Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1997 Mar;179(6):1962–1973. doi: 10.1128/jb.179.6.1962-1973.1997

Identification and characterization of the ccdA gene, required for cytochrome c synthesis in Bacillus subtilis.

T Schiött 1, C von Wachenfeldt 1, L Hederstedt 1
PMCID: PMC178920  PMID: 9068642

Abstract

The gram-positive, endospore-forming bacterium Bacillus subtilis contains several membrane-bound c-type cytochromes. We have isolated a mutant pleiotropically deficient in cytochromes c. The responsible mutation resides in a gene which we have named ccdA (cytochrome c defective). This gene is located at 173 degrees on the B. subtilis chromosome. The ccdA gene was found to be specifically required for synthesis of cytochromes of the c type. CcdA is a predicted 26-kDa integral membrane protein with no clear similarity to any known cytochrome c biogenesis protein but seems to be related to a part of Escherichia coli DipZ/DsbD. The ccdA gene is cotranscribed with two other genes. These genes encode a putative 13.5-kDa single-domain response regulator, similar to B. subtilis CheY and Spo0F, and a predicted 18-kDa hydrophobic protein with no similarity to any protein in databases, respectively. Inactivation of the three genes showed that only ccdA is required for cytochrome c synthesis. The results also demonstrated that cytochromes of the c type are not needed for growth of B. subtilis.

Full Text

The Full Text of this article is available as a PDF (600.2 KB).

Selected References

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

  1. Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403–410. doi: 10.1016/S0022-2836(05)80360-2. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. Azevedo V., Alvarez E., Zumstein E., Damiani G., Sgaramella V., Ehrlich S. D., Serror P. An ordered collection of Bacillus subtilis DNA segments cloned in yeast artificial chromosomes. Proc Natl Acad Sci U S A. 1993 Jul 1;90(13):6047–6051. doi: 10.1073/pnas.90.13.6047. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bairoch A., Apweiler R. The SWISS-PROT protein sequence data bank and its new supplement TREMBL. Nucleic Acids Res. 1996 Jan 1;24(1):21–25. doi: 10.1093/nar/24.1.21. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  6. 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]
  7. Bischoff D. S., Ordal G. W. Sequence and characterization of Bacillus subtilis CheB, a homolog of Escherichia coli CheY, and its role in a different mechanism of chemotaxis. J Biol Chem. 1991 Jul 5;266(19):12301–12305. [PubMed] [Google Scholar]
  8. Chamberlain J. P. Fluorographic detection of radioactivity in polyacrylamide gels with the water-soluble fluor, sodium salicylate. Anal Biochem. 1979 Sep 15;98(1):132–135. doi: 10.1016/0003-2697(79)90716-4. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dingman D. W., Sonenshein A. L. Purification of aconitase from Bacillus subtilis and correlation of its N-terminal amino acid sequence with the sequence of the citB gene. J Bacteriol. 1987 Jul;169(7):3062–3067. doi: 10.1128/jb.169.7.3062-3067.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Ferguson S. J. The functions and synthesis of bacterial c-type cytochromes with particular reference to Paracoccus denitrificans and Rhodobacter capsulatus. Biochim Biophys Acta. 1991 May 23;1058(1):17–20. doi: 10.1016/s0005-2728(05)80259-2. [DOI] [PubMed] [Google Scholar]
  13. Fleischmann R. D., Adams M. D., White O., Clayton R. A., Kirkness E. F., Kerlavage A. R., Bult C. J., Tomb J. F., Dougherty B. A., Merrick J. M. Whole-genome random sequencing and assembly of Haemophilus influenzae Rd. Science. 1995 Jul 28;269(5223):496–512. doi: 10.1126/science.7542800. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Fraser C. M., Gocayne J. D., White O., Adams M. D., Clayton R. A., Fleischmann R. D., Bult C. J., Kerlavage A. R., Sutton G., Kelley J. M. The minimal gene complement of Mycoplasma genitalium. Science. 1995 Oct 20;270(5235):397–403. doi: 10.1126/science.270.5235.397. [DOI] [PubMed] [Google Scholar]
  16. Fridén H., Hederstedt L. Role of His residues in Bacillus subtilis cytochrome b558 for haem binding and assembly of succinate: quinone oxidoreductase (complex II). Mol Microbiol. 1990 Jun;4(6):1045–1056. doi: 10.1111/j.1365-2958.1990.tb00677.x. [DOI] [PubMed] [Google Scholar]
  17. Green G. N., Gennis R. B. Isolation and characterization of an Escherichia coli mutant lacking cytochrome d terminal oxidase. J Bacteriol. 1983 Jun;154(3):1269–1275. doi: 10.1128/jb.154.3.1269-1275.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. 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]
  20. Haldenwang W. G. The sigma factors of Bacillus subtilis. Microbiol Rev. 1995 Mar;59(1):1–30. doi: 10.1128/mr.59.1.1-30.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hansson M., Rutberg L., Schröder I., Hederstedt L. The Bacillus subtilis hemAXCDBL gene cluster, which encodes enzymes of the biosynthetic pathway from glutamate to uroporphyrinogen III. J Bacteriol. 1991 Apr;173(8):2590–2599. doi: 10.1128/jb.173.8.2590-2599.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. Henriques A. O., Beall B. W., Moran C. P., Jr CotM of Bacillus subtilis, a member of the alpha-crystallin family of stress proteins, is induced during development and participates in spore outer coat formation. J Bacteriol. 1997 Mar;179(6):1887–1897. doi: 10.1128/jb.179.6.1887-1897.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Holmgren E., Hederstedt L., Rutberg L. Role of heme in synthesis and membrane binding of succinic dehydrogenase in Bacillus subtilis. J Bacteriol. 1979 May;138(2):377–382. doi: 10.1128/jb.138.2.377-382.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Iida A., Teshiba S., Mizobuchi K. Identification and characterization of the tktB gene encoding a second transketolase in Escherichia coli K-12. J Bacteriol. 1993 Sep;175(17):5375–5383. doi: 10.1128/jb.175.17.5375-5383.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Ish-Horowicz D., Burke J. F. Rapid and efficient cosmid cloning. Nucleic Acids Res. 1981 Jul 10;9(13):2989–2998. doi: 10.1093/nar/9.13.2989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Ito J., Spizizen J. Increased rate of asporogenous mutations following treatment of Bacillus subtilis spores with ethyl methanesulfonate. Mutat Res. 1971 Sep;13(1):93–96. doi: 10.1016/0027-5107(71)90130-8. [DOI] [PubMed] [Google Scholar]
  28. Izard J. W., Kendall D. A. Signal peptides: exquisitely designed transport promoters. Mol Microbiol. 1994 Sep;13(5):765–773. doi: 10.1111/j.1365-2958.1994.tb00469.x. [DOI] [PubMed] [Google Scholar]
  29. Kaneko T., Tanaka A., Sato S., Kotani H., Sazuka T., Miyajima N., Sugiura M., Tabata S. Sequence analysis of the genome of the unicellular cyanobacterium Synechocystis sp. strain PCC6803. I. Sequence features in the 1 Mb region from map positions 64% to 92% of the genome. DNA Res. 1995 Aug 31;2(4):153-66, 191-8. doi: 10.1093/dnares/2.4.153. [DOI] [PubMed] [Google Scholar]
  30. Loferer H., Hennecke H. Protein disulphide oxidoreductases in bacteria. Trends Biochem Sci. 1994 Apr;19(4):169–171. doi: 10.1016/0968-0004(94)90279-8. [DOI] [PubMed] [Google Scholar]
  31. 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]
  32. 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]
  33. Murray C. L., Rabinowitz J. C. Nucleotide sequences of transcription and translation initiation regions in Bacillus phage phi 29 early genes. J Biol Chem. 1982 Jan 25;257(2):1053–1062. [PubMed] [Google Scholar]
  34. Neville D. M., Jr Molecular weight determination of protein-dodecyl sulfate complexes by gel electrophoresis in a discontinuous buffer system. J Biol Chem. 1971 Oct 25;246(20):6328–6334. [PubMed] [Google Scholar]
  35. Niaudet B., Goze A., Ehrlich S. D. Insertional mutagenesis in Bacillus subtilis: mechanism and use in gene cloning. Gene. 1982 Oct;19(3):277–284. doi: 10.1016/0378-1119(82)90017-8. [DOI] [PubMed] [Google Scholar]
  36. Nikkola M., Lindqvist Y., Schneider G. Refined structure of transketolase from Saccharomyces cerevisiae at 2.0 A resolution. J Mol Biol. 1994 May 6;238(3):387–404. doi: 10.1006/jmbi.1994.1299. [DOI] [PubMed] [Google Scholar]
  37. Ohné M. Regulation of aconitase synthesis in Bacillus subtilis: induction, feedback repression, and catabolite repression. J Bacteriol. 1974 Mar;117(3):1295–1305. doi: 10.1128/jb.117.3.1295-1305.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Page M. D., Ferguson S. J. Apo forms of cytochrome c550 and cytochrome cd1 are translocated to the periplasm of Paracoccus denitrificans in the absence of haem incorporation caused either mutation or inhibition of haem synthesis. Mol Microbiol. 1990 Jul;4(7):1181–1192. doi: 10.1111/j.1365-2958.1990.tb00693.x. [DOI] [PubMed] [Google Scholar]
  39. Peterson S. N., Hu P. C., Bott K. F., Hutchison C. A., 3rd A survey of the Mycoplasma genitalium genome by using random sequencing. J Bacteriol. 1993 Dec;175(24):7918–7930. doi: 10.1128/jb.175.24.7918-7930.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Philipp W. J., Poulet S., Eiglmeier K., Pascopella L., Balasubramanian V., Heym B., Bergh S., Bloom B. R., Jacobs W. R., Jr, Cole S. T. An integrated map of the genome of the tubercle bacillus, Mycobacterium tuberculosis H37Rv, and comparison with Mycobacterium leprae. Proc Natl Acad Sci U S A. 1996 Apr 2;93(7):3132–3137. doi: 10.1073/pnas.93.7.3132. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. 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]
  42. Raleigh E. A., Wilson G. Escherichia coli K-12 restricts DNA containing 5-methylcytosine. Proc Natl Acad Sci U S A. 1986 Dec;83(23):9070–9074. doi: 10.1073/pnas.83.23.9070. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. 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]
  44. Resnekov O., Rutberg L., von Gabain A. Changes in the stability of specific mRNA species in response to growth stage in Bacillus subtilis. Proc Natl Acad Sci U S A. 1990 Nov;87(21):8355–8359. doi: 10.1073/pnas.87.21.8355. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Riethdorf S., Völker U., Gerth U., Winkler A., Engelmann S., Hecker M. Cloning, nucleotide sequence, and expression of the Bacillus subtilis lon gene. J Bacteriol. 1994 Nov;176(21):6518–6527. doi: 10.1128/jb.176.21.6518-6527.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. 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]
  47. 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]
  48. Rose M., Entian K. D. New genes in the 170 degrees region of the Bacillus subtilis genome encode DNA gyrase subunits, a thioredoxin, a xylanase and an amino acid transporter. Microbiology. 1996 Nov;142(Pt 11):3097–3101. doi: 10.1099/13500872-142-11-3097. [DOI] [PubMed] [Google Scholar]
  49. Rosenkrantz M. S., Dingman D. W., Sonenshein A. L. Bacillus subtilis citB gene is regulated synergistically by glucose and glutamine. J Bacteriol. 1985 Oct;164(1):155–164. doi: 10.1128/jb.164.1.155-164.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. 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]
  51. 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]
  52. Sambongi Y., Ferguson S. J. Synthesis of holo Paracoccus denitrificans cytochrome c550 requires targeting to the periplasm whereas that of holo Hydrogenobacter thermophilus cytochrome c552 does not. Implications for c-type cytochrome biogenesis. FEBS Lett. 1994 Feb 28;340(1-2):65–70. doi: 10.1016/0014-5793(94)80174-6. [DOI] [PubMed] [Google Scholar]
  53. 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]
  54. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Spizizen J. TRANSFORMATION OF BIOCHEMICALLY DEFICIENT STRAINS OF BACILLUS SUBTILIS BY DEOXYRIBONUCLEATE. Proc Natl Acad Sci U S A. 1958 Oct 15;44(10):1072–1078. doi: 10.1073/pnas.44.10.1072. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Sprenger G. A. Nucleotide sequence of the Escherichia coli K-12 transketolase (tkt) gene. Biochim Biophys Acta. 1993 Nov 16;1216(2):307–310. doi: 10.1016/0167-4781(93)90161-6. [DOI] [PubMed] [Google Scholar]
  57. Sundström M., Lindqvist Y., Schneider G., Hellman U., Ronne H. Yeast TKL1 gene encodes a transketolase that is required for efficient glycolysis and biosynthesis of aromatic amino acids. J Biol Chem. 1993 Nov 15;268(32):24346–24352. [PubMed] [Google Scholar]
  58. 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]
  59. 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]
  60. Tinoco I., Jr, Borer P. N., Dengler B., Levin M. D., Uhlenbeck O. C., Crothers D. M., Bralla J. Improved estimation of secondary structure in ribonucleic acids. Nat New Biol. 1973 Nov 14;246(150):40–41. doi: 10.1038/newbio246040a0. [DOI] [PubMed] [Google Scholar]
  61. 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]
  62. Yoshikawa H., Kazami J., Yamashita S., Chibazakura T., Sone H., Kawamura F., Oda M., Isaka M., Kobayashi Y., Saito H. Revised assignment for the Bacillus subtilis spo0F gene and its homology with spo0A and with two Escherichia coli genes. Nucleic Acids Res. 1986 Jan 24;14(2):1063–1072. doi: 10.1093/nar/14.2.1063. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. 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]
  64. 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]
  65. 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]
  66. von Wachenfeldt C., Hederstedt L. Molecular biology of Bacillus subtilis cytochromes. FEMS Microbiol Lett. 1992 Dec 15;100(1-3):91–100. doi: 10.1111/j.1574-6968.1992.tb14025.x. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES