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. 2013 Apr 10;18(11):1273-95.
doi: 10.1089/ars.2012.4686. Epub 2012 Oct 18.

S-bacillithiolation protects conserved and essential proteins against hypochlorite stress in firmicutes bacteria

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

S-bacillithiolation protects conserved and essential proteins against hypochlorite stress in firmicutes bacteria

Bui Khanh Chi et al. Antioxid Redox Signal. .

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.

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Figures

FIG. 1.
FIG. 1.
NaOCl stress causes widespread S-bacillithiolations in four Bacillus species and Staphylococcus carnosus as shown by nonreducing BSH-specific immunoblot analysis. (A, C) IAM-alkylated protein extracts of B. subtilis wild-type and bshA mutant cells harvested before (co) and at different times after NaOCl (A) or diamide stress (C) were subjected to nonreducing SDS-PAGE and BSH-specific immunoblot analysis. (B) IAM-alkylated protein extracts of B. subtilis wild-type cells exposed to 80 μM NaOCl in the BMM and to 4 mM NaOCl in an LB medium were analyzed by BSH-specific immunoblot analysis. MetE is only expressed in BMM and identified as *MetE-SSB. (D) IAM-alkylated protein extracts of B. subtilis (B. sub), B. pumilus (B. pum), B. amyloliquefaciens (B. amy), B. megaterium (B. meg), and S. carnosus (S. carn) harvested before (co) and 10 min after NaOCl stress were analyzed by BSH-specific immunoblot analysis. In all Bacillus strains, the methionine synthase MetE is the most abundant S-bacillithiolated protein as indicated by the asterisks. BMM, Belitsky minimal medium; BSH, bacillithiol; IAM, iodoacetamide; LB, Luria Broth; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate.
FIG. 2.
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.
FIG. 2.
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.
FIG. 2.
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.
FIG. 3.
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.)
FIG. 4.
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.
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