Transcriptome analysis of glucose starved Bacillus subtilis cells
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ABSTRACT:
ORGANISM(S): Bacillus subtilis subsp. subtilis str. 168
SUBMITTER: Ulrike Mäder
PROVIDER: E-GEOD-24058 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:In this study, temporal changes in the proteome, transcriptome and extracellular metabolome of B. subtilis caused by glucose starvation were monitored. For proteomic profiling, a combination of in vivo metabolic labeling and shot gun mass spectrometric analysis was carried out for five different proteomic subfractions (i. cytosolic, ii. extracellular, iii. membrane, iv. integral membrane, and v. surface proteome fraction) leading to the identification of about 52% of the predicted proteome of B. subtilis. Acquired proteomic and transcriptomic data were analyzed using Voronoi treemaps which link functional classifications and relative expression changes of gene products according to their fate in the stationary phase. The data obtained enables for in-depth analysis of major physiological processes including protein degradation and is the first comprehensive profiling of changes in the membrane subfraction.
2010-12-01 | GSE24058 | GEO
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:Spx is a global regulator of genes that are induced by disulfide stress in Bacillus subtilis. Most of the Spx-regulated genes (SRGs) are of unknown function, but many encode products conserved in low %GC Gram positive bacteria. Using a gene-disruption library of B. subtilis genomic mutations, the SRGs were screened for phenotypes related to Spx-controlled activities, such as growth in minimal medium and sensitivity to methylglyoxal, but nearly all of the SRG mutations showed little if any phenotype. To uncover SRG function, the mutations were rescreened in an spx mutant background to determine which mutant SRG allele would enhance the spx mutant phenotype. One of the SRGs, ytpQ, was the site of a mutation that, when combined with an spx null mutation, elevated the severity of the Spx mutant phenotype, as shown by reduced growth in a minimal medium and by hypersensitivity to methylglyoxal. Proteomic and transcriptomic data indicated that the ytpQ mutation caused the derepression of the Fur and PerR regulons, as well as heightened LexA-controlled gene expression. Our study suggests that the ytpQ gene, encoding a conserved DUF1444 protein, functions directly or indirectly in repairing or stabilizing Fe-bearing proteins, which are sensitive to thiol reactive agents and, therefore, likely influenced by Spx-controlled genes. The wild type parent, ytpQ and spx mutants were grown in a glucose minimal medium, with and without 2.8 mM methylglyoxal, to mid-log phase. Cells were harvested and RNA was extracted for microarray hybridization analysis to determine if any changes in the transcriptome could be detected that were attributable to the ytpQ mutation. Microarray hybridizations were performed with RNA from three biological replicates.
2011-10-24 | E-GEOD-28872 | biostudies-arrayexpress
Project description:In this study, we introduce for the first time a growth chamber system suitable for physical plasma treatment of bacteria in liquid medium. Bacillus subtilis 168 cells were treated with argon plasma in order to investigate their specific stress response usong a proteomic and transcriptomic approach. The treatment with three different discharge voltages revealed not only growth differences, but also clear cellular stress responses. B. subtilis faces severe cell wall stress, which was made visible alsoelectron microscopy, DNA damages and oxidative stress. The biological findings could be supported by the reactive plasma species, found by plasma diagnostics, i.e. optical emission spectroscopy (OES) and Fourier transformed infrared spectroscopy (FTIR). Cells were grown in LB medium. At OD540 of 0.5, argon plasma treatment was set for 15 min with a discharge power of 5 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.
2011-06-01 | E-GEOD-27113 | biostudies-arrayexpress
Project description:The field of systems biology has been rapidly increasing in the past decade. However, the data produced by “omics” approaches is lagging behind the requirements of this field, especially when it comes to absolute abundances of membrane proteins. In the present study, a novel approach for large-scale absolute quantification of this challenging subset of proteins has been established and evaluated using osmotic stress management in the Gram-positive model bacterium Bacillus subtilis as proof of principle precedent. Selected membrane proteins were labelled using a SNAP-tag, which allowed to visually inspect the enrichment of the membrane fraction by immunoassays. Absolute membrane protein concentrations were determined via shotgun proteomics by spiking crude membrane extracts of chromosomally SNAP-tagged and wild-type B. subtilis strains with protein standards of known concentration. Shotgun data was subsequently calibrated by targeted mass spectrometry using SNAP as an anchor protein, and an enrichment factor was calculated in order to obtain membrane protein copy numbers/µm2. The presented approach enabled the accurate determination of physiological changes resulting from imposed hyperosmotic stress, thereby offering a clear visualization of alterations in membrane protein arrangements and shedding light on putative membrane complexes. This straightforward and cost-effective methodology for quantitative proteome studies can be implemented by any research group with mass-spectrometry expertise. Importantly, it can be applied to the full spectrum of physiologically relevant conditions, ranging from environmental stresses to the biotechnological production of small molecules and proteins, which relies heavily on B. subtilis.
2019-08-26 | PXD014272 | Pride
Project description:Investigation of whole genome gene expression level changes in sporulating Bacillus subtilis 168 delta-prpE mutant, compared to the wild-type strain. The mutation engineered into this strain results in impaired germination of spores. A six chip study using total RNA extracted from three separate wild-type cultures of sporulating Bacillus subtilis 168 and three separate cultures of sporulating mutant strain, Bacillus subtilis 168 delta-prpE, in which prpE (yjbP BSU11630) gene coding for a protein phosphatase is deleted entirely. Each chip consists of four fields able to measure the expression level of 4,104 genes from Bacillus subtilis subsp. subtilis strain 168 NC_000964 with eight 60-mer probe pairs (PM/MM) per gene, with two-fold technical redundancy.
2013-07-08 | E-GEOD-44125 | biostudies-arrayexpress
Project description:Whole-genome microarray technology and state-of-the-art proteomic techniques were applied to provide a global and time-resolved picture of the physiological response of B. subtilis cells exposed to a severe and sudden osmotic up-shift. This combined experimental approach provided quantitative data for 3961 mRNA profiles, 590 expression profiles of proteins detected in the cytosol and 383 expression profiles of proteins detected in the membrane fraction. Our study uncovered a well-coordinated induction of gene expression subsequent to an osmotic up-shift that involves large parts of the SigB, SigW, SigM and SigX regulons and additionally osmotic up-regulation of a large number of genes that do not belong to these regulons. In total, osmotic up-regulation of about 500 B. subtilis genes was observed. Our data provide an unprecedented rich basis for further in-depth investigation on the physiological and genetic responses of B. subtilis to hyperosmotic stress. Cells were grown in a minimal medium to early exponential phase and were then exposed to a strong osmotic up-shift by the addition of 6% (w/v) NaCl. Samples were taken before and 10, 30, 60 and 120 min subsequent to the addition of NaCl. Microarray hybridizations were performed with RNA from three biological replicates. The individual samples were labeled with Cy5; a reference pool consisting of equal amounts of RNA from all 15 samples was labeled with Cy3 (common reference design). After intensity-dependent (Lowess) normalization, the ratios of duplicate spots were averaged resulting in three biologically independent expression values per gene and time point.
2010-05-19 | E-GEOD-18345 | biostudies-arrayexpress
Project description:The genome sequence is the “blue-print of life”, but proteomics is required to relate this blue-print of life to the actual physiology of living cells. Because of their low complexity – only about 2.000 proteins make a Staphylococcus aureus cell viable – bacteria are excellent model systems to identify the entire protein assembly of a living organism. Many of the studies on physiological proteomics of bacteria, however, still rely on 2D gel-based technologies that visualize only a minor part of the proteome. In this study it will be shown that the majority of proteins expressed in the Gram-positive human pathogen S. aureus can be identified and even quantified by a combination of gel-based and gel-free approaches, the latter having undergone formidable improvements over the last 10 years. S. aureus has been the model of choice for our proteomic studies, because it combines high pathogenicity with increasing antibiotic resistance, which calls for new leads in the development of anti-staphylococcal therapy. On the way towards the entire proteome of S. aureus, we analysed four subproteomic fractions: cytosolic proteins, membrane-bound proteins, cell-surface associated and extracellular proteins, the two latter fractions containing most of the virulence factors. To obtain quantitative results, we compared growing cells and non-growing/stationary phase cells using the 15N metabolic labelling approach. With almost 2.000 proteins, 80 % of the expressed proteome was identified and even quantified in growing and non-growing cells. A comprehensive inventory of the entire protein assembly during the two physiologically distinct states is presented, which integrates data ranging from gene expression to subcellular localization. Three major protein classes were distinguished: (i) Proteins induced in the stationary phase only (ii) Vegetative proteins, no longer synthesized but stable in the stationary phase (iii) Vegetative proteins, no longer required in non-growing cells and finally degraded. This quantitative proteomics approach for growing/non-growing cells, which represents a proof-of-principle for whole-cell physiological proteomics, can now be extended to address particular infection-relevant physiological states. Importantly, the present model study represents the highest coverage of a bacterial proteome so far (except low complexity bacteria). It thus paves the way towards a new understanding of cell physiology and pathophysiology of S. aureus and related pathogenic bacteria, opening a new era in infection-related research on this crucial pathogen. For the DNA-microarray experiment RNA was extracted from two independently grown cultures. One was grown in 14N labelled BioExpress medium and the other in 15N labelled BioExpress medium. Samples of exponentially growing cells (OD600=0.5) and of cell at 5h after entry into stationary phase were harvested from every culture. Equal amounts of all four RNAs were pooled and the pool was used as common reference (Cy3-labeld) for the four sample RNAs (Cy3). Thus, in total four hybridizations, one for each sample versus the common reference, were performed.
2009-11-11 | E-GEOD-15060 | biostudies-arrayexpress
Project description:RNA processing and degradation is initiated by endonucleolytic cleavage of the target RNAs. In many bacteria, this activity is performed by RNase E which is not present in Bacillus subtilis and other Gram-positive bacteria. Recently, the essential endoribonuclease RNase Y has been discovered in B. subtilis. This RNase is involved in the degradation of bulk mRNA suggesting a major role in mRNA metabolism. However, only a few targets of RNase Y have been identified so far. In order to assess the global impact of RNase Y, we compared the transcriptomes of strains expressing RNase Y or depleted for RNase Y. Our results indicate that processing by RNase Y results in accumulation of about 80 mRNAs. Some of these targets were substantially stabilized by RNase Y depletion, resulting in half-lives in the range of an hour. Moreover, about 40 mRNAs were less abundant when RNase Y was depleted among them the mRNAs of the operons required for biofilm formation. Interestingly, overexpression of RNase Y was sufficient to induce biofilm formation. The results emphasize the importance of RNase Y for B. subtilis and are in support of the idea that RNase Y is the functional equivalent of RNase E. To study the global function of RNase Y, a microarray analysis was performed with a B. subtilis strain allowing controlled depletion of RNase Y. Strain GP193 (Commichau et al., 2009, Mol. Cell. Proteomics 8: 1350-1360) expressing the rny gene under the control of a xylose-inducible promoter was cultivated in CSE minimal medium in the presence or absence of the inducer xylose. The transcriptomes of the two cultures (i.e. expressing RNase Y and depleted for RNase Y) were compared at a timepoint at which the reduced RNase Y amounts already affected the mRNA turnover whereas the growth rates of the two cultures were still almost identical.
2011-08-31 | E-GEOD-30430 | biostudies-arrayexpress
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