Comprehensively Characterizing the Thioredoxin Interactome In Vivo Highlights the Central Role Played by This Ubiquitous Oxidoreductase in Redox Control
2016; Elsevier BV; Volume: 15; Issue: 6 Linguagem: Inglês
10.1074/mcp.m115.056440
ISSN1535-9484
AutoresIsabelle S. Arts, Didier Vertommen, Francesca Baldin, Géraldine Laloux, Jean‐François Collet,
Tópico(s)Metalloenzymes and iron-sulfur proteins
ResumoThioredoxin (Trx) is a ubiquitous oxidoreductase maintaining protein-bound cysteine residues in the reduced thiol state. Here, we combined a well-established method to trap Trx substrates with the power of bacterial genetics to comprehensively characterize the in vivo Trx redox interactome in the model bacterium Escherichia coli. Using strains engineered to optimize trapping, we report the identification of a total 268 Trx substrates, including 201 that had never been reported to depend on Trx for reduction. The newly identified Trx substrates are involved in a variety of cellular processes, ranging from energy metabolism to amino acid synthesis and transcription. The interaction between Trx and two of its newly identified substrates, a protein required for the import of most carbohydrates, PtsI, and the bacterial actin homolog MreB was studied in detail. We provide direct evidence that PtsI and MreB contain cysteine residues that are susceptible to oxidation and that participate in the formation of an intermolecular disulfide with Trx. By considerably expanding the number of Trx targets, our work highlights the role played by this major oxidoreductase in a variety of cellular processes. Moreover, as the dependence on Trx for reduction is often conserved across species, it also provides insightful information on the interactome of Trx in organisms other than E. coli. Thioredoxin (Trx) is a ubiquitous oxidoreductase maintaining protein-bound cysteine residues in the reduced thiol state. Here, we combined a well-established method to trap Trx substrates with the power of bacterial genetics to comprehensively characterize the in vivo Trx redox interactome in the model bacterium Escherichia coli. Using strains engineered to optimize trapping, we report the identification of a total 268 Trx substrates, including 201 that had never been reported to depend on Trx for reduction. The newly identified Trx substrates are involved in a variety of cellular processes, ranging from energy metabolism to amino acid synthesis and transcription. The interaction between Trx and two of its newly identified substrates, a protein required for the import of most carbohydrates, PtsI, and the bacterial actin homolog MreB was studied in detail. We provide direct evidence that PtsI and MreB contain cysteine residues that are susceptible to oxidation and that participate in the formation of an intermolecular disulfide with Trx. By considerably expanding the number of Trx targets, our work highlights the role played by this major oxidoreductase in a variety of cellular processes. Moreover, as the dependence on Trx for reduction is often conserved across species, it also provides insightful information on the interactome of Trx in organisms other than E. coli. Thioredoxins (Trxs) 1The abbreviations used are:TrxThioredoxinA22S-(3,4-dichlorobenzyl) isothioureaBCPBacterioferritin comigratory proteinDMDDimedoneGrxGlutaredoxinHOClHypochlorous acidH2O2Hydrogen peroxideIAMIodoacetamideIPTGIsopropyl β-D-1-thiogalactopyranosideLBLysogeny brothMalPEGMethoxyl polyethylene glycol maleimideMsrMethionine sulfoxide reductaseNEMN-ethylmaleimidePAPS reductase3′-phosphoadenosine 5′-phosphosulfate reductasePtsIPhosphoenolpyruvate-protein phosphotransferase (enzyme I)RFPRed fluorescent proteinRNRRibonucleotide reductaseTpxThiol peroxidase. are small antioxidant enzymes that catalyze the reduction of disulfide bonds that form in substrate proteins either as part of a catalytic cycle (see below) or following exposure to reactive oxygen species (ROS). As such, Trxs are involved in many different cellular processes, ranging from the defense against oxidative stress to the regulation of numerous signal transduction pathways and the modulation of the inflammatory response (1.Collet J.F. Messens J. Structure, function, and mechanism of thioredoxin proteins.Antiox. Redox Signal. 2010; 13: 1205-1216Crossref PubMed Scopus (247) Google Scholar, 2.Nakamura T. Nakamura H. Hoshino T. Ueda S. Wada H. Yodoi J. Redox regulation of lung inflammation by thioredoxin.Antiox. Redox Signal. 2005; 7: 60-71Crossref PubMed Scopus (0) Google Scholar). Thioredoxin S-(3,4-dichlorobenzyl) isothiourea Bacterioferritin comigratory protein Dimedone Glutaredoxin Hypochlorous acid Hydrogen peroxide Iodoacetamide Isopropyl β-D-1-thiogalactopyranoside Lysogeny broth Methoxyl polyethylene glycol maleimide Methionine sulfoxide reductase N-ethylmaleimide 3′-phosphoadenosine 5′-phosphosulfate reductase Phosphoenolpyruvate-protein phosphotransferase (enzyme I) Red fluorescent protein Ribonucleotide reductase Thiol peroxidase. Trxs have been identified in most living organisms, including archaea, bacteria, plants and mammals (1.Collet J.F. Messens J. Structure, function, and mechanism of thioredoxin proteins.Antiox. Redox Signal. 2010; 13: 1205-1216Crossref PubMed Scopus (247) Google Scholar). They all share a canonical WCGPC catalytic motif located on a highly conserved fold, which consists of five β-strands surrounded by four α-helices (Fig. 1A) (3.Holmgren A. Soderberg B.O. Eklund H. Branden C.I. Three-dimensional structure of Escherichia coli thioredoxin-S2 to 2.8 A resolution.Proc. Natl. Acad. Sci. U.S.A. 1975; 72: 2305-2309Crossref PubMed Google Scholar). The cysteine residues of the WCGPC motif are the key players used by Trxs to break disulfide bonds in substrate proteins. In this reaction, the first cysteine performs a nucleophilic attack on an oxidized substrate, which results in the formation of a mixed-disulfide intermediate between Trx and its substrate. This intermediate is then resolved by a nucleophilic attack of the second cysteine of the WCGPC motif, leading to the oxidation of thioredoxin and the release of the reduced substrate (1.Collet J.F. Messens J. Structure, function, and mechanism of thioredoxin proteins.Antiox. Redox Signal. 2010; 13: 1205-1216Crossref PubMed Scopus (247) Google Scholar) (Fig. 1B). The catalytic cysteine residues are then converted back to the reduced state by the flavoenzyme thioredoxin reductase at the expense of the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH). Escherichia coli Trx1, encoded by the gene trxA, is the first Trx that was discovered (4.Laurent T.C. Moore E.C. Reichard P. Enzymatic synthesis of deoxyribonucleotides. IV. Isolation and characterization of thioredoxin, the hydrogen donor from Escherichia coli B.J. Biol. Chem. 1964; 239: 3436-3444Abstract Full Text PDF PubMed Google Scholar, 5.Moore E.C. Reichard P. Thelander L. Enzymatic synthesis of deoxyribonucleotides.V. Purification and properties of thioredoxin reductase from Escherichia coli B.J. Biol. Chem. 1964; 239: 3445-3452Abstract Full Text PDF PubMed Google Scholar). Being constitutively expressed in the cytoplasm, it reduces disulfide bonds that form during the catalytic cycle of important enzymes such as ribonucleotide reductase (RNR) (4.Laurent T.C. Moore E.C. 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Sulfenic acid formation and overoxidation of essential CYS61.J. Biol. Chem. 2003; 278: 9203-9211Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar) and BCP (13.Reeves S.A. Parsonage D. Nelson K.J. Poole L.B. Kinetic and thermodynamic features reveal that Escherichia coli BCP is an unusually versatile peroxiredoxin.Biochemistry. 2011; 50: 8970-8981Crossref PubMed Scopus (34) Google Scholar). In addition to its role in enzyme recycling, Trx1 also controls the redox state of proteins with cysteine residues prone to oxidation. When exposed to ROS, sensitive cysteines are first modified to sulfenic acids (-SOH), which then often react with another thiol present in the vicinity to form a disulfide (14.Roos G. Messens J. Protein sulfenic acid formation: from cellular damage to redox regulation.Free Radic. Biol. Med. 2011; 51: 314-326Crossref PubMed Scopus (173) Google Scholar). Being able to reduce both sulfenic acids and disulfides (1.Collet J.F. Messens J. Structure, function, and mechanism of thioredoxin proteins.Antiox. Redox Signal. 2010; 13: 1205-1216Crossref PubMed Scopus (247) Google Scholar, 15.Nandi D.L. Horowitz P.M. Westley J. Rhodanese as a thioredoxin oxidase.Int. J. Biochem. Cell Biol. 2000; 32: 465-473Crossref PubMed Scopus (0) Google Scholar), Trx1 plays an active role in the protection of proteins from oxidative damages. Differential thiol trapping experiments led to the identification of a handful of proteins that depend on this protective activity of Trx1 (16.Leichert L.I. Jakob U. Protein thiol modifications visualized in vivo.PLos Biol. 2004; 2: e333Crossref PubMed Scopus (187) Google Scholar). Although a number of proteins that interact with Trx1 have been identified using copurification experiments (17.Kumar J.K. Tabor S. Richardson C.C. Proteomic analysis of thioredoxin-targeted proteins in Escherichia coli.Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 3759-3764Crossref PubMed Scopus (138) Google Scholar), a comprehensive survey of proteins that depend on this oxidoreductase for reduction is still missing. The goal of the present study was to fully grasp the importance of Trx1 in controlling the redox state of intracellular proteins by characterizing the redox interactome of this major oxidoreductase. A powerful approach to identify Trx substrates consists in trapping the covalent intermediates that form between this protein and its substrates when the second catalytic cysteine of the WCGPC motif is mutated to an alanine. In this case, dissociation of the mixed-disulfide intermediate is prevented, stabilizing the complexes between thioredoxin and its substrates (1.Collet J.F. Messens J. Structure, function, and mechanism of thioredoxin proteins.Antiox. Redox Signal. 2010; 13: 1205-1216Crossref PubMed Scopus (247) Google Scholar). This approach, which has never been applied to E. coli Trx1, already led to the identification of putative Trx substrates in a variety of organisms, including Chlorobaculum tepidum and Synechocystis sp (18.Hosoya-Matsuda N. Inoue K. Hisabori T. Roles of thioredoxins in the obligate anaerobic green sulfur photosynthetic bacterium Chlorobaculum tepidum.Mol. Plant. 2009; 2: 336-343Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar, 19.Lindahl M. Florencio F.J. Thioredoxin-linked processes in cyanobacteria are as numerous as in chloroplasts, but targets are different.Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 16107-16112Crossref PubMed Scopus (128) Google Scholar, 20.Perez-Perez M.E. Florencio F.J. Lindahl M. Selecting thioredoxins for disulphide proteomics: target proteomes of three thioredoxins from the cyanobacterium Synechocystis sp. PCC 6803.Proteomics. 2006; 6: S186-195Crossref PubMed Google Scholar, 21.Mata-Cabana A. Florencio F.J. Lindahl M. Membrane proteins from the cyanobacterium Synechocystis sp. PCC 6803 interacting with thioredoxin.Proteomics. 2007; 7: 3953-3963Crossref PubMed Scopus (48) Google Scholar), algae and plants (22.Motohashi K. Kondoh A. Stumpp M.T. Hisabori T. Comprehensive survey of proteins targeted by chloroplast thioredoxin.Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 11224-11229Crossref PubMed Scopus (319) Google Scholar, 23.Balmer Y. Koller A. del Val G. Manieri W. Schurmann P. Buchanan B.B. Proteomics gives insight into the regulatory function of chloroplast thioredoxins.Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 370-375Crossref PubMed Scopus (339) Google Scholar, 24.Balmer Y. Vensel W.H. Tanaka C.K. Hurkman W.J. Gelhaye E. Rouhier N. Jacquot J.P. Manieri W. Schurmann P. Droux M. Buchanan B.B. Thioredoxin links redox to the regulation of fundamental processes of plant mitochondria.Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 2642-2647Crossref PubMed Scopus (254) Google Scholar, 25.Yamazaki D. Motohashi K. Kasama T. Hara Y. Hisabori T. Target proteins of the cytosolic thioredoxins in Arabidopsis thaliana.Plant Cell Physiol. 2004; 45: 18-27Crossref PubMed Scopus (167) Google Scholar, 26.Yoshida K. Noguchi K. Motohashi K. Hisabori T. Systematic exploration of thioredoxin target proteins in plant mitochondria.Plant Cell Physiol. 2013; 54: 875-892Crossref PubMed Scopus (75) Google Scholar, 27.Wong J.H. Cai N. Balmer Y. Tanaka C.K. Vensel W.H. Hurkman W.J. Buchanan B.B. Thioredoxin targets of developing wheat seeds identified by complementary proteomic approaches.Phytochemistry. 2004; 65: 1629-1640Crossref PubMed Scopus (134) Google Scholar, 28.Lemaire S.D. Guillon B. Le Marechal P. Keryer E. Miginiac-Maslow M. Decottignies P. New thioredoxin targets in the unicellular photosynthetic eukaryote Chlamydomonas reinhardtii.Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 7475-7480Crossref PubMed Scopus (192) Google Scholar, 29.Marchand C. Le Marechal P. Meyer Y. Decottignies P. 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Collin V. Issakidis-Bourguet E. Marechal P.L. Decottignies P. Thioredoxin targets in Arabidopsis roots.Proteomics. 2010; 10: 2418-2428Crossref PubMed Scopus (41) Google Scholar, 34.Rey P. Cuine S. Eymery F. Garin J. Court M. Jacquot J.P. Rouhier N. Broin M. Analysis of the proteins targeted by CDSP32, a plastidic thioredoxin participating in oxidative stress responses.Plant J. 2005; 41: 31-42Crossref PubMed Scopus (120) Google Scholar), parasites (35.Sturm N. Jortzik E. Mailu B.M. Koncarevic S. Deponte M. Forchhammer K. Rahlfs S. Becker K. Identification of proteins targeted by the thioredoxin superfamily in Plasmodium falciparum.PLoS Pathog. 2009; 5: e1000383Crossref PubMed Scopus (62) Google Scholar, 36.Kawazu S. Takemae H. Komaki-Yasuda K. Kano S. Target proteins of the cytosolic thioredoxin in Plasmodium falciparum.Parasitol. Int. 2010; 59: 298-302Crossref PubMed Scopus (14) Google Scholar, 37.Schlosser S. Leitsch D. Duchene M. Entamoeba histolytica: identification of thioredoxin-targeted proteins and analysis of serine acetyltransferase-1 as a prototype example.Biochem. J. 2013; 451: 277-288Crossref PubMed Scopus (26) Google Scholar), and mammals (38.Wu C. Jain M.R. Li Q. Oka S. Li W. Kong A.N. Nagarajan N. Sadoshima J. Simmons W.J. Li H. Identification of novel nuclear targets of human thioredoxin 1.Mol. Cell. Proteomics. 2014; 13: 3507-3518Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). However, to our knowledge, in all studies carried out so far, the Trx mutants were used to capture potential target proteins in cellular extracts, being sometimes immobilized on a resin (18.Hosoya-Matsuda N. Inoue K. Hisabori T. Roles of thioredoxins in the obligate anaerobic green sulfur photosynthetic bacterium Chlorobaculum tepidum.Mol. Plant. 2009; 2: 336-343Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar, 19.Lindahl M. Florencio F.J. Thioredoxin-linked processes in cyanobacteria are as numerous as in chloroplasts, but targets are different.Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 16107-16112Crossref PubMed Scopus (128) Google Scholar, 20.Perez-Perez M.E. Florencio F.J. Lindahl M. Selecting thioredoxins for disulphide proteomics: target proteomes of three thioredoxins from the cyanobacterium Synechocystis sp. PCC 6803.Proteomics. 2006; 6: S186-195Crossref PubMed Google Scholar, 22.Motohashi K. Kondoh A. Stumpp M.T. Hisabori T. Comprehensive survey of proteins targeted by chloroplast thioredoxin.Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 11224-11229Crossref PubMed Scopus (319) Google Scholar, 23.Balmer Y. Koller A. del Val G. Manieri W. Schurmann P. Buchanan B.B. Proteomics gives insight into the regulatory function of chloroplast thioredoxins.Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 370-375Crossref PubMed Scopus (339) Google Scholar, 24.Balmer Y. Vensel W.H. Tanaka C.K. Hurkman W.J. Gelhaye E. Rouhier N. Jacquot J.P. Manieri W. Schurmann P. Droux M. Buchanan B.B. Thioredoxin links redox to the regulation of fundamental processes of plant mitochondria.Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 2642-2647Crossref PubMed Scopus (254) Google Scholar, 25.Yamazaki D. Motohashi K. Kasama T. Hara Y. Hisabori T. Target proteins of the cytosolic thioredoxins in Arabidopsis thaliana.Plant Cell Physiol. 2004; 45: 18-27Crossref PubMed Scopus (167) Google Scholar, 26.Yoshida K. Noguchi K. Motohashi K. Hisabori T. Systematic exploration of thioredoxin target proteins in plant mitochondria.Plant Cell Physiol. 2013; 54: 875-892Crossref PubMed Scopus (75) Google Scholar, 27.Wong J.H. Cai N. Balmer Y. Tanaka C.K. Vensel W.H. Hurkman W.J. Buchanan B.B. Thioredoxin targets of developing wheat seeds identified by complementary proteomic approaches.Phytochemistry. 2004; 65: 1629-1640Crossref PubMed Scopus (134) Google Scholar, 28.Lemaire S.D. Guillon B. Le Marechal P. Keryer E. Miginiac-Maslow M. Decottignies P. New thioredoxin targets in the unicellular photosynthetic eukaryote Chlamydomonas reinhardtii.Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 7475-7480Crossref PubMed Scopus (192) Google Scholar, 29.Marchand C. Le Marechal P. Meyer Y. Decottignies P. Comparative proteomic approaches for the isolation of proteins interacting with thioredoxin.Proteomics. 2006; 6: 6528-6537Crossref PubMed Scopus (86) Google Scholar, 30.Alkhalfioui F. Renard M. Vensel W.H. Wong J. Tanaka C.K. Hurkman W.J. Buchanan B.B. Montrichard F. Thioredoxin-linked proteins are reduced during germination of Medicago truncatula seeds.Plant Physiol. 2007; 144: 1559-1579Crossref PubMed Scopus (120) Google Scholar, 31.Balmer Y. Vensel W.H. Cai N. Manieri W. Schurmann P. Hurkman W.J. Buchanan B.B. A complete ferredoxin/thioredoxin system regulates fundamental processes in amyloplasts.Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 2988-2993Crossref PubMed Scopus (142) Google Scholar, 32.Hall M. Mata-Cabana A. Akerlund H.E. Florencio F.J. Schroder W.P. Lindahl M. Kieselbach T. Thioredoxin targets of the plant chloroplast lumen and their implications for plastid function.Proteomics. 2010; 10: 987-1001Crossref PubMed Scopus (68) Google Scholar, 33.Marchand C.H. Vanacker H. Collin V. Issakidis-Bourguet E. Marechal P.L. Decottignies P. Thioredoxin targets in Arabidopsis roots.Proteomics. 2010; 10: 2418-2428Crossref PubMed Scopus (41) Google Scholar, 34.Rey P. Cuine S. Eymery F. Garin J. Court M. Jacquot J.P. Rouhier N. Broin M. Analysis of the proteins targeted by CDSP32, a plastidic thioredoxin participating in oxidative stress responses.Plant J. 2005; 41: 31-42Crossref PubMed Scopus (120) Google Scholar, 35.Sturm N. Jortzik E. Mailu B.M. Koncarevic S. Deponte M. Forchhammer K. Rahlfs S. Becker K. Identification of proteins targeted by the thioredoxin superfamily in Plasmodium falciparum.PLoS Pathog. 2009; 5: e1000383Crossref PubMed Scopus (62) Google Scholar, 36.Kawazu S. Takemae H. Komaki-Yasuda K. Kano S. Target proteins of the cytosolic thioredoxin in Plasmodium falciparum.Parasitol. Int. 2010; 59: 298-302Crossref PubMed Scopus (14) Google Scholar, 37.Schlosser S. Leitsch D. Duchene M. Entamoeba histolytica: identification of thioredoxin-targeted proteins and analysis of serine acetyltransferase-1 as a prototype example.Biochem. J. 2013; 451: 277-288Crossref PubMed Scopus (26) Google Scholar). Hence, substrate trapping occurred in vitro. As proteins that normally do not get oxidized in vivo may become oxidatively damaged following cell lysis, in vitro trapping is likely to lead to the identification of nonphysiological targets. Moreover, if certain physiological substrates unfold following overoxidation during extract preparation, they may not be recognized by the Trx trapping mutant and will be missed. In this study, we decided to take advantage of E. coli being easily amenable to genetic manipulation and protein expression to comprehensively identify the substrates of Trx1 in vivo. To that end, we expressed a Trx1WCGPA mutant in cells that were genetically engineered to optimize trapping by alteration of the cytoplasmic reducing pathways and that were exposed or not to an exogenous oxidative stress. This led us to the identification of a total of 268 putative substrates of Trx1, including 201 that had never been reported to depend on Trx for reduction and 78 that were found to interact with Trx1 only under severe oxidative stress conditions. Two of the new proteins involved in a disulfide complex with Trx1WCGPA, the bacterial actin homolog MreB and the protein involved in sugar import PtsI, were studied in more detail. Our study significantly expands the number of Trx targets, further highlighting the role played by this major oxidoreductase in a variety of cellular processes. The dependence of a given protein on the Trx system for reduction is usually conserved. For instance, RNR, MsrA, and MsrB are Trx substrates in organisms ranging from bacteria and fungi to plants and mammals (39.Lu J. Holmgren A. The thioredoxin antioxidant system.Free Radic. Biol. Med. 2014; 66: 75-87Crossref PubMed Scopus (811) Google Scholar). Therefore, our work also provides insightful information on the interactome of Trx in organisms other than E. coli. Bacterial strains and plasmids used in this study are listed in supplemental Table S1. Bacterial strains are E. coli MC4100 derivatives (40.Casadaban M.J. Transposition and fusion of the lac genes to selected promoters in Escherichia coli using bacteriophage lambda and Mu.J. Mol. Biol. 1976; 104: 541-555Crossref PubMed Scopus (1265) Google Scholar). All alleles, unless indicated, were moved by P1 transduction using standard procedures (41.Miller J.H. A Short Course in Bacterial Genetics: Laboratory Manual (Cold Spring Harbor Laboratory, ed). Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1992Google Scholar). Strains IA195 and IA198, where ptsI was replaced by ptsIC502S or ptsIC272AC324MC575S on the chromosome, were constructed as follows. First, a cat-sacB cassette, encoding chloramphenicol acetyl transferase (cat) and SacB, a protein conferring sensitivity to sucrose, was amplified from strain CH1990 using primers ptsI-catsacB_Fw and ptsI-catsacB_Rv. The resulting PCR product shared a 40-bp homology to the 5′ UTR and 3′ UTR of ptsI at its 5′ and 3′ ends, respectively, and was used for lambda-red recombineering in strain IA186 as previously described (42.Yu D. Ellis H.M. Lee E.C. Jenkins N.A. Copeland N.G. Court D.L. An efficient recombination system for chromosome engineering in Escherichia coli.Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 5978-5983Crossref PubMed Scopus (1284) Google Scholar). We verified that the cat-sacB cassette replaced the ptsI gene in the resulting strain (IA190) by sequencing across the junctions. The cat-sacB cassette was subsequently replaced by one of the mutated versions of ptsI (ptsIC502S or ptsIC272AC324MC575S) using the same technique. Briefly, ptsIC502S and ptsIC272AC324MC575S were amplified from plasmids pIA4 and pIA5 using primers catsacB-ptsI_Fw and catsacB-ptsIC502S_Rv, or catsacB-ptsI_Fw and catsacB-ptsIC272AC324MC575S_Rv, respectively, and the resulting PCR products were used for a second round of lambda-red recombineering in strain IA192 (IA190 transformed with pSIM5-Tet vector). Loss of the cassette in the resulting IA195 and IA198 strains was verified by positive (sucrose resistance) and negative (chloramphenicol sensitivity) selection and by PCR. Unless indicated, bacteria were grown aerobically at 37 °C in LB medium. When necessary, growth media were supplemented with ampicillin (200 μg/ml), kanamycin (50 μg/ml) or chloramphenicol (10 μg/ml). For testing the ability to ferment glucose, MacConkey medium, supplemented with 0.5% glucose, was used. The plasmids and primers used in the present study are listed in supplemental Tables S1 and S2. The pFB1 expression vector was constructed as follows. The region encoding trxA was amplified from the chromosome using primers Trx1_Fw and Trx1_Rv and inserted into the pHE43 vector restricted with NcoI and BglII (yciMHis was excised from the plasmid and replaced by trxA, allowing Trx1 to be His-tagged at the C terminus). The second cysteine of the WCGPC motif of Trx1 was mutated into an alanine by site-directed mutagenesis of pFB1 using primers Trx1WCGPA_Fw and Trx1WCGPA_Rv, generating plasmid pFB2. The pIA1 expression vector was constructed as follows. The region containing trxC was amplified from the chromosome using primers Trx2_Fw and Trx2_Rv and inserted into the pBAD-HisB vector restricted with NcoI and BglII. The second cysteine of the WCGPC motif of Trx2 was mutated into an alanine by site-directed mutagenesis of pIA1 using primers Trx2WCGPA_Fw and Trx2WCGPA_Rv, generating plasmid pIA2. The pIA3 expression vector was constructed as follows. ptsI was amplified from the chromosome using primers PtsI_Fw and PtsI_Rv and inserted into the pET28a vector. The cysteines of PtsI were mutated into serine, alanine, or methionine by site-directed mutagenesis of pIA3 using primers PtsIC502S_Fw and PtsIC502S_Rv, and primers PtsIC272A_Fw, PtsIC272A_Rv, PtsIC324M_Fw, PtsIC324M_Rv, PtsIC575S_Fw, and PtsIC575S_Rv, generating plasmids pIA4 and pIA5. Strains FB32, IA170 and IA370 were grown in one liter LB medium at 37 °C to reach an A600 of 0.5. Expression of Trx1WCGPAwas induced by addition of 0.2% l-arabinose. After 2 h, the culture was treated, or not, with 2 mm hypochlorous acid (HOCl) for 10 min and then precipitated with 10% trichloroacetic acid (TCA) and placed at 4 °C overnight. Cells were then centrifuged at 11,325 × g for 45 min. 50 ml of cold 5% TCA were added on the pellet and another centrifugation was performed at 17,696 × g for 20 min. The proteins were then resuspended in 25 ml of 100 mm NaPi pH 8.0, 300 mm NaCl, 0.3% SDS, 8 m urea, and 10 mm iodoacetamide (IAM) to prevent any further disulfide bond rearrangement. The lysate was homogenized on a roller during 20 min and centrifuged at 23,708 × g for 45 min. The cleared lysate was finally diluted three times and loaded onto a 1 ml HisTrap FF column (GE Healthcare) equilibrated with 50 mm NaPi pH 8.0, 300 mm NaCl, and 0.3% SDS (buffer A). After washing with buffer A, proteins were eluted with a linear gradient from 0 to 300 mm imidazole in buffer A. Only one peak eluted from the column. This fraction was concentrated to 1.5 ml (using a Vivaspin Turbo 15 device (Sartorius, Goettingen, Germany)) and proteins were resolved on SDS-PAGE under nonreducing conditions (first dimension). The gel lane was cut, incubated in 20 ml of a buffer containing 10% SDS, 0.3 m Tris pH 7.5, 100 mm dithiothreitol (DTT), and 50% glycerol for 1 h, and placed on top of a second SDS-PAGE gel. After electrophoresis, proteins were visualized with PageBlue Protein Staining Solution (Thermo Scientific). Spots of interest were excised from the SDS-PAGE gel, digested with trypsin, and analyzed by liquid chromatographic tandem mass spectrometry (LC-MS/MS) using an LTQ XL as described (43.Arts I.S. Ball G. Leverrier P. Garvis S. Nicolaes V. Vertommen D. Ize B. Tamu Dufe V. Messens J. Voulhoux R. Collet J.F. Dissecting the machinery that introduces disulfide bonds in Pseudomonas aeruginosa.mBio. 2013; 4: e00912-00913Crossref PubMed Scopus (35) Google Scholar). Briefly, peptides were separated by an acetonitrile gradient on a C18 column and the MS scan routine was set to analyze by MS/MS the five most intense ions of
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