S-Cysteinylation Is a General Mechanism for Thiol Protection of Bacillus subtilis Proteins after Oxidative Stress

Abstract

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...

Diamide Triggers Mainly S Thiolations in the Cytoplasmic Proteomes of Bacillus subtilis and Staphylococcus aureus

“…S thiolations prevent protein thiols from irreversible oxidation to sulfinic and sulfonic acids, but also affect enzyme activity (35,47) and signal transduction (39,42). In B. subtilis, we have identified a few cytoplasmic proteins that are S cysteinylated (33). In addition, the organic peroxide sensor OhrR was inactivated by an S bacillithiolation in B. subtilis (42).…”

“…However, more recent findings demonstrate that diamide stress leads also to S thiolations: formation of disulfide bonds between proteins and LMW thiols (8,13,33). S thiolations prevent protein thiols from irreversible oxidation to sulfinic and sulfonic acids, but also affect enzyme activity (35,47) and signal transduction (39,42).…”

Cadmium Toxicity in Glutathione Mutants ofEscherichia coli

“…GSH is synthesized from glutamate, cysteine, and glycine in two ATP-consuming steps (involving GshA and GshB) via the intermediate ␥-glutamylcysteine (␥EC). The latter is the major thiol in aerobic phototrophic halobacteria (archaea) (14), while cysteine may substitute for GSH in some functions in Bacillus subtilis (24).…”

The Paralogous MarR/DUF24-Family Repressors YodB and CatR Control Expression of the Catechol Dioxygenase CatE inBacillus subtilis

“…Quinones and diamide deplete the cellular pool of low-molecular-weight (LMW) thiols via different mechanisms. Diamide leads to an increase in reversible thiol modifications, such as inter-and intramolecular disulfides and S thiolations (disulfides between proteins and LMW thiols) (9,10,27). Quinones can act as oxidants or electrophiles (13,20,25).…”