Global impact of protein arginine phosphorylation on the physiology of Bacillus subtilis
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ABSTRACT:
ORGANISM(S): Bacillus subtilis subsp. subtilis str. 168
SUBMITTER: Ulrike MM-CM-$der
PROVIDER: E-GEOD-31249 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Reversible protein phosphorylation is an important and ubiquitous protein modification in all living cells. We report that protein arginine phosphorylation plays a physiological significant role for the regulation of protein activity. We detected 121 arginine phospho-sites for 87 proteins in the Gram-positive model organism Bacillus subtilis in vivo. Moreover, we provide evidences that arginine phosphorylations are involved in the fine-tuned signal transduction of many critical cellular processes, such as protein degradation, motility, competence, stringent and stress response. Our results suggest that in B. subtilis the activity of a protein arginine phosphatase allows a fast regulation of protein activity by protein arginine kinases and that protein arginine phosphorylations play an important role as a reversible post-translational modification in bacteria.
2012-04-01 | GSE31249 | GEO
Project description:Here, we investigated for the first time the systems-wide response of B. subtilis to different simultaneous stresses, i.e. nutrient limitation and high osmolarity. To address the anticipated complexity of the cellular response networks, we combined chemostat experiments under conditions of carbon limitation, salt stress and osmoprotection with multi-omics analyses at the transcriptome, proteome, metabolome and fluxome levels. Our results indicate that the flux through central carbon and energy metabolism is very robust under all conditions studied. The key to achieve this robustness is the adjustment of the biocatalytic machinery to compensate for solvent-induced impairment of enzymatic activities during osmotic stress. The accumulation of the exogenously provided osmoprotectant glycine betaine helps the cell to rescue enzyme activities in the presence of high salt. A major effort of the cell during osmotic stress is the production of the compatible solute proline. This is achieved by the concerted adjustment of multiple reactions around the 2-oxoglutarate node, which drives metabolism towards the proline precursor glutamate. The fine-tuning of the transcriptional and metabolic networks involves functional modules that overarch the individual pathways. We applied transcriptomic, mass spectrometry-based protein, metabolite and 13C-metabolic flux analysis techniques to B. subtilis cells grown under well-controlled conditions in a glucose-limited chemostat at a growth rate of 0.1 h-1 under i) reference conditions, ii) in the presence of 1.2 M NaCl, and iii) in the presence of 1.2 M NaCl and 1 mM GB. Microarray hybridizations were performed with RNA from three biological replicates. The individual samples were labeled with Cy5; a reference pool containing equal amounts of RNA from all 9 samples was labeled with Cy3.
2014-03-31 | E-GEOD-53333 | biostudies-arrayexpress
Project description:In this study, we present a follow up investigation on the effects of argon plasma on vegetative growing Bacillus subtilis 168 (see GSE27113). A growth chamber system suitable for low temperature gas plasma treatment of bacteria in liquid medium enabled a first glimpse on the complex cellular response. In order to gain further knowledge, a second kind of plasma treatment was applied and combined proteomic and transcriptomic analyses were used to investigate the specific stress response of B. subtilis cells to treatment with not only argon but also air plasma. Besides similar cellular responses, due to both argon and air plasma treatment, a variety of different responses such as growth retardation and morphological changes were observed. Furthermore, we analyzed the specific response to DNA damages and oxidative stress. The biological findings such as oxidative stress responses were supported by the detection of reactive plasma species by OES and FTIR measurements. Cells were grown in LB medium. At OD540 of 0.5, air plasma treatment was set for 15 min with a discharge power of 7.4 W. Samples for mircroarray analysis were taken 5 min after the 15 min plasma treatment. Microarray hybridizations were performed with RNA from three biological replicates. The individual samples were labeled with Cy5; a reference pool containing equal amounts of RNA from all samples was labeled with Cy3.
2013-08-22 | E-GEOD-32865 | biostudies-arrayexpress
Project description:Bacillus subtilis is exposed to a wide range of transitory stress and starvation conditions. Here we investigate the expression changes observed in the B. subtilis wild type strain 168 and its isogenic sigB mutant(BSM29) with respect to each stress condition tested. Gene expression was queried for the stress conditions: ethanol-, butanol-, osmotic- and oxidative stress, heat shock, low temperature growth, glucose as well as oxygen limitation. For butanol-, ethanol-, osmotic-, and oxidative stress as well as heat shock : time points (0min, 5min, 10min, 15min and 20min) ; for glucose limitation and oxygen limitation : time points (0min, 15min, 30min, 45min, 60min or 90min) and for low temperature growth, samples for recording of expression values were taken during mid-exponential growth at OD540 0.9 and 1.0.
2012-03-14 | E-GEOD-32895 | biostudies-arrayexpress
Project description:Reversible protein phosphorylation is one of the major mechanisms in the regulation of protein expression and protein activity, controlling physiological functions of the important human pathogen Staphylococcus aureus. Phosphorylations at serine, threonine and tyrosine are known to influence for example protein activity in central metabolic pathways and the more energy-rich phosphorylations at histidine, aspartate or cysteine can be found as part of two component system sensor domains or mediating bacterial virulence. In addition to these well-known phosphorylations, the phosphorylation at arginine residues plays an essential role. Hence, the deletion mutant S. aureus COL ΔptpB (protein tyrosine phosphatase B) was studied since the protein PtpB is assumed to be an arginine phosphatase. A gel-free approach was applied to analyse the changes in the phosphoproteome of the deletion mutant ΔptpB and the wild type in growing cells, thereby focusing on the occurrence of phosphorylation on arginine residues. In order to enhance the reliability of identified phosphorylation sites at arginine residues, a subset of arginine phosphorylated peptides was chemically synthesised. Combined spectral libraries based on phosphoenriched samples, synthetic arginine phosphorylated peptides and classical proteome samples provide a sophisticated tool for the analysis of arginine phosphorylations. This way, 212 proteins phosphorylated on serine, threonine, tyrosine or arginine residues were identified within the mutant ∆ptpB and 102 in wild type samples. Among them, 207 arginine phosphosites were identified exclusively within the mutant ∆ptpB, widely distributed along the whole bacterial metabolism. This identification of putative targets of PtpB allows further investigation of the physiological relevance of arginine phosphorylations and provides the basis for reliable quantification of arginine phosphorylations in bacteria.
2017-12-06 | PXD007167 | Pride
Project description:One of the first steps in bacterial cell division is the polymerization of the tubulin-like protein FtsZ at midcell. The dynamics of FtsZ polymerization is regulated by a set of proteins among which ZapA. A zapA mutation does not result in a clear phenotype in Bacillus subtilis. In this study we used a synthetic-lethal screen to find genes that become essential when ZapA is absent. Three transposon insertions were found in yvcL. Deletion of yvcL in a wild type background had only a mild effect on growth, but a yvcL zapA double mutant is very filamentous and sick. This filamentation is caused by a strong reduction in FtsZ polymerization, suggesting that YvcL is involved in an early stage of cell division. YvcL is 25 % identical and 50 % similar to the Streptomyces coelicolor transcription factor WhiA. WhiA is required for septation of aerial hyphae during sporulation. Using GFP fusions, we show that YvcL localizes at the nucleoid. Surprisingly, transcriptome analyses in combination with a ChIP on chip assay did not provide clear evidence that YvcL functions as a transcription factor. To gain more insight into the function of YvcL, we searched for suppressors of the filamentous phenotype of a M-bM-^HM-^FyvcL M-bM-^HM-^FzapA mutant. Transposon insertions in gtaB and pgcA restored normal cell division of the double mutant. The corresponding proteins have been implemented in the metabolic sensing of cell division. We conclude that YvcL (WhiA) is involved in cell division in B. subtilis through an as yet unknown mechanism. Comparing wild tpe Bascillus subtilus (n=3) with Bascillus Subtilis KS400 (n=2) and Bascillus subtilis KS696 (n=2)
2013-11-28 | E-GEOD-45824 | biostudies-arrayexpress
Project description:Protein S-thiolation is a post-translational thiol-modification that controls redox-sensing transcription factors and protects active site cysteine residues against irreversible oxidation. In B. subtilis, the MarR-type repressor OhrR was shown to sense organic hydroperoxides via formation of mixed disulfides with the redox buffer bacillithiol (Cys-GlcN-Malate) termed as S-bacillithiolation. We have studied changes in the transcriptome and redox proteome caused by the strong oxidant hypochloric acid (NaOCl), the active component of house-hold bleach. The OhrR-controlled peroxiredoxin OhrA was most strongly up-regulated by NaOCl stress and conferred specific protection against NaOCl. Inactivation of the OhrR repressor was caused by S-bacillithiolation of the redox-sensing Cys15 residue in response to NaOCl. Two cobalamin-independent methionine synthases MetE and YxjG were identified as S-bacillithiolated at their essential active site cysteines resulting in hypochlorite-induced methionine limitation. In summary, our studies show that S-bacillithiolation of OhrR and the methionine synthases is the major mechanism in protection against hypochlorite stress in B. subtilis. The B. subtilis 168 wild type strain was grown in minimal medium to OD500 of 0.4 and harvested before and 10 minutes after exposure to 50 µM NaOCl. Microarray hybridizations were performed in triplicate using RNA isolated from independent cultures.
2011-07-04 | E-GEOD-27637 | biostudies-arrayexpress
Project description:Ever since the identification of the first protein-arginine kinase, McsB, in B. subtilis arginine phosphorylation gained more attention. However, the analysis of phosphorylations especially phosphormaidates comes along with several challenges. The sub-stoichiometric nature of protein phosphorylation requires proper enrichment strategies prior to LC-MS/MS analysis and the acid instability of phosphoramidates was long though to impede those enrichments. Further good spectral quality is required which can be reduced by the presence of neutral losses of phosphoric acid upon higher energy collision induced dissociation. Adaptation of common enrichment strategies to less acidic conditions and the use of electron-transfer dissociation allowed the identification of more than 200 pArg sites in B. subtilis and S. aureus so far. Here we show that pArg is stable enough for commonly used Fe3+-IMAC enrichment followed by LC-MS/MS and that HCD is still the gold standard for phosphoproteomics. By profiling a serine/ threonine kinase (Stk1) and phosphatase (Stp1) mutant from a methicillin-resistant S. aureus mutant library, we identified 1062 pArg sites and thus the most comprehensive arginine phosphoproteome to date. Using synthetic phosphoarginine peptides we validated the presence and localization of arginine phosphorylation in S. aureus and lastly, we could show that Stp1 is influencing the arginine phosphoproteom without being a protein-arginine phosphatase.
2022-06-09 | PXD026981 | Pride
Project description:How cells dynamically respond to fluctuating environmental conditions depends on the architecture and noise of the underlying genetic circuits. Most work characterizing stress pathways in the model bacterium Bacillus subtilis has been performed on bulk cultures using ensemble assays. However, investigating the single cell response to stress is important since noise might generate significant phenotypic heterogeneity. Here, we study a single stress response (glucose starvation) to allow comparison of both population- and single cell data. Using a top-down approach, we investigate the transcriptional dynamics of various stress-related genes of B. subtilis in response to glucose starvation. Our data reveal that most stress-induced pathways respond highly heterogeneously to glucose starvation. Bacteria are commonly thought to exhibit phenotypic heterogeneity to increase their survival under changing environments by always having a subpopulation pre-adapted to adverse conditions. Interestingly, growth experiments with cultures that were either pre-adapted for glucose starvation or non-starved cells showed that non-starved cells demonstrate a significant growth advantage when challenged with a new stress. This suggests that once cells engage in a certain stress pathway they are only optimised for this specific condition. Hence it is important for the population to always maintain a 'naM-CM-/ve' subpopulation in case of fluctuating environments. Our data indicate the presence of performance-based selection, by which only cells with a sufficiently high-energy status or cellular fitness are able to diversify. The transcriptome of glucose-starved and non-starved B. subtilis 168 trp+ cultures was compared using 3 biological replicates.
2012-06-30 | E-GEOD-33506 | biostudies-arrayexpress
Project description:OmpR is a DNA binding protein belonging to the OmpR/EnvZ two component system. This system is known to sense changes in osmolarity in Escherichia coli. Recently, OmpR in Salmonella enterica serovar Typhimurium was found to be activated by acidic pH and DNA relaxation. In this study, ChIP-on-chip was employed to ascertain the genome-wide distribution of OmpR in Salmonella Typhimurium and Escherichia coli in acidic and neutral pH. In addition we investigated the affect of DNA relaxation on OmpR binding in Salmonella Typhimurium. Analysis of OmpR binding at pH 7 and pH 4.5 in E-minimal medium in both SL1344 and CSH50. Three independent biological replicates at each pH 7 and pH 4.5 was performed. This was done in each strain background. DNA was immunopreciptated using an anti-FLAG anitbody which binds to flag-tagged OmpR in both strains. Two control M-bM-^@M-^XmockM-bM-^@M-^Y experiments were performed under the same culture condtions in each strain background; DNA was immunopreciptated using normal mouse IgG antibody. To analyse the effect of DNA relaxation onOmpR binding SL1344 was maintained in the exponential growth phase in LB broth with and without treatment with the drug novobiocin (25 M-NM-<g/ml) for 40 min. This was peformed on two independent occasions. In all cases the experimental immunoprecipitated DNA was hybridised against the input DNA.
2014-03-27 | E-GEOD-49914 | biostudies-arrayexpress