Project description:PhyR is an unusual type of response regulator consisting of a receiver domain and an extracytoplasmic function (ECF) sigma factor-like domain. It was recently described as a master regulator of general stress response in Methylobacterium extorquens. Orthologues of this regulator are present in essentially all free-living Alphaproteobacteria. In most of them, phyR is genetically closely linked to a gene encoding an ECF sigma factor. Here, we investigate the role of these two regulators in the soybean symbiont Bradyrhizobium japonicum USDA110. Using deletion mutants and phenotypic assays, we showed that PhyR and the ECF sigma factor sigma(EcfG) are involved in heat shock and desiccation resistance upon carbon starvation. Both mutants had symbiotic defects on the plant hosts Glycine max (soybean) and Vigna radiata (mungbean). They induced fewer nodules than the wild type and these nodules were smaller, less pigmented, and their specific nitrogenase activity was drastically reduced 2 or 3 weeks after inoculation. Four weeks after infection, soybean nodule development caught up to a large extent whereas most mungbean nodules remained defective even 5 weeks after infection. Remarkably, both mutants triggered aberrant nodules on the different host plants with ectopically emerging roots. Microarray analysis revealed that PhyR and sigma(EcfG) control congruent regulons suggesting both regulators are part of the same signalling cascade. This finding was further substantiated by in vitro protein-protein interaction studies which are in line with a partner-switching mechanism controlling gene regulation triggered by phosphorylation of PhyR. The large number of genes of unknown function present in the PhyR/sigma(EcfG) regulon and the conspicuous symbiotic phenotype suggest that these regulators are involved in the Bradyrhizobium-legume interaction via yet undisclosed mechanisms. Comparative analysis of the B. japonicum phyR mutant 8402, ecfG mutant 8404 mutant and the wild type during exponential growth in rich medium (PSY) and after 24 hours starvation in carbon source-free minimal medium
Project description:PhyR is an unusual type of response regulator consisting of a receiver domain and an extracytoplasmic function (ECF) sigma factor-like domain. It was recently described as a master regulator of general stress response in Methylobacterium extorquens. Orthologues of this regulator are present in essentially all free-living Alphaproteobacteria. In most of them, phyR is genetically closely linked to a gene encoding an ECF sigma factor. Here, we investigate the role of these two regulators in the soybean symbiont Bradyrhizobium japonicum USDA110. Using deletion mutants and phenotypic assays, we showed that PhyR and the ECF sigma factor sigma(EcfG) are involved in heat shock and desiccation resistance upon carbon starvation. Both mutants had symbiotic defects on the plant hosts Glycine max (soybean) and Vigna radiata (mungbean). They induced fewer nodules than the wild type and these nodules were smaller, less pigmented, and their specific nitrogenase activity was drastically reduced 2 or 3 weeks after inoculation. Four weeks after infection, soybean nodule development caught up to a large extent whereas most mungbean nodules remained defective even 5 weeks after infection. Remarkably, both mutants triggered aberrant nodules on the different host plants with ectopically emerging roots. Microarray analysis revealed that PhyR and sigma(EcfG) control congruent regulons suggesting both regulators are part of the same signalling cascade. This finding was further substantiated by in vitro protein-protein interaction studies which are in line with a partner-switching mechanism controlling gene regulation triggered by phosphorylation of PhyR. The large number of genes of unknown function present in the PhyR/sigma(EcfG) regulon and the conspicuous symbiotic phenotype suggest that these regulators are involved in the Bradyrhizobium-legume interaction via yet undisclosed mechanisms.
Project description:In Rhodobacter sphaeroides a transcriptional response to the reactive oxygen species singlet oxygen is controlled by the group IV sigma factor RpoE and the anti-sigma factor ChrR. In this study, we integrated various large datasets to identify genes within the singlet oxygen stress response that contain RpoE-dependent promoters within R. sphaeroides. Transcript pattern clustering and a RpoE-binding sequence model were used to predict candidate promoters that respond to singlet oxygen stress in R. sphaeroides. These candidate promoters were experimentally validated to nine R. sphaeroides RpoE-dependent promoters that control the transcription of 15 genes activated by singlet oxygen.
Project description:In Rhodobacter sphaeroides a transcriptional response to the reactive oxygen species singlet oxygen is controlled by the group IV sigma factor RpoE and the anti-sigma factor ChrR. In this study, we integrated various large datasets to identify genes within the singlet oxygen stress response that contain RpoE-dependent promoters within R. sphaeroides. Transcript pattern clustering and a RpoE-binding sequence model were used to predict candidate promoters that respond to singlet oxygen stress in R. sphaeroides. These candidate promoters were experimentally validated to nine R. sphaeroides RpoE-dependent promoters that control the transcription of 15 genes activated by singlet oxygen. DNA immunoprecipitated with polyclonal antibodies against RpoE or the Beta' subunit of RNA polymerase was labelled with Cy5 and hybridized on two-color tilling arrays (triplicates for each) with genomic DNA as an input control labelled with Cy3.
Project description:part of GSE8478: Genome-wide transcript analysis of Bradyrhizobium japonicum bacteroids in soybean root nodules This SuperSeries is composed of the SubSeries listed below.
Project description:The purpose of the study is to identify Irr-responsive genes in the bacterium Bradyrhizobium japonicum. Parent strain LO was compared to irr mutant strain LODTM5 by whole genome microarray analysis. Both cell types were grown in iron-limited media. Keywords: Comparison of B. japonicum wild type and mutant cells
Project description:DISCLAIMER: This project actually contains two separate and independent assays by mistake. They should be not be considered together.</br></br>Assay 9769 - Bradyrhizobium japonicum proteomic reference map PMID : 20806226</br>Assay 15318 - Vigna mungo leaf proteome map PMID : 23587433
Project description:Analysis of a Bradyrhizobium japonicum pmtA mutant. PmtA catalyzes the first of three consecutive methylation reactions leading to phosphatidylcholine (PC) formation in B. japonicum. Disruption of the pmtA gene results in a significantly reduced PC content causing a defect in symbiosis with the soybean host. This study provides the first insight into global transcriptomic changes of a bacterial phosphatidylcholine biosynthesis mutant. Cells of the pmtA mutant and the wild type were grown to mid-exponential phase in full medium (PSY) under aerobic culture conditions. Keywords: genetic modification Comparative analyis of the B. japonicum pmtA mutant and the wild type grown under aerobic culture conditions.
Project description:Analysis of a Bradyrhizobium japonicum pmtA mutant. PmtA catalyzes the first of three consecutive methylation reactions leading to phosphatidylcholine (PC) formation in B. japonicum. Disruption of the pmtA gene results in a significantly reduced PC content causing a defect in symbiosis with the soybean host. This study provides the first insight into global transcriptomic changes of a bacterial phosphatidylcholine biosynthesis mutant. Cells of the pmtA mutant and the wild type were grown to mid-exponential phase in full medium (PSY) under aerobic culture conditions. Keywords: genetic modification
Project description:The growth and persistence of rhizobia and bradyrhizobia in soils are negatively impacted by drought conditions. In this study, we used genome-wide transcriptional analyses to obtain a comprehensive understanding of the response of Bradyrhizobium japonicum to drought. Desiccation of cells resulted in the differential expression of 15 to 20% of the 8,480 B. japonicum open reading frames, with considerable differentiation between early (after 4 h) and late (after 24 and 72 h) expressed genes. While 225 genes were universally up-regulated at all three incubation times in response to desiccation, an additional 43 and 403 up-regulated genes were common to the 4/24- and 24/72-h incubation times, respectively. Desiccating conditions resulted in the significant induction (>2.0-fold) of the trehalose-6-phosphate synthetase (otsA), trehalose-6-phosphate phosphatase (otsB), and trehalose synthase (treS) genes, which encode two of the three trehalose synthesis pathways found in B. japonicum. Gene induction was correlated with an elevated intracellular concentration of trehalose and increased activity of trehalose-6-phosphate synthetase, collectively supporting the hypothesis that this disaccharide plays a prominent and important role in promoting desiccation tolerance in B. japonicum. Microarray data also indicated that sigma(54)- and sigma(24)-associated transcriptional regulators and genes encoding isocitrate lyase, oxidative stress responses, the synthesis and transport of exopolysaccharides, heat shock response proteins, enzymes for the modification and repair of nucleic acids, and the synthesis of pili and flagella are also involved in the response of B. japonicum to desiccation. Polyethylene glycol-generated osmotic stress induced significantly fewer genes than those transcriptionally activated by desiccation. However, 67 genes were commonly induced under both conditions. Taken together, these results suggest that B. japonicum directly responds to desiccation by adapting to changes imparted by reduced water activity, such as the synthesis of trehalose and polysaccharides and, secondarily, by the induction of a wide variety of proteins involved in protection of the cell membrane, repair of DNA damage, stability and integrity of proteins, and oxidative stress responses. Keywords: stress response; time course