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

The bacterial strains used were B. subtilis 168 (trpC2) (1) and a B. subtilis strain expressing gshF_Lm (trpC2 amyE::lacI gshF Spec^r). All B. subtilis strains were cultivated in a synthetic medium (60) at 37°C under vigorous agitation. S. aureus UAMS-1 (28) was cultivated in a synthetic medium described earlier (26). However, MOPS (morpholinepropanesulfonic acid) and glycine were omitted and all amino acids were at 1 mM. If needed, cysteine was substituted by 1 mM Na₂S₂O₃. Spectinomycin was used for B. subtilis gshF_Lm at 100 μg/ml. GSH production in the B. subtilis gshF_Lm strain was induced with 40 μM IPTG (isopropyl-β-d-thiogalactopyranoside) at an optical density at 500 nm (OD₅₀₀) of 0.2. All strains were grown to the exponential growth phase (OD₅₀₀ of 0.5) and exposed to 1 mM (B. subtilis) or 2 mM (S. aureus) diamide. The B. subtilis gshF_Lm strain was exposed to 50 μM Paraquat and 100 μM H₂O₂. Controls were taken at an OD₅₀₀ of 0.5.

Growth curves were performed by measuring OD₅₀₀ at different time points. For survival tests, samples from bacterial cultivations were diluted and 100 μl was plated onto LB agar plates. The plates were incubated over night at 37°C, colonies were counted, and subsequently the weighted arithmetic mean was determined. For calculations of the dry cell weight (DCW), 20 ml of B. subtilis cells was collected by centrifugation at different time points as replicates. At these time points, the OD₅₀₀ was measured with different dilutions of the culture. The cells were washed twice with distilled water and transferred to a constant-preweight glass vial. The glass vial was dried in an oven (60°C) to constant weight. The OD₅₀₀ was plotted against the net weight of the glass vials. The analysis of the regression resulted in the following relationship: DCW (mg/ml culture) = 0.2107 × OD₅₀₀ + 0.0043, with R ² = 0.995.

A 2.43-kb fragment of Listeria monocytogenes EGD-e DNA, containing the gshF gene (lmo2770) including its putative ribosomal binding site, was amplified by PCR employing a forward SphI primer, 5′-TATA GCATGC TTATTTAAAACCCCTGAGGTG-3′, and a reverse SphI primer, 5′-ATAT GCATGC AAATGGTGAAATTGGATTG-3′. The underlined letters show the SphI restriction site. The purified fragment digested with SphI was sequenced to verify identity to the deposited sequence and cloned into SphI sites of the integration vector pDR79 (amyE::PspaC-spc; a gift from Sigal Ben-Yehuda, The Hebrew University, Jerusalem, Israel). The resulting construct, containing a proper PspaC-gshF fusion, was designated pDR79-gshF. A linearized pDR79-gshF DNA was transformed into competent B. subtilis 168 cells and integrated into the amyE gene. Transformants were selected for spectinomycin resistance (100 μg/ml) and designated the B. subtilis gshF_Lm strain, which contained a disrupted amyE gene with an intact GSH synthase gene fused to the IPTG-inducible PspaC promoter.

Cells were cultured as described above. Two milliliters of culture was centrifuged for 1 min and washed with 50 mM Tris-HCl (pH 8.0). The pellet was resuspended in 50 μl extraction buffer (50% [vol/vol] acetonitrile [ACN] in 20 mM Tris-HCl, pH 8.0) containing 2 mM monobromobimane (mBBr; Molecular Probes). As an internal standard, 1 μM penicillamine was added to each sample. The suspension was incubated for 15 min at 60°C in the dark. Control samples were incubated for 10 min with 5 mM N-ethylmaleimide (NEM; Sigma-Aldrich) under the same conditions before the addition of mBBr (2 mM). The cellular debris was removed by centrifugation, and the reaction was stopped by addition of 5 mM aqueous methane sulfonic acid. Thiol standards were prepared as described previously (21).

A derivatized sample was separated by high-performance liquid chromatography (HPLC) (51), and peak recognition was performed with a fluorescence detector (Shimadzu RF-10AXL). Briefly, the CoA method was used, which constituted a tetrabutylammonium phosphate (TBAP; Sigma-Aldrich) ion-pairing protocol designed for the separation of CoA-bimane derivatives (51). A C₈ RP column (AccQ-Tag, 3.0 μm, 150 mm; Waters) at a flow rate of 0.8 ml min⁻¹ was used in a column oven at 25°C. The chromatographic protocol employed the following solvents and gradients: solvent A, 10% (vol/vol) methanol, 0.25% (vol/vol) acetic acid, and 10 mM TBAP, pH 3.4; solvent B, 90% (vol/vol) methanol, 0.25% (vol/vol) acetic acid, and 10 mM TBAP. At time zero, 0% solvent B was used, followed by 30 min with 40% solvent B, 40 min with 75% solvent B, and 50 min with 100% solvent B.

Cells were grown and treated with diamide as described above. To determine the amounts of LMW thiols, 50 ml of the culture was centrifuged and the pellet was resuspended in 600 μl extraction buffer. The suspension was incubated for 10 min at 60°C, and subsequently the cell debris was removed by centrifugation. Five hundred microliters of supernatant was mixed with 10 μl 100 mM 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) in dimethyl sulfoxide. The pellet was again incubated with 600 μl extraction buffer, as described above, and 500 μl was added to the same tube used before. The A ₄₁₂ was measured, and the LMW thiol content was calculated.

To determine the amount of protein thiols, the pellet harvested from 50 ml of culture was resuspended in 1,000 μl denaturing buffer, consisting of 8 M urea, 1% CHAPS {3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate}, 1 mM EDTA, 200 mM Tris-HCl (pH 8.0), and 10 μl of 100 mM DTNB was added directly. The suspension was incubated for 15 min and centrifuged. The supernatant was diluted, A ₄₁₂ was measured, and the protein thiol content was calculated.

Cells were cultured and treated with diamide as described above. Forty milliliters of the culture was centrifuged, and the pellet was resuspended in 400 μl extraction buffer. For GSSG determination, the buffer contained 3% scavenger reagent (GSH/GSSG assay kit from Oxford Biomedical Research). The suspension was incubated for 15 min at 60°C, and subsequently the cell debris was removed by centrifugation. Different dilutions were used for the assay according to the manufacturer's instructions.

Cells were harvested by centrifugation at 10,000 × g for 10 min at 4°C. The pellet was resuspended in denaturing buffer containing 100 mM iodoacetamide (IAM). Cells were disrupted by sonication, and the lysate was incubated in the dark for 20 min to allow alkylation of thiol groups. Proteins were precipitated in a fourfold volume of pure acetone. The resulting pellet was washed twice with acetone, dried in a SpeedVac, and resuspended in denaturing buffer without IAM.

The two-dimensional (2D) gel-based FALKO assay was performed as described by Hochgräfe et al. (32). Proteins were isolated with alkylated thiol groups as described above. The protein concentration was measured according to Bradford et al. (9), and equal protein amounts of stressed and control samples were used. All reversible thiol modifications (disulfide bonds and mixed disulfides) were reduced with Tris-(2-carboxyethyl-)-phosphine (TCEP; Molecular Probes) and subsequently labeled with the fluorescence dye BODIPY FL C₁-IA [N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-yl)-methyl)-iodoacetamide; Molecular Probes] as described previously (32). The labeling reaction was stopped by centrifugation through a Bio-Rad Micro Bio-Spin 6 column. 2D polyacrylamide gel electrophoresis (PAGE), gel imaging, data analysis, and protein identification were performed as described elsewhere (32).

Proteins were isolated with alkylated thiol groups as described above. The concentration of protein was measured according to Bradford et al. (9), and equal protein amounts of all samples were applied. Samples were separated by SDS-PAGE under nonreducing conditions. The lanes of the SDS gel were cut and incubated in sample buffer (2% SDS, 62.5 mM Tris-HCl, pH 8.0) containing 50 mM dithiothreitol to reduce disulfide bonds. Subsequently, the lanes were incubated in sample buffer containing 100 mM IAM or 100 μM BODIPY FL C₁-IA, respectively. Each lane of the SDS gel was positioned vertically and separated again by SDS-PAGE. The resulting diagonal SDS gels were stained with SYPRO Ruby and scanned. Subsequently, proteins were stained with Coomassie brilliant blue as described previously (20) and scanned. Proteins that did not migrate on the diagonal were identified as described previously (20). For control purposes, SDS lanes were separated under reducing conditions in the first and second dimensions. Proteins that did not comigrate with the diagonal were omitted from further studies.

The monitoring of S thiolations by [³⁵S]cysteine was performed as described previously (33). In brief, protein synthesis was inhibited by addition of chloramphenicol for 30 min (B. subtilis gshF_Lm strain) and fusidic acid for 60 min (S. aureus). The culture was supplemented with [³⁵S]cysteine for 30 min, and subsequently cells were treated with diamide for 30 min or harvested as control samples. The proteins were isolated as described above. Equal protein amounts of all samples were applied to filter disks, and the radioactivity was measured in a scintillation counter. Aliquots of protein samples were reduced with TCEP prior to filter disk application to prove reducible S thiolations.

Analyses of intracellular metabolites by gas chromatography-mass spectrometry (GC-MS) were performed as described by Liebeke et al. (46). Briefly, samples were harvested by fast filtration, and subsequently the metabolism was quenched with an ethanol solution and liquid nitrogen. After cell disruption and metabolite extraction, samples were analyzed by GC-MS. Samples to detect possible disulfides and NADP(H) as well as ppGpp were analyzed by liquid chromatography (LC)-MS as described before (4, 18). An aminopropyl column (Phenomenex Luna NH₂, 250 mm by 2 mm, 5 μm) was used with solvent A (20 mM ammonium acetate, 20 mM ammonium hydroxide, 5% [vol/vol] ACN in water; pH 9.45) and solvent B (pure ACN). The flow rate was 0.2 ml·min⁻¹, with the following gradient: t = 0 min, 85% solvent B; t = 15 min, 0% solvent B; t = 38 min, 0% solvent B; t = 40 min, 85% solvent B; and t = 50 min, 85% solvent B. The mass spectrometer was adjusted as described by Donat et al. (18).

Gel electrophoresis LC-MS (GeLC-MS) was carried out as described by Hochgräfe et al. (33). In brief, protein extracts of cells, stressed with diamide for 30 min, were isolated as described above. However, protein precipitation by acetone was omitted. The protein extracts were separated by nonreducing, one-dimensional SDS-PAGE. Gel lanes were sliced and each slice was digested with trypsin. The resulting peptide mixtures were separated by reversed-phase HPLC directly coupled to an LTQ Orbitrap mass spectrometer for subsequent tandem-MS (MS/MS) analysis. Extracts of B. subtilis ghsF_Lm cells were searched against database of B. subtilis 168 extracted from SubtiList (http://genolist.pasteur.fr/SubtiList ). Extracts of S. aureus UAMS-1 cells were searched against a fusion protein database of all accessible S. aureus strains, including phage proteins and plasmid-encoded proteins.

The cysteine synthase CysM has a crucial role in the regulation of cysteine biosynthesis and stress response of S. aureus to diamide (48, 59), suggesting an important role for cysteine or derivatives of cysteine. Recently, the LMW thiols cysteine, CoA, and BSH could be identified as abundant thiols in bacilli and S. aureus (52). We found S thiolations based on cysteine in B. subtilis in six proteins (33) and have extended these studies to analyze S thiolations in S. aureus. We used a [³⁵S]cysteine-based assay described by Hochgräfe et al. (33). In brief, protein synthesis was inhibited by fusidic acid and [³⁵S]cysteine was supplemented. Proteins were isolated from cells before and after exposure to diamide. The protein-associated radioactivity was measured before and after treatment with a reducing agent. A cysteine-free medium is required for the assay to ensure sufficient [³⁵S]cysteine incorporation in S thiolations. S. aureus strains COL, UAMS-1, Newman, and NCTC 8325 and 6850 were examined for growth with different sulfur sources, including methionine, sulfate, thiosulfate, or thiocyanate. S. aureus UAMS-1 was the only strain that could assimilate thiosulfate and was therefore used for further investigations.

Exposure of cells to diamide led to 2.8-fold-higher protein-bound radioactivity compared to the untreated sample (Fig. 1). Reduction of the protein extract of diamide treated cells by TCEP caused a loss of protein-bound radioactivity of almost 70%, indicating that most of the [³⁵S]cysteine was part of protein S thiolations. Protein extracts of S. aureus UAMS-1 exposed to diamide were subjected to GeLC-MS to identify LMW thiols responsible for S thiolations. Two proteins were identified that are modified by S cysteinylation: the acetoin reductase (SAR0129) is S cysteinylated at C-155 (see Fig. S1A in the supplemental material), and the hypothetical lipoprotein SAR0761 is S cysteinylated at C-133 (see Fig. S1B in the supplemental material). Results at a lower confidence level suggest S cysteinylations in a glycyl-tRNA synthetase (SAR1642) at C-344, a phosphoribosylaminoimidazole-succinocarboxamide synthase (SAR1040) at C-196, a pyruvate kinase (SAR1776) at C8, a triosephosphate isomerase (SAR0830) at C-40, and SarA (SAR0625) at C-9. Because all modified proteins were identified from the annotated genome of S. aureus MRSA252, we combined letters from this strain name into a prefix for our designations: “SAR.” Concluding these MS results, S. aureus uses S cysteinylation for protein thiol modification during disulfide stress.

Diamide stress leads to a massive increase of reversible oxidations of protein thiols in B. subtilis and S. aureus (32, 64). These modifications were identified as S cysteinylations in different proteins of B. subtilis (33) and S. aureus as shown here. We next analyzed the extent of newly formed inter- and intramolecular disulfide bonds in response to disulfide stress. For this purpose, the diagonal nonreducing/reducing SDS gel electrophoresis approach was used (57) for separation of untreated samples and those exposed to diamide.

Under control conditions, most proteins migrate along the diagonal, indicating their reduced state. The identification of proteins from randomly chosen gel pieces from the diagonal revealed proteins with and without cysteine residues in B. subtilis (Fig. 2A [e.g., KatA, Cah, Hag, GapA, and PurC]) and S. aureus (Fig. 3A [e.g., Asp23, RplD, and SAR0872]). This indicates that most proteins do not form intra- or intermolecular disulfide bonds under control conditions. Proteins that migrate below the diagonal include FusA, ThiC, IlvC, HisI, HisB, YkrT, YumC, and AhpC in B. subtilis (Fig. 2A) and AhpC, MetN-2, HutG, GlyQS, NrdE/Rir1, PurH, and a DeoD homologue in S. aureus (Fig. 3A). These proteins most likely form intermolecular disulfide bonds even under control conditions. In S. aureus and B. subtilis, proteins above the diagonal were detected, indicating formation of intramolecular disulfide bonds (Fig. 2A and 3A). Six proteins of B. subtilis were identified above the diagonal as MetE, GltB, GuaB, ArgF, YumC, and Tpx. These contain at least two cysteines required for intramolecular disulfide bond generation. However, we have to consider that some proteins with intramolecular disulfide bonds might migrate close to the diagonal.

Diagonal gel electrophoresis of protein extracts from diamide-treated B. subtilis cells identified YceE with a newly formed intramolecular disulfide bond above the diagonal (Fig. 2B). Two proteins with newly formed intermolecular disulfide bonds were identified below the diagonal as the β-ketoacyl-acyl carrier protein synthase II FabF and the α subunit of the oxygen-dependent ribonucleotide diphosphate reductase NrdE (Fig. 2B). FabF, NrdE, and YceE are not induced after exposure to diamide (45). Thus, we can exclude the appearance of these spots due to increased amounts of protein. Diamide also leads to an increase in disulfide bonds in S. aureus. We detected four proteins with newly formed intermolecular disulfide bonds (Fig. 3B [Y1 to Y4]) and three proteins with newly formed intramolecular disulfide bonds (Fig. 3B [Z1 to Z3]).

The diagonal gel electrophoresis approach does not visualize proteins which are S thiolated. To monitor all proteins with reversible thiol modifications, including S thiolations, we combined the diagonal gel electrophoresis approach with a thiol-specific fluorescent labeling. In brief, we separated the protein extracts with chemically IAM-alkylated thiol groups under nonreducing conditions. The excised gel lanes were reduced, and those proteins with reversible thiol oxidations were stained with the fluorescent dye BODIPY-FL C₁ IA. Proteins that are S thiolated migrate along the diagonal but are now fluorescently labeled. Many fluorescence signals were detected along the diagonal under control conditions (Fig. 2C and 3C), which increased after diamide stress (Fig. 2D and 3D) in B. subtilis and in S. aureus. Comparison of the images from the original approach and this combined approach did not result in the detection of additional protein spots apart from the diagonal. These findings further support the formation of S thiolations in B. subtilis and S. aureus after diamide stress.

We investigated the global intracellular metabolite pool—the metabolome—to follow changes in the level of LMW thiols and other compounds after disulfide stress in response to diamide.

The cysteine pool decreased after exposure to diamide by about 15-fold (Fig. 4A). However, we did not detect formation of cystine, the cysteine disulfide, or other disulfides at the given sample points. We also monitored cysteine precursors, O-acetylserine, serine, and methionine. Methionine levels decreased about fourfold during disulfide stress (Fig. 4A). O-Acetylserine levels increased up to 20-fold, whereas serine levels did not change significantly (Fig. 4A). The pool of glycine, a serine precursor, was depleted up to 75% in diamide-treated cells (Fig. 4A). Additionally, serine can be synthesized via pyruvate or the phospho-serine-pathway (56). Both use glyceraldehyde-3-phosphate as a precursor. The concentration of glyceraldehyde-3-phosphate decreased up to 50% during disulfide stress (Fig. 4A). The levels of cysteine, methionine, O-acetylserine, glycine, and glyceraldehyde-3-phosphate were restored to normal 120 min after exposure to diamide. This restoration of the metabolite pool occurred in parallel to the restoration of growth (45).

The metabolomic data further showed increased amounts of threonine (14-fold) after exposure to diamide (Fig. 4B). Threonine is a precursor for the synthesis of the branched-chain amino acids leucine, isoleucine, and valine. Diamide leads to a decrease in the levels of valine (5.5-fold) and isoleucine (4-fold), while the amount of leucine decreased later, and not as markedly (Fig. 4B). Additionally, synthesis of ppGpp, the alarmon of the stringent response, could be observed during disulfide stress (Fig. 4B).

These observations revealed significant modifications in the cysteine and branched-chain amino acid biosynthesis pathways in response to diamide. The drastic depletion of cysteine and its precursors supports the idea of S thiolation of protein thiols after exposure to diamide.

The results described above suggest that B. subtilis and S. aureus possess a thiol redox buffer based on cysteine. However, neither of these bacteria is able to produce GSH. The gshF gene of Listeria monocytogenes, a close relative of B. subtilis, encodes a multidomain GSH synthase (29). By heterologous expression of L. monocytogenes gshF in a B. subtilis gene, we engineered the B. subtilis gshF_Lm strain.

To determine expression of GshF in the B. subtilis gshF_Lm strain, protein extracts from IPTG-induced and noninduced cells were separated by 2D PAGE. The GshF (lmo2770) protein could be identified in the gel of the IPTG-induced B. subtilis gshF_Lm strain (Fig. 5B). Growth of the B. subtilis gshF_Lm strain was not inhibited by IPTG addition (Fig. 5A). To assess production of GSH in the B. subtilis gshF_Lm strain, whole-cell lysates were labeled with the thiol-reactive compound mBBr and separated by HPLC. In parallel, control samples were incubated with NEM prior to exposure to mBBr. NEM binds to thiols and thus inhibits the subsequent binding of mBBr. Hence, only peaks in the mBBr sample account for thiols that are not present in the NEM sample. The IPTG-induced B. subtilis gshF_Lm strain contained GSH at a concentration of 2.63 μmol/g DCW and γ-glutamylcysteine (γ-GC), the immediate precursor of GSH, at a concentration of 0.74 μmol/g DCW (Fig. 6B and F). GSH and γ-GC were hardly detectable in the noninduced B. subtilis gshF_Lm strain cultures. Cysteine and CoA were present under noninducing conditions at concentrations of 0.25 and 0.87 μmol/g DCW, respectively (Fig. 6D and F). In response to GSH production, cysteine (0.31 μmol/g DCW) and CoA (0.8 μmol/g DCW) were detected at similar concentrations (Fig. 6B and F). Analyses of the whole LMW thiol amount by the DTNB method showed that it exceeded the sum of thiols analyzed by this HPLC method (Fig. 6F and G). Not all thiols could be quantified by the HPLC method, and differences in the preparation methods might explain this observation. We found whole LMW thiol amounts of 5.42 μmol/g DCW in the GSH-free B. subtilis gshF_Lm strain and 13.39 μmol/g DCW in the GSH-producing B. subtilis gshF_Lm strain (Fig. 6G).

We were interested in whether GSH produced by the B. subtilis gshF_Lm strain contributes to the thiol redox buffer. Phenotypical experiments revealed that GSH production had no discernible effect on growth or survival under thiol-specific stress conditions (Fig. 5C). Additionally, we analyzed this strain for oxidative stress resistance induced by H₂O₂ and the superoxide-generating agent methyl viologen. During peroxide stress, no significant changes could be detected in growth or survival due to production of GSH (see Fig. S2A in the supplemental material). However, during superoxide stress, cells without GSH recovered slightly faster than cells with GSH (see Fig. S2B in the supplemental material).

Analyses of the whole LMW thiol amount by the DTNB method revealed no significant difference between noninduced (1.33 μmol/g DCW) and induced (1.91 μmol/g DCW) B. subtilis gshF_Lm strain cells after exposure to diamide (Fig. 6G). This suggests a strong oxidation of GSH, which could be verified by measuring the GSH/GSSG ratio (Fig. 6I). In the exponential growth phase, GSH was present at 8.67 μmol/g DCW and GSSG at 0.03 μmol/g DCW, giving a ratio of 267.67:1. Under diamide stress, the concentration of GSH was 1.95 μmol/g DCW and that of GSSG was 2.61 μmol/g DCW, resulting in a ratio of 1:1.34.

Next, we analyzed the thiol redox proteome of the B. subtilis gshF_Lm strain using the FALKO assay, the diagonal gel electrophoresis methodology, and the S thiolation assay based on [³⁵S]cysteine to detect differences in reversible thiol modifications of proteins. Proteins of GSH-producing and GSH-free B. subtilis gshF_Lm strain cells were analyzed before and after exposure to diamide. Analyses of reversible thiol modifications using the FALKO assay showed no significant differences based on GSH production, either under control conditions or in response to diamide stress (see Fig. S3 in the supplemental material). Furthermore, comparison of the diagonal electrophoretic patterns from noninduced and GSH-producing, B. subtilis gshF_Lm strain cells did not show significant changes in the formation of disulfide bonds before and after diamide stress (see Fig. S4 in the supplemental material). This is supported by quantitative analyses of protein thiols in the B. subtilis gshF_Lm strain (Fig. 6H). We measured 12.89 μmol/g DCW in GSH-free cells and 12.98 μmol/g DCW in GSH-producing cells under control conditions. Diamide led to a loss of 60% of reduced protein thiols in GSH-free (5.26 μmol/g DCW) and GSH-producing (5.17 μmol/g DCW) cells. Thus, GSH production did not affect the level of reduced protein thiols. Remarkably, [³⁵S]cysteine incorporation of GSH-producing B. subtilis gshF_Lm strain cells decreased 2.8-fold compared to the level in the noninduced strain (Fig. 7A and B, left columns). This difference might result from a higher pool of LMW thiols in the GSH-producing strain (Fig. 6F and H). Exposure to diamide resulted in a comparable increase of [³⁵S]cysteine incorporation in both GSH-producing (5.3-fold) and nonproducing (5.9-fold) cultures. Reduction of samples treated with diamide by TCEP resulted in an 80% loss of radioactivity in the GSH-free sample and 65% loss in the GSH-containing sample (Fig. 7A and B). The marked loss of radioactivity following protein reduction indicates S thiolations.

To assess whether S glutathionylation of proteins occurs, and if in such instances the proteins were previously known to be S thiolated, we analyzed the protein extracts of diamide-treated GSH-producing B. subtilis gshF_Lm strain cells by GeLC-MS. We found S glutathionylation in YwaA at C-104 (see Fig. S5 in the supplemental material). YwaA was previously shown to be S cysteinylated in B. subtilis wild-type cells (33). Preliminary data show further examples of S glutathionylations in YjcI (C-128), Tsf (C-239), PurB (C-199), YjdL (C-177), and MetE (C-719). Although cysteine is present in GSH-producing B. subtilis gshF_Lm strain cells, we did not find any S cysteinylations.