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. 1990 May;172(5):2392–2400. doi: 10.1128/jb.172.5.2392-2400.1990

Paracoccus denitrificans cytochrome c1 gene replacement mutants.

E Gerhus 1, P Steinrücke 1, B Ludwig 1
PMCID: PMC208874  PMID: 2158969

Abstract

We describe the construction and characterization of gene replacement mutants for the respiratory chain component cytochrome c1 in the bacterium Paracoccus denitrificans. Its structural gene (fbcC) was inactivated by insertion of the kanamycin resistance gene, introduced into a suicide vector, and conjugated into Paracoccus; chromosomal mutants obtained by homologous recombination were selected by antibiotic resistance screening and further characterized biochemically. They showed the complete spectral, enzymatic, and immunological loss of the fbcC gene product together with a serious defect in the assembly of the two other gene products of the fbc operon, cytochrome b and the FeS protein. A possible role of the cytochrome c1 in the assembly process for the enzyme complex is discussed. A functional restoration to wild-type phenotype was achieved by complementing in trans with a newly constructed broad-host-range vector carrying the fbcC gene cassette. When the complete fbc operon was present on this vector, overexpression of complex III subunits was observed. Apart from their physiological significance, such mutants are a prerequisite for probing structure-function relationships by site-directed mutagenesis in order to understand molecular details of electron transport and energy transduction processes of this respiratory enzyme in bacteria and in mitochondria.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Anraku Y. Bacterial electron transport chains. Annu Rev Biochem. 1988;57:101–132. doi: 10.1146/annurev.bi.57.070188.000533. [DOI] [PubMed] [Google Scholar]
  2. Beck E., Ludwig G., Auerswald E. A., Reiss B., Schaller H. Nucleotide sequence and exact localization of the neomycin phosphotransferase gene from transposon Tn5. Gene. 1982 Oct;19(3):327–336. doi: 10.1016/0378-1119(82)90023-3. [DOI] [PubMed] [Google Scholar]
  3. Beckmann J. D., Ljungdahl P. O., Trumpower B. L. Mutational analysis of the mitochondrial Rieske iron-sulfur protein of Saccharomyces cerevisiae. I. Construction of a RIP1 deletion strain and isolation of temperature-sensitive mutants. J Biol Chem. 1989 Mar 5;264(7):3713–3722. [PubMed] [Google Scholar]
  4. Berry E. A., Trumpower B. L. Isolation of ubiquinol oxidase from Paracoccus denitrificans and resolution into cytochrome bc1 and cytochrome c-aa3 complexes. J Biol Chem. 1985 Feb 25;260(4):2458–2467. [PubMed] [Google Scholar]
  5. Crivellone M. D., Wu M. A., Tzagoloff A. Assembly of the mitochondrial membrane system. Analysis of structural mutants of the yeast coenzyme QH2-cytochrome c reductase complex. J Biol Chem. 1988 Oct 5;263(28):14323–14333. [PubMed] [Google Scholar]
  6. Davidson E., Daldal F. Primary structure of the bc1 complex of Rhodopseudomonas capsulata. Nucleotide sequence of the pet operon encoding the Rieske cytochrome b, and cytochrome c1 apoproteins. J Mol Biol. 1987 May 5;195(1):13–24. doi: 10.1016/0022-2836(87)90323-8. [DOI] [PubMed] [Google Scholar]
  7. Ditta G., Schmidhauser T., Yakobson E., Lu P., Liang X. W., Finlay D. R., Guiney D., Helinski D. R. Plasmids related to the broad host range vector, pRK290, useful for gene cloning and for monitoring gene expression. Plasmid. 1985 Mar;13(2):149–153. doi: 10.1016/0147-619x(85)90068-x. [DOI] [PubMed] [Google Scholar]
  8. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  9. Gabellini N. Organization and structure of the genes for the cytochrome b/c1 complex in purple photosynthetic bacteria. A phylogenetic study describing the homology of the b/c1 subunits between prokaryotes, mitochondria, and chloroplasts. J Bioenerg Biomembr. 1988 Feb;20(1):59–83. doi: 10.1007/BF00762138. [DOI] [PubMed] [Google Scholar]
  10. Gabellini N., Sebald W. Nucleotide sequence and transcription of the fbc operon from Rhodopseudomonas sphaeroides. Evaluation of the deduced amino acid sequences of the FeS protein, cytochrome b and cytochrome c1. Eur J Biochem. 1986 Feb 3;154(3):569–579. doi: 10.1111/j.1432-1033.1986.tb09437.x. [DOI] [PubMed] [Google Scholar]
  11. Haltia T., Finel M., Harms N., Nakari T., Raitio M., Wikström M., Saraste M. Deletion of the gene for subunit III leads to defective assembly of bacterial cytochrome oxidase. EMBO J. 1989 Dec 1;8(12):3571–3579. doi: 10.1002/j.1460-2075.1989.tb08529.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Haltia T., Puustinen A., Finel M. The Paracoccus denitrificans cytochrome aa3 has a third subunit. Eur J Biochem. 1988 Mar 15;172(3):543–546. doi: 10.1111/j.1432-1033.1988.tb13923.x. [DOI] [PubMed] [Google Scholar]
  13. Hase T., Harabayashi M., Kawai K., Matsubara H. Expression of modified cytochrome c1 genes and restoration of the respiratory function in a yeast mutant lacking the nuclear cytochrome c1 gene. J Biochem. 1987 Aug;102(2):401–410. doi: 10.1093/oxfordjournals.jbchem.a122067. [DOI] [PubMed] [Google Scholar]
  14. Hauska G., Hurt E., Gabellini N., Lockau W. Comparative aspects of quinol-cytochrome c/plastocyanin oxidoreductases. Biochim Biophys Acta. 1983 Jul 15;726(2):97–133. doi: 10.1016/0304-4173(83)90002-2. [DOI] [PubMed] [Google Scholar]
  15. Hauska G., Nitschke W., Herrmann R. G. Amino acid identities in the three redox center-carrying polypeptides of cytochrome bc1/b6f complexes. J Bioenerg Biomembr. 1988 Apr;20(2):211–228. doi: 10.1007/BF00768395. [DOI] [PubMed] [Google Scholar]
  16. Henry M. F., Vignais P. M. Induction by cyanide of cytochrome d in the plasma membrane of Paracoccus denitrificans. FEBS Lett. 1979 Apr 1;100(1):41–46. doi: 10.1016/0014-5793(79)81127-8. [DOI] [PubMed] [Google Scholar]
  17. Howell N., Gilbert K. Mutational analysis of the mouse mitochondrial cytochrome b gene. J Mol Biol. 1988 Oct 5;203(3):607–618. doi: 10.1016/0022-2836(88)90195-7. [DOI] [PubMed] [Google Scholar]
  18. John P., Whatley F. R. The bioenergetics of Paracoccus denitrificans. Biochim Biophys Acta. 1977 Oct 5;463(2):129–153. doi: 10.1016/0304-4173(77)90006-4. [DOI] [PubMed] [Google Scholar]
  19. Kay R., McPherson J. Hybrid pUC vectors for addition of new restriction enzyme sites to the ends of DNA fragments. Nucleic Acids Res. 1987 Mar 25;15(6):2778–2778. doi: 10.1093/nar/15.6.2778. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kranz R. G. Isolation of mutants and genes involved in cytochromes c biosynthesis in Rhodobacter capsulatus. J Bacteriol. 1989 Jan;171(1):456–464. doi: 10.1128/jb.171.1.456-464.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kurowski B., Ludwig B. The genes of the Paracoccus denitrificans bc1 complex. Nucleotide sequence and homologies between bacterial and mitochondrial subunits. J Biol Chem. 1987 Oct 5;262(28):13805–13811. [PubMed] [Google Scholar]
  22. Lang F. S., Oesterhelt D. Gene transfer system for Rhodopseudomonas viridis. J Bacteriol. 1989 Aug;171(8):4425–4435. doi: 10.1128/jb.171.8.4425-4435.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Leary J. J., Brigati D. J., Ward D. C. Rapid and sensitive colorimetric method for visualizing biotin-labeled DNA probes hybridized to DNA or RNA immobilized on nitrocellulose: Bio-blots. Proc Natl Acad Sci U S A. 1983 Jul;80(13):4045–4049. doi: 10.1073/pnas.80.13.4045. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Ludwig B. Cytochrome c oxidase from Paracoccus denitrificans. Methods Enzymol. 1986;126:153–159. doi: 10.1016/s0076-6879(86)26017-6. [DOI] [PubMed] [Google Scholar]
  25. Ludwig B., Schatz G. A two-subunit cytochrome c oxidase (cytochrome aa3) from Paracoccus dentrificans. Proc Natl Acad Sci U S A. 1980 Jan;77(1):196–200. doi: 10.1073/pnas.77.1.196. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Ludwig B., Suda K., Cerletti N. Cytochrome c1 from Paracoccus denitrificans. Eur J Biochem. 1983 Dec 15;137(3):597–602. doi: 10.1111/j.1432-1033.1983.tb07867.x. [DOI] [PubMed] [Google Scholar]
  27. Marck C. 'DNA Strider': a 'C' program for the fast analysis of DNA and protein sequences on the Apple Macintosh family of computers. Nucleic Acids Res. 1988 Mar 11;16(5):1829–1836. doi: 10.1093/nar/16.5.1829. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Messing J., Crea R., Seeburg P. H. A system for shotgun DNA sequencing. Nucleic Acids Res. 1981 Jan 24;9(2):309–321. doi: 10.1093/nar/9.2.309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Messing J. New M13 vectors for cloning. Methods Enzymol. 1983;101:20–78. doi: 10.1016/0076-6879(83)01005-8. [DOI] [PubMed] [Google Scholar]
  30. Ohnishi T., Schägger H., Meinhardt S. W., LoBrutto R., Link T. A., von Jagow G. Spatial organization of redox active centers in the bovine heart ubiquinol-cytochrome c oxidoreductase. J Biol Chem. 1989 Jan 15;264(2):735–744. [PubMed] [Google Scholar]
  31. Prentki P., Krisch H. M. In vitro insertional mutagenesis with a selectable DNA fragment. Gene. 1984 Sep;29(3):303–313. doi: 10.1016/0378-1119(84)90059-3. [DOI] [PubMed] [Google Scholar]
  32. Puustinen A., Finel M., Virkki M., Wikström M. Cytochrome o (bo) is a proton pump in Paracoccus denitrificans and Escherichia coli. FEBS Lett. 1989 Jun 5;249(2):163–167. doi: 10.1016/0014-5793(89)80616-7. [DOI] [PubMed] [Google Scholar]
  33. Raitio M., Jalli T., Saraste M. Isolation and analysis of the genes for cytochrome c oxidase in Paracoccus denitrificans. EMBO J. 1987 Sep;6(9):2825–2833. doi: 10.1002/j.1460-2075.1987.tb02579.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Rich P. R. Electron and proton transfers through quinones and cytochrome bc complexes. Biochim Biophys Acta. 1984 Apr 9;768(1):53–79. doi: 10.1016/0304-4173(84)90007-7. [DOI] [PubMed] [Google Scholar]
  35. Saraste M. Location of haem-binding sites in the mitochondrial cytochrome b. FEBS Lett. 1984 Jan 30;166(2):367–372. doi: 10.1016/0014-5793(84)80114-3. [DOI] [PubMed] [Google Scholar]
  36. Schoppink P. J., Hemrika W., Reynen J. M., Grivell L. A., Berden J. A. Yeast ubiquinol: cytochrome c oxidoreductase is still active after inactivation of the gene encoding the 17-kDa subunit VI. Eur J Biochem. 1988 Apr 5;173(1):115–122. doi: 10.1111/j.1432-1033.1988.tb13974.x. [DOI] [PubMed] [Google Scholar]
  37. Simon R. High frequency mobilization of gram-negative bacterial replicons by the in vitro constructed Tn5-Mob transposon. Mol Gen Genet. 1984;196(3):413–420. doi: 10.1007/BF00436188. [DOI] [PubMed] [Google Scholar]
  38. Steinrücke P., Steffens G. C., Panskus G., Buse G., Ludwig B. Subunit II of cytochrome c oxidase from Paracoccus denitrificans. DNA sequence, gene expression and the protein. Eur J Biochem. 1987 Sep 15;167(3):431–439. doi: 10.1111/j.1432-1033.1987.tb13356.x. [DOI] [PubMed] [Google Scholar]
  39. Steinrücke P., Steffens G. C., Panskus G., Buse G., Ludwig B. Subunit II of cytochrome c oxidase from Paracoccus denitrificans. DNA sequence, gene expression and the protein. Eur J Biochem. 1987 Sep 15;167(3):431–439. doi: 10.1111/j.1432-1033.1987.tb13356.x. [DOI] [PubMed] [Google Scholar]
  40. Telser J., Hoffman B. M., LoBrutto R., Ohnishi T., Tsai A. L., Simpkin D., Palmer G. Evidence for N coordination to Fe in the [2Fe-2S] center in yeast mitochondrial complex III. Comparison with similar findings for analogous bacterial [2Fe-2S] proteins. FEBS Lett. 1987 Apr 6;214(1):117–121. doi: 10.1016/0014-5793(87)80024-8. [DOI] [PubMed] [Google Scholar]
  41. Van Verseveld H. W., Stouthamer A. H. Electron-transport chain and coupled oxidative phosphorylation in methanol-grown Paracoccus denitrificans. Arch Microbiol. 1978 Jul;118(1):13–20. doi: 10.1007/BF00406068. [DOI] [PubMed] [Google Scholar]
  42. Vandeyar M. A., Weiner M. P., Hutton C. J., Batt C. A. A simple and rapid method for the selection of oligodeoxynucleotide-directed mutants. Gene. 1988 May 15;65(1):129–133. doi: 10.1016/0378-1119(88)90425-8. [DOI] [PubMed] [Google Scholar]
  43. Vieira J., Messing J. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene. 1982 Oct;19(3):259–268. doi: 10.1016/0378-1119(82)90015-4. [DOI] [PubMed] [Google Scholar]
  44. Widger W. R., Cramer W. A., Herrmann R. G., Trebst A. Sequence homology and structural similarity between cytochrome b of mitochondrial complex III and the chloroplast b6-f complex: position of the cytochrome b hemes in the membrane. Proc Natl Acad Sci U S A. 1984 Feb;81(3):674–678. doi: 10.1073/pnas.81.3.674. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Wikström M., Krab K., Saraste M. Proton-translocating cytochrome complexes. Annu Rev Biochem. 1981;50:623–655. doi: 10.1146/annurev.bi.50.070181.003203. [DOI] [PubMed] [Google Scholar]
  46. Woese C. R. Bacterial evolution. Microbiol Rev. 1987 Jun;51(2):221–271. doi: 10.1128/mr.51.2.221-271.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Yang X. H., Trumpower B. L. Protonmotive Q cycle pathway of electron transfer and energy transduction in the three-subunit ubiquinol-cytochrome c oxidoreductase complex of Paracoccus denitrificans. J Biol Chem. 1988 Aug 25;263(24):11962–11970. [PubMed] [Google Scholar]
  48. Yang X. H., Trumpower B. L. Purification of a three-subunit ubiquinol-cytochrome c oxidoreductase complex from Paracoccus denitrificans. J Biol Chem. 1986 Sep 15;261(26):12282–12289. [PubMed] [Google Scholar]
  49. di Rago J. P., Colson A. M. Molecular basis for resistance to antimycin and diuron, Q-cycle inhibitors acting at the Qi site in the mitochondrial ubiquinol-cytochrome c reductase in Saccharomyces cerevisiae. J Biol Chem. 1988 Sep 5;263(25):12564–12570. [PubMed] [Google Scholar]
  50. von Jagow G., Link T. A. Use of specific inhibitors on the mitochondrial bc1 complex. Methods Enzymol. 1986;126:253–271. doi: 10.1016/s0076-6879(86)26026-7. [DOI] [PubMed] [Google Scholar]

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