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Title: $Copper Stress Causes an in Vivo Requirement for the Escherichia coli Disulfide Isomerase DsbC
Abstract: In Escherichia coli, the periplasmic disulfide oxidoreductase DsbA is thought to be a powerful but nonspecific oxidant, joining cysteines together the moment they enter the periplasm. DsbC, the primary disulfide isomerase, likely resolves incorrect disulfides. Given the reliance of protein function on correct disulfide bonds, it is surprising that no phenotype has been established for null mutations in dsbC. Here we demonstrate that mutations in the entire DsbC disulfide isomerization pathway cause an increased sensitivity to the redox-active metal copper. We find that copper catalyzes periplasmic disulfide bond formation under aerobic conditions and that copper catalyzes the formation of disulfide-bonded oligomers in vitro, which DsbC can resolve. Our data suggest that the copper sensitivity of dsbC– strains arises from the inability of the cell to rearrange copper-catalyzed non-native disulfides in the absence of functional DsbC. Absence of functional DsbA augments the deleterious effects of copper on a dsbC– strain, even though the dsbA– single mutant is unaffected by copper. This may indicate that DsbA successfully competes with copper and forms disulfide bonds more accurately than copper does. These findings lead us to a model in which DsbA may be significantly more accurate in disulfide oxidation than previously thought, and in which the primary role of DsbC may be to rearrange incorrect disulfide bonds that are formed during certain oxidative stresses. In Escherichia coli, the periplasmic disulfide oxidoreductase DsbA is thought to be a powerful but nonspecific oxidant, joining cysteines together the moment they enter the periplasm. DsbC, the primary disulfide isomerase, likely resolves incorrect disulfides. Given the reliance of protein function on correct disulfide bonds, it is surprising that no phenotype has been established for null mutations in dsbC. Here we demonstrate that mutations in the entire DsbC disulfide isomerization pathway cause an increased sensitivity to the redox-active metal copper. We find that copper catalyzes periplasmic disulfide bond formation under aerobic conditions and that copper catalyzes the formation of disulfide-bonded oligomers in vitro, which DsbC can resolve. Our data suggest that the copper sensitivity of dsbC– strains arises from the inability of the cell to rearrange copper-catalyzed non-native disulfides in the absence of functional DsbC. Absence of functional DsbA augments the deleterious effects of copper on a dsbC– strain, even though the dsbA– single mutant is unaffected by copper. This may indicate that DsbA successfully competes with copper and forms disulfide bonds more accurately than copper does. These findings lead us to a model in which DsbA may be significantly more accurate in disulfide oxidation than previously thought, and in which the primary role of DsbC may be to rearrange incorrect disulfide bonds that are formed during certain oxidative stresses. Most periplasmic Escherichia coli proteins contain at least two cysteine residues and many are stable and active only when these cysteines form their native disulfide bond pairings (1Hiniker A. Bardwell J.C. J. Biol. Chem. 2004; 279: 12967-12973Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). In E. coli, a family of thiol-disulfide oxidoreductases ensures that periplasmic and secreted proteins form correct disulfide bonds. DsbA is the primary disulfide oxidant in the periplasm. It rapidly donates its disulfide directly to substrate proteins and oxidizes them (2Bardwell J.C. McGovern K. Beckwith J. Cell. 1991; 67: 581-589Abstract Full Text PDF PubMed Scopus (846) Google Scholar). DsbA is believed to act as a relatively nonspecific oxidant, joining any two cysteines that approach each other (3Berkmen M. Boyd D. Beckwith J. J. Biol. Chem. 2005; PubMed Google Scholar). A dsbA– strain shows several in vivo phenotypes, including attenuated virulence and loss of motility, because of the absence of disulfide bonds in proteins involved in these pathways (4Missiakas D. Georgopoulos C. Raina S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7084-7088Crossref PubMed Scopus (206) Google Scholar, 5Watarai M. Tobe T. Yoshikawa M. Sasakawa C. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4927-4931Crossref PubMed Scopus (100) Google Scholar). DsbC, a second periplasmic thiol-disulfide oxidoreductase, appears to function as a disulfide isomerase both in vitro and in vivo. In vitro, DsbC has been shown to rearrange non-native disulfides in well studied isomerization substrates such as BPTI and RNase A (6Zapun A. Missiakas D. Raina S. Creighton T.E. Biochemistry. 1995; 34: 5075-5089Crossref PubMed Scopus (222) Google Scholar, 7Bader M.W. Xie T. Yu C.A. Bardwell J.C. J. Biol. Chem. 2000; 275: 26082-26088Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). In vivo, DsbC is required for full activity of a handful of proteins containing at least one non-consecutive disulfide bond (1Hiniker A. Bardwell J.C. J. Biol. Chem. 2004; 279: 12967-12973Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). We have found that the periplasmic proteins RNase I (four disulfides, one non-consecutive) and MepA (three disulfides, two non-consecutive) require DsbC for their stability and, in the case of RNase I, in vivo activity (1Hiniker A. Bardwell J.C. J. Biol. Chem. 2004; 279: 12967-12973Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Berkmen et al. (3Berkmen M. Boyd D. Beckwith J. J. Biol. Chem. 2005; PubMed Google Scholar) recently showed that the folding of Agp (three consecutive disulfides) becomes DsbC-dependent with the introduction of a non-consecutive disulfide bond. These results suggest that the principal role of DsbC under non-stress conditions is to rearrange disulfide bonds that DsbA forms incorrectly. A key question remaining in disulfide biology is the relative importance of disulfide oxidation and disulfide isomerization during in vivo protein folding. The disulfide oxidation pathway in the E. coli periplasm is well characterized, with about 25 proteins identified that require DsbA for correct folding and functioning (2Bardwell J.C. McGovern K. Beckwith J. Cell. 1991; 67: 581-589Abstract Full Text PDF PubMed Scopus (846) Google Scholar, 8Kadokura H. Tian H. Zander T. Bardwell J.C. Beckwith J. Science. 2004; 303: 534-537Crossref PubMed Scopus (189) Google Scholar). In contrast, only three in vivo substrates have been found for DsbC, and none have been found for the second periplasmic disulfide isomerase DsbG (1Hiniker A. Bardwell J.C. J. Biol. Chem. 2004; 279: 12967-12973Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 3Berkmen M. Boyd D. Beckwith J. J. Biol. Chem. 2005; PubMed Google Scholar). Whereas a dsbA– strain shows pleiotropic phenotypes, no consistent phenotype has yet been found for a dsbC– or dsbG– strain. This suggests that disulfide isomerization may be less important under non-stress conditions than previously believed. Here we show that mutants lacking any part of the DsbC disulfide isomerization pathway are less viable than isogenic wild-type strains under oxidative copper stress conditions. We show that copper, a redox-active metal, catalyzes the formation of disulfide bonds in vivo, and appears to introduce incorrect disulfides more frequently than DsbA does. Our data suggest that the copper sensitivity of dsbC– strains arises from the inability of the cell to rearrange copper-catalyzed non-native disulfide bonds in the absence of functional DsbC. Intriguingly, this may indicate a role for DsbC in combating periplasmic oxidative stress. Bacterial Strains and Growth Conditions—The bacterial strains used in this study are listed in TABLE ONE. For copper sensitivity assays, bacteria were grown in Brain Heart Infusion (BHI) 3The abbreviations used are: BHI, brain heart infusion; DTT, dithiothreitol; X-gal, 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside; drRNase A, denatured and reduced RNase A. media (Difco). For motility assays, malF-lacZ assays, and the alkaline phosphatase assay, an M63 minimal media was used (13.6 g of KH2PO4/2 g of (NH4)2SO4/0.5 mg FeSO4·7H2O, pH 7.0) with 0.4% glucose, 0.1% casamino acids, 1 mm MgSO4, 2 μg/ml thiamine, 2 μg/ml biotin, 2 μg/ml nicotinamide, and 0.2 μg/ml riboflavin. Media was supplemented with 100 μg/ml X-gal and/or CuCl2, AgNO3, MnCl2, ZnCl2, NiSO4, CoCl2, or iron(III) citrate to a final concentration between 0.05 mm and 30 mm as required for the assays. Strains were grown at 34 °C rather than 37 °C as we found that the copper phenotype was more pronounced at this temperature. The BL21 dsbC– strain AH131 was created by P1 transduction of dsbC::kan moved out of SR3324 (MC4100 dsbC::kan obtained from George Georgiou) into wild-type BL21. AH358 (AH50 ΔdsbC::cm dsbA::kan1) was created by P1 transduction of dsbA::kan1 out of JCB571 (Bardwell laboratory strain) into AH65 (AH50 ΔdsbC::cm). Anaerobic growth was performed in an anaerobic chamber using BD BBL GasPak Anaerobic System Envelope and BBL GasPak Anaerobic System Indicator.TABLE ONEStrains used in this studyStrainRelevant genotypeSourceAH50MC1000 phoR Δara714 leu+ phoA68Ref. 9Rietsch A. Belin D. Martin N. Beckwith J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13048-13053Crossref PubMed Scopus (244) Google ScholarAH55AH50 dsbA::kan1Ref. 9Rietsch A. Belin D. Martin N. Beckwith J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13048-13053Crossref PubMed Scopus (244) Google ScholarAH65AH50 ΔdsbC::cmRef. 9Rietsch A. Belin D. Martin N. Beckwith J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13048-13053Crossref PubMed Scopus (244) Google ScholarAH66AH50 dsbG::kanRef. 18Bessette P.H. Cotto J.J. Gilbert H.F. Georgiou G. J. Biol. Chem. 1999; 274: 7784-7792Abstract Full Text Full Text PDF PubMed Scopus (139) Google ScholarAH358AH50 ΔdsbC::cm dsbA::kan1This studyAH392AH50 dsbD::cmRef. 9Rietsch A. Belin D. Martin N. Beckwith J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13048-13053Crossref PubMed Scopus (244) Google ScholarAH396AH50 dsbD::cm, dsbA::kan1Ref. 15Rietsch A. Bessette P. Georgiou G. Beckwith J. J. Bacteriol. 1997; 179: 6602-6608Crossref PubMed Scopus (196) Google ScholarJCB169MC4100Lab strainAH51MC4100 dsbC::kanThis studyBL21F- ompT hsdSB(rB-mB-) gal dcm lonNovagenAH131BL21 dsbC::kanRef. 1Hiniker A. Bardwell J.C. J. Biol. Chem. 2004; 279: 12967-12973Abstract Full Text Full Text PDF PubMed Scopus (138) Google ScholarJCB816MC1000 phoR λ102Ref. 2Bardwell J.C. McGovern K. Beckwith J. Cell. 1991; 67: 581-589Abstract Full Text PDF PubMed Scopus (846) Google ScholarJCB817JCB 816 dsbA::kan1Ref. 2Bardwell J.C. McGovern K. Beckwith J. Cell. 1991; 67: 581-589Abstract Full Text PDF PubMed Scopus (846) Google ScholarJCB818JCB 816 dsbA::kan1 dsbB::kanRef. 2Bardwell J.C. McGovern K. Beckwith J. Cell. 1991; 67: 581-589Abstract Full Text PDF PubMed Scopus (846) Google Scholar Open table in a new tab Alkaline Phosphatase Assay—Alkaline phosphatase assays were performed as previously described (9Rietsch A. Belin D. Martin N. Beckwith J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13048-13053Crossref PubMed Scopus (244) Google Scholar). The values shown represent the average of at least three separate experiments. In Vitro Copper Oxidation—DsbC was purified as described previously using nickel chromatography via a His6 tag (10Bader M.W. Hiniker A. Regeimbal J. Goldstone D. Haebel P.W. Riemer J. Metcalf P. Bardwell J.C. EMBO J. 2001; 20: 1555-1562Crossref PubMed Scopus (92) Google Scholar). It was reduced for 10 min on ice with 10 mm dithiothreitol (DTT), and the DTT was removed by buffer exchange into 25 mm Hepes pH 7.5 using a Nap 5 column (Amersham Biosciences). Bovine pancreatic RNase A was purchased from Sigma, reduced and denatured in 7 m guanidinium chloride, 40 mm Hepes (pH 7.5), 120 mm dithiothreitol, and 0.2 mm EDTA at 25 °C for 2 h, and buffer-exchanged into 0.1% acetic acid. Denatured and reduced RNase A (drRNase A) was quantified using ϵ275.5 = 9,300 m–1 cm–1, whereas native RNase A was quantified using ϵ275.5 = 9,800 m–1 cm–1 (Swiss Protein Database). The thiol content of reduced DsbC and drRNase A was measured with 5,5′-dithiobis(2-nitrobenzoic acid) as described, and both were found to be >90% reduced (11Riddles P.W. Blakeley R.L. Zerner B. Methods Enzymol. 1983; 91: 49-60Crossref PubMed Scopus (1090) Google Scholar). Proteins were flash-frozen and stored at ≤ –20 °C until use. For oxidation by copper/H2O2, native RNase A or drRNase A was diluted into 300 mm NaCl, 50 mm sodium phosphate, pH 6 to a final concentration of 50 μm in the presence of 50 μm CuCl2, 2 mm H2O2, 50 μm CuCl2/2 mm H2O2, or buffer alone and incubated at 25 °C for 30 min. CuCl2/H2O2 was removed and buffer was exchanged by gel filtration to 300 mm NaCl, 50 mm sodium phosphate, pH 7.5. The protein concentration after gel filtration was measured by a Bradford assay (12Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (222164) Google Scholar); the thiol oxidation status was measured with 5,5′-dithiobis(2-nitrobenzoic acid). Samples were diluted to 20 μm, incubated with or without 20 μm reduced DsbC for 2 h at 25°C, and run under reducing and non-reducing conditions on a 14% Tris-glycine gel (Invitrogen). To test the ability of DsbC to restore activity to copper-oxidized RNase A, 200-μl aliquots of the above samples were added to 4 mm cCMP (final concentration) in 400 μl (final volume) of 300 mm NaCl, 50 mm sodium phosphate, pH 7.5. The hydrolysis of cCMP was followed at 296 nm for 300 s and the initial hydrolysis rate for the first 30 s of the measurement was recorded. The initial hydrolysis rate of an equimolar amount of native RNase A was set to 100% activity and all other samples expressed as percent native RNase A activity. The DsbC Disulfide Isomerization Pathway Is Involved in Copper Resistance—Given the importance of proper protein folding to the well being of the cell, it is surprising that no clear phenotype has yet been found for null mutations in the E. coli principal disulfide isomerase, DsbC. Missiakas et al. (13Missiakas D. Georgopoulos C. Raina S. EMBO J. 1994; 13: 2013-2020Crossref PubMed Scopus (197) Google Scholar, 14Missiakas D. Schwager F. Raina S. EMBO J. 1995; 14: 3415-3424Crossref PubMed Scopus (171) Google Scholar) reported that dsbC– strains are benzylpenicillin and DTT-sensitive. However, we and others have been unable to repeat the DTT sensitivity (data not shown). 4G. Georgiou, personal communication. We can reproduce dsbC– benzylpenicillin sensitivity but only in certain strain backgrounds (data not shown). Previous work by Rietsch et al. (9Rietsch A. Belin D. Martin N. Beckwith J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13048-13053Crossref PubMed Scopus (244) Google Scholar) demonstrated that dsbD– strains are sensitive to the redox-active metal copper and that thioredoxin mutants are also sensitive to copper, but less so than a dsbD– strain. It is known that thioredoxin passes electrons to DsbD, maintaining DsbD in reduced and active form (15Rietsch A. Bessette P. Georgiou G. Beckwith J. J. Bacteriol. 1997; 179: 6602-6608Crossref PubMed Scopus (196) Google Scholar). DsbD then passes electrons to a number of periplasmic proteins, including DsbC (16Porat A. Cho S.H. Beckwith J. Res. Microbiol. 2004; 155: 617-622Crossref PubMed Scopus (39) Google Scholar). Thus, it seemed possible that the entire disulfide isomerization pathway might be involved in copper resistance and that dsbC– strains would also be copper-sensitive, despite previous reports that did not observe dsbC– copper sensitivity (13Missiakas D. Georgopoulos C. Raina S. EMBO J. 1994; 13: 2013-2020Crossref PubMed Scopus (197) Google Scholar). To test this, we compared the ability of wild-type strains and strains lacking individual dsb genes to grow on various concentrations of CuCl2 (strains listed in TABLE ONE). In agreement with Rietsch et al. (9Rietsch A. Belin D. Martin N. Beckwith J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13048-13053Crossref PubMed Scopus (244) Google Scholar), we found that a mutation in dsbD causes increased sensitivity to copper (TABLE TWO). Whereas the MC1000-derived wild-type strain AH50 grew on BHI-copper plates at 11 mm CuCl2, the isogenic dsbD– strain, AH392, was unable to form single colonies at 8 mm CuCl2. Importantly, we found that dsbC– strains were also copper-sensitive, exhibiting the same copper-sensitive phenotype as the dsbD– strain. To ensure that our result was not dependent on strain background, we compared the copper sensitivity of wild-type and dsbC– MC4100 and BL21 strains and found that the dsbC– mutant was copper-sensitive in all three strain backgrounds tested (data not shown). We also reintroduced wild-type DsbC expressed from the pBAD33 plasmid into the dsbC– strain, and found that DsbC expressed from this plasmid restored copper resistance to wild-type levels. We note that our strongest phenotype is found when the strains are grown on brain heart infusion (BHI) agar, a very rich media. The experiments of Metheringham et al. (17Metheringham R. Tyson K.L. Crooke H. Missiakas D. Raina S. Cole J.A. Mol. Gen. Genet. 1996; 253: 95-102PubMed Google Scholar) that failed to show dsbC– copper sensitivity were done on LB agar, where we see a weaker, but still clear, phenotype.TABLE TWOCopper sensitivities of null mutations in the E. coli periplasmic disulfide oxidoreductasesStrainGenotype8 mm CuCl24 mm CuCl2AH50Wild-type++++aViability relative to wild type, ranging from wild-type-sized single colonies (++++) to not viable (-).++++aViability relative to wild type, ranging from wild-type-sized single colonies (++++) to not viable (-).AH55dsbA-++++++++AH392dsbD--++++AH65dsbC--++++AH66dsbG-++++++++AH396dsbA- dsbD---AH358dsbA- dsbC---a Viability relative to wild type, ranging from wild-type-sized single colonies (++++) to not viable (-). Open table in a new tab DsbG is a second putative periplasmic disulfide isomerase that is 30% identical to DsbC at the amino acid level (18Bessette P.H. Cotto J.J. Gilbert H.F. Georgiou G. J. Biol. Chem. 1999; 274: 7784-7792Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). We compared the copper sensitivity of dsbG– and wild-type strains and found no appreciable difference between the wild-type and dsbG– cells, both in MC1000 and in BL21. At 11 mm CuCl2, dsbG– colonies were slightly smaller than wild-type colonies; however, the effect was much less dramatic than for dsbC– strains, which failed to grow at all under these conditions. When wild-type DsbG was overexpressed from the pBAD33 plasmid in the dsbC– strain, DsbG could only partially rescue dsbC– copper sensitivity. We conclude that of the two disulfide isomerases, DsbC is chiefly responsible for maintaining wild-type levels of copper resistance in vivo, but that DsbG may play a small role as well. To determine whether a dsbC– strain shows increased sensitivity to other metals, the relative growth of isogenic dsbC– and wild-type strains were tested on BHI plates supplemented with silver(I) nitrate, manganese(II) chloride, iron(III) citrate, zinc(II) chloride, nickel(II) sulfate, or cobalt(II) chloride. All metals were tested in increments of 1 mm or less until the lethal concentration of the metal in the wild-type strain was reached. AH65 (dsbC–) showed no increased sensitivity to any of these metals relative to AH50, the isogenic wild type. Additionally, under anaerobic conditions, wild-type and dsbC– strains were equally resistant to copper treatment. Copper Catalyzes Disulfide Bond Formation in Vivo—Two mechanisms can explain the requirement for the DsbC disulfide isomerization pathway under conditions of copper stress. One possibility is that there exists at least one DsbC substrate protein that becomes essential under copper stress conditions but that is not essential during normal growth. In this case, the substrate would always require disulfide isomerization by DsbC for its correct folding but would only become essential in the presence of copper. Copper is a toxic metal, so periplasmic or membrane proteins involved in copper homeostasis are appropriate candidates. To be consistent with our observations that dsbC– strains are not copper-sensitive when grown anaerobically and are not sensitive to other metals tested, the substrate should be important to copper resistance under aerobic but not anaerobic conditions and should not be involved in resistance to any of the other metals tested. To require DsbC for folding, the substrate should have three or more cysteine residues and need disulfide isomerization to achieve its native structure. A number of proteins are involved in copper resistance (see Rensing and Grass for a review, Ref. 19Rensing C. Grass G. FEMS Microbiol. Rev. 2003; 27: 197-213Crossref PubMed Scopus (568) Google Scholar), but we have not found any that meet these criteria. Many copper resistance proteins are important both aerobically and anaerobically (such as the P-type ATPase CopA), and others are involved in resistance to multiple metals (the Cus proteins are important for silver as well as copper resistance). The multi-copper oxidase CueO functions in copper resistance only under aerobic conditions and contains three cysteine residues. However, the crystal structure of this protein has been solved, and reveals no disulfide bonds (20Roberts S.A. Weichsel A. Grass G. Thakali K. Hazzard J.T. Tollin G. Rensing C. Montfort W.R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 2766-2771Crossref PubMed Scopus (289) Google Scholar). While it is possible that DsbC does act on a copper homeostasis protein, we have been unable to find a likely candidate among the known copper resistance proteins. A second possibility is that copper stress may lead to incorrect disulfide bonds in proteins that do not normally require disulfide isomerization. In this case, copper stress might cause DsbC to become essential because copper is catalyzing the formation of non-native disulfide bonds. It has recently been shown that copper can form in vitro disulfides in protein substrates (21Kachur A.V. Koch C.J. Biaglow J.E. Free Radic Res. 1999; 31: 23-34Crossref PubMed Scopus (98) Google Scholar, 22Matsui Lee I.S. Suzuki M. Hayashi N. Hu J. Van Eldik L.J. Titani K. Nishikimi M. Arch. Biochem. Biophys. 2000; 374: 137-141Crossref PubMed Scopus (25) Google Scholar). To examine whether copper can catalyze disulfide bonds in vivo, we looked at the ability of copper to complement a dsbA– phenotype. A dsbA– strain exhibits a number of phenotypes caused by a generalized loss of disulfide bond formation. On minimal media, a dsbA– strain is unable to form the single disulfide in the flagellar motor protein FlgI that is required for FlgI folding. Therefore, dsbA– strains are not motile while wild-type strains are motile (23Dailey F.E. Berg H.C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1043-1047Crossref PubMed Scopus (212) Google Scholar). We tested the ability of copper to restore motility to a dsbA– strain. In the absence of copper, only the dsbA+ strain JCB816 exhibited motility, while JCB817, the dsbA– strain, was completely non-motile (TABLE THREE). In the presence of 0.2 mm copper, however, the dsbA– strain became nearly as motile as wild type, showing that copper was able to form the single disulfide in FlgI and restore motility.TABLE THREECopper complements mutations in the DsbA-DsbB pathwayStrainGenotypePhenotype on minimal mediaPhenotype on minimal media + CuCl2MotilityMalF-βgalMotilityMalF-βgalJCB816Wild-type+ + + +aMotility relative to wild type, ranging from wild-type levels of motility (+ + + +) to non-motile (—).White+ + + +aMotility relative to wild type, ranging from wild-type levels of motility (+ + + +) to non-motile (—).WhiteJCB817dsbA——Blue+ + +WhiteJCB818dsbA— dsbB——Blue+ + +Whitea Motility relative to wild type, ranging from wild-type levels of motility (+ + + +) to non-motile (—). Open table in a new tab The ability of copper to introduce disulfides in FlgI is possibly specific to this protein and may not apply to other periplasmic or membrane proteins. To determine whether copper introduces disulfides more widely, we looked at disulfide formation in a disulfide detector protein, a malF-lacZ fusion construct (23Dailey F.E. Berg H.C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1043-1047Crossref PubMed Scopus (212) Google Scholar). This disulfide detector consists of β-galactosidase, normally a cytoplasmic protein with four thiol groups, fused to the inner membrane protein MalF (24Silhavy T.J. Casadaban M.J. Shuman H.A. Beckwith J.R. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 3423-3427Crossref PubMed Scopus (56) Google Scholar). In the presence of functional DsbA, it is believed that non-native disulfide bonds are formed in β-galactosidase, causing it to be inactive (25Tian H. Boyd D. Beckwith J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4730-4735Crossref PubMed Scopus (70) Google Scholar). In the absence of a periplasmic disulfide oxidant, β-galactosidase retains its reduced thiol groups and can fold to its active form (25Tian H. Boyd D. Beckwith J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4730-4735Crossref PubMed Scopus (70) Google Scholar). Consistent with previous work, JCB816, a wild-type strain expressing the MalF-LacZ fusion protein, was white on X-gal, indicating that β-galactosidase was inactive in this strain and thus contained non-native disulfides (TABLE THREE). In contrast, JCB817, a dsbA– strain isogenic with JCB816, was blue on X-gal, illustrating that disulfide bonds could not form. In the presence of 0.1 mm CuCl2, the dsbA– strain JCB817 became white on X-gal, suggesting that copper was catalyzing disulfide bond formation in the MalF-LacZ protein. To examine whether copper rescue of disulfide bond formation depends on DsbB, the native reoxidant of DsbA, we repeated the motility and MalF-LacZ assays using a dsbA– dsbB– double mutant, JCB818. JCB818 was non-motile on minimal motility medium but also nearly as motile as wild type in the presence of 0.2 mm copper (TABLE THREE). Similarly, JCB818 was blue on X-gal alone and white on X-gal-0.1 mm CuCl2 plates. These findings indicate that copper can bypass the entire DsbA-DsbB pathway and form de novo disulfides in periplasmic proteins. Disulfide Bond Formation and DsbC– Sensitivity Are Unique to Aerobic Copper Stress—The ability of copper to complement a dsbA–dsbB– strain demonstrates that copper forms de novo disulfide bonds in vivo. If copper-catalyzed disulfide formation causes dsbC– copper sensitivity, then metals that do not cause dsbC– sensitivity should not catalyze disulfide bond formation. We therefore tested the metals that did not cause dsbC– sensitivity for their ability to form disulfide bonds by measuring their ability to both inhibit MalF-LacZ activity and restore motility in a dsbA– strain. Silver(I) nitrate, manganese(II) chloride, iron(III) citrate, zinc(II) chloride, nickel(II) sulfate, and cobalt(II) chloride were each examined at a range of concentrations from 0 mm to the lethal concentration for that metal. Addition of silver(I) nitrate, manganese(II) chloride, or iron(III) citrate did not inhibit β-galactosidase activity at any concentration tested. Although zinc(II) chloride, nickel(II) sulfate, and cobalt(II) chloride were able to inhibit β-galactosidase activity in the MalF-LacZ assay, none of the metals could restore motility in the dsbA– strain at any concentration tested. Furthermore, the wild-type strain remained motile in the presence of these metals. Of the metals tested, only copper could both abolish MalF-LacZ activity and rescue motility in a dsbA– strain. This indicates that in vivo disulfide bond formation is not a universal property of metal cations. We postulated that copper complementation of a dsbA– strain could be due to the specific redox properties of copper. The conversion of Cu2+ to Cu1+ has a redox potential of 0.153 V, likely allowing Cu2+ to oxidize free thiol groups and then become re-oxidized by oxygen species. Most other metals have properties that do not allow them to redox cycle: they either have redox potentials that do not allow re-oxidation by oxygen species, do not have multiple redox states, or are largely insoluble under physiologic conditions. Redox cycling of copper likely requires the presence of molecular oxygen species in order to re-oxidize copper after it has oxidized thiol groups. We tested the ability of copper to restore disulfide bond formation in the absence of oxygen species by testing motility under anaerobic conditions. Under anaerobic conditions, copper could not restore motility to a dsbA– strain at any copper concentration tested, supporting the idea that in vivo disulfide bond formation by copper relies on the presence of molecular oxygen. This was in good agreement with our finding that, under anaerobic conditions, wild-type and dsbC– strains were equally resistant to copper treatment. Copper Forms Non-native Disulfide Bonds in Vivo—Our motility and MalF-LacZ assays indicate that copper catalyzes periplasmic disulfide bond formation. To address whether copper forms a high proportion of non-native disulfide bonds in the cell, we examined the effect of copper on a strain with an increased number of periplasmic proteins harboring exposed thiol groups. If copper forms incorrect disulfide bonds between any free thiol groups, this strain might be especially sensitive to the effects of copper in the absence of a disulfide isomerase such as DsbC. A dsbA– strain exactly fits these criteria because it lacks DsbA and therefore acquires disulfides more slowly than a wild-type strain (2Bardwell J.C. McGovern K. Beckwith J. Cell. 1991; 67: 581-589Abstract Full T