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The alternative sigma factor σ(B) is the master regulator of the general stress regulon that comprises approximately 200 genes whose products confer a comprehensive stress resistance to Bacillus subtilis. The characterization of MgsR (modulator of the general stress response) revealed that the activation and induction of σ(B) are a prerequisite but not sufficient for a full expression of all general stress genes. MgsR is a paralogue of the global regulator of the diamide stress response, Spx, and controls a subregulon of the general stress response. Here we demonstrate that MgsR activity is controlled at multiple levels. These mechanisms include a positive autoregulatory loop on mgsR transcription, a post-translational redox-sensitive activation step by an intramolecular disulfide bond formation in response to ethanol stress in vivo, as well as rapid proteolytic degradation of MgsR by the ClpXP and ClpCP proteases. Our results indicate an elaborate regulatory network integrating secondary oxidative stress signals into a σ(B) -mediated regulatory cascade that is aimed at rapid and finely tuned target gene expression to coordinately fulfil the physiological needs of the cell in the face of multiple environmental changes., (© 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.)

CtsR, the global heat shock repressor in low GC, Gram+ bacteria, regulates a crucial subset of genes involved in protein quality control. CtsR de-repression occurs not only during heat stress but also during a variety of other environmental stresses, most notably thiol-specific oxidative stress. Here we report that McsA acts as a molecular redox switch that regulates CtsR de-repression via the activation of McsB. Once critical thiols of McsA become oxidized, the strong interaction between McsA and McsB is interrupted and free McsB is no longer inhibited by McsA, resulting in the inactivation of CtsR. This mechanism differs significantly from inactivation of CtsR during heat stress demonstrating a dual activity control of CtsR. Moreover, we show that in those low GC, Gram+ bacteria, which lack the McsA/McsB complex, the Zn finger protein ClpE is able to sense and respond to oxidative stress, also resulting in CtsR inactivation., (© 2011 Blackwell Publishing Ltd.)

Glutathione constitutes a key player in the thiol redox buffer in many organisms. However, the gram-positive bacteria Bacillus subtilis and Staphylococcus aureus lack this low-molecular-weight thiol. Recently, we identified S-cysteinylated proteins in B. subtilis after treatment of cells with the disulfide-generating electrophile diamide. S cysteinylation is thought to protect protein thiols against irreversible oxidation to sulfinic and sulfonic acids. Here we show that S thiolation occurs also in S. aureus proteins after exposure to diamide. We further analyzed the formation of inter- and intramolecular disulfide bonds in cytoplasmic proteins using diagonal nonreducing/reducing sodium dodecyl sulfate gel electrophoresis. However, only a few proteins were identified that form inter- or intramolecular disulfide bonds under control and diamide stress conditions in B. subtilis and S. aureus. Depletion of the cysteine pool was concomitantly measured in B. subtilis using a metabolomics approach. Thus, the majority of reversible thiol modifications that were previously detected by two-dimensional gel fluorescence-based thiol modification assay are most likely based on S thiolations. Finally, we found that a glutathione-producing B. subtilis strain which expresses the Listeria monocytogenes gshF gene did not show enhanced oxidative stress resistance compared to the wild type.

Summary: Quinones are highly toxic naturally occurring thiol-reactive compounds. We have previously described novel pathways for quinone detoxification in the Gram-positive bacterium Bacillus subtilis. In this study, we have investigated the extent of irreversible and reversible thiol modifications caused in vivo by electrophilic quinones. Exposure to toxic benzoquinone (BQ) concentrations leads to depletion of numerous Cys-rich cytoplasmic proteins in the proteome of B. subtilis. Mass spectrometry and immunoblot analyses demonstrated that these BQ-depleted proteins represent irreversibly damaged BQ aggregates that escape the two-dimensional gel separation. This enabled us to quantify the depletion of thiol-containing proteins which are the in vivo targets for thiol-(S)-alkylation by toxic quinone compounds. Metabolomic approaches confirmed that protein depletion is accompanied by depletion of the low-molecular-weight (LMW) thiol cysteine. Finally, no increased formation of disulphide bonds was detected in the thiol-redox proteome in response to sublethal quinone concentrations. The glyceraldehyde-3-phosphate dehydrogenase (GapA) was identified as the only new target for reversible thiol modifications after exposure to toxic quinones. Together our data show that the thiol-(S)-alkylation reaction with protein and non-protein thiols is the in vivo mechanism for thiol depletion and quinone toxicity in B. subtilis and most likely also in other bacteria.

S-Thiolation is crucial for protection and regulation of thiol-containing proteins during oxidative stress and is frequently achieved by the formation of mixed disulfides with glutathione. However, many Gram-positive bacteria including Bacillus subtilis lack the low molecular weight (LMW) thiol glutathione. Here we provide evidence that S-thiolation by the LMW thiol cysteine represents a general mechanism in B. subtilis. In vivo labeling of proteins with [(35)S]cysteine and nonreducing two-dimensional PAGE analyses revealed that a large subset of proteins previously identified as having redox-sensitive thiols are modified by cysteine in response to treatment with the thiol-specific oxidant diamide. By means of multidimensional shotgun proteomics, the sites of S-cysteinylation for six proteins could be identified, three of which are known to be S-glutathionylated in other organisms.