S-Bacillithiolation Protects Conserved and Essential Proteins Against Hypochlorite Stress in Firmicutes Bacteria

Bui Khanh Chi; Alexandra A Roberts; Tran Thi Thanh Huyen; Katrin Bäsell; Dörte Becher; Dirk Albrecht; Chris J Hamilton; Haike Antelmann

doi:10.1089/ars.2012.4686

. 2013 Apr 10;18(11):1273–1295. doi: 10.1089/ars.2012.4686

S-Bacillithiolation Protects Conserved and Essential Proteins Against Hypochlorite Stress in Firmicutes Bacteria

Bui Khanh Chi ¹, Alexandra A Roberts ², Tran Thi Thanh Huyen ¹, Katrin Bäsell ¹, Dörte Becher ¹, Dirk Albrecht ¹, Chris J Hamilton ², Haike Antelmann ^1,^✉

PMCID: PMC3584511 PMID: 22938038

Abstract

Aims: Protein S-bacillithiolations are mixed disulfides between protein thiols and the bacillithiol (BSH) redox buffer that occur in response to NaOCl in Bacillus subtilis. We used BSH-specific immunoblots, shotgun liquid chromatography (LC)–tandem mass spectrometry (MS/MS) analysis and redox proteomics to characterize the S-bacillithiolomes of B. subtilis, B. megaterium, B. pumilus, B. amyloliquefaciens, and Staphylococcus carnosus and also measured the BSH/oxidized bacillithiol disulfide (BSSB) redox ratio after NaOCl stress. Results: In total, 54 proteins with characteristic S-bacillithiolation (SSB) sites were identified, including 29 unique proteins and eight proteins conserved in two or more of these bacteria. The methionine synthase MetE is the most abundant S-bacillithiolated protein in Bacillus species after NaOCl exposure. Further, S-bacillithiolated proteins include the translation elongation factor EF-Tu and aminoacyl-tRNA synthetases (ThrS), the DnaK and GrpE chaperones, the two-Cys peroxiredoxin YkuU, the ferredoxin–NADP⁺ oxidoreductase YumC, the inorganic pyrophosphatase PpaC, the inosine-5′-monophosphate dehydrogenase GuaB, proteins involved in thiamine biosynthesis (ThiG and ThiM), queuosine biosynthesis (QueF), biosynthesis of aromatic amino acids (AroA and AroE), serine (SerA), branched-chain amino acids (YwaA), and homocysteine (LuxS and MetI). The thioredoxin-like proteins, YphP and YtxJ, are S-bacillithiolated at their active sites, suggesting a function in the de-bacillithiolation process. S-bacillithiolation is accompanied by a two-fold increase in the BSSB level and a decrease in the BSH/BSSB redox ratio in B. subtilis. Innovation: Many essential and conserved proteins, including the dominant MetE, were identified in the S-bacillithiolome of different Bacillus species and S. carnosus using shotgun-LC-MS/MS analyses. Conclusion: S-bacillithiolation is a widespread redox control mechanism among Firmicutes bacteria that protects conserved metabolic enzymes and essential proteins against overoxidation. Antioxid. Redox Signal. 18, 1273–1295.

Introduction

The reduced cytoplasm is maintained by low-molecular-weight (LMW) thiol-redox buffers, such as glutathione (GSH), present in eukaryotes and in Escherichia coli (34). Redox buffers protect bacteria against reactive oxygen species (ROS) generated during respiration or produced by activated macrophages during infections (1). ROS cause reversible intramolecular and intermolecular disulfides of proteins as well as mixed disulfides between protein thiols and LMW thiols (S-thiolations). In eukaryotes, protein S-glutathionylation is the most important post-translational thiol modification caused by ROS, and has been shown to control redox-sensing transcription factors and to protect active-site cysteine (Cys) residues of essential metabolic enzymes against irreversible oxidation to Cys-sulfinic or sulfonic acids (8, 13, 51). As an effective redox control mechanism, S-glutathionylation must meet several criteria: (i) reversibility, (ii) specificity to active site Cys, (iii) change in protein function/activity, and (iv) induction by ROS or reactive nitrogen species (RNS). S-glutathionylation also serves as a form of GSH storage to prevent the export of oxidized glutathione disulfide (GSSG) under oxidative stress (8). Many eukaryotic proteins such as α-ketoglutarate dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase, ornithine δ-aminotransferase, pyruvate kinase, heat-specific chaperones, and regulatory proteins (c-Jun and NF-κB) are reversibly inactivated or activated by S-glutathionylation (8, 21). In E. coli, the oxidative stress-responsive regulator OxyR is activated by S-glutathionylation in vitro (22), while the activities of glyceraldehyde-3-phosphate dehydrogenase, methionine synthase, and 3′-phosphoadenosine-5′-phosphosulfate reductase are inhibited by S-glutathionylation in E. coli (3, 19, 29).

Innovation.

Recently, we identified six S-bacillithiolated proteins in Bacillus subtilis in response to NaOCl stress (7). The methionine synthase MetE was inactivated by S-bacillithiolation causing methionine auxotrophy. Here, we have studied the conserved S-bacillithiolome among four different Bacillus species and Staphylococcus carnosus using bacillithiol (BSH)-specific immunoblots, shotgun liquid chromatography–tandem mass spectrometry analysis, and redox proteomics. We identified 54 S-bacillithiolated proteins, including eight conserved proteins (MetE, AroA, GuaB, TufA, PpaC, SerA, YphP, and YumC) and 29 unique proteins involved in amino acid and cofactor biosynthesis, nucleotide metabolism, translation, protein quality control, redox, and antioxidant functions. S-bacillithiolation is accompanied by an increased oxidized bacillithiol disulfide (BSSB) level and a decreased BSH/BSSB ratio. Together, our data support a major role of the BSH redox buffer in redox control and protection of conserved and essential proteins against irreversible oxidation by S-bacillithiolations in Firmicutes bacteria.

Gram-positive bacteria do not produce GSH. Among the actinomycetes, mycothiol is the predominant LMW thiol that serves analogous roles to GSH (20, 39). In low-GC Gram-positive bacteria, such as Bacillus and Staphylococcus species, bacillithiol (BSH) serves as a redox buffer that was structurally characterized as Cys-GlcN-malate (12, 16, 40). Mutants with defects in BSH biosynthesis displayed strong sensitivities to the epoxide antibiotic fosfomycin and to NaOCl stress (7, 12). In Bacillus subtilis, the redox-sensing MarR-type repressor OhrR forms mixed disulfides with BSH (S-bacillithiolations) in response to organic hydroperoxides and NaOCl stress (7, 26). S-bacillithiolation of OhrR alleviates repression of transcription of the ohrA peroxiredoxin gene as a protection mechanism against NaOCl toxicity (7). Besides OhrR, we have recently identified by liquid chromatography (LC)–tandem mass spectrometry (MS/MS) analysis five cytoplasmic proteins with S-bacillithiolations after NaOCl treatment (7). Hypochloric acid shows very fast reaction rates with Cys residues (k₂=3×10⁷ M⁻¹ s⁻¹) and rapidly leads to irreversible oxidation products, such as sulfinic and sulfonic acids (9, 15, 42, 50). Thus, S-bacillithiolation serves to protect active-site Cys residues of key metabolic enzymes against overoxidation. S-bacillithiolation of the methionine synthase MetE at the active site Cys730 caused a methionine starvation phenotype in NaOCl-treated cells, presumably to slow down translation while the OhrA peroxiredoxin removes the toxic oxidant (7).

In this work, we have identified proteome-wide conserved S-bacillithiolations in various industrially important Bacillus and Staphylococcus species. We used BSH-specific immunoblot analyses, redox proteomic approaches, and shotgun-LC-MS/MS analyses to identify essential and conserved proteins in the S-bacillithiolome that have been previously identified in the S-glutathionylome in eukaryotes. The protein bacillithiolations were accompanied by an increased level of oxidized bacillithiol disulfide (BSSB) and a decreased BSH/BSSB redox ratio in B. subtilis.

Results

Defining the conditions of NaOCl stress to induce S-bacillithiolations in the proteome of B. amyloliquefaciens, B. pumilus, B. megaterium, and S. carnosus

To describe more comprehensively the S-bacillithiolome, we focused on four industrially important Bacillus species: B. subtilis 168, B. pumilus SBUG1799, B. amyloliquefaciens FZB42 (5), B. megaterium SBUG1152 (36), and S. carnosus TM300 (43). The B. subtilis, B. pumilus, and B. megaterium strains were able to grow in the Belitsky minimal medium (BMM), and low NaOCl concentrations could be applied to reduce the growth rate: 75 μM NaOCl for B. subtilis (7), 125 μM for B. pumilus, and 80 μM for B. megaterium (Supplementary Fig. S1B, C; Supplementary Data are available online at www.liebertpub.com/ars). For the growth of B. amyloliquefaciens FZB42, we used the BMM with 0.1% casamino acids, and 275–300 μM NaOCl showed a growth-inhibitory effect (Supplementary Fig. S1A). For S. carnosus, growth was only optimal in a complete medium, and 3.25–3.5 mM NaOCl reduced the growth rate in a Luria Broth (LB) medium (Supplementary Fig. S1D). Since amino acids and peptides present in LB also react with NaOCl, much higher NaOCl concentrations were required to inhibit the growth of S. carnosus in the LB medium.

MetE is the major target for S-bacillithiolation in Bacillus species as revealed by nonreducing BSH-specific immunoblot analysis

To monitor the extent of S-bacillithiolation after NaOCl stress, BSH-specific antibodies were used to test protein extracts from NaOCl-treated B. subtilis wild-type and bshA-mutant cells in nonreducing immunoblot analyses. The BSH immunoblots showed a very strong 90-kDa band after NaOCl stress, corresponding to MetE-SSB (Fig. 1A). The identity of MetE was confirmed by LC-MS/MS analysis of the specific band from nonreducing sodium dodecyl sulfate (SDS) gels (Supplementary Table S2). S-bacillithiolated MetE, modified at Cys719 and Cys730, was previously identified as the most strongly oxidized protein in the redox proteome of B. subtilis (7). We further confirmed S-bacillithiolation of MetE by comparison of extracts from NaOCl-treated B. subtilis cells grown in the BMM and LB media. In a rich LB medium, MetE is repressed, and thus the MetE-SSB modification is not visible in the BSH immunoblots (Fig. 1B). The MetE-SSB band was also not visible in NaOCl-exposed bshA-mutant cells or in reducing BSH immunoblots (Supplementary Fig. S2). Besides MetE, there were a few weaker bands in the 45–55-kDa range, which represent other protein BSH-mixed disulfides (protein-SSB), since these are absent in the bshA mutant cells. These BSH immunoblot results show that MetE is the most abundant S-bacillithiolated protein in NaOCl-treated B. subtilis cells.

Furthermore, BSH immunoblots indicate that MetE is also the major BSH-modified protein in diamide-treated cells (Fig. 1C). In addition, many other BSH-modified proteins are visible after diamide stress, indicating that S-bacillithiolation is also a significant thiol modification in diamide-treated cells.

Next, we analyzed the S-bacillithiolome of NaOCl-treated cells from B. amyloliquefaciens, B. pumilus, B. megaterium, and S. carnosus (Fig. 1D). The protein extracts of all NaOCl-treated Bacillus strains showed MetE as the most abundant protein-SSB, indicating that S-bacillithiolation of MetE is a common protection mechanism against oxidative stress among Bacillus species. In B. megaterium, MetE-SSB was observed at a higher molecular weight and confirmed by LC-MS/MS analysis (Supplementary Table S2). In S. carnosus grown in LB medium, various protein-SSBs were visible, indicating that NaOCl also causes S-bacillithiolations in Staphylococcus. Because MetE synthesis is strongly repressed in LB medium, MetE-SSB was not detected in S. carnosus using BSH-specific immunoblots.

Identification of proteins with S-bacillithiolations in NaOCl-treated cells of B. subtilis, B. amyloliquefaciens, B. pumilus, B. megaterium, and S. carnosus using redox proteomics and shotgun-LC-MS/MS analyses

Previously, we have used redox proteomics based on the reduction of disulfides and labeling of released thiols with the fluorescence dye BODIPY FL C₁-IA to identify proteins oxidized by NaOCl stress in B. subtilis (7). We applied our redox proteome approach for comparison of B. subtilis, B. amyloliquefaciens, B. megaterium, B. pumilus, and S. carnosus to identify proteins with disulfides after NaOCl stress, including those with potential SSB sites. The BODIPY-fluorescence image was overlayed with the Coomassie-stained protein amount image, and the oxidation ratios were calculated versus protein quantities (Fig. 2A, B and Supplementary Figs. S3–S7 and Table 1). The reversibly oxidized proteins were identified using MALDI-TOF/TOF mass spectrometry analysis (Supplementary Table S1), and the oxidation ratios were used for hierarchical cluster analyses (Supplementary Figs. S3–S7).

FIG. 2. — Summary of NaOCl-sensitive proteins (A) and constitutively oxidized proteins (B) in the thiol redox proteomes of four different *Bacillus* strains and *S. carnosus* including unique and conserved S-bacillithiolated proteins **(D)** that were identified by LC-MS/MS analysis (E). **(A)** Close-ups of conserved NaOCl-sensitive proteins including S-bacillithiolated proteins identified in the redox proteomes of *B. subtilis*, *B. amyloliquefaciens*, *B. pumilus*, *B. megaterium*, and *S. carnosus*. Reversibly oxidized proteins in the redox proteome after NaOCl treatment are shown that have been identified with specific and conserved SSB sites (*) using LC-MS/MS analysis. Overlay images represent the redox proteome (red) compared to the Coomassie-stained protein amount image (green) at control conditions (co) and 10 and 30 min after NaOCl exposure (1–3). Column 4 shows the overlay redox proteome control *versus* redox proteome 30-min NaOCl stress. The identified proteins and their fluorescence/protein quantity ratios are listed in Table 1, and the complete redox proteomes of all strains are given in Supplementary Figures S3–S7. **(B)** Examples of conserved redox-controlled proteins that are oxidized under control conditions to intermolecular or intramolecular disulfides representing thiol-dependent peroxiredoxins (AhpC, YkuU, and YgaF) and thiol peroxidases (Tpx), PdhD, and Adk. Overlay images represent the redox proteome (red) compared to the Coomassie-stained protein amount image (green) of *B. subtilis*, *B. amyloliquefaciens*, *B. pumilus*, *B. megaterium*, and *S. carnosus* at control conditions (co) and 10 and 30 min after NaOCl exposure (1–3). Column 4 shows the overlay redox proteome control *versus* redox proteome 30-min NaOCl stress. The identified proteins and their fluorescence/protein quantity ratios are listed in Table 1, and the complete redox proteomes of all strains are listed in Supplementary Figures S3–S7. **(C)** Diagonal nonreducing/reducing SDS-PAGE confirmed intermolecular disulfides for AhpC homologs in *B. subtilis*, *B. amyloliquefaciens*, *B.megaterium*, *S. carnosus* and for YkuU in *B. pumilus*. The IAM-alkylated proteins extracts (100 μg) of NaOCl-treated cells of all strains were separated by 2D nonreducing/reducing diagonal SDS-PAGE analysis as described previously (41). The AhpC homologs are encircled in the diagonal assays of *B. subtilis* (AhpC-Bsub), *B. amyloliquefaciens* (AhpC-Bam), *S. carnosus* (AhpC-Scar), *B. megaterium* (AhpC-Bmeg), and *B. pumilus* (YkuU-Bpum). **(D)** Unique and conserved S-bacillithiolated proteins identified in the redox proteomes of different *Bacillus* strains and *S. carnosus*. The number of the identified reversibly oxidized proteins in the redox proteomes of the different strains is shown in brackets. Unique and conserved proteins with identified SSB sites are listed by names that are oxidized in the redox proteome. **(E)** Unique and conserved S-bacillithiolated proteins identified using LC-MS/MS analysis in different *Bacillus* strains and *S. carnosus*. The number of proteins with identified SSB sites after NaOCl stress using LC-MS/MS analysis in the different *Bacillus* strains and *S. carnosus* is shown in brackets. Unique and conserved proteins with SSB sites are listed by names. LC, liquid chromatography; MS/MS, tandem mass spectrometry.

Table 1.

Identification of Reversibly Oxidized Proteins in the Redox Proteome in Bacillus subtilis, B. amyloliquefaciens, B. pumilus, B. megaterium, and Staphylococcus carnosus

	Redox ratios BODIPY fluorescence/protein amount
	B. subtilis			B. pumilus			B. amyloliquefaciens			B. megaterium			S. carnosus
Proteins	co	10′	30′	co	10′	30′	co	10′	30′	co	10′	30′	co	10′	30′	Protein function	AC-No. (Uniprot)	Function of conserved Cys	Post-translational Cys-modifications
AbrB BPUM_0021				2.1	2.4	1.5										Possible transcriptional regulator	A8F904	Cys54 essential for DNA-binding activity
AccB	1.9	3.6	3.2													Biotin carboxyl-carrier protein of acetyl-CoA carboxylase	P49786	Cys117 conserved
AccD				0.8	2.5	2.8										Acetyl-CoA carboxylase, carboxyl transferase, beta-subunit	B4AMC5	Cys33,35,52,54 Zn binding
AcnA/CitB										1.2	1.2	1.4	1.3	1.6	1.5	Aconitate hydratase (Aconitase)	D5DFZ0/B9DP54	Cys444, 510, 513 conserved: 4Fe-4S-cluster-binding site	Homolog Cys404 oxidized by NaOCl in E. coli
Adk^*	5.4	4.7	4.3	2.3	1.5	1.5	1.8	1.8	1.8	2.2	2.5	4.9	2.6	4.5	2.3	Adenylate kinase	F4E1S4	Cys130, 133, 150 conserved, Zn-finger motif	Cys130–133 disulfide; Cys150^-S-SB by NaOCl in S. carn*
AhpC	5.4	2.7	2.9				5.4	3.6	3.8	4.0	3.7	3.8	4.1	4.9	4.8	Alkylhydroperoxide reductase small-subunit	A7ZAJ9	Cys47, 166 conserved; Cys47 active site	Cys47-Cys166 form catalytic disulfide
AldA													0.4	1.3	1.1	Putative aldehyde dehydrogenase	B9DKD6	Cys290-conserved active site, nucleophile
ArcA													0.7	0.9	0.9	Arginine deiminase 1	B9DKE2	Cys401-conserved amidino-cysteine-intermediate active site
ArcC													2.2	1.8	1.4	Carbamate kinase 1	B9DJ94	No Cys conserved
ArgG				0.4	2.4	1.9										Argininosuccinate synthase	B4AMD7	No Cys conserved	Cys187 S-SCys by diamide in Bsub
AroA^*	0.4	1.1	1.3	0.5	1.2	1.6	0.6	3.4	1.1	0.7	1.0	1.8				Bifunctional 3-deoxy-7-phosphoheptulonate synthase/chorismate mutase	A7Z7R9	Cys126, 259 conserved	Cys126^-SSB by NaOCl in B. sub., B .pum., B. amy., S. carn*.
ArsC (Sca_0451)													1.3	5.4	6.2	Putative ArsC/Spx family protein	B9DJH2	Cys10, 13 conserved: form redox-active disulfide bond by oxidative stress
BMD_4393										0.9	1.2	0.8				Acetyl-CoA acetyltransferase	D5DL95	Active-site Cys90 acetylated by AcetylCoA; Cys381 important for proton exchange
CheW				0.3	1.7	2.2										Chemotaxis protein CheW	A8FDA9	No Cys conserved
CheY				0.6	0.5	0.6										Response regulator CheY	A8FD98	No Cys conserved
CysH	1.6	1.1	1.6													Phosphoadenosine phosphosulfate reductase	P94498	Cys120, 121, 203, 206, 229 conserved	Cys239 forms mixed disulfide with Cys32 of Trx in E. coli
CysI				3.4	1.7	2.6										Sulfite reductase [NADPH] hemoprotein beta-component	B4AD94	Cys436, 442, 481, 485 conserved: 4Fe-4S-cluster-binding site
CysJ (BAT_2248)	1.0	1.3	0.9	1.4	1.4	1.6	2.7	2.0	2.0							Sulfite reductase [NADPH] flavoprotein, alpha-component	B4AD95	Cys166, 559 conserved	Cys428–432 intramolecular disulfide
DapA							0.4	2.3	2.4							Dihydrodipicolinate synthase	F4EJJ2	No Cys conserved
Dat							2.0	1.2	1.3							d-alanine aminotransferase	A7Z2X8	Cys141 close to the active-site Lys144
DeoD							4.3	3.7	4.3							Purine nucleoside phosphorylase DeoD-type	A7Z5M8	No Cys conserved
DhbE							1.7	1.9	1.5							Putative 2,3-dihydroxybenzoate-AMP ligase DhbE	A7Z8A8	Cys218, 324, 473 conserved
DnaJ													2.6	3.7	3.3	Chaperone protein DnaJ	B9DNJ9	Cys147, 150, 204, 207 Zn-finger1; Cys164, 167, 190, 193 Zn-finger2
DnaK^*													0.7	0.5	0.6	Chaperone protein DnaK	B9DNK0	Cys15 not conserved	Cys15^-S-SB by NaOCl in S. carn*.
Eno	0.7	0.8	0.6													Enolase	P37869	No Cys conserved
FabH													0.5	1.6	1.5	3-oxoacyl-[acyl-carrier-protein] synthase 3	B9DIT1	Cys112 conserved active site
FabZ													4.0	5.3	6.3	(3R)-hydroxymyristoyl-[acyl-carrier-protein] dehydratase	B9DMG5	Cys94, 134 conserved
FdhL (Sca_1802)													2.9	3.3	3.0	Putative formate dehydrogenase	B9DLU9	Cys37, 48, 51, 63: 2Fe-2S; Cys99, 102, 109: 4Fe-4S; Cys147, 150, 153, 157: 4Fe-4S; Cys190, 193, 196, 200: 4Fe-4S; Cys264, 267, 271, 299: 4Fe-4S
FolE2													1.0	2.3	2.7	GTP cyclohydrolase	B9DKT0	Cys176 conserved, catalytically important site, ligand for Zn²⁺
FusA										0.5	0.7	0.5	1.0	0.6	0.7	Elongation factor G	D5D9Q2	Cys258, 389 conserved
GapA	0.8	2.9	3.6	0.5	2.3	1.6	0.4	2.4	2.8	0.4	1.0	1.2	0.7	0.9	0.9	Glyceraldehyde-3-phosphate dehydrogenase	F4E0S2	Cys152 conserved active site	Cys152–156 intramolecular disulfide
GlmS													0.3	0.7	0.6	Putative glucosamine-fructose-6-phosphate aminotransferase	B9DMA1	Cys2 catalytic site, forms γ-glutamyl-thioester intermediate during Gln hydrolysis
GlnA													1.6	2.4	2.2	Glutamine synthetase	B9DPA6	Cys96 conserved
GltB				5.7	5.6	4.0										Glutamate synthase (NADPH) small subunit	A8FE17	Cys46, 49, 54, 67, 106, 110, 114, 307 conserved: Two 4Fe-4S cluster
GltX				1.8	1.8	2.6	2.3	1.4	1.7				0.4	0.7	0.8	Glutamyl-tRNA synthetase	A7Z0L4	Cys108, 110 conserved, form Zn binding site
GlyA				0.7	0.8	0.4										Serine hydroxymethyl transferase	A8FIC1	Cys64 conserved	Cys66 oxidized by NaOCl in E. coli
GlyQS													0.8	0.7	0.8	Glycyl-tRNA synthetase	B9DNL1	Cys230, 328, 344, 422 conserved, close to ATP binding sites
GrpE^*													0.2	2.6	2.8	HSP-70 cofactor protein GrpE	B9DNK1	No Cys conserved	Cys95^-S-SB by NaOCl in S. carn*.
GuaA				2.7	1.8	1.3							1.2	0.7	0.9	GMP synthase [glutamine-hydrolyzing]	A8FAH5	Cys85 conserved active site, nucleophilic thiolate
GuaB^*	4.9	3.6	3.8	1.1	2.2	2.1	0.8	2.2	2.5	1.4	2.0	1.7	1.3	3.8	3.6	Inosine-5′-monophosphate dehydrogenase	F4EQ59	Cys308 conserved- active site, forms thioimidate intermediate	Cys308^-S-SB by NaOCl in B. sub, B. amy., B. meg*.
GuaC													3.4	5.2	4.6	GMP reductase	B9DP67	Cys174 conserved active site, forms thioimidate intermediate
Hit							4.7	3.1	3.5							Cell cycle regulation histidine triad (Hit) protein	G0IF34	No Cys conserved
Hom (BAT_0329)				0.6	2.5	3.4										Homoserine dehydrogenase	B4AG18	No Cys conserved
Icd				0.4	1.4	1.3				0.4	0.6	0.6				Isocitrate dehydrogenase [NADP]	B4AMB6	Cys118, 411 conserved; Cys118 in active substrate binding site
IlvB										1.8	1.6	1.1				Acetolactate synthase, large subunit, biosynthetic type	D5DTM7	No Cys conserved
IlvC	1.5	1.7	2.0	0.6	0.6	0.6	0.7	1.7	1.6	2.5	2.7	1.7				Ketol-acid reductoisomerase	G0IJS5	Cys199, 227 conserved
IlvD										2.1	2.5	1.9				Dihydroxy-acid dehydratase	D5DER1	Cys122, 189, 192, 195: 4Fe-4S cluster binding
IspA							1.9	1.5	1.3							Major intracellular serine protease	A7Z3U2	No Cys conserved
IspG							5.6	2.1	2.6							4-Hydroxy-3-methylbut-2-en-1-yl diphosphate synthase	F4ESF5	Cys268, 271, 303 conserved: 4Fe-4S-cluster-binding site
Ldh^*													2.7	4.4	4.3	l-lactate dehydrogenase	B9DN51	Cys72 conserved	Cys72^-S-SB by NaOCl in S. carn*.
LeuC	1.1	1.3	1.6	4.0	3.4	2.7				2.7	3.4	3.2				3-Isopropylmalate dehydratase large subunit	B4AM28	Cys346, 406, 409 conserved: 4Fe-4S cluster binding
LuxS^*				0.7	2.1	4.5	6.9	2.7	3.2				0.3	2.9	2.7	S-ribosyl homocysteine lyase (B. pumilus) (LuxS)	B4ANL1	Cys84, 126 conserved: Cys84 essential for catalysis; Cys126 Fe-binding site	Cys41^-S-SB by NaOCl stress in B. pum*.
LysC				0.6	1.8	1.9										Aspartokinase	A8FG00	Cys169 conserved
LysC (BAT_1429)				0.5	1.4	1.3										Aspartokinase	B4AM64	No Cys conserved
MenB													2.2	1.4	1.9	Dihydroxynaphtoic acid synthetase	B9DQ83	Cys71, 128, 174, 205 conserved; Cys71 in substrate-binding site
MetE^*	0.3	1.5	1.4	0.4	1.8	1.6	0.3	1.7	2.0	0.8	1.8	1.2				Methionine synthase	A7Z3U1	Cys647, 730 essential-Zinc binding active site	Cys730^-S-SB; Cys719^-SSB
MetK							0.6	0.6	0.5	0.7	0.6	0.4				S-Adenosylmethionine synthase	F4EA25	Cys22, 44 conserved, near active metal (Mg²⁺, K⁺)-binding site
MsrA													1.7	1.9	2.1	Peptide methionine sulfoxide reductase	B9DNX6	Cys10, 13 conserved; Cys 10 active site	Cys10-Cys13 disulfide
MsrB													2.6	2.2	3.0	Peptide methionine sulfoxide reductase	B9DNY9	Cys114-conserved nucleophile active site
MtnA	1.7	2.3	2.6													Methylthioribose-1-phosphate isomerase	O31662	Cys160 conserved, in active site, transition state stabilizer
MtnW				0.4	1.1	1.8										2,3-Diketo-5-methylthiopentyl-1-phosphate enolase	A8FCG8	No Cys conserved
MurA										1.4	1.4	1.1				UDP-N-acetylglucosamine 1-carboxyvinyltransferase 1	D5DM45	Cys117-conserved proton donor active site, binds UDP-GlcNAc
Ndh (Sca_0546)				0.7	0.6	0.5				4.5	4.2	4.6	2.5	1.4	1.4	NADH dehydrogenase ndh	B4AHV4	No Cys conserved
NifS (BAT_1509)				3.6	4.7	3.1										Cysteine desulfurase	B4ALY7	Cys325-conserved active site
OhrA (BAT_2845)				0.2	6.7	6.7										Organic hydroperoxide resistance protein	B4AEW1	Cys59-,123-conserved Cys	Catalytic Cys59–Cys123 disulfide
PdhC (BAT_2691)				1.2	0.6	0.8										Dihydrolipoyl-lysine-residue acetyl-transferase component of PDH (E2)	B4AEF9	No Cys conserved
PdhD	2.7	1.5	1.3	1.3	0.8	1.0	1.4	1.4	1.2	1.1	0.9	1.1	1.2	1.0	1.2	Dihydrolipoyl dehydrogenase, E3 component of PDH complex	A7Z482	Cys47-, 52-conserved, redox-active disulfide	Cys47–52 disulfide by NaOCl
PdxS													4.2	4.6	4.6	Pyridoxal biosynthesis lyase PdxS	B9DKX7	Cys128, 130 conserved
PfkA										3.3	3.9	2.8				6-Phosphofructokinase 2	D5DPB6	Cys73, 119 conserved in Bacillus species
PheT													2.0	1.6	1.8	Phenylalanyl-tRNA synthetase beta-chain	B9DPU9	Cys65, 79, 120 conserved, in tRNA-binding domain
PpaC^*	2.4	2.7	2.8	1.0	1.5	2.1	3.8	1.7	3.9	2.0	4.2	3.2				Manganese-dependent inorganic pyrophosphatase	A7ZAR2	Cys18, 118, 158 conserved	Cys158^-S-SB by NaOCl in B. sub.*
Prs				1.0	0.9	1.0							1.9	1.6	1.6	Ribose-phosphate pyrophosphokinase	A8F918	Cys57, 59, 251, 252 widely conserved
PtsI													0.9	0.5	0.8	Phosphoenolpyruvate-protein phosphotransferase	P23533	Cys364, 503, 556 conserved; Cys503 proton donor active site
PurB										4.7	2.7	2.2				Adenylosuccinate lyase	D5DWW6	Cys31 conserved
PurL				1.4	1.5	1.1										Phosphoribosylformylglycinamidine synthase 2	A8FAM7	Cys55, 164, 190, 259, 527 conserved
PurQ	0.7	4.8	4.6	2.0	3.0	3.8										Phosphoribosylformylglycinamidine synthase 1	B4AI84	Cys13, 86 conserved, Cys86 nucleophilic active site
PyrAB										1.4	1.0	0.9				Carbamoyl-phosphate synthase large chain 2	D5DJR1	Cys34, 182, 204, 232, 281, 525, 1034 conserved
PyrE				0.9	1.3	0.6										Orotate phosphoribosyltransferase	B4AE64	No Cys conserved
PyrG							2.1	2.3	2.1							CTP synthase	A7Z9T4	Cys381 conserved active site
PyrH													3.3	2.1	2.3	Putative uridylate kinase	B9DPH0	Cys207 conserved
QueC (Sca_0360)													3.7	3.8	3.8	Putative ExsB family protein (7-cyano-7-deazaguanine synthase)	B9DK41	Cys191, 200, 203, 206 conserved Zn binding site
QueF^*				0.5	1.2	1.9	1.0	5.0	4.8				1.4	5.4	4.9	NADPH-dependent 7-cyano-7-deazaguanine reductase	B4AEQ3	Cys56 in active site (binds Zn)	Cys56^-S-SB by NaOCl in B. sub*.
Sat				1.2	1.1	1.2										Sulfate adenylyltransferase	B4AE60	Cys319, 322, 331 conserved, close to active site that binds APS
Sca_1381^*													2.9	2.5	2.5	Putative transaldolase	B9DN38	Cys200 conserved	Cys200^-S-SB by NaOCl in S. carn*.
Sca_1429													1.2	4.9	5.1	Putative tRNA (cytidine/uridine-2′-O-)-methyltransferase	B9DMX6	Cys23 conserved
Sca_1464													1.9	2.3	2.9	CobB/CobQ-like glutamine amidotransferase domain protein	B9DMV5	Cys94 conserved nucleophilic active site
Sca_2233													2.9	2.2	4.0	Putative short-chain dehydrogenase	B9DJB3	No Cys conserved
SecA													0.7	1.2	1.5	Protein translocase subunit	P47994	Cys828, 830, 839, 840 conserved Zn-binding site
SerA^* (BMD_2386)	1.3	1.5	4.1	1.8	1.1	1.3				0.6	0.6	0.9				Phosphoglycerate dehydrogenase	B4AKF2	No Cys conserved	Cys410^-S-SB by NaOCl in B. sub*.
SerS							0.6	1.4	2.5							Seryl-tRNA synthetase 2	Q659K7	Cys260, 353 conserved in ATP-binding active site
SodA										0.3	2.0	1.9				Superoxide dismutase (Mn)	D5DMJ9	Cys not conserved
SpxA													1.5	3.4	4.7	Regulatory protein Spx	B9DIR2	Cys10, 13 conserved redox-active disulfide
Ssb	2.7	3.4	2.5	2.4	1.7	1.5							1.8	2.1	1.8	Single-stranded DNA-binding protein	A8FJE6	No Cys conserved
SucC							1.2	1.0	0.8				1.3	1.0	1.2	Succinyl-CoA ligase [ADP-forming] subunit-beta	F4EIU4	Cys102 in ATP-binding site, Cys193 in Mg/Mn-binding site
SufC										0.6	1.3	2.1				FeS assembly ATPase SufC	D5DM25	No Cys conserved
TenA (BAT_2971)	1.7	2.1	2.3	0.9	1.0	0.9										Tena/thi-4 family (Thiaminase II)	B4AF84	Cys135-conserved catalytic active site
TenA-2 BPUM_3574				1.9	1.5	1.4										Thiaminase	A8FJ01	Cys135-conserved catalytic active site
ThiC										2.9	3.4	2.8				Phosphomethylpyrimidine synthase	D5DA39	Cys538, 541, 546: 4Fe-4S-S-AdoMet cluster
ThiD				2.1	2.1	1.7							0.3	2.3	2.5	Phosphomethylpyrimidine kinase	A8FIN1	No Cys conserved
ThiG^*				0.6	2.5	2.1										Thiazole synthase	B4AF80	Cys92 conserved	Cys102^-S-SB by NaOCl in B. pum.*
ThrS													1.1	1.8	1.5	Threonyl-tRNA synthetase	B9DNC9	Cys336-conserved, catalytic Zn-binding site
TpiA				0.7	0.7	0.6	1.0	0.8	0.8	2.2	2.3	1.8	0.8	0.6	0.6	Triosephosphate isomerase	B4AGH8	Cys126 conserved, required for folding and stability
Tpx	7.2	4.6	4.1	7.0	3.7	5.8	8.6	4.2	3.3	5.0	6.2	5.6	4.9	5.0	5.8	Probable thiol peroxidase	B4AME1	Cys60, 95 conserved active site	Cys60-Cys95 intra-molecular disulfide
TrxA_Bs/Trx_Bp	1.5	3.9	3.6	6.9	10.3	7.8										Thioredoxin BAT_3297 not conserved in B. subtilis	B4AP00	Cys62 conserved in alkaliphilic Bacilli
Tsf				0.5	0.4	0.3	0.6	0.5	0.5	0.4	1.0	1.2	0.8	0.5	0.6	Elongation factor Ts	A7Z4S1	Cys22 conserved
Tuf^*	0.6	0.5	0.5	0.6	1.1	1.2	0.3	0.9	0.8	0.5	0.8	0.7	0.4	0.5	0.5	Elongation factor Tu	A7Z0N5	Cys83 conserved, in GTP-binding site	Cys83^-S-SB by NaOCl in B. sub, B. meg, B. pum*
TypA													1.1	0.6	0.6	Putative GTP-binding protein family protein TypA	B9DQ05	Cys400 conserved
UspA (Sca_1315)													1.8	1.3	1.2	Putative universal stress protein	B9DNA5	Cys111, 137 conserved in UspA-homologs
YceC (TerC)	3.1	3.5	3.8				1.8	2.2	2.7							Tellurium resistance protein	A7Z138	No Cys conserved
YceD (TerD)				1.8	2.4	3.6	2.0	1.9	1.7							Tellurium resistance protein	G0IK82	No Cys conserved
YceE (TerE)							1.2	2.0	2.2							Tellurium resistance protein TerE	A7Z140	No Cys conserved
YdjL (BAT_1651)				1.7	1.4	1.5										Zinc-containing alcohol dehydrogenase	B4ALJ8	Cys34,37 probably Zn-binding
YerC				9.3	4.2	4.1										Sporulation protein YerC	A8FAP4	Cys27, 35 conserved
YflT (Sca_0046)													3.2	4.2	2.3	Putative uncharacterized protein	B9DM00	No Cys conserved
YgaF				6.4	4.7	4.7										Peroxiredoxin	A8FB93	Cys45, 50 conserved; Cys45 active site
YisY (Sca_0638)													1.5	0.7	1.2	Putative hydrolase of alpha-/beta-fold family	B9DQ91	Cys226 conserved
YitJ				1.2	2.4	3.1										Methylenetetrahydrofolate reductase	A8FBU4	Cys200, 265, 266 conserved Zn-binding site
YjcI (MetI)^*							1.3	1.3	1.3							Cystathionine gamma-synthase/O-acetylhomoserine (thiol)-lyase	A7Z3I1	Cys338 conserved	Cys369^-SSB by NaOCl in B. pum*.
YjgF (Sca_0147)													1.2	5.0	4.6	Putative purine regulatory protein	B9DLD4	Cys103 conserved, in homotrimer interaction site
YjoA							3.8	2.0	2.6							Uncharacterized protein YjoA	A7Z3N1	No Cys conserved
YkuU^*				6.9	4.7	5.6										Peroxiredoxin YkuU	A8FCN5	Cys52, 169 conserved; Cys52 active site	Cys52-Cys169 catalytic disulfide bond; Cys169^*-SSB
YneT				2.2	1.8	1.7										CoA-binding protein	A8FDR9	No Cys conserved
YodC							4.1	2.7	3.1							Nitroreductase YodC	A7Z5L7	Cys153 conserved, close to FMN-binding site
YphP^*							6.3	2.6	4.2							Thioredoxin-like protein UPF0403 family protein	A7Z5T6	Cys53, 55, 144 conserved; Cys53 redox-active site	Cys53^-SSB by NaOCl in B. sub.*
YtxJ (Sca_0389)^*													1.1	3.0	5.4	Thioredoxin-like protein DUF2847 family protein	B9DJN8	Cys30 conserved (TCPIS motif), Cys30 redox active Cys	Cys30^-SSB by NaOCl in S. carn*.
YvdA (Sca_1457)													1.6	6.1	3.9	Putative carbonic anhydrase	B9DMU8	Cys38, 99, 103 conserved catalytic Zn-binding site
YxjG^*	1.0	1.9	2.2				1.4	2.6	3.0							Methionine synthase homolog	A7ZAB5	Cys251, 346 homolog to Zn-binding Cys647, 730 in MetE	Cys346^-SSB by NaOCl in B. sub*.
YybR (HypR)	7.4	6.1	12.0													MarR/DUF24-family regulator; responds to disulfide stress	P37486	Cys14 conserved in MarR/DUF24 family, redox-sensitive Cys	Cys14-Cys49' intersubunit disulfide by NaOCl

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The proteins were cut from Coomassie-stained 2D-gels, tryptic in-gel-digested, and identified using MALDI-TOF-TOF MS/MS. The table lists the oxidation ratios/protein quantities of all identified reversibly oxidized proteins at control (co) and 10 and 30 min after NaOCl stress as average values of two biological replicate experiments (Exp1 and Exp2), their protein names and functions, Uniprot-accession numbers, and information about conserved Cys residues as revealed by the Conserved Domain Database CDD (33) (http://ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). The Mascot search results with protein and peptide scores and the oxidation ratios/protein quantities of reversibly oxidized proteins at control (co) and 10 and 30 min after NaOCl stress of two biological replicate experiments (Exp1 and Exp2) with standard deviations are given in Supplementary Table S1. The redox ratios/protein quantities for the B. subtilis proteins were taken from the previous study (7). When a protein is identified in more than one Bacillus species and/or S. carnosus, the accession number is related to one species corresponding to the information about Cys conservation and functions. The asterisk (^*) indicates that these proteins have been identified with peptide-SSB sites using LC-MS/MS analysis, and the BSH-modified Cys site is indicated in the last column. Proteins with oxidation/protein amount ratios that are more than 1.5-fold induced after NaOCl stress are highlighted in gray.

BSH, bacillithiol; Cys, cysteine; MS/MS, tandem mass spectrometry.

In total, 71 different proteins were identified in all five strains that are constitutively oxidized at either control or NaOCl stress conditions. Several conserved redox-sensitive proteins that coordinate metal ions (Zn, Fe-S-cluster) or form disulfide bonds for catalysis were identified in untreated cells, including the adenylate kinase Adk, the alkyl hydroperoxide reductase small-subunit AhpC, the 2-Cys peroxiredoxins YkuU and YgaF (in B. pumilus), the thiol peroxidase Tpx, the dihydrolipoamide dehydrogenase E3 subunit of the pyruvate dehydrogenase complex PdhD, and the sulfite reductase flavoprotein alpha-subunit CysJ (Fig. 2B). The intramolecular disulfide bonds were confirmed previously for PdhD, Adk, and CysJ in B. subtilis (7). In B. pumilus, which does not possess the AhpC-AhpF peroxiredoxin/reductase system (14), the YkuU 2-Cys peroxiredoxin is strongly oxidized similar to AhpC in other Bacillus strains, suggesting that YkuU might replace AhpC in peroxide detoxification. The intermolecular disulfides of AhpC homologs and YkuU were confirmed for all Bacillus strains and S. carnosus using diagonal nonreducing/reducing SDS–polyacrylamide gel electrophoresis (PAGE) analysis (Fig. 2C). Notably, in S. carnosus, the NADH dehydrogenase Sca_0546 and the universal stress protein UspA are among the most abundant proteins with strong oxidation ratios under control conditions (Supplementary Fig. S7).

Exposure of Bacillus strains and S. carnosus to sublethal NaOCl concentrations caused increased oxidation ratios of 57 different NaOCl-sensitive proteins (Table 1). The information about the conservations of Cys residues was obtained from the CDD database (www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) (33). Many NaOCl-sensitive proteins harbor active-site Cys residues that are nucleophilic, catalytically important, bind substrate intermediates or serve as Zn or Fe ligands. These NaOCl-sensitive proteins are associated with a variety of cellular functions, such as ROS scavenging, amino acid biosynthesis, fatty acid biosynthesis, purine biosynthesis, translation, and protein modification/folding factors. Examples are the methionine sulfoxide reductases (MsrA and MsrB) and two ArsC family proteins (SpxA and Sca_0451) that are known to form intramolecular disulfides by oxidative stress (7, 38) (Supplementary Fig. S7 and Fig. 2A). In B. megaterium, major NaOCl-sensitive proteins were identified as the Mn-dependent superoxide dismutase SodA and the Fe-S-cluster assembly ATPase SufC (Supplementary Fig. S6 and Fig. 2A). The SufA chaperone was identified with an SSB-site at the conserved Cys120 within the Fe-S-cluster site in B. amyloliquefaciens (Table 2). The glyceraldehyde-3-phosphate dehydrogenase (GapA) was among the most strongly de novo oxidized proteins by NaOCl stress in all redox proteomes. The redox-active intramolecular disulfide in GapA's active site has been mapped (7, 27).

Table 2.

Identification of S-Bacillithiolated Proteins in the Proteomes of B. subtilis, B. amyloliquefaciens, B. pumilus, B. megaterium, and S. carnosus Using Liquid Chromatography-MS/MS Analysis

Accession	Protein	Function	Essential	Redox-sensing Cys	Peptide-SSB or -SSCys sequence	m/z precursor	m/z prec-134 (-malate)	Peptide neutral MW	Charge
B. subtilis
O34777	OhrR^*	Organic hydroperoxide resistance repressor (control of the OhrA peroxiredoxin)	No	C15 redox sensing (cons.)	(K)LENQLC₁₅(+BSH)FLLYASSR(E)	684.9800	640.6500	2051.9183	3
P80877	MetE^*	5-methyltetrahydropteroyltriglutamate-homocysteine methyltransferase (methionine synthase)	No	C647,730 Zn-binding active site (cons.)	(R)FWVNPDC₇₃₀(+BSH)GLK(T)	787.8297	720.9900	1573.6449	2
					(R)VPSTEEMYNIIVDALAVC₇₁₉(+BSH)PTDR(F)	944.7584	900.4600	2831.2533	3
P42318	YxjG^*	Methionine synthase homolog	No	C346 Zn-binding active site (cons.)	(R)YVSLDQLC₃₄₁(+IAM)LSPQC₃₄₆(+BSH)GFASTEEGNK(L)	981.4173	936.8900	2941.2301	3
P37487	PpaC^*	Manganese-dependent inorganic pyrophosphatase	Yes	C158 cons.	(K)SPTC₁₅₈(+BSH)TDQDVAAAK(E)	851.8443	785.1000	1701.6740	2
P35136	SerA^*	d-3-phosphoglycerate dehydrogenase (serine biosynthesis)	No	C410 cons. in Bacillus species	(K)ISSSESGYDNC₄₁₀(+BSH)ISVK(V)	992.9086	926.0500	1983.8026	2
P39912	AroA	Phospho-2-dehydro-3-deoxyheptonate aldolase/chorismate mutase (chorismate biosynthesis)	No	C126 cons.	(R)FIVGPC₁₂₆(+BSH)AVESYEQVAEVAAAAK(K)	883.4080	839.4800	2647.2022	3
P33166	Tuf	Elongation factor Tu	Yes	C83 in GTP-binding site 82–86 (cons.)	(R)HYAHVDC₈₃(+BSH)PGHADYVK(N)	527.7176	494.4700	2106.8413	4
P21879	GuaB	Inosine-5′-monophosphate dehydrogenase (GMP biosynthesis)	Yes	C308 active site (cons.)	(K)VGIGPGSIC₃₀₈(+BSH)TTR(V)	519.5691	475.0800	1555.6854	3
P54170	*YphP^,^#**	UPF0403 protein, thioredoxin-like protein	No	C53 active site (cons.)	(K)AEGTTLVVVNSVC₅₃(+BSH)GC₅₅(+IAM)AAGLAR(P)	815.3758	771.1400	2443.1056	3
O05268	YumC	Ferredoxin—NADP reductase 2 (FAD-dependent pyridine nucleotide-disulfide oxidoreductase)	Yes	C85 probably active site (cons.)	(K)FDQTIC₈₅(+Cys)LEQAVESVEK(Q)	653.3026	n.d.	1956.8860	3
P18255	ThrS	Threonine—tRNA ligase 1	No	C338 Zn-binding; C573 not cons.	(R)LQC₅₇₃(+BSH)EGLR(V)	607.7535	540.8300	1213.4925	2
O31678	QueF	NADPH-dependent 7-cyano-7-deazaguanine reductase (tRNA-Queuosine biosynthesis)	No	C56 active Zn-binding site (cons.)	(K)FNC₄₉(+IAM)PEFTSLC₅₆(+BSH)PK(T)	613.5820	569.1400	1837.7241	3
B. amyloliquefaciens
A7Z3U1	MetE	5-methyltetrahydropteroyltriglutamate-homocysteine methyltransferase (methionine synthase)	No	C647,730 Zn-binding active site (cons.)	(R)FWVNPDC₇₃₀(+BSH)GLK(T)	787.8297	721.0100	1573.6448	2
					(R)FWVNPDC₇₃₀(+Cys)GLK(T)	433.1948	n.d.	1296.5626	3
					(R)VPATEEIYQIIDDALEVC₇₁₉(+BSH)PTDR(F)	962.7668	918.2900	2885.2787	3
A7ZAR2	PpaC	Manganese-dependent inorganic pyrophosphatase	Yes	C158 cons.	(K)SPTC₁₅₈(+Cys)TEQDIAAAK(E)	727.3190	n.d.	1452.6235	2
A7Z657	SerA	d-3-phosphoglycerate dehydrogenase (serine biosynthesis)	No	C410 cons.	(K)ISSNESGYDNC₄₁₀(+BSH)ISVK(V)	671.2758	626.8900	2010.8054	3
A7Z7R9	AroA	Phospho-2-dehydro-3-deoxyheptonate aldolase/chorismatemutase (chorismate biosynthesis)	No	C126 cons.	(R)FIVGPC₁₂₆(+BSH)AVESYEQVAEVAAAAK(K)	883.4087	n.d.	2647.2042	3
A7Z612	AroE	3-phosphoshikimate 1-carboxyvinyltransferase (chorismate biosynthesis)	No	C79 not cons.	(K)GIDALC₇₉(+BSH)EPDSLLDVGNSGTTIR(L)	881.4009	836.7600	2641.1808	3
					(K)GIDALC₇₉(+Cys)EPDSLLDVGNSGTTIR(L)	789.0375	n.d.	2364.0906	3
A7Z0D1	GuaB	Inosine-5′-monophosphate dehydrogenase (GMP biosynthesis)	Yes	C308 active site (cons.) forms thioimidate intermediate	(K)VGIGPGSIC₃₀₈(+BSH)TTR(V)	778.8491	712.0500	1555.6837	2
A7Z8C1	YumC	Ferredoxin—NADP reductase 2 (FAD-dependent Pyridine nucleotide-disulfide oxidoreductase)	Yes	C85 probably active site (cons.)	(K)FDQTIC₈₅(+Cys)LEQAVESVEK(Q)	653.3023	n.d.	1956.8851	3
					(K)FDQTIC₈₅(+BSH)LEQAVESVEK(Q)	745.6595	701.2400	2233.9568	3
A7Z7N3	ThiI	Probable tRNA sulfur transferase (YtbJ homolog)	No	C81 not cons.	(K)C₈₁(+BSH)ESKLEDIK(K)	730.8164	n.d.	1459.6183	2
		(Thiamine biosynthesis)
A7Z4X4	CotE	Spore coat protein E	No	C113 cons.	(K)VLQQPNC₁₁₃(+Cys)LEVTISPNGNK(I)	691.6771	n.d.	2072.0094	3
F4EAC4	SufA	Chaperone involved in Fe-S cluster assembly	No	C102,104,120 cons: Fe-S-cluster	(K)NAGTPEEC₁₂₀(+BSH)(-)	608.7031	541.5800	1215.3916	2
A7Z1M1	Alr	Alanine racemase 1 (cell wall biosynthesis)	Yes	C63 not cons.	(K)AALEAGASC₆₃(+BSH)LAVAILDEAISLR(K)	851.7523	807.5500	2552.2351	3
B. pumilus
B4AEV7	MetE	5-methyltetrahydropteroyltriglutamate-homocysteine methyltransferase (methionine synthase)	No	C647,730 Zn-binding active site (cons.)	(R)FWVNPDC₇₃₀(+BSH)GLK(T)	787.8287	721.0000	1573.6428	2
A8FIT1	YwaA	Branched-chain amino-acid aminotransferase (valine, leucine, isoleucine biosynthesis)	No	C104 S-SCys by diamide in B. sub.	(R)LC₁₀₄(+BSH)IPQIDTETVLEGLNELIR(I)	889.1032	844.8300	2664.2878	3
A8FGB2	AroA	Phospho-2-dehydro-3-deoxyheptonate aldolase/chorismatemutase (chorismate biosynthesis)	No	C126 cons.	(R)FIVGPC₁₂₆(+BSH)AVESYEQVAEVAAAAK(Q)	883.4035	n.d.	2647.1887	3
B4AN33	Tuf	Elongation factor Tu	Yes	C83 in GTP-binding site 82–86 (cons.)	(R)HYAHVDC₈₃(+BSH)PGHADYVK(N)	527.7175	494.4800	2106.8408	4
B4AF61	MetI	Cystathionine-gamma synthase (CGS) (O-succinylhomoserine(thiol)-lyase) (homocysteine biosynthesis)	No	C335 cons.	(R)IANGVC₃₆₉(+BSH)NK(L)	607.7542	540.7300	1213.4939	2
B4ANL1	LuxS	S-ribosylhomocysteine lyase (homocysteine biosynthesis)	No	C84 essential for catalysis (cons.) C126 Fe-binding site (cons.)	(R)FC₄₁(+BSH)QPNK(Q)	566.7175	499.7200	1131.4204	2
B4AF80	ThiG	Thiazole synthase (Thiamine biosynthesis)	No	C92 cons.	(K)VEVIGC₁₀₂(+BSH)SR(S)	629.7681	562.9200	1257.5217	2
B4AFT4	KatX2	Catalase	?	No Cys cons.	(K)LLAIC₄₆₁(+BSH)NFYR(A)	503.5634	459.1000	1507.6684	3
A8FCN5	YkuU	2-Cys peroxiredoxin	?	C52, 169 cons.; C52 active site; C52–C169 catalytic disulfide	(R)VLQALQTGGLC₁₆₉(+BSH)PANWKPGQK(T)	835.7413	791.4100	2504.2022	3
A8FHX4	FliW	Flagellar assembly factor	No	C144 not cons.	(K)HLLEVASSC₁₄₄(+BSH)	677.7782	610.9300	1353.5418	2
A8FG49	CitZ	Citrate (Si)-synthase II (TCA cycle)	No	C195 cons.	(R)VC₁₉₅(+BSH)VATLSDIYSGVTAAIGALK(G)	816.7327	772.5300	2447.1762	3
B. megaterium
D5DIR6	MetE	5-methyltetrahydropteroyltriglutamate-homocysteine methyltransferase (methionine synthase)	No	C649,732 Zn-binding active site	(R)ALQVLDPALFWINPDC₇₃₂(+BSH)GLK(T)	837.0709	792.9500	2508.1908	3
D5DER3	PpaC	Manganese-dependent inorganic pyrophosphatase	Yes	C158 cons.	(K)SPTC₁₅₈(+BSH)TDQDVAAAK(E)	568.2314	523.7300	1701.6723	3
					(K)SPTC₁₅₈(+Cys)TDQDVAAAK(E)	713.3030	n.d.	1424.5915	2
D5D924	GuaB	Inosine-5′-monophosphate dehydrogenase (GMP biosynthesis)	Yes	C308 active site, forms thioimidate intermediate (cons.)	(K)VGIGPGSIC₃₀₈(+BSH)TTR(V)	778.8499	712.1000	1555.6853	2
S. carnosus
B9DN54	AroA	Phospho-2-dehydro-3-deoxyheptonate aldolase/chorismate mutase (chorismate biosynthesis)	No	C83, 125 cons; C83 catalytic site; C125 Fe-binding site	(K)SFIFGPC₁₂₅(+BSH)SVESQEQVDK(V)	765.9924	721.8800	2294.9553	3
B9DKV8	Tuf	Elongation factor Tu	No	C82 in GTP-binding site 81–85	(R)HYAHVDC₈₂(+BSH)PGHADYVK(N)	527.7177	494.5300	2106.8417	4
B9DM03	GuaB	Inosine-5′-monophosphate dehydrogenase, (GMP biosynthesis)	No	C307 active site, forms thioimidate intermediate (cons.)	(K)VGIGPGSIC₃₀₇(+BSH)TTR(I)	519.5693	475.0700	1555.6862	3
B9DNY0	YphP	UPF0403 protein (thioredoxin-like protein)	No	C54 active site (CxC-motiv) cons.	(K)NVGKDETTFVVINSTC₅₄(+BSH)GC₅₆(+IAM)AAGLAR(P)	720.5766	687.4300	2878.2773	4
B9DJN8	YtxJ	DUF2847 protein (thioredoxin-like protein)	No	C30 active site (TCPI-motiv) cons.	(K)HSNTC₃₀(+BSH)PISANAYDQFNK(F)	769.3155	724.9500	2304.9246	3
B9DNK0	DnaK	Chaperone protein	No	No Cys cons.	(K)IIGIDLGTTNSC₁₅(+BSH)VAVLEGDEPK(V)	880.7465	836.5500	2639.2176	3
B9DNK1	GrpE	Chaperone protein	No	No Cys cons.	(K)TYQAQC₉₅(+BSH)VLTDILPTIDNIER(A)	901.4220	856.9600	2701.2442	3
B9DN51	Ldh	l-lactate dehydrogenase	No	C72 cons.	(K)AGSYEDC₇₂(+BSH)SDADLVVITAGAPQKPGETR(L)	787.3511	754.4000	3145.3684	4
Q9RGS6	ThiM	Hydroxyethylthiazole kinase (thiamine biosynthesis)	No	C258 not cons.	(R)IDSDAVAENC₂₅₈(+BSH)NLEEVK(-)	715.6331	671.2800	2143.8775	3
B9DMD5	Sca_1625	Putative aldehyde dehydrogenase family protein (similar to GbsA betaine aldehyde dehydrogenase)	?	C279 active site (cons.)	(K)VYNNTGQVC₂₇₉(+BSH)TAGTR(T)	627.2645	582.8600	1878.7716	3
					(K)VYNNTGQVC₂₇₉(+Cys)TAGTR(T)	534.9047	n.d.	1601.6922	3
B9DN59	YtpR	Similar to Phe-tRNA synthetase (YtpR homolog)	No	C126,167 cons.	(K)VNVGNEELQIVC₁₂₆(+BSH)GAPNVEAGQK(V)	888.7393	844.1200	2663.1961	3
					(K)VNVGNEELQIVC₁₂₆(+Cys)GAPNVEAGQK(V)	796.3812	n.d.	2386.1218	3
B9DNG1	Dtd	d-tyrosyl-tRNA(Tyr) deacylase (YrvI homolog)	No	No Cys cons.	(R)LYEAFNEALC₁₁₃(+Cys)QYGVEVK(T)	698.9884	n.d.	2093.9434	3
B9DLW2	SceB	SceB precursor (SsaA homolog)	?	C166 cons	(R)TSSGANYYTAGQC₁₆₆(+BSH)TYYAFDR(A)	879.0096	834.6200	2634.0069	3
					(R)TSSGANYYTAGQC₁₆₆(+Cys)TYYAFDR(A)	786.6523	n.d.	2356.9352	3
B9DN38	Sca_1381	Putative transaldolase	?	C200 cons.	(R)ELLNVIQADEIGADIITC₂₀₀(+BSH)PSGVISK(I)	998.8192	954.2100	2993.4358	3
B9DQ05	TypA	Putative GTP-binding protein family protein (YlaG homolog)	No	C408 not cons.	(R)VQC₄₀₈(+BSH)EVPQENAGAVIESLGQR(K)	841.7123	n.d.	2522.1150	3
B9DML0	Sca_1554	Putative ABC transporter ATP-binding protein	?	No Cys cons.	(R)QIENC₅₈₃(+IAM)EAEIEAC₅₉₀(+BSH)EQK(I)	730.6216	686.2800	2188.8431	3
B9DM56	Adk	Adenylate kinase	Yes	C130, 133, 150 cons., Zn-finger motif	(K)VDGVC₁₅₀(+BSH)DLDGGK(L)	491.8620	447.3800	1472.5642	3

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Cytoplasmic protein extracts of B. subtilis, B. amyloliquefaciens, B. pumilus, B. megaterium, and S. carnosus were prepared in an urea-IAM-buffer, tryptic in-gel-digested, and analyzed in a LTQ Orbitrap-Velos™ mass spectrometer as described in the Materials and Methods section. Peptides with S-bacillithiolations were identified by the additional mass of 396 Da at Cys residues and the diagnostic neutral malate loss fragment ions that appeared as abundant ions in the fragment ion MS/MS spectra. Peptides with S-cysteinylation were identified with a mass difference of 119 Da at Cys residues. The table includes the Uniprot accession numbers and protein functions as derived from the UniprotKB database (http://uniprot.org/) and information about conserved Cys residues and Cys functions as revealed by the Conserved Domain Database CDD (34) (http://ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). The table also includes the m/z of the precursor ions, m/z of the precursor malate loss ions (−134 Da), and the neutral molecular mass of the BSH-modified peptide and peptide charges. The Xcorr, ΔCn scores, and mass deviations of the S-bacillithiolated peptides and complete collision-induced diffraction (CID) MS/MS spectrum of all peptides and the b and y fragment ion series are given in Supplementary Figures S8 and S9 as follows: OhrR (S8A), MetE (S8B), YxjG (S8C), PpaC (S8D), SerA (S8E), AroA (S8H), Tuf (S8I), GuaB (S8J), YphP (S8F), YtxJ (S8G), YumC (S8K), ThrS (S8L), QueF (S8M), YwaA (S8N), MetI (S8O), LuxS (S8P), ThiG (S8U), KatX2 (S8S), YkuU (S8T), FliW (S8Q), CitZ (S8R), AroE (S8V), CotE (S8W), SufA (S8X), Alr (S8Y), ThiI (S8Z), DnaK (S9A), GrpE (S9B), Ldh (S9C), ThiM (S9D), Sca_1625 (S9E), YtpR (S9F), Dtd (S9G), SceB (S9H), Sca_1381 (S9I), TypA (S9J), Sca_1554 (S9K), and Adk (S9L). ^* indicates that these proteins with SSB sites have been identified in the previous study in B. subtilis (7) and are listed here for comparison. Bold texts indicate conserved S-bacillithiolated proteins that are identified in more than two of the four Bacillus species and S. carnosus in this study. ^#The YphP protein was identified in untreated and NaOCl-treated B. subtilis cells. ? indicates that it is unknown if this protein is essential. n.d. indicates that the malate-loss precursor ion was not determined.

IAM, iodoacetamide; LTQ, linear-trap quadrupole; MW, molecular weight.

We were interested to identify proteins with NaOCl-sensitive thiols that form SSB sites in Bacillus spp. and S. carnosus. Tryptic digests of protein extracts from control and NaOCl-treated cells were analyzed for S-bacillithiolations using Orbitrap-Velos LC-MS/MS analysis. The CID MS/MS spectra of the BSH-modified peptides show characteristic and abundant malate loss precursor ions (precursor-134 Da) that were used as diagnostic fragment ions to search for BSH-modified peptides and labeled in the MS/MS spectra (Table 2, Supplementary Tables S2 and Supplementary Figs. S8 and S9) (7). In our previous study, we found S-bacillithiolation sites for the thioredoxin-like protein YphP in B. subtilis under control conditions (7), but no further SSB sites were identified in Bacillus species and S. carnosus. In response to NaOCl stress, we could identify in total 54 proteins with characteristic SSB sites, including 29 unique proteins and eight conserved proteins with identical SSB sites in two or more Bacillus strains and S. carnosus. The eight proteins with conserved SSB sites include the methionine synthase (MetE), the inorganic pyrophosphatase (PpaC), the 3-D-phosphoglycerate dehydrogenase (SerA), the bifunctional 3-deoxy-7-phosphoheptulonate synthase/chorismate mutase (AroA), the translation elongation factor EF-Tu (TufA), the inosine 5′-monophosphate (IMP) dehydrogenase (GuaB), the thioredoxin-like protein (YphP), and the ferredoxin–NADP⁺ oxidoreductase (YumC) (Fig. 2A, E and Supplementary Figs. S8 and S9).

The methionine synthase MetE was oxidized most strongly and specifically by NaOCl stress in the redox proteome of all Bacillus strains (Fig. 2A and Supplementary Figs. S3–S7), and the conserved Cys730-SSB and Cys719-SSB sites were mapped by LC-MS/MS analyses (Supplementary Fig. S8). The YxjG methionine synthase paralog was only oxidized by NaOCl stress in B. subtilis and B. amyloliquefaciens (Supplementary Figs. S3 and S4). Further, NaOCl-sensitive enzymes with SSB sites at nonconserved Cys369 and Cys41, respectively, include the cystathionine-gamma synthase (MetI) and S-ribosylhomocysteine lyase (LuxS) of B. pumilus involved in homocysteine biosynthesis (Supplementary Figs. S8 and S9). The conserved proteins SerA with Cys410-SSB and PpaC with Cys158-SSB modifications (7) are constitutively oxidized in the redox proteomes of all strains, suggesting that basal S-thiolation is observed for selected proteins during aerobic growth (Fig. 2A and Supplementary Figs. S3–S7). The AroA protein was strongly oxidized in all redox proteomes by NaOCl, and the conserved Cys126-SSB site was identified by LC-MS/MS analysis (Fig. 2A and Supplementary Figs. S3–S7 and Table 2). AroA functions in the first step of chorismate synthesis catalyzing the production of 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP), and thus its S-bacillithiolation could regulate the biosynthesis of aromatic amino acids (Fig. 3). In B. amyloliquefaciens, the AroE protein that functions in the later steps of chorismate biosynthesis was also identified with a Cys79-SSB site. The essential GuaB protein that functions in guanosine 5′-phosphate biosynthesis showed increased oxidation ratios by NaOCl stress in most Bacillus spp., and the active-site Cys308 was identified as a conserved SSB site (Figs. 2A and 3 and Supplementary Figs. S3–S7 and Table 2). Enzymes for thiamine biosynthesis also harbor NaOCl-sensitive thiols, such as the thiazole synthase ThiG in B. pumilus that was identified with a Cys102-SSB site. The hydroxyethylthiazole kinase ThiM of S. carnosus was identified with a Cys258-SSB modification (Figs. 2A and 3 and Supplementary Figs. S3–S7 and Table 2). The NADPH-dependent 7-cyano-7-deazaguanine reductase QueF was identified with an SSB site at the active site Zn-binding Cys55, and was NaOCl-oxidized in the redox proteomes of B. amyloliquefaciens and B. pumilus. QueF is involved in the biosynthesis of queuosine, a hypermodified base found in the wobble positions of tRNAs (25). Additional proteins with increased oxidation ratios and identified SSB sites include the translation elongation factor EF-Tu, aminoacyl-tRNA synthetases (ThrS, YtpR, and TypA), and the DnaK and GrpE chaperones (Fig. 2A, Tables 1 and 2). Oxidation of DnaK and GrpE was only observed in the S. carnosus redox proteome, and the SSB sites were mapped at Cys15 and Cys95, respectively (Fig. 2A and Supplementary Figs. S3–S7 and Table 2).

FIG. 3. — **Metabolic enzymes with SSB sites were identified within the biosynthetic pathways for methionine, Cys, chorismate, thiamine, GMP, and queuosine.** The translation and protein quality control machinery is another target of S-bacillithiolation. The schematics of the metabolic pathways refer to the annotated genes of *B. subtilis* modified from the SubtiPathways database (http://subtiwiki.uni-goettingen.de/subtipathways.html), and possible inhibitory steps in these pathways are shown by the identified S-bacillithiolated proteins SerA, PpaC, MetE, YxjG, MetI, LuxS (Met, Cys biosynthesis); AroA, AroE (Chorismate biosynthesis); ThiG, ThiM (Thiamine biosynthesis); GuaB and QueF (GMP, queuosine biosynthesis). The schematic for queuosine biosynthesis is adapted from (37), and the Met and Cys biosynthesis pathway is from (48). Cys, cysteine; GMP, guanosine 5′-phosphate. (To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars.)

Among the redox and antioxidant proteins, the thioredoxin-like proteins YphP and YtxJ (Sca_0389) were identified with SSB sites at their active-site Cys53 and Cys30, respectively, and oxidized in the redox proteome of S. carnosus by NaOCl stress (Fig. 2A and Supplementary Figs. S8 and S9). The ferredoxin–NADP⁺ oxidoreductase YumC (23) was identified with an SSB modification at the active-site Cys85.

In S. carnosus, the lactate dehydrogenase Ldh and the putative transaldolase Sca_1381 are constitutively oxidized in the redox proteome, and the Cys72-SSB and Cys200-SSB sites, respectively, were identified by LC-MS/MS (Fig. 2A; Table 2). The aldehyde dehydrogenase AldA and the arginine deiminase ArcA are NaOCl-sensitive proteins in the redox proteome of S. carnosus that also possess active-site Cys residues.

NaOCl stress caused a decreased BSH/BSSB redox ratio in B. subtilis as shown by thiol metabolomics

In B. subtilis, grown in an LB medium, similar amounts of BSH (0.6–1 μmol/g dry weight [DW]) and Cys (0.6 μmol/g DW) have been quantified previously (12, 40). The redox ratios were calculated previously as ∼400:1 for BSH/BSSB and ∼120:1 for Cys/Cystine in LB-grown B. subtilis cells (40). We quantified the change of the BSH/BSSB and Cys/Cystine redox ratios in B. subtilis cells grown in the BMM after NaOCl stress (Fig. 4 and Supplementary Fig. S10). The BSH level was 2.2 μmol/g DW in growing cells (OD₅₀₀=0.4) and increased to 2.7 μmol/g DW directly after NaOCl stress and to 3.2 μmol/g DW during postexponential growth (Fig. 4A). BSH synthesis is about two-fold increased by NaOCl stress compared to control cells. The BSSB level was 0.06 μmol/g DW in growing cells, and NaOCl exposure resulted in a rapid two-fold increase to 0.11 μmol/g DW after 15 min (Fig. 4B). The BSSB level decreased within 60 min after NaOCl stress to control levels. The BSH/BSSB redox ratio was calculated as 40–50:1 during exponential growth, dropped two-fold shortly after NaOCl exposure, and recovered to 50–60:1 within 2 h (Fig. 4C, D). Approximately 3% (0.06 μmol/g DW) of the total cellular BSH content was bound as protein-SSB during growth and increased two-fold after exposure to 120 μM NaOCl (Fig. 4E). The BSH level is four- to eight-fold higher than Cys, and the levels of Cys (0.4 μmol/g DW), Cystine (0.05 μmol/g DW), and the Cys/Cystine redox ratio (∼10:1) were not affected by NaOCl stress. These data confirm that BSH is used as major redox buffer and oxidized to BSSB by NaOCl stress, causing a decreased BSH/BSSB redox ratio and S-bacillithiolation in B. subtilis cells.

FIG. 4. — **The BSH/BSSB redox ratio and BSH protein-mixed disulfides are changed in *B. subtilis* after NaOCl exposure.** *B. subtilis* cells grown in BMM were exposed to 120 μM NaOCl stress at OD₆₀₀=0.4 and harvested immediately before (0 min) and 15, 60, and 120 min post-NaOCl stress. The reduced thiol redox buffer (RSH) including BSH and Cys **(A)**, the oxidized thiol redox buffer (RSSR) including BSSB and Cystine **(B)**, and protein BSH contents **(E)** were measured as described in the Materials and Methods section. The RSH/RSSR ratios are calculated for control cells **(C)** and NaOCl-treated cells **(D)** in relation to the growth curve. The time point of NaOCl exposure is indicated by an arrow. The combined growth curves of untreated and NaOCl-treated cells are also shown for comparison **(F)**. Experiments were performed in triplicate, and the error bars are given as standard error of the means values. Representing peaks for BSH, Cys, BSSB, Cystine and protein BSH are shown in the high performance liquid chromatography chromatograms in Supplementary Figure S10. BSSB, oxidized bacillithiol disulfide.

Discussion

Previously, we have shown that BSH forms mixed protein disulfides (S-bacillithiolations) with essential catalytic Cys residues of enzymes involved in the methionine biosynthesis pathway and with the redox-sensing OhrR repressor after NaOCl stress in B. subtilis (7). Thus, S-bacillithiolation acts as a redox-switch under hypochlorite stress to induce methionine starvation and up-regulation of the OhrA peroxiredoxin for detoxification. In this work, S-bacillithiolation sites in 54 proteins have been identified among four different industrially important Bacillus species and S. carnosus after exposure to NaOCl. We applied our previous LC-MS/MS analysis to directly identify S-bacillithiolation sites and proved the MS/MS spectra by their characteristic malate loss precursor ions. The number of S-bacillithiolated proteins is lower than that of glutathionylated proteins identified in eukaryotes. This discrepancy could be due to the labile nature of the Cys-GlcN-Mal moiety that limits its detection, and also because the amount of BSH is much lower (2–3 μmol/g DW) compared to GSH in E. coli (19 μmol/g DW) or mycothiol (MSH) in Mycobacterium smegmatis (40 μmol/g DW) (40). Thus, selective thiol-trapping and enrichment tools need to be developed to quantify S-bacillithiolated proteins more comprehensively. To identify S-glutathionylated proteins in the proteome of eukaryotic organisms, redox proteomic approaches have been developed that use glutaredoxin (Grx) for specific de-glutathionylation of cell extracts, followed by biotin-N-ethylmaleimide (NEM) alkylation and affinity purification (e.g., the biotin-switch assay) (21, 28, 30, 31). By analogy, large-scale identification and quantification of BSH-mixed protein disulfides would require selective reduction by bacilliredoxins (Brxs), followed by alkylation and purification protocols. We identified possible candidates for putative Brxs (YphP and YtxJ) that were S-bacillithiolated at their active-site Cys residues. Current attempts are directed to develop Brx-based redox proteomics methods to further characterize and quantify the S-bacillithiolome, and to analyze the substrate specificities of these putative Brxs.

The fluorescence-based redox proteomics approach was used to quantify all oxidized proteins, and many conserved S-bacillithiolated proteins show increased oxidation ratios upon NaOCl exposure (e.g., MetE, YxjG, AroA, and ThiG). However, some conserved S-bacillithiolated proteins like TufA, GuaB, PpaC, and SerA did not show increased oxidation ratios by NaOCl stress in the redox proteomes of most Bacillus spp., indicating that S-bacillithiolation also occurs under nonstress aerobic growth conditions. This confirms results of S-glutathionylations in malaria parasites, human, and yeast cells, where the basal level S-thiolation occurs even under nonstress conditions as a result of aerobic growth (21, 24, 30). In Plasmodium falciparum, more than 400 S-glutathionylated proteins were identified in nonstressed cells (21). The authors further demonstrated in vitro that the activities of only a few identified metabolic enzymes were inhibited by S-glutathionylation. The physiological role and verification of the predicted basal level S-bacillithiolation of essential proteins in our study require further detailed investigations, since we could not identify S-bacillithiolations of these proteins in untreated cells using LC-MS/MS analysis. Overall, our combined redox proteomics and LC-MS/MS analyses showed a similar composition of the S-bacillithiolome compared to the S-glutathionylomes described for eukaryotes in oxidatively stressed cells (8, 31). The S-bacillithiolome contains mainly biosynthetic enzymes for amino acids, cofactors, nucleotides, as well as translation factors, chaperones, and redox and antioxidant proteins. Most proteins are enzymes involved in amino acid biosynthesis pathways, such as methionine, Cys (MetE, YxjG, MetI, LuxS, and PpaC), serine (SerA), aromatic amino acids (AroA and AroE), branched chain amino acids (YwaA), GTP synthesis (GuaB), queousine biosynthesis (QueF), and thiamine biosynthesis (ThiG, ThiI, and ThiM). We further identified S-bacillithiolated proteins involved in translation, such as elongation factor EF-Tu and aminoacyl-tRNA-synthetases; the heat-specific chaperones (DnaK and GrpE); and redox and antioxidant proteins, including thioredoxin-like proteins (YphP and YtxJ), the ferredoxin-NADP⁺ oxidoreductase (YumC), and peroxiredoxins (YkuU), which could function in the de-bacillithiolation process or require BSH for their regeneration.

The identified S-bacillithiolated proteins include eight conserved proteins identified in B. subtilis (MetE, GuaB, PpaC, SerA, AroA, Tuf, YphP, and YumC), which could be confirmed as S-bacillithiolated proteins in other species (Fig. 2D, E). Most conserved S-bacillithiolated proteins are abundant proteins oxidized in the redox proteomes of Bacillus and Staphylococcus species (e.g., MetE, Tuf, GuaB, PpaC, SerA, and AroA) and have conserved SSB peptides (Fig. 2A and Table 2). In addition, we detected 29 unique proteins that were S-bacillithiolated only in one Bacillus species or S. carnosus. The most unique proteins with SSB sites were identified in S. carnosus (12 SSB sites) and B. pumilus (8 SSB sites). These proteins are less abundant in the gel-based proteome and were only detected by LC-MS/MS analysis. For example, ThiM and ThiG proteins were found as specific targets for S-bacillithiolation in S. carnosus and B. pumilus, respectively. Novel S-bacillithiolated proteins were mapped in S. carnosus as the putative aldehyde dehydrogenase (Sca_1625), the transaldolase (Sca_1381), and the lactate dehydrogenases (Ldh), as well as the SceB precursor. These proteins are not conserved in Bacillus species, but homologs are present in the pathogen S. aureus. Interestingly, the chaperones GrpE and DnaK were S-bacillithiolated in S. carnosus, but Cys residues are absent in the GrpE and DnaK homologs of Bacillus species. The detection of some unique SSB-peptides could be also due to better peptide ionization properties of the tryptic peptides of less-conserved proteins.

Using BSH-specific immunoblots, we could demonstrate that MetE is the most abundantly S-bacillithiolated protein in all Bacillus species. MetE is most strongly oxidized by NaOCl stress in the redox proteome of B. subtilis wild-type and bshA mutant cells and is S-cysteinylated in the bshA mutant (7). Similar MetE oxidation ratios were calculated after 30 and 60 min post NaOCl stress in the wild-type and bshA mutant cells, but reduction of MetE took significant longer in the bshA mutant (Supplementary Fig. S11). Similarly, AroA was S-bacillithiolated in the wild-type cells and displays similar oxidation ratios in the wild-type and bshA mutant cells within the first hour, but is more slowly reduced in the bshA mutant. This may suggest that S-cysteinylation can fully compensate for S-bacillithiolation in the absence of BSH, but the reduction of these mixed disulfides occurs with different efficiencies.

The physiological importance of MetE inhibition by S-thiolation has previously been demonstrated. Methionine auxotrophy is one common phenotype in bacteria resulting from S-thiolation of MetE in B. subtilis and E. coli in response to oxidative stress (7, 19). In all Bacillus species, the conserved, active-site Cys730 and the nonconserved Cys719 were identified as SSB sites for MetE. The observed S-bacillithiolation of LuxS and MetI could be required to inhibit homocysteine biosynthesis after NaOCl stress, since homocysteine is toxic and accumulates when Met biosynthesis is inhibited, due to MetE-SSB modification (Fig. 3).

Among the conserved S-bacillithiolated proteins, the chorismate synthase AroA was identified, which catalyzes the synthesis of DAHP. Notably, the DAHP synthase Aro4p of yeast cells was found as a target for the Grx2 and showed increased thiol redox ratios at Cys76 and Cys244 in the grx2 mutant (35). The essential IMP dehydrogenase GuaB and the pyrophosphatase PpaC have been previously found to be S-glutathionylated in human T-lymphocytes and endothelial cells under diamide stress conditions (11, 30).

An important finding of this study is the identification of S-bacillithiolated proteins involved in translation and protein quality control, such as elongation factor EF-Tu and the DnaK and GrpE chaperones. The heat-specific Hsp70 and Hsp90 chaperones are well represented in the S-glutathionylomes of human and yeast cells (11, 45). The elongation factor TufA promotes GTP-dependent binding of aminoacyl-tRNAs to the A-sites of the ribosome, but also has chaperone functions (4, 47, 49). The TufA protein was oxidized at Cys256 by NaOCl stress in E. coli, and a tufA mutant was highly sensitive to NaOCl (27). In the Bacillus spp. and S. carnosus TufA proteins, we identified the conserved Cys82-SSB modification in the GTP-binding site. Since TufA is essential in B. subtilis, its S-thiolation in oxidatively stressed cells could cause inhibition of protein synthesis during oxidative stress as previously shown in yeast cells (45). Thus, the fact that amino acid biosynthetic enzymes and translation proteins dominate in the conserved S-bacillithiolome suggests that S-bacillithiolation could control protein synthesis during oxidative stress. The strong repression of the stringent controlled RelA regulon, including genes for ribosomal proteins, by NaOCl stress in our previous microarray analysis further indicates a decreased protein synthesis that could be caused by S-bacillithiolation (7). The activation of the stringent response by ROS and RNS has also been shown in Salmonella (2, 17). Specifically, the stringent response regulatory DnaK suppressor protein (DksA) was shown to be play a role in controlling GSH biosynthesis and other metabolic pathways associated with the generation of reducing power under oxidative stress conditions (17).

The next question is how the reversibility of this redox mechanism (protein de-bacillithiolation) might be mediated. Redoxins catalyzing the reversible S-thiolation/reduction of S-glutathionylated proteins include Grxs, thioredoxins, glutathione S-transferases, and GSH disulfide reductases that were identified in large-scale redox proteomics studies (21). The mechanisms of protein de-glutathionylation by mono- and dithiol Grx have been studied in plants and mammalian systems (13, 52). In both mechanisms, the nucleophilic active-site Cys first attacks the GSH-mixed protein disulfide, resulting in substrate de-glutathionylation and formation of a Grx-SSG intermediate (52). In plant dithiol Grx, this Grx-SSB intermediate is then attacked by a second reactive Cys not present in the CXXC motif that leads to Grx intramolecular disulfide formation. This Grx disulfide is finally resolved by an NADPH-dependent ferredoxin:thioredoxin reductase. In the monothiol mechanism, the Grx-SSG intermediate is regenerated by GSH that is oxidized to GSSG and requires the GSH reductase to restore the reduced GSH pool (52). The thioredoxin-like proteins YphP and YtxJ could possibly function as putative Brxs. YphP is a DUF1094 family protein whose previously solved crystal structure adopts a thioredoxin-fold, has a conserved C₅₃XC₅₅ motif (with a reduction potential of −130 mV), and was demonstrated to display some weak disulfide isomerase activity (10). Under NaOCl stress, YphP is S-bacillithiolated at its more solvent-exposed active-site Cys53. The DUF2847 family Brx protein YtxJ is another thioredoxin-like protein with a conserved TCPIS motif reminiscent of the same redox-active motif found in many monothiol Grxs (18). YtxJ was identified in S. carnosus, S-bacillithiolated in its TCPIS motif. The detection of S-bacillithiolated YphP and YtxJ during NaOCl stress could represent the trapping of transient intermediates of Brx-mediated de-bacillithiolation pathways. The distribution of YphP and YtxJ homologs among bacteria encoding BSH biosynthesis genes (12) further suggests their roles as Brx candidates. Our current experiments are directed to further investigate the functions of these putative Brx proteins in the de-bacillithiolation pathway.

We further analyzed whether the BSH/BSSB redox ratio is affected by NaOCl stress that could contribute to S-bacillithiolation of proteins. In E. coli cells, a four-fold decrease in the GSH/GSSG redox ratio from 370:1 to 87:1 was observed in menadione-treated cells (46). The two-fold increase of BSSB post NaOCl stress is comparable to the increase of GSSG in E. coli cells exposed to menadione (46). The increased GSSG levels result in a decreased GSH/GSSG ratio that could contribute to protein S-glutathionylation spontaneously via thiol–disulfide exchange between GSSG and protein thiols (13). However, this thiol–disulfide exchange is kinetically too slow and very unlikely in vivo, since it would require a drop in the GSH:GSSG ratio from 100:1 to 1:1 for 50% conversion of protein thiols to protein GSH-mixed disulfides (13, 52). In our studies, the BSH:BSSB redox ratio dropped two-fold, confirming that BSH is oxidized to BSSB by NaOCl. This BSH:BSSB ratio of 25:1 is unlikely to be sufficient for spontaneous formation of protein-SSB via a direct thiol–disulfide exchange reaction between protein-SH and BSSB. Instead, S-bacillithiolation first requires oxidation of reactive Cys residues to activated thiol derivatives, such as sulfenic acids (9, 15). NaOCl leads to chlorination of Cys residues to unstable sulfenylchloride intermediates that rapidly react further to form disulfides (50). Thus, we suggest that sulfenylchloride formation at reactive Cys residues by NaOCl and the decreased BSH/BSSB redox ratio contribute to the mechanism of S-bacillithiolation in Firmicutes bacteria.

Finally, the question arises if hypochlorite is a physiologically relevant oxidant that mediates protein S-bacillithiolation in Bacillus and Staphylococcus species. Hypochlorite is encountered by soil bacteria and pathogenic Bacillus and Staphylococcus species, since it is present in household bleach and other disinfectants, and it is also produced by activated macrophages during the infection process by the enzyme myeloperoxidase. The observed S-bacillithiolations in soil-dwelling Bacillus species after NaOCl stress might mimic the protective response to toxic ROS, such as hydroxyl radicals, which are generated during respiration, explaining the conservation of some of the protein targets across different species.

Materials and Methods

Bacterial strains and growth conditions

The bacterial strains used were B. subtilis wild-type strains 168 (trpC2) and CU1065 (trpC2 pheA1) and bshA-mutant strain HB11002 (CU1065 trpC2 and bshA::mls^r) (12), B. amyloliquefaciens FZB42 (5), B. pumilus SBUG1799, B. megaterium SBUG1152 (36), and S. carnosus TM300 (43). Cultivation was performed as detailed in the Supplementary Materials and Methods section.

Thiol redox proteome analysis and protein identification using MALDI-TOF-TOF MS/MS

The thiol redox proteome analysis was performed as described (7). Cells were harvested at control and NaOCl stress conditions, sonicated, alkylated in 8 M urea/1% chaps/100 mM iodoacetamide (IAM), and acetone-precipitated. The pellet was resolved in an urea/chaps buffer without IAM, and 200 μg of the protein extract was reduced with 10 mM Tris-(2-carboxyethyl)-phosphine and labeled with BODIPY FL C₁-IA [N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-yl)-methyl)-iodoacetamide] (Invitrogen, Eugene, OR). The fluorescence-labeled protein extract was separated using 2D PAGE, scanned for BODIPY fluorescence, and stained with Coomassie as described (7). Quantitative image analysis was performed with DECODON Delta 2D software (www.decodon.com). The oxidation ratios were calculated as fluorescence/protein amount ratios and are listed in Supplementary Table S1. The log2 oxidation ratios were used for hierarchical clustering analysis using Multi-Experiment Viewer (MeV v.4.8) software (http://mev.tm4.org/) (32). Tryptic digestion of proteins, peptide spotting, and MALDI-TOF-TOF measurement were performed as described in the Supplementary Materials and Methods section.

Linear-trap quadrupole–Orbitrap Velos mass spectrometry

Bacterial cells were sonicated in an urea/chaps alkylation buffer with 100 mM IAM and separated by 15% nonreducing SDS-PAGE. In-gel tryptic digests and Orbitrap Velos LC-MS/MS analysis were performed as described in the Supplementary Materials and Methods section. S-bacillithiolated peptides were identified by searching all MS/MS spectra in .dta format against B. subtilis 168, B. megaterium, B. amyloliquefaciens FZB42, B. pumilus, and S. carnosus TM300 target-decoy protein sequence databases extracted from UniprotKB release 12.7 using Sorcerer^™-SEQUEST^® according to the parameters and SEQUEST filter criteria as described in the Supplementary Materials and Methods section.

Preparation of keyhole limpet hemocyanin-BSH for generation of BSH-antibodies

Two milligrams of synthetic BSH (44) was conjugated to 10 mg keyhole limpet hemocyanin (KLH) using the Maleimide-Activated KLH Conjugation Kit (Sigma) according to the details described in the Supplementary Materials and Methods section.

Nonreducing BSH-immunoblot analysis and nonreducing/reducing diagonal SDS-PAGE

Polyclonal BSH antisera were used at 1:500 dilution, and 25 μg IAM-alkylated protein extracts was used for nonreducing 15% SDS-PAGE and immunoblot analysis as described (6). Nonreducing/reducing diagonal SDS-PAGE analysis was performed as previously described (6).

Quantification of reduced thiol redox buffer (RSH), oxidized thiol redox buffer (RSSR), and protein BSH

B. subtilis CU1065 was grown in BMM to an OD₆₀₀ of 0.44 and harvested before and after NaOCl stress for subsequent RSH, RSSR, and protein RSH quantification as described in the Supplementary Materials and Methods section.

Supplementary Material

Supplemental data

Supp_Data.pdf^{(11.2MB, pdf)}

Supplemental data

Supp_Table2.pdf^{(168.2KB, pdf)}

Supplemental data

Supp_Table1.pdf^{(246.6KB, pdf)}

Abbreviations Used

APS: adenosine-5′-phosphosulfate
BMM: Belitsky minimal medium
Brx: bacilliredoxin
BSH: bacillithiol
BSSB: oxidized bacillithiol disulfide
CDG: 7-carboxy-7-deazaguanine
CPH4: 6-carboxy-5,6,7,8-tetrahydropterin
Cys: cysteine
DAHP: 3-deoxy-d-arabino-heptulosonate 7-phosphate
DW: dry weight
DxP: 1-deoxy-d-xylulose 5-phosphate
EPSP: 5-enolpyruvylshikimate-3-phosphate
GMP: guanosine 5′-phosphate
Grx: glutaredoxin
GSH: glutathione
GSSG: oxidized glutathione disulfide
GST: glutathione S-transferase
H2NTP: 7,8-dihydroneopterin triphosphate
HET-P: 4-methyl-5-(beta-hydroxyethyl)thiazole monophosphate
HMP: 2-methyl-4-amino-5-hydroxymethyl pyrimidine
HMP-PP: 2-methyl-4-amino-5-hydroxymethyl pyrimidine pyrophosphate
IAM: iodoacetamide
IMP: inosine 5′-phosphate
KLH: keyhole limpet hemocyanin
LB: Luria broth
LC: liquid chromatography
LMW: low molecular weight
LTQ: linear-trap quadrupole
Met: methionine
MS/MS: tandem mass spectrometry
MW: molecular weight
N5,N10-THF: 5,10-methylenetetrahydrofolate
N5-THF: 5-methyltetrahydrofolate
OAS: O-acetylserine
PAPS: 3′-phosphoadenosine-5′-phosphosulfate
PEP: phosphoenol pyruvate
PAGE: polyacrylamide gel electrophoresis
PP_i: inorganic pyrophosphate
PPP: pentose phosphate pathway
preQ0: 7-cyano-7-deazaguanine
preQ1: 7-aminomethyl-7-deazaguanine
protein-SSB: BSH protein-mixed disulfide
PRPP: 5-phospho-alpha-d-ribose 1-diphosphate
Pyr: pyruvate
Q: queuosine
RNS: reactive nitrogen species
ROS: reactive oxygen species
RSH: reduced thiol redox buffer
RSSR: oxidized thiol redox buffer
SAM: S-adenosyl methionine
SDS: sodium dodecyl sulfate
THF: tetrahydrofolate
TMP: thiamine monophosphate
TPP: thiamine pyrophosphate
XMP: xanthosine 5′-phosphate

Acknowledgments

We thank the Decodon Company for support with Decodon Delta 2D software, Frieder Schauer for providing strains B. pumilus SBUG1799 and B. megaterium SBUG1152, and Dana Clausen for excellent technical assistance. We are very grateful to Ahmed Gaballa and John D. Helmann for discussion of the results before publication and for providing the B. subtilis bshA mutant. This work was supported by grants from the Deutsche Forschungsgemeinschaft (AN746/2-1 and AN746/3-1) to H.A., by project TRIG A from the University of Science, Hanoi National University to T.T.T.H., and by a grant from the Biotechnology and Biological Science Research Council (BB/H013504/1) to C.J.H.

Author Disclosure Statement

No competing financial interests exist.

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Associated Data

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Supplementary Materials

Supplemental data

Supp_Data.pdf^{(11.2MB, pdf)}

Supplemental data

Supp_Table2.pdf^{(168.2KB, pdf)}

Supplemental data

Supp_Table1.pdf^{(246.6KB, pdf)}

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PERMALINK

S-Bacillithiolation Protects Conserved and Essential Proteins Against Hypochlorite Stress in Firmicutes Bacteria

Bui Khanh Chi

Alexandra A Roberts

Tran Thi Thanh Huyen

Katrin Bäsell

Dörte Becher

Dirk Albrecht

Chris J Hamilton

Haike Antelmann

Abstract

Introduction

Innovation.

Results

Defining the conditions of NaOCl stress to induce S-bacillithiolations in the proteome of B. amyloliquefaciens, B. pumilus, B. megaterium, and S. carnosus

MetE is the major target for S-bacillithiolation in Bacillus species as revealed by nonreducing BSH-specific immunoblot analysis

FIG. 1.

Identification of proteins with S-bacillithiolations in NaOCl-treated cells of B. subtilis, B. amyloliquefaciens, B. pumilus, B. megaterium, and S. carnosus using redox proteomics and shotgun-LC-MS/MS analyses

FIG. 2.

Table 1.

Table 2.

FIG. 3.

NaOCl stress caused a decreased BSH/BSSB redox ratio in B. subtilis as shown by thiol metabolomics

FIG. 4.

Discussion

Materials and Methods

Bacterial strains and growth conditions

Thiol redox proteome analysis and protein identification using MALDI-TOF-TOF MS/MS

Linear-trap quadrupole–Orbitrap Velos mass spectrometry

Preparation of keyhole limpet hemocyanin-BSH for generation of BSH-antibodies

Nonreducing BSH-immunoblot analysis and nonreducing/reducing diagonal SDS-PAGE

Quantification of reduced thiol redox buffer (RSH), oxidized thiol redox buffer (RSSR), and protein BSH

Supplementary Material

Abbreviations Used

Acknowledgments

Author Disclosure Statement

References

Associated Data

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