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• W2148222884 abstract "Protein S-thiolation is a post-translational thiol-modification that controls redox-sensing transcription factors and protects active site cysteine residues against irreversible oxidation. In Bacillus subtilis the MarR-type repressor OhrR was shown to sense organic hydroperoxides via formation of mixed disulfides with the redox buffer bacillithiol (Cys-GlcN-Malate, BSH), termed as S-bacillithiolation. Here we have studied changes in the transcriptome and redox proteome caused by the strong oxidant hypochloric acid in B. subtilis. The expression profile of NaOCl stress is indicative of disulfide stress as shown by the induction of the thiol- and oxidative stress-specific Spx, CtsR, and PerR regulons. Thiol redox proteomics identified only few cytoplasmic proteins with reversible thiol-oxidations in response to NaOCl stress that include GapA and MetE. Shotgun-liquid chromatography-tandem MS analyses revealed that GapA, Spx, and PerR are oxidized to intramolecular disulfides by NaOCl stress. Furthermore, we identified six S-bacillithiolated proteins in NaOCl-treated cells, including the OhrR repressor, two methionine synthases MetE and YxjG, the inorganic pyrophosphatase PpaC, the 3-d-phosphoglycerate dehydrogenase SerA, and the putative bacilliredoxin YphP. S-bacillithiolation of the OhrR repressor leads to up-regulation of the OhrA peroxiredoxin that confers together with BSH specific protection against NaOCl. S-bacillithiolation of MetE, YxjG, PpaC and SerA causes hypochlorite-induced methionine starvation as supported by the induction of the S-box regulon. The mechanism of S-glutathionylation of MetE has been described in Escherichia coli also leading to enzyme inactivation and methionine auxotrophy. In summary, our studies discover an important role of the bacillithiol redox buffer in protection against hypochloric acid by S-bacillithiolation of the redox-sensing regulator OhrR and of four enzymes of the methionine biosynthesis pathway. Protein S-thiolation is a post-translational thiol-modification that controls redox-sensing transcription factors and protects active site cysteine residues against irreversible oxidation. In Bacillus subtilis the MarR-type repressor OhrR was shown to sense organic hydroperoxides via formation of mixed disulfides with the redox buffer bacillithiol (Cys-GlcN-Malate, BSH), termed as S-bacillithiolation. Here we have studied changes in the transcriptome and redox proteome caused by the strong oxidant hypochloric acid in B. subtilis. The expression profile of NaOCl stress is indicative of disulfide stress as shown by the induction of the thiol- and oxidative stress-specific Spx, CtsR, and PerR regulons. Thiol redox proteomics identified only few cytoplasmic proteins with reversible thiol-oxidations in response to NaOCl stress that include GapA and MetE. Shotgun-liquid chromatography-tandem MS analyses revealed that GapA, Spx, and PerR are oxidized to intramolecular disulfides by NaOCl stress. Furthermore, we identified six S-bacillithiolated proteins in NaOCl-treated cells, including the OhrR repressor, two methionine synthases MetE and YxjG, the inorganic pyrophosphatase PpaC, the 3-d-phosphoglycerate dehydrogenase SerA, and the putative bacilliredoxin YphP. S-bacillithiolation of the OhrR repressor leads to up-regulation of the OhrA peroxiredoxin that confers together with BSH specific protection against NaOCl. S-bacillithiolation of MetE, YxjG, PpaC and SerA causes hypochlorite-induced methionine starvation as supported by the induction of the S-box regulon. The mechanism of S-glutathionylation of MetE has been described in Escherichia coli also leading to enzyme inactivation and methionine auxotrophy. In summary, our studies discover an important role of the bacillithiol redox buffer in protection against hypochloric acid by S-bacillithiolation of the redox-sensing regulator OhrR and of four enzymes of the methionine biosynthesis pathway. Reactive oxygen species (ROS) 1The abbreviations used are: ROSreactive oxygen speciesBSHbacillithiolBrxbacilliredoxinCHPcumene hydroperoxideCyscysteineGlcNAcN-acetyl glucoseamineGSHglutathioneGSTglutathione S-transferaseIAMiodoacetamideIPimmunoprecipitationLMWlow molecular weightMalmalateMSHmycothiolMetmethionineMGmethylglyoxalMHQmethylhydroquinoneN5-THF5-methyltetrahydrofolateN5,N10-THF5,10-methylenetetrahydrofolatePAPS3′-phosphoadenosine-5′-phosphosulfatePPiinorganic pyrophosphateROOHorganic hydroperoxideROHorganic alcoholRESreactive electrophilic speciesRuMPribulose-5-monophosphateTHFtetrahydrofolateTrxABthioredoxin/thioredoxin reductaseRNSreactive nitrogen speciesFOXferrous oxidation xylenol orange. 1The abbreviations used are: ROSreactive oxygen speciesBSHbacillithiolBrxbacilliredoxinCHPcumene hydroperoxideCyscysteineGlcNAcN-acetyl glucoseamineGSHglutathioneGSTglutathione S-transferaseIAMiodoacetamideIPimmunoprecipitationLMWlow molecular weightMalmalateMSHmycothiolMetmethionineMGmethylglyoxalMHQmethylhydroquinoneN5-THF5-methyltetrahydrofolateN5,N10-THF5,10-methylenetetrahydrofolatePAPS3′-phosphoadenosine-5′-phosphosulfatePPiinorganic pyrophosphateROOHorganic hydroperoxideROHorganic alcoholRESreactive electrophilic speciesRuMPribulose-5-monophosphateTHFtetrahydrofolateTrxABthioredoxin/thioredoxin reductaseRNSreactive nitrogen speciesFOXferrous oxidation xylenol orange. are an unavoidable consequence of the aerobic lifestyle of many organisms (1Imlay J.A. Pathways of oxidative damage.Annu. Rev. Microbiol. 2003; 57: 395-418Crossref PubMed Scopus (1636) Google Scholar, 2Imlay J.A. Cellular defenses against superoxide and hydrogen peroxide.Annu. Rev. Biochem. 2008; 77: 755-776Crossref PubMed Scopus (1110) Google Scholar). ROS can be generated by incomplete reduction of molecular oxygen during the respiratory chain. Pathogenic bacteria encounter ROS, such as hydrogen peroxide (H2O2), superoxide anion and hypochloric acid as defense of the innate immune response during host-pathogen interactions. Upon bacterial infection, myeloperoxidase is released from activated macrophages to produce the strong oxidant hypochloric acid from H2O2 and Cl− (3Davies M.J. Myeloperoxidase-derived oxidation: mechanisms of biological damage and its prevention.J. Clin. Biochem. Nutr. 2011; 48: 8-19Crossref PubMed Scopus (286) Google Scholar, 4Hawkins C.L. Pattison D.I. Davies M.J. Hypochlorite-induced oxidation of amino acids, peptides and proteins.Amino Acids. 2003; 25: 259-274Crossref PubMed Scopus (480) Google Scholar). reactive oxygen species bacillithiol bacilliredoxin cumene hydroperoxide cysteine N-acetyl glucoseamine glutathione glutathione S-transferase iodoacetamide immunoprecipitation low molecular weight malate mycothiol methionine methylglyoxal methylhydroquinone 5-methyltetrahydrofolate 5,10-methylenetetrahydrofolate 3′-phosphoadenosine-5′-phosphosulfate inorganic pyrophosphate organic hydroperoxide organic alcohol reactive electrophilic species ribulose-5-monophosphate tetrahydrofolate thioredoxin/thioredoxin reductase reactive nitrogen species ferrous oxidation xylenol orange. reactive oxygen species bacillithiol bacilliredoxin cumene hydroperoxide cysteine N-acetyl glucoseamine glutathione glutathione S-transferase iodoacetamide immunoprecipitation low molecular weight malate mycothiol methionine methylglyoxal methylhydroquinone 5-methyltetrahydrofolate 5,10-methylenetetrahydrofolate 3′-phosphoadenosine-5′-phosphosulfate inorganic pyrophosphate organic hydroperoxide organic alcohol reactive electrophilic species ribulose-5-monophosphate tetrahydrofolate thioredoxin/thioredoxin reductase reactive nitrogen species ferrous oxidation xylenol orange. ROS can damage all cellular macromolecules, such as proteins, lipids, carbohydrates, and nucleotides (2Imlay J.A. Cellular defenses against superoxide and hydrogen peroxide.Annu. Rev. Biochem. 2008; 77: 755-776Crossref PubMed Scopus (1110) Google Scholar, 5Faulkner M.J. Helmann J.D. Peroxide stress elicits adaptive changes in bacterial metal ion homeostasis.Antioxid Redox Signal. 2011; doi 10.1089/ars.2010.3682Crossref PubMed Scopus (104) Google Scholar). Cells activate the expression of antioxidant mechanisms to detoxify ROS and to repair the damage. The response of bacteria to H2O2 and organic hydroperoxides (ROOH) involves heme-cofactor containing catalases and thiol-dependent peroxidases as detoxification mechanisms (5Faulkner M.J. Helmann J.D. Peroxide stress elicits adaptive changes in bacterial metal ion homeostasis.Antioxid Redox Signal. 2011; doi 10.1089/ars.2010.3682Crossref PubMed Scopus (104) Google Scholar, 6Mongkolsuk S. Helmann J.D. Regulation of inducible peroxide stress responses.Mol. Microbiol. 2002; 45: 9-15Crossref PubMed Scopus (225) Google Scholar). Peroxidases use conserved redox-active cysteine residues that function in reduction of peroxides. These oxidative stress defense mechanisms are often controlled by redox-sensitive transcription factors that undergo post-translational thiol-modifications upon challenge with ROS leading either to activation or inactivation of the transcription factors (7Antelmann H. Helmann J.D. Thiol-based Redox Switches and Gene Regulation.Antioxid Redox Signal. 2011; 14: 1049-1063Crossref PubMed Scopus (275) Google Scholar). Post-translational thiol-modifications implicated in redox-sensing mechanisms are known as thiol-disulfide redox-switches and include in most cases inter- or intramolecular disulfides and mixed disulfides with low molecular weight (LMW) thiols (S-thiolations). The best studied examples for peroxide-sensing thiol-based transcription factors are Escherichia coli OxyR (8Zheng M. Aslund F. Storz G. Activation of the OxyR transcription factor by reversible disulfide bond formation.Science. 1998; 279: 1718-1721Crossref PubMed Scopus (970) Google Scholar, 9Lee C. Lee S.M. Mukhopadhyay P. Kim S.J. Lee S.C. Ahn W.S. Yu M.H. Storz G. Ryu S.E. Redox regulation of OxyR requires specific disulfide bond formation involving a rapid kinetic reaction path.Nat. Struct. Mol. Biol. 2004; 11: 1179-1185Crossref PubMed Scopus (203) Google Scholar, 10Choi H. Kim S. Mukhopadhyay P. Cho S. Woo J. Storz G. Ryu S.E. Structural basis of the redox switch in the OxyR transcription factor.Cell. 2001; 105: 103-113Abstract Full Text Full Text PDF PubMed Scopus (434) Google Scholar, 11Kim S.O. Merchant K. Nudelman R. Beyer Jr., W.F. Keng T. DeAngelo J. Hausladen A. Stamler J.S. OxyR: a molecular code for redox-related signaling.Cell. 2002; 109: 383-396Abstract Full Text Full Text PDF PubMed Scopus (388) Google Scholar) and yeast Yap1 transcription factor (7Antelmann H. Helmann J.D. Thiol-based Redox Switches and Gene Regulation.Antioxid Redox Signal. 2011; 14: 1049-1063Crossref PubMed Scopus (275) Google Scholar, 12D'Autréaux B. Toledano M.B. ROS as signalling molecules: mechanisms that generate specificity in ROS homeostasis.Nat. Rev. Mol. Cell Biol. 2007; 8: 813-824Crossref PubMed Scopus (2481) Google Scholar, 13Brandes N. Schmitt S. Jakob U. Thiol-based redox switches in eukaryotic proteins.Antioxid Redox Signal. 2009; 11: 997-1014Crossref PubMed Scopus (279) Google Scholar) that are activated by intramolecular disulfide bond formation to induce the antioxidant defense mechanisms. In Bacillus subtilis, the major detoxification enzymes for peroxides are catalase (KatA) and alkylhydroperoxide reductase (AhpCF) that are controlled of the peroxide-sensing Fur family metalloregulatory PerR repressor (5Faulkner M.J. Helmann J.D. Peroxide stress elicits adaptive changes in bacterial metal ion homeostasis.Antioxid Redox Signal. 2011; doi 10.1089/ars.2010.3682Crossref PubMed Scopus (104) Google Scholar). PerR harbors a structural Zn-binding site coordinated by four cysteine residues and a regulatory Fe or Mn binding site consisting of His and Asp residues. Inactivation of PerR is caused by Fe-catalyzed oxidation of His37 and His91 to 2-oxohistidine in the regulatory site (5Faulkner M.J. Helmann J.D. Peroxide stress elicits adaptive changes in bacterial metal ion homeostasis.Antioxid Redox Signal. 2011; doi 10.1089/ars.2010.3682Crossref PubMed Scopus (104) Google Scholar, 14Lee J.W. Helmann J.D. The PerR transcription factor senses H2O2 by metal-catalysed histidine oxidation.Nature. 2006; 440: 363-367Crossref PubMed Scopus (421) Google Scholar, 15Traoré D.A. El Ghazouani A. Jacquamet L. Borel F. Ferrer J.L. Lascoux D. Ravanat J.L. Jaquinod M. Blondin G. Caux-Thang C. Duarte V. Latour J.M. Structural and functional characterization of 2-oxo-histidine in oxidized PerR protein.Nat. Chem. Biol. 2009; 5: 53-59Crossref PubMed Scopus (91) Google Scholar, 16Jacquamet L. Traoré D.A. Ferrer J.L. Proux O. Testemale D. Hazemann J.L. Nazarenko E. El Ghazouani A. Caux-Thang C. Duarte V. Latour J.M. Structural characterization of the active form of PerR: insights into the metal-induced activation of PerR and Fur proteins for DNA binding.Mol. Microbiol. 2009; 73: 20-31Crossref PubMed Scopus (87) Google Scholar). The response to ROOH involves the MarR-type repressor OhrR in B. subtilis that is conserved in many other bacteria (6Mongkolsuk S. Helmann J.D. Regulation of inducible peroxide stress responses.Mol. Microbiol. 2002; 45: 9-15Crossref PubMed Scopus (225) Google Scholar, 7Antelmann H. Helmann J.D. Thiol-based Redox Switches and Gene Regulation.Antioxid Redox Signal. 2011; 14: 1049-1063Crossref PubMed Scopus (275) Google Scholar). OhrR-like proteins control a thiol-dependent peroxiredoxin that converts ROOH to organic alcohols. OhrR proteins can be devided into the one and two-Cys families of redox sensing repressors. The OhrR protein of Xanthomonas campestris belongs to the two-Cys family that is oxidized to a Cys22-Cys127‘ intermolecular disulfide between both subunits of the OhrR dimer (17Panmanee W. Vattanaviboon P. Poole L.B. Mongkolsuk S. Novel organic hydroperoxide-sensing and responding mechanisms for OhrR, a major bacterial sensor and regulator of organic hydroperoxide stress.J. Bacteriol. 2006; 188: 1389-1395Crossref PubMed Scopus (97) Google Scholar). One-Cys OhrR proteins harbor one conserved N-terminal Cys with the prototype of B. subtilis OhrR or Staphylococcus aureus SarZ and MgrA (7Antelmann H. Helmann J.D. Thiol-based Redox Switches and Gene Regulation.Antioxid Redox Signal. 2011; 14: 1049-1063Crossref PubMed Scopus (275) Google Scholar, 18Chen P.R. Brugarolas P. He C. Redox Signaling in Human Pathogens.Antioxid Redox Signal. 2011; 14: 1107-1118Crossref PubMed Scopus (49) Google Scholar). Cumene hydroperoxide (CHP) leads to Cys15 oxidation to sulfenic acid that is rapidly oxidized to S-thiolated OhrR containing cysteine or the redox buffer bacillithiol (BSH) (19Lee J.W. Soonsanga S. Helmann J.D. A complex thiolate switch regulates the Bacillus subtilis organic peroxide sensor OhrR.Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 8743-8748Crossref PubMed Scopus (205) Google Scholar, 20Newton G.L. Rawat M. La Clair J.J. Jothivasan V.K. Budiarto T. Hamilton C.J. Claiborne A. Helmann J.D. Fahey R.C. Bacillithiol is an antioxidant thiol produced in Bacilli.Nat. Chem. Biol. 2009; 5: 625-627Crossref PubMed Scopus (213) Google Scholar). Thus, B. subtilis OhrR is controlled by S-cysteinylation and S-bacillithiolation as redox-switch mechanism leading to inactivation of the OhrR repressor function and derepression of ohrA transcription. In previous studies, we investigated the global response, post-translational modifications and specific regulatory mechanisms that are induced by reactive electrophilic species (RES) in B. subtilis, such as diamide, quinones, or aldehydes. RES deplete the cellular redox buffer cysteine leading to induction of the Spx-regulon that controls thiol-disulfide oxidoreductases (TrxAB) to restore the redox homeostasis (21Liebeke M. Pöther D.C. van Duy N. Albrecht D. Becher D. Hochgräfe F. Lalk M. Hecker M. Antelmann H. Depletion of thiol-containing proteins in response to quinones in Bacillus subtilis.Mol. Microbiol. 2008; 69: 1513-1529Crossref PubMed Scopus (81) Google Scholar, 22Pöther D.C. Liebeke M. Hochgräfe F. Antelmann H. Becher D. Lalk M. Lindequist U. Borovok I. Cohen G. Aharonowitz Y. Hecker M. Diamide triggers mainly S Thiolations in the cytoplasmic proteomes of Bacillus subtilis and Staphylococcus aureus.J. Bacteriol. 2009; 191: 7520-7530Crossref PubMed Scopus (57) Google Scholar, 23Nguyen T.T. Eiamphungporn W. Mäder U. Liebeke M. Lalk M. Hecker M. Helmann J.D. Antelmann H. Genome-wide responses to carbonyl electrophiles in Bacillus subtilis: control of the thiol-dependent formaldehyde dehydrogenase AdhA and cysteine proteinase YraA by the MerR-family regulator YraB (AdhR).Mol. Microbiol. 2009; 71: 876-894Crossref PubMed Scopus (81) Google Scholar). B. subtilis encodes specific redox-sensing regulators of the MarR/DUF24-family that sense RES, but not ROS (7Antelmann H. Helmann J.D. Thiol-based Redox Switches and Gene Regulation.Antioxid Redox Signal. 2011; 14: 1049-1063Crossref PubMed Scopus (275) Google Scholar). These include the paralogous repressors YodB and CatR that are inactivated via intermolecular disulfide formation by diamide and quinones resulting in derepression of the azoreductase (AzoR1), nitroreductase (YodC), and thiol-dependent dioxygenase (CatE) catalyzing the detoxification of the electrophiles (24Chi B.K. Albrecht D. Gronau K. Becher D. Hecker M. Antelmann H. The redox-sensing regulator YodB senses quinones and diamide via a thiol-disulfide switch in Bacillus subtilis.Proteomics. 2010; 10: 3155-3164Crossref PubMed Scopus (33) Google Scholar, 25Chi B.K. Kobayashi K. Albrecht D. Hecker M. Antelmann H. The paralogous MarR/DUF24-family repressors YodB and CatR control expression of the catechol dioxygenase CatE in Bacillus subtilis.J. Bacteriol. 2010; 192: 4571-4581Crossref PubMed Scopus (25) Google Scholar, 26Leelakriangsak M. Huyen N.T. Töwe S. van Duy N. Becher D. Hecker M. Antelmann H. Zuber P. Regulation of quinone detoxification by the thiol stress sensing DUF24/MarR-like repressor, YodB in Bacillus subtilis.Mol. Microbiol. 2008; 67: 1108-1124Crossref PubMed Scopus (64) Google Scholar). Other proteins of the MarR/DUF24-family (HxlR) and of the MerR/NmlR-family (AdhR) sense specifically aldehydes, such as formaldehyde and methylglyoxal (23Nguyen T.T. Eiamphungporn W. Mäder U. Liebeke M. Lalk M. Hecker M. Helmann J.D. Antelmann H. Genome-wide responses to carbonyl electrophiles in Bacillus subtilis: control of the thiol-dependent formaldehyde dehydrogenase AdhA and cysteine proteinase YraA by the MerR-family regulator YraB (AdhR).Mol. Microbiol. 2009; 71: 876-894Crossref PubMed Scopus (81) Google Scholar). In this study, we were interested in the global response, regulatory mechanisms, and post-translational thiol-modifications that contribute to the resistance of B. subtilis to the strong oxidant hypochloric acid. Hypochloric acid is the active component of household bleach and widely used as antimicrobial disinfectant to clean surfaces. The bactericidal effect of hypochloric acid has been proposed to involve generation of ROS, such as superoxide anion and hydroxyl radical formation in E. coli (27Touati D. Jacques M. Tardat B. Bouchard L. Despied S. Lethal oxidative damage and mutagenesis are generated by iron in delta fur mutants of Escherichia coli: protective role of superoxide dismutase.J. Bacteriol. 1995; 177: 2305-2314Crossref PubMed Scopus (376) Google Scholar). Recent redox proteomics studies in E. coli using the OxICAT approach have shown that bleach causes strong disulfide formation and protein aggregation in a different set of proteins than H2O2 (28Leichert L.I. Gehrke F. Gudiseva H.V. Blackwell T. Ilbert M. Walker A.K. Strahler J.R. Andrews P.C. Jakob U. Quantifying changes in the thiol redox proteome upon oxidative stress in vivo.Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 8197-8202Crossref PubMed Scopus (413) Google Scholar). As defense mechanism against NaOCl stress, E. coli uses the redox controlled chaperone Hsp33 that is activated by NaOCl by the formation of intramolecular disulfides in the Zn-redox switch centers resulting in Zn release, oxidative unfolding and dimerization (29Winter J. Ilbert M. Graf P.C. Ozcelik D. Jakob U. Bleach activates a redox-regulated chaperone by oxidative protein unfolding.Cell. 2008; 135: 691-701Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar). Hsp33 protects cells against NaOCl-induced protein aggregation. The mode of action has been also studied using transcriptome analyses in pathogenic E. coli O157:H7 outbreak strains and the food-borne pathogen B. cereus ATCC14579 (30Wang S. Deng K. Zaremba S. Deng X. Lin C. Wang Q. Tortorello M.L. Zhang W. Transcriptomic response of Escherichia coli O157:H7 to oxidative stress.Appl. Environ. Microbiol. 2009; 75: 6110-6123Crossref PubMed Scopus (122) Google Scholar, 31Ceragioli M. Mols M. Moezelaar R. Ghelardi E. Senesi S. Abee T. Comparative transcriptomic and phenotypic analysis of the responses of Bacillus cereus to various disinfectant treatments.Appl. Environ. Microbiol. 2010; 76: 3352-3360Crossref PubMed Scopus (80) Google Scholar). Both transcriptome analyses suggest a major oxidative stress response mechanism of NaOCl. Regulons involved in the biosynthesis of sulfur and sulfur-containing amino acids were up-regulated by NaOCl in both genome-wide studies. However, the mode of action of hypochloric acid has not yet been investigated in B. subtilis. We have used transcriptomic and redox proteomic approaches coupled with shotgun-LC-MS/MS analyses to analyze the mode of action and reversible thiol-modifications by NaOCl stress in B. subtilis. We discovered that the major resistant determinant to NaOCl is the OhrA peroxiredoxin that conferred specific protection against NaOCl toxicity. Moreover, we identified S-bacillithiolations of the OhrR repressor, two methionine synthases MetE and YxjG, the inorganic pyrophosphatase PpaC, and the 3-d-phosphoglycerate dehydrogenase SerA as major protection mechanisms against hypochlorite stress in B. subtilis. The bacterial strains used were B. subtilis wild-type strains 168 (trpC2), JH642 (trpC2 attSPβ), and CU1065 (trpC2 pheA1) and mutant strains Δspx (trpC2,spx::neor) (32Nakano M.M. Hajarizadeh F. Zhu Y. Zuber P. Loss-of-function mutations in yjbD result in ClpX- and ClpP-independent competence development of Bacillus subtilis.Mol. Microbiol. 2001; 42: 383-394Crossref PubMed Scopus (77) Google Scholar), ΔohrR (trpC2,ohrR::cmr), ΔohrA (trpC2, ohrA::cmr), ΔsigB (trpC2,sigB::cmr), ΔperR (trpC2,perR::cmr) (33Hayashi K. Kensuke T. Kobayashi K. Ogasawara N. Ogura M. Bacillus subtilis RghR (YvaN) represses rapG and rapH, which encode inhibitors of expression of the srfA operon.Mol. Microbiol. 2006; 59: 1714-1729Crossref PubMed Scopus (56) Google Scholar), HB9121 (CU1065 trpC2,ohrR::kmr ohrR-FLAG (Spcr) ohrA-cat lacZ (Neor) (19Lee J.W. Soonsanga S. Helmann J.D. A complex thiolate switch regulates the Bacillus subtilis organic peroxide sensor OhrR.Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 8743-8748Crossref PubMed Scopus (205) Google Scholar), HB2048 (CU1065 SPβc2Δ2::Tn917::(ohrA-cat-lacZ)ohrR::kan,thrC::pXTohrRC15S) (34Fuangthong M. Helmann J.D. The OhrR repressor senses organic hydroperoxides by reversible formation of a cysteine-sulfenic acid derivative.Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 6690-6695Crossref PubMed Scopus (183) Google Scholar), HB11002 (CU1065 trpC2, bshA::mlsr), and HB11053 (CU1065 trpC2, bshB1:: spcr bshB2::cmr) (35Gaballa A. Newton G.L. Antelmann H. Parsonage D. Upton H. Rawat M. Claiborne A. Fahey R.C. Helmann J.D. Biosynthesis and functions of bacillithiol, a major low-molecular-weight thiol in Bacilli.Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 6482-6486Crossref PubMed Scopus (180) Google Scholar). B. subtilis strains were cultivated under vigorous agitation at 37 °C in Belitsky minimal medium (BMM) as described previously (36Stülke J. Hanschke R. Hecker M. Temporal activation of beta-glucanase synthesis in Bacillus subtilis is mediated by the GTP pool.J. Gen. Microbiol. 1993; 139: 2041-2045Crossref PubMed Scopus (201) Google Scholar). The antibiotics were used at the following concentrations: 1 μg/ml erythromycin, 25 μg/ml lincomycin, 5 μg/ml chloramphenicol, 10 μg/ml kanamycin, and 100 μg/ml spectinomycin. Sodium hypochlorite (15% stock solution), diamide, and cumene hydroperoxide were purchased from Sigma Aldrich. For NaOCl stress exposure, cells were grown in BMM to an optical density at 500 nm (OD500) of 0.4 and treated with 50, 75, or 100 μm NaOCl diluted freshly in destilled water. The growth experiments in the presence of methionine were performed by addition of 75 μm methionine either after inoculation of the culture or 30 and 60 min after NaOCl stress exposure. Gene deletions for construction of the ohrA mutant were generated using long-flanking-homology polymerase chain reaction (LFH-PCR) as described previously (25Chi B.K. Kobayashi K. Albrecht D. Hecker M. Antelmann H. The paralogous MarR/DUF24-family repressors YodB and CatR control expression of the catechol dioxygenase CatE in Bacillus subtilis.J. Bacteriol. 2010; 192: 4571-4581Crossref PubMed Scopus (25) Google Scholar). Primers ohrA-F1 (5′-TGCAGCTGATTGAGGATACG-3′) and ohrA-F2 (5′-GTTATCCGCTCACAATTCGCGGTCTGATGAAATGACCT-3′) were used to amplify the up fragment and primers ohrA-R1 (5′-CGTCGTGACTGGGAAAACGGTGTGACGCTGCAAGTAAA-5′) and ohrA-R2 (5′-CCCTTCAATCTCCGAATCAA-3′) to amplify the down fragment, respectively. Fragments were amplified and joined together with the chloramphenicol cassette using Pfusion DNA polymerase (Invitrogen, Carlsbad, CA) as described (33Hayashi K. Kensuke T. Kobayashi K. Ogasawara N. Ogura M. Bacillus subtilis RghR (YvaN) represses rapG and rapH, which encode inhibitors of expression of the srfA operon.Mol. Microbiol. 2006; 59: 1714-1729Crossref PubMed Scopus (56) Google Scholar). Integration and deletion of the ohrA gene were confirmed by PCR and by Northern blot analysis using digoxigenin-labeled RNA probes of the corresponding gene. The concentrations of the remaining NaOCl in the culture supernatants were determined using the FOX assay (37Nourooz-Zadeh J. Tajaddini-Sarmadi J. Wolff S.P. Measurement of plasma hydroperoxide concentrations by the ferrous oxidation-xylenol orange assay in conjunction with triphenylphosphine.Anal. Biochem. 1994; 220: 403-409Crossref PubMed Scopus (610) Google Scholar). FOX reagent was prepared by mixing 100 ml FOX I (100 mm sorbitol, 125 μm xylenol orange) and 1 ml FOX II (25 mm ammonious ferrous(II)sulfate in 2.5 m H2SO4). It was not possible to measure any NaOCl concentrations in BMM with tryptophane and glutamate. Thus, cells were grown to an OD500 of 0.4 in BMM, centrifuged and resuspended in BMM without tryptophane and glutamate before the addition of 75 μm NaOCl. Samples of 500 μl medium were taken at different time points after NaOCl addition, mixed with 500 μl FOX reagent and incubated at room temperature for 60 min. The absorbance was measured at 560 nm. Calibration curves were generated using NaOCl concentrations in the range from 0 to 100 μm diluted in BMM without tryptophane and glutamate. The thiol redox proteome analysis was performed as described previously (38Hochgräfe F. Mostertz J. Albrecht D. Hecker M. Fluorescence thiol modification assay: oxidatively modified proteins in Bacillus subtilis.Mol. Microbiol. 2005; 58: 409-425Crossref PubMed Scopus (73) Google Scholar) with the modifications as explained (21Liebeke M. Pöther D.C. van Duy N. Albrecht D. Becher D. Hochgräfe F. Lalk M. Hecker M. Antelmann H. Depletion of thiol-containing proteins in response to quinones in Bacillus subtilis.Mol. Microbiol. 2008; 69: 1513-1529Crossref PubMed Scopus (81) Google Scholar). Cells were harvested before (control conditions) and 10, 20, and 30 min after exposure to 50 μm NaOCl stress, resuspended in urea/CHAPS alkylation buffer (8 M urea; 1% CHAPS; 1 mm EDTA; 200 mm Tris-HCl pH 8,0; 100 mm iodoacetamide (IAM)), sonicated, alkylated for 20 min in the dark, precipitated with 100% acetone, washed several times with 80% acetone and dried. After resolving in urea/CHAPS buffer without IAM, 200 μg of the protein extract were reduced with 10 mm Tris-(2-carboxyethyl)-phosphine (TCEP) 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 as described (21Liebeke M. Pöther D.C. van Duy N. Albrecht D. Becher D. Hochgräfe F. Lalk M. Hecker M. Antelmann H. Depletion of thiol-containing proteins in response to quinones in Bacillus subtilis.Mol. Microbiol. 2008; 69: 1513-1529Crossref PubMed Scopus (81) Google Scholar). The two-dimensional (2D) gels were scanned using a Typhoon 9400 variable mode imager (Amersham Biosciences, Freiburg, Germany) for BODIPY-fluorescence and then stained with Colloidal Coomassie for protein amounts. Quantitative image analysis was performed with the DECODON Delta 2D software (http://www.decodon.com). The first alkylation protocol (38Hochgräfe F. Mostertz J. Albrecht D. Hecker M. Fluorescence thiol modification assay: oxidatively modified proteins in Bacillus subtilis.Mol. Microbiol. 2005; 58: 409-425Crossref PubMed Scopus (73) Google Scholar) that applied the TCA-precipitation step to harvest cells to stop thiol-disulfide exchanges was changed previously (21Liebeke M. Pöther D.C. van Duy N. Albrecht D. Becher D. Hochgräfe F. Lalk M. Hecker M. Antelmann H. Depletion of thiol-containing proteins in response to quinones in Bacillus subtilis.Mol. Microbiol. 2008; 69: 1513-1529Crossref PubMed Scopus (81) Google Scholar) for two reasons: (1) The 2D gels were of bad quality and exhibited protein streaking during the isoelectric focusing (IEF) and (2) GapA was oxidized artificially by this TCA precipitation step under control conditions but not if this TCA step was omitted (38Hochg" @default.
• W2148222884 created "2016-06-24" @default.
• W2148222884 creator A5008320479 @default.
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• W2148222884 date "2011-11-01" @default.
• W2148222884 modified "2023-10-12" @default.
• W2148222884 title "S-Bacillithiolation Protects Against Hypochlorite Stress in Bacillus subtilis as Revealed by Transcriptomics and Redox Proteomics" @default.
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