to correlate with their increased ethanol tolerance and ability to restrain plasma membrane permeabilization induced throughout high gravity fermentation. Conclusions PDR18 gene over-expression increases yeast ethanol tolerance and fermentation performance leading to the production of highly inhibitory concentrations of ethanol. PDR18 overexpression in industrial yeast strains appears to be a promising approach to improve alcoholic fermentation performance for sustainable bio-ethanol production. PMID:22839110

Mutations in sit B and sit D genes affect manganese-growth requirements in Sinorhizobium meliloti.

PubMed

Platero, Raúl A; Jaureguy, Melina; Battistoni, Federico J; Fabiano, Elena R

2003-01-21

Two transposon-induced mutants of Sinorhizobium meliloti 242 were isolated based on their inability to grow on rich medium supplemented with the metal chelator ethylenediamine di-o-hydroxyphenylacetic acid (EDDHA) and either heme-compounds or siderophores as iron sources. Tagged loci of these mutants were identified as sit B and sit D genes. These genes encode components of an ABC (ATP-binding cassette) metal-type permease in several Gram-negative bacteria. In this work, the phenotypes of these two mutants were compared with those of two siderophore-mediated iron transport mutants. The results strongly implicate a role of the sit genes in manganese acquisition when this metal is limiting in S. meliloti.

Diversity of ABC transporter genes across the plant kingdom and their potential utility in biotechnology.

PubMed

Lane, Thomas S; Rempe, Caroline S; Davitt, Jack; Staton, Margaret E; Peng, Yanhui; Soltis, Douglas Edward; Melkonian, Michael; Deyholos, Michael; Leebens-Mack, James H; Chase, Mark; Rothfels, Carl J; Stevenson, Dennis; Graham, Sean W; Yu, Jun; Liu, Tao; Pires, J Chris; Edger, Patrick P; Zhang, Yong; Xie, Yinlong; Zhu, Ying; Carpenter, Eric; Wong, Gane Ka-Shu; Stewart, C Neal

2016-05-31

The ATP-binding cassette (ABC) transporter gene superfamily is ubiquitous among extant organisms and prominently represented in plants. ABC transporters act to transport compounds across cellular membranes and are involved in a diverse range of biological processes. Thus, the applicability to biotechnology is vast, including cancer resistance in humans, drug resistance among vertebrates, and herbicide and other xenobiotic resistance in plants. In addition, plants appear to harbor the highest diversity of ABC transporter genes compared with any other group of organisms. This study applied transcriptome analysis to survey the kingdom-wide ABC transporter diversity in plants and suggest biotechnology applications of this diversity. We utilized sequence similarity-based informatics techniques to infer the identity of ABC transporter gene candidates from 1295 phylogenetically-diverse plant transcriptomes. A total of 97,149 putative (approximately 25 % were full-length) ABC transporter gene members were identified; each RNA-Seq library (plant sample) had 88 ± 30 gene members. As expected, simpler organisms, such as algae, had fewer unique members than vascular land plants. Differences were also noted in the richness of certain ABC transporter subfamilies. Land plants had more unique ABCB, ABCC, and ABCG transporter gene members on average (p < 0.005), and green algae, red algae, and bryophytes had significantly more ABCF transporter gene members (p < 0.005). Ferns had significantly fewer ABCA transporter gene members than all other plant groups (p < 0.005). We present a transcriptomic overview of ABC transporter gene members across all major plant groups. An increase in the number of gene family members present in the ABCB, ABCC, and ABCD transporter subfamilies may indicate an expansion of the ABC transporter superfamily among green land plants, which include all crop species. The striking difference between the number of ABCA subfamily transporter gene members between ferns and other plant taxa is surprising and merits further investigation. Discussed is the potential exploitation of ABC transporters in plant biotechnology, with an emphasis on crops.

Selenium-Dependent Biogenesis of Formate Dehydrogenase in Campylobacter jejuni Is Controlled by the fdhTU Accessory Genes

PubMed Central

Shaw, Frances L.; Mulholland, Francis; Le Gall, Gwénaëlle; Porcelli, Ida; Hart, Dave J.; Pearson, Bruce M.

2012-01-01

The food-borne bacterial pathogen Campylobacter jejuni efficiently utilizes organic acids such as lactate and formate for energy production. Formate is rapidly metabolized via the activity of the multisubunit formate dehydrogenase (FDH) enzyme, of which the FdhA subunit is predicted to contain a selenocysteine (SeC) amino acid. In this study we investigated the function of the cj1500 and cj1501 genes of C. jejuni, demonstrate that they are involved in selenium-controlled production of FDH, and propose the names fdhT and fdhU, respectively. Insertional inactivation of fdhT or fdhU in C. jejuni resulted in the absence of FdhA and FdhB protein expression, reduced fdhABC RNA levels, the absence of FDH enzyme activity, and the lack of formate utilization, as assessed by 1H nuclear magnetic resonance. The fdhABC genes are transcribed from a single promoter located two genes upstream of fdhA, and the decrease in fdhABC RNA levels in the fdhU mutant is mediated at the posttranscriptional level. FDH activity and the ability to utilize formate were restored by genetic complementation with fdhU and by supplementation of the growth media with selenium dioxide. Disruption of SeC synthesis by inactivation of the selA and selB genes also resulted in the absence of FDH activity, which could not be restored by selenium supplementation. Comparative genomic analysis suggests a link between the presence of selA and fdhTU orthologs and the predicted presence of SeC in FdhA. The fdhTU genes encode accessory proteins required for FDH expression and activity in C. jejuni, possibly by contributing to acquisition or utilization of selenium. PMID:22609917

Regulation of the CgPdr1 Transcription Factor from the Pathogen Candida glabrata ▿

PubMed Central

Paul, Sanjoy; Schmidt, Jennifer A.; Moye-Rowley, W. Scott

2011-01-01

Candida glabrata is an opportunistic human pathogen that is increasingly associated with candidemia, owing in part to the intrinsic and acquired high tolerance the organism exhibits for the important clinical antifungal drug fluconazole. This elevated fluconazole resistance often develops through gain-of-function mutations in the zinc cluster-containing transcriptional regulator C. glabrata Pdr1 (CgPdr1). CgPdr1 induces the expression of an ATP-binding cassette (ABC) transporter-encoding gene, CgCDR1. Saccharomyces cerevisiae has two CgPdr1 homologues called ScPdr1 and ScPdr3. These factors control the expression of an ABC transporter-encoding gene called ScPDR5, which encodes a homologue of CgCDR1. Loss of the mitochondrial genome (ρ0 cell) or overexpression of the mitochondrial enzyme ScPsd1 induces ScPDR5 expression in a strictly ScPdr3-dependent fashion. ScPdr3 requires the presence of a transcriptional Mediator subunit called Gal11 (Med15) to fully induce ScPDR5 transcription in response to ρ0 signaling. ScPdr1 does not respond to either ρ0 signals or ScPsd1 overproduction. In this study, we employed transcriptional fusions between CgPdr1 target promoters, like CgCDR1, to demonstrate that CgPdr1 stimulates gene expression via binding to elements called pleiotropic drug response elements (PDREs). Deletion mapping and electrophoretic mobility shift assays demonstrated that a single PDRE in the CgCDR1 promoter was capable of supporting ρ0-induced gene expression. Removal of one of the two ScGal11 homologues from C. glabrata caused a major defect in drug-induced expression of CgCDR1 but had a quantitatively minor effect on ρ0-stimulated transcription. These data demonstrate that CgPdr1 appears to combine features of ScPdr1 and ScPdr3 to produce a transcription factor with chimeric regulatory properties. PMID:21131438

Inactivation and deficiency of core proteins of photosystems I and II caused by genetical phylloquinone and plastoquinone deficiency but retained lamellar structure in a T-DNA mutant of Arabidopsis.

PubMed

Shimada, Hiroshi; Ohno, Ryoichi; Shibata, Masaru; Ikegami, Isamu; Onai, Kiyoshi; Ohto, Masa-aki; Takamiya, Ken-ichiro

2005-02-01

Phylloquinone, a substituted 1,4-naphthoquinone with an 18-carbon-saturated phytyl tail, functions as a bound one-electron carrier cofactor at the A1 site of photosystem I (PSI). A Feldmann tag line mutant, no. 2755 (designated as abc4 hereafter), showed pale-green young leaves and white old leaves. The mutated nuclear gene encoded 1,4-dihydroxy-2-naphtoic acid phytyltransferase, an enzyme of phylloquinone biosynthesis, and high-performance liquid chromatography analysis revealed that the abc4 mutant contained no phylloquinone, and only about 3% plastoquinone. Photooxidation of P700 of PSI in the abc4 mutant was not observed, and reduced-versus-oxidized difference spectroscopy indicated that the abc4 mutant had no P700. The maximum quantum yield of photosystem II (PSII) in the abc4 mutant was much decreased, and the electron transfer from PSII to PSI in the abc4 mutant did not occur. For the pale-green leaves of the abc4 mutant plant, the ultrastructure of the chloroplasts was almost the same as that of the wild-type plant. However, the chloroplasts in the albino leaves of the mutant were smaller and had a lot of grana thylakoids and few stroma thylakoids. The amounts of PSI and PSII core subunits in the abc4 mutant were significantly decreased compared with those in the wild type. These results suggested that a deficiency of phylloquinone in PSI caused the abolishment of PSI and a partial defect of PSII due to a significant decrease of plastoquinone, but did not influence the ultrastructure of the chloroplasts in young leaves.

mRMR-ABC: A Hybrid Gene Selection Algorithm for Cancer Classification Using Microarray Gene Expression Profiling

PubMed Central

Alshamlan, Hala; Badr, Ghada; Alohali, Yousef

2015-01-01

An artificial bee colony (ABC) is a relatively recent swarm intelligence optimization approach. In this paper, we propose the first attempt at applying ABC algorithm in analyzing a microarray gene expression profile. In addition, we propose an innovative feature selection algorithm, minimum redundancy maximum relevance (mRMR), and combine it with an ABC algorithm, mRMR-ABC, to select informative genes from microarray profile. The new approach is based on a support vector machine (SVM) algorithm to measure the classification accuracy for selected genes. We evaluate the performance of the proposed mRMR-ABC algorithm by conducting extensive experiments on six binary and multiclass gene expression microarray datasets. Furthermore, we compare our proposed mRMR-ABC algorithm with previously known techniques. We reimplemented two of these techniques for the sake of a fair comparison using the same parameters. These two techniques are mRMR when combined with a genetic algorithm (mRMR-GA) and mRMR when combined with a particle swarm optimization algorithm (mRMR-PSO). The experimental results prove that the proposed mRMR-ABC algorithm achieves accurate classification performance using small number of predictive genes when tested using both datasets and compared to previously suggested methods. This shows that mRMR-ABC is a promising approach for solving gene selection and cancer classification problems. PMID:25961028

mRMR-ABC: A Hybrid Gene Selection Algorithm for Cancer Classification Using Microarray Gene Expression Profiling.

PubMed

Alshamlan, Hala; Badr, Ghada; Alohali, Yousef

2015-01-01

An artificial bee colony (ABC) is a relatively recent swarm intelligence optimization approach. In this paper, we propose the first attempt at applying ABC algorithm in analyzing a microarray gene expression profile. In addition, we propose an innovative feature selection algorithm, minimum redundancy maximum relevance (mRMR), and combine it with an ABC algorithm, mRMR-ABC, to select informative genes from microarray profile. The new approach is based on a support vector machine (SVM) algorithm to measure the classification accuracy for selected genes. We evaluate the performance of the proposed mRMR-ABC algorithm by conducting extensive experiments on six binary and multiclass gene expression microarray datasets. Furthermore, we compare our proposed mRMR-ABC algorithm with previously known techniques. We reimplemented two of these techniques for the sake of a fair comparison using the same parameters. These two techniques are mRMR when combined with a genetic algorithm (mRMR-GA) and mRMR when combined with a particle swarm optimization algorithm (mRMR-PSO). The experimental results prove that the proposed mRMR-ABC algorithm achieves accurate classification performance using small number of predictive genes when tested using both datasets and compared to previously suggested methods. This shows that mRMR-ABC is a promising approach for solving gene selection and cancer classification problems.

The Yersinia pestis siderophore, yersiniabactin, and the ZnuABC system both contribute to zinc acquisition and the development of lethal septicaemic plague in mice.

PubMed

Bobrov, Alexander G; Kirillina, Olga; Fetherston, Jacqueline D; Miller, M Clarke; Burlison, Joseph A; Perry, Robert D

2014-08-01

Bacterial pathogens must overcome host sequestration of zinc (Zn(2+) ), an essential micronutrient, during the infectious disease process. While the mechanisms to acquire chelated Zn(2+) by bacteria are largely undefined, many pathogens rely upon the ZnuABC family of ABC transporters. Here we show that in Yersinia pestis, irp2, a gene encoding the synthetase (HMWP2) for the siderophore yersiniabactin (Ybt) is required for growth under Zn(2+) -deficient conditions in a strain lacking ZnuABC. Moreover, growth stimulation with exogenous, purified apo-Ybt provides evidence that Ybt may serve as a zincophore for Zn(2+) acquisition. Studies with the Zn(2+) -dependent transcriptional reporter znuA::lacZ indicate that the ability to synthesize Ybt affects the levels of intracellular Zn(2+) . However, the outer membrane receptor Psn and TonB as well as the inner membrane (IM) ABC transporter YbtPQ, which are required for Fe(3+) acquisition by Ybt, are not needed for Ybt-dependent Zn(2+) uptake. In contrast, the predicted IM protein YbtX, a member of the Major Facilitator Superfamily, was essential for Ybt-dependent Zn(2+) uptake. Finally, we show that the ZnuABC system and the Ybt synthetase HMWP2, presumably by Ybt synthesis, both contribute to the development of a lethal infection in a septicaemic plague mouse model. © 2014 John Wiley & Sons Ltd.

The human gastric pathogen Helicobacter pylori has a potential acetone carboxylase that enhances its ability to colonize mice

PubMed Central

Brahmachary, Priyanka; Wang, Ge; Benoit, Stéphane L; Weinberg, Michael V; Maier, Robert J; Hoover, Timothy R

2008-01-01

Background Helicobacter pylori colonizes the human stomach and is the etiological agent of peptic ulcer disease. All three H. pylori strains that have been sequenced to date contain a potential operon whose products share homology with the subunits of acetone carboxylase (encoded by acxABC) from Xanthobacter autotrophicus strain Py2 and Rhodobacter capsulatus strain B10. Acetone carboxylase catalyzes the conversion of acetone to acetoacetate. Genes upstream of the putative acxABC operon encode enzymes that convert acetoacetate to acetoacetyl-CoA, which is metabolized further to generate two molecules of acetyl-CoA. Results To determine if the H. pylori acxABC operon has a role in host colonization the acxB homolog in the mouse-adapted H. pylori SS1 strain was inactivated with a chloramphenicol-resistance (cat) cassette. In mouse colonization studies the numbers of H. pylori recovered from mice inoculated with the acxB:cat mutant were generally one to two orders of magnitude lower than those recovered from mice inoculated with the parental strain. A statistical analysis of the data using a Wilcoxin Rank test indicated the differences in the numbers of H. pylori isolated from mice inoculated with the two strains were significant at the 99% confidence level. Levels of acetone associated with gastric tissue removed from uninfected mice were measured and found to range from 10–110 μmols per gram wet weight tissue. Conclusion The colonization defect of the acxB:cat mutant suggests a role for the acxABC operon in survival of the bacterium in the stomach. Products of the H. pylori acxABC operon may function primarily in acetone utilization or may catalyze a related reaction that is important for survival or growth in the host. H. pylori encounters significant levels of acetone in the stomach which it could use as a potential electron donor for microaerobic respiration. PMID:18215283

[Molecular cloning and characterization of cDNA of the rpc10+ gene encoding the smallest subunit of nuclear RNA polymerases of Schizosaccharomyces pombe].

PubMed

Shpakovskiĭ, G V; Lebedenko, E N

1997-05-01

The full-length cDNA of the rpc10+ gene encoding mini-subunit Rpc10, which is common for all three nuclear RNA polymerases of the fission yeast Schizosaccharomyces pombe, was cloned and sequenced. The Rpc10 subunit of Sz. pombe and its homologs from S. cerevisiae and H. sapiens are positively charged proteins with a highly conserved C-terminal region and an invariant zinc-binding domain (Zn-finger) of a typical amino acid composition: YxCx2Cx12RCx2CGxR. Functional tests of heterospecific complementation, using tetrad analysis or plasmid shuffling, showed that the Rpc10 subunit of Sz. pombe can successfully replace the homologous ABC10 alpha subunit in nuclear RNA polymerases I-III of S. cerevisiae.

Transient Hypermutagenesis Accelerates the Evolution of Legume Endosymbionts following Horizontal Gene Transfer

PubMed Central

Remigi, Philippe; Capela, Delphine; Clerissi, Camille; Tasse, Léna; Torchet, Rachel; Bouchez, Olivier; Batut, Jacques; Cruveiller, Stéphane; Rocha, Eduardo P. C.; Masson-Boivin, Catherine

2014-01-01

Horizontal gene transfer (HGT) is an important mode of adaptation and diversification of prokaryotes and eukaryotes and a major event underlying the emergence of bacterial pathogens and mutualists. Yet it remains unclear how complex phenotypic traits such as the ability to fix nitrogen with legumes have successfully spread over large phylogenetic distances. Here we show, using experimental evolution coupled with whole genome sequencing, that co-transfer of imuABC error-prone DNA polymerase genes with key symbiotic genes accelerates the evolution of a soil bacterium into a legume symbiont. Following introduction of the symbiotic plasmid of Cupriavidus taiwanensis, the Mimosa symbiont, into pathogenic Ralstonia solanacearum we challenged transconjugants to become Mimosa symbionts through serial plant-bacteria co-cultures. We demonstrate that a mutagenesis imuABC cassette encoded on the C. taiwanensis symbiotic plasmid triggered a transient hypermutability stage in R. solanacearum transconjugants that occurred before the cells entered the plant. The generated burst in genetic diversity accelerated symbiotic adaptation of the recipient genome under plant selection pressure, presumably by improving the exploration of the fitness landscape. Finally, we show that plasmid imuABC cassettes are over-represented in rhizobial lineages harboring symbiotic plasmids. Our findings shed light on a mechanism that may have facilitated the dissemination of symbiotic competency among α- and β-proteobacteria in natura and provide evidence for the positive role of environment-induced mutagenesis in the acquisition of a complex lifestyle trait. We speculate that co-transfer of complex phenotypic traits with mutagenesis determinants might frequently enhance the ecological success of HGT. PMID:25181317

Constitutive expression of catABC genes in the aniline-assimilating bacterium Rhodococcus species AN-22: production, purification, characterization and gene analysis of CatA, CatB and CatC

PubMed Central

Matsumura, Eitaro; Sakai, Masashi; Hayashi, Katsuaki; Murakami, Shuichiro; Takenaka, Shinji; Aoki, Kenji

2005-01-01

The aniline-assimilating bacterium Rhodococcus sp. AN-22 was found to constitutively synthesize CatB (cis,cis-muconate cycloisomerase) and CatC (muconolactone isomerase) in its cells growing on non-aromatic substrates, in addition to the previously reported CatA (catechol 1,2-dioxygenase). The bacterium maintained the specific activity of the three enzymes at an almost equal level during cultivation on succinate. CatB and CatC were purified to homogeneity and characterized. CatB was a monomer with a molecular mass of 44 kDa. The enzyme was activated by Mn2+, Co2+ and Mg2+. Native CatC was a homo-octamer with a molecular mass of 100 kDa. The enzyme was stable between pH 7.0 and 10.5 and was resistant to heating up to 90 °C. Genes coding for CatA, CatB and CatC were cloned and named catA, catB and catC respectively. The catABC genes were transcribed as one operon. The deduced amino acid sequences of CatA, CatB and CatC showed high identities with those from other Gram-positive micro-organisms. A regulator gene such as catR encoding a regulatory protein was not observed around the cat gene cluster of Rhodococcus sp. AN-22, but a possible relic of catR was found in the upstream region of catA. Reverse transcriptase-PCR and primer extension analyses showed that the transcriptional start site of the cat gene cluster was located 891 bp upstream of the catA initiation codon in the AN-22 strain growing on both aniline and succinate. Based on these data, we concluded that the bacterium constitutively transcribed the catABC genes and translated its mRNA into CatA, CatB and CatC. PMID:16156722

The severity of hereditary porphyria is modulated by the porphyrin exporter and Lan antigen ABCB6

PubMed Central

Fukuda, Yu; Cheong, Pak Leng; Lynch, John; Brighton, Cheryl; Frase, Sharon; Kargas, Vasileios; Rampersaud, Evadnie; Wang, Yao; Sankaran, Vijay G.; Yu, Bing; Ney, Paul A.; Weiss, Mitchell J.; Vogel, Peter; Bond, Peter J.; Ford, Robert C.; Trent, Ronald J.; Schuetz, John D.

2016-01-01

Hereditary porphyrias are caused by mutations in genes that encode haem biosynthetic enzymes with resultant buildup of cytotoxic metabolic porphyrin intermediates. A long-standing open question is why the same causal porphyria mutations exhibit widely variable penetrance and expressivity in different individuals. Here we show that severely affected porphyria patients harbour variant alleles in the ABCB6 gene, also known as Lan, which encodes an ATP-binding cassette (ABC) transporter. Plasma membrane ABCB6 exports a variety of disease-related porphyrins. Functional studies show that most of these ABCB6 variants are expressed poorly and/or have impaired function. Accordingly, homozygous disruption of the Abcb6 gene in mice exacerbates porphyria phenotypes in the Fechm1Pas mouse model, as evidenced by increased porphyrin accumulation, and marked liver injury. Collectively, these studies support ABCB6 role as a genetic modifier of porphyria and suggest that porphyrin-inducing drugs may produce excessive toxicities in individuals with the rare Lan(−) blood type. PMID:27507172

Caenorhabditis elegans ABCRNAi transporters interact genetically with rde-2 and mut-7.

PubMed

Sundaram, Prema; Han, Wang; Cohen, Nancy; Echalier, Benjamin; Albin, John; Timmons, Lisa

2008-02-01

RNA interference (RNAi) mechanisms are conserved and consist of an interrelated network of activities that not only respond to exogenous dsRNA, but also perform endogenous functions required in the fine tuning of gene expression and in maintaining genome integrity. Not surprisingly, RNAi functions have widespread influences on cellular function and organismal development. Previously, we observed a reduced capacity to mount an RNAi response in nine Caenorhabditis elegans mutants that are defective in ABC transporter genes (ABC(RNAi) mutants). Here, we report an exhaustive study of mutants, collectively defective in 49 different ABC transporter genes, that allowed for the categorization of one additional transporter into the ABC(RNAi) gene class. Genetic complementation tests reveal functions for ABC(RNAi) transporters in the mut-7/rde-2 branch of the RNAi pathway. These second-site noncomplementation interactions suggest that ABC(RNAi) proteins and MUT-7/RDE-2 function together in parallel pathways and/or as multiprotein complexes. Like mut-7 and rde-2, some ABC(RNAi) mutants display transposon silencing defects. Finally, our analyses reveal a genetic interaction network of ABC(RNAi) gene function with respect to this part of the RNAi pathway. From our results, we speculate that the coordinated activities of ABC(RNAi) transporters, through their effects on endogenous RNAi-related mechanisms, ultimately affect chromosome function and integrity.

Gene Cluster Responsible for Secretion of and Immunity to Multiple Bacteriocins, the NKR-5-3 Enterocins

PubMed Central

Ishibashi, Naoki; Himeno, Kohei; Masuda, Yoshimitsu; Perez, Rodney Honrada; Iwatani, Shun; Wilaipun, Pongtep; Leelawatcharamas, Vichien; Nakayama, Jiro; Sonomoto, Kenji

2014-01-01

Enterococcus faecium NKR-5-3, isolated from Thai fermented fish, is characterized by the unique ability to produce five bacteriocins, namely, enterocins NKR-5-3A, -B, -C, -D, and -Z (Ent53A, Ent53B, Ent53C, Ent53D, and Ent53Z). Genetic analysis with a genome library revealed that the bacteriocin structural genes (enkA [ent53A], enkC [ent53C], enkD [ent53D], and enkZ [ent53Z]) that encode these peptides (except for Ent53B) are located in close proximity to each other. This NKR-5-3ACDZ (Ent53ACDZ) enterocin gene cluster (approximately 13 kb long) includes certain bacteriocin biosynthetic genes such as an ABC transporter gene (enkT), two immunity genes (enkIaz and enkIc), a response regulator (enkR), and a histidine protein kinase (enkK). Heterologous-expression studies of enkT and ΔenkT mutant strains showed that enkT is responsible for the secretion of Ent53A, Ent53C, Ent53D, and Ent53Z, suggesting that EnkT is a wide-range ABC transporter that contributes to the effective production of these bacteriocins. In addition, EnkIaz and EnkIc were found to confer self-immunity to the respective bacteriocins. Furthermore, bacteriocin induction assays performed with the ΔenkRK mutant strain showed that EnkR and EnkK are regulatory proteins responsible for bacteriocin production and that, together with Ent53D, they constitute a three-component regulatory system. Thus, the Ent53ACDZ gene cluster is essential for the biosynthesis and regulation of NKR-5-3 enterocins, and this is, to our knowledge, the first report that demonstrates the secretion of multiple bacteriocins by an ABC transporter. PMID:25149515

Identification of a lineage specific zinc responsive genomic island in Mycobacterium avium ssp. paratuberculosis.

PubMed

Eckelt, Elke; Jarek, Michael; Frömke, Cornelia; Meens, Jochen; Goethe, Ralph

2014-12-06

Maintenance of metal homeostasis is crucial in bacterial pathogenicity as metal starvation is the most important mechanism in the nutritional immunity strategy of host cells. Thus, pathogenic bacteria have evolved sensitive metal scavenging systems to overcome this particular host defence mechanism. The ruminant pathogen Mycobacterium avium ssp. paratuberculosis (MAP) displays a unique gut tropism and causes a chronic progressive intestinal inflammation. MAP possesses eight conserved lineage specific large sequence polymorphisms (LSP), which distinguish MAP from its ancestral M. avium ssp. hominissuis or other M. avium subspecies. LSP14 and LSP15 harbour many genes proposed to be involved in metal homeostasis and have been suggested to substitute for a MAP specific, impaired mycobactin synthesis. In the present study, we found that a LSP14 located putative IrtAB-like iron transporter encoded by mptABC was induced by zinc but not by iron starvation. Heterologous reporter gene assays with the lacZ gene under control of the mptABC promoter in M. smegmatis (MSMEG) and in a MSMEG∆furB deletion mutant revealed a zinc dependent, metalloregulator FurB mediated expression of mptABC via a conserved mycobacterial FurB recognition site. Deep sequencing of RNA from MAP cultures treated with the zinc chelator TPEN revealed that 70 genes responded to zinc limitation. Remarkably, 45 of these genes were located on a large genomic island of approximately 90 kb which harboured LSP14 and LSP15. Thirty-five of these genes were predicted to be controlled by FurB, due to the presence of putative binding sites. This clustering of zinc responsive genes was exclusively found in MAP and not in other mycobacteria. Our data revealed a particular genomic signature for MAP given by a unique zinc specific locus, thereby suggesting an exceptional relevance of zinc for the metabolism of MAP. MAP seems to be well adapted to maintain zinc homeostasis which might contribute to the peculiarity of MAP pathogenicity.

ABC-B transporter genes in Dirofilaria immitis.

PubMed

Bourguinat, Catherine; Che, Hua; Mani, Thangadurai; Keller, Kathy; Prichard, Roger K

2016-08-01

Dirofilaria immitis is a filarial nematode causing infection and heartworm disease in dogs and other canids, cats, and occasionally in humans. Prevention with macrocyclic lactones (ML) is recommended during the mosquito transmission season. Recently, ML resistance has been reported. ABC-B transporter genes are thought to be involved in the mechanism of ML resistance in other nematodes. This study aimed to identify all the ABC-B transporter genes in D. immitis using as a reference the nDi.2.2 D. immitis whole genome, which is not completely annotated. Using bioinformatic tools and PCR amplification on pooled D. immitis genomic DNA and on pooled cDNA, nine ABC transporter genes including one pseudogene were characterized. Bioinformatic and phylogenetic analyses allowed identification of three P-glycoproteins (Pgps) (Dim-pgp-3 Dim-pgp-10, Dim-pgp-11), of two ABC-B half transporter genes (one ortholog of Cel-haf-4 and Cel-haf-9; and one ortholog of Cel-haf-1 and Cel-haf-3), of one ABC half transporter gene (ortholog of Cel-haf-5) that contained an ABC-C motif, and of one additional half transporter that would require functional study for characterization. The number of ABC-B transporter genes identified was lower than in Caenorhabditis elegans and Haemonchus contortus. Further studies are needed to understand their possible role in ML resistance in D. immitis. These ABC transporters constitute a base for ML resistance investigation in D. immitis and advance our understanding of the molecular biology of this parasite. Copyright © 2016. Published by Elsevier Ltd.

Molecular cloning and expression profile of an ATP-binding cassette (ABC) transporter gene from the hemipteran insect Nilaparvata lugens.

PubMed

Zha, W J; Li, S H; Zhou, L; Chen, Z J; Liu, K; Yang, G C; Hu, G; He, G C; You, A Q

2015-03-30

The ATP-binding cassette (ABC) transporters belong to a large superfamily of proteins that have important physiological functions in all living organisms. In insects, ABC transporters have important functions in the transport of molecules, and are also involved in insecticide resistance, metabolism, and development. In this study, the Nilaparvata lugens Stal (Hemiptera: Delphacidae) ABCG (NlABCG) gene was identified and characterized. The complete mRNA sequence of NlABCG was 2608-bp long, with an open reading frame of 2064 bp encoding a protein comprised of 687 amino acids. The conserved regions include three N-glycosylation and 34 phosphorylation sites, as well as seven transmembrane domains. The amino acid identity with the closely related species Acyrthosiphon pisum was 42.8%. Developmental expression analysis using quantitative real-time reverse transcriptase PCR suggested that the NlABCG transcript was expressed at all developmental stages of N. lugens. The lowest expression of NlABCG was in the 1st instar, and levels increased with larval growth. The transcript profiles of NlABCG were analyzed in various tissues from a 5th instar nymph, and the highest expression was observed in the midgut. These results suggest that the sequence, characteristics, and expression of NlABCG are highly conserved, and basic information is provided for its functional analysis.

[Cloning of cDNA for RNA polymerase subunit from the fission yeast Schizosaccharomyces pombe by heterospecific complementation in Saccharomyces cerevisiae].

PubMed

Shpakovskiĭ, G V; Lebedenko, E N; Thuriaux, P

1997-02-01

The rpb10 cDNA of the fission yeast Schizosaccharomyces pombe, encoding one of the five small subunits common to all three nuclear DNA-dependent RNA polymerases, was isolated from an expression cDNA library by two independent approaches: PCR-based screening and direct suppression by means of heterospecific complementation of a temperature-sensitive mutant defective in the corresponding gene of Saccharomyces cerevisiae. The cloned Sz. pombe cDNA encodes a protein Rpb10 of 71 amino acids with an M of 8,275 Da, sharing 51 amino acids (71% identity) with the subunit ABC10 beta of RNA polymerases I-III from S. cerevisiae. All eukaryotic members of this protein family have the same general organization featuring two highly conserved motifs (RCFT/SCGK and RYCCRRM) around an atypical zinc finger and an additional invariant HVDLIEK motif toward the C-terminal end. The last motif is only characteristics for homologs from eukaryotes. In keeping with this remarkable structural conservation, the Sz. pombe cDNA also fully complemented a S. cerevisiae deletion mutant lacking subunit ABC10 beta (null allele rpb10-delta 1::HIS3).

The Homogentisate Pathway: a Central Catabolic Pathway Involved in the Degradation of l-Phenylalanine, l-Tyrosine, and 3-Hydroxyphenylacetate in Pseudomonas putida

PubMed Central

Arias-Barrau, Elsa; Olivera, Elías R.; Luengo, José M.; Fernández, Cristina; Galán, Beatriz; García, José L.; Díaz, Eduardo; Miñambres, Baltasar

2004-01-01

Pseudomonas putida metabolizes Phe and Tyr through a peripheral pathway involving hydroxylation of Phe to Tyr (PhhAB), conversion of Tyr into 4-hydroxyphenylpyruvate (TyrB), and formation of homogentisate (Hpd) as the central intermediate. Homogentisate is then catabolized by a central catabolic pathway that involves three enzymes, homogentisate dioxygenase (HmgA), fumarylacetoacetate hydrolase (HmgB), and maleylacetoacetate isomerase (HmgC), finally yielding fumarate and acetoacetate. Whereas the phh, tyr, and hpd genes are not linked in the P. putida genome, the hmgABC genes appear to form a single transcriptional unit. Gel retardation assays and lacZ translational fusion experiments have shown that hmgR encodes a specific repressor that controls the inducible expression of the divergently transcribed hmgABC catabolic genes, and homogentisate is the inducer molecule. Footprinting analysis revealed that HmgR protects a region in the Phmg promoter that spans a 17-bp palindromic motif and an external direct repetition from position −16 to position 29 with respect to the transcription start site. The HmgR protein is thus the first IclR-type regulator that acts as a repressor of an aromatic catabolic pathway. We engineered a broad-host-range mobilizable catabolic cassette harboring the hmgABC, hpd, and tyrB genes that allows heterologous bacteria to use Tyr as a unique carbon and energy source. Remarkably, we show here that the catabolism of 3-hydroxyphenylacetate in P. putida U funnels also into the homogentisate central pathway, revealing that the hmg cluster is a key catabolic trait for biodegradation of a small number of aromatic compounds. PMID:15262943

The homogentisate pathway: a central catabolic pathway involved in the degradation of L-phenylalanine, L-tyrosine, and 3-hydroxyphenylacetate in Pseudomonas putida.

PubMed

Arias-Barrau, Elsa; Olivera, Elías R; Luengo, José M; Fernández, Cristina; Galán, Beatriz; García, José L; Díaz, Eduardo; Miñambres, Baltasar

2004-08-01

Pseudomonas putida metabolizes Phe and Tyr through a peripheral pathway involving hydroxylation of Phe to Tyr (PhhAB), conversion of Tyr into 4-hydroxyphenylpyruvate (TyrB), and formation of homogentisate (Hpd) as the central intermediate. Homogentisate is then catabolized by a central catabolic pathway that involves three enzymes, homogentisate dioxygenase (HmgA), fumarylacetoacetate hydrolase (HmgB), and maleylacetoacetate isomerase (HmgC), finally yielding fumarate and acetoacetate. Whereas the phh, tyr, and hpd genes are not linked in the P. putida genome, the hmgABC genes appear to form a single transcriptional unit. Gel retardation assays and lacZ translational fusion experiments have shown that hmgR encodes a specific repressor that controls the inducible expression of the divergently transcribed hmgABC catabolic genes, and homogentisate is the inducer molecule. Footprinting analysis revealed that HmgR protects a region in the Phmg promoter that spans a 17-bp palindromic motif and an external direct repetition from position -16 to position 29 with respect to the transcription start site. The HmgR protein is thus the first IclR-type regulator that acts as a repressor of an aromatic catabolic pathway. We engineered a broad-host-range mobilizable catabolic cassette harboring the hmgABC, hpd, and tyrB genes that allows heterologous bacteria to use Tyr as a unique carbon and energy source. Remarkably, we show here that the catabolism of 3-hydroxyphenylacetate in P. putida U funnels also into the homogentisate central pathway, revealing that the hmg cluster is a key catabolic trait for biodegradation of a small number of aromatic compounds.

Expression Profile of Drug and Nutrient Absorption Related Genes in Madin-Darby Canine Kidney (MDCK) Cells Grown under Differentiation Conditions.

PubMed

Quan, Yong; Jin, Yisheng; Faria, Teresa N; Tilford, Charles A; He, Aiqing; Wall, Doris A; Smith, Ronald L; Vig, Balvinder S

2012-06-18

The expression levels of genes involved in drug and nutrient absorption were evaluated in the Madin-Darby Canine Kidney (MDCK) in vitro drug absorption model. MDCK cells were grown on plastic surfaces (for 3 days) or on Transwell® membranes (for 3, 5, 7, and 9 days). The expression profile of genes including ABC transporters, SLC transporters, and cytochrome P450 (CYP) enzymes was determined using the Affymetrix® Canine GeneChip®. Expression of genes whose probe sets passed a stringent confirmation process was examined. Expression of a few transporter (MDR1, PEPT1 and PEPT2) genes in MDCK cells was confirmed by RT-PCR. The overall gene expression profile was strongly influenced by the type of support the cells were grown on. After 3 days of growth, expression of 28% of the genes was statistically different (1.5-fold cutoff, p < 0.05) between the cells grown on plastic and Transwell® membranes. When cells were differentiated on Transwell® membranes, large changes in gene expression profile were observed during the early stages, which then stabilized after 5-7 days. Only a small number of genes encoding drug absorption related SLC, ABC, and CYP were detected in MDCK cells, and most of them exhibited low hybridization signals. Results from this study provide valuable reference information on endogenous gene expression in MDCK cells that could assist in design of drug-transporter and/or drug-enzyme interaction studies, and help interpret the contributions of various transporters and metabolic enzymes in studies with MDCK cells.

Expression Profile of Drug and Nutrient Absorption Related Genes in Madin-Darby Canine Kidney (MDCK) Cells Grown under Differentiation Conditions

PubMed Central

Quan, Yong; Jin, Yisheng; Faria, Teresa N.; Tilford, Charles A.; He, Aiqing; Wall, Doris A.; Smith, Ronald L.; Vig, Balvinder S.

2012-01-01

The expression levels of genes involved in drug and nutrient absorption were evaluated in the Madin-Darby Canine Kidney (MDCK) in vitro drug absorption model. MDCK cells were grown on plastic surfaces (for 3 days) or on Transwell® membranes (for 3, 5, 7, and 9 days). The expression profile of genes including ABC transporters, SLC transporters, and cytochrome P450 (CYP) enzymes was determined using the Affymetrix® Canine GeneChip®. Expression of genes whose probe sets passed a stringent confirmation process was examined. Expression of a few transporter (MDR1, PEPT1 and PEPT2) genes in MDCK cells was confirmed by RT-PCR. The overall gene expression profile was strongly influenced by the type of support the cells were grown on. After 3 days of growth, expression of 28% of the genes was statistically different (1.5-fold cutoff, p < 0.05) between the cells grown on plastic and Transwell® membranes. When cells were differentiated on Transwell® membranes, large changes in gene expression profile were observed during the early stages, which then stabilized after 5–7 days. Only a small number of genes encoding drug absorption related SLC, ABC, and CYP were detected in MDCK cells, and most of them exhibited low hybridization signals. Results from this study provide valuable reference information on endogenous gene expression in MDCK cells that could assist in design of drug-transporter and/or drug-enzyme interaction studies, and help interpret the contributions of various transporters and metabolic enzymes in studies with MDCK cells. PMID:24300234

Mu-Like Prophage in Serogroup B Neisseria meningitidis Coding for Surface-Exposed Antigens

PubMed Central

Masignani, Vega; Giuliani, Marzia Monica; Tettelin, Hervé; Comanducci, Maurizio; Rappuoli, Rino; Scarlato, Vincenzo

2001-01-01

Sequence analysis of the genome of Neisseria meningititdis serogroup B revealed the presence of an ∼35-kb region inserted within a putative gene coding for an ABC-type transporter. The region contains 46 open reading frames, 29 of which are colinear and homologous to the genes of Escherichia coli Mu phage. Two prophages with similar organizations were also found in serogroup A meningococcus, and one was found in Haemophilus influenzae. Early and late phage functions are well preserved in this family of Mu-like prophages. Several regions of atypical nucleotide content were identified. These likely represent genes acquired by horizontal transfer. Three of the acquired genes are shown to code for surface-associated antigens, and the encoded proteins are able to induce bactericidal antibodies. PMID:11254622

Phosphite Utilization by the Globally Important Marine Diazotroph Trichodesmium

NASA Astrophysics Data System (ADS)

Polyviou, D.; Hitchcock, A.; Baylay, A. J.; Moore, C. M.; Bibby, T. S.

2016-02-01

The marine cyanobacterium Trichodesmium is responsible for a significant fraction of oceanic nitrogen fixation and plays a key role in biogeochemical processes in the contemporary ocean. It has recently been shown that it also contributes to an emerging oceanic phosphorus (P) redox cycle. This is of interest as the availability of P constrains the growth of Trichodesmium in large expanses of the tropical and subtropical oceans. A four-gene cluster (ptxABCD) encodes a putative ABC transporter (ptxABC) and NAD-dependent dehydrogenase (ptxD) and is suggested to be involved in utilisation of the reduced inorganic compound phosphite. The gene cluster is identified in the Trichodesmium erythraeum IMS101 genome (Tery_0365-0368). Here, we report the occurrence of these genes in available in-situ metagenomic and metatranscriptomic datasets confirming the presence and expression of ptxABCD in diverse Trichodesmium species in the field. We also demonstrate that T.erythraeum IMS101 in culture can grow on phosphite as its sole source of P. The current lack of an established system for genetic manipulation of this organism inhibits direct functional characterisation of ptxABCD. To circumvent this we exploit the model cyanobacteria Synechocystis PCC6803 as a vehicle for the heterologous expression of the Trichodesmium genes. We demonstrate that only combined expression of both the transporter and the dehydrogenase enables Synechocystis to utilize phosphite, confirming the function of Tery_0365-0367 as a phosphite uptake system (PtxABC) and Tery_0368 as a phosphite dehydrogenase (PtxD). Our findings suggest that previously reported uptake of phosphite by Trichodesmium consortia in the field likely reflects an active biological process by Trichodesmium itself. These results highlight the diversity of phosphorus sources available to Trichodesmium in a resource-limited ocean and also the power of using heterologous gene expression to determine the function of genes identified in marine microbes.

A novel approach for dimension reduction of microarray.

PubMed

Aziz, Rabia; Verma, C K; Srivastava, Namita

2017-12-01

This paper proposes a new hybrid search technique for feature (gene) selection (FS) using Independent component analysis (ICA) and Artificial Bee Colony (ABC) called ICA+ABC, to select informative genes based on a Naïve Bayes (NB) algorithm. An important trait of this technique is the optimization of ICA feature vector using ABC. ICA+ABC is a hybrid search algorithm that combines the benefits of extraction approach, to reduce the size of data and wrapper approach, to optimize the reduced feature vectors. This hybrid search technique is facilitated by evaluating the performance of ICA+ABC on six standard gene expression datasets of classification. Extensive experiments were conducted to compare the performance of ICA+ABC with the results obtained from recently published Minimum Redundancy Maximum Relevance (mRMR) +ABC algorithm for NB classifier. Also to check the performance that how ICA+ABC works as feature selection with NB classifier, compared the combination of ICA with popular filter techniques and with other similar bio inspired algorithm such as Genetic Algorithm (GA) and Particle Swarm Optimization (PSO). The result shows that ICA+ABC has a significant ability to generate small subsets of genes from the ICA feature vector, that significantly improve the classification accuracy of NB classifier compared to other previously suggested methods. Copyright © 2017 Elsevier Ltd. All rights reserved.

Genome-Wide Identification and Expression Profiling of ATP-Binding Cassette (ABC) Transporter Gene Family in Pineapple (Ananas comosus (L.) Merr.) Reveal the Role of AcABCG38 in Pollen Development

PubMed Central

Chen, Piaojuan; Li, Yi; Zhao, Lihua; Hou, Zhimin; Yan, Maokai; Hu, Bingyan; Liu, Yanhui; Azam, Syed Muhammad; Zhang, Ziyan; Rahman, Zia ur; Liu, Liping; Qin, Yuan

2017-01-01

Pineapple (Ananas comosus L.) cultivation commonly relies on asexual reproduction which is easily impeded by many factors in agriculture production. Sexual reproduction might be a novel approach to improve the pineapple planting. However, genes controlling pineapple sexual reproduction are still remain elusive. In different organisms a conserved superfamily proteins known as ATP binding cassette (ABC) participate in various biological processes. Whereas, till today the ABC gene family has not been identified in pineapple. Here 100 ABC genes were identified in the pineapple genome and grouped into eight subfamilies (5 ABCAs, 20 ABCBs, 16 ABCCs, 2 ABCDs, one ABCEs, 5 ABCFs, 42 ABCGs and 9 ABCIs). Gene expression profiling revealed the dynamic expression pattern of ABC gene family in various tissues and different developmental stages. AcABCA5, AcABCB6, AcABCC4, AcABCC7, AcABCC9, AcABCG26, AcABCG38 and AcABCG42 exhibited preferential expression in ovule and stamen. Over-expression of AcABCG38 in the Arabidopsis double mutant abcg1-2abcg16-2 partially restored its pollen abortion defects, indicating that AcABCG38 plays important roles in pollen development. Our study on ABC gene family in pineapple provides useful information for developing sexual pineapple plantation which could be utilized to improve pineapple agricultural production. PMID:29312399

Genome-Wide Identification and Expression Profiling of ATP-Binding Cassette (ABC) Transporter Gene Family in Pineapple (Ananas comosus (L.) Merr.) Reveal the Role of AcABCG38 in Pollen Development.

PubMed

Chen, Piaojuan; Li, Yi; Zhao, Lihua; Hou, Zhimin; Yan, Maokai; Hu, Bingyan; Liu, Yanhui; Azam, Syed Muhammad; Zhang, Ziyan; Rahman, Zia Ur; Liu, Liping; Qin, Yuan

2017-01-01

Pineapple ( Ananas comosus L .) cultivation commonly relies on asexual reproduction which is easily impeded by many factors in agriculture production. Sexual reproduction might be a novel approach to improve the pineapple planting. However, genes controlling pineapple sexual reproduction are still remain elusive. In different organisms a conserved superfamily proteins known as ATP binding cassette (ABC) participate in various biological processes. Whereas, till today the ABC gene family has not been identified in pineapple. Here 100 ABC genes were identified in the pineapple genome and grouped into eight subfamilies (5 ABCAs , 20 ABCB s, 16 ABCCs , 2 ABCDs , one ABCEs , 5 ABCFs , 42 ABCGs and 9 ABCIs ). Gene expression profiling revealed the dynamic expression pattern of ABC gene family in various tissues and different developmental stages. AcABCA5, AcABCB6, AcABCC4 , AcABCC7 , AcABCC9 , AcABCG26 , AcABCG38 and AcABCG42 exhibited preferential expression in ovule and stamen. Over-expression of AcABCG38 in the Arabidopsis double mutant abcg1-2abcg16-2 partially restored its pollen abortion defects, indicating that AcABCG38 plays important roles in pollen development. Our study on ABC gene family in pineapple provides useful information for developing sexual pineapple plantation which could be utilized to improve pineapple agricultural production.

The Novel Helicobacter pylori CznABC Metal Efflux Pump Is Required for Cadmium, Zinc, and Nickel Resistance, Urease Modulation, and Gastric Colonization

PubMed Central

Stähler, Frank Nils; Odenbreit, Stefan; Haas, Rainer; Wilrich, Julia; Vliet, Arnoud H. M. Van; Kusters, Johannes G.; Kist, Manfred; Bereswill, Stefan

2006-01-01

Maintaining metal homeostasis is crucial for the adaptation of Helicobacter pylori to the gastric environment. Iron, copper, and nickel homeostasis has recently been demonstrated to be required for the establishment of H. pylori infection in animal models. Here we demonstrate that the HP0969-0971 gene cluster encoding the Czc-type metal export pump homologs HP0969, HP0970, and the H. pylori-specific protein HP0971 forms part of a novel H. pylori metal resistance determinant, which is required for gastric colonization and for the modulation of urease activity. Insertional mutagenesis of the HP0971, HP0970, or HP0969 genes in H. pylori reference strain 26695 resulted in increased sensitivity to cadmium, zinc, and nickel (czn), suggesting that the encoded proteins constitute a metal-specific export pump. Accordingly, the genes were designated cznC (HP0971), cznB (HP0970), and cznA (HP0969). The CznC and CznA proteins play a predominant role in nickel homeostasis, since only the cznC and cznA mutants but not the cznB mutant displayed an 8- to 10-fold increase in urease activity. Nickel-specific affinity chromatography demonstrated that recombinant versions of CznC and CznB can bind to nickel and that the purified CznB protein interacted with cadmium and zinc, since both metals competitively inhibited nickel binding. Finally, single cznA, cznB, and cznC mutants did not colonize the stomach in a Mongolian gerbil-based animal model. This demonstrates that the metal export functions of H. pylori cznABC are essential for gastric colonization and underlines the extraordinary importance of metal ion homeostasis for the survival of H. pylori in the gastric environment. PMID:16790756

A PhoPQ-Regulated ABC Transporter System Exports Tetracycline in Pseudomonas aeruginosa.

PubMed

Chen, Lin; Duan, Kangmin

2016-05-01

Pseudomonas aeruginosa is an important human pathogen whose infections are difficult to treat due to its high intrinsic resistance to many antibiotics. Here, we show that the disruption of PA4456, encoding the ATP binding component of a putative ATP-binding cassette (ABC) transporter, increased the bacterium's susceptible to tetracycline and other antibiotics or toxic chemicals. Fluorescence spectroscopy and antibiotic accumulation tests showed that the interruption of the ABC transporter caused increased intracellular accumulation of tetracycline, demonstrating a role of the ABC transporter in tetracycline expulsion. Site-directed mutagenesis proved that the conserved residues of E170 in the Walker B motif and H203 in the H-loop, which are important for ATP hydrolysis, were essential for the function of PA4456. Through a genome-wide search, the PhoPQ two-component system was identified as a regulator of the computationally predicted PA4456-4452 operon that encodes the ABC transporter system. A >5-fold increase of the expression of this operon was observed in the phoQ mutant. The results obtained also show that the expression of the phzA1B1C1D1E1 operon and the production of pyocyanin were significantly higher in the ABC transporter mutant, signifying a connection between the ABC transporter and pyocyanin production. These results indicated that the PhoPQ-regulated ABC transporter is associated with intrinsic resistance to antibiotics and other adverse compounds in P. aeruginosa, probably by extruding them out of the cell. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Global transcriptomic response of Anoxybacillus sp. SK 3-4 to aluminum exposure.

PubMed

Lim, Jia Chun; Thevarajoo, Suganthi; Selvaratnam, Chitra; Goh, Kian Mau; Shamsir, Mohd Shahir; Ibrahim, Zaharah; Chong, Chun Shiong

2017-02-01

Anoxybacillus sp. SK 3-4 is a Gram-positive, rod-shaped bacterium and a member of family Bacillaceae. We had previously reported that the strain is an aluminum resistant thermophilic bacterium. This is the first report to provide a detailed analysis of the global transcriptional response of Anoxybacillus when the cells were exposed to 600 mg L -1 of aluminum. The transcriptome was sequenced using Illumina MiSeq sequencer. Total of 708 genes were differentially expressed (fold change >2.00) with 316 genes were up-regulated while 347 genes were down-regulated, in comparing to control with no aluminum added in the culture. Based on Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, the majority of genes encoding for cell metabolism such as glycolysis, sulfur metabolism, cysteine and methionine metabolism were up-regulated; while most of the gene associated with tricarboxylic acid cycle (TCA cycle) and valine, leucine and isoleucine metabolism were down-regulated. In addition, a significant number of the genes encoding ABC transporters, metal ions transporters, and some stress response proteins were also differentially expressed following aluminum exposure. The findings provide further insight and help us to understand on the resistance of Anoxybacillus sp. SK 3-4 toward aluminium. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

ATP-binding cassette (ABC) proteins in aquatic invertebrates: Evolutionary significance and application in marine ecotoxicology.

PubMed

Jeong, Chang-Bum; Kim, Hui-Su; Kang, Hye-Min; Lee, Jae-Seong

2017-04-01

The ATP-binding cassette (ABC) protein superfamily is known to play a fundamental role in biological processes and is highly conserved across animal taxa. The ABC proteins function as active transporters for multiple substrates across the cellular membrane by ATP hydrolysis. As this superfamily is derived from a common ancestor, ABC genes have evolved via lineage-specific duplications through the process of adaptation. In this review, we summarized information about the ABC gene families in aquatic invertebrates, considering their evolution and putative functions in defense mechanisms. Phylogenetic analysis was conducted to examine the evolutionary significance of ABC gene families in aquatic invertebrates. Particularly, a massive expansion of multixenobiotic resistance (MXR)-mediated efflux transporters was identified in the absence of the ABCG2 (BCRP) gene in Ecdysozoa and Platyzoa, suggesting that a loss of Abcg2 gene occurred sporadically in these species during divergence of Protostome to Lophotrochozoa. Furthermore, in aquatic invertebrates, the ecotoxicological significance of MXR is discussed while considering the role of MXR-mediated efflux transporters in response to various environmental pollutants. Copyright © 2017 Elsevier B.V. All rights reserved.

Cloning and Expression of the Benzoate Dioxygenase Genes from Rhodococcus sp. Strain 19070

PubMed Central

Haddad, Sandra; Eby, D. Matthew; Neidle, Ellen L.

2001-01-01

The bopXYZ genes from the gram-positive bacterium Rhodococcus sp. strain 19070 encode a broad-substrate-specific benzoate dioxygenase. Expression of the BopXY terminal oxygenase enabled Escherichia coli to convert benzoate or anthranilate (2-aminobenzoate) to a nonaromatic cis-diol or catechol, respectively. This expression system also rapidly transformed m-toluate (3-methylbenzoate) to an unidentified product. In contrast, 2-chlorobenzoate was not a good substrate. The BopXYZ dioxygenase was homologous to the chromosomally encoded benzoate dioxygenase (BenABC) and the plasmid-encoded toluate dioxygenase (XylXYZ) of gram-negative acinetobacters and pseudomonads. Pulsed-field gel electrophoresis failed to identify any plasmid in Rhodococcus sp. strain 19070. Catechol 1,2- and 2,3-dioxygenase activity indicated that strain 19070 possesses both meta- and ortho-cleavage degradative pathways, which are associated in pseudomonads with the xyl and ben genes, respectively. Open reading frames downstream of bopXYZ, designated bopL and bopK, resembled genes encoding cis-diol dehydrogenases and benzoate transporters, respectively. The bop genes were in the same order as the chromosomal ben genes of P. putida PRS2000. The deduced sequences of BopXY were 50 to 60% identical to the corresponding proteins of benzoate and toluate dioxygenases. The reductase components of these latter dioxygenases, BenC and XylZ, are 201 residues shorter than the deduced BopZ sequence. As predicted from the sequence, expression of BopZ in E. coli yielded an approximately 60-kDa protein whose presence corresponded to increased cytochrome c reductase activity. While the N-terminal region of BopZ was approximately 50% identical in sequence to the entire BenC or XylZ reductases, the C terminus was unlike other known protein sequences. PMID:11375157

Temporal dynamics of the ABC transporter response to insecticide treatment: insights from the malaria vector Anopheles stephensi

NASA Astrophysics Data System (ADS)

Epis, Sara; Porretta, Daniele; Mastrantonio, Valentina; Urbanelli, Sandra; Sassera, Davide; De Marco, Leone; Mereghetti, Valeria; Montagna, Matteo; Ricci, Irene; Favia, Guido; Bandi, Claudio

2014-12-01

In insects, ABC transporters have been shown to contribute to defence/resistance to insecticides by reducing toxic concentrations in cells/tissues. Despite the extensive studies about this detoxifying mechanism, the temporal patterns of ABC transporter activation have been poorly investigated. Using the malaria vector Anopheles stephensi as a study system, we investigated the expression profile of ABC genes belonging to different subfamilies in permethrin-treated larvae at different time points (30 min to 48 h). Our results showed that the expression of ABCB and ABCG subfamily genes was upregulated at 1 h after treatment, with the highest expression observed at 6 h. Therefore, future investigations on the temporal dynamics of ABC gene expression will allow a better implementation of insecticide treatment regimens, including the use of specific inhibitors of ABC efflux pumps.

Metabolism of a plant derived galactose‐containing polysaccharide by Bifidobacterium breve UCC2003

PubMed Central

O'Connell Motherway, Mary; Fitzgerald, Gerald F.; van Sinderen, Douwe

2011-01-01

Summary In this study, we describe the functional characterization of the Bifidobacterium breve UCC2003 gal locus, which is dedicated to the utilization of galactan, a plant‐derived polysaccharide. Using a combination of molecular approaches we conclude that the galA gene of B. breve UCC2003 encodes a β‐1,4‐endogalactanase producing galacto‐oligosaccharides, which are specifically internalized by an ABC transport system, encoded by galBCDE, and which are then hydrolysed to galactose moieties by a dedicated intracellular β‐galactosidase, specified by galG. The generated galactose molecules are presumed to be fed into the fructose‐6‐phosphate phosphoketolase pathway via the Leloir pathway, thereby allowing B. breve UCC2003 to use galactan as its sole carbon and energy source. In addition to these findings we demonstrate that GalR is a LacI‐type DNA‐binding protein, which not only appears to control transcription of the galCDEGR operon, but also that of the galA gene. PMID:21375716

GH51 Arabinofuranosidase and Its Role in the Methylglucuronoarabinoxylan Utilization System in Paenibacillus sp. Strain JDR-2

PubMed Central

Sawhney, Neha

2014-01-01

Methylglucuronoarabinoxylan (MeGAXn) from agricultural residues and energy crops is a significant yet underutilized biomass resource for production of biofuels and chemicals. Mild thermochemical pretreatment of bagasse yields MeGAXn requiring saccharifying enzymes for conversion to fermentable sugars. A xylanolytic bacterium, Paenibacillus sp. strain JDR-2, produces an extracellular cell-associated GH10 endoxylanse (XynA1) which efficiently depolymerizes methylglucuronoxylan (MeGXn) from hardwoods coupled with assimilation of oligosaccharides for further processing by intracellular GH67 α-glucuronidase, GH10 endoxylanase, and GH43 β-xylosidase. This process has been ascribed to genes that comprise a xylan utilization regulon that encodes XynA1 and includes a gene cluster encoding transcriptional regulators, ABC transporters, and intracellular enzymes that convert assimilated oligosaccharides to fermentable sugars. Here we show that Paenibacillus sp. JDR-2 utilized MeGAXn without accumulation of oligosaccharides in the medium. The Paenibacillus sp. JDR-2 growth rate on MeGAXn was 3.1-fold greater than that on oligosaccharides generated from MeGAXn by XynA1. Candidate genes encoding GH51 arabinofuranosidases with potential roles were identified. Following growth on MeGAXn, quantitative reverse transcription-PCR identified a cluster of genes encoding a GH51 arabinofuranosidase (AbfB) and transcriptional regulators which were coordinately expressed along with the genes comprising the xylan utilization regulon. The action of XynA1 on MeGAXn generated arabinoxylobiose, arabinoxylotriose, xylobiose, xylotriose, and methylglucuronoxylotriose. Recombinant AbfB processed arabinoxylooligosaccharides to xylooligosaccharides and arabinose. MeGAXn processing by Paenibacillus sp. JDR-2 may be achieved by extracellular depolymerization by XynA1 coupled to assimilation of oligosaccharides and further processing by intracellular enzymes, including AbfB. Paenibacillus sp. JDR-2 provides a GH10/GH67 system complemented with genes encoding intracellular GH51 arabinofuranosidases for efficient utilization of MeGAXn. PMID:25063665

Phylogenetic relatedness determined between antibiotic resistance and 16S rRNA genes in actinobacteria.

PubMed

Sagova-Mareckova, Marketa; Ulanova, Dana; Sanderova, Petra; Omelka, Marek; Kamenik, Zdenek; Olsovska, Jana; Kopecky, Jan

2015-04-01

Distribution and evolutionary history of resistance genes in environmental actinobacteria provide information on intensity of antibiosis and evolution of specific secondary metabolic pathways at a given site. To this day, actinobacteria producing biologically active compounds were isolated mostly from soil but only a limited range of soil environments were commonly sampled. Consequently, soil remains an unexplored environment in search for novel producers and related evolutionary questions. Ninety actinobacteria strains isolated at contrasting soil sites were characterized phylogenetically by 16S rRNA gene, for presence of erm and ABC transporter resistance genes and antibiotic production. An analogous analysis was performed in silico with 246 and 31 strains from Integrated Microbial Genomes (JGI_IMG) database selected by the presence of ABC transporter genes and erm genes, respectively. In the isolates, distances of erm gene sequences were significantly correlated to phylogenetic distances based on 16S rRNA genes, while ABC transporter gene distances were not. The phylogenetic distance of isolates was significantly correlated to soil pH and organic matter content of isolation sites. In the analysis of JGI_IMG datasets the correlation between phylogeny of resistance genes and the strain phylogeny based on 16S rRNA genes or five housekeeping genes was observed for both the erm genes and ABC transporter genes in both actinobacteria and streptomycetes. However, in the analysis of sequences from genomes where both resistance genes occurred together the correlation was observed for both ABC transporter and erm genes in actinobacteria but in streptomycetes only in the erm gene. The type of erm resistance gene sequences was influenced by linkage to 16S rRNA gene sequences and site characteristics. The phylogeny of ABC transporter gene was correlated to 16S rRNA genes mainly above the genus level. The results support the concept of new specific secondary metabolite scaffolds occurring more likely in taxonomically distant producers but suggest that the antibiotic selection of gene pools is also influenced by site conditions.

Characterization and expression profiling of ATP-binding cassette transporter genes in the diamondback moth, Plutella xylostella (L.).

PubMed

Qi, Weiping; Ma, Xiaoli; He, Weiyi; Chen, Wei; Zou, Mingmin; Gurr, Geoff M; Vasseur, Liette; You, Minsheng

2016-09-27

ATP-binding cassette (ABC) transporters are one of the major transmembrane protein families found in all organisms and play important roles in transporting a variety of compounds across intra and extra cellular membranes. In some species, ABC transporters may be involved in the detoxification of substances such as insecticides. The diamondback moth, Plutella xylostella (L.), a destructive pest of cruciferous crops worldwide, is an important species to study as it is resistant to many types of insecticides as well as biological control Bacillus thuringiensis toxins. A total of 82 ABC genes were identified from our published P. xylostella genome, and grouped into eight subfamilies (ABCA-H) based on phylogenetic analysis. Genes of subfamilies ABCA, ABCC and ABCH were found to be expanded in P. xylostella compared with those in Bombyx mori, Manduca sexta, Heliconius melpomene, Danaus plexippus, Drosophila melanogaster, Tetranychus urticae and Homo sapiens. Phylogenetic analysis indicated that many of the ABC transporters in P. xylostella are orthologous to the well-studied ABC transporter genes in the seven other species. Transcriptome- and qRT-PCR-based analysis elucidated physiological effects of ABC gene expressions of P. xylostella which were developmental stage- and tissue-specific as well as being affected by whether or not the insects were from an insecticide-resistant strain. Two ABCC and one ABCA genes were preferentially expressed in midgut of the 4th-instar larvae of a susceptible strain (Fuzhou-S) suggesting their potential roles in metabolizing plant defensive chemicals. Most of the highly expressed genes in insecticide-resistant strains were also predominantly expressed in the tissues of Malpighian tubules and midgut. This is the most comprehensive study on identification, characterization and expression profiling of ABC transporter genes in P. xylostella to date. The diversified features and expression patterns of this gene family may be associated with the evolutionary capacity of this species to develop resistance to a wide range of insecticides and biological toxins. Our findings provide a solid foundation for future functional studies on specific ABC transporter genes in P. xylostella, and for further understanding of their physiological roles and regulatory pathways in insecticide resistance.

Flexible exportation mechanisms of arthrofactin in Pseudomonas sp. MIS38.

PubMed

Lim, S P; Roongsawang, N; Washio, K; Morikawa, M

2009-07-01

To obtain further insights into transportation mechanisms of a most effective biosurfactant, arthrofactin in Pseudomonas sp. MIS38. A cluster genes arfA/B/C encodes an arthrofactin synthetase complex (ArfA/B/C). Downstream of the arfA/B/C lie genes encoding a putative periplasmic protein (ArfD, 362 aa) and a putative ATP-binding cassette transporter (ArfE, 651 aa), namely arfD and arfE, respectively. The arfA/B/C, arfD, and arfE form an operon suggesting their functional connection. Gene knockout mutants ArfD:Km, ArfE:Km, ArfD:Tc/ArfE:Km, and gene overexpression strains MIS38(pME6032_arfD/E) and ArfE:Km(pME6032_arfD/E) were prepared and analysed for arthrofactin production profiles. It was found that the production levels of arthrofactin were temporally reduced in the mutants or increased in the gene overexpression strains, but they eventually became similar level to that of MIS38. Addition of ABC transporter inhibitors, glibenclamide and sodium ortho-vanadate dramatically reduced the production levels of arthrofactin. This excludes a possibility that arthrofactin is exported by diffusion with the aid of its own high surfactant activity. ArfD/E is not an exclusive but a primary exporter of arthrofactin during early growth stage. Reduction in the arthrofactin productivity of arfD and arfE knockout mutants was eventually rescued by another ABC transporter system. Effects of arfD and arfE overexpression were evident only for 1-day cultivation. Multiple ATP dependent active transporter systems are responsible for the production of arthrofactin. Pseudomonas bacteria are characterized to be endued with multiple exporter and efflux systems for secondary metabolites including antibiotics, plant toxins, and biosurfactants. The present work demonstrates exceptionally flexible and highly controlled transportation mechanisms of a most effective lipopeptide biosurfactant, arthrofactin in Pseudomonas sp. MIS38. Because lipopeptide biosurfactants are known to enhance efficacy of bioactive compounds and arfA/B/C/D/E orthologous genes are also found in plant pathogenic P. fluorescens and P. syringae strains, the knowledge would also contribute to develop a technology controlling plant diseases.

IMG-ABC: A Knowledge Base To Fuel Discovery of Biosynthetic Gene Clusters and Novel Secondary Metabolites.

PubMed

Hadjithomas, Michalis; Chen, I-Min Amy; Chu, Ken; Ratner, Anna; Palaniappan, Krishna; Szeto, Ernest; Huang, Jinghua; Reddy, T B K; Cimermančič, Peter; Fischbach, Michael A; Ivanova, Natalia N; Markowitz, Victor M; Kyrpides, Nikos C; Pati, Amrita

2015-07-14

In the discovery of secondary metabolites, analysis of sequence data is a promising exploration path that remains largely underutilized due to the lack of computational platforms that enable such a systematic approach on a large scale. In this work, we present IMG-ABC (https://img.jgi.doe.gov/abc), an atlas of biosynthetic gene clusters within the Integrated Microbial Genomes (IMG) system, which is aimed at harnessing the power of "big" genomic data for discovering small molecules. IMG-ABC relies on IMG's comprehensive integrated structural and functional genomic data for the analysis of biosynthetic gene clusters (BCs) and associated secondary metabolites (SMs). SMs and BCs serve as the two main classes of objects in IMG-ABC, each with a rich collection of attributes. A unique feature of IMG-ABC is the incorporation of both experimentally validated and computationally predicted BCs in genomes as well as metagenomes, thus identifying BCs in uncultured populations and rare taxa. We demonstrate the strength of IMG-ABC's focused integrated analysis tools in enabling the exploration of microbial secondary metabolism on a global scale, through the discovery of phenazine-producing clusters for the first time in Alphaproteobacteria. IMG-ABC strives to fill the long-existent void of resources for computational exploration of the secondary metabolism universe; its underlying scalable framework enables traversal of uncovered phylogenetic and chemical structure space, serving as a doorway to a new era in the discovery of novel molecules. IMG-ABC is the largest publicly available database of predicted and experimental biosynthetic gene clusters and the secondary metabolites they produce. The system also includes powerful search and analysis tools that are integrated with IMG's extensive genomic/metagenomic data and analysis tool kits. As new research on biosynthetic gene clusters and secondary metabolites is published and more genomes are sequenced, IMG-ABC will continue to expand, with the goal of becoming an essential component of any bioinformatic exploration of the secondary metabolism world. Copyright © 2015 Hadjithomas et al.

The ASK1 gene regulates B function gene expression in cooperation with UFO and LEAFY in Arabidopsis.

PubMed

Zhao, D; Yu, Q; Chen, M; Ma, H

2001-07-01

The Arabidopsis floral regulatory genes APETALA3 (AP3) and PISTILLATA (PI) are required for the B function according to the ABC model for floral organ identity. AP3 and PI expression are positively regulated by the LEAFY (LFY) and UNUSUAL FLORAL ORGANS (UFO) genes. UFO encodes an F-box protein, and we have shown previously that UFO genetically interacts with the ASK1 gene encoding a SKP1 homologue; both the F-box containing protein and SKP1 are subunits of ubiquitin ligases. We show here that the ask1-1 mutation can enhance the floral phenotypes of weak lfy and ap3 mutants; therefore, like UFO, ASK1 also interacts with LFY and AP3 genetically. Furthermore, our results from RNA in situ hybridizations indicate that ASK1 regulates early AP3 and PI expression. These results support the idea that UFO and ASK1 together positively regulate AP3 and PI expression. We propose that the UFO and ASK1 proteins are components of a ubiquitin ligase that mediates the proteolysis of a repressor of AP3 and PI expression. Our genetic studies also indicate that ASK1 and UFO play a role in regulating the number of floral organ primordia, and we discuss possible mechanisms for such a regulation.

Loss of plastoglobule kinases ABC1K1 and ABC1K3 causes conditional degreening, modified prenyl-lipids, and recruitment of the jasmonic acid pathway

USDA-ARS?s Scientific Manuscript database

Plastoglobules (PGs) are plastid lipid-protein particles. This study examines the function of PG-localized kinases ABC1K1 and ABC1K3 in Arabidopsis thaliana. Several lines of evidence suggested that ABC1K1 and ABC1K3 form a protein complex. Null mutants for both genes (abc1k1 and abc1k3) and the dou...

Epstein-Barr virus latent gene sequences as geographical markers of viral origin: unique EBNA3 gene signatures identify Japanese viruses as distinct members of the Asian virus family.

PubMed

Sawada, Akihisa; Croom-Carter, Deborah; Kondo, Osamu; Yasui, Masahiro; Koyama-Sato, Maho; Inoue, Masami; Kawa, Keisei; Rickinson, Alan B; Tierney, Rosemary J

2011-05-01

Polymorphisms in Epstein-Barr virus (EBV) latent genes can identify virus strains from different human populations and individual strains within a population. An Asian EBV signature has been defined almost exclusively from Chinese viruses, with little information from other Asian countries. Here we sequenced polymorphic regions of the EBNA1, 2, 3A, 3B, 3C and LMP1 genes of 31 Japanese strains from control donors and EBV-associated T/NK-cell lymphoproliferative disease (T/NK-LPD) patients. Though identical to Chinese strains in their dominant EBNA1 and LMP1 alleles, Japanese viruses were subtly different at other loci. Thus, while Chinese viruses mainly fall into two families with strongly linked 'Wu' or 'Li' alleles at EBNA2 and EBNA3A/B/C, Japanese viruses all have the consensus Wu EBNA2 allele but fall into two families at EBNA3A/B/C. One family has variant Li-like sequences at EBNA3A and 3B and the consensus Li sequence at EBNA3C; the other family has variant Wu-like sequences at EBNA3A, variants of a low frequency Chinese allele 'Sp' at EBNA3B and a consensus Sp sequence at EBNA3C. Thus, EBNA3A/B/C allelotypes clearly distinguish Japanese from Chinese strains. Interestingly, most Japanese viruses also lack those immune-escape mutations in the HLA-A11 epitope-encoding region of EBNA3B that are so characteristic of viruses from the highly A11-positive Chinese population. Control donor-derived and T/NK-LPD-derived strains were similarly distributed across allelotypes and, by using allelic polymorphisms to track virus strains in patients pre- and post-haematopoietic stem-cell transplant, we show that a single strain can induce both T/NK-LPD and B-cell-lymphoproliferative disease in the same patient.

Effect of SOS-induced levels of imuABC on spontaneous and damage-induced mutagenesis in Caulobacter crescentus.

PubMed

Alves, Ingrid R; Lima-Noronha, Marco A; Silva, Larissa G; Fernández-Silva, Frank S; Freitas, Aline Luiza D; Marques, Marilis V; Galhardo, Rodrigo S

2017-11-01

imuABC (imuAB dnaE2) genes are responsible for SOS-mutagenesis in Caulobacter crescentus and other bacterial species devoid of umuDC. In this work, we have constructed operator-constitutive mutants of the imuABC operon. We used this genetic tool to investigate the effect of SOS-induced levels of these genes upon both spontaneous and damage-induced mutagenesis. We showed that constitutive expression of imuABC does not increase spontaneous or damage-induced mutagenesis, nor increases cellular resistance to DNA-damaging agents. Nevertheless, the presence of the operator-constitutive mutation rescues mutagenesis in a recA background, indicating that imuABC are the only genes required at SOS-induced levels for translesion synthesis (TLS) in C. crescentus. Furthermore, these data also show that TLS mediated by ImuABC does not require RecA, unlike umuDC-dependent mutagenesis in E. coli. Copyright © 2017 Elsevier B.V. All rights reserved.

ABC transporters are involved in defense against permethrin insecticide in the malaria vector Anopheles stephensi.

PubMed

Epis, Sara; Porretta, Daniele; Mastrantonio, Valentina; Comandatore, Francesco; Sassera, Davide; Rossi, Paolo; Cafarchia, Claudia; Otranto, Domenico; Favia, Guido; Genchi, Claudio; Bandi, Claudio; Urbanelli, Sandra

2014-07-29

Proteins from the ABC family (ATP-binding cassette) represent the largest known group of efflux pumps, responsible for transporting specific molecules across lipid membranes in both prokaryotic and eukaryotic organisms. In arthropods they have been shown to play a role in insecticide defense/resistance. The presence of ABC transporters and their possible association with insecticide transport have not yet been investigated in the mosquito Anopheles stephensi, the major vector of human malaria in the Middle East and South Asian regions. Here we investigated the presence and role of ABCs in transport of permethrin insecticide in a susceptible strain of this mosquito species. To identify ABC transporter genes we obtained a transcriptome from untreated larvae of An. stephensi and then compared it with the annotated transcriptome of Anopheles gambiae. To analyse the association between ABC transporters and permethrin we conducted bioassays with permethrin alone and in combination with an ABC inhibitor, and then we investigated expression profiles of the identified genes in larvae exposed to permethrin. Bioassays showed an increased mortality of mosquitoes when permethrin was used in combination with the ABC-transporter inhibitor. Genes for ABC transporters were detected in the transcriptome, and five were selected (AnstABCB2, AnstABCB3, AnstABCB4, AnstABCmember6 and AnstABCG4). An increased expression in one of them (AnstABCG4) was observed in larvae exposed to the LD50 dose of permethrin. Contrary to what was found in other insect species, no up-regulation was observed in the AnstABCB genes. Our results show for the first time the involvement of ABC transporters in larval defense against permethrin in An. stephensi and, more in general, confirm the role of ABC transporters in insecticide defense. The differences observed with previous studies highlight the need of further research as, despite the growing number of studies on ABC transporters in insects, the heterogeneity of the results available at present does not allow us to infer general trends in ABC transporter-insecticide interactions.

Paenibacillus polymyxa PKB1 produces variants of polymyxin B-type antibiotics.

PubMed

Shaheen, Mohamed; Li, Jingru; Ross, Avena C; Vederas, John C; Jensen, Susan E

2011-12-23

Polymyxins are cationic lipopeptide antibiotics active against many species of Gram-negative bacteria. We sequenced the gene cluster for polymyxin biosynthesis from Paenibacillus polymyxa PKB1. The 40.8 kb gene cluster comprises three nonribosomal peptide synthetase-encoding genes and two ABC transporter-like genes. Disruption of a peptide synthetase gene abolished all antibiotic production, whereas deletion of one or both transporter genes only reduced antibiotic production. Computational analysis of the peptide synthetase modules suggested that the enzyme system produces variant forms of polymyxin B (1 and 2), with D-2,4-diaminobutyrate instead of L-2,4-diaminobutyrate in amino acid position 3. Two antibacterial metabolites were resolved by HPLC and identified by high-resolution mass spectrometry and MS/MS sequencing as the expected variants 3 and 4 of polymyxin B(1) (1) and B(2) (2). Stereochemical analysis confirmed the presence of both D-2,4-diaminobutyrate and L-2,4-diaminobutyrate residues. Copyright © 2011 Elsevier Ltd. All rights reserved.

Role of the Twin-Arginine Translocation Pathway in Staphylococcus▿ †

PubMed Central

Biswas, Lalitha; Biswas, Raja; Nerz, Christiane; Ohlsen, Knut; Schlag, Martin; Schäfer, Tina; Lamkemeyer, Tobias; Ziebandt, Anne-Kathrin; Hantke, Klaus; Rosenstein, Ralf; Götz, Friedrich

2009-01-01

In Staphylococcus, the twin-arginine translocation (Tat) pathway is present only in some species and is composed of TatA and TatC. The tatAC operon is associated with the fepABC operon, which encodes homologs to an iron-binding lipoprotein, an iron-dependent peroxidase (FepB), and a high-affinity iron permease. The FepB protein has a typical twin-arginine (RR) signal peptide. The tat and fep operons constitute an entity that is not present in all staphylococcal species. Our analysis was focused on Staphylococcus aureus and S. carnosus strains. Tat deletion mutants (ΔtatAC) were unable to export active FepB, indicating that this enzyme is a Tat substrate. When the RR signal sequence from FepB was fused to prolipase and protein A, their export became Tat dependent. Since no other protein with a Tat signal could be detected, the fepABC-tatAC genes comprise not only a genetic but also a functional unit. We demonstrated that FepABC drives iron import, and in a mouse kidney abscess model, the bacterial loads of ΔtatAC and Δtat-fep mutants were decreased. For the first time, we show that the Tat pathway in S. aureus is functional and serves to translocate the iron-dependent peroxidase FepB. PMID:19633084

Molecular identification of an ABC transporter complex for manganese: analysis of a cyanobacterial mutant strain impaired in the photosynthetic oxygen evolution process.

PubMed Central

Bartsevich, V V; Pakrasi, H B

1995-01-01

During photosynthesis, the photosystem II (PSII) pigment-protein complex catalyzes oxygen evolution, a reaction in which a four-manganese ensemble plays a crucial role. Using a newly developed selection scheme, we have isolated BP13, a random photosynthesis-deficient mutant strain of the cyanobacterium, Synechocystis 6803. This mutant grew slowly under photoautotrophic conditions, and had a low oxygen evolution activity. Biochemical analysis revealed that the lesion in this mutant strain had specifically affected the Mn ensemble in PSII. Interestingly, incubation of BP13 cells with micromolar levels of added Mn induced rapid recovery of oxygen evolution activity. The mutant could be complemented with a fragment of wild-type chromosomal DNA containing three closely linked genes, mntA, mntB and mntC. These gene products showed significant sequence similarities with polypeptide components of bacterial permeases that are members of the 'ABC (ATP binding cassette) superfamily' of transporter proteins. We determined that in the BP13 strain, a single nucleotide change had resulted in the replacement of an alanine by an aspartic acid residue in MntA, a soluble protein containing ATP binding motifs. These results suggest that the mntCAB gene cluster encodes polypeptide components of a Mn transporter, the first such protein complex identified in any organism. PMID:7743991

Enhancement of Microbial Biodesulfurization via Genetic Engineering and Adaptive Evolution

PubMed Central

Wang, Jia; Butler, Robert R.; Wu, Fan; Pombert, Jean-François; Kilbane, John J.; Stark, Benjamin C.

2017-01-01

In previous work from our laboratories a synthetic gene encoding a peptide (“Sulpeptide 1” or “S1”) with a high proportion of methionine and cysteine residues had been designed to act as a sulfur sink and was inserted into the dsz (desulfurization) operon of Rhodococcus erythropolis IGTS8. In the work described here this construct (dszAS1BC) and the intact dsz operon (dszABC) cloned into vector pRESX under control of the (Rhodococcus) kstD promoter were transformed into the desulfurization-negative strain CW25 of Rhodococcus qingshengii. The resulting strains (CW25[pRESX-dszABC] and CW25[pRESX-dszAS1BC]) were subjected to adaptive selection by repeated passages at log phase (up to 100 times) in minimal medium with dibenzothiophene (DBT) as sole sulfur source. For both strains DBT metabolism peaked early in the selection process and then decreased, eventually averaging four times that of the initial transformed cells; the maximum specific activity achieved by CW25[pRESX-dszAS1BC] exceeded that of CW25[pRESX-dszABC]. Growth rates increased by 7-fold (CW25[pRESX-dszABC]) and 13-fold (CW25[pRESX-dszAS1BC]) and these increases were stable. The adaptations of CW25[pRESX-dszAS1BC] were correlated with a 3-5X increase in plasmid copy numbers from those of the initial transformed cells; whole genome sequencing indicated that during its selection processes no mutations occurred to any of the dsz, S1, or other genes and promoters involved in sulfur metabolism, stress response, or DNA methylation, and that the effect of the sulfur sink produced by S1 is likely very small compared to the cells’ overall cysteine and methionine requirements. Nevertheless, a combination of genetic engineering using sulfur sinks and increasing Dsz capability with adaptive selection may be a viable strategy to increase biodesulfurization ability. PMID:28060828

Comparative genomic analysis of Acinetobacter strains isolated from murine colonic crypts.

PubMed

Saffarian, Azadeh; Touchon, Marie; Mulet, Céline; Tournebize, Régis; Passet, Virginie; Brisse, Sylvain; Rocha, Eduardo P C; Sansonetti, Philippe J; Pédron, Thierry

2017-07-11

A restricted set of aerobic bacteria dominated by the Acinetobacter genus was identified in murine intestinal colonic crypts. The vicinity of such bacteria with intestinal stem cells could indicate that they protect the crypt against cytotoxic and genotoxic signals. Genome analyses of these bacteria were performed to better appreciate their biodegradative capacities. Two taxonomically different clusters of Acinetobacter were isolated from murine proximal colonic crypts, one was identified as A. modestus and the other as A. radioresistens. Their identification was performed through biochemical parameters and housekeeping gene sequencing. After selection of one strain of each cluster (A. modestus CM11G and A. radioresistens CM38.2), comparative genomic analysis was performed on whole-genome sequencing data. The antibiotic resistance pattern of these two strains is different, in line with the many genes involved in resistance to heavy metals identified in both genomes. Moreover whereas the operon benABCDE involved in benzoate metabolism is encoded by the two genomes, the operon antABC encoding the anthranilate dioxygenase, and the phenol hydroxylase gene cluster are absent in the A. modestus genomic sequence, indicating that the two strains have different capacities to metabolize xenobiotics. A common feature of the two strains is the presence of a type IV pili system, and the presence of genes encoding proteins pertaining to secretion systems such as Type I and Type II secretion systems. Our comparative genomic analysis revealed that different Acinetobacter isolated from the same biological niche, even if they share a large majority of genes, possess unique features that could play a specific role in the protection of the intestinal crypt.

Genome-wide identification of ATP-binding cassette (ABC) transporters and their roles in response to polycyclic aromatic hydrocarbons (PAHs) in the copepod Paracyclopina nana.

PubMed

Jeong, Chang-Bum; Kim, Duck-Hyun; Kang, Hye-Min; Lee, Young Hwan; Kim, Hui-Su; Kim, Il-Chan; Lee, Jae-Seong

2017-02-01

The ATP-binding cassette (ABC) protein superfamily is one of the largest gene families and is highly conserved in all domains. The ABC proteins play roles in several biological processes, including multi-xenobiotic resistance (MXR), by functioning as transporters in the cellular membrane. They also mediate the cellular efflux of a wide range of substrates against concentration gradients. In this study, 37 ABC genes belonging to eight distinct subfamilies were identified in the marine copepod Paracyclopina nana and annotated based on a phylogenetic analysis. Also, the functions of P-glycoproteins (P-gp) and multidrug resistance-associated proteins (MRPs), conferring MXR, were verified using fluorescent substrates and specific inhibitors. The activities of MXR-mediated ABC proteins and their transcriptional level were examined in response to polyaromatic hydrocarbons (PAHs), main components of the water-accommodated fraction. This study increases the understanding of the protective role of MXR in response to PAHs over the comparative evolution of ABC gene families. Copyright © 2016 Elsevier B.V. All rights reserved.

Independent activity of the homologous small regulatory RNAs AbcR1 and AbcR2 in the legume symbiont Sinorhizobium meliloti.

PubMed

Torres-Quesada, Omar; Millán, Vicenta; Nisa-Martínez, Rafael; Bardou, Florian; Crespi, Martín; Toro, Nicolás; Jiménez-Zurdo, José I

2013-01-01

The legume symbiont Sinorhizobium meliloti expresses a plethora of small noncoding RNAs (sRNAs) whose function is mostly unknown. Here, we have functionally characterized two tandemly encoded S. meliloti Rm1021 sRNAs that are similar in sequence and structure. Homologous sRNAs (designated AbcR1 and AbcR2) have been shown to regulate several ABC transporters in the related α-proteobacteria Agrobacterium tumefaciens and Brucella abortus. In Rm1021, AbcR1 and AbcR2 exhibit divergent unlinked regulation and are stabilized by the RNA chaperone Hfq. AbcR1 is transcribed in actively dividing bacteria, either in culture, rhizosphere or within the invasion zone of mature alfalfa nodules. Conversely, AbcR2 expression is induced upon entry into stationary phase and under abiotic stress. Only deletion of AbcR1 resulted into a discrete growth delay in rich medium, but both are dispensable for symbiosis. Periplasmic proteome profiling revealed down-regulation of the branched-chain amino acid binding protein LivK by AbcR1, but not by AbcR2. A double-plasmid reporter assay confirmed the predicted specific targeting of the 5'-untranslated region of the livK mRNA by AbcR1 in vivo. Our findings provide evidences of independent regulatory functions of these sRNAs, probably to fine-tune nutrient uptake in free-living and undifferentiated symbiotic rhizobia.

Independent Activity of the Homologous Small Regulatory RNAs AbcR1 and AbcR2 in the Legume Symbiont Sinorhizobium meliloti

PubMed Central

Torres-Quesada, Omar; Millán, Vicenta; Nisa-Martínez, Rafael; Bardou, Florian; Crespi, Martín; Toro, Nicolás; Jiménez-Zurdo, José I.

2013-01-01

The legume symbiont Sinorhizobium meliloti expresses a plethora of small noncoding RNAs (sRNAs) whose function is mostly unknown. Here, we have functionally characterized two tandemly encoded S. meliloti Rm1021 sRNAs that are similar in sequence and structure. Homologous sRNAs (designated AbcR1 and AbcR2) have been shown to regulate several ABC transporters in the related α-proteobacteria Agrobacterium tumefaciens and Brucella abortus. In Rm1021, AbcR1 and AbcR2 exhibit divergent unlinked regulation and are stabilized by the RNA chaperone Hfq. AbcR1 is transcribed in actively dividing bacteria, either in culture, rhizosphere or within the invasion zone of mature alfalfa nodules. Conversely, AbcR2 expression is induced upon entry into stationary phase and under abiotic stress. Only deletion of AbcR1 resulted into a discrete growth delay in rich medium, but both are dispensable for symbiosis. Periplasmic proteome profiling revealed down-regulation of the branched-chain amino acid binding protein LivK by AbcR1, but not by AbcR2. A double-plasmid reporter assay confirmed the predicted specific targeting of the 5′-untranslated region of the livK mRNA by AbcR1 in vivo. Our findings provide evidences of independent regulatory functions of these sRNAs, probably to fine-tune nutrient uptake in free-living and undifferentiated symbiotic rhizobia. PMID:23869210

Cloning and expression analysis of the ATP-binding cassette transporter gene MFABC1 and the alternative oxidase gene MfAOX1 from Monilinia fructicola.

PubMed

Schnabel, Guido; Dait, Qun; Paradkar, Manjiri R

2003-10-01

Brown rot, caused by Moniliniafructicola (G Wint) Honey, is a serious disease of peach in all commercial peach production areas in the USA, including South Carolina where it has been primarily controlled by pre-harvest application of 14-alpha demethylation (DMI) fungicides for more than 15 years. Recently, the Qo fungicide azoxystrobin was registered for brown rot control and is currently being investigated for its potential as a DMI fungicide rotation partner because of its different mode of action. In an effort to investigate molecular mechanisms of DMI and Qo fungicide resistance in M fructicola, the ABC transporter gene MfABC1 and the alternative oxidase gene MfAOX1 were cloned to study their potential role in conferring fungicide resistance. The MfABC1 gene was 4380 bp in length and contained one intron of 71 bp. The gene revealed high amino acid homologies with atrB from Aspergillus nidulans (Eidam) Winter, an ABC transporter conferring resistance to many fungicides, including DMI fungicides. MfABC1 gene expression was induced after myclobutanil and propiconazole treatment in isolates with low sensitivity to the same fungicides, and in an isolate with high sensitivity to propiconazole. The results suggest that the MfABC1 gene may be a DMI fungicide resistance determinant in M fructicola. The alternative oxidase gene MfAOX1 from M fructicola was cloned and gene expression was analyzed. The MfAOX1 gene was 1077 bp in length and contained two introns of 54 and 67 bp. The amino acid sequence was 63.8, 63.8 and 57.7% identical to alternative oxidases from Venturia inaequalis (Cooke) Winter, Aspergillus niger van Teighem and A nidulans, respectively. MfAOX1 expression in some but not all M fructicola isolates was induced in mycelia treated with azoxystrobin. Azoxystrobin at 2 microg ml(-1) significantly induced MfAOX1 expression in isolates with low MfAOX1 constitutive expression levels.

Enterococcus faecalis utilizes maltose by connecting two incompatible metabolic routes via a novel maltose-6’-phosphate phosphatase (MapP)

PubMed Central

Mokhtari, Abdelhamid; Blancato, Víctor S.; Repizo, Guillermo; Henry, Céline; Pikis, Andreas; Bourand, Alexa; de Fátima Álvarez, María; Immel, Stefan; Mechakra-Maza, Aicha; Hartke, Axel; Thompson, John; Magni, Christian; Deutscher, Josef

2013-01-01

Summary Similar to Bacillus subtilis, Enterococcus faecalis transports and phosphorylates maltose via a phosphoenolpyruvate (PEP):maltose phosphotransferase system (PTS). The maltose-specific PTS permease is encoded by the malT gene. However, E. faecalis lacks a malA gene encoding a 6-phospho-α-glucosidase which in B. subtilis hydrolyses maltose-6’-P into glucose and glucose-6-P. Instead, an operon encoding a maltose phosphorylase (MalP), a phosphoglucomutase and a mutarotase starts upstream from malT. MalP was suggested to split maltose-6-P into glucose-1-P and glucose-6-P. However, purified MalP phosphorolyses maltose but not maltose-6’-P. We discovered that the gene downstream from malT encodes a novel enzyme (MapP) that dephosphorylates maltose-6’-P formed by the PTS. The resulting intracellular maltose is cleaved by MalP into glucose and glucose-1-P. Slow uptake of maltose probably via a maltodextrin ABC transporter allows poor growth for the mapP but not the malP mutant. Synthesis of MapP in a B. subtilis mutant accumulating maltose-6’-P restored growth on maltose. MapP catalyzes the dephosphorylation of intracellular maltose-6’-P, and the resulting maltose is converted by the B. subtilis maltose phosphorylase into glucose and glucose-1-P. MapP therefore connects PTS-mediated maltose uptake to maltose phosphorylase-catalyzed metabolism. Dephosphorylation assays with a wide variety of phospho-substrates revealed that MapP preferably dephosphorylates disaccharides containing an O-α-glycosyl linkage. PMID:23490043

Metabolism of a plant derived galactose-containing polysaccharide by Bifidobacterium breve UCC2003.

PubMed

O'Connell Motherway, Mary; Fitzgerald, Gerald F; van Sinderen, Douwe

2011-05-01

In this study, we describe the functional characterization of the Bifidobacterium breve UCC2003 gal locus, which is dedicated to the utilization of galactan, a plant-derived polysaccharide. Using a combination of molecular approaches we conclude that the galA gene of B. breve UCC2003 encodes a β-1,4-endogalactanase producing galacto-oligosaccharides, which are specifically internalized by an ABC transport system, encoded by galBCDE, and which are then hydrolysed to galactose moieties by a dedicated intracellular β-galactosidase, specified by galG. The generated galactose molecules are presumed to be fed into the fructose-6-phosphate phosphoketolase pathway via the Leloir pathway, thereby allowing B. breve UCC2003 to use galactan as its sole carbon and energy source. In addition to these findings we demonstrate that GalR is a LacI-type DNA-binding protein, which not only appears to control transcription of the galCDEGR operon, but also that of the galA gene. © 2010 University College Cork. Journal compilation © 2010 Society for Applied Microbiology and Blackwell Publishing Ltd.

Seasonal Expression of the Picocyanobacterial Phosphonate Transporter Gene phnD in the Sargasso Sea

PubMed Central

Ilikchyan, Irina N.; McKay, Robert Michael L.; Kutovaya, Olga A.; Condon, Rob; Bullerjahn, George S.

2010-01-01

In phosphorus-limited marine environments, picocyanobacteria (Synechococcus and Prochlorococcus spp.) can hydrolyze naturally occurring phosphonates as a P source. Utilization of 2-aminoethylphosphonate (2-AEP) is dependent on expression of the phn genes, encoding functions required for uptake, and C–P bond cleavage. Prior work has indicated that expression of picocyanobacterial phnD, encoding the phosphonate binding protein of the phosphonate ABC transporter, is a proxy for the assimilation of phosphonates in natural assemblages of Synechococcus spp. and Prochlorococcus spp (Ilikchyan et al., 2009). In this study, we expand this work to assess seasonal phnD expression in the Sargasso Sea. By RT-PCR, our data confirm that phnD expression is constitutive for the Prochlorococcus spp. detected, but in Synechococcus spp. phnD transcription follows patterns of phosphorus availability in the mixed layer. Specifically, our data suggest that phnD is repressed in the spring when P is bioavailable following deep winter mixing. In the fall, phnD expression follows a depth-dependent pattern reflecting depleted P at the surface following summertime drawdown, and elevated P at depth. PMID:21687717

[Process and mechanism of plants in overcoming acid soil aluminum stress].

PubMed

Zhao, Tian-Long; Xie, Guang-Ning; Zhang, Xiao-Xia; Qiu, Lin-Quan; Wang, Na; Zhang, Su-Zhi

2013-10-01

Aluminum (Al) stress is one of the most important factors affecting the plant growth on acid soil. Currently, global soil acidification further intensifies the Al stress. Plants can detoxify Al via the chelation of ionic Al and organic acids to store the ionic Al in vacuoles and extrude it from roots. The Al extrusion is mainly performed by the membrane-localized anion channel proteins Al(3+)-activated malate transporter (ALMT) and multi-drug and toxin extrusion (MATE). The genes encoding ABC transporter and zinc-finger protein conferred plant Al tolerance have also been found. The identification of these Al-resistant genes makes it possible to increase the Al resistance of crop plants and enhance their production by the biological methods such as gene transformation and mark-associated breeding. The key problems needed to be solved and the possible directions in the researches of plant Al stress resistance were proposed.

Discovering Novel Bile Protection Systems in Bifidobacterium breve UCC2003 through Functional Genomics

PubMed Central

Ruiz, Lorena; Zomer, Aldert; O'Connell-Motherway, Mary; van Sinderen, Douwe

2012-01-01

Tolerance of gut commensals to bile salt exposure is an important feature for their survival in and colonization of the intestinal environment. A transcriptomic approach was employed to study the response of Bifidobacterium breve UCC2003 to bile, allowing the identification of a number of bile-induced genes with a range of predicted functions. The potential roles of a selection of these bile-inducible genes in bile protection were analyzed following heterologous expression in Lactococcus lactis. Genes encoding three transport systems belonging to the major facilitator superfamily (MFS), Bbr_0838, Bbr_0832, and Bbr_1756, and three ABC-type transporters, Bbr_0406-0407, Bbr_1804-1805, and Bbr_1826-1827, were thus investigated and shown to provide enhanced resistance and survival to bile exposure. This work significantly improves our understanding as to how bifidobacteria respond to and survive bile exposure. PMID:22156415

Overexpression of the ABC transporter AvtAB increases avermectin production in Streptomyces avermitilis.

PubMed

Qiu, Jingfan; Zhuo, Ying; Zhu, Dongqing; Zhou, Xiufen; Zhang, Lixin; Bai, Linquan; Deng, Zixin

2011-10-01

Avermectins are 16-membered macrocyclic polyketides with potent antiparasitic activities, produced by Streptomyces avermitilis. Upstream of the avermectin biosynthetic gene cluster, there is the avtAB operon encoding the ABC transporter AvtAB, which is highly homologous to the mammalian multidrug efflux pump P-glycoprotein (Pgp). Inactivation of avtAB had no effect, but increasing the concentration of avtAB mRNA 30-500-fold, using a multi-copy plasmid in S. avermitilis, enhanced avermectin production about two-fold both in the wild-type and in a high-yield producer strain on agar plates. In liquid industrial fermentation medium, the overall productivity of avermectin B1a in the engineered high-yield producer was improved for about 50%, from 3.3 to 4.8 g/l. In liquid YMG medium, moreover, the ratio of intracellular to extracellular accumulation of avermectin B1a was dropped from 6:1 to 4.5:1 in response to multiple copies of avtAB. Additionally, the overexpression of avtAB did not cause any increased expression of the avermectin biosynthetic genes through RT-PCR analysis. We propose that the AvtAB transporter exports avermectin, and thus reduces the feedback inhibition on avermectin production inside the cell. This strategy may be useful for enhancing the production of other antibiotics.

Transcriptional Analysis of Prebiotic Uptake and Catabolism by Lactobacillus acidophilus NCFM

PubMed Central

Andersen, Joakim Mark; Barrangou, Rodolphe; Hachem, Maher Abou; Lahtinen, Sampo J.; Goh, Yong-Jun; Svensson, Birte; Klaenhammer, Todd R.

2012-01-01

The human gastrointestinal tract can be positively modulated by dietary supplementation of probiotic bacteria in combination with prebiotic carbohydrates. Here differential transcriptomics and functional genomics were used to identify genes in Lactobacillus acidophilus NCFM involved in the uptake and catabolism of 11 potential prebiotic compounds consisting of α- and β- linked galactosides and glucosides. These oligosaccharides induced genes encoding phosphoenolpyruvate-dependent sugar phosphotransferase systems (PTS), galactoside pentose hexuronide (GPH) permease, and ATP-binding cassette (ABC) transporters. PTS systems were upregulated primarily by di- and tri-saccharides such as cellobiose, isomaltose, isomaltulose, panose and gentiobiose, while ABC transporters were upregulated by raffinose, Polydextrose, and stachyose. A single GPH transporter was induced by lactitol and galactooligosaccharides (GOS). The various transporters were associated with a number of glycoside hydrolases from families 1, 2, 4, 13, 32, 36, 42, and 65, involved in the catabolism of various α- and β-linked glucosides and galactosides. Further subfamily specialization was also observed for different PTS-associated GH1 6-phospho-β-glucosidases implicated in the catabolism of gentiobiose and cellobiose. These findings highlight the broad oligosaccharide metabolic repertoire of L. acidophilus NCFM and establish a platform for selection and screening of both probiotic bacteria and prebiotic compounds that may positively influence the gastrointestinal microbiota. PMID:23028535

Efficient extracellular production of type I secretion pathway-dependent Pseudomonas fluorescens lipase in recombinant Escherichia coli by heterologous ABC protein exporters.

PubMed

Eom, Gyeong Tae; Lee, Seung Hwan; Oh, Young Hoon; Choi, Ji Eun; Park, Si Jae; Song, Jae Kwang

2014-10-01

Heterologous ABC protein exporters, the apparatus of type I secretion pathway in Gram-negative bacteria, were used for extracellular production of Pseudomonas fluorescens lipase (TliA) in recombinant Escherichia coli. The effect of the expression of different ABC protein exporter gene clusters (P. fluorescens tliDEF, Pseudomonas aeruginosa aprDEF, Erwinia chrysanthemi prtDEF, and Serratia marcescens lipBCD genes) was examined on the secretion of TliA at growth temperatures of 20, 25, 30 and 35 °C. TliA secretion in recombinant E. coli XL10-Gold varied depending upon type of ABC protein exporter and culture temperature. E. coli expressing S. marcescens lipBCD genes showed the highest secretion level of TliA (122.8 U ml(-1)) when cultured at 25 °C. Thus, optimized culture conditions for efficient extracellular production of lipase in recombinant E. coli can be designed by changing the type of ABC protein exporter and the growth temperature.

IMG-ABC. A knowledge base to fuel discovery of biosynthetic gene clusters and novel secondary metabolites

DOE PAGES

Hadjithomas, Michalis; Chen, I-Min Amy; Chu, Ken; ...

2015-07-14

In the discovery of secondary metabolites, analysis of sequence data is a promising exploration path that remains largely underutilized due to the lack of computational platforms that enable such a systematic approach on a large scale. In this work, we present IMG-ABC (https://img.jgi.doe.gov/abc), an atlas of biosynthetic gene clusters within the Integrated Microbial Genomes (IMG) system, which is aimed at harnessing the power of “big” genomic data for discovering small molecules. IMG-ABC relies on IMG’s comprehensive integrated structural and functional genomic data for the analysis of biosynthetic gene clusters (BCs) and associated secondary metabolites (SMs). SMs and BCs serve asmore » the two main classes of objects in IMG-ABC, each with a rich collection of attributes. A unique feature of IMG-ABC is the incorporation of both experimentally validated and computationally predicted BCs in genomes as well as metagenomes, thus identifying BCs in uncultured populations and rare taxa. We demonstrate the strength of IMG-ABC’s focused integrated analysis tools in enabling the exploration of microbial secondary metabolism on a global scale, through the discovery of phenazine-producing clusters for the first time in lphaproteobacteria. IMG-ABC strives to fill the long-existent void of resources for computational exploration of the secondary metabolism universe; its underlying scalable framework enables traversal of uncovered phylogenetic and chemical structure space, serving as a doorway to a new era in the discovery of novel molecules. IMG-ABC is the largest publicly available database of predicted and experimental biosynthetic gene clusters and the secondary metabolites they produce. The system also includes powerful search and analysis tools that are integrated with IMG’s extensive genomic/metagenomic data and analysis tool kits. As new research on biosynthetic gene clusters and secondary metabolites is published and more genomes are sequenced, IMG-ABC will continue to expand, with the goal of becoming an essential component of any bioinformatic exploration of the secondary metabolism world.« less

Differential Adipose Tissue Gene Expression Profiles in Abacavir Treated Patients That May Contribute to the Understanding of Cardiovascular Risk: A Microarray Study

PubMed Central

Shahmanesh, Mohsen; Phillips, Kenneth; Boothby, Meg; Tomlinson, Jeremy W.

2015-01-01

Objective To compare changes in gene expression by microarray from subcutaneous adipose tissue from HIV treatment naïve patients treated with efavirenz based regimens containing abacavir (ABC), tenofovir (TDF) or zidovidine (AZT). Design Subcutaneous fat biopsies were obtained before, at 6- and 18–24-months after treatment, and from HIV negative controls. Groups were age, ethnicity, weight, biochemical profile, and pre-treatment CD4 count matched. Microarray data was generated using the Agilent Whole Human Genome Microarray. Identification of differentially expressed genes and genomic response pathways was performed using limma and gene set enrichment analysis. Results There were significant divergences between ABC and the other two groups 6 months after treatment in genes controlling cell adhesion and environmental information processing, with some convergence at 18–24 months. Compared to controls the ABC group, but not AZT or TDF showed enrichment of genes controlling adherence junction, at 6 months and 18–24 months (adjusted p<0.05) and focal adhesions and tight junction at 6 months (p<0.5). Genes controlling leukocyte transendothelial migration (p<0.05) and ECM-receptor interactions (p = 0.04) were over-expressed in ABC compared to TDF and AZT at 6 months but not at 18–24 months. Enrichment of pathways and individual genes controlling cell adhesion and environmental information processing were specifically dysregulated in the ABC group in comparison with other treatments. There was little difference between AZT and TDF. Conclusion After initiating treatment, there is divergence in the expression of genes controlling cell adhesion and environmental information processing between ABC and both TDF and AZT in subcutaneous adipose tissue. If similar changes are also taking place in other tissues including the coronary vasculature they may contribute to the increased risk of cardiovascular events reported in patients recently started on abacavir-containing regimens. PMID:25617630

Enhancement of antibiotic productions by engineered nitrate utilization in actinomycetes.

PubMed

Meng, Sitong; Wu, Hang; Wang, Lei; Zhang, Buchang; Bai, Linquan

2017-07-01

Nitrate is necessary for primary and secondary metabolism of actinomycetes and stimulates the production of a few antibiotics, such as lincomycin and rifamycin. However, the mechanism of this nitrate-stimulating effect was not fully understood. Two putative ABC-type nitrate transporters were identified in Streptomyces lincolnensis NRRL2936 and verified to be involved in lincomycin biosynthesis. With nitrate supplementation, the transcription of nitrogen assimilation genes, nitrate-specific ABC1 transporter genes, and lincomycin exporter gene lmrA was found to be enhanced and positively regulated by the global regulator GlnR, whose expression was also improved. Moreover, heterologous expression of ABC2 transporter genes in Streptomyces coelicolor M145 resulted in an increased actinorhodin production. Further incorporation of a nitrite-specific transporter gene nirC, as in nirC-ABC2 cassette, led to an even higher actinorhodin production. Similarly, the titers of salinomycin, ansamitocin, lincomycin, and geldanamycin were increased with the integration of this cassette to Streptomyces albus BK3-25, Actinosynnema pretiosum ATCC31280, S. lincolnensis LC-G, and Streptomyces hygroscopicus XM201, respectively. Our work expanded the nitrate-stimulating effect to many antibiotic producers by utilizing the nirC-ABC2 cassette for enhanced nitrate utilization, which could become a general tool for titer increase of antibiotics in actinomycetes.

Tolerance to quaternary ammonium compound disinfectants may enhance growth of Listeria monocytogenes in the food industry.

PubMed

Møretrø, Trond; Schirmer, Bjørn C T; Heir, Even; Fagerlund, Annette; Hjemli, Pernille; Langsrud, Solveig

2017-01-16

The antibacterial effect of disinfectants is crucial for the control of Listeria monocytogenes in food processing environments. Tolerance of L. monocytogenes to sublethal levels of disinfectants based on quaternary ammonium compounds (QAC) is conferred by the resistance determinants qacH and bcrABC. The presence and distribution of these genes have been anticipated to have a role in the survival and growth of L. monocytogenes in food processing environments where QAC based disinfectants are in common use. In this study, a panel of 680 L. monocytogenes from nine Norwegian meat- and salmon processing plants were grouped into 36 MLVA profiles. The presence of qacH and bcrABC was determined in 101 isolates from the 26 most common MLVA profiles. Five MLVA profiles contained qacH and two contained bcrABC. Isolates with qacH and bcrABC showed increased tolerance to the QAC Benzalkonium chloride (BC), with minimal inhibitory concentrations (MICs) of 5-12, 10-13 and <5ppm for strains with qacH (two allele variants observed), bcrABC, and neither gene, respectively. Isolates with qacH or bcrABC were not more tolerant to BC in bactericidal tests in suspension or in biofilms compared with isolates lacking the genes. Water residue samples collected from surfaces in meat processing plants after QAC disinfection had bactericidal effect against L. monocytogenes when the sample BC levels were high (>100ppm). A sample with lower BC concentrations (14ppm of chain length C-12 and 2.7ppm of chain length C-14) inhibited growth of L. monocytogenes not containing bcrABC or qacH, compared to strains with these genes. The study has shown that L. monocytogenes harbouring the QAC resistance genes qacH and bcrABC are prevalent in the food industry and that residuals of QAC may be present in concentrations after sanitation in the industry that result in a growth advantage for bacteria with such resistance genes. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

The cabABC Operon Essential for Biofilm and Rugose Colony Development in Vibrio vulnificus

PubMed Central

Park, Jin Hwan; Jo, Youmi; Jang, Song Yee; Kwon, Haenaem; Irie, Yasuhiko; Parsek, Matthew R.; Kim, Myung Hee; Choi, Sang Ho

2015-01-01

A transcriptome analysis identified Vibrio vulnificus cabABC genes which were preferentially expressed in biofilms. The cabABC genes were transcribed as a single operon. The cabA gene was induced by elevated 3′,5′-cyclic diguanylic acid (c-di-GMP) and encoded a calcium-binding protein CabA. Comparison of the biofilms produced by the cabA mutant and its parent strain JN111 in microtiter plates using crystal-violet staining demonstrated that CabA contributed to biofilm formation in a calcium-dependent manner under elevated c-di-GMP conditions. Genetic and biochemical analyses revealed that CabA was secreted to the cell exterior through functional CabB and CabC, distributed throughout the biofilm matrix, and produced as the biofilm matured. These results, together with the observation that CabA also contributes to the development of rugose colony morphology, indicated that CabA is a matrix-associated protein required for maturation, rather than adhesion involved in the initial attachment, of biofilms. Microscopic comparison of the structure of biofilms produced by JN111 and the cabA mutant demonstrated that CabA is an extracellular matrix component essential for the development of the mature biofilm structures in flow cells and on oyster shells. Exogenously providing purified CabA restored the biofilm- and rugose colony-forming abilities of the cabA mutant when calcium was available. Circular dichroism and size exclusion analyses revealed that calcium binding induces CabA conformational changes which may lead to multimerization. Extracellular complementation experiments revealed that CabA can assemble a functional matrix only when exopolysaccharides coexist. Consequently, the combined results suggested that CabA is a structural protein of the extracellular matrix and multimerizes to a conformation functional in building robust biofilms, which may render V. vulnificus to survive in hostile environments and reach a concentrated infective dose. PMID:26406498

A Conserved UDP-Glucose Dehydrogenase Encoded outside the hasABC Operon Contributes to Capsule Biogenesis in Group A Streptococcus

PubMed Central

Cole, Jason N.; Aziz, Ramy K.; Kuipers, Kirsten; Timmer, Anjuli M.; Nizet, Victor

2012-01-01

Group A Streptococcus (GAS) is a human-specific bacterial pathogen responsible for serious morbidity and mortality worldwide. The hyaluronic acid (HA) capsule of GAS is a major virulence factor, contributing to bloodstream survival through resistance to neutrophil and antimicrobial peptide killing and to in vivo pathogenicity. Capsule biosynthesis has been exclusively attributed to the ubiquitous hasABC hyaluronan synthase operon, which is highly conserved across GAS serotypes. Previous reports indicate that hasA, encoding hyaluronan synthase, and hasB, encoding UDP-glucose 6-dehydrogenase, are essential for capsule production in GAS. Here, we report that precise allelic exchange mutagenesis of hasB in GAS strain 5448, a representative of the globally disseminated M1T1 serotype, did not abolish HA capsule synthesis. In silico whole-genome screening identified a putative HasB paralog, designated HasB2, with 45% amino acid identity to HasB at a distant location in the GAS chromosome. In vitro enzymatic assays demonstrated that recombinant HasB2 is a functional UDP-glucose 6-dehydrogenase enzyme. Mutagenesis of hasB2 alone slightly decreased capsule abundance; however, a ΔhasB ΔhasB2 double mutant became completely acapsular. We conclude that HasB is not essential for M1T1 GAS capsule biogenesis due to the presence of a newly identified HasB paralog, HasB2, which most likely resulted from gene duplication. The identification of redundant UDP-glucose 6-dehydrogenases underscores the importance of HA capsule expression for M1T1 GAS pathogenicity and survival in the human host. PMID:22961854

Amino Acid Transporters and Release of Hydrophobic Amino Acids in the Heterocyst-Forming Cyanobacterium Anabaena sp. Strain PCC 7120.

PubMed

Pernil, Rafael; Picossi, Silvia; Herrero, Antonia; Flores, Enrique; Mariscal, Vicente

2015-04-23

Anabaena sp. strain PCC 7120 is a filamentous cyanobacterium that can use inorganic compounds such as nitrate or ammonium as nitrogen sources. In the absence of combined nitrogen, it can fix N2 in differentiated cells called heterocysts. Anabaena also shows substantial activities of amino acid uptake, and three ABC-type transporters for amino acids have been previously characterized. Seven new loci encoding predicted amino acid transporters were identified in the Anabaena genomic sequence and inactivated. Two of them were involved in amino acid uptake. Locus alr2535-alr2541 encodes the elements of a hydrophobic amino acid ABC-type transporter that is mainly involved in the uptake of glycine. ORF all0342 encodes a putative transporter from the dicarboxylate/amino acid:cation symporter (DAACS) family whose inactivation resulted in an increased uptake of a broad range of amino acids. An assay to study amino acid release from Anabaena filaments to the external medium was set up. Net release of the alanine analogue α-aminoisobutyric acid (AIB) was observed when transport system N-I (a hydrophobic amino acid ABC-type transporter) was engaged in the uptake of a specific substrate. The rate of AIB release was directly proportional to the intracellular AIB concentration, suggesting leakage from the cells by diffusion.

Syntrophomonas wolfei Uses an NADH-Dependent, Ferredoxin-Independent [FeFe]-Hydrogenase To Reoxidize NADH

PubMed Central

Losey, Nathaniel A.; Mus, Florence; Peters, John W.; Le, Huynh M.

2017-01-01

ABSTRACT Syntrophomonas wolfei syntrophically oxidizes short-chain fatty acids (four to eight carbons in length) when grown in coculture with a hydrogen- and/or formate-using methanogen. The oxidation of 3-hydroxybutyryl-coenzyme A (CoA), formed during butyrate metabolism, results in the production of NADH. The enzyme systems involved in NADH reoxidation in S. wolfei are not well understood. The genome of S. wolfei contains a multimeric [FeFe]-hydrogenase that may be a mechanism for NADH reoxidation. The S. wolfei genes for the multimeric [FeFe]-hydrogenase (hyd1ABC; SWOL_RS05165, SWOL_RS05170, SWOL_RS05175) and [FeFe]-hydrogenase maturation proteins (SWOL_RS05180, SWOL_RS05190, SWOL_RS01625) were coexpressed in Escherichia coli, and the recombinant Hyd1ABC was purified and characterized. The purified recombinant Hyd1ABC was a heterotrimer with an αβγ configuration and a molecular mass of 115 kDa. Hyd1ABC contained 29.2 ± 1.49 mol of Fe and 0.7 mol of flavin mononucleotide (FMN) per mole enzyme. The purified, recombinant Hyd1ABC reduced NAD+ and oxidized NADH without the presence of ferredoxin. The HydB subunit of the S. wolfei multimeric [FeFe]-hydrogenase lacks two iron-sulfur centers that are present in known confurcating NADH- and ferredoxin-dependent [FeFe]-hydrogenases. Hyd1ABC is a NADH-dependent hydrogenase that produces hydrogen from NADH without the need of reduced ferredoxin, which differs from confurcating [FeFe]-hydrogenases. Hyd1ABC provides a mechanism by which S. wolfei can reoxidize NADH produced during syntrophic butyrate oxidation when low hydrogen partial pressures are maintained by a hydrogen-consuming microorganism. IMPORTANCE Our work provides mechanistic understanding of the obligate metabolic coupling that occurs between hydrogen-producing fatty and aromatic acid-degrading microorganisms and their hydrogen-consuming partners in the process called syntrophy (feeding together). The multimeric [FeFe]-hydrogenase used NADH without the involvement of reduced ferredoxin. The multimeric [FeFe]-hydrogenase would produce hydrogen from NADH only when hydrogen concentrations were low. Hydrogen production from NADH by Syntrophomonas wolfei would likely cease before any detectable amount of cell growth occurred. Thus, continual hydrogen production requires the presence of a hydrogen-consuming partner to keep hydrogen concentrations low and explains, in part, the obligate requirement that S. wolfei has for a hydrogen-consuming partner organism during growth on butyrate. We have successfully expressed genes encoding a multimeric [FeFe]-hydrogenase in E. coli, demonstrating that such an approach can be advantageous to characterize complex redox proteins from difficult-to-culture microorganisms. PMID:28802265

Functional characterization of two SOS-regulated genes involved in mitomycin C resistance in Caulobacter crescentus.

PubMed

Lopes-Kulishev, Carina O; Alves, Ingrid R; Valencia, Estela Y; Pidhirnyj, María I; Fernández-Silva, Frank S; Rodrigues, Ticiane R; Guzzo, Cristiane R; Galhardo, Rodrigo S

2015-09-01

The SOS response is a universal bacterial regulon involved in the cellular response to DNA damage and other forms of stress. In Caulobacter crescentus, previous work has identified a plethora of genes that are part of the SOS regulon, but the biological roles of several of them remain to be determined. In this study, we report that two genes, hereafter named mmcA and mmcB, are involved in the defense against DNA damage caused by mitomycin C (MMC), but not against lesions induced by other common DNA damaging agents, such as UVC light, methyl methanesulfonate (MMS) and hydrogen peroxide. mmcA is a conserved gene that encodes a member of the glyoxalases/dioxygenases protein family, and acts independently of known DNA repair pathways. On the other hand, epistasis analysis showed that mmcB acts in the same pathway as imuC (dnaE2), and is required specifically for MMC-induced mutagenesis, but not for that induced by UV light, suggesting a role for MmcB in translesion synthesis-dependent repair of MMC damage. We show that the lack of MMC-induced mutability in the mmcB strain is not caused by lack of proper SOS induction of the imuABC operon, involved in translesion synthesis (TLS) in C. crescentus. Based on this data and on structural analysis of a close homolog, we propose that MmcB is an endonuclease which creates substrates for ImuABC-mediated TLS patches. Copyright © 2015 Elsevier B.V. All rights reserved.

Multidrug resistance in fungi: regulation of transporter-encoding gene expression

PubMed Central

Paul, Sanjoy; Moye-Rowley, W. Scott

2014-01-01

A critical risk to the continued success of antifungal chemotherapy is the acquisition of resistance; a risk exacerbated by the few classes of effective antifungal drugs. Predictably, as the use of these drugs increases in the clinic, more resistant organisms can be isolated from patients. A particularly problematic form of drug resistance that routinely emerges in the major fungal pathogens is known as multidrug resistance. Multidrug resistance refers to the simultaneous acquisition of tolerance to a range of drugs via a limited or even single genetic change. This review will focus on recent progress in understanding pathways of multidrug resistance in fungi including those of most medical relevance. Analyses of multidrug resistance in Saccharomyces cerevisiae have provided the most detailed outline of multidrug resistance in a eukaryotic microorganism. Multidrug resistant isolates of S. cerevisiae typically result from changes in the activity of a pair of related transcription factors that in turn elicit overproduction of several target genes. Chief among these is the ATP-binding cassette (ABC)-encoding gene PDR5. Interestingly, in the medically important Candida species, very similar pathways are involved in acquisition of multidrug resistance. In both C. albicans and C. glabrata, changes in the activity of transcriptional activator proteins elicits overproduction of a protein closely related to S. cerevisiae Pdr5 called Cdr1. The major filamentous fungal pathogen, Aspergillus fumigatus, was previously thought to acquire resistance to azole compounds (the principal antifungal drug class) via alterations in the azole drug target-encoding gene cyp51A. More recent data indicate that pathways in addition to changes in the cyp51A gene are important determinants in A. fumigatus azole resistance. We will discuss findings that suggest azole resistance in A. fumigatus and Candida species may share more mechanistic similarities than previously thought. PMID:24795641

Clostridium botulinum: a bug with beauty and weapon.

PubMed

Shukla, H D; Sharma, S K

2005-01-01

Clostridium botulinum, a Gram-positive, anaerobic spore-forming bacteria, is distinguished by its significant clinical applications as well as its potential to be used as bioterror agent. Growing cells secrete botulinum neurotoxin (BoNT), the most poisonous of all known poisons. While BoNT is the causative agent of deadly neuroparalytic botulism, it also serves as a remarkably effective treatment for involuntary muscle disorders such as blepharospasm, strabismus, hemifacial spasm, certain types of spasticity in children, and other ailments. BoNT is also used in cosmetology for the treatment of glabellar lines, and is well-known as the active component of the anti-aging medications Botox and Dysport. In addition, recent reports show that botulinum neurotoxin can be used as a tool for pharmaceutical drug delivery. However, BoNT remains the deadliest of all toxins, and is viewed by biodefense researchers as a possible agent of bioterrorism (BT). Among seven serotypes, C. botulinum type A is responsible for the highest mortality rate in botulism, and thus has the greatest potential to act as biological weapon. Genome sequencing of C. botulinum type A Hall strain (ATCC 3502) is now complete, and has shown the genome size to be 3.89 Mb with a G+C content of approximately 28.2%. The bacterium harbors a 16.3 kb plasmid with a 26.8% G+C content--slightly lower than that of the chromosome. Most of the virulence factors in C. botulinum are chromosomally encoded; bioinformatic analysis of the genome sequence has shown that the plasmid does not harbor toxin genes or genes for related virulence factors. Interestingly, the plasmid does harbor genes essential to replication, including dnaE, which encodes the alpha subunit of DNA polymerase III which has close similarity with its counterpart in C. perfringens strain 13. The plasmid also contains similar genes to those that encode the ABC-type multidrug transport ATPase, and permease. The presence of ABC-type multidrug transport ATPase, and permease suggests putative involvement of efflux pumps in bacteriocin production, modification, and export in C. botulinum. The C. botulinum plasmid additionally harbors genes for LambdaBa04 prophage and site-specific recombinase that are similar to those found in the Ames strain of Bacillus anthracis; these genes and their products may play a role in genomic rearrangement. Completion of genome sequencing for C. botulinum will provide an opportunity to design genomic and proteomic-based systems for detecting different serotypes of C. botulinum strains in the environment. The completed sequence may also facilitate identification of potential virulence factors and drug targets, as well as help characterize neurotoxin-complexing proteins, their polycistronic expression, and phylogenetic relationships between different serotypes.

DNA sequence of a ColV plasmid and prevalence of selected plasmid-encoded virulence genes among avian Escherichia coli strains.

PubMed

Johnson, Timothy J; Siek, Kylie E; Johnson, Sara J; Nolan, Lisa K

2006-01-01

ColV plasmids have long been associated with the virulence of Escherichia coli, despite the fact that their namesake trait, ColV production, does not appear to contribute to virulence. Such plasmids or their associated sequences appear to be quite common among avian pathogenic E. coli (APEC) and are strongly linked to the virulence of these organisms. In the present study, a 180-kb ColV plasmid was sequenced and analyzed. This plasmid, pAPEC-O2-ColV, possesses a 93-kb region containing several putative virulence traits, including iss, tsh, and four putative iron acquisition and transport systems. The iron acquisition and transport systems include those encoding aerobactin and salmochelin, the sit ABC iron transport system, and a putative iron transport system novel to APEC, eit. In order to determine the prevalence of the virulence-associated genes within this region among avian E. coli strains, 595 APEC and 199 avian commensal E. coli isolates were examined for genes of this region using PCR. Results indicate that genes contained within a portion of this putative virulence region are highly conserved among APEC and that the genes of this region occur significantly more often in APEC than in avian commensal E. coli. The region of pAPEC-O2-ColV containing genes that are highly prevalent among APEC appears to be a distinguishing trait of APEC strains.

DNA Sequence of a ColV Plasmid and Prevalence of Selected Plasmid-Encoded Virulence Genes among Avian Escherichia coli Strains

PubMed Central

Johnson, Timothy J.; Siek, Kylie E.; Johnson, Sara J.; Nolan, Lisa K.

2006-01-01

ColV plasmids have long been associated with the virulence of Escherichia coli, despite the fact that their namesake trait, ColV production, does not appear to contribute to virulence. Such plasmids or their associated sequences appear to be quite common among avian pathogenic E. coli (APEC) and are strongly linked to the virulence of these organisms. In the present study, a 180-kb ColV plasmid was sequenced and analyzed. This plasmid, pAPEC-O2-ColV, possesses a 93-kb region containing several putative virulence traits, including iss, tsh, and four putative iron acquisition and transport systems. The iron acquisition and transport systems include those encoding aerobactin and salmochelin, the sit ABC iron transport system, and a putative iron transport system novel to APEC, eit. In order to determine the prevalence of the virulence-associated genes within this region among avian E. coli strains, 595 APEC and 199 avian commensal E. coli isolates were examined for genes of this region using PCR. Results indicate that genes contained within a portion of this putative virulence region are highly conserved among APEC and that the genes of this region occur significantly more often in APEC than in avian commensal E. coli. The region of pAPEC-O2-ColV containing genes that are highly prevalent among APEC appears to be a distinguishing trait of APEC strains. PMID:16385064

Genetic basis of pyrethroid resistance in a population of Anopheles arabiensis, the primary malaria vector in Lower Moshi, north-eastern Tanzania.

PubMed

Matowo, Johnson; Jones, Christopher M; Kabula, Bilali; Ranson, Hilary; Steen, Keith; Mosha, Franklin; Rowland, Mark; Weetman, David

2014-06-19

Pyrethroid resistance has been slower to emerge in Anopheles arabiensis than in An. gambiae s.s and An. funestus and, consequently, studies are only just beginning to unravel the genes involved. Permethrin resistance in An. arabiensis in Lower Moshi, Tanzania has been linked to elevated levels of both P450 monooxygenases and β-esterases. We have conducted a gene expression study to identify specific genes linked with metabolic resistance in the Lower Moshi An. arabiensis population. Microarray experiments employing an An. gambiae whole genome expression chip were performed on An. arabiensis, using interwoven loop designs. Permethrin-exposed survivors were compared to three separate unexposed mosquitoes from the same or a nearby population. A subsection of detoxification genes were chosen for subsequent quantitative real-time PCR (qRT-PCR). Microarray analysis revealed significant over expression of 87 probes and under expression of 85 probes (in pairwise comparisons between permethrin survivors and unexposed sympatric and allopatric samples from Dar es Salaam (controls). For qRT-PCR we targeted over expressed ABC transporter genes (ABC '2060'), a glutathione-S-transferase, P450s and esterases. Design of efficient, specific primers was successful for ABC '2060'and two P450s (CYP6P3, CYP6M2). For the CYP4G16 gene, we used the primers that were previously used in a microarray study of An. arabiensis from Zanzibar islands. Over expression of CYP4G16 and ABC '2060' was detected though with contrasting patterns in pairwise comparisons between survivors and controls. CYP4G16 was only up regulated in survivors, whereas ABC '2060' was similar in survivors and controls but over expressed in Lower Moshi samples compared to the Dar es Salaam samples. Increased transcription of CYP4G16 and ABC '2060' are linked directly and indirectly respectively, with permethrin resistance in Lower Moshi An. arabiensis. Increased transcription of a P450 (CYP4G16) and an ABC transporter (ABC 2060) are linked directly and indirectly respectively, with permethrin resistance in Lower Moshi An. arabiensis. Our study provides replication of CYP4G16 as a candidate gene for pyrethroid resistance in An. arabiensis, although its role may not be in detoxification, and requires further investigation.

Relevance of Peptide Uptake Systems to the Physiology and Virulence of Streptococcus agalactiae

PubMed Central

Samen, Ulrike; Gottschalk, Birgit; Eikmanns, Bernhard J.; Reinscheid, Dieter J.

2004-01-01

Streptococcus agalactiae is a major cause of invasive infections in human newborns. To satisfy its growth requirements, S. agalactiae takes up 9 of the 20 proteinogenic amino acids from the environment. Defined S. agalactiae mutants in one or several of four putative peptide permease systems were constructed and tested for peptide uptake, growth in various media, and expression of virulence traits. Oligopeptide uptake by S. agalactiae was shown to be mediated by the ABC transporter OppA1-F, which possesses two substrate-binding proteins (OppA1 and OppA2) with overlapping substrate specificities. Dipeptides were found to be taken up in parallel by the oligopeptide permease OppA1-F, by the dipeptide ABC transporter DppA-E, and by the dipeptide symporter DpsA. Reverse transcription-PCR analysis revealed a polycistronic organization of the genes oppA1-F and dppA-E and a monocistronic organization of dpsA in S. agalactiae. The results of quantitative real-time PCR revealed a medium-dependent expression of the operons dppA-E and oppA1-F in S. agalactiae. Growth of S. agalactiae in human amniotic fluid was shown to require an intact dpsA gene, indicating an important role of DpsA during the infection of the amniotic cavity by S. agalactiae. Deletion of the oppB gene reduced the adherence of S. agalactiae to epithelial cells by 26%, impaired its adherence to fibrinogen and fibronectin by 42 and 33%, respectively, and caused a 35% reduction in expression of the fbsA gene, which encodes a fibrinogen-binding protein in S. agalactiae. These data indicate that the oligopeptide permease is involved in modulating virulence traits and virulence gene expression in S. agalactiae. PMID:14973032

The Mannitol Utilization System of the Marine Bacterium Zobellia galactanivorans

PubMed Central

Groisillier, Agnès; Labourel, Aurore; Michel, Gurvan

2014-01-01

Mannitol is a polyol that occurs in a wide range of living organisms, where it fulfills different physiological roles. In particular, mannitol can account for as much as 20 to 30% of the dry weight of brown algae and is likely to be an important source of carbon for marine heterotrophic bacteria. Zobellia galactanivorans (Flavobacteriia) is a model for the study of pathways involved in the degradation of seaweed carbohydrates. Annotation of its genome revealed the presence of genes potentially involved in mannitol catabolism, and we describe here the biochemical characterization of a recombinant mannitol-2-dehydrogenase (M2DH) and a fructokinase (FK). Among the observations, the M2DH of Z. galactanivorans was active as a monomer, did not require metal ions for catalysis, and featured a narrow substrate specificity. The FK characterized was active on fructose and mannose in the presence of a monocation, preferentially K+. Furthermore, the genes coding for these two proteins were adjacent in the genome and were located directly downstream of three loci likely to encode an ATP binding cassette (ABC) transporter complex, suggesting organization into an operon. Gene expression analysis supported this hypothesis and showed the induction of these five genes after culture of Z. galactanivorans in the presence of mannitol as the sole source of carbon. This operon for mannitol catabolism was identified in only 6 genomes of Flavobacteriaceae among the 76 publicly available at the time of the analysis. It is not conserved in all Bacteroidetes; some species contain a predicted mannitol permease instead of a putative ABC transporter complex upstream of M2DH and FK ortholog genes. PMID:25548051

The Role of the Photoreceptor ABC Transporter ABCA4 in Lipid Transport and Stargardt Macular Degeneration

PubMed Central

Molday, Robert S.; Zhong, Ming; Quazi, Faraz

2009-01-01

ABCA4 is a member of the ABCA subfamily of ATP binding cassette (ABC) transporters that is expressed in rod and cone photoreceptors of the vertebrate retina. ABCA4, also known as the Rim protein and ABCR, is a large 2273 amino acid glycoprotein organized as two tandem halves, each containing a single membrane spanning segment followed sequentially by a large exocytoplasmic domain, a multispanning membrane domain and a nucleotide binding domain. Over 500 mutations in the gene encoding ABCA4 are associated with a spectrum of related autosomal recessive retinal degenerative diseases including Stargardt macular degeneration, cone-rod dystrophy and a subset of retinitis pigmentosa. Biochemical studies on the purified ABCA4 together with analysis of abca4 knockout mice and patients with Stargardt disease have implicated ABCA4 as a retinylidene-phosphatidylethanolamine transporter that facilitates the removal of potentially reactive retinal derivatives from photoreceptors following photoexcitation. Knowledge of the genetic and molecular basis for ABCA4 related retinal degenerative diseases is being used to develop rationale therapeutic treatments for this set of disorders. PMID:19230850

[The ABC transporters of Saccharomyces cerevisiae].

PubMed

Wawrzycka, Donata

2011-01-01

The ABC transporters (ATP Binding Cassette) compose one of the bigest protein family with the great medical, industrial and economical impact. They are found in all organism from bacteria to man. ABC proteins are responsible for resistance of microorganism to antibiotics and fungicides and multidrug resistance of cancer cells. Mutations in ABC transporters genes cause seriuos deseases like cystic fibrosis, adrenoleucodystrophy or ataxia. Transport catalized by ABC proteins is charged with energy from the ATP hydrolysis. The ABC superfamily contains transporters, canals, receptors. Analysis of the Saccharomyces cerevisiae genome allowed to distinguish 30 potential ABC proteins which are classified into 6 subfamilies. The structural and functional similarity of the yeast and human ABC proteins allowes to use the S. cerevisiae as a model organism for ABC transporters characterisation. In this work the present state of knowleadge on yeast S. cerevisiae ABC proteins was summarised.

Dual system to reinforce biological containment of recombinant bacteria designed for rhizoremediation.

PubMed

Ronchel, M C; Ramos, J L

2001-06-01

Active biological containment (ABC) systems have been designed to control at will the survival or death of a bacterial population. These systems are based on the use of a killing gene, e.g., a porin-inducing protein such as the one encoded by the Escherichia coli gef gene, and a regulatory circuit that controls expression of the killing gene in response to the presence or absence of environmental signals. An ABC system for recombinant microorganisms that degrade a model pollutant was designed on the basis of the Pseudomonas putida TOL plasmid meta-cleavage regulatory circuit. The system consists of a fusion of the Pm promoter to lacI, whose expression is controlled by XylS with 3-methylbenzoate, and a fusion of a synthetic P(lac) promoter to gef. In the presence of the model pollutant, bacterial cells survived and degraded the target compound, whereas in the absence of the aromatic carboxylic acid cell death was induced. The system had two main drawbacks: (i) the slow death of the bacterial cells in soil versus the fast killing rate in liquid cultures in laboratory assays, and (ii) the appearance of mutants, at a rate of about 10(-8) per cell and generation, that did not die after the pollutant had been exhausted. We reinforced the ABC system by including it in a Deltaasd P. putida background. A P. putida Deltaasd mutant is viable only in complex medium supplemented with diaminopimelic acid, methionine, lysine, and threonine. We constructed a P. putida Deltaasd strain, called MCR7, with a Pm::asd fusion in the host chromosome. This strain was viable in the presence of 3-methylbenzoate because synthesis of the essential metabolites was achieved through XylS-dependent induction. In the P. putida MCR7 strain, an ABC system (Pm::lacI, xylS, P(lac)::gef) was incorporated into the host chromosome to yield strain MCR8. The number of MCR8 mutants that escaped killing was below our detection limit (<10(-9) mutants per cell and generation). The MCR8 strain survived and colonized rhizosphere soil with 3-methylbenzoate at a level similar to that of the wild-type strain. However, it disappeared in less than 20 to 25 days in soils without the pollutant, whereas an asd(+), biologically contained counterpart such as P. putida CMC4 was still detectable in soils after 100 days.

A Ferredoxin- and F420H2-Dependent, Electron-Bifurcating, Heterodisulfide Reductase with Homologs in the Domains Bacteria and Archaea

PubMed Central

Yan, Zhen

2017-01-01

ABSTRACT Heterodisulfide reductases (Hdr) of the HdrABC class are ancient enzymes and a component of the anaerobic core belonging to the prokaryotic common ancestor. The ancient origin is consistent with the widespread occurrence of genes encoding putative HdrABC homologs in metabolically diverse prokaryotes predicting diverse physiological functions; however, only one HdrABC has been characterized and that was from a narrow metabolic group of obligate CO2-reducing methanogenic anaerobes (methanogens) from the domain Archaea. Here we report the biochemical characterization of an HdrABC homolog (HdrA2B2C2) from the acetate-utilizing methanogen Methanosarcina acetivorans with unusual properties structurally and functionally distinct from the only other HdrABC characterized. Homologs of the HdrA2B2C2 archetype are present in phylogenetically and metabolically diverse species from the domains Bacteria and Archaea. The expression of the individual HdrA2, HdrB2, and HdrB2C2 enzymes in Escherichia coli, and reconstitution of an active HdrA2B2C2 complex, revealed an intersubunit electron transport pathway dependent on ferredoxin or coenzyme F420 (F420H2) as an electron donor. Remarkably, HdrA2B2C2 couples the previously unknown endergonic oxidation of F420H2 and reduction of ferredoxin with the exergonic oxidation of F420H2 and reduction of the heterodisulfide of coenzyme M and coenzyme B (CoMS-SCoB). The unique electron bifurcation predicts a role for HdrA2B2C2 in Fe(III)-dependent anaerobic methane oxidation (ANME) by M. acetivorans and uncultured species from ANME environments. HdrA2B2C2, ubiquitous in acetotrophic methanogens, was shown to participate in electron transfer during acetotrophic growth of M. acetivorans and proposed to be essential for growth in the environment when acetate is limiting. PMID:28174314

Mycoplasma agalactiae MAG_5040 is a Mg2+-Dependent, Sugar-Nonspecific SNase Recognised by the Host Humoral Response during Natural Infection

PubMed Central

Cacciotto, Carla; Addis, Maria Filippa; Coradduzza, Elisabetta; Carcangiu, Laura; Nuvoli, Anna Maria; Tore, Gessica; Dore, Gian Mario; Pagnozzi, Daniela; Uzzau, Sergio; Chessa, Bernardo; Pittau, Marco; Alberti, Alberto

2013-01-01

In this study the enzymatic activity of Mycoplasma agalactiae MAG_5040, a magnesium-dependent nuclease homologue to the staphylococcal SNase was characterized and its antigenicity during natural infections was established. A UGA corrected version of MAG_5040, lacking the region encoding the signal peptide, was expressed in Escherichia coli as a GST fusion protein. Recombinant GST-MAG_5040 exhibits nuclease activity similar to typical sugar-nonspecific endo- and exonucleases, with DNA as the preferred substrate and optimal activity in the presence of 20 mM MgCl2 at temperatures ranging from 37 to 45°C. According to in silico analyses, the position of the gene encoding MAG_5040 is consistently located upstream an ABC transporter, in most sequenced mycoplasmas belonging to the Mycoplasma hominis group. In M. agalactiae, MAG_5040 is transcribed in a polycistronic RNA together with the ABC transporter components and with MAG_5030, which is predicted to be a sugar solute binding protein by 3D modeling and homology search. In a natural model of sheep and goats infection, anti-MAG_5040 antibodies were detected up to 9 months post infection. Taking into account its enzymatic activity, MAG_5040 could play a key role in Mycoplasma agalactiae survival into the host, contributing to host pathogenicity. The identification of MAG_5040 opens new perspectives for the development of suitable tools for the control of contagious agalactia in small ruminants. PMID:23469065

Site-Specific Mobilization of Vinyl Chloride Respiration Islands by a Mechanism Common in Dehalococcoides

PubMed Central

2011-01-01

Background Vinyl chloride is a widespread groundwater pollutant and Group 1 carcinogen. A previous comparative genomic analysis revealed that the vinyl chloride reductase operon, vcrABC, of Dehalococcoides sp. strain VS is embedded in a horizontally-acquired genomic island that integrated at the single-copy tmRNA gene, ssrA. Results We targeted conserved positions in available genomic islands to amplify and sequence four additional vcrABC -containing genomic islands from previously-unsequenced vinyl chloride respiring Dehalococcoides enrichments. We identified a total of 31 ssrA-specific genomic islands from Dehalococcoides genomic data, accounting for 47 reductive dehalogenase homologous genes and many other non-core genes. Sixteen of these genomic islands contain a syntenic module of integration-associated genes located adjacent to the predicted site of integration, and among these islands, eight contain vcrABC as genetic 'cargo'. These eight vcrABC -containing genomic islands are syntenic across their ~12 kbp length, but have two phylogenetically discordant segments that unambiguously differentiate the integration module from the vcrABC cargo. Using available Dehalococcoides phylogenomic data we estimate that these ssrA-specific genomic islands are at least as old as the Dehalococcoides group itself, which in turn is much older than human civilization. Conclusions The vcrABC -containing genomic islands are a recently-acquired subset of a diverse collection of ssrA-specific mobile elements that are a major contributor to strain-level diversity in Dehalococcoides, and may have been throughout its evolution. The high similarity between vcrABC sequences is quantitatively consistent with recent horizontal acquisition driven by ~100 years of industrial pollution with chlorinated ethenes. PMID:21635780

Characterization and expression of the ABC family (G group) in 'Dangshansuli' pear (Pyrus bretschneideri Rehd.) and its russet mutant.

PubMed

Hou, Zhaoqi; Jia, Bing; Li, Fei; Liu, Pu; Liu, Li; Ye, Zhenfeng; Zhu, Liwu; Wang, Qi; Heng, Wei

2018-01-01

The plant genes encoding ABCGs that have been identified to date play a role in suberin formation in response to abiotic and biotic stress. In the present study, 80 ABCG genes were identified in 'Dangshansuli' Chinese white pear and designated as PbABCGs. Based on the structural characteristics and phylogenetic analysis, the PbABCG family genes could be classified into seven main groups: classes A-G. Segmental and dispersed duplications were the primary forces underlying the PbABCG gene family expansion in 'Dangshansuli' pear. Most of the PbABCG duplicated gene pairs date to the recent whole-genome duplication that occurred 30~45 million years ago. Purifying selection has also played a critical role in the evolution of the ABCG genes. Ten PbABCG genes screened in the transcriptome of 'Dangshansuli' pear and its russet mutant 'Xiusu' were validated, and the expression levels of the PbABCG genes exhibited significant differences at different stages. The results presented here will undoubtedly be useful for better understanding of the complexity of the PbABCG gene family and will facilitate the functional characterization of suberin formation in the russet mutant.

Characterization of SiaA, a streptococcal heme-binding protein associated with a heme ABC transport system.

PubMed

Sook, Brian R; Block, Darci R; Sumithran, Suganya; Montañez, Griselle E; Rodgers, Kenton R; Dawson, John H; Eichenbaum, Zehava; Dixon, Dabney W

2008-02-26

Many pathogenic bacteria require heme and obtain it from their environment. Heme transverses the cytoplasmic membrane via an ATP binding cassette (ABC) pathway. Although a number of heme ABC transport systems have been described in pathogenic bacteria, there is as yet little biophysical characterization of the proteins in these systems. The sia (hts) gene cluster encodes a heme ABC transporter in the Gram positive Streptococcus pyogenes. The lipoprotein-anchored heme binding protein (HBP) of this transporter is SiaA (HtsA). In the current study, resonance Raman (rR), magnetic circular dichroism (MCD), and nuclear magnetic resonance (NMR) spectroscopies were used to determine the coordination state and spin state of both the ferric and ferrous forms of this protein. Identifiers from these techniques suggest that the heme is six-coordinate and low-spin in both oxidation states of the protein, with methionine and histidine as axial ligands. SiaA has a pKa of 9.7 +/- 0.1, attributed to deprotonation of the axial histidine. Guanidinium titration studies show that the ferric state is less stable than the ferrous state, with DeltaG(H2O) values for the oxidized and reduced proteins of 7.3 +/- 0.8 and 16.0 +/- 3.6 kcal mol-1, respectively. The reductive and oxidative midpoint potentials determined via spectroelectrochemistry are 83 +/- 3 and 64 +/- 3 mV, respectively; the irreversibility of heme reduction suggests that redox cycling of the heme is coupled to a kinetically sluggish change in structure or conformation. The biophysical characterization described herein will significantly advance our understanding of structure-function relationships in HBP.

eUnaG: a new ligand-inducible fluorescent reporter to detect drug transporter activity in live cells

PubMed Central

Yeh, Johannes T.-H.; Nam, Kwangho; Yeh, Joshua T.-H.; Perrimon, Norbert

2017-01-01

The absorption, distribution, metabolism and excretion (ADME) of metabolites and toxic organic solutes are orchestrated by the ATP-binding cassette (ABC) transporters and the organic solute carrier family (SLC) proteins. A large number of ABC and SLC transpoters exist; however, only a small number have been well characterized. To facilitate the analysis of these transporters, which is important for drug safety and physiological studies, we developed a sensitive genetically encoded bilirubin (BR)-inducible fluorescence sensor (eUnaG) to detect transporter-coupled influx/efflux of organic compounds. This sensor can be used in live cells to measure transporter activity, as excretion of BR depends on ABC and SLC transporters. Applying eUnaG in functional RNAi screens, we characterize l(2)03659 as a Drosophila multidrug resistant-associated ABC transporter. PMID:28176814

Comparative Sequence Analysis of the Plasmid-Encoded Regulator of Enteropathogenic Escherichia coli Strains

PubMed Central

Okeke, Iruka N.; Borneman, Jade A.; Shin, Sooan; Mellies, Jay L.; Quinn, Laura E.; Kaper, James B.

2001-01-01

Enteropathogenic Escherichia coli (EPEC) strains that carry the EPEC adherence factor (EAF) plasmid were screened for the presence of different EAF sequences, including those of the plasmid-encoded regulator (per). Considerable variation in gene content of EAF plasmids from different strains was seen. However, bfpA, the gene encoding the structural subunit for the bundle-forming pilus, bundlin, and per genes were found in 96.8% of strains. Sequence analysis of the per operon and its promoter region from 15 representative strains revealed that it is highly conserved. Most of the variation occurs in the 5′ two-thirds of the perA gene. In contrast, the C-terminal portion of the predicted PerA protein that contains the DNA-binding helix-turn-helix motif is 100% conserved in all strains that possess a full-length gene. In a minority of strains including the O119:H2 and canine isolates and in a subset of O128:H2 and O142:H6 strains, frameshift mutations in perA leading to premature truncation and consequent inactivation of the gene were identified. Cloned perA, -B, and -C genes from these strains, unlike those from strains with a functional operon, failed to activate the LEE1 operon and bfpA transcriptional fusions or to complement a per mutant in reference strain E2348/69. Furthermore, O119, O128, and canine strains that carry inactive per operons were deficient in virulence protein expression. The context in which the perABC operon occurs on the EAF plasmid varies. The sequence upstream of the per promoter region in EPEC reference strains E2348/69 and B171-8 was present in strains belonging to most serogroups. In a subset of O119:H2, O128:H2, and O142:H6 strains and in the canine isolate, this sequence was replaced by an IS1294-homologous sequence. PMID:11500429

Characterization of the response to zinc deficiency in the cyanobacterium Anabaena sp. strain PCC 7120.

PubMed

Napolitano, Mauro; Rubio, Miguel Ángel; Santamaría-Gómez, Javier; Olmedo-Verd, Elvira; Robinson, Nigel J; Luque, Ignacio

2012-05-01

Zur regulators control zinc homeostasis by repressing target genes under zinc-sufficient conditions in a wide variety of bacteria. This paper describes how part of a survey of duplicated genes led to the identification of the open reading frame all2473 as the gene encoding the Zur regulator of the cyanobacterium Anabaena sp. strain PCC 7120. All2473 binds to DNA in a zinc-dependent manner, and its DNA-binding sequence was characterized, which allowed us to determine the relative contribution of particular nucleotides to Zur binding. A zur mutant was found to be impaired in the regulation of zinc homeostasis, showing sensitivity to elevated concentrations of zinc but not other metals. In an effort to characterize the Zur regulon in Anabaena, 23 genes containing upstream putative Zur-binding sequences were identified and found to be regulated by Zur. These genes are organized in six single transcriptional units and six operons, some of them containing multiple Zur-regulated promoters. The identities of genes of the Zur regulon indicate that Anabaena adapts to conditions of zinc deficiency by replacing zinc metalloproteins with paralogues that fulfill the same function but presumably with a lower zinc demand, and with inducing putative metallochaperones and membrane transport systems likely being involved in the scavenging of extracellular zinc, including plasma membrane ABC transport systems and outer membrane TonB-dependent receptors. Among the Zur-regulated genes, the ones showing the highest induction level encode proteins of the outer membrane, suggesting a primary role for components of this cell compartment in the capture of zinc cations from the extracellular medium.

Role of the Caenorhabditis elegans multidrug resistance gene, mrp-4, in gut granule differentiation.

PubMed

Currie, Erin; King, Brian; Lawrenson, Andrea L; Schroeder, Lena K; Kershner, Aaron M; Hermann, Greg J

2007-11-01

Caenorhabditis elegans gut granules are lysosome-related organelles with birefringent contents. mrp-4, which encodes an ATP-binding cassette (ABC) transporter homologous to mammalian multidrug resistance proteins, functions in the formation of gut granule birefringence. mrp-4(-) embryos show a delayed appearance of birefringent material in the gut granule but otherwise appear to form gut granules properly. mrp-4(+) activity is required for the extracellular mislocalization of birefringent material, body-length retraction, and NaCl sensitivity, phenotypes associated with defective gut granule biogenesis exhibited by embryos lacking the activity of GLO-1/Rab38, a putative GLO-1 guanine nucleotide exchange factor GLO-4, and the AP-3 complex. Multidrug resistance protein (MRP)-4 localizes to the gut granule membrane, consistent with it playing a direct role in the transport of molecules that compose and/or facilitate the formation of birefringent crystals within the gut granule. However, MRP-4 is also present in oocytes and early embryos, and our genetic analyses indicate that its site of action in the formation of birefringent material may not be limited to just the gut granule in embryos. In a search for genes that function similarly to mrp-4(+), we identified WHT-2, another ABC transporter that acts in parallel to MRP-4 for the formation of birefringent material in the gut granule.

The involvement of the nif-associated ferredoxin-like genes fdxA and fdxN of Herbaspirillum seropedicae in nitrogen fixation.

PubMed

Souza, André L F; Invitti, Adriana L; Rego, Fabiane G M; Monteiro, Rose A; Klassen, Giseli; Souza, Emanuel M; Chubatsu, Leda S; Pedrosa, Fábio O; Rigo, Liu U

2010-02-01

The pathway of electron transport to nitrogenase in the endophytic beta-Proteobacterium Herbaspirillum seropedicae has not been characterized. We have generated mutants in two nif-associated genes encoding putative ferredoxins, fdxA and fdxN. The fdxA gene is part of the operon nifHDKENXorf1orf2fdxAnifQmodABC and is transcribed from the nifH promoter, as revealed by lacZ gene fusion. The fdxN gene is probably cotranscribed with the nifB gene. Mutational analysis suggests that the FdxA protein is essential for maximum nitrogenase activity, since the nitrogenase activity of the fdxA mutant strain was reduced to about 30% of that of the wild-type strain. In addition, the fdxA mutation had no effect on the nitrogenase switch-off in response to ammonium. Nitrogenase activity of a mutant strain lacking the fdxN gene was completely abolished. This phenotype was reverted by complementation with fdxN expressed under lacZ promoter control. The results suggest that the products of both the fdxA and fdxN genes are probably involved in electron transfer during nitrogen fixation.

Heterotopic expression of class B floral homeotic genes supports a modified ABC model for tulip (Tulipa gesneriana).

PubMed

Kanno, Akira; Saeki, Hiroshi; Kameya, Toshiaki; Saedler, Heinz; Theissen, Günter

2003-07-01

In higher eudicotyledonous angiosperms the floral organs are typically arranged in four different whorls, containing sepals, petals, stamens and carpels. According to the ABC model, the identity of these organs is specified by floral homeotic genes of class A, A+B, B+C and C, respectively. In contrast to the sepal and petal whorls of eudicots, the perianths of many plants from the Liliaceae family have two outer whorls of almost identical petaloid organs, called tepals. To explain the Liliaceae flower morphology, van Tunen et al. (1993) proposed a modified ABC model, exemplified with tulip. According to this model, class B genes are not only expressed in whorls 2 and 3, but also in whorl 1. Thus the organs of both whorls 1 and 2 express class A plus class B genes and, therefore, get the same petaloid identity. To test this modified ABC model we have cloned and characterized putative class B genes from tulip. Two DEF- and one GLO-like gene were identified, named TGDEFA, TGDEFB and TGGLO. Northern hybridization analysis showed that all of these genes are expressed in whorls 1, 2 and 3 (outer and inner tepals and stamens), thus corroborating the modified ABC model. In addition, these experiments demonstrated that TGGLO is also weakly expressed in carpels, leaves, stems and bracts. Gel retardation assays revealed that TGGLO alone binds to DNA as a homodimer. In contrast, TGDEFA and TGDEFB cannot homodimerize, but make heterodimers with PI. Homodimerization of GLO-like protein has also been reported for lily, suggesting that this phenomenon is conserved within Liliaceae plants or even monocot species.

Targeted inactivation of the murine Abca3 gene leads to respiratory failure in newborns with defective lamellar bodies

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hammel, Markus; Michel, Geert; Hoefer, Christina

2007-08-10

Mutations in the human ABCA3 gene, encoding an ABC-transporter, are associated with respiratory failure in newborns and pediatric interstitial lung disease. In order to study disease mechanisms, a transgenic mouse model with a disrupted Abca3 gene was generated by targeting embryonic stem cells. While heterozygous animals developed normally and were fertile, individuals homozygous for the altered allele (Abca3-/-) died within one hour after birth from respiratory failure, ABCA3 protein being undetectable. Abca3-/- newborns showed atelectasis of the lung in comparison to a normal gas content in unaffected or heterozygous littermates. Electron microscopy demonstrated the absence of normal lamellar bodies inmore » type II pneumocytes. Instead, condensed structures with apparent absence of lipid content were found. We conclude that ABCA3 is required for the formation of lamellar bodies and lung surfactant function. The phenotype of respiratory failure immediately after birth corresponds to the clinical course of severe ABCA3 mutations in human newborns.« less

Glutamine-rich toxic proteins GrtA, GrtB and GrtC together with the antisense RNA AsgR constitute a toxin-antitoxin-like system in Corynebacterium glutamicum.

PubMed

Maeda, Tomoya; Tanaka, Yuya; Inui, Masayuki

2018-06-01

The Corynebacterium glutamicum R grtA (cgR_2936), grtB (cgR_2934) and grtC (cgR_2933) genes were identified as paralogs encoding glutamine-rich toxic proteins. We also identified a new antisense small RNA AsgR (antisense sRNA for grtA) that overlaps the 3' end of the grtA gene. Single over-expressions of grtA, grtB and grtC resulted in complete inhibition of Escherichia coli cell growth. This growth was rescued by co-expression of AsgR. Similar effects were observed in C. glutamicum, although the toxicities of these proteins were moderate. Inhibition of AsgR transcription resulted in increased levels and prolonged half-lives of grtA, grtB and grtC mRNAs. We also found that the expression levels of grtA, grtB and grtC were increased in an RNase III deletion mutant. Primer extension analysis revealed the RNase III cleavage site to be in the 3' untranslated region (3'-UTR) of the grtA mRNA. The expression levels of grtA, grtB and grtC were increased after exposure to several stresses, including heat shock, treatment with penicillin G, lysozyme or H 2 O 2 . The deletions of grtABC and asgR genes resulted in decreased survival rate under several stresses. These results indicate that GrtABC and AsgR constitute a type I toxin-antitoxin-like system in C. glutamicum. © 2018 John Wiley & Sons Ltd.

IMG-ABC: An Atlas of Biosynthetic Gene Clusters to Fuel the Discovery of Novel Secondary Metabolites

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, I-Min; Chu, Ken; Ratner, Anna

2014-10-28

In the discovery of secondary metabolites (SMs), large-scale analysis of sequence data is a promising exploration path that remains largely underutilized due to the lack of relevant computational resources. We present IMG-ABC (https://img.jgi.doe.gov/abc/) -- An Atlas of Biosynthetic gene Clusters within the Integrated Microbial Genomes (IMG) system1. IMG-ABC is a rich repository of both validated and predicted biosynthetic clusters (BCs) in cultured isolates, single-cells and metagenomes linked with the SM chemicals they produce and enhanced with focused analysis tools within IMG. The underlying scalable framework enables traversal of phylogenetic dark matter and chemical structure space -- serving as a doorwaymore » to a new era in the discovery of novel molecules.« less

Mechanistic Study of Utilization of Water-Insoluble Saccharomyces cerevisiae Glucans by Bifidobacterium breve Strain JCM1192

PubMed Central

Keung, Hoi Yee; Li, Tsz Kai; Sham, Lok To; Cheung, Man Kit; Cheung, Peter Chi Keung

2017-01-01

ABSTRACT Bifidobacteria exert beneficial effects on hosts and are extensively used as probiotics. However, due to the genetic inaccessibility of these bacteria, little is known about their mechanisms of carbohydrate utilization and regulation. Bifidobacterium breve strain JCM1192 can grow on water-insoluble yeast (Saccharomyces cerevisiae) cell wall glucans (YCWG), which were recently considered as potential prebiotics. According to the results of 1H nuclear magnetic resonance (NMR) spectrometry, the YCWG were composed of highly branched (1→3,1→6)-β-glucans and (1→4,1→6)-α-glucans. Although the YCWG were composed of 78.3% β-glucans and 21.7% α-glucans, only α-glucans were consumed by the B. breve strain. The ABC transporter (malEFG1) and pullulanase (aapA) genes were transcriptionally upregulated in the metabolism of insoluble yeast glucans, suggesting their potential involvement in the process. A nonsense mutation identified in the gene encoding an ABC transporter ATP-binding protein (MalK) led to growth failure of an ethyl methanesulfonate-generated mutant with yeast glucans. Coculture of the wild-type strain and the mutant showed that this protein was responsible for the import of yeast glucans or their breakdown products, rather than the export of α-glucan-catabolizing enzymes. Further characterization of the carbohydrate utilization of the mutant and three of its revertants indicated that this mutation was pleiotropic: the mutant could not grow with maltose, glycogen, dextrin, raffinose, cellobiose, melibiose, or turanose. We propose that insoluble yeast α-glucans are hydrolyzed by extracellular pullulanase into maltose and/or maltooligosaccharides, which are then transported into the cell by the ABC transport system composed of MalEFG1 and MalK. The mechanism elucidated here will facilitate the development of B. breve and water-insoluble yeast glucans as novel synbiotics. IMPORTANCE In general, Bifidobacterium strains are genetically intractable. Coupling classic forward genetics with next-generation sequencing, here we identified an ABC transporter ATP-binding protein (MalK) responsible for the import of insoluble yeast glucan breakdown products by B. breve JCM1192. We demonstrated the pleiotropic effects of the ABC transporter ATP-binding protein in maltose/maltooligosaccharide, raffinose, cellobiose, melibiose, and turanose transport. With the addition of transcriptional analysis, we propose that insoluble yeast glucans are broken down by extracellular pullulanase into maltose and/or maltooligosaccharides, which are then transported into the cell by the ABC transport system composed of MalEFG1 and MalK. The mechanism elucidated here will facilitate the development of B. breve and water-insoluble yeast glucans as novel synbiotics. PMID:28115383

Mechanistic Study of Utilization of Water-Insoluble Saccharomyces cerevisiae Glucans by Bifidobacterium breve Strain JCM1192.

PubMed

Keung, Hoi Yee; Li, Tsz Kai; Sham, Lok To; Cheung, Man Kit; Cheung, Peter Chi Keung; Kwan, Hoi Shan

2017-04-01

Bifidobacteria exert beneficial effects on hosts and are extensively used as probiotics. However, due to the genetic inaccessibility of these bacteria, little is known about their mechanisms of carbohydrate utilization and regulation. Bifidobacterium breve strain JCM1192 can grow on water-insoluble yeast ( Saccharomyces cerevisiae ) cell wall glucans (YCWG), which were recently considered as potential prebiotics. According to the results of 1 H nuclear magnetic resonance (NMR) spectrometry, the YCWG were composed of highly branched (1→3,1→6)-β-glucans and (1→4,1→6)-α-glucans. Although the YCWG were composed of 78.3% β-glucans and 21.7% α-glucans, only α-glucans were consumed by the B. breve strain. The ABC transporter ( malEFG1 ) and pullulanase ( aapA ) genes were transcriptionally upregulated in the metabolism of insoluble yeast glucans, suggesting their potential involvement in the process. A nonsense mutation identified in the gene encoding an ABC transporter ATP-binding protein (MalK) led to growth failure of an ethyl methanesulfonate-generated mutant with yeast glucans. Coculture of the wild-type strain and the mutant showed that this protein was responsible for the import of yeast glucans or their breakdown products, rather than the export of α-glucan-catabolizing enzymes. Further characterization of the carbohydrate utilization of the mutant and three of its revertants indicated that this mutation was pleiotropic: the mutant could not grow with maltose, glycogen, dextrin, raffinose, cellobiose, melibiose, or turanose. We propose that insoluble yeast α-glucans are hydrolyzed by extracellular pullulanase into maltose and/or maltooligosaccharides, which are then transported into the cell by the ABC transport system composed of MalEFG1 and MalK. The mechanism elucidated here will facilitate the development of B. breve and water-insoluble yeast glucans as novel synbiotics. IMPORTANCE In general, Bifidobacterium strains are genetically intractable. Coupling classic forward genetics with next-generation sequencing, here we identified an ABC transporter ATP-binding protein (MalK) responsible for the import of insoluble yeast glucan breakdown products by B. breve JCM1192. We demonstrated the pleiotropic effects of the ABC transporter ATP-binding protein in maltose/maltooligosaccharide, raffinose, cellobiose, melibiose, and turanose transport. With the addition of transcriptional analysis, we propose that insoluble yeast glucans are broken down by extracellular pullulanase into maltose and/or maltooligosaccharides, which are then transported into the cell by the ABC transport system composed of MalEFG1 and MalK. The mechanism elucidated here will facilitate the development of B. breve and water-insoluble yeast glucans as novel synbiotics. Copyright © 2017 American Society for Microbiology.

Novel Mutation in the ATP-Binding Cassette Transporter A3 (ABCA3) Encoding Gene Causes Respiratory Distress Syndrome in A Term Newborn in Southwest Iran

PubMed Central

Rezaei, Farideh; Shafiei, Mohammad; Shariati, Gholamreza; Dehdashtian, Ali; Mohebbi, Maryam; Galehdari, Hamid

2016-01-01

Introduction ABCA3 glycoprotein belongs to the ATP-binding cassette (ABC) superfamily of transporters, which utilize the energy derived from hydrolysis of ATP for the translocation of a wide variety of substrates across the plasma membrane. Mutations in the ABCA3 gene are knowingly causative for fatal surfactant deficiency, particularly respiratory distress syndrome (RDS) in term babies. Case Presentation In this study, Sanger sequencing of the whole ABCA3 gene (NCBI NM_001089) was performed in a neonatal boy with severe RDS. A homozygous mutation has been identified in the patient. Parents were heterozygous for the same missense mutation GGA > AGA at position 202 in exon 6 of the ABCA3 gene (c.604G > A; p.G202R). Furthermore, 70 normal individuals have been analyzed for the mentioned change with negative results. Conclusions Regarding Human Genome Mutation Database (HGMD) and other literature recherche, the detected change is a novel mutation and has not been reported before. Bioinformatics mutation predicting tools prefer it as pathogenic. PMID:27437095

Indole-Induced Activities of β-Lactamase and Efflux Pump Confer Ampicillin Resistance in Pseudomonas putida KT2440

PubMed Central

Kim, Jisun; Shin, Bora; Park, Chulwoo; Park, Woojun

2017-01-01

Indole, which is widespread in microbial communities, has received attention because of its effects on bacterial physiology. Pseudomonas putida and Pseudomonas aeruginosa can acquire ampicillin (Amp) resistance during growth on indole-Amp agar. Transcriptome, mutant, and inhibitor studies have suggested that Amp resistance induced by indole can be attributed to increased gene expression of ttgAB encoding two genes of RND-type multidrug efflux operons and an ampC encoding β-lactamase. Expression, enzyme activities, and mutational analyses indicated that AmpC β-lactamase is important for acquiring Amp resistance of P. putida in the presence of indole. Here, we show, for the first time, that volatile indole increased Amp-resistant cells. Consistent with results of the volatile indole assay, a low concentration of indole in liquid culture promoted growth initially, but led to mutagenesis after indole was depleted, which could not be observed at high indole concentrations. Interestingly, ttgAB and ampC gene expression levels correlate with the concentration of indole, which might explain the low number of Amp-mutated cells in high indole concentrations. The expression levels of genes involved in mutagenesis, namely rpoS, recA, and mutS, were also modulated by indole. Our data indicates that indole reduces Amp-induced heterogeneity by promoting expression of TtgABC or MexAB-OprM efflux pumps and the indole-induced β-lactamase in P. putida and P. aeruginosa. PMID:28352264

ABCdb: an online resource for ABC transporter repertories from sequenced archaeal and bacterial genomes.

PubMed

Fichant, Gwennaele; Basse, Marie-Jeanne; Quentin, Yves

2006-03-01

The ATP-binding cassette (ABC) transporters are one of the major classes of active transporters. They are widespread in archaea, bacteria, and eukaryota, indicating that they have arisen early in evolution. They are involved in many essential physiological processes, but the majority import or export a wide variety of compounds across cellular membranes. These systems share a common architecture composed of four (exporters) or five (importers) domains. To identify and reconstruct functional ABC transporters encoded by archaeal and bacterial genomes, we have developed a bioinformatic strategy. Cross-reference to the transport classification system is used to predict the type of compound transported. A high quality of annotation is achieved by manual verification of the predictions. However, in order to face the rapid increase in the number of published genomes, we also include analyses of genomes issuing directly from the automated strategy. Querying the database (http://www-abcdb.biotoul.fr) allows to easily retrieve ABC transporter repertories and related data. Additional query tools have been developed for the analysis of the ABC family from both functional and evolutionary perspectives.

Peatland Acidobacteria with a dissimilatory sulfur metabolism.

PubMed

Hausmann, Bela; Pelikan, Claus; Herbold, Craig W; Köstlbacher, Stephan; Albertsen, Mads; Eichorst, Stephanie A; Glavina Del Rio, Tijana; Huemer, Martin; Nielsen, Per H; Rattei, Thomas; Stingl, Ulrich; Tringe, Susannah G; Trojan, Daniela; Wentrup, Cecilia; Woebken, Dagmar; Pester, Michael; Loy, Alexander

2018-02-23

Sulfur-cycling microorganisms impact organic matter decomposition in wetlands and consequently greenhouse gas emissions from these globally relevant environments. However, their identities and physiological properties are largely unknown. By applying a functional metagenomics approach to an acidic peatland, we recovered draft genomes of seven novel Acidobacteria species with the potential for dissimilatory sulfite (dsrAB, dsrC, dsrD, dsrN, dsrT, dsrMKJOP) or sulfate respiration (sat, aprBA, qmoABC plus dsr genes). Surprisingly, the genomes also encoded DsrL, which so far was only found in sulfur-oxidizing microorganisms. Metatranscriptome analysis demonstrated expression of acidobacterial sulfur-metabolism genes in native peat soil and their upregulation in diverse anoxic microcosms. This indicated an active sulfate respiration pathway, which, however, might also operate in reverse for dissimilatory sulfur oxidation or disproportionation as proposed for the sulfur-oxidizing Desulfurivibrio alkaliphilus. Acidobacteria that only harbored genes for sulfite reduction additionally encoded enzymes that liberate sulfite from organosulfonates, which suggested organic sulfur compounds as complementary energy sources. Further metabolic potentials included polysaccharide hydrolysis and sugar utilization, aerobic respiration, several fermentative capabilities, and hydrogen oxidation. Our findings extend both, the known physiological and genetic properties of Acidobacteria and the known taxonomic diversity of microorganisms with a DsrAB-based sulfur metabolism, and highlight new fundamental niches for facultative anaerobic Acidobacteria in wetlands based on exploitation of inorganic and organic sulfur molecules for energy conservation.

Contribution of tumor endothelial cells to drug resistance: anti-angiogenic tyrosine kinase inhibitors act as p-glycoprotein antagonists.

PubMed

Bani, MariaRosa; Decio, Alessandra; Giavazzi, Raffaella; Ghilardi, Carmen

2017-05-01

Tumor endothelial cells (TEC) differ from the normal counterpart, in both gene expression and functionality. TEC may acquire drug resistance, a characteristic that is maintained in vitro. There is evidence that TEC are more resistant to chemotherapeutic drugs, substrates of ATP-binding cassette (ABC) transporters. TEC express p-glycoprotein (encoded by ABCB1), while no difference in other ABC transporters was revealed compared to normal endothelia. A class of tyrosine kinase inhibitors (TKI), used as angiostatic compounds, interferes with the ATPase activity of p-glycoprotein, thus impairing its functionality. The exposure of ovarian adenocarcinoma TEC to the TKIs sunitinib or sorafenib was found to abrogate resistance (proliferation and motility) to doxorubicin and paclitaxel in vitro, increasing intracellular drug accumulation. A similar effect has been reported by the p-glycoprotein inhibitor verapamil. No beneficial effect was observed in combination with cytotoxic drugs that are not p-glycoprotein substrates. The current paper reviews the mechanisms of TEC chemoresistance and shows the role of p-glycoprotein in mediating such resistance. Inhibition of p-glycoprotein by anti-angiogenic TKI might contribute to the beneficial effect of these small molecules, when combined with chemotherapy, in counteracting acquired drug resistance.

PdSlt2 Penicillium digitatum mitogen-activated-protein kinase controls sporulation and virulence during citrus fruit infection.

PubMed

de Ramón-Carbonell, Marta; Sánchez-Torres, Paloma

2017-12-01

The Slt2 mitogen-activated protein (MAP) kinase homologue of Penicillium digitatum, the most relevant pathogen-producing citrus green mould decay during postharvest, was identified and explored. The P. digitatum Slt2-MAPK coding gene (PdSlt2) was functionally characterized by homologous gene elimination and transcriptomic evaluation. The absence of PdSlt2 gene resulted in significantly reduced virulence during citrus infection. The ΔPdSlt2 mutants were also defective in asexual reproduction, showing impairment of sporulation during citrus infection. Gene expression analysis revealed that PdSlt2 was highly induced during citrus fruit infection at early stages (1 dpi). Moreover, PdSlt2 deletion altered gene expression profiles. The relative gene expression (RGE) of fungicide resistance- and fungal virulence-related genes showed that PdSlt2 acts as negative regulator of several transporter encoding genes (ABC and MFS transporters) and a positive regulator of two sterol demethylases. This study indicates that PdSlt2 MAPK is functionally preserved in P. digitatum and highlights the relevant role of the PdSlt2 MAP kinase-mediated signalling pathway in regulating diverse genes crucial for infection and asexual reproduction. Copyright © 2017 British Mycological Society. Published by Elsevier Ltd. All rights reserved.

The ABC Model and its Applicability to Basal Angiosperms

PubMed Central

Soltis, Douglas E.; Chanderbali, André S.; Kim, Sangtae; Buzgo, Matyas; Soltis, Pamela S.

2007-01-01

Background Although the flower is the central feature of the angiosperms, little is known of its origin and subsequent diversification. The ABC model has long been the unifying paradigm for floral developmental genetics, but it is based on phylogenetically derived eudicot models. Synergistic research involving phylogenetics, classical developmental studies, genomics and developmental genetics has afforded valuable new insights into floral evolution in general, and the early flower in particular. Scope and Conclusions Genomic studies indicate that basal angiosperms, and by inference the earliest angiosperms, had a rich tool kit of floral genes. Homologues of the ABCE floral organ identity genes are also present in basal angiosperm lineages; however, C-, E- and particularly B-function genes are more broadly expressed in basal lineages. There is no single model of floral organ identity that applies to all angiosperms; there are multiple models that apply depending on the phylogenetic position and floral structure of the group in question. The classic ABC (or ABCE) model may work well for most eudicots. However, modifications are needed for basal eudicots and, the focus of this paper, basal angiosperms. We offer ‘fading borders’ as a testable hypothesis for the basal-most angiosperms and, by inference, perhaps some of the earliest (now extinct) angiosperms. PMID:17616563

Structural Diversification of Lyngbyatoxin A by Host-Dependent Heterologous Expression of the tleABC Biosynthetic Gene Cluster.

PubMed

Zhang, Lihan; Hoshino, Shotaro; Awakawa, Takayoshi; Wakimoto, Toshiyuki; Abe, Ikuro

2016-08-03

Natural products have enormous structural diversity, yet little is known about how such diversity is achieved in nature. Here we report the structural diversification of a cyanotoxin-lyngbyatoxin A-and its biosynthetic intermediates by heterologous expression of the Streptomyces-derived tleABC biosynthetic gene cluster in three different Streptomyces hosts: S. lividans, S. albus, and S. avermitilis. Notably, the isolated lyngbyatoxin derivatives, including four new natural products, were biosynthesized by crosstalk between the heterologous tleABC gene cluster and the endogenous host enzymes. The simple strategy described here has expanded the structural diversity of lyngbyatoxin A and its biosynthetic intermediates, and provides opportunities for investigation of the currently underestimated hidden biosynthetic crosstalk. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Aldouronate utilization in Paenibacillus sp. strain JDR-2: Physiological and enzymatic evidence for coupling of extracellular depolymerization and intracellular metabolism.

PubMed

Nong, Guang; Rice, John D; Chow, Virginia; Preston, James F

2009-07-01

Paenibacillus sp. strain JDR-2, an aggressively xylanolytic bacterium isolated from decaying sweet gum wood, secretes a multimodular glycohydrolase family GH10 endoxylanase (XynA1) anchored to the cell surface. The gene encoding XynA1 is part of a xylan utilization regulon that includes an aldouronate utilization gene cluster with genes encoding a GH67 alpha-glucuronidase (AguA), a GH10 endoxylanase (XynA2), and a GH43 arabinofuranosidase/beta-xylosidase (XynB). Here we show that this Paenibacillus sp. strain is able to utilize methylglucuronoxylose (MeGAX(1)), an aldobiuronate product that accumulates during acid pretreatment of lignocellulosic biomass, and methylglucuronoxylotriose (MeGAX(3)), the product of the extracellular XynA1 acting on methylglucuronoxylan (MeGAX(n)). The average rates of utilization of MeGAX(n), MeGAX(1), and MeGAX(3) were 149.8, 59.4, and 54.3 microg xylose equivalents.ml(-1).h(-1), respectively, and were proportional to the specific growth rates on the substrates. AguA was active with MeGAX(1) and MeGAX(3), releasing 4-O-methyl-d-glucuronate alpha-1,2 linked to a nonreducing terminal xylose residue. XynA2 converted xylotriose, generated by the action of AguA on MeGAX(3), to xylose and xylobiose. The ability to utilize MeGAX(1) provides a novel metabolic potential for bioconversion of acid hydrolysates of lignocellulosics. The 2.8-fold-greater rate of utilization of polymeric MeGAX(n) than that of MeGAX(3) indicates that there is coupling of extracellular depolymerization, assimilation, and intracellular metabolism, allowing utilization of lignocellulosics with minimal pretreatment. Along with adjacent genes encoding transcriptional regulators and ABC transporter proteins, the aguA and xynA2 genes in the cluster described above contribute to the efficient utilization of aldouronates derived from dilute acid and/or enzyme pretreatment protocols applied to the conversion of hemicellulose to biofuels and chemicals.

Characterization and Heterologous Expression of the Genes Encoding Enterocin A Production, Immunity, and Regulation in Enterococcus faecium DPC1146

PubMed Central

O’Keeffe, Triona; Hill, Colin; Ross, R. Paul

1999-01-01

Enterocin A is a small, heat-stable, antilisterial bacteriocin produced by Enterococcus faecium DPC1146. The sequence of a 10,879-bp chromosomal region containing at least 12 open reading frames (ORFs), 7 of which are predicted to play a role in enterocin biosynthesis, is presented. The genes entA, entI, and entF encode the enterocin A prepeptide, the putative immunity protein, and the induction factor prepeptide, respectively. The deduced proteins EntK and EntR resemble the histidine kinase and response regulator proteins of two-component signal transducing systems of the AgrC-AgrA type. The predicted proteins EntT and EntD are homologous to ABC (ATP-binding cassette) transporters and accessory factors, respectively, of several other bacteriocin systems and to proteins implicated in the signal-sequence-independent export of Escherichia coli hemolysin A. Immediately downstream of the entT and entD genes are two ORFs, the product of one of which, ORF4, is very similar to the product of the yteI gene of Bacillus subtilis and to E. coli protease IV, a signal peptide peptidase known to be involved in outer membrane lipoprotein export. Another potential bacteriocin is encoded in the opposite direction to the other genes in the enterocin cluster. This putative bacteriocin-like peptide is similar to LafX, one of the components of the lactacin F complex. A deletion which included one of two direct repeats upstream of the entA gene abolished enterocin A activity, immunity, and ability to induce bacteriocin production. Transposon insertion upstream of the entF gene also had the same effect, but this mutant could be complemented by exogenously supplied induction factor. The putative EntI peptide was shown to be involved in the immunity to enterocin A. Cloning of a 10.5-kb amplicon comprising all predicted ORFs and regulatory regions resulted in heterologous production of enterocin A and induction factor in Enterococcus faecalis, while a four-gene construct (entAITD) under the control of a constitutive promoter resulted in heterologous enterocin A production in both E. faecalis and Lactococcus lactis. PMID:10103244

Effect of β-elemene on the kinetics of intracellular transport of d-luciferin potassium salt (ABC substrate) in doxorubicin-resistant breast cancer cells and the associated molecular mechanism.

PubMed

Tang, Chao-Yuan; Zhu, Li-Xin; Yu, Jian-Dong; Chen, Zhi; Gu, Man-Cang; Mu, Chao-Feng; Liu, Qi; Xiong, Yang

2018-07-30

In order to explore the mechanism of the reversing multidrug resistance (MDR) phenotypes by β-elemene (β-ELE) in doxorubicin (DOX)-resistant breast cancer cells (MCF-7/DOX), both the functionality and quantity of the ABC transporters in MCF-7/DOX were studied. Bioluminescence imaging (BLI) was used to study the efflux of d-luciferin potassium salt, the substrate of ATP-binding cassette transporters (ABC transporters), in MCF-7/DOX cells treated by β-ELE. At the same time three major ABC transport proteins and genes-related MDR, P-glycoprotein (P-gp, ABCB1) and multidrug resistance-associated protein 1 (MRP, ABCC1) as well as breast cancer resistance protein (BCRP, ABCG2) were analyzed by q-PCR and Western blot. To investigate the efflux functionality of ABC transporters, MCF-7/DOX Fluc cell line with stably-overexpressed luciferase was established. BLI was then used to real-time monitor the efflux kinetics of d-luciferin potassium salt before and after MCF-7/DOX Fluc cells being treated with β-ELE or not. The results showed that the efflux of d-luciferin potassium salt from MCF-7/DOX Fluc was lessened when pretreated with β-ELE, which means that β-ELE may dampen the functionality of ABC transporters, thus decrease the efflux of d-fluorescein potassium or other chemotherapies which also serve as the substrates of ABC transporters. As the effect of β-ELE on the expression of ABC transporters, the results of q-PCR and Western blot showed that gene and protein expression of ABC transporters such as P-gp, MRP, and BCRP were down-regulated after the treatment of β-ELE. To verify the efficacy of β-ELE on reversing MDR, MCF-7/DOX cells were treated with the combination of DOX and β-ELE. MTT assay showed that β-ELE increased the inhibitory effect of DOX on the proliferation of MCF-7/DOX, and the IC 50 of the combination group was much lower than that of the single DOX or β-ELE treatment. In all, β-ELE may reverse MDR through the substrates of ABC transporters by two ways, to lessen the ABC protein efflux by weakening their functionality, or to reduce the quantity of ABC gene and protein expression. Copyright © 2018. Published by Elsevier B.V.

Comparative transcriptomic evidence for Tween80-enhanced biodegradation of phenanthrene by Sphingomonas sp. GY2B.

PubMed

Liu, Shasha; Guo, Chuling; Lin, Weijia; Wu, Fengji; Lu, Guining; Lu, Jing; Dang, Zhi

2017-12-31

Previous study of the effects of surfactants on the biodegradation of phenanthrene focused on investigating alterations of the cell characteristics of Sphingomonas sp. GY2B. However, genes regulation associated with biodegradation and biological processes in response to the presence of surfactants, remains unclear. In this study, comparative transcriptome analysis was conducted to observe the gene expression of GY2B during phenanthrene biodegradation in the presence and absence of Tween80. A diverse set of genes was regulated by Tween80, leading to increased biodegradation of phenanthrene by GY2B: (i) Tween80 increased expression of genes related to H + transport in the plasma membrane to provide a driving force (i.e., ATP) for accelerating transmembrane transport of phenanthrene with increasing Tween80 concentrations, thereby enhancing the uptake and degradation of phenanthrene by GY2B; (ii) Tween80 (1 and 8 CMC) promoted intracellular biodegradation of phenanthrene by stimulating expression of genes encoding dioxygenases and monooxygenase, increasing expression of genes involved in intracellular metabolic processes (e.g., TCA cycle); and (iii) Tween80 likely increased GY2B vitality and growth by inducing expression of genes associated with ABC transporters and protein transport, regulating genes involved in other biological processes (e.g., transcription, translation). Copyright © 2017. Published by Elsevier B.V.

Mdr65 decreases toxicity of multiple insecticides in Drosophila melanogaster.

PubMed

Sun, Haina; Buchon, Nicolas; Scott, Jeffrey G

2017-10-01

ABC transporters are ubiquitous membrane-bound proteins, present in both prokaryotes and eukaryotes. The major function of eukaryotic ABC transporters is to mediate the efflux of a variety of substrates (including xenobiotics) out of cells. ABC transporters have been widely investigated in humans, particularly for their involvement in multidrug resistance (MDR). Considerably less is known about their roles in transport and/or excretion in insects. ABC transporters are only known to function as exporters in insects. Drosophila melanogaster has 56 ABC transporter genes, including eight which are phylogenetically most similar to the human Mdr genes (ABCB1 clade). We investigated the role of ABC transporters in the ABCB1 clade in modulating the susceptibility to insecticides. We took advantage of the GAL4/UAS system in D. melanogaster to knockdown the expression levels of Mdr65, Mdr50, Mdr49 and ABCB6 using transgenic UAS-RNAi lines and conditional driver lines. The most notable effects were increased sensitivities to nine different insecticides by silencing of Mdr65. Furthermore, a null mutation of Mdr65 decreased the malathion, malaoxon and fipronil LC 50 values by a factor of 1.9, 2.1 and 3.9, respectively. Altogether, this data demonstrates the critical role of ABC transporters, particularly Mdr65, in altering the toxicity of specific, structurally diverse, insecticides in D. melanogaster. Copyright © 2017 Elsevier Ltd. All rights reserved.

fbpABC gene cluster in Neisseria meningitidis is transcribed as an operon.

PubMed

Khun, H H; Deved, V; Wong, H; Lee, B C

2000-12-01

The neisserial fbpABC locus has been proposed to constitute a single transcriptional unit. To confirm this operonic arrangement, transcription assays using reverse transcriptase PCR amplification were conducted with Neisseria meningitidis. The presence of fbpAB and fbpBC transcripts obtained by priming cDNA synthesis with an fbpC-sequence-specific oligonucleotide indicates that fbpABC is organized as a single expression unit. The ratio of fbpA to fbpABC mRNA was approximately between 10- to 20-fold, as determined by real-time quantitative PCR.

fbpABC Gene Cluster in Neisseria meningitidis Is Transcribed as an Operon

PubMed Central

Khun, Heng H.; Deved, Vinay; Wong, Howard; Lee, B. Craig

2000-01-01

The neisserial fbpABC locus has been proposed to constitute a single transcriptional unit. To confirm this operonic arrangement, transcription assays using reverse transcriptase PCR amplification were conducted with Neisseria meningitidis. The presence of fbpAB and fbpBC transcripts obtained by priming cDNA synthesis with an fbpC-sequence-specific oligonucleotide indicates that fbpABC is organized as a single expression unit. The ratio of fbpA to fbpABC mRNA was approximately between 10- to 20-fold, as determined by real-time quantitative PCR. PMID:11083849

A LuxR homolog in a cottonwood tree endophyte that activates gene expression in response to a plant signal or specific peptides

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schaefer, Amy L.; Oda, Yasuhiro; Coutinho, Bruna Goncalves

Homologs of the LuxR acyl-homoserine lactone (AHL) quorum-sensing signal receptor are prevalent in Proteobacteria isolated from roots of the Eastern cottonwood tree, Populus deltoides. Many of these isolates possess an orphan LuxR homolog, closely related to OryR from the rice pathogen Xanthomonas oryzae. OryR does not respond to AHL signals but, instead, responds to an unknown plant compound. We discovered an OryR homolog, PipR, in the cottonwood endophyte Pseudomonas sp. strain GM79. The genes adjacent to pipR encode a predicted ATP-binding cassette (ABC) peptide transporter and peptidases. We purified the putative peptidases, PipA and AapA, and confirmed their predicted activities.more » A transcriptional pipA-gfp reporter was responsive to PipR in the presence of plant leaf macerates, but it was not influenced by AHLs, similar to findings with OryR. We found that PipR also responded to protein hydrolysates to activate pipA-gfp expression. Among many peptides tested, the tripeptide Ser-His-Ser showed inducer activity but at relatively high concentrations. An ABC peptide transporter mutant failed to respond to leaf macerates, peptone, or Ser-His-Ser, while peptidase mutants expressed higher-than-wild-type levels of pipA-gfp in response to any of these signals. Our studies are consistent with a model where active transport of a peptidelike signal is required for the signal to interact with PipR, which then activates peptidase gene expression. As a result, the identification of a peptide ligand for PipR sets the stage to identify plant-derived signals for the OryR family of orphan LuxR proteins.« less

Sustainable growth of Dehalococcoides mccartyi 195 by corrinoid salvaging and remodeling in defined lactate-fermenting consortia.

PubMed

Men, Yujie; Seth, Erica C; Yi, Shan; Allen, Robert H; Taga, Michiko E; Alvarez-Cohen, Lisa

2014-04-01

Corrinoids are essential cofactors of reductive dehalogenases in Dehalococcoides mccartyi, an important bacterium in bioremediation, yet sequenced D. mccartyi strains do not possess the complete pathway for de novo corrinoid biosynthesis. Pelosinus sp. and Desulfovibrio sp. have been detected in dechlorinating communities enriched from contaminated groundwater without exogenous cobalamin corrinoid. To investigate the corrinoid-related interactions among key members of these communities, we constructed consortia by growing D. mccartyi strain 195 (Dhc195) in cobalamin-free, trichloroethene (TCE)- and lactate-amended medium in cocultures with Desulfovibrio vulgaris Hildenborough (DvH) or Pelosinus fermentans R7 (PfR7) and with both in tricultures. Only the triculture exhibited sustainable dechlorination and cell growth when a physiological level of 5,6-dimethylbenzimidazole (DMB), the lower ligand of cobalamin, was provided. In the triculture, DvH provided hydrogen while PfR7 provided corrinoids to Dhc195, and the initiation of dechlorination and Dhc195 cell growth was highly dependent on the growth of PfR7. Corrinoid analysis indicated that Dhc195 imported and remodeled the phenolic corrinoids produced by PfR7 into cobalamin in the presence of DMB. Transcriptomic analyses of Dhc195 showed the induction of the CbiZ-dependent corrinoid-remodeling pathway and BtuFCD corrinoid ABC transporter genes during corrinoid salvaging and remodeling. In contrast, another operon annotated to encode a putative iron/cobalamin ABC transporter (DET1174-DET1176) was induced when cobalamin was exogenously provided. Interestingly, a global upregulation of phage-related genes was observed when PfR7 was present. These findings provide insights into both the gene regulation of corrinoid salvaging and remodeling in Dhc195 when it is grown without exogenous cobalamin and microbe-to-microbe interactions in dechlorinating microbial communities.

A LuxR homolog in a cottonwood tree endophyte that activates gene expression in response to a plant signal or specific peptides

DOE PAGES

Schaefer, Amy L.; Oda, Yasuhiro; Coutinho, Bruna Goncalves; ...

2016-08-02

Homologs of the LuxR acyl-homoserine lactone (AHL) quorum-sensing signal receptor are prevalent in Proteobacteria isolated from roots of the Eastern cottonwood tree, Populus deltoides. Many of these isolates possess an orphan LuxR homolog, closely related to OryR from the rice pathogen Xanthomonas oryzae. OryR does not respond to AHL signals but, instead, responds to an unknown plant compound. We discovered an OryR homolog, PipR, in the cottonwood endophyte Pseudomonas sp. strain GM79. The genes adjacent to pipR encode a predicted ATP-binding cassette (ABC) peptide transporter and peptidases. We purified the putative peptidases, PipA and AapA, and confirmed their predicted activities.more » A transcriptional pipA-gfp reporter was responsive to PipR in the presence of plant leaf macerates, but it was not influenced by AHLs, similar to findings with OryR. We found that PipR also responded to protein hydrolysates to activate pipA-gfp expression. Among many peptides tested, the tripeptide Ser-His-Ser showed inducer activity but at relatively high concentrations. An ABC peptide transporter mutant failed to respond to leaf macerates, peptone, or Ser-His-Ser, while peptidase mutants expressed higher-than-wild-type levels of pipA-gfp in response to any of these signals. Our studies are consistent with a model where active transport of a peptidelike signal is required for the signal to interact with PipR, which then activates peptidase gene expression. As a result, the identification of a peptide ligand for PipR sets the stage to identify plant-derived signals for the OryR family of orphan LuxR proteins.« less

Investigating the compatibility of the biocontrol agent Clonostachys rosea IK726 with prodigiosin-producing Serratia rubidaea S55 and phenazine-producing Pseudomonas chlororaphis ToZa7.

PubMed

Kamou, Nathalie N; Dubey, Mukesh; Tzelepis, Georgios; Menexes, Georgios; Papadakis, Emmanouil N; Karlsson, Magnus; Lagopodi, Anastasia L; Jensen, Dan Funck

2016-05-01

This study was carried out to assess the compatibility of the biocontrol fungus Clonostachys rosea IK726 with the phenazine-producing Pseudomonas chlororaphis ToZa7 or with the prodigiosin-producing Serratia rubidaea S55 against Fusarium oxysporum f. sp. radicis-lycopersici. The pathogen was inhibited by both strains in vitro, whereas C. rosea displayed high tolerance to S. rubidaea but not to P. chlororaphis. We hypothesized that this could be attributed to the ATP-binding cassette (ABC) proteins. The results of the reverse transcription quantitative PCR showed an induction of seven genes (abcB1, abcB20, abcB26, abcC12, abcC12, abcG8 and abcG25) from subfamilies B, C and G. In planta experiments showed a significant reduction in foot and root rot on tomato plants inoculated with C. rosea and P. chlororaphis. This study demonstrates the potential for combining different biocontrol agents and suggests an involvement of ABC transporters in secondary metabolite tolerance in C. rosea.

Multidrug ATP-binding cassette transporters are essential for hepatic development of Plasmodium sporozoites.

PubMed

Rijpma, Sanna R; van der Velden, Maarten; González-Pons, Maria; Annoura, Takeshi; van Schaijk, Ben C L; van Gemert, Geert-Jan; van den Heuvel, Jeroen J M W; Ramesar, Jai; Chevalley-Maurel, Severine; Ploemen, Ivo H; Khan, Shahid M; Franetich, Jean-Francois; Mazier, Dominique; de Wilt, Johannes H W; Serrano, Adelfa E; Russel, Frans G M; Janse, Chris J; Sauerwein, Robert W; Koenderink, Jan B; Franke-Fayard, Blandine M

2016-03-01

Multidrug resistance-associated proteins (MRPs) belong to the C-family of ATP-binding cassette (ABC) transport proteins and are known to transport a variety of physiologically important compounds and to be involved in the extrusion of pharmaceuticals. Rodent malaria parasites encode a single ABC transporter subfamily C protein, whereas human parasites encode two: MRP1 and MRP2. Although associated with drug resistance, their biological function and substrates remain unknown. To elucidate the role of MRP throughout the parasite life cycle, Plasmodium berghei and Plasmodium falciparum mutants lacking MRP expression were generated. P. berghei mutants lacking expression of the single MRP as well as P. falciparum mutants lacking MRP1, MRP2 or both proteins have similar blood stage growth kinetics and drug-sensitivity profiles as wild type parasites. We show that MRP1-deficient parasites readily invade primary human hepatocytes and develop into mature liver stages. In contrast, both P. falciparum MRP2-deficient parasites and P. berghei mutants lacking MRP protein expression abort in mid to late liver stage development, failing to produce mature liver stages. The combined P. berghei and P. falciparum data are the first demonstration of a critical role of an ABC transporter during Plasmodium liver stage development. © 2015 John Wiley & Sons Ltd.

Genome tailoring powered production of isobutanol in continuous CO2/H2 blend fermentation using engineered acetogen biocatalyst.

PubMed

Gak, Eugene; Tyurin, Michael; Kiriukhin, Michael

2014-05-01

The cell energy fraction that powered maintenance and expression of genes encoding pro-phage elements, pta-ack cluster, early sporulation, sugar ABC transporter periplasmic proteins, 6-phosphofructokinase, pyruvate kinase, and fructose-1,6-disphosphatase in acetogen Clostridium sp. MT871 was re-directed to power synthetic operon encoding isobutanol biosynthesis at the expense of these genes achieved via their elimination. Genome tailoring decreased cell duplication time by 7.0 ± 0.1 min (p < 0.05) compared to the parental strain, with intact genome and cell duplication time of 68 ± 1 min (p < 0.05). Clostridium sp. MT871 with tailored genome was UVC-mutated to withstand 6.1 % isobutanol in fermentation broth to prevent product inhibition in an engineered commercial biocatalyst producing 5 % (674.5 mM) isobutanol during two-step continuous fermentation of CO2/H2 gas blend. Biocatalyst Clostridium sp. MT871RG- 11IBR6 was engineered to express six copies of synthetic operon comprising optimized synthetic format dehydrogenase, pyruvate formate lyase, acetolactate synthase, acetohydroxyacid reductoisomerase, 2,3-dihydroxy-isovalerate dehydratase, branched-chain alpha-ketoacid decarboxylase gene, aldehyde dehydrogenase, and alcohol dehydrogenase, regaining cell duplication time of 68 ± 1 min (p < 0.05) for the parental strain. This is the first report on isobutanol production by an engineered acetogen biocatalyst suitable for commercial manufacturing of this chemical/fuel using continuous fermentation of CO2/H2 blend thus contributing to the reversal of global warming.

Identification and characterization of the nickel uptake system for urease biogenesis in Streptococcus salivarius 57.I.

PubMed

Chen, Yi-Ywan M; Burne, Robert A

2003-12-01

Ureases are multisubunit enzymes requiring Ni(2+) for activity. The low pH-inducible urease gene cluster in Streptococcus salivarius 57.I is organized as an operon, beginning with ureI, followed by ureABC (structural genes), and ureEFGD (accessory genes). Urease biogenesis also requires a high-affinity Ni(2+) uptake system. By searching the partial genome sequence of a closely related organism, Streptococcus thermophilus LMG18311, three open reading frame (ORFs) homologous to those encoding proteins involved in cobalamin biosynthesis and cobalt transport (cbiMQO) were identified immediately 3' to the ure operon. To determine whether these genes were involved in urease biogenesis by catalyzing Ni(2+) uptake in S. salivarius, regions 3' to ureD were amplified by PCRs from S. salivarius by using primers identical to the S. thermophilus sequences. Sequence analysis of the products revealed three ORFs. Reverse transcriptase PCR was used to demonstrate that the ORFs are transcribed as part of the ure operon. Insertional inactivation of ORF1 with a polar kanamycin marker completely abolished urease activity and the ability to accumulate (63)Ni(2+) during growth. Supplementation of the growth medium with NiCl(2) at concentrations as low as 2.5 micro M partially restored urease activity in the mutant. Both wild-type and mutant strains showed enhanced urease activity when exogenous Ni(2+) was provided at neutral pH. Enhancement of urease activity by adding nickel was regulated at the posttranslational level. Thus, ORF1, ORF2, and ORF3 are part of the ure operon, and these genes, designated ureM, ureQ, and ureO, respectively, likely encode a Ni(2+)-specific ATP-binding cassette transporter.

Induction of hepatic ABC transporter expression is part of the PPARalpha-mediated fasting response in the mouse.

PubMed

Kok, Tineke; Wolters, Henk; Bloks, Vincent W; Havinga, Rick; Jansen, Peter L M; Staels, Bart; Kuipers, Folkert

2003-01-01

Fatty acids are natural ligands of the peroxisome proliferator-activated receptor alpha (PPARalpha). Synthetic ligands of this nuclear receptor, i.e., fibrates, induce the hepatic expression of the multidrug resistance 2 gene (Mdr2), encoding the canalicular phospholipid translocator, and affect hepatobiliary lipid transport. We tested whether fasting-associated fatty acid release from adipose tissues alters hepatic transporter expression and bile formation in a PPARalpha-dependent manner. A 24-hour fasting/48-hour refeeding schedule was used in wild-type and Pparalpha((-/-)) mice. Expression of genes involved in the control of bile formation was determined and related to secretion rates of biliary components. Expression of Pparalpha, farnesoid X receptor, and liver X receptor alpha genes encoding nuclear receptors that control hepatic bile salt and sterol metabolism was induced on fasting in wild-type mice only. The expression of Mdr2 was 5-fold increased in fasted wild-type mice and increased only marginally in Pparalpha((-/-)) mice, and it normalized on refeeding. Mdr2 protein levels and maximal biliary phospholipid secretion rates were clearly increased in fasted wild-type mice. Hepatic expression of the liver X receptor target genes ATP binding cassette transporter a1 (Abca1), Abcg5, and Abcg8, implicated in hepatobiliary cholesterol transport, was induced in fasted wild-type mice only. However, the maximal biliary cholesterol secretion rate was reduced by approximately 50%. Induction of Mdr2 expression and function is part of the PPARalpha-mediated fasting response in mice. Fasting also induces expression of the putative hepatobiliary cholesterol transport genes Abca1, Abcg5, and Abcg8, but, nonetheless, maximal biliary cholesterol excretion is decreased after fasting.

Phylum-wide analysis of genes/proteins related to the last steps of assembly and export of extracellular polymeric substances (EPS) in cyanobacteria

NASA Astrophysics Data System (ADS)

Pereira, Sara B.; Mota, Rita; Vieira, Cristina P.; Vieira, Jorge; Tamagnini, Paula

2015-10-01

Many cyanobacteria produce extracellular polymeric substances (EPS) with particular characteristics (e.g. anionic nature and presence of sulfate) that make them suitable for industrial processes such as bioremediation of heavy metals or thickening, suspending or emulsifying agents. Nevertheless, their biosynthetic pathway(s) are still largely unknown, limiting their utilization. In this work, a phylum-wide analysis of genes/proteins putatively involved in the assembly and export of EPS in cyanobacteria was performed. Our results demonstrated that most strains harbor genes encoding proteins related to the three main pathways: Wzy-, ABC transporter-, and Synthase-dependent, but often not the complete set defining one pathway. Multiple gene copies are mainly correlated to larger genomes, and the strains with reduced genomes (e.g. the clade of marine unicellular Synechococcus and Prochlorococcus), seem to have lost most of the EPS-related genes. Overall, the distribution of the different genes/proteins within the cyanobacteria phylum raises the hypothesis that cyanobacterial EPS production may not strictly follow one of the pathways previously characterized. Moreover, for the proteins involved in EPS polymerization, amino acid patterns were defined and validated constituting a novel and robust tool to identify proteins with similar functions and giving a first insight to which polymer biosynthesis they are related to.

The genome of the social amoeba Dictyostelium discoideum

PubMed Central

Eichinger, L.; Pachebat, J.A.; Glöckner, G.; Rajandream, M.-A.; Sucgang, R.; Berriman, M.; Song, J.; Olsen, R.; Szafranski, K.; Xu, Q.; Tunggal, B.; Kummerfeld, S.; Madera, M.; Konfortov, B. A.; Rivero, F.; Bankier, A. T.; Lehmann, R.; Hamlin, N.; Davies, R.; Gaudet, P.; Fey, P.; Pilcher, K.; Chen, G.; Saunders, D.; Sodergren, E.; Davis, P.; Kerhornou, A.; Nie, X.; Hall, N.; Anjard, C.; Hemphill, L.; Bason, N.; Farbrother, P.; Desany, B.; Just, E.; Morio, T.; Rost, R.; Churcher, C.; Cooper, J.; Haydock, S.; van Driessche, N.; Cronin, A.; Goodhead, I.; Muzny, D.; Mourier, T.; Pain, A.; Lu, M.; Harper, D.; Lindsay, R.; Hauser, H.; James, K.; Quiles, M.; Babu, M. Madan; Saito, T.; Buchrieser, C.; Wardroper, A.; Felder, M.; Thangavelu, M.; Johnson, D.; Knights, A.; Loulseged, H.; Mungall, K.; Oliver, K.; Price, C.; Quail, M.A.; Urushihara, H.; Hernandez, J.; Rabbinowitsch, E.; Steffen, D.; Sanders, M.; Ma, J.; Kohara, Y.; Sharp, S.; Simmonds, M.; Spiegler, S.; Tivey, A.; Sugano, S.; White, B.; Walker, D.; Woodward, J.; Winckler, T.; Tanaka, Y.; Shaulsky, G.; Schleicher, M.; Weinstock, G.; Rosenthal, A.; Cox, E.C.; Chisholm, R. L.; Gibbs, R.; Loomis, W. F.; Platzer, M.; Kay, R. R.; Williams, J.; Dear, P. H.; Noegel, A. A.; Barrell, B.; Kuspa, A.

2005-01-01

The social amoebae are exceptional in their ability to alternate between unicellular and multicellular forms. Here we describe the genome of the best-studied member of this group, Dictyostelium discoideum. The gene-dense chromosomes encode ~12,500 predicted proteins, a high proportion of which have long repetitive amino acid tracts. There are many genes for polyketide synthases and ABC transporters, suggesting an extensive secondary metabolism for producing and exporting small molecules. The genome is rich in complex repeats, one class of which is clustered and may serve as centromeres. Partial copies of the extrachromosomal rDNA element are found at the ends of each chromosome, suggesting a novel telomere structure and the use of a common mechanism to maintain both the rDNA and chromosomal termini. A proteome-based phylogeny shows that the amoebozoa diverged from the animal/fungal lineage after the plant/animal split, but Dictyostelium appears to have retained more of the diversity of the ancestral genome than either of these two groups. PMID:15875012

Functional Genomics of the Aeromonas salmonicida Lipopolysaccharide O-Antigen and A-Layer from Typical and Atypical Strains

PubMed Central

Merino, Susana; de Mendoza, Elena; Canals, Rocío; Tomás, Juan M.

2015-01-01

The A. salmonicida A450 LPS O-antigen, encoded by the wbsalmo gene cluster, is exported through an ABC-2 transporter-dependent pathway. It represents the first example of an O-antigen LPS polysaccharide with three different monosaccharides in their repeating unit assembled by this pathway. Until now, only repeating units with one or two different monosaccharides have been described. Functional genomic analysis of this wbsalmo region is mostly in agreement with the LPS O-antigen structure of acetylated l-rhamnose (Rha), d-glucose (Glc), and 2-amino-2-deoxy-d-mannose (ManN). Between genes of the wbsalmo we found the genes responsible for the biosynthesis and assembly of the S-layer (named A-layer in these strains). Through comparative genomic analysis and in-frame deletions of some of the genes, we concluded that all the A. salmonicida typical and atypical strains, other than A. salmonicida subsp. pectinolytica strains, shared the same wbsalmo and presence of A-layer. A. salmonicida subsp. pectinolytica strains lack wbsalmo and A-layer, two major virulence factors, and this could be the reason they are the only ones not found as fish pathogens. PMID:26082990

Neuronal glycosylation differentials in normal, injured and chondroitinase-treated environments

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kilcoyne, Michelle; Sharma, Shashank; McDevitt, Niamh

2012-04-13

Highlights: Black-Right-Pointing-Pointer Carbohydrates are important in the CNS and ChABC has been used for spinal cord injury (SCI) treatment. Black-Right-Pointing-Pointer Neuronal glycosylation in injury and after ChABC treatment is unknown. Black-Right-Pointing-Pointer In silico mining verified that glyco-related genes were differentially regulated after SCI. Black-Right-Pointing-Pointer In vitro model system revealed abnormal sialylation in an injured environment. Black-Right-Pointing-Pointer The model indicated a return to normal neuronal glycosylation after ChABC treatment. -- Abstract: Glycosylation is found ubiquitously throughout the central nervous system (CNS). Chondroitin sulphate proteoglycans (CSPGs) are a group of molecules heavily substituted with glycosaminoglycans (GAGs) and are found in the extracellularmore » matrix (ECM) and cell surfaces. Upon CNS injury, a glial scar is formed, which is inhibitory for axon regeneration. Several CSPGs are up-regulated within the glial scar, including NG2, and these CSPGs are key inhibitory molecules of axonal regeneration. Treatment with chondroitinase ABC (ChABC) can neutralise the inhibitory nature of NG2. A gene expression dataset was mined in silico to verify differentially regulated glycosylation-related genes in neurons after spinal cord injury and identify potential targets for further investigation. To establish the glycosylation differential of neurons that grow in a healthy, inhibitory and ChABC-treated environment, we established an indirect co-culture system where PC12 neurons were grown with primary astrocytes, Neu7 astrocytes (which overexpress NG2) and Neu7 astrocytes treated with ChABC. After 1, 4 and 8 days culture, lectin cytochemistry of the neurons was performed using five fluorescently-labelled lectins (ECA MAA, PNA, SNA-I and WFA). Usually {alpha}-(2,6)-linked sialylation scarcely occurs in the CNS but this motif was observed on the neurons in the injured environment only at day 8. Treatment with ChABC was successful in returning neuronal glycosylation to normal conditions at all timepoints for MAA, PNA and SNA-I staining, and by day 8 in the case of WFA. This study demonstrated neuronal cell surface glycosylation changes in an inhibitory environment and indicated a return to normal glycosylation after treatment with ChABC, which may be promising for identifying potential therapies for neuronal regeneration strategies.« less

Multihospital Occurrence of Pan-Resistant Klebsiella pneumoniae Sequence Type 147 with an ISEcp1-Directed blaOXA-181 Insertion in the mgrB Gene in the United Arab Emirates

PubMed Central

Sonnevend, Ágnes; Ghazawi, Akela; Hashmey, Rayhan; Haidermota, Aliasgher; Girgis, Safinaz; Alfaresi, Mubarak; Omar, Mohammed; Paterson, David L.; Zowawi, Hosam M.

2017-01-01

ABSTRACT The emergence of pan-resistant Klebsiella pneumoniae strains is an increasing concern. In the present study, we describe a cluster of 9 pan-resistant K. pneumoniae sequence type 147 (ST147) isolates encountered in 4 patients over nearly 1 year in 3 hospitals of the United Arab Emirates (UAE). The isolates exhibited highly similar genotypes. All produced chromosomally encoded OXA-181, and the majority also produced the NDM-5 carbapenemase. As with the previously described single isolate from the UAE, MS6671, the mgrB was disrupted by a functional, ISEcp1-driven blaOXA-181 insertion causing resistance to carbapenems. The mutation was successfully complemented with an intact mgrB gene, indicating that it was responsible for colistin resistance. blaNDM-5 was located within a resistance island of an approximately 100-kb IncFII plasmid carrying ermB, mph(A), blaTEM-1B, rmtB, blaNDM-5, sul1, aadA2, and dfrA12 resistance genes. Sequencing this plasmid (pABC143-NDM) revealed that its backbone was nearly identical to that of plasmid pMS6671E from which several resistance genes, including blaNDM-5, had been deleted. More extensive similarities of the backbone and the resistance island were found between pABC143C-NDM and the blaNDM-5-carrying IncFII plasmids of two K. pneumoniae ST147 isolates from South Korea, one of which was colistin resistant, and both also produced OXA-181. Notably, one of these strains was isolated from a patient transferred from the UAE. Our data show that this pan-resistant clone has an alarming capacity to maintain itself over an extended period of time and is even likely to be transmitted internationally. PMID:28438945

Multihospital Occurrence of Pan-Resistant Klebsiella pneumoniae Sequence Type 147 with an ISEcp1-Directed blaOXA-181 Insertion in the mgrB Gene in the United Arab Emirates.

PubMed

Sonnevend, Ágnes; Ghazawi, Akela; Hashmey, Rayhan; Haidermota, Aliasgher; Girgis, Safinaz; Alfaresi, Mubarak; Omar, Mohammed; Paterson, David L; Zowawi, Hosam M; Pál, Tibor

2017-07-01

The emergence of pan-resistant Klebsiella pneumoniae strains is an increasing concern. In the present study, we describe a cluster of 9 pan-resistant K. pneumoniae sequence type 147 (ST147) isolates encountered in 4 patients over nearly 1 year in 3 hospitals of the United Arab Emirates (UAE). The isolates exhibited highly similar genotypes. All produced chromosomally encoded OXA-181, and the majority also produced the NDM-5 carbapenemase. As with the previously described single isolate from the UAE, MS6671, the mgrB was disrupted by a functional, IS Ecp1 -driven bla OXA-181 insertion causing resistance to carbapenems. The mutation was successfully complemented with an intact mgrB gene, indicating that it was responsible for colistin resistance. bla NDM-5 was located within a resistance island of an approximately 100-kb IncFII plasmid carrying ermB , mph (A), bla TEM-1B , rmtB , bla NDM-5 , sul1 , aadA2 , and dfrA12 resistance genes. Sequencing this plasmid (pABC143-NDM) revealed that its backbone was nearly identical to that of plasmid pMS6671E from which several resistance genes, including bla NDM-5 , had been deleted. More extensive similarities of the backbone and the resistance island were found between pABC143C-NDM and the bla NDM-5 -carrying IncFII plasmids of two K. pneumoniae ST147 isolates from South Korea, one of which was colistin resistant, and both also produced OXA-181. Notably, one of these strains was isolated from a patient transferred from the UAE. Our data show that this pan-resistant clone has an alarming capacity to maintain itself over an extended period of time and is even likely to be transmitted internationally. Copyright © 2017 American Society for Microbiology.

Conversion of chlorobiphenyls into phenylhexadienoates and benzoates by the enzymes of the upper pathway for polychlorobiphenyl degradation encoded by the bph locus of Pseudomonas sp. strain LB400.

PubMed Central

Seeger, M; Timmis, K N; Hofer, B

1995-01-01

Metabolism of 21 chlorobiphenyls by the enzymes of the upper biphenyl catabolic pathway encoded by the bph locus of Pseudomonas sp. strain LB400 was investigated by using recombinant strains harboring gene cassettes containing bphABC or bphABCD. The enzymes of the upper pathway were generally able to metabolize mono- and dichlorinated biphenyls but only partially transform most trichlorinated congeners investigated: 14 of 15 mono- and dichlorinated and 2 of 6 trichlorinated congeners were converted into benzoates. All mono- and at least 8 of 12 dichlorinated congeners were attacked by the bphA-encoded biphenyl dioxygenase virtually exclusively at ortho and meta carbons. This enzyme exhibited a high degree of selectivity for the aromatic ring to be attacked, with the order of ring preference being non- > ortho- > meta- > para-substituted for mono- and dichlorinated congeners. The influence of the chlorine substitution pattern of the metabolized ring on benzoate formation resembled its influence on the reactivity of initial dioxygenation, suggesting that the rate of benzoate formation may frequently be determined by the rate of initial attack. The absorption spectra of phenylhexadienoates formed correlated with the presence or absence of a chlorine substituent at an ortho position. PMID:7618878

A 38-Kilobase Pathogenicity Island Specific for Mycobacterium avium subsp. paratuberculosis Encodes Cell Surface Proteins Expressed in the Host

PubMed Central

Stratmann, Janin; Strommenger, Birgit; Goethe, Ralph; Dohmann, Karen; Gerlach, Gerald-F.; Stevenson, Karen; Li, Ling-ling; Zhang, Qing; Kapur, Vivek; Bull, Tim J.

2004-01-01

We have used representational difference analysis to identify a novel Mycobacterium avium subsp. paratuberculosis-specific ABC transporter operon (mpt), which comprises six open reading frames designated mptA to -F and is immediately preceded by two putative Fur boxes. Functional genomics revealed that the mpt operon is flanked on one end by a fep cluster encoding proteins involved in the uptake of Fe3+ and on the other end by a sid cluster encoding non-ribosome-dependent heterocyclic siderophore synthases. Together these genes form a 38-kb M. avium subsp. paratuberculosis-specific locus flanked by an insertion sequence similar to IS1110. Expression studies using Western blot analyses showed that MptC is present in the envelope fraction of M. avium subsp. paratuberculosis. The MptD protein was shown to be surface exposed, using a specific phage (fMptD) isolated from a phage-peptide library, by differential screening of Mycobacterium smegmatis transformants. The phage fMptD-derived peptide could be used in a peptide-mediated capture PCR with milk from infected dairy herds, thereby showing surface-exposed expression of the MptD protein in the host. Together, these data suggest that the 38-kb locus constitutes an M. avium subsp. paratuberculosis pathogenicity island. PMID:14977927

Evolution of Mycolic Acid Biosynthesis Genes and Their Regulation during Starvation in Mycobacterium tuberculosis

PubMed Central

Jamet, Stevie; Quentin, Yves; Coudray, Coralie; Texier, Pauline; Laval, Françoise; Daffé, Mamadou

2015-01-01

ABSTRACT Mycobacterium tuberculosis, the etiological agent of tuberculosis, is a Gram-positive bacterium with a unique cell envelope composed of an essential outer membrane. Mycolic acids, which are very-long-chain (up to C100) fatty acids, are the major components of this mycomembrane. The enzymatic pathways involved in the biosynthesis and transport of mycolates are fairly well documented and are the targets of the major antituberculous drugs. In contrast, only fragmented information is available on the expression and regulation of the biosynthesis genes. In this study, we report that the hadA, hadB, and hadC genes, which code for the mycolate biosynthesis dehydratase enzymes, are coexpressed with three genes that encode proteins of the translational apparatus. Consistent with the well-established control of the translation potential by nutrient availability, starvation leads to downregulation of the hadABC genes along with most of the genes required for the synthesis, modification, and transport of mycolates. The downregulation of a subset of the biosynthesis genes is partially dependent on RelMtb, the key enzyme of the stringent response. We also report the phylogenetic evolution scenario that has shaped the current genetic organization, characterized by the coregulation of the hadABC operon with genes of the translational apparatus and with genes required for the modification of the mycolates. IMPORTANCE Mycobacterium tuberculosis infects one-third of the human population worldwide, and despite the available therapeutic arsenal, it continues to kill millions of people each year. There is therefore an urgent need to identify new targets and develop a better understanding of how the bacterium is adapting itself to host defenses during infection. A prerequisite of this understanding is knowledge of how this adaptive skill has been implanted by evolution. Nutrient scarcity is an environmental condition the bacterium has to cope with during infection. In many bacteria, adaptation to starvation relies partly on the stringent response. M. tuberculosis's unique outer membrane layer, the mycomembrane, is crucial for its viability and virulence. Despite its being the target of the major antituberculosis drugs, only scattered information exists on how the genes required for biosynthesis of the mycomembrane are expressed and regulated during starvation. This work has addressed this issue as a step toward the identification of new targets in the fight against M. tuberculosis. PMID:26416833

Analysis of the APETALA3- and PISTILLATA-like genes in Hedyosmum orientale (Chloranthaceae) provides insight into the evolution of the floral homeotic B-function in angiosperms

PubMed Central

Liu, Shujun; Sun, Yonghua; Du, Xiaoqiu; Xu, Qijiang; Wu, Feng; Meng, Zheng

2013-01-01

Background and Aims According to the floral ABC model, B-function genes appear to play a key role in the origin and diversification of the perianth during the evolution of angiosperms. The basal angiosperm Hedyosmum orientale (Chloranthaceae) has unisexual inflorescences associated with a seemingly primitive reproductive morphology and a reduced perianth structure in female flowers. The aim of this study was to investigate the nature of the perianth and the evolutionary state of the B-function programme in this species. Methods A series of experiments were conducted to characterize B-gene homologues isolated from H. orientale, including scanning electron microscopy to observe the development of floral organs, phylogenetic analysis to reconstruct gene evolutionary history, reverse transcription–PCR, quantitative real-time PCR and in situ hybridization to identify gene expression patterns, the yeast two-hybrid assay to explore protein dimerization affinities, and transgenic analyses in Arabidopsis thaliana to determine activities of the encoded proteins. Key Results The expression of HoAP3 genes was restricted to stamens, whereas HoPI genes were broadly expressed in all floral organs. HoAP3 was able to partially restore the stamen but not petal identity in Arabidopsis ap3-3 mutants. In contrast, HoPI could rescue aspects of both stamen and petal development in Arabidopsis pi-1 mutants. When the complete C-terminal sequence of HoPI was deleted, however, no or weak transgenic phenotypes were observed and homodimerization capability was completely abolished. Conclusions The results suggest that Hedyosmum AP3-like genes have an ancestral function in specifying male reproductive organs, and that the activity of the encoded PI-like proteins is highly conserved between Hedyosmum and Arabidopsis. Moreover, there is evidence that the C-terminal region is important for the function of HoPI. Our findings indicate that the development of the proposed perianth in Hedyosmum does not rely on the B homeotic function. PMID:23956161

Identification of the heme acquisition system in Vibrio vulnificus M2799.

PubMed

Kawano, Hiroaki; Miyamoto, Katsushiro; Yasunobe, Megumi; Murata, Masahiro; Yamahata, Eri; Yamaguchi, Ryo; Miyaki, Yuta; Tsuchiya, Takahiro; Tanabe, Tomotaka; Funahashi, Tatsuya; Tsujibo, Hiroshi

2018-04-01

Vibrio vulnificus, the causative agent of serious, often fatal, infections in humans, requires iron for its pathogenesis. As such, it obtains iron via both vulnibactin and heme-mediated iron-uptake systems. In this study, we identified the heme acquisition system in V. vulnificus M2799. The nucleotide sequences of the genes encoding heme receptors HupA and HvtA and the ATP-binding cassette (ABC) transport system proteins HupB, HupC, and HupD were determined, and then used in the construction of deletion mutants developed from a Δics strain, which could not synthesize vulnibactin. Growth experiments using these mutants indicated that HupA and HvtA are major and minor heme receptors, respectively. The expressions of two proteins were analyzed by the quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR). Furthermore, complementation analyses confirmed that the HupBCD proteins are the only ABC transport system shared by both the HupA and HvtA receptors. This is the first genetic evidence that the HupBCD proteins are essential for heme acquisition by V. vulnificus. Further investigation showed that hupA, hvtA, and hupBCD are regulated by Fur. The qRT-PCR analysis of the heme receptor genes revealed that HupR, a LysR-family positive transcriptional activator, upregulates the expression of hupA, but not hvtA. In addition, ptrB was co-transcribed with hvtA, and PtrB had no influence on growth in low-iron CM9 medium supplemented with hemin, hemoglobin, or cytochrome C. Copyright © 2018 Elsevier Ltd. All rights reserved.

The ABC transporter ABCG29 is involved in H2O2 tolerance and biocontrol traits in the fungus Clonostachys rosea.

PubMed

Dubey, Mukesh; Jensen, Dan Funck; Karlsson, Magnus

2016-04-01

For successful biocontrol interactions, biological control organisms must tolerate toxic metabolites produced by themselves or plant pathogens during mycoparasitic/antagonistic interactions, by host plant during colonization of the plant, and xenobiotics present in the environment. ATP-binding cassette (ABC) transporters can play a significant role in tolerance of toxic compounds by mediating active transport across the cellular membrane. This paper reports on functional characterization of an ABC transporter ABCG29 in the biocontrol fungus Clonostachys rosea strain IK726. Gene expression analysis showed induced expression of abcG29 during exposure to the Fusarium spp. mycotoxin zearalenone (ZEA) and the fungicides Cantus, Chipco Green and Apron. Expression of abcG29 in C. rosea was significantly higher during C. rosea-C. rosea (Cr-Cr) interaction or in exposure to C. rosea culture filtrate for 2 h, compared to interaction with Fusarium graminearum or 2 h exposure to F. graminearum culture filtrate. In contrast with gene expression data, ΔabcG29 strains did not display reduced tolerance towards ZEA, fungicides or chemical agents known for inducing oxidative, cell wall or osmotic stress, compared to C. rosea WT. The exception was a significant reduction in tolerance to H2O2 (10 mM) in ΔabcG29 strains when conidia were used as an inoculum. The antagonistic ability of ΔabcG29 strains towards F. graminearum, Fusarium oxysporum or Botrytis cinerea in dual plate assays were not different compared with WT. However, in biocontrol assays ΔabcG29 strains displayed reduced ability to protect Arabidopsis thaliana leaves from B. cinerea, and barley seedling from F. graminearum as measured by an A. thaliana detached leaf assay and a barley foot rot disease assay, respectively. These data show that the ABCG29 is dispensable for ZEA and fungicides tolerance, and antagonism but not H2O2 tolerance and biocontrol effects in C. rosea.

Functional Dependence between Septal Protein SepJ from Anabaena sp. Strain PCC 7120 and an Amino Acid ABC-Type Uptake Transporter.

PubMed

Escudero, Leticia; Mariscal, Vicente; Flores, Enrique

2015-08-01

In the diazotrophic filaments of heterocyst-forming cyanobacteria, two different cell types, the CO2-fixing vegetative cells and the N2-fixing heterocysts, exchange nutrients, including some amino acids. In the model organism Anabaena sp. strain PCC 7120, the SepJ protein, composed of periplasmic and integral membrane (permease) sections, is located at the intercellular septa joining adjacent cells in the filament. The unicellular cyanobacterium Synechococcus elongatus strain PCC 7942 bears a gene, Synpcc7942_1024 (here designated dmeA), encoding a permease homologous to the SepJ permease domain. Synechococcus strains lacking dmeA or lacking dmeA and expressing Anabaena sepJ were constructed. The Synechococcus dmeA mutant showed a significant 22 to 32% decrease in the uptake of aspartate, glutamate, and glutamine, a phenotype that could be partially complemented by Anabaena sepJ. Synechococcus mutants of an ATP-binding-cassette (ABC)-type transporter for polar amino acids showed >98% decreased uptake of glutamate irrespective of the presence of dmeA or Anabaena sepJ in the same strain. Thus, Synechococcus DmeA or Anabaena SepJ is needed to observe full (or close to full) activity of the ABC transporter. An Anabaena sepJ deletion mutant was significantly impaired in glutamate and aspartate uptake, which also in this cyanobacterium requires the activity of an ABC-type transporter for polar amino acids. SepJ appears therefore to generally stimulate the activity of cyanobacterial ABC-type transporters for polar amino acids. Conversely, an Anabaena mutant of three ABC-type transporters for amino acids was impaired in the intercellular transfer of 5-carboxyfluorescein, a SepJ-related property. Our results unravel possible functional interactions in transport elements important for diazotrophic growth. Membrane transporters are essential for many aspects of cellular life, from uptake and export of substances in unicellular organisms to intercellular molecular exchange in multicellular organisms. Heterocyst-forming cyanobacteria such as Anabaena represent a unique case of multicellularity, in which two cell types exchange nutrients and regulators. The SepJ protein located at the intercellular septa in the filaments of Anabaena contains a permease domain of the drug/metabolite transporter (DMT) superfamily that somehow contributes to intercellular molecular transfer. In this work, we have found that SepJ stimulates the activity of a polar amino acid uptake transporter of the ATP-binding-cassette (ABC) superfamily, which could itself affect an intercellular transfer activity related to SepJ, thus unraveling possible functional interactions between these different transporters. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

NEMO Binding Domain peptide inhibits constitutive NF-κB activity and reduces tumor burden in a canine model of relapsed, refractory Diffuse Large B-Cell Lymphoma

PubMed Central

Gaurnier-Hausser, Anita; Patel, Reema; Baldwin, Albert S.; May, Michael J.; Mason, Nicola J.

2011-01-01

Purpose Activated B-Cell Diffuse Large B-Cell Lymphoma (ABC-DLBCL) is an aggressive, poorly chemoresponsive lymphoid malignancy characterized by constitutive canonical NF-κB activity that promotes lymphomagenesis and chemotherapy resistance via over-expression of anti-apoptotic NF-κB target genes. Inhibition of the canonical NF-κB pathway may therefore have therapeutic relevance in ABC-DLBCL. Here we set out to determine whether dogs with spontaneous DLBCL have comparative aberrant constitutive NF-κB activity and to determine the therapeutic relevance of NF-κB inhibition in dogs with relapsed, resistant DLBCL. Experimental Design Canonical NF-κB activity was evaluated by electrophoretic mobility shift assays and immunoblot analyses, and NF-κB target gene expression was measured by qRT-PCR. Primary malignant canine B lymphocytes were treated with the selective IKK complex inhibitor Nemo Binding Domain (NBD) peptide, and evaluated for NF-κB activity and apoptosis. NBD peptide was administered intra-nodally to dogs with relapsed B-cell lymphoma and NF-κB target gene expression and tumor burden were evaluated pre and post treatment. Results Constitutive canonical NF-κB activity and increased NF-κB target gene expression was detected in primary DLBCL tissue. NBD peptide inhibited this activity and induced apoptosis of primary malignant B cells in vitro. Intra-tumoral injections of NBD peptide to dogs with relapsed DLBCL inhibited NF-κB target gene expression and reduced tumor burden. Conclusions This work shows that dogs with spontaneous DLBCL represent a clinically relevant, spontaneous, large animal model for human ABC-DLBCL and demonstrates the therapeutic relevance of NF-κB inhibition in the treatment of ABC-DLBCL. These results have important translational relevance for ABC-DLBCL treatment in human patients. PMID:21610150

Hierarchy of Iron Uptake Systems: Yfu and Yiu Are Functional in Yersinia pestis▿

PubMed Central

Kirillina, Olga; Bobrov, Alexander G.; Fetherston, Jacqueline D.; Perry, Robert D.

2006-01-01

In addition to the yersiniabactin (Ybt) siderophore-dependent system, two inorganic iron ABC transport systems of Yersinia pestis, Yfe and Yfu, have been characterized. Here we show that the Yfu system functions in Y. pestis: a Ybt− Yfe− Yfu− mutant exhibited a greater growth defect under iron-deficient conditions than its Ybt− Yfe− parental strain. We also demonstrate that another putative Y. pestis iron uptake system, Yiu, which potentially encodes an outer membrane receptor, YiuR, and an ABC iron transport cassette, YiuABC, is functional. The cloned yiuABC operon restored growth of an enterobactin-deficient mutant Escherichia coli strain, 1017, under iron-chelated conditions. Iron uptake by the Yiu system in Y. pestis was demonstrated only when the Ybt, Yfe, and Yfu systems were mutated. Using a yiuA::lacZ fusion, we show that the yiuABC promoter is repressed by iron through Fur. A mouse model of bubonic plague failed to show a significant role for the Yiu system in the disease process. These results demonstrate that two additional iron transporters are functional in Y. pestis and indicate that there is a hierarchy of iron transporters, with Ybt being most effective and Yiu being the least effective of those systems which have been characterized. PMID:16954402

Genetic and Genomic Insights into the Role of Benzoate-Catabolic Pathway Redundancy in Burkholderia xenovorans LB400†

PubMed Central

Denef, V. J.; Klappenbach, J. A.; Patrauchan, M. A.; Florizone, C.; Rodrigues, J. L. M.; Tsoi, T. V.; Verstraete, W.; Eltis, L. D.; Tiedje, J. M.

2006-01-01

Transcriptomic and proteomic analyses of Burkholderia xenovorans LB400, a potent polychlorinated biphenyl (PCB) degrader, have implicated growth substrate- and phase-dependent expression of three benzoate-catabolizing pathways: a catechol ortho cleavage (ben-cat) pathway and two benzoyl-coenzyme A pathways, encoded by gene clusters on the large chromosome (boxC) and the megaplasmid (boxM). To elucidate the significance of this apparent redundancy, we constructed mutants with deletions of the ben-cat pathway (the ΔbenABCD::kan mutant), the boxC pathway (the ΔboxABC::kan mutant), and both pathways (the ΔbenABCDΔ boxABC::kan mutant). All three mutants oxidized benzoate in resting-cell assays. However, the ΔbenABCD::kan and ΔbenABCD ΔboxABC::kan mutants grew at reduced rates on benzoate and displayed increased lag phases. By contrast, growth on succinate, on 4-hydroxybenzoate, and on biphenyl was unaffected. Microarray and proteomic analyses revealed that cells of the ΔbenABCD::kan mutant growing on benzoate expressed both box pathways. Overall, these results indicate that all three pathways catabolize benzoate. Deletion of benABCD abolished the ability of LB400 to grow using 3-chlorobenzoate. None of the benzoate pathways could degrade 2- or 4-chlorobenzoate, indicating that the pathway redundancy does not directly contribute to LB400's PCB-degrading capacities. Finally, an extensive sigmaE-regulated oxidative stress response not present in wild-type LB400 grown on benzoate was detected in these deletion mutants, supporting our earlier suggestion that the box pathways are preferentially active under reduced oxygen tension. Our data further substantiate the expansive network of tightly interconnected and complexly regulated aromatic degradation pathways in LB400. PMID:16391095

Contribution of Efflux Pumps, Porins, and β-Lactamases to Multidrug Resistance in Clinical Isolates of Acinetobacter baumannii

PubMed Central

Rumbo, C.; Gato, E.; López, M.; Ruiz de Alegría, C.; Fernández-Cuenca, F.; Martínez-Martínez, L.; Vila, J.; Pachón, J.; Cisneros, J. M.; Rodríguez-Baño, J.; Pascual, A.

2013-01-01

We investigated the mechanisms of resistance to carbapenems, aminoglycosides, glycylcyclines, tetracyclines, and quinolones in 90 multiresistant clinical strains of Acinetobacter baumannii isolated from two genetically unrelated A. baumannii clones: clone PFGE-ROC-1 (53 strains producing the OXA-58 β-lactamase enzyme and 18 strains with the OXA-24 β-lactamase) and clone PFGE-HUI-1 (19 strains susceptible to carbapenems). We used real-time reverse transcriptase PCR to correlate antimicrobial resistance (MICs) with expression of genes encoding chromosomal β-lactamases (AmpC and OXA-51), porins (OmpA, CarO, Omp33, Dcap-like, OprB, Omp25, OprC, OprD, and OmpW), and proteins integral to six efflux systems (AdeABC, AdeIJK, AdeFGH, CraA, AbeM, and AmvA). Overexpression of the AdeABC system (level of expression relative to that by A. baumannii ATCC 17978, 30- to 45-fold) was significantly associated with resistance to tigecycline, minocycline, and gentamicin and other biological functions. However, hyperexpression of the AdeIJK efflux pump (level of expression relative to that by A. baumannii ATCC 17978, 8- to 10-fold) was significantly associated only with resistance to tigecycline and minocycline (to which the TetB efflux system also contributed). TetB and TetA(39) efflux pumps were detected in clinical strains and were associated with resistance to tetracyclines and doxycycline. The absence of the AdeABC system and the lack of expression of other mechanisms suggest that tigecycline-resistant strains of the PFGE-HUI-1 clone may be associated with a novel resistance-nodulation-cell efflux pump (decreased MICs in the presence of the inhibitor Phe-Arg β-naphthylamide dihydrochloride) and the TetA(39) system. PMID:23939894

Hydrogenase activity in the foodborne pathogen Campylobacter jejuni depends upon a novel ABC-type nickel transporter (NikZYXWV) and is SlyD-independent.

PubMed

Howlett, Robert M; Hughes, Bethan M; Hitchcock, Andrew; Kelly, David J

2012-06-01

Campylobacter jejuni is a human pathogen of worldwide significance. It is commensal in the gut of many birds and mammals, where hydrogen is a readily available electron donor. The bacterium possesses a single membrane-bound, periplasmic-facing NiFe uptake hydrogenase that depends on the acquisition of environmental nickel for activity. The periplasmic binding protein Cj1584 (NikZ) of the ATP binding cassette (ABC) transporter encoded by the cj1584c-cj1580c (nikZYXWV) operon in C. jejuni strain NCTC 11168 was found to be nickel-repressed and to bind free nickel ions with a submicromolar K(d) value, as measured by fluorescence spectroscopy. Unlike the Escherichia coli NikA protein, NikZ did not bind EDTA-chelated nickel and lacks key conserved residues implicated in metallophore interaction. A C. jejuni cj1584c null mutant strain showed an approximately 22-fold decrease in intracellular nickel content compared with the wild-type strain and a decreased rate of uptake of (63)NiCl(2). The inhibition of residual nickel uptake at higher nickel concentrations in this mutant by hexa-ammine cobalt (III) chloride or magnesium ions suggests that low-affinity uptake occurs partly through the CorA magnesium transporter. Hydrogenase activity was completely abolished in the cj1584c mutant after growth in unsupplemented media, but was fully restored after growth with 0.5 mM nickel chloride. Mutation of the putative metallochaperone gene slyD (cj0115) had no effect on either intracellular nickel accumulation or hydrogenase activity. Our data reveal a strict dependence of hydrogenase activity in C. jejuni on high-affinity nickel uptake through an ABC transporter that has distinct properties compared with the E. coli Nik system.

Genomic evidence of geographically widespread effect of gene flow from polar bears into brown bears

PubMed Central

Cahill, James A; Stirling, Ian; Kistler, Logan; Salamzade, Rauf; Ersmark, Erik; Fulton, Tara L; Stiller, Mathias; Green, Richard E; Shapiro, Beth

2015-01-01

Polar bears are an arctic, marine adapted species that is closely related to brown bears. Genome analyses have shown that polar bears are distinct and genetically homogeneous in comparison to brown bears. However, these analyses have also revealed a remarkable episode of polar bear gene flow into the population of brown bears that colonized the Admiralty, Baranof and Chichagof islands (ABC islands) of Alaska. Here, we present an analysis of data from a large panel of polar bear and brown bear genomes that includes brown bears from the ABC islands, the Alaskan mainland and Europe. Our results provide clear evidence that gene flow between the two species had a geographically wide impact, with polar bear DNA found within the genomes of brown bears living both on the ABC islands and in the Alaskan mainland. Intriguingly, while brown bear genomes contain up to 8.8% polar bear ancestry, polar bear genomes appear to be devoid of brown bear ancestry, suggesting the presence of a barrier to gene flow in that direction. PMID:25490862

Genomic evidence of geographically widespread effect of gene flow from polar bears into brown bears.

PubMed

Cahill, James A; Stirling, Ian; Kistler, Logan; Salamzade, Rauf; Ersmark, Erik; Fulton, Tara L; Stiller, Mathias; Green, Richard E; Shapiro, Beth

2015-03-01

Polar bears are an arctic, marine adapted species that is closely related to brown bears. Genome analyses have shown that polar bears are distinct and genetically homogeneous in comparison to brown bears. However, these analyses have also revealed a remarkable episode of polar bear gene flow into the population of brown bears that colonized the Admiralty, Baranof and Chichagof islands (ABC islands) of Alaska. Here, we present an analysis of data from a large panel of polar bear and brown bear genomes that includes brown bears from the ABC islands, the Alaskan mainland and Europe. Our results provide clear evidence that gene flow between the two species had a geographically wide impact, with polar bear DNA found within the genomes of brown bears living both on the ABC islands and in the Alaskan mainland. Intriguingly, while brown bear genomes contain up to 8.8% polar bear ancestry, polar bear genomes appear to be devoid of brown bear ancestry, suggesting the presence of a barrier to gene flow in that direction. © 2014 The Authors. Molecular Ecology Published by John Wiley & Sons Ltd.

A Novel Phenanthrene Dioxygenase from Nocardioides sp. Strain KP7: Expression in Escherichia coli

PubMed Central

Saito, Atsushi; Iwabuchi, Tokuro; Harayama, Shigeaki

2000-01-01

Nocardioides sp. strain KP7 grows on phenanthrene but not on naphthalene. This organism degrades phenanthrene via 1-hydroxy-2-naphthoate, o-phthalate, and protocatechuate. The genes responsible for the degradation of phenanthrene to o-phthalate (phd) were found by Southern hybridization to reside on the chromosome. A 10.6-kb DNA fragment containing eight phd genes was cloned and sequenced. The phdA, phdB, phdC, and phdD genes, which encode the α and β subunits of the oxygenase component, a ferredoxin, and a ferredoxin reductase, respectively, of phenanthrene dioxygenase were identified. The gene cluster, phdAB, was located 8.3 kb downstream of the previously characterized phdK gene, which encodes 2-carboxybenzaldehyde dehydrogenase. The phdCD gene cluster was located 2.9 kb downstream of the phdB gene. PhdA and PhdB exhibited moderate (less than 60%) sequence identity to the α and β subunits of other ring-hydroxylating dioxygenases. The PhdC sequence showed features of a [3Fe-4S] or [4Fe-4S] type of ferredoxin, not of the [2Fe-2S] type of ferredoxin that has been found in most of the reported ring-hydroxylating dioxygenases. PhdD also showed moderate (less than 40%) sequence identity to known reductases. The phdABCD genes were expressed poorly in Escherichia coli, even when placed under the control of strong promoters. The introduction of a Shine-Dalgarno sequence upstream of each initiation codon of the phdABCD genes improved their expression in E. coli. E. coli cells carrying phdBCD or phdACD exhibited no phenanthrene-degrading activity, and those carrying phdABD or phdABC exhibited phenanthrene-degrading activity which was significantly less than that in cells carrying the phdABCD genes. It was thus concluded that all of the phdABCD genes are necessary for the efficient expression of phenanthrene-degrading activity. The genetic organization of the phd genes, the phylogenetically diverged positions of these genes, and an unusual type of ferredoxin component suggest phenanthrene dioxygenase in Nocardioides sp. strain KP7 to be a new class of aromatic ring-hydroxylating dioxygenases. PMID:10735855

Decreased detoxification genes and genome size make the human body louse an efficient model to study xenobiotic metabolism

PubMed Central

Lee, Si Hyeock; Kang, Jae Soon; Min, Jee Sun; Yoon, Kyong Sup; Strycharz, Joseph P.; Johnson, Reed; Mittapalli, Omprakash; Margam, Venu M.; Sun, Weilin; Li, Hong-Mei; Xie, Jun; Wu, Jing; Kirkness, Ewen F.; Berenbaum, May R.; Pittendrigh, Barry R.; Clark, J. Marshall

2010-01-01

The human body louse, Pediculus humanus humanus, has one of the smallest insect genomes, containing ~10,775 annotated genes (Kirkness et al. 2010). Annotation of detoxification [cytochrome P450 monooxygenase (P450), glutathione-S-transferase (GST), esterase (Est), and ATP-binding cassette transporter (ABC transporter)] genes revealed that they are dramatically reduced in P. h. humanus compared to other insects except for Apis mellifera. There are 37 P450, 13 GST and 17 Est genes present in P. h. humanus, approximately half of that found in Drosophila melanogaster and Anopheles gambiae. The number of putatively functional ABC transporter genes in P. h. humanus and A. mellifera are the same (36) but both have fewer than An. gambiae (44) or D. melanogaster (65). The reduction of detoxification genes in P. h. humanus may be due to their simple life history, where they do not encounter a wide variety of xenobiotics. Neuronal component genes are highly conserved across different insect species as expected due to their critical function. Although reduced in number, P. h. humanus still retains at least a minimum repertoire of genes known to confer metabolic or toxicokinetic resistance to xenobiotics (e.g., Cyp3 clade P450s, Delta GSTs, B clade Ests and B/C subfamily ABC transporters), suggestive of its high potential for resistance development. PMID:20561088

Likelihood analysis of the chalcone synthase genes suggests the role of positive selection in morning glories (Ipomoea).

PubMed

Yang, Ji; Gu, Hongya; Yang, Ziheng

2004-01-01

Chalcone synthase (CHS) is a key enzyme in the biosynthesis of flavonoides, which are important for the pigmentation of flowers and act as attractants to pollinators. Genes encoding CHS constitute a multigene family in which the copy number varies among plant species and functional divergence appears to have occurred repeatedly. In morning glories (Ipomoea), five functional CHS genes (A-E) have been described. Phylogenetic analysis of the Ipomoea CHS gene family revealed that CHS A, B, and C experienced accelerated rates of amino acid substitution relative to CHS D and E. To examine whether the CHS genes of the morning glories underwent adaptive evolution, maximum-likelihood models of codon substitution were used to analyze the functional sequences in the Ipomoea CHS gene family. These models used the nonsynonymous/synonymous rate ratio (omega = d(N)/ d(S)) as an indicator of selective pressure and allowed the ratio to vary among lineages or sites. Likelihood ratio test suggested significant variation in selection pressure among amino acid sites, with a small proportion of them detected to be under positive selection along the branches ancestral to CHS A, B, and C. Positive Darwinian selection appears to have promoted the divergence of subfamily ABC and subfamily DE and is at least partially responsible for a rate increase following gene duplication.

A Silent ABC Transporter Isolated from Streptomyces rochei F20 Induces Multidrug Resistance

PubMed Central

Fernández-Moreno, Miguel A.; Carbó, Lázaro; Cuesta, Trinidad; Vallín, Carlos; Malpartida, Francisco

1998-01-01

In the search for heterologous activators for actinorhodin production in Streptomyces lividans, 3.4 kb of DNA from Streptomyces rochei F20 (a streptothricin producer) were characterized. Subcloning experiments showed that the minimal DNA fragment required for activation was 0.4 kb in size. The activation is mediated by increasing the levels of transcription of the actII-ORF4 gene. Sequencing of the minimal activating fragment did not reveal any clues about its mechanism; nevertheless, it was shown to overlap the 3′ end of two convergent genes, one of whose translated products (ORF2) strongly resembles that of other genes belonging to the ABC transporter superfamily. Computer-assisted analysis of the 3.4-kb DNA sequence showed the 3′ terminus of an open reading frame (ORF), i.e., ORFA, and three complete ORFs (ORF1, ORF2, and ORFB). Searches in the databases with their respective gene products revealed similarities for ORF1 and ORF2 with ATP-binding proteins and transmembrane proteins, respectively, which are found in members of the ABC transporter superfamily. No similarities for ORFA and ORFB were found in the databases. Insertional inactivation of ORF1 and ORF2, their transcription analysis, and their cloning in heterologous hosts suggested that these genes were not expressed under our experimental conditions; however, cloning of ORF1 and ORF2 together (but not separately) under the control of an expressing promoter induced resistance to several chemically different drugs: oleandomycin, erythromycin, spiramycin, doxorubicin, and tetracycline. Thus, this genetic system, named msr, is a new bacterial multidrug ABC transporter. PMID:9696745

Aldouronate Utilization in Paenibacillus sp. Strain JDR-2: Physiological and Enzymatic Evidence for Coupling of Extracellular Depolymerization and Intracellular Metabolism ▿

PubMed Central

Nong, Guang; Rice, John D.; Chow, Virginia; Preston, James F.

2009-01-01

Paenibacillus sp. strain JDR-2, an aggressively xylanolytic bacterium isolated from decaying sweet gum wood, secretes a multimodular glycohydrolase family GH10 endoxylanase (XynA1) anchored to the cell surface. The gene encoding XynA1 is part of a xylan utilization regulon that includes an aldouronate utilization gene cluster with genes encoding a GH67 α-glucuronidase (AguA), a GH10 endoxylanase (XynA2), and a GH43 arabinofuranosidase/β-xylosidase (XynB). Here we show that this Paenibacillus sp. strain is able to utilize methylglucuronoxylose (MeGAX1), an aldobiuronate product that accumulates during acid pretreatment of lignocellulosic biomass, and methylglucuronoxylotriose (MeGAX3), the product of the extracellular XynA1 acting on methylglucuronoxylan (MeGAXn). The average rates of utilization of MeGAXn, MeGAX1, and MeGAX3 were 149.8, 59.4, and 54.3 μg xylose equivalents·ml−1·h−1, respectively, and were proportional to the specific growth rates on the substrates. AguA was active with MeGAX1 and MeGAX3, releasing 4-O-methyl-d-glucuronate α-1,2 linked to a nonreducing terminal xylose residue. XynA2 converted xylotriose, generated by the action of AguA on MeGAX3, to xylose and xylobiose. The ability to utilize MeGAX1 provides a novel metabolic potential for bioconversion of acid hydrolysates of lignocellulosics. The 2.8-fold-greater rate of utilization of polymeric MeGAXn than that of MeGAX3 indicates that there is coupling of extracellular depolymerization, assimilation, and intracellular metabolism, allowing utilization of lignocellulosics with minimal pretreatment. Along with adjacent genes encoding transcriptional regulators and ABC transporter proteins, the aguA and xynA2 genes in the cluster described above contribute to the efficient utilization of aldouronates derived from dilute acid and/or enzyme pretreatment protocols applied to the conversion of hemicellulose to biofuels and chemicals. PMID:19395566

Environmental Conditions Influence Induction of Key ABC-Transporter Genes Affecting Glyphosate Resistance Mechanism in Conyza canadensis.

PubMed

Tani, Eleni; Chachalis, Demosthenis; Travlos, Ilias S; Bilalis, Dimitrios

2016-04-20

Conyza canadensis has been reported to be the most frequent weed species that evolved resistance to glyphosate in various parts of the world. The objective of the present study was to investigate the effect of environmental conditions (temperature and light) on the expression levels of the EPSPS gene and two major ABC-transporter genes (M10 and M11) on glyphosate susceptible (GS) and glyphosate resistant (GR) horseweed populations, collected from several regions across Greece. Real-time PCR was conducted to determine the expression level of the aforementioned genes when glyphosate was applied at normal (1×; 533 g·a.e.·ha(-1)) and high rates (4×, 8×), measured at an early one day after treatment (DAT) and a later stage (four DAT) of expression. Plants were exposed to light or dark conditions, at three temperature regimes (8, 25, 35 °C). GR plants were made sensitive when exposed to 8 °C with light; those sensitized plants behaved biochemically (shikimate accumulation) and molecularly (expression of EPSPS and ABC-genes) like the GS plants. Results from the current study show the direct link between the environmental conditions and the induction level of the above key genes that likely affect the efficiency of the proposed mechanism of glyphosate resistance.

Environmental Conditions Influence Induction of Key ABC-Transporter Genes Affecting Glyphosate Resistance Mechanism in Conyza canadensis

PubMed Central

Tani, Eleni; Chachalis, Demosthenis; Travlos, Ilias S.; Bilalis, Dimitrios

2016-01-01

Conyza canadensis has been reported to be the most frequent weed species that evolved resistance to glyphosate in various parts of the world. The objective of the present study was to investigate the effect of environmental conditions (temperature and light) on the expression levels of the EPSPS gene and two major ABC-transporter genes (M10 and M11) on glyphosate susceptible (GS) and glyphosate resistant (GR) horseweed populations, collected from several regions across Greece. Real-time PCR was conducted to determine the expression level of the aforementioned genes when glyphosate was applied at normal (1×; 533 g·a.e.·ha−1) and high rates (4×, 8×), measured at an early one day after treatment (DAT) and a later stage (four DAT) of expression. Plants were exposed to light or dark conditions, at three temperature regimes (8, 25, 35 °C). GR plants were made sensitive when exposed to 8 °C with light; those sensitized plants behaved biochemically (shikimate accumulation) and molecularly (expression of EPSPS and ABC-genes) like the GS plants. Results from the current study show the direct link between the environmental conditions and the induction level of the above key genes that likely affect the efficiency of the proposed mechanism of glyphosate resistance. PMID:27104532

Bfr1p is responsible for tributyltin resistance in Schizosaccharomyces pombe.

PubMed

Akiyama, Koichi; Iwaki, Tomoko; Sugimoto, Naoko; Chardwiriyapreecha, Soracom; Kawano, Miyuki; Nishimoto, Sogo; Sugahara, Takuya; Sekito, Takayuki; Kakinuma, Yoshimi

2011-01-01

ATP-binding cassette (ABC) transporter plays an important role for resistance against xenobiotics. There are eleven ABC transporter genes in the genome of fission yeast Schizosaccharomyces pombe. We examined the role of ABC transporter against the toxicity of tributyltin chloride (TBT), a widespread environmental pollutant, in cell growth. Among individual ABC transporter mutants, the growth of a mutant deficient in Bfr1p, a plasma membrane-embedded transporter, was extremely sensitive to TBT. The lethal TBT concentration inducing 50% of cell death (LC(50)) was 25 µM for the parent strain and 10.2 µM for the bfr1∆ mutant. Thus, Bfr1p was responsible for TBT resistance in S. pombe.

The Reverse Transcription Inhibitor Abacavir Shows Anticancer Activity in Prostate Cancer Cell Lines

PubMed Central

Molinari, Agnese; Parisi, Chiara; Bozzuto, Giuseppina; Toccacieli, Laura; Formisano, Giuseppe; De Orsi, Daniela; Paradisi, Silvia; Grober, OlÌ Maria Victoria; Ravo, Maria; Weisz, Alessandro; Arcieri, Romano; Vella, Stefano; Gaudi, Simona

2010-01-01

Background Transposable Elements (TEs) comprise nearly 45% of the entire genome and are part of sophisticated regulatory network systems that control developmental processes in normal and pathological conditions. The retroviral/retrotransposon gene machinery consists mainly of Long Interspersed Nuclear Elements (LINEs-1) and Human Endogenous Retroviruses (HERVs) that code for their own endogenous reverse transcriptase (RT). Interestingly, RT is typically expressed at high levels in cancer cells. Recent studies report that RT inhibition by non-nucleoside reverse transcriptase inhibitors (NNRTIs) induces growth arrest and cell differentiation in vitro and antagonizes growth of human tumors in animal model. In the present study we analyze the anticancer activity of Abacavir (ABC), a nucleoside reverse transcription inhibitor (NRTI), on PC3 and LNCaP prostate cancer cell lines. Principal Findings ABC significantly reduces cell growth, migration and invasion processes, considerably slows S phase progression, induces senescence and cell death in prostate cancer cells. Consistent with these observations, microarray analysis on PC3 cells shows that ABC induces specific and dose-dependent changes in gene expression, involving multiple cellular pathways. Notably, by quantitative Real-Time PCR we found that LINE-1 ORF1 and ORF2 mRNA levels were significantly up-regulated by ABC treatment. Conclusions Our results demonstrate the potential of ABC as anticancer agent able to induce antiproliferative activity and trigger senescence in prostate cancer cells. Noteworthy, we show that ABC elicits up-regulation of LINE-1 expression, suggesting the involvement of these elements in the observed cellular modifications. PMID:21151977

The FlxABCD-HdrABC proteins correspond to a novel NADH dehydrogenase/heterodisulfide reductase widespread in anaerobic bacteria and involved in ethanol metabolism in Desulfovibrio vulgaris Hildenborough.

PubMed

Ramos, Ana Raquel; Grein, Fabian; Oliveira, Gonçalo P; Venceslau, Sofia S; Keller, Kimberly L; Wall, Judy D; Pereira, Inês A C

2015-07-01

Flavin-based electron bifurcation (FBEB) is an important mechanism for the energy metabolism of anaerobes. A new family of NADH dehydrogenases, the flavin oxidoreductase (FlxABCD, previously called FloxABCD), was proposed to perform FBEB in sulphate-reducing organisms coupled with heterodisulfide reductase (HdrABC). We found that the hdrABC-flxABCD gene cluster is widespread among anaerobic bacteria, pointing to a general and important role in their bioenergetics. In this work, we studied FlxABCD of Desulfovibrio vulgaris Hildenborough. The hdr-flx genes are part of the same transcriptional unit and are increased in transcription during growth in ethanol-sulfate, and to a less extent during pyruvate fermentation. Two mutant strains were generated: one where expression of the hdr-flx genes was interrupted and another lacking the flxA gene. Both strains were unable to grow with ethanol-sulfate, whereas growth was restored in a flxA-complemented strain. The mutant strains also produced very reduced amounts of ethanol compared with the wild type during pyruvate fermentation. Our results show that in D. vulgaris, the FlxABCD-HdrABC proteins are essential for NADH oxidation during growth on ethanol, probably involving a FBEB mechanism that leads to reduction of ferredoxin and the small protein DsrC, while in fermentation they operate in reverse, reducing NAD(+) for ethanol production. © 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.

Rooting and Other Characteristics of a Transgenic Walnut Hybrid (Juglans hindsii x J. regia) Rootstock Expressing rolABC

Treesearch

Kourosh Vahdati; James R. McKenna; Abhaya M. Dandekar; Charles A. Leslie; Sandie L. Uratsu; Wesley P. Hackett; Paola Negri; Gale H. McGranahan

2002-01-01

Walnuts (Juglans spp.) are difficult-to-root woody plants. The rolABC genes (rolA + rolB + rolC), derived from the bacteria Agrobacterium rhizogenes, have been shown to increase the rooting potential of other difficult-to-root woody plants. We inserted the...

[Biological characteristics of an enteroinvasive Escherichia coli strain with tatABC deletion].

PubMed

Gong, Zhaolong; Ye, Changyun; Liu, Xiaobing; Zhang, Min; Zhuo, Qin

2013-05-04

To study the relationship between twin-arginine translocation system (Tat) system with the biological characteristics of enteroinvasive Escherichia coli (EIEC). Through homologous recombination, we constructed EIEC's tatABC gene deletion strain and complementary strain, and explored their impact on bacterial form, substrate transport function as well as on HeLa cells and guinea pig's corneal invasion force. The tatABC gene deletion strain had apparent changes in bacterial form, loss of substrate transporter function, and significant weakened bacterial invasion force (the number of the deletion strain invading into HeLa cells was decreased significantly, and the ability of its corneal lesion capacity of the guinea pig was significantly weakened), while the complementary strain was similar to the wild strain in the above respects. EIEC's Tat protein transport system is closely related with the biological characteristics of EIEC.

Genomic Evidence for Island Population Conversion Resolves Conflicting Theories of Polar Bear Evolution

PubMed Central

Cahill, James A.; Green, Richard E.; Fulton, Tara L.; Stiller, Mathias; Jay, Flora; Ovsyanikov, Nikita; Salamzade, Rauf; St. John, John; Stirling, Ian; Slatkin, Montgomery; Shapiro, Beth

2013-01-01

Despite extensive genetic analysis, the evolutionary relationship between polar bears (Ursus maritimus) and brown bears (U. arctos) remains unclear. The two most recent comprehensive reports indicate a recent divergence with little subsequent admixture or a much more ancient divergence followed by extensive admixture. At the center of this controversy are the Alaskan ABC Islands brown bears that show evidence of shared ancestry with polar bears. We present an analysis of genome-wide sequence data for seven polar bears, one ABC Islands brown bear, one mainland Alaskan brown bear, and a black bear (U. americanus), plus recently published datasets from other bears. Surprisingly, we find clear evidence for gene flow from polar bears into ABC Islands brown bears but no evidence of gene flow from brown bears into polar bears. Importantly, while polar bears contributed <1% of the autosomal genome of the ABC Islands brown bear, they contributed 6.5% of the X chromosome. The magnitude of sex-biased polar bear ancestry and the clear direction of gene flow suggest a model wherein the enigmatic ABC Island brown bears are the descendants of a polar bear population that was gradually converted into brown bears via male-dominated brown bear admixture. We present a model that reconciles heretofore conflicting genetic observations. We posit that the enigmatic ABC Islands brown bears derive from a population of polar bears likely stranded by the receding ice at the end of the last glacial period. Since then, male brown bear migration onto the island has gradually converted these bears into an admixed population whose phenotype and genotype are principally brown bear, except at mtDNA and X-linked loci. This process of genome erosion and conversion may be a common outcome when climate change or other forces cause a population to become isolated and then overrun by species with which it can hybridize. PMID:23516372

Genomic evidence for island population conversion resolves conflicting theories of polar bear evolution.

PubMed

Cahill, James A; Green, Richard E; Fulton, Tara L; Stiller, Mathias; Jay, Flora; Ovsyanikov, Nikita; Salamzade, Rauf; St John, John; Stirling, Ian; Slatkin, Montgomery; Shapiro, Beth

2013-01-01

Despite extensive genetic analysis, the evolutionary relationship between polar bears (Ursus maritimus) and brown bears (U. arctos) remains unclear. The two most recent comprehensive reports indicate a recent divergence with little subsequent admixture or a much more ancient divergence followed by extensive admixture. At the center of this controversy are the Alaskan ABC Islands brown bears that show evidence of shared ancestry with polar bears. We present an analysis of genome-wide sequence data for seven polar bears, one ABC Islands brown bear, one mainland Alaskan brown bear, and a black bear (U. americanus), plus recently published datasets from other bears. Surprisingly, we find clear evidence for gene flow from polar bears into ABC Islands brown bears but no evidence of gene flow from brown bears into polar bears. Importantly, while polar bears contributed <1% of the autosomal genome of the ABC Islands brown bear, they contributed 6.5% of the X chromosome. The magnitude of sex-biased polar bear ancestry and the clear direction of gene flow suggest a model wherein the enigmatic ABC Island brown bears are the descendants of a polar bear population that was gradually converted into brown bears via male-dominated brown bear admixture. We present a model that reconciles heretofore conflicting genetic observations. We posit that the enigmatic ABC Islands brown bears derive from a population of polar bears likely stranded by the receding ice at the end of the last glacial period. Since then, male brown bear migration onto the island has gradually converted these bears into an admixed population whose phenotype and genotype are principally brown bear, except at mtDNA and X-linked loci. This process of genome erosion and conversion may be a common outcome when climate change or other forces cause a population to become isolated and then overrun by species with which it can hybridize.

In-vivo-induced antigenic determinants of Fusobacterium nucleatum subsp. nucleatum.

PubMed

Lee, H-R; Rhyu, I-C; Kim, H-D; Jun, H-K; Min, B-M; Lee, S-H; Choi, B-K

2011-04-01

Fusobacterium nucleatum plays a pivotal role in dental plaque biofilm formation and is known to be involved in chronic inflammatory systemic disease. However, limited knowledge of F. nucleatum genes expressed in vivo interferes with our understanding of pathogenesis. In this study, we identified F. nucleatum genes induced in vivo using in-vivo-induced antigen technology (IVIAT). Among 30,000 recombinant clones screened, 87 reacted reproducibly with pooled sera from 10 patients with periodontitis. The clones encoded for 32 different proteins, of which 28 could be assigned to their functions, which were categorized in translation, transcription, transport, energy metabolism, cell envelope, cellular process, fatty acid and phospholipid metabolism, transposition, cofactor biosynthesis, amino acid biosynthesis, and DNA replication. Putative virulence factors detected were ABC transporter, butyrate-acetoacetate CoA-transferase, hemin receptor, hemolysin, hemolysin-related protein, LysR family transcriptional regulator, serine protease, and transposase. Analysis of immune responses to the in-vivo-induced (ivi) antigens in five patients demonstrated that most were reactive to these proteins, confirming results with pooled sera. IVIAT-identified F. nucleatum genes in this study may accelerate the elucidation of F. nucleatum-mediated molecular pathogenesis. © 2011 John Wiley & Sons A/S.

Midgut transcriptome response to a Cry toxin in the diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae).

PubMed

Lei, Yanyuan; Zhu, Xun; Xie, Wen; Wu, Qingjun; Wang, Shaoli; Guo, Zhaojiang; Xu, Baoyun; Li, Xianchun; Zhou, Xuguo; Zhang, Youjun

2014-01-01

To investigate the response of Plutella xylostella transcriptome in defending against a Bt toxin, high-throughput RNA-sequencing was carried out to examine Cry1Ac-susceptible and -resistant strains. The comparative analysis indentified over 2900 differentially expressed unigenes (DEUs) between these two strains. Gene Ontology analysis placed these unigenes primarily into cell, cell part, organelle, binding, catalytic, cellular process, metabolic process, and response to stimulus categories. Based on pathway analyses, DEUs were enriched in oxidoreductase activity and membrane lipid metabolic processes, and they were also significantly enriched in pathways related to the metabolic and biosynthesis of secondary metabolites. Most of the unigenes involved in the metabolic pathway were up-regulated in resistant strains. Within the ABC transporter pathway, majority of the down-regulated unigenes belong to ABCC2 and ABCC10, respectively, while up-regulated unigenes were mainly categorized as ABCG2. Furthermore, two aminopeptidases, and four cadherins encoding genes were significantly elevated as well. This study provides a transcriptome foundation for the identification and functional characterization of genes involved in the Bt resistance in an agriculturally important insect pest, P. xylostella. © 2013 Elsevier B.V. All rights reserved.

Deciphering the Routes of invasion of Drosophila suzukii by Means of ABC Random Forest.

PubMed

Fraimout, Antoine; Debat, Vincent; Fellous, Simon; Hufbauer, Ruth A; Foucaud, Julien; Pudlo, Pierre; Marin, Jean-Michel; Price, Donald K; Cattel, Julien; Chen, Xiao; Deprá, Marindia; François Duyck, Pierre; Guedot, Christelle; Kenis, Marc; Kimura, Masahito T; Loeb, Gregory; Loiseau, Anne; Martinez-Sañudo, Isabel; Pascual, Marta; Polihronakis Richmond, Maxi; Shearer, Peter; Singh, Nadia; Tamura, Koichiro; Xuéreb, Anne; Zhang, Jinping; Estoup, Arnaud

2017-04-01

Deciphering invasion routes from molecular data is crucial to understanding biological invasions, including identifying bottlenecks in population size and admixture among distinct populations. Here, we unravel the invasion routes of the invasive pest Drosophila suzukii using a multi-locus microsatellite dataset (25 loci on 23 worldwide sampling locations). To do this, we use approximate Bayesian computation (ABC), which has improved the reconstruction of invasion routes, but can be computationally expensive. We use our study to illustrate the use of a new, more efficient, ABC method, ABC random forest (ABC-RF) and compare it to a standard ABC method (ABC-LDA). We find that Japan emerges as the most probable source of the earliest recorded invasion into Hawaii. Southeast China and Hawaii together are the most probable sources of populations in western North America, which then in turn served as sources for those in eastern North America. European populations are genetically more homogeneous than North American populations, and their most probable source is northeast China, with evidence of limited gene flow from the eastern US as well. All introduced populations passed through bottlenecks, and analyses reveal five distinct admixture events. These findings can inform hypotheses concerning how this species evolved between different and independent source and invasive populations. Methodological comparisons indicate that ABC-RF and ABC-LDA show concordant results if ABC-LDA is based on a large number of simulated datasets but that ABC-RF out-performs ABC-LDA when using a comparable and more manageable number of simulated datasets, especially when analyzing complex introduction scenarios. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Decreased detoxification genes and genome size make the human body louse an efficient model to study xenobiotic metabolism.

PubMed

Lee, S H; Kang, J S; Min, J S; Yoon, K S; Strycharz, J P; Johnson, R; Mittapalli, O; Margam, V M; Sun, W; Li, H-M; Xie, J; Wu, J; Kirkness, E F; Berenbaum, M R; Pittendrigh, B R; Clark, J M

2010-10-01

The human body louse, Pediculus humanus humanus, has one of the smallest insect genomes, containing ∼10 775 annotated genes. Annotation of detoxification [cytochrome P450 monooxygenase (P450), glutathione-S-transferase (GST), esterase (Est) and ATP-binding cassette transporter (ABC transporter)] genes revealed that they are dramatically reduced in P. h. humanus compared to other insects except for Apis mellifera. There are 37 P450, 13 GST and 17 Est genes present in P. h. humanus, approximately half the number found in Drosophila melanogaster and Anopheles gambiae. The number of putatively functional ABC transporter genes in P. h. humanus and Ap. mellifera are the same (36) but both have fewer than An. gambiae (44) or Dr. melanogaster (65). The reduction of detoxification genes in P. h. humanus may be a result of this louse's simple life history, in which it does not encounter a wide variety of xenobiotics. Neuronal component genes are highly conserved across different insect species as expected because of their critical function. Although reduced in number, P. h. humanus still retains at least a minimum repertoire of genes known to confer metabolic or toxicokinetic resistance to xenobiotics (eg Cyp3 clade P450s, Delta GSTs, B clade Ests and B/C subfamily ABC transporters), suggestive of its high potential for resistance development. © 2010 The Authors. Insect Molecular Biology © 2010 The Royal Entomological Society.

Transcriptomic analysis of Saccharomyces cerevisiae upon honokiol treatment.

PubMed

Zhu, Xiaolong; Zou, Shenshen; Li, Youbin; Liang, Yongheng

2017-09-01

Honokiol (HNK), one of the main medicinal components in Magnolia officinalis, possesses antimicrobial activity against a variety of pathogenic bacteria and fungi. However, little is known of the molecular mechanisms underpinning the antimicrobial activity. To explore the molecular mechanism of its antifungal activity, we determined the effects of HNK on the mRNA expression profile of Saccharomyces cerevisiae using a DNA microarray approach. HNK markedly induced the expression of genes related to iron uptake and homeostasis. Conversely, genes associated with respiratory electron transport were downregulated, mirroring the effects of iron starvation. Meanwhile, HNK-induced growth deficiency was partly rescued by iron supplementation and HNK reacted with iron, producing iron complexes that depleted iron. These results suggest that HNK treatment induced iron starvation. Additionally, HNK treatment resulted in the upregulation of genes involved in protein synthesis and drug resistance networks. Furthermore, the deletion of PDR5, a gene encoding the plasma membrane ATP binding cassette (ABC) transporter, conferred sensitivity to HNK. Overexpression of PDR5 enhanced resistance of WT and pdr5Δ strains to HNK. Taken together, these findings suggest that HNK, which can be excluded by overexpression of Pdr5, functions in multiple cellular processes in S. cerevisiae, particularly in inducing iron starvation to inhibit cell growth. Copyright © 2017 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

γ-Lindane Increases Microcystin Synthesis in Microcystis aeruginosa PCC7806

PubMed Central

Ceballos-Laita, Laura; Calvo-Begueria, Laura; Lahoz, Jessica; Bes, María-Teresa; Fillat, María F.; Peleato, María-Luisa

2015-01-01

HCH factories, and the waste dumpsites associated to its production, have become a global environmental concern, and their runoff could pollute ground and surface waters with high levels of the pollutant. In this study, the influence of lindane (γ-HCH) on microcystin production has been investigated in Microcystis aeruginosa PCC7806. This toxic cyanobacterium is highly tolerant to γ-lindane (20 mg/L), and produces more toxin (microcystin) in the presence of the pollutant. Microcystis degrades γ-lindane and presence of γ-lindane induces genes involved in its own degradation (nirA). RT-PCRsq has been used to monitor changes in levels of transcripts encoded by the mcy operon (mcyD, mcyH and mcyJ), responsible for the microcystin synthesis machinery, as well as other genes involved in its transcriptional regulation, such as ntcA and fur family members. The presence of lindane in the culture media induces mcyD expression, as well as ntcA gene transcription, while other genes, such as mcyH, (putative ABC transporter), are downregulated. The amount of microcystin found in the cells and the culture media is higher when M. aeruginosa is treated with γ-lindane than in control cells. The results suggest that in a lindane polluted environment, Microcystis toxic strains may enhance their microcystin synthesis. PMID:26404326

OsAGSW1, an ABC1-like kinase gene, is involved in the regulation of grain size and weight in rice.

PubMed

Li, Tao; Jiang, Jieming; Zhang, Shengchun; Shu, Haoran; Wang, Yaqin; Lai, Jianbin; Du, Jinju; Yang, Chengwei

2015-09-01

Grain shape and weight are two determining agronomic traits of rice yield. ABC1 (Activity of bc1 complex) is a newly found atypical kinase in plants. Here, we report on an ABC1 protein kinase gene, OsAGSW1 (ABC1-like kinase related to Grain size and Weight). Expression of OsAGSW1-GFP in rice revealed that OsAGSW1 is localized to the chloroplasts in rice. Analysis of OsAGSW1 promoter::β-glucuronidase transgenic rice indicated that this gene was highly expressed in vascular bundles in shoot, hull and caryopsis. Furthermore, OsAGSW1-RNAi and overexpressed transgenic rice lines were generated. Stable transgenic lines overexpressing OsAGSW1 exhibited a phenotype with a significant increase in grain size, grain weight, grain filling rate and 1000-grain weight compared with the wild-type and RNAi transgenic plants. Microscopy analysis showed that spikelet hulls just before heading were different in the OsAGSW1-overexpressed plants compared with wild-type and OsAGSW1 RNAi rice. Further cytological analysis showed that the number of external parenchyma cells in rice hulls of OsAGSW1-overexpressed plants increased, leading to wider and longer spikelet hulls than those of the wild-type and OsAGSW1-RNAi plants. The vascular cross-sectional area in lemma, carpopodium and ovules also strikingly increased and area of both xylem and phloem were enlarged in the OsAGSW1-overexpressed plants. Thus, our results demonstrated that OsAGSW1 plays an important role in seed shape and size of rice by regulating the number of external parenchyma cells and the development of vascular bundles, providing a new insight into the functions of ABC1 genes in plants. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

RCN1/OsABCG5, an ATP-binding cassette (ABC) transporter, is required for hypodermal suberization of roots in rice (Oryza sativa).

PubMed

Shiono, Katsuhiro; Ando, Miho; Nishiuchi, Shunsaku; Takahashi, Hirokazu; Watanabe, Kohtaro; Nakamura, Motoaki; Matsuo, Yuichi; Yasuno, Naoko; Yamanouchi, Utako; Fujimoto, Masaru; Takanashi, Hideki; Ranathunge, Kosala; Franke, Rochus B; Shitan, Nobukazu; Nishizawa, Naoko K; Takamure, Itsuro; Yano, Masahiro; Tsutsumi, Nobuhiro; Schreiber, Lukas; Yazaki, Kazufumi; Nakazono, Mikio; Kato, Kiyoaki

2014-10-01

Suberin is a complex polymer composed of aliphatic and phenolic compounds. It is a constituent of apoplastic plant interfaces. In many plant species, including rice (Oryza sativa), the hypodermis in the outer part of roots forms a suberized cell wall (the Casparian strip and/or suberin lamellae), which inhibits the flow of water and ions and protects against pathogens. To date, there is no genetic evidence that suberin forms an apoplastic transport barrier in the hypodermis. We discovered that a rice reduced culm number1 (rcn1) mutant could not develop roots longer than 100 mm in waterlogged soil. The mutated gene encoded an ATP-binding cassette (ABC) transporter named RCN1/OsABCG5. RCN1/OsABCG5 gene expression in the wild type was increased in most hypodermal and some endodermal roots cells under stagnant deoxygenated conditions. A GFP-RCN1/OsABCG5 fusion protein localized at the plasma membrane of the wild type. Under stagnant deoxygenated conditions, well suberized hypodermis developed in wild types but not in rcn1 mutants. Under stagnant deoxygenated conditions, apoplastic tracers (periodic acid and berberine) were blocked at the hypodermis in the wild type but not in rcn1, indicating that the apoplastic barrier in the mutant was impaired. The amount of the major aliphatic suberin monomers originating from C(28) and C(30) fatty acids or ω-OH fatty acids was much lower in rcn1 than in the wild type. These findings suggest that RCN1/OsABCG5 has a role in the suberization of the hypodermis of rice roots, which contributes to formation of the apoplastic barrier. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.

Comparison of transcriptional profiles of Clostridium thermocellum grown on cellobiose and pretreated yellow poplar using RNA-Seq

PubMed Central

Wei, Hui; Fu, Yan; Magnusson, Lauren; Baker, John O.; Maness, Pin-Ching; Xu, Qi; Yang, Shihui; Bowersox, Andrew; Bogorad, Igor; Wang, Wei; Tucker, Melvin P.; Himmel, Michael E.; Ding, Shi-You

2014-01-01

The anaerobic, thermophilic bacterium, Clostridium thermocellum, secretes multi-protein enzyme complexes, termed cellulosomes, which synergistically interact with the microbial cell surface and efficiently disassemble plant cell wall biomass. C. thermocellum has also been considered a potential consolidated bioprocessing (CBP) organism due to its ability to produce the biofuel products, hydrogen, and ethanol. We found that C. thermocellum fermentation of pretreated yellow poplar (PYP) produced 30 and 39% of ethanol and hydrogen product concentrations, respectively, compared to fermentation of cellobiose. RNA-seq was used to analyze the transcriptional profiles of these cells. The PYP-grown cells taken for analysis at the late stationary phase showed 1211 genes up-regulated and 314 down-regulated by more than two-fold compared to the cellobiose-grown cells. These affected genes cover a broad spectrum of specific functional categories. The transcriptional analysis was further validated by sub-proteomics data taken from the literature; as well as by quantitative reverse transcription-PCR (qRT-PCR) analyses of selected genes. Specifically, 47 cellulosomal protein-encoding genes, genes for 4 pairs of SigI-RsgI for polysaccharide sensing, 7 cellodextrin ABC transporter genes, and a set of NAD(P)H hydogenase and alcohol dehydrogenase genes were up-regulated for cells growing on PYP compared to cellobiose. These genes could be potential candidates for future studies aimed at gaining insight into the regulatory mechanism of this organism as well as for improvement of C. thermocellum in its role as a CBP organism. PMID:24782837

LrABCF1, a GCN-type ATP-binding cassette transporter from lilium regale, is involved in defense responses against viral and fungal pathogens

USDA-ARS?s Scientific Manuscript database

ATP-binding cassette (ABC) transporters are essential for membrane translocation in diverse biological processes, such as plant development and defense response. Here, a general control non-derepressible (GCN)-type ABC transporter gene, designated LrABCF1, was identified from Cucumber mosaic virus (...

The genome of Syntrophorhabdus aromaticivorans strain UI provides new insights for syntrophic aromatic compound metabolism and electron flow.

PubMed

Nobu, Masaru K; Narihiro, Takashi; Hideyuki, Tamaki; Qiu, Yan-Ling; Sekiguchi, Yuji; Woyke, Tanja; Goodwin, Lynne; Davenport, Karen W; Kamagata, Yoichi; Liu, Wen-Tso

2015-12-01

How aromatic compounds are degraded in various anaerobic ecosystems (e.g. groundwater, sediments, soils and wastewater) is currently poorly understood. Under methanogenic conditions (i.e. groundwater and wastewater treatment), syntrophic metabolizers are known to play an important role. This study explored the draft genome of Syntrophorhabdus aromaticivorans strain UI and identified the first syntrophic phenol-degrading phenylphosphate synthase (PpsAB) and phenylphosphate carboxylase (PpcABCD) and syntrophic terephthalate-degrading decarboxylase complexes. The strain UI genome also encodes benzoate degradation through hydration of the dienoyl-coenzyme A intermediate as observed in Geobacter metallireducens and Syntrophus aciditrophicus. Strain UI possesses electron transfer flavoproteins, hydrogenases and formate dehydrogenases essential for syntrophic metabolism. However, the biochemical mechanisms for electron transport between these H2 /formate-generating proteins and syntrophic substrate degradation remain unknown for many syntrophic metabolizers, including strain UI. Analysis of the strain UI genome revealed that heterodisulfide reductases (HdrABC), which are poorly understood electron transfer genes, may contribute to syntrophic H2 and formate generation. The genome analysis further identified a putative ion-translocating ferredoxin : NADH oxidoreductase (IfoAB) that may interact with HdrABC and dissimilatory sulfite reductase gamma subunit (DsrC) to perform novel electron transfer mechanisms associated with syntrophic metabolism. © 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.

A class of tricyclic compounds blocking malaria parasite oocyst development and transmission.

PubMed

Eastman, Richard T; Pattaradilokrat, Sittiporn; Raj, Dipak K; Dixit, Saurabh; Deng, Bingbing; Miura, Kazutoyo; Yuan, Jing; Tanaka, Takeshi Q; Johnson, Ronald L; Jiang, Hongying; Huang, Ruili; Williamson, Kim C; Lambert, Lynn E; Long, Carole; Austin, Christopher P; Wu, Yimin; Su, Xin-Zhuan

2013-01-01

Malaria is a deadly infectious disease in many tropical and subtropical countries. Previous efforts to eradicate malaria have failed, largely due to the emergence of drug-resistant parasites, insecticide-resistant mosquitoes and, in particular, the lack of drugs or vaccines to block parasite transmission. ATP-binding cassette (ABC) transporters are known to play a role in drug transport, metabolism, and resistance in many organisms, including malaria parasites. To investigate whether a Plasmodium falciparum ABC transporter (Pf14_0244 or PfABCG2) modulates parasite susceptibility to chemical compounds or plays a role in drug resistance, we disrupted the gene encoding PfABCG2, screened the recombinant and the wild-type 3D7 parasites against a library containing 2,816 drugs approved for human or animal use, and identified an antihistamine (ketotifen) that became less active against the PfABCG2-disrupted parasite in culture. In addition to some activity against asexual stages and gametocytes, ketotifen was highly potent in blocking oocyst development of P. falciparum and the rodent parasite Plasmodium yoelii in mosquitoes. Tests of structurally related tricyclic compounds identified additional compounds with similar activities in inhibiting transmission. Additionally, ketotifen appeared to have some activity against relapse of Plasmodium cynomolgi infection in rhesus monkeys. Further clinical evaluation of ketotifen and related compounds, including synthetic new derivatives, in blocking malaria transmission may provide new weapons for the current effort of malaria eradication.

Characterization and regulation of the resistance-nodulation-cell division-type multidrug efflux pumps MdtABC and MdtUVW from the fire blight pathogen Erwinia amylovora.

PubMed

Pletzer, Daniel; Weingart, Helge

2014-07-11

The Gram-negative bacterium Erwinia amylovora is the causal agent of the devastating disease fire blight in rosaceous plants such as apple, pear, quince, raspberry, and cotoneaster. In order to survive and multiply in a host, microbes must be able to circumvent the toxic effects of antimicrobial plant compounds, such as flavonoids and tannins. E. amylovora uses multidrug efflux transporters that recognize and actively export toxic compounds out of the cells. Here, two heterotrimeric resistance-nodulation-cell division (RND)-type multidrug efflux pumps, MdtABC and MdtUVW, from E. amylovora were identified. These RND systems are unusual in that they contain two different RND proteins forming a functional pump. To find the substrate specificities of the two efflux systems, we overexpressed the transporters in a hypersensitive mutant lacking the major RND pump AcrB. Both transporters mediated resistance to several flavonoids, fusidic acid and novobiocin. Additionally, MdtABC mediated resistance towards josamycin, bile salts and silver nitrate, and MdtUVW towards clotrimazole. The ability of the mdtABC- and mdtUVW-deficient mutants to multiply in apple rootstock was reduced. Quantitative RT-PCR analyses revealed that the expression of the transporter genes was induced during infection of apple rootstock. The polyphenolic plant compound tannin, as well as the heavy metal salt tungstate was found to induce the expression of mdtABC. Finally, the expression of the mdtABC genes was shown to be regulated by BaeR, the response regulator of the two-component system BaeSR, a cell envelope stress response system that controls the adaptive responses to changes in the environment. The expression of MdtABC and MdtUVW is induced during growth of E. amylovora in planta. We identified the plant polyphenol tannin as inducer of mdtABC expression. The reduced ability of the mdtABC- and mdtUVW-deficient mutants to multiply in apple rootstock suggests that the efflux pumps are involved in resistance to plant antimicrobials, maybe including flavonoids, which were identified as substrates of both pumps. Furthermore, we found that the mdtABC operon belongs to the regulon of the two-component regulator BaeR suggesting a role of this RND transporter in the cell envelope stress response of E. amylovora.

Characterization and regulation of the Resistance-Nodulation-Cell Division-type multidrug efflux pumps MdtABC and MdtUVW from the fire blight pathogen Erwinia amylovora

PubMed Central

2014-01-01

Background The Gram-negative bacterium Erwinia amylovora is the causal agent of the devastating disease fire blight in rosaceous plants such as apple, pear, quince, raspberry, and cotoneaster. In order to survive and multiply in a host, microbes must be able to circumvent the toxic effects of antimicrobial plant compounds, such as flavonoids and tannins. E. amylovora uses multidrug efflux transporters that recognize and actively export toxic compounds out of the cells. Here, two heterotrimeric resistance-nodulation-cell division (RND)-type multidrug efflux pumps, MdtABC and MdtUVW, from E. amylovora were identified. These RND systems are unusual in that they contain two different RND proteins forming a functional pump. Results To find the substrate specificities of the two efflux systems, we overexpressed the transporters in a hypersensitive mutant lacking the major RND pump AcrB. Both transporters mediated resistance to several flavonoids, fusidic acid and novobiocin. Additionally, MdtABC mediated resistance towards josamycin, bile salts and silver nitrate, and MdtUVW towards clotrimazole. The ability of the mdtABC- and mdtUVW-deficient mutants to multiply in apple rootstock was reduced. Quantitative RT-PCR analyses revealed that the expression of the transporter genes was induced during infection of apple rootstock. The polyphenolic plant compound tannin, as well as the heavy metal salt tungstate was found to induce the expression of mdtABC. Finally, the expression of the mdtABC genes was shown to be regulated by BaeR, the response regulator of the two-component system BaeSR, a cell envelope stress response system that controls the adaptive responses to changes in the environment. Conclusions The expression of MdtABC and MdtUVW is induced during growth of E. amylovora in planta. We identified the plant polyphenol tannin as inducer of mdtABC expression. The reduced ability of the mdtABC- and mdtUVW-deficient mutants to multiply in apple rootstock suggests that the efflux pumps are involved in resistance to plant antimicrobials, maybe including flavonoids, which were identified as substrates of both pumps. Furthermore, we found that the mdtABC operon belongs to the regulon of the two-component regulator BaeR suggesting a role of this RND transporter in the cell envelope stress response of E. amylovora. PMID:25012600

Two single-nucleotide polymorphisms of the RELN gene and symptom-based and developmental deficits among children and adolescents with autistic spectrum disorders in the Tianjin, China.

PubMed

Wang, Geng-Fu; Ye, Sheng; Gao, Lei; Han, Yu; Guo, Xuan; Dong, Xiao-Peng; Su, Yuan-Yuan; Zhang, Xin

2018-05-10

Increasing evidence has revealed that genetic variants in Reelin (RELN) gene, especially single-nucleotide polymorphisms (SNPs), correlate with autistic spectrum disorders (ASD) risk; however, no consensus have been reached. This study aimed to provide additional evidence for the association between two SNPs of RELN (i.e., rs736707, rs2229864) and ASD risk, as well as the relationship between RELN gene and symptom-based and developmental deficits of ASD patients in Chinese Han children and adolescents. 157 ASD subjects and 256 typical development (TD) controls were genotyped by TaqMan® genotyping assay. ASD patients were assessed by Childhood Autism Rating Scale (CARS), Autism Behavior Checklist (ABC), and Early Childhood Development Questionnaire (ECDQ). We found that SNP rs2229864 was associated with the genetic predisposition of ASD, whereas a negative association between SNP rs2229864 and symptom-based and developmental features was detected. In contrast, RELN rs736707 correlated with the sensory subscale of the ABC, the relating subscale of the ABC and the total score of ABC, although we did not detect a significant association between SNP rs736707 and ASD risk. Furthermore, a significant rs736707-rs2229864 haplotype was detected. Individuals with a CC haplotype were more likely to have ASD, but individuals with a CT haplotype had more chance be TD controls. Further studies using more samples and including more gene variants in RELN are warranted to confirm our results. Copyright © 2018 Elsevier B.V. All rights reserved.

Conservation of nodulation genes between Rhizobium meliloti and a slow-growing Rhizobium strain that nodulates a nonlegume host

PubMed Central

Marvel, Deborah J.; Kuldau, Gretchen; Hirsch, Ann; Richards, Eric; Torrey, John G.; Ausubel, Frederick M.

1985-01-01

Parasponia, a woody member of the elm family, is the only nonlegume genus whose members are known to form an effective nitrogen-fixing symbiosis with a Rhizobium species. The bacterial strain RP501 is a slow-growing strain of Rhizobium isolated from Parasponia nodules. Strain RP501 also nodulates the legumes siratro (Macroptilium atropurpureum) and cowpea (Vigna unguiculata). Using a cosmid clone bank of RP501 DNA, we isolated a 13.4-kilobase (kb) EcoRI fragment that complemented insertion and point mutations in three contiguous nodulation genes (nodABC) of Rhizobium meliloti, the endosymbiont of alfalfa (Medicago sativa). The complemented R. meliloti nod mutants induced effective nitrogen-fixing nodules on alfalfa seedlings but not on siratro, cowpeas, or Parasponia. The cloned RP501 nodulation locus hybridized to DNA fragments carrying the R. meliloti nodABC genes. A 3-kb cluster of Tn5 insertion mutations on the RP501 13.4-kb EcoRI fragment prevented complementation of R. meliloti nodABC mutations. Images PMID:16593600

Classification of a Haemophilus influenzae ABC Transporter HI1470/71 through Its Cognate Molybdate Periplasmic Binding Protein, MolA

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tirado-Lee, Leidamarie; Lee, Allen; Rees, Douglas C.

2014-10-02

molA (HI1472) from H. influenzae encodes a periplasmic binding protein (PBP) that delivers substrate to the ABC transporter MolB{sub 2}C{sub 2} (formerly HI1470/71). The structures of MolA with molybdate and tungstate in the binding pocket were solved to 1.6 and 1.7 {angstrom} resolution, respectively. The MolA-binding protein binds molybdate and tungstate, but not other oxyanions such as sulfate and phosphate, making it the first class III molybdate-binding protein structurally solved. The {approx}100 {mu}M binding affinity for tungstate and molybdate is significantly lower than observed for the class II ModA molybdate-binding proteins that have nanomolar to low micromolar affinity for molybdate.more » The presence of two molybdate loci in H. influenzae suggests multiple transport systems for one substrate, with molABC constituting a low-affinity molybdate locus.« less

Induction of multixenobiotic defense mechanisms in resistant Daphnia magna clones as a general cellular response to stress.

PubMed

Jordão, Rita; Campos, Bruno; Lemos, Marco F L; Soares, Amadeu M V M; Tauler, Romà; Barata, Carlos

2016-06-01

Multixenobiotic resistance mechanisms (MXR) were recently identified in Daphnia magna. Previous results characterized gene transcripts of genes encoding and efflux activities of four putative ABCB1 and ABCC transporters that were chemically induced but showed low specificity against model transporter substrates and inhibitors, thus preventing us from distinguishing between activities of different efflux transporter types. In this study we report on the specificity of induction of ABC transporters and of the stress protein hsp70 in clones selected to be genetically resistant to ABCB1 chemical substrates. Clones resistant to mitoxantrone, ivermectin and pentachlorophenol showed distinctive transcriptional responses of transporter protein coding genes and of putative transporter dye activities. Expression of hsp70 proteins also varied across resistant clones. Clones resistant to mitoxantrone and pentachlorophenol showed high constitutive levels of hsp70. Transcriptional levels of the abcb1 gene transporter and of putative dye transporter activity were also induced to a greater extent in the pentachlorophenol resistant clone. Observed higher dye transporter activities in individuals from clones resistant to mitoxantrone and ivermectin were unrelated with transcriptional levels of the studied four abcc and abcb1 transporter genes. These findings suggest that Abcb1 induction in D. magna may be a part of a general cellular stress response. Copyright © 2016 Elsevier B.V. All rights reserved.

Cap 'n' collar C regulates genes responsible for imidacloprid resistance in the Colorado potato beetle, Leptinotarsa decemlineata.

PubMed

Gaddelapati, Sharath Chandra; Kalsi, Megha; Roy, Amit; Palli, Subba Reddy

2018-08-01

The Colorado potato beetle (CPB), Leptinotarsa decemlineata developed resistance to imidacloprid after exposure to this insecticide for multiple generations. Our previous studies showed that xenobiotic transcription factor, cap 'n' collar isoform C (CncC) regulates the expression of multiple cytochrome P450 genes, which play essential roles in resistance to plant allelochemicals and insecticides. In this study, we sought to obtain a comprehensive picture of the genes regulated by CncC in imidacloprid-resistant CPB. We performed sequencing of RNA isolated from imidacloprid-resistant CPB treated with dsRNA targeting CncC or gene coding for green fluorescent protein (control). Comparative transcriptome analysis showed that CncC regulated the expression of 1798 genes, out of which 1499 genes were downregulated in CncC knockdown beetles. Interestingly, expression of 79% of imidacloprid induced P450 genes requires CncC. We performed quantitative real-time PCR to verify the reduction in the expression of 20 genes including those coding for detoxification enzymes (P450s, glutathione S-transferases, and esterases) and ABC transporters. The genes coding for ABC transporters are induced in insecticide resistant CPB and require CncC for their expression. Knockdown of genes coding for ABC transporters simultaneously or individually caused an increase in imidacloprid-induced mortality in resistant beetles confirming their contribution to insecticide resistance. These studies identified CncC as a transcription factor involved in regulation of genes responsible for imidacloprid resistance. Small molecule inhibitors of CncC or suppression of CncC by RNAi could provide effective synergists for pest control or management of insecticide resistance. Copyright © 2018 Elsevier Ltd. All rights reserved.

Determination of Flower Structure in Elaeis guineensis: Do Palms use the Same Homeotic Genes as Other Species?

PubMed Central

Adam, Helene; Jouannic, Stefan; Morcillo, Fabienne; Verdeil, Jean-Luc; Duval, Yves; Tregear, James W.

2007-01-01

Aims In this article a review is made of data recently obtained on the structural diversity and possible functions of MADS box genes in the determination of flower structure in the African oil palm (Elaeis guineensis). MADS box genes play a dominant role in the ABC model established to explain how floral organ identity is determined in model dicotyledon species such as Arabidopsis thaliana and Antirrhinum majus. In the monocotyledons, although there appears to be a broad general conservation of ABC gene functions, the model itself needs to be adapted in some cases, notably for certain species which produce flowers with sepals and petals of similar appearance. For the moment, ABC genes remain unstudied in a number of key monocot clades, so only a partial picture is available for the Liliopsida as a whole. The aim of this article is to summarize data recently obtained for the African oil palm Elaeis guineensis, a member of the family Arecaceae (Arecales), and to discuss their significance with respect to knowledge gained from other Angiosperm groups, particularly within the monocotyledons. Scope The essential details of reproductive development in oil palm are discussed and an overview is provided of the structural and functional characterization of MADS box genes likely to play a homeotic role in flower development in this species. Conclusions The structural and functional data provide evidence for a general conservation of the generic ‘ABC’ model in oil palm, rather than the ‘modified ABC model’ proposed for some other monocot species which produce homochlamydeous flowers (i.e. with morphologically similar organs in both perianth whorls), such as members of the Liliales. Our oil palm data therefore follow a similar pattern to those obtained for other Commelinid species in the orders Commelinales and Poales. The significance of these findings is discussed. PMID:17355996

Evidence for cryptic northern refugia in the last glacial period in Cryptomeria japonica

PubMed Central

Kimura, Megumi K.; Uchiyama, Kentaro; Nakao, Katsuhiro; Moriguchi, Yoshinari; San Jose-Maldia, Lerma; Tsumura, Yoshihiko

2014-01-01

Background and Aims Distribution shifts and natural selection during past climatic changes are important factors in determining the genetic structure of forest species. In particular, climatic fluctuations during the Quaternary appear to have caused changes in the distribution ranges of plants, and thus strongly affected their genetic structure. This study was undertaken to identify the responses of the conifer Cryptomeria japonica, endemic to the Japanese Archipelago, to past climatic changes using a combination of phylogeography and species distribution modelling (SDM) methods. Specifically, this study focused on the locations of refugia during the last glacial maximum (LGM). Methods Genetic diversity and structure were examined using 20 microsatellite markers in 37 populations of C. japonica. The locations of glacial refugia were assessed using STRUCTURE analysis, and potential habitats under current and past climate conditions were predicted using SDM. The process of genetic divergence was also examined using the approximate Bayesian computation procedure (ABC) in DIY ABC to test the divergence time between the gene pools detected by the STRUCTURE analysis. Key Results STRUCTURE analysis identified four gene pools: northern Tohoku district; from Chubu to Chugoku district; from Tohoku to Shikoku district on the Pacific Ocean side of the Archipelago; and Yakushima Island. DIY ABC analysis indicated that the four gene pools diverged at the same time before the LGM. SDM also indicated potential northern cryptic refugia. Conclusions The combined evidence from microsatellites and SDM clearly indicates that climatic changes have shaped the genetic structure of C. japonica. The gene pool detected in northern Tohoku district is likely to have been established by cryptic northern refugia on the coast of the Japan Sea to the west of the Archipelago. The gene pool in Yakushima Island can probably be explained simply by long-term isolation from the other gene pools since the LGM. These results are supported by those of SDM and the predicted divergence time determined using ABC analysis. PMID:25355521

Mutation in the peb1A Locus of Campylobacter jejuni Reduces Interactions with Epithelial Cells and Intestinal Colonization of Mice

PubMed Central

Pei, Zhiheng; Burucoa, Christophe; Grignon, Bernadette; Baqar, Shahida; Huang, Xiao-Zhe; Kopecko, Dennis J.; Bourgeois, A. L.; Fauchere, Jean-Louis; Blaser, Martin J.

1998-01-01

Campylobacter jejuni is one of the leading causes of bacterial diarrhea throughout the world. We previously found that PEB1 is a homolog of cluster 3 binding proteins of bacterial ABC transporters and that a C. jejuni adhesin, cell-binding factor 1 (CBF1), if not identical to, contains PEB1. A single protein migrating at approximately 27 to 28 kDa was recognized by anti-CBF1 and anti-PEB1. To determine the role that the operon encoding PEB1 plays in C. jejuni adherence, peb1A, the gene encoding PEB1, was disrupted in strain 81-176 by insertion of a kanamycin resistance gene through homologous recombination. Inactivation of this operon completely abolished expression of CBF1, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. In comparison to the wild-type strain, the mutant strain showed 50- to 100-fold less adherence to and 15-fold less invasion of epithelial cells in culture. Mouse challenge studies showed that the rate and duration of intestinal colonization by the mutant were significantly lower and shorter than with the wild-type strain. In summary, PEB1 is identical to a previously identified cell-binding factor, CBF1, in C. jejuni, and the peb1A locus plays an important role in epithelial cell interactions and in intestinal colonization in a mouse model. PMID:9488379

A NodD-like protein activates transcription of genes involved with naringenin degradation in a flavonoid-dependent manner in Herbaspirillum seropedicae.

PubMed

Wassem, R; Marin, A M; Daddaoua, A; Monteiro, R A; Chubatsu, L S; Ramos, J L; Deakin, W J; Broughton, W J; Pedrosa, F O; Souza, E M

2017-03-01

Herbaspirillum seropedicae is an associative, endophytic non-nodulating diazotrophic bacterium that colonises several grasses. An ORF encoding a LysR-type transcriptional regulator, very similar to NodD proteins of rhizobia, was identified in its genome. This nodD-like gene, named fdeR, is divergently transcribed from an operon encoding enzymes involved in flavonoid degradation (fde operon). Apigenin, chrysin, luteolin and naringenin strongly induce transcription of the fde operon, but not that of the fdeR, in an FdeR-dependent manner. The intergenic region between fdeR and fdeA contains several generic LysR consensus sequences (T-N 11 -A) and we propose a binding site for FdeR, which is conserved in other bacteria. DNase I foot-printing revealed that the interaction with the FdeR binding site is modified by the four flavonoids that stimulate transcription of the fde operon. Moreover, FdeR binds naringenin and chrysin as shown by isothermal titration calorimetry. Interestingly, FdeR also binds in vitro to the nod-box from the nodABC operon of Rhizobium sp. NGR234 and is able to activate its transcription in vivo. These results show that FdeR exhibits two features of rhizobial NodD proteins: nod-box recognition and flavonoid-dependent transcription activation, but its role in H. seropedicae and related organisms seems to have evolved to control flavonoid metabolism. © 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.

The Components of the Unique Zur Regulon of Cupriavidus metallidurans Mediate Cytoplasmic Zinc Handling

PubMed Central

Bütof, Lucy; Schmidt-Vogler, Christopher; Herzberg, Martin; Große, Cornelia

2017-01-01

ABSTRACT Zinc is an essential trace element, yet it is toxic at high concentrations. In the betaproteobacterium Cupriavidus metallidurans, the highly efficient removal of surplus zinc from the periplasm is responsible for the outstanding metal resistance of the organism. Rather than having a typical Zur-dependent, high-affinity ATP-binding cassette transporter of the ABC protein superfamily for zinc uptake at low concentrations, C. metallidurans has the secondary zinc importer ZupT of the zinc-regulated transporter, iron-regulated transporter (ZRT/IRT)-like protein (ZIP) family. It is important to understand, therefore, how this zinc-resistant bacterium copes with exposure to low zinc concentrations. Members of the Zur regulon in C. metallidurans were identified by comparing the transcriptomes of a Δzur mutant and its parent strain. The consensus sequence of the Zur-binding box was derived for the zupTp promoter-regulatory region by use of a truncation assay. The motif was used to predict possible Zur boxes upstream of Zur regulon members. The binding of Zur to these boxes was confirmed. Two Zur boxes upstream of the cobW1 gene, encoding a putative zinc chaperone, proved to be required for complete repression of cobW1 and its downstream genes in cells cultivated in mineral salts medium. A Zur box upstream of each of zur-cobW2, cobW3, and zupT permitted both low expression levels of these genes and their upregulation under conditions of zinc starvation. This demonstrates a compartmentalization of zinc homeostasis in C. metallidurans, where the periplasm is responsible for the removal of surplus zinc, cytoplasmic components are responsible for the management of zinc as an essential cofactor, and the two compartments are connected by ZupT. IMPORTANCE Elucidating zinc homeostasis is necessary for understanding both host-pathogen interactions and the performance of free-living bacteria in their natural environments. Escherichia coli acquires zinc under conditions of low zinc concentrations via the Zur-controlled ZnuABC importer of the ABC superfamily, and this was also the paradigm for other bacteria. In contrast, the heavy-metal-resistant bacterium C. metallidurans achieves high tolerance to zinc through sophisticated zinc handling and efflux systems operating on periplasmic zinc ions, so that removal of surplus zinc is a periplasmic feature in this bacterium. It is shown here that this process is augmented by the management of zinc by cytoplasmic zinc chaperones, whose synthesis is controlled by the Zur regulator. This demonstrates a new mechanism, involving compartmentalization, for organizing zinc homeostasis. PMID:28808127

The components of the unique Zur regulon of Cupriavidus metallidurans mediate cytoplasmic zinc handling.

PubMed

Bütof, Lucy; Schmidt-Vogler, Christopher; Herzberg, Martin; Große, Cornelia; Nies, Dietrich H

2017-08-14

Zinc is an essential trace element and at the same time it is toxic at high concentrations. In the beta-proteobacterium Cupriavidus metallidurans the highly efficient removal of surplus zinc from the periplasm is responsible for its outstanding metal resistance. Rather than having a typical Zur-dependent, high-affinity ATP-binding cassette transporter of the ABC protein superfamily for zinc uptake at low concentrations, C. metallidurans instead has the secondary zinc importer ZupT of the ZRT/IRT (ZIP) family. It is important to understand, therefore, how this zinc-resistant bacterium copes when it is exposed to low zinc concentrations. Members of the Zur regulon in C. metallidurans were identified by comparing the transcriptomes of a Δ zur mutant and its parent strain. The consensus sequence of the Zur-binding box was derived for the zupTp promoter-regulatory region using a truncation assay. The motif was used to predict possible Zur-boxes upstream of Zur regulon members. Binding of Zur to these boxes was confirmed. Two Zur-boxes upstream of the cobW 1 gene, encoding a putative zinc chaperone, proved to be required for complete repression of cobW 1 and its downstream genes in cells cultivated in mineral salts medium. A Zur box upstream of each of zur-cobW 2 , cobW 3 and zupT permitted low-expression level of these genes plus their up-regulation under zinc starvation conditions. This demonstrates a compartmentalization of zinc homeostasis in C. metallidurans with the periplasm being responsible for removal of surplus zinc and cytoplasmic components for management of zinc as an essential co-factor, with both compartments connected by ZupT. Importance Elucidating zinc homeostasis is necessary to understand both host-pathogen interactions and performance of free-living bacteria in their natural environment. Escherichia coli acquires zinc under low zinc concentrations by the Zur-controlled ZnuABC importer of the ABC superfamily, and this was also the paradigm for other bacteria. In contrast, the heavy metal-resistant bacterium C. metallidurans achieves high tolerance to zinc due to sophisticated zinc handling and efflux systems operating on periplasmic zinc ions, so that removal of surplus zinc is a periplasmic feature in this bacterium. It is shown here that this process is augmented by management of zinc through cytoplasmic zinc chaperones, whose syntheses are controlled by the Zur regulator. This demonstrates a new mechanism to organize zinc homeostasis through compartmentalization. Copyright © 2017 American Society for Microbiology.

A Novel Zn2-Cys6 Transcription Factor AtrR Plays a Key Role in an Azole Resistance Mechanism of Aspergillus fumigatus by Co-regulating cyp51A and cdr1B Expressions

PubMed Central

Shimizu, Kiminori; Paul, Sanjoy; Ohba, Ayumi; Gonoi, Tohru; Watanabe, Akira; Gomi, Katsuya

2017-01-01

Successful treatment of aspergillosis caused by Aspergillus fumigatus is threatened by an increasing incidence of drug resistance. This situation is further complicated by the finding that strains resistant to azoles, the major antifungal drugs for aspergillosis, have been widely disseminated across the globe. To elucidate mechanisms underlying azole resistance, we identified a novel transcription factor that is required for normal azole resistance in Aspergillus fungi including A. fumigatus, Aspergillus oryzae, and Aspergillus nidulans. This fungal-specific Zn2-Cys6 type transcription factor AtrR was found to regulate expression of the genes related to ergosterol biosynthesis, including cyp51A that encodes a target protein of azoles. The atrR deletion mutant showed impaired growth under hypoxic conditions and attenuation of virulence in murine infection model for aspergillosis. These results were similar to the phenotypes for a mutant strain lacking SrbA that is also a direct regulator for the cyp51A gene. Notably, AtrR was responsible for the expression of cdr1B that encodes an ABC transporter related to azole resistance, whereas SrbA was not involved in the regulation. Chromatin immunoprecipitation assays indicated that AtrR directly bound both the cyp51A and cdr1B promoters. In the clinically isolated itraconazole resistant strain that harbors a mutant Cyp51A (G54E), deletion of the atrR gene resulted in a hypersensitivity to the azole drugs. Together, our results revealed that AtrR plays a pivotal role in a novel azole resistance mechanism by co-regulating the drug target (Cyp51A) and putative drug efflux pump (Cdr1B). PMID:28052140

Characterization of Haemophilus ducreyi cdtA, cdtB, and cdtC Mutants in In Vitro and In Vivo Systems

PubMed Central

Lewis, David A.; Stevens, Marla K.; Latimer, Jo L.; Ward, Christine K.; Deng, Kaiping; Blick, Robert; Lumbley, Sheryl R.; Ison, Catherine A.; Hansen, Eric J.

2001-01-01

Haemophilus ducreyi expresses a soluble cytolethal distending toxin (CDT) that is encoded by the cdtABC gene cluster and can be detected in culture supernatant fluid by its ability to kill HeLa cells. The cdtA, cdtB, and cdtC genes of H. ducreyi were cloned independently into plasmid vectors, and their encoded proteins expressed singly or in various combinations in an Escherichia coli background. All three gene products had to be expressed in order for E. coli-derived culture supernatant fluids to demonstrate cytotoxicity for HeLa cells. Isogenic H. ducreyi cdtA and cdtB mutants were constructed and used in combination with the wild-type parent strain and a previously described H. ducreyi cdtC mutant (M. K. Stevens, J. L. Latimer, S. R. Lumbley, C. K. Ward, L. D. Cope, T. Lagergard, and E. J. Hansen, Infect. Immun. 67:3900–3908, 1999) to determine the relative contributions of the CdtA, CdtB, and CdtC proteins to CDT activity. Expression of CdtA, CdtB, and CdtC appeared necessary for H. ducreyi-derived culture supernatant fluid to exhibit cytotoxicity for HeLa cells. Whole-cell sonicates and periplasmic extracts from the cdtB and cdtC mutants had no effect on HeLa cells, whereas these same fractions from a cdtA mutant had a very modest cytotoxic effect on these same human cells. CdtA appeared to be primarily associated with the H. ducreyi cell envelope, whereas both CdtB and CdtC were present primarily in the soluble fraction from sonicated cells. Both the cdtA mutant and the cdtB mutant were found to be fully virulent in the temperature-dependent rabbit model for experimental chancroid. PMID:11500438

Transcriptional responses in Lactococcus lactis subsp. cremoris to the changes in oxygen and redox potential during milk acidification.

PubMed

Larsen, N; Brøsted Werner, B; Jespersen, L

2016-08-01

Milk acidification and metabolic activity of the starter cultures are affected by oxygen; however, molecular factors related to the redox changes are poorly defined. The objective of the study was to investigate transcriptional responses in Lactococcus lactis subsp. cremoris CHCCO2 grown in milk to the shifts of oxygen and redox potential (Eh7 ). Transcriptomic studies were performed with the use of Illumina HiSeq 2000 mRNA sequencing and validated by the real-time quantitative PCR. In total 105 differentially expressed genes were assigned functional gene names. Most of the differentially expressed genes were detected during aerobic reduction phase. Upregulated genes were implicated in lactose utilization, glycogen biosynthesis, amino sugar metabolism, oxidation-reduction, pyrimidine biosynthesis and DNA integration processes. Genes of purine nucleotide biosynthesis and genes encoding amino acid, multidrug resistance and ion ABC transporters were mostly downregulated, while oligopeptide transporter genes were reduced during oxygen depletion and induced at minimum Eh7 . Understanding of gene responses in starter cultures to the changes of oxidation-reduction state is important for the better control and reproducibility of dairy fermentations. We applied mRNA sequencing by Illumina HiSeq 2000 to investigate gene expression profile in a dairy strain of Lactococcus lactis subsp. cremoris during milk acidification. Novelty of this study lies in linking transcriptional responses to oxygen depletion and the changes of redox potential with the fermentation kinetics and clarification of molecular factors specifically expressed in milk which might be essential for bacterial performance and the final quality of cheeses. © 2016 The Society for Applied Microbiology.

Comparative transcriptome analysis of Sogatella furcifera (Horváth) exposed to different insecticides.

PubMed

Zhou, Cao; Yang, Hong; Wang, Zhao; Long, Gui-Yun; Jin, Dao-Chao

2018-06-08

White-backed planthopper, Sogatella furcifera (Horváth) (Hemiptera: Delphacidae), one of the main agricultural insect pests in China, is resistant to a wide variety of insecticides. We used transcriptome analysis to compare the expression patterns of resistance- and stress-response genes in S. furcifera subjected to imidacloprid, deltamethrin, and triazophos stress, to determine the molecular mechanisms of resistance to these insecticides. A comparative analysis of gene expression under imidacloprid, deltamethrin, and triazophos stress revealed 1,123, 841, and 316 upregulated unigenes, respectively, compared to the control. These upregulated genes included seven P450s (two CYP2 clade, three CYP3 clade, and two CYP4 clade), one GST, one ABC transporter (ABCF), and seven Hsps (one 90 and six Hsp70s) under imidacloprid stress; one P450 (CYP3 clade), two ABC transporters (one ABCF and one ABCD), and one Hsp (Hsp90) under deltamethrin stress; one P450 (CYP3 clade) and one ABC transporter (ABCF) under triazophos stress. In addition, 80 genes were commonly upregulated in response to the three insecticide treatments, including laminin, larval cuticle protein, and fasciclin, which are associated with epidermal formation. These results provide a valuable resource for the molecular characterisation of insecticide action in S. furcifera, especially the molecular characteristics of insecticide cross resistance.

Quantification and in situ localisation of abcb1 and abcc9genes in toxicant-exposed sea urchin embryos.

PubMed

Bošnjak, Ivana; Pleić, Ivana Lepen; Borra, Marco; Mladineo, Ivona

2013-12-01

A multixenobiotic resistance (MXR) mechanism mediated by ABC binding cassette (ABC) transport proteins is an efficient chemical defence mechanism in sea urchin embryos. The aim of our work was to evidence whether exposure to sub-lethal doses of specific contaminants (oxybenzone (OXI), mercuric chloride (HgCl2) and trybutiltin (TBT)) would induce MXR transporter activity during embryonic development (from zygote to blastula stage) in purple sea urchin (Paracentrotus lividus) embryos. Further, we present data on molecular identification, transport function, expression levels and gene localisation of two ABC efflux transporters-P-glycoprotein (ABCB1/P-gp) and sulfonylurea-receptor-like protein (ABCC9/SUR-like). Partial cDNA sequences of abcb1 and abcc9 were identified and quantitative PCR (qPCR) evidenced an increase in mRNA transcript levels of both ABC transporters during the two-cell, as well as an overall decrease during the blastulae stage. Calcein-AM efflux activity assay indicated the activation of multidrug resistance-associated protein/ABCC-like transport in the presence of HgCl2 and TBT in exposed blastulae. The in situ hybridisation of the two-cell and blastula stages showed ubiquitous localisation of both transcripts within cells, supporting qPCR data. In conclusion, ABCB1 and ABCC9 are constitutive, as are HgCl2, TBT and OXI-inducible ABC membrane transporters, coexpressed in the zygote, two-cell and blastula stages of the P. lividus. Their ubiquitous cell localisation further fortifies their protective role in early embryonic development.

RiArsB and RiMT-11: Two novel genes induced by arsenate in arbuscular mycorrhiza.

PubMed

Maldonado-Mendoza, Ignacio E; Harrison, Maria J

Plants associated with arbuscular mycorrhizal fungi (AMF) increase their tolerance to arsenic-polluted soils. This study aims to investigate the genes involved in the AMF molecular response to arsenic pollution. Genes encoding proteins involved in arsenic metabolism were identified and their expression assessed by PCR or RT-qPCR. The As-inducible gene GiArsA (R. irregularis ABC ATPase component of the ArsAB arsenite efflux pump) and two new genes, an arsenate/arsenite permease component of ArsAB (RiArsB) and a methyltransferase type 11 (RiMT-11) were induced when arsenate was added to two-compartment in vitro monoxenic cultures of R. irregularis-transformed carrot roots. RiArsB and RiMT-11 expression in extraradical hyphae in response to arsenate displayed maximum induction 4-6 h after addition of 350 μM arsenate. Their expression was also detected in colonized root tissues grown in pots, or in the root-fungus compartment of two-compartment in vitro systems. We used a Medicago truncatula double mutant (mtpt4/mtpt8) to demonstrate that RiMT-11 and RiArsB transcripts accumulate in response to the addition of arsenate but not in response to phosphate. These results suggest that these genes respond to arsenate addition regardless of non-functional Pi symbiotic transport, and that RiMT-11 may be involved in arsenate detoxification by methylation in AMF-colonized tissues. Copyright © 2017 British Mycological Society. All rights reserved.

Effect of Periodontal Pathogens on the Metatranscriptome of a Healthy Multispecies Biofilm Model

PubMed Central

Duran-Pinedo, Ana

2012-01-01

Oral bacterial biofilms are highly complex microbial communities with up to 700 different bacterial taxa. We report here the use of metatranscriptomic analysis to study patterns of community gene expression in a multispecies biofilm model composed of species found in healthy oral biofilms (Actinomyces naeslundii, Lactobacillus casei, Streptococcus mitis, Veillonella parvula, and Fusobacterium nucleatum) and the same biofilm plus the periodontopathogens Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans. The presence of the periodontopathogens altered patterns in gene expression, and data indicate that transcription of protein-encoding genes and small noncoding RNAs is stimulated. In the healthy biofilm hypothetical proteins, transporters and transcriptional regulators were upregulated while chaperones and cell division proteins were downregulated. However, when the pathogens were present, chaperones were highly upregulated, probably due to increased levels of stress. We also observed a significant upregulation of ABC transport systems and putative transposases. Changes in Clusters of Orthologous Groups functional categories as well as gene set enrichment analysis based on Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways showed that in the absence of pathogens, only sets of proteins related to transport and secondary metabolism were upregulated, while in the presence of pathogens, proteins related to growth and division as well as a large portion of transcription factors were upregulated. Finally, we identified several small noncoding RNAs whose predicted targets were genes differentially expressed in the open reading frame libraries. These results show the importance of pathogens controlling gene expression of a healthy oral community and the usefulness of metatranscriptomic techniques to study gene expression profiles in complex microbial community models. PMID:22328675

Structural elucidation of transmembrane domain zero (TMD0) of EcdL: A multidrug resistance-associated protein (MRP) family of ATP-binding cassette transporter protein revealed by atomistic simulation.

PubMed

Bera, Krishnendu; Rani, Priyanka; Kishor, Gaurav; Agarwal, Shikha; Kumar, Antresh; Singh, Durg Vijay

2017-09-20

ATP-Binding cassette (ABC) transporters play an extensive role in the translocation of diverse sets of biologically important molecules across membrane. EchnocandinB (antifungal) and EcdL protein of Aspergillus rugulosus are encoded by the same cluster of genes. Co-expression of EcdL and echinocandinB reflects tightly linked biological functions. EcdL belongs to Multidrug Resistance associated Protein (MRP) subfamily of ABC transporters with an extra transmembrane domain zero (TMD0). Complete structure of MRP subfamily comprising of TMD0 domain, at atomic resolution is not known. We hypothesized that the transportation of echonocandinB is mediated via EcdL protein. Henceforth, it is pertinent to know the topological arrangement of TMD0, with other domains of protein and its possible role in transportation of echinocandinB. Absence of effective template for TMD0 domain lead us to model by I-TASSER, further structure has been refined by multiple template modelling using homologous templates of remaining domains (TMD1, NBD1, TMD2, NBD2). The modelled structure has been validated for packing, folding and stereochemical properties. MD simulation for 0.1 μs has been carried out in the biphasic environment for refinement of modelled protein. Non-redundant structures have been excavated by clustering of MD trajectory. The structural alignment of modelled structure has shown Z-score -37.9; 31.6, 31.5 with RMSD; 2.4, 4.2, 4.8 with ABC transporters; PDB ID 4F4C, 4M1 M, 4M2T, respectively, reflecting the correctness of structure. EchinocandinB has been docked to the modelled as well as to the clustered structures, which reveals interaction of echinocandinB with TMD0 and other TM helices in the translocation path build of TMDs.

A new human lung adenocarcinoma cell line harboring the EML4-ALK fusion gene.

PubMed

Isozaki, Hideko; Yasugi, Masayuki; Takigawa, Nagio; Hotta, Katsuyuki; Ichihara, Eiki; Taniguchi, Akihiko; Toyooka, Shinichi; Hashida, Shinsuke; Sendo, Toshiaki; Tanimoto, Mitsune; Kiura, Katsuyuki

2014-10-01

The echinoderm microtubule associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) fusion gene was identified in patients with non-small cell lung cancer. To the best of our knowledge, there are only three cell lines harboring the EML4-ALK fusion gene, which have contributed to the development of therapeutic strategies. Therefore, we tried to establish a new lung cancer cell line harboring EML4-ALK. A 61-year-old Japanese female presented with chest discomfort. She was diagnosed with left lung adenocarcinoma with T4N3M1 Stage IV. Although she was treated with chemotherapy, her disease progressed with massive pleural effusion. Because the EML4-ALK rearrangement was found in a biopsied specimen using fluorescence in situ hybridization, she was treated with crizotinib. She did well for 3 months. Tumor cells were obtained from the malignant pleural effusion before treatment with crizotinib. Cells continued to proliferate substantially for several weeks. The cell line was designated ABC-11. The EML4-ALK fusion protein and genes were identified in ABC-11 cells using fluorescence in situ hybridization and immunohistochemistry, respectively. ABC-11 cells were sensitive to crizotinib and next-generation ALK inhibitors (ceritinib and AP26113), as determined by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Phosphorylated ALK protein and its downstream signaling were suppressed by treatment with crizotinib in western blotting. Furthermore, we could transplant ABC-11 cells subcutaneously into BALB/c nu/nu mice. We successfully established a new lung adenocarcinoma cell line harboring the EML4-ALK fusion gene. This cell line could contribute to future research of EML4-ALK-positive lung cancer both in vivo and in vitro. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Targeting the chromatin remodeling enzyme BRG1 increases the efficacy of chemotherapy drugs in breast cancer cells

PubMed Central

Wu, Qiong; Sharma, Soni; Cui, Hang; LeBlanc, Scott E.; Zhang, Hong; Muthuswami, Rohini; Nickerson, Jeffrey A.; Imbalzano, Anthony N.

2016-01-01

Brahma related gene product 1 (BRG1) is an ATPase that drives the catalytic activity of a subset of the mammalian SWI/SNF chromatin remodeling enzymes. BRG1 is overexpressed in most human breast cancer tumors without evidence of mutation and is required for breast cancer cell proliferation. We demonstrate that knockdown of BRG1 sensitized triple negative breast cancer cells to chemotherapeutic drugs used to treat breast cancer. An inhibitor of the BRG1 bromodomain had no effect on breast cancer cell viability, but an inhibitory molecule that targets the BRG1 ATPase activity recapitulated the increased drug efficacy observed in the presence of BRG1 knockdown. We further demonstrate that inhibition of BRG1 ATPase activity blocks the induction of ABC transporter genes by these chemotherapeutic drugs and that BRG1 binds to ABC transporter gene promoters. This inhibition increased intracellular concentrations of the drugs, providing a likely mechanism for the increased chemosensitivity. Since ABC transporters and their induction by chemotherapy drugs are a major cause of chemoresistance and treatment failure, these results support the idea that targeting the enzymatic activity of BRG1 would be an effective adjuvant therapy for breast cancer. PMID:27029062

Genes Important for Catalase Activity in Enterococcus faecalis

PubMed Central

Baureder, Michael; Hederstedt, Lars

2012-01-01

Little in general is known about how heme proteins are assembled from their constituents in cells. The Gram-positive bacterium Enterococcus faecalis cannot synthesize heme and does not depend on it for growth. However, when supplied with heme in the growth medium the cells can synthesize two heme proteins; catalase (KatA) and cytochrome bd (CydAB). To identify novel factors important for catalase biogenesis libraries of E. faecalis gene insertion mutants were generated using two different types of transposons. The libraries of mutants were screened for clones deficient in catalase activity using a colony zymogram staining procedure. Analysis of obtained clones identified, in addition to katA (encoding the catalase enzyme protein), nine genes distributed over five different chromosomal loci. No factors with a dedicated essential role in catalase biogenesis or heme trafficking were revealed, but the results indicate the RNA degradosome (srmB, rnjA), an ABC-type oligopeptide transporter (oppBC), a two-component signal transducer (etaR), and NADH peroxidase (npr) as being important for expression of catalase activity in E. faecalis. It is demonstrated that catalase biogenesis in E. faecalis is independent of the CydABCD proteins and that a conserved proline residue in the N-terminal region of KatA is important for catalase assembly. PMID:22590595

Identification of a new biocontrol gene in Trichoderma atroviride: the role of an ABC transporter membrane pump in the interaction with different plant-pathogenic fungi.

PubMed

Ruocco, Michelina; Lanzuise, Stefania; Vinale, Francesco; Marra, Roberta; Turrà, David; Woo, Sheridan Lois; Lorito, Matteo

2009-03-01

Successful biocontrol interactions often require that the beneficial microbes involved are resistant or tolerant to a variety of toxicants, including antibiotics produced by themselves or phytopathogens, plant antimicrobial compounds, and synthetic chemicals or contaminants. The ability of Trichoderma spp., the most widely applied biocontrol fungi, to withstand different chemical stresses, including those associated with mycoparasitism, is well known. In this work, we identified an ATP-binding cassette transporter cell membrane pump as an important component of the above indicated resistance mechanisms that appears to be supported by an extensive and powerful cell detoxification system. The encoding gene, named Taabc2, was cloned from a strain of Trichoderma atroviride and characterized. Its expression was found to be upregulated in the presence of pathogen-secreted metabolites, specific mycotoxins and some fungicides, and in conditions that stimulate the production in Trichoderma spp. of antagonism-related factors (toxins and enzymes). The key role of this gene in antagonism and biocontrol was demonstrated by the characterization of the obtained deletion mutants. They suffered an increased susceptibility to inhibitory compounds either secreted by pathogenic fungi or possibly produced by the biocontrol microbe itself and lost, partially or entirely, the ability to protect tomato plants from Pythium ultimum and Rhizoctonia solani attack.

Unveiling the metabolic potential of two soil-derived microbial consortia selected on wheat straw

PubMed Central

Jiménez, Diego Javier; Chaves-Moreno, Diego; van Elsas, Jan Dirk

2015-01-01

Based on the premise that plant biomass can be efficiently degraded by mixed microbial cultures and/or enzymes, we here applied a targeted metagenomics-based approach to explore the metabolic potential of two forest soil-derived lignocellulolytic microbial consortia, denoted RWS and TWS (bred on wheat straw). Using the metagenomes of three selected batches of two experimental systems, about 1.2 Gb of sequence was generated. Comparative analyses revealed an overrepresentation of predicted carbohydrate transporters (ABC, TonB and phosphotransferases), two-component sensing systems and β-glucosidases/galactosidases in the two consortia as compared to the forest soil inoculum. Additionally, “profiling” of carbohydrate-active enzymes showed significant enrichments of several genes encoding glycosyl hydrolases of families GH2, GH43, GH92 and GH95. Sequence analyses revealed these to be most strongly affiliated to genes present on the genomes of Sphingobacterium, Bacteroides, Flavobacterium and Pedobacter spp. Assembly of the RWS and TWS metagenomes generated 16,536 and 15,902 contigs of ≥10 Kb, respectively. Thirteen contigs, containing 39 glycosyl hydrolase genes, constitute novel (hemi)cellulose utilization loci with affiliation to sequences primarily found in the Bacteroidetes. Overall, this study provides deep insight in the plant polysaccharide degrading capabilities of microbial consortia bred from forest soil, highlighting their biotechnological potential. PMID:26343383

Trans-Cinnamaldehyde and Eugenol Increase Acinetobacter baumannii Sensitivity to Beta-Lactam Antibiotics.

PubMed

Karumathil, Deepti P; Nair, Meera Surendran; Gaffney, James; Kollanoor-Johny, Anup; Venkitanarayanan, Kumar

2018-01-01

Multi-drug resistant (MDR) Acinetobacter baumannii is a major nosocomial pathogen causing a wide range of clinical conditions with significant mortality rates. A. baumannii strains are equipped with a multitude of antibiotic resistance mechanisms, rendering them resistant to most of the currently available antibiotics. Thus, there is a critical need to explore novel strategies for controlling antibiotic resistance in A. baumannii . This study investigated the efficacy of two food-grade, plant-derived antimicrobials (PDAs), namely trans -cinnamaldehyde (TC) and eugenol (EG) in decreasing A. baumannii 's resistance to seven β-lactam antibiotics, including ampicillin, methicillin, meropenem, penicillin, aztreonam, amoxicillin, and piperacillin. Two MDR A. baumannii isolates (ATCC 17978 and AB 251847) were separately cultured in tryptic soy broth (∼6 log CFU/ml) containing the minimum inhibitory concentration (MIC) of TC or EG with or without the MIC of each antibiotic at 37°C for 18 h. A. baumannii strains not exposed to the PDAs or antibiotics served as controls. Following incubation, A. baumannii counts were determined by broth dilution assay. In addition, the effect of PDAs on the permeability of outer membrane and efflux pumps in A. baumannii was measured. Further, the effect of TC and EG on the expression of A. baumannii genes encoding resistance to β-lactam antibiotics ( blaP ), efflux pumps ( adeABC ), and multi-drug resistant protein ( mdrp ) was studied using real-time quantitative PCR (RT-qPCR). The experiment was replicated three times with duplicate samples of each treatment and control. The results from broth dilution assay indicated that both TC and EG in combination with antibiotics increased the sensitivity of A. baumannii to all the tested antibiotics ( P < 0.05). The two PDAs inhibited the function of A. baumannii efflux pump, (AdeABC), but did not increase the permeability of its outer membrane. Moreover, RT-qPCR data revealed that TC and EG down-regulated the expression of majority of the genes associated with β-lactam antibiotic resistance, especially blaP and adeABC ( P < 0.05). The results suggest that TC and EG could potentially be used along with β-lactam antibiotics for controlling MDR A. baumannii infections; however, their clinical significance needs to be determined using in vivo studies.

Photorhabdus luminescens genes induced upon insect infection

PubMed Central

Münch, Anna; Stingl, Lavinia; Jung, Kirsten; Heermann, Ralf

2008-01-01

Background Photorhabdus luminescens is a Gram-negative luminescent enterobacterium and a symbiote to soil nematodes belonging to the species Heterorhabditis bacteriophora. P.luminescens is simultaneously highly pathogenic to insects. This bacterium exhibits a complex life cycle, including one symbiotic stage characterized by colonization of the upper nematode gut, and a pathogenic stage, characterized by release from the nematode into the hemocoel of insect larvae, resulting in rapid insect death caused by bacterial toxins. P. luminescens appears to sense and adapt to the novel host environment upon changing hosts, which facilitates the production of factors involved in survival within the host, host-killing, and -exploitation. Results A differential fluorescence induction (DFI) approach was applied to identify genes that are up-regulated in the bacterium after infection of the insect host Galleria mellonella. For this purpose, a P. luminescens promoter-trap library utilizing the mCherry fluorophore as a reporter was constructed, and approximately 13,000 clones were screened for fluorescence induction in the presence of a G. mellonella larvae homogenate. Since P. luminescens has a variety of regulators that potentially sense chemical molecules, like hormones, the screen for up-regulated genes or operons was performed in vitro, excluding physicochemical signals like oxygen, temperature or osmolarity as variables. Clones (18) were obtained exhibiting at least 2.5-fold induced fluorescence and regarded as specific responders to insect homogenate. In combination with a bioinformatics approach, sequence motifs were identified in these DNA-fragments that are similar to 29 different promoters within the P. luminescens genome. By cloning each of the predicted promoters upstream of the reporter gene, induction was verified for 27 promoters in vitro, and for 24 promoters in viable G. mellonella larvae. Among the validated promoters are some known to regulate the expression of toxin genes, including tccC1 (encoding an insecticidal toxin complex), and others encoding putative toxins. A comparably high number of metabolic genes or operons were observed to be induced upon infection; among these were eutABC, hutUH, and agaZSVCD, which encode proteins involved in ethanolamine, histidine and tagatose degradation, respectively. The results reflect rearrangements in metabolism and the use of other metabolites available from the insect. Furthermore, enhanced activity of promoters controlling the expression of genes encoding enzymes linked to antibiotic production and/or resistance was observed. Antibiotic production and resistance may influence competition with other bacteria, and thus might be important for a successful infection. Lastly, several genes of unknown function were identified that may represent novel pathogenicity factors. Conclusion We show that a DFI screen is useful for identifying genes or operons induced by chemical stimuli, such as diluted insect homogenate. A bioinformatics comparison of motifs similar to known promoters is a powerful tool for identifying regulated genes or operons. We conclude that signals for the regulation of those genes or operons induced in P. luminescens upon insect infection may represent a wide variety of compounds that make up the insect host. Our results provide insight into the complex response to the host that occurs in a bacterial pathogen, particularly reflecting the potential for metabolic shifts and other specific changes associated with virulence. PMID:18489737

Gene selection for cancer classification with the help of bees.

PubMed

Moosa, Johra Muhammad; Shakur, Rameen; Kaykobad, Mohammad; Rahman, Mohammad Sohel

2016-08-10

Development of biologically relevant models from gene expression data notably, microarray data has become a topic of great interest in the field of bioinformatics and clinical genetics and oncology. Only a small number of gene expression data compared to the total number of genes explored possess a significant correlation with a certain phenotype. Gene selection enables researchers to obtain substantial insight into the genetic nature of the disease and the mechanisms responsible for it. Besides improvement of the performance of cancer classification, it can also cut down the time and cost of medical diagnoses. This study presents a modified Artificial Bee Colony Algorithm (ABC) to select minimum number of genes that are deemed to be significant for cancer along with improvement of predictive accuracy. The search equation of ABC is believed to be good at exploration but poor at exploitation. To overcome this limitation we have modified the ABC algorithm by incorporating the concept of pheromones which is one of the major components of Ant Colony Optimization (ACO) algorithm and a new operation in which successive bees communicate to share their findings. The proposed algorithm is evaluated using a suite of ten publicly available datasets after the parameters are tuned scientifically with one of the datasets. Obtained results are compared to other works that used the same datasets. The performance of the proposed method is proved to be superior. The method presented in this paper can provide subset of genes leading to more accurate classification results while the number of selected genes is smaller. Additionally, the proposed modified Artificial Bee Colony Algorithm could conceivably be applied to problems in other areas as well.

Ectoine and 5-hydroxyectoine accumulation in the halophile Virgibacillus halodenitrificans PDB-F2 in response to salt stress.

PubMed

Tao, Ping; Li, Hui; Yu, Yunjiang; Gu, Jidong; Liu, Yongdi

2016-08-01

The moderately halophilic bacterium Virgibacillus halodenitrificans PDB-F2 copes with salinity by synthesizing or taking up compatible solutes. The main compatible solutes in this strain were ectoine and hydroxyectoine, as determined by (1)H nuclear magnetic resonance spectroscopy ((1)H-NMR). A high-performance liquid chromatography (HPLC) analysis showed that ectoine was the major solute that was synthesized in response to elevated salinity, while hydroxyectoine was a minor solute. However, the hydroxyectoine/ectoine ratio increased from 0.04 at 3 % NaCl to 0.45 at 15 % NaCl in the late exponential growth phase. A cluster of ectoine biosynthesis genes was identified, including three genes in the order of ectA, ectB, and ectC. The hydroxyectoine biosynthesis gene ectD was not part of the ectABC gene cluster. Reverse transcription-quantitative polymerase chain reactions (RT-qPCR) showed that the expression of the ect genes was salinity dependent. The expression of ectABC reached a maximum at 12 % NaCl, while ectD expression increased up to 15 % NaCl. Ectoine and hydroxyectoine production was growth phase dependent. The hydroxyectoine/ectoine ratio increased from 0.018 in the early exponential phase to 0.11 in the stationary phase at 5 % NaCl. Hydroxyectoine biosynthesis started much later than ectoine biosynthesis after osmotic shock, and the temporal expression of the ect genes differed under these conditions, with the ectABC genes being expressed first, followed by ectD gene. Increased culture salinity triggered ectoine or hydroxyectoine uptake when they were added to the medium. Hydroxyectoine was accumulated preferentially when both ectoine and hydroxyectoine were provided exogenously.

ABC transporters affect the elimination and toxicity of CdTe quantum dots in liver and kidney cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Mingli; Yin, Huancai; Bai, Pengli

This paper aimed to investigate the role of adenosine triphosphate-binding cassette (ABC) transporters on the efflux and the toxicity of nanoparticles in liver and kidney cells. In this study, we synthesized CdTe quantum dots (QDs) that were monodispersed and emitted green fluorescence (maximum peak at 530 nm). Such QDs tended to accumulate in human hepatocellular carcinoma cells (HepG2), human kidney cells 2 (HK-2), and Madin-Darby canine kidney (MDCK) cells, and cause significant toxicity in all the three cell lines. Using specific inhibitors and inducers of P-glycoprotein (Pgp) and multidrug resistance associated proteins (Mrps), the cellular accumulation and subsequent toxicity ofmore » QDs in HepG2 and HK-2 cells were significantly affected, while only slight changes appeared in MDCK cells, corresponding well with the functional expressions of ABC transporters in cells. Moreover, treatment of QDs caused concentration- and time- dependent induction of ABC transporters in HepG2 and HK-2 cells, but such phenomenon was barely found in MDCK cells. Furthermore, the effects of CdTe QDs on ABC transporters were found to be greater than those of CdCl{sub 2} at equivalent concentrations of cadmium, indicating that the effects of QDs should be a combination of free Cd{sup 2+} and specific properties of QDs. Overall, these results indicated a strong dependence between the functional expressions of ABC transporters and the efflux of QDs, which could be an important reason for the modulation of QDs toxicity by ABC transporters. - Highlights: • ABC transporters contributed actively to the cellular efflux of CdTe quantum dots. • ABC transporters affected the cellular toxicity of CdTe quantum dots. • Treatment of CdTe quantum dots induced the gene expression of ABC transporters. • Free Cd{sup 2+} should be partially involved in the effects of QDs on ABC transporters. • Cellular efflux of quantum dots could be an important modulator for its toxicity.« less

Genetic Bee Colony (GBC) algorithm: A new gene selection method for microarray cancer classification.

PubMed

Alshamlan, Hala M; Badr, Ghada H; Alohali, Yousef A

2015-06-01

Naturally inspired evolutionary algorithms prove effectiveness when used for solving feature selection and classification problems. Artificial Bee Colony (ABC) is a relatively new swarm intelligence method. In this paper, we propose a new hybrid gene selection method, namely Genetic Bee Colony (GBC) algorithm. The proposed algorithm combines the used of a Genetic Algorithm (GA) along with Artificial Bee Colony (ABC) algorithm. The goal is to integrate the advantages of both algorithms. The proposed algorithm is applied to a microarray gene expression profile in order to select the most predictive and informative genes for cancer classification. In order to test the accuracy performance of the proposed algorithm, extensive experiments were conducted. Three binary microarray datasets are use, which include: colon, leukemia, and lung. In addition, another three multi-class microarray datasets are used, which are: SRBCT, lymphoma, and leukemia. Results of the GBC algorithm are compared with our recently proposed technique: mRMR when combined with the Artificial Bee Colony algorithm (mRMR-ABC). We also compared the combination of mRMR with GA (mRMR-GA) and Particle Swarm Optimization (mRMR-PSO) algorithms. In addition, we compared the GBC algorithm with other related algorithms that have been recently published in the literature, using all benchmark datasets. The GBC algorithm shows superior performance as it achieved the highest classification accuracy along with the lowest average number of selected genes. This proves that the GBC algorithm is a promising approach for solving the gene selection problem in both binary and multi-class cancer classification. Copyright © 2015 Elsevier Ltd. All rights reserved.

Chromosome-level genome assembly and transcriptome of the green alga Chromochloris zofingiensis illuminates astaxanthin production.

PubMed

Roth, Melissa S; Cokus, Shawn J; Gallaher, Sean D; Walter, Andreas; Lopez, David; Erickson, Erika; Endelman, Benjamin; Westcott, Daniel; Larabell, Carolyn A; Merchant, Sabeeha S; Pellegrini, Matteo; Niyogi, Krishna K

2017-05-23

Microalgae have potential to help meet energy and food demands without exacerbating environmental problems. There is interest in the unicellular green alga Chromochloris zofingiensis , because it produces lipids for biofuels and a highly valuable carotenoid nutraceutical, astaxanthin. To advance understanding of its biology and facilitate commercial development, we present a C. zofingiensis chromosome-level nuclear genome, organelle genomes, and transcriptome from diverse growth conditions. The assembly, derived from a combination of short- and long-read sequencing in conjunction with optical mapping, revealed a compact genome of ∼58 Mbp distributed over 19 chromosomes containing 15,274 predicted protein-coding genes. The genome has uniform gene density over chromosomes, low repetitive sequence content (∼6%), and a high fraction of protein-coding sequence (∼39%) with relatively long coding exons and few coding introns. Functional annotation of gene models identified orthologous families for the majority (∼73%) of genes. Synteny analysis uncovered localized but scrambled blocks of genes in putative orthologous relationships with other green algae. Two genes encoding beta-ketolase ( BKT ), the key enzyme synthesizing astaxanthin, were found in the genome, and both were up-regulated by high light. Isolation and molecular analysis of astaxanthin-deficient mutants showed that BKT1 is required for the production of astaxanthin. Moreover, the transcriptome under high light exposure revealed candidate genes that could be involved in critical yet missing steps of astaxanthin biosynthesis, including ABC transporters, cytochrome P450 enzymes, and an acyltransferase. The high-quality genome and transcriptome provide insight into the green algal lineage and carotenoid production.

Chromosome-level genome assembly and transcriptome of the green alga Chromochloris zofingiensis illuminates astaxanthin production

DOE PAGES

Roth, Melissa S.; Cokus, Shawn J.; Gallaher, Sean D.; ...

2017-05-08

Microalgae have potential to help meet energy and food demands without exacerbating environmental problems. There is interest in the unicellular green alga Chromochloris zofingiensis, because it produces lipids for biofuels and a highly valuable carotenoid nutraceutical, astaxanthin. Here, to advance understanding of its biology and facilitate commercial development, we present a C. zofingiensis chromosome-level nuclear genome, organelle genomes, and transcriptome from diverse growth conditions. The assembly, derived from a combination of short- and long-read sequencing in conjunction with optical mapping, revealed a compact genome of ~58 Mbp distributed over 19 chromosomes containing 15,274 predicted protein-coding genes. The genome has uniformmore » gene density over chromosomes, low repetitive sequence content (~6%), and a high fraction of protein-coding sequence (~39%) with relatively long coding exons and few coding introns. Functional annotation of gene models identified orthologous families for the majority (~73%) of genes. Synteny analysis uncovered localized but scrambled blocks of genes in putative orthologous relationships with other green algae. Two genes encoding beta-ketolase (BKT), the key enzyme synthesizing astaxanthin, were found in the genome, and both were up-regulated by high light. Isolation and molecular analysis of astaxanthin-deficient mutants showed that BKT1 is required for the production of astaxanthin. Moreover, the transcriptome under high light exposure revealed candidate genes that could be involved in critical yet missing steps of astaxanthin biosynthesis, including ABC transporters, cytochrome P450 enzymes, and an acyltransferase. Finally, the high-quality genome and transcriptome provide insight into the green algal lineage and carotenoid production.« less

Chromosome-level genome assembly and transcriptome of the green alga Chromochloris zofingiensis illuminates astaxanthin production

DOE Office of Scientific and Technical Information (OSTI.GOV)

Roth, Melissa S.; Cokus, Shawn J.; Gallaher, Sean D.

Microalgae have potential to help meet energy and food demands without exacerbating environmental problems. There is interest in the unicellular green alga Chromochloris zofingiensis, because it produces lipids for biofuels and a highly valuable carotenoid nutraceutical, astaxanthin. Here, to advance understanding of its biology and facilitate commercial development, we present a C. zofingiensis chromosome-level nuclear genome, organelle genomes, and transcriptome from diverse growth conditions. The assembly, derived from a combination of short- and long-read sequencing in conjunction with optical mapping, revealed a compact genome of ~58 Mbp distributed over 19 chromosomes containing 15,274 predicted protein-coding genes. The genome has uniformmore » gene density over chromosomes, low repetitive sequence content (~6%), and a high fraction of protein-coding sequence (~39%) with relatively long coding exons and few coding introns. Functional annotation of gene models identified orthologous families for the majority (~73%) of genes. Synteny analysis uncovered localized but scrambled blocks of genes in putative orthologous relationships with other green algae. Two genes encoding beta-ketolase (BKT), the key enzyme synthesizing astaxanthin, were found in the genome, and both were up-regulated by high light. Isolation and molecular analysis of astaxanthin-deficient mutants showed that BKT1 is required for the production of astaxanthin. Moreover, the transcriptome under high light exposure revealed candidate genes that could be involved in critical yet missing steps of astaxanthin biosynthesis, including ABC transporters, cytochrome P450 enzymes, and an acyltransferase. Finally, the high-quality genome and transcriptome provide insight into the green algal lineage and carotenoid production.« less

Chromosome-level genome assembly and transcriptome of the green alga Chromochloris zofingiensis illuminates astaxanthin production

PubMed Central

Roth, Melissa S.; Cokus, Shawn J.; Gallaher, Sean D.; Walter, Andreas; Lopez, David; Erickson, Erika; Endelman, Benjamin; Westcott, Daniel; Larabell, Carolyn A.; Merchant, Sabeeha S.; Pellegrini, Matteo

2017-01-01

Microalgae have potential to help meet energy and food demands without exacerbating environmental problems. There is interest in the unicellular green alga Chromochloris zofingiensis, because it produces lipids for biofuels and a highly valuable carotenoid nutraceutical, astaxanthin. To advance understanding of its biology and facilitate commercial development, we present a C. zofingiensis chromosome-level nuclear genome, organelle genomes, and transcriptome from diverse growth conditions. The assembly, derived from a combination of short- and long-read sequencing in conjunction with optical mapping, revealed a compact genome of ∼58 Mbp distributed over 19 chromosomes containing 15,274 predicted protein-coding genes. The genome has uniform gene density over chromosomes, low repetitive sequence content (∼6%), and a high fraction of protein-coding sequence (∼39%) with relatively long coding exons and few coding introns. Functional annotation of gene models identified orthologous families for the majority (∼73%) of genes. Synteny analysis uncovered localized but scrambled blocks of genes in putative orthologous relationships with other green algae. Two genes encoding beta-ketolase (BKT), the key enzyme synthesizing astaxanthin, were found in the genome, and both were up-regulated by high light. Isolation and molecular analysis of astaxanthin-deficient mutants showed that BKT1 is required for the production of astaxanthin. Moreover, the transcriptome under high light exposure revealed candidate genes that could be involved in critical yet missing steps of astaxanthin biosynthesis, including ABC transporters, cytochrome P450 enzymes, and an acyltransferase. The high-quality genome and transcriptome provide insight into the green algal lineage and carotenoid production. PMID:28484037

Inter-phylum HGT has shaped the metabolism of many mesophilic and anaerobic bacteria.

PubMed

Caro-Quintero, Alejandro; Konstantinidis, Konstantinos T

2015-03-17

Genome sequencing has revealed that horizontal gene transfer (HGT) is a major evolutionary process in bacteria. Although it is generally assumed that closely related organisms engage in genetic exchange more frequently than distantly related ones, the frequency of HGT among distantly related organisms and the effect of ecological relatedness on the frequency has not been rigorously assessed. Here, we devised a novel bioinformatic pipeline, which minimized the effect of over-representation of specific taxa in the available databases and other limitations of homology-based approaches by analyzing genomes in standardized triplets, to quantify gene exchange between bacterial genomes representing different phyla. Our analysis revealed the existence of networks of genetic exchange between organisms with overlapping ecological niches, with mesophilic anaerobic organisms showing the highest frequency of exchange and engaging in HGT twice as frequently as their aerobic counterparts. Examination of individual cases suggested that inter-phylum HGT is more pronounced than previously thought, affecting up to ∼ 16% of the total genes and ∼ 35% of the metabolic genes in some genomes (conservative estimation). In contrast, ribosomal and other universal protein-coding genes were subjected to HGT at least 150 times less frequently than genes encoding the most promiscuous metabolic functions (for example, various dehydrogenases and ABC transport systems), suggesting that the species tree based on the former genes may be reliable. These results indicated that the metabolic diversity of microbial communities within most habitats has been largely assembled from preexisting genetic diversity through HGT and that HGT accounts for the functional redundancy among phyla.

A global analysis of protein expression profiles in Sinorhizobium meliloti: discovery of new genes for nodule occupancy and stress adaptation.

PubMed

Djordjevic, Michael A; Chen, Han Cai; Natera, Siria; Van Noorden, Giel; Menzel, Christian; Taylor, Scott; Renard, Clotilde; Geiger, Otto; Weiller, Georg F

2003-06-01

A proteomic examination of Sinorhizobium meliloti strain 1021 was undertaken using a combination of 2-D gel electrophoresis, peptide mass fingerprinting, and bioinformatics. Our goal was to identify (i) putative symbiosis- or nutrient-stress-specific proteins, (ii) the biochemical pathways active under different conditions, (iii) potential new genes, and (iv) the extent of posttranslational modifications of S. meliloti proteins. In total, we identified the protein products of 810 genes (13.1% of the genome's coding capacity). The 810 genes generated 1,180 gene products, with chromosomal genes accounting for 78% of the gene products identified (18.8% of the chromosome's coding capacity). The activity of 53 metabolic pathways was inferred from bioinformatic analysis of proteins with assigned Enzyme Commission numbers. Of the remaining proteins that did not encode enzymes, ABC-type transporters composed 12.7% and regulatory proteins 3.4% of the total. Proteins with up to seven transmembrane domains were identified in membrane preparations. A total of 27 putative nodule-specific proteins and 35 nutrient-stress-specific proteins were identified and used as a basis to define genes and describe processes occurring in S. meliloti cells in nodules and under stress. Several nodule proteins from the plant host were present in the nodule bacteria preparations. We also identified seven potentially novel proteins not predicted from the DNA sequence. Post-translational modifications such as N-terminal processing could be inferred from the data. The posttranslational addition of UMP to the key regulator of nitrogen metabolism, PII, was demonstrated. This work demonstrates the utility of combining mass spectrometry with protein arraying or separation techniques to identify candidate genes involved in important biological processes and niche occupations that may be intransigent to other methods of gene expression profiling.

[Detection of putative polysaccharide biosynthesis genes in Azospirillum brasilense strains from serogroups I and II].

PubMed

Petrova, L P; Prilipov, A G; Katsy, E I

2017-01-01

It is known that in Azospirillum brasilense strains Sp245 and SR75 included in serogroup I, the repeat units of their O-polysaccharides consist of five residues of D-rhamnose, and in strain SR15, of four; and the heteropolymeric O-polysaccharide of A. brasilense type strain Sp7 from serogroup II contains not less than five types of repeat units. In the present work, a complex of nondegenerate primers to the genes of A. brasilense Sp245 plasmids AZOBR_p6, AZOBR_p3, and AZOBR_p2, which encode putative enzymes for the biosynthesis of core oligosaccharide and O-polysaccharide of lipopolysaccharide, capsular polysaccharides, and exopolysaccharides, was proposed. By using the designed primers, products of the expected sizes were synthesized in polymerase chain reactions on genomic DNA of A. brasilense Sp245, SR75, SR15, and Sp7 in 36, 29, 23, and 12 cases, respectively. As a result of sequencing of a number of amplicons, a high (86–99%) level of identity of the corresponding putative polysaccharide biosynthesis genes in three A. brasilense strains from serogroup I was detected. In a blotting-hybridization reaction with the biotin-labeled DNA of the A. brasilense gene AZOBR_p60122 coding for putative permease of the ABC transporter of polysaccharides, localization of the homologous gene in ~120-MDa plasmids of the bacteria A. brasilense SR15 and SR75 was revealed.

Transcriptional regulation of NAD metabolism in bacteria: genomic reconstruction of NiaR (YrxA) regulon

PubMed Central

Rodionov, Dmitry A.; Li, Xiaoqing; Rodionova, Irina A.; Yang, Chen; Sorci, Leonardo; Dervyn, Etienne; Martynowski, Dariusz; Zhang, Hong; Gelfand, Mikhail S.; Osterman, Andrei L.

2008-01-01

A comparative genomic approach was used to reconstruct transcriptional regulation of NAD biosynthesis in bacteria containing orthologs of Bacillus subtilis gene yrxA, a previously identified niacin-responsive repressor of NAD de novo synthesis. Members of YrxA family (re-named here NiaR) are broadly conserved in the Bacillus/Clostridium group and in the deeply branching Fusobacteria and Thermotogales lineages. We analyzed upstream regions of genes associated with NAD biosynthesis to identify candidate NiaR-binding DNA motifs and assess the NiaR regulon content in these species. Representatives of the two distinct types of candidate NiaR-binding sites, characteristic of the Firmicutes and Thermotogales, were verified by an electrophoretic mobility shift assay. In addition to transcriptional control of the nadABC genes, the NiaR regulon in some species extends to niacin salvage (the pncAB genes) and includes uncharacterized membrane proteins possibly involved in niacin transport. The involvement in niacin uptake proposed for one of these proteins (re-named NiaP), encoded by the B. subtilis gene yceI, was experimentally verified. In addition to bacteria, members of the NiaP family are conserved in multicellular eukaryotes, including human, pointing to possible NaiP involvement in niacin utilization in these organisms. Overall, the analysis of the NiaR and NrtR regulons (described in the accompanying paper) revealed mechanisms of transcriptional regulation of NAD metabolism in nearly a hundred diverse bacteria. PMID:18276644

BraLTP1, a Lipid Transfer Protein Gene Involved in Epicuticular Wax Deposition, Cell Proliferation and Flower Development in Brassica napus

PubMed Central

Liu, Fang; Xiong, Xiaojuan; Wu, Lei; Fu, Donghui; Hayward, Alice; Zeng, Xinhua; Cao, Yinglong; Wu, Yuhua; Li, Yunjing; Wu, Gang

2014-01-01

Plant non-specific lipid transfer proteins (nsLTPs) constitute large multigene families that possess complex physiological functions, many of which remain unclear. This study isolated and characterized the function of a lipid transfer protein gene, BraLTP1 from Brassica rapa, in the important oilseed crops Brassica napus. BraLTP1 encodes a predicted secretory protein, in the little known VI Class of nsLTP families. Overexpression of BnaLTP1 in B. napus caused abnormal green coloration and reduced wax deposition on leaves and detailed wax analysis revealed 17–80% reduction in various major wax components, which resulted in significant water-loss relative to wild type. BnaLTP1 overexpressing leaves exhibited morphological disfiguration and abaxially curled leaf edges, and leaf cross-sections revealed cell overproliferation that was correlated to increased cytokinin levels (tZ, tZR, iP, and iPR) in leaves and high expression of the cytokinin biosynthsis gene IPT3. BnaLTP1-overexpressing plants also displayed morphological disfiguration of flowers, with early-onset and elongated carpel development and outwardly curled stamen. This was consistent with altered expression of a a number of ABC model genes related to flower development. Together, these results suggest that BraLTP1 is a new nsLTP gene involved in wax production or deposition, with additional direct or indirect effects on cell division and flower development. PMID:25314222

Global Screening of Salmonella enterica Serovar Typhimurium Genes for Desiccation Survival

PubMed Central

Mandal, Rabindra K.; Kwon, Young M.

2017-01-01

Salmonella spp., one of the most common foodborne bacterial pathogens, has the ability to survive under desiccation conditions in foods and food processing facilities for years. This raises the concerns of Salmonella infection in humans associated with low water activity foods. Salmonella responds to desiccation stress via complex pathways involving immediate physiological actions as well as coordinated genetic responses. However, the exact mechanisms of Salmonella to resist desiccation stress remain to be fully elucidated. In this study, we screened a genome-saturating transposon (Tn5) library of Salmonella Typhimurium (S. Typhimurium) 14028s under the in vitro desiccation stress using transposon sequencing (Tn-seq). We identified 61 genes and 6 intergenic regions required to overcome desiccation stress. Salmonella desiccation resistance genes were mostly related to energy production and conversion; cell wall/membrane/envelope biogenesis; inorganic ion transport and metabolism; regulation of biological process; DNA metabolic process; ABC transporters; and two component system. More than 20% of the Salmonella desiccation resistance genes encode either putative or hypothetical proteins. Phenotypic evaluation of 12 single gene knockout mutants showed 3 mutants (atpH, atpG, and corA) had significantly (p < 0.02) reduced survival as compared to the wild type during desiccation survival. Thus, our study provided new insights into the molecular mechanisms utilized by Salmonella for survival against desiccation stress. The findings might be further exploited to develop effective control strategies against Salmonella contamination in low water activity foods and food processing facilities. PMID:28943871

A Ferredoxin- and F 420 H 2 -Dependent, Electron-Bifurcating, Heterodisulfide Reductase with Homologs in the Domains Bacteria and Archaea

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yan, Zhen; Wang, Mingyu; Ferry, James G.

Heterodisulfide reductases (Hdr) of the HdrABC class are ancient enzymes and a component of the anaerobic core belonging to the prokaryotic common ancestor. The ancient origin is consistent with the widespread occurrence of genes encoding putative HdrABC homologs in metabolically diverse prokaryotes predicting diverse physiological functions; but, only one HdrABC has been characterized and that was from a narrow metabolic group of obligate CO 2-reducing methanogenic anaerobes (methanogens) from the domainArchaea. Here we report the biochemical characterization of an HdrABC homolog (HdrA2B2C2) from the acetate-utilizing methanogenMethanosarcina acetivoranswith unusual properties structurally and functionally distinct from the only other HdrABC characterized. Homologsmore » of the HdrA2B2C2 archetype are present in phylogenetically and metabolically diverse species from the domainsBacteriaandArchaea. The expression of the individual HdrA2, HdrB2, and HdrB2C2 enzymes inEscherichia coli, and reconstitution of an active HdrA2B2C2 complex, revealed an intersubunit electron transport pathway dependent on ferredoxin or coenzyme F 420(F 420H 2) as an electron donor. Remarkably, HdrA2B2C2 couples the previously unknown endergonic oxidation of F 420H 2and reduction of ferredoxin with the exergonic oxidation of F 420H 2and reduction of the heterodisulfide of coenzyme M and coenzyme B (CoMS-SCoB). The unique electron bifurcation predicts a role for HdrA2B2C2 in Fe(III)-dependent anaerobic methane oxidation (ANME) byM. acetivoransand uncultured species from ANME environments. HdrA2B2C2, ubiquitous in acetotrophic methanogens, was shown to participate in electron transfer during acetotrophic growth ofM. acetivoransand proposed to be essential for growth in the environment when acetate is limiting.IMPORTANCEDiscovery of the archetype HdrA2B2C2 heterodisulfide reductase with categorically unique properties extends the understanding of this ancient family beyond CO 2-reducing methanogens to include diverse prokaryotes from the domainsBacteriaandArchaea. Furthermore, the unprecedented coenzyme F 420-dependent electron bifurcation, an emerging fundamental principle of energy conservation, predicts a role for HdrA2B2C2 in diverse metabolisms, including anaerobic CH 4-oxidizing pathways. Our results document an electron transport role for HdrA2B2C2 in acetate-utilizing methanogens responsible for at least two-thirds of the methane produced in Earth’s biosphere. The previously unavailable heterologous production of individual subunits and the reconstitution of HdrA2B2C2 with activity have provided an understanding of intersubunit electron transfer in the HdrABC class and a platform for investigating the principles of electron bifurcation.« less

A Ferredoxin- and F 420 H 2 -Dependent, Electron-Bifurcating, Heterodisulfide Reductase with Homologs in the Domains Bacteria and Archaea

DOE PAGES

Yan, Zhen; Wang, Mingyu; Ferry, James G.; ...

2017-02-07

Heterodisulfide reductases (Hdr) of the HdrABC class are ancient enzymes and a component of the anaerobic core belonging to the prokaryotic common ancestor. The ancient origin is consistent with the widespread occurrence of genes encoding putative HdrABC homologs in metabolically diverse prokaryotes predicting diverse physiological functions; but, only one HdrABC has been characterized and that was from a narrow metabolic group of obligate CO 2-reducing methanogenic anaerobes (methanogens) from the domainArchaea. Here we report the biochemical characterization of an HdrABC homolog (HdrA2B2C2) from the acetate-utilizing methanogenMethanosarcina acetivoranswith unusual properties structurally and functionally distinct from the only other HdrABC characterized. Homologsmore » of the HdrA2B2C2 archetype are present in phylogenetically and metabolically diverse species from the domainsBacteriaandArchaea. The expression of the individual HdrA2, HdrB2, and HdrB2C2 enzymes inEscherichia coli, and reconstitution of an active HdrA2B2C2 complex, revealed an intersubunit electron transport pathway dependent on ferredoxin or coenzyme F 420(F 420H 2) as an electron donor. Remarkably, HdrA2B2C2 couples the previously unknown endergonic oxidation of F 420H 2and reduction of ferredoxin with the exergonic oxidation of F 420H 2and reduction of the heterodisulfide of coenzyme M and coenzyme B (CoMS-SCoB). The unique electron bifurcation predicts a role for HdrA2B2C2 in Fe(III)-dependent anaerobic methane oxidation (ANME) byM. acetivoransand uncultured species from ANME environments. HdrA2B2C2, ubiquitous in acetotrophic methanogens, was shown to participate in electron transfer during acetotrophic growth ofM. acetivoransand proposed to be essential for growth in the environment when acetate is limiting.IMPORTANCEDiscovery of the archetype HdrA2B2C2 heterodisulfide reductase with categorically unique properties extends the understanding of this ancient family beyond CO 2-reducing methanogens to include diverse prokaryotes from the domainsBacteriaandArchaea. Furthermore, the unprecedented coenzyme F 420-dependent electron bifurcation, an emerging fundamental principle of energy conservation, predicts a role for HdrA2B2C2 in diverse metabolisms, including anaerobic CH 4-oxidizing pathways. Our results document an electron transport role for HdrA2B2C2 in acetate-utilizing methanogens responsible for at least two-thirds of the methane produced in Earth’s biosphere. The previously unavailable heterologous production of individual subunits and the reconstitution of HdrA2B2C2 with activity have provided an understanding of intersubunit electron transfer in the HdrABC class and a platform for investigating the principles of electron bifurcation.« less

A Ferredoxin- and F420H2-Dependent, Electron-Bifurcating, Heterodisulfide Reductase with Homologs in the Domains Bacteria and Archaea.

PubMed

Yan, Zhen; Wang, Mingyu; Ferry, James G

2017-02-07

Heterodisulfide reductases (Hdr) of the HdrABC class are ancient enzymes and a component of the anaerobic core belonging to the prokaryotic common ancestor. The ancient origin is consistent with the widespread occurrence of genes encoding putative HdrABC homologs in metabolically diverse prokaryotes predicting diverse physiological functions; however, only one HdrABC has been characterized and that was from a narrow metabolic group of obligate CO 2 -reducing methanogenic anaerobes (methanogens) from the domain Archaea Here we report the biochemical characterization of an HdrABC homolog (HdrA2B2C2) from the acetate-utilizing methanogen Methanosarcina acetivorans with unusual properties structurally and functionally distinct from the only other HdrABC characterized. Homologs of the HdrA2B2C2 archetype are present in phylogenetically and metabolically diverse species from the domains Bacteria and Archaea The expression of the individual HdrA2, HdrB2, and HdrB2C2 enzymes in Escherichia coli, and reconstitution of an active HdrA2B2C2 complex, revealed an intersubunit electron transport pathway dependent on ferredoxin or coenzyme F 420 (F 420 H 2 ) as an electron donor. Remarkably, HdrA2B2C2 couples the previously unknown endergonic oxidation of F 420 H 2 and reduction of ferredoxin with the exergonic oxidation of F 420 H 2 and reduction of the heterodisulfide of coenzyme M and coenzyme B (CoMS-SCoB). The unique electron bifurcation predicts a role for HdrA2B2C2 in Fe(III)-dependent anaerobic methane oxidation (ANME) by M. acetivorans and uncultured species from ANME environments. HdrA2B2C2, ubiquitous in acetotrophic methanogens, was shown to participate in electron transfer during acetotrophic growth of M. acetivorans and proposed to be essential for growth in the environment when acetate is limiting. Discovery of the archetype HdrA2B2C2 heterodisulfide reductase with categorically unique properties extends the understanding of this ancient family beyond CO 2 -reducing methanogens to include diverse prokaryotes from the domains Bacteria and Archaea The unprecedented coenzyme F 420 -dependent electron bifurcation, an emerging fundamental principle of energy conservation, predicts a role for HdrA2B2C2 in diverse metabolisms, including anaerobic CH 4 -oxidizing pathways. The results document an electron transport role for HdrA2B2C2 in acetate-utilizing methanogens responsible for at least two-thirds of the methane produced in Earth's biosphere. The previously unavailable heterologous production of individual subunits and the reconstitution of HdrA2B2C2 with activity have provided an understanding of intersubunit electron transfer in the HdrABC class and a platform for investigating the principles of electron bifurcation. Copyright © 2017 Yan et al.

TaABC1, a member of the activity of bc1 complex protein kinase family from common wheat, confers enhanced tolerance to abiotic stresses in Arabidopsis

PubMed Central

Wang, Caixiang; Jing, Ruilian; Mao, Xinguo; Chang, Xiaoping; Li, Ang

2011-01-01

Abiotic stresses such as drought, salinity, and low temperature have drastic effects on plant growth and development. However, the molecular mechanisms regulating biochemical and physiological changes in response to stresses are not well understood. Protein kinases are major signal transduction factors among the reported molecular mechanisms mediating acclimation to environmental changes. Protein kinase ABC1 (activity of bc1 complex) is involved in regulating coenzyme Q biosynthesis in mitochondria in yeast (Saccharomyces cersvisiae), and in balancing oxidative stress in chloroplasts in Arabidopsis thaliana. In the current study, TaABC1 (Triticum aestivum L. activity of bc1 complex) protein kinase was localized to the cell membrane, cytoplasm, and nucleus. The effects of overexpressing TaABC1 in transgenic Arabidopsis plants on responses to drought, salt, and cold stress were further investigated. Transgenic Arabidopsis overexpressing the TaABC1 protein showed lower water loss and higher osmotic potential, photochemistry efficiency, and chlorophyll content, while cell membrane stability and controlled reactive oxygen species homeostasis were maintained. In addition, overexpression of TaABC1 increased the expression of stress-responsive genes, such as DREB1A, DREB2A, RD29A, ABF3, KIN1, CBF1, LEA, and P5CS, detected by real-time PCR analysis. The results suggest that TaABC1 overexpression enhances drought, salt, and cold stress tolerance in Arabidopsis, and imply that TaABC1 may act as a regulatory factor involved in a multiple stress response pathways. PMID:21115661

An ATP-binding cassette subfamily G full transporter is essential for the retention of leaf water in both wild barley and rice.

PubMed

Chen, Guoxiong; Komatsuda, Takao; Ma, Jian Feng; Nawrath, Christiane; Pourkheirandish, Mohammad; Tagiri, Akemi; Hu, Yin-Gang; Sameri, Mohammad; Li, Xinrong; Zhao, Xin; Liu, Yubing; Li, Chao; Ma, Xiaoying; Wang, Aidong; Nair, Sudha; Wang, Ning; Miyao, Akio; Sakuma, Shun; Yamaji, Naoki; Zheng, Xiuting; Nevo, Eviatar

2011-07-26

Land plants have developed a cuticle preventing uncontrolled water loss. Here we report that an ATP-binding cassette (ABC) subfamily G (ABCG) full transporter is required for leaf water conservation in both wild barley and rice. A spontaneous mutation, eibi1.b, in wild barley has a low capacity to retain leaf water, a phenotype associated with reduced cutin deposition and a thin cuticle. Map-based cloning revealed that Eibi1 encodes an HvABCG31 full transporter. The gene was highly expressed in the elongation zone of a growing leaf (the site of cutin synthesis), and its gene product also was localized in developing, but not in mature tissue. A de novo wild barley mutant named "eibi1.c," along with two transposon insertion lines of rice mutated in the ortholog of HvABCG31 also were unable to restrict water loss from detached leaves. HvABCG31 is hypothesized to function as a transporter involved in cutin formation. Homologs of HvABCG31 were found in green algae, moss, and lycopods, indicating that this full transporter is highly conserved in the evolution of land plants.

Benzene degradation in a denitrifying biofilm reactor: activity and microbial community composition.

PubMed

van der Waals, Marcelle J; Atashgahi, Siavash; da Rocha, Ulisses Nunes; van der Zaan, Bas M; Smidt, Hauke; Gerritse, Jan

2017-06-01

Benzene is an aromatic compound and harmful for the environment. Biodegradation of benzene can reduce the toxicological risk after accidental or controlled release of this chemical in the environment. In this study, we further characterized an anaerobic continuous biofilm culture grown for more than 14 years on benzene with nitrate as electron acceptor. We determined steady state degradation rates, microbial community composition dynamics in the biofilm, and the initial anaerobic benzene degradation reactions. Benzene was degraded at a rate of 0.15 μmol/mg protein/day and a first-order rate constant of 3.04/day which was fourfold higher than rates reported previously. Bacteria belonging to the Peptococcaceae were found to play an important role in this anaerobic benzene-degrading biofilm culture, but also members of the Anaerolineaceae were predicted to be involved in benzene degradation or benzene metabolite degradation based on Illumina MiSeq analysis of 16S ribosomal RNA genes. Biomass retention in the reactor using a filtration finger resulted in reduction of benzene degradation capacity. Detection of the benzene carboxylase encoding gene, abcA, and benzoic acid in the culture vessel indicated that benzene degradation proceeds through an initial carboxylation step.

An ATP-binding cassette subfamily G full transporter is essential for the retention of leaf water in both wild barley and rice

PubMed Central

Chen, Guoxiong; Komatsuda, Takao; Ma, Jian Feng; Nawrath, Christiane; Pourkheirandish, Mohammad; Tagiri, Akemi; Hu, Yin-Gang; Sameri, Mohammad; Li, Xinrong; Zhao, Xin; Liu, Yubing; Li, Chao; Ma, Xiaoying; Wang, Aidong; Nair, Sudha; Wang, Ning; Miyao, Akio; Sakuma, Shun; Yamaji, Naoki; Zheng, Xiuting; Nevo, Eviatar

2011-01-01

Land plants have developed a cuticle preventing uncontrolled water loss. Here we report that an ATP-binding cassette (ABC) subfamily G (ABCG) full transporter is required for leaf water conservation in both wild barley and rice. A spontaneous mutation, eibi1.b, in wild barley has a low capacity to retain leaf water, a phenotype associated with reduced cutin deposition and a thin cuticle. Map-based cloning revealed that Eibi1 encodes an HvABCG31 full transporter. The gene was highly expressed in the elongation zone of a growing leaf (the site of cutin synthesis), and its gene product also was localized in developing, but not in mature tissue. A de novo wild barley mutant named “eibi1.c,” along with two transposon insertion lines of rice mutated in the ortholog of HvABCG31 also were unable to restrict water loss from detached leaves. HvABCG31 is hypothesized to function as a transporter involved in cutin formation. Homologs of HvABCG31 were found in green algae, moss, and lycopods, indicating that this full transporter is highly conserved in the evolution of land plants. PMID:21737747

Transcriptome Analysis of the Brucella abortus BvrR/BvrS Two-Component Regulatory System

PubMed Central

Viadas, Cristina; Rodríguez, María C.; Sangari, Felix J.; Gorvel, Jean-Pierre; García-Lobo, Juan M.; López-Goñi, Ignacio

2010-01-01

Background The two-component BvrR/BvrS system is essential for Brucella abortus virulence. It was shown previously that its dysfunction alters the expression of some major outer membrane proteins and the pattern of lipid A acylation. To determine the genes regulated by BvrR/BvrS, we performed a whole-genome microarray analysis using B. abortus RNA obtained from wild type and bvrR mutant cells grown in the same conditions. Methodology/Principal Findings A total of 127 differentially expressed genes were found: 83 were over expressed and 44 were less expressed in the bvrR mutant. Two operons, the phosphotransferase system and the maltose transport system, were down-regulated. Several genes involved in cell envelope or outer membrane biogenesis were differentially expressed: genes for outer membrane proteins (omp25a, omp25d), lipoproteins, LPS and fatty acid biosynthesis, stress response proteins, chaperones, flagellar genes, and twelve genes encoding ABC transport systems. Ten genes related with carbon metabolism (pckA and fumB among others) were up-regulated in the bvrR mutant, and denitrification genes (nirK, norC and nosZ) were also regulated. Notably, seven transcriptional regulators were affected, including VjbR, ExoR and OmpR that were less expressed in the bvrR mutant. Finally, the expression of eleven genes which have been previously related with Brucella virulence was also altered. Conclusions/Significance All these data corroborate the impact of BvrR/BvrS on cell envelope modulation, confirm that this system controls the carbon and nitrogen metabolism, and suggest a cross-talk among some regulators to adjust the Brucella physiology to the shift expected to occur during the transit from the extracellular to the intracellular niche. PMID:20422049

NtPDR3, an iron-deficiency inducible ABC transporter in Nicotiana tabacum.

PubMed

Ducos, Eric; Fraysse, Staffan; Boutry, Marc

2005-12-19

In plants, the ABC transporter PDR (pleiotropic drug resistance) subfamily is composed of approximately 15 genes, few of which have been analyzed. We have identified NtPDR3, a Nicotiana tabacum PDR gene belonging to a cluster for which no functional data was previously available. NtPDR3 was found to be induced in suspension cells treated with methyl jasmonate, salicylic acid, 1-naphthalene acetic acid, or cembrene, a macrocyclic diterpene. In agreement with the identification of a putative iron deficiency element in the NtPDR3 transcription promoter region, we found that iron deficiency in the culture medium induced NtPDR3 expression, thus suggesting a new function of the PDR transporter family.

Characterization of Multi-Drug Resistant Enterococcus faecalis Isolated from Cephalic Recording Chambers in Research Macaques (Macaca spp.).

PubMed

Woods, Stephanie E; Lieberman, Mia T; Lebreton, Francois; Trowel, Elise; de la Fuente-Núñez, César; Dzink-Fox, Joanne; Gilmore, Michael S; Fox, James G

2017-01-01

Nonhuman primates are commonly used for cognitive neuroscience research and often surgically implanted with cephalic recording chambers for electrophysiological recording. Aerobic bacterial cultures from 25 macaques identified 72 bacterial isolates, including 15 Enterococcus faecalis isolates. The E. faecalis isolates displayed multi-drug resistant phenotypes, with resistance to ciprofloxacin, enrofloxacin, trimethoprim-sulfamethoxazole, tetracycline, chloramphenicol, bacitracin, and erythromycin, as well as high-level aminoglycoside resistance. Multi-locus sequence typing showed that most belonged to two E. faecalis sequence types (ST): ST 4 and ST 55. The genomes of three representative isolates were sequenced to identify genes encoding antimicrobial resistances and other traits. Antimicrobial resistance genes identified included aac(6')-aph(2"), aph(3')-III, str, ant(6)-Ia, tetM, tetS, tetL, ermB, bcrABR, cat, and dfrG, and polymorphisms in parC (S80I) and gyrA (S83I) were observed. These isolates also harbored virulence factors including the cytolysin toxin genes in ST 4 isolates, as well as multiple biofilm-associated genes (esp, agg, ace, SrtA, gelE, ebpABC), hyaluronidases (hylA, hylB), and other survival genes (ElrA, tpx). Crystal violet biofilm assays confirmed that ST 4 isolates produced more biofilm than ST 55 isolates. The abundance of antimicrobial resistance and virulence factor genes in the ST 4 isolates likely relates to the loss of CRISPR-cas. This macaque colony represents a unique model for studying E. faecalis infection associated with indwelling devices, and provides an opportunity to understand the basis of persistence of this pathogen in a healthcare setting.

[Polymorphisms of killer cell immunoglobulin-like receptor and its ligand HLA-I gene among northern Chinese Han population].

PubMed

Wu, Lingyan; Xie, Zhengde; Liu, Yali; Ai, Junhong; Liu, Chunyan; Shen, Kunling

2015-10-01

OBJECTIVE To investigate the distribution of killer cell immunoglobulin-like receptors (KIR) and their specific ligands human leukocyte antigen-I (HLA-I) gene in northern China. METHODS One hundred and eighty-four unrelated northern Chinese Han individuals were recruited. Genotypes of the KIR and HLA-ABC genes were studied by sequence-specific primer polymerase chain reaction (SSP-PCR). RESULTS Sixteen KIR genes were detected among the 184 unrelated individuals. In all individuals, the four framework genes were present. The frequencies for those carrying the remaining 12 KIR genes have ranged from 16.3% to 99.5%. Twenty-four KIR genotypes were identified, for which half were detected in a single individual. A new genotype comprised of KIR2DL3, 3DL1, 2DP1 and the framework genes was detected in one subject. Respectively, 12, 27 and 11 specificities of HLA alleles were identified on the HLA-A, B, C loci. CONCLUSION The distribution of polymorphisms of KIR and its ligand HLA-ABC genes among northern Chinese Han population have been ascertained. The frequencies of 9 KIR/HLA combinations in the above population have been determined for the first time.

Novel organization of the common nodulation genes in Rhizobium leguminosarum bv. phaseoli strains.

PubMed Central

Vázquez, M; Dávalos, A; de las Peñas, A; Sánchez, F; Quinto, C

1991-01-01

Nodulation by Rhizobium, Bradyrhizobium, and Azorhizobium species in the roots of legumes and nonlegumes requires the proper expression of plant genes and of both common and specific bacterial nodulation genes. The common nodABC genes form an operon or are physically mapped together in all species studied thus far. Rhizobium leguminosarum bv. phaseoli strains are classified in two groups. The type I group has reiterated nifHDK genes and a narrow host range of nodulation. The type II group has a single copy of the nifHDK genes and a wide host range of nodulation. We have found by genetic and nucleotide sequence analysis that in type I strain CE-3, the functional common nodA gene is separated from the nodBC genes by 20 kb and thus is transcriptionally separated from the latter genes. This novel organization could be the result of a complex rearrangement, as we found zones of identity between the two separated nodA and nodBC regions. Moreover, this novel organization of the common nodABC genes seems to be a general characteristic of R. leguminosarum bv. phaseoli type I strains. Despite the separation, the coordination of the expression of these genes seems not to be altered. PMID:1991718

Accumulation of murine amyloid-β mimics early Alzheimer's disease.

PubMed

Krohn, Markus; Bracke, Alexander; Avchalumov, Yosef; Schumacher, Toni; Hofrichter, Jacqueline; Paarmann, Kristin; Fröhlich, Christina; Lange, Cathleen; Brüning, Thomas; von Bohlen Und Halbach, Oliver; Pahnke, Jens

2015-08-01

Amyloidosis mouse models of Alzheimer's disease are generally established by transgenic approaches leading to an overexpression of mutated human genes that are known to be involved in the generation of amyloid-β in Alzheimer's families. Although these models made substantial contributions to the current knowledge about the 'amyloid hypothesis' of Alzheimer's disease, the overproduction of amyloid-β peptides mimics only inherited (familiar) Alzheimer's disease, which accounts for <1% of all patients with Alzheimer's disease. The inherited form is even regarded a 'rare' disease according to the regulations for funding of the European Union (www.erare.eu). Here, we show that mice that are double-deficient for neprilysin (encoded by Mme), one major amyloid-β-degrading enzyme, and the ABC transporter ABCC1, a major contributor to amyloid-β clearance from the brain, develop various aspects of sporadic Alzheimer's disease mimicking the clinical stage of mild cognitive impairment. Using behavioural tests, electrophysiology and morphological analyses, we compared different ABC transporter-deficient animals and found that alterations are most prominent in neprilysin × ABCC1 double-deficient mice. We show that these mice have a reduced probability to survive, show increased anxiety in new environments, and have a reduced working memory performance. Furthermore, we detected morphological changes in the hippocampus and amygdala, e.g. astrogliosis and reduced numbers of synapses, leading to defective long-term potentiation in functional measurements. Compared to human, murine amyloid-β is poorly aggregating, due to changes in three amino acids at N-terminal positions 5, 10, and 13. Interestingly, our findings account for the action of early occurring amyloid-β species/aggregates, i.e. monomers and small amyloid-β oligomers. Thus, neprilysin × ABCC1 double-deficient mice present a new model for early effects of amyloid-β-related mild cognitive impairment that allows investigations without artificial overexpression of inherited Alzheimer's disease genes. © The Author (2015). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

A novel transformation system using a bleomycin resistance marker with chemosensitizers for Aspergillus oryzae.

PubMed

Suzuki, Satoshi; Tada, Sawaki; Fukuoka, Mari; Taketani, Hiroko; Tsukakoshi, Yoshiki; Matsushita, Mayumi; Oda, Kosuke; Kusumoto, Ken-Ichi; Kashiwagi, Yutaka; Sugiyama, Masanori

2009-05-22

Aspergillus oryzae is resistant to many kinds of antibiotics, which hampers their use to select transformants. In fact, the fungus is resistant to over 200microg/ml of bleomycin (Bm). By enhancing the susceptibility of A. oryzae to Bm using Triton X-100 as a detergent and an ATP-binding cassette (ABC) pump inhibitor, chlorpromazine, to the growing medium, we established a novel transformation system by Bm selection for A. oryzae. In a medium containing these reagents, A. oryzae showed little growth even in the presence of 30microg Bm/ml. Based on these findings, we constructed a Bm-resistance expression cassette (BmR), in which blmB encoding Bm N-acetyltransferase from Bm-producing Streptomyces verticillus was expressed under the control of a fungal promoter. We obtained a gene knockout mutant efficiently by Bm selection, i.e., the chromosomal ligD coding region was successfully replaced by BmR using ligD disruption cassette consisted of ligD flanking sequence and BmR through homologous recombination.

In vivo and in vitro acquisition of resistance to voriconazole by Candida krusei.

PubMed

Ricardo, Elisabete; Miranda, Isabel M; Faria-Ramos, Isabel; Silva, Raquel M; Rodrigues, Acácio Gonçalves; Pina-Vaz, Cidália

2014-08-01

Candida krusei is an important agent of opportunistic infections that often displays resistance to several antifungals. We describe here the in vivo acquisition of resistance to voriconazole (VRC) by C. krusei isolates recovered from a leukemia patient during a long period of VRC therapy. In order to mimic the in vivo development of VRC resistance, a susceptible C. krusei isolate was exposed daily to 1 μg/ml of VRC in vitro. Interestingly, after 5 days of exposure to VRC, a MIC of 4 μg/ml was achieved; this value remained constant after 25 additional days of treatment with VRC and also after 30 consecutive days of incubation in VRC-free medium. Our objective was to determine the associated molecular resistance mechanisms, such as expression of efflux pump genes and ERG11 gene mutations, among the resistant strains. Synergistic effects between the efflux blocker tacrolimus (FK506) and VRC were found in all of the resistant strains. Moreover, ABC1 gene expression increased over time in both the in vivo- and in vitro-induced resistant strains, in contrast to the ABC2 and ERG11 genes, whose expression was invariably lower and constant. ERG11 gene sequencing showed two different types of mutations, i.e., heterozygosity at T1389T/C, corresponding to synonymous mutations, in C. krusei strains and a missense mutation at position T418C, resulting in a change from Tyr to His, among resistant C. krusei clinical isolates. This study highlights the relevance of ATP-dependent efflux pump (namely, Abc1p) activity in VRC resistance and describes new mutations in the ERG11 gene among resistant C. krusei clinical isolates. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Down-regulation of a novel ABC transporter gene (Pxwhite) is associated with Cry1Ac resistance in the diamondback moth, Plutella xylostella (L.).

PubMed

Guo, Zhaojiang; Kang, Shi; Zhu, Xun; Xia, Jixing; Wu, Qingjun; Wang, Shaoli; Xie, Wen; Zhang, Youjun

2015-04-01

Biopesticides or transgenic crops based on Cry toxins from the soil bacterium Bacillus thuringiensis (Bt) effectively control agricultural insect pests. The sustainable use of Bt biopesticides and Bt crops is threatened, however, by the development of Cry resistance in the target pests. The diamondback moth, Plutella xylostella (L.), is the first pest that developed resistance to a Bt biopesticide in the field, and a recent study has shown that the resistance of P. xylostella to Cry1Ac is caused by a mutation in an ATP-binding cassette (ABC) transporter gene (ABCC2). In this study, we report that down-regulation of a novel ABC transporter gene from ABCG subfamily (Pxwhite) is associated with Cry1Ac resistance in P. xylostella. The full-length cDNA sequence of Pxwhite was cloned and analyzed. Spatial-temporal expression detection revealed that Pxwhite was expressed in all tissues and developmental stages, and highest expressed in Malpighian tubule tissue and in egg stage. Sequence variation analysis of Pxwhite indicated the absence of constant non-synonymous mutations between susceptible and resistant strains, whereas midgut transcript analysis showed that Pxwhite was remarkably reduced in all resistant strains and further reduced when larvae of the moderately resistant SZ-R strain were subjected to selection with Cry1Ac toxin. Furthermore, RNA interference (RNAi)-mediated suppression of Pxwhite gene expression significantly reduced larval susceptibility to Cry1Ac toxin, and genetic linkage analysis confirmed that down-regulation of Pxwhite gene is tightly linked to Cry1Ac resistance in P. xylostella. To our knowledge, this is the first report indicating that Pxwhite gene is involved in Cry1Ac resistance in P. xylostella. Copyright © 2015 Elsevier Ltd. All rights reserved.

The Composite 259-kb Plasmid of Martelella mediterranea DSM 17316T–A Natural Replicon with Functional RepABC Modules from Rhodobacteraceae and Rhizobiaceae

PubMed Central

Bartling, Pascal; Brinkmann, Henner; Bunk, Boyke; Overmann, Jörg; Göker, Markus; Petersen, Jörn

2017-01-01

A multipartite genome organization with a chromosome and many extrachromosomal replicons (ECRs) is characteristic for Alphaproteobacteria. The best investigated ECRs of terrestrial rhizobia are the symbiotic plasmids for legume root nodulation and the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens. RepABC plasmids represent the most abundant alphaproteobacterial replicon type. The currently known homologous replication modules of rhizobia and Rhodobacteraceae are phylogenetically distinct. In this study, we surveyed type-strain genomes from the One Thousand Microbial Genomes (KMG-I) project and identified a roseobacter-specific RepABC-type operon in the draft genome of the marine rhizobium Martelella mediterranea DSM 17316T. PacBio genome sequencing demonstrated the presence of three circular ECRs with sizes of 593, 259, and 170-kb. The rhodobacteral RepABC module is located together with a rhizobial equivalent on the intermediate sized plasmid pMM259, which likely originated in the fusion of a pre-existing rhizobial ECR with a conjugated roseobacter plasmid. Further evidence for horizontal gene transfer (HGT) is given by the presence of a roseobacter-specific type IV secretion system on the 259-kb plasmid and the rhodobacteracean origin of 62% of the genes on this plasmid. Functionality tests documented that the genuine rhizobial RepABC module from the Martelella 259-kb plasmid is only maintained in A. tumefaciens C58 (Rhizobiaceae) but not in Phaeobacter inhibens DSM 17395 (Rhodobacteraceae). Unexpectedly, the roseobacter-like replication system is functional and stably maintained in both host strains, thus providing evidence for a broader host range than previously proposed. In conclusion, pMM259 is the first example of a natural plasmid that likely mediates genetic exchange between roseobacters and rhizobia. PMID:28983283

DOE Office of Scientific and Technical Information (OSTI.GOV)

Niu, Mingshan; Jiangsu Key Laboratory of Bone Marrow Stem Cell, Xuzhou Medical College, Xuzhou, Jiangsu; Department of Hematology, Affiliated Hospital of Xuzhou Medical College, Xuzhou, Jiangsu

Constitutive NF-κB activation is required for survival of activated B cell-like subtype of diffuse large B cell lymphoma (ABC-DLBCL). However, current NF-κB targeting strategies lack cancer cell specificity. Here, we identified a novel inhibitor, piperlongumine, features direct binding to NF-κB p65 subunit and suppression of p65 nuclear import. This was accompanied by NF-κB reporter activity suppression and NF-κB target gene downregulation. Moreover, mutation of Cys{sup 38} to Ser in p65 abolished this effect of piperlongumine on inhibition of p65 nuclear import. Furthermore, we show that piperlongumine selectively inhibited proliferation and induced apoptosis of ABC-DLBCL cells. Most notably, it has beenmore » reported that piperlongumine did not affect normal cells even at high doses and was nontoxic to animals. Hence, our current study provides new insight into piperlongumine's mechanism of action and novel approach to ABC-DLBCL target therapy. - Highlights: • Current NF-κB targeting strategies lack cancer cell specificity. • Piperlongumine inhibits NF-κB p65 subunit nuclear import via directly binding to p65. • Piperlongumine selectively inhibits proliferation of ABC-DLBCL cells. • This study provides a novel approach to ABC-DLBCL target therapy.« less

Redefining C and D in the petunia ABC.

PubMed

Heijmans, Klaas; Ament, Kai; Rijpkema, Anneke S; Zethof, Jan; Wolters-Arts, Mieke; Gerats, Tom; Vandenbussche, Michiel

2012-06-01

According to the ABC(DE) model for flower development, C-genes are required for stamen and carpel development and floral determinacy, and D-genes were proposed to play a unique role in ovule development. Both C- and D-genes belong to the AGAMOUS (AG) subfamily of MADS box transcription factors. We show that the petunia (Petunia hybrida) C-clade genes PETUNIA MADS BOX GENE3 and FLORAL BINDING PROTEIN6 (FBP6) largely overlap in function, both in floral organ identity specification and floral determinacy, unlike the pronounced subfunctionalization observed in Arabidopsis thaliana and snapdragon (Antirrhinum majus). Some specialization has also evolved, since FBP6 plays a unique role in the development of the style and stigma. Furthermore, we show that the D-genes FBP7 and FBP11 are not essential to confer ovule identity. Instead, this function is redundantly shared among all AG members. In turn, the D-genes also participate in floral determinacy. Gain-of-function analyses suggest the presence of a posttranscriptional C-repression mechanism in petunia, most likely not existing in Arabidopsis. Finally, we show that expression maintenance of the paleoAPETALA3-type B-gene TOMATO MADS BOX GENE6 depends on the activity of C-genes. Taken together, this demonstrates considerable variation in the molecular control of floral development between eudicot species.

Redefining C and D in the Petunia ABC[W

PubMed Central

Heijmans, Klaas; Ament, Kai; Rijpkema, Anneke S.; Zethof, Jan; Wolters-Arts, Mieke; Gerats, Tom; Vandenbussche, Michiel

2012-01-01

According to the ABC(DE) model for flower development, C-genes are required for stamen and carpel development and floral determinacy, and D-genes were proposed to play a unique role in ovule development. Both C- and D-genes belong to the AGAMOUS (AG) subfamily of MADS box transcription factors. We show that the petunia (Petunia hybrida) C-clade genes PETUNIA MADS BOX GENE3 and FLORAL BINDING PROTEIN6 (FBP6) largely overlap in function, both in floral organ identity specification and floral determinacy, unlike the pronounced subfunctionalization observed in Arabidopsis thaliana and snapdragon (Antirrhinum majus). Some specialization has also evolved, since FBP6 plays a unique role in the development of the style and stigma. Furthermore, we show that the D-genes FBP7 and FBP11 are not essential to confer ovule identity. Instead, this function is redundantly shared among all AG members. In turn, the D-genes also participate in floral determinacy. Gain-of-function analyses suggest the presence of a posttranscriptional C-repression mechanism in petunia, most likely not existing in Arabidopsis. Finally, we show that expression maintenance of the paleoAPETALA3-type B-gene TOMATO MADS BOX GENE6 depends on the activity of C-genes. Taken together, this demonstrates considerable variation in the molecular control of floral development between eudicot species. PMID:22706285

[A report of atypical hypomyelinating leukodystrophy with atrophy of the basal ganglia and cerebellum caused by a de novo mutation in tubulin beta 4A (TUBB4A) gene and literature review].

PubMed

Du, Y; Li, C; Guo, J; Guo, P; Li, Z Y; Zhang, W

2017-06-01

Objective: To explore the clinical symptoms and neuroimaging features of a patient with atypical hypomyelinating leukodystrophy with atrophy of the basal ganglia and cerebellum (H-ABC) caused by a novel TUBB4A mutation. Methods: We analyzed the clinical data, imaging features and the result of genetic testing of a case diagnosed as atypical H-ABC. Results: The initial symptoms were progressive spasticity, mild cerebellar ataxia and mild cognitive impairment. MRI showed regional blurring of slight high signal on T(2)-weight and FLAIR image in white matter of the bilateral midbrain ventral, internal capsule, posteior horn of lateral ventricle and centrum semiovale, with normal bilateral cerebellar and caudoputamen nucleus. Compared with normal subjects of the same age and gender, hypometabolism was found by (18)F-FDG-PET in brainstem, cerebellar and caudoputamen nucleus in the patient. Genetic testing revealed a de novo pathogenic exome missense heterozygous mutations c. 70G>A in TUBB4A, which was not reported in the human gene mutation database (HGMDpro) and was assessed to be a pathogenic mutation by pathogenic mutation prediction software. Conclusions: The diversity of TUBB4A gene mutations may cause different functional and/or structural impairment in subcortical white matter, cerebellar and caudoputamen nucleus, leading to atypical symptoms and neuroimaging features. Genetic testing for pathogenic mutation in TUBB 4A gene is a key for the diagnosis of H - ABC .

Suppression of c-Myc is involved in multi-walled carbon nanotubes' down-regulation of ATP-binding cassette transporters in human colon adenocarcinoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Zhaojing; Xu, Yonghong; Meng, Xiangning

Over-expression of ATP-binding cassette (ABC) transporters, a large family of integral membrane proteins that decrease cellular drug uptake and accumulation by active extrusion, is one of the major causes of cancer multi-drug resistance (MDR) that frequently leads to failure of chemotherapy. Carbon nanotubes (CNTs)-based drug delivery devices hold great promise in enhancing the efficacy of cancer chemotherapy. However, CNTs' effects on the ABC transporters remain under-investigated. In this study, we found that multiwalled carbon nanotubes (MWCNTs) reduced transport activity and expression of ABC transporters including ABCB1/Pgp and ABCC4/MRP4 in human colon adenocarcinoma Caco-2 cells. Proto-oncogene c-Myc, which directly regulates ABCmore » gene expression, was concurrently decreased in MWCNT-treated cells and forced over-expression of c-Myc reversed MWCNTs' inhibitory effects on ABCB1 and ABCC4 expression. MWCNT-cell membrane interaction and cell membrane oxidative damage were observed. However, antioxidants such as vitamin C, β-mecaptoethanol and dimethylthiourea failed to antagonize MWCNTs' down-regulation of ABC transporters. These data suggest that MWCNTs may act on c-Myc, but not through oxidative stress, to down-regulate ABC transporter expression. Our findings thus shed light on CNTs' novel cellular effects that may be utilized to develop CNTs-based drug delivery devices to overcome ABC transporter-mediated cancer chemoresistance.« less

Downregulation of miR-29a/b/c in placenta accreta inhibits apoptosis of implantation site intermediate trophoblast cells by targeting MCL1.

PubMed

Gu, Yongzhong; Bian, Yuehong; Xu, Xiaofei; Wang, Xietong; Zuo, Changting; Meng, Jinlai; Li, Hongyan; Zhao, Shigang; Ning, Yunnan; Cao, Yongzhi; Huang, Tao; Yan, Junhao; Chen, Zi-Jiang

2016-12-01

Placenta accreta is defined as abnormal adhesion of placental villi to the uterine myometrium. Although this condition has become more common as a result of the increasing rate of cesarean sections, the underlying causative mechanism(s) remain elusive. Because microRNA-29a/b/c (miR-29a/b/c) have been shown to play important roles in placental development, this study evaluated the roles of these microRNAs in placenta accreta. Expression of miR-29a/b/c and myeloid cell leukemia-1 (MCL1) were quantified in patient tissues and HTR8/SVneo trophoblast cells using the real-time quantitative polymerase chain reaction. Western blotting was used to analyze expression of the MCL1 protein in HTR8/SVneo trophoblast cells with altered expression of miR-29a/b/c. To determine their role in apoptosis, miR-29a/b/c were overexpressed in HTR-8/SVneo cells, and levels of apoptosis were analyzed by flow cytometry. Luciferase activity assays were used to determine whether MCL1 is a target gene of miR-29a/b/c. Expression of miR-29a/b/c was significantly lower in creta sites compared to noncreta sites (p = 0.018, 0.041, and 0.022, respectively), but expression of MCL1 was upregulated in creta sites (p = 0.039). MCL1 expression was significantly downregulated in HTR-8/SVneo cells overexpressing miR-29a/b/c (p = 0.002, 0.008, and 0.013, respectively). Luciferase activity assays revealed that miR-29a/b/c directly target the 3' untranslated region of MCL1 in 293T cells. Over-expression of miR-29a/b/c induced apoptosis in the HTR-8/SVneo trophoblast cell line. Moreover, histopathological evaluation revealed that the number of implantation site intermediate trophoblast (ISIT) cells was increased in creta sites and that these cells were positive for MCL1. Our results demonstrate that in placenta accreta, miR-29a/b/c inhibits apoptosis of ISIT cells by targeting MCL1. These findings provide new insights into the pathogenesis of placenta accreta. Copyright © 2016 Elsevier Ltd. All rights reserved.

A Novel Anoxic Pathway for Urea and Cyanate in Marine Oxygen Deficient Zones Revealed by Combined Microbiological and Biogeochemical Tools

NASA Astrophysics Data System (ADS)

Widner, B.; Fuchsman, C. A.; Babbin, A. R.; Ji, Q.; Mulholland, M. R.

2016-02-01

Urea and cyanate are reduced nitrogen compounds that can serve as nitrogen and carbon sources for marine microbes, and cyanate forms from decomposition of urea. Some marine bacteria, including cyanobacteria, possess genes encoding an ABC-type cyanate transporter and an intracellular cyanate hydratase, and genes for urea uptake and assimilation are widespread. To investigate cyanate distribution and availability in the ocean, we recently developed a nanomolar cyanate assay specific to seawater. In an oxygenated water column, urea and cyanate concentrations are generally low in surface waters and exhibit a concentration maximum near the base of the euphotic zone likely due to production from organic matter degradation. Below the euphotic zone, urea and cyanate concentrations decrease, likely due to oxidation reactions. It has been suggested that simple organic nitrogen compounds may support anaerobic ammonium oxidation (anammox) in oxygen deficient zones (ODZs). We mapped urea and cyanate distributions and used stable isotope-labeled urea and cyanate to measure their potential support of anammox and their uptake within the Eastern Tropical North and South Pacific ODZs. We also employed metagenomic techniques to determine the abundance and distribution of genes for the uptake and assimilation of urea and cyanate. The combined data indicate that, in ODZs, urea is used primarily as a nitrogen source while cyanate is used as both a nitrogen source and to generate energy.

Divergence of the Floral A-Function between an Asterid and a Rosid Species[OPEN

PubMed Central

Heijmans, Klaas; Rozier, Frédérique; Zethof, Jan; Chamot, Sophy

2017-01-01

The ABC model is widely used as a genetic framework for understanding floral development and evolution. In this model, the A-function is required for the development of sepals and petals and to antagonize the C-function in the outer floral whorls. In the rosid species Arabidopsis thaliana, the AP2-type AP2 transcription factor represents a major A-function protein, but how the A-function is encoded in other species is not well understood. Here, we show that in the asterid species petunia (Petunia hybrida), AP2B/BLIND ENHANCER (BEN) confines the C-function to the inner petunia floral whorls, in parallel with the microRNA BLIND. BEN belongs to the TOE-type AP2 gene family, members of which control flowering time in Arabidopsis. In turn, we demonstrate that the petunia AP2-type REPRESSOR OF B-FUNCTION (ROB) genes repress the B-function (but not the C-function) in the first floral whorl, together with BEN. We propose a combinatorial model for patterning the B- and C-functions, leading to the homeotic conversion of sepals into petals, carpels, or stamens, depending on the genetic context. Combined with earlier results, our findings suggest that the molecular mechanisms controlling the spatial restriction of the floral organ identity genes are more diverse than the well-conserved B and C floral organ identity functions. PMID:28646074

Cpe1786/IscR of Clostridium perfringens represses expression of genes involved in Fe-S cluster biogenesis.

PubMed

André, Gaelle; Haudecoeur, Elise; Courtois, Emmanuelle; Monot, Marc; Dupuy, Bruno; Rodionov, Dmitry A; Martin-Verstraete, Isabelle

2017-05-01

Cpe1786 of Clostridium perfringens is an Rrf2-type regulator containing the three-cysteine residues coordinating a Fe-S in IscR, the repressor controlling Fe-S homeostasis in enterobacteria. The cpe1786 gene formed an operon with iscSU involved in Fe-S biogenesis and tmrU. This operon was transcribed from a σ A -dependent promoter. We showed that in the heterologous host Bacillus subtilis, Cpe1786, renamed IscR Cp , negatively controlled its own transcription. We constructed an iscR mutant in C. perfringens. We then compared the expression profile of strain 13 and of the iscR mutant. IscR Cp controlled expression of genes involved in Fe-S biogenesis, in amino acid or sugar metabolisms, in fermentation pathways and in host compound utilization. We then demonstrated, using a ChIP-PCR experiment, that IscR Cp interacted with its promoter region in vivo in C. perfringens and with the promoter of cpe2093 encoding an amino acid ABC transporter. We utilized a comparative genomic approach to infer a candidate IscR binding motif and reconstruct IscR regulons in clostridia. We showed that point mutations in the conserved motif of 29 bp identified upstream of iscR decreased the cysteine-dependent repression of iscR mediated by IscR Cp . Copyright © 2016 Institut Pasteur. All rights reserved.

Solitary BioY Proteins Mediate Biotin Transport into Recombinant Escherichia coli

PubMed Central

Finkenwirth, Friedrich; Kirsch, Franziska

2013-01-01

Energy-coupling factor (ECF) transporters form a large group of vitamin uptake systems in prokaryotes. They are composed of highly diverse, substrate-specific, transmembrane proteins (S units), a ubiquitous transmembrane protein (T unit), and homo- or hetero-oligomeric ABC ATPases. Biotin transporters represent a special case of ECF-type systems. The majority of the biotin-specific S units (BioY) is known or predicted to interact with T units and ABC ATPases. About one-third of BioY proteins, however, are encoded in organisms lacking any recognizable T unit. This finding raises the question of whether these BioYs function as transporters in a solitary state, a feature ascribed to certain BioYs in the past. To address this question in living cells, an Escherichia coli K-12 derivative deficient in biotin synthesis and devoid of its endogenous high-affinity biotin transporter was constructed as a reference strain. This organism is particularly suited for this purpose because components of ECF transporters do not naturally occur in E. coli K-12. The double mutant was viable in media containing either high levels of biotin or a precursor of the downstream biosynthetic path. Importantly, it was nonviable on trace levels of biotin. Eight solitary bioY genes of proteobacterial origin were individually expressed in the reference strain. Each of the BioYs conferred biotin uptake activity on the recombinants, which was inferred from uptake assays with [3H]biotin and growth of the cells on trace levels of biotin. The results underscore that solitary BioY transports biotin across the cytoplasmic membrane. PMID:23836870

Heteroresistance to Fluconazole Is a Continuously Distributed Phenotype among Candida glabrata Clinical Strains Associated with In Vivo Persistence.

PubMed

Ben-Ami, Ronen; Zimmerman, Offer; Finn, Talya; Amit, Sharon; Novikov, Anna; Wertheimer, Noa; Lurie-Weinberger, Mor; Berman, Judith

2016-08-02

Candida glabrata causes persistent infections in patients treated with fluconazole and often acquires resistance following exposure to the drug. Here we found that clinical strains of C. glabrata exhibit cell-to-cell variation in drug response (heteroresistance). We used population analysis profiling (PAP) to assess fluconazole heteroresistance (FLC(HR)) and to ask if it is a binary trait or a continuous phenotype. Thirty (57.6%) of 52 fluconazole-sensitive clinical C. glabrata isolates met accepted dichotomous criteria for FLC(HR) However, quantitative grading of FLC(HR) by using the area under the PAP curve (AUC) revealed a continuous distribution across a wide range of values, suggesting that all isolates exhibit some degree of heteroresistance. The AUC correlated with rhodamine 6G efflux and was associated with upregulation of the CDR1 and PDH1 genes, encoding ATP-binding cassette (ABC) transmembrane transporters, implying that HetR populations exhibit higher levels of drug efflux. Highly FLC(HR) C. glabrata was recovered more frequently than nonheteroresistant C. glabrata from hematogenously infected immunocompetent mice following treatment with high-dose fluconazole (45.8% versus 15%, P = 0.029). Phylogenetic analysis revealed some phenotypic clustering but also variations in FLC(HR) within clonal groups, suggesting both genetic and epigenetic determinants of heteroresistance. Collectively, these results establish heteroresistance to fluconazole as a graded phenotype associated with ABC transporter upregulation and fluconazole efflux. Heteroresistance may explain the propensity of C. glabrata for persistent infection and the emergence of breakthrough resistance to fluconazole. Heteroresistance refers to variability in the response to a drug within a clonal cell population. This phenomenon may have crucial importance for the way we look at antimicrobial resistance, as heteroresistant strains are not detected by standard laboratory susceptibility testing and may be associated with failure of antimicrobial therapy. We describe for the first time heteroresistance to fluconazole in C. glabrata, a finding that may explain the propensity of this pathogen to acquire resistance following exposure to fluconazole and to persist despite treatment. We found that, rather than being a binary all-or-none trait, heteroresistance was a continuously distributed phenotype associated with increased expression of genes that encode energy-dependent drug efflux transporters. Moreover, we show that heteroresistance is associated with failure of fluconazole to clear infection with C. glabrata Together, these findings provide an empirical framework for determining and quantifying heteroresistance in C. glabrata. Copyright © 2016 Ben-Ami et al.

The calcineruin inhibitor cyclosporine a synergistically enhances the susceptibility of Candida albicans biofilms to fluconazole by multiple mechanisms.

PubMed

Jia, Wei; Zhang, Haiyun; Li, Caiyun; Li, Gang; Liu, Xiaoming; Wei, Jun

2016-06-18

Biofilms produced by Candida albicans (C. albicans) are intrinsically resistant to fungicidal agents, which are a main cause of the pathogenesis of catheter infections. Several lines of evidence have demonstrated that calcineurin inhibitor FK506 or cyclosporine A (CsA) can remarkably enhance the antifungal activity of fluconazole (FLC) against biofilm-producing C. albicans strain infections. The aim of present study is thus to interrogate the mechanism underpinning the synergistic effect of FLC and calcineurin inhibitors. Twenty four clinical C. albicans strains isolated from bloodstream showed a distinct capacity of biofilm formation. A combination of calcineurin inhibitor CsA and FLC exhibited a dose-dependent synergistic antifungal effect on the growth and biofilm formation of C. albicans isolates as determined by a XTT assay and fluorescent microscopy assay. The synergistic effect was accompanied with a significantly down-regulated expression of adhesion-related genes ALS3, hypha-related genes HWP1, ABC transporter drug-resistant genes CDR1 and MDR1, and FLC targeting gene, encoding sterol 14alpha-demethylase (ERG11) in clinical C. albicans isolates. Furthermore, an addition of CsA significantly reduced the cellular surface hydrophobicity but increased intracellular calcium concentration as determined by a flow cytometry assay (p < 0.05). The results presented in this report demonstrated that the synergistic effect of CsA and FLC on inhibited C. albicans biofilm formation and enhanced susceptibility to FLC was in part through a mechanism involved in suppressing the expression of biofilm related and drug-resistant genes, and reducing cellular surface hydrophobicity, as well as evoking intracellular calcium concentration.

Quorum-dependent transfer of the opine-catabolic plasmid pAoF64/95 is regulated by a novel mechanism involving inhibition of the TraR antiactivator TraM.

PubMed

Wetzel, Margaret E; Asenstorfer, Robert E; Tate, Max E; Farrand, Stephen K

2018-04-10

We previously described a plasmid of Agrobacterium spp., pAoF64/95, in which the quorum-sensing system that controls conjugative transfer is induced by the opine mannopine. We also showed that the quorum-sensing regulators TraR, TraM, and TraI function similarly to their counterparts in other repABC plasmids. However, traR, unlike its counterpart on Ti plasmids, is monocistronic and not located in an operon that is inducible by the conjugative opine. Here, we report that both traR and traM are expressed constitutively and not regulated by growth with mannopine. We report two additional regulatory genes, mrtR and tmsP, that are involved in a novel mechanism of control of TraR activity. Both genes are located in the distantly linked region of pAoF64/95 encoding mannopine utilization. MrtR, in the absence of mannopine, represses the four-gene mocC operon as well as tmsP, which is the distal gene of the eight-gene motA operon. As judged by a bacterial two-hybrid analysis, TmsP, which shows amino acid sequence relatedness with the TraM-binding domain of TraR, interacts with the antiactivator. We propose a model in which mannopine, acting through the repressor MrtR, induces expression of TmsP which then titrates the levels of TraM thereby freeing TraR to activate the tra regulon. © 2018 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

Identification of accessory genome regions in poultry Clostridium perfringens isolates carrying the netB plasmid.

PubMed

Lepp, D; Gong, J; Songer, J G; Boerlin, P; Parreira, V R; Prescott, J F

2013-03-01

Necrotic enteritis (NE) is an economically important disease of poultry caused by certain Clostridium perfringens type A strains. NE pathogenesis involves the NetB toxin, which is encoded on a large conjugative plasmid within a 42-kb pathogenicity locus. Recent multilocus sequence type (MLST) studies have identified two predominant NE-associated clonal groups, suggesting that host genes are also involved in NE pathogenesis. We used microarray comparative genomic hybridization (CGH) to assess the gene content of 54 poultry isolates from birds that were healthy or that suffered from NE. A total of 400 genes were variably present among the poultry isolates and nine nonpoultry strains, many of which had putative functions related to nutrient uptake and metabolism and cell wall and capsule biosynthesis. The variable genes were organized into 142 genomic regions, 49 of which contained genes significantly associated with netB-positive isolates. These regions included three previously identified NE-associated loci as well as several apparent fitness-related loci, such as a carbohydrate ABC transporter, a ferric-iron siderophore uptake system, and an adhesion locus. Additional loci were related to plasmid maintenance. Cluster analysis of the CGH data grouped all of the netB-positive poultry isolates into two major groups, separated according to two prevalent clonal groups based on MLST analysis. This study identifies chromosomal loci associated with netB-positive poultry strains, suggesting that the chromosomal background can confer a selective advantage to NE-causing strains, possibly through mechanisms involving iron acquisition, carbohydrate metabolism, and plasmid maintenance.

Regulators of floral fragrance production and their target genes in petunia are not exclusively active in the epidermal cells of petals.

PubMed

Van Moerkercke, Alex; Galván-Ampudia, Carlos S; Verdonk, Julian C; Haring, Michel A; Schuurink, Robert C

2012-05-01

In which cells of the flower volatile biosynthesis takes place is unclear. In rose and snapdragon, some enzymes of the volatile phenylpropanoid/benzenoid pathway have been shown to be present in the epidermal cells of petals. It is therefore generally believed that the production of these compounds occurs in these cells. However, whether the entire pathway is active in these cells and whether it is exclusively active in these cells remains to be proven. Cell-specific transcription factors activating these genes will determine in which cells they are expressed. In petunia, the transcription factor EMISSION OF BENZENOIDS II (EOBII) activates the ODORANT1 (ODO1) promoter and the promoter of the biosynthetic gene isoeugenol synthase (IGS). The regulator ODO1 in turn activates the promoter of the shikimate gene 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). Here the identification of a new target gene of ODO1, encoding an ABC transporter localized on the plasma membrane, PhABCG1, which is co-expressed with ODO1, is described. PhABCG1 expression is up-regulated in petals overexpressing ODO1 through activation of the PhABCG1 promoter. Interestingly, the ODO1, PhABCG1, and IGS promoters were active in petunia protoplasts originating from both epidermal and mesophyll cell layers of the petal, suggesting that the volatile phenylpropanoid/benzenoid pathway in petunia is active in these different cell types. Since volatile release occurs from epidermal cells, trafficking of (volatile) compounds between cell layers must be involved, but the exact function of PhABCG1 remains to be resolved.

The most conserved genome segments for life detection on Earth and other planets.

PubMed

Isenbarger, Thomas A; Carr, Christopher E; Johnson, Sarah Stewart; Finney, Michael; Church, George M; Gilbert, Walter; Zuber, Maria T; Ruvkun, Gary

2008-12-01

On Earth, very simple but powerful methods to detect and classify broad taxa of life by the polymerase chain reaction (PCR) are now standard practice. Using DNA primers corresponding to the 16S ribosomal RNA gene, one can survey a sample from any environment for its microbial inhabitants. Due to massive meteoritic exchange between Earth and Mars (as well as other planets), a reasonable case can be made for life on Mars or other planets to be related to life on Earth. In this case, the supremely sensitive technologies used to study life on Earth, including in extreme environments, can be applied to the search for life on other planets. Though the 16S gene has become the standard for life detection on Earth, no genome comparisons have established that the ribosomal genes are, in fact, the most conserved DNA segments across the kingdoms of life. We present here a computational comparison of full genomes from 13 diverse organisms from the Archaea, Bacteria, and Eucarya to identify genetic sequences conserved across the widest divisions of life. Our results identify the 16S and 23S ribosomal RNA genes as well as other universally conserved nucleotide sequences in genes encoding particular classes of transfer RNAs and within the nucleotide binding domains of ABC transporters as the most conserved DNA sequence segments across phylogeny. This set of sequences defines a core set of DNA regions that have changed the least over billions of years of evolution and provides a means to identify and classify divergent life, including ancestrally related life on other planets.

Analysis of rice PDR-like ABC transporter genes in sheath blight resistance

USDA-ARS?s Scientific Manuscript database

Sheath blight caused by Rhizoctonia solani is one of the most damaging diseases of rice worldwide. To understand the molecular mechanism of resistance, we identified 450 differentially expressed genes in a resistant rice cultivar Jasmine 85 after R. solani infection with a combination of DNA microar...

Unusual Genomic Traits Suggest Methylocystis bryophila S285 to Be Well Adapted for Life in Peatlands

PubMed Central

Han, Dongfei; Dedysh, Svetlana N

2018-01-01

Abstract The genus Methylocystis belongs to the class Alphaproteobacteria, the family Methylocystaceae, and encompasses aerobic methanotrophic bacteria with the serine pathway of carbon assimilation. All Methylocystis species are able to fix dinitrogen and several members of this genus are also capable of using acetate or ethanol in the absence of methane, which explains their wide distribution in various habitats. One additional trait that enables their survival in the environment is possession of two methane-oxidizing isozymes, the conventional particulate methane monooxygenase (pMMO) with low-affinity to substrate (pMMO1) and the high-affinity enzyme (pMMO2). Here, we report the finished genome sequence of Methylocystis bryophila S285, a pMMO2-possessing methanotroph from a Sphagnum-dominated wetland, and compare it to the genome of Methylocystis sp. strain SC2, which is the first methanotroph with confirmed high-affinity methane oxidation potential. The complete genome of Methylocystis bryophila S285 consists of a 4.53 Mb chromosome and one plasmid, 175 kb in size. The genome encodes two types of particulate MMO (pMMO1 and pMMO2), soluble MMO and, in addition, contains a pxmABC-like gene cluster similar to that present in some gammaproteobacterial methanotrophs. The full set of genes related to the serine pathway, the tricarboxylic acid cycle as well as the ethylmalonyl-CoA pathway is present. In contrast to most described methanotrophs including Methylocystis sp. strain SC2, two different types of nitrogenases, that is, molybdenum–iron and vanadium–iron types, are encoded in the genome of strain S285. This unique combination of genome-based traits makes Methylocystis bryophila well adapted to the fluctuation of carbon and nitrogen sources in wetlands. PMID:29390143

Switching between nitrogen and glucose limitation: Unraveling transcriptional dynamics in Escherichia coli.

PubMed

Löffler, Michael; Simen, Joana Danica; Müller, Jan; Jäger, Günter; Laghrami, Salaheddine; Schäferhoff, Karin; Freund, Andreas; Takors, Ralf

2017-09-20

Transcriptional control under nitrogen and carbon-limitation conditions have been well analyzed for Escherichia coli. However, the transcriptional dynamics that underlie the shift in regulatory programs from nitrogen to carbon limitation is not well studied. In the present study, cells were cultivated at steady state under nitrogen (ammonia)-limited conditions then shifted to carbon (glucose) limitation to monitor changes in transcriptional dynamics. Nitrogen limitation was found to be dominated by sigma 54 (RpoN) and sigma 38 (RpoS), whereas the "housekeeping" sigma factor 70 (RpoD) and sigma 38 regulate cellular status under glucose limitation. During the transition, nitrogen-mediated control was rapidly redeemed and mRNAs that encode active uptake systems, such as ptsG and manXYZ, were quickly amplified. Next, genes encoding facilitators such as lamB were overexpressed, followed by high affinity uptake systems such as mglABC and non-specific porins such as ompF. These regulatory programs are complex and require well-equilibrated and superior control. At the metabolome level, 2-oxoglutarate is the likely component that links carbon- and nitrogen-mediated regulation by interacting with major regulatory elements. In the case of dual glucose and ammonia limitation, sigma 24 (RpoE) appears to play a key role in orchestrating these complex regulatory networks. Copyright © 2017 Elsevier B.V. All rights reserved.

Study of formation of green eggshell color in ducks through global gene expression.

PubMed

Xu, Fa Qiong; Li, Ang; Lan, Jing Jing; Wang, Yue Ming; Yan, Mei Jiao; Lian, Sen Yang; Wu, Xu

2018-01-01

The green eggshell color produced by ducks is a threshold trait that can be influenced by various factors, such as hereditary, environment and nutrition. The aim of this study was to investigate the genetic regulation of the formation of eggs with green shells in Youxian ducks. We performed integrative analysis of mRNAs and miRNAs expression profiling in the shell gland samples from ducks by RNA-Seq. We found 124 differentially expressed genes that were associated with various pathways, such as the ATP-binding cassette (ABC) transporter and solute carrier supper family pathways. A total of 31 differentially expressed miRNAs were found between ducks laying green eggs and white eggs. KEGG pathway analysis of the predicted miRNA target genes also indicated the functional characteristics of these miRNAs; they were involved in the ABC transporter pathway and the solute carrier (SLC) supper family. Analysis with qRT-PCR was applied to validate the results of global gene expression, which showed a correlation between results obtained by RNA-seq and RT-qPCR. Moreover, a miRNA-mRNA interaction network was established using correlation analysis of differentially expressed mRNA and miRNA. Compared to ducks that lay white eggs, ducks that lay green eggs include six up-regulated miRNAs that had regulatory effects on 35 down-regulated genes, and seven down-regulated miRNAs which influenced 46 up-regulated genes. For example, the ABC transporter pathway could be regulated by expressing gga-miR-144-3p (up-regulated) with ABCG2 (up-regulated) and other miRNAs and genes. This study provides valuable information about mRNA and miRNA regulation in duck shell gland tissues, and provides foundational information for further study on the eggshell color formation and marker-assisted selection for Youxian duck breeding.

Role of the ganSPQAB Operon in Degradation of Galactan by Bacillus subtilis.

PubMed

Watzlawick, Hildegard; Morabbi Heravi, Kambiz; Altenbuchner, Josef

2016-10-15

Bacillus subtilis possesses different enzymes for the utilization of plant cell wall polysaccharides. This includes a gene cluster containing galactan degradation genes (ganA and ganB), two transporter component genes (ganQ and ganP), and the sugar-binding lipoprotein-encoding gene ganS (previously known as cycB). These genes form an operon that is regulated by GanR. The degradation of galactan by B. subtilis begins with the activity of extracellular GanB. GanB is an endo-β-1,4-galactanase and is a member of glycoside hydrolase (GH) family 53. This enzyme was active on high-molecular-weight arabinose-free galactan and mainly produced galactotetraose as well as galactotriose and galactobiose. These galacto-oligosaccharides may enter the cell via the GanQP transmembrane proteins of the galactan ABC transporter. The specificity of the galactan ABC transporter depends on the sugar-binding lipoprotein, GanS. Purified GanS was shown to bind galactotetraose and galactotriose using thermal shift assay. The energy for this transport is provided by MsmX, an ATP-binding protein. The transported galacto-oligosaccharides are further degraded by GanA. GanA is a β-galactosidase that belongs to GH family 42. The GanA enzyme was able to hydrolyze short-chain β-1,4-galacto-oligosaccharides as well as synthetic β-galactopyranosides into galactose. Thermal shift assay as well as electrophoretic mobility shift assay demonstrated that galactobiose is the inducer of the galactan operon regulated by GanR. DNase I footprinting revealed that the GanR protein binds to an operator overlapping the -35 box of the σ(A)-type promoter of Pgan, which is located upstream of ganS IMPORTANCE: Bacillus subtilis is a Gram-positive soil bacterium that utilizes different types of carbohydrates, such as pectin, as carbon sources. So far, most of the pectin degradation systems and enzymes have been thoroughly studied in B. subtilis Nevertheless, the B. subtilis utilization system of galactan, which is found as the side chain of the rhamnogalacturonan type I complex in pectin, has remained partially studied. Here, we investigated the galactan utilization system consisting of the ganSPQAB operon and its regulator ganR This study improves our knowledge of the carbohydrate degradation systems of B. subtilis, especially the pectin degradation systems. Moreover, the galactan-degrading enzymes may be exploited for the production of galacto-oligosaccharides, which are used as prebiotic substances in the food industry. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Species-specific serine-threonine protein kinase Pkb2 of Bifidobacterium longum subsp. longum: Genetic environment and substrate specificity.

PubMed

Nezametdinova, V Z; Mavletova, D A; Alekseeva, M G; Chekalina, M S; Zakharevich, N V; Danilenko, V N

2018-06-01

The objective of this study was to determine for phosphorylated substrates of the species-specific serine-threonine protein kinase (STPK) Pkb2 from Bifidobacterium longum subsp. longum GT15. Two approaches were employed: analyses of phosphorylated membrane vesicles protein spectra following kinase reactions and analyses of the genes surrounding pkb2. A bioinformatics analysis of the genes surrounding pkb2 found a species-specific gene cluster PFNA in the genomes of 34 different bifidobacterial species. The identified cluster consisted of 5-8 genes depending on the species. The first five genes are characteristic for all considered species. These are the following genes encoding serine-threonine protein kinase (pkb2), fibronectin type III domain-containing protein (fn3), AAA-ATPase (aaa-atp), hypothetical protein with DUF58 domain (duf58) and transglutaminase (tgm). The sixth (protein phosphatase, prpC), seventh (hypothetical protein, BLGT_RS02790), and eighth (FHA domain-containing protein, fha) genes are included in this cluster, but they are not found in all species. The operon organization of the PFNA gene cluster was confirmed with transcriptional analysis. AAA-ATPase, which is encoded by a gene of the PFNA gene cluster, was found to be a substrate of the STPK Pkb2. Fourteen AAA-ATPase sites (seven serine, six threonine, and one tyrosine) phosphorylated by STPK Pkb2 were revealed. Analysis of the spectra of phosphorylated membrane vesicles proteins allowed us to identify eleven proteins that were considered as possible Pkb2 substrates. They belong to several functional classes: proteins involved in transcription and translation; proteins of the F1-domain of the FoF1-ATPase; ABC-transporters; molecular chaperone GroEL; and glutamine synthase, GlnA1. All identified proteins were considered moonlighting proteins. Three out of 11 proteins (glutamine synthetase GlnA1 and FoF1-ATPase alpha and beta subunits) were selected for further in vitro phosphorylation assays and were shown to be phosphorylated by Pkb2. Four phosphorylated substrates of the species-specific STPK Pkb2 from B. longum subsp. longum GT15 were identified for the first time. They included the moonlighting protein glutamine synthase GlnA, FoF1-ATPase alpha and beta subunits, and the chaperone MoxR family of AAA-ATPase. The ability of bifidobacterial STPK to phosphorylate the substrate on serine, threonine, and tyrosine residues was shown for the first time. Copyright © 2018 Elsevier Ltd. All rights reserved.

Translational control plays an important role in the adaptive heat-shock response of Streptomyces coelicolor

PubMed Central

Pothi, Radhika; Hesketh, Andrew; Möller-Levet, Carla; Hodgson, David A; Laing, Emma E; Stewart, Graham R; Smith, Colin P

2018-01-01

Abstract Stress-induced adaptations require multiple levels of regulation in all organisms to repair cellular damage. In the present study we evaluated the genome-wide transcriptional and translational changes following heat stress exposure in the soil-dwelling model actinomycete bacterium, Streptomyces coelicolor. The combined analysis revealed an unprecedented level of translational control of gene expression, deduced through polysome profiling, in addition to transcriptional changes. Our data show little correlation between the transcriptome and ‘translatome’; while an obvious downward trend in genome wide transcription was observed, polysome associated transcripts following heat-shock showed an opposite upward trend. A handful of key protein players, including the major molecular chaperones and proteases were highly induced at both the transcriptional and translational level following heat-shock, a phenomenon known as ‘potentiation’. Many other transcripts encoding cold-shock proteins, ABC-transporter systems, multiple transcription factors were more highly polysome-associated following heat stress; interestingly, these protein families were not induced at the transcriptional level and therefore were not previously identified as part of the stress response. Thus, stress coping mechanisms at the level of gene expression in this bacterium go well beyond the induction of a relatively small number of molecular chaperones and proteases in order to ensure cellular survival at non-physiological temperatures. PMID:29746664

Translational control plays an important role in the adaptive heat-shock response of Streptomyces coelicolor.

PubMed

Bucca, Giselda; Pothi, Radhika; Hesketh, Andrew; Möller-Levet, Carla; Hodgson, David A; Laing, Emma E; Stewart, Graham R; Smith, Colin P

2018-05-09

Stress-induced adaptations require multiple levels of regulation in all organisms to repair cellular damage. In the present study we evaluated the genome-wide transcriptional and translational changes following heat stress exposure in the soil-dwelling model actinomycete bacterium, Streptomyces coelicolor. The combined analysis revealed an unprecedented level of translational control of gene expression, deduced through polysome profiling, in addition to transcriptional changes. Our data show little correlation between the transcriptome and 'translatome'; while an obvious downward trend in genome wide transcription was observed, polysome associated transcripts following heat-shock showed an opposite upward trend. A handful of key protein players, including the major molecular chaperones and proteases were highly induced at both the transcriptional and translational level following heat-shock, a phenomenon known as 'potentiation'. Many other transcripts encoding cold-shock proteins, ABC-transporter systems, multiple transcription factors were more highly polysome-associated following heat stress; interestingly, these protein families were not induced at the transcriptional level and therefore were not previously identified as part of the stress response. Thus, stress coping mechanisms at the level of gene expression in this bacterium go well beyond the induction of a relatively small number of molecular chaperones and proteases in order to ensure cellular survival at non-physiological temperatures.

Serum Factors from Pseudoxanthoma Elasticum Patients Alter Elastic Fiber Formation In Vitro

PubMed Central

Le Saux, Olivier; Bunda, Severa; VanWart, Christopher M.; Douet, Vanessa; Got, Laurence; Martin, Ludovic; Hinek, Aleksander

2017-01-01

Pseudoxanthoma elasticum (PXE) is a heritable disorder mainly characterized by calcified elastic fibers in cutaneous, ocular, and vascular tissues. PXE is caused by mutations in ABCC6, a gene encoding an ABC transporter predominantly expressed in liver and kidneys. The functional relationship between ABCC6 and elastic fiber calcification is unknown. We speculated that ABCC6 deficiency in PXE patients induces a persistent imbalance in circulating metabolite(s), which may impair the synthetic abilities of normal elastoblasts or specifically alter elastic fiber assembly. Therefore, we compared the deposition of elastic fiber proteins in cultures of fibroblasts derived from PXE and unaffected individuals. PXE fibroblasts cultured with normal human serum expressed and deposited increased amounts of proteins, but structurally normal elastic fibers. Interestingly, normal and PXE fibroblasts as well as normal smooth muscle cells deposited abnormal aggregates of elastic fibers when maintained in the presence of serum from PXE patients. The expression of tropoelastin and other elastic fiber-associated genes was not significantly modulated by the presence of PXE serum. These results indicated that certain metabolites present in PXE sera interfered with the normal assembly of elastic fibers in vitro and suggested that PXE is a primary metabolic disorder with secondary connective tissue manifestations. PMID:16543900

Rhizobium symbiotic genes required for nodulation of legume and nonlegume hosts

PubMed Central

Marvel, Deborah J.; Torrey, John G.; Ausubel, Frederick M.

1987-01-01

Parasponia, a woody member of the elm family, is the only nonlegume genus whose members are known to form an effective nitrogen-fixing symbiosis with Bradyrhizobium or Rhizobium species. The Bradyrhizobium strain Rp501, isolated from Parasponia nodules, also nodulates the legumes siratro (Macroptilium atropurpureum) and cowpea (Vigna unguiculata). To test whether some of the same genes are involved in the early stages of legume and nonlegume nodulation, we generated transposon Tn5 insertions in the region of three evolutionarily conserved genes (nodA, nodB, and nodC) required for legume nodulation in several Rhizobium and Bradyrhizobium species. Assays of these mutant Rp501 strains on legume hosts and Parasponia seedlings established that nodABC are required for nodulation of legume and nonlegume hosts, indicating that nonlegumes and legumes can respond to the same bacterial signal(s). In addition, a strain carrying a Tn5 insertion adjacent to the nodABC genes vigorously nodulated Rp501 legume hosts but was incapable of nodulating Parasponia, possibly identifying a nonlegume-specific nodulation function. Images PMID:16593814

Analysis of LexA binding sites and transcriptomics in response to genotoxic stress in Leptospira interrogans.

PubMed

Schons-Fonseca, Luciane; da Silva, Josefa B; Milanez, Juliana S; Domingos, Renan H; Smith, Janet L; Nakaya, Helder I; Grossman, Alan D; Ho, Paulo L; da Costa, Renata M A

2016-02-18

We determined the effects of DNA damage caused by ultraviolet radiation on gene expression in Leptospira interrogans using DNA microarrays. These data were integrated with DNA binding in vivo of LexA1, a regulator of the DNA damage response, assessed by chromatin immunoprecipitation and massively parallel DNA sequencing (ChIP-seq). In response to DNA damage, Leptospira induced expression of genes involved in DNA metabolism, in mobile genetic elements and defective prophages. The DNA repair genes involved in removal of photo-damage (e.g. nucleotide excision repair uvrABC, recombinases recBCD and resolvases ruvABC) were not induced. Genes involved in various metabolic pathways were down regulated, including genes involved in cell growth, RNA metabolism and the tricarboxylic acid cycle. From ChIP-seq data, we observed 24 LexA1 binding sites located throughout chromosome 1 and one binding site in chromosome 2. Expression of many, but not all, genes near those sites was increased following DNA damage. Binding sites were found as far as 550 bp upstream from the start codon, or 1 kb into the coding sequence. Our findings indicate that there is a shift in gene expression following DNA damage that represses genes involved in cell growth and virulence, and induces genes involved in mutagenesis and recombination. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

ABC transporter activity linked to radiation resistance and molecular subtype in pediatric medulloblastoma

PubMed Central

2013-01-01

Background Resistance to radiation treatment remains a major clinical problem for patients with brain cancer. Medulloblastoma is the most common malignant brain tumor of childhood, and occurs in the cerebellum. Though radiation treatment has been critical in increasing survival rates in recent decades, the presence of resistant cells in a substantial number of medulloblastoma patients leads to relapse and death. Methods Using the established medulloblastoma cell lines UW228 and Daoy, we developed a novel model system to enrich for and study radiation tolerant cells early after radiation exposure. Using fluorescence-activated cell sorting, dead cells and cells that had initiated apoptosis were removed, allowing surviving cells to be investigated before extensive proliferation took place. Results Isolated surviving cells were tumorigenic in vivo and displayed elevated levels of ABCG2, an ABC transporter linked to stem cell behavior and drug resistance. Further investigation showed another family member, ABCA1, was also elevated in surviving cells in these lines, as well as in early passage cultures from pediatric medulloblastoma patients. We discovered that the multi-ABC transporter inhibitors verapamil and reserpine sensitized cells from particular patients to radiation, suggesting that ABC transporters have a functional role in cellular radiation protection. Additionally, verapamil had an intrinsic anti-proliferative effect, with transient exposure in vitro slowing subsequent in vivo tumor formation. When expression of key ABC transporter genes was assessed in medulloblastoma tissue from 34 patients, levels were frequently elevated compared with normal cerebellum. Analysis of microarray data from independent cohorts (n = 428 patients) showed expression of a number of ABC transporters to be strongly correlated with certain medulloblastoma subtypes, which in turn are associated with clinical outcome. Conclusions ABC transporter inhibitors are already being trialed clinically, with the aim of decreasing chemotherapy resistance. Our findings suggest that the inhibition of ABC transporters could also increase the efficacy of radiation treatment for medulloblastoma patients. Additionally, the finding that certain family members are associated with particular molecular subtypes (most notably high ABCA8 and ABCB4 expression in Sonic Hedgehog pathway driven tumors), along with cell membrane location, suggests ABC transporters are worthy of consideration for the diagnostic classification of medulloblastoma. PMID:24219920

Characterization of a lactose-responsive promoter of ATP-binding cassette (ABC) transporter gene from Lactobacillus acidophilus 05-172.

PubMed

Zeng, Zhu; Zuo, Fanglei; Yu, Rui; Zhang, Bo; Ma, Huiqin; Chen, Shangwu

2017-09-01

A novel lactose-responsive promoter of the ATP-binding cassette (ABC) transporter gene Lba1680 of Lactobacillus acidophilus strain 05-172 isolated from a traditionally fermented dairy product koumiss was characterized. In L. acidophilus 05-172, expression of Lba1680 was induced by lactose, with lactose-induced transcription of Lba1680 being 6.1-fold higher than that induced by glucose. This is in contrast to L. acidophilus NCFM, a strain isolated from human feces, in which expression of Lba1680 and Lba1679 is induced by glucose. Both gene expression and enzyme activity assays in L. paracasei transformed with a vector containing the inducible Lba1680 promoter (PLba1680) of strain 05-172 and a heme-dependent catalase gene as reporter confirmed that PLba1680 is specifically induced by lactose. Its regulatory expression could not be repressed by glucose, and was independent of cAMP receptor protein. This lactose-responsive promoter might be used in the expression of functional genes in L. paracasei incorporated into a lactose-rich environment, such as dairy products. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Purification and biochemical characterization of NpABCG5/NpPDR5, a plant pleiotropic drug resistance transporter expressed in Nicotiana tabacum BY-2 suspension cells.

PubMed

Toussaint, Frédéric; Pierman, Baptiste; Bertin, Aurélie; Lévy, Daniel; Boutry, Marc

2017-05-04

Pleiotropic drug resistance (PDR) transporters belong to the ABCG subfamily of ATP-binding cassette (ABC) transporters and are involved in the transport of various molecules across plasma membranes. During evolution, PDR genes appeared independently in fungi and in plants from a duplication of a half-size ABC gene. The enzymatic properties of purified PDR transporters from yeast have been characterized. This is not the case for any plant PDR transporter, or, incidentally, for any purified plant ABC transporter. Yet, plant PDR transporters play important roles in plant physiology such as hormone signaling or resistance to pathogens or herbivores. Here, we describe the expression, purification, enzymatic characterization and 2D analysis by electron microscopy of NpABCG5/NpPDR5 from Nicotiana plumbaginifolia , which has been shown to be involved in the plant defense against herbivores. We constitutively expressed NpABCG5/NpPDR5, provided with a His-tag in a homologous system: suspension cells from Nicotiana tabacum (Bright Yellow 2 line). NpABCG5/NpPDR5 was targeted to the plasma membrane and was solubilized by dodecyl maltoside and purified by Ni-affinity chromatography. The ATP-hydrolyzing specific activity (27 nmol min -1  mg -1 ) was stimulated seven-fold in the presence of 0.1% asolectin. Electron microscopy analysis indicated that NpABCG5/NpPDR5 is monomeric and with dimensions shorter than those of known ABC transporters. Enzymatic data (optimal pH and sensitivity to inhibitors) confirmed that plant and fungal PDR transporters have different properties. These data also show that N. tabacum suspension cells are a convenient host for the purification and biochemical characterization of ABC transporters. © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

Polymorphisms in ABC Transporter Genes and Concentrations of Mercury in Newborns – Evidence from Two Mediterranean Birth Cohorts

PubMed Central

Llop, Sabrina; Engström, Karin; Ballester, Ferran; Franforte, Elisa; Alhamdow, Ayman; Pisa, Federica; Tratnik, Janja Snoj; Mazej, Datja; Murcia, Mario; Rebagliato, Marisa; Bustamante, Mariona; Sunyer, Jordi; Sofianou-Katsoulis, Αikaterini; Prasouli, Alexia; Antonopoulou, Eleni; Antoniadou, Ioanna; Nakou, Sheena; Barbone, Fabio; Horvat, Milena; Broberg, Karin

2014-01-01

Background The genetic background may influence methylmercury (MeHg) metabolism and neurotoxicity. ATP binding cassette (ABC) transporters actively transport various xenobiotics across biological membranes. Objective To investigate the role of ABC polymorphisms as modifiers of prenatal exposure to MeHg. Methods The study population consisted of participants (n = 1651) in two birth cohorts, one in Italy and Greece (PHIME) and the other in Spain (INMA). Women were recruited during pregnancy in Italy and Spain, and during the perinatal period in Greece. Total mercury concentrations were measured in cord blood samples by atomic absorption spectrometry. Maternal fish intake during pregnancy was determined from questionnaires. Polymorphisms (n = 5) in the ABC genes ABCA1, ABCB1, ABCC1 and ABCC2 were analysed in both cohorts. Results ABCB1 rs2032582, ABCC1 rs11075290, and ABCC2 rs2273697 modified the associations between maternal fish intake and cord blood mercury concentrations. The overall interaction coefficient between rs2032582 and log2-transformed fish intake was negative for carriers of GT (β = −0.29, 95%CI −0.47, −0.12) and TT (β = −0.49, 95%CI −0.71, −0.26) versus GG, meaning that for a doubling in fish intake of the mothers, children with the rs2032582 GG genotype accumulated 35% more mercury than children with TT. For rs11075290, the interaction coefficient was negative for carriers of TC (β = −0.12, 95%CI −0.33, 0.09), and TT (β = −0.28, 95%CI −0.51, −0.06) versus CC. For rs2273697, the interaction coefficient was positive when combining GA+AA (β = 0.16, 95%CI 0.01, 0.32) versus GG. Conclusion The ABC transporters appear to play a role in accumulation of MeHg during early development. PMID:24831289

ABCA Transporter Gene Expression and Poor Outcome in Epithelial Ovarian Cancer

PubMed Central

Hedditch, Ellen L.; Gao, Bo; Russell, Amanda J.; Lu, Yi; Emmanuel, Catherine; Beesley, Jonathan; Johnatty, Sharon E.; Chen, Xiaoqing; Harnett, Paul; George, Joshy; Williams, Rebekka T.; Flemming, Claudia; Lambrechts, Diether; Despierre, Evelyn; Lambrechts, Sandrina; Vergote, Ignace; Karlan, Beth; Lester, Jenny; Orsulic, Sandra; Walsh, Christine; Fasching, Peter; Beckmann, Matthias W.; Ekici, Arif B.; Hein, Alexander; Matsuo, Keitaro; Hosono, Satoyo; Nakanishi, Toru; Yatabe, Yasushi; Pejovic, Tanja; Bean, Yukie; Heitz, Florian; Harter, Philipp; du Bois, Andreas; Schwaab, Ira; Hogdall, Estrid; Kjaer, Susan K.; Jensen, Allan; Hogdall, Claus; Lundvall, Lene; Engelholm, Svend Aage; Brown, Bob; Flanagan, James; Metcalf, Michelle D; Siddiqui, Nadeem; Sellers, Thomas; Fridley, Brooke; Cunningham, Julie; Schildkraut, Joellen; Iversen, Ed; Weber, Rachel P.; Berchuck, Andrew; Goode, Ellen; Bowtell, David D.; Chenevix-Trench, Georgia; deFazio, Anna; Norris, Murray D.; MacGregor, Stuart; Haber, Michelle; Henderson, Michelle J.

2014-01-01

Background ATP-binding cassette (ABC) transporters play various roles in cancer biology and drug resistance, but their association with outcomes in serous epithelial ovarian cancer (EOC) is unknown. Methods The relationship between clinical outcomes and ABC transporter gene expression in two independent cohorts of high-grade serous EOC tumors was assessed with real-time quantitative polymerase chain reaction, analysis of expression microarray data, and immunohistochemistry. Associations between clinical outcomes and ABCA transporter gene single nucleotide polymorphisms were tested in a genome-wide association study. Impact of short interfering RNA–mediated gene suppression was determined by colony forming and migration assays. Association with survival was assessed with Kaplan–Meier analysis and log-rank tests. All statistical tests were two-sided. Results Associations with outcome were observed with ABC transporters of the “A” subfamily, but not with multidrug transporters. High-level expression of ABCA1, ABCA6, ABCA8, and ABCA9 in primary tumors was statistically significantly associated with reduced survival in serous ovarian cancer patients. Low levels of ABCA5 and the C-allele of rs536009 were associated with shorter overall survival (hazard ratio for death = 1.50; 95% confidence interval [CI] =1.26 to 1.79; P = 6.5e−6). The combined expression pattern of ABCA1, ABCA5, and either ABCA8 or ABCA9 was associated with particularly poor outcome (mean overall survival in group with adverse ABCA1, ABCA5 and ABCA9 gene expression = 33.2 months, 95% CI = 26.4 to 40.1; vs 55.3 months in the group with favorable ABCA gene expression, 95% CI = 49.8 to 60.8; P = .001), independently of tumor stage or surgical debulking status. Suppression of cholesterol transporter ABCA1 inhibited ovarian cancer cell growth and migration in vitro, and statin treatment reduced ovarian cancer cell migration. Conclusions Expression of ABCA transporters was associated with poor outcome in serous ovarian cancer, implicating lipid trafficking as a potentially important process in EOC. PMID:24957074

Systematic Analysis and Comparison of Nucleotide-Binding Site Disease Resistance Genes in a Diploid Cotton Gossypium raimondii

PubMed Central

Wei, Hengling; Li, Wei; Sun, Xiwei; Zhu, Shuijin; Zhu, Jun

2013-01-01

Plant disease resistance genes are a key component of defending plants from a range of pathogens. The majority of these resistance genes belong to the super-family that harbors a Nucleotide-binding site (NBS). A number of studies have focused on NBS-encoding genes in disease resistant breeding programs for diverse plants. However, little information has been reported with an emphasis on systematic analysis and comparison of NBS-encoding genes in cotton. To fill this gap of knowledge, in this study, we identified and investigated the NBS-encoding resistance genes in cotton using the whole genome sequence information of Gossypium raimondii. Totally, 355 NBS-encoding resistance genes were identified. Analyses of the conserved motifs and structural diversity showed that the most two distinct features for these genes are the high proportion of non-regular NBS genes and the high diversity of N-termini domains. Analyses of the physical locations and duplications of NBS-encoding genes showed that gene duplication of disease resistance genes could play an important role in cotton by leading to an increase in the functional diversity of the cotton NBS-encoding genes. Analyses of phylogenetic comparisons indicated that, in cotton, the NBS-encoding genes with TIR domain not only have their own evolution pattern different from those of genes without TIR domain, but also have their own species-specific pattern that differs from those of TIR genes in other plants. Analyses of the correlation between disease resistance QTL and NBS-encoding resistance genes showed that there could be more than half of the disease resistance QTL associated to the NBS-encoding genes in cotton, which agrees with previous studies establishing that more than half of plant resistance genes are NBS-encoding genes. PMID:23936305

Genome-wide comparative analysis of NBS-encoding genes between Brassica species and Arabidopsis thaliana.

PubMed

Yu, Jingyin; Tehrim, Sadia; Zhang, Fengqi; Tong, Chaobo; Huang, Junyan; Cheng, Xiaohui; Dong, Caihua; Zhou, Yanqiu; Qin, Rui; Hua, Wei; Liu, Shengyi

2014-01-03

Plant disease resistance (R) genes with the nucleotide binding site (NBS) play an important role in offering resistance to pathogens. The availability of complete genome sequences of Brassica oleracea and Brassica rapa provides an important opportunity for researchers to identify and characterize NBS-encoding R genes in Brassica species and to compare with analogues in Arabidopsis thaliana based on a comparative genomics approach. However, little is known about the evolutionary fate of NBS-encoding genes in the Brassica lineage after split from A. thaliana. Here we present genome-wide analysis of NBS-encoding genes in B. oleracea, B. rapa and A. thaliana. Through the employment of HMM search and manual curation, we identified 157, 206 and 167 NBS-encoding genes in B. oleracea, B. rapa and A. thaliana genomes, respectively. Phylogenetic analysis among 3 species classified NBS-encoding genes into 6 subgroups. Tandem duplication and whole genome triplication (WGT) analyses revealed that after WGT of the Brassica ancestor, NBS-encoding homologous gene pairs on triplicated regions in Brassica ancestor were deleted or lost quickly, but NBS-encoding genes in Brassica species experienced species-specific gene amplification by tandem duplication after divergence of B. rapa and B. oleracea. Expression profiling of NBS-encoding orthologous gene pairs indicated the differential expression pattern of retained orthologous gene copies in B. oleracea and B. rapa. Furthermore, evolutionary analysis of CNL type NBS-encoding orthologous gene pairs among 3 species suggested that orthologous genes in B. rapa species have undergone stronger negative selection than those in B .oleracea species. But for TNL type, there are no significant differences in the orthologous gene pairs between the two species. This study is first identification and characterization of NBS-encoding genes in B. rapa and B. oleracea based on whole genome sequences. Through tandem duplication and whole genome triplication analysis in B. oleracea, B. rapa and A. thaliana genomes, our study provides insight into the evolutionary history of NBS-encoding genes after divergence of A. thaliana and the Brassica lineage. These results together with expression pattern analysis of NBS-encoding orthologous genes provide useful resource for functional characterization of these genes and genetic improvement of relevant crops.

Genome-wide profiling of Hfq-binding RNAs uncovers extensive post-transcriptional rewiring of major stress response and symbiotic regulons in Sinorhizobium meliloti.

PubMed

Torres-Quesada, Omar; Reinkensmeier, Jan; Schlüter, Jan-Philip; Robledo, Marta; Peregrina, Alexandra; Giegerich, Robert; Toro, Nicolás; Becker, Anke; Jiménez-Zurdo, Jose I

2014-01-01

The RNA chaperone Hfq is a global post-transcriptional regulator in bacteria. Here, we used RNAseq to analyze RNA populations from the legume symbiont Sinorhizobium meliloti that were co-immunoprecipitated (CoIP-RNA) with a FLAG-tagged Hfq in five growth/stress conditions. Hfq-bound transcripts (1315) were largely identified in stressed bacteria and derived from small RNAs (sRNAs), both trans-encoded (6.4%) and antisense (asRNAs; 6.3%), and mRNAs (86%). Pull-down with Hfq recovered a small proportion of annotated S. meliloti sRNAs (14% of trans-sRNAs and 2% of asRNAs) suggesting a discrete impact of this protein in sRNA pathways. Nonetheless, Hfq selectively stabilized CoIP-enriched sRNAs, anticipating that these interactions are functionally significant. Transcription of 26 Hfq-bound sRNAs was predicted to occur from promoters recognized by the major stress σ factors σ(E2) or σ(H1/2). Recovery rates of sRNAs in each of the CoIP-RNA libraries suggest a large impact of Hfq-assisted riboregulation in S. meliloti osmoadaptation. Hfq directly targeted 18% of the predicted S. meliloti mRNAs, which encode functionally diverse proteins involved in transport and metabolism, σ(E2)-dependent stress responses, quorum sensing, flagella biosynthesis, ribosome, and membrane assembly or symbiotic nitrogen fixation. Canonical targeting of the 5' regions of two of the ABC transporter mRNAs by the homologous Hfq-binding AbcR1 and AbcR2 sRNAs leading to inhibition of protein synthesis was confirmed in vivo. We therefore provide a comprehensive resource for the systems-level deciphering of hitherto unexplored S. meliloti stress and symbiotic post-transcriptional regulons and the identification of Hfq-dependent sRNA-mRNA regulatory pairs.

Genome-wide profiling of Hfq-binding RNAs uncovers extensive post-transcriptional rewiring of major stress response and symbiotic regulons in Sinorhizobium meliloti

PubMed Central

Torres-Quesada, Omar; Reinkensmeier, Jan; Schlüter, Jan-Philip; Robledo, Marta; Peregrina, Alexandra; Giegerich, Robert; Toro, Nicolás; Becker, Anke; Jiménez-Zurdo, Jose I

2014-01-01

The RNA chaperone Hfq is a global post-transcriptional regulator in bacteria. Here, we used RNAseq to analyze RNA populations from the legume symbiont Sinorhizobium meliloti that were co-immunoprecipitated (CoIP-RNA) with a FLAG-tagged Hfq in five growth/stress conditions. Hfq-bound transcripts (1315) were largely identified in stressed bacteria and derived from small RNAs (sRNAs), both trans-encoded (6.4%) and antisense (asRNAs; 6.3%), and mRNAs (86%). Pull-down with Hfq recovered a small proportion of annotated S. meliloti sRNAs (14% of trans-sRNAs and 2% of asRNAs) suggesting a discrete impact of this protein in sRNA pathways. Nonetheless, Hfq selectively stabilized CoIP-enriched sRNAs, anticipating that these interactions are functionally significant. Transcription of 26 Hfq-bound sRNAs was predicted to occur from promoters recognized by the major stress σ factors σE2 or σH1/2. Recovery rates of sRNAs in each of the CoIP–RNA libraries suggest a large impact of Hfq-assisted riboregulation in S. meliloti osmoadaptation. Hfq directly targeted 18% of the predicted S. meliloti mRNAs, which encode functionally diverse proteins involved in transport and metabolism, σE2-dependent stress responses, quorum sensing, flagella biosynthesis, ribosome, and membrane assembly or symbiotic nitrogen fixation. Canonical targeting of the 5′ regions of two of the ABC transporter mRNAs by the homologous Hfq-binding AbcR1 and AbcR2 sRNAs leading to inhibition of protein synthesis was confirmed in vivo. We therefore provide a comprehensive resource for the systems-level deciphering of hitherto unexplored S. meliloti stress and symbiotic post-transcriptional regulons and the identification of Hfq-dependent sRNA–mRNA regulatory pairs. PMID:24786641

Sublingual immunization with the phosphate-binding-protein (PstS) reduces oral colonization by Streptococcus mutans.

PubMed

Ferreira, E L; Batista, M T; Cavalcante, R C M; Pegos, V R; Passos, H M; Silva, D A; Balan, A; Ferreira, L C S; Ferreira, R C C

2016-10-01

Bacterial ATP-binding cassette (ABC) transporters play a crucial role in the physiology and pathogenicity of different bacterial species. Components of ABC transporters have also been tested as target antigens for the development of vaccines against different bacterial species, such as those belonging to the Streptococcus genus. Streptococcus mutans is the etiological agent of dental caries, and previous studies have demonstrated that deletion of the gene encoding PstS, the substrate-binding component of the phosphate uptake system (Pst), reduced the adherence of the bacteria to abiotic surfaces. In the current study, we generated a recombinant form of the S. mutans PstS protein (rPstS) with preserved structural features, and we evaluated the induction of antibody responses in mice after sublingual mucosal immunization with a formulation containing the recombinant protein and an adjuvant derived from the heat-labile toxin from enterotoxigenic Escherichia coli strains. Mice immunized with rPstS exhibited systemic and secreted antibody responses, measured by the number of immunoglobulin A-secreting cells in draining lymph nodes. Serum antibodies raised in mice immunized with rPstS interfered with the adhesion of bacteria to the oral cavity of naive mice challenged with S. mutans. Similarly, mice actively immunized with rPstS were partially protected from oral colonization after challenge with the S. mutans NG8 strain. Therefore, our results indicate that S. mutans PstS is a potential target antigen capable of inducing specific and protective antibody responses after sublingual administration. Overall, these observations raise interesting perspectives for the development of vaccines to prevent dental caries. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Myostatin propeptide mutation of the hypermuscular Compact mice decreases the formation of myostatin and improves insulin sensitivity.

PubMed

Kocsis, Tamas; Trencsenyi, Gyorgy; Szabo, Kitti; Baan, Julia Aliz; Muller, Geza; Mendler, Luca; Garai, Ildiko; Reinauer, Hans; Deak, Ferenc; Dux, Laszlo; Keller-Pinter, Aniko

2017-03-01

The TGFβ family member myostatin (growth/differentiation factor-8) is a negative regulator of skeletal muscle growth. The hypermuscular Compact mice carry the 12-bp Mstn(Cmpt-dl1Abc) deletion in the sequence encoding the propeptide region of the precursor promyostatin, and additional modifier genes of the Compact genetic background contribute to determine the full expression of the phenotype. In this study, by using mice strains carrying mutant or wild-type myostatin alleles with the Compact genetic background and nonmutant myostatin with the wild-type background, we studied separately the effect of the Mstn(Cmpt-dl1Abc) mutation or the Compact genetic background on morphology, metabolism, and signaling. We show that both the Compact myostatin mutation and Compact genetic background account for determination of skeletal muscle size. Despite the increased musculature of Compact s, the absolute size of heart and kidney is not influenced by myostatin mutation; however, the Compact genetic background increases them. Both Compact myostatin and genetic background exhibit systemic metabolic effects. The Compact mutation decreases adiposity and improves whole body glucose uptake, insulin sensitivity, and 18 FDG uptake of skeletal muscle and white adipose tissue, whereas the Compact genetic background has the opposite effect. Importantly, the mutation does not prevent the formation of mature myostatin; however, a decrease in myostatin level was observed, leading to altered activation of Smad2, Smad1/5/8, and Akt, and an increased level of p-AS160, a Rab-GTPase-activating protein responsible for GLUT4 translocation. Based on our analysis, the Compact genetic background strengthens the effect of myostatin mutation on muscle mass, but those can compensate for each other when systemic metabolic effects are compared. Copyright © 2017 the American Physiological Society.

ABC transporter content diversity in Streptococcus pneumoniae impacts competence regulation and bacteriocin production.

PubMed

Wang, Charles Y; Patel, Nisha; Wholey, Wei-Yun; Dawid, Suzanne

2018-06-19

The opportunistic pathogen Streptococcus pneumoniae (pneumococcus) uses natural genetic competence to increase its adaptability through horizontal gene transfer. One method of acquiring DNA is through predation of neighboring strains with antimicrobial peptides called "bacteriocins." Competence and production of the major family of pneumococcal bacteriocins, pneumocins, are regulated by the quorum-sensing systems com and blp , respectively. In the classical paradigm, the ABC transporters ComAB and BlpAB each secretes its own system's signaling pheromone and in the case of BlpAB also secretes the pneumocins. While ComAB is found in all pneumococci, only 25% of strains encode an intact version of BlpAB [BlpAB(+)] while the rest do not [BlpAB(-)]. Contrary to the classical paradigm, it was previously shown that BlpAB(-) strains can activate blp through ComAB-mediated secretion of the blp pheromone during brief periods of competence. To better understand the full extent of com - blp crosstalk, we examined the contribution of each transporter to competence development and pneumocin secretion. We found that BlpAB(+) strains have a greater capacity for competence activation through BlpAB-mediated secretion of the com pheromone. Similarly, we show that ComAB and BlpAB are promiscuous and both can secrete pneumocins. Consequently, differences in pneumocin secretion between BlpAB(+) and BlpAB(-) strains derive from the regulation and kinetics of transporter expression rather than substrate specificity. We speculate that BlpAB(-) strains (opportunists) use pneumocins mainly in a narrowly tailored role for DNA acquisition and defense during competence while BlpAB(+) strains (aggressors) expand their use for the general inhibition of rival strains. Copyright © 2018 the Author(s). Published by PNAS.

Structure, function, and evolution of bacterial ATP-binding cassette systems

DOE Office of Scientific and Technical Information (OSTI.GOV)

Davidson, A.L.; Dassa, E.; Orelle, C.

2010-07-27

The ATP-binding cassette (ABC) systems constitute one of the largest superfamilies of paralogous sequences. All ABC systems share a highly conserved ATP-hydrolyzing domain or protein (the ABC; also referred to as a nucleotide-binding domain [NBD]) that is unequivocally characterized by three short sequence motifs (Fig. 1): these are the Walker A and Walker B motifs, indicative of the presence of a nucleotide-binding site, and the signature motif, unique to ABC proteins, located upstream of the Walker B motif (426). Other motifs diagnostic of ABC proteins are also indicated in Fig. 1. The biological significance of these motifs is discussed inmore » Structure, Function, and Dynamics of the ABC. ABC systems are widespread among living organisms and have been detected in all genera of the three kingdoms of life, with remarkable conservation in the primary sequence of the cassette and in the organization of the constitutive domains or subunits (203, 420). ABC systems couple the energy of ATP hydrolysis to an impressively large variety of essential biological phenomena, comprising not only transmembrane (TM) transport, for which they are best known, but also several non-transport-related processes, such as translation elongation (62) and DNA repair (174). Although ABC systems deserve much attention because they are involved in severe human inherited diseases (107), they were first discovered and characterized in detail in prokaryotes, as early as the 1970s (13, 148, 238, 468). The most extensively analyzed systems were the high-affinity histidine and maltose uptake systems of Salmonella enterica serovar Typhimurium and Escherichia coli. Over 2 decades ago, after the completion of the nucleotide sequences encoding these transporters in the respective laboratories of Giovanna Ames and Maurice Hofnung, Hiroshi Nikaido and colleagues noticed that the two systems displayed a global similarity in the nature of their components and, moreover, that the primary sequences of MalK and HisP, the proteins suspected to energize these transporters, shared as much as 32% identity in amino acid residues when their sequences were aligned (171). Later, it was found that several bacterial proteins involved in uptake of nutrients, export of toxins, cell division, bacterial nodulation of plants, and DNA repair displayed the same similarity in their sequences (127, 196). This led to the notion that the conserved protein, which had been shown to bind ATP (198, 201), would probably energize the systems mentioned above by coupling the energy of ATP hydrolysis to transport. The latter was demonstrated with the maltose and histidine transporters by use of isolated membrane vesicles (105, 379) and purified transporters reconstituted into proteoliposomes (30, 98). The determination of the sequence of the first eukaryotic protein strongly similar to these bacterial transporters (the P-glycoprotein, involved in resistance of cancer cells to multiple drugs) (169, 179) demonstrated that these proteins were not restricted to prokaryotes. Two names, 'traffic ATPases' (15) and the more accepted name 'ABC transporters' (193, 218), were proposed for members of this new superfamily. ABC systems can be divided into three main functional categories, as follows. Importers mediate the uptake of nutrients in prokaryotes. The nature of the substrates that are transported is very wide, including mono- and oligosaccharides, organic and inorganic ions, amino acids, peptides, ironsiderophores, metals, polyamine cations, opines, and vitamins. Exporters are involved in the secretion of various molecules, such as peptides, lipids, hydrophobic drugs, polysaccharides, and proteins, including toxins such as hemolysin. The third category of systems is apparently not involved in transport, with some members being involved in translation of mRNA and in DNA repair. Despite the large, diverse population of substrates handled and the difference in the polarity of transport, importers and exporters share a common organization made of two hydrophobic membrane-spanning or integral membrane (IM) domains and two hydrophilic domains carrying the ABC peripherally associated with the IM domains on the cytosolic side of the membrane (26). In importers, these four domains are almost always independent polypeptide chains that come together to form a multimeric complex. In most exporters, including the E. coli hemolysin exporter HlyB, the N-terminal IM and the C-terminal ABC domains are fused as a single polypeptide chain (IM-ABC). An inverted organization in which the IM domain is C-terminal with respect to the ABC domain (ABC-IM) exists, such as in the MacB protein, involved in macrolide resistance in E. coli. No IM domain partners have been identified for ABC proteins falling into the third category, and these proteins consist of two ABCs fused together (ABC2).« less

Determination of multidrug resistance mechanisms in Clostridium perfringens type A isolates using RNA sequencing and 2D-electrophoresis.

PubMed

Ma, Yu-Hua; Ye, Gui-Sheng

2018-06-11

In this study, we screened differentially expressed genes in a multidrug-resistant isolate strain of Clostridium perfringens by RNA sequencing. We also separated and identified differentially expressed proteins (DEPs) in the isolate strain by two-dimensional electrophoresis (2-DE) and mass spectrometry (MS). The RNA sequencing results showed that, compared with the control strain, 1128 genes were differentially expressed in the isolate strain, and these included 227 up-regulated genes and 901 down-regulated genes. Bioinformatics analysis identified the following genes and gene categories that are potentially involved in multidrug resistance (MDR) in the isolate strain: drug transport, drug response, hydrolase activity, transmembrane transporter, transferase activity, amidase transmembrane transporter, efflux transmembrane transporter, bacterial chemotaxis, ABC transporter, and others. The results of the 2-DE showed that 70 proteins were differentially expressed in the isolate strain, 45 of which were up-regulated and 25 down-regulated. Twenty-seven DEPs were identified by MS and these included the following protein categories: ribosome, antimicrobial peptide resistance, and ABC transporter, all of which may be involved in MDR in the isolate strain of C. perfringens. The results provide reference data for further investigations on the drug resistant molecular mechanisms of C. perfringens.

Topological and organizational properties of the products of house-keeping and tissue-specific genes in protein-protein interaction networks.

PubMed

Lin, Wen-Hsien; Liu, Wei-Chung; Hwang, Ming-Jing

2009-03-11

Human cells of various tissue types differ greatly in morphology despite having the same set of genetic information. Some genes are expressed in all cell types to perform house-keeping functions, while some are selectively expressed to perform tissue-specific functions. In this study, we wished to elucidate how proteins encoded by human house-keeping genes and tissue-specific genes are organized in human protein-protein interaction networks. We constructed protein-protein interaction networks for different tissue types using two gene expression datasets and one protein-protein interaction database. We then calculated three network indices of topological importance, the degree, closeness, and betweenness centralities, to measure the network position of proteins encoded by house-keeping and tissue-specific genes, and quantified their local connectivity structure. Compared to a random selection of proteins, house-keeping gene-encoded proteins tended to have a greater number of directly interacting neighbors and occupy network positions in several shortest paths of interaction between protein pairs, whereas tissue-specific gene-encoded proteins did not. In addition, house-keeping gene-encoded proteins tended to connect with other house-keeping gene-encoded proteins in all tissue types, whereas tissue-specific gene-encoded proteins also tended to connect with other tissue-specific gene-encoded proteins, but only in approximately half of the tissue types examined. Our analysis showed that house-keeping gene-encoded proteins tend to occupy important network positions, while those encoded by tissue-specific genes do not. The biological implications of our findings were discussed and we proposed a hypothesis regarding how cells organize their protein tools in protein-protein interaction networks. Our results led us to speculate that house-keeping gene-encoded proteins might form a core in human protein-protein interaction networks, while clusters of tissue-specific gene-encoded proteins are attached to the core at more peripheral positions of the networks.

Draft genome sequence of Actinotignum schaalii DSM 15541T: Genetic insights into the lifestyle, cell fitness and virulence.

PubMed

Yassin, Atteyet F; Langenberg, Stefan; Huntemann, Marcel; Clum, Alicia; Pillay, Manoj; Palaniappan, Krishnaveni; Varghese, Neha; Mikhailova, Natalia; Mukherjee, Supratim; Reddy, T B K; Daum, Chris; Shapiro, Nicole; Ivanova, Natalia; Woyke, Tanja; Kyrpides, Nikos C

2017-01-01

The permanent draft genome sequence of Actinotignum schaalii DSM 15541T is presented. The annotated genome includes 2,130,987 bp, with 1777 protein-coding and 58 rRNA-coding genes. Genome sequence analysis revealed absence of genes encoding for: components of the PTS systems, enzymes of the TCA cycle, glyoxylate shunt and gluconeogensis. Genomic data revealed that A. schaalii is able to oxidize carbohydrates via glycolysis, the nonoxidative pentose phosphate and the Entner-Doudoroff pathways. Besides, the genome harbors genes encoding for enzymes involved in the conversion of pyruvate to lactate, acetate and ethanol, which are found to be the end products of carbohydrate fermentation. The genome contained the gene encoding Type I fatty acid synthase required for de novo FAS biosynthesis. The plsY and plsX genes encoding the acyltransferases necessary for phosphatidic acid biosynthesis were absent from the genome. The genome harbors genes encoding enzymes responsible for isoprene biosynthesis via the mevalonate (MVA) pathway. Genes encoding enzymes that confer resistance to reactive oxygen species (ROS) were identified. In addition, A. schaalii harbors genes that protect the genome against viral infections. These include restriction-modification (RM) systems, type II toxin-antitoxin (TA), CRISPR-Cas and abortive infection system. A. schaalii genome also encodes several virulence factors that contribute to adhesion and internalization of this pathogen such as the tad genes encoding proteins required for pili assembly, the nanI gene encoding exo-alpha-sialidase, genes encoding heat shock proteins and genes encoding type VII secretion system. These features are consistent with anaerobic and pathogenic lifestyles. Finally, resistance to ciprofloxacin occurs by mutation in chromosomal genes that encode the subunits of DNA-gyrase (GyrA) and topisomerase IV (ParC) enzymes, while resistant to metronidazole was due to the frxA gene, which encodes NADPH-flavin oxidoreductase.

Describing the role of Drosophila melanogaster ABC transporters in insecticide biology using CRISPR-Cas9 knockouts.

PubMed

Denecke, Shane; Fusetto, Roberto; Batterham, Philip

2017-12-01

ABC transporters have a well-established role in drug resistance, effluxing xenobiotics from cells and tissues within the organism. More recently, research has been dedicated to understanding the role insect ABC transporters play in insecticide toxicity, but progress in understanding the contribution of specific transporters has been hampered by the lack of functional genetic tools. Here, we report knockouts of three Drosophila melanogaster ABC transporter genes, Mdr49, Mdr50, and Mdr65, that are homologous to the well-studied mammalian ABCB1 (P-glycoprotein). Each knockout mutant was created in the same wild type background and tested against a panel of insecticides representing different chemical classes. Mdr65 knockouts were more susceptible to all neuroactive insecticides tested, but Mdr49 and Mdr50 knockouts showed increased susceptibility or resistance depending on the insecticide used. Mdr65 was chosen for further analysis. Calculation of LC 50 values for the Mdr65 knockout allowed the substrate specificity of this transporter to be examined. No obvious distinguishing structural features were shared among MDR65 substrates. A role for Mdr65 in insecticide transport was confirmed by testing the capacity of the knockout to synergize with the ABC inhibitor verapamil and by measuring the levels of insecticide retained in the body of knockout flies. These data unambiguously establish the influence of ABC transporters on the capacity of wild type D. melanogaster to tolerate insecticide exposure and suggest that both tissue and substrate specificity underpin this capacity. Copyright © 2017 Elsevier Ltd. All rights reserved.

Divergence of the Floral A-Function between an Asterid and a Rosid Species.

PubMed

Morel, Patrice; Heijmans, Klaas; Rozier, Frédérique; Zethof, Jan; Chamot, Sophy; Bento, Suzanne Rodrigues; Vialette-Guiraud, Aurélie; Chambrier, Pierre; Trehin, Christophe; Vandenbussche, Michiel

2017-07-01

The ABC model is widely used as a genetic framework for understanding floral development and evolution. In this model, the A-function is required for the development of sepals and petals and to antagonize the C-function in the outer floral whorls. In the rosid species Arabidopsis thaliana , the AP2-type AP2 transcription factor represents a major A-function protein, but how the A-function is encoded in other species is not well understood. Here, we show that in the asterid species petunia ( Petunia hybrida ), AP2B/BLIND ENHANCER ( BEN ) confines the C-function to the inner petunia floral whorls, in parallel with the microRNA BLIND BEN belongs to the TOE-type AP2 gene family, members of which control flowering time in Arabidopsis. In turn, we demonstrate that the petunia AP2-type REPRESSOR OF B-FUNCTION ( ROB ) genes repress the B-function (but not the C-function) in the first floral whorl, together with BEN We propose a combinatorial model for patterning the B- and C-functions, leading to the homeotic conversion of sepals into petals, carpels, or stamens, depending on the genetic context. Combined with earlier results, our findings suggest that the molecular mechanisms controlling the spatial restriction of the floral organ identity genes are more diverse than the well-conserved B and C floral organ identity functions. © 2017 American Society of Plant Biologists. All rights reserved.

Identification of transcriptome involved in atrazine detoxification and degradation in alfalfa (Medicago sativa) exposed to realistic environmental contamination.

PubMed

Zhang, Jing Jing; Lu, Yi Chen; Zhang, Shu Hao; Lu, Feng Fan; Yang, Hong

2016-08-01

Plants are constantly exposed to a variety of toxic compounds (or xenobiotics) such as pesticides (or herbicides). Atrazine (ATZ) as herbicide has become one of the environmental contaminants due to its intensive use during crop production. Plants have evolved strategies to cope with the adverse impact of ATZ. However, the mechanism for ATZ degradation and detoxification in plants is largely unknown. Here we employed a global RNA-sequencing (RNA-Seq) strategy to dissect transcriptome variation in alfalfa (Medicago sativa) exposed to ATZ. Four libraries were constructed including Root-ATZ (root control, ATZ-free), Shoot-ATZ, Root+ATZ (root treated with ATZ) and Shoot+ATZ. Hierarchical clustering was performed to display the expression patterns for all differentially expressed genes (DEGs) under ATZ exposure. Transcripts involved in ATZ detoxification, stress responses (e.g. oxidation and reduction, conjugation and hydrolytic reactions), and regulations of cysteine biosynthesis were identified. Several genes encoding glycosyltransferases, glutathione S-transferases or ABC transporters were up-regulated notably. Also, many other genes involved in oxidation-reduction, conjugation, and hydrolysis for herbicide degradation were differentially expressed. These results suggest that ATZ in alfalfa can be detoxified or degraded through different pathways. The expression patterns of some DEGs by high-throughput sequencing were well confirmed by qRT-PCR. Our results not only highlight the transcriptional complexity in alfalfa exposed to ATZ but represent a major improvement for analyzing transcriptional changes on a large scale as well. Copyright © 2016 Elsevier Inc. All rights reserved.

Determining Roles of Accessory Genes in Denitrification by Mutant Fitness Analyses

PubMed Central

Vaccaro, Brian J.; Thorgersen, Michael P.; Lancaster, W. Andrew; Price, Morgan N.; Wetmore, Kelly M.; Poole, Farris L.; Deutschbauer, Adam; Arkin, Adam P.

2015-01-01

Enzymes of the denitrification pathway play an important role in the global nitrogen cycle, including release of nitrous oxide, an ozone-depleting greenhouse gas. In addition, nitric oxide reductase, maturation factors, and proteins associated with nitric oxide detoxification are used by pathogens to combat nitric oxide release by host immune systems. While the core reductases that catalyze the conversion of nitrate to dinitrogen are well understood at a mechanistic level, there are many peripheral proteins required for denitrification whose basic function is unclear. A bar-coded transposon DNA library from Pseudomonas stutzeri strain RCH2 was grown under denitrifying conditions, using nitrate or nitrite as an electron acceptor, and also under molybdenum limitation conditions, with nitrate as the electron acceptor. Analysis of sequencing results from these growths yielded gene fitness data for 3,307 of the 4,265 protein-encoding genes present in strain RCH2. The insights presented here contribute to our understanding of how peripheral proteins contribute to a fully functioning denitrification pathway. We propose a new low-affinity molybdate transporter, OatABC, and show that differential regulation is observed for two MoaA homologs involved in molybdenum cofactor biosynthesis. We also propose that NnrS may function as a membrane-bound NO sensor. The dominant HemN paralog involved in heme biosynthesis is identified, and a CheR homolog is proposed to function in nitrate chemotaxis. In addition, new insights are provided into nitrite reductase redundancy, nitric oxide reductase maturation, nitrous oxide reductase maturation, and regulation. PMID:26452555

The durable wheat disease resistance gene Lr34 confers common rust and northern corn leaf blight resistance in maize.

PubMed

Sucher, Justine; Boni, Rainer; Yang, Ping; Rogowsky, Peter; Büchner, Heike; Kastner, Christine; Kumlehn, Jochen; Krattinger, Simon G; Keller, Beat

2017-04-01

Maize (corn) is one of the most widely grown cereal crops globally. Fungal diseases of maize cause significant economic damage by reducing maize yields and by increasing input costs for disease management. The most sustainable control of maize diseases is through the release and planting of maize cultivars with durable disease resistance. The wheat gene Lr34 provides durable and partial field resistance against multiple fungal diseases of wheat, including three wheat rust pathogens and wheat powdery mildew. Because of its unique qualities, Lr34 became a cornerstone in many wheat disease resistance programmes. The Lr34 resistance is encoded by a rare variant of an ATP-binding cassette (ABC) transporter that evolved after wheat domestication. An Lr34-like disease resistance phenotype has not been reported in other cereal species, including maize. Here, we transformed the Lr34 resistance gene into the maize hybrid Hi-II. Lr34-expressing maize plants showed increased resistance against the biotrophic fungal disease common rust and the hemi-biotrophic disease northern corn leaf blight. Furthermore, the Lr34-expressing maize plants developed a late leaf tip necrosis phenotype, without negative impact on plant growth. With this and previous reports, it could be shown that Lr34 is effective against various biotrophic and hemi-biotrophic diseases that collectively parasitize all major cereal crop species. © 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

Regulators of floral fragrance production and their target genes in petunia are not exclusively active in the epidermal cells of petals

PubMed Central

Van Moerkercke, Alex; Galván-Ampudia, Carlos S.; Verdonk, Julian C.; Haring, Michel A.; Schuurink, Robert C.

2012-01-01

In which cells of the flower volatile biosynthesis takes place is unclear. In rose and snapdragon, some enzymes of the volatile phenylpropanoid/benzenoid pathway have been shown to be present in the epidermal cells of petals. It is therefore generally believed that the production of these compounds occurs in these cells. However, whether the entire pathway is active in these cells and whether it is exclusively active in these cells remains to be proven. Cell-specific transcription factors activating these genes will determine in which cells they are expressed. In petunia, the transcription factor EMISSION OF BENZENOIDS II (EOBII) activates the ODORANT1 (ODO1) promoter and the promoter of the biosynthetic gene isoeugenol synthase (IGS). The regulator ODO1 in turn activates the promoter of the shikimate gene 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). Here the identification of a new target gene of ODO1, encoding an ABC transporter localized on the plasma membrane, PhABCG1, which is co-expressed with ODO1, is described. PhABCG1 expression is up-regulated in petals overexpressing ODO1 through activation of the PhABCG1 promoter. Interestingly, the ODO1, PhABCG1, and IGS promoters were active in petunia protoplasts originating from both epidermal and mesophyll cell layers of the petal, suggesting that the volatile phenylpropanoid/benzenoid pathway in petunia is active in these different cell types. Since volatile release occurs from epidermal cells, trafficking of (volatile) compounds between cell layers must be involved, but the exact function of PhABCG1 remains to be resolved. PMID:22345641

The Transcriptional Response of Listeria monocytogenes during Adaptation to Growth on Lactate and Diacetate Includes Synergistic Changes That Increase Fermentative Acetoin Production▿†

PubMed Central

Stasiewicz, Matthew J.; Wiedmann, Martin; Bergholz, Teresa M.

2011-01-01

The organic acids lactate and diacetate are commonly used in combination in ready-to-eat foods because they show synergistic ability to inhibit the growth of Listeria monocytogenes. Full-genome microarrays were used to investigate the synergistic transcriptomic responses of two L. monocytogenes strains, H7858 (serotype 4b) and F6854 (serotype 1/2a), to these two organic acids under conditions representing osmotic and cold stress encountered in foods. Strains were exposed to brain heart infusion (BHI) broth at 7°C with 4.65% water-phase (w.p.) NaCl at pH 6.1 with (i) 2% w.p. potassium lactate, (ii) 0.14% w.p. sodium diacetate, (iii) the combination of both at the same levels, or (iv) no organic acids as a control. RNA was extracted 8 h after exposure, during lag phase, to capture gene transcription changes during adaptation to the organic acid stress. Significant differential transcription of 1,041 genes in H7858 and 640 genes in F6854 was observed in at least one pair of the 4 different treatments. The effects of combined treatment with lactate and diacetate included (i) synergistic transcription differences for 474 and 209 genes in H7858 and F6854, respectively, (ii) differential transcription of genes encoding cation transporters and ABC transporters of metals, and (iii) altered metabolism, including induction of a nutrient-limiting stress response, reduction of menaquinone biosynthesis, and a shift from fermentative production of acetate and lactate to energetically less favorable, neutral acetoin. These data suggest that additional treatments that interfere with cellular energy generation processes could more efficiently inhibit the growth of L. monocytogenes. PMID:21666015

Enhanced gravi- and phototropism in plant mdr mutants mislocalizing the auxin efflux protein PIN1.

PubMed

Noh, Bosl; Bandyopadhyay, Anindita; Peer, Wendy Ann; Spalding, Edgar P; Murphy, Angus S

2003-06-26

Many aspects of plant growth and development are dependent on the flow of the hormone auxin down the plant from the growing shoot tip where it is synthesized. The direction of auxin transport in stems is believed to result from the basal localization within cells of the PIN1 membrane protein, which controls the efflux of the auxin anion. Mutations in two genes homologous to those encoding the P-glycoprotein ABC transporters that are especially abundant in multidrug-resistant tumour cells in animals were recently shown to block polar auxin transport in the hypocotyls of Arabidopsis seedlings. Here we show that the mdr mutants display faster and greater gravitropism and enhanced phototropism instead of the impaired curvature development expected in mutants lacking polar auxin transport. We find that these phenotypes result from a disruption of the normal accumulation of PIN1 protein along the basal end of hypocotyl cells associated with basipetal auxin flow. Lateral auxin conductance becomes relatively larger as a result, enhancing the growth differentials responsible for tropic responses.

Growth of desferrioxamine-deficient Streptomyces mutants through xenosiderophore piracy of airborne fungal contaminations.

PubMed

Arias, Anthony Argüelles; Lambert, Stéphany; Martinet, Loïc; Adam, Delphine; Tenconi, Elodie; Hayette, Marie-Pierre; Ongena, Marc; Rigali, Sébastien

2015-07-01

Due to the necessity of iron for housekeeping functions, nutrition, morphogenesis and secondary metabolite production, siderophore piracy could be a key strategy in soil and substrate colonization by microorganisms. Here we report that mutants of bacterium Streptomyces coelicolor unable to produce desferrioxamine siderophores could recover growth when the plates were contaminated by indoor air spores of a Penicillium species and Engyodontium album. UPLC-ESI-MS analysis revealed that the HPLC fractions with the extracellular 'resuscitation' factors of the Penicillium isolate were only those that contained siderophores, i.e. Fe-dimerum acid, ferrichrome, fusarinine C and coprogen. The restored growth of the Streptomyces mutants devoid of desferrioxamine is most likely mediated through xenosiderophore uptake as the cultivability depends on the gene encoding the ABC-transporter-associated DesE siderophore-binding protein. That a filamentous fungus allows the growth of desferrioxamine non-producing Streptomyces in cocultures confirms that xenosiderophore piracy plays a vital role in nutritional interactions between these taxonomically unrelated filamentous microorganisms. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Comparative vesicle proteomics reveals selective regulation of protein expression in chestnut blight fungus by a hypovirus.

PubMed

Wang, Jinzi; Wang, Fangzhen; Feng, Youjun; Mi, Ke; Chen, Qi; Shang, Jinjie; Chen, Baoshan

2013-01-14

The chestnut blight fungus (Cryphonectria parasitica) and hypovirus constitute a model system to study fungal pathogenesis and mycovirus-host interaction. Knowledge in this field has been gained largely from investigations at gene transcription level so far. Here we report a systematic analysis of the vesicle proteins of the host fungus with/without hypovirus infection. Thirty-three differentially expressed protein spots were identified in the purified vesicle protein samples by two-dimensional electrophoresis and mass spectrometry. Down-regulated proteins were mostly cargo proteins involved in primary metabolism and energy generation and up-regulated proteins were mostly vesicle associated proteins and ABC transporter. A virus-encoded protein p48 was found to have four forms with different molecular mass in vesicles from the virus-infected strain. While a few of the randomly selected differentially expressed proteins were in accordance with their transcription profiles, majority were not in agreement with their mRNA accumulation patterns, suggesting that an extensive post-transcriptional regulation may have occurred in the host fungus upon a hypovirus infection. Copyright © 2012 Elsevier B.V. All rights reserved.

Nucleotide sequences of two genomic DNAs encoding peroxidase of Arabidopsis thaliana.

PubMed

Intapruk, C; Higashimura, N; Yamamoto, K; Okada, N; Shinmyo, A; Takano, M

1991-02-15

The peroxidase (EC 1.11.1.7)-encoding gene of Arabidopsis thaliana was screened from a genomic library using a cDNA encoding a neutral isozyme of horseradish, Armoracia rusticana, peroxidase (HRP) as a probe, and two positive clones were isolated. From the comparison with the sequences of the HRP-encoding genes, we concluded that two clones contained peroxidase-encoding genes, and they were named prxCa and prxEa. Both genes consisted of four exons and three introns; the introns had consensus nucleotides, GT and AG, at the 5' and 3' ends, respectively. The lengths of each putative exon of the prxEa gene were the same as those of the HRP-basic-isozyme-encoding gene, prxC3, and coded for 349 amino acids (aa) with a sequence homology of 89% to that encoded by prxC3. The prxCa gene was very close to the HRP-neutral-isozyme-encoding gene, prxC1b, and coded for 354 aa with 91% homology to that encoded by prxC1b. The aa sequence homology was 64% between the two peroxidases encoded by prxCa and prxEa.

A Truncated AdeS Kinase Protein Generated by ISAba1 Insertion Correlates with Tigecycline Resistance in Acinetobacter baumannii

PubMed Central

Sun, Jun-Ren; Perng, Cherng-Lih; Chan, Ming-Chin; Morita, Yuji; Lin, Jung-Chung; Su, Chih-Mao; Wang, Wei-Yao; Chang, Tein-Yao; Chiueh, Tzong-Shi

2012-01-01

Over-expression of AdeABC efflux pump stimulated continuously by the mutated AdeRS two component system has been found to result in antimicrobial resistance, even tigecycline (TGC) resistance, in multidrug-resistant Acinetobacter baumannii (MRAB). Although the insertion sequence, ISAba1, contributes to one of the AdeRS mutations, the detail mechanism remains unclear. In the present study we collected 130 TGC-resistant isolates from 317 carbapenem resistant MRAB (MRAB-C) isolates, and 38 of them were characterized with ISAba1 insertion in the adeS gene. The relationship between the expression of AdeABC efflux pump and TGC resistant was verified indirectly by successfully reducing TGC resistance with NMP, an efflux pump inhibitor. Further analysis showed that the remaining gene following the ISAba1 insertion was still transcribed to generate a truncated AdeS protein by the Pout promoter on ISAba1 instead of frame shift or pre-termination. Through introducing a series of recombinant adeRS constructs into a adeRS knockout strain, we demonstrated the truncated AdeS protein was constitutively produced and stimulating the expression of AdeABC efflux pump via interaction with AdeR. Our findings suggest a mechanism of antimicrobial resistance induced by an aberrant cytoplasmic sensor derived from an insertion element. PMID:23166700

Spaceflight Enhances Cell Aggregation and Random Budding in Candida albicans

PubMed Central

Woolley, Christine M.; Barrila, Jennifer; Buchanan, Kent; McCracken, James; Inglis, Diane O.; Searles, Stephen C.; Nelman-Gonzalez, Mayra A.; Ott, C. Mark; Wilson, James W.; Pierson, Duane L.; Stefanyshyn-Piper, Heidemarie M.; Hyman, Linda E.; Nickerson, Cheryl A.

2013-01-01

This study presents the first global transcriptional profiling and phenotypic characterization of the major human opportunistic fungal pathogen, Candida albicans, grown in spaceflight conditions. Microarray analysis revealed that C. albicans subjected to short-term spaceflight culture differentially regulated 452 genes compared to synchronous ground controls, which represented 8.3% of the analyzed ORFs. Spaceflight-cultured C. albicans–induced genes involved in cell aggregation (similar to flocculation), which was validated by microscopic and flow cytometry analysis. We also observed enhanced random budding of spaceflight-cultured cells as opposed to bipolar budding patterns for ground samples, in accordance with the gene expression data. Furthermore, genes involved in antifungal agent and stress resistance were differentially regulated in spaceflight, including induction of ABC transporters and members of the major facilitator family, downregulation of ergosterol-encoding genes, and upregulation of genes involved in oxidative stress resistance. Finally, downregulation of genes involved in actin cytoskeleton was observed. Interestingly, the transcriptional regulator Cap1 and over 30% of the Cap1 regulon was differentially expressed in spaceflight-cultured C. albicans. A potential role for Cap1 in the spaceflight response of C. albicans is suggested, as this regulator is involved in random budding, cell aggregation, and oxidative stress resistance; all related to observed spaceflight-associated changes of C. albicans. While culture of C. albicans in microgravity potentiates a global change in gene expression that could induce a virulence-related phenotype, no increased virulence in a murine intraperitoneal (i.p.) infection model was observed under the conditions of this study. Collectively, our data represent an important basis for the assessment of the risk that commensal flora could play during human spaceflight missions. Furthermore, since the low fluid-shear environment of microgravity is relevant to physical forces encountered by pathogens during the infection process, insights gained from this study could identify novel infectious disease mechanisms, with downstream benefits for the general public. PMID:24324620

Degradation of Benzene by Pseudomonas veronii 1YdBTEX2 and 1YB2 Is Catalyzed by Enzymes Encoded in Distinct Catabolism Gene Clusters.

PubMed

de Lima-Morales, Daiana; Chaves-Moreno, Diego; Wos-Oxley, Melissa L; Jáuregui, Ruy; Vilchez-Vargas, Ramiro; Pieper, Dietmar H

2016-01-01

Pseudomonas veronii 1YdBTEX2, a benzene and toluene degrader, and Pseudomonas veronii 1YB2, a benzene degrader, have previously been shown to be key players in a benzene-contaminated site. These strains harbor unique catabolic pathways for the degradation of benzene comprising a gene cluster encoding an isopropylbenzene dioxygenase where genes encoding downstream enzymes were interrupted by stop codons. Extradiol dioxygenases were recruited from gene clusters comprising genes encoding a 2-hydroxymuconic semialdehyde dehydrogenase necessary for benzene degradation but typically absent from isopropylbenzene dioxygenase-encoding gene clusters. The benzene dihydrodiol dehydrogenase-encoding gene was not clustered with any other aromatic degradation genes, and the encoded protein was only distantly related to dehydrogenases of aromatic degradation pathways. The involvement of the different gene clusters in the degradation pathways was suggested by real-time quantitative reverse transcription PCR. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Serum albumin promotes ATP-binding cassette transporter-dependent sterol uptake in yeast.

PubMed

Marek, Magdalena; Silvestro, Daniele; Fredslund, Maria D; Andersen, Tonni G; Pomorski, Thomas G

2014-12-01

Sterol uptake in fungi is a multistep process that involves interaction between external sterols and the cell wall, incorporation of sterol molecules into the plasma membrane, and subsequent integration into intracellular membranes for turnover. ATP-binding cassette (ABC) transporters have been implicated in sterol uptake, but key features of their activity remain to be elucidated. Here, we apply fluorescent cholesterol (NBD-cholesterol) to monitor sterol uptake under anaerobic and aerobic conditions in two fungal species, Candida glabrata (Cg) and Saccharomyces cerevisiae (Sc). We found that in both fungal species, ABC transporter-dependent uptake of cholesterol under anaerobic conditions and in mutants lacking HEM1 gene is promoted in the presence of the serum protein albumin that is able to bind the sterol molecule. Furthermore, the C. glabrata ABC transporter CgAus1p expressed in S. cerevisiae requires the presence of serum or albumin for efficient cholesterol uptake. These results suggest that albumin can serve as sterol donor in ABC transporter-dependent sterol uptake, a process potentially important for growth of C. glabrata inside infected humans. © 2014 The Authors. FEMS Yeast Research published by John Wiley & Sons Ltd on behalf of Federation of European Microbiological Societies.

Trichothecene resistance in wheat: Development of molecular markers for PDR-type ABC transporter genes.

PubMed

Mitterbauer, R; Heinrich, M; Rauscher, R; Lemmens, M; Bürstmayr, H; Adam, G

2003-03-01

Infection withFusarium graminearum andF. culmorum not only causes severe yield and quality losses, the most relevant concern is the contamination of cereal foods and feeds with trichothecenes (e.g. deoxynivalenol, DON). The ability to synthesize trichothecenes has been shown to be a virulence factor ofF. graminearum on wheat and, on the other hand, toxin resistance is most likely an important component of field resistance. Our hypothesis is that pleiotropic drug resistance mediated by PDR-type ABC transporter proteins (acting as membrane located drug efflux pumps) is a relevant mechanism of DON resistance not only in yeast but also in wheat. Goal of this project is the development of molecular markers for this gene family for use in marker-assisted plant breeding programs. The technical difficulties caused by the large size of the PDR-family are discussed.

Differential Protein Expression in Streptococcus uberis under Planktonic and Biofilm Growth Conditions ▿ †

PubMed Central

Crowley, R. C.; Leigh, J. A.; Ward, P. N.; Lappin-Scott, H. M.; Bowler, L. D.

2011-01-01

The bovine pathogen Streptococcus uberis was assessed for biofilm growth. The transition from planktonic to biofilm growth in strain 0140J correlated with an upregulation of several gene products that have been shown to be important for pathogenesis, including a glutamine ABC transporter (SUB1152) and a lactoferrin binding protein (gene lbp; protein SUB0145). PMID:21075893

Regulation of ATP-binding Cassette Transporters and Cholesterol Efflux by Glucose in Primary Human Monocytes and Murine Bone Marrow-derived Macrophages

PubMed Central

Spartano, N. L.; Lamon-Fava, S.; Matthan, N. R.; Ronxhi, J.; Greenberg, A. S.; Obin, M. S.; Lichtenstein, A. H.

2014-01-01

Purpose Individuals with type 2 diabetes mellitus are at increased risk of developing atherosclerosis. This may be partially attributable to suppression of macrophage ATP-binding cassette (ABC) transporter mediated cholesterol efflux by sustained elevated blood glucose concentrations. 2 models were used to assess this potential relationship: human monocytes/leukocytes and murine bone marrow-derived macrophages (BMDM). Methods 10 subjects (4 F/6 M, 50–85 years, BMI 25–35 kg/m2) underwent an oral glucose challenge. Baseline and 1- and 2-h post-challenge ABC-transporter mRNA expression was determined in monocytes, leukocytes and peripheral blood mononuclear cells (PBMC). In a separate study, murine-BMDM were exposed to 5 mmol/L D-glucose (control) or additional 20 mmol/L D-or L-glucose and 25 ug/mL oxidized low density lipoprotein (oxLDL). High density lipoprotein (HDL)-mediated cholesterol efflux and ABC-transporter (ABCA1 and ABCG1) expression were determined. Results Baseline ABCA1and ABCG1 expression was lower (> 50 %) in human monocytes and PBMC than leukocytes (p < 0.05). 1 h post-challenge leukocyte ABCA1 and ABCG1 expression increased by 37 % and 30 %, respectively (p < 0.05), and began to return to baseline thereafter. There was no significant change in monocyte ABC-transporter expression. In murine BMDM, higher glucose concentrations suppressed HDL-mediated cholesterol efflux (10 %; p < 0.01) without significantly affecting ABCA1 and ABCG1 expression. Data demonstrate that leukocytes are not a reliable indicator of monocyte ABC-transporter expression. Conclusions Human monocyte ABC-transporter gene expression was unresponsive to a glucose challenge. Correspondingly, in BMDM, hyperglycemia attenuated macrophage cholesterol efflux in the absence of altered ABC-transporter expression, suggesting that hyperglycemia, per se, suppresses cholesterol transporter activity. This glucose-related impairment in cholesterol efflux may potentially contribute to diabetes-associated atherosclerosis. PMID:24838154

Growth promotion of the opportunistic human pathogen, Staphylococcus lugdunensis, by heme, hemoglobin, and coculture with Staphylococcus aureus

PubMed Central

Brozyna, Jeremy R; Sheldon, Jessica R; Heinrichs, David E

2014-01-01

Staphylococcus lugdunensis is both a commensal of humans and an opportunistic pathogen. Little is currently known about the molecular mechanisms underpinning the virulence of this bacterium. Here, we demonstrate that in contrast to S. aureus,S. lugdunensis makes neither staphyloferrin A (SA) nor staphyloferrin B (SB) in response to iron deprivation, owing to the absence of the SB gene cluster, and a large deletion in the SA biosynthetic gene cluster. As a result, the species grows poorly in serum-containing media, and this defect was complemented by introduction of the S. aureusSA gene cluster into S. lugdunensis. S. lugdunensis expresses the HtsABC and SirABC transporters for SA and SB, respectively; the latter gene set is found within the isd (heme acquisition) gene cluster. An isd deletion strain was significantly debilitated for iron acquisition from both heme and hemoglobin, and was also incapable of utilizing ferric-SB as an iron source, while an hts mutant could not grow on ferric-SA as an iron source. In iron-restricted coculture experiments, S. aureus significantly enhanced the growth of S. lugdunensis, in a manner dependent on staphyloferrin production by S. aureus, and the expression of the cognate transporters by S. lugdunensis. PMID:24515974

Caenorhabditis elegans ABCRNAi Transporters Interact Genetically With rde-2 and mut-7

PubMed Central

Sundaram, Prema; Han, Wang; Cohen, Nancy; Echalier, Benjamin; Albin, John; Timmons, Lisa

2008-01-01

RNA interference (RNAi) mechanisms are conserved and consist of an interrelated network of activities that not only respond to exogenous dsRNA, but also perform endogenous functions required in the fine tuning of gene expression and in maintaining genome integrity. Not surprisingly, RNAi functions have widespread influences on cellular function and organismal development. Previously, we observed a reduced capacity to mount an RNAi response in nine Caenorhabditis elegans mutants that are defective in ABC transporter genes (ABCRNAi mutants). Here, we report an exhaustive study of mutants, collectively defective in 49 different ABC transporter genes, that allowed for the categorization of one additional transporter into the ABCRNAi gene class. Genetic complementation tests reveal functions for ABCRNAi transporters in the mut-7/rde-2 branch of the RNAi pathway. These second-site noncomplementation interactions suggest that ABCRNAi proteins and MUT-7/RDE-2 function together in parallel pathways and/or as multiprotein complexes. Like mut-7 and rde-2, some ABCRNAi mutants display transposon silencing defects. Finally, our analyses reveal a genetic interaction network of ABCRNAi gene function with respect to this part of the RNAi pathway. From our results, we speculate that the coordinated activities of ABCRNAi transporters, through their effects on endogenous RNAi-related mechanisms, ultimately affect chromosome function and integrity. PMID:18245353

MADS-box genes and floral development: the dark side.

PubMed

Heijmans, Klaas; Morel, Patrice; Vandenbussche, Michiel

2012-09-01

The origin of the flower during evolution has been a crucial step in further facilitating plants to colonize a wide range of different niches on our planet. The >250 000 species of flowering plants existing today display an astonishing diversity in floral architecture. For this reason, the flower is a very attractive subject for evolutionary developmental (evo-devo) genetics studies. Research during the last two decades has provided compelling evidence that the origin and functional diversification of MIKC(c) MADS-box transcription factors has played a critical role during evolution of flowering plants. As master regulators of floral organ identity, MADS-box proteins are at the heart of the classic ABC model for floral development. Despite the enormous progress made in the field of floral development, there still remain aspects that are less well understood. Here we highlight some of the dark corners within our current knowledge on MADS-box genes and flower development, which would be worthwhile investigating in more detail in future research. These include the general question of to what extent MADS-box gene functions are conserved between species, the function of TM8-clade MADS-box genes which so far have remained uncharacterized, the divergence within the A-function, and post-transcriptional regulation of the ABC-genes.

An ATP-binding cassette transporter and two rRNA methyltransferases are involved in resistance to avilamycin in the producer organism Streptomyces viridochromogenes Tü57.

PubMed

Weitnauer, G; Gaisser, S; Trefzer, A; Stockert, S; Westrich, L; Quiros, L M; Mendez, C; Salas, J A; Bechthold, A

2001-03-01

Three different resistance factors from the avilamycin biosynthetic gene cluster of Streptomyces viridochromogenes Tü57, which confer avilamycin resistance when expressed in Streptomyces lividans TK66, were isolated. Analysis of the deduced amino acid sequences showed that AviABC1 is similar to a large family of ATP-binding transporter proteins and that AviABC2 resembles hydrophobic transmembrane proteins known to act jointly with the ATP-binding proteins. The deduced amino acid sequence of aviRb showed similarity to those of other rRNA methyltransferases, and AviRa did not resemble any protein in the databases. Independent expression in S. lividans TK66 of aviABC1 plus aviABC2, aviRa, or aviRb conferred different levels of resistance to avilamycin: 5, 10, or 250 microg/ml, respectively. When either aviRa plus aviRb or aviRa plus aviRb plus aviABC1 plus aviABC2 was coexpressed in S. lividans TK66, avilamycin resistance levels reached more than 250 microg/ml. Avilamycin A inhibited poly(U)-directed polyphenylalanine synthesis in an in vitro system using ribosomes of S. lividans TK66(pUWL201) (GWO), S. lividans TK66(pUWL201-Ra) (GWRa), or S. lividans TK66(pUWL201-Rb) (GWRb), whereas ribosomes of S. lividans TK66 containing pUWL201-Ra+Rb (GWRaRb) were highly resistant. aviRa and aviRb were expressed in Escherichia coli, and both enzymes were purified as fusion proteins to near homogeneity. Both enzymes showed rRNA methyltransferase activity using a mixture of 16S and 23S rRNAs from E. coli as the substrate. Coincubation experiments revealed that the enzymes methylate different positions of rRNA.

Manipulating the ABCs of self-assembly via low-χ block polymer design

PubMed Central

Chang, Alice B.; Lee, Byeongdu; Garland, Carol M.; Jones, Simon C.; Matsen, Mark W.

2017-01-01

Block polymer self-assembly typically translates molecular chain connectivity into mesoscale structure by exploiting incompatible blocks with large interaction parameters (χij). In this article, we demonstrate that the converse approach, encoding low-χ interactions in ABC bottlebrush triblock terpolymers (χAC ≲ 0), promotes organization into a unique mixed-domain lamellar morphology, which we designate LAMP. Transmission electron microscopy indicates that LAMP exhibits ACBC domain connectivity, in contrast to conventional three-domain lamellae (LAM3) with ABCB periods. Complementary small-angle X-ray scattering experiments reveal a strongly decreasing domain spacing with increasing total molar mass. Self-consistent field theory reinforces these observations and predicts that LAMP is thermodynamically stable below a critical χAC, above which LAM3 emerges. Both experiments and theory expose close analogies to ABA′ triblock copolymer phase behavior, collectively suggesting that low-χ interactions between chemically similar or distinct blocks intimately influence self-assembly. These conclusions provide fresh opportunities for block polymer design with potential consequences spanning all self-assembling soft materials. PMID:28588139

Terminal oxidase diversity and function in "Metallosphaera yellowstonensis": gene expression and protein modeling suggest mechanisms of Fe(II) oxidation in the sulfolobales.

PubMed

Kozubal, M A; Dlakic, M; Macur, R E; Inskeep, W P

2011-03-01

"Metallosphaera yellowstonensis" is a thermoacidophilic archaeon isolated from Yellowstone National Park that is capable of autotrophic growth using Fe(II), elemental S, or pyrite as electron donors. Analysis of the draft genome sequence from M. yellowstonensis strain MK1 revealed seven different copies of heme copper oxidases (subunit I) in a total of five different terminal oxidase complexes, including doxBCEF, foxABCDEFGHIJ, soxABC, and the soxM supercomplex, as well as a novel hypothetical two-protein doxB-like polyferredoxin complex. Other genes found in M. yellowstonensis with possible roles in S and or Fe cycling include a thiosulfate oxidase (tqoAB), a sulfite oxidase (som), a cbsA cytochrome b(558/566), several small blue copper proteins, and a novel gene sequence coding for a putative multicopper oxidase (Mco). Results from gene expression studies, including reverse transcriptase (RT) quantitative PCR (qPCR) of cultures grown autotrophically on either Fe(II), pyrite, or elemental S showed that the fox gene cluster and mco are highly expressed under conditions where Fe(II) is an electron donor. Metagenome sequence and gene expression studies of Fe-oxide mats confirmed the importance of fox genes (e.g., foxA and foxC) and mco under Fe(II)-oxidizing conditions. Protein modeling of FoxC suggests a novel lysine-lysine or lysine-arginine heme B binding domain, indicating that it is likely the cytochrome component of a heterodimer complex with foxG as a ferredoxin subunit. Analysis of mco shows that it encodes a novel multicopper blue protein with two plastocyanin type I copper domains that may play a role in the transfer of electrons within the Fox protein complex. An understanding of metabolic pathways involved in aerobic iron and sulfur oxidation in Sulfolobales has broad implications for understanding the evolution and niche diversification of these thermophiles as well as practical applications in fields such as bioleaching of trace metals from pyritic ores.

Genetic Separation of Listeria monocytogenes Causing Central Nervous System Infections in Animals

PubMed Central

Aguilar-Bultet, Lisandra; Nicholson, Pamela; Rychener, Lorenz; Dreyer, Margaux; Gözel, Bulent; Origgi, Francesco C.; Oevermann, Anna; Frey, Joachim; Falquet, Laurent

2018-01-01

Listeria monocytogenes is a foodborne pathogen that causes abortion, septicemia, gastroenteritis and central nervous system (CNS) infections in ruminants and humans. L. monocytogenes strains mainly belong to two distinct phylogenetic groups, named lineages I and II. In general, clinical cases in humans and animals, in particular CNS infections, are caused by lineage I strains, while most of the environmental and food strains belong to lineage II. Little is known about why lineage I is more virulent than lineage II, even though various molecular factors and mechanisms associated with pathogenesis are known. In this study, we have used a variety of whole genome sequence analyses and comparative genomic tools in order to find characteristics that distinguish lineage I from lineage II strains and CNS infection strains from non-CNS strains. We analyzed 225 strains and identified single nucleotide variants between lineages I and II, as well as differences in the gene content. Using a novel approach based on Reads Per Kilobase per Million Mapped (RPKM), we identified 167 genes predominantly absent in lineage II but present in lineage I. These genes are mostly encoding for membrane-associated proteins. Additionally, we found 77 genes that are largely absent in the non-CNS associated strains, while 39 genes are especially lacking in our defined “non-clinical” group. Based on the RPKM analysis and the metadata linked to the L. monocytogenes strains, we identified 6 genes potentially associated with CNS cases, which include a transcriptional regulator, an ABC transporter and a non-coding RNA. Although there is not a clear separation between pathogenic and non-pathogenic strains based on phylogenetic lineages, the presence of the genes identified in our study reveals potential pathogenesis traits in ruminant L. monocytogenes strains. Ultimately, the differences that we have found in our study will help steer future studies in understanding the virulence mechanisms of the most pathogenic L. monocytogenes strains. PMID:29459888

Transcript and proteomic analysis of developing white lupin (Lupinus albus L.) roots

PubMed Central

Tian, Li; Peel, Gregory J; Lei, Zhentian; Aziz, Naveed; Dai, Xinbin; He, Ji; Watson, Bonnie; Zhao, Patrick X; Sumner, Lloyd W; Dixon, Richard A

2009-01-01

Background White lupin (Lupinus albus L.) roots efficiently take up and accumulate (heavy) metals, adapt to phosphate deficiency by forming cluster roots, and secrete antimicrobial prenylated isoflavones during development. Genomic and proteomic approaches were applied to identify candidate genes and proteins involved in antimicrobial defense and (heavy) metal uptake and translocation. Results A cDNA library was constructed from roots of white lupin seedlings. Eight thousand clones were randomly sequenced and assembled into 2,455 unigenes, which were annotated based on homologous matches in the NCBInr protein database. A reference map of developing white lupin root proteins was established through 2-D gel electrophoresis and peptide mass fingerprinting. High quality peptide mass spectra were obtained for 170 proteins. Microsomal membrane proteins were separated by 1-D gel electrophoresis and identified by LC-MS/MS. A total of 74 proteins were putatively identified by the peptide mass fingerprinting and the LC-MS/MS methods. Genomic and proteomic analyses identified candidate genes and proteins encoding metal binding and/or transport proteins, transcription factors, ABC transporters and phenylpropanoid biosynthetic enzymes. Conclusion The combined EST and protein datasets will facilitate the understanding of white lupin's response to biotic and abiotic stresses and its utility for phytoremediation. The root ESTs provided 82 perfect simple sequence repeat (SSR) markers with potential utility in breeding white lupin for enhanced agronomic traits. PMID:19123941

Prochlorococcus Genetic Transformation and the Genomics of Nitrogen Metabolism

DTIC Science & Technology

2005-09-01

MIT9313 and MED4 have ABC-type urea transporters and urease genes. Prochlorococcus PCC 9511 urease activity is independent of the nitrogen source in the...medium (Palinska et al., 2000), suggesting that the urease genes lack genetic regulation. MIT9313 has genes for nitrite transport and utilization...cyanobacterium, synthesizes the smallest urease ." Microbiology 146 Pt 12: 3099-107. Palinska, K. A., W. Laloui, et al. (2002). "The signal transducer P-Il and

AAV vector-mediated secretion of chondroitinase provides a sensitive tracer for axonal arborisations.

PubMed

Alves, João Nuno; Muir, Elizabeth M; Andrews, Melissa R; Ward, Anneliese; Michelmore, Nicholas; Dasgupta, Debayan; Verhaagen, Joost; Moloney, Elizabeth B; Keynes, Roger J; Fawcett, James W; Rogers, John H

2014-04-30

As part of a project to express chondroitinase ABC (ChABC) in neurons of the central nervous system, we have inserted a modified ChABC gene into an adeno-associated viral (AAV) vector and injected it into the vibrissal motor cortex in adult rats to determine the extent and distribution of expression of the enzyme. A similar vector for expression of green fluorescent protein (GFP) was injected into the same location. For each vector, two versions with minor differences were used, giving similar results. After 4 weeks, the brains were stained to show GFP and products of chondroitinase digestion. Chondroitinase was widely expressed, and the AAV-ChABC and AAV-GFP vectors gave similar expression patterns in many respects, consistent with the known projections from the directly transduced neurons in vibrissal motor cortex and adjacent cingulate cortex. In addition, diffusion of vector to deeper neuronal populations led to labelling of remote projection fields which was much more extensive with AAV-ChABC than with AAV-GFP. The most notable of these populations are inferred to be neurons of cortical layer 6, projecting widely in the thalamus, and neurons of the anterior pole of the hippocampus, projecting through most of the hippocampus. We conclude that, whereas GFP does not label the thinnest axonal branches of some neuronal types, chondroitinase is efficiently secreted from these arborisations and enables their extent to be sensitively visualised. After 12 weeks, chondroitinase expression was undiminished. Copyright © 2014 Elsevier B.V. All rights reserved.

Defining the ABC of gene essentiality in streptococci.

PubMed

Charbonneau, Amelia R L; Forman, Oliver P; Cain, Amy K; Newland, Graham; Robinson, Carl; Boursnell, Mike; Parkhill, Julian; Leigh, James A; Maskell, Duncan J; Waller, Andrew S

2017-05-31

Utilising next generation sequencing to interrogate saturated bacterial mutant libraries provides unprecedented information for the assignment of genome-wide gene essentiality. Exposure of saturated mutant libraries to specific conditions and subsequent sequencing can be exploited to uncover gene essentiality relevant to the condition. Here we present a barcoded transposon directed insertion-site sequencing (TraDIS) system to define an essential gene list for Streptococcus equi subsp. equi, the causative agent of strangles in horses, for the first time. The gene essentiality data for this group C Streptococcus was compared to that of group A and B streptococci. Six barcoded variants of pGh9:ISS1 were designed and used to generate mutant libraries containing between 33,000-66,000 unique mutants. TraDIS was performed on DNA extracted from each library and data were analysed separately and as a combined master pool. Gene essentiality determined that 19.5% of the S. equi genome was essential. Gene essentialities were compared to those of group A and group B streptococci, identifying concordances of 90.2% and 89.4%, respectively and an overall concordance of 83.7% between the three species. The use of barcoded pGh9:ISS1 to generate mutant libraries provides a highly useful tool for the assignment of gene function in S. equi and other streptococci. The shared essential gene set of group A, B and C streptococci provides further evidence of the close genetic relationships between these important pathogenic bacteria. Therefore, the ABC of gene essentiality reported here provides a solid foundation towards reporting the functional genome of streptococci.

Another Brick in the Wall: a Rhamnan Polysaccharide Trapped inside Peptidoglycan of Lactococcus lactis.

PubMed

Sadovskaya, Irina; Vinogradov, Evgeny; Courtin, Pascal; Armalyte, Julija; Meyrand, Mickael; Giaouris, Efstathios; Palussière, Simon; Furlan, Sylviane; Péchoux, Christine; Ainsworth, Stuart; Mahony, Jennifer; van Sinderen, Douwe; Kulakauskas, Saulius; Guérardel, Yann; Chapot-Chartier, Marie-Pierre

2017-09-12

Polysaccharides are ubiquitous components of the Gram-positive bacterial cell wall. In Lactococcus lactis , a polysaccharide pellicle (PSP) forms a layer at the cell surface. The PSP structure varies among lactococcal strains; in L. lactis MG1363, the PSP is composed of repeating hexasaccharide phosphate units. Here, we report the presence of an additional neutral polysaccharide in L. lactis MG1363 that is a rhamnan composed of α-l-Rha trisaccharide repeating units. This rhamnan is still present in mutants devoid of the PSP, indicating that its synthesis can occur independently of PSP synthesis. High-resolution magic-angle spinning nuclear magnetic resonance (HR-MAS NMR) analysis of whole bacterial cells identified a PSP at the surface of wild-type cells. In contrast, rhamnan was detected only at the surface of PSP-negative mutant cells, indicating that rhamnan is located underneath the surface-exposed PSP and is trapped inside peptidoglycan. The genetic determinants of rhamnan biosynthesis appear to be within the same genetic locus that encodes the PSP biosynthetic machinery, except the gene tagO encoding the initiating glycosyltransferase. We present a model of rhamnan biosynthesis based on an ABC transporter-dependent pathway. Conditional mutants producing reduced amounts of rhamnan exhibit strong morphological defects and impaired division, indicating that rhamnan is essential for normal growth and division. Finally, a mutation leading to reduced expression of lcpA , encoding a protein of the LytR-CpsA-Psr (LCP) family, was shown to severely affect cell wall structure. In lcpA mutant cells, in contrast to wild-type cells, rhamnan was detected by HR-MAS NMR, suggesting that LcpA participates in the attachment of rhamnan to peptidoglycan. IMPORTANCE In the cell wall of Gram-positive bacteria, the peptidoglycan sacculus is considered the major structural component, maintaining cell shape and integrity. It is decorated with other glycopolymers, including polysaccharides, the roles of which are not fully elucidated. In the ovococcus Lactococcus lactis , a polysaccharide with a different structure between strains forms a layer at the bacterial surface and acts as the receptor for various bacteriophages that typically exhibit a narrow host range. The present report describes the identification of a novel polysaccharide in the L. lactis cell wall, a rhamnan that is trapped inside the peptidoglycan and covalently bound to it. We propose a model of rhamnan synthesis based on an ABC transporter-dependent pathway. Rhamnan appears as a conserved component of the lactococcal cell wall playing an essential role in growth and division, thus highlighting the importance of polysaccharides in the cell wall integrity of Gram-positive ovococci. Copyright © 2017 Sadovskaya et al.

Detoxification and stress response genes expressed in a western North American bumble bee, Bombus huntii (Hymenoptera: Apidae).

PubMed

Xu, Junhuan; Strange, James P; Welker, Dennis L; James, Rosalind R

2013-12-12

The Hunt bumble bee (Bombus huntii Greene, Hymenoptera: Apidae) is a holometabolous, social insect important as a pollinator in natural and agricultural ecosystems in western North America. Bumble bees spend a significant amount of time foraging on a wide variety of flowering plants, and this activity exposes them to both plant toxins and pesticides, posing a threat to individual and colony survival. Little is known about what detoxification pathways are active in bumble bees, how the expression of detoxification genes changes across life stages, or how the number of detoxification genes expressed in B. huntii compares to other insects. We found B. huntii expressed at least 584 genes associated with detoxification and stress responses. The expression levels of some of these genes, such as those encoding the cytochrome P450s, glutathione S-transferases (GSTs) and glycosidases, vary among different life stages to a greater extent than do other genes. We also found that the number of P450s, GSTs and esterase genes expressed by B. huntii is similar to the number of these genes found in the genomes of other bees, namely Bombus terrestris, Bombus impatiens, Apis mellifera and Megachile rotundata, but many fewer than are found in the fly Drosophila melanogaster. Bombus huntii has transcripts for a large number of detoxification and stress related proteins, including oxidation and reduction enzymes, conjugation enzymes, hydrolytic enzymes, ABC transporters, cadherins, and heat shock proteins. The diversity of genes expressed within some detoxification pathways varies among the life stages and castes, and we typically identified more genes in the adult females than in larvae, pupae, or adult males, for most pathways. Meanwhile, we found the numbers of detoxification and stress genes expressed by B. huntii to be more similar to other bees than to the fruit fly. The low number of detoxification genes, first noted in the honey bee, appears to be a common phenomenon among bees, and perhaps results from their symbiotic relationship with plants. Many flowering plants benefit from pollinators, and thus offer these insects rewards (such as nectar) rather than defensive plant toxins.

Gene discovery for enzymes involved in limonene modification or utilization by the mountain pine beetle-associated pathogen Grosmannia clavigera.

PubMed

Wang, Ye; Lim, Lynette; Madilao, Lina; Lah, Ljerka; Bohlmann, Joerg; Breuil, Colette

2014-08-01

To successfully colonize and eventually kill pine trees, Grosmannia clavigera (Gs cryptic species), the main fungal pathogen associated with the mountain pine beetle (Dendroctonus ponderosae), has developed multiple mechanisms to overcome host tree chemical defenses, of which terpenoids are a major component. In addition to a monoterpene efflux system mediated by a recently discovered ABC transporter, Gs has genes that are highly induced by monoterpenes and that encode enzymes that modify or utilize monoterpenes [especially (+)-limonene]. We showed that pine-inhabiting Ophiostomale fungi are tolerant to monoterpenes, but only a few, including Gs, are known to utilize monoterpenes as a carbon source. Gas chromatography-mass spectrometry (GC-MS) revealed that Gs can modify (+)-limonene through various oxygenation pathways, producing carvone, p-mentha-2,8-dienol, perillyl alcohol, and isopiperitenol. It can also degrade (+)-limonene through the C-1-oxygenated pathway, producing limonene-1,2-diol as the most abundant intermediate. Transcriptome sequencing (RNA-seq) data indicated that Gs may utilize limonene 1,2-diol through beta-oxidation and then valine and tricarboxylic acid (TCA) metabolic pathways. The data also suggested that at least two gene clusters, located in genome contigs 108 and 161, were highly induced by monoterpenes and may be involved in monoterpene degradation processes. Further, gene knockouts indicated that limonene degradation required two distinct Baeyer-Villiger monooxygenases (BVMOs), an epoxide hydrolase and an enoyl coenzyme A (enoyl-CoA) hydratase. Our work provides information on enzyme-mediated limonene utilization or modification and a more comprehensive understanding of the interaction between an economically important fungal pathogen and its host's defense chemicals.

Gene Discovery for Enzymes Involved in Limonene Modification or Utilization by the Mountain Pine Beetle-Associated Pathogen Grosmannia clavigera

PubMed Central

Wang, Ye; Lim, Lynette; Madilao, Lina; Lah, Ljerka; Bohlmann, Joerg

2014-01-01

To successfully colonize and eventually kill pine trees, Grosmannia clavigera (Gs cryptic species), the main fungal pathogen associated with the mountain pine beetle (Dendroctonus ponderosae), has developed multiple mechanisms to overcome host tree chemical defenses, of which terpenoids are a major component. In addition to a monoterpene efflux system mediated by a recently discovered ABC transporter, Gs has genes that are highly induced by monoterpenes and that encode enzymes that modify or utilize monoterpenes [especially (+)-limonene]. We showed that pine-inhabiting Ophiostomale fungi are tolerant to monoterpenes, but only a few, including Gs, are known to utilize monoterpenes as a carbon source. Gas chromatography-mass spectrometry (GC-MS) revealed that Gs can modify (+)-limonene through various oxygenation pathways, producing carvone, p-mentha-2,8-dienol, perillyl alcohol, and isopiperitenol. It can also degrade (+)-limonene through the C-1-oxygenated pathway, producing limonene-1,2-diol as the most abundant intermediate. Transcriptome sequencing (RNA-seq) data indicated that Gs may utilize limonene 1,2-diol through beta-oxidation and then valine and tricarboxylic acid (TCA) metabolic pathways. The data also suggested that at least two gene clusters, located in genome contigs 108 and 161, were highly induced by monoterpenes and may be involved in monoterpene degradation processes. Further, gene knockouts indicated that limonene degradation required two distinct Baeyer-Villiger monooxygenases (BVMOs), an epoxide hydrolase and an enoyl coenzyme A (enoyl-CoA) hydratase. Our work provides information on enzyme-mediated limonene utilization or modification and a more comprehensive understanding of the interaction between an economically important fungal pathogen and its host's defense chemicals. PMID:24837377

Genomic Characterization of Haemophilus parasuis SH0165, a Highly Virulent Strain of Serovar 5 Prevalent in China

PubMed Central

Zhou, Rui; Jin, Qi; Fan, Yang; Bei, Weicheng; Chen, Huanchun

2011-01-01

Haemophilus parasuis can be either a commensal bacterium of the porcine respiratory tract or an opportunistic pathogen causing Glässer's disease, a severe systemic disease that has led to significant economical losses in the pig industry worldwide. We determined the complete genomic sequence of H. parasuis SH0165, a highly virulent strain of serovar 5, which was isolated from a hog pen in North China. The single circular chromosome was 2,269,156 base pairs in length and contained 2,031 protein-coding genes. Together with the full spectrum of genes detected by the analysis of metabolic pathways, we confirmed that H. parasuis generates ATP via both fermentation and respiration, and possesses an intact TCA cycle for anabolism. In addition to possessing the complete pathway essential for the biosynthesis of heme, this pathogen was also found to be well-equipped with different iron acquisition systems, such as the TonB system and ABC-type transport complexes, to overcome iron limitation during infection and persistence. We identified a number of genes encoding potential virulence factors, such as type IV fimbriae and surface polysaccharides. Analysis of the genome confirmed that H. parasuis is naturally competent, as genes related to DNA uptake are present. A nine-mer DNA uptake signal sequence (ACAAGCGGT), identical to that found in Actinobacillus pleuropneumoniae and Mannheimia haemolytica, followed by similar downstream motifs, was identified in the SH0165 genome. Genomic and phylogenetic comparisons with other Pasteurellaceae species further indicated that H. parasuis was closely related to another swine pathogenic bacteria A. pleuropneumoniae. The comprehensive genetic analysis presented here provides a foundation for future research on the metabolism, natural competence and virulence of H. parasuis. PMID:21611187

Discovery of a widespread prokaryotic 5-oxoprolinase that was hiding in plain sight.

PubMed

Niehaus, Thomas D; Elbadawi-Sidhu, Mona; de Crécy-Lagard, Valérie; Fiehn, Oliver; Hanson, Andrew D

2017-09-29

5-Oxoproline (OP) is well-known as an enzymatic intermediate in the eukaryotic γ-glutamyl cycle, but it is also an unavoidable damage product formed spontaneously from glutamine and other sources. Eukaryotes metabolize OP via an ATP-dependent 5-oxoprolinase; most prokaryotes lack homologs of this enzyme (and the γ-glutamyl cycle) but are predicted to have some way to dispose of OP if its spontaneous formation in vivo is significant. Comparative analysis of prokaryotic genomes showed that the gene encoding pyroglutamyl peptidase, which removes N-terminal OP residues, clusters in diverse genomes with genes specifying homologs of a fungal lactamase (renamed p rokaryotic 5-o x oprolinase A , pxpA ) and homologs of allophanate hydrolase subunits (renamed pxpB and pxpC ). Inactivation of Bacillus subtilis pxpA , pxpB , or pxpC genes slowed growth, caused OP accumulation in cells and medium, and prevented use of OP as a nitrogen source. Assays of cell lysates showed that ATP-dependent 5-oxoprolinase activity disappeared when pxpA , pxpB , or pxpC was inactivated. 5-Oxoprolinase activity could be reconstituted in vitro by mixing recombinant B. subtilis PxpA, PxpB, and PxpC proteins. In addition, overexpressing Escherichia coli pxpABC genes in E. coli increased 5-oxoprolinase activity in lysates ≥1700-fold. This work shows that OP is a major universal metabolite damage product and that OP disposal systems are common in all domains of life. Furthermore, it illustrates how easily metabolite damage and damage-control systems can be overlooked, even for central metabolites in model organisms. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

ABCA transporter gene expression and poor outcome in epithelial ovarian cancer.

PubMed

Hedditch, Ellen L; Gao, Bo; Russell, Amanda J; Lu, Yi; Emmanuel, Catherine; Beesley, Jonathan; Johnatty, Sharon E; Chen, Xiaoqing; Harnett, Paul; George, Joshy; Williams, Rebekka T; Flemming, Claudia; Lambrechts, Diether; Despierre, Evelyn; Lambrechts, Sandrina; Vergote, Ignace; Karlan, Beth; Lester, Jenny; Orsulic, Sandra; Walsh, Christine; Fasching, Peter; Beckmann, Matthias W; Ekici, Arif B; Hein, Alexander; Matsuo, Keitaro; Hosono, Satoyo; Nakanishi, Toru; Yatabe, Yasushi; Pejovic, Tanja; Bean, Yukie; Heitz, Florian; Harter, Philipp; du Bois, Andreas; Schwaab, Ira; Hogdall, Estrid; Kjaer, Susan K; Jensen, Allan; Hogdall, Claus; Lundvall, Lene; Engelholm, Svend Aage; Brown, Bob; Flanagan, James; Metcalf, Michelle D; Siddiqui, Nadeem; Sellers, Thomas; Fridley, Brooke; Cunningham, Julie; Schildkraut, Joellen; Iversen, Ed; Weber, Rachel P; Berchuck, Andrew; Goode, Ellen; Bowtell, David D; Chenevix-Trench, Georgia; deFazio, Anna; Norris, Murray D; MacGregor, Stuart; Haber, Michelle; Henderson, Michelle J

2014-07-01

ATP-binding cassette (ABC) transporters play various roles in cancer biology and drug resistance, but their association with outcomes in serous epithelial ovarian cancer (EOC) is unknown. The relationship between clinical outcomes and ABC transporter gene expression in two independent cohorts of high-grade serous EOC tumors was assessed with real-time quantitative polymerase chain reaction, analysis of expression microarray data, and immunohistochemistry. Associations between clinical outcomes and ABCA transporter gene single nucleotide polymorphisms were tested in a genome-wide association study. Impact of short interfering RNA-mediated gene suppression was determined by colony forming and migration assays. Association with survival was assessed with Kaplan-Meier analysis and log-rank tests. All statistical tests were two-sided. Associations with outcome were observed with ABC transporters of the "A" subfamily, but not with multidrug transporters. High-level expression of ABCA1, ABCA6, ABCA8, and ABCA9 in primary tumors was statistically significantly associated with reduced survival in serous ovarian cancer patients. Low levels of ABCA5 and the C-allele of rs536009 were associated with shorter overall survival (hazard ratio for death = 1.50; 95% confidence interval [CI] =1.26 to 1.79; P = 6.5e-6). The combined expression pattern of ABCA1, ABCA5, and either ABCA8 or ABCA9 was associated with particularly poor outcome (mean overall survival in group with adverse ABCA1, ABCA5 and ABCA9 gene expression = 33.2 months, 95% CI = 26.4 to 40.1; vs 55.3 months in the group with favorable ABCA gene expression, 95% CI = 49.8 to 60.8; P = .001), independently of tumor stage or surgical debulking status. Suppression of cholesterol transporter ABCA1 inhibited ovarian cancer cell growth and migration in vitro, and statin treatment reduced ovarian cancer cell migration. Expression of ABCA transporters was associated with poor outcome in serous ovarian cancer, implicating lipid trafficking as a potentially important process in EOC. © The Author 2014. Published by Oxford University Press. All rights reserved.

Mollusk genes encoding lysine tRNA (UUU) contain introns.

PubMed

Matsuo, M; Abe, Y; Saruta, Y; Okada, N

1995-11-20

New intron-containing genes encoding tRNAs were discovered when genomic DNA isolated from various animal species was amplified by the polymerase chain reaction (PCR) with primers based on sequences of rabbit tRNA(Lys). From sequencing analysis of the products of PCR, we found that introns are present in several genes encoding tRNA(Lys) in mollusks, such as Loligo bleekeri (squid) and Octopus vulgaris (octopus). These introns were specific to genes encoding tRNA(Lys)(CUU) and were not present in genes encoding tRNA(Lys)(CUU). In addition, the sequences of the introns were different from one another. To confirm the results of our initial experiments, we isolated and sequenced genes encoding tRNA(Lys)(CUU) and tRNA(Lys)(UUU). The gene for tRNA(Lys)(UUU) from squid contained an intron, whose sequence was the same as that identified by PCR, and the gene formed a cluster with a corresponding pseudogene. Several DNA regions of 2.1 kb containing this cluster appeared to be tandemly arrayed in the squid genome. By contrast, the gene encoding tRNA(Lys)(CUU) did not contain an intron, as shown also by PCR. The tRNA(Lys)(UUU) that corresponded to the analyzed gene was isolated and characterized. The present study provides the first example of an intron-containing gene encoding a tRNA in mollusks and suggests the universality of introns in such genes in higher eukaryotes.

Human AZU-1 gene, variants thereof and expressed gene products

DOEpatents

Chen, Huei-Mei; Bissell, Mina

2004-06-22

A human AZU-1 gene, mutants, variants and fragments thereof. Protein products encoded by the AZU-1 gene and homologs encoded by the variants of AZU-1 gene acting as tumor suppressors or markers of malignancy progression and tumorigenicity reversion. Identification, isolation and characterization of AZU-1 and AZU-2 genes localized to a tumor suppressive locus at chromosome 10q26, highly expressed in nonmalignant and premalignant cells derived from a human breast tumor progression model. A recombinant full length protein sequences encoded by the AZU-1 gene and nucleotide sequences of AZU-1 and AZU-2 genes and variant and fragments thereof. Monoclonal or polyclonal antibodies specific to AZU-1, AZU-2 encoded protein and to AZU-1, or AZU-2 encoded protein homologs.

Comparative symbiotic plasmid analysis indicates that symbiosis gene ancestor type affects plasmid genetic evolution.

PubMed

Wang, X; Zhao, L; Zhang, L; Wu, Y; Chou, M; Wei, G

2018-07-01

Rhizobial symbiotic plasmids play vital roles in mutualistic symbiosis with legume plants by executing the functions of nodulation and nitrogen fixation. To explore the gene composition and genetic constitution of rhizobial symbiotic plasmids, comparison analyses of 24 rhizobial symbiotic plasmids derived from four rhizobial genera was carried out. Results illustrated that rhizobial symbiotic plasmids had higher proportion of functional genes participating in amino acid transport and metabolism, replication; recombination and repair; carbohydrate transport and metabolism; energy production and conversion and transcription. Mesorhizobium amorphae CCNWGS0123 symbiotic plasmid - pM0123d had similar gene composition with pR899b and pSNGR234a. All symbiotic plasmids shared 13 orthologous genes, including five nod and eight nif/fix genes which participate in the rhizobia-legume symbiosis process. These plasmids contained nod genes from four ancestors and fix genes from six ancestors. The ancestral type of pM0123d nod genes was similar with that of Rhizobium etli plasmids, while the ancestral type of pM0123d fix genes was same as that of pM7653Rb. The phylogenetic trees constructed based on nodCIJ and fixABC displayed different topological structures mainly due to nodCIJ and fixABC ancestral type discordance. The study presents valuable insights into mosaic structures and the evolution of rhizobial symbiotic plasmids. This study compared 24 rhizobial symbiotic plasmids that included four genera and 11 species, illuminating the functional gene composition and symbiosis gene ancestor types of symbiotic plasmids from higher taxonomy. It provides valuable insights into mosaic structures and the evolution of symbiotic plasmids. © 2018 The Society for Applied Microbiology.

The mitochondrial gene encoding ribosomal protein S12 has been translocated to the nuclear genome in Oenothera.

PubMed Central

Grohmann, L; Brennicke, A; Schuster, W

1992-01-01

The Oenothera mitochondrial genome contains only a gene fragment for ribosomal protein S12 (rps12), while other plants encode a functional gene in the mitochondrion. The complete Oenothera rps12 gene is located in the nucleus. The transit sequence necessary to target this protein to the mitochondrion is encoded by a 5'-extension of the open reading frame. Comparison of the amino acid sequence encoded by the nuclear gene with the polypeptides encoded by edited mitochondrial cDNA and genomic sequences of other plants suggests that gene transfer between mitochondrion and nucleus started from edited mitochondrial RNA molecules. Mechanisms and requirements of gene transfer and activation are discussed. Images PMID:1454526

The multidrug ABC transporter BmrC/BmrD of Bacillus subtilis is regulated via a ribosome-mediated transcriptional attenuation mechanism

PubMed Central

Reilman, Ewoud; Mars, Ruben A. T.; van Dijl, Jan Maarten; Denham, Emma L.

2014-01-01

Expression of particular drug transporters in response to antibiotic pressure is a critical element in the development of bacterial multidrug resistance, and represents a serious concern for human health. To obtain a better understanding of underlying regulatory mechanisms, we have dissected the transcriptional activation of the ATP-binding cassette (ABC) transporter BmrC/BmrD of the Gram-positive model bacterium Bacillus subtilis. By using promoter-GFP fusions and live cell array technology, we demonstrate a temporally controlled transcriptional activation of the bmrCD genes in response to antibiotics that target protein synthesis. Intriguingly, bmrCD expression only occurs during the late-exponential and stationary growth stages, irrespective of the timing of the antibiotic challenge. We show that this is due to tight transcriptional control by the transition state regulator AbrB. Moreover, our results show that the bmrCD genes are co-transcribed with bmrB (yheJ), a small open reading frame immediately upstream of bmrC that harbors three alternative stem-loop structures. These stem-loops are apparently crucial for antibiotic-induced bmrCD transcription. Importantly, the antibiotic-induced bmrCD expression requires translation of bmrB, which implies that BmrB serves as a regulatory leader peptide. Altogether, we demonstrate for the first time that a ribosome-mediated transcriptional attenuation mechanism can control the expression of a multidrug ABC transporter. PMID:25217586

Hfq Influences Multiple Transport Systems and Virulence in the Plant Pathogen Agrobacterium tumefaciens

PubMed Central

Wilms, Ina; Möller, Philip; Stock, Anna-Maria; Gurski, Rosemarie; Lai, Erh-Min

2012-01-01

The Hfq protein mediates gene regulation by small RNAs (sRNAs) in about 50% of all bacteria. Depending on the species, phenotypic defects of an hfq mutant range from mild to severe. Here, we document that the purified Hfq protein of the plant pathogen and natural genetic engineer Agrobacterium tumefaciens binds to the previously described sRNA AbcR1 and its target mRNA atu2422, which codes for the substrate binding protein of an ABC transporter taking up proline and γ-aminobutyric acid (GABA). Several other ABC transporter components were overproduced in an hfq mutant compared to their levels in the parental strain, suggesting that Hfq plays a major role in controlling the uptake systems and metabolic versatility of A. tumefaciens. The hfq mutant showed delayed growth, altered cell morphology, and reduced motility. Although the DNA-transferring type IV secretion system was produced, tumor formation by the mutant strain was attenuated, demonstrating an important contribution of Hfq to plant transformation by A. tumefaciens. PMID:22821981

Novel, non-symbiotic isolates of Neorhizobium from a dryland agricultural soil.

PubMed

Soenens, Amalia; Imperial, Juan

2018-01-01

Semi-selective enrichment, followed by PCR screening, resulted in the successful direct isolation of fast-growing Rhizobia from a dryland agricultural soil. Over 50% of these isolates belong to the genus Neorhizobium , as concluded from partial rpoB and near-complete 16S rDNA sequence analysis. Further genotypic and genomic analysis of five representative isolates confirmed that they form a coherent group within Neorhizobium , closer to N. galegae than to the remaining Neorhizobium species, but clearly differentiated from the former, and constituting at least one new genomospecies within Neorhizobium. All the isolates lacked nod and nif symbiotic genes but contained a repABC replication/maintenance region, characteristic of rhizobial plasmids, within large contigs from their draft genome sequences. These repABC sequences were related, but not identical, to repABC sequences found in symbiotic plasmids from N. galegae , suggesting that the non-symbiotic isolates have the potential to harbor symbiotic plasmids. This is the first report of non-symbiotic members of Neorhizobium from soil.

Involvement of LeMRP, an ATP-binding cassette transporter, in shikonin transport and biosynthesis in Lithospermum erythrorhizon.

PubMed

Zhu, Y; Chu, S-J; Luo, Y-L; Fu, J-Y; Tang, C-Y; Lu, G-H; Pang, Y-J; Wang, X-M; Yang, R-W; Qi, J-L; Yang, Y-H

2018-03-01

Shikonin and its derivatives are important medicinal secondary metabolites accumulating in roots of Lithospermum erythrorhizon. Although some membrane proteins have been identified as transporters of secondary metabolites, the mechanisms underlying shikonin transport and accumulation in L. erythrorhizon cells still remain largely unknown. In this study, we isolated a cDNA encoding LeMRP, an ATP-binding cassette transporter from L. erythrorhizon, and further investigated its functions in the transport and biosynthesis of shikonin using the yeast transformation and transgenic hairy root methods, respectively. Real-time PCR was applied for expression analyses of LeMRP and shikonin biosynthetic enzyme genes. Functional analysis of LeMRP using the heterologous yeast cell expression system showed that LeMRP could be involved in shikonin transport. Transgenic hairy roots of L. erythrorhizon demonstrated that LeMRP overexpressing hairy roots produced more shikonin than the empty vector (EV) control. Real-time PCR results revealed that the enhanced shikonin biosynthesis in the overexpression lines was mainly caused by highly up-regulated expression of genes coding key enzymes (LePAL, HMGR, Le4CL and LePGT) involved in shikonin biosynthesis. Conversely, LeMRP RNAi decreased the accumulation of shikonin and effectively down-regulated expression level of the above genes. Typical inhibitors of ABC proteins, such as azide and buthionine sulphoximine, dramatically inhibited accumulation of shikonin in hairy roots. Our findings provide evidence for the important direct or indirect role of LeMRP in transmembrane transport and biosynthesis of shikonin. © 2017 German Society for Plant Sciences and The Royal Botanical Society of the Netherlands.

Determining Roles of Accessory Genes in Denitrification by Mutant Fitness Analyses

DOE PAGES

Vaccaro, Brian J.; Thorgersen, Michael P.; Lancaster, W. Andrew; ...

2015-10-09

Enzymes of the denitrification pathway play an important role in the global nitrogen cycle, including release of nitrous oxide, an ozone-depleting greenhouse gas. In addition, nitric oxide reductase, maturation factors, and proteins associated with nitric oxide detoxification are used by pathogens to combat nitric oxide release by host immune systems. While the core reductases that catalyze the conversion of nitrate to dinitrogen are well understood at a mechanistic level, there are many peripheral proteins required for denitrification whose basic function is unclear. A bar-coded transposon DNA library fromPseudomonas stutzeristrain RCH2 was grown under denitrifying conditions, using nitrate or nitrite asmore » an electron acceptor, and also under molybdenum limitation conditions, with nitrate as the electron acceptor. Analysis of sequencing results from these growths yielded gene fitness data for 3,307 of the 4,265 protein-encoding genes present in strain RCH2. The insights presented here contribute to our understanding of how peripheral proteins contribute to a fully functioning denitrification pathway. We propose a new low-affinity molybdate transporter, OatABC, and show that differential regulation is observed for two MoaA homologs involved in molybdenum cofactor biosynthesis. We also propose that NnrS may function as a membrane-bound NO sensor. Finally, the dominant HemN paralog involved in heme biosynthesis is identified, and a CheR homolog is proposed to function in nitrate chemotaxis. In addition, new insights are provided into nitrite reductase redundancy, nitric oxide reductase maturation, nitrous oxide reductase maturation, and regulation.« less

Expression of the CDR1 efflux pump in clinical Candida albicans isolates is controlled by a negative regulatory element

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gaur, Naseem Akhtar; Manoharlal, Raman; Saini, Preeti

2005-06-24

Resistance to azole antifungal drugs in clinical isolates of the human fungal pathogen Candida albicans is often caused by constitutive overexpression of the CDR1 gene, which encodes a multidrug efflux pump of the ABC transporter superfamily. To understand the relevance of a recently identified negative regulatory element (NRE) in the CDR1 promoter for the control of CDR1 expression in the clinical scenario, we investigated the effect of mutation or deletion of the NRE on CDR1 expression in two matched pairs of azole-sensitive and resistant clinical isolates of C. albicans. Expression of GFP or lacZ reporter genes from the wild typemore » CDR1 promoter was much higher in the azole-resistant C. albicans isolates than in the azole-susceptible isolates, reflecting the known differences in CDR1 expression in these strains. Deletion or mutation of the NRE resulted in enhanced reporter gene expression in azole-sensitive strains, but did not further increase the already high CDR1 promoter activity in the azole-resistant strains. In agreement with these findings, electrophoretic mobility shift assays showed a reduced binding to the NRE of nuclear extracts from the resistant C. albicans isolates as compared with extracts from the sensitive isolates. These results demonstrate that the NRE is involved in maintaining CDR1 expression at basal levels and that this repression is overcome in azole-resistant clinical C. albicans isolates, resulting in constitutive CDR1 overexpression and concomitant drug resistance.« less

A Comprehensive Survey of Sequence Variation in the ABCA4 (ABCR) Gene in Stargardt Disease and Age-Related Macular Degeneration

PubMed Central

Rivera, Andrea; White, Karen; Stöhr, Heidi; Steiner, Klaus; Hemmrich, Nadine; Grimm, Timo; Jurklies, Bernhard; Lorenz, Birgit; Scholl, Hendrik P. N.; Apfelstedt-Sylla, Eckhart; Weber, Bernhard H. F.

2000-01-01

Stargardt disease (STGD) is a common autosomal recessive maculopathy of early and young-adult onset and is caused by alterations in the gene encoding the photoreceptor-specific ATP-binding cassette (ABC) transporter (ABCA4). We have studied 144 patients with STGD and 220 unaffected individuals ascertained from the German population, to complete a comprehensive, population-specific survey of the sequence variation in the ABCA4 gene. In addition, we have assessed the proposed role for ABCA4 in age-related macular degeneration (AMD), a common cause of late-onset blindness, by studying 200 affected individuals with late-stage disease. Using a screening strategy based primarily on denaturing gradient gel electrophoresis, we have identified in the three study groups a total of 127 unique alterations, of which 90 have not been previously reported, and have classified 72 as probable pathogenic mutations. Of the 288 STGD chromosomes studied, mutations were identified in 166, resulting in a detection rate of ∼58%. Eight different alleles account for 61% of the identified disease alleles, and at least one of these, the L541P-A1038V complex allele, appears to be a founder mutation in the German population. When the group with AMD and the control group were analyzed with the same methodology, 18 patients with AMD and 12 controls were found to harbor possible disease-associated alterations. This represents no significant difference between the two groups; however, for detection of modest effects of rare alleles in complex diseases, the analysis of larger cohorts of patients may be required. PMID:10958763

A comprehensive survey of sequence variation in the ABCA4 (ABCR) gene in Stargardt disease and age-related macular degeneration.

PubMed

Rivera, A; White, K; Stöhr, H; Steiner, K; Hemmrich, N; Grimm, T; Jurklies, B; Lorenz, B; Scholl, H P; Apfelstedt-Sylla, E; Weber, B H

2000-10-01

Stargardt disease (STGD) is a common autosomal recessive maculopathy of early and young-adult onset and is caused by alterations in the gene encoding the photoreceptor-specific ATP-binding cassette (ABC) transporter (ABCA4). We have studied 144 patients with STGD and 220 unaffected individuals ascertained from the German population, to complete a comprehensive, population-specific survey of the sequence variation in the ABCA4 gene. In addition, we have assessed the proposed role for ABCA4 in age-related macular degeneration (AMD), a common cause of late-onset blindness, by studying 200 affected individuals with late-stage disease. Using a screening strategy based primarily on denaturing gradient gel electrophoresis, we have identified in the three study groups a total of 127 unique alterations, of which 90 have not been previously reported, and have classified 72 as probable pathogenic mutations. Of the 288 STGD chromosomes studied, mutations were identified in 166, resulting in a detection rate of approximately 58%. Eight different alleles account for 61% of the identified disease alleles, and at least one of these, the L541P-A1038V complex allele, appears to be a founder mutation in the German population. When the group with AMD and the control group were analyzed with the same methodology, 18 patients with AMD and 12 controls were found to harbor possible disease-associated alterations. This represents no significant difference between the two groups; however, for detection of modest effects of rare alleles in complex diseases, the analysis of larger cohorts of patients may be required.

Molecular characterization of the pSinB plasmid of the arsenite oxidizing, metallotolerant Sinorhizobium sp. M14 - insight into the heavy metal resistome of sinorhizobial extrachromosomal replicons.

PubMed

Romaniuk, Krzysztof; Dziewit, Lukasz; Decewicz, Przemyslaw; Mielnicki, Sebastian; Radlinska, Monika; Drewniak, Lukasz

2017-01-01

Sinorhizobium sp. M14 is an As(III)-oxidizing, psychrotolerant strain, capable of growth in the presence of extremely high concentrations of arsenic and many other heavy metals. Metallotolerant abilities of the M14 strain depend upon the presence of two extrachromosomal replicons: pSinA (∼ 109 kb) and pSinB (∼ 300 kb). The latter was subjected to complex analysis. The performed analysis demonstrated that the plasmid pSinB is a narrow-host-range repABC-type replicon, which is fully stabilized by the phd-vapC-like toxin-antitoxin stabilizing system. In silico analysis showed that among the phenotypic gene clusters of the plasmid pSinB, eight modules are potentially involved in heavy metals resistance (HMR). These modules carry genes encoding efflux pumps, permeases, transporters and copper oxidases, which provide resistance to arsenic, cadmium, cobalt, copper, iron, mercury, nickel, silver and zinc. The functional analysis revealed that the HMR modules are active and have an effect on the minimal inhibitory concentration (MIC) values observed for the heterological host cells. The phenotype was manifested by an increase or decrease of the MICs of heavy metals and it was strain specific. The analysis of distribution of the heavy metal resistance genes, i.e. resistome, in Sinorhizobium spp. plasmids, revealed that the HMR modules are common in these replicons. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Gene Flow Across Genus Barriers – Conjugation of Dinoroseobacter shibae’s 191-kb Killer Plasmid into Phaeobacter inhibens and AHL-mediated Expression of Type IV Secretion Systems

PubMed Central

Patzelt, Diana; Michael, Victoria; Päuker, Orsola; Ebert, Matthias; Tielen, Petra; Jahn, Dieter; Tomasch, Jürgen; Petersen, Jörn; Wagner-Döbler, Irene

2016-01-01

Rhodobacteraceae harbor a conspicuous wealth of extrachromosomal replicons (ECRs) and therefore the exchange of genetic material via horizontal transfer has been supposed to be a major evolutionary driving force. Many plasmids in this group encode type IV secretion systems (T4SS) that are expected to mediate transfer of proteins and/or DNA into host cells, but no experimental evidence of either has yet been provided. Dinoroseobacter shibae, a species of the Roseobacter group within the Rhodobacteraceae family, contains five ECRs that are crucial for anaerobic growth, survival under starvation and the pathogenicity of this model organism. Here we tagged two syntenous but compatible RepABC-type plasmids of 191 and 126-kb size, each encoding a T4SS, with antibiotic resistance genes and demonstrated their conjugational transfer into a distantly related Roseobacter species, namely Phaeobacter inhibens. Pulsed field gel electrophoresis showed transfer of those replicons into the recipient both individually but also together documenting the efficiency of conjugation. We then studied the influence of externally added quorum sensing (QS) signals on the expression of the T4SS located on the sister plasmids. A QS deficient D. shibae null mutant (ΔluxI1) lacking synthesis of N-acyl-homoserine lactones (AHLs) was cultivated with a wide spectrum of chemically diverse long-chain AHLs. All AHLs with lengths of the acid side-chain ≥14 reverted the ΔluxI1 phenotype to wild-type. Expression of the T4SS was induced up to log2 ∼3fold above wild-type level. We hypothesize that conjugation in roseobacters is QS-controlled and that the QS system may detect a wide array of long-chain AHLs at the cell surface. PMID:27303368

Enzymology and structure of the GH13_31 glucan 1,6-α-glucosidase that confers isomaltooligosaccharide utilization in the probiotic Lactobacillus acidophilus NCFM.

PubMed

Møller, Marie S; Fredslund, Folmer; Majumder, Avishek; Nakai, Hiroyuki; Poulsen, Jens-Christian N; Lo Leggio, Leila; Svensson, Birte; Abou Hachem, Maher

2012-08-01

Isomaltooligosaccharides (IMO) have been suggested as promising prebiotics that stimulate the growth of probiotic bacteria. Genomes of probiotic lactobacilli from the acidophilus group, as represented by Lactobacillus acidophilus NCFM, encode α-1,6 glucosidases of the family GH13_31 (glycoside hydrolase family 13 subfamily 31) that confer degradation of IMO. These genes reside frequently within maltooligosaccharide utilization operons, which include an ATP-binding cassette transporter and α-glucan active enzymes, e.g., maltogenic amylases and maltose phosphorylases, and they also occur separated from any carbohydrate transport or catabolism genes on the genomes of some acidophilus complex members, as in L. acidophilus NCFM. Besides the isolated locus encoding a GH13_31 enzyme, the ABC transporter and another GH13 in the maltooligosaccharide operon were induced in response to IMO or maltotetraose, as determined by reverse transcription-PCR (RT-PCR) transcriptional analysis, suggesting coregulation of α-1,6- and α-1,4-glucooligosaccharide utilization loci in L. acidophilus NCFM. The L. acidophilus NCFM GH13_31 (LaGH13_31) was produced recombinantly and shown to be a glucan 1,6-α-glucosidase active on IMO and dextran and product-inhibited by glucose. The catalytic efficiency of LaGH13_31 on dextran and the dextran/panose (trisaccharide) efficiency ratio were the highest reported for this class of enzymes, suggesting higher affinity at distal substrate binding sites. The crystal structure of LaGH13_31 was determined to a resolution of 2.05 Å and revealed additional substrate contacts at the +2 subsite in LaGH13_31 compared to the GH13_31 from Streptococcus mutans (SmGH13_31), providing a possible structural rationale to the relatively high affinity for dextran. A comprehensive phylogenetic and activity motif analysis mapped IMO utilization enzymes from gut microbiota to rationalize preferential utilization of IMO by gut residents.

Enzymology and Structure of the GH13_31 Glucan 1,6-α-Glucosidase That Confers Isomaltooligosaccharide Utilization in the Probiotic Lactobacillus acidophilus NCFM

PubMed Central

Møller, Marie S.; Fredslund, Folmer; Majumder, Avishek; Nakai, Hiroyuki; Poulsen, Jens-Christian N.; Lo Leggio, Leila; Svensson, Birte

2012-01-01

Isomaltooligosaccharides (IMO) have been suggested as promising prebiotics that stimulate the growth of probiotic bacteria. Genomes of probiotic lactobacilli from the acidophilus group, as represented by Lactobacillus acidophilus NCFM, encode α-1,6 glucosidases of the family GH13_31 (glycoside hydrolase family 13 subfamily 31) that confer degradation of IMO. These genes reside frequently within maltooligosaccharide utilization operons, which include an ATP-binding cassette transporter and α-glucan active enzymes, e.g., maltogenic amylases and maltose phosphorylases, and they also occur separated from any carbohydrate transport or catabolism genes on the genomes of some acidophilus complex members, as in L. acidophilus NCFM. Besides the isolated locus encoding a GH13_31 enzyme, the ABC transporter and another GH13 in the maltooligosaccharide operon were induced in response to IMO or maltotetraose, as determined by reverse transcription-PCR (RT-PCR) transcriptional analysis, suggesting coregulation of α-1,6- and α-1,4-glucooligosaccharide utilization loci in L. acidophilus NCFM. The L. acidophilus NCFM GH13_31 (LaGH13_31) was produced recombinantly and shown to be a glucan 1,6-α-glucosidase active on IMO and dextran and product-inhibited by glucose. The catalytic efficiency of LaGH13_31 on dextran and the dextran/panose (trisaccharide) efficiency ratio were the highest reported for this class of enzymes, suggesting higher affinity at distal substrate binding sites. The crystal structure of LaGH13_31 was determined to a resolution of 2.05 Å and revealed additional substrate contacts at the +2 subsite in LaGH13_31 compared to the GH13_31 from Streptococcus mutans (SmGH13_31), providing a possible structural rationale to the relatively high affinity for dextran. A comprehensive phylogenetic and activity motif analysis mapped IMO utilization enzymes from gut microbiota to rationalize preferential utilization of IMO by gut residents. PMID:22685275

Four subunits that are shared by the three classes of RNA polymerase are functionally interchangeable between Homo sapiens and Saccharomyces cerevisiae.

PubMed Central

Shpakovski, G V; Acker, J; Wintzerith, M; Lacroix, J F; Thuriaux, P; Vigneron, M

1995-01-01

Four cDNAs encoding human polypeptides hRPB7.0, hRPB7.6, hRPB17, and hRPB14.4 (referred to as Hs10 alpha, Hs10 beta, Hs8, and Hs6, respectively), homologous to the ABC10 alpha, ABC10 beta, ABC14.5, and ABC23 RNA polymerase subunits (referred to as Sc10 alpha, Sc10 beta, Sc8, and Sc6, respectively) of Saccharomyces cerevisiae, were cloned and characterized for their ability to complement defective yeast mutants. Hs10 alpha and the corresponding Sp10 alpha of Schizosaccharomyces pombe can complement an S. cerevisiae mutant (rpc10-delta::HIS3) defective in Sc10 alpha. The peptide sequences are highly conserved in their carboxy-terminal halves, with an invariant motif CX2CX12RCX2CGXR corresponding to a canonical zinc-binding domain. Hs10 beta, Sc10 beta, and the N subunit of archaeal RNA polymerase are homologous. An invariant CX2CGXnCCR motif presumably forms an atypical zinc-binding domain. Hs10 beta, but not the archaeal subunit, complemented an S. cerevisiae mutant (rpb10-delta 1::HIS3) lacking Sc10 beta. Hs8 complemented a yeast mutant (rpb8-delta 1::LYS2) defective in the corresponding Sc8 subunit, although with a strong thermosensitive phenotype. Interspecific complementation also occurred with Hs6 and with the corresponding Dm6 cDNA of Drosophila melanogaster. Hs6 cDNA and the Sp6 cDNA of S. pombe are dosage-dependent suppressors of rpo21-4, a mutation generating a slowly growing yeast defective in the largest subunit of RNA polymerase II. Finally, a doubly chimeric S. cerevisiae strain bearing the Sp6 cDNA and the human Hs10 beta cDNA was also viable. No interspecific complementation was observed for the human hRPB25 (Hs5) homolog of the yeast ABC27 (Sc5) subunit. PMID:7651387

Modification of glucose import capacity in Escherichia coli: physiologic consequences and utility for improving DNA vaccine production

PubMed Central

2013-01-01

Background The bacterium Escherichia coli can be grown employing various carbohydrates as sole carbon and energy source. Among them, glucose affords the highest growth rate. This sugar is nowadays widely employed as raw material in industrial fermentations. When E. coli grows in a medium containing non-limiting concentrations of glucose, a metabolic imbalance occurs whose main consequence is acetate secretion. The production of this toxic organic acid reduces strain productivity and viability. Solutions to this problem include reducing glucose concentration by substrate feeding strategies or the generation of mutant strains with impaired glucose import capacity. In this work, a collection of E. coli strains with inactive genes encoding proteins involved in glucose transport where generated to determine the effects of reduced glucose import capacity on growth rate, biomass yield, acetate and production of an experimental plasmid DNA vaccine (pHN). Results A group of 15 isogenic derivatives of E. coli W3110 were generated with single and multiple deletions of genes encoding glucose, mannose, beta-glucoside, maltose and N-acetylglucosamine components of the phosphoenolpyruvate:sugar phosphotransferase system (PTS), as well as the galactose symporter and the Mgl galactose/glucose ABC transporter. These strains were characterized by growing them in mineral salts medium supplemented with 2.5 g/L glucose. Maximum specific rates of glucose consumption (qs) spanning from 1.33 to 0.32 g/g h were displayed by the group of mutants and W3110, which resulted in specific growth rates ranging from 0.65-0.18 h-1. Acetate accumulation was reduced or abolished in cultures with all mutant strains. W3110 and five selected mutant derivatives were transformed with pHN. A 3.2-fold increase in pHN yield on biomass was observed in cultures of a mutant strain with deletion of genes encoding the glucose and mannose PTS components, as well as Mgl. Conclusions The group of E. coli mutants generated in this study displayed a reduction or elimination of overflow metabolism and a linear correlation between qs and the maximum specific growth rate as well as the acetate production rate. By comparing DNA vaccine production parameters among some of these mutants, it was possible to identify a near-optimal glucose import rate value for this particular application. The strains employed in this study should be a useful resource for studying the effects of different predefined qs values on production capacity for various biotechnological products. PMID:23638701

The Functional Network of the Arabidopsis Plastoglobule Proteome Based on Quantitative Proteomics and Genome-Wide Coexpression Analysis1[C][W][OA

PubMed Central

Lundquist, Peter K.; Poliakov, Anton; Bhuiyan, Nazmul H.; Zybailov, Boris; Sun, Qi; van Wijk, Klaas J.

2012-01-01

Plastoglobules (PGs) in chloroplasts are thylakoid-associated monolayer lipoprotein particles containing prenyl and neutral lipids and several dozen proteins mostly with unknown functions. An integrated view of the role of the PG is lacking. Here, we better define the PG proteome and provide a conceptual framework for further studies. The PG proteome from Arabidopsis (Arabidopsis thaliana) leaf chloroplasts was determined by mass spectrometry of isolated PGs and quantitative comparison with the proteomes of unfractionated leaves, thylakoids, and stroma. Scanning electron microscopy showed the purity and size distribution of the isolated PGs. Compared with previous PG proteome analyses, we excluded several proteins and identified six new PG proteins, including an M48 metallopeptidase and two Absence of bc1 complex (ABC1) atypical kinases, confirmed by immunoblotting. This refined PG proteome consisted of 30 proteins, including six ABC1 kinases and seven fibrillins together comprising more than 70% of the PG protein mass. Other fibrillins were located predominantly in the stroma or thylakoid and not in PGs; we discovered that this partitioning can be predicted by their isoelectric point and hydrophobicity. A genome-wide coexpression network for the PG genes was then constructed from mRNA expression data. This revealed a modular network with four distinct modules that each contained at least one ABC1K and/or fibrillin gene. Each module showed clear enrichment in specific functions, including chlorophyll degradation/senescence, isoprenoid biosynthesis, plastid proteolysis, and redox regulators and phosphoregulators of electron flow. We propose a new testable model for the PGs, in which sets of genes are associated with specific PG functions. PMID:22274653

SatR Is a Repressor of Fluoroquinolone Efflux Pump SatAB

PubMed Central

Escudero, Jose Antonio; San Millan, Alvaro; Montero, Natalia; Gutierrez, Belen; Ovejero, Cristina Martinez; Carrilero, Laura

2013-01-01

Streptococcus suis is an emerging zoonotic agent responsible for high-mortality outbreaks among the human population in China. In this species, the ABC transporter SatAB mediates fluoroquinolone resistance when overexpressed. Here, we describe and characterize satR, an open reading frame (ORF) encoding a MarR superfamily regulator that acts as a repressor of satAB. satR is cotranscribed with satAB, and its interruption entails the overexpression of the pump, leading to a clinically relevant increase in resistance to fluoroquinolones. PMID:23650171

The organization of the fuc regulon specifying L-fucose dissimilation in Escherichia coli K12 as determined by gene cloning.

PubMed

Chen, Y M; Zhu, Y; Lin, E C

1987-12-01

In Escherichia coli the six known genes specifying the utilization of L-fucose as carbon and energy source cluster at 60.2 min and constitute a regulon. These genes include fucP (encoding L-fucose permease), fucI (encoding L-fucose isomerase), fucK (encoding L-fuculose kinase), fucA (encoding L-fuculose 1-phosphate aldolase), fucO (encoding L-1,2-propanediol oxidoreductase), and fucR (encoding the regulatory protein). In this study the fuc genes were cloned and their positions on the chromosome were established by restriction endonuclease and complementation analyses. Clockwise, the gene order is: fucO-fucA-fucP-fucI-fucK-fucR. The operons comprising the structural genes and the direction of transcription were determined by complementation analysis and Southern blot hybridization. The fucPIK and fucA operons are transcribed clockwise. The fucO operon is transcribed counterclockwise. The fucR gene product activates the three structural operons in trans.

Evolutionary Characteristics of Missing Proteins: Insights into the Evolution of Human Chromosomes Related to Missing-Protein-Encoding Genes.

PubMed

Xu, Aishi; Li, Guang; Yang, Dong; Wu, Songfeng; Ouyang, Hongsheng; Xu, Ping; He, Fuchu

2015-12-04

Although the "missing protein" is a temporary concept in C-HPP, the biological information for their "missing" could be an important clue in evolutionary studies. Here we classified missing-protein-encoding genes into two groups, the genes encoding PE2 proteins (with transcript evidence) and the genes encoding PE3/4 proteins (with no transcript evidence). These missing-protein-encoding genes distribute unevenly among different chromosomes, chromosomal regions, or gene clusters. In the view of evolutionary features, PE3/4 genes tend to be young, spreading at the nonhomology chromosomal regions and evolving at higher rates. Interestingly, there is a higher proportion of singletons in PE3/4 genes than the proportion of singletons in all genes (background) and OTCSGs (organ, tissue, cell type-specific genes). More importantly, most of the paralogous PE3/4 genes belong to the newly duplicated members of the paralogous gene groups, which mainly contribute to special biological functions, such as "smell perception". These functions are heavily restricted into specific type of cells, tissues, or specific developmental stages, acting as the new functional requirements that facilitated the emergence of the missing-protein-encoding genes during evolution. In addition, the criteria for the extremely special physical-chemical proteins were first set up based on the properties of PE2 proteins, and the evolutionary characteristics of those proteins were explored. Overall, the evolutionary analyses of missing-protein-encoding genes are expected to be highly instructive for proteomics and functional studies in the future.

[Genetic instability of probiotic characteristics in the Bifidobacterium longum subsp. longum B379M strain during cultivation and maintenance].

PubMed

Averina, O V; Nezametdinova, V Z; Alekseeva, M G; Danilenko, V N

2012-11-01

The stability of inheriting several genes in the Russian commercial strain Bifidobacterium longum subsp. longum B379M during cultivation and maintenance under laboratory conditions has been studied. The examined genes code for probiotic characteristics, such as utilization of several sugars (lacA2 gene, encoding beta-galactosidase; ara gene, encoding arabinosidase; and galA gene, encoding arabinogalactan endo-beta-galactosidase); synthesis of bacteriocins (lans gene, encoding lanthionine synthetase); and mobile gene tet(W), conferring resistance to the antibiotic tetracycline. The other gene families studied include the genes responsible for signal transduction and adaptation to stress conditions in the majority of bacteria (serine/threonine protein kinases and the toxin-antitoxin systems of MazEF and RelBE types) and transcription regulators (genes encoding WhiB family proteins). Genomic DNA was analyzed by PCR using specially selected primers. A loss of the genes galA and tet(W) has been shown. It is proposed to expand the requirements on probiotic strains, namely, to control retention of the key probiotic genes using molecular biological methods.

Isolated gene encoding an enzyme with UDP-glucose pyrophosphorylase and phosphoglucomutase activities from Cyclotella cryptica

DOEpatents

Jarvis, Eric E.; Roessler, Paul G.

1999-01-01

The present invention relates to a cloned gene which encodes an enzyme, the purified enzyme, and the applications and products resulting from the use of the gene and enzyme. The gene, isolated from Cyclotella cryptica, encodes a multifunctional enzyme that has both UDP-glucose pyrophosphorylase and phosphoglucomutase activities.

Human Genomic Signatures of Brain Oscillations During Memory Encoding.

PubMed

Berto, Stefano; Wang, Guang-Zhong; Germi, James; Lega, Bradley C; Konopka, Genevieve

2018-05-01

Memory encoding is an essential step for all learning. However, the genetic and molecular mechanisms underlying human memory encoding remain poorly understood, and how this molecular framework permits the emergence of specific patterns of brain oscillations observed during mnemonic processing is unknown. Here, we directly compare intracranial electroencephalography recordings from the neocortex in individuals performing an episodic memory task with human gene expression from the same areas. We identify genes correlated with oscillatory memory effects across 6 frequency bands. These genes are enriched for autism-related genes and have preferential expression in neurons, in particular genes encoding synaptic proteins and ion channels, supporting the idea that the genes regulating voltage gradients are involved in the modulation of oscillatory patterns during successful memory encoding across brain areas. Memory-related genes are distinct from those correlated with other forms of cognitive processing and resting state fMRI. These data are the first to identify correlations between gene expression and active human brain states as well as provide a molecular window into memory encoding oscillations in the human brain.

Implications of ESR1 Mutations in Hormone Receptor-Positive Breast Cancer.

PubMed

Reinert, Tomás; Gonçalves, Rodrigo; Bines, José

2018-04-17

Endocrine treatment resistance eventually develops during adjuvant and even more often during hormonal treatment for advanced breast cancer (ABC). An ESR1 gene mutation, which encodes for the estrogen receptor (ER) protein, is one of the potential mechanisms of therapy resistance. The ESR1 mutations result in conformational changes in the ER leading to subsequent estrogen-independent transcriptional activity. These mutations are found at a lower level in early stage when compared to metastatic BC, more often through selective pressure after aromatase inhibitor (AI) treatment. Recent studies have explored the role of ESR1 mutations as potential prognostic and predictive biomarkers and showed that ESR1 mutations are likely associated with a more aggressive disease. However, definitive associations with outcome in order to make a specific treatment recommendation are yet to be found. The development of targeted therapy directed to ESR1-mutated clones is an appealing concept, and preclinical and clinical works are in progress. ESR1 mutations represent an exciting field with a rapidly increasing number of recent publications that will likely advance the knowledge of treatment resistance mechanisms and pave the way into more individualized patient endocrine treatment.

Rv1458c: a new diagnostic marker for identification of Mycobacterium tuberculosis complex in a novel duplex PCR assay.

PubMed

Shrivastava, Kamal; Garima, Kushal; Narang, Anshika; Bhattacharyya, Kausik; Vishnoi, Ekta; Singh, Roshan Kumar; Chaudhry, Anil; Prasad, Rajendra; Bose, Mridula; Varma-Basil, Mandira

2017-03-01

We explored the efficiency of Rv1458c, the gene encoding a putative ABC drug transporter specific for the Mycobacterium tuberculosis complex (MTBC), as a diagnostic marker. A 190 bp region of Rv1458c and a 300 bp region of hsp65 were targeted in a novel duplex PCR assay and the results were compared with those for PCR restriction analysis(PRA) using the restriction enzymes NruI and BamHI. Species identification of a subset of the isolates (n=50) was confirmed by sequencing. Clinical isolates of M. tuberculosis (n=426) obtained from clinically suspected patients of pulmonary tuberculosis and mycobacterial (n=13) and non-mycobacterial (n=8) reference strains were included in the study. The duplex PCR assay correctly identified 320/426 isolates as MTBC and 106/426 isolates as non-tuberculous mycobacteria(NTM). The test was 100 % specific and sensitive when compared with NruI/BamHI PCR restriction analysis and highlighted the use of Rv1458c as a diagnostic marker for MTBC. The duplex PCR assay could be developed for use as a screening test to identify MTBC in clinical specimens in peripheral laboratories with limited resources.

The Yersiniabactin-Associated ATP Binding Cassette Proteins YbtP and YbtQ Enhance Escherichia coli Fitness during High-Titer Cystitis

PubMed Central

Koh, Eun-Ik; Hung, Chia S.

2016-01-01

The Yersinia high-pathogenicity island (HPI) is common to multiple virulence strategies used by Escherichia coli strains associated with urinary tract infection (UTI). Among the genes in this island are ybtP and ybtQ, encoding distinctive ATP binding cassette (ABC) proteins associated with iron(III)-yersiniabactin import in Yersinia pestis. In this study, we compared the impact of ybtPQ on a model E. coli cystitis strain during in vitro culture and experimental murine infections. A ybtPQ-null mutant exhibited no growth defect under standard culture conditions, consistent with nonessentiality in this background. A growth defect phenotype was observed and genetically complemented in vitro during iron(III)-yersiniabactin-dependent growth. Following inoculation into the bladders of C3H/HEN and C3H/HeOuJ mice, this strain exhibited a profound, 106-fold competitive infection defect in the subgroup of mice that progressed to high-titer bladder infections. These results identify a virulence role for YbtPQ in the highly inflammatory microenvironment characteristic of high-titer cystitis. The profound competitive defect may relate to the apparent selection of Yersinia HPI-positive E. coli in uncomplicated clinical UTIs. PMID:26883590

ATP-Binding Cassette Efflux Transporters in Human Placenta

PubMed Central

Ni, Zhanglin; Mao, Qingcheng

2010-01-01

Pregnant women are often complicated with diseases including viral or bacterial infections, epilepsy, hypertension, or pregnancy-induced conditions such as depression and gestational diabetes that require treatment with medication. In addition, substance abuse during pregnancy remains a major public health problem. Many drugs used by pregnant women are off label without the necessary dose, efficacy, and safety data required for rational dosing regimens of these drugs. Thus, a major concern arising from the widespread use of drugs by pregnant women is the transfer of drugs across the placental barrier, leading to potential toxicity to the developing fetus. Knowledge regarding the ATP-binding cassette (ABC) efflux transporters, which play an important role in drug transfer across the placental barrier, is absolutely critical for optimizing the therapeutic strategy to treat the mother while protecting the fetus during pregnancy. Such transporters include P-glycoprotein (P-gp, gene symbol ABCB1), the breast cancer resistance protein (BCRP, gene symbol ABCG2), and the multidrug resistance proteins (MRPs, gene symbol ABCCs). In this review, we summarize the current knowledge with respect to developmental expression and regulation, membrane localization, functional significance, and genetic polymorphisms of these ABC transporters in the placenta and their relevance to fetal drug exposure and toxicity. PMID:21118087

Effects of lithium on growth, maturation, reproduction and gene expression in the nematode Caenorhabditis elegans.

PubMed

Inokuchi, Ayako; Yamamoto, Ryoko; Morita, Fumiyo; Takumi, Shota; Matsusaki, Hiromi; Ishibashi, Hiroshi; Tominaga, Nobuaki; Arizono, Koji

2015-09-01

Lithium (Li) has been widely used to treat bipolar disorder, and industrial use of Li has been increasing; thus, environmental pollution and ecological impacts of Li have become a concern. This study was conducted to clarify the potential biological effects of LiCl and Li(2)CO(3) on a nematode, Caenorhabditis elegans as a model system for evaluating soil contaminated with Li. Exposure of C. elegans to LiCl and Li(2)CO(3) decreased growth/maturation and reproduction. The lowest observed effect concentrations for growth, maturation and reproduction were 1250, 313 and 10 000 µm, respectively, for LiCl and 750, 750 and 3000 µm, respectively, for Li(2)CO(3). We also investigated the physiological function of LiCl and LiCO(3) in C. elegans using DNA microarray analysis as an eco-toxicogenomic approach. Among approximately 300 unique genes, including metabolic genes, the exposure to 78 µm LiCl downregulated the expression of 36 cytochrome P450, 16 ABC transporter, 10 glutathione S-transferase, 16 lipid metabolism and two vitellogenin genes. On the other hand, exposure to 375 µm Li(2)CO(3) downregulated the expression of 11 cytochrome P450, 13 ABC transporter, 13 lipid metabolism and one vitellogenin genes. No gene was upregulated by LiCl or Li(2)CO(3). These results suggest that LiCl and Li(2)CO(3) potentially affect the biological and physiological function in C. elegans associated with alteration of the gene expression such as metabolic genes. Our data also provide experimental support for the utility of toxicogenomics by integrating gene expression profiling into a toxicological study of an environmentally important organism such as C. elegans. Copyright © 2015 John Wiley & Sons, Ltd.

An ATP-Binding Cassette Transporter and Two rRNA Methyltransferases Are Involved in Resistance to Avilamycin in the Producer Organism Streptomyces viridochromogenes Tü57

PubMed Central

Weitnauer, Gabriele; Gaisser, Sibylle; Trefzer, Axel; Stockert, Sigrid; Westrich, Lucy; Quiros, Luis M.; Mendez, Carmen; Salas, Jose A.; Bechthold, Andreas

2001-01-01

Three different resistance factors from the avilamycin biosynthetic gene cluster of Streptomyces viridochromogenes Tü57, which confer avilamycin resistance when expressed in Streptomyces lividans TK66, were isolated. Analysis of the deduced amino acid sequences showed that AviABC1 is similar to a large family of ATP-binding transporter proteins and that AviABC2 resembles hydrophobic transmembrane proteins known to act jointly with the ATP-binding proteins. The deduced amino acid sequence of aviRb showed similarity to those of other rRNA methyltransferases, and AviRa did not resemble any protein in the databases. Independent expression in S. lividans TK66 of aviABC1 plus aviABC2, aviRa, or aviRb conferred different levels of resistance to avilamycin: 5, 10, or 250 μg/ml, respectively. When either aviRa plus aviRb or aviRa plus aviRb plus aviABC1 plus aviABC2 was coexpressed in S. lividans TK66, avilamycin resistance levels reached more than 250 μg/ml. Avilamycin A inhibited poly(U)-directed polyphenylalanine synthesis in an in vitro system using ribosomes of S. lividans TK66(pUWL201) (GWO), S. lividans TK66(pUWL201-Ra) (GWRa), or S. lividans TK66(pUWL201-Rb) (GWRb), whereas ribosomes of S. lividans TK66 containing pUWL201-Ra+Rb (GWRaRb) were highly resistant. aviRa and aviRb were expressed in Escherichia coli, and both enzymes were purified as fusion proteins to near homogeneity. Both enzymes showed rRNA methyltransferase activity using a mixture of 16S and 23S rRNAs from E. coli as the substrate. Coincubation experiments revealed that the enzymes methylate different positions of rRNA. PMID:11181344

Isolated gene encoding an enzyme with UDP-glucose pyrophosphorylase and phosphoglucomutase activities from Cyclotella cryptica

DOEpatents

Jarvis, E.E.; Roessler, P.G.

1999-07-27

The present invention relates to a cloned gene which encodes an enzyme, the purified enzyme, and the applications and products resulting from the use of the gene and enzyme. The gene, isolated from Cyclotella cryptica, encodes a multifunctional enzyme that has both UDP-glucose pyrophosphorylase and phosphoglucomutase activities. 8 figs.

Activation of the STAT3 Signaling Pathway Is Associated With Poor Survival in Diffuse Large B-Cell Lymphoma Treated With R-CHOP

PubMed Central

Huang, Xin; Meng, Bin; Iqbal, Javeed; Ding, B. Belinda; Perry, Anamarija M.; Cao, Wenfeng; Smith, Lynette M.; Bi, Chengfeng; Jiang, Chunsun; Greiner, Timothy C.; Weisenburger, Dennis D.; Rimsza, Lisa; Rosenwald, Andreas; Ott, German; Delabie, Jan; Campo, Elias; Braziel, Rita M.; Gascoyne, Randy D.; Cook, James R.; Tubbs, Raymond R.; Jaffe, Elaine S.; Armitage, James O.; Vose, Julie M.; Staudt, Louis M.; McKeithan, Timothy W.; Chan, Wing C.; Ye, B. Hilda; Fu, Kai

2013-01-01

Purpose We previously reported that constitutive STAT3 activation is a prominent feature of the activated B-cell subtype of diffuse large B-cell lymphomas (ABC-DLBCL). In this study, we investigated whether STAT3 activation can risk stratify patients with DLBCL. Patients and Methods By an immunohistochemical method, we investigated phosphotyrosine STAT3 (PY-STAT3) expression from 185 patients with DLBCL treated with R-CHOP (rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone). Cell line-based siRNA experiments were also performed to generate an 11-gene, PY-STAT3 activation signature, which was used to study a previously published cohort of 222 patients with DLBCL. The STAT3 activation status determined by these two methods and by STAT3 mRNA levels were then correlated with survival. Results PY-STAT3 was detected in 37% of DLBCL and enriched in ABC-DLBCL cases (P = .03). PY-STAT3 positivity significantly correlated with poor overall survival (OS; P = .01) and event-free survival (EFS; P = .006). Similar observations were made for high levels of STAT3 mRNA. In multivariable analysis, PY-STAT3 status (P = .02), International Prognostic Index (P = .02), and BCL2 expression (P = .046) were independent prognosticators of OS in this cohort. Among the cell-of-origin subgroups, PY-STAT3 was associated with poor EFS among non–germinal center B-cell DLBCL cases only (P = .027). Similarly, the 11-gene STAT3 activation signature correlated with poor survival in the entire DLBCL cohort (OS, P < .001; EFS, P < .001) as well as the ABC-DLBCL subgroup (OS, P = .029; EFS, P = .025). Conclusion STAT3 activation correlated with poor survival in patients with DLBCL treated with R-CHOP, especially those with tumors of the ABC-DLBCL subtype. PMID:24220563

Polymorphisms in ATP-binding cassette transporters associated with maternal methylmercury disposition and infant neurodevelopment in mother-infant pairs in the Seychelles Child Development Study

PubMed Central

Engström, Karin; Love, Tanzy M; Watson, Gene E; Zareba, Grazyna; Yeates, Alison; Wahlberg, Karin; Alhamdow, Ayman; Thurston, Sally W; Mulhern, Maria; McSorley, Emeir M; Strain, JJ; Davidson, Philip W; Shamlaye, Conrad F; Myers, GJ; Rand, Matthew D; van Wijngaarden, Edwin; Broberg, Karin

2016-01-01

Background ATP-binding cassette (ABC) transporters have been associated with methylmercury (MeHg) toxicity in experimental animal models. Aims To evaluate the association of single nucleotide polymorphisms (SNPs) in maternal ABC transporter genes with 1) maternal hair MeHg concentrations during pregnancy and 2) child neurodevelopmental outcomes. Materials and methods Nutrition Cohort 2 (NC2) is an observational mother-child cohort recruited in the Republic of Seychelles from 2008–2011. Total mercury (Hg) was measured in maternal hair growing during pregnancy as a biomarker for prenatal MeHg exposure (N=1313) (mean 3.9 ppm). Infants completed developmental assessments by Bayley Scales of Infant Development II (BSID-II) at 20 months of age (N=1331). Genotyping for fifteen SNPs in ABCC1, ABCC2 and ABCB1 was performed for the mothers. Results Seven of fifteen ABC SNPs (ABCC1 rs11075290, rs212093, and rs215088; ABCC2 rs717620; ABCB1 rs10276499, rs1202169, and rs2032582) were associated with concentrations of maternal hair Hg (p<0.001 to 0.013). One SNP (ABCC1 rs11075290) was also significantly associated with neurodevelopment; children born to mothers with rs11075290 CC genotype (mean hair Hg 3.6 ppm) scored on average 2 points lower on the Mental Development Index (MDI) and 3 points lower on the Psychomotor Development Index (PDI) than children born to mothers with TT genotype (mean hair Hg 4.7 ppm) while children with the CT genotype (mean hair Hg 4.0 ppm) had intermediate BSID scores. Discussion Genetic variation in ABC transporter genes was associated with maternal hair Hg concentrations. The implications for MeHg dose in the developing child and neurodevelopmental outcomes need to be further investigated. PMID:27262785

Polymorphisms in ATP-binding cassette transporters associated with maternal methylmercury disposition and infant neurodevelopment in mother-infant pairs in the Seychelles Child Development Study.

PubMed

Engström, Karin; Love, Tanzy M; Watson, Gene E; Zareba, Grazyna; Yeates, Alison; Wahlberg, Karin; Alhamdow, Ayman; Thurston, Sally W; Mulhern, Maria; McSorley, Emeir M; Strain, J J; Davidson, Philip W; Shamlaye, Conrad F; Myers, G J; Rand, Matthew D; van Wijngaarden, Edwin; Broberg, Karin

2016-09-01

ATP-binding cassette (ABC) transporters have been associated with methylmercury (MeHg) toxicity in experimental animal models. To evaluate the association of single nucleotide polymorphisms (SNPs) in maternal ABC transporter genes with 1) maternal hair MeHg concentrations during pregnancy and 2) child neurodevelopmental outcomes. Nutrition Cohort 2 (NC2) is an observational mother-child cohort recruited in the Republic of Seychelles from 2008-2011. Total mercury (Hg) was measured in maternal hair growing during pregnancy as a biomarker for prenatal MeHg exposure (N=1313) (mean 3.9ppm). Infants completed developmental assessments by Bayley Scales of Infant Development II (BSID-II) at 20months of age (N=1331). Genotyping for fifteen SNPs in ABCC1, ABCC2 and ABCB1 was performed for the mothers. Seven of fifteen ABC SNPs (ABCC1 rs11075290, rs212093, and rs215088; ABCC2 rs717620; ABCB1 rs10276499, rs1202169, and rs2032582) were associated with concentrations of maternal hair Hg (p<0.001 to 0.013). One SNP (ABCC1 rs11075290) was also significantly associated with neurodevelopment; children born to mothers with rs11075290 CC genotype (mean hair Hg 3.6ppm) scored on average 2 points lower on the Mental Development Index (MDI) and 3 points lower on the Psychomotor Development Index (PDI) than children born to mothers with TT genotype (mean hair Hg 4.7ppm) while children with the CT genotype (mean hair Hg 4.0ppm) had intermediate BSID scores. Genetic variation in ABC transporter genes was associated with maternal hair Hg concentrations. The implications for MeHg dose in the developing child and neurodevelopmental outcomes need to be further investigated. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Evolutionary analysis of hydrophobin gene family in two wood-degrading basidiomycetes, Phlebia brevispora and Heterobasidion annosum s.l.

PubMed Central

2013-01-01

Background Hydrophobins are small secreted cysteine-rich proteins that play diverse roles during different phases of fungal life cycle. In basidiomycetes, hydrophobin-encoding genes often form large multigene families with up to 40 members. The evolutionary forces driving hydrophobin gene expansion and diversification in basidiomycetes are poorly understood. The functional roles of individual genes within such gene families also remain unclear. The relationship between the hydrophobin gene number, the genome size and the lifestyle of respective fungal species has not yet been thoroughly investigated. Here, we present results of our survey of hydrophobin gene families in two species of wood-degrading basidiomycetes, Phlebia brevispora and Heterobasidion annosum s.l. We have also investigated the regulatory pattern of hydrophobin-encoding genes from H. annosum s.s. during saprotrophic growth on pine wood as well as on culture filtrate from Phlebiopsis gigantea using micro-arrays. These data are supplemented by results of the protein structure modeling for a representative set of hydrophobins. Results We have identified hydrophobin genes from the genomes of two wood-degrading species of basidiomycetes, Heterobasidion irregulare, representing one of the microspecies within the aggregate H. annosum s.l., and Phlebia brevispora. Although a high number of hydrophobin-encoding genes were observed in H. irregulare (16 copies), a remarkable expansion of these genes was recorded in P. brevispora (26 copies). A significant expansion of hydrophobin-encoding genes in other analyzed basidiomycetes was also documented (1–40 copies), whereas contraction through gene loss was observed among the analyzed ascomycetes (1–11 copies). Our phylogenetic analysis confirmed the important role of gene duplication events in the evolution of hydrophobins in basidiomycetes. Increased number of hydrophobin-encoding genes appears to have been linked to the species’ ecological strategy, with the non-pathogenic fungi having increased numbers of hydrophobins compared with their pathogenic counterparts. However, there was no significant relationship between the number of hydrophobin-encoding genes and genome size. Furthermore, our results revealed significant differences in the expression levels of the 16 H. annosum s.s. hydrophobin-encoding genes which suggest possible differences in their regulatory patterns. Conclusions A considerable expansion of the hydrophobin-encoding genes in basidiomycetes has been observed. The distribution and number of hydrophobin-encoding genes in the analyzed species may be connected to their ecological preferences. Results of our analysis also have shown that H. annosum s.l. hydrophobin-encoding genes may be under positive selection. Our gene expression analysis revealed differential expression of H. annosum s.s. hydrophobin genes under different growth conditions, indicating their possible functional diversification. PMID:24188142

Glutathione S-transferase-encoding gene as a potential probe for environmental bacterial isolates capable of degrading polycyclic aromatic hydrocarbons.

PubMed Central

Lloyd-Jones, G; Lau, P C

1997-01-01

Homologs of the glutathione S-transferase (GST)-encoding gene were identified in a collection of aromatic hydrocarbon-degrading Sphingomonas spp. isolated from New Zealand, Antarctica, and the United States by using PCR primers designed from the GST-encoding gene of Sphingomonas paucimobilis EPA505. Sequence analysis of PCR fragments generated from these isolates and of the GST gene amplified from DNA extracted from polycyclic aromatic hydrocarbon (PAH)-contaminated soil revealed a high degree of conservation, which may make the GST-encoding gene a potentially useful marker for PAH-degrading bacteria. PMID:9251217

Enterotoxin-encoding genes in Staphylococcus spp. from bulk goat milk.

PubMed

Lyra, Daniele G; Sousa, Francisca G C; Borges, Maria F; Givisiez, Patrícia E N; Queiroga, Rita C R E; Souza, Evandro L; Gebreyes, Wondwossen A; Oliveira, Celso J B

2013-02-01

Although Staphylococcus aureus has been implicated as the main Staphylococcus species causing human food poisoning, recent studies have shown that coagulase-negative Staphylococcus could also harbor enterotoxin-encoding genes. Such organisms are often present in goat milk and are the most important mastitis-causing agents. Therefore, this study aimed to investigate the occurrence of enterotoxin-encoding genes among coagulase-positive (CoPS) and coagulase-negative (CoNS) staphylococci isolated from raw goat milk produced in the semi-arid region of Paraiba, the most important region for goat milk production in Brazil. Enterotoxin-encoding genes were screened in 74 staphylococci isolates (30 CoPS and 44 CoNS) by polymerase chain reaction targeting the genes sea, seb, sec, sed, see, seg, seh, and sei. Enterotoxin-encoding genes were found in nine (12.2%) isolates, and four different genes (sea, sec, seg, and sei) were identified amongst the isolates. The most frequent genes were seg and sei, which were often found simultaneously in 44.5% of the isolates. The gene sec was the most frequent among the classical genes, and sea was found only in one isolate. All CoPS isolates (n=7) harboring enterotoxigenic genes were identified as S. aureus. The two coagulase-negative isolates were S. haemolyticus and S. hominis subsp. hominis and they harbored sei and sec genes, respectively. A higher frequency of enterotoxin-encoding genes was observed amongst CoPS (23.3%) than CoNS (4.5%) isolates (p<0.05), reinforcing the importance of S. aureus as a potential foodborne agent. However, the potential risk posed by CoNS in goat milk should not be ignored because it has a higher occurrence in goat milk and enterotoxin-encoding genes were detected in some isolates.

Biodegradation of PuEDTA and Impacts on Pu Mobility

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bolton, H., Jr.; Rai, D.; Xun, L.

The contamination of many DOE sites by Pu presents a long-term problem because of its long half-life (240,000 yrs) and the low drinking water standard (<10{sup -12} M). EDTA was co-disposed with radionuclides (e.g., Pu, {sup 60}Co), formed strong complexes, and enhanced radionuclide transport at several DOE sites. Biodegradation of EDTA should decrease Pu mobility. One objective of this project was to determine the biodegradation of EDTA in the presence of PuEDTA complexes. The aqueous system investigated at pH 7 (10{sup -4} M EDTA and 10{sup -6} M Pu) contained predominantly Pu(OH){sub 2}EDTA{sup 2-}. The EDTA was degraded at amore » faster rate in the presence of Pu. As the total concentration of both EDTA and PuEDTA decreased (i.e., 10{sup -5} M EDTA and 10{sup -7} M PuEDTA), the presence of Pu decreased the biodegradation rate of the EDTA. It is currently unclear why the concentration of Pu directly affects the increase/decrease in rate of EDTA biodegradation. The soluble Pu concentration decreased, in agreement with thermodynamic predictions, as the EDTA was biodegraded, indicating that biodegradation of EDTA will decrease Pu mobility when the Pu is initially present as Pu(IV)EDTA. A second objective was to investigate how the presence of competing metals, commonly encountered in geologic media, will influence the speciation and biodegradation of Pu(IV)-EDTA. Studies on the solubilities of Fe(OH){sub 3}(s) and of Fe(OH){sub 3}(s) plus PuO{sub 2}(am) in the presence of EDTA and as a function of pH showed that Fe(III) out competes the Pu(IV) for the EDTA complex, thereby showing that Pu(IV) will not form stable complexes with EDTA for enhanced transport of Pu in Fe(III) dominated subsurface systems. A third objective is to investigate the genes and enzymes involved in EDTA biodegradation. BNC1 can use EDTA and another synthetic chelating agent nitrilotriacetate (NTA) as sole carbon and nitrogen sources. The same catabolic enzymes are responsible for both EDTA and NTA degradation except that additional enzymes are required for EDTA degradation. When the catabolic genes were cloned and sequenced, the gene cluster also contained genes encoding a hypothetical ABC-type transporter. RT-PCR analysis showed that the transporter genes and EDTA monooxygenase gene (emoA) are co-transcribed. EppA is one of the transporter genes, and it codes for a periplasmic binding protein responsible for binding to the substrate before transport across the membrane can occur. EppA was cloned, expressed, and purified in Escherichia coli and found to bind, MgEDTA, CaEDTA, Fe(III)EDTA, MgNTA, CaNTA, and Fe(III)NTA. Our data also suggest that BNC1 uses the same ABC-type transporter for both EDTA and NTA uptake. Results from these studies can provide mechanistic understanding and approaches to assist in the bioremediate PuEDTA and other radionuclide-EDTA complexes at DOE sites.« less

The multidrug ABC transporter BmrC/BmrD of Bacillus subtilis is regulated via a ribosome-mediated transcriptional attenuation mechanism.

PubMed

Reilman, Ewoud; Mars, Ruben A T; van Dijl, Jan Maarten; Denham, Emma L

2014-10-01

Expression of particular drug transporters in response to antibiotic pressure is a critical element in the development of bacterial multidrug resistance, and represents a serious concern for human health. To obtain a better understanding of underlying regulatory mechanisms, we have dissected the transcriptional activation of the ATP-binding cassette (ABC) transporter BmrC/BmrD of the Gram-positive model bacterium Bacillus subtilis. By using promoter-GFP fusions and live cell array technology, we demonstrate a temporally controlled transcriptional activation of the bmrCD genes in response to antibiotics that target protein synthesis. Intriguingly, bmrCD expression only occurs during the late-exponential and stationary growth stages, irrespective of the timing of the antibiotic challenge. We show that this is due to tight transcriptional control by the transition state regulator AbrB. Moreover, our results show that the bmrCD genes are co-transcribed with bmrB (yheJ), a small open reading frame immediately upstream of bmrC that harbors three alternative stem-loop structures. These stem-loops are apparently crucial for antibiotic-induced bmrCD transcription. Importantly, the antibiotic-induced bmrCD expression requires translation of bmrB, which implies that BmrB serves as a regulatory leader peptide. Altogether, we demonstrate for the first time that a ribosome-mediated transcriptional attenuation mechanism can control the expression of a multidrug ABC transporter. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Translocation of the ABC transporter ABCD4 from the endoplasmic reticulum to lysosomes requires the escort protein LMBD1.

PubMed

Kawaguchi, Kosuke; Okamoto, Takumi; Morita, Masashi; Imanaka, Tsuneo

2016-07-26

We previously demonstrated that ABCD4 does not localize to peroxisomes but rather, the endoplasmic reticulum (ER), because it lacks the NH2-terminal hydrophilic region required for peroxisomal targeting. It was recently reported that mutations in ABCD4 result in a failure to release vitamin B12 from lysosomes. A similar phenotype is caused by mutations in LMBRD1, which encodes the lysosomal membrane protein LMBD1. These findings suggested to us that ABCD4 translocated from the ER to lysosomes in association with LMBD1. In this report, it is demonstrated that ABCD4 interacts with LMBD1 and then localizes to lysosomes, and this translocation depends on the lysosomal targeting ability of LMBD1. Furthermore, endogenous ABCD4 was localized to both lysosomes and the ER, and its lysosomal localization was disturbed by knockout of LMBRD1. To the best of our knowledge, this is the first report demonstrating that the subcellular localization of the ABC transporter is determined by its association with an adaptor protein.

Manipulating the ABCs of self-assembly via low-χ block polymer design

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chang, Alice B.; Bates, Christopher M.; Lee, Byeongdu

Block polymer self-assembly typically translates molecular chain connectivity into mesoscale structure by exploiting incompatible blocks with large interaction parameters (χ ij). In this report, we demonstrate that the converse approach, encoding low-χ interactions in ABC bottlebrush triblock terpolymers (χ AC ≲ 0), promotes organization into a unique mixed-domain lamellar morphology which we designate LAM P. Transmission electron microscopy indicates that LAMP exhibits ACBC domain connectivity, in contrast to conventional three-domain lamellae (LAM 3) with ABCB periods. Complementary small angle X-ray scattering experiments reveal a strongly decreasing domain spacing with increasing total molar mass. Self-consistent field theory reinforces these observations andmore » predicts that LAM P is thermodynamically stable below a critical χ AC, above which LAM 3 emerges. Both experiments and theory expose close analogies to ABA triblock copolymer phase behavior, collectively suggesting that low-χ interactions between chemically similar or distinct blocks intimately influence self-assembly. Furthermore, these conclusions provide new opportunities in block polymer design with potential consequences spanning all self-assembling soft materials.« less

Manipulating the ABCs of self-assembly via low-χ block polymer design

DOE PAGES

Chang, Alice B.; Bates, Christopher M.; Lee, Byeongdu; ...

2017-06-06

Block polymer self-assembly typically translates molecular chain connectivity into mesoscale structure by exploiting incompatible blocks with large interaction parameters (χ ij). In this report, we demonstrate that the converse approach, encoding low-χ interactions in ABC bottlebrush triblock terpolymers (χ AC ≲ 0), promotes organization into a unique mixed-domain lamellar morphology which we designate LAM P. Transmission electron microscopy indicates that LAMP exhibits ACBC domain connectivity, in contrast to conventional three-domain lamellae (LAM 3) with ABCB periods. Complementary small angle X-ray scattering experiments reveal a strongly decreasing domain spacing with increasing total molar mass. Self-consistent field theory reinforces these observations andmore » predicts that LAM P is thermodynamically stable below a critical χ AC, above which LAM 3 emerges. Both experiments and theory expose close analogies to ABA triblock copolymer phase behavior, collectively suggesting that low-χ interactions between chemically similar or distinct blocks intimately influence self-assembly. Furthermore, these conclusions provide new opportunities in block polymer design with potential consequences spanning all self-assembling soft materials.« less

Combining Constitutively Active Rheb Expression and Chondroitinase Promotes Functional Axonal Regeneration after Cervical Spinal Cord Injury.

PubMed

Wu, Di; Klaw, Michelle C; Connors, Theresa; Kholodilov, Nikolai; Burke, Robert E; Côté, Marie-Pascale; Tom, Veronica J

2017-12-06

After spinal cord injury (SCI), severed axons in the adult mammalian CNS are unable to mount a robust regenerative response. In addition, the glial scar at the lesion site further restricts the regenerative potential of axons. We hypothesized that a combinatorial approach coincidentally targeting these obstacles would promote axonal regeneration. We combined (1) transplantation of a growth-permissive peripheral nerve graft (PNG) into an incomplete, cervical lesion cavity; (2) transduction of neurons rostral to the SCI site to express constitutively active Rheb (caRheb; a Ras homolog enriched in brain), a GTPase that directly activates the growth-promoting pathway mammalian target of rapamycin (mTOR) via AAV-caRheb injection; and (3) digestion of growth-inhibitory chondroitin sulfate proteoglycans within the glial scar at the distal PNG interface using the bacterial enzyme chondroitinase ABC (ChABC). We found that expressing caRheb in neurons post-SCI results in modestly yet significantly more axons regenerating across a ChABC-treated distal graft interface into caudal spinal cord than either treatment alone. Excitingly, we found that caRheb+ChABC treatment significantly potentiates the formation of synapses in the host spinal cord and improves the animals' ability to use the affected forelimb. Thus, this combination strategy enhances functional axonal regeneration following a cervical SCI. Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

Three copies of a single protein II-encoding sequence in the genome of Neisseria gonorrhoeae JS3: evidence for gene conversion and gene duplication.

PubMed

van der Ley, P

1988-11-01

Gonococci express a family of related outer membrane proteins designated protein II (P.II). These surface proteins are subject to both phase variation and antigenic variation. The P.II gene repertoire of Neisseria gonorrhoeae strain JS3 was found to consist of at least ten genes, eight of which were cloned. Sequence analysis and DNA hybridization studies revealed that one particular P.II-encoding sequence is present in three distinct, but almost identical, copies in the JS3 genome. These genes encode the P.II protein that was previously identified as P.IIc. Comparison of their sequences shows that the multiple copies of this P.IIc-encoding gene might have been generated by both gene conversion and gene duplication.

Analysis of the promoters involved in enterocin AS-48 expression.

PubMed

Cebrián, Rubén; Rodríguez-Ruano, Sonia; Martínez-Bueno, Manuel; Valdivia, Eva; Maqueda, Mercedes; Montalbán-López, Manuel

2014-01-01

The enterocin AS-48 is the best characterized antibacterial circular protein in prokaryotes. It is a hydrophobic and cationic bacteriocin, which is ribosomally synthesized by enterococcal cells and post-translationally cyclized by a head-to-tail peptide bond. The production of and immunity towards AS-48 depend upon the coordinated expression of ten genes organized in two operons, as-48ABC (where genes encoding enzymes with processing, secretion, and immunity functions are adjacent to the structural as-48A gene) and as-48C1DD1EFGH. The current study describes the identification of the promoters involved in AS-48 expression. Seven putative promoters have been here amplified, and separately inserted into the promoter-probe vector pTLR1, to create transcriptional fusions with the mCherry gene used as a reporter. The activity of these promoter regions was assessed measuring the expression of the fluorescent mCherry protein using the constitutive pneumococcal promoter PX as a reference. Our results revealed that only three promoters PA, P2(2) and PD1 were recognized in Enterococcus faecalis, Lactococcus lactis and Escherichia coli, in the conditions tested. The maximal fluorescence was obtained with PX in all the strains, followed by the P2(2) promoter, which level of fluorescence was 2-fold compared to PA and 4-fold compared to PD1. Analysis of putative factors influencing the promoter activity in single and double transformants in E. faecalis JH2-2 demonstrated that, in general, a better expression was achieved in presence of pAM401-81. In addition, the P2(2) promoter could be regulated in a negative fashion by genes existing in the native pMB-2 plasmid other than those of the as-48 cluster, while the pH seems to affect differently the as-48 promoter expression.

In silico Prediction, in vitro Antibacterial Spectrum, and Physicochemical Properties of a Putative Bacteriocin Produced by Lactobacillus rhamnosus Strain L156.4

PubMed Central

Oliveira, Letícia de C.; Silveira, Aline M. M.; Monteiro, Andréa de S.; dos Santos, Vera L.; Nicoli, Jacques R.; Azevedo, Vasco A. de C.; Soares, Siomar de C.; Dias-Souza, Marcus V.; Nardi, Regina M. D.

2017-01-01

A bacteriocinogenic Lactobacillus rhamnosus L156.4 strain isolated from the feces of NIH mice was identified by 16S rRNA gene sequencing and MALDI-TOF mass spectrometry. The entire genome was sequenced using Illumina, annotated in the PGAAP, and RAST servers, and deposited. Conserved genes associated with bacteriocin synthesis were predicted using BAGEL3, leading to the identification of an open reading frame (ORF) that shows homology with the L. rhamnosus GG (ATCC 53103) prebacteriocin gene. The encoded protein contains a conserved protein motif associated a structural gene of the Enterocin A superfamily. We found ORFs related to the prebacteriocin, immunity protein, ABC transporter proteins, and regulatory genes with 100% identity to those of L. rhamnosus HN001. In this study, we provide evidence of a putative bacteriocin produced by L. rhamnosus L156.4 that was further confirmed by in vitro assays. The antibacterial activity of the substances produced by this strain was evaluated using the deferred agar-spot and spot-on-the lawn assays, and a wide antimicrobial activity spectrum against human and foodborne pathogens was observed. The physicochemical characterization of the putative bacteriocin indicated that it was sensitive to proteolytic enzymes, heat stable and maintained its antibacterial activity in a pH ranging from 3 to 9. The activity against Lactobacillus fermentum, which was used as an indicator strain, was detected during bacterial logarithmic growth phase, and a positive correlation was confirmed between bacterial growth and production of the putative bacteriocin. After a partial purification from cell-free supernatant by salt precipitation, the putative bacteriocin migrated as a diffuse band of approximately 1.0–3.0 kDa by SDS-PAGE. Additional studies are being conducted to explore its use in the food industry for controlling bacterial growth and for probiotic applications. PMID:28579977

The Zur regulon of Corynebacterium glutamicum ATCC 13032

PubMed Central

2010-01-01

Background Zinc is considered as an essential element for all living organisms, but it can be toxic at large concentrations. Bacteria therefore tightly regulate zinc metabolism. The Cg2502 protein of Corynebacterium glutamicum was a candidate to control zinc metabolism in this species, since it was classified as metalloregulator of the zinc uptake regulator (Zur) subgroup of the ferric uptake regulator (Fur) family of DNA-binding transcription regulators. Results The cg2502 (zur) gene was deleted in the chromosome of C. glutamicum ATCC 13032 by an allelic exchange procedure to generate the zur-deficient mutant C. glutamicum JS2502. Whole-genome DNA microarray hybridizations and real-time RT-PCR assays comparing the gene expression in C. glutamicum JS2502 with that of the wild-type strain detected 18 genes with enhanced expression in the zur mutant. The expression data were combined with results from cross-genome comparisons of shared regulatory sites, revealing the presence of candidate Zur-binding sites in the mapped promoter regions of five transcription units encoding components of potential zinc ABC-type transporters (cg0041-cg0042/cg0043; cg2911-cg2912-cg2913), a putative secreted protein (cg0040), a putative oxidoreductase (cg0795), and a putative P-loop GTPase of the COG0523 protein family (cg0794). Enhanced transcript levels of the respective genes in C. glutamicum JS2502 were verified by real-time RT-PCR, and complementation of the mutant with a wild-type zur gene reversed the effect of differential gene expression. The zinc-dependent expression of the putative cg0042 and cg2911 operons was detected in vivo with a gfp reporter system. Moreover, the zinc-dependent binding of purified Zur protein to double-stranded 40-mer oligonucleotides containing candidate Zur-binding sites was demonstrated in vitro by DNA band shift assays. Conclusion Whole-genome expression profiling and DNA band shift assays demonstrated that Zur directly represses in a zinc-dependent manner the expression of nine genes organized in five transcription units. Accordingly, the Zur (Cg2502) protein is the key transcription regulator for genes involved in zinc homeostasis in C. glutamicum. PMID:20055984

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vaccaro, Brian J.; Thorgersen, Michael P.; Lancaster, W. Andrew

Enzymes of the denitrification pathway play an important role in the global nitrogen cycle, including release of nitrous oxide, an ozone-depleting greenhouse gas. In addition, nitric oxide reductase, maturation factors, and proteins associated with nitric oxide detoxification are used by pathogens to combat nitric oxide release by host immune systems. While the core reductases that catalyze the conversion of nitrate to dinitrogen are well understood at a mechanistic level, there are many peripheral proteins required for denitrification whose basic function is unclear. A bar-coded transposon DNA library fromPseudomonas stutzeristrain RCH2 was grown under denitrifying conditions, using nitrate or nitrite asmore » an electron acceptor, and also under molybdenum limitation conditions, with nitrate as the electron acceptor. Analysis of sequencing results from these growths yielded gene fitness data for 3,307 of the 4,265 protein-encoding genes present in strain RCH2. The insights presented here contribute to our understanding of how peripheral proteins contribute to a fully functioning denitrification pathway. We propose a new low-affinity molybdate transporter, OatABC, and show that differential regulation is observed for two MoaA homologs involved in molybdenum cofactor biosynthesis. We also propose that NnrS may function as a membrane-bound NO sensor. Finally, the dominant HemN paralog involved in heme biosynthesis is identified, and a CheR homolog is proposed to function in nitrate chemotaxis. In addition, new insights are provided into nitrite reductase redundancy, nitric oxide reductase maturation, nitrous oxide reductase maturation, and regulation.« less

Cellular engineering of Artemisia annua and Artemisia dubia with the rol ABC genes for enhanced production of potent anti-malarial drug artemisinin.

PubMed

Kiani, Bushra Hafeez; Suberu, John; Mirza, Bushra

2016-05-04

Malaria is causing more than half of a million deaths and 214 million clinical cases annually. Despite tremendous efforts for the control of malaria, the global morbidity and mortality have not been significantly changed in the last 50 years. Artemisinin, extracted from the medicinal plant Artemisia sp. is an effective anti-malarial drug. In 2015, elucidation of the effectiveness of artemisinin as a potent anti-malarial drug was acknowledged with a Nobel prize. Owing to the tight market and low yield of artemisinin, an economical way to increase its production is to increase its content in Artemisia sp. through different biotechnological approaches including genetic transformation. Artemisia annua and Artemisia dubia were transformed with rol ABC genes through Agrobacterium tumefacienes and Agrobacterium rhizogenes methods. The artemisinin content was analysed and compared between transformed and untransformed plants with the help of LC-MS/MS. Expression of key genes [Cytochrome P450 (CYP71AV1), aldehyde dehydrogenase 1 (ALDH1), amorpha-4, 11 diene synthase (ADS)] in the biosynthetic pathway of artemisinin and gene for trichome development and sesquiterpenoid biosynthetic (TFAR1) were measured using Quantitative real time PCR (qRT-PCR). Trichome density was analysed using confocal microscope. Artemisinin content was significantly increased in transformed material of both Artemisia species when compared to un-transformed plants. The artemisinin content within leaves of transformed lines was increased by a factor of nine, indicating that the plant is capable of synthesizing much higher amounts than has been achieved so far through traditional breeding. Expression of all artemisinin biosynthesis genes was significantly increased, although variation between the genes was observed. CYP71AV1 and ALDH1 expression levels were higher than that of ADS. Levels of the TFAR1 expression were also increased in all transgenic lines. Trichome density was also significantly increased in the leaves of transformed plants, but no trichomes were found in control roots or transformed roots. The detection of significantly raised levels of expression of the genes involved in artemisinin biosynthesis in transformed roots correlated with the production of significant amounts of artemisinin in these tissues. This suggests that synthesis is occurring in tissues other than the trichomes, which contradicts previous theories. Transformation of Artemisia sp. with rol ABC genes can lead to the increased production of artemisinin, which will help to meet the increasing demand of artemisinin because of its diverse pharmacological and anti-malarial importance.

Workflow to study genetic biodiversity of aflatoxigenic Aspergillus spp. in Georgia, USA

USDA-ARS?s Scientific Manuscript database

Peanut seeds were sampled from the entire state of Georgia in 2014. More than 600 isolates of Aspergillus spp. were collected using modified-dichloran rose Bengal (MDRB) medium, 240 of those isolates were fingerprinted with 25 InDel markers within the aflatoxin-biosynthesis gene cluster (ABC). Clust...

CRTP-13: ABC-GCB Expression Signatures in Human B-cell Lymphoma on the NanoString Platform | Frederick National Laboratory for Cancer Research

Cancer.gov

The CLIA Molecular Diagnostics Laboratory within the Cancer Research Technology Program will perform messenger RNA isolation and expression analysis specific to a 20-gene panel on formalin-fixed paraffin-embedded (FFPE) patient samples using the Nano

Prevention of egg contamination by Salmonella Enteritidis after oral vaccination of laying hens with Salmonella Enteritidis ΔtolC and ΔacrABacrEFmdtABC mutants.

PubMed

Kilroy, Sofie; Raspoet, Ruth; Haesebrouck, Freddy; Ducatelle, Richard; Van Immerseel, Filip

2016-08-12

Vaccination of laying hens has been successfully used to reduce egg contamination by Salmonella Enteritidis, decreasing human salmonellosis cases worldwide. Currently used vaccines for layers are either inactivated vaccines or live attenuated strains produced by mutagenesis. Targeted gene deletion mutants hold promise for future vaccines, because specific bacterial functions can be removed that may improve safety and allow differentiation from field strains. In this study, the efficacy of Salmonella Enteritidis ΔtolC and ΔacrABacrEFmdtABC strains in laying hens as live vaccines was evaluated. The mutants are deficient in either the membrane channel TolC (ΔtolC) or the multi-drug efflux systems acrAB, acrEF and mdtABC (ΔacrABacrEFmdtABC). These strains have a decreased ability for gut and tissue colonization and are unable to survive in egg white, the latter preventing transmission of the vaccine strains to humans. Two groups of 30 laying hens were orally inoculated at day 1, 6 weeks and 16 weeks of age with 10(8) cfu of either vaccine strain, while a third group was left unvaccinated. At 24 weeks of age, the birds were intravenously challenged with 5 × 10(7) cfu Salmonella Enteritidis PT4 S1400/94. The vaccine strains were not shed or detected in the gut, internal organs or eggs, 2 weeks after the third vaccination. The strains significantly protected against gut and internal organ colonization, and completely prevented egg contamination by Salmonella Enteritidis under the conditions of this study. This indicates that Salmonella Enteritidis ΔtolC and ΔacrABacrEFmdtABC strains might be valuable strains for vaccination of layers against Salmonella Enteritidis.

Understanding the Role of the Master Regulator XYR1 in Trichoderma reesei by Global Transcriptional Analysis

PubMed Central

dos Santos Castro, Lilian; de Paula, Renato G.; Antoniêto, Amanda C. C.; Persinoti, Gabriela F.; Silva-Rocha, Rafael; Silva, Roberto N.

2016-01-01

We defined the role of the transcriptional factor—XYR1—in the filamentous fungus Trichoderma reesei during cellulosic material degradation. In this regard, we performed a global transcriptome analysis using RNA-Seq of the Δxyr1 mutant strain of T. reesei compared with the parental strain QM9414 grown in the presence of cellulose, sophorose, and glucose as sole carbon sources. We found that 5885 genes were expressed differentially under the three tested carbon sources. Of these, 322 genes were upregulated in the presence of cellulose, while 367 and 188 were upregulated in sophorose and glucose, respectively. With respect to genes under the direct regulation of XYR1, 30 and 33 are exclusive to cellulose and sophorose, respectively. The most modulated genes in the Δxyr1 belong to Carbohydrate-Active Enzymes (CAZymes), transcription factors, and transporters families. Moreover, we highlight the downregulation of transporters belonging to the MFS and ABC transporter families. Of these, MFS members were mostly downregulated in the presence of cellulose. In sophorose and glucose, the expression of these transporters was mainly upregulated. Our results revealed that MFS and ABC transporters could be new players in cellulose degradation and their role was shown to be carbon source-dependent. Our findings contribute to a better understanding of the regulatory mechanisms of XYR1 to control cellulase gene expression in T. reesei in the presence of cellulosic material, thereby potentially enhancing its application in several biotechnology fields. PMID:26909077

Understanding the Role of the Master Regulator XYR1 in Trichoderma reesei by Global Transcriptional Analysis.

PubMed

Dos Santos Castro, Lilian; de Paula, Renato G; Antoniêto, Amanda C C; Persinoti, Gabriela F; Silva-Rocha, Rafael; Silva, Roberto N

2016-01-01

We defined the role of the transcriptional factor-XYR1-in the filamentous fungus Trichoderma reesei during cellulosic material degradation. In this regard, we performed a global transcriptome analysis using RNA-Seq of the Δxyr1 mutant strain of T. reesei compared with the parental strain QM9414 grown in the presence of cellulose, sophorose, and glucose as sole carbon sources. We found that 5885 genes were expressed differentially under the three tested carbon sources. Of these, 322 genes were upregulated in the presence of cellulose, while 367 and 188 were upregulated in sophorose and glucose, respectively. With respect to genes under the direct regulation of XYR1, 30 and 33 are exclusive to cellulose and sophorose, respectively. The most modulated genes in the Δxyr1 belong to Carbohydrate-Active Enzymes (CAZymes), transcription factors, and transporters families. Moreover, we highlight the downregulation of transporters belonging to the MFS and ABC transporter families. Of these, MFS members were mostly downregulated in the presence of cellulose. In sophorose and glucose, the expression of these transporters was mainly upregulated. Our results revealed that MFS and ABC transporters could be new players in cellulose degradation and their role was shown to be carbon source-dependent. Our findings contribute to a better understanding of the regulatory mechanisms of XYR1 to control cellulase gene expression in T. reesei in the presence of cellulosic material, thereby potentially enhancing its application in several biotechnology fields.

[Expression changes of major outer membrane protein antigens in Leptospira interrogans during infection and its mechanism].

PubMed

Zheng, Linli; Ge, Yumei; Hu, Weilin; Yan, Jie

2013-03-01

To determine expression changes of major outer membrane protein(OMP) antigens of Leptospira interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai during infection of human macrophages and its mechanism. OmpR encoding genes and OmpR-related histidine kinase (HK) encoding gene of L.interrogans strain Lai and their functional domains were predicted using bioinformatics technique. mRNA level changes of the leptospiral major OMP-encoding genes before and after infection of human THP-1 macrophages were detected by real-time fluorescence quantitative RT-PCR. Effects of the OmpR-encoding genes and HK-encoding gene on the expression of leptospiral OMPs during infection were determined by HK-peptide antiserum block assay and closantel inhibitive assays. The bioinformatics analysis indicated that LB015 and LB333 were referred to OmpR-encoding genes of the spirochete, while LB014 might act as a OmpR-related HK-encoding gene. After the spirochete infecting THP-1 cells, mRNA levels of leptospiral lipL21, lipL32 and lipL41 genes were rapidly and persistently down-regulated (P <0.01), whereas mRNA levels of leptospiral groEL, mce, loa22 and ligB genes were rapidly but transiently up-regulated (P<0.01). The treatment with closantel and HK-peptide antiserum partly reversed the infection-based down-regulated mRNA levels of lipL21 and lipL48 genes (P <0.01). Moreover, closantel caused a decrease of the infection-based up-regulated mRNA levels of groEL, mce, loa22 and ligB genes (P <0.01). Expression levels of L.interrogans strain Lai major OMP antigens present notable changes during infection of human macrophages. There is a group of OmpR-and HK-encoding genes which may play a major role in down-regulation of expression levels of partial OMP antigens during infection.

A new evolutionary variant of the streptogramin A resistance protein, Vga(A)LC, from Staphylococcus haemolyticus with shifted substrate specificity towards lincosamides.

PubMed

Novotna, G; Janata, J

2006-12-01

We found a new variant of the streptogramin A resistance gene, vga(A)LC, in clinical isolates of Staphylococcus haemolyticus resistant to lincomycin and clindamycin but susceptible to erythromycin and in which no relevant lincosamide resistance gene was detected. The gene vga(A)LC, differing from the gene vga(A) at the protein level by seven amino acid substitutions, was present exclusively in S. haemolyticus strains resistant to both lincosamides and streptogramin A (LS(A) phenotype). Antibiotic resistance profiles of the ATP-binding cassette (ABC) proteins Vga(A)(LC) and Vga(A) in the antibiotic-susceptible host S. aureus RN4220 were compared. It was shown that Vga(A)LC conferred resistance to both lincosamides and streptogramin A, while Vga(A) conferred significant resistance to streptogramin A only. Detailed analysis of the seven amino acid substitutions, distinguishing the two related ABC proteins with different substrate specificities, identified the substrate-recognizing site: four clustered substitutions (L212S, G219V, A220T, and G226S) in the spacer between the two ATP-binding cassettes altered the substrate specificity and constituted the lincosamide-streptogramin A resistance phenotype. A transport experiment with radiolabeled lincomycin demonstrated that the mechanism of lincosamide resistance in S. haemolyticus was identical to that of the reported macrolide-streptogramin B resistance conferred by Msr(A).

A New Evolutionary Variant of the Streptogramin A Resistance Protein, Vga(A)LC, from Staphylococcus haemolyticus with Shifted Substrate Specificity towards Lincosamides▿

PubMed Central

Novotna, G.; Janata, J.

2006-01-01

We found a new variant of the streptogramin A resistance gene, vga(A)LC, in clinical isolates of Staphylococcus haemolyticus resistant to lincomycin and clindamycin but susceptible to erythromycin and in which no relevant lincosamide resistance gene was detected. The gene vga(A)LC, differing from the gene vga(A) at the protein level by seven amino acid substitutions, was present exclusively in S. haemolyticus strains resistant to both lincosamides and streptogramin A (LSA phenotype). Antibiotic resistance profiles of the ATP-binding cassette (ABC) proteins Vga(A)LC and Vga(A) in the antibiotic-susceptible host S. aureus RN4220 were compared. It was shown that Vga(A)LC conferred resistance to both lincosamides and streptogramin A, while Vga(A) conferred significant resistance to streptogramin A only. Detailed analysis of the seven amino acid substitutions, distinguishing the two related ABC proteins with different substrate specificities, identified the substrate-recognizing site: four clustered substitutions (L212S, G219V, A220T, and G226S) in the spacer between the two ATP-binding cassettes altered the substrate specificity and constituted the lincosamide-streptogramin A resistance phenotype. A transport experiment with radiolabeled lincomycin demonstrated that the mechanism of lincosamide resistance in S. haemolyticus was identical to that of the reported macrolide-streptogramin B resistance conferred by Msr(A). PMID:17015629

Molecular and genetic characterization of the rhizopine catabolism (mocABRC) genes of Rhizobium meliloti L5-30.

PubMed

Rossbach, S; Kulpa, D A; Rossbach, U; de Bruijn, F J

1994-10-17

Rhizopine (L-3-O-methyl-scyllo-inosamine, 3-O-MSI) is a symbiosis-specific compound, which is synthesized in nitrogen-fixing nodules of Medicago sativa induced by Rhizobium meliloti strain L5-30. 3-O-MSI is thought to function as an unusual growth substrate for R. meliloti L5-30, which carries a locus (mos) responsible for its synthesis closely linked to a locus (moc) responsible for its degradation. Here, the essential moc genes were delimited by Tn5 mutagenesis and shown to be organized into two regions, separated by 3 kb of DNA. The DNA sequence of a 9-kb fragment spanning the two moc regions was determined, and four genes were identified that play an essential role in rhizopine catabolism (mocABC and mocR). The analysis of the DNA sequence and the amino acid sequence of the deduced protein products revealed that MocA resembles NADH-dependent dehydrogenases. MocB exhibits characteristic features of periplasmic-binding proteins that are components of high-affinity transport systems. MocC does not share significant homology with any protein in the database. MocR shows homology with the GntR class of bacterial regulator proteins. These results suggest that the mocABC genes are involved in the uptake and subsequent degradation of rhizopine, whereas mocR is likely to play a regulatory role.

Transcriptomic analysis of Arabidopsis developing stems: a close-up on cell wall genes

PubMed Central

Minic, Zoran; Jamet, Elisabeth; San-Clemente, Hélène; Pelletier, Sandra; Renou, Jean-Pierre; Rihouey, Christophe; Okinyo, Denis PO; Proux, Caroline; Lerouge, Patrice; Jouanin, Lise

2009-01-01

Background Different strategies (genetics, biochemistry, and proteomics) can be used to study proteins involved in cell biogenesis. The availability of the complete sequences of several plant genomes allowed the development of transcriptomic studies. Although the expression patterns of some Arabidopsis thaliana genes involved in cell wall biogenesis were identified at different physiological stages, detailed microarray analysis of plant cell wall genes has not been performed on any plant tissues. Using transcriptomic and bioinformatic tools, we studied the regulation of cell wall genes in Arabidopsis stems, i.e. genes encoding proteins involved in cell wall biogenesis and genes encoding secreted proteins. Results Transcriptomic analyses of stems were performed at three different developmental stages, i.e., young stems, intermediate stage, and mature stems. Many genes involved in the synthesis of cell wall components such as polysaccharides and monolignols were identified. A total of 345 genes encoding predicted secreted proteins with moderate or high level of transcripts were analyzed in details. The encoded proteins were distributed into 8 classes, based on the presence of predicted functional domains. Proteins acting on carbohydrates and proteins of unknown function constituted the two most abundant classes. Other proteins were proteases, oxido-reductases, proteins with interacting domains, proteins involved in signalling, and structural proteins. Particularly high levels of expression were established for genes encoding pectin methylesterases, germin-like proteins, arabinogalactan proteins, fasciclin-like arabinogalactan proteins, and structural proteins. Finally, the results of this transcriptomic analyses were compared with those obtained through a cell wall proteomic analysis from the same material. Only a small proportion of genes identified by previous proteomic analyses were identified by transcriptomics. Conversely, only a few proteins encoded by genes having moderate or high level of transcripts were identified by proteomics. Conclusion Analysis of the genes predicted to encode cell wall proteins revealed that about 345 genes had moderate or high levels of transcripts. Among them, we identified many new genes possibly involved in cell wall biogenesis. The discrepancies observed between results of this transcriptomic study and a previous proteomic study on the same material revealed post-transcriptional mechanisms of regulation of expression of genes encoding cell wall proteins. PMID:19149885

The short version of the Activities-specific Balance Confidence (ABC) scale: its validity, reliability, and relationship to balance impairment and falls in older adults.

PubMed

Schepens, Stacey; Goldberg, Allon; Wallace, Melissa

2010-01-01

A shortened version of the ABC 16-item scale (ABC-16), the ABC-6, has been proposed as an alternative balance confidence measure. We investigated whether the ABC-6 is a valid and reliable measure of balance confidence and examined its relationship to balance impairment and falls in older adults. Thirty-five community-dwelling older adults completed the ABC-16, including the 6 questions of the ABC-6. They also completed the following clinical balance tests: unipedal stance time (UST), functional reach (FR), Timed Up and Go (TUG), and maximum step length (MSL). Participants reported 12-month falls history. Balance confidence on the ABC-6 was significantly lower than on the ABC-16, however scores were highly correlated. Fallers reported lower balance confidence than non-fallers as measured by the ABC-6 scale, but confidence did not differ between the groups with the ABC-16. The ABC-6 significantly correlated with all balance tests assessed and number of falls. The ABC-16 significantly correlated with all balance tests assessed, but not with number of falls. Test-retest reliability for the ABC-16 and ABC-6 was good to excellent. The ABC-6 is a valid and reliable measure of balance confidence in community-dwelling older adults, and shows stronger relationships to falls than does the ABC-16. The ABC-6 may be a more useful balance confidence assessment tool than the ABC-16. Copyright 2009 Elsevier Ireland Ltd. All rights reserved.

Identification of ABC transporter genes conferring combined pleuromutilin-lincosamide-streptogramin A resistance in bovine methicillin-resistant Staphylococcus aureus and coagulase-negative staphylococci.

PubMed

Wendlandt, Sarah; Kadlec, Kristina; Feßler, Andrea T; Schwarz, Stefan

2015-06-12

The aim of this study was to investigate the genetic basis of combined pleuromutilin-lincosamide-streptogramin A resistance in 26 unrelated methicillin-resistant Staphylococcus aureus (MRSA) and coagulase-negative staphylococci (CoNS) from dairy cows suffering from mastitis. The 26 pleuromutilin-resistant staphylococcal isolates were screened for the presence of the genes vga(A), vga(B), vga(C), vga(E), vga(E) variant, sal(A), vmlR, cfr, lsa(A), lsa(B), lsa(C), and lsa(E) by PCR. None of the 26 isolates carried the genes vga(B), vga(C), vga(E), vga(E) variant, vmlR, cfr, lsa(A), lsa(B), or lsa(C). Two Staphylococcus haemolyticus and single Staphylococcus xylosus, Staphylococcus lentus, and Staphylococcus hominis were vga(A)-positive. Twelve S. aureus, two Staphylococcus warneri, as well as single S. lentus and S. xylosus carried the lsa(E) gene. Moreover, single S. aureus, S. haemolyticus, S. xylosus, and Staphylococcus epidermidis were positive for both genes, vga(A) and lsa(E). The sal(A) gene was found in a single Staphylococcus sciuri. All ABC transporter genes were located in the chromosomal DNA, except for a plasmid-borne vga(A) gene in the S. epidermidis isolate. The genetic environment of the lsa(E)-positive isolates was analyzed using previously described PCR assays. Except for the S. warneri and S. xylosus, all lsa(E)-positive isolates harbored a part of the previously described enterococcal multiresistance gene cluster. This is the first report of the novel lsa(E) gene in the aforementioned bovine CoNS species. This is also the first identification of the sal(A) gene in a S. sciuri from a case of bovine mastitis. Moreover, the sal(A) gene was shown to also confer pleuromutilin resistance. Copyright © 2015 Elsevier B.V. All rights reserved.

Diversity and role of plasmids in adaptation of bacteria inhabiting the Lubin copper mine in Poland, an environment rich in heavy metals.

PubMed

Dziewit, Lukasz; Pyzik, Adam; Szuplewska, Magdalena; Matlakowska, Renata; Mielnicki, Sebastian; Wibberg, Daniel; Schlüter, Andreas; Pühler, Alfred; Bartosik, Dariusz

2015-01-01

The Lubin underground mine, is one of three mining divisions in the Lubin-Glogow Copper District in Lower Silesia province (Poland). It is the source of polymetallic ore that is rich in copper, silver and several heavy metals. Black shale is also significantly enriched in fossil organic matter in the form of long-chain hydrocarbons, polycyclic aromatic hydrocarbons, organic acids, esters, thiophenes and metalloporphyrins. Biological analyses have revealed that this environment is inhabited by extremophilic bacteria and fungi. Kupfershiefer black shale and samples of water, bottom and mineral sediments from the underground (below 600 m) Lubin mine were taken and 20 bacterial strains were isolated and characterized. All exhibited multi-resistant and hypertolerant phenotypes to heavy metals. We analyzed the plasmidome of these strains in order to evaluate the diversity and role of mobile DNA in adaptation to the harsh conditions of the mine environment. Experimental and bioinformatic analyses of 11 extrachromosomal replicons were performed. Three plasmids, including a broad-host-range replicon containing a Tn3 family transposon, carried genes conferring resistance to arsenic, cadmium, cobalt, mercury and zinc. Functional analysis revealed that the resistance modules exhibit host specificity, i.e., they may increase or decrease tolerance to toxic ions depending on the host strain. The other identified replicons showed diverse features. Among them we identified a catabolic plasmid encoding enzymes involved in the utilization of histidine and vanillate, a putative plasmid-like prophage carrying genes responsible for NAD biosynthesis, and two repABC-type plasmids containing virulence-associated genes. These findings provide an unique molecular insight into the pool of extrachromosomal replicons and highlight their role in the biology and adaptation of extremophilic bacteria inhabiting terrestrial deep subsurface.

The SloR Metalloregulator is Involved in the Streptococcus mutans Oxidative Stress Response

PubMed Central

Crepps, Sarah C.; Fields, Emily E.; Galan, Diego; Corbett, John P.; Von Hasseln, Elizabeth R.; Spatafora, Grace A.

2015-01-01

SUMMARY A 25kDa SloR metalloregulatory protein in Streptococcus mutans modulates the expression of multiple genes, including the sloABC operon that encodes essential Mn2+ transport and genes that promote cariogenesis. In this study, we report on SloC- and SloR-deficient strains of S. mutans (GMS284 and GMS584, respectively) that demonstrate compromised survivorship compared to their UA159 wildtype progenitor and their complemented strains (GMS285 and GMS585, respectively), when challenged with streptonigrin and/or in growth competition experiments. The results of streptonigrin assays revealed significantly larger zones of inhibition for GMS584 than for either UA159 or GMS585, indicating weakened S. mutans survivorship in the absence of SloR. Competition assays revealed a compromised ability for GMS284 and GMS584 to survive peroxide challenge compared with their SloC- and SloR-proficient counterparts. These findings are consistent with a role for SloC and SloR in S. mutans aerotolerance. We also predicted differential expression of oxidative stress tolerance genes in GMS584 versus UA159 and GMS585 when grown aerobically. The results of qRT-PCR experiments revealed S. mutans sod, tpx, and sloC expression that was up-regulated in GMS584 compared to UA159 and GMS585, indicating that the impact of oxidative stress on S. mutans is more severe in the absence of SloR than in its presence. The results of electrophoretic mobility shift assays indicate that SloR does not bind to the sod or tpx promoter regions directly, implicating intermediaries that may arbitrate the SloR response to oxidative stress. PMID:26577188

The SloR metalloregulator is involved in the Streptococcus mutans oxidative stress response.

PubMed

Crepps, S C; Fields, E E; Galan, D; Corbett, J P; Von Hasseln, E R; Spatafora, G A

2016-12-01

SloR, a 25-kDa metalloregulatory protein in Streptococcus mutans modulates the expression of multiple genes, including the sloABC operon that encodes essential Mn 2+ transport and genes that promote cariogenesis. In this study, we report on SloC- and SloR-deficient strains of S. mutans (GMS284 and GMS584, respectively) that demonstrate compromised survivorship compared with their UA159 wild-type progenitor and their complemented strains (GMS285 and GMS585, respectively), when challenged with streptonigrin and/or in growth competition experiments. The results of streptonigrin assays revealed significantly larger zones of inhibition for GMS584 than for either UA159 or GMS585, indicating weakened S. mutans survivorship in the absence of SloR. Competition assays revealed a compromised ability for GMS284 and GMS584 to survive peroxide challenge compared with their SloC- and SloR-proficient counterparts. These findings are consistent with a role for SloC and SloR in S. mutans aerotolerance. We also predicted differential expression of oxidative stress tolerance genes in GMS584 versus UA159 and GMS585 when grown aerobically. The results of quantitative RT-PCR experiments revealed S. mutans sod, tpx, and sloC expression that was upregulated in GMS584 compared with UA159 and GMS585, indicating that the impact of oxidative stress on S. mutans is more severe in the absence of SloR than in its presence. The results of electrophoretic mobility shift assays indicate that SloR does not bind to the sod or tpx promoter regions directly, implicating intermediaries that may arbitrate the SloR response to oxidative stress. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Trehalose/2-sulfotrehalose biosynthesis and glycine-betaine uptake are widely spread mechanisms for osmoadaptation in the Halobacteriales

PubMed Central

Youssef, Noha H; Savage-Ashlock, Kristen N; McCully, Alexandra L; Luedtke, Brandon; Shaw, Edward I; Hoff, Wouter D; Elshahed, Mostafa S

2014-01-01

We investigated the mechanisms of osmoadaptation in the order Halobacteriales, with special emphasis on Haladaptatus paucihalophilus, known for its ability to survive in low salinities. H. paucihalophilus genome contained genes for trehalose synthesis (trehalose-6-phosphate synthase/trehalose-6-phosphatase (OtsAB pathway) and trehalose glycosyl-transferring synthase pathway), as well as for glycine betaine uptake (BCCT family of secondary transporters and QAT family of ABC transporters). H. paucihalophilus cells synthesized and accumulated ∼1.97–3.72 μmol per mg protein of trehalose in a defined medium, with its levels decreasing with increasing salinities. When exogenously supplied, glycine betaine accumulated intracellularly with its levels increasing at higher salinities. RT-PCR analysis strongly suggested that H. paucihalophilus utilizes the OtsAB pathway for trehalose synthesis. Out of 83 Halobacteriales genomes publicly available, genes encoding the OtsAB pathway and glycine betaine BCCT family transporters were identified in 38 and 60 genomes, respectively. Trehalose (or its sulfonated derivative) production and glycine betaine uptake, or lack thereof, were experimentally verified in 17 different Halobacteriales species. Phylogenetic analysis suggested that trehalose synthesis is an ancestral trait within the Halobacteriales, with its absence in specific lineages reflecting the occurrence of gene loss events during Halobacteriales evolution. Analysis of multiple culture-independent survey data sets demonstrated the preference of trehalose-producing genera to saline and low salinity habitats, and the dominance of genera lacking trehalose production capabilities in permanently hypersaline habitats. This study demonstrates that, contrary to current assumptions, compatible solutes production and uptake represent a common mechanism of osmoadaptation within the Halobacteriales. PMID:24048226

Cellular Response of Shewanella oneidensis to Strontium Stress†

PubMed Central

Brown, Steven D.; Martin, Madhavi; Deshpande, Sameer; Seal, Sudipta; Huang, Katherine; Alm, Eric; Yang, Yunfeng; Wu, Liyou; Yan, Tingfen; Liu, Xueduan; Arkin, Adam; Chourey, Karuna; Zhou, Jizhong; Thompson, Dorothea K.

2006-01-01

The physiology and transcriptome dynamics of the metal ion-reducing bacterium Shewanella oneidensis strain MR-1 in response to nonradioactive strontium (Sr) exposure were investigated. Studies indicated that MR-1 was able to grow aerobically in complex medium in the presence of 180 mM SrCl2 but showed severe growth inhibition at levels above that concentration. Temporal gene expression profiles were generated from aerobically grown, mid-exponential-phase MR-1 cells shocked with 180 mM SrCl2 and analyzed for significant differences in mRNA abundance with reference to data for nonstressed MR-1 cells. Genes with annotated functions in siderophore biosynthesis and iron transport were among the most highly induced (>100-fold [P < 0.05]) open reading frames in response to acute Sr stress, and a mutant (SO3032::pKNOCK) defective in siderophore production was found to be hypersensitive to SrCl2 exposure, compared to parental and wild-type strains. Transcripts encoding multidrug and heavy metal efflux pumps, proteins involved in osmotic adaptation, sulfate ABC transporters, and assimilative sulfur metabolism enzymes also were differentially expressed following Sr exposure but at levels that were several orders of magnitude lower than those for iron transport genes. Precipitate formation was observed during aerobic growth of MR-1 in broth cultures amended with 50, 100, or 150 mM SrCl2 but not in cultures of the SO3032::pKNOCK mutant or in the abiotic control. Chemical analysis of this precipitate using laser-induced breakdown spectroscopy and static secondary ion mass spectrometry indicated extracellular solid-phase sequestration of Sr, with at least a portion of the heavy metal associated with carbonate phases. PMID:16391131

Differentially expressed microRNAs associated with changes of transcript levels in detoxification pathways and DDT-resistance in the Drosophila melanogaster strain 91-R.

PubMed

Seong, Keon Mook; Coates, Brad S; Kim, Do-Hyup; Hansen, Allison K; Pittendrigh, Barry R

2018-01-01

Dichloro-diphenyl-trichloroethane (DDT) resistance among arthropod species is a model for understanding the molecular adaptations in response to insecticide exposures. Previous studies reported that DDT resistance may involve one or multiple detoxification genes, such as cytochrome P450 monooxygenases (P450s), glutathione S-transferases (GSTs), esterases, and ATP binding cassette (ABC) transporters, or changes in the voltage-sensitive sodium channel. However, the possible involvement of microRNAs (miRNAs) in the post-transcriptional regulation of genes associated with DDT resistance in the Drosophila melanogaster strain 91-R remains poorly understood. In this study, the majority of the resulting miRNAs discovered in small RNA libraries from 91-R and the susceptible control strain, 91-C, ranged from 16-25 nt, and contained 163 precursors and 256 mature forms of previously-known miRNAs along with 17 putative novel miRNAs. Quantitative analyses predicted the differential expression of ten miRNAs between 91-R and 91-C, and, based on Gene Ontology and pathway analysis, these ten miRNAs putatively target transcripts encoding proteins involved in detoxification mechanisms. RT-qPCR validated an inverse correlation between levels of differentially-expressed miRNAs and their putatively targeted transcripts, which implies a role of these miRNAs in the differential regulation of detoxification pathways in 91-R compared to 91-C. This study provides evidence associating the differential expression of miRNAs in response to multigenerational DDT selection in Drosophila melanogaster and provides important clues for understanding the possible roles of miRNAs in mediating insecticide resistance traits.

Trichoderma genes

DOEpatents

Foreman, Pamela [Los Altos, CA; Goedegebuur, Frits [Vlaardingen, NL; Van Solingen, Pieter [Naaldwijk, NL; Ward, Michael [San Francisco, CA

2012-06-19

Described herein are novel gene sequences isolated from Trichoderma reesei. Two genes encoding proteins comprising a cellulose binding domain, one encoding an arabionfuranosidase and one encoding an acetylxylanesterase are described. The sequences, CIP1 and CIP2, contain a cellulose binding domain. These proteins are especially useful in the textile and detergent industry and in pulp and paper industry.

The rice blast resistance gene Ptr encodes an atypical protein required for broad spectrum disease resistance

USDA-ARS?s Scientific Manuscript database

Plant resistance (R) genes typically encode proteins with nucleotide binding site-leucine rich repeat (NLR) domains. We identified a novel, broad-spectrum rice blast R gene, Ptr, encoding a non-NLR protein with four Armadillo repeats. Ptr was originally identified by fast neutron mutagenesis as a ...

Selenium Pretreatment Alleviated LPS-Induced Immunological Stress Via Upregulation of Several Selenoprotein Encoding Genes in Murine RAW264.7 Cells.

PubMed

Wang, Longqiong; Jing, Jinzhong; Yan, Hui; Tang, Jiayong; Jia, Gang; Liu, Guangmang; Chen, Xiaoling; Tian, Gang; Cai, Jingyi; Shang, Haiying; Zhao, Hua

2018-04-18

This study was conducted to profile selenoprotein encoding genes in mouse RAW264.7 cells upon lipopolysaccharide (LPS) challenge and integrate their roles into immunological regulation in response to selenium (Se) pretreatment. LPS was used to develop immunological stress in macrophages. Cells were pretreated with different levels of Se (0, 0.5, 1.0, 1.5, 2.0 μmol Se/L) for 2 h, followed by LPS (100 ng/mL) stimulation for another 3 h. The mRNA expression of 24 selenoprotein encoding genes and 9 inflammation-related genes were investigated. The results showed that LPS (100 ng/mL) effectively induced immunological stress in RAW264.7 cells with induced inflammation cytokines, IL-6 and TNF-α, mRNA expression, and cellular secretion. LPS increased (P < 0.05) mRNA profiles of 9 inflammation-related genes in cells, while short-time Se pretreatment modestly reversed (P < 0.05) the LPS-induced upregulation of 7 genes (COX-2, ICAM-1, IL-1β, IL-6, IL-10, iNOS, and MCP-1) and further increased (P < 0.05) expression of IFN-β and TNF-α in stressed cells. Meanwhile, LPS decreased (P < 0.05) mRNA levels of 18 selenoprotein encoding genes and upregulated mRNA levels of TXNRD1 and TXNRD3 in cells. Se pretreatment recovered (P < 0.05) expression of 3 selenoprotein encoding genes (GPX1, SELENOH, and SELENOW) in a dose-dependent manner and increased (P < 0.05) expression of another 5 selenoprotein encoding genes (SELENOK, SELENOM, SELENOS, SELENOT, and TXNRD2) only at a high level (2.0 μmol Se/L). Taken together, LPS-induced immunological stress in RAW264.7 cells accompanied with the global downregulation of selenoprotein encoding genes and Se pretreatment alleviated immunological stress via upregulation of a subset of selenoprotein encoding genes.

Disruption of the psbA gene by the copy correction mechanism reveals that the expression of plastid-encoded genes is regulated by photosynthesis activity.

PubMed

Khan, Muhammad Sarwar; Hameed, Waqar; Nozoe, Mikio; Shiina, Takashi

2007-05-01

The functional analysis of genes encoded by the chloroplast genome of tobacco by reverse genetics is routine. Nevertheless, for a small number of genes their deletion generates heteroplasmic genotypes, complicating their analysis. There is thus the need for additional strategies to develop deletion mutants for these genes. We have developed a homologous copy correction-based strategy for deleting/mutating genes encoded on the chloroplast genome. This system was used to produce psbA knockouts. The resulting plants are homoplasmic and lack photosystem II (PSII) activity. Further, the deletion mutants exhibit a distinct phenotype; young leaves are green, whereas older leaves are bleached, irrespective of light conditions. This suggests that senescence is promoted by the absence of psbA. Analysis of the transcript levels indicates that NEP (nuclear-encoded plastid RNA polymerase)-dependent plastid genes are up regulated in the psbA deletion mutants, whereas the bleached leaves retain plastid-encoded plastid RNA polymerase activity. Hence, the expression of NEP-dependent plastid genes may be regulated by photosynthesis, either directly or indirectly.

GeneFisher-P: variations of GeneFisher as processes in Bio-jETI

PubMed Central

Lamprecht, Anna-Lena; Margaria, Tiziana; Steffen, Bernhard; Sczyrba, Alexander; Hartmeier, Sven; Giegerich, Robert

2008-01-01

Background PCR primer design is an everyday, but not trivial task requiring state-of-the-art software. We describe the popular tool GeneFisher and explain its recent restructuring using workflow techniques. We apply a service-oriented approach to model and implement GeneFisher-P, a process-based version of the GeneFisher web application, as a part of the Bio-jETI platform for service modeling and execution. We show how to introduce a flexible process layer to meet the growing demand for improved user-friendliness and flexibility. Results Within Bio-jETI, we model the process using the jABC framework, a mature model-driven, service-oriented process definition platform. We encapsulate remote legacy tools and integrate web services using jETI, an extension of the jABC for seamless integration of remote resources as basic services, ready to be used in the process. Some of the basic services used by GeneFisher are in fact already provided as individual web services at BiBiServ and can be directly accessed. Others are legacy programs, and are made available to Bio-jETI via the jETI technology. The full power of service-based process orientation is required when more bioinformatics tools, available as web services or via jETI, lead to easy extensions or variations of the basic process. This concerns for instance variations of data retrieval or alignment tools as provided by the European Bioinformatics Institute (EBI). Conclusions The resulting service- and process-oriented GeneFisher-P demonstrates how basic services from heterogeneous sources can be easily orchestrated in the Bio-jETI platform and lead to a flexible family of specialized processes tailored to specific tasks. PMID:18460174

Association of HS6ST3 gene polymorphisms with obesity and triglycerides: gene x gender interaction.

PubMed

Wang, Ke-Sheng; Wang, Liang; Liu, Xuefeng; Zeng, Min

2013-12-01

The heparan sulfate 6-O-sulfotransferase 3 (HS6ST3) gene is involved in heparan sulphate and heparin metabolism, and has been reported to be associated with diabetic retinopathy in type 2 diabetes.We hypothesized that HS6ST3 gene polymorphisms might play an important role in obesity and related phenotypes (such as triglycerides). We examined genetic associations of 117 single-nucleotide polymorphisms (SNPs) within the HS6ST3 gene with obesity and triglycerides using two Caucasian samples: the Marshfield sample (1442 obesity cases and 2122 controls), and the Health aging and body composition (Health ABC) sample (305 cases and 1336 controls). Logistic regression analysis of obesity as a binary trait and linear regression analysis of triglycerides as a continuous trait, adjusted for age and sex, were performed using PLINK. Single marker analysis showed that six SNPs in the Marshfield sample and one SNP in the Health ABC sample were associated with obesity (P < 0.05). SNP rs535812 revealed a stronger association with obesity in meta-analysis of these two samples (P = 0.0105). The T-A haplotype from rs878950 and rs9525149 revealed significant association with obesity in the Marshfield sample (P = 0.012). Moreover, nine SNPs showed associations with triglycerides in the Marshfield sample (P < 0.05) and the best signal was rs1927796 (P = 0.00858). In addition, rs7331762 showed a strong gene x gender interaction (P = 0.00956) for obesity while rs1927796 showed a strong gene x gender interaction (P = 0.000625) for triglycerides in the Marshfield sample. These findings contribute new insights into the pathogenesis of obesity and triglycerides and demonstrate the importance of gender differences in the aetiology.

Genome-Wide Identification and Mapping of NBS-Encoding Resistance Genes in Solanum tuberosum Group Phureja

PubMed Central

Lozano, Roberto; Ponce, Olga; Ramirez, Manuel; Mostajo, Nelly; Orjeda, Gisella

2012-01-01

The majority of disease resistance (R) genes identified to date in plants encode a nucleotide-binding site (NBS) and leucine-rich repeat (LRR) domain containing protein. Additional domains such as coiled-coil (CC) and TOLL/interleukin-1 receptor (TIR) domains can also be present. In the recently sequenced Solanum tuberosum group phureja genome we used HMM models and manual curation to annotate 435 NBS-encoding R gene homologs and 142 NBS-derived genes that lack the NBS domain. Highly similar homologs for most previously documented Solanaceae R genes were identified. A surprising ∼41% (179) of the 435 NBS-encoding genes are pseudogenes primarily caused by premature stop codons or frameshift mutations. Alignment of 81.80% of the 577 homologs to S. tuberosum group phureja pseudomolecules revealed non-random distribution of the R-genes; 362 of 470 genes were found in high density clusters on 11 chromosomes. PMID:22493716

Spontaneous and evolutionary changes in the antibiotic resistance of Burkholderia cenocepacia observed by global gene expression analysis.

PubMed

Sass, Andrea; Marchbank, Angela; Tullis, Elizabeth; Lipuma, John J; Mahenthiralingam, Eshwar

2011-07-22

Burkholderia cenocepacia is a member of the Burkholderia cepacia complex group of bacteria that cause infections in individuals with cystic fibrosis. B. cenocepacia isolate J2315 has been genome sequenced and is representative of a virulent, epidemic CF strain (ET12). Its genome encodes multiple antimicrobial resistance pathways and it is not known which of these is important for intrinsic or spontaneous resistance. To map these pathways, transcriptomic analysis was performed on: (i) strain J2315 exposed to sub-inhibitory concentrations of antibiotics and the antibiotic potentiator chlorpromazine, and (ii) on spontaneous mutants derived from J2315 and with increased resistance to the antibiotics amikacin, meropenem and trimethoprim-sulfamethoxazole. Two pan-resistant ET12 outbreak isolates recovered two decades after J2315 were also compared to identify naturally evolved gene expression changes. Spontaneous resistance in B. cenocepacia involved more gene expression changes and different subsets of genes than those provoked by exposure to sub inhibitory concentrations of each antibiotic. The phenotype and altered gene expression in the resistant mutants was also stable irrespective of the presence of the priming antibiotic. Both known and novel genes involved in efflux, antibiotic degradation/modification, membrane function, regulation and unknown functions were mapped. A novel role for the phenylacetic acid (PA) degradation pathway genes was identified in relation to spontaneous resistance to meropenem and glucose was found to repress their expression. Subsequently, 20 mM glucose was found to produce greater that 2-fold reductions in the MIC of multiple antibiotics against B. cenocepacia J2315. Mutation of an RND multidrug efflux pump locus (BCAM0925-27) and squalene-hopene cyclase gene (BCAS0167), both upregulated after chlorpromazine exposure, confirmed their role in resistance. The recently isolated outbreak isolates had altered the expression of multiple genes which mirrored changes seen in the antibiotic resistant mutants, corroborating the strategy used to model resistance. Mutation of an ABC transporter gene (BCAS0081) upregulated in both outbreak strains, confirmed its role in B. cenocepacia resistance. Global mapping of the genetic pathways which mediate antibiotic resistance in B. cenocepacia has revealed that they are multifactorial, identified potential therapeutic targets and also demonstrated that putative catabolite repression of genes by glucose can improve antibiotic efficacy.

Jasmonate and ethylene dependent defence gene expression and suppression of fungal virulence factors: two essential mechanisms of Fusarium head blight resistance in wheat?

PubMed Central

2012-01-01

Background Fusarium head blight (FHB) caused by Fusarium species like F. graminearum is a devastating disease of wheat (Triticum aestivum) worldwide. Mycotoxins such as deoxynivalenol produced by the fungus affect plant and animal health, and cause significant reductions of grain yield and quality. Resistant varieties are the only effective way to control this disease, but the molecular events leading to FHB resistance are still poorly understood. Transcriptional profiling was conducted for the winter wheat cultivars Dream (moderately resistant) and Lynx (susceptible). The gene expressions at 32 and 72 h after inoculation with Fusarium were used to trace possible defence mechanisms and associated genes. A comparative qPCR was carried out for selected genes to analyse the respective expression patterns in the resistant cultivars Dream and Sumai 3 (Chinese spring wheat). Results Among 2,169 differentially expressed genes, two putative main defence mechanisms were found in the FHB-resistant Dream cultivar. Both are defined base on their specific mode of resistance. A non-specific mechanism was based on several defence genes probably induced by jasmonate and ethylene signalling, including lipid-transfer protein, thionin, defensin and GDSL-like lipase genes. Additionally, defence-related genes encoding jasmonate-regulated proteins were up-regulated in response to FHB. Another mechanism based on the targeted suppression of essential Fusarium virulence factors comprising proteases and mycotoxins was found to be an essential, induced defence of general relevance in wheat. Moreover, similar inductions upon fungal infection were frequently observed among FHB-responsive genes of both mechanisms in the cultivars Dream and Sumai 3. Conclusions Especially ABC transporter, UDP-glucosyltransferase, protease and protease inhibitor genes associated with the defence mechanism against fungal virulence factors are apparently active in different resistant genetic backgrounds, according to reports on other wheat cultivars and barley. This was further supported in our qPCR experiments on seven genes originating from this mechanism which revealed similar activities in the resistant cultivars Dream and Sumai 3. Finally, the combination of early-stage and steady-state induction was associated with resistance, while transcript induction generally occurred later and temporarily in the susceptible cultivars. The respective mechanisms are attractive for advanced studies aiming at new resistance and toxin management strategies. PMID:22857656

Molecular characteristics of Multidrug Resistant Acinetobacter baumannii Isolates from US soldiers from Iraq at the National Naval Medical Center

USDA-ARS?s Scientific Manuscript database

Background: Infections with A. baumannii-calcoaceticus complex (ABC) have complicated the care of combat casualties. The majority of A. baumannii isolates cultured from injured personnel from OIF and OEF have been multi drug resistant (MDR). Therefore, the genes causing MDR and genotypes related to ...

CGI-58, a key regulator of lipid homeostasis and signaling in plants, also regulates polyamine metabolism

USDA-ARS?s Scientific Manuscript database

Comparative Gene Identification-58 (CGI-58) is an alpha/beta hydrolase-type protein that regulates lipid homeostasis and signaling in eukaryotes by interacting with and stimulating the activity of several different types of proteins, including a lipase in mammalian cells and a peroxisomal ABC transp...

Genetic polymorphisms of ATP-binding cassette (ABC) proteins, overall survival and drug toxicity in patients with Acute Myeloid Leukemia

PubMed Central

Hampras, Shalaka S; Sucheston, Lara; Weiss, Joli; Baer, Maria R; Zirpoli, Gary; Singh, Prashant K; Wetzler, Meir; Chennamaneni, Raj; Blanco, Javier G; Ford, LaurieAnn; Moysich, Kirsten B

2010-01-01

The overall survival of patients with acute myeloid leukemia (AML) remains poor due to both intrinsic and acquired chemotherapy resistance. Over expression of ATP binding cassette (ABC) proteins in AML cells has been suggested as a putative mechanism of drug resistance. Genetic variation among individuals affecting the expression or function of these proteins may contribute to inter-individual variation in treatment outcomes. DNA from pre-treatment bone marrow or blood samples from 261 patients age 20-85 years, who received cytarabine and anthracycline-based therapy at Roswell Park Cancer Institute between 1994 and 2006, was genotyped for eight non-synonymous single nucleotide polymorphisms in the ABCB1, ABCC1 and ABCG2 drug transporter genes. Heterozygous (AG) or homozygous (AA) variant genotypes for rs2231137 (G34A) in the ABCG2 (BRCP) gene, compared to the wild type (GG) genotype were associated with both significantly improved survival (HR=0.44, 95%CI=0.25-0.79), and increased odds for toxicity (OR=8.41, 95%CI= 1.10-64.28). Thus genetic polymorphisms in the ABCG2 (BRCP) gene may contribute to differential survival outcomes and toxicities in AML patients via a mechanism of decreased drug efflux in both, AML cells and normal progenitors. PMID:21311724

Transformation of Lettuce with rol ABC Genes: Extracts Show Enhanced Antioxidant, Analgesic, Anti-Inflammatory, Antidepressant, and Anticoagulant Activities in Rats.

PubMed

Ismail, Hammad; Dilshad, Erum; Waheed, Mohammad Tahir; Mirza, Bushra

2017-03-01

Lettuce is an edible crop that is well known for dietary and antioxidant benefits. The present study was conducted to investigate the effects of rol ABC genes on antioxidant and medicinal potential of lettuce by Agrobacterium-mediated transformation. Transgene integration and expression was confirmed through PCR and real-time RT-PCR, respectively. The transformed plants showed 91-102 % increase in total phenolic contents and 53-65 % increase in total flavonoid contents compared to untransformed plants. Total antioxidant capacity and total reducing power increased up to 112 and 133 % in transformed plants, respectively. Results of DPPH assay showed maximum 51 % increase, and lipid peroxidation assay exhibited 20 % increase in antioxidant activity of transformed plants compared to controls. Different in vivo assays were carried out in rats. The transgenic plants showed up to 80 % inhibition in both hot plate analgesic assay and carrageenan-induced hind paw edema test, while untransformed plants showed only 45 % inhibition. Antidepressant and anticoagulant potential of transformed plants was also significantly enhanced compared to untransformed plants. Taken together, the present work highlights the use of rol genes to enhance the secondary metabolite production in lettuce and improve its analgesic, anti-inflammatory, antidepressant, and anticoagulatory properties.

Combination of Ibrutinib and ABT-199 in Diffuse Large B-Cell Lymphoma and Follicular Lymphoma.

PubMed

Kuo, Hsu-Ping; Ezell, Scott A; Schweighofer, Karl J; Cheung, Leo W K; Hsieh, Sidney; Apatira, Mutiah; Sirisawad, Mint; Eckert, Karl; Hsu, Ssucheng J; Chen, Chun-Te; Beaupre, Darrin M; Versele, Matthias; Chang, Betty Y

2017-07-01

Diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma are the most prevalent B-lymphocyte neoplasms in which abnormal activation of the Bruton tyrosine kinase (BTK)-mediated B-cell receptor signaling pathway contributes to pathogenesis. Ibrutinib is an oral covalent BTK inhibitor that has shown some efficacy in both indications. To improve ibrutinib efficacy through combination therapy, we first investigated differential gene expression in parental and ibrutinib-resistant cell lines to better understand the mechanisms of resistance. Ibrutinib-resistant TMD8 cells had higher BCL2 gene expression and increased sensitivity to ABT-199, a BCL-2 inhibitor. Consistently, clinical samples from ABC-DLBCL patients who experienced poorer response to ibrutinib had higher BCL2 gene expression. We further demonstrated synergistic growth suppression by ibrutinib and ABT-199 in multiple ABC-DLBCL, GCB-DLBCL, and follicular lymphoma cell lines. The combination of both drugs also reduced colony formation, increased apoptosis, and inhibited tumor growth in a TMD8 xenograft model. A synergistic combination effect was also found in ibrutinib-resistant cells generated by either genetic mutation or drug treatment. Together, these findings suggest a potential clinical benefit from ibrutinib and ABT-199 combination therapy. Mol Cancer Ther; 16(7); 1246-56. ©2017 AACR . ©2017 American Association for Cancer Research.

The candidate histocompatibility locus of a Basal chordate encodes two highly polymorphic proteins.

PubMed

Nydam, Marie L; Netuschil, Nikolai; Sanders, Erin; Langenbacher, Adam; Lewis, Daniel D; Taketa, Daryl A; Marimuthu, Arumugapradeep; Gracey, Andrew Y; De Tomaso, Anthony W

2013-01-01

The basal chordate Botryllus schlosseri undergoes a natural transplantation reaction governed by a single, highly polymorphic locus called the fuhc. Our initial characterization of this locus suggested it encoded a single gene alternatively spliced into two transcripts: a 555 amino acid-secreted form containing the first half of the gene, and a full-length, 1008 amino acid transmembrane form, with polymorphisms throughout the ectodomain determining outcome. We have now found that the locus encodes two highly polymorphic genes which are separated by a 227 bp intergenic region: first, the secreted form as previously described, and a second gene encoding a 531 amino acid membrane-bound gene containing three extracellular immunoglobulin domains. While northern blotting revealed only these two mRNAs, both PCR and mRNA-seq detect a single capped and polyadenylated transcript that encodes processed forms of both genes linked by the intergenic region, as well as other transcripts in which exons of the two genes are spliced together. These results might suggest that the two genes are expressed as an operon, during which both genes are co-transcribed and then trans-spliced into two separate messages. This type of transcriptional regulation has been described in tunicates previously; however, the membrane-bound gene does not encode a typical Splice Leader (SL) sequence at the 5' terminus that usually accompanies trans-splicing. Thus, the presence of stable transcripts encoding both genes may suggest a novel mechanism of regulation, or conversely may be rare but stable transcripts in which the two mRNAs are linked due to a small amount of read-through by RNA polymerase. Both genes are highly polymorphic and co-expressed on tissues involved in histocompatibility. In addition, polymorphisms on both genes correlate with outcome, although we have found a case in which it appears that the secreted form may be major allorecognition determinant.

Development of a gene synthesis platform for the efficient large scale production of small genes encoding animal toxins.

PubMed

Sequeira, Ana Filipa; Brás, Joana L A; Guerreiro, Catarina I P D; Vincentelli, Renaud; Fontes, Carlos M G A

2016-12-01

Gene synthesis is becoming an important tool in many fields of recombinant DNA technology, including recombinant protein production. De novo gene synthesis is quickly replacing the classical cloning and mutagenesis procedures and allows generating nucleic acids for which no template is available. In addition, when coupled with efficient gene design algorithms that optimize codon usage, it leads to high levels of recombinant protein expression. Here, we describe the development of an optimized gene synthesis platform that was applied to the large scale production of small genes encoding venom peptides. This improved gene synthesis method uses a PCR-based protocol to assemble synthetic DNA from pools of overlapping oligonucleotides and was developed to synthesise multiples genes simultaneously. This technology incorporates an accurate, automated and cost effective ligation independent cloning step to directly integrate the synthetic genes into an effective Escherichia coli expression vector. The robustness of this technology to generate large libraries of dozens to thousands of synthetic nucleic acids was demonstrated through the parallel and simultaneous synthesis of 96 genes encoding animal toxins. An automated platform was developed for the large-scale synthesis of small genes encoding eukaryotic toxins. Large scale recombinant expression of synthetic genes encoding eukaryotic toxins will allow exploring the extraordinary potency and pharmacological diversity of animal venoms, an increasingly valuable but unexplored source of lead molecules for drug discovery.

Comprehensive search for accessory proteins encoded with archaeal and bacterial type III CRISPR-cas gene cassettes reveals 39 new cas gene families.

PubMed

Shah, Shiraz A; Alkhnbashi, Omer S; Behler, Juliane; Han, Wenyuan; She, Qunxin; Hess, Wolfgang R; Garrett, Roger A; Backofen, Rolf

2018-06-19

A study was undertaken to identify conserved proteins that are encoded adjacent to cas gene cassettes of Type III CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats - CRISPR associated) interference modules. Type III modules have been shown to target and degrade dsDNA, ssDNA and ssRNA and are frequently intertwined with cofunctional accessory genes, including genes encoding CRISPR-associated Rossman Fold (CARF) domains. Using a comparative genomics approach, and defining a Type III association score accounting for coevolution and specificity of flanking genes, we identified and classified 39 new Type III associated gene families. Most archaeal and bacterial Type III modules were seen to be flanked by several accessory genes, around half of which did not encode CARF domains and remain of unknown function. Northern blotting and interference assays in Synechocystis confirmed that one particular non-CARF accessory protein family was involved in crRNA maturation. Non-CARF accessory genes were generally diverse, encoding nuclease, helicase, protease, ATPase, transporter and transmembrane domains with some encoding no known domains. We infer that additional families of non-CARF accessory proteins remain to be found. The method employed is scalable for potential application to metagenomic data once automated pipelines for annotation of CRISPR-Cas systems have been developed. All accessory genes found in this study are presented online in a readily accessible and searchable format for researchers to audit their model organism of choice: http://accessory.crispr.dk .

Disruption of the ABC transporter genes PDR5, YOR1, and SNQ2, and their participation in improved fermentative activity of a sake yeast mutant showing pleiotropic drug resistance.

PubMed

Watanabe, M; Mizoguchi, H; Nishimura, A

2000-01-01

Clotrimazole-resistant mutants from sake yeasts show improved fermentative activity in sake mash and pleiotropic drug resistance (PDR). The PDR mechanism is interpreted by overexpression of ATP-binding cassette (ABC) transporters, which extrude various kinds of drugs out of a cell. In a clotrimazole-resistant mutant, CTZ21, isolated from the haploid sake yeast HL69, the levels of mRNA for three major ABC transporter genes, PDR5, SNQ2, and YOR1, markedly increased. These three genes of CTZ21 were disrupted to investigate which participated in the improved fermentative activity of CTZ21. The fermentative activities of deltapdr5 and deltasnq2 strains of CTZ21 were reduced to that of HL69 in the initial and middle stages of fermentation. In the last stage, however, the sake meter [(1/gravity - 1) x 1443] of the deltapdr5 and deltasnq2 strains rose faster than that of HL69. On the other hand, a deltayor1 strain of CTZ21 fermented sake mash in a manner nearly identical to that of CTZ21 until the last stage of fermentation. But in the last stage, fermentation of the deltayor1 slowed down compared with that of CTZ21. A deltayor1 strain of HL69 also exhibited much reduced fermentative activity in the middle and last fermentation stages. The YOR1 gene seems necessary for sake fermentation to be completed efficiently. The ATP content in sake mash brewed with CTZ21 was drastically decreased throughout the whole fermentation period. This low ATP level was restored to a medium level in the cases of both the deltapdr5 and deltasnq2 strains of CTZ21. In contrast, the deltayor1 of CTZ21 exhibited a low ATP level in sake mash in the same manner as CTZ21. These results suggest that the low ATP level of CTZ21 contributes to a certain extent its improved fermentative activity in the initial and middle stages of sake fermentation.

Modularity of Plant Metabolic Gene Clusters: A Trio of Linked Genes That Are Collectively Required for Acylation of Triterpenes in Oat[W][OA

PubMed Central

Mugford, Sam T.; Louveau, Thomas; Melton, Rachel; Qi, Xiaoquan; Bakht, Saleha; Hill, Lionel; Tsurushima, Tetsu; Honkanen, Suvi; Rosser, Susan J.; Lomonossoff, George P.; Osbourn, Anne

2013-01-01

Operon-like gene clusters are an emerging phenomenon in the field of plant natural products. The genes encoding some of the best-characterized plant secondary metabolite biosynthetic pathways are scattered across plant genomes. However, an increasing number of gene clusters encoding the synthesis of diverse natural products have recently been reported in plant genomes. These clusters have arisen through the neo-functionalization and relocation of existing genes within the genome, and not by horizontal gene transfer from microbes. The reasons for clustering are not yet clear, although this form of gene organization is likely to facilitate co-inheritance and co-regulation. Oats (Avena spp) synthesize antimicrobial triterpenoids (avenacins) that provide protection against disease. The synthesis of these compounds is encoded by a gene cluster. Here we show that a module of three adjacent genes within the wider biosynthetic gene cluster is required for avenacin acylation. Through the characterization of these genes and their encoded proteins we present a model of the subcellular organization of triterpenoid biosynthesis. PMID:23532069

Purifying, cloning and characterizing a novel dehalogenase from Bacillus sp. GZT to enhance the biodegradation of 2,4,6-tribromophenol in water.

PubMed

Liang, Zhishu; Li, Guiying; An, Taicheng

2017-06-01

2,4,6-Tribromophenol (TBP), an intermediate of brominated flame retardants, can easily release to environment and recalcitrant to degradation. Previously, Bacillus sp. GZT, a pure aerobic strain capable of simultaneously debrominating and mineralizing TBP, was successfully isolated by us. To further obtain a practical application and dig up its TBP degradation mechanism, a total of 46.7-fold purification of a novel dehalogenase with a final specific activity of 18.9 U mg -1 and a molecular mass of 63.4 kDa was achieved. Under optimal conditions (35 °C and 200 rpm), up to 80% degradation efficiencies were achieved within 120 min. Adding H 2 O 2 , NADPH, Mn 2+ and Mg 2+ promoted enzyme reaction effectively; while EDTA, methyl viologen, Ni 2+ , Cu 2+ , Ca 2+ and Fe 2+ strongly inhibited reaction activities. The debromination of TBP was catalyzed by the enzyme at a Km of 78 μM and a Vmax of 0.65 min -1  mg protein -1 , which indicated that this dehalogenase could specifically eliminate TBP with a high efficiency and stability. Based on MALDI-TOF/TOF analysis, the dehalogenase shared 98% identity with peptide ABC transporter substrate-binding protein. One open reading frame (ORF) encoding this peptide was found in Strain GZT genome, subjected to clone and expressed in Escherichia coli (E. coli) to characterize the encoding gene. Result showed that this recombinant strain could also remove as similar amount of TBP as Bacillus sp. GZT under the identical condition. Based on these results, we suggest that this newly-isolated TBP dehalogenase highlights a new approach for remediating TBP pollution. Copyright © 2017 Elsevier Ltd. All rights reserved.

Reconstruction of the bifidobacterial pan-secretome reveals the network of extracellular interactions between bifidobacteria and the infant gut.

PubMed

Lugli, Gabriele Andrea; Mancino, Walter; Milani, Christian; Duranti, Sabrina; Turroni, Francesca; van Sinderen, Douwe; Ventura, Marco

2018-06-08

The repertoire of secreted proteins decoded by a microorganism represents proteins released from or associated with the cell's surface. In gut commensals, such as bifidobacteria, these proteins are perceived to be functionally relevant as they regulate the interaction with the gut environment. In the current study, we have screened the predicted proteome of over 300 bifidobacterial strains amongst the currently recognized bifidobacterial species to generate a comprehensive database encompassing bifidobacterial extracellular proteins. A glycobiome analysis of this predicted bifidobacterial secretome revealed that a correlation exists between particular bifidobacterial species and their capability to hydrolyze HMOs and intestinal glyconjugates such as mucin. Furthermore, exploration of metatranscriptomic datasets of the infant gut microbiota allowed the evaluation of the expression of bifidobacterial genes encoding extracellular proteins, represented by ABC transporter substrate-binding proteins and glycoside hydrolases enzymes involved in the degradation of human milk oligosaccharides and mucin. Overall, this study provides insights into how bifidobacteria interact with their natural yet highly complex environment, the infant gut. Importance The ecological success of bifidobacteria relies on the activity of extracellular proteins that are involved in the metabolism of nutrients and the interaction with the environment. To date, information on secreted proteins encoded by bifidobacteria are incomplete and just related to few species. In this study, we reconstructed the bifidobacterial pan-secretome, revealing extracellular proteins that modulate the interaction of bifidobacteria with their natural environment. Furthermore, a survey of secretion system between bifidobacterial genomes allowed the identification of a conserved Sec-dependent secretion machinery in all the analyzed genomes and the Tat protein translocation system in the chromosomes of 23 strains belonging to Bifidobacterium longum subsp. longum and Bifidobacterium aesculapii . Copyright © 2018 American Society for Microbiology.

Detection with synthetic oligonucleotide probes of nucleotide sequence variations in the genes encoding enterotoxins of Escherichia coli.

PubMed Central

Nishibuchi, M; Murakami, A; Arita, M; Jikuya, H; Takano, J; Honda, T; Miwatani, T

1989-01-01

We examined variations in the genes encoding heat-stable enterotoxin (ST) and heat-labile enterotoxin (LT) in 88 strains of Escherichia coli isolated from individuals with traveler's diarrhea to find suitable sequences for use as oligonucleotide probes. Four oligonucleotide probes of the gene encoding ST of human origin (STIb or STh), one oligonucleotide probe of the gene encoding ST of porcine origin (STIa or STp), and three oligonucleotide probes of the gene encoding LT of human origin (LTIh) were used in DNA colony hybridization tests. In 15 of 22 strains possessing the STh gene and 28 of 42 strains producing LT, the sequences of all regions tested were identical to the published sequences. One region in the STh gene examined with a 18-mer probe was relatively well conserved and was shown to be closely associated with the enterotoxicity of the E. coli strains in suckling mice. This oligonucleotide, however, hybridized with strains of Vibrio cholerae O1, V. parahaemolyticus, and Yersinia enterocolitica that gave negative results in the suckling mouse assay. PMID:2685027

Cyclic stretch-induced the cytoskeleton rearrangement and gene expression of cytoskeletal regulators in human periodontal ligament cells.

PubMed

Wu, Yaqin; Zhuang, Jiabao; Zhao, Dan; Zhang, Fuqiang; Ma, Jiayin; Xu, Chun

2017-10-01

This study aimed to explore the mechanism of the stretch-induced cell realignment and cytoskeletal rearrangement by identifying several mechanoresponsive genes related to cytoskeletal regulators in human PDL cells. After the cells were stretched by 1, 10 and 20% strains for 0.5, 1, 2, 4, 6, 12 or 24 h, the changes of the morphology and content of microfilaments were recorded and calculated. Meanwhile, the expression of 84 key genes encoding cytoskeletal regulators after 6 and 24 h stretches with 20% strain was detected by using real-time PCR array. Western blot was applied to identify the protein expression level of several cytoskeletal regulators encoded by these differentially expressed genes. The confocal fluorescent staining results confirmed that stretch-induced realignment of cells and rearrangement of microfilaments. Among the 84 genes screened, one gene was up-regulated while two genes were down-regulated after 6 h stretch. Meanwhile, three genes were up-regulated while two genes were down-regulated after 24 h stretch. These genes displaying differential expression included genes regulating polymerization/depolymerization of microfilaments (CDC42EP2, FNBP1L, NCK2, PIKFYVE, WASL), polymerization/depolymerization of microtubules (STMN1), interacting between microfilaments and microtubules (MACF1), as well as a phosphatase (PPP1R12B). Among the proteins encoded by these genes, the protein expression level of Cdc42 effector protein-2 (encoded by CDC42EP2) and Stathmin-1 (encoded by STMN1) was down-regulated, while the protein expression level of N-WASP (encoded by WASL) was up-regulated. The present study confirmed the cyclic stretch-induced cellular realignment and rearrangement of microfilaments in the human PDL cells and indicated several force-sensitive genes with regard to cytoskeletal regulators.

A High-Resolution Gene Map of the Chloroplast Genome of the Red Alga Porphyra purpurea.

PubMed Central

Reith, M; Munholland, J

1993-01-01

Extensive DNA sequencing of the chloroplast genome of the red alga Porphyra purpurea has resulted in the detection of more than 125 genes. Fifty-eight (approximately 46%) of these genes are not found on the chloroplast genomes of land plants. These include genes encoding 17 photosynthetic proteins, three tRNAs, and nine ribosomal proteins. In addition, nine genes encoding proteins related to biosynthetic functions, six genes encoding proteins involved in gene expression, and at least five genes encoding miscellaneous proteins are among those not known to be located on land plant chloroplast genomes. The increased coding capacity of the P. purpurea chloroplast genome, along with other characteristics such as the absence of introns and the conservation of ancestral operons, demonstrate the primitive nature of the P. purpurea chloroplast genome. In addition, evidence for a monophyletic origin of chloroplasts is suggested by the identification of two groups of genes that are clustered in chloroplast genomes but not in cyanobacteria. PMID:12271072

Genome-Wide Architecture of Disease Resistance Genes in Lettuce

PubMed Central

Christopoulou, Marilena; Wo, Sebastian Reyes-Chin; Kozik, Alex; McHale, Leah K.; Truco, Maria-Jose; Wroblewski, Tadeusz; Michelmore, Richard W.

2015-01-01

Genome-wide motif searches identified 1134 genes in the lettuce reference genome of cv. Salinas that are potentially involved in pathogen recognition, of which 385 were predicted to encode nucleotide binding-leucine rich repeat receptor (NLR) proteins. Using a maximum-likelihood approach, we grouped the NLRs into 25 multigene families and 17 singletons. Forty-one percent of these NLR-encoding genes belong to three families, the largest being RGC16 with 62 genes in cv. Salinas. The majority of NLR-encoding genes are located in five major resistance clusters (MRCs) on chromosomes 1, 2, 3, 4, and 8 and cosegregate with multiple disease resistance phenotypes. Most MRCs contain primarily members of a single NLR gene family but a few are more complex. MRC2 spans 73 Mb and contains 61 NLRs of six different gene families that cosegregate with nine disease resistance phenotypes. MRC3, which is 25 Mb, contains 22 RGC21 genes and colocates with Dm13. A library of 33 transgenic RNA interference tester stocks was generated for functional analysis of NLR-encoding genes that cosegregated with disease resistance phenotypes in each of the MRCs. Members of four NLR-encoding families, RGC1, RGC2, RGC21, and RGC12 were shown to be required for 16 disease resistance phenotypes in lettuce. The general composition of MRCs is conserved across different genotypes; however, the specific repertoire of NLR-encoding genes varied particularly of the rapidly evolving Type I genes. These tester stocks are valuable resources for future analyses of additional resistance phenotypes. PMID:26449254

An ABC estimate of pedigree error rate: application in dog, sheep and cattle breeds.

PubMed

Leroy, G; Danchin-Burge, C; Palhiere, I; Baumung, R; Fritz, S; Mériaux, J C; Gautier, M

2012-06-01

On the basis of correlations between pairwise individual genealogical kinship coefficients and allele sharing distances computed from genotyping data, we propose an approximate Bayesian computation (ABC) approach to assess pedigree file reliability through gene-dropping simulations. We explore the features of the method using simulated data sets and show precision increases with the number of markers. An application is further made with five dog breeds, four sheep breeds and one cattle breed raised in France and displaying various characteristics and population sizes, using microsatellite or SNP markers. Depending on the breeds, pedigree error estimations range between 1% and 9% in dog breeds, 1% and 10% in sheep breeds and 4% in cattle breeds. © 2011 The Authors, Animal Genetics © 2011 Stichting International Foundation for Animal Genetics.

Cloning, characterization, expression analysis and inhibition studies of a novel gene encoding Bowman-Birk type protease inhibitor from rice bean

USDA-ARS?s Scientific Manuscript database

This paper presents the first study describing the isolation, cloning and characterization of a full length gene encoding Bowman-Birk protease inhibitor (RbTI) from rice bean (Vigna umbellata). A full-length protease inhibitor gene with complete open reading frame of 327bp encoding 109 amino acids w...

Cytochrome b5 gene and protein of Candida tropicalis and methods relating thereto

DOEpatents

Craft, David L.; Madduri, Krishna M.; Loper, John C.

2003-01-01

A novel gene has been isolated which encodes cytochrome b5 (CYTb5) protein of the .omega.-hydroxylase complex of C. tropicalis 20336. Vectors including this gene, and transformed host cells are provided. Methods of increasing the production of a CYTb5 protein are also provided which involve transforming a host cell with a gene encoding this protein and culturing the cells. Methods of increasing the production of a dicarboxylic acid are also provided which involve increasing in the host cell the number of genes encoding this protein.

Genome complexity in the coelacanth is reflected in its adaptive immune system

USGS Publications Warehouse

Saha, Nil Ratan; Ota, Tatsuya; Litman, Gary W.; Hansen, John; Parra, Zuly; Hsu, Ellen; Buonocore, Francesco; Canapa, Adriana; Cheng, Jan-Fang; Amemiya, Chris T.

2014-01-01

We have analyzed the available genome and transcriptome resources from the coelacanth in order to characterize genes involved in adaptive immunity. Two highly distinctive IgW-encoding loci have been identified that exhibit a unique genomic organization, including a multiplicity of tandemly repeated constant region exons. The overall organization of the IgW loci precludes typical heavy chain class switching. A locus encoding IgM could not be identified either computationally or by using several different experimental strategies. Four distinct sets of genes encoding Ig light chains were identified. This includes a variant sigma-type Ig light chain previously identified only in cartilaginous fishes and which is now provisionally denoted sigma-2. Genes encoding α/β and γ/δ T-cell receptors, and CD3, CD4, and CD8 co-receptors also were characterized. Ig heavy chain variable region genes and TCR components are interspersed within the TCR α/δ locus; this organization previously was reported only in tetrapods and raises questions regarding evolution and functional cooption of genes encoding variable regions. The composition, organization and syntenic conservation of the major histocompatibility complex locus have been characterized. We also identified large numbers of genes encoding cytokines and their receptors, and other genes associated with adaptive immunity. In terms of sequence identity and organization, the adaptive immune genes of the coelacanth more closely resemble orthologous genes in tetrapods than those in teleost fishes, consistent with current phylogenomic interpretations. Overall, the work reported described herein highlights the complexity inherent in the coelacanth genome and provides a rich catalog of immune genes for future investigations.

ABC transporters and the proteasome complex are implicated in susceptibility to Stevens-Johnson syndrome and toxic epidermal necrolysis across multiple drugs.

PubMed

Nicoletti, Paola; Bansal, Mukesh; Lefebvre, Celine; Guarnieri, Paolo; Shen, Yufeng; Pe'er, Itsik; Califano, Andrea; Floratos, Aris

2015-01-01

Stevens-Johnson syndrome (SJS) and Toxic Epidermal Necrolysis (TEN) represent rare but serious adverse drug reactions (ADRs). Both are characterized by distinctive blistering lesions and significant mortality rates. While there is evidence for strong drug-specific genetic predisposition related to HLA alleles, recent genome wide association studies (GWAS) on European and Asian populations have failed to identify genetic susceptibility alleles that are common across multiple drugs. We hypothesize that this is a consequence of the low to moderate effect size of individual genetic risk factors. To test this hypothesis we developed Pointer, a new algorithm that assesses the aggregate effect of multiple low risk variants on a pathway using a gene set enrichment approach. A key advantage of our method is the capability to associate SNPs with genes by exploiting physical proximity as well as by using expression quantitative trait loci (eQTLs) that capture information about both cis- and trans-acting regulatory effects. We control for known bias-inducing aspects of enrichment based analyses, such as: 1) gene length, 2) gene set size, 3) presence of biologically related genes within the same linkage disequilibrium (LD) region, and, 4) genes shared among multiple gene sets. We applied this approach to publicly available SJS/TEN genome-wide genotype data and identified the ABC transporter and Proteasome pathways as potentially implicated in the genetic susceptibility of non-drug-specific SJS/TEN. We demonstrated that the innovative SNP-to-gene mapping phase of the method was essential in detecting the significant enrichment for those pathways. Analysis of an independent gene expression dataset provides supportive functional evidence for the involvement of Proteasome pathways in SJS/TEN cutaneous lesions. These results suggest that Pointer provides a useful framework for the integrative analysis of pharmacogenetic GWAS data, by increasing the power to detect aggregate effects of multiple low risk variants. The software is available for download at https://sourceforge.net/projects/pointergsa/.

The Bacillus subtilis ywjI (glpX) gene encodes a class II fructose-1,6-bisphosphatase, functionally equivalent to the class III Fbp enzyme.

PubMed

Jules, Matthieu; Le Chat, Ludovic; Aymerich, Stéphane; Le Coq, Dominique

2009-05-01

We present here experimental evidence that the Bacillus subtilis ywjI gene encodes a class II fructose-1,6-bisphosphatase, functionally equivalent to the fbp-encoded class III enzyme, and constitutes with the upstream gene, murAB, an operon transcribed at the same level under glycolytic or gluconeogenic conditions.

The Bacillus subtilis ywjI (glpX) Gene Encodes a Class II Fructose-1,6-Bisphosphatase, Functionally Equivalent to the Class III Fbp Enzyme▿

PubMed Central

Jules, Matthieu; Le Chat, Ludovic; Aymerich, Stéphane; Le Coq, Dominique

2009-01-01

We present here experimental evidence that the Bacillus subtilis ywjI gene encodes a class II fructose-1,6-bisphosphatase, functionally equivalent to the fbp-encoded class III enzyme, and constitutes with the upstream gene, murAB, an operon transcribed at the same level under glycolytic or gluconeogenic conditions. PMID:19270101

Dietary xenosterols lead to infertility and loss of abdominal adipose tissue in sterolin-deficient mice[S

PubMed Central

Solca, Curzio; Tint, G. Stephen; Patel, Shailendra B.

2013-01-01

The investigation of the human disease sitosterolemia (MIM 210250) has shed light not only on the pathways by which dietary sterols may traffic but also on how the mammalian body rids itself of cholesterol and defends against xenosterols. Two genes, ABCG5 and ABCG8, located at the sitosterolemia locus, each encodes a membrane-bound ABC half-transporter and constitutes a functional unit whose activity has now been shown to account for biliary and intestinal sterol excretion. Knockout mice deficient in Abcg5 or Abcg8 recapitulate many of the phenotypic features of sitosterolemia. During the course of our studies to characterize these knockout mice, we noted that these mice, raised on normal rodent chow, exhibited infertility as well as loss of abdominal fat. We show that, although sitosterolemia does not lead to any structural defects or to any overt endocrine defects, fertility could be restored if xenosterols are specifically blocked from entry and that the loss of fat is also reversed by a variety of maneuvers that limit xenosterol accumulation. These studies show that xenosterols may have a significant biological impact on normal mammalian physiology and that the Abcg5 or Abcg8 knockout mouse model may prove useful in investigating the role of xenosterols on mammalian physiology. PMID:23180829

Garvicin A, a Novel Class IId Bacteriocin from Lactococcus garvieae That Inhibits Septum Formation in L. garvieae Strains

PubMed Central

Cárdenas, Nivia; Martínez, Beatriz; Ruiz-Barba, José Luis; Fernández-Garayzábal, José F.; Rodríguez, Juan M.; Gibello, Alicia

2013-01-01

Lactococcus garvieae 21881, isolated in a human clinical case, produces a novel class IId bacteriocin, garvicin A (GarA), which is specifically active against other L. garvieae strains, including fish- and bovine-pathogenic isolates. Purification from active supernatants, sequence analyses, and plasmid-curing experiments identified pGL5, one of the five plasmids found in L. garvieae [M. Aguado-Urda et al., PLoS One 7(6):e40119, 2012], as the coding plasmid for the structural gene of GarA (lgnA), its putative immunity protein (lgnI), and the ABC transporter and its accessory protein (lgnC and lgnD). Interestingly, pGL5-cured strains were still resistant to GarA. Other putative bacteriocins encoded by the remaining plasmids were not detected during purification, pointing to GarA as the main inhibitor secreted by L. garvieae 21881. Mode-of-action studies revealed a potent bactericidal activity of GarA. Moreover, transmission microscopy showed that GarA seems to act by inhibiting septum formation in L. garvieae cells. This potent and species-specific inhibition by GarA holds promise for applications in the prevention or treatment of infections caused by pathogenic strains of L. garvieae in both veterinary and clinical settings. PMID:23666326

Molecular Cloning and Characterization of a P-Glycoprotein from the Diamondback Moth, Plutella xylostella (Lepidoptera: Plutellidae)

PubMed Central

Tian, Lixia; Yang, Jiaqiang; Hou, Wenjie; Xu, Baoyun; Xie, Wen; Wang, Shaoli; Zhang, Youjun; Zhou, Xuguo; Wu, Qingjun

2013-01-01

Macrocyclic lactones such as abamectin and ivermectin constitute an important class of broad-spectrum insecticides. Widespread resistance to synthetic insecticides, including abamectin and ivermectin, poses a serious threat to the management of diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae), a major pest of cruciferous plants worldwide. P-glycoprotein (Pgp), a member of the ABC transporter superfamily, plays a crucial role in the removal of amphiphilic xenobiotics, suggesting a mechanism for drug resistance in target organisms. In this study, PxPgp1, a putative Pgp gene from P. xylostella, was cloned and characterized. The open reading frame (ORF) of PxPgp1 consists of 3774 nucleotides, which encodes a 1257-amino acid peptide. The deduced PxPgp1 protein possesses structural characteristics of a typical Pgp, and clusters within the insect ABCB1. PxPgp1 was expressed throughout all developmental stages, and showed the highest expression level in adult males. PxPgp1 was highly expressed in midgut, malpighian tubules and testes. Elevated expression of PxPgp1 was observed in P. xylostella strains after they were exposed to the abamectin treatment. In addition, the constitutive expressions of PxPgp1 were significantly higher in laboratory-selected and field-collected resistant strains in comparison to their susceptible counterpart. PMID:24264038

Vital and dispensable roles of Plasmodium multidrug resistance transporters during blood- and mosquito-stage development.

PubMed

Rijpma, Sanna R; van der Velden, Maarten; Annoura, Takeshi; Matz, Joachim M; Kenthirapalan, Sanketha; Kooij, Taco W A; Matuschewski, Kai; van Gemert, Geert-Jan; van de Vegte-Bolmer, Marga; Siebelink-Stoter, Rianne; Graumans, Wouter; Ramesar, Jai; Klop, Onny; Russel, Frans G M; Sauerwein, Robert W; Janse, Chris J; Franke-Fayard, Blandine M; Koenderink, Jan B

2016-07-01

Multidrug resistance (MDR) proteins belong to the B subfamily of the ATP Binding Cassette (ABC) transporters, which export a wide range of compounds including pharmaceuticals. In this study, we used reverse genetics to study the role of all seven Plasmodium MDR proteins during the life cycle of malaria parasites. Four P. berghei genes (encoding MDR1, 4, 6 and 7) were refractory to deletion, indicating a vital role during blood stage multiplication and validating them as potential targets for antimalarial drugs. Mutants lacking expression of MDR2, MDR3 and MDR5 were generated in both P. berghei and P. falciparum, indicating a dispensable role for blood stage development. Whereas P. berghei mutants lacking MDR3 and MDR5 had a reduced blood stage multiplication in vivo, blood stage growth of P. falciparum mutants in vitro was not significantly different. Oocyst maturation and sporozoite formation in Plasmodium mutants lacking MDR2 or MDR5 was reduced. Sporozoites of these P. berghei mutants were capable of infecting mice and life cycle completion, indicating the absence of vital roles during liver stage development. Our results demonstrate vital and dispensable roles of MDR proteins during blood stages and an important function in sporogony for MDR2 and MDR5 in both Plasmodium species. © 2016 John Wiley & Sons Ltd.

The ABCs of flower development: mutational analysis of AP1/FUL-like genes in rice provides evidence for a homeotic (A)-function in grasses.

PubMed

Wu, Feng; Shi, Xiaowei; Lin, Xuelei; Liu, Yuan; Chong, Kang; Theißen, Günter; Meng, Zheng

2017-01-01

The well-known ABC model describes the combinatorial interaction of homeotic genes in specifying floral organ identities. While the B- and C-functions are highly conserved throughout flowering plants and even in gymnosperms, the A-function, which specifies the identity of perianth organs (sepals and petals in eudicots), remains controversial. One reason for this is that in most plants that have been investigated thus far, with Arabidopsis being a remarkable exception, one does not find recessive mutants in which the identity of both types of perianth organs is affected. Here we report a comprehensive mutational analysis of all four members of the AP1/FUL-like subfamily of MADS-box genes in rice (Oryza sativa). We demonstrate that OsMADS14 and OsMADS15, in addition to their function of specifying meristem identity, are also required to specify palea and lodicule identities. Because these two grass-specific organs are very likely homologous to sepals and petals of eudicots, respectively, we conclude that there is a floral homeotic (A)-function in rice as defined previously. Together with other recent findings, our data suggest that AP1/FUL-like genes were independently recruited to fulfil the (A)-function in grasses and some eudicots, even though other scenarios cannot be excluded and are discussed. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

Molecular comparison of the structural proteins encoding gene clusters of two related Lactobacillus delbrueckii bacteriophages.

PubMed Central

Vasala, A; Dupont, L; Baumann, M; Ritzenthaler, P; Alatossava, T

1993-01-01

Virulent phage LL-H and temperate phage mv4 are two related bacteriophages of Lactobacillus delbrueckii. The gene clusters encoding structural proteins of these two phages have been sequenced and further analyzed. Six open reading frames (ORF-1 to ORF-6) were detected. Protein sequencing and Western immunoblotting experiments confirmed that ORF-3 (g34) encoded the main capsid protein Gp34. The presence of a putative late promoter in front of the phage LL-H g34 gene was suggested by primer extension experiments. Comparative sequence analysis between phage LL-H and phage mv4 revealed striking similarities in the structure and organization of this gene cluster, suggesting that the genes encoding phage structural proteins belong to a highly conservative module. Images PMID:8497043

Inhibition of the Human ABC Efflux Transporters P-gp and BCRP by the BDE-47 Hydroxylated Metabolite 6-OH-BDE-47: Considerations for Human Exposure

EPA Science Inventory

High body burdens of polybrominated diphenyl ethers (PBDEs) in infants and young children have led to increased concern over their potential impact on human development. PBDE exposure can alter the expression of genes involved in thyroid homeostasis, including those of ATP-bindin...

Lévy flight artificial bee colony algorithm

NASA Astrophysics Data System (ADS)

Sharma, Harish; Bansal, Jagdish Chand; Arya, K. V.; Yang, Xin-She

2016-08-01

Artificial bee colony (ABC) optimisation algorithm is a relatively simple and recent population-based probabilistic approach for global optimisation. The solution search equation of ABC is significantly influenced by a random quantity which helps in exploration at the cost of exploitation of the search space. In the ABC, there is a high chance to skip the true solution due to its large step sizes. In order to balance between diversity and convergence in the ABC, a Lévy flight inspired search strategy is proposed and integrated with ABC. The proposed strategy is named as Lévy Flight ABC (LFABC) has both the local and global search capability simultaneously and can be achieved by tuning the Lévy flight parameters and thus automatically tuning the step sizes. In the LFABC, new solutions are generated around the best solution and it helps to enhance the exploitation capability of ABC. Furthermore, to improve the exploration capability, the numbers of scout bees are increased. The experiments on 20 test problems of different complexities and five real-world engineering optimisation problems show that the proposed strategy outperforms the basic ABC and recent variants of ABC, namely, Gbest-guided ABC, best-so-far ABC and modified ABC in most of the experiments.

Bacillus subtilis 168 Contains Two Differentially Regulated Genes Encoding l-Asparaginase

PubMed Central

Fisher, Susan H.; Wray, Lewis V.

2002-01-01

Expression of the two Bacillus subtilis genes encoding l-asparaginase is controlled by independent regulatory factors. The ansZ gene (formerly yccC) was shown by mutational analysis to encode a functional l-asparaginase, the expression of which is activated during nitrogen-limited growth by the TnrA transcription factor. Gel mobility shift and DNase I footprinting experiments indicate that TnrA regulates ansZ expression by binding to a DNA site located upstream of the ansZ promoter. The expression of the ansA gene, which encodes the second l-asparaginase, was found to be induced by asparagine. The ansA repressor, AnsR, was shown to negatively regulate its own expression. PMID:11914346

Bacillus subtilis 168 contains two differentially regulated genes encoding L-asparaginase.

PubMed

Fisher, Susan H; Wray, Lewis V

2002-04-01

Expression of the two Bacillus subtilis genes encoding L-asparaginase is controlled by independent regulatory factors. The ansZ gene (formerly yccC) was shown by mutational analysis to encode a functional L-asparaginase, the expression of which is activated during nitrogen-limited growth by the TnrA transcription factor. Gel mobility shift and DNase I footprinting experiments indicate that TnrA regulates ansZ expression by binding to a DNA site located upstream of the ansZ promoter. The expression of the ansA gene, which encodes the second L-asparaginase, was found to be induced by asparagine. The ansA repressor, AnsR, was shown to negatively regulate its own expression.

Recombinant DNA encoding a desulfurization biocatalyst

DOEpatents

Rambosek, John; Piddington, Chris S.; Kovacevich, Brian R.; Young, Kevin D.; Denome, Sylvia A.

1994-01-01

This invention relates to a recombinant DNA molecule containing a gene or genes which encode a biocatalyst capable of desulfurizing a fossil fuel which contains organic sulfur molecules. For example, the present invention encompasses a recombinant DNA molecule containing a gene or genes of a strain of Rhodococcus rhodochrous.

Sites Inferred by Metabolic Background Assertion Labeling (SIMBAL): adapting the Partial Phylogenetic Profiling algorithm to scan sequences for signatures that predict protein function

PubMed Central

2010-01-01

Background Comparative genomics methods such as phylogenetic profiling can mine powerful inferences from inherently noisy biological data sets. We introduce Sites Inferred by Metabolic Background Assertion Labeling (SIMBAL), a method that applies the Partial Phylogenetic Profiling (PPP) approach locally within a protein sequence to discover short sequence signatures associated with functional sites. The approach is based on the basic scoring mechanism employed by PPP, namely the use of binomial distribution statistics to optimize sequence similarity cutoffs during searches of partitioned training sets. Results Here we illustrate and validate the ability of the SIMBAL method to find functionally relevant short sequence signatures by application to two well-characterized protein families. In the first example, we partitioned a family of ABC permeases using a metabolic background property (urea utilization). Thus, the TRUE set for this family comprised members whose genome of origin encoded a urea utilization system. By moving a sliding window across the sequence of a permease, and searching each subsequence in turn against the full set of partitioned proteins, the method found which local sequence signatures best correlated with the urea utilization trait. Mapping of SIMBAL "hot spots" onto crystal structures of homologous permeases reveals that the significant sites are gating determinants on the cytosolic face rather than, say, docking sites for the substrate-binding protein on the extracellular face. In the second example, we partitioned a protein methyltransferase family using gene proximity as a criterion. In this case, the TRUE set comprised those methyltransferases encoded near the gene for the substrate RF-1. SIMBAL identifies sequence regions that map onto the substrate-binding interface while ignoring regions involved in the methyltransferase reaction mechanism in general. Neither method for training set construction requires any prior experimental characterization. Conclusions SIMBAL shows that, in functionally divergent protein families, selected short sequences often significantly outperform their full-length parent sequence for making functional predictions by sequence similarity, suggesting avenues for improved functional classifiers. When combined with structural data, SIMBAL affords the ability to localize and model functional sites. PMID:20102603

Comparative Genomics of DNA Recombination and Repair in Cyanobacteria: Biotechnological Implications

PubMed Central

Cassier-Chauvat, Corinne; Veaudor, Théo; Chauvat, Franck

2016-01-01

Cyanobacteria are fascinating photosynthetic prokaryotes that are regarded as the ancestors of the plant chloroplast; the purveyors of oxygen and biomass for the food chain; and promising cell factories for an environmentally friendly production of chemicals. In colonizing most waters and soils of our planet, cyanobacteria are inevitably challenged by environmental stresses that generate DNA damages. Furthermore, many strains engineered for biotechnological purposes can use DNA recombination to stop synthesizing the biotechnological product. Hence, it is important to study DNA recombination and repair in cyanobacteria for both basic and applied research. This review reports what is known in a few widely studied model cyanobacteria and what can be inferred by mining the sequenced genomes of morphologically and physiologically diverse strains. We show that cyanobacteria possess many E. coli-like DNA recombination and repair genes, and possibly other genes not yet identified. E. coli-homolog genes are unevenly distributed in cyanobacteria, in agreement with their wide genome diversity. Many genes are extremely well conserved in cyanobacteria (mutMS, radA, recA, recFO, recG, recN, ruvABC, ssb, and uvrABCD), even in small genomes, suggesting that they encode the core DNA repair process. In addition to these core genes, the marine Prochlorococcus and Synechococcus strains harbor recBCD (DNA recombination), umuCD (mutational DNA replication), as well as the key SOS genes lexA (regulation of the SOS system) and sulA (postponing of cell division until completion of DNA reparation). Hence, these strains could possess an E. coli-type SOS system. In contrast, several cyanobacteria endowed with larger genomes lack typical SOS genes. For examples, the two studied Gloeobacter strains lack alkB, lexA, and sulA; and Synechococcus PCC7942 has neither lexA nor recCD. Furthermore, the Synechocystis PCC6803 lexA product does not regulate DNA repair genes. Collectively, these findings indicate that not all cyanobacteria have an E. coli-type SOS system. Also interestingly, several cyanobacteria possess multiple copies of E. coli-like DNA repair genes, such as Acaryochloris marina MBIC11017 (2 alkB, 3 ogt, 7 recA, 3 recD, 2 ssb, 3 umuC, 4 umuD, and 8 xerC), Cyanothece ATCC51142 (2 lexA and 4 ruvC), and Nostoc PCC7120 (2 ssb and 3 xerC). PMID:27881980

Transcriptome of the inflorescence meristems of the biofuel plant Jatropha curcas treated with cytokinin.

PubMed

Pan, Bang-Zhen; Chen, Mao-Sheng; Ni, Jun; Xu, Zeng-Fu

2014-11-17

Jatropha curcas, whose seed content is approximately 30-40% oil, is an ideal feedstock for producing biodiesel and bio-jet fuels. However, Jatropha plants have a low number of female flowers, which results in low seed yield that cannot meet the needs of the biofuel industry. Thus, increasing the number of female flowers is critical for the improvement of Jatropha seed yield. Our previous findings showed that cytokinin treatment can increase the flower number and female to male ratio and also induce bisexual flowers in Jatropha. The mechanisms underlying the influence of cytokinin on Jatropha flower development and sex determination, however, have not been clarified. This study examined the transcriptional levels of genes involved in the response to cytokinin in Jatropha inflorescence meristems at different time points after cytokinin treatment by 454 sequencing, which gave rise to a total of 294.6 Mb of transcript sequences. Up-regulated and down-regulated annotated and novel genes were identified, and the expression levels of the genes of interest were confirmed by qRT-PCR. The identified transcripts include those encoding genes involved in the biosynthesis, metabolism, and signaling of cytokinin and other plant hormones, flower development and cell division, which may be related to phenotypic changes of Jatropha in response to cytokinin treatment. Our analysis indicated that Jatropha orthologs of the floral organ identity genes known as ABCE model genes, JcAP1,2, JcPI, JcAG, and JcSEP1,2,3, were all significantly repressed, with an exception of one B-function gene JcAP3 that was shown to be up-regulated by BA treatment, indicating different mechanisms to be involved in the floral organ development of unisexual flowers of Jatropha and bisexual flowers of Arabidopsis. Several cell division-related genes, including JcCycA3;2, JcCycD3;1, JcCycD3;2 and JcTSO1, were up-regulated, which may contribute to the increased flower number after cytokinin treatment. This study presents the first report of global expression patterns of cytokinin-regulated transcripts in Jatropha inflorescence meristems. This report laid the foundation for further mechanistic studies on Jatropha and other non-model plants responding to cytokinin. Moreover, the identification of functional candidate genes will be useful for generating superior varieties of high-yielding transgenic Jatropha.

A highly divergent gene cluster in honey bees encodes a novel silk family.

PubMed

Sutherland, Tara D; Campbell, Peter M; Weisman, Sarah; Trueman, Holly E; Sriskantha, Alagacone; Wanjura, Wolfgang J; Haritos, Victoria S

2006-11-01

The pupal cocoon of the domesticated silk moth Bombyx mori is the best known and most extensively studied insect silk. It is not widely known that Apis mellifera larvae also produce silk. We have used a combination of genomic and proteomic techniques to identify four honey bee fiber genes (AmelFibroin1-4) and two silk-associated genes (AmelSA1 and 2). The four fiber genes are small, comprise a single exon each, and are clustered on a short genomic region where the open reading frames are GC-rich amid low GC intergenic regions. The genes encode similar proteins that are highly helical and predicted to form unusually tight coiled coils. Despite the similarity in size, structure, and composition of the encoded proteins, the genes have low primary sequence identity. We propose that the four fiber genes have arisen from gene duplication events but have subsequently diverged significantly. The silk-associated genes encode proteins likely to act as a glue (AmelSA1) and involved in silk processing (AmelSA2). Although the silks of honey bees and silkmoths both originate in larval labial glands, the silk proteins are completely different in their primary, secondary, and tertiary structures as well as the genomic arrangement of the genes encoding them. This implies independent evolutionary origins for these functionally related proteins.

Photocontrol of the expression of genes encoding chlorophyll a/b binding proteins and small subunit of ribulose-1,5-bisphosphate carboxylase in etiolated seedlings of Lycopersicon esculentum (L. ) and Nicotiana tabacum (L. )

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wehmeyer, B.; Cashmore, A.R.; Schaefer, E.

Phytochrome and the blue ultraviolet-A photoreceptor control light-induced expression of genes encoding the chlorophyll a/b binding protein of photosystem II and photosystem I and the genes for the small subunit of the ribulose-1,5-bisphosphate carboxylase in etiolated seedlings of Lycopersicon esculentum (tomato) and Nicotiana tabacum (tobacco). A high irradiance response also controls the induction of these genes. Genes encoding photosystem II- and I-associated chlorophyll a/b binding proteins both exhibit a transient rapid increase in expression in response to light pulse or to continuous irradiation. In contrast, genes encoding the small subunit exhibit a continuous increase in expression in response to light.more » These distinct expression characteristics are shown to reflect differences at the level of transcription.« less

Reactive oxygen species and autophagy play a role in survival and differentiation of the phytopathogen Moniliophthora perniciosa.

PubMed

Pungartnik, C; Melo, S C O; Basso, T S; Macena, W G; Cascardo, J C M; Brendel, M

2009-01-01

The hemibiotrophic basidiomycete Moniliophthora perniciosa causes "witches' broom disease" in cacao (Theobroma cacao). During plant infection, M. perniciosa changes from mono to dikaryotic life form, an event which could be triggered by changes in plant nutritional offer and plant defense molecules, i.e., from high to low content of glycerol and hydrogen peroxide. We have recently shown that in vitro glycerol induces oxidative stress resistance in dikaryotic M. perniciosa. In order to understand under which conditions in parasite-plant interaction M. perniciosa changes from intercellular monokaryotic to intracellular dikaryotic growth phase we studied the role of glycerol on mutagen-induced oxidative stress resistance of basidiospores and monokaryotic hyphae; we also studied the role of H(2)O(2) as a signaling molecule for in vitro dikaryotization and whether changes in nutritional offer by the plant could be compensated by inducible fungal autophagy. Mono-/dikaryotic glycerol or glucose-grown cells and basidiospores were exposed to the oxidative stress-inducing mutagens H(2)O(2) and Paraquat as well as to pre-dominantly DNA damaging 4-nitroquinoline-1-oxide and UVC irradiation. Basidiospores showed highest resistance to all treatments and glycerol-grown monokaryotic hyphae were more resistant than dikaryotic hyphae. Monokaryotic cells exposed to 1microM of H(2)O(2) in glycerol-media induced formation of clamp connections within 2 days while 1mM H(2)O(2) did not within a week in the same medium; no clamp connections were formed in H(2)O(2)-containing glucose media within a week. Lower concentrations of H(2)O(2) and glycerol, when occurring in parallel, are shown to be two signals for dikaryotization in vitro and may be also during the course of infection. Q-PCR studies of glycerol-grown dikaryotic cells exposed to oxidative stress (10mM H(2)O(2)) showed high expression of MpSOD2 and transient induction of ABC cytoplasmic membrane transporter gene MpYOR1 and autophagy-related gene MpATG8. Expression of a second ABC transporter gene MpSNQ2 was 14-fold induced after H(2)O(2) exposure in glucose as compared to glycerol-grown hyphae while MpYOR1 did not show strong variation of expression under similar conditions. Glucose-grown dikaryotic cells showed elevated expression of MpATG8, especially after exposure to H(2)O(2) and 4-nitroquinoline-1-oxide. During different stages preceding basidiocarp formation MpATG8 and the two catalase-encoding genes MpCTA1 and MpCTT1 were expressed continuously. We have compiled our results and literature data in a model graph, which compares the in vitro and in planta development and differentiation of M. perniciosa with the help of physiological and morphological landmarks.

Inhibition of cholesterol absorption associated with a PPAR alpha-dependent increase in ABC binding cassette transporter A1 in mice.

PubMed

Knight, Brian L; Patel, Dilip D; Humphreys, Sandy M; Wiggins, David; Gibbons, Geoffrey F

2003-11-01

Dietary supplementation with the peroxisome proliferator-activated receptor alpha (PPAR alpha) ligand WY 14,643 gave rise to a 4- to 5-fold increase in the expression of mRNA for the ATP binding cassette transporter A1 (ABCA1) in the intestine of normal mice. There was no effect in the intestine of PPAR alpha-null mice. Consumption of a high-cholesterol diet also increased intestinal ABCA1 expression. The effects of WY 14,643 and the high-cholesterol diet were not additive. WY 14,643 feeding reduced intestinal absorption of cholesterol in the normal mice, irrespective of the dietary cholesterol concentration, and this resulted in lower diet-derived cholesterol and cholesteryl ester concentrations in plasma and liver. At each concentration of dietary cholesterol, there was a similar significant inverse correlation between intestinal ABCA1 mRNA content and the amount of cholesterol absorbed. The fibrate-induced changes in the intestines of the normal mice were accompanied by an increased concentration of the mRNA encoding the sterol-regulatory element binding protein-1c gene (SREBP-1c), a known target gene for the oxysterol receptor liver X receptor alpha (LXR alpha). There was a correlation between intestinal ABCA1 mRNA and SREBP-1c mRNA contents, but not between SREBP-1c mRNA content and cholesterol absorption. These results suggest that PPAR alpha influences cholesterol absorption through modulating ABCA1 activity in the intestine by a mechanism involving LXR alpha.

The evolution of genes encoding for green fluorescent proteins: insights from cephalochordates (amphioxus)

NASA Astrophysics Data System (ADS)

Yue, Jia-Xing; Holland, Nicholas D.; Holland, Linda Z.; Deheyn, Dimitri D.

2016-06-01

Green Fluorescent Protein (GFP) was originally found in cnidarians, and later in copepods and cephalochordates (amphioxus) (Branchiostoma spp). Here, we looked for GFP-encoding genes in Asymmetron, an early-diverged cephalochordate lineage, and found two such genes closely related to some of the Branchiostoma GFPs. Dim fluorescence was found throughout the body in adults of Asymmetron lucayanum, and, as in Branchiostoma floridae, was especially intense in the ripe ovaries. Spectra of the fluorescence were similar between Asymmetron and Branchiostoma. Lineage-specific expansion of GFP-encoding genes in the genus Branchiostoma was observed, largely driven by tandem duplications. Despite such expansion, purifying selection has strongly shaped the evolution of GFP-encoding genes in cephalochordates, with apparent relaxation for highly duplicated clades. All cephalochordate GFP-encoding genes are quite different from those of copepods and cnidarians. Thus, the ancestral cephalochordates probably had GFP, but since GFP appears to be lacking in more early-diverged deuterostomes (echinoderms, hemichordates), it is uncertain whether the ancestral cephalochordates (i.e. the common ancestor of Asymmetron and Branchiostoma) acquired GFP by horizontal gene transfer (HGT) from copepods or cnidarians or inherited it from the common ancestor of copepods and deuterostomes, i.e. the ancestral bilaterians.

Flower Development and Perianth Identity Candidate Genes in the Basal Angiosperm Aristolochia fimbriata (Piperales: Aristolochiaceae)

PubMed Central

Pabón-Mora, Natalia; Suárez-Baron, Harold; Ambrose, Barbara A.; González, Favio

2015-01-01

Aristolochia fimbriata (Aristolochiaceae: Piperales) exhibits highly synorganized flowers with a single convoluted structure forming a petaloid perianth that surrounds the gynostemium, putatively formed by the congenital fusion between stamens and the upper portion of the carpels. Here we present the flower development and morphology of A. fimbriata, together with the expression of the key regulatory genes that participate in flower development, particularly those likely controlling perianth identity. A. fimbriata is a member of the magnoliids, and thus gene expression detected for all ABCE MADS-box genes in this taxon, can also help to elucidate patterns of gene expression prior the independent duplications of these genes in eudicots and monocots. Using both floral development and anatomy in combination with the isolation of MADS-box gene homologs, gene phylogenetic analyses and expression studies (both by reverse transcription PCR and in situ hybridization), we present hypotheses on floral organ identity genes involved in the formation of this bizarre flower. We found that most MADS-box genes were expressed in vegetative and reproductive tissues with the exception of AfimSEP2, AfimAGL6, and AfimSTK transcripts that are only found in flowers and capsules but are not detected in leaves. Two genes show ubiquitous expression; AfimFUL that is found in all floral organs at all developmental stages as well as in leaves and capsules, and AfimAG that has low expression in leaves and is found in all floral organs at all stages with a considerable reduction of expression in the limb of anthetic flowers. Our results indicate that expression of AfimFUL is indicative of pleiotropic roles and not of a perianth identity specific function. On the other hand, expression of B-class genes, AfimAP3 and AfimPI, suggests their conserved role in stamen identity and corroborates that the perianth is sepal and not petal-derived. Our data also postulates an AGL6 ortholog as a candidate gene for sepal identity in the Aristolochiaceae and provides testable hypothesis for a modified ABCE model in synorganized magnoliid flowers. PMID:26697047

Recombinant DNA encoding a desulfurization biocatalyst

DOEpatents

Rambosek, J.; Piddington, C.S.; Kovacevich, B.R.; Young, K.D.; Denome, S.A.

1994-10-18

This invention relates to a recombinant DNA molecule containing a gene or genes which encode a biocatalyst capable of desulfurizing a fossil fuel which contains organic sulfur molecules. For example, the present invention encompasses a recombinant DNA molecule containing a gene or genes of a strain of Rhodococcus rhodochrous. 13 figs.