Transcriptome sequencing and metabolic pathways of astaxanthin accumulated in Haematococcus pluvialis mutant under 15% CO2.

PubMed

Cheng, Jun; Li, Ke; Zhu, Yanxia; Yang, Weijuan; Zhou, Junhu; Cen, Kefa

2017-03-01

Transcriptome sequencing and annotation was performed on Haematococcus pluvialis mutant red cells induced with high light under 15% CO 2 to demonstrate why astaxanthin yield of the mutant was 1.7 times higher than that of a wild strain. It was found that 56% of 1947 differentially expressed genes were upregulated in mutant cells. Most significant differences were found in unigenes related to photosynthesis, carotenoid biosynthesis and fatty acid biosynthesis pathways. The pyruvate kinase increased by 3.5-fold in mutant cells. Thus, more pyruvate, which was beneficial to carotenoids and fatty acid biosynthesis, was generated. Phytoene synthase, zeta-carotene desaturase, lycopene beta-cyclase involved in β-carotene biosynthesis in mutant cells were upregulated by 10.4-, 4.4-, and 5.8-fold, respectively. Beta-carotene 3-hydroxylase catalyzing conversion of β-carotene into astaxanthin was upregulated by 18.4-fold. The fatty acid biosynthesis was promoted because of the upregulation of acetyl-CoA synthetase and acetyl-CoA carboxylase, thus increasing astaxanthin esterification and accumulation in mutant cells. Copyright © 2016 Elsevier Ltd. All rights reserved.

Transcription analysis of peloric mutants of Phalaenopsis orchids derived from tissue culture.

PubMed

Chen, Ya Huei; Tsai, Yi Jung; Huang, Jian Zhi; Chen, Fure Chyi

2005-08-01

Tissue culture has been widely used for mass propagation of Phalaenopsis. However, somaclonal variation occurred during micropropagation process posed a severe problem by affecting product quality. In this study, wild type and peloric flower buds of Phalaenopsis hybrids derived from flower stalk nodal culture were used for cDNA-RAPD and cDNA suppression subtractive hybridization analyses in order to study their genetic difference in terms of expressed sequence tags. A total of 209 ESTs from normal flower buds and 230 from mutants were sequenced. These ESTs sequences can be grouped into several functional categories involved in different cellular processes including metabolism, signal transduction, transcription, cell growth and division, protein synthesis, and protein localization, and into a subcategory of proteins with unknown function. Cymbidium mosaic virus transcript was surprisingly found expressed frequently in the peloric mutant of P. Little Mary. Real-time RT-PCR analysis on selected ESTs showed that in mutant flower buds, a bZIP transcription factor (TGA1a-like protein) was down-regulated, while up-regulated genes include auxin-regulated protein kinase, cyclophilin, and TCP-like genes. A retroelement clone was also preferentially expressed in the peloric mutant flowers. On the other hand, ESTs involved in DNA methylation, chromatin remodeling and post-transcriptional regulation, such as DNA methyltransferase, histone acetyltransferase, ERECTA, and DEAD/DEAH RNA helicase, were enriched in normal flower buds than the mutants. The enriched transcripts in the wild type indicate the down regulation of these transcripts in the mutants, and vice versa. The potential roles of the analyzed transcripts in the development of Phalaenopsis flowers are discussed.

Identification of Novel Virulence Determinants in Mycobacterium paratuberculosis by Screening a Library of Insertional Mutants†

PubMed Central

Shin, Sung Jae; Wu, Chia-wei; Steinberg, Howard; Talaat, Adel M.

2006-01-01

Johne's disease, caused by Mycobacterium paratuberculosis infection, is a worldwide problem for the dairy industry and has a possible involvement in Crohn's disease in humans. To identify virulence determinants of this economically important pathogen, a library of 5,060 transposon mutants was constructed using Tn5367 insertion mutagenesis, followed by large-scale sequencing to identify disrupted genes. In this report, 1,150 mutants were analyzed and 970 unique insertion sites were identified. Sequence analysis of the disrupted genes indicated that the insertion of Tn5367 was more prevalent in genomic regions with G+C content (50.5 to 60.5%) lower than the average G+C content (69.3%) of the rest of the genome. Phenotypic screening of the library identified disruptions of genes involved in iron, tryptophan, or mycolic acid metabolic pathways that displayed unique growth characteristics. Bioinformatic analysis of disrupted genes identified a list of potential virulence determinants for further testing with animals. Mouse infection studies showed a significant decrease in tissue colonization by mutants with a disruption in the gcpE, pstA, kdpC, papA2, impA, umaA1, or fabG2_2 gene. Attenuation phenotypes were tissue specific (e.g., for the umaA1 mutant) as well as time specific (e.g., for the impA mutant), suggesting that those genes may be involved in different virulence mechanisms. The identified potential virulence determinants represent novel functional classes that could be necessary for mycobacterial survival during infection and could provide suitable targets for vaccine and drug development against Johne's and Crohn's diseases. PMID:16790754

Post-irradiation somatic mutation and clonal stabilisation time in the human colon.

PubMed Central

Campbell, F; Williams, G T; Appleton, M A; Dixon, M F; Harris, M; Williams, E D

1996-01-01

BACKGROUND: Colorectal crypts are clonal units in which somatic mutation of marker genes in stem cells leads to crypt restricted phenotypic conversion initially involving part of the crypt, later the whole crypt. Studies in mice show that the time taken for the great majority of mutated crypts to be completely converted, the clonal stabilisation time, is four weeks in the colon and 21 weeks in the ileum. Differences in the clonal stabilisation time between tissues and species are thought to reflect differences in stem cell organisation and crypt kinetics. AIM: To study the clonal stabilisation time in the human colorectum. METHODS: Stem cell mutation can lead to crypt restricted loss of O-acetylation of sialomucins in subjects heterozygous for O-acetyltransferase gene activity. mPAS histochemistry was used to visualise and quantify crypts partially or wholly involved by the mutant phenotype in 21 informative cases who had undergone colectomy up to 34 years after radiotherapy. RESULTS: Radiotherapy was followed by a considerable increase in the discordant crypt frequency that remained significantly increased for many years. The proportion of discordant crypts showing partial involvement was initially high but fell to normal levels about 12 months after irradiation. CONCLUSIONS: Crypts wholly involved by a mutant phenotype are stable and persistent while partially involved crypts are transient. The clonal stabilisation time is approximately one year in the human colon compared with four weeks in the mouse. The most likely reason for this is a difference in the number of stem cells in a crypt stem cell niche, although differences in stem cell cycle time and crypt fission may also contribute. These findings are of relevance to colorectal gene therapy and carcinogenesis in stem cell systems. PMID:8944567

Effective non-denaturing purification method for improving the solubility of recombinant actin-binding proteins produced by bacterial expression.

PubMed

Chung, Jeong Min; Lee, Sangmin; Jung, Hyun Suk

2017-05-01

Bacterial expression is commonly used to produce recombinant and truncated mutant eukaryotic proteins. However, heterologous protein expression may render synthesized proteins insoluble. The conventional method used to express a poorly soluble protein, which involves denaturation and refolding, is time-consuming and inefficient. There are several non-denaturing approaches that can increase the solubility of recombinant proteins that include using different bacterial cell strains, altering the time of induction, lowering the incubation temperature, and employing different detergents for purification. In this study, we compared several non-denaturing protocols to express and purify two insoluble 34 kDa actin-bundling protein mutants. The solubility of the mutant proteins was not affected by any of the approaches except for treatment with the detergent sarkosyl. These results indicate that sarkosyl can effectively improve the solubility of insoluble proteins during bacterial expression. Copyright © 2016. Published by Elsevier Inc.

The New Rga Locus Encodes a Negative Regulator of Gibberellin Response in Arabidopsis Thaliana

PubMed Central

Silverstone, A. L.; Mak, PYA.; Martinez, E. C.; Sun, T.

1997-01-01

We have identified a new locus involved in gibberellin (GA) signal transduction by screening for suppressors of the Arabidopsis thaliana GA biosynthetic mutant ga1-3. The locus is named RGA for repressor of ga1-3. Based on the recessive phenotype of the digenic rga/ga1-3 mutant, the wild-type gene product of RGA is probably a negative regulator of GA responses. Our screen for suppressors of ga1-3 identified 17 mutant alleles of RGA as well as 10 new mutant alleles at the previously identified SPY locus. The digenic (double homozygous) rga/ga1-3 mutants are able to partially repress several defects of ga1-3 including stem growth, leaf abaxial trichome initiation, flowering time, and apical dominance. The phenotype of the trigenic mutant (triple homozygous) rga/spy/ga1-3 shows that rga and spy have additive effects regulating flowering time, abaxial leaf trichome initiation and apical dominance. This trigenic mutant is similar to wild type with respect to each of these developmental events. Because rga/spy/ga1-3 is almost insensitive to GA for hypocotyl growth and its bolting stem is taller than the wild-type plant, the combined effects of the rga and spy mutations appear to allow GA-independent stem growth. Our studies indicate that RGA lies on a separate branch of the GA signal transduction pathway from SPY, which leads us to propose a modified model of the GA response pathway. PMID:9215910

NUA Activities at the Plant Nuclear Pore

PubMed Central

Xu, Xianfeng Morgan; Rose, Annkatrin

2007-01-01

NUA (Nuclear Pore Anchor), the Arabidopsis homolog of Tpr (Translocated Promoter Region), is one of the few nuclear pore proteins conserved between animals, yeast and plants. In the May issue of Plant Cell, we report that null mutants of NUA show a pleiotropic, early flowering phenotype accompanied by changes in SUMo and RNA homeostasis. We have shown that the early flowering phenotype is caused by changed abundances of flowering time regulators involved in several pathways. Arabidopsis nua mutants phenocopy mutants lacking the ESD4 (EARlY IN ShoRT DAYS 4) SUMo protease, similar to mutants of their respective yeast homologs. however, in contrast to the comparable yeast mutants, ESD4 does not appear to be delocalized from the nuclear pore in nua mutants. Taken together, our experimental data suggests a role for NUA in controlling mRNA export from the nucleus as well as SUMo protease activity at the nuclear pore, comparable but not identical to its homologs in other eukaryotes. Furthermore, characterization of NUA illustrates a potential link at the nuclear pore between SUMo modification, RNA homeostasis and plant developmental control. PMID:19704557

A laboratory simulation of Arabidopsis seed dormancy cycling provides new insight into its regulation by clock genes and the dormancy-related genes DOG1, MFT, CIPK23 and PHYA.

PubMed

Footitt, Steven; Ölçer-Footitt, Hülya; Hambidge, Angela J; Finch-Savage, William E

2017-08-01

Environmental signals drive seed dormancy cycling in the soil to synchronize germination with the optimal time of year, a process essential for species' fitness and survival. Previous correlation of transcription profiles in exhumed seeds with annual environmental signals revealed the coordination of dormancy-regulating mechanisms with the soil environment. Here, we developed a rapid and robust laboratory dormancy cycling simulation. The utility of this simulation was tested in two ways: firstly, using mutants in known dormancy-related genes [DELAY OF GERMINATION 1 (DOG1), MOTHER OF FLOWERING TIME (MFT), CBL-INTERACTING PROTEIN KINASE 23 (CIPK23) and PHYTOCHROME A (PHYA)] and secondly, using further mutants, we test the hypothesis that components of the circadian clock are involved in coordination of the annual seed dormancy cycle. The rate of dormancy induction and relief differed in all lines tested. In the mutants, dog1-2 and mft2, dormancy induction was reduced but not absent. DOG1 is not absolutely required for dormancy. In cipk23 and phyA dormancy, induction was accelerated. Involvement of the clock in dormancy cycling was clear when mutants in the morning and evening loops of the clock were compared. Dormancy induction was faster when the morning loop was compromised and delayed when the evening loop was compromised. © 2017 The Authors Plant, Cell & Environment Published by John Wiley & Sons Ltd.

Involvement of complex sphingolipids and phosphatidylserine in endosomal trafficking in yeast Saccharomyces cerevisiae.

PubMed

Tani, Motohiro; Kuge, Osamu

2012-12-01

Sphingolipids play critical roles in many physiologically important events in the yeast Saccharomyces cerevisiae. In this study, we found that csg2Δ mutant cells defective in the synthesis of mannosylinositol phosphorylceramide exhibited abnormal intracellular accumulation of an exocytic v-SNARE, Snc1, under phosphatidylserine synthase gene (PSS1)-repressive conditions, although in wild-type cells, Snc1 was known to cycle between plasma membranes and the late Golgi via post-Golgi endosomes. The mislocalized Snc1 was co-localized with an endocytic marker dye, FM4-64, upon labelling for a short time. The abnormal distribution of Snc1 was suppressed by deletion of GYP2 encoding a GTPase-activating protein that negatively regulates endosomal vesicular trafficking, or expression of GTP-restricted form of Ypt32 GTPase. Furthermore, an endocytosis-deficient mutant of Snc1 was localized to plasma membranes in PSS1-repressed csg2Δ mutant cells as well as wild-type cells. Thus, the PSS1-repressed csg2Δ mutant cells were indicated to be defective in the trafficking of Snc1 from post-Golgi endosomes to the late Golgi. In contrast, the vesicular trafficking pathways via pre-vacuolar endosomes in the PSS1-repressed csg2Δ mutant cells seemed to be normal. These results suggested that specific complex sphingolipids and phosphatidylserine are co-ordinately involved in specific vesicular trafficking pathway. © 2012 Blackwell Publishing Ltd.

Involvement of Clostridium botulinum ATCC 3502 Sigma Factor K in Early-Stage Sporulation

PubMed Central

Kirk, David G.; Dahlsten, Elias; Zhang, Zhen; Korkeala, Hannu

2012-01-01

A key survival mechanism of Clostridium botulinum, the notorious neurotoxic food pathogen, is the ability to form heat-resistant spores. While the genetic mechanisms of sporulation are well understood in the model organism Bacillus subtilis, nothing is known about these mechanisms in C. botulinum. Using the ClosTron gene-knockout tool, sigK, encoding late-stage (stage IV) sporulation sigma factor K in B. subtilis, was disrupted in C. botulinum ATCC 3502 to produce two different mutants with distinct insertion sites and orientations. Both mutants were unable to form spores, and their elongated cell morphology suggested that the sporulation pathway was blocked at an early stage. In contrast, sigK-complemented mutants sporulated successfully. Quantitative real-time PCR analysis of sigK in the parent strain revealed expression at the late log growth phase in the parent strain. Analysis of spo0A, encoding the sporulation master switch, in the sigK mutant and the parent showed significantly reduced relative levels of spo0A expression in the sigK mutant compared to the parent strain. Similarly, sigF showed significantly lower relative transcription levels in the sigK mutant than the parent strain, suggesting that the sporulation pathway was blocked in the sigK mutant at an early stage. We conclude that σK is essential for early-stage sporulation in C. botulinum ATCC 3502, rather than being involved in late-stage sporulation, as reported for the sporulation model organism B. subtilis. Understanding the sporulation mechanism of C. botulinum provides keys to control the public health risks that the spores of this dangerous pathogen cause through foods. PMID:22544236

Involvement of Clostridium botulinum ATCC 3502 sigma factor K in early-stage sporulation.

PubMed

Kirk, David G; Dahlsten, Elias; Zhang, Zhen; Korkeala, Hannu; Lindström, Miia

2012-07-01

A key survival mechanism of Clostridium botulinum, the notorious neurotoxic food pathogen, is the ability to form heat-resistant spores. While the genetic mechanisms of sporulation are well understood in the model organism Bacillus subtilis, nothing is known about these mechanisms in C. botulinum. Using the ClosTron gene-knockout tool, sigK, encoding late-stage (stage IV) sporulation sigma factor K in B. subtilis, was disrupted in C. botulinum ATCC 3502 to produce two different mutants with distinct insertion sites and orientations. Both mutants were unable to form spores, and their elongated cell morphology suggested that the sporulation pathway was blocked at an early stage. In contrast, sigK-complemented mutants sporulated successfully. Quantitative real-time PCR analysis of sigK in the parent strain revealed expression at the late log growth phase in the parent strain. Analysis of spo0A, encoding the sporulation master switch, in the sigK mutant and the parent showed significantly reduced relative levels of spo0A expression in the sigK mutant compared to the parent strain. Similarly, sigF showed significantly lower relative transcription levels in the sigK mutant than the parent strain, suggesting that the sporulation pathway was blocked in the sigK mutant at an early stage. We conclude that σ(K) is essential for early-stage sporulation in C. botulinum ATCC 3502, rather than being involved in late-stage sporulation, as reported for the sporulation model organism B. subtilis. Understanding the sporulation mechanism of C. botulinum provides keys to control the public health risks that the spores of this dangerous pathogen cause through foods.

The impact of the IGF-1 system of cancer cells on radiation response - An in vitro study.

PubMed

Venkatachalam, Senthiladipan; Mettler, Esther; Fottner, Christian; Miederer, Matthias; Kaina, Bernd; Weber, Matthias M

2017-12-01

Overexpression of the insulin-like growth factor-1 receptor (IGF-1R) is associated with increased cell proliferation, differentiation, transformation, and tumorigenicity. Additionally, signaling involved in the resistance of cancer cells to radiotherapy originates from IGF-1R. The purpose of this study was to investigate the role of the IGF-1 system in the radiation response and further evaluate its effect on the expression of DNA repair pathway genes. To inhibit the IGF-1 system, we stably transfected the Caco-2 cell line to express a kinase-deficient IGF-1R mutant. We then studied the effects of this mutation on cell growth, the response to radiation, and clonogenic survival, as well as using a cell viability assay to examine DNA damage and repair. Finally, we performed immunofluorescence for γ-H2AX to examine double-strand DNA breaks and evaluated the expression of 84 key genes involved in DNA repair with a real-time PCR array. Mutant IGF-1R cells exhibited significantly blunted cell growth and viability, compared to wild-type cells, as well as reduced clonogenic survival after γ-irradiation. However, mutant IGF-1R cells did not show any significant delays in the repair of radiation-induced DNA double-strand breaks. Furthermore, expression of mutant IGF-1R significantly down-regulated the mRNA levels of BRCA2, a major protein involved in homologous recombination DNA repair. These results indicate that blocking the IGF-1R-mediated signaling cascade, through the expression of a kinase-deficient IGF-1R mutant, reduces cell growth and sensitizes cancer cells to ionizing radiation. Therefore, the IGF-1R system could be a potential target to enhance radio-sensitivity and the efficacy of cancer treatments.

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

Identification of genes involved in serum tolerance in the clinical strain Cronobacter sakazakii ES5.

PubMed

Schwizer, Sarah; Tasara, Taurai; Zurfluh, Katrin; Stephan, Roger; Lehner, Angelika

2013-02-15

Cronobacter spp. are opportunistic pathogens that can cause septicemia and infections of the central nervous system primarily in premature, low-birth weight and/or immune-compromised neonates. Serum resistance is a crucial virulence factor for the development of systemic infections, including bacteremia. It was the aim of the current study to identify genes involved in serum tolerance in a selected Cronobacter sakazakii strain of clinical origin. Screening of 2749 random transposon knock out mutants of a C. sakazakii ES 5 library for modified serum tolerance (compared to wild type) revealed 10 mutants showing significantly increased/reduced resistance to serum killing. Identification of the affected sites in mutants displaying reduced serum resistance revealed genes encoding for surface and membrane proteins as well as regulatory elements or chaperones. By this approach, the involvement of the yet undescribed Wzy_C superfamily domain containing coding region in serum tolerance was observed and experimentally confirmed. Additionally, knock out mutants with enhanced serum tolerance were observed. Examination of respective transposon insertion loci revealed regulatory (repressor) elements, coding regions for chaperones and efflux systems as well as the coding region for the protein YbaJ. Real time expression analysis experiments revealed, that knock out of the gene for this protein negatively affects the expression of the fimA gene, which is a key structural component of the formation of fimbriae. Fimbriae are structures of high immunogenic potential and it is likely that absence/truncation of the ybaJ gene resulted in a non-fimbriated phenotype accounting for the enhanced survival of this mutant in human serum. By using a transposon knock out approach we were able to identify genes involved in both increased and reduced serum tolerance in Cronobacter sakazakii ES5. This study reveals first insights in the complex nature of serum tolerance of Cronobacter spp.

Melatonin induction and its role in high light stress tolerance in Arabidopsis thaliana.

PubMed

Lee, Hyoung Yool; Back, Kyoungwhan

2018-05-16

In plants, melatonin is a potent bioactive molecule involved in the response against various biotic and abiotic stresses. However, little is known of its defensive role against high light (HL) stress. In this study, we found that melatonin was transiently induced in response to HL stress in Arabidopsis thaliana with a simultaneous increase in the expression of melatonin biosynthetic genes, including serotonin N-acetyltransferase1 (SNAT1). Transient induction of melatonin was also observed in the flu mutant, a singlet oxygen ( 1 O 2 )-producing mutant, upon light exposure, suggestive of melatonin induction by chloroplastidic 1 O 2 against HL stress. An Arabidopsis snat1 mutant was devoid of melatonin induction upon HL stress, resulting in high susceptibility to HL stress. Exogenous melatonin treatment mitigated damage caused by HL stress in the snat1 mutant by reducing O 2 - production and increasing the expression of various ROS-responsive genes. In analogy, an Arabidopsis SNAT1-overexpressing line showed increased tolerance of HL stress concomitant with a reduction in malondialdehyde and ion leakage. A complementation line expressing an Arabidopsis SNAT1 genomic fragment in the snat1 mutant completely restored HL stress susceptibility in the snat1 mutant to levels comparable to that of wild-type Col-0 plants. The results of the analysis of several Arabidopsis genetic lines reveal for the first time at the genetic level that melatonin is involved in conferring HL stress tolerance in plants. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Transcription Analysis of the Beta-Glucosidase Precursor in Wild-Type and l-4i Mutant Bombyx mori (Lepidoptera: Bombycidae).

PubMed

Kang, Lequn; Huang, Fei; Wu, Fan; Zhao, Qiaoling

2015-01-01

Lethal fourth-instar larvae (l-4i) mutant of Bombyx mori, a recently discovered novel mutant, die from energy depletion due to genetic mutation. Beta-glucosidase is a common digestive enzyme that hydrolyzes cellulose in the diet to provide energy. In this study, the mRNA expression profiles of B. mori beta-glucosidase precursor (BmpreBG) were characterized by reverse transcription polymerase chain reaction and quantitative real-time polymerase chain reaction. The transcription level of BmpreBG varied in different tissues and developmental stages, except in the pupa and moth, which are the no-diet period. Remarkably, the mRNA expression level of BmpreBG was sharply reduced in l-4i but not in the wild type, which suggested that the digestive function of the mutant was severely damaged. This was consistent with the l-4i phenotypic traits of not eating mulberries, lack of energy, and ultimate death. 5'-rapid amplification of cDNA ends showed, for the first time, that BmpreBG has a 160-bp 5'-untranslated region. These findings suggested that B. mori β-glucosidase precursor was involved in the death process of l-4i mutant larvae. © The Author 2015. Published by Oxford University Press on behalf of the Entomological Society of America.

White - cGMP Interaction Promotes Fast Locomotor Recovery from Anoxia in Adult Drosophila

PubMed Central

2017-01-01

Increasing evidence indicates that the white (w) gene in Drosophila possesses extra-retinal functions in addition to its classical role in eye pigmentation. We have previously shown that w+ promotes fast and consistent locomotor recovery from anoxia, but how w+ modulates locomotor recovery is largely unknown. Here we show that in the absence of w+, several PDE mutants, especially cyclic guanosine monophosphate (cGMP)-specific PDE mutants, display wildtype-like fast locomotor recovery from anoxia, and that during the night time, locomotor recovery was light-sensitive in white-eyed mutant w1118, and light-insensitive in PDE mutants under w1118 background. Data indicate the involvement of cGMP in the modulation of recovery timing and presumably, light-evoked cGMP fluctuation is associated with light sensitivity of locomotor recovery. This was further supported by the observations that w-RNAi-induced delay of locomotor recovery was completely eliminated by upregulation of cGMP through multiple approaches, including PDE mutation, simultaneous overexpression of an atypical soluble guanylyl cyclase Gyc88E, or sildenafil feeding. Lastly, prolonged sildenafil feeding promoted fast locomotor recovery from anoxia in w1118. Taken together, these data suggest that a White-cGMP interaction modulates the timing of locomotor recovery from anoxia. PMID:28060942

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

The YJR127C/ZMS1 gene product is involved in glycerol-based respiratory growth of the yeast Saccharomyces cerevisiae.

PubMed

Lu, Lin; Roberts, George G; Oszust, Cynthia; Hudson, Alan P

2005-10-01

A putative yeast mitochondrial upstream activating sequence (UAS) was used in a one-hybrid screening procedure that identified the YJR127C ORF on chromosome X. This gene was previously designated ZMS1 and is listed as a transcription factor on the SGD website. Real time RT-PCR assays showed that expression of YJR127C/ZMS1 was glucose-repressible, and a deletion mutant for the gene showed a growth defect on glycerol-based but not on glucose- or ethanol-based medium. Real time RT-PCR analyses identified severely attenuated transcript levels from GUT1 and GUT2 to be the source of that growth defect, the products of GUT1 and GUT2 are required for glycerol utilization. mRNA levels from a large group of mitochondria- and respiration-related nuclear genes also were shown to be attenuated in the deletion mutant. Importantly, transcript levels from the mitochondrial OLI1 gene, which has an associated organellar UAS, were attenuated in the DeltaYJR127C mutant during glycerol-based growth, but those from COX3 (OXI2), which lacks an associated mitochondrial UAS, were not. Transcriptome analysis of the glycerol-grown deletion mutant showed that genes in several metabolic and other categories are affected by loss of this gene product, including protein transport, signal transduction, and others. Thus, the product of YJR127C/ZMS1 is involved in transcriptional control for genes in both cellular genetic compartments, many of which specify products required for glycerol-based growth, respiration, and other functions.

Cross-species microarray hybridization to identify developmentally regulated genes in the filamentous fungus Sordaria macrospora.

PubMed

Nowrousian, Minou; Ringelberg, Carol; Dunlap, Jay C; Loros, Jennifer J; Kück, Ulrich

2005-04-01

The filamentous fungus Sordaria macrospora forms complex three-dimensional fruiting bodies that protect the developing ascospores and ensure their proper discharge. Several regulatory genes essential for fruiting body development were previously isolated by complementation of the sterile mutants pro1, pro11 and pro22. To establish the genetic relationships between these genes and to identify downstream targets, we have conducted cross-species microarray hybridizations using cDNA arrays derived from the closely related fungus Neurospora crassa and RNA probes prepared from wild-type S. macrospora and the three developmental mutants. Of the 1,420 genes which gave a signal with the probes from all the strains used, 172 (12%) were regulated differently in at least one of the three mutants compared to the wild type, and 17 (1.2%) were regulated differently in all three mutant strains. Microarray data were verified by Northern analysis or quantitative real time PCR. Among the genes that are up- or down-regulated in the mutant strains are genes encoding the pheromone precursors, enzymes involved in melanin biosynthesis and a lectin-like protein. Analysis of gene expression in double mutants revealed a complex network of interaction between the pro gene products.

Functional genomics of fuzzless-lintless mutant of Gossypium hirsutum L. cv. MCU5 reveal key genes and pathways involved in cotton fibre initiation and elongation

PubMed Central

2012-01-01

Background Fuzzless-lintless cotton mutants are considered to be the ideal material to understand the molecular mechanisms involved in fibre cell development. Although there are few reports on transcriptome and proteome analyses in cotton at fibre initiation and elongation stages, there is no comprehensive comparative transcriptome analysis of fibre-bearing and fuzzless-lintless cotton ovules covering fibre initiation to secondary cell wall (SCW) synthesis stages. In the present study, a comparative transcriptome analysis was carried out using G. hirsutum L. cv. MCU5 wild-type (WT) and it’s near isogenic fuzzless-lintless (fl) mutant at fibre initiation (0 dpa/days post anthesis), elongation (5, 10 and 15 dpa) and SCW synthesis (20 dpa) stages. Results Scanning electron microscopy study revealed the delay in the initiation of fibre cells and lack of any further development after 2 dpa in the fl mutant. Transcriptome analysis showed major down regulation of transcripts (90%) at fibre initiation and early elongation (5 dpa) stages in the fl mutant. Majority of the down regulated transcripts at fibre initiation stage in the fl mutant represent calcium and phytohormone mediated signal transduction pathways, biosynthesis of auxin and ethylene and stress responsive transcription factors (TFs). Further, transcripts involved in carbohydrate and lipid metabolisms, mitochondrial electron transport system (mETS) and cell wall loosening and elongation were highly down-regulated at fibre elongation stage (5–15 dpa) in the fl mutant. In addition, cellulose synthases and sucrose synthase C were down-regulated at SCW biosynthesis stage (15–20 dpa). Interestingly, some of the transcripts (~50%) involved in phytohormone signalling and stress responsive transcription factors that were up-regulated at fibre initiation stage in the WT were found to be up-regulated at much later stage (15 dpa) in fl mutant. Conclusions Comparative transcriptome analysis of WT and its near isogenic fl mutant revealed key genes and pathways involved at various stages of fibre development. Our data implicated the significant role of mitochondria mediated energy metabolism during fibre elongation process. The delayed expression of genes involved in phytohormone signalling and stress responsive TFs in the fl mutant suggests the need for a coordinated expression of regulatory mechanisms in fibre cell initiation and differentiation. PMID:23151214

Reconstruction of extended Petri nets from time series data and its application to signal transduction and to gene regulatory networks

PubMed Central

2011-01-01

Background Network inference methods reconstruct mathematical models of molecular or genetic networks directly from experimental data sets. We have previously reported a mathematical method which is exclusively data-driven, does not involve any heuristic decisions within the reconstruction process, and deliveres all possible alternative minimal networks in terms of simple place/transition Petri nets that are consistent with a given discrete time series data set. Results We fundamentally extended the previously published algorithm to consider catalysis and inhibition of the reactions that occur in the underlying network. The results of the reconstruction algorithm are encoded in the form of an extended Petri net involving control arcs. This allows the consideration of processes involving mass flow and/or regulatory interactions. As a non-trivial test case, the phosphate regulatory network of enterobacteria was reconstructed using in silico-generated time-series data sets on wild-type and in silico mutants. Conclusions The new exact algorithm reconstructs extended Petri nets from time series data sets by finding all alternative minimal networks that are consistent with the data. It suggested alternative molecular mechanisms for certain reactions in the network. The algorithm is useful to combine data from wild-type and mutant cells and may potentially integrate physiological, biochemical, pharmacological, and genetic data in the form of a single model. PMID:21762503

Disrupting the cinnamyl alcohol dehydrogenase 1 gene (BdCAD1) leads to altered lignification and improved saccharification in Brachypodium distachyon.

PubMed

Bouvier d'Yvoire, Madeleine; Bouchabke-Coussa, Oumaya; Voorend, Wannes; Antelme, Sébastien; Cézard, Laurent; Legée, Frédéric; Lebris, Philippe; Legay, Sylvain; Whitehead, Caragh; McQueen-Mason, Simon J; Gomez, Leonardo D; Jouanin, Lise; Lapierre, Catherine; Sibout, Richard

2013-02-01

Brachypodium distachyon (Brachypodium) has been proposed as a model for grasses, but there is limited knowledge regarding its lignins and no data on lignin-related mutants. The cinnamyl alcohol dehydrogenase (CAD) genes involved in lignification are promising targets to improve the cellulose-to-ethanol conversion process. Down-regulation of CAD often induces a reddish coloration of lignified tissues. Based on this observation, we screened a chemically induced population of Brachypodium mutants (Bd21-3 background) for red culm coloration. We identified two mutants (Bd4179 and Bd7591), with mutations in the BdCAD1 gene. The mature stems of these mutants displayed reduced CAD activity and lower lignin content. Their lignins were enriched in 8-O-4- and 4-O-5-coupled sinapaldehyde units, as well as resistant inter-unit bonds and free phenolic groups. By contrast, there was no increase in coniferaldehyde end groups. Moreover, the amount of sinapic acid ester-linked to cell walls was measured for the first time in a lignin-related CAD grass mutant. Functional complementation of the Bd4179 mutant with the wild-type BdCAD1 allele restored the wild-type phenotype and lignification. Saccharification assays revealed that Bd4179 and Bd7591 lines were more susceptible to enzymatic hydrolysis than wild-type plants. Here, we have demonstrated that BdCAD1 is involved in lignification of Brachypodium. We have shown that a single nucleotide change in BdCAD1 reduces the lignin level and increases the degree of branching of lignins through incorporation of sinapaldehyde. These changes make saccharification of cells walls pre-treated with alkaline easier without compromising plant growth. © 2012 The Authors The Plant Journal © 2012 Blackwell Publishing Ltd.

Characterization and Complementation of a Chlorophyll-Less Dominant Mutant GL1 in Lagerstroemia indica.

PubMed

Wang, Shu'an; Wang, Peng; Gao, Lulu; Yang, Rutong; Li, Linfang; Zhang, Enliang; Wang, Qing; Li, Ya; Yin, Zengfang

2017-05-01

Crape myrtle (Lagerstroemia indica) is a woody ornamental plant popularly grown because of its long-lasting, midsummer blooms and beautiful colors. The GL1 dominant mutant is the first chlorophyll-less mutant identified in crape myrtle. It was obtained from a natural yellow leaf bud mutation. We previously revealed that leaf color of the GL1 mutant is affected by light intensity. However, the mechanism of the GL1 mutant on light response remained unclear. The acclimation response of mutant and wild-type (WT) plants was assessed in a time series after transferring from low light (LL) to high light (HL) by analyzing chlorophyll synthesis precursor content, photosynthetic performance, and gene expression. In LL conditions, coproporphyrinogen III (Coprogen III) content had the greatest amount of accumulation in the mutant compared with WT, increasing by 100%. This suggested that the yellow leaf phenotype of the GL1 dominant mutant might be caused by disruption of coproporphyrinogen III oxidase (CPO) biosynthesis. Furthermore, the candidate gene, oxygen-independent CPO (HEMN), might only affect expression of upstream genes involved in chlorophyll metabolism in the mutant. Moreover, two genes, photosystem II (PSII) 10 kDa protein (psbR) and chlorophyll a/b binding protein gene (CAB1), had decreased mRNA levels in the GL1 mutant within the first 96 h following LL/HL transfer compared with the WT. Hierarchical clustering revealed that these two genes shared a similar expression trend as the oxygen-dependent CPO (HEMF). These findings provide evidence that GL1 is highly coordinated with PSII stability and chloroplast biogenesis.

ICP22 and the UL13 Protein Kinase Are both Required for Herpes Simplex Virus-Induced Modification of the Large Subunit of RNA Polymerase II

PubMed Central

Long, Melissa C.; Leong, Vivian; Schaffer, Priscilla A.; Spencer, Charlotte A.; Rice, Stephen A.

1999-01-01

Herpes simplex virus type 1 (HSV-1) infection alters the phosphorylation of the large subunit of RNA polymerase II (RNAP II), resulting in the depletion of the hypophosphorylated and hyperphosphorylated forms of this polypeptide (known as IIa and IIo, respectively) and induction of a novel, alternatively phosphorylated form (designated IIi). We previously showed that the HSV-1 immediate-early protein ICP22 is involved in this phenomenon, since induction of IIi and depletion of IIa are deficient in cells infected with 22/n199, an HSV-1 ICP22 nonsense mutant (S. A. Rice, M. C. Long, V. Lam, P. A. Schaffer, and C. A. Spencer, J. Virol. 69:5550–5559, 1995). However, depletion of IIo still occurs in 22/n199-infected cells. This suggests either that another viral gene product affects the RNAP II large subunit or that the truncated ICP22 polypeptide encoded by 22/n199 retains residual activity which leads to IIo depletion. To distinguish between these possibilities, we engineered an HSV-1 ICP22 null mutant, d22-lacZ, and compared it to 22/n199. The two mutants are indistinguishable in their effects on the RNAP II large subunit, suggesting that an additional viral gene product is involved in altering RNAP II. Two candidates are UL13, a protein kinase which has been implicated in ICP22 phosphorylation, and the virion host shutoff (Vhs) factor, the expression of which is positively regulated by ICP22 and UL13. To test whether UL13 is involved, a UL13-deficient viral mutant, d13-lacZ, was engineered. This mutant was defective in IIi induction and IIa depletion, displaying a phenotype very similar to that of d22-lacZ. In contrast, a Vhs mutant had effects that were indistinguishable from wild-type HSV-1. Therefore, UL13 but not the Vhs function plays a role in modifying the RNAP II large subunit. To study the potential role of UL13 in viral transcription, we carried out nuclear run-on transcription analyses in infected human embryonic lung cells. Infections with either UL13 or ICP22 mutants led to significantly reduced amounts of viral genome transcription at late times after infection. Together, our results suggest that ICP22 and UL13 are involved in a common pathway that alters RNAP II phosphorylation and that in some cell lines this change promotes viral late transcription. PMID:10364308

Construction of brewing-wine Aspergillus oryzae pyrG- mutant by pyrG gene deletion and its application in homology transformation.

PubMed

Du, Yu; Xie, Guizhen; Yang, Chunfa; Fang, Baishan; Chen, Hongwen

2014-06-01

pyrG(-) host cells are indispensable for pyrG(-) based transformation system. Isolations of pyrG(-) host cells by random mutations are limited by time-consuming, unclear genetic background and potential interferences of homogenous recombination. The purpose of this study was to construct brewing-wine Aspergillus oryzae pyrG(-) mutant by site-directed mutation of pyrG gene deletion which would be used as a host for further transformation. pMD-pyrGAB, a vector carrying pyrG deletion cassette, was used to construct pyrG(-) mutant of A. oryzae. Three stable pyrG deletion mutants of A. oryzae were isolated by resistant to 5-fluoroorotic acid and confirmed by polymerase chain reaction analysis, indicating that pyrG was completely excised. The ΔpyrG mutants were applied as pyrG(-) host cells to disrupt xdh gene encoding xylitol dehydrogenase, which involves in xylitol production of A. oryzae. The xdh disruption mutants were efficiently constructed by transforming a pMD-pyrG-xdh disruption plasmid carrying pyrG, and the produced xylitol concentration of the Δxdh mutant was three times as much as that of the ΔpyrG recipient. Site-directed pyrG gene deletion is thus an effective way for the isolation of pyrG(-) host cells, and the established host-vector system could be applied in further functional genomics analysis and molecular breeding of A. oryzae. © The Author 2014. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.

Reduced mycorrhizal colonization (rmc) tomato mutant lacks expression of SymRK signaling pathway genes

PubMed Central

Nair, Aswathy; Bhargava, Sujata

2012-01-01

Comparison of the expression of 13 genes involved in arbuscular mycorrhizal (AM) symbiosis was performed in a wild type tomato (Solanum lycopersicum cv 76R) and its reduced mycorrhizal colonization mutant rmc in response to colonization with Glomus fasiculatum. Four defense-related genes were induced to a similar extent in the mutant and wild type AM colonized plants, indicating a systemic response to AM colonization. Genes related to nutrient exchange between the symbiont partners showed higher expression in the AM roots of wild type plants than the mutant plants, which correlated with their arbuscular frequency. A symbiosis receptor kinase that is involved in both nodulation and AM symbiosis was not expressed in the rmc mutant. The fact that some colonization was observed in rmc was suggestive of the existence of an alternate colonization signaling pathway for AM symbiosis in this mutant. PMID:23221680

SGR2, a Phospholipase-Like Protein, and ZIG/SGR4, a SNARE, Are Involved in the Shoot Gravitropism of Arabidopsis

PubMed Central

Kato, Takehide; Morita, Miyo Terao; Fukaki, Hidehiro; Yamauchi, Yoshiro; Uehara, Michiko; Niihama, Mitsuru; Tasaka, Masao

2002-01-01

In higher plants, the shoot and the root generally show negative and positive gravitropism, respectively. To elucidate the molecular mechanisms involved in gravitropism, we have isolated many shoot gravitropism mutants in Arabidopsis. The sgr2 and zig/sgr4 mutants exhibited abnormal gravitropism in both inflorescence stems and hypocotyls. These genes probably are involved in the early step(s) of the gravitropic response. The sgr2 mutants also had misshapen seed and seedlings, whereas the stem of the zig/sgr4 mutants elongated in a zigzag fashion. The SGR2 gene encodes a novel protein that may be part of a gene family represented by bovine phosphatidic acid–preferring phospholipase A1 containing a putative transmembrane domain. This gene family has been reported only in eukaryotes. The ZIG gene was found to encode AtVTI11, a protein that is homologous with yeast VTI1 and is involved in vesicle transport. Our observations suggest that the two genes may be involved in a vacuolar membrane system that affects shoot gravitropism. PMID:11826297

NUCLEAR PORE ANCHOR, the Arabidopsis Homolog of Tpr/Mlp1/Mlp2/Megator, Is Involved in mRNA Export and SUMO Homeostasis and Affects Diverse Aspects of Plant Development[W

PubMed Central

Xu, Xianfeng Morgan; Rose, Annkatrin; Muthuswamy, Sivaramakrishnan; Jeong, Sun Yong; Venkatakrishnan, Sowmya; Zhao, Qiao; Meier, Iris

2007-01-01

Vertebrate Tpr and its yeast homologs Mlp1/Mlp2, long coiled-coil proteins of nuclear pore inner basket filaments, are involved in mRNA export, telomere organization, spindle pole assembly, and unspliced RNA retention. We identified Arabidopsis thaliana NUCLEAR PORE ANCHOR (NUA) encoding a 237-kD protein with similarity to Tpr. NUA is located at the inner surface of the nuclear envelope in interphase and in the vicinity of the spindle in prometaphase. Four T-DNA insertion lines were characterized, which comprise an allelic series of increasing severity for several correlating phenotypes, such as early flowering under short days and long days, increased abundance of SUMO conjugates, altered expression of several flowering regulators, and nuclear accumulation of poly(A)+ RNA. nua mutants phenocopy mutants of EARLY IN SHORT DAYS4 (ESD4), an Arabidopsis SUMO protease concentrated at the nuclear periphery. nua esd4 double mutants resemble nua and esd4 single mutants, suggesting that the two proteins act in the same pathway or complex, supported by yeast two-hybrid interaction. Our data indicate that NUA is a component of nuclear pore-associated steps of sumoylation and mRNA export in plants and that defects in these processes affect the signaling events of flowering time regulation and additional developmental processes. PMID:17513499

[Cloning, mutagenesis and symbiotic phenotype of three lipid transfer protein encoding genes from Mesorhizobium huakuii 7653R].

PubMed

Li, Yanan; Zeng, Xiaobo; Zhou, Xuejuan; Li, Youguo

2016-12-04

Lipid transfer protein superfamily is involved in lipid transport and metabolism. This study aimed to construct mutants of three lipid transfer protein encoding genes in Mesorhizobium huakuii 7653R, and to study the phenotypes and function of mutations during symbiosis with Astragalus sinicus. We used bioinformatics to predict structure characteristics and biological functions of lipid transfer proteins, and conducted semi-quantitative and fluorescent quantitative real-time PCR to analyze the expression levels of target genes in free-living and symbiotic conditions. Using pK19mob insertion mutagenesis to construct mutants, we carried out pot plant experiments to observe symbiotic phenotypes. MCHK-5577, MCHK-2172 and MCHK-2779 genes encoding proteins belonged to START/RHO alpha_C/PITP/Bet_v1/CoxG/CalC (SRPBCC) superfamily, involved in lipid transport or metabolism, and were identical to M. loti at 95% level. Gene relative transcription level of the three genes all increased compared to free-living condition. We obtained three mutants. Compared with wild-type 7653R, above-ground biomass of plants and nodulenitrogenase activity induced by the three mutants significantly decreased. Results indicated that lipid transfer protein encoding genes of Mesorhizobium huakuii 7653R may play important roles in symbiotic nitrogen fixation, and the mutations significantly affected the symbiotic phenotypes. The present work provided a basis to study further symbiotic function mechanism associated with lipid transfer proteins from rhizobia.

SalB inactivation modulates culture supernatant exoproteins and affects autolysis and viability in Enterococcus faecalis OG1RF.

PubMed

Shankar, Jayendra; Walker, Rachel G; Wilkinson, Mark C; Ward, Deborah; Horsburgh, Malcolm J

2012-07-01

The culture supernatant fraction of an Enterococcus faecalis gelE mutant of strain OG1RF contained elevated levels of the secreted antigen SalB. Using differential fluorescence gel electrophoresis (DIGE) the salB mutant was shown to possess a unique complement of exoproteins. Differentially abundant exoproteins were identified using matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. Stress-related proteins including DnaK, Dps family protein, SOD, and NADH peroxidase were present in greater quantity in the OG1RF salB mutant culture supernatant. Moreover, several proteins involved in cell wall synthesis and cell division, including d-Ala-d-Lac ligase and EzrA, were present in reduced quantity in OG1RF salB relative to the parent strain. The salB mutant displayed reduced viability and anomalous cell division, and these phenotypes were exacerbated in a gelE salB double mutant. An epistatic relationship between gelE and salB was not identified with respect to increased autolysis and cell morphological changes observed in the salB mutant. SalB was purified as a six-histidine-tagged protein to investigate peptidoglycan hydrolytic activity; however, activity was not evident. High-pressure liquid chromatography (HPLC) analysis of reduced muropeptides from peptidoglycan digested with mutanolysin revealed that the salB mutant and OG1RF were indistinguishable.

Bacillus subtilis Mutants with Knockouts of the Genes Encoding Ribonucleases RNase Y and RNase J1 Are Viable, with Major Defects in Cell Morphology, Sporulation, and Competence

PubMed Central

Figaro, Sabine; Durand, Sylvain; Gilet, Laetitia; Cayet, Nadège; Sachse, Martin

2013-01-01

The genes encoding the ribonucleases RNase J1 and RNase Y have long been considered essential for Bacillus subtilis cell viability, even before there was concrete knowledge of their function as two of the most important enzymes for RNA turnover in this organism. Here we show that this characterization is incorrect and that ΔrnjA and Δrny mutants are both viable. As expected, both strains grow relatively slowly, with doubling times in the hour range in rich medium. Knockout mutants have major defects in their sporulation and competence development programs. Both mutants are hypersensitive to a wide range of antibiotics and have dramatic alterations to their cell morphologies, suggestive of cell envelope defects. Indeed, RNase Y mutants are significantly smaller in diameter than wild-type strains and have a very disordered peptidoglycan layer. Strains lacking RNase J1 form long filaments in tight spirals, reminiscent of mutants of the actin-like proteins (Mre) involved in cell shape determination. Finally, we combined the rnjA and rny mutations with mutations in other components of the degradation machinery and show that many of these strains are also viable. The implications for the two known RNA degradation pathways of B. subtilis are discussed. PMID:23504012

Identification of a transporter Slr0982 involved in ethanol tolerance in cyanobacterium Synechocystis sp. PCC 6803

PubMed Central

Zhang, Yanan; Niu, Xiangfeng; Shi, Mengliang; Pei, Guangsheng; Zhang, Xiaoqing; Chen, Lei; Zhang, Weiwen

2015-01-01

Cyanobacteria have been engineered to produce ethanol through recent synthetic biology efforts. However, one major challenge to the cyanobacterial systems for high-efficiency ethanol production is their low tolerance to the ethanol toxicity. With a major goal to identify novel transporters involved in ethanol tolerance, we constructed gene knockout mutants for 58 transporter-encoding genes of Synechocystis sp. PCC 6803 and screened their tolerance change under ethanol stress. The efforts allowed discovery of a mutant of slr0982 gene encoding an ATP-binding cassette transporter which grew poorly in BG11 medium supplemented with 1.5% (v/v) ethanol when compared with the wild type, and the growth loss could be recovered by complementing slr0982 in the Δslr0982 mutant, suggesting that slr0982 is involved in ethanol tolerance in Synechocystis. To decipher the tolerance mechanism involved, a comparative metabolomic and network-based analysis of the wild type and the ethanol-sensitive Δslr0982 mutant was performed. The analysis allowed the identification of four metabolic modules related to slr0982 deletion in the Δslr0982 mutant, among which metabolites like sucrose and L-pyroglutamic acid which might be involved in ethanol tolerance, were found important for slr0982 deletion in the Δslr0982 mutant. This study reports on the first transporter related to ethanol tolerance in Synechocystis, which could be a useful target for further tolerance engineering. In addition, metabolomic and network analysis provides important findings for better understanding of the tolerance mechanism to ethanol stress in Synechocystis. PMID:26052317

Helicobacter pylori Urease Activity is Influenced by Ferric Uptake Regulator

PubMed Central

Lee, Jong Seung; Lee, Ji Hyuk; Lee, Hye Jin; Lee, Jee Hyun; Choi, Young Ok

2010-01-01

Purpose The role of the Ferric Uptake Regulator (FUR) in the acid resistance of Helicobacter pylori (H. pylori) has been thought to be independent of urease. However, we demonstrated in this study that Fur influences urease activity. Materials and Methods A fur knockout mutant of H. pylori was constructed by replacing the Fur gene with a kanamycin resistant marker gene. The wild-type H. pylori and fur mutant were compared for survival. The integrity of the inner membrane of the bacteria was evaluated by confocal microscopy using membrane-permeant and -impermeant fluorescent DNA probes. Urease activity of intact H. pylori was measured between pH 3 and 8. Real time PCR of both strains was performed for urease genes including ureI, ureE, ureF, ureG, and ureH. Results The fur deletion affected the survival of H. pylori at pH 4. The urease activity curve of the intact fur mutant showed the same shape as the wild-type but was 3-fold lower than the wild-type at a pH of less than 5. Real time PCR revealed that the expression of all genes was consistently down-regulated in the fur mutant. Conclusion The results of this study showed that fur appears to be involved in acid resistant H. pylori urease activity. PMID:20046512

Computational and Experimental Study of Neuroglobin and Mutants

NASA Astrophysics Data System (ADS)

Nelson, Lauren; Cho, Samuel; Kim-Shaprio, Daniel

Neuroglobin (Ngb) is a hexacoordinated heme protein that is closely related to hemoglobin and myoglobin and normally found in the brain and nervous systems. It is involved in cellular oxygen homeostasis and reversibly binds to oxygen with a higher binding affinity than hemoglobin. To protect the brain tissue from hypoxic or ischemic conditions, Ngb increases oxygen availability. We have previously shown that a mutant form of Ngb reduces nitrite to nitric oxide 50x faster than myoglobin and 500x faster than hemoglobin. It also tightly binds to carbon monoxide (CO) with an association rate that is 500x faster than hemoglobin. To analyze the structure of neuroglobin and the characteristics causing these phenomena, we performed 3 sets of 1 microsecond molecular dynamic (MD) simulations of wild-type oxidized and reduced human Ngb and their C46A, C55A, H64L, and H64Q mutants. We also directly compare our MD simulations with time-resolved absorption spectroscopy. These studies will help identify treatments for diseases involving low nitric oxide availability and carbon monoxide poisoning. This research was supported by an NIH NSRA predoctoral fellowship in the Structural and Computational Biophysics Program training Grant (T32GM095440-05).

Mutations in Ran system affected telomere silencing in Saccharomyces cerevisiae

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

Hayashi, Naoyuki; Department of Molecular Oncology, Cancer Research Institute, Kanazawa University, Kanazawa, Ishikawa 920-0934; Kobayashi, Masahiko

The Ran GTPase system regulates the direction and timing of several cellular events, such as nuclear-cytosolic transport, centrosome formation, and nuclear envelope assembly in telophase. To gain insight into the Ran system's involvement in chromatin formation, we investigated gene silencing at the telomere in several mutants of the budding yeast Saccharomyces cerevisiae, which had defects in genes involved in the Ran system. A mutation of the RanGAP gene, rna1-1, caused reduced silencing at the telomere, and partial disruption of the nuclear Ran binding factor, yrb2-{delta}2, increased this silencing. The reduced telomere silencing in rna1-1 cells was suppressed by a highmore » dosage of the SIR3 gene or the SIT4 gene. Furthermore, hyperphosphorylated Sir3 protein accumulated in the rna1-1 mutant. These results suggest that RanGAP is required for the heterochromatin structure at the telomere in budding yeast.« less

ECB deacylase mutants

DOEpatents

Arnold, Frances H.; Shao, Zhixin; Zhao, Huimin; Giver, Lorraine J.

2002-01-01

A method for in vitro mutagenesis and recombination of polynucleotide sequences based on polymerase-catalyzed extension of primer oligonucleotides is disclosed. The method involves priming template polynucleotide(s) with random-sequences or defined-sequence primers to generate a pool of short DNA fragments with a low level of point mutations. The DNA fragments are subjected to denaturization followed by annealing and further enzyme-catalyzed DNA polymerization. This procedure is repeated a sufficient number of times to produce full-length genes which comprise mutants of the original template polynucleotides. These genes can be further amplified by the polymerase chain reaction and cloned into a vector for expression of the encoded proteins.

Genetic regulation by NLA and microRNA827 for maintaining nitrate-dependent phosphate homeostasis in arabidopsis.

PubMed

Kant, Surya; Peng, Mingsheng; Rothstein, Steven J

2011-03-01

Plants need abundant nitrogen and phosphorus for higher yield. Improving plant genetics for higher nitrogen and phosphorus use efficiency would save potentially billions of dollars annually on fertilizers and reduce global environmental pollution. This will require knowledge of molecular regulators for maintaining homeostasis of these nutrients in plants. Previously, we reported that the NITROGEN LIMITATION ADAPTATION (NLA) gene is involved in adaptive responses to low-nitrogen conditions in Arabidopsis, where nla mutant plants display abrupt early senescence. To understand the molecular mechanisms underlying NLA function, two suppressors of the nla mutation were isolated that recover the nla mutant phenotype to wild type. Map-based cloning identified these suppressors as the phosphate (Pi) transport-related genes PHF1 and PHT1.1. In addition, NLA expression is shown to be regulated by the low-Pi induced microRNA miR827. Pi analysis revealed that the early senescence in nla mutant plants was due to Pi toxicity. These plants accumulated over five times the normal Pi content in shoots specifically under low nitrate and high Pi but not under high nitrate conditions. Also the Pi overaccumulator pho2 mutant shows Pi toxicity in a nitrate-dependent manner similar to the nla mutant. Further, the nitrate and Pi levels are shown to have an antagonistic crosstalk as displayed by their differential effects on flowering time. The results demonstrate that NLA and miR827 have pivotal roles in regulating Pi homeostasis in plants in a nitrate-dependent fashion.

Protein tyrosine phosphatase of liver regeneration-1 is required for normal timing of cell cycle progression during liver regeneration

PubMed Central

Jiao, Yang; Ye, Diana Z.; Li, Zhaoyu; Teta-Bissett, Monica; Peng, Yong; Taub, Rebecca; Greenbaum, Linda E.

2014-01-01

Protein tyrosine phosphatase of liver regeneration-1 (Prl-1) is an immediate-early gene that is significantly induced during liver regeneration. Several in vitro studies have suggested that Prl-1 is important for the regulation of cell cycle progression. To evaluate its function in liver regeneration, we ablated the Prl-1 gene specifically in mouse hepatocytes using the Cre-loxP system. Prl-1 mutant mice (Prl-1loxP/loxP;AlfpCre) appeared normal and fertile. Liver size and metabolic function in Prl-1 mutants were comparable to controls, indicating that Prl-1 is dispensable for liver development, postnatal growth, and hepatocyte differentiation. Mutant mice demonstrated a delay in DNA synthesis after 70% partial hepatectomy, although ultimate liver mass restoration was not affected. At 40 h posthepatectomy, reduced protein levels of the cell cycle regulators cyclin E, cyclin A2, cyclin B1, and cyclin-dependent kinase 1 were observed in Prl-1 mutant liver. Investigation of the major signaling pathways involved in liver regeneration demonstrated that phosphorylation of protein kinase B (AKT) and signal transducer and activator of transcription (STAT) 3 were significantly reduced at 40 h posthepatectomy in Prl-1 mutants. Taken together, this study provides evidence that Prl-1 is required for proper timing of liver regeneration after partial hepatectomy. Prl-1 promotes G1/S progression via modulating expression of several cell cycle regulators through activation of the AKT and STAT3 signaling pathway. PMID:25377314

Identification of genes associated with growth cessation and bud dormancy entrance using a dormancy-incapable tree mutant.

PubMed

Jiménez, Sergio; Li, Zhigang; Reighard, Gregory L; Bielenberg, Douglas G

2010-02-09

In many tree species the perception of short days (SD) can trigger growth cessation, dormancy entrance, and the establishment of a chilling requirement for bud break. The molecular mechanisms connecting photoperiod perception, growth cessation and dormancy entrance in perennials are not clearly understood. The peach [Prunus persica (L.) Batsch] evergrowing (evg) mutant fails to cease growth and therefore cannot enter dormancy under SD. We used the evg mutant to filter gene expression associated with growth cessation after exposure to SD. Wild-type and evg plants were grown under controlled conditions of long days (16 h/8 h) followed by transfer to SD (8 h/16 h) for eight weeks. Apical tissues were sampled at zero, one, two, four, and eight weeks of SD and suppression subtractive hybridization was performed between genotypes at the same time points. We identified 23 up-regulated genes in the wild-type with respect to the mutant during SD exposure. We used quantitative real-time PCR to verify the expression of the differentially expressed genes in wild-type tissues following the transition to SD treatment. Three general expression patterns were evident: one group of genes decreased at the time of growth cessation (after 2 weeks in SD), another that increased immediately after the SD exposure and then remained steady, and another that increased throughout SD exposure. The use of the dormancy-incapable mutant evg has allowed us to reduce the number of genes typically detected by differential display techniques for SD experiments. These genes are candidates for involvement in the signalling pathway leading from photoperiod perception to growth cessation and dormancy entrance and will be the target of future investigations.

Generation of mariner-based transposon insertion mutant library of Bacillus sphaericus 2297 and investigation of genes involved in sporulation and mosquito-larvicidal crystal protein synthesis.

PubMed

Wu, Yiming; Hu, Xiaomin; Ge, Yong; Zheng, Dasheng; Yuan, Zhiming

2012-05-01

Bacillus sphaericus has been used with great success in mosquito control programs worldwide. Under conditions of nutrient limitation, it undergoes sporulation via a series of well defined morphological stages. However, only a small number of genes involved in sporulation have been identified. To identify genes associated with sporulation, and to understand the relationship between sporulation and crystal protein synthesis, a random mariner-based transposon insertion mutant library of B. sphaericus strain 2297 was constructed and seven sporulation-defective mutants were selected. Sequencing of the DNA flanking of the transposon insertion identified several genes involved in sporulation. The morphologies of mutants were determined by electron microscopy and synthesis of crystal proteins was analyzed by SDS-PAGE and Western blot. Four mutants blocked at early stages of sporulation failed to produce crystal proteins and had lower larvicidal activity. However, the other three mutants were blocked at later stages and were able to form crystal proteins, and the larvicidal activity was similar to wild type. These results indicated that crystal protein synthesis in B. sphaericus is dependent on sporulation initiation. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Transposon mutagenesis of probiotic Lactobacillus casei identifies asnH, an asparagine synthetase gene involved in its immune-activating capacity.

PubMed

Ito, Masahiro; Kim, Yun-Gi; Tsuji, Hirokazu; Takahashi, Takuya; Kiwaki, Mayumi; Nomoto, Koji; Danbara, Hirofumi; Okada, Nobuhiko

2014-01-01

Lactobacillus casei ATCC 27139 enhances host innate immunity, and the J1 phage-resistant mutants of this strain lose the activity. A transposon insertion mutant library of L. casei ATCC 27139 was constructed, and nine J1 phage-resistant mutants out of them were obtained. Cloning and sequencing analyses identified three independent genes that were disrupted by insertion of the transposon element: asnH, encoding asparagine synthetase, and dnaJ and dnaK, encoding the molecular chaperones DnaJ and DnaK, respectively. Using an in vivo mouse model of Listeria infection, only asnH mutant showed deficiency in their ability to enhance host innate immunity, and complementation of the mutation by introduction of the wild-type asnH in the mutant strain recovered the immuno-augmenting activity. AsnH protein exhibited asparagine synthetase activity when the lysozyme-treated cell wall extracts of L. casei ATCC 27139 was added as substrate. The asnH mutants lost the thick and rigid peptidoglycan features that are characteristic to the wild-type cells, indicating that AsnH of L. casei is involved in peptidoglycan biosynthesis. These results indicate that asnH is required for the construction of the peptidoglycan composition involved in the immune-activating capacity of L. casei ATCC 27139.

Molecular mechanisms associated with leukemic transformation of MPL-mutant myeloproliferative neoplasms

PubMed Central

Beer, Philip A.; Ortmann, Christina A.; Stegelmann, Frank; Guglielmelli, Paola; Reilly, John T.; Larsen, Thomas S.; Hasselbalch, Hans C.; Vannucchi, Alessandro M.; Möller, Peter; Döhner, Konstanze; Green, Anthony R.

2010-01-01

Somatic activating mutations in MPL, the thrombopoietin receptor, occur in the myeloproliferative neoplasms, although virtually nothing is known about their role in evolution to acute myeloid leukemia. In this study, the MPL T487A mutation, identified in de novo acute myeloid leukemia, was not detected in 172 patients with a myeloproliferative neoplasm. In patients with a prior MPL W515L-mutant myeloproliferative neoplasm, leukemic transformation was accompanied by MPL-mutant leukemic blasts, was seen in the absence of prior cytoreductive therapy and often involved loss of wild-type MPL by mitotic recombination. Moreover, clonal analysis of progenitor colonies at the time of leukemic transformation revealed the presence of multiple genetically distinct but phylogenetically-related clones bearing different TP53 mutations, implying a mutator-phenotype and indicating that leukemic transformation may be preceded by the parallel expansion of diverse hematopoietic clones. PMID:20823136

Cellular Prion Protein Combined with Galectin-3 and -6 Affects the Infectivity Titer of an Endogenous Retrovirus Assayed in Hippocampal Neuronal Cells

PubMed Central

Shin, Hae-Young; Goto, Joy J.; Carp, Richard I.; Choi, Eun-Kyoung; Kim, Yong-Sun

2016-01-01

Prion diseases are infectious and fatal neurodegenerative diseases which require the cellular prion protein, PrPC, for development of diseases. The current study shows that the PrPC augments infectivity and plaque formation of a mouse endogenous retrovirus, MuLV. We have established four neuronal cell lines expressing mouse PrPC, PrP+/+; two express wild type PrPC (MoPrPwild) and the other two express mutant PrPC (MoPrPmut). Infection of neuronal cells from various PrP+/+ and PrP-/- (MoPrPKO) lines with MuLV yielded at least three times as many plaques in PrP+/+ than in PrP-/-. Furthermore, among the four PrP+/+ lines, one mutant line, P101L, had at least 2.5 times as many plaques as the other three PrP+/+ lines. Plaques in P101L were four times larger than those in other PrP+/+ lines. Colocalization of PrP and CAgag was seen in MuLV-infected PrP+/+ cells. In the PrP-MuLV interaction, the involvement of galectin-3 and -6 was observed by immunoprecipitation with antibody to PrPC. These results suggest that PrPC combined with galectin-3 and -6 can act as a receptor for MuLV. P101L, the disease form of mutant PrPC results suggest the genetic mutant form of PrPC may be more susceptible to viral infection. PMID:27936017

Caenorhabditis elegans syndecan (SDN-1) is required for normal egg laying and associates with the nervous system and the vulva.

PubMed

Minniti, Alicia N; Labarca, Mariana; Hurtado, Claudia; Brandan, Enrique

2004-10-01

In Caenorhabditis elegans, the identification of many enzymes involved in the synthesis and modification of glycosaminoglycans (GAGs), essential components of proteoglycans, has attained special attention in recent years. Mutations in all the genes that encode for GAG biosynthetic enzymes show defects in the development of the vulva, specifically in the invagination of the vulval epithelium. Mutants for certain heparan sulfate modifying enzymes present axonal and cellular guidance defects in specific neuronal classes. Although most of the enzymes involved in the biosynthesis and modification of heparan sulfate have been characterized in C. elegans, little is known regarding the core proteins to which these GAGs covalently bind in proteoglycans. A single syndecan homologue (sdn-1) has been identified in the C. elegans genome through sequence analysis. In the present study, we show that C. elegans synthesizes sulfated proteoglycans, seen as three distinct species in western blot analysis. In the sdn-1 (ok449) deletion mutant allele we observed the lack of one species, which corresponds to a 50 kDa product after heparitinase treatment. The expression of sdn-1 mRNA and sequencing revealed that sdn-1 (ok449) deletion mutants lack two glycosylation sites. Hence, the missing protein in the western blot analysis probably corresponds to SDN-1. In addition, we show that SDN-1 localizes to the C. elegans nerve ring, nerve cords and to the vulva. SDN-1 is found specifically phosphorylated in nerve ring neurons and in the vulva, in both wild-type worms and sdn-1 (ok449) deletion mutants. These mutants show a defective egg-laying phenotype. Our results show for the first time, the identification, localization and some functional aspects of syndecan in the nematode C. elegans.

A repetitive mutation and selection system for bacterial evolution to increase the specific affinity to pancreatic cancer cells.

PubMed

Osawa, Masaki

2018-01-01

It is difficult to target and kill cancer cells. One possible approach is to mutate bacteria to enhance their binding to cancer cells. In the present study, Gram-negative Escherichia coli and Gram-positive Bacillus subtilis were randomly mutated, and then were positively and negatively selected for binding cancer vs normal cells. With repetitive mutation and selection both bacteria successfully evolved to increase affinity to the pancreatic cancer cell line (Mia PaCa-2) but not normal cells (HPDE: immortalized human pancreatic ductal epithelial cells). The mutant E. coli and B. subtilis strains bound to Mia PaCa-2 cells about 10 and 25 times more than to HPDE cells. The selected E. coli strain had mutations in biofilm-related genes and the regulatory region for a type I pilus gene. Consistent with type I pili involvement, mannose could inhibit the binding to cells. The results suggest that weak but specific binding is involved in the initial step of adhesion. To test their ability to kill Mia PaCa-2 cells, hemolysin was expressed in the mutant strain. The hemolysin released from the mutant strain was active and could kill Mia PaCa-2 cells. In the case of B. subtilis, the initial binding to the cells was a weak interaction of the leading pole of the motile bacteria. The frequency of this interaction to Mia PaCa-2 cells dramatically increased in the evolved mutant strain. This mutant strain could also specifically invade beneath Mia PaCa-2 cells and settle there. This type of mutation/selection strategy may be applicable to other combinations of cancer cells and bacterial species.

Isolation and Characterization of Mms-Sensitive Mutants of SACCHAROMYCES CEREVISIAE

PubMed Central

Prakash, Louise; Prakash, Satya

1977-01-01

We have isolated mutants sensitive to methyl methanesulfonate (MMS) in Saccharomyces cerevisiae. Alleles of rad1, rad4, rad6, rad52, rad55 and rad57 were found among these mms mutants. Twenty-nine of the mms mutants which complement the existing radiation-sensitive (rad and rev ) mutants belong to 22 new complementation groups. Mutants from five complementation groups are sensitive only to MMS. Mutants of 11 complementation groups are sensitive to UV or X rays in addition to MMS, mutants of six complementation groups are sensitive to all three agents. The cross-sensitivities of these mms mutants to UV and X rays are discussed in terms of their possible involvement in DNA repair. Sporulation is reduced or absent in homozygous diploids of mms mutants from nine complementation groups. PMID:195865

Germination-defective mutant of Neurospora crassa that responds to siderophores

NASA Technical Reports Server (NTRS)

Charlang, G.; Williams, N. P.

1977-01-01

A conditionally germination-defective mutant of Neurospora crassa has been found to be partially curable by ferricrocin and other siderophores. The mutant conidia rapidly lose their membrane-bound siderophores when suspended in buffer or growth media. Germination is consequently delayed unless large numbers of conidia are present (positive population effect). This indicates that the mutant has a membrane defect involving the siderophore attachment site.

Development and characterization of rice mutants for functional genomic studies and breeding

USDA-ARS?s Scientific Manuscript database

Mutagenesis is a powerful tool for creating genetic materials for studying functional genomics, breeding, and understanding the molecular basis of disease resistance. Approximately 100,000 putative mutants of rice (Oryza sativa L.) have been generated with mutagens. Numerous mutant genes involved in...

Optimisation of nutritional requirements for dopamine synthesis by calcium alginate-entrapped mutant strain of Aspergillus oryzae EMS-6.

PubMed

Ali, Sikander; Nawaz, Wajeeha

2017-02-01

The optimisation of nutritional requirements for dopamine (DA) synthesis by calcium alginate-entrapped mutant variant of Aspergillus oryzae EMS-6 using submerged fermentation technique was investigated. A total of 13 strains were isolated from soil. Isolate I-2 was selected as a better producer of DA and improved by exposing with ethyl methylsulphonate (EMS). EMS-6 was selected as it exhibited 43 μg/mL DA activity. The mutant variable was further treated with low levels of l-cysteine HCl to make it resistant against diversion and environmental stress. The conidiospores of mutant variant were entrapped in calcium alginate beads for stable product formation. EMS-6 gave maximum DA activity (124 μg/mL) when supplemented with 0.1% peptone and 0.2% sucrose, under optimised parameters viz. pH 3, temperature of 55 °C and incubation time of 70 min. The study involves the high profile of DA activity and is needed, as DA is capable to control numerous neurogenic disorders.

The Mediator subunit SFR6/MED16 controls defence gene expression mediated by salicylic acid and jasmonate responsive pathways.

PubMed

Wathugala, Deepthi L; Hemsley, Piers A; Moffat, Caroline S; Cremelie, Pieter; Knight, Marc R; Knight, Heather

2012-07-01

• Arabidopsis SENSITIVE TO FREEZING6 (SFR6) controls cold- and drought-inducible gene expression and freezing- and osmotic-stress tolerance. Its identification as a component of the MEDIATOR transcriptional co-activator complex led us to address its involvement in other transcriptional responses. • Gene expression responses to Pseudomonas syringae, ultraviolet-C (UV-C) irradiation, salicylic acid (SA) and jasmonic acid (JA) were investigated in three sfr6 mutant alleles by quantitative real-time PCR and susceptibility to UV-C irradiation and Pseudomonas infection were assessed. • sfr6 mutants were more susceptible to both Pseudomonas syringae infection and UV-C irradiation. They exhibited correspondingly weaker PR (pathogenesis-related) gene expression than wild-type Arabidopsis following these treatments or after direct application of SA, involved in response to both UV-C and Pseudomonas infection. Other genes, however, were induced normally in the mutants by these treatments. sfr6 mutants were severely defective in expression of plant defensin genes in response to JA; ectopic expression of defensin genes was provoked in wild-type but not sfr6 by overexpression of ERF5. • SFR6/MED16 controls both SA- and JA-mediated defence gene expression and is necessary for tolerance of Pseudomonas syringae infection and UV-C irradiation. It is not, however, a universal regulator of stress gene transcription and is likely to mediate transcriptional activation of specific regulons only. © 2012 The Authors. New Phytologist © 2012 New Phytologist Trust.

Poly(ADP-ribose)polymerases are involved in microhomology mediated back-up non-homologous end joining in Arabidopsis thaliana.

PubMed

Jia, Qi; den Dulk-Ras, Amke; Shen, Hexi; Hooykaas, Paul J J; de Pater, Sylvia

2013-07-01

Besides the KU-dependent classical non-homologous end-joining (C-NHEJ) pathway, an alternative NHEJ pathway first identified in mammalian systems, which is often called the back-up NHEJ (B-NHEJ) pathway, was also found in plants. In mammalian systems PARP was found to be one of the essential components in B-NHEJ. Here we investigated whether PARP1 and PARP2 were also involved in B-NHEJ in Arabidopsis. To this end Arabidopsis parp1, parp2 and parp1parp2 (p1p2) mutants were isolated and functionally characterized. The p1p2 double mutant was crossed with the C-NHEJ ku80 mutant resulting in the parp1parp2ku80 (p1p2k80) triple mutant. As expected, because of their role in single strand break repair (SSBR) and base excision repair (BER), the p1p2 and p1p2k80 mutants were shown to be sensitive to treatment with the DNA damaging agent MMS. End-joining assays in cell-free leaf protein extracts of the different mutants using linear DNA substrates with different ends reflecting a variety of double strand breaks were performed. The results showed that compatible 5'-overhangs were accurately joined in all mutants, that KU80 protected the ends preventing the formation of large deletions and that PARP proteins were involved in microhomology mediated end joining (MMEJ), one of the characteristics of B-NHEJ.

Involvement of NADH Oxidase in Biofilm Formation in Streptococcus sanguinis

PubMed Central

Ge, Xiuchun; Shi, Xiaoli; Shi, Limei; Liu, Jinlin; Stone, Victoria; Kong, Fanxiang; Kitten, Todd; Xu, Ping

2016-01-01

Biofilms play important roles in microbial communities and are related to infectious diseases. Here, we report direct evidence that a bacterial nox gene encoding NADH oxidase is involved in biofilm formation. A dramatic reduction in biofilm formation was observed in a Streptococcus sanguinis nox mutant under anaerobic conditions without any decrease in growth. The membrane fluidity of the mutant bacterial cells was found to be decreased and the fatty acid composition altered, with increased palmitic acid and decreased stearic acid and vaccenic acid. Extracellular DNA of the mutant was reduced in abundance and bacterial competence was suppressed. Gene expression analysis in the mutant identified two genes with altered expression, gtfP and Idh, which were found to be related to biofilm formation through examination of their deletion mutants. NADH oxidase-related metabolic pathways were analyzed, further clarifying the function of this enzyme in biofilm formation. PMID:26950587

Naringenin Regulates Expression of Genes Involved in Cell Wall Synthesis in Herbaspirillum seropedicae▿

PubMed Central

Tadra-Sfeir, M. Z.; Souza, E. M.; Faoro, H.; Müller-Santos, M.; Baura, V. A.; Tuleski, T. R.; Rigo, L. U.; Yates, M. G.; Wassem, R.; Pedrosa, F. O.; Monteiro, R. A.

2011-01-01

Five thousand mutants of Herbaspirillum seropedicae SmR1 carrying random insertions of transposon pTnMod-OGmKmlacZ were screened for differential expression of LacZ in the presence of naringenin. Among the 16 mutants whose expression was regulated by naringenin were genes predicted to be involved in the synthesis of exopolysaccharides, lipopolysaccharides, and auxin. These loci are probably involved in establishing interactions with host plants. PMID:21257805

Naringenin regulates expression of genes involved in cell wall synthesis in Herbaspirillum seropedicae.

PubMed

Tadra-Sfeir, M Z; Souza, E M; Faoro, H; Müller-Santos, M; Baura, V A; Tuleski, T R; Rigo, L U; Yates, M G; Wassem, R; Pedrosa, F O; Monteiro, R A

2011-03-01

Five thousand mutants of Herbaspirillum seropedicae SmR1 carrying random insertions of transposon pTnMod-OGmKmlacZ were screened for differential expression of LacZ in the presence of naringenin. Among the 16 mutants whose expression was regulated by naringenin were genes predicted to be involved in the synthesis of exopolysaccharides, lipopolysaccharides, and auxin. These loci are probably involved in establishing interactions with host plants.

Computing border bases using mutant strategies

NASA Astrophysics Data System (ADS)

Ullah, E.; Abbas Khan, S.

2014-01-01

Border bases, a generalization of Gröbner bases, have actively been addressed during recent years due to their applicability to industrial problems. In cryptography and coding theory a useful application of border based is to solve zero-dimensional systems of polynomial equations over finite fields, which motivates us for developing optimizations of the algorithms that compute border bases. In 2006, Kehrein and Kreuzer formulated the Border Basis Algorithm (BBA), an algorithm which allows the computation of border bases that relate to a degree compatible term ordering. In 2007, J. Ding et al. introduced mutant strategies bases on finding special lower degree polynomials in the ideal. The mutant strategies aim to distinguish special lower degree polynomials (mutants) from the other polynomials and give them priority in the process of generating new polynomials in the ideal. In this paper we develop hybrid algorithms that use the ideas of J. Ding et al. involving the concept of mutants to optimize the Border Basis Algorithm for solving systems of polynomial equations over finite fields. In particular, we recall a version of the Border Basis Algorithm which is actually called the Improved Border Basis Algorithm and propose two hybrid algorithms, called MBBA and IMBBA. The new mutants variants provide us space efficiency as well as time efficiency. The efficiency of these newly developed hybrid algorithms is discussed using standard cryptographic examples.

Analysis of nodule senescence in pea (Pisum sativum L.) using laser microdissection, real-time PCR, and ACC immunolocalization.

PubMed

Serova, Tatiana A; Tikhonovich, Igor A; Tsyganov, Viktor E

2017-05-01

A delay in the senescence of symbiotic nodules could prolong active nitrogen fixation, resulting in improved crop yield and a reduced need for chemical fertilizers. The molecular genetic mechanisms underlying nodule senescence have not been extensively studied with a view to breeding varieties with delayed nodule senescence. In such studies, plant mutants with the phenotype of premature degradation of symbiotic structures are useful models to elucidate the genetic basis of nodule senescence. Using a dataset from transcriptome analysis of Medicago truncatula Gaertn. nodules and previous studies on pea (Pisum sativum L.) nodules, we developed a set of molecular markers based on genes that are known to be activated during nodule senescence. These genes encode cysteine proteases, a thiol protease, a bZIP transcription factor, enzymes involved in the biosynthesis of ethylene (ACS2 for ACC synthase and ACO1 for ACC oxidase) and ABA (AO3 for aldehyde oxidase), and an enzyme involved in catabolism of gibberellins (GA 2-oxidase). We analyzed the transcript levels of these genes in the nodules of two pea wild-types (cv. Sparkle and line Sprint-2) and two mutant lines, one showing premature nodule senescence (E135F (sym13)) and one showing no morphological signs of symbiotic structure degradation (Sprint-2Fix - (sym31)). Real-time PCR analyses revealed that all of the selected genes showed increased transcript levels during nodule aging in all phenotypes. Remarkably, at 4 weeks after inoculation (WAI), the transcript levels of all analyzed genes were significantly higher in the early senescent nodules of the mutant line E135F (sym13) and in nodules of the mutant Sprint-2Fix - (sym31) than in the active nitrogen-fixing nodules of wild-types. In contrast, the transcript levels of the same genes of both wild-types were significantly increased only at 6 WAI. We evaluated the expression of selected markers in the different histological nodule zones of pea cv. Sparkle and its mutant line E135F (sym13) by laser capture microdissection analysis. Finally, we analyzed ACC by immunolocalization in the nodules of both wild-type pea and their mutants. Together, the results indicate that nodule senescence is a general plant response to nodule ineffectiveness. Copyright © 2017 Elsevier GmbH. All rights reserved.

Trehalose synthesis in Aspergillus niger: characterization of six homologous genes, all with conserved orthologs in related species

PubMed Central

2014-01-01

Background The disaccharide trehalose is a major component of fungal spores and is released upon germination. Moreover, the sugar is well known for is protective functions, e.g. against thermal stress and dehydration. The properties and synthesis of trehalose have been well investigated in the bakers’ yeast Saccharomyces cerevisiae. In filamentous fungi, such knowledge is limited, although several gene products have been identified. Results Using Aspergillus niger as a model fungus, the aim of this study was to provide an overview of all genes involved in trehalose synthesis. This fungus has three potential trehalose-6-phosphate synthase encoding genes, tpsA-C, and three putative trehalose phosphate phosphatase encoding genes, tppA-C, of which two have not previously been identified. Expression of all six genes was confirmed using real-time PCR, and conserved orthologs could be identified in related Aspergilli. Using a two-hybrid approach, there is a strong indication that four of the proteins physically interact, as has previously been shown in S. cerevisiae. When creating null mutants of all the six genes, three of them, ΔtpsA, ΔtppA and ΔtppB, had lower internal trehalose contents. The only mutant with a pronounced morphological difference was ΔtppA, in which sporulation was severely reduced with abnormal conidiophores. This was also the only mutant with accumulated levels of trehalose-6-phosphate, indicating that the encoded protein is the main phosphatase under normal conditions. Besides ΔtppA, the most studied deletion mutant in this work was ΔtppB. This gene encodes a protein conserved in filamentous Ascomycota. The ΔtppB mutant displayed a low, but not depleted, internal trehalose content, and conidia were more susceptible to thermal stress. Conclusion A. niger contains at least 6 genes putatively involved in trehalose synthesis. Gene expressions related to germination have been quantified and deletion mutants characterized: Mutants lacking tpsA, tppA or tppB have reduced internal trehalose contents. Furthermore, tppA, under normal conditions, encodes the functional trehalose-6-phosphate-phosphatase. PMID:24725382

Selection of Salmonella enterica Serovar Typhi Genes Involved during Interaction with Human Macrophages by Screening of a Transposon Mutant Library

PubMed Central

Sabbagh, Sébastien C.; Lepage, Christine; McClelland, Michael; Daigle, France

2012-01-01

The human-adapted Salmonella enterica serovar Typhi (S. Typhi) causes a systemic infection known as typhoid fever. This disease relies on the ability of the bacterium to survive within macrophages. In order to identify genes involved during interaction with macrophages, a pool of approximately 105 transposon mutants of S. Typhi was subjected to three serial passages of 24 hours through human macrophages. Mutants recovered from infected macrophages (output) were compared to the initial pool (input) and those significantly underrepresented resulted in the identification of 130 genes encoding for cell membrane components, fimbriae, flagella, regulatory processes, pathogenesis, and many genes of unknown function. Defined deletions in 28 genes or gene clusters were created and mutants were evaluated in competitive and individual infection assays for uptake and intracellular survival during interaction with human macrophages. Overall, 26 mutants had defects in the competitive assay and 14 mutants had defects in the individual assay. Twelve mutants had defects in both assays, including acrA, exbDB, flhCD, fliC, gppA, mlc, pgtE, typA, waaQGP, SPI-4, STY1867-68, and STY2346. The complementation of several mutants by expression of plasmid-borne wild-type genes or gene clusters reversed defects, confirming that the phenotypic impairments within macrophages were gene-specific. In this study, 35 novel phenotypes of either uptake or intracellular survival in macrophages were associated with Salmonella genes. Moreover, these results reveal several genes encoding molecular mechanisms not previously known to be involved in systemic infection by human-adapted typhoidal Salmonella that will need to be elucidated. PMID:22574205

Genes Associated with Desiccation and Osmotic Stress in Listeria monocytogenes as Revealed by Insertional Mutagenesis

PubMed Central

Hingston, Patricia A.; Piercey, Marta J.

2015-01-01

Listeria monocytogenes is a foodborne pathogen whose survival in food processing environments may be associated with its tolerance to desiccation. To probe the molecular mechanisms used by this bacterium to adapt to desiccation stress, a transposon library of 11,700 L. monocytogenes mutants was screened, using a microplate assay, for strains displaying increased or decreased desiccation survival (43% relative humidity, 15°C) in tryptic soy broth (TSB). The desiccation phenotypes of selected mutants were subsequently assessed on food-grade stainless steel (SS) coupons in TSB plus 1% glucose (TSB-glu). Single transposon insertions in mutants exhibiting a change in desiccation survival of >0.5 log CFU/cm2 relative to that of the wild type were determined by sequencing arbitrary PCR products. Strain morphology, motility, and osmotic stress survival (in TSB-glu plus 20% NaCl) were also analyzed. The initial screen selected 129 desiccation-sensitive (DS) and 61 desiccation-tolerant (DT) mutants, out of which secondary screening on SS confirmed 15 DT and 15 DS mutants. Among the DT mutants, seven immotile and flagellum-less strains contained transposons in genes involved in flagellum biosynthesis (fliP, flhB, flgD, flgL) and motor control (motB, fliM, fliY), while others harbored transposons in genes involved in membrane lipid biosynthesis, energy production, potassium uptake, and virulence. The genes that were interrupted in the 15 DS mutants included those involved in energy production, membrane transport, protein metabolism, lipid biosynthesis, oxidative damage control, and putative virulence. Five DT and 14 DS mutants also demonstrated similar significantly (P < 0.05) different survival relative to that of the wild type when exposed to osmotic stress, demonstrating that some genes likely have similar roles in allowing the organism to survive the two water stresses. PMID:26025900

Murine models of VACTERL syndrome: Role of sonic hedgehog signaling pathway.

PubMed

Kim, P C; Mo, R; Hui Cc, C

2001-02-01

VACTERL syndrome is a common surgical condition affecting the development of many midaxial organs. The etiology, embryology, and pathogenesis of the VACTERL syndrome are not known. The authors report here new mouse models of VACTERL syndrome involving the Sonic hedgehog (Shh) signaling pathway. Mutant mice involving Shh signaling, the Shh transcription factors Gli2-/- and Gli3-/-, Gli2-/-;Gli3+/- double heterozygotes, and Shh-/- were analyzed. In addition to reported vertebral, anal, tracheoesophageal, and limb anomalies, mutant mice display cardiac, renal, and associated anomalies, namely congenital diaphragmatic hernia and omphalocele, known to be associated in VACTERL syndrome. The Shh transcription factors Gli2 and Gli3 have specific and overlapping roles in the induction of VACTERL phenotypes in a gene-dose dependent manner in these mutants. To the authors' knowledge, these mutant mice represent the first animal model that mimics the human VACTERL syndrome, and suggests that aberrations in Shh signaling might be involved in the VACTERL syndrome.

Loss of the starvation-induced gene Rack1 leads to glycogen deficiency and impaired autophagic responses in Drosophila.

PubMed

Erdi, Balázs; Nagy, Péter; Zvara, Agnes; Varga, Agnes; Pircs, Karolina; Ménesi, Dalma; Puskás, László G; Juhász, Gábor

2012-07-01

Autophagy delivers cytoplasmic material for lysosomal degradation in eukaryotic cells. Starvation induces high levels of autophagy to promote survival in the lack of nutrients. We compared genome-wide transcriptional profiles of fed and starved control, autophagy-deficient Atg7 and Atg1 null mutant Drosophila larvae to search for novel regulators of autophagy. Genes involved in catabolic processes including autophagy were transcriptionally upregulated in all cases. We also detected repression of genes involved in DNA replication in autophagy mutants compared with control animals. The expression of Rack1 (receptor of activated protein kinase C 1) increased 4.1- to 5.5-fold during nutrient deprivation in all three genotypes. The scaffold protein Rack1 plays a role in a wide range of processes including translation, cell adhesion and migration, cell survival and cancer. Loss of Rack1 led to attenuated autophagic response to starvation, and glycogen stores were decreased 11.8-fold in Rack1 mutant cells. Endogenous Rack1 partially colocalized with GFP-Atg8a and early autophagic structures on the ultrastructural level, suggesting its involvement in autophagosome formation. Endogenous Rack1 also showed a high degree of colocalization with glycogen particles in the larval fat body, and with Shaggy, the Drosophila homolog of glycogen synthase kinase 3B (GSK-3B). Our results, for the first time, demonstrated the fundamental role of Rack1 in autophagy and glycogen synthesis.

Involvement of NADPH oxidase isoforms in the production of O2− manipulated by ABA in the senescing leaves of early-senescence-leaf (esl) mutant rice (Oryza sativa)

PubMed Central

Wang, Fubiao; Zhao, Qian; Liu, Jianchao; Cheng, Fangmin

2018-01-01

In this study, the differences in reactive oxygen species (ROS) generation and abscisic acid (ABA) accumulation in senescing leaves were investigated by early-senescence-leaf (esl) mutant and its wild type, to clarify the relationship among ABA levels, ROS generation, and NADPH oxidase (Nox) in senescing leaves of rice (Oryza sativa). The temporal expression levels of OsNox isoforms in senescing leaves and their expression patterns in response to ABA treatment were determined through quantitative real-time reverse transcription PCR (qRT-PCR). Results showed that the flag leaf of the esl mutant generated more O2- concentrations and accumulated higher ABA levels than the wild-type cultivar did in the grain-filling stage. Exogenous ABA treatment induced O2- generation; however, it was depressed by diphenyleneiodonium chloride (DPI) pretreatment in the detached leaf segments. This finding suggested the involvement of NADPH oxidase in ABA-induced O2- generation. The esl mutant exhibited significantly higher expression of OsNox2, OsNox5, OsNox6, and OsNox7 in the initial of grain-filling stage, followed by sharply decrease. The transcriptional levels of OsNox1, OsNox3, and OsFR07 in the flag leaf of the esl mutant were significantly lower than those in the wild-type cultivar. The expression levels of OsNox2, OsNox5, OsNox6, and OsNox7 were significantly enhanced by exogenous ABA treatments. The enhanced expression levels of OsNox2 and OsNox6 were dependent on the duration of ABA treatment. The inducible expression levels of OsNox5 and OsNox7 were dependent on ABA concentrations. By contrast, exogenous ABA treatment severely repressed the transcripts of OsNox1, OsNox3, and OsFR07 in the detached leaf segments. Therefore, OsNox2, OsNox5, OsNox6, and OsNox7 were probably involved in the ABA-induced O2- generation in the initial stage of leaf senescence. Subsequently, other oxidases activated in deteriorating cells were associated with ROS generation and accumulation in the senescing leaves of the esl mutant. Conversely, OsNox1, OsNox3, and OsFR07 were not associated with ABA-induced O2- generation during leaf senescence. PMID:29309410

Involvement of NADPH oxidase isoforms in the production of O2- manipulated by ABA in the senescing leaves of early-senescence-leaf (esl) mutant rice (Oryza sativa).

PubMed

Li, Zhaowei; Wang, Fubiao; Zhao, Qian; Liu, Jianchao; Cheng, Fangmin

2018-01-01

In this study, the differences in reactive oxygen species (ROS) generation and abscisic acid (ABA) accumulation in senescing leaves were investigated by early-senescence-leaf (esl) mutant and its wild type, to clarify the relationship among ABA levels, ROS generation, and NADPH oxidase (Nox) in senescing leaves of rice (Oryza sativa). The temporal expression levels of OsNox isoforms in senescing leaves and their expression patterns in response to ABA treatment were determined through quantitative real-time reverse transcription PCR (qRT-PCR). Results showed that the flag leaf of the esl mutant generated more O2- concentrations and accumulated higher ABA levels than the wild-type cultivar did in the grain-filling stage. Exogenous ABA treatment induced O2- generation; however, it was depressed by diphenyleneiodonium chloride (DPI) pretreatment in the detached leaf segments. This finding suggested the involvement of NADPH oxidase in ABA-induced O2- generation. The esl mutant exhibited significantly higher expression of OsNox2, OsNox5, OsNox6, and OsNox7 in the initial of grain-filling stage, followed by sharply decrease. The transcriptional levels of OsNox1, OsNox3, and OsFR07 in the flag leaf of the esl mutant were significantly lower than those in the wild-type cultivar. The expression levels of OsNox2, OsNox5, OsNox6, and OsNox7 were significantly enhanced by exogenous ABA treatments. The enhanced expression levels of OsNox2 and OsNox6 were dependent on the duration of ABA treatment. The inducible expression levels of OsNox5 and OsNox7 were dependent on ABA concentrations. By contrast, exogenous ABA treatment severely repressed the transcripts of OsNox1, OsNox3, and OsFR07 in the detached leaf segments. Therefore, OsNox2, OsNox5, OsNox6, and OsNox7 were probably involved in the ABA-induced O2- generation in the initial stage of leaf senescence. Subsequently, other oxidases activated in deteriorating cells were associated with ROS generation and accumulation in the senescing leaves of the esl mutant. Conversely, OsNox1, OsNox3, and OsFR07 were not associated with ABA-induced O2- generation during leaf senescence.

Genetic Regulation by NLA and MicroRNA827 for Maintaining Nitrate-Dependent Phosphate Homeostasis in Arabidopsis

PubMed Central

Kant, Surya; Peng, Mingsheng; Rothstein, Steven J.

2011-01-01

Plants need abundant nitrogen and phosphorus for higher yield. Improving plant genetics for higher nitrogen and phosphorus use efficiency would save potentially billions of dollars annually on fertilizers and reduce global environmental pollution. This will require knowledge of molecular regulators for maintaining homeostasis of these nutrients in plants. Previously, we reported that the NITROGEN LIMITATION ADAPTATION (NLA) gene is involved in adaptive responses to low-nitrogen conditions in Arabidopsis, where nla mutant plants display abrupt early senescence. To understand the molecular mechanisms underlying NLA function, two suppressors of the nla mutation were isolated that recover the nla mutant phenotype to wild type. Map-based cloning identified these suppressors as the phosphate (Pi) transport-related genes PHF1 and PHT1.1. In addition, NLA expression is shown to be regulated by the low-Pi induced microRNA miR827. Pi analysis revealed that the early senescence in nla mutant plants was due to Pi toxicity. These plants accumulated over five times the normal Pi content in shoots specifically under low nitrate and high Pi but not under high nitrate conditions. Also the Pi overaccumulator pho2 mutant shows Pi toxicity in a nitrate-dependent manner similar to the nla mutant. Further, the nitrate and Pi levels are shown to have an antagonistic crosstalk as displayed by their differential effects on flowering time. The results demonstrate that NLA and miR827 have pivotal roles in regulating Pi homeostasis in plants in a nitrate-dependent fashion. PMID:21455488

A defect in carbon catabolite repression associated with uncontrollable and excessive maltose uptake.

PubMed

Entian, K D

1980-01-01

The previously isolated recessive mutant allele hex2-3 of Saccharomyces cerevisiae caused a defect in carbon catabolite repression of maltase, invertase, malate dehydrogenase, and respiration but at the same time led to an extreme sensitivity to maltose (Zimmerman and Scheel, 1977; Entian and Zimmermann, 1980). Addition of maltose to a growing culture of a hex2-3 mutant resulted within 60 to 90 min in an inhibition of growth, glycolysis, and de novo protein synthesis. This was not accompanied by any abnormal levels of glycolysis metabolites or glycolytic enzyme activities. However, inhibitory effects coincided with a dramatic increase in intracellular glucose up to 150 mM relative to cell water as opposed to 2.5 mM in wild-type cells. This abnormal behavior is interpreted as a result of an uncontrolled maltose uptake in hex2 mutants, which in combination with increasing maltase activity results in an accumulation of intracellular glucose. Obviously the amount of available glucose surpassed glycolytic capacity in hex2 mutants. Properties of mutant alleles hex2 and hex1 (see Entian and Zimmermann, 1980) clearly show, that specific gene functions are involved in adapting the rate of sugar uptake into the cell to the actual glycolytic capacity.

Metabolic switches and adaptations deduced from the proteomes of Streptomyces coelicolor wild type and phoP mutant grown in batch culture.

PubMed

Thomas, Louise; Hodgson, David A; Wentzel, Alexander; Nieselt, Kay; Ellingsen, Trond E; Moore, Jonathan; Morrissey, Edward R; Legaie, Roxane; Wohlleben, Wolfgang; Rodríguez-García, Antonio; Martín, Juan F; Burroughs, Nigel J; Wellington, Elizabeth M H; Smith, Margaret C M

2012-02-01

Bacteria in the genus Streptomyces are soil-dwelling oligotrophs and important producers of secondary metabolites. Previously, we showed that global messenger RNA expression was subject to a series of metabolic and regulatory switches during the lifetime of a fermentor batch culture of Streptomyces coelicolor M145. Here we analyze the proteome from eight time points from the same fermentor culture and, because phosphate availability is an important regulator of secondary metabolite production, compare this to the proteome of a similar time course from an S. coelicolor mutant, INB201 (ΔphoP), defective in the control of phosphate utilization. The proteomes provide a detailed view of enzymes involved in central carbon and nitrogen metabolism. Trends in protein expression over the time courses were deduced from a protein abundance index, which also revealed the importance of stress pathway proteins in both cultures. As expected, the ΔphoP mutant was deficient in expression of PhoP-dependent genes, and several putatively compensatory metabolic and regulatory pathways for phosphate scavenging were detected. Notably there is a succession of switches that coordinately induce the production of enzymes for five different secondary metabolite biosynthesis pathways over the course of the batch cultures.

Ferritin, an iron source in meat for Staphylococcus xylosus?

PubMed

Vermassen, Aurore; Talon, Régine; Leroy, Sabine

2016-05-16

Staphylococcus xylosus is frequently isolated from food of animal origin. Moreover, this species is one of the major starter cultures used for meat fermentation. Iron is a key element for growth and survival of bacteria. Meat is particularly rich in haemic (myoglobin and haemoglobin) and non-haemic (ferritin and transferrin) iron sources. Ferritin is a storage protein able to capture large quantities of iron. It is highly resistant to microbial attack and few microorganisms can use it as an iron source. Surprisingly, we found that the S. xylosus C2a strain grows in the presence of ferritin as a sole iron source. A three-cistron operon was highly overexpressed under ferritin iron growth conditions. We generated a deletion-insertion in the first gene of the operon and evaluated the phenotype of the mutant. The mutant showed decreased growth because it was less able to acquire iron from ferritin. Transcriptional analysis of the mutant revealed downregulation of several genes involved in the response to oxidative stress. This study characterized for the first time the capacity of a Staphylococcus to use iron from ferritin and revealed that a potential reductive pathway was involved in this acquisition. We hypothesize that this ability could give an advantage to S. xylosus in meat products. Copyright © 2016 Elsevier B.V. All rights reserved.

Huntingtin-interacting protein 1 influences worm and mouse presynaptic function and protects Caenorhabditis elegans neurons against mutant polyglutamine toxicity.

PubMed

Parker, J Alex; Metzler, Martina; Georgiou, John; Mage, Marilyne; Roder, John C; Rose, Ann M; Hayden, Michael R; Néri, Christian

2007-10-10

Huntingtin-interacting protein 1 (HIP1) was identified through its interaction with htt (huntingtin), the Huntington's disease (HD) protein. HIP1 is an endocytic protein that influences transport and function of AMPA and NMDA receptors in the brain. However, little is known about its contribution to neuronal dysfunction in HD. We report that the Caenorhabditis elegans HIP1 homolog hipr-1 modulates presynaptic activity and the abundance of synaptobrevin, a protein involved in synaptic vesicle fusion. Presynaptic function was also altered in hippocampal brain slices of HIP1-/- mice demonstrating delayed recovery from synaptic depression and a reduction in paired-pulse facilitation, a form of presynaptic plasticity. Interestingly, neuronal dysfunction in transgenic nematodes expressing mutant N-terminal huntingtin was specifically enhanced by hipr-1 loss of function. A similar effect was observed with several other mutant proteins that are expressed at the synapse and involved in endocytosis, such as unc-11/AP180, unc-26/synaptojanin, and unc-57/endophilin. Thus, HIP1 is involved in presynaptic nerve terminal activity and modulation of mutant polyglutamine-induced neuronal dysfunction. Moreover, synaptic proteins involved in endocytosis may protect neurons against amino acid homopolymer expansion.

Assessing stress responses to atmospheric cold plasma exposure using Escherichia coli knock-out mutants.

PubMed

Han, L; Boehm, D; Patil, S; Cullen, P J; Bourke, P

2016-08-01

This study investigated the effect of atmospheric cold plasma (ACP) exposure-induced stress on microbial inactivation patterns and the regulation of genes involved in the microbial stress response in conjunction with key processing parameters of exposure time and post-treatment storage time. Cell suspensions of Escherichia coli BW 25113 and its isogenic knock-out mutants in rpoS, soxR, soxS, oxyR and dnaK genes were treated with high-voltage ACP in a sealed package for 1, 3 and 5 min followed by 0-, 1- and 24-h post-treatment storage. Reactive oxygen species (ROS) densities and colony formation were determined. ΔrpoS strain showed higher microbial reduction and greater cell permeability than other mutants, while ΔoxyR only showed this effect after 5 min of treatment. With increased post-treatment storage time, ΔsoxS and ΔsoxR had increased sensitivity and resistance respectively. ΔdnaK cell suspensions had much higher ROS than other strains and showed increased sensitivity with 24 h post-treatment storage. RpoS and oxyR genes have both short-term and long-term regulatory effects under plasma stress. However, knocking out dnaK gene had an immediate response on ROS scavenging and long-term repairing mechanisms. ΔsoxR and ΔsoxS had different responses to ACP treatment with the increase in post-treatment time in relation to clearance of reactive species implying the different characteristics and functions as subunits. By comparing the response of mutants under ACP exposure to key processing parameters, the mechanism of microbial inactivation was partly revealed with respect to cellular regulation and repairing genes. © 2016 The Society for Applied Microbiology.

ADP/ATP mitochondrial carrier MD simulations to shed light on the structural-dynamical events that, after an additional mutation, restore the function in a pathological single mutant.

PubMed

Di Marino, Daniele; Oteri, Francesco; Morozzo Della Rocca, Blasco; Chillemi, Giovanni; Falconi, Mattia

2010-12-01

Molecular dynamics simulations of the wild type bovine ADP/ATP mitochondrial carrier, and of the single Ala113Pro and double Ala113Pro/Val180Met mutants, embedded in a lipid bilayer, have been carried out for 30ns to shed light on the structural-dynamical changes induced by the Val180Met mutation restoring the carrier function in the Ala113Pro pathologic mutant. Principal component analysis indicates that, for the three systems, the protein dynamics is mainly characterized by the motion of the matrix loops and of the odd-numbered helices having a conserved proline in their central region. Analysis of the motions shows a different behaviour of single pathological mutant with respect of the other two systems. The single mutation induces a regularization and rigidity of the H3 helix, lost upon the introduction of the second mutation. This is directly correlated to the salt bridge distribution involving residues Arg79, Asp134 and Arg234, hypothesized to interact with the substrate. In fact, in the wild type simulation two stable inter-helices salt bridges, crucial for substrate binding, are present almost over all the simulation time. In line with the impaired ADP transport, one salt interaction is lost in the single mutant trajectory but reappears in the double mutant simulation, where a salt bridge network matching the wild type is restored. Other important structural-dynamical properties, such as the trans-membrane helices mobility, analyzed via the principal component analysis, are similar for the wild type and double mutant while are different for the single mutant, providing a mechanistic explanation for their different functional properties. Copyright © 2010 Elsevier Inc. All rights reserved.

Akt mediated ROS-dependent selective targeting of mutant KRAS tumors.

PubMed

Iskandar, Kartini; Rezlan, Majidah; Pervaiz, Shazib

2014-10-01

Reactive oxygen species (ROS) play a critical role in a variety of cellular processes, ranging from cell survival and proliferation to cell death. Previously, we reported the ability of a small molecule compound, C1, to induce ROS dependent autophagy associated apoptosis in human cancer cell lines and primary tumor cells (Wong C. et al. 2010). Our ongoing investigations have unraveled a hitherto undefined novel signaling network involving hyper-phosphorylation of Akt and Akt-mediated ROS production in cancer cell lines. Interestingly, drug-induced Akt activation is selectively seen in cell lines that carry mutant KRAS; HCT116 cells that carry the V13D KRAS mutation respond favorably to C1 while HT29 cells expressing wild type KRAS are relatively resistant. Of note, not only does the compound target mutant KRAS expressing cells but also induces RAS activation as evidenced by the PAK pull down assay. Corroborating this, pharmacological inhibition as well as siRNA mediated silencing of KRAS or Akt, blocked C1-induced ROS production and rescued tumor colony forming ability in HCT116 cells. To further confirm the involvement of KRAS, we made use of mutant KRAS transformed RWPE-1 prostate epithelial cells. Notably, drug-induced ROS generation and death sensitivity was significantly higher in RWPE-1-KRAS cells than the RWPE-1-vector cells, thus confirming the results obtained with mutant KRAS colorectal carcinoma cell line. Lastly, we made use of HCT116 mutant KRAS knockout cells (KO) where the mutant KRAS allele had been deleted, thus expressing a single wild-type KRAS allele. Exposure of the KO cells to C1 failed to induce Akt activation and mitochondrial ROS production. Taken together, results show the involvement of activated Akt in ROS-mediated selective targeting of mutant KRAS expressing tumors, which could have therapeutic implications given the paucity of chemotherapeutic strategies specifically targeting KRAS mutant cancers. Copyright © 2014. Published by Elsevier Inc.

Characterization of RACK7 as a Novel Factor Involved in BRCA1 Mutation Mediated Breast Cancer

DTIC Science & Technology

2012-10-01

Characterization of RACK7 as a Novel Factor Involved in BRCA1 Mutation Mediated Breast Cancer Inder Verma, Quan Zhu, Martin Preyer, and Amy Rommel Salk...samples from 5 more human BRCA1 mutant tumors. We continue to collect more human BRCA1-mutant tumors from Dr. Petra Nederlof at the Netherland Cancer...damage pathway. Abstract: "The Novel Zinc-finger Protein ZMYND8 Is Involved in the Stabilization of Stalled Replication Forks", Martin Preyer and

The Kynurenine 3-Monooxygenase Encoding Gene, BcKMO, Is Involved in the Growth, Development, and Pathogenicity of Botrytis cinerea

PubMed Central

Zhang, Kang; Yuan, Xuemei; Zang, Jinping; Wang, Min; Zhao, Fuxin; Li, Peifen; Cao, Hongzhe; Han, Jianmin; Xing, Jihong; Dong, Jingao

2018-01-01

A pathogenic mutant, BCG183, was obtained by screening the T-DNA insertion library of Botrytis cinerea. A novel pathogenicity-related gene BcKMO, which encodes kynurenine 3-monooxygenase (KMO), was isolated and identified via thermal asymmetric interlaced PCR, bioinformatics analyses, and KMO activity measurement. The mutant BCG183 grew slowly, did not produce conidia and sclerotia, had slender hyphae, and presented enhanced pathogenicity. The phenotype and pathogenicity of the BcKMO-complementing mutant (BCG183/BcKMO) were similar to those of the wild-type (WT) strain. The activities of polymethylgalacturonase, polygalacturonase, and toxins were significantly higher, whereas acid production was significantly decreased in the mutant BCG183, when compared with those in the WT and BCG183/BcKMO. Moreover, the sensitivity of mutant BCG183 to NaCl and KCl was remarkably increased, whereas that to fluconazole, Congo Red, menadione, H2O2, and SQ22536 and U0126 [cAMP-dependent protein kinase (cAMP) and mitogen-activated protein kinase (MAPK) signaling pathways inhibitors, respectively] were significantly decreased compared with the other strains. Furthermore, the key genes involved in the cAMP and MAPK signaling pathways, Pka1, Pka2, PkaR, Bcg2, Bcg3, bmp1, and bmp3, were significantly upregulated or downregulated in the mutant BCG183. BcKMO expression levels were also upregulated or downregulated in the RNAi mutants of the key genes involved in the cAMP and MAPK signaling pathways. These findings indicated that BcKMO positively regulates growth and development, but negatively regulates pathogenicity of B. cinerea. Furthermore, BcKMO was found to be involved in controlling cell wall degrading enzymes activity, toxins activity, acid production, and cell wall integrity, and participate in cAMP and MAPK signaling pathways of B. cinerea. PMID:29867912

The Kynurenine 3-Monooxygenase Encoding Gene, BcKMO, Is Involved in the Growth, Development, and Pathogenicity of Botrytis cinerea.

PubMed

Zhang, Kang; Yuan, Xuemei; Zang, Jinping; Wang, Min; Zhao, Fuxin; Li, Peifen; Cao, Hongzhe; Han, Jianmin; Xing, Jihong; Dong, Jingao

2018-01-01

A pathogenic mutant, BCG183, was obtained by screening the T-DNA insertion library of Botrytis cinerea . A novel pathogenicity-related gene BcKMO , which encodes kynurenine 3-monooxygenase (KMO), was isolated and identified via thermal asymmetric interlaced PCR, bioinformatics analyses, and KMO activity measurement. The mutant BCG183 grew slowly, did not produce conidia and sclerotia, had slender hyphae, and presented enhanced pathogenicity. The phenotype and pathogenicity of the BcKMO -complementing mutant (BCG183/ BcKMO ) were similar to those of the wild-type (WT) strain. The activities of polymethylgalacturonase, polygalacturonase, and toxins were significantly higher, whereas acid production was significantly decreased in the mutant BCG183, when compared with those in the WT and BCG183/ BcKMO . Moreover, the sensitivity of mutant BCG183 to NaCl and KCl was remarkably increased, whereas that to fluconazole, Congo Red, menadione, H 2 O 2 , and SQ22536 and U0126 [cAMP-dependent protein kinase (cAMP) and mitogen-activated protein kinase (MAPK) signaling pathways inhibitors, respectively] were significantly decreased compared with the other strains. Furthermore, the key genes involved in the cAMP and MAPK signaling pathways, Pka1 , Pka2 , PkaR , Bcg2 , Bcg3 , bmp1 , and bmp3, were significantly upregulated or downregulated in the mutant BCG183. BcKMO expression levels were also upregulated or downregulated in the RNAi mutants of the key genes involved in the cAMP and MAPK signaling pathways. These findings indicated that BcKMO positively regulates growth and development, but negatively regulates pathogenicity of B. cinerea . Furthermore, BcKMO was found to be involved in controlling cell wall degrading enzymes activity, toxins activity, acid production, and cell wall integrity, and participate in cAMP and MAPK signaling pathways of B. cinerea .

Histone H2B monoubiquitination is involved in the regulation of cutin and wax composition in Arabidopsis thaliana.

PubMed

Ménard, Rozenn; Verdier, Gaëtan; Ors, Mareva; Erhardt, Mathieu; Beisson, Fred; Shen, Wen-Hui

2014-02-01

The plant cuticle is a chemically heterogeneous lipophilic layer composed of a cutin polymer matrix and waxes which covers the aerial parts of plants. This layer plays an essential role in the survival of plants by protecting them from desiccation and (a)biotic stresses. Knowledge on the gene networks and mechanisms regulating the synthesis of cuticle components during organ expansion or stress response remains limited however. Here, using five loss-of-function mutants for histone monoubiquitination, we report on the role of two RING E3 ligases, namely HISTONE MONOUBIQUITINATION 1 and 2 (HUB1 and HUB2), in the selective transcriptional activation of four cuticle biosynthesis genes in Arabidopsis thaliana. Microscopy observations showed that in hub1-6 and hub2-2 mutants irregular epidermal cells and disorganized cuticle layers were present in rosette leaves. Water loss measurements on excised rosettes demonstrated that cuticular permeability was significantly increased in the mutants. Chemical analysis of cuticle components revealed that the wax composition was changed and that cutin 16:0 dicarboxylic acid was significantly reduced in all hub mutants. Analysis of transcript levels of selected genes indicated that LACS2, ATT1 and HOTHEAD involved in cutin biosynthesis and CER1 involved in wax biosynthesis were down-regulated in the hub mutants, while the expression of LACERATA, CER3, CER6 and CER10 remained unchanged. Chromatin immunoprecipitation assays further showed that hub mutants are impaired in dynamic changes of histone H2B monoubiquitination at several loci of down-regulated genes. Taken together, these data establish that the regulation of cuticle composition involves chromatin remodeling by H2B monoubiquitination.

Functional Inactivation of Putative Photosynthetic Electron Acceptor Ferredoxin C2 (FdC2) Induces Delayed Heading Date and Decreased Photosynthetic Rate in Rice

PubMed Central

Ruan, Banpu; Kang, Shujing; He, Lei; Zhang, Sen; Dong, Guojun; Hu, Jiang; Zeng, Dali; Zhang, Guangheng; Gao, Zhenyu; Ren, Deyong; Hu, Xingming; Chen, Guang; Guo, Longbiao; Qian, Qian; Zhu, Li

2015-01-01

Ferredoxin (Fd) protein as unique electron acceptor, involved in a variety of fundamental metabolic and signaling processes, which is indispensable for plant growth. The molecular mechanisms of Fd such as regulation of electron partitioning, impact of photosynthetic rate and involvement in the carbon fixing remain elusive in rice. Here we reported a heading date delay and yellowish leaf 1 (hdy1) mutant derived from Japonica rice cultivar “Nipponbare” subjected to EMS treatment. In the paddy field, the hdy1 mutant appeared at a significantly late heading date and had yellow-green leaves during the whole growth stage. Further investigation indicated that the abnormal phenotype of hdy1 was connected with depressed pigment content and photosynthetic rate. Genetic analysis results showed that the hdy1 mutant phenotype was caused by a single recessive nuclear gene mutation. Map-based cloning revealed that OsHDY1 is located on chromosome 3 and encodes an ortholog of the AtFdC2 gene. Complementation and overexpression, transgenic plants exhibited the mutant phenotype including head date, leaf color and the transcription levels of the FdC2 were completely rescued by transformation with OsHDY1. Real-time PCR revealed that the expression product of OsHDY1 was detected in almost all of the organs except root, whereas highest expression levels were observed in seeding new leaves. The lower expression levels of HDY1 and content of iron were detected in hdy1 than WT’s. The FdC2::GFP was detected in the chloroplasts of rice. Real-time PCR results showed that the expression of many photosynthetic electron transfer related genes in hdy1 were higher than WT. Our results suggest that OsFdC2 plays an important role in photosynthetic rate and development of heading date by regulating electron transfer and chlorophyll content in rice. PMID:26598971

Time-resolved spectroscopic studies of photosynthetic reaction centers and tetrapyrrole chromophores for biomedical and solar-energy applications

NASA Astrophysics Data System (ADS)

Kee, Hooi Ling

2008-10-01

The photophysical properties of diverse tetrapyrrole chromophores as well as energy and electron transfer processes in tetrapyrrole dyads are investigated using static and time-resolved (femtoseconds to seconds) absorption and fluorescence spectroscopy. The goal of these studies is to elucidate the molecular design principals necessary to construct chromophores with the specific and tunable properties that will enhance applications in optical molecular imaging, photodynamic therapy, and solar-energy conversion. The kinetic properties of the transient intermediate P+H B- involving the bacteriopheophytin molecule HB on the normally inactive (B) cofactor branch of the bacterial photosynthetic reaction center are examined in Rhodobacter capsulatus mutants. Using nanosecond flash photolysis and F(L181)Y/Y(M208)F/L(M212)H mutant, the decay pathways and yields of P+HB- were measured, giving an overall yield of 13% for B-side charge separation P* → P+HB- → P+ QB- in this mutant. The goal of these studies is to understand the fundamental differences in the rates, yields, and mechanisms of charge separation and charge recombination along the two parallel electron-transport chains in the bacterial reaction center.

Molecular genetic analysis of plant gravitropism

NASA Technical Reports Server (NTRS)

Lomax, T. L.

1997-01-01

The analysis of mutants is a powerful approach for elucidating the components of complex biological processes. A growing number of mutants have been isolated which affect plant gravitropism and the classes of mutants found thus far provide important information about the gravity response mechanism. The wide variety of mutants isolated, especially in Arabidopsis, indicates that gravitropism is a complex, multi-step process. The existence of mutants altered in either root gravitropism alone, shoot gravitropism alone, or both indicates that the root and shoot gravitropic mechanisms have both separate and common steps. Reduced starch mutants have confirmed the role of amyloplasts in sensing the gravity signal. The hormone auxin is thought to act as the transducing signal between the sites of gravity perception (the starch parenchyma cells surrounding the vascular tissue in shoots and the columella cells of root caps) and asymmetric growth (the epidermal cells of the elongation zone(s) of each organ). To date, all mutants that are resistant to high concentrations of auxin have also been found to exhibit a reduced gravitropic response, thus supporting the role of auxin. Not all gravitropic mutants are auxin-resistant, however, indicating that there are additional steps which do not involve auxin. Studies with mutants of tomato which exhibit either reduced or reversed gravitropic responses further support the role of auxin redistribution in gravitropism and suggest that both red light and cytokinin interact with gravitropism through controlling lateral auxin transport. Plant responses to gravity thus likely involve changes in both auxin transport and sensitivity.

A large inversion in the linear chromosome of Streptomyces griseus caused by replicative transposition of a new Tn3 family transposon.

PubMed

Murata, M; Uchida, T; Yang, Y; Lezhava, A; Kinashi, H

2011-04-01

We have comprehensively analyzed the linear chromosomes of Streptomyces griseus mutants constructed and kept in our laboratory. During this study, macrorestriction analysis of AseI and DraI fragments of mutant 402-2 suggested a large chromosomal inversion. The junctions of chromosomal inversion were cloned and sequenced and compared with the corresponding target sequences in the parent strain 2247. Consequently, a transposon-involved mechanism was revealed. Namely, a transposon originally located at the left target site was replicatively transposed to the right target site in an inverted direction, which generated a second copy and at the same time caused a 2.5-Mb chromosomal inversion. The involved transposon named TnSGR was grouped into a new subfamily of the resolvase-encoding Tn3 family transposons based on its gene organization. At the end, terminal diversity of S. griseus chromosomes is discussed by comparing the sequences of strains 2247 and IFO13350.

Mutant p53 proteins alter cancer cell secretome and tumour microenvironment: Involvement in cancer invasion and metastasis.

PubMed

Cordani, Marco; Pacchiana, Raffaella; Butera, Giovanna; D'Orazi, Gabriella; Scarpa, Aldo; Donadelli, Massimo

2016-07-01

An ever-increasing number of studies highlight the role of mutant p53 proteins in the alteration of cancer cell secretome and in the modification of tumour microenvironment, sustaining an invasive phenotype of cancer cell. The knowledge of the molecular mechanisms underlying the interplay between mutant p53 proteins and the microenvironment is becoming fundamental for the identification of both efficient anticancer therapeutic strategies and novel serum biomarkers. In this review, we summarize the novel findings concerning the regulation of secreted molecules by cancer cells bearing mutant TP53 gene. In particular, we highlight data from available literature, suggesting that mutant p53 proteins are able to (i) alter the secretion of enzymes involved in the modulation of extracellular matrix components; (ii) alter the secretion of inflammatory cytokines; (iii) increase the extracellular acidification; and (iv) regulate the crosstalk between cancer and stromal cells. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Homology-dependent repair is involved in 45S rDNA loss in plant CAF-1 mutants

PubMed Central

Muchová, Veronika; Amiard, Simon; Mozgová, Iva; Dvořáčková, Martina; Gallego, Maria E; White, Charles; Fajkus, Jiří

2015-01-01

Arabidopsis thaliana mutants in FAS1 and FAS2 subunits of chromatin assembly factor 1 (CAF1) show progressive loss of 45S rDNA copies and telomeres. We hypothesized that homology-dependent DNA damage repair (HDR) may contribute to the loss of these repeats in fas mutants. To test this, we generated double mutants by crossing fas mutants with knock-out mutants in RAD51B, one of the Rad51 paralogs of A. thaliana. Our results show that the absence of RAD51B decreases the rate of rDNA loss, confirming the implication of RAD51B-dependent recombination in rDNA loss in the CAF1 mutants. Interestingly, this effect is not observed for telomeric repeat loss, which thus differs from that acting in rDNA loss. Involvement of DNA damage repair in rDNA dynamics in fas mutants is further supported by accumulation of double-stranded breaks (measured as γ-H2AX foci) in 45S rDNA. Occurrence of the foci is not specific for S-phase, and is ATM-independent. While the foci in fas mutants occur both in the transcribed (intranucleolar) and non-transcribed (nucleoplasmic) fraction of rDNA, double fas rad51b mutants show a specific increase in the number of the intranucleolar foci. These results suggest that the repair of double-stranded breaks present in the transcribed rDNA region is RAD51B dependent and that this contributes to rDNA repeat loss in fas mutants, presumably via the single-stranded annealing recombination pathway. Our results also highlight the importance of proper chromatin assembly in the maintenance of genome stability. PMID:25359579

Priming of anti-herbivore defense in tomato by arbuscular mycorrhizal fungus and involvement of the jasmonate pathway.

PubMed

Song, Yuan Yuan; Ye, Mao; Li, Chuan You; Wang, Rui Long; Wei, Xiao Chen; Luo, Shi Ming; Zeng, Ren Sen

2013-07-01

Mycorrhizas play a vital role in soil fertility, plant nutrition, and resistance to environmental stresses. However, mycorrhizal effects on plant resistance to herbivorous insects and the related mechanisms are poorly understood. This study evaluated effects of root colonization of tomato (Solanum lycopersicum Mill.) by arbuscular mycorrhizal fungi (AMF) Glomus mosseae on plant defense responses against a chewing caterpillar Helicoverpa arimigera. Mycorrhizal inoculation negatively affected larval performance. Real time RT-PCR analyses showed that mycorrhizal inoculation itself did not induce transcripts of most genes tested. However, insect feeding on AMF pre-inoculated plants resulted in much stronger defense response induction of four defense-related genes LOXD, AOC, PI-I, and PI-II in the leaves of tomato plants relative to non-inoculated plants. Four tomato genotypes: a wild-type (WT) plant, a jasmonic acid (JA) biosynthesis mutant (spr2), a JA-signaling perception mutant (jai1), and a JA-overexpressing 35S::PS plant were used to determine the role of the JA pathway in AMF-primed defense. Insect feeding on mycorrhizal 35S::PS plants led to higher induction of defense-related genes relative to WT plants. However, insect feeding on mycorrhizal spr2 and jai1 mutant plants did not induce transcripts of these genes. Bioassays showed that mycorrhizal inoculation on spr2 and jai1 mutants did not change plant resistance against H. arimigera. These results indicates that mycorrhizal colonization could prime systemic defense responses in tomato upon herbivore attack, and that the JA pathway is involved in defense priming by AMF.

The genetics of green thorax, a new larval colour mutant, non-linked with ruby - eye locus in the malaria mosquito, Anopheles stephensi.

PubMed

Sanil, D; Shetty, N J

2009-06-01

Anopheles stephensi, an important vector of malaria continues to be distributed widely in the Indian subcontinent. The natural vigour of the species combined with its new tolerance, indeed resistance to insecticides has made it obligatory that we look for control methods involving genetic manipulation. Hence, there is an immediate need for greater understanding of the genetics of this vector species. One of the requirements for such genetic studies is the establishment of naturally occurring mutants, establishment of the genetic basis for the same and use of such mutants in the genetic transformation studies and other genetic control programme(s). This paper describes the isolation and genetic studies of a larval colour mutant, green thorax (gt), and linkage studies involving another autosomal recessive mutant ruby- eye (ru) in An. stephensi. After the initial discovery, the mutant green thorax was crossed inter se and pure homozygous stock of the mutant was established. The stock of the mutant ruby- eye, which has been maintained as a pure stock in the laboratory. Crosses were made between the wild type and mutant, green thorax to determine the mode of inheritance of green thorax. For linkage studies, crosses were made between the mutant green thorax and another autosomal recessive mutant ruby-eye. The percentage cross-over was calculated for the genes linkage relationship for gt and gt ru. Results of crosses between mutant and wild type showed that the inheritance of green thorax (gt) in An. stephensi is monofactorial in nature. The gt allele is recessive to wild type and is autosomal. The linkage studies showed no linkage between ru and gt. The mutant gt represents an excellent marker for An. stephensi as it is expressed in late III instar stage of larvae and is prominent in IV instar and pupal stages with complete penetrance and high viability. The said mutant could be easily identified without the aid of a microscope. This mutant can be used extensively to conduct basic and applied research. The mutant has been maintained in two large cages in our laboratory.

Parallel analysis of tagged deletion mutants efficiently identifies genes involved in endoplasmic reticulum biogenesis.

PubMed

Wright, Robin; Parrish, Mark L; Cadera, Emily; Larson, Lynnelle; Matson, Clinton K; Garrett-Engele, Philip; Armour, Chris; Lum, Pek Yee; Shoemaker, Daniel D

2003-07-30

Increased levels of HMG-CoA reductase induce cell type- and isozyme-specific proliferation of the endoplasmic reticulum. In yeast, the ER proliferations induced by Hmg1p consist of nuclear-associated stacks of smooth ER membranes known as karmellae. To identify genes required for karmellae assembly, we compared the composition of populations of homozygous diploid S. cerevisiae deletion mutants following 20 generations of growth with and without karmellae. Using an initial population of 1,557 deletion mutants, 120 potential mutants were identified as a result of three independent experiments. Each experiment produced a largely non-overlapping set of potential mutants, suggesting that differences in specific growth conditions could be used to maximize the comprehensiveness of similar parallel analysis screens. Only two genes, UBC7 and YAL011W, were identified in all three experiments. Subsequent analysis of individual mutant strains confirmed that each experiment was identifying valid mutations, based on the mutant's sensitivity to elevated HMG-CoA reductase and inability to assemble normal karmellae. The largest class of HMG-CoA reductase-sensitive mutations was a subset of genes that are involved in chromatin structure and transcriptional regulation, suggesting that karmellae assembly requires changes in transcription or that the presence of karmellae may interfere with normal transcriptional regulation. Copyright 2003 John Wiley & Sons, Ltd.

Starch Biosynthesis in Guard Cells But Not in Mesophyll Cells Is Involved in CO2-Induced Stomatal Closing1[OPEN

PubMed Central

Stephan, Aaron B.; Schroeder, Julian I.

2016-01-01

Starch metabolism is involved in stomatal movement regulation. However, it remains unknown whether starch-deficient mutants affect CO2-induced stomatal closing and whether starch biosynthesis in guard cells and/or mesophyll cells is rate limiting for high CO2-induced stomatal closing. Stomatal responses to [CO2] shifts and CO2 assimilation rates were compared in Arabidopsis (Arabidopsis thaliana) mutants that were either starch deficient in all plant tissues (ADP-Glc-pyrophosphorylase [ADGase]) or retain starch accumulation in guard cells but are starch deficient in mesophyll cells (plastidial phosphoglucose isomerase [pPGI]). ADGase mutants exhibited impaired CO2-induced stomatal closure, but pPGI mutants did not, showing that starch biosynthesis in guard cells but not mesophyll functions in CO2-induced stomatal closing. Nevertheless, starch-deficient ADGase mutant alleles exhibited partial CO2 responses, pointing toward a starch biosynthesis-independent component of the response that is likely mediated by anion channels. Furthermore, whole-leaf CO2 assimilation rates of both ADGase and pPGI mutants were lower upon shifts to high [CO2], but only ADGase mutants caused impairments in CO2-induced stomatal closing. These genetic analyses determine the roles of starch biosynthesis for high CO2-induced stomatal closing. PMID:27208296

Identifying RNA splicing factors using IFT genes in Chlamydomonas reinhardtii.

PubMed

Lin, Huawen; Zhang, Zhengyan; Iomini, Carlo; Dutcher, Susan K

2018-03-01

Intraflagellar transport moves proteins in and out of flagella/cilia and it is essential for the assembly of these organelles. Using whole-genome sequencing, we identified splice site mutations in two IFT genes, IFT81 ( fla9 ) and IFT121 ( ift121-2 ), which lead to flagellar assembly defects in the unicellular green alga Chlamydomonas reinhardtii The splicing defects in these ift mutants are partially corrected by mutations in two conserved spliceosome proteins, DGR14 and FRA10. We identified a dgr14 deletion mutant, which suppresses the 3' splice site mutation in IFT81 , and a frameshift mutant of FRA10 , which suppresses the 5' splice site mutation in IFT121 Surprisingly, we found dgr14-1 and fra10 mutations suppress both splice site mutations. We suggest these two proteins are involved in facilitating splice site recognition/interaction; in their absence some splice site mutations are tolerated. Nonsense mutations in SMG1 , which is involved in nonsense-mediated decay, lead to accumulation of aberrant transcripts and partial restoration of flagellar assembly in the ift mutants. The high density of introns and the conservation of noncore splicing factors, together with the ease of scoring the ift mutant phenotype, make Chlamydomonas an attractive organism to identify new proteins involved in splicing through suppressor screening. © 2018 The Authors.

Involvement of Penicillium digitatum PdSUT1 in fungicide sensitivity and virulence during citrus fruit infection.

PubMed

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

2017-10-01

A putative sucrose transporter PdSUT1 included in the same clade that Sut1p from Schizosaccharomyces pombe was identified in Penicillium digitatum, the major citrus postharvest pathogen. PdSUT1 gene was characterized using target gene disruption and gene overexpression. The ΔPdSUT1 mutants generated by gene elimination showed reduction in fungal virulence during citrus fruit infection assayed in mature fruit at 20°C. However, the overexpression mutants did not increased disease severity neither in the mutants coming from a high virulent nor from a low virulent P. digitatum progenitor strains. Moreover, fungicide sensitivity was affected in the deletant mutants but not in the overexpression transformants. The expression analysis of several genes involved in fungicide resistance showed an intensification of MFS transporters and a decrease of sterol demethylases transcriptional abundance in the ΔPdSUT1 mutants compare to the parental wild type strain. PdSUT1 appear not to be directly involved in fungicide resistance although can affect the gene expression of fungicide related genes. These results indicate that PdSUT1 contribute to P. digitatum fungal virulence and influence fungicide sensitivity through carbohydrate uptake and MFS transporters gene activation. Copyright © 2017 Elsevier GmbH. All rights reserved.

Molecular evidence that the eukaryotic THO/TREX complex is required for efficient transcription elongation.

PubMed

Rondón, Ana G; Jimeno, Sonia; García-Rubio, María; Aguilera, Andrés

2003-10-03

THO/TREX is a conserved eukaryotic complex formed by the core THO complex plus proteins involved in mRNA metabolism and export such as Sub2 and Yra1. Mutations in any of the THO/TREX structural genes cause pleiotropic phenotypes such as transcription impairment, increased transcription-associated recombination, and mRNA export defects. To assay the relevance of THO/TREX complex in transcription, we performed in vitro transcription elongation assays in mutant cell extracts using supercoiled DNA templates containing two G-less cassettes. With these assays, we demonstrate that hpr1delta, tho2delta, and mft1delta mutants of the THO complex and sub2 mutants show significant reductions in the efficiency of transcription elongation. The mRNA expression defect of hpr1delta mutants was not due to an increase in mRNA decay, as determined by mRNA half-life measurements and mRNA time course accumulation experiments in the absence of Rrp6p exoribonuclease. This work demonstrates that THO and Sub2 are required for efficient transcription elongation, providing further evidence for the coupling between transcription and mRNA metabolism and export.

The conflict between cell proliferation and expansion primarily affects stem organogenesis in Arabidopsis.

PubMed

Maeda, Saori; Gunji, Shizuka; Hanai, Kenya; Hirano, Tomonari; Kazama, Yusuke; Ohbayashi, Iwai; Abe, Tomoko; Sawa, Shinichiro; Tsukaya, Hirokazu; Ferjani, Ali

2014-11-01

Plant shoot organs such as stems, leaves and flowers are derived from specialized groups of stem cells organized at the shoot apical meristem (SAM). Organogenesis involves two major processes, namely cell proliferation and differentiation, whereby the former contributes to increasing the cell number and the latter involves substantial increases in cell volume through cell expansion. Co-ordination between the above processes in time and space is essential for proper organogenesis. To identify regulatory factors involved in proper organogenesis, heavy-ion beam-irradiated de-etiolated (det) 3-1 seeds have been used to identify striking phenotypes in the A#26-2; det3-1 mutant. In addition to the stunted plant stature mimicking det3-1, the A#26-2; det3-1 mutant exhibited stem thickening, increased floral organ number and a fruit shape reminiscent of clavata (clv) mutants. DNA sequencing analysis demonstrated that A#26-2; det3-1 harbors a mutation in the CLV3 gene. Importantly, A#26-2; det3-1 displayed cracks that randomly occurred on the main stem with a frequency of approximately 50%. Furthermore, the double mutants clv3-8 det3-1, clv1-4 det3-1 and clv2-1 det3-1 consistently showed stem cracks with frequencies of approximately 97, 38 and 35%, respectively. Cross-sections of stems further revealed an increase in vascular bundle number, cell number and size in the pith of clv3-8 det3-1 compared with det3-1. These findings suggest that the stem inner volume increase due to clv mutations exerts an outward mechanical stress; that in a det3-1 background (defective in cell expansion) resulted in cracking of the outermost layer of epidermal cells. © The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

The time of day effects of warm temperature on flowering time involve PIF4 and PIF5

PubMed Central

Thines, Bryan C.; Duarte, Maritza I.; Harmon, Frank G.

2014-01-01

Warm temperature promotes flowering in Arabidopsis thaliana and this response involves multiple signalling pathways. To understand the temporal dynamics of temperature perception, tests were carried out to determine if there was a daily window of enhanced sensitivity to warm temperature (28 °C). Warm temperature applied during daytime, night-time, or continuously elicited earlier flowering, but the effects of each treatment were unequal. Plants exposed to warm night (WN) conditions flowered nearly as early as those in constant warm (CW) conditions, while treatment with warm days (WD) caused later flowering than either WN or CW. Flowering in each condition relied to varying degrees on the activity of CO , FT , PIF4 , and PIF5 , as well as the action of unknown genes. The combination of signalling pathways involved in flowering depended on the time of the temperature cue. WN treatments caused a significant advance in the rhythmic expression waveform of CO, which correlated with pronounced up-regulation of FT expression, while WD caused limited changes in CO expression and no stimulation of FT expression. WN- and WD-induced flowering was partially CO independent and, unexpectedly, dependent on PIF4 and PIF5 . pif4-2, pif5-3, and pif4-2 pif5-3 mutants had delayed flowering under all three warm conditions. The double mutant was also late flowering in control conditions. In addition, WN conditions alone imposed selective changes to PIF4 and PIF5 expression. Thus, the PIF4 and PIF5 transcription factors promote flowering by at least two means: inducing FT expression in WN and acting outside of FT by an unknown mechanism in WD. PMID:24574484

Attenuated Signature-Tagged Mutagenesis Mutants of Brucella melitensis Identified during the Acute Phase of Infection in Mice

PubMed Central

Lestrate, P.; Dricot, A.; Delrue, R.-M.; Lambert, C.; Martinelli, V.; De Bolle, X.; Letesson, J.-J.; Tibor, A.

2003-01-01

For this study, we screened 1,152 signature-tagged mutagenesis mutants of Brucella melitensis 16M in a mouse model of infection and found 36 of them to be attenuated in vivo. Molecular characterization of transposon insertion sites showed that for four mutants, the affected genes were only present in Rhizobiaceae. Another mutant contained a disruption in a gene homologous to mosA, which is involved in rhizopine biosynthesis in some strains of Rhizobium, suggesting that this sugar may be involved in Brucella pathogenicity. A mutant was disrupted in a gene homologous to fliF, a gene potentially coding for the MS ring, a basal component of the flagellar system. Surprisingly, a mutant was affected in the rpoA gene, coding for the essential α-subunit of the RNA polymerase. This disruption leaves a partially functional protein, impaired for the activation of virB transcription, as demonstrated by the absence of induction of the virB promoter in the Tn5::rpoA background. The results presented here highlight the fact that the ability of Brucella to induce pathogenesis shares similarities with the molecular mechanisms used by both Rhizobium and Agrobacterium to colonize their hosts. PMID:14638795

The Agr communication system provides a benefit to the populations of Listeria monocytogenes in soil.

PubMed

Vivant, Anne-Laure; Garmyn, Dominique; Gal, Laurent; Piveteau, Pascal

2014-01-01

In this study, we investigated whether the Agr communication system of the pathogenic bacterium Listeria monocytogenes was involved in adaptation and competitiveness in soil. Alteration of the ability to communicate, either by deletion of the gene coding the response regulator AgrA (response-negative mutant) or the signal pro-peptide AgrD (signal-negative mutant), did not affect population dynamics in soil that had been sterilized but survival was altered in biotic soil suggesting that the Agr system of L. monocytogenes was involved to face the complex soil biotic environment. This was confirmed by a set of co-incubation experiments. The fitness of the response-negative mutant was lower either in the presence or absence of the parental strain but the fitness of the signal-negative mutant depended on the strain with which it was co-incubated. The survival of the signal-negative mutant was higher when co-cultured with the parental strain than when co-cultured with the response-negative mutant. These results showed that the ability to respond to Agr communication provided a benefit to listerial cells to compete. These results might also indicate that in soil, the Agr system controls private goods rather than public goods.

A Presynaptic Regulatory System Acts Transsynaptically via Mon1 to Regulate Glutamate Receptor Levels in Drosophila.

PubMed

Deivasigamani, Senthilkumar; Basargekar, Anagha; Shweta, Kumari; Sonavane, Pooja; Ratnaparkhi, Girish S; Ratnaparkhi, Anuradha

2015-10-01

Mon1 is an evolutionarily conserved protein involved in the conversion of Rab5 positive early endosomes to late endosomes through the recruitment of Rab7. We have identified a role for Drosophila Mon1 in regulating glutamate receptor levels at the larval neuromuscular junction. We generated mutants in Dmon1 through P-element excision. These mutants are short-lived with strong motor defects. At the synapse, the mutants show altered bouton morphology with several small supernumerary or satellite boutons surrounding a mature bouton; a significant increase in expression of GluRIIA and reduced expression of Bruchpilot. Neuronal knockdown of Dmon1 is sufficient to increase GluRIIA levels, suggesting its involvement in a presynaptic mechanism that regulates postsynaptic receptor levels. Ultrastructural analysis of mutant synapses reveals significantly smaller synaptic vesicles. Overexpression of vglut suppresses the defects in synaptic morphology and also downregulates GluRIIA levels in Dmon1 mutants, suggesting that homeostatic mechanisms are not affected in these mutants. We propose that DMon1 is part of a presynaptically regulated transsynaptic mechanism that regulates GluRIIA levels at the larval neuromuscular junction. Copyright © 2015 by the Genetics Society of America.

Phytohormonal Networks Promote Differentiation of Fiber Initials on Pre-Anthesis Cotton Ovules Grown In Vitro and In Planta

PubMed Central

Kim, Hee Jin; Hinchliffe, Doug J.; Triplett, Barbara A.; Chen, Z. Jeffrey; Stelly, David M.; Yeater, Kathleen M.; Moon, Hong S.; Gilbert, Matthew K.; Thyssen, Gregory N.; Turley, Rickie B.; Fang, David D.

2015-01-01

The number of cotton (Gossypium sp.) ovule epidermal cells differentiating into fiber initials is an important factor affecting cotton yield and fiber quality. Despite extensive efforts in determining the molecular mechanisms regulating fiber initial differentiation, only a few genes responsible for fiber initial differentiation have been discovered. To identify putative genes directly involved in the fiber initiation process, we used a cotton ovule culture technique that controls the timing of fiber initial differentiation by exogenous phytohormone application in combination with comparative expression analyses between wild type and three fiberless mutants. The addition of exogenous auxin and gibberellins to pre-anthesis wild type ovules that did not have visible fiber initials increased the expression of genes affecting auxin, ethylene, ABA and jasmonic acid signaling pathways within 1 h after treatment. Most transcripts expressed differentially by the phytohormone treatment in vitro were also differentially expressed in the ovules of wild type and fiberless mutants that were grown in planta. In addition to MYB25-like, a gene that was previously shown to be associated with the differentiation of fiber initials, several other differentially expressed genes, including auxin/indole-3-acetic acid (AUX/IAA) involved in auxin signaling, ACC oxidase involved in ethylene biosynthesis, and abscisic acid (ABA) 8'-hydroxylase an enzyme that controls the rate of ABA catabolism, were co-regulated in the pre-anthesis ovules of both wild type and fiberless mutants. These results support the hypothesis that phytohormonal signaling networks regulate the temporal expression of genes responsible for differentiation of cotton fiber initials in vitro and in planta. PMID:25927364

Role of Tir and Intimin in the Virulence of Rabbit Enteropathogenic Escherichia coli Serotype O103:H2

PubMed Central

Marchès, Olivier; Nougayrède, Jean-Philippe; Boullier, Séverine; Mainil, Jacques; Charlier, Gérard; Raymond, Isabelle; Pohl, Pierre; Boury, Michèle; De Rycke, Jean; Milon, Alain; Oswald, Eric

2000-01-01

Attaching and effacing (A/E) rabbit enteropathogenic Escherichia coli (REPEC) strains belonging to serogroup O103 are an important cause of diarrhea in weaned rabbits. Like human EPEC strains, they possess the locus of enterocyte effacement clustering the genes involved in the formation of the A/E lesions. In addition, pathogenic REPEC O103 strains produce an Esp-dependent but Eae (intimin)-independent alteration of the host cell cytoskeleton characterized by the formation of focal adhesion complexes and the reorganization of the actin cytoskeleton into bundles of stress fibers. To investigate the role of intimin and its translocated coreceptor (Tir) in the pathogenicity of REPEC, we have used a newly constructed isogenic tir null mutant together with a previously described eae null mutant. When human HeLa epithelial cells were infected, the tir mutant was still able to induce the formation of stress fibers as previously reported for the eae null mutant. When the rabbit epithelial cell line RK13 was used, REPEC O103 produced a classical fluorescent actin staining (FAS) effect, whereas both the eae and tir mutants were FAS negative. In a rabbit ligated ileal loop model, neither mutant was able to induce A/E lesions. In contrast to the parental strain, which intimately adhered to the enterocytes and destroyed the brush border microvilli, bacteria of both mutants were clustered in the mucus without reaching and damaging the microvilli. The role of intimin and Tir was then analyzed in vivo by oral inoculation of weaned rabbits. Although both mutants were still present in the intestinal flora of the rabbits 3 weeks after oral inoculation, neither mutant strain induced any clinical signs or significant weight loss in the inoculated rabbits whereas the parental strain caused the death of 90% of the inoculated rabbits. Nevertheless, an inflammatory infiltrate was present in the lamina propria of the rabbits infected with both mutants, with an inflammatory response greater for the eae null mutant. In conclusion, we have confirmed the role of intimin in virulence, and we have shown, for the first time, that Tir is also a key factor in vivo for pathogenicity. PMID:10722617

Role of tir and intimin in the virulence of rabbit enteropathogenic Escherichia coli serotype O103:H2.

PubMed

Marchès, O; Nougayrède, J P; Boullier, S; Mainil, J; Charlier, G; Raymond, I; Pohl, P; Boury, M; De Rycke, J; Milon, A; Oswald, E

2000-04-01

Attaching and effacing (A/E) rabbit enteropathogenic Escherichia coli (REPEC) strains belonging to serogroup O103 are an important cause of diarrhea in weaned rabbits. Like human EPEC strains, they possess the locus of enterocyte effacement clustering the genes involved in the formation of the A/E lesions. In addition, pathogenic REPEC O103 strains produce an Esp-dependent but Eae (intimin)-independent alteration of the host cell cytoskeleton characterized by the formation of focal adhesion complexes and the reorganization of the actin cytoskeleton into bundles of stress fibers. To investigate the role of intimin and its translocated coreceptor (Tir) in the pathogenicity of REPEC, we have used a newly constructed isogenic tir null mutant together with a previously described eae null mutant. When human HeLa epithelial cells were infected, the tir mutant was still able to induce the formation of stress fibers as previously reported for the eae null mutant. When the rabbit epithelial cell line RK13 was used, REPEC O103 produced a classical fluorescent actin staining (FAS) effect, whereas both the eae and tir mutants were FAS negative. In a rabbit ligated ileal loop model, neither mutant was able to induce A/E lesions. In contrast to the parental strain, which intimately adhered to the enterocytes and destroyed the brush border microvilli, bacteria of both mutants were clustered in the mucus without reaching and damaging the microvilli. The role of intimin and Tir was then analyzed in vivo by oral inoculation of weaned rabbits. Although both mutants were still present in the intestinal flora of the rabbits 3 weeks after oral inoculation, neither mutant strain induced any clinical signs or significant weight loss in the inoculated rabbits whereas the parental strain caused the death of 90% of the inoculated rabbits. Nevertheless, an inflammatory infiltrate was present in the lamina propria of the rabbits infected with both mutants, with an inflammatory response greater for the eae null mutant. In conclusion, we have confirmed the role of intimin in virulence, and we have shown, for the first time, that Tir is also a key factor in vivo for pathogenicity.

Molecular genetic analysis of activation-tagged transcription factors thought to be involved in photomorphogenesis

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

Neff, Michael M.

This is a final report for Department of Energy Grant No. DE-FG02-08ER15927 entitled “Molecular Genetic Analysis of Activation-Tagged Transcription Factors Thought to be Involved in Photomorphogenesis”. Based on our preliminary photobiological and genetic analysis of the sob1-D mutant, we hypothesized that OBP3 is a transcription factor involved in both phytochrome and cryptochrome-mediated signal transduction. In addition, we hypothesized that OBP3 is involved in auxin signaling and root development. Based on our preliminary photobiological and genetic analysis of the sob2-D mutant, we also hypothesized that a related gene, LEP, is involved in hormone signaling and seedling development.

Role of active site rigidity in activity: MD simulation and fluorescence study on a lipase mutant.

PubMed

Kamal, Md Zahid; Mohammad, Tabrez Anwar Shamim; Krishnamoorthy, G; Rao, Nalam Madhusudhana

2012-01-01

Relationship between stability and activity of enzymes is maintained by underlying conformational flexibility. In thermophilic enzymes, a decrease in flexibility causes low enzyme activity while in less stable proteins such as mesophiles and psychrophiles, an increase in flexibility is associated with enhanced enzyme activity. Recently, we identified a mutant of a lipase whose stability and activity were enhanced simultaneously. In this work, we probed the conformational dynamics of the mutant and the wild type lipase, particularly flexibility of their active site using molecular dynamic simulations and time-resolved fluorescence techniques. In contrast to the earlier observations, our data show that active site of the mutant is more rigid than wild type enzyme. Further investigation suggests that this lipase needs minimal reorganization/flexibility of active site residues during its catalytic cycle. Molecular dynamic simulations suggest that catalytically competent active site geometry of the mutant is relatively more preserved than wild type lipase, which might have led to its higher enzyme activity. Our study implies that widely accepted positive correlation between conformation flexibility and enzyme activity need not be stringent and draws attention to the possibility that high enzyme activity can still be accomplished in a rigid active site and stable protein structures. This finding has a significant implication towards better understanding of involvement of dynamic motions in enzyme catalysis and enzyme engineering through mutations in active site.

Visualization of reticulophagy in living cells using an endoplasmic reticulum-targeted p62 mutant.

PubMed

Wang, Liang; Liu, Lei; Qin, Lingsong; Luo, Qingming; Zhang, Zhihong

2017-04-01

Reticulophagy is a type of selective autophagy in which protein aggregate-containing and/or damaged endoplasmic reticulum (ER) fragments are engulfed for lysosomal degradation, which is important for ER homeostasis. Several chemical drugs and mutant proteins that promote protein aggregate formation within the ER lumen can efficiently induce reticulophagy in mammalian cells. However, the exact mechanism and cellular localization of reticulophagy remain unclear. In this report, we took advantage of the self-oligomerization property of p62/SQSTM1, an adaptor for selective autophagy, and developed a novel reticulophagy system based on an ER-targeted p62 mutant to investigate the process of reticulophagy in living cells. LC3 conversion analysis via western blot suggested that p62 mutant aggregate-induced ER stress triggered a cellular autophagic response. Confocal imaging showed that in cells with moderate aggregation conditions, the aggregates of ER-targeted p62 mutants were efficiently sequestered by autophagosomes, which was characterized by colocalization with the autophagosome precursor marker ATG16L1, the omegasome marker DFCP1, and the late autophagosomal marker LC3/GATE-16. Moreover, time-lapse imaging data demonstrated that the LC3- or DFCP1-positive protein aggregates are tightly associated with the reticular structures of the ER, thereby suggesting that reticulophagy occurs at the ER and that omegasomes may be involved in this process.

Identification of a peroxisomal-targeted aldolase involved in chlorophyll biosynthesis and sugar metabolism in rice.

PubMed

Zhang, Fei; Zhang, Pan; Zhang, Yu; Wang, Shouchuang; Qu, Lianghuan; Liu, Xianqing; Luo, Jie

2016-09-01

Chlorophyll plays remarkable and critical roles in photosynthetic light-harvesting, energy transduction and plant development. In this study, we identified a rice Chl-deficient mutant, ygdl-1 (yellow green and droopy leaf-1), which showed yellow-green leaves throughout plant development with decreased content of Chls and carotene and an increased Chl a/b ratio. The ygdl-1 mutant also exhibited severe defects in chloroplast development, including disorganized grana stacks. Sequence analysis revealed that the mutant contained a T-DNA insertion within the promoter of a fructose-1,6-bisphosphate aldolase (OsAld-Y), which dramatically reduced the OsAld-Y mRNA level, and its identity was verified by transgenic complementation. Real-time PCR analysis showed that the expression levels of genes associated with chlorophyll biosynthesis and chloroplast development were concurrently altered in the ygdl-1 mutant. The expression of OsAld-Y-GFP fusion protein in tobacco epidermal cells showed that OsAld-Y was localized to the peroxisome. In addition, the analysis of primary carbon metabolites revealed the significantly reduced levels of sucrose and fructose in the mutant leaves, while the glucose content was similar to wild-type plants. Our results suggest that the OsAld-Y participates in Chl accumulation, chloroplast development and plant growth by influencing the photosynthetic rate of leaves and the sugar metabolism of rice. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

A potential functional association between mutant BMPR2 and primary ovarian insufficiency.

PubMed

Patiño, Liliana Catherine; Silgado, Daniel; Laissue, Paul

2017-06-01

Primary ovarian insufficiency (POI) affects ~1% of women in the general population. Despite numerous attempts at identifying POI genetic aetiology, coding mutations in only a few genes have been functionally related to POI pathogenesis. It has been suggested that mutant BMPR2 might contribute towards the phenotype. Several BMP15 (a BMPR2 ligand) coding mutations in human species have been related to POI pathogenesis. The BMPR2 p.Ser987Phe mutation, previously identified in a woman with POI, might therefore lead to cellular dysfunction contributing to the phenotype. To explore such an assumption, the present study assessed potential pathogenic subcellular localization/aggregation patterns associated with the p.Ser987Phe mutant form of BMPR2 in a relevant model for studying ovarian function. A significant increase in protein-like aggregation patterns was identified at the endoplasmic reticulum (ER) which permitted us to establish, for the first time, a potential functional association between mutant BMPR2 and POI aetiology. Since BMPR2 mutant forms were previously related to idiopathic pulmonary arterial hypertension, BMPR2 mutations may be related to an as-yet-to-be described syndromic form of POI involving pulmonary dysfunction. Additional assays are necessary to confirm that BMPR2 abnormal subcellular patterns are composed by aggregates. POI: primary ovarian insufficiency; ER: endoplasmic reticulum; NGS: next generation sequencing.

Arabidopsis DNA polymerase lambda mutant is mildly sensitive to DNA double strand breaks but defective in integration of a transgene

PubMed Central

Furukawa, Tomoyuki; Angelis, Karel J.; Britt, Anne B.

2015-01-01

The DNA double-strand break (DSB) is a critical type of damage, and can be induced by both endogenous sources (e.g., errors of oxidative metabolism, transposable elements, programmed meiotic breaks, or perturbation of the DNA replication fork) and exogenous sources (e.g., ionizing radiation or radiomimetic chemicals). Although higher plants, like mammals, are thought to preferentially repair DSBs via nonhomologous end joining (NHEJ), much remains unclear about plant DSB repair pathways. Our reverse genetic approach suggests that DNA polymerase λ is involved in DSB repair in Arabidopsis. The Arabidopsis T-DNA insertion mutant (atpolλ-1) displayed sensitivity to both gamma-irradiation and treatment with radiomimetic reagents, but not to other DNA damaging treatments. The atpolλ-1 mutant showed a moderate sensitivity to DSBs, while Arabidopsis Ku70 and DNA ligase 4 mutants (atku70-3 and atlig4-2), both of which play critical roles in NHEJ, exhibited a hypersensitivity to these treatments. The atpolλ-1/atlig4-2 double mutant exhibited a higher sensitivity to DSBs than each single mutant, but the atku70/atpolλ-1 showed similar sensitivity to the atku70-3 mutant. We showed that transcription of the DNA ligase 1, DNA ligase 6, and Wee1 genes was quickly induced by BLM in several NHEJ deficient mutants in contrast to wild-type. Finally, the T-DNA transformation efficiency dropped in NHEJ deficient mutants and the lowest transformation efficiency was scored in the atpolλ-1/atlig4-2 double mutant. These results imply that AtPolλ is involved in both DSB repair and DNA damage response pathway. PMID:26074930

Dissecting the Role of CHITINASE-LIKE1 in Nitrate-Dependent Changes in Root Architecture1[C][W

PubMed Central

Hermans, Christian; Porco, Silvana; Vandenbussche, Filip; Gille, Sascha; De Pessemier, Jérôme; Van Der Straeten, Dominique; Verbruggen, Nathalie; Bush, Daniel R.

2011-01-01

The root phenotype of an Arabidopsis (Arabidopsis thaliana) mutant of CHITINASE-LIKE1 (CTL1), called arm (for anion-related root morphology), was previously shown to be conditional on growth on high nitrate, chloride, or sucrose. Mutants grown under restrictive conditions displayed inhibition of primary root growth, radial swelling, proliferation of lateral roots, and increased root hair density. We found here that the spatial pattern of CTL1 expression was mainly in the root and root tips during seedling development and that the protein localized to the cell wall. Fourier-transform infrared microspectroscopy of mutant root tissues indicated differences in spectra assigned to linkages in cellulose and pectin. Indeed, root cell wall polymer composition analysis revealed that the arm mutant contained less crystalline cellulose and reduced methylesterification of pectins. We also explored the implication of growth regulators on the phenotype of the mutant response to the nitrate supply. Exogenous abscisic acid application inhibited more drastically primary root growth in the arm mutant but failed to repress lateral branching compared with the wild type. Cytokinin levels were higher in the arm root, but there were no changes in mitotic activity, suggesting that cytokinin is not directly involved in the mutant phenotype. Ethylene production was higher in arm but inversely proportional to the nitrate concentration in the medium. Interestingly, eto2 and eto3 ethylene overproduction mutants mimicked some of the conditional root characteristics of the arm mutant on high nitrate. Our data suggest that ethylene may be involved in the arm mutant phenotype, albeit indirectly, rather than functioning as a primary signal. PMID:21949212

Characterization of CpSte11, a MAPKKK gene of Cryphonectria parasitica, and initial evidence of its involvement in the pheromone response pathway.

PubMed

Park, Jin-Ah; Kim, Jung-Mi; Park, Seung-Moon; Kim, Dae-Hyuk

2012-04-01

The gene CpSte11 of Cryphonectria parasitica, which encodes a yeast Ste11 homologue, was cloned and characterized. Gene replacement analysis revealed a high frequency of CpSte11 null mutants. When compared with the wild-type parent strain, CpSte11 null mutants showed no difference in terms of growth rate or pigmentation. However, CpSte11 null mutants showed a marked decrease in both the number and size of stromal pustules on chestnut twigs. The virulence test showed that, in comparison with those of the wild-type and virus-infected hypovirulent strains, CpSte11 null mutants produced necrotic areas of intermediate size. Disruption of the CpSte11 gene also resulted in defects in female fertility. Down-regulation of transcripts for the mating pheromone precursor gene, Mf2/2, and mating response transcription factors, such as cpst12 and pro1, was observed in CpSte11 null mutants. The down-regulation of Mf2/2, cpst12 and pro1 was also observed in the mutant phenotype of Cpmk2, a mating response Fus3-like mitogen-activated protein kinase (MAPK) gene, but not in the mutant of Cpmk1, a high-osmolarity glycerol Hog1-like MAPK gene. These results indicate that the cloned CpSte11 gene is functionally involved in the mating response pathway and acts through downstream targets, including Cpmk2, cpst12, pro1 and Mf2/2. However, the characteristics of the CpSte11 null mutant were fully phenocopied only in the cpst12 null mutant, but not in other studied null mutants of components of the putative mating response pathway. © 2011 THE AUTHORS. MOLECULAR PLANT PATHOLOGY © 2011 BSPP AND BLACKWELL PUBLISHING LTD.

The mitochondrial cytochrome c peroxidase Ccp1 of Saccharomyces cerevisiae is involved in conveying an oxidative stress signal to the transcription factor Pos9 (Skn7).

PubMed

Charizanis, C; Juhnke, H; Krems, B; Entian, K D

1999-10-01

In Saccharomyces cerevisiae two transcription factors, Pos9 (Skn7) and Yap1, are involved in the response to oxidative stress. Fusion of the Pos9 response-regulator domain to the Gal4 DNA-binding domain results in a transcription factor which renders the expression of a GAL1-lacZ reporter gene dependent on oxidative stress. To identify genes which are involved in the oxygen-dependent activation of the Gal4-Pos9 hybrid protein we screened for mutants that failed to induce the heterologous test system upon oxidative stress (fap mutants for factors activating Pos9). We isolated several respiration-deficient and some respiration-competent mutants by this means. We selected for further characterization only those mutants which also displayed an oxidative-stress-sensitive phenotype. One of the respiration-deficient mutants (complementation groupfap6) could be complemented by the ISM1 gene, which encodes mitochondrial isoleucyl tRNA synthetase, suggesting that respiration competence was important for signalling of oxidative stress. In accordance with this notion a rho0 strain and a wild-type strain in which respiration had been blocked (by treatment with antimycin A or with cyanide) also failed to activate Gal4-Pos9 upon imposition of oxidative stress. Another mutant, fap24, which was respiration-competent, could be complemented by CCP1, which encodes the mitochondrial cytochrome c peroxidase. Mitochondrial cytochrome c peroxidase degrades reactive oxygen species within the mitochondria. This suggested a possible sensor function for the enzyme in the oxidative stress response. To test this we used the previously described point mutant ccp1 W191F, which is characterized by a 10(4)-fold decrease in electron flux between cytochrome c and cytochrome c peroxidase. The Ccp1W191F mutant was still capable of activating the Pos9 transcriptional activation domain, suggesting that the signalling function of Ccp1 is independent of electron flux rates.

Genes Associated with Desiccation and Osmotic Stress in Listeria monocytogenes as Revealed by Insertional Mutagenesis.

PubMed

Hingston, Patricia A; Piercey, Marta J; Truelstrup Hansen, Lisbeth

2015-08-15

Listeria monocytogenes is a foodborne pathogen whose survival in food processing environments may be associated with its tolerance to desiccation. To probe the molecular mechanisms used by this bacterium to adapt to desiccation stress, a transposon library of 11,700 L. monocytogenes mutants was screened, using a microplate assay, for strains displaying increased or decreased desiccation survival (43% relative humidity, 15°C) in tryptic soy broth (TSB). The desiccation phenotypes of selected mutants were subsequently assessed on food-grade stainless steel (SS) coupons in TSB plus 1% glucose (TSB-glu). Single transposon insertions in mutants exhibiting a change in desiccation survival of >0.5 log CFU/cm(2) relative to that of the wild type were determined by sequencing arbitrary PCR products. Strain morphology, motility, and osmotic stress survival (in TSB-glu plus 20% NaCl) were also analyzed. The initial screen selected 129 desiccation-sensitive (DS) and 61 desiccation-tolerant (DT) mutants, out of which secondary screening on SS confirmed 15 DT and 15 DS mutants. Among the DT mutants, seven immotile and flagellum-less strains contained transposons in genes involved in flagellum biosynthesis (fliP, flhB, flgD, flgL) and motor control (motB, fliM, fliY), while others harbored transposons in genes involved in membrane lipid biosynthesis, energy production, potassium uptake, and virulence. The genes that were interrupted in the 15 DS mutants included those involved in energy production, membrane transport, protein metabolism, lipid biosynthesis, oxidative damage control, and putative virulence. Five DT and 14 DS mutants also demonstrated similar significantly (P < 0.05) different survival relative to that of the wild type when exposed to osmotic stress, demonstrating that some genes likely have similar roles in allowing the organism to survive the two water stresses. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

A CheR/CheB fusion protein is involved in cyst cell development and chemotaxis in Azospirillum brasilense Sp7.

PubMed

Wu, Lixian; Cui, Yanhua; Hong, Yuanyuan; Chen, Sanfeng

2011-12-20

We here report the sequence and functional analysis of cstB of Azospirillum brasilense Sp7. The predicted cstB contains C-terminal two PAS domains and N-terminal part which has similarity with CheB-CheR fusion protein. cstB mutants had reduced swarming ability compared to that of A. brasilense wild-type strain, implying that cstB was involved in chemotaxis in A. brasilense. A microscopic analysis revealed that cstB mutants developed mature cyst cells more quickly than wild type, indicating that cstB is involved in cyst formation. cstB mutants were affected in colony morphology and the production of exopolysaccharides (EPS) which are essential for A. brasilense cells to differentiate into cyst-like forms. These observations suggested that cstB was a multi-effector involved in cyst development and chemotaxis in A. brasilense. Copyright © 2010 Elsevier GmbH. All rights reserved.

Gravity persistent signal 1 reveals a novel cytochrome P450 involved in gravitropic signal transduction

NASA Astrophysics Data System (ADS)

Wyatt, Sarah

Understanding gene expression that occurs during gravitopism is important for studying the processes that link the perception of gravity to the growth response. Arabidopsis plants with a mutation in the GRAVITY PERSISTENT SIGNAL (GPS)1 locus show a "no response" phenotype during gravistimulation experiments. Basepital auxin transport in gps1 mutant was unaffected by the mutation, but auxin was not laterally redistributed after gravistimulation. GPS1 encodes CYP705A22, a cytochrome P450 protein (P450) of unknown function. The wild type CYP705A22 gene was transformed into the gps1 mutant background and successfully rescued the mutant phenotype. Data mining of microarray data collected from gravistimulated root tips of Arabidopsis indicated that although CYP705A22 was not expressed in roots, a family member CYP705A5 was up-regulated within 3 minutes after gravistimulation. Expression profiling of CYP705A5, using real-time quantitative PCR, showed that CYP705A5 was up-regulated nearly five fold within minutes of gravity stimulation. And reporter gene fusions that link the CYP705A5 gene to the green fluorescent protein showed that CYP705A5 was expressed in the root zones of elongation and maturation. Computer modeling of the catalytic domain of CYP705A22 and CYP705A5 and in silico substrate docking simulations generated a list of 130 compounds that are potential substrates of the P450s. Many of the compounds are phenylpropanoid derivatives. Heterologous expression of CYP705A5 in baculovirus and Type 1 binding studies indicate the substrate of the P450 may be quercitin or myricetin. A mutation affecting CYP705A5 expression resulted in a delayed gravity response in roots. The mutant phenotype could be chemically complemented, and DPBA staining in the CYP705A5 mutant indicated a 1.5 fold accumulation of quercetin in mutant roots as compared to WT. These data, taken together, may indicate that we have identified a flavonoid pathway that regulates auxin distribution and thus is involved in gravitropic signal transduction. (Partially support by NSF: 0618506 to SEW)

The role of mitochondria in carbon catabolite repression in yeast.

PubMed

Haussmann, P; Zimmermann, F K

1976-10-18

The role of mitochondria in carbon catabolite repression in Saccharomyces cerevisiae was investigated by comparing normal, respiratory competent (RHO) strains with their mitochondrially inherited, respiratory deficient mutant derivatives (rho). Formation of maltase and invertase was used as an indicator system for the effect of carbon catabolite repression on carbon catabolic reactions. Fermentation rates for glucose, maltose and sucrose were the same in RHO and rho strains. Specific activities of maltase and invertase were usually higher in the rho-mutants. A very pronounced difference in invertase levels was observed when cells were grown on maltose; rho-mutants had around 30 times more invertase than their RHO parent strains. The fact that rho-mutants were much less sensitive to carbon catabolite repression of invertase synthesis than their RHO parents was used to search for the mitochondrial factor(s) or function(s) involved in carbon catabolite repression. A possible metabolic influence of mitochondria on this system of regulation was tested after growth of RHO strains under anaerobic conditions (no respiration nor oxidative phosphorylation), in the presence of KCN (respiration inhibited), dinitrophenol (uncoupling of oxidative phosphorylation) and of both inhibitors anaerobic conditions and dinitrophenol had no effect on the extent of invertase repression. KCN reduced the degree of repression but not to the level found in rho-mutants. A combination of both inhibitors gave the same results as with KCN alone. Erythromycin and chloramphenicol were used as specific inhibitors of mitochondrial protein synthesis. Erythromycin prevented the formation of mitochondrial respiratory systems but did not induce rho-mutants under the conditions used. However, repression of invertase was as strong as in the absence of the inhibitor. Chloramphenicol led only to a slight reduction of the respiratory systems and did not affect invertase levels. A combination of both antibiotics had about the same effect as growth in the presence of KCN. The results showed that mitochondria are involved in carbon catabolite repression and they cause an increase in the degree of repression. These effects cannot be due to mere metabolic activities nor to factors made on the mitochondrial protein synthesizing machinery. This regulatory role of mitochondria is observed as long as an intact mitochondrial genome is maintained.

Mutation design of a thermophilic Rubisco based on three-dimensional structure enhances its activity at ambient temperature.

PubMed

Fujihashi, Masahiro; Nishitani, Yuichi; Kiriyama, Tomohiro; Aono, Riku; Sato, Takaaki; Takai, Tomoyuki; Tagashira, Kenta; Fukuda, Wakao; Atomi, Haruyuki; Imanaka, Tadayuki; Miki, Kunio

2016-10-01

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) plays a central role in carbon dioxide fixation on our planet. Rubisco from a hyperthermophilic archaeon Thermococcus kodakarensis (Tk-Rubisco) shows approximately twenty times the activity of spinach Rubisco at high temperature, but only one-eighth the activity at ambient temperature. We have tried to improve the activity of Tk-Rubisco at ambient temperature, and have successfully constructed several mutants which showed higher activities than the wild-type enzyme both in vitro and in vivo. Here, we designed new Tk-Rubisco mutants based on its three-dimensional structure and a sequence comparison of thermophilic and mesophilic plant Rubiscos. Four mutations were introduced to generate new mutants based on this strategy, and one of the four mutants, T289D, showed significantly improved activity compared to that of the wild-type enzyme. The crystal structure of the Tk-Rubisco T289D mutant suggested that the increase in activity was due to mechanisms distinct from those involved in the improvement in activity of Tk-Rubisco SP8, a mutant protein previously reported to show the highest activity at ambient temperature. Combining the mutations of T289D and SP8 successfully generated a mutant protein (SP8-T289D) with the highest activity to date both in vitro and in vivo. The improvement was particularly pronounced for the in vivo activity of SP8-T289D when introduced into the mesophilic, photosynthetic bacterium Rhodopseudomonas palustris, which resulted in a strain with nearly two-fold higher specific growth rates compared to that of a strain harboring the wild-type enzyme at ambient temperature. Proteins 2016; 84:1339-1346. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Acidithiobacillus caldus Sulfur Oxidation Model Based on Transcriptome Analysis between the Wild Type and Sulfur Oxygenase Reductase Defective Mutant

PubMed Central

Chen, Linxu; Ren, Yilin; Lin, Jianqun; Liu, Xiangmei; Pang, Xin; Lin, Jianqiang

2012-01-01

Background Acidithiobacillus caldus (A. caldus) is widely used in bio-leaching. It gains energy and electrons from oxidation of elemental sulfur and reduced inorganic sulfur compounds (RISCs) for carbon dioxide fixation and growth. Genomic analyses suggest that its sulfur oxidation system involves a truncated sulfur oxidation (Sox) system (omitting SoxCD), non-Sox sulfur oxidation system similar to the sulfur oxidation in A. ferrooxidans, and sulfur oxygenase reductase (SOR). The complexity of the sulfur oxidation system of A. caldus generates a big obstacle on the research of its sulfur oxidation mechanism. However, the development of genetic manipulation method for A. caldus in recent years provides powerful tools for constructing genetic mutants to study the sulfur oxidation system. Results An A. caldus mutant lacking the sulfur oxygenase reductase gene (sor) was created and its growth abilities were measured in media using elemental sulfur (S0) and tetrathionate (K2S4O6) as the substrates, respectively. Then, comparative transcriptome analysis (microarrays and real-time quantitative PCR) of the wild type and the Δsor mutant in S0 and K2S4O6 media were employed to detect the differentially expressed genes involved in sulfur oxidation. SOR was concluded to oxidize the cytoplasmic elemental sulfur, but could not couple the sulfur oxidation with the electron transfer chain or substrate-level phosphorylation. Other elemental sulfur oxidation pathways including sulfur diooxygenase (SDO) and heterodisulfide reductase (HDR), the truncated Sox pathway, and the S4I pathway for hydrolysis of tetrathionate and oxidation of thiosulfate in A. caldus are proposed according to expression patterns of sulfur oxidation genes and growth abilities of the wild type and the mutant in different substrates media. Conclusion An integrated sulfur oxidation model with various sulfur oxidation pathways of A. caldus is proposed and the features of this model are summarized. PMID:22984393

Identification of a two-component signal transduction system involved in fimbriation of Porphyromonas gingivalis.

PubMed

Hayashi, J; Nishikawa, K; Hirano, R; Noguchi, T; Yoshimura, F

2000-01-01

Porphyromonas gingivalis, a periodontopathogen, is an oral anaerobic gram-negative bacterium with numerous fimbriae on the cell surface. Fimbriae have been considered to be an important virulence factor in this organism. We analyzed the genomic DNA of transposon-induced, fimbria-deficient mutants derived from ATCC 33277 and found that seven independent mutants had transposon insertions within the same restriction fragment. Cloning and sequencing of the disrupted region from one of the mutants revealed two adjacent open reading frames (ORFs) which seemed to encode a two-component signal transduction system. We also found that six of the mutants had insertions in a gene, fimS, a homologue of the genes encoding sensor kinase, and that the insertion in the remaining one disrupted the gene immediately downstream, fimR, a homologue of the response regulator genes in other bacteria. These findings suggest that this two-component regulatory system is involved in fimbriation of P. gingivalis.

Genes and signaling pathways involved in memory enhancement in mutant mice

PubMed Central

2014-01-01

Mutant mice have been used successfully as a tool for investigating the mechanisms of memory at multiple levels, from genes to behavior. In most cases, manipulating a gene expressed in the brain impairs cognitive functions such as memory and their underlying cellular mechanisms, including synaptic plasticity. However, a remarkable number of mutations have been shown to enhance memory in mice. Understanding how to improve a system provides valuable insights into how the system works under normal conditions, because this involves understanding what the crucial components are. Therefore, more can be learned about the basic mechanisms of memory by studying mutant mice with enhanced memory. This review will summarize the genes and signaling pathways that are altered in the mutants with enhanced memory, as well as their roles in synaptic plasticity. Finally, I will discuss how knowledge of memory-enhancing mechanisms could be used to develop treatments for cognitive disorders associated with impaired plasticity. PMID:24894914

Evidence for ACD5 ceramide kinase activity involvement in Arabidopsis response to cold stress.

PubMed

Dutilleul, Christelle; Chavarria, Heidy; Rézé, Nathalie; Sotta, Bruno; Baudouin, Emmanuel; Guillas, Isabelle

2015-12-01

Although sphingolipids emerged as important signals for plant response to low temperature, investigations have been limited so far to the function of long-chain base intermediates. The formation and function of ceramide phosphates (Cer-Ps) in chilled Arabidopsis were explored. Cer-Ps were analysed by thin layer chromatography (TLC) following in vivo metabolic radiolabelling. Ceramide kinase activity, gene expression and growth phenotype were determined in unstressed and cold-stressed wild type (WT) and Arabidopsis ceramide kinase mutant acd5. A rapid and transient formation of Cer-P occurs in cold-stressed WT Arabidopsis plantlets and cultured cells, which is strongly impaired in acd5 mutant. Although concomitant, Cer-P formation is independent of long-chain base phosphate (LCB-P) formation. No variation of ceramide kinase activity was measured in vitro in WT plantlets upon cold stress but the activity in acd5 mutant was further reduced by cold stress. At the seedling stage, acd5 response to cold was similar to that of WT. Nevertheless, acd5 seed germination was hypersensitive to cold and abscisic acid (ABA), and ABA-dependent gene expression was modified in acd5 seeds when germinated at low temperature. Our data involve for the first time Cer-P and ACD5 in low temperature response and further underline the complexity of sphingolipid signalling operating during cold stress. © 2015 John Wiley & Sons Ltd.

Interleukin-6 is an essential determinant of on-time parturition in the mouse.

PubMed

Robertson, Sarah A; Christiaens, Inge; Dorian, Camilla L; Zaragoza, Dean B; Care, Alison S; Banks, Anke M; Olson, David M

2010-08-01

IL-6 abundance in amniotic fluid and uterine tissues increases in late gestation or with infection-associated preterm labor. A role in regulation of labor onset is suggested by observations that IL-6 increases expression of genes controlling prostaglandin synthesis and signaling in isolated uterine cells, but whether IL-6 is essential for normal parturition is unknown. To evaluate the physiological role of IL-6 in parturition in mice, we investigated the effect of Il6 null mutation on the timing of parturition and expression of genes associated with uterine activation. Il6 null mutant mice delivered 24 h later than wild-type mice, although circulating progesterone fell similarly in both genotypes during the prepartal period. Il6 null mutant mice were also refractory to low doses of lipopolysaccharide sufficient to induce preterm delivery in wild-type mice. The characteristic late-gestation elevation in uterine expression of Oxtr mRNA encoding oxytocin receptor, and peripartal increases in Ptgfr and Ptgs2 mRNAs regulating prostaglandin synthesis and signaling were delayed by 24 h in Il6 null mutant mice. Conversely, Ptger4 mRNA encoding the prostaglandin E receptor-4 was abnormally elevated in late-gestation in Il6 null mutant mice. Administration of recombinant IL-6 from d 11.5 postcoitum until term restored the normal timing of delivery and normalized Ptger4 mRNA expression in late gestation. We conclude that IL-6 has a key role in controlling the progression of events culminating in parturition and that it acts downstream of luteolysis in the uterus to regulate genes involved in the prostaglandin-mediated uterine activation cascade.

A Cold-Inducible DEAD-Box RNA Helicase from Arabidopsis thaliana Regulates Plant Growth and Development under Low Temperature.

PubMed

Liu, Yuelin; Tabata, Daisuke; Imai, Ryozo

2016-01-01

DEAD-box RNA helicases comprise a large family and are involved in a range of RNA processing events. Here, we identified one of the Arabidopsis thaliana DEAD-box RNA helicases, AtRH7, as an interactor of Arabidopsis COLD SHOCK DOMAIN PROTEIN 3 (AtCSP3), which is an RNA chaperone involved in cold adaptation. Promoter:GUS transgenic plants revealed that AtRH7 is expressed ubiquitously and that its levels of the expression are higher in rapidly growing tissues. Knockout mutant lines displayed several morphological alterations such as disturbed vein pattern, pointed first true leaves, and short roots, which resemble ribosome-related mutants of Arabidopsis. In addition, aberrant floral development was also observed in rh7 mutants. When the mutants were germinated at low temperature (12°C), both radicle and first leaf emergence were severely delayed; after exposure of seedlings to a long period of cold, the mutants developed aberrant, fewer, and smaller leaves. RNA blots and circular RT-PCR revealed that 35S and 18S rRNA precursors accumulated to higher levels in the mutants than in WT under both normal and cold conditions, suggesting the mutants are partially impaired in pre-rRNA processing. Taken together, the results suggest that AtRH7 affects rRNA biogenesis and plays an important role in plant growth under cold.

Disruption of LACCASE4 and 17 Results in Tissue-Specific Alterations to Lignification of Arabidopsis thaliana Stems[W

PubMed Central

Berthet, Serge; Demont-Caulet, Nathalie; Pollet, Brigitte; Bidzinski, Przemyslaw; Cézard, Laurent; Le Bris, Phillipe; Borrega, Nero; Hervé, Jonathan; Blondet, Eddy; Balzergue, Sandrine; Lapierre, Catherine; Jouanin, Lise

2011-01-01

Peroxidases have been shown to be involved in the polymerization of lignin precursors, but it remains unclear whether laccases (EC 1.10.3.2) participate in constitutive lignification. We addressed this issue by studying laccase T-DNA insertion mutants in Arabidopsis thaliana. We identified two genes, LAC4 and LAC17, which are strongly expressed in stems. LAC17 was mainly expressed in the interfascicular fibers, whereas LAC4 was expressed in vascular bundles and interfascicular fibers. We produced two double mutants by crossing the LAC17 (lac17) mutant with two LAC4 mutants (lac4-1 and lac4-2). The single and double mutants grew normally in greenhouse conditions. The single mutants had moderately low lignin levels, whereas the stems of lac4-1 lac17 and lac4-2 lac17 mutants had lignin contents that were 20 and 40% lower than those of the control, respectively. These lower lignin levels resulted in higher saccharification yields. Thioacidolysis revealed that disrupting LAC17 principally affected the deposition of G lignin units in the interfascicular fibers and that complementation of lac17 with LAC17 restored a normal lignin profile. This study provides evidence that both LAC4 and LAC17 contribute to the constitutive lignification of Arabidopsis stems and that LAC17 is involved in the deposition of G lignin units in fibers. PMID:21447792

Gr and hp-1 tomato mutants unveil unprecedented interactions between arbuscular mycorrhizal symbiosis and fruit ripening.

PubMed

Chialva, Matteo; Zouari, Inès; Salvioli, Alessandra; Novero, Mara; Vrebalov, Julia; Giovannoni, James J; Bonfante, Paola

2016-07-01

Systemic responses to an arbuscular mycorrhizal fungus reveal opposite phenological patterns in two tomato ripening mutants depending whether ethylene or light reception is involved. The availability of tomato ripening mutants has revealed many aspects of the genetics behind fleshy fruit ripening, plant hormones and light signal reception. Since previous analyses revealed that arbuscular mycorrhizal symbiosis influences tomato berry ripening, we wanted to test the hypothesis that an interplay might occur between root symbiosis and fruit ripening. With this aim, we screened seven tomato mutants affected in the ripening process for their responsiveness to the arbuscular mycorrhizal fungus Funneliformis mosseae. Following their phenological responses we selected two mutants for a deeper analysis: Green ripe (Gr), deficient in fruit ethylene perception and high-pigment-1 (hp-1), displaying enhanced light signal perception throughout the plant. We investigated the putative interactions between ripening processes, mycorrhizal establishment and systemic effects using biochemical and gene expression tools. Our experiments showed that both mutants, notwithstanding a normal mycorrhizal phenotype at root level, exhibit altered arbuscule functionality. Furthermore, in contrast to wild type, mycorrhization did not lead to a higher phosphate concentration in berries of both mutants. These results suggest that the mutations considered interfere with arbuscular mycorrhiza inducing systemic changes in plant phenology and fruits metabolism. We hypothesize a cross talk mechanism between AM and ripening processes that involves genes related to ethylene and light signaling.

Genetic Screening Identifies Cyanogenesis-Deficient Mutants of Lotus japonicus and Reveals Enzymatic Specificity in Hydroxynitrile Glucoside Metabolism[W][OA

PubMed Central

Takos, Adam; Lai, Daniela; Mikkelsen, Lisbeth; Abou Hachem, Maher; Shelton, Dale; Motawia, Mohammed Saddik; Olsen, Carl Erik; Wang, Trevor L.; Martin, Cathie; Rook, Fred

2010-01-01

Cyanogenesis, the release of hydrogen cyanide from damaged plant tissues, involves the enzymatic degradation of amino acid–derived cyanogenic glucosides (α-hydroxynitrile glucosides) by specific β-glucosidases. Release of cyanide functions as a defense mechanism against generalist herbivores. We developed a high-throughput screening method and used it to identify cyanogenesis deficient (cyd) mutants in the model legume Lotus japonicus. Mutants in both biosynthesis and catabolism of cyanogenic glucosides were isolated and classified following metabolic profiling of cyanogenic glucoside content. L. japonicus produces two cyanogenic glucosides: linamarin (derived from Val) and lotaustralin (derived from Ile). Their biosynthesis may involve the same set of enzymes for both amino acid precursors. However, in one class of mutants, accumulation of lotaustralin and linamarin was uncoupled. Catabolic mutants could be placed in two complementation groups, one of which, cyd2, encoded the β-glucosidase BGD2. Despite the identification of nine independent cyd2 alleles, no mutants involving the gene encoding a closely related β-glucosidase, BGD4, were identified. This indicated that BGD4 plays no role in cyanogenesis in L. japonicus in vivo. Biochemical analysis confirmed that BGD4 cannot hydrolyze linamarin or lotaustralin and in L. japonicus is specific for breakdown of related hydroxynitrile glucosides, such as rhodiocyanoside A. By contrast, BGD2 can hydrolyze both cyanogenic glucosides and rhodiocyanosides. Our genetic analysis demonstrated specificity in the catabolic pathways for hydroxynitrile glucosides and implied specificity in their biosynthetic pathways as well. In addition, it has provided important tools for elucidating and potentially modifying cyanogenesis pathways in plants. PMID:20453117

The ntrB and ntrC Genes Are Involved in the Regulation of Poly-3-Hydroxybutyrate Biosynthesis by Ammonia in Azospirillum brasilense Sp7

PubMed Central

Sun, Jun; Peng, Xuan; Van Impe, Jan; Vanderleyden, Jos

2000-01-01

Azospirillum brasilense Sp7 and its ntrA (rpoN), ntrBC, and ntrC mutants have been evaluated for their capabilities of poly-3-hydroxybutyrate (PHB) accumulation in media with high and low ammonia concentrations. It was observed that the ntrBC and ntrC mutants can produce PHB in both low- and high-C/N-ratio media, while no significant PHB production was observed for the wild type or the ntrA mutant in low-C/N-ratio media. Further investigation by fermentation analysis indicated that the ntrBC and ntrC mutants were able to grow and accumulate PHB simultaneously in the presence of a high concentration of ammonia in the medium, while little PHB was produced in the wild type and ntrA (rpoN) mutant during active growth phase. These results provide the first genetic evidence that the ntrB and ntrC genes are involved in the regulation of PHB synthesis by ammonia in A. brasilense Sp7. PMID:10618211

Retinoblastoma protein (pRB) was significantly phosphorylated through a Ras-to-MAPK pathway in mutant K-ras stably transfected human adrenocortical cells.

PubMed

Chen, Y-F; Chiu, H-H; Wu, C-H; Wang, J-Y; Chen, F-M; Tzou, W-H; Shin, S-J; Lin, S-R

2003-10-01

Our previous studies have shown that the cell proliferation rate, mRNA levels of p450scc, p450c17, and 3betaHSD, and secretion of cortisol were significantly increased in human adrenocortical cells stably transfected with mutated K-ras expression plasmid "pK568MRSV" after being inducted with IPTG. In addition, the increased level was a time-dependent manner. However, the levels of p450, p450scc, p450c17, 3betaHSD, cortisol, and cell proliferation rate were inhibited by a MEK phospholation inhibitor, PD098059. The above results prove that mutated K-ras oncogene is able to regulate tumorigenesis and steroidogenesis through a Ras-RAF-MEK-MAPK signal transduction pathway. The aim of this study was to investigate regulated factors in this pathway and also examine whether the other signal transduction pathways or other moles involved in tumorigenesis or steroidogenesis. In the first year, we analyzed gene profiles of mutant K-ras-transfected adrenocortical cells by DNA microarray to determine the gene expression related to cell cycle, signal transduction, apoptosis, tumorigenesis, steroidogenesis, and other expressed sequence tag. After being affected by the K-ras mutant, gene expression was significantly increased in some upregulated genes. Human zinc-finger protein 22 increased by 28.5 times, Osteopontin increased by 5.8 times, LIM domain Kinase 2 (LIMK2) increased by 3.3 times, Homo sapiens dual-specificity tyrosine-(Y)-phosphorylation regulated Kinase 2 (DYRK2) increased by 2.2 times, and human syntaxin 3 increased by two times. On the other hand, significant decreases in gene expression were also observed in some downregulated genes. Retinoblastoma binding protein 1 (RBBP1) decreased by four times, Homo sapiens craniofacial development protein 1 (CFDP1) decreased by 2.4 times, DAP Kinase-related apoptosis-inducing protein Kinase 1 (DRAK1) decreased by 2.3 times, SKI-interacting protein (SKIP) decreased by 2.2 times, and human poly(A)-Binding protein (PABP) decreased by 2.1 times. In all significant differentially expressed genes, preliminary analysis by bioinformatics revealed that after induced K-ras mutant expression by isopropyl thiogalctoside (IPTG), the downregulation of RBBP1 gene was most correlated to cell proliferation. RBBP1 can bind with RB/E2F to form a mSIN3-HDAC complex, which induces cell cycle arrest in the G1/G0 stage by repressing transcription of E2F-regulated genes. The result of a Northern blot showed that RBBP1 were inhibited after an induction of IPTG for 36 h. Another Northern blot analysis proved that mRNA levels of cyclin D1 and c-myc increased in proportion to K-ras expression. Finally, Western blot was carried out, and the results showed that phosphorylated pRB also increased. Taken together, we infer that the mutant K-ras oncogene promoted the cells to proceed to the G1/S stage by the inhibiting the formation of RB/RBBP1-dependent repressor complex from binding with the SIN3-HDAC complex, which resulted in the acetylation of histone to active transcription of E2F-regulated genes. However, the roles of the other differentially expressed genes involved in cell proliferation, cell morphologic change, tumorigenesis, or steroidogenesis still need further investigation.

Transcript profiling by microarray and marker analysis of the short cotton (Gossypium hirsutum L.) fiber mutant Ligon lintless-1 (Li1).

PubMed

Gilbert, Matthew K; Turley, Rickie B; Kim, Hee Jin; Li, Ping; Thyssen, Gregory; Tang, Yuhong; Delhom, Christopher D; Naoumkina, Marina; Fang, David D

2013-06-17

Cotton fiber length is very important to the quality of textiles. Understanding the genetics and physiology of cotton fiber elongation can provide valuable tools to the cotton industry by targeting genes or other molecules responsible for fiber elongation. Ligon Lintless-1 (Li1) is a monogenic mutant in Upland cotton (Gossypium hirsutum) which exhibits an early cessation of fiber elongation resulting in very short fibers (< 6 mm) at maturity. This presents an excellent model system for studying the underlying molecular and cellular processes involved with cotton fiber elongation. Previous reports have characterized Li1 at early cell wall elongation and during later secondary cell wall synthesis, however there has been very limited analysis of the transition period between these developmental time points. Physical and morphological measurements of the Li1 mutant fibers were conducted, including measurement of the cellulose content during development. Affymetrix microarrays were used to analyze transcript profiles at the critical developmental time points of 3 days post anthesis (DPA), the late elongation stage of 12 DPA and the early secondary cell wall synthesis stage of 16 DPA. The results indicated severe disruption to key hormonal and other pathways related to fiber development, especially pertaining to the transition stage from elongation to secondary cell wall synthesis. Gene Ontology enrichment analysis identified several key pathways at the transition stage that exhibited altered regulation. Genes involved in ethylene biosynthesis and primary cell wall rearrangement were affected, and a primary cell wall-related cellulose synthase was transcriptionally repressed. Linkage mapping using a population of 2,553 F2 individuals identified SSR markers associated with the Li1 genetic locus on chromosome 22. Linkage mapping in combination with utilizing the diploid G. raimondii genome sequences permitted additional analysis of the region containing the Li1 gene. The early termination of fiber elongation in the Li1 mutant is likely controlled by an early upstream regulatory factor resulting in the altered regulation of hundreds of downstream genes. Several elongation-related genes that exhibited altered expression profiles in the Li1 mutant were identified. Molecular markers closely associated with the Li1 locus were developed. Results presented here will lay the foundation for further investigation of the genetic and molecular mechanisms of fiber elongation.

Gene expression analyses in tomato near isogenic lines provide evidence for ethylene and abscisic acid biosynthesis fine-tuning during arbuscular mycorrhiza development.

PubMed

Fracetto, Giselle Gomes Monteiro; Peres, Lázaro Eustáquio Pereira; Lambais, Marcio Rodrigues

2017-07-01

Plant responses to the environment and microorganisms, including arbuscular mycorrhizal fungi, involve complex hormonal interactions. It is known that abscisic acid (ABA) and ethylene may be involved in the regulation of arbuscular mycorrhiza (AM) and that part of the detrimental effects of ABA deficiency in plants is due to ethylene overproduction. In this study, we aimed to determine whether the low susceptibility to mycorrhizal colonization in ABA-deficient mutants is due to high levels of ethylene and whether AM development is associated with changes in the steady-state levels of transcripts of genes involved in the biosynthesis of ethylene and ABA. For that, tomato (Solanum lycopersicum) ethylene overproducer epinastic (epi) mutant and the ABA-deficient notabilis (not) and sitiens (sit) mutants, in the same Micro-Tom (MT) genetic background, were inoculated with Rhizophagus clarus, and treated with the ethylene biosynthesis inhibitor aminoethoxyvinylglycine (AVG). The development of AM, as well as the steady-state levels of transcripts involved in ethylene (LeACS2, LeACO1 and LeACO4) and ABA (LeNCED) biosynthesis, was determined. The intraradical colonization in epi, not and sit mutants was significantly reduced compared to MT. The epi mutant completely restored the mycorrhizal colonization to the levels of MT with the application of 10 µM of AVG, probably due to the inhibition of the ACC synthase gene expression. The steady-state levels of LeACS2 and LeACO4 transcripts were induced in mycorrhizal roots of MT, whereas the steady-state levels of LeACO1 and LeACO4 transcripts were significantly induced in sit, and the steady-state levels of LeNCED transcripts were significantly induced in all genotypes and in mycorrhizal roots of epi mutants treated with AVG. The reduced mycorrhizal colonization in sit mutants seems not to be limited by ethylene production via ACC oxidase regulation. Both ethylene overproduction and ABA deficiency impaired AM fungal colonization in tomato roots, indicating that, besides hormonal interactions, a fine-tuning of each hormone level is required for AM development.

Bacterial cell motility of Burkholderia gut symbiont is required to colonize the insect gut.

PubMed

Lee, Jun Beom; Byeon, Jin Hee; Jang, Ho Am; Kim, Jiyeun Kate; Yoo, Jin Wook; Kikuchi, Yoshitomo; Lee, Bok Luel

2015-09-14

We generated a Burkholderia mutant, which is deficient of an N-acetylmuramyl-l-alanine amidase, AmiC, involved in peptidoglycan degradation. When non-motile ΔamiC mutant Burkholderia cells harboring chain form were orally administered to Riptortus insects, ΔamiC mutant cells were unable to establish symbiotic association. But, ΔamiC mutant complemented with amiC gene restored in vivo symbiotic association. ΔamiC mutant cultured in minimal medium restored their motility with single-celled morphology. When ΔamiC mutant cells harboring single-celled morphology were administered to the host insect, this mutant established normal symbiotic association, suggesting that bacterial motility is essential for the successful symbiosis between host insect and Burkholderia symbiont. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Sex Reversal in Zebrafish fancl Mutants Is Caused by Tp53-Mediated Germ Cell Apoptosis

PubMed Central

Rodríguez-Marí, Adriana; Cañestro, Cristian; BreMiller, Ruth A.; Nguyen-Johnson, Alexandria; Asakawa, Kazuhide; Kawakami, Koichi; Postlethwait, John H.

2010-01-01

The molecular genetic mechanisms of sex determination are not known for most vertebrates, including zebrafish. We identified a mutation in the zebrafish fancl gene that causes homozygous mutants to develop as fertile males due to female-to-male sex reversal. Fancl is a member of the Fanconi Anemia/BRCA DNA repair pathway. Experiments showed that zebrafish fancl was expressed in developing germ cells in bipotential gonads at the critical time of sexual fate determination. Caspase-3 immunoassays revealed increased germ cell apoptosis in fancl mutants that compromised oocyte survival. In the absence of oocytes surviving through meiosis, somatic cells of mutant gonads did not maintain expression of the ovary gene cyp19a1a and did not down-regulate expression of the early testis gene amh; consequently, gonads masculinized and became testes. Remarkably, results showed that the introduction of a tp53 (p53) mutation into fancl mutants rescued the sex-reversal phenotype by reducing germ cell apoptosis and, thus, allowed fancl mutants to become fertile females. Our results show that Fancl function is not essential for spermatogonia and oogonia to become sperm or mature oocytes, but instead suggest that Fancl function is involved in the survival of developing oocytes through meiosis. This work reveals that Tp53-mediated germ cell apoptosis induces sex reversal after the mutation of a DNA–repair pathway gene by compromising the survival of oocytes and suggests the existence of an oocyte-derived signal that biases gonad fate towards the female developmental pathway and thereby controls zebrafish sex determination. PMID:20661450

Application of signature-tagged mutagenesis to the study of virulence of Erwinia amylovora.

PubMed

Wang, Limei; Beer, Steven V

2006-12-01

To identify genes that contribute to the virulence of Erwinia amylovora in plants, 1892 mutants were created and screened in pools of < or =96 mutants using signature-tagged mutagenesis. Nineteen mutants were not recovered from apple shoots following inoculation, which suggested that the insertions in these mutants affected genes important for bacterial survival in planta. DNA flanking the Tn5 insertions in the 19 mutants was sequenced and analysed by blast. One mutant had a Tn5 insertion in amsE, a gene involved in the biosynthesis of exopolysaccaride (EPS). Fourteen mutants had insertions in loci that were implicated in biosynthesis or transport of particular amino acids or nucleotides, a site-specific recombinase active during cell division and several putative proteins of unknown function; the flanking DNA of the remaining four mutants lacked significant homology with any DNA in the database. When inoculated individually to hosts, 10 of the 19 mutants caused significantly less disease and multiplied less, as compared with the wild-type strain.

A Single Mutation at the Sheet Switch Region Results in Conformational Changes Favoring 6 Light-Chain Fibrillogenesis

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

Hernández-Santoyo, A.; Del Pozo Yauner, L; Fuentes-Silva, D

Systemic amyloid light-chain (LC) amyloidosis is a disease process characterized by the pathological deposition of monoclonal LCs in tissue. All LC subtypes are capable of fibril formation although {lambda} chains, particularly those belonging to the {lambda}6 type, are overrepresented. Here, we report the thermodynamic and in vitro fibrillogenic properties of several mutants of the {lambda}6 protein 6aJL2 in which Pro7 and/or His8 was substituted by Ser or Pro. The H8P and H8S mutants were almost as stable as the wild-type protein and were poorly fibrillogenic. In contrast, the P7S mutation decreased the thermodynamic stability of 6aJL2 and greatly enhanced itsmore » capacity to form amyloid-like fibrils in vitro. The crystal structure of the P7S mutant showed that the substitution induced both local and long-distance effects, such as the rearrangement of the VL (variable region of the light chain)-VL interface. This mutant crystallized in two orthorhombic polymorphs, P2{sub 1}2{sub 1}2{sub 1} and C222{sub 1}. In the latter, a monomer that was not arranged in the typical Bence-Jones dimer was observed for the first time. Crystal-packing analysis of the C222{sub 1} lattice showed the establishment of intermolecular {beta}-{beta} interactions that involved the N-terminus and {beta}-strand B and that these could be relevant in the mechanism of LC fibril formation. Our results strongly suggest that Pro7 is a key residue in the conformation of the N-terminal sheet switch motif and, through long-distance interactions, is also critically involved in the contacts that stabilized the VL interface in {lambda}6 LCs.« less

A Plant Cryptochrome Controls Key Features of the Chlamydomonas Circadian Clock and Its Life Cycle.

PubMed

Müller, Nico; Wenzel, Sandra; Zou, Yong; Künzel, Sandra; Sasso, Severin; Weiß, Daniel; Prager, Katja; Grossman, Arthur; Kottke, Tilman; Mittag, Maria

2017-05-01

Cryptochromes are flavin-binding proteins that act as blue light receptors in bacteria, fungi, plants, and insects and are components of the circadian oscillator in mammals. Animal and plant cryptochromes are evolutionarily divergent, although the unicellular alga Chlamydomonas reinhardtii ( Chlamydomonas throughout) has both an animal-like cryptochrome and a plant cryptochrome (pCRY; formerly designated CPH1). Here, we show that the pCRY protein accumulates at night as part of a complex. Functional characterization of pCRY was performed based on an insertional mutant that expresses only 11% of the wild-type pCRY level. The pcry mutant is defective for central properties of the circadian clock. In the mutant, the period is lengthened significantly, ultimately resulting in arrhythmicity, while blue light-based phase shifts show large deviations from what is observed in wild-type cells. We also show that pCRY is involved in gametogenesis in Chlamydomonas pCRY is down-regulated in pregametes and gametes, and in the pcry mutant, there is altered transcript accumulation under blue light of the strictly light-dependent, gamete-specific gene GAS28 pCRY acts as a negative regulator for the induction of mating ability in the light and for the loss of mating ability in the dark. Moreover, pCRY is necessary for light-dependent germination, during which the zygote undergoes meiosis that gives rise to four vegetative cells. In sum, our data demonstrate that pCRY is a key blue light receptor in Chlamydomonas that is involved in both circadian timing and life cycle progression. © 2017 American Society of Plant Biologists. All Rights Reserved.

Identification of genes associated with resilience/vulnerability to sleep deprivation and starvation in Drosophila.

PubMed

Thimgan, Matthew S; Seugnet, Laurent; Turk, John; Shaw, Paul J

2015-05-01

Flies mutant for the canonical clock protein cycle (cyc(01)) exhibit a sleep rebound that is ∼10 times larger than wild-type flies and die after only 10 h of sleep deprivation. Surprisingly, when starved, cyc(01) mutants can remain awake for 28 h without demonstrating negative outcomes. Thus, we hypothesized that identifying transcripts that are differentially regulated between waking induced by sleep deprivation and waking induced by starvation would identify genes that underlie the deleterious effects of sleep deprivation and/or protect flies from the negative consequences of waking. We used partial complementary DNA microarrays to identify transcripts that are differentially expressed between cyc(01) mutants that had been sleep deprived or starved for 7 h. We then used genetics to determine whether disrupting genes involved in lipid metabolism would exhibit alterations in their response to sleep deprivation. Laboratory. Drosophila melanogaster. Sleep deprivation and starvation. We identified 84 genes with transcript levels that were differentially modulated by 7 h of sleep deprivation and starvation in cyc(01) mutants and were confirmed in independent samples using quantitative polymerase chain reaction. Several of these genes were predicted to be lipid metabolism genes, including bubblegum, cueball, and CG4500, which based on our data we have renamed heimdall (hll). Using lipidomics we confirmed that knockdown of hll using RNA interference significantly decreased lipid stores. Importantly, genetically modifying bubblegum, cueball, or hll resulted in sleep rebound alterations following sleep deprivation compared to genetic background controls. We have identified a set of genes that may confer resilience/vulnerability to sleep deprivation and demonstrate that genes involved in lipid metabolism modulate sleep homeostasis. © 2015 Associated Professional Sleep Societies, LLC.

Period 2 gene deletion abolishes β-endorphin neuronal response to ethanol

PubMed Central

Agapito, Maria; Mian, Nadia; Boyadjieva, Nadka I.; Sarkar, Dipak K.

2010-01-01

Background Ethanol exposure during early life has been shown to permanently alter the circadian expression of clock regulatory genes and the β-endorphin precursor proopiomelanocortin (POMC) gene in the hypothalamus. Ethanol also alters the stress- and immune-regulatory functions of β-endorphin neurons in laboratory rodents. Our aim was to determine whether the circadian clock regulatory Per2 gene modulates the action of ethanol on β-endorphin neurons in mice. Methods Per2 mutant (mPer2Brdml) and wild type (C57BL/6J) mice were used to determine the effect of Per2 mutation on ethanol-regulated β-endorphin neuronal activity during neonatal period using an in vitro mediobasal hypothalamic (MBH) cell culture model and an in vivo milk formula feeding animal model. The β-endorphin neuronal activity following acute and chronic ethanol treatments, was evaluated by measuring the peptide released from cultured cells or peptide levels in the MBH tissues, using enzyme-linked immunosorbent assay (ELISA). Results Per2 mutant mice showed a higher basal level of β-endorphin release from cultured MBH cells and a moderate increase in the peptide content in the MBH in comparison to control mice. However, unlike wild type mice, Per2 mutant mice showed no stimulatory or inhibitory β-endorphin secretory responses to acute and chronic ethanol challenges in vitro. Furthermore, Per2 mutant mice, but not wild type mice, failed to show the stimulatory and inhibitory responses of MBH β-endorphin levels to acute and chronic ethanol challenges in vivo. Conclusions These results suggest for the first time that the Per2 gene may be critically involved in regulating β-endorphin neuronal function. Furthermore, the data revealed an involvement of the Per2 gene in regulating β-endorphin neuronal responses to ethanol. PMID:20586752

A comparative proteomic analysis of Vibrio cholerae O1 wild-type cells versus a phoB mutant showed that the PhoB/PhoR system is required for full growth and rpoS expression under inorganic phosphate abundance.

PubMed

Lery, Letícia M S; Goulart, Carolina L; Figueiredo, Felipe R; Verdoorn, Karine S; Einicker-Lamas, Marcelo; Gomes, Fabio M; Machado, Ednildo A; Bisch, Paulo M; von Kruger, Wanda M A

2013-06-28

PhoB/PhoR is a two-component system originally described as involved in inorganic phosphate (Pi) transport and metabolism under Pi limitation. In order to disclose other roles of this system, a proteomic analysis of Vibrio cholerae 569BSR and its phoB/phoR mutant under high Pi levels was performed. Most of the proteins downregulated by the mutant have roles in energy production and conversion and in amino acid transport and metabolism. In contrast, the phoB/phoR mutant upregulated genes mainly involved in adaptation to atypical conditions, indicating that the absence of a functional PhoB/PhoR caused increased expression of a number of genes from distinct stress response pathways. This might be a strategy to overcome the lack of RpoS, whose expression in the stationary phase cells of V. cholerae seems to be controlled by PhoB/PhoR. Moreover, compared to the wild-type strain the phoB/phoR mutant presented a reduced cell density at stationary phase of culture in Pi abundance, lower resistance to acid shock, but higher tolerance to thermal and osmotic stresses. Together our findings show, for the first time, the requirement of PhoB/PhoR for full growth under high Pi level and for the accumulation of RpoS, indicating that PhoB/PhoR is a fundamental system for the biology of V. cholerae. Certain V. cholerae strains are pathogenic to humans, causing cholera, an acute dehydrating diarrhoeal disease endemic in Southern Asia, parts of Africa and Latin America, where it has been responsible for significant mortality and economical damage. Its ability to grow within distinct niches is dependent on gene expression regulation. PhoB/PhoR is a two-component system originally described as involved in inorganic phosphate (Pi) transport and metabolism under Pi limitation. However, Pho regulon genes also play roles in virulence, motility and biofilm formation, among others. In this paper we report that the absence of a functional PhoB/PhoR caused increased expression of a number of genes from distinct stress response pathways, in Pi abundance. Moreover, we showed, for the first time, that the interrelationship between PhoB-RpoS-(p)ppGpp-poly(P) in V. cholerae, is somewhat diverse from the model of inter-regulation between those systems, described in Escherichia coli. The V. cholerae dependence on PhoB/PhoR for the RpoS mediated stress response and cellular growth under Pi abundance, suggests that this system's roles are broader than previously thought. Copyright © 2013. Published by Elsevier B.V.

No Significant Increase in the Δ4- and Δ7-Dafachronic Acid Concentration in the Long-Lived glp-1 Mutant, nor in the Mutants Defective in Dauer Formation.

PubMed

Li, Tie-Mei; Liu, Weilong; Lu, Shan; Zhang, Yan-Ping; Jia, Le-Mei; Chen, Jie; Li, Xiangke; Lei, Xiaoguang; Dong, Meng-Qiu

2015-05-12

The steroid hormone dafachronic acid (DA) regulates dauer formation and lifespan in Caenorhabditis elegans by binding to the nuclear receptor DAF-12. However, little is known about how DA concentrations change under various physiologic conditions and about how DA/DAF-12 signaling interacts with other signaling pathways that also regulate dauer formation and lifespan. Using a sensitive bioanalytical method, we quantified the endogenous DA concentrations in a long-lived germline-less glp-1 mutant and in the Dauer formation-defective (Daf-d) mutants daf-12, daf-16, daf-5, and daf-3. We found that the DA concentration in the glp-1 mutant was similar to that in the wild type (WT). This result is contrary to the long-held belief that germline loss-induced longevity involves increased DA production and suggests instead that this type of longevity involves an enhanced response to DA. We also found evidence suggesting that increased DA sensitivity underlies lifespan extension triggered by exogenous DA. At the L2/L3 stage, the DA concentration in a daf-12 null mutant decreased to 22% of the WT level. This finding is consistent with the previously proposed positive feedback regulation between DAF-12 and DA production. Surprisingly, the DA concentrations in the daf-16, daf-5, and daf-3 mutants were only 19-34% of the WT level at the L2/L3 stage, slightly greater than those in the Dauer formation-constitutive (Daf-c) mutants at the pre-dauer stage (4-15% of the WT L2 control). Our experimental evidence suggested that the positive feedback between DA and DAF-12 was partially induced in the three Daf-d mutants. Copyright © 2015 Li et al.

Changes in transcript levels of starch hydrolysis genes and raising citric acid production via carbon ion irradiation mutagenesis of Aspergillus niger.

PubMed

Hu, Wei; Li, Wenjian; Chen, Hao; Liu, Jing; Wang, Shuyang; Chen, Jihong

2017-01-01

The filamentous ascomycete Aspergillus niger is well known for its ability to accumulate citric acid for the hydrolysis of starchy materials. To improve citric acid productivity, heavy ion beam mutagenesis was utilized to produce mutant A.niger strains with enhanced production of citric acid in this work. It was demonstrated that a mutant HW2 with high concentration of citric acid was isolated after carbon ion irradiation with the energy of 80Mev/μ, which was obvious increase higher than the original strain from liquefied corn starch as a feedstock. More importantly, with the evidence from the expression profiles of key genes and enzyme activity involved in the starch hydrolysis process between original strain and various phenotype mutants, our results confirmed that different transcript levels of key genes involving in starch hydrolysis process between original strain and mutants could be a significant contributor to different citric acid concentration in A.niger, such as, amyR and glaA, which therefore opened a new avenue for constructing genetically engineered A.niger mutants for high-yield citric acid accumulation in the future. As such, this work demonstrated that heavy ion beam mutagenesis presented an efficient alternative strategy to be developed to generate various phenotype microbe species mutants for functional genes research.

Mutants of Saccharomyces Cerevisiae with Defects in Acetate Metabolism: Isolation and Characterization of Acn(-) Mutants

PubMed Central

McCammon, M. T.

1996-01-01

The two carbon compounds, ethanol and acetate, can be oxidatively metabolized as well as assimilated into carbohydrate in the yeast Saccharomyces cerevisiae. The distribution of acetate metabolic enzymes among several cellular compartments, mitochondria, peroxisomes, and cytoplasm makes it an intriguing system to study complex metabolic interactions. To investigate the complex process of carbon catabolism and assimilation, mutants unable to grow on acetate were isolated. One hundred five Acn(-) (``ACetate Nonutilizing'') mutants were sorted into 21 complementation groups with an additional 20 single mutants. Five of the groups have defects in TCA cycle enzymes: MDH1, CIT1, ACO1, IDH1, and IDH2. A defect in RTG2, involved in the retrograde communication between the mitochondrion and the nucleus, was also identified. Four genes encode enzymes of the glyoxylate cycle and gluconeogenesis: ICL1, MLS1, MDH2, and PCK1. Five other genes appear to be defective in regulating metabolic activity since elevated levels of enzymes in several metabolic pathways, including the glyoxylate cycle, gluconeogenesis, and acetyl-CoA metabolism, were detected in these mutants: ACN8, ACN9, ACN17, ACN18, and ACN42. In summary, this analysis has identified at least 22 and as many as 41 different genes involved in acetate metabolism. PMID:8878673

The Agr communication system provides a benefit to the populations of Listeria monocytogenes in soil

PubMed Central

Vivant, Anne-Laure; Garmyn, Dominique; Gal, Laurent; Piveteau, Pascal

2014-01-01

In this study, we investigated whether the Agr communication system of the pathogenic bacterium Listeria monocytogenes was involved in adaptation and competitiveness in soil. Alteration of the ability to communicate, either by deletion of the gene coding the response regulator AgrA (response-negative mutant) or the signal pro-peptide AgrD (signal-negative mutant), did not affect population dynamics in soil that had been sterilized but survival was altered in biotic soil suggesting that the Agr system of L. monocytogenes was involved to face the complex soil biotic environment. This was confirmed by a set of co-incubation experiments. The fitness of the response-negative mutant was lower either in the presence or absence of the parental strain but the fitness of the signal-negative mutant depended on the strain with which it was co-incubated. The survival of the signal-negative mutant was higher when co-cultured with the parental strain than when co-cultured with the response-negative mutant. These results showed that the ability to respond to Agr communication provided a benefit to listerial cells to compete. These results might also indicate that in soil, the Agr system controls private goods rather than public goods. PMID:25414837

Role for the epidermal growth factor receptor in chemotherapy-induced alopecia.

PubMed

Bichsel, Kyle J; Gogia, Navdeep; Malouff, Timothy; Pena, Zachary; Forney, Eric; Hammiller, Brianna; Watson, Patrice; Hansen, Laura A

2013-01-01

Treatment of cancer patients with chemotherapeutics like cyclophosphamide often causes alopecia as a result of premature and aberrant catagen. Because the epidermal growth factor receptor (EGFR) signals anagen hair follicles to enter catagen, we hypothesized that EGFR signaling may be involved in cyclophosphamide-induced alopecia. To test this hypothesis, skin-targeted Egfr mutant mice were generated by crossing floxed Egfr and Keratin 14 promoter-driven Cre recombinase mice. Cyclophosphamide treatment of control mice resulted in alopecia while Egfr mutant skin was resistant to cyclophosphamide-induced alopecia. Egfr mutant skin entered catagen normally, as indicated by dermal papilla condensation and decreased follicular proliferation, but did not progress to telogen as did Egfr wild type follicles. Egfr mutant follicles responded with less proliferation, apoptosis, and fewer p53-positive cells after cyclophosphamide. Treatment of control mice with the EGFR inhibitors erlotinib or gefitinib similarly suppressed alopecia and catagen progression by cyclophosphamide. Secondary analysis of clinical trials utilizing EGFR-targeted therapies and alopecia-inducing chemotherapy also revealed evidence for involvement of EGFR in chemotherapy-induced alopecia. Taken together, our results demonstrated the involvement of EGFR signaling in chemotherapy-induced alopecia, which will help in the design of novel therapeutic regimens to minimize chemotherapy-induced alopecia.

Adaptation of Arabidopsis to nitrogen limitation involves induction of anthocyanin synthesis which is controlled by the NLA gene

PubMed Central

Peng, Mingsheng; Hudson, Darryl; Schofield, Andrew; Tsao, Rong; Yang, Raymond; Gu, Honglan; Bi, Yong-Mei; Rothstein, Steven. J.

2008-01-01

Plants can survive a limiting nitrogen (N) supply by developing a set of N limitation adaptive responses. However, the Arabidopsis nla (nitrogen limitation adaptation) mutant fails to produce such responses, and cannot adapt to N limitation. In this study, the nla mutant was utilized to understand further the effect of NLA on Arabidopsis adaptation to N limitation. Grown with limiting N, the nla mutant could not accumulate anthocyanins and instead produced an N limitation-induced early senescence phenotype. In contrast, when supplied with limiting N and limiting phosphorus (Pi), the nla mutants accumulated abundant anthocyanins and did not show the N limitation-induced early senescence phenotype. These results support the hypothesis that Arabidopsis has a specific pathway to control N limitation-induced anthocyanin synthesis, and the nla mutation disrupts this pathway. However, the nla mutation does not affect the Pi limitation-induced anthocyanin synthesis pathway. Therefore, Pi limitation induced the nla mutant to accumulate anthocyanins under N limitation and allowed this mutant to adapt to N limitation. Under N limitation, the nla mutant had a significantly down-regulated expression of many genes functioning in anthocyanin synthesis, and an enhanced expression of genes involved in lignin production. Correspondingly, the nla mutant grown with limiting N showed a significantly lower production of anthocyanins (particularly cyanidins) and an increase in lignin contents compared with wild-type plants. These data suggest that NLA controls Arabidopsis adaptability to N limitation by channelling the phenylpropanoid metabolic flux to the induced anthocyanin synthesis, which is important for Arabidopsis to adapt to N limitation. PMID:18552353

Effect of the N-terminal residues on the quaternary dynamics of human adult hemoglobin

NASA Astrophysics Data System (ADS)

Chang, Shanyan; Mizuno, Misao; Ishikawa, Haruto; Mizutani, Yasuhisa

2016-05-01

The protein dynamics of human hemoglobin following ligand photolysis was studied by time-resolved resonance Raman spectroscopy. The time-resolved spectra of two kinds of recombinant hemoglobin expressed in Escherichia coli, normal recombinant hemoglobin and the α(V1M)/β(V1M) double mutant, were compared with those of human adult hemoglobin (HbA) purified from blood. A frequency shift of the iron-histidine stretching [ν(Fe-His)] band was observed in the time-resolved spectra of all three hemoglobin samples, indicative of tertiary and quaternary changes in the protein following photolysis. The spectral changes of the α(V1M)/β(V1M) double mutant were distinct from those of HbA in the tens of microseconds region, whereas the spectral changes of normal recombinant hemoglobin were similar to those of HbA isolated from blood. These results demonstrated that a structural change in the N-termini is involved in the second step of the quaternary structure change of hemoglobin. We discuss the implications of these results for understanding the allosteric pathway of HbA.

Mutant with diphtheria toxin receptor and acidification function but defective in entry of toxin

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

Kohno, Kenji; Hayes, H.; Mekada, Eisuke

1987-09-01

A mutant of Chinese hamster ovary cells, GE1, that is highly resistant to diphtheria toxin was isolated. The mutant contains 50% ADP-ribosylatable elongation factor 2, but its protein synthesis was not inhibited by the toxin even at concentrations above 100 {mu}g/ml. {sup 125}I-labeled diphtheria toxin was associated with GE1 cells as well as with the parent cells but did not block protein synthesis of GE1 cells even when the cells were exposed to low pH in the presence or absence of NH{sub 4}Cl. The infections of GE1 cells and the parent cells by vesicular stomatitis virus were similar. GE1 cellsmore » were cross-resistant to Pseudomonas aeruginosa exotoxin A and so were about 1,000 times more resistant to this toxin than the parent cells. Hybrids of GE1 cells and the parent cells or mutant cells lacking a functional receptor were more sensitive to diphtheria toxin than GE1 cells. These results suggest that entry of diphtheria toxin into cells requires a cellular factor(s) in addition to those involved in receptor function and acidification of endosomes and that GE1 cells do not express this cellular factor. This character is recessive in GE1 cells.« less

Autophagy-linked FYVE protein (Alfy) promotes autophagic removal of misfolded proteins involved in amyotrophic lateral sclerosis (ALS).

PubMed

Han, Huihui; Wei, Wanyi; Duan, Weisong; Guo, Yansu; Li, Yi; Wang, Jie; Bi, Yue; Li, Chunyan

2015-03-01

Autophagy-linked FYVE (Alfy) is a protein implicated in the selective degradation of aggregated proteins. In our present study, we found that Alfy was recruited into the aggregated G93A-SOD1 in transgenic mice with amyotrophic lateral sclerosis (ALS). We demonstrated that Alfy overexpression could decrease the expression of mutant proteins via the autophagosome-lysosome pathway, and thereby, the toxicity of mutant proteins was reduced. The clearance of the mutant proteins in NSC34 cells was significantly inhibited in an Alfy knockdown cellular model. We therefore deduced that Alfy translocalization likely is involved in the pathogenesis of ALS. Alfy may be developed into a useful target for ALS therapy.

Improvement of acetic acid tolerance of Saccharomyces cerevisiae using a zinc-finger-based artificial transcription factor and identification of novel genes involved in acetic acid tolerance.

PubMed

Ma, Cui; Wei, Xiaowen; Sun, Cuihuan; Zhang, Fei; Xu, Jianren; Zhao, Xinqing; Bai, Fengwu

2015-03-01

Acetic acid is present in cellulosic hydrolysate as a potent inhibitor, and the superior acetic acid tolerance of Saccharomyces cerevisiae ensures good cell viability and efficient ethanol production when cellulosic raw materials are used as substrates. In this study, a mutant strain of S. cerevisiae ATCC4126 (Sc4126-M01) with improved acetic acid tolerance was obtained through screening strains transformed with an artificial zinc finger protein transcription factor (ZFP-TF) library. Further analysis indicated that improved acetic acid tolerance was associated with improved catalase (CAT) activity. The ZFP coding sequence associated with the improved phenotype was identified, and real-time RT-PCR analysis revealed that three of the possible genes involved in the enhanced acetic acid tolerance regulated by this ZFP-TF, namely YFL040W, QDR3, and IKS1, showed decreased transcription levels in Sc4126-M01 in the presence of acetic acid, compared to those in the control strain. Sc4126-M01 mutants having QDR3 and IKS1 deletion (ΔQDR3 and ΔIKS1) exhibited higher acetic acid tolerance than the wild-type strain under acetic acid treatment. Glucose consumption rate and ethanol productivity in the presence of 5 g/L acetic acid were improved in the ΔQDR3 mutant compared to the wild-type strain. Our studies demonstrated that the synthetic ZFP-TF library can be used to improve acetic acid tolerance of S. cerevisiae and that the employment of an artificial transcription factor can facilitate the exploration of novel functional genes involved in stress tolerance of S. cerevisiae.

Involvement of the Vacuoles of the Endodermis in the Early Process of Shoot Gravitropism in Arabidopsis

PubMed Central

Morita, Miyo Terao; Kato, Takehide; Nagafusa, Kiyoshi; Saito, Chieko; Ueda, Takashi; Nakano, Akihiko; Tasaka, Masao

2002-01-01

The endodermal cells of the shoot are thought to be the gravity-sensing cells in Arabidopsis. The amyloplasts in the endodermis that sediment in the direction of gravity may act as statoliths. Endodermis-specific expression of SGR2 and ZIG using the SCR promoter could complement the abnormal shoot gravitropism of the sgr2 and zig mutants, respectively. The abnormalities in amyloplast sedimentation observed in both mutants recovered simultaneously. These results indicate that both genes in the endodermal cell layer are crucial for shoot gravitropism. ZIG encodes AtVTI11, which is a SNARE involved in vesicle transport to the vacuole. The fusion protein of SGR2 and green fluorescent protein localized to the vacuole and small organelles. These observations indicate that ZIG and SGR2 are involved in the formation and function of the vacuole, a notion supported by the results of subcellular analysis of the sgr2 and zig mutants with electron microscopy. These results strongly suggest that the vacuole participates in the early events of gravitropism and that SGR2 and ZIG functions are involved. PMID:11826298

Life without complex I: proteome analyses of an Arabidopsis mutant lacking the mitochondrial NADH dehydrogenase complex

PubMed Central

Fromm, Steffanie; Senkler, Jennifer; Eubel, Holger; Peterhänsel, Christoph; Braun, Hans-Peter

2016-01-01

The mitochondrial NADH dehydrogenase complex (complex I) is of particular importance for the respiratory chain in mitochondria. It is the major electron entry site for the mitochondrial electron transport chain (mETC) and therefore of great significance for mitochondrial ATP generation. We recently described an Arabidopsis thaliana double-mutant lacking the genes encoding the carbonic anhydrases CA1 and CA2, which both form part of a plant-specific ‘carbonic anhydrase domain’ of mitochondrial complex I. The mutant lacks complex I completely. Here we report extended analyses for systematically characterizing the proteome of the ca1ca2 mutant. Using various proteomic tools, we show that lack of complex I causes reorganization of the cellular respiration system. Reduced electron entry into the respiratory chain at the first segment of the mETC leads to induction of complexes II and IV as well as alternative oxidase. Increased electron entry at later segments of the mETC requires an increase in oxidation of organic substrates. This is reflected by higher abundance of proteins involved in glycolysis, the tricarboxylic acid cycle and branched-chain amino acid catabolism. Proteins involved in the light reaction of photosynthesis, the Calvin cycle, tetrapyrrole biosynthesis, and photorespiration are clearly reduced, contributing to the significant delay in growth and development of the double-mutant. Finally, enzymes involved in defense against reactive oxygen species and stress symptoms are much induced. These together with previously reported insights into the function of plant complex I, which were obtained by analysing other complex I mutants, are integrated in order to comprehensively describe ‘life without complex I’. PMID:27122571

Analysis of a Range of Catabolic Mutants Provides Evidence That Phytanoyl-Coenzyme A Does Not Act as a Substrate of the Electron-Transfer Flavoprotein/Electron-Transfer Flavoprotein:Ubiquinone Oxidoreductase Complex in Arabidopsis during Dark-Induced Senescence1[W][OA

PubMed Central

Araújo, Wagner L.; Ishizaki, Kimitsune; Nunes-Nesi, Adriano; Tohge, Takayuki; Larson, Tony R.; Krahnert, Ina; Balbo, Ilse; Witt, Sandra; Dörmann, Peter; Graham, Ian A.; Leaver, Christopher J.; Fernie, Alisdair R.

2011-01-01

The process of dark-induced senescence in plants is not fully understood, however, the functional involvement of an electron-transfer flavoprotein/electron-transfer flavoprotein:ubiquinone oxidoreductase (ETF/ETFQO), has been demonstrated. Recent studies have revealed that the enzymes isovaleryl-coenzyme A (CoA) dehydrogenase and 2-hydroxyglutarate dehydrogenase act as important electron donors to this complex. In addition both enzymes play a role in the breakdown of cellular carbon storage reserves with isovaleryl-CoA dehydrogenase being involved in degradation of the branched-chain amino acids, phytol, and lysine while 2-hydroxyglutarate dehydrogenase is exclusively involved in lysine degradation. Given that the chlorophyll breakdown intermediate phytanoyl-CoA accumulates dramatically both in knockout mutants of the ETF/ETFQO complex and of isovaleryl-CoA dehydrogenase following growth in extended dark periods we have investigated the direct importance of chlorophyll breakdown for the supply of carbon and electrons during this process. For this purpose we isolated three independent Arabidopsis (Arabidopsis thaliana) knockout mutants of phytanoyl-CoA 2-hydroxylase and grew them under the same extended darkness regime as previously used. Despite the fact that these mutants accumulated phytanoyl-CoA and also 2-hydroxyglutarate they exhibited no morphological changes in comparison to the other mutants previously characterized. These results are consistent with a single entry point of phytol breakdown into the ETF/ETFQO system and furthermore suggest that phytol is not primarily metabolized by this pathway. Furthermore analysis of isovaleryl-CoA dehydrogenase/2-hydroxyglutarate dehydrogenase double mutants generated here suggest that these two enzymes essentially account for the entire electron input via the ETF complex. PMID:21788362

Enterococcus faecalis Constitutes an Unusual Bacterial Model in Lysozyme Resistance▿

PubMed Central

Hébert, Laurent; Courtin, Pascal; Torelli, Riccardo; Sanguinetti, Maurizio; Chapot-Chartier, Marie-Pierre; Auffray, Yanick; Benachour, Abdellah

2007-01-01

Lysozyme is an important and widespread compound of the host constitutive defense system, and it is assumed that Enterococcus faecalis is one of the few bacteria that are almost completely lysozyme resistant. On the basis of the sequence analysis of the whole genome of E. faecalis V583 strain, we identified two genes that are potentially involved in lysozyme resistance, EF_0783 and EF_1843. Protein products of these two genes share significant homology with Staphylococcus aureus peptidoglycan O-acetyltransferase (OatA) and Streptococcus pneumoniae N-acetylglucosamine deacetylase (PgdA), respectively. In order to determine whether EF_0783 and EF_1843 are involved in lysozyme resistance, we constructed their corresponding mutants and a double mutant. The ΔEF_0783 mutant and ΔEF_0783 ΔEF_1843 double mutant were shown to be more sensitive to lysozyme than the parental E. faecalis JH2-2 strain and ΔEF_1843 mutant were. However, compared to other bacteria, such as Listeria monocytogenes or S. pneumoniae, the tolerance of ΔEF_0783 and ΔEF_0783 ΔEF_1843 mutants towards lysozyme remains very high. Peptidoglycan structure analysis showed that EF_0783 modifies the peptidoglycan by O acetylation of N-acetyl muramic acid, while the EF_1843 deletion has no obvious effect on peptidoglycan structure under the same conditions. Moreover, the EF_0783 and EF_1843 deletions seem to significantly affect the ability of E. faecalis to survive within murine macrophages. In all, while EF_0783 is currently involved in the lysozyme resistance of E. faecalis, peptidoglycan O acetylation and de-N-acetylation are not the main mechanisms conferring high levels of lysozyme resistance to E. faecalis. PMID:17785473

Characterization of low phosphorus insensitive Mutants Reveals a Crosstalk between Low Phosphorus-Induced Determinate Root Development and the Activation of Genes Involved in the Adaptation of Arabidopsis to Phosphorus Deficiency1

PubMed Central

Sánchez-Calderón, Lenin; López-Bucio, José; Chacón-López, Alejandra; Gutiérrez-Ortega, Abel; Hernández-Abreu, Esmeralda; Herrera-Estrella, Luis

2006-01-01

Low phosphorus (P) availability is one of the most limiting factors for plant productivity in many natural and agricultural ecosystems. Plants display a wide range of adaptive responses to cope with low P stress, which generally serve to enhance P availability in the soil and to increase its uptake by roots. In Arabidopsis (Arabidopsis thaliana), primary root growth inhibition and increased lateral root formation have been reported to occur in response to P limitation. To gain knowledge of the genetic mechanisms that regulate root architectural responses to P availability, we designed a screen for identifying Arabidopsis mutants that fail to arrest primary root growth when grown under low P conditions. Eleven low phosphorus insensitive (lpi) mutants that define at least four different complementation groups involved in primary root growth responses to P availability were identified. The lpi mutants do not show the typical determinate developmental program induced by P stress in the primary root. Other root developmental aspects of the low P rescue system, including increased root hair elongation and anthocyanin accumulation, remained unaltered in lpi mutants. In addition to the insensitivity of primary root growth inhibition, when subjected to P deprivation, lpi mutants show a reduced induction in the expression of several genes involved in the P starvation rescue system (PHOSPHATE TRANSPORTER 1 and 2, PURPLE ACID PHOSPHATASE 1, ACID PHOSPHATASE 5, and INDUCED BY PHOSPHATE STARVATION 1). Our results provide genetic support for the role of P as an important signal for postembryonic root development and root meristem maintenance and show a crosstalk in developmental and biochemical responses to P deprivation. PMID:16443695

Reduction of spontaneous somatic mutation frequency by a low-dose X irradiation of Drosophila larvae and possible involvement of DNA single-strand damage repair.

PubMed

Koana, Takao; Takahashi, Takashi; Tsujimura, Hidenobu

2012-03-01

The third instar larvae of Drosophila were irradiated with X rays, and the somatic mutation frequency in their wings was measured after their eclosion. In the flies with normal DNA repair and apoptosis functions, 0.2 Gy irradiation at 0.05 Gy/min reduced the frequency of the so-called small spot (mutant cell clone with reduced reproductive activity) compared with that in the sham-irradiated flies. When apoptosis was suppressed using the baculovirus p35 gene, the small spot frequency increased four times in the sham-irradiated control group, but the reduction by the 0.2-Gy irradiation was still evident. In a non-homologous end joining-deficient mutant, the small spot frequency was also reduced by 0.2 Gy radiation. In a mutant deficient in single-strand break repair, no reduction in the small spot frequency by 0.2 Gy radiation was observed, and the small spot frequency increased with the radiation dose. Large spot (mutant cell clone with normal reproductive activity) frequency was not affected by suppression of apoptosis and increased monotonically with radiation dose in wild-type larvae and in mutants for single- or double-strand break repair. It is hypothesized that some of the small spots resulted from single-strand damage and, in wild-type larvae, 0.2 Gy radiation activated the normal single-strand break repair gene, which reduced the background somatic mutation frequency.

Screen for leukotoxin mutants in Aggregatibacter actinomycetemcomitans: genes of the phosphotransferase system are required for leukotoxin biosynthesis.

PubMed

Isaza, Maria P; Duncan, Matthew S; Kaplan, Jeffrey B; Kachlany, Scott C

2008-08-01

Aggregatibacter (formerly Actinobacillus) actinomycetemcomitans is a pathogen that causes localized aggressive periodontitis and extraoral infections including infective endocarditis. Recently, we reported that A. actinomycetemcomitans is beta-hemolytic on certain growth media due to the production of leukotoxin (LtxA). Based on this observation and our ability to generate random transposon insertions in A. actinomycetemcomitans, we developed and carried out a rapid screen for LtxA mutants. Using PCR, we mapped several of the mutations to genes that are known or predicted to be required for LtxA production, including ltxA, ltxB, ltxD, and tdeA. In addition, we identified an insertion in a gene previously not recognized to be involved in LtxA biosynthesis, ptsH. ptsH encodes the protein HPr, a phosphocarrier protein that is part of the sugar phosphotransferase system. HPr results in the phosphorylation of other proteins and ultimately in the activation of adenylate cyclase and cyclic AMP (cAMP) production. The ptsH mutant showed only partial hemolysis on blood agar and did not produce LtxA. The phenotype was complemented by supplying wild-type ptsH in trans, and real-time PCR analysis showed that the ptsH mutant produced approximately 10-fold less ltxA mRNA than the wild-type strain. The levels of cAMP in the ptsH mutant were significantly lower than in the wild-type strain, and LtxA production could be restored by adding exogenous cAMP to the culture.

Intact long-type DupA protein in Helicobacter pylori is an ATPase involved in multifunctional biological activities.

PubMed

Wang, Ming-yi; Chen, Cheng; Shao, Chen; Wang, Shao-bo; Wang, Ai-chu; Yang, Ya-chao; Yuan, Xiao-yan; Shao, Shi-he

2015-04-01

The function of intact long-type DupA protein in Helicobacter pylori was analyzed using immunoblotting and molecular biology techniques in the study. After cloning, expression and purification, ATPase activity of DupA protein was detected. Antibody was produced for localization and interaction proteins analysis. The dupA-deleted mutant was generated for adhesion and CagA protein translocation assay, susceptibility to different pH, IL-8 secretion assay, cytotoxicity to MKN-45 cells and proteins-involved apoptosis analysis. DupA protein exhibited an ATPase activity (129.5±17.8 U/mgprot) and located in bacterial membrane, while it did not involve the adhesion and CagA protein delivery of H. pylori. DupA protein involved the urease secretion as the interaction proteins. The wild type strain had a stronger growth in low pH than the dupA-deleted mutant (p < 0.001). IL-8 productions from GES-1 cells infected with the wild type strain were significantly higher than from those with the mutant (p < 0.001). The amounts of vital MKN-45 cells were decreased and the numbers of apoptotic cells were increased with the wild type strain, compared to those with the mutant after 12 h (p < 0.05). The increase of cleaved Caspase-3 and Bax was significantly higher and the decrease of Bcl-2 was more obvious in MKN-45 cells exposed to the wild type strain than that exposed to the mutant after 6 h. We demonstrate that intact long-type DupA protein located in membrane as ATPase is a true virulence factor associated with duodenal ulcer development involving the IL-8 induction and urease secretion, while it inhibits gastric cancer cell growth in vitro by activating the mitochondria-mediated apoptotic pathway. Copyright © 2015 Elsevier Ltd. All rights reserved.

Fast Homozygosity Mapping and Identification of a Zebrafish ENU-Induced Mutation by Whole-Genome Sequencing

PubMed Central

Voz, Marianne L.; Coppieters, Wouter; Manfroid, Isabelle; Baudhuin, Ariane; Von Berg, Virginie; Charlier, Carole; Meyer, Dirk; Driever, Wolfgang; Martial, Joseph A.; Peers, Bernard

2012-01-01

Forward genetics using zebrafish is a powerful tool for studying vertebrate development through large-scale mutagenesis. Nonetheless, the identification of the molecular lesion is still laborious and involves time-consuming genetic mapping. Here, we show that high-throughput sequencing of the whole zebrafish genome can directly locate the interval carrying the causative mutation and at the same time pinpoint the molecular lesion. The feasibility of this approach was validated by sequencing the m1045 mutant line that displays a severe hypoplasia of the exocrine pancreas. We generated 13 Gb of sequence, equivalent to an eightfold genomic coverage, from a pool of 50 mutant embryos obtained from a map-cross between the AB mutant carrier and the WIK polymorphic strain. The chromosomal region carrying the causal mutation was localized based on its unique property to display high levels of homozygosity among sequence reads as it derives exclusively from the initial AB mutated allele. We developed an algorithm identifying such a region by calculating a homozygosity score along all chromosomes. This highlighted an 8-Mb window on chromosome 5 with a score close to 1 in the m1045 mutants. The sequence analysis of all genes within this interval revealed a nonsense mutation in the snapc4 gene. Knockdown experiments confirmed the assertion that snapc4 is the gene whose mutation leads to exocrine pancreas hypoplasia. In conclusion, this study constitutes a proof-of-concept that whole-genome sequencing is a fast and effective alternative to the classical positional cloning strategies in zebrafish. PMID:22496837

Ozone-Sensitive Arabidopsis Mutants with Deficiencies in Photorespiratory Enzymes.

PubMed

Saji, Shoko; Bathula, Srinivas; Kubo, Akihiro; Tamaoki, Masanori; Aono, Mitsuko; Sano, Tomoharu; Tobe, Kazuo; Timm, Stefan; Bauwe, Hermann; Nakajima, Nobuyoshi; Saji, Hikaru

2017-05-01

An ozone-sensitive mutant was isolated from T-DNA-tagged lines of Arabidopsis thaliana. The T-DNA was inserted at a locus on chromosome 3, where two genes encoding glycolate oxidases, GOX1 and GOX2, peroxisomal enzymes involved in photorespiration, reside contiguously. The amounts of the mutant's foliar transcripts for these genes were reduced, and glycolate oxidase activity was approximately 60% of that of the wild-type plants. No difference in growth and appearance was observed between the mutant and the wild-type plants under normal conditions with ambient air under a light intensity of 100 µmol photons m-2 s-1. However, signs of severe damage, such as chlorosis and ion leakage from the tissue, rapidly appeared in mutant leaves in response to ozone treatment at a concentration of 0.2 µl l-1 under a higher light intensity of 350 µmol photons m-2 s-1 that caused no such symptoms in the wild-type plant. The mutant also exhibited sensitivity to sulfur dioxide and long-term high-intensity light. Arabidopsis mutants with deficiencies in other photorespiratory enzymes such as glutamate:glyoxylate aminotransferase and hydroxypyruvate reductase also exhibited ozone sensitivities. Therefore, photorespiration appears to be involved in protection against photooxidative stress caused by ozone and other abiotic factors under high-intensity light. © The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

DJ-1/PARK7, But Not Its L166P Mutant Linked to Autosomal Recessive Parkinsonism, Modulates the Transcriptional Activity of the Orphan Nuclear Receptor Nurr1 In Vitro and In Vivo.

PubMed

Lu, Lingling; Zhao, Shasha; Gao, Ge; Sun, Xiaohong; Zhao, Huanying; Yang, Hui

2016-12-01

Although mutations of DJ-1 have been linked to autosomal recessive Parkinsonism for years, its physiological function and the pathological mechanism of its mutants are not well understood. We report for the first time that exogenous application of DJ-1, but not its L166P mutant, enhances the nuclear translocation and the transcriptional activity of Nurr1, a transcription factor essential for dopaminergic neuron development and maturation, both in vitro and in vivo. Knockdown of DJ-1 attenuates Nurr1 activity. Further investigation showed that signaling of Raf/MEK/ERK MAPKs is involved in this regulatory process and that activation induced by exogenous DJ-1 is antagonized by U0126, an ERK pathway inhibitor, indicating that DJ-1 modulates Nurr1 activity via the Raf/MEK/ERK pathway. Our findings shed light on the novel function of DJ-1 to enhance Nurr1 activity and provide the first insight into the molecular mechanism by which DJ-1 enhances Nurr1 activity.

Reduced Gut Acidity Induces an Obese-Like Phenotype in Drosophila melanogaster and in Mice

PubMed Central

Yen, Jui-Hung; Kuo, Ping-Chang; Yeh, Sheng-Rong; Lin, Hung-Yu; Fu, Tsai-Feng; Wu, Ming-Shiang; Wang, Horng-Dar; Wang, Pei-Yu

2015-01-01

In order to identify genes involved in stress and metabolic regulation, we carried out a Drosophila P-element-mediated mutagenesis screen for starvation resistance. We isolated a mutant, m2, that showed a 23% increase in survival time under starvation conditions. The P-element insertion was mapped to the region upstream of the vha16-1 gene, which encodes the c subunit of the vacuolar-type H+-ATPase. We found that vha16-1 is highly expressed in the fly midgut, and that m2 mutant flies are hypomorphic for vha16-1 and also exhibit reduced midgut acidity. This deficit is likely to induce altered metabolism and contribute to accelerated aging, since vha16-1 mutant flies are short-lived and display increases in body weight and lipid accumulation. Similar phenotypes were also induced by pharmacological treatment, through feeding normal flies and mice with a carbonic anhydrase inhibitor (acetazolamide) or proton pump inhibitor (PPI, lansoprazole) to suppress gut acid production. Our study may thus provide a useful model for investigating chronic acid suppression in patients. PMID:26436771

Down regulation of miR-124 in both Werner syndrome DNA helicase mutant mice and mutant Caenorhabditis elegans wrn-1 reveals the importance of this microRNA in accelerated aging

PubMed Central

Dallaire, Alexandra; Garand, Chantal; Paquet, Eric R.; Mitchell, Sarah J.; de Cabo, Rafael; Simard, Martin J.

2012-01-01

Small non-coding microRNAs are believed to be involved in the mechanism of aging but nothing is known on the impact of microRNAs in the progeroid disorder Werner syndrome (WS). WS is a premature aging disorder caused by mutations in a RecQ-like DNA helicase. Mice lacking the helicase domain of the WRN ortholog exhibit many phenotypic features of WS, including a pro-oxidant status and a shorter mean life span. Caenorhabditis elegans (C. elegans) with a nonfunctional wrn-1 DNA helicase also exhibit a shorter life span. Thus, both models are relevant to study the expression of microRNAs involved in WS. In this study, we show that miR-124 expression is lost in the liver of Wrn helicase mutant mice. Interestingly, the expression of this conserved miR-124 in whole wrn-1 mutant worms is also significantly reduced. The loss of mir-124 in C. elegans increases reactive oxygen species formation and accumulation of the aging marker lipofuscin, reduces whole body ATP levels and results in a reduction in life span. Finally, supplementation of vitamin C normalizes the median life span of wrn-1 and mir-124 mutant worms. These results suggest that biological pathways involving WRN and miR-124 are conserved in the aging process across different species. PMID:23075628

Partial deficiency of isoleucine impairs root development and alters transcript levels of the genes involved in branched-chain amino acid and glucosinolate metabolism in Arabidopsis

PubMed Central

Liu, Dong

2013-01-01

Isoleucine is one of the branched-chain amino acids (BCAAs) that are essential substrates for protein synthesis in all organisms. Although the metabolic pathway for isoleucine has been well characterized in higher plants, it is not known whether it plays a specific role in plant development. In this study, an Arabidopsis mutant, lib (low isoleucine biosynthesis), that has defects in both cell proliferation and cell expansion processes during root development, was characterized. The lib mutant carries a T-DNA insertion in the last exon of the OMR1 gene that encodes a threonine deaminase/dehydratase (TD). TD catalyses the deamination and dehydration of threonine, which is the first and also the committed step in the biosynthesis of isoleucine. This T-DNA insertion results in a partial deficiency of isoleucine in lib root tissues but it does not affect its total protein content. Application of exogenous isoleucine or introduction of a wild-type OMR1 gene into the lib mutant can completely rescue the mutant phenotypes. These results reveal an important role for isoleucine in plant development. In addition, microarray analysis indicated that the partial deficiency of isoleucine in the lib mutant triggers a decrease in transcript levels of the genes encoding the major enzymes involved in the BCAA degradation pathway; the analysis also indicated that many genes involved in the biosynthesis of methionine-derived glucosinolates are up-regulated. PMID:23230023

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.

IDC1, a pezizomycotina-specific gene that belongs to the PaMpk1 MAP kinase transduction cascade of the filamentous fungus Podospora anserina.

PubMed

Jamet-Vierny, Corinne; Debuchy, Robert; Prigent, Magali; Silar, Philippe

2007-12-01

Components involved in the activation of the MAPK cascades in filamentous fungi are not well known. Here, we provide evidence that IDC1, a pezizomycotina-specific gene is involved along with the PaNox1 NADPH oxidase in the nuclear localization of the PaMpk1 MAP kinase, a prerequisite for MAPK activity. Mutants of IDC1 display the same phenotypes as mutants in PaNox1 and PaMpk1, i.e., lack of pigment and of aerial hyphae, female sterility, impairment in hyphal interference and inability to develop Crippled Growth cell degeneration. As observed for the PaNox1 mutant, IDC1 mutants are hypostatic to PaMpk1 mutants. IDC1 seems to play a key role in sexual reproduction. Indeed, fertility is diminished in strains with lower level of IDC1. In strains over-expressing IDC1, protoperithecia reach a later stage of development towards perithecia without fertilization; however, upon fertilization maturation of fertile perithecia is diminished and delayed. In addition, heterokaryon construction shows that IDC1 is necessary together with PaNox1 in the perithecial envelope but not in the dikaryon resulting from fertilization.

Transposon mutagenesis reveals differential pathogenesis of Ralstonia solanacearum on tomato and Arabidopsis.

PubMed

Lin, Yu-Mei; Chou, I-Chun; Wang, Jaw-Fen; Ho, Fang-I; Chu, Yu-Ju; Huang, Pei-Cheng; Lu, Der-Kang; Shen, Hwei-Ling; Elbaz, Mounira; Huang, Shu-Mei; Cheng, Chiu-Ping

2008-09-01

Ralstonia solanacearum causes a deadly wilting disease on a wide range of crops. To elucidate pathogenesis of this bacterium in different host plants, we set out to identify R. solanacearum genes involved in pathogenesis by screening random transposon insertion mutants of a highly virulent strain, Pss190, on tomato and Arabidopsis thaliana. Mutants exhibiting various decreased virulence levels on these two hosts were identified. Sequence analysis showed that most, but not all, of the identified pathogenesis genes are conserved among distinct R. solanacearum strains. A few of the disrupted loci were not reported previously as being involved in R. solanacearum pathogenesis. Notably, a group of mutants exhibited differential pathogenesis on tomato and Arabidopsis. These results were confirmed by characterizing allelic mutants in one other R. solanacearum strain of the same phylotype. The significantly decreased mutants' colonization in Arabidopsis was found to be correlated with differential pathogenesis on these two plants. Differential requirement of virulence genes suggests adaptation of this bacterium in different host environments. Together, this study reveals commonalities and differences of R. solanacearum pathogenesis on single solanaceous and nonsolanaceous hosts, and provides important new insights into interactions between R. solanacearum and different host plants.

The endosomal sorting complex required for transport (ESCRT) is required for the sensitivity of yeast cells to nickel ions in Saccharomyces cerevisiae.

PubMed

Luo, Chong; Cao, Chunlei; Jiang, Linghuo

2016-05-01

Nickel is one of the toxic environment metal pollutants and is linked to various human diseases. In this study, through a functional genomics approach we have identified 16 nickel-sensitive and 22 nickel-tolerant diploid deletion mutants of budding yeast genes, many of which are novel players in the regulation of nickel homeostasis. The 16 nickel-sensitive mutants are of genes mainly involved in the protein folding, modification and destination and the cellular transport processes, while the 22 nickel-tolerant mutants are of genes encoding components of ESCRT complexes as well as protein factors involved in both the cell wall integrity maintenance and the vacuolar protein sorting process. In consistence with their phenotypes, most of these nickel-sensitive mutants show reduced intracellular nickel contents, while the majority of these nickel-tolerant mutants show elevated intracellular nickel contents, as compared to the wild type in response to nickel stress. Our data provides a basis for our understanding the regulation of nickel homeostasis and molecular mechanisms of nickel-induced human pathogenesis. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Multimodal imaging of lung cancer and its microenvironment (Conference Presentation)

NASA Astrophysics Data System (ADS)

Hariri, Lida P.; Niederst, Matthew J.; Mulvey, Hillary; Adams, David C.; Hu, Haichuan; Chico Calero, Isabel; Szabari, Margit V.; Vakoc, Benjamin J.; Hasan, Tayyaba; Bouma, Brett E.; Engelman, Jeffrey A.; Suter, Melissa J.

2016-03-01

Despite significant advances in targeted therapies for lung cancer, nearly all patients develop drug resistance within 6-12 months and prognosis remains poor. Developing drug resistance is a progressive process that involves tumor cells and their microenvironment. We hypothesize that microenvironment factors alter tumor growth and response to targeted therapy. We conducted in vitro studies in human EGFR-mutant lung carcinoma cells, and demonstrated that factors secreted from lung fibroblasts results in increased tumor cell survival during targeted therapy with EGFR inhibitor, gefitinib. We also demonstrated that increased environment stiffness results in increased tumor survival during gefitinib therapy. In order to test our hypothesis in vivo, we developed a multimodal optical imaging protocol for preclinical intravital imaging in mouse models to assess tumor and its microenvironment over time. We have successfully conducted multimodal imaging of dorsal skinfold chamber (DSC) window mice implanted with GFP-labeled human EGFR mutant lung carcinoma cells and visualized changes in tumor development and microenvironment facets over time. Multimodal imaging included structural OCT to assess tumor viability and necrosis, polarization-sensitive OCT to measure tissue birefringence for collagen/fibroblast detection, and Doppler OCT to assess tumor vasculature. Confocal imaging was also performed for high-resolution visualization of EGFR-mutant lung cancer cells labeled with GFP, and was coregistered with OCT. Our results demonstrated that stromal support and vascular growth are essential to tumor progression. Multimodal imaging is a useful tool to assess tumor and its microenvironment over time.

Adherence to abiotic surface induces SOS response in Escherichia coli K-12 strains under aerobic and anaerobic conditions.

PubMed

Costa, Suelen B; Campos, Ana Carolina C; Pereira, Ana Claudia M; de Mattos-Guaraldi, Ana Luiza; Júnior, Raphael Hirata; Rosa, Ana Cláudia P; Asad, Lídia M B O

2014-09-01

During the colonization of surfaces, Escherichia coli bacteria often encounter DNA-damaging agents and these agents can induce several defence mechanisms. Base excision repair (BER) is dedicated to the repair of oxidative DNA damage caused by reactive oxygen species (ROS) generated by chemical and physical agents or by metabolism. In this work, we have evaluated whether the interaction with an abiotic surface by mutants derived from E. coli K-12 deficient in some enzymes that are part of BER causes DNA damage and associated filamentation. Moreover, we studied the role of endonuclease V (nfi gene; 1506 mutant strain) in biofilm formation. Endonuclease V is an enzyme that is involved in DNA repair of nitrosative lesions. We verified that endonuclease V is involved in biofilm formation. Our results showed more filamentation in the xthA mutant (BW9091) and triple xthA nfo nth mutant (BW535) than in the wild-type strain (AB1157). By contrast, the mutant nfi did not present filamentation in biofilm, although its wild-type strain (1466) showed rare filaments in biofilm. The filamentation of bacterial cells attaching to a surface was a consequence of SOS induction measured by the SOS chromotest. However, biofilm formation depended on the ability of the bacteria to induce the SOS response since the mutant lexA Ind(-) did not induce the SOS response and did not form any biofilm. Oxygen tension was an important factor for the interaction of the BER mutants, since these mutants exhibited decreased quantitative adherence under anaerobic conditions. However, our results showed that the presence or absence of oxygen did not affect the viability of BW9091 and BW535 strains. The nfi mutant and its wild-type did not exhibit decreased biofilm formation under anaerobic conditions. Scanning electron microscopy was also performed on the E. coli K-12 strains that had adhered to the glass, and we observed the presence of a structure similar to an extracellular matrix that depended on the oxygen tension. In conclusion, it was proven that bacterial interaction with abiotic surfaces can lead to SOS induction and associated filamentation. Moreover, we verified that endonuclease V is involved in biofilm formation. © 2014 The Authors.

Degradation of misfolded proteins by autophagy: is it a strategy for Huntington's disease treatment?

PubMed

Lin, Fang; Qin, Zheng-Hong

2013-01-01

Autophagy is a degradation pathway for long-lived cytoplasmic proteins, protein complexes, or damaged organelles. The accumulation and aggregation of misfolded proteins are hallmarks of several neurodegenerative diseases. Many researchers have reported that autophagy degrades disease-causing misfolded and aggregated proteins, including mutant huntingtin (Htt) in Huntington's disease, mutant synuclein in familial Parkingson's disease, mutant Cu, Zn-Superoxide dismutase (SOD1) in familial amyotrophic lateral sclerosis. In this review, we will bring up new evidence to elucidate the involvement of autophagy in degradation of mutant Htt, discuss the mechanisms regulating the degradation of mutant Htt by autophagy and the therapeutic effects of drugs that enhance autophagy to improve clearance of mutant Htt. We propose that enhancement of autophagy by drugs may be a strategy to treat or retard progression of Huntington's disease.

Transcriptome and proteome analysis of nitrogen starvation responses in Synechocystis 6803 ΔglgC, a mutant incapable of glycogen storage

DOE PAGES

Carrieri, Damian; Lombardi, Thomas; Paddock, Troy; ...

2016-11-17

Molecular mechanisms that regulate carbon flux are poorly understood in algae. The ΔglgC mutant of the cyanobacterium Synechocystis sp. PCC 6803 is incapable of glycogen storage and displays an array of physiological responses under nitrogen starvation that are different from wild-type (WT). These include non-bleaching phenotype and the redirection of photosynthetically fixed carbon towards excreted organic acids (overflow metabolism) without biomass growth. To understand the role of gene/protein expression in these responses, we followed the time course of transcripts by genome-scale microarrays and proteins by shotgun proteomics in ΔglgC and WT cells upon nitrogen starvation. Compared to WT, the degradationmore » of phycobilisome rod proteins was delayed and attenuated in the mutant, and the core proteins were less degraded; both contributed to the non-bleaching appearance despite the induction of nblA genes, suggesting the presence of a break in regulation of the phycobilisome degradation pathway downstream of nblA induction. The mutant displayed NtcA-mediated transcriptional response to nitrogen starvation, indicating that it is able to sense nitrogen status. Furthermore, some responses to nitrogen starvation appear to be stronger in the mutant, as shown by the increases in transcripts for the transcriptional regulator, rre37, which regulates central carbon metabolism. Accordingly, multiple proteins involved in photosynthesis, central carbon metabolism, and carbon storage and utilization showed lower abundance in the mutant. Furthermore, these results indicate that the transition in the central carbon metabolism from growth to overflow metabolism in ΔglgC does not require increases in expression of the overflow pathway enzymes; the transition and non-bleaching phenotype are likely regulated instead at the metabolite level.« less

Transcriptome and proteome analysis of nitrogen starvation responses in Synechocystis 6803 ΔglgC, a mutant incapable of glycogen storage

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

Carrieri, Damian; Lombardi, Thomas; Paddock, Troy

Molecular mechanisms that regulate carbon flux are poorly understood in algae. The ΔglgC mutant of the cyanobacterium Synechocystis sp. PCC 6803 is incapable of glycogen storage and displays an array of physiological responses under nitrogen starvation that are different from wild-type (WT). These include non-bleaching phenotype and the redirection of photosynthetically fixed carbon towards excreted organic acids (overflow metabolism) without biomass growth. To understand the role of gene/protein expression in these responses, we followed the time course of transcripts by genome-scale microarrays and proteins by shotgun proteomics in ΔglgC and WT cells upon nitrogen starvation. Compared to WT, the degradationmore » of phycobilisome rod proteins was delayed and attenuated in the mutant, and the core proteins were less degraded; both contributed to the non-bleaching appearance despite the induction of nblA genes, suggesting the presence of a break in regulation of the phycobilisome degradation pathway downstream of nblA induction. The mutant displayed NtcA-mediated transcriptional response to nitrogen starvation, indicating that it is able to sense nitrogen status. Furthermore, some responses to nitrogen starvation appear to be stronger in the mutant, as shown by the increases in transcripts for the transcriptional regulator, rre37, which regulates central carbon metabolism. Accordingly, multiple proteins involved in photosynthesis, central carbon metabolism, and carbon storage and utilization showed lower abundance in the mutant. Furthermore, these results indicate that the transition in the central carbon metabolism from growth to overflow metabolism in ΔglgC does not require increases in expression of the overflow pathway enzymes; the transition and non-bleaching phenotype are likely regulated instead at the metabolite level.« less

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

Chan, R.K.; Otte, C.A.

Saccharomyces cerevisiae MATa cells carrying mutations in either sst1 or sst2 are supersensitive to the G1 arrest induced by ..cap alpha.. factor pheromone. When sst1 mutants were mixed with normal SST/sup +/ cells, the entire population recovered together from ..cap alpha.. factor arrest, suggesting that SST/sup +/ cells helped sst1 mutants to recover. Complementation tests and linkage analysis showed that sst1 and bar1, a mutation which eliminates the ability of MATa cells to act as a ''barrier'' to the diffusion of ..cap alpha.. factor, were lesions in the same genes. These findings suggest that sst1 mutants are defective in recoverymore » from ..cap alpha.. factor arrest because they are unable to degrade the pheromone. In contrast, recovery of sst2 mutants was not potentiated by the presence of SST/sup +/ cells in mixing experiments. When either normal MATa cells or mutant cells carrying defects in sst1 or sst2 were exposed to ..cap alpha.. factor for 1 h and then washed free of the pheromone, the sst2 cells subsequently remained arrested in the absence of ..cap alpha.. factor for a much longer time than SST/sup +/ or sst1 cells. These observations suggest that the defect in sst2 mutants is intrinsic to the cell and is involved in the mechanism of ..cap alpha.. factor action at some step after the initial interaction of the pheromone with the cell. The presence of an sst2 mutation appears to cause a growth debility, since repeated serial subculture of haploid sst2-1 strains led to the accumulation of faster-growing revertants that were pheromone resistant and were mating defective (''sterile'').« less

Use of the Hungate anaerobic technique in the isolation of phloroglucinol-negative mutants of Coprococcus species.

PubMed Central

Thompson, L A; Gates, D M; Ingledew, W M; Jones, G A

1976-01-01

The Hungate anaerobic technique was used with a standard procedure for bacterial mutagenesis employing N-methyl-N-nitro-N'-nitrosoguanidine to obtain mutants of an obligate anaerobe. Three mutant strains were derived from a Coprococcus sp., strain Pe15, a rumen anaerobe capable of growing on phloroglucinol. The mutants did not grow on phloroglucinol but did degrade the compound in anaerobic washed-cell suspensions, producing the same end products in approximately the same proportions as the wild type. It was concluded that the mutants were blocked in a unique step or steps necessary for carbon skeleton or energy synthesis from phloroglucinol and not in formation of an enzyme involved in the pathway of phloroglucinol degradation. PMID:782358

Use of the Hungate anaerobic technique in the isolation of phloroglucinol-negative mutants of Coprococcus species.

PubMed

Thompson, L A; Gates, D M; Ingledew, W M; Jones, G A

1976-01-01

The Hungate anaerobic technique was used with a standard procedure for bacterial mutagenesis employing N-methyl-N-nitro-N'-nitrosoguanidine to obtain mutants of an obligate anaerobe. Three mutant strains were derived from a Coprococcus sp., strain Pe15, a rumen anaerobe capable of growing on phloroglucinol. The mutants did not grow on phloroglucinol but did degrade the compound in anaerobic washed-cell suspensions, producing the same end products in approximately the same proportions as the wild type. It was concluded that the mutants were blocked in a unique step or steps necessary for carbon skeleton or energy synthesis from phloroglucinol and not in formation of an enzyme involved in the pathway of phloroglucinol degradation.

Mechanical properties of elytra from Tribolium castaneum wild-type and body color mutant strains

USDA-ARS?s Scientific Manuscript database

Cuticle tanning in insects involves simultaneous cuticular hardening and pigmentation. The dynamic mechanical properties of the highly modified and cuticle-rich forewings (elytra) from Tribolium castaneum (red flour beetle) body color mutant strains were investigated to determine the relationship b...

Structural, molecular motions, and free-energy landscape of Leishmania sterol-14α-demethylase wild type and drug resistant mutant: a comparative molecular dynamics study.

PubMed

Vijayakumar, Saravanan; Das, Pradeep

2018-04-18

Sterol-14α-demethylase (CYP51) is an ergosterol pathway enzyme crucial for the survival of infectious Leishmania parasite. Recent high-throughput metabolomics and whole genome sequencing study revealed amphotericin B resistance in Leishmania is indeed due to mutation in CYP51. The residue of mutation (asparagine 176) is conserved across the kinetoplastidae and not in yeast or humans, portraying its functional significance. In order to understand the possible cause for the resistance, knowledge of structural changes due to mutation is of high importance. To shed light on the structural changes of wild and mutant CYP51, we conducted comparative molecular dynamics simulation study. The active site, substrate biding cavity, substrate channel entrance (SCE), and cavity involving the mutated site were studied based on basic parameters and large concerted molecular motions derived from essential dynamics analyses of 100 ns simulation. Results indicated that mutant CYP51 is stable and less compact than the wild type. Correspondingly, the solvent accessible surface area (SASA) of the mutant was found to be increased, especially in active site and cavities not involving the mutation site. Free-energy landscape analysis disclosed mutant to have a rich conformational diversity than wild type, with various free-energy conformations of mutant having SASA greater than wild type with SCE open. More residues were found to interact with the mutant CYP51 upon docking of substrate to both the wild and mutant CYP51. These results indicate that, relative to wild type, the N176I mutation of CYP51 in Leishmania mexicana could possibly favor increased substrate binding efficiency.

The RNA Chaperone Hfq Is Involved in Stress Tolerance and Virulence in Uropathogenic Proteus mirabilis

PubMed Central

Wang, Min-Cheng; Liaw, Shwu-Jen

2014-01-01

Hfq is a bacterial RNA chaperone involved in the riboregulation of diverse genes via small noncoding RNAs. Here, we show that Hfq is critical for the uropathogenic Proteus mirabilis to effectively colonize the bladder and kidneys in a murine urinary tract infection (UTI) model and to establish burned wound infection of the rats. In this regard, we found the hfq mutant induced higher IL-8 and MIF levels of uroepithelial cells and displayed reduced intra-macrophage survival. The loss of hfq affected bacterial abilities to handle H2O2 and osmotic pressures and to grow at 50°C. Relative to wild-type, the hfq mutant had reduced motility, fewer flagella and less hemolysin expression and was less prone to form biofilm and to adhere to and invade uroepithelial cells. The MR/P fimbrial operon was almost switched to the off phase in the hfq mutant. In addition, we found the hfq mutant exhibited an altered outer membrane profile and had higher RpoE expression, which indicates the hfq mutant may encounter increased envelope stress. With the notion of envelope disturbance in the hfq mutant, we found increased membrane permeability and antibiotic susceptibilities in the hfq mutant. Finally, we showed that Hfq positively regulated the RpoS level and tolerance to H2O2 in the stationary phase seemed largely mediated through the Hfq-dependent RpoS expression. Together, our data indicate that Hfq plays a critical role in P. mirabilis to establish UTIs by modulating stress responses, surface structures and virulence factors. This study suggests Hfq may serve as a scaffold molecule for development of novel anti-P. mirabilis drugs and P. mirabilis hfq mutant is a vaccine candidate for preventing UTIs. PMID:24454905

Signature-tagged mutagenesis screening revealed a novel smooth-to-rough transition determinant of Salmonella enterica serovar Enteritidis.

PubMed

Jiao, Yang; Guo, Rongxian; Tang, Peipei; Kang, Xilong; Yin, Junlei; Wu, Kaiyue; Geng, Shizhong; Li, Qiuchun; Sun, Jun; Xu, Xiulong; Zhou, Xiaohui; Gan, Junji; Jiao, Xinan; Liu, Xiufan; Pan, Zhiming

2017-03-03

Salmonella enterica serovar Enteritidis (S. Enteritidis) has emerged as one of the most important food-borne pathogens for humans. Lipopolysaccharide (LPS), as a component of the outer membrane, is responsible for the virulence and smooth-to-rough transition in S. Enteritidis. In this study, we screened S. Enteritidis signature-tagged transposon mutant library using monoclonal antibody against somatic O 9 antigen (O 9 MAb) and O 9 factor rabbit antiserum to identify novel genes that are involved in smooth-to-rough transition. A total of 480 mutants were screened and one mutant with transposon insertion in rfbG gene had smooth-to-rough transition phenotype. In order to verify the role of rfbG gene, an rfbG insertion or deletion mutant was constructed using λ-Red recombination system. Phenotypic and biological analysis revealed that rfbG insertion or deletion mutants were similar to the wild-type strain in growth rate and biochemical properties, but the swimming motility was reduced. SE Slide Agglutination test and ELISA test showed that rfbG mutants do not stimulate animals to produce agglutinating antibody. In addition, the half-lethal dose (LD 50 ) of the rfbG deletion mutant strain was 10 6.6 -fold higher than that of the parent strain in a mouse model when injected intraperitoneally. These data indicate that the rfbG gene is involved in smooth-to-rough transition, swimming motility and virulence of S. Enteritidis. Furthermore, somatic O-antigen antibody-based approach to screen signature-tagged transposon mutants is feasible to clarify LPS biosynthesis and to find suitable markers in DIVA-vaccine research.

Mesophilic Aeromonas sp. serogroup O:11 resistance to complement-mediated killing.

PubMed Central

Merino, S; Rubires, X; Aguilar, A; Albertí, S; Hernandez-Allés, S; Benedí, V J; Tomas, J M

1996-01-01

The complement activation by and resistance to complement-mediated killing of Aeromonas sp. strains from serogroup O:11 were investigated by using different wild-type strains (with an S-layer characteristic of this serogroup) and their isogenic mutants characterized for their surface components (S-layer and lipopolysaccharide [LPS]). All of the Aeromonas sp. serogroup O:11 wild-type strains are unable to activate complement, which suggested that the S-layer completely covered the LPS molecules. We found that the classical complement pathway is involved in serum killing of susceptible Aeromonas sp. mutant strains of serogroup O11, while the alternative complement pathway seems not to be involved, and that the complement activation seems to be independent of antibody. The smooth mutant strains devoid of the S-layer (S-layer isogenic mutants) or isogenic LPS mutant strains with a complete or rather complete LPS core (also without the S-layer) are able to activate complement but are resistant to complement-mediated killing. The reasons for this resistance are that C3b is rapidly degraded, and therefore the lytic membrane attack complex (C5b-9) is not formed. Isogenic LPS rough mutants with an incomplete LPS core are serum sensitive because they bind more C3b than the resistant strains, the C3b is not completely degraded, and therefore the lytic complex (C5b-9) is formed. PMID:8945581

Increased production of biomass-degrading enzymes by double deletion of creA and creB genes involved in carbon catabolite repression in Aspergillus oryzae.

PubMed

Ichinose, Sakurako; Tanaka, Mizuki; Shintani, Takahiro; Gomi, Katsuya

2018-02-01

In a previous study, we reported that a double gene deletion mutant for CreA and CreB, which constitute the regulatory machinery involved in carbon catabolite repression, exhibited improved production of α-amylase compared with the wild-type strain and single creA or creB deletion mutants in Aspergillus oryzae. Because A. oryzae can also produce biomass-degrading enzymes, such as xylolytic and cellulolytic enzymes, we examined the production levels of those enzymes in deletion mutants in this study. Xylanase and β-glucosidase activities in the wild-type were hardly detected in submerged culture containing xylose as the carbon source, whereas those enzyme activities were significantly increased in the single creA deletion (ΔcreA) and double creA and creB deletion (ΔcreAΔcreB) mutants. In particular, the ΔcreAΔcreB mutant exhibited >100-fold higher xylanase and β-glucosidase activities than the wild-type. Moreover, in solid-state culture, the β-glucosidase activity of the double deletion mutant was >7-fold higher than in the wild-type. These results suggested that deletion of both creA and creB genes could also efficiently improve the production levels of biomass-degrading enzymes in A. oryzae. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Changes in transcript levels of starch hydrolysis genes and raising citric acid production via carbon ion irradiation mutagenesis of Aspergillus niger

PubMed Central

Li, Wenjian; Chen, Hao; Liu, Jing; Wang, Shuyang; Chen, Jihong

2017-01-01

The filamentous ascomycete Aspergillus niger is well known for its ability to accumulate citric acid for the hydrolysis of starchy materials. To improve citric acid productivity, heavy ion beam mutagenesis was utilized to produce mutant A.niger strains with enhanced production of citric acid in this work. It was demonstrated that a mutant HW2 with high concentration of citric acid was isolated after carbon ion irradiation with the energy of 80Mev/μ, which was obvious increase higher than the original strain from liquefied corn starch as a feedstock. More importantly, with the evidence from the expression profiles of key genes and enzyme activity involved in the starch hydrolysis process between original strain and various phenotype mutants, our results confirmed that different transcript levels of key genes involving in starch hydrolysis process between original strain and mutants could be a significant contributor to different citric acid concentration in A.niger, such as, amyR and glaA, which therefore opened a new avenue for constructing genetically engineered A.niger mutants for high-yield citric acid accumulation in the future. As such, this work demonstrated that heavy ion beam mutagenesis presented an efficient alternative strategy to be developed to generate various phenotype microbe species mutants for functional genes research. PMID:28650980

Controlled synthesis of the DSF cell–cell signal is required for biofilm formation and virulence in Xanthomonas campestris

PubMed Central

Torres, Pablo S; Malamud, Florencia; Rigano, Luciano A; Russo, Daniela M; Marano, María Rosa; Castagnaro, Atilio P; Zorreguieta, Angeles; Bouarab, Kamal; Dow, John Maxwell; Vojnov, Adrián A

2007-01-01

Virulence of the black rot pathogen Xanthomonas campestris pv. campestris (Xcc) is regulated by cell–cell signalling involving the diffusible signal factor DSF. Synthesis and perception of DSF require products of genes within the rpf cluster (for regulation of pathogenicity factors). RpfF directs DSF synthesis whereas RpfC and RpfG are involved in DSF perception. Here we have examined the role of the rpf/DSF system in biofilm formation in minimal medium using confocal laser-scanning microscopy of GFP-labelled bacteria. Wild-type Xcc formed microcolonies that developed into a structured biofilm. In contrast, an rpfF mutant (DSF-minus) and an rpfC mutant (DSF overproducer) formed only unstructured arrangements of bacteria. A gumB mutant, defective in xanthan biosynthesis, was also unable to develop the typical wild-type biofilm. Mixed cultures of gumB and rpfF mutants formed a typical biofilm in vitro. In contrast, in mixed cultures the rpfC mutant prevented the formation of the structured biofilm by the wild-type and did not restore wild-type biofilm phenotypes to gumB or rpfF mutants. These effects on structured biofilm formation were correlated with growth and disease development by Xcc strains in Nicotiana benthamiana leaves. These findings suggest that DSF signalling is finely balanced during both biofilm formation and virulence. PMID:17635553

The Drosophila TRPA channel, Painless, regulates sexual receptivity in virgin females

PubMed Central

Sakai, Takaomi; Kasuya, Junko; Kitamoto, Toshihiro; Aigaki, Toshiro

2009-01-01

Transient receptor potential (TRP) channels play crucial roles in sensory perception. Expression of the Drosophila painless (pain) gene, a homolog of the mammalian TRPA1/ANKTM1 gene, in the peripheral nervous system is required for avoidance behavior of noxious heat or wasabi. Here we report a novel role of the Pain TRP channel expressed in the nervous system in the sexual receptivity in Drosophila virgin females. Compared with wild-type females, pain mutant females copulated with wild-type males significantly earlier. Wild-type males showed comparable courtship latency and courtship index toward wild-type and pain mutant females. Therefore, the early copulation observed in wild-type male and pain mutant female pairs is the result of enhanced sexual receptivity in pain mutant females. Involvement of pain in enhanced female sexual receptivity was confirmed by rescue experiments in which expression of a pain transgene in a pain mutant background restored the female sexual receptivity to the wild-type level. Targeted expression of pain RNAi in putative cholinergic or GABAergic neurons phenocopied the mutant phenotype of pain females. On the other hand, target expression of pain RNAi in dopaminergic neurons did not affect female sexual receptivity. In addition, conditional suppression of neurotransmission in putative GABAergic neurons resulted in a similar enhanced sexual receptivity. Our results suggest that Pain TRP channels expressed in cholinergic and/or GABAergic neurons are involved in female sexual receptivity. PMID:19531155

The Drosophila TRPA channel, Painless, regulates sexual receptivity in virgin females.

PubMed

Sakai, T; Kasuya, J; Kitamoto, T; Aigaki, T

2009-07-01

Transient receptor potential (TRP) channels play crucial roles in sensory perception. Expression of the Drosophila painless (pain) gene, a homolog of the mammalian TRPA1/ANKTM1 gene, in the peripheral nervous system is required for avoidance behavior of noxious heat or wasabi. In this study, we report a novel role of the Pain TRP channel expressed in the nervous system in the sexual receptivity in Drosophila virgin females. Compared with wild-type females, pain mutant females copulated with wild-type males significantly earlier. Wild-type males showed comparable courtship latency and courtship index toward wild-type and pain mutant females. Therefore, the early copulation observed in wild-type male and pain mutant female pairs is the result of enhanced sexual receptivity in pain mutant females. Involvement of pain in enhanced female sexual receptivity was confirmed by rescue experiments in which expression of a pain transgene in a pain mutant background restored the female sexual receptivity to the wild-type level. Targeted expression of pain RNA interference (RNAi) in putative cholinergic or GABAergic neurons phenocopied the mutant phenotype of pain females. However, target expression of pain RNAi in dopaminergic neurons did not affect female sexual receptivity. In addition, conditional suppression of neurotransmission in putative GABAergic neurons resulted in a similar enhanced sexual receptivity. Our results suggest that Pain TRP channels expressed in cholinergic and/or GABAergic neurons are involved in female sexual receptivity.

MLLT1 YEATS domain mutations in clinically distinctive Favourable Histology Wilms tumours

PubMed Central

Perlman, Elizabeth J.; Gadd, Samantha; Arold, Stefan T.; Radhakrishnan, Anand; Gerhard, Daniela S.; Jennings, Lawrence; Huff, Vicki; Guidry Auvil, Jaime M.; Davidsen, Tanja M.; Dome, Jeffrey S.; Meerzaman, Daoud; Hsu, Chih Hao; Nguyen, Cu; Anderson, James; Ma, Yussanne; Mungall, Andrew J.; Moore, Richard A.; Marra, Marco A.; Mullighan, Charles G.; Ma, Jing; Wheeler, David A.; Hampton, Oliver A.; Gastier-Foster, Julie M.; Ross, Nicole; Smith, Malcolm A.

2015-01-01

Wilms tumour is an embryonal tumour of childhood that closely resembles the developing kidney. Genomic changes responsible for the development of the majority of Wilms tumours remain largely unknown. Here we identify recurrent mutations within Wilms tumours that involve the highly conserved YEATS domain of MLLT1 (ENL), a gene known to be involved in transcriptional elongation during early development. The mutant MLLT1 protein shows altered binding to acetylated histone tails. Moreover, MLLT1-mutant tumours show an increase in MYC gene expression and HOX dysregulation. Patients with MLLT1-mutant tumours present at a younger age and have a high prevalence of precursor intralobar nephrogenic rests. These data support a model whereby activating MLLT1 mutations early in renal development result in the development of Wilms tumour. PMID:26635203

Embryonic expression of shuttle craft, a Drosophila gene involved in neuron development, is associated with adult lifespan.

PubMed

Roshina, Natalia V; Symonenko, Alexander V; Krementsova, Anna V; Trostnikov, Mikhail V; Pasyukova, Elena G

2014-12-01

Despite the progress in aging research that highlights the role of the nervous system in longevity, whether genes that control development and consequently structure of the nervous system affect lifespan is unclear. We demonstrated that a mutation inshuttle craft, a gene involved in the nervous system development, increased the lifespan of unmated females and decreased the lifespan of mated females, without affecting males. Precise reversions of the mutation lead to the restoration of the lifespan specific to control females. In mutant unmated females, increased lifespan was associated with elevated locomotion at older ages, indicating slowed aging. In mutant mated females, reproduction was decreased compared to controls, indicating a lack of tradeoff between this trait and lifespan. No differences in shuttle craft transcription were observed between whole bodies, ovaries, and brains of mutant and control females of different ages, either unmated or mated. The amount of shuttle craft transcript appeared to be substantially decreased in mutant embryos. Our results demonstrated that a gene that regulates development of the nervous system might also influence longevity, and thus expanded the spectrum of genes involved in lifespan control. We hypothesize that this "carry-over" effect might be the result of transcription regulation in embryos.

The C2H2-type transcription factor, FlbC, is involved in the transcriptional regulation of Aspergillus oryzae glucoamylase and protease genes specifically expressed in solid-state culture.

PubMed

Tanaka, Mizuki; Yoshimura, Midori; Ogawa, Masahiro; Koyama, Yasuji; Shintani, Takahiro; Gomi, Katsuya

2016-07-01

Aspergillus oryzae produces a large amount of secreted proteins in solid-state culture, and some proteins such as glucoamylase (GlaB) and acid protease (PepA) are specifically produced in solid-state culture, but rarely in submerged culture. From the disruption mutant library of A. oryzae transcriptional regulators, we successfully identified a disruption mutant showing an extremely low production level of GlaB but a normal level of α-amylase production. This strain was a disruption mutant of the C2H2-type transcription factor, FlbC, which is reported to be involved in the regulation of conidiospore development. Disruption mutants of other upstream regulators comprising a conidiation regulatory network had no apparent effect on GlaB production in solid-state culture. In addition to GlaB, the production of acid protease in solid-state culture was also markedly decreased by flbC disruption. Northern blot analyses revealed that transcripts of glaB and pepA were significantly decreased in the flbC disruption strain. These results suggested that FlbC is involved in the transcriptional regulation of genes specifically expressed under solid-state cultivation conditions, possibly independent of the conidiation regulatory network.

Metabolic characterization of isocitrate dehydrogenase (IDH) mutant and IDH wildtype gliomaspheres uncovers cell type-specific vulnerabilities.

PubMed

Garrett, Matthew; Sperry, Jantzen; Braas, Daniel; Yan, Weihong; Le, Thuc M; Mottahedeh, Jack; Ludwig, Kirsten; Eskin, Ascia; Qin, Yue; Levy, Rachelle; Breunig, Joshua J; Pajonk, Frank; Graeber, Thomas G; Radu, Caius G; Christofk, Heather; Prins, Robert M; Lai, Albert; Liau, Linda M; Coppola, Giovanni; Kornblum, Harley I

2018-01-01

There is considerable interest in defining the metabolic abnormalities of IDH mutant tumors to exploit for therapy. While most studies have attempted to discern function by using cell lines transduced with exogenous IDH mutant enzyme, in this study, we perform unbiased metabolomics to discover metabolic differences between a cohort of patient-derived IDH1 mutant and IDH wildtype gliomaspheres. Using both our own microarray and the TCGA datasets, we performed KEGG analysis to define pathways differentially enriched in IDH1 mutant and IDH wildtype cells and tumors. Liquid chromatography coupled to mass spectrometry analysis with labeled glucose and deoxycytidine tracers was used to determine differences in overall cellular metabolism and nucleotide synthesis. Radiation-induced DNA damage and repair capacity was assessed using a comet assay. Differences between endogenous IDH1 mutant metabolism and that of IDH wildtype cells transduced with the IDH1 (R132H) mutation were also investigated. Our KEGG analysis revealed that IDH wildtype cells were enriched for pathways involved in de novo nucleotide synthesis, while IDH1 mutant cells were enriched for pathways involved in DNA repair. LC-MS analysis with fully labeled 13 C-glucose revealed distinct labeling patterns between IDH1 mutant and wildtype cells. Additional LC-MS tracing experiments confirmed increased de novo nucleotide synthesis in IDH wildtype cells relative to IDH1 mutant cells. Endogenous IDH1 mutant cultures incurred less DNA damage than IDH wildtype cultures and sustained better overall growth following X-ray radiation. Overexpression of mutant IDH1 in a wildtype line did not reproduce the range of metabolic differences observed in lines expressing endogenous mutations, but resulted in depletion of glutamine and TCA cycle intermediates, an increase in DNA damage following radiation, and a rise in intracellular ROS. These results demonstrate that IDH1 mutant and IDH wildtype cells are easily distinguishable metabolically by analyzing expression profiles and glucose consumption. Our results also highlight important differences in nucleotide synthesis utilization and DNA repair capacity that could be exploited for therapy. Altogether, this study demonstrates that IDH1 mutant gliomas are a distinct subclass of glioma with a less malignant, but also therapy-resistant, metabolic profile that will likely require distinct modes of therapy.

Metabolomic homeostasis shifts after callus formation and shoot regeneration in tomato

PubMed Central

Kumari, Alka; Ray, Kamalika; Sadhna, Sadhna; Pandey, Arun Kumar; Sreelakshmi, Yellamaraju; Sharma, Rameshwar

2017-01-01

Plants can regenerate from a variety of tissues on culturing in appropriate media. However, the metabolic shifts involved in callus formation and shoot regeneration are largely unknown. The metabolic profiles of callus generated from tomato (Solanum lycopersicum) cotyledons and that of shoot regenerated from callus were compared with the pct1-2 mutant that exhibits enhanced polar auxin transport and the shr mutant that exhibits elevated nitric oxide levels. The transformation from cotyledon to callus involved a major shift in metabolite profiles with denser metabolic networks in the callus. In contrast, the transformation from callus to shoot involved minor changes in the networks. The metabolic networks in pct1-2 and shr mutants were distinct from wild type and were rewired with shifts in endogenous hormones and metabolite interactions. The callus formation was accompanied by a reduction in the levels of metabolites involved in cell wall lignification and cellular immunity. On the contrary, the levels of monoamines were upregulated in the callus and regenerated shoot. The callus formation and shoot regeneration were accompanied by an increase in salicylic acid in wild type and mutants. The transformation to the callus and also to the shoot downregulated LST8 and upregulated TOR transcript levels indicating a putative linkage between metabolic shift and TOR signalling pathway. The network analysis indicates that shift in metabolite profiles during callus formation and shoot regeneration is governed by a complex interaction between metabolites and endogenous hormones. PMID:28481937

Involvement of signal peptidase I in Streptococcus sanguinis biofilm formation

PubMed Central

Ge, Xiuchun; Stone, Victoria; Zhu, Bin; Kitten, Todd

2017-01-01

Biofilm accounts for 65–80 % of microbial infections in humans. Considerable evidence links biofilm formation by oral microbiota to oral disease and consequently systemic infections. Streptococcus sanguinis, a Gram-positive bacterium, is one of the most abundant species of the oral microbiota and it contributes to biofilm development in the oral cavity. Due to its altered biofilm formation, we investigated a biofilm mutant, ΔSSA_0351, that is deficient in type I signal peptidase (SPase) in this study. Although the growth curve of the ΔSSA_0351 mutant showed no significant difference from that of the wild-type strain SK36, biofilm assays using both microtitre plate assay and confocal laser scanning microscopy (CLSM) confirmed a sharp reduction in biofilm formation in the mutant compared to the wild-type strain and the paralogous mutant ΔSSA_0849. Scanning electron microscopy (SEM) revealed remarkable differences in the cell surface morphologies and chain length of the ΔSSA_0351 mutant compared with those of the wild-type strain. Transcriptomic and proteomic assays using RNA sequencing and mass spectrometry, respectively, were conducted on the ΔSSA_0351 mutant to evaluate the functional impact of SPase on biofilm formation. Subsequently, bioinformatics analysis revealed a number of proteins that were differentially regulated in the ΔSSA_0351 mutant, narrowing down the list of SPase substrates involved in biofilm formation to lactate dehydrogenase (SSA_1221) and a short-chain dehydrogenase (SSA_0291). With further experimentation, this list defined the link between SSA_0351-encoded SPase, cell wall biosynthesis and biofilm formation. PMID:28869408

Investigating the Association between Flowering Time and Defense in the Arabidopsis thaliana-Fusarium oxysporum Interaction.

PubMed

Lyons, Rebecca; Rusu, Anca; Stiller, Jiri; Powell, Jonathan; Manners, John M; Kazan, Kemal

2015-01-01

Plants respond to pathogens either by investing more resources into immunity which is costly to development, or by accelerating reproductive processes such as flowering time to ensure reproduction occurs before the plant succumbs to disease. In this study we explored the link between flowering time and pathogen defense using the interaction between Arabidopsis thaliana and the root infecting fungal pathogen Fusarium oxysporum. We report that F. oxysporum infection accelerates flowering time and regulates transcription of a number of floral integrator genes, including FLOWERING LOCUS C (FLC), FLOWERING LOCUS T (FT) and GIGANTEA (GI). Furthermore, we observed a positive correlation between late flowering and resistance to F. oxysporum in A. thaliana natural ecotypes. Late-flowering gi and autonomous pathway mutants also exhibited enhanced resistance to F. oxysporum, supporting the association between flowering time and defense. However, epistasis analysis showed that accelerating flowering time by deletion of FLC in fve-3 or fpa-7 mutants did not alter disease resistance, suggesting that the effect of autonomous pathway on disease resistance occurs independently from flowering time. Indeed, RNA-seq analyses suggest that fve-3 mediated resistance to F. oxysporum is most likely a result of altered defense-associated gene transcription. Together, our results indicate that the association between flowering time and pathogen defense is complex and can involve both pleiotropic and direct effects.

Investigating the Association between Flowering Time and Defense in the Arabidopsis thaliana-Fusarium oxysporum Interaction

PubMed Central

Lyons, Rebecca; Rusu, Anca; Stiller, Jiri; Powell, Jonathan; Manners, John M.; Kazan, Kemal

2015-01-01

Plants respond to pathogens either by investing more resources into immunity which is costly to development, or by accelerating reproductive processes such as flowering time to ensure reproduction occurs before the plant succumbs to disease. In this study we explored the link between flowering time and pathogen defense using the interaction between Arabidopsis thaliana and the root infecting fungal pathogen Fusarium oxysporum. We report that F. oxysporum infection accelerates flowering time and regulates transcription of a number of floral integrator genes, including FLOWERING LOCUS C (FLC), FLOWERING LOCUS T (FT) and GIGANTEA (GI). Furthermore, we observed a positive correlation between late flowering and resistance to F. oxysporum in A. thaliana natural ecotypes. Late-flowering gi and autonomous pathway mutants also exhibited enhanced resistance to F. oxysporum, supporting the association between flowering time and defense. However, epistasis analysis showed that accelerating flowering time by deletion of FLC in fve-3 or fpa-7 mutants did not alter disease resistance, suggesting that the effect of autonomous pathway on disease resistance occurs independently from flowering time. Indeed, RNA-seq analyses suggest that fve-3 mediated resistance to F. oxysporum is most likely a result of altered defense-associated gene transcription. Together, our results indicate that the association between flowering time and pathogen defense is complex and can involve both pleiotropic and direct effects. PMID:26034991

Isolation and partial characterization of a mutant of Penicillium funiculosum for the saccharification of straw

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

Hoffman, R.M.; Wood, T.M.

1985-01-01

Clearing of agar plates containing ball-milled, delignified straw has been used for screening mutants of Penicillium funiculosum IMI 87160 III. The effects of glycerol and a number of sugars on the clearing were investigated for selecting derepressed mutants. The ..beta..-glucosidase synthesis by one such mutant, C22c, in shake flasks containing straw was not repressed by 5% glycerol, whereas activities on filter paper, CM-cellulose, and p-nitrophenyl-..beta..-xylosidase were only partially derepressed; xylanase was extensively derepressed. The evidence for separate control of the enzymes involved in the solubilization of straw is discussed. 23 references.

Characterization of Escherichia coli d-Cycloserine Transport and Resistant Mutants

PubMed Central

Baisa, Gary; Stabo, Nicholas J.

2013-01-01

d-Cycloserine (DCS) is a broad-spectrum antibiotic that inhibits d-alanine ligase and alanine racemase activity. When Escherichia coli K-12 or CFT073 is grown in minimal glucose or glycerol medium, CycA transports DCS into the cell. E. coli K-12 cycA and CFT073 cycA mutant strains display increased DCS resistance when grown in minimal medium. However, the cycA mutants exhibit no change in DCS sensitivity compared to their parental strains when grown in LB (CFT073 and K-12) or human urine (CFT073 only). These data suggest that cycA does not participate in DCS sensitivity when strains are grown in a non-minimal medium. The small RNA GvcB acts as a negative regulator of E. coli K-12 cycA expression when grown in LB. Three E. coli K-12 gcvB mutant strains failed to demonstrate a change in DCS sensitivity when grown in LB. This further suggests a limited role for cycA in DCS sensitivity. To aid in the identification of E. coli genes involved in DCS sensitivity when grown on complex media, the Keio K-12 mutant collection was screened for DCS-resistant strains. dadA, pnp, ubiE, ubiF, ubiG, ubiH, and ubiX mutant strains showed elevated DCS resistance. The phenotypes associated with these mutants were used to further define three previously characterized E. coli DCS-resistant strains (χ316, χ444, and χ453) isolated by Curtiss and colleagues (R. Curtiss, III, L. J. Charamella, C. M. Berg, and P. E. Harris, J. Bacteriol. 90:1238–1250, 1965). A dadA mutation was identified in both χ444 and χ453. In addition, results are presented that indicate for the first time that DCS can antagonize d-amino acid dehydrogenase (DadA) activity. PMID:23316042

The RAD24 (= Rs1) Gene Product of Saccharomyces cerevisiae Participates in Two Different Pathways of DNA Repair

PubMed Central

Eckardt-Schupp, Friederike; Siede, Wolfram; Game, John C.

1987-01-01

The moderately UV- and X-ray-sensitive mutant of Saccharomyces cerevisiae originally designated rs1 complements all rad and mms mutants available. Therefore, the new nomination rad24-1 according to the RAD nomenclature is suggested. RAD24 maps on chromosome V, close to RAD3 (1.3 cM). In order to associate the RAD24 gene with one of the three repair pathways, double mutants of rad24 and various representative genes of each pathway were constructed. The UV and X-ray sensitivities of the double mutants compared to the single mutants indicate that RAD24 is involved in excision repair of UV damage (RAD3 epistasis group), as well as in recombination repair of UV and X-ray damage (RAD52 epistasis group). Properties of the mutant are discussed which hint at the control of late steps in the pathways. PMID:3549445

Brucella melitensis MucR, an orthologue of Sinorhizobium meliloti MucR, is involved in resistance to oxidative, detergent, and saline stresses and cell envelope modifications.

PubMed

Mirabella, A; Terwagne, M; Zygmunt, M S; Cloeckaert, A; De Bolle, X; Letesson, J J

2013-02-01

Brucella spp. and Sinorhizobium meliloti are alphaproteobacteria that share not only an intracellular lifestyle in their respective hosts, but also a crucial requirement for cell envelope components and their timely regulation for a successful infectious cycle. Here, we report the characterization of Brucella melitensis mucR, which encodes a zinc finger transcriptional regulator that has previously been shown to be involved in cellular and mouse infections at early time points. MucR modulates the surface properties of the bacteria and their resistance to environmental stresses (i.e., oxidative stress, cationic peptide, and detergents). We show that B. melitensis mucR is a functional orthologue of S. meliloti mucR, because it was able to restore the production of succinoglycan in an S. meliloti mucR mutant, as detected by calcofluor staining. Similar to S. meliloti MucR, B. melitensis MucR also represses its own transcription and flagellar gene expression via the flagellar master regulator ftcR. More surprisingly, we demonstrate that MucR regulates a lipid A core modification in B. melitensis. These changes could account for the attenuated virulence of a mucR mutant. These data reinforce the idea that there is a common conserved circuitry between plant symbionts and animal pathogens that regulates the relationship they have with their hosts.

The RNA silencing enzyme RNA polymerase v is required for plant immunity.

PubMed

López, Ana; Ramírez, Vicente; García-Andrade, Javier; Flors, Victor; Vera, Pablo

2011-12-01

RNA-directed DNA methylation (RdDM) is an epigenetic control mechanism driven by small interfering RNAs (siRNAs) that influence gene function. In plants, little is known of the involvement of the RdDM pathway in regulating traits related to immune responses. In a genetic screen designed to reveal factors regulating immunity in Arabidopsis thaliana, we identified NRPD2 as the OVEREXPRESSOR OF CATIONIC PEROXIDASE 1 (OCP1). NRPD2 encodes the second largest subunit of the plant-specific RNA Polymerases IV and V (Pol IV and Pol V), which are crucial for the RdDM pathway. The ocp1 and nrpd2 mutants showed increases in disease susceptibility when confronted with the necrotrophic fungal pathogens Botrytis cinerea and Plectosphaerella cucumerina. Studies were extended to other mutants affected in different steps of the RdDM pathway, such as nrpd1, nrpe1, ago4, drd1, rdr2, and drm1drm2 mutants. Our results indicate that all the mutants studied, with the exception of nrpd1, phenocopy the nrpd2 mutants; and they suggest that, while Pol V complex is required for plant immunity, Pol IV appears dispensable. Moreover, Pol V defective mutants, but not Pol IV mutants, show enhanced disease resistance towards the bacterial pathogen Pseudomonas syringae DC3000. Interestingly, salicylic acid (SA)-mediated defenses effective against PsDC3000 are enhanced in Pol V defective mutants, whereas jasmonic acid (JA)-mediated defenses that protect against fungi are reduced. Chromatin immunoprecipitation analysis revealed that, through differential histone modifications, SA-related defense genes are poised for enhanced activation in Pol V defective mutants and provide clues for understanding the regulation of gene priming during defense. Our results highlight the importance of epigenetic control as an additional layer of complexity in the regulation of plant immunity and point towards multiple components of the RdDM pathway being involved in plant immunity based on genetic evidence, but whether this is a direct or indirect effect on disease-related genes is unclear.

A second gene for type I signal peptidase in Bradyrhizobium japonicum, sipF, is located near genes involved in RNA processing and cell division.

PubMed

Bairl, A; Müller, P

1998-11-01

The TnphoA-induced Bradyrhizobium japonicum mutant 184 shows slow growth and aberrant colonization of soybean nodules. Using a DNA fragment adjacent to the transposon insertion site as a probe, a 3.4-kb BglII fragment of B. japonicum 110spc4 DNA was identified and cloned. Sequence analysis indicated that two truncated ORFs and three complete ORFs were encoded on this fragment. A database search revealed homologies to several other prokaryotic proteins: PdxJ (an enzyme involved in vitamin B6 biosynthesis), AcpS (acyl carrier protein synthase), Lep or Sip (prokaryotic type I signal peptidase), RNase III (an endoribonuclease which processes double-stranded rRNA precursors and mRNA) and Era (a GTP-binding protein required for cell division). The mutation in strain 184 was found to lie within the signal peptidase gene, which was designated sipF. Therefore, sipF is located in a region that encodes gene products involved in posttranscriptional and posttranslational processing processes. By complementation of the lep(ts) E. coli mutant strain IT41 it was demonstrated that sipF indeed encodes a functional signal peptidase, and genetic complementation of B. japonicum mutant 184 by a 2.8-kb SalI fragment indicated that sipF is expressed from a promoter located directly upstream of sipF. Using a non-polar kanamycin resistance cassette, a specific sipF mutant was constructed which exhibited defects in symbiosis similar to those of the original mutant 184.

Transcriptome profile analysis of floral sex determination in cucumber.

PubMed

Wu, Tao; Qin, Zhiwei; Zhou, Xiuyan; Feng, Zhuo; Du, Yalin

2010-07-15

Cucumber has been widely studied as a model for floral sex determination. In this investigation, we performed genome-wide transcriptional profiling of apical tissue of a gynoecious mutant (Csg-G) and the monoecious wild-type (Csg-M) of cucumber in an attempt to isolate genes involved in sex determination, using the Solexa technology. The profiling analysis revealed numerous changes in gene expression attributable to the mutation, which resulted in the down-regulation of 600 genes and the up-regulation of 143 genes. The Solexa data were confirmed by reverse transcription polymerase chain reaction (RT-PCR) and real-time quantitative RT-PCR (qRT-PCR). Gene ontology (GO) analysis revealed that the differentially expressed genes were mainly involved in biogenesis, transport and organization of cellular component, macromolecular and cellular biosynthesis, localization, establishment of localization, translation and other processes. Furthermore, the expression of some of these genes depended upon the tissue and the developmental stage of the flowers of gynoecious mutant. The results of this study suggest two important concepts, which govern sex determination in cucumber. First, the differential expression of genes involved in plant hormone signaling pathways, such as ACS, Asr1, CsIAA2, CS-AUX1 and TLP, indicate that phytohormones and their crosstalk might play a critical role in the sex determination. Second, the regulation of some transcription factors, including EREBP-9, may also be involved in this developmental process. Copyright (c) 2010 Elsevier GmbH. All rights reserved.

Effects of the histone-like protein HU on cellulose degradation and biofilm formation of Cytophaga hutchinsonii.

PubMed

Guan, Zhiwei; Wang, Ying; Gao, Lijuan; Zhang, Weican; Lu, Xuemei

2018-06-06

Cytophaga hutchinsonii, belonging to Bacteroidetes, is speculated to use a novel cell-contact mode to digest cellulose. In this study, we identified a histone-like protein HU, CHU_2750, in C. hutchinsonii, whose transcription could be induced by crystalline but not amorphous cellulose. We constructed a CHU_2750-deleted mutant and expressed CHU_2750 in Escherichia coli to study the gene's functions. Our results showed that although the deletion of CHU_2750 was not lethal to C. hutchinsonii, the mutant displayed an abnormal filamentous morphology, loose nucleoid, and obvious defects in the degradation of crystalline cellulose and cell motility. Further study indicated that the mutant displayed significantly decreased cell surface and intracellular endoglucanase activities but with β-glucosidase activities similar to the wild-type strain. Analyses by real-time quantitative PCR revealed that the transcription levels of many genes involved in cellulose degradation and/or cell motility were significantly downregulated in the mutant. In addition, we found that CHU_2750 was important for biofilm formation of C. hutchinsonii. The main extracellular components of the biofilm were analyzed, and the results showed that the mutant yielded significantly less exopolysaccharide but more extracellular DNA and protein than the wild-type strain. Collectively, our findings demonstrated that CHU_2750 is important for cellulose degradation, cell motility, and biofilm formation of C. hutchinsonii by modulating transcription of certain related genes, and it is the first identified transcriptional regulator in these processes of C. hutchinsonii. Our study shed more light on the mechanisms of cellulose degradation, cell motility, and biofilm formation by C. hutchinsonii.

Screen for Leukotoxin Mutants in Aggregatibacter actinomycetemcomitans: Genes of the Phosphotransferase System Are Required for Leukotoxin Biosynthesis▿

PubMed Central

Isaza, Maria P.; Duncan, Matthew S.; Kaplan, Jeffrey B.; Kachlany, Scott C.

2008-01-01

Aggregatibacter (formerly Actinobacillus) actinomycetemcomitans is a pathogen that causes localized aggressive periodontitis and extraoral infections including infective endocarditis. Recently, we reported that A. actinomycetemcomitans is beta-hemolytic on certain growth media due to the production of leukotoxin (LtxA). Based on this observation and our ability to generate random transposon insertions in A. actinomycetemcomitans, we developed and carried out a rapid screen for LtxA mutants. Using PCR, we mapped several of the mutations to genes that are known or predicted to be required for LtxA production, including ltxA, ltxB, ltxD, and tdeA. In addition, we identified an insertion in a gene previously not recognized to be involved in LtxA biosynthesis, ptsH. ptsH encodes the protein HPr, a phosphocarrier protein that is part of the sugar phosphotransferase system. HPr results in the phosphorylation of other proteins and ultimately in the activation of adenylate cyclase and cyclic AMP (cAMP) production. The ptsH mutant showed only partial hemolysis on blood agar and did not produce LtxA. The phenotype was complemented by supplying wild-type ptsH in trans, and real-time PCR analysis showed that the ptsH mutant produced approximately 10-fold less ltxA mRNA than the wild-type strain. The levels of cAMP in the ptsH mutant were significantly lower than in the wild-type strain, and LtxA production could be restored by adding exogenous cAMP to the culture. PMID:18541661

Involvement of the pituitary-specific transcription factor pit-1 in somatolactotrope cell growth and death: an approach using dominant-negative pit-1 mutants.

PubMed

Pellegrini, Isabelle; Roche, Cathy; Quentien, Marie-Helene; Ferrand, Mireille; Gunz, Ginette; Thirion, Sylvie; Bagnis, Claude; Enjalbert, Alain; Franc, Jean-Louis

2006-12-01

The anterior pituitary-specific transcription factor Pit-1 was initially identified and cloned as a transactivator of the prolactin (PRL) and GH genes and later as a regulator of the TSHb gene. It was found to be a major developmental regulator, because natural Pit-1 gene mutations cause a dwarf phenotype in mice and cause combined pituitary hormone deficiency associated with pituitary hypoplasia in humans. To further investigate the growth-promoting effects of Pit-1, we used a strategy based on the use of dominant-negative Pit-1 mutants as an alternative means of inactivating endogenous Pit-1 functions. R271W, a Pit-1 mutant identified in one allele in patients with severe combined pituitary hormone deficiency, and Pit-1Delta1-123, a deletion mutant in which only the DNA binding domain of Pit-1 is conserved, were generated, and their ability to abolish the effects of the endogenous native Pit-1 in the differentiated proliferating somatolactotrope GH4C1 cell line was investigated. Enforced expression of the dominant-negative mutants in GH4C1 cells using recombinant lentiviral vectors decreased the levels of expression of known Pit-1 target genes such as PRL and GH, abolished the hormone release, and reduced cell viability by decreasing the growth rate and inducing apoptosis via a caspase-independent pathway. These results show for the first time that the growth-promoting effects of Pit-1 are at least partly due to the fact that this transcription factor prevents apoptotic cell death.

Effect of sporophytic PIRL9 genotype on post-meiotic expression of the Arabidopsis pirl1;pirl9 mutant pollen phenotype.

PubMed

Forsthoefel, Nancy R; Vernon, Daniel M

2011-02-01

Plant intracellular ras-group-related leucine-rich repeat proteins (PIRLs) are a novel class of plant leucine-rich repeat (LRR) proteins structurally related to animal ras-group LRRs involved in cell signaling and gene regulation. Gene knockout analysis has shown that two members of the Arabidopsis thaliana PIRL gene family, PIRL1 and PIRL9, are redundant and essential for pollen development and viability: pirl1;pirl9 microspores produced by pirl1/PIRL1;pirl9 plants consistently abort just before pollen mitosis I. qrt1 tetrad analysis demonstrated that the genes become essential after meiosis, during anther stage 10. In this study, we characterized the phenotype of pirl1;pirl9 pollen produced by plants heterozygous for pirl9 (pirl1;pirl9/PIRL9). Alexander's staining, scanning electron microscopy, and fluorescence microscopy indicated that pirl1;pirl9 double mutants produced by pirl9 heterozygotes have a less severe phenotype and more variable morphology than pirl1;pirl9 pollen from pirl1/PIRL1;pirl9 plants. Mutant pollen underwent developmental arrest with variable timing, often progressing beyond pollen mitosis I and arresting at the binucleate stage. Thus, although the pirl1 and pirl9 mutations act post-meiosis, the timing and expressivity of the pirl1;pirl9 pollen phenotype depends on the pirl9 genotype of the parent plant. These results suggest a continued requirement for PIRL1 and PIRL9 beyond the initiation of pollen mitosis. Furthermore, they reveal a modest but novel sporophytic effect in which parent plant genotype influences a mutant phenotype expressed in the haploid generation.

Systematic gene deletions evidences that laccases are involved in several stages of wood degradation in the filamentous fungus Podospora anserina.

PubMed

Xie, Ning; Chapeland-Leclerc, Florence; Silar, Philippe; Ruprich-Robert, Gwenaël

2014-01-01

Transformation of plant biomass into biofuels may supply environmentally friendly alternative biological sources of energy. Laccases are supposed to be involved in the lysis of lignin, a prerequisite step for efficient breakdown of cellulose into fermentable sugars. The role in development and plant biomass degradation of the nine canonical laccases belonging to three different subfamilies and one related multicopper oxidase of the Ascomycota fungus Podospora anserina was investigated by targeted gene deletion. The 10 genes were inactivated singly, and multiple mutants were constructed by genetic crosses. lac6(Δ), lac8(Δ) and mco(Δ) mutants were significantly reduced in their ability to grow on lignin-containing materials, but also on cellulose and plastic. Furthermore, lac8(Δ), lac7(Δ), mco(Δ) and lac6(Δ) mutants were defective towards resistance to phenolic substrates and H2 O2 , which may also impact lignocellulose breakdown. Double and multiple mutants were generally more affected than single mutants, evidencing redundancy of function among laccases. Our study provides the first genetic evidences that laccases are major actors of wood utilization in a fungus and that they have multiple roles during this process apart from participation in lignin lysis. © 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.

Arabidopsis ECERIFERUM9 involvement in cuticle formation and maintenance of plant water status

USDA-ARS?s Scientific Manuscript database

A unique set of allelic Arabidopsis mutants are described that exhibit either suppressed or completely inhibited expression of a gene designated ECERIFERUM9 (CER9). These mutants exhibit a dramatic elevation in the total amount of leaf cutin monomers, and a dramatic shift in the leaf cuticular wax p...

PDF receptor signaling in Caenorhabditis elegans modulates locomotion and egg-laying.

PubMed

Meelkop, Ellen; Temmerman, Liesbet; Janssen, Tom; Suetens, Nick; Beets, Isabel; Van Rompay, Liesbeth; Shanmugam, Nilesh; Husson, Steven J; Schoofs, Liliane

2012-09-25

In Caenorhabditis elegans, pdfr-1 encodes three receptors of the secretin receptor family. These G protein-coupled receptors are activated by three neuropeptides, pigment dispersing factors 1a, 1b and 2, which are encoded by pdf-1 and pdf-2. We isolated a PDF receptor loss-of-function allele (lst34) by means of a mutagenesis screen and show that the PDF signaling system is involved in locomotion and egg-laying. We demonstrate that the pdfr-1 mutant phenocopies the defective locomotor behavior of the pdf-1 mutant and that pdf-1 and pdf-2 behave antagonistically. All three PDF receptor splice variants are involved in the regulation of locomotor behavior. Cell specific rescue experiments show that this pdf mediated behavior is regulated by neurons rather than body wall muscles. We also show that egg-laying patterns of pdf-1 and pdf-2 mutants are affected, but not those of pdfr-1 mutants, pointing to a novel role for the PDF-system in the regulation of egg-laying. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Cell Transformation by PTP1B Truncated Mutants Found in Human Colon and Thyroid Tumors.

PubMed

Mei, Wenhan; Wang, Kemin; Huang, Jian; Zheng, Xinmin

2016-01-01

Expression of wild-type protein tyrosine phosphatase (PTP) 1B may act either as a tumor suppressor by dysregulation of protein tyrosine kinases or a tumor promoter through Src dephosphorylation at Y527 in human breast cancer cells. To explore whether mutated PTP1B is involved in human carcinogenesis, we have sequenced PTP1B cDNAs from human tumors and found splice mutations in ~20% of colon and thyroid tumors. The PTP1BΔE6 mutant expressed in these two tumor types and another PTP1BΔE5 mutant expressed in colon tumor were studied in more detail. Although PTP1BΔE6 revealed no phosphatase activity compared with wild-type PTP1B and the PTP1BΔE5 mutant, its expression induced oncogenic transformation of rat fibroblasts without Src activation, indicating that it involved signaling pathways independent of Src. The transformed cells were tumourigenic in nude mice, suggesting that the PTP1BΔE6 affected other molecule(s) in the human tumors. These observations may provide a novel therapeutic target for colon and thyroid cancer.

Disruption of the Arabidopsis Defense Regulator Genes SAG101, EDS1, and PAD4 Confers Enhanced Freezing Tolerance

PubMed Central

Chen, Qin-Fang; Xu, Le; Tan, Wei-Juan; Chen, Liang; Qi, Hua; Xie, Li-Juan; Chen, Mo-Xian; Liu, Bin-Yi; Yu, Lu-Jun; Yao, Nan; Zhang, Jian-Hua; Shu, Wensheng; Xiao, Shi

2017-01-01

In Arabidopsis, three lipase-like regulators, SAG101, EDS1, and PAD4, act downstream of resistance protein-associated defense signaling. Although the roles of SAG101, EDS1, and PAD4 in biotic stress have been extensively studied, little is known about their functions in plant responses to abiotic stresses. Here, we show that SAG101, EDS1, and PAD4 are involved in the regulation of freezing tolerance in Arabidopsis. With or without cold acclimation, the sag101, eds1, and pad4 single mutants, as well as their double mutants exhibited similarly enhanced tolerance to freezing temperatures. Upon cold exposure, the sag101, eds1, and pad4 mutants showed increased transcript levels of C-REPEAT/DRE BINDING FACTORs and their regulons, compared with wild type. Moreover, freezing-induced cell death and accumulation of hydrogen peroxide were ameliorated in sag101, eds1, and pad4 mutants. The sag101, eds1, and pad4 mutants had much lower salicylic acid (SA) and diacylglycerol (DAG) contents than wild type and exogenous application of SA and DAG compromised the freezing tolerance of the mutants. Furthermore, SA suppressed the cold-induced expression of DGATs and DGKs in wild-type leaves. These findings indicate that SAG101, EDS1, and PAD4 are involved in freezing response in Arabidopsis, at least in part, by modulating the homeostasis of SA and DAG. PMID:26149542

A combinatorial strategy for treating KRAS mutant lung cancer

PubMed Central

Manchado, Eusebio; Weissmueller, Susann; Morris, John P.; Chen, Chi-Chao; Wullenkord, Ramona; Lujambio, Amaia; de Stanchina, Elisa; Poirier, John T.; Gainor, Justin F.; Corcoran, Ryan B.; Engelman, Jeffrey A.; Rudin, Charles M.; Rosen, Neal; Lowe, Scott W.

2016-01-01

Therapeutic targeting of KRAS-mutant lung adenocarcinoma represents a major goal of clinical oncology. KRAS itself has proven difficult to inhibit, and the effectiveness of agents that target key KRAS effectors has been thwarted by activation of compensatory or parallel pathways that limit their efficacy as single agents. Here we take a systematic approach towards identifying combination targets for trametinib, an FDA-approved MEK inhibitor that acts downstream of KRAS to suppress signaling through the mitogen-activated protein kinase (MAPK) cascade. Informed by a short-hairpin RNA (shRNA) screen, we show that trametinib provokes a compensatory response involving the fibroblast growth factor receptor 1 (FGFR1) that leads to signaling rebound and adaptive drug resistance. As a consequence, genetic or pharmacologic inhibition of FGFR1 in combination with trametinib enhances tumor cell death in vitro and in vivo. This compensatory response shows distinct specificities – it is dominated by FGFR1 in KRAS mutant lung and pancreatic cancer cells, but is not activated or involves other mechanisms in KRAS wild-type lung and KRAS-mutant colon cancer cells. Importantly, KRAS-mutant lung cancer cells and patient tumors treated with trametinib show an increase in FRS2 phosphorylation, a biomarker of FGFR activation; this increase is abolished by FGFR1 inhibition and correlates with sensitivity to trametinib and FGFR inhibitor combinations. These results demonstrate that FGFR1 can mediate adaptive resistance to trametinib and validate a combinatorial approach for treating KRAS-mutant lung cancer. PMID:27338794

Heterotrimeric G proteins-mediated resistance to necrotrophic pathogens includes mechanisms independent of salicylic acid-, jasmonic acid/ethylene- and abscisic acid-mediated defense signaling.

PubMed

Trusov, Yuri; Sewelam, Nasser; Rookes, James Edward; Kunkel, Matt; Nowak, Ekaterina; Schenk, Peer Martin; Botella, José Ramón

2009-04-01

Heterotrimeric G proteins are involved in the defense response against necrotrophic fungi in Arabidopsis. In order to elucidate the resistance mechanisms involving heterotrimeric G proteins, we analyzed the effects of the Gβ (subunit deficiency in the mutant agb1-2 on pathogenesis-related gene expression, as well as the genetic interaction between agb1-2 and a number of mutants of established defense pathways. Gβ-mediated signaling suppresses the induction of salicylic acid (SA)-, jasmonic acid (JA)-, ethylene (ET)- and abscisic acid (ABA)-dependent genes during the initial phase of the infection with Fusarium oxysporum (up to 48 h after inoculation). However, at a later phase it enhances JA/ET-dependent genes such as PDF1.2 and PR4. Quantification of the Fusarium wilt symptoms revealed that Gβ- and SA-deficient mutants were more susceptible than wild-type plants, whereas JA- and ET-insensitive and ABA-deficient mutants demonstrated various levels of resistance. Analysis of the double mutants showed that the Gβ-mediated resistance to F. oxysporum and Alternaria brassicicola was mostly independent of all of the previously mentioned pathways. However, the progressive decay of agb1-2 mutants was compensated by coi1-21 and jin1-9 mutations, suggesting that at this stage of F. oxysporum infection Gβ acts upstream of COI1 and ATMYC2 in JA signaling. © 2008 The Authors. Journal compilation © 2008 Blackwell Publishing Ltd.

Loss of DDB1 Leads to Transcriptional p53 Pathway Activation in Proliferating Cells, Cell Cycle Deregulation, and Apoptosis in Zebrafish Embryos.

PubMed

Hu, Zhilian; Holzschuh, Jochen; Driever, Wolfgang

2015-01-01

DNA damage-binding protein 1 (DDB1) is a large subunit of the heterodimeric DDB complex that recognizes DNA lesions and initiates the nucleotide excision repair process. DDB1 is also a component of the CUL4 E3 ligase complex involved in a broad spectrum of cellular processes by targeted ubiquitination of key regulators. Functions of DDB1 in development have been addressed in several model organisms, however, are not fully understood so far. Here we report an ENU induced mutant ddb1 allele (ddb1m863) identified in zebrafish (Danio rerio), and analyze its effects on development. Zebrafish ddb1 is expressed broadly, both maternally and zygotically, with enhanced expression in proliferation zones. The (ddb1m863 mutant allele affects the splice acceptor site of exon 20, causing a splicing defect that results in truncation of the 1140 amino acid protein after residue 800, lacking part of the β-propeller domain BPC and the C-terminal helical domain CTD. ddb1m863 zygotic mutant embryos have a pleiotropic phenotype, including smaller and abnormally shaped brain, head skeleton, eyes, jaw, and branchial arches, as well as reduced dopaminergic neuron groups. However, early forming tissues develop normally in zygotic ddb1m863 mutant embryos, which may be due to maternal rescue. In ddb1m863 mutant embryos, pcna-expressing proliferating cell populations were reduced, concurrent with increased apoptosis. We also observed a concomitant strong up-regulation of transcripts of the tumor suppressor p53 (tp53) and the cell cycle inhibitor cdkn1a (p21a/bCIP1/WAF1) in proliferating tissues. In addition, transcription of cyclin genes ccna2 and ccnd1 was deregulated in ddb1m863 mutants. Reduction of p53 activity by anti-sense morpholinos alleviated the apoptotic phenotype in ddb1m863 mutants. These results imply that Ddb1 may be involved in maintaining proper cell cycle progression and viability of dividing cells during development through transcriptional mechanisms regulating genes involved in cell cycle control and cell survival.

Identification of Proteus mirabilis Mutants with Increased Sensitivity to Antimicrobial Peptides

PubMed Central

McCoy, Andrea J.; Liu, Hongjian; Falla, Timothy J.; Gunn, John S.

2001-01-01

Antimicrobial peptides (APs) are important components of the innate defenses of animals, plants, and microorganisms. However, some bacterial pathogens are resistant to the action of APs. For example, Proteus mirabilis is highly resistant to the action of APs, such as polymyxin B (PM), protegrin, and the synthetic protegrin analog IB-367. To better understand this resistance, a transposon mutagenesis approach was used to generate P. mirabilis mutants sensitive to APs. Four unique PM-sensitive mutants of P. mirabilis were identified (these mutants were >2 to >128 times more sensitive than the wild type). Two of these mutants were also sensitive to IB-367 (16 and 128 times more sensitive than the wild type). Lipopolysaccharide (LPS) profiles of the PM- and protegrin-sensitive mutants demonstrated marked differences in both the lipid A and O-antigen regions, while the PM-sensitive mutants appeared to have alterations of either lipid A or O antigen. Matrix-assisted laser desorption ionization–time of flight mass spectrometry analysis of the wild-type and PM-sensitive mutant lipid A showed species with one or two aminoarabinose groups, while lipid A from the PM- and protegrin-sensitive mutants was devoid of aminoarabinose. When the mutants were streaked on an agar-containing medium, the swarming motility of the PM- and protegrin-sensitive mutants was completely inhibited and the swarming motility of the mutants sensitive to only PM was markedly decreased. DNA sequence analysis of the mutagenized loci revealed similarities to an O-acetyltransferase (PM and protegrin sensitive) and ATP synthase and sap loci (PM sensitive). These data further support the role of LPS modifications as an elaborate mechanism in the resistance of certain bacterial species to APs and suggest that LPS surface charge alterations may play a role in P. mirabilis swarming motility. PMID:11408219

Lysis Delay and Burst Shrinkage of Coliphage T7 by Deletion of Terminator Tφ Reversed by Deletion of Early Genes

PubMed Central

Nguyen, Huong Minh

2014-01-01

ABSTRACT Bacteriophage T7 terminator Tφ is a class I intrinsic terminator coding for an RNA hairpin structure immediately followed by oligo(U), which has been extensively studied in terms of its transcription termination mechanism, but little is known about its physiological or regulatory functions. In this study, using a T7 mutant phage, where a 31-bp segment of Tφ was deleted from the genome, we discovered that deletion of Tφ from T7 reduces the phage burst size but delays lysis timing, both of which are disadvantageous for the phage. The burst downsizing could directly result from Tφ deletion-caused upregulation of gene 17.5, coding for holin, among other Tφ downstream genes, because infection of gp17.5-overproducing Escherichia coli by wild-type T7 phage showed similar burst downsizing. However, the lysis delay was not associated with cellular levels of holin or lysozyme or with rates of phage adsorption. Instead, when allowed to evolve spontaneously in five independent adaptation experiments, the Tφ-lacking mutant phage, after 27 or 29 passages, recovered both burst size and lysis time reproducibly by deleting early genes 0.5, 0.6, and 0.7 of class I, among other mutations. Deletion of genes 0.5 to 0.7 from the Tφ-lacking mutant phage decreased expression of several Tφ downstream genes to levels similar to that of the wild-type phage. Accordingly, phage T7 lysis timing is associated with cellular levels of Tφ downstream gene products. This suggests the involvement of unknown factor(s) besides the known lysis proteins, lysozyme and holin, and that Tφ plays a role of optimizing burst size and lysis time during T7 infection. IMPORTANCE E. coli PMID:24335287

Identification of opsA, a Gene Involved in Solute Stress Mitigation and Survival in Soil, in the Polycyclic Aromatic Hydrocarbon-Degrading Bacterium Novosphingobium sp. Strain LH128

PubMed Central

Fida, Tekle Tafese; Breugelmans, Philip; Lavigne, Rob; van der Meer, Jan Roelof; De Mot, René; Vaysse, Pierre-Joseph

2014-01-01

The aim of this study was to identify genes involved in solute and matric stress mitigation in the polycyclic aromatic hydrocarbon (PAH)-degrading Novosphingobium sp. strain LH128. The genes were identified using plasposon mutagenesis and by selection of mutants that showed impaired growth in a medium containing 450 mM NaCl as a solute stress or 10% (wt/vol) polyethylene glycol (PEG) 6000 as a matric stress. Eleven and 14 mutants showed growth impairment when exposed to solute and matric stresses, respectively. The disrupted sequences were mapped on a draft genome sequence of strain LH128, and the corresponding gene functions were predicted. None of them were shared between solute and matric stress-impacted mutants. One NaCl-affected mutant (i.e., NA7E1) with a disruption in a gene encoding a putative outer membrane protein (OpsA) was susceptible to lower NaCl concentrations than the other mutants. The growth of NA7E1 was impacted by other ions and nonionic solutes and by sodium dodecyl sulfate (SDS), suggesting that opsA is involved in osmotic stress mitigation and/or outer membrane stability in strain LH128. NA7E1 was also the only mutant that showed reduced growth and less-efficient phenanthrene degradation in soil compared to the wild type. Moreover, the survival of NA7E1 in soil decreased significantly when the moisture content was decreased but was unaffected when soluble solutes from sandy soil were removed by washing. opsA appears to be important for the survival of strain LH128 in soil, especially in the case of reduced moisture content, probably by mitigating the effects of solute stress and retaining membrane stability. PMID:24657861

Identification and analysis of novel genes involved in gravitropism of Arabidopsis thaliana.

NASA Astrophysics Data System (ADS)

Morita, Miyo T.; Tasaka, Masao; Masatoshi Taniguchi, .

2012-07-01

Gravitropism is a continuous control with regard to the orientation and juxtaposition of the various parts of the plant body in response to gravity. In higher plants, the relative directional change of gravity is mainly suscepted in specialized cells called statocytes, followed by signal conversion from physical information into physiological information within the statocytes. We have studied the early process of shoot gravitropism, gravity sensing and signaling process, mainly by molecular genetic approach. In Arabidopsis shoot, statocytes are the endodermal cells. sgr1/scarcrow (scr) and sgr7/short-root (shr) mutants fail to form the endodermis and to respond to gravity in their inflorescence stems. Since both SGR1/SCR and SGR7/SHR are transcriptional factors, at least a subset of their downstream genes can be expected to be involved in gravitropism. In addition, eal1 (endodermal-amyloplast less 1), which exhibits no gravitropism in inflorescence stem but retains ability to form endodermis, is a hypomorphic allele of sgr7/shr. Take advantage of these mutants, we performed DNA microarray analysis and compared gene expression profiles between wild type and the mutants. We found that approx. 40 genes were commonly down-regulated in these mutants and termed them DGE (DOWN-REGULATED GENE IN EAL1) genes. DGE1 has sequence similarity to Oryza sativa LAZY1 that is involved in shoot gravitropism of rice. DGE2 has a short region homologous to DGE1. DTL (DGE TWO-LIKE}) that has 54% identity to DGE2 is found in Arabidopsis genome. All three genes are conserved in angiosperm but have no known functional domains or motifs. We analyzed T-DNA insertion for these genes in single or multiple combinations. In dge1 dge2 dtl triple mutant, gravitropic response of shoot, hypocotyl and root dramatically reduced. Now we are carrying out further physiological and molecular genetic analysis of the triple mutant.

BMP-Mediated Functional Cooperation between Dlx5;Dlx6 and Msx1;Msx2 during Mammalian Limb Development

PubMed Central

Vieux-Rochas, Maxence; Bouhali, Kamal; Mantero, Stefano; Garaffo, Giulia; Provero, Paolo; Astigiano, Simonetta; Barbieri, Ottavia; Caratozzolo, Mariano F.; Tullo, Apollonia; Guerrini, Luisa; Lallemand, Yvan; Robert, Benoît

2013-01-01

The Dlx and Msx homeodomain transcription factors play important roles in the control of limb development. The combined disruption of Msx1 and Msx2, as well as that of Dlx5 and Dlx6, lead to limb patterning defects with anomalies in digit number and shape. Msx1;Msx2 double mutants are characterized by the loss of derivatives of the anterior limb mesoderm which is not observed in either of the simple mutants. Dlx5;Dlx6 double mutants exhibit hindlimb ectrodactyly. While the morphogenetic action of Msx genes seems to involve the BMP molecules, the mode of action of Dlx genes still remains elusive. Here, examining the limb phenotypes of combined Dlx and Msx mutants we reveal a new Dlx-Msx regulatory loop directly involving BMPs. In Msx1;Dlx5;Dlx6 triple mutant mice (TKO), beside the expected ectrodactyly, we also observe the hallmark morphological anomalies of Msx1;Msx2 double mutants suggesting an epistatic role of Dlx5 and Dlx6 over Msx2. In Msx2;Dlx5;Dlx6 TKO mice we only observe an aggravation of the ectrodactyly defect without changes in the number of the individual components of the limb. Using a combination of qPCR, ChIP and bioinformatic analyses, we identify two Dlx/Msx regulatory pathways: 1) in the anterior limb mesoderm a non-cell autonomous Msx-Dlx regulatory loop involves BMP molecules through the AER and 2) in AER cells and, at later stages, in the limb mesoderm the regulation of Msx2 by Dlx5 and Dlx6 occurs also cell autonomously. These data bring new elements to decipher the complex AER-mesoderm dialogue that takes place during limb development and provide clues to understanding the etiology of congenital limb malformations. PMID:23382810

BMP-mediated functional cooperation between Dlx5;Dlx6 and Msx1;Msx2 during mammalian limb development.

PubMed

Vieux-Rochas, Maxence; Bouhali, Kamal; Mantero, Stefano; Garaffo, Giulia; Provero, Paolo; Astigiano, Simonetta; Barbieri, Ottavia; Caratozzolo, Mariano F; Tullo, Apollonia; Guerrini, Luisa; Lallemand, Yvan; Robert, Benoît; Levi, Giovanni; Merlo, Giorgio R

2013-01-01

The Dlx and Msx homeodomain transcription factors play important roles in the control of limb development. The combined disruption of Msx1 and Msx2, as well as that of Dlx5 and Dlx6, lead to limb patterning defects with anomalies in digit number and shape. Msx1;Msx2 double mutants are characterized by the loss of derivatives of the anterior limb mesoderm which is not observed in either of the simple mutants. Dlx5;Dlx6 double mutants exhibit hindlimb ectrodactyly. While the morphogenetic action of Msx genes seems to involve the BMP molecules, the mode of action of Dlx genes still remains elusive. Here, examining the limb phenotypes of combined Dlx and Msx mutants we reveal a new Dlx-Msx regulatory loop directly involving BMPs. In Msx1;Dlx5;Dlx6 triple mutant mice (TKO), beside the expected ectrodactyly, we also observe the hallmark morphological anomalies of Msx1;Msx2 double mutants suggesting an epistatic role of Dlx5 and Dlx6 over Msx2. In Msx2;Dlx5;Dlx6 TKO mice we only observe an aggravation of the ectrodactyly defect without changes in the number of the individual components of the limb. Using a combination of qPCR, ChIP and bioinformatic analyses, we identify two Dlx/Msx regulatory pathways: 1) in the anterior limb mesoderm a non-cell autonomous Msx-Dlx regulatory loop involves BMP molecules through the AER and 2) in AER cells and, at later stages, in the limb mesoderm the regulation of Msx2 by Dlx5 and Dlx6 occurs also cell autonomously. These data bring new elements to decipher the complex AER-mesoderm dialogue that takes place during limb development and provide clues to understanding the etiology of congenital limb malformations.

Role for the Ankyrin eukaryotic-like genes of Legionella pneumophila in parasitism of protozoan hosts and human macrophages.

PubMed

Habyarimana, Fabien; Al-Khodor, Souhaila; Kalia, Awdhesh; Graham, James E; Price, Christopher T; Garcia, Maria Teresa; Kwaik, Yousef Abu

2008-06-01

Legionella pneumophila is a ubiquitous organism in the aquatic environment where it is capable of invasion and intracellular proliferation within various protozoan species and is also capable of causing pneumonia in humans. In silico analysis showed that the three sequenced L. pneumophila genomes each contained a common multigene family of 11 ankyrin (ank) genes encoding proteins with approximately 30-35 amino acid tandem Ankyrin repeats that are involved in protein-protein interactions in eukaryotic cells. To examine whether the ank genes are involved in tropism of protozoan hosts, we have constructed isogenic mutants of L. pneumophila in ten of the ank genes. Among the mutants, the DeltaankH and DeltaankJ mutants exhibit significant defects in robust intracellular replication within A. polyphaga, Hartmanella vermiformis and Tetrahymena pyriformis. A similar defect is also exhibited in human macrophages. Most of the ank genes are upregulated by L. pneumophila upon growth transition into the post-exponential phase in vitro and within Acanthamoeba polyphaga, and this upregulation is mediated, at least in part, by RpoS. Single-cell analyses have shown that upon co-infection of the wild-type strain with the ankH or ankJ mutant, the replication defect of the mutant is rescued within communal phagosomes harbouring the wild-type strain, similar to dot/icm mutants. Therefore, at least two of the L. pneumophila eukaryotic-like Ank proteins play a role in intracellular replication of L. pneumophila within amoeba, ciliated protozoa and human macrophages. The Ank proteins may not be involved in host tropism in the aquatic environment. Many of the L. pneumophila eukaryotic-like ank genes are triggered upon growth transition into post-exponential phase in vitro as well as within A. polyphaga. Our data suggest a role for AnkH and AnkJ in modulation of phagosome biogenesis by L. pneumophila independent of evasion of lysosomal fusion and recruitment of the rough endoplasmic reticulum.

A cell–cell signaling sensor is required for virulence and insect transmission of Xylella fastidiosa

PubMed Central

Chatterjee, Subhadeep; Wistrom, Christina; Lindow, Steven E.

2008-01-01

Cell–cell signaling in Xylella fastidiosa, a xylem-colonizing plant pathogenic bacterium, mediated by a fatty acid Diffusible Signaling Factor (DSF), is required to colonize insect vectors and to suppress virulence to grape. Here, we show that a hybrid two-component regulatory protein RpfC is involved in negative regulation of DSF synthesis by RpfF in X. fastidiosa. X. fastidiosa rpfC mutants hyperexpress rpfF and overproduce DSF and are deficient in virulence and movement in the xylem vessels of grape. The expression of the genes encoding the adhesins FimA, HxfA, and HxfB is much higher in rpfC mutants, which also exhibit a hyperattachment phenotype in culture that is associated with their inability to migrate in xylem vessels and cause disease. rpfF mutants deficient in DSF production have the opposite phenotypes for all of these traits. RpfC is also involved in the regulation of other signaling components including rpfG, rpfB, a GGDEF domain protein that may be involved in intracellular signaling by modulating the levels of cyclic-di-GMP, and the virulence factors tolC and pglA required for disease. rpfC mutants are able to colonize the mouthparts of insect vectors and wild-type strains but are not transmitted as efficiently to new host plants, apparently because of their high levels of adhesiveness. Because of the conflicting contributions of adhesiveness and other traits to movement within plants and vectoring to new host plants, X. fastidiosa apparently coordinates these traits in a population-size-dependent fashion involving accumulation of DSF. PMID:18268318

A cell-cell signaling sensor is required for virulence and insect transmission of Xylella fastidiosa.

PubMed

Chatterjee, Subhadeep; Wistrom, Christina; Lindow, Steven E

2008-02-19

Cell-cell signaling in Xylella fastidiosa, a xylem-colonizing plant pathogenic bacterium, mediated by a fatty acid Diffusible Signaling Factor (DSF), is required to colonize insect vectors and to suppress virulence to grape. Here, we show that a hybrid two-component regulatory protein RpfC is involved in negative regulation of DSF synthesis by RpfF in X. fastidiosa. X. fastidiosa rpfC mutants hyperexpress rpfF and overproduce DSF and are deficient in virulence and movement in the xylem vessels of grape. The expression of the genes encoding the adhesins FimA, HxfA, and HxfB is much higher in rpfC mutants, which also exhibit a hyperattachment phenotype in culture that is associated with their inability to migrate in xylem vessels and cause disease. rpfF mutants deficient in DSF production have the opposite phenotypes for all of these traits. RpfC is also involved in the regulation of other signaling components including rpfG, rpfB, a GGDEF domain protein that may be involved in intracellular signaling by modulating the levels of cyclic-di-GMP, and the virulence factors tolC and pglA required for disease. rpfC mutants are able to colonize the mouthparts of insect vectors and wild-type strains but are not transmitted as efficiently to new host plants, apparently because of their high levels of adhesiveness. Because of the conflicting contributions of adhesiveness and other traits to movement within plants and vectoring to new host plants, X. fastidiosa apparently coordinates these traits in a population-size-dependent fashion involving accumulation of DSF.

Isolation of peroxisome-deficient mutants of Saccharomyces cerevisiae.

PubMed Central

Erdmann, R; Veenhuis, M; Mertens, D; Kunau, W H

1989-01-01

Two mutants of Saccharomyces cerevisiae affected in peroxisomal assembly (pas mutants) have been isolated and characterized. Each strain contains a single mutation that results in (i) the inability to grow on oleic acid, (ii) accumulation of peroxisomal matrix enzymes in the cytosol, and (iii) absence of detectable peroxisomes at the ultrastructural level. These lesions (pas1-1 and pas2) are shown to be nonallelic and recessive. Crossing of pas1-1 and pas2 strains resulted in diploid cells that had regained the ability to grow on oleic acid as sole carbon source and to form peroxisomes. These pas mutants may provide useful tools for future studies on the molecular mechanisms involved in peroxisomal assembly. Images PMID:2568633

Arabidopsis thickvein mutation affects vein thickness and organ vascularization, and resides in a provascular cell-specific spermine synthase involved in vein definition and in polar auxin transport.

PubMed

Clay, Nicole K; Nelson, Timothy

2005-06-01

Polar auxin transport has been implicated in the induction of vascular tissue and in the definition of vein positions. Leaves treated with chemical inhibitors of polar auxin transport exhibited vascular phenotypes that include increased vein thickness and vascularization. We describe a recessive mutant, thickvein (tkv), which develops thicker veins in leaves and in inflorescence stems. The increased vein thickness is attributable to an increased number of vascular cells. Mutant plants have smaller leaves and shorter inflorescence stems, and this reduction in organ size and height is accompanied by an increase in organ vascularization, which appears to be attributable to an increase in the recruitment of cells into veins. Furthermore, although floral development is normal, auxin transport in the inflorescence stem is significantly reduced in the mutant, suggesting that the defect in auxin transport is responsible for the vascular phenotypes. In the primary root, the veins appear morphologically normal, but root growth in the tkv mutant is hypersensitive to exogenous cytokinin. The tkv mutation was found to reside in the ACL5 gene, which encodes a spermine synthase and whose expression is specific to provascular cells. We propose that ACL5/TKV is involved in vein definition (defining the boundaries between veins and nonvein regions) and in polar auxin transport, and that polyamines are involved in this process.

Arabidopsis thickvein Mutation Affects Vein Thickness and Organ Vascularization, and Resides in a Provascular Cell-Specific Spermine Synthase Involved in Vein Definition and in Polar Auxin Transport1

PubMed Central

Clay, Nicole K.; Nelson, Timothy

2005-01-01

Polar auxin transport has been implicated in the induction of vascular tissue and in the definition of vein positions. Leaves treated with chemical inhibitors of polar auxin transport exhibited vascular phenotypes that include increased vein thickness and vascularization. We describe a recessive mutant, thickvein (tkv), which develops thicker veins in leaves and in inflorescence stems. The increased vein thickness is attributable to an increased number of vascular cells. Mutant plants have smaller leaves and shorter inflorescence stems, and this reduction in organ size and height is accompanied by an increase in organ vascularization, which appears to be attributable to an increase in the recruitment of cells into veins. Furthermore, although floral development is normal, auxin transport in the inflorescence stem is significantly reduced in the mutant, suggesting that the defect in auxin transport is responsible for the vascular phenotypes. In the primary root, the veins appear morphologically normal, but root growth in the tkv mutant is hypersensitive to exogenous cytokinin. The tkv mutation was found to reside in the ACL5 gene, which encodes a spermine synthase and whose expression is specific to provascular cells. We propose that ACL5/TKV is involved in vein definition (defining the boundaries between veins and nonvein regions) and in polar auxin transport, and that polyamines are involved in this process. PMID:15894745

Identification of Genes Associated with Resilience/Vulnerability to Sleep Deprivation and Starvation in Drosophila

PubMed Central

Thimgan, Matthew S.; Seugnet, Laurent; Turk, John; Shaw, Paul J.

2015-01-01

Background and Study Objectives: Flies mutant for the canonical clock protein cycle (cyc01) exhibit a sleep rebound that is ∼10 times larger than wild-type flies and die after only 10 h of sleep deprivation. Surprisingly, when starved, cyc01 mutants can remain awake for 28 h without demonstrating negative outcomes. Thus, we hypothesized that identifying transcripts that are differentially regulated between waking induced by sleep deprivation and waking induced by starvation would identify genes that underlie the deleterious effects of sleep deprivation and/or protect flies from the negative consequences of waking. Design: We used partial complementary DNA microarrays to identify transcripts that are differentially expressed between cyc01 mutants that had been sleep deprived or starved for 7 h. We then used genetics to determine whether disrupting genes involved in lipid metabolism would exhibit alterations in their response to sleep deprivation. Setting: Laboratory. Patients or Participants: Drosophila melanogaster. Interventions: Sleep deprivation and starvation. Measurements and Results: We identified 84 genes with transcript levels that were differentially modulated by 7 h of sleep deprivation and starvation in cyc01 mutants and were confirmed in independent samples using quantitative polymerase chain reaction. Several of these genes were predicted to be lipid metabolism genes, including bubblegum, cueball, and CG4500, which based on our data we have renamed heimdall (hll). Using lipidomics we confirmed that knockdown of hll using RNA interference significantly decreased lipid stores. Importantly, genetically modifying bubblegum, cueball, or hll resulted in sleep rebound alterations following sleep deprivation compared to genetic background controls. Conclusions: We have identified a set of genes that may confer resilience/vulnerability to sleep deprivation and demonstrate that genes involved in lipid metabolism modulate sleep homeostasis. Citation: Thimgan MS, Seugnet L, Turk J, Shaw PJ. Identification of genes associated with resilience/vulnerability to sleep deprivation and starvation in Drosophila. SLEEP 2015;38(5):801–814. PMID:25409104

High-throughput measurement of recombination rates and genetic interference in Saccharomyces cerevisiae.

PubMed

Raffoux, Xavier; Bourge, Mickael; Dumas, Fabrice; Martin, Olivier C; Falque, Matthieu

2018-06-01

Allelic recombination owing to meiotic crossovers is a major driver of genome evolution, as well as a key player for the selection of high-performing genotypes in economically important species. Therefore, we developed a high-throughput and low-cost method to measure recombination rates and crossover patterning (including interference) in large populations of the budding yeast Saccharomyces cerevisiae. Recombination and interference were analysed by flow cytometry, which allows time-consuming steps such as tetrad microdissection or spore growth to be avoided. Moreover, our method can also be used to compare recombination in wild-type vs. mutant individuals or in different environmental conditions, even if the changes in recombination rates are small. Furthermore, meiotic mutants often present recombination and/or pairing defects affecting spore viability but our method does not involve growth steps and thus avoids filtering out non-viable spores. Copyright © 2018 John Wiley & Sons, Ltd.

Non-cell autonomous cell death caused by transmission of Huntingtin aggregates in Drosophila.

PubMed

Babcock, Daniel T; Ganetzky, Barry

2015-01-01

Recent evidence indicates that protein aggregates can spread between neurons in several neurodegenerative diseases but much remains unknown regarding the underlying mechanisms responsible for this spreading and its role in disease progression. We recently demonstrated that mutant Huntingtin aggregates spread between cells within the Drosophila brain resulting in non-cell autonomous loss of a pair of large neurons in the posterior protocerebrum. However, the full extent of neuronal loss throughout the brain was not determined. Here we examine the effects of driving expression of mutant Huntingtin in Olfactory Receptor Neurons (ORNs) by using a marker for cleaved caspase activity to monitor neuronal apoptosis as a function of age. We find widespread caspase activity in various brain regions over time, demonstrating that non-cell autonomous damage is widespread. Improved understanding of which neurons are most vulnerable and why should be useful in developing treatment strategies for neurodegenerative diseases that involve transcellular spreading of aggregates.

Elongator promotes germination and early post-germination growth.

PubMed

Woloszynska, Magdalena; Gagliardi, Olimpia; Vandenbussche, Filip; Van Lijsebettens, Mieke

2018-01-02

The Elongator complex interacts with RNA polymerase II and via histone acetylation and DNA demethylation facilitates epigenetically the transcription of genes involved in diverse processes in plants, including growth, development, and immune response. Recently, we have shown that the Elongator complex promotes hypocotyl elongation and photomorphogenesis in Arabidopsis thaliana by regulating the photomorphogenesis and growth-related gene network that converges on genes implicated in cell wall biogenesis and hormone signaling. Here, we report that germination in the elo mutant was delayed by 6 h in the dark when compared to the wild type in a time lapse and germination assay. A number of germination-correlated genes were down-regulated in the elo transcriptome, suggesting a transcriptional regulation by Elongator. We also show that the hypocotyl elongation defect observed in the elo mutants in darkness originates very early in the post-germination development and is independent from the germination delay.

Sin1, a Mutation Affecting Female Fertility in Arabidopsis, Interacts with Mod1, Its Recessive Modifier

PubMed Central

Lang, J. D.; Ray, S.; Ray, A.

1994-01-01

In Arabidopsis thaliana, a mutation in the SIN1 gene causes aberrant ovule development and female-specific sterility. The effect of the sin1 mutation is polymorphic and pleiotropic in different genetic backgrounds. The polymorphism concerns morphology of the mutant ovules. The pleiotropism involves internodal distance and inflorescence initiation time. The particular ovule phenotype and the length of internodes are dependent on an interaction of sin1 with a second recessive gene, which we term mod1. The recessive mod1 allele in a homozygous sin1 mutant plant reduces internode length and ovule integument size. The mutation sin1, but not mod1, has a demonstrable effect on ovule morphology when acting idependently. In our crosses mod1 was inseparably linked to the well known mutation erecta that is known to cause a reduction in internode and pedicel lengths. PMID:7982564

Neutron Radiation Affects the Expression of Genes Involved in the Response to Auxin, Senescence and Oxidative Stress in Arabidopsis

NASA Astrophysics Data System (ADS)

Fortunati, A.; Tassone, P.; Migliaccio, F.

2008-06-01

Researches were conducted on the effect of neutron radiation on the expression of genes auxin activated or connected with the process of senescence in Arabidopsis plants. The research was done by applying the real-time polymerase chain reaction (PCR) technique. The results indicated that the auxin response factors (ARFs) genes are clearly downregulated, whereas the indolacetic acid-induced (Aux/IAAs) genes in some cases were upregulated. By contrast in the mutants for auxin transport aux1 and eir1 the ARFs genes were upregulated. In addition, both in the wildtype and mutants, some already known genes activated by stress and senescence were significantly upregulated. On the basis of these researches we conclude that the process of senescence induced by irradiation is, at least in part, controlled by the physiology of the hormone auxin.

The Cysteine Protease CEP1, a Key Executor Involved in Tapetal Programmed Cell Death, Regulates Pollen Development in Arabidopsis[W][OPEN

PubMed Central

Zhang, Dandan; Liu, Di; Lv, Xiaomeng; Wang, Ying; Xun, Zhili; Liu, Zhixiong; Li, Fenglan; Lu, Hai

2014-01-01

Tapetal programmed cell death (PCD) is a prerequisite for pollen grain development in angiosperms, and cysteine proteases are the most ubiquitous hydrolases involved in plant PCD. We identified a papain-like cysteine protease, CEP1, which is involved in tapetal PCD and pollen development in Arabidopsis thaliana. CEP1 is expressed specifically in the tapetum from stages 5 to 11 of anther development. The CEP1 protein first appears as a proenzyme in precursor protease vesicles and is then transported to the vacuole and transformed into the mature enzyme before rupture of the vacuole. cep1 mutants exhibited aborted tapetal PCD and decreased pollen fertility with abnormal pollen exine. A transcriptomic analysis revealed that 872 genes showed significantly altered expression in the cep1 mutants, and most of them are important for tapetal cell wall organization, tapetal secretory structure formation, and pollen development. CEP1 overexpression caused premature tapetal PCD and pollen infertility. ELISA and quantitative RT-PCR analyses confirmed that the CEP1 expression level showed a strong relationship to the degree of tapetal PCD and pollen fertility. Our results reveal that CEP1 is a crucial executor during tapetal PCD and that proper CEP1 expression is necessary for timely degeneration of tapetal cells and functional pollen formation. PMID:25035401

The cysteine protease CEP1, a key executor involved in tapetal programmed cell death, regulates pollen development in Arabidopsis.

PubMed

Zhang, Dandan; Liu, Di; Lv, Xiaomeng; Wang, Ying; Xun, Zhili; Liu, Zhixiong; Li, Fenglan; Lu, Hai

2014-07-01

Tapetal programmed cell death (PCD) is a prerequisite for pollen grain development in angiosperms, and cysteine proteases are the most ubiquitous hydrolases involved in plant PCD. We identified a papain-like cysteine protease, CEP1, which is involved in tapetal PCD and pollen development in Arabidopsis thaliana. CEP1 is expressed specifically in the tapetum from stages 5 to 11 of anther development. The CEP1 protein first appears as a proenzyme in precursor protease vesicles and is then transported to the vacuole and transformed into the mature enzyme before rupture of the vacuole. cep1 mutants exhibited aborted tapetal PCD and decreased pollen fertility with abnormal pollen exine. A transcriptomic analysis revealed that 872 genes showed significantly altered expression in the cep1 mutants, and most of them are important for tapetal cell wall organization, tapetal secretory structure formation, and pollen development. CEP1 overexpression caused premature tapetal PCD and pollen infertility. ELISA and quantitative RT-PCR analyses confirmed that the CEP1 expression level showed a strong relationship to the degree of tapetal PCD and pollen fertility. Our results reveal that CEP1 is a crucial executor during tapetal PCD and that proper CEP1 expression is necessary for timely degeneration of tapetal cells and functional pollen formation. © 2014 American Society of Plant Biologists. All rights reserved.

Cj1386 is an ankyrin-containing protein involved in heme trafficking to catalase in Campylobacter jejuni.

PubMed

Flint, Annika; Sun, Yi-Qian; Stintzi, Alain

2012-01-01

Campylobacter jejuni, a microaerophilic bacterium, is the most frequent cause of human bacterial gastroenteritis. C. jejuni is exposed to harmful reactive oxygen species (ROS) produced during its own normal metabolic processes and during infection from the host immune system and from host intestinal microbiota. These ROS will damage DNA and proteins and cause peroxidation of lipids. Consequently, identifying ROS defense mechanisms is important for understanding how Campylobacter survives this environmental stress during infection. Construction of a ΔCj1386 isogenic deletion mutant and phenotypic assays led to its discovery as a novel oxidative stress defense gene. The ΔCj1386 mutant has an increased sensitivity toward hydrogen peroxide. The Cj1386 gene is located directly downstream from katA (catalase) in the C. jejuni genome. A ΔkatAΔ Cj1386 double deletion mutant was constructed and exhibited a sensitivity to hydrogen peroxide similar to that seen in the ΔCj1386 and ΔkatA single deletion mutants. This observation suggests that Cj1386 may be involved in the same detoxification pathway as catalase. Despite identical KatA abundances, catalase activity assays showed that the ΔCj1386 mutant had a reduced catalase activity relative to that of wild-type C. jejuni. Heme quantification of KatA protein from the ΔCj1386 mutant revealed a significant decrease in heme concentration. This indicates an important role for Cj1386 in heme trafficking to KatA within C. jejuni. Interestingly, the ΔCj1386 mutant had a reduced ability to colonize the ceca of chicks and was outcompeted by the wild-type strain for colonization of the gastrointestinal tract of neonate piglets. These results indicate an important role for Cj1386 in Campylobacter colonization and pathogenesis.

Identification of the residues involved in stabilization of the semiquinone radical in the high-affinity ubiquinone binding site in cytochrome bo(3) from Escherichia coli by site-directed mutagenesis and EPR spectroscopy.

PubMed

Hellwig, Petra; Yano, Takahiro; Ohnishi, Tomoko; Gennis, Robert B

2002-08-27

During turnover of cytochrome bo(3) from Escherichia coli, a semiquinone radical is stabilized in a high-affinity binding site. To identify binding partners of this radical, site-directed mutants have been designed on the basis of a recently modeled quinone binding site (Abramson et al., 2000). The R71H, H98F, D75H, and I102W mutant enzymes were found to show very little or no quinol oxidase activity. The thermodynamic and EPR spectroscopic properties of semiquinone radicals in these mutants were characterized. For the H98F and the R71H mutants, no EPR signal of the semiquinone radical was observed in the redox potential range from -100 to 250 mV. During potentiometric titration of the D75H mutant enzyme, a semiquinone signal was detected in the same potential range as that of the wild-type enzyme. However, the EPR spectrum of the D75H mutant lacks the characteristic hyperfine structure of the semiquinone radical signal observed in the wild-type oxidase, indicating that D75 or the introduced His, interacts with the semiquinone radical. For the I102W mutant, a free radical signal was observed with a redox midpoint potential downshifted by about 200 mV. On the basis of these observations, it is suggested that R71, D75, and H98 residues are involved in the stabilization of the semiquinone state in the high-affinity binding site. Details of the possible binding motif and mechanistic implications are discussed.

Mutational Evidence for the Critical Role of CBF Transcription Factors in Cold Acclimation in Arabidopsis1

PubMed Central

Zhang, Zhengjing; Li, Yuanya

2016-01-01

The three tandemly arranged CBF genes, CBF1, CBF2, and CBF3, are involved in cold acclimation. Due to the lack of stable loss-of-function Arabidopsis (Arabidopsis thaliana) mutants deficient in all three CBF genes, it is still unclear whether the CBF genes are essential for freezing tolerance and whether they may have other functions besides cold acclimation. In this study, we used the CRISPR/Cas9 system to generate cbf single, double, and triple mutants. Compared to the wild type, the cbf triple mutants are extremely sensitive to freezing after cold acclimation, demonstrating that the three CBF genes are essential for cold acclimation. Our results show that the three CBF genes also contribute to basal freezing tolerance. Unexpectedly, we found that the cbf triple mutants are defective in seedling development and salt stress tolerance. Transcript profiling revealed that the CBF genes regulate 414 cold-responsive (COR) genes, of which 346 are CBF-activated genes and 68 are CBF-repressed genes. The analysis suggested that CBF proteins are extensively involved in the regulation of carbohydrate and lipid metabolism, cell wall modification, and gene transcription. Interestingly, like the triple mutants, cbf2 cbf3 double mutants are more sensitive to freezing after cold acclimation compared to the wild type, but cbf1 cbf3 double mutants are more resistant, suggesting that CBF2 is more important than CBF1 and CBF3 in cold acclimation-dependent freezing tolerance. Our results not only demonstrate that the three CBF genes together are required for cold acclimation and freezing tolerance, but also reveal that they are important for salt tolerance and seedling development. PMID:27252305

Cj1386 Is an Ankyrin-Containing Protein Involved in Heme Trafficking to Catalase in Campylobacter jejuni

PubMed Central

Flint, Annika; Sun, Yi-Qian

2012-01-01

Campylobacter jejuni, a microaerophilic bacterium, is the most frequent cause of human bacterial gastroenteritis. C. jejuni is exposed to harmful reactive oxygen species (ROS) produced during its own normal metabolic processes and during infection from the host immune system and from host intestinal microbiota. These ROS will damage DNA and proteins and cause peroxidation of lipids. Consequently, identifying ROS defense mechanisms is important for understanding how Campylobacter survives this environmental stress during infection. Construction of a ΔCj1386 isogenic deletion mutant and phenotypic assays led to its discovery as a novel oxidative stress defense gene. The ΔCj1386 mutant has an increased sensitivity toward hydrogen peroxide. The Cj1386 gene is located directly downstream from katA (catalase) in the C. jejuni genome. A ΔkatAΔ Cj1386 double deletion mutant was constructed and exhibited a sensitivity to hydrogen peroxide similar to that seen in the ΔCj1386 and ΔkatA single deletion mutants. This observation suggests that Cj1386 may be involved in the same detoxification pathway as catalase. Despite identical KatA abundances, catalase activity assays showed that the ΔCj1386 mutant had a reduced catalase activity relative to that of wild-type C. jejuni. Heme quantification of KatA protein from the ΔCj1386 mutant revealed a significant decrease in heme concentration. This indicates an important role for Cj1386 in heme trafficking to KatA within C. jejuni. Interestingly, the ΔCj1386 mutant had a reduced ability to colonize the ceca of chicks and was outcompeted by the wild-type strain for colonization of the gastrointestinal tract of neonate piglets. These results indicate an important role for Cj1386 in Campylobacter colonization and pathogenesis. PMID:22081390

The surfactant of Legionella pneumophila Is secreted in a TolC-dependent manner and is antagonistic toward other Legionella species.

PubMed

Stewart, Catherine R; Burnside, Denise M; Cianciotto, Nicholas P

2011-11-01

When Legionella pneumophila grows on agar plates, it secretes a surfactant that promotes flagellum- and pilus-independent "sliding" motility. We isolated three mutants that were defective for surfactant. The first two had mutations in genes predicted to encode cytoplasmic enzymes involved in lipid metabolism. These genes mapped to two adjacent operons that we designated bbcABCDEF and bbcGHIJK. Backcrossing and complementation confirmed the importance of the bbc genes and suggested that the Legionella surfactant is lipid containing. The third mutant had an insertion in tolC. TolC is the outer membrane part of various trimolecular complexes involved in multidrug efflux and type I protein secretion. Complementation of the tolC mutant restored sliding motility. Mutants defective for an inner membrane partner of TolC also lacked a surfactant, confirming that TolC promotes surfactant secretion. L. pneumophila (lspF) mutants lacking type II protein secretion (T2S) are also impaired for a surfactant. When the tolC and lspF mutants were grown next to each other, the lsp mutant secreted surfactant, suggesting that TolC and T2S conjoin to mediate surfactant secretion, with one being the conduit for surfactant export and the other the exporter of a molecule that is required for induction or maturation of surfactant synthesis/secretion. Although the surfactant was not required for the extracellular growth, intracellular infection, and intrapulmonary survival of L. pneumophila, it exhibited antimicrobial activity toward seven other species of Legionella but not toward various non-Legionella species. These data suggest that the surfactant provides L. pneumophila with a selective advantage over other legionellae in the natural environment.

The Surfactant of Legionella pneumophila Is Secreted in a TolC-Dependent Manner and Is Antagonistic toward Other Legionella Species ▿†

PubMed Central

Stewart, Catherine R.; Burnside, Denise M.; Cianciotto, Nicholas P.

2011-01-01

When Legionella pneumophila grows on agar plates, it secretes a surfactant that promotes flagellum- and pilus-independent “sliding” motility. We isolated three mutants that were defective for surfactant. The first two had mutations in genes predicted to encode cytoplasmic enzymes involved in lipid metabolism. These genes mapped to two adjacent operons that we designated bbcABCDEF and bbcGHIJK. Backcrossing and complementation confirmed the importance of the bbc genes and suggested that the Legionella surfactant is lipid containing. The third mutant had an insertion in tolC. TolC is the outer membrane part of various trimolecular complexes involved in multidrug efflux and type I protein secretion. Complementation of the tolC mutant restored sliding motility. Mutants defective for an inner membrane partner of TolC also lacked a surfactant, confirming that TolC promotes surfactant secretion. L. pneumophila (lspF) mutants lacking type II protein secretion (T2S) are also impaired for a surfactant. When the tolC and lspF mutants were grown next to each other, the lsp mutant secreted surfactant, suggesting that TolC and T2S conjoin to mediate surfactant secretion, with one being the conduit for surfactant export and the other the exporter of a molecule that is required for induction or maturation of surfactant synthesis/secretion. Although the surfactant was not required for the extracellular growth, intracellular infection, and intrapulmonary survival of L. pneumophila, it exhibited antimicrobial activity toward seven other species of Legionella but not toward various non-Legionella species. These data suggest that the surfactant provides L. pneumophila with a selective advantage over other legionellae in the natural environment. PMID:21890700

RodZ and PgsA Play Intertwined Roles in Membrane Homeostasis of Bacillus subtilis and Resistance to Weak Organic Acid Stress

PubMed Central

van Beilen, Johan; Blohmke, Christoph J.; Folkerts, Hendrik; de Boer, Richard; Zakrzewska, Anna; Kulik, Wim; Vaz, Fred M.; Brul, Stanley; Ter Beek, Alexander

2016-01-01

Weak organic acids like sorbic and acetic acid are widely used to prevent growth of spoilage organisms such as Bacilli. To identify genes involved in weak acid stress tolerance we screened a transposon mutant library of Bacillus subtilis for sorbic acid sensitivity. Mutants of the rodZ (ymfM) gene were found to be hypersensitive to the lipophilic weak organic acid. RodZ is involved in determining the cell’s rod-shape and believed to interact with the bacterial actin-like MreB cytoskeleton. Since rodZ lies upstream in the genome of the essential gene pgsA (phosphatidylglycerol phosphate synthase) we hypothesized that expression of the latter might also be affected in rodZ mutants and hence contribute to the phenotype observed. We show that both genes are co-transcribed and that both the rodZ::mini-Tn10 mutant and a conditional pgsA mutant, under conditions of minimal pgsA expression, were sensitive to sorbic and acetic acid. Both strains displayed a severely altered membrane composition. Compared to the wild-type strain, phosphatidylglycerol and cardiolipin levels were lowered and the average acyl chain length was elongated. Induction of rodZ expression from a plasmid in our transposon mutant led to no recovery of weak acid susceptibility comparable to wild-type levels. However, pgsA overexpression in the same mutant partly restored sorbic acid susceptibility and fully restored acetic acid sensitivity. A construct containing both rodZ and pgsA as on the genome led to some restored growth as well. We propose that RodZ and PgsA play intertwined roles in membrane homeostasis and tolerance to weak organic acid stress. PMID:27818647

Amino acid sequence surrounding the chondroitin sulfate attachment site of thrombomodulin regulates chondroitin polymerization.

PubMed

Izumikawa, Tomomi; Kitagawa, Hiroshi

2015-05-01

Thrombomodulin (TM) is a cell-surface glycoprotein and a critical mediator of endothelial anticoagulant function. TM exists as both a chondroitin sulfate (CS) proteoglycan (PG) form and a non-PG form lacking a CS chain (α-TM); therefore, TM can be described as a part-time PG. Previously, we reported that α-TM bears an immature, truncated linkage tetrasaccharide structure (GlcAβ1-3Galβ1-3Galβ1-4Xyl). However, the biosynthetic mechanism to generate part-time PGs remains unclear. In this study, we used several mutants to demonstrate that the amino acid sequence surrounding the CS attachment site influences the efficiency of chondroitin polymerization. In particular, the presence of acidic residues surrounding the CS attachment site was indispensable for the elongation of CS. In addition, mutants defective in CS elongation did not exhibit anti-coagulant activity, as in the case with α-TM. Together, these data support a model for CS chain assembly in which specific core protein determinants are recognized by a key biosynthetic enzyme involved in chondroitin polymerization. Copyright © 2015 Elsevier Inc. All rights reserved.

sae is essential for expression of the staphylococcal adhesins Eap and Emp.

PubMed

Harraghy, Niamh; Kormanec, Jan; Wolz, Christiane; Homerova, Dagmar; Goerke, Christiane; Ohlsen, Knut; Qazi, Saara; Hill, Philip; Herrmann, Mathias

2005-06-01

Eap and Emp are two Staphylococcus aureus adhesins initially described as extracellular matrix binding proteins. Eap has since emerged as being important in adherence to and invasion of eukaryotic cells, as well as being described as an immunomodulator and virulence factor in chronic infections. This paper describes the mapping of the transcription start point of the eap and emp promoters. Moreover, using reporter-gene assays and real-time PCR in defined regulatory mutants, environmental conditions and global regulators affecting expression of eap and emp were investigated. Marked differences were found in expression of eap and emp between strain Newman and the 8325 derivatives SH1000 and 8325-4. Moreover, both genes were repressed in the presence of glucose. Analysis of expression of both genes in various regulatory mutants revealed that sarA and agr were involved in their regulation, but the data suggested that there were additional regulators of both genes. In a sae mutant, expression of both genes was severely repressed. sae expression was also reduced in the presence of glucose, suggesting that repression of eap and emp in glucose-containing medium may, in part, be a consequence of a decrease in expression of sae.

Molecular pathways: targeting RAC-p21-activated serine-threonine kinase signaling in RAS-driven cancers.

PubMed

Baker, Nicole M; Yee Chow, Hoi; Chernoff, Jonathan; Der, Channing J

2014-09-15

Cancers driven by oncogenic Ras proteins encompass some of the most deadly human cancer types, and there is a pressing need to develop therapies for these diseases. Although recent studies suggest that mutant Ras proteins may yet be druggable, the most promising and advanced efforts involve inhibitors of Ras effector signaling. Most efforts to target Ras signaling have been aimed at the ERK mitogen-activated protein kinase and the phosphoinositide 3-kinase signaling networks. However, to date, no inhibitors of these Ras effector pathways have been effective against RAS-mutant cancers. This ineffectiveness is due, in part, to the involvement of additional effectors in Ras-dependent cancer growth, such as the Rac small GTPase and the p21-activated serine-threonine kinases (PAK). PAK proteins are involved in many survival, cell motility, and proliferative pathways in the cell and may present a viable new target in Ras-driven cancers. In this review, we address the role and therapeutic potential of Rac and group I PAK proteins in driving mutant Ras cancers. ©2014 American Association for Cancer Research.

Use of a nonhomologous end joining deficient strain (Deltaku70) of the ergot fungus Claviceps purpurea for identification of a nonribosomal peptide synthetase gene involved in ergotamine biosynthesis.

PubMed

Haarmann, Thomas; Lorenz, Nicole; Tudzynski, Paul

2008-01-01

The ergot fungus Claviceps purpurea uses mainly the nonhomologous-end-joining (NHEJ) system for integration of exogenous DNA, leading to a low frequency of homologous integration (1-2%). To improve gene targeting efficiency we deleted the C. purpurea ku70 gene in two different strains: the pathogenic strain 20.1 and the apathogenic, ergot alkaloid producing strain P1. The mutants were not impaired in vegetative and pathogenic development nor alkaloid production. Gene targeting efficiency was significantly increased (50-60%) in the Deltaku70 mutants. The P1 Deltaku70 strain (producing ergotamine and ergocryptine) was used for targeted deletion of lpsA1, one of the two trimodular NRPS genes present in the alkaloid gene cluster, encoding D-lysergyl peptide synthetases involved in formation of the tripeptide moiety of ergopeptines. Mutants lacking the lpsA1 gene were shown to be incapable of producing ergotamine but were still able to produce ergocryptine, proving that LpsA1 is involved in ergotamine biosynthesis.

Actin dynamics involved in gravity perception in Arabidopsis inflorescense stem

NASA Astrophysics Data System (ADS)

Tasaka, Masao; Nakamura, Moritaka; Morita, Miyo T.

The amyloplasts sedimentation in the endodermal cells is important for gravity perception in Arabidopsis shoot. Our previous study suggests that SGR5(SHOOT GRAVITROPISM 5) and SGR9 are synergistically involved in regulation of amyloplast movement in these cells, and shows that sgr5 sgr9 double mutant completely loses gravitropic response. SGR5 encodes putative transcription factor and SGR9 encodes a ring finger containing protein, which surrounds amyloplasts. It has been reported that amyloplasts are surrounded by actin microfilaments (MFs), and that treatment with actin polymerization inhibitor enhances gravitropic organ curvature. However, not only the molecular link between amyolplasts and MFs, but also regulatory role of MFs in gravitropic response is still unclear. Here, we found that treatment with actin polymerization inhibitor restored gravitropic response of sgr5 sgr9 double mutant stems. The result suggests that abnormal amyloplasts movement in the double mutant could result from inhibition of MFs depolymerization, leading to abnormal gravitropism. We are investigating whether SGR5 and SGR9 are involved in amyloplasts movement by regulating actin remodeling in gravity perceptive cells.

Isolation and characterization of acyclovir-resistant mutants of herpes simplex virus.

PubMed

Field, H J; Darby, G; Wildy, P

1980-07-01

Mutants of HSV which are resistant to acyclovir (acycloguanosine) have been isolated following serial passages of several herpes simplex virus (HSV) strains in the presence of the drug. The majority of the mutants isolated are defective in induction of thymidine kinase (TK) and this is consistent with the observation that independently isolated TK- viruses are naturally resistant to ACV. One mutant is described (SC16 R9C2) which is resistant in biochemically transformed cells which express HSV TK. This suggests that its resistance resides at a level other than TK. It is also resistant to phosphonoacetic acid, suggesting that the DNA polymerase locus may be involved. A further mutant is described [Cl (101) P2C5] which induces normal levels of TK, although the nature of resistance of this virus is not yet elucidated.

Characterization of a Novel Drosophila SERT Mutant: Insights on the Contribution of the Serotonin Neural System to Behaviors.

PubMed

Hidalgo, Sergio; Molina-Mateo, Daniela; Escobedo, Pía; Zárate, Rafaella V; Fritz, Elsa; Fierro, Angélica; Perez, Edwin G; Iturriaga-Vasquez, Patricio; Reyes-Parada, Miguel; Varas, Rodrigo; Fuenzalida-Uribe, Nicolás; Campusano, Jorge M

2017-10-18

A better comprehension on how different molecular components of the serotonergic system contribute to the adequate regulation of behaviors in animals is essential in the interpretation on how they are involved in neuropsychiatric and pathological disorders. It is possible to study these components in "simpler" animal models including the fly Drosophila melanogaster, given that most of the components of the serotonergic system are conserved between vertebrates and invertebrates. Here we decided to advance our understanding on how the serotonin plasma membrane transporter (SERT) contributes to serotonergic neurotransmission and behaviors in Drosophila. In doing this, we characterized for the first time a mutant for Drosophila SERT (dSERT) and additionally used a highly selective serotonin-releasing drug, 4-methylthioamphetamine (4-MTA), whose mechanism of action involves the SERT protein. Our results show that dSERT mutant animals exhibit an increased survival rate in stress conditions, increased basal motor behavior, and decreased levels in an anxiety-related parameter, centrophobism. We also show that 4-MTA increases the negative chemotaxis toward a strong aversive odorant, benzaldehyde. Our neurochemical data suggest that this effect is mediated by dSERT and depends on the 4-MTA-increased release of serotonin in the fly brain. Our in silico data support the idea that these effects are explained by specific interactions between 4-MTA and dSERT. In sum, our neurochemical, in silico, and behavioral analyses demonstrate the critical importance of the serotonergic system and particularly dSERT functioning in modulating several behaviors in Drosophila.

Murine GRPR and Stathmin Control in Opposite Directions both Cued Fear Extinction and Neural Activities of the Amygdala and Prefrontal Cortex

PubMed Central

Martel, Guillaume; Hevi, Charles; Wong, Alexandra; Zushida, Ko; Uchida, Shusaku; Shumyatsky, Gleb P.

2012-01-01

Extinction is an integral part of normal healthy fear responses, while it is compromised in several fear-related mental conditions in humans, such as post-traumatic stress disorder (PTSD). Although much research has recently been focused on fear extinction, its molecular and cellular underpinnings are still unclear. The development of animal models for extinction will greatly enhance our approaches to studying its neural circuits and the mechanisms involved. Here, we describe two gene-knockout mouse lines, one with impaired and another with enhanced extinction of learned fear. These mutant mice are based on fear memory-related genes, stathmin and gastrin-releasing peptide receptor (GRPR). Remarkably, both mutant lines showed changes in fear extinction to the cue but not to the context. We performed indirect imaging of neuronal activity on the second day of cued extinction, using immediate-early gene c-Fos. GRPR knockout mice extinguished slower (impaired extinction) than wildtype mice, which was accompanied by an increase in c-Fos activity in the basolateral amygdala and a decrease in the prefrontal cortex. By contrast, stathmin knockout mice extinguished faster (enhanced extinction) and showed a decrease in c-Fos activity in the basolateral amygdala and an increase in the prefrontal cortex. At the same time, c-Fos activity in the dentate gyrus was increased in both mutant lines. These experiments provide genetic evidence that the balance between neuronal activities of the amygdala and prefrontal cortex defines an impairment or facilitation of extinction to the cue while the hippocampus is involved in the context-specificity of extinction. PMID:22312434

Murine GRPR and stathmin control in opposite directions both cued fear extinction and neural activities of the amygdala and prefrontal cortex.

PubMed

Martel, Guillaume; Hevi, Charles; Wong, Alexandra; Zushida, Ko; Uchida, Shusaku; Shumyatsky, Gleb P

2012-01-01

Extinction is an integral part of normal healthy fear responses, while it is compromised in several fear-related mental conditions in humans, such as post-traumatic stress disorder (PTSD). Although much research has recently been focused on fear extinction, its molecular and cellular underpinnings are still unclear. The development of animal models for extinction will greatly enhance our approaches to studying its neural circuits and the mechanisms involved. Here, we describe two gene-knockout mouse lines, one with impaired and another with enhanced extinction of learned fear. These mutant mice are based on fear memory-related genes, stathmin and gastrin-releasing peptide receptor (GRPR). Remarkably, both mutant lines showed changes in fear extinction to the cue but not to the context. We performed indirect imaging of neuronal activity on the second day of cued extinction, using immediate-early gene c-Fos. GRPR knockout mice extinguished slower (impaired extinction) than wildtype mice, which was accompanied by an increase in c-Fos activity in the basolateral amygdala and a decrease in the prefrontal cortex. By contrast, stathmin knockout mice extinguished faster (enhanced extinction) and showed a decrease in c-Fos activity in the basolateral amygdala and an increase in the prefrontal cortex. At the same time, c-Fos activity in the dentate gyrus was increased in both mutant lines. These experiments provide genetic evidence that the balance between neuronal activities of the amygdala and prefrontal cortex defines an impairment or facilitation of extinction to the cue while the hippocampus is involved in the context-specificity of extinction.

Isolation and characterization of ultraviolet light-sensitive mutants of the blue-green alga Anacystis nidulans.

NASA Technical Reports Server (NTRS)

Asato, Y.

1972-01-01

Three independently isolated ultraviolet light sensitive (uvs) mutants of Anacystis nidulans were characterized. Strain uvs-1 showed the highest sensitivity to UV by its greatly reduced photoreactivation capacity following irradiation. Pretreatment with caffeine suppressed the dark-survival curve of strain uvs-1, thus indicating the presence of excision enzymes involved in dark repair. Under 'black' and 'white' illumination, strain uvs-1 shows photorecovery properties comparable with wild-type cultures. Results indicate that strains uvs-1, uvs-35, and uvs-88 are probably genetically distinct UV-sensitive mutants.

Net light-induced oxygen evolution in photosystem I deletion mutants of the cyanobacterium Synechocystis sp. PCC 6803.

PubMed

Wang, Qing Jun; Singh, Abhay; Li, Hong; Nedbal, Ladislav; Sherman, Louis A; Govindjee; Whitmarsh, John

2012-05-01

Oxygenic photosynthesis in cyanobacteria, algae, and plants requires photosystem II (PSII) to extract electrons from H(2)O and depends on photosystem I (PSI) to reduce NADP(+). Here we demonstrate that mixotrophically-grown mutants of the cyanobacterium Synechocystis sp. PCC 6803 that lack PSI (ΔPSI) are capable of net light-induced O(2) evolution in vivo. The net light-induced O(2) evolution requires glucose and can be sustained for more than 30 min. Utilizing electron transport inhibitors and chlorophyll a fluorescence measurements, we show that in these mutants PSII is the source of the light-induced O(2) evolution, and that the plastoquinone pool is reduced by PSII and subsequently oxidized by an unidentified electron acceptor that does not involve the plastoquinol oxidase site of the cytochrome b(6)f complex. Moreover, both O(2) evolution and chlorophyll a fluorescence kinetics of the ΔPSI mutants are highly sensitive to KCN, indicating the involvement of a KCN-sensitive enzyme(s). Experiments using (14)C-labeled bicarbonate show that the ΔPSI mutants assimilate more CO(2) in the light compared to the dark. However, the rate of the light-minus-dark CO(2) assimilation accounts for just over half of the net light-induced O(2) evolution rate, indicating the involvement of unidentified terminal electron acceptors. Based on these results we suggest that O(2) evolution in ΔPSI cells can be sustained by an alternative electron transport pathway that results in CO(2) assimilation and that includes PSII, the platoquinone pool, and a KCN-sensitive enzyme. Copyright © 2012 Elsevier B.V. All rights reserved.

A new mutant of Arabidopsis disturbed in its roots, right-handed slanting, and gravitropism defines a gene that encodes a heat-shock factor.

PubMed

Fortunati, A; Piconese, S; Tassone, P; Ferrari, S; Migliaccio, F

2008-01-01

A new mutant of Arabidopsis named rha1 is characterized and the gene involved cloned. In roots, the mutant shows minimal right-handed slanting, reduced gravitropic response, notable resistance to 2,4-D, but scarce resistance to IAA and NAA. The roots also show a clear resistance to the auxin transport inhibitors TIBA and NPA, and to ethylene. Other characteristics are a reduced number of lateral roots and reduced size of shoot and root in the seedlings. The gene, cloned through TAIL-PCR, was found to be a heat-shock factor that maps on chromosome 5, close to and above the RFLP marker m61. The rha1 structure, mRNA, and translation product are reported. Since, so far, no other gravitropic mutant has been described as mutated in a heat-shock factor, rha1 belongs to a new group of mutants disturbed in slanting, gravitropism, and auxin physiology. As shown through the RT-PCR analyses of its expression, the gene retains the function connected with heat shock. If the characteristics connected with auxin physiology are considered, however, it is also likely that the gene, as a transcription factor, could be involved in root circumnutation, gravitropic response, and hormonal control of differentiation. Since GUS staining under the gene promoter was localized mainly in the mature tissues, rha1 does not seem to be involved in the first steps of gravitropism, but is rather related to the general response to auxin. The alterations in slanting (mainly due to reduced chiral circumnutation) and gravitropism lead to the supposition that the two processes may have, at least in part, common origins.

Metabolic pathways of Pseudomonas aeruginosa involved in competition with respiratory bacterial pathogens

PubMed Central

Beaume, Marie; Köhler, Thilo; Fontana, Thierry; Tognon, Mikael; Renzoni, Adriana; van Delden, Christian

2015-01-01

Background: Chronic airway infection by Pseudomonas aeruginosa considerably contributes to lung tissue destruction and impairment of pulmonary function in cystic-fibrosis (CF) patients. Complex interplays between P. aeruginosa and other co-colonizing pathogens including Staphylococcus aureus, Burkholderia sp., and Klebsiella pneumoniae may be crucial for pathogenesis and disease progression. Methods: We generated a library of PA14 transposon insertion mutants to identify P. aeruginosa genes required for exploitative and direct competitions with S. aureus, Burkholderia cenocepacia, and K. pneumoniae. Results: Whereas wild-type PA14 inhibited S. aureus growth, two transposon insertions located in pqsC and carB, resulted in reduced growth inhibition. PqsC is involved in the synthesis of 4-hydroxy-2-alkylquinolines (HAQs), a family of molecules having antibacterial properties, while carB is a key gene in pyrimidine biosynthesis. The carB mutant was also unable to grow in the presence of B. cepacia and K. pneumoniae but not Escherichia coli and S. epidermidis. We further identified a transposon insertion in purF, encoding a key enzyme of purine metabolism. This mutant displayed a severe growth deficiency in the presence of Gram-negative but not of Gram-positive bacteria. We identified a beneficial interaction in a bioA transposon mutant, unable to grow on rich medium. This growth defect could be restored either by addition of biotin or by co-culturing the mutant in the presence of K. pneumoniae or E. coli. Conclusion: Complex interactions take place between the various bacterial species colonizing CF-lungs. This work identified both detrimental and beneficial interactions occurring between P. aeruginosa and three other respiratory pathogens involving several major metabolic pathways. Manipulating these pathways could be used to interfere with bacterial interactions and influence the colonization by respiratory pathogens. PMID:25954256

Transposon Mutagenesis of the Plant-Associated Bacillus amyloliquefaciens ssp. plantarum FZB42 Revealed That the nfrA and RBAM17410 Genes Are Involved in Plant-Microbe-Interactions

PubMed Central

Dietel, Kristin; Beator, Barbara; Dolgova, Olga; Fan, Ben; Bleiss, Wilfrid; Ziegler, Jörg; Schmid, Michael; Hartmann, Anton; Borriss, Rainer

2014-01-01

Bacillus amyloliquefaciens ssp. plantarum FZB42 represents the prototype of Gram-positive plant growth promoting and biocontrol bacteria. In this study, we applied transposon mutagenesis to generate a transposon library, which was screened for genes involved in multicellular behavior and biofilm formation on roots as a prerequisite of plant growth promoting activity. Transposon insertion sites were determined by rescue-cloning followed by DNA sequencing. As in B. subtilis, the global transcriptional regulator DegU was identified as an activator of genes necessary for swarming and biofilm formation, and the DegU-mutant of FZB42 was found impaired in efficient root colonization. Direct screening of 3,000 transposon insertion mutants for plant-growth-promotion revealed the gene products of nfrA and RBAM_017140 to be essential for beneficial effects exerted by FZB42 on plants. We analyzed the performance of GFP-labeled wild-type and transposon mutants in the colonization of lettuce roots using confocal laser scanning microscopy. While the wild-type strain heavily colonized root surfaces, the nfrA mutant did not colonize lettuce roots, although it was not impaired in growth in laboratory cultures, biofilm formation and swarming motility on agar plates. The RBAM17410 gene, occurring in only a few members of the B. subtilis species complex, was directly involved in plant growth promotion. None of the mutant strains were affected in producing the plant growth hormone auxin. We hypothesize that the nfrA gene product is essential for overcoming the stress caused by plant response towards bacterial root colonization. PMID:24847778

Ca2+ binding and conformational changes in a calmodulin domain.

PubMed

Evenäs, J; Malmendal, A; Thulin, E; Carlström, G; Forsén, S

1998-09-29

Calcium activation of the C-terminal domain of calmodulin was studied using 1H and 15N NMR spectroscopy. The important role played by the conserved bidentate glutamate Ca2+ ligand in the binding loops is emphasized by the striking effects resulting from a mutation of this glutamic acid to a glutamine, i.e. E104Q in loop III and E140Q in loop IV. The study involves determination of Ca2+ binding constants, assignments, and structural characterizations of the apo, (Ca2+)1, and (Ca2+)2 states of the E104Q mutant and comparisons to the wild-type protein and the E140Q mutant [Evenäs et al. (1997) Biochemistry 36, 3448-3457]. NMR titration data show sequential Ca2+ binding in the E104Q mutant. The first Ca2+ binds to loop IV and the second to loop III, which is the order reverse to that observed for the E140Q mutant. In both mutants, the major structural changes occur upon Ca2+ binding to loop IV, which implies a different response to Ca2+ binding in the N- and C-terminal EF-hands. Spectral characteristics show that the (Ca2+)1 and (Ca2+)2 states of the E104Q mutant undergo global exchange on a 10-100 micros time scale between conformations seemingly similar to the closed and open structures of this domain in wild-type calmodulin, paralleling earlier observations for the (Ca2+)2 state of the E140Q mutant, indicating that both glutamic acid residues, E104 and E140, are required for stabilization of the open conformation in the (Ca2+)2 state. To verify that the NOE constraints cannot be fulfilled in a single structure, solution structures of the (Ca2+)2 state of the E104Q mutant are calculated. Within the ensemble of structures the precision is good. However, the clearly dynamic nature of the state, a large number of violated distance restraints, ill-defined secondary structural elements, and comparisons to the structures of calmodulin indicate that the ensemble does not provide a good picture of the (Ca2+)2 state of the E104Q mutant but rather represents the distance-averaged structure of at least two distinct different conformations.

The Regulatory Protein RosR Affects Rhizobium leguminosarum bv. trifolii Protein Profiles, Cell Surface Properties, and Symbiosis with Clover

PubMed Central

Rachwał, Kamila; Boguszewska, Aleksandra; Kopcińska, Joanna; Karaś, Magdalena; Tchórzewski, Marek; Janczarek, Monika

2016-01-01

Rhizobium leguminosarum bv. trifolii is capable of establishing a symbiotic relationship with plants from the genus Trifolium. Previously, a regulatory protein encoded by rosR was identified and characterized in this bacterium. RosR possesses a Cys2-His2-type zinc finger motif and belongs to Ros/MucR family of rhizobial transcriptional regulators. Transcriptome profiling of the rosR mutant revealed a role of this protein in several cellular processes, including the synthesis of cell-surface components and polysaccharides, motility, and bacterial metabolism. Here, we show that a mutation in rosR resulted in considerable changes in R. leguminosarum bv. trifolii protein profiles. Extracellular, membrane, and periplasmic protein profiles of R. leguminosarum bv. trifolii wild type and the rosR mutant were examined, and proteins with substantially different abundances between these strains were identified. Compared with the wild type, extracellular fraction of the rosR mutant contained greater amounts of several proteins, including Ca2+-binding cadherin-like proteins, a RTX-like protein, autoaggregation protein RapA1, and flagellins FlaA and FlaB. In contrast, several proteins involved in the uptake of various substrates were less abundant in the mutant strain (DppA, BraC, and SfuA). In addition, differences were observed in membrane proteins of the mutant and wild-type strains, which mainly concerned various transport system components. Using atomic force microscopy (AFM) imaging, we characterized the topography and surface properties of the rosR mutant and wild-type cells. We found that the mutation in rosR gene also affected surface properties of R. leguminosarum bv. trifolii. The mutant cells were significantly more hydrophobic than the wild-type cells, and their outer membrane was three times more permeable to the hydrophobic dye N-phenyl-1-naphthylamine. The mutation of rosR also caused defects in bacterial symbiotic interaction with clover plants. Compared with the wild type, the rosR mutant infected host plant roots much less effectively and its nodule occupation was disturbed. At the ultrastructural level, the most striking differences between the mutant and the wild-type nodules concerned the structure of infection threads, release of bacteria, and bacteroid differentiation. This confirms an essential role of RosR in establishment of successful symbiotic interaction of R. leguminosarum bv. trifolii with clover plants. PMID:27602024

The Regulatory Protein RosR Affects Rhizobium leguminosarum bv. trifolii Protein Profiles, Cell Surface Properties, and Symbiosis with Clover.

PubMed

Rachwał, Kamila; Boguszewska, Aleksandra; Kopcińska, Joanna; Karaś, Magdalena; Tchórzewski, Marek; Janczarek, Monika

2016-01-01

Rhizobium leguminosarum bv. trifolii is capable of establishing a symbiotic relationship with plants from the genus Trifolium. Previously, a regulatory protein encoded by rosR was identified and characterized in this bacterium. RosR possesses a Cys2-His2-type zinc finger motif and belongs to Ros/MucR family of rhizobial transcriptional regulators. Transcriptome profiling of the rosR mutant revealed a role of this protein in several cellular processes, including the synthesis of cell-surface components and polysaccharides, motility, and bacterial metabolism. Here, we show that a mutation in rosR resulted in considerable changes in R. leguminosarum bv. trifolii protein profiles. Extracellular, membrane, and periplasmic protein profiles of R. leguminosarum bv. trifolii wild type and the rosR mutant were examined, and proteins with substantially different abundances between these strains were identified. Compared with the wild type, extracellular fraction of the rosR mutant contained greater amounts of several proteins, including Ca(2+)-binding cadherin-like proteins, a RTX-like protein, autoaggregation protein RapA1, and flagellins FlaA and FlaB. In contrast, several proteins involved in the uptake of various substrates were less abundant in the mutant strain (DppA, BraC, and SfuA). In addition, differences were observed in membrane proteins of the mutant and wild-type strains, which mainly concerned various transport system components. Using atomic force microscopy (AFM) imaging, we characterized the topography and surface properties of the rosR mutant and wild-type cells. We found that the mutation in rosR gene also affected surface properties of R. leguminosarum bv. trifolii. The mutant cells were significantly more hydrophobic than the wild-type cells, and their outer membrane was three times more permeable to the hydrophobic dye N-phenyl-1-naphthylamine. The mutation of rosR also caused defects in bacterial symbiotic interaction with clover plants. Compared with the wild type, the rosR mutant infected host plant roots much less effectively and its nodule occupation was disturbed. At the ultrastructural level, the most striking differences between the mutant and the wild-type nodules concerned the structure of infection threads, release of bacteria, and bacteroid differentiation. This confirms an essential role of RosR in establishment of successful symbiotic interaction of R. leguminosarum bv. trifolii with clover plants.

Morphogenetic Pathway of Spore Wall Assembly in Saccharomyces cerevisiae

PubMed Central

Coluccio, Alison; Bogengruber, Edith; Conrad, Michael N.; Dresser, Michael E.; Briza, Peter; Neiman, Aaron M.

2004-01-01

The Saccharomyces cerevisiae spore is protected from environmental damage by a multilaminar extracellular matrix, the spore wall, which is assembled de novo during spore formation. A set of mutants defective in spore wall assembly were identified in a screen for mutations causing sensitivity of spores to ether vapor. The spore wall defects in 10 of these mutants have been characterized in a variety of cytological and biochemical assays. Many of the individual mutants are defective in the assembly of specific layers within the spore wall, leading to arrests at discrete stages of assembly. The localization of several of these gene products has been determined and distinguishes between proteins that likely are involved directly in spore wall assembly and probable regulatory proteins. The results demonstrate that spore wall construction involves a series of dependent steps and provide the outline of a morphogenetic pathway for assembly of a complex extracellular structure. PMID:15590821

Pharmacodynamic Model To Describe the Concentration-Dependent Selection of Cefotaxime-Resistant Escherichia coli

PubMed Central

Olofsson, Sara K.; Geli, Patricia; Andersson, Dan I.; Cars, Otto

2005-01-01

Antibiotic dosing regimens may vary in their capacity to select mutants. Our hypothesis was that selection of a more resistant bacterial subpopulation would increase with the time within a selective window (SW), i.e., when drug concentrations fall between the MICs of two strains. An in vitro kinetic model was used to study the selection of two Escherichia coli strains with different susceptibilities to cefotaxime. The bacterial mixtures were exposed to cefotaxime for 24 h and SWs of 1, 2, 4, 8, and 12 h. A mathematical model was developed that described the selection of preexisting and newborn mutants and the post-MIC effect (PME) as functions of pharmacokinetic parameters. Our main conclusions were as follows: (i) the selection between preexisting mutants increased with the time within the SW; (ii) the emergence and selection of newborn mutants increased with the time within the SW (with a short time, only 4% of the preexisting mutants were replaced by newborn mutants, compared to the longest times, where 100% were replaced); and (iii) PME increased with the area under the concentration-time curve (AUC) and was slightly more pronounced with a long elimination half-life (T1/2) than with a short T1/2 situation, when AUC is fixed. We showed that, in a dynamic competition between strains with different levels of resistance, the appearance of newborn high-level resistant mutants from the parental strains and the PME can strongly affect the outcome of the selection and that pharmacodynamic models can be used to predict the outcome of resistance development. PMID:16304176

Gα proteins Gvm2 and Gvm3 regulate vegetative growth, asexual development, and pathogenicityon apple in Valsa mali.

PubMed

Song, Na; Dai, Qingqing; Zhu, Baitao; Wu, Yuxing; Xu, Ming; Voegele, Ralf Thomas; Gao, Xiaoning; Kang, Zhensheng; Huang, Lili

2017-01-01

In fungi, heterotrimeric guanine-nucleotide binding proteins (G-proteins) are key elements of signal transduction pathways, which control growth, asexual and sexual development, as well as virulence. In this study, we have identified two genes encoding heterotrimeric G protein alpha subunits, named Gvm2 and Gvm3, from Valsa mali, the causal agent of apple Valsa canker. Characterization of Gvm2 and Gvm3 mutants indicates that Gvm3 may be a crucial regulator of vegetative growth. Deletion of the corresponding gene results in a 20% reduction in growth rate. Besides, Gvm2 and Gvm3 seem to be involved in asexual reproduction, and mutants are hypersensitive to oxidative and cell membrane stresses. Interestingly, both G protein alpha subunits were most probably involved in V. mali virulence. In infection assays using Malus domestica cv. 'Fuji' leaves and twigs, the size of lesions caused by deletion mutants △Gvm2, or △Gvm3 are significantly reduced. Furthermore, many genes encoding hydrolytic enzymes-important virulence factors in V. mali-are expressed at a lower level in these deletion mutants. Our results suggest that Gvm2 and Gvm3 play an important role in virulence probably by regulation of expression of cell wall degrading enzymes. △Gvm2, and △Gvm3 mutants were further analyzed with respect to their impact on the transcript levels of genes in the cAMP/PKA pathway. The expression of the genes encoding adenylate cyclase VmAC, protein kinase A (PKA) regulatory subunit VmPKR, and PKA catalytic subunit VmPKA1 are down-regulated in both mutants. Further analyses indicated that intracellular cAMP level and PKA activity are down-regulated in the △Gvm3 mutant, but are basically unchanged in the △Gvm2 mutant. Overall, our findings indicate that both Gvm2 and Gvm3 play diverse roles in the modulation of vegetative growth, asexual development, and virulence in V. mali.

Imidacloprid does not induce Cyp genes involved in insecticide resistance of a mutant Drosophila melanogaster line.

PubMed

Kalajdzic, Predrag; Markaki, Maria; Oehler, Stefan; Savakis, Charalambos

2013-10-01

Certain xenobiotics have the capacity to induce the expression of genes involved in various biological phenomena, including insecticide resistance. The induction potential of different chemicals, among them different insecticides, has been documented for a number of insect species. In this study, we have analyzed the induction potential of Imidacloprid, a widely used member of the neonicotinoid insecticide family. Genes Cyp6g1 and Cyp6a2, known to be involved in the resistance of mutant Drosophila melanogaster line MiT[W⁻]3R2 to Imidacloprid and DDT were included in the analyzed sample. We find that Imidacloprid does not induce expression of the analyzed genes. Copyright © 2013 Elsevier Ltd. All rights reserved.

The Klebsiella pneumoniae wabG Gene: Role in Biosynthesis of the Core Lipopolysaccharide and Virulence

PubMed Central

Izquierdo, Luis; Coderch, Núria; Piqué, Nuria; Bedini, Emiliano; Michela Corsaro, Maria; Merino, Susana; Fresno, Sandra; Tomás, Juan M.; Regué, Miguel

2003-01-01

To determine the function of the wabG gene in the biosynthesis of the core lipopolysaccharide (LPS) of Klebsiella pneumoniae, we constructed wabG nonpolar mutants. Data obtained from the comparative chemical and structural analysis of LPS samples obtained from the wild type, the mutant strain, and the complemented mutant demonstrated that the wabG gene is involved in attachment to α-l-glycero-d-manno-heptopyranose II (l,d-HeppII) at the O-3 position of an α-d-galactopyranosyluronic acid (α-d-GalAp) residue. K. pneumoniae nonpolar wabG mutants were devoid of the cell-attached capsular polysaccharide but were still able to produce capsular polysaccharide. Similar results were obtained with K. pneumoniae nonpolar waaC and waaF mutants, which produce shorter LPS core molecules than do wabG mutants. Other outer core K. pneumoniae nonpolar mutants in the waa gene cluster were encapsulated. K. pneumoniae waaC, waaF, and wabG mutants were avirulent when tested in different animal models. Furthermore, these mutants were more sensitive to some hydrophobic compounds than the wild-type strains. All these characteristics were rescued by reintroduction of the waaC, waaF, and wabG genes from K. pneumoniae. PMID:14645282

Mutants in the mouse NuRD/Mi2 component P66alpha are embryonic lethal.

PubMed

Marino, Susan; Nusse, Roel

2007-06-13

The NuRD/Mi2 chromatin complex is involved in histone modifications and contains a large number of subunits, including the p66 protein. There are two mouse and human p66 paralogs, p66alpha and p66beta. The functions of these genes are not clear, in part because there are no mutants available, except in invertebrate model systems. We made loss of function mutants in the mouse p66alpha gene (mp66alpha, official name Gatad2a, MGI:2384585). We found that mp66alpha is essential for development, as mutant embryos die around day 10 of embryogenesis. The gene is not required for normal blastocyst development or for implantation. The phenotype of mutant embryos and the pattern of gene expression in mutants are consistent with a role of mp66alpha in gene silencing. mp66alpha is an essential gene, required for early mouse development. The lethal phenotype supports a role in execution of methylated DNA silencing.

Mutants in three novel complementation groups inhibit membrane protein insertion into and soluble protein translocation across the endoplasmic reticulum membrane of Saccharomyces cerevisiae

PubMed Central

1992-01-01

We have isolated mutants that inhibit membrane protein insertion into the ER membrane of Saccharomyces cerevisiae. The mutants were contained in three complementation groups, which we have named SEC70, SEC71, and SEC72. The mutants also inhibited the translocation of soluble proteins into the lumen of the ER, indicating that they pleiotropically affect protein transport across and insertion into the ER membrane. Surprisingly, the mutants inhibited the translocation and insertion of different proteins to drastically different degrees. We have also shown that mutations in SEC61 and SEC63, which were previously isolated as mutants inhibiting the translocation of soluble proteins, also affect the insertion of membrane proteins into the ER. Taken together our data indicate that the process of protein translocation across the ER membrane involves a much larger number of gene products than previously appreciated. Moreover, different translocation substrates appear to have different requirements for components of the cellular targeting and translocation apparatus. PMID:1730771

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

Asp30 of Aspergillus oryzae cutinase CutL1 is involved in the ionic interaction with fungal hydrophobin RolA.

PubMed

Terauchi, Yuki; Kim, Yoon-Kyung; Tanaka, Takumi; Nanatani, Kei; Takahashi, Toru; Abe, Keietsu

2017-07-01

Aspergillus oryzae hydrophobin RolA adheres to the biodegradable polyester polybutylene succinate-co-adipate (PBSA) and promotes PBSA degradation by interacting with A. oryzae polyesterase CutL1 and recruiting it to the PBSA surface. In our previous studies, we found that positively charged amino acid residues (H32, K34) of RolA and negatively charged residues (E31, D142, D171) of CutL1 are important for the cooperative ionic interaction between RolA and CutL1, but some other charged residues in the triple mutant CutL1-E31S/D142S/D171S are also involved. In the present study, on the basis of the 3D-structure of CutL1, we hypothesized that D30 is also involved in the CutL1-RolA interaction. We substituted D30 with serine and performed kinetic analysis of the interaction between wild-type RolA and the single mutant CutL1-D30S or quadruple mutant CutL1-D30S/E31S/D142S/D171S by using quartz crystal microbalance. Our results indicate that D30 is a novel residue involved in the ionic interaction between RolA and CutL1.

A fixed-time diffusion analysis method determines that the three cheV genes of Helicobacter pylori differentially affect motility

PubMed Central

Lowenthal, Andrew C.; Simon, Christopher; Fair, Amber S.; Mehmood, Khalid; Terry, Karianne; Anastasia, Stephanie; Ottemann, Karen M.

2009-01-01

Helicobacter pylori is a chemotactic bacterium that has three CheV proteins in its predicted chemotaxis signal transduction system. CheV proteins contain both CheW- and response-regulator-like domains. To determine the function of these proteins, we developed a fixed-time diffusion method that would quantify bacterial direction change without needing to define particular behaviours, to deal with the many behaviours that swimming H. pylori exhibit. We then analysed mutants that had each cheV gene deleted individually and found that the behaviour of each mutant differed substantially from wild-type and the other mutants. cheV1 and cheV2 mutants displayed smooth swimming behaviour, consistent with decreased cellular CheY-P, similar to a cheW mutant. In contrast, the cheV3 mutation had the opposite effect and the mutant cells appeared to change direction frequently. Additional analysis showed that the cheV mutants displayed aberrant behaviour as compared to the wild-type in the soft-agar chemotaxis assay. The soft-agar assay phenotype was less extreme compared to that seen in the fixed-time diffusion model, suggesting that the cheV mutants are able to partially compensate for their defects under some conditions. Each cheV mutant furthermore had defects in mouse colonization that ranged from severe to modest, consistent with a role in chemotaxis. These studies thus show that the H. pylori CheV proteins each differently affect swimming behaviour. PMID:19332820

Possible involvement of inefficient cleavage of preprovasopressin by signal peptidase as a cause for familial central diabetes insipidus.

PubMed Central

Ito, M; Oiso, Y; Murase, T; Kondo, K; Saito, H; Chinzei, T; Racchi, M; Lively, M O

1993-01-01

A transition of G to A at nucleotide position 279 in exon 1 of the vasopressin gene has been identified in patients with familial central diabetes insipidus. The mutation predicts an amino acid substitution of Thr (ACG) for Ala (GCG) at the COOH terminus of the signal peptide in preprovasopression (preproVP). Translation in vitro of wild-type and mutant mRNAs produced 19-kD preproVPs. When translated in the presence of canine pancreatic rough microsomes, wild-type preproVP was converted to a 21-kD protein, whereas the mutant mRNA produced proteins of 21 kD and 23 kD. NH2-terminal amino acid sequence analysis revealed that the 21-kD proteins from the wild-type and the mutants were proVPs generated by the proteolytic cleavage of the 19-residue signal peptide and the addition of carbohydrate. Accordingly, mutant preproVP was cleaved at the correct site after Thr-19, but the efficiency of cleavage by signal peptidase was < 25% that observed for the wild-type preproVP, resulting in the formation of a predominant glycosylated but uncleaved 23-kD product. These data suggest that inefficient processing of preproVP produced by the mutant allele is possibly involved in the pathogenesis of diabetes insipidus in the affected individuals. Images PMID:8514868

Proteins Exported via the PrsD-PrsE Type I Secretion System and the Acidic Exopolysaccharide Are Involved in Biofilm Formation by Rhizobium leguminosarum

PubMed Central

Russo, Daniela M.; Williams, Alan; Edwards, Anne; Posadas, Diana M.; Finnie, Christine; Dankert, Marcelo; Downie, J. Allan; Zorreguieta, Angeles

2006-01-01

The type I protein secretion system of Rhizobium leguminosarum bv. viciae encoded by the prsD and prsE genes is responsible for secretion of the exopolysaccharide (EPS)-glycanases PlyA and PlyB. The formation of a ring of biofilm on the surface of the glass in shaken cultures by both the prsD and prsE secretion mutants was greatly affected. Confocal laser scanning microscopy analysis of green-fluorescent-protein-labeled bacteria showed that during growth in minimal medium, R. leguminosarum wild type developed microcolonies, which progress to a characteristic three-dimensional biofilm structure. However, the prsD and prsE secretion mutants were able to form only an immature biofilm structure. A mutant disrupted in the EPS-glycanase plyB gene showed altered timing of biofilm formation, and its structure was atypical. A mutation in an essential gene for EPS synthesis (pssA) or deletion of several other pss genes involved in EPS synthesis completely abolished the ability of R. leguminosarum to develop a biofilm. Extracellular complementation studies of mixed bacterial cultures confirmed the role of the EPS and the modulation of the biofilm structure by the PrsD-PrsE secreted proteins. Protein analysis identified several additional proteins secreted by the PrsD-PrsE secretion system, and N-terminal sequencing revealed peptides homologous to the N termini of proteins from the Rap family (Rhizobium adhering proteins), which could have roles in cellular adhesion in R. leguminosarum. We propose a model for R. leguminosarum in which synthesis of the EPS leads the formation of a biofilm and several PrsD-PrsE secreted proteins are involved in different aspects of biofilm maturation, such as modulation of the EPS length or mediating attachment between bacteria. PMID:16740954

Identification and Phenotypic Characterization of ZEBRA LEAF16 Encoding a β-Hydroxyacyl-ACP Dehydratase in Rice

PubMed Central

Liu, Ziwen; Wang, Zhiyuan; Gu, Han; You, Jia; Hu, Manman; Zhang, Yujun; Zhu, Ze; Wang, Yihua; Liu, Shijia; Chen, Liangming; Liu, Xi; Tian, Yunlu; Zhou, Shirong; Jiang, Ling; Liu, Linglong; Wan, Jianmin

2018-01-01

The chloroplast is a self-independent organelle and contains its own transcription and translation systems. The establishment of genetic systems is vital for normal plant growth and development. We isolated a rice zebra leaf 16 (zl16) mutant derived from rice cultivar 9311. The zl16 mutant showed chlorotic abnormalities in the transverse sectors of the young leaves of seedlings. The use of transmission electron microscopy (TEM) demonstrated that dramatic defects occurred in variegated zl16 leaves during the early development of a chloroplast. Map-based cloning revealed that ZL16 encodes a β-hydroxyacyl-ACP dehydratase (HAD) involved in de novo fatty acid synthesis. Compared with the wild type, a missense mutation (Arg164Trp) in the zl16 mutant was identified, which significantly reduced enzymatic activity and altered the three-dimensional modeling structure of the putative protein. ZL16 was ubiquitously expressed in various plant organs, with a pronounced level in the young leaf. A subcellular localization experiment indicated that ZL16 was targeted in the chloroplast. Furthermore, we analyzed the expression of some nuclear genes involved in chloroplast development, and found they were altered in the zl16 mutant. RNA-Seq analysis indicated that some genes related to cell membrane constituents were downregulated in the mutant. An in vivo metabolic assay revealed that the total fatty acid content in the mutant was significantly decreased relative to the wild type. Our results indicate that HAD is essential for the development of chloroplasts by regulating the synthesis of fatty acids in rice. PMID:29946330

α2-COP is involved in early secretory traffic in Arabidopsis and is required for plant growth

PubMed Central

Gimeno-Ferrer, Fátima; Pastor-Cantizano, Noelia; Bernat-Silvestre, César; Selvi-Martínez, Pilar; Vera-Sirera, Francisco; Gao, Caiji; Perez-Amador, Miguel Angel; Jiang, Liwen; Aniento, Fernando

2017-01-01

Abstract COP (coat protein) I-coated vesicles mediate intra-Golgi transport and retrograde transport from the Golgi to the endoplasmic reticulum. These vesicles form through the action of the small GTPase ADP-ribosylation factor 1 (ARF1) and the COPI heptameric protein complex (coatomer), which consists of seven subunits (α-, β-, β′-, γ-, δ-, ε- and ζ-COP). In contrast to mammals and yeast, several isoforms for coatomer subunits, with the exception of γ and δ, have been identified in Arabidopsis. To understand the role of COPI proteins in plant biology, we have identified and characterized a loss-of-function mutant of α2-COP, an Arabidopsis α-COP isoform. The α2-cop mutant displayed defects in plant growth, including small rosettes, stems and roots and mislocalization of p24δ5, a protein of the p24 family containing a C-terminal dilysine motif involved in COPI binding. The α2-cop mutant also exhibited abnormal morphology of the Golgi apparatus. Global expression analysis of the α2-cop mutant revealed altered expression of plant cell wall-associated genes. In addition, a strong upregulation of SEC31A, which encodes a subunit of the COPII coat, was observed in the α2-cop mutant; this also occurs in a mutant of a gene upstream of COPI assembly, GNL1, which encodes an ARF-guanine nucleotide exchange factor (GEF). These findings suggest that loss of α2-COP affects the expression of secretory pathway genes. PMID:28025315

Isolation and characterization of gallium resistant Pseudomonas aeruginosa mutants.

PubMed

García-Contreras, Rodolfo; Lira-Silva, Elizabeth; Jasso-Chávez, Ricardo; Hernández-González, Ismael L; Maeda, Toshinari; Hashimoto, Takahiro; Boogerd, Fred C; Sheng, Lili; Wood, Thomas K; Moreno-Sánchez, Rafael

2013-12-01

Pseudomonas aeruginosa PA14 cells resistant to the novel antimicrobial gallium nitrate (Ga) were developed using transposon mutagenesis and by selecting spontaneous mutants. The mutants showing the highest growth in the presence of Ga were selected for further characterization. These mutants showed 4- to 12-fold higher Ga minimal inhibitory growth concentrations and a greater than 8-fold increase in the minimum biofilm eliminating Ga concentration. Both types of mutants produced Ga resistant biofilms whereas the formation of wild-type biofilms was strongly inhibited by Ga. The gene interrupted in the transposon mutant was hitA, which encodes a periplasmic iron binding protein that delivers Fe³⁺ to the HitB iron permease; complementation of the mutant with the hitA gene restored the Ga sensitivity. This hitA mutant showed a 14-fold decrease in Ga internalization versus the wild-type strain, indicating that the HitAB system is also involved in the Ga uptake. Ga uptake in the spontaneous mutant was also lower, although no mutations were found in the hitAB genes. Instead, this mutant harbored 64 non-silent mutations in several genes including those of the phenazine pyocyanin biosynthesis. The spontaneous mutant produced 2-fold higher pyocyanin basal levels than the wild-type; the addition of this phenazine to wild-type cultures protected them from the Ga bacteriostatic effect. The present data indicate that mutations affecting Ga transport and probably pyocyanin biosynthesis enable cells to develop resistance to Ga. Copyright © 2013 Elsevier GmbH. All rights reserved.

The MADS Box Genes ABS, SHP1, and SHP2 Are Essential for the Coordination of Cell Divisions in Ovule and Seed Coat Development and for Endosperm Formation in Arabidopsis thaliana

PubMed Central

Tekleyohans, Dawit G.; Wittkop, Benjamin; Snowdon, Rod J.

2016-01-01

Seed formation is a pivotal process in plant reproduction and dispersal. It begins with megagametophyte development in the ovule, followed by fertilization and subsequently coordinated development of embryo, endosperm, and maternal seed coat. Two closely related MADS-box genes, SHATTERPROOF 1 and 2 (SHP1 and SHP2) are involved in specifying ovule integument identity in Arabidopsis thaliana. The MADS box gene ARABIDOPSIS BSISTER (ABS or TT16) is required, together with SEEDSTICK (STK) for the formation of endothelium, part of the seed coat and innermost tissue layer formed by the maternal plant. Little is known about the genetic interaction of SHP1 and SHP2 with ABS and the coordination of endosperm and seed coat development. In this work, mutant and expression analysis shed light on this aspect of concerted development. Triple tt16 shp1 shp2 mutants produce malformed seedlings, seed coat formation defects, fewer seeds, and mucilage reduction. While shp1 shp2 mutants fail to coordinate the timely development of ovules, tt16 mutants show less peripheral endosperm after fertilization. Failure in coordinated division of the innermost integument layer in early ovule stages leads to inner seed coat defects in tt16 and tt16 shp1 shp2 triple mutant seeds. An antagonistic action of ABS and SHP1/SHP2 is observed in inner seed coat layer formation. Expression analysis also indicates that ABS represses SHP1, SHP2, and FRUITFUL expression. Our work shows that the evolutionary conserved Bsister genes are required not only for endothelium but also for endosperm development and genetically interact with SHP1 and SHP2 in a partially antagonistic manner. PMID:27776173

High-Throughput Genetic Screens Identify a Large and Diverse Collection of New Sporulation Genes in Bacillus subtilis.

PubMed

Meeske, Alexander J; Rodrigues, Christopher D A; Brady, Jacqueline; Lim, Hoong Chuin; Bernhardt, Thomas G; Rudner, David Z

2016-01-01

The differentiation of the bacterium Bacillus subtilis into a dormant spore is among the most well-characterized developmental pathways in biology. Classical genetic screens performed over the past half century identified scores of factors involved in every step of this morphological process. More recently, transcriptional profiling uncovered additional sporulation-induced genes required for successful spore development. Here, we used transposon-sequencing (Tn-seq) to assess whether there were any sporulation genes left to be discovered. Our screen identified 133 out of the 148 genes with known sporulation defects. Surprisingly, we discovered 24 additional genes that had not been previously implicated in spore formation. To investigate their functions, we used fluorescence microscopy to survey early, middle, and late stages of differentiation of null mutants from the B. subtilis ordered knockout collection. This analysis identified mutants that are delayed in the initiation of sporulation, defective in membrane remodeling, and impaired in spore maturation. Several mutants had novel sporulation phenotypes. We performed in-depth characterization of two new factors that participate in cell-cell signaling pathways during sporulation. One (SpoIIT) functions in the activation of σE in the mother cell; the other (SpoIIIL) is required for σG activity in the forespore. Our analysis also revealed that as many as 36 sporulation-induced genes with no previously reported mutant phenotypes are required for timely spore maturation. Finally, we discovered a large set of transposon insertions that trigger premature initiation of sporulation. Our results highlight the power of Tn-seq for the discovery of new genes and novel pathways in sporulation and, combined with the recently completed null mutant collection, open the door for similar screens in other, less well-characterized processes.

High-Throughput Genetic Screens Identify a Large and Diverse Collection of New Sporulation Genes in Bacillus subtilis

PubMed Central

Brady, Jacqueline; Lim, Hoong Chuin; Bernhardt, Thomas G.; Rudner, David Z.

2016-01-01

The differentiation of the bacterium Bacillus subtilis into a dormant spore is among the most well-characterized developmental pathways in biology. Classical genetic screens performed over the past half century identified scores of factors involved in every step of this morphological process. More recently, transcriptional profiling uncovered additional sporulation-induced genes required for successful spore development. Here, we used transposon-sequencing (Tn-seq) to assess whether there were any sporulation genes left to be discovered. Our screen identified 133 out of the 148 genes with known sporulation defects. Surprisingly, we discovered 24 additional genes that had not been previously implicated in spore formation. To investigate their functions, we used fluorescence microscopy to survey early, middle, and late stages of differentiation of null mutants from the B. subtilis ordered knockout collection. This analysis identified mutants that are delayed in the initiation of sporulation, defective in membrane remodeling, and impaired in spore maturation. Several mutants had novel sporulation phenotypes. We performed in-depth characterization of two new factors that participate in cell–cell signaling pathways during sporulation. One (SpoIIT) functions in the activation of σE in the mother cell; the other (SpoIIIL) is required for σG activity in the forespore. Our analysis also revealed that as many as 36 sporulation-induced genes with no previously reported mutant phenotypes are required for timely spore maturation. Finally, we discovered a large set of transposon insertions that trigger premature initiation of sporulation. Our results highlight the power of Tn-seq for the discovery of new genes and novel pathways in sporulation and, combined with the recently completed null mutant collection, open the door for similar screens in other, less well-characterized processes. PMID:26735940

Bioconversion of glycerol to ethanol by a mutant Enterobacter aerogenes

PubMed Central

2012-01-01

The main objective of this research is to develop, by adaptive evolution, mutant strains of Enterobacter aerogenes ATCC 13048 that are capable of withstanding high glycerol concentration as well as resisting ethanol-inhibition. The mutant will be used for high ethanol fermentation from glycerol feedstock. Ethanol production from pure (P-) and recovered (R-) glycerol using the stock was evaluated. A six-tube-subculture-generations method was used for developing the mutant. This involved subculturing the organism six consecutive times in tubes containing the same glycerol and ethanol concentrations at the same culture conditions. Then, the glycerol and/or ethanol concentration was increased and the six subculture generations were repeated. A strain capable of growing in 200 g/L glycerol and 30 g/L ethanol was obtained. The ability of this mutant, vis-à-vis the original strain, in utilizing glycerol in a high glycerol containing medium, with the concomitant ethanol yield, was assessed. Tryptic soy broth without dextrose (TSB) was used as the fermentation medium. Fermentation products were analyzed using HPLC. In a 20 g/L glycerol TSB, E. aerogenes ATCC 13048 converted 18.5 g/L P-glycerol and 17.8 g/L R-glycerol into 12 and 12.8 g/L ethanol, respectively. In a 50 g/L P-glycerol TSB, it utilized only 15.6 g/L glycerol; but the new strain used up 39 g/L, yielding 20 g/L ethanol after 120 h, an equivalence of 1.02 mol ethanol/mol-glycerol. This is the highest ethanol yield reported from glycerol bioconversion. The result of this P-glycerol fermentation can be duplicated using the R-glycerol from biodiesel production. PMID:22455837

The mouse lymphoma assay detects recombination, deletion, and aneuploidy.

PubMed

Wang, Jianyong; Sawyer, Jeffrey R; Chen, Ling; Chen, Tao; Honma, Masamitsu; Mei, Nan; Moore, Martha M

2009-05-01

The mouse lymphoma assay (MLA) uses the thymidine kinase (Tk) gene of the L5178Y/Tk(+/-)-3.7.2C mouse lymphoma cell line as a reporter gene to evaluate the mutagenicity of chemical and physical agents. The MLA is recommended by both the United States Food and Drug Administration and the United States Environmental Protection Agency as the preferred in vitro mammalian cell mutation assay for genetic toxicology screening because it detects a wide range of genetic alterations, including both point mutations and chromosomal mutations. However, the specific types of chromosomal mutations that can be detected by the MLA need further clarification. For this purpose, three chemicals, including two clastogens and an aneugen (3'-azido-3'-deoxythymidine, mitomycin C, and taxol), were used to induce Tk mutants. Loss of heterozygosity (LOH) analysis was used to select mutants that could be informative as to whether they resulted from deletion, mitotic recombination, or aneuploidy. A combination of additional methods, G-banding analysis, chromosome painting, and a real-time PCR method to detect the copy number (CN) of the Tk gene was then used to provide a detailed analysis. LOH involving at least 25% of chromosome 11, a normal karyotype, and a Tk CN of 2 would indicate that the mutant resulted from recombination, whereas LOH combined with a karyotypically visible deletion of chromosome 11 and a Tk CN of 1 would indicate a deletion. Aneuploidy was confirmed using G-banding combined with chromosome painting analysis for mutants showing LOH at every microsatellite marker on chromosome 11. From this analysis, it is clear that mouse lymphoma Tk mutants can result from recombination, deletion, and aneuploidy.

The Arthroderma benhamiae hydrophobin HypA mediates hydrophobicity and influences recognition by human immune effector cells.

PubMed

Heddergott, Christoph; Bruns, Sandra; Nietzsche, Sandor; Leonhardt, Ines; Kurzai, Oliver; Kniemeyer, Olaf; Brakhage, Axel A

2012-05-01

Dermatophytes are the most common cause of superficial mycoses in humans and animals. They can coexist with their hosts for many years without causing significant symptoms but also cause highly inflammatory diseases. To identify mechanisms involved in the modulation of the host response during infection caused by the zoophilic dermatophyte Arthroderma benhamiae, cell wall-associated surface proteins were studied. By two-dimensional gel electrophoresis, we found that a hydrophobin protein designated HypA was the dominant cell surface protein. HypA was also detected in the supernatant during the growth and conidiation of the fungus. The A. benhamiae genome harbors only a single hydrophobin gene, designated hypA. A hypA deletion mutant was generated, as was a complemented hypA mutant strain (hypA(C)). In contrast to the wild type and the complemented strain, the hypA deletion mutant exhibited "easily wettable" mycelia and conidia, indicating the loss of surface hydrophobicity of both morphotypes. Compared with the wild type, the hypA deletion mutant triggered an increased activation of human neutrophil granulocytes and dendritic cells, characterized by an increased release of the immune mediators interleukin-6 (IL-6), IL-8, IL-10, and tumor necrosis factor alpha (TNF-α). For the first time, we observed the formation of neutrophil extracellular traps against dermatophytes, whose level of formation was increased by the ΔhypA mutant compared with the wild type. Furthermore, conidia of the ΔhypA strain were killed more effectively by neutrophils. Our data suggest that the recognition of A. benhamiae by the cellular immune defense system is notably influenced by the presence of the surface rodlet layer formed by the hydrophobin HypA.

The Arthroderma benhamiae Hydrophobin HypA Mediates Hydrophobicity and Influences Recognition by Human Immune Effector Cells

PubMed Central

Heddergott, Christoph; Bruns, Sandra; Nietzsche, Sandor; Leonhardt, Ines; Kurzai, Oliver; Kniemeyer, Olaf

2012-01-01

Dermatophytes are the most common cause of superficial mycoses in humans and animals. They can coexist with their hosts for many years without causing significant symptoms but also cause highly inflammatory diseases. To identify mechanisms involved in the modulation of the host response during infection caused by the zoophilic dermatophyte Arthroderma benhamiae, cell wall-associated surface proteins were studied. By two-dimensional gel electrophoresis, we found that a hydrophobin protein designated HypA was the dominant cell surface protein. HypA was also detected in the supernatant during the growth and conidiation of the fungus. The A. benhamiae genome harbors only a single hydrophobin gene, designated hypA. A hypA deletion mutant was generated, as was a complemented hypA mutant strain (hypAC). In contrast to the wild type and the complemented strain, the hypA deletion mutant exhibited “easily wettable” mycelia and conidia, indicating the loss of surface hydrophobicity of both morphotypes. Compared with the wild type, the hypA deletion mutant triggered an increased activation of human neutrophil granulocytes and dendritic cells, characterized by an increased release of the immune mediators interleukin-6 (IL-6), IL-8, IL-10, and tumor necrosis factor alpha (TNF-α). For the first time, we observed the formation of neutrophil extracellular traps against dermatophytes, whose level of formation was increased by the ΔhypA mutant compared with the wild type. Furthermore, conidia of the ΔhypA strain were killed more effectively by neutrophils. Our data suggest that the recognition of A. benhamiae by the cellular immune defense system is notably influenced by the presence of the surface rodlet layer formed by the hydrophobin HypA. PMID:22408226

Pro-Apoptotic Role of the Human YPEL5 Gene Identified by Functional Complementation of a Yeast moh1Δ Mutation.

PubMed

Lee, Ji Young; Jun, Do Youn; Park, Ju Eun; Kwon, Gi Hyun; Kim, Jong-Sik; Kim, Young Ho

2017-03-28

To examine the pro-apoptotic role of the human ortholog (YPEL5) of the Drosophila Yippee protein, the cell viability of Saccharomyces cerevisiae mutant strain with deleted MOH1 , the yeast ortholog, was compared with that of the wild-type (WT)- MOH1 strain after exposure to different apoptogenic stimulants, including UV irradiation, methyl methanesulfonate (MMS), camptothecin (CPT), heat shock, and hyperosmotic shock. The moh1 Δ mutant exhibited enhanced cell viability compared with the WT- MOH1 strain when treated with lethal UV irradiation, 1.8 mM MMS, 100 µ CPT, heat shock at 50°C, or 1.2 M KCl. At the same time, the level of Moh1 protein was commonly up-regulated in the WT- MOH1 strain as was that of Ynk1 protein, which is known as a marker for DNA damage. Although the enhanced UV resistance of the moh1 Δ mutant largely disappeared following transformation with the yeast MOH1 gene or one of the human YPEL1-YPEL5 genes, the transformant bearing pYES2- YPEL5 was more sensitive to lethal UV irradiation and its UV sensitivity was similar to that of the WT- MOH1 strain. Under these conditions, the UV irradiation-induced apoptotic events, such as FITC-Annexin V stainability, mitochondrial membrane potential (ΔΨm) loss, and metacaspase activation, occurred to a much lesser extent in the moh1 Δ mutant compared with the WT- MOH1 strain and the mutant strain bearing pYES2- MOH1 or pYES2- YPEL5 . These results demonstrate the functional conservation between yeast Moh1 and human YPEL5, and their involvement in mitochondria-dependent apoptosis induced by DNA damage.

The RNA Silencing Enzyme RNA Polymerase V Is Required for Plant Immunity

PubMed Central

López, Ana; Ramírez, Vicente; García-Andrade, Javier; Flors, Victor; Vera, Pablo

2011-01-01

RNA–directed DNA methylation (RdDM) is an epigenetic control mechanism driven by small interfering RNAs (siRNAs) that influence gene function. In plants, little is known of the involvement of the RdDM pathway in regulating traits related to immune responses. In a genetic screen designed to reveal factors regulating immunity in Arabidopsis thaliana, we identified NRPD2 as the OVEREXPRESSOR OF CATIONIC PEROXIDASE 1 (OCP1). NRPD2 encodes the second largest subunit of the plant-specific RNA Polymerases IV and V (Pol IV and Pol V), which are crucial for the RdDM pathway. The ocp1 and nrpd2 mutants showed increases in disease susceptibility when confronted with the necrotrophic fungal pathogens Botrytis cinerea and Plectosphaerella cucumerina. Studies were extended to other mutants affected in different steps of the RdDM pathway, such as nrpd1, nrpe1, ago4, drd1, rdr2, and drm1drm2 mutants. Our results indicate that all the mutants studied, with the exception of nrpd1, phenocopy the nrpd2 mutants; and they suggest that, while Pol V complex is required for plant immunity, Pol IV appears dispensable. Moreover, Pol V defective mutants, but not Pol IV mutants, show enhanced disease resistance towards the bacterial pathogen Pseudomonas syringae DC3000. Interestingly, salicylic acid (SA)–mediated defenses effective against PsDC3000 are enhanced in Pol V defective mutants, whereas jasmonic acid (JA)–mediated defenses that protect against fungi are reduced. Chromatin immunoprecipitation analysis revealed that, through differential histone modifications, SA–related defense genes are poised for enhanced activation in Pol V defective mutants and provide clues for understanding the regulation of gene priming during defense. Our results highlight the importance of epigenetic control as an additional layer of complexity in the regulation of plant immunity and point towards multiple components of the RdDM pathway being involved in plant immunity based on genetic evidence, but whether this is a direct or indirect effect on disease-related genes is unclear. PMID:22242006

Involvement of Two Plasmids in the Degradation of Carbaryl by Arthrobacter sp. Strain RC100

PubMed Central

Hayatsu, Masahito; Hirano, Motoko; Nagata, Tadahiro

1999-01-01

A bacterium capable of utilizing carbaryl (1-naphthyl N-methylcarbamate) as the sole carbon source was isolated from carbaryl-treated soil. This bacterium was characterized taxonomically as Arthrobacter and was designated strain RC100. RC100 hydrolyzes the N-methylcarbamate linkage to 1-naphthol, which was further metabolized via salicylate and gentisate. Strain RC100 harbored three plasmids (designated pRC1, pRC2, and pRC3). Mutants unable to degrade carbaryl arose at a high frequency after treating the culture with mitomycin C. All carbaryl-hydrolysis-deficient mutants (Cah−) lacked pRC1, and all 1-naphthol-utilization-deficient mutants (Nat−) lacked pRC2. The plasmid-free strain RC107 grew on gentisate as a carbon source. These two plasmids could be transferred to Cah− mutants or Nat− mutants by conjugation, resulting in the restoration of the Cah and Nah phenotypes. PMID:10049857

Improvement of DNA transfer frequency and transposon mutagenesis of Erwinia carotovora subsp. betavasculorum.

PubMed Central

Rella, M; Axelrood, P E; Weinhold, A R; Schroth, M N

1989-01-01

The production of antibiotics and their role in microbial competition under natural conditions can be readily studied by the use of transposon mutants. Several antibiotic-producing strains of Erwinia carotovora subsp. betavasculorum were unable to accept foreign DNA. A plasmid delivery system was developed, using ethyl methanesulfonate mutagenesis, which entailed isolating E. carotovora subsp. betavasculorum mutants able to accept foreign DNA and transfer it to other strains. This enabled transposon mutagenesis of a wild-type antibiotic-producing strain of E. carotovora subsp. betavasculorum. Twelve antibiotic-negative mutants were isolated, and one of these showed a reduction in antibiotic production in vitro. Many of these mutants also showed a reduction in their ability to macerate potato tissue. The mutants were classified into four genetic groups on the basis of their genetic and phenotypic characteristics, indicating that several genes are involved in antibiotic biosynthesis by E. carotovora subsp. betavasculorum. PMID:2543291

A detailed study of gerJ mutants of Bacillus subtilis.

PubMed

Warburg, R J; Buchanan, C E; Parent, K; Halvorson, H O

1986-08-01

A total of nine gerJ mutants have now been isolated in Bacillus subtilis. All are defective in their spore germination properties, being blocked at an intermediate (phase grey) stage. The dormant spores are sensitive to heating at 90 degrees C and two of the mutants (generated by transposon insertion) produce spores sensitive at 80 degrees C. The spores of these two more extreme mutants had a visibly defective cortex when studied by electron microscopy, as did some of the other mutants. During sporulation, the acquisition of spore resistance properties and the appearance of the sporulation-specific penicillin-binding protein PBP5* were delayed. A strain probably carrying a lacZ fusion to the gerJ promoter demonstrated increased expression between t2 and t4. We propose that the gerJ locus is involved in the control of one or more sporulation-specific genes.

SP600125 has a remarkable anticancer potential against undifferentiated thyroid cancer through selective action on ROCK and p53 pathways.

PubMed

Grassi, Elisa Stellaria; Vezzoli, Valeria; Negri, Irene; Lábadi, Árpád; Fugazzola, Laura; Vitale, Giovanni; Persani, Luca

2015-11-03

Thyroid cancer is the most common endocrine malignancy with increasing incidence worldwide.The majority of thyroid cancer cases are well differentiated with favorable outcome. However, undifferentiated thyroid cancers are one of the most lethal human malignancies because of their invasiveness, metastatization and refractoriness even to the most recently developed therapies.In this study we show for the first time a significant hyperactivation of ROCK/HDAC6 pathway in thyroid cancer tissues, and its negative correlation with p53 DNA binding ability.We demonstrate that a small compound, SP600125 (SP), is able to induce cell death selectively in undifferentiated thyroid cancer cell lines by specifically acting on the pathogenic pathways of cancer development. In detail, SP acts on the ROCK/HDAC6 pathway involved in dedifferentiation and invasiveness of undifferentiated human cancers, by restoring its physiological activity level. As main consequence, cancer cell migration is inhibited and, at the same time, cell death is induced through the mitotic catastrophe. Moreover, SP exerts a preferential action on the mutant p53 by increasing its DNA binding ability. In TP53-mutant cells that survive mitotic catastrophe this process results in p21 induction and eventually lead to premature senescence. In conclusion, SP has been proved to be able to simultaneously block cell replication and migration, the two main processes involved in cancer development and dissemination, making it an ideal candidate for developing new drugs against anaplastic thyroid cancer.

Serratia marcescens arn, a PhoP-regulated locus necessary for polymyxin B resistance.

PubMed

Lin, Quei Yen; Tsai, Yi-Lin; Liu, Ming-Che; Lin, Wei-Cheng; Hsueh, Po-Ren; Liaw, Shwu-Jen

2014-09-01

Polymyxins, which are increasingly being used to treat infections caused by multidrug-resistant bacteria, perform poorly against Serratia marcescens. To investigate the underlying mechanisms, Tn5 mutagenesis was performed and two mutants exhibiting increased polymyxin B (PB) susceptibility were isolated. The mutants were found to have Tn5 inserted into the arnB and arnC genes. In other bacteria, arnB and arnC belong to the seven-gene arn operon, which is involved in lipopolysaccharide (LPS) modification. LPSs of arn mutants had greater PB-binding abilities than that of wild-type LPS. Further, we identified PhoP, a bacterial two-component response regulator, as a regulator of PB susceptibility in S. marcescens. By the reporter assay, we found PB- and low-Mg2+-induced expression of phoP and arn in the wild-type strain but not in the phoP mutant. Complementation of the phoP mutant with the full-length phoP gene restored the PB MIC and induction by PB and low Mg2+ levels, as in the wild type. An electrophoretic mobility shift assay (EMSA) further demonstrated that PhoP bound directly to the arn promoter. The PB challenge test confirmed that pretreatment with PB and low Mg2+ levels protected S. marcescens from a PB challenge in the wild-type strain but not in the phoP mutant. Real-time reverse transcriptase-PCR also indicated that PB serves as a signal to regulate expression of ugd, a gene required for LPS modification, in S. marcescens through a PhoP-dependent pathway. Finally, we found that PB-resistant clinical isolates displayed greater expression of arnA upon exposure to PB than did susceptible isolates. This is the first report to describe the role of S. marcescens arn in PB resistance and its modulation by PB and Mg2+ through the PhoP protein. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Serratia marcescens arn, a PhoP-Regulated Locus Necessary for Polymyxin B Resistance

PubMed Central

Lin, Quei Yen; Tsai, Yi-Lin; Liu, Ming-Che; Lin, Wei-Cheng; Hsueh, Po-Ren

2014-01-01

Polymyxins, which are increasingly being used to treat infections caused by multidrug-resistant bacteria, perform poorly against Serratia marcescens. To investigate the underlying mechanisms, Tn5 mutagenesis was performed and two mutants exhibiting increased polymyxin B (PB) susceptibility were isolated. The mutants were found to have Tn5 inserted into the arnB and arnC genes. In other bacteria, arnB and arnC belong to the seven-gene arn operon, which is involved in lipopolysaccharide (LPS) modification. LPSs of arn mutants had greater PB-binding abilities than that of wild-type LPS. Further, we identified PhoP, a bacterial two-component response regulator, as a regulator of PB susceptibility in S. marcescens. By the reporter assay, we found PB- and low-Mg2+-induced expression of phoP and arn in the wild-type strain but not in the phoP mutant. Complementation of the phoP mutant with the full-length phoP gene restored the PB MIC and induction by PB and low Mg2+ levels, as in the wild type. An electrophoretic mobility shift assay (EMSA) further demonstrated that PhoP bound directly to the arn promoter. The PB challenge test confirmed that pretreatment with PB and low Mg2+ levels protected S. marcescens from a PB challenge in the wild-type strain but not in the phoP mutant. Real-time reverse transcriptase-PCR also indicated that PB serves as a signal to regulate expression of ugd, a gene required for LPS modification, in S. marcescens through a PhoP-dependent pathway. Finally, we found that PB-resistant clinical isolates displayed greater expression of arnA upon exposure to PB than did susceptible isolates. This is the first report to describe the role of S. marcescens arn in PB resistance and its modulation by PB and Mg2+ through the PhoP protein. PMID:24957827

The interaction between glucose and cytokinin signaling in controlling Arabidopsis thaliana seedling root growth and development

PubMed Central

Kushwah, Sunita

2017-01-01

ABSTRACT Cytokinin (CK) and glucose (GLC) control several common responses in plants. There is an extensive overlap between CK and GLC signal transduction pathways in Arabidopsis. Physiologically, both GLC and CK could regulate root length in light. CK interacts with GLC via HXK1 dependent pathway for root length control. Wild-type (WT) roots cannot elongate in the GLC free medium while CK-receptor mutant ARABIDOPSIS HISTIDINE KINASE4 (ahk4) and type B ARR triple mutant ARABIDOPSIS RESPONSE REGULATOR1, 10,11 (arr1, 10,11) roots could elongate even in the absence of GLC as compared with the WT. The root hair initiation was also found defective in CK signaling mutants ahk4, arr1,10,11 and arr3,4,5,6,8,9 on increasing GLC concentration (up to 3%); and lesser number of root hairs were visible even at 5% GLC as compared with the WT. Out of 941 BAP regulated genes, 103 (11%) genes were involved in root growth and development. Out of these 103 genes, 60 (58%) genes were also regulated by GLC. GLC could regulate 5736 genes, which include 327 (6%) genes involved in root growth and development. Out of these 327 genes, 60 (18%) genes were also regulated by BAP. Both GLC and CK signaling cannot alter root length in light in auxin signaling mutant AUXIN RESPONSE3/INDOLE-3-ACETIC ACID17 (axr3/iaa17) suggesting that they may involve auxin signaling component as a nodal point. Therefore CK- and GLC- signaling are involved in controlling different aspects of root growth and development such as root length, with auxin signaling components working as downstream target. PMID:28467152

The interaction between glucose and cytokinin signaling in controlling Arabidopsis thaliana seedling root growth and development.

PubMed

Kushwah, Sunita; Laxmi, Ashverya

2017-05-04

Cytokinin (CK) and glucose (GLC) control several common responses in plants. There is an extensive overlap between CK and GLC signal transduction pathways in Arabidopsis. Physiologically, both GLC and CK could regulate root length in light. CK interacts with GLC via HXK1 dependent pathway for root length control. Wild-type (WT) roots cannot elongate in the GLC free medium while CK-receptor mutant ARABIDOPSIS HISTIDINE KINASE4 (ahk4) and type B ARR triple mutant ARABIDOPSIS RESPONSE REGULATOR1, 10,11 (arr1, 10,11) roots could elongate even in the absence of GLC as compared with the WT. The root hair initiation was also found defective in CK signaling mutants ahk4, arr1,10,11 and arr3,4,5,6,8,9 on increasing GLC concentration (up to 3%); and lesser number of root hairs were visible even at 5% GLC as compared with the WT. Out of 941 BAP regulated genes, 103 (11%) genes were involved in root growth and development. Out of these 103 genes, 60 (58%) genes were also regulated by GLC. GLC could regulate 5736 genes, which include 327 (6%) genes involved in root growth and development. Out of these 327 genes, 60 (18%) genes were also regulated by BAP. Both GLC and CK signaling cannot alter root length in light in auxin signaling mutant AUXIN RESPONSE3/INDOLE-3-ACETIC ACID17 (axr3/iaa17) suggesting that they may involve auxin signaling component as a nodal point. Therefore CK- and GLC- signaling are involved in controlling different aspects of root growth and development such as root length, with auxin signaling components working as downstream target.

Evidence from in vivo manipulations of lipid composition in mutants that the delta 3-trans-hexadecenoic acid-containing phosphatidylglycerol is involved in the biogenesis of the light-harvesting chlorophyll a/b-protein complex of Chlamydomonas reinhardtii.

PubMed

Dubertret, G; Mirshahi, A; Mirshahi, M; Gerard-Hirne, C; Tremolieres, A

1994-12-01

The phosphatidylglycerol containing the unusual delta 3-trans hexadecenoic fatty acid is specifically found in photosynthetic membranes of eukaryotic organisms. Its involvement in the biogenesis and the structure of the light-harvesting chlorophyll a/b-protein complex has been evidenced by in vivo targeting this lipid to photosynthetic membranes of Chlamydomonas reinhardtii mutants lacking this lipid. In the mf1 and mf2 mutants, this deficiency results in (a) the absence of the oligomeric light-harvesting complex of photosystem 2; (b) an extensive destacking of thylakoid membranes; (c) a very low 77-K fluorescence emission in the photosystem-2 region. We show in this paper that these deficiencies result from modifications in the pigment and polypeptide compositions of the photosystem-2 light-harvesting complex; it contains less chlorophyll b and some of its constitutive polypeptides are absent or reduced in amount, while immunologically related polypeptides of lower molecular mass accumulate. The direct involvement of the lack of trans-C16: 1-phosphatidylglycerol in these deficiencies is evidenced by the partial restoration of normal characteristics of the light-harvesting complex (pigment and polypeptide composition, oligomerization) after liposome-mediated, in vivo incorporation of this lipid into the photosynthetic membranes of the mf2 mutant. Trans-C16:1-phosphatidylglycerol, therefore, is involved in the biogenesis of the photosystem-2 light-harvesting chlorophyll a/b-protein complex through a mechanism that may prevent degradation processes. Its contribution to the structural conformation of neosynthesized monomers and to their organization into stable oligomeric form is discussed.

THE REELIN RECEPTORS VLDLR AND ApoER2 REGULATE SENSORIMOTOR GATING IN MICE

PubMed Central

Barr, Alasdair M.; Fish, Kenneth N.; Markou, Athina

2007-01-01

Summary Postmortem brain loss of reelin is noted in schizophrenia patients. Accordingly, heterozygous reeler mutant mice have been proposed as a putative model of this disorder. Little is known, however, about the involvement of the two receptors for reelin, Very-Low-Density Lipoprotein Receptor (VLDLR) and Apolipoprotein E Receptor 2 (ApoER2), on pre-cognitive processes of relevance to deficits seen in schizophrenia. Thus, we evaluated sensorimotor gating in mutant mice heterozygous or homozygous for the two reelin receptors. Mutant mice lacking one of these reelin receptors were tested for prepulse inhibition (PPI) of the acoustic startle reflex prior to and following puberty, and on a crossmodal PPI task, involving the presentation of acoustic and tactile stimuli. Furthermore, because schizophrenia patients show increased sensitivity to N-methyl-D-aspartate (NMDA) receptor blockade, we assessed the sensitivity of these mice to the PPI-disruptive effects of the NMDA receptor antagonist phencyclidine. The results demonstrated that acoustic PPI did not differ between mutant and wildtype mice. However, VLDLR homozygous mice displayed significant deficits in crossmodal PPI, while ApoER2 heterozygous and homozygous mice displayed significantly increased crossmodal PPI. Both ApoER2 and VLDLR heterozygous and homozygous mice exhibited greater sensitivity to the PPI-disruptive effects of phencyclidine than wildtype mice. These results indicate that partial or complete loss of either one of the reelin receptors results in a complex pattern of alterations in PPI function that include alterations in crossmodal, but not acoustic, PPI and increased sensitivity to NMDA receptor blockade. Thus, reelin receptor function appears to be critically involved in crossmodal PPI and the modulation of the PPI response by NMDA receptors. These findings have relevance to a range of neuropsychiatric disorders that involve sensorimotor gating deficits, including schizophrenia.. PMID:17261317

Multiple virulence factors regulated by quorum sensing may help in establishment and colonisation of urinary tract by Pseudomonas aeruginosa during experimental urinary tract infection.

PubMed

Gupta, P; Gupta, R K; Harjai, K

2013-01-01

Damage caused by an organism during infection is attributed to production of virulence factors. Different virulence factors produced by the organism contribute to its pathogenicity, individually. During infectious conditions, role of virulence factors produced by the pathogen is different, depending upon the site of involvement. Pseudomonas aeruginosa is an opportunistic nosocomial pathogen known to cause infections of the respiratory tract, burn wound, urinary tract and eye. Importance of virulence factors produced by P. Aeruginosa during infections such as keratitis, burn wound and respiratory tract is known. The present study was designed to understand the importance of different virulence factors of P. aeruginosa in urinary tract infection in vivo. An ascending urinary tract infection model was established in mice using standard parent strain PAO1 and its isogenic mutant, JP2. Mice were sacrificed at different time intervals and renal tissue homogenates were used for estimation of renal bacterial load and virulence factors. Both parent and mutant strains were able to reach the renal tissue. PAO 1 PAO1 was isolated from renal tissue till day 5 post-infection. However, the mutant strain was unable to colonise the renal tissue. Failure of mutant strain to colonise was attributed to its inability to produce protease, elastase and rhamnolipid. This study suggests that protease, elastase and rhamnolipid contribute to pathogenesis and survival of P. aeruginosa during urinary tract infection.

Amino acid substitution converts WEREWOLF function from an activator to a repressor of Arabidopsis non-hair cell development.

PubMed

Tominaga-Wada, Rumi; Nukumizu, Yuka; Wada, Takuji

2012-02-01

Root hair cell or non-hair cell fate determination in Arabidopsis thaliana root epidermis is model system for plant cell development. Two types of MYB transcription factors, the R2R3-type MYB, WEREWOLF (WER), and an R3-type MYB, CAPRICE (CPC), are involved in this cell fate determination process. To study the molecular basis of this process, we analyzed the functional relationship of WER and CPC. WER-CPC chimeric constructs were made from WER where all or parts of the MYB R3 region were replaced with the corresponding regions from CPC R3, and the constructs were introduced into the cpc-2 mutant. Although, the WER gene did not rescue the cpc-2 mutant 'small number of root hairs' phenotype, the WER-CPC chimera with two amino acids substitution (WC6) completely rescued the cpc-2 mutant phenotype. Furthermore, the WER-CPC chimera with 37 amino acids substitution (WC5) excessively rescued the cpc-2 mutant and induced 2.5 times more root hairs than wild-type. Consistent with this phenotype, GL2 gene expression was strongly reduced in WC5 in a cpc-2 background. Our results suggest that swapping at least two amino acids is sufficient to convert WER to CPC function. Therefore, these key residues may have strongly contributed to the selection of these important functions over evolution. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Astrocytes expressing ALS‐linked mutant FUS induce motor neuron death through release of tumor necrosis factor‐alpha

PubMed Central

Kia, Azadeh; McAvoy, Kevin; Krishnamurthy, Karthik; Trotti, Davide

2018-01-01

Mutations in fused in sarcoma (FUS) are linked to amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease affecting both upper and lower motor neurons. While it is established that astrocytes contribute to the death of motor neurons in ALS, the specific contribution of mutant FUS (mutFUS) through astrocytes has not yet been studied. Here, we used primary astrocytes expressing a N‐terminally GFP tagged R521G mutant or wild‐type FUS (WTFUS) and show that mutFUS‐expressing astrocytes undergo astrogliosis, damage co‐cultured motor neurons via activation of an inflammatory response and produce conditioned medium (ACM) that is toxic to motor neurons in isolation. Time lapse imaging shows that motor neuron cultures exposed to mutFUS ACM, but not WTFUS ACM, undergo significant cell loss, which is preceded by progressive degeneration of neurites. We found that Tumor Necrosis Factor‐Alpha (TNFα) is secreted into ACM of mutFUS‐expressing astrocytes. Accordingly, mutFUS astrocyte‐mediated motor neuron toxicity is blocked by targeting soluble TNFα with neutralizing antibodies. We also found that mutant astrocytes trigger changes to motor neuron AMPA receptors (AMPAR) that render them susceptible to excitotoxicity and AMPAR‐mediated cell death. Our data provide the first evidence of astrocytic involvement in FUS‐ALS, identify TNFα as a mediator of this toxicity, and provide several potential therapeutic targets to protect motor neurons in FUS‐linked ALS. PMID:29380416

Functional and proteomic analyses reveal that wxcB is involved in virulence, motility, detergent tolerance, and biofilm formation in Xanthomonas campestris pv. vesicatoria.

PubMed

Park, Hye-Jee; Jung, Ho Won; Han, Sang-Wook

2014-09-26

The bacterial envelope possesses diverse functions, including protection against environmental stress and virulence factors for host infection. Here, we report the function of wxcB in Xanthomonas campestris pv. vesicatoria (Xcv), a causal agent of bacterial leaf spot disease in tomato and pepper. To characterize roles of wxcB, we generated a knockout mutant (XcvΔwxcB) and found that the virulence of the mutant was weaker than that of the wild type in tomato plants. To predict the mechanism affected by wxcB, we compared protein expressions between the wild type and the mutant. Expression of 152 proteins showed a greater than 2-fold difference. Proteins involved in motility and cell wall/membrane were the most abundant. Through phenotypic assays, we further demonstrated that the mutant displayed reduced motility and tolerance to treatment, but it showed increased biofilm formation. Interestingly, the LPS profile was unchanged. These results lead to new insights into the functions of wxcB that is associated with cell wall/membrane functions, which contributes to pathogen virulence. Copyright © 2014 Elsevier Inc. All rights reserved.

Reptin/Ruvbl2 is a Lrrc6/Seahorse interactor essential for cilia motility

PubMed Central

Zhao, Lu; Yuan, Shiaulou; Cao, Ying; Kallakuri, Sowjanya; Li, Yuanyuan; Kishimoto, Norihito; DiBella, Linda; Sun, Zhaoxia

2013-01-01

Primary ciliary dyskinesia (PCD) is an autosomal recessive disease caused by defective cilia motility. The identified PCD genes account for about half of PCD incidences and the underlying mechanisms remain poorly understood. We demonstrate that Reptin/Ruvbl2, a protein known to be involved in epigenetic and transcriptional regulation, is essential for cilia motility in zebrafish. We further show that Reptin directly interacts with the PCD protein Lrrc6/Seahorse and this interaction is critical for the in vivo function of Lrrc6/Seahorse in zebrafish. Moreover, whereas the expression levels of multiple dynein arm components remain unchanged or become elevated, the density of axonemal dynein arms is reduced in reptinhi2394 mutants. Furthermore, Reptin is highly enriched in the cytosol and colocalizes with Lrrc6/Seahorse. Combined, these results suggest that the Reptin-Lrrc6/Seahorse complex is involved in dynein arm formation. We also show that although the DNA damage response is induced in reptinhi2394 mutants, it remains unchanged in cilia biogenesis mutants and lrrc6/seahorse mutants, suggesting that increased DNA damage response is not intrinsic to ciliary defects and that in vertebrate development, Reptin functions in multiple processes, both cilia specific and cilia independent. PMID:23858445

Reptin/Ruvbl2 is a Lrrc6/Seahorse interactor essential for cilia motility.

PubMed

Zhao, Lu; Yuan, Shiaulou; Cao, Ying; Kallakuri, Sowjanya; Li, Yuanyuan; Kishimoto, Norihito; DiBella, Linda; Sun, Zhaoxia

2013-07-30

Primary ciliary dyskinesia (PCD) is an autosomal recessive disease caused by defective cilia motility. The identified PCD genes account for about half of PCD incidences and the underlying mechanisms remain poorly understood. We demonstrate that Reptin/Ruvbl2, a protein known to be involved in epigenetic and transcriptional regulation, is essential for cilia motility in zebrafish. We further show that Reptin directly interacts with the PCD protein Lrrc6/Seahorse and this interaction is critical for the in vivo function of Lrrc6/Seahorse in zebrafish. Moreover, whereas the expression levels of multiple dynein arm components remain unchanged or become elevated, the density of axonemal dynein arms is reduced in reptin(hi2394) mutants. Furthermore, Reptin is highly enriched in the cytosol and colocalizes with Lrrc6/Seahorse. Combined, these results suggest that the Reptin-Lrrc6/Seahorse complex is involved in dynein arm formation. We also show that although the DNA damage response is induced in reptin(hi2394) mutants, it remains unchanged in cilia biogenesis mutants and lrrc6/seahorse mutants, suggesting that increased DNA damage response is not intrinsic to ciliary defects and that in vertebrate development, Reptin functions in multiple processes, both cilia specific and cilia independent.

Exit of Plasmodium Sporozoites from Oocysts Is an Active Process That Involves the Circumsporozoite Protein

PubMed Central

Wang, Qian; Fujioka, Hisashi; Nussenzweig, Victor

2005-01-01

Plasmodium sporozoites develop within oocysts residing in the mosquito midgut. Mature sporozoites exit the oocysts, enter the hemolymph, and invade the salivary glands. The circumsporozoite (CS) protein is the major surface protein of salivary gland and oocyst sporozoites. It is also found on the oocyst plasma membrane and on the inner surface of the oocyst capsule. CS protein contains a conserved motif of positively charged amino acids: region II-plus, which has been implicated in the initial stages of sporozoite invasion of hepatocytes. We investigated the function of region II-plus by generating mutant parasites in which the region had been substituted with alanines. Mutant parasites produced normal numbers of sporozoites in the oocysts, but the sporozoites were unable to exit the oocysts. In in vitro as well, there was a profound delay, upon trypsin treatment, in the release of mutant sporozoites from oocysts. We conclude that the exit of sporozoites from oocysts is an active process that involves the region II-plus of CS protein. In addition, the mutant sporozoites were not infective to young rats. These findings provide a new target for developing reagents that interfere with the transmission of malaria. PMID:16201021

Identification of Residues Involved in Substrate Specificity and Cytotoxicity of Two Closely Related Cutinases from Mycobacterium tuberculosis

PubMed Central

Dedieu, Luc; Serveau-Avesque, Carole; Canaan, Stéphane

2013-01-01

The enzymes belonging to the cutinase family are serine enzymes active on a large panel of substrates such as cutin, triacylglycerols, and phospholipids. In the M. tuberculosis H37Rv genome, seven genes coding for cutinase-like proteins have been identified with strong immunogenic properties suggesting a potential role as vaccine candidates. Two of these enzymes which are secreted and highly homologous, possess distinct substrates specificities. Cfp21 is a lipase and Cut4 is a phospholipase A2, which has cytotoxic effects on macrophages. Structural overlay of their three-dimensional models allowed us to identify three areas involved in the substrate binding process and to shed light on this substrate specificity. By site-directed mutagenesis, residues present in these Cfp21 areas were replaced by residues occurring in Cut4 at the same location. Three mutants acquired phospholipase A1 and A2 activities and the lipase activities of two mutants were 3 and 15 fold greater than the Cfp21 wild type enzyme. In addition, contrary to mutants with enhanced lipase activity, mutants that acquired phospholipase B activities induced macrophage lysis as efficiently as Cut4 which emphasizes the relationship between apparent phospholipase A2 activity and cytotoxicity. Modification of areas involved in substrate specificity, generate recombinant enzymes with higher activity, which may be more immunogenic than the wild type enzymes and could therefore constitute promising candidates for antituberculous vaccine production. PMID:23843969

Identification of residues involved in substrate specificity and cytotoxicity of two closely related cutinases from Mycobacterium tuberculosis.

PubMed

Dedieu, Luc; Serveau-Avesque, Carole; Canaan, Stéphane

2013-01-01

The enzymes belonging to the cutinase family are serine enzymes active on a large panel of substrates such as cutin, triacylglycerols, and phospholipids. In the M. tuberculosis H37Rv genome, seven genes coding for cutinase-like proteins have been identified with strong immunogenic properties suggesting a potential role as vaccine candidates. Two of these enzymes which are secreted and highly homologous, possess distinct substrates specificities. Cfp21 is a lipase and Cut4 is a phospholipase A2, which has cytotoxic effects on macrophages. Structural overlay of their three-dimensional models allowed us to identify three areas involved in the substrate binding process and to shed light on this substrate specificity. By site-directed mutagenesis, residues present in these Cfp21 areas were replaced by residues occurring in Cut4 at the same location. Three mutants acquired phospholipase A1 and A2 activities and the lipase activities of two mutants were 3 and 15 fold greater than the Cfp21 wild type enzyme. In addition, contrary to mutants with enhanced lipase activity, mutants that acquired phospholipase B activities induced macrophage lysis as efficiently as Cut4 which emphasizes the relationship between apparent phospholipase A2 activity and cytotoxicity. Modification of areas involved in substrate specificity, generate recombinant enzymes with higher activity, which may be more immunogenic than the wild type enzymes and could therefore constitute promising candidates for antituberculous vaccine production.

S-nitrosoglutathione promotes cell wall remodelling, alters the transcriptional profile and induces root hair formation in the hairless root hair defective 6 (rhd6) mutant of Arabidopsis thaliana.

PubMed

Moro, Camila Fernandes; Gaspar, Marilia; da Silva, Felipe Rodrigues; Pattathil, Sivakumar; Hahn, Michael G; Salgado, Ione; Braga, Marcia Regina

2017-03-01

Nitric oxide (NO) exerts pleiotropic effects on plant development; however, its involvement in cell wall modification during root hair formation (RHF) has not yet been addressed. Here, mutants of Arabidopsis thaliana with altered root hair phenotypes were used to assess the involvement of S-nitrosoglutathione (GSNO), the primary NO source, in cell wall dynamics and gene expression in roots induced to form hairs. GSNO and auxin restored the root hair phenotype of the hairless root hair defective 6 (rhd6) mutant. A positive correlation was observed between increased NO production and RHF induced by auxin in rhd6 and transparent testa glabra (ttg) mutants. Deposition of an epitope within rhamnogalacturonan-I recognized by the CCRC-M2 antibody was delayed in root hair cells (trichoblasts) compared with nonhair cells (atrichoblasts). GSNO, but not auxin, restored the wild-type root glycome and transcriptome profiles in rhd6, modulating the expression of a large number of genes related to cell wall composition and metabolism, as well as those encoding ribosomal proteins, DNA and histone-modifying enzymes and proteins involved in post-translational modification. Our results demonstrate that NO plays a key role in cell wall remodelling in trichoblasts and suggest that it also participates in chromatin modification in root cells of A. thaliana. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

Pseudo-constitutivity of nitrate-responsive genes in nitrate reductase mutants

PubMed Central

Schinko, Thorsten; Gallmetzer, Andreas; Amillis, Sotiris; Strauss, Joseph

2013-01-01

In fungi, transcriptional activation of genes involved in NO3- assimilation requires the presence of an inducer (nitrate or nitrite) and low intracellular concentrations of the pathway products ammonium or glutamine. In Aspergillus nidulans, the two transcription factors NirA and AreA act synergistically to mediate nitrate/nitrite induction and nitrogen metabolite derepression, respectively. In all studied fungi and in plants, mutants lacking nitrate reductase (NR) activity express nitrate-metabolizing enzymes constitutively without the addition of inducer molecules. Based on their work in A. nidulans, Cove and Pateman proposed an “autoregulation control” model for the synthesis of nitrate metabolizing enzymes in which the functional nitrate reductase molecule would act as co-repressor in the absence and as co-inducer in the presence of nitrate. However, NR mutants could simply show “pseudo-constitutivity” due to induction by nitrate which accumulates over time in NR-deficient strains. Here we examined this possibility using strains which lack flavohemoglobins (fhbs), and are thus unable to generate nitrate internally, in combination with nitrate transporter mutations (nrtA, nrtB) and a GFP-labeled NirA protein. Using different combinations of genotypes we demonstrate that nitrate transporters are functional also in NR null mutants and show that the constitutive phenotype of NR mutants is not due to nitrate accumulation from intracellular sources but depends on the activity of nitrate transporters. However, these transporters are not required for nitrate signaling because addition of external nitrate (10 mM) leads to standard induction of nitrate assimilatory genes in the nitrate transporter double mutants. We finally show that NR does not regulate NirA localization and activity, and thus the autoregulation model, in which NR would act as a co-repressor of NirA in the absence of nitrate, is unlikely to be correct. Results from this study instead suggest that transporter-mediated NO3- accumulation in NR deficient mutants, originating from traces of nitrate in the media, is responsible for the constitutive expression of NirA-regulated genes, and the associated phenotype is thus termed “pseudo-constitutive”. PMID:23454548

Characterization of Escherichia coli Type 1 Pilus Mutants with Altered Binding Specificities

PubMed Central

Harris, Sandra L.; Spears, Patricia A.; Havell, Edward A.; Hamrick, Terri S.; Horton, John R.; Orndorff, Paul E.

2001-01-01

PCR mutagenesis and a unique enrichment scheme were used to obtain two mutants, each with a single lesion in fimH, the chromosomal gene that encodes the adhesin protein (FimH) of Escherichia coli type 1 pili. These mutants were noteworthy in part because both were altered in the normal range of cell types bound by FimH. One mutation altered an amino acid at a site previously shown to be involved in temperature-dependent binding, and the other altered an amino acid lining the predicted FimH binding pocket. PMID:11395476

Pyridoxine and its relation to lipids. Studies with pyridoxineless mutants of Aspergillus nidulans.

PubMed

Mohana, K; Shanmugasundaram, E R

1978-01-01

The effect of pyridoxine deficiency on fat metabolism was studied using mutant strains of Aspergillus nidulans requiring pyridoxine for growth. Under pyridoxine deficiency the mutants exhibited increased levels of total lipid, sterols, phospholipids, and triacylglycerols. Total fatty acids were found to decrease with pyridoxine deficiency. An increase in saturated fatty acids and decrease in unsaturated fatty acids were seen with deficiency. Pyridoxine deficiency also increased lower carbon chain fatty acids. A possible involvement of pyridoxine in the elongation of fatty acid chain and in the desaturation of fatty acids in Aspergillus nidulans is suggested.

New genes of Xanthomonas citri subsp. citri involved in pathogenesis and adaptation revealed by a transposon-based mutant library

PubMed Central

2009-01-01

Background Citrus canker is a disease caused by the phytopathogens Xanthomonas citri subsp. citri, Xanthomonas fuscans subsp. aurantifolli and Xanthomonas alfalfae subsp. citrumelonis. The first of the three species, which causes citrus bacterial canker type A, is the most widely spread and severe, attacking all citrus species. In Brazil, this species is the most important, being found in practically all areas where citrus canker has been detected. Like most phytobacterioses, there is no efficient way to control citrus canker. Considering the importance of the disease worldwide, investigation is needed to accurately detect which genes are related to the pathogen-host adaptation process and which are associated with pathogenesis. Results Through transposon insertion mutagenesis, 10,000 mutants of Xanthomonas citri subsp. citri strain 306 (Xcc) were obtained, and 3,300 were inoculated in Rangpur lime (Citrus limonia) leaves. Their ability to cause citrus canker was analyzed every 3 days until 21 days after inoculation; a set of 44 mutants showed altered virulence, with 8 presenting a complete loss of causing citrus canker symptoms. Sequencing of the insertion site in all 44 mutants revealed that 35 different ORFs were hit, since some ORFs were hit in more than one mutant, with mutants for the same ORF presenting the same phenotype. An analysis of these ORFs showed that some encoded genes were previously known as related to pathogenicity in phytobacteria and, more interestingly, revealed new genes never implicated with Xanthomonas pathogenicity before, including hypothetical ORFs. Among the 8 mutants with no canker symptoms are the hrpB4 and hrpX genes, two genes that belong to type III secretion system (TTSS), two hypothetical ORFS and, surprisingly, the htrA gene, a gene reported as involved with the virulence process in animal-pathogenic bacteria but not described as involved in phytobacteria virulence. Nucleic acid hybridization using labeled cDNA probes showed that some of the mutated genes are differentially expressed when the bacterium is grown in citrus leaves. Finally, comparative genomic analysis revealed that 5 mutated ORFs are in new putative pathogenicity islands. Conclusion The identification of these new genes related with Xcc infection and virulence is a great step towards the understanding of plant-pathogen interactions and could allow the development of strategies to control citrus canker. PMID:19149882

Genetic control of enhanced mutability of mitochondrial DNA and gamma-ray sensitivity in Saccharomyces cerevisiae.

PubMed Central

Foury, F; Goffeau, A

1979-01-01

Five nuclear mutants enhancing the spontaneous mutation rate of mtDNA have been isolated in Saccharomyces cerevisiae. These mutators fall into five complementation groups and are located at five genetic loci different from rad50 to rad57 loci. Three mutants (gam1, gam2, and gam4), insensitive or weakly sensitive to gamma-rays, exhibit increased frequency of spontaneous production of mutants with large deletions of the mtDNA (p-) and of all tested mitochondrial drug-resistant mutants. Two other mutants (gam3 and gam5), highly sensitive to gamma-rays, increase only the mutation rate of particular alleles of the mtDNA. The mutant gam5 enhances only the production of p- and erythromycin-resistant clones. The mutant gam3 exhibits an enhanced rate of oligomycin-resistant clones as well as a collateral increase of nuclear mutability. The existence of gam3 and gam5 mutants indicates that at least two common steps control both nuclear DNA repair and the mutability of particular alleles of the mtDNA. However, the general spontaneous mutability of the mtDNA includes at least three steps not involved in the repair of nuclear DNA, as revealed by the gam1, gam2, and gam4 mutations. PMID:392521

RNA-Seq analysis of global transcriptomic changes suggests a roles for the MAPK pathway and carbon metabolism in cell wall maintenance in a Saccharomyces cerevisiae FKS1 mutant.

PubMed

Huang, Cong; Zhao, Fengguang; Lin, Ying; Zheng, Suiping; Liang, Shuli; Han, Shuangyan

2018-06-07

FKS1 encodes a β-1,3-glucan synthase, which is a key player in cell wall assembly in Saccharomyces cerevisiae. Here we analyzed the global transcriptomic changes in the FKS1 mutant to establish a correlation between the changes in the cell wall of the FKS1 mutant and the molecular mechanism of cell wall maintenance. These transcriptomic profiles showed that there are 1151 differentially expressed genes (DEGs) in the FKS1 mutant. Through KEGG pathway analysis of the DEGs, the MAPK pathway and seven pathways involved in carbon metabolism were significantly enriched. We found that the MAPK pathway is activated for FKS1 mutant survival and the synthesis of cell wall components are reinforced in the FKS1 mutant. Our results confirm that the FKS1 mutant has a β-1,3-glucan defect that affects the cell wall and partly elucidate the molecular mechanism responsible for cell wall synthesis. Our greater understanding of these mechanisms helps to explain how the FKS1 mutant survives, has useful implications for the study of similar pathways in other fungi, and increases the theoretical foundation for the regulation of the cell wall in S. cerevisiae. Copyright © 2018 Elsevier Inc. All rights reserved.

Tryptophan biosynthetic enzymes of Staphylococcus aureus.

PubMed

Proctor, A R; Kloos, W E

1973-04-01

Tryptophan biosynthetic enzymes were assayed in various tryptophan mutants of Staphylococcus aureus strain 655 and the wild-type parent. All mutants, except trpB mutants, lacked only the activity corresponding to the particular biosynthetic block, as suggested previously by analysis of accumulated intermediates and auxonography. Tryptophan synthetase A was not detected in extracts of either trpA or trpB mutants but appeared normal in other mutants. Mutants in certain other classes exhibited partial loss of another particular tryptophan enzyme activity. Tryptophan synthetase B activity was not detected in cell extract preparations but was detected in whole cells. The original map order proposed for the S. aureus tryptophan gene cluster was clarified by the definition of trpD (phosphoribosyl transferase(-)) and trpF (phosphoribosyl anthranilate isomerase(-)) mutants. These mutants were previously unresolved and designated as trp(DF) mutants (anthranilate accumulators). Phosphoribosyl anthranilate isomerase and indole-3-glycerol phosphate synthetase enzymes were separable by molecular sieve chromatography, suggesting that these functions are coded by separate loci. Molecular sieve chromatography failed to reveal aggregates involving anthranilate synthetase, phosphoribosyl transferase, phosphoribosyl anthranilate isomerase, and indole-3-glycerol phosphate synthetase, and this procedure provided an estimate of the molecular weights of these enzymes. Tryptophan was shown to repress synthesis of all six tryptophan biosynthetic enzymes, and derepression of all six activities was incident upon tryptophan starvation. Tryptophan inhibited the activity of anthranilate synthetase, the first enzyme of the pathway.

Intact interval timing in circadian CLOCK mutants.

PubMed

Cordes, Sara; Gallistel, C R

2008-08-28

While progress has been made in determining the molecular basis for the circadian clock, the mechanism by which mammalian brains time intervals measured in seconds to minutes remains a mystery. An obvious question is whether the interval-timing mechanism shares molecular machinery with the circadian timing mechanism. In the current study, we trained circadian CLOCK +/- and -/- mutant male mice in a peak-interval procedure with 10 and 20-s criteria. The mutant mice were more active than their wild-type littermates, but there were no reliable deficits in the accuracy or precision of their timing as compared with wild-type littermates. This suggests that expression of the CLOCK protein is not necessary for normal interval timing.

The RING finger E3 ligase STRF1 is involved in membrane trafficking and modulates salt-stress response in Arabidopsis thaliana.

PubMed

Tian, Miaomiao; Lou, Lijuan; Liu, Lijing; Yu, Feifei; Zhao, Qingzhen; Zhang, Huawei; Wu, Yaorong; Tang, Sanyuan; Xia, Ran; Zhu, Baoge; Serino, Giovanna; Xie, Qi

2015-04-01

Salt stress is a detrimental factor for plant growth and development. The response to salt stress has been shown to involve components in the intracellular trafficking system, as well as components of the ubiquitin-proteasome system (UPS). In this article, we have identified in Arabidopsis thaliana a little reported ubiquitin ligase involved in salt-stress response, which we named STRF1 (Salt Tolerance RING Finger 1). STRF1 is a member of RING-H2 finger proteins and we demonstrate that it has ubiquitin ligase activity in vitro. We also show that STRF1 localizes mainly at the plasma membrane and at the intracellular endosomes. strf1-1 loss-of-function mutant seedlings exhibit accelerated endocytosis in roots, and have altered expression of several genes involved in the membrane trafficking system. Moreover, protein trafficking inhibitor, brefeldin A (BFA), treatment has increased BFA bodies in strf1-1 mutant. This mutant also showed increased tolerance to salt, ionic and osmotic stresses, reduced accumulation of reactive oxygen species during salt stress, and increased expression of AtRbohD, which encodes a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase involved in H2 O2 production. We conclude that STRF1 is a membrane trafficking-related ubiquitin ligase, which helps the plant to respond to salt stress by monitoring intracellular membrane trafficking and reactive oxygen species (ROS) production. © 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.

Cell wall composition contributes to the control of transpiration efficiency in Arabidopsis thaliana.

PubMed

Liang, Yun-Kuan; Xie, Xiaodong; Lindsay, Shona E; Wang, Yi Bing; Masle, Josette; Williamson, Lisa; Leyser, Ottoline; Hetherington, Alistair M

2010-11-01

To identify loci in Arabidopsis involved in the control of transpirational water loss and transpiration efficiency (TE) we carried out an infrared thermal imaging-based screen. We report the identification of a new allele of the Arabidopsis CesA7 cellulose synthase locus designated AtCesA7(irx3-5) involved in the control of TE. Leaves of the AtCesA7(irx3-5) mutant are warmer than the wild type (WT). This is due to reduced stomatal pore widths brought about by guard cells that are significantly smaller than the WT. The xylem of the AtCesA7(irx3-5) mutant is also partially collapsed, and we suggest that the small guard cells in the mutant result from decreased water supply to the developing leaf. We used carbon isotope discrimination to show that TE is increased in AtCesA7(irx3-5) when compared with the WT. Our work identifies a new class of genes that affects TE and raises the possibility that other genes involved in cell wall biosynthesis will have an impact on water use efficiency. © 2010 The Authors. The Plant Journal © 2010 Blackwell Publishing Ltd.

Mutant Copper-Zinc Superoxide Dismutase (SOD1) Induces Protein Secretion Pathway Alterations and Exosome Release in Astrocytes

PubMed Central

Basso, Manuela; Pozzi, Silvia; Tortarolo, Massimo; Fiordaliso, Fabio; Bisighini, Cinzia; Pasetto, Laura; Spaltro, Gabriella; Lidonnici, Dario; Gensano, Francesco; Battaglia, Elisa; Bendotti, Caterina; Bonetto, Valentina

2013-01-01

Amyotrophic lateral sclerosis is the most common motor neuron disease and is still incurable. The mechanisms leading to the selective motor neuron vulnerability are still not known. The interplay between motor neurons and astrocytes is crucial in the outcome of the disease. We show that mutant copper-zinc superoxide dismutase (SOD1) overexpression in primary astrocyte cultures is associated with decreased levels of proteins involved in secretory pathways. This is linked to a general reduction of total secreted proteins, except for specific enrichment in a number of proteins in the media, such as mutant SOD1 and valosin-containing protein (VCP)/p97. Because there was also an increase in exosome release, we can deduce that astrocytes expressing mutant SOD1 activate unconventional secretory pathways, possibly as a protective mechanism. This may help limit the formation of intracellular aggregates and overcome mutant SOD1 toxicity. We also found that astrocyte-derived exosomes efficiently transfer mutant SOD1 to spinal neurons and induce selective motor neuron death. We conclude that the expression of mutant SOD1 has a substantial impact on astrocyte protein secretion pathways, contributing to motor neuron pathology and disease spread. PMID:23592792

Reduced gravitropic sensitivity in roots of a starch-deficient mutant of Nicotiana sylvestris

NASA Technical Reports Server (NTRS)

Kiss, J. Z.; Sack, F. D.

1989-01-01

Gravitropism was studied in seedlings of Nicotiana sylvestris Speg. et Comes wild-type (WT) and mutant NS 458 which has a defective plastid phosphoglucomutase (EC 2.7.5.1.). Starch was greatly reduced in NS 458 compared to the WT, but small amounts of starch were detected in rootcap columella cells in NS 458 by light and electron microscopy. The roots of WT are more sensitive to gravity than mutant NS 458 roots since: (1) in mutant roots, curvature was reduced and delayed in the time course of curvature; (2) curvature of mutant roots was 24-56% that of WT roots over the range of induction periods tested; (3) in intermittent-stimulation experiments, curvature of mutant roots was 37% or less than that of WT roots in all treatments tested. The perception time, determined by intermittent-stimulation experiments, was < or = 5 s for WT roots and 30-60 s for mutant roots. The growth rates for WT and NS 458 roots were essentially equal. These results and our previous results with WT and starchless mutant Arabidopsis roots (Kiss et al. 1989, Planta 177, 198-206) support the conclusions that a full complement of starch is necessary for full gravitropic sensitivity and that amyloplasts function in gravity perception. Since a presumed relatively small increase in plastid buoyant mass (N. sylvestris mutant versus Arabidopsis mutant) significantly improves the orientation of the N. sylvestris mutant roots, we suggest that plastids are the likeliest candidates to be triggering gravity perception in roots of both mutants.

Key Enzymes of the Semiphosphorylative Entner-Doudoroff Pathway in the Haloarchaeon Haloferax volcanii: Characterization of Glucose Dehydrogenase, Gluconate Dehydratase, and 2-Keto-3-Deoxy-6-Phosphogluconate Aldolase.

PubMed

Sutter, Jan-Moritz; Tästensen, Julia-Beate; Johnsen, Ulrike; Soppa, Jörg; Schönheit, Peter

2016-08-15

The halophilic archaeon Haloferax volcanii has been proposed to degrade glucose via the semiphosphorylative Entner-Doudoroff (spED) pathway. So far, the key enzymes of this pathway, glucose dehydrogenase (GDH), gluconate dehydratase (GAD), and 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase (KDPGA), have not been characterized, and their functional involvement in glucose degradation has not been demonstrated. Here we report that the genes HVO_1083 and HVO_0950 encode GDH and KDPGA, respectively. The recombinant enzymes show high specificity for glucose and KDPG and did not convert the corresponding C4 epimers galactose and 2-keto-3-deoxy-6-phosphogalactonate at significant rates. Growth studies of knockout mutants indicate the functional involvement of both GDH and KDPGA in glucose degradation. GAD was purified from H. volcanii, and the encoding gene, gad, was identified as HVO_1488. GAD catalyzed the specific dehydration of gluconate and did not utilize galactonate at significant rates. A knockout mutant of GAD lost the ability to grow on glucose, indicating the essential involvement of GAD in glucose degradation. However, following a prolonged incubation period, growth of the Δgad mutant on glucose was recovered. Evidence is presented that under these conditions, GAD was functionally replaced by xylonate dehydratase (XAD), which uses both xylonate and gluconate as substrates. Together, the characterization of key enzymes and analyses of the respective knockout mutants present conclusive evidence for the in vivo operation of the spED pathway for glucose degradation in H. volcanii The work presented here describes the identification and characterization of the key enzymes glucose dehydrogenase, gluconate dehydratase, and 2-keto-3-deoxy-6-phosphogluconate aldolase and their encoding genes of the proposed semiphosphorylative Entner-Doudoroff pathway in the haloarchaeon Haloferax volcanii The functional involvement of the three enzymes was proven by analyses of the corresponding knockout mutants. These results provide evidence for the in vivo operation of the semiphosphorylative Entner-Doudoroff pathway in haloarchaea and thus expand our understanding of the unusual sugar degradation pathways in the domain Archaea. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Identification of Bicarbonate as a Trigger and Genes Involved with Extracellular DNA Export in Mycobacterial Biofilms.

PubMed

Rose, Sasha J; Bermudez, Luiz E

2016-12-06

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. Many bacteria contain extracellular DNA (eDNA) in their biofilm matrix, as it has various biological and physical functions. We recently reported that nontuberculous mycobacteria (NTM) can contain significant quantities of eDNA in their biofilms. In some bacteria, eDNA is derived from dead cells, but that does not appear to be the case for all eDNA-containing organisms, including NTM. In this study, we found that eDNA export in NTM is conditionally dependent on the molecules to which the bacteria are exposed and that bicarbonate positively influences eDNA export. We also identified genes and proteins important for eDNA export, which begins to piece together a description of a mechanism for eDNA. Better understanding of eDNA export can give new targets for the development of antivirulence drugs, which are attractive because resistance to classical antibiotics is currently a significant problem. Copyright © 2016 Rose and Bermudez.

Mutant fatty acid desaturase and methods for directed mutagenesis

DOEpatents

Shanklin, John [Shoreham, NY; Whittle, Edward J [Greenport, NY

2008-01-29

The present invention relates to methods for producing fatty acid desaturase mutants having a substantially increased activity towards substrates with fewer than 18 carbon atom chains relative to an unmutagenized precursor desaturase having an 18 carbon chain length specificity, the sequences encoding the desaturases and to the desaturases that are produced by the methods. The present invention further relates to a method for altering a function of a protein, including a fatty acid desaturase, through directed mutagenesis involving identifying candidate amino acid residues, producing a library of mutants of the protein by simultaneously randomizing all amino acid candidates, and selecting for mutants which exhibit the desired alteration of function. Candidate amino acids are identified by a combination of methods. Enzymatic, binding, structural and other functions of proteins can be altered by the method.

Identification of new developmentally regulated genes involved in Streptomyces coelicolor sporulation.

PubMed

Salerno, Paola; Persson, Jessica; Bucca, Giselda; Laing, Emma; Ausmees, Nora; Smith, Colin P; Flärdh, Klas

2013-12-05

The sporulation of aerial hyphae of Streptomyces coelicolor is a complex developmental process. Only a limited number of the genes involved in this intriguing morphological differentiation programme are known, including some key regulatory genes. The aim of this study was to expand our knowledge of the gene repertoire involved in S. coelicolor sporulation. We report a DNA microarray-based investigation of developmentally controlled gene expression in S. coelicolor. By comparing global transcription patterns of the wild-type parent and two mutants lacking key regulators of aerial hyphal sporulation, we found a total of 114 genes that had significantly different expression in at least one of the two mutants compared to the wild-type during sporulation. A whiA mutant showed the largest effects on gene expression, while only a few genes were specifically affected by whiH mutation. Seven new sporulation loci were investigated in more detail with respect to expression patterns and mutant phenotypes. These included SCO7449-7451 that affect spore pigment biogenesis; SCO1773-1774 that encode an L-alanine dehydrogenase and a regulator-like protein and are required for maturation of spores; SCO3857 that encodes a protein highly similar to a nosiheptide resistance regulator and affects spore maturation; and four additional loci (SCO4421, SCO4157, SCO0934, SCO1195) that show developmental regulation but no overt mutant phenotype. Furthermore, we describe a new promoter-probe vector that takes advantage of the red fluorescent protein mCherry as a reporter of cell type-specific promoter activity. Aerial hyphal sporulation in S. coelicolor is a technically challenging process for global transcriptomic investigations since it occurs only as a small fraction of the colony biomass and is not highly synchronized. Here we show that by comparing a wild-type to mutants lacking regulators that are specifically affecting processes in aerial hypha, it is possible to identify previously unknown genes with important roles in sporulation. The transcriptomic data reported here should also serve as a basis for identification of further developmentally important genes in future functional studies.

Molecular characterization of three 3-ketosteroid-Δ(1)-dehydrogenase isoenzymes of Rhodococcus ruber strain Chol-4.

PubMed

Fernández de las Heras, Laura; van der Geize, Robert; Drzyzga, Oliver; Perera, Julián; María Navarro Llorens, Juana

2012-11-01

Rhodococcus ruber strain Chol-4 isolated from a sewage sludge sample is able to grow on minimal medium supplemented with steroids, showing a broad catabolic capacity. This paper reports the characterization of three different 3-ketosteroid-Δ(1)-dehydrogenases (KstDs) in the genome of R. ruber strain Chol-4. The genome of this strain does not contain any homologues of a 3-keto-5α-steroid-Δ(4)-dehydrogenase (Kst4d or TesI) that appears in the genomes of Rhodococcus erythropolis SQ1 or Comamonas testosteroni. Growth experiments with kstD2 mutants, either a kstD2 single mutant, kstD2 double mutants in combination with kstD1 or kstD3, or the triple kstD1,2,3 mutant, proved that KstD2 is involved in the transformation of 4-androstene-3,17-dione (AD) to 1,4-androstadiene-3,17-dione (ADD) and in the conversion of 9α-hydroxy-4-androstene-3,17-dione (9OHAD) to 9α-hydroxy-1,4-androstadiene-3,17-dione (9OHADD). kstD2,3 and kstD1,2,3 R. ruber mutants (both lacking KstD2 and KstD3) did not grow in minimal medium with cholesterol as the only carbon source, thus demonstrating the involvement of KstD2 and KstD3 in cholesterol degradation. In contrast, mutation of kstD1 does not alter the bacterial growth on the steroids tested in this study and therefore, the role of this protein still remains unclear. The absence of a functional KstD2 in R. ruber mutants provoked in all cases an accumulation of 9OHAD, as a branch product probably formed by the action of a 3-ketosteroid-9α-hydroxylase (KshAB) on the AD molecule. Therefore, KstD2 is a key enzyme in the AD catabolism pathway of R. ruber strain Chol-4 while KstD3 is involved in cholesterol catabolism. Copyright © 2012 Elsevier Ltd. All rights reserved.

Rapid Conversion of Mutant IDH1 from Driver to Passenger in a Model of Human Gliomagenesis

PubMed Central

Johannessen, Tor-Christian Aase; Mukherjee, Joydeep; Viswanath, Pavithra; Ohba, Shigeo; Ronen, Sabrina M.; Bjerkvig, Rolf; Pieper, Russell O.

2016-01-01

Missense mutations in the active site of isocitrate dehydrogenase 1 (IDH1) biologically and diagnostically distinguish low-grade gliomas and secondary glioblastomas from primary glioblastomas. IDH1 mutations lead to the formation of the oncometabolite 2-hydroxyglutarate (2-HG) from the reduction of α-ketoglutarate (α-KG), which in turn facilitates tumorigenesis by modifying DNA and histone methylation as well blocking differentiation processes. While mutant IDH1 expression is thought to drive the gliomagenesis process, the extent to which it remains a viable therapeutic target remains unknown. To address this question we exposed immortalized (p53/pRb-deficient), untransformed human astrocytes to the mutant IDH1 inhibitor AGI-5198 prior to, concomitant with, or at intervals after, introduction of transforming mutant IDH1, then measured effects on 2-HG levels, histone methylation (H3K4me3, H3K9me2, H3K9me3 or H3K27me3) and growth in soft-agar. Addition of AGI-5198 prior to, or concomitant with, introduction of mutant IDH1 blocked all mutant IDH1-driven changes including cellular transformation. Addition at time intervals as short as 4 days following introduction of mutant IDH1 also suppressed 2-HG levels, but had minimal effects on histone methylation, and lost the ability to suppress clonogenicity in a time-dependent manner. Furthermore, in two different models of mutant IDH1-driven gliomagenesis, AGI-5198 exposures that abolished production of 2-HG also failed to decrease histone methylation, adherent cell growth, or anchorage-independent growth in soft-agar over a prolonged period. These studies show although mutant IDH1 expression drives gliomagenesis, mutant IDH1 itself rapidly converts from driver to passenger. Implications Agents that target mutant IDH may be effective for a narrow time and may require further optimization or additional therapeutics in glioma. PMID:27430238

EMK protein kinase-null mice: dwarfism and hypofertility associated with alterations in the somatotrope and prolactin pathways.

PubMed

Bessone, S; Vidal, F; Le Bouc, Y; Epelbaum, J; Bluet-Pajot, M T; Darmon, M

1999-10-01

Gene trapping was used in embryonic stem (ES) cells in an attempt to inactivate genes involved in development. The Emk (ELKL motif kinase) gene has been disrupted and a mutant mouse line derived. Previous work had shown that EMK kinases, called MARK in the rat, exert a major control on microtubule stability by phosphorylating microtubule-associated proteins and that genes homologous to Emk in yeast or Caenorhabditis elegans are essential for cell and embryonic polarity. Although we found the Emk gene to be active in the preimplantation mouse embryo and then to show a widespread expression, Emk-null mice had no embryonic defect and were viable. They show an overall proportionate dwarfism and a peculiar hypofertility: homozygotes are not fertile when intercrossed, but are fertile in other types of crosses. Insulin-like growth factor I (IGF I) and IGF-binding protein 3 (IGFBP3) were reduced in the plasma of homozygotes of both sexes. A direct implication of the EMK kinase in IGF I plasmatic production is unlikely because the Emk gene does not seem to be expressed in hepatocytes. Nevertheless, GH assayed at arbitrary times in plasma did not show differences between genotypes and GH concentrations in pituitary extracts were not found to be altered in homozygotes. Our results, though, do not exclude the possibility that in the mutants the overall quantity of GH secreted daily is reduced. Our observation of a smaller size of the pituitaries of the mutants is in favor of this hypothesis. The prolactin concentration in the pituitaries was much lowered in homozygous females, but it was normal in males. The possible involvement of EMK protein kinase in hormone secretion in the pituitary and/or the hypothalamus, via the microtubule network, is discussed. Copyright 1999 Academic Press.

Positional cloning of a Bombyx pink-eyed white egg locus reveals the major role of cardinal in ommochrome synthesis

PubMed Central

Osanai-Futahashi, M; Tatematsu, K-i; Futahashi, R; Narukawa, J; Takasu, Y; Kayukawa, T; Shinoda, T; Ishige, T; Yajima, S; Tamura, T; Yamamoto, K; Sezutsu, H

2016-01-01

Ommochromes are major insect pigments involved in coloration of compound eyes, eggs, epidermis and wings. In the silkworm Bombyx mori, adult compound eyes and eggs contain a mixture of the ommochrome pigments such as ommin and xanthommatin. Here, we identified the gene involved in ommochrome biosynthesis by positional cloning of B. mori egg and eye color mutant pink-eyed white egg (pe). The recessive homozygote of pe has bright red eyes and white or pale pink eggs instead of a normal dark coloration due to the decrease of dark ommochrome pigments. By genetic linkage analysis, we narrowed down the pe-linked region to ~258 kb, containing 17 predicted genes. RNA sequencing analyses showed that the expression of one candidate gene, the ortholog of Drosophila haem peroxidase cardinal, coincided with egg pigmentation timing, similar to other ommochrome-related genes such as Bm-scarlet and Bm-re. In two pe strains, a common missense mutation was found within a conserved motif of B. mori cardinal homolog (Bm-cardinal). RNA interference-mediated knockdown and transcription activator-like effector nuclease (TALEN)-mediated knockout of the Bm-cardinal gene produced the same phenotype as pe in terms of egg, adult eye and larval epidermis coloration. A complementation test of the pe mutant with the TALEN-mediated Bm-cardinal-deficient strain showed that the mutant phenotype could not be rescued, indicating that Bm-cardinal is responsible for pe. Moreover, knockdown of the cardinal homolog in Tribolium castaneum also induced red compound eyes. Our results indicate that cardinal plays a major role in ommochrome synthesis of holometabolous insects. PMID:26328757

Positional cloning of a Bombyx pink-eyed white egg locus reveals the major role of cardinal in ommochrome synthesis.

PubMed

Osanai-Futahashi, M; Tatematsu, K-I; Futahashi, R; Narukawa, J; Takasu, Y; Kayukawa, T; Shinoda, T; Ishige, T; Yajima, S; Tamura, T; Yamamoto, K; Sezutsu, H

2016-02-01

Ommochromes are major insect pigments involved in coloration of compound eyes, eggs, epidermis and wings. In the silkworm Bombyx mori, adult compound eyes and eggs contain a mixture of the ommochrome pigments such as ommin and xanthommatin. Here, we identified the gene involved in ommochrome biosynthesis by positional cloning of B. mori egg and eye color mutant pink-eyed white egg (pe). The recessive homozygote of pe has bright red eyes and white or pale pink eggs instead of a normal dark coloration due to the decrease of dark ommochrome pigments. By genetic linkage analysis, we narrowed down the pe-linked region to ~258 kb, containing 17 predicted genes. RNA sequencing analyses showed that the expression of one candidate gene, the ortholog of Drosophila haem peroxidase cardinal, coincided with egg pigmentation timing, similar to other ommochrome-related genes such as Bm-scarlet and Bm-re. In two pe strains, a common missense mutation was found within a conserved motif of B. mori cardinal homolog (Bm-cardinal). RNA interference-mediated knockdown and transcription activator-like effector nuclease (TALEN)-mediated knockout of the Bm-cardinal gene produced the same phenotype as pe in terms of egg, adult eye and larval epidermis coloration. A complementation test of the pe mutant with the TALEN-mediated Bm-cardinal-deficient strain showed that the mutant phenotype could not be rescued, indicating that Bm-cardinal is responsible for pe. Moreover, knockdown of the cardinal homolog in Tribolium castaneum also induced red compound eyes. Our results indicate that cardinal plays a major role in ommochrome synthesis of holometabolous insects.

Characterization of multilocus lesions in human cells exposed to X radiation and radon

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

Chaudhry, M.A.; Jiang, Q.; Ricanati, M.

Human TK6 lymphoblasts were exposed to X radiation or radon, and thymidine kinase negative (TK{sup -/-}) mutants were selected, isolated and harvested for analysis of structural changes in the TK gene. A large majority (82%) of the radon-induced mutants, 74% of the X-radiation-induced mutants and 45% of the spontaneous mutants lost the entire active TK allele. To analyze these mutants further we measured the loss of heterozygosity at several loci neighboring the TK locus on chromosome 17q. A greater proportion (61%) of the radon-induced mutants than X-radiation-induced or spontaneous mutants harbored the smaller lesions involving the TK allele alone ormore » extending from the TK locus to one or both of the closest neighboring sequences tested. Further, 21% of the X-radiation-induced mutants but only 5% of the radon-induced mutants lost heterozygosity at the col1A1 locus, 31 Mb from the TK gene. These results are in agreement with a recent analysis of radon- and X-radiation-induced lesions inactivating the HPRT gene of TK6 cells, in which we reported that a lower percentage of radon- than X-radiation-induced mutants showed lesions extending to markers 800 kb or more from the HPRT gene on the X chromosome. In the present study, we observed that the percentage of slowly growing and very slowly growing TK{sup -/-} mutants was greater after treatment with radon than after treatment with X radiation, regardless of the type of lesion present. It is possible, therefore, that the radon-induced lesions are complex and/or less easily repaired, leading to slow growth in a large proportion of the surviving mutant cells. 36 refs., 6 figs., 2 tabs.« less

Anaerobic Respiration Using a Complete Oxidative TCA Cycle Drives Multicellular Swarming in Proteus mirabilis

PubMed Central

Alteri, Christopher J.; Himpsl, Stephanie D.; Engstrom, Michael D.; Mobley, Harry L. T.

2012-01-01

ABSTRACT Proteus mirabilis rapidly migrates across surfaces using a periodic developmental process of differentiation alternating between short swimmer cells and elongated hyperflagellated swarmer cells. To undergo this vigorous flagellum-mediated motility, bacteria must generate a substantial proton gradient across their cytoplasmic membranes by using available energy pathways. We sought to identify the link between energy pathways and swarming differentiation by examining the behavior of defined central metabolism mutants. Mutations in the tricarboxylic acid (TCA) cycle (fumC and sdhB mutants) caused altered patterns of swarming periodicity, suggesting an aerobic pathway. Surprisingly, the wild-type strain swarmed on agar containing sodium azide, which poisons aerobic respiration; the fumC TCA cycle mutant, however, was unable to swarm on azide. To identify other contributing energy pathways, we screened transposon mutants for loss of swarming on sodium azide and found insertions in the following genes that involved fumarate metabolism or respiration: hybB, encoding hydrogenase; fumC, encoding fumarase; argH, encoding argininosuccinate lyase (generates fumarate); and a quinone hydroxylase gene. These findings validated the screen and suggested involvement of anaerobic electron transport chain components. Abnormal swarming periodicity of fumC and sdhB mutants was associated with the excretion of reduced acidic fermentation end products. Bacteria lacking SdhB were rescued to wild-type pH and periodicity by providing fumarate, independent of carbon source but dependent on oxygen, while fumC mutants were rescued by glycerol, independent of fumarate only under anaerobic conditions. These findings link multicellular swarming patterns with fumarate metabolism and membrane electron transport using a previously unappreciated configuration of both aerobic and anaerobic respiratory chain components. PMID:23111869

Revitalization of a Forward Genetic Screen Identifies Three New Regulators of Fungal Secondary Metabolism in the Genus Aspergillus

PubMed Central

Pfannenstiel, Brandon T.; Zhao, Xixi; Wortman, Jennifer; Throckmorton, Kurt; Spraker, Joseph E.; Luo, Xingyu; Lindner, Daniel L.; Lim, Fang Yun; Knox, Benjamin P.; Haas, Brian; Fischer, Gregory J.; Choera, Tsokyi; Butchko, Robert A. E.; Bok, Jin-Woo; Affeldt, Katharyn J.

2017-01-01

ABSTRACT The study of aflatoxin in Aspergillus spp. has garnered the attention of many researchers due to aflatoxin’s carcinogenic properties and frequency as a food and feed contaminant. Significant progress has been made by utilizing the model organism Aspergillus nidulans to characterize the regulation of sterigmatocystin (ST), the penultimate precursor of aflatoxin. A previous forward genetic screen identified 23 A. nidulans mutants involved in regulating ST production. Six mutants were characterized from this screen using classical mapping (five mutations in mcsA) and complementation with a cosmid library (one mutation in laeA). The remaining mutants were backcrossed and sequenced using Illumina and Ion Torrent sequencing platforms. All but one mutant contained one or more sequence variants in predicted open reading frames. Deletion of these genes resulted in identification of mutant alleles responsible for the loss of ST production in 12 of the 17 remaining mutants. Eight of these mutations were in genes already known to affect ST synthesis (laeA, mcsA, fluG, and stcA), while the remaining four mutations (in laeB, sntB, and hamI) were in previously uncharacterized genes not known to be involved in ST production. Deletion of laeB, sntB, and hamI in A. flavus results in loss of aflatoxin production, confirming that these regulators are conserved in the aflatoxigenic aspergilli. This report highlights the multifaceted regulatory mechanisms governing secondary metabolism in Aspergillus. Additionally, these data contribute to the increasing number of studies showing that forward genetic screens of fungi coupled with whole-genome resequencing is a robust and cost-effective technique. PMID:28874473

Role of cyclic di-GMP in Xylella fastidiosa biofilm formation, plant virulence, and insect transmission.

PubMed

Chatterjee, Subhadeep; Killiny, Nabil; Almeida, Rodrigo P P; Lindow, Steven E

2010-10-01

Xylella fastidiosa must coordinately regulate a variety of traits contributing to biofilm formation, host plant and vector colonization, and transmission between plants. Traits such as production of extracellular polysaccharides (EPS), adhesins, extracellular enzymes, and pili are expressed in a cell-density-dependent fashion mediated by a cell-to-cell signaling system involving a fatty acid diffusible signaling factor (DSF). The expression of gene PD0279 (which has a GGDEF domain) is downregulated in the presence of DSF and may be involved in intracellular signaling by modulating the levels of cyclic di-GMP. PD0279, designated cyclic di-GMP synthase A (cgsA), is required for biofilm formation, plant virulence, and vector transmission. cgsA mutants exhibited a hyperadhesive phenotype in vitro and overexpressed gumJ, hxfA, hxfB, xadA, and fimA, which promote attachment of cells to surfaces and, hence, biofilm formation. The mutants were greatly reduced in virulence to grape albeit still transmissible by insect vectors, although at a reduced level compared with transmission rates of the wild-type strain, despite the fact that similar numbers of cells of the cgsA mutant were acquired by the insects from infected plants. High levels of EPS were measured in cgsA mutants compared with wild-type strains, and scanning electron microscopy analysis also revealed a thicker amorphous layer surrounding the mutants. Overexpression of cgsA in a cgsA-complemented mutant conferred the opposite phenotypes in vitro. These results suggest that decreases of cyclic di-GMP result from the accumulation of DSF as cell density increases, leading to a phenotypic transition from a planktonic state capable of colonizing host plants to an adhesive state that is insect transmissible.

bdhA-patD operon as a virulence determinant, revealed by a novel large-scale approach for identification of Legionella pneumophila mutants defective for amoeba infection.

PubMed

Aurass, P; Pless, B; Rydzewski, K; Holland, G; Bannert, N; Flieger, A

2009-07-01

Legionella pneumophila, the causative agent of Legionnaires' disease, is an intracellular parasite of eukaryotic cells. In the environment, it colonizes amoebae. After being inhaled into the human lung, the bacteria infect and damage alveolar cells in a way that is mechanistically similar to the amoeba infection. Several L. pneumophila traits, among those the Dot/Icm type IVB protein secretion machinery, are essential for exploiting host cells. In our search for novel Legionella virulence factors, we developed an agar plate assay, designated the scatter screen, which allowed screening for mutants deficient in infecting Acanthamoeba castellanii amoebae. Likewise, an L. pneumophila clone bank consisting of 23,000 transposon mutants was investigated here, and 19 different established Legionella virulence genes, for example, dot/icm genes, were identified. Importantly, 70 novel virulence-associated genes were found. One of those is L. pneumophila bdhA, coding for a protein with homology to established 3-hydroxybutyrate dehydrogenases involved in poly-3-hydroxybutyrate metabolism. Our study revealed that bdhA is cotranscribed with patD, encoding a patatin-like protein of L. pneumophila showing phospholipase A and lysophospholipase A activities. In addition to strongly reduced lipolytic activities and increased poly-3-hydroxybutyrate levels, the L. pneumophila bdhA-patD mutant showed a severe replication defect in amoebae and U937 macrophages. Our data suggest that the operon is involved in poly-3-hydroxybutyrate utilization and phospholipolysis and show that the bdhA-patD operon is a virulence determinant of L. pneumophila. In summary, the screen for amoeba-sensitive Legionella clones efficiently isolated mutants that do not grow in amoebae and, in the case of the bdhA-patD mutant, also human cells.

Comprehensive Gene Expression Analysis of Rice Aleurone Cells: Probing the Existence of an Alternative Gibberellin Receptor1

PubMed Central

Yano, Kenji; Aya, Koichiro; Hirano, Ko; Ordonio, Reynante Lacsamana; Ueguchi-Tanaka, Miyako; Matsuoka, Makoto

2015-01-01

Current gibberellin (GA) research indicates that GA must be perceived in plant nuclei by its cognate receptor, GIBBERELLIN INSENSITIVE DWARF1 (GID1). Recognition of GA by GID1 relieves the repression mediated by the DELLA protein, a model known as the GID1-DELLA GA perception system. There have been reports of potential GA-binding proteins in the plasma membrane that perceive GA and induce α-amylase expression in cereal aleurone cells, which is mechanistically different from the GID1-DELLA system. Therefore, we examined the expression of the rice (Oryza sativa) α-amylase genes in rice mutants impaired in the GA receptor (gid1) and the DELLA repressor (slender rice1; slr1) and confirmed their lack of response to GA in gid1 mutants and constitutive expression in slr1 mutants. We also examined the expression of GA-regulated genes by genome-wide microarray and quantitative reverse transcription-polymerase chain reaction analyses and confirmed that all GA-regulated genes are modulated by the GID1-DELLA system. Furthermore, we studied the regulatory network involved in GA signaling by using a set of mutants defective in genes involved in GA perception and gene expression, namely gid1, slr1, gid2 (a GA-related F-box protein mutant), and gamyb (a GA-related trans-acting factor mutant). Almost all GA up-regulated genes were regulated by the four named GA-signaling components. On the other hand, GA down-regulated genes showed different expression patterns with respect to GID2 and GAMYB (e.g. a considerable number of genes are not controlled by GAMYB or GID2 and GAMYB). Based on these observations, we present a comprehensive discussion of the intricate network of GA-regulated genes in rice aleurone cells. PMID:25511432

Dissection of Symbiosis and Organ Development by Integrated Transcriptome Analysis of Lotus japonicus Mutant and Wild-Type Plants

PubMed Central

Høgslund, Niels; Radutoiu, Simona; Krusell, Lene; Voroshilova, Vera; Hannah, Matthew A.; Goffard, Nicolas; Sanchez, Diego H.; Lippold, Felix; Ott, Thomas; Sato, Shusei; Tabata, Satoshi; Liboriussen, Poul; Lohmann, Gitte V.; Schauser, Leif; Weiller, Georg F.; Udvardi, Michael K.; Stougaard, Jens

2009-01-01

Genetic analyses of plant symbiotic mutants has led to the identification of key genes involved in Rhizobium-legume communication as well as in development and function of nitrogen fixing root nodules. However, the impact of these genes in coordinating the transcriptional programs of nodule development has only been studied in limited and isolated studies. Here, we present an integrated genome-wide analysis of transcriptome landscapes in Lotus japonicus wild-type and symbiotic mutant plants. Encompassing five different organs, five stages of the sequentially developed determinate Lotus root nodules, and eight mutants impaired at different stages of the symbiotic interaction, our data set integrates an unprecedented combination of organ- or tissue-specific profiles with mutant transcript profiles. In total, 38 different conditions sampled under the same well-defined growth regimes were included. This comprehensive analysis unravelled new and unexpected patterns of transcriptional regulation during symbiosis and organ development. Contrary to expectations, none of the previously characterized nodulins were among the 37 genes specifically expressed in nodules. Another surprise was the extensive transcriptional response in whole root compared to the susceptible root zone where the cellular response is most pronounced. A large number of transcripts predicted to encode transcriptional regulators, receptors and proteins involved in signal transduction, as well as many genes with unknown function, were found to be regulated during nodule organogenesis and rhizobial infection. Combining wild type and mutant profiles of these transcripts demonstrates the activation of a complex genetic program that delineates symbiotic nitrogen fixation. The complete data set was organized into an indexed expression directory that is accessible from a resource database, and here we present selected examples of biological questions that can be addressed with this comprehensive and powerful gene expression data set. PMID:19662091

Characterization of the yrbA Gene of Bacillus subtilis, Involved in Resistance and Germination of Spores

PubMed Central

Takamatsu, Hiromu; Kodama, Takeko; Nakayama, Tatsuo; Watabe, Kazuhito

1999-01-01

Insertional inactivation of the yrbA gene of Bacillus subtilis reduced the resistance of the mutant spores to lysozyme. The yrbA mutant spores lost their optical density at the same rate as the wild-type spores upon incubation with l-alanine but became only phase gray and did not swell. The response of the mutant spores to a combination of asparagine, glucose, fructose, and KCl was also extremely poor; in this medium yrbA spores exhibited only a small loss in optical density and gave a mixture of phase-bright, -gray, and -dark spores. Northern blot analysis of yrbA transcripts in various sig mutants indicated that yrbA was transcribed by RNA polymerase with ςE beginning at 2 h after the start of sporulation. The yrbA promoter was localized by primer extension analysis, and the sequences of the −35 (TCATAAC) and −10 (CATATGT) regions were similar to the consensus sequences of genes recognized by ςE. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of proteins solubilized from intact yrbA mutant spores showed an alteration in the protein profile, as 31- and 36-kDa proteins, identified as YrbA and CotG, respectively, were absent, along with some other minor changes. Electron microscopic examination of yrbA spores revealed changes in the spore coat, including a reduction in the density and thickness of the outer layer and the appearance of an inner coat layer-like structure around the outside of the coat. This abnormal coat structure was also observed on the outside of the developing forespores of the yrbA mutant. These results suggest that YrbA is involved in assembly of some coat proteins which have roles in both spore lysozyme resistance and germination. PMID:10438771

Isolation and characterization of OmpC porin mutants with altered pore properties

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

Misra, R.; Benson, S.A.

1988-02-01

The LamB protien is normally required for the uptake of maltodextrins. Starting with a LamB/sup -/ OmpF/sup -/ strain, we have isolated mutants that will grow on maltodextrins. The mutation conferring the Dex/sup +/ phenotype in the majority of these mutants has been mapped to the ompC locus. These mutants, unlike LamB/sup -/ OmpF/sup -/ strains, grew on maltotriose and maltotetraose, but not on maltopentaose, and showed a significantly higher rate of (/sup 14/C) maltose uptake than the parent strain did. In addition, these mutants showed increased sensitivity to certain ..beta..-lactam antibiotics and sodium dodecyl sulfate, but did not exhibitmore » an increase in sensitivity to other antibiotics and detergents. The nucleotide sequence of these mutants has been determined. In all cases, residue 74 (arginine) of the mature OmpC protein was affected. The results suggest that this region of the OmpC protein is involved in the pore domain and that the alterations lead to an increased pore size.« less

Mutants in the Mouse NuRD/Mi2 Component P66α Are Embryonic Lethal

PubMed Central

Marino, Susan; Nusse, Roel

2007-01-01

Background The NuRD/Mi2 chromatin complex is involved in histone modifications and contains a large number of subunits, including the p66 protein. There are two mouse and human p66 paralogs, p66α and p66β. The functions of these genes are not clear, in part because there are no mutants available, except in invertebrate model systems. Methodology We made loss of function mutants in the mouse p66α gene (mp66α, official name Gatad2a, MGI:2384585). We found that mp66α is essential for development, as mutant embryos die around day 10 of embryogenesis. The gene is not required for normal blastocyst development or for implantation. The phenotype of mutant embryos and the pattern of gene expression in mutants are consistent with a role of mp66α in gene silencing. Conclusion mp66α is an essential gene, required for early mouse development. The lethal phenotype supports a role in execution of methylated DNA silencing. PMID:17565372

[Pigment-protein complexes nd the number of the reaction photosystem centers in pea chlorophyll mutants].

PubMed

Ladygin, V G

2004-01-01

We studied fluorescent and absorption properties of the chloroplasts and pigment-protein complexes isolated by gel electrophoresis from the leaves of pea, the initial cultivar Torsdag and mutants chlorotica 2004 and 2014. Specific maxima of fluorescence and chlorophyll forms in individual complexes have been determined from the absorption and fluorescence spectra of the chloroplast chlorophyll and their secondary derivatives at 23 and -196 degrees C. Chlorotica 2004 mutant proved to have an increased intensity of a long-wave band at both 23 degrees C (745 nm) and -196 degrees C (728 nm) of the light-harvesting complex I. At the same time, this mutant featured a decreased accumulation of chlorophyll forms at 690, 697, and 708 nm forming the nearest-neighbor antenna of PSI reaction center. No spectral differences have been revealed between chlorotica 2014 mutant and the initial cultivar. Gel electrophoresis demonstrated synthesis of all chlorophyll-protein complexes in both mutants. At the same time, analysis of photochemical activity of PSI and PSII reaction centers and evaluation of the light-harvesting antenna as well as the number of reaction centers of the photosystems suggest that chlorotica 2004 mutant has 1.7 times less PSI reaction centers due to a mutation-disturbed chlorophyll a-protein complex of PSI. The primary effect of chlorotica 2014 mutation remains unclear. The proportional changes in the photosystem complexes in this mutant suggest that they are secondary and result from a 50% decrease in chlorophyll content.

Participation of Candida albicans Transcription Factor RLM1 in Cell Wall Biogenesis and Virulence

PubMed Central

Delgado-Silva, Yolanda; Vaz, Catarina; Carvalho-Pereira, Joana; Carneiro, Catarina; Nogueira, Eugénia; Correia, Alexandra; Carreto, Laura; Silva, Sónia; Faustino, Augusto; Pais, Célia; Oliveira, Rui; Sampaio, Paula

2014-01-01

Candida albicans cell wall is important for growth and interaction with the environment. RLM1 is one of the putative transcription factors involved in the cell wall integrity pathway, which plays an important role in the maintenance of the cell wall integrity. In this work we investigated the involvement of RLM1 in the cell wall biogenesis and in virulence. Newly constructed C. albicans Δ/Δrlm1 mutants showed typical cell wall weakening phenotypes, such as hypersensitivity to Congo Red, Calcofluor White, and caspofungin (phenotype reverted in the presence of sorbitol), confirming the involvement of RLM1 in the cell wall integrity. Additionally, the cell wall of C. albicans Δ/Δrlm1 showed a significant increase in chitin (213%) and reduction in mannans (60%), in comparison with the wild-type, results that are consistent with cell wall remodelling. Microarray analysis in the absence of any stress showed that deletion of RLM1 in C. albicans significantly down-regulated genes involved in carbohydrate catabolism such as DAK2, GLK4, NHT1 and TPS1, up-regulated genes involved in the utilization of alternative carbon sources, like AGP2, SOU1, SAP6, CIT1 or GAL4, and genes involved in cell adhesion like ECE1, ALS1, ALS3, HWP1 or RBT1. In agreement with the microarray results adhesion assays showed an increased amount of adhering cells and total biomass in the mutant strain, in comparison with the wild-type. C. albicans mutant Δ/Δrlm1 strain was also found to be less virulent than the wild-type and complemented strains in the murine model of disseminated candidiasis. Overall, we showed that in the absence of RLM1 the modifications in the cell wall composition alter yeast interaction with the environment, with consequences in adhesion ability and virulence. The gene expression findings suggest that this gene participates in the cell wall biogenesis, with the mutant rearranging its metabolic pathways to allow the use of alternative carbon sources. PMID:24466000

Capturing the metabolomic diversity of KRAS mutants in non-small-cell lung cancer cells

PubMed Central

Marabese, Mirko; Broggini, Massimo; Pastorelli, Roberta

2014-01-01

In non-small-cell lung cancer (NSCLC), one-fifth of patients have KRAS mutations, which are considered a negative predictive factor to first-line therapy. Evidence is emerging that not all KRAS mutations have the same biological activities and possible remodeling of cell metabolism by KRAS activation might complicate the scenario. An open question is whether different KRAS mutations at codon-12 affect cellular metabolism differently with possible implications for different responses to cancer treatments. We applied an explorative mass spectrometry-based untargeted metabolomics strategy to characterize the largest possible number of metabolites that might distinguish isogenic NSCLC cells overexpressing mutated forms of KRAS at codon-12 (G12C, G12D, G12V) and the wild-type. The glutamine deprivation assay and real-time PCR were used to confirm the involvement of some of the metabolic pathways highlighted. Cell clones indicated distinct metabolomic profiles in KRAS wild-type and mutants. Clones harboring different KRAS mutations at codon-12 also had different metabolic remodeling, such as a different redox buffering system and different glutamine-dependency not driven by the transcriptional state of enzymes involved in glutaminolysis. These findings indicate that KRAS mutations at codon-12 are associated with different metabolomic profiles that might affect the responses to cancer treatments. PMID:24952473

Identification and Characterization of an Acinetobacter baumannii Biofilm-Associated Protein▿

PubMed Central

Loehfelm, Thomas W.; Luke, Nicole R.; Campagnari, Anthony A.

2008-01-01

We have identified a homologue to the staphylococcal biofilm-associated protein (Bap) in a bloodstream isolate of Acinetobacter baumannii. The fully sequenced open reading frame is 25,863 bp and encodes a protein with a predicted molecular mass of 854 kDa. Analysis of the nucleotide sequence reveals a repetitive structure consistent with bacterial cell surface adhesins. Bap-specific monoclonal antibody (MAb) 6E3 was generated to an epitope conserved among 41% of A. baumannii strains isolated during a recent outbreak in the U.S. military health care system. Flow cytometry confirms that the MAb 6E3 epitope is surface exposed. Random transposon mutagenesis was used to generate A. baumannii bap1302::EZ-Tn5, a mutant negative for surface reactivity to MAb 6E3 in which the transposon disrupts the coding sequence of bap. Time course confocal laser scanning microscopy and three-dimensional image analysis of actively growing biofilms demonstrates that this mutant is unable to sustain biofilm thickness and volume, suggesting a role for Bap in supporting the development of the mature biofilm structure. This is the first identification of a specific cell surface protein directly involved in biofilm formation by A. baumannii and suggests that Bap is involved in intercellular adhesion within the mature biofilm. PMID:18024522

Transcriptome alteration in Phytophthora infestans in response to phenazine-1-carboxylic acid production by Pseudomonas fluorescens strain LBUM223.

PubMed

Roquigny, Roxane; Novinscak, Amy; Arseneault, Tanya; Joly, David L; Filion, Martin

2018-06-19

Phytophthora infestans is responsible for late blight, one of the most important potato diseases. Phenazine-1-carboxylic acid (PCA)-producing Pseudomonas fluorescens strain LBUM223 isolated in our laboratory shows biocontrol potential against various plant pathogens. To characterize the effect of LBUM223 on the transcriptome of P. infestans, we conducted an in vitro time-course study. Confrontational assay was performed using P. infestans inoculated alone (control) or with LBUM223, its phzC- isogenic mutant (not producing PCA), or exogenically applied PCA. Destructive sampling was performed at 6, 9 and 12 days and the transcriptome of P. infestans was analysed using RNA-Seq. The expression of a subset of differentially expressed genes was validated by RT-qPCR. Both LBUM223 and exogenically applied PCA significantly repressed P. infestans' growth at all times. Compared to the control treatment, transcriptomic analyses showed that the percentages of all P. infestans' genes significantly altered by LBUM223 and exogenically applied PCA increased as time progressed, from 50 to 61% and from to 32 to 46%, respectively. When applying an absolute cut-off value of 3 fold change or more for all three harvesting times, 207 genes were found significantly differentially expressed by PCA, either produced by LBUM223 or exogenically applied. Gene ontology analysis revealed that both treatments altered the expression of key functional genes involved in major functions like phosphorylation mechanisms, transmembrane transport and oxidoreduction activities. Interestingly, even though no host plant tissue was present in the in vitro system, PCA also led to the overexpression of several genes encoding effectors. The mutant only slightly repressed P. infestans' growth and barely altered its transcriptome. Our study suggests that PCA is involved in P. infestans' growth repression and led to important transcriptomic changes by both up- and down-regulating gene expression in P. infestans over time. Different metabolic functions were altered and many effectors were found to be upregulated, suggesting their implication in biocontrol.

Gene expression profile analysis of Ligon lintless-1 (Li1) mutant reveals important genes and pathways in cotton leaf and fiber development.

PubMed

Ding, Mingquan; Jiang, Yurong; Cao, Yuefen; Lin, Lifeng; He, Shae; Zhou, Wei; Rong, Junkang

2014-02-10

Ligon lintless-1 (Li1) is a monogenic dominant mutant of Gossypium hirsutum (upland cotton) with a phenotype of impaired vegetative growth and short lint fibers. Despite years of research involving genetic mapping and gene expression profile analysis of Li1 mutant ovule tissues, the gene remains uncloned and the underlying pathway of cotton fiber elongation is still unclear. In this study, we report the whole genome-level deep-sequencing analysis of leaf tissues of the Li1 mutant. Differentially expressed genes in leaf tissues of mutant versus wild-type (WT) plants are identified, and the underlying pathways and potential genes that control leaf and fiber development are inferred. The results show that transcription factors AS2, YABBY5, and KANDI-like are significantly differentially expressed in mutant tissues compared with WT ones. Interestingly, several fiber development-related genes are found in the downregulated gene list of the mutant leaf transcriptome. These genes include heat shock protein family, cytoskeleton arrangement, cell wall synthesis, energy, H2O2 metabolism-related genes, and WRKY transcription factors. This finding suggests that the genes are involved in leaf morphology determination and fiber elongation. The expression data are also compared with the previously published microarray data of Li1 ovule tissues. Comparative analysis of the ovule transcriptomes of Li1 and WT reveals that a number of pathways important for fiber elongation are enriched in the downregulated gene list at different fiber development stages (0, 6, 9, 12, 15, 18dpa). Differentially expressed genes identified in both leaf and fiber samples are aligned with cotton whole genome sequences and combined with the genetic fine mapping results to identify a list of candidate genes for Li1. Copyright © 2013 Elsevier B.V. All rights reserved.

Conservative Tryptophan Mutants of the Protein Tyrosine Phosphatase YopH Exhibit Impaired WPD-Loop Function and Crystallize with Divanadate Esters in Their Active Sites

PubMed Central

Moise, Gwendolyn; Gallup, Nathan M.; Alexandrova, Anastassia N.; Hengge, Alvan C.; Johnson, Sean J.

2016-01-01

Catalysis in protein tyrosine phosphatases (PTPs) involves movement of a protein loop called the WPD loop that brings a conserved aspartic acid into the active site to function as a general acid. Mutation of the tryptophan in the WPD loop of the PTP YopH to any other residue with a planar, aromatic side chain (phenylalanine, tyrosine, or histidine) disables general acid catalysis. Crystal structures reveal these conservative mutations leave this critical loop in a catalytically unproductive, quasi-open position. Although the loop positions in crystal structures are similar for all three conservative mutants, the reasons inhibiting normal loop closure differ for each mutant. In the W354F and W354Y mutants, steric clashes result from six-membered rings occupying the position of the five-membered ring of the native indole side chain. The histidine mutant dysfunction results from new hydrogen bonds stabilizing the unproductive position. The results demonstrate how even modest modifications can disrupt catalytically important protein dynamics. Crystallization of all the catalytically compromised mutants in the presence of vanadate gave rise to vanadate dimers at the active site. In W354Y and W354H, a divanadate ester with glycerol is observed. Such species have precedence in solution and are known from the small molecule crystal database. Such species have not been observed in the active site of a phosphatase, as a functional phosphatase would rapidly catalyze their decomposition. The compromised functionality of the mutants allows the trapping of species that undoubtedly form in solution and are capable of binding at the active sites of PTPs, and, presumably, other phosphatases. In addition to monomeric vanadate, such higher-order vanadium-based molecules are likely involved in the interaction of vanadate with PTPs in solution. PMID:26445170

Sterol Methyl Oxidases Affect Embryo Development via Auxin-Associated Mechanisms.

PubMed

Zhang, Xia; Sun, Shuangli; Nie, Xiang; Boutté, Yohann; Grison, Magali; Li, Panpan; Kuang, Susu; Men, Shuzhen

2016-05-01

Sterols are essential molecules for multiple biological processes, including embryogenesis, cell elongation, and endocytosis. The plant sterol biosynthetic pathway is unique in the involvement of two distinct sterol 4α-methyl oxidase (SMO) families, SMO1 and SMO2, which contain three and two isoforms, respectively, and are involved in sequential removal of the two methyl groups at C-4. In this study, we characterized the biological functions of members of the SMO2 gene family. SMO2-1 was strongly expressed in most tissues during Arabidopsis (Arabidopsis thaliana) development, whereas SMO2-2 showed a more specific expression pattern. Although single smo2 mutants displayed no obvious phenotype, the smo2-1 smo2-2 double mutant was embryonic lethal, and the smo2-1 smo2-2/+ mutant was dwarf, whereas the smo2-1/+ smo2-2 mutant exhibited a moderate phenotype. The phenotypes of the smo2 mutants resembled those of auxin-defective mutants. Indeed, the expression of DR5rev:GFP, an auxin-responsive reporter, was reduced and abnormal in smo2-1 smo2-2 embryos. Furthermore, the expression and subcellular localization of the PIN1 auxin efflux facilitator also were altered. Consistent with these observations, either the exogenous application of auxin or endogenous auxin overproduction (YUCCA9 overexpression) partially rescued the smo2-1 smo2-2 embryonic lethality. Surprisingly, the dwarf phenotype of smo2-1 smo2-2/+ was completely rescued by YUCCA9 overexpression. Gas chromatography-mass spectrometry analysis revealed a substantial accumulation of 4α-methylsterols, substrates of SMO2, in smo2 heterozygous double mutants. Together, our data suggest that SMO2s are important for correct sterol composition and function partially through effects on auxin accumulation, auxin response, and PIN1 expression to regulate Arabidopsis embryogenesis and postembryonic development. © 2016 American Society of Plant Biologists. All Rights Reserved.

Sterol Methyl Oxidases Affect Embryo Development via Auxin-Associated Mechanisms1

PubMed Central

Zhang, Xia; Sun, Shuangli; Nie, Xiang; Boutté, Yohann; Grison, Magali; Li, Panpan; Kuang, Susu

2016-01-01

Sterols are essential molecules for multiple biological processes, including embryogenesis, cell elongation, and endocytosis. The plant sterol biosynthetic pathway is unique in the involvement of two distinct sterol 4α-methyl oxidase (SMO) families, SMO1 and SMO2, which contain three and two isoforms, respectively, and are involved in sequential removal of the two methyl groups at C-4. In this study, we characterized the biological functions of members of the SMO2 gene family. SMO2-1 was strongly expressed in most tissues during Arabidopsis (Arabidopsis thaliana) development, whereas SMO2-2 showed a more specific expression pattern. Although single smo2 mutants displayed no obvious phenotype, the smo2-1 smo2-2 double mutant was embryonic lethal, and the smo2-1 smo2-2/+ mutant was dwarf, whereas the smo2-1/+ smo2-2 mutant exhibited a moderate phenotype. The phenotypes of the smo2 mutants resembled those of auxin-defective mutants. Indeed, the expression of DR5rev:GFP, an auxin-responsive reporter, was reduced and abnormal in smo2-1 smo2-2 embryos. Furthermore, the expression and subcellular localization of the PIN1 auxin efflux facilitator also were altered. Consistent with these observations, either the exogenous application of auxin or endogenous auxin overproduction (YUCCA9 overexpression) partially rescued the smo2-1 smo2-2 embryonic lethality. Surprisingly, the dwarf phenotype of smo2-1 smo2-2/+ was completely rescued by YUCCA9 overexpression. Gas chromatography-mass spectrometry analysis revealed a substantial accumulation of 4α-methylsterols, substrates of SMO2, in smo2 heterozygous double mutants. Together, our data suggest that SMO2s are important for correct sterol composition and function partially through effects on auxin accumulation, auxin response, and PIN1 expression to regulate Arabidopsis embryogenesis and postembryonic development. PMID:27006488

delta. -aminolevulinic acid dehydratase deficiency can cause. delta. -aminolevulinate auxotrophy in Escherichia coli

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

O'Neill, G.P.; Michelsen, U.; Soll, D.

Ethylmethane sulfonate-induced mutants of several Escherichia coli strains that required {delta}-aminolevulinic acid (ALA) for growth were isolated by penicillin enrichment or by selection for respiratory-defective strains resistant to the aminoglycoside antibiotic kanamycin. Three classes of mutants were obtained. Two-thirds of the strains were mutants in hemA. Representative of a third of the mutations was the hem-201 mutation. This mutation was mapped to min 8.6 to 8.7. Complementation of the auxotrophic phenotype by wild-type DNA from the corresponding phage 8F10 allowed the isolation of the gene. DNA sequence analysis revealed that the hem-201 gene encoded ALA dehydratase and was similar tomore » a known hemB gene of E. coli. Complementation studies of hem-201 and hemB1 mutant strains with various hem-201 gene subfragments showed that hem-201 and the previously reported hemB1 mutation are in the same gene and that no other gene is required to complement the hem-201 mutant. ALA-forming activity from glutamate could not be detected by in vitro or in vivo assays. Extracts of hem-201 cells had drastically reduce ALA dehydratase levels, while cells transformed with the plasmid-encoded wild-type gene possessed highly elevated enzyme levels. The ALA requirement for growth, the lack of any ALA-forming enzymatic activity, and greatly reduced ALA dehydratase activity of the hem-201 strain suggest that a diffusible product of an enzyme in the heme biosynthetic pathway after ALA formation is involved in positive regulation of ALA biosynthesis. Analysis of another class of ALA-requiring mutants showed that the auxotrophy of the hem-205 mutant could be relieved by either methionine or cysteine and that the mutation maps in the cysG gene, which encodes uroporphyrinogen III methylase. The properties of these nonleaky ALA-requiring strains suggest that ALA is involved more extensively in E. coli intermediary metabolism than has been appreciated to date.« less

A C2H2-type zinc finger protein, SGR5, is involved in early events of gravitropism in Arabidopsis inflorescence stems.

PubMed

Morita, Miyo T; Sakaguchi, Keitaro; Kiyose, Shin-Ichiro; Taira, Kensuke; Kato, Takehide; Nakamura, Moritaka; Tasaka, Masao

2006-08-01

Plants can sense the direction of gravity and change the growth orientation of their organs. To elucidate the molecular mechanisms of gravity perception and the signal transduction of gravitropism, we have characterized a number of shoot gravitropism (sgr) mutants of Arabidopsis. The sgr5-1 mutant shows reduced gravitropism in the inflorescence stem but its root and hypocotyl have normal gravitropism. SGR5 encodes a zinc finger protein with a coiled-coil motif. The SGR5-GFP fusion protein is localized in the nucleus of Arabidopsis protoplasts, suggesting that SGR5 may act as a transcription factor. Analysis of GUS expression under the control of the SGR5 promoter revealed that SGR5 is mainly expressed in the endodermis, the gravity-sensing tissue in inflorescence stems. Furthermore, the observation that endodermis-specific expression of SGR5 using the SCR promoter in the sgr5-1 mutant restores shoot gravitropism indicates that it could function in the gravity-sensing endodermal cell layer. In contrast to other sgr mutants reported previously, almost all amyloplasts in the endodermal cells of the sgr5-1 mutant sedimented in the direction of gravity. Taken together, our results suggest that SGR5 may be involved in an early event in shoot gravitropism such as gravity perception and/or a signaling process subsequent to amyloplast sedimentation as a putative transcription factor in gravity-perceptive cells.

Stamina pistilloida, the Pea ortholog of Fim and UFO, is required for normal development of flowers, inflorescences, and leaves.

PubMed

Taylor, S; Hofer, J; Murfet, I

2001-01-01

Isolation and characterization of two severe alleles at the Stamina pistilloida (Stp) locus reveals that Stp is involved in a wide range of developmental processes in the garden pea. The most severe allele, stp-4, results in flowers consisting almost entirely of sepals and carpels. Production of ectopic secondary flowers in stp-4 plants suggests that Stp is involved in specifying floral meristem identity in pea. The stp mutations also reduce the complexity of the compound pea leaf, and primary inflorescences often terminate prematurely in an aberrant sepaloid flower. In addition, stp mutants were shorter than their wild-type siblings due to a reduction in cell number in their internodes. Fewer cells were also found in the epidermis of the leaf rachis of stp mutants. Examination of the effects of stp-4 in double mutant combinations with af, tl, det, and veg2-2-mutations known to influence leaf, inflorescence, and flower development in pea-suggests that Stp function is independent of these genes. A synergistic interaction between weak mutant alleles at Stp and Uni indicated that these two genes act together, possibly to regulate primordial growth. Molecular analysis revealed that Stp is the pea homolog of the Antirrhinum gene Fimbriata (Fim) and of UNUSUAL FLORAL ORGANS (UFO) from Arabidopsis. Differences between Fim/UFO and Stp mutant phenotypes and expression patterns suggest that expansion of Stp activity into the leaf was an important step during evolution of the compound leaf in the garden pea.

Mutational and Functional Analysis of the β-Carotene Ketolase Involved in the Production of Canthaxanthin and Astaxanthin

PubMed Central

Ye, Rick W.; Stead, Kristen J.; Yao, Henry; He, Hongxian

2006-01-01

Biosynthesis of the commercial carotenoids canthaxanthin and astaxanthin requires β-carotene ketolase. The functional importance of the conserved amino acid residues of this enzyme from Paracoccus sp. strain N81106 (formerly classified as Agrobacterium aurantiacum) was analyzed by alanine-scanning mutagenesis. Mutations in the three highly conserved histidine motifs involved in iron coordination abolished its ability to catalyze the formation of ketocarotenoids. This supports the hypothesis that the CrtW ketolase belongs to the family of iron-dependent integral membrane proteins. Most of the mutations generated at other highly conserved residues resulted in partial activity. All partially active mutants showed a higher amount of adonixanthin accumulation than did the wild type when expressed in Escherichia coli cells harboring the zeaxanthin biosynthetic gene cluster. Some of the partially active mutants also produced a significant amount of echinenone when expressed in cells producing β-carotene. In fact, expression of a mutant carrying D117A resulted in the accumulation of echinenone as the predominant carotenoid. These observations indicate that partial inactivation of the CrtW ketolase can often lead to the production of monoketolated intermediates. In order to improve the conversion rate of astaxanthin catalyzed by the CrtW ketolase, a color screening system was developed. Three randomly generated mutants, carrying L175M, M99V, and M99I, were identified to have improved activity. These mutants are potentially useful in pathway engineering for the production of astaxanthin. PMID:16957201

Identification of two new genes, mukE and mukF, involved in chromosome partitioning in Escherichia coli.

PubMed

Yamanaka, K; Ogura, T; Niki, H; Hiraga, S

1996-02-25

We have previously reported that the MukB protein is essential for chromosome partitioning in Escherichia coli and that mukB mutants produce anucleate cells and are temperature-sensitive for colony formation. The mukB gene maps at 21 min on the E. coli chromosome and smtA-mukF-mukE-mukB genes might comprise an operon, which is transcribed in a clockwise direction. Here, we report that mukF and mukE null mutants are both temperature-sensitive for colony formation and produce anucleate cells even at the permissive temperature. These phenotypes are the same as those observed in the mukB null mutant. The primary sequence of MukF includes a leucine zipper structure and an acidic domain. Mutational analysis revealed that both are required for MukF function. When the MukF protein was overproduced in the wild-type strain, anucleate cells were produced. In contrast, overproduction of either MukE or MukB did not cause the defect. In null mutants for the mukF, mukE, and mukB genes, the synchronous initiation of chromosome replication was not affected. The mini-F plasmid was as stably maintained in these mutants as in the wild-type strain. These results indicate that the MukF, MukE, and MukB proteins are involved in the chromosome partitioning steps, but are not required for mini-F plasmid partitioning.

Identification of the 2-Hydroxyglutarate and Isovaleryl-CoA Dehydrogenases as Alternative Electron Donors Linking Lysine Catabolism to the Electron Transport Chain of Arabidopsis Mitochondria[W][OA

PubMed Central

Araújo, Wagner L.; Ishizaki, Kimitsune; Nunes-Nesi, Adriano; Larson, Tony R.; Tohge, Takayuki; Krahnert, Ina; Witt, Sandra; Obata, Toshihiro; Schauer, Nicolas; Graham, Ian A.; Leaver, Christopher J.; Fernie, Alisdair R.

2010-01-01

The process of dark-induced senescence in plants is relatively poorly understood, but a functional electron-transfer flavoprotein/electron-transfer flavoprotein:ubiquinone oxidoreductase (ETF/ETFQO) complex, which supports respiration during carbon starvation, has recently been identified. Here, we studied the responses of Arabidopsis thaliana mutants deficient in the expression of isovaleryl-CoA dehydrogenase and 2-hydroxyglutarate dehydrogenase to extended darkness and other environmental stresses. Evaluations of the mutant phenotypes following carbon starvation induced by extended darkness identify similarities to those exhibited by mutants of the ETF/ETFQO complex. Metabolic profiling and isotope tracer experimentation revealed that isovaleryl-CoA dehydrogenase is involved in degradation of the branched-chain amino acids, phytol, and Lys, while 2-hydroxyglutarate dehydrogenase is involved exclusively in Lys degradation. These results suggest that isovaleryl-CoA dehydrogenase is the more critical for alternative respiration and that a series of enzymes, including 2-hydroxyglutarate dehydrogenase, plays a role in Lys degradation. Both physiological and metabolic phenotypes of the isovaleryl-CoA dehydrogenase and 2-hydroxyglutarate dehydrogenase mutants were not as severe as those observed for mutants of the ETF/ETFQO complex, indicating some functional redundancy of the enzymes within the process. Our results aid in the elucidation of the pathway of plant Lys catabolism and demonstrate that both isovaleryl-CoA dehydrogenase and 2-hydroxyglutarate dehydrogenase act as electron donors to the ubiquinol pool via an ETF/ETFQO-mediated route. PMID:20501910

The microtubule associated protein END BINDING 1 represses root responses to mechanical cues.

PubMed

Gleeson, Laura; Squires, Shannon; Bisgrove, Sherryl R

2012-05-01

The ability of roots to navigate around rocks and other debris as they grow through the soil requires a mechanism for detecting and responding to input from both touch and gravity sensing systems. The microtubule associated protein END BINDING 1b (EB1b) is involved in this process as mutants have defects responding to combinations of touch and gravity cues. This study investigates the role of EB1b in root responses to mechanical cues. We find that eb1b-1 mutant roots exhibit an increase over wild type in their response to touch and that the expression of EB1b genes in transgenic mutants restores the response to wild type levels, indicating that EB1b is an inhibitor of the response. Mutant roots are also hypersensitive to increased levels of mechanical stimulation, revealing the presence of another process that activates the response. These findings are supported by analyses of double mutants between eb1b-1 and seedlings carrying mutations in PHOSPHOGLUCOMUTASE (PGM), ALTERED RESPONSE TO GRAVITY1 (ARG1), or TOUCH3 (TCH3), genes that encode proteins involved in gravity sensing, signaling, or touch responses, respectively. A model is proposed in which root responses to mechanical cues are modulated by at least two competing regulatory processes, one that promotes touch-mediated growth and another, regulated by EB1b, which dampens root responses to touch and enhances gravitropism. © 2012. Published by Elsevier Ireland Ltd. All rights reserved.

Mendel's green cotyledon gene encodes a positive regulator of the chlorophyll-degrading pathway

PubMed Central

Sato, Yutaka; Morita, Ryouhei; Nishimura, Minoru; Yamaguchi, Hiroyasu; Kusaba, Makoto

2007-01-01

Mutants that retain greenness of leaves during senescence are known as “stay-green” mutants. The most famous stay-green mutant is Mendel's green cotyledon pea, one of the mutants used in determining the law of genetics. Pea plants homozygous for this recessive mutation (known as i at present) retain greenness of the cotyledon during seed maturation and of leaves during senescence. We found tight linkage between the I locus and stay-green gene originally found in rice, SGR. Molecular analysis of three i alleles including one with no SGR expression confirmed that the I gene encodes SGR in pea. Functional analysis of sgr mutants in pea and rice further revealed that leaf functionality is lowered despite a high chlorophyll a (Chl a) and chlorophyll b (Chl b) content in the late stage of senescence, suggesting that SGR is primarily involved in Chl degradation. Consistent with this observation, a wide range of Chl–protein complexes, but not the ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit, were shown to be more stable in sgr than wild-type plants. The expression of OsCHL and NYC1, which encode the first enzymes in the degrading pathways of Chl a and Chl b, respectively, was not affected by sgr in rice. The results suggest that SGR might be involved in activation of the Chl-degrading pathway during leaf senescence through translational or posttranslational regulation of Chl-degrading enzymes. PMID:17709752

Mendel's green cotyledon gene encodes a positive regulator of the chlorophyll-degrading pathway.

PubMed

Sato, Yutaka; Morita, Ryouhei; Nishimura, Minoru; Yamaguchi, Hiroyasu; Kusaba, Makoto

2007-08-28

Mutants that retain greenness of leaves during senescence are known as "stay-green" mutants. The most famous stay-green mutant is Mendel's green cotyledon pea, one of the mutants used in determining the law of genetics. Pea plants homozygous for this recessive mutation (known as i at present) retain greenness of the cotyledon during seed maturation and of leaves during senescence. We found tight linkage between the I locus and stay-green gene originally found in rice, SGR. Molecular analysis of three i alleles including one with no SGR expression confirmed that the I gene encodes SGR in pea. Functional analysis of sgr mutants in pea and rice further revealed that leaf functionality is lowered despite a high chlorophyll a (Chl a) and chlorophyll b (Chl b) content in the late stage of senescence, suggesting that SGR is primarily involved in Chl degradation. Consistent with this observation, a wide range of Chl-protein complexes, but not the ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit, were shown to be more stable in sgr than wild-type plants. The expression of OsCHL and NYC1, which encode the first enzymes in the degrading pathways of Chl a and Chl b, respectively, was not affected by sgr in rice. The results suggest that SGR might be involved in activation of the Chl-degrading pathway during leaf senescence through translational or posttranslational regulation of Chl-degrading enzymes.

Beyond Tryptophan Synthase: Identification of Genes That Contribute to Chlamydia trachomatis Survival during Gamma Interferon-Induced Persistence and Reactivation

PubMed Central

Muramatsu, Matthew K.; Brothwell, Julie A.; Stein, Barry D.; Putman, Timothy E.; Rockey, Daniel D.

2016-01-01

Chlamydia trachomatis can enter a viable but nonculturable state in vitro termed persistence. A common feature of C. trachomatis persistence models is that reticulate bodies fail to divide and make few infectious progeny until the persistence-inducing stressor is removed. One model of persistence that has relevance to human disease involves tryptophan limitation mediated by the host enzyme indoleamine 2,3-dioxygenase, which converts l-tryptophan to N-formylkynurenine. Genital C. trachomatis strains can counter tryptophan limitation because they encode a tryptophan-synthesizing enzyme. Tryptophan synthase is the only enzyme that has been confirmed to play a role in interferon gamma (IFN-γ)-induced persistence, although profound changes in chlamydial physiology and gene expression occur in the presence of persistence-inducing stressors. Thus, we screened a population of mutagenized C. trachomatis strains for mutants that failed to reactivate from IFN-γ-induced persistence. Six mutants were identified, and the mutations linked to the persistence phenotype in three of these were successfully mapped. One mutant had a missense mutation in tryptophan synthase; however, this mutant behaved differently from previously described synthase null mutants. Two hypothetical genes of unknown function, ctl0225 and ctl0694, were also identified and may be involved in amino acid transport and DNA damage repair, respectively. Our results indicate that C. trachomatis utilizes functionally diverse genes to mediate survival during and reactivation from persistence in HeLa cells. PMID:27430273

Plant hormone jasmonate prioritizes defense over growth by interfering with gibberellin signaling cascade.

PubMed

Yang, Dong-Lei; Yao, Jian; Mei, Chuan-Sheng; Tong, Xiao-Hong; Zeng, Long-Jun; Li, Qun; Xiao, Lang-Tao; Sun, Tai-ping; Li, Jigang; Deng, Xing-Wang; Lee, Chin Mei; Thomashow, Michael F; Yang, Yinong; He, Zuhua; He, Sheng Yang

2012-05-08

Plants must effectively defend against biotic and abiotic stresses to survive in nature. However, this defense is costly and is often accompanied by significant growth inhibition. How plants coordinate the fluctuating growth-defense dynamics is not well understood and remains a fundamental question. Jasmonate (JA) and gibberellic acid (GA) are important plant hormones that mediate defense and growth, respectively. Binding of bioactive JA or GA ligands to cognate receptors leads to proteasome-dependent degradation of specific transcriptional repressors (the JAZ or DELLA family of proteins), which, at the resting state, represses cognate transcription factors involved in defense (e.g., MYCs) or growth [e.g. phytochrome interacting factors (PIFs)]. In this study, we found that the coi1 JA receptor mutants of rice (a domesticated monocot crop) and Arabidopsis (a model dicot plant) both exhibit hallmark phenotypes of GA-hypersensitive mutants. JA delays GA-mediated DELLA protein degradation, and the della mutant is less sensitive to JA for growth inhibition. Overexpression of a selected group of JAZ repressors in Arabidopsis plants partially phenocopies GA-associated phenotypes of the coi1 mutant, and JAZ9 inhibits RGA (a DELLA protein) interaction with transcription factor PIF3. Importantly, the pif quadruple (pifq) mutant no longer responds to JA-induced growth inhibition, and overexpression of PIF3 could partially overcome JA-induced growth inhibition. Thus, a molecular cascade involving the COI1-JAZ-DELLA-PIF signaling module, by which angiosperm plants prioritize JA-mediated defense over growth, has been elucidated.

Identification of the 2-hydroxyglutarate and isovaleryl-CoA dehydrogenases as alternative electron donors linking lysine catabolism to the electron transport chain of Arabidopsis mitochondria.

PubMed

Araújo, Wagner L; Ishizaki, Kimitsune; Nunes-Nesi, Adriano; Larson, Tony R; Tohge, Takayuki; Krahnert, Ina; Witt, Sandra; Obata, Toshihiro; Schauer, Nicolas; Graham, Ian A; Leaver, Christopher J; Fernie, Alisdair R

2010-05-01

The process of dark-induced senescence in plants is relatively poorly understood, but a functional electron-transfer flavoprotein/electron-transfer flavoprotein:ubiquinone oxidoreductase (ETF/ETFQO) complex, which supports respiration during carbon starvation, has recently been identified. Here, we studied the responses of Arabidopsis thaliana mutants deficient in the expression of isovaleryl-CoA dehydrogenase and 2-hydroxyglutarate dehydrogenase to extended darkness and other environmental stresses. Evaluations of the mutant phenotypes following carbon starvation induced by extended darkness identify similarities to those exhibited by mutants of the ETF/ETFQO complex. Metabolic profiling and isotope tracer experimentation revealed that isovaleryl-CoA dehydrogenase is involved in degradation of the branched-chain amino acids, phytol, and Lys, while 2-hydroxyglutarate dehydrogenase is involved exclusively in Lys degradation. These results suggest that isovaleryl-CoA dehydrogenase is the more critical for alternative respiration and that a series of enzymes, including 2-hydroxyglutarate dehydrogenase, plays a role in Lys degradation. Both physiological and metabolic phenotypes of the isovaleryl-CoA dehydrogenase and 2-hydroxyglutarate dehydrogenase mutants were not as severe as those observed for mutants of the ETF/ETFQO complex, indicating some functional redundancy of the enzymes within the process. Our results aid in the elucidation of the pathway of plant Lys catabolism and demonstrate that both isovaleryl-CoA dehydrogenase and 2-hydroxyglutarate dehydrogenase act as electron donors to the ubiquinol pool via an ETF/ETFQO-mediated route.

Involvement of multiple stressors induced by non-thermal plasma-charged aerosols during inactivation of airborne bacteria

PubMed Central

Vaze, Nachiket D.; Park, Sin; Brooks, Ari D.; Fridman, Alexander; Joshi, Suresh G.

2017-01-01

A lab-scale, tunable, single-filament, point-to-point nonthermal dieletric-barrier discharge (DBD) plasma device was built to study the mechanisms of inactivation of aerosolized bacterial pathogens. The system inactivates airborne antibiotic-resistant pathogens efficiently. Nebulization mediated pre-optimized (4 log and 7 log) bacterial loads were challenged to plasma-charged aerosols, and lethal and sublethal doses determined using colony assay, and cell viability assay; and the loss of membrane potential and cellular respiration were determined using cell membrane potential assay and XTT assay. Using the strategies of Escherichia coli wildtype, over-expression mutant, deletion mutants, and peroxide and heat stress scavenging, we analyzed activation of intracellular reactive oxygen species (ROS) and heat shock protein (hsp) chaperons. Superoxide dismutase deletion mutants (ΔsodA, ΔsodB, ΔsodAΔsodB) and catalase mutants ΔkatG and ΔkatEΔkatG did not show significant difference from wildtype strain, and ΔkatE and ΔahpC was found significantly more susceptible to cell death than wildtype. The oxyR regulon was found to mediate plasma-charged aerosol-induced oxidative stress in bacteria. Hsp deficient E. coli (ΔhtpG, ΔgroEL, ΔclpX, ΔgrpE) showed complete inactivation of cells at ambient temperature, and the treatment at cold temperature (4°C) significantly protected hsp deletion mutants and wildtype cells, and indicate a direct involvement of hsp in plasma-charged aerosol mediated E. coli cell death. PMID:28166240

Proliferation of smooth muscle cells stimulated by Porphyromonas gingivalis is inhibited by apple polyphenol.

PubMed

Inaba, Hiroaki; Tagashira, Motoyuki; Kanda, Tomomasa; Amano, Atsuo

2011-11-01

Porphyromonas gingivalis (Pg) is thought to be involved in the progression of occlusive arterial lesions, whereas vascular smooth muscle cell (SMC) proliferation is considered to be involved in occlusive arterial disease. We previously showed that bacteremia caused by Pg infection induced proliferation of mouse aortic SMCs. Furthermore, human SMCs stimulated with human plasma incubated with Pg showed a marked transformation from the contractile to proliferative phenotype. In the present study, we examine the involvement of Pg gingipains and fimbriae in induction of the SMC transformation and proliferation, and effective inhibitors. Pg strains including gingipain- and fimbria-null mutants were incubated in human plasma, after which the bacteria were removed and the supernatants were added to cultured SMCs. To evaluate the effects of inhibitors, Pg organisms were incubated in plasma in the presence of apple polyphenol (AP), epigallocatechin gallate, KYT-1 (Arg-gingipain inhibitor), and KYT-36 (Lys-gingipain inhibitor). Plasma supernatants from wild-type and fimbria-mutant cultures markedly stimulated cellular proliferation, whereas those containing gingipain-null mutants showed negligible effects. SMC proliferation was also induced by plasma treated with trypsin. Furthermore, plasma supernatants cultured in the presence of KYT-1/KYT-36 and AP showed significant inhibitory effects on SMC proliferation, whereas cultures with epigallocatechin gallate did not. Our results suggest that Pg gingipains are involved in the induction of SMC transformation and proliferation, whereas this was inhibited by AP.

Integrative transcriptome, proteome, phosphoproteome and genetic mapping reveals new aspects in a fiberless mutant of cotton

PubMed Central

Ma, Qi-Feng; Wu, Chun-Hui; Wu, Man; Pei, Wen-Feng; Li, Xing-Li; Wang, Wen-Kui; Zhang, Jinfa; Yu, Ji-Wen; Yu, Shu-Xun

2016-01-01

To investigate the molecular mechanisms of fiber initiation in cotton (Gossypium spp.), an integrated approach combining transcriptome, iTRAQ-based proteome and genetic mapping was taken to compare the ovules of the Xuzhou 142 wild type (WT) with its fuzzless-lintless (fl) mutant at −3 and 0 day post-anthesis. A total of 1,953 mRNAs, 187 proteins, and 131 phosphoproteins were differentially expressed (DE) between WT and fl, and the levels of transcripts and their encoded proteins and phosphoproteins were highly congruent. A functional analysis suggested that the abundance of proteins were mainly involved in amino sugar, nucleotide sugar and fatty acid metabolism, one carbon pool for folate metabolism and flavonoid biosynthesis. qRT-PCR, Western blotting, and enzymatic assays were performed to confirm the regulation of these transcripts and proteins. A molecular mapping located the lintless gene li3 in the fl mutant on chromosome 26 for the first time. A further in-silico physical mapping of DE genes with sequence variations between fl and WT identified one and four candidate genes in the li3 and n2 regions, respectively. Taken together, the transcript abundance, phosphorylation status of proteins at the fiber initiation stage and candidate genes have provided insights into regulatory processes underlying cotton fiber initiation. PMID:27075604

Mutagenic effects of heavy ion radiation in plants

NASA Astrophysics Data System (ADS)

Mei, M.; Deng, H.; Lu, Y.; Zhuang, C.; Liu, Z.; Qiu, Q.; Qiu, Y.; Yang, T. C.

1994-10-01

Genetic and developmental effects of heavy ions in maize and rice were investigated. Heavy particles with various charges and energies were accelerated at the BEVALAC. The frequency of occurence of white-yellow stripes on leaves of plants developed from irradiated maize seeds increased linearly with dose, and high-LET heavy charged particles, e.g., neon, argon, and iron, were 2-12 times as effective as gamma rays in inducing this type of mutation. The effectiveness of high-LET heavy ion in (1) inhibiting rice seedling growth, (2) reducing plant fertility, (3) inducing chromosome aberration and micronuclei in root tip cells and pollen mother cells of the first generation plants developed from exposed seeds, and (4) inducing mutation in the second generation, were greater than that of low-LET gamma rays. All effects observed were dose-dependent; however, there appeared to be an optimal range of doses for inducing certain types of mutation, for example, for argon ions (400 MeV/u) at 90-100 Gy, several valuable mutant lines with favorable characters, such as semidwarf, early maturity and high yield ability, were obtained. Experimental results suggest that the potential application of heavy ions in crop improvement is promising. RFLP analysis of two semidwarf mutants induced by argon particles revealed that large DNA alterations might be involved in these mutants.

Involvement of polyubiquitin chains via specific chain linkages in stress response in mammalian cells.

PubMed

Fujimuro, Masahiro; Nishiya, Tadashi; Nomura, Yasuyuki; Yokosawa, Hideyoshi

2005-12-01

Polyubiquitination plays key roles in various proteasome-dependent and independent cellular events. To elucidate roles in stress response of polyubiquitin chains formed via specific chain linkages in mammalian cells, we established NIH3T3 stable cell lines that are capable of conditionally expressing K29R, K48R and K63R ubiquitin mutants, in which the Lys29, Lys48 and Lys63 residues of ubiquitin had been changed to Arg, and we examined the effects of various stresses on their cell viabilities. The expression of K63R ubiquitin mutant decreased viability of the cells post-exposed to ethanol, H(2)O(2) and methyl methanesulfonate (MMS), while that of K48R mutant decreased viability of the cells post-exposed to heat shock as well as ethanol, H(2)O(2) and MMS. Thus, these results suggest that polyubiquitin chains formed via specific chain linkages are involved in the respective stress responses in mammalian cells.

Isolation and Characterization of Sex-Linked Female-Sterile Mutants in DROSOPHILA MELANOGASTER

PubMed Central

Gans, Madeleine; Audit, Claudie; Masson, Michele

1975-01-01

The purpose of the experiments described was to identify X chromosome genes functioning mainly or exclusively during oogenesis. Two mutagenesis experiments were carried out with ethyl methane sulfonate. Following treatment inducing 60% lethals, 9% of the treated X chromosomes carried a female sterility mutation which did not otherwise seriously affect viability. Among —95 isolated mutants, 19 were heat-sensitive and 5 cold-sensitive. The mutants have been classified as follows: I (16 mutants; 12 complementation groups): the females laid few or no eggs; the defect concerned either ovulation or oogenesis. II (37 mutants; 18 complementation groups): the female laid morphologically abnormal eggs, often with increased membrane permeability. III A (13 mutants; at least 8 complementation groups): the homozygous females were sterile if mated to mutant males; their progeny (homo- and hemizygous) died at a late embryonic stage (11 mutants), at the larval stage (1 mutant) or at the pupal stage (1 mutant). However fertility was partly restored by breeding to wild-type males as shown by survival of some heterozygous descendants. III B (29 mutants; 22 complementation groups): the fertility of the females was not restored by breeding to a wild-type male. Most of the eggs of 13 of the mutants died at a late stage of embryogenesis. The eggs of the others ceased development earlier or, perhaps, remained unfertilized. The distribution of the number of mutants per complementation group led to an estimation of a total of about 150 X-linked genes involved in female fertility. The females of three mutants, heat-sensitive and totally sterile at 29°, produced at a lower temperature descendants morphologically abnormal or deprived of germ cells. Three other mutants not described in detail showed a reduction in female fertility with many descendants lacking germ cells. A desirable mutant which was not recovered was one with normal fertile females producing descendants which, regardless of their genotype, bore specific morphological abnormalities. The value of the mutants isolated for analysis of the complex processes leading to egg formation and initiation of development is discussed. PMID:814037

Transcriptomic and molecular genetic analysis of the cell wall salvage response of Aspergillus niger to the absence of galactofuranose synthesis.

PubMed

Park, Joohae; Hulsman, Mark; Arentshorst, Mark; Breeman, Matthijs; Alazi, Ebru; Lagendijk, Ellen L; Rocha, Marina C; Malavazi, Iran; Nitsche, Benjamin M; van den Hondel, Cees A M J J; Meyer, Vera; Ram, Arthur F J

2016-09-01

The biosynthesis of cell surface-located galactofuranose (Galf)-containing glycostructures such as galactomannan, N-glycans and O-glycans in filamentous fungi is important to secure the integrity of the cell wall. UgmA encodes an UDP-galactopyranose mutase, which is essential for the formation of Galf. Consequently, the ΔugmA mutant lacks Galf-containing molecules. Our previous work in Aspergillus niger work suggested that loss of function of ugmA results in activation of the cell wall integrity (CWI) pathway which is characterized by increased expression of the agsA gene, encoding an α-glucan synthase. In this study, the transcriptional response of the ΔugmA mutant was further linked to the CWI pathway by showing the induced and constitutive phosphorylation of the CWI-MAP kinase in the ΔugmA mutant. To identify genes involved in cell wall remodelling in response to the absence of galactofuranose biosynthesis, a genome-wide expression analysis was performed using RNAseq. Over 400 genes were higher expressed in the ΔugmA mutant compared to the wild-type. These include genes that encode enzymes involved in chitin (gfaB, gnsA, chsA) and α-glucan synthesis (agsA), and in β-glucan remodelling (bgxA, gelF and dfgC), and also include several glycosylphosphatidylinositol (GPI)-anchored cell wall protein-encoding genes. In silico analysis of the 1-kb promoter regions of the up-regulated genes in the ΔugmA mutant indicated overrepresentation of genes with RlmA, MsnA, PacC and SteA-binding sites. The importance of these transcription factors for survival of the ΔugmA mutant was analysed by constructing the respective double mutants. The ΔugmA/ΔrlmA and ΔugmA/ΔmsnA double mutants showed strong synthetic growth defects, indicating the importance of these transcription factors to maintain cell wall integrity in the absence of Galf biosynthesis. © 2016 The Authors Cellular Microbiology Published by John Wiley & Sons Ltd.

Intact Interval Timing in Circadian CLOCK Mutants

PubMed Central

Cordes, Sara; Gallistel, C. R.

2008-01-01

While progress has been made in determining the molecular basis for the circadian clock, the mechanism by which mammalian brains time intervals measured in seconds to minutes remains a mystery. An obvious question is whether the interval timing mechanism shares molecular machinery with the circadian timing mechanism. In the current study, we trained circadian CLOCK +/− and −/− mutant male mice in a peak-interval procedure with 10 and 20-s criteria. The mutant mice were more active than their wild-type littermates, but there were no reliable deficits in the accuracy or precision of their timing as compared with wild-type littermates. This suggests that expression of the CLOCK protein is not necessary for normal interval timing. PMID:18602902

Mammalian Cardiovascular Patterning as Determined by Hemodynamic Forces and Blood Vessel Genetics

NASA Astrophysics Data System (ADS)

Anderson, Gregory Arthur

Cardiovascular development is a process that involves the timing of multiple molecular events, and numerous subtle three-dimensional conformational changes. Traditional developmental biology techniques have provided large quantities of information as to how these complex organ systems develop. However, the major drawback of the majority of current developmental biological imaging is that they are two-dimensional in nature. It is now well recognized that circulation of blood is required for normal patterning and remodeling of blood vessels. Normal blood vessel formation is dependent upon a complex network of signaling pathways, and genetic mutations in these pathways leads to impaired vascular development, heart failure, and lethality. As such, it is not surprising that mutant mice with aberrant cardiovascular patterning are so common, since normal development requires proper coordination between three systems: the heart, the blood, and the vasculature. This thesis describes the implementation of a three-dimensional imaging technique, optical projection tomography (OPT), in conjunction with a computer-based registration algorithm to statistically analyze developmental differences in groups of wild-type mouse embryos. Embryos that differ by only a few hours' gestational time are shown to have developmental differences in blood vessel formation and heart development progression that can be discerned. This thesis describes how we analyzed mouse models of cardiovascular perturbation by OPT to detect morphological differences in embryonic development in both qualitative and quantitative ways. Both a blood vessel specific mutation and a cardiac specific mutation were analyzed, providing evidence that developmental defects of these types can be quantified. Finally, we describe the implementation of OPT imaging to identify statistically significant phenotypes from three different mouse models of cardiovascular perturbation across a range of developmental time points. Image registration methods, combined with intensity- and deformation-based analyses are described and utilized to fully characterize myosin light chain 2a (Mlc2a), delta-like ligand 4 (Dll4), and Endoglin (Eng) mutant mouse embryos. We show that Eng mutant embryos are statistically similar to the Mlc2a phenotype, confirming that these mouse mutants suffer from a primary cardiac developmental defect. Thus, a loss of hemodynamic force caused by defective pumping of the heart is the primary developmental defect affecting these mice.

Magic Pools: Parallel Assessment of Transposon Delivery Vectors in Bacteria

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

Liu, Hualan; Price, Morgan N.; Waters, Robert Jordan

Transposon mutagenesis coupled to next-generation sequencing (TnSeq) is a powerful approach for discovering the functions of bacterial genes. However, the development of a suitable TnSeq strategy for a given bacterium can be costly and time-consuming. To meet this challenge, we describe a part-based strategy for constructing libraries of hundreds of transposon delivery vectors, which we term “magic pools.” Within a magic pool, each transposon vector has a different combination of upstream sequences (promoters and ribosome binding sites) and antibiotic resistance markers as well as a random DNA barcode sequence, which allows the tracking of each vector during mutagenesis experiments. Tomore » identify an efficient vector for a given bacterium, we mutagenize it with a magic pool and sequence the resulting insertions; we then use this efficient vector to generate a large mutant library. We used the magic pool strategy to construct transposon mutant libraries in five genera of bacteria, including three genera of the phylumBacteroidetes. IMPORTANCEMolecular genetics is indispensable for interrogating the physiology of bacteria. However, the development of a functional genetic system for any given bacterium can be time-consuming. Here, we present a streamlined approach for identifying an effective transposon mutagenesis system for a new bacterium. Our strategy first involves the construction of hundreds of different transposon vector variants, which we term a “magic pool.” The efficacy of each vector in a magic pool is monitored in parallel using a unique DNA barcode that is introduced into each vector design. Using archived DNA “parts,” we next reassemble an effective vector for making a whole-genome transposon mutant library that is suitable for large-scale interrogation of gene function using competitive growth assays. Here, we demonstrate the utility of the magic pool system to make mutant libraries in five genera of bacteria.« less

Magic Pools: Parallel Assessment of Transposon Delivery Vectors in Bacteria

DOE PAGES

Liu, Hualan; Price, Morgan N.; Waters, Robert Jordan; ...

2018-01-16

Transposon mutagenesis coupled to next-generation sequencing (TnSeq) is a powerful approach for discovering the functions of bacterial genes. However, the development of a suitable TnSeq strategy for a given bacterium can be costly and time-consuming. To meet this challenge, we describe a part-based strategy for constructing libraries of hundreds of transposon delivery vectors, which we term “magic pools.” Within a magic pool, each transposon vector has a different combination of upstream sequences (promoters and ribosome binding sites) and antibiotic resistance markers as well as a random DNA barcode sequence, which allows the tracking of each vector during mutagenesis experiments. Tomore » identify an efficient vector for a given bacterium, we mutagenize it with a magic pool and sequence the resulting insertions; we then use this efficient vector to generate a large mutant library. We used the magic pool strategy to construct transposon mutant libraries in five genera of bacteria, including three genera of the phylumBacteroidetes. IMPORTANCEMolecular genetics is indispensable for interrogating the physiology of bacteria. However, the development of a functional genetic system for any given bacterium can be time-consuming. Here, we present a streamlined approach for identifying an effective transposon mutagenesis system for a new bacterium. Our strategy first involves the construction of hundreds of different transposon vector variants, which we term a “magic pool.” The efficacy of each vector in a magic pool is monitored in parallel using a unique DNA barcode that is introduced into each vector design. Using archived DNA “parts,” we next reassemble an effective vector for making a whole-genome transposon mutant library that is suitable for large-scale interrogation of gene function using competitive growth assays. Here, we demonstrate the utility of the magic pool system to make mutant libraries in five genera of bacteria.« less

Quantitative phosphoproteomics identifies SnRK2 protein kinase substrates and reveals the effectors of abscisic acid action.

PubMed

Wang, Pengcheng; Xue, Liang; Batelli, Giorgia; Lee, Shinyoung; Hou, Yueh-Ju; Van Oosten, Michael J; Zhang, Huiming; Tao, W Andy; Zhu, Jian-Kang

2013-07-02

Sucrose nonfermenting 1 (SNF1)-related protein kinase 2s (SnRK2s) are central components of abscisic acid (ABA) signaling pathways. The snrk2.2/2.3/2.6 triple-mutant plants are nearly completely insensitive to ABA, suggesting that most of the molecular actions of ABA are triggered by the SnRK2s-mediated phosphorylation of substrate proteins. Only a few substrate proteins of the SnRK2s are known. To identify additional substrate proteins of the SnRK2s and provide insight into the molecular actions of ABA, we used quantitative phosphoproteomics to compare the global changes in phosphopeptides in WT and snrk2.2/2.3/2.6 triple mutant seedlings in response to ABA treatment. Among the 5,386 unique phosphorylated peptides identified in this study, we found that ABA can increase the phosphorylation of 166 peptides and decrease the phosphorylation of 117 peptides in WT seedlings. In the snrk2.2/2.3/2.6 triple mutant, 84 of the 166 peptides, representing 58 proteins, could not be phosphorylated, or phosphorylation was not increased under ABA treatment. In vitro kinase assays suggest that most of the 58 proteins can serve as substrates of the SnRK2s. The SnRK2 substrates include proteins involved in flowering time regulation, RNA and DNA binding, miRNA and epigenetic regulation, signal transduction, chloroplast function, and many other cellular processes. Consistent with the SnRK2 phosphorylation of flowering time regulators, the snrk2.2/2.3/2.6 triple mutant flowered significantly earlier than WT. These results shed new light on the role of the SnRK2 protein kinases and on the downstream effectors of ABA action, and improve our understanding of plant responses to adverse environments.

In vitro susceptibility of Trichophyton rubrum isolates to griseofulvin and tioconazole. Induction and isolation of a resistant mutant to both antimycotic drugs. Mutant of Trichophyton rubrum resistant to griseofulvin and tioconazole.

PubMed

Fachin, A L; Maffei, C M; Martinez-Rossi, N M

1996-01-01

The in vitro susceptibility of three clinical Trichophyton rubrum isolates to griseofulvin and tioconazole, determined by the minimal inhibitory concentration (MIC), was 2 and 0.5 to 1.0 micrograms/ml, respectively. One mutant (gril) obtained after mutagenic treatment of one of these isolates was selected and showed simultaneous resistance to griseofulvin (MIC > 2000 micrograms/ml) and tioconazole (MIC = 1.0 microgram/ml). The clinical importance and the possibility of a multidrug resistance (MDR)-type mechanism being involved in this event is discussed.

Genetic Enhancer Analysis Reveals that FLORAL ORGAN NUMBER2 and OsMADS3 Co-operatively Regulate Maintenance and Determinacy of the Flower Meristem in Rice.

PubMed

Yasui, Yukiko; Tanaka, Wakana; Sakamoto, Tomoaki; Kurata, Tetsuya; Hirano, Hiro-Yuki

2017-05-01

Meristems such as the shoot apical meristem and flower meristem (FM) act as a reservoir of stem cells, which reproduce themselves and supply daughter cells for the differentiation of lateral organs. In Oryza sativa (rice), the FLORAL ORGAN NUMBER2 (FON2) gene, which is similar to Arabidopsis CLAVATA3, is involved in meristem maintenance. In fon2 mutants, the numbers of floral organs are increased due to an enlargement of the FM. To identify new factors regulating meristem maintenance in rice, we performed a genetic screening of mutants that enhanced the fon2 mutation, and found a mutant line (2B-424) in which pistil number was dramatically increased. By using a map-based approach and next-generation sequencing, we found that the line 2B-424 had a complete loss-of-function mutation (a large deletion) in OsMADS3, a class C MADS-box gene that is known to be involved in stamen specification. Disruption of OsMADS3 in the fon2 mutant by CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9) technology caused a flower phenotype similar to that of 2B-424, confirming that the gene responsible for enhancement of fon2 was OsMADS3. Morphological analysis showed that the fon2 and osmads3 mutations synergistically affected pistil development and FM determinacy. We also found that whorl 3 was duplicated in mature flowers and the FM was enlarged at an early developmental stage in severe osmads3 single mutants. These findings suggest that OsMADS3 is involved not only in FM determinacy in late flower development but also in FM activity in early flower development. © The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Role of immune cells in animal models for inherited neuropathies: facts and visions.

PubMed

Mäurer, Mathias; Kobsar, Igor; Berghoff, Martin; Schmid, Christoph D; Carenini, Stefano; Martini, Rudolf

2002-04-01

Mice heterozygously deficient in the peripheral myelin adhesion molecule P0 (P0+/- mice) are models for some forms of Charcot-Marie-Tooth (CMT) neuropathies. In addition to the characteristic hallmarks of demyelination, elevated numbers of CD8-positive T-lymphocytes and F4/80-positive macrophages are striking features in the nerves of these mice. These immune cells increase in number with age and progress of demyelination, suggesting that they might be functionally related to myelin damage. In order to investigate the pathogenetic role of lymphocytes, the myelin mutants were cross-bred with recombination activating gene 1 (RAG-1)-deficient mice, which lack mature T- and B-lymphocytes. The immunodeficient myelin mutants showed a less severe myelin degeneration. The beneficial effect of lymphocyte-deficiency was reversible, since demyelination worsened in immunodeficient myelin-mutants when reconstituted with bone marrow from wild-type mice. Ultrastructural analysis revealed macrophages in close apposition to myelin and demyelinated axons. We therefore cross-bred the P0+/- mice with spontaneous osteopetrotic (op) mutants deficient in the macrophage colony-stimulating factor (M-CSF), hence displaying impaired macrophage activation. In the corresponding double mutants the numbers of macrophages were not elevated in the peripheral nerves, and the demyelinating phenotype was less severe than in the genuine P0+/- mice, demonstrating that macrophages are also functionally involved in the pathogenesis of genetically mediated demyelination. We also examined other models for inherited neuropathies for a possible involvement of immune cells. We chose mice deficient in the gap junction component connexin 32, a model for the X-linked form of CMT. Similar to P0-deficient mice, T-lymphocytes and macrophages were elevated and macrophages showed a close apposition to degenerating myelin. We conclude that the involvement of T-lymphocytes and macrophages is a common pathogenetic feature in various forms of slowly progressive inherited neuropathies.

Comparative transcriptomic analysis of key genes involved in flavonoid biosynthetic pathway and identification of a flavonol synthase from Artemisia annua L.

PubMed

Liu, S; Liu, L; Tang, Y; Xiong, S; Long, J; Liu, Z; Tian, N

2017-07-01

The regulatory mechanism of flavonoids, which synergise anti-malarial and anti-cancer compounds in Artemisia annua, is still unclear. In this study, an anthocyanidin-accumulating mutant callus was induced from A. annua and comparative transcriptomic analysis of wild-type and mutant calli performed, based on the next-generation Illumina/Solexa sequencing platform and de novo assembly. A total of 82,393 unigenes were obtained and 34,764 unigenes were annotated in the public database. Among these, 87 unigenes were assigned to 14 structural genes involved in the flavonoid biosynthetic pathway and 37 unigenes were assigned to 17 structural genes related to metabolism of flavonoids. More than 30 unigenes were assigned to regulatory genes, including R2R3-MYB, bHLH and WD40, which might regulate flavonoid biosynthesis. A further 29 unigenes encoding flavonoid biosynthetic enzymes or transcription factors were up-regulated in the mutant, while 19 unigenes were down-regulated, compared with the wild type. Expression levels of nine genes involved in the flavonoid pathway were compared using semi-quantitative RT-PCR, and results were consistent with comparative transcriptomic analysis. Finally, a putative flavonol synthase gene (AaFLS1) was identified from enzyme assay in vitro and in vivo through heterogeneous expression, and confirmed comparative transcriptomic analysis of wild-type and mutant callus. The present work has provided important target genes for the regulation of flavonoid biosynthesis in A. annua. © 2017 German Botanical Society and The Royal Botanical Society of the Netherlands.

Experimental validation of a predicted feedback loop in the multi-oscillator clock of Arabidopsis thaliana

PubMed Central

Locke, James C W; Kozma-Bognár, László; Gould, Peter D; Fehér, Balázs; Kevei, Éva; Nagy, Ferenc; Turner, Matthew S; Hall, Anthony; Millar, Andrew J

2006-01-01

Our computational model of the circadian clock comprised the feedback loop between LATE ELONGATED HYPOCOTYL (LHY), CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and TIMING OF CAB EXPRESSION 1 (TOC1), and a predicted, interlocking feedback loop involving TOC1 and a hypothetical component Y. Experiments based on model predictions suggested GIGANTEA (GI) as a candidate for Y. We now extend the model to include a recently demonstrated feedback loop between the TOC1 homologues PSEUDO-RESPONSE REGULATOR 7 (PRR7), PRR9 and LHY and CCA1. This three-loop network explains the rhythmic phenotype of toc1 mutant alleles. Model predictions fit closely to new data on the gi;lhy;cca1 mutant, which confirm that GI is a major contributor to Y function. Analysis of the three-loop network suggests that the plant clock consists of morning and evening oscillators, coupled intracellularly, which may be analogous to coupled, morning and evening clock cells in Drosophila and the mouse. PMID:17102804

Zebrafish embryology and cartilage staining protocols for high school students.

PubMed

Emran, Farida; Brooks, Jacqueline M; Zimmerman, Steven R; Johnson, Susan L; Lue, Robert A

2009-06-01

The Life Sciences-Howard Hughes Medical Institute Outreach Program at Harvard University supports high school science education by offering an on-campus program for students and their teachers to participate in investigative, hands-on laboratory sessions. The outreach program has recently designed and launched a successful zebrafish embryology protocol that we present here. The main objectives of this protocol are to introduce students to zebrafish as a model research organism and to provide students with direct experience with current techniques used in embryological research. The content of the lab is designed to generate discussions on embryology, genetics, fertilization, natural selection, and animal adaptation. The protocol produces reliable results in a time-efficient manner using a minimum of reagents. The protocol presented here consists of three sections: observations of live zebrafish larvae at different developmental stages, cartilage staining of zebrafish larvae, and a mutant hunt involving identification of two zebrafish mutants (nacre and chokh). Here, we describe the protocol, show the results obtained for each section, and suggest possible alternatives for different lab settings.

Root graviresponsiveness and columella cell structure in carotenoid-deficient seedlings of Zea mays

NASA Technical Reports Server (NTRS)

Moore, R.; McClelen, C. E.

1985-01-01

Root graviresponsiveness in normal and carotenoid-deficient mutant seedlings of Zea mays was not significantly different. Columella cells in roots of mutant seedlings were characterized by fewer, smaller, and a reduced relative volume of plastids as compared to columella cells of normal seedlings. Plastids in columella cells of mutant seedlings possessed reduced amounts of starch. Although approximately 10 per cent of the columella cells in mutant seedlings lacked starch, their plastids were located at the bottom of the cell. These results suggest that (i) carotenoids are not necessary for root gravitropism, (ii) graviresponsiveness is not necessarily proportional to the size, number, or relative volume of plastids in columella cells, and (iii) sedimentation of plastids in columella cells may not result directly from their increased density due to starch content. Plastids in columella cells of normal and mutant seedlings were associated with bands of microtubule-like structures, suggesting that these structures may be involved in 'positioning' plastids in the cell.

Cold-sensitive mutants G680V and G691C of Dictyostelium myosin II confer dramatically different biochemical defects.

PubMed

Patterson, B; Ruppel, K M; Wu, Y; Spudich, J A

1997-10-31

Cold-sensitive myosin mutants represent powerful tools for dissecting discrete deficiencies in myosin function. Biochemical characterization of two such mutants, G680V and G691C, has allowed us to identify separate facets of myosin motor function perturbed by each alteration. Compared with wild type, the G680V myosin exhibits a substantially enhanced affinity for several nucleotides, decreased ATPase activity, and overoccupancy or creation of a novel strongly actin-binding state. The properties of the novel strong binding state are consistent with a partial arrest or pausing at the onset of the mechanical stroke. The G691C mutant, on the other hand, exhibits an elevated basal ATPase indicative of premature phosphate release. By releasing phosphate without a requirement for actin binding, the G691C can bypass the part of the cycle involving the mechanical stroke. The two mutants, despite having alterations in glycine residues separated by only 11 residues, have dramatically different consequences on the mechanochemical cycle.

Generation of a glucose de-repressed mutant of Trichoderma reesei using disparity mutagenesis.

PubMed

Iwakuma, Hidekazu; Koyama, Yoshiyuki; Miyachi, Ayako; Nasukawa, Masashi; Matsumoto, Hitoshi; Yano, Shuntaro; Ogihara, Jun; Kasumi, Takafumi

2016-01-01

We obtained a novel glucose de-repressed mutant of Trichoderma reesei using disparity mutagenesis. A plasmid containing DNA polymerase δ lacking proofreading activity, and AMAI, an autonomously replicating sequence was introduced into T. reesei ATCC66589. The rate of mutation evaluated with 5-fluoroorotic acid resistance was approximately 30-fold higher than that obtained by UV irradiation. The transformants harboring incompetent DNA polymerase δ were then selected on 2-deoxyglucose agar plates with hygromycin B. The pNP-lactoside hydrolyzing activities of mutants were 2 to 5-fold higher than the parent in liquid medium containing glucose. Notably, the amino acid sequence of cre1, a key gene involved in glucose repression, was identical in the mutant and parent strains, and further, the cre1 expression levels was not abolished in the mutant. Taken together, these results demonstrate that the strains of T. reesei generated by disparity mutagenesis are glucose de-repressed variants that contain mutations in yet-unidentified factors other than cre1.

THE USE OF RADIATION-INDUCED MUTATIONS IN CROP BREEDING IN LATIN AMERICA AND SOME BIOLOGICAL EFFECTS OF RADIATION IN COFFEE

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

Moh, C.C.

1962-07-01

Results are summarized from a study on the genetic effects of radiation in coffee as observed in R/sub 1/ plants grown from seeds exposed to x radiation, gamma radiation, or thermal neutrons. A high frequency of morphological mutants was observed in the young plants. Possible reaction mechanisms involved in the induction of the mutants are discussed. (C.H.)

Corynebacterium diphtheriae invasion-associated protein (DIP1281) is involved in cell surface organization, adhesion and internalization in epithelial cells

PubMed Central

2010-01-01

Background Corynebacterium diphtheriae, the causative agent of diphtheria, is well-investigated in respect to toxin production, while little is known about C. diphtheriae factors crucial for colonization of the host. In this study, we investigated the function of surface-associated protein DIP1281, previously annotated as hypothetical invasion-associated protein. Results Microscopic inspection of DIP1281 mutant strains revealed an increased size of the single cells in combination with an altered less club-like shape and formation of chains of cells rather than the typical V-like division forms or palisades of growing C. diphtheriae cells. Cell viability was not impaired. Immuno-fluorescence microscopy, SDS-PAGE and 2-D PAGE of surface proteins revealed clear differences of wild-type and mutant protein patterns, which were verified by atomic force microscopy. DIP1281 mutant cells were not only altered in shape and surface structure but completely lack the ability to adhere to host cells and consequently invade these. Conclusions Our data indicate that DIP1281 is predominantly involved in the organization of the outer surface protein layer rather than in the separation of the peptidoglycan cell wall of dividing bacteria. The adhesion- and invasion-negative phenotype of corresponding mutant strains is an effect of rearrangements of the outer surface. PMID:20051108

Genome-wide analysis of bacterial determinants of plant growth promotion and induced systemic resistance by Pseudomonas fluorescens.

PubMed

Cheng, Xu; Etalo, Desalegn W; van de Mortel, Judith E; Dekkers, Ester; Nguyen, Linh; Medema, Marnix H; Raaijmakers, Jos M

2017-11-01

Pseudomonas fluorescens strain SS101 (Pf.SS101) promotes growth of Arabidopsis thaliana, enhances greening and lateral root formation, and induces systemic resistance (ISR) against the bacterial pathogen Pseudomonas syringae pv. tomato (Pst). Here, targeted and untargeted approaches were adopted to identify bacterial determinants and underlying mechanisms involved in plant growth promotion and ISR by Pf.SS101. Based on targeted analyses, no evidence was found for volatiles, lipopeptides and siderophores in plant growth promotion by Pf.SS101. Untargeted, genome-wide analyses of 7488 random transposon mutants of Pf.SS101 led to the identification of 21 mutants defective in both plant growth promotion and ISR. Many of these mutants, however, were auxotrophic and impaired in root colonization. Genetic analysis of three mutants followed by site-directed mutagenesis, genetic complementation and plant bioassays revealed the involvement of the phosphogluconate dehydratase gene edd, the response regulator gene colR and the adenylsulfate reductase gene cysH in both plant growth promotion and ISR. Subsequent comparative plant transcriptomics analyses strongly suggest that modulation of sulfur assimilation, auxin biosynthesis and transport, steroid biosynthesis and carbohydrate metabolism in Arabidopsis are key mechanisms linked to growth promotion and ISR by Pf.SS101. © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

The post-transcriptional regulatory system CSR controls the balance of metabolic pools in upper glycolysis of Escherichia coli.

PubMed

Morin, Manon; Ropers, Delphine; Letisse, Fabien; Laguerre, Sandrine; Portais, Jean-Charles; Cocaign-Bousquet, Muriel; Enjalbert, Brice

2016-05-01

Metabolic control in Escherichia coli is a complex process involving multilevel regulatory systems but the involvement of post-transcriptional regulation is uncertain. The post-transcriptional factor CsrA is stated as being the only regulator essential for the use of glycolytic substrates. A dozen enzymes in the central carbon metabolism (CCM) have been reported as potentially controlled by CsrA, but its impact on the CCM functioning has not been demonstrated. Here, a multiscale analysis was performed in a wild-type strain and its isogenic mutant attenuated for CsrA (including growth parameters, gene expression levels, metabolite pools, abundance of enzymes and fluxes). Data integration and regulation analysis showed a coordinated control of the expression of glycolytic enzymes. This also revealed the imbalance of metabolite pools in the csrA mutant upper glycolysis, before the phosphofructokinase PfkA step. This imbalance is associated with a glucose-phosphate stress. Restoring PfkA activity in the csrA mutant strain suppressed this stress and increased the mutant growth rate on glucose. Thus, the carbon storage regulator system is essential for the effective functioning of the upper glycolysis mainly through its control of PfkA. This work demonstrates the pivotal role of post-transcriptional regulation to shape the carbon metabolism. © 2016 John Wiley & Sons Ltd.

Pollen embryogenesis to induce, detect, and analyze mutants

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

Constantin, M.J.

The development of fully differentiated plants from individual pollen grains through a series of developmental phases that resemble embryogenesis beginning with the zygote was demonstrated during the mid-1960's. This technology opened the door to the use of haploid plants (sporophytes with the gametic number of chromosomes) for plant breeding and genetic studies, biochemical and metabolic studies, and the selection of mutations. Although pollen embryogenesis has been demonstrated successfully in numerous plant genera, the procedure cannot as yet be used routinely to generate large populations of plants for experiments. Practical results from use of the technology in genetic toxicology research tomore » detect mutations have failed to fully realize the theoretical potential; further developments of the technology could overcome the limitations. Pollen embryogenesis could be used to develop plants from mutant pollen grains to verify that genetic changes are involved. Through either spontaneous or induced chromosome doubling, these plants can be made homozygous and used to analyze genetically the mutants involved. The success of this approach will depend on the mutant frequency relative to the fraction of pollen grains that undergo embryogenesis; these two factors will dictate population size needed for success. Research effort is needed to further develop pollen embryogenesis for use in the detection of genotoxins under both laboratory and in situ conditions.« less

LitR of Vibrio salmonicida Is a Salinity-Sensitive Quorum-Sensing Regulator of Phenotypes Involved in Host Interactions and Virulence

PubMed Central

Bjelland, Ane Mohn; Sørum, Henning; Tegegne, Daget Ayana; Winther-Larsen, Hanne C.; Willassen, Nils Peder

2012-01-01

Vibrio (Aliivibrio) salmonicida is the causal agent of cold-water vibriosis, a fatal bacterial septicemia primarily of farmed salmonid fish. The molecular mechanisms of invasion, colonization, and growth of V. salmonicida in the host are still largely unknown, and few virulence factors have been identified. Quorum sensing (QS) is a cell-to-cell communication system known to regulate virulence and other activities in several bacterial species. The genome of V. salmonicida LFI1238 encodes products presumably involved in several QS systems. In this study, the gene encoding LitR, a homolog of the master regulator of QS in V. fischeri, was deleted. Compared to the parental strain, the litR mutant showed increased motility, adhesion, cell-to-cell aggregation, and biofilm formation. Furthermore, the litR mutant produced less cryptic bioluminescence, whereas production of acylhomoserine lactones was unaffected. Our results also indicate a salinity-sensitive regulation of LitR. Finally, reduced mortality was observed in Atlantic salmon infected with the litR mutant, implying that the fish were more susceptible to infection with the wild type than with the mutant strain. We hypothesize that LitR inhibits biofilm formation and favors planktonic growth, with the latter being more adapted for pathogenesis in the fish host. PMID:22371373

Identification of genes and gene clusters involved in mycotoxin synthesis

USDA-ARS?s Scientific Manuscript database

Research methods to identify and characterize genes involved in mycotoxin biosynthetic pathways have evolved considerably over the years. Before whole genome sequences were available (e.g. pre-genomics), work focused primarily on chemistry, biosynthetic mutant strains and molecular analysis of sing...

Circulation of Pneumocystis dihydropteroate synthase mutants in France.

PubMed

Le Gal, Solène; Damiani, Céline; Perrot, Maëla; Rouillé, Amélie; Virmaux, Michèle; Quinio, Dorothée; Moalic, Elodie; Saliou, Philippe; Berthou, Christian; Le Meur, Yann; Totet, Anne; Nevez, Gilles

2012-10-01

Data on the prevalence of Pneumocystis jirovecii (P. jirovecii) dihydropteroate synthase (DHPS) mutants in France are still limited. In this study, mutant prevalence in the Brest region (western France) was determined. Archival pulmonary specimens from 85 patients infected with P. jirovecii and admitted to our institution (University Hospital, Brest) from October 2007 to February 2010 were retrospectively typed at the DHPS locus using a polymerase chain reaction-restriction fragment length polymorphism assay. Type identification was successful in 66 of 85 patients. Sixty-four patients were infected with a wild type, whereas mutants were found in 2 patients (2/66, 3%). Medical chart analysis revealed that these 2 patients usually lived in Paris. Another patient usually lived on the French Riviera, whereas 63 patients were from the city of Brest. Thus, the corrected prevalence of mutants in patients who effectively lived in our geographic area was 0% (0/63). Taking into account that i) Paris is characterized by a high prevalence of mutants from 18.5% to 40%, ii) infection diagnoses were performed in the 2 Parisians during their vacation <30 days, iii) infection incubation is assumed to last about 2 months, the results provide evidence of mutant circulation from Paris to Brest through infected vacationers. The study shows that the usual city of patient residence, rather than the city of infection diagnosis, is a predictor of mutants and that P. jirovecii infections involving mutants do not represent a public health issue in western France. Copyright © 2012 Elsevier Inc. All rights reserved.

Characterization of a Putative Spindle Assembly Checkpoint Kinase Mps1, Suggests Its Involvement in Cell Division, Morphogenesis and Oxidative Stress Tolerance in Candida albicans

PubMed Central

Ruhela, Deepa; Kamthan, Ayushi; Maiti, Protiti; Datta, Asis

2014-01-01

In Saccharomyces cerevisiae MPS1 is one of the major protein kinase that governs the spindle checkpoint pathway. The S. cerevisiae structural homolog of opportunistic pathogen Candida albicans CaMPS1, is indispensable for the cell viability. The essentiality of Mps1 was confirmed by Homozygote Trisome test. To determine its biological function in this pathogen conditional mutant was generated through regulatable MET3 promoter. Examination of heterozygous and conditional (+Met/Cys) mps1 mutants revealed a mitosis specific arrest phenotype, where mutants showed large buds with undivided nuclei. Flowcytometry analysis revealed abnormal ploidy levels in mps1mutant. In presence of anti-microtubule drug Nocodazole, mps1 mutant showed a dramatic loss of viability suggesting a role of Mps1 in Spindle Assembly Checkpoint (SAC) activation. These mutants were also defective in microtubule organization. Moreover, heterozygous mutant showed defective in-vitro yeast to hyphae morphological transition. Growth defect in heterozygous mutant suggest haploinsufficiency of this gene. qRT PCR analysis showed around 3 fold upregulation of MPS1 in presence of serum. This expression of MPS1 is dependent on Efg1and is independent of other hyphal regulators like Ras1 and Tpk2. Furthermore, mps1 mutants were also sensitive to oxidative stress. Heterozygous mps1 mutant did not undergo morphological transition and showed 5-Fold reduction in colony forming units in response to macrophage. Thus, the vital checkpoint kinase, Mps1 besides cell division also has a role in morphogenesis and oxidative stress tolerance, in this pathogenic fungus. PMID:25025778

Characterization of a putative spindle assembly checkpoint kinase Mps1, suggests its involvement in cell division, morphogenesis and oxidative stress tolerance in Candida albicans.

PubMed

Kamthan, Mohan; Nalla, Vijaya Kumar; Ruhela, Deepa; Kamthan, Ayushi; Maiti, Protiti; Datta, Asis

2014-01-01

In Saccharomyces cerevisiae MPS1 is one of the major protein kinase that governs the spindle checkpoint pathway. The S. cerevisiae structural homolog of opportunistic pathogen Candida albicans CaMPS1, is indispensable for the cell viability. The essentiality of Mps1 was confirmed by Homozygote Trisome test. To determine its biological function in this pathogen conditional mutant was generated through regulatable MET3 promoter. Examination of heterozygous and conditional (+Met/Cys) mps1 mutants revealed a mitosis specific arrest phenotype, where mutants showed large buds with undivided nuclei. Flowcytometry analysis revealed abnormal ploidy levels in mps1 mutant. In presence of anti-microtubule drug Nocodazole, mps1 mutant showed a dramatic loss of viability suggesting a role of Mps1 in Spindle Assembly Checkpoint (SAC) activation. These mutants were also defective in microtubule organization. Moreover, heterozygous mutant showed defective in-vitro yeast to hyphae morphological transition. Growth defect in heterozygous mutant suggest haploinsufficiency of this gene. qRT PCR analysis showed around 3 fold upregulation of MPS1 in presence of serum. This expression of MPS1 is dependent on Efg1 and is independent of other hyphal regulators like Ras1 and Tpk2. Furthermore, mps1 mutants were also sensitive to oxidative stress. Heterozygous mps1 mutant did not undergo morphological transition and showed 5-Fold reduction in colony forming units in response to macrophage. Thus, the vital checkpoint kinase, Mps1 besides cell division also has a role in morphogenesis and oxidative stress tolerance, in this pathogenic fungus.

ARABIDOPSIS HOMOLOG of TRITHORAX1 (ATX1) is required for cell production, patterning, and morphogenesis in root development

PubMed Central

Napsucialy-Mendivil, Selene; Alvarez-Venegas, Raúl; Shishkova, Svetlana; Dubrovsky, Joseph G.

2014-01-01

ARABIDOPSIS HOMOLOG of TRITHORAX1 (ATX1/SDG27), a known regulator of flower development, encodes a H3K4histone methyltransferase that maintains a number of genes in an active state. In this study, the role of ATX1 in root development was evaluated. The loss-of-function mutant atx1-1 was impaired in primary root growth. The data suggest that ATX1 controls root growth by regulating cell cycle duration, cell production, and the transition from cell proliferation in the root apical meristem (RAM) to cell elongation. In atx1-1, the quiescent centre (QC) cells were irregular in shape and more expanded than those of the wild type. This feature, together with the atypical distribution of T-divisions, the presence of oblique divisions, and the abnormal cell patterning in the RAM, suggests a lack of coordination between cell division and cell growth in the mutant. The expression domain of QC-specific markers was expanded both in the primary RAM and in the developing lateral root primordia of atx1-1 plants. These abnormalities were independent of auxin-response gradients. ATX1 was also found to be required for lateral root initiation, morphogenesis, and emergence. The time from lateral root initiation to emergence was significantly extended in the atx1-1 mutant. Overall, these data suggest that ATX1 is involved in the timing of root development, stem cell niche maintenance, and cell patterning during primary and lateral root development. Thus, ATX1 emerges as an important player in root system architecture. PMID:25205583

Arabidopsis genomes uncoupled 5 (GUN5) mutant reveals the involvement of Mg-chelatase H subunit in plastid-to-nucleus signal transduction

PubMed Central

Mochizuki, Nobuyoshi; Brusslan, Judy A.; Larkin, Robert; Nagatani, Akira; Chory, Joanne

2001-01-01

A plastid-derived signal plays an important role in the coordinated expression of both nuclear- and chloroplast-localized genes that encode photosynthesis-related proteins. Arabidopsis GUN (genomes uncoupled) loci have been identified as components of plastid-to-nucleus signal transduction. Unlike wild-type plants, gun mutants have nuclear Lhcb1 expression in the absence of chloroplast development. We observed a synergistic phenotype in some gun double-mutant combinations, suggesting there are at least two independent pathways in plastid-to-nucleus signal transduction. There is a reduction of chlorophyll accumulation in gun4 and gun5 mutant plants, and a gun4gun5 double mutant shows an albino phenotype. We cloned the GUN5 gene, which encodes the ChlH subunit of Mg-chelatase. We also show that gun2 and gun3 are alleles of the known photomorphogenic mutants, hy1 and hy2, which are required for phytochromobilin synthesis from heme. These findings suggest that certain perturbations of the tetrapyrrole biosynthetic pathway generate a signal from chloroplasts that causes transcriptional repression of nuclear genes encoding plastid-localized proteins. The comparison of mutant phenotypes of gun5 and another Mg-chelatase subunit (ChlI) mutant suggests a specific function for ChlH protein in the plastid-signaling pathway. PMID:11172074

Analysis of dofA, a fruA-dependent developmental gene, and its homologue, dofB, in Myxococcus xanthus.

PubMed

Horiuchi, Takayuki; Akiyama, Takuya; Inouye, Sumiko; Komano, Teruya

2002-12-01

The developmentally regulated gene dofA, identified from pulse-labeling experiments by two-dimensional gel electrophoresis, and its homologue, dofB, were cloned and characterized in Myxococcus xanthus. Deletion of dofA and dofB did not affect the vegetative growth and development of M. xanthus. dofA was specifically expressed during development, while dofB expression was observed during vegetative growth and development. The dofA-lacZ fusion was introduced into a fruA mutant and A, B, C, D, and E extracellular signal mutants. The pattern of dofA expression in the C signal mutant was similar to that of the wild-type strain, while dofA expression was not detected in the fruA mutant. These results are consistent with those of the pulse-labeling experiments. dofA expression was reduced in A and E signal mutants, whereas dofA expression was delayed in B and D signal mutants. The patterns of expression of the dofA gene in the fruA mutant and the five signal mutants are strikingly similar to that of the tps gene, which encodes protein S, a major component of the outer surface of the myxospore; this result suggests that the dofA and tps genes are similarly regulated. The involvement of a highly GC-rich inverted repeat sequence (underlined), CGGCCCCCGATTCGTCGGGGGCCG, in developmentally regulated dofA expression is suggested.

Candidate genes for panhypopituitarism identified by gene expression profiling

PubMed Central

Mortensen, Amanda H.; MacDonald, James W.; Ghosh, Debashis

2011-01-01

Mutations in the transcription factors PROP1 and PIT1 (POU1F1) lead to pituitary hormone deficiency and hypopituitarism in mice and humans. The dysmorphology of developing Prop1 mutant pituitaries readily distinguishes them from those of Pit1 mutants and normal mice. This and other features suggest that Prop1 controls the expression of genes besides Pit1 that are important for pituitary cell migration, survival, and differentiation. To identify genes involved in these processes we used microarray analysis of gene expression to compare pituitary RNA from newborn Prop1 and Pit1 mutants and wild-type littermates. Significant differences in gene expression were noted between each mutant and their normal littermates, as well as between Prop1 and Pit1 mutants. Otx2, a gene critical for normal eye and pituitary development in humans and mice, exhibited elevated expression specifically in Prop1 mutant pituitaries. We report the spatial and temporal regulation of Otx2 in normal mice and Prop1 mutants, and the results suggest Otx2 could influence pituitary development by affecting signaling from the ventral diencephalon and regulation of gene expression in Rathke's pouch. The discovery that Otx2 expression is affected by Prop1 deficiency provides support for our hypothesis that identifying molecular differences in mutants will contribute to understanding the molecular mechanisms that control pituitary organogenesis and lead to human pituitary disease. PMID:21828248

Calpains are involved in asexual and sexual development, cell wall integrity and pathogenicity of the rice blast fungus

PubMed Central

Liu, Xiao-Hong; Ning, Guo-Ao; Huang, Lu-Yao; Zhao, Ya-Hui; Dong, Bo; Lu, Jian-Ping; Lin, Fu-Cheng

2016-01-01

Calpains are ubiquitous and well-conserved proteins that belong to the calcium-dependent, non-lysosomal cysteine protease family. In this study, 8 putative calpains were identified using Pfam domain analysis and BlastP searches in M. oryzae. Three single gene deletion mutants (ΔMocapn7, ΔMocapn9 and ΔMocapn14) and two double gene deletion mutants (ΔMocapn4ΔMocapn7 and ΔMocapn9ΔMocapn7) were obtained using the high-throughput gene knockout system. The calpain disruption mutants showed defects in colony characteristics, conidiation, sexual reproduction and cell wall integrity. The mycelia of the ΔMocapn7, ΔMocapn4ΔMocapn7 and ΔMocapn9ΔMocapn7 mutants showed reduced pathogenicity on rice and barley. PMID:27502542

FGF receptors ubiquitylation: dependence on tyrosine kinase activity and role in downregulation.

PubMed

Monsonego-Ornan, E; Adar, R; Rom, E; Yayon, A

2002-09-25

A crucial aspect of ligand-mediated receptor activation and shut-down is receptor internalization and degradation. Here we compared the ubiquitylation of either wild type or a K508A 'kinase-dead' mutant of fibroblast growth factor receptor 3 (FGFR3) with that of its naturally occurring overactive mutants, G380R as in achondroplasia, or K650E involved in thanatophoric dysplasia. Fibroblast growth factor receptors ubiquitylation was found to be directly proportional to their intrinsic tyrosine kinase activity, both of which could be blocked using kinase inhibitors. Despite excessive ubiquitylation, both overactive mutants failed to be efficiently degraded, even when challenged with ligand or overexpression of c-Cbl, a putative E3 ligase. We conclude that phosphorylation is essential for FGFR3 ubiquitylation, but is not sufficient to induce downregulation of its internalization resistant mutants.

The essential requirement of an animal heme peroxidase protein during the wing maturation process in Drosophila.

PubMed

Bailey, Dondra; Basar, Mohammed Abul; Nag, Sanjay; Bondhu, Nivedita; Teng, Shaloei; Duttaroy, Atanu

2017-01-11

Thus far, a handful of genes have been shown to be related to the wing maturation process in insects. A novel heme peroxidase enzyme known as curly suppressor (Cysu)(formerly CG5873), have been characterized in this report because it is involved in wing morphogenesis. Using bioinformatics tools we found that Cysu is remarkably conserved in the genus Drosophila (>95%) as well as in invertebrates (>70%), although its vertebrate orthologs show poor homology. Time-lapse imaging and histochemical analyses have confirmed that the defective wing phenotype of Cysu is not a result of any underlying cellular alterations; instead, its wings fail to expand in mature adults. The precise requirement of Cysu in wings was established by identifying a bona fide mutant of Cysu from the Bloomington Drosophila Stock Centre collection. Its requirement in the wing has also been shown by RNA knockdown of the gene. Subsequent transgenic rescue of the mutant wing phenotype with the wild-type gene confirmed the phenotype resulting from Cysu mutant. With appropriate GAL4 driver like engrailed-GAL4, the Cysu phenotype was compartmentalized, which raises a strong possibility that Cysu is not localized in the extracellular matrix (ECM); hence, Cysu is not engaged in bonding the dorsal and ventral cuticular layers. Finally, shortened lifespan of the Cysu mutant suggests it is functionally essential for other biological processes as well. Cysu, a peroxinectin-like gene, is required during the wing maturation process in Drosophila because as a heme peroxidase, Cysu is capable of utilizing H 2 O 2 , which plays an essential role in post-eclosion wing morphogenesis.

Helicobacter pylori gene silencing in vivo demonstrates urease is essential for chronic infection

PubMed Central

Walton, Senta M.; Liao, Tingting; Stubbs, Keith A.; Marshall, Barry J.; Fulurija, Alma; Benghezal, Mohammed

2017-01-01

Helicobacter pylori infection causes chronic active gastritis that after many years of infection can develop into peptic ulceration or gastric adenocarcinoma. The bacterium is highly adapted to surviving in the gastric environment and a key adaptation is the virulence factor urease. Although widely postulated, the requirement of urease expression for persistent infection has not been elucidated experimentally as conventional urease knockout mutants are incapable of colonization. To overcome this constraint, conditional H. pylori urease mutants were constructed by adapting the tetracycline inducible expression system that enabled changing the urease phenotype of the bacteria during established infection. Through tight regulation we demonstrate that urease expression is not only required for establishing initial colonization but also for maintaining chronic infection. Furthermore, successful isolation of tet-escape mutants from a late infection time point revealed the strong selective pressure on this gastric pathogen to continuously express urease in order to maintain chronic infection. In addition to mutations in the conditional gene expression system, escape mutants were found to harbor changes in other genes including the alternative RNA polymerase sigma factor, fliA, highlighting the genetic plasticity of H. pylori to adapt to a changing niche. The tet-system described here opens up opportunities to studying genes involved in the chronic stage of H. pylori infection to gain insight into bacterial mechanisms promoting immune escape and life-long infection. Furthermore, this genetic tool also allows for a new avenue of inquiry into understanding the importance of various virulence determinants in a changing biological environment when the bacterium is put under duress. PMID:28644872

Helicobacter pylori gene silencing in vivo demonstrates urease is essential for chronic infection.

PubMed

Debowski, Aleksandra W; Walton, Senta M; Chua, Eng-Guan; Tay, Alfred Chin-Yen; Liao, Tingting; Lamichhane, Binit; Himbeck, Robyn; Stubbs, Keith A; Marshall, Barry J; Fulurija, Alma; Benghezal, Mohammed

2017-06-01

Helicobacter pylori infection causes chronic active gastritis that after many years of infection can develop into peptic ulceration or gastric adenocarcinoma. The bacterium is highly adapted to surviving in the gastric environment and a key adaptation is the virulence factor urease. Although widely postulated, the requirement of urease expression for persistent infection has not been elucidated experimentally as conventional urease knockout mutants are incapable of colonization. To overcome this constraint, conditional H. pylori urease mutants were constructed by adapting the tetracycline inducible expression system that enabled changing the urease phenotype of the bacteria during established infection. Through tight regulation we demonstrate that urease expression is not only required for establishing initial colonization but also for maintaining chronic infection. Furthermore, successful isolation of tet-escape mutants from a late infection time point revealed the strong selective pressure on this gastric pathogen to continuously express urease in order to maintain chronic infection. In addition to mutations in the conditional gene expression system, escape mutants were found to harbor changes in other genes including the alternative RNA polymerase sigma factor, fliA, highlighting the genetic plasticity of H. pylori to adapt to a changing niche. The tet-system described here opens up opportunities to studying genes involved in the chronic stage of H. pylori infection to gain insight into bacterial mechanisms promoting immune escape and life-long infection. Furthermore, this genetic tool also allows for a new avenue of inquiry into understanding the importance of various virulence determinants in a changing biological environment when the bacterium is put under duress.

Using co-expression analysis and stress-based screens to uncover Arabidopsis peroxisomal proteins involved in drought response

DOE PAGES

Li, Jiying; Hu, Jianping; Bassham, Diane

2015-09-14

Peroxisomes are essential organelles that house a wide array of metabolic reactions important for plant growth and development. However, our knowledge regarding the role of peroxisomal proteins in various biological processes, including plant stress response, is still incomplete. Recent proteomic studies of plant peroxisomes significantly increased the number of known peroxisomal proteins and greatly facilitated the study of peroxisomes at the systems level. The objectives of this study were to determine whether genes that encode peroxisomal proteins with related functions are co-expressed in Arabidopsis and identify peroxisomal proteins involved in stress response using in silico analysis and mutant screens. Usingmore » microarray data from online databases, we performed hierarchical clustering analysis to generate a comprehensive view of transcript level changes for Arabidopsis peroxisomal genes during development and under abiotic and biotic stress conditions. Many genes involved in the same metabolic pathways exhibited co-expression, some genes known to be involved in stress response are regulated by the corresponding stress conditions, and function of some peroxisomal proteins could be predicted based on their coexpression pattern. Since drought caused expression changes to the highest number of genes that encode peroxisomal proteins, we subjected a subset of Arabidopsis peroxisomal mutants to a drought stress assay. Mutants of the LON2 protease and the photorespiratory enzyme hydroxypyruvate reductase 1 (HPR1) showed enhanced susceptibility to drought, suggesting the involvement of peroxisomal quality control and photorespiration in drought resistance. Lastly, our study provided a global view of how genes that encode peroxisomal proteins respond to developmental and environmental cues and began to reveal additional peroxisomal proteins involved in stress response, thus opening up new avenues to investigate the role of peroxisomes in plant adaptation to environmental stresses.« less

AtRH57, a DEAD-box RNA helicase, is involved in feedback inhibition of glucose-mediated abscisic acid accumulation during seedling development and additively affects pre-ribosomal RNA processing with high glucose.

PubMed

Hsu, Yi-Feng; Chen, Yun-Chu; Hsiao, Yu-Chun; Wang, Bing-Jyun; Lin, Shih-Yun; Cheng, Wan-Hsing; Jauh, Guang-Yuh; Harada, John J; Wang, Co-Shine

2014-01-01

The Arabidopsis thaliana T-DNA insertion mutant rh57-1 exhibited hypersensitivity to glucose (Glc) and abscisic acid (ABA). The other two rh57 mutants also showed Glc hypersensitivity similar to rh57-1, strongly suggesting that the Glc-hypersensitive feature of these mutants results from mutation of AtRH57. rh57-1 and rh57-3 displayed severely impaired seedling growth when grown in Glc concentrations higher than 3%. The gene, AtRH57 (At3g09720), was expressed in all Arabidopsis organs and its transcript was significantly induced by ABA, high Glc and salt. The new AtRH57 belongs to class II DEAD-box RNA helicase gene family. Transient expression of AtRH57-EGFP (enhanced green fluorescent protein) in onion cells indicated that AtRH57 was localized in the nucleus and nucleolus. Purified AtRH57-His protein was shown to unwind double-stranded RNA independent of ATP in vitro. The ABA biosynthesis inhibitor fluridone profoundly redeemed seedling growth arrest mediated by sugar. rh57-1 showed increased ABA levels when exposed to high Glc. Quantitative real time polymerase chain reaction analysis showed that AtRH57 acts in a signaling network downstream of HXK1. A feedback inhibition of ABA accumulation mediated by AtRH57 exists within the sugar-mediated ABA signaling. AtRH57 mutation and high Glc conditions additively caused a severe defect in small ribosomal subunit formation. The accumulation of abnormal pre-rRNA and resistance to protein synthesis-related antibiotics were observed in rh57 mutants and in the wild-type Col-0 under high Glc conditions. These results suggested that AtRH57 plays an important role in rRNA biogenesis in Arabidopsis and participates in response to sugar involving Glc- and ABA signaling during germination and seedling growth. © 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.

Combinatorial Regulation of Meiotic Holliday Junction Resolution in C. elegans by HIM-6 (BLM) Helicase, SLX-4, and the SLX-1, MUS-81 and XPF-1 Nucleases

PubMed Central

Sonneville, Remi; Jagut, Marlène; Woglar, Alexander; Blow, Julian; Jantsch, Verena; Gartner, Anton

2013-01-01

Holliday junctions (HJs) are cruciform DNA structures that are created during recombination events. It is a matter of considerable importance to determine the resolvase(s) that promote resolution of these structures. We previously reported that C. elegans GEN-1 is a symmetrically cleaving HJ resolving enzyme required for recombinational repair, but we could not find an overt role in meiotic recombination. Here we identify C. elegans proteins involved in resolving meiotic HJs. We found no evidence for a redundant meiotic function of GEN-1. In contrast, we discovered two redundant HJ resolution pathways likely coordinated by the SLX-4 scaffold protein and also involving the HIM-6/BLM helicase. SLX-4 associates with the SLX-1, MUS-81 and XPF-1 nucleases and has been implicated in meiotic recombination in C. elegans. We found that C. elegans [mus-81; xpf-1], [slx-1; xpf-1], [mus-81; him-6] and [slx-1; him-6] double mutants showed a similar reduction in survival rates as slx-4. Analysis of meiotic diakinesis chromosomes revealed a distinct phenotype in these double mutants. Instead of wild-type bivalent chromosomes, pairs of “univalents” linked by chromatin bridges occur. These linkages depend on the conserved meiosis-specific transesterase SPO-11 and can be restored by ionizing radiation, suggesting that they represent unresolved meiotic HJs. This suggests the existence of two major resolvase activities, one provided by XPF-1 and HIM-6, the other by SLX-1 and MUS-81. In all double mutants crossover (CO) recombination is reduced but not abolished, indicative of further redundancy in meiotic HJ resolution. Real time imaging revealed extensive chromatin bridges during the first meiotic division that appear to be eventually resolved in meiosis II, suggesting back-up resolution activities acting at or after anaphase I. We also show that in HJ resolution mutants, the restructuring of chromosome arms distal and proximal to the CO still occurs, suggesting that CO initiation but not resolution is likely to be required for this process. PMID:23901331

Combinatorial regulation of meiotic holliday junction resolution in C. elegans by HIM-6 (BLM) helicase, SLX-4, and the SLX-1, MUS-81 and XPF-1 nucleases.

PubMed

Agostinho, Ana; Meier, Bettina; Sonneville, Remi; Jagut, Marlène; Woglar, Alexander; Blow, Julian; Jantsch, Verena; Gartner, Anton

2013-01-01

Holliday junctions (HJs) are cruciform DNA structures that are created during recombination events. It is a matter of considerable importance to determine the resolvase(s) that promote resolution of these structures. We previously reported that C. elegans GEN-1 is a symmetrically cleaving HJ resolving enzyme required for recombinational repair, but we could not find an overt role in meiotic recombination. Here we identify C. elegans proteins involved in resolving meiotic HJs. We found no evidence for a redundant meiotic function of GEN-1. In contrast, we discovered two redundant HJ resolution pathways likely coordinated by the SLX-4 scaffold protein and also involving the HIM-6/BLM helicase. SLX-4 associates with the SLX-1, MUS-81 and XPF-1 nucleases and has been implicated in meiotic recombination in C. elegans. We found that C. elegans [mus-81; xpf-1], [slx-1; xpf-1], [mus-81; him-6] and [slx-1; him-6] double mutants showed a similar reduction in survival rates as slx-4. Analysis of meiotic diakinesis chromosomes revealed a distinct phenotype in these double mutants. Instead of wild-type bivalent chromosomes, pairs of "univalents" linked by chromatin bridges occur. These linkages depend on the conserved meiosis-specific transesterase SPO-11 and can be restored by ionizing radiation, suggesting that they represent unresolved meiotic HJs. This suggests the existence of two major resolvase activities, one provided by XPF-1 and HIM-6, the other by SLX-1 and MUS-81. In all double mutants crossover (CO) recombination is reduced but not abolished, indicative of further redundancy in meiotic HJ resolution. Real time imaging revealed extensive chromatin bridges during the first meiotic division that appear to be eventually resolved in meiosis II, suggesting back-up resolution activities acting at or after anaphase I. We also show that in HJ resolution mutants, the restructuring of chromosome arms distal and proximal to the CO still occurs, suggesting that CO initiation but not resolution is likely to be required for this process.

Effect of the deletion of the C region on the structure and hydration of insulin-like growth factor 1: a molecular dynamics investigation

NASA Astrophysics Data System (ADS)

Degreve, Leo; Silva, Luciene B.

The structure and hydration of insulin-like growth factor 1 and an inactive mutant lacking the C region have been investigated in aqueous solution by molecular dynamics simulation. The overall structures of the two polypeptide resemble those determined by NMR spectroscopy. The deletion of the C region in the wild polypeptide introduces structural stability in the mutant, leading to a better definition of the secondary structure elements. A detailed hydration analysis was performed using the radial distribution functions and energy distributions. The backbone of the mutant is in general more solvent accessible than the wild polypeptide backbone. The structural rearrangements induced in the mutant led to changes in the solvent exposition of Tyr24 and Tyr60, which are residues important for ligand-receptor complex formation. Tyr24 exhibited a similar degree of solvent exposition in both IGF-1 and in the mutant; however, its hydroxyl group in the wild polypeptide is better solvated than in the mutant. Tyr60 was found to be solvent exposed in the wild protein, while in the mutant the involvement of its hydroxyl group in intramolecular hydrogen bonds led to it being buried away from the solvent.

Arabidopsis ILITHYIA protein is necessary for proper chloroplast biogenesis and root development independent of eIF2α phosphorylation.

PubMed

Faus, I; Niñoles, R; Kesari, V; Llabata, P; Tam, E; Nebauer, S G; Santiago, J; Hauser, M T; Gadea, J

One of the main mechanisms blocking translation after stress situations is mediated by phosphorylation of the α-subunit of the eukaryotic initiation factor 2 (eIF2), performed in Arabidopsis by the protein kinase GCN2 which interacts and is activated by ILITHYIA(ILA). ILA is involved in plant immunity and its mutant lines present phenotypes not shared by the gcn2 mutants. The functional link between these two genes remains elusive in plants. In this study, we show that, although both ILA and GCN2 genes are necessary to mediate eIF2α phosphorylation upon treatments with the aromatic amino acid biosynthesis inhibitor glyphosate, their mutants develop distinct root and chloroplast phenotypes. Electron microscopy experiments reveal that ila mutants, but not gcn2, are affected in chloroplast biogenesis, explaining the macroscopic phenotype previously observed for these mutants. ila3 mutants present a complex transcriptional reprogramming affecting defense responses, photosynthesis and protein folding, among others. Double mutant analyses suggest that ILA has a distinct function which is independent of GCN2 and eIF2α phosphorylation. These results suggest that these two genes may have common but also distinct functions in Arabidopsis. Copyright © 2018 Elsevier GmbH. All rights reserved.

Differential effects of lesion mimic mutants in barley on disease development by facultative pathogens

PubMed Central

McGrann, Graham R. D.; Steed, , Andrew; Burt, Christopher; Nicholson, Paul; Brown, James K. M.

2015-01-01

Lesion mimic mutants display spontaneous necrotic spots and chlorotic leaves as a result of mis-regulated cell death programmes. Typically these mutants have increased resistance to biotrophic pathogens but their response to facultative fungi that cause necrotrophic diseases is less well studied. The effect of altered cell death regulation on the development of disease caused by Ramularia collo-cygni, Fusarium culmorum and Oculimacula yallundae was explored using a collection of barley necrotic (nec) lesion mimic mutants. nec8 mutants displayed lower levels of all three diseases compared to nec9 mutants, which had increased R. collo-cygni but decreased F. culmorum disease symptoms. nec1 mutants reduced disease development caused by both R. collo-cygni and F. culmorum. The severity of the nec1-induced lesion mimic phenotype and F. culmorum symptom development was reduced by mutation of the negative cell death regulator MLO. The significant reduction in R. collo-cygni symptoms caused by nec1 was completely abolished in the presence of the mlo-5 allele and both symptoms and fungal biomass were greater than in the wild-type. These results indicate that physiological pathways involved in regulation of cell death interact with one another in their effects on different fungal pathogens. PMID:25873675

Identification of genes involved in low aminoglycoside-induced SOS response in Vibrio cholerae: a role for transcription stalling and Mfd helicase

PubMed Central

Baharoglu, Zeynep; Babosan, Anamaria; Mazel, Didier

2014-01-01

Sub-inhibitory concentrations (sub-MIC) of antibiotics play a very important role in selection and development of resistances. Unlike Escherichia coli, Vibrio cholerae induces its SOS response in presence of sub-MIC aminoglycosides. A role for oxidized guanine residues was observed, but the mechanisms of this induction remained unclear. To select for V. cholerae mutants that do not induce low aminoglycoside-mediated SOS induction, we developed a genetic screen that renders induction of SOS lethal. We identified genes involved in this pathway using two strategies, inactivation by transposition and gene overexpression. Interestingly, we obtained mutants inactivated for the expression of proteins known to destabilize the RNA polymerase complex. Reconstruction of the corresponding mutants confirmed their specific involvement in induction of SOS by low aminoglycoside concentrations. We propose that DNA lesions formed on aminoglycoside treatment are repaired through the formation of single-stranded DNA intermediates, inducing SOS. Inactivation of functions that dislodge RNA polymerase leads to prolonged stalling on these lesions, which hampers SOS induction and repair and reduces viability under antibiotic stress. The importance of these mechanisms is illustrated by a reduction of aminoglycoside sub-MIC. Our results point to a central role for transcription blocking at DNA lesions in SOS induction, so far underestimated. PMID:24319148

Identification of genes involved in low aminoglycoside-induced SOS response in Vibrio cholerae: a role for transcription stalling and Mfd helicase.

PubMed

Baharoglu, Zeynep; Babosan, Anamaria; Mazel, Didier

2014-02-01

Sub-inhibitory concentrations (sub-MIC) of antibiotics play a very important role in selection and development of resistances. Unlike Escherichia coli, Vibrio cholerae induces its SOS response in presence of sub-MIC aminoglycosides. A role for oxidized guanine residues was observed, but the mechanisms of this induction remained unclear. To select for V. cholerae mutants that do not induce low aminoglycoside-mediated SOS induction, we developed a genetic screen that renders induction of SOS lethal. We identified genes involved in this pathway using two strategies, inactivation by transposition and gene overexpression. Interestingly, we obtained mutants inactivated for the expression of proteins known to destabilize the RNA polymerase complex. Reconstruction of the corresponding mutants confirmed their specific involvement in induction of SOS by low aminoglycoside concentrations. We propose that DNA lesions formed on aminoglycoside treatment are repaired through the formation of single-stranded DNA intermediates, inducing SOS. Inactivation of functions that dislodge RNA polymerase leads to prolonged stalling on these lesions, which hampers SOS induction and repair and reduces viability under antibiotic stress. The importance of these mechanisms is illustrated by a reduction of aminoglycoside sub-MIC. Our results point to a central role for transcription blocking at DNA lesions in SOS induction, so far underestimated.

Effect of hypergravity on lignin formation and expression of lignin-related genes in inflorescence stems of an ethylene-insensitive Arabidopsis mutant ein3-1

NASA Astrophysics Data System (ADS)

Karahara, Ichirou; Kobayashi, Mai; Tamaoki, Daisuke; Kamisaka, Seiichiro

Our previous studies have shown that hypergravity inhibits growth and promotes lignin forma-tion in inflorescence stems of Arabidopsis thaliana by up-regulation of genes involved in lignin biosynthesis (Tamaoki et al. 2006, 2009). In the present study, we have examined whether ethylene is involved in these responses using an ethylene-insensitive Arabidopsis mutant ein3-1. Our results revealed that hypergravity treatment at 300 G for 24 h significantly inhibited growth of inflorescence stems, promoted both deposition of acetyl bromide extractable lignin and gene expression involved in lignin formation in inflorescence stems of wild type plants. Growth inhibition of inflorescence stems was also observed in ein3-1. However, the effects of hypergravity on the promotion of the deposition of acetyl bromide lignin and the expression of genes involved in lignin formation were not observed in ein3-1, indicating that ethylene sig-naling is involved in the up-regulation of the expression of lignin-related genes as well as the promotion of deposition of lignin by hypergravity in Arabidopsis inflorescence stems.

Transcript profiling of crown rootless1 mutant stem base reveals new elements associated with crown root development in rice

PubMed Central

2011-01-01

Background In rice, the major part of the post-embryonic root system is made of stem-derived roots named crown roots (CR). Among the few characterized rice mutants affected in root development, crown rootless1 mutant is unable to initiate crown root primordia. CROWN ROOTLESS1 (CRL1) is induced by auxin and encodes an AS2/LOB-domain transcription factor that acts upstream of the gene regulatory network controlling CR development. Results To identify genes involved in CR development, we compared global gene expression profile in stem bases of crl1 mutant and wild-type (WT) plants. Our analysis revealed that 250 and 236 genes are down- and up-regulated respectively in the crl1 mutant. Auxin induces CRL1 expression and consequently it is expected that auxin also alters the expression of genes that are early regulated by CRL1. To identify genes under the early control of CRL1, we monitored the expression kinetics of a selected subset of genes, mainly chosen among those exhibiting differential expression, in crl1 and WT following exogenous auxin treatment. This analysis revealed that most of these genes, mainly related to hormone, water and nutrient, development and homeostasis, were likely not regulated directly by CRL1. We hypothesized that the differential expression for these genes observed in the crl1 mutant is likely a consequence of the absence of CR formation. Otherwise, three CRL1-dependent auxin-responsive genes: FSM (FLATENNED SHOOT MERISTEM)/FAS1 (FASCIATA1), GTE4 (GENERAL TRANSCRIPTION FACTOR GROUP E4) and MAP (MICROTUBULE-ASSOCIATED PROTEIN) were identified. FSM/FAS1 and GTE4 are known in rice and Arabidopsis to be involved in the maintenance of root meristem through chromatin remodelling and cell cycle regulation respectively. Conclusion Our data showed that the differential regulation of most genes in crl1 versus WT may be an indirect consequence of CRL1 inactivation resulting from the absence of CR in the crl1 mutant. Nevertheless some genes, FAS1/FSM, GTE4 and MAP, require CRL1 to be induced by auxin suggesting that they are likely directly regulated by CRL1. These genes have a function related to polarized cell growth, cell cycle regulation or chromatin remodelling. This suggests that these genes are controlled by CRL1 and involved in CR initiation in rice. PMID:21806801

Transcriptional mutagenesis: causes and involvement in tumor development

PubMed Central

Brégeon, Damien; Doetsch, Paul W.

2013-01-01

The majority of normal cells in a human do not multiply continuously but are quiescent and devote most of their energy to gene transcription. When DNA damages in the transcribed strand of an active gene are bypassed by an RNA polymerase, they can miscode at the damaged site and produce mutant transcripts. This process known as transcriptional mutagenesis can lead to the production of mutant proteins that could be important in tumor development. PMID:21346784

A Direct Screening Procedure for Gravitropism Mutants in Arabidopsis thaliana (L.) Heynh. 1

PubMed Central

Bullen, Bertha L.; Best, Thérèse R.; Gregg, Mary M.; Barsel, Sara-Ellen; Poff, Kenneth L.

1990-01-01

In order to isolate gravitropism mutants of Arabidopsis thaliana (L.) Heynh. var Estland for the genetic dissection of the gravitropism pathway, a direct screening procedure has been developed in which mutants are selected on the basis of their gravitropic response. Variability in hypocotyl curvature was dependent on the germination time of each seed stock, resulting in the incorrect identification of several lines as gravitropism mutants when a standard protocol for the potentiation of germination was used. When the protocol was adjusted to allow for differences in germination time, these lines were eliminated from the collection. Out of the 60,000 M2 seedlings screened, 0.3 to 0.4% exhibited altered gravitropism. In approximately 40% of these mutant lines, only gravitropism by the root or the hypocotyl was altered, while the response of the other organ was unaffected. These data support the hypothesis that root and hypocotyl gravitropism are genetically separable. PMID:11537704

A direct screening procedure for gravitropism mutants in Arabidopsis thaliana (L.) Heynh

NASA Technical Reports Server (NTRS)

Bullen, B. L.; Best, T. R.; Gregg, M. M.; Poff, K. L.; Barsel, S-E (Principal Investigator)

1990-01-01

In order to isolate gravitropism mutants of Arabidopsis thaliana (L.) Heynh. var Estland for the genetic dissection of the gravitropism pathway, a direct screening procedure has been developed in which mutants are selected on the basis of their gravitropic response. Variability in hypocotyl curvature was dependent on the germination time of each seed stock, resulting in the incorrect identification of several lines as gravitropism mutants when a standard protocol for the potentiation of germination was used. When the protocol was adjusted to allow for differences in germination time, these lines were eliminated from the collection. Out of the 60,000 M2 seedlings screened, 0.3 to 0.4% exhibited altered gravitropism. In approximately 40% of these mutant lines, only gravitropism by the root or the hypocotyl was altered, while the response of the other organ was unaffected. These data support the hypothesis that root and hypocotyl gravitropism are genetically separable.

Working-for-Food Behaviors: A Preclinical Study in Prader-Willi Mutant Mice.

PubMed

Lassi, Glenda; Maggi, Silvia; Balzani, Edoardo; Cosentini, Ilaria; Garcia-Garcia, Celina; Tucci, Valter

2016-11-01

Abnormal feeding behavior is one of the main symptoms of Prader-Willi syndrome (PWS). By studying a PWS mouse mutant line, which carries a paternally inherited deletion of the small nucleolar RNA 116 (Snord116), we observed significant changes in working-for-food behavioral responses at various timescales. In particular, we report that PWS mutant mice show a significant delay compared to wild-type littermate controls in responding to both hour-scale and seconds-to-minutes-scale time intervals. This timing shift in mutant mice is associated with better performance in the working-for-food task, and results in better decision making in these mutant mice. The results of our study reveal a novel aspect of the organization of feeding behavior, and advance the understanding of the interplay between the metabolic functions and cognitive mechanisms of PWS. Copyright © 2016 by the Genetics Society of America.

A link among DNA replication, recombination, and gene expression revealed by genetic and genomic analysis of TEBICHI gene of Arabidopsis thaliana.

PubMed

Inagaki, Soichi; Nakamura, Kenzo; Morikami, Atsushi

2009-08-01

Spatio-temporal regulation of gene expression during development depends on many factors. Mutations in Arabidopsis thaliana TEBICHI (TEB) gene encoding putative helicase and DNA polymerase domains-containing protein result in defects in meristem maintenance and correct organ formation, as well as constitutive DNA damage response and a defect in cell cycle progression; but the molecular link between these phenotypes of teb mutants is unknown. Here, we show that mutations in the DNA replication checkpoint pathway gene, ATR, but not in ATM gene, enhance developmental phenotypes of teb mutants, although atr suppresses cell cycle defect of teb mutants. Developmental phenotypes of teb mutants are also enhanced by mutations in RAD51D and XRCC2 gene, which are involved in homologous recombination. teb and teb atr double mutants exhibit defects in adaxial-abaxial polarity of leaves, which is caused in part by the upregulation of ETTIN (ETT)/AUXIN RESPONSIVE FACTOR 3 (ARF3) and ARF4 genes. The Helitron transposon in the upstream of ETT/ARF3 gene is likely to be involved in the upregulation of ETT/ARF3 in teb. Microarray analysis indicated that teb and teb atr causes preferential upregulation of genes nearby the Helitron transposons. Furthermore, interestingly, duplicated genes, especially tandemly arrayed homologous genes, are highly upregulated in teb or teb atr. We conclude that TEB is required for normal progression of DNA replication and for correct expression of genes during development. Interplay between these two functions and possible mechanism leading to altered expression of specific genes will be discussed.

Methionine biosynthesis is essential for infection in the rice blast fungus Magnaporthe oryzae.

PubMed

Saint-Macary, Marie Emmanuelle; Barbisan, Crystel; Gagey, Marie Josèphe; Frelin, Océane; Beffa, Roland; Lebrun, Marc Henri; Droux, Michel

2015-01-01

Methionine is a sulfur amino acid standing at the crossroads of several biosynthetic pathways. In fungi, the last step of methionine biosynthesis is catalyzed by a cobalamine-independent methionine synthase (Met6, EC 2.1.1.14). In the present work, we studied the role of Met6 in the infection process of the rice blast fungus, Magnaporthe oryzae. To this end MET6 null mutants were obtained by targeted gene replacement. On minimum medium, MET6 null mutants were auxotrophic for methionine. Even when grown in presence of excess methionine, these mutants displayed developmental defects, such as reduced mycelium pigmentation, aerial hypha formation and sporulation. They also displayed characteristic metabolic signatures such as increased levels of cysteine, cystathionine, homocysteine, S-adenosylmethionine, S-adenosylhomocysteine while methionine and glutathione levels remained unchanged. These metabolic perturbations were associated with the over-expression of MgCBS1 involved in the reversed transsulfuration pathway that metabolizes homocysteine into cysteine and MgSAM1 and MgSAHH1 involved in the methyl cycle. This suggests a physiological adaptation of M. oryzae to metabolic defects induced by the loss of Met6, in particular an increase in homocysteine levels. Pathogenicity assays showed that MET6 null mutants were non-pathogenic on both barley and rice leaves. These mutants were defective in appressorium-mediated penetration and invasive infectious growth. These pathogenicity defects were rescued by addition of exogenous methionine and S-methylmethionine. These results show that M. oryzae cannot assimilate sufficient methionine from plant tissues and must synthesize this amino acid de novo to fulfill its sulfur amino acid requirement during infection.

GOLDEN2-LIKE transcription factors coordinate the tolerance to Cucumber mosaic virus in Arabidopsis

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

Han, Xue-Ying; Li, Peng-Xu; Zou, Li-Juan

Arabidopsis thaliana GOLDEN2-LIKE (GLKs) transcription factors play important roles in regulation of photosynthesis-associated nuclear genes, as well as participate in chloroplast development. However, the involvement of GLKs in plants resistance to virus remains largely unknown. Here, the relationship between GLKs and Cucumber mosaic virus (CMV) stress response was investigated. Our results showed that the Arabidopsis glk1glk2 double-mutant was more susceptible to CMV infection and suffered more serious damages (such as higher oxidative damages, more compromised in PSII photochemistry and more reactive oxygen species accumulation) when compared with the wild-type plants. Interestingly, there was little difference between single mutant (glk1 ormore » glk2) and wild-type plants in response to CMV infection, suggesting GLK1 and GLK2 might function redundant in virus resistance in Arabidopsis. Furthermore, the induction of antioxidant system and defense-associated genes expression in the double mutant were inhibited when compared with single mutant or wild-type plants after CMV infection. Further evidences showed that salicylic acid (SA) and jasmonic acid (JA) might be involved in GLKs-mediated virus resistance, as SA or JA level and synthesis-related genes transcription were impaired in glk1glk2 mutant. Taken together, our results indicated that GLKs played a positively role in virus resistance in Arabidopsis. - Highlights: • GLKs play a positive role in CMV resistance in Arabidopsis. • Defective of GLKs suffered more ROS accumulation. • Arabidopsis lacking GLKs have damaged photosynthesis. • Arabidopsis lacking GLKs show low SA and JA accumulation.« less

Stamina pistilloida, the Pea Ortholog of Fim and UFO, Is Required for Normal Development of Flowers, Inflorescences, and Leaves

PubMed Central

Taylor, Scott; Hofer, Julie; Murfet, Ian

2001-01-01

Isolation and characterization of two severe alleles at the Stamina pistilloida (Stp) locus reveals that Stp is involved in a wide range of developmental processes in the garden pea. The most severe allele, stp-4, results in flowers consisting almost entirely of sepals and carpels. Production of ectopic secondary flowers in stp-4 plants suggests that Stp is involved in specifying floral meristem identity in pea. The stp mutations also reduce the complexity of the compound pea leaf, and primary inflorescences often terminate prematurely in an aberrant sepaloid flower. In addition, stp mutants were shorter than their wild-type siblings due to a reduction in cell number in their internodes. Fewer cells were also found in the epidermis of the leaf rachis of stp mutants. Examination of the effects of stp-4 in double mutant combinations with af, tl, det, and veg2-2—mutations known to influence leaf, inflorescence, and flower development in pea—suggests that Stp function is independent of these genes. A synergistic interaction between weak mutant alleles at Stp and Uni indicated that these two genes act together, possibly to regulate primordial growth. Molecular analysis revealed that Stp is the pea homolog of the Antirrhinum gene Fimbriata (Fim) and of UNUSUAL FLORAL ORGANS (UFO) from Arabidopsis. Differences between Fim/UFO and Stp mutant phenotypes and expression patterns suggest that expansion of Stp activity into the leaf was an important step during evolution of the compound leaf in the garden pea. PMID:11158527

Hydrogen is a preferred intermediate in the energy-conserving electron transport chain of Methanosarcina barkeri.

PubMed

Kulkarni, Gargi; Kridelbaugh, Donna M; Guss, Adam M; Metcalf, William W

2009-09-15

Methanogens use an unusual energy-conserving electron transport chain that involves reduction of a limited number of electron acceptors to methane gas. Previous biochemical studies suggested that the proton-pumping F(420)H(2) dehydrogenase (Fpo) plays a crucial role in this process during growth on methanol. However, Methanosarcina barkeri Delta fpo mutants constructed in this study display no measurable phenotype on this substrate, indicating that Fpo plays a minor role, if any. In contrast, Delta frh mutants lacking the cytoplasmic F(420)-reducing hydrogenase (Frh) are severely affected in their ability to grow and make methane from methanol, and double Delta fpo/Delta frh mutants are completely unable to use this substrate. These data suggest that the preferred electron transport chain involves production of hydrogen gas in the cytoplasm, which then diffuses out of the cell, where it is reoxidized with transfer of electrons into the energy-conserving electron transport chain. This hydrogen-cycling metabolism leads directly to production of a proton motive force that can be used by the cell for ATP synthesis. Nevertheless, M. barkeri does have the flexibility to use the Fpo-dependent electron transport chain when needed, as shown by the poor growth of the Delta frh mutant. Our data suggest that the rapid enzymatic turnover of hydrogenases may allow a competitive advantage via faster growth rates in this freshwater organism. The mutant analysis also confirms the proposed role of Frh in growth on hydrogen/carbon dioxide and suggests that either Frh or Fpo is needed for aceticlastic growth of M. barkeri.

Characterization of the hyperrecombination phenotype of the pol3-t mutation of Saccharomyces cerevisiae.

PubMed

Galli, Alvaro; Cervelli, Tiziana; Schiestl, Robert H

2003-05-01

The DNA polymerase delta (Pol3p/Cdc2p) allele pol3-t of Saccharomyces cerevisiae has previously been shown to increase the frequency of deletions between short repeats (several base pairs), between homologous DNA sequences separated by long inverted repeats, and between distant short repeats, increasing the frequency of genomic deletions. We found that the pol3-t mutation increased intrachromosomal recombination events between direct DNA repeats up to 36-fold and interchromosomal recombination 14-fold. The hyperrecombination phenotype of pol3-t was partially dependent on the Rad52p function but much more so on Rad1p. However, in the double-mutant rad1 Delta rad52 Delta, the pol3-t mutation still increased spontaneous intrachromosomal recombination frequencies, suggesting that a Rad1p Rad52p-independent single-strand annealing pathway is involved. UV and gamma-rays were less potent inducers of recombination in the pol3-t mutant, indicating that Pol3p is partly involved in DNA-damage-induced recombination. In contrast, while UV- and gamma-ray-induced intrachromosomal recombination was almost completely abolished in the rad52 or the rad1 rad52 mutant, there was still good induction in those mutants in the pol3-t background, indicating channeling of lesions into the above-mentioned Rad1p Rad52p-independent pathway. Finally, a heterozygous pol3-t/POL3 mutant also showed an increased frequency of deletions and MMS sensitivity at the restrictive temperature, indicating that even a heterozygous polymerase delta mutation might increase the frequency of genetic instability.

Deficiency of Arabidopsis thaliana frataxin alters activity of mitochondrial Fe-S proteins and induces oxidative stress.

PubMed

Busi, Maria V; Maliandi, María V; Valdez, Hugo; Clemente, Marina; Zabaleta, Eduardo J; Araya, Alejandro; Gomez-Casati, Diego F

2006-12-01

Frataxin, a protein crucial for the biogenesis of mitochondria in different organisms, was recently identified in Arabidopsis thaliana. To investigate the role of frataxin in higher plants, we analyze two knock-out and one knock-down T-DNA insertion mutants. The knock-out mutants present an embryo-lethal phenotype, indicating an essential role for frataxin. The knock-down mutant has reduced frataxin mRNA and protein levels. This mutant also presents retarded growth, reduced fresh weight of fruits and reduced number of seeds per fruit. Surprisingly, transcription of aconitase and the Fe-S subunit of succinate dehydrogenase (SDH2-1) are increased in mutant plants; however, the activity of these proteins is reduced, indicating a role for frataxin in Fe-S cluster assembly or insertion of Fe-S clusters into proteins. Mutant plants also have increased CO(2) assimilation rates, exhibit increased formation of reactive oxygen species (ROS) and have increased levels of transcripts for proteins known to be involved in the ROS stress responses. These results indicate that frataxin is an essential protein in plants, required for full activity of mitochondrial Fe-S proteins and playing a protective role against oxidative damage.

Assembly of the mitochondrial membrane system: mutations in the pho2 locus of the mitochondrial genome of Saccharomyces cerevisiae.

PubMed

Coruzzi, G; Trembath, M K; Tzagoloff, A

1978-12-01

Two mutants of Saccharomyces cerevisiae which show a loss of mitochondrial rutamycin-sensitive ATPase activity are described. Although phenotypically similar to mutants of the mitochondrial locus pho1 [F. Foury and A. Tzagoloff (1976) Eur. J. Biochem. 68, 113-119], these mutants define a second ATPase locus on the mitochondrial DNA (designated pho2), which is genetically unlinked to pho1. Analysis of recombination in crosses involving multiple antibiotic resistance markers indicates that the locus is in the segment of the genome between ery1 and oli2, very close to oli1. In fact it is proposed that the oli1 and pho2 mutations are in the same gene. Supporting evidence for this proposal includes: 1. The analysis of marker retention in petite mutants shows that the oli1 and pho2 loci were either retained or lost together in all cases. 2. Recombination frequencies of 0.05% or less are observed in crosses between the oli1 and pho2 loci. 3. When rho+ revertants are isolated from the pho2 mutants they frequently are oligomycin resistant. 4. pho2 mutants have an altered subunit 9 of the ATPase complex.

Genetic analysis of the roles of agaA, agaI, and agaS genes in the N-acetyl-D-galactosamine and D-galactosamine catabolic pathways in Escherichia coli strains O157:H7 and C

PubMed Central

2013-01-01

Background The catabolic pathways of N-acetyl-D-galactosamine (Aga) and D-galactosamine (Gam) in E. coli were proposed from bioinformatic analysis of the aga/gam regulon in E. coli K-12 and later from studies using E. coli C. Of the thirteen genes in this cluster, the roles of agaA, agaI, and agaS predicted to code for Aga-6-P-deacetylase, Gam-6-P deaminase/isomerase, and ketose-aldolase isomerase, respectively, have not been experimentally tested. Here we study their roles in Aga and Gam utilization in E. coli O157:H7 and in E. coli C. Results Knockout mutants in agaA, agaI, and agaS were constructed to test their roles in Aga and Gam utilization. Knockout mutants in the N-acetylglucosamine (GlcNAc) pathway genes nagA and nagB coding for GlcNAc-6-P deacetylase and glucosamine-6-P deaminase/isomerase, respectively, and double knockout mutants ΔagaA ΔnagA and ∆agaI ∆nagB were also constructed to investigate if there is any interplay of these enzymes between the Aga/Gam and the GlcNAc pathways. It is shown that Aga utilization was unaffected in ΔagaA mutants but ΔagaA ΔnagA mutants were blocked in Aga and GlcNAc utilization. E. coli C ΔnagA could not grow on GlcNAc but could grow when the aga/gam regulon was constitutively expressed. Complementation of ΔagaA ΔnagA mutants with either agaA or nagA resulted in growth on both Aga and GlcNAc. It was also found that ΔagaI, ΔnagB, and ∆agaI ΔnagB mutants were unaffected in utilization of Aga and Gam. Importantly, ΔagaS mutants were blocked in Aga and Gam utilization. Expression analysis of relevant genes in these strains with different genetic backgrounds by real time RT-PCR supported these observations. Conclusions Aga utilization was not affected in ΔagaA mutants because nagA was expressed and substituted for agaA. Complementation of ΔagaA ΔnagA mutants with either agaA or nagA also showed that both agaA and nagA can substitute for each other. The ∆agaI, ∆nagB, and ∆agaI ∆nagB mutants were not affected in Aga and Gam utilization indicating that neither agaI nor nagB is involved in the deamination and isomerization of Gam-6-P. We propose that agaS codes for Gam-6-P deaminase/isomerase in the Aga/Gam pathway. PMID:23634833

Genome-wide screen for inositol auxotrophy in Saccharomyces cerevisiae implicates lipid metabolism in stress response signaling.

PubMed

Villa-García, Manuel J; Choi, Myung Sun; Hinz, Flora I; Gaspar, María L; Jesch, Stephen A; Henry, Susan A

2011-02-01

Inositol auxotrophy (Ino(-) phenotype) in budding yeast has classically been associated with misregulation of INO1 and other genes involved in lipid metabolism. To identify all non-essential yeast genes that are necessary for growth in the absence of inositol, we carried out a genome-wide phenotypic screening for deletion mutants exhibiting Ino(-) phenotypes under one or more growth conditions. We report the identification of 419 genes, including 385 genes not previously reported, which exhibit this phenotype when deleted. The identified genes are involved in a wide range of cellular processes, but are particularly enriched in those affecting transcription, protein modification, membrane trafficking, diverse stress responses, and lipid metabolism. Among the Ino(-) mutants involved in stress response, many exhibited phenotypes that are strengthened at elevated temperature and/or when choline is present in the medium. The role of inositol in regulation of lipid metabolism and stress response signaling is discussed.

Hypertonicity-induced transmitter release at Drosophila neuromuscular junctions is partly mediated by integrins and cAMP/protein kinase A

NASA Technical Reports Server (NTRS)

Suzuki, Kazuhiro; Grinnell, Alan D.; Kidokoro, Yoshiaki

2002-01-01

The frequency of quantal transmitter release increases upon application of hypertonic solutions. This effect bypasses the Ca(2+) triggering step, but requires the presence of key molecules involved in vesicle fusion, and hence could be a useful tool for dissecting the molecular process of vesicle fusion. We have examined the hypertonicity response at neuromuscular junctions of Drosophila embryos in Ca(2+)-free saline. Relative to wild-type, the response induced by puff application of hypertonic solution was enhanced in a mutant, dunce, in which the cAMP level is elevated, or in wild-type embryos treated with forskolin, an activator of adenylyl cyclase, while protein kinase A (PKA) inhibitors decreased it. The response was also smaller in a mutant, DC0, which lacks the major subunit of PKA. Thus the cAMP/PKA cascade is involved in the hypertonicity response. Peptides containing the sequence Arg-Gly-Asp (RGD), which inhibit binding of integrins to natural ligands, reduced the response, whereas a peptide containing the non-binding sequence Arg-Gly-Glu (RGE) did not. A reduced response persisted in a mutant, myospheroid, which expresses no integrins, and the response in DC0 was unaffected by RGD peptides. These data indicate that there are at lease two components in the hypertonicity response: one that is integrin mediated and involves the cAMP/PKA cascade, and another that is not integrin mediated and does not involve the cAMP/PKA cascade.

Disparate Fluorescence Properties of 2-[4′-(Iodoacetamido)anilino]-Naphthalene-6-Sulfonic Acid Attached to Cys-84 and Cys-35 of Troponin C in Cardiac Muscle Troponin

PubMed Central

Dong, Wen-Ji; Wang, Chien-Kao; Gordon, Albert M.; Cheung, Herbert C.

1997-01-01

Two monocysteine mutants of cardiac muscle troponin C, cTnC(C35S) and cTnC(C84S), were genetically generated and labeled with the fluorescent probe 2-[4′-(iodoacetamido)anilino]naphthalene-6-sulfonic acid (IAANS) at Cys-84 and Cys-35, respectively. Cys-84 is located on helix D in the regulatory N-domain, and Cys-35 is at the -y position of the inactive 12-residue loop of site I. These labeled mutants were studied by a variety of steady-state and time-resolved fluorescence methods. In the absence of divalent cation, the fluorescence of the attached IAANS indicated an exposed environment at Cys-35 and a relatively less-exposed environment at Cys-84. The binding of Ca2+ to the single regulatory site elicited a large enhancement of the emission of IAANS attached to Cys-84, but only marginal fluorescence changes of the probe at Cys-35. Upon reconstitution of the labeled cTnC mutants with troponin I and troponin T to form the three-subunit troponin, the fluorescence of IAANS-Cys-84 in apo-troponin was spectrally similar to that observed with the Ca2+-loaded uncomplexed cTnC mutant. Only very moderate changes in the fluorescence of IAANS-Cys-84 were observed when the regulatory site in reconstituted troponin was saturated. The exposed Cys-35 environment of the uncomplexed cTnC mutant became considerably less exposed and less polar when the mutant was incorporated into apo-troponin. In contrast to the Cys-84 site, saturation of the regulatory site II by Ca2+ in reconstituted troponin resulted in a reversal of the environment of the Cys-35 site toward a more exposed and more polar environment. These results indicated involvement of the inactive loop I in the Ca2+ trigger mechanism in cardiac muscle. The fluorescence of IAANS at both Cys-84 and Cys-35 was sensitive to phosphorylation of cTnI in reconstituted troponin, and the sensitivity was observed with both apo-troponin and Ca2+-loaded troponin. PMID:9017210

Insights into the activity change of spore photoproduct lyase induced by mutations at a peripheral glycine residue

NASA Astrophysics Data System (ADS)

Yang, Linlin; Li, Lei

2017-03-01

UV radiation triggers the formation of 5-thyminyl-5,6-dihydrothymine, i.e. the spore photoproduct (SP), in the genomic DNA of bacterial endospores. These SPs, if not repaired in time, may lead to genome instability and cell death. SP is mainly repaired by spore photoproduct lyase (SPL) during spore outgrowth via an unprecedented protein-harbored radical transfer pathway that is composed of at least a cysteine and two tyrosine residues. This mechanism is consistent with the recently solved SPL structure that shows all three residues are located in proximity and thus able to participate in the radical transfer process during the enzyme catalysis. In contrast, an earlier in vivo mutational study identified a glycine to arginine mutation at the position 168 on the B. subtilis SPL that was later found to be > 15 Å away from the enzyme active site. This mutation appears to abolish the enzyme activity because endospores carrying this mutant were sensitive to UV light. To understand the molecular basis for this rendered enzyme activity, we constructed two SPL mutations G168A and G168R, examined their repair of dinucleotide SP TpT, and found that both mutants exhibit reduced enzyme activity. Comparing with the wildtype (WT) SPL enzyme, the G168A mutant slows down the SP TpT repair by 3 4 fold while the G168R mutant by 80 fold. Both mutants exhibit a smaller apparent (DV) kinetic isotope effect (KIE) but a bigger competitive (DV/K) KIE than that by the WT SPL. Moreover, the G168R mutant also produces a large portion of the abortive repair product TpT-SO2-; the formation of which indicates that cysteine 141 is no longer well positioned as the H-donor to the thymine allylic radical intermediate. All these data imply that the mutation at the remote glycine 168 residue alters the enzyme 3D structure, subsequently reducing the SPL activity by changing the positions of the essential amino acids involved in the radical transfer process.

Monitoring humans for somatic mutation in the endogenous PIG-a gene using red blood cells.

PubMed

Dobrovolsky, Vasily N; Elespuru, Rosalie K; Bigger, C Anita H; Robison, Timothy W; Heflich, Robert H

2011-12-01

The endogenous X-linked PIG-A gene is involved in the synthesis of glycosyl phosphatidyl inositol (GPI) anchors that tether specific protein markers to the exterior of mammalian cell cytoplasmic membranes. Earlier studies in rodent models indicate that Pig-a mutant red blood cells (RBCs) can be induced in animals treated with genotoxic agents, and that flow cytometry can be used to identify rare RBCs deficient in the GPI-anchored protein, CD59, as a marker of Pig-a gene mutation. We investigated if a similar approach could be used for detecting gene mutation in humans. We first determined the frequency of spontaneous CD59-deficient RBCs (presumed PIG-A mutants) in 97 self-identified healthy volunteers. For most subjects, the frequency of CD59-deficient RBCs was low (average of 5.1 ± 4.9 × 10(-6) ; median of 3.8 × 10(-6) and mutant frequency less than 8 × 10(-6) for 75% of subjects), with a statistically significant difference in median mutant frequencies between males and females. PIG-A RBC mutant frequency displayed poor correlation with the age and no correlation with the smoking status of the subjects. Also, two individuals had markedly increased CD59-deficient RBC frequencies of ∼300 × 10(-6) and ∼100 × 10(-6) . We then monitored PIG-A mutation in 10 newly diagnosed cancer patients undergoing chemotherapy with known genotoxic drugs. The frequency of CD59-deficient RBCs in the blood of the patients was measured before the start of chemotherapy and three times over a period of ∼6 months while on/after chemotherapy. Responses were generally weak, most observations being less than the median mutant frequency for both males and females; the greatest response was an approximate three-fold increase in the frequency of CD59-deficient RBCs in one patient treated with a combination of cisplatin and etoposide. These results suggest that the RBC PIG-A assay can be adopted to measuring somatic cell mutation in humans. Further research is necessary to determine the assay's sensitivity in detecting mutations induced by genotoxic agents acting via different mechanisms. Copyright © 2011 Wiley-Liss, Inc.

Sensitivity of human cells expressing low-fidelity or weak-catalytic-activity variants of DNA polymerase ζ to genotoxic stresses.

PubMed

Suzuki, Tetsuya; Grúz, Petr; Honma, Masamitsu; Adachi, Noritaka; Nohmi, Takehiko

2016-09-01

Translesion DNA polymerases (TLS pols) play critical roles in defense mechanisms against genotoxic agents. The defects or mutations of TLS pols are predicted to result in hypersensitivity of cells to environmental mutagens. In this study, human cells expressing DNA polymerase ζ (Pol ζ) variants with low fidelity or weak catalytic activity have been established with Nalm-6-MSH+ cells and their sensitivity to mutagenicity and cytotoxicity of benzo[a]pyrene diol epoxide (BPDE) and ultraviolet-C light (UV-C) was examined. The low-fidelity mutants were engineered by knocking-in DNA sequences that direct changes of leucine 2618 to either phenylalanine (L2618F) or methionine (L2618M) of Pol ζ. The weak-catalytic-activity mutants were generated by knocking-in DNA sequences that direct changes of either tyrosine 2779 to phenylalanine (Y2779F) or aspartate 2781 to asparagine (D2781N). In addition, a +1 frameshift mutation, i.e., CCC to CCCC, was introduced in the coding region of the TK1 gene to measure the mutant frequencies. Doubling time and spontaneous TK mutant frequencies of the established cell lines were similar to those of the wild-type cells. The low-fidelity mutants displayed, however, higher sensitivity to the mutagenicity of BPDE and UV-C than the wild-type cells although their cytotoxic sensitivity was not changed. In contrast, the weak-catalytic-activity mutants were more sensitive to the cytotoxicity of BPDE and UV-C than the wild-type cells, and displayed much higher sensitivity to the clastogenicity of BPDE than the wild-type cells in an in vitro micronucleus assay. These results indicate that human Pol ζ is involved in TLS across DNA lesions induced by BPDE and UV-C and also that the TLS plays important roles in induction of mutations, clastogenicity and in cellular survival of the damaged human cells. Similarities and differences in in vivo roles of yeast and human Pol ζ in genome integrity are discussed. Copyright © 2016 Elsevier B.V. All rights reserved.

A Candida guilliermondii lysine hyperproducer capable of elevated citric acid production.

PubMed

West, Thomas P

2016-05-01

A mutant of the yeast Candida guilliermondii ATCC 9058 exhibiting elevated citric acid production was isolated based upon its ability to overproduce lysine. This method involved the use of a solid medium containing a combination of lysine analogues to identify a mutant that produced a several-fold higher lysine level compared to its parent strain using glucose or glycerol as a carbon source. The mutant strain was also capable of producing more than a fivefold higher citric acid level on glycerol as a carbon source compared to its parent strain. It was concluded that the screening of yeast lysine hyperproducer strains could provide a rapid approach to isolate yeast citric acid hyperproducer strains.

Thermo-labile stability of sigmaH (Spo0H) in temperature-sensitive spo0H mutants of Bacillus subtilis can be suppressed by mutations in RNA polymerase beta subunit.

PubMed

Ohashi, Y; Sugimaru, K; Nanamiya, H; Sebata, T; Asai, K; Yoshikawa, H; Kawamura, F

1999-03-18

We isolated novel temperature-sensitive mutants of spo0H, spo0H1 and spo0H5, having E61K and G30E amino-acid substitutions within the sigmaH protein, respectively, and located in the highly conserved region, "2", among prokaryotic sigma factors that participates in binding to core enzyme of RNA polymerase. These mutants showed a sporulation-deficient phenotype at 43 degrees C. Moreover, we successfully isolated suppressor mutants that were spontaneously generated from the spo0H mutants. Our genetic analysis of these suppressor mutations revealed that the suppressor mutations are within the rpoB gene coding for the beta subunit of RNA polymerase. The mutations caused single amino-acid substitutions, E857A and P1055S, in rpoB18 and rpoB532 mutants that were generated from spo0H1 and spo0H5, respectively. Whereas the sigmaH-dependent expression of a spo0A-bgaB fusion was greatly reduced in both spo0H mutants, their expression was partially restored in the suppressor mutants at 43 degrees C. Western blot analysis showed that the level of sigmaH protein in the wild type increased between T0 and T2 and decreased after T3, while the level of sigmaH protein in spo0H mutants was greatly reduced throughout growth, indicating that the mutant sigmaH proteins were rapidly degraded by some unknown proteolytic enzyme(s). The analysis of the half-life of sigmaH protein showed that the short life of sigmaH in spo0H mutants is prolonged in the suppressor mutants. These findings suggest that, at least to some extent, the process of E-sigmaH formation may be involved in stabilization of sigmaH at the onset of sporulation.

Molecular Mechanism of Terbinafine Resistance in Saccharomyces cerevisiae

PubMed Central

Leber, Regina; Fuchsbichler, Sandra; Klobučníková, Vlasta; Schweighofer, Natascha; Pitters, Eva; Wohlfarter, Kathrin; Lederer, Mojca; Landl, Karina; Ruckenstuhl, Christoph; Hapala, Ivan; Turnowsky, Friederike

2003-01-01

Ten mutants of the yeast Saccharomyces cerevisiae resistant to the antimycotic terbinafine were isolated after chemical or UV mutagenesis. Molecular analysis of these mutants revealed single base pair exchanges in the ERG1 gene coding for squalene epoxidase, the target of terbinafine. The mutants did not show cross-resistance to any of the substrates of various pleiotropic drug resistance efflux pumps tested. The ERG1 mRNA levels in the mutants did not differ from those in the wild-type parent strains. Terbinafine resistance was transmitted with the mutated alleles in gene replacement experiments, proving that single amino acid substitutions in the Erg1 protein were sufficient to confer the resistance phenotype. The amino acid changes caused by the point mutations were clustered in two regions of the Erg1 protein. Seven mutants carried the amino acid substitutions F402L (one mutant), F420L (one mutant), and P430S (five mutants) in the C-terminal part of the protein; and three mutants carried an L251F exchange in the central part of the protein. Interestingly, all exchanges identified involved amino acids which are conserved in the squalene epoxidases of yeasts and mammals. Two mutations that were generated by PCR mutagenesis of the ERG1 gene and that conferred terbinafine resistance mapped in the same regions of the Erg1 protein, with one resulting in an L251F exchange and the other resulting in an F433S exchange. The results strongly indicate that these regions are responsible for the interaction of yeast squalene epoxidase with terbinafine. PMID:14638499

Role of the hydrophilic channels of simian virus 40 T-antigen helicase in DNA replication.

PubMed

Wang, Weiping; Manna, David; Simmons, Daniel T

2007-05-01

The simian virus 40 (SV40) hexameric helicase consists of a central channel and six hydrophilic channels located between adjacent large tier domains within each hexamer. To study the function of the hydrophilic channels in SV40 DNA replication, a series of single-point substitutions were introduced at sites not directly involved in protein-protein contacts. The mutants were characterized biochemically in various ways. All mutants oligomerized normally in the absence of DNA. Interestingly, 8 of the 10 mutants failed to unwind an origin-containing DNA fragment and nine of them were totally unable to support SV40 DNA replication in vitro. The mutants fell into four classes based on their biochemical properties. Class A mutants bound DNA normally and had normal ATPase and helicase activities but failed to unwind origin DNA and support SV40 DNA replication. Class B mutants were compromised in single-stranded DNA and origin DNA binding at low protein concentrations. They were defective in helicase activity and unwinding of the origin and in supporting DNA replication. Class C and D mutants possessed higher-than-normal single-stranded DNA binding activity at low protein concentrations. The class C mutants failed to separate origin DNA and support DNA replication. The class D mutants unwound origin DNA normally but were compromised in their ability to support DNA replication. Taken together, these results suggest that the hydrophilic channels have an active role in the unwinding of SV40 DNA from the origin and the placement of the resulting single strands within the helicase.

A proteomic analysis of ferulic acid metabolism in Amycolatopsis sp. ATCC 39116.

PubMed

Meyer, Florian; Netzer, Julius; Meinert, Christina; Voigt, Birgit; Riedel, Katharina; Steinbüchel, Alexander

2018-05-16

The pseudonocardiate Amycolatopsis sp. ATCC 39116 is used for the biotechnical production of natural vanillin from ferulic acid. Our laboratory has performed genetic modifications of this strain previously, but there are still many gaps in our knowledge regarding its vanillin tolerance and the general metabolism. We performed cultivations with this bacterium and compared the proteomes of stationary phase cells before ferulic acid feeding with those during ferulic acid feeding. Thereby, we identified 143 differently expressed proteins. Deletion mutants were constructed and characterized to analyze the function of nine corresponding genes. Using these mutants, we identified an active ferulic acid β-oxidation pathway and the enzymes which constitute this pathway. A combined deletion mutant in which the β-oxidation as well as non-β-oxidation pathways of ferulic acid degradation were deleted was unable to grow on ferulic acid as the sole source of carbon and energy. This mutant differs from the single deletion mutants and was unable to grow on ferulic acid. Furthermore, we showed that the non-β-oxidation pathway is involved in caffeic acid degradation; however, its deletion is complemented even in the double deletion mutant. This shows that both pathways can complement each other. The β-oxidation deletion mutant produced significantly reduced amounts of vanillic acid (0.12 instead of 0.35 g/l). Therefore, the resulting mutant could be used as an improved production strain. The quinone oxidoreductase deletion mutant (ΔytfG) degraded ferulic acid slower at first but produced comparable amounts of vanillin and significantly less vanillyl alcohol when compared to the parent strain.

In Vitro Selection of ramR and soxR Mutants Overexpressing Efflux Systems by Fluoroquinolones as Well as Cefoxitin in Klebsiella pneumoniae▿

PubMed Central

Bialek-Davenet, Suzanne; Marcon, Estelle; Leflon-Guibout, Véronique; Lavigne, Jean-Philippe; Bert, Frédéric; Moreau, Richard; Nicolas-Chanoine, Marie-Hélène

2011-01-01

The relationship between efflux system overexpression and cross-resistance to cefoxitin, quinolones, and chloramphenicol has recently been reported in Klebsiella pneumoniae. In 3 previously published clinical isolates and 17 in vitro mutants selected with cefoxitin or fluoroquinolones, mutations in the potential regulator genes of the AcrAB efflux pump (acrR, ramR, ramA, marR, marA, soxR, soxS, and rob) were searched, and their impacts on efflux-related antibiotic cross-resistance were assessed. All mutants but 1, and 2 clinical isolates, overexpressed acrB. No mutation was detected in the regulator genes studied among the clinical isolates and 8 of the mutants. For the 9 remaining mutants, a mutation was found in the ramR gene in 8 of them and in the soxR gene in the last one, resulting in overexpression of ramA and soxS, respectively. Transformation of the ramR mutants and the soxR mutant with the wild-type ramR and soxR genes, respectively, abolished overexpression of acrB and ramA in the ramR mutants and of soxS in the soxR mutant, as well as antibiotic cross-resistance. Resistance due to efflux system overexpression was demonstrated for 4 new antibiotics: cefuroxime, cefotaxime, ceftazidime, and ertapenem. This study shows that the ramR and soxR genes control the expression of efflux systems in K. pneumoniae and suggests the existence of efflux pumps other than AcrAB and of other loci involved in the regulation of AcrAB expression. PMID:21464248

Genetic control of anastomosis in Podospora anserina.

PubMed

Tong, Laetitia Chan Ho; Silar, Philippe; Lalucque, Hervé

2014-09-01

We developed a new microscopy procedure to study anastomoses in the model ascomycete Podospora anserina and compared it with the previous method involving the formation of balanced heterokaryons. Both methods showed a good correlation. Heterokaryon formation was less quantifiable, but enabled to observe very rare events. Microscopic analysis evidenced that anastomoses were greatly influence by growth conditions and were severely impaired in the IDC mutants of the PaMpk1, PaMpk2, IDC1 and PaNox1 pathways. Yet some mutants readily formed heterokaryons, albeit with a delay when compared to the wild type. We also identified IDC(821), a new mutant presenting a phenotype similar to the other IDC mutants, including lack of anastomosis. Complete genome sequencing revealed that IDC(821) was affected in the orthologue of the Neurospora crassa So gene known to control anastomosis in several other ascomycetes. Copyright © 2014 Elsevier Inc. All rights reserved.

Ethylene signaling triggered by low concentrations of ascorbic acid regulates biomass accumulation in Arabidopsis thaliana.

PubMed

Caviglia, M; Mazorra Morales, L M; Concellón, A; Gergoff Grozeff, G E; Wilson, M; Foyer, C H; Bartoli, C G

2018-02-02

Ascorbic acid (AA) is a major redox buffer in plant cells. The role of ethylene in the redox signaling pathways that influence photosynthesis and growth was explored in two independent AA deficient Arabidopsis thaliana mutants (vtc2-1 and vtc2-4). Both mutants, which are defective in the AA biosynthesis gene GDP-L-galactose phosphorylase, produce higher amounts of ethylene than wt plants. In contrast to the wt, the inhibition of ethylene signaling increased leaf conductance, photosynthesis and dry weight in both vtc2 mutant lines. The AA-deficient mutants showed altered expression of genes encoding proteins involved in the synthesis/responses to phytohormones that control growth, particularly auxin, cytokinins, abscisic acid, brassinosterioids, ethylene and salicylic acid. These results demonstrate that AA deficiency modifies hormone signaling in plants, redox-ethylene interactions providing a regulatory node controlling shoot biomass accumulation. Copyright © 2018 Elsevier Inc. All rights reserved.

The Arabidopsis mutant cev1 links cell wall signaling to jasmonate and ethylene responses.

PubMed

Ellis, Christine; Karafyllidis, Ioannis; Wasternack, Claus; Turner, John G

2002-07-01

Biotic and abiotic stresses stimulate the synthesis of jasmonates and ethylene, which, in turn, induce the expression of genes involved in stress response and enhance defense responses. The cev1 mutant has constitutive expression of stress response genes and has enhanced resistance to fungal pathogens. Here, we show that cev1 plants have increased production of jasmonate and ethylene and that its phenotype is suppressed by mutations that interrupt jasmonate and ethylene signaling. Genetic mapping, complementation analysis, and sequence analysis revealed that CEV1 is the cellulose synthase CeSA3. CEV1 was expressed predominantly in root tissues, and cev1 roots contained less cellulose than wild-type roots. Significantly, the cev1 mutant phenotype could be reproduced by treating wild-type plants with cellulose biosynthesis inhibitors, and the cellulose synthase mutant rsw1 also had constitutive expression of VSP. We propose that the cell wall can signal stress responses in plants.

In vivo and in vitro analyses of a Bombyx mori nucleopolyhedrovirus mutant lacking functional vfgf.

PubMed

Katsuma, Susumu; Horie, Satoshi; Daimon, Takaaki; Iwanaga, Masashi; Shimada, Toru

2006-11-10

All lepidopteran baculovirus genomes sequenced to date encode a viral fibroblast growth factor homolog (vfgf), suggesting that vfgf may play an important role in the infection cycle of lepidopteran baculoviruses. Here, we describe the characterization of a Bombyx mori nucleopolyhedrovirus (BmNPV) mutant lacking functional vfgf. We constructed a vfgf deletion mutant (BmFGFD) and characterized it in BmN cells and B. mori larvae. We observed that budded virus (BV) production was reduced in BmFGFD-infected BmN cells and B. mori larvae. The larval bioassays also revealed that deletion of vfgf did not reduce the infectivity; however, the mutant virus did take 20 h longer to kill B. mori larvae than wild-type BmNPV, when tested either by BV injection or by polyhedrin-inclusion body ingestion. These results suggest that BmNPV vfgf is involved in efficient virus production in BmN cells and B. mori larvae.

IMP2, a nuclear gene controlling the mitochondrial dependence of galactose, maltose and raffinose utilization in Saccharomyces cerevisiae.

PubMed

Donnini, C; Lodi, T; Ferrero, I; Puglisi, P P

1992-02-01

The IMP2 gene of Saccharomyces cerevisiae is involved in the nucleo-mitochondrial control of maltose, galactose and raffinose utilization as shown by the inability of imp2 mutants to grow on these carbon sources in respiratory-deficient conditions or in the presence of ethidium bromide and erythromycin. The negative phenotype cannot be scored in the presence of inhibitors of respiration and oxidative phosphorylation, indicating that the role of the mitochondria in the utilization of the above-mentioned carbon sources in imp2 mutants is not at the energetical level. Mutations in the IMP2 gene also confer many phenotypic alterations in respiratory-sufficient conditions, e.g. leaky phenotype on oxidizable carbon sources, sensitivity to heat shock and sporulation deficiency. The IMP2 gene has been cloned, sequenced and disrupted. The phenotype of null imp2 mutants is indistinguishable from that of the originally isolated mutant.

NMR studies of structure, hydrogen exchange, and main-chain dynamics in a disrupted-core mutant of thioredoxin.

PubMed Central

De Lorimier, R.; Hellinga, H. W.; Spicer, L. D.

1996-01-01

Core-packing mutants of proteins often approach molten globule states, and hence may have attributes of folding intermediates. We have studied a core-packing mutant of thioredoxin, L78K, in which a leucine residue is substituted by lysine, using 15N heteronuclear two- and three-dimensional NMR. Chemical shift differences between the mutant and wild-type main-chain resonances reveal that structural changes caused by the mutation are localized within 12 A of the altered side chain. The majority of resonances are unchanged, as are many 1H-1H NOEs indicative of the main-chain fold, suggesting that the structure of L78K is largely similar to wild type. Hydrogen exchange studies reveal that residues comprising the central beta-sheet of both mutant and wild-type proteins constitute a local unfolding unit, but with the unfolding/folding equilibrium approximately 12 times larger in L78K. The dynamics of main-chain NH bonds in L78K were studied by 15N spin relaxation and compared with a previous study of wild type. Order parameters for angular motion of NH bonds in the mutant are on average lower than in wild type, suggesting greater spatial freedom on a rapid time scale, but may also be related to different rotational correlation times in the two proteins. There is also evidence of greater conformational exchange in the mutant. Differences between mutant and wild type in hydrogen exchange and main-chain dynamics are not confined to the vicinity of the mutation. We infer that mispacking of the protein core in one location affects local dynamics and stability throughout. PMID:8976564

Ultrasensitive Quantification of Hepatitis B Virus A1762T/G1764A Mutant by a SimpleProbe PCR Using a Wild-Type-Selective PCR Blocker and a Primer-Blocker-Probe Partial-Overlap Approach ▿

PubMed Central

Nie, Hui; Evans, Alison A.; London, W. Thomas; Block, Timothy M.; Ren, Xiangdong David

2011-01-01

Hepatitis B virus (HBV) carrying the A1762T/G1764A double mutation in the basal core promoter (BCP) region is associated with HBe antigen seroconversion and increased risk of liver cirrhosis and hepatocellular carcinoma (HCC). Quantification of the mutant viruses may help in predicting the risk of HCC. However, the viral genome tends to have nucleotide polymorphism, which makes it difficult to design hybridization-based assays including real-time PCR. Ultrasensitive quantification of the mutant viruses at the early developmental stage is even more challenging, as the mutant is masked by excessive amounts of the wild-type (WT) viruses. In this study, we developed a selective inhibitory PCR (siPCR) using a locked nucleic acid-based PCR blocker to selectively inhibit the amplification of the WT viral DNA but not the mutant DNA. At the end of siPCR, the proportion of the mutant could be increased by about 10,000-fold, making the mutant more readily detectable by downstream applications such as real-time PCR and DNA sequencing. We also describe a primer-probe partial overlap approach which significantly simplified the melting curve patterns and minimized the influence of viral genome polymorphism on assay accuracy. Analysis of 62 patient samples showed a complete match of the melting curve patterns with the sequencing results. More than 97% of HBV BCP sequences in the GenBank database can be correctly identified by the melting curve analysis. The combination of siPCR and the SimpleProbe real-time PCR enabled mutant quantification in the presence of a 100,000-fold excess of the WT DNA. PMID:21562108

The circling mutant Pcdh15roda is a new mouse model for hearing loss.

PubMed

Torres, Adriana Amorim; Rzadzinska, Agnieszka K; Ribeiro, Andrea Frozino; Silva, Daniel Almeida da Silva E; Guénet, Jean-Louis; Massironi, Sílvia Maria Gomes; Godard, Ana Lúcia Brunialti

2013-01-01

Mouse mutagenesis is a key tool for studying gene function and several mutant alleles have been described and constitute mouse models for human hereditary diseases. Genetic hearing loss represents over 50% of all hearing loss cases in children and, due to the heterogeneity of the disorder, there is still a demand for the isolation and characterization of new genes and alleles. Here we report phenotypic and molecular characterization of a new mouse model for hereditary hearing loss. The mutant rodador, isolated by Massironi and colleagues in 2006, presents an autosomal recessive disorder characterized by deafness and balance dysfunction associated with abnormal stereocilia in the inner ear. The mutation was mapped to mouse chromosome 10, and characterization of the gene Pcdh15 revealed an AT-to-GC transition in intron 23 of mutant animals. The alteration led to the switch of a dinucleotide ApA for ApG, creating a novel intronic acceptor splice site, which leads to incorporation of eight intronic bases into the processed mRNA and alteration of the downstream reading frame. In silico analysis indicated that the mutated protein is truncated and lacks two cadherin domains, and the transmembrane and cytoplasmic domains. Real Time PCR analyses revealed a significantly reduced Pcdh15 mRNA level in the brain of mutant mice, which might be due to the mechanism of non-sense mediated decay. In man, mutations in the orthologue PCDH15 cause non-syndromic deafness and Usher Syndrome Type 1F, a genetic disorder characterized by hearing loss and retinitis pigmentosa. Rodador mouse constitutes a new model for studying deafness in these conditions and may help in the comprehension of the pathogeneses of the disease, as well as of the mechanisms involved in the morphogenesis and function of inner ear stereocilia. This is a new ENU-induced allele and the first isolated in a BALB/c background. Copyright © 2013 Elsevier B.V. All rights reserved.

Mutation of the Erwinia amylovora argD Gene Causes Arginine Auxotrophy, Nonpathogenicity in Apples, and Reduced Virulence in Pears

PubMed Central

Ramos, Laura S.; Lehman, Brian L.; Peter, Kari A.

2014-01-01

Fire blight is caused by Erwinia amylovora and is the most destructive bacterial disease of apples and pears worldwide. In this study, we found that E. amylovora argD(1000)::Tn5, an argD Tn5 transposon mutant that has the Tn5 transposon inserted after nucleotide 999 in the argD gene-coding region, was an arginine auxotroph that did not cause fire blight in apple and had reduced virulence in immature pear fruits. The E. amylovora argD gene encodes a predicted N-acetylornithine aminotransferase enzyme, which is involved in the production of the amino acid arginine. A plasmid-borne copy of the wild-type argD gene complemented both the nonpathogenic and the arginine auxotrophic phenotypes of the argD(1000)::Tn5 mutant. However, even when mixed with virulent E. amylovora cells and inoculated onto immature apple fruit, the argD(1000)::Tn5 mutant still failed to grow, while the virulent strain grew and caused disease. Furthermore, the pCR2.1-argD complementation plasmid was stably maintained in the argD(1000)::Tn5 mutant growing in host tissues without any antibiotic selection. Therefore, the pCR2.1-argD complementation plasmid could be useful for the expression of genes, markers, and reporters in E. amylovora growing in planta, without concern about losing the plasmid over time. The ArgD protein cannot be considered an E. amylovora virulence factor because the argD(1000)::Tn5 mutant was auxotrophic and had a primary metabolism defect. Nevertheless, these results are informative about the parasitic nature of the fire blight disease interaction, since they indicate that E. amylovora cannot obtain sufficient arginine from apple and pear fruit tissues or from apple vegetative tissues, either at the beginning of the infection process or after the infection has progressed to an advanced state. PMID:25172854

Mutation of the Erwinia amylovora argD gene causes arginine auxotrophy, nonpathogenicity in apples, and reduced virulence in pears.

PubMed

Ramos, Laura S; Lehman, Brian L; Peter, Kari A; McNellis, Timothy W

2014-11-01

Fire blight is caused by Erwinia amylovora and is the most destructive bacterial disease of apples and pears worldwide. In this study, we found that E. amylovora argD(1000)::Tn5, an argD Tn5 transposon mutant that has the Tn5 transposon inserted after nucleotide 999 in the argD gene-coding region, was an arginine auxotroph that did not cause fire blight in apple and had reduced virulence in immature pear fruits. The E. amylovora argD gene encodes a predicted N-acetylornithine aminotransferase enzyme, which is involved in the production of the amino acid arginine. A plasmid-borne copy of the wild-type argD gene complemented both the nonpathogenic and the arginine auxotrophic phenotypes of the argD(1000)::Tn5 mutant. However, even when mixed with virulent E. amylovora cells and inoculated onto immature apple fruit, the argD(1000)::Tn5 mutant still failed to grow, while the virulent strain grew and caused disease. Furthermore, the pCR2.1-argD complementation plasmid was stably maintained in the argD(1000)::Tn5 mutant growing in host tissues without any antibiotic selection. Therefore, the pCR2.1-argD complementation plasmid could be useful for the expression of genes, markers, and reporters in E. amylovora growing in planta, without concern about losing the plasmid over time. The ArgD protein cannot be considered an E. amylovora virulence factor because the argD(1000)::Tn5 mutant was auxotrophic and had a primary metabolism defect. Nevertheless, these results are informative about the parasitic nature of the fire blight disease interaction, since they indicate that E. amylovora cannot obtain sufficient arginine from apple and pear fruit tissues or from apple vegetative tissues, either at the beginning of the infection process or after the infection has progressed to an advanced state. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Investigation of structural dynamics of Thrombocytopenia Cargeeg mutants of human apoptotic cytochrome c: A molecular dynamics simulation approach.

PubMed

Muneeswaran, Gurusamy; Kartheeswaran, Subramanian; Pandiaraj, Manickam; Muthukumar, Kaliappan; Sankaralingam, Muniyandi; Arunachalam, Saravanavadivu

2017-11-01

Naturally occurring mutations to cytochrome c (cyt-c) have been identified recently in patients with mild autosomal dominant thrombocytopenia (low platelet levels), which yield cyt-c mutants with enhanced apoptotic activity. However, the molecular mechanism underlying this low platelet production and enhanced apoptosis remain unclear. Therefore, an attempt is made herein for the first time to investigate the effects of mutations of glycine 41 by serine (G41S) and tyrosine 48 by histidine (Y48H) on the conformational and dynamic changes of apoptotic (Fe 3+ ) cyt-c using all atom molecular dynamics (MD) simulations in explicit water solvent. Our 30ns MD simulations demonstrate considerable structural differences in G41S and Y48H compared to wild type (WT) cyt-c, such as increasing distances between the critical electron transfer residues results in open conformation at the heme active site, large fluctuations in β-turns and α-helices. Additionally, although the β-sheets remain mostly unaffected in all the three cyt-c simulations, the α-helices undergo conformational switch to β-turns in both the mutant simulations. Importantly, this conformational switch of α-helix to β-turn around heme active site should attributes to the loss of intraprotein H-bonds in the mutant simulations especially between NE2 (His26) and O (Pro44) in agreement with the experimental report. Further, essential dynamics analysis reveals that overall motions of WT cyt-c is mainly involved only in the first eigenvector, but in G41S and Y48H the overall motions are mainly in three and two eigenvectors respectively. Overall, the detailed atomistic level information provide a unifying description for the molecular mechanism of structural destabilization, disregulation of platelet formation and enhanced peroxidase activity of the mutant cyt-c's in the pathology of intrinsic apoptosis. Copyright © 2017 Elsevier B.V. All rights reserved.

Xenobiotic metabolism in the fourth dimension: PARtners in time.

PubMed

Green, Carla B; Takahashi, Joseph S

2006-07-01

A significant portion of the transcriptome in mammals, including the PAR bZIP transcription factors DBP, HLF, and TEF, is under circadian clock control. In this issue of Cell Metabolism, Gachon and colleagues (Gachon et al., 2006) show that disruption of these three genes in mice alters gene expression patterns of many proteins involved in drug metabolism and in liver and kidney responses to xenobiotic agents. Triple mutant mice have severe physiological deficits, including increased hypersensitivity to xenobiotic agents and premature aging, highlighting the profound effect the circadian clock has on this important response system.

Mutant HNF-1{alpha} and mutant HNF-1{beta} identified in MODY3 and MODY5 downregulate DPP-IV gene expression in Caco-2 cells

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

Gu Ning; Laboratory of Neurochemistry, Graduate School of Human and Environmental Studies, Kyoto University, Kyoto; Adachi, Tetsuya

2006-08-04

Dipeptidylpeptidase IV (DPP-IV) is a well-documented drug target for the treatment of type 2 diabetes. Hepatocyte nuclear factors (HNF)-1{alpha} and HNF-1{beta}, known as the causal genes of MODY3 and MODY5, respectively, have been reported to be involved in regulation of DPP-IV gene expression. But, it is not completely clear (i) that they play roles in regulation of DPP-IV gene expression, and (ii) whether DPP-IV gene activity is changed by mutant HNF-1{alpha} and mutant HNF-1{beta} in MODY3 and MODY5. To explore these questions, we investigated transactivation effects of wild HNF-1{alpha} and 13 mutant HNF-1{alpha}, as well as wild HNF-1{beta} and 2more » mutant HNF-1{beta}, on DPP-IV promoter luciferase gene in Caco-2 cells by means of a transient experiment. Both wild HNF-1{alpha} and wild HNF-1{beta} significantly transactivated DPP-IV promoter, but mutant HNF-1{alpha} and mutant HNF-1{beta} exhibited low transactivation activity. Moreover, to study whether mutant HNF-1{alpha} and mutant HNF-1{beta} change endogenous DPP-IV enzyme activity, we produced four stable cell lines from Caco-2 cells, in which wild HNF-1{alpha} or wild HNF-1{beta}, or else respective dominant-negative mutant HNF-1{alpha}T539fsdelC or dominant-negative mutant HNF-1{beta}R177X, was stably expressed. We found that DPP-IV gene expression and enzyme activity were significantly increased in wild HNF-1{alpha} cells and wild HNF-1{beta} cells, whereas they decreased in HNF-1{alpha}T539fsdelC cells and HNF-1{beta}R177X cells, compared with DPP-IV gene expression and enzyme activity in Caco-2 cells. These results suggest that both wild HNF-1{alpha} and wild HNF-1{beta} have a stimulatory effect on DPP-IV gene expression, but that mutant HNF-1{alpha} and mutant HNF-1{beta} attenuate the stimulatory effect.« less

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

SNF1-Related Protein Kinases Type 2 Are Involved in Plant Responses to Cadmium Stress1[C][W

PubMed Central

Kulik, Anna; Anielska-Mazur, Anna; Bucholc, Maria; Koen, Emmanuel; Szymańska, Katarzyna; Żmieńko, Agnieszka; Krzywińska, Ewa; Wawer, Izabela; McLoughlin, Fionn; Ruszkowski, Dariusz; Figlerowicz, Marek; Testerink, Christa; Skłodowska, Aleksandra; Wendehenne, David; Dobrowolska, Grażyna

2012-01-01

Cadmium ions are notorious environmental pollutants. To adapt to cadmium-induced deleterious effects plants have developed sophisticated defense mechanisms. However, the signaling pathways underlying the plant response to cadmium are still elusive. Our data demonstrate that SnRK2s (for SNF1-related protein kinase2) are transiently activated during cadmium exposure and are involved in the regulation of plant response to this stress. Analysis of tobacco (Nicotiana tabacum) Osmotic Stress-Activated Protein Kinase activity in tobacco Bright Yellow 2 cells indicates that reactive oxygen species (ROS) and nitric oxide, produced mainly via an l-arginine-dependent process, contribute to the kinase activation in response to cadmium. SnRK2.4 is the closest homolog of tobacco Osmotic Stress-Activated Protein Kinase in Arabidopsis (Arabidopsis thaliana). Comparative analysis of seedling growth of snrk2.4 knockout mutants versus wild-type Arabidopsis suggests that SnRK2.4 is involved in the inhibition of root growth triggered by cadmium; the mutants were more tolerant to the stress. Measurements of the level of three major species of phytochelatins (PCs) in roots of plants exposed to Cd2+ showed a similar (PC2, PC4) or lower (PC3) concentration in snrk2.4 mutants in comparison to wild-type plants. These results indicate that the enhanced tolerance of the mutants does not result from a difference in the PCs level. Additionally, we have analyzed ROS accumulation in roots subjected to Cd2+ treatment. Our data show significantly lower Cd2+-induced ROS accumulation in the mutants’ roots. Concluding, the obtained results indicate that SnRK2s play a role in the regulation of plant tolerance to cadmium, most probably by controlling ROS accumulation triggered by cadmium ions. PMID:22885934

Genome-Scale Analysis of Mycoplasma agalactiae Loci Involved in Interaction with Host Cells

PubMed Central

Skapski, Agnès; Hygonenq, Marie-Claude; Sagné, Eveline; Guiral, Sébastien; Citti, Christine; Baranowski, Eric

2011-01-01

Mycoplasma agalactiae is an important pathogen of small ruminants, in which it causes contagious agalactia. It belongs to a large group of “minimal bacteria” with a small genome and reduced metabolic capacities that are dependent on their host for nutrients. Mycoplasma survival thus relies on intimate contact with host cells, but little is known about the factors involved in these interactions or in the more general infectious process. To address this issue, an assay based on goat epithelial and fibroblastic cells was used to screen a M. agalactiae knockout mutant library. Mutants with reduced growth capacities in cell culture were selected and 62 genomic loci were identified as contributing to this phenotype. As expected for minimal bacteria, “transport and metabolism” was the functional category most commonly implicated in this phenotype, but 50% of the selected mutants were disrupted in coding sequences (CDSs) with unknown functions, with surface lipoproteins being most commonly represented in this category. Since mycoplasmas lack a cell wall, lipoproteins are likely to be important in interactions with the host. A few intergenic regions were also identified that may act as regulatory sequences under co-culture conditions. Interestingly, some mutants mapped to gene clusters that are highly conserved across mycoplasma species but located in different positions. One of these clusters was found in a transcriptionally active region of the M. agalactiae chromosome, downstream of a cryptic promoter. A possible scenario for the evolution of these loci is discussed. Finally, several CDSs identified here are conserved in other important pathogenic mycoplasmas, and some were involved in horizontal gene transfer with phylogenetically distant species. These results provide a basis for further deciphering functions mediating mycoplasma-host interactions. PMID:21966487

Catabolism of N-Acetylneuraminic Acid, a Fitness Function of the Food-Borne Lactic Acid Bacterium Lactobacillus sakei, Involves Two Newly Characterized Proteins

PubMed Central

Chaillou, Stéphane; Zagorec, Monique; Champomier-Vergès, Marie-Christine

2013-01-01

In silico analysis of the genome sequence of the meat-borne lactic acid bacterium (LAB) Lactobacillus sakei 23K has revealed a repertoire of potential functions related to the adaptation of this bacterium to the meat environment. Among these functions, the ability to use N-acetyl-neuraminic acid (NANA) as a carbon source could provide a competitive advantage for growth on meat in which this amino sugar is present. In this work, we proposed to analyze the functionality of a gene cluster encompassing nanTEAR and nanK (nanTEAR-nanK). We established that this cluster encoded a pathway allowing transport and early steps of the catabolism of NANA in this genome. We also demonstrated that this cluster was absent from the genome of other L. sakei strains that were shown to be unable to grow on NANA. Moreover, L. sakei 23K nanA, nanT, nanK, and nanE genes were able to complement Escherichia coli mutants. Construction of different mutants in L. sakei 23K ΔnanR, ΔnanT, and ΔnanK and the double mutant L. sakei 23K Δ(nanA-nanE) made it possible to show that all were impaired for growth on NANA. In addition, two genes located downstream from nanK, lsa1644 and lsa1645, are involved in the catabolism of sialic acid in L. sakei 23K, as a L. sakei 23K Δlsa1645 mutant was no longer able to grow on NANA. All these results demonstrate that the gene cluster nanTEAR-nanK-lsa1644-lsa1645 is indeed involved in the use of NANA as an energy source by L. sakei. PMID:23335758

The Fusarium oxysporum gnt2, Encoding a Putative N-Acetylglucosamine Transferase, Is Involved in Cell Wall Architecture and Virulence

PubMed Central

López-Fernández, Loida; Ruiz-Roldán, Carmen; Pareja-Jaime, Yolanda; Prieto, Alicia; Khraiwesh, Husam; Roncero, M. Isabel G.

2013-01-01

With the aim to decipher the molecular dialogue and cross talk between Fusarium oxysporum f.sp. lycopersci and its host during infection and to understand the molecular bases that govern fungal pathogenicity, we analysed genes presumably encoding N-acetylglucosaminyl transferases, involved in glycosylation of glycoproteins, glycolipids, proteoglycans or small molecule acceptors in other microorganisms. In silico analysis revealed the existence of seven putative N-glycosyl transferase encoding genes (named gnt) in F. oxysporum f.sp. lycopersici genome. gnt2 deletion mutants showed a dramatic reduction in virulence on both plant and animal hosts. Δgnt2 mutants had αalterations in cell wall properties related to terminal αor β-linked N-acetyl glucosamine. Mutant conidia and germlings also showed differences in structure and physicochemical surface properties. Conidial and hyphal aggregation differed between the mutant and wild type strains, in a pH independent manner. Transmission electron micrographs of germlings showed strong cell-to-cell adherence and the presence of an extracellular chemical matrix. Δgnt2 cell walls presented a significant reduction in N-linked oligosaccharides, suggesting the involvement of Gnt2 in N-glycosylation of cell wall proteins. Gnt2 was localized in Golgi-like sub-cellular compartments as determined by fluorescence microscopy of GFP::Gnt2 fusion protein after treatment with the antibiotic brefeldin A or by staining with fluorescent sphingolipid BODIPY-TR ceramide. Furthermore, density gradient ultracentrifugation allowed co-localization of GFP::Gnt2 fusion protein and Vps10p in subcellular fractions enriched in Golgi specific enzymatic activities. Our results suggest that N-acetylglucosaminyl transferases are key components for cell wall structure and influence interactions of F. oxysporum with both plant and animal hosts during pathogenicity. PMID:24416097

A temperature-sensitive dcw1 mutant of Saccharomyces cerevisiae is cell cycle arrested with small buds which have aberrant cell walls.

PubMed

Kitagaki, Hiroshi; Ito, Kiyoshi; Shimoi, Hitoshi

2004-10-01

Dcw1p and Dfg5p in Saccharomyces cerevisiae are homologous proteins that were previously shown to be involved in cell wall biogenesis and to be essential for growth. Dcw1p was found to be a glycosylphosphatidylinositol-anchored membrane protein. To investigate the roles of these proteins in cell wall biogenesis and cell growth, we constructed mutant alleles of DCW1 by random mutagenesis, introduced them into a Deltadcw1 Deltadfg5 background, and isolated a temperature-sensitive mutant, DC61 (dcw1-3 Deltadfg5). When DC61 cells were incubated at 37 degrees C, most cells had small buds, with areas less than 20% of those of the mother cells. This result indicates that DC61 cells arrest growth with small buds at 37 degrees C. At 37 degrees C, fewer DC61 cells had 1N DNA content and most of them still had a single nucleus located apart from the bud neck. In addition, in DC61 cells incubated at 37 degrees C, bipolar spindles were not formed. These results indicate that DC61 cells, when incubated at 37 degrees C, are cell cycle arrested after DNA replication and prior to the separation of spindle pole bodies. The small buds of DC61 accumulated chitin in the bud cortex, and some of them were lysed, which indicates that they had aberrant cell walls. A temperature-sensitive dfg5 mutant, DF66 (Deltadcw1 dfg5-29), showed similar phenotypes. DCW1 and DFG5 mRNA levels peaked in the G1 and S phases, respectively. These results indicate that Dcw1p and Dfg5p are involved in bud formation through their involvement in biogenesis of the bud cell wall.

Effects of Combined Low Glutathione with Mild Oxidative and Low Phosphorus Stress on the Metabolism of Arabidopsis thaliana

PubMed Central

Fukushima, Atsushi; Iwasa, Mami; Nakabayashi, Ryo; Kobayashi, Makoto; Nishizawa, Tomoko; Okazaki, Yozo; Saito, Kazuki; Kusano, Miyako

2017-01-01

Plants possess highly sensitive mechanisms that monitor environmental stress levels for a dose-dependent fine-tuning of their growth and development. Differences in plant responses to severe and mild abiotic stresses have been recognized. Although many studies have revealed that glutathione can contribute to plant tolerance to various environmental stresses, little is known about the relationship between glutathione and mild abiotic stress, especially the effect of stress-induced altered glutathione levels on the metabolism. Here, we applied a systems biology approach to identify key pathways involved in the gene-to-metabolite networks perturbed by low glutathione content under mild abiotic stress in Arabidopsis thaliana. We used glutathione synthesis mutants (cad2-1 and pad2-1) and plants overexpressing the gene encoding γ-glutamylcysteine synthetase, the first enzyme of the glutathione biosynthetic pathway. The plants were exposed to two mild stress conditions—oxidative stress elicited by methyl viologen and stress induced by the limited availability of phosphate. We observed that the mutants and transgenic plants showed similar shoot growth as that of the wild-type plants under mild abiotic stress. We then selected the synthesis mutants and performed multi-platform metabolomics and microarray experiments to evaluate the possible effects on the overall metabolome and the transcriptome. As a common oxidative stress response, several flavonoids that we assessed showed overaccumulation, whereas the mild phosphate stress resulted in increased levels of specific kaempferol- and quercetin-glycosides. Remarkably, in addition to a significant increased level of sugar, osmolytes, and lipids as mild oxidative stress-responsive metabolites, short-chain aliphatic glucosinolates over-accumulated in the mutants, whereas the level of long-chain aliphatic glucosinolates and specific lipids decreased. Coordinated gene expressions related to glucosinolate and flavonoid biosynthesis also supported the metabolite responses in the pad2-1 mutant. Our results suggest that glutathione synthesis mutants accelerate transcriptional regulatory networks to control the biosynthetic pathways involved in glutathione-independent scavenging metabolites, and that they might reconfigure the metabolic networks in primary and secondary metabolism, including lipids, glucosinolates, and flavonoids. This work provides a basis for the elucidation of the molecular mechanisms involved in the metabolic and transcriptional regulatory networks in response to combined low glutathione content with mild oxidative and nutrient stress in A. thaliana. PMID:28894456

Dicer and Argonaute Genes Involved in RNA Interference in the Entomopathogenic Fungus Metarhizium robertsii.

PubMed

Meng, Huimin; Wang, Zhangxun; Wang, Yulong; Zhu, Hong; Huang, Bo

2017-04-01

RNA interference (RNAi) is a gene-silencing mechanism that plays an important role in gene regulation in a number of eukaryotic organisms. Two core components, Dicer and Argonaute, are central in the RNAi machinery. However, the physiological roles of Dicer and Argonaute in the entomopathogenic fungus Metarhizium robertsii have remained unclear. Here, the roles of genes encoding Dicer ( M. robertsii dcl1 [ Mrdcl1 ] and Mrdcl2 ) and Argonaute ( Mrago1 and Mrago2 ) proteins in M. robertsii were investigated. The results showed that the Dicer-like protein MrDCL2 and Argonaute protein MrAGO1 are the major components of the RNAi process occurring in M. robertsii The Dicer and Argonaute genes were not involved in the regulation of growth and diverse abiotic stress response in M. robertsii under the tested conditions. Moreover, our results showed that the Dicer and Argonaute gene mutants demonstrated reduced abilities to produce conidia, compared to the wild type (WT) and the gene-rescued mutant. In particular, the conidial yields in the Δ dcl2 and Δ ago1 mutants were reduced by 55.8% and 59.3%, respectively, compared with those from the control strains. Subsequently, for the WT and Δ dcl2 mutant strains, digital gene expression (DGE) profiling analysis of the stage of mycelium growth and conidiogenesis revealed that modest changes occur in development or metabolism processes, which may explain the reduction in conidiation in the Δ dcl2 mutant. In addition, we further applied high-throughput sequencing technology to identify small RNAs (sRNAs) that are differentially expressed in the WT and the Δ dcl2 mutant and found that 4 known microRNA-like small RNAs (milRNAs) and 8 novel milRNAs were Mrdcl2 dependent in M. robertsii IMPORTANCE The identification and characterization of components in RNAi have contributed significantly to our understanding of the mechanism and functions of RNAi in eukaryotes. Here, we found that Dicer and Argonaute genes play an important role in regulating conidiation in M. robertsii Our study also demonstrates that diverse small RNA pathways exist in M. robertsii The study provides a theoretical platform for exploration of the functions of Dicer and Argonaute genes involved in RNAi in fungi. Copyright © 2017 American Society for Microbiology.

A high constitutive catalase activity confers resistance to methyl viologen-promoted oxidative stress in a mutant of the cyanobacterium Nostoc punctiforme ATCC 29133.

PubMed

Moirangthem, Lakshmipyari Devi; Bhattacharya, Sudeshna; Stensjö, Karin; Lindblad, Peter; Bhattacharya, Jyotirmoy

2014-04-01

A spontaneous methyl viologen (MV)-resistant mutant of the nitrogen-fixing cyanobacterium Nostoc punctiforme ATCC 29133 was isolated and the major enzymatic antioxidants involved in combating MV-induced oxidative stress were evaluated. The mutant displayed a high constitutive catalase activity as a consequence of which, the intracellular level of reactive oxygen species in the mutant was lower than the wild type (N. punctiforme) in the presence of MV. The superoxide dismutase (SOD) activity that consisted of a SodA (manganese-SOD) and a SodB (iron-SOD) was not suppressed in the mutant following MV treatment. The mutant was, however, characterised by a lower peroxidase activity compared with its wild type, and its improved tolerance to externally added H₂O₂ could only be attributed to enhanced catalase activity. Furthermore, MV-induced toxic effects on the wild type such as (1) loss of photosynthetic performance assessed as maximal quantum yield of photosystem II, (2) nitrogenase inactivation, and (3) filament fragmentation and cell lysis were not observed in the mutant. These findings highlight the importance of catalase in preventing MV-promoted oxidative damage and cell death in the cyanobacterium N. punctiforme. Such oxidative stress resistant mutants of cyanobacteria are likely to be a better source of biofertilisers, as they can grow and fix nitrogen in an unhindered manner in agricultural fields that are often contaminated with the herbicide MV, also commonly known as paraquat.

Multiple ABC transporters are involved in the acquisition of petrobactin in Bacillus anthracis

PubMed Central

Dixon, Shandee D.; Janes, Brian K.; Bourgis, Alexandra; Carlson, Paul E.; Hanna, Philip C.

2012-01-01

Summary In Bacillus anthracis the siderophore petrobactin is vital for iron acquisition and virulence. The petrobactin-binding receptor FpuA is required for these processes. Here additional components of petrobactin reacquisition are described. To identify these proteins, mutants of candidate permease and ATPase genes were generated allowing for characterization of multiple petrobactin ATP-binding cassette (ABC)-import systems. Either of two distinct permeases, FpuB or FatCD, are required for iron acquisition and play redundant roles in petrobactin transport. A mutant strain lacking both permeases, ΔfpuBΔfatCD, was incapable of using petrobactin as an iron source and exhibited attenuated virulence in a murine model of inhalational anthrax infection. ATPase mutants were generated in either of the permease mutant backgrounds to identify the ATPase(s) interacting with each individual permease channel. Mutants lacking the FpuB permease and FatE ATPase (ΔfpuBΔfatE) and a mutant lacking the distinct ATPases FpuC and FpuD generated in the ΔfatCD background (ΔfatCDΔfpuCΔfpuD) displayed phenotypic characteristics of a mutant deficient in petrobactin import. A mutant lacking all three of the identified ATPases (ΔfatEΔfpuCΔfpuD) exhibited the same growth defect in iron-depleted conditions. Taken together, these results provide the first description of the permease and ATPase proteins required for the import of petrobactin in B. anthracis. PMID:22429808

Analysis of dofA, a fruA-Dependent Developmental Gene, and Its Homologue, dofB, in Myxococcus xanthus

PubMed Central

Horiuchi, Takayuki; Akiyama, Takuya; Inouye, Sumiko; Komano, Teruya

2002-01-01

The developmentally regulated gene dofA, identified from pulse-labeling experiments by two-dimensional gel electrophoresis, and its homologue, dofB, were cloned and characterized in Myxococcus xanthus. Deletion of dofA and dofB did not affect the vegetative growth and development of M. xanthus. dofA was specifically expressed during development, while dofB expression was observed during vegetative growth and development. The dofA-lacZ fusion was introduced into a fruA mutant and A, B, C, D, and E extracellular signal mutants. The pattern of dofA expression in the C signal mutant was similar to that of the wild-type strain, while dofA expression was not detected in the fruA mutant. These results are consistent with those of the pulse-labeling experiments. dofA expression was reduced in A and E signal mutants, whereas dofA expression was delayed in B and D signal mutants. The patterns of expression of the dofA gene in the fruA mutant and the five signal mutants are strikingly similar to that of the tps gene, which encodes protein S, a major component of the outer surface of the myxospore; this result suggests that the dofA and tps genes are similarly regulated. The involvement of a highly GC-rich inverted repeat sequence (underlined), CGGCCCCCGATTCGTCGGGGGCCG, in developmentally regulated dofA expression is suggested. PMID:12446630

Characterization of oxidative phosphorylation enzymes in Euglena gracilis and its white mutant strain W(gm)ZOflL.

PubMed

Krnáčová, Katarína; Rýdlová, Ivana; Vinarčíková, Michaela; Krajčovič, Juraj; Vesteg, Matej; Horváth, Anton

2015-03-12

The enzymes involved in Euglena oxidative phosphorylation (OXPHOS) were characterized in this study. We have demonstrated that Euglena gracilis strain Z and its stable bleached non-photosynthetic mutant strain WgmZOflL both possess fully functional OXPHOS apparatus as well as pathways requiring terminal alternative oxidase(s) and alternative mitochondrial NADH-dehydrogenase(s). Light (or dark) and plastid (non)functionality seem to have little effect on oxygen consumption, the activities of the enzymes involved in OXPHOS and the action of respiration inhibitors in Euglena. This study also demonstrates biochemical properties of complex III (cytochrome c reductase) in Euglena. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

A novel protein involved in heart development in Ambystoma mexicanum is localized in endoplasmic reticulum.

PubMed

Jia, P; Zhang, C; Huang, X P; Poda, M; Akbas, F; Lemanski, S L; Erginel-Unaltuna, N; Lemanski, L F

2008-11-01

The discovery of the naturally occurring cardiac non-function (c) animal strain in Ambystoma mexicanum (axolotl) provides a valuable animal model to study cardiomyocyte differentiation. In homozygous mutant animals (c/c), rhythmic contractions of the embryonic heart are absent due to a lack of organized myofibrils. We have previously cloned a partial sequence of a peptide cDNA (N1) from an anterior-endoderm-conditioned-medium RNA library that had been shown to be able to rescue the mutant phenotype. In the current studies we have fully cloned the N1 full length cDNA sequence from the library. N1 protein has been detected in both adult heart and skeletal muscle but not in any other adult tissues. GFP-tagged expression of the N1 protein has revealed localization of the N1 protein in the endoplasmic reticulum (ER). Results from in situ hybridization experiments have confirmed the dramatic decrease of expression of N1 mRNA in mutant (c/c) embryos indicating that the N1 gene is involved in heart development.

Identification of new adventitious rooting mutants amongst suppressors of the Arabidopsis thaliana superroot2 mutation.

PubMed

Pacurar, Daniel Ioan; Pacurar, Monica Lacramioara; Bussell, John Desmond; Schwambach, Joseli; Pop, Tiberia Ioana; Kowalczyk, Mariusz; Gutierrez, Laurent; Cavel, Emilie; Chaabouni, Salma; Ljung, Karin; Fett-Neto, Arthur Germano; Pamfil, Doru; Bellini, Catherine

2014-04-01

The plant hormone auxin plays a central role in adventitious rooting and is routinely used with many economically important, vegetatively propagated plant species to promote adventitious root initiation and development on cuttings. Nevertheless the molecular mechanisms through which it acts are only starting to emerge. The Arabidopsis superroot2-1 (sur2-1) mutant overproduces auxin and, as a consequence, develops excessive adventitious roots in the hypocotyl. In order to increase the knowledge of adventitious rooting and of auxin signalling pathways and crosstalk, this study performed a screen for suppressors of superroot2-1 phenotype. These suppressors provide a new resource for discovery of genetic players involved in auxin signalling pathways or at the crosstalk of auxin and other hormones or environmental signals. This study reports the identification and characterization of 26 sur2-1 suppressor mutants, several of which were identified as mutations in candidate genes involved in either auxin biosynthesis or signalling. In addition to confirming the role of auxin as a central regulator of adventitious rooting, superroot2 suppressors indicated possible crosstalk with ethylene signalling in this process.

Hfq is a global regulator that controls the pathogenicity of Staphylococcus aureus.

PubMed

Liu, Yu; Wu, Na; Dong, Jie; Gao, Yaping; Zhang, Xin; Mu, Chunhua; Shao, Ningsheng; Yang, Guang

2010-09-29

The Hfq protein is reported to be an RNA chaperone, which is involved in the stress response and the virulence of several pathogens. In E. coli, Hfq can mediate the interaction between some sRNAs and their target mRNAs. But it is controversial whether Hfq plays an important role in S. aureus. In this study, we found that the deletion of hfq gene in S. aureus 8325-4 can increase the surface carotenoid pigments. The hfq mutant was more resistant to oxidative stress but the pathogenicity of the mutant was reduced. We reveal that the Hfq protein can be detected only in some S. aureus strains. Using microarray and qRT-PCR, we identified 116 genes in the hfq mutant which had differential expression from the wild type, most of which are related to the phenotype and virulence of S. aureus. Among the 116 genes, 49 mRNAs can specifically bind Hfq protein, which indicates that Hfq also acts as an RNA binding protein in S. aureus. Our data suggest that Hfq protein of S. aureus is a multifunctional regulator involved in stress and virulence.

Sigma-1 receptor agonist PRE084 is protective against mutant huntingtin-induced cell degeneration: involvement of calpastatin and the NF-κB pathway

PubMed Central

Hyrskyluoto, A; Pulli, I; Törnqvist, K; Huu Ho, T; Korhonen, L; Lindholm, D

2013-01-01

Alterations in mitochondria and increased oxidative stress are associated with the disease progression in Huntington's disease (HD). Endoplasmic reticulum (ER) stress and oxidative damage are linked through the close communication between the ER and mitochondria. Sigma-1 receptor (Sig-1R) is a chaperone protein in the ER that is involved in ER stress regulation, but little is known about its role in HD or the mechanisms for cell protection. Here we show that the Sig-1R agonist, PRE084 increases cell survival and counteracts the deleterious effects caused by N-terminal mutant huntingtin proteins in neuronal PC6.3 cells. Particularly, PRE084 increased the levels of cellular antioxidants by activating the NF-κB pathway that is compromised by the expression of mutant huntingtin proteins. These results show that the Sig-1R agonist has beneficial effects in models of HD and that compounds affecting the Sig-1R may be promising targets for future drug development in HD. PMID:23703391

Inviability of a DNA2 deletion mutant is due to the DNA damage checkpoint.

PubMed

Budd, Martin E; Antoshechkin, Igor A; Reis, Clara; Wold, Barbara J; Campbell, Judith L

2011-05-15

Dna2 is a dual polarity exo/endonuclease, and 5' to 3' DNA helicase involved in Okazaki Fragment Processing (OFP) and Double-Strand Break (DSB) Repair. In yeast, DNA2 is an essential gene, as expected for a DNA replication protein. Suppression of the lethality of dna2Δ mutants has been found to occur by two mechanisms: overexpression of RAD27 (scFEN1) , encoding a 5' to 3' exo/endo nuclease that processes Okazaki fragments (OFs) for ligation, or deletion of PIF1, a 5' to 3' helicase involved in mitochondrial recombination, telomerase inhibition and OFP. Mapping of a novel, spontaneously arising suppressor of dna2Δ now reveals that mutation of rad9 and double mutation of rad9 mrc1 can also suppress the lethality of dna2Δ mutants. Interaction of dna2Δ and DNA damage checkpoint mutations provides insight as to why dna2Δ is lethal but rad27Δ is not, even though evidence shows that Rad27 (ScFEN1) processes most of the Okazaki fragments, while Dna2 processes only a subset.

Mouse Tmem135 mutation reveals a mechanism involving mitochondrial dynamics that leads to age-dependent retinal pathologies

PubMed Central

Lee, Wei-Hua; Higuchi, Hitoshi; Ikeda, Sakae; Macke, Erica L; Takimoto, Tetsuya; Pattnaik, Bikash R; Liu, Che; Chu, Li-Fang; Siepka, Sandra M; Krentz, Kathleen J; Rubinstein, C Dustin; Kalejta, Robert F; Thomson, James A; Mullins, Robert F; Takahashi, Joseph S; Pinto, Lawrence H; Ikeda, Akihiro

2016-01-01

While the aging process is central to the pathogenesis of age-dependent diseases, it is poorly understood at the molecular level. We identified a mouse mutant with accelerated aging in the retina as well as pathologies observed in age-dependent retinal diseases, suggesting that the responsible gene regulates retinal aging, and its impairment results in age-dependent disease. We determined that a mutation in the transmembrane 135 (Tmem135) is responsible for these phenotypes. We observed localization of TMEM135 on mitochondria, and imbalance of mitochondrial fission and fusion in mutant Tmem135 as well as Tmem135 overexpressing cells, indicating that TMEM135 is involved in the regulation of mitochondrial dynamics. Additionally, mutant retina showed higher sensitivity to oxidative stress. These results suggest that the regulation of mitochondrial dynamics through TMEM135 is critical for protection from environmental stress and controlling the progression of retinal aging. Our study identified TMEM135 as a critical link between aging and age-dependent diseases. DOI: http://dx.doi.org/10.7554/eLife.19264.001 PMID:27863209

Global gene expression during stringent response in Corynebacterium glutamicum in presence and absence of the rel gene encoding (p)ppGpp synthase

PubMed Central

Brockmann-Gretza, Olaf; Kalinowski, Jörn

2006-01-01

Background The stringent response is the initial reaction of microorganisms to nutritional stress. During stringent response the small nucleotides (p)ppGpp act as global regulators and reprogram bacterial transcription. In this work, the genetic network controlled by the stringent response was characterized in the amino acid-producing Corynebacterium glutamicum. Results The transcriptome of a C. glutamicum rel gene deletion mutant, unable to synthesize (p)ppGpp and to induce the stringent response, was compared with that of its rel-proficient parent strain by microarray analysis. A total of 357 genes were found to be transcribed differentially in the rel-deficient mutant strain. In a second experiment, the stringent response was induced by addition of DL-serine hydroxamate (SHX) in early exponential growth phase. The time point of the maximal effect on transcription was determined by real-time RT-PCR using the histidine and serine biosynthetic genes. Transcription of all of these genes reached a maximum at 10 minutes after SHX addition. Microarray experiments were performed comparing the transcriptomes of SHX-induced cultures of the rel-proficient strain and the rel mutant. The differentially expressed genes were grouped into three classes. Class A comprises genes which are differentially regulated only in the presence of an intact rel gene. This class includes the non-essential sigma factor gene sigB which was upregulated and a large number of genes involved in nitrogen metabolism which were downregulated. Class B comprises genes which were differentially regulated in response to SHX in both strains, independent of the rel gene. A large number of genes encoding ribosomal proteins fall into this class, all being downregulated. Class C comprises genes which were differentially regulated in response to SHX only in the rel mutant. This class includes genes encoding putative stress proteins and global transcriptional regulators that might be responsible for the complex transcriptional patterns detected in the rel mutant when compared directly with its rel-proficient parent strain. Conclusion In C. glutamicum the stringent response enfolds a fast answer to an induced amino acid starvation on the transcriptome level. It also showed some significant differences to the transcriptional reactions occuring in Escherichia coli and Bacillus subtilis. Notable are the rel-dependent regulation of the nitrogen metabolism genes and the rel-independent regulation of the genes encoding ribosomal proteins. PMID:16961923

The deubiquitinating enzyme AMSH1 is required for rhizobial infection and nodule organogenesis in Lotus japonicus.

PubMed

Małolepszy, Anna; Urbański, Dorian Fabian; James, Euan K; Sandal, Niels; Isono, Erika; Stougaard, Jens; Andersen, Stig Uggerhøj

2015-08-01

Legume-rhizobium symbiosis contributes large quantities of fixed nitrogen to both agricultural and natural ecosystems. This global impact and the selective interaction between rhizobia and legumes culminating in development of functional root nodules have prompted detailed studies of the underlying mechanisms. We performed a screen for aberrant nodulation phenotypes using the Lotus japonicus LORE1 insertion mutant collection. Here, we describe the identification of amsh1 mutants that only develop small nodule primordia and display stunted shoot growth, and show that the aberrant nodulation phenotype caused by LORE1 insertions in the Amsh1 gene may be separated from the shoot phenotype. In amsh1 mutants, rhizobia initially became entrapped in infection threads with thickened cells walls. Some rhizobia were released into plant cells much later than observed for the wild-type; however, no typical symbiosome structures were formed. Furthermore, cytokinin treatment only very weakly induced nodule organogenesis in amsh1 mutants, suggesting that AMSH1 function is required downstream of cytokinin signaling. Biochemical analysis showed that AMSH1 is an active deubiquitinating enzyme, and that AMSH1 specifically cleaves K63-linked ubiquitin chains. Post-translational ubiquitination and deubiquitination processes involving the AMSH1 deubiquitinating enzyme are thus involved in both infection and organogenesis in Lotus japonicus. © 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.

The zebrafish mutant vps18 as a model for vesicle-traffic related hypopigmentation diseases.

PubMed

Maldonado, Ernesto; Hernandez, Fabiola; Lozano, Carlos; Castro, Marta E; Navarro, Rosa E

2006-08-01

Hypopigmentation is a characteristic of several diseases associated with vesicle traffic defects, like the Hermansky-Pudlak, Chediak-Higashi, and Griscelli syndromes. Hypopigmentation is also a characteristic of the zebrafish mutant vps18(hi2499A), which is affected in the gene vps18, a component of the homotypic fusion and protein sorting complex that is involved in tethering during vesicular traffic. Vps18, as part of this complex, participates in the formation of early endosomes, late endosomes, and lysosomes. Here, we show that Vps18 is also involved in the formation of melanosomes. In the zebrafish mutant vps18(hi2499A) the retroviral insertion located at exon 4 of vps18, leads to the formation of two abnormal splicing variants lacking the coding sequence for the clathrin repeat and the RING finger conserved domains. A deficiency of Vps18 in zebrafish larvae results in hepatomegaly and skin hypopigmentation. We also observed a drastic reduction in the number of melanosomes in the eye's retinal pigmented epithelium along with the accumulation of immature melanosomes. A significant reduction in the vps18(hi2499A) larvae visual system capacity was found using the optokinetic response assay. We propose that the insertional mutant vps18(hi2499A) can be used as a model for studying hypopigmentation diseases in which vesicle traffic problems exist.

Partial Müllerian Duct Retention in Smad4 Conditional Mutant Male Mice.

PubMed

Petit, Fabrice G; Deng, Chuxia; Jamin, Soazik P

2016-01-01

Müllerian duct regression is a complex process which involves the AMH signalling pathway. We have previously demonstrated that besides AMH and its specific type II receptor (AMHRII), BMPR-IA and Smad5 are two essential factors implicated in this mechanism. Mothers against decapentaplegic homolog 4 (Smad4) is a transcription factor and the common Smad (co-Smad) involved in transforming growth factor beta (TGF-β) signalling pathway superfamily. Since Smad4 null mutants die early during gastrulation, we have inactivated Smad4 in the Müllerian duct mesenchyme. Specific inactivation of Smad4 in the urogenital ridge leads to the partial persistence of the Müllerian duct in adult male mice. Careful examination of the urogenital tract reveals that the Müllerian duct retention is randomly distributed either on one side or both sides. Histological analysis shows a uterus-like structure, which is confirmed by the expression of estrogen receptor α. As previously described in a β-catenin conditional mutant mouse model, β-catenin contributes to Müllerian duct regression. In our mutant male embryos, it appears that β-catenin expression is locally reduced along the urogenital ridge as compared to control mice. Moreover, the expression pattern is similar to those observed in control female mice. This study shows that reduced Smad4 expression disrupts the Wnt/β-catenin signalling leading to the partial persistence of Müllerian duct.

Life-span extension by dietary restriction is mediated by NLP-7 signaling and coelomocyte endocytosis in C. elegans.

PubMed

Park, Sang-Kyu; Link, Christopher D; Johnson, Thomas E

2010-02-01

Recent studies have shown that the rate of aging can be modulated by diverse interventions. Dietary restriction is the most widely used intervention to promote longevity; however, the mechanisms underlying the effect of dietary restriction remain elusive. In a previous study, we identified two novel genes, nlp-7 and cup-4, required for normal longevity in Caenorhabditis elegans. nlp-7 is one of a set of neuropeptide-like protein genes; cup-4 encodes an ion-channel involved in endocytosis by coelomocytes. Here, we assess whether nlp-7 and cup-4 mediate longevity increases by dietary restriction. RNAi of nlp-7 or cup-4 significantly reduces the life span of the eat-2 mutant, a genetic model of dietary restriction, but has no effect on the life span of long-lived mutants resulting from reduced insulin/IGF-1 signaling or dysfunction of the mitochondrial electron transport chain. The life-span extension observed in wild-type N2 worms by dietary restriction using bacterial dilution is prevented significantly in nlp-7 and cup-4 mutants. RNAi knockdown of genes encoding candidate receptors of NLP-7 and genes involved in endocytosis by coelomocytes also specifically shorten the life span of the eat-2 mutant. We conclude that two novel pathways, NLP-7 signaling and endocytosis by coelomocytes, are required for life extension under dietary restriction in C. elegans.

Gibberellin modulates anther development in rice via the transcriptional regulation of GAMYB.

PubMed

Aya, Koichiro; Ueguchi-Tanaka, Miyako; Kondo, Maki; Hamada, Kazuki; Yano, Kentaro; Nishimura, Mikio; Matsuoka, Makoto

2009-05-01

Gibberellins (GAs) play important roles in regulating reproductive development, especially anther development. Our previous studies revealed that the MYB transcriptional factor GAMYB, an important component of GA signaling in cereal aleurone cells, is also important for anther development. Here, we examined the physiological functions of GA during anther development through phenotypic analyses of rice (Oryza sativa) GA-deficient, GA-insensitive, and gamyb mutants. The mutants exhibited common defects in programmed cell death (PCD) of tapetal cells and formation of exine and Ubisch bodies. Microarray analysis using anther RNAs of these mutants revealed that rice GAMYB is involved in almost all instances of GA-regulated gene expression in anthers. Among the GA-regulated genes, we focused on two lipid metabolic genes, a cytochrome P450 hydroxylase CYP703A3 and beta-ketoacyl reductase, both of which might be involved in providing a substrate for exine and Ubisch body. GAMYB specifically interacted with GAMYB binding motifs in the promoter regions in vitro, and mutation of these motifs in promoter-beta-glucuronidase (GUS) transformants caused reduced GUS expression in anthers. Furthermore, a knockout mutant for CYP703A3 showed gamyb-like defects in exine and Ubisch body formation. Together, these results suggest that GA regulates exine formation and the PCD of tapetal cells and that direct activation of CYP703A3 by GAMYB is key to exine formation.

Comparative proteomics of a tor inducible Aspergillus fumigatus mutant reveals involvement of the Tor kinase in iron regulation.

PubMed

Baldin, Clara; Valiante, Vito; Krüger, Thomas; Schafferer, Lukas; Haas, Hubertus; Kniemeyer, Olaf; Brakhage, Axel A

2015-07-01

The Tor (target of rapamycin) kinase is one of the major regulatory nodes in eukaryotes. Here, we analyzed the Tor kinase in Aspergillus fumigatus, which is the most important airborne fungal pathogen of humans. Because deletion of the single tor gene was apparently lethal, we generated a conditional lethal tor mutant by replacing the endogenous tor gene by the inducible xylp-tor gene cassette. By both 2DE and gel-free LC-MS/MS, we found that Tor controls a variety of proteins involved in nutrient sensing, stress response, cell cycle progression, protein biosynthesis and degradation, but also processes in mitochondria, such as respiration and ornithine metabolism, which is required for siderophore formation. qRT-PCR analyses indicated that mRNA levels of ornithine biosynthesis genes were increased under iron limitation. When tor was repressed, iron regulation was lost. In a deletion mutant of the iron regulator HapX also carrying the xylp-tor cassette, the regulation upon iron deprivation was similar to that of the single tor inducible mutant strain. In line, hapX expression was significantly reduced when tor was repressed. Thus, Tor acts either upstream of HapX or independently of HapX as a repressor of the ornithine biosynthesis genes and thereby regulates the production of siderophores. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Putrescine Is Involved in Arabidopsis Freezing Tolerance and Cold Acclimation by Regulating Abscisic Acid Levels in Response to Low Temperature1

PubMed Central

Cuevas, Juan C.; López-Cobollo, Rosa; Alcázar, Rubén; Zarza, Xavier; Koncz, Csaba; Altabella, Teresa; Salinas, Julio; Tiburcio, Antonio F.; Ferrando, Alejandro

2008-01-01

The levels of endogenous polyamines have been shown to increase in plant cells challenged with low temperature; however, the functions of polyamines in the regulation of cold stress responses are unknown. Here, we show that the accumulation of putrescine under cold stress is essential for proper cold acclimation and survival at freezing temperatures because Arabidopsis (Arabidopsis thaliana) mutants defective in putrescine biosynthesis (adc1, adc2) display reduced freezing tolerance compared to wild-type plants. Genes ADC1 and ADC2 show different transcriptional profiles upon cold treatment; however, they show similar and redundant contributions to cold responses in terms of putrescine accumulation kinetics and freezing sensitivity. Our data also demonstrate that detrimental consequences of putrescine depletion during cold stress are due, at least in part, to alterations in the levels of abscisic acid (ABA). Reduced expression of NCED3, a key gene involved in ABA biosynthesis, and down-regulation of ABA-regulated genes are detected in both adc1 and adc2 mutant plants under cold stress. Complementation analysis of adc mutants with ABA and reciprocal complementation tests of the aba2-3 mutant with putrescine support the conclusion that putrescine controls the levels of ABA in response to low temperature by modulating ABA biosynthesis and gene expression. PMID:18701673

Molecular Basis for Antioxidant Enzymes in Mediating Copper Detoxification in the Nematode Caenorhabditis elegans

PubMed Central

Song, Shaojuan; Zhang, Xueyao; Wu, Haihua; Han, Yan; Zhang, Jianzhen; Ma, Enbo; Guo, Yaping

2014-01-01

Antioxidant enzymes play a major role in defending against oxidative damage by copper. However, few studies have been performed to determine which antioxidant enzymes respond to and are necessary for copper detoxification. In this study, we examined both the activities and mRNA levels of SOD, CAT, and GPX under excessive copper stress in Caenorhabditis elegans, which is a powerful model for toxicity studies. Then, taking advantage of the genetics of this model, we assessed the lethal concentration (LC50) values of copper for related mutant strains. The results showed that the SOD, CAT, and GPX activities were significantly greater in treated groups than in controls. The mRNA levels of sod-3, sod-5, ctl-1, ctl-2, and almost all gpx genes were also significantly greater in treated groups than in controls. Among tested mutants, the sod-5, ctl-1, gpx-3, gpx-4, and gpx-6 variants exhibited hypersensitivity to copper. The strains with SOD or CAT over expression were reduced sensitive to copper. Mutations in daf-2 and age-1, which are involved in the insulin/insulin-like growth factor-1 signaling pathway, result in reduced sensitivity to stress. Here, we showed that LC50 values for copper in daf-2 and age-1 mutants were significantly greater than in N2 worms. However, the LC50 values in daf-16;daf-2 and daf-16;age-1 mutants were significantly reduced than in daf-2 and age-1 mutants, implying that reduced copper sensitivity is influenced by DAF-16-related functioning. SOD, CAT, and GPX activities and the mRNA levels of the associated copper responsive genes were significantly increased in daf-2 and age-1 mutants compared to N2. Additionally, the activities of SOD, CAT, and GPX were greater in these mutants than in N2 when treated with copper. Our results not only support the theory that antioxidant enzymes play an important role in copper detoxification but also identify the response and the genes involved in these processes. PMID:25243607

The ftsH gene of Bacillus subtilis is involved in major cellular processes such as sporulation, stress adaptation and secretion.

PubMed

Deuerling, E; Mogk, A; Richter, C; Purucker, M; Schumann, W

1997-03-01

The ftsH gene of Bacillus subtilis has been identified as a general stress gene which is transiently induced after thermal or osmotic upshift. The FtsH protein exhibits 70.1% homology to FtsH of Escherichia coli which constitutes an essential ATP- and Zn(2+)-dependent protease anchored in the cytoplasmic membrane via two N-terminal transmembrane domains. This paper describes the isolation and functional characterization of an ftsH null mutant which was obtained by integration of a cat-cassette near the 5' end of ftsH, thereby preventing the synthesis of FtsH protein. In contrast to the situation in E. coli, ftsH is dispensable in B. subtilis but results in a pleiotropic phenotype. While the mutant cells grew mostly as large filaments under physiological conditions, they turned out to be extremely sensitive to heat and salt stress. Although ftsH is necessary for adaptation to heat, it is not involved in the regulation of the heat-shock response. The induction profiles of representative genes of the CIRCE and sigma-B regulon and class III heat-shock genes ion and clpC were identical in the wild type and the ftsH null mutant. Furthermore, the ftsH knockout strain was unable to sporulate, and this failure was probably due to the absence of Spo0A protein which is essential for entry into the sporulation programme. In addition, secretion of bulk exoproteins was severely impaired in the ftsH null mutant after entry into stationary phase. The alpha-amylase and subtilisin activity in the supernatant was specifically tested. Whereas the activity of alpha-amylase increased after entry into stationary phase in both the wild type and the ftsH mutant strain, that of subtilisin encoded by aprE was prevented at the level of transcription in the mutant. Most of these results can be explained by the failure to synthesize appropriate amounts of Spo0A protein in the ftsH null mutant and point to ftsH as a developmental checkpoint.

Type 1 fimbriae and extracellular polysaccharides are preeminent uropathogenic Escherichia coli virulence determinants in the murine urinary tract.

PubMed

Bahrani-Mougeot, Farah K; Buckles, Eric L; Lockatell, C V; Hebel, J R; Johnson, D E; Tang, C M; Donnenberg, M S

2002-08-01

Escherichia coli is the leading cause of urinary tract infections (UTIs). Despite the association of numerous bacterial factors with uropathogenic E. coli (UPEC), few such factors have been proved to be required for UTI in animal models. Previous investigations of urovirulence factors have relied on prior identification of phenotypic characteristics. We used signature-tagged mutagenesis (STM) in an unbiased effort to identify genes that are essential for UPEC survival within the murine urinary tract. A library of 2049 transposon mutants of the prototypic UPEC strain CFT073 was constructed using mini-Tn5km2 carrying 92 unique tags and screened in a murine model of ascending UTI. After initial screening followed by confirmation in co-infection experiments, 19 survival-defective mutants were identified. These mutants were recovered in numbers 101- to 106-fold less than the wild type in the bladder, kidneys or urine or at more than one site. The transposon junctions from each attenuated mutant were sequenced and analysed. Mutations were found in: (i) the type 1 fimbrial operon; (ii) genes involved in the biosyn-thesis of extracellular polysaccharides including group I capsule, group II capsule and enterobacterial common antigen; (iii) genes involved in metabolic pathways; and (iv) genes with unknown function. Five of the genes identified are absent from the genome of the E. coli K-12 strain. Mutations in type 1 fimbrial genes resulted in severely attenuated colonization, even in the case of a mutant with an insertion upstream of the fim operon that affected the rate of fimbrial switching from the 'off' to the 'on' phase. Three mutants had insertions in a new type II capsule biosynthesis locus on a pathogenicity island and were impaired in the production of capsule in vivo. An additional mutant with an insertion in wecE was unable to synthesize enterobacterial common antigen. These results confirm the pre-eminence of type 1 fimbriae, establish the importance of extracellular polysaccharides in the pathogenesis of UTI and identify new urovirulence determinants.

Cellulose Synthesis in Agrobacterium tumefaciens

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

Alan R. White; Ann G. Matthysse

2004-07-31

We have cloned the celC gene and its homologue from E. coli, yhjM, in an expression vector and expressed the both genes in E. coli; we have determined that the YhjM protein is able to complement in vitro cellulose synthesis by extracts of A. tumefaciens celC mutants, we have purified the YhjM protein product and are currently examining its enzymatic activity; we have examined whole cell extracts of CelC and various other cellulose mutants and wild type bacteria for the presence of cellulose oligomers and cellulose; we have examined the ability of extracts of wild type and cellulose mutants includingmore » CelC to incorporate UDP-14C-glucose into cellulose and into water-soluble, ethanol-insoluble oligosaccharides; we have made mutants which synthesize greater amounts of cellulose than the wild type; and we have examined the role of cellulose in the formation of biofilms by A. tumefaciens. In addition we have examined the ability of a putative cellulose synthase gene from the tunicate Ciona savignyi to complement an A. tumefaciens celA mutant. The greatest difference between our knowledge of bacterial cellulose synthesis when we started this project and current knowledge is that in 1999 when we wrote the original grant very few bacteria were known to synthesize cellulose and genes involved in this synthesis were sequenced only from Acetobacter species, A. tumefaciens and Rhizobium leguminosarum. Currently many bacteria are known to synthesize cellulose and genes that may be involved have been sequenced from more than 10 species of bacteria. This additional information has raised the possibility of attempting to use genes from one bacterium to complement mutants in another bacterium. This will enable us to examine the question of which genes are responsible for the three dimensional structure of cellulose (since this differs among bacterial species) and also to examine the interactions between the various proteins required for cellulose synthesis. We have carried out one preliminary experiment of this type and have successfully complemented an A. tumefaciens CelC mutant with the homologous gene (yhjM) from E. coli.« less

A disruption of ctpA encoding carboxy-terminal protease attenuates Burkholderia mallei and induces partial protection in CD1 mice.

PubMed

Bandara, Aloka B; DeShazer, David; Inzana, Thomas J; Sriranganathan, Nammalwar; Schurig, Gerhardt G; Boyle, Stephen M

2008-09-01

Burkholderia mallei is the etiologic agent of glanders in solipeds (horses, mules and donkeys), and incidentally in carnivores and humans. Little is known about the molecular mechanisms of B. mallei pathogenesis. The putative carboxy-terminal processing protease (CtpA) of B. mallei is a member of a novel family of endoproteases involved in the maturation of proteins destined for the cell envelope. All species and isolates of Burkholderia carry a highly conserved copy of ctpA. We studied the involvement of CtpA on growth, cell morphology, persistence, and pathogenicity of B. mallei. A sucrose-resistant strain of B. mallei was constructed by deleting a major portion of the sacB gene of the wild type strain ATCC 23344 by gene replacement, and designated as strain 23344DeltasacB. A portion of the ctpA gene (encoding CtpA) of strain 23344DeltasacB was deleted by gene replacement to generate strain 23344DeltasacBDeltactpA. In contrast to the wild type ATCC 23344 or the sacB mutant 23344DeltasacB, the ctpA mutant 23344DeltasacBDeltactpA displayed altered cell morphologies with partially or fully disintegrated cell envelopes. Furthermore, relative to the wild type, the ctpA mutant displayed slower growth in vitro and less ability to survive in J774.2 murine macrophages. The expression of mRNA of adtA, the gene downstream of ctpA was similar among the three strains suggesting that disruption of ctpA did not induce any polar effects. As with the wild type or the sacB mutant, the ctpA mutant exhibited a dose-dependent lethality when inoculated intraperitoneally into CD1 mice. The CD1 mice inoculated with a non-lethal dose of the ctpA mutant produced specific serum immunoglobulins IgG1 and IgG2a and were partially protected against challenge with wild type B. mallei ATCC 23344. These findings suggest that CtpA regulates in vitro growth, cell morphology and intracellular survival of B. mallei, and a ctpA mutant protects CD1 mice against glanders.

FLOWERING LOCUS C (FLC) regulates development pathways throughout the life cycle of Arabidopsis

PubMed Central

Deng, Weiwei; Ying, Hua; Helliwell, Chris A.; Taylor, Jennifer M.; Peacock, W. James; Dennis, Elizabeth S.

2011-01-01

FLOWERING LOCUS C (FLC) has a key role in the timing of the initiation of flowering in Arabidopsis. FLC binds and represses two genes that promote flowering, FT and SOC1. We show that FLC binds to many other genes, indicating that it has regulatory roles other than the repression of flowering. We identified 505 FLC binding sites, mostly located in the promoter regions of genes and containing at least one CArG box, the motif known to be associated with MADS-box proteins such as FLC. We examined 40 of the target genes, and 20 showed increased transcript levels in an flc mutant compared with the wild type. Five genes showed decreased expression in the mutant, indicating that FLC binding can result in either transcriptional repression or activation. The genes we identified as FLC targets are involved in developmental pathways throughout the life history of the plant, many of which are associated with reproductive development. FLC is also involved in vegetative development, as evidenced by its binding to SPL15, delaying the progression from juvenile to adult phase. Some of the FLC target genes are also bound by two other MADS-box proteins, AP1 and SEP3, suggesting that MADS-box genes may operate in a network of control at different stages of the life cycle, many ultimately contributing to the development of the reproductive phase of the plant. PMID:21464308

FLOWERING LOCUS C (FLC) regulates development pathways throughout the life cycle of Arabidopsis.

PubMed

Deng, Weiwei; Ying, Hua; Helliwell, Chris A; Taylor, Jennifer M; Peacock, W James; Dennis, Elizabeth S

2011-04-19

FLOWERING LOCUS C (FLC) has a key role in the timing of the initiation of flowering in Arabidopsis. FLC binds and represses two genes that promote flowering, FT and SOC1. We show that FLC binds to many other genes, indicating that it has regulatory roles other than the repression of flowering. We identified 505 FLC binding sites, mostly located in the promoter regions of genes and containing at least one CArG box, the motif known to be associated with MADS-box proteins such as FLC. We examined 40 of the target genes, and 20 showed increased transcript levels in an flc mutant compared with the wild type. Five genes showed decreased expression in the mutant, indicating that FLC binding can result in either transcriptional repression or activation. The genes we identified as FLC targets are involved in developmental pathways throughout the life history of the plant, many of which are associated with reproductive development. FLC is also involved in vegetative development, as evidenced by its binding to SPL15, delaying the progression from juvenile to adult phase. Some of the FLC target genes are also bound by two other MADS-box proteins, AP1 and SEP3, suggesting that MADS-box genes may operate in a network of control at different stages of the life cycle, many ultimately contributing to the development of the reproductive phase of the plant.

Activation of cyclic electron flow by hydrogen peroxide in vivo

DOE PAGES

Strand, Deserah D.; Livingston, Aaron K.; Satoh-Cruz, Mio; ...

2015-04-13

Cyclic electron flow (CEF) around photosystem I is thought to balance the ATP/NADPH energy budget of photosynthesis, requiring that its rate be finely regulated. The mechanisms of this regulation are not well understood. We observed that mutants that exhibited constitutively high rates of CEF also showed elevated production of H 2O 2. We thus tested the hypothesis that CEF can be activated by H 2O 2 in vivo. CEF was strongly increased by H 2O 2 both by infiltration or in situ production by chloroplast-localized glycolate oxidase, implying that H 2O 2 can activate CEF either directly by redox modulationmore » of key enzymes, or indirectly by affecting other photosynthetic processes. CEF appeared with a half time of about 20 min after exposure to H 2O 2, suggesting activation of previously expressed CEF-related machinery. H 2O 2-dependent CEF was not sensitive to antimycin A or loss of PGR5, indicating that increased CEF probably does not involve the PGR5-PGRL1 associated pathway. In contrast, the rise in CEF was not observed in a mutant deficient in the chloroplast NADPH:PQ reductase (NDH), supporting the involvement of this complex in CEF activated by H 2O 2. In conclusion, we propose that H 2O 2 is a missing link between environmental stress, metabolism, and redox regulation of CEF in higher plants.« less

Mycobacterium avium Genes Associated with the Ability To Form a Biofilm

PubMed Central

Yamazaki, Yoshitaka; Danelishvili, Lia; Wu, Martin; MacNab, Molly; Bermudez, Luiz E.

2006-01-01

Mycobacterium avium is widely distributed in the environment, and it is chiefly found in water and soil. M. avium, as well as Mycobacterium smegmatis, has been recognized to produce a biofilm or biofilm-like structure. We screened an M. avium green fluorescent protein (GFP) promoter library in M. smegmatis for genes involved in biofilm formation on polyvinyl chloride (PVC) plates. Clones associated with increased GFP expression ≥2.0-fold over the baseline were sequenced. Seventeen genes, most encoding proteins of the tricarboxylic acid (TCA) cycle and GDP-mannose and fatty acid biosynthesis, were identified. Their regulation in M. avium was confirmed by examining the expression of a set of genes by real-time PCR after incubation on PVC plates. In addition, screening of 2,000 clones of a transposon mutant bank constructed using M. avium strain A5, a mycobacterial strain with the ability to produce large amounts of biofilm, revealed four mutants with an impaired ability to form biofilm. Genes interrupted by transposons were homologues of M. tuberculosis 6-oxodehydrogenase (sucA), enzymes of the TCA cycle, protein synthetase (pstB), enzymes of glycopeptidolipid (GPL) synthesis, and Rv1565c (a hypothetical membrane protein). In conclusion, it appears that GPL biosynthesis, including the GDP-mannose biosynthesis pathway, is the most important pathway involved in the production of M. avium biofilm. PMID:16391123

Regulation of Sleep by Insulin-like Peptide System in Drosophila melanogaster.

PubMed

Cong, Xiaona; Wang, Haili; Liu, Zhenxing; He, Chunxia; An, Chunju; Zhao, Zhangwu

2015-07-01

Most organisms have behavioral and physiological circadian rhythms, which are controlled by an endogenous clock. Although genetic analysis has revealed the intracellular mechanism of the circadian clock, the manner in which this clock communicates its temporal information to produce systemic regulation is still largely unknown. Sleep behavior was measured using the Drosophila Activity Monitoring System (DAMS) monitor under a 12 h light:12 h dark cycle and constant darkness (DD), and 5 min without recorded activity were defined as a bout of sleep. Here we show that Drosophila insulin-like peptides (DILPs) and their receptor (DInR) regulate sleep behavior. All mutants of the seven dilps and the mutant of their receptor exhibit decreases of total sleep except dilp4 mutants, whereas upregulation of DILP and DInR in the nervous system led to increased sleep. Histological analysis identified four previously unidentified neurons expressing DILP: D1, P1, L1, and L2, of which L1 and L2 belong to the LNd and LNv clock neurons that separately regulate different times of sleep. In addition, dilp2 levels significantly decrease when flies were fasted, which is consistent with a previous report that starvation inhibits sleep, further indicating that the dilp system is involved in sleep regulation. Taken together, the results indicate that the Drosophila insulin-like peptide system is a crucial regulator of sleep. © 2015 Associated Professional Sleep Societies, LLC.

Atomic Force Microscopy Reveals a Morphological Differentiation of Chromobacterium violaceum Cells Associated with Biofilm Development and Directed by N-Hexanoyl-L-Homoserine Lactone

PubMed Central

Kamaeva, Anara A.; Vasilchenko, Alexey S.; Deryabin, Dmitry G.

2014-01-01

Chromobacterium violaceum abounds in soil and water ecosystems in tropical and subtropical regions and occasionally causes severe and often fatal human and animal infections. The quorum sensing (QS) system and biofilm formation are essential for C. violaceum's adaptability and pathogenicity, however, their interrelation is still unknown. C. violaceum's cell and biofilm morphology were examined by atomic force microscopy (AFM) in comparison with growth rates, QS-dependent violacein biosynthesis and biofilm biomass quantification. To evaluate QS regulation of these processes, the wild-type strain C. violaceum ATCC 31532 and its mini-Tn5 mutant C. violaceum NCTC 13274, cultivated with and without the QS autoinducer N-hexanoyl-L-homoserine lactone (C6-HSL), were used. We report for the first time the unusual morphological differentiation of C. violaceum cells, associated with biofilm development and directed by the QS autoinducer. AFM revealed numerous invaginations of the external cytoplasmic membrane of wild-type cells, which were repressed in the mutant strain and restored by exogenous C6-HSL. With increasing bacterial growth, polymer matrix extrusions formed in place of invaginations, whereas mutant cells were covered with a diffusely distributed extracellular substance. Thus, quorum sensing in C. violaceum involves a morphological differentiation that organises biofilm formation and leads to a highly differentiated matrix structure. PMID:25111599

Transcriptome Analysis of a Premature Leaf Senescence Mutant of Common Wheat (Triticum aestivum L.)

PubMed Central

Xia, Chuan; Zhang, Lichao; Dong, Chunhao; Liu, Xu; Kong, Xiuying

2018-01-01

Leaf senescence is an important agronomic trait that affects both crop yield and quality. In this study, we characterized a premature leaf senescence mutant of wheat (Triticum aestivum L.) obtained by ethylmethane sulfonate (EMS) mutagenesis, named m68. Genetic analysis showed that the leaf senescence phenotype of m68 is controlled by a single recessive nuclear gene. We compared the transcriptome of wheat leaves between the wild type (WT) and the m68 mutant at four time points. Differentially expressed gene (DEG) analysis revealed many genes that were closely related to senescence genes. Gene Ontology (GO) enrichment analysis suggested that transcription factors and protein transport genes might function in the beginning of leaf senescence, while genes that were associated with chlorophyll and carbon metabolism might function in the later stage. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that the genes that are involved in plant hormone signal transduction were significantly enriched. Through expression pattern clustering of DEGs, we identified 1012 genes that were induced during senescence, and we found that the WRKY family and zinc finger transcription factors might be more important than other transcription factors in the early stage of leaf senescence. These results will not only support further gene cloning and functional analysis of m68, but also facilitate the study of leaf senescence in wheat. PMID:29534430

Evaluation of nigrostriatal dopaminergic function in adult +/+ and +/- BDNF mutant mice.

PubMed

Dluzen, D E; Gao, X; Story, G M; Anderson, L I; Kucera, J; Walro, J M

2001-07-01

Deletion of a single copy of the BDNF gene has been shown to affect the nigrostriatal dopaminergic system of young adult BDNF mice. In the present report we evaluated various indices of nigrostriatal dopaminergic function between 9-month-old wild-type (+/+) and heterozygous (+/-) BDNF mutant mice. Performance in a sensorimotor beam walking task was significantly decreased in +/- mice as indicated by increased times required to traverse both a wide (21 mm) and narrow (6 mm) beam. No differences in spontaneous locomotor behavior were observed between the +/+ and +/- mice. Amphetamine-stimulated (5 mg/kg) locomotor behavior was increased to a greater degree in the +/- mice, with the number of movements performed by these mice being significantly greater than their +/+ controls. Corpus striatal dopamine concentrations were significantly greater in the +/- BDNF mice. The absence of any significant differences for dopamine concentrations within the hypothalamus and olfactory bulb of these mice, as well as an absence of any difference in striatal norepinephrine concentrations, suggested a relative specificity of these effects to the corpus striatum. Both the +/- and +/+ mice showed similar reductions in striatal dopamine concentrations in response to a neurotoxic regimen of methamphetamine (20 mg/kg). Collectively these data show increased levels of striatal dopamine concentrations associated with altered behavioral responses involving the nigrostriatal dopaminergic system within the heterozygous BDNF mutant mice. Copyright 2001 Academic Press.

Behavioral consequences of dopamine deficiency in the Drosophila central nervous system

PubMed Central

Riemensperger, Thomas; Isabel, Guillaume; Coulom, Hélène; Neuser, Kirsa; Seugnet, Laurent; Kume, Kazuhiko; Iché-Torres, Magali; Cassar, Marlène; Strauss, Roland; Preat, Thomas; Hirsh, Jay; Birman, Serge

2011-01-01

The neuromodulatory function of dopamine (DA) is an inherent feature of nervous systems of all animals. To learn more about the function of neural DA in Drosophila, we generated mutant flies that lack tyrosine hydroxylase, and thus DA biosynthesis, selectively in the nervous system. We found that DA is absent or below detection limits in the adult brain of these flies. Despite this, they have a lifespan similar to WT flies. These mutants show reduced activity, extended sleep time, locomotor deficits that increase with age, and they are hypophagic. Whereas odor and electrical shock avoidance are not affected, aversive olfactory learning is abolished. Instead, DA-deficient flies have an apparently “masochistic” tendency to prefer the shock-associated odor 2 h after conditioning. Similarly, sugar preference is absent, whereas sugar stimulation of foreleg taste neurons induces normal proboscis extension. Feeding the DA precursor l-DOPA to adults substantially rescues the learning deficit as well as other impaired behaviors that were tested. DA-deficient flies are also defective in positive phototaxis, without alteration in visual perception and optomotor response. Surprisingly, visual tracking is largely maintained, and these mutants still possess an efficient spatial orientation memory. Our findings show that flies can perform complex brain functions in the absence of neural DA, whereas specific behaviors involving, in particular, arousal and choice require normal levels of this neuromodulator. PMID:21187381

PORPHOBILINOGEN DEAMINASE Deficiency Alters Vegetative and Reproductive Development and Causes Lesions in Arabidopsis

PubMed Central

Quesada, Víctor; Hricová, Andrea; Ponce, María Rosa; Micol, José Luis

2013-01-01

The Arabidopsis rugosa1 (rug1) mutant has irregularly shaped leaves and reduced growth. In the absence of pathogens, leaves of rug1 plants have spontaneous lesions reminiscent of those seen in lesion-mimic mutants; rug1 plants also express cytological and molecular markers associated with defence against pathogens. These rug1 phenotypes are made stronger by dark/light transitions. The rug1 mutant also has delayed flowering time, upregulation of the floral repressor FLOWERING LOCUS C (FLC) and downregulation of the flowering promoters FT and SOC1/AGL20. Vernalization suppresses the late flowering phenotype of rug1 by repressing FLC. Microarray analysis revealed that 280 nuclear genes are differentially expressed between rug1 and wild type; almost a quarter of these genes are involved in plant defence. In rug1, the auxin response is also affected and several auxin-responsive genes are downregulated. We identified the RUG1 gene by map-based cloning and found that it encodes porphobilinogen deaminase (PBGD), also known as hydroxymethylbilane synthase, an enzyme of the tetrapyrrole biosynthesis pathway, which produces chlorophyll, heme, siroheme and phytochromobilin in plants. PBGD activity is reduced in rug1 plants, which accumulate porphobilinogen. Our results indicate that Arabidopsis PBGD deficiency impairs the porphyrin pathway and triggers constitutive activation of plant defence mechanisms leading to leaf lesions and affecting vegetative and reproductive development. PMID:23308205

Mutagenic effects of heavy ion radiation in plants

NASA Technical Reports Server (NTRS)

Mei, M.; Deng, H.; Lu, Y.; Zhuang, C.; Liu, Z.; Qiu, Q.; Qiu, Y.; Yang, T. C.

1994-01-01

Genetic and developmental effects of heavy ions in maize and rice were investigated. Heavy particles with various charges and energies were accelerated at the BEVALAC. The frequency of occurrence of white-yellow stripes on leaves of plants developed from irradiated maize seeds increased linearly with dose, and high Linear Energy Transfer (LET) heavy charged particles, e.g., neon, argon, and iron, were 2-12 times as effective as gamma rays in inducing this type of mutation. The effectiveness of high-LET heavy ion in (1) inhibiting rice seedling growth, (2) reducing plant fertility, (3) inducing chromosome aberration and micronuclei in root tip cells and pollen mother cells of the first generation plants developed from exposed seeds, and (4) inducing mutation in the second generation, were greater than that of low-LET gamma rays. All effects observed were dose-dependent; however, there appeared to be an optimal range of doses for inducing certain types of mutation, for example, for argon ions (400 MeV/u) at 90-100 Gy, several valuable mutant lines with favorable characters, such as semidwarf, early maturity and high yield ability, were obtained. Experimental results suggest that the potential application of heavy ions in crop improvement is promising. Restriction-fragment-length-polymorphism (RFLP) analysis of two semidwarf mutants induced by argon particles revealed that large DNA alterations might be involved in these mutants.

Atomic force microscopy reveals a morphological differentiation of chromobacterium violaceum cells associated with biofilm development and directed by N-hexanoyl-L-homoserine lactone.

PubMed

Kamaeva, Anara A; Vasilchenko, Alexey S; Deryabin, Dmitry G

2014-01-01

Chromobacterium violaceum abounds in soil and water ecosystems in tropical and subtropical regions and occasionally causes severe and often fatal human and animal infections. The quorum sensing (QS) system and biofilm formation are essential for C. violaceum's adaptability and pathogenicity, however, their interrelation is still unknown. C. violaceum's cell and biofilm morphology were examined by atomic force microscopy (AFM) in comparison with growth rates, QS-dependent violacein biosynthesis and biofilm biomass quantification. To evaluate QS regulation of these processes, the wild-type strain C. violaceum ATCC 31532 and its mini-Tn5 mutant C. violaceum NCTC 13274, cultivated with and without the QS autoinducer N-hexanoyl-L-homoserine lactone (C6-HSL), were used. We report for the first time the unusual morphological differentiation of C. violaceum cells, associated with biofilm development and directed by the QS autoinducer. AFM revealed numerous invaginations of the external cytoplasmic membrane of wild-type cells, which were repressed in the mutant strain and restored by exogenous C6-HSL. With increasing bacterial growth, polymer matrix extrusions formed in place of invaginations, whereas mutant cells were covered with a diffusely distributed extracellular substance. Thus, quorum sensing in C. violaceum involves a morphological differentiation that organises biofilm formation and leads to a highly differentiated matrix structure.

A Mutation in γ-Tubulin Alters Microtubule Dynamics and Organization and Is Synthetically Lethal with the Kinesin-like Protein Pkl1pV⃞

PubMed Central

Paluh, Janet L.; Nogales, Eva; Oakley, Berl R.; McDonald, Kent; Pidoux, Alison L.; Cande, W. Z.

2000-01-01

Mitotic segregation of chromosomes requires spindle pole functions for microtubule nucleation, minus end organization, and regulation of dynamics. γ-Tubulin is essential for nucleation, and we now extend its role to these latter processes. We have characterized a mutation in γ-tubulin that results in cold-sensitive mitotic arrest with an elongated bipolar spindle but impaired anaphase A. At 30°C cytoplasmic microtubule arrays are abnormal and bundle into single larger arrays. Three-dimensional time-lapse video microscopy reveals that microtubule dynamics are altered. Localization of the mutant γ-tubulin is like the wild-type protein. Prediction of γ-tubulin structure indicates that non-α/β-tubulin protein–protein interactions could be affected. The kinesin-like protein (klp) Pkl1p localizes to the spindle poles and spindle and is essential for viability of the γ-tubulin mutant and in multicopy for normal cell morphology at 30°C. Localization and function of Pkl1p in the mutant appear unaltered, consistent with a redundant function for this protein in wild type. Our data indicate a broader role for γ-tubulin at spindle poles in regulating aspects of microtubule dynamics and organization. We propose that Pkl1p rescues an impaired function of γ-tubulin that involves non-tubulin protein–protein interactions, presumably with a second motor, MAP, or MTOC component. PMID:10749926

The F-Box Protein Rcy1p Is Involved in Endocytic Membrane Traffic and Recycling Out of an Early Endosome in Saccharomyces cerevisiae

PubMed Central

Wiederkehr, Andreas; Avaro, Sandrine; Prescianotto-Baschong, Cristina; Haguenauer-Tsapis, Rosine; Riezman, Howard

2000-01-01

In Saccharomyces cerevisiae, endocytic material is transported through different membrane-bound compartments before it reaches the vacuole. In a screen for mutants that affect membrane trafficking along the endocytic pathway, we have identified a novel mutant disrupted for the gene YJL204c that we have renamed RCY1 (recycling 1). Deletion of RCY1 leads to an early block in the endocytic pathway before the intersection with the vacuolar protein sorting pathway. Mutation of RCY1 leads to the accumulation of an enlarged compartment that contains the t-SNARE Tlg1p and lies close to areas of cell expansion. In addition, endocytic markers such as Ste2p and the fluorescent dyes, Lucifer yellow and FM4-64, were found in a similar enlarged compartment after their internalization. To determine whether rcy1Δ is defective for recycling, we have developed an assay that measures the recycling of previously internalized FM4-64. This method enables us to follow the recycling pathway in yeast in real time. Using this assay, it could be demonstrated that recycling of membranes is rapid in S. cerevisiae and that a major fraction of internalized FM4-64 is secreted back into the medium within a few minutes. The rcy1Δ mutant is strongly defective in recycling. PMID:10769031

Phenotypic Suppression of the Gibberellin-Insensitive Mutant (gai) of Arabidopsis.

PubMed Central

Wilson, R. N.; Somerville, C. R.

1995-01-01

The semidominant gibberellin-insensitive (gai) mutant of Arabidopsis thaliana shows impairment in multiple responses to the plant hormone gibberellin A3, which include effects on seed germination, stem elongation, apical dominance, and rapid flowering in short days. Results presented here show that the gai mutation also interferes with development of fertile flowers in continuous light. Mu-tagenesis of the gai mutant resulted in recovery of 17 independent mutants in which the gibberellin-insensitive phenotype is partially or completely suppressed. Sixteen of the suppressor mutations act semidominantly to restore gibberellin responsiveness. One representative of this class, the gar1 mutation, could not be genetically separated from the gai locus and is proposed to cause inactivation of the gai gene. The exceptional gar2 mutation partially suppresses the gai phenotype, is completely dominant, and is not linked to the gai locus. The gar2 mutation may define a new gene involved in gibberellin signaling. A recessive allele of the spindly (SPY) locus, spy-5, was also found to partially suppress the gai mutant phenotype. PMID:12228487