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. 2001 Apr 1;355(Pt 1):51–58. doi: 10.1042/0264-6021:3550051

The Escherichia coli CcmG protein fulfils a specific role in cytochrome c assembly.

E Reid 1, J Cole 1, D J Eaves 1
PMCID: PMC1221711  PMID: 11256948

Abstract

In Escherichia coli K-12, c-type cytochromes are synthesized only during anaerobic growth with trimethylamine-N-oxide, nitrite or low concentrations of nitrate as the terminal electron acceptor. A thioredoxin-like protein, CcmG, is one of 12 proteins required for their assembly in the periplasm. Its postulated function is to reduce disulphide bonds formed between correctly paired cysteine residues in the cytochrome c apoproteins prior to haem attachment by CcmF and CcmH. We report that loss of CcmG synthesis by mutation was not compensated by a second mutation in disulphide-bond-forming proteins, DsbA or DsbB, or by the chemical reductant, 2-mercaptoethanesulphonic acid. An anti-CcmG polyclonal antibody was used in Western-blot analysis to probe the redox state of CcmG in mutants defective in the synthesis of other proteins essential for cytochrome c assembly. The oxidized form of CcmG accumulated not only in trxA or dipZ mutants defective in the transfer of electrons from the cytoplasm for disulphide isomerization and reduction reactions in the periplasm, but also in ccmF and ccmH mutants. The requirement of both CcmF and CcmH for the reduction of the disulphide bond in CcmG indicates that CcmG functions later than CcmF and CcmH in cytochrome c assembly, rather than in electron transfer from the membrane-associated DipZ (also known as DsbD) to CcmH. The data support a model proposed by others in which CcmG catalyses one of the last reactions specific to cytochrome c assembly.

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Selected References

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  1. Bader M., Muse W., Ballou D. P., Gassner C., Bardwell J. C. Oxidative protein folding is driven by the electron transport system. Cell. 1999 Jul 23;98(2):217–227. doi: 10.1016/s0092-8674(00)81016-8. [DOI] [PubMed] [Google Scholar]
  2. Bardwell J. C., Lee J. O., Jander G., Martin N., Belin D., Beckwith J. A pathway for disulfide bond formation in vivo. Proc Natl Acad Sci U S A. 1993 Feb 1;90(3):1038–1042. doi: 10.1073/pnas.90.3.1038. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Beckman D. L., Kranz R. G. Cytochromes c biogenesis in a photosynthetic bacterium requires a periplasmic thioredoxin-like protein. Proc Natl Acad Sci U S A. 1993 Mar 15;90(6):2179–2183. doi: 10.1073/pnas.90.6.2179. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chung J., Chen T., Missiakas D. Transfer of electrons across the cytoplasmic membrane by DsbD, a membrane protein involved in thiol-disulphide exchange and protein folding in the bacterial periplasm. Mol Microbiol. 2000 Mar;35(5):1099–1109. doi: 10.1046/j.1365-2958.2000.01778.x. [DOI] [PubMed] [Google Scholar]
  5. Crooke H., Cole J. The biogenesis of c-type cytochromes in Escherichia coli requires a membrane-bound protein, DipZ, with a protein disulphide isomerase-like domain. Mol Microbiol. 1995 Mar;15(6):1139–1150. doi: 10.1111/j.1365-2958.1995.tb02287.x. [DOI] [PubMed] [Google Scholar]
  6. Darwin A., Hussain H., Griffiths L., Grove J., Sambongi Y., Busby S., Cole J. Regulation and sequence of the structural gene for cytochrome c552 from Escherichia coli: not a hexahaem but a 50 kDa tetrahaem nitrite reductase. Mol Microbiol. 1993 Sep;9(6):1255–1265. doi: 10.1111/j.1365-2958.1993.tb01255.x. [DOI] [PubMed] [Google Scholar]
  7. Eaves D. J., Grove J., Staudenmann W., James P., Poole R. K., White S. A., Griffiths I., Cole J. A. Involvement of products of the nrfEFG genes in the covalent attachment of haem c to a novel cysteine-lysine motif in the cytochrome c552 nitrite reductase from Escherichia coli. Mol Microbiol. 1998 Apr;28(1):205–216. doi: 10.1046/j.1365-2958.1998.00792.x. [DOI] [PubMed] [Google Scholar]
  8. Einsle O., Messerschmidt A., Stach P., Bourenkov G. P., Bartunik H. D., Huber R., Kroneck P. M. Structure of cytochrome c nitrite reductase. Nature. 1999 Jul 29;400(6743):476–480. doi: 10.1038/22802. [DOI] [PubMed] [Google Scholar]
  9. Fabianek R. A., Hennecke H., Thöny-Meyer L. The active-site cysteines of the periplasmic thioredoxin-like protein CcmG of Escherichia coli are important but not essential for cytochrome c maturation in vivo. J Bacteriol. 1998 Apr;180(7):1947–1950. doi: 10.1128/jb.180.7.1947-1950.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Fabianek R. A., Hofer T., Thöny-Meyer L. Characterization of the Escherichia coli CcmH protein reveals new insights into the redox pathway required for cytochrome c maturation. Arch Microbiol. 1999 Jan;171(2):92–100. doi: 10.1007/s002030050683. [DOI] [PubMed] [Google Scholar]
  11. Fabianek R. A., Huber-Wunderlich M., Glockshuber R., Künzler P., Hennecke H., Thöny-Meyer L. Characterization of the Bradyrhizobium japonicum CycY protein, a membrane-anchored periplasmic thioredoxin that may play a role as a reductant in the biogenesis of c-type cytochromes. J Biol Chem. 1997 Feb 14;272(7):4467–4473. doi: 10.1074/jbc.272.7.4467. [DOI] [PubMed] [Google Scholar]
  12. Gordon E. H., Page M. D., Willis A. C., Ferguson S. J. Escherichia coli DipZ: anatomy of a transmembrane protein disulphide reductase in which three pairs of cysteine residues, one in each of three domains, contribute differentially to function. Mol Microbiol. 2000 Mar;35(6):1360–1374. doi: 10.1046/j.1365-2958.2000.01796.x. [DOI] [PubMed] [Google Scholar]
  13. Grovc J., Busby S., Cole J. The role of the genes nrf EFG and ccmFH in cytochrome c biosynthesis in Escherichia coli. Mol Gen Genet. 1996 Sep 13;252(3):332–341. doi: 10.1007/BF02173779. [DOI] [PubMed] [Google Scholar]
  14. Grove J., Tanapongpipat S., Thomas G., Griffiths L., Crooke H., Cole J. Escherichia coli K-12 genes essential for the synthesis of c-type cytochromes and a third nitrate reductase located in the periplasm. Mol Microbiol. 1996 Feb;19(3):467–481. doi: 10.1046/j.1365-2958.1996.383914.x. [DOI] [PubMed] [Google Scholar]
  15. Hussain H., Grove J., Griffiths L., Busby S., Cole J. A seven-gene operon essential for formate-dependent nitrite reduction to ammonia by enteric bacteria. Mol Microbiol. 1994 Apr;12(1):153–163. doi: 10.1111/j.1365-2958.1994.tb01004.x. [DOI] [PubMed] [Google Scholar]
  16. Iobbi-Nivol C., Crooke H., Griffiths L., Grove J., Hussain H., Pommier J., Mejean V., Cole J. A. A reassessment of the range of c-type cytochromes synthesized by Escherichia coli K-12. FEMS Microbiol Lett. 1994 Jun 1;119(1-2):89–94. doi: 10.1111/j.1574-6968.1994.tb06872.x. [DOI] [PubMed] [Google Scholar]
  17. Kobayashi T., Ito K. Respiratory chain strongly oxidizes the CXXC motif of DsbB in the Escherichia coli disulfide bond formation pathway. EMBO J. 1999 Mar 1;18(5):1192–1198. doi: 10.1093/emboj/18.5.1192. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kobayashi T., Kishigami S., Sone M., Inokuchi H., Mogi T., Ito K. Respiratory chain is required to maintain oxidized states of the DsbA-DsbB disulfide bond formation system in aerobically growing Escherichia coli cells. Proc Natl Acad Sci U S A. 1997 Oct 28;94(22):11857–11862. doi: 10.1073/pnas.94.22.11857. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kranz R., Lill R., Goldman B., Bonnard G., Merchant S. Molecular mechanisms of cytochrome c biogenesis: three distinct systems. Mol Microbiol. 1998 Jul;29(2):383–396. doi: 10.1046/j.1365-2958.1998.00869.x. [DOI] [PubMed] [Google Scholar]
  20. Lang S. E., Jenney F. E., Jr, Daldal F. Rhodobacter capsulatus CycH: a bipartite gene product with pleiotropic effects on the biogenesis of structurally different c-type cytochromes. J Bacteriol. 1996 Sep;178(17):5279–5290. doi: 10.1128/jb.178.17.5279-5290.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Loferer H., Bott M., Hennecke H. Bradyrhizobium japonicum TlpA, a novel membrane-anchored thioredoxin-like protein involved in the biogenesis of cytochrome aa3 and development of symbiosis. EMBO J. 1993 Sep;12(9):3373–3383. doi: 10.1002/j.1460-2075.1993.tb06011.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Macdonald H., Cole J. Molecular cloning and functional analysis of the cysG and nirB genes of Escherichia coli K12, two closely-linked genes required for NADH-dependent nitrite reductase activity. Mol Gen Genet. 1985;200(2):328–334. doi: 10.1007/BF00425444. [DOI] [PubMed] [Google Scholar]
  23. Metheringham R., Griffiths L., Crooke H., Forsythe S., Cole J. An essential role for DsbA in cytochrome c synthesis and formate-dependent nitrite reduction by Escherichia coli K-12. Arch Microbiol. 1995 Oct;164(4):301–307. doi: 10.1007/BF02529965. [DOI] [PubMed] [Google Scholar]
  24. Metheringham R., Tyson K. L., Crooke H., Missiakas D., Raina S., Cole J. A. Effects of mutations in genes for proteins involved in disulphide bond formation in the periplasm on the activities of anaerobically induced electron transfer chains in Escherichia coli K12. Mol Gen Genet. 1996 Nov 27;253(1-2):95–102. doi: 10.1007/pl00013815. [DOI] [PubMed] [Google Scholar]
  25. Missiakas D., Georgopoulos C., Raina S. Identification and characterization of the Escherichia coli gene dsbB, whose product is involved in the formation of disulfide bonds in vivo. Proc Natl Acad Sci U S A. 1993 Aug 1;90(15):7084–7088. doi: 10.1073/pnas.90.15.7084. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Missiakas D., Raina S. Protein folding in the bacterial periplasm. J Bacteriol. 1997 Apr;179(8):2465–2471. doi: 10.1128/jb.179.8.2465-2471.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Missiakas D., Schwager F., Raina S. Identification and characterization of a new disulfide isomerase-like protein (DsbD) in Escherichia coli. EMBO J. 1995 Jul 17;14(14):3415–3424. doi: 10.1002/j.1460-2075.1995.tb07347.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Monika E. M., Goldman B. S., Beckman D. L., Kranz R. G. A thioreduction pathway tethered to the membrane for periplasmic cytochromes c biogenesis; in vitro and in vivo studies. J Mol Biol. 1997 Sep 5;271(5):679–692. doi: 10.1006/jmbi.1997.1227. [DOI] [PubMed] [Google Scholar]
  29. Méjean V., Iobbi-Nivol C., Lepelletier M., Giordano G., Chippaux M., Pascal M. C. TMAO anaerobic respiration in Escherichia coli: involvement of the tor operon. Mol Microbiol. 1994 Mar;11(6):1169–1179. doi: 10.1111/j.1365-2958.1994.tb00393.x. [DOI] [PubMed] [Google Scholar]
  30. Page M. D., Ferguson S. J. Paracoccus denitrificans CcmG is a periplasmic protein-disulphide oxidoreductase required for c- and aa3-type cytochrome biogenesis; evidence for a reductase role in vivo. Mol Microbiol. 1997 Jun;24(5):977–990. doi: 10.1046/j.1365-2958.1997.4061775.x. [DOI] [PubMed] [Google Scholar]
  31. Page M. D., Sambongi Y., Ferguson S. J. Contrasting routes of c-type cytochrome assembly in mitochondria, chloroplasts and bacteria. Trends Biochem Sci. 1998 Mar;23(3):103–108. doi: 10.1016/s0968-0004(98)01173-6. [DOI] [PubMed] [Google Scholar]
  32. Pope N. R., Cole J. A. Generation of a membrane potential by one of two independent pathways for nitrite reduction by Escherichia coli. J Gen Microbiol. 1982 Jan;128(1):219–222. doi: 10.1099/00221287-128-1-219. [DOI] [PubMed] [Google Scholar]
  33. Pope N. R., Cole J. A. Pyruvate and ethanol as electron donors for nitrite reduction by Escherichia coli K12. J Gen Microbiol. 1984 May;130(5):1279–1284. doi: 10.1099/00221287-130-5-1279. [DOI] [PubMed] [Google Scholar]
  34. Raina S., Missiakas D. Making and breaking disulfide bonds. Annu Rev Microbiol. 1997;51:179–202. doi: 10.1146/annurev.micro.51.1.179. [DOI] [PubMed] [Google Scholar]
  35. Reid E., Eaves D. J., Cole J. A. The CcmE protein from Escherichia coli is a haem-binding protein. FEMS Microbiol Lett. 1998 Sep 15;166(2):369–375. doi: 10.1111/j.1574-6968.1998.tb13914.x. [DOI] [PubMed] [Google Scholar]
  36. Rietsch A., Belin D., Martin N., Beckwith J. An in vivo pathway for disulfide bond isomerization in Escherichia coli. Proc Natl Acad Sci U S A. 1996 Nov 12;93(23):13048–13053. doi: 10.1073/pnas.93.23.13048. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Rietsch A., Bessette P., Georgiou G., Beckwith J. Reduction of the periplasmic disulfide bond isomerase, DsbC, occurs by passage of electrons from cytoplasmic thioredoxin. J Bacteriol. 1997 Nov;179(21):6602–6608. doi: 10.1128/jb.179.21.6602-6608.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Sambongi Y., Ferguson S. J. Specific thiol compounds complement deficiency in c-type cytochrome biogenesis in Escherichia coli carrying a mutation in a membrane-bound disulphide isomerase-like protein. FEBS Lett. 1994 Oct 24;353(3):235–238. doi: 10.1016/0014-5793(94)01053-6. [DOI] [PubMed] [Google Scholar]
  39. Schulz H., Fabianek R. A., Pellicioli E. C., Hennecke H., Thöny-Meyer L. Heme transfer to the heme chaperone CcmE during cytochrome c maturation requires the CcmC protein, which may function independently of the ABC-transporter CcmAB. Proc Natl Acad Sci U S A. 1999 May 25;96(11):6462–6467. doi: 10.1073/pnas.96.11.6462. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Schulz H., Hennecke H., Thöny-Meyer L. Prototype of a heme chaperone essential for cytochrome c maturation. Science. 1998 Aug 21;281(5380):1197–1200. doi: 10.1126/science.281.5380.1197. [DOI] [PubMed] [Google Scholar]
  41. Stewart E. J., Katzen F., Beckwith J. Six conserved cysteines of the membrane protein DsbD are required for the transfer of electrons from the cytoplasm to the periplasm of Escherichia coli. EMBO J. 1999 Nov 1;18(21):5963–5971. doi: 10.1093/emboj/18.21.5963. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Thomas P. E., Ryan D., Levin W. An improved staining procedure for the detection of the peroxidase activity of cytochrome P-450 on sodium dodecyl sulfate polyacrylamide gels. Anal Biochem. 1976 Sep;75(1):168–176. doi: 10.1016/0003-2697(76)90067-1. [DOI] [PubMed] [Google Scholar]
  43. Throne-Holst M., Thöny-Meyer L., Hederstedt L. Escherichia coli ccm in-frame deletion mutants can produce periplasmic cytochrome b but not cytochrome c. FEBS Lett. 1997 Jun 30;410(2-3):351–355. doi: 10.1016/s0014-5793(97)00656-x. [DOI] [PubMed] [Google Scholar]
  44. Thöny-Meyer L. Biogenesis of respiratory cytochromes in bacteria. Microbiol Mol Biol Rev. 1997 Sep;61(3):337–376. doi: 10.1128/mmbr.61.3.337-376.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Thöny-Meyer L., Fischer F., Künzler P., Ritz D., Hennecke H. Escherichia coli genes required for cytochrome c maturation. J Bacteriol. 1995 Aug;177(15):4321–4326. doi: 10.1128/jb.177.15.4321-4326.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Thöny-Meyer L., Künzler P. Translocation to the periplasm and signal sequence cleavage of preapocytochrome c depend on sec and lep, but not on the ccm gene products. Eur J Biochem. 1997 Jun 15;246(3):794–799. doi: 10.1111/j.1432-1033.1997.t01-1-00794.x. [DOI] [PubMed] [Google Scholar]
  47. Vargas C., Wu G., Davies A. E., Downie J. A. Identification of a gene encoding a thioredoxin-like product necessary for cytochrome c biosynthesis and symbiotic nitrogen fixation in Rhizobium leguminosarum. J Bacteriol. 1994 Jul;176(13):4117–4123. doi: 10.1128/jb.176.13.4117-4123.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Zeng H., Snavely I., Zamorano P., Javor G. T. Low ubiquinone content in Escherichia coli causes thiol hypersensitivity. J Bacteriol. 1998 Jul;180(14):3681–3685. doi: 10.1128/jb.180.14.3681-3685.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]

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