Cysteine Is Exported from the Escherichia coliCytoplasm by CydDC, an ATP-binding Cassette-type Transporter Required for Cytochrome Assembly
2002; Elsevier BV; Volume: 277; Issue: 51 Linguagem: Inglês
10.1074/jbc.m205615200
ISSN1083-351X
AutoresMarc S. Pittman, Hazel Corker, Guanghui Wu, Marie Binet, Arthur J.G. Moir, Robert K. Poole,
Tópico(s)Enzyme Structure and Function
ResumoAssembly of Escherichia colicytochrome bd and periplasmic cytochromes requires the ATP-binding cassette transporter CydDC, whose substrate is unknown. Two-dimensional SDS-PAGE comparison of periplasm from wild-type andcydD mutant strains revealed that the latter was deficient in several periplasmic transport binding proteins, but no single major protein was missing in the cydD periplasm. Instead, CydDC exports from cytoplasm to periplasm the amino acid cysteine, demonstrated using everted membrane vesicles that transported radiolabeled cysteine inward in an ATP-dependent, uncoupler-independent manner. New pleiotropic cydDphenotypes are reported, including sensitivity to benzylpenicillin and dithiothreitol, and loss of motility, consistent with periplasmic defects in disulfide bond formation. Exogenous cysteine reversed these phenotypes and affected levels of periplasmic c-type cytochromes in cydD and wild-type strains but did not restore cytochrome d. Consistent with CydDC being a cysteine exporter, cydD mutant growth was hypersensitive to high cysteine concentrations and accumulated higher cytoplasmic cysteine levels, as did a mutant defective inorf299, encoding a transporter of the major facilitator superfamily. A cydD orf299 double mutant was extremely cysteine-sensitive and had higher cytoplasmic cysteine levels, whereas CydDC overexpression conferred resistance to high extracellular cysteine concentrations. We propose that CydDC exports cysteine, crucial for redox homeostasis in the periplasm. Assembly of Escherichia colicytochrome bd and periplasmic cytochromes requires the ATP-binding cassette transporter CydDC, whose substrate is unknown. Two-dimensional SDS-PAGE comparison of periplasm from wild-type andcydD mutant strains revealed that the latter was deficient in several periplasmic transport binding proteins, but no single major protein was missing in the cydD periplasm. Instead, CydDC exports from cytoplasm to periplasm the amino acid cysteine, demonstrated using everted membrane vesicles that transported radiolabeled cysteine inward in an ATP-dependent, uncoupler-independent manner. New pleiotropic cydDphenotypes are reported, including sensitivity to benzylpenicillin and dithiothreitol, and loss of motility, consistent with periplasmic defects in disulfide bond formation. Exogenous cysteine reversed these phenotypes and affected levels of periplasmic c-type cytochromes in cydD and wild-type strains but did not restore cytochrome d. Consistent with CydDC being a cysteine exporter, cydD mutant growth was hypersensitive to high cysteine concentrations and accumulated higher cytoplasmic cysteine levels, as did a mutant defective inorf299, encoding a transporter of the major facilitator superfamily. A cydD orf299 double mutant was extremely cysteine-sensitive and had higher cytoplasmic cysteine levels, whereas CydDC overexpression conferred resistance to high extracellular cysteine concentrations. We propose that CydDC exports cysteine, crucial for redox homeostasis in the periplasm. Escherichia coli possesses two major membrane-bound terminal respiratory oxidases, namely cytochromes bo′("bo 3" encoded by cyoABCDE) andbd. The latter comprises two polypeptide subunits (encoded by cydA and cydB) and hemesb 558, b 595, andd (1Ingledew W.J. Poole R.K. Microbiol. Rev. 1984; 48: 222-271Crossref PubMed Google Scholar, 2Gennis R.B. Stewart V. Niedhardt F.C. Curtis R. Ingraham J.R. Lin E.C.C. Low K.B. Magasanik B. Reznikoff W.S. Riley M. Schaechter M. Umbarger H.E. Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology. 2nd Ed. American Society for Microbiology, Washington, D. C.1996: 217-261Google Scholar, 3Jünemann S. Biochim. Biophys. Acta. 1997; 1321: 107-127Crossref PubMed Scopus (227) Google Scholar). Both oxidases catalyze ubiquinol oxidation and oxygen reduction but differ in the efficiency with which electron transfer is coupled to proton translocation (2Gennis R.B. Stewart V. Niedhardt F.C. Curtis R. Ingraham J.R. Lin E.C.C. Low K.B. Magasanik B. Reznikoff W.S. Riley M. Schaechter M. Umbarger H.E. Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology. 2nd Ed. American Society for Microbiology, Washington, D. C.1996: 217-261Google Scholar, 4Poole R.K. Cook G.M. Poole R.K. Advances in Microbial Physiology. 43. Academic Press, London2000: 165-224Google Scholar), and the pattern of expression in response to environment (1Ingledew W.J. Poole R.K. Microbiol. Rev. 1984; 48: 222-271Crossref PubMed Google Scholar, 2Gennis R.B. Stewart V. Niedhardt F.C. Curtis R. Ingraham J.R. Lin E.C.C. Low K.B. Magasanik B. Reznikoff W.S. Riley M. Schaechter M. Umbarger H.E. Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology. 2nd Ed. American Society for Microbiology, Washington, D. C.1996: 217-261Google Scholar, 4Poole R.K. Cook G.M. Poole R.K. Advances in Microbial Physiology. 43. Academic Press, London2000: 165-224Google Scholar). Significantly, cytochrome bd is required for resistance to a number of environmental stresses and its loss attenuates virulence in certain bacteria (5Way S.S. Sallustio S. Magliozzo R.S. Goldberg M.B. J. Bacteriol. 1999; 181: 1229-1237Crossref PubMed Google Scholar, 6Endley S. McMurray D. Ficht T.A. J. Bacteriol. 2001; 183: 2454-2462Crossref PubMed Scopus (119) Google Scholar). Assembly of cytochrome bd is dependent not only on the structural genes cydAB, but also on the unlinkedcydDC operon (7Georgiou C.D. Fang H. Gennis R.B. J. Bacteriol. 1987; 169: 2107-2112Crossref PubMed Google Scholar, 8Poole R.K. Williams H.D. Downie J.A. Gibson F. J. Gen. Microbiol. 1989; 135: 1865-1874PubMed Google Scholar, 9Poole R.K. Hatch L. Cleeter M.W.J. Gibson F. Cox G.B. Wu G. Mol. Microbiol. 1993; 10: 421-430Crossref Scopus (73) Google Scholar). The latter genes are predicted to encode a heterodimeric ABC 1The abbreviations used are: ABC, ATP-binding cassette; DTT, dithiothreitol; MOPS, 3-(N-morpholino)propanesulfonic acid; IPG, immobilized pH gradient; LB, Luria-Bertani; EDDHA, ethylenediamine di(o-hydroxyphenylacetic acid); CCCP, carbonyl cyanidep-chlorophenylhydrazone; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid1The abbreviations used are: ABC, ATP-binding cassette; DTT, dithiothreitol; MOPS, 3-(N-morpholino)propanesulfonic acid; IPG, immobilized pH gradient; LB, Luria-Bertani; EDDHA, ethylenediamine di(o-hydroxyphenylacetic acid); CCCP, carbonyl cyanidep-chlorophenylhydrazone; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid-type transporter (traffic ATPase) (9Poole R.K. Hatch L. Cleeter M.W.J. Gibson F. Cox G.B. Wu G. Mol. Microbiol. 1993; 10: 421-430Crossref Scopus (73) Google Scholar) with an unknown export function (10Poole R.K. Gibson F. Wu G. FEMS Lett. 1994; 117: 217-224Crossref Scopus (79) Google Scholar, 11Saurin W. Hofnung M. Dassa E. J. Mol. Evol. 1999; 48: 22-41Crossref PubMed Scopus (258) Google Scholar). Unlike traffic ATPases involved in uptake, CydDC is thought not to interact with a cognate periplasmic-binding protein. Strains defective in eithercydD or cydC display complex phenotypes in addition to loss of cytochrome bd. These include loss of periplasmic b- and c-type cytochromes (10Poole R.K. Gibson F. Wu G. FEMS Lett. 1994; 117: 217-224Crossref Scopus (79) Google Scholar, 12Goldman B.S. Gabbert K.K. Kranz R.G. J. Bacteriol. 1996; 178: 6338-6347Crossref PubMed Google Scholar); increased sensitivity to high temperature, H2O2, azide, and Zn2+ ions (8Poole R.K. Williams H.D. Downie J.A. Gibson F. J. Gen. Microbiol. 1989; 135: 1865-1874PubMed Google Scholar, 12Goldman B.S. Gabbert K.K. Kranz R.G. J. Bacteriol. 1996; 178: 6338-6347Crossref PubMed Google Scholar,13Delaney J.M. Ang D. Georgopoulos C. J. Bacteriol. 1992; 174: 1240-1247Crossref PubMed Google Scholar); and inability to exit stationary phase at 37 °C under aerobic conditions (14Siegele D.A. Imlay K.R.C. Imlay J.A. J. Bacteriol. 1996; 178: 6091-6096Crossref PubMed Google Scholar). We hypothesized that the substrate of CydDC might be heme (9Poole R.K. Hatch L. Cleeter M.W.J. Gibson F. Cox G.B. Wu G. Mol. Microbiol. 1993; 10: 421-430Crossref Scopus (73) Google Scholar, 10Poole R.K. Gibson F. Wu G. FEMS Lett. 1994; 117: 217-224Crossref Scopus (79) Google Scholar) that would be assembled into apocytochromes following export to the periplasm. However, the assembly of heme into heterologous apoproteins (e.g. Ascarishemoglobin) exported to the periplasm of E. coli does not require cydC (12Goldman B.S. Gabbert K.K. Kranz R.G. J. Bacteriol. 1996; 178: 6338-6347Crossref PubMed Google Scholar), suggesting that outward transport of heme is not absolutely dependent on CydDC. Furthermore, transport studies using inside-out vesicles derived from wild-type and cydDmutant strains revealed no discernible differences between the two strains in association of radiolabeled heme with, or transport by, vesicle membranes (15Cook G.M. Poole RK Microbiology. 2000; 146: 527-536Crossref PubMed Scopus (35) Google Scholar). An important clue to the function of CydDC was the finding (12Goldman B.S. Gabbert K.K. Kranz R.G. J. Bacteriol. 1996; 178: 6338-6347Crossref PubMed Google Scholar) that the periplasm of a cydC mutant is more oxidizing, as assayed using 5,5′-dithiobis(2-nitrobenzoic acid), than that of a wild-type strain. This suggests that CydDC exports a reducing molecule to the periplasm and therefore contributes to the maintenance of the balanced redox conditions required for cytochrome c biogenesis in the periplasm. CcmH, containing a conserved CXXC motif, is required in E. coli for keeping the heme-binding site of apocytochrome c in a reduced form for subsequent heme ligation (16Fabianek R.A. Hofer T. Thöny-Meyer L. Arch. Microbiol. 1999; 171: 92-100Crossref PubMed Scopus (80) Google Scholar). Several other protein thiol:disulfide oxidoreductases are required for cytochrome c maturation; loss of DsbA, DsbB, or DsbD (DipZ) each results in a loss of c-type cytochromes (17Crooke H. Cole J. Mol. Microbiol. 1995; 15: 1139-1150Crossref PubMed Scopus (114) Google Scholar, 18Sambongi Y. Ferguson S.J. FEBS Lett. 1994; 353: 235-238Crossref PubMed Scopus (64) Google Scholar, 19Sambongi Y. Ferguson S.J. FEBS Lett. 1996; 398: 265-268Crossref PubMed Scopus (45) Google Scholar). DsbA and DsbB are involved in the formation of disulfide bonds in various periplasmic proteins (20Bardwell J.C. McGovern K. Beckwith J. Cell. 1991; 67: 581-589Abstract Full Text PDF PubMed Scopus (829) Google Scholar, 21Bardwell J.C.A. Lee J.-O. Jander G. Martin N. Belin D. Beckwith J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1038-1042Crossref PubMed Scopus (359) Google Scholar), whereas DsbD translocates electrons from the cytoplasm to the periplasm (22Stewart E.J. Katzen F. Beckwith J. EMBO J. 1999; 18: 5963-5971Crossref PubMed Scopus (127) Google Scholar), thereby providing a source of reducing power to an otherwise oxidized environment. The aim of this work was to identify the substrate exported by CydDC. We failed to find an obvious protein candidate, but show instead that CydDC exports cysteine to the periplasm, the first demonstration of ATP-driven l-cysteine export. Support for this conclusion comes from: (a) direct demonstration that everted membrane vesicles take up cysteine in an ATP- and CydDC-dependent manner, corresponding to export in vivo; (b) correction by exogenous l-cysteine of newly reportedcydD phenotypes, specifically loss of motility and increased sensitivity to benzylpenicillin; (c) detection of higher cytoplasmic levels of cysteine in cydD mutant cells; (d) susceptibility of cydD mutants to growth inhibition by external cysteine; and (e) increased resistance to cytotoxic levels of cysteine by strains that overexpress CydDC. E. coli strain AN2343 carrying the mutant cydD1 allele and its isogenic wild-type parent strain AN2342 have been described before (8Poole R.K. Williams H.D. Downie J.A. Gibson F. J. Gen. Microbiol. 1989; 135: 1865-1874PubMed Google Scholar). Strains RKP4611 and RKP4612 were constructed by P1vir transduction (23Silhavy T.J. Berman M.L. Enquist L.W. Experiments with Gene Fusions. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1984Google Scholar) of theorf299::KmR allele from strain MC4100Δ299 (24Dassler T. Maier T. Winterhalter C. Böck A. Mol. Microbiol. 2000; 36: 1101-1112Crossref PubMed Scopus (131) Google Scholar) into strains AN2342 and AN2343, respectively. Strains RKP2634 and RKP2005 were obtained by transformation of the wild-type and cydD mutant strain, respectively, with plasmid pRP33 (9Poole R.K. Hatch L. Cleeter M.W.J. Gibson F. Cox G.B. Wu G. Mol. Microbiol. 1993; 10: 421-430Crossref Scopus (73) Google Scholar) that has thecydDC + operon cloned into vector pBR328. Cells were grown in Luria-Bertani (LB) broth (pH 7.0) (25Miller J.H. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1972Google Scholar), or in MOPS-buffered minimal medium (pH 7.4) (26Stewart V. Parales J. J. Bacteriol. 1988; 170: 1589-1597Crossref PubMed Google Scholar) supplemented with 40 mm lactose plus 10% (v/v) LB. Kanamycin and benzylpenicillin (penicillin G) were added to give final concentrations of 30 and 20 μg ml−1, respectively. l-Cysteine was added as a filter-sterilized 100 mm stock solution to media, giving the final concentrations in the text. Aerated cultures were grown in Erlenmeyer flasks containing one fifth of their volume by shaking (200 rpm) at 30 °C or 37 °C. Anaerobically grown cultures were obtained by filling growth vessels to the brim with LB (supplemented with 20 mm KNO3) and incubating without shaking at 37 °C for 14 h. Cells were grown to stationary phase in LB broth at 30 °C, and 5-μl drops were spotted onto semi-solid LB medium (0.3% Difco agar). The cells were incubated at 30 °C for up to 3 days, and the diameter of the resultant swarm of growth was measured. Cells were grown to stationary phase in LB broth at 37 °C, and serial dilutions in 1-ml aliquots were made. Portions (5 μl) of serially diluted suspensions were drop-plated onto solid LB medium containing benzylpenicillin (20 μg ml−1) and 0.5, 1, 1.5, or 2 mm cysteine. Plates were incubated overnight at 37 °C, and the colonies were counted. Periplasmic fractions were isolated using a modified procedure of Willis et al.(27Willis R.C. Morris R.G. Cirakoglu C. Schellenberg G.D. Gerber N.H. Furlong C.E. Arch. Biochem. Biophys. 1974; 161: 64-75Crossref Scopus (58) Google Scholar). In brief, 200 ml of culture was conditioned for osmotic shock by the addition of 6 ml of 1 m NaCl and 6 ml of 1m Tris-HCl buffer (pH 7.3). An equal volume of a 40% (w/v) sucrose solution containing 33 mm Tris-HCl (pH 7.3) and 2 mm EDTA was added, and incubated at room temperature for 20 min. Cells were harvested, and to each pellet 6 ml of ice-cold water was added. After 45 s on ice, MgCl2 was added to 1 mm and the cells kept on ice for 10 min. Finally, the periplasmic fraction was obtained by centrifugation (10,000 ×g for 5 min) at 4 °C to remove cell debris and stored at 4 °C until ready for use. The cytoplasmic fraction for enzyme assays was produced from the pellet (spheroplasts), which was resuspended in a buffer (6 ml) that contained (final concentration) 20% (w/v) sucrose, 200 mm Tris-HCl (pH 7.5), and 1 mm Na EDTA. Sonication (15 μm amplitude, four or five 15-s bursts, with 30-s breaks) on ice was followed by centrifugation (100,000 ×g for 70 min), and the resulting supernatant (cytoplasm) was stored at 4 °C. For assay of cysteine in the cytoplasm, 400 ml of culture was used and the spheroplasts were suspended in 1 ml of water. Centrifugation after sonication was at 200,000 × g for 2 h. Assays of β-galactosidase (25Miller J.H. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1972Google Scholar) and alkaline phosphatase (28Brickman E. Beckwith J. J. Mol. Biol. 1975; 96: 307-316Crossref PubMed Scopus (320) Google Scholar, 29Michaelis S. Inouye H. Oliver D. Beckwith J. J. Bacteriol. 1983; 154: 366-374Crossref PubMed Google Scholar) were used to determine the purity of periplasmic and cytoplasmic fractions. Activities were measured at room temperature by monitoring at 420 nm the hydrolysis ofo-nitrophenyl-β-d-galactopyranoside or 4-nitrophenyl phosphate, respectively. Periplasmic samples were concentrated ∼2-fold with a Centricon YM-3 centrifugal filter device (Amicon Bioseparations-Millipore Corp.) with a maximum volume of 2 ml and a molecular mass cut-off of 3,000 Da. A portion (2 ml) of each sample was spun (5,000 × g for 120 min) without the retentate vial. An additional 2 ml of sample was centrifuged exactly as above, and samples were pooled. Concentrated periplasm (∼0.2 mg of protein) was included in 125 μl (total volume) of rehydration solution (8 m urea, 2% (w/v) CHAPS, 0.5% (v/v) IPG buffer pH 3–10 (non-linear) (Amersham Biosciences), 0.28% dithiothreitol, and a few grains of bromphenol blue) and applied to a 7-cm IPG strip. After rehydration (18–20 h), two-dimensional gel electrophoresis was carried out using a Multiphor II horizontal unit with immobilized pH gradients (pre-cast IPG strip, pH 3–10, non-linear) in the first dimension and a sodium dodecyl sulfate (SDS)-polyacrylamide gel (8–18% polyacrylamide) in the second dimension, according to the instructions from the manufacturer (Amersham Biosciences). Gels were stained with Coomassie Blue. Proteins were electroblotted onto ProBlott (Applied Biosystems) membranes at 400–500 mA for 1.5–2 h before staining with Coomassie Blue. The N-terminal sequences of the protein spots were determined by sequential Edman degradation (30Qi S.-Y. Moir A.J.G. O'Connor C.D. J. Bacteriol. 1996; 178: 12032-12038Crossref Google Scholar). Sequence identity was computed using the Colibri web site (genolist.pasteur.fr/colibri/) FASTA function (31Pearson W.R. Lipman D.J. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 2444-2448Crossref PubMed Scopus (9380) Google Scholar). Further information on sequenced proteins was found on the SWISS-PROT web site (www.expasy.ch/). This was carried out using the method of Gaitonde (32Gaitonde M.K. Biochem. J. 1967; 104: 627-633Crossref PubMed Scopus (1003) Google Scholar). A standard curve (0–0.5 μmol of cysteine-HCl) was prepared, and used to quantify cysteine levels in cytoplasmic fractions, which had been treated with acetic acid and acid ninhydrin "Reagent 2" (250 mg ninhydrin dissolved in a mixture of 6 ml of acetic acid and 4 ml of HCl). Samples were heated in a boiling water bath for 10 min, then cooled rapidly in water before dilution to 5 or 10 ml using 95% ethanol. After 30 min at room temperature, the reaction products were measured at 561 nm. To correct for interference by other ninhydrin-reactive components that contributed to a sloping base line in the absorbance spectra of dilute cytoplasmic fractions,A 561 was measured relative to a baseline drawn between 530 and 590 nm. Cytochrome d was quantified in cells grown aerobically to stationary phase in 50 ml of LB and harvested at 6000 × g for 15 min. Cells were washed with 100 mm potassium phosphate buffer (pH 7.2) and used to record reduced minus oxidized difference spectra and CO + reduced minus reduced difference spectra at room temperature as before (8Poole R.K. Williams H.D. Downie J.A. Gibson F. J. Gen. Microbiol. 1989; 135: 1865-1874PubMed Google Scholar), except that a SDB4 dual wavelength scanning spectrophotometer (33Kalnenieks U. Galinina N. Bringer-Meyer S. Poole R.K. FEMS Microbiol. Lett. 1998; 168: 91-97PubMed Google Scholar) was used. For cytochrome d, an absorption coefficient ε (622 minus 644 nm) of 12.6 mm−1 cm−1 (34Kita K. Konishi K. Anraku Y. J. Biol. Chem. 1984; 259: 3368-3374Abstract Full Text PDF PubMed Google Scholar) was used in CO difference spectra. For c-type cytochromes, periplasmic fractions were isolated as described above to minimize interference by other cytochromes with overlapping spectral features. Reducedminus oxidized difference spectra at room temperature were recorded as in Ref. 10Poole R.K. Gibson F. Wu G. FEMS Lett. 1994; 117: 217-224Crossref Scopus (79) Google Scholar but in the SDB4 dual wavelength scanning spectrophotometer. Correction for base-line drift in the Soret region was accomplished by dropping a vertical from the absorption peak at ∼423 nm (NrfA has a maximum in absolute spectra at 420.5 nm; Ref. 35Bamford V.A. Angove H.C. Seward H.E. Thomson A.J. Cole J.A. Butt J.N. Hemmings A.M. Richardson D.J. Biochemistry. 2002; 41: 2921-2931Crossref PubMed Scopus (134) Google Scholar) to a base line drawn between 404 and 450 nm. The absorption coefficient ε used was 146 mm−1 cm−1 (10Poole R.K. Gibson F. Wu G. FEMS Lett. 1994; 117: 217-224Crossref Scopus (79) Google Scholar), determined by using the absorption coefficient ε551–540for the α-band (10Poole R.K. Gibson F. Wu G. FEMS Lett. 1994; 117: 217-224Crossref Scopus (79) Google Scholar) and a γ/α ratio of 7.5 measured in spectra of concentrated periplasmic fractions. Protein contents of cell suspensions and periplasmic fractions were assayed using the method of Markwell et al. (36Markwell M.A. Haas S.M. Bieber L.L. Tolbert N.E. Anal. Biochem. 1978; 87: 206-210Crossref PubMed Scopus (5327) Google Scholar). Up to 6 liters of culture was grown aerobically at 37 °C to the mid-exponential phase of growth (A 600 = 0.6) in MOPS minimal medium supplemented with lactose and LB. Cells were harvested by centrifugation and the cell pellet washed with pre-cooled 10 mm Tris-HCl (pH 7.5), containing 140 mm choline chloride, 0.5 mm dithiothreitol, and 10% glycerol (v/v) followed by resuspension in the same buffer (5 vol/g of wet cells). Everted vesicles were prepared by the method of Ambudkar et al. (37Ambudkar S.V. Zlotnick G.W. Rosen B.P. J. Biol. Chem. 1984; 259: 6142-6145Abstract Full Text PDF PubMed Google Scholar). In brief, cells were disrupted by a single passage through a French pressure cell at 4000 p.s.i. (34.5 megapascals). Pancreatic DNase and MgCl2 were added at final concentrations of 0.1 mg ml−1 and 2.5 mm, respectively, and the mixture was incubated on ice for 1 h or until the viscosity decreased significantly. After centrifugation at 10,000 × g for 10 min, vesicles were sedimented from the supernatant by centrifugation at 150,000 × g for 1 h. Vesicles were gently washed once in the same buffer, collected by centrifuging and resuspended to15–20 mg of protein ml−1. Aliquots (100 μl) were diluted with an equal volume of glycerol before snap-freezing and storage at −20 °C. [14C]Lactose (2109 MBq mmol−1) and [35S]cysteine (3145 MBq mmol−1; Amersham Biosciences) were added to final concentrations of 0.06 and 0.5 mm, respectively, in the transport assay. In addition, non-labeled lactose and cysteine were added at final concentrations of 1.94 and 0.5 mm, respectively. Everted vesicles were thawed slowly on ice and diluted to 1.0 mg of protein ml−1 in 10 mm Tris-HCl (pH 8.0) containing 140 mm choline chloride and 5 mm MgCl2. Vesicles were added to glass tubes containing buffer (pre-equilibrated at 30 °C) to a final volume of 200 μl, and were incubated at 30 °C for 15 min without shaking.to initiate [14C]lactose transport, vesicles were energized for 15 min prior to lactose addition with 20 mm d-lactate. [35S]Cysteine transport was initiated by the addition of cysteine for 5 min prior to the addition of 10 mm ATP. Vesicles were de-energized with either CCCP (2 μm) to dissipate the proton gradient (15Cook G.M. Poole RK Microbiology. 2000; 146: 527-536Crossref PubMed Scopus (35) Google Scholar), or sodium orthovanadate (50 μm), an analogue of inorganic phosphate that mimics the γ-phosphate of ATP in the transition state for ATP hydrolysis (38Davidson A.L. J. Bacteriol. 2002; 184: 1225-1233Crossref PubMed Scopus (93) Google Scholar). Transport was terminated by rapidly pouring the contents onto cellulose-nitrate filters (0.45-μm pore size), which were washed twice with 4 ml of 100 mm LiCl, and dried. Radioactivity was measured by liquid scintillation counting. To minimize nonspecific binding of substrate to filters, the filters were pre-soaked in 100 mm LiCl. The periplasm of a cydC mutant is more oxidized than that of a wild-type strain (12Goldman B.S. Gabbert K.K. Kranz R.G. J. Bacteriol. 1996; 178: 6338-6347Crossref PubMed Google Scholar). It seems plausible, therefore, that candidate substrates for the CydDC transporter are any reducing or oxygen-scavenging agents. Interestingly, the cydDC operon is adjacent to thetrxB (thioredoxin reductase) gene (9Poole R.K. Hatch L. Cleeter M.W.J. Gibson F. Cox G.B. Wu G. Mol. Microbiol. 1993; 10: 421-430Crossref Scopus (73) Google Scholar, 39Blattner F.R. Plunkett G. Bloch C.A. Perna N.T. Burland V. Riley M. Collado-Vides J. Glasner J.D. Rode C.K. Mayhew G.F. Gregor J. Davis N.W. Kirkpatrick H.A. Goeden M.A. Rose D.J. Mau B. Shao Y. Science. 1997; 277: 1453-1462Crossref PubMed Scopus (6019) Google Scholar) on the E. coli chromosome but trxB mutants do synthesize cytochromes c and bd (10Poole R.K. Gibson F. Wu G. FEMS Lett. 1994; 117: 217-224Crossref Scopus (79) Google Scholar), ruling out TrxB as a candidate substrate. Mutants defective in trxA (encoding thioredoxin) and grx (glutaredoxin) also synthesize cytochrome bd (15Cook G.M. Poole RK Microbiology. 2000; 146: 527-536Crossref PubMed Scopus (35) Google Scholar). Although trxA mutants are unable to assemble c-type cytochromes unless complemented with 2-mercaptoethanesulfonic acid (40Reid E. Eaves D.J. Cole J.A. FEMS Microbiol. Lett. 1998; 166: 369-375Crossref PubMed Scopus (41) Google Scholar), this demonstrates that TrxA is not essential for cytochrome bd assembly either. However, a redox protein other than TrxB, thioredoxin, or glutaredoxin remains an intriguing candidate, as this would explain the plethora of redox-associated phenotypes of cydDC mutants. We therefore sought a protein that might be transported by CydDC by using two-dimensional SDS-PAGE and N-terminal sequencing to analyze periplasmic fractions of wild-type and cydD strains. Marker enzyme assays on both periplasmic and cytoplasmic fractions revealed <5% contamination by cytoplasmic and periplasmic enzymes, respectively (results not shown). Comparison of two-dimensional gels (Fig.1 and TableI) revealed several major differences, and, of the spots chosen for excision and subsequent Edman degradation, all were found to be periplasmic proteins, the determined sequences of which began after a signal sequence. This strongly suggests that all proteins identified were exported from the cytoplasm to the periplasm by a Sec-dependent mechanism (41Pugsley A.P. Microbiol. Rev. 1993; 57: 50-108Crossref PubMed Google Scholar). The proteins represented by spots 1 and 9 were identified as OppA (42Andrews J.C. Blevins T.C. Short S.A. J. Bacteriol. 1986; 165: 428-433Crossref PubMed Google Scholar) and AnsB (43Bonthon D.T. Gene (Amst.). 1990; 91: 101-105Crossref PubMed Scopus (25) Google Scholar), respectively, and were expressed at significantly higher levels in the periplasm of the wild type than that of the mutant (Fig. 1). A minor spot (number 8) was also OppA and may result from post-translational alteration or modification of lysine residues during electrophoresis (44Gooley A.A. Packer N.H. Proteome Research: New Frontiers in Functional Genomics. Springer, Berlin1997: 63-69Google Scholar). Proteins OsmY (45Yim H.H. Villarejo M. J. Bacteriol. 1992; 174: 3637-3644Crossref PubMed Scopus (116) Google Scholar) and HisJ (46Kustu S.G. McFarland N.C. Hiu S.P. Esmon B. Ames G.F. J. Bacteriol. 1979; 138: 218-234Crossref PubMed Google Scholar) (spots 5 and 6, respectively) were expressed at slightly more elevated levels in the cydDmutant periplasm compared with that of the wild type (Fig 1). The remaining five sequenced proteins (MalE, GlnH, ProX, HisJ, and DppA) were expressed at slightly higher levels in the wild type compared with the cydD mutant periplasm and are the periplasmic binding-proteins of secondary type transport systems in E. coli (see Ref. 47Boos W. Lucht J.M. Neidhardt F.C. Curtiss R. Ingraham J.L. Lin E.C.C. Low K.B. Magasanik B. Reznikoff W.S. Riley M. Schaechter M. Umbarger H.E. Escherichia coli and Salmonella: Cellular and Molecular Biology. 2nd Ed. American Society for Microbiology Press, Washington, D. C.1996: 1175-1209Google Scholar and references therein). Transport mechanisms for all of these proteins are already established, so it seems unlikely that they are substrates of the CydDC transporter.Table IN-terminal sequences of proteins extracted from selected spots in two-dimensional PAGE gels of periplasmic fractionsProteinSequence (with amino acid residue nos.)Enzyme functionpI1-aLiterature values.M r1-aLiterature values.Accession nos.kDaSpot 1 (OppA)27ADVPAGVTLAEK38Periplasmic oligopeptide-binding protein precursor5.8558.36P23843Spot 2 (DppA)29KTLVYXSEGDPE40Periplasmic dipeptide transport protein precursor (dipeptide-binding protein)5.7557.41P23847Spot 3 (MalE)27KIEEGKLVIWIN38Maltose-binding periplasmic protein precursor5.2240.71P02928Spot 4 (ProX)22ADLPGKGITVNPVQ35High affinity glycine betaine-binding protein5.6533.73P14177Spot 5 (OsmY)32TTNESAGQKND42Periplasmic, sigma S-dependent protein5.4218.16P27291Spot 6 (HisJ)23AIPQNIRI30Histidine-binding protein of high affinity5.1726.23P39182Spot 7Not availableNear 5.8∼50Spot 8 (OppA)27ADVPAG32Same as spot 15.8558.36P23843Spot 9 (AnsB)23LPNITILA30l-Asparaginase II (precursor)5.6634.59P00805Spot 10 (GlnH)23ADKKLVVAT31Glutamine-binding periplasmic protein6.8724.96P103441-a Literature values. Open table in a new tab Many of the well documented phenotypes associated with loss of CydDC are actually attributable to the consequent loss of cytochrome bd (48Goldman B.S. Gabber
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