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• W2068861782 abstract "In Escherichia coli, two pathways use NADPH to reduce disulfide bonds that form in some cytoplasmic enzymes during catalysis: the thioredoxin system, which consists of thioredoxin reductase and thioredoxin, and the glutaredoxin system, composed of glutathione reductase, glutathione, and three glutaredoxins. These systems may also reduce disulfide bonds which form spontaneously in cytoplasmic proteins when E. coli is grown aerobically. We have investigated the role of both systems in determining the thiol-disulfide balance in the cytoplasm by determining the ability of protein disulfide bonds to form in mutants missing components of these systems. We find that both the thioredoxin and glutaredoxin systems contribute to reducing disulfide bonds in cytoplasmic proteins. In addition, these systems can partially substitute for each otherin vivo since double mutants missing parts of both systems generally allow substantially more disulfide bond formation than mutants missing components of just one system. Some of these double mutants were found to require the addition of a disulfide reductant to the medium to grow well aerobically. Thus, E. coli requires either a functional thioredoxin or glutaredoxin system to reduce disulfide bonds which appear after each catalytic cycle in the essential enzyme ribonucleotide reductase and perhaps to reduce non-native disulfide bonds in cytoplasmic proteins. Our results suggest the existence of a novel thioredoxin in E. coli. In Escherichia coli, two pathways use NADPH to reduce disulfide bonds that form in some cytoplasmic enzymes during catalysis: the thioredoxin system, which consists of thioredoxin reductase and thioredoxin, and the glutaredoxin system, composed of glutathione reductase, glutathione, and three glutaredoxins. These systems may also reduce disulfide bonds which form spontaneously in cytoplasmic proteins when E. coli is grown aerobically. We have investigated the role of both systems in determining the thiol-disulfide balance in the cytoplasm by determining the ability of protein disulfide bonds to form in mutants missing components of these systems. We find that both the thioredoxin and glutaredoxin systems contribute to reducing disulfide bonds in cytoplasmic proteins. In addition, these systems can partially substitute for each otherin vivo since double mutants missing parts of both systems generally allow substantially more disulfide bond formation than mutants missing components of just one system. Some of these double mutants were found to require the addition of a disulfide reductant to the medium to grow well aerobically. Thus, E. coli requires either a functional thioredoxin or glutaredoxin system to reduce disulfide bonds which appear after each catalytic cycle in the essential enzyme ribonucleotide reductase and perhaps to reduce non-native disulfide bonds in cytoplasmic proteins. Our results suggest the existence of a novel thioredoxin in E. coli. Disulfide bridges play an important structural role in many proteins (1Doig A.J. Williams D.H. J. Mol. Biol. 1991; 217: 389-398Crossref PubMed Scopus (166) Google Scholar, 2Creighton T.E. Protein Folding. W. H. Freeman and Co., New York1992Google Scholar). While these bonds often occur in extracytoplasmic proteins, they are rarely found in cytoplasmic proteins (3Schultz G.E. Schirmer R.H. Principles of Protein Structure. Springer-Verlag, New York1979Crossref Google Scholar, 4Thornton J.M. J. Mol. Biol. 1981; 151: 261-287Crossref PubMed Scopus (677) Google Scholar). It has been suggested that the cytoplasm is too reducing for many disulfide bonds to form (5Ziegler D.M. Poulsen L.L. Trends Biochem. Sci. 1977; 2: 79-81Abstract Full Text PDF Scopus (80) Google Scholar, 6Gilbert H.F. Adv. Enzymol. Relat. Areas Mol. Biol. 1990; 63: 69-172PubMed Google Scholar). In fact, when many exported proteins that ordinarily form disulfide bonds are expressed in the cytoplasm, they do not form these bonds (7Pollitt S. Zalkin H. J. Bacteriol. 1983; 153: 27-32Crossref PubMed Google Scholar, 8Derman A.I. Beckwith J. J. Bacteriol. 1991; 173: 7719-7723Crossref PubMed Scopus (163) Google Scholar) (for an exception, see Ref. 9Nilsson B. Berman-Marks C. Kuntz I.D. Anderson S. J. Biol. Chem. 1991; 266: 2970-2977Abstract Full Text PDF PubMed Google Scholar). A number of factors are thought to determine the thiol-disulfide balance in the cytoplasm of Escherichia coli. The principle thiol-disulfide redox buffer in the cytoplasm is constituted by the cysteine containing tripeptide glutathione. E. coli contains high levels of glutathione (the intracellular concentration is approximately 5 mm) that is kept almost entirely reduced (10Kosower N.S. Kosower E.M. Int. Rev. Cytol. 1978; 54: 109-160Crossref PubMed Scopus (1080) Google Scholar). The ratio of reduced to oxidized glutathione in the E. coli cytoplasm is roughly 50:1 to 200:1 (11Hwang C. Sinsky A.J. Lodish H.F. Science. 1992; 257: 1496-1502Crossref PubMed Scopus (1608) Google Scholar). In vitro, similar levels of oxidized and reduced glutathione are not conducive to disulfide bond formation in many proteins (11Hwang C. Sinsky A.J. Lodish H.F. Science. 1992; 257: 1496-1502Crossref PubMed Scopus (1608) Google Scholar, 12Saxena V.P. Wetlaufer D.B. Biochemistry. 1970; 9: 5015-5023Crossref PubMed Scopus (363) Google Scholar, 13Lyles M.M. Gilbert H.F. Biochemistry. 1991; 30: 613-619Crossref PubMed Scopus (354) Google Scholar, 14Walker K.W. Gilbert H.F. J. Biol. Chem. 1994; 269: 28487-28493Abstract Full Text PDF PubMed Google Scholar). In addition to glutathione, the E. coli cytoplasm contains at least four thiol-disulfide oxidoreductases that may help reduce protein disulfide bonds in the cytoplasm via their redox active disulfides: thioredoxin, glutaredoxin 1, glutaredoxin 2, and glutaredoxin 3 (15Holmgren A. Annu. Rev. Biochem. 1985; 54: 237-271Crossref PubMed Google Scholar, 16Holmgren A. J. Biol. Chem. 1989; 264: 13963-13966Abstract Full Text PDF PubMed Google Scholar, 17Åslund F. Ehn B. Miranda-Vizuete A. Pueyo C. Holmgren A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9813-9817Crossref PubMed Scopus (164) Google Scholar). Thioredoxin is the best characterized of these and belongs to a superfamily of proteins that contain an active site CX1X2C motif and share a similar fold, in those cases where structure is known (18Martin J.L. Structure. 1995; 3: 245-250Abstract Full Text Full Text PDF PubMed Scopus (694) Google Scholar). The redox potential of thioredoxin is low (−270 mV) (19Krause G. Lundström J. Barea J.L. Pueyo de la Cuesta C. Holmgren A. J. Biol. Chem. 1991; 266: 9494-9500Abstract Full Text PDF PubMed Google Scholar) and, in vitro, thioredoxin efficiently reduces disulfide bonds in a wide variety of proteins (15Holmgren A. Annu. Rev. Biochem. 1985; 54: 237-271Crossref PubMed Google Scholar,20Holmgren A. Methods Enzymol. 1984; 107: 295-300Crossref PubMed Scopus (118) Google Scholar). Although they have not been as extensively tested as thioredoxin, the glutaredoxins are generally less efficient reductants of disulfide bonds than thioredoxin (16Holmgren A. J. Biol. Chem. 1989; 264: 13963-13966Abstract Full Text PDF PubMed Google Scholar, 21Åslund F. Nordstrand K. Berndt K.D. Nikkola M. Bergman T. Ponstingl H. Jörnvall H. Otting G. Holmgren A. J. Biol. Chem. 1996; 271: 6736-6745Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). This may be partially explained by the higher redox potentials of the glutaredoxins (glutaredoxin 1 = −233 mV; glutaredoxin 3 = −198 mV). 1F. Åslund, K. D. Berndt, and A. Holmgren, submitted for publication. Upon reducing disulfide bonds, the thiol-disulfide oxidoreductases become oxidized. In order for these proteins to be functional, they in turn must be kept reduced. The flavoenzyme thioredoxin reductase uses NADPH to reduce thioredoxin but is unable to reduce any of the glutaredoxins (16Holmgren A. J. Biol. Chem. 1989; 264: 13963-13966Abstract Full Text PDF PubMed Google Scholar, 22Holmgren A. J. Biol. Chem. 1979; 254: 9113-9119Abstract Full Text PDF PubMed Google Scholar). 2F. Åslund and A. Holmgren, unpublished results. Instead, the glutaredoxins are reduced by glutathione, which in turn is reduced by glutathione reductase. Like thioredoxin reductase, glutathione reductase is a flavoenzyme that uses NADPH to reduce its substrate. Since glutathione does not efficiently reduce thioredoxin (22Holmgren A. J. Biol. Chem. 1979; 254: 9113-9119Abstract Full Text PDF PubMed Google Scholar) and the glutaredoxins are not substrates of thioredoxin reductase, it has been presumed that E. coli has two separate pathways for using NADPH to reduce disulfide bonds in the cytoplasm: the thioredoxin system (which consists of thioredoxin reductase and thioredoxin) and the glutaredoxin system (glutathione reductase, glutathione, and the three glutaredoxins) (Fig. 1) (16Holmgren A. J. Biol. Chem. 1989; 264: 13963-13966Abstract Full Text PDF PubMed Google Scholar, 23Gleason F.K. Holmgren A. FEMS Microbiol. Rev. 1988; 54: 271-298Crossref Google Scholar). Similar systems are thought to reduce protein disulfide bonds in the cytoplasm of eukaryotic cells as well. Most eukaryotic cells contain high levels of reduced glutathione as well as thioredoxin and glutaredoxin (6Gilbert H.F. Adv. Enzymol. Relat. Areas Mol. Biol. 1990; 63: 69-172PubMed Google Scholar, 15Holmgren A. Annu. Rev. Biochem. 1985; 54: 237-271Crossref PubMed Google Scholar, 24Holmgren A. Björnstedt M. Methods Enzymol. 1995; 252: 199-208Crossref PubMed Scopus (820) Google Scholar, 25Holmgren A. Åslund F. Methods Enzymol. 1995; 252: 283-292Crossref PubMed Scopus (298) Google Scholar). In E. coli, the thioredoxin and glutaredoxin systems are known to participate in the reduction of disulfide bonds in essential cytoplasmic enzymes which require this step to complete their catalytic cycles. These include ribonucleotide reductase, PAPS 3The abbreviations used are: PAPS, 3′-phosphoadenosine-5′-phosphosulfate; DTT, dithiothreitol; Km, kanamycin resistance; Cm, chloramphenicol resistance; Tc, tetracyclin resistance; AP, alkaline phosphatase. reductase, and methionine sulfoxide reductase (16Holmgren A. J. Biol. Chem. 1989; 264: 13963-13966Abstract Full Text PDF PubMed Google Scholar). However, the ability of the thioredoxin and glutaredoxin systems to function in vivo as general reductants of cytoplasmic protein disulfide bonds has not been fully determined. We have previously demonstrated that the thioredoxin system has a role in this process (26Derman A.I. Prinz W.A. Belin D. Beckwith J. Science. 1993; 262: 1744-1747Crossref PubMed Scopus (377) Google Scholar). In this study, we have investigated the role of the glutaredoxin system and both systems together in determining the thiol-disulfide equilibrium in the cytoplasm. Media and chemical reagents were prepared or purchased as described previously (27Derman A.I. Puziss J.W. Bassford Jr., P.J. Beckwith J. EMBO J. 1993; 12: 879-888Crossref PubMed Scopus (176) Google Scholar). Diazenedicarboxylic acid bis(N,N′-dimethylamide) (diamide) and DTT were purchased from Sigma. The strains and plasmids used in this study are listed in Table I. Strains were constructed by P1 transduction (28Miller J.H. A Short Course in Bacterial Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1992Google Scholar). The mutant alleles of the genes encoding components of the thioredoxin and glutaredoxin systems (Fig. 1) used to construct the strains for this study are:trxB::Km (29Russel M. Model P. Holmgren A. Brändén C.I. Jörnvall H. Sjöberg B.-M. Thioredoxin and Glutaredoxin Systems: Structure and Function. Raven Press, New York1986: 331-337Google Scholar), ΔtrxA (30Russel M. Model P. J. Biol Chem. 1986; 261: 14997-15005Abstract Full Text PDF PubMed Google Scholar),gor522 (lab collection),gshA20::Tn10Km (31Greenberg J.T. Demple B. J. Bacteriol. 1986; 168: 1026-1029Crossref PubMed Google Scholar),grxA::Km (32Russel M. Holmgren A. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 990-994Crossref PubMed Scopus (48) Google Scholar), and grxC::Cm (this study). Three of the mutants (WP861, WP863, and WP864) containtrxB36 (26Derman A.I. Prinz W.A. Belin D. Beckwith J. Science. 1993; 262: 1744-1747Crossref PubMed Scopus (377) Google Scholar) in place of trxB::Km.Table IStrains and plasmids used in this workPlasmidsDescriptionSource or referencepΔA-uPAΔA-uPA under control of tac promoterD. BelinpAID135APΔ2–22 under control of tac promoter27Derman A.I. Puziss J.W. Bassford Jr., P.J. Beckwith J. EMBO J. 1993; 12: 879-888Crossref PubMed Scopus (176) Google ScholarpGrxCpGEM-3Z containing a 1723-bp fragment of the E. coli chromosome which includesgrxCF. ÅslundpGrxC::CmpGrxC but withgrxC::CmThis workStrainsRelevant genotypeSource or referenceDHB4F′ lac-pro lacI Q /Δ(ara-leu)7697 araD139 ΔlacX74 galE galK rpsL phoR Δ(phoA)PvuII ΔmalF3 thiLab collectionWP551DHB4 + pAID135This workWP591WP551ΔtrxAThis workWP552WP551trxB::KmThis workWP838WP551grxA::KmThis workWP823WP551grxC::CmThis workWP776WP551gshA20::Tn10KmThis workWP841WP551gor522… . mini-Tn10TcThis workWP898WP551gshA20::Tn10Km ΔtrxAThis workWP786WP551 gshA20::Tn10KmtrxB::Km… . Tn10This workWP843WP551 gor522… . mini-Tn10TcΔtrxAThis workWP782WP551gor522… . mini-Tn10TctrxB::KmThis workWP861WP551trxB36 grxA::KmThis workWP824WP551trxB::Km grxC::CmThis workWP592WP551 trxB::KmΔtrxAThis workWP863WP551 trxB36 grxA::Km grxC::CmThis workWP826WP551 trxB::KmgrxC::Cm ΔtrxAThis workWP864WP551 trxB36 grxA::KmgrxC::Cm ΔtrxAThis workWP839WP551 ΔtrxA grxA::KmThis workWP825WP551 ΔtrxA grxC::CmThis workWP860WP551 ΔtrxA grxA::KmgrxC::CmThis workWP859WP551grxA::Km grxC::CmThis workWP759DHB4 gshA20::Tn10KmtrxB::Km… . Tn10This workWP778DHB4 gor522… . mini-Tn10TctrxB::KmThis workWP613DHB4trxB36 Δ 1 fbp… . Tn10KmLab collectionAD494DHB4 trxB::KmThis workWP840DHB4 gor522… . mini-Tn10TcThis workWP822DHB4 ΔtrxA grxA::KmgrxC::CmThis workWP758DHB4gshA20::KmThis workWP843DHB4ΔtrxA gor522… . mini-Tn10TcThis workWP612DHB4 ΔtrxA gshA20::KmThis workJCB495recDJ. BardwellWP522DHB4gor522… . mini-Tn10TcLab collectionJTG10gshA20::Tn10Km31Greenberg J.T. Demple B. J. Bacteriol. 1986; 168: 1026-1029Crossref PubMed Google ScholarA304trxB::Km29Russel M. Model P. Holmgren A. Brändén C.I. Jörnvall H. Sjöberg B.-M. Thioredoxin and Glutaredoxin Systems: Structure and Function. Raven Press, New York1986: 331-337Google ScholarA307ΔtrxA30Russel M. Model P. J. Biol Chem. 1986; 261: 14997-15005Abstract Full Text PDF PubMed Google ScholarA407grxA::Km32Russel M. Holmgren A. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 990-994Crossref PubMed Scopus (48) Google ScholarA305trxB::Km… . Tn10M. Russel Open table in a new tab A strain in which the coding region of grxC, which encodes glutaredoxin 3, was replaced with a gene encoding chloramphenicol resistance (grxC::Cm) was constructed by linear transformation (33Russel C.B. Thaler D.S. Dahlquist F.W. J. Bacteriol. 1989; 171: 2609-2613Crossref PubMed Google Scholar). For this purpose, we constructed a plasmid pGrxC::Cm, which contains grxC::Cm and the chromosomal DNA flanking grxC. We made pGrxC::Cm from pGrxC, which contains a 1723-base pair fragment of chromosomal DNA that includes grxC cloned into the EcoRI site of pGEM-3Z. 4F. Åslund, K. D. Berndt, G. Spyrou, and A. Holmgren, manuscript in preparation. pGrxC was cut with BalI and BamHI, which removes all but 20 base pairs of the coding sequence of grxC. ABamHI linker was added to the BalI site and the ends of the resulting fragment were ligated together. Into theBamHI site of the resulting plasmid, we cloned DNA encoding chloramphenicol resistance obtained from pHP45Ω-Cm (34Fellay R. Frey J. Krisch H. Gene ( Amst. ). 1987; 52: 147-154Crossref PubMed Scopus (567) Google Scholar) after digestion with BamHI. The resulting plasmid is called pGrxC::Cm. The fragment of chromosomal DNA containinggrxC::Cm obtained from pGrxC::Cm after digestion with EcoRI was then used to transform JCB495 (recD) to chloramphenicol resistance. The replacement ofgrxC by grxC::Cm in the resulting strain was confirmed by P1 transduction, polymerase chain reaction, and Western blot analysis using anti-glutaredoxin 3 antibody (data not shown). Two of the strains constructed for this study, WP759 (trxB gshA) and WP778 (trxB gor), grew very poorly unless they were grown in medium that contains a disulfide reductant like DTT. To construct WP759, WP758 (gshA) was transduced with P1 grown on A305 (trxB::K m … . Tn10) and plated on NZ-amine-A plates containing 20 μg/ml tetracycline. A 1-cm filter disk containing 25 μl of 1 mDTT was placed on the plate. After incubated for 24 h at 37 °C, two types of colonies were observed: large colonies evenly distributed on the plate and small colonies that were only present roughly 1 to 2 cm from the filter disk. The large colonies did not require DTT for growth and were found by P1 transduction not to containtrxB::Km. The small colonies required DTT for growth and contained trxB::Km (by P1 transduction). The same procedure was used to construct WP778 except that AD494 (trxB::Km) was transducted to tetracycline resistance with P1 grown on WP522 (gor522… . mini-Tn10Tc). WP759 and WP778 were grown on NZ-amine-A plates with 8 mmDTT. 4 and 2 mm DTT was used to grow these strains in liquid NZ-amine-A and liquid M63 minimal medium, respectively. In minimal medium, these strains require supplementation with cysteine. Growth rates of WP759 and WP778 (Fig. 2) were determined as follows. Overnight cultures of both strains and DHB4 were grown at 37 °C in NZ-amine-A medium supplemented with 4 mm DTT. The cultures were diluted 1:100 in the same medium and their growth followed for 3 h by determining their optical density at 600 nm. The cells were then pelleted and resuspended in NZ-amide-A medium without DTT and their growth was followed for another 3 h. Growth rates were calculated from the average of three determinations and differed from one another by less than 1%. WP759 and WP778 accumulate suppressing mutations that allow them to grow about as rapidly as DHB4 (wild-type) in media that does not contain a disulfide reductant. Since these mutations arise at a high frequency (approximately 10−6 or 10−7), it was necessary to monitor the growth rate of these strains when they were grown without DTT (e.g. to determine AP activity or urokinase activity) to make sure that they grew slowly. Cultures that did not grow as slowly as those shown in Fig. 2 B were discarded. The strains shown in Table IIwere grown at 37 °C in M63 minimal medium containing 0.2% glucose, 50 μg/ml each of all amino acids except methionine, 200 μg/ml ampicillin, and 5 mmisopropyl-1-thio-β-d-galactopyranoside to a final optical density at 600 nm of approximately 0.4. They were then incubated on ice for 20 min in the presence of 100 mm iodoacetamide. The remainder of the assay was performed as described in Ref. 27Derman A.I. Puziss J.W. Bassford Jr., P.J. Beckwith J. EMBO J. 1993; 12: 879-888Crossref PubMed Scopus (176) Google Scholar, except that 100 mm iodoacetamide was used instead of 1 mm iodoacetamide in the wash buffer. The assays were performed in duplicate and varied by less than 5%.Table IIAP activity of various strains expressing APΔ2–22StrainRelevant genotypeAP activityWP551Wild-type90WP552trxB870WP591trxA130WP841gor260WP776gshA130WP838grxA80WP823grxC290WP592trxB trxA310WP861trxB grxA420WP824trxB grxC860WP843trxA gor860WP898trxA gshA760WP839trxA grxA63WP825trxA grxC32WP863trxB trxA grxA230WP826trxB trxA grxC140WP860trxA grxA grxC200WP864trxB trxA grxA grxC290Strains were grown in M63 minimal medium containing 0.2% glucose, 200 μg/ml ampicillin, 5 mmisopropyl-thio-β-d-galactopyranoside, and 50 μg/ml each of all amino acids except methionine. Open table in a new tab Strains were grown in M63 minimal medium containing 0.2% glucose, 200 μg/ml ampicillin, 5 mmisopropyl-thio-β-d-galactopyranoside, and 50 μg/ml each of all amino acids except methionine. The strains shown in Table III were grown at 37 °C to an optical density at 600 nm of approximately 0.6 in NZ-amine-A containing 200 μg/ml ampicillin and 4 mm DTT. They were pelleted, resuspended in NZ-amine-A containing 200 μg/ml ampicillin and 5 mm isopropyl-1-thio-β-d-galactopyranoside, and grown at 37 °C for 3 h. The AP assays were then performed as above.Table IIIAP activity of strains expressing APΔ2–22 after growth for 3 h without DTTStrainRelevant genotypeAP activityWP551Wild-type38WP552trxB240WP782trxB gor1200WP786trxB gshA1700Strains were grown as described under “Experimental Procedures” Open table in a new tab Strains were grown as described under “Experimental Procedures” Cells were grown in NZ-amine-A plus 200 μg/ml ampicillin and 4 mm DTT at 37 °C to an optical density at 600 nm of approximately 0.6. They were pelleted and resuspended in NZ-amine-A plus 200 μg/ml ampicillin and 5 mm isopropyl-1-thio-β-d-galactopyranoside and grown for 3 h at 37 °C. Zymography using casein plasminogen agar underlays was performed as described previously (26Derman A.I. Prinz W.A. Belin D. Beckwith J. Science. 1993; 262: 1744-1747Crossref PubMed Scopus (377) Google Scholar) except that after incubation of the samples on ice for the times indicated in Fig.3, iodoacetamide was added to 100 mm and the samples were incubated for an additional 20 min on ice. To determine the affect of diazenedicarboxylic acid bis(N,N′-dimethylamide) (diamide) on growth rate, cells were grown to an optical density of 0.2 at 600 nm in NZ-amine-A medium at 37 °C and diamide was then added to a final concentration of 250 mm. The growth of the cells was then followed by determining their optical density at 600 nm. To investigate the role of the thioredoxin and glutaredoxin systems in maintaining the thiol-disulfide balance in the cytoplasm, we constructed a set of mutants missing various components of these systems. We then assessed the ability of disulfide bonds to form in the cytoplasm of these strains by determining the extent to which E. coli alkaline phosphatase (AP) is able to form disulfide bonds in the cytoplasm of the mutants. AP is a periplasmic homodimeric enzyme that contains two intrachain disulfide bonds in each monomer (35Kim E.E. Wyckoff H.W. Clin. Chim. Acta. 1989; 186: 175-188Crossref Scopus (171) Google Scholar). These bonds are required for AP to be enzymatically active. AP is synthesized with an N-terminal signal sequence which targets it for export to the periplasm. When AP is expressed with a defective or missing signal sequence, it is not exported to the periplasm, but remains in the cytoplasm (36Michaelis S. Inouye H. Oliver D. Beckwith J. J. Bacteriol. 1983; 154: 366-374Crossref PubMed Google Scholar). In this compartment, AP does not form disulfide bonds and cannot fold into an enzymatically active conformation (8Derman A.I. Beckwith J. J. Bacteriol. 1991; 173: 7719-7723Crossref PubMed Scopus (163) Google Scholar). However, in the cytoplasm of E. colimutants missing thioredoxin reductase a fraction of a signal sequenceless version of AP (APΔ2-22) does forms disulfide bonds and folds into an active conformation (26Derman A.I. Prinz W.A. Belin D. Beckwith J. Science. 1993; 262: 1744-1747Crossref PubMed Scopus (377) Google Scholar). Thus, APΔ2-22 can be used to assess the potential for disulfide bond formation in the cytoplasm of the mutants missing components of the thioredoxin and glutaredoxin systems. Some mutants lacking components of the thioredoxin and glutaredoxin systems have been previously constructed. However, these mutants are in different genetic backgrounds. To facilitate comparisons between the mutants, a set of isogenic strains was constructed by P1 transduction (Table I). The mutant alleles of each gene used in the construction of these strains do not make any detectable functional product of that gene (Refs. 29Russel M. Model P. Holmgren A. Brändén C.I. Jörnvall H. Sjöberg B.-M. Thioredoxin and Glutaredoxin Systems: Structure and Function. Raven Press, New York1986: 331-337Google Scholar, 31Greenberg J.T. Demple B. J. Bacteriol. 1986; 168: 1026-1029Crossref PubMed Google Scholar, and37Miranda-Vizuete A. Martinez-Galisteo E. Åslund F. Lopez-Barea J. Pueyo C. Holmgren A. J. Biol. Chem. 1994; 269: 16631-16637Abstract Full Text PDF PubMed Google Scholar; data not shown). In addition to the previously described mutants lacking glutaredoxin 1 (encoded by grxA), we wished to obtain mutants lacking either glutaredoxin 2 or glutaredoxin 3. A mutant missing glutaredoxin 2 has only recently been constructed, 5A. Vlamis-Gardikas and A. Holmgren, manuscript in preparation. and was not used in these studies. We constructed a strain missing glutaredoxin 3 using linear transformation (33Russel C.B. Thaler D.S. Dahlquist F.W. J. Bacteriol. 1989; 171: 2609-2613Crossref PubMed Google Scholar). The plasmid pGrxC contains a fragment of chromosomal DNA that includes grxC (which encodes glutaredoxin 3). The coding region of grxC in this plasmid was removed and replaced with DNA from pHP45Ω-Cm (34Fellay R. Frey J. Krisch H. Gene ( Amst. ). 1987; 52: 147-154Crossref PubMed Scopus (567) Google Scholar) which confers chloramphenicol resistance. The fragment of chromosomal DNA was then removed from the resulting plasmid and used to transform arecD strain (JCB495) to chloramphenicol resistance. The replacement of grxC with grxC::Cm was confirmed by polymerase chain reaction and P1 transduction (data not shown). Western blot analysis revealed thatgrxC::Cm cells contained no detectable glutaredoxin 3 (data not shown). grxC is in an operon withsecB, which encodes an export-specific cytoplasmic chaperone. Western blots revealed that strains containinggrxC::Cm did not make detectable amounts of SecB (data not shown). Seven mutants constructed for this study could not grow in M9 or M63 minimal medium unless it was supplemented with cysteine or glutathione: WP839 (trxA grxA), WP860 (trxA grxA grxC), WP864 (trxB trxA grxA grxC), WP843 (trxA gor), WP898 (trxA gshA), WP824 (trxB grxA), and WP863 (trxB grxA grxC) (data not shown). DHB4, the wild-type parent of these strains, does not require these supplements. However, these additives were not required if the M9 medium was prepared with sulfite instead of sulfate (data not shown). None of these strains required cysteine or glutathione to grow in NZ-amine-A medium. Similar findings have been reported for trxB grxA and trxA grxA mutants and were attributed to the inability of these strains to reduce PAPS reductase, an enzyme that is required for growth when sulfate is the only source of sulfur in the medium (32Russel M. Holmgren A. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 990-994Crossref PubMed Scopus (48) Google Scholar, 38Kren B. Parsell D. Fuchs J.A. J. Bacteriol. 1988; 170: 308-315Crossref PubMed Google Scholar, 39Russel M. Model P. Holmgren A. J. Bacteriol. 1990; 172: 1923-1929Crossref PubMed Scopus (105) Google Scholar). Two strains constructed for this study, WP759 (trxB gshA) and WP778 (trxB gor), grew extremely poorly. They formed microscopic colonies on rich medium (NZ-amine-A) after incubation for 1 day and small colonies after 4 days at 37 °C (wild-type cells form large colonies in 1 day). Since we suspected that these strains grew poorly because they were not able to efficiently reduce cytoplasmic protein disulfide bonds, we determined whether their growth rate could be improved by adding a disulfide reductant to the medium. Both strains grew at about the same rate as wild-type cells when 4 mmDTT was added to the medium (Fig. 2). The doubling time of WP759 and WP778 in NZ-amine-A medium without DTT is about 300 min, while this rate increases to 30 min when 4 mm DTT is added to the medium. The growth defect of these mutants could also be complemented by the addition of β-mercaptoethanol, but not oxidized DTT, to the medium (data not shown). Interestingly, WP759 and WP778 were able to grow about as rapidly as wild-type cells, even in medium that did not contain DTT or another disulfide reductant, when the cells were grown anaerobically (data not shown). Mutations that allow WP759 and WP778 to grow well aerobically without a disulfide reductant in the medium arose at a relatively high frequency (approximately 10−6 or 10−7). These mutations have not been characterized further. APΔ2-22 becomes active in the cytoplasm of the mutants missing components of either the thioredoxin or the glutaredoxin systems (Table II). We confirmed that the AP activity in some of these mutants in fact reflects an increase in the extent of disulfide bond formation in APΔ2-22 by using the finding that oxidized and reduced APΔ2-22 can be separated with nonreducing SDS-PAGE (8Derman A.I. Beckwith J. J. Bacteriol. 1991; 173: 7719-7723Crossref PubMed Scopus (163) Google Scholar) to determine the redox status of APΔ2-22 in the mutants (data not shown). As previously reported, mutants missing thioredoxin reductase allow a substantial amount of APΔ2-22 (approximately 25%) to form disulfide bonds in the cytoplasm. Cells missing thioredoxin or one of the components of the glutaredoxin system allow less APΔ2-22 to form disulfide bonds. The amount of AP activity in many of these mutants is only slightly higher than that found in wild-type cells. However, even these small differences probably indicate a substantial increase in the ability of APΔ2-22 to form disulfide bonds in the cytoplasm of these mutants. We suggest this since, as far as we can tell, all of the AP activity in wild-type cells (WP551) reflects the 1–2% of APΔ2-22 that is exported to the periplasm; no disulfide bonds form in APΔ2-22 in the cytoplasm of wild-type cells. Thus, the increase in activity in the mutant" @default.
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