Cloning and Molecular Analysis of a Mannitol Operon of Phosphoenolpyruvate-dependent Phosphotransferase (PTS) type From Vibrio cholerae O395

PubMed Central

Kumar, Sanath; Smith, Kenneth P.; Floyd, Jody L.; Varela, Manuel F.

2010-01-01

A putative mannitol operon of the phosphoenolpyruvate phosphotransferase (PTS) type was cloned from Vibrio cholerae O395 and its activity studied in Escherichia coli. The 3.9 kb operon comprising of three genes is organized as mtlADR. Based on the sequence analysis, these were identified as genes encoding a putative mannitol-specific enzyme IICBA (EIIMtl) component (MtlA), a mannitol-1-phosphate dehydrogenase (MtlD) and a mannitol operon repressor (MtlR). The transport of [3H]mannitol by the cloned mannitol operon in E. coli was 13.8±1.4 nmol/min/mg protein. The insertional inactivation of EIIMtl abolished mannitol and sorbitol transport in V. cholerae O395. Comparison of the mannitol utilization apparatus of V. cholerae with those of Gram-negative and Gram positive bacteria suggests highly conserved nature of the system. MtlA and MtlD exhibit 75% similarity with corresponding sequences of E. coli mannitol operon genes, while MtlR has 63% similarity with MtlR of E. coli. The cloning of V. cholerae mannitol utilization system in an E. coli background will help in elucidating the functional properties of this operon. PMID:21184218

A synthetic system for expression of components of a bacterial microcompartment.

PubMed

Sargent, Frank; Davidson, Fordyce A; Kelly, Ciarán L; Binny, Rachelle; Christodoulides, Natasha; Gibson, David; Johansson, Emelie; Kozyrska, Katarzyna; Lado, Lucia Licandro; Maccallum, Jane; Montague, Rachel; Ortmann, Brian; Owen, Richard; Coulthurst, Sarah J; Dupuy, Lionel; Prescott, Alan R; Palmer, Tracy

2013-11-01

In general, prokaryotes are considered to be single-celled organisms that lack internal membrane-bound organelles. However, many bacteria produce proteinaceous microcompartments that serve a similar purpose, i.e. to concentrate specific enzymic reactions together or to shield the wider cytoplasm from toxic metabolic intermediates. In this paper, a synthetic operon encoding the key structural components of a microcompartment was designed based on the genes for the Salmonella propanediol utilization (Pdu) microcompartment. The genes chosen included pduA, -B, -J, -K, -N, -T and -U, and each was shown to produce protein in an Escherichia coli chassis. In parallel, a set of compatible vectors designed to express non-native cargo proteins was also designed and tested. Engineered hexa-His tags allowed isolation of the components of the microcompartments together with co-expressed, untagged, cargo proteins. Finally, an in vivo protease accessibility assay suggested that a PduD-GFP fusion could be protected from proteolysis when co-expressed with the synthetic microcompartment operon. This work gives encouragement that it may be possible to harness the genes encoding a non-native microcompartment for future biotechnological applications.

Specific DNA binding of a potential transcriptional regulator, inosine 5'-monophosphate dehydrogenase-related protein VII, to the promoter region of a methyl coenzyme m reductase I-encoding operon retrieved from Methanothermobacter thermautotrophicus strain DeltaH.

PubMed

Shinzato, Naoya; Enoki, Miho; Sato, Hiroaki; Nakamura, Kohei; Matsui, Toru; Kamagata, Yoichi

2008-10-01

Two methyl coenzyme M reductases (MCRs) encoded by the mcr and mrt operons of the hydrogenotrophic methanogen Methanothermobacter thermautotrophicus DeltaH are expressed in response to H(2) availability. In the present study, cis elements and trans-acting factors responsible for the gene expression of MCRs were investigated by using electrophoretic mobility shift assay (EMSA) and affinity particle purification. A survey of their operator regions by EMSA with protein extracts from mrt-expressing cultures restricted them to 46- and 41-bp-long mcr and mrt upstream regions, respectively. Affinity particle purification of DNA-binding proteins conjugated with putative operator regions resulted in the retrieval of a protein attributed to IMP dehydrogenase-related protein VII (IMPDH VII). IMPDH VII is predicted to have a winged helix-turn-helix DNA-binding motif and two cystathionine beta-synthase domains, and it has been suspected to be an energy-sensing module. EMSA with oligonucleotide probes with unusual sequences showed that the binding site of IMPDH VII mostly overlaps the factor B-responsible element-TATA box of the mcr operon. The results presented here suggest that IMPDH VII encoded by MTH126 is a plausible candidate for the transcriptional regulator of the mcr operon in this methanogen.

LacR Is a Repressor of lacABCD and LacT Is an Activator of lacTFEG, Constituting the lac Gene Cluster in Streptococcus pneumoniae

PubMed Central

Afzal, Muhammad; Shafeeq, Sulman

2014-01-01

Comparison of the transcriptome of Streptococcus pneumoniae strain D39 grown in the presence of either lactose or galactose with that of the strain grown in the presence of glucose revealed the elevated expression of various genes and operons, including the lac gene cluster, which is organized into two operons, i.e., lac operon I (lacABCD) and lac operon II (lacTFEG). Deletion of the DeoR family transcriptional regulator lacR that is present downstream of the lac gene cluster revealed elevated expression of lac operon I even in the absence of lactose. This suggests a function of LacR as a transcriptional repressor of lac operon I, which encodes enzymes involved in the phosphorylated tagatose pathway in the absence of lactose or galactose. Deletion of lacR did not affect the expression of lac operon II, which encodes a lactose-specific phosphotransferase. This finding was further confirmed by β-galactosidase assays with PlacA-lacZ and PlacT-lacZ in the presence of either lactose or glucose as the sole carbon source in the medium. This suggests the involvement of another transcriptional regulator in the regulation of lac operon II, which is the BglG-family transcriptional antiterminator LacT. We demonstrate the role of LacT as a transcriptional activator of lac operon II in the presence of lactose and CcpA-independent regulation of the lac gene cluster in S. pneumoniae. PMID:24951784

LOV Histidine Kinase Modulates the General Stress Response System and Affects the virB Operon Expression in Brucella abortus

PubMed Central

Sycz, Gabriela; Carrica, Mariela Carmen; Tseng, Tong-Seung; Bogomolni, Roberto A.; Briggs, Winslow R.; Goldbaum, Fernando A.; Paris, Gastón

2015-01-01

Brucella is the causative agent of the zoonotic disease brucellosis, and its success as an intracellular pathogen relies on its ability to adapt to the harsh environmental conditions that it encounters inside the host. The Brucella genome encodes a sensor histidine kinase containing a LOV domain upstream from the kinase, LOVHK, which plays an important role in light-regulated Brucella virulence. In this report we study the intracellular signaling pathway initiated by the light sensor LOVHK using an integrated biochemical and genetic approach. From results of bacterial two-hybrid assays and phosphotransfer experiments we demonstrate that LOVHK functionally interacts with two response regulators: PhyR and LovR, constituting a functional two-component signal-transduction system. LOVHK contributes to the activation of the General Stress Response (GSR) system in Brucella via PhyR, while LovR is proposed to be a phosphate-sink for LOVHK, decreasing its phosphorylation state. We also show that in the absence of LOVHK the expression of the virB operon is down-regulated. In conclusion, our results suggest that LOVHK positively regulates the GSR system in vivo, and has an effect on the expression of the virB operon. The proposed regulatory network suggests a similar role for LOVHK in other microorganisms. PMID:25993430

Coordinated Regulation of the EIIMan and fruRKI Operons of Streptococcus mutans by Global and Fructose-Specific Pathways

PubMed Central

Zeng, Lin; Chakraborty, Brinta; Farivar, Tanaz

2017-01-01

ABSTRACT The glucose/mannose-phosphotransferase system (PTS) permease EIIMan encoded by manLMN in the dental caries pathogen Streptococcus mutans has a dominant influence on sugar-specific, CcpA-independent catabolite repression (CR). Mutations in manL affect energy metabolism and virulence-associated traits, including biofilm formation, acid tolerance, and competence. Using promoter::reporter fusions, expression of the manLMN and the fruRKI operons, encoding a transcriptional regulator, a fructose-1-phosphate kinase and a fructose-PTS permease EIIFru, respectively, was monitored in response to carbohydrate source and in mutants lacking CcpA, FruR, and components of EIIMan. Expression of genes for EIIMan and EIIFru was directly regulated by CcpA and CR, as evinced by in vivo and in vitro methods. Unexpectedly, not only was the fruRKI operon negatively regulated by FruR, but also so was manLMN. Carbohydrate transport by EIIMan had a negative influence on expression of manLMN but not fruRKI. In agreement with the proposed role of FruR in regulating these PTS operons, loss of fruR or fruK substantially altered growth on a number of carbohydrates, including fructose. RNA deep sequencing revealed profound changes in gene regulation caused by deletion of fruK or fruR. Collectively, these findings demonstrate intimate interconnection of the regulation of two major PTS permeases in S. mutans and reveal novel and important contributions of fructose metabolism to global regulation of gene expression. IMPORTANCE The ability of Streptococcus mutans and other streptococcal pathogens to survive and cause human diseases is directly dependent upon their capacity to metabolize a variety of carbohydrates, including glucose and fructose. Our research reveals that metabolism of fructose has broad influences on the regulation of utilization of glucose and other sugars, and mutants with changes in certain genes involved in fructose metabolism display profoundly different abilities to grow and express virulence-related traits. Mutants lacking the FruR regulator or a particular phosphofructokinase, FruK, display changes in expression of a large number of genes encoding transcriptional regulators, enzymes required for energy metabolism, biofilm development, biosynthetic and degradative processes, and tolerance of a spectrum of environmental stressors. Since fructose is a major component of the modern human diet, the results have substantial significance in the context of oral health and the development of dental caries. PMID:28821551

Sequence and features of the tryptophan operon of Vibrio parahemolyticus.

PubMed

Crawford, I P; Han, C Y; Silverman, M

1991-01-01

The nucleotide sequence of the trp operon of the marine enteric bacterium Vibrio parahemolyticus is presented. The gene order E, G, D, C(F), B, A is identical to that of other enterics. The structural genes of the operon are preceded by a long leader region encoding a 41-residue peptide containing five tryptophan residues. The organization of the leader region suggests that transcription of the operon is subject to attenuation control. The promoter-operator region of the V. parahemolyticus trp operon is almost identical to the corresponding promoter-operator of E. coli. The similarities suggest that promoter strength and operator function are identical in the two species, and that transcription initiation is regulated by repression. The operon appears to lack the internal promoter within trpD that is common in terrestrial enteric species.

Meta-Analyses of Dehalococcoides mccartyi Strain 195 Transcriptomic Profiles Identify a Respiration Rate-Related Gene Expression Transition Point and Interoperon Recruitment of a Key Oxidoreductase Subunit

PubMed Central

Mansfeldt, Cresten B.; Rowe, Annette R.; Heavner, Gretchen L. W.; Zinder, Stephen H.

2014-01-01

A cDNA-microarray was designed and used to monitor the transcriptomic profile of Dehalococcoides mccartyi strain 195 (in a mixed community) respiring various chlorinated organics, including chloroethenes and 2,3-dichlorophenol. The cultures were continuously fed in order to establish steady-state respiration rates and substrate levels. The organization of array data into a clustered heat map revealed two major experimental partitions. This partitioning in the data set was further explored through principal component analysis. The first two principal components separated the experiments into those with slow (1.6 ± 0.6 μM Cl−/h)- and fast (22.9 ± 9.6 μM Cl−/h)-respiring cultures. Additionally, the transcripts with the highest loadings in these principal components were identified, suggesting that those transcripts were responsible for the partitioning of the experiments. By analyzing the transcriptomes (n = 53) across experiments, relationships among transcripts were identified, and hypotheses about the relationships between electron transport chain members were proposed. One hypothesis, that the hydrogenases Hup and Hym and the formate dehydrogenase-like oxidoreductase (DET0186-DET0187) form a complex (as displayed by their tight clustering in the heat map analysis), was explored using a nondenaturing protein separation technique combined with proteomic sequencing. Although these proteins did not migrate as a single complex, DET0112 (an FdhB-like protein encoded in the Hup operon) was found to comigrate with DET0187 rather than with the catalytic Hup subunit DET0110. On closer inspection of the genome annotations of all Dehalococcoides strains, the DET0185-to-DET0187 operon was found to lack a key subunit, an FdhB-like protein. Therefore, on the basis of the transcriptomic, genomic, and proteomic evidence, the place of the missing subunit in the DET0185-to-DET0187 operon is likely filled by recruiting a subunit expressed from the Hup operon (DET0112). PMID:25063656

An Inducible Operon Is Involved in Inulin Utilization in Lactobacillus plantarum Strains, as Revealed by Comparative Proteogenomics and Metabolic Profiling.

PubMed

Buntin, Nirunya; Hongpattarakere, Tipparat; Ritari, Jarmo; Douillard, François P; Paulin, Lars; Boeren, Sjef; Shetty, Sudarshan A; de Vos, Willem M

2017-01-15

The draft genomes of Lactobacillus plantarum strains isolated from Asian fermented foods, infant feces, and shrimp intestines were sequenced and compared to those of well-studied strains. Among 28 strains of L. plantarum, variations in the genomic features involved in ecological adaptation were elucidated. The genome sizes ranged from approximately 3.1 to 3.5 Mb, of which about 2,932 to 3,345 protein-coding sequences (CDS) were predicted. The food-derived isolates contained a higher number of carbohydrate metabolism-associated genes than those from infant feces. This observation correlated to their phenotypic carbohydrate metabolic profile, indicating their ability to metabolize the largest range of sugars. Surprisingly, two strains (P14 and P76) isolated from fermented fish utilized inulin. β-Fructosidase, the inulin-degrading enzyme, was detected in the supernatants and cell wall extracts of both strains. No activity was observed in the cytoplasmic fraction, indicating that this key enzyme was either membrane-bound or extracellularly secreted. From genomic mining analysis, a predicted inulin operon of fosRABCDXE, which encodes β-fructosidase and many fructose transporting proteins, was found within the genomes of strains P14 and P76. Moreover, pts1BCA genes, encoding sucrose-specific IIBCA components involved in sucrose transport, were also identified. The proteomic analysis revealed the mechanism and functional characteristic of the fosRABCDXE operon involved in the inulin utilization of L. plantarum The expression levels of the fos operon and pst genes were upregulated at mid-log phase. FosE and the LPXTG-motif cell wall anchored β-fructosidase were induced to a high abundance when inulin was present as a carbon source. Inulin is a long-chain carbohydrate that may act as a prebiotic, which provides many health benefits to the host by selectively stimulating the growth and activity of beneficial bacteria in the colon. While certain lactobacilli can catabolize inulin, this has not yet been described for Lactobacillus plantarum, and an associated putative inulin operon has not been reported in this species. By using comparative and functional genomics, we showed that two L. plantarum strains utilized inulin and identified functional inulin operons in their genomes. The proteogenomic data revealed that inulin degradation and uptake routes, which related to the fosRABCDXE operon and pstBCA genes, were widely expressed among L. plantarum strains. The present work provides a novel understanding of gene regulation and mechanisms of inulin utilization in probiotic L. plantarum generating opportunities for synbiotic product development. Copyright © 2016 American Society for Microbiology.

An Inducible Operon Is Involved in Inulin Utilization in Lactobacillus plantarum Strains, as Revealed by Comparative Proteogenomics and Metabolic Profiling

PubMed Central

Buntin, Nirunya; Hongpattarakere, Tipparat; Ritari, Jarmo; Douillard, François P.; Paulin, Lars; Boeren, Sjef; Shetty, Sudarshan A.

2016-01-01

ABSTRACT The draft genomes of Lactobacillus plantarum strains isolated from Asian fermented foods, infant feces, and shrimp intestines were sequenced and compared to those of well-studied strains. Among 28 strains of L. plantarum, variations in the genomic features involved in ecological adaptation were elucidated. The genome sizes ranged from approximately 3.1 to 3.5 Mb, of which about 2,932 to 3,345 protein-coding sequences (CDS) were predicted. The food-derived isolates contained a higher number of carbohydrate metabolism-associated genes than those from infant feces. This observation correlated to their phenotypic carbohydrate metabolic profile, indicating their ability to metabolize the largest range of sugars. Surprisingly, two strains (P14 and P76) isolated from fermented fish utilized inulin. β-Fructosidase, the inulin-degrading enzyme, was detected in the supernatants and cell wall extracts of both strains. No activity was observed in the cytoplasmic fraction, indicating that this key enzyme was either membrane-bound or extracellularly secreted. From genomic mining analysis, a predicted inulin operon of fosRABCDXE, which encodes β-fructosidase and many fructose transporting proteins, was found within the genomes of strains P14 and P76. Moreover, pts1BCA genes, encoding sucrose-specific IIBCA components involved in sucrose transport, were also identified. The proteomic analysis revealed the mechanism and functional characteristic of the fosRABCDXE operon involved in the inulin utilization of L. plantarum. The expression levels of the fos operon and pst genes were upregulated at mid-log phase. FosE and the LPXTG-motif cell wall anchored β-fructosidase were induced to a high abundance when inulin was present as a carbon source. IMPORTANCE Inulin is a long-chain carbohydrate that may act as a prebiotic, which provides many health benefits to the host by selectively stimulating the growth and activity of beneficial bacteria in the colon. While certain lactobacilli can catabolize inulin, this has not yet been described for Lactobacillus plantarum, and an associated putative inulin operon has not been reported in this species. By using comparative and functional genomics, we showed that two L. plantarum strains utilized inulin and identified functional inulin operons in their genomes. The proteogenomic data revealed that inulin degradation and uptake routes, which related to the fosRABCDXE operon and pstBCA genes, were widely expressed among L. plantarum strains. The present work provides a novel understanding of gene regulation and mechanisms of inulin utilization in probiotic L. plantarum generating opportunities for synbiotic product development. PMID:27815279

The sim Operon Facilitates the Transport and Metabolism of Sucrose Isomers in Lactobacillus casei ATCC 334▿

PubMed Central

Thompson, John; Jakubovics, Nicholas; Abraham, Bindu; Hess, Sonja; Pikis, Andreas

2008-01-01

Inspection of the genome sequence of Lactobacillus casei ATCC 334 revealed two operons that might dissimilate the five isomers of sucrose. To test this hypothesis, cells of L. casei ATCC 334 were grown in a defined medium supplemented with various sugars, including each of the five isomeric disaccharides. Extracts prepared from cells grown on the sucrose isomers contained high levels of two polypeptides with Mrs of ∼50,000 and ∼17,500. Neither protein was present in cells grown on glucose, maltose or sucrose. Proteomic, enzymatic, and Western blot analyses identified the ∼50-kDa protein as an NAD+- and metal ion-dependent phospho-α-glucosidase. The oligomeric enzyme was purified, and a catalytic mechanism is proposed. The smaller polypeptide represented an EIIA component of the phosphoenolpyruvate-dependent sugar phosphotransferase system. Phospho-α-glucosidase and EIIA are encoded by genes at the LSEI_0369 (simA) and LSEI_0374 (simF) loci, respectively, in a block of seven genes comprising the sucrose isomer metabolism (sim) operon. Northern blot analyses provided evidence that three mRNA transcripts were up-regulated during logarithmic growth of L. casei ATCC 334 on sucrose isomers. Internal simA and simF gene probes hybridized to ∼1.5- and ∼1.3-kb transcripts, respectively. A 6.8-kb mRNA transcript was detected by both probes, which was indicative of cotranscription of the entire sim operon. PMID:18310337

Comparison of the overlapping frd and ampC operons of Escherichia coli with the corresponding DNA sequences in other gram-negative bacteria.

PubMed

Bergström, S; Lindberg, F P; Olsson, O; Normark, S

1983-09-01

Specific DNA probes from Escherichia coli K-12 were used to analyze the sequence divergence of the frd and ampC operons in various species of gram-negative bacteria. These operons code for the fumarate reductase complex and the chromosomal beta-lactamase, respectively. We demonstrate that the two operons show the same general pattern of divergence, although the frd operon is considerably more conserved than is the ampC operon. The major exception is Salmonella typhimurium LT2, which shows a strong homology to the E. coli frd probe but none to the E. coli ampC probe. The operons from Citrobacter freundii and Shigella sonnei were cloned and characterized by physical mapping, Southern hybridization, and protein synthesis in minicells. In S. sonnei, as in E. coli K-12, the frd and ampC operons overlap (T. Grundström and B. Jaurin, Proc. Natl. Acad. Sci. U.S.A. 79:1111-1115, 1982). Only minor discrepancies between the two operons were found over the entire frd-ampC region. In C. freundii, the ampC and frd operons do not overlap, being separated by about 1,100 base pairs. Presumably the inducible property of the C. freundii chromosomal beta-lactamase is encoded by this 1,100-base-pair DNA segment.

IncC of broad-host-range plasmid RK2 modulates KorB transcriptional repressor activity In vivo and operator binding in vitro.

PubMed

Jagura-Burdzy, G; Kostelidou, K; Pole, J; Khare, D; Jones, A; Williams, D R; Thomas, C M

1999-05-01

The korAB operon of broad-host-range plasmid RK2 encodes five genes, two of which, incC and korB, belong to the parA and parB families, respectively, of genome partitioning functions. Both korB and a third gene, korA, are responsible for coordinate regulation of operons encoding replication, transfer, and stable inheritance functions. Overexpression of incC alone caused rapid displacement of RK2. Using two different reporter systems, we show that incC modulates the action of KorB. Using promoter fusions to the reporter gene xylE, we show that incC potentiates the repression of transcription by korB. This modulation of korB activity was only observed with incC1, which encodes the full-length IncC (364 amino acids [aa]), whereas no effect was observed with incC2, which encodes a polypeptide of 259 aa that lacks the N-terminal 105 aa. Using bacterial extracts with IncC1 and IncC2 or IncC1 purified through the use of a His6 tail and Ni-agarose chromatography, we showed that IncC1 potentiates the binding of KorB to DNA at representative KorB operators. The ability of IncC to stabilize KorB-DNA complexes suggests that these two proteins work together in the global regulation of many operons on the IncP-1 genomes, as well in plasmid partitioning.

Hierarchical regulation of photosynthesis gene expression by the oxygen-responsive PrrBA and AppA-PpsR systems of Rhodobacter sphaeroides.

PubMed

Gomelsky, Larissa; Moskvin, Oleg V; Stenzel, Rachel A; Jones, Denise F; Donohue, Timothy J; Gomelsky, Mark

2008-12-01

In the facultatively phototrophic proteobacterium Rhodobacter sphaeroides, formation of the photosynthetic apparatus is oxygen dependent. When oxygen tension decreases, the response regulator PrrA of the global two-component PrrBA system is believed to directly activate transcription of the puf, puh, and puc operons, encoding structural proteins of the photosynthetic complexes, and to indirectly upregulate the photopigment biosynthesis genes bch and crt. Decreased oxygen also results in inactivation of the photosynthesis-specific repressor PpsR, bringing about derepression of the puc, bch, and crt operons. We uncovered a hierarchical relationship between these two regulatory systems, earlier thought to function independently. We also more accurately assessed the spectrum of gene targets of the PrrBA system. First, expression of the appA gene, encoding the PpsR antirepressor, is PrrA dependent, which establishes one level of hierarchical dominance of the PrrBA system over AppA-PpsR. Second, restoration of the appA transcript to the wild-type level is insufficient for rescuing phototrophic growth impairment of the prrA mutant, whereas inactivation of ppsR is sufficient. This suggests that in addition to controlling appA transcription, PrrA affects the activity of the AppA-PpsR system via an as yet unidentified mechanism(s). Third, PrrA directly activates several bch and crt genes, traditionally considered to be the PpsR targets. Therefore, in R. sphaeroides, the global PrrBA system regulates photosynthesis gene expression (i) by rigorous control over the photosynthesis-specific AppA-PpsR regulatory system and (ii) by extensive direct transcription activation of genes encoding structural proteins of photosynthetic complexes as well as genes encoding photopigment biosynthesis enzymes.

BadR and BadM Proteins Transcriptionally Regulate Two Operons Needed for Anaerobic Benzoate Degradation by Rhodopseudomonas palustris

PubMed Central

Hirakawa, Hidetada; Hirakawa, Yuko; Greenberg, E. Peter

2015-01-01

The bacterium Rhodopseudomonas palustris grows with the aromatic acid benzoate and the alicyclic acid cyclohexanecarboxylate (CHC) as sole carbon sources. The enzymatic steps in an oxygen-independent pathway for CHC degradation have been elucidated, but it was unknown how the CHC operon (badHI aliAB badK) encoding the enzymes for CHC degradation was regulated. aliA and aliB encode enzymes for the conversion of CHC to cyclohex-1-enecarboxyl–coenzyme A (CHene-CoA). At this point, the pathway for CHC degradation merges with the pathway for anaerobic benzoate degradation, as CHene-CoA is an intermediate in both degradation pathways. Three enzymes, encoded by badK, badH, and badI, prepare and cleave the alicyclic ring of CHene-CoA to yield pimelyl-CoA. Here, we show that the MarR transcription factor family member, BadR, represses transcription of the CHC operon by binding near the transcription start site of badH. 2-Ketocyclohexane-1-carboxyl–CoA, an intermediate of CHC and benzoate degradation, interacts with BadR to abrogate repression. We also present evidence that the transcription factor BadM binds to the promoter of the badDEFGAB (Bad) operon for the anaerobic conversion of benzoate to CHene-CoA to repress its expression. Contrary to previous reports, BadR does not appear to control expression of the Bad operon. These data enhance our view of the transcriptional regulation of anaerobic benzoate degradation by R. palustris. PMID:25888170

Uptake of chitosan-derived D-glucosamine oligosaccharides in Streptomyces coelicolor A3(2).

PubMed

Viens, Pascal; Dubeau, Marie-Pierre; Kimura, Akane; Desaki, Yoshitake; Shinya, Tomonori; Shibuya, Naoto; Saito, Akihiro; Brzezinski, Ryszard

2015-05-01

The csnR gene, localized at the beginning of an operon, csnR-K, which organization is conserved through many actinomycete genomes, was previously shown to repress the transcription of the chitosanase gene csnA in Streptomyces lividans. However, knowledge on the function of the whole csnR-K operon in the metabolism of chitosan (an N-deacetylated derivative of chitin) remained limited. Mutants of S. coelicolor A3(2) harboring partial or total deletions of the csnR-K operon were analyzed for their capacity to uptake glucosamine oligosaccharides (GlcN)n. The csnR-K operon was autoregulated by CsnR repressor and its transcription was inducible by GlcN oligosaccharides. The operon controlled the uptake of GlcN oligosaccharides in S. coelicolor A3(2), with a minor contribution to the consumption of monomeric GlcN but not chitin-related N-acetylated derivatives. The deletion of the whole operon abolished the uptake of GlcN oligosaccharides. The CsnEFG transporter encoded by this operon is the front door for the assimilation of chitosan-derived hydrolysis products in S. coelicolor A3(2). The ATP-binding component MsiK was essential for CsnEFG transport function. Also, deletion of msiK abolished the induction of csnA transcription by GlcN oligosaccharides. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Carbon source-dependent expansion of the genetic code in bacteria

PubMed Central

Prat, Laure; Heinemann, Ilka U.; Aerni, Hans R.; Rinehart, Jesse; O’Donoghue, Patrick; Söll, Dieter

2012-01-01

Despite the fact that the genetic code is known to vary between organisms in rare cases, it is believed that in the lifetime of a single cell the code is stable. We found Acetohalobium arabaticum cells grown on pyruvate genetically encode 20 amino acids, but in the presence of trimethylamine (TMA), A. arabaticum dynamically expands its genetic code to 21 amino acids including pyrrolysine (Pyl). A. arabaticum is the only known organism that modulates the size of its genetic code in response to its environment and energy source. The gene cassette pylTSBCD, required to biosynthesize and genetically encode UAG codons as Pyl, is present in the genomes of 24 anaerobic archaea and bacteria. Unlike archaeal Pyl-decoding organisms that constitutively encode Pyl, we observed that A. arabaticum controls Pyl encoding by down-regulating transcription of the entire Pyl operon under growth conditions lacking TMA, to the point where no detectable Pyl-tRNAPyl is made in vivo. Pyl-decoding archaea adapted to an expanded genetic code by minimizing TAG codon frequency to typically ∼5% of ORFs, whereas Pyl-decoding bacteria (∼20% of ORFs contain in-frame TAGs) regulate Pyl-tRNAPyl formation and translation of UAG by transcriptional deactivation of genes in the Pyl operon. We further demonstrate that Pyl encoding occurs in a bacterium that naturally encodes the Pyl operon, and identified Pyl residues by mass spectrometry in A. arabaticum proteins including two methylamine methyltransferases. PMID:23185002

Coordinated Regulation of the EIIMan and fruRKI Operons of Streptococcus mutans by Global and Fructose-Specific Pathways.

PubMed

Zeng, Lin; Chakraborty, Brinta; Farivar, Tanaz; Burne, Robert A

2017-11-01

The glucose/mannose-phosphotransferase system (PTS) permease EII Man encoded by manLMN in the dental caries pathogen Streptococcus mutans has a dominant influence on sugar-specific, CcpA-independent catabolite repression (CR). Mutations in manL affect energy metabolism and virulence-associated traits, including biofilm formation, acid tolerance, and competence. Using promoter::reporter fusions, expression of the manLMN and the fruRKI operons, encoding a transcriptional regulator, a fructose-1-phosphate kinase and a fructose-PTS permease EII Fru , respectively, was monitored in response to carbohydrate source and in mutants lacking CcpA, FruR, and components of EII Man Expression of genes for EII Man and EII Fru was directly regulated by CcpA and CR, as evinced by in vivo and in vitro methods. Unexpectedly, not only was the fruRKI operon negatively regulated by FruR, but also so was manLMN Carbohydrate transport by EII Man had a negative influence on expression of manLMN but not fruRKI In agreement with the proposed role of FruR in regulating these PTS operons, loss of fruR or fruK substantially altered growth on a number of carbohydrates, including fructose. RNA deep sequencing revealed profound changes in gene regulation caused by deletion of fruK or fruR Collectively, these findings demonstrate intimate interconnection of the regulation of two major PTS permeases in S. mutans and reveal novel and important contributions of fructose metabolism to global regulation of gene expression. IMPORTANCE The ability of Streptococcus mutans and other streptococcal pathogens to survive and cause human diseases is directly dependent upon their capacity to metabolize a variety of carbohydrates, including glucose and fructose. Our research reveals that metabolism of fructose has broad influences on the regulation of utilization of glucose and other sugars, and mutants with changes in certain genes involved in fructose metabolism display profoundly different abilities to grow and express virulence-related traits. Mutants lacking the FruR regulator or a particular phosphofructokinase, FruK, display changes in expression of a large number of genes encoding transcriptional regulators, enzymes required for energy metabolism, biofilm development, biosynthetic and degradative processes, and tolerance of a spectrum of environmental stressors. Since fructose is a major component of the modern human diet, the results have substantial significance in the context of oral health and the development of dental caries. Copyright © 2017 American Society for Microbiology.

Synergic role of the two ars operons in arsenic tolerance in Pseudomonas putida KT2440.

PubMed

Fernández, Matilde; Udaondo, Zulema; Niqui, José-Luis; Duque, Estrella; Ramos, Juan-Luis

2014-10-01

The chromosome of Pseudomonas putida KT2440 carries two clusters of genes, denoted ars1 and ars2, that are annotated as putative arsenic resistance operons. In this work, we present evidence that both operons encode functional arsenic-response regulatory genes as well as arsenic extrusion systems that confer resistance to both arsenite [As(III)] and arsenate [As(V)]. Transcriptional fusions of P(ars1) and P(ars2) to lacZ revealed that expression of both operons was induced by arsenite and arsenate. We generated single mutants in ars1 and ars2, which showed lower resistance to arsenic than the wild-type strain. A double ars1/ars2 was found to be highly sensitive to arsenic. Minimum inhibitory concentrations (MICs) for single mutants decreased two- to fourfold with respect to the parental strain, while in the double mutant the MIC decreased 128-fold for arsenite and 32-fold for arsenate. Bioinformatic analysis revealed that the ars2 resistance operon is part of the core genome of P. putida, while the ars1 operon appears to only occur in the KT2440 strain, suggesting that ars1 was acquired by horizontal gene transfer. The presence of ars1 in KT2440 may explain why it exhibits higher resistance to arsenic than other P. putida strains, which bear only the ars2 operon.

Identification in Marinomonas mediterranea of a novel quinoprotein with glycine oxidase activity.

PubMed

Campillo-Brocal, Jonatan Cristian; Lucas-Elio, Patricia; Sanchez-Amat, Antonio

2013-08-01

A novel enzyme with lysine-epsilon oxidase activity was previously described in the marine bacterium Marinomonas mediterranea. This enzyme differs from other l-amino acid oxidases in not being a flavoprotein but containing a quinone cofactor. It is encoded by an operon with two genes lodA and lodB. The first one codes for the oxidase, while the second one encodes a protein required for the expression of the former. Genome sequencing of M. mediterranea has revealed that it contains two additional operons encoding proteins with sequence similarity to LodA. In this study, it is shown that the product of one of such genes, Marme_1655, encodes a protein with glycine oxidase activity. This activity shows important differences in terms of substrate range and sensitivity to inhibitors to other glycine oxidases previously described which are flavoproteins synthesized by Bacillus. The results presented in this study indicate that the products of the genes with different degrees of similarity to lodA detected in bacterial genomes could constitute a reservoir of different oxidases. © 2013 The Authors. Microbiology Open published by John Wiley & Sons Ltd.

Molecular Diversity of Plasmids Bearing Genes That Encode Toluene and Xylene Metabolism in Pseudomonas Strains Isolated from Different Contaminated Sites in Belarus

PubMed Central

Sentchilo, Vladimir S.; Perebituk, Alexander N.; Zehnder, Alexander J. B.; van der Meer, Jan Roelof

2000-01-01

Twenty different Pseudomonas strains utilizing m-toluate were isolated from oil-contaminated soil samples near Minsk, Belarus. Seventeen of these isolates carried plasmids ranging in size from 78 to about 200 kb (assigned pSVS plasmids) and encoding the meta cleavage pathway for toluene metabolism. Most plasmids were conjugative but of unknown incompatibility groups, except for one, which belonged to the IncP9 group. The organization of the genes for toluene catabolism was determined by restriction analysis and hybridization with xyl gene probes of pWW0. The majority of the plasmids carried xyl-type genes highly homologous to those of pWW53 and organized in a similar manner (M. T. Gallegos, P. A. Williams, and J. L. Ramos, J. Bacteriol. 179:5024–5029, 1997), with two distinguishable meta pathway operons, one upper pathway operon, and three xylS-homologous regions. All of these plasmids also possessed large areas of homologous DNA outside the catabolic genes, suggesting a common ancestry. Two other pSVS plasmids carried only one meta pathway operon, one upper pathway operon, and one copy each of xylS and xylR. The backbones of these two plasmids differed greatly from those of the others. Whereas these parts of the plasmids, carrying the xyl genes, were mostly conserved between plasmids of each group, the noncatabolic parts had undergone intensive DNA rearrangements. DNA sequencing of specific regions near and within the xylTE and xylA genes of the pSVS plasmids confirmed the strong homologies to the xyl genes of pWW53 and pWW0. However, several recombinations were discovered within the upper pathway operons of the pSVS plasmids and pWW0. The main genetic mechanisms which are thought to have resulted in the present-day configuration of the xyl operons are discussed in light of the diversity analysis carried out on the pSVS plasmids. PMID:10877777

Expression of the Kdp ATPase Is Consistent with Regulation by Turgor Pressure

PubMed Central

Malli, Ravi; Epstein, Wolfgang

1998-01-01

The kdpFABC operon of Escherichia coli encodes the four protein subunits of the Kdp K+ transport system. Kdp is expressed when growth is limited by the availability of K+. Expression of Kdp is dependent on the products of the adjacent kdpDE operon, which encodes a pair of two-component regulators. Studies with kdp-lac fusions led to the suggestion that change in turgor pressure acts as the signal to express Kdp (L. A. Laimins, D. B. Rhoads, and W. Epstein, Proc. Natl. Acad. Sci. USA 78:464–468, 1981). More recently, effects of compatible solutes, among others, have been interpreted as inconsistent with the turgor model (H. Asha and J. Gowrishankar, J. Bacteriol. 175:4528–4537, 1993). We re-examined the effects of compatible solutes and of medium pH on expression of Kdp in studies in which growth rate was also measured. In all cases, Kdp expression correlated with the K+ concentration when growth began to slow. Making the reasonable but currently untestable assumptions that the reduction in growth rate by K+ limitation is due to a reduction in turgor and that addition of betaine does not increase turgor, we concluded that all of the data on Kdp expression are consistent with control by turgor pressure. PMID:9748442

Phenotypical Analysis of the Lactobacillus rhamnosus GG Fimbrial spaFED Operon: Surface Expression and Functional Characterization of Recombinant SpaFED Pili in Lactococcus lactis

PubMed Central

Kant, Ravi; Palva, Airi; von Ossowski, Ingemar

2014-01-01

A noticeable genomic feature of many piliated Gram-positive bacterial species is the presence of more than one pilus-encoding operon. Paradigmatically, the gut-adapted Lactobacillus rhamnosus GG strain contains two different fimbrial operons in its genome. However, whereas one of these operons (called spaCBA) is encoding for the functionally mucus-/collagen-binding SpaCBA pilus, for the other operon (called spaFED) any native expression of the SpaFED-called pili is still the subject of some uncertainty. Irrespective of such considerations, we decided it would be of relevance or interest to decipher the gross structure of this pilus type, and as well assess its functional capabilities for cellular adhesion and immunostimulation. For this, and by following the approach we had used previously to explicate the immuno-properties of SpaCBA pili, we constructed nisin-inducible expression clones producing either wild-type or SpaF pilin-deleted surface-assembled L. rhamnosus GG SpaFED pili on Lactococcus lactis cells. Using these piliated lactococcal constructs, we found that the pilin-polymerized architecture of a recombinant-produced SpaFED pilus coincides with sequence-based functional predictions of the related pilins, and in fact is prototypical of those other sortase-dependent pilus-like structures thus far characterized for piliated Gram-positive bacteria. Moreover, we confirmed that among the different pilin subunits encompassing spaFED operon-encoded pili, the SpaF pilin is a main adhesion determinant, and when present in the assembled structure can mediate pilus binding to mucus, certain extracellular matrix proteins, and different gut epithelial cell lines. However, somewhat unexpectedly, when recombinant SpaFED pili are surface-attached, we found that they could not potentiate the existing lactococcal cell-induced immune responses so elicited from intestinal- and immune-related cells, but rather instead, they could dampen them. Accordingly, we have now provided the first phenotypical description of a spaFED pilus operon, and with that furthered the functional understanding of surface piliation for a particular gut-commensalic genre of piliated Gram-positive bacteria. PMID:25415357

Phenotypical analysis of the Lactobacillus rhamnosus GG fimbrial spaFED operon: surface expression and functional characterization of recombinant SpaFED pili in Lactococcus lactis.

PubMed

Rintahaka, Johanna; Yu, Xia; Kant, Ravi; Palva, Airi; von Ossowski, Ingemar

2014-01-01

A noticeable genomic feature of many piliated Gram-positive bacterial species is the presence of more than one pilus-encoding operon. Paradigmatically, the gut-adapted Lactobacillus rhamnosus GG strain contains two different fimbrial operons in its genome. However, whereas one of these operons (called spaCBA) is encoding for the functionally mucus-/collagen-binding SpaCBA pilus, for the other operon (called spaFED) any native expression of the SpaFED-called pili is still the subject of some uncertainty. Irrespective of such considerations, we decided it would be of relevance or interest to decipher the gross structure of this pilus type, and as well assess its functional capabilities for cellular adhesion and immunostimulation. For this, and by following the approach we had used previously to explicate the immuno-properties of SpaCBA pili, we constructed nisin-inducible expression clones producing either wild-type or SpaF pilin-deleted surface-assembled L. rhamnosus GG SpaFED pili on Lactococcus lactis cells. Using these piliated lactococcal constructs, we found that the pilin-polymerized architecture of a recombinant-produced SpaFED pilus coincides with sequence-based functional predictions of the related pilins, and in fact is prototypical of those other sortase-dependent pilus-like structures thus far characterized for piliated Gram-positive bacteria. Moreover, we confirmed that among the different pilin subunits encompassing spaFED operon-encoded pili, the SpaF pilin is a main adhesion determinant, and when present in the assembled structure can mediate pilus binding to mucus, certain extracellular matrix proteins, and different gut epithelial cell lines. However, somewhat unexpectedly, when recombinant SpaFED pili are surface-attached, we found that they could not potentiate the existing lactococcal cell-induced immune responses so elicited from intestinal- and immune-related cells, but rather instead, they could dampen them. Accordingly, we have now provided the first phenotypical description of a spaFED pilus operon, and with that furthered the functional understanding of surface piliation for a particular gut-commensalic genre of piliated Gram-positive bacteria.

Cation-induced transcriptional regulation of the dlt operon of Staphylococcus aureus.

PubMed

Koprivnjak, Tomaz; Mlakar, Vid; Swanson, Lindsey; Fournier, Benedicte; Peschel, Andreas; Weiss, Jerrold P

2006-05-01

Lipoteichoic and wall teichoic acids (TA) are highly anionic cell envelope-associated polymers containing repeating polyglycerol/ribitol phosphate moieties. Substitution of TA with D-alanine is important for modulation of many cell envelope-dependent processes, such as activity of autolytic enzymes, binding of divalent cations, and susceptibility to innate host defenses. D-Alanylation of TA is diminished when bacteria are grown in medium containing increased NaCl concentrations, but the effects of increased salt concentration on expression of the dlt operon encoding proteins mediating D-alanylation of TA are unknown. We demonstrate that Staphylococcus aureus transcriptionally represses dlt expression in response to high concentrations of Na(+) and moderate concentrations of Mg(2+) and Ca(2+) but not sucrose. Changes in dlt mRNA are induced within 15 min and sustained for several generations of growth. Mg(2+)-induced dlt repression depends on the ArlSR two-component system. Northern blotting, reverse transcription-PCR, and SMART-RACE analyses suggest that the dlt transcript begins 250 bp upstream of the dltA start codon and includes an open reading frame immediately upstream of dltA. Chloramphenicol transacetylase transcriptional fusions indicate that a region encompassing the 171 to 325 bp upstream of dltA is required for expression and Mg(2+)-induced repression of the dlt operon in S. aureus.

Cation-Induced Transcriptional Regulation of the dlt Operon of Staphylococcus aureus

PubMed Central

Koprivnjak, Tomaz; Mlakar, Vid; Swanson, Lindsey; Fournier, Benedicte; Peschel, Andreas; Weiss, Jerrold P.

2006-01-01

Lipoteichoic and wall teichoic acids (TA) are highly anionic cell envelope-associated polymers containing repeating polyglycerol/ribitol phosphate moieties. Substitution of TA with d-alanine is important for modulation of many cell envelope-dependent processes, such as activity of autolytic enzymes, binding of divalent cations, and susceptibility to innate host defenses. d-Alanylation of TA is diminished when bacteria are grown in medium containing increased NaCl concentrations, but the effects of increased salt concentration on expression of the dlt operon encoding proteins mediating d-alanylation of TA are unknown. We demonstrate that Staphylococcus aureus transcriptionally represses dlt expression in response to high concentrations of Na+ and moderate concentrations of Mg2+ and Ca2+ but not sucrose. Changes in dlt mRNA are induced within 15 min and sustained for several generations of growth. Mg2+-induced dlt repression depends on the ArlSR two-component system. Northern blotting, reverse transcription-PCR, and SMART-RACE analyses suggest that the dlt transcript begins 250 bp upstream of the dltA start codon and includes an open reading frame immediately upstream of dltA. Chloramphenicol transacetylase transcriptional fusions indicate that a region encompassing the 171 to 325 bp upstream of dltA is required for expression and Mg2+-induced repression of the dlt operon in S. aureus. PMID:16672616

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.

Transcription of the extended hyp-operon in Nostoc sp. strain PCC 7120

PubMed Central

Agervald, Åsa; Stensjö, Karin; Holmqvist, Marie; Lindblad, Peter

2008-01-01

Background The maturation of hydrogenases into active enzymes is a complex process and e.g. a correctly assembled active site requires the involvement of at least seven proteins, encoded by hypABCDEF and a hydrogenase specific protease, encoded either by hupW or hoxW. The N2-fixing cyanobacterium Nostoc sp. strain PCC 7120 may contain both an uptake and a bidirectional hydrogenase. The present study addresses the presence and expression of hyp-genes in Nostoc sp. strain PCC 7120. Results RT-PCRs demonstrated that the six hyp-genes together with one ORF may be transcribed as a single operon. Transcriptional start points (TSPs) were identified 280 bp upstream from hypF and 445 bp upstream of hypC, respectively, demonstrating the existence of several transcripts. In addition, five upstream ORFs located in between hupSL, encoding the small and large subunits of the uptake hydrogenase, and the hyp-operon, and two downstream ORFs from the hyp-genes were shown to be part of the same transcript unit. A third TSP was identified 45 bp upstream of asr0689, the first of five ORFs in this operon. The ORFs are annotated as encoding unknown proteins, with the exception of alr0692 which is identified as a NifU-like protein. Orthologues of the four ORFs asr0689-alr0692, with a highly conserved genomic arrangement positioned between hupSL, and the hyp genes are found in several other N2-fixing cyanobacteria, but are absent in non N2-fixing cyanobacteria with only the bidirectional hydrogenase. Short conserved sequences were found in six intergenic regions of the extended hyp-operon, appearing between 11 and 79 times in the genome. Conclusion This study demonstrated that five ORFs upstream of the hyp-gene cluster are co-transcribed with the hyp-genes, and identified three TSPs in the extended hyp-gene cluster in Nostoc sp. strain PCC 7120. This may indicate a function related to the assembly of a functional uptake hydrogenase, hypothetically in the assembly of the small subunit of the enzyme. PMID:18442387

Novel Twin Streptolysin S-Like Peptides Encoded in the sag Operon Homologue of Beta-Hemolytic Streptococcus anginosus

PubMed Central

Tabata, Atsushi; Nakano, Kota; Ohkura, Kazuto; Tomoyasu, Toshifumi; Kikuchi, Ken; Whiley, Robert A.

2013-01-01

Streptococcus anginosus is a member of the anginosus group streptococci, which form part of the normal human oral flora. In contrast to the pyogenic group streptococci, our knowledge of the virulence factors of the anginosus group streptococci, including S. anginosus, is not sufficient to allow a clear understanding of the basis of their pathogenicity. Generally, hemolysins are thought to be important virulence factors in streptococcal infections. In the present study, a sag operon homologue was shown to be responsible for beta-hemolysis in S. anginosus strains by random gene knockout. Interestingly, contrary to pyogenic group streptococci, beta-hemolytic S. anginosus was shown to have two tandem sagA homologues, encoding streptolysin S (SLS)-like peptides, in the sag operon homologue. Gene deletion and complementation experiments revealed that both genes were functional, and these SLS-like peptides were essential for beta-hemolysis in beta-hemolytic S. anginosus. Furthermore, the amino acid sequence of these SLS-like peptides differed from that of the typical SLS of S. pyogenes, especially in their propeptide domain, and an amino acid residue indicated to be important for the cytolytic activity of SLS in S. pyogenes was deleted in both S. anginosus homologues. These data suggest that SLS-like peptides encoded by two sagA homologues in beta-hemolytic S. anginosus may be potential virulence factors with a different structure essential for hemolytic activity and/or the maturation process compared to the typical SLS present in pyogenic group streptococci. PMID:23292771

Identification and characterization of the gltK gene encoding a membrane-associated glucose transport protein of pseudomonas aeruginosa.

PubMed

Adewoye, L O; Worobec, E A

2000-08-08

The Pseudomonas aeruginosa oprB gene encodes the carbohydrate-selective OprB porin, which translocates substrate molecules across the outer membrane to the periplasmic glucose-binding protein. We identified and cloned two open reading frames (ORFs) flanking the oprB gene but are not in operonic arrangement with the oprB gene. The downstream ORF encodes a putative polypeptide homologous to members of a family of transcriptional repressors, whereas the oprB gene is preceded by an ORF encoding a putative product, which exhibits strong homology to several carbohydrate transport ATP-binding cassette (ABC) proteins. The genomic copy of the upstream ORF was mutagenized by homologous recombination. Analysis of the deletion mutant in comparison with the wild type revealed a significant reduction in [14C] glucose transport activity in the mutant strain, suggesting that this ORF likely encodes the inner membrane component of the glucose ABC transporter. It is thus designated gltK gene to reflect its homology to the Pseudomona fluorescens mtlK and its involvement in the high-affinity glucose transport system. Multiple alignment analysis revealed that the P. aeruginosa gltK gene product is a member of the MalK subfamily of ABC proteins.

AguR, a Transmembrane Transcription Activator of the Putrescine Biosynthesis Operon in Lactococcus lactis, Acts in Response to the Agmatine Concentration.

PubMed

Linares, Daniel M; Del Rio, Beatriz; Redruello, Begoña; Ladero, Victor; Martin, M Cruz; de Jong, Anne; Kuipers, Oscar P; Fernandez, Maria; Alvarez, Miguel A

2015-09-01

Dairy industry fermentative processes mostly use Lactococcus lactis as a starter. However, some dairy L. lactis strains produce putrescine, a biogenic amine that raises food safety and spoilage concerns, via the agmatine deiminase (AGDI) pathway. The enzymatic activities responsible for putrescine biosynthesis in this bacterium are encoded by the AGDI gene cluster. The role of the catabolic genes aguB, aguD, aguA, and aguC has been studied, but knowledge regarding the role of aguR (the first gene in the cluster) remains limited. In the present work, aguR was found to be a very low level constitutively expressed gene that is essential for putrescine biosynthesis and is transcribed independently of the polycistronic mRNA encoding the catabolic genes (aguBDAC). In response to agmatine, AguR acts as a transcriptional activator of the aguB promoter (PaguB), which drives the transcription of the aguBDAC operon. Inverted sequences required for PaguB activity were identified by deletion analysis. Further work indicated that AguR is a transmembrane protein which might function as a one-component signal transduction system that senses the agmatine concentration of the medium and, accordingly, regulates the transcription of the aguBDAC operon through a C-terminal cytoplasmic DNA-binding domain typically found in LuxR-like proteins. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

AguR, a Transmembrane Transcription Activator of the Putrescine Biosynthesis Operon in Lactococcus lactis, Acts in Response to the Agmatine Concentration

PubMed Central

Linares, Daniel M.; del Rio, Beatriz; Redruello, Begoña; Martin, M. Cruz; de Jong, Anne; Kuipers, Oscar P.; Fernandez, Maria

2015-01-01

Dairy industry fermentative processes mostly use Lactococcus lactis as a starter. However, some dairy L. lactis strains produce putrescine, a biogenic amine that raises food safety and spoilage concerns, via the agmatine deiminase (AGDI) pathway. The enzymatic activities responsible for putrescine biosynthesis in this bacterium are encoded by the AGDI gene cluster. The role of the catabolic genes aguB, aguD, aguA, and aguC has been studied, but knowledge regarding the role of aguR (the first gene in the cluster) remains limited. In the present work, aguR was found to be a very low level constitutively expressed gene that is essential for putrescine biosynthesis and is transcribed independently of the polycistronic mRNA encoding the catabolic genes (aguBDAC). In response to agmatine, AguR acts as a transcriptional activator of the aguB promoter (PaguB), which drives the transcription of the aguBDAC operon. Inverted sequences required for PaguB activity were identified by deletion analysis. Further work indicated that AguR is a transmembrane protein which might function as a one-component signal transduction system that senses the agmatine concentration of the medium and, accordingly, regulates the transcription of the aguBDAC operon through a C-terminal cytoplasmic DNA-binding domain typically found in LuxR-like proteins. PMID:26116671

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

An Iron-Regulated Autolysin Remodels the Cell Wall To Facilitate Heme Acquisition in Staphylococcus lugdunensis

PubMed Central

Farrand, Allison J.; Haley, Kathryn P.; Lareau, Nichole M.; Heilbronner, Simon; McLean, John A.; Foster, Timothy

2015-01-01

Bacteria alter their cell surface in response to changing environments, including those encountered upon invasion of a host during infection. One alteration that occurs in several Gram-positive pathogens is the presentation of cell wall-anchored components of the iron-regulated surface determinant (Isd) system, which extracts heme from host hemoglobin to fulfill the bacterial requirement for iron. Staphylococcus lugdunensis, an opportunistic pathogen that causes infective endocarditis, encodes an Isd system. Unique among the known Isd systems, S. lugdunensis contains a gene encoding a putative autolysin located adjacent to the Isd operon. To elucidate the function of this putative autolysin, here named IsdP, we investigated its contribution to Isd protein localization and hemoglobin-dependent iron acquisition. S. lugdunensis IsdP was found to be iron regulated and cotranscribed with the Isd operon. IsdP is a specialized peptidoglycan hydrolase that cleaves the stem peptide and pentaglycine crossbridge of the cell wall and alters processing and anchoring of a major Isd system component, IsdC. Perturbation of IsdC localization due to isdP inactivation results in a hemoglobin utilization growth defect. These studies establish IsdP as an autolysin that functions in heme acquisition and describe a role for IsdP in cell wall reorganization to accommodate nutrient uptake systems during infection. PMID:26123800

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

Molecular Characterization and Regulation of the aguBA Operon, Responsible for Agmatine Utilization in Pseudomonas aeruginosa PAO1

PubMed Central

Nakada, Yuji; Jiang, Ying; Nishijyo, Takayuki; Itoh, Yoshifumi; Lu, Chung-Dar

2001-01-01

Pseudomonas aeruginosa PAO1 utilizes agmatine as the sole carbon and nitrogen source via two reactions catalyzed successively by agmatine deiminase (encoded by aguA; also called agmatine iminohydrolase) and N-carbamoylputrescine amidohydrolase (encoded by aguB). The aguBA and adjacent aguR genes were cloned and characterized. The predicted AguB protein (Mr 32,759; 292 amino acids) displayed sequence similarity (≤60% identity) to enzymes of the β-alanine synthase/nitrilase family. While the deduced AguA protein (Mr 41,190; 368 amino acids) showed no significant similarity to any protein of known function, assignment of agmatine deiminase to AguA in this report discovered a new family of carbon-nitrogen hydrolases widely distributed in organisms ranging from bacteria to Arabidopsis. The aguR gene encoded a putative regulatory protein (Mr 24,424; 221 amino acids) of the TetR protein family. Measurements of agmatine deiminase and N-carbamoylputrescine amidohydrolase activities indicated the induction effect of agmatine and N-carbamoylputrescine on expression of the aguBA operon. The presence of an inducible promoter for the aguBA operon in the aguR-aguB intergenic region was demonstrated by lacZ fusion experiments, and the transcription start of this promoter was localized 99 bp upstream from the initiation codon of aguB by S1 nuclease mapping. Experiments with knockout mutants of aguR established that expression of the aguBA operon became constitutive in the aguR background. Interaction of AguR overproduced in Escherichia coli with the aguBA regulatory region was demonstrated by gel retardation assays, supporting the hypothesis that AguR serves as the negative regulator of the aguBA operon, and binding of agmatine and N-carbamoylputrescine to AguR would antagonize its repressor function. PMID:11673419

Two-component regulators involved in the global control of virulence in Erwinia carotovora subsp. carotovora.

PubMed

Eriksson, A R; Andersson, R A; Pirhonen, M; Palva, E T

1998-08-01

Production of extracellular, plant cell wall degrading enzymes, the main virulence determinants of the plant pathogen Erwinia carotovora subsp. carotovora, is coordinately controlled by a complex regulatory network. Insertion mutants in the exp (extracellular enzyme production) loci exhibit pleiotropic defects in virulence and the growth-phase-dependent transcriptional activation of genes encoding extracellular enzymes. Two new exp mutations, designated expA and expS, were characterized. Introduction of the corresponding wild-type alleles to the mutants complemented both the lack of virulence and the impaired production of plant cell wall degrading enzymes. The expA gene was shown to encode a 24-kDa polypeptide that is structurally and functionally related to the uvrY gene product of Escherichia coli and the GacA response regulator of Pseudomonas fluorescens. Functional similarity of expA and uvrY was demonstrated by genetic complementation. The expA gene is organized in an operon together with a uvrC-like gene, identical to the organization of uvrY and uvrC in E. coli. The unlinked expS gene encodes a putative sensor kinase that shows 92% identity to the recently described rpfA gene product from another E. carotovora subsp. carotovora strain. Our data suggest that ExpS and ExpA are members of two-component sensor kinase and response regulator families, respectively. These two proteins might interact in controlling virulence gene expression in E. carotovora subsp. carotovora.

The CopRS Two-Component System Is Responsible for Resistance to Copper in the Cyanobacterium Synechocystis sp. PCC 68031[C][W][OA

PubMed Central

Giner-Lamia, Joaquín; López-Maury, Luis; Reyes, José C.; Florencio, Francisco J.

2012-01-01

Photosynthetic organisms need copper for cytochrome oxidase and for plastocyanin in the fundamental processes of respiration and photosynthesis. However, excess of free copper is detrimental inside the cells and therefore organisms have developed homeostatic mechanisms to tightly regulate its acquisition, sequestration, and efflux. Herein we show that the CopRS two-component system (also known as Hik31-Rre34) is essential for copper resistance in Synechocystis sp. PCC 6803. It regulates expression of a putative heavy-metal efflux-resistance nodulation and division type copper efflux system (encoded by copBAC) as well as its own expression (in the copMRS operon) in response to the presence of copper in the media. Mutants in this two-component system or the efflux system render cells more sensitive to the presence of copper in the media and accumulate more intracellular copper than the wild type. Furthermore, CopS periplasmic domain is able to bind copper, suggesting that CopS could be able to detect copper directly. Both operons (copMRS and copBAC) are also induced by the photosynthetic inhibitor 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone but this induction requires the presence of copper in the media. The reduced response of two mutant strains to copper, one lacking plastocyanin and a second one impaired in copper transport to the thylakoid, due to the absence of the PI-type ATPases PacS and CtaA, suggests that CopS can detect intracellular copper. In addition, a tagged version of CopS with a triple HA epitope localizes to both the plasma and the thylakoid membranes, suggesting that CopS could be involved in copper detection in both the periplasm and the thylakoid lumen. PMID:22715108

The CopRS two-component system is responsible for resistance to copper in the cyanobacterium Synechocystis sp. PCC 6803.

PubMed

Giner-Lamia, Joaquín; López-Maury, Luis; Reyes, José C; Florencio, Francisco J

2012-08-01

Photosynthetic organisms need copper for cytochrome oxidase and for plastocyanin in the fundamental processes of respiration and photosynthesis. However, excess of free copper is detrimental inside the cells and therefore organisms have developed homeostatic mechanisms to tightly regulate its acquisition, sequestration, and efflux. Herein we show that the CopRS two-component system (also known as Hik31-Rre34) is essential for copper resistance in Synechocystis sp. PCC 6803. It regulates expression of a putative heavy-metal efflux-resistance nodulation and division type copper efflux system (encoded by copBAC) as well as its own expression (in the copMRS operon) in response to the presence of copper in the media. Mutants in this two-component system or the efflux system render cells more sensitive to the presence of copper in the media and accumulate more intracellular copper than the wild type. Furthermore, CopS periplasmic domain is able to bind copper, suggesting that CopS could be able to detect copper directly. Both operons (copMRS and copBAC) are also induced by the photosynthetic inhibitor 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone but this induction requires the presence of copper in the media. The reduced response of two mutant strains to copper, one lacking plastocyanin and a second one impaired in copper transport to the thylakoid, due to the absence of the P(I)-type ATPases PacS and CtaA, suggests that CopS can detect intracellular copper. In addition, a tagged version of CopS with a triple HA epitope localizes to both the plasma and the thylakoid membranes, suggesting that CopS could be involved in copper detection in both the periplasm and the thylakoid lumen.

Bacterial cellulose biosynthesis: diversity of operons, subunits, products, and functions.

PubMed

Römling, Ute; Galperin, Michael Y

2015-09-01

Recent studies of bacterial cellulose biosynthesis, including structural characterization of a functional cellulose synthase complex, provided the first mechanistic insight into this fascinating process. In most studied bacteria, just two subunits, BcsA and BcsB, are necessary and sufficient for the formation of the polysaccharide chain in vitro. Other subunits - which differ among various taxa - affect the enzymatic activity and product yield in vivo by modulating (i) the expression of the biosynthesis apparatus, (ii) the export of the nascent β-D-glucan polymer to the cell surface, and (iii) the organization of cellulose fibers into a higher-order structure. These auxiliary subunits play key roles in determining the quantity and structure of resulting biofilms, which is particularly important for the interactions of bacteria with higher organisms - leading to rhizosphere colonization and modulating the virulence of cellulose-producing bacterial pathogens inside and outside of host cells. We review the organization of four principal types of cellulose synthase operon found in various bacterial genomes, identify additional bcs genes that encode components of the cellulose biosynthesis and secretion machinery, and propose a unified nomenclature for these genes and subunits. We also discuss the role of cellulose as a key component of biofilms and in the choice between acute infection and persistence in the host. Copyright © 2015 Elsevier Ltd. All rights reserved.

Ancient Origin of the Tryptophan Operon and the Dynamics of Evolutionary Change†

PubMed Central

Xie, Gary; Keyhani, Nemat O.; Bonner; Jensen, Roy A.

2003-01-01

The seven conserved enzymatic domains required for tryptophan (Trp) biosynthesis are encoded in seven genetic regions that are organized differently (whole-pathway operons, multiple partial-pathway operons, and dispersed genes) in prokaryotes. A comparative bioinformatics evaluation of the conservation and organization of the genes of Trp biosynthesis in prokaryotic operons should serve as an excellent model for assessing the feasibility of predicting the evolutionary histories of genes and operons associated with other biochemical pathways. These comparisons should provide a better understanding of possible explanations for differences in operon organization in different organisms at a genomics level. These analyses may also permit identification of some of the prevailing forces that dictated specific gene rearrangements during the course of evolution. Operons concerned with Trp biosynthesis in prokaryotes have been in a dynamic state of flux. Analysis of closely related organisms among the Bacteria at various phylogenetic nodes reveals many examples of operon scission, gene dispersal, gene fusion, gene scrambling, and gene loss from which the direction of evolutionary events can be deduced. Two milestone evolutionary events have been mapped to the 16S rRNA tree of Bacteria, one splitting the operon in two, and the other rejoining it by gene fusion. The Archaea, though less resolved due to a lesser genome representation, appear to exhibit more gene scrambling than the Bacteria. The trp operon appears to have been an ancient innovation; it was already present in the common ancestor of Bacteria and Archaea. Although the operon has been subjected, even in recent times, to dynamic changes in gene rearrangement, the ancestral gene order can be deduced with confidence. The evolutionary history of the genes of the pathway is discernible in rough outline as a vertical line of descent, with events of lateral gene transfer or paralogy enriching the analysis as interesting features that can be distinguished. As additional genomes are thoroughly analyzed, an increasingly refined resolution of the sequential evolutionary steps is clearly possible. These comparisons suggest that present-day trp operons that possess finely tuned regulatory features are under strong positive selection and are able to resist the disruptive evolutionary events that may be experienced by simpler, poorly regulated operons. PMID:12966138

Permanent draft genome of Thiobacillus thioparus DSM 505T, an obligately chemolithoautotrophic member of the Betaproteobacteria

DOE PAGES

Hutt, Lee P.; Huntemann, Marcel; Clum, Alicia; ...

2017-01-19

Thiobacillus thioparus DSM 505 T is one of first two isolated strains of inorganic sulfur-oxidising Bacteria. The original strain of T. thioparus was lost almost 100 years ago and the working type strain is Culture C T (=DSM 505 T = ATCC 8158 T ) isolated by Starkey in 1934 from agricultural soil at Rutgers University, New Jersey, USA. It is an obligate chemolithoautotroph that conserves energy from the oxidation of reduced inorganic sulfur compounds using the Kelly-Trudinger pathway and uses it to fix carbon dioxide It is not capable of heterotrophic or mixotrophic growth. The strain has a genomemore » size of 3,201,518 bp. Here we report the genome sequence, annotation and characteristics. The genome contains 3,135 protein coding and 62 RNA coding genes. Genes encoding the transaldolase variant of the Calvin-Benson-Bassham cycle were also identified and an operon encoding carboxysomes, along with Smith's biosynthetic horseshoe in lieu of Krebs' cycle sensu stricto. Terminal oxidases were identified, viz. cytochrome c oxidase (cbb3, EC 1.9.3.1) and ubiquinol oxidase (bd, EC 1.10.3.10). There is a partial sox operon of the Kelly-Friedrich pathway of inorganic sulfur-oxidation that contains soxXYZAB genes but lacking soxCDEF, there is also a lack of the DUF302 gene previously noted in the sox operon of other members of the 'Proteobacteria' that can use trithionate as an energy source. In spite of apparently not growing anaerobically with denitrification, the nar, nir, nor and nos operons encoding enzymes of denitrification are found in the T. thioparus genome, in the same arrangements as in the true denitrifier T. denitrificans.« less

Permanent draft genome of Thiobacillus thioparus DSM 505T, an obligately chemolithoautotrophic member of the Betaproteobacteria

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

Hutt, Lee P.; Huntemann, Marcel; Clum, Alicia

Thiobacillus thioparus DSM 505 T is one of first two isolated strains of inorganic sulfur-oxidising Bacteria. The original strain of T. thioparus was lost almost 100 years ago and the working type strain is Culture C T (=DSM 505 T = ATCC 8158 T ) isolated by Starkey in 1934 from agricultural soil at Rutgers University, New Jersey, USA. It is an obligate chemolithoautotroph that conserves energy from the oxidation of reduced inorganic sulfur compounds using the Kelly-Trudinger pathway and uses it to fix carbon dioxide It is not capable of heterotrophic or mixotrophic growth. The strain has a genomemore » size of 3,201,518 bp. Here we report the genome sequence, annotation and characteristics. The genome contains 3,135 protein coding and 62 RNA coding genes. Genes encoding the transaldolase variant of the Calvin-Benson-Bassham cycle were also identified and an operon encoding carboxysomes, along with Smith's biosynthetic horseshoe in lieu of Krebs' cycle sensu stricto. Terminal oxidases were identified, viz. cytochrome c oxidase (cbb3, EC 1.9.3.1) and ubiquinol oxidase (bd, EC 1.10.3.10). There is a partial sox operon of the Kelly-Friedrich pathway of inorganic sulfur-oxidation that contains soxXYZAB genes but lacking soxCDEF, there is also a lack of the DUF302 gene previously noted in the sox operon of other members of the 'Proteobacteria' that can use trithionate as an energy source. In spite of apparently not growing anaerobically with denitrification, the nar, nir, nor and nos operons encoding enzymes of denitrification are found in the T. thioparus genome, in the same arrangements as in the true denitrifier T. denitrificans.« less

The three-dimensional structure of TrmB, a transcriptional regulator of dual function in the hyperthermophilic archaeon Pyrococcus furiosus in complex with sucrose

PubMed Central

Krug, Michael; Lee, Sung-Jae; Boos, Winfried; Diederichs, Kay; Welte, Wolfram

2013-01-01

TrmB is a repressor that binds maltose, maltotriose, and sucrose, as well as other α-glucosides. It recognizes two different operator sequences controlling the TM (Trehalose/Maltose) and the MD (Maltodextrin) operon encoding the respective ABC transporters and sugar-degrading enzymes. Binding of maltose to TrmB abrogates repression of the TM operon but maintains the repression of the MD operon. On the other hand, binding of sucrose abrogates repression of the MD operon but maintains repression of the TM operon. The three-dimensional structure of TrmB in complex with sucrose was solved and refined to a resolution of 3.0 Å. The structure shows the N-terminal DNA binding domain containing a winged-helix-turn-helix (wHTH) domain followed by an amphipathic helix with a coiled-coil motif. The latter promotes dimerization and places the symmetry mates of the putative recognition helix in the wHTH motif about 30 Å apart suggesting a canonical binding to two successive major grooves of duplex palindromic DNA. This suggests that the structure resembles the conformation of TrmB recognizing the pseudopalindromic TM promoter but not the conformation recognizing the nonpalindromic MD promoter. PMID:23576322

Shikimate Induced Transcriptional Activation of Protocatechuate Biosynthesis Genes by QuiR, a LysR-Type Transcriptional Regulator, in Listeria monocytogenes.

PubMed

Prezioso, Stephanie M; Xue, Kevin; Leung, Nelly; Gray-Owen, Scott D; Christendat, Dinesh

2018-04-27

Listeria monocytogenes is a common foodborne bacterial pathogen that contaminates plant and animal consumable products. The persistent nature of L. monocytogenes is associated with millions of dollars in food recalls annually. Here, we describe the role of shikimate in directly modulating the expression of genes encoding enzymes for the conversion of quinate and shikimate metabolites to protocatechuate. In L. monocytogenes, these genes are found within two operons, named qui1 and qui2. In addition, a gene named quiR, encoding a LysR-Type Transcriptional Regulator (QuiR), is located immediately upstream of the qui1 operon. Transcriptional lacZ-promoter fusion experiments show that QuiR induces gene expression of both qui1 and qui2 operons in the presence of shikimate. Furthermore, co-crystallization of the QuiR effector binding domain in complex with shikimate provides insights into the mechanism of activation of this regulator. Together these data show that upon shikimate accumulation, QuiR activates the transcription of genes encoding enzymes involved in shikimate and quinate utilization for the production of protocatechuate. Furthermore, the accumulation of protocatechuate leads to the inhibition of Listeria growth. Since protocatechuate is not known to be utilized by Listeria, its role is distinct from those established in other bacteria. Copyright © 2018 Elsevier Ltd. All rights reserved.

Stable zymomonas mobilis xylose and arabinose fermenting strains

DOEpatents

Zhang, Min [Lakewood, CO; Chou, Yat-Chen [Taipei, TW

2008-04-08

The present invention briefly includes a transposon for stable insertion of foreign genes into a bacterial genome, comprising at least one operon having structural genes encoding enzymes selected from the group consisting of xylAxylB, araBAD and tal/tkt, and at least one promoter for expression of the structural genes in the bacterium, a pair of inverted insertion sequences, the operons contained inside the insertion sequences, and a transposase gene located outside of the insertion sequences. A plasmid shuttle vector for transformation of foreign genes into a bacterial genome, comprising at least one operon having structural genes encoding enzymes selected from the group consisting of xylAxylB, araBAD and tal/tkt, at least one promoter for expression of the structural genes in the bacterium, and at least two DNA fragments having homology with a gene in the bacterial genome to be transformed, is also provided.The transposon and shuttle vectors are useful in constructing significantly different Zymomonas mobilis strains, according to the present invention, which are useful in the conversion of the cellulose derived pentose sugars into fuels and chemicals, using traditional fermentation technology, because they are stable for expression in a non-selection medium.

Transcript analysis of the extended hyp-operon in the cyanobacteria Nostoc sp. strain PCC 7120 and Nostoc punctiforme ATCC 29133

PubMed Central

2011-01-01

Background Cyanobacteria harbor two [NiFe]-type hydrogenases consisting of a large and a small subunit, the Hup- and Hox-hydrogenase, respectively. Insertion of ligands and correct folding of nickel-iron hydrogenases require assistance of accessory maturation proteins (encoded by the hyp-genes). The intergenic region between the structural genes encoding the uptake hydrogenase (hupSL) and the accessory maturation proteins (hyp genes) in the cyanobacteria Nostoc PCC 7120 and N. punctiforme were analysed using molecular methods. Findings The five ORFs, located in between the uptake hydrogenase structural genes and the hyp-genes, can form a transcript with the hyp-genes. An identical genomic localization of these ORFs are found in other filamentous, N2-fixing cyanobacterial strains. In N. punctiforme and Nostoc PCC 7120 the ORFs upstream of the hyp-genes showed similar transcript level profiles as hupS (hydrogenase structural gene), nifD (nitrogenase structural gene), hypC and hypF (accessory hydrogenase maturation genes) after nitrogen depletion. In silico analyzes showed that these ORFs in N. punctiforme harbor the same conserved regions as their homologues in Nostoc PCC 7120 and that they, like their homologues in Nostoc PCC 7120, can be transcribed together with the hyp-genes forming a larger extended hyp-operon. DNA binding studies showed interactions of the transcriptional regulators CalA and CalB to the promoter regions of the extended hyp-operon in N. punctiforme and Nostoc PCC 7120. Conclusions The five ORFs upstream of the hyp-genes in several filamentous N2-fixing cyanobacteria have an identical genomic localization, in between the genes encoding the uptake hydrogenase and the maturation protein genes. In N. punctiforme and Nostoc PCC 7120 they are transcribed as one operon and may form transcripts together with the hyp-genes. The expression pattern of the five ORFs within the extended hyp-operon in both Nostoc punctiforme and Nostoc PCC 7120 is similar to the expression patterns of hupS, nifD, hypF and hypC. CalA, a known transcription factor, interacts with the promoter region between hupSL and the five ORFs in the extended hyp-operon in both Nostoc strains. PMID:21672234

Functional Analysis of Genes for Biosynthesis of Pyocyanin and Phenazine-1-Carboxamide from Pseudomonas aeruginosa PAO1

PubMed Central

Mavrodi, Dmitri V.; Bonsall, Robert F.; Delaney, Shannon M.; Soule, Marilyn J.; Phillips, Greg; Thomashow, Linda S.

2001-01-01

Two seven-gene phenazine biosynthetic loci were cloned from Pseudomonas aeruginosa PAO1. The operons, designated phzA1B1C1D1E1F1G1 and phzA2B2C2D2E2F2G2, are homologous to previously studied phenazine biosynthetic operons from Pseudomonas fluorescens and Pseudomonas aureofaciens. Functional studies of phenazine-nonproducing strains of fluorescent pseudomonads indicated that each of the biosynthetic operons from P. aeruginosa is sufficient for production of a single compound, phenazine-1-carboxylic acid (PCA). Subsequent conversion of PCA to pyocyanin is mediated in P. aeruginosa by two novel phenazine-modifying genes, phzM and phzS, which encode putative phenazine-specific methyltransferase and flavin-containing monooxygenase, respectively. Expression of phzS alone in Escherichia coli or in enzymes, pyocyanin-nonproducing P. fluorescens resulted in conversion of PCA to 1-hydroxyphenazine. P. aeruginosa with insertionally inactivated phzM or phzS developed pyocyanin-deficient phenotypes. A third phenazine-modifying gene, phzH, which has a homologue in Pseudomonas chlororaphis, also was identified and was shown to control synthesis of phenazine-1-carboxamide from PCA in P. aeruginosa PAO1. Our results suggest that there is a complex pyocyanin biosynthetic pathway in P. aeruginosa consisting of two core loci responsible for synthesis of PCA and three additional genes encoding unique enzymes involved in the conversion of PCA to pyocyanin, 1-hydroxyphenazine, and phenazine-1-carboxamide. PMID:11591691

The transcriptional terminator sequences downstream of the covR gene terminate covR/S operon transcription to generate covR monocistronic transcripts in Streptococcus pyogenes.

PubMed

Chiang-Ni, Chuan; Tsou, Chih-Cheng; Lin, Yee-Shin; Chuang, Woei-Jer; Lin, Ming-T; Liu, Ching-Chuan; Wu, Jiunn-Jong

2008-12-31

CovR/S is an important two component regulatory system, which regulates about 15% of the gene expression in Streptococcus pyogenes. The covR/S locus was identified as an operon generating an RNA transcript around 2.5-kb in size. In this study, we found the covR/S operon produced three RNA transcripts (around 2.5-, 1.0-, and 0.8-kb in size). Using RNA transcriptional terminator sequence prediction and transcriptional terminator analysis, we identified two atypical rho-independent terminator sequences downstream of the covR gene and showed these terminator sequences terminate RNA transcription efficiently. These results indicate that covR/S operon generates covR/S transcript and monocistronic covR transcripts.

Transcriptional Regulation and Characterization of a Novel β-Fructofuranosidase-Encoding Gene from Bifidobacterium breve UCC2003

PubMed Central

Ryan, Sinéad M.; Fitzgerald, Gerald F.; van Sinderen, Douwe

2005-01-01

An operon involved in fructooligosaccharide breakdown was identified in the genome of Bifidobacterium breve UCC2003. This 2.6-kb transcriptional unit was comprised of three genes that encoded a putative permease, a conserved hypothetical protein, and a β-fructofuranosidase. Active transcription of the operon was observed when B. breve UCC2003 was grown on sucrose or Actilight, while transcription appeared to be repressed when the organism was grown on glucose, fructose, a combination of glucose and sucrose, or a combination of fructose and sucrose. The β-fructofuranosidase encoded by this operon was purified and biochemically characterized. The optimum pH and temperature for catalytic activity were determined to be pH 6.0 and 37°C, respectively, and there was a dependence on bivalent cations, particularly manganese. The Km and Vmax values for sucrose hydrolysis were determined to be 25 ± 2 mM and 24 ± 3 μmol min−1 mg−1, respectively. Interestingly, the enzyme was shown to specifically catalyze cleavage of the β(2-1) glycosidic bond between glucose and its neighboring fructose moiety in sucrose and other fructooligosaccharides with a relatively low degree of polymerization, and there was no detectable activity towards the β(2-1) glycosidic bond between two fructose moieties within the same substrate. To our knowledge, such an enzymatic activity has not previously been described in bifidobacteria or other gram-positive bacteria. PMID:16000751

Bacterial cellulose biosynthesis: diversity of operons, subunits, products and functions

PubMed Central

Römling, Ute; Galperin, Michael Y.

2015-01-01

Summary Recent studies of bacterial cellulose biosynthesis, including structural characterization of a functional cellulose synthase complex, provided the first mechanistic insight into this fascinating process. In most studied bacteria, just two subunits, BcsA and BcsB, are necessary and sufficient for the formation of the polysaccharide chain in vitro. Other subunits – which differ among various taxa – affect the enzymatic activity and product yield in vivo by modulating expression of biosynthesis apparatus, export of the nascent β-D-glucan polymer to the cell surface, and the organization of cellulose fibers into a higher-order structure. These auxiliary subunits play key roles in determining the quantity and structure of the resulting biofilm, which is particularly important for interactions of bacteria with higher organisms that lead to rhizosphere colonization and modulate virulence of cellulose-producing bacterial pathogens inside and outside of host cells. Here we review the organization of four principal types of cellulose synthase operons found in various bacterial genomes, identify additional bcs genes that encode likely components of the cellulose biosynthesis and secretion machinery, and propose a unified nomenclature for these genes and subunits. We also discuss the role of cellulose as a key component of biofilms formed by a variety of free-living and pathogenic bacteria and, for the latter, in the choice between acute infection and persistence in the host. PMID:26077867

Bordetella pertussis risA, but Not risS, Is Required for Maximal Expression of Bvg-Repressed Genes

PubMed Central

Stenson, Trevor H.; Allen, Andrew G.; al-Meer, Jehan A.; Maskell, Duncan; Peppler, Mark S.

2005-01-01

Expression of virulence determinants by Bordetella pertussis, the primary etiological agent of whooping cough, is regulated by the BvgAS two-component regulatory system. The role of a second two-component regulatory system, encoded by risAS, in this process is not defined. Here, we show that mutation of B. pertussis risA does not affect Bvg-activated genes or proteins. However, mutation of risA resulted in greatly diminished expression of Bvg-repressed antigens and decreased transcription of Bvg-repressed genes. In contrast, mutation of risS had no effect on the expression of Bvg-regulated molecules. Mutation of risA also resulted in decreased bacterial invasion in a HeLa cell model. However, decreased invasion could not be attributed to the decreased expression of Bvg-repressed products, suggesting that mutation of risA may affect the expression of a variety of genes. Unlike the risAS operons in B. parapertussis and B. bronchiseptica, B. pertussis risS is a pseudogene that encodes a truncated RisS sensor. Deletion of the intact part of the B. pertussis risS gene does not affect the expression of risA-dependent, Bvg-repressed genes. These observations suggest that RisA activation occurs through cross-regulation by a heterologous system. PMID:16113320

Method for metabolizing carbazole in petroleum

DOEpatents

Kayser, Kevin J.; Kilbane, II, John J.

2005-09-13

A method for selective cleavage of C--N bonds genes that encode for at least one enzyme suitable for conversion of carbazole to 2-aminobiphenyl-2,3-diol are combined with a gene encoding an amidase suitable for selectively cleaving a C--N bond in 2-aminobiphenyl-2,3-diol, forming an operon that encodes for cleavage of both C--N bonds of said carbazole. The operon is inserted into a host culture which, in turn, is contacted with the carbazole, resulting in selective cleavage of both C--N bonds of the carbazole. Also disclosed is a new microorganism that expresses a carbazole degradation trait constitutively and a method for degrading carbazole employing this microorganism.

Characterization of Hydrogen Metabolism in the Multicellular Green Alga Volvox carteri.

PubMed

Cornish, Adam J; Green, Robin; Gärtner, Katrin; Mason, Saundra; Hegg, Eric L

2015-01-01

Hydrogen gas functions as a key component in the metabolism of a wide variety of microorganisms, often acting as either a fermentative end-product or an energy source. The number of organisms reported to utilize hydrogen continues to grow, contributing to and expanding our knowledge of biological hydrogen processes. Here we demonstrate that Volvox carteri f. nagariensis, a multicellular green alga with differentiated cells, evolves H2 both when supplied with an abiotic electron donor and under physiological conditions. The genome of Volvox carteri contains two genes encoding putative [FeFe]-hydrogenases (HYDA1 and HYDA2), and the transcripts for these genes accumulate under anaerobic conditions. The HYDA1 and HYDA2 gene products were cloned, expressed, and purified, and both are functional [FeFe]-hydrogenases. Additionally, within the genome the HYDA1 and HYDA2 genes cluster with two putative genes which encode hydrogenase maturation proteins. This gene cluster resembles operon-like structures found within bacterial genomes and may provide further insight into evolutionary relationships between bacterial and algal [FeFe]-hydrogenase genes.

Characterization of Hydrogen Metabolism in the Multicellular Green Alga Volvox carteri

PubMed Central

Cornish, Adam J.; Green, Robin; Gärtner, Katrin; Mason, Saundra; Hegg, Eric L.

2015-01-01

Hydrogen gas functions as a key component in the metabolism of a wide variety of microorganisms, often acting as either a fermentative end-product or an energy source. The number of organisms reported to utilize hydrogen continues to grow, contributing to and expanding our knowledge of biological hydrogen processes. Here we demonstrate that Volvox carteri f. nagariensis, a multicellular green alga with differentiated cells, evolves H2 both when supplied with an abiotic electron donor and under physiological conditions. The genome of Volvox carteri contains two genes encoding putative [FeFe]-hydrogenases (HYDA1 and HYDA2), and the transcripts for these genes accumulate under anaerobic conditions. The HYDA1 and HYDA2 gene products were cloned, expressed, and purified, and both are functional [FeFe]-hydrogenases. Additionally, within the genome the HYDA1 and HYDA2 genes cluster with two putative genes which encode hydrogenase maturation proteins. This gene cluster resembles operon-like structures found within bacterial genomes and may provide further insight into evolutionary relationships between bacterial and algal [FeFe]-hydrogenase genes. PMID:25927230

Characterization of Hydrogen Metabolism in the Multicellular Green Alga Volvox carteri

DOE PAGES

Cornish, Adam J.; Green, Robin; Gärtner, Katrin; ...

2015-04-30

Hydrogen gas functions as a key component in the metabolism of a wide variety of microorganisms, often acting as either a fermentative end-product or an energy source. The number of organisms reported to utilize hydrogen continues to grow, contributing to and expanding our knowledge of biological hydrogen processes. Here we demonstrate that Volvox carteri f. nagariensis, a multicellular green alga with differentiated cells, evolves H 2 both when supplied with an abiotic electron donor and under physiological conditions. The genome of Volvox carteri contains two genes encoding putative [FeFe]-hydrogenases (HYDA1 and HYDA2), and the transcripts for these genes accumulate undermore » anaerobic conditions. The HYDA1 and HYDA2 gene products were cloned, expressed, and purified, and both are functional [FeFe]-hydrogenases. Additionally, within the genome the HYDA1 and HYDA2 genes cluster with two putative genes which encode hydrogenase maturation proteins. This gene cluster resembles operon-like structures found within bacterial genomes and may provide further insight into evolutionary relationships between bacterial and algal [FeFe]-hydrogenase genes.« less

The groESL Chaperone Operon of Lactobacillus johnsonii†

PubMed Central

Walker, D. Carey; Girgis, Hany S.; Klaenhammer, Todd R.

1999-01-01

The Lactobacillus johnsonii VPI 11088 groESL operon was localized on the chromosome near the insertion element IS1223. The operon was initially cloned as a series of three overlapping PCR fragments, which were sequenced and used to design primers to amplify the entire operon. The amplified fragment was used as a probe to recover the chromosomal copy of the groESL operon from a partial library of L. johnsonii VPI 11088 (NCK88) DNA, cloned in the shuttle vector pTRKH2. The 2,253-bp groESL fragment contained three putative open reading frames, two of which encoded the ubiquitous GroES and GroEL chaperone proteins. Analysis of the groESL promoter region revealed three transcription initiation sites, as well as three sets of inverted repeats (IR) positioned between the transcription and translation start sites. Two of the three IR sets bore significant homology to the CIRCE elements, implicated in negative regulation of the heat shock response in many bacteria. Northern analysis and primer extension revealed that multiple temperature-sensitive promoters preceded the groESL chaperone operon, suggesting that stress protein production in L. johnsonii is strongly regulated. Maximum groESL transcription activity was observed following a shift to 55°C, and a 15 to 30-min exposure of log-phase cells to this temperature increased the recovery of freeze-thawed L. johnsonii VPI 11088. These results suggest that a brief, preconditioning heat shock can be used to trigger increased chaperone production and provide significant cross-protection from the stresses imposed during the production of frozen culture concentrates. PMID:10388700

Isoprenoid Pyrophosphate-Dependent Transcriptional Regulation of Carotenogenesis in Corynebacterium glutamicum

PubMed Central

Henke, Nadja A.; Heider, Sabine A. E.; Hannibal, Silvin; Wendisch, Volker F.; Peters-Wendisch, Petra

2017-01-01

Corynebacterium glutamicum is a natural producer of the C50 carotenoid decaprenoxanthin. The crtEcg0722crtBIYEb operon comprises most of its genes for terpenoid biosynthesis. The MarR-type regulator encoded upstream and in divergent orientation of the carotenoid biosynthesis operon has not yet been characterized. This regulator, named CrtR in this study, is encoded in many actinobacterial genomes co-occurring with terpenoid biosynthesis genes. CrtR was shown to repress the crt operon of C. glutamicum since DNA microarray experiments revealed that transcript levels of crt operon genes were increased 10 to 70-fold in its absence. Transcriptional fusions of a promoter-less gfp gene with the crt operon and crtR promoters confirmed that CrtR represses its own gene and the crt operon. Gel mobility shift assays with purified His-tagged CrtR showed that CrtR binds to a region overlapping with the −10 and −35 promoter sequences of the crt operon. Isoprenoid pyrophosphates interfered with binding of CrtR to its target DNA, a so far unknown mechanism for regulation of carotenogenesis. The molecular details of protein-ligand interactions remain to be studied. Decaprenoxanthin synthesis by C. glutamicum wild type was enhanced 10 to 30-fold upon deletion of crtR and was decreased 5 to 6-fold as result of crtR overexpression. Moreover, deletion of crtR was shown as metabolic engineering strategy to improve production of native and non-native carotenoids including lycopene, β-carotene, C.p. 450 and sarcinaxanthin. PMID:28484430

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.

The uncharacterized transcription factor YdhM is the regulator of the nemA gene, encoding N-ethylmaleimide reductase.

PubMed

Umezawa, Yoshimasa; Shimada, Tomohiro; Kori, Ayako; Yamada, Kayoko; Ishihama, Akira

2008-09-01

N-ethylmaleimide (NEM) has been used as a specific reagent of Cys modification in proteins and thus is toxic for cell growth. On the Escherichia coli genome, the nemA gene coding for NEM reductase is located downstream of the gene encoding an as-yet-uncharacterized transcription factor, YdhM. Disruption of the ydhM gene results in reduction of nemA expression even in the induced state, indicating that the two genes form a single operon. After in vitro genomic SELEX screening, one of the target recognition sequences for YdhM was identified within the promoter region for this ydhM-nemA operon. Both YdhM binding in vitro to the ydhM promoter region and transcription repression in vivo of the ydhM-nemA operon by YdhM were markedly reduced by the addition of NEM. Taken together, we propose that YdhM is the repressor for the nemA gene, thus hereafter designated NemR. The repressor function of NemR was inactivated by the addition of not only NEM but also other Cys modification reagents, implying that Cys modification of NemR renders it inactive. This is an addition to the mode of controlling activity of transcription factors by alkylation with chemical agents.

The pkI gene encoding pyruvate kinase I links to the luxZ gene which enhances bioluminescence of the lux operon from Photobacterium leiognathi.

PubMed

Lin, J W; Lu, H C; Chen, H Y; Weng, S F

1997-10-09

Partial 3'-end nucleotide sequence of the pkI gene (GenBank accession No. AF019143) from Photobacterium leiognathi ATCC 25521 has been determined, and the encoded pyruvate kinase I is deduced. Pyruvate kinase I is the key enzyme of glycolysis, which converts phosphoenol pyruvate to pyruvate. Alignment and comparison of pyruvate kinase Is from P. leiognathi, E. coli and Salmonella typhimurium show that they are homologous. Nucleotide sequence reveals that the pkI gene is linked to the luxZ gene that enhances bioluminescence of the lux operon from P. leiognathi. The gene order of the pkI and luxZ genes is-pk1-ter-->-R&R"-luxZ-ter"-->, whereas ter is transcriptional terminator for the pkI and related genes, and R&R" is the regulatory region and ter" is transcriptional terminator for the luxZ gene. It clearly elicits that the pkI gene and luxZ gene are divided to two operons. Functional analysis confirms that the potential hairpin loop omega T is the transcriptional terminator for the pkI and related genes. It infers that the pkI and related genes are simply linked to the luxZ gene in P. leiognathi genome.

Nonhemolytic Streptococcus pyogenes Isolates That Lack Large Regions of the sag Operon Mediating Streptolysin S Production▿

PubMed Central

Yoshino, Miho; Murayama, Somay Y.; Sunaoshi, Katsuhiko; Wajima, Takeaki; Takahashi, Miki; Masaki, Junko; Kurokawa, Iku; Ubukata, Kimiko

2010-01-01

Among nonhemolytic Streptococcus pyogenes (group A streptococcus) strains (n = 9) isolated from patients with pharyngitis or acute otitis media, we identified three deletions in the region from the epf gene, encoding the extracellular matrix binding protein, to the sag operon, mediating streptolysin S production. PMID:20018818

Two-component signal transduction systems of Xanthomonas spp.: a lesson from genomics.

PubMed

Qian, Wei; Han, Zhong-Ji; He, Chaozu

2008-02-01

The two-component signal transduction systems (TCSTSs), consisting of a histidine kinase sensor (HK) and a response regulator (RR), are the dominant molecular mechanisms by which prokaryotes sense and respond to environmental stimuli. Genomes of Xanthomonas generally contain a large repertoire of TCSTS genes (approximately 92 to 121 for each genome), which encode diverse structural groups of HKs and RRs. Among them, although a core set of 70 TCSTS genes (about two-thirds in total) which accumulates point mutations with a slow rate are shared by these genomes, the other genes, especially hybrid HKs, experienced extensive genetic recombination, including genomic rearrangement, gene duplication, addition or deletion, and fusion or fission. The recombinations potentially promote the efficiency and complexity of TCSTSs in regulating gene expression. In addition, our analysis suggests that a co-evolutionary model, rather than a selfish operon model, is the major mechanism for the maintenance and microevolution of TCSTS genes in the genomes of Xanthomonas. Genomic annotation, secondary protein structure prediction, and comparative genomic analyses of TCSTS genes reviewed here provide insights into our understanding of signal networks in these important phytopathogenic bacteria.

Spectroscopy of Cu(II)-PcoC and the multicopper oxidase function of PcoA, two essential components of Escherichia coli pco copper resistance operon.

PubMed

Huffman, David L; Huyett, Jennifer; Outten, F Wayne; Doan, Peter E; Finney, Lydia A; Hoffman, Brian M; O'Halloran, Thomas V

2002-08-06

The plasmid-encoded pco copper resistance operon in Escherichia coli consists of seven genes that are expressed from two pco promoters in response to elevated copper; however, little is known about how they mediate resistance to excess environmental copper. Two of the genes encode the soluble periplasmic proteins PcoA and PcoC. We show here that inactivation of PcoC, and PcoA to a lesser extent, causes cells to become more sensitive to copper than wild-type nonresistant strains, consistent with a tightly coupled detoxification pathway. Periplasmic extracts show copper-inducible oxidase activity, attributed to the multicopper oxidase function of PcoA. PcoC, a much smaller protein than PcoA, binds one Cu(II) and exhibits a weak electronic transition characteristic of a type II copper center. ENDOR and ESEEM spectroscopy of Cu(II)-PcoC and the (15)N- and Met-CD(3)-labeled samples are consistent with a tetragonal ligand environment of three nitrogens and one aqua ligand "in the plane". A weakly associated S-Met and aqua are likely axial ligands. At least one N is a histidine and is likely trans to the in-plane aqua ligand. The copper chemistry of PcoC and the oxidase function of PcoA are consistent with the emerging picture of the chromosomally encoded copper homeostasis apparatus in the E. coli cell envelope [Outten, F. W., Huffman, D. L., Hale, J. A., and O'Halloran, T. V. (2001) J. Biol. Chem. 276, 30670-30677]. We propose a model for the plasmid system in which Cu(I)-PcoC functions in this copper efflux pathway as a periplasmic copper binding protein that docks with the multiple repeats of Met-rich domains in PcoA to effect oxidation of Cu(I) to the less toxic Cu(II) form. The solvent accessibility of the Cu(II) in PcoC may allow for metal transfer to other plasmid and chromosomal factors and thus facilitate removal of Cu(II) from the cell envelope.

Regulation of the yjjQ-bglJ Operon, Encoding LuxR-Type Transcription Factors, and the Divergent yjjP Gene by H-NS and LeuO▿ †

PubMed Central

Stratmann, Thomas; Madhusudan, S.; Schnetz, Karin

2008-01-01

The yjjQ and bglJ genes encode LuxR-type transcription factors conserved in several enterobacterial species. YjjQ is a potential virulence factor in avian pathogenic Escherichia coli. BglJ counteracts the silencing of the bgl (β-glucoside) operon by H-NS in E. coli K-12. Here we show that yjjQ and bglJ form an operon carried by E. coli K-12, whose expression is repressed by the histone-like nucleoid structuring (H-NS) protein. The LysR-type transcription factor LeuO counteracts this repression. Furthermore, the yjjP gene, encoding a membrane protein of unknown function and located upstream in divergent orientation to the yjjQ-bglJ operon, is likewise repressed by H-NS. Mapping of the promoters as well as the H-NS and LeuO binding sites within the 555-bp intergenic region revealed that H-NS binds to the center of the AT-rich regulatory region and distal to the divergent promoters. LeuO sites map to the center and to positions distal to the yjjQ promoters, while one LeuO binding site overlaps with the divergent yjjP promoter. This latter LeuO site is required for full derepression of the yjjQ promoters. The arrangement of regulatory sites suggests that LeuO restructures the nucleoprotein complex formed by H-NS. Furthermore, the data support the conclusion that LeuO, whose expression is likewise repressed by H-NS and which is a virulence factor in Salmonella enterica, is a master regulator that among other loci, also controls the yjjQ-bglJ operon and thus indirectly the presumptive targets of YjjQ and BglJ. PMID:18055596

Modulation of anaerobic energy metabolism of Bacillus subtilis by arfM (ywiD).

PubMed

Marino, M; Ramos, H C; Hoffmann, T; Glaser, P; Jahn, D

2001-12-01

Bacillus subtilis grows under anaerobic conditions utilizing nitrate ammonification and various fermentative processes. The two-component regulatory system ResDE and the redox regulator Fnr are the currently known parts of the regulatory system for anaerobic adaptation. Mutation of the open reading frame ywiD located upstream of the respiratory nitrate reductase operon narGHJI resulted in elimination of the contribution of nitrite dissimilation to anaerobic nitrate respiratory growth. Significantly reduced nitrite reductase (NasDE) activity was detected, while respiratory nitrate reductase activity was unchanged. Anaerobic induction of nasDE expression was found to be significantly dependent on intact ywiD, while anaerobic narGHJI expression was ywiD independent. Anaerobic transcription of hmp, encoding a flavohemoglobin-like protein, and of the fermentative operons lctEP and alsSD, responsible for lactate and acetoin formation, was partially dependent on ywiD. Expression of pta, encoding phosphotransacetylase involved in fermentative acetate formation, was not influenced by ywiD. Transcription of the ywiD gene was anaerobically induced by the redox regulator Fnr via the conserved Fnr-box (TGTGA-6N-TCACT) centered 40.5 bp upstream of the transcriptional start site. Anaerobic induction of ywiD by resDE was found to be indirect via resDE-dependent activation of fnr. The ywiD gene is subject to autorepression and nitrite repression. These results suggest a ResDE --> Fnr --> YwiD regulatory cascade for the modulation of genes involved in the anaerobic metabolism of B. subtilis. Therefore, ywiD was renamed arfM for anaerobic respiration and fermentation modulator.

The Antisense RNA As1_flv4 in the Cyanobacterium Synechocystis sp. PCC 6803 Prevents Premature Expression of the flv4-2 Operon upon Shift in Inorganic Carbon Supply*

PubMed Central

Eisenhut, Marion; Georg, Jens; Klähn, Stephan; Sakurai, Isamu; Mustila, Henna; Zhang, Pengpeng; Hess, Wolfgang R.; Aro, Eva-Mari

2012-01-01

The functional relevance of natural cis-antisense transcripts is mostly unknown. Here we have characterized the association of three antisense RNAs and one intergenically encoded noncoding RNA with an operon that plays a crucial role in photoprotection of photosystem II under low carbon conditions in the cyanobacterium Synechocystis sp. PCC 6803. Cyanobacteria show strong gene expression dynamics in response to a shift of cells from high carbon to low levels of inorganic carbon (Ci), but the regulatory mechanisms are poorly understood. Among the most up-regulated genes in Synechocystis are flv4, sll0218, and flv2, which are organized in the flv4-2 operon. The flavodiiron proteins encoded by this operon open up an alternative electron transfer route, likely starting from the QB site in photosystem II, under photooxidative stress conditions. Our expression analysis of cells shifted from high carbon to low carbon demonstrated an inversely correlated transcript accumulation of the flv4-2 operon mRNA and one antisense RNA to flv4, designated as As1_flv4. Overexpression of As1_flv4 led to a decrease in flv4-2 mRNA. The promoter activity of as1_flv4 was transiently stimulated by Ci limitation and negatively regulated by the AbrB-like transcription regulator Sll0822, whereas the flv4-2 operon was positively regulated by the transcription factor NdhR. The results indicate that the tightly regulated antisense RNA As1_flv4 establishes a transient threshold for flv4-2 expression in the early phase after a change in Ci conditions. Thus, it prevents unfavorable synthesis of the proteins from the flv4-2 operon. PMID:22854963

A copper-induced quinone degradation pathway provides protection against combined copper/quinone stress in Lactococcus lactis IL1403.

PubMed

Mancini, Stefano; Abicht, Helge K; Gonskikh, Yulia; Solioz, Marc

2015-02-01

Quinones are ubiquitous in the environment. They occur naturally but are also in widespread use in human and industrial activities. Quinones alone are relatively benign to bacteria, but in combination with copper, they become toxic by a mechanism that leads to intracellular thiol depletion. Here, it was shown that the yahCD-yaiAB operon of Lactococcus lactis IL1403 provides resistance to combined copper/quinone stress. The operon is under the control of CopR, which also regulates expression of the copRZA copper resistance operon as well as other L. lactis genes. Expression of the yahCD-yaiAB operon is induced by copper but not by quinones. Two of the proteins encoded by the operon appear to play key roles in alleviating quinone/copper stress: YaiB is a flavoprotein that converts p-benzoquinones to less toxic hydroquinones, using reduced nicotinamide adenine dinucleotide phosphate (NADPH) as reductant; YaiA is a hydroquinone dioxygenase that converts hydroquinone putatively to 4-hydroxymuconic semialdehyde in an oxygen-consuming reaction. Hydroquinone and methylhydroquinone are both substrates of YaiA. Deletion of yaiB causes increased sensitivity of L. lactis to quinones and complete growth arrest under combined quinone and copper stress. Copper induction of the yahCD-yaiAB operon offers protection to copper/quinone toxicity and could provide a growth advantage to L. lactis in some environments. © 2014 John Wiley & Sons Ltd.

Role of fimV in type II secretion system-dependent protein secretion of Pseudomonas aeruginosa on solid medium.

PubMed

Michel, Gérard P F; Aguzzi, Anthony; Ball, Geneviève; Soscia, Chantal; Bleves, Sophie; Voulhoux, Romé

2011-07-01

Although classical type II secretion systems (T2SSs) are widely present in Gram-negative bacteria, atypical T2SSs can be found in some species. In Pseudomonas aeruginosa, in addition to the classical T2SS Xcp, it was reported that two genes, xphA and xqhA, located outside the xcp locus were organized in an operon (PaQa) which encodes the orphan PaQa subunit. This subunit is able to associate with other components of the classical Xcp machinery to form a functional hybrid T2SS. In the present study, using a transcriptional lacZ fusion, we found that the PaQa operon was more efficiently expressed (i) on solid LB agar than in liquid LB medium, (ii) at 25 °C than at 37 °C and (iii) at an early stage of growth. These results suggested an adaptation of the hybrid system to particular environmental conditions. Transposon mutagenesis led to the finding that vfr and fimV genes are required for optimal expression of the orphan PaQa operon in the defined growth conditions used. Using an original culturing device designed to monitor secretion on solid medium, the ring-plate system, we found that T2SS-dependent secretion of exoproteins, namely the elastase LasB, was affected in a fimV deletion mutant. Our findings led to the discovery of an interplay between FimV and the global regulator Vfr triggering the modulation of the level of Vfr and consequently the modulation of T2SS-dependent secretion on solid medium.

Screening of In Vivo Activated Genes in Enterococcus faecalis during Insect and Mouse Infections and Growth in Urine

PubMed Central

Hanin, Aurelie; Sava, Irina; Bao, YinYin; Huebner, Johannes; Hartke, Axel; Auffray, Yanick; Sauvageot, Nicolas

2010-01-01

Enterococcus faecalis is part of the commensal microbiota of humans and its main habitat is the gastrointestinal tract. Although harmless in healthy individuals, E. faecalis has emerged as a major cause of nosocomial infections. In order to better understand the transformation of a harmless commensal into a life-threatening pathogen, we developed a Recombination-based In Vivo Expression Technology for E. faecalis. Two R-IVET systems with different levels of sensitivity have been constructed in a E. faecalis V583 derivative strain and tested in the insect model Galleria mellonella, during growth in urine, in a mouse bacteremia and in a mouse peritonitis model. Our combined results led to the identification of 81 in vivo activated genes. Among them, the ef_3196/7 operon was shown to be strongly induced in the insect host model. Deletion of this operonic structure demonstrated that this two-component system was essential to the E. faecalis pathogenic potential in Galleria. Gene ef_0377, induced in insect and mammalian models, has also been further analyzed and it has been demonstrated that this ankyrin-encoding gene was also involved in E. faecalis virulence. Thus these R-IVET screenings led to the identification of new E. faecalis factors implied in in vivo persistence and pathogenic potential of this opportunistic pathogen. PMID:20686694

Structural features of PhoX, one of the phosphate-binding proteins from Pho regulon of Xanthomonas citri

PubMed Central

Pegos, Vanessa R.; Santos, Rodrigo M. L.; Medrano, Francisco J.

2017-01-01

In Escherichia coli, the ATP-Binding Cassette transporter for phosphate is encoded by the pstSCAB operon. PstS is the periplasmic component responsible for affinity and specificity of the system and has also been related to a regulatory role and chemotaxis during depletion of phosphate. Xanthomonas citri has two phosphate-binding proteins: PstS and PhoX, which are differentially expressed under phosphate limitation. In this work, we focused on PhoX characterization and comparison with PstS. The PhoX three-dimensional structure was solved in a closed conformation with a phosphate engulfed in the binding site pocket between two domains. Comparison between PhoX and PstS revealed that they originated from gene duplication, but despite their similarities they show significant differences in the region that interacts with the permeases. PMID:28542513

The CsoR-like sulfurtransferase repressor (CstR) is a persulfide sensor in Staphylococcus aureus.

PubMed

Luebke, Justin L; Shen, Jiangchuan; Bruce, Kevin E; Kehl-Fie, Thomas E; Peng, Hui; Skaar, Eric P; Giedroc, David P

2014-12-01

How cells regulate the bioavailability of utilizable sulfur while mitigating the effects of hydrogen sulfide toxicity is poorly understood. CstR [Copper-sensing operon repressor (CsoR)-like sulfurtransferase repressor] represses the expression of the cst operon encoding a putative sulfide oxidation system in Staphylococcus aureus. Here, we show that the cst operon is strongly and transiently induced by cellular sulfide stress in an acute phase and specific response and that cst-encoded genes are necessary to mitigate the effects of sulfide toxicity. Growth defects are most pronounced when S. aureus is cultured in chemically defined media with thiosulfate (TS) as a sole sulfur source, but are also apparent when cystine is used or in rich media. Under TS growth conditions, cells fail to grow as a result of either unregulated expression of the cst operon in a ΔcstR strain or transformation with a non-inducible C31A/C60A CstR that blocks cst induction. This suggests that the cst operon contributes to cellular sulfide homeostasis. Tandem high-resolution mass spectrometry reveals derivatization of CstR by both inorganic tetrasulfide and an organic persulfide, glutathione persulfide, to yield a mixture of Cys31-Cys60' interprotomer cross-links, including di-, tri- and tetrasulfide bonds, which allosterically inhibit cst operator DNA binding by CstR. © 2014 John Wiley & Sons Ltd.

The CsoR-like sulfurtransferase repressor (CstR) is a persulfide sensor in Staphylococcus aureus

PubMed Central

Luebke, Justin L.; Shen, Jiangchuan; Bruce, Kevin E.; Kehl-Fie, Thomas E.; Peng, Hui; Skaar, Eric P.; Giedroc, David P.

2014-01-01

How cells regulate the bioavailability of utilizable sulfur while mitigating the effects of hydrogen sulfide toxicity is poorly understood. CstR (Copper-sensing operon repressor (CsoR)-like sulfurtransferase repressor) represses the expression of the cst operon encoding a putative sulfide oxidation system in Staphylococcus aureus. Here, we show that the cst operon is strongly and transiently induced by cellular sulfide stress in an acute phase and specific response and that cst-encoded genes are necessary to mitigate the effects of sulfide toxicity. Growth defects are most pronounced when S. aureus is cultured in chemically defined media with thiosulfate (TS) as a sole sulfur source, but are also apparent when cystine is used or in rich media. Under TS growth conditions, cells fail to grow as a result of either unregulated expression of the cst operon in a ΔcstR strain or transformation with a non-inducible C31A/C60A CstR that blocks cst induction. This suggests that the cst operon contributes to cellular sulfide homeostasis. Tandem high resolution mass spectrometry reveals derivatization of CstR by both inorganic tetrasulfide and an organic persulfide, glutathione persulfide, to yield a mixture of Cys31-Cys60’ interprotomer crosslinks, including di-, tri- and tetrasulfide bonds, which allosterically inhibit cst operator DNA binding by CstR. PMID:25318663

A mutant crp allele that differentially activates the operons of the fuc regulon in Escherichia coli.

PubMed

Zhu, Y; Lin, E C

1988-05-01

L-Fucose is used by Escherichia coli through an inducible pathway mediated by a fucP-encoded permease, a fucI-encoded isomerase, a fucK-encoded kinase, and a fucA-encoded aldolase. The adolase catalyzes the formation of dihydroxyacetone phosphate and L-lactaldehyde. Anaerobically, lactaldehyde is converted by a fucO-encoded oxidoreductase to L-1,2-propanediol, which is excreted. The fuc genes belong to a regulon comprising four linked operons: fucO, fucA, fucPIK, and fucR. The positive regulator encoded by fucR responds to fuculose 1-phosphate as the effector. Mutants serially selected for aerobic growth on propanediol became constitutive in fucO and fucA [fucO(Con) fucA(Con)], but noninducible in fucPIK [fucPIK(Non)]. An external suppressor mutation that restored growth on fucose caused constitutive expression of fucPIK. Results from this study indicate that this suppressor mutation occurred in crp, which encodes the cyclic AMP-binding (or receptor) protein. When the suppressor allele (crp-201) was transduced into wild-type strains, the recipient became fucose negative and fucose sensitive (with glycerol as the carbon and energy source) because of impaired expression of fucA. The fucPIK operon became hyperinducible. The growth rate on maltose was significantly reduced, but growth on L-rhamnose, D-galactose, L-arabinose, glycerol, or glycerol 3-phosphate was close to normal. Lysogenization of fuc+ crp-201 cells by a lambda bacteriophage bearing crp+ restored normal growth ability on fucose. In contrast, lysogenization of [fucO(Con)fucA(Con)fucPIK(Non)crp-201] cells by the same phage retarded their growth on fucose.

A mutant crp allele that differentially activates the operons of the fuc regulon in Escherichia coli.

PubMed Central

Zhu, Y; Lin, E C

1988-01-01

L-Fucose is used by Escherichia coli through an inducible pathway mediated by a fucP-encoded permease, a fucI-encoded isomerase, a fucK-encoded kinase, and a fucA-encoded aldolase. The adolase catalyzes the formation of dihydroxyacetone phosphate and L-lactaldehyde. Anaerobically, lactaldehyde is converted by a fucO-encoded oxidoreductase to L-1,2-propanediol, which is excreted. The fuc genes belong to a regulon comprising four linked operons: fucO, fucA, fucPIK, and fucR. The positive regulator encoded by fucR responds to fuculose 1-phosphate as the effector. Mutants serially selected for aerobic growth on propanediol became constitutive in fucO and fucA [fucO(Con) fucA(Con)], but noninducible in fucPIK [fucPIK(Non)]. An external suppressor mutation that restored growth on fucose caused constitutive expression of fucPIK. Results from this study indicate that this suppressor mutation occurred in crp, which encodes the cyclic AMP-binding (or receptor) protein. When the suppressor allele (crp-201) was transduced into wild-type strains, the recipient became fucose negative and fucose sensitive (with glycerol as the carbon and energy source) because of impaired expression of fucA. The fucPIK operon became hyperinducible. The growth rate on maltose was significantly reduced, but growth on L-rhamnose, D-galactose, L-arabinose, glycerol, or glycerol 3-phosphate was close to normal. Lysogenization of fuc+ crp-201 cells by a lambda bacteriophage bearing crp+ restored normal growth ability on fucose. In contrast, lysogenization of [fucO(Con)fucA(Con)fucPIK(Non)crp-201] cells by the same phage retarded their growth on fucose. PMID:2834341

Role of BkdR, a Transcriptional Activator of the SigL-Dependent Isoleucine and Valine Degradation Pathway in Bacillus subtilis

PubMed Central

Debarbouille, Michel; Gardan, Rozenn; Arnaud, Maryvonne; Rapoport, George

1999-01-01

A new gene, bkdR (formerly called yqiR), encoding a regulator with a central (catalytic) domain was found in Bacillus subtilis. This gene controls the utilization of isoleucine and valine as sole nitrogen sources. Seven genes, previously called yqiS, yqiT, yqiU, yqiV, bfmBAA, bfmBAB, and bfmBB and now referred to as ptb, bcd, buk, lpd, bkdA1, bkdA2, and bkdB, are located downstream from the bkdR gene in B. subtilis. The products of these genes are similar to phosphate butyryl coenzyme A transferase, leucine dehydrogenase, butyrate kinase, and four components of the branched-chain keto acid dehydrogenase complex: E3 (dihydrolipoamide dehydrogenase), E1α (dehydrogenase), E1β (decarboxylase), and E2 (dihydrolipoamide acyltransferase). Isoleucine and valine utilization was abolished in bcd and bkdR null mutants of B. subtilis. The seven genes appear to be organized as an operon, bkd, transcribed from a −12, −24 promoter. The expression of the bkd operon was induced by the presence of isoleucine or valine in the growth medium and depended upon the presence of the sigma factor SigL, a member of the sigma 54 family. Transcription of this operon was abolished in strains containing a null mutation in the regulatory gene bkdR. Deletion analysis showed that upstream activating sequences are involved in the expression of the bkd operon and are probably the target of bkdR. Transcription of the bkd operon is also negatively controlled by CodY, a global regulator of gene expression in response to nutritional conditions. PMID:10094682

Identification of an operon, Pil-Chp, that controls twitching motility and virulence in Xylella fastidiosa.

PubMed

Cursino, Luciana; Galvani, Cheryl D; Athinuwat, Dusit; Zaini, Paulo A; Li, Yaxin; De La Fuente, Leonardo; Hoch, Harvey C; Burr, Thomas J; Mowery, Patricia

2011-10-01

Xylella fastidiosa is an important phytopathogenic bacterium that causes many serious plant diseases, including Pierce's disease of grapevines. Disease manifestation by X. fastidiosa is associated with the expression of several factors, including the type IV pili that are required for twitching motility. We provide evidence that an operon, named Pil-Chp, with genes homologous to those found in chemotaxis systems, regulates twitching motility. Transposon insertion into the pilL gene of the operon resulted in loss of twitching motility (pilL is homologous to cheA genes encoding kinases). The X. fastidiosa mutant maintained the type IV pili, indicating that the disrupted pilL or downstream operon genes are involved in pili function, and not biogenesis. The mutated X. fastidiosa produced less biofilm than wild-type cells, indicating that the operon contributes to biofilm formation. Finally, in planta the mutant produced delayed and less severe disease, indicating that the Pil-Chp operon contributes to the virulence of X. fastidiosa, presumably through its role in twitching motility.

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.

A trans-acting leader RNA from a Salmonella virulence gene

PubMed Central

Choi, Eunna; Han, Yoontak; Cho, Yong-Joon; Nam, Daesil; Lee, Eun-Jin

2017-01-01

Bacteria use flagella to move toward nutrients, find its host, or retract from toxic substances. Because bacterial flagellum is one of the ligands that activate the host innate immune system, its synthesis should be tightly regulated during host infection, which is largely unknown. Here, we report that a bacterial leader mRNA from the mgtCBR virulence operon in the intracellular pathogen Salmonella enterica serovar Typhimurium binds to the fljB coding region of mRNAs in the fljBA operon encoding the FljB phase 2 flagellin, a main component of bacterial flagella and the FljA repressor for the FliC phase 1 flagellin, and degrades fljBA mRNAs in an RNase E-dependent fashion during infection. A nucleotide substitution of the fljB flagellin gene that prevents the mgtC leader RNA-mediated down-regulation increases the fljB-encoded flagellin synthesis, leading to a hypermotile phenotype inside macrophages. Moreover, the fljB nucleotide substitution renders Salmonella hypervirulent, indicating that FljB-based motility must be compromised in the phagosomal compartment where Salmonella resides. This suggests that this pathogen promotes pathogenicity by producing a virulence protein and limits locomotion by a trans-acting leader RNA from the same virulence gene during infection. PMID:28874555

A predictive biophysical model of translational coupling to coordinate and control protein expression in bacterial operons

PubMed Central

Tian, Tian; Salis, Howard M.

2015-01-01

Natural and engineered genetic systems require the coordinated expression of proteins. In bacteria, translational coupling provides a genetically encoded mechanism to control expression level ratios within multi-cistronic operons. We have developed a sequence-to-function biophysical model of translational coupling to predict expression level ratios in natural operons and to design synthetic operons with desired expression level ratios. To quantitatively measure ribosome re-initiation rates, we designed and characterized 22 bi-cistronic operon variants with systematically modified intergenic distances and upstream translation rates. We then derived a thermodynamic free energy model to calculate de novo initiation rates as a result of ribosome-assisted unfolding of intergenic RNA structures. The complete biophysical model has only five free parameters, but was able to accurately predict downstream translation rates for 120 synthetic bi-cistronic and tri-cistronic operons with rationally designed intergenic regions and systematically increased upstream translation rates. The biophysical model also accurately predicted the translation rates of the nine protein atp operon, compared to ribosome profiling measurements. Altogether, the biophysical model quantitatively predicts how translational coupling controls protein expression levels in synthetic and natural bacterial operons, providing a deeper understanding of an important post-transcriptional regulatory mechanism and offering the ability to rationally engineer operons with desired behaviors. PMID:26117546

Escherichia coli mutant with altered respiratory control of the frd operon.

PubMed Central

Iuchi, S; Kuritzkes, D R; Lin, E C

1985-01-01

In wild-type Escherichia coli, fumarate reductase encoded by the frd operon is inducible by its substrate in the absence of molecular oxygen and nitrate. Synthesis of this enzyme under permissive conditions requires the fnr+ gene product, which is believed to be a pleiotropic regulatory protein that activates transcription. A spontaneous mutant was isolated in which the expression of the frd operon no longer depended on the presence of fumarate or the fnr+ gene product. Aerobic repression of the operon was abolished, but nitrate repression remained intact. Transductional analysis showed that the mutation was closely linked to the frd locus. The mutant phenotype strongly suggests that repression by molecular oxygen and nitrate is mediated by different mechanisms. PMID:3882660

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

Functional Characterization of Key Enzymes involved in l-Glutamate Synthesis and Degradation in the Thermotolerant and Methylotrophic Bacterium Bacillus methanolicus

PubMed Central

Krog, Anne; Heggeset, Tonje Marita Bjerkan; Ellingsen, Trond Erling

2013-01-01

Bacillus methanolicus wild-type strain MGA3 secretes 59 g/liter−1 of l-glutamate in fed-batch methanol cultivations at 50°C. We recently sequenced the MGA3 genome, and we here characterize key enzymes involved in l-glutamate synthesis and degradation. One glutamate dehydrogenase (GDH) that is encoded by yweB and two glutamate synthases (GOGATs) that are encoded by the gltAB operon and by gltA2 were found, in contrast to Bacillus subtilis, which has two different GDHs and only one GOGAT. B. methanolicus has a glutamine synthetase (GS) that is encoded by glnA and a 2-oxoglutarate dehydrogenase (OGDH) that is encoded by the odhAB operon. The yweB, gltA, gltB, and gltA2 gene products were purified and characterized biochemically in vitro. YweB has a low Km value for ammonium (10 mM) and a high Km value for l-glutamate (250 mM), and the Vmax value is 7-fold higher for l-glutamate synthesis than for the degradation reaction. GltA and GltA2 displayed similar Km values (1 to 1.4 mM) and Vmax values (4 U/mg) for both l-glutamate and 2-oxoglutarate as the substrates, and GltB had no effect on the catalytic activities of these enzymes in vitro. Complementation assays indicated that GltA and not GltA2 is dependent on GltB for GOGAT activity in vivo. To our knowledge, this is the first report describing the presence of two active GOGATs in a bacterium. In vivo experiments indicated that OGDH activity and, to some degree, GOGAT activity play important roles in regulating l-glutamate production in this organism. PMID:23811508

Functional characterization of key enzymes involved in L-glutamate synthesis and degradation in the thermotolerant and methylotrophic bacterium Bacillus methanolicus.

PubMed

Krog, Anne; Heggeset, Tonje Marita Bjerkan; Ellingsen, Trond Erling; Brautaset, Trygve

2013-09-01

Bacillus methanolicus wild-type strain MGA3 secretes 59 g/liter(-1) of l-glutamate in fed-batch methanol cultivations at 50°C. We recently sequenced the MGA3 genome, and we here characterize key enzymes involved in l-glutamate synthesis and degradation. One glutamate dehydrogenase (GDH) that is encoded by yweB and two glutamate synthases (GOGATs) that are encoded by the gltAB operon and by gltA2 were found, in contrast to Bacillus subtilis, which has two different GDHs and only one GOGAT. B. methanolicus has a glutamine synthetase (GS) that is encoded by glnA and a 2-oxoglutarate dehydrogenase (OGDH) that is encoded by the odhAB operon. The yweB, gltA, gltB, and gltA2 gene products were purified and characterized biochemically in vitro. YweB has a low Km value for ammonium (10 mM) and a high Km value for l-glutamate (250 mM), and the Vmax value is 7-fold higher for l-glutamate synthesis than for the degradation reaction. GltA and GltA2 displayed similar Km values (1 to 1.4 mM) and Vmax values (4 U/mg) for both l-glutamate and 2-oxoglutarate as the substrates, and GltB had no effect on the catalytic activities of these enzymes in vitro. Complementation assays indicated that GltA and not GltA2 is dependent on GltB for GOGAT activity in vivo. To our knowledge, this is the first report describing the presence of two active GOGATs in a bacterium. In vivo experiments indicated that OGDH activity and, to some degree, GOGAT activity play important roles in regulating l-glutamate production in this organism.

Role of the parCBA Operon of the Broad-Host-Range Plasmid RK2 in Stable Plasmid Maintenance

PubMed Central

Easter, Carla L.; Schwab, Helmut; Helinski, Donald R.

1998-01-01

The par region of the stably maintained broad-host-range plasmid RK2 is organized as two divergent operons, parCBA and parDE, and a cis-acting site. parDE encodes a postsegregational killing system, and parCBA encodes a resolvase (ParA), a nuclease (ParB), and a protein of unknown function (ParC). The present study was undertaken to further delineate the role of the parCBA region in the stable maintenance of RK2 by first introducing precise deletions in the three genes and then assessing the abilities of the different constructs to stabilize RK2 in three strains of Escherichia coli and two strains of Pseudomonas aeruginosa. The intact parCBA operon was effective in stabilizing a conjugation-defective RK2 derivative in E. coli MC1061K and RR1 but was relatively ineffective in E. coli MV10Δlac. In the two strains in which the parCBA operon was effective, deletions in parB, parC, or both parB and parC caused an approximately twofold reduction in the stabilizing ability of the operon, while a deletion in the parA gene resulted in a much greater loss of parCBA activity. For P. aeruginosa PAO1161Rifr, the parCBA operon provided little if any plasmid stability, but for P. aeruginosa PAC452Rifr, the RK2 plasmid was stabilized to a substantial extent by parCBA. With this latter strain, parA and res alone were sufficient for stabilization. The cer resolvase system of plasmid ColE1 and the loxP/Cre system of plasmid P1 were tested in comparison with the parCBA operon. We found that, not unlike what was previously observed with MC1061K, cer failed to stabilize the RK2 plasmid with par deletions in strain MV10Δlac, but this multimer resolution system was effective in stabilizing the plasmid in strain RR1. The loxP/Cre system, on the other hand, was very effective in stabilizing the plasmid in all three E. coli strains. These observations indicate that the parA gene, along with its res site, exhibits a significant level of plasmid stabilization in the absence of the parC and parB genes but that in at least one E. coli strain, all three genes are required for maximum stabilization. It cannot be determined from these results whether or not the stabilization effects seen with parCBA or the cer and loxP/Cre systems are strictly due to a reduction in the level of RK2 dimers and an increase in the number of plasmid monomer units or if these systems play a role in a more complex process of plasmid stabilization that requires as an essential step the resolution of plasmid dimers. PMID:9811663

Genetic characterization of moaB mutants of Escherichia coli

PubMed Central

Kozmin, Stanislav G.; Schaaper, Roel M.

2013-01-01

The moaABCDE operon of Escherichia coli encodes enzymes essential for the biosynthesis of the molybdenum cofactor (Moco). However, the role of the moaB gene within this operon has remained enigmatic. Here, we have investigated the effect of moaB defects on two phenotypes diagnostic for Moco-deficiency: chlorate-resistance and sensitivity to the base analog 6-N-hydroxylaminopurine (HAP). We found that transposon insertions in moaB caused partial Moco-deficiency associated with chlorate-resistance, but not for HAP-sensitivity. On the other hand, in-frame deletions of moaB, or moaB overexpression, had no effect on either phenotype. Our combined data are consistent with the lack of any role for MoaB in Moco biosynthesis in E. coli. PMID:23680484

A Copper-Activated Two-Component System Interacts with Zinc and Imipenem Resistance in Pseudomonas aeruginosa▿

PubMed Central

Caille, Olivier; Rossier, Claude; Perron, Karl

2007-01-01

The effects of copper (Cu) on trace metal and antibiotic resistance of Pseudomonas aeruginosa have been investigated. Cu treatments induced resistance not only to this metal but also, surprisingly, to zinc (Zn). Quantitative reverse transcription-PCR (qRT-PCR) revealed that after Cu treatment the transcription of the czcRS two-component system (TCS) operon was enhanced as well as that of the czcCBA operon encoding an efflux pump specific for zinc, cadmium, and cobalt. Cu treatments at the same time caused a decrease in the production of OprD porin, resulting in resistance to the carbapenem antibiotic imipenem. The CzcR regulator was known to repress oprD. However, Cu was still able to decrease the production of OprD and induce imipenem resistance in a czcRS knockout mutant. This strongly suggested that another Cu-dependent regulatory system was acting negatively on oprD expression. TCS regulator genes copR-copS have been shown to be involved in Cu tolerance in P. aeruginosa. qRT-PCR showed that overproduction of the CopR or of the CzcR regulator resulted in increased transcription of the czcC gene as well as in a decrease in oprD gene transcription, either in the wild-type strain or in the czcRS knockout mutant. Overproduction experiments suggest that a metal-dependent mechanism operates at the posttranscriptional level to control the production of the CzcCBA efflux pump. This study shows that CopR is a new negative regulator of OprD porin and that it links Zn, Cu, and imipenem resistances by interacting with the CzcRS TCS. PMID:17449606

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.

Sulfate-Dependent Repression of Genes That Function in Organosulfur Metabolism in Bacillus subtilis Requires Spx

PubMed Central

Erwin, Kyle N.; Nakano, Shunji; Zuber, Peter

2005-01-01

Oxidative stress in Bacillus subtilis results in the accumulation of Spx protein, which exerts both positive and negative transcriptional control over a genome-wide scale through its interaction with the RNA polymerase α subunit. Previous microarray transcriptome studies uncovered a unique class of genes that are controlled by Spx-RNA polymerase interaction under normal growth conditions that do not promote Spx overproduction. These genes were repressed by Spx when sulfate was present as a sole sulfur source. The genes include those of the ytmI, yxeI, and ssu operons, which encode products resembling proteins that function in the uptake and desulfurization of organic sulfur compounds. Primer extension and analysis of operon-lacZ fusion expression revealed that the operons are repressed by sulfate and cysteine; however, Spx functioned only in sulfate-dependent repression. Both the ytmI operon and the divergently transcribed ytlI, encoding a LysR-type regulator that positively controls ytmI operon transcription, are repressed by Spx in sulfate-containing media. The CXXC motif of Spx, which is necessary for redox sensitive control of Spx activity in response to oxidative stress, is not required for sulfate-dependent repression. The yxeL-lacZ and ssu-lacZ fusions were also repressed in an Spx-dependent manner in media containing sulfate as the sole sulfur source. This work uncovers a new role for Spx in the control of sulfur metabolism in a gram-positive bacterium under nonstressful growth conditions. PMID:15937167

Origin and Evolution of the Sodium -Pumping NADH: Ubiquinone Oxidoreductase

PubMed Central

Reyes-Prieto, Adrian; Barquera, Blanca; Juárez, Oscar

2014-01-01

The sodium -pumping NADH: ubiquinone oxidoreductase (Na+-NQR) is the main ion pump and the primary entry site for electrons into the respiratory chain of many different types of pathogenic bacteria. This enzymatic complex creates a transmembrane gradient of sodium that is used by the cell to sustain ionic homeostasis, nutrient transport, ATP synthesis, flagellum rotation and other essential processes. Comparative genomics data demonstrate that the nqr operon, which encodes all Na+-NQR subunits, is found in a large variety of bacterial lineages with different habitats and metabolic strategies. Here we studied the distribution, origin and evolution of this enzymatic complex. The molecular phylogenetic analyses and the organizations of the nqr operon indicate that Na+-NQR evolved within the Chlorobi/Bacteroidetes group, after the duplication and subsequent neofunctionalization of the operon that encodes the homolog RNF complex. Subsequently, the nqr operon dispersed through multiple horizontal transfer events to other bacterial lineages such as Chlamydiae, Planctomyces and α, β, γ and δ -proteobacteria. Considering the biochemical properties of the Na+-NQR complex and its physiological role in different bacteria, we propose a detailed scenario to explain the molecular mechanisms that gave rise to its novel redox- dependent sodium -pumping activity. Our model postulates that the evolution of the Na+-NQR complex involved a functional divergence from its RNF homolog, following the duplication of the rnf operon, the loss of the rnfB gene and the recruitment of the reductase subunit of an aromatic monooxygenase. PMID:24809444

The Bacillus subtilis GntR family repressor YtrA responds to cell wall antibiotics.

PubMed

Salzberg, Letal I; Luo, Yun; Hachmann, Anna-Barbara; Mascher, Thorsten; Helmann, John D

2011-10-01

The transglycosylation step of cell wall synthesis is a prime antibiotic target because it is essential and specific to bacteria. Two antibiotics, ramoplanin and moenomycin, target this step by binding to the substrate lipid II and the transglycosylase enzyme, respectively. Here, we compare the ramoplanin and moenomycin stimulons in the Gram-positive model organism Bacillus subtilis. Ramoplanin strongly induces the LiaRS two-component regulatory system, while moenomycin almost exclusively induces genes that are part of the regulon of the extracytoplasmic function (ECF) σ factor σ(M). Ramoplanin additionally induces the ytrABCDEF and ywoBCD operons, which are not part of a previously characterized antibiotic-responsive regulon. Cluster analysis reveals that these two operons are selectively induced by a subset of cell wall antibiotics that inhibit lipid II function or recycling. Repression of both operons requires YtrA, which recognizes an inverted repeat in front of its own operon and in front of ywoB. These results suggest that YtrA is an additional regulator of cell envelope stress responses.

The Bacillus subtilis GntR Family Repressor YtrA Responds to Cell Wall Antibiotics▿§

PubMed Central

Salzberg, Letal I.; Luo, Yun; Hachmann, Anna-Barbara; Mascher, Thorsten; Helmann, John D.

2011-01-01

The transglycosylation step of cell wall synthesis is a prime antibiotic target because it is essential and specific to bacteria. Two antibiotics, ramoplanin and moenomycin, target this step by binding to the substrate lipid II and the transglycosylase enzyme, respectively. Here, we compare the ramoplanin and moenomycin stimulons in the Gram-positive model organism Bacillus subtilis. Ramoplanin strongly induces the LiaRS two-component regulatory system, while moenomycin almost exclusively induces genes that are part of the regulon of the extracytoplasmic function (ECF) σ factor σM. Ramoplanin additionally induces the ytrABCDEF and ywoBCD operons, which are not part of a previously characterized antibiotic-responsive regulon. Cluster analysis reveals that these two operons are selectively induced by a subset of cell wall antibiotics that inhibit lipid II function or recycling. Repression of both operons requires YtrA, which recognizes an inverted repeat in front of its own operon and in front of ywoB. These results suggest that YtrA is an additional regulator of cell envelope stress responses. PMID:21856850

The BatR/BatS Two-Component Regulatory System Controls the Adaptive Response of Bartonella henselae during Human Endothelial Cell Infection ▿ † ‡

PubMed Central

Quebatte, Maxime; Dehio, Michaela; Tropel, David; Basler, Andrea; Toller, Isabella; Raddatz, Guenter; Engel, Philipp; Huser, Sonja; Schein, Hermine; Lindroos, Hillevi L.; Andersson, Siv G. E.; Dehio, Christoph

2010-01-01

Here, we report the first comprehensive study of Bartonella henselae gene expression during infection of human endothelial cells. Expression of the main cluster of upregulated genes, comprising the VirB type IV secretion system and its secreted protein substrates, is shown to be under the positive control of the transcriptional regulator BatR. We demonstrate binding of BatR to the promoters of the virB operon and a substrate-encoding gene and provide biochemical evidence that BatR and BatS constitute a functional two-component regulatory system. Moreover, in contrast to the acid-inducible (pH 5.5) homologs ChvG/ChvI of Agrobacterium tumefaciens, BatR/BatS are optimally activated at the physiological pH of blood (pH 7.4). By conservation analysis of the BatR regulon, we show that BatR/BatS are uniquely adapted to upregulate a genus-specific virulence regulon during hemotropic infection in mammals. Thus, we propose that BatR/BatS two-component system homologs represent vertically inherited pH sensors that control the expression of horizontally transmitted gene sets critical for the diverse host-associated life styles of the alphaproteobacteria. PMID:20418395

Horizontal gene transfer of chromosomal Type II toxin-antitoxin systems of Escherichia coli.

PubMed

Ramisetty, Bhaskar Chandra Mohan; Santhosh, Ramachandran Sarojini

2016-02-01

Type II toxin-antitoxin systems (TAs) are small autoregulated bicistronic operons that encode a toxin protein with the potential to inhibit metabolic processes and an antitoxin protein to neutralize the toxin. Most of the bacterial genomes encode multiple TAs. However, the diversity and accumulation of TAs on bacterial genomes and its physiological implications are highly debated. Here we provide evidence that Escherichia coli chromosomal TAs (encoding RNase toxins) are 'acquired' DNA likely originated from heterologous DNA and are the smallest known autoregulated operons with the potential for horizontal propagation. Sequence analyses revealed that integration of TAs into the bacterial genome is unique and contributes to variations in the coding and/or regulatory regions of flanking host genome sequences. Plasmids and genomes encoding identical TAs of natural isolates are mutually exclusive. Chromosomal TAs might play significant roles in the evolution and ecology of bacteria by contributing to host genome variation and by moderation of plasmid maintenance. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Transcriptional Profiling of Caulobacter crescentus during Growth on Complex and Minimal Media

PubMed Central

Hottes, Alison K.; Meewan, Maliwan; Yang, Desiree; Arana, Naomi; Romero, Pedro; McAdams, Harley H.; Stephens, Craig

2004-01-01

Microarray analysis was used to examine gene expression in the freshwater oligotrophic bacterium Caulobacter crescentus during growth on three standard laboratory media, including peptone-yeast extract medium (PYE) and minimal salts medium with glucose or xylose as the carbon source. Nearly 400 genes (approximately 10% of the genome) varied significantly in expression between at least two of these media. The differentially expressed genes included many encoding transport systems, most notably diverse TonB-dependent outer membrane channels of unknown substrate specificity. Amino acid degradation pathways constituted the largest class of genes induced in PYE. In contrast, many of the genes upregulated in minimal media encoded enzymes for synthesis of amino acids, including incorporation of ammonia and sulfate into glutamate and cysteine. Glucose availability induced expression of genes encoding enzymes of the Entner-Doudoroff pathway, which was demonstrated here through mutational analysis to be essential in C. crescentus for growth on glucose. Xylose induced expression of genes encoding several hydrolytic exoenzymes as well as an operon that may encode a novel pathway for xylose catabolism. A conserved DNA motif upstream of many xylose-induced genes was identified and shown to confer xylose-specific expression. Xylose is an abundant component of xylan in plant cell walls, and the microarray data suggest that in addition to serving as a carbon source for growth of C. crescentus, this pentose may be interpreted as a signal to produce enzymes associated with plant polymer degradation. PMID:14973021

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

A Shigella flexneri Virulence Plasmid Encoded Factor Controls Production of Outer Membrane Vesicles

PubMed Central

Sidik, Saima; Kottwitz, Haila; Benjamin, Jeremy; Ryu, Julie; Jarrar, Ameer; Garduno, Rafael; Rohde, John R.

2014-01-01

Shigella spp. use a repertoire of virulence plasmid-encoded factors to cause shigellosis. These include components of a Type III Secretion Apparatus (T3SA) that is required for invasion of epithelial cells and many genes of unknown function. We constructed an array of 99 deletion mutants comprising all genes encoded by the virulence plasmid (excluding those known to be required for plasmid maintenance) of Shigella flexneri. We screened these mutants for their ability to bind the dye Congo red: an indicator of T3SA function. This screen focused our attention on an operon encoding genes that modify the cell envelope including virK, a gene of partially characterized function. We discovered that virK is required for controlled release of proteins to the culture supernatant. Mutations in virK result in a temperature-dependent overproduction of outer membrane vesicles (OMVs). The periplasmic chaperone/protease DegP, a known regulator of OMV production in Escherichia coli (encoded by a chromosomal gene), was found to similarly control OMV production in S. flexneri. Both virK and degP show genetic interactions with mxiD, a structural component of the T3SA. Our results are consistent with a model in which VirK and DegP relieve the periplasmic stress that accompanies assembly of the T3SA. PMID:25378474

Role of the Mce1 transporter in the lipid homeostasis of Mycobacterium tuberculosis.

PubMed

Forrellad, Marina Andrea; McNeil, Michael; Santangelo, María de la Paz; Blanco, Federico Carlos; García, Elizabeth; Klepp, Laura Inés; Huff, Jason; Niederweis, Michael; Jackson, Mary; Bigi, Fabiana

2014-03-01

Tuberculosis is one of the leading causes of mortality throughout the world. Mycobacterium tuberculosis, the causative agent of human tuberculosis, has developed several strategies involving proteins and other compounds known collectively as virulence factors to subvert human host defences and invade the human host. The Mce proteins are among these virulence-related proteins and are encoded by the mce1, mce2, mce3 and mce4 operons in the genome of M. tuberculosis. It has been proposed that these operons encode ABC-like lipid transporters; however, the nature of their substrates has only been revealed in the case of the Mce4 proteins. Here we found that the knockout of the mce1 operon alters the lipid profile of M. tuberculosis H37Rv and the uptake of palmitic acid. Thin layer chromatography and liquid chromatography-mass spectrometry analysis showed that the mce1 mutant accumulates more mycolic acids than the wild type and complemented strains. Interestingly, this accumulation of mycolic acid is exacerbated when bacteria are cultured in the presence of palmitic acid or arachidonic acid. These results suggest that the mce1 operon may serve as a mycolic acid re-importer. Copyright © 2013 Elsevier Ltd. All rights reserved.

The excludon: a new concept in bacterial antisense RNA-mediated gene regulation.

PubMed

Sesto, Nina; Wurtzel, Omri; Archambaud, Cristel; Sorek, Rotem; Cossart, Pascale

2013-02-01

In recent years, non-coding RNAs have emerged as key regulators of gene expression. Among these RNAs, the antisense RNAs (asRNAs) are particularly abundant, but in most cases the function and mechanism of action for a particular asRNA remains elusive. Here, we highlight a recently discovered paradigm termed the excludon, which defines a genomic locus encoding an unusually long asRNA that spans divergent genes or operons with related or opposing functions. Because these asRNAs can inhibit the expression of one operon while functioning as an mRNA for the adjacent operon, they act as fine-tuning regulatory switches in bacteria.

Activation of both acfA and acfD transcription by Vibrio cholerae ToxT requires binding to two centrally located DNA sites in an inverted repeat conformation.

PubMed

Withey, Jeffrey H; DiRita, Victor J

2005-05-01

The Gram-negative bacterium Vibrio cholerae is the infectious agent responsible for the disease Asiatic cholera. The genes required for V. cholerae virulence, such as those encoding the cholera toxin (CT) and toxin-coregulated pilus (TCP), are controlled by a cascade of transcriptional activators. Ultimately, the direct transcriptional activator of the majority of V. cholerae virulence genes is the AraC/XylS family member ToxT protein, the expression of which is activated by the ToxR and TcpP proteins. Previous studies have identified the DNA sites to which ToxT binds upstream of the ctx operon, encoding CT, and the tcpA operon, encoding, among other products, the major subunit of the TCP. These known ToxT binding sites are seemingly dissimilar in sequence other than being A/T rich. Further results suggested that ctx and tcpA each has a pair of ToxT binding sites arranged in a direct repeat orientation upstream of the core promoter elements. In this work, using both transcriptional lacZ fusions and in vitro copper-phenanthroline footprinting experiments, we have identified the ToxT binding sites between the divergently transcribed acfA and acfD genes, which encode components of the accessory colonization factor required for efficient intestinal colonization by V. cholerae. Our results indicate that ToxT binds to a pair of DNA sites between acfA and acfD in an inverted repeat orientation. Moreover, a mutational analysis of the ToxT binding sites indicates that both binding sites are required by ToxT for transcriptional activation of both acfA and acfD. Using copper-phenanthroline footprinting to assess the occupancy of ToxT on DNA having mutations in one of these binding sites, we found that protection by ToxT of the unaltered binding site was not affected, whereas protection by ToxT of the mutant binding site was significantly reduced in the region of the mutations. The results of further footprinting experiments using DNA templates having +5 bp and +10 bp insertions between the two ToxT binding sites indicate that both binding sites are occupied by ToxT regardless of their positions relative to each other. Based on these results, we propose that ToxT binds independently to two DNA sites between acfA and acfD to activate transcription of both genes.

Regulation of nrf operon expression in pathogenic enteric bacteria: sequence divergence reveals new regulatory complexity

PubMed Central

Godfrey, Rita E.; Lee, David J.; Busby, Stephen J. W.

2017-01-01

Summary The Escherichia coli K‐12 nrf operon encodes a periplasmic nitrite reductase, the expression of which is driven from a single promoter, pnrf. Expression from pnrf is activated by the FNR transcription factor in response to anaerobiosis and further increased in response to nitrite by the response regulator proteins, NarL and NarP. FNR‐dependent transcription is suppressed by the binding of two nucleoid associated proteins, IHF and Fis. As Fis levels increase in cells grown in rich medium, the positioning of its binding site, overlapping the promoter −10 element, ensures that pnrf is sharply repressed. Here, we investigate the expression of the nrf operon promoter from various pathogenic enteric bacteria. We show that pnrf from enterohaemorrhagic E. coli is more active than its K‐12 counterpart, exhibits substantial FNR‐independent activity and is insensitive to nutrient quality, due to an improved −10 element. We also demonstrate that the Salmonella enterica serovar Typhimurium core promoter is more active than previously thought, due to differences around the transcription start site, and that its expression is repressed by downstream sequences. We identify the CsrA RNA binding protein as being responsible for this, and show that CsrA differentially regulates the E. coli K‐12 and Salmonella nrf operons. PMID:28211111

ThrR, a DNA-binding transcription factor involved in controlling threonine biosynthesis in Bacillus subtilis.

PubMed

Rosenberg, Jonathan; Müller, Peter; Lentes, Sabine; Thiele, Martin J; Zeigler, Daniel R; Tödter, Dominik; Paulus, Henry; Brantl, Sabine; Stülke, Jörg; Commichau, Fabian M

2016-09-01

The threonine dehydratase IlvA is part of the isoleucine biosynthesis pathway in the Gram-positive model bacterium Bacillus subtilis. Consequently, deletion of ilvA causes isoleucine auxotrophy. It has been reported that ilvA pseudo-revertants having a derepressed hom-thrCB operon appear in the presence of threonine. Here we have characterized two classes of ilvA pseudo-revertants. In the first class the hom-thrCB operon was derepressed unmasking the threonine dehydratase activity of the threonine synthase ThrC. In the second class of mutants, threonine biosynthesis was more broadly affected. The first class of ilvA pseudo-revertants had a mutation in the Phom promoter (P*hom ), resulting in constitutive expression of the hom-thrCB operon. In the second class of ilvA pseudo-revertants, the thrR gene encoding a putative DNA-binding protein was inactivated, also resulting in constitutive expression of the hom-thrCB operon. Here we demonstrate that ThrR is indeed a DNA-binding transcription factor that regulates the hom-thrCB operon and the thrD aspartokinase gene. DNA binding assays uncovered the DNA-binding site of ThrR and revealed that the repressor competes with the RNA polymerase for DNA binding. This study also revealed that ThrR orthologs are ubiquitous in genomes from the Gram-positive phylum Firmicutes and in some Gram-negative bacteria. © 2016 John Wiley & Sons Ltd.

The effector gene xopAE of Xanthomonas euvesicatoria 85-10 is part of an operon and encodes an E3 ubiquitin ligase.

PubMed

Popov, Georgy; Majhi, Bharat Bhusan; Sessa, Guido

2018-05-21

The type III effector XopAE from the Xanthomonas euvesicatoria strain 85-10 ( Xe 85-10) was previously shown to inhibit plant immunity and enhance pathogen-induced disease symptoms. Evolutionary analysis of 60 xopAE alleles ( AEal ) revealed that the xopAE locus is conserved in multiple Xanthomonas species. The majority of xopAE alleles (55 out of 60) encodes a single ORF ( xopAE ), while in 5 alleles, including AEal 37 of the Xe 85-10 strain, a frame-shift splits the locus into two ORFs ( hpaF and a truncated xopAE ). To test whether the second ORF of AEal 37 ( xopAE 85-10 ) is translated, we examined expression of YFP fused downstream to truncated or mutant forms of the locus in Xanthomonas bacteria. YFP fluorescence was detected at maximal levels when the reporter was in proximity of an internal ribosome-binding site upstream to a rare ATT start codon in the xopAE 85-10 ORF, but severely reduced when these elements were abolished. In agreement with the notion that xopAE 85- 10 is a functional gene, its protein product was translocated into plant cells by the type III secretion system and translocation was dependent on its upstream ORF hpaF. Homology modeling predicted that XopAE 85-10 contains an E3 ligase XL-box domain at the C-terminus, and in vitro assays demonstrated that this domain displays mono-ubiquitination activity. Remarkably, the XL-box was essential for XopAE 85-10 to inhibit PAMP-induced gene expression in Arabidopsis protoplasts. Together, these results indicate that the xopAE 85-10 gene resides in a functional operon, which utilizes the alternative start codon ATT, and encodes a novel XL-box E3 ligase. Importance Xanthomonas bacteria utilize a type III secretion system to cause disease in many crops. This study provides insights into evolution, translocation and biochemical function of the XopAE type III secreted effector contributing to the understanding of Xanthomonas-host interactions. We establish XopAE as core effector of seven Xanthomonas species and elucidate evolution of the Xanthomonas euvesicatoria xopAE locus, which contains an operon encoding a truncated effector. Our findings indicate that this operon evolved from the split of a multi-domains gene into two ORFs that conserved the original domain function. Analysis of xopAE 85-10 translation provides the first evidence for translation initiation from an ATT codon in Xanthomonas Our data demonstrate that XopAE 85-10 is an XL-box E3 ubiquitin ligase and provide insights into structure and function of this effector family. Copyright © 2018 American Society for Microbiology.

Aureusimines in Staphylococcus aureus are not involved in virulence.

PubMed

Sun, Fei; Cho, Hoonsik; Jeong, Do-Won; Li, Chunling; He, Chuan; Bae, Taeok

2010-12-29

Recently, dipeptide aureusimines were reported to activate expression of staphylococcal virulence genes, such as alpha-hemolysin, and increase S. aureus virulence. Surprisingly, most of the virulence genes affected by aureusimines form part of the regulon of the SaeRS two component system (TCS), raising the possibility that SaeRS might be directly or indirectly involved in the aureusimine-dependent signaling process. Using HPLC analyses, we confirmed that a transposon mutant of ausA, the gene encoding the aureusimine dipeptide synthesis enzyme, does not produce dipeptides. However, the transposon mutant showed normal hemolysis activity and alpha-hemolysin/SaeP production. Furthermore, the P1 promoter of the sae operon, one of the targets of the SaeRS TCS, showed normal transcription activity. Moreover, in contrast to the original report, the ausA transposon mutant did not exhibit attenuated virulence in an animal infection model. DNA sequencing revealed that the ausA deletion mutant used in the original study has an 83 nt-duplication in saeS. Hemolysis activity of the original mutant was restored by a plasmid carrying the sae operon. A mutant of the sae operon showed elevated resistance to chloramphenicol and erythromycin, two antibiotics widely used during staphylococcal mutagenesis. At 43°C in the presence of erythromycin and aeration, the conditions typically employed for staphylococcal mutagenesis, an saeR transposon mutant grew much faster than a control mutant and the saeR mutant was highly enriched in a mixed culture experiment. Our results show that the previously reported roles of aureusimines in staphylococcal gene regulation and virulence were due to an unintended mutation in saeS, which was likely selected due to elevated resistance of the mutant to environmental stresses. Thus, there is no evidence indicating that the dipeptide aureusimines play a role in sae-mediated virulence factor production or contribute to staphylococcal virulence.

Dechlorination of lindane by the cyanobacterium Anabaena sp. strain PCC7120 depends on the function of the nir operon.

PubMed Central

Kuritz, T; Bocanera, L V; Rivera, N S

1997-01-01

Nitrate is essential for lindane dechlorination by the cyanobacteria Anabaena sp. strain PCC7120 and Nostoc ellipsosporum, as it is for dechlorination of other organic compounds by heterotrophic microorganisms. Based on analyses of mutants and effects of environmental factors, we conclude that lindane dechlorination by Anabaena sp. requires a functional nir operon that encodes the enzymes for nitrate utilization. PMID:9150239

Expression of the Arginine Deiminase Pathway Genes in Lactobacillus sakei Is Strain Dependent and Is Affected by the Environmental pH

PubMed Central

Rimaux, T.; Rivière, A.; Illeghems, K.; Weckx, S.; De Vuyst, L.

2012-01-01

The adaptation of Lactobacillus sakei to a meat environment is reflected in its metabolic potential. For instance, the ability to utilize arginine through the arginine deiminase (ADI) pathway, resulting in additional ATP, represents a competitive benefit. In L. sakei CTC 494, the arc operon (arcABCTDR) shows the same gene order and organization as that in L. sakei 23K, the genome sequence of which is known. However, differences in relative gene expression were found, and these seemed to be optimal in different growth phases, namely, the highest relative gene expression level was in the end exponential growth phase in the case of L. sakei CTC 494 and in the mid-exponential growth phase of L. sakei 23K. Also, the environmental pH influenced the relative expression level of the arc operon, as shown for L. sakei CTC 494, with the highest relative expression level occurring at the optimal pH for growth (pH 6.0). Deviations from this optimal pH (pH 5.0 and pH 7.0) resulted in an overall decline of the relative expression level of all genes of the arc operon. Furthermore, a differential relative expression of the individual genes of the arc operon was found, with the highest relative gene expression occurring for the first two genes of the arc operon (arcA and arcB). Finally, it was shown that some L. sakei strains were able to convert agmatine into putrescine, suggesting an operational agmatine deiminase pathway in these strains, a metabolic trait that is undesirable in meat fermentations. This study shows that this metabolic trait is most probably encoded by a previously erroneously annotated second putative arc operon. PMID:22544250

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

Cellulose as an extracellular matrix component present in Enterobacter sakazakii biofilms.

PubMed

Grimm, Maya; Stephan, Roger; Iversen, Carol; Manzardo, Giuseppe G G; Rattei, Thomas; Riedel, Kathrin; Ruepp, Andreas; Frishman, Dmitrij; Lehner, Angelika

2008-01-01

Cellulose was identified and characterized as an extracellular matrix component present in the biofilm of an Enterobacter sakazakii clinical isolate grown in nutrient-deficient (M9) medium. Using a bacterial artificial cloning approach in Escherichia coli and subsequent screening of transformants for fluorescence on calcofluor plates, nine genes organized in two operons were identified as putatively responsible for the biosynthesis of cellulose. In addition to the genes already described for cellulose production, two more genes were identified, putatively transcribed together with the genes from the first operon. Putative cellulose in E. sakazakii ES5 biofilm grown on glass coverslips was visualized by calcofluor staining and confocal fluorescence laser scanning microscopy. For the first time, the presence of cellulose in biofilms produced by E. sakazakii was confirmed by methylation analysis.

Cloning and characterization of ftsZ and pyrF from the archaeon Thermoplasma acidophilum

NASA Technical Reports Server (NTRS)

Yaoi, T.; Laksanalamai, P.; Jiemjit, A.; Kagawa, H. K.; Alton, T.; Trent, J. D.

2000-01-01

To characterize cytoskeletal components of archaea, the ftsZ gene from Thermoplasma acidophilum was cloned and sequenced. In T. acidophilum ftsZ, which is involved in cell division, was found to be in an operon with the pyrF gene, which encodes orotidine-5'-monophosphate decarboxylase (ODC), an essential enzyme in pyrimidine biosynthesis. Both ftsZ and pyrF from T. acidophilum were expressed in Escherichia coli and formed functional proteins. FtsZ expression in wild-type E. coli resulted in the filamentous phenotype characteristic of ftsZ mutants. T. acidophilum pyrF expression in an E. coli mutant lacking pyrF complemented the mutation and rescued the strain. Sequence alignments of ODCs from archaea, bacteria, and eukarya reveal five conserved regions, two of which have homology to 3-hexulose-6-phosphate synthase (HPS), suggesting a common substrate recognition and binding motif. Copyright 2000 Academic Press.

Paracoccus denitrificans possesses two BioR homologs having a role in regulation of biotin metabolism.

PubMed

Feng, Youjun; Kumar, Ritesh; Ravcheev, Dmitry A; Zhang, Huimin

2015-08-01

Recently, we determined that BioR, the GntR family of transcription factor, acts as a repressor for biotin metabolism exclusively distributed in certain species of α-proteobacteria, including the zoonotic agent Brucella melitensis and the plant pathogen Agrobacterium tumefaciens. However, the scenario is unusual in Paracoccus denitrificans, another closely related member of the same phylum α-proteobacteria featuring with denitrification. Not only does it encode two BioR homologs Pden_1431 and Pden_2922 (designated as BioR1 and BioR2, respectively), but also has six predictive BioR-recognizable sites (the two bioR homolog each has one site, whereas the two bio operons (bioBFDAGC and bioYB) each contains two tandem BioR boxes). It raised the possibility that unexpected complexity is present in BioR-mediated biotin regulation. Here we report that this is the case. The identity of the purified BioR proteins (BioR1 and BioR2) was confirmed with LC-QToF-MS. Phylogenetic analyses combined with GC percentage raised a possibility that the bioR2 gene might be acquired by horizontal gene transfer. Gel shift assays revealed that the predicted BioR-binding sites are functional for the two BioR homologs, in much similarity to the scenario seen with the BioR site of A. tumefaciens bioBFDAZ. Using the A. tumefaciens reporter system carrying a plasmid-borne LacZ fusion, we revealed that the two homologs of P. denitrificans BioR are functional repressors for biotin metabolism. As anticipated, not only does the addition of exogenous biotin stimulate efficiently the expression of bioYB operon encoding biotin transport/uptake system BioY, but also inhibits the transcription of the bioBFDAGC operon resembling the de novo biotin synthetic pathway. EMSA-based screening failed to demonstrate that the biotin-related metabolite is involved in BioR-DNA interplay, which is consistent with our former observation with Brucella BioR. Our finding defined a complex regulatory network for biotin metabolism in P. denitrificans by two BioR proteins. © 2015 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

Paracoccus denitrificans possesses two BioR homologs having a role in regulation of biotin metabolism

PubMed Central

Feng, Youjun; Kumar, Ritesh; Ravcheev, Dmitry A; Zhang, Huimin

2015-01-01

Recently, we determined that BioR, the GntR family of transcription factor, acts as a repressor for biotin metabolism exclusively distributed in certain species of α-proteobacteria, including the zoonotic agent Brucella melitensis and the plant pathogen Agrobacterium tumefaciens. However, the scenario is unusual in Paracoccus denitrificans, another closely related member of the same phylum α-proteobacteria featuring with denitrification. Not only does it encode two BioR homologs Pden_1431 and Pden_2922 (designated as BioR1 and BioR2, respectively), but also has six predictive BioR-recognizable sites (the two bioR homolog each has one site, whereas the two bio operons (bioBFDAGC and bioYB) each contains two tandem BioR boxes). It raised the possibility that unexpected complexity is present in BioR-mediated biotin regulation. Here we report that this is the case. The identity of the purified BioR proteins (BioR1 and BioR2) was confirmed with LC-QToF-MS. Phylogenetic analyses combined with GC percentage raised a possibility that the bioR2 gene might be acquired by horizontal gene transfer. Gel shift assays revealed that the predicted BioR-binding sites are functional for the two BioR homologs, in much similarity to the scenario seen with the BioR site of A. tumefaciens bioBFDAZ. Using the A. tumefaciens reporter system carrying a plasmid-borne LacZ fusion, we revealed that the two homologs of P. denitrificans BioR are functional repressors for biotin metabolism. As anticipated, not only does the addition of exogenous biotin stimulate efficiently the expression of bioYB operon encoding biotin transport/uptake system BioY, but also inhibits the transcription of the bioBFDAGC operon resembling the de novo biotin synthetic pathway. EMSA-based screening failed to demonstrate that the biotin-related metabolite is involved in BioR-DNA interplay, which is consistent with our former observation with Brucella BioR. Our finding defined a complex regulatory network for biotin metabolism in P. denitrificans by two BioR proteins. PMID:26037461

Bistability and Biofilm Formation in Bacillus subtilis

PubMed Central

Chai, Yunrong; Chu, Frances; Kolter, Roberto; Losick, Richard

2008-01-01

Summary Biofilms of Bacillus subtilis consist of long chains of cells that are held together in bundles by an extracellular matrix of exopolysaccharide and the protein TasA. The exopolysaccharide is produced by enzymes encoded by the epsA-O operon and the gene encoding TasA is located in the yqxM-sipW-tasA operon. Both operons are under the control of the repressor SinR. Derepression is mediated by the antirepressor SinI, which binds to SinR with a 1:1 stoichiometry. Paradoxically, in medium promoting derepression of the matrix operons, the overall concentration of SinR in the culture greatly exceeded that of SinI. We show that under biofilm-promoting conditions sinI, which is under the control of the response regulator Spo0A, was expressed only in a small subpopulation of cells, whereas sinR was expressed in almost all cells. Activation of Spo0A is known to be subject to a bistable switch, and we infer that SinI reaches levels sufficient to trigger matrix production only in the subpopulation of cells in which Spo0A is active. Additionally, evidence suggests that sinI is expressed at intermediate, but not low or high, levels of Spo0A activity, which may explain why certain nutritional conditions are more effective in promoting biofilm formation than others. PMID:18047568

Production of haemolysins by strains of the Actinobacillus minor/"porcitonsillarum" complex.

PubMed

Arya, Gitanjali; Niven, Donald F

2010-03-24

Actinobacillus minor and "Actinobacillus porcitonsillarum" are distinguished by their haemolytic activities, the latter organism being haemolytic and the former, non-haemolytic. Analysis of a whole genome shotgun sequence, however, revealed that A. minor strain 202, like "A. porcitonsillarum", possesses a haemolysin-encoding apxII operon. The purpose of this study was therefore to investigate haemolysin production by this organism and also by three additional members of the A. minor/"porcitonsillarum" complex, strains 33PN and 7ATS and A. minor strain NM305(T). Primers based on sequences within the apxII genes of strain 202 allowed the amplification of appropriately sized fragments from DNA from strain 33PN suggesting that this organism also possesses an apxII operon. Analysis of a whole genome shotgun sequence failed to reveal any trace of an apxII operon in strain NM305(T) and attempts to amplify apxII genes from DNA from strain 7ATS also failed. Strains 202 and 33PN, and surprisingly, the type strain of A. minor and strain 7ATS, were all found to be haemolysin-positive as growth media from cultures of these organisms could promote the lysis of erythrocytes in suspension. The erythrocyte specificities of the haemolysins produced by strains 202 and 33PN indicated that the haemolytic activities exhibited by these organisms were due to ApxII. In keeping with the apparent lack of apxII genes in strains NM305(T) and 7ATS, the haemolysins produced by these organisms were not erythrocyte-specific and with both organisms, haemolytic activity appeared to be due to a combination of heat-stable and heat-labile components. The identities of these components, however, remain unknown. Copyright 2009 Elsevier B.V. All rights reserved.

A novel endo-hydrogenase activity recycles hydrogen produced by nitrogen fixation.

PubMed

Ng, Gordon; Tom, Curtis G S; Park, Angela S; Zenad, Lounis; Ludwig, Robert A

2009-01-01

Nitrogen (N(2)) fixation also yields hydrogen (H(2)) at 1:1 stoichiometric amounts. In aerobic diazotrophic (able to grow on N(2) as sole N-source) bacteria, orthodox respiratory hupSL-encoded hydrogenase activity, associated with the cell membrane but facing the periplasm (exo-hydrogenase), has nevertheless been presumed responsible for recycling such endogenous hydrogen. As shown here, for Azorhizobium caulinodans diazotrophic cultures open to the atmosphere, exo-hydrogenase activity is of no consequence to hydrogen recycling. In a bioinformatic analysis, a novel seven-gene A. caulinodans hyq cluster encoding an integral-membrane, group-4, Ni,Fe-hydrogenase with homology to respiratory complex I (NADH: quinone dehydrogenase) was identified. By analogy, Hyq hydrogenase is also integral to the cell membrane, but its active site faces the cytoplasm (endo-hydrogenase). An A. caulinodans in-frame hyq operon deletion mutant, constructed by "crossover PCR", showed markedly decreased growth rates in diazotrophic cultures; normal growth was restored with added ammonium--as expected of an H(2)-recycling mutant phenotype. Using A. caulinodans hyq merodiploid strains expressing beta-glucuronidase as promoter-reporter, the hyq operon proved strongly and specifically induced in diazotrophic culture; as well, hyq operon induction required the NIFA transcriptional activator. Therefore, the hyq operon is constituent of the nif regulon. Representative of aerobic N(2)-fixing and H(2)-recycling alpha-proteobacteria, A. caulinodans possesses two respiratory Ni,Fe-hydrogenases: HupSL exo-hydrogenase activity drives exogenous H(2) respiration, and Hyq endo-hydrogenase activity recycles endogenous H(2), specifically that produced by N(2) fixation. To benefit human civilization, H(2) has generated considerable interest as potential renewable energy source as its makings are ubiquitous and its combustion yields no greenhouse gases. As such, the reversible, group-4 Ni,Fe-hydrogenases, such as the A. caulinodans Hyq endo-hydrogenase, offer promise as biocatalytic agents for H(2) production and/or consumption.

Lactate Utilization Is Regulated by the FadR-Type Regulator LldR in Pseudomonas aeruginosa

PubMed Central

Gao, Chao; Hu, Chunhui; Zheng, Zhaojuan; Jiang, Tianyi; Dou, Peipei; Zhang, Wen; Che, Bin; Wang, Yujiao; Lv, Min

2012-01-01

NAD-independent l-lactate dehydrogenase (l-iLDH) and NAD-independent d-lactate dehydrogenase (d-iLDH) activities are induced coordinately by either enantiomer of lactate in Pseudomonas strains. Inspection of the genomic sequences of different Pseudomonas strains revealed that the lldPDE operon comprises 3 genes, lldP (encoding a lactate permease), lldD (encoding an l-iLDH), and lldE (encoding a d-iLDH). Cotranscription of lldP, lldD, and lldE in Pseudomonas aeruginosa strain XMG starts with the base, C, that is located 138 bp upstream of the lldP ATG start codon. The lldPDE operon is located adjacent to lldR (encoding an FadR-type regulator, LldR). The gel mobility shift assays revealed that the purified His-tagged LldR binds to the upstream region of lldP. An XMG mutant strain that constitutively expresses d-iLDH and l-iLDH was found to contain a mutation in lldR that leads to an Ile23-to-serine substitution in the LldR protein. The mutated protein, LldRM, lost its DNA-binding activity. A motif with a hyphenated dyad symmetry (TGGTCTTACCA) was identified as essential for the binding of LldR to the upstream region of lldP by using site-directed mutagenesis. l-Lactate and d-lactate interfered with the DNA-binding activity of LldR. Thus, l-iLDH and d-iLDH were expressed when the operon was induced in the presence of l-lactate or d-lactate. PMID:22408166

Pseudomonas aeruginosa type IV minor pilins and PilY1 regulate virulence by modulating FimS-AlgR activity.

PubMed

Marko, Victoria A; Kilmury, Sara L N; MacNeil, Lesley T; Burrows, Lori L

2018-05-18

Type IV pili are expressed by a wide range of prokaryotes, including the opportunistic pathogen Pseudomonas aeruginosa. These flexible fibres mediate twitching motility, biofilm maturation, surface adhesion, and virulence. The pilus is composed mainly of major pilin subunits while the low abundance minor pilins FimU-PilVWXE and the putative adhesin PilY1 prime pilus assembly and are proposed to form the pilus tip. The minor pilins and PilY1 are encoded in an operon that is positively regulated by the FimS-AlgR two-component system. Independent of pilus assembly, PilY1 was proposed to be a mechanosensory component that-in conjunction with minor pilins-triggers up-regulation of acute virulence phenotypes upon surface attachment. Here, we investigated the link between the minor pilins/PilY1 and virulence. pilW, pilX, and pilY1 mutants had reduced virulence towards Caenorhabditis elegans relative to wild type or a major pilin mutant, implying a role in pathogenicity that is independent of pilus assembly. We hypothesized that loss of specific minor pilins relieves feedback inhibition on FimS-AlgR, increasing transcription of the AlgR regulon and delaying C. elegans killing. Reporter assays confirmed that FimS-AlgR were required for increased expression of the minor pilin operon upon loss of select minor pilins. Overexpression of AlgR or its hyperactivation via a phosphomimetic mutation reduced virulence, and the virulence defects of pilW, pilX, and pilY1 mutants required FimS-AlgR expression and activation. We propose that PilY1 and the minor pilins inhibit their own expression, and that loss of these proteins leads to FimS-mediated activation of AlgR that suppresses expression of acute-phase virulence factors and delays killing. This mechanism could contribute to adaptation of P. aeruginosa in chronic lung infections, as mutations in the minor pilin operon result in the loss of piliation and increased expression of AlgR-dependent virulence factors-such as alginate-that are characteristic of such infections.

Identification and Characterization of Cronobacter Iron Acquisition Systems

PubMed Central

Grim, C. J.; Kothary, M. H.; Gopinath, G.; Jarvis, K. G.; Beaubrun, J. Jean-Gilles; McClelland, M.; Tall, B. D.

2012-01-01

Cronobacter spp. are emerging pathogens that cause severe infantile meningitis, septicemia, or necrotizing enterocolitis. Contaminated powdered infant formula has been implicated as the source of Cronobacter spp. in most cases, but questions still remain regarding the natural habitat and virulence potential for each strain. The iron acquisition systems in 231 Cronobacter strains isolated from different sources were identified and characterized. All Cronobacter spp. have both the Feo and Efe systems for acquisition of ferrous iron, and all plasmid-harboring strains (98%) have the aerobactin-like siderophore, cronobactin, for transport of ferric iron. All Cronobacter spp. have the genes encoding an enterobactin-like siderophore, although it was not functional under the conditions tested. Furthermore, all Cronobacter spp. have genes encoding five receptors for heterologous siderophores. A ferric dicitrate transport system (fec system) is encoded specifically by a subset of Cronobacter sakazakii and C. malonaticus strains, of which a high percentage were isolated from clinical samples. Phylogenetic analysis confirmed that the fec system is most closely related to orthologous genes present in human-pathogenic bacterial strains. Moreover, all strains of C. dublinensis and C. muytjensii encode two receptors, FcuA and Fct, for heterologous siderophores produced by plant pathogens. Identification of putative Fur boxes and expression of the genes under iron-depleted conditions revealed which genes and operons are components of the Fur regulon. Taken together, these results support the proposition that C. sakazakii and C. malonaticus may be more associated with the human host and C. dublinensis and C. muytjensii with plants. PMID:22706064

Identification of Surprisingly Diverse Type IV Pili, across a Broad Range of Gram-Positive Bacteria

PubMed Central

Roos, David S.; Pohlschröder, Mechthild

2011-01-01

Background In Gram-negative bacteria, type IV pili (TFP) have long been known to play important roles in such diverse biological phenomena as surface adhesion, motility, and DNA transfer, with significant consequences for pathogenicity. More recently it became apparent that Gram-positive bacteria also express type IV pili; however, little is known about the diversity and abundance of these structures in Gram-positives. Computational tools for automated identification of type IV pilins are not currently available. Results To assess TFP diversity in Gram-positive bacteria and facilitate pilin identification, we compiled a comprehensive list of putative Gram-positive pilins encoded by operons containing highly conserved pilus biosynthetic genes (pilB, pilC). A surprisingly large number of species were found to contain multiple TFP operons (pil, com and/or tad). The N-terminal sequences of predicted pilins were exploited to develop PilFind, a rule-based algorithm for genome-wide identification of otherwise poorly conserved type IV pilins in any species, regardless of their association with TFP biosynthetic operons (http://signalfind.org). Using PilFind to scan 53 Gram-positive genomes (encoding >187,000 proteins), we identified 286 candidate pilins, including 214 in operons containing TFP biosynthetic genes (TBG+ operons). Although trained on Gram-positive pilins, PilFind identified 55 of 58 manually curated Gram-negative pilins in TBG+ operons, as well as 53 additional pilin candidates in operons lacking biosynthetic genes in ten species (>38,000 proteins), including 27 of 29 experimentally verified pilins. False positive rates appear to be low, as PilFind predicted only four pilin candidates in eleven bacterial species (>13,000 proteins) lacking TFP biosynthetic genes. Conclusions We have shown that Gram-positive bacteria contain a highly diverse set of type IV pili. PilFind can be an invaluable tool to study bacterial cellular processes known to involve type IV pilus-like structures. Its use in combination with other currently available computational tools should improve the accuracy of predicting the subcellular localization of bacterial proteins. PMID:22216142

The Use of Amino Sugars by Bacillus subtilis: Presence of a Unique Operon for the Catabolism of Glucosamine

PubMed Central

Gaugué, Isabelle; Oberto, Jacques; Putzer, Harald; Plumbridge, Jacqueline

2013-01-01

B. subtilis grows more rapidly using the amino sugar glucosamine as carbon source, than with N-acetylglucosamine. Genes for the transport and metabolism of N-acetylglucosamine (nagP and nagAB) are found in all the sequenced Bacilli (except Anoxybacillus flavithermus). In B. subtilis there is an additional operon (gamAP) encoding second copies of genes for the transport and catabolism of glucosamine. We have developed a method to make multiple deletion mutations in B. subtilis employing an excisable spectinomycin resistance cassette. Using this method we have analysed the contribution of the different genes of the nag and gam operons for their role in utilization of glucosamine and N-acetylglucosamine. Faster growth on glucosamine is due to the presence of the gamAP operon, which is strongly induced by glucosamine. Although the gamA and nagB genes encode isozymes of GlcN6P deaminase, catabolism of N-acetylglucosamine relies mostly upon the gamA gene product. The genes for use of N-acetylglucosamine, nagAB and nagP, are repressed by YvoA (NagR), a GntR family regulator, whose gene is part of the nagAB yvoA(nagR) operon. The gamAP operon is repressed by YbgA, another GntR family repressor, whose gene is expressed divergently from gamAP. The nagAB yvoA synton is found throughout the Bacilli and most firmicutes. On the other hand the ybgA-gamAP synton, which includes the ybgB gene for a small protein of unknown provenance, is only found in B. subtilis (and a few very close relatives). The origin of ybgBA-gamAP grouping is unknown but synteny analysis suggests lateral transfer from an unidentified donor. The presence of gamAP has enabled B. subtilis to efficiently use glucosamine as carbon source. PMID:23667565

hisT is part of a multigene operon in Escherichia coli K-12.

PubMed Central

Marvel, C C; Arps, P J; Rubin, B C; Kammen, H O; Penhoet, E E; Winkler, M E

1985-01-01

The Escherichia coli K-12 hisT gene has been cloned, and its organization and expression have been analyzed on multicopy plasmids. The hisT gene, which encodes tRNA pseudouridine synthase I (PSUI), was isolated on a Clarke-Carbon plasmid known to contain the purF gene. The presence of the hisT gene on this plasmid was suggested by its ability to restore both production of PSUI enzymatic activity and suppression of amber mutations in a hisT mutant strain. A 2.3-kilobase HindIII-ClaI restriction fragment containing the hisT gene was subcloned into plasmid pBR322, and the resulting plasmid (designated psi 300) was mapped with restriction enzymes. Complementation analysis with different kinds of hisT mutations and tRNA structural analysis confirmed that plasmid psi 300 contained the hisT structural gene. Enzyme assays showed that plasmid psi 300 overproduced PSUI activity by ca. 20-fold compared with the wild-type level. Subclones containing restriction fragments from plasmid psi 300 inserted downstream from the lac promoter established that the hisT gene is oriented from the HindIII site toward the ClaI site. Other subclones and derivatives of plasmid psi 300 containing insertion or deletion mutations were constructed and assayed for production of PSUI activity and production of proteins in minicells. These experiments showed that: (i) the proximal 1.3-kilobase HindIII-BssHII restriction fragment contains a promoter for the hisT gene and encodes a 45,000-dalton polypeptide that is not PSUI; (ii) the distal 1.0-kilobase BssHII-ClaI restriction fragment encodes the 31,000-dalton PSUI polypeptide; (iii) the 45,000-dalton polypeptide is synthesized in an approximately eightfold excess compared with PSUI; and (iv) synthesis of the two polypeptides is coupled, suggesting that the two genes are part of an operon. Insertion of mini-Mu d1 (lac Km) phage into plasmid psi 300 confirmed that the hisT gene is the downstream gene in the operon. Images PMID:2981810

GamR, the LysR-Type Galactose Metabolism Regulator, Regulates hrp Gene Expression via Transcriptional Activation of Two Key hrp Regulators, HrpG and HrpX, in Xanthomonas oryzae pv. oryzae.

PubMed

Rashid, M Mamunur; Ikawa, Yumi; Tsuge, Seiji

2016-07-01

Xanthomonas oryzae pv. oryzae is the causal agent of bacterial leaf blight of rice. For the virulence of the bacterium, the hrp genes, encoding components of the type III secretion system, are indispensable. The expression of hrp genes is regulated by two key hrp regulators, HrpG and HrpX: HrpG regulates hrpX, and HrpX regulates other hrp genes. Several other regulators have been shown to be involved in the regulation of hrp genes. Here, we found that a LysR-type transcriptional regulator that we named GamR, encoded by XOO_2767 of X. oryzae pv. oryzae strain MAFF311018, positively regulated the transcription of both hrpG and hrpX, which are adjacent to each other but have opposite orientations, with an intergenic upstream region in common. In a gel electrophoresis mobility shift assay, GamR bound directly to the middle of the upstream region common to hrpG and hrpX The loss of either GamR or its binding sites decreased hrpG and hrpX expression. Also, GamR bound to the upstream region of either a galactose metabolism-related gene (XOO_2768) or a galactose metabolism-related operon (XOO_2768 to XOO_2771) located next to gamR itself and positively regulated the genes. The deletion of the regulator gene resulted in less bacterial growth in a synthetic medium with galactose as a sole sugar source. Interestingly, induction of the galactose metabolism-related gene was dependent on galactose, while that of the hrp regulator genes was galactose independent. Our results indicate that the LysR-type transcriptional regulator that regulates the galactose metabolism-related gene(s) also acts in positive regulation of two key hrp regulators and the following hrp genes in X. oryzae pv. oryzae. The expression of hrp genes encoding components of the type III secretion system is essential for the virulence of many plant-pathogenic bacteria, including Xanthomonas oryzae pv. oryzae. It is specifically induced during infection. Research has revealed that in this bacterium, hrp gene expression is controlled by two key hrp regulators, HrpG and HrpX, along with several other regulators in the complex regulatory network, but the details remain unclear. Here, we found that a novel LysR-type transcriptional activator, named GamR, functions as an hrp regulator by directly activating the transcription of both hrpG and hrpX Interestingly, GamR also regulates a galactose metabolism-related gene (or operon) in a galactose-dependent manner, while the regulation of hrpG and hrpX is independent of the sugar. Our finding of a novel hrp regulator that directly and simultaneously regulates two key hrp regulators provides new insights into an important and complex regulation system of X. oryzae pv. oryzae hrp genes. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Redundant phenazine operons in Pseudomonas aeruginosa exhibit environment-dependent expression and differential roles in pathogenicity

PubMed Central

Recinos, David A.; Sekedat, Matthew D.; Hernandez, Adriana; Cohen, Taylor Sitarik; Sakhtah, Hassan; Prince, Alice S.; Price-Whelan, Alexa; Dietrich, Lars E. P.

2012-01-01

Evolutionary biologists have postulated that several fitness advantages may be conferred by the maintenance of duplicate genes, including environmental adaptation resulting from differential regulation. We examined the expression and physiological contributions of two redundant operons in the adaptable bacterium Pseudomonas aeruginosa PA14. These operons, phzA1-G1 (phz1) and phzA2-G2 (phz2), encode nearly identical sets of proteins that catalyze the synthesis of phenazine-1-carboxylic acid, the precursor for several phenazine derivatives. Phenazines perform diverse roles in P. aeruginosa physiology and act as virulence factors during opportunistic infections of plant and animal hosts. Although reports have indicated that phz1 is regulated by the Pseudomonas quinolone signal, factors controlling phz2 expression have not been identified, and the relative contributions of these redundant operons to phenazine biosynthesis have not been evaluated. We found that in liquid cultures, phz1 was expressed at higher levels than phz2, although phz2 showed a greater contribution to phenazine production. In colony biofilms, phz2 was expressed at high levels, whereas phz1 expression was not detectable, and phz2 was responsible for virtually all phenazine production. Analysis of mutants defective in quinolone signal synthesis revealed a critical role for 4-hydroxy-2-heptylquinoline in phz2 induction. Finally, deletion of phz2, but not of phz1, decreased lung colonization in a murine model of infection. These results suggest that differential regulation of the redundant phz operons allows P. aeruginosa to adapt to diverse environments. PMID:23129634

The Mercury Resistance Operon: From an Origin in a Geothermal Environment to an Efficient Detoxification Machine

PubMed Central

Boyd, Eric S.; Barkay, Tamar

2012-01-01

Mercuric mercury (Hg[II]) is a highly toxic and mobile element that is likely to have had a pronounced and adverse effect on biology since Earth’s oxygenation ∼2.4 billion years ago due to its high affinity for protein sulfhydryl groups, which upon binding destabilize protein structure and decrease enzyme activity, resulting in a decreased organismal fitness. The central enzyme in the microbial mercury detoxification system is the mercuric reductase (MerA) protein, which catalyzes the reduction of Hg(II) to volatile Hg(0). In addition to MerA, mer operons encode for proteins involved in regulation, Hg binding, and organomercury degradation. Mer-mediated approaches have had broad applications in the bioremediation of mercury-contaminated environments and industrial waste streams. Here, we examine the composition of 272 individual mer operons and quantitatively map the distribution of mer-encoded functions on both taxonomic SSU rRNA gene and MerA phylogenies. The results indicate an origin and early evolution of MerA among thermophilic bacteria and an overall increase in the complexity of mer operons through evolutionary time, suggesting continual gene recruitment and evolution leading to an improved efficiency and functional potential of the Mer detoxification system. Consistent with a positive relationship between the evolutionary history and topology of MerA and SSU rRNA gene phylogenies (Mantel R = 0.81, p < 0.01), the distribution of the majority of mer functions, when mapped on these phylograms, indicates an overall tendency to inherit mer-encoded functions through vertical descent. However, individual mer functions display evidence of a variable degree of vertical inheritance, with several genes exhibiting strong evidence for acquisition via lateral gene transfer and/or gene loss. Collectively, these data suggest that (i) mer has evolved from a simple system in geothermal environments to a widely distributed and more complex and efficient detoxification system, and (ii) merA is a suitable biomarker for examining the functional diversity of Hg detoxification and for predicting the composition of mer operons in natural environments. PMID:23087676

Role of P27 -P55 operon from Mycobacterium tuberculosis in the resistance to toxic compounds

PubMed Central

2011-01-01

Background The P27-P55 (lprG-Rv1410c) operon is crucial for the survival of Mycobacterium tuberculosis, the causative agent of human tuberculosis, during infection in mice. P55 encodes an efflux pump that has been shown to provide Mycobacterium smegmatis and Mycobacterium bovis BCG with resistance to several drugs, while P27 encodes a mannosylated glycoprotein previously described as an antigen that modulates the immune response against mycobacteria. The objective of this study was to determine the individual contribution of the proteins encoded in the P27-P55 operon to the resistance to toxic compounds and to the cell wall integrity of M. tuberculosis. Method In order to test the susceptibility of a mutant of M. tuberculosis H37Rv in the P27-P55 operon to malachite green, sodium dodecyl sulfate, ethidium bromide, and first-line antituberculosis drugs, this strain together with the wild type strain and a set of complemented strains were cultivated in the presence and in the absence of these drugs. In addition, the malachite green decolorization rate of each strain was obtained from decolorization curves of malachite green in PBS containing bacterial suspensions. Results The mutant strain decolorized malachite green faster than the wild type strain and was hypersensitive to both malachite green and ethidium bromide, and more susceptible to the first-line antituberculosis drugs: isoniazid and ethambutol. The pump inhibitor reserpine reversed M. tuberculosis resistance to ethidium bromide. These results suggest that P27-P55 functions through an efflux-pump like mechanism. In addition, deletion of the P27-P55 operon made M. tuberculosis susceptible to sodium dodecyl sulfate, suggesting that the lack of both proteins causes alterations in the cell wall permeability of the bacterium. Importantly, both P27 and P55 are required to restore the wild type phenotypes in the mutant. Conclusions The results clearly indicate that P27 and P55 are functionally connected in processes that involve the preservation of the cell wall and the transport of toxic compounds away from the cells. PMID:21762531

Hyper Accumulation of Arsenic in Mutants of Ochrobactrum tritici Silenced for Arsenite Efflux Pumps

PubMed Central

Piedade, Ana Paula; Morais, Paula V.

2015-01-01

Ochrobactrum tritici SCII24T is a highly As-resistant bacterium, with two previously described arsenic resistance operons, ars1 and ars2. Among a large number of genes, these operons contain the arsB and Acr3 genes that encode the arsenite efflux pumps responsible for arsenic resistance. Exploring the genome of O. tritici SCII24T, an additional putative operon (ars3) was identified and revealed the presence of the Acr3_2 gene that encodes for an arsenite efflux protein but which came to prove to not be required for full As resistance. The genes encoding for arsenite efflux pumps, identified in this strain, were inactivated to develop microbial accumulators of arsenic as new tools for bioremediation. Six different mutants were produced, studied and three were more useful as biotools. O. tritici wild type and the Acr3-mutants showed the highest resistance to As(III), being able to grow up to 50 mM of arsenite. On the other hand, arsB-mutants were not able to grow at concentrations higher than 1 mM As(III), and were the most As(III) sensitive mutants. In the presence of 1 mM As(III), the strain with arsB and Acr3_1 mutated showed the highest intracellular arsenic concentration (up to 17 ng(As)/mg protein), while in assays with 5 mM As(III), the single arsB-mutant was able to accumulate the highest concentration of arsenic (up to 10 ng(As)/mg protein). Therefore, arsB is the main gene responsible for arsenite resistance in O. tritici. However, both genes arsB and Acr3_1 play a crucial role in the resistance mechanism, depending on the arsenite concentration in the medium. In conclusion, at moderate arsenite concentrations, the double arsB- and Acr3_1-mutant exhibited a great ability to accumulate arsenite and can be seen as a promising bioremediation tool for environmental arsenic detoxification. PMID:26132104

Cloning, sequencing, and characterization of the Bacillus subtilis biotin biosynthetic operon.

PubMed

Bower, S; Perkins, J B; Yocum, R R; Howitt, C L; Rahaim, P; Pero, J

1996-07-01

A 10-kb region of the Bacillus subtilis genome that contains genes involved in biotin-biosynthesis was cloned and sequenced. DNA sequence analysis indicated that B. subtilis contains homologs of the Escherichia coli and Bacillus sphaericus bioA, bioB, bioD, and bioF genes. These four genes and a homolog of the B. sphaericus bioW gene are arranged in a single operon in the order bioWAFDR and are followed by two additional genes, bioI and orf2. bioI and orf2 show no similarity to any other known biotin biosynthetic genes. The bioI gene encodes a protein with similarity to cytochrome P-450s and was able to complement mutations in either bioC or bioH of E. coli. Mutations in bioI caused B. subtilis to grow poorly in the absence of biotin. The bradytroph phenotype of bioI mutants was overcome by pimelic acid, suggesting that the product of bioI functions at a step prior to pimelic acid synthesis. The B. subtilis bio operon is preceded by a putative vegetative promoter sequence and contains just downstream a region of dyad symmetry with homology to the bio regulatory region of B. sphaericus. Analysis of a bioW-lacZ translational fusion indicated that expression of the biotin operon is regulated by biotin and the B. subtilis birA gene.

Cloning, sequencing, and characterization of the Bacillus subtilis biotin biosynthetic operon.

PubMed Central

Bower, S; Perkins, J B; Yocum, R R; Howitt, C L; Rahaim, P; Pero, J

1996-01-01

A 10-kb region of the Bacillus subtilis genome that contains genes involved in biotin-biosynthesis was cloned and sequenced. DNA sequence analysis indicated that B. subtilis contains homologs of the Escherichia coli and Bacillus sphaericus bioA, bioB, bioD, and bioF genes. These four genes and a homolog of the B. sphaericus bioW gene are arranged in a single operon in the order bioWAFDR and are followed by two additional genes, bioI and orf2. bioI and orf2 show no similarity to any other known biotin biosynthetic genes. The bioI gene encodes a protein with similarity to cytochrome P-450s and was able to complement mutations in either bioC or bioH of E. coli. Mutations in bioI caused B. subtilis to grow poorly in the absence of biotin. The bradytroph phenotype of bioI mutants was overcome by pimelic acid, suggesting that the product of bioI functions at a step prior to pimelic acid synthesis. The B. subtilis bio operon is preceded by a putative vegetative promoter sequence and contains just downstream a region of dyad symmetry with homology to the bio regulatory region of B. sphaericus. Analysis of a bioW-lacZ translational fusion indicated that expression of the biotin operon is regulated by biotin and the B. subtilis birA gene. PMID:8763940

Pseudomonas aeruginosa uses a cyclic-di-GMP-regulated adhesin to reinforce the biofilm extracellular matrix

PubMed Central

Borlee, Bradley R; Goldman, Aaron D; Murakami, Keiji; Samudrala, Ram; Wozniak, Daniel J; Parsek, Matthew R

2010-01-01

Pseudomonas aeruginosa, the principal pathogen of cystic fibrosis patients, forms antibiotic-resistant biofilms promoting chronic colonization of the airways. The extracellular (EPS) matrix is a crucial component of biofilms that provides the community multiple benefits. Recent work suggests that the secondary messenger, cyclic-di-GMP, promotes biofilm formation. An analysis of factors specifically expressed in P. aeruginosa under conditions of elevated c-di-GMP, revealed functions involved in the production and maintenance of the biofilm extracellular matrix. We have characterized one of these components, encoded by the PA4625 gene, as a putative adhesin and designated it cdrA. CdrA shares structural similarities to extracellular adhesins that belong to two-partner secretion systems. The cdrA gene is in a two gene operon that also encodes a putative outer membrane transporter, CdrB. The cdrA gene encodes a 220 KDa protein that is predicted to be rod-shaped protein harbouring a β-helix structural motif. Western analysis indicates that the CdrA is produced as a 220 kDa proprotein and processed to 150 kDa before secretion into the extracellular medium. We demonstrated that cdrAB expression is minimal in liquid culture, but is elevated in biofilm cultures. CdrAB expression was found to promote biofilm formation and auto-aggregation in liquid culture. Aggregation mediated by CdrA is dependent on the Psl polysaccharide and can be disrupted by adding mannose, a key structural component of Psl. Immunoprecipitation of Psl present in culture supernatants resulted in co-immunoprecipitation of CdrA, providing additional evidence that CdrA directly binds to Psl. A mutation in cdrA caused a decrease in biofilm biomass and resulted in the formation of biofilms exhibiting decreased structural integrity. Psl-specific lectin staining suggests that CdrA either cross-links Psl polysaccharide polymers and/or tethers Psl to the cells, resulting in increased biofilm structural stability. Thus, this study identifies a key protein structural component of the P. aeruginosa EPS matrix. PMID:20088866

A Eukaryotic-Type Serine/Threonine Protein Kinase Is Required for Biofilm Formation, Genetic Competence, and Acid Resistance in Streptococcus mutans

PubMed Central

Hussain, Haitham; Branny, Pavel; Allan, Elaine

2006-01-01

We report an operon encoding a eukaryotic-type serine/threonine protein kinase (STPK) and its cognate phosphatase (STPP) in Streptococcus mutans. Mutation of the gene encoding the STPK produced defects in biofilm formation, genetic competence, and acid resistance, determinants important in caries pathogenesis. PMID:16452447

Identification of genetic loci that contribute to Campylobacter resistance to fowlicidin-1, a chicken host defense peptide

PubMed Central

Hoang, Ky Van; Wang, Ying; Lin, Jun

2012-01-01

Antimicrobial peptides (AMPs) are critical components of host defense limiting bacterial infections at the gastrointestinal mucosal surface. Bacterial pathogens have co-evolved with host innate immunity and developed means to counteract the effect of endogenous AMPs. However, molecular mechanisms of AMP resistance in Campylobacter, an important human food-borne pathogen with poultry as a major reservoir, are still largely unknown. In this study, random transposon mutagenesis and targeted site-directed mutagenesis approaches were used to identify genetic loci contributing Campylobacter resistance to fowlicidin-1, a chicken AMP belonging to cathelicidin family. An efficient transposon mutagenesis approach (EZ::TN™ Transposome) in conjunction with a microtiter plate screening identified three mutants whose susceptibilities to fowlicidin-1 were significantly increased. Backcrossing of the transposon mutations into parent strain confirmed that the AMP-sensitive phenotype in each mutant was linked to the specific transposon insertion. Direct sequencing showed that these mutants have transposon inserted in the genes encoding two-component regulator CbrR, transporter CjaB, and putative trigger factor Tig. Genomic analysis also revealed an operon (Cj1580c-1584c) that is homologous to sapABCDF, an operon conferring resistance to AMP in other pathogens. Insertional inactivation of Cj1583c (sapB) significantly increased susceptibility of Campylobacter to fowlicidin-1. The sapB as well as tig and cjaB mutants were significantly impaired in their ability to compete with their wild-type strain 81–176 to colonize the chicken cecum. Together, this study identified four genetic loci in Campylobacter that will be useful for characterizing molecular basis of Campylobacter resistance to AMPs, a significant knowledge gap in Campylobacter pathogenesis. PMID:22919624

The PhoBR two-component system regulates antibiotic biosynthesis in Serratia in response to phosphate

PubMed Central

2009-01-01

Background Secondary metabolism in Serratia sp. ATCC 39006 (Serratia 39006) is controlled via a complex network of regulators, including a LuxIR-type (SmaIR) quorum sensing (QS) system. Here we investigate the molecular mechanism by which phosphate limitation controls biosynthesis of two antibiotic secondary metabolites, prodigiosin and carbapenem, in Serratia 39006. Results We demonstrate that a mutation in the high affinity phosphate transporter pstSCAB-phoU, believed to mimic low phosphate conditions, causes upregulation of secondary metabolism and QS in Serratia 39006, via the PhoBR two-component system. Phosphate limitation also activated secondary metabolism and QS in Serratia 39006. In addition, a pstS mutation resulted in upregulation of rap. Rap, a putative SlyA/MarR-family transcriptional regulator, shares similarity with the global regulator RovA (regulator of virulence) from Yersina spp. and is an activator of secondary metabolism in Serratia 39006. We demonstrate that expression of rap, pigA-O (encoding the prodigiosin biosynthetic operon) and smaI are controlled via PhoBR in Serratia 39006. Conclusion Phosphate limitation regulates secondary metabolism in Serratia 39006 via multiple inter-linked pathways, incorporating transcriptional control mediated by three important global regulators, PhoB, SmaR and Rap. PMID:19476633

Induction of the Nitrate Assimilation nirA Operon and Protein-Protein Interactions in the Maturation of Nitrate and Nitrite Reductases in the Cyanobacterium Anabaena sp. Strain PCC 7120.

PubMed

Frías, José E; Flores, Enrique

2015-07-01

Nitrate is widely used as a nitrogen source by cyanobacteria, in which the nitrate assimilation structural genes frequently constitute the so-called nirA operon. This operon contains the genes encoding nitrite reductase (nirA), a nitrate/nitrite transporter (frequently an ABC-type transporter; nrtABCD), and nitrate reductase (narB). In the model filamentous cyanobacterium Anabaena sp. strain PCC 7120, which can fix N2 in specialized cells termed heterocysts, the nirA operon is expressed at high levels only in media containing nitrate or nitrite and lacking ammonium, a preferred nitrogen source. Here we examined the genes downstream of the nirA operon in Anabaena and found that a small open reading frame of unknown function, alr0613, can be cotranscribed with the operon. The next gene in the genome, alr0614 (narM), showed an expression pattern similar to that of the nirA operon, implying correlated expression of narM and the operon. A mutant of narM with an insertion mutation failed to produce nitrate reductase activity, consistent with the idea that NarM is required for the maturation of NarB. Both narM and narB mutants were impaired in the nitrate-dependent induction of the nirA operon, suggesting that nitrite is an inducer of the operon in Anabaena. It has previously been shown that the nitrite reductase protein NirA requires NirB, a protein likely involved in protein-protein interactions, to attain maximum activity. Bacterial two-hybrid analysis confirmed possible NirA-NirB and NarB-NarM interactions, suggesting that the development of both nitrite reductase and nitrate reductase activities in cyanobacteria involves physical interaction of the corresponding enzymes with their cognate partners, NirB and NarM, respectively. Nitrate is an important source of nitrogen for many microorganisms that is utilized through the nitrate assimilation system, which includes nitrate/nitrite membrane transporters and the nitrate and nitrite reductases. Many cyanobacteria assimilate nitrate, but regulation of the nitrate assimilation system varies in different cyanobacterial groups. In the N2-fixing, heterocyst-forming cyanobacteria, the nirA operon, which includes the structural genes for the nitrate assimilation system, is expressed in the presence of nitrate or nitrite if ammonium is not available to the cells. Here we studied the genes required for production of an active nitrate reductase, providing information on the nitrate-dependent induction of the operon, and found evidence for possible protein-protein interactions in the maturation of nitrate reductase and nitrite reductase. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

The Dickeya dadantii biofilm matrix consists of cellulose nanofibres, and is an emergent property dependent upon the type III secretion system and the cellulose synthesis operon.

PubMed

Jahn, Courtney E; Selimi, Dija A; Barak, Jeri D; Charkowski, Amy O

2011-10-01

Dickeya dadantii is a plant-pathogenic bacterium that produces cellulose-containing biofilms, called pellicles, at the air-liquid interface of liquid cultures. D. dadantii pellicle formation appears to be an emergent property dependent upon at least three gene clusters, including cellulose synthesis, type III secretion system (T3SS) and flagellar genes. The D. dadantii cellulose synthesis operon is homologous to that of Gluconacetobacter xylinus, which is used for industrial cellulose production, and the cellulose nanofibres produced by D. dadantii were similar in diameter and branching pattern to those produced by G. xylinus. Salmonella enterica, an enterobacterium closely related to D. dadantii, encodes a second type of cellulose synthesis operon, and it produced biofilm strands that differed in width and branching pattern from those of D. dadantii and G. xylinus. Unlike any previously described cellulose fibre, the D. dadantii cellulose nanofibres were decorated with bead-like structures. Mutation of the cellulose synthesis operon genes resulted in loss of cellulose synthesis and production of a cellulase-resistant biofilm. Mutation of other genes required for pellicle formation, including those encoding FliA (a sigma factor that regulates flagella production), HrpL (a sigma factor that regulates the T3SS), and AdrA, a GGDEF protein, affected both biofilm and cell morphology. Mutation of the cellulose synthase bcsA or of bcsC resulted in decreased accumulation of the T3SS-secreted protein HrpN.

Contribution of Resistance-Nodulation-Division Efflux Pump Operon smeU1-V-W-U2-X to Multidrug Resistance of Stenotrophomonas maltophilia ▿

PubMed Central

Chen, Chao-Hsien; Huang, Chiang-Ching; Chung, Tsao-Chuen; Hu, Rouh-Mei; Huang, Yi-Wei; Yang, Tsuey-Ching

2011-01-01

KJ09C, a multidrug-resistant mutant of Stenotrophomonas maltophilia KJ, was generated by in vitro selection with chloramphenicol. The multidrug-resistant phenotype of KJ09C was attributed to overexpression of a resistance nodulation division (RND)-type efflux system encoded by an operon consisting of five genes: smeU1, smeV, smeW, smeU2, and smeX. Proteins encoded by smeV, smeW, and smeX were similar to the membrane fusion protein, RND transporter, and outer membrane protein, respectively, of known RND-type systems. The proteins encoded by smeU1 and smeU2 were found to belong to the family of short-chain dehydrogenases/reductases. Mutant KJ09C exhibited increased resistance to chloramphenicol, quinolones, and tetracyclines and susceptibility to aminoglycosides; susceptibility to β-lactams and erythromycin was not affected. The expression of the smeU1-V-W-U2-X operon was regulated by the divergently transcribed LysR-type regulator gene smeRv. Overexpression of the SmeVWX pump contributed to the acquired resistance to chloramphenicol, quinolones, and tetracyclines. Inactivation of smeV and smeW completely abolished the activity of the SmeVWX pump, whereas inactivation of smeX alone decreased the activity of the SmeVWX pump. The enhanced aminoglycoside susceptibility observed in KJ09C resulted from SmeX overexpression. PMID:21930878

The rpoE operon regulates heat stress response in Burkholderia pseudomallei.

PubMed

Vanaporn, Muthita; Vattanaviboon, Paiboon; Thongboonkerd, Visith; Korbsrisate, Sunee

2008-07-01

Burkholderia pseudomallei is a gram-negative bacterium and the causative agent of melioidosis, one of the important lethal diseases in tropical regions. In this article, we demonstrate the crucial role of the B. pseudomallei rpoE locus in the response to heat stress. The rpoE operon knockout mutant exhibited growth retardation and reduced survival when exposed to a high temperature. Expression analysis using rpoH promoter-lacZ fusion revealed that heat stress induction of rpoH, which encodes heat shock sigma factor (sigma(H)), was abolished in the B. pseudomallei rpoE mutant. Analysis of the rpoH promoter region revealed sequences sharing high homology to the consensus sequence of sigma(E)-dependent promoters. Moreover, the putative heat-induced sigma(H)-regulated heat shock proteins (i.e. GroEL and HtpG) were also absent in the rpoE operon mutant. Altogether, our data suggest that the rpoE operon regulates B. pseudomallei heat stress response through the function of rpoH.

Increased dipicolinic acid production with an enhanced spoVF operon in Bacillus subtilis and medium optimization.

PubMed

Takahashi, Fumikazu; Sumitomo, Nobuyuki; Hagihara, Hiroshi; Ozaki, Katsuya

2015-01-01

Dipicolinic acid (DPA) is a multi-functional agent for cosmetics, antimicrobial products, detergents, and functional polymers. The aim of this study was to design a new method for producing DPA from renewable material. The Bacillus subtilis spoVF operon encodes enzymes for DPA synthase and the part of lysine biosynthetic pathway. However, DPA is only synthesized in the sporulation phase, so the productivity of DPA is low level. Here, we report that DPA synthase was expressed in vegetative cells, and DPA was produced in the culture medium by replacement of the spoVFA promoter with other highly expressed promoter in B. subtilis vegetative cells, such as spoVG promoter. DPA levels were increased in the culture medium of genetically modified strains. DPA productivity was significantly improved up to 29.14 g/L in 72 h culture by improving the medium composition using a two-step optimization technique with the Taguchi methodology.

yadBC of Yersinia pestis, a new virulence determinant for bubonic plague.

PubMed

Forman, Stanislav; Wulff, Christine R; Myers-Morales, Tanya; Cowan, Clarissa; Perry, Robert D; Straley, Susan C

2008-02-01

In all Yersinia pestis strains examined, the adhesin/invasin yadA gene is a pseudogene, yet Y. pestis is invasive for epithelial cells. To identify potential surface proteins that are structurally and functionally similar to YadA, we searched the Y. pestis genome for open reading frames with homology to yadA and found three: the bicistronic operon yadBC (YPO1387 and YPO1388 of Y. pestis CO92; y2786 and y2785 of Y. pestis KIM5), which encodes two putative surface proteins, and YPO0902, which lacks a signal sequence and likely is nonfunctional. In this study we characterized yadBC regulation and tested the importance of this operon for Y. pestis adherence, invasion, and virulence. We found that loss of yadBC caused a modest loss of invasiveness for epithelioid cells and a large decrease in virulence for bubonic plague but not for pneumonic plague in mice.

Identification and Characterization of Non-Cellulose-Producing Mutants of Gluconacetobacter hansenii Generated by Tn5 Transposon Mutagenesis

PubMed Central

Deng, Ying; Nagachar, Nivedita; Xiao, Chaowen; Tien, Ming

2013-01-01

The acs operon of Gluconacetobacter is thought to encode AcsA, AcsB, AcsC, and AcsD proteins that constitute the cellulose synthase complex, required for the synthesis and secretion of crystalline cellulose microfibrils. A few other genes have been shown to be involved in this process, but their precise role is unclear. We report here the use of Tn5 transposon insertion mutagenesis to identify and characterize six non-cellulose-producing (Cel−) mutants of Gluconacetobacter hansenii ATCC 23769. The genes disrupted were acsA, acsC, ccpAx (encoding cellulose-complementing protein [the subscript “Ax” indicates genes from organisms formerly classified as Acetobacter xylinum]), dgc1 (encoding guanylate dicyclase), and crp-fnr (encoding a cyclic AMP receptor protein/fumarate nitrate reductase transcriptional regulator). Protein blot analysis revealed that (i) AcsB and AcsC were absent in the acsA mutant, (ii) the levels of AcsB and AcsC were significantly reduced in the ccpAx mutant, and (iii) the level of AcsD was not affected in any of the Cel− mutants. Promoter analysis showed that the acs operon does not include acsD, unlike the organization of the acs operon of several strains of closely related Gluconacetobacter xylinus. Complementation experiments confirmed that the gene disrupted in each Cel− mutant was responsible for the phenotype. Quantitative real-time PCR and protein blotting results suggest that the transcription of bglAx (encoding β-glucosidase and located immediately downstream from acsD) was strongly dependent on Crp/Fnr. A bglAx knockout mutant, generated via homologous recombination, produced only ∼16% of the wild-type cellulose level. Since the crp-fnr mutant did not produce any cellulose, Crp/Fnr may regulate the expression of other gene(s) involved in cellulose biosynthesis. PMID:24013627

Export of l-Isoleucine from Corynebacterium glutamicum: a Two-Gene-Encoded Member of a New Translocator Family

PubMed Central

Kennerknecht, Nicole; Sahm, Hermann; Yen, Ming-Ren; Pátek, Miroslav; Saier, Jr., Milton H.; Eggeling, Lothar

2002-01-01

Bacteria possess amino acid export systems, and Corynebacterium glutamicum excretes l-isoleucine in a process dependent on the proton motive force. In order to identify the system responsible for l-isoleucine export, we have used transposon mutagenesis to isolate mutants of C. glutamicum sensitive to the peptide isoleucyl-isoleucine. In one such mutant, strong peptide sensitivity resulted from insertion into a gene designated brnF encoding a hydrophobic protein predicted to possess seven transmembrane spanning helices. brnE is located downstream of brnF and encodes a second hydrophobic protein with four putative membrane-spanning helices. A mutant deleted of both genes no longer exports l-isoleucine, whereas an overexpressing strain exports this amino acid at an increased rate. BrnF and BrnE together are also required for the export of l-leucine and l-valine. BrnFE is thus a two-component export permease specific for aliphatic hydrophobic amino acids. Upstream of brnFE and transcribed divergently is an Lrp-like regulatory gene required for active export. Searches for homologues of BrnFE show that this type of exporter is widespread in prokaryotes but lacking in eukaryotes and that both gene products which together comprise the members of a novel family, the LIV-E family, generally map together within a single operon. Comparisons of the BrnF and BrnE phylogenetic trees show that gene duplication events in the early bacterial lineage gave rise to multiple paralogues that have been retained in α-proteobacteria but not in other prokaryotes analyzed. PMID:12081967

Transcription of the Streptococcus pyogenes Hyaluronic Acid Capsule Biosynthesis Operon Is Regulated by Previously Unknown Upstream Elements

PubMed Central

Falaleeva, Marina; Zurek, Oliwia W.; Watkins, Robert L.; Reed, Robert W.; Ali, Hadeel; Sumby, Paul; Voyich, Jovanka M.

2014-01-01

The important human pathogen Streptococcus pyogenes (group A Streptococcus [GAS]) produces a hyaluronic acid (HA) capsule that plays critical roles in immune evasion. Previous studies showed that the hasABC operon encoding the capsule biosynthesis enzymes is under the control of a single promoter, P1, which is negatively regulated by the two-component regulatory system CovR/S. In this work, we characterize the sequence upstream of P1 and identify a novel regulatory region controlling transcription of the capsule biosynthesis operon in the M1 serotype strain MGAS2221. This region consists of a promoter, P2, which initiates transcription of a novel small RNA, HasS, an intrinsic transcriptional terminator that inefficiently terminates HasS, permitting read-through transcription of hasABC, and a putative promoter which lies upstream of P2. Electrophoretic mobility shift assays, quantitative reverse transcription-PCR, and transcriptional reporter data identified CovR as a negative regulator of P2. We found that the P1 and P2 promoters are completely repressed by CovR, and capsule expression is regulated by the putative promoter upstream of P2. Deletion of hasS or of the terminator eliminates CovR-binding sequences, relieving repression and increasing read-through, hasA transcription, and capsule production. Sequence analysis of 44 GAS genomes revealed a high level of polymorphism in the HasS sequence region. Most of the HasS variations were located in the terminator sequences, suggesting that this region is under strong selective pressure. We discovered that the terminator deletion mutant is highly resistant to neutrophil-mediated killing and is significantly more virulent in a mouse model of GAS invasive disease than the wild-type strain. Together, these results are consistent with the naturally occurring mutations in this region modulating GAS virulence. PMID:25287924

Strategy of Pseudomonas pseudoalcaligenes C70 for effective degradation of phenol and salicylate

PubMed Central

Heinaru, Eeva; Naanuri, Eve; Mehike, Maris; Leito, Ivo; Heinaru, Ain

2017-01-01

Phenol- and naphthalene-degrading indigenous Pseudomonas pseudoalcaligenes strain C70 has great potential for the bioremediation of polluted areas. It harbours two chromosomally located catechol meta pathways, one of which is structurally and phylogenetically very similar to the Pseudomonas sp. CF600 dmp operon and the other to the P. stutzeri AN10 nah lower operon. The key enzymes of the catechol meta pathway, catechol 2,3-dioxygenase (C23O) from strain C70, PheB and NahH, have an amino acid identity of 85%. The metabolic and regulatory phenotypes of the wild-type and the mutant strain C70ΔpheB lacking pheB were evaluated. qRT-PCR data showed that in C70, the expression of pheB- and nahH-encoded C23O was induced by phenol and salicylate, respectively. We demonstrate that strain C70 is more effective in the degradation of phenol and salicylate, especially at higher substrate concentrations, when these compounds are present as a mixture; i.e., when both pathways are expressed. Moreover, NahH is able to substitute for the deleted PheB in phenol degradation when salicylate is also present in the growth medium. The appearance of a yellow intermediate 2-hydroxymuconic semialdehyde was followed by the accumulation of catechol in salicylate-containing growth medium, and lower expression levels and specific activities of the C23O of the sal operon were detected. However, the excretion of the toxic intermediate catechol to the growth medium was avoided when the growth medium was supplemented with phenol, seemingly due to the contribution of the second meta pathway encoded by the phe genes. PMID:28257519

A novel l-isoleucine-4′-dioxygenase and l-isoleucine dihydroxylation cascade in Pantoea ananatis

PubMed Central

Smirnov, Sergey V; Sokolov, Pavel M; Kotlyarova, Veronika A; Samsonova, Natalya N; Kodera, Tomohiro; Sugiyama, Masakazu; Torii, Takayoshi; Hibi, Makoto; Shimizu, Sakayu; Yokozeki, Kenzo; Ogawa, Jun

2013-01-01

A unique operon structure has been identified in the genomes of several plant- and insect-associated bacteria. The distinguishing feature of this operon is the presence of tandem hilA and hilB genes encoding dioxygenases belonging to the PF13640 and PF10014 (BsmA) Pfam families, respectively. The genes encoding HilA and HilB from Pantoea ananatis AJ13355 were cloned and expressed in Escherichia coli. The culturing of E. coli cells expressing hilA (E. coli-HilA) or both hilA and hilB (E. coli-HilAB) in the presence of l-isoleucine resulted in the conversion of l-isoleucine into two novel biogenic compounds: l-4′-isoleucine and l-4,4′-dihydroxyisoleucine, respectively. In parallel, two novel enzymatic activities were detected in the crude cell lysates of the E. coli-HilA and E. coli-HilAB strains: l-isoleucine, 2-oxoglutarate: oxygen oxidoreductase (4′-hydroxylating) (HilA) and l-4′-hydroxyisoleucine, 2-oxoglutarate: oxygen oxidoreductase (4-hydroxylating) (HilB), respectively. Two hypotheses regarding the physiological significance of C-4(4′)-hydroxylation of l-isoleucine in bacteria are also discussed. According to first hypothesis, the l-isoleucine dihydroxylation cascade is involved in synthesis of dipeptide antibiotic in P. ananatis. Another unifying hypothesis is that the C-4(4′)-hydroxylation of l-isoleucine in bacteria could result in the synthesis of signal molecules belonging to two classes: 2(5H)-furanones and analogs of N-acyl homoserine lactone. PMID:23554367

Identification and Analysis of a Novel Gene Cluster Involves in Fe2+ Oxidation in Acidithiobacillus ferrooxidans ATCC 23270, a Typical Biomining Acidophile.

PubMed

Ai, Chenbing; Liang, Yuting; Miao, Bo; Chen, Miao; Zeng, Weimin; Qiu, Guanzhou

2018-07-01

Iron-oxidizing Acidithiobacillus spp. are applied worldwide in biomining industry to extract metals from sulfide minerals. They derive energy for survival through Fe 2+ oxidation and generate Fe 3+ for the dissolution of sulfide minerals. However, molecular mechanisms of their iron oxidation still remain elusive. A novel two-cytochrome-encoding gene cluster (named tce gene cluster) encoding a high-molecular-weight cytochrome c (AFE_1428) and a c 4 -type cytochrome c 552 (AFE_1429) in A. ferrooxidans ATCC 23270 was first identified in this study. Bioinformatic analysis together with transcriptional study showed that AFE_1428 and AFE_1429 were the corresponding paralog of Cyc2 (AFE_3153) and Cyc1 (AFE_3152) which were encoded by the extensively studied rus operon and had been proven involving in ferrous iron oxidation. Both AFE_1428 and AFE_1429 contained signal peptide and the classic heme-binding motif(s) as their corresponding paralog. The modeled structure of AFE_1429 showed high resemblance to Cyc1. AFE_1428 and AFE_1429 were preferentially transcribed as their corresponding paralogs in the presence of ferrous iron as sole energy source as compared with sulfur. The tce gene cluster is highly conserved in the genomes of four phylogenetic-related A. ferrooxidans strains that were originally isolated from different sites separated with huge geographical distance, which further implies the importance of this gene cluster. Collectively, AFE_1428 and AFE_1429 involve in Fe 2+ oxidation like their corresponding paralog by integrating with the metalloproteins encoded by rus operon. This study provides novel insights into the Fe 2+ oxidation mechanism in Fe 2+ -oxidizing A. ferrooxidans ssp.

Molecular Characterization of Enterococcus faecalis N06-0364 with Low-Level Vancomycin Resistance Harboring a Novel d-Ala-d-Ser Gene Cluster, vanL▿

PubMed Central

Boyd, David A.; Willey, Barbara M.; Fawcett, Darlene; Gillani, Nazira; Mulvey, Michael R.

2008-01-01

Enterococcus faecalis N06-0364, exhibiting a vancomycin MIC of 8 μg/ml, was found to harbor a novel d-Ala-d-Ser gene cluster, designated vanL. The vanL gene cluster was similar in organization to the vanC operon, but the VanT serine racemase was encoded by two separate genes, vanTmL (membrane binding) and vanTrL (racemase). PMID:18458129

Characterization of the regulation of a plant polysaccharide utilization operon and its role in biofilm formation in Bacillus subtilis

PubMed Central

Habib, Cameron; Yu, Yiyang; Gozzi, Kevin; Ching, Carly; Shemesh, Moshe

2017-01-01

The soil bacterium Bacillus subtilis is often found in association with plants in the rhizosphere. Previously, plant polysaccharides have been shown to stimulate formation of root-associated multicellular communities, or biofilms, in this bacterium, yet the underlying mechanism is not fully understood. A five-gene gan operon (ganSPQAB) in B. subtilis has recently been shown to be involved in utilization of the plant-derived polysaccharide galactan. Despite these findings, molecular details about the regulation of the operon and the role of the operon in biofilm formation remain elusive. In this study, we performed comprehensive genetic analyses on the regulation of the gan operon. We show that this operon is regulated both by a LacI-like transcription repressor (GanR), which directly binds to pairs of inverted DNA repeats in the promoter region of the operon, and by the catabolite control protein A (CcpA). Derepression can be triggered by the presence of the inducer β-1,4-galactobiose, a hydrolysis product of galactan, or in situ when B. subtilis cells are associated with plant roots. In addition to the transcriptional regulation, the encoded ß-galactosidase GanA (by ganA), which hydrolyzes ß-1,4-galactobiose into galactose, is inhibited at the enzymatic level by the catalytic product galactose. Thus, the galactan utilization pathway is under complex regulation involving both positive and negative feedback mechanisms in B. subtilis. We discuss about the biological significance of such complex regulation as well as a hypothesis of biofilm induction by galactan via multiple mechanisms. PMID:28617843

Dissecting the role of NtrC and RpoN in the expression of assimilatory nitrate and nitrite reductases in Bradyrhizobium diazoefficiens.

PubMed

López, María F; Cabrera, Juan J; Salas, Ana; Delgado, María J; López-García, Silvina L

2017-04-01

Bradyrhizobium diazoefficiens, a nitrogen-fixing endosymbiont of soybeans, is a model strain for studying rhizobial denitrification. This bacterium can also use nitrate as the sole nitrogen (N) source during aerobic growth by inducing an assimilatory nitrate reductase encoded by nasC located within the narK-bjgb-flp-nasC operon along with a nitrite reductase encoded by nirA at a different chromosomal locus. The global nitrogen two-component regulatory system NtrBC has been reported to coordinate the expression of key enzymes in nitrogen metabolism in several bacteria. In this study, we demonstrate that disruption of ntrC caused a growth defect in B. diazoefficiens cells in the presence of nitrate or nitrite as the sole N source and a decreased activity of the nitrate and nitrite reductase enzymes. Furthermore, the expression of narK-lacZ or nirA-lacZ transcriptional fusions was significantly reduced in the ntrC mutant after incubation under nitrate assimilation conditions. A B. diazoefficiens rpoN 1/2 mutant, lacking both copies of the gene encoding the alternative sigma factor σ 54 , was also defective in aerobic growth with nitrate as the N source as well as in nitrate and nitrite reductase expression. These results demonstrate that the NtrC regulator is required for expression of the B. diazoefficiens nasC and nirA genes and that the sigma factor RpoN is also involved in this regulation.

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

The dual-function chaperone HycH improves assembly of the formate hydrogenlyase complex.

PubMed

Lindenstrauß, Ute; Skorupa, Philipp; McDowall, Jennifer S; Sargent, Frank; Pinske, Constanze

2017-08-11

The assembly of multi-protein complexes requires the concerted synthesis and maturation of its components and subsequently their co-ordinated interaction. The membrane-bound formate hydrogenlyase (FHL) complex is the primary hydrogen-producing enzyme in Escherichia coli and is composed of seven subunits mostly encoded within the hycA-I operon for [NiFe]-hydrogenase-3 (Hyd-3). The HycH protein is predicted to have an accessory function and is not part of the final structural FHL complex. In this work, a mutant strain devoid of HycH was characterised and found to have significantly reduced FHL activity due to the instability of the electron transfer subunits. HycH was shown to interact specifically with the unprocessed species of HycE, the catalytic hydrogenase subunit of the FHL complex, at different stages during the maturation and assembly of the complex. Variants of HycH were generated with the aim of identifying interacting residues and those that influence activity. The R70/71/K72, the Y79, the E81 and the Y128 variant exchanges interrupt the interaction with HycE without influencing the FHL activity. In contrast, FHL activity, but not the interaction with HycE, was negatively influenced by H37 exchanges with polar residues. Finally, a HycH Y30 variant was unstable. Surprisingly, an overlapping function between HycH with its homologous counterpart HyfJ from the operon encoding [NiFe]-hydrogenase-4 (Hyd-4) was identified and this is the first example of sharing maturation machinery components between Hyd-3 and Hyd-4 complexes. The data presented here show that HycH has a novel dual role as an assembly chaperone for a cytoplasmic [NiFe]-hydrogenase. © 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

Sorbitol-Fermenting Enterohemorrhagic Escherichia coli O157:H− Isolates from Czech Patients with Novel Plasmid Composition Not Previously Seen in German Isolates

PubMed Central

Bauwens, Andreas; Marejková, Monika; Middendorf-Bauchart, Barbara; Prager, Rita; Kossow, Annelene; Zhang, Wenlan; Karch, Helge

2017-01-01

ABSTRACT Sorbitol-fermenting (SF) enterohemorrhagic Escherichia coli (EHEC) O157:H− strains, first identified in Germany, have emerged as important pathogens throughout Europe. Besides chromosomally encoded Shiga toxin 2a (the major virulence factor), several putative virulence loci, including the hly, etp, and sfp operons, encoding EHEC hemolysin, type II secretion system proteins, and Sfp fimbriae, respectively, are located on the 121-kb plasmid pSFO157 in German strains. Here we report novel SF EHEC O157:H− strains isolated from patients in the Czech Republic. These strains share the core genomes and chromosomal virulence loci encoding toxins (stx2a and the cdtV-ABC operon) and adhesins (eae-γ, efa1, lpfAO157OI-141, and lpfAO157OI-154) with German strains but differ essentially in their plasmids. In contrast to all previously detected SF EHEC O157:H− strains, the Czech strains carry two plasmids, of 79 kb and 86 kb. The 79-kb plasmid harbors the sfp operon, but neither of the plasmids contains the hly and etp operons. Sequence analyses demonstrated that the 79-kb plasmid (pSFO157 258/98-1) evolved from pSFO157 of German strains by deletion of a 41,534-bp region via homologous recombination, resulting in loss of the hly and etp operons. The 86-kb plasmid (pSFO157 258/98-2) displays 98% sequence similarity to a 92.7-kb plasmid of an extraintestinal pathogenic E. coli bloodstream isolate. Our finding of this novel plasmid composition in SF EHEC O157:H− strains extends the evolutionary history of EHEC O157 plasmids. Moreover, the unique molecular plasmid characteristics permit the identification of such strains, thereby facilitating further investigations of their geographic distribution, clinical significance, and epidemiology. IMPORTANCE Since their first identification in Germany in 1989, sorbitol-fermenting enterohemorrhagic Escherichia coli O157:H− (nonmotile) strains have emerged as important causes of the life-threatening disease hemolytic-uremic syndrome in Europe. They account for 10 to 20% of sporadic cases of this disease and have caused several large outbreaks. The strains isolated throughout Europe share conserved chromosomal and plasmid characteristics. Here we identified novel sorbitol-fermenting enterohemorrhagic E. coli O157:H− patient isolates in the Czech Republic which differ from all such strains reported previously by their unique plasmid characteristics, including plasmid number, composition of plasmid-carried virulence genes, and plasmid origins. Our findings contribute substantially to understanding the evolution of E. coli O157 strains and their plasmids. In practical terms, they enable the identification of strains with these novel plasmid characteristics in patient stool samples and thus the investigation of their roles as human pathogens in other geographic areas. PMID:28970221

Sorbitol-Fermenting Enterohemorrhagic Escherichia coli O157:H- Isolates from Czech Patients with Novel Plasmid Composition Not Previously Seen in German Isolates.

PubMed

Bauwens, Andreas; Marejková, Monika; Middendorf-Bauchart, Barbara; Prager, Rita; Kossow, Annelene; Zhang, Wenlan; Karch, Helge; Mellmann, Alexander; Bielaszewska, Martina

2017-12-01

Sorbitol-fermenting (SF) enterohemorrhagic Escherichia coli (EHEC) O157:H - strains, first identified in Germany, have emerged as important pathogens throughout Europe. Besides chromosomally encoded Shiga toxin 2a (the major virulence factor), several putative virulence loci, including the hly , etp , and sfp operons, encoding EHEC hemolysin, type II secretion system proteins, and Sfp fimbriae, respectively, are located on the 121-kb plasmid pSFO157 in German strains. Here we report novel SF EHEC O157:H - strains isolated from patients in the Czech Republic. These strains share the core genomes and chromosomal virulence loci encoding toxins ( stx 2a and the cdtV -ABC operon) and adhesins ( eae -γ, efa1 , lpfA O157OI-141 , and lpfA O157OI-154 ) with German strains but differ essentially in their plasmids. In contrast to all previously detected SF EHEC O157:H - strains, the Czech strains carry two plasmids, of 79 kb and 86 kb. The 79-kb plasmid harbors the sfp operon, but neither of the plasmids contains the hly and etp operons. Sequence analyses demonstrated that the 79-kb plasmid (pSFO157 258/98-1) evolved from pSFO157 of German strains by deletion of a 41,534-bp region via homologous recombination, resulting in loss of the hly and etp operons. The 86-kb plasmid (pSFO157 258/98-2) displays 98% sequence similarity to a 92.7-kb plasmid of an extraintestinal pathogenic E. coli bloodstream isolate. Our finding of this novel plasmid composition in SF EHEC O157:H - strains extends the evolutionary history of EHEC O157 plasmids. Moreover, the unique molecular plasmid characteristics permit the identification of such strains, thereby facilitating further investigations of their geographic distribution, clinical significance, and epidemiology. IMPORTANCE Since their first identification in Germany in 1989, sorbitol-fermenting enterohemorrhagic Escherichia coli O157:H - (nonmotile) strains have emerged as important causes of the life-threatening disease hemolytic-uremic syndrome in Europe. They account for 10 to 20% of sporadic cases of this disease and have caused several large outbreaks. The strains isolated throughout Europe share conserved chromosomal and plasmid characteristics. Here we identified novel sorbitol-fermenting enterohemorrhagic E. coli O157:H - patient isolates in the Czech Republic which differ from all such strains reported previously by their unique plasmid characteristics, including plasmid number, composition of plasmid-carried virulence genes, and plasmid origins. Our findings contribute substantially to understanding the evolution of E. coli O157 strains and their plasmids. In practical terms, they enable the identification of strains with these novel plasmid characteristics in patient stool samples and thus the investigation of their roles as human pathogens in other geographic areas. Copyright © 2017 American Society for Microbiology.

A Novel Pheromone Quorum-Sensing System Controls the Development of Natural Competence in Streptococcus thermophilus and Streptococcus salivarius▿ †

PubMed Central

Fontaine, Laetitia; Boutry, Céline; de Frahan, Marie Henry; Delplace, Brigitte; Fremaux, Christophe; Horvath, Philippe; Boyaval, Patrick; Hols, Pascal

2010-01-01

In streptococcal species, the key step of competence development is the transcriptional induction of comX, which encodes the alternative sigma factor σX, which positively regulates genes necessary for DNA transformation. In Streptococcus species belonging to the mitis and mutans groups, induction of comX relies on the activation of a three-component system consisting of a secreted pheromone, a histidine kinase, and a response regulator. In Streptococcus thermophilus, a species belonging to the salivarius group, the oligopeptide transporter Ami is essential for comX expression under competence-inducing conditions. This suggests a different regulation pathway of competence based on the production and reimportation of a signal peptide. The objective of our work was to identify the main actors involved in the early steps of comX induction in S. thermophilus LMD-9. Using a transcriptomic approach, four highly induced early competence operons were identified. Among them, we found a Rgg-like regulator (Ster_0316) associated with a nonannotated gene encoding a 24-amino-acid hydrophobic peptide (Shp0316). Through genetic deletions, we showed that these two genes are essential for comX induction. Moreover, addition to the medium of synthetic peptides derived from the C-terminal part of Shp0316 restored comX induction and transformation of a Shp0316-deficient strain. These peptides also induced competence in S. thermophilus and Streptococcus salivarius strains that are poorly transformable or not transformable. Altogether, our results show that Ster_0316 and Shp0316, renamed ComRS, are the two members of a novel quorum-sensing system responsible for comX induction in species from the salivarius group, which differs from the classical phosphorelay three-component system identified previously in streptococci. PMID:20023010

Integration of a complex regulatory cascade involving the SirA/BarA and Csr global regulatory systems that controls expression of the Salmonella SPI-1 and SPI-2 virulence regulons through HilD.

PubMed

Martínez, Luary C; Yakhnin, Helen; Camacho, Martha I; Georgellis, Dimitris; Babitzke, Paul; Puente, José L; Bustamante, Víctor H

2011-06-01

Salmonella pathogenicity islands 1 and 2 (SPI-1 and SPI-2) play key roles in the pathogenesis of Salmonella enterica. Previously, we showed that when Salmonella grows in Luria-Bertani medium, HilD, encoded in SPI-1, first induces the expression of hilA, located in SPI-1, and subsequently of the ssrAB operon, located in SPI-2. These genes code for HilA and the SsrA/B two-component system, the positive regulators of the SPI-1 and SPI-2 regulons respectively. In this study, we demonstrate that CsrA, a global regulatory RNA binding protein, post-transcriptionally regulates hilD expression by directly binding near the Shine-Dalgarno and translation initiation codon sequences of the hilD mRNA, preventing its translation and leading to its accelerated turnover. Negative regulation is counteracted by the global SirA/BarA two-component system, which directly activates the expression of CsrB and CsrC, two non-coding regulatory RNAs that sequester CsrA, thereby preventing it from binding to its target mRNAs. Our results illustrate the integration of global and specific regulators into a multifactorial regulatory cascade controlling the expression of virulence genes acquired by horizontal transfer events. © 2011 Blackwell Publishing Ltd.

Identification of VanN-type vancomycin resistance in an Enterococcus faecium isolate from chicken meat in Japan.

PubMed

Nomura, Takahiro; Tanimoto, Koichi; Shibayama, Keigo; Arakawa, Yoshichika; Fujimoto, Shuhei; Ike, Yasuyoshi; Tomita, Haruyoshi

2012-12-01

Five VanN-type vancomycin-resistant Enterococcus faecium strains were isolated from a sample of domestic chicken meat in Japan. All isolates showed low-level resistance to vancomycin (MIC, 12 mg/liter) and had the same pulsed-field gel electrophoresis profile. The vancomycin resistance was encoded on a large plasmid (160 kbp) and was expressed constitutively. The VanN-type resistance operon was identical to the first resistance operon to be reported, with the exception of a 1-bp deletion in vanT(N) and a 1-bp substitution in vanS(N).

Agrobacterium tumefaciens can obtain sulphur from an opine that is synthesized by octopine synthase using S-methylmethionine as a substrate.

PubMed

Flores-Mireles, Ana Lidia; Eberhard, Anatol; Winans, Stephen C

2012-06-01

Agrobacterium tumefaciens incites plant tumours that produce nutrients called opines, which are utilized by the bacteria during host colonization. Various opines provide sources of carbon, nitrogen and phosphorous, but virtually nothing was previously known about how A. tumefaciens acquires sulphur during colonization. Some strains encode an operon required for the catabolism of the opine octopine. This operon contains a gene, msh, that is predicted to direct the conversion of S-methylmethionine (SMM) and homocysteine (HCys) to two equivalents of methionine. Purified Msh carried out this reaction, suggesting that SMM could be an intermediate in opine catabolism. Purified octopine synthase (Ocs, normally expressed in plant tumours) utilized SMM and pyruvate to produce a novel opine, designated sulfonopine, whose catabolism by the bacteria would regenerate SMM. Sulfonopine was produced by tobacco and Arabidopsis when colonized by A. tumefaciens and was utilized as sole source of sulphur by A. tumefaciens. Purified Ocs also used 13 other proteogenic and non-proteogenic amino acids as substrates, including three that contain sulphur. Sulfonopine and 11 other opines were tested for induction of octopine catabolic operon and all were able to do so. This is the first study of the acquisition of sulphur, an essential element, by this pathogen. © 2012 Blackwell Publishing Ltd.

Identification of the Operon for the Sorbitol (Glucitol) Phosphoenolpyruvate:Sugar Phosphotransferase System in Streptococcus mutans

PubMed Central

Boyd, David A.; Thevenot, Tracy; Gumbmann, Markus; Honeyman, Allen L.; Hamilton, Ian R.

2000-01-01

Transposon mutagenesis and marker rescue were used to isolate and identify an 8.5-kb contiguous region containing six open reading frames constituting the operon for the sorbitol P-enolpyruvate phosphotransferase transport system (PTS) of Streptococcus mutans LT11. The first gene, srlD, codes for sorbitol-6-phosphate dehydrogenase, followed downstream by srlR, coding for a transcriptional regulator; srlM, coding for a putative activator; and the srlA, srlE, and srlB genes, coding for the EIIC, EIIBC, and EIIA components of the sorbitol PTS, respectively. Among all sorbitol PTS operons characterized to date, the srlD gene is found after the genes coding for the EII components; thus, the location of the gene in S. mutans is unique. The SrlR protein is similar to several transcriptional regulators found in Bacillus spp. that contain PTS regulator domains (J. Stülke, M. Arnaud, G. Rapoport, and I. Martin-Verstraete, Mol. Microbiol. 28:865–874, 1998), and its gene overlaps the srlM gene by 1 bp. The arrangement of these two regulatory genes is unique, having not been reported for other bacteria. PMID:10639465

Quantifying translational coupling in E. coli synthetic operons using RBS modulation and fluorescent reporters.

PubMed

Levin-Karp, Ayelet; Barenholz, Uri; Bareia, Tasneem; Dayagi, Michal; Zelcbuch, Lior; Antonovsky, Niv; Noor, Elad; Milo, Ron

2013-06-21

Translational coupling is the interdependence of translation efficiency of neighboring genes encoded within an operon. The degree of coupling may be quantified by measuring how the translation rate of a gene is modulated by the translation rate of its upstream gene. Translational coupling was observed in prokaryotic operons several decades ago, but the quantitative range of modulation translational coupling leads to and the factors governing this modulation were only partially characterized. In this study, we systematically quantify and characterize translational coupling in E. coli synthetic operons using a library of plasmids carrying fluorescent reporter genes that are controlled by a set of different ribosome binding site (RBS) sequences. The downstream gene expression level is found to be enhanced by the upstream gene expression via translational coupling with the enhancement level varying from almost no coupling to over 10-fold depending on the upstream gene's sequence. Additionally, we find that the level of translational coupling in our system is similar between the second and third locations in the operon. The coupling depends on the distance between the stop codon of the upstream gene and the start codon of the downstream gene. This study is the first to systematically and quantitatively characterize translational coupling in a synthetic E. coli operon. Our analysis will be useful in accurate manipulation of gene expression in synthetic biology and serves as a step toward understanding the mechanisms involved in translational expression modulation.

Participation of the arcRACME protein in self-activation of the arc operon located in the arginine catabolism mobile element in pandemic clone USA300.

PubMed

Rozo, Zayda Lorena Corredor; Márquez-Ortiz, Ricaurte Alejandro; Castro, Betsy Esperanza; Gómez, Natasha Vanegas; Escobar-Pérez, Javier

2017-07-01

Staphylococcus aureus pandemic clone USA300 has, in addition to its constitutive arginine catabolism (arc) gene cluster, an arginine catabolism mobile element (ACME) carrying another such cluster, which gives this clone advantages in colonisation and infection. Gene arcR, which encodes an oxygen-sensitive transcriptional regulator, is inside ACME and downstream of the constitutive arc gene cluster, and this situation may have an impact on its activation. Different relative expression behaviours are proven here for arcRACME and the arcACME operon compared to the constitutive ones. We also show that the artificially expressed recombinant ArcRACME protein binds to the promoter region of the arcACME operon; this mechanism can be related to a positive feedback model, which may be responsible for increased anaerobic survival of the USA300 clone during infection-related processes.

Molecular and cellular characterisation of the zinc uptake (Znu) system of Nostoc punctiforme.

PubMed

Hudek, Lee; Pearson, Leanne A; Michalczyk, Agnes; Neilan, Brett A; Ackland, M Leigh

2013-11-01

Metal homoeostasis in cyanobacteria is based on uptake and export systems that are controlled by their own regulators. This study characterises the zinc uptake (Znu) system in Nostoc punctiforme. The system was found to comprise of three subunits in an ACB operon: a Zn(2+)-binding protein (ZnuA18), a transmembrane domain (ZnuB) and an ATPase (ZnuC). These proteins are encoded within the znu operon regulated by a zinc uptake transcription repressor (Zur). Interestingly, a second Zn(2+)-binding protein (ZnuA08) was also identified at a distal genomic location. Interactions between components of the ZnuACB system were investigated using knockouts of the individual genes. The znuA08(-), znuA18(-), znuB(-) and znuC(-) mutants displayed overall reduced znuACB transcript levels, suggesting that all system components are required for normal expression of znu genes. Zinc uptake assays in the Zn(2+)-binding protein mutant strains showed that the disruption of znuA18 had a greater negative effect on zinc uptake than disruption of znuA08. Complementation studies in Escherichia coli indicated that both znuA08 and znuA18 were able to restore zinc uptake in a znuA(-) mutant, with znuA18 permitting the highest zinc uptake rate. The N. punctiforme zur was also able to complement the E. coli zur(-) mutant. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

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.

Virulence factors encoded by Legionella longbeachae identified on the basis of the genome sequence analysis of clinical isolate D-4968.

PubMed

Kozak, Natalia A; Buss, Meghan; Lucas, Claressa E; Frace, Michael; Govil, Dhwani; Travis, Tatiana; Olsen-Rasmussen, Melissa; Benson, Robert F; Fields, Barry S

2010-02-01

Legionella longbeachae causes most cases of legionellosis in Australia and may be underreported worldwide due to the lack of L. longbeachae-specific diagnostic tests. L. longbeachae displays distinctive differences in intracellular trafficking, caspase 1 activation, and infection in mouse models compared to Legionella pneumophila, yet these two species have indistinguishable clinical presentations in humans. Unlike other legionellae, which inhabit freshwater systems, L. longbeachae is found predominantly in moist soil. In this study, we sequenced and annotated the genome of an L. longbeachae clinical isolate from Oregon, isolate D-4968, and compared it to the previously published genomes of L. pneumophila. The results revealed that the D-4968 genome is larger than the L. pneumophila genome and has a gene order that is different from that of the L. pneumophila genome. Genes encoding structural components of type II, type IV Lvh, and type IV Icm/Dot secretion systems are conserved. In contrast, only 42/140 homologs of genes encoding L. pneumophila Icm/Dot substrates have been found in the D-4968 genome. L. longbeachae encodes numerous proteins with eukaryotic motifs and eukaryote-like proteins unique to this species, including 16 ankyrin repeat-containing proteins and a novel U-box protein. We predict that these proteins are secreted by the L. longbeachae Icm/Dot secretion system. In contrast to the L. pneumophila genome, the L. longbeachae D-4968 genome does not contain flagellar biosynthesis genes, yet it contains a chemotaxis operon. The lack of a flagellum explains the failure of L. longbeachae to activate caspase 1 and trigger pyroptosis in murine macrophages. These unique features of L. longbeachae may reflect adaptation of this species to life in soil.

Fnr, NarP, and NarL Regulation of Escherichia coli K-12 napF (Periplasmic Nitrate Reductase) Operon Transcription In Vitro

PubMed Central

Darwin, Andrew J.; Ziegelhoffer, Eva C.; Kiley, Patricia J.; Stewart, Valley

1998-01-01

The expression of several Escherichia coli operons is activated by the Fnr protein during anaerobic growth and is further controlled in response to nitrate and nitrite by the homologous response regulators, NarL and NarP. Among these operons, the napF operon, encoding a periplasmic nitrate reductase, has unique features with respect to its Fnr-, NarL-, and NarP-dependent regulation. First, the Fnr-binding site is unusually located compared to the control regions of most other Fnr-activated operons, suggesting different Fnr-RNA polymerase contacts during transcriptional activation. Second, nitrate and nitrite activation is solely dependent on NarP but is antagonized by the NarL protein. In this study, we used DNase I footprint analysis to confirm our previous assignment of the unusual location of the Fnr-binding site in the napF control region. In addition, the in vivo effects of Fnr-positive control mutations on napF operon expression indicate that the napF promoter is atypical with respect to Fnr-mediated activation. The transcriptional regulation of napF was successfully reproduced in vitro by using a supercoiled plasmid template and purified Fnr, NarL, and NarP proteins. These in vitro transcription experiments demonstrate that, in the presence of Fnr, the NarP protein causes efficient transcription activation whereas the NarL protein does not. This suggests that Fnr and NarP may act synergistically to activate napF operon expression. As observed in vivo, this activation by Fnr and NarP is antagonized by the addition of NarL in vitro. PMID:9696769

Evaluation of the Role of the opgGH Operon in Yersinia pseudotuberculosis and Its Deletion during the Emergence of Yersinia pestis

PubMed Central

Quintard, Kévin; Dewitte, Amélie; Reboul, Angéline; Madec, Edwige; Bontemps-Gallo, Sébastien; Dondeyne, Jacqueline; Marceau, Michaël; Simonet, Michel

2015-01-01

The opgGH operon encodes glucosyltransferases that synthesize osmoregulated periplasmic glucans (OPGs) from UDP-glucose, using acyl carrier protein (ACP) as a cofactor. OPGs are required for motility, biofilm formation, and virulence in various bacteria. OpgH also sequesters FtsZ in order to regulate cell size according to nutrient availability. Yersinia pestis (the agent of flea-borne plague) lost the opgGH operon during its emergence from the enteropathogen Yersinia pseudotuberculosis. When expressed in OPG-negative strains of Escherichia coli and Dickeya dadantii, opgGH from Y. pseudotuberculosis restored OPGs synthesis, motility, and virulence. However, Y. pseudotuberculosis did not produce OPGs (i) under various growth conditions or (ii) when overexpressing its opgGH operon, its galUF operon (governing UDP-glucose), or the opgGH operon or Acp from E. coli. A ΔopgGH Y. pseudotuberculosis strain showed normal motility, biofilm formation, resistance to polymyxin and macrophages, and virulence but was smaller. Consistently, Y. pestis was smaller than Y. pseudotuberculosis when cultured at ≥37°C, except when the plague bacillus expressed opgGH. Y. pestis expressing opgGH grew normally in serum and within macrophages and was fully virulent in mice, suggesting that small cell size was not advantageous in the mammalian host. Lastly, Y. pestis expressing opgGH was able to infect Xenopsylla cheopis fleas normally. Our results suggest an evolutionary scenario whereby an ancestral Yersinia strain lost a factor required for OPG biosynthesis but kept opgGH (to regulate cell size). The opgGH operon was presumably then lost because OpgH-dependent cell size control became unnecessary. PMID:26150539

Bacillus subtilis IolQ (DegA) is a transcriptional repressor of iolX encoding NAD+-dependent scyllo-inositol dehydrogenase.

PubMed

Kang, Dong-Min; Michon, Christophe; Morinaga, Tetsuro; Tanaka, Kosei; Takenaka, Shinji; Ishikawa, Shu; Yoshida, Ken-Ichi

2017-07-11

Bacillus subtilis is able to utilize at least three inositol stereoisomers as carbon sources, myo-, scyllo-, and D-chiro-inositol (MI, SI, and DCI, respectively). NAD + -dependent SI dehydrogenase responsible for SI catabolism is encoded by iolX. Even in the absence of functional iolX, the presence of SI or MI in the growth medium was found to induce the transcription of iolX through an unknown mechanism. Immediately upstream of iolX, there is an operon that encodes two genes, yisR and iolQ (formerly known as degA), each of which could encode a transcriptional regulator. Here we performed an inactivation analysis of yisR and iolQ and found that iolQ encodes a repressor of the iolX transcription. The coding sequence of iolQ was expressed in Escherichia coli and the gene product was purified as a His-tagged fusion protein, which bound to two sites within the iolX promoter region in vitro. IolQ is a transcriptional repressor of iolX. Genetic evidences allowed us to speculate that SI and MI might possibly be the intracellular inducers, however they failed to antagonize DNA binding of IolQ in in vitro experiments.

Structural, Bioinformatic, and In Vivo Analyses of Two Treponema pallidum Lipoproteins Reveal a Unique TRAP Transporter

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

Deka, Ranjit K.; Brautigam, Chad A.; Goldberg, Martin

2012-05-25

Treponema pallidum, the bacterial agent of syphilis, is predicted to encode one tripartite ATP-independent periplasmic transporter (TRAP-T). TRAP-Ts typically employ a periplasmic substrate-binding protein (SBP) to deliver the cognate ligand to the transmembrane symporter. Herein, we demonstrate that the genes encoding the putative TRAP-T components from T. pallidum, tp0957 (the SBP), and tp0958 (the symporter), are in an operon with an uncharacterized third gene, tp0956. We determined the crystal structure of recombinant Tp0956; the protein is trimeric and perforated by a pore. Part of Tp0956 forms an assembly similar to those of 'tetratricopeptide repeat' (TPR) motifs. The crystal structure ofmore » recombinant Tp0957 was also determined; like the SBPs of other TRAP-Ts, there are two lobes separated by a cleft. In these other SBPs, the cleft binds a negatively charged ligand. However, the cleft of Tp0957 has a strikingly hydrophobic chemical composition, indicating that its ligand may be substantially different and likely hydrophobic. Analytical ultracentrifugation of the recombinant versions of Tp0956 and Tp0957 established that these proteins associate avidly. This unprecedented interaction was confirmed for the native molecules using in vivo cross-linking experiments. Finally, bioinformatic analyses suggested that this transporter exemplifies a new subfamily of TPATs (TPR-protein-associated TRAP-Ts) that require the action of a TPR-containing accessory protein for the periplasmic transport of a potentially hydrophobic ligand(s).« less

Hierarchical mutational events compensate for glutamate auxotrophy of a Bacillus subtilis gltC mutant.

PubMed

Dormeyer, Miriam; Lübke, Anastasia L; Müller, Peter; Lentes, Sabine; Reuß, Daniel R; Thürmer, Andrea; Stülke, Jörg; Daniel, Rolf; Brantl, Sabine; Commichau, Fabian M

2017-06-01

Glutamate is the major donor of nitrogen for anabolic reactions. The Gram-positive soil bacterium Bacillus subtilis either utilizes exogenously provided glutamate or synthesizes it using the gltAB-encoded glutamate synthase (GOGAT). In the absence of glutamate, the transcription factor GltC activates expression of the GOGAT genes for glutamate production. Consequently, a gltC mutant strain is auxotrophic for glutamate. Using a genetic selection and screening system, we could isolate and differentiate between gltC suppressor mutants in one step. All mutants had acquired the ability to synthesize glutamate, independent of GltC. We identified (i) gain-of-function mutations in the gltR gene, encoding the transcription factor GltR, (ii) mutations in the promoter of the gltAB operon and (iii) massive amplification of the genomic locus containing the gltAB operon. The mutants belonging to the first two classes constitutively expressed the gltAB genes and produced sufficient glutamate for growth. By contrast, mutants that belong to the third class appeared most frequently and solved glutamate limitation by increasing the copy number of the poorly expressed gltAB genes. Thus, glutamate auxotrophy of a B. subtilis gltC mutant can be relieved in multiple ways. Moreover, recombination-dependent amplification of the gltAB genes is the predominant mutational event indicating a hierarchy of mutations. © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

Prediction of operon-like gene clusters in the Arabidopsis thaliana genome based on co-expression analysis of neighboring genes.

PubMed

Wada, Masayoshi; Takahashi, Hiroki; Altaf-Ul-Amin, Md; Nakamura, Kensuke; Hirai, Masami Y; Ohta, Daisaku; Kanaya, Shigehiko

2012-07-15

Operon-like arrangements of genes occur in eukaryotes ranging from yeasts and filamentous fungi to nematodes, plants, and mammals. In plants, several examples of operon-like gene clusters involved in metabolic pathways have recently been characterized, e.g. the cyclic hydroxamic acid pathways in maize, the avenacin biosynthesis gene clusters in oat, the thalianol pathway in Arabidopsis thaliana, and the diterpenoid momilactone cluster in rice. Such operon-like gene clusters are defined by their co-regulation or neighboring positions within immediate vicinity of chromosomal regions. A comprehensive analysis of the expression of neighboring genes therefore accounts a crucial step to reveal the complete set of operon-like gene clusters within a genome. Genome-wide prediction of operon-like gene clusters should contribute to functional annotation efforts and provide novel insight into evolutionary aspects acquiring certain biological functions as well. We predicted co-expressed gene clusters by comparing the Pearson correlation coefficient of neighboring genes and randomly selected gene pairs, based on a statistical method that takes false discovery rate (FDR) into consideration for 1469 microarray gene expression datasets of A. thaliana. We estimated that A. thaliana contains 100 operon-like gene clusters in total. We predicted 34 statistically significant gene clusters consisting of 3 to 22 genes each, based on a stringent FDR threshold of 0.1. Functional relationships among genes in individual clusters were estimated by sequence similarity and functional annotation of genes. Duplicated gene pairs (determined based on BLAST with a cutoff of E<10(-5)) are included in 27 clusters. Five clusters are associated with metabolism, containing P450 genes restricted to the Brassica family and predicted to be involved in secondary metabolism. Operon-like clusters tend to include genes encoding bio-machinery associated with ribosomes, the ubiquitin/proteasome system, secondary metabolic pathways, lipid and fatty-acid metabolism, and the lipid transfer system. Copyright © 2012 Elsevier B.V. All rights reserved.

Construction and expression of immunogenic hybrid enterotoxigenic Escherichia coli CFA/I and CS2 colonization fimbriae for use in vaccines.

PubMed

Tobias, Joshua; Svennerholm, Ann-Mari; Holmgren, Jan; Lebens, Michael

2010-07-01

Enterotoxigenic Escherichia coli (ETEC) are an important cause of diarrheal morbidity in developing countries, especially in children and also of traveler's diarrhea. Colonization factors (CFs) of ETEC, like CFA/I and CS2 which are genetically and structurally related, play a substantial role in pathogenicity, and since intestinal-mucosal immune responses against CFs appear to be protective, much effort has focused on the development of a CF-based ETEC vaccine. We have constructed hybrid operons in which the major CS2 subunit-encoding cotA gene was inserted into the CFA/I operon, either replacing (hybrid I) or being added to the major CFA/I subunit-encoding cfaB gene (hybrid II). Using specific monoclonal antibodies against the major subunits of CFA/I and CS2, high levels of surface expression of both fimbrial subunits were shown in E. coli carrying the hybrid II operon. Oral immunization of mice with formalin-killed bacteria expressing hybrid II fimbriae induced strong CFA/I- and CS2-specific serum IgG + IgM and fecal IgA antibody responses, which were higher than those achieved by similar immunization with the reference strains. Bacteria expressing hybrid fimbriae are potential candidate strains in an oral-killed CF-ETEC vaccine, and the approach represents an attractive and novel means of producing a broad-spectrum ETEC vaccine.

Cross-Regulation between the phz1 and phz2 Operons Maintain a Balanced Level of Phenazine Biosynthesis in Pseudomonas aeruginosa PAO1

PubMed Central

Jiang, Bei; Xiao, Bo; Liu, Linde; Ge, Yihe; Hu, Xiaomei

2016-01-01

Gene duplication often provides selective advantages for the survival of microorganisms in adapting to varying environmental conditions. P. aeruginosa PAO1 possesses two seven-gene operons [phz1 (phzA1B1C1D1E1F1G1) and phz2 (phzA2B2C2D2E2F2G2)] that are involved in the biosynthesis of phenazine-1-carboxylic acid and its derivatives. Although the two operons are highly homologous and their functions are well known, it is unclear how the two phz operons coordinate their expressions to maintain the phenazine biosynthesis. By constructing single and double deletion mutants of the two phz operons, we found that the phz1-deletion mutant produced the same or less amount of phenazine-1-carboxylic acid and pyocyanin in GA medium than the phz2-knockout mutant while the phz1-phz2 double knockout mutant did not produce any phenazines. By generating phzA1 and phzA2 translational and transcriptional fusions with a truncated lacZ reporter, we found that the expression of the phz1 operon increased significantly at the post-transcriptional level and did not alter at the transcriptional level in the absence of the phz2 operon. Surprisingly, the expression the phz2 operon increased significantly at the post-transcriptional level and only moderately at the transcriptional level in the absence of the phz1 operon. Our findings suggested that a complex cross-regulation existed between the phz1 and phz2 operons. By mediating the upregulation of one phz operon expression while the other was deleted, this crosstalk would maintain the homeostatic balance of phenazine biosynthesis in P. aeruginosa PAO1. PMID:26735915

Haloarcula marismortui cytochrome b-561 is encoded by the narC gene in the dissimilatory nitrate reductase operon.

PubMed

Yoshimatsu, Katsuhiko; Araya, Osamu; Fujiwara, Taketomo

2007-01-01

The composition of membrane-bound electron-transferring proteins from denitrifying cells of Haloarcula marismortui was compared with that from the aerobic cells. Accompanying nitrate reductase catalytic NarGH subcomplex, cytochrome b-561, cytochrome b-552, and halocyanin-like blue copper protein were induced under denitrifying conditions. Cytochrome b-561 was purified to homogeneity and was shown to be composed of a polypeptide with a molecular mass of 40 kDa. The cytochrome was autooxidizable and its redox potential was -27 mV. The N-terminal sequence of the cytochrome was identical to the deduced amino acid sequence of the narC gene product encoded in the third ORF of the nitrate reductase operon with a unique arrangement of ORFs. The sequence of the cytochrome was homologous with that of the cytochrome b subunit of respiratory cytochrome bc. A possibility that the cytochrome bc and the NarGH constructed a supercomplex was discussed.

The thiostrepton-resistance-encoding gene in Streptomyces laurentii is located within a cluster of ribosomal protein operons.

PubMed

Smith, T M; Jiang, Y F; Shipley, P; Floss, H G

1995-10-16

A common approach to identify and clone biosynthetic gene from an antibiotic-producing streptomycete is to clone the resistance gene for the antibiotic of interest and then use that gene to clone DNA that is linked to it. As a first step toward cloning the genes responsible for the biosynthesis of thiostrepton (Th) in Streptomyces laurentii (Sl), the Th resistance-encoding gene (tsnR) was cloned as a 1.5-kb BamHI-PvuII fragment in Escherichia coli (Ec), and shown to confer Th resistance when introduced into S. lividans TK24. The tsnR-containing DNA fragment was used as a probe to isolate clones from cosmid libraries of DNA in the Ec cosmid vector SuperCos, and pOJ446 (an Ec/streptomycete) cosmid vector. Sequence and genetic analysis of the DNA flanking the tsnR indicates that the Sl tsnR is not closely linked to biosynthetic genes. Instead it is located within a cluster of ribosomal protein operons.

Interplay of Noisy Gene Expression and Dynamics Explains Patterns of Bacterial Operon Organization

NASA Astrophysics Data System (ADS)

Igoshin, Oleg

2011-03-01

Bacterial chromosomes are organized into operons -- sets of genes co-transcribed into polycistronic messenger RNA. Hypotheses explaining the emergence and maintenance of operons include proportional co-regulation, horizontal transfer of intact ``selfish'' operons, emergence via gene duplication, and co-production of physically interacting proteins to speed their association. We hypothesized an alternative: operons can reduce or increase intrinsic gene expression noise in a manner dependent on the post-translational interactions, thereby resulting in selection for or against operons in depending on the network architecture. We devised five classes of two-gene network modules and show that the effects of operons on intrinsic noise depend on class membership. Two classes exhibit decreased noise with co-transcription, two others reveal increased noise, and the remaining one does not show a significant difference. To test our modeling predictions we employed bioinformatic analysis to determine the relationship gene expression noise and operon organization. The results confirm the overrepresentation of noise-minimizing operon architectures and provide evidence against other hypotheses. Our results thereby suggest a central role for gene expression noise in selecting for or maintaining operons in bacterial chromosomes. This demonstrates how post-translational network dynamics may provide selective pressure for organizing bacterial chromosomes, and has practical consequences for designing synthetic gene networks. This work is supported by National Institutes of Health grant 1R01GM096189-01.

The rec A operon: a novel stress response gene cluster in Bacteroides fragilis

PubMed Central

Nicholson, Samantha A; Smalley, Darren; Smith, C. Jeffrey; Abratt, Valerie R

2014-01-01

Bacteroides fragilis, an opportunistic pathogen of humans, is a leading cause of bacteraemias and anaerobic abscesses which are often treated with metronidazole, a drug which damages DNA. This study investigated the responses of the B. fragilis recA three gene operon to the stress experienced during metronidazole treatment and exposure to reactive oxygen species simulating those generated by the host immune system during infection. A transcriptionally regulated response was observed using quantitative RT-PCR after metronidazole and hydrogen peroxide treatment, with all three genes being upregulated under stress conditions. In vivo and in vitro analysis of the functional role of the second gene of the operon was done using heterologous complementation and protein expression (in Escherichia coli), with subsequent biochemical assay. This gene encoded a functional bacterioferritin co-migratory protein (BCP) which was thiol-specific and had antioxidant properties, including protection of the glutamine synthetase III enzyme. This in vitro data supports the hypothesis that the genes of the operon may be involved in protection of the bacteria from the oxidative burst during tissue invasion and may play a significant role in bacterial survival and metronidazole resistance during treatment of B. fragilis infections. PMID:24703997

Amplification of the groESL operon in Pseudomonas putida increases siderophore gene promoter activity.

PubMed

Venturi, V; Wolfs, K; Leong, J; Weisbeek, P J

1994-10-17

Pseudobactin 358 is the yellow-green fluorescent siderophore [microbial iron(III) transport agent] produced by Pseudomonas putida WCS358 under iron-limiting conditions. The genes encoding pseudobactin 358 biosynthesis are iron-regulated at the level of transcription. In this study, the molecular characterization is reported of a cosmid clone of WCS358 DNA that can stimulate, in an iron-dependent manner, the activity of a WCS358 siderophore gene promoter in the heterologous Pseudomonas strain A225. The functional region in the clone was identified by subcloning, transposon mutagenesis and DNA sequencing as the groESL operon of strain WCS358. This increase in promoter activity was not observed when the groESL genes of strain WCS358 were integrated via a transposon vector into the genome of Pseudomonas A225, indicating that multiple copies of the operon are necessary for the increase in siderophore gene promoter activity. Amplification of the Escherichia coli and WCS358 groESL genes also increased iron-regulated promoter activity in the parent strain WCS358. The groESL operon codes for the chaperone proteins GroES and GroEL, which are responsible for mediating the folding and assembly of many proteins.

Functional genomics of corrinoid starvation in the organohalide-respiring bacterium Dehalobacter restrictus strain PER-K23

PubMed Central

Rupakula, Aamani; Lu, Yue; Kruse, Thomas; Boeren, Sjef; Holliger, Christof; Smidt, Hauke; Maillard, Julien

2015-01-01

De novo corrinoid biosynthesis represents one of the most complicated metabolic pathways in nature. Organohalide-respiring bacteria (OHRB) have developed different strategies to deal with their need of corrinoid, as it is an essential cofactor of reductive dehalogenases, the key enzymes in OHR metabolism. In contrast to Dehalococcoides mccartyi, the genome of Dehalobacter restrictus strain PER-K23 contains a complete set of corrinoid biosynthetic genes, of which cbiH appears to be truncated and therefore non-functional, possibly explaining the corrinoid auxotrophy of this obligate OHRB. Comparative genomics within Dehalobacter spp. revealed that one (operon-2) of the five distinct corrinoid biosynthesis associated operons present in the genome of D. restrictus appeared to be present only in that particular strain, which encodes multiple members of corrinoid transporters and salvaging enzymes. Operon-2 was highly up-regulated upon corrinoid starvation both at the transcriptional (346-fold) and proteomic level (46-fold on average), in line with the presence of an upstream cobalamin riboswitch. Together, these data highlight the importance of this operon in corrinoid homeostasis in D. restrictus and the augmented salvaging strategy this bacterium adopted to cope with the need for this essential cofactor. PMID:25610435

Transcriptional regulation of fatty acid biosynthesis in mycobacteria

PubMed Central

Mondino, S.; Gago, G.; Gramajo, H.

2013-01-01

SUMMARY The main purpose of our study is to understand how mycobacteria exert control over the biosynthesis of their membrane lipids and find out the key components of the regulatory network that control fatty acid biosynthesis at the transcriptional level. In this paper we describe the identification and purification of FasR, a transcriptional regulator from Mycobacterium sp. that controls the expression of the fatty acid synthase (fas) and the 4-phosphopantetheinyl transferase (acpS) encoding genes, whose products are involved in the fatty acid and mycolic acid biosynthesis pathways. In vitro studies demonstrated that fas and acpS genes are part of the same transcriptional unit and that FasR specifically binds to three conserved operator sequences present in the fas-acpS promoter region (Pfas). The construction and further characterization of a fasR conditional mutant confirmed that FasR is a transcriptional activator of the fas-acpS operon and that this protein is essential for mycobacteria viability. Furthermore, the combined used of Pfas-lacZ fusions in different fasR backgrounds and electrophoretic mobility shift assays experiments, strongly suggested that long-chain acyl-CoAs are the effector molecules that modulate the affinity of FasR for its DNA binding sequences and therefore the expression of the essential fas-acpS operon. PMID:23721164

An extracytoplasmic function sigma factor-dependent periplasmic glutathione peroxidase is involved in oxidative stress response of Shewanella oneidensis

DOE PAGES

Dai, Jingcheng; Wei, Hehong; Tian, Chunyuan; ...

2015-01-01

Background: Bacteria use alternative sigma factors (σs) to regulate condition-specific gene expression for survival and Shewanella harbors multiple ECF (extracytoplasmic function) σ genes and cognate anti-sigma factor genes. Here we comparatively analyzed two of the rpoE-like operons in the strain MR-1: rpoE-rseA-rseB-rseC and rpoE2-chrR. Results: RpoE was important for bacterial growth at low and high temperatures, in the minimal medium, and high salinity. The degP/htrA orthologue, required for growth of Escherichia coli and Pseudomonas aeruginosa at high temperature, is absent in Shewanella, while the degQ gene is RpoE-regulated and is required for bacterial growth at high temperature. RpoE2 was essentialmore » for the optimal growth in oxidative stress conditions because the rpoE2 mutant was sensitive to hydrogen peroxide and paraquat. The operon encoding a ferrochelatase paralogue (HemH2) and a periplasmic glutathione peroxidase (PgpD) was identified as RpoE2-dependent. PgpD exhibited higher activities and played a more important role in the oxidative stress responses than the cytoplasmic glutathione peroxidase CgpD under tested conditions. The rpoE2-chrR operon and the identified regulon genes, including pgpD and hemH2, are coincidently absent in several psychrophilic and/or deep-sea Shewanella strains. Conclusion: In S. oneidensis MR-1, the RpoE-dependent degQ gene is required for optimal growth under high temperature. The rpoE2 and RpoE2-dependent pgpD gene encoding a periplasmic glutathione peroxidase are involved in oxidative stress responses. But rpoE2 is not required for bacterial growth at low temperature and it even affected bacterial growth under salt stress, indicating that there is a tradeoff between the salt resistance and RpoE2-mediated oxidative stress responses.« less

An extracytoplasmic function sigma factor-dependent periplasmic glutathione peroxidase is involved in oxidative stress response of Shewanella oneidensis

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

Dai, Jingcheng; Wei, Hehong; Tian, Chunyuan

Background: Bacteria use alternative sigma factors (σs) to regulate condition-specific gene expression for survival and Shewanella harbors multiple ECF (extracytoplasmic function) σ genes and cognate anti-sigma factor genes. Here we comparatively analyzed two of the rpoE-like operons in the strain MR-1: rpoE-rseA-rseB-rseC and rpoE2-chrR. Results: RpoE was important for bacterial growth at low and high temperatures, in the minimal medium, and high salinity. The degP/htrA orthologue, required for growth of Escherichia coli and Pseudomonas aeruginosa at high temperature, is absent in Shewanella, while the degQ gene is RpoE-regulated and is required for bacterial growth at high temperature. RpoE2 was essentialmore » for the optimal growth in oxidative stress conditions because the rpoE2 mutant was sensitive to hydrogen peroxide and paraquat. The operon encoding a ferrochelatase paralogue (HemH2) and a periplasmic glutathione peroxidase (PgpD) was identified as RpoE2-dependent. PgpD exhibited higher activities and played a more important role in the oxidative stress responses than the cytoplasmic glutathione peroxidase CgpD under tested conditions. The rpoE2-chrR operon and the identified regulon genes, including pgpD and hemH2, are coincidently absent in several psychrophilic and/or deep-sea Shewanella strains. Conclusion: In S. oneidensis MR-1, the RpoE-dependent degQ gene is required for optimal growth under high temperature. The rpoE2 and RpoE2-dependent pgpD gene encoding a periplasmic glutathione peroxidase are involved in oxidative stress responses. But rpoE2 is not required for bacterial growth at low temperature and it even affected bacterial growth under salt stress, indicating that there is a tradeoff between the salt resistance and RpoE2-mediated oxidative stress responses.« less

Increased flexibility in the use of exogenous lipoic acid by Staphylococcus aureus.

PubMed

Laczkovich, Irina; Teoh, Wei Ping; Flury, Sarah; Grayczyk, James P; Zorzoli, Azul; Alonzo, Francis

2018-04-16

Lipoic acid is a cofactor required for intermediary metabolism that is either synthesized de novo or acquired from environmental sources. The bacterial pathogen Staphylococcus aureus encodes enzymes required for de novo biosynthesis, but also encodes two ligases, LplA1 and LplA2, that are sufficient for lipoic acid salvage during infection. S. aureus also encodes two H proteins, GcvH of the glycine cleavage system and the homologous GcvH-L encoded in an operon with LplA2. GcvH is a recognized conduit for lipoyl transfer to α-ketoacid dehydrogenase E2 subunits, while the function of GcvH-L remains unclear. The potential to produce two ligases and two H proteins is an unusual characteristic of S. aureus that is unlike most other Gram positive Firmicutes and might allude to an expanded pathway of lipoic acid acquisition in this microorganism. Here, we demonstrate that LplA1 and LplA2 facilitate lipoic acid salvage by differentially targeting lipoyl domain-containing proteins; LplA1 targets H proteins and LplA2 targets α-ketoacid dehydrogenase E2 subunits. Furthermore, GcvH and GcvH-L both facilitate lipoyl relay to E2 subunits. Altogether, these studies identify an expanded mode of lipoic acid salvage used by S. aureus and more broadly underscore the importance of bacterial adaptations when faced with nutritional limitation. © 2018 John Wiley & Sons Ltd.

Interactions of Kid-Kis toxin-antitoxin complexes with the parD operator-promoter region of plasmid R1 are piloted by the Kis antitoxin and tuned by the stoichiometry of Kid-Kis oligomers.

PubMed

Monti, Maria C; Hernández-Arriaga, Ana M; Kamphuis, Monique B; López-Villarejo, Juan; Heck, Albert J R; Boelens, Rolf; Díaz-Orejas, Ramón; van den Heuvel, Robert H H

2007-01-01

The parD operon of Escherichia coli plasmid R1 encodes a toxin-antitoxin system, which is involved in plasmid stabilization. The toxin Kid inhibits cell growth by RNA degradation and its action is neutralized by the formation of a tight complex with the antitoxin Kis. A fascinating but poorly understood aspect of the kid-kis system is its autoregulation at the transcriptional level. Using macromolecular (tandem) mass spectrometry and DNA binding assays, we here demonstrate that Kis pilots the interaction of the Kid-Kis complex in the parD regulatory region and that two discrete Kis-binding regions are present on parD. The data clearly show that only when the Kis concentration equals or exceeds the Kid concentration a strong cooperative effect exists between strong DNA binding and Kid2-Kis2-Kid2-Kis2 complex formation. We propose a model in which transcriptional repression of the parD operon is tuned by the relative molar ratio of the antitoxin and toxin proteins in solution. When the concentration of the toxin exceeds that of the antitoxin tight Kid2-Kis2-Kid2 complexes are formed, which only neutralize the lethal activity of Kid. Upon increasing the Kis concentration, (Kid2-Kis2)n complexes repress the kid-kis operon.

PhzA/B catalyzes the formation of the tricycle in phenazine biosynthesis.

USDA-ARS?s Scientific Manuscript database

Phenazines are redox-active bacterial secondary metabolites that participate in important biological processes such as the generation of toxic reactive oxygen species and the reduction of environmental iron. Their biosynthesis from chorismic acid depends on enzymes encoded by the phz operon, but man...

X-Prolyl Dipeptidyl Aminopeptidase Gene (pepX) Is Part of the glnRA Operon in Lactobacillus rhamnosus

PubMed Central

Varmanen, Pekka; Savijoki, Kirsi; Åvall, Silja; Palva, Airi; Tynkkynen, Soile

2000-01-01

A peptidase gene expressing X-prolyl dipeptidyl aminopeptidase (PepX) activity was cloned from Lactobacillus rhamnosus 1/6 by using the chromogenic substrate l-glycyl-l-prolyl-β-naphthylamide for screening of a genomic library in Escherichia coli. The nucleotide sequence of a 3.5-kb HindIII fragment expressing the peptidase activity revealed one complete open reading frame (ORF) of 2,391 nucleotides. The 797-amino-acid protein encoded by this ORF was shown to be 40, 39, and 36% identical with PepXs from Lactobacillus helveticus, Lactobacillus delbrueckii, and Lactococcus lactis, respectively. By Northern analysis with a pepX-specific probe, transcripts of 4.5 and 7.0 kb were detected, indicating that pepX is part of a polycistronic operon in L. rhamnosus. Cloning and sequencing of the upstream region of pepX revealed the presence of two ORFs of 360 and 1,338 bp that were shown to be able to encode proteins with high homology to GlnR and GlnA proteins, respectively. By multiple primer extension analyses, the only functional promoter in the pepX region was located 25 nucleotides upstream of glnR. Northern analysis with glnA- and pepX-specific probes indicated that transcription from glnR promoter results in a 2.0-kb dicistronic glnR-glnA transcript and also in a longer read-through polycistronic transcript of 7.0 kb that was detected with both probes in samples from cells in exponential growth phase. The glnA gene was disrupted by a single-crossover recombinant event using a nonreplicative plasmid carrying an internal part of glnA. In the disruption mutant, glnRA-specific transcription was derepressed 10-fold compared to the wild type, but the 7.0-kb transcript was no longer detectable with either the glnA- or pepX-specific probe, demonstrating that pepX is indeed part of glnRA operon in L. rhamnosus. Reverse transcription-PCR analysis further supported this operon structure. An extended stem-loop structure was identified immediately upstream of pepX in the glnA-pepX intergenic region, a sequence that showed homology to a 23S-5S intergenic spacer and to several other L. rhamnosus-related entries in data banks. PMID:10613874

X-prolyl dipeptidyl aminopeptidase gene (pepX) is part of the glnRA operon in Lactobacillus rhamnosus.

PubMed

Varmanen, P; Savijoki, K; Avall, S; Palva, A; Tynkkynen, S

2000-01-01

A peptidase gene expressing X-prolyl dipeptidyl aminopeptidase (PepX) activity was cloned from Lactobacillus rhamnosus 1/6 by using the chromogenic substrate L-glycyl-L-prolyl-beta-naphthylamide for screening of a genomic library in Escherichia coli. The nucleotide sequence of a 3.5-kb HindIII fragment expressing the peptidase activity revealed one complete open reading frame (ORF) of 2,391 nucleotides. The 797-amino-acid protein encoded by this ORF was shown to be 40, 39, and 36% identical with PepXs from Lactobacillus helveticus, Lactobacillus delbrueckii, and Lactococcus lactis, respectively. By Northern analysis with a pepX-specific probe, transcripts of 4.5 and 7.0 kb were detected, indicating that pepX is part of a polycistronic operon in L. rhamnosus. Cloning and sequencing of the upstream region of pepX revealed the presence of two ORFs of 360 and 1,338 bp that were shown to be able to encode proteins with high homology to GlnR and GlnA proteins, respectively. By multiple primer extension analyses, the only functional promoter in the pepX region was located 25 nucleotides upstream of glnR. Northern analysis with glnA- and pepX-specific probes indicated that transcription from glnR promoter results in a 2.0-kb dicistronic glnR-glnA transcript and also in a longer read-through polycistronic transcript of 7.0 kb that was detected with both probes in samples from cells in exponential growth phase. The glnA gene was disrupted by a single-crossover recombinant event using a nonreplicative plasmid carrying an internal part of glnA. In the disruption mutant, glnRA-specific transcription was derepressed 10-fold compared to the wild type, but the 7.0-kb transcript was no longer detectable with either the glnA- or pepX-specific probe, demonstrating that pepX is indeed part of glnRA operon in L. rhamnosus. Reverse transcription-PCR analysis further supported this operon structure. An extended stem-loop structure was identified immediately upstream of pepX in the glnA-pepX intergenic region, a sequence that showed homology to a 23S-5S intergenic spacer and to several other L. rhamnosus-related entries in data banks.

High-quality draft genome sequence of Ensifer meliloti Mlalz-1, a microsymbiont of Medicago laciniata (L.) miller collected in Lanzarote, Canary Islands, Spain

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

Osman, Wan Adnawani Meor; van Berkum, Peter; León-Barrios, Milagros

Ensifer meliloti Mlalz-1 (INSDC = ATZD00000000) is an aerobic, motile, Gram-negative, non-spore-forming rod that was isolated from an effective nitrogen-fixing nodule of Medicago laciniata (L.) Miller from a soil sample collected near the town of Guatiza on the island of Lanzarote, the Canary Islands, Spain. This strain nodulates and forms an effective symbiosis with the highly specific host M. laciniata. This rhizobial genome was sequenced as part of the DOE Joint Genome Institute 2010 Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria (GEBA-RNB) sequencing project. Here in this paper, the features of E. meliloti Mlalz-1 are described, together with high-qualitymore » permanent draft genome sequence information and annotation. The 6,664,116 bp high-quality draft genome is arranged in 99 scaffolds of 100 contigs, containing 6314 protein-coding genes and 74 RNA-only encoding genes. Strain Mlalz-1 is closely related to Ensifer meliloti IAM 12611 T, Ensifer medicae A 321T and Ensifer numidicus ORS 1407 T, based on 16S rRNA gene sequences. gANI values of ≥98.1% support the classification of strain Mlalz-1 as E. meliloti . Nodulation of M. laciniata requires a specific nodC allele, and the nodC gene of strain Mlalz-1 shares ≥98% sequence identity with nodC of M. laciniata-nodulating Ensifer strains, but ≤93% with nodC of Ensifer strains that nodulate other Medicago species. Strain Mlalz-1 is unique among sequenced E. meliloti strains in possessing genes encoding components of a T2SS and in having two versions of the adaptive acid tolerance response lpiA-acvB operon. In E. medicae strain WSM419, lpiA is essential for enhancing survival in lethal acid conditions. The second copy of the lpiA-acvB operon of strain Mlalz-1 has highest sequence identity (> 96%) with that of E. medicae strains, which suggests genetic recombination between strain Mlalz-1 and E. medicae and the horizontal gene transfer of lpiA-acvB.« less

High-quality draft genome sequence of Ensifer meliloti Mlalz-1, a microsymbiont of Medicago laciniata (L.) miller collected in Lanzarote, Canary Islands, Spain

DOE PAGES

Osman, Wan Adnawani Meor; van Berkum, Peter; León-Barrios, Milagros; ...

2017-09-25

Ensifer meliloti Mlalz-1 (INSDC = ATZD00000000) is an aerobic, motile, Gram-negative, non-spore-forming rod that was isolated from an effective nitrogen-fixing nodule of Medicago laciniata (L.) Miller from a soil sample collected near the town of Guatiza on the island of Lanzarote, the Canary Islands, Spain. This strain nodulates and forms an effective symbiosis with the highly specific host M. laciniata. This rhizobial genome was sequenced as part of the DOE Joint Genome Institute 2010 Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria (GEBA-RNB) sequencing project. Here in this paper, the features of E. meliloti Mlalz-1 are described, together with high-qualitymore » permanent draft genome sequence information and annotation. The 6,664,116 bp high-quality draft genome is arranged in 99 scaffolds of 100 contigs, containing 6314 protein-coding genes and 74 RNA-only encoding genes. Strain Mlalz-1 is closely related to Ensifer meliloti IAM 12611 T, Ensifer medicae A 321T and Ensifer numidicus ORS 1407 T, based on 16S rRNA gene sequences. gANI values of ≥98.1% support the classification of strain Mlalz-1 as E. meliloti . Nodulation of M. laciniata requires a specific nodC allele, and the nodC gene of strain Mlalz-1 shares ≥98% sequence identity with nodC of M. laciniata-nodulating Ensifer strains, but ≤93% with nodC of Ensifer strains that nodulate other Medicago species. Strain Mlalz-1 is unique among sequenced E. meliloti strains in possessing genes encoding components of a T2SS and in having two versions of the adaptive acid tolerance response lpiA-acvB operon. In E. medicae strain WSM419, lpiA is essential for enhancing survival in lethal acid conditions. The second copy of the lpiA-acvB operon of strain Mlalz-1 has highest sequence identity (> 96%) with that of E. medicae strains, which suggests genetic recombination between strain Mlalz-1 and E. medicae and the horizontal gene transfer of lpiA-acvB.« less

Genomic and Transcriptomic Analysis of Escherichia coli Strains Associated with Persistent and Transient Bovine Mastitis and the Role of Colanic Acid.

PubMed

Lippolis, John D; Holman, Devin B; Brunelle, Brian W; Thacker, Tyler C; Bearson, Bradley L; Reinhardt, Timothy A; Sacco, Randy E; Casey, Thomas A

2018-01-01

Escherichia coli is a leading cause of bacterial mastitis in dairy cattle. It is most often transient in nature, causing an infection that lasts 2 to 3 days. However, E. coli has been shown to cause a persistent infection in a minority of cases. Mechanisms that allow for a persistent E. coli infection are not fully understood. The goal of this work was to determine differences between E. coli strains originally isolated from dairy cattle with transient and persistent mastitis. Using RNA sequencing, we show gene expression differences in nearly 200 genes when bacteria from the two clinical phenotypes are compared. We sequenced the genomes of the E. coli strains and report genes unique to the two phenotypes. Differences in the wca operon, which encodes colanic acid, were identified by DNA as well as RNA sequencing and differentiated the two phenotypes. Previous work demonstrated that E. coli strains that cause persistent infections were more motile than those that cause transient infections. Deletion of genes in the wca operon from a persistent-infection strain resulted in a reduction of motility as measured in swimming and swarming assays. Furthermore, colanic acid has been shown to protect bacteria from complement-mediated killing. We show that transient-infection E. coli strains were more sensitive to complement-mediated killing. The deletion of genes from the wca operon caused a persistent-infection E. coli strain to become sensitive to complement-mediated killing. This work identifies important differences between E. coli strains that cause persistent and transient mammary infections in dairy cattle. This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.

The VirSR Two-Component Signal Transduction System Regulates NetB Toxin Production in Clostridium perfringens▿

PubMed Central

Cheung, Jackie K.; Keyburn, Anthony L.; Carter, Glen P.; Lanckriet, Anouk L.; Van Immerseel, Filip; Moore, Robert J.; Rood, Julian I.

2010-01-01

Clostridium perfringens causes several diseases in domestic livestock, including necrotic enteritis in chickens, which is of concern to the poultry industry due to its health implications and associated economic cost. The novel pore-forming toxin NetB is a critical virulence factor in the pathogenesis of this disease. In this study, we have examined the regulation of NetB toxin production. In C. perfringens, the quorum sensing-dependent VirSR two-component signal transduction system regulates genes encoding several toxins and extracellular enzymes. Analysis of the sequence upstream of the netB gene revealed the presence of potential DNA binding sites, or VirR boxes, that are recognized by the VirR response regulator. In vitro binding experiments showed that purified VirR was able to recognize and bind to these netB-associated VirR boxes. Furthermore, using a reporter gene assay, the netB VirR boxes were shown to be functional. Mutation of the virR gene in two avian C. perfringens strains was shown to significantly reduce the production of the NetB toxin; culture supernatants derived from these strains were no longer cytotoxic to Leghorn male hepatoma cells. Complementation with the virRS operon restored the toxin phenotypes to wild type. The results also showed that the VirSR two-component system regulates the expression of netB at the level of transcription. We postulate that in the gastrointestinal tract of infected birds, NetB production is upregulated when the population of C. perfringens cells reaches a threshold level that leads to activation of the VirSR system. PMID:20457789

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.

Dynamical behavior of psb gene transcripts in greening wheat seedlings. I. Time course of accumulation of the pshA through psbN gene transcripts during light-induced greening.

PubMed

Kawaguchi, H; Fukuda, I; Shiina, T; Toyoshima, Y

1992-11-01

The time course of the accumulation of the transcripts from 13 psb genes encoding a major part of the proteins composing photosystem II during light-induced greening of dark-grown wheat seedlings was examined focusing on early stages of plastid development (0.5 h through 72 h). The 13 genes can be divided into three groups. (1) The psbA gene is transcribed as a single transcript of 1.3 kb in the dark-grown seedlings, but its level increases 5- to 7-fold in response to light due to selective increase in RNA stability as well as in transcription activity. (2) The psbE-F-L-J operon, psbM and psbN genes are transcribed as a single transcript of 1.1 kb, two transcripts of 0.5 and 0.7 kb and a single transcript of 0.3 kb, respectively, in the dark-grown seedlings. The levels of accumulation of every transcript remain unchanged or rather decrease during plastid development under illumination. (3) The psbK-I-D-C gene cluster and psbB-H operon exhibit fairly complicated northern hybridization patterns during the greening process. When a psbC or psbD gene probe was used for northern hybridization, five transcripts differing in length were detected in the etioplasts from 5-day old dark-grown seedlings. After 2 h illumination, two new transcripts of different length appeared. Light induction of new transcripts was also observed in the psbB-H operon.

Function analysis of 5'-UTR of the cellulosomal xyl-doc cluster in Clostridium papyrosolvens.

PubMed

Zou, Xia; Ren, Zhenxing; Wang, Na; Cheng, Yin; Jiang, Yuanyuan; Wang, Yan; Xu, Chenggang

2018-01-01

Anaerobic, mesophilic, and cellulolytic Clostridium papyrosolvens produces an efficient cellulolytic extracellular complex named cellulosome that hydrolyzes plant cell wall polysaccharides into simple sugars. Its genome harbors two long cellulosomal clusters: cip - cel operon encoding major cellulosome components (including scaffolding) and xyl - doc gene cluster encoding hemicellulases. Compared with works on cip - cel operon, there are much fewer studies on xyl - doc mainly due to its rare location in cellulolytic clostridia. Sequence analysis of xyl - doc revealed that it harbors a 5' untranslated region (5'-UTR) which potentially plays a role in the regulation of downstream gene expression. Here, we analyzed the function of 5'-UTR of xyl - doc cluster in C. papyrosolvens in vivo via transformation technology developed in this study. In this study, we firstly developed an electrotransformation method for C. papyrosolvens DSM 2782 before the analysis of 5'-UTR of xyl - doc cluster. In the optimized condition, a field with an intensity of 7.5-9.0 kV/cm was applied to a cuvette (0.2 cm gap) containing a mixture of plasmid and late cell suspended in exponential phase to form a 5 ms pulse in a sucrose-containing buffer. Afterwards, the putative promoter and the 5'-UTR of xyl - doc cluster were determined by sequence alignment. It is indicated that xyl - doc possesses a long conservative 5'-UTR with a complex secondary structure encompassing at least two perfect stem-loops which are potential candidates for controlling the transcriptional termination. In the last step, we employed an oxygen-independent flavin-based fluorescent protein (FbFP) as a quantitative reporter to analyze promoter activity and 5'-UTR function in vivo. It revealed that 5'-UTR significantly blocked transcription of downstream genes, but corn stover can relieve its suppression. In the present study, our results demonstrated that 5'-UTR of the cellulosomal xyl - doc cluster blocks the transcriptional activity of promoter. However, some substrates, such as corn stover, can relieve the effect of depression of 5'-UTR. Thus, it is speculated that 5'-UTR of xyl - doc was a putative riboswitch to regulate the expression of downstream cellulosomal genes, which is helpful to understand the complex regulation of cellulosome.

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.

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.

Defense Islands in Bacterial and Archaeal Genomes and Prediction of Novel Defense Systems ▿†‡

PubMed Central

Makarova, Kira S.; Wolf, Yuri I.; Snir, Sagi; Koonin, Eugene V.

2011-01-01

The arms race between cellular life forms and viruses is a major driving force of evolution. A substantial fraction of bacterial and archaeal genomes is dedicated to antivirus defense. We analyzed the distribution of defense genes and typical mobilome components (such as viral and transposon genes) in bacterial and archaeal genomes and demonstrated statistically significant clustering of antivirus defense systems and mobile genes and elements in genomic islands. The defense islands are enriched in putative operons and contain numerous overrepresented gene families. A detailed sequence analysis of the proteins encoded by genes in these families shows that many of them are diverged variants of known defense system components, whereas others show features, such as characteristic operonic organization, that are suggestive of novel defense systems. Thus, genomic islands provide abundant material for the experimental study of bacterial and archaeal antivirus defense. Except for the CRISPR-Cas systems, different classes of defense systems, in particular toxin-antitoxin and restriction-modification systems, show nonrandom clustering in defense islands. It remains unclear to what extent these associations reflect functional cooperation between different defense systems and to what extent the islands are genomic “sinks” that accumulate diverse nonessential genes, particularly those acquired via horizontal gene transfer. The characteristics of defense islands resemble those of mobilome islands. Defense and mobilome genes are nonrandomly associated in islands, suggesting nonadaptive evolution of the islands via a preferential attachment-like mechanism underpinned by the addictive properties of defense systems such as toxins-antitoxins and an important role of horizontal mobility in the evolution of these islands. PMID:21908672

Defense islands in bacterial and archaeal genomes and prediction of novel defense systems.

PubMed

Makarova, Kira S; Wolf, Yuri I; Snir, Sagi; Koonin, Eugene V

2011-11-01

The arms race between cellular life forms and viruses is a major driving force of evolution. A substantial fraction of bacterial and archaeal genomes is dedicated to antivirus defense. We analyzed the distribution of defense genes and typical mobilome components (such as viral and transposon genes) in bacterial and archaeal genomes and demonstrated statistically significant clustering of antivirus defense systems and mobile genes and elements in genomic islands. The defense islands are enriched in putative operons and contain numerous overrepresented gene families. A detailed sequence analysis of the proteins encoded by genes in these families shows that many of them are diverged variants of known defense system components, whereas others show features, such as characteristic operonic organization, that are suggestive of novel defense systems. Thus, genomic islands provide abundant material for the experimental study of bacterial and archaeal antivirus defense. Except for the CRISPR-Cas systems, different classes of defense systems, in particular toxin-antitoxin and restriction-modification systems, show nonrandom clustering in defense islands. It remains unclear to what extent these associations reflect functional cooperation between different defense systems and to what extent the islands are genomic "sinks" that accumulate diverse nonessential genes, particularly those acquired via horizontal gene transfer. The characteristics of defense islands resemble those of mobilome islands. Defense and mobilome genes are nonrandomly associated in islands, suggesting nonadaptive evolution of the islands via a preferential attachment-like mechanism underpinned by the addictive properties of defense systems such as toxins-antitoxins and an important role of horizontal mobility in the evolution of these islands.

Novel two-component transmembrane transcription control: regulation of iron dicitrate transport in Escherichia coli K-12.

PubMed

Van Hove, B; Staudenmaier, H; Braun, V

1990-12-01

Citrate and iron have to enter only the periplasmic space in order to induce the citrate-dependent iron(III) transport system of Escherichia coli. The five transport genes fecABCDE form an operon and are transcribed from fecA to fecE. Two genes, termed fecI and fecR, that mediate induction by iron(III) dicitrate have been identified upstream of fecA. The fecI gene encodes a protein of 173 amino acids (molecular weight, 19,478); the fecR gene encodes a protein of 317 amino acids (molecular weight, 35,529). Chromosomal fecI::Mu d1 mutants were unable to grow with iron(III) dicitrate as the sole iron source and synthesized no FecA outer membrane receptor protein. Growth was restored by transformation with plasmids encoding fecI or fecI and fecR. FecA and beta-galactosidase syntheses under transcription control of the fecB gene (fecB::Mu d1) were constitutive in fecI transformants and were regulated by iron(III) dicitrate in fecI fecR transformants. The amino acid sequence of the FecI protein contains a region close to the carboxy-terminal end for which a helix-turn-helix motif is predicted, which is typical for DNA-binding regulatory proteins. The FecI protein was found in the membrane, and the FecR protein was found in the periplasmic fraction. It is proposed that the FecR protein is the sensor that recognizes iron(III) dicitrate in the periplasm. The FecI protein activates fec gene expression by binding to the fec operator region. In the absence of citrate, FecR inactivates FecI. The lack of sequence homologies to other transmembrane signaling proteins and the location of the two proteins suggest a new type of transmembrane control mechanism.

The Streptococcus pyogenes serotype M49 Nra-Ralp3 transcriptional regulatory network and its control of virulence factor expression from the novel eno ralp3 epf sagA pathogenicity region.

PubMed

Kreikemeyer, Bernd; Nakata, Masanobu; Köller, Thomas; Hildisch, Hendrikje; Kourakos, Vassilios; Standar, Kerstin; Kawabata, Shigetada; Glocker, Michael O; Podbielski, Andreas

2007-12-01

Many Streptococcus pyogenes (group A streptococcus [GAS]) virulence factor- and transcriptional regulator-encoding genes cluster together in discrete genomic regions. Nra is a central regulator of the FCT region. Previous studies exclusively described Nra as a transcriptional repressor of adhesin and toxin genes. Here transcriptome and proteome analysis of a serotype M49 GAS strain and an isogenic Nra mutant of this strain revealed the complete Nra regulon profile. Nra is active in all growth phases tested, with the largest regulon in the transition phase. Almost exclusively, virulence factor-encoding genes are repressed by Nra; these genes include the GAS pilus operon, the capsule synthesis operon, the cytolysin-mediated translocation system genes, all Mga region core virulence genes, and genes encoding other regulators, like the Ihk/Irr system, Rgg, and two additional RofA-like protein family regulators. Surprisingly, our experiments revealed that Nra additionally acts as a positive regulator, mostly for genes encoding proteins and enzymes with metabolic functions. Epidemiological investigations revealed strong genetic linkage of one particular Nra-repressed regulator, Ralp3 (SPy0735), with a gene encoding Epf (extracellular protein factor from Streptococcus suis). In a serotype-specific fashion, this ralp3 epf gene block is integrated, most likely via transposition, into the eno sagA virulence gene block, which is present in all GAS serotypes. In GAS serotypes M1, M4, M12, M28, and M49 this novel discrete genetic region is therefore designated the eno ralp3 epf sagA (ERES) pathogenicity region. Functional experiments showed that Epf is a novel GAS plasminogen-binding protein and revealed that Ralp3 activity counteracts Nra and MsmR regulatory activity. In addition to the Mga and FCT regions, the ERES region is the third discrete chromosomal pathogenicity region. All of these regions are transcriptionally linked, adding another level of complexity to the known GAS growth phase-dependent regulatory network.

The Streptococcus pyogenes Serotype M49 Nra-Ralp3 Transcriptional Regulatory Network and Its Control of Virulence Factor Expression from the Novel eno ralp3 epf sagA Pathogenicity Region▿ †

PubMed Central

Kreikemeyer, Bernd; Nakata, Masanobu; Köller, Thomas; Hildisch, Hendrikje; Kourakos, Vassilios; Standar, Kerstin; Kawabata, Shigetada; Glocker, Michael O.; Podbielski, Andreas

2007-01-01

Many Streptococcus pyogenes (group A streptococcus [GAS]) virulence factor- and transcriptional regulator-encoding genes cluster together in discrete genomic regions. Nra is a central regulator of the FCT region. Previous studies exclusively described Nra as a transcriptional repressor of adhesin and toxin genes. Here transcriptome and proteome analysis of a serotype M49 GAS strain and an isogenic Nra mutant of this strain revealed the complete Nra regulon profile. Nra is active in all growth phases tested, with the largest regulon in the transition phase. Almost exclusively, virulence factor-encoding genes are repressed by Nra; these genes include the GAS pilus operon, the capsule synthesis operon, the cytolysin-mediated translocation system genes, all Mga region core virulence genes, and genes encoding other regulators, like the Ihk/Irr system, Rgg, and two additional RofA-like protein family regulators. Surprisingly, our experiments revealed that Nra additionally acts as a positive regulator, mostly for genes encoding proteins and enzymes with metabolic functions. Epidemiological investigations revealed strong genetic linkage of one particular Nra-repressed regulator, Ralp3 (SPy0735), with a gene encoding Epf (extracellular protein factor from Streptococcus suis). In a serotype-specific fashion, this ralp3 epf gene block is integrated, most likely via transposition, into the eno sagA virulence gene block, which is present in all GAS serotypes. In GAS serotypes M1, M4, M12, M28, and M49 this novel discrete genetic region is therefore designated the eno ralp3 epf sagA (ERES) pathogenicity region. Functional experiments showed that Epf is a novel GAS plasminogen-binding protein and revealed that Ralp3 activity counteracts Nra and MsmR regulatory activity. In addition to the Mga and FCT regions, the ERES region is the third discrete chromosomal pathogenicity region. All of these regions are transcriptionally linked, adding another level of complexity to the known GAS growth phase-dependent regulatory network. PMID:17893125

The sps Gene Products Affect the Germination, Hydrophobicity, and Protein Adsorption of Bacillus subtilis Spores

PubMed Central

Cangiano, Giuseppina; Sirec, Teja; Panarella, Cristina; Isticato, Rachele; Baccigalupi, Loredana; De Felice, Maurilio

2014-01-01

The multilayered surface of the Bacillus subtilis spore is composed of proteins and glycans. While over 70 different proteins have been identified as surface components, carbohydrates associated with the spore surface have not been characterized in detail yet. Bioinformatic data suggest that the 11 products of the sps operon are involved in the synthesis of polysaccharides present on the spore surface, but an experimental validation is available only for the four distal genes of the operon. Here, we report a transcriptional analysis of the sps operon and a functional study performed by constructing and analyzing two null mutants lacking either all or only the promoter-proximal gene of the operon. Our results show that both sps mutant spores apparently have normal coat and crust but have a small germination defect and are more hydrophobic than wild-type spores. We also show that spores lacking all Sps proteins are highly adhesive and form extensive clumps. In addition, sps mutant spores have an increased efficiency in adsorbing a heterologous enzyme, suggesting that hydrophobic force is a major determinant of spore adsorption and indicating that a deep understanding of the surface properties of the spore is essential for its full development as a surface display platform. PMID:25239894

A super-family of transcriptional activators regulates bacteriophage packaging and lysis in Gram-positive bacteria

PubMed Central

Quiles-Puchalt, Nuria; Tormo-Más, María Ángeles; Campoy, Susana; Toledo-Arana, Alejandro; Monedero, Vicente; Lasa, Íñigo; Novick, Richard P.; Christie, Gail E.; Penadés, José R.

2013-01-01

The propagation of bacteriophages and other mobile genetic elements requires exploitation of the phage mechanisms involved in virion assembly and DNA packaging. Here, we identified and characterized four different families of phage-encoded proteins that function as activators required for transcription of the late operons (morphogenetic and lysis genes) in a large group of phages infecting Gram-positive bacteria. These regulators constitute a super-family of proteins, here named late transcriptional regulators (Ltr), which share common structural, biochemical and functional characteristics and are unique to this group of phages. They are all small basic proteins, encoded by genes present at the end of the early gene cluster in their respective phage genomes and expressed under cI repressor control. To control expression of the late operon, the Ltr proteins bind to a DNA repeat region situated upstream of the terS gene, activating its transcription. This involves the C-terminal part of the Ltr proteins, which control specificity for the DNA repeat region. Finally, we show that the Ltr proteins are the only phage-encoded proteins required for the activation of the packaging and lysis modules. In summary, we provide evidence that phage packaging and lysis is a conserved mechanism in Siphoviridae infecting a wide variety of Gram-positive bacteria. PMID:23771138

Genetic Locus for Streptolysin S Production by Group A Streptococcus

PubMed Central

Nizet, Victor; Beall, Bernard; Bast, Darrin J.; Datta, Vivekananda; Kilburn, Laurie; Low, Donald E.; De Azavedo, Joyce C. S.

2000-01-01

Group A streptococcus (GAS) is an important human pathogen that causes pharyngitis and invasive infections, including necrotizing fasciitis. Streptolysin S (SLS) is the cytolytic factor that creates the zone of beta-hemolysis surrounding GAS colonies grown on blood agar. We recently reported the discovery of a potential genetic determinant involved in SLS production, sagA, encoding a small peptide of 53 amino acids (S. D. Betschel, S. M. Borgia, N. L. Barg, D. E. Low, and J. C. De Azavedo, Infect. Immun. 66:1671–1679, 1998). Using transposon mutagenesis, chromosomal walking steps, and data from the GAS genome sequencing project (www.genome.ou.edu/strep.html), we have now identified a contiguous nine-gene locus (sagA to sagI) involved in SLS production. The sag locus is conserved among GAS strains regardless of M protein type. Targeted plasmid integrational mutagenesis of each gene in the sag operon resulted in an SLS-negative phenotype. Targeted integrations (i) upstream of the sagA promoter and (ii) downstream of a terminator sequence after sagI did not affect SLS production, establishing the functional boundaries of the operon. A rho-independent terminator sequence between sagA and sagB appears to regulate the amount of sagA transcript produced versus transcript for the entire operon. Reintroduction of the nine-gene sag locus on a plasmid vector restored SLS activity to the nonhemolytic sagA knockout mutant. Finally, heterologous expression of the intact sag operon conferred the SLS beta-hemolytic phenotype to the nonhemolytic Lactococcus lactis. We conclude that gene products of the GAS sag operon are both necessary and sufficient for SLS production. Sequence homologies of sag operon gene products suggest that SLS is related to the bacteriocin family of microbial toxins. PMID:10858242

Genetic and functional properties of uncultivated thermophilic crenarchaeotes from a subsurface gold mine as revealed by analysis of genome fragments.

PubMed

Nunoura, Takuro; Hirayama, Hisako; Takami, Hideto; Oida, Hanako; Nishi, Shinro; Shimamura, Shigeru; Suzuki, Yohey; Inagaki, Fumio; Takai, Ken; Nealson, Kenneth H; Horikoshi, Koki

2005-12-01

Within a phylum Crenarchaeota, only some members of the hyperthermophilic class Thermoprotei, have been cultivated and characterized. In this study, we have constructed a metagenomic library from a microbial mat formation in a subsurface hot water stream of the Hishikari gold mine, Japan, and sequenced genome fragments of two different phylogroups of uncultivated thermophilic Crenarchaeota: (i) hot water crenarchaeotic group (HWCG) I (41.2 kb), and (ii) HWCG III (49.3 kb). The genome fragment of HWCG I contained a 16S rRNA gene, two tRNA genes and 35 genes encoding proteins but no 23S rRNA gene. Among the genes encoding proteins, several genes for putative aerobic-type carbon monoxide dehydrogenase represented a potential clue with regard to the yet unknown metabolism of HWCG I Archaea. The genome fragment of HWCG III contained a 16S/23S rRNA operon and 44 genes encoding proteins. In the 23S rRNA gene, we detected a homing-endonuclease encoding a group I intron similar to those detected in hyperthermophilic Crenarchaeota and Bacteria, as well as eukaryotic organelles. The reconstructed phylogenetic tree based on the 23S rRNA gene sequence reinforced the intermediate phylogenetic affiliation of HWCG III bridging the hyperthermophilic and non-thermophilic uncultivated Crenarchaeota.

High levels of bioplastic are produced in fertile transplastomic tobacco plants engineered with a synthetic operon for the production of polyhydroxybutyrate.

PubMed

Bohmert-Tatarev, Karen; McAvoy, Susan; Daughtry, Sean; Peoples, Oliver P; Snell, Kristi D

2011-04-01

An optimized genetic construct for plastid transformation of tobacco (Nicotiana tabacum) for the production of the renewable, biodegradable plastic polyhydroxybutyrate (PHB) was designed using an operon extension strategy. Bacterial genes encoding the PHB pathway enzymes were selected for use in this construct based on their similarity to the codon usage and GC content of the tobacco plastome. Regulatory elements with limited homology to the host plastome yet known to yield high levels of plastidial recombinant protein production were used to enhance the expression of the transgenes. A partial transcriptional unit, containing genes of the PHB pathway and a selectable marker gene encoding spectinomycin resistance, was flanked at the 5' end by the host plant's psbA coding sequence and at the 3' end by the host plant's 3' psbA untranslated region. This design allowed insertion of the transgenes into the plastome as an extension of the psbA operon, rendering the addition of a promoter to drive the expression of the transgenes unnecessary. Transformation of the optimized construct into tobacco and subsequent spectinomycin selection of transgenic plants yielded T0 plants that were capable of producing up to 18.8% dry weight PHB in samples of leaf tissue. These plants were fertile and produced viable seed. T1 plants producing up to 17.3% dry weight PHB in samples of leaf tissue and 8.8% dry weight PHB in the total biomass of the plant were also isolated.

Specific binding of the Xanthomonas campestris pv. vesicatoria AraC-type transcriptional activator HrpX to plant-inducible promoter boxes.

PubMed

Koebnik, Ralf; Krüger, Antje; Thieme, Frank; Urban, Alexander; Bonas, Ulla

2006-11-01

The pathogenicity of the plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria depends on a type III secretion system which is encoded by the 23-kb hrp (hypersensitive response and pathogenicity) gene cluster. Expression of the hrp operons is strongly induced in planta and in a special minimal medium and depends on two regulatory proteins, HrpG and HrpX. In this study, DNA affinity enrichment was used to demonstrate that the AraC-type transcriptional activator HrpX binds to a conserved cis-regulatory element, the plant-inducible promoter (PIP) box (TTCGC-N(15)-TTCGC), present in the promoter regions of four hrp operons. No binding of HrpX was observed when DNA fragments lacking a PIP box were used. HrpX also bound to a DNA fragment containing an imperfect PIP box (TTCGC-N(8)-TTCGT). Dinucleotide replacements in each half-site of the PIP box strongly decreased binding of HrpX, while simultaneous dinucleotide replacements in both half-sites completely abolished binding. Based on the complete genome sequence of Xanthomonas campestris pv. vesicatoria, putative plant-inducible promoters consisting of a PIP box and a -10 promoter motif were identified in the promoter regions of almost all HrpX-activated genes. Bioinformatic analyses and reverse transcription-PCR experiments revealed novel HrpX-dependent genes, among them a NUDIX hydrolase gene and several genes with a predicted role in the degradation of the plant cell wall. We conclude that HrpX is the most downstream component of the hrp regulatory cascade, which is proposed to directly activate most genes of the hrpX regulon via binding to corresponding PIP boxes.

Genetic and Biochemical Characterization of a Gene Operon for trans-Aconitic Acid, a Novel Nematicide from Bacillus thuringiensis.

PubMed

Du, Cuiying; Cao, Shiyun; Shi, Xiangyu; Nie, Xiangtao; Zheng, Jinshui; Deng, Yun; Ruan, Lifang; Peng, Donghai; Sun, Ming

2017-02-24

trans -Aconitic acid (TAA) is an isomer of cis -aconitic acid (CAA), an intermediate of the tricarboxylic acid cycle that is synthesized by aconitase. Although TAA production has been detected in bacteria and plants for many years and is known to be a potent inhibitor of aconitase, its biosynthetic origins and the physiological relevance of its activity have remained unclear. We have serendipitously uncovered key information relevant to both of these questions. Specifically, in a search for novel nematicidal factors from Bacillus thuringiensis , a significant nematode pathogen harboring many protein virulence factors, we discovered a high yielding component that showed activity against the plant-parasitic nematode Meloidogyne incognita and surprisingly identified it as TAA. Comparison with CAA, which displayed a much weaker nematicidal effect, suggested that TAA is specifically synthesized by B. thuringiensis as a virulence factor. Analysis of mutants deficient in plasmids that were anticipated to encode virulence factors allowed us to isolate a TAA biosynthesis-related ( tbr ) operon consisting of two genes, tbrA and tbrB We expressed the corresponding proteins, TbrA and TbrB, and characterized them as an aconitate isomerase and TAA transporter, respectively. Bioinformatics analysis of the TAA biosynthetic gene cluster revealed the association of the TAA genes with transposable elements relevant for horizontal gene transfer as well as a distribution across B. cereus bacteria and other B. thuringiensis strains, suggesting a general role for TAA in the interactions of B. cereus group bacteria with nematode hosts in the soil environment. This study reveals new bioactivity for TAA and the TAA biosynthetic pathway, improving our understanding of virulence factors employed by B. thuringiensis pathogenesis and providing potential implications for nematode management applications. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Roles of Salmonella typhimurium umuDC and samAB in UV mutagenesis and UV sensitivity.

PubMed Central

Nohmi, T; Yamada, M; Watanabe, M; Murayama, S Y; Sofuni, T

1992-01-01

Expression of the umuDC operon is required for UV mutagenesis and most chemical mutagenesis in Escherichia coli. The closely related species Salmonella typhimurium has two sets of umuDC-like operons; the samAB operon is located in a 60-MDa cryptic plasmid, while the S. typhimurium umuDC (umuDCST) operon resides in a chromosome. The roles of these two umuDC-like operons in UV mutagenesis and UV sensitivity of S. typhimurium were investigated. A pBR322-derived plasmid carrying the samAB operon more efficiently restored UV mutability to a umuD44 strain and a umuC122::Tn5 strain of E. coli than a plasmid carrying the umuDCST operon did. When the umuDCST operon was specifically deleted from the chromosome of S. typhimurium TA2659, the resulting strain was not UV mutable and was more sensitive to the killing effect of UV irradiation than the parent strain was. Curing of the 60-MDa cryptic plasmid carrying the samAB operon did not influence the UV mutability of strain TA2659 but did increase its resistance to UV killing. A pSC101-derived plasmid carrying the samAB operon did not restore UV mutability to a umuD44 strain of E. coli, whereas pBR322- or pBluescript-derived plasmids carrying the samAB operon efficiently did restore UV mutability. We concluded that the umuDCST operon plays a major role in UV mutagenesis in S. typhimurium and that the ability of the samAB operon to promote UV mutagenesis is strongly affected by gene dosage. Possible reasons for the poor ability of samAB to promote UV mutagenesis when it is present on low-copy-number plasmids are discussed. Images PMID:1400244

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.

Discovery and characterization of a sulfoquinovose mutarotase using kinetic analysis at equilibrium by exchange spectroscopy

PubMed Central

Abayakoon, Palika; Lingford, James P.; Jin, Yi; Bengt, Christopher; Davies, Gideon J.; Yao, Shenggen; Goddard-Borger, Ethan D.

2018-01-01

Bacterial sulfoglycolytic pathways catabolize sulfoquinovose (SQ), or glycosides thereof, to generate a three-carbon metabolite for primary cellular metabolism and a three-carbon sulfonate that is expelled from the cell. Sulfoglycolytic operons encoding an Embden–Meyerhof–Parnas-like or Entner–Doudoroff (ED)-like pathway harbor an uncharacterized gene (yihR in Escherichia coli; PpSQ1_00415 in Pseudomonas putida) that is up-regulated in the presence of SQ, has been annotated as an aldose-1-epimerase and which may encode an SQ mutarotase. Our sequence analyses and structural modeling confirmed that these proteins possess mutarotase-like active sites with conserved catalytic residues. We overexpressed the homolog from the sulfo-ED operon of Herbaspirillum seropedicaea (HsSQM) and used it to demonstrate SQ mutarotase activity for the first time. This was accomplished using nuclear magnetic resonance exchange spectroscopy, a method that allows the chemical exchange of magnetization between the two SQ anomers at equilibrium. HsSQM also catalyzed the mutarotation of various aldohexoses with an equatorial 2-hydroxy group, including d-galactose, d-glucose, d-glucose-6-phosphate (Glc-6-P), and d-glucuronic acid, but not d-mannose. HsSQM displayed only 5-fold selectivity in terms of efficiency (kcat/KM) for SQ versus the glycolysis intermediate Glc-6-P; however, its proficiency [kuncat/(kcat/KM)] for SQ was 17 000-fold better than for Glc-6-P, revealing that HsSQM preferentially stabilizes the SQ transition state. PMID:29535276

Problem-Solving Test: Tryptophan Operon Mutants

ERIC Educational Resources Information Center

Szeberenyi, Jozsef

2010-01-01

This paper presents a problem-solving test that deals with the regulation of the "trp" operon of "Escherichia coli." Two mutants of this operon are described: in mutant A, the operator region of the operon carries a point mutation so that it is unable to carry out its function; mutant B expresses a "trp" repressor protein unable to bind…

Expression of Fap amyloids in Pseudomonas aeruginosa, P. fluorescens, and P. putida results in aggregation and increased biofilm formation

PubMed Central

Dueholm, Morten S; Søndergaard, Mads T; Nilsson, Martin; Christiansen, Gunna; Stensballe, Allan; Overgaard, Michael T; Givskov, Michael; Tolker-Nielsen, Tim; Otzen, Daniel E; Nielsen, Per H

2013-01-01

The fap operon, encoding functional amyloids in Pseudomonas (Fap), is present in most pseudomonads, but so far the expression and importance for biofilm formation has only been investigated for P. fluorescens strain UK4. In this study, we demonstrate the capacity of P. aeruginosa PAO1, P. fluorescens Pf-5, and P. putida F1 to express Fap fibrils, and investigated the effect of Fap expression on aggregation and biofilm formation. The fap operon in all three Pseudomonas species conferred the ability to express Fap fibrils as shown using a recombinant approach. This Fap overexpression consistently resulted in highly aggregative phenotypes and in increased biofilm formation. Detailed biophysical investigations of purified fibrils confirmed FapC as the main fibril monomer and supported the role of FapB as a minor, nucleating constituent as also indicated by bioinformatic analysis. Bioinformatics analysis suggested FapF and FapD as a potential β-barrel membrane pore and protease, respectively. Manipulation of the fap operon showed that FapA affects monomer composition of the final amyloid fibril, and that FapB is an amyloid protein, probably a nucleator for FapC polymerization. Our study highlights the fap operon as a molecular machine for functional amyloid formation. PMID:23504942

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

Oxalic acid biosynthesis is encoded by an operon in Burkholderia glumae

USDA-ARS?s Scientific Manuscript database

Although the biosynthesis of oxalic acid is known to occur in a number of bacteria, the mechanism(s) regulating its production remains largely unknown. To date, there is no report on the identification of an oxalic acid biosynthetic pathway gene from bacteria. In an attempt to identify such a gene...

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

Rajeev, Lara; Chen, Amy; Kazakov, Alexey E.

Sulfate-reducing bacteria (SRB) are sensitive to low concentrations of nitrite, and nitrite has been used to control SRB-related biofouling in oil fields. Desulfovibrio vulgaris Hildenborough, a model SRB, carries a cytochrome c-type nitrite reductase (nrfHA) that confers resistance to low concentrations of nitrite. The regulation of this nitrite reductase has not been directly examined to date. In this study, we show that DVU0621 (NrfR), a sigma54-dependent two-component system response regulator, is the positive regulator for this operon. NrfR activates the expression of the nrfHA operon in response to nitrite stress. We also show that nrfR is needed for fitness atmore » low cell densities in the presence of nitrite because inactivation of nrfR affects the rate of nitrite reduction. We also predict and validate the binding sites for NrfR upstream of the nrfHA operon using purified NrfR in gel shift assays. Here we discuss possible roles for NrfR in regulating nitrate reductase genes in nitrate-utilizing Desulfovibrio spp. The NrfA nitrite reductase is prevalent across several bacterial phyla and required for dissimilatory nitrite reduction. However, regulation of the nrfA gene has been studied in only a few nitrate-utilizing bacteria. Here, we show that in D. vulgaris, a bacterium that does not respire nitrate, the expression of nrfHA is induced by NrfR upon nitrite stress. This is the first report of regulation of nrfA by a sigma54-dependent two-component system. Finally, our study increases our knowledge of nitrite stress responses and possibly of the regulation of nitrate reduction in SRB.« less

Analysis of the vhoGAC and vhtGAC operons from Methanosarcina mazei strain Gö1, both encoding a membrane-bound hydrogenase and a cytochrome b.

PubMed

Deppenmeier, U; Blaut, M; Lentes, S; Herzberg, C; Gottschalk, G

1995-01-15

DNA encompassing the structural genes of two membrane-bound hydrogenases from Methanosarcina mazei Gö1 was cloned and sequenced. The genes, arranged in the order vhoG and vhoA as well as vhtG and vhtA, were identified as those encoding the small and the large subunits of the NiFe hydrogenases [Deppenmeier, U., Blaut, M., Schmidt, B. & Gottschalk, G. (1992) Arch. Microbiol. 157, 505-511]. Northern-blot analysis revealed that the structural genes formed part of two operons, both containing one additional open reading frame (vhoC and vhtC) which codes for a cytochrome b. This conclusion was drawn from the homology of the deduced N-terminal amino acid sequences of vhoC and vhtC and the N-terminus of a 27-kDa cytochrome isolated from Ms. mazei C16. VhoC and VhtC contain four tentative hydrophobic segments which might span the cytoplasmic membrane. Hydropathy plots suggest that His23 and His50 are involved in heme coordination. The comparison of the sequencing data of vhoG and vhtG with the experimentally determined N-terminus of the small subunit indicate the presence of a 48-amino-acid leader peptide in front of the polypeptides. VhoA and VhtA contained the conserved sequence DPCXXC in the C-terminal region, which excludes the presence of a selenocysteine residue in these hydrogenases. Promoter sequences were found upstream of vhoG and vhtG, respectively. Downstream of vhoC, a putative terminator sequence was identified. Alignments of the deduced amino acid sequences of the gene clusters vhoGAC and vhtGAC showed 92-97% identity. Only the C-termini of VhoC and VhtC were not similar.

Interactions of Kid–Kis toxin–antitoxin complexes with the parD operator-promoter region of plasmid R1 are piloted by the Kis antitoxin and tuned by the stoichiometry of Kid–Kis oligomers

PubMed Central

Monti, Maria C.; Hernández-Arriaga, Ana M.; Kamphuis, Monique B.; López-Villarejo, Juan; Heck, Albert J. R.; Boelens, Rolf; Díaz-Orejas, Ramón; van den Heuvel, Robert H. H.

2007-01-01

The parD operon of Escherichia coli plasmid R1 encodes a toxin–antitoxin system, which is involved in plasmid stabilization. The toxin Kid inhibits cell growth by RNA degradation and its action is neutralized by the formation of a tight complex with the antitoxin Kis. A fascinating but poorly understood aspect of the kid–kis system is its autoregulation at the transcriptional level. Using macromolecular (tandem) mass spectrometry and DNA binding assays, we here demonstrate that Kis pilots the interaction of the Kid–Kis complex in the parD regulatory region and that two discrete Kis-binding regions are present on parD. The data clearly show that only when the Kis concentration equals or exceeds the Kid concentration a strong cooperative effect exists between strong DNA binding and Kid2–Kis2–Kid2–Kis2 complex formation. We propose a model in which transcriptional repression of the parD operon is tuned by the relative molar ratio of the antitoxin and toxin proteins in solution. When the concentration of the toxin exceeds that of the antitoxin tight Kid2–Kis2–Kid2 complexes are formed, which only neutralize the lethal activity of Kid. Upon increasing the Kis concentration, (Kid2–Kis2)n complexes repress the kid–kis operon. PMID:17317682

Molecular Analysis of the Locus Responsible for Production of Plantaricin S, a Two-Peptide Bacteriocin Produced by Lactobacillus plantarum LPCO10

PubMed Central

Stephens, Sarah K.; Floriano, Belén; Cathcart, Declan P.; Bayley, Susan A.; Witt, Valerie F.; Jiménez-Díaz, Rufino; Warner, Philip J.; Ruiz-Barba, José Luis

1998-01-01

A 4.5-kb region of chromosomal DNA carrying the locus responsible for the production of plantaricin S, a two-peptide bacteriocin produced by Lactobacillus plantarum LPCO10 (R. Jiménez-Díaz, J. L. Ruiz-Barba, D. P. Cathcart, H. Holo, I. F. Nes, K. H. Sletten, and P. J. Warner, Appl. Environ. Microbiol. 61:4459–4463, 1995), has been cloned, and the nucleotide sequence has been elucidated. Two genes, designated plsA and plsB and encoding peptides α and β, respectively, of plantaricin S, plus an open reading frame (ORF), ORF2, were found to be organized in an operon. Northern blot analysis showed that these genes are cotranscribed, giving a ca. 0.7-kb mRNA, whose transcription start point was determined by primer extension. Nucleotide sequences of plsA and plsB revealed that both genes are translated as bacteriocin precursors which include N-terminal leader sequences of the double-glycine type. The role of ORF2 is unknown at the moment, although it might be expected to encode an immunity protein of the type described for other bacteriocin operons. In addition, several other potential ORFs have been found, including some which may be responsible for the regulation of bacteriocin production. Two of them, ORF8 and ORF14, show strong homology with histidine protein kinase and response regulator genes, respectively, which have been found to be involved in the regulation of the production of other bacteriocins from lactic acid bacteria. A third ORF, ORF5, shows homology with gene agrB from Staphylococcus aureus, which is involved in the mechanism of regulation of the virulence phenotype in this species. Thus, an agr-like regulatory system for the production of plantaricin S is postulated. PMID:9572965

HemR is an OmpR/PhoB-like response regulator from Leptospira, which simultaneously effects transcriptional activation and repression of key haem metabolism genes.

PubMed

Morero, Natalia R; Botti, Horacio; Nitta, Kazuhiro R; Carrión, Federico; Obal, Gonzalo; Picardeau, Mathieu; Buschiazzo, Alejandro

2014-10-01

Several Leptospira species cause leptospirosis, the most extended zoonosis worldwide. In bacteria, two-component systems constitute key signalling pathways, some of which are involved in pathogenesis. The physiological roles of two-component systems in Leptospira are largely unknown, despite identifying several dozens within their genomes. Biochemical confirmation of an operative phosphorelaying two-component system has been obtained so far only for the Hklep/Rrlep pair. It is known that hklep/rrlep knockout strains of Leptospira biflexa result in haem auxotrophy, although their de novo biosynthesis machinery remains fully functional. Haem is essential for Leptospira, but information about Hklep/Rrlep effector function(s) and target(s) is still lacking. We are now reporting a thorough molecular characterization of this system, which we rename HemK/HemR. The DNA HemR-binding motif was determined, and found within the genomes of saprophyte and pathogenic Leptospira. In this way, putative HemR-regulated genes were pinpointed, including haem catabolism-related (hmuO - haem oxygenase) and biosynthesis-related (the hemA/C/D/B/L/E/N/G operon). Specific HemR binding to these two promoters was quantified, and a dual function was observed in vivo, inversely repressing the hmuO, while activating the hemA operon transcription. The crystal structure of HemR receiver domain was determined, leading to a mechanistic model for its dual regulatory role. © 2014 John Wiley & Sons Ltd.

Mutational Activation of the AmgRS Two-Component System in Aminoglycoside-Resistant Pseudomonas aeruginosa

PubMed Central

Lau, Calvin Ho-Fung; Fraud, Sebastien; Jones, Marcus; Peterson, Scott N.; Poole, Keith

2013-01-01

The amgRS operon encodes a presumed membrane stress-responsive two-component system linked to intrinsic aminoglycoside resistance in Pseudomonas aeruginosa. Genome sequencing of a lab isolate showing modest pan-aminoglycoside resistance, strain K2979, revealed a number of mutations, including a substitution in amgS that produced an R182C change in the AmgS sensor kinase product of this gene. Introduction of this mutation into an otherwise wild-type strain recapitulated the resistance phenotype, while correcting the mutation in the resistant mutant abrogated the resistant phenotype, confirming that the amgS mutation is responsible for the aminoglycoside resistance of strain K2979. The amgSR182 mutation promoted an AmgR-dependent, 2- to 3-fold increase in expression of the AmgRS target genes htpX and PA5528, mirroring the impact of aminoglycoside exposure of wild-type cells on htpX and PA5528 expression. This suggests that amgSR182 is a gain-of-function mutation that activates AmgS and the AmgRS two-component system in promoting modest resistance to aminoglycosides. Screening of several pan-aminoglycoside-resistant clinical isolates of P. aeruginosa revealed three that showed elevated htpX and PA5528 expression and harbored single amino acid-altering mutations in amgS (V121G or D106N) and no mutations in amgR. Introduction of the amgSV121G mutation into wild-type P. aeruginosa generated a resistance phenotype reminiscent of the amgSR182 mutant and produced a 2- to 3-fold increase in htpX and PA5528 expression, confirming that it, too, is a gain-of-function aminoglycoside resistance-promoting mutation. These results highlight the contribution of amgS mutations and activation of the AmgRS two-component system to acquired aminoglycoside resistance in lab and clinical isolates of P. aeruginosa. PMID:23459488

Global Analysis of Photosynthesis Transcriptional Regulatory Networks

PubMed Central

Imam, Saheed; Noguera, Daniel R.; Donohue, Timothy J.

2014-01-01

Photosynthesis is a crucial biological process that depends on the interplay of many components. This work analyzed the gene targets for 4 transcription factors: FnrL, PrrA, CrpK and MppG (RSP_2888), which are known or predicted to control photosynthesis in Rhodobacter sphaeroides. Chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) identified 52 operons under direct control of FnrL, illustrating its regulatory role in photosynthesis, iron homeostasis, nitrogen metabolism and regulation of sRNA synthesis. Using global gene expression analysis combined with ChIP-seq, we mapped the regulons of PrrA, CrpK and MppG. PrrA regulates ∼34 operons encoding mainly photosynthesis and electron transport functions, while CrpK, a previously uncharacterized Crp-family protein, regulates genes involved in photosynthesis and maintenance of iron homeostasis. Furthermore, CrpK and FnrL share similar DNA binding determinants, possibly explaining our observation of the ability of CrpK to partially compensate for the growth defects of a ΔFnrL mutant. We show that the Rrf2 family protein, MppG, plays an important role in photopigment biosynthesis, as part of an incoherent feed-forward loop with PrrA. Our results reveal a previously unrealized, high degree of combinatorial regulation of photosynthetic genes and significant cross-talk between their transcriptional regulators, while illustrating previously unidentified links between photosynthesis and the maintenance of iron homeostasis. PMID:25503406

Global analysis of photosynthesis transcriptional regulatory networks.

PubMed

Imam, Saheed; Noguera, Daniel R; Donohue, Timothy J

2014-12-01

Photosynthesis is a crucial biological process that depends on the interplay of many components. This work analyzed the gene targets for 4 transcription factors: FnrL, PrrA, CrpK and MppG (RSP_2888), which are known or predicted to control photosynthesis in Rhodobacter sphaeroides. Chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) identified 52 operons under direct control of FnrL, illustrating its regulatory role in photosynthesis, iron homeostasis, nitrogen metabolism and regulation of sRNA synthesis. Using global gene expression analysis combined with ChIP-seq, we mapped the regulons of PrrA, CrpK and MppG. PrrA regulates ∼34 operons encoding mainly photosynthesis and electron transport functions, while CrpK, a previously uncharacterized Crp-family protein, regulates genes involved in photosynthesis and maintenance of iron homeostasis. Furthermore, CrpK and FnrL share similar DNA binding determinants, possibly explaining our observation of the ability of CrpK to partially compensate for the growth defects of a ΔFnrL mutant. We show that the Rrf2 family protein, MppG, plays an important role in photopigment biosynthesis, as part of an incoherent feed-forward loop with PrrA. Our results reveal a previously unrealized, high degree of combinatorial regulation of photosynthetic genes and significant cross-talk between their transcriptional regulators, while illustrating previously unidentified links between photosynthesis and the maintenance of iron homeostasis.

Toxin-antitoxin systems and regulatory mechanisms in Mycobacterium tuberculosis.

PubMed

Slayden, Richard A; Dawson, Clinton C; Cummings, Jason E

2018-06-01

There has been a significant reduction in annual tuberculosis incidence since the World Health Organization declared tuberculosis a global health threat. However, treatment of M. tuberculosis infections requires lengthy multidrug therapeutic regimens to achieve a durable cure. The development of new drugs that are active against resistant strains and phenotypically diverse organisms continues to present the greatest challenge in the future. Numerous phylogenomic analyses have revealed that the Mtb genome encodes a significantly expanded repertoire of toxin-antitoxin (TA) loci that makes up the Mtb TA system. A TA loci is a two-gene operon encoding a 'toxin' protein that inhibits bacterial growth and an interacting 'antitoxin' partner that neutralizes the inhibitory activity of the toxin. The presence of multiple chromosomally encoded TA loci in Mtb raises important questions in regard to expansion, regulation and function. Thus, the functional roles of TA loci in Mtb pathogenesis have received considerable attention over the last decade. The cumulative results indicate that they are involved in regulating adaptive responses to stresses associated with the host environment and drug treatment. Here we review the TA families encoded in Mtb, discuss the duplication of TA loci in Mtb, regulatory mechanism of TA loci, and phenotypic heterogeneity and pathogenesis.

AmgRS-mediated envelope stress-inducible expression of the mexXY multidrug efflux operon of Pseudomonas aeruginosa

PubMed Central

Lau, Calvin Ho-Fung; Krahn, Thomas; Gilmour, Christie; Mullen, Erin; Poole, Keith

2015-01-01

AmgRS is an envelope stress-responsive two-component system and aminoglycoside resistance determinant in Pseudomonas aeruginosa that is proposed to protect cells from membrane damage caused by aminoglycoside-generated mistranslated polypeptides. Consistent with this, a ΔamgR strain showed increased aminoglycoside-promoted membrane damage, damage that was largely absent in AmgRS-activated amgS-mutant strains. Intriguingly, one such mutation, V121G, while providing for enhanced resistance to aminoglycosides, rendered P. aeruginosa susceptible to several ribosome-targeting nonaminoglycoside antimicrobials that are inducers and presumed substrates of the MexXY-OprM multidrug efflux system. Surprisingly, the amgSV121G mutation increased mexXY expression threefold, suggesting that export of these nonaminoglycosides was compromised in the amgSV121G mutant. Nonetheless, a link was established between AmgRS activation and mexXY expression and this was confirmed in studies showing that aminoglycoside-promoted mexXY expression is dependent on AmgRS. While nonaminoglycosides also induced mexXY expression, this was not AmgRS-dependent, consistent with these agents not generating mistranslated polypeptides and not activating AmgRS. The aminoglycoside inducibility of mexXY was abrogated in a mutant lacking the AmgRS target genes htpX and PA5528, encoding a presumed cytoplasmic membrane-associated protease and a membrane protein of unknown function, respectively. Thus, aminoglycoside induction of mexXY is a response to membrane damage and activation of the AmgRS two-component system. PMID:25450797

High Levels of Bioplastic Are Produced in Fertile Transplastomic Tobacco Plants Engineered with a Synthetic Operon for the Production of Polyhydroxybutyrate1[C][OA

PubMed Central

Bohmert-Tatarev, Karen; McAvoy, Susan; Daughtry, Sean; Peoples, Oliver P.; Snell, Kristi D.

2011-01-01

An optimized genetic construct for plastid transformation of tobacco (Nicotiana tabacum) for the production of the renewable, biodegradable plastic polyhydroxybutyrate (PHB) was designed using an operon extension strategy. Bacterial genes encoding the PHB pathway enzymes were selected for use in this construct based on their similarity to the codon usage and GC content of the tobacco plastome. Regulatory elements with limited homology to the host plastome yet known to yield high levels of plastidial recombinant protein production were used to enhance the expression of the transgenes. A partial transcriptional unit, containing genes of the PHB pathway and a selectable marker gene encoding spectinomycin resistance, was flanked at the 5′ end by the host plant’s psbA coding sequence and at the 3′ end by the host plant’s 3′ psbA untranslated region. This design allowed insertion of the transgenes into the plastome as an extension of the psbA operon, rendering the addition of a promoter to drive the expression of the transgenes unnecessary. Transformation of the optimized construct into tobacco and subsequent spectinomycin selection of transgenic plants yielded T0 plants that were capable of producing up to 18.8% dry weight PHB in samples of leaf tissue. These plants were fertile and produced viable seed. T1 plants producing up to 17.3% dry weight PHB in samples of leaf tissue and 8.8% dry weight PHB in the total biomass of the plant were also isolated. PMID:21325565

A taxonomy of bacterial microcompartment loci constructed by a novel scoring method

DOE PAGES

Axen, Seth D.; Erbilgin, Onur; Kerfeld, Cheryl A.; ...

2014-10-23

Bacterial microcompartments (BMCs) are proteinaceous organelles involved in both autotrophic and heterotrophic metabolism. All BMCs share homologous shell proteins but differ in their complement of enzymes; these are typically encoded adjacent to shell protein genes in genetic loci, or operons. To enable the identification and prediction of functional (sub)types of BMCs, we developed LoClass, an algorithm that finds putative BMC loci and inventories, weights, and compares their constituent pfam domains to construct a locus similarity network and predict locus (sub)types. In addition to using LoClass to analyze sequences in the Non-redundant Protein Database, we compared predicted BMC loci found inmore » seven candidate bacterial phyla (six from single-cell genomic studies) to the LoClass taxonomy. Together, these analyses resulted in the identification of 23 different types of BMCs encoded in 30 distinct locus (sub)types found in 23 bacterial phyla. These include the two carboxysome types and a divergent set of metabolosomes, BMCs that share a common catalytic core and process distinct substrates via specific signature enzymes. Furthermore, many Candidate BMCs were found that lack one or more core metabolosome components, including one that is predicted to represent an entirely new paradigm for BMC-associated metabolism, joining the carboxysome and metabolosome. By placing these results in a phylogenetic context, we provide a framework for understanding the horizontal transfer of these loci, a starting point for studies aimed at understanding the evolution of BMCs. This comprehensive taxonomy of BMC loci, based on their constituent protein domains, foregrounds the functional diversity of BMCs and provides a reference for interpreting the role of BMC gene clusters encoded in isolate, single cell, and metagenomic data. Many loci encode ancillary functions such as transporters or genes for cofactor assembly; this expanded vocabulary of BMC-related functions should be useful for design of genetic modules for introducing BMCs in bioengineering applications.« less

A Taxonomy of Bacterial Microcompartment Loci Constructed by a Novel Scoring Method

PubMed Central

Kerfeld, Cheryl A.

2014-01-01

Bacterial microcompartments (BMCs) are proteinaceous organelles involved in both autotrophic and heterotrophic metabolism. All BMCs share homologous shell proteins but differ in their complement of enzymes; these are typically encoded adjacent to shell protein genes in genetic loci, or operons. To enable the identification and prediction of functional (sub)types of BMCs, we developed LoClass, an algorithm that finds putative BMC loci and inventories, weights, and compares their constituent pfam domains to construct a locus similarity network and predict locus (sub)types. In addition to using LoClass to analyze sequences in the Non-redundant Protein Database, we compared predicted BMC loci found in seven candidate bacterial phyla (six from single-cell genomic studies) to the LoClass taxonomy. Together, these analyses resulted in the identification of 23 different types of BMCs encoded in 30 distinct locus (sub)types found in 23 bacterial phyla. These include the two carboxysome types and a divergent set of metabolosomes, BMCs that share a common catalytic core and process distinct substrates via specific signature enzymes. Furthermore, many Candidate BMCs were found that lack one or more core metabolosome components, including one that is predicted to represent an entirely new paradigm for BMC-associated metabolism, joining the carboxysome and metabolosome. By placing these results in a phylogenetic context, we provide a framework for understanding the horizontal transfer of these loci, a starting point for studies aimed at understanding the evolution of BMCs. This comprehensive taxonomy of BMC loci, based on their constituent protein domains, foregrounds the functional diversity of BMCs and provides a reference for interpreting the role of BMC gene clusters encoded in isolate, single cell, and metagenomic data. Many loci encode ancillary functions such as transporters or genes for cofactor assembly; this expanded vocabulary of BMC-related functions should be useful for design of genetic modules for introducing BMCs in bioengineering applications. PMID:25340524

A global analysis of adaptive evolution of operons in cyanobacteria.

PubMed

Memon, Danish; Singh, Abhay K; Pakrasi, Himadri B; Wangikar, Pramod P

2013-02-01

Operons are an important feature of prokaryotic genomes. Evolution of operons is hypothesized to be adaptive and has contributed significantly towards coordinated optimization of functions. Two conflicting theories, based on (i) in situ formation to achieve co-regulation and (ii) horizontal gene transfer of functionally linked gene clusters, are generally considered to explain why and how operons have evolved. Furthermore, effects of operon evolution on genomic traits such as intergenic spacing, operon size and co-regulation are relatively less explored. Based on the conservation level in a set of diverse prokaryotes, we categorize the operonic gene pair associations and in turn the operons as ancient and recently formed. This allowed us to perform a detailed analysis of operonic structure in cyanobacteria, a morphologically and physiologically diverse group of photoautotrophs. Clustering based on operon conservation showed significant similarity with the 16S rRNA-based phylogeny, which groups the cyanobacterial strains into three clades. Clade C, dominated by strains that are believed to have undergone genome reduction, shows a larger fraction of operonic genes that are tightly packed in larger sized operons. Ancient operons are in general larger, more tightly packed, better optimized for co-regulation and part of key cellular processes. A sub-clade within Clade B, which includes Synechocystis sp. PCC 6803, shows a reverse trend in intergenic spacing. Our results suggest that while in situ formation and vertical descent may be a dominant mechanism of operon evolution in cyanobacteria, optimization of intergenic spacing and co-regulation are part of an ongoing process in the life-cycle of operons.

Genotypic and phenotypic characterization of multidrug resistant Salmonella Typhimurium and Salmonella Kentucky strains recovered from chicken carcasses

PubMed Central

Grant, Ar’Quette; Choi, Seon Young; Alam, M. Samiul; Bell, Rebecca; Cavanaugh, Christopher; Balan, Kannan V.; Babu, Uma S.

2017-01-01

Abstract Salmonella Typhimurium is the leading cause of human non-typhoidal gastroenteritis in the US. S. Kentucky is one the most commonly recovered serovars from commercially processed poultry carcasses. This study compared the genotypic and phenotypic properties of two Salmonella enterica strains Typhimurium (ST221_31B) and Kentucky (SK222_32B) recovered from commercially processed chicken carcasses using whole genome sequencing, phenotype characterizations and an intracellular killing assay. Illumina MiSeq platform was used for sequencing of two Salmonella genomes. Phylogenetic analysis employing homologous alignment of a 1,185 non-duplicated protein-coding gene in the Salmonella core genome demonstrated fully resolved bifurcating patterns with varying levels of diversity that separated ST221_31B and SK222_32B genomes into distinct monophyletic serovar clades. Single nucleotide polymorphism (SNP) analysis identified 2,432 (ST19) SNPs within 13 Typhimurium genomes including ST221_31B representing Sequence Type ST19 and 650 (ST152) SNPs were detected within 13 Kentucky genomes including SK222_32B representing Sequence Type ST152. In addition to serovar-specific conserved coding sequences, the genomes of ST221_31B and SK222_32B harbor several genomic regions with significant genetic differences. These included phage and phage-like elements, carbon utilization or transport operons, fimbriae operons, putative membrane associated protein-encoding genes, antibiotic resistance genes, siderophore operons, and numerous hypothetical protein-encoding genes. Phenotype microarray results demonstrated that ST221_31B is capable of utilizing certain carbon compounds more efficiently as compared to SK222_3B; namely, 1,2-propanediol, M-inositol, L-threonine, α-D-lactose, D-tagatose, adonitol, formic acid, acetoacetic acid, and L-tartaric acid. ST221_31B survived for 48 h in macrophages, while SK222_32B was mostly eliminated. Further, a 3-fold growth of ST221_31B was observed at 24 hours post-infection in chicken granulosa cells while SK222_32B was unable to replicate in these cells. These results suggest that Salmonella Typhimurium can survive host defenses better and could be more invasive than Salmonella Kentucky and provide some insights into the genomic determinants responsible for these differences. PMID:28481935

Genotypic and phenotypic characterization of multidrug resistant Salmonella Typhimurium and Salmonella Kentucky strains recovered from chicken carcasses.

PubMed

Tasmin, Rizwana; Hasan, Nur A; Grim, Christopher J; Grant, Ar'Quette; Choi, Seon Young; Alam, M Samiul; Bell, Rebecca; Cavanaugh, Christopher; Balan, Kannan V; Babu, Uma S; Parveen, Salina

2017-01-01

Salmonella Typhimurium is the leading cause of human non-typhoidal gastroenteritis in the US. S. Kentucky is one the most commonly recovered serovars from commercially processed poultry carcasses. This study compared the genotypic and phenotypic properties of two Salmonella enterica strains Typhimurium (ST221_31B) and Kentucky (SK222_32B) recovered from commercially processed chicken carcasses using whole genome sequencing, phenotype characterizations and an intracellular killing assay. Illumina MiSeq platform was used for sequencing of two Salmonella genomes. Phylogenetic analysis employing homologous alignment of a 1,185 non-duplicated protein-coding gene in the Salmonella core genome demonstrated fully resolved bifurcating patterns with varying levels of diversity that separated ST221_31B and SK222_32B genomes into distinct monophyletic serovar clades. Single nucleotide polymorphism (SNP) analysis identified 2,432 (ST19) SNPs within 13 Typhimurium genomes including ST221_31B representing Sequence Type ST19 and 650 (ST152) SNPs were detected within 13 Kentucky genomes including SK222_32B representing Sequence Type ST152. In addition to serovar-specific conserved coding sequences, the genomes of ST221_31B and SK222_32B harbor several genomic regions with significant genetic differences. These included phage and phage-like elements, carbon utilization or transport operons, fimbriae operons, putative membrane associated protein-encoding genes, antibiotic resistance genes, siderophore operons, and numerous hypothetical protein-encoding genes. Phenotype microarray results demonstrated that ST221_31B is capable of utilizing certain carbon compounds more efficiently as compared to SK222_3B; namely, 1,2-propanediol, M-inositol, L-threonine, α-D-lactose, D-tagatose, adonitol, formic acid, acetoacetic acid, and L-tartaric acid. ST221_31B survived for 48 h in macrophages, while SK222_32B was mostly eliminated. Further, a 3-fold growth of ST221_31B was observed at 24 hours post-infection in chicken granulosa cells while SK222_32B was unable to replicate in these cells. These results suggest that Salmonella Typhimurium can survive host defenses better and could be more invasive than Salmonella Kentucky and provide some insights into the genomic determinants responsible for these differences.

Lvr, a Signaling System That Controls Global Gene Regulation and Virulence in Pathogenic Leptospira.

PubMed

Adhikarla, Haritha; Wunder, Elsio A; Mechaly, Ariel E; Mehta, Sameet; Wang, Zheng; Santos, Luciane; Bisht, Vimla; Diggle, Peter; Murray, Gerald; Adler, Ben; Lopez, Francesc; Townsend, Jeffrey P; Groisman, Eduardo; Picardeau, Mathieu; Buschiazzo, Alejandro; Ko, Albert I

2018-01-01

Leptospirosis is an emerging zoonotic disease with more than 1 million cases annually. Currently there is lack of evidence for signaling pathways involved during the infection process of Leptospira . In our comprehensive genomic analysis of 20 Leptospira spp. we identified seven pathogen-specific Two-Component System (TCS) proteins. Disruption of two these TCS genes in pathogenic Leptospira strain resulted in loss-of-virulence in a hamster model of leptospirosis. Corresponding genes lvrA and lvrB (leptospira virulence regulator ) are juxtaposed in an operon and are predicted to encode a hybrid histidine kinase and a hybrid response regulator, respectively. Transcriptome analysis of lvr mutant strains with disruption of one ( lvrB ) or both genes ( lvrA/B ) revealed global transcriptional regulation of 850 differentially expressed genes. Phosphotransfer assays demonstrated that LvrA phosphorylates LvrB and predicted further signaling downstream to one or more DNA-binding response regulators, suggesting that it is a branched pathway. Phylogenetic analyses indicated that lvrA and lvrB evolved independently within different ecological lineages in Leptospira via gene duplication. This study uncovers a novel-signaling pathway that regulates virulence in pathogenic Leptospira (Lvr), providing a framework to understand the molecular bases of regulation in this life-threatening bacterium.

Lvr, a Signaling System That Controls Global Gene Regulation and Virulence in Pathogenic Leptospira

PubMed Central

Adhikarla, Haritha; Wunder, Elsio A.; Mechaly, Ariel E.; Mehta, Sameet; Wang, Zheng; Santos, Luciane; Bisht, Vimla; Diggle, Peter; Murray, Gerald; Adler, Ben; Lopez, Francesc; Townsend, Jeffrey P.; Groisman, Eduardo; Picardeau, Mathieu; Buschiazzo, Alejandro; Ko, Albert I.

2018-01-01

Leptospirosis is an emerging zoonotic disease with more than 1 million cases annually. Currently there is lack of evidence for signaling pathways involved during the infection process of Leptospira. In our comprehensive genomic analysis of 20 Leptospira spp. we identified seven pathogen-specific Two-Component System (TCS) proteins. Disruption of two these TCS genes in pathogenic Leptospira strain resulted in loss-of-virulence in a hamster model of leptospirosis. Corresponding genes lvrA and lvrB (leptospira virulence regulator) are juxtaposed in an operon and are predicted to encode a hybrid histidine kinase and a hybrid response regulator, respectively. Transcriptome analysis of lvr mutant strains with disruption of one (lvrB) or both genes (lvrA/B) revealed global transcriptional regulation of 850 differentially expressed genes. Phosphotransfer assays demonstrated that LvrA phosphorylates LvrB and predicted further signaling downstream to one or more DNA-binding response regulators, suggesting that it is a branched pathway. Phylogenetic analyses indicated that lvrA and lvrB evolved independently within different ecological lineages in Leptospira via gene duplication. This study uncovers a novel-signaling pathway that regulates virulence in pathogenic Leptospira (Lvr), providing a framework to understand the molecular bases of regulation in this life-threatening bacterium. PMID:29600195

The Mannitol Operon Repressor MTIR belongs to a new class of transcription regulators in bacteria.

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

Tan, K.; Borovilos, M.; Zhou, M

2009-12-25

Many bacteria express phosphoenolpyruvate-dependent phosphotransferase systems (PTS). The mannitol-specific PTS catalyze the uptake and phosphorylation of d-mannitol. The uptake system comprises several genes encoded in the single operon. The expression of the mannitol operon is regulated by a proposed transcriptional factor, mannitol operon repressor (MtlR) that was first studied in Escherichia coli. Here we report the first crystal structures of MtlR from Vibrio parahemeolyticus (Vp-MtlR) and its homolog YggD protein from Shigella flexneri (Sf-YggD). MtlR and YggD belong to the same protein family (Pfam05068). Although Vp-MtlR and Sf-YggD share low sequence identity (22%), their overall structures are very similar, representingmore » a novel all {alpha}-helical fold, and indicate similar function. However, their lack of any known DNA-binding structural motifs and their unfavorable electrostatic properties imply that MtlR/YggD are unlikely to bind a specific DNA operator directly as proposed earlier. This structural observation is further corroborated by in vitro DNA-binding studies of E. coli MtlR (Ec-MtlR), which detected no interaction of Ec-MtlR with the well characterized mannitol operator/promoter region. Therefore, MtlR/YggD belongs to a new class of transcription factors in bacteria that may regulate gene expression indirectly as a part of a larger transcriptional complex.« less

Expression of Fap amyloids in Pseudomonas aeruginosa, P. fluorescens, and P. putida results in aggregation and increased biofilm formation.

PubMed

Dueholm, Morten S; Søndergaard, Mads T; Nilsson, Martin; Christiansen, Gunna; Stensballe, Allan; Overgaard, Michael T; Givskov, Michael; Tolker-Nielsen, Tim; Otzen, Daniel E; Nielsen, Per H

2013-06-01

The fap operon, encoding functional amyloids in Pseudomonas (Fap), is present in most pseudomonads, but so far the expression and importance for biofilm formation has only been investigated for P. fluorescens strain UK4. In this study, we demonstrate the capacity of P. aeruginosa PAO1, P. fluorescens Pf-5, and P. putida F1 to express Fap fibrils, and investigated the effect of Fap expression on aggregation and biofilm formation. The fap operon in all three Pseudomonas species conferred the ability to express Fap fibrils as shown using a recombinant approach. This Fap overexpression consistently resulted in highly aggregative phenotypes and in increased biofilm formation. Detailed biophysical investigations of purified fibrils confirmed FapC as the main fibril monomer and supported the role of FapB as a minor, nucleating constituent as also indicated by bioinformatic analysis. Bioinformatics analysis suggested FapF and FapD as a potential β-barrel membrane pore and protease, respectively. Manipulation of the fap operon showed that FapA affects monomer composition of the final amyloid fibril, and that FapB is an amyloid protein, probably a nucleator for FapC polymerization. Our study highlights the fap operon as a molecular machine for functional amyloid formation. © 2013 The Authors. Microbiology Open published by John Wiley & Sons Ltd.

Blocking phosphatidylcholine utilization in Pseudomonas aeruginosa, via mutagenesis of fatty acid, glycerol and choline degradation pathways, confirms the importance of this nutrient source in vivo.

PubMed

Sun, Zhenxin; Kang, Yun; Norris, Michael H; Troyer, Ryan M; Son, Mike S; Schweizer, Herbert P; Dow, Steven W; Hoang, Tung T

2014-01-01

Pseudomonas aeruginosa can grow to very high-cell-density (HCD) during infection of the cystic fibrosis (CF) lung. Phosphatidylcholine (PC), the major component of lung surfactant, has been hypothesized to support HCD growth of P. aeruginosa in vivo. The phosphorylcholine headgroup, a glycerol molecule, and two long-chain fatty acids (FAs) are released by enzymatic cleavage of PC by bacterial phospholipase C and lipases. Three different bacterial pathways, the choline, glycerol, and fatty acid degradation pathways, are then involved in the degradation of these PC components. Here, we identified five potential FA degradation (Fad) related fadBA-operons (fadBA1-5, each encoding 3-hydroxyacyl-CoA dehydrogenase and acyl-CoA thiolase). Through mutagenesis and growth analyses, we showed that three (fadBA145) of the five fadBA-operons are dominant in medium-chain and long-chain Fad. The triple fadBA145 mutant also showed reduced ability to degrade PC in vitro. We have previously shown that by partially blocking Fad, via mutagenesis of fadBA5 and fadDs, we could significantly reduce the ability of P. aeruginosa to replicate on FA and PC in vitro, as well as in the mouse lung. However, no studies have assessed the ability of mutants, defective in choline and/or glycerol degradation in conjunction with Fad, to grow on PC or in vivo. Hence, we constructed additional mutants (ΔfadBA145ΔglpD, ΔfadBA145ΔbetAB, and ΔfadBA145ΔbetABΔglpD) significantly defective in the ability to degrade FA, choline, and glycerol and, therefore, PC. The analysis of these mutants in the BALB/c mouse lung infection model showed significant inability to utilize PC in vitro, resulted in decreased replication fitness and competitiveness in vivo compared to the complement strain, although there was little to no variation in typical virulence factor production (e.g., hemolysin, lipase, and protease levels). This further supports the hypothesis that lung surfactant PC serves as an important nutrient for P. aeruginosa during CF lung infection.

Structural Insight into the Clostridium difficile Ethanolamine Utilisation Microcompartment

PubMed Central

Faulds-Pain, Alexandra; Lewis, Richard J.; Marles-Wright, Jon

2012-01-01

Bacterial microcompartments form a protective proteinaceous barrier around metabolic enzymes that process unstable or toxic chemical intermediates. The genome of the virulent, multidrug-resistant Clostridium difficile 630 strain contains an operon, eut, encoding a bacterial microcompartment with genes for the breakdown of ethanolamine and its utilisation as a source of reduced nitrogen and carbon. The C. difficile eut operon displays regulatory genetic elements and protein encoding regions in common with homologous loci found in the genomes of other bacteria, including the enteric pathogens Salmonella enterica and Enterococcus faecalis. The crystal structures of two microcompartment shell proteins, CD1908 and CD1918, and an uncharacterised protein with potential enzymatic activity, CD1925, were determined by X-ray crystallography. CD1908 and CD1918 display the same protein fold, though the order of secondary structure elements is permuted in CD1908 and this protein displays an N-terminal β-strand extension. These proteins form hexamers with molecules related by crystallographic and non-crystallographic symmetry. The structure of CD1925 has a cupin β-barrel fold and a putative active site that is distinct from the metal-ion dependent catalytic cupins. Thin-section transmission electron microscopy of Escherichia coli over-expressing eut proteins indicates that CD1918 is capable of self-association into arrays, suggesting an organisational role for CD1918 in the formation of this microcompartment. The work presented provides the basis for further study of the architecture and function of the C. difficile eut microcompartment, its role in metabolism and the wider consequences of intestinal colonisation and virulence in this pathogen. PMID:23144756

A Heme-responsive Regulator Controls Synthesis of Staphyloferrin B in Staphylococcus aureus*♦

PubMed Central

Laakso, Holly A.; Marolda, Cristina L.; Pinter, Tyler B.; Stillman, Martin J.; Heinrichs, David E.

2016-01-01

Staphylococcus aureus possesses a multitude of mechanisms by which it can obtain iron during growth under iron starvation conditions. It expresses an effective heme acquisition system (the iron-regulated surface determinant system), it produces two carboxylate-type siderophores staphyloferrin A and staphyloferrin B (SB), and it expresses transporters for many other siderophores that it does not synthesize. The ferric uptake regulator protein regulates expression of genes encoding all of these systems. Mechanisms of fine-tuning expression of iron-regulated genes, beyond simple iron regulation via ferric uptake regulator, have not been uncovered in this organism. Here, we identify the ninth gene of the sbn operon, sbnI, as encoding a ParB/Spo0J-like protein that is required for expression of genes in the sbn operon from sbnD onward. Expression of sbnD–I is drastically decreased in an sbnI mutant, and the mutant does not synthesize detectable SB during early phases of growth. Thus, SB-mediated iron acquisition is impaired in an sbnI mutant strain. We show that the protein forms dimers and tetramers in solution and binds to DNA within the sbnC coding region. Moreover, we show that SbnI binds heme and that heme-bound SbnI does not bind DNA. Finally, we show that providing exogenous heme to S. aureus growing in an iron-free medium results in delayed synthesis of SB. This is the first study in S. aureus that identifies a DNA-binding regulatory protein that senses heme to control gene expression for siderophore synthesis. PMID:26534960

Regulation of Nitrite Stress Response in Desulfovibrio vulgaris Hildenborough, a Model Sulfate-Reducing Bacterium

DOE PAGES

Rajeev, Lara; Chen, Amy; Kazakov, Alexey E.; ...

2015-08-17

Sulfate-reducing bacteria (SRB) are sensitive to low concentrations of nitrite, and nitrite has been used to control SRB-related biofouling in oil fields. Desulfovibrio vulgaris Hildenborough, a model SRB, carries a cytochrome c-type nitrite reductase (nrfHA) that confers resistance to low concentrations of nitrite. The regulation of this nitrite reductase has not been directly examined to date. In this study, we show that DVU0621 (NrfR), a sigma54-dependent two-component system response regulator, is the positive regulator for this operon. NrfR activates the expression of the nrfHA operon in response to nitrite stress. We also show that nrfR is needed for fitness atmore » low cell densities in the presence of nitrite because inactivation of nrfR affects the rate of nitrite reduction. We also predict and validate the binding sites for NrfR upstream of the nrfHA operon using purified NrfR in gel shift assays. Here we discuss possible roles for NrfR in regulating nitrate reductase genes in nitrate-utilizing Desulfovibrio spp. The NrfA nitrite reductase is prevalent across several bacterial phyla and required for dissimilatory nitrite reduction. However, regulation of the nrfA gene has been studied in only a few nitrate-utilizing bacteria. Here, we show that in D. vulgaris, a bacterium that does not respire nitrate, the expression of nrfHA is induced by NrfR upon nitrite stress. This is the first report of regulation of nrfA by a sigma54-dependent two-component system. Finally, our study increases our knowledge of nitrite stress responses and possibly of the regulation of nitrate reduction in SRB.« less

Involvement of the C-terminal extension of the alpha polypeptide and of the PucC protein in LH2 complex biosynthesis in Rubrivivax gelatinosus.

PubMed

Steunou, Anne-Soisig; Ouchane, Soufian; Reiss-Husson, Françoise; Astier, Chantal

2004-05-01

The facultative phototrophic nonsulfur bacterium Rubrivivax gelatinosus exhibits several differences from other species of purple bacteria in the organization of its photosynthetic genes. In particular, the puc operon contains only the pucB and pucA genes encoding the beta and alpha polypeptides of the light-harvesting 2 (LH2) complex. Downstream of the pucBA operon is the pucC gene in the opposite transcriptional orientation. The transcription of pucBA and pucC has been studied. No pucC transcript was detected either by Northern blotting or by reverse transcription-PCR analysis. The initiation site of pucBA transcription was determined by primer extension, and Northern blot analysis revealed the presence of two transcripts of 0.8 and 0.65 kb. The half-lives of both transcripts are longer in cells grown semiaerobically than in photosynthetically grown cells, and the small transcript is the less stable. It was reported that the alpha polypeptide, encoded by the pucA gene, presents a C-terminal extension which is not essential for LH2 function in vitro. The biological role of this alanine- and proline-rich C-terminal extension in vivo has been investigated. Two mutants with C-terminal deletions of 13 and 18 residues have been constructed. Both present the two pucBA transcripts, while their phenotypes are, respectively, LH2+ and LH2-, suggesting that a minimal length of the C-terminal extension is required for LH2 biogenesis. Another important factor involved in the LH2 biogenesis is the PucC protein. To gain insight into the function of this protein in R. gelatinosus, we constructed and characterized a PucC mutant. The mutant is devoid of LH2 complex under semiaerobiosis but still produces a small amount of these antennae under photosynthetic growth conditions. This conditional phenotype suggests the involvement of another factor in LH2 biogenesis.

Characterization of Staphylococcus aureus mutants expressing reduced susceptibility to common house-cleaners

PubMed Central

Davis, A.O.; O’Leary, J.O.; Muthaiyan, A.; Langevin, M.J.; Delgado, A.; Abalos, A.T.; Fajardo, A.R.; Marek, J.; Wilkinson, B.J.; Gustafson, J.E.

2013-01-01

Aims To characterize mutants of Staphylococcus aureus expressing reduced susceptibility to house cleaners (HC), assess the impact of the alternative sigma factor SigB on HC susceptibility, and determine the MIC of clinical methicillin-resistant S. aureus (MRSA) to a HC. Methods and Results Susceptibility to HC, HC components, H2O2, vancomycin and oxacillin and physiological parameters were determined for HC-reduced susceptibility (HCRS) mutants, parent strain COL and COLsigB::kan. HCRS mutants selected with three HC expressed reduced susceptibility to multiple HC, HC components, H2O2 and vancomycin. Two unique HCRS mutants also lost the methicillin resistance determinant. In addition, all HCRS mutants exhibited better growth at two temperatures, and one HCRS mutant expressed reduced carotenoid production. COLsigB::kan demonstrated increased susceptibility to all HC and many HC components. sigB operon mutations were not detected in one HCRS mutant background. Of 76 clinical MRSA, 20 exhibited reduced susceptibility to a HC. Conclusions HCRS mutants demonstrate altered susceptibility to multiple antimicrobials. While sigB is required for full HC resistance, one HCRS mechanism does not involve sigB operon mutations. Clinical MRSA expressing reduced susceptibility to a common HC were detected. Significance and Impact of the Study This study suggests that HCRS mutants are not protected against, nor selected by, practical HC concentrations. PMID:15659191

Awakening sleeping beauty: production of propionic acid in Escherichia coli through the sbm operon requires the activity of a methylmalonyl-CoA epimerase.

PubMed

Gonzalez-Garcia, Ricardo Axayacatl; McCubbin, Tim; Wille, Annalena; Plan, Manuel; Nielsen, Lars Keld; Marcellin, Esteban

2017-07-17

Propionic acid is used primarily as a food preservative with smaller applications as a chemical building block for the production of many products including fabrics, cosmetics, drugs, and plastics. Biological production using propionibacteria would be competitive against chemical production through hydrocarboxylation of ethylene if native producers could be engineered to reach near-theoretical yield and good productivity. Unfortunately, engineering propionibacteria has proven very challenging. It has been suggested that activation of the sleeping beauty operon in Escherichia coli is sufficient to achieve propionic acid production. Optimising E. coli production should be much easier than engineering propionibacteria if tolerance issues can be addressed. Propionic acid is produced in E. coli via the sleeping beauty mutase operon under anaerobic conditions in rich medium via amino acid degradation. We observed that the sbm operon enhances amino acids degradation to propionic acid and allows E. coli to degrade isoleucine. However, we show here that the operon lacks an epimerase reaction that enables propionic acid production in minimal medium containing glucose as the sole carbon source. Production from glucose can be restored by engineering the system with a methylmalonyl-CoA epimerase from Propionibacterium acidipropionici (0.23 ± 0.02 mM). 1-Propanol production was also detected from the promiscuous activity of the native alcohol dehydrogenase (AdhE). We also show that aerobic conditions are favourable for propionic acid production. Finally, we increase titre 65 times using a combination of promoter engineering and process optimisation. The native sbm operon encodes an incomplete pathway. Production of propionic acid from glucose as sole carbon source is possible when the pathway is complemented with a methylmalonyl-CoA epimerase. Although propionic acid via the restored succinate dissimilation pathway is considered a fermentative process, the engineered pathway was shown to be functional under anaerobic and aerobic conditions.

Genomic, proteomic, and biochemical analysis of the organohalide respiratory pathway in Desulfitobacterium dehalogenans.

PubMed

Kruse, Thomas; van de Pas, Bram A; Atteia, Ariane; Krab, Klaas; Hagen, Wilfred R; Goodwin, Lynne; Chain, Patrick; Boeren, Sjef; Maphosa, Farai; Schraa, Gosse; de Vos, Willem M; van der Oost, John; Smidt, Hauke; Stams, Alfons J M

2015-03-01

Desulfitobacterium dehalogenans is able to grow by organohalide respiration using 3-chloro-4-hydroxyphenyl acetate (Cl-OHPA) as an electron acceptor. We used a combination of genome sequencing, biochemical analysis of redox active components, and shotgun proteomics to study elements of the organohalide respiratory electron transport chain. The genome of Desulfitobacterium dehalogenans JW/IU-DC1(T) consists of a single circular chromosome of 4,321,753 bp with a GC content of 44.97%. The genome contains 4,252 genes, including six rRNA operons and six predicted reductive dehalogenases. One of the reductive dehalogenases, CprA, is encoded by a well-characterized cprTKZEBACD gene cluster. Redox active components were identified in concentrated suspensions of cells grown on formate and Cl-OHPA or formate and fumarate, using electron paramagnetic resonance (EPR), visible spectroscopy, and high-performance liquid chromatography (HPLC) analysis of membrane extracts. In cell suspensions, these components were reduced upon addition of formate and oxidized after addition of Cl-OHPA, indicating involvement in organohalide respiration. Genome analysis revealed genes that likely encode the identified components of the electron transport chain from formate to fumarate or Cl-OHPA. Data presented here suggest that the first part of the electron transport chain from formate to fumarate or Cl-OHPA is shared. Electrons are channeled from an outward-facing formate dehydrogenase via menaquinones to a fumarate reductase located at the cytoplasmic face of the membrane. When Cl-OHPA is the terminal electron acceptor, electrons are transferred from menaquinones to outward-facing CprA, via an as-yet-unidentified membrane complex, and potentially an extracellular flavoprotein acting as an electron shuttle between the quinol dehydrogenase membrane complex and CprA. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Genomic, Proteomic, and Biochemical Analysis of the Organohalide Respiratory Pathway in Desulfitobacterium dehalogenans

PubMed Central

van de Pas, Bram A.; Atteia, Ariane; Krab, Klaas; Hagen, Wilfred R.; Goodwin, Lynne; Chain, Patrick; Boeren, Sjef; Maphosa, Farai; Schraa, Gosse; de Vos, Willem M.; van der Oost, John; Smidt, Hauke

2014-01-01

Desulfitobacterium dehalogenans is able to grow by organohalide respiration using 3-chloro-4-hydroxyphenyl acetate (Cl-OHPA) as an electron acceptor. We used a combination of genome sequencing, biochemical analysis of redox active components, and shotgun proteomics to study elements of the organohalide respiratory electron transport chain. The genome of Desulfitobacterium dehalogenans JW/IU-DC1T consists of a single circular chromosome of 4,321,753 bp with a GC content of 44.97%. The genome contains 4,252 genes, including six rRNA operons and six predicted reductive dehalogenases. One of the reductive dehalogenases, CprA, is encoded by a well-characterized cprTKZEBACD gene cluster. Redox active components were identified in concentrated suspensions of cells grown on formate and Cl-OHPA or formate and fumarate, using electron paramagnetic resonance (EPR), visible spectroscopy, and high-performance liquid chromatography (HPLC) analysis of membrane extracts. In cell suspensions, these components were reduced upon addition of formate and oxidized after addition of Cl-OHPA, indicating involvement in organohalide respiration. Genome analysis revealed genes that likely encode the identified components of the electron transport chain from formate to fumarate or Cl-OHPA. Data presented here suggest that the first part of the electron transport chain from formate to fumarate or Cl-OHPA is shared. Electrons are channeled from an outward-facing formate dehydrogenase via menaquinones to a fumarate reductase located at the cytoplasmic face of the membrane. When Cl-OHPA is the terminal electron acceptor, electrons are transferred from menaquinones to outward-facing CprA, via an as-yet-unidentified membrane complex, and potentially an extracellular flavoprotein acting as an electron shuttle between the quinol dehydrogenase membrane complex and CprA. PMID:25512312

Methicillin-Susceptible Teicoplanin-Resistant Staphylococcus haemolyticus Isolate from a Bloodstream Infection with Novel Mutations in the tcaRAB Teicoplanin Resistance Operon.

PubMed

Bakthavatchalam, Yamuna Devi; Sudarsanam, Thambu David; Babu, Priyanka; Munuswamy, Elakkiya; Muthuirulandi Sethuvel, Dhiviya Prabaa; Devanga Ragupathi, Naveen Kumar; Veeraraghavan, Balaji

2017-07-24

Staphylococcus haemolyticus is a coagulase-negative staphylococcus that is frequently isolated from blood cultures. Here, we report a case of methicillin-susceptible S. haemolyticus that is resistant to teicoplanin (TEC) and heteroresistant to vancomycin (VAN). The isolate was susceptible to cefoxitin and resistant to TEC by Etest. Population analysis profile-area under the curve analysis confirmed the presence of a VAN heteroresistant subpopulation. Next-generation sequencing analysis of the genome revealed the presence of blaZ and msr(A), which encode cross-resistance to macrolide, lincosamide, and streptogramin B, and the quinolone resistance-conferring gene norA. In addition, several amino acid substitutions were observed in the TEC resistance operon tcaRAB, including I3N, I390N, and L450I in tcaA and L44V, G52V, and S87P in tcaR, as well as in the transpeptidase encoding gene walK (D336Y, R375L, and V404A) and L315 and P316 in graS. We hypothesized that this combination of mutations could confer TEC resistance and reduced VAN susceptibility.

Identification of a conserved branched RNA structure that functions as a factor-independent terminator.

PubMed

Johnson, Christopher M; Chen, Yuqing; Lee, Heejin; Ke, Ailong; Weaver, Keith E; Dunny, Gary M

2014-03-04

Anti-Q is a small RNA encoded on pCF10, an antibiotic resistance plasmid of Enterococcus faecalis, which negatively regulates conjugation of the plasmid. In this study we sought to understand how Anti-Q is generated relative to larger transcripts of the same operon. We found that Anti-Q folds into a branched structure that functions as a factor-independent terminator. In vitro and in vivo, termination is dependent on the integrity of this structure as well as the presence of a 3' polyuridine tract, but is not dependent on other downstream sequences. In vitro, terminated transcripts are released from RNA polymerase after synthesis. In vivo, a mutant with reduced termination efficiency demonstrated loss of tight control of conjugation function. A search of bacterial genomes revealed the presence of sequences that encode Anti-Q-like RNA structures. In vitro and in vivo experiments demonstrated that one of these functions as a terminator. This work reveals a previously unappreciated flexibility in the structure of factor-independent terminators and identifies a mechanism for generation of functional small RNAs; it should also inform annotation of bacterial sequence features, such as terminators, functional sRNAs, and operons.

Identification of a conserved branched RNA structure that functions as a factor-independent terminator

PubMed Central

Johnson, Christopher M.; Chen, Yuqing; Lee, Heejin; Ke, Ailong; Weaver, Keith E.; Dunny, Gary M.

2014-01-01

Anti-Q is a small RNA encoded on pCF10, an antibiotic resistance plasmid of Enterococcus faecalis, which negatively regulates conjugation of the plasmid. In this study we sought to understand how Anti-Q is generated relative to larger transcripts of the same operon. We found that Anti-Q folds into a branched structure that functions as a factor-independent terminator. In vitro and in vivo, termination is dependent on the integrity of this structure as well as the presence of a 3′ polyuridine tract, but is not dependent on other downstream sequences. In vitro, terminated transcripts are released from RNA polymerase after synthesis. In vivo, a mutant with reduced termination efficiency demonstrated loss of tight control of conjugation function. A search of bacterial genomes revealed the presence of sequences that encode Anti-Q–like RNA structures. In vitro and in vivo experiments demonstrated that one of these functions as a terminator. This work reveals a previously unappreciated flexibility in the structure of factor-independent terminators and identifies a mechanism for generation of functional small RNAs; it should also inform annotation of bacterial sequence features, such as terminators, functional sRNAs, and operons. PMID:24550474

Genetic modification of Bacillus subtilis for production of D-chiro-inositol, an investigational drug candidate for treatment of type 2 diabetes and polycystic ovary syndrome.

PubMed

Yoshida, Ken-ichi; Yamaguchi, Masanori; Morinaga, Tetsuro; Ikeuchi, Maya; Kinehara, Masaki; Ashida, Hitoshi

2006-02-01

D-chiro-inositol (DCI) is a drug candidate for the treatment of type 2 diabetes and polycystic ovary syndrome, since it improves the efficiency with which the body uses insulin and also promotes ovulation. Here, we report genetic modification of Bacillus subtilis for production of DCI from myo-inositol (MI). The B. subtilis iolABCDEFGHIJ operon encodes enzymes for the multiple steps of the MI catabolic pathway. In the first and second steps, MI is converted to 2-keto-MI (2KMI) by IolG and then to 3D-(3,5/4)-trihydroxycyclohexane-1,2-dione by IolE. In this study, we identified iolI encoding inosose isomerase, which converts 2KMI to 1-keto-D-chiro-inositol (1KDCI), and found that IolG reduces 1KDCI to DCI. Inactivation of iolE in a mutant constitutively expressing the iol operon blocked the MI catabolic pathway to accumulate 2KMI, which was converted to DCI via the activity of IolI and IolG. The mutant was able to convert at least 6% of input MI in the culture medium to DCI.

Expression of acrA and acrB Genes in Esherichia coli Mutants with or without marR or acrR Mutations.

PubMed

Pourahmad Jaktaji, Razieh; Jazayeri, Nasim

2013-12-01

The major antibiotic efflux pump of Esherichia coli is AcrAB-TolC. The first part of the pump, AcrAB, is encoded by acrAB operon. The expression of this operon can be kept elevated by overexpression of an activator, MarA following inactivation of MarR and AcrR repressors due to mutation in encoding genes, marR and acrR, respectively. The aims of this research were to use E. coli mutants with or without mutation in marR to search for the presence of possible mutation in acrR and to quantify the expression of acrAB. The DNA binding region of acrR gene in these mutants were amplified by PCR and sequenced. The relative expression of acrA and acrB were determined by real time PCR. RESULTS showed that W26 and C14 had the same mutation in acrR, but none of the mutants overexpressed acrA and acrB in comparison with wild type strain. The effect of marR or acrR mutation on acrAB overexpression is dependent on levels of resistance to tetracycline and ciprofloxacin.

A molecular sensor that allows a gut commensal to control its nutrient foundation in a competitive ecosystem

PubMed Central

Hooper, Lora V.; Xu, Jian; Falk, Per G.; Midtvedt, Tore; Gordon, Jeffrey I.

1999-01-01

Little is known about how members of the indigenous microflora interact with their mammalian hosts to establish mutually beneficial relationships. We have used a gnotobiotic mouse model to show that Bacteroides thetaiotaomicron, a component of the intestinal microflora of mice and humans, uses a repressor, FucR, as a molecular sensor of l-fucose availability. FucR coordinates expression of an operon encoding enzymes in the l-fucose metabolic pathway with expression of another locus that regulates production of fucosylated glycans in intestinal enterocytes. Genetic and biochemical studies indicate that FucR does this by using fucose as an inducer at one locus and as a corepressor at the other locus. Coordinating this commensal’s immediate nutritional requirements with production of a host-derived energy source is consistent with its need to enter and persist within a competitive ecosystem. PMID:10449780

High-quality draft genome sequence of Ensifer meliloti Mlalz-1, a microsymbiont of Medicago laciniata (L.) miller collected in Lanzarote, Canary Islands, Spain.

PubMed

Osman, Wan Adnawani Meor; van Berkum, Peter; León-Barrios, Milagros; Velázquez, Encarna; Elia, Patrick; Tian, Rui; Ardley, Julie; Gollagher, Margaret; Seshadri, Rekha; Reddy, T B K; Ivanova, Natalia; Woyke, Tanja; Pati, Amrita; Markowitz, Victor; Baeshen, Mohamed N; Baeshen, Naseebh Nabeeh; Kyrpides, Nikos; Reeve, Wayne

2017-01-01

10.1601/nm.1335 Mlalz-1 (INSDC = ATZD00000000) is an aerobic, motile, Gram-negative, non-spore-forming rod that was isolated from an effective nitrogen-fixing nodule of Medicago laciniata (L.) Miller from a soil sample collected near the town of Guatiza on the island of Lanzarote, the Canary Islands, Spain. This strain nodulates and forms an effective symbiosis with the highly specific host M. laciniata . This rhizobial genome was sequenced as part of the DOE Joint Genome Institute 2010 Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria (GEBA-RNB) sequencing project. Here the features of 10.1601/nm.1335 Mlalz-1 are described, together with high-quality permanent draft genome sequence information and annotation. The 6,664,116 bp high-quality draft genome is arranged in 99 scaffolds of 100 contigs, containing 6314 protein-coding genes and 74 RNA-only encoding genes. Strain Mlalz-1 is closely related to 10.1601/nm.1335 10.1601/strainfinder?urlappend=%3Fid%3DIAM+12611 T , 10.1601/nm.1334 A 321 T and 10.1601/nm.17831 10.1601/strainfinder?urlappend=%3Fid%3DORS+1407 T , based on 16S rRNA gene sequences. gANI values of ≥98.1% support the classification of strain Mlalz-1 as 10.1601/nm.1335. Nodulation of M. laciniata requires a specific nodC allele, and the nodC gene of strain Mlalz-1 shares ≥98% sequence identity with nodC of M. laciniata -nodulating 10.1601/nm.1328 strains, but ≤93% with nodC of 10.1601/nm.1328 strains that nodulate other Medicago species. Strain Mlalz-1 is unique among sequenced 10.1601/nm.1335 strains in possessing genes encoding components of a T2SS and in having two versions of the adaptive acid tolerance response lpiA-acvB operon. In 10.1601/nm.1334 strain 10.1601/strainfinder?urlappend=%3Fid%3DWSM+419, lpiA is essential for enhancing survival in lethal acid conditions. The second copy of the lpiA-acvB operon of strain Mlalz-1 has highest sequence identity (> 96%) with that of 10.1601/nm.1334 strains, which suggests genetic recombination between strain Mlalz-1 and 10.1601/nm.1334 and the horizontal gene transfer of lpiA-acvB .

Solution NMR structure of hypothetical protein CV_2116 encoded by a viral prophage element in Chromobacterium violaceum.

PubMed

Yang, Yunhuang; Ramelot, Theresa A; Cort, John R; Garcia, Maite; Yee, Adelinda; Arrowsmith, Cheryl H; Kennedy, Michael A

2012-01-01

CV_2116 is a small hypothetical protein of 82 amino acids from the Gram-negative coccobacillus Chromobacterium violaceum. A PSI-BLAST search using the CV_2116 sequence as a query identified only one hit (E = 2e(-07)) corresponding to a hypothetical protein OR16_04617 from Cupriavidus basilensis OR16, which failed to provide insight into the function of CV_2116. The CV_2116 gene was cloned into the p15TvLic expression plasmid, transformed into E. coli, and (13)C- and (15)N-labeled NMR samples of CV_2116 were overexpressed in E. coli and purified for structure determination using NMR spectroscopy. The resulting high-quality solution NMR structure of CV_2116 revealed a novel α + β fold containing two anti-parallel β-sheets in the N-terminal two-thirds of the protein and one α-helix in the C-terminal third of the protein. CV_2116 does not belong to any known protein sequence family and a Dali search indicated that no similar structures exist in the protein data bank. Although no function of CV_2116 could be derived from either sequence or structural similarity searches, the neighboring genes of CV_2116 encode various proteins annotated as similar to bacteriophage tail assembly proteins. Interestingly, C. violaceum exhibits an extensive network of bacteriophage tail-like structures that likely result from lateral gene transfer by incorporation of viral DNA into its genome (prophages) due to bacteriophage infection. Indeed, C. violaceum has been shown to contain four prophage elements and CV_2116 resides in the fourth of these elements. Analysis of the putative operon in which CV_2116 resides indicates that CV_2116 might be a component of the bacteriophage tail-like assembly that occurs in C. violaceum.

Detection and genomic characterization of motility in Lactobacillus curvatus: confirmation of motility in a species outside the Lactobacillus salivarius clade.

PubMed

Cousin, Fabien J; Lynch, Shónagh M; Harris, Hugh M B; McCann, Angela; Lynch, Denise B; Neville, B Anne; Irisawa, Tomohiro; Okada, Sanae; Endo, Akihito; O'Toole, Paul W

2015-02-01

Lactobacillus is the largest genus within the lactic acid bacteria (LAB), with almost 180 species currently identified. Motility has been reported for at least 13 Lactobacillus species, all belonging to the Lactobacillus salivarius clade. Motility in lactobacilli is poorly characterized. It probably confers competitive advantages, such as superior nutrient acquisition and niche colonization, but it could also play an important role in innate immune system activation through flagellin–Toll-like receptor 5 (TLR5) interaction. We now report strong evidence of motility in a species outside the L. salivarius clade, Lactobacillus curvatus (strain NRIC0822). The motility of L. curvatus NRIC 0822 was revealed by phase-contrast microscopy and soft-agar motility assays. Strain NRIC 0822 was motile at temperatures between 15 °C and 37 °C, with a range of different carbohydrates, and under varying atmospheric conditions. We sequenced the L. curvatus NRIC 0822 genome, which revealed that the motility genes are organized in a single operon and that the products are very similar (>98.5% amino acid similarity over >11,000 amino acids) to those encoded by the motility operon of Lactobacillus acidipiscis KCTC 13900 (shown for the first time to be motile also). Moreover, the presence of a large number of mobile genetic elements within and flanking the motility operon of L. curvatus suggests recent horizontal transfer between members of two distinct Lactobacillus clades: L. acidipiscis in the L. salivarius clade and L. curvatus inthe L. sakei clade. This study provides novel phenotypic, genetic, and phylogenetic insights into flagellum-mediated motility in lactobacilli.

Unique aspects of fiber degradation by the ruminal ethanologen Ruminococcus albus 7 revealed by physiological and transcriptomic analysis

DOE PAGES

Christopherson, Melissa R.; Dawson, John A.; Stevenson, David M.; ...

2014-12-04

Bacteria in the genus Ruminococcus are ubiquitous members of the mammalian gastrointestinal tract. In particular, they are important in ruminants where they digest a wide range of plant cell wall polysaccharides. For example, Ruminococcus albus 7 is a primary cellulose degrader that produces acetate usable by its bovine host. Moreover, it is one of the few organisms that ferments cellulose to form ethanol at mesophilic temperatures in vitro. The mechanism of cellulose degradation by R. albus 7 is not well-defined and is thought to involve pilin-like proteins, unique carbohydrate-binding domains, a glycocalyx, and cellulosomes. We used a combination of comparativemore » genomics, fermentation analyses, and transcriptomics to further clarify the cellulolytic and fermentative potential of R. albus 7. A comparison of the R. albus 7 genome sequence against the genome sequences of related bacteria that either encode or do not encode cellulosomes revealed that R. albus 7 does not encode for most canonical cellulosomal components. Fermentation analysis of R. albus 7 revealed the ability to produce ethanol and acetate on a wide range of fibrous substrates in vitro. Global transcriptomic analysis of R. albus 7 grown at identical dilution rates on cellulose and cellobiose in a chemostat showed that this bacterium, when growing on cellulose, utilizes a carbohydrate-degrading strategy that involves increased transcription of the rare carbohydrate-binding module (CBM) family 37 domain and the tryptophan biosynthetic operon. Our data suggest that R. albus 7 does not use canonical cellulosomal components to degrade cellulose, but rather up-regulates the expression of CBM37-containing enzymes and tryptophan biosynthesis. This study contributes to a revised model of carbohydrate degradation by this key member of the rumen ecosystem.« less

The coxBAC Operon Encodes a Cytochrome c Oxidase Required for Heterotrophic Growth in the Cyanobacterium Anabaena variabilis Strain ATCC 29413

PubMed Central

Schmetterer, Georg; Valladares, Ana; Pils, Dietmar; Steinbach, Susanne; Pacher, Margit; Muro-Pastor, Alicia M.; Flores, Enrique; Herrero, Antonia

2001-01-01

Three genes, coxB, coxA, and coxC, found in a clone from a gene library of the cyanobacterium Anabaena variabilis strain ATCC 29413, were identified by hybridization with an oligonucleotide specific for aa3-type cytochrome c oxidases. Deletion of these genes from the genome of A. variabilis strain ATCC 29413 FD yielded strain CSW1, which displayed no chemoheterotrophic growth and an impaired cytochrome c oxidase activity. Photoautotrophic growth of CSW1, however, was unchanged, even with dinitrogen as the nitrogen source. A higher cytochrome c oxidase activity was detected in membrane preparations from dinitrogen-grown CSW1 than from nitrate-grown CSW1, but comparable activities of respiratory oxygen uptake were found in the wild type and in CSW1. Our data indicate that the identified cox gene cluster is essential for fructose-dependent growth in the dark, but not for growth on dinitrogen, and that other terminal respiratory oxidases are expressed in this cyanobacterium. Transcription analysis showed that coxBAC constitutes an operon which is expressed from two transcriptional start points. The use of one of them was stimulated by fructose. PMID:11591688

An homolog of the Frz Phosphoenolpyruvate:carbohydrate phosphoTransferase System of extraintestinal pathogenic Escherichia coli is encoded on a genomic island in specific lineages of Streptococcus agalactiae.

PubMed

Patron, Kévin; Gilot, Philippe; Camiade, Emilie; Mereghetti, Laurent

2015-06-01

We identified a Streptococcus agalactiae metabolic region (fru2) coding for a Phosphoenolpyruvate:carbohydrate phosphoTransferase System (PTS) homologous to the Frz system of extraintestinal pathogenic Escherichia coli strains. The Frz system is involved in environmental sensing and regulation of the expression of adaptation and virulence genes in E. coli. The S. agalactiae fru2 region codes three subunits of a PTS transporter of the fructose-mannitol family, a transcriptional activator of PTSs of the MtlR family, an allulose-6 phosphate-3-epimerase, a transaldolase and a transketolase. We demonstrated that all these genes form an operon. The fru2 operon is present in a 17494-bp genomic island. We analyzed by multilocus sequence typing a population of 492 strains representative of the S. agalactiae population and we showed that the presence of the fru2 operon is linked to the phylogeny of S. agalactiae. The fru2 operon is always present within strains of clonal complexes CC 1, CC 7, CC 10, CC 283 and singletons ST 130 and ST 288, but never found in other CCs and STs. Our results indicate that the fru2 operon was acquired during the evolution of the S. agalactiae species from a common ancestor before the divergence of CC 1, CC 7, CC 10, CC 283, ST 130 and ST 288. As S. agalactiae strains of CC 1 and CC 10 are frequently isolated from adults with invasive disease, we hypothesize that the S. agalactiae Fru2 system senses the environment to allow the bacterium to adapt to new conditions encountered during the infection of adults. Copyright © 2015 Elsevier B.V. All rights reserved.

Cellulose synthesizing Complexes in Vascular Plants andProcaryotes

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

Brown, Richard M, Jr; Saxena, Inder Mohan

2009-07-07

Continuing the work initiated under DE-FG03-94ER20145, the following major accomplishments were achieved under DE-FG02-03ER15396 from 2003-2007: (a) we purified the acsD gene product of the Acetobacter cellulose synthase operon as well as transferred the CesA cellulose gene from Gossypium into E. coli in an attempt to crystallize this protein for x-ray diffraction structural analysis; however, crystallization attempts proved unsuccessful; (b) the Acetobacter cellulose synthase operon was successfully incorporated into Synechococcus, a cyanobacterium2; (c) this operon in Synechococcus was functionally expressed; (d) we successfully immunolabeled Vigna cellulose and callose synthase components and mapped their distribution before and after wounding; (e) wemore » developed a novel method to produce replicas of cellulose synthases in tobacco BY-2 cells, and we demonstrated the cytoplasmic domain of the rosette TC; (f) from the moss Physcomitrella, we isolated two full-length cDNA sequences of cellulose synthase (PpCesA1 and PpCesA2) and attempted to obtain full genomic DNA sequences; (g) we examined the detailed molecular structure of a new form of non-crystalline cellulose known as nematic ordered cellulose (=NOC)3.« less

A system-level model for the microbial regulatory genome.

PubMed

Brooks, Aaron N; Reiss, David J; Allard, Antoine; Wu, Wei-Ju; Salvanha, Diego M; Plaisier, Christopher L; Chandrasekaran, Sriram; Pan, Min; Kaur, Amardeep; Baliga, Nitin S

2014-07-15

Microbes can tailor transcriptional responses to diverse environmental challenges despite having streamlined genomes and a limited number of regulators. Here, we present data-driven models that capture the dynamic interplay of the environment and genome-encoded regulatory programs of two types of prokaryotes: Escherichia coli (a bacterium) and Halobacterium salinarum (an archaeon). The models reveal how the genome-wide distributions of cis-acting gene regulatory elements and the conditional influences of transcription factors at each of those elements encode programs for eliciting a wide array of environment-specific responses. We demonstrate how these programs partition transcriptional regulation of genes within regulons and operons to re-organize gene-gene functional associations in each environment. The models capture fitness-relevant co-regulation by different transcriptional control mechanisms acting across the entire genome, to define a generalized, system-level organizing principle for prokaryotic gene regulatory networks that goes well beyond existing paradigms of gene regulation. An online resource (http://egrin2.systemsbiology.net) has been developed to facilitate multiscale exploration of conditional gene regulation in the two prokaryotes. © 2014 The Authors. Published under the terms of the CC BY 4.0 license.

Ribosomal RNA and ribosomal proteins in corynebacteria.

PubMed

Martín, Juan F; Barreiro, Carlos; González-Lavado, Eva; Barriuso, Mónica

2003-09-04

Ribosomal RNAs (rRNAs) (16S, 23S, 5S) encoded by the rrn operons and ribosomal proteins play a very important role in the formation of ribosomes and in the control of translation. Five copies of the rrn operon were reported by hybridization studies in Brevibacterium (Corynebacterium) lactofermentum but the genome sequence of Corynebacterium glutamicum provided evidence for six rrn copies. All six copies of the C. glutamicum 16S rRNA have a size of 1523 bp and each of the six copies of the 5S contain 120 bp whereas size differences are found between the six copies of the 23S rRNA. The anti-Shine-Dalgarno sequence at the 3'-end of the 16S rRNA was 5'-CCUCCUUUC-3'. Each rrn operon is transcribed as a large precursor rRNA (pre-rRNA) that is processed by RNaseIII and other RNases at specific cleavage boxes that have been identified in the C. glutamicum pre-rRNA. A secondary structure of the C. glutamicum 16S rRNA is proposed. The 16S rRNA sequence has been used as a molecular evolution clock allowing the deduction of a phylogenetic tree of all Corynebacterium species. In C. glutamicum, there are 11 ribosomal protein gene clusters encoding 42 ribosomal proteins. The organization of some of the ribosomal protein gene cluster is identical to that of Escherichia coli whereas in other clusters the organization of the genes is rather different. Some specific ribosomal protein genes are located in a different cluster in C. glutamicum when compared with E. coli, indicating that the control of expression of these genes is different in E. coli and C. glutamicum.

Genetic organization and regulation of a meta cleavage pathway for catechols produced from catabolism of toluene, benzene, phenol, and cresols by Pseudomonas pickettii PKO1.

PubMed Central

Kukor, J J; Olsen, R H

1991-01-01

Plasmid pRO1957 contains a 26.5-kb BamHI restriction endonuclease-cleaved DNA fragment cloned from the chromosome of Pseudomonas pickettii PKO1 that allows P. aeruginosa PAO1c to grow on toluene, benzene, phenol, or m-cresol as the sole carbon source. The genes encoding enzymes for meta cleavage of catechol or 3-methylcatechol, derived from catabolism of these substrates, were subcloned from pRO1957 and were shown to be organized into a single operon with the promoter proximal to tbuE. Deletion and analysis of subclones demonstrated that the order of genes in the meta cleavage operon was tbuEFGKIHJ, which encoded catechol 2,3-dioxygenase, 2-hydroxymuconate semialdehyde hydrolase, 2-hydroxymuconate semialdehyde dehydrogenase, 4-hydroxy-2-oxovalerate aldolase, 4-oxalocrotonate decarboxylase, 4-oxalocrotonate isomerase, and 2-hydroxypent-2,4-dienoate hydratase, respectively. The regulatory gene for the tbuEFGKIHJ operon, designated tbuS, was subcloned into vector plasmid pRO2317 from pRO1957 as a 1.3-kb PstI fragment, designated pRO2345. When tbuS was not present, meta pathway enzyme expression was partially derepressed, but these activity levels could not be fully induced. However, when tbuS was present in trans with tbuEFGKIHJ, meta pathway enzymes were repressed in the absence of an effector and were fully induced when an effector was present. This behavior suggests that the gene product of tbuS acts as both a repressor and an activator. Phenol and m-cresol were inducers of meta pathway enzymatic activity. Catechol, 3-methylcatechol, 4-methylcatechol, o-cresol, and p-cresol were not inducers but could be metabolized by cells previously induced by phenol or m-cresol. PMID:1856161

Control of copper resistance and inorganic sulfur metabolism by paralogous regulators in Staphylococcus aureus.

PubMed

Grossoehme, Nicholas; Kehl-Fie, Thomas E; Ma, Zhen; Adams, Keith W; Cowart, Darin M; Scott, Robert A; Skaar, Eric P; Giedroc, David P

2011-04-15

All strains of Staphylococcus aureus encode a putative copper-sensitive operon repressor (CsoR) and one other CsoR-like protein of unknown function. We show here that NWMN_1991 encodes a bona fide Cu(I)-inducible CsoR of a genetically unlinked copA-copZ copper resistance operon in S. aureus strain Newman. In contrast, an unannotated open reading frame found between NWMN_0027 and NWMN_0026 (denoted NWMN_0026.5) encodes a CsoR-like regulator that represses expression of adjacent genes by binding specifically to a pair of canonical operator sites positioned in the NWMN_0027-0026.5 intergenic region. Inspection of these regulated genes suggests a role in assimilation of inorganic sulfur from thiosulfate and vectorial sulfur transfer, and we designate NWMN_0026.5 as CstR (CsoR-like sulfur transferase repressor). Expression analysis demonstrates that CsoR and CstR control their respective regulons in response to distinct stimuli with no overlap in vivo. Unlike CsoR, CstR does not form a stable complex with Cu(I); operator binding is instead inhibited by oxidation of the intersubunit cysteine pair to a mixture of disulfide and trisulfide linkages by a likely metabolite of thiosulfate assimilation, sulfite. CsoR is unreactive toward sulfite under the same conditions. We conclude that CsoR and CstR are paralogs in S. aureus that function in the same cytoplasm to control distinct physiological processes.

Control of Copper Resistance and Inorganic Sulfur Metabolism by Paralogous Regulators in Staphylococcus aureus*

PubMed Central

Grossoehme, Nicholas; Kehl-Fie, Thomas E.; Ma, Zhen; Adams, Keith W.; Cowart, Darin M.; Scott, Robert A.; Skaar, Eric P.; Giedroc, David P.

2011-01-01

All strains of Staphylococcus aureus encode a putative copper-sensitive operon repressor (CsoR) and one other CsoR-like protein of unknown function. We show here that NWMN_1991 encodes a bona fide Cu(I)-inducible CsoR of a genetically unlinked copA-copZ copper resistance operon in S. aureus strain Newman. In contrast, an unannotated open reading frame found between NWMN_0027 and NWMN_0026 (denoted NWMN_0026.5) encodes a CsoR-like regulator that represses expression of adjacent genes by binding specifically to a pair of canonical operator sites positioned in the NWMN_0027–0026.5 intergenic region. Inspection of these regulated genes suggests a role in assimilation of inorganic sulfur from thiosulfate and vectorial sulfur transfer, and we designate NWMN_0026.5 as CstR (CsoR-like sulfur transferase repressor). Expression analysis demonstrates that CsoR and CstR control their respective regulons in response to distinct stimuli with no overlap in vivo. Unlike CsoR, CstR does not form a stable complex with Cu(I); operator binding is instead inhibited by oxidation of the intersubunit cysteine pair to a mixture of disulfide and trisulfide linkages by a likely metabolite of thiosulfate assimilation, sulfite. CsoR is unreactive toward sulfite under the same conditions. We conclude that CsoR and CstR are paralogs in S. aureus that function in the same cytoplasm to control distinct physiological processes. PMID:21339296

The pyrimidine nucleotide biosynthetic pathway modulates production of biofilm determinants in Escherichia coli.

PubMed

Garavaglia, Marco; Rossi, Elio; Landini, Paolo

2012-01-01

Bacteria are often found in multicellular communities known as biofilms, which constitute a resistance form against environmental stresses. Extracellular adhesion and cell aggregation factors, responsible for bacterial biofilm formation and maintenance, are tightly regulated in response to physiological and environmental cues. We show that, in Escherichia coli, inactivation of genes belonging to the de novo uridine monophosphate (UMP) biosynthetic pathway impairs production of curli fibers and cellulose, important components of the bacterial biofilm matrix, by inhibiting transcription of the csgDEFG operon, thus preventing production of the biofilm master regulator CsgD protein. Supplementing growth media with exogenous uracil, which can be converted to UMP through the pyrimidine nucleotide salvage pathway, restores csgDEFG transcription and curli production. In addition, however, exogenous uracil triggers cellulose production, particularly in strains defective in either carB or pyrB genes, which encode enzymes catalyzing the first steps of de novo UMP biosynthesis. Our results indicate the existence of tight and complex links between pyrimidine metabolism and curli/cellulose production: transcription of the csgDEFG operon responds to pyrimidine nucleotide availability, while cellulose production is triggered by exogenous uracil in the absence of active de novo UMP biosynthesis. We speculate that perturbations in the UMP biosynthetic pathways allow the bacterial cell to sense signals such as starvation, nucleic acids degradation, and availability of exogenous pyrimidines, and to adapt the production of the extracellular matrix to the changing environmental conditions.

Chromosomal Rearrangements in Salmonella enterica Serotype Typhi Affecting Molecular Typing in Outbreak Investigations

PubMed Central

Echeita, M. A.; Usera, M. A.

1998-01-01

Salmonella enterica serotype Typhi strains belonging to eight different outbreaks of typhoid fever that occurred in Spain between 1989 and 1994 were analyzed by ribotyping and pulsed-field gel electrophoresis. For three outbreaks, two different patterns were detected for each outbreak. The partial digestion analysis by the intron-encoded endonuclease I-CeuI of the two different strains from each outbreak provided an excellent tool for examining the organization of the genomes of epidemiologically related strains. S. enterica serotype Typhi seems to be more susceptible than other serotypes to genetic rearrangements produced by homologous recombinations between rrn operons; these rearrangements do not substantially alter the stability or survival of the bacterium. We conclude that genetic rearrangements can occur during the emergence of an outbreak. PMID:9650981

CopM is a novel copper-binding protein involved in copper resistance in Synechocystis sp. PCC 6803.

PubMed

Giner-Lamia, Joaquín; López-Maury, Luis; Florencio, Francisco J

2015-02-01

Copper resistance system in the cyanobacterium Synechocystis sp. PCC 6803 comprises two operons, copMRS and copBAC, which are expressed in response to copper in the media. copBAC codes for a heavy-metal efflux-resistance nodulation and division (HME-RND) system, while copMRS codes for a protein of unknown function, CopM, and a two-component system CopRS, which controls the expression of these two operons. Here, we report that CopM is a periplasmic protein able to bind Cu(I) with high affinity (KD ~3 × 10(-16) ). Mutants lacking copM showed a sensitive copper phenotype similar to mutants affected in copB, but lower than mutants of the two-component system CopRS, suggesting that CopBAC and CopM constitute two independent resistance mechanisms. Moreover, constitutive expression of copM is able to partially suppress the copper sensitivity of the copR mutant strain, pointing out that CopM per se is able to confer copper resistance. Furthermore, constitutive expression of copM was able to reduce total cellular copper content of the copR mutant to the levels determined in the wild-type (WT) strain. Finally, CopM was localized not only in the periplasm but also in the extracellular space, suggesting that CopM can also prevent copper accumulation probably by direct copper binding outside the cell. © 2014 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

CopM is a novel copper-binding protein involved in copper resistance in Synechocystis sp. PCC 6803

PubMed Central

Giner-Lamia, Joaquín; López-Maury, Luis; Florencio, Francisco J

2015-01-01

Copper resistance system in the cyanobacterium Synechocystis sp. PCC 6803 comprises two operons, copMRS and copBAC, which are expressed in response to copper in the media. copBAC codes for a heavy-metal efflux–resistance nodulation and division (HME-RND) system, while copMRS codes for a protein of unknown function, CopM, and a two-component system CopRS, which controls the expression of these two operons. Here, we report that CopM is a periplasmic protein able to bind Cu(I) with high affinity (KD ∼3 × 10−16). Mutants lacking copM showed a sensitive copper phenotype similar to mutants affected in copB, but lower than mutants of the two-component system CopRS, suggesting that CopBAC and CopM constitute two independent resistance mechanisms. Moreover, constitutive expression of copM is able to partially suppress the copper sensitivity of the copR mutant strain, pointing out that CopM per se is able to confer copper resistance. Furthermore, constitutive expression of copM was able to reduce total cellular copper content of the copR mutant to the levels determined in the wild-type (WT) strain. Finally, CopM was localized not only in the periplasm but also in the extracellular space, suggesting that CopM can also prevent copper accumulation probably by direct copper binding outside the cell. PMID:25545960

Pseudomonas fluorescens ATCC 13525 Containing an Artificial Oxalate Operon and Vitreoscilla Hemoglobin Secretes Oxalic Acid and Solubilizes Rock Phosphate in Acidic Alfisols

PubMed Central

Archana, G.; Naresh Kumar, G.

2014-01-01

Oxalate secretion was achieved in Pseudomonas fluorescens ATCC 13525 by incorporation of genes encoding Aspergillus niger oxaloacetate acetyl hydrolase (oah), Fomitopsis plaustris oxalate transporter (FpOAR) and Vitreoscilla hemoglobin (vgb) in various combinations. Pf (pKCN2) transformant containing oah alone accumulated 19 mM oxalic acid intracellularly but secreted 1.2 mM. However, in the presence of an artificial oxalate operon containing oah and FpOAR genes in plasmid pKCN4, Pf (pKCN4) secreted 13.6 mM oxalate in the medium while 3.6 mM remained inside. This transformant solubilized 509 μM of phosphorus from rock phosphate in alfisol which is 4.5 fold higher than the Pf (pKCN2) transformant. Genomic integrants of P. fluorescens (Pf int1 and Pf int2) containing artificial oxalate operon (plac-FpOAR-oah) and artificial oxalate gene cluster (plac-FpOAR-oah, vgb, egfp) secreted 4.8 mM and 5.4 mM oxalic acid, released 329 μM and 351 μM P, respectively, in alfisol. The integrants showed enhanced root colonization, improved growth and increased P content of Vigna radiata plants. This study demonstrates oxalic acid secretion in P. fluorescens by incorporation of an artificial operon constituted of genes for oxalate synthesis and transport, which imparts mineral phosphate solubilizing ability to the organism leading to enhanced growth and P content of V. radiata in alfisol soil. PMID:24705024

Cloning and identification of Group 1 mrp operon encoding a novel monovalent cation/proton antiporter system from the moderate halophile Halomonas zhaodongensis.

PubMed

Meng, Lin; Hong, Shan; Liu, Henan; Huang, Haipeng; Sun, Hao; Xu, Tong; Jiang, Juquan

2014-11-01

The novel species Halomonas zhaodongensis NEAU-ST10-25(T) recently identified by our group is a moderate halophile which can grow at the range of 0-2.5 M NaCl (optimum 0.5 M) and pH 6-12 (optimum pH 9). To explore its halo-alkaline tolerant mechanism, genomic DNA was screened from NEAU-ST10-25(T) in this study for Na(+)(Li(+))/H(+) antiporter genes by selection in Escherichia coli KNabc lacking three major Na(+)(Li(+))/H(+) antiporters. One mrp operon could confer tolerance of E. coli KNabc to 0.8 M NaCl and 100 mM LiCl, and an alkaline pH. This operon was previously mainly designated mrp (also mnh, pha or sha) due to its multiple resistance and pH-related activity. Here, we will also use mrp to designate the homolog from H. zhaodongensis (Hz_mrp). Sequence analysis and protein alignment showed that Hz_mrp should belong to Group 1 mrp operons. Further phylogenetic analysis reveals that Hz_Mrp system should represent a novel sub-class of Group 1 Mrp systems. This was confirmed by a significant difference in pH-dependent activity profile or the specificity and affinity for the transported monovalent cations between Hz_Mrp system and all the known Mrp systems. Therefore, we propose that Hz_Mrp should be categorized as a novel Group 1 Mrp system.

Effect of metal sulfide pulp density on gene expression of electron transporters in Acidithiobacillus sp. FJ2.

PubMed

Fatemi, Faezeh; Miri, Saba; Jahani, Samaneh

2017-05-01

In Acidithiobacillus ferrooxidans, one of the most important bioleaching bacterial species, the proteins encoded by the rus operon are involved in the electron transfer from Fe 2+ to O 2 . To obtain further knowledge about the mechanism(s) involved in the adaptive responses of the bacteria to growth on the different uranium ore pulp densities, we analyzed the expression of the four genes from the rus operon by real-time PCR, when Acidithiobacillus sp. FJ2 was grown in the presence of different uranium concentrations. The uranium bioleaching results showed the inhibitory effects of the metal pulp densities on the oxidation activity of the bacteria which can affect Eh, pH, Fe oxidation and uranium extractions. Gene expression analysis indicated that Acidithiobacillus sp. FJ2 tries to survive in the stress with increasing in the expression levels of cyc2, cyc1, rus and coxB, but the metal toxicity has a negative effect on the gene expression in different pulp densities. These results indicated that Acidithiobacillus sp. FJ2 could leach the uranium even in high pulp density (50%) by modulation in rus operon gene responses.

Restoration of GABA production machinery in Lactobacillus brevis by accessible carbohydrates, anaerobiosis and early acidification.

PubMed

Wu, Qinglong; Shah, Nagendra P

2018-02-01

Lactobacillus brevis is an efficient cell factory for producing bioactive γ-aminobutyric acid (GABA) by its gad operon-encoded glutamic acid decarboxylase (GAD) system. However, little mechanistic insights have been reported on the effects of carbohydrate, oxygen and early acidification on GABA production machinery in Lb. brevis. In the present study, GABA production from Lb. brevis was enhanced by accessible carbohydrates. Fast growth of this organism was stimulated by maltose and xylose. However, its GABA production was highly suppressed by oxygen exposure, but was fully restored by anaerobiosis that up-regulated the expression of gad operon in Lb. brevis cells. Although the level of cytosolic acidity was suitable for the functioning of GadA and GadB, early acidification of the medium (ipH 5 and ipH 4) restored GABA synthesis strictly in aerated cells of Lb. brevis because the expression of gad operon was not up-regulated in them. We conclude that GABA production machinery in Lb. brevis could be restored by accessible carbohydrates, anaerobiosis and early acidification. This will be of interest for controlling fermentation for synthesis of GABA and manufacturing GABA-rich fermented vegetables. Copyright © 2017. Published by Elsevier Ltd.

Two groups of phenylalanine biosynthetic operon leader peptides genes: a high level of apparently incidental frameshifting in decoding Escherichia coli pheL

PubMed Central

Gurvich, Olga L.; Näsvall, S. Joakim; Baranov, Pavel V.; Björk, Glenn R.; Atkins, John F.

2011-01-01

The bacterial pheL gene encodes the leader peptide for the phenylalanine biosynthetic operon. Translation of pheL mRNA controls transcription attenuation and, consequently, expression of the downstream pheA gene. Fifty-three unique pheL genes have been identified in sequenced genomes of the gamma subdivision. There are two groups of pheL genes, both of which are short and contain a run(s) of phenylalanine codons at an internal position. One group is somewhat diverse and features different termination and 5′-flanking codons. The other group, mostly restricted to Enterobacteria and including Escherichia coli pheL, has a conserved nucleotide sequence that ends with UUC_CCC_UGA. When these three codons in E. coli pheL mRNA are in the ribosomal E-, P- and A-sites, there is an unusually high level, 15%, of +1 ribosomal frameshifting due to features of the nascent peptide sequence that include the penultimate phenylalanine. This level increases to 60% with a natural, heterologous, nascent peptide stimulator. Nevertheless, studies with different tRNAPro mutants in Salmonella enterica suggest that frameshifting at the end of pheL does not influence expression of the downstream pheA. This finding of incidental, rather than utilized, frameshifting is cautionary for other studies of programmed frameshifting. PMID:21177642

Two Paralogous Families of a Two-Gene Subtilisin Operon Are Widely Distributed in Oral Treponemes

PubMed Central

Correia, Frederick F.; Plummer, Alvin R.; Ellen, Richard P.; Wyss, Chris; Boches, Susan K.; Galvin, Jamie L.; Paster, Bruce J.; Dewhirst, Floyd E.

2003-01-01

Certain oral treponemes express a highly proteolytic phenotype and have been associated with periodontal diseases. The periodontal pathogen Treponema denticola produces dentilisin, a serine protease of the subtilisin family. The two-gene operon prcA-prtP is required for expression of active dentilisin (PrtP), a putative lipoprotein attached to the treponeme's outer membrane or sheath. The purpose of this study was to examine the diversity and structure of treponemal subtilisin-like proteases in order to better understand their distribution and function. The complete sequences of five prcA-prtP operons were determined for Treponema lecithinolyticum, “Treponema vincentii,” and two canine species. Partial operon sequences were obtained for T. socranskii subsp. 04 as well as 450- to 1,000-base fragments of prtP genes from four additional treponeme strains. Phylogenetic analysis demonstrated that the sequences fall into two paralogous families. The first family includes the sequence from T. denticola. Treponemes possessing this operon family express chymotrypsin-like protease activity and can cleave the substrate N-succinyl-alanyl-alanyl-prolyl-phenylalanine-p-nitroanilide (SAAPFNA). Treponemes possessing the second paralog family do not possess chymotrypsin-like activity or cleave SAAPFNA. Despite examination of a range of protein and peptide substrates, the specificity of the second protease family remains unknown. Each of the fully sequenced prcA and prtP genes contains a 5′ hydrophobic leader sequence with a treponeme lipobox. The two paralogous families of treponeme subtilisins represent a new subgroup within the subtilisin family of proteases and are the only subtilisin lipoprotein family. The present study demonstrated that the subtilisin paralogs comprising a two-gene operon are widely distributed among treponemes. PMID:14617650

Antimicrobial Tolerance of Pseudomonas aeruginosa Biofilms Is Activated during an Early Developmental Stage and Requires the Two-Component Hybrid SagS

PubMed Central

Gupta, Kajal; Marques, Cláudia N. H.; Petrova, Olga E.

2013-01-01

A hallmark characteristic of biofilms is their extraordinary tolerance to antimicrobial agents. While multiple factors are thought to contribute to the high level of antimicrobial tolerance of biofilms, little is known about the timing of induction of biofilm tolerance. Here, we asked when over the course of their development do biofilms gain their tolerance to antimicrobial agents? We demonstrate that in Pseudomonas aeruginosa, biofilm tolerance is linked to biofilm development, with transition to the irreversible attachment stage regulated by the two-component hybrid SagS, marking the timing when biofilms switch to the high-level tolerance phenotype. Inactivation of sagS rendered biofilms but not planktonic cells more susceptible to tobramycin, norfloxacin, and hydrogen peroxide. Moreover, inactivation of sagS also eliminated the recalcitrance of biofilms to killing by bactericidal antimicrobial agents, a phenotype comparable to that observed upon inactivation of brlR, which encodes a MerR-like transcriptional regulator required for biofilm tolerance. Multicopy expression of brlR in a ΔsagS mutant restored biofilm resistance and recalcitrance to killing by bactericidal antibiotics to wild-type levels. In contrast, expression of sagS did not restore the susceptibility phenotype of ΔbrlR mutant biofilms to wild-type levels, indicating that BrlR functions downstream of SagS. Inactivation of sagS correlated with reduced BrlR levels in biofilms, with the produced BrlR being impaired in binding to the previously described BrlR-activated promoters of the two multidrug efflux pump operons mexAB-oprM and mexEF-oprN. Our findings demonstrate that biofilm tolerance is linked to early biofilm development and SagS, with SagS contributing indirectly to BrlR activation. PMID:23995639

Analysis of Two-Component Systems in Group B Streptococcus Shows That RgfAC and the Novel FspSR Modulate Virulence and Bacterial Fitness

PubMed Central

Faralla, Cristina; Metruccio, Matteo M.; De Chiara, Matteo; Mu, Rong; Patras, Kathryn A.; Muzzi, Alessandro; Grandi, Guido; Margarit, Immaculada; Doran, Kelly S.

2014-01-01

ABSTRACT Group B Streptococcus (GBS), in the transition from commensal organisms to pathogens, will encounter diverse host environments and, thus, require coordinated control of the transcriptional responses to these changes. This work was aimed at better understanding the role of two-component signal transduction systems (TCS) in GBS pathophysiology through a systematic screening procedure. We first performed a complete inventory and sensory mechanism classification of all putative GBS TCS by genomic analysis. Five TCS were further investigated by the generation of knockout strains, and in vitro transcriptome analysis identified genes regulated by these systems, ranging from 0.1% to 3% of the genome. Interestingly, two sugar phosphotransferase systems appeared to be differentially regulated in the TCS-16 knockout strain (TCS loci were numbered in order of their appearance on the chromosome), suggesting an involvement in monitoring carbon source availability. High-throughput analysis of bacterial growth on different carbon sources showed that TCS-16 was necessary for the growth of GBS on fructose-6-phosphate. Additional transcriptional analysis provided further evidence for a stimulus-response circuit where extracellular fructose-6-phosphate leads to autoinduction of TCS-16, with concomitant dramatic upregulation of the adjacent operon, which encodes a phosphotransferase system. The TCS-16-deficient strain exhibited decreased persistence in a model of vaginal colonization. All mutant strains were also characterized in a murine model of systemic infection, and inactivation of TCS-17 (also known as RgfAC) resulted in hypervirulence. Our data suggest a role for the previously unknown TCS-16, here named FspSR, in bacterial fitness and carbon metabolism during host colonization, and the data also provide experimental evidence for TCS-17/RgfAC involvement in virulence. PMID:24846378

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

Comparative genomic analysis and characterization of incompatibility group FIB plasmid encoded virulence factors of Salmonella enterica isolated from food sources.

PubMed

Khajanchi, Bijay K; Hasan, Nur A; Choi, Seon Young; Han, Jing; Zhao, Shaohua; Colwell, Rita R; Cerniglia, Carl E; Foley, Steven L

2017-08-02

The degree to which the chromosomal mediated iron acquisition system contributes to virulence of many bacterial pathogens is well defined. However, the functional roles of plasmid encoded iron acquisition systems, specifically Sit and aerobactin, have yet to be determined for Salmonella spp. In a recent study, Salmonella enterica strains isolated from different food sources were sequenced on the Illumina MiSeq platform and found to harbor the incompatibility group (Inc) FIB plasmid. In this study, we examined sequence diversity and the contribution of factors encoded on the IncFIB plasmid to the virulence of S. enterica. Whole genome sequences of seven S. enterica isolates were compared to genomes of serovars of S. enterica isolated from food, animal, and human sources. SeqSero analysis predicted that six strains were serovar Typhimurium and one was Heidelberg. Among the S. Typhimurium strains, single nucleotide polymorphism (SNP)-based phylogenetic analyses revealed that five of the isolates clustered as a single monophyletic S. Typhimurium subclade, while one of the other strains branched with S. Typhimurium from a bovine source. DNA sequence based phylogenetic diversity analyses showed that the IncFIB plasmid-encoded Sit and aerobactin iron acquisition systems are conserved among bacterial species including S. enterica. The IncFIB plasmid was transferred to an IncFIB plasmid deficient strain of S. enterica by conjugation. The transconjugant SE819::IncFIB persisted in human intestinal epithelial (Caco-2) cells at a higher rate than the recipient SE819. Genes of the Sit and aerobactin operons in the IncFIB plasmid were differentially expressed in iron-rich and iron-depleted growth media. Minimal sequence diversity was detected in the Sit and aerobactin operons in the IncFIB plasmids present among different bacterial species, including foodborne Salmonella strains. IncFIB plasmid encoded factors play a role during infection under low-iron conditions in host cells.

Hydrogen Sulfide Sensing through Reactive Sulfur Species (RSS) and Nitroxyl (HNO) in Enterococcus faecalis.

PubMed

Shen, Jiangchuan; Walsh, Brenna J C; Flores-Mireles, Ana Lidia; Peng, Hui; Zhang, Yifan; Zhang, Yixiang; Trinidad, Jonathan C; Hultgren, Scott J; Giedroc, David P

2018-05-17

Recent studies of hydrogen sulfide (H 2 S) signaling implicate low molecular weight (LMW) thiol persulfides and other reactive sulfur species (RSS) as signaling effectors. Here, we show that a CstR protein from the human pathogen Enterococcus faecalis ( E. faecalis), previously identified in Staphylococcus aureus ( S. aureus), is an RSS-sensing repressor that transcriptionally regulates a cst-like operon in response to both exogenous sulfide stress and Angeli's salt, a precursor of nitroxyl (HNO). E. faecalis CstR reacts with coenzyme A persulfide (CoASSH) to form interprotomer disulfide and trisulfide bridges between C32 and C61', which negatively regulate DNA binding to a consensus CstR DNA operator. A Δ cstR strain exhibits deficiency in catheter colonization in a catheter-associated urinary tract infection (CAUTI) mouse model, suggesting sulfide regulation and homeostasis is critical for pathogenicity. Cellular polysulfide metabolite profiling of sodium sulfide-stressed E. faecalis confirms an increase in both inorganic polysulfides and LMW thiols and persulfides sensed by CstR. The cst-like operon encodes two authentic thiosulfate sulfurtransferases and an enzyme we characterize here as an NADH and FAD-dependent coenzyme A (CoA) persulfide reductase (CoAPR) that harbors an N-terminal CoA disulfide reductase (CDR) domain and a C-terminal rhodanese homology domain (RHD). Both cysteines in the CDR (C42) and RHD (C508) domains are required for CoAPR activity and complementation of a sulfide-induced growth phenotype of a S. aureus strain lacking cstB, encoding a nonheme Fe II persulfide dioxygenase. We propose that S. aureus CstB and E. faecalis CoAPR employ orthogonal chemistries to lower CoASSH that accumulates under conditions of cellular sulfide toxicity and signaling.

The Arsenic Detoxification System in Corynebacteria: Basis and Application for Bioremediation and Redox Control.

PubMed

Mateos, Luis M; Villadangos, Almudena F; de la Rubia, Alfonso G; Mourenza, Alvaro; Marcos-Pascual, Laura; Letek, Michal; Pedre, Brandán; Messens, Joris; Gil, Jose A

2017-01-01

Arsenic (As) is widespread in the environment and highly toxic. It has been released by volcanic and anthropogenic activities and causes serious health problems worldwide. To survive arsenic-rich environments, soil and saprophytic microorganisms have developed molecular detoxification mechanisms to survive arsenic-rich environments, mainly by the enzymatic conversion of inorganic arsenate (As V ) to arsenite (As III ) by arsenate reductases, which is then extruded by arsenite permeases. One of these Gram-positive bacteria, Corynebacterium glutamicum, the workhorse of biotechnological research, is also resistant to arsenic. To sanitize contaminated soils and waters, C. glutamicum strains were modified to work as arsenic "biocontainers." Two chromosomally encoded ars operons (ars1 and ars2) are responsible for As resistance. The genes within these operons encode for metalloregulatory proteins (ArsR1/R2), arsenite permeases (Acr3-1/-2), and arsenate reductases (ArsC1/C2/C1'). ArsC1/C2 arsenate reductases are coupled to the low molecular weight thiol mycothiol (MSH) and to the recently discovered mycoredoxin-1 (Mrx-1) present in most Actinobacteria. This MSH/Mrx-1 redox system protects cells against different forms of stress, including reactive oxygen species (ROS), metals, and antibiotics. ROS can modify functional sulfur cysteines by oxidizing the thiol (-SH) to a sulfenic acid (-SOH). These oxidation-sensitive protein cysteine thiols are redox regulated by the MSH/Mrx-1 couple in Corynebacterium and Mycobacterium. In summary, the molecular mechanisms involved in arsenic resistance system in C. glutamicum have paved the way for understanding the cellular response against oxidative stress in Actinobacteria. Copyright © 2017 Elsevier Inc. All rights reserved.

In Silico Analysis of Usher Encoding Genes in Klebsiella pneumoniae and Characterization of Their Role in Adhesion and Colonization

PubMed Central

Khater, Fida; Balestrino, Damien; Charbonnel, Nicolas; Dufayard, Jean François; Brisse, Sylvain; Forestier, Christiane

2015-01-01

Chaperone/usher (CU) assembly pathway is used by a wide range of Enterobacteriaceae to assemble adhesive surface structures called pili or fimbriae that play a role in bacteria-host cell interactions. In silico analysis revealed that the genome of Klebsiella pneumoniae LM21 harbors eight chromosomal CU loci belonging to γκп and ϭ clusters. Of these, only two correspond to previously described operons, namely type 1 and type 3-encoding operons. Isogenic usher deletion mutants of K. pneumoniae LM21 were constructed for each locus and their role in adhesion to animal (Intestine 407) and plant (Arabidopsis thaliana) cells, biofilm formation and murine intestinal colonization was investigated. Type 3 pili usher deleted mutant was impaired in all assays, whereas type 1 pili usher deleted mutant only showed attenuation in adhesion to plant cells and in intestinal colonization. The LM21ΔkpjC mutant was impaired in its capacity to adhere to Arabidopsis cells and to colonize the murine intestine, either alone or in co-inoculation experiments. Deletion of LM21kpgC induced a significant decrease in biofilm formation, in adhesion to animal cells and in colonization of the mice intestine. The LM21∆kpaC and LM21∆kpeC mutants were only attenuated in biofilm formation and the adhesion abilities to Arabidopsis cells, respectively. No clear in vitro or in vivo effect was observed for LM21∆kpbC and LM21∆kpdC mutants. The multiplicity of CU loci in K. pneumoniae genome and their specific adhesion pattern probably reflect the ability of the bacteria to adhere to different substrates in its diverse ecological niches. PMID:25751658

An operon encoding three glycolytic enzymes in Lactobacillus delbrueckii subsp. bulgaricus: glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase and triosephosphate isomerase.

PubMed

Branny, P; de la Torre, F; Garel, J R

1998-04-01

The structural genes gap, pgk and tpi encoding three glycolytic enzymes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 3-phosphoglycerate kinase (PGK) and triosephosphate isomerase (TPI), respectively, have been cloned and sequenced from Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus). The genes were isolated after screening genomic sublibraries with specific gap and pgk probes obtained by PCR amplification of chromosomal DNA with degenerate primers corresponding to amino acid sequences highly conserved in GAPDHs and PGKs. Nucleotide sequencing revealed that the three genes were organized in the order gap-pgk-tpi. The translation start codons of the three genes were identified by alignment of the N-terminal sequences. These genes predicted polypeptide chains of 338, 403 and 252 amino acids for GAPDH, PGK and TPI, respectively, and they were separated by 96 bp between gap and pgk, and by only 18 bp between pgk and tpi. The codon usage in gap, pgk, tpi and three other glycolytic genes from L. bulgaricus differed, noticeably from that in other chromosomal genes. The site of transcriptional initiation was located by primer extension, and a probable promoter was identified for the gap-pgk-tpi operon. Northern hybridization of total RNA with specific probes showed two transcripts, an mRNA of 1.4 kb corresponding to the gap gene, and a less abundant mRNA of 3.4 kb corresponding to the gap-pgk-tpi cluster. The absence of a visible terminator in the 3'-end of the shorter transcript and the location of this 3'-end inside the pgk gene indicated that this shorter transcript was produced by degradation of the longer one, rather than by an early termination of transcription after the gap gene.

Identification of a Gene Cluster Enabling Lactobacillus casei BL23 To Utilize myo-Inositol▿ †

PubMed Central

Yebra, María Jesús; Zúñiga, Manuel; Beaufils, Sophie; Pérez-Martínez, Gaspar; Deutscher, Josef; Monedero, Vicente

2007-01-01

Genome analysis of Lactobacillus casei BL23 revealed that, compared to L. casei ATCC 334, it carries a 12.8-kb DNA insertion containing genes involved in the catabolism of the cyclic polyol myo-inositol (MI). Indeed, L. casei ATCC 334 does not ferment MI, whereas strain BL23 is able to utilize this carbon source. The inserted DNA consists of an iolR gene encoding a DeoR family transcriptional repressor and a divergently transcribed iolTABCDG1G2EJK operon, encoding a complete MI catabolic pathway, in which the iolK gene probably codes for a malonate semialdehyde decarboxylase. The presence of iolK suggests that L. casei has two alternative pathways for the metabolism of malonic semialdehyde: (i) the classical MI catabolic pathway in which IolA (malonate semialdehyde dehydrogenase) catalyzes the formation of acetyl-coenzyme A from malonic semialdehyde and (ii) the conversion of malonic semialdehyde to acetaldehyde catalyzed by the product of iolK. The function of the iol genes was verified by the disruption of iolA, iolT, and iolD, which provided MI-negative strains. By contrast, the disruption of iolK resulted in a strain with no obvious defect in MI utilization. Transcriptional analyses conducted with different mutant strains showed that the iolTABCDG1G2EJK cluster is regulated by substrate-specific induction mediated by the inactivation of the transcriptional repressor IolR and by carbon catabolite repression mediated by the catabolite control protein A (CcpA). This is the first example of an operon for MI utilization in lactic acid bacteria and illustrates the versatility of carbohydrate utilization in L. casei BL23. PMID:17449687

Pseudomonas syringae pv. tomato DC3000 CmaL (PSPTO4723), a DUF1330 Family Member, Is Needed To Produce l-allo-Isoleucine, a Precursor for the Phytotoxin Coronatine

PubMed Central

Worley, Jay N.; Russell, Alistair B.; Wexler, Aaron G.; Bronstein, Philip A.; Kvitko, Brian H.; Krasnoff, Stuart B.; Munkvold, Kathy R.; Swingle, Bryan

2013-01-01

Pseudomonas syringae pv. tomato DC3000 produces the phytotoxin coronatine, a major determinant of the leaf chlorosis associated with DC3000 pathogenesis. The DC3000 PSPTO4723 (cmaL) gene is located in a genomic region encoding type III effectors; however, it promotes chlorosis in the model plant Nicotiana benthamiana in a manner independent of type III secretion. Coronatine is produced by the ligation of two moieties, coronafacic acid (CFA) and coronamic acid (CMA), which are produced by biosynthetic pathways encoded in separate operons. Cross-feeding experiments, performed in N. benthamiana with cfa, cma, and cmaL mutants, implicate CmaL in CMA production. Furthermore, analysis of bacterial supernatants under coronatine-inducing conditions revealed that mutants lacking either the cma operon or cmaL accumulate CFA rather than coronatine, supporting a role for CmaL in the regulation or biosynthesis of CMA. CmaL does not appear to regulate CMA production, since the expression of proteins with known roles in CMA production is unaltered in cmaL mutants. Rather, CmaL is needed for the first step in CMA synthesis, as evidenced by the fact that wild-type levels of coronatine production are restored to a ΔcmaL mutant when it is supplemented with 50 μg/ml l-allo-isoleucine, the starting unit for CMA production. cmaL is found in all other sequenced P. syringae strains with coronatine biosynthesis genes. This characterization of CmaL identifies a critical missing factor in coronatine production and provides a foundation for further investigation of a member of the widespread DUF1330 protein family. PMID:23144243

The Global Redox Responding RegB/RegA Signal Transduction System Regulates the Genes Involved in Ferrous Iron and Inorganic Sulfur Compound Oxidation of the Acidophilic Acidithiobacillus ferrooxidans.

PubMed

Moinier, Danielle; Byrne, Deborah; Amouric, Agnès; Bonnefoy, Violaine

2017-01-01

The chemical attack of ore by ferric iron and/or sulfuric acid releases valuable metals. The products of these reactions are recycled by iron and sulfur oxidizing microorganisms. These acidophilic chemolithotrophic prokaryotes, among which Acidithiobacillus ferrooxidans , grow at the expense of the energy released from the oxidation of ferrous iron and/or inorganic sulfur compounds (ISCs). In At. ferrooxidans , it has been shown that the expression of the genes encoding the proteins involved in these respiratory pathways is dependent on the electron donor and that the genes involved in iron oxidation are expressed before those responsible for ISCs oxidation when both iron and sulfur are present. Since the redox potential increases during iron oxidation but remains stable during sulfur oxidation, we have put forward the hypothesis that the global redox responding two components system RegB/RegA is involved in this regulation. To understand the mechanism of this system and its role in the regulation of the aerobic respiratory pathways in At. ferrooxidans , the binding of different forms of RegA (DNA binding domain, wild-type, unphosphorylated and phosphorylated-like forms of RegA) on the regulatory region of different genes/operons involved in ferrous iron and ISC oxidation has been analyzed. We have shown that the four RegA forms are able to bind specifically the upstream region of these genes. Interestingly, the phosphorylation of RegA did not change its affinity for its cognate DNA. The transcriptional start site of these genes/operons has been determined. In most cases, the RegA binding site(s) was (were) located upstream from the -35 (or -24) box suggesting that RegA does not interfere with the RNA polymerase binding. Based on the results presented in this report, the role of the RegB/RegA system in the regulation of the ferrous iron and ISC oxidation pathways in At. ferrooxidans is discussed.

The Glucuronic Acid Utilization Gene Cluster from Bacillus stearothermophilus T-6

PubMed Central

Shulami, Smadar; Gat, Orit; Sonenshein, Abraham L.; Shoham, Yuval

1999-01-01

A λ-EMBL3 genomic library of Bacillus stearothermophilus T-6 was screened for hemicellulolytic activities, and five independent clones exhibiting β-xylosidase activity were isolated. The clones overlap each other and together represent a 23.5-kb chromosomal segment. The segment contains a cluster of xylan utilization genes, which are organized in at least three transcriptional units. These include the gene for the extracellular xylanase, xylanase T-6; part of an operon coding for an intracellular xylanase and a β-xylosidase; and a putative 15.5-kb-long transcriptional unit, consisting of 12 genes involved in the utilization of α-d-glucuronic acid (GlcUA). The first four genes in the potential GlcUA operon (orf1, -2, -3, and -4) code for a putative sugar transport system with characteristic components of the binding-protein-dependent transport systems. The most likely natural substrate for this transport system is aldotetraouronic acid [2-O-α-(4-O-methyl-α-d-glucuronosyl)-xylotriose] (MeGlcUAXyl3). The following two genes code for an intracellular α-glucuronidase (aguA) and a β-xylosidase (xynB). Five more genes (kdgK, kdgA, uxaC, uxuA, and uxuB) encode proteins that are homologous to enzymes involved in galacturonate and glucuronate catabolism. The gene cluster also includes a potential regulatory gene, uxuR, the product of which resembles repressors of the GntR family. The apparent transcriptional start point of the cluster was determined by primer extension analysis and is located 349 bp from the initial ATG codon. The potential operator site is a perfect 12-bp inverted repeat located downstream from the promoter between nucleotides +170 and +181. Gel retardation assays indicated that UxuR binds specifically to this sequence and that this binding is efficiently prevented in vitro by MeGlcUAXyl3, the most likely molecular inducer. PMID:10368143

Zymomonas pentose-sugar fermenting strains and uses thereof

DOEpatents

Zhang, Min [Lakewood, CO; Chou, Yat-Chen [Golden, CO; Howe, William [Golden, CO; Eddy, Christine [Golden, CO; Evans, Kent [Littleton, CO; Mohagheghi, Ali [Northglenn, CO

2007-05-29

Disclosed in the present invention is a Zymomonas integrant and derivatives of these integrants that posses the ability to ferment pentose into ethanol. The genetic sequences encoding for the pentose-fermenting enzymes are integrated into the Zymomonas in a two-integration event of homologous recombination and transposition. Each operon includes more than one pentose-reducing enzyme encoding sequence. The integrant in some embodiments includes enzyme sequences encoding xylose isomerase, xylulokinase, transketolase and transketolase. The Zymomonas integrants are highly stable, and retain activity for producing the pentose-fermenting enzyme for between 80 to 160 generations. The integrants are also resistant to acetate inhibition, as the integrants demonstrate efficient ethanol production even in the presence of 8 up to 16 grams acetate per liter media. These stably integrated sequences provide a unique Zymomonas that may then be used for the efficient conversion of pentose sugars (xylose, arabinose) to ethanol. Method of using the Zymomonas integrants and derivatives thereof in production of ethanol from cellulosic feedstock is also disclosed. The invention also provides a method for preparing a Zymomonas integrant as part of the present invention. The host Zymomonas strain found particularly useful in the creation of these compositions and methods is Zymomonas mobilis 31821.

Identification and Analysis of the Biosynthetic Gene Cluster Encoding the Thiopeptide Antibiotic Cyclothiazomycin in Streptomyces hygroscopicus 10-22▿ †

PubMed Central

Wang, Jiang; Yu, Yi; Tang, Kexuan; Liu, Wen; He, Xinyi; Huang, Xi; Deng, Zixin

2010-01-01

Thiopeptide antibiotics are an important class of natural products resulting from posttranslational modifications of ribosomally synthesized peptides. Cyclothiazomycin is a typical thiopeptide antibiotic that has a unique bridged macrocyclic structure derived from an 18-amino-acid structural peptide. Here we reported cloning, sequencing, and heterologous expression of the cyclothiazomycin biosynthetic gene cluster from Streptomyces hygroscopicus 10-22. Remarkably, successful heterologous expression of a 22.7-kb gene cluster in Streptomyces lividans 1326 suggested that there is a minimum set of 15 open reading frames that includes all of the functional genes required for cyclothiazomycin production. Six genes of these genes, cltBCDEFG flanking the structural gene cltA, were predicted to encode the enzymes required for the main framework of cyclothiazomycin, and two enzymes encoded by a putative operon, cltMN, were hypothesized to participate in the tailoring step to generate the tertiary thioether, leading to the final cyclization of the bridged macrocyclic structure. This rigorous bioinformatics analysis based on heterologous expression of cyclothiazomycin resulted in an ideal biosynthetic model for us to understand the biosynthesis of thiopeptides. PMID:20154110

Engineering a synthetic anaerobic respiration for reduction of xylose to xylitol using NADH output of glucose catabolism by Escherichia coli AI21.

PubMed

Iverson, Andrew; Garza, Erin; Manow, Ryan; Wang, Jinhua; Gao, Yuanyuan; Grayburn, Scott; Zhou, Shengde

2016-04-16

Anaerobic rather than aerobic fermentation is preferred for conversion of biomass derived sugars to high value redox-neutral and reduced commodities. This will likely result in a higher yield of substrate to product conversion and decrease production cost since substrate often accounts for a significant portion of the overall cost. To this goal, metabolic pathway engineering has been used to optimize substrate carbon flow to target products. This approach works well for the production of redox neutral products such as lactic acid from redox neutral sugars using the reducing power NADH (nicotinamide adenine dinucleotide, reduced) generated from glycolysis (2 NADH per glucose equivalent). Nevertheless, greater than two NADH per glucose catabolized is needed for the production of reduced products (such as xylitol) from redox neutral sugars by anaerobic fermentation. The Escherichia coli strain AI05 (ΔfrdBC ΔldhA ΔackA Δ(focA-pflB) ΔadhE ΔptsG ΔpdhR::pflBp 6-(aceEF-lpd)), previously engineered for reduction of xylose to xylitol using reducing power (NADH equivalent) of glucose catabolism, was further engineered by 1) deleting xylAB operon (encoding for xylose isomerase and xylulokinase) to prevent xylose from entering the pentose phosphate pathway; 2) anaerobically expressing the sdhCDAB-sucABCD operon (encoding for succinate dehydrogenase, α-ketoglutarate dehydrogenase and succinyl-CoA synthetase) to enable an anaerobically functional tricarboxcylic acid cycle with a theoretical 10 NAD(P)H equivalent per glucose catabolized. These reducing equivalents can be oxidized by synthetic respiration via xylose reduction, producing xylitol. The resulting strain, AI21 (pAI02), achieved a 96 % xylose to xylitol conversion, with a yield of 6 xylitol per glucose catabolized (molar yield of xylitol per glucose consumed (YRPG) = 6). This represents a 33 % improvement in xylose to xylitol conversion, and a 63 % increase in xylitol yield per glucose catabolized over that achieved by AI05 (pAI02). Increasing reducing power (NADH equivalent) output per glucose catabolized was achieved by anaerobic expression of both the pdh operon (pyruvate dehydrogenase) and the sdhCDAB-sucABCD operon, resulting in a strain capable of generating 10 NADH equivalent per glucose under anaerobic condition. The new E. coli strain AI21 (pAI02) achieved an actual 96 % conversion of xylose to xylitol (via synthetic respiration), and 6 xylitol (from xylose) per glucose catabolized (YRPG = 6, the highest known value). This strategy can be used to engineer microbial strains for the production of other reduced products from redox neutral sugars using glucose as a source of reducing power.

Oral vaccination with salmonella simultaneously expressing Yersinia pestis F1 and V antigens protects against bubonic and pneumonic plague.

PubMed

Yang, Xinghong; Hinnebusch, B Joseph; Trunkle, Theresa; Bosio, Catharine M; Suo, Zhiyong; Tighe, Mike; Harmsen, Ann; Becker, Todd; Crist, Kathryn; Walters, Nancy; Avci, Recep; Pascual, David W

2007-01-15

The gut provides a large area for immunization enabling the development of mucosal and systemic Ab responses. To test whether the protective Ags to Yersinia pestis can be orally delivered, the Y. pestis caf1 operon, encoding the F1-Ag and virulence Ag (V-Ag) were cloned into attenuated Salmonella vaccine vectors. F1-Ag expression was controlled under a promoter from the caf1 operon; two different promoters (P), PtetA in pV3, PphoP in pV4, as well as a chimera of the two in pV55 were tested. F1-Ag was amply expressed; the chimera in the pV55 showed the best V-Ag expression. Oral immunization with Salmonella-F1 elicited elevated secretory (S)-IgA and serum IgG titers, and Salmonella-V-Ag(pV55) elicited much greater S-IgA and serum IgG Ab titers than Salmonella-V-Ag(pV3) or Salmonella-V-Ag(pV4). Hence, a new Salmonella vaccine, Salmonella-(F1+V)Ags, made with a single plasmid containing the caf1 operon and the chimeric promoter for V-Ag allowed the simultaneous expression of F1 capsule and V-Ag. Salmonella-(F1+V)Ags elicited elevated Ab titers similar to their monotypic derivatives. For bubonic plague, mice dosed with Salmonella-(F1+V)Ags and Salmonella-F1-Ag showed similar efficacy (>83% survival) against approximately 1000 LD(50) Y. pestis. For pneumonic plague, immunized mice required immunity to both F1- and V-Ags because the mice vaccinated with Salmonella-(F1+V)Ags protected against 100 LD(50) Y. pestis. These results show that a single Salmonella vaccine can deliver both F1- and V-Ags to effect both systemic and mucosal immune protection against Y. pestis.

Structural Basis of Low-Affinity Nickel Binding to the Nickel-Responsive Transcription Factor NikR from Escherichia coli†‡

PubMed Central

2010-01-01

Escherichia coli NikR regulates cellular nickel uptake by binding to the nik operon in the presence of nickel and blocking transcription of genes encoding the nickel uptake transporter. NikR has two binding affinities for the nik operon: a nanomolar dissociation constant with stoichiometric nickel and a picomolar dissociation constant with excess nickel [Bloom, S. L., and Zamble, D. B. (2004) Biochemistry 43, 10029−10038; Chivers, P. T., and Sauer, R. T. (2002) Chem. Biol. 9, 1141−1148]. While it is known that the stoichiometric nickel ions bind at the NikR tetrameric interface [Schreiter, E. R., et al. (2003) Nat. Struct. Biol. 10, 794−799; Schreiter, E. R., et al. (2006) Proc. Natl. Acad. Sci. U.S.A. 103, 13676−13681], the binding sites for excess nickel ions have not been fully described. Here we have determined the crystal structure of NikR in the presence of excess nickel to 2.6 Å resolution and have obtained nickel anomalous data (1.4845 Å) in the presence of excess nickel for both NikR alone and NikR cocrystallized with a 30-nucleotide piece of double-stranded DNA containing the nik operon. These anomalous data show that excess nickel ions do not bind to a single location on NikR but instead reveal a total of 22 possible low-affinity nickel sites on the NikR tetramer. These sites, for which there are six different types, are all on the surface of NikR, and most are found in both the NikR alone and NikR−DNA structures. Using a combination of crystallographic data and molecular dynamics simulations, the nickel sites can be described as preferring octahedral geometry, utilizing one to three protein ligands (typically histidine) and at least two water molecules. PMID:20704276

Deregulation of the arginine deiminase (arc) operon in penicillin-tolerant mutants of Streptococcus gordonii.

PubMed

Caldelari, I; Loeliger, B; Langen, H; Glauser, M P; Moreillon, P

2000-10-01

Penicillin tolerance is an incompletely understood phenomenon that allows bacteria to resist drug-induced killing. Tolerance was studied with independent Streptococcus gordonii mutants generated by cyclic exposure to 500 times the MIC of penicillin. Parent cultures lost 4 to 5 log(10) CFU/ml of viable counts/24 h. In contrast, each of four independent mutant cultures lost < or =2 log(10) CFU/ml/24 h. The mutants had unchanged penicillin-binding proteins but contained increased amounts of two proteins with respective masses of ca. 50 and 45 kDa. One mutant (Tol1) was further characterized. The two proteins showing increased levels were homologous to the arginine deiminase and ornithine carbamoyl transferase of other gram-positive bacteria and were encoded by an operon that was >80% similar to the arginine-deiminase (arc) operon of these organisms. Partial nucleotide sequencing and insertion inactivation of the S. gordonii arc locus indicated that tolerance was not a direct consequence of arc alteration. On the other hand, genetic transformation of tolerance by Tol1 DNA always conferred arc deregulation. In nontolerant recipients, arc was repressed during exponential growth and up-regulated during postexponential growth. In tolerant transformants, arc was constitutively expressed. Tol1 DNA transformed tolerance at the same rate as transformation of a point mutation (10(-2) to 10(-3)). The tolerance mutation mapped on a specific chromosomal fragment but was physically distant from arc. Importantly, arc deregulation was observed in most (6 of 10) of additional independent penicillin-tolerant mutants. Thus, although not exclusive, the association between arc deregulation and tolerance was not fortuitous. Since penicillin selection mimicked the antibiotic pressure operating in the clinical environment, arc deregulation might be an important correlate of naturally occurring tolerance and help in understanding the mechanism(s) underlying this clinically problematic phenotype.

Transcriptional Regulation of Aggregatibacter actinomycetemcomitans lsrACDBFG and lsrRK Operons and Their Role in Biofilm Formation

PubMed Central

Torres-Escobar, Ascención; Juárez-Rodríguez, María Dolores; Lamont, Richard J.

2013-01-01

Autoinducer-2 (AI-2) is required for biofilm formation and virulence of the oral pathogen Aggregatibacter actinomycetemcomitans, and we previously showed that lsrB codes for a receptor for AI-2. The lsrB gene is expressed as part of the lsrACDBFG operon, which is divergently transcribed from an adjacent lsrRK operon. In Escherichia coli, lsrRK encodes a repressor and AI-2 kinase that function to regulate lsrACDBFG. To determine if lsrRK controls lsrACDBFG expression and influences biofilm growth of A. actinomycetemcomitans, we first defined the promoters for each operon. Transcriptional reporter plasmids containing the 255-bp lsrACDBFG-lsrRK intergenic region (IGR) fused to lacZ showed that essential elements of lsrR promoter reside 89 to 255 bp upstream from the lsrR start codon. Two inverted repeat sequences that represent potential binding sites for LsrR and two sequences resembling the consensus cyclic AMP receptor protein (CRP) binding site were identified in this region. Using electrophoretic mobility shift assay (EMSA), purified LsrR and CRP proteins were shown to bind probes containing these sequences. Surprisingly, the 255-bp IGR did not contain the lsrA promoter. Instead, a fragment encompassing nucleotides +1 to +159 of lsrA together with the 255-bp IGR was required to promote lsrA transcription. This suggests that a region within the lsrA coding sequence influences transcription, or alternatively that the start codon of A. actinomycetemcomitans lsrA has been incorrectly annotated. Transformation of ΔlsrR, ΔlsrK, ΔlsrRK, and Δcrp deletion mutants with lacZ reporters containing the lsrA or lsrR promoter showed that LsrR negatively regulates and CRP positively regulates both lsrACDBFG and lsrRK. However, in contrast to what occurs in E. coli, deletion of lsrK had no effect on the transcriptional activity of the lsrA or lsrR promoters, suggesting that another kinase may be capable of phosphorylating AI-2 in A. actinomycetemcomitans. Finally, biofilm formation of the ΔlsrR, ΔlsrRK, and Δcrp mutants was significantly reduced relative to that of the wild type, indicating that proper regulation of the lsr locus is required for optimal biofilm growth by A. actinomycetemcomitans. PMID:23104800

DltX of Bacillus thuringiensis Is Essential for D-Alanylation of Teichoic Acids and Resistance to Antimicrobial Response in Insects

PubMed Central

Kamar, Rita; Réjasse, Agnès; Jéhanno, Isabelle; Attieh, Zaynoun; Courtin, Pascal; Chapot-Chartier, Marie-Pierre; Nielsen-Leroux, Christina; Lereclus, Didier; el Chamy, Laure; Kallassy, Mireille; Sanchis-Borja, Vincent

2017-01-01

The dlt operon of Gram-positive bacteria is required for the incorporation of D-alanine esters into cell wall-associated teichoic acids (TAs). Addition of D-alanine to TAs reduces the negative charge of the cell envelope thereby preventing cationic antimicrobial peptides (CAMPs) from reaching their target of action on the bacterial surface. In most gram-positive bacteria, this operon consists of five genes dltXABCD but the involvement of the first ORF (dltX) encoding a small protein of unknown function, has never been investigated. The aim of this study was to establish whether this protein is involved in the D-alanylation process in Bacillus thuringiensis. We, therefore constructed an in frame deletion mutant of dltX, without affecting the expression of the other genes of the operon. The growth characteristics of the dltX mutant and those of the wild type strain were similar under standard in vitro conditions. However, disruption of dltX drastically impaired the resistance of B. thuringiensis to CAMPs and significantly attenuated its virulence in two insect species. Moreover, high-performance liquid chromatography studies showed that the dltX mutant was devoid of D-alanine, and electrophoretic mobility measurements indicated that the cells carried a higher negative surface charge. Scanning electron microscopy experiments showed morphological alterations of these mutant bacteria, suggesting that depletion of D-alanine from TAs affects cell wall structure. Our findings suggest that DltX is essential for the incorporation of D-alanyl esters into TAs. Therefore, DltX plays a direct role in the resistance to CAMPs, thus contributing to the survival of B. thuringiensis in insects. To our knowledge, this work is the first report examining the involvement of dltX in the D-alanylation of TAs. PMID:28824570

Detection and Genomic Characterization of Motility in Lactobacillus curvatus: Confirmation of Motility in a Species outside the Lactobacillus salivarius Clade

PubMed Central

Cousin, Fabien J.; Lynch, Shónagh M.; Harris, Hugh M. B.; McCann, Angela; Lynch, Denise B.; Neville, B. Anne; Irisawa, Tomohiro; Okada, Sanae; Endo, Akihito

2014-01-01

Lactobacillus is the largest genus within the lactic acid bacteria (LAB), with almost 180 species currently identified. Motility has been reported for at least 13 Lactobacillus species, all belonging to the Lactobacillus salivarius clade. Motility in lactobacilli is poorly characterized. It probably confers competitive advantages, such as superior nutrient acquisition and niche colonization, but it could also play an important role in innate immune system activation through flagellin–Toll-like receptor 5 (TLR5) interaction. We now report strong evidence of motility in a species outside the L. salivarius clade, Lactobacillus curvatus (strain NRIC 0822). The motility of L. curvatus NRIC 0822 was revealed by phase-contrast microscopy and soft-agar motility assays. Strain NRIC 0822 was motile at temperatures between 15°C and 37°C, with a range of different carbohydrates, and under varying atmospheric conditions. We sequenced the L. curvatus NRIC 0822 genome, which revealed that the motility genes are organized in a single operon and that the products are very similar (>98.5% amino acid similarity over >11,000 amino acids) to those encoded by the motility operon of Lactobacillus acidipiscis KCTC 13900 (shown for the first time to be motile also). Moreover, the presence of a large number of mobile genetic elements within and flanking the motility operon of L. curvatus suggests recent horizontal transfer between members of two distinct Lactobacillus clades: L. acidipiscis in the L. salivarius clade and L. curvatus in the L. sakei clade. This study provides novel phenotypic, genetic, and phylogenetic insights into flagellum-mediated motility in lactobacilli. PMID:25501479

Genes for 2,4,5-Trichlorophenoxyacetic Acid Metabolism in Burkholderia cepacia AC1100: Characterization of the tftC and tftD Genes and Locations of the tft Operons on Multiple Replicons

PubMed Central

Hübner, Anette; Danganan, Clyde E.; Xun, Luying; Chakrabarty, A. M.; Hendrickson, William

1998-01-01

Burkholderia cepacia AC1100 uses the chlorinated aromatic compound 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) as a sole source of carbon and energy. The enzyme which converts the first intermediate in the pathway, 2,4,5-trichlorophenol, to 5-chlorohydroquinone has been purified and consists of two subunits of 58 and 22 kDa, encoded by the tftC and tftD genes (48). A degenerate primer was designed from the N terminus of the 58-kDa polypeptide and used to isolate a clone containing the tftC and tftD genes from a genomic library of AC1100. The derived amino acid sequences of tftC and tftD show significant homology to the two-component monooxygenases HadA of Burkholderia pickettii, HpaBC of Escherichia coli, and HpaAH of Klebsiella pneumonia. Expression of the tftC and tftD genes appeared to be induced when they were grown in the presence of 2,4,5-T, as shown by RNA slot blot and primer extension analyses. Three sets of cloned tft genes were used as probes to explore the genomic organization of the pathway. Pulsed-field gel electrophoresis analyses of whole chromosomes of B. cepacia AC1100 demonstrated that the genome is comprised of five replicons of 4.0, 2.7, 0.53, 0.34, and 0.15 Mbp, designated I to V, respectively. The tft genes are located on the smaller replicons: the tftAB cluster is on replicon IV, tftEFGH is on replicon III, and copies of the tftC and the tftCD operons are found on both replicons III and IV. When cells were grown in the absence of 2,4,5-T, the genes were lost at high frequency by chromosomal deletions and rearrangements to produce 2,4,5-T-negative mutants. In one mutant, the tftA and tftB genes translocated from one replicon to another, with the concomitant loss of tftEFGH and one copy of tftCD. PMID:9603818

Metabolic engineering of Pseudomonas putida for the utilization of parathion as a carbon and energy source.

PubMed

Walker, Andy W; Keasling, Jay D

2002-06-30

Pseudomonas putida KT2442 was engineered to use the organophosphate pesticide parathion, a compound similar to other organophosphate pesticides and chemical warfare agents, as a source of carbon and energy. The initial step in the engineered degradation pathway was parathion hydrolysis by organophosphate hydrolase (OPH) to p-nitrophenol (PNP) and diethyl thiophosphate, compounds that cannot be metabolized by P. putida KT2442. The gene encoding the native OPH (opd), with and without the secretory leader sequence, was cloned into broad-host-range plasmids under the control of tac and taclac promoters. Expression of opd from the tac promoter resulted in high OPH activity, whereas expression from the taclac promoter resulted in low activity. A plasmid-harboring operons encoding enzymes for p-nitrophenol transformation to beta-ketoadipate was transformed into P. putida allowing the organism to use 0.5 mM PNP as a carbon and energy source. Transformation of P. putida with the plasmids harboring opd and the PNP operons allowed the organism to utilize 0.8 mM parathion as a source of carbon and energy. Degradation studies showed that parathion formed a separate dense, non-aqueous phase liquid phase but was still bioavailable. Copyright 2002 Wiley Periodicals, Inc.

Expression of acrA and acrB Genes in Esherichia coli Mutants with or without marR or acrR Mutations

PubMed Central

Pourahmad Jaktaji, Razieh; Jazayeri, Nasim

2013-01-01

Objective(s): The major antibiotic efflux pump of Esherichia coli is AcrAB-TolC. The first part of the pump, AcrAB, is encoded by acrAB operon. The expression of this operon can be kept elevated by overexpression of an activator, MarA following inactivation of MarR and AcrR repressors due to mutation in encoding genes, marR and acrR, respectively. The aims of this research were to use E. coli mutants with or without mutation in marR to search for the presence of possible mutation in acrR and to quantify the expression of acrAB. Materials and Methods: The DNA binding region of acrR gene in these mutants were amplified by PCR and sequenced. The relative expression of acrA and acrB were determined by real time PCR. Results: Results showed that W26 and C14 had the same mutation in acrR, but none of the mutants overexpressed acrA and acrB in comparison with wild type strain. Conclusions: The effect of marR or acrR mutation on acrAB overexpression is dependent on levels of resistance to tetracycline and ciprofloxacin. PMID:24570831

Expression of arsenic resistance genes in the obligate anaerobe Bacteroides vulgatus ATCC 8482, a gut microbiome bacterium

PubMed Central

Li, Jiaojiao; Mandal, Goutam; Rosen, Barry P.

2016-01-01

The response of the obligate anaerobe Bacteroides vulgatus ATCC 8482, a common human gut microbiota, to arsenic was determined. B. vulgatus ATCC 8482 is highly resistant to pentavalent As(V) and methylarsenate (MAs(V)). It is somewhat more sensitive to trivalent inorganic As(III) but 100-fold more sensitive to methylarsenite (MAs(III)) than to As(III). B. vulgatus ATCC 8482 has eight continuous genes in its genome that we demonstrate form an arsenical-inducible transcriptional unit. The first gene of this ars operon, arsR, encodes a putative ArsR As(III)-responsive transcriptional repressor. The next three genes encode proteins of unknown function. The remaining genes, arsDABC, have well-characterized roles in detoxification of inorganic arsenic, but there are no known genes for MAs(III) resistance. Expression of each gene after exposure to trivalent and pentavalent inorganic and methylarsenicals was analyzed. MAs(III) was the most effective inducer. The arsD gene was the most highly expressed of the ars operon genes. These results demonstrate that this anaerobic microbiome bacterium has arsenic-responsive genes that confer resistance to inorganic arsenic and may be responsible for the organism's ability to maintain its prevalence in the gut following dietary exposure to inorganic arsenic. PMID:27040269

Zinc-Responsive Regulation of Alternative Ribosomal Protein Genes in Streptomyces coelicolor Involves Zur and σR▿ †

PubMed Central

Owen, Gillian A.; Pascoe, Ben; Kallifidas, Dimitris; Paget, Mark S. B.

2007-01-01

Streptomyces coelicolor contains paralogous versions of seven ribosomal proteins (S14, S18, L28, L31, L32, L33, and L36), which differ in their potential to bind structural zinc. The paralogues are termed C+ or C− on the basis of the presence or absence of putative cysteine ligands. Here, mutational studies suggest that the C− version of L31 can functionally replace its C+ paralogue only when expressed at an artificially elevated level. We show that the level of expression of four transcriptional units encoding C− proteins is elevated under conditions of zinc deprivation. Zur controls the expression of three transcriptional units (including rpmG2, rpmE2, rpmB2, rpsN2, rpmF2, and possibly rpsR2). Zur also controls the expression of the znuACB operon, which is predicted to encode a high-affinity zinc transport system. Surprisingly, the zinc-responsive control of the rpmG3-rpmJ2 operon is dictated by σR, a sigma factor that was previously shown to control the response to disulfide stress in S. coelicolor. The induction of σR activity during zinc limitation establishes an important link between thiol-disulfide metabolism and zinc homeostasis. PMID:17400736

Zinc-responsive regulation of alternative ribosomal protein genes in Streptomyces coelicolor involves zur and sigmaR.

PubMed

Owen, Gillian A; Pascoe, Ben; Kallifidas, Dimitris; Paget, Mark S B

2007-06-01

Streptomyces coelicolor contains paralogous versions of seven ribosomal proteins (S14, S18, L28, L31, L32, L33, and L36), which differ in their potential to bind structural zinc. The paralogues are termed C(+) or C(-) on the basis of the presence or absence of putative cysteine ligands. Here, mutational studies suggest that the C(-) version of L31 can functionally replace its C(+) paralogue only when expressed at an artificially elevated level. We show that the level of expression of four transcriptional units encoding C(-) proteins is elevated under conditions of zinc deprivation. Zur controls the expression of three transcriptional units (including rpmG2, rpmE2, rpmB2, rpsN2, rpmF2, and possibly rpsR2). Zur also controls the expression of the znuACB operon, which is predicted to encode a high-affinity zinc transport system. Surprisingly, the zinc-responsive control of the rpmG3-rpmJ2 operon is dictated by sigma(R), a sigma factor that was previously shown to control the response to disulfide stress in S. coelicolor. The induction of sigma(R) activity during zinc limitation establishes an important link between thiol-disulfide metabolism and zinc homeostasis.

Genome analysis of the freshwater planktonic Vulcanococcus limneticus sp. nov. reveals horizontal transfer of nitrogenase operon and alternative pathways of nitrogen utilization.

PubMed

Di Cesare, Andrea; Cabello-Yeves, Pedro J; Chrismas, Nathan A M; Sánchez-Baracaldo, Patricia; Salcher, Michaela M; Callieri, Cristiana

2018-04-16

Many cyanobacteria are capable of fixing atmospheric nitrogen, playing a crucial role in biogeochemical cycling. Little is known about freshwater unicellular cyanobacteria Synechococcus spp. at the genomic level, despite being recognised of considerable ecological importance in aquatic ecosystems. So far, it has not been shown whether these unicellular picocyanobacteria have the potential for nitrogen fixation. Here, we present the draft-genome of the new pink-pigmented Synechococcus-like strain Vulcanococcus limneticus. sp. nov., isolated from the volcanic Lake Albano (Central Italy). The novel species Vulcanococcus limneticus sp. nov. falls inside the sub-cluster 5.2, close to the estuarine/marine strains in a maximum-likelihood phylogenetic tree generated with 259 marker genes with representatives from marine, brackish, euryhaline and freshwater habitats. V.limneticus sp. nov. possesses a complete nitrogenase and nif operon. In an experimental setup under nitrogen limiting and non-limiting conditions, growth was observed in both cases. However, the nitrogenase genes (nifHDK) were not transcribed, i.e., V.limneticus sp. nov. did not fix nitrogen, but instead degraded the phycobilisomes to produce sufficient amounts of ammonia. Moreover, the strain encoded many other pathways to incorporate ammonia, nitrate and sulphate, which are energetically less expensive for the cell than fixing nitrogen. The association of the nif operon to a genomic island, the relatively high amount of mobile genetic elements (52 transposases) and the lower observed GC content of V.limneticus sp. nov. nif operon (60.54%) compared to the average of the strain (68.35%) support the theory that this planktonic strain may have obtained, at some point of its evolution, the nif operon by horizontal gene transfer (HGT) from a filamentous or heterocystous cyanobacterium. In this study, we describe the novel species Vulcanococcus limneticus sp. nov., which possesses a complete nif operon for nitrogen fixation. The finding that in our experimental conditions V.limneticus sp. nov. did not express the nifHDK genes led us to reconsider the actual ecological meaning of these accessory genes located in genomic island that have possibly been acquired via HGT.

Glycerol metabolism of Lactobacillus rhamnosus ATCC 7469: cloning and expression of two glycerol kinase genes.

PubMed

Alvarez, María de Fátima; Medina, Roxana; Pasteris, Sergio E; Strasser de Saad, Ana M; Sesma, Fernando

2004-01-01

Lactobacillus rhamnosus ATCC 7469 was able to grow in glycerol as the sole source of energy in aerobic conditions, producing lactate, acetate, and diacetyl. A biphasic growth was observed in the presence of glucose. In this condition, glycerol consumption began after glucose was exhausted from the culture medium. Glycerol kinase activity was detected in L. rhamnosus ATCC 7469, a characteristic of microorganisms which catabolize glycerol in aerobic conditions. Genetic analysis revealed that this strain possesses two glycerol kinase genes: gykA and glpK, that encode for two different glycerol kinases GykA and GlpK, respectively. The glpK geneis associated in an operon with alpha-glycerophosphate oxidase (glpO) and glycerol facilitator (glpF) genes. Transcriptional analysis revealed that only glpK is expressed when L. rhamnosus was grown on glycerol. Copyright 2004 S. Karger AG, Basel

Crystal structures of MW1337R and lin2004: Representatives of a novel protein family that adopt a four-helical bundle fold

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

Kozbial, Piotr; Xu, Qingping; Chiu, Hsiu-Ju

2009-08-28

To extend the structural coverage of proteins with unknown functions, we targeted a novel protein family (Pfam accession number PF08807, DUF1798) for which we proposed and determined the structures of two representative members. The MW1337R gene of Staphylococcus aureus subsp. aureus Rosenbach (Wood 46) encodes a protein with a molecular weight of 13.8 kDa (residues 1-116) and a calculated isoelectric point of 5.15. The lin2004 gene of the nonspore-forming bacterium Listeria innocua Clip11262 encodes a protein with a molecular weight of 14.6 kDa (residues 1-121) and a calculated isoelectric point of 5.45. MW1337R and lin2004, as well as their homologs,more » which, so far, have been found only in Bacillus, Staphylococcus, Listeria, and related genera (Geobacillus, Exiguobacterium, and Oceanobacillus), have unknown functions and are annotated as hypothetical proteins. The genomic contexts of MW1337R and lin2004 are similar and conserved in related species. In prokaryotic genomes, most often, functionally interacting proteins are coded by genes, which are colocated in conserved operons. Proteins from the same operon as MW1337R and lin2004 either have unknown functions (i.e., belong to DUF1273, Pfam accession number PF06908) or are similar to ypsB from Bacillus subtilis. The function of ypsB is unclear, although it has a strong similarity to the N-terminal region of DivIVA, which was characterized as a bifunctional protein with distinct roles during vegetative growth and sporulation. In addition, members of the DUF1273 family display distant sequence similarity with the DprA/Smf protein, which acts downstream of the DNA uptake machinery, possibly in conjunction with RecA. The RecA activities in Bacillus subtilis are modulated by RecU Holliday-junction resolvase. In all analyzed cases, the gene coding for RecU is in the vicinity of MW1337R, lin2004, or their orthologs, but on a different operon located in the complementary DNA strand. Here, we report the crystal structures of MW1337R and lin2004, which were determined using the semiautomated, high-throughput pipeline of the Joint Center for Structural Genomics (JCSG), part of the National Institute of General Medical Sciences Protein Structure Initiative.« less

Distinct Residues Contribute to Motility Repression and Autoregulation in the Proteus mirabilis Fimbria-Associated Transcriptional Regulator AtfJ.

PubMed

Bode, Nadine J; Chan, Kun-Wei; Kong, Xiang-Peng; Pearson, Melanie M

2016-08-01

Proteus mirabilis contributes to a significant number of catheter-associated urinary tract infections, where coordinated regulation of adherence and motility is critical for ascending disease progression. Previously, the mannose-resistant Proteus-like (MR/P) fimbria-associated transcriptional regulator MrpJ has been shown to both repress motility and directly induce the transcription of its own operon; in addition, it affects the expression of a wide range of cellular processes. Interestingly, 14 additional mrpJ paralogs are included in the P. mirabilis genome. Looking at a selection of MrpJ paralogs, we discovered that these proteins, which consistently repress motility, also have nonidentical functions that include cross-regulation of fimbrial operons. A subset of paralogs, including AtfJ (encoded by the ambient temperature fimbrial operon), Fim8J, and MrpJ, are capable of autoinduction. We identified an element of the atf promoter extending from 487 to 655 nucleotides upstream of the transcriptional start site that is responsive to AtfJ, and we found that AtfJ directly binds this fragment. Mutational analysis of AtfJ revealed that its two identified functions, autoregulation and motility repression, are not invariably linked. Residues within the DNA-binding helix-turn-helix domain are required for motility repression but not necessarily autoregulation. Likewise, the C-terminal domain is dispensable for motility repression but is essential for autoregulation. Supported by a three-dimensional (3D) structural model, we hypothesize that the C-terminal domain confers unique regulatory capacities on the AtfJ family of regulators. Balancing adherence with motility is essential for uropathogens to successfully establish a foothold in their host. Proteus mirabilis uses a fimbria-associated transcriptional regulator to switch between these antagonistic processes by increasing fimbrial adherence while simultaneously downregulating flagella. The discovery of multiple related proteins, many of which also function as motility repressors, encoded in the P. mirabilis genome has raised considerable interest as to their functionality and potential redundancy in this organism. This study provides an important advance in this field by elucidating the nonidentical effects of these paralogs on a molecular level. Our mechanistic studies of one member of this group, AtfJ, shed light on how these differing functions may be conferred despite the limited sequence variety exhibited by the paralogous proteins. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Functional analysis of aromatic biosynthetic pathways in Pseudomonas putida KT2440

PubMed Central

Molina‐Henares, M. Antonia; García‐Salamanca, Adela; Molina‐Henares, A. Jesús; De La Torre, Jesús; Herrera, M. Carmen; Ramos, Juan L.; Duque, Estrella

2009-01-01

Summary Pseudomonas putida KT2440 is a non‐pathogenic prototrophic bacterium with high potential for biotechnological applications. Despite all that is known about this strain, the biosynthesis of essential chemicals has not been fully analysed and auxotroph mutants are scarce. We carried out massive mini‐Tn5 random mutagenesis and screened for auxotrophs that require aromatic amino acids. The biosynthesis of aromatic amino acids was analysed in detail including physical and transcriptional organization of genes, complementation assays and feeding experiments to establish pathway intermediates. There is a single pathway from chorismate leading to the biosynthesis of tryptophan, whereas the biosynthesis of phenylalanine and tyrosine is achieved through multiple convergent pathways. Genes for tryptophan biosynthesis are grouped in unlinked regions with the trpBA and trpGDE genes organized as operons and the trpI, trpE and trpF genes organized as single transcriptional units. The pheA and tyrA gene‐encoding multifunctional enzymes for phenylalanine and tyrosine biosynthesis are linked in the chromosome and form an operon with the serC gene involved in serine biosynthesis. The last step in the biosynthesis of these two amino acids requires an amino transferase activity for which multiple tyrB‐like genes are present in the host chromosome. PMID:21261884

Identification and characterization of novel multiple bacteriocins produced by Leuconostoc pseudomesenteroides QU 15.

PubMed

Sawa, N; Okamura, K; Zendo, T; Himeno, K; Nakayama, J; Sonomoto, K

2010-07-01

To characterize novel multiple bacteriocins produced by Leuconostoc pseudomesenteroides QU 15. Leuconostoc pseudomesenteroides QU 15 isolated from Nukadoko (rice bran bed) produced novel bacteriocins. By using three purification steps, four antimicrobial peptides termed leucocin A (ΔC7), leucocin A-QU 15, leucocin Q and leucocin N were purified from the culture supernatant. The amino acid sequences of leucocin A (ΔC7) and leucocin A-QU 15 were identical to that of leucocin A-UAL 187 belonging to class IIa bacteriocins, but leucocin A (ΔC7) was deficient in seven C-terminal residues. Leucocin Q and leucocin N are novel class IId bacteriocins. Moreover, the DNA sequences encoding three bacteriocins, leucocin A-QU 15, leucocin Q and leucocin N were obtained. These bacteriocins including two novel bacteriocins were identified from Leuc. pseudomesenteroides QU 15. They showed similar antimicrobial spectra, but their intensities differed. The C-terminal region of leucocin A-QU 15 was important for its antimicrobial activity. Leucocins Q and N were encoded by adjacent open reading frames (ORFs) in the same operon, but leucocin A-QU 15 was not. These leucocins were produced concomitantly by the same strain. Although the two novel bacteriocins were encoded by adjacent ORFs, a characteristic of class IIb bacteriocins, they did not show synergistic activity. © 2010 The Authors. Journal compilation © 2010 The Society for Applied Microbiology.

A novel photosynthetic strategy for adaptation to low-iron aquatic environments

USGS Publications Warehouse

Chauhan, D.; Folea, I.M.; Jolley, C.C.; Kouril, R.; Lubner, C.E.; Lin, S.; Kolber, D.; Wolfe-Simon, Felisa; Golbeck, J.H.; Boekema, E.J.; Fromme, P.

2011-01-01

Iron (Fe) availability is a major limiting factor for primary production in aquatic environments. Cyanobacteria respond to Fe deficiency by derepressing the isiAB operon, which encodes the antenna protein IsiA and flavodoxin. At nanomolar Fe concentrations, a PSI-IsiA supercomplex forms, comprising a PSI trimer encircled by two complete IsiA rings. This PSI-IsiA supercomplex is the largest photosynthetic membrane protein complex yet isolated. This study presents a detailed characterization of this complex using transmission electron microscopy and ultrafast fluorescence spectroscopy. Excitation trapping and electron transfer are highly efficient, allowing cyanobacteria to avoid oxidative stress. This mechanism may be a major factor used by cyanobacteria to successfully adapt to modern low-Fe environments. ?? 2010 American Chemical Society.

Complete genome sequence of Streptococcus troglodytae TKU31 isolated from the oral cavity of a chimpanzee (Pan troglodytes).

PubMed

Okamoto, Masaaki; Naito, Mariko; Miyanohara, Mayu; Imai, Susumu; Nomura, Yoshiaki; Saito, Wataru; Momoi, Yasuko; Takada, Kazuko; Miyabe-Nishiwaki, Takako; Tomonaga, Masaki; Hanada, Nobuhiro

2016-12-01

Streptococcus troglodytae TKU31 was isolated from the oral cavity of a chimpanzee (Pan troglodytes) and was found to be the most closely related species of the mutans group streptococci to Streptococcus mutans. The complete sequence of TKU31 genome consists of a single circular chromosome that is 2,097,874 base pairs long and has a G + C content of 37.18%. It possesses 2082 coding sequences (CDSs), 65 tRNAs and five rRNA operons (15 rRNAs). Two clustered regularly interspaced short palindromic repeats, six insertion sequences and two predicted prophage elements were identified. The genome of TKU31 harbors some putative virulence associated genes, including gtfB, gtfC and gtfD genes encoding glucosyltransferase and gbpA, gbpB, gbpC and gbpD genes encoding glucan-binding cell wall-anchored protein. The deduced amino acid identity of the rhamnose-glucose polysaccharide F gene (rgpF), which is one of the serotype determinants, is 91% identical with that of S. mutans LJ23 (serotype k) strain. However, two other virulence-associated genes cnm and cbm, which encode the collagen-binding proteins, were not found in the TKU31 genome. The complete genome sequence of S. troglodytae TKU31 has been deposited at DDBJ/European Nucleotide Archive/GenBank under the accession no. AP014612. © 2016 The Societies and John Wiley & Sons Australia, Ltd.

In vitro expression of Escherichia coli ribosomal protein genes: autogenous inhibition of translation.

PubMed Central

Yates, J L; Arfsten, A E; Nomura, M

1980-01-01

Escherichia coli ribosomal protein L1 (0.5 micro M) was found to inhibit the synthesis of both proteins of the L11 operon, L11 and L1, but not the synthesis of other proteins directed by lambda rifd 18 DNA. Similarly, S4 (1 micro M) selectively inhibited the synthesis of three proteins of the alpha operon, S13, S11, and S4, directed by lambda spcI DNA or a restriction enzyme fragment obtained from this DNA. S8 (3.6 micro M) also showed preferential inhibitory effects on the synthesis of some proteins encoded in the spc operon, L24 and L5 (and probably S14 and S8), directed by lambda spcl DNA or a restriction enzyme fragment carrying the genes for these proteins. The inhibitory effect of L1 was observed only with L1 and not with other proteins examined, including S4 and S8. Similarly, the effect of S4 was not observed with L1 or S8, and that of S8 was not seen with L1 or S4. Inhibition was shown to take place at the level of translation rather than transcription. Thus, at least some ribosomal proteins (L1 S4, and S8) have the ability to cause selective translational inhibition of the synthesis of certain ribosomal proteins whose genes are in the same operon as their own. These results support the hypothesis that certain free ribosomal proteins not assembled into ribosomes act as "autogenous" feedback inhibitors to regulate the synthesis of ribosomal proteins. Images PMID:6445562

Transcriptional activation of the tad type IVb pilus operon by PypB in Yersinia enterocolitica.

PubMed

Schilling, Jennifer; Wagner, Karin; Seekircher, Stephanie; Greune, Lilo; Humberg, Verena; Schmidt, M Alexander; Heusipp, Gerhard

2010-07-01

Type IV pili are virulence factors in various bacteria and mediate, among other functions, the colonization of diverse surfaces. Various subclasses of type IV pili have been identified, but information on pilus expression, biogenesis, and the associated phenotypes is sparse for the genus Yersinia. We recently described the identification of PypB as a transcriptional regulator in Yersinia enterocolitica. Here we show that the pypB gene is associated with the tad locus, a genomic island that is widespread among bacterial and archaeal species. The genetic linkage of pypB with the tad locus is conserved throughout the yersiniae but is not found among other bacteria carrying the tad locus. We show that the genes of the tad locus form an operon in Y. enterocolitica that is controlled by PypB and that pypB is part of this operon. The tad genes encode functions necessary for the biogenesis of the Flp subfamily of type IVb pili initially described for Aggregatibacter actinomycetemcomitans to mediate a tight-adherence phenotype. In Y. enterocolitica, the Flp pilin protein shows some peculiarities in its amino acid sequence that imply similarities as well as differences compared to typical motifs found in the Flp subtype of type IVb pili. Flp is expressed and processed after PypB overproduction, resulting in microcolony formation but not in increased adherence to biotic or abiotic surfaces. Our data describe the transcriptional regulation of the tad type IVb pilus operon by PypB in Y. enterocolitica but fail to show most previously described phenotypes associated with this type of pilus in other bacteria.

Characterization of two genes encoding the Mycobacterium tuberculosis ribonucleotide reductase small subunit.

PubMed Central

Yang, F; Curran, S C; Li, L S; Avarbock, D; Graf, J D; Chua, M M; Lu, G; Salem, J; Rubin, H

1997-01-01

Two nrdF genes, nrdF1 and nrdF2, encoding the small subunit (R2) of ribonucleotide reductase (RR) from Mycobacterium tuberculosis have 71% identity at the amino acid level and are both highly homologous with Salmonella typhimurium R2F. The calculated molecular masses of R2-1 and R2-2 are 36,588 (322 amino acids [aa]) and 36,957 (324 aa) Da, respectively. Western blot analysis of crude M. tuberculosis extracts indicates that both R2s are expressed in vivo. Recombinant R2-2 is enzymatically active when assayed with pure recombinant M. tuberculosis R1 subunit. Both ATP and dATP are activators for CDP reduction up to 2 and 1 mM, respectively. The gene encoding M. tuberculosis R2-1, nrdF1, is not linked to nrdF2, nor is either gene linked to the gene encoding the large subunit, M. tuberculosis nrdE. The gene encoding MTP64 was found downstream from nrdF1, and the gene encoding alcohol dehydrogenase was found downstream from nrdF2. A nrdA(Ts) strain of E. coli (E101) could be complemented by simultaneous transformation with M. tuberculosis nrdE and nrdF2. An M. tuberculosis nrdF2 variant in which the codon for the catalytically necessary tyrosine was replaced by the phenylalanine codon did not complement E101 when cotransformed with M. tuberculosis nrdE. Similarly, M. tuberculosis nrdF1 and nrdE did not complement E101. Activity of recombinant M. tuberculosis RR was inhibited by incubating the enzyme with a peptide corresponding to the 7 C-terminal amino acid residues of the R2-2 subunit. M. tuberculosis is a species in which a nrdEF system appears to encode the biologically active species of RR and also the only bacterial species identified so far in which class I RR subunits are not arranged on an operon. PMID:9335290

High accuracy operon prediction method based on STRING database scores.

PubMed

Taboada, Blanca; Verde, Cristina; Merino, Enrique

2010-07-01

We present a simple and highly accurate computational method for operon prediction, based on intergenic distances and functional relationships between the protein products of contiguous genes, as defined by STRING database (Jensen,L.J., Kuhn,M., Stark,M., Chaffron,S., Creevey,C., Muller,J., Doerks,T., Julien,P., Roth,A., Simonovic,M. et al. (2009) STRING 8-a global view on proteins and their functional interactions in 630 organisms. Nucleic Acids Res., 37, D412-D416). These two parameters were used to train a neural network on a subset of experimentally characterized Escherichia coli and Bacillus subtilis operons. Our predictive model was successfully tested on the set of experimentally defined operons in E. coli and B. subtilis, with accuracies of 94.6 and 93.3%, respectively. As far as we know, these are the highest accuracies ever obtained for predicting bacterial operons. Furthermore, in order to evaluate the predictable accuracy of our model when using an organism's data set for the training procedure, and a different organism's data set for testing, we repeated the E. coli operon prediction analysis using a neural network trained with B. subtilis data, and a B. subtilis analysis using a neural network trained with E. coli data. Even for these cases, the accuracies reached with our method were outstandingly high, 91.5 and 93%, respectively. These results show the potential use of our method for accurately predicting the operons of any other organism. Our operon predictions for fully-sequenced genomes are available at http://operons.ibt.unam.mx/OperonPredictor/.

Microbial cycling of isoprene, the most abundantly produced biological volatile organic compound on Earth.

PubMed

McGenity, Terry J; Crombie, Andrew T; Murrell, J Colin

2018-04-01

Isoprene (2-methyl-1,3-butadiene), the most abundantly produced biogenic volatile organic compound (BVOC) on Earth, is highly reactive and can have diverse and often detrimental atmospheric effects, which impact on climate and health. Most isoprene is produced by terrestrial plants, but (micro)algal production is important in aquatic environments, and the relative bacterial contribution remains unknown. Soils are a sink for isoprene, and bacteria that can use isoprene as a carbon and energy source have been cultivated and also identified using cultivation-independent methods from soils, leaves and coastal/marine environments. Bacteria belonging to the Actinobacteria are most frequently isolated and identified, and Proteobacteria have also been shown to degrade isoprene. In the freshwater-sediment isolate, Rhodococcus strain AD45, initial oxidation of isoprene to 1,2-epoxy-isoprene is catalyzed by a multicomponent isoprene monooxygenase encoded by the genes isoABCDEF. The resultant epoxide is converted to a glutathione conjugate by a glutathione S-transferase encoded by isoI, and further degraded by enzymes encoded by isoGHJ. Genome sequence analysis of actinobacterial isolates belonging to the genera Rhodococcus, Mycobacterium and Gordonia has revealed that isoABCDEF and isoGHIJ are linked in an operon, either on a plasmid or the chromosome. In Rhodococcus strain AD45 both isoprene and epoxy-isoprene induce a high level of transcription of 22 contiguous genes, including isoABCDEF and isoGHIJ. Sequence analysis of the isoA gene, encoding the large subunit of the oxygenase component of isoprene monooxygenase, from isolates has facilitated the development of PCR primers that are proving valuable in investigating the ecology of uncultivated isoprene-degrading bacteria.

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

Deletion of Lipoteichoic Acid Synthase Impacts Expression of Genes Encoding Cell Surface Proteins in Lactobacillus acidophilus

PubMed Central

Selle, Kurt; Goh, Yong J.; Johnson, Brant R.; O’Flaherty, Sarah; Andersen, Joakim M.; Barrangou, Rodolphe; Klaenhammer, Todd R.

2017-01-01

Lactobacillus acidophilus NCFM is a well-characterized probiotic microorganism, supported by a decade of genomic and functional phenotypic investigations. L. acidophilus deficient in lipoteichoic acid (LTA), a major immunostimulant in Gram-positive bacteria, has been shown to shift immune system responses in animal disease models. However, the pleiotropic effects of removing LTA from the cell surface in lactobacilli are unknown. In this study, we surveyed the global transcriptional and extracellular protein profiles of two strains of L. acidophilus deficient in LTA. Twenty-four differentially expressed genes specific to the LTA-deficient strains were identified, including a predicted heavy metal resistance operon and several putative peptidoglycan hydrolases. Cell morphology and manganese sensitivity phenotypes were assessed in relation to the putative functions of differentially expressed genes. LTA-deficient L. acidophilus exhibited elongated cellular morphology and their growth was severely inhibited by elevated manganese concentrations. Exoproteomic surveys revealed distinct changes in the composition and relative abundances of several extracellular proteins and showed a bias of intracellular proteins in LTA-deficient strains of L. acidophilus. Taken together, these results elucidate the impact of ltaS deletion on the transcriptome and extracellular proteins of L. acidophilus, suggesting roles of LTA in cell morphology and ion homeostasis as a structural component of the Gram positive cell wall. PMID:28443071

Deletion of Lipoteichoic Acid Synthase Impacts Expression of Genes Encoding Cell Surface Proteins in Lactobacillus acidophilus.

PubMed

Selle, Kurt; Goh, Yong J; Johnson, Brant R; O'Flaherty, Sarah; Andersen, Joakim M; Barrangou, Rodolphe; Klaenhammer, Todd R

2017-01-01

Lactobacillus acidophilus NCFM is a well-characterized probiotic microorganism, supported by a decade of genomic and functional phenotypic investigations. L. acidophilus deficient in lipoteichoic acid (LTA), a major immunostimulant in Gram-positive bacteria, has been shown to shift immune system responses in animal disease models. However, the pleiotropic effects of removing LTA from the cell surface in lactobacilli are unknown. In this study, we surveyed the global transcriptional and extracellular protein profiles of two strains of L. acidophilus deficient in LTA. Twenty-four differentially expressed genes specific to the LTA-deficient strains were identified, including a predicted heavy metal resistance operon and several putative peptidoglycan hydrolases. Cell morphology and manganese sensitivity phenotypes were assessed in relation to the putative functions of differentially expressed genes. LTA-deficient L. acidophilus exhibited elongated cellular morphology and their growth was severely inhibited by elevated manganese concentrations. Exoproteomic surveys revealed distinct changes in the composition and relative abundances of several extracellular proteins and showed a bias of intracellular proteins in LTA-deficient strains of L. acidophilus . Taken together, these results elucidate the impact of ltaS deletion on the transcriptome and extracellular proteins of L. acidophilus , suggesting roles of LTA in cell morphology and ion homeostasis as a structural component of the Gram positive cell wall.

Genome-wide analysis of trans-splicing in the nematode Pristionchus pacificus unravels conserved gene functions for germline and dauer development in divergent operons.

PubMed

Sinha, Amit; Langnick, Claudia; Sommer, Ralf J; Dieterich, Christoph

2014-09-01

Discovery of trans-splicing in multiple metazoan lineages led to the identification of operon-like gene organization in diverse organisms, including trypanosomes, tunicates, and nematodes, but the functional significance of such operons is not completely understood. To see whether the content or organization of operons serves similar roles across species, we experimentally defined operons in the nematode model Pristionchus pacificus. We performed affinity capture experiments on mRNA pools to specifically enrich for transcripts that are trans-spliced to either the SL1- or SL2-spliced leader, using spliced leader-specific probes. We obtained distinct trans-splicing patterns from the analysis of three mRNA pools (total mRNA, SL1 and SL2 fraction) by RNA-seq. This information was combined with a genome-wide analysis of gene orientation and spacing. We could confirm 2219 operons by RNA-seq data out of 6709 candidate operons, which were predicted by sequence information alone. Our gene order comparison of the Caenorhabditis elegans and P. pacificus genomes shows major changes in operon organization in the two species. Notably, only 128 out of 1288 operons in C. elegans are conserved in P. pacificus. However, analysis of gene-expression profiles identified conserved functions such as an enrichment of germline-expressed genes and higher expression levels of operonic genes during recovery from dauer arrest in both species. These results provide support for the model that a necessity for increased transcriptional efficiency in the context of certain developmental processes could be a selective constraint for operon evolution in metazoans. Our method is generally applicable to other metazoans to see if similar functional constraints regulate gene organization into operons. © 2014 Sinha et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

A two-component regulatory system, pehR-pehS, controls endopolygalacturonase production and virulence in the plant pathogen Erwinia carotovora subsp. carotovora.

PubMed

Flego, D; Marits, R; Eriksson, A R; Kõiv, V; Karlsson, M B; Heikinheimo, R; Palva, E T

2000-04-01

Genes coding for the main virulence determinants of the plant pathogen Erwinia carotovora subsp. carotovora, the plant cell wall-degrading enzymes, are under the coordinate control of global regulator systems including both positive and negative factors. In addition to this global control, some virulence determinants are subject to specific regulation. We have previously shown that mutations in the pehR locus result in reduced virulence and impaired production of one of these enzymes, an endopolygalacturonase (PehA). In contrast, these pehR strains produce essentially wild-type levels of other extracellular enzymes including pectate lyases and cellulases. In this work, we characterized the pehR locus and showed that the DNA sequence is composed of two genes, designated pehR and pehS, present in an operon. Mutations in either pehR or pehS caused a Peh-negative phenotype and resulted in reduced virulence on tobacco seedlings. Complementation experiments indicated that both genes are required for transcriptional activation of the endopolygalacturonase gene, pehA, as well as restoration of virulence. Structural characterization of the pehR-pehS operon demonstrated that the corresponding polypeptides are highly similar to the two-component transcriptional regulators PhoP-PhoQ of both Escherichia coli and Salmonella typhimurium. Functional similarity of PehR-PehS with PhoP-PhoQ of E. coli and S. typhimurium was demonstrated by genetic complementation.

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

Functional amyloid in Pseudomonas.

PubMed

Dueholm, Morten S; Petersen, Steen V; Sønderkær, Mads; Larsen, Poul; Christiansen, Gunna; Hein, Kim L; Enghild, Jan J; Nielsen, Jeppe L; Nielsen, Kåre L; Nielsen, Per H; Otzen, Daniel E

2010-08-01

Amyloids are highly abundant in many microbial biofilms and may play an important role in their architecture. Nevertheless, little is known of the amyloid proteins. We report the discovery of a novel functional amyloid expressed by a Pseudomonas strain of the P. fluorescens group. The amyloid protein was purified and the amyloid-like structure verified. Partial sequencing by MS/MS combined with full genomic sequencing of the Pseudomonas strain identified the gene coding for the major subunit of the amyloid fibril, termed fapC. FapC contains a thrice repeated motif that differs from those previously found in curli fimbrins and prion proteins. The lack of aromatic residues in the repeat shows that aromatic side chains are not needed for efficient amyloid formation. In contrast, glutamine and asparagine residues seem to play a major role in amyloid formation as these are highly conserved in curli, prion proteins and FapC. fapC is conserved in many Pseudomonas strains including the opportunistic pathogen P. aeruginosa and is situated in a conserved operon containing six genes, of which one encodes a fapC homologue. Heterologous expression of the fapA-F operon in Escherichia coli BL21(DE3) resulted in a highly aggregative phenotype, showing that the operon is involved in biofilm formation. © 2010 Blackwell Publishing Ltd.

The SdiA-Regulated Gene srgE Encodes a Type III Secreted Effector

PubMed Central

Habyarimana, Fabien; Sabag-Daigle, Anice

2014-01-01

Salmonella enterica serovar Typhimurium is a food-borne pathogen that causes severe gastroenteritis. The ability of Salmonella to cause disease depends on two type III secretion systems (T3SSs) encoded in two distinct Salmonella pathogenicity islands, 1 and 2 (SPI1 and SPI2, respectively). S. Typhimurium encodes a solo LuxR homolog, SdiA, which can detect the acyl-homoserine lactones (AHLs) produced by other bacteria and upregulate the rck operon and the srgE gene. SrgE is predicted to encode a protein of 488 residues with a coiled-coil domain between residues 345 and 382. In silico studies have provided conflicting predictions as to whether SrgE is a T3SS substrate. Therefore, in this work, we tested the hypothesis that SrgE is a T3SS effector by two methods, a β-lactamase activity assay and a split green fluorescent protein (GFP) complementation assay. SrgE with β-lactamase fused to residue 40, 100, 150, or 300 was indeed expressed and translocated into host cells, but SrgE with β-lactamase fused to residue 400 or 488 was not expressed, suggesting interference by the coiled-coil domain. Similarly, SrgE with GFP S11 fused to residue 300, but not to residue 488, was expressed and translocated into host cells. With both systems, translocation into host cells was dependent upon SPI2. A phylogenetic analysis indicated that srgE is found only within Salmonella enterica subspecies. It is found sporadically within both typhoidal and nontyphoidal serovars, although the SrgE protein sequences found within typhoidal serovars tend to cluster separately from those found in nontyphoidal serovars, suggesting functional diversification. PMID:24727228

Biochemical localization of a protein involved in Gluconacetobacter hansenii cellulose synthesis

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

Iyer, Prashanti R; Catchmark, Jeffrey M; Brown, Nicole Robitaille

2011-02-08

Using subcellular fractionation and Western blot methods, we have shown that AcsD, one of the proteins encoded by the Acetobacter cellulose synthase (acs) operon, is localized in the periplasmic region of the cell. AcsD protein was heterologously expressed in Escherichia coli and purified using histidine tag affinity methods. The purified protein was used to obtain rabbit polyclonal antibodies. The purity of the subcellular fractions was assessed by marker enzyme assays.

Metabolic engineering of the shikimate pathway

DOEpatents

Juminaga, Darmawi; Keasling, Jay D.

2017-01-10

The present disclosure relates to engineered microorganisms that produce amino acids and amino acid intermediates. In particular, the disclosure relates to recombinant nucleic acids encoding operons that increase production of aromatic amino acids and the aromatic amino acid intermediate shikimate; microorganisms with increased production of aromatic amino acids and the aromatic amino acid intermediate shikimate; and methods related to the production of aromatic amino acids, the aromatic amino acid intermediate shikimate, and commodity chemicals derived therefrom.

PspG, a New Member of the Yersinia enterocolitica Phage Shock Protein Regulon

PubMed Central

Green, Rebecca C.; Darwin, Andrew J.

2004-01-01

The Yersinia enterocolitica phage shock protein (Psp) system is induced when the Ysc type III secretion system is produced or when only the YscC secretin component is synthesized. Some psp null mutants have a growth defect when YscC is produced and a severe virulence defect in animals. The Y. enterocolitica psp locus is made up of two divergently transcribed cistrons, pspF and pspABCDycjXF. pspA operon expression is dependent on RpoN (σ54) and the enhancer-binding protein PspF. Previous data indicated that PspF also controls at least one gene that is not part of the psp locus. In this study we describe the identification of pspG, a new member of the PspF regulon. Predicted RpoN-binding sites upstream of the pspA genes from different bacteria have a common divergence from the consensus sequence, which may be a signature of PspF-dependent promoters. The Y. enterocolitica pspG gene was identified because its promoter also has this signature. Like the pspA operon, pspG is positively regulated by PspF, negatively regulated by PspA, and induced in response to the production of secretins. Purified His6-PspF protein specifically interacts with the pspA and pspG control regions. A pspA operon deletion mutant has a growth defect when the YscC secretin is produced and a virulence defect in a mouse model of infection. These phenotypes were exacerbated by a pspG null mutation. Therefore, PspG is the missing component of the Y. enterocolitica Psp regulon that was previously predicted to exist. PMID:15262928

Cloning and expression of clostridium acetobutylicum ATCC 824 acetoacetyl-coenzyme A:acetate/butyrate:coenzyme A-transferase in Escherichia coli

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

Cary, J.W.; Petersen, D.J.; Bennett, G.N.

1990-06-01

Coenzyme A (CoA)-transferase (acetoacetyl-CoA:acetate/butyrate:CoA-transferase (butyrate-acetoacetate CoA-transferase) (EC 2.8.3.9)) of Clostridium acetobutylicum ATCC 824 is an important enzyme in the metabolic shift between the acid-producing and solvent-forming states of this organism. The genes encoding the two subunits of this enzyme have been cloned and subsequent subcloning experiments established the position of the structural genes for CoA-transferase. Complementation of Escherichia coli ato mutants with the recombinant plasmid pCoAT4 (pUC19 carrying a 1.8-kilobase insert of C. acetobutylicum DNA encoding CoA-transferase activity) enabled the transformants to grow on butyrate as a sole carbon source. Despite the ability of CoA-transferase to complement the ato defectmore » in E. coli mutants, Southern blot and Western blot (immunoblot) analyses showed showed that neither the C. acetobutylicum genes encoding CoA-transferase nor the enzyme itself shared any apparent homology with its E. coli counterpart. Polypeptides of M{sub r} of the purified CoA-transferase subunits were observed by Western blot and maxicell analysis of whole-cell extracts of E.coli harboring pCoAT4. The proximity and orientation of the genes suggest that the genes encoding the two subunits of CoA-transferase may form an operon similar to that found in E. coli. In the plasmid, however, transcription appears to be primarily from the lac promoter of the vector.« less

Determination of grain size distribution function using two-dimensional Fourier transforms of tone pulse encoded images

NASA Technical Reports Server (NTRS)

Generazio, E. R.

1986-01-01

Microstructural images may be tone pulse encoded and subsequently Fourier transformed to determine the two-dimensional density of frequency components. A theory is developed relating the density of frequency components to the density of length components. The density of length components corresponds directly to the actual grain size distribution function from which the mean grain shape, size, and orientation can be obtained.

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

Prediction and characterization of enzymatic activities guided by sequence similarity and genome neighborhood networks

DOE PAGES

Zhao, Suwen; Sakai, Ayano; Zhang, Xinshuai; ...

2014-06-30

Metabolic pathways in eubacteria and archaea often are encoded by operons and/or gene clusters (genome neighborhoods) that provide important clues for assignment of both enzyme functions and metabolic pathways. We describe a bioinformatic approach (genome neighborhood network; GNN) that enables large scale prediction of the in vitro enzymatic activities and in vivo physiological functions (metabolic pathways) of uncharacterized enzymes in protein families. We demonstrate the utility of the GNN approach by predicting in vitro activities and in vivo functions in the proline racemase superfamily (PRS; InterPro IPR008794). The predictions were verified by measuring in vitro activities for 51 proteins inmore » 12 families in the PRS that represent ~85% of the sequences; in vitro activities of pathway enzymes, carbon/nitrogen source phenotypes, and/or transcriptomic studies confirmed the predicted pathways. The synergistic use of sequence similarity networks3 and GNNs will facilitate the discovery of the components of novel, uncharacterized metabolic pathways in sequenced genomes.« less

VraT/YvqF Is Required for Methicillin Resistance and Activation of the VraSR Regulon in Staphylococcus aureus

PubMed Central

Yin, Shouhui; Jo, Dae Sun; Montgomery, Christopher P.; Daum, Robert S.

2013-01-01

Staphylococcus aureus infections caused by strains that are resistant to all forms of penicillin, so-called methicillin-resistant S. aureus (MRSA) strains, have become common. One strategy to counter MRSA infections is to use compounds that resensitize MRSA to methicillin. S. aureus responds to diverse classes of cell wall-inhibitory antibiotics, like methicillin, using the two-component regulatory system VraSR (vra) to up- or downregulate a set of genes (the cell wall stimulon) that presumably facilitates resistance to these antibiotics. Accordingly, VraS and VraR mutations decrease resistance to methicillin, vancomycin, and daptomycin cell wall antimicrobials. vraS and vraR are encoded together on a transcript downstream of two other genes, which we call vraU and vraT (previously called yvqF). By producing nonpolar deletions in vraU and vraT in a USA300 MRSA clinical isolate, we demonstrate that vraT is essential for optimal expression of methicillin resistance in vitro, whereas vraU is not required for this phenotype. The deletion of vraT also improved the outcomes of oxacillin therapy in mouse models of lung and skin infection. Since vraT expressed in trans did not complement a vra operon deletion, we conclude that VraT does not inactivate the antimicrobial. Genome-wide transcriptional microarray experiments reveal that VraT facilitates resistance by playing a necessary regulatory role in the VraSR-mediated cell wall stimulon. Our data prove that VraTSR comprise a novel three-component regulatory system required to facilitate resistance to cell wall agents in S. aureus. We also provide the first in vivo proof of principle for using VraT as a sole target to resensitize MRSA to β-lactams. PMID:23070169

Lactococcus lactis LMG2081 Produces Two Bacteriocins, a Nonlantibiotic and a Novel Lantibiotic

PubMed Central

Mirkovic, Nemanja; Polovic, Natalija; Vukotic, Goran; Jovcic, Branko; Miljkovic, Marija; Radulovic, Zorica; Diep, Dzung B.

2016-01-01

Bacteriocin producers normally possess dedicated immunity systems to protect themselves from their own bacteriocins. Lactococcus lactis strains LMG2081 and BGBM50 are known as lactococcin G producers. However, BGBM50 was sensitive to LMG2081, which indicated that LMG2081 might produce additional bacteriocins that are not present in BGBM50. Therefore, whole-genome sequencing of the two strains was performed, and a lantibiotic operon (called lctLMG) was identified in LMG2081 but not in BGBM50. The lctLMG operon contains six open reading frames; the first three genes, lmgA, lmgM, and lmgT, are involved in the biosynthesis and export of bacteriocin, while the other three genes, lmgF, lmgE, and lmgG, are involved in lantibiotic immunity. Mutational analysis confirmed that the lctLMG operon is responsible for the additional antimicrobial activity. Specifically, site-directed mutation within this operon rendered LMG2081 inactive toward BGBM50. Subsequent purification and electrospray ionization–time of flight mass spectrometric analysis confirmed that the lantibiotic bacteriocin called lacticin LMG is exported as a 25-amino-acid peptide. Lacticin LMG is highly similar to the lacticin 481 group. It is interesting that a bacteriocin producer produces two different classes of bacteriocins, whose operons are located in the chromosome and a plasmid. PMID:26896142

Transcription of All amoC Copies Is Associated with Recovery ofNitrosomonas europaea from Ammonia Starvation

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

Berube, Paul M.; Samudrala, Ram; Stahl, David A.

2007-09-21

The chemolithotrophic ammonia-oxidizing bacteriumNitrosomonas europaea is known to be highly resistant to starvationconditions. The transcriptional response of N. europaea to ammoniaaddition following short- and long-term starvation was examined by primerextension and S1 nuclease protection analyses of genes encoding enzymesfor ammonia oxidation (amoCAB operons) and CO2 fixation (cbbLS), a third,lone copy of amoC (amoC3), and two representative housekeeping genes(glyA and rpsJ). Primer extension analysis of RNA isolated from growing,starved, and recovering cells revealed two differentially regulatedpromoters upstream of the two amoCAB operons. The distal sigma 70 typeamoCAB promoter was constitutively active in the presence of ammonia, butthe proximal promoter was onlymore » active when cells were recovering fromammonia starvation. The lone, divergent copy of amoC (amoC3) wasexpressed only during recovery. Both the proximal amoC1,2 promoter andthe amoC3 promoter are similar to gram-negative sigma E promoters, thusimplicating sigma E in the regulation of the recovery response. Althoughmodeling of subunit interactions suggested that a nonconservative prolinesubstitution in AmoC3 may modify the activity of the holoenzyme,characterization of a Delta amoC3 strain showed no significant differencein starvation recovery under conditions evaluated. In contrast to the amotranscripts, a delayed appearance of transcripts for a gene required forCO2 fixation (cbbL) suggested that its transcription is retarded untilsufficient energy is available. Overall, these data revealed a programmedexit from starvation likely involving regulation by sigma E and thecoordinated regulation of catabolic and anabolic genes.« less

Photosynthetic Versatility in the Genome of Geitlerinema sp. PCC 9228 (Formerly Oscillatoria limnetica 'Solar Lake'), a Model Anoxygenic Photosynthetic Cyanobacterium.

PubMed

Grim, Sharon L; Dick, Gregory J

2016-01-01

Anoxygenic cyanobacteria that use sulfide as the electron donor for photosynthesis are a potentially influential but poorly constrained force on Earth's biogeochemistry. Their versatile metabolism may have boosted primary production and nitrogen cycling in euxinic coastal margins in the Proterozoic. In addition, they represent a biological mechanism for limiting the accumulation of atmospheric oxygen, especially before the Great Oxidation Event and in the low-oxygen conditions of the Proterozoic. In this study, we describe the draft genome sequence of Geitlerinema sp. PCC 9228, formerly Oscillatoria limnetica 'Solar Lake', a mat-forming diazotrophic cyanobacterium that can switch between oxygenic photosynthesis and sulfide-based anoxygenic photosynthesis (AP). Geitlerinema possesses three variants of psbA , which encodes protein D1, a core component of the photosystem II reaction center. Phylogenetic analyses indicate that one variant is closely affiliated with cyanobacterial psbA genes that code for a D1 protein used for oxygen-sensitive processes. Another version is phylogenetically similar to cyanobacterial psbA genes that encode D1 proteins used under microaerobic conditions, and the third variant may be cued to high light and/or elevated oxygen concentrations. Geitlerinema has the canonical gene for sulfide quinone reductase (SQR) used in cyanobacterial AP and a putative transcriptional regulatory gene in the same operon. Another operon with a second, distinct sqr and regulatory gene is present, and is phylogenetically related to sqr genes used for high sulfide concentrations. The genome has a comprehensive nif gene suite for nitrogen fixation, supporting previous observations of nitrogenase activity. Geitlerinema possesses a bidirectional hydrogenase rather than the uptake hydrogenase typically used by cyanobacteria in diazotrophy. Overall, the genome sequence of Geitlerinema sp. PCC 9228 highlights potential cyanobacterial strategies to cope with fluctuating redox gradients and nitrogen availability that occur in benthic mats over a diel cycle. Such dynamic geochemical conditions likely also challenged Proterozoic cyanobacteria, modulating oxygen production. The genetic repertoire that underpins flexible oxygenic/anoxygenic photosynthesis in cyanobacteria provides a foundation to explore the regulation, evolutionary context, and biogeochemical implications of these co-occurring metabolisms in Earth history.

Photosynthetic Versatility in the Genome of Geitlerinema sp. PCC 9228 (Formerly Oscillatoria limnetica ‘Solar Lake’), a Model Anoxygenic Photosynthetic Cyanobacterium

PubMed Central

Grim, Sharon L.; Dick, Gregory J.

2016-01-01

Anoxygenic cyanobacteria that use sulfide as the electron donor for photosynthesis are a potentially influential but poorly constrained force on Earth’s biogeochemistry. Their versatile metabolism may have boosted primary production and nitrogen cycling in euxinic coastal margins in the Proterozoic. In addition, they represent a biological mechanism for limiting the accumulation of atmospheric oxygen, especially before the Great Oxidation Event and in the low-oxygen conditions of the Proterozoic. In this study, we describe the draft genome sequence of Geitlerinema sp. PCC 9228, formerly Oscillatoria limnetica ‘Solar Lake’, a mat-forming diazotrophic cyanobacterium that can switch between oxygenic photosynthesis and sulfide-based anoxygenic photosynthesis (AP). Geitlerinema possesses three variants of psbA, which encodes protein D1, a core component of the photosystem II reaction center. Phylogenetic analyses indicate that one variant is closely affiliated with cyanobacterial psbA genes that code for a D1 protein used for oxygen-sensitive processes. Another version is phylogenetically similar to cyanobacterial psbA genes that encode D1 proteins used under microaerobic conditions, and the third variant may be cued to high light and/or elevated oxygen concentrations. Geitlerinema has the canonical gene for sulfide quinone reductase (SQR) used in cyanobacterial AP and a putative transcriptional regulatory gene in the same operon. Another operon with a second, distinct sqr and regulatory gene is present, and is phylogenetically related to sqr genes used for high sulfide concentrations. The genome has a comprehensive nif gene suite for nitrogen fixation, supporting previous observations of nitrogenase activity. Geitlerinema possesses a bidirectional hydrogenase rather than the uptake hydrogenase typically used by cyanobacteria in diazotrophy. Overall, the genome sequence of Geitlerinema sp. PCC 9228 highlights potential cyanobacterial strategies to cope with fluctuating redox gradients and nitrogen availability that occur in benthic mats over a diel cycle. Such dynamic geochemical conditions likely also challenged Proterozoic cyanobacteria, modulating oxygen production. The genetic repertoire that underpins flexible oxygenic/anoxygenic photosynthesis in cyanobacteria provides a foundation to explore the regulation, evolutionary context, and biogeochemical implications of these co-occurring metabolisms in Earth history. PMID:27790189

Differential transcriptional control of the two tRNA(fMet) genes of Escherichia coli K-12.

PubMed

Nagase, T; Ishii, S; Imamoto, F

1988-07-15

The metZ gene of Escherichia coli, which encodes the tRNA(f1Met), was cloned. Using the nucleotide sequence, in vitro transcription, and S1 nuclease mapping analyses, we identified the promoter region, transcriptional start point, the two tandem tRNA(f1Met) structural genes separated by an intergenic space of 33 bp, and the two Rho-independent transcriptional termination sites, in that order. We compared the promoter region of the metZ gene with that of the metY gene, which encodes the tRNA(f2Met) and is located in the promoter-proximal portion of the nusA operon. A G + C-rich sequence (5'-GCGCATCCAC-3'), similar to the corresponding sequence of the rrn promoters that are under stringent control, was found between the Pribnow box and the transcriptional start point of the metZ promoter, but not in the metY promoter region. We therefore examined the effect of guanosine 3'-diphosphate, 5'-diphosphate (ppGpp), the chemical mediator of stringent control, and found that ppGpp inhibited the transcription of the metZ gene, but not that of the metY gene. These data suggested that the promoters for metZ and metY have different physiological functions and are regulated by different mechanisms.

Macrolide resistance in Legionella pneumophila: the role of LpeAB efflux pump.

PubMed

Massip, Clémence; Descours, Ghislaine; Ginevra, Christophe; Doublet, Patricia; Jarraud, Sophie; Gilbert, Christophe

2017-05-01

A previous study on 12 in vitro -selected azithromycin-resistant Legionella pneumophila lineages showed that ribosomal mutations were major macrolide resistance determinants. In addition to these mechanisms that have been well described in many species, mutations upstream of lpeAB operon, homologous to acrAB in Escherichia coli , were identified in two lineages. In this study, we investigated the role of LpeAB and of these mutations in macrolide resistance of L. pneumophila . The role of LpeAB was studied by testing the antibiotic susceptibility of WT, deleted and complemented L. pneumophila Paris strains. Translational fusion experiments using GFP as a reporter were conducted to investigate the consequences of the mutations observed in the upstream sequence of lpeAB operon. We demonstrated the involvement of LpeAB in an efflux pump responsible for a macrolide-specific reduced susceptibility of L. pneumophila Paris strain. Mutations in the upstream sequence of lpeAB operon were associated with an increased protein expression. Increased expression was also observed under sub-inhibitory macrolide concentrations in strains with both mutated and WT promoting regions. LpeAB are components of an efflux pump, which is a macrolide resistance determinant in L. pneumophila Paris strain. Mutations observed in the upstream sequence of lpeAB operon in resistant lineages led to an overexpression of this efflux pump. Sub-inhibitory concentrations of macrolides themselves participated in upregulating this efflux and could constitute a first step in the acquisition of a high macrolide resistance level. © The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Identification of Bicarbonate as a Trigger and Genes Involved with Extracellular DNA Export in Mycobacterial Biofilms

PubMed Central

Rose, Sasha J.

2016-01-01

ABSTRACT Extracellular DNA (eDNA) is an integral biofilm matrix component of numerous pathogens, including nontuberculous mycobacteria (NTM). Cell lysis is the source of eDNA in certain bacteria, but the source of eDNA remains unidentified for NTM, as well as for other eDNA-containing bacterial species. In this study, conditions affecting eDNA export were examined, and genes involved with the eDNA export mechanism were identified. After a method for monitoring eDNA in real time in undisturbed biofilms was established, different conditions affecting eDNA were investigated. Bicarbonate positively influenced eDNA export in a pH-independent manner in Mycobacterium avium, M. abscessus, and M. chelonae. The surface-exposed proteome of M. avium in eDNA-containing biofilms revealed abundant carbonic anhydrases. Chemical inhibition of carbonic anhydrases with ethoxzolamide significantly reduced eDNA export. An unbiased transposon mutant library screen for eDNA export in M. avium identified many severely eDNA-attenuated mutants, including one not expressing a unique FtsK/SpoIIIE-like DNA-transporting pore, two with inactivation of carbonic anhydrases, and nine with inactivation of genes belonging to a unique genomic region, as well as numerous mutants involved in metabolism and energy production. Complementation of nine mutants that included the FtsK/SpoIIIE and carbonic anhydrase significantly restored eDNA export. Interestingly, several attenuated eDNA mutants have mutations in genes encoding proteins that were found with the surface proteomics, and many more mutations are localized in operons potentially encoding surface proteins. Collectively, our data strengthen the evidence of eDNA export being an active mechanism that is activated by the bacterium responding to bicarbonate. PMID:27923918

The morphogenetic MreBCD proteins of Escherichia coli form an essential membrane-bound complex.

PubMed

Kruse, Thomas; Bork-Jensen, Jette; Gerdes, Kenn

2005-01-01

MreB proteins of Escherichia coli, Bacillus subtilis and Caulobacter crescentus form actin-like cables lying beneath the cell surface. The cables are required to guide longitudinal cell wall synthesis and their absence leads to merodiploid spherical and inflated cells prone to cell lysis. In B. subtilis and C. crescentus, the mreB gene is essential. However, in E. coli, mreB was inferred not to be essential. Using a tight, conditional gene depletion system, we systematically investigated whether the E. coli mreBCD-encoded components were essential. We found that cells depleted of mreBCD became spherical, enlarged and finally lysed. Depletion of each mre gene separately conferred similar gross changes in cell morphology and viability. Thus, the three proteins encoded by mreBCD are all essential and function in the same morphogenetic pathway. Interestingly, the presence of a multicopy plasmid carrying the ftsQAZ genes suppressed the lethality of deletions in the mre operon. Using GFP and cell fractionation methods, we showed that the MreC and MreD proteins were associated with the cell membrane. Using a bacterial two-hybrid system, we found that MreC interacted with both MreB and MreD. In contrast, MreB and MreD did not interact in this assay. Thus, we conclude that the E. coli MreBCD form an essential membrane-bound complex. Curiously, MreB did not form cables in cell depleted for MreC, MreD or RodA, indicating a mutual interdependency between MreB filament morphology and cell shape. Based on these and other observations we propose a model in which the membrane-associated MreBCD complex directs longitudinal cell wall synthesis in a process essential to maintain cell morphology.

Regulation of Bacteriocin Production and Cell Death by the VicRK Signaling System in Streptococcus mutans

PubMed Central

Senadheera, D. B.; Cordova, M.; Ayala, E. A.; Chávez de Paz, L. E.; Singh, K.; Downey, J. S.; Svensäter, G.; Goodman, S. D.

2012-01-01

The VicRK two-component signaling system modulates biofilm formation, genetic competence, and stress tolerance in Streptococcus mutans. We show here that the VicRK modulates bacteriocin production and cell viability, in part by direct modulation of competence-stimulating peptide (CSP) production in S. mutans. Global transcriptome and real-time transcriptional analysis of the VicK-deficient mutant (SmuvicK) revealed significant modulation of several bacteriocin-related loci, including nlmAB, nlmC, and nlmD (P < 0.001), suggesting a role for the VicRK in producing mutacins IV, V, and VI. Bacteriocin overlay assays revealed an altered ability of the vic mutants to kill related species. Since a well-conserved VicR binding site (TGTWAH-N5-TGTWAH) was identified within the comC coding region, we confirmed VicR binding to this sequence using DNA footprinting. Overexpression of the vic operon caused growth-phase-dependent repression of comC, comDE, and comX. In the vic mutants, transcription of nlmC/cipB encoding mutacin V, previously linked to CSP-dependent cell lysis, as well as expression of its putative immunity factor encoded by immB, were significantly affected relative to the wild type (P < 0.05). In contrast to previous reports that proposed a hyper-resistant phenotype for the VicK mutant in cell viability, the release of extracellular genomic DNA was significantly enhanced in SmuvicK (P < 0.05), likely as a result of increased autolysis compared with the parent. The drastic influence of VicRK on cell viability was also demonstrated using vic mutant biofilms. Taken together, we have identified a novel regulatory link between the VicRK and ComDE systems to modulate bacteriocin production and cell viability of S. mutans. PMID:22228735

The Transcription Factor AlsR Binds and Regulates the Promoter of the alsSD Operon Responsible for Acetoin Formation in Bacillus subtilis

PubMed Central

Frädrich, Claudia; March, Anika; Fiege, Kerstin; Hartmann, Anja; Jahn, Dieter

2012-01-01

Bacillus subtilis forms acetoin under anaerobic fermentative growth conditions and as a product of the aerobic carbon overflow metabolism. Acetoin formation from pyruvate requires α-acetolactate synthase and acetolactate decarboxylase, both encoded by the alsSD operon. The alsR gene, encoding the LysR-type transcriptional regulator AlsR, was found to be essential for the in vivo expression of alsSD in response to anaerobic acetate accumulation, the addition of acetate, low pH, and the aerobic stationary phase. The expressions of the alsSD operon and the alsR regulatory gene were independent of other regulators of the anaerobic regulatory network, including ResDE, Fnr, and ArfM. A negative autoregulation of alsR was observed. In vitro transcription from the alsSD promoter using purified B. subtilis RNA polymerase required AlsR. DNA binding studies with purified recombinant AlsR in combination with promoter mutagenesis experiments identified a 19-bp high-affinity palindromic binding site (TAAT-N11-ATTA) at positions −76 to −58 (regulatory binding site [RBS]) and a low-affinity site (AT-N11-AT) at positions −41 to −27 (activator binding site [ABS]) upstream of the transcriptional start site of alsSD. The RBS and ABS were found to be essential for in vivo alsSD transcription. AlsR binding to both sites induced the formation of higher-order, transcription-competent complexes. The AlsR protein carrying the S100A substitution at the potential coinducer binding site still bound to the RBS and ABS. However, AlsR(S100A) failed to form the higher-order complex and to initiate in vivo and in vitro transcription. A model for AlsR promoter binding and transcriptional activation was deduced. PMID:22178965

Genome sequence analyses of two isolates from the recent Escherichia coli outbreak in Germany reveal the emergence of a new pathotype: Entero-Aggregative-Haemorrhagic Escherichia coli (EAHEC).

PubMed

Brzuszkiewicz, Elzbieta; Thürmer, Andrea; Schuldes, Jörg; Leimbach, Andreas; Liesegang, Heiko; Meyer, Frauke-Dorothee; Boelter, Jürgen; Petersen, Heiko; Gottschalk, Gerhard; Daniel, Rolf

2011-12-01

The genome sequences of two Escherichia coli O104:H4 strains derived from two different patients of the 2011 German E. coli outbreak were determined. The two analyzed strains were designated E. coli GOS1 and GOS2 (German outbreak strain). Both isolates comprise one chromosome of approximately 5.31 Mbp and two putative plasmids. Comparisons of the 5,217 (GOS1) and 5,224 (GOS2) predicted protein-encoding genes with various E. coli strains, and a multilocus sequence typing analysis revealed that the isolates were most similar to the entero-aggregative E. coli (EAEC) strain 55989. In addition, one of the putative plasmids of the outbreak strain is similar to pAA-type plasmids of EAEC strains, which contain aggregative adhesion fimbrial operons. The second putative plasmid harbors genes for extended-spectrum β-lactamases. This type of plasmid is widely distributed in pathogenic E. coli strains. A significant difference of the E. coli GOS1 and GOS2 genomes to those of EAEC strains is the presence of a prophage encoding the Shiga toxin, which is characteristic for enterohemorrhagic E. coli (EHEC) strains. The unique combination of genomic features of the German outbreak strain, containing characteristics from pathotypes EAEC and EHEC, suggested that it represents a new pathotype Entero-Aggregative-Haemorrhagic E scherichia c oli (EAHEC).

Epsilon-toxin production by Clostridium perfringens type D strain CN3718 is dependent upon the agr operon but not the VirS/VirR two-component regulatory system.

PubMed

Chen, Jianming; Rood, Julian I; McClane, Bruce A

2011-01-01

Clostridium perfringens type B and D strains cause enterotoxemias and enteritis in livestock after proliferating in the intestines and producing epsilon-toxin (ETX), alpha-toxin (CPA), and, usually, perfringolysin O (PFO). Although ETX is one of the most potent bacterial toxins, the regulation of ETX production by type B or D strains remains poorly understood. The present work determined that the type D strain CN3718 upregulates production of ETX upon close contact with enterocyte-like Caco-2 cells. This host cell-induced upregulation of ETX expression was mediated at the transcriptional level. Using an isogenic agrB null mutant and complemented strain, the agr operon was shown to be required when CN3718 produces ETX in broth culture or, via a secreted signal consistent with a quorum-sensing (QS) effect, upregulates ETX production upon contact with host cells. These findings provide the first insights into the regulation of ETX production, as well as additional evidence that the Agr-like QS system functions as a global regulator of C. perfringens toxin production. Since it was proposed previously that the Agr-like QS system regulates C. perfringens gene expression via the VirS/VirR two-component regulatory system, an isogenic virR null mutant of CN3718 was constructed to evaluate the importance of VirS/VirR for CN3718 toxin production. This mutation affected production of CPA and PFO, but not ETX, by CN3718. These results provide the first indication that C. perfringens toxin expression regulation by the Agr-like quorum-sensing system may not always act via the VirS/VirR two-component system. IMPORTANCE Mechanisms by which Clostridium perfringens type B and D strains regulate production of epsilon-toxin (ETX), a CDC class B select toxin, are poorly understood. Production of several other toxins expressed by C. perfringens is wholly or partially regulated by both the Agr-like quorum-sensing (QS) system and the VirS/VirR two-component regulatory system, so the present study tested whether ETX expression by type D strain CN3718 also requires these regulatory systems. The agr operon was shown to be essential for signaling CN3718 to produce ETX in broth culture or to upregulate ETX production upon close contact with enterocyte-like Caco-2 cells, which may have pathogenic relevance since ETX is produced intestinally. However, ETX production remained at wild-type levels after inactivation of the VirS/VirR system in CN3718. These findings provide the first information regarding regulation of ETX production and suggest Agr-like QS toxin production regulation in C. perfringens does not always require the VirS/VirR system.

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

Finding New Enzymes from Bacterial Physiology: A Successful Approach Illustrated by the Detection of Novel Oxidases in Marinomonas mediterranea

PubMed Central

Sanchez-Amat, Antonio; Solano, Francisco; Lucas-Elío, Patricia

2010-01-01

The identification and study of marine microorganisms with unique physiological traits can be a very powerful tool discovering novel enzymes of possible biotechnological interest. This approach can complement the enormous amount of data concerning gene diversity in marine environments offered by metagenomic analysis, and can help to place the activities associated with those sequences in the context of microbial cellular metabolism and physiology. Accordingly, the detection and isolation of microorganisms that may be a good source of enzymes is of great importance. Marinomonas mediterranea, for example, has proven to be one such useful microorganism. This Gram-negative marine bacterium was first selected because of the unusually high amounts of melanins synthesized in media containing the amino acid l-tyrosine. The study of its molecular biology has allowed the cloning of several genes encoding oxidases of biotechnological interest, particularly in white and red biotechnology. Characterization of the operon encoding the tyrosinase responsible for melanin synthesis revealed that a second gene in that operon encodes a protein, PpoB2, which is involved in copper transfer to tyrosinase. This finding made PpoB2 the first protein in the COG5486 group to which a physiological role has been assigned. Another enzyme of interest described in M. mediterranea is a multicopper oxidase encoding a membrane-associated enzyme that shows oxidative activity on a wide range of substrates typical of both laccases and tyrosinases. Finally, an enzyme very specific for l-lysine, which oxidises this amino acid in epsilon position and that has received a new EC number (1.4.3.20), has also been described for M. mediterranea. Overall, the studies carried out on this bacterium illustrate the power of exploring the physiology of selected microorganisms to discover novel enzymes of biotechnological relevance. PMID:20411113

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.

Identification and characterization of transcripts from the biotin biosynthetic operon of Bacillus subtilis.

PubMed Central

Perkins, J B; Bower, S; Howitt, C L; Yocum, R R; Pero, J

1996-01-01

Northern (RNA) blot analysis of the Bacillus subtilis biotin operon, bioWAFDBIorf2, detected at least two steady-state polycistronic transcripts initiated from a putative vegetative (Pbio) promoter that precedes the operon, i.e., a full-length 7.2-kb transcript covering the entire operon and a more abundant 5.1-kb transcript covering just the first five genes of the operon. Biotin and the B. subtilis birA gene product regulated synthesis of the transcripts. Moreover, replacing the putative Pbio promoter and regulatory sequence with a constitutive SP01 phage promoter resulted in higher-level constitutive synthesis. Removal of a rho-independent terminator-like sequence located between the fifth (bioB) and sixth (bioI) genes prevented accumulation of the 5.1-kb transcript, suggesting that the putative terminator functions to limit expression of bioI, which is thought to be involved in an early step in biotin synthesis. PMID:8892842

Identification and characterization of transcripts from the biotin biosynthetic operon of Bacillus subtilis.

PubMed

Perkins, J B; Bower, S; Howitt, C L; Yocum, R R; Pero, J

1996-11-01

Northern (RNA) blot analysis of the Bacillus subtilis biotin operon, bioWAFDBIorf2, detected at least two steady-state polycistronic transcripts initiated from a putative vegetative (Pbio) promoter that precedes the operon, i.e., a full-length 7.2-kb transcript covering the entire operon and a more abundant 5.1-kb transcript covering just the first five genes of the operon. Biotin and the B. subtilis birA gene product regulated synthesis of the transcripts. Moreover, replacing the putative Pbio promoter and regulatory sequence with a constitutive SP01 phage promoter resulted in higher-level constitutive synthesis. Removal of a rho-independent terminator-like sequence located between the fifth (bioB) and sixth (bioI) genes prevented accumulation of the 5.1-kb transcript, suggesting that the putative terminator functions to limit expression of bioI, which is thought to be involved in an early step in biotin synthesis.

Incorporation of a horizontally transferred gene into an operon during cnidarian evolution.

PubMed

Dana, Catherine E; Glauber, Kristine M; Chan, Titus A; Bridge, Diane M; Steele, Robert E

2012-01-01

Genome sequencing has revealed examples of horizontally transferred genes, but we still know little about how such genes are incorporated into their host genomes. We have previously reported the identification of a gene (flp) that appears to have entered the Hydra genome through horizontal transfer. Here we provide additional evidence in support of our original hypothesis that the transfer was from a unicellular organism, and we show that the transfer occurred in an ancestor of two medusozoan cnidarian species. In addition we show that the gene is part of a bicistronic operon in the Hydra genome. These findings identify a new animal phylum in which trans-spliced leader addition has led to the formation of operons, and define the requirements for evolution of an operon in Hydra. The identification of operons in Hydra also provides a tool that can be exploited in the construction of transgenic Hydra strains.

Conditional-suicide containment system for bacteria which mineralize aromatics

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

Contreras, A.; Ramos, J.L.; Molin, S.

A model conditional-suicide system to control genetically engineered microorganisms able to degrade substituted benzoates is reported. The system is based on two elements. One element consists of a fusion between the promoter of the Pseudomonas putide TOL plasmid-encoded meta-cleavage pathway operon (P{sub m}) and the lacI gene encoding Lac repressor plus sylS, coding for the positive regulator of P{sub m}. The other element carries a fusion between the P{sub tac} promoter and the gef gene, which encodes a killing function. In the absence of effectors, expression of the P{sub tac}::gef cassette is no longer prevented and a high rate ofmore » cell killing is observed. The substitution of XylS for XylSthr45, a mutant regulator with altered effector specificity and increased affinity for benzoates, allows the control of populations able to degrade a wider range of benzoates at micromolar substrate concentrations. Given the wide effector specificity of the key regulators, the wild-type and mutant ZylS proteins, the system should allow the control of populations able to metabolize benzoate; methyl-, dimethyl-, chloro-, dichloro-, ethyl-, and methoxybenzoates; salicylate; and methyl- and chlorosalicylates. A small population of genetically engineered microorganisms became Gef resistant; however, the mechanism of such survival remains unknown.« less

Respiratory arsenate reductase as a bidirectional enzyme

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

Richey, Christine; Chovanec, Peter; Department of Chemistry and Biochemistry, Duquesne University, Pittsburgh, PA 15282

2009-05-01

The haloalkaliphilic bacterium Alkalilimnicola ehrlichii is capable of anaerobic chemolithoautotrophic growth by coupling the oxidation of arsenite (As(III)) to the reduction of nitrate and carbon dioxide. Analysis of its complete genome indicates that it lacks a conventional arsenite oxidase (Aox), but instead possesses two operons that each encode a putative respiratory arsenate reductase (Arr). Here we show that one homolog is expressed under chemolithoautotrophic conditions and exhibits both arsenite oxidase and arsenate reductase activity. We also demonstrate that Arr from two arsenate respiring bacteria, Alkaliphilus oremlandii and Shewanella sp. strain ANA-3, is also biochemically reversible. Thus Arr can function asmore » a reductase or oxidase. Its physiological role in a specific organism, however, may depend on the electron potentials of the molybdenum center and [Fe-S] clusters, additional subunits, or constitution of the electron transfer chain. This versatility further underscores the ubiquity and antiquity of microbial arsenic metabolism.« less

Respiratory arsenate reductase as a bidirectional enzyme

USGS Publications Warehouse

Richey, C.; Chovanec, P.; Hoeft, S.E.; Oremland, R.S.; Basu, P.; Stolz, J.F.

2009-01-01

The haloalkaliphilic bacterium Alkalilimnicola ehrlichii is capable of anaerobic chemolithoautotrophic growth by coupling the oxidation of arsenite (As(III)) to the reduction of nitrate and carbon dioxide. Analysis of its complete genome indicates that it lacks a conventional arsenite oxidase (Aox), but instead possesses two operons that each encode a putative respiratory arsenate reductase (Arr). Here we show that one homolog is expressed under chemolithoautotrophic conditions and exhibits both arsenite oxidase and arsenate reductase activity. We also demonstrate that Arr from two arsenate respiring bacteria, Alkaliphilus oremlandii and Shewanella sp. strain ANA-3, is also biochemically reversible. Thus Arr can function as a reductase or oxidase. Its physiological role in a specific organism, however, may depend on the electron potentials of the molybdenum center and [Fe–S] clusters, additional subunits, or constitution of the electron transfer chain. This versatility further underscores the ubiquity and antiquity of microbial arsenic metabolism.

Divergent Roles of RPA Homologs of the Model Archaeon Halobacterium salinarum in Survival of DNA Damage.

PubMed

Evans, Jessica J; Gygli, Patrick E; McCaskill, Julienne; DeVeaux, Linda C

2018-04-20

The haloarchaea are unusual in possessing genes for multiple homologs to the ubiquitous single-stranded DNA binding protein (SSB or replication protein A, RPA) found in all three domains of life. Halobacterium salinarum contains five homologs: two are eukaryotic in organization, two are prokaryotic and are encoded on the minichromosomes, and one is uniquely euryarchaeal. Radiation-resistant mutants previously isolated show upregulation of one of the eukaryotic-type RPA genes. Here, we have created deletions in the five RPA operons. These deletion mutants were exposed to DNA-damaging conditions: ionizing radiation, UV radiation, and mitomycin C. Deletion of the euryarchaeal homolog, although not lethal as in Haloferax volcanii , causes severe sensitivity to all of these agents. Deletion of the other RPA/SSB homologs imparts a variable sensitivity to these DNA-damaging agents, suggesting that the different RPA homologs have specialized roles depending on the type of genomic insult encountered.

Salmonella enterica serotype Typhimurium Std fimbriae bind terminal alpha(1,2)fucose residues in the cecal mucosa.

PubMed

Chessa, Daniela; Winter, Maria G; Jakomin, Marcello; Bäumler, Andreas J

2009-02-01

The std operon encodes a fimbrial adhesin of Salmonella enterica serotype Typhimurium that is required for attachment to intestinal epithelial cells and for cecal colonization in the mouse. To study the mechanism by which this virulence factor contributes to colonization we characterized its binding specificity. Std-mediated binding to human colonic epithelial (Caco-2) cells could be abrogated by removing N-linked glycans. Adherence of Std fimbriated S. Typhimurium to Caco-2 cells could be blocked by co-incubation with H type 2 oligosaccharide (Fucalpha1-2Galbeta1-4GlcNAc) or by pretreatment of cells with alpha1-2 fucosidase. In contrast, pretreatment of Caco-2 cells with neuraminidase or co-incubation with the type 2 disaccharide precursor (Galbeta1-4GlcNAc) did not reduce adherence of Std fimbriated S. Typhimurium. Binding of purified Std fimbriae to Fucalpha1-2Galbeta1-4GlcNAc in a solid phase binding assay was competitively inhibited by Ulex europaeus agglutinin-I (UEA-I), a lectin specific for Fucalpha1-2 moieties. Purified Std fimbriae and UEA both bound to a receptor localized in the mucus layer of the murine cecum. These data suggest that the std operon encodes an adhesin that binds an alpha1-2 fucosylated receptor(s) present in the cecal mucosa.

Insights from the draft genome into the pathogenicity of a clinical isolate of Elizabethkingia meningoseptica Em3.

PubMed

Chen, Shicheng; Soehnlen, Marty; Downes, Frances P; Walker, Edward D

2017-01-01

Elizabethkingia meningoseptica is an emerging, healthcare-associated pathogen causing a high mortality rate in immunocompromised patients. We report the draft genome sequence of E. meningoseptica Em3, isolated from sputum from a patient with multiple underlying diseases. The genome has a length of 4,037,922 bp, a GC-content 36.4%, and 3673 predicted protein-coding sequences. Average nucleotide identity analysis (>95%) assigned the bacterium to the species E. meningoseptica. Genome analysis showed presence of the curli formation and assembly operon and a gene encoding hemagglutinins, indicating ability to form biofilm. In vitro biofilm assays demonstrated that E. meningoseptica Em3 formed more biofilm than E. anophelis Ag1 and E. miricola Emi3, both lacking the curli operon. A gene encoding thiol-activated cholesterol-dependent cytolysin in E. meningoseptica Em3 (potentially involved in lysing host immune cells) was also absent in E. anophelis Ag1 and E. miricola Emi3. Strain Em3 showed α-hemolysin activity on blood agar medium, congruent with presence of hemolysin and cytolysin genes. Furthermore, presence of heme uptake and utilization genes demonstrated adaptations for bloodstream infections. Strain Em3 contained 12 genes conferring resistance to β-lactams, including β-lactamases class A, class B, and metallo-β-lactamases. Results of comparative genomic analysis here provide insights into the evolution of E. meningoseptica Em3 as a pathogen.

Salmonella enterica serotype Typhimurium Std fimbriae bind terminal α (1,2)fucose residues in the cecal mucosa

PubMed Central

Chessa, Daniela; Winter, Maria G.; Jakomin, Marcello; Bäumler, Andreas J.

2013-01-01

SUMMARY The std operon encodes a fimbrial adhesin of Salmonella enterica serotype Typhimurium that is required for attachment to intestinal epithelial cells and for cecal colonization in the mouse. To study the mechanism by which this virulence factor contributes to colonization we characterized its binding specificity. Std-mediated binding to human colonic epithelial (Caco-2) cells could be abrogated by removing N-linked glycans. Adherence of Std fimbriated S. Typhimurium to Caco-2 cells could be blocked by co-incubation with H type 2 oligosaccharide (Fucα1-2Galβ1-4GlcNAc) or by pretreatment of cells with α1-2 fucosidase. In contrast, pretreatment of Caco-2 cells with neuraminidase or co-incubation with the type 2 disaccharide precursor (Galβ1-4GlcNAc) did not reduce adherence of Std fimbriated S. Typhimurium. Binding of purified Std fimbriae to Fucα1-2Galβ1-4GlcNAc in a solid phase binding assay was competitively inhibited by Ulex europaeus agglutinin-I (UEA-I), a lectin specific for Fucα1-2 moieties. Purified Std fimbriae and UEA both bound to a receptor localized in the mucus layer of the murine cecum. These data suggest that the std operon encodes an adhesin that binds an α1-2 fucosylated receptor(s) present in the cecal mucosa. PMID:19183274

A Set of Activators and Repressors Control Peripheral Glucose Pathways in Pseudomonas putida To Yield a Common Central Intermediate▿

PubMed Central

del Castillo, Teresa; Duque, Estrella; Ramos, Juan L.

2008-01-01

Pseudomonas putida KT2440 channels glucose to the central Entner-Doudoroff intermediate 6-phosphogluconate through three convergent pathways. The genes for these convergent pathways are clustered in three independent regions on the host chromosome. A number of monocistronic units and operons coexist within each of these clusters, favoring coexpression of catabolic enzymes and transport systems. Expression of the three pathways is mediated by three transcriptional repressors, HexR, GnuR, and PtxS, and by a positive transcriptional regulator, GltR-2. In this study, we generated mutants in each of the regulators and carried out transcriptional assays using microarrays and transcriptional fusions. These studies revealed that HexR controls the genes that encode glucokinase/glucose 6-phosphate dehydrogenase that yield 6-phosphogluconate; the genes for the Entner-Doudoroff enzymes that yield glyceraldehyde-3-phosphate and pyruvate; and gap-1, which encodes glyceraldehyde-3-phosphate dehydrogenase. GltR-2 is the transcriptional regulator that controls specific porins for the entry of glucose into the periplasmic space, as well as the gtsABCD operon for glucose transport through the inner membrane. GnuR is the repressor of gluconate transport and gluconokinase responsible for the conversion of gluconate into 6-phosphogluconate. PtxS, however, controls the enzymes for oxidation of gluconate to 2-ketogluconate, its transport and metabolism, and a set of genes unrelated to glucose metabolism. PMID:18245293

Insight on specificity of uracil permeases of the NAT/NCS2 family from analysis of the transporter encoded in the pyrimidine utilization operon of Escherichia coli.

PubMed

Botou, Maria; Lazou, Panayiota; Papakostas, Konstantinos; Lambrinidis, George; Evangelidis, Thomas; Mikros, Emmanuel; Frillingos, Stathis

2018-04-01

The uracil permease UraA of Escherichia coli is a structurally known prototype for the ubiquitous Nucleobase-Ascorbate Transporter (NAT) or Nucleobase-Cation Symporter-2 (NCS2) family and represents a well-defined subgroup of bacterial homologs that remain functionally unstudied. Here, we analyze four of these homologs, including RutG of E. coli which shares 35% identity with UraA and is encoded in the catabolic rut (pyrimidine utilization) operon. Using amplified expression in E. coli K-12, we show that RutG is a high-affinity permease for uracil, thymine and, at low efficiency, xanthine and recognizes also 5-fluorouracil and oxypurinol. In contrast, UraA and the homologs from Acinetobacter calcoaceticus and Aeromonas veronii are permeases specific for uracil and 5-fluorouracil. Molecular docking indicates that thymine is hindered from binding to UraA by a highly conserved Phe residue which is absent in RutG. Site-directed replacement of this Phe with Ala in the three uracil-specific homologs allows high-affinity recognition and/or transport of thymine, emulating the RutG profile. Furthermore, all RutG orthologs from enterobacteria retain an Ala at this position, implying that they can use both uracil and thymine and, possibly, xanthine as substrates and provide the bacterial cell with a range of catabolizable nucleobases. © 2018 John Wiley & Sons Ltd.

Conditions for the Evolution of Gene Clusters in Bacterial Genomes

PubMed Central

Ballouz, Sara; Francis, Andrew R.; Lan, Ruiting; Tanaka, Mark M.

2010-01-01

Genes encoding proteins in a common pathway are often found near each other along bacterial chromosomes. Several explanations have been proposed to account for the evolution of these structures. For instance, natural selection may directly favour gene clusters through a variety of mechanisms, such as increased efficiency of coregulation. An alternative and controversial hypothesis is the selfish operon model, which asserts that clustered arrangements of genes are more easily transferred to other species, thus improving the prospects for survival of the cluster. According to another hypothesis (the persistence model), genes that are in close proximity are less likely to be disrupted by deletions. Here we develop computational models to study the conditions under which gene clusters can evolve and persist. First, we examine the selfish operon model by re-implementing the simulation and running it under a wide range of conditions. Second, we introduce and study a Moran process in which there is natural selection for gene clustering and rearrangement occurs by genome inversion events. Finally, we develop and study a model that includes selection and inversion, which tracks the occurrence and fixation of rearrangements. Surprisingly, gene clusters fail to evolve under a wide range of conditions. Factors that promote the evolution of gene clusters include a low number of genes in the pathway, a high population size, and in the case of the selfish operon model, a high horizontal transfer rate. The computational analysis here has shown that the evolution of gene clusters can occur under both direct and indirect selection as long as certain conditions hold. Under these conditions the selfish operon model is still viable as an explanation for the evolution of gene clusters. PMID:20168992

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.

Bioconversion of methanol to value-added mevalonate by engineered Methylobacterium extorquens AM1 containing an optimized mevalonate pathway.

PubMed

Zhu, Wen-Liang; Cui, Jin-Yu; Cui, Lan-Yu; Liang, Wei-Fan; Yang, Song; Zhang, Chong; Xing, Xin-Hui

2016-03-01

Methylotrophic biosynthesis using methanol as a feedstock is a promising and attractive method to solve the over-dependence of the bioindustry on sugar feedstocks derived from grains that are used for food. In this study, we introduced and engineered the mevalonate pathway into Methylobacterium extorquens AM1 to achieve high mevalonate production from methanol, which could be a platform for terpenoid synthesis. We first constructed a natural operon (MVE) harboring the mvaS and mvaE genes from Enterococcus faecalis as well as an artificial operon (MVH) harboring the hmgcs1 gene from Blattella germanica and the tchmgr gene from Trypanosoma cruzi that encoded enzymes with the highest reported activities. We achieved mevalonate titers of 56 and 66 mg/L, respectively, in flask cultivation. Introduction of the phaA gene from Ralstonia eutropha into the operon MVH increased the mevalonate titer to 180 mg/L, 3.2-fold higher than that of the natural operon MVE. Further modification of the expression level of the phaA gene by regulating the strength of the ribosomal binding site resulted in an additional 20 % increase in mevalonate production to 215 mg/L. A fed-batch fermentation of the best-engineered strain yielded a mevalonate titer of 2.22 g/L, which was equivalent to an overall yield and productivity of 28.4 mg mevalonate/g methanol and 7.16 mg/L/h, respectively. The production of mevalonate from methanol, which is the initial, but critical step linking methanol with valuable terpenoids via methylotrophic biosynthesis, represents a proof of concept for pathway engineering in M. extorquens AM1.

Cluster of Genes That Encode Positive and Negative Elements Influencing Filament Length in a Heterocyst-Forming Cyanobacterium

PubMed Central

Merino-Puerto, Victoria; Herrero, Antonia

2013-01-01

The filamentous, heterocyst-forming cyanobacteria perform oxygenic photosynthesis in vegetative cells and nitrogen fixation in heterocysts, and their filaments can be hundreds of cells long. In the model heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120, the genes in the fraC-fraD-fraE operon are required for filament integrity mainly under conditions of nitrogen deprivation. The fraC operon transcript partially overlaps gene all2395, which lies in the opposite DNA strand and ends 1 bp beyond fraE. Gene all2395 produces transcripts of 1.35 kb (major transcript) and 2.2 kb (minor transcript) that overlap fraE and whose expression is dependent on the N-control transcription factor NtcA. Insertion of a gene cassette containing transcriptional terminators between fraE and all2395 prevented production of the antisense RNAs and resulted in an increased length of the cyanobacterial filaments. Deletion of all2395 resulted in a larger increase of filament length and in impaired growth, mainly under N2-fixing conditions and specifically on solid medium. We denote all2395 the fraF gene, which encodes a protein restricting filament length. A FraF-green fluorescent protein (GFP) fusion protein accumulated significantly in heterocysts. Similar to some heterocyst differentiation-related proteins such as HglK, HetL, and PatL, FraF is a pentapeptide repeat protein. We conclude that the fraC-fraD-fraE←fraF gene cluster (where the arrow indicates a change in orientation), in which cis antisense RNAs are produced, regulates morphology by encoding proteins that influence positively (FraC, FraD, FraE) or negatively (FraF) the length of the filament mainly under conditions of nitrogen deprivation. This gene cluster is often conserved in heterocyst-forming cyanobacteria. PMID:23813733

Plasmid-Encoded Phthalate Catabolic Pathway in Arthrobacter keyseri 12B†

PubMed Central

Eaton, Richard W.

2001-01-01

Several 2-substituted benzoates (including 2-trifluoromethyl-, 2-chloro-, 2-bromo-, 2-iodo-, 2-nitro-, 2-methoxy-, and 2-acetyl-benzoates) were converted by phthalate-grown Arthrobacter keyseri (formerly Micrococcus sp.) 12B to the corresponding 2-substituted 3,4-dihydroxybenzoates (protocatechuates). Because these products lack a carboxyl group at the 2 position, they were not substrates for the next enzyme of the phthalate catabolic pathway, 3,4-dihydroxyphthalate 2-decarboxylase, and accumulated. When these incubations were carried out in iron-containing minimal medium, the products formed colored chelates. This chromogenic response was subsequently used to identify recombinant Escherichia coli strains carrying genes encoding the responsible enzymes, phthalate 3,4-dioxygenase and 3,4-dihydroxy-3,4-dihydrophthalate dehydrogenase, from the 130-kbp plasmid pRE1 of strain 12B. Beginning with the initially cloned 8.14-kbp PstI fragment of pRE824 as a probe to identify recombinant plasmids carrying overlapping fragments, a DNA segment of 33.5 kbp was cloned from pRE1 on several plasmids and mapped using restriction endonucleases. From these plasmids, the sequence of 26,274 contiguous bp was determined. Sequenced DNA included several genetic units: tnpR, pcm operon, ptr genes, pehA, norA fragment, and pht operon, encoding a transposon resolvase, catabolism of protocatechuate (3,4-dihydroxybenzoate), a putative ATP-binding cassette transporter, a possible phthalate ester hydrolase, a fragment of a norfloxacin resistance-like transporter, and the conversion of phthalate to protocatechuate, respectively. Activities of the eight enzymes involved in the catabolism of phthalate through protocatechuate to pyruvate and oxaloacetate were demonstrated in cells or cell extracts of recombinant E. coli strains. PMID:11371533

Growth rate regulation of Escherichia coli acetyl coenzyme A carboxylase, which catalyzes the first committed step of lipid biosynthesis.

PubMed Central

Li, S J; Cronan, J E

1993-01-01

Acetyl coenzyme A (CoA) carboxylase catalyzes the synthesis of malonyl-CoA, the first intermediate of fatty acid synthesis. The Escherichia coli enzyme is encoded by four subunits located at three different positions on the E. coli chromosome. The accBC genes lie in a small operon at min 72, whereas accA and accD are located at min 4.3 and 50, respectively. We examined the expression of the genes that encode the E. coli acetyl-CoA carboxylase subunits (accA, accBC, and accD) under a variety of growth conditions by quantitative Northern (RNA) blot analysis. We found a direct correlation between the levels of transcription of the acc genes and the rate of cellular growth. Consistent results were also obtained upon nutritional upshift and downshift experiments and upon dilution of stationary-phase cultures into fresh media. We also determined the 5' end of the accA and accD mRNAs by primer extension and did transcriptional fusion analysis of the previously reported accBC promoter. Several interesting features were found in the promoter regions of these genes, including a bent DNA sequence and an open reading frame within the unusually long leader mRNA of the accBC operon, potential stem-loop structures in the accA and accD mRNA leader regions, and a stretch of GC-rich sequences followed by AT-rich sequences common to all three promoters. In addition, both accA and accD are located in complex gene clusters. For example, the accA promoter was localized within the upstream polC gene (which encodes the DNA polymerase III catalytic subunit), suggesting that additional regulatory mechanisms exist. Images PMID:7678242

The Xylella fastidosa RTX operons: evidence for the evolution of protein mosaics through novel genetic exchanges.

PubMed

Gambetta, Gregory A; Matthews, Mark A; Syvanen, Michael

2018-05-04

Xylella fastidiosa (Xf) is a gram negative bacterium inhabiting the plant vascular system. In most species this bacterium lives as a benign symbiote, but in several agriculturally important plants (e.g. coffee, citrus, grapevine) Xf is pathogenic. Xf has four loci encoding homologues to hemolysin RTX proteins, virulence factors involved in a wide range of plant pathogen interactions. We show that all four genes are expressed during pathogenesis in grapevine. The sequences from these four genes have a complex repetitive structure. At the C-termini, sequence diversity between strains is what would be expected from orthologous genes. However, within strains there is no N-terminal homology, indicating these loci encode RTXs of different functions and/or specificities. More striking is that many of the orthologous loci between strains share this extreme variation at the N-termini. Thus these RTX orthologues are most easily visualized as fusions between the orthologous C-termini and different N-termini. Further, the four genes are found in operons having a peculiar structure with an extensively duplicated module encoding a small protein with homology to the N-terminal region of the full length RTX. Surprisingly, some of these small peptides are most similar not to their corresponding full length RTX, but to the N-termini of RTXs from other Xf strains, and even other remotely related species. These results demonstrate that these genes are expressed in planta during pathogenesis. Their structure suggests extensive evolutionary restructuring through horizontal gene transfers and heterologous recombination mechanisms. The sum of the evidence suggests these repetitive modules are a novel kind of mobile genetic element.

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

Lactococcus lactis LMG2081 Produces Two Bacteriocins, a Nonlantibiotic and a Novel Lantibiotic.

PubMed

Mirkovic, Nemanja; Polovic, Natalija; Vukotic, Goran; Jovcic, Branko; Miljkovic, Marija; Radulovic, Zorica; Diep, Dzung B; Kojic, Milan

2016-04-01

Bacteriocin producers normally possess dedicated immunity systems to protect themselves from their own bacteriocins.Lactococcus lactis strains LMG2081 and BGBM50 are known as lactococcin G producers. However, BGBM50 was sensitive to LMG2081, which indicated that LMG2081 might produce additional bacteriocins that are not present in BGBM50. Therefore, whole-genome sequencing of the two strains was performed, and a lantibiotic operon (called lctLMG) was identified in LMG2081 but not in BGBM50. The lctLMG operon contains six open reading frames; the first three genes,lmgA ,lmgM, and lmgT, are involved in the biosynthesis and export of bacteriocin, while the other three genes,lmgF,lmgE, and lmgG, are involved in lantibiotic immunity. Mutational analysis confirmed that the lctLMG operon is responsible for the additional antimicrobial activity. Specifically, site-directed mutation within this operon rendered LMG2081 inactive toward BGBM50. Subsequent purification and electrospray ionization-time of flight mass spectrometric analysis confirmed that the lantibiotic bacteriocin called lacticin LMG is exported as a 25-amino-acid peptide. Lacticin LMG is highly similar to the lacticin 481 group. It is interesting that a bacteriocin producer produces two different classes of bacteriocins, whose operons are located in the chromosome and a plasmid. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Endoribonuclease type II toxin-antitoxin systems: functional or selfish?

PubMed

Ramisetty, Bhaskar Chandra Mohan; Santhosh, Ramachandran Sarojini

2017-07-01

Most bacterial genomes have multiple type II toxin-antitoxin systems (TAs) that encode two proteins which are referred to as a toxin and an antitoxin. Toxins inhibit a cellular process, while the interaction of the antitoxin with the toxin attenuates the toxin's activity. Endoribonuclease-encoding TAs cleave RNA in a sequence-dependent fashion, resulting in translational inhibition. To account for their prevalence and retention by bacterial genomes, TAs are credited with clinically significant phenomena, such as bacterial programmed cell death, persistence, biofilms and anti-addiction to plasmids. However, the programmed cell death and persistence hypotheses have been challenged because of conceptual, methodological and/or strain issues. In an alternative view, chromosomal TAs seem to be retained by virtue of addiction at two levels: via a poison-antidote combination (TA proteins) and via transcriptional reprogramming of the downstream core gene (due to integration). Any perturbation in the chromosomal TA operons could cause fitness loss due to polar effects on the downstream genes and hence be detrimental under natural conditions. The endoribonucleases encoding chromosomal TAs are most likely selfish DNA as they are retained by bacterial genomes, even though TAs do not confer a direct advantage via the TA proteins. TAs are likely used by various replicons as 'genetic arms' that allow the maintenance of themselves and associated genetic elements. TAs seem to be the 'selfish arms' that make the best use of the 'arms race' between bacterial genomes and plasmids.

[Regulation of sporulation by two-component system YvcPQ in Bacillus thuringiensis].

PubMed

Fan, Qingyun; Zhang, Shumeng; Gong, Yujing; He, Jin

2017-01-04

To study the regulation of sporulation controlled by two-component system (TCS) YvcPQ. β-galactosidase experiment was used to verify the regulation of YvcP on kapD expression; bacterial one-hybrid assay, EMSA and RT-qPCR were applied to study the regulation of AbrB on yvcPQ expression; markerless gene deletion coupled with spore count was used to reveal the influence of yvcPQ and kapD expressions on sporulation. transcriptional regulator AbrB up-regulated the expression of yvcPQ; YvcP promoted the expression of kapD to inhibit sporulation. AbrB up-regulated the transcription of yvcPQ operon, then the increased YvcP strengthened the transcriptional acitivation of sporulation inhibitor gene kapD, and subsequently inhibited sporulation.

Regulation of the Salmonella enterica std fimbrial operon by DNA adenine methylation, SeqA, and HdfR.

PubMed

Jakomin, Marcello; Chessa, Daniela; Bäumler, Andreas J; Casadesús, Josep

2008-11-01

DNA adenine methylase (dam) mutants of Salmonella enterica serovar Typhimurium grown under laboratory conditions express the std fimbrial operon, which is tightly repressed in the wild type. Here, we show that uncontrolled production of Std fimbriae in S. enterica serovar Typhimurium dam mutants contributes to attenuation in mice, as indicated by the observation that an stdA dam strain is more competitive than a dam strain upon oral infection. Dam methylation appears to regulate std transcription, rather than std mRNA stability or turnover. A genetic screen for std regulators showed that the GATC-binding protein SeqA directly or indirectly represses std expression, while the poorly characterized yifA gene product serves as an std activator. YifA encodes a putative LysR-like protein and has been renamed HdfR, like its Escherichia coli homolog. Activation of std expression by HdfR is observed only in dam and seqA backgrounds. These data suggest that HdfR directly or indirectly activates std transcription. Since SeqA is unable to bind nonmethylated DNA, it is possible that std operon derepression in dam and seqA mutants may result from unconstrained HdfR-mediated activation of std transcription. Derepression of std in dam and seqA mutants of S. enterica occurs in only a fraction of the bacterial population, suggesting the occurrence of either bistable expression or phase variation.

Crystal structure of the toxin Msmeg_6760, the structural homolog of Mycobacterium tuberculosis Rv2035, a novel type II toxin involved in the hypoxic response

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

Bajaj, R. Alexandra; Arbing, Mark A.; Shin, Annie

The structure of Msmeg_6760, a protein of unknown function, has been determined. Biochemical and bioinformatics analyses determined that Msmeg_6760 interacts with a protein encoded in the same operon, Msmeg_6762, and predicted that the operon is a toxin–antitoxin (TA) system. Structural comparison of Msmeg_6760 with proteins of known function suggests that Msmeg_6760 binds a hydrophobic ligand in a buried cavity lined by large hydrophobic residues. Access to this cavity could be controlled by a gate–latch mechanism. The function of the Msmeg_6760 toxin is unknown, but structure-based predictions revealed that Msmeg_6760 and Msmeg_6762 are homologous to Rv2034 and Rv2035, a predicted novelmore » TA system involved inMycobacterium tuberculosislatency during macrophage infection. The Msmeg_6760 toxin fold has not been previously described for bacterial toxins and its unique structural features suggest that toxin activation is likely to be mediated by a novel mechanism.« less

The Myxococcus xanthus two-component system CorSR regulates expression of a gene cluster involved in maintaining copper tolerance during growth and development.

PubMed

Sánchez-Sutil, María Celestina; Pérez, Juana; Gómez-Santos, Nuria; Shimkets, Lawrence J; Moraleda-Muñoz, Aurelio; Muñoz-Dorado, José

2013-01-01

Myxococcus xanthus is a soil-dwelling member of the δ-Proteobacteria that exhibits a complex developmental cycle upon starvation. Development comprises aggregation and differentiation into environmentally resistant myxospores in an environment that includes fluctuations in metal ion concentrations. While copper is essential for M. xanthus cells because several housekeeping enzymes use it as a cofactor, high copper concentrations are toxic. These opposing effects force cells to maintain a tight copper homeostasis. A plethora of paralogous genes involved in copper detoxification, all of which are differentially regulated, have been reported in M. xanthus. The use of in-frame deletion mutants and fusions with the reporter gene lacZ has allowed the identification of a two-component system, CorSR, that modulates the expression of an operon termed curA consisting of nine genes whose expression slowly increases after metal addition, reaching a plateau. Transcriptional regulation of this operon is complex because transcription can be initiated at different promoters and by different types of regulators. These genes confer copper tolerance during growth and development. Copper induces carotenoid production in a ΔcorSR mutant at lower concentrations than with the wild-type strain due to lack of expression of a gene product resembling subunit III of cbb3-type cytochrome c oxidase. This data may explain why copper induces carotenoid biosynthesis at suboptimal rather than optimal growth conditions in wild-type strains.

The Myxococcus xanthus Two-Component System CorSR Regulates Expression of a Gene Cluster Involved in Maintaining Copper Tolerance during Growth and Development

PubMed Central

Sánchez-Sutil, María Celestina; Pérez, Juana; Gómez-Santos, Nuria; Shimkets, Lawrence J.; Moraleda-Muñoz, Aurelio; Muñoz-Dorado, José

2013-01-01

Myxococcus xanthus is a soil-dwelling member of the δ–Proteobacteria that exhibits a complex developmental cycle upon starvation. Development comprises aggregation and differentiation into environmentally resistant myxospores in an environment that includes fluctuations in metal ion concentrations. While copper is essential for M. xanthus cells because several housekeeping enzymes use it as a cofactor, high copper concentrations are toxic. These opposing effects force cells to maintain a tight copper homeostasis. A plethora of paralogous genes involved in copper detoxification, all of which are differentially regulated, have been reported in M. xanthus. The use of in-frame deletion mutants and fusions with the reporter gene lacZ has allowed the identification of a two-component system, CorSR, that modulates the expression of an operon termed curA consisting of nine genes whose expression slowly increases after metal addition, reaching a plateau. Transcriptional regulation of this operon is complex because transcription can be initiated at different promoters and by different types of regulators. These genes confer copper tolerance during growth and development. Copper induces carotenoid production in a ΔcorSR mutant at lower concentrations than with the wild-type strain due to lack of expression of a gene product resembling subunit III of cbb3-type cytochrome c oxidase. This data may explain why copper induces carotenoid biosynthesis at suboptimal rather than optimal growth conditions in wild-type strains. PMID:23874560

Molecular cloning and characterization of two genes for the biotin carboxylase and carboxyltransferase subunits of acetyl coenzyme A carboxylase in Myxococcus xanthus.

PubMed

Kimura, Y; Miyake, R; Tokumasu, Y; Sato, M

2000-10-01

We have cloned a DNA fragment from a genomic library of Myxococcus xanthus using an oligonucleotide probe representing conserved regions of biotin carboxylase subunits of acetyl coenzyme A (acetyl-CoA) carboxylases. The fragment contained two open reading frames (ORF1 and ORF2), designated the accB and accA genes, capable of encoding a 538-amino-acid protein of 58.1 kDa and a 573-amino-acid protein of 61.5 kDa, respectively. The protein (AccA) encoded by the accA gene was strikingly similar to biotin carboxylase subunits of acetyl-CoA and propionyl-CoA carboxylases and of pyruvate carboxylase. The putative motifs for ATP binding, CO(2) fixation, and biotin binding were found in AccA. The accB gene was located upstream of the accA gene, and they formed a two-gene operon. The protein (AccB) encoded by the accB gene showed high degrees of sequence similarity with carboxyltransferase subunits of acetyl-CoA and propionyl-CoA carboxylases and of methylmalonyl-CoA decarboxylase. Carboxybiotin-binding and acyl-CoA-binding domains, which are conserved in several carboxyltransferase subunits of acyl-CoA carboxylases, were found in AccB. An accA disruption mutant showed a reduced growth rate and reduced acetyl-CoA carboxylase activity compared with the wild-type strain. Western blot analysis indicated that the product of the accA gene was a biotinylated protein that was expressed during the exponential growth phase. Based on these results, we propose that this M. xanthus acetyl-CoA carboxylase consists of two subunits, which are encoded by the accB and accA genes, and occupies a position between prokaryotic and eukaryotic acetyl-CoA carboxylases in terms of evolution.

The CsrR/CsrS two-component system of group A Streptococcus responds to environmental Mg2+.

PubMed

Gryllos, Ioannis; Levin, James C; Wessels, Michael R

2003-04-01

Group A streptococci control expression of key virulence determinants via the two-component sensorregulator system CsrRCsrS. The membrane-bound sensor CsrS is thought to respond to previously unknown environmental signal(s) by controlling phosphorylation of its cognate regulator component CsrR. Phosphorylation of CsrR increases its affinity for binding to the promoter regions of Csr-regulated genes to repress transcription. Here we show that environmental Mg(2+) concentration is a potent and specific stimulus for CsrRCsrS-mediated regulation. We studied the effect of divalent cations on expression of the Csr-regulated hyaluronic acid capsule genes (hasABC) by measuring chloramphenicol acetyltransferase (CAT) activity in a reporter strain of group A Streptococcus carrying a has operon promoter-cat fusion. Addition of Mg(2+), but not of Ca(2+), Mn(2+), or Zn(2+), repressed capsule gene expression by up to 80% in a dose-dependent fashion. The decrease in capsule gene transcription was associated with a marked reduction in cell-associated capsular polysaccharide. RNA hybridization analysis demonstrated reduced expression of the Csr-regulated hasABC operon, streptokinase (ska), and streptolysin S (sagA) during growth in the presence of 15 mM Mg(2+) for the wild-type strain 003CAT but not for an isogenic csrS mutant. We propose that Mg(2+) binds to CsrS to induce phosphorylation of CsrR and subsequent repression of virulence gene expression. The low concentration of Mg(2+) in extracellular body fluids predicts that the CsrRCsrS system is maintained in the inactive state during infection, thereby allowing maximal expression of critical virulence determinants in the human host.

Parallel Evolution and Horizontal Gene Transfer of the pst Operon in Firmicutes from Oligotrophic Environments

PubMed Central

Moreno-Letelier, Alejandra; Olmedo, Gabriela; Eguiarte, Luis E.; Martinez-Castilla, Leon; Souza, Valeria

2011-01-01

The high affinity phosphate transport system (pst) is crucial for phosphate uptake in oligotrophic environments. Cuatro Cienegas Basin (CCB) has extremely low P levels and its endemic Bacillus are closely related to oligotrophic marine Firmicutes. Thus, we expected the pst operon of CCB to share the same evolutionary history and protein similarity to marine Firmicutes. Orthologs of the pst operon were searched in 55 genomes of Firmicutes and 13 outgroups. Phylogenetic reconstructions were performed for the pst operon and 14 concatenated housekeeping genes using maximum likelihood methods. Conserved domains and 3D structures of the phosphate-binding protein (PstS) were also analyzed. The pst operon of Firmicutes shows two highly divergent clades with no correlation to the type of habitat nor a phylogenetic congruence, suggesting horizontal gene transfer. Despite sequence divergence, the PstS protein had a similar 3D structure, which could be due to parallel evolution after horizontal gene transfer events. PMID:21461370

Engineering of photosynthetic mannitol biosynthesis from CO2 in a cyanobacterium.

PubMed

Jacobsen, Jacob H; Frigaard, Niels-Ulrik

2014-01-01

D-Mannitol (hereafter denoted mannitol) is used in the medical and food industry and is currently produced commercially by chemical hydrogenation of fructose or by extraction from seaweed. Here, the marine cyanobacterium Synechococcus sp. PCC 7002 was genetically modified to photosynthetically produce mannitol from CO2 as the sole carbon source. Two codon-optimized genes, mannitol-1-phosphate dehydrogenase (mtlD) from Escherichia coli and mannitol-1-phosphatase (mlp) from the protozoan chicken parasite Eimeria tenella, in combination encoding a biosynthetic pathway from fructose-6-phosphate to mannitol, were expressed in the cyanobacterium resulting in accumulation of mannitol in the cells and in the culture medium. The mannitol biosynthetic genes were expressed from a single synthetic operon inserted into the cyanobacterial chromosome by homologous recombination. The mannitol biosynthesis operon was constructed using a novel uracil-specific excision reagent (USER)-based polycistronic expression system characterized by ligase-independent, directional cloning of the protein-encoding genes such that the insertion site was regenerated after each cloning step. Genetic inactivation of glycogen biosynthesis increased the yield of mannitol presumably by redirecting the metabolic flux to mannitol under conditions where glycogen normally accumulates. A total mannitol yield equivalent to 10% of cell dry weight was obtained in cell cultures synthesizing glycogen while the yield increased to 32% of cell dry weight in cell cultures deficient in glycogen synthesis; in both cases about 75% of the mannitol was released from the cells into the culture medium by an unknown mechanism. The highest productivity was obtained in a glycogen synthase deficient culture that after 12 days showed a mannitol concentration of 1.1 g mannitol L(-1) and a production rate of 0.15 g mannitol L(-1) day(-1). This system may be useful for biosynthesis of valuable sugars and sugar derivatives from CO2 in cyanobacteria. © 2013 International Metabolic Engineering Society Published by International Metabolic Engineering Society All rights reserved.

Arsenic Detoxification by Geobacter Species.

PubMed

Dang, Yan; Walker, David J F; Vautour, Kaitlin E; Dixon, Steven; Holmes, Dawn E

2017-02-15

Insight into the mechanisms for arsenic detoxification by Geobacter species is expected to improve the understanding of global cycling of arsenic in iron-rich subsurface sedimentary environments. Analysis of 14 different Geobacter genomes showed that all of these species have genes coding for an arsenic detoxification system (ars operon), and several have genes required for arsenic respiration (arr operon) and methylation (arsM). Genes encoding four arsenic repressor-like proteins were detected in the genome of G. sulfurreducens; however, only one (ArsR1) regulated transcription of the ars operon. Elimination of arsR1 from the G. sulfurreducens chromosome resulted in enhanced transcription of genes coding for the arsenic efflux pump (Acr3) and arsenate reductase (ArsC). When the gene coding for Acr3 was deleted, cells were not able to grow in the presence of either the oxidized or reduced form of arsenic, while arsC deletion mutants could grow in the presence of arsenite but not arsenate. These studies shed light on how Geobacter influences arsenic mobility in anoxic sediments and may help us develop methods to remediate arsenic contamination in the subsurface. This study examines arsenic transformation mechanisms utilized by Geobacter, a genus of iron-reducing bacteria that are predominant in many anoxic iron-rich subsurface environments. Geobacter species play a major role in microbially mediated arsenic release from metal hydroxides in the subsurface. This release raises arsenic concentrations in drinking water to levels that are high enough to cause major health problems. Therefore, information obtained from studies of Geobacter should shed light on arsenic cycling in iron-rich subsurface sedimentary environments, which may help reduce arsenic-associated illnesses. These studies should also help in the development of biosensors that can be used to detect arsenic contaminants in anoxic subsurface environments. We examined 14 different Geobacter genomes and found that all of these species possess genes coding for an arsenic detoxification system (ars operon), and some also have genes required for arsenic respiration (arr operon) and arsenic methylation (arsM). Copyright © 2017 American Society for Microbiology.

Arsenic Detoxification by Geobacter Species

PubMed Central

Walker, David J. F.; Vautour, Kaitlin E.; Dixon, Steven

2016-01-01

ABSTRACT Insight into the mechanisms for arsenic detoxification by Geobacter species is expected to improve the understanding of global cycling of arsenic in iron-rich subsurface sedimentary environments. Analysis of 14 different Geobacter genomes showed that all of these species have genes coding for an arsenic detoxification system (ars operon), and several have genes required for arsenic respiration (arr operon) and methylation (arsM). Genes encoding four arsenic repressor-like proteins were detected in the genome of G. sulfurreducens; however, only one (ArsR1) regulated transcription of the ars operon. Elimination of arsR1 from the G. sulfurreducens chromosome resulted in enhanced transcription of genes coding for the arsenic efflux pump (Acr3) and arsenate reductase (ArsC). When the gene coding for Acr3 was deleted, cells were not able to grow in the presence of either the oxidized or reduced form of arsenic, while arsC deletion mutants could grow in the presence of arsenite but not arsenate. These studies shed light on how Geobacter influences arsenic mobility in anoxic sediments and may help us develop methods to remediate arsenic contamination in the subsurface. IMPORTANCE This study examines arsenic transformation mechanisms utilized by Geobacter, a genus of iron-reducing bacteria that are predominant in many anoxic iron-rich subsurface environments. Geobacter species play a major role in microbially mediated arsenic release from metal hydroxides in the subsurface. This release raises arsenic concentrations in drinking water to levels that are high enough to cause major health problems. Therefore, information obtained from studies of Geobacter should shed light on arsenic cycling in iron-rich subsurface sedimentary environments, which may help reduce arsenic-associated illnesses. These studies should also help in the development of biosensors that can be used to detect arsenic contaminants in anoxic subsurface environments. We examined 14 different Geobacter genomes and found that all of these species possess genes coding for an arsenic detoxification system (ars operon), and some also have genes required for arsenic respiration (arr operon) and arsenic methylation (arsM). PMID:27940542

A four-gene operon in Bacillus cereus produces two rare spore-decorating sugars

PubMed Central

Li, Zi; Mukherjee, Thiya; Bowler, Kyle; Namdari, Sholeh; Snow, Zachary; Prestridge, Sarah; Carlton, Alexandra; Bar-Peled, Maor

2017-01-01

Bacterial glycan structures on cell surfaces are critical for cell-cell recognition and adhesion and in host-pathogen interactions. Accordingly, unraveling the sugar composition of bacterial cell surfaces can shed light on bacterial growth and pathogenesis. Here, we found that two rare sugars with a 3-C-methyl-6-deoxyhexose structure were linked to spore glycans in Bacillus cereus ATCC 14579 and ATCC 10876. Moreover, we identified a four-gene operon in B. cereus ATCC 14579 that encodes proteins with the following sequential enzyme activities as determined by mass spectrometry and one- and two-dimensional NMR methods: CTP:glucose-1-phosphate cytidylyltransferase, CDP-Glc 4,6-dehydratase, NADH-dependent SAM:C-methyltransferase, and NADPH-dependent CDP-3-C-methyl-6-deoxyhexose 4-reductase. The last enzyme predominantly yielded CDP-3-C-methyl-6-deoxygulose (CDP-cereose) and likely generated a 4-epimer CDP-3-C-methyl-6-deoxyallose (CDP-cillose). Some members of the B. cereus sensu lato group produce CDP-3-C-methyl-6-deoxy sugars for the formation of cereose-containing glycans on spores, whereas others such as Bacillus anthracis do not. Gene knockouts of the Bacillus C-methyltransferase and the 4-reductase confirmed their involvement in the formation of cereose-containing glycan on B. cereus spores. We also found that cereose represented 0.2–1% spore dry weight. Moreover, mutants lacking cereose germinated faster than the wild type, yet the mutants exhibited no changes in sporulation or spore resistance to heat. The findings reported here may provide new insights into the roles of the uncommon 3-C-methyl-6-deoxy sugars in cell-surface recognition and host-pathogen interactions of the genus Bacillus. PMID:28298443

A four-gene operon in Bacillus cereus produces two rare spore-decorating sugars.

PubMed

Li, Zi; Mukherjee, Thiya; Bowler, Kyle; Namdari, Sholeh; Snow, Zachary; Prestridge, Sarah; Carlton, Alexandra; Bar-Peled, Maor

2017-05-05

Bacterial glycan structures on cell surfaces are critical for cell-cell recognition and adhesion and in host-pathogen interactions. Accordingly, unraveling the sugar composition of bacterial cell surfaces can shed light on bacterial growth and pathogenesis. Here, we found that two rare sugars with a 3- C -methyl-6-deoxyhexose structure were linked to spore glycans in Bacillus cereus ATCC 14579 and ATCC 10876. Moreover, we identified a four-gene operon in B. cereus ATCC 14579 that encodes proteins with the following sequential enzyme activities as determined by mass spectrometry and one- and two-dimensional NMR methods: CTP:glucose-1-phosphate cytidylyltransferase, CDP-Glc 4,6-dehydratase, NADH-dependent SAM: C -methyltransferase, and NADPH-dependent CDP-3- C -methyl-6-deoxyhexose 4-reductase. The last enzyme predominantly yielded CDP-3- C -methyl-6-deoxygulose (CDP-cereose) and likely generated a 4-epimer CDP-3- C -methyl-6-deoxyallose (CDP-cillose). Some members of the B. cereus sensu lato group produce CDP-3- C -methyl-6-deoxy sugars for the formation of cereose-containing glycans on spores, whereas others such as Bacillus anthracis do not. Gene knockouts of the Bacillus C -methyltransferase and the 4-reductase confirmed their involvement in the formation of cereose-containing glycan on B. cereus spores. We also found that cereose represented 0.2-1% spore dry weight. Moreover, mutants lacking cereose germinated faster than the wild type, yet the mutants exhibited no changes in sporulation or spore resistance to heat. The findings reported here may provide new insights into the roles of the uncommon 3- C -methyl-6-deoxy sugars in cell-surface recognition and host-pathogen interactions of the genus Bacillus . © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Altered Regulation of Escherichia coli Biotin Biosynthesis in BirA Superrepressor Mutant Strains

PubMed Central

Chakravartty, Vandana

2012-01-01

Transcription of the Escherichia coli biotin (bio) operon is directly regulated by the biotin protein ligase BirA, the enzyme that covalently attaches biotin to its cognate acceptor proteins. Binding of BirA to the bio operator requires dimerization of the protein, which is triggered by BirA-catalyzed synthesis of biotinoyl-adenylate (biotinoyl-5′-AMP), the obligatory intermediate of the ligation reaction. Although several aspects of this regulatory system are well understood, no BirA superrepressor mutant strains had been isolated. Such superrepressor BirA proteins would repress the biotin operon transcription in vivo at biotin concentrations well below those needed for repression by wild-type BirA. We isolated mutant strains having this phenotype by a combined selection-screening approach and resolved multiple mutations to give several birA superrepressor alleles, each having a single mutation, all of which showed repression dominant over that of the wild-type allele. All of these mutant strains repressed bio operon transcription in vivo at biotin concentrations that gave derepression of the wild-type strain and retained sufficient ligation activity for growth when overexpressed. All of the strains except that encoding G154D BirA showed derepression of bio operon transcription upon overproduction of a biotin-accepting protein. In BirA, G154D was a lethal mutation in single copy, and the purified protein was unable to transfer biotin from enzyme-bound biotinoyl-adenylate either to the natural acceptor protein or to a biotin-accepting peptide sequence. Consistent with the transcriptional repression data, each of the purified mutant proteins showed increased affinity for the biotin operator DNA in electrophoretic mobility shift assays. Surprisingly, although most of the mutations were located in the catalytic domain, all of those tested, except G154D BirA, had normal ligase activity. Most of the mutations that gave superrepressor phenotypes altered residues located close to the dimerization interface of BirA. However, two mutations were located at sites well removed from the interface. The properties of the superrepressor mutants strengthen and extend other data indicating that BirA function entails extensive interactions among the three domains of the protein and show that normal ligase activity does not ensure normal DNA binding. PMID:22210766

Phosphate assimilation in Rhizobium (Sinorhizobium) meliloti: identification of a pit-like gene.

PubMed

Bardin, S D; Voegele, R T; Finan, T M

1998-08-01

Rhizobium meliloti mutants defective in the phoCDET-encoded phosphate transport system form root nodules on alfalfa plants that fail to fix nitrogen (Fix-). We have previously reported that two classes of second-site mutations can suppress the Fix- phenotype of phoCDET mutants to Fix+. Here we show that one of these suppressor loci (sfx1) contains two genes, orfA and pit, which appear to form an operon transcribed in the order orfA-pit. The Pit protein is homologous to various phosphate transporters, and we present evidence that three suppressor mutations arose from a single thymidine deletion in a hepta-thymidine sequence centered 54 nucleotides upstream of the orfA transcription start site. This mutation increased the level of orfA-pit transcription. These data, together with previous biochemical evidence, show that the orfA-pit genes encode a Pi transport system that is expressed in wild-type cells grown with excess Pi but repressed in cells under conditions of Pi limitation. In phoCDET mutant cells, orfA-pit expression is repressed, but this repression is alleviated by the second-site suppressor mutations. Suppression increases orfA-pit expression compensating for the deficiencies in phosphate assimilation and symbiosis of the phoCDET mutants.

Conjugative Plasmid Transfer in Xylella fastidiosa Is Dependent on tra and trb Operon Functions

PubMed Central

Van Horn, Christopher R.

2017-01-01

ABSTRACT The insect-transmitted plant pathogen Xylella fastidiosa is capable of efficient horizontal gene transfer (HGT) and recombination. Natural transformation occurs at high rates in X. fastidiosa, but there also is evidence that certain strains of X. fastidiosa carry native plasmids equipped with transfer and mobilization genes, suggesting conjugation as an additional mechanism of HGT in some instances. Two operons, tra and trb, putatively encoding a conjugative type IV secretion system, are found in some but not all X. fastidiosa isolates, often on native plasmids. X. fastidiosa strains that carry the conjugative transfer genes can belong to different subspecies and frequently differ in host ranges. Using X. fastidiosa strain M23 (X. fastidiosa subsp. fastidiosa) or Dixon (X. fastidiosa subsp. multiplex) as the donor strain and Temecula (X. fastidiosa subsp. fastidiosa) as the recipient strain, plasmid transfer was characterized using the mobilizable broad-host-range vector pBBR5pemIK. Transfer of plasmid pBBR5pemIK was observed under in vitro conditions with both donor strains and was dependent on both tra and trb operon functions. A conjugative mechanism likely contributes to gene transfer between diverse strains of X. fastidiosa, possibly facilitating adaptation to new environments or different hosts. IMPORTANCE Xylella fastidiosa is an important plant pathogen worldwide, infecting a wide range of different plant species. The emergence of new diseases caused by X. fastidiosa, or host switching of existing strains, is thought to be primarily due to the high frequency of HGT and recombination in this pathogen. Transfer of plasmids by a conjugative mechanism enables movement of larger amounts of genetic material at one time, compared with other routes of gene transfer such as natural transformation. Establishing the prevalence and functionality of this mechanism in X. fastidiosa contributes to a better understanding of HGT, adaptation, and disease emergence in this diverse pathogen. PMID:28808128

Conjugative Plasmid Transfer in Xylella fastidiosa Is Dependent on tra and trb Operon Functions.

PubMed

Burbank, Lindsey P; Van Horn, Christopher R

2017-11-01

The insect-transmitted plant pathogen Xylella fastidiosa is capable of efficient horizontal gene transfer (HGT) and recombination. Natural transformation occurs at high rates in X. fastidiosa , but there also is evidence that certain strains of X. fastidiosa carry native plasmids equipped with transfer and mobilization genes, suggesting conjugation as an additional mechanism of HGT in some instances. Two operons, tra and trb , putatively encoding a conjugative type IV secretion system, are found in some but not all X. fastidiosa isolates, often on native plasmids. X. fastidiosa strains that carry the conjugative transfer genes can belong to different subspecies and frequently differ in host ranges. Using X. fastidiosa strain M23 ( X. fastidiosa subsp. fastidiosa ) or Dixon ( X. fastidiosa subsp. multiplex ) as the donor strain and Temecula ( X. fastidiosa subsp. fastidiosa ) as the recipient strain, plasmid transfer was characterized using the mobilizable broad-host-range vector pBBR5pemIK. Transfer of plasmid pBBR5pemIK was observed under in vitro conditions with both donor strains and was dependent on both tra and trb operon functions. A conjugative mechanism likely contributes to gene transfer between diverse strains of X. fastidiosa , possibly facilitating adaptation to new environments or different hosts. IMPORTANCE Xylella fastidiosa is an important plant pathogen worldwide, infecting a wide range of different plant species. The emergence of new diseases caused by X. fastidiosa , or host switching of existing strains, is thought to be primarily due to the high frequency of HGT and recombination in this pathogen. Transfer of plasmids by a conjugative mechanism enables movement of larger amounts of genetic material at one time, compared with other routes of gene transfer such as natural transformation. Establishing the prevalence and functionality of this mechanism in X. fastidiosa contributes to a better understanding of HGT, adaptation, and disease emergence in this diverse pathogen.

Regulation of Multiple Carbon Monoxide Consumption Pathways in Anaerobic Bacteria

PubMed Central

Techtmann, Stephen M.; Colman, Albert S.; Murphy, Michael B.; Schackwitz, Wendy S.; Goodwin, Lynne A.; Robb, Frank T.

2011-01-01

Carbon monoxide (CO), well known as a toxic gas, is increasingly recognized as a key metabolite and signaling molecule. Microbial utilization of CO is quite common, evidenced by the rapid escalation in description of new species of CO-utilizing bacteria and archaea. Carbon monoxide dehydrogenase (CODH), the protein complex that enables anaerobic CO-utilization, has been well-characterized from an increasing number of microorganisms, however the regulation of multiple CO-related gene clusters in single isolates remains unexplored. Many species are extraordinarily resistant to high CO concentrations, thriving under pure CO at more than one atmosphere. We hypothesized that, in strains that can grow exclusively on CO, both carbon acquisition via the CODH/acetyl CoA synthase complex and energy conservation via a CODH-linked hydrogenase must be differentially regulated in response to the availability of CO. The CO-sensing transcriptional activator, CooA is present in most CO-oxidizing bacteria. Here we present a genomic and phylogenetic survey of CODH operons and cooA genes found in CooA-containing bacteria. Two distinct groups of CooA homologs were found: one clade (CooA-1) is found in the majority of CooA-containing bacteria, whereas the other clade (CooA-2) is found only in genomes that encode multiple CODH clusters, suggesting that the CooA-2 might be important for cross-regulation of competing CODH operons. Recombinant CooA-1 and CooA-2 regulators from the prototypical CO-utilizing bacterium Carboxydothermus hydrogenoformans were purified, and promoter binding analyses revealed that CooA-1 specifically regulates the hydrogenase-linked CODH, whereas CooA-2 is able to regulate both the hydrogenase-linked CODH and the CODH/ACS operons. These studies point to the ability of dual CooA homologs to partition CO into divergent CO-utilizing pathways resulting in efficient consumption of a single limiting growth substrate available across a wide range of concentrations. PMID:21808633

Characterization of an Aldolase Involved in Cholesterol Side Chain Degradation in Mycobacterium tuberculosis.

PubMed

Gilbert, Stephanie; Hood, LaChae; Seah, Stephen Y K

2018-01-15

The heteromeric acyl coenzyme A (acyl-CoA) dehydrogenase FadE28-FadE29 and the enoyl-CoA hydratase ChsH1-ChsH2, encoded by genes within the intracellular growth ( igr ) operon of Mycobacterium tuberculosis , catalyze the dehydrogenation of the cholesterol metabolite 3-oxo-4-pregnene-20-carboxyl-CoA (3-OPC-CoA), with a 3-carbon side chain, and subsequent hydration of the product 3-oxo-4,17-pregnadiene-20-carboxyl-CoA (3-OPDC-CoA) to form 17-hydroxy-3-oxo-4-pregnene-20-carboxyl-CoA (17-HOPC-CoA). The gene downstream of chsH2 , i.e., ltp2 , was expressed in recombinant Rhodococcus jostii RHA1 in combination with other genes within the igr operon. His-tagged Ltp2 copurified with untagged ChsH1-ChsH2, ChsH2, or the C-terminal domain of ChsH2, which contains a domain of unknown function (DUF35). Ltp2 in association with ChsH1-ChsH2 or just the DUF35 domain of ChsH2 was shown to catalyze the retroaldol cleavage of 17-HOPC-CoA to form androst-4-ene-3,17-dione and propionyl-CoA. Steady-state kinetic analysis using the Ltp2-DUF35 complex showed that the aldolase had optimal activity at pH 7.5, with a K m of 6.54 ± 0.90 μM and a k cat of 159 ± 8.50 s -1 ChsH1-ChsH2 could hydrate only about 30% of 3-OPDC-CoA, but this unfavorable equilibrium could be overcome when the aldolase was present to remove the hydrated product, providing a rationale for the close association of the aldolase with the hydratase. Homologs of ChsH1, ChsH2, and Ltp2 are found in steroid-degrading Gram-positive and Gram-negative bacteria, suggesting that side chains of diverse steroids may be cleaved by aldolases in the bacteria. IMPORTANCE The C-C bond cleavage of the D-ring side chain of cholesterol was shown to be catalyzed by an aldolase. The aldolase associates with the hydratase that catalyzes the preceding reaction in the cholesterol side chain degradation pathway. These enzymes are encoded by genes within the intracellular growth ( igr ) operon of M. tuberculosis , and the operon was demonstrated previously to be linked to the pathogenicity and persistence of the bacteria in macrophages and in mice. Copyright © 2017 American Society for Microbiology.

The Global Redox Responding RegB/RegA Signal Transduction System Regulates the Genes Involved in Ferrous Iron and Inorganic Sulfur Compound Oxidation of the Acidophilic Acidithiobacillus ferrooxidans

PubMed Central

Moinier, Danielle; Byrne, Deborah; Amouric, Agnès; Bonnefoy, Violaine

2017-01-01

The chemical attack of ore by ferric iron and/or sulfuric acid releases valuable metals. The products of these reactions are recycled by iron and sulfur oxidizing microorganisms. These acidophilic chemolithotrophic prokaryotes, among which Acidithiobacillus ferrooxidans, grow at the expense of the energy released from the oxidation of ferrous iron and/or inorganic sulfur compounds (ISCs). In At. ferrooxidans, it has been shown that the expression of the genes encoding the proteins involved in these respiratory pathways is dependent on the electron donor and that the genes involved in iron oxidation are expressed before those responsible for ISCs oxidation when both iron and sulfur are present. Since the redox potential increases during iron oxidation but remains stable during sulfur oxidation, we have put forward the hypothesis that the global redox responding two components system RegB/RegA is involved in this regulation. To understand the mechanism of this system and its role in the regulation of the aerobic respiratory pathways in At. ferrooxidans, the binding of different forms of RegA (DNA binding domain, wild-type, unphosphorylated and phosphorylated-like forms of RegA) on the regulatory region of different genes/operons involved in ferrous iron and ISC oxidation has been analyzed. We have shown that the four RegA forms are able to bind specifically the upstream region of these genes. Interestingly, the phosphorylation of RegA did not change its affinity for its cognate DNA. The transcriptional start site of these genes/operons has been determined. In most cases, the RegA binding site(s) was (were) located upstream from the −35 (or −24) box suggesting that RegA does not interfere with the RNA polymerase binding. Based on the results presented in this report, the role of the RegB/RegA system in the regulation of the ferrous iron and ISC oxidation pathways in At. ferrooxidans is discussed. PMID:28747899

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

Price, Morgan N.; Arkin, Adam P.; Alm, Eric J.

Operons are a major feature of all prokaryotic genomes, but how and why operon structures vary is not well understood. To elucidate the life-cycle of operons, we compared gene order between Escherichia coli K12 and its relatives and identified the recently formed and destroyed operons in E. coli. This allowed us to determine how operons form, how they become closely spaced, and how they die. Our findings suggest that operon evolution is driven by selection on gene expression patterns. First, both operon creation and operon destruction lead to large changes in gene expression patterns. For example, the removal of lysAmore » and ruvA from ancestral operons that contained essential genes allowed their expression to respond to lysine levels and DNA damage, respectively. Second, some operons have undergone accelerated evolution, with multiple new genes being added during a brief period. Third, although most operons are closely spaced because of a neutral bias towards deletion and because of selection against large overlaps, highly expressed operons tend to be widely spaced because of regulatory fine-tuning by intervening sequences. Although operon evolution seems to be adaptive, it need not be optimal: new operons often comprise functionally unrelated genes that were already in proximity before the operon formed.« less

Kinetics of nif Gene Expression in a Nitrogen-Fixing Bacterium

PubMed Central

Poza-Carrión, César; Jiménez-Vicente, Emilio; Navarro-Rodríguez, Mónica; Echavarri-Erasun, Carlos

2014-01-01

Nitrogen fixation is a tightly regulated trait. Switching from N2 fixation-repressing conditions to the N2-fixing state is carefully controlled in diazotrophic bacteria mainly because of the high energy demand that it imposes. By using quantitative real-time PCR and quantitative immunoblotting, we show here how nitrogen fixation (nif) gene expression develops in Azotobacter vinelandii upon derepression. Transient expression of the transcriptional activator-encoding gene, nifA, was followed by subsequent, longer-duration waves of expression of the nitrogenase biosynthetic and structural genes. Importantly, expression timing, expression levels, and NifA dependence varied greatly among the nif operons. Moreover, the exact concentrations of Nif proteins and their changes over time were determined for the first time. Nif protein concentrations were exquisitely balanced, with FeMo cofactor biosynthetic proteins accumulating at levels 50- to 100-fold lower than those of the structural proteins. Mutants lacking nitrogenase structural genes or impaired in FeMo cofactor biosynthesis showed overenhanced responses to derepression that were proportional to the degree of nitrogenase activity impairment, consistent with the existence of at least two negative-feedback regulatory mechanisms. The first such mechanism responded to the levels of fixed nitrogen, whereas the second mechanism appeared to respond to the levels of the mature NifDK component. Altogether, these findings provide a framework to engineer N2 fixation in nondiazotrophs. PMID:24244007

Kinetics of Nif gene expression in a nitrogen-fixing bacterium.

PubMed

Poza-Carrión, César; Jiménez-Vicente, Emilio; Navarro-Rodríguez, Mónica; Echavarri-Erasun, Carlos; Rubio, Luis M

2014-02-01

Nitrogen fixation is a tightly regulated trait. Switching from N2 fixation-repressing conditions to the N2-fixing state is carefully controlled in diazotrophic bacteria mainly because of the high energy demand that it imposes. By using quantitative real-time PCR and quantitative immunoblotting, we show here how nitrogen fixation (nif) gene expression develops in Azotobacter vinelandii upon derepression. Transient expression of the transcriptional activator-encoding gene, nifA, was followed by subsequent, longer-duration waves of expression of the nitrogenase biosynthetic and structural genes. Importantly, expression timing, expression levels, and NifA dependence varied greatly among the nif operons. Moreover, the exact concentrations of Nif proteins and their changes over time were determined for the first time. Nif protein concentrations were exquisitely balanced, with FeMo cofactor biosynthetic proteins accumulating at levels 50- to 100-fold lower than those of the structural proteins. Mutants lacking nitrogenase structural genes or impaired in FeMo cofactor biosynthesis showed overenhanced responses to derepression that were proportional to the degree of nitrogenase activity impairment, consistent with the existence of at least two negative-feedback regulatory mechanisms. The first such mechanism responded to the levels of fixed nitrogen, whereas the second mechanism appeared to respond to the levels of the mature NifDK component. Altogether, these findings provide a framework to engineer N2 fixation in nondiazotrophs.

Lipopolysaccharide O-Antigen Prevents Phagocytosis of Vibrio anguillarum by Rainbow Trout (Oncorhynchus mykiss) Skin Epithelial Cells

PubMed Central

Lindell, Kristoffer; Fahlgren, Anna; Hjerde, Erik; Willassen, Nils-Peder; Fällman, Maria; Milton, Debra L.

2012-01-01

Colonization of host tissues is a first step taken by many pathogens during the initial stages of infection. Despite the impact of bacterial disease on wild and farmed fish, only a few direct studies have characterized bacterial factors required for colonization of fish tissues. In this study, using live-cell and confocal microscopy, rainbow trout skin epithelial cells, the main structural component of the skin epidermis, were demonstrated to phagocytize bacteria. Mutant analyses showed that the fish pathogen Vibrio anguillarum required the lipopolysaccharide O-antigen to evade phagocytosis and that O-antigen transport required the putative wzm-wzt-wbhA operon, which encodes two ABC polysaccharide transporter proteins and a methyltransferase. Pretreatment of the epithelial cells with mannose prevented phagocytosis of V. anguillarum suggesting that a mannose receptor is involved in the uptake process. In addition, the O-antigen transport mutants could not colonize the skin but they did colonize the intestines of rainbow trout. The O-antigen polysaccharides were also shown to aid resistance to the antimicrobial factors, lysozyme and polymyxin B. In summary, rainbow trout skin epithelial cells play a role in the fish innate immunity by clearing bacteria from the skin epidermis. In defense, V. anguillarum utilizes O-antigen polysaccharides to evade phagocytosis by the epithelial cells allowing it to colonize rapidly fish skin tissues. PMID:22662189

Identification of a Third Osmoprotectant Transport System, the OsmU System, in Salmonella enterica

PubMed Central

Frossard, Stephen M.; Khan, Aftab A.; Warrick, Eric C.; Gately, Jonathan M.; Hanson, Andrew D.; Oldham, Michael L.; Sanders, David Avram

2012-01-01

The growth of Salmonella enterica serovar Typhimurium mutants lacking the ProP and ProU osmoprotectant transport systems is stimulated by glycine betaine in high-osmolarity media, suggesting that this organism has an additional osmoprotectant transport system. Bioinformatic analysis revealed that the genome of this organism contains a hitherto-unidentified operon, designated osmU, consisting of four genes whose products show high similarity to ABC-type transport systems for osmoprotectants in other bacteria. The osmU operon was inactivated by a site-directed deletion, which abolished the ability of glycine betaine to alleviate the inhibitory effect of high osmolarity and eliminated the accumulation of [14C]glycine betaine and [14C]choline-O-sulfate in high-osmolarity media in a strain lacking the ProP and ProU systems. Although the OsmU system can take up glycine betaine and choline-O-sulfate, these two osmoprotectants are recognized at low affinity by this transporter, suggesting that there might be more efficient substrates that are yet to be discovered. The transcription of osmU is induced 23-fold by osmotic stress (0.3 M NaCl). The osmU operon is present in the genomes of a number of Enterobacteriaceae, and orthologs of the OsmU system can be recognized in a wide variety of Bacteria and Archaea. The structure of the periplasmic binding protein component of this transporter, OsmX, was modeled on the crystallographic structure of the glycine betaine-binding protein ProX of Archaeoglobus fulgidus; the resultant model indicated that the amino acids that constitute substrate-binding site, including an “aromatic cage” made up of four tyrosines, are conserved between these two proteins. PMID:22609924

Characteristics of a ugp-encoded and phoB-dependent glycerophosphoryl diester phosphodiesterase which is physically dependent on the ugp transport system of Escherichia coli.

PubMed

Brzoska, P; Boos, W

1988-09-01

The ugp-encoded transport system of Escherichia coli accumulates sn-glycerol-3-phosphate with high affinity; it is binding protein mediated and part of the pho regulon. Here, we report that glycerophosphoryl diesters (deacylated phospholipids) are also high-affinity substrates for the ugp-encoded system. The diesters are not taken up in an unaltered form but are hydrolyzed during transport to sn-glycerol-3-phosphate plus the corresponding alcohols. The enzyme responsible for this reaction is not essential for the translocation of sn-glycerol-3-phosphate or for the glycerophosphoryl diesters but can only hydrolyze diesters that are in the process of being transported. Diesters in the periplasm or in the cytoplasm were not recognized, and no enzymatic activity could be detected in cellular extracts. The enzyme is encoded by the last gene in the ugp operon, termed ugpQ. The product of the ugpQ gene, expressed in minicells, has an apparent molecular weight of 17,500. We present evidence that only one major phoB-dependent promoter controls all ugp genes.

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

Evolutionary dynamics of nematode operons: easy come, slow go.

PubMed

Qian, Wenfeng; Zhang, Jianzhi

2008-03-01

Operons are widespread in prokaryotes, but are uncommon in eukaryotes, except nematode worms, where approximately 15% of genes reside in over 1100 operons in the model organism Caenorhabditis elegans. It is unclear how operons have become abundant in nematode genomes. The "one-way street" hypothesis asserts that once formed by chance, operons are very difficult to break, because the breakage would leave downstream genes in an operon without a promoter, and hence, unexpressed. To test this hypothesis, we analyzed the presence and absence of C. elegans operons in Caenorhabditis briggsae, Caenorhabditis remanei, and Caenorhabditis brenneri, using Pristionchus pacificus and Brugia malayi as outgroups, and identified numerous operon gains and losses. Coupled with experimental examination of trans-splicing patterns, our comparative genomic analysis revealed diverse molecular mechanisms of operon losses, including inversion, insertion, and relocation, but the presence of internal promoters was not found to facilitate operon losses. In several cases, the data allowed inference of mechanisms by which downstream genes are expressed after operon breakage. We found that the rate of operon gain is approximately 3.3 times that of operon loss. Thus, the evolutionary dynamics of nematode operons is better described as "easy come, slow go," rather than a "one-way street." Based on a mathematic model of operon gains and losses and additional assumptions, we projected that the number of operons in C. elegans will continue to rise by 6%-18% in future evolution before reaching equilibrium between operon gains and losses.

GxySBA ABC Transporter of Agrobacterium tumefaciens and Its Role in Sugar Utilization and vir Gene Expression

PubMed Central

Zhao, Jinlei

2014-01-01

Monosaccharides available in the extracellular milieu of Agrobacterium tumefaciens can be transported into the cytoplasm, or via the periplasmic sugar binding protein, ChvE, play a critical role in controlling virulence gene expression. The ChvE-MmsAB ABC transporter is involved in the utilization of a wide range of monosaccharide substrates but redundant transporters are likely given the ability of a chvE-mmsAB deletion strain to grow, albeit more slowly, in the presence of particular monosaccharides. In this study, a putative ABC transporter encoded by the gxySBA operon is identified and shown to be involved in the utilization of glucose, xylose, fucose, and arabinose, which are also substrates for the ChvE-MmsAB ABC transporter. Significantly, GxySBA is also shown to be the first characterized glucosamine ABC transporter. The divergently transcribed gene gxyR encodes a repressor of the gxySBA operon, the function of which can be relieved by a subset of the transported sugars, including glucose, xylose, and glucosamine, and this substrate-induced expression can be repressed by glycerol. Furthermore, deletion of the transporter can increase the sensitivity of the virulence gene expression system to certain sugars that regulate it. Collectively, the results reveal a remarkably diverse set of substrates for the GxySBA transporter and its contribution to the repression of sugar sensitivity by the virulence-controlling system, thereby facilitating the capacity of the bacterium to distinguish between the soil and plant environments. PMID:24957625

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

The Vibrio cholerae Mrp system: cation/proton antiport properties and enhancement of bile salt resistance in a heterologous host.

PubMed

Dzioba-Winogrodzki, Judith; Winogrodzki, Olga; Krulwich, Terry A; Boin, Markus A; Häse, Claudia C; Dibrov, Pavel

2009-01-01

The mrp operon from Vibrio cholerae encoding a putative multisubunit Na(+)/H(+) antiporter was cloned and functionally expressed in the antiporter-deficient strain of Escherichia coli EP432. Cells of EP432 expressing Vc-Mrp exhibited resistance to Na(+) and Li(+) as well as to natural bile salts such as sodium cholate and taurocholate. When assayed in everted membrane vesicles of the E. coli EP432 host, Vc-Mrp had sufficiently high antiport activity to facilitate the first extensive analysis of Mrp system from a Gram-negative bacterium encoded by a group 2 mrp operon. Vc-Mrp was found to exchange protons for Li(+), Na(+), and K(+) ions in pH-dependent manner with maximal activity at pH 9.0-9.5. Exchange was electrogenic (more than one H(+) translocated per cation moved in opposite direction). The apparent K(m) at pH 9.0 was 1.08, 1.30, and 68.5 mM for Li(+), Na(+), and K(+), respectively. Kinetic analyses suggested that Vc-Mrp operates in a binding exchange mode with all cations and protons competing for binding to the antiporter. The robust ion antiport activity of Vc-Mrp in sub-bacterial vesicles and its effect on bile resistance of the heterologous host make Vc-Mrp an attractive experimental model for the further studies of biochemistry and physiology of Mrp systems. Copyright 2008 S. Karger AG, Basel.

Localization, cloning, and sequence determination of the conjugative plasmid ColB2 pilin gene.

PubMed Central

Finlay, B B; Frost, L S; Paranchych, W

1984-01-01

ColB2 is a colicin-producing, 96-kilobase plasmid which encodes a conjugative system that is similar, but not identical, to F. A restriction map of this plasmid was generated, and DNA homology studies between F and ColB2 plasmids revealed homology only between their transfer operons. The locations of the ColB2 transfer operon and ColB2 pilin gene were localized on this restriction map. The gene encoding ColB2 pilin, traA, was cloned and sequenced. The pilin protein of ColB2 is identical to F, except at the amino terminus, where ala-gln of ColB2 pilin corresponds to Ala-Gly-Ser-Ser of F pilin. This is due to a 6-base-pair deletion in the ColB2 pilin gene. Biochemical studies on tryptic peptides derived from ColB2 pilin demonstrate the location of this gene to be correct. There is a putative signal peptidase cleavage site after the sequence Ala-Met-Ala, giving a signal peptide of 51 amino acids and a mature pilin protein of 68 amino acids (7,000 daltons). The amino terminus is blocked, probably with an acetyl group. A chimera containing the ColB2 pilin gene was able to complement an F traA mutant, demonstrating that the pilus assembly proteins of F can utilize the ColB2 pilin protein to form a pilus. Images PMID:6090427

The nucleoid-associated protein Fis directly modulates the synthesis of cellulose, an essential component of pellicle-biofilms in the phytopathogenic bacterium Dickeya dadantii.

PubMed

Prigent-Combaret, Claire; Zghidi-Abouzid, Ouafa; Effantin, Géraldine; Lejeune, Philippe; Reverchon, Sylvie; Nasser, William

2012-10-01

Bacteria use biofilm structures to colonize surfaces and to survive in hostile conditions, and numerous bacteria produce cellulose as a biofilm matrix polymer. Hence, expression of the bcs operon, responsible for cellulose biosynthesis, must be finely regulated in order to allow bacteria to adopt the proper surface-associated behaviours. Here we show that in the phytopathogenic bacterium, Dickeya dadantii, production of cellulose is required for pellicle-biofilm formation and resistance to chlorine treatments. Expression of the bcs operon is growth phase-regulated and is stimulated in biofilms. Furthermore, we unexpectedly found that the nucleoid-associated protein and global regulator of virulence functions, Fis, directly represses bcs operon expression by interacting with an operator that is absent from the bcs operon of animal pathogenic bacteria and the plant pathogenic bacterium Pectobacterium. Moreover, production of cellulose enhances plant surface colonization by D. dadantii. Overall, these data suggest that cellulose production and biofilm formation may be important factors for surface colonization by D. dadantii and its subsequent survival in hostile environments. This report also presents a new example of how bacteria can modulate the action of a global regulator to co-ordinate basic metabolism, virulence and modifications of lifestyle. © 2012 Blackwell Publishing Ltd.

Mycobacterium ahvazicum sp. nov., the nineteenth species of the Mycobacterium simiae complex.

PubMed

Bouam, Amar; Heidarieh, Parvin; Shahraki, Abodolrazagh Hashemi; Pourahmad, Fazel; Mirsaeidi, Mehdi; Hashemzadeh, Mohamad; Baptiste, Emeline; Armstrong, Nicholas; Levasseur, Anthony; Robert, Catherine; Drancourt, Michel

2018-03-07

Four slowly growing mycobacteria isolates were isolated from the respiratory tract and soft tissue biopsies collected in four unrelated patients in Iran. Conventional phenotypic tests indicated that these four isolates were identical to Mycobacterium lentiflavum while 16S rRNA gene sequencing yielded a unique sequence separated from that of M. lentiflavum. One representative strain AFP-003 T was characterized as comprising a 6,121,237-bp chromosome (66.24% guanosine-cytosine content) encoding for 5,758 protein-coding genes, 50 tRNA and one complete rRNA operon. A total of 2,876 proteins were found to be associated with the mobilome, including 195 phage proteins. A total of 1,235 proteins were found to be associated with virulence and 96 with toxin/antitoxin systems. The genome of AFP-003 T has the genetic potential to produce secondary metabolites, with 39 genes found to be associated with polyketide synthases and non-ribosomal peptide syntases and 11 genes encoding for bacteriocins. Two regions encoding putative prophages and three OriC regions separated by the dnaA gene were predicted. Strain AFP-003 T genome exhibits 86% average nucleotide identity with Mycobacterium genavense genome. Genetic and genomic data indicate that strain AFP-003 T is representative of a novel Mycobacterium species that we named Mycobacterium ahvazicum, the nineteenth species of the expanding Mycobacterium simiae complex.

Operon Formation is Driven by Co-Regulation and Not by Horizontal Gene Transfer

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

Price, Morgan N.; Huang, Katherine H.; Arkin, Adam P.

Although operons are often subject to horizontal gene transfer (HGT), non-HGT genes are particularly likely to be in operons. To resolve this apparent discrepancy and to determine whether HGT is involved in operon formation, we examined the evolutionary history of the genes and operons in Escherichia coli K12. We show that genes that have homologs in distantly related bacteria but not in close relatives of E. coli (indicating HGTi) form new operons at about the same rates as native genes. Furthermore, genes in new operons are no more likely than other genes to have phylogenetic trees that are inconsistent withmore » the species tree. In contrast, essential genes and ubiquitous genes without paralogs (genes believed to undergo HGT rarely) often form new operons. We conclude that HGT is not associated with operon formation, but instead promotes the prevalence of pre-existing operons. To explain operon formation, we propose that new operons reduce the amount of regulatory information required to specify optimal expression patterns. Consistent with this hypothesis, operons have greater amounts of conserved regulatory sequences than do individually transcribed genes.« less

Induction of surfactin production in Bacillus subtilis by gsp, a gene located upstream of the gramicidin S operon in Bacillus brevis.

PubMed Central

Borchert, S; Stachelhaus, T; Marahiel, M A

1994-01-01

The deduced amino acid sequence of the gsp gene, located upstream of the 5' end of the gramicidin S operon (grs operon) in Bacillus brevis, showed a high degree of similarity to the sfp gene product, which is located downstream of the srfA operon in B. subtilis. The gsp gene complemented in trans a defect in the sfp gene (sfpO) and promoted production of the lipopeptide antibiotic surfactin. The functional homology of Gsp and Sfp and the sequence similarity of these two proteins to EntD suggest that the three proteins represent a new class of proteins involved in peptide secretion, in support of a hypothesis published previously (T. H. Grossman, M. Tuckman, S. Ellestad, and M. S. Osburne, J. Bacteriol. 175:6203-6211, 1993). Images PMID:7512553

The Catabolite Control Protein E (CcpE) Affects Virulence Determinant Production and Pathogenesis of Staphylococcus aureus*

PubMed Central

Hartmann, Torsten; Baronian, Grégory; Nippe, Nadine; Voss, Meike; Schulthess, Bettina; Wolz, Christiane; Eisenbeis, Janina; Schmidt-Hohagen, Kerstin; Gaupp, Rosmarie; Sunderkötter, Cord; Beisswenger, Christoph; Bals, Robert; Somerville, Greg A.; Herrmann, Mathias; Molle, Virginie; Bischoff, Markus

2014-01-01

Carbon metabolism and virulence determinant production are often linked in pathogenic bacteria, and several regulatory elements have been reported to mediate this linkage in Staphylococcus aureus. Previously, we described a novel protein, catabolite control protein E (CcpE) that functions as a regulator of the tricarboxylic acid cycle. Here we demonstrate that CcpE also regulates virulence determinant biosynthesis and pathogenesis. Specifically, deletion of ccpE in S. aureus strain Newman revealed that CcpE affects transcription of virulence factors such as capA, the first gene in the capsule biosynthetic operon; hla, encoding α-toxin; and psmα, encoding the phenol-soluble modulin cluster α. Electrophoretic mobility shift assays demonstrated that CcpE binds to the hla promoter. Mice challenged with S. aureus strain Newman or its isogenic ΔccpE derivative revealed increased disease severity in the ΔccpE mutant using two animal models; an acute lung infection model and a skin infection model. Complementation of the mutant with the ccpE wild-type allele restored all phenotypes, demonstrating that CcpE is negative regulator of virulence in S. aureus. PMID:25193664

Crystal structure of the Vibrio cholerae colonization factor TcpF and identification of a functional immunogenic site.

PubMed

Megli, Christina J; Yuen, Alex S W; Kolappan, Subramaniapillai; Richardson, Malcolm R; Dharmasena, Madushini N; Krebs, Shelly J; Taylor, Ronald K; Craig, Lisa

2011-06-03

Vibrio cholerae relies on two main virulence factors--toxin-coregulated pilus (TCP) and cholera toxin--to cause the gastrointestinal disease cholera. TCP is a type IV pilus that mediates bacterial autoagglutination and colonization of the intestine. TCP is encoded by the tcp operon, which also encodes TcpF, a protein of unknown function that is secreted by V. cholerae in a TCP-dependent manner. Although TcpF is not required for TCP biogenesis, a tcpF mutant has a colonization defect in the infant mouse cholera model that is as severe as a pilus mutant. Furthermore, TcpF antisera protect against V. cholerae infection. TcpF has no apparent sequence homology to any known protein. Here, we report the de novo X-ray crystal structure of TcpF and the identification of an epitope that is critical for its function as a colonization factor. A monoclonal antibody recognizing this epitope is protective against V. cholerae challenge and adds to the protection provided by an anti-TcpA antibody. These data suggest that TcpF has a novel function in V. cholerae colonization and define a region crucial for this function. Copyright © 2011 Elsevier Ltd. All rights reserved.

Staphylococcus aureus CstB is a novel multidomain persulfide dioxygenase-sulfurtransferase involved in hydrogen sulfide detoxification

PubMed Central

Shen, Jiangchuan; Keithly, Mary E.; Armstrong, Richard N.; Higgins, Khadine A.; Edmonds, Katherine A.; Giedroc, David P.

2016-01-01

Hydrogen sulfide (H2S) is both a lethal gas and an emerging gasotransmitter in humans, suggesting that cellular H2S level must be tightly regulated. CstB is encoded by the cst operon of the major human pathogen Staphylococcus aureus (S. aureus) and is under the transcriptional control of the persulfide sensor CstR and H2S. Here we show that CstB is a multifunctional Fe(II)-containing persulfide dioxygenase (PDO), analogous to the vertebrate protein ETHE1 (Ethylmalonic Encephalopathy Protein 1). Chromosomal deletion of ethe1 is fatal in vertebrates. In the presence of molecular oxygen (O2), hETHE1 oxidizes glutathione persulfide (GSSH) to generate sulfite and reduced glutathione. In contrast, CstB oxidizes major cellular low molecular weight (LMW) persulfide substrates from S. aureus, coenzyme A persulfide (CoASSH) and bacillithiol persulfide (BSSH), directly to generate thiosulfate (TS) and reduced thiols, thereby avoiding the cellular toxicity of sulfite. Both Cys201 in the N-terminal PDO domain (CstBPDO) and Cys408 in the C-terminal rhodanese domain (CstBRhod) strongly enhance the TS generating activity of CstB. CstB also possesses persulfide transferase (PT; reverse rhodanese) activity which generates TS when provided with LMW persulfides and sulfite, as well as conventional thiosulfate transferase (TST; rhodanese) activity; both activities require Cys408. CstB protects S. aureus against H2S toxicity with C201S and C408S cstB genes unable to rescue a NaHS-induced ΔcstB growth phenotype. Induction of the cst operon by NaHS reveals that functional CstB impacts the cellular TS concentrations. These data collectively suggest that CstB may have evolved to facilitate the clearance of LMW persulfides that occur upon the elevation of the level of cellular H2S and hence may have an impact on bacterial viability under H2S stress, in concert with the other enzymes encoded by the cst operon. PMID:26177047

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.

Biochemical and Genetic Characterization of Coagulin, a New Antilisterial Bacteriocin in the Pediocin Family of Bacteriocins, Produced by Bacillus coagulans I4

PubMed Central

Le Marrec, Claire; Hyronimus, Bertrand; Bressollier, Philippe; Verneuil, Bernard; Urdaci, Maria C.

2000-01-01

A plasmid-linked antimicrobial peptide, named coagulin, produced by Bacillus coagulans I4 has recently been reported (B. Hyronimus, C. Le Marrec and M. C. Urdaci, J. Appl. Microbiol. 85:42–50, 1998). In the present study, the complete, unambiguous primary amino acid sequence of the peptide was obtained by a combination of both N-terminal sequencing of purified peptide and the complete sequence deduced from the structural gene harbored by plasmid I4. Data revealed that this peptide of 44 residues has an amino acid sequence similar to that described for pediocins AcH and PA-1, produced by different Pediococcus acidilactici strains and 100% identical. Coagulin and pediocin differed only by a single amino acid at their C terminus. Analysis of the genetic determinants revealed the presence, on the pI4 DNA, of the entire 3.5-kb operon of four genes described for pediocin AcH and PA-1 production. No extended homology was observed between pSMB74 from P. acidilactici and pI4 when analyzing the regions upstream and downstream of the operon. An oppositely oriented gene immediately dowstream of the bacteriocin operon specifies a 474-amino-acid protein which shows homology to Mob-Pre (plasmid recombination enzyme) proteins encoded by several small plasmids extracted from gram-positive bacteria. This is the first report of a pediocin-like peptide appearing naturally in a non-lactic acid bacterium genus. PMID:11097892

Additional regulatory activities of MrkH for the transcriptional expression of the Klebsiella pneumoniae mrk genes: Antagonist of H-NS and repressor.

PubMed

Ares, Miguel A; Fernández-Vázquez, José L; Pacheco, Sabino; Martínez-Santos, Verónica I; Jarillo-Quijada, Ma Dolores; Torres, Javier; Alcántar-Curiel, María D; González-Y-Merchand, Jorge A; De la Cruz, Miguel A

2017-01-01

Klebsiella pneumoniae is a common opportunistic pathogen causing nosocomial infections. One of the main virulence determinants of K. pneumoniae is the type 3 pilus (T3P). T3P helps the bacterial interaction to both abiotic and biotic surfaces and it is crucial for the biofilm formation. T3P is genetically organized in three transcriptional units: the mrkABCDF polycistronic operon, the mrkHI bicistronic operon and the mrkJ gene. MrkH is a regulatory protein encoded in the mrkHI operon, which positively regulates the mrkA pilin gene and its own expression. In contrast, the H-NS nucleoid protein represses the transcriptional expression of T3P. Here we reported that MrkH and H-NS positively and negatively regulate mrkJ expression, respectively, by binding to the promoter of mrkJ. MrkH protein recognized a sequence located at position -63.5 relative to the transcriptional start site of mrkJ gene. Interestingly, our results show that, in addition to its known function as classic transcriptional activator, MrkH also positively controls the expression of mrk genes by acting as an anti-repressor of H-NS; moreover, our results support the notion that high levels of MrkH repress T3P expression. Our data provide new insights about the complex regulatory role of the MrkH protein on the transcriptional control of T3P in K. pneumoniae.

Additional regulatory activities of MrkH for the transcriptional expression of the Klebsiella pneumoniae mrk genes: Antagonist of H-NS and repressor

PubMed Central

Ares, Miguel A.; Fernández-Vázquez, José L.; Pacheco, Sabino; Martínez-Santos, Verónica I.; Jarillo-Quijada, Ma. Dolores; Torres, Javier; Alcántar-Curiel, María D.; González-y-Merchand, Jorge A.; De la Cruz, Miguel A.

2017-01-01

Klebsiella pneumoniae is a common opportunistic pathogen causing nosocomial infections. One of the main virulence determinants of K. pneumoniae is the type 3 pilus (T3P). T3P helps the bacterial interaction to both abiotic and biotic surfaces and it is crucial for the biofilm formation. T3P is genetically organized in three transcriptional units: the mrkABCDF polycistronic operon, the mrkHI bicistronic operon and the mrkJ gene. MrkH is a regulatory protein encoded in the mrkHI operon, which positively regulates the mrkA pilin gene and its own expression. In contrast, the H-NS nucleoid protein represses the transcriptional expression of T3P. Here we reported that MrkH and H-NS positively and negatively regulate mrkJ expression, respectively, by binding to the promoter of mrkJ. MrkH protein recognized a sequence located at position -63.5 relative to the transcriptional start site of mrkJ gene. Interestingly, our results show that, in addition to its known function as classic transcriptional activator, MrkH also positively controls the expression of mrk genes by acting as an anti-repressor of H-NS; moreover, our results support the notion that high levels of MrkH repress T3P expression. Our data provide new insights about the complex regulatory role of the MrkH protein on the transcriptional control of T3P in K. pneumoniae. PMID:28278272

Crosstalk between virulence loci: regulation of Salmonella enterica pathogenicity island 1 (SPI-1) by products of the std fimbrial operon.

PubMed

López-Garrido, Javier; Casadesús, Josep

2012-01-01

Invasion of intestinal epithelial cells is a critical step in Salmonella infection and requires the expression of genes located in Salmonella pathogenicity island 1 (SPI-1). A key factor for SPI-1 expression is DNA adenine (Dam) methylation, which activates synthesis of the SPI-1 transcriptional activator HilD. Dam-dependent regulation of hilD is postranscriptional (and therefore indirect), indicating the involvement of unknown cell functions under Dam methylation control. A genetic screen has identified the std fimbrial operon as the missing link between Dam methylation and SPI-1. We show that all genes in the std operon are part of a single transcriptional unit, and describe three previously uncharacterized ORFs (renamed stdD, stdE, and stdF). We present evidence that two such loci (stdE and stdF) are involved in Dam-dependent control of Salmonella SPI-1: in a Dam(-) background, deletion of stdE or stdF suppresses SPI-1 repression; in a Dam(+) background, constitutive expression of StdE and/or StdF represses SPI-1. Repression of SPI-1 by products of std operon explains the invasion defect of Salmonella Dam(-) mutants, which constitutively express the std operon. Dam-dependent repression of std in the ileum may be required to permit invasion, as indicated by two observations: constitutive expression of StdE and StdF reduces invasion of epithelial cells in vitro (1,000 fold) and attenuates Salmonella virulence in the mouse model (>60 fold). In turn, crosstalk between std and SPI-1 may play a role in intestinal infections by preventing expression of SPI-1 in the caecum, an intestinal compartment in which the std operon is known to be expressed.

Altered Regulation of the Diguanylate Cyclase YaiC Reduces Production of Type 1 Fimbriae in a Pst Mutant of Uropathogenic Escherichia coli CFT073

PubMed Central

Crépin, Sébastien; Porcheron, Gaëlle; Houle, Sébastien; Harel, Josée

2017-01-01

ABSTRACT The pst gene cluster encodes the phosphate-specific transport (Pst) system. Inactivation of the Pst system constitutively activates the two-component regulatory system PhoBR and attenuates the virulence of pathogenic bacteria. In uropathogenic Escherichia coli strain CFT073, attenuation by inactivation of pst is predominantly attributed to the decreased expression of type 1 fimbriae. However, the molecular mechanisms connecting the Pst system and type 1 fimbriae are unknown. To address this, a transposon library was constructed in the pst mutant, and clones were tested for a regain in type 1 fimbrial production. Among them, the diguanylate cyclase encoded by yaiC (adrA in Salmonella) was identified to connect the Pst system and type 1 fimbrial expression. In the pst mutant, the decreased expression of type 1 fimbriae is connected by the induction of yaiC. This is predominantly due to altered expression of the FimBE-like recombinase genes ipuA and ipbA, affecting at the same time the inversion of the fim promoter switch (fimS). In the pst mutant, inactivation of yaiC restored fim-dependent adhesion to bladder cells and virulence. Interestingly, the expression of yaiC was activated by PhoB, since transcription of yaiC was linked to the PhoB-dependent phoA-psiF operon. As YaiC is involved in cyclic di-GMP (c-di-GMP) biosynthesis, an increased accumulation of c-di-GMP was observed in the pst mutant. Hence, the results suggest that one mechanism by which deletion of the Pst system reduces the expression of type 1 fimbriae is through PhoBR-mediated activation of yaiC, which in turn increases the accumulation of c-di-GMP, represses the fim operon, and, consequently, attenuates virulence in the mouse urinary tract infection model. IMPORTANCE Urinary tract infections (UTIs) are common bacterial infections in humans. They are mainly caused by uropathogenic Escherichia coli (UPEC). We previously showed that interference with phosphate homeostasis decreases the expression of type 1 fimbriae and attenuates UPEC virulence. Herein, we identified that alteration of the phosphate metabolism increases production of the signaling molecule c-di-GMP, which in turn decreases the expression of type 1 fimbriae. We also determine the regulatory cascade leading to the accumulation of c-di-GMP and identify the Pho regulon as new players in c-di-GMP-mediated cell signaling. By understanding the molecular mechanisms leading to the expression of virulence factors, we will be in a better position to develop new therapeutics. PMID:28924030

Altered Regulation of the Diguanylate Cyclase YaiC Reduces Production of Type 1 Fimbriae in a Pst Mutant of Uropathogenic Escherichia coli CFT073.

PubMed

Crépin, Sébastien; Porcheron, Gaëlle; Houle, Sébastien; Harel, Josée; Dozois, Charles M

2017-12-15

The pst gene cluster encodes the phosphate-specific transport (Pst) system. Inactivation of the Pst system constitutively activates the two-component regulatory system PhoBR and attenuates the virulence of pathogenic bacteria. In uropathogenic Escherichia coli strain CFT073, attenuation by inactivation of pst is predominantly attributed to the decreased expression of type 1 fimbriae. However, the molecular mechanisms connecting the Pst system and type 1 fimbriae are unknown. To address this, a transposon library was constructed in the pst mutant, and clones were tested for a regain in type 1 fimbrial production. Among them, the diguanylate cyclase encoded by yaiC ( adrA in Salmonella ) was identified to connect the Pst system and type 1 fimbrial expression. In the pst mutant, the decreased expression of type 1 fimbriae is connected by the induction of yaiC This is predominantly due to altered expression of the FimBE-like recombinase genes ipuA and ipbA , affecting at the same time the inversion of the fim promoter switch ( fimS ). In the pst mutant, inactivation of yaiC restored fim -dependent adhesion to bladder cells and virulence. Interestingly, the expression of yaiC was activated by PhoB, since transcription of yaiC was linked to the PhoB-dependent phoA-psiF operon. As YaiC is involved in cyclic di-GMP (c-di-GMP) biosynthesis, an increased accumulation of c-di-GMP was observed in the pst mutant. Hence, the results suggest that one mechanism by which deletion of the Pst system reduces the expression of type 1 fimbriae is through PhoBR-mediated activation of yaiC , which in turn increases the accumulation of c-di-GMP, represses the fim operon, and, consequently, attenuates virulence in the mouse urinary tract infection model. IMPORTANCE Urinary tract infections (UTIs) are common bacterial infections in humans. They are mainly caused by uropathogenic Escherichia coli (UPEC). We previously showed that interference with phosphate homeostasis decreases the expression of type 1 fimbriae and attenuates UPEC virulence. Herein, we identified that alteration of the phosphate metabolism increases production of the signaling molecule c-di-GMP, which in turn decreases the expression of type 1 fimbriae. We also determine the regulatory cascade leading to the accumulation of c-di-GMP and identify the Pho regulon as new players in c-di-GMP-mediated cell signaling. By understanding the molecular mechanisms leading to the expression of virulence factors, we will be in a better position to develop new therapeutics. Copyright © 2017 American Society for Microbiology.

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

Novel Accurate Bacterial Discrimination by MALDI-Time-of-Flight MS Based on Ribosomal Proteins Coding in S10-spc-alpha Operon at Strain Level S10-GERMS

NASA Astrophysics Data System (ADS)

Tamura, Hiroto; Hotta, Yudai; Sato, Hiroaki

2013-08-01

Matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is one of the most widely used mass-based approaches for bacterial identification and classification because of the simple sample preparation and extremely rapid analysis within a few minutes. To establish the accurate MALDI-TOF MS bacterial discrimination method at strain level, the ribosomal subunit proteins coded in the S 10-spc-alpha operon, which encodes half of the ribosomal subunit protein and is highly conserved in eubacterial genomes, were selected as reliable biomarkers. This method, named the S10-GERMS method, revealed that the strains of genus Pseudomonas were successfully identified and discriminated at species and strain levels, respectively; therefore, the S10-GERMS method was further applied to discriminate the pathovar of P. syringae. The eight selected biomarkers (L24, L30, S10, S12, S14, S16, S17, and S19) suggested the rapid discrimination of P. syringae at the strain (pathovar) level. The S10-GERMS method appears to be a powerful tool for rapid and reliable bacterial discrimination and successful phylogenetic characterization. In this article, an overview of the utilization of results from the S10-GERMS method is presented, highlighting the characterization of the Lactobacillus casei group and discrimination of the bacteria of genera Bacillus and Sphingopyxis despite only two and one base difference in the 16S rRNA gene sequence, respectively.

Transcriptional Analysis of the MrpJ Network: Modulation of Diverse Virulence-Associated Genes and Direct Regulation of mrp Fimbrial and flhDC Flagellar Operons in Proteus mirabilis

PubMed Central

Bode, Nadine J.; Debnath, Irina; Kuan, Lisa; Schulfer, Anjelique; Ty, Maureen

2015-01-01

The enteric bacterium Proteus mirabilis is associated with a significant number of catheter-associated urinary tract infections (UTIs). Strict regulation of the antagonistic processes of adhesion and motility, mediated by fimbriae and flagella, respectively, is essential for disease progression. Previously, the transcriptional regulator MrpJ, which is encoded by the mrp fimbrial operon, has been shown to repress both swimming and swarming motility. Here we show that MrpJ affects an array of cellular processes beyond adherence and motility. Microarray analysis found that expression of mrpJ mimicking levels observed during UTIs leads to differential expression of 217 genes related to, among other functions, bacterial virulence, type VI secretion, and metabolism. We probed the molecular mechanism of transcriptional regulation by MrpJ using transcriptional reporters and chromatin immunoprecipitation (ChIP). Binding of MrpJ to two virulence-associated target gene promoters, the promoters of the flagellar master regulator flhDC and mrp itself, appears to be affected by the condensation state of the native chromosome, although both targets share a direct MrpJ binding site proximal to the transcriptional start. Furthermore, an mrpJ deletion mutant colonized the bladders of mice at significantly lower levels in a transurethral model of infection. Additionally, we observed that mrpJ is widely conserved in a collection of recent clinical isolates. Altogether, these findings support a role of MrpJ as a global regulator of P. mirabilis virulence. PMID:25847961

The civRT operon is important for Campylobacter jejuni strain 81-176 host cell interactions through regulation of the formate dehydrogenase operon

USDA-ARS?s Scientific Manuscript database

C. jejuni colonizes the intestinal mucosa, and the severity of disease in different strains is correlated with host cell interaction and invasion. A microarray screen to identify genes differentially regulated during C. jejuni interaction with tissue culture cells revealed the up-regulation of a two...

Molecular cloning and nucleotide sequences of the genes for two essential proteins constituting a novel enzyme system for heptaprenyl diphosphate synthesis.

PubMed

Koike-Takeshita, A; Koyama, T; Obata, S; Ogura, K

1995-08-04

The genes encoding two dissociable components essential for Bacillus stearothermophilus heptaprenyl diphosphate synthase (all-trans-hexparenyl-diphosphate:isopentenyl-diphosphate hexaprenyl-trans-transferase, EC 2.5.1.30) were cloned, and their nucleotide sequences were determined. Sequence analyses revealed the presence of three open reading frames within 2,350 base pairs, designated as ORF-1, ORF-2, and ORF-3 in order of nucleotide sequence, which encode proteins of 220, 234, and 323 amino acids, respectively. Deletion experiments have shown that expression of the enzymatic activity requires the presence of ORF-1 and ORF-3, but ORF-2 is not essential. As a result, this enzyme was proved genetically to consist of two different protein compounds with molecular masses of 25 kDa (Component I) and 36 kDa (Component II), encoded by two of the three tandem genes. The protein encoded by ORF-1 has no similarity to any protein so far registered. However, the protein encoded by ORF-3 shows a 32% similarity to the farnesyl diphosphate synthase of the same bacterium and has seven highly conserved regions that have been shown typical in prenyltransferases (Koyama, T., Obata, S., Osabe, M., Takeshita, A., Yokoyama, K., Uchida, M., Nishino, T., and Ogura, K. (1993) J. Biochem. (Tokyo) 113, 355-363).

A FRET-Based DNA Biosensor Tracks OmpR-Dependent Acidification of Salmonella during Macrophage Infection

PubMed Central

Chakraborty, Smarajit; Mizusaki, Hideaki; Kenney, Linda J.

2015-01-01

In bacteria, one paradigm for signal transduction is the two-component regulatory system, consisting of a sensor kinase (usually a membrane protein) and a response regulator (usually a DNA binding protein). The EnvZ/OmpR two-component system responds to osmotic stress and regulates expression of outer membrane proteins. In Salmonella, EnvZ/OmpR also controls expression of another two-component system SsrA/B, which is located on Salmonella Pathogenicity Island (SPI) 2. SPI-2 encodes a type III secretion system, which functions as a nanomachine to inject bacterial effector proteins into eukaryotic cells. During the intracellular phase of infection, Salmonella switches from assembling type III secretion system structural components to secreting effectors into the macrophage cytoplasm, enabling Salmonella to replicate in the phagocytic vacuole. Major questions remain regarding how bacteria survive the acidified vacuole and how acidification affects bacterial secretion. We previously reported that EnvZ sensed cytoplasmic signals rather than extracellular ones, as intracellular osmolytes altered the dynamics of a 17-amino-acid region flanking the phosphorylated histidine. We reasoned that the Salmonella cytoplasm might acidify in the macrophage vacuole to activate OmpR-dependent transcription of SPI-2 genes. To address these questions, we employed a DNA-based FRET biosensor (“I-switch”) to measure bacterial cytoplasmic pH and immunofluorescence to monitor effector secretion during infection. Surprisingly, we observed a rapid drop in bacterial cytoplasmic pH upon phagocytosis that was not predicted by current models. Cytoplasmic acidification was completely dependent on the OmpR response regulator, but did not require known OmpR-regulated genes such as ompC, ompF, or ssaC (SPI-2). Microarray analysis highlighted the cadC/BA operon, and additional experiments confirmed that it was repressed by OmpR. Acidification was blocked in the ompR null background in a Cad-dependent manner. Acid-dependent activation of OmpR stimulated type III secretion; blocking acidification resulted in a neutralized cytoplasm that was defective for SPI-2 secretion. Based upon these findings, we propose that Salmonella infection involves an acid-dependent secretion process in which the translocon SseB moves away from the bacterial cell surface as it associates with the vacuolar membrane, driving the secretion of SPI-2 effectors such as SseJ. New steps in the SPI-2 secretion process are proposed. PMID:25875623

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

Shatsky, Maxim; Allen, Simon; Gold, Barbara

Numerous affinity purification – mass-spectrometry (AP-MS) and yeast two hybrid (Y2H) screens have each defined thousands of pairwise protein-protein interactions (PPIs), most between functionally unrelated proteins. The accuracy of these networks, however, is under debate. Here we present an AP-MS survey of the bacterium Desulfovibrio vulgaris together with a critical reanalysis of nine published bacterial Y2H and AP-MS screens. We have identified 459 high confidence PPIs from D. vulgaris and 391 from Escherichia coli. Compared to the nine published interactomes, our two networks are smaller; are much less highly connected; have significantly lower false discovery rates; and are much moremore » enriched in protein pairs that are encoded in the same operon, have similar functions, and are reproducibly detected in other physical interaction assays. Lastly, our work establishes more stringent benchmarks for the properties of protein interactomes and suggests that bona fide PPIs much more frequently involve protein partners that are annotated with similar functions or that can be validated in independent assays than earlier studies suggested.« less

Processing of the 5'-UTR and existence of protein factors that regulate translation of tobacco chloroplast psbN mRNA.

PubMed

Kuroda, Hiroshi; Sugiura, Masahiro

2014-12-01

The chloroplast psbB operon includes five genes encoding photosystem II and cytochrome b 6 /f complex components. The psbN gene is located on the opposite strand. PsbN is localized in the thylakoid and is present even in the dark, although its level increases upon illumination and then decreases. However, the translation mechanism of the psbN mRNA remains unclear. Using an in vitro translation system from tobacco chloroplasts and a green fluorescent protein as a reporter protein, we show that translation occurs from a tobacco primary psbN 5'-UTR of 47 nucleotides (nt). Unlike many other chloroplast 5'-UTRs, the psbN 5'-UTR has two processing sites, at -39 and -24 upstream from the initiation site. Processing at -39 enhanced the translation rate fivefold. In contrast, processing at -24 did not affect the translation rate. These observations suggest that the two distinct processing events regulate, at least in part, the level of PsbN during development. The psbN 5'-UTR has no Shine-Dalgarno (SD)-like sequence. In vitro translation assays with excess amounts of the psbN 5'-UTR or with deleted psbN 5'-UTR sequences demonstrated that protein factors are required for translation and that their binding site is an 18 nt sequence in the 5'-UTR. Mobility shift assays using 10 other chloroplast 5'-UTRs suggested that common or similar proteins are involved in translation of a set of mRNAs lacking SD-like sequences.

Naringenin degradation by the endophytic diazotroph Herbaspirillum seropedicae SmR1.

PubMed

Marin, A M; Souza, E M; Pedrosa, F O; Souza, L M; Sassaki, G L; Baura, V A; Yates, M G; Wassem, R; Monteiro, R A

2013-01-01

Several bacteria are able to degrade flavonoids either to use them as carbon sources or as a detoxification mechanism. Degradation pathways have been proposed for several bacteria, but the genes responsible are not known. We identified in the genome of the endophyte Herbaspirillum seropedicae SmR1 an operon potentially associated with the degradation of aromatic compounds. We show that this operon is involved in naringenin degradation and that its expression is induced by naringenin and chrysin, two closely related flavonoids. Mutation of fdeA, the first gene of the operon, and fdeR, its transcriptional activator, abolished the ability of H. seropedicae to degrade naringenin.

Detecting uber-operons in prokaryotic genomes.

PubMed

Che, Dongsheng; Li, Guojun; Mao, Fenglou; Wu, Hongwei; Xu, Ying

2006-01-01

We present a study on computational identification of uber-operons in a prokaryotic genome, each of which represents a group of operons that are evolutionarily or functionally associated through operons in other (reference) genomes. Uber-operons represent a rich set of footprints of operon evolution, whose full utilization could lead to new and more powerful tools for elucidation of biological pathways and networks than what operons have provided, and a better understanding of prokaryotic genome structures and evolution. Our prediction algorithm predicts uber-operons through identifying groups of functionally or transcriptionally related operons, whose gene sets are conserved across the target and multiple reference genomes. Using this algorithm, we have predicted uber-operons for each of a group of 91 genomes, using the other 90 genomes as references. In particular, we predicted 158 uber-operons in Escherichia coli K12 covering 1830 genes, and found that many of the uber-operons correspond to parts of known regulons or biological pathways or are involved in highly related biological processes based on their Gene Ontology (GO) assignments. For some of the predicted uber-operons that are not parts of known regulons or pathways, our analyses indicate that their genes are highly likely to work together in the same biological processes, suggesting the possibility of new regulons and pathways. We believe that our uber-operon prediction provides a highly useful capability and a rich information source for elucidation of complex biological processes, such as pathways in microbes. All the prediction results are available at our Uber-Operon Database: http://csbl.bmb.uga.edu/uber, the first of its kind.

Detecting uber-operons in prokaryotic genomes

PubMed Central

Che, Dongsheng; Li, Guojun; Mao, Fenglou; Wu, Hongwei; Xu, Ying

2006-01-01

We present a study on computational identification of uber-operons in a prokaryotic genome, each of which represents a group of operons that are evolutionarily or functionally associated through operons in other (reference) genomes. Uber-operons represent a rich set of footprints of operon evolution, whose full utilization could lead to new and more powerful tools for elucidation of biological pathways and networks than what operons have provided, and a better understanding of prokaryotic genome structures and evolution. Our prediction algorithm predicts uber-operons through identifying groups of functionally or transcriptionally related operons, whose gene sets are conserved across the target and multiple reference genomes. Using this algorithm, we have predicted uber-operons for each of a group of 91 genomes, using the other 90 genomes as references. In particular, we predicted 158 uber-operons in Escherichia coli K12 covering 1830 genes, and found that many of the uber-operons correspond to parts of known regulons or biological pathways or are involved in highly related biological processes based on their Gene Ontology (GO) assignments. For some of the predicted uber-operons that are not parts of known regulons or pathways, our analyses indicate that their genes are highly likely to work together in the same biological processes, suggesting the possibility of new regulons and pathways. We believe that our uber-operon prediction provides a highly useful capability and a rich information source for elucidation of complex biological processes, such as pathways in microbes. All the prediction results are available at our Uber-Operon Database: , the first of its kind. PMID:16682449

Linearmycins Activate a Two-Component Signaling System Involved in Bacterial Competition and Biofilm Morphology

PubMed Central

2017-01-01

ABSTRACT Bacteria use two-component signaling systems to adapt and respond to their competitors and changing environments. For instance, competitor bacteria may produce antibiotics and other bioactive metabolites and sequester nutrients. To survive, some species of bacteria escape competition through antibiotic production, biofilm formation, or motility. Specialized metabolite production and biofilm formation are relatively well understood for bacterial species in isolation. How bacteria control these functions when competitors are present is not well studied. To address fundamental questions relating to the competitive mechanisms of different species, we have developed a model system using two species of soil bacteria, Bacillus subtilis and Streptomyces sp. strain Mg1. Using this model, we previously found that linearmycins produced by Streptomyces sp. strain Mg1 cause lysis of B. subtilis cells and degradation of colony matrix. We identified strains of B. subtilis with mutations in the two-component signaling system yfiJK operon that confer dual phenotypes of specific linearmycin resistance and biofilm morphology. We determined that expression of the ATP-binding cassette (ABC) transporter yfiLMN operon, particularly yfiM and yfiN, is necessary for biofilm morphology. Using transposon mutagenesis, we identified genes that are required for YfiLMN-mediated biofilm morphology, including several chaperones. Using transcriptional fusions, we found that YfiJ signaling is activated by linearmycins and other polyene metabolites. Finally, using a truncated YfiJ, we show that YfiJ requires its transmembrane domain to activate downstream signaling. Taken together, these results suggest coordinated dual antibiotic resistance and biofilm morphology by a single multifunctional ABC transporter promotes competitive fitness of B. subtilis. IMPORTANCE DNA sequencing approaches have revealed hitherto unexplored diversity of bacterial species in a wide variety of environments that includes the gastrointestinal tract of animals and the rhizosphere of plants. Interactions between different species in bacterial communities have impacts on our health and industry. However, many approaches currently used to study whole bacterial communities do not resolve mechanistic details of interspecies interactions, including how bacteria sense and respond to their competitors. Using a competition model, we have uncovered dual functions for a previously uncharacterized two-component signaling system involved in specific antibiotic resistance and biofilm morphology. Insights gleaned from signaling within interspecies interaction models build a more complete understanding of gene functions important for bacterial communities and will enhance community-level analytical approaches. PMID:28461449

Genetics of digalactoside-binding adhesin from a uropathogenic Escherichia coli strain.

PubMed Central

Normark, S; Lark, D; Hull, R; Norgren, M; Båga, M; O'Hanley, P; Schoolnik, G; Falkow, S

1983-01-01

The uropathogenic strain Escherichia coli J96 mediates mannose-resistant hemagglutination owing to production of a digalactoside-binding adhesin. A cosmid clone from this strain has been isolated that, when harbored in E. coli K-12, expressed Pap pili and this adhesin (R. Hull et al., Infect. Immun. 33:933-938, 1981). By transposon mutagenesis and by the construction of a number of hybrid plasmid derivatives, we have demonstrated that about 8.5 kilobases of DNA is required to generate a mannose-resistant hemagglutination-positive phenotype in E. coli K-12 strain P678-54. The structural gene for the Pap pili monomer, papA, has been identified and mapped close to the promotor-proximal end of the Pap operon. Although strain P678-54 that harbored a Tn5 insertion within papA showed a mannose-resistant hemagglutination-positive phenotype, it was negative in a competitive enzyme-linked immunosorbent assay with anti-Pap pilus serum. This could mean that a Pap adhesin is encoded by a region on the Pap operon that is distinct from papA. Images PMID:6136465

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.

The LysR-type transcription factor PacR is a global regulator of photosynthetic carbon assimilation in Anabaena.

PubMed

Picossi, Silvia; Flores, Enrique; Herrero, Antonia

2015-09-01

Cyanobacteria perform water-splitting photosynthesis and are important primary producers impacting the carbon and nitrogen cycles at global scale. They fix CO2 through ribulose-bisphosphate carboxylase/oxygenase (RuBisCo) and have evolved a distinct CO2 concentrating mechanism (CCM) that builds high CO2 concentrations in the vicinity of RuBisCo favouring its carboxylase activity. Filamentous cyanobacteria such as Anabaena fix CO2 in photosynthetic vegetative cells, which donate photosynthate to heterocysts that rely on a heterotrophic metabolism to fix N2 . CCM elements are induced in response to inorganic carbon limitation, a cue that exposes the photosynthetic apparatus to photodamage by over-reduction. An Anabaena mutant lacking the LysR-type transcription factor All3953 grew poorly and dies under high light. The rbcL operon encoding RuBisCo was induced upon carbon limitation in the wild type but not in the mutant. ChIP-Seq analysis was used to globally identify All3953 targets under carbon limitation. Targets include, besides rbcL, genes encoding CCM elements, photorespiratory pathway- photosystem- and electron transport-related components, and factors, including flavodiiron proteins, with a demonstrated or putative function in photoprotection. Quantitative reverse transcription polymerase chain reaction analysis of selected All3953 targets showed regulation in the wild type but not in the mutant. All3953 (PacR) is a global regulator of carbon assimilation in an oxygenic photoautotroph. © 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.

Discovery of an operon that participates in agmatine metabolism and regulates biofilm formation in Pseudomonas aeruginosa

PubMed Central

Williams, Bryan J.; Du, Rui-Hong; Calcutt, M. Wade; Abdolrasulnia, Rasul; Christman, Brian W.; Blackwell, Timothy S.

2013-01-01

Summary Agmatine is the decarboxylation product of arginine and a number of bacteria have devoted enzymatic pathways for its metabolism. Pseudomonas aeruginosa harbours the aguBA operon that metabolizes agmatine to putrescine, which can be subsequently converted into other polyamines or shunted into the TCA cycle for energy production. We discovered an alternate agmatine operon in the P. aeruginosa strain PA14 named agu2ABCA′ that contains two genes for agmatine deiminases (agu2A and agu2A′). This operon was found to be present in 25% of clinical P. aeruginosa isolates. Agu2A′ contains a twin-arginine translocation signal at its N-terminus and site-directed mutagenesis and cell fractionation experiments confirmed this protein is secreted to the periplasm. Analysis of the agu2ABCA′ promoter demonstrates that agmatine induces expression of the operon during the stationary phase of growth and during biofilm growth and agu2ABCA′ provides only weak complementation of aguBA, which is induced during log phase. Biofilm assays of mutants of all three agmatine deiminase genes in PA14 revealed that deletion of agu2ABCA′, specifically its secreted product Agu2A′, reduces biofilm production of PA14 following addition of exogenous agmatine. Together, these findings reveal a novel role for the agu2ABCA′ operon in the biofilm development of P. aeruginosa. PMID:20149107

Cloning and characterization of the Bacillus subtilis birA gene encoding a repressor of the biotin operon.

PubMed

Bower, S; Perkins, J; Yocum, R R; Serror, P; Sorokin, A; Rahaim, P; Howitt, C L; Prasad, N; Ehrlich, S D; Pero, J

1995-05-01

The Bacillus subtilis birA gene, which regulates biotin biosynthesis, has been cloned and characterized. The birA gene maps at 202 degrees on the B. subtilis chromosome and encodes a 36,200-Da protein that is 27% identical to Escherichia coli BirA protein. Three independent mutations in birA that lead to deregulation of biotin synthesis alter single amino acids in the amino-terminal end of the protein. The amino-terminal region that is affected by these three birA mutations shows sequence similarity to the helix-turn-helix DNA binding motif previously identified in E. coli BirA protein. B. subtilis BirA protein also possesses biotin-protein ligase activity, as judged by its ability to complement a conditional lethal birA mutant of E. coli.

Cloning and characterization of the Bacillus subtilis birA gene encoding a repressor of the biotin operon.

PubMed Central

Bower, S; Perkins, J; Yocum, R R; Serror, P; Sorokin, A; Rahaim, P; Howitt, C L; Prasad, N; Ehrlich, S D; Pero, J

1995-01-01

The Bacillus subtilis birA gene, which regulates biotin biosynthesis, has been cloned and characterized. The birA gene maps at 202 degrees on the B. subtilis chromosome and encodes a 36,200-Da protein that is 27% identical to Escherichia coli BirA protein. Three independent mutations in birA that lead to deregulation of biotin synthesis alter single amino acids in the amino-terminal end of the protein. The amino-terminal region that is affected by these three birA mutations shows sequence similarity to the helix-turn-helix DNA binding motif previously identified in E. coli BirA protein. B. subtilis BirA protein also possesses biotin-protein ligase activity, as judged by its ability to complement a conditional lethal birA mutant of E. coli. PMID:7730294

Structure and Function of AmtR in Mycobacterium smegmatis: Implications for Post-Transcriptional Regulation of Urea Metabolism through a Small Antisense RNA.

PubMed

Petridis, Michael; Vickers, Chelsea; Robson, Jennifer; McKenzie, Joanna L; Bereza, Magdalena; Sharrock, Abigail; Aung, Htin Lin; Arcus, Vickery L; Cook, Gregory M

2016-10-23

Soil-dwelling bacteria of the phylum actinomycetes generally harbor either GlnR or AmtR as a global regulator of nitrogen metabolism. Mycobacterium smegmatis harbors both of these canonical regulators; GlnR regulates the expression of key genes involved in nitrogen metabolism, while the function and signal transduction pathway of AmtR in M. smegmatis remains largely unknown. Here, we report the structure and function of the M. smegmatis AmtR and describe the role of AmtR in the regulation of nitrogen metabolism in response to nitrogen availability. To determine the function of AmtR in M. smegmatis, we performed genome-wide expression profiling comparing the wild-type versus an ∆amtR mutant and identified significant changes in the expression of 11 genes, including an operon involved in urea degradation. An AmtR consensus-binding motif (CTGTC-N 4 -GACAG) was identified in the promoter region of this operon, and ligand-independent, high-affinity AmtR binding was validated by both electrophoretic mobility shift assays and surface plasmon resonance measurements. We confirmed the transcription of a cis-encoded small RNA complementary to the gene encoding AmtR under nitrogen excess, and we propose a post-transcriptional regulatory mechanism for AmtR. The three-dimensional X-ray structure of AmtR at 2.0Å revealed an overall TetR-like dimeric structure, and the alignment of the M. smegmatis AmtR and Corynebacterium glutamicum AmtR regulatory domains showed poor structural conservation, providing a potential explanation for the lack of M. smegmatis AmtR interaction with the adenylylated P II protein. Taken together, our data suggest an AmtR (repressor)/GlnR (activator) competitive binding mechanism for transcriptional regulation of urea metabolism that is controlled by a cis-encoded small antisense RNA. Copyright © 2016 Elsevier Ltd. All rights reserved.

Arsenic biotransformation potential of microbial arsH responses in the biogeochemical cycling of arsenic-contaminated groundwater.

PubMed

Chang, Jin-Soo; Yoon, In-Ho; Kim, Kyoung-Woong

2018-01-01

ArsH encodes an oxidoreductase, an NAD(P)H-dependent mononucleotide reductase, with an unknown function, frequently within an ars operon, and is widely distributed in bacteria. Novel arsenite-oxidizing bacteria have been isolated from arsenic-contaminated groundwater and surface soil in Vietnam. We found that ArsH gene activity, with arsenite oxidase in the periplasm; it revealed arsenic oxidation potential of the arsH system. Batch experiment results revealed Citrobacter freundii strain VTan4 (DQ481466) and Pseudomonas putida strain VTw33 (DQ481482) completely oxidized 1 mM of arsenite to arsenate within 30-50 h. High concentrations of arsenic were detected in groundwater and surrounding soil obtained from Vinh Tru village in Ha Nam province (groundwater: 11.0 μg/L to 37.0 μg/L; and soil: 2.5 mg/kg, 390.1 mg/kg), respectively. An arsH gene encoding an organoarsenical oxidase protein was observed in arsenite-oxidizing Citrobacter freundii strain VTan4 (DQ481466), whereas arsB, arsH, and arsH were detected in Pseudomonas putida strain VTw33 (DQ481482). arsH gene in bacteria was first reported from Vietnam for resistance and arsenite oxidase. We proposed that residues, Ser 43, Arg 45, Ser 48, and Tyr 49 are required for arsenic binding and activation of arsH. The ars-mediated biotransformation strongly influenced potential arsenite oxidase enzyme of the operon encoding a homogeneous arsH. Results suggest that the further study of arsenite-oxidizing bacteria may lead to a better understanding of arsenite oxidase responses, such as those of arsH, that may be applied to control biochemical properties; for example, speciation, detoxification, bioremediation, biotransformation, and mobilization of arsenic in contaminated groundwater. Copyright © 2017 Elsevier Ltd. All rights reserved.

Modular Engineering of l-Tyrosine Production in Escherichia coli

PubMed Central

Juminaga, Darmawi; Baidoo, Edward E. K.; Redding-Johanson, Alyssa M.; Batth, Tanveer S.; Burd, Helcio; Mukhopadhyay, Aindrila; Petzold, Christopher J.

2012-01-01

Efficient biosynthesis of l-tyrosine from glucose is necessary to make biological production economically viable. To this end, we designed and constructed a modular biosynthetic pathway for l-tyrosine production in E. coli MG1655 by encoding the enzymes for converting erythrose-4-phosphate (E4P) and phosphoenolpyruvate (PEP) to l-tyrosine on two plasmids. Rational engineering to improve l-tyrosine production and to identify pathway bottlenecks was directed by targeted proteomics and metabolite profiling. The bottlenecks in the pathway were relieved by modifications in plasmid copy numbers, promoter strength, gene codon usage, and the placement of genes in operons. One major bottleneck was due to the bifunctional activities of quinate/shikimate dehydrogenase (YdiB), which caused accumulation of the intermediates dehydroquinate (DHQ) and dehydroshikimate (DHS) and the side product quinate; this bottleneck was relieved by replacing YdiB with its paralog AroE, resulting in the production of over 700 mg/liter of shikimate. Another bottleneck in shikimate production, due to low expression of the dehydroquinate synthase (AroB), was alleviated by optimizing the first 15 codons of the gene. Shikimate conversion to l-tyrosine was improved by replacing the shikimate kinase AroK with its isozyme, AroL, which effectively consumed all intermediates formed in the first half of the pathway. Guided by the protein and metabolite measurements, the best producer, consisting of two medium-copy-number, dual-operon plasmids, was optimized to produce >2 g/liter l-tyrosine at 80% of the theoretical yield. This work demonstrates the utility of targeted proteomics and metabolite profiling in pathway construction and optimization, which should be applicable to other metabolic pathways. PMID:22020510

Operon-mapper: A Web Server for Precise Operon Identification in Bacterial and Archaeal Genomes.

PubMed

Taboada, Blanca; Estrada, Karel; Ciria, Ricardo; Merino, Enrique

2018-06-19

Operon-mapper is a web server that accurately, easily, and directly predicts the operons of any bacterial or archaeal genome sequence. The operon predictions are based on the intergenic distance of neighboring genes as well as the functional relationships of their protein-coding products. To this end, Operon-mapper finds all the ORFs within a given nucleotide sequence, along with their genomic coordinates, orthology groups, and functional relationships. We believe that Operon-mapper, due to its accuracy, simplicity and speed, as well as the relevant information that it generates, will be a useful tool for annotating and characterizing genomic sequences. http://biocomputo.ibt.unam.mx/operon_mapper/.

Heat Shock Response of Archaeoglobus fulgidus†

PubMed Central

Rohlin, Lars; Trent, Jonathan D.; Salmon, Kirsty; Kim, Unmi; Gunsalus, Robert P.; Liao, James C.

2005-01-01

The heat shock response of the hyperthermophilic archaeon Archaeoglobus fulgidus strain VC-16 was studied using whole-genome microarrays. On the basis of the resulting expression profiles, approximately 350 of the 2,410 open reading frames (ORFs) (ca. 14%) exhibited increased or decreased transcript abundance. These span a range of cell functions, including energy production, amino acid metabolism, and signal transduction, where the majority are uncharacterized. One ORF called AF1298 was identified that contains a putative helix-turn-helix DNA binding motif. The gene product, HSR1, was expressed and purified from Escherichia coli and was used to characterize specific DNA recognition regions upstream of two A. fulgidus genes, AF1298 and AF1971. The results indicate that AF1298 is autoregulated and is part of an operon with two downstream genes that encode a small heat shock protein, Hsp20, and cdc48, an AAA+ ATPase. The DNase I footprints using HSR1 suggest the presence of a cis-binding motif upstream of AF1298 consisting of CTAAC-N5-GTTAG. Since AF1298 is negatively regulated in response to heat shock and encodes a protein only distantly related to the N-terminal DNA binding domain of Phr of Pyrococcus furiosus, these results suggest that HSR1 and Phr may belong to an evolutionarily diverse protein family involved in heat shock regulation in hyperthermophilic and mesophilic Archaea organisms. PMID:16109946

The Identification and Functional Characterization of WxL Proteins from Enterococcus faecium Reveal Surface Proteins Involved in Extracellular Matrix Interactions

PubMed Central

Galloway-Peña, Jessica R.; Liang, Xiaowen; Singh, Kavindra V.; Yadav, Puja; Chang, Chungyu; La Rosa, Sabina Leanti; Shelburne, Samuel; Ton-That, Hung; Höök, Magnus

2014-01-01

The WxL domain recently has been identified as a novel cell wall binding domain found in numerous predicted proteins within multiple Gram-positive bacterial species. However, little is known about the function of proteins containing this novel domain. Here, we identify and characterize 6 Enterococcus faecium proteins containing the WxL domain which, by reverse transcription-PCR (RT-PCR) and genomic analyses, are located in three similarly organized operons, deemed WxL loci A, B, and C. Western blotting, electron microscopy, and enzyme-linked immunosorbent assays (ELISAs) determined that genes of WxL loci A and C encode antigenic, cell surface proteins exposed at higher levels in clinical isolates than in commensal isolates. Secondary structural analyses of locus A recombinant WxL domain-containing proteins found they are rich in β-sheet structure and disordered segments. Using Biacore analyses, we discovered that recombinant WxL proteins from locus A bind human extracellular matrix proteins, specifically type I collagen and fibronectin. Proteins encoded by locus A also were found to bind to each other, suggesting a novel cell surface complex. Furthermore, bile salt survival assays and animal models using a mutant from which all three WxL loci were deleted revealed the involvement of WxL operons in bile salt stress and endocarditis pathogenesis. In summary, these studies extend our understanding of proteins containing the WxL domain and their potential impact on colonization and virulence in E. faecium and possibly other Gram-positive bacterial species. PMID:25512313

Identification and Disruption of the proBA Locus in Listeria monocytogenes: Role of Proline Biosynthesis in Salt Tolerance and Murine Infection

PubMed Central

Sleator, Roy D.; Gahan, Cormac G. M.; Hill, Colin

2001-01-01

Intracellular accumulation of the amino acid proline has previously been linked to the salt tolerance and virulence potential of a number of bacteria. Taking advantage of the proBA mutant Escherichia coli CSH26, we identified a listerial proBA operon coding for enzymes functionally similar to the glutamyl kinase (GK) and glutamylphosphate reductase (GPR) enzyme complex which catalyzes the first and second steps of proline biosynthesis in E. coli. The first gene of the operon, proB, is predicted to encode GK, a 276-residue protein with a calculated molecular mass of 30.03 kDa and pl of 5.2. Distal to the promoter and overlapping the 3′ end of proB by 17 bp is proA, which encodes GPR, a 415-residue protein with a calculated molecular mass of 45.50 kDa (pl 5.3). Using this information, we created a chromosomal deletion mutant by allelic exchange which is auxotrophic for proline. This mutant was used to assess the contribution of proline anabolism to osmotolerance and virulence. While inactivation of proBA had no significant effect on virulence in mouse assays (either perorally or intraperitoneally), growth at low (2 to 4% NaCl) and high (>6% NaCl) salt concentrations in complex media was significantly reduced in the absence of efficient proline synthesis. We conclude that while proline biosynthesis plays little, if any, role in the intracellular life cycle and infectious nature of Listeria monocytogenes, it can play an important role in survival in osmolyte-depleted environments of elevated osmolarity. PMID:11375165

Differential Substrate Specificity and Kinetic Behavior of Escherichia coli YfdW and Oxalobacter formigenes Formyl Coenzyme A Transferase▿ †

PubMed Central

Toyota, Cory G.; Berthold, Catrine L.; Gruez, Arnaud; Jónsson, Stefán; Lindqvist, Ylva; Cambillau, Christian; Richards, Nigel G. J.

2008-01-01

The yfdXWUVE operon appears to encode proteins that enhance the ability of Escherichia coli MG1655 to survive under acidic conditions. Although the molecular mechanisms underlying this phenotypic behavior remain to be elucidated, findings from structural genomic studies have shown that the structure of YfdW, the protein encoded by the yfdW gene, is homologous to that of the enzyme that mediates oxalate catabolism in the obligate anaerobe Oxalobacter formigenes, O. formigenes formyl coenzyme A transferase (FRC). We now report the first detailed examination of the steady-state kinetic behavior and substrate specificity of recombinant, wild-type YfdW. Our studies confirm that YfdW is a formyl coenzyme A (formyl-CoA) transferase, and YfdW appears to be more stringent than the corresponding enzyme (FRC) in Oxalobacter in employing formyl-CoA and oxalate as substrates. We also report the effects of replacing Trp-48 in the FRC active site with the glutamine residue that occupies an equivalent position in the E. coli protein. The results of these experiments show that Trp-48 precludes oxalate binding to a site that mediates substrate inhibition for YfdW. In addition, the replacement of Trp-48 by Gln-48 yields an FRC variant for which oxalate-dependent substrate inhibition is modified to resemble that seen for YfdW. Our findings illustrate the utility of structural homology in assigning enzyme function and raise the question of whether oxalate catabolism takes place in E. coli upon the up-regulation of the yfdXWUVE operon under acidic conditions. PMID:18245280

Extrachromosomal Nucleolus-Like Compartmentalization by a Plasmid-Borne Ribosomal RNA Operon and Its Role in Nucleoid Compaction.

PubMed

Mata Martin, Carmen; Sun, Zhe; Zhou, Yan Ning; Jin, Ding Jun

2018-01-01

In the fast-growing Escherichia coli cells, RNA polymerase (RNAP) molecules are concentrated and form foci at clusters of ribosomal RNA (rRNA) operons resembling eukaryotic nucleolus. The bacterial nucleolus-like organization, spatially compartmentalized at the surface of the compact bacterial chromosome (nucleoid), serves as transcription factories for rRNA synthesis and ribosome biogenesis, which influences the organization of the nucleoid. Unlike wild type that has seven rRNA operons in the genome in a mutant that has six (Δ6 rrn ) rRNA operons deleted in the genome, there are no apparent transcription foci and the nucleoid becomes uncompacted, indicating that formation of RNAP foci requires multiple copies of rRNA operons clustered in space and is critical for nucleoid compaction. It has not been determined, however, whether a multicopy plasmid-borne rRNA operon (p rrnB ) could substitute the multiple chromosomal rRNA operons for the organization of the bacterial nucleolus-like structure in the mutants of Δ6 rrn and Δ7 rrn that has all seven rRNA operons deleted in the genome. We hypothesized that extrachromosomal nucleolus-like structures are similarly organized and functional in trans from p rrnB in these mutants. In this report, using multicolor images of three-dimensional superresolution Structured Illumination Microscopy (3D-SIM), we determined the distributions of both RNAP and NusB that are a transcription factor involved in rRNA synthesis and ribosome biogenesis, p rrnB clustering, and nucleoid structure in these two mutants in response to environmental cues. Our results found that the extrachromosomal nucleolus-like organization tends to be spatially located at the poles of the mutant cells. In addition, formation of RNAP foci at the extrachromosomal nucleolus-like structure condenses the nucleoid, supporting the idea that active transcription at the nucleolus-like organization is a driving force in nucleoid compaction.

Relatively slow stochastic gene-state switching in the presence of positive feedback significantly broadens the region of bimodality through stabilizing the uninduced phenotypic state.

PubMed

Ge, Hao; Wu, Pingping; Qian, Hong; Xie, Xiaoliang Sunney

2018-03-01

Within an isogenic population, even in the same extracellular environment, individual cells can exhibit various phenotypic states. The exact role of stochastic gene-state switching regulating the transition among these phenotypic states in a single cell is not fully understood, especially in the presence of positive feedback. Recent high-precision single-cell measurements showed that, at least in bacteria, switching in gene states is slow relative to the typical rates of active transcription and translation. Hence using the lac operon as an archetype, in such a region of operon-state switching, we present a fluctuating-rate model for this classical gene regulation module, incorporating the more realistic operon-state switching mechanism that was recently elucidated. We found that the positive feedback mechanism induces bistability (referred to as deterministic bistability), and that the parameter range for its occurrence is significantly broadened by stochastic operon-state switching. We further show that in the absence of positive feedback, operon-state switching must be extremely slow to trigger bistability by itself. However, in the presence of positive feedback, which stabilizes the induced state, the relatively slow operon-state switching kinetics within the physiological region are sufficient to stabilize the uninduced state, together generating a broadened parameter region of bistability (referred to as stochastic bistability). We illustrate the opposite phenotype-transition rate dependence upon the operon-state switching rates in the two types of bistability, with the aid of a recently proposed rate formula for fluctuating-rate models. The rate formula also predicts a maximal transition rate in the intermediate region of operon-state switching, which is validated by numerical simulations in our model. Overall, our findings suggest a biological function of transcriptional "variations" among genetically identical cells, for the emergence of bistability and transition between phenotypic states.

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

Selfish operons: the evolutionary impact of gene clustering in prokaryotes and eukaryotes.

PubMed

Lawrence, J

1999-12-01

The Selfish Operon Model postulates that the organization of bacterial genes into operons is beneficial to the constituent genes in that proximity allows horizontal cotransfer of all genes required for a selectable phenotype; eukaryotic operons formed for very different reasons. Horizontal transfer of selfish operons most probably promotes bacterial diversification.

Molecular cloning and expression of a gene that controls the high-temperature regulon of Escherichia coli.

PubMed Central

Neidhardt, F C; VanBogelen, R A; Lau, E T

1983-01-01

The high-temperature production (HTP) regulon of Escherichia coli consists of a set of operons that are induced coordinately by a shift to a high temperature under the control of a single chromosomal gene called htpR or hin. To identify more components of this regulon, the rates of synthesis of many polypeptides resolved on two-dimensional polyacrylamide gels were measured in various strains by pulse-labeling after a temperature shift-up. A total of 13 polypeptides were found to be heat inducible only in cells bearing a normal htpR gene on the chromosome or on a plasmid; on this basis these polypeptides were designated products of the HTP regulon. Several hybrid plasmids that contain segments of the E. coli chromosome in the 75-min region were found to carry the htpR gene. A restriction map of this region was constructed, and selected fragments were subcloned and tested for the ability to complement an htpR mutant. The polypeptides encoded by these fragments were detected by permitting expression in maxicells, minicells, and chloramphenicol-treated cells. Complementation was accompanied by production of a polypeptide having a molecular weight of approximately 33,000. This polypeptide, designated F33.4, was markedly reduced in amount in an htpR mutant expected to contain very little htpR gene product. Polypeptide F33.4 is postulated to be the product of htpR and to be an effector that controls heat induction of the HTP regulon. Images PMID:6337122

Roles of relSpn in Stringent Response, Global Regulation, and Virulence of Serotype 2 Streptococcus pneumoniae D39

PubMed Central

Kazmierczak, Krystyna M.; Wayne, Kyle J.; Rechtsteiner, Andreas; Winkler, Malcolm E.

2009-01-01

Summary RelA/ SpoT homolog (RSH) proteins have (p)ppGpp synthetase and hydrolase activities that mediate major global responses to nutrient limitation and other stresses. RSH proteins are conserved in most bacteria and play diverse roles in bacterial pathogenesis. We report here that the RSH protein of S. pneumoniae, RelSpn, can be deleted and is the primary source of (p)ppGpp synthesis in virulent strain D39 under some conditions. A D39 ΔrelSpn mutant grew well in complex medium, but did not grow in chemically defined medium unless supplemented with the metals copper and manganese. Transcriptome analysis of D39 rel+Spn and ΔreSpn strains treated with mupirocin revealed relSpn-independent (translation stress), relSpn-dependent (stringent response), and ΔrelSpn-dependent changes suggesting that relSpn and (p)ppGpp amount play wide-ranging homeostatic roles in pneumococcal physiology, besides adjusting macromolecular synthesis and transport in response to nutrient availability. Notably, the relSpn-dependent response included significant up-regulation of the ply operon encoding pneumolysin toxin, whereas the ΔrelSpn-dependent response affected expression linked to the VicRK and CiaRH two component systems. Finally, a D39 ΔrelSpn mutant was severely attenuated and displayed a significantly altered course of disease progression in a mouse model of infection, which was restored to normal by an ectopic copy of rel+Spn. PMID:19426208

Structural Analysis of the Regulatory Domain of ExsA, a Key Transcriptional Regulator of the Type Three Secretion System in Pseudomonas aeruginosa

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

Shrestha, Manisha; Xiao, Yi; Robinson, Howard

Pseudomonas aeruginosa employs a type three secretion system to facilitate infections in mammalian hosts. The operons encoding genes of structural components of the secretion machinery and associated virulence factors are all under the control of the AraC-type transcriptional activator protein, ExsA. ExsA belongs to a unique subfamily of AraC-proteins that is regulated through protein-protein contacts rather than small molecule ligands. Prior to infection, ExsA is inhibited through a direct interaction with the anti-activator ExsD. To activate ExsA upon host cell contact this interaction is disrupted by the anti-antiactivator protein ExsC. Here we report the crystal structure of the regulatory domainmore » of ExsA, which is known to mediate ExsA dimerization as well as ExsD binding. The crystal structure suggests two models for the ExsA dimer. Both models confirmed the previously shown involvement of helix α-3 in ExsA dimerization but one also suggest a role for helix α-2. These structural data are supported by the observation that a mutation in α-2 greatly diminished the ability of ExsA to activate transcription in vitro. Lastly, additional in vitro transcription studies revealed that a conserved pocket, used by AraC and the related ToxT protein for the binding of small molecule regulators, although present in ExsA is not involved in binding of ExsD.« less

Structural Analysis of the Regulatory Domain of ExsA, a Key Transcriptional Regulator of the Type Three Secretion System in Pseudomonas aeruginosa

DOE PAGES

Shrestha, Manisha; Xiao, Yi; Robinson, Howard; ...

2015-08-28

Pseudomonas aeruginosa employs a type three secretion system to facilitate infections in mammalian hosts. The operons encoding genes of structural components of the secretion machinery and associated virulence factors are all under the control of the AraC-type transcriptional activator protein, ExsA. ExsA belongs to a unique subfamily of AraC-proteins that is regulated through protein-protein contacts rather than small molecule ligands. Prior to infection, ExsA is inhibited through a direct interaction with the anti-activator ExsD. To activate ExsA upon host cell contact this interaction is disrupted by the anti-antiactivator protein ExsC. Here we report the crystal structure of the regulatory domainmore » of ExsA, which is known to mediate ExsA dimerization as well as ExsD binding. The crystal structure suggests two models for the ExsA dimer. Both models confirmed the previously shown involvement of helix α-3 in ExsA dimerization but one also suggest a role for helix α-2. These structural data are supported by the observation that a mutation in α-2 greatly diminished the ability of ExsA to activate transcription in vitro. Lastly, additional in vitro transcription studies revealed that a conserved pocket, used by AraC and the related ToxT protein for the binding of small molecule regulators, although present in ExsA is not involved in binding of ExsD.« less

Evidence against the selfish operon theory.

PubMed

Pál, Csaba; Hurst, Laurence D

2004-06-01

According to the selfish operon hypothesis, the clustering of genes and their subsequent organization into operons is beneficial for the constituent genes because it enables the horizontal gene transfer of weakly selected, functionally coupled genes. The majority of these are expected to be non-essential genes. From our analysis of the Escherichia coli genome, we conclude that the selfish operon hypothesis is unlikely to provide a general explanation for clustering nor can it account for the gene composition of operons. Contrary to expectations, essential genes with related functions have an especially strong tendency to cluster, even if they are not in operons. Moreover, essential genes are particularly abundant in operons.

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

Eckert, C. A.; Sullivan, R.; Johnson, C.

CO2 and H2 are promising feedstocks for production of valuable biocompounds. Ralstonia eutropha utilizes these feedstocks to generate energy (ATP) and reductant (NAD(P)H) via oxidation of H2 by a membrane-bound (MBH) and a soluble hydrogenase (SH) for CO2 fixation by the Calvin-Benson-Bassham (CBB) cycle. Increased expression of the enzyme that fixes CO2 (RubisCO) resulted in 6-fold activity improvement in vitro, while increased expression of the MBH operon or the SH operon plus MBH operon maturation factors necessary for activity resulted in a 10-fold enhancement. Current research involves genetic manipulation of two endogenous cbb operons for increased expression, analysis of expressionmore » and activity of CBB/MBH/SH, cofactor ratios, and downstream products during autotrophic growth in control versus enhanced strains, and development of strategies for long-term, optimal overexpression. These studies will improve our understanding of autotrophic metabolism and provide a chassis strain for autotrophic production of biodiesel and other valuable carbon biocompounds.« less

Organization and post-transcriptional processing of the psb B operon from chloroplasts of Populus deltoides.

PubMed

Dixit, R; Trivedi, P K; Nath, P; Sane, P V

1999-09-01

Chloroplast genes are typically organized into polycistronic transcription units that give rise to complex sets of mono- and oligo-cistronic overlapping RNAs through a series of processing steps. The psbB operon contains genes for the PSII (psbB, psbT, psbH) and cytochrome b(6)f (petB and petD) complexes which are needed in different amounts during chloroplast biogenesis. The functional significance of gene organization in this polycistronic unit, containing information for two different complexes, is not known and is of interest. To determine the organization and expression of these complexes, studies have been carried out on crop plants by different groups, but not much information is known about trees. We present the nucleotide sequences of PSII genes and RNA profiles of the genes located in the psbB operon from Populus deltoides, a tree species. Although the gene organization of this operon in P. deltoides is similar to that in other species, a few variations have been observed in the processing scheme.

1,3-Propanediol dehydrogenases in Lactobacillus reuteri: impact on central metabolism and 3-hydroxypropionaldehyde production.

PubMed

Stevens, Marc J A; Vollenweider, Sabine; Meile, Leo; Lacroix, Christophe

2011-08-03

Lactobacillus reuteri metabolizes glycerol to 3-hydroxypropionaldehyde (3-HPA) and further to 1,3-propanediol (1,3-PDO), the latter step catalysed by a propanediol dehydrogenase (PDH). The last step in this pathway regenerates NAD+ and enables therefore the energetically more favourable production of acetate over ethanol during growth on glucose. A search throughout the genome of L. reuteri DSM 20016 revealed two putative PDHs encoded by ORFs lr_0030 and lr_1734. ORF lr_1734 is situated in the pdu operon encoding the glycerol conversion machinery and therefore likely involved in 1,3-PDO formation. ORF lr_0030 has not been associated with PDH-activity so far. To elucidate the role of these two PDHs, gene deletion mutant strains were constructed. Growth behaviour on glucose was comparable between the wild type and both mutant strains. However, on glucose + glycerol, the exponential growth rate of Δlr_0030 was lower compared to the wild type and the lr_1734 mutant. Furthermore, glycerol addition resulted in decreased ethanol production in the wild type and Δlr_1734, but not in Δlr_0030. PDH activity measurements using 3-HPA as a substrate revealed lower activity of Δlr_0030 extracts from exponential growing cells compared to wild type and Δlr_1734 extracts.During biotechnological 3-HPA production using non-growing cells, the ratio 3-HPA to 1,3-PDO was approximately 7 in the wild type and Δlr_0030, whereas this ratio was 12.5 in the mutant Δlr_1734. The enzyme encoded by lr_0030 plays a pivotal role in 3-HPA conversion in exponential growing L. reuteri cells. The enzyme encoded by lr_1734 is active during 3-HPA production by non-growing cells and this enzyme is a useful target to enhance 3-HPA production and minimize formation of the by-product 1,3-PDO.

Four Proteins Encoded in the gspB-secY2A2 Operon of Streptococcus gordonii Mediate the Intracellular Glycosylation of the Platelet-Binding Protein GspB

PubMed Central

Takamatsu, Daisuke; Bensing, Barbara A.; Sullam, Paul M.

2004-01-01

Platelet binding by Streptococcus gordonii strain M99 is mediated predominantly by the cell surface glycoprotein GspB. This adhesin consists of a putative N-terminal signal peptide, two serine-rich regions (SRR1 and SRR2), a basic region between SRR1 and SRR2, and a C-terminal cell wall anchoring domain. The glycosylation of GspB is mediated at least in part by Gly and Nss, which are encoded in the secY2A2 locus immediately downstream of gspB. This region also encodes two proteins (Gtf and Orf4) that are required for the expression of GspB but whose functions have not been delineated. In this study, we further characterized the roles of Gly, Nss, Gtf, and Orf4 by investigating the expression and glycosylation of a series of glutathione S-transferase-GspB fusion proteins in M99 and in gly, nss, gtf, and orf4 mutants. Compared with fusion proteins expressed in the wild-type background, fusion proteins expressed in the mutant strain backgrounds showed altered electrophoretic mobility. In addition, the fusion proteins formed insoluble aggregates in protoplasts of the gtf and orf4 mutants. Glycan detection and lectin blot analysis revealed that SRR1 and SRR2 were glycosylated but that the basic region was unmodified. When the fusion protein was expressed in Escherichia coli, glycosylation of this protein was observed only in the presence of both gtf and orf4. These results demonstrate that Gly, Nss, Gtf, and Orf4 are all involved in the intracellular glycosylation of SRRs. Moreover, Gtf and Orf4 are essential for glycosylation, which in turn is important for the solubility of GspB. PMID:15489421

The Genome Sequence of the Tomato-Pathogenic Actinomycete Clavibacter michiganensis subsp. michiganensis NCPPB382 Reveals a Large Island Involved in Pathogenicity▿ †

PubMed Central

Gartemann, Karl-Heinz; Abt, Birte; Bekel, Thomas; Burger, Annette; Engemann, Jutta; Flügel, Monika; Gaigalat, Lars; Goesmann, Alexander; Gräfen, Ines; Kalinowski, Jörn; Kaup, Olaf; Kirchner, Oliver; Krause, Lutz; Linke, Burkhard; McHardy, Alice; Meyer, Folker; Pohle, Sandra; Rückert, Christian; Schneiker, Susanne; Zellermann, Eva-Maria; Pühler, Alfred; Eichenlaub, Rudolf; Kaiser, Olaf; Bartels, Daniela

2008-01-01

Clavibacter michiganensis subsp. michiganensis is a plant-pathogenic actinomycete that causes bacterial wilt and canker of tomato. The nucleotide sequence of the genome of strain NCPPB382 was determined. The chromosome is circular, consists of 3.298 Mb, and has a high G+C content (72.6%). Annotation revealed 3,080 putative protein-encoding sequences; only 26 pseudogenes were detected. Two rrn operons, 45 tRNAs, and three small stable RNA genes were found. The two circular plasmids, pCM1 (27.4 kbp) and pCM2 (70.0 kbp), which carry pathogenicity genes and thus are essential for virulence, have lower G+C contents (66.5 and 67.6%, respectively). In contrast to the genome of the closely related organism Clavibacter michiganensis subsp. sepedonicus, the genome of C. michiganensis subsp. michiganensis lacks complete insertion elements and transposons. The 129-kb chp/tomA region with a low G+C content near the chromosomal origin of replication was shown to be necessary for pathogenicity. This region contains numerous genes encoding proteins involved in uptake and metabolism of sugars and several serine proteases. There is evidence that single genes located in this region, especially genes encoding serine proteases, are required for efficient colonization of the host. Although C. michiganensis subsp. michiganensis grows mainly in the xylem of tomato plants, no evidence for pronounced genome reduction was found. C. michiganensis subsp. michiganensis seems to have as many transporters and regulators as typical soil-inhabiting bacteria. However, the apparent lack of a sulfate reduction pathway, which makes C. michiganensis subsp. michiganensis dependent on reduced sulfur compounds for growth, is probably the reason for the poor survival of C. michiganensis subsp. michiganensis in soil. PMID:18192381

A Bacillus subtilis Gene Induced by Cold Shock Encodes a Membrane Phospholipid Desaturase

PubMed Central

Aguilar, Pablo S.; Cronan, John E.; de Mendoza, Diego

1998-01-01

Bacillus subtilis grown at 37°C synthesizes saturated fatty acids with only traces of unsaturated fatty acids (UFAs). However, when cultures growing at 37°C are transferred to 20°C, UFA synthesis is induced. We report the identification and characterization of the gene encoding the fatty acid desaturase of B. subtilis. This gene, called des, was isolated by complementation of Escherichia coli strains with mutations in either of two different genes of UFA synthesis. The des gene encodes a polypeptide of 352 amino acid residues containing the three conserved histidine cluster motifs and two putative membrane-spanning domains characteristic of the membrane-bound desaturases of plants and cyanobacteria. Expression of the des gene in E. coli resulted in desaturation of palmitic acid moieties of the membrane phospholipids to give the novel mono-UFA cis-5-hexadecenoic acid, indicating that the B. subtilis des gene product is a Δ5 acyl-lipid desaturase. The des gene was disrupted, and the resulting null mutant strains were unable to synthesize UFAs upon a shift to low growth temperatures. The des null mutant strain grew as well as its congenic parent at 20 or 37°C but showed severely reduced survival during stationary phase. Analysis of operon fusions in which the des promoter directed the synthesis of a lacZ reporter gene showed that des expression is repressed at 37°C, but a shift of cultures from 37 to 20°C resulted in a 10- to 15-fold increase in transcription. This is the first report of a membrane phospholipid desaturase in a nonphotosynthetic organism and the first direct evidence for cold induction of a desaturase. PMID:9555904

N-terminal truncations in the FhlA protein result in formate- and MoeA-independent expression of the hyc (formate hydrogenlyase) operon of Escherichia coli.

PubMed

Self, W T; Hasona, A; Shanmugam, K T

2001-11-01

The formate hydrogenlyase complex of Escherichia coli catalyses the cleavage of formate to CO2 and H2 and consists of a molybdoenzyme formate dehydrogenase-H, hydrogenase 3 and intermediate electron carriers. The structural genes of this enzyme complex are activated by the FhlA protein in the presence of both formate and molybdate; ModE-Mo serves as a secondary activator. Mutational analysis of the FhlA protein established that the unique N-terminal region of this protein was responsible for formate- and molybdenum-dependent transcriptional control of the hyc operon. Analysis of the N-terminal sequence of the FhlA protein revealed a unique motif (amino acids 7-37), which is also found in ATPases associated with several members of the ABC-type transporter family. A deletion derivative of FhlA lacking these amino acids (FhlA9-2) failed to activate the hyc operon in vivo, although the FhlA9-2 did bind to hyc promoter DNA in vitro. The ATPase activity of the FhlA9-2-DNA-formate complex was at least three times higher than that of the native protein-DNA-formate complex, and this degree of activity was achieved at a lower formate level. Extending the deletion to amino acid 117 (FhlA167) not only reversed the FhlA(-) phenotype of FhlA9-2, but also led to both molybdenum- and formate-independence. Deleting the entire N-terminal domain (between amino acids 5 and 374 of the 692 amino acid protein) also led to an effector-independent transcriptional activator (FhlA165), which had a twofold higher level of hyc operon expression than the native protein. Both FhlA165 and FhlA167 still required ModE-Mo as a secondary activator for an optimal level of hyc-lac expression. The FhlA165 protein also had a twofold higher affinity to hyc promoter DNA than the native FhlA protein, while the FhlA167 protein had a significantly lower affinity for hyc promoter DNA in vitro. Although the ATPase activity of the native protein was increased by formate, the ATPase activity of neither FhlA165 or FhlA167 responded to formate. Removal of the first 117 amino acids of the FhlA protein appears to result in a constitutive, effector-independent activation of transcription of the genes encoding the components of the formate hydrogenlyase complex. The sequence similarity to ABC-ATPases, combined with the properties of the FhlA deletion proteins, led to the proposal that the N-terminal region of the native FhlA protein interacts with formate transport proteins, both as a formate transport facilitator and as a cytoplasmic acceptor.

Co-spread of metal and antibiotic resistance within ST3-IncHI2 plasmids from E. coli isolates of food-producing animals.

PubMed

Fang, Liangxing; Li, Xingping; Li, Liang; Li, Shumin; Liao, Xiaoping; Sun, Jian; Liu, Yahong

2016-05-04

Concerns have been raised in recent years regarding co-selection for antibiotic resistance among bacteria exposed to heavy metals, particularly copper and zinc, used as growth promoters for some livestock species. In this study, 25 IncHI2 plasmids harboring oqxAB (20/25)/blaCTX-M (18/25) were found with sizes ranging from ∼260 to ∼350 kb and 22 belonged to the ST3-IncHI2 group. In addition to blaCTX-M and oqxAB, pcoA-E (5/25) and silE-P (5/25), as well as aac(6')-Ib-cr (18/25), floR (16/25), rmtB (6/25), qnrS1(3/25) and fosA3 (2/25), were also identified on these IncHI2 plasmids. The plasmids carried pco and sil contributed to increasing in the MICs of CuSO4 and AgNO3. The genetic context surrounding the two operons was well conserved except some variations within the pco operon. The ~32 kb region containing the two operons identified in the IncHI2 plasmids was also found in chromosomes of different Enterobacteriaceae species. Further, phylogenetic analysis of this structure showed that Tn7-like transposon might play an important role in cross-genus transfer of the sil and pco operons among Enterobacteriaceae. In conclusion, co-existence of the pco and sil operons, and oqxAB/blaCTX-M as well as other antibiotic resistance genes on IncHI2 plasmids may promote the development of multidrug-resistant bacteria.

Stabilization of the Virulence Plasmid pSLT of Salmonella Typhimurium by Three Maintenance Systems and Its Evaluation by Using a New Stability Test.

PubMed

Lobato-Márquez, Damián; Molina-García, Laura; Moreno-Córdoba, Inma; García-Del Portillo, Francisco; Díaz-Orejas, Ramón

2016-01-01

Certain Salmonella enterica serovars belonging to subspecies I carry low-copy-number virulence plasmids of variable size (50-90 kb). All of these plasmids share the spv operon, which is important for systemic infection. Virulence plasmids are present at low copy numbers. Few copies reduce metabolic burden but suppose a risk of plasmid loss during bacterial division. This drawback is counterbalanced by maintenance modules that ensure plasmid stability, including partition systems and toxin-antitoxin (TA) loci. The low-copy number virulence pSLT plasmid of Salmonella enterica serovar Typhimurium encodes three auxiliary maintenance systems: one partition system ( parAB ) and two TA systems ( ccdAB ST and vapBC2 ST ). The TA module ccdAB ST has previously been shown to contribute to pSLT plasmid stability and vapBC2 ST to bacterial virulence. Here we describe a novel assay to measure plasmid stability based on the selection of plasmid-free cells following elimination of plasmid-containing cells by ParE toxin, a DNA gyrase inhibitor. Using this new maintenance assay we confirmed a crucial role of parAB in pSLT maintenance. We also showed that vapBC2 ST , in addition to contribute to bacterial virulence, is important for plasmid stability. We have previously shown that ccdAB ST encodes an inactive CcdB ST toxin. Using our new stability assay we monitored the contribution to plasmid stability of a ccdAB ST variant containing a single mutation (R99W) that restores the toxicity of CcdB ST . The "activation" of CcdB ST (R99W) did not increase pSLT stability by ccdAB ST . In contrast, ccdAB ST behaves as a canonical type II TA system in terms of transcriptional regulation. Of interest, ccdAB ST was shown to control the expression of a polycistronic operon in the pSLT plasmid. Collectively, these results show that the contribution of the CcdB ST toxin to pSLT plasmid stability may depend on its role as a co-repressor in coordination with CcdA ST antitoxin more than on its toxic activity.

FnrL and Three Dnr Regulators Are Used for the Metabolic Adaptation to Low Oxygen Tension in Dinoroseobacter shibae

PubMed Central

Ebert, Matthias; Laaß, Sebastian; Thürmer, Andrea; Roselius, Louisa; Eckweiler, Denitsa; Daniel, Rolf; Härtig, Elisabeth; Jahn, Dieter

2017-01-01

The heterotrophic marine bacterium Dinoroseobacter shibae utilizes aerobic respiration and anaerobic denitrification supplemented with aerobic anoxygenic photosynthesis for energy generation. The aerobic to anaerobic transition is controlled by four Fnr/Crp family regulators in a unique cascade-type regulatory network. FnrL is utilizing an oxygen-sensitive Fe-S cluster for oxygen sensing. Active FnrL is inducing most operons encoding the denitrification machinery and the corresponding heme biosynthesis. Activation of gene expression of the high oxygen affinity cbb3-type and repression of the low affinity aa3-type cytochrome c oxidase is mediated by FnrL. Five regulator genes including dnrE and dnrF are directly controlled by FnrL. Multiple genes of the universal stress protein (USP) and cold shock response are further FnrL targets. DnrD, most likely sensing NO via a heme cofactor, co-induces genes of denitrification, heme biosynthesis, and the regulator genes dnrE and dnrF. DnrE is controlling genes for a putative Na+/H+ antiporter, indicating a potential role of a Na+ gradient under anaerobic conditions. The formation of the electron donating primary dehydrogenases is coordinated by FnrL and DnrE. Many plasmid encoded genes were DnrE regulated. DnrF is controlling directly two regulator genes including the Fe-S cluster biosynthesis regulator iscR, genes of the electron transport chain and the glutathione metabolism. The genes for nitrate reductase and CO dehydrogenase are repressed by DnrD and DnrF. Both regulators in concert with FnrL are inducing the photosynthesis genes. One of the major denitrification operon control regions, the intergenic region between nirS and nosR2, contains one Fnr/Dnr binding site. Using regulator gene mutant strains, lacZ-reporter gene fusions in combination with promoter mutagenesis, the function of the single Fnr/Dnr binding site for FnrL-, DnrD-, and partly DnrF-dependent nirS and nosR2 transcriptional activation was shown. Overall, the unique regulatory network of the marine bacterium D. shibae for the transition from aerobic to anaerobic growth composed of four Crp/Fnr family regulators was elucidated. PMID:28473807

Genome Sequence of Thermotoga sp Strain RQ2, a Hyperthermophilic Bacterium Isolated from a Geothermally Heated Region of the Seafloor near Ribeira Quente, the Azores

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

Swithers, Kristen S; DiPippo, Jonathan L; Bruce, David

2011-01-01

Thermotoga sp. strain RQ2 is probably a strain of Thermotoga maritima. Its complete genome sequence allows for an examination of the extent and consequences of gene flow within Thermotoga species and strains. Thermotoga sp. RQ2 differs from T. maritima in its genes involved in myo-inositol metabolism. Its genome also encodes an apparent fructose phosphotransferase system (PTS) sugar transporter. This operon is also found in Thermotoga naphthophila strain RKU-10 but no other Thermotogales. These are the first reported PTS transporters in the Thermotogales.

Genome of the opportunistic pathogen Streptococcus sanguinis.

PubMed

Xu, Ping; Alves, Joao M; Kitten, Todd; Brown, Arunsri; Chen, Zhenming; Ozaki, Luiz S; Manque, Patricio; Ge, Xiuchun; Serrano, Myrna G; Puiu, Daniela; Hendricks, Stephanie; Wang, Yingping; Chaplin, Michael D; Akan, Doruk; Paik, Sehmi; Peterson, Darrell L; Macrina, Francis L; Buck, Gregory A

2007-04-01

The genome of Streptococcus sanguinis is a circular DNA molecule consisting of 2,388,435 bp and is 177 to 590 kb larger than the other 21 streptococcal genomes that have been sequenced. The G+C content of the S. sanguinis genome is 43.4%, which is considerably higher than the G+C contents of other streptococci. The genome encodes 2,274 predicted proteins, 61 tRNAs, and four rRNA operons. A 70-kb region encoding pathways for vitamin B(12) biosynthesis and degradation of ethanolamine and propanediol was apparently acquired by horizontal gene transfer. The gene complement suggests new hypotheses for the pathogenesis and virulence of S. sanguinis and differs from the gene complements of other pathogenic and nonpathogenic streptococci. In particular, S. sanguinis possesses a remarkable abundance of putative surface proteins, which may permit it to be a primary colonizer of the oral cavity and agent of streptococcal endocarditis and infection in neutropenic patients.

Hypothesis Formation and Qualitative Reasoning in Molecular Biology

DTIC Science & Technology

1989-06-01

presents studies of the trp operon in the bacterium S . Marcescens . In vitro transcription studies showed that transcription termination does occur in...observed was that there are two 4.4. ANNOTATED CHRONOLOGY OF THE RESEARCH 135 translation-start codons in the S . marcescens leader region. The authors...of leader-region mRNA secondary structures in attenuation in the S . marcescens trp operon. A different bac- terium was used because it included

Non-contiguous genome sequence of Mycobacterium simiae strain DSM 44165(T.).

PubMed

Sassi, Mohamed; Robert, Catherine; Raoult, Didier; Drancourt, Michel

2013-01-01

Mycobacterium simiae is a non-tuberculosis mycobacterium causing pulmonary infections in both immunocompetent and imunocompromized patients. We announce the draft genome sequence of M. simiae DSM 44165(T). The 5,782,968-bp long genome with 65.15% GC content (one chromosome, no plasmid) contains 5,727 open reading frames (33% with unknown function and 11 ORFs sizing more than 5000 -bp), three rRNA operons, 52 tRNA, one 66-bp tmRNA matching with tmRNA tags from Mycobacterium avium, Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium microti, Mycobacterium marinum, and Mycobacterium africanum and 389 DNA repetitive sequences. Comparing ORFs and size distribution between M. simiae and five other Mycobacterium species M. simiae clustered with M. abscessus and M. smegmatis. A 40-kb prophage was predicted in addition to two prophage-like elements, 7-kb and 18-kb in size, but no mycobacteriophage was seen after the observation of 10(6) M. simiae cells. Fifteen putative CRISPRs were found. Three genes were predicted to encode resistance to aminoglycosides, betalactams and macrolide-lincosamide-streptogramin B. A total of 163 CAZYmes were annotated. M. simiae contains ESX-1 to ESX-5 genes encoding for a type-VII secretion system. Availability of the genome sequence may help depict the unique properties of this environmental, opportunistic pathogen.

Analysis, Characterization, and Loci of the tuf Genes in Lactobacillus and Bifidobacterium Species and Their Direct Application for Species Identification

PubMed Central

Ventura, Marco; Canchaya, Carlos; Meylan, Valèrie; Klaenhammer, Todd R.; Zink, Ralf

2003-01-01

We analyzed the tuf gene, encoding elongation factor Tu, from 33 strains representing 17 Lactobacillus species and 8 Bifidobacterium species. The tuf sequences were aligned and used to infer phylogenesis among species of lactobacilli and bifidobacteria. We demonstrated that the synonymous substitution affecting this gene renders elongation factor Tu a reliable molecular clock for investigating evolutionary distances of lactobacilli and bifidobacteria. In fact, the phylogeny generated by these tuf sequences is consistent with that derived from 16S rRNA analysis. The investigation of a multiple alignment of tuf sequences revealed regions conserved among strains belonging to the same species but distinct from those of other species. PCR primers complementary to these regions allowed species-specific identification of closely related species, such as Lactobacillus casei group members. These tuf gene-based assays developed in this study provide an alternative to present methods for the identification for lactic acid bacterial species. Since a variable number of tuf genes have been described for bacteria, the presence of multiple genes was examined. Southern analysis revealed one tuf gene in the genomes of lactobacilli and bifidobacteria, but the tuf gene was arranged differently in the genomes of these two taxa. Our results revealed that the tuf gene in bifidobacteria is flanked by the same gene constellation as the str operon, as originally reported for Escherichia coli. In contrast, bioinformatic and transcriptional analyses of the DNA region flanking the tuf gene in four Lactobacillus species indicated the same four-gene unit and suggested a novel tuf operon specific for the genus Lactobacillus. PMID:14602655

Transcriptional analysis of the MrpJ network: modulation of diverse virulence-associated genes and direct regulation of mrp fimbrial and flhDC flagellar operons in Proteus mirabilis.

PubMed

Bode, Nadine J; Debnath, Irina; Kuan, Lisa; Schulfer, Anjelique; Ty, Maureen; Pearson, Melanie M

2015-06-01

The enteric bacterium Proteus mirabilis is associated with a significant number of catheter-associated urinary tract infections (UTIs). Strict regulation of the antagonistic processes of adhesion and motility, mediated by fimbriae and flagella, respectively, is essential for disease progression. Previously, the transcriptional regulator MrpJ, which is encoded by the mrp fimbrial operon, has been shown to repress both swimming and swarming motility. Here we show that MrpJ affects an array of cellular processes beyond adherence and motility. Microarray analysis found that expression of mrpJ mimicking levels observed during UTIs leads to differential expression of 217 genes related to, among other functions, bacterial virulence, type VI secretion, and metabolism. We probed the molecular mechanism of transcriptional regulation by MrpJ using transcriptional reporters and chromatin immunoprecipitation (ChIP). Binding of MrpJ to two virulence-associated target gene promoters, the promoters of the flagellar master regulator flhDC and mrp itself, appears to be affected by the condensation state of the native chromosome, although both targets share a direct MrpJ binding site proximal to the transcriptional start. Furthermore, an mrpJ deletion mutant colonized the bladders of mice at significantly lower levels in a transurethral model of infection. Additionally, we observed that mrpJ is widely conserved in a collection of recent clinical isolates. Altogether, these findings support a role of MrpJ as a global regulator of P. mirabilis virulence. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Bacterial genes involved in incorporation of nickel into a hydrogenase enzyme.

PubMed Central

Fu, C; Javedan, S; Moshiri, F; Maier, R J

1994-01-01

Nickel is an essential component of all H2-uptake hydrogenases. A fragment of DNA that complements a H2-uptake-deficient but nickel-cured mutant strain (JHK7) of Bradyrhizobium japonicum was isolated and sequenced. This 4.5-kb DNA fragment contains four open reading frames designated as ORF1, hupN, hupO, and hupP, which encode polypeptides with predicted masses of 17, 40, 19, and 63.5 kDa, respectively. The last three open reading frames (hupNOP) are most likely organized as an operon with a putative sigma 54-type promoter. Based on its hydropathy profile, HupN is predicted to be a transmembrane protein. It has 56% identity to the previously described HoxN (high-affinity nickel transport protein) of Alcaligenes eutrophus. A subclone (pJF23) containing the hupNOP genes excluding ORF1 completely complemented (in trans) strain JHK7 for hydrogenase activity in low nickel conditions. pJF26 containing only a functional hupN complemented the hydrogenase activity of mutant strain JHK7 to 30-55% of the wild-type level. Mutant strain JHK70, with a chromosomal deletion in hupP but with an intact hupNO, showed greater activities than pJF26-complemented JHK7 but still had lower activities than the wild type at all nickel levels tested. pJF25, containing the entire hupO and hupP, but without hupN (a portion of hupN was deleted), did not complement hydrogenase activity of mutant strain JHK7. The results suggest that the products of the hupNOP operon are all involved in nickel incorporation/metabolism into the hydrogenase apoprotein. Based on (previous) nickel transport studies of strain JHK7, the hupNOP genes appear not to be involved in nickel transport by whole cells. The deleterious effects on hydrogenase expression are most pronounced by lack of the HupN product. PMID:8197192

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

Control of photosynthetic membrane assembly in Rhodobacter sphaeroides mediated by puhA and flanking sequences.

PubMed Central

Sockett, R E; Donohue, T J; Varga, A R; Kaplan, S

1989-01-01

A reaction center H- strain (RCH-) of Rhodobacter sphaeroides, PUHA1, was made by in vitro deletion of an XhoI restriction endonuclease fragment from the puhA gene coupled with insertion of a kanamycin resistance gene cartridge. The resulting construct was delivered to R. sphaeroides wild-type 2.4.1, with the defective puhA gene replacing the wild-type copy by recombination, followed by selection for kanamycin resistance. When grown under conditions known to induce intracytoplasmic membrane development, PUHA1 synthesized a pigmented intracytoplasmic membrane. Spectral analysis of this membrane showed that it was deficient in B875 spectral complexes as well as functional reaction centers and that the level of B800-850 spectral complexes was greater than in the wild type. The RCH- strain was photosythetically incompetent, but photosynthetic growth was restored by complementation with a 1.45-kilobase (kb) BamHI restriction endonuclease fragment containing the puhA gene carried in trans on plasmid pRK404. B875 spectral complexes were not restored by complementation with the 1.45-kb BamHI restriction endonuclease fragment containing the puhA gene but were restored along with photosynthetic competence by complementation with DNA from a cosmid carrying the puhA gene, as well as a flanking DNA sequence. Interestingly, B875 spectral complexes, but not photosynthetic competence, were restored to PUHA1 by introduction in trans of a 13-kb BamHI restriction endonuclease fragment carrying genes encoding the puf operon region of the DNA. The effect of the puhA deletion was further investigated by an examination of the levels of specific mRNA species derived from the puf and puc operons, as well as by determinations of the relative abundances of polypeptides associated with various spectral complexes by immunological methods. The roles of puhA and other genetic components in photosynthetic gene expression and membrane assembly are discussed. Images PMID:2644200

[Investigation of biofilm formation properties of staphylococcus isolates].

PubMed

Öcal, Duygu Nilüfer; Dolapçı, İştar; Karahan, Zeynep Ceren; Tekeli, Alper

2017-01-01

Biofilm production is an important virulence factor which allows staphylococci to adhere to medical devices. The principal component of biofilm is a "polysaccharide intercellular adhesin (PIA)" which is composed of a beta-1,6-N-acetylglucosamine polymer synthesized by an enzyme (N-acetylglucosamine transferase) encoded by the ica operon found on the bacterial chromosome. This operon is composed of four genes (A, B, C, and D), and a transposable element IS256. In this study, we aimed to determine the biofilm production characteristics of invasive/non-invasive staphylococcus isolates and different staphylococcus species. Biofilm production of 166 staphylococci was phenotypically investigated on Congo Red Agar (CRA); the presence of icaA, icaD and IS256 genes were investigated by polymerase chain reaction (PCR). 74 of the isolates (44.6%) were identified as methicillin resistant Staphylococcus aureus (MRSA), 25 (15.1%) as methicillin sensitive S.aureus (MSSA), 25 (37.3%) as Staphylococcus hominis, 20 (12%) as S.epidermidis, ten (15%) as Staphylococcus haemolyticus, nine (13.4%) as Staphylococcus capitis, two (3%) Staphylococcus saprophyticus and one (1.5%) as Staphylococcus warnerii. Of the MRSA strains, 52 were isolated from blood and 22 from nose; all MSSA strains were isolated from nose cultures. Coagulase-negative staphylococci (CoNS) strains were composed of invasive and non-invasive strains isolated from nose, catheter tip and blood cultures from patients with catheter. Production with CRA method was found to be statistically significant in invasive isolates (p< 0.001). It is concluded that; as the biofilm formation capacity of invasive isolates can cause refractory infections and the importance of carriage and hospital infections of these bacteria, it is important to prevent the spread of these isolates. A combination of phenotypic and genotypic tests is recommended for the investigation of biofilm formation in staphylococci. 40.3% of the CoNS isolates, and 85.8% of S.aureus isolates produced biofilm on CRA (p< 0.001) and with PCR method the ratio of carrying three genes was found to be statistically important in S.aureus when compared with CoNS. Carriage of three genes and biofilm formation capacity of invasive isolates can cause refractory infections and the importance of carriage and hospital infections of these bacteria, it is important to prevent the spread of these isolates. A combination of phenotypic and genotypic tests is recommended for the investigation of biofilm formation in staphylococci.

A multicopper oxidase is essential for manganese oxidation and laccase-like activity in Pedomicrobium sp. ACM 3067.

PubMed

Ridge, Justin P; Lin, Marianne; Larsen, Eloise I; Fegan, Mark; McEwan, Alastair G; Sly, Lindsay I

2007-04-01

Pedomicrobium sp. ACM 3067 is a budding-hyphal bacterium belonging to the alpha-Proteobacteria which is able to oxidize soluble Mn2+ to insoluble manganese oxide. A cosmid, from a whole-genome library, containing the putative genes responsible for manganese oxidation was identified and a primer-walking approach yielded 4350 bp of novel sequence. Analysis of this sequence showed the presence of a predicted three-gene operon, moxCBA. The moxA gene product showed homology to multicopper oxidases (MCOs) and contained the characteristic four copper-binding motifs (A, B, C and D) common to MCOs. An insertion mutation of moxA showed that this gene was essential for both manganese oxidation and laccase-like activity. The moxB gene product showed homology to a family of outer membrane proteins which are essential for Type I secretion in Gram-negative bacteria. moxBA has not been observed in other manganese-oxidizing bacteria but homologues were identified in the genomes of several bacteria including Sinorhizobium meliloti 1021 and Agrobacterium tumefaciens C58. These results suggest that moxBA and its homologues constitute a family of genes encoding an MCO and a predicted component of the Type I secretion system.

Positions of Trp Codons in the Leader Peptide-Coding Region of the at Operon Influence Anti-Trap Synthesis and trp Operon Expression in Bacillus licheniformis▿

PubMed Central

Levitin, Anastasia; Yanofsky, Charles

2010-01-01

Tryptophan, phenylalanine, tyrosine, and several other metabolites are all synthesized from a common precursor, chorismic acid. Since tryptophan is a product of an energetically expensive biosynthetic pathway, bacteria have developed sensing mechanisms to downregulate synthesis of the enzymes of tryptophan formation when synthesis of the amino acid is not needed. In Bacillus subtilis and some other Gram-positive bacteria, trp operon expression is regulated by two proteins, TRAP (the tryptophan-activated RNA binding protein) and AT (the anti-TRAP protein). TRAP is activated by bound tryptophan, and AT synthesis is increased upon accumulation of uncharged tRNATrp. Tryptophan-activated TRAP binds to trp operon leader RNA, generating a terminator structure that promotes transcription termination. AT binds to tryptophan-activated TRAP, inhibiting its RNA binding ability. In B. subtilis, AT synthesis is upregulated both transcriptionally and translationally in response to the accumulation of uncharged tRNATrp. In this paper, we focus on explaining the differences in organization and regulatory functions of the at operon's leader peptide-coding region, rtpLP, of B. subtilis and Bacillus licheniformis. Our objective was to correlate the greater growth sensitivity of B. licheniformis to tryptophan starvation with the spacing of the three Trp codons in its at operon leader peptide-coding region. Our findings suggest that the Trp codon location in rtpLP of B. licheniformis is designed to allow a mild charged-tRNATrp deficiency to expose the Shine-Dalgarno sequence and start codon for the AT protein, leading to increased AT synthesis. PMID:20061467

Mutations affecting transport of the hexitols D-mannitol, D-glucitol, and galactitol in Escherichia coli K-12: isolation and mapping.

PubMed Central

Lengeler, J

1975-01-01

Mutants of Escherichia coli K-12 unable to grow on any of the three naturally occurring hexitols D-manitol, D-glucitol, and galactitol and, among these specifically, mutants with altered transport and phosphorylating activity have been isolated. Different isolation procedures have been utilized, including suicide by D-[3H]mannitol, chemotaxis, and resistance to the toxic hexitol analogue 2-deoxy-arabino-hexitol. Mutations thus obtained have been mapped in four distinct operons. (i) Mutations affecting an enzyme II-complexmt1 activity of the phosphoenolpyruvate-dependent phosphotransferase system all map in gene mtlA. This gene has previously been shown (Solomon and Lin, 1972) to be part of an operon, mtl, located at 71 min on the E. coli linkage map containing, in addition to mtlA, the cis-dominant regulatory gene mtlC and mtlD, the structural gene for the enzyme D-mannitol-1-phosphate dehydrogenase. The gene order in this operon, induced by D-mannitol, is mtlC A D. (ii) Mutations in gene gutA affecting a second enzyme II-complexgut of the phosphotransferase system map at 51 min, clustered in operon gutC A D together with the cis-dominant regulatory gene gutC and the structural gene gutD for the enzyme D-glucitol-6-phosphate dehydrogenase. The gut operon, previously called sbl or srl, is induced by D-glucitol. (iii) Mutations affecting the transport and catabolism of galactitol are clustered in a third operon, gatC A D, located at 40.5 min. This operon again contains a cis-dominant regulatory gene, gatC, the structural gene gatD for galactitol-1-phosphate dehydrogenase, and gene gatA coding for a thrid hexitol-specific enzyme II-complexgat. Other genes coding for two additional enzymes involved in galactitol catabolism apparently are not linked to gatC A D. (iv) A fourth class of mutants pleiotropically negative for hexitol growth and transport maps in the pts operon. Triple-negative mutants (mtlA gutA gatA) do not have further transport or phosphorylating activity for any of the three hexitols. PMID:1100602

Transport of 5-aminolevulinic acid by the dipeptide permease in Salmonella typhimurium.

PubMed

Elliott, T

1993-01-01

In a previous search for mutants of Salmonella typhimurium that are defective in heme synthesis, one class that is apparently defective in 5-aminolevulinic acid (ALA) uptake (alu) was found. Here, I describe the characterization of these mutations. The mutations all map to a single locus near 77.5 min on the genetic map, which is transcribed counterclockwise. Nutritional tests, genetic and physical mapping, and partial DNA sequence analysis revealed that alu mutants are defective in a periplasmic binding protein-dependent permease that also transports dipeptides, encoded by the dpp operon. The uptake of labeled ALA is defective in dpp mutants and is markedly increased in a strain that has elevated transcription of the dpp locus. Unlabeled L-leucyl-glycine competes with labeled ALA for uptake. In a strain carrying both a dpp-lac operon fusion and a functional copy of the dpp locus, the expression of beta-galactosidase is not induced by ALA, nor, in a hemL mutant, does expression of dpp change substantially during starvation for ALA. The dipeptide permease displays a relaxed substrate specificity that allows transport of the important nonpeptide nutrient ALA, whose structure is closely related to that of glycyl-glycine.

Regulating Toxin-Antitoxin Expression: Controlled Detonation of Intracellular Molecular Timebombs

PubMed Central

Hayes, Finbarr; Kędzierska, Barbara

2014-01-01

Genes for toxin-antitoxin (TA) complexes are widely disseminated in bacteria, including in pathogenic and antibiotic resistant species. The toxins are liberated from association with the cognate antitoxins by certain physiological triggers to impair vital cellular functions. TAs also are implicated in antibiotic persistence, biofilm formation, and bacteriophage resistance. Among the ever increasing number of TA modules that have been identified, the most numerous are complexes in which both toxin and antitoxin are proteins. Transcriptional autoregulation of the operons encoding these complexes is key to ensuring balanced TA production and to prevent inadvertent toxin release. Control typically is exerted by binding of the antitoxin to regulatory sequences upstream of the operons. The toxin protein commonly works as a transcriptional corepressor that remodels and stabilizes the antitoxin. However, there are notable exceptions to this paradigm. Moreover, it is becoming clear that TA complexes often form one strand in an interconnected web of stress responses suggesting that their transcriptional regulation may prove to be more intricate than currently understood. Furthermore, interference with TA gene transcriptional autoregulation holds considerable promise as a novel antibacterial strategy: artificial release of the toxin factor using designer drugs is a potential approach to induce bacterial suicide from within. PMID:24434949

Structure of the detoxification catalyst mercuric ion reductase from Bacillus sp. strain RC607

NASA Astrophysics Data System (ADS)

Schiering, N.; Kabsch, W.; Moore, M. J.; Distefano, M. D.; Walsh, C. T.; Pai, E. F.

1991-07-01

SEVERAL hundred million tons of toxic mercurials are dispersed in the biosphere1. Microbes can detoxify organo-mercurials and mercury salts through sequential action of two enzymes, organomercury lyase2 and mercuric ion reductase (MerA) 3-5. The latter, a homodimer with homology to the FAD-dependent disulphide oxidoreductases6, catalyses the reaction NADPH + Hg(II) --> NADP+ + H+Hg(0), one of the very rare enzymic reactions with metal substrates. Human glutathione reductase7,8 serves as a reference molecule for FAD-dependent disulphide reductases and between its primary structure9 and that of MerA from Tn501 (Pseudomonas), Tn21 (Shigella), pI258 (Staphylococcus) and Bacillus, 25-30% of the residues have been conserved10,11. All MerAs have a C-terminal extension about 15 residues long but have very varied N termini. Although the enzyme from Streptomyces lividans has no addition, from Pseudomonas aeruginosa Tn5Ol and Bacillus sp. strain RC607 it has one and two copies respectively of a domain of 80-85 residues, highly homologous to MerP, the periplasmic component of proteins encoded by the mer operon11. These domains can be proteolytically cleaved off without changing the catalytic efficiency3. We report here the crystal structure of MerA from the Gram-positive bacterium Bacillus sp. strain RC607. Analysis of its complexes with nicotinamide dinucleotide substrates and the inhibitor Cd(II) reveals how limited structural changes enable an enzyme to accept as substrate what used to be a dangerous inhibitor. Knowledge of the mode of mercury ligation is a prerequisite for understanding this unique detoxification mechanism.

Gene encoding γ-carbonic anhydrase is cotranscribed with argC and induced in response to stationary phase and high CO2 in Azospirillum brasilense Sp7

PubMed Central

2010-01-01

Background Carbonic anhydrase (CA) is a ubiquitous enzyme catalyzing the reversible hydration of CO2 to bicarbonate, a reaction underlying diverse biochemical and physiological processes. Gamma class carbonic anhydrases (γ-CAs) are widespread in prokaryotes but their physiological roles remain elusive. At present, only γ-CA of Methanosarcina thermophila (Cam) has been shown to have CA activity. Genome analysis of a rhizobacterium Azospirillum brasilense, revealed occurrence of ORFs encoding one β-CA and two γ-CAs. Results One of the putative γ-CA encoding genes of A. brasilense was cloned and overexpressed in E. coli. Electrometric assays for CA activity of the whole cell extracts overexpressing recombinant GCA1 did not show CO2 hydration activity. Reverse transcription-PCR analysis indicated that gca1 in A. brasilense is co-transcribed with its upstream gene annotated as argC, which encodes a putative N-acetyl-γ-glutamate-phosphate reductase. 5'-RACE also demonstrated that there was no transcription start site between argC and gca1, and the transcription start site located upstream of argC transcribed both the genes (argC-gca1). Using transcriptional fusions of argC-gca1 upstream region with promoterless lacZ, we further demonstrated that gca1 upstream region did not have any promoter and its transcription occurred from a promoter located in the argC upstream region. The transcription of argC-gca1 operon was upregulated in stationary phase and at elevated CO2 atmosphere. Conclusions This study shows lack of CO2 hydration activity in a recombinant protein expressed from a gene predicted to encode a γ-carbonic anhydrase in A. brasilense although it cross reacts with anti-Cam antibody raised against a well characterized γ-CA. The organization and regulation of this gene along with the putative argC gene suggests its involvement in arginine biosynthetic pathway instead of the predicted CO2 hydration. PMID:20598158

Gene encoding gamma-carbonic anhydrase is cotranscribed with argC and induced in response to stationary phase and high CO2 in Azospirillum brasilense Sp7.

PubMed

Kaur, Simarjot; Mishra, Mukti N; Tripathi, Anil K

2010-07-04

Carbonic anhydrase (CA) is a ubiquitous enzyme catalyzing the reversible hydration of CO2 to bicarbonate, a reaction underlying diverse biochemical and physiological processes. Gamma class carbonic anhydrases (gamma-CAs) are widespread in prokaryotes but their physiological roles remain elusive. At present, only gamma-CA of Methanosarcina thermophila (Cam) has been shown to have CA activity. Genome analysis of a rhizobacterium Azospirillum brasilense, revealed occurrence of ORFs encoding one beta-CA and two gamma-CAs. One of the putative gamma-CA encoding genes of A. brasilense was cloned and overexpressed in E. coli. Electrometric assays for CA activity of the whole cell extracts overexpressing recombinant GCA1 did not show CO2 hydration activity. Reverse transcription-PCR analysis indicated that gca1 in A. brasilense is co-transcribed with its upstream gene annotated as argC, which encodes a putative N-acetyl-gamma-glutamate-phosphate reductase. 5'-RACE also demonstrated that there was no transcription start site between argC and gca1, and the transcription start site located upstream of argC transcribed both the genes (argC-gca1). Using transcriptional fusions of argC-gca1 upstream region with promoterless lacZ, we further demonstrated that gca1 upstream region did not have any promoter and its transcription occurred from a promoter located in the argC upstream region. The transcription of argC-gca1 operon was upregulated in stationary phase and at elevated CO2 atmosphere. This study shows lack of CO2 hydration activity in a recombinant protein expressed from a gene predicted to encode a gamma-carbonic anhydrase in A. brasilense although it cross reacts with anti-Cam antibody raised against a well characterized gamma-CA. The organization and regulation of this gene along with the putative argC gene suggests its involvement in arginine biosynthetic pathway instead of the predicted CO2 hydration.

moxFG region encodes four polypeptides in the methanol-oxidizing bacterium Methylobacterium sp. strain AM1

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

Anderson, D.J.; Lidstrom, M.E.

The polypeptides encoded by a putative methanol oxidation (mox) operon of Methylobacterium sp. strain AM1 were expressed in Escherichia coli, using a coupled in vivo T7 RNA polymerase/promoter gene expression system. Two mox genes had been previously mapped to this region: moxF, the gene encoding the methanol dehydrogenase (MeDH) polypeptide; and moxG, a gene believed to encode a soluble type c cytochrome, cytochrome c/sub L/. In this study, four polypeptides of M/sub r/, 60,000, 30,000, 20,000, and 12,000 were found to be encoded by the moxFG region and were tentatively designated moxF, -J, -G, and -I, respectively. The arrangement ofmore » the genes (5' to 3') was found to be moxFJGI. The identities of three of the four polypeptides were determined by protein immunoblot analysis. The product of moxF, the M/sub r/-60,000 polypeptide, was confirmed to be the MeDH polypeptide. The product of moxG, the M/sub r/-20,000 polypeptide, was identified as mature cytochrome c/sub L/, and the product of moxI, the M/sub r/-12,000 polypeptide, was identified as a MeDH-associated polypeptide that copurifies with the holoenzyme. The identity of the M/sub r/-30,000 polypeptide (the moxJ gene product) could not be determined. The function of the M/sub r/-12,000 MeDH-associated polypeptide is not yet clear. However, it is not present in mutants that lack the M/sub r/-60,000 MeDH subunit, and it appears that the stability of the MeDH-associated polypeptide is dependent on the presence of the M/sub r/-60,000 MeDH polypeptide. Our data suggest that both the M/sub r/-30,000 and -12,000 polypeptides are involved in methanol oxidation, which would bring to 12 the number of mox genes in Methylobacterium sp. strain AM1.« less

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.

Characterization of the biotin uptake system encoded by the biotin-inducible bioYMN operon of Corynebacterium glutamicum

PubMed Central

2012-01-01

Background The amino acid-producing Gram-positive Corynebacterium glutamicum is auxotrophic for biotin although biotin ring assembly starting from the precursor pimeloyl-CoA is still functional. It possesses AccBC, the α-subunit of the acyl-carboxylases involved in fatty acid and mycolic acid synthesis, and pyruvate carboxylase as the only biotin-containing proteins. Comparative genome analyses suggested that the putative transport system BioYMN encoded by cg2147, cg2148 and cg2149 might be involved in biotin uptake by C. glutamicum. Results By comparison of global gene expression patterns of cells grown with limiting or excess supply of biotin or with dethiobiotin as supplement replacing biotin revealed that expression of genes coding for enzymes of biotin ring assembly and for the putative uptake system was regulated according to biotin availability. RT-PCR and 5'-RACE experiments demonstrated that the genes bioY, bioM, and bioN are transcribed from one promoter as a single transcript. Biochemical analyses revealed that BioYMN catalyzes the effective uptake of biotin with a concentration of 60 nM biotin supporting a half-maximal transport rate. Maximal biotin uptake rates were at least five fold higher in biotin-limited cells as compared to cells grown with excess biotin. Overexpression of bioYMN led to an at least 50 fold higher biotin uptake rate as compared to the empty vector control. Overproduction of BioYMN alleviated biotin limitation and interfered with triggering L-glutamate production by biotin limitation. Conclusions The operon bioYMN from C. glutamicum was shown to be induced by biotin limitation. Transport assays with radio-labeled biotin revealed that BioYMN functions as a biotin uptake system. Overexpression of bioYMN affected L-glutamate production triggered by biotin limitation. PMID:22243621

Sequence analysis of the phs operon in Salmonella typhimurium and the contribution of thiosulfate reduction to anaerobic energy metabolism.

PubMed Central

Heinzinger, N K; Fujimoto, S Y; Clark, M A; Moreno, M S; Barrett, E L

1995-01-01

The phs chromosomal locus of Salmonella typhimurium is essential for the dissimilatory anaerobic reduction of thiosulfate to hydrogen sulfide. Sequence analysis of the phs region revealed a functional operon with three open reading frames, designated phsA, phsB, and phsC, which encode peptides of 82.7, 21.3, and 28.5 kDa, respectively. The predicted products of phsA and phsB exhibited significant homology with the catalytic and electron transfer subunits of several other anaerobic molybdoprotein oxidoreductases, including Escherichia coli dimethyl sulfoxide reductase, nitrate reductase, and formate dehydrogenase. Simultaneous comparison of PhsA to seven homologous molybdoproteins revealed numerous similarities among all eight throughout the entire frame, hence, significant amino acid conservation among molybdoprotein oxidoreductases. Comparison of PhsB to six other homologous sequences revealed four highly conserved iron-sulfur clusters. The predicted phsC product was highly hydrophobic and similar in size to the hydrophobic subunits of the molybdoprotein oxidoreductases containing subunits homologous to phsA and phsB. Thus, phsABC appears to encode thiosulfate reductase. Single-copy phs-lac translational fusions required both anaerobiosis and thiosulfate for full expression, whereas multicopy phs-lac translational fusions responded to either thiosulfate or anaerobiosis, suggesting that oxygen and thiosulfate control of phs involves negative regulation. A possible role for thiosulfate reduction in anaerobic respiration was examined. Thiosulfate did not significantly augment the final densities of anaerobic cultures grown on any of the 18 carbon sources tested. on the other hand, washed stationary-phase cells depleted of ATP were shown to synthesize small amounts of ATP on the addition of the formate and thiosulfate, suggesting that the thiosulfate reduction plays a unique role in anaerobic energy conservation by S typhimurium. PMID:7751291

Defining the disulphide stress response in Streptomyces coelicolor A3(2): identification of the sigmaR regulon.

PubMed

Paget, M S; Molle, V; Cohen, G; Aharonowitz, Y; Buttner, M J

2001-11-01

In the Gram-positive, antibiotic-producing bacterium Streptomyces coelicolor A3(2), the thiol-disulphide status of the hyphae is controlled by a novel regulatory system consisting of a sigma factor, sigmaR, and its cognate anti-sigma factor, RsrA. Oxidative stress induces intramolecular disulphide bond formation in RsrA, which causes it to lose affinity for sigmaR, thereby releasing sigmaR to activate transcription of the thioredoxin operon, trxBA. Here, we exploit a preliminary consensus sequence for sigmaR target promoters to identify 27 new sigmaR target genes and operons, thereby defining the global response to disulphide stress in this organism. Target genes related to thiol metabolism encode a second thioredoxin (TrxC), a glutaredoxin-like protein and enzymes involved in the biosynthesis of the low-molecular-weight thiol-containing compounds cysteine and molybdopterin. In addition, the level of the major actinomycete thiol buffer, mycothiol, was fourfold lower in a sigR null mutant, although no candidate mycothiol biosynthetic genes were identified among the sigmaR targets. Three sigmaR target genes encode ribosome-associated products (ribosomal subunit L31, ppGpp synthetase and tmRNA), suggesting that the translational machinery is modified by disulphide stress. The product of another sigmaR target gene was found to be a novel RNA polymerase-associated protein, RbpA, suggesting that the transcriptional machinery may also be modified in response to disulphide stress. We present DNA sequence evidence that many of the targets identified in S. coelicolor are also under the control of the sigmaR homologue in the actinomycete pathogen Mycobacterium tuberculosis.

Characterization of the biotin uptake system encoded by the biotin-inducible bioYMN operon of Corynebacterium glutamicum.

PubMed

Schneider, Jens; Peters-Wendisch, Petra; Stansen, K Corinna; Götker, Susanne; Maximow, Stanislav; Krämer, Reinhard; Wendisch, Volker F

2012-01-13

The amino acid-producing Gram-positive Corynebacterium glutamicum is auxotrophic for biotin although biotin ring assembly starting from the precursor pimeloyl-CoA is still functional. It possesses AccBC, the α-subunit of the acyl-carboxylases involved in fatty acid and mycolic acid synthesis, and pyruvate carboxylase as the only biotin-containing proteins. Comparative genome analyses suggested that the putative transport system BioYMN encoded by cg2147, cg2148 and cg2149 might be involved in biotin uptake by C. glutamicum. By comparison of global gene expression patterns of cells grown with limiting or excess supply of biotin or with dethiobiotin as supplement replacing biotin revealed that expression of genes coding for enzymes of biotin ring assembly and for the putative uptake system was regulated according to biotin availability. RT-PCR and 5'-RACE experiments demonstrated that the genes bioY, bioM, and bioN are transcribed from one promoter as a single transcript. Biochemical analyses revealed that BioYMN catalyzes the effective uptake of biotin with a concentration of 60 nM biotin supporting a half-maximal transport rate. Maximal biotin uptake rates were at least five fold higher in biotin-limited cells as compared to cells grown with excess biotin. Overexpression of bioYMN led to an at least 50 fold higher biotin uptake rate as compared to the empty vector control. Overproduction of BioYMN alleviated biotin limitation and interfered with triggering L-glutamate production by biotin limitation. The operon bioYMN from C. glutamicum was shown to be induced by biotin limitation. Transport assays with radio-labeled biotin revealed that BioYMN functions as a biotin uptake system. Overexpression of bioYMN affected L-glutamate production triggered by biotin limitation.

Working memory component processes: isolating BOLD signal changes.

PubMed

Motes, Michael A; Rypma, Bart

2010-01-15

The chronology of the component processes subserving working memory (WM) and hemodynamic response lags has hindered the use of fMRI for exploring neural substrates of WM. In the present study, however, participants completed full trials that involved encoding two or six letters, maintaining the memory set over a delay, and then deciding whether a probe was in the memory set or not. Additionally, they completed encode-only, encode-and-maintain, and encode-and-decide partial trials intermixed with the full trials. The inclusion of partial trials allowed for the isolation of BOLD signal changes to the different trial periods. The results showed that only lateral and medial prefrontal cortex regions differentially responded to the 2- and 6-letter memory sets over the trial periods, showing greater activation to 6-letter sets during the encode and maintain trial periods. Thus, the data showed the differential involvement of PFC in the encoding and maintenance of supra- and sub-capacity memory sets and show the efficacy of using fMRI partial trial methods to study WM component processes.

Working Memory Component Processes: Isolating BOLD Signal-Changes

PubMed Central

Motes, Michael A.; Rypma, Bart

2009-01-01

The chronology of the component processes subserving working memory (WM) and hemodynamic response lags have hindered the use of fMRI for exploring neural substrates of WM. In the present study, however, participants completed full trials that involved encoding two or six letters, maintaining the memory-set over a delay, and then deciding whether a probe was in the memory-set or not. Additionally, they completed encode only, encode and maintain, and encode and decide partial-trials intermixed with the full-trials. The inclusion of partial-trials allowed for the isolation of BOLD signal-changes to the different trial-periods. The results showed that only lateral and medial prefrontal cortex regions differentially responded to the 2- and 6-letter memory-sets over the trial-periods, showing greater activation to 6-letter sets during the encode and maintain trial-periods. Thus, the data showed the differential involvement of PFC in the encoding and maintenance of supra- and sub-capacity memory-sets and show the efficacy of using fMRI partial-trial methods to study WM component processes. PMID:19732840