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. 1997 Jul;179(14):4523–4529. doi: 10.1128/jb.179.14.4523-4529.1997

Bacillus subtilis CcdA-defective mutants are blocked in a late step of cytochrome c biogenesis.

T Schiött 1, M Throne-Holst 1, L Hederstedt 1
PMCID: PMC179287  PMID: 9226261

Abstract

Cytochromes of the c type contain covalently bound heme. In bacteria, they are located on the outside of the cytoplasmic membrane. Cytochrome c synthesis involves export of heme and apocytochrome across the cytoplasmic membrane followed by ligation of heme to the polypeptide. Using radioactive protoheme IX produced in Escherichia coli, we show that Bacillus subtilis can use heme from the growth medium for cytochrome c synthesis. The B. subtilis ccdA gene encodes a 26-kDa integral membrane protein which is required for cytochrome c synthesis (T. Schiött et al., J. Bacteriol. 179:1962-1973, 1997). In this work, we analyzed the stage at which cytochrome c synthesis is blocked in a ccdA deletion mutant. The following steps were found to be normal in the mutant: (i) transcription and translation of cytochrome c structural genes, (ii) translocation of apocytochrome across the cytoplasmic membrane, and (iii) heme transport from the cytoplasm to cytochrome polypeptide on the outer side of the cytoplasmic membrane. It is concluded that CcdA is required for a late step in the cytochrome c synthesis pathway.

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Selected References

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  1. Arwert F., Venema G. Transformation in Bacillus subtilis. Fate of newly introduced transforming DNA. Mol Gen Genet. 1973;123(2):185–198. doi: 10.1007/BF00267334. [DOI] [PubMed] [Google Scholar]
  2. Bardwell J. C., McGovern K., Beckwith J. Identification of a protein required for disulfide bond formation in vivo. Cell. 1991 Nov 1;67(3):581–589. doi: 10.1016/0092-8674(91)90532-4. [DOI] [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. Beckman D. L., Trawick D. R., Kranz R. G. Bacterial cytochromes c biogenesis. Genes Dev. 1992 Feb;6(2):268–283. doi: 10.1101/gad.6.2.268. [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. Derman A. I., Beckwith J. Escherichia coli alkaline phosphatase fails to acquire disulfide bonds when retained in the cytoplasm. J Bacteriol. 1991 Dec;173(23):7719–7722. doi: 10.1128/jb.173.23.7719-7722.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Fortnagel P., Freese E. Analysis of sporulation mutants. II. Mutants blocked in the citric acid cycle. J Bacteriol. 1968 Apr;95(4):1431–1438. doi: 10.1128/jb.95.4.1431-1438.1968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Friedmann H. C., Baldwin E. T. Reverse-phase purification and silica gel thin-layer chromatography of porphyrin carboxylic acids. Anal Biochem. 1984 Mar;137(2):473–480. doi: 10.1016/0003-2697(84)90115-5. [DOI] [PubMed] [Google Scholar]
  9. Goldman B. S., Gabbert K. K., Kranz R. G. Use of heme reporters for studies of cytochrome biosynthesis and heme transport. J Bacteriol. 1996 Nov;178(21):6338–6347. doi: 10.1128/jb.178.21.6338-6347.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. Haima P., Bron S., Venema G. The effect of restriction on shotgun cloning and plasmid stability in Bacillus subtilis Marburg. Mol Gen Genet. 1987 Sep;209(2):335–342. doi: 10.1007/BF00329663. [DOI] [PubMed] [Google Scholar]
  12. Hamada K., Bethge P. H., Mathews F. S. Refined structure of cytochrome b562 from Escherichia coli at 1.4 A resolution. J Mol Biol. 1995 Apr 14;247(5):947–962. doi: 10.1006/jmbi.1995.0192. [DOI] [PubMed] [Google Scholar]
  13. Hansson M., Hederstedt L. Purification and characterisation of a water-soluble ferrochelatase from Bacillus subtilis. Eur J Biochem. 1994 Feb 15;220(1):201–208. doi: 10.1111/j.1432-1033.1994.tb18615.x. [DOI] [PubMed] [Google Scholar]
  14. Hederstedt L. Molecular properties, genetics, and biosynthesis of Bacillus subtilis succinate dehydrogenase complex. Methods Enzymol. 1986;126:399–414. doi: 10.1016/s0076-6879(86)26040-1. [DOI] [PubMed] [Google Scholar]
  15. Hägerhäll C., Fridén H., Aasa R., Hederstedt L. Transmembrane topology and axial ligands to hemes in the cytochrome b subunit of Bacillus subtilis succinate:menaquinone reductase. Biochemistry. 1995 Sep 5;34(35):11080–11089. doi: 10.1021/bi00035a013. [DOI] [PubMed] [Google Scholar]
  16. Jacobs M., Andersen J. B., Kontinen V., Sarvas M. Bacillus subtilis PrsA is required in vivo as an extracytoplasmic chaperone for secretion of active enzymes synthesized either with or without pro-sequences. Mol Microbiol. 1993 May;8(5):957–966. doi: 10.1111/j.1365-2958.1993.tb01640.x. [DOI] [PubMed] [Google Scholar]
  17. Kutoh E., Sone N. Quinol-cytochrome c oxidoreductase from the thermophilic bacterium PS3. Purification and properties of a cytochrome bc1(b6f) complex. J Biol Chem. 1988 Jun 25;263(18):9020–9026. [PubMed] [Google Scholar]
  18. Lauraeus M., Haltia T., Saraste M., Wikström M. Bacillus subtilis expresses two kinds of haem-A-containing terminal oxidases. Eur J Biochem. 1991 May 8;197(3):699–705. doi: 10.1111/j.1432-1033.1991.tb15961.x. [DOI] [PubMed] [Google Scholar]
  19. Louie G. V., Brownlie P. D., Lambert R., Cooper J. B., Blundell T. L., Wood S. P., Warren M. J., Woodcock S. C., Jordan P. M. Structure of porphobilinogen deaminase reveals a flexible multidomain polymerase with a single catalytic site. Nature. 1992 Sep 3;359(6390):33–39. doi: 10.1038/359033a0. [DOI] [PubMed] [Google Scholar]
  20. MacGregor C. H. Biosynthesis of membrane-bound nitrate reductase in Escherichia coli: evidence for a soluble precursor. J Bacteriol. 1976 Apr;126(1):122–131. doi: 10.1128/jb.126.1.122-131.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Magnusson K., Hederstedt L., Rutberg L. Cloning and expression in Escherichia coli of sdhA, the structural gene for cytochrome b558 of the Bacillus subtilis succinate dehydrogenase complex. J Bacteriol. 1985 Jun;162(3):1180–1185. doi: 10.1128/jb.162.3.1180-1185.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Merchante R., Pooley H. M., Karamata D. A periplasm in Bacillus subtilis. J Bacteriol. 1995 Nov;177(21):6176–6183. doi: 10.1128/jb.177.21.6176-6183.1995. [DOI] [PMC free article] [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. 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]
  25. Moore G. R., Williams R. J., Peterson J., Thomson A. J., Mathews F. S. A spectroscopic investigation of the structure and redox properties of Escherichia coli cytochrome b-562. Biochim Biophys Acta. 1985 May 20;829(1):83–96. doi: 10.1016/0167-4838(85)90071-8. [DOI] [PubMed] [Google Scholar]
  26. Nikkila H., Gennis R. B., Sligar S. G. Cloning and expression of the gene encoding the soluble cytochrome b562 of Escherichia coli. Eur J Biochem. 1991 Dec 5;202(2):309–313. doi: 10.1111/j.1432-1033.1991.tb16377.x. [DOI] [PubMed] [Google Scholar]
  27. Poole R. K., Gibson F., Wu G. The cydD gene product, component of a heterodimeric ABC transporter, is required for assembly of periplasmic cytochrome c and of cytochrome bd in Escherichia coli. FEMS Microbiol Lett. 1994 Apr 1;117(2):217–223. doi: 10.1111/j.1574-6968.1994.tb06768.x. [DOI] [PubMed] [Google Scholar]
  28. Privé G. G., Verner G. E., Weitzman C., Zen K. H., Eisenberg D., Kaback H. R. Fusion proteins as tools for crystallization: the lactose permease from Escherichia coli. Acta Crystallogr D Biol Crystallogr. 1994 Jul 1;50(Pt 4):375–379. doi: 10.1107/S0907444993014301. [DOI] [PubMed] [Google Scholar]
  29. Ramseier T. M., Winteler H. V., Hennecke H. Discovery and sequence analysis of bacterial genes involved in the biogenesis of c-type cytochromes. J Biol Chem. 1991 Apr 25;266(12):7793–7803. [PubMed] [Google Scholar]
  30. Ritz D., Bott M., Hennecke H. Formation of several bacterial c-type cytochromes requires a novel membrane-anchored protein that faces the periplasm. Mol Microbiol. 1993 Aug;9(4):729–740. doi: 10.1111/j.1365-2958.1993.tb01733.x. [DOI] [PubMed] [Google Scholar]
  31. Ritz D., Thöny-Meyer L., Hennecke H. The cycHJKL gene cluster plays an essential role in the biogenesis of c-type cytochromes in Bradyrhizobium japonicum. Mol Gen Genet. 1995 Apr 10;247(1):27–38. doi: 10.1007/BF00425818. [DOI] [PubMed] [Google Scholar]
  32. Sambongi Y., Crooke H., Cole J. A., Ferguson S. J. A mutation blocking the formation of membrane or periplasmic endogenous and exogenous c-type cytochromes in Escherichia coli permits the cytoplasmic formation of Hydrogenobacter thermophilus holo cytochrome c552. FEBS Lett. 1994 May 16;344(2-3):207–210. doi: 10.1016/0014-5793(94)00399-8. [DOI] [PubMed] [Google Scholar]
  33. Sambongi Y., Stoll R., Ferguson S. J. Alteration of haem-attachment and signal-cleavage sites for Paracoccus denitrificans cytochrome C550 probes pathway of c-type cytochrome biogenesis in Escherichia coli. Mol Microbiol. 1996 Mar;19(6):1193–1204. doi: 10.1111/j.1365-2958.1996.tb02465.x. [DOI] [PubMed] [Google Scholar]
  34. Santana M., Kunst F., Hullo M. F., Rapoport G., Danchin A., Glaser P. Molecular cloning, sequencing, and physiological characterization of the qox operon from Bacillus subtilis encoding the aa3-600 quinol oxidase. J Biol Chem. 1992 May 25;267(15):10225–10231. [PubMed] [Google Scholar]
  35. Schiött T., von Wachenfeldt C., Hederstedt L. Identification and characterization of the ccdA gene, required for cytochrome c synthesis in Bacillus subtilis. J Bacteriol. 1997 Mar;179(6):1962–1973. doi: 10.1128/jb.179.6.1962-1973.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Schägger H., von Jagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem. 1987 Nov 1;166(2):368–379. doi: 10.1016/0003-2697(87)90587-2. [DOI] [PubMed] [Google Scholar]
  37. Sone N., Fujiwara Y. Effects of aeration during growth of Bacillus stearothermophilus on proton pumping activity and change of terminal oxidases. J Biochem. 1991 Dec;110(6):1016–1021. doi: 10.1093/oxfordjournals.jbchem.a123671. [DOI] [PubMed] [Google Scholar]
  38. Sone N., Tsuchiya N., Inoue M., Noguchi S. Bacillus stearothermophilus qcr operon encoding rieske FeS protein, cytochrome b6, and a novel-type cytochrome c1 of quinol-cytochrome c reductase. J Biol Chem. 1996 May 24;271(21):12457–12462. doi: 10.1074/jbc.271.21.12457. [DOI] [PubMed] [Google Scholar]
  39. Steinrücke P., Gerhus E., Ludwig B. Paracoccus denitrificans mutants deleted in the gene for subunit II of cytochrome c oxidase also lack subunit I. J Biol Chem. 1991 Apr 25;266(12):7676–7681. [PubMed] [Google Scholar]
  40. Tanaka T., Inoue M., Sakamoto J., Sone N. Intra- and inter-complex cross-linking of subunits in the quinol oxidase super-complex from thermophilic Bacillus PS3. J Biochem. 1996 Mar;119(3):482–486. doi: 10.1093/oxfordjournals.jbchem.a021267. [DOI] [PubMed] [Google Scholar]
  41. 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]
  42. Thöny-Meyer L., Ritz D., Hennecke H. Cytochrome c biogenesis in bacteria: a possible pathway begins to emerge. Mol Microbiol. 1994 Apr;12(1):1–9. doi: 10.1111/j.1365-2958.1994.tb00988.x. [DOI] [PubMed] [Google Scholar]
  43. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]
  44. Yu J., Hederstedt L., Piggot P. J. The cytochrome bc complex (menaquinone:cytochrome c reductase) in Bacillus subtilis has a nontraditional subunit organization. J Bacteriol. 1995 Dec;177(23):6751–6760. doi: 10.1128/jb.177.23.6751-6760.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. van der Oost J., von Wachenfeld C., Hederstedt L., Saraste M. Bacillus subtilis cytochrome oxidase mutants: biochemical analysis and genetic evidence for two aa3-type oxidases. Mol Microbiol. 1991 Aug;5(8):2063–2072. doi: 10.1111/j.1365-2958.1991.tb00829.x. [DOI] [PubMed] [Google Scholar]
  46. von Wachenfeldt C., Hederstedt L. Bacillus subtilis 13-kilodalton cytochrome c-550 encoded by cccA consists of a membrane-anchor and a heme domain. J Biol Chem. 1990 Aug 15;265(23):13939–13948. [PubMed] [Google Scholar]
  47. von Wachenfeldt C., Hederstedt L. Bacillus subtilis holo-cytochrome c-550 can be synthesised in aerobic Escherichia coli. FEBS Lett. 1990 Sep 17;270(1-2):147–151. doi: 10.1016/0014-5793(90)81255-m. [DOI] [PubMed] [Google Scholar]
  48. von Wachenfeldt C., Hederstedt L. Physico-chemical characterisation of membrane-bound and water-soluble forms of Bacillus subtilis cytochrome c-550. Eur J Biochem. 1993 Mar 1;212(2):499–509. doi: 10.1111/j.1432-1033.1993.tb17687.x. [DOI] [PubMed] [Google Scholar]

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