S-Thiolation is crucial for protection and regulation of thiolcontaining 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 twodimensional 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 thiolspecific 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.Protein S-thiolation by low molecular weight (LMW) 3 thiols prevents the irreversible oxidation of cysteine residues during oxidative stress and plays a pivotal role in the redox regulation of thiol-containing proteins. For example, the formation of mixed disulfides between target proteins and the LMW thiol glutathione is a key event in the regulation of the eukaryotic proteins, including c-Jun, thioredoxin, and glyceraldehyde-3-phosphate dehydrogenase (1-5). S-Glutathionylation has also been reported to modulate the activity of Escherichia coli methionine synthase (MetE), phosphoadenosine phosphosulfate reductase (CysH), and the oxidative stress-specific transcription factor OxyR (6 -8).Because many Gram-positive bacteria lack glutathione, the nature of S-thiolation in these organisms remains elusive (9). In the Gram-positive model organism Bacillus subtilis, cysteine represents the most abundant LMW thiol (9). One of the most obvious responses of B. subtilis to disulfide stress is the strong induction of cysteine biosynthesis genes (10). Although the origins of this effect are unclear, it might reflect a consumption of free cysteine by oxidation to cystine and the formation of mixed disulfides with proteins.In a previous proteome analysis, we reported that reversible thiol oxidation occurs in a number of B. subtilis proteins after oxidative stress indicating a general thiol-protection mechanism in this organism (11). We also showed that overoxidation of cysteine residues to sulfonic acid caused by high level peroxide stress results in protein damage and in the irreversible loss of protein activity in B. subtilis and Staphylococcus aureus (11,12). However, the nature of the reversible thiol modifications was not apparent and likely reflected a mixture of intra-or intermolecular protein disulfides as well as mixed disulfides with LMW thiols. Recently, Lee et al. (13) demonstrated that the organic peroxide-sensing repressor OhrR of B. subtilis is reversibly inactivated by S-thiolation by cysteine, an unknown thiol of 398 Da, or coenzyme A (CoASH) under conditions of oxidative stress. S-Thiolation by CoASH also has been shown to occur dur...

SummaryQuinones are highly toxic naturally occurring thiolreactive 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 thiolcontaining 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 lowmolecular-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.

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.Cysteine thiols in proteins fulfill an important and diverse set of cellular functions. In particular, they participate in enzymatic catalysis; in metal coordination, such as in the generation of Fe-S-clusters; and in determining the spatial structure of proteins via disulfide bond formation (3,22,23,38). Cysteines are strong nucleophiles amenable to posttranslational modifications by reactive oxygen species (ROS) and reactive nitrogen species, leading to disulfides; to sulfenic, sulfinic, or sulfonic acids; mixed disulfides with low-molecular-weight (LMW) thiols (S thiolations); and S nitrosylations (7,16,17,27).The redox status of the cytoplasm is under physiological conditions in a reduced state. Thus, most cysteines are present as free thiols (6). Because aerobic organisms have to cope with oxidative stress caused by ROS, such as superoxide anions, hydrogen peroxide, or hydroxyl radicals, they need to employ effective mechanisms that maintain the reduced state. In gramnegative bacteria, the thiol-disulfide balance is accomplished by the glutathione (GSH) system, a thiol-based redox buffer. The GSH system consists of glutaredoxin (Grx), GSH (␥-glutamylcysteinyl glycine), GSH reductase, and GSH peroxidase (34). Reduction of disulfides occurs via sequential electron transfer from glutaredoxin and reduced GSH; oxidized GSH (GSSG) is reduced by the NADPH-dependent GSH reductase. GSH peroxidase enables the direct detoxification of ROS by GSH oxidation.However, many gram-positive bacteria lack genes for GSH biosynthesis. Actinomycetes instead use a thiol redox buffer based on mycothiol (50). Bacillus subtilis, Staphylococcus aureus, and other gram-pos...

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The oxidative stress response of Bacillus licheniformis after treatment with hydrogen peroxide was investigated at the transcriptome, proteome and metabolome levels. In this comprehensive study, 84 proteins and 467 transcripts were found to be up or downregulated in response to the stressor. Among the upregulated genes were many that are known to have important functions in the oxidative stress response of other organisms, such as catalase, alkylhydroperoxide reductase or the thioredoxin system. Many of these genes could be grouped into putative regulons by genomic mining. The occurrence of oxidative damage to proteins was analyzed by a 2-DE-based approach. In addition, we report the induction of genes with hitherto unknown functions, which may be important for the specific oxidative stress response of B. licheniformis. The genes BLi04114 and BLi04115, that are located adjacent to the catalase gene, were massively induced during peroxide stress. Furthermore, the genes BLi04207 and BLi04208, which encode proteins homologous to glyoxylate cycle enzymes, were also induced by peroxide. Metabolomic analyses support the induction of the glyoxylate cycle during oxidative stress in B. licheniformis.