Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1998 May 1;331(Pt 3):897–904. doi: 10.1042/bj3310897

Periplasmic nitrate-reducing system of the phototrophic bacterium Rhodobacter sphaeroides DSM 158: transcriptional and mutational analysis of the napKEFDABC gene cluster.

F Reyes 1, M Gavira 1, F Castillo 1, C Moreno-Vivián 1
PMCID: PMC1219433  PMID: 9560320

Abstract

The phototrophic bacterium Rhodobacter sphaeroides DSM 158 is able to reduce nitrate to nitrite by means of a periplasmic nitrate reductase which is induced by nitrate and is not repressed by ammonium or oxygen. Recently, a 6.8 kb PstI DNA fragment carrying the napABC genes coding for this periplasmic nitrate-reducing system was cloned [Reyes, Roldán, Klipp, Castillo and Moreno-Vivián (1996) Mol. Microbiol. 19, 1307-1318]. Further sequence and genetic analyses of the DNA region upstream from the napABC genes reveal the presence of four additional nap genes. All these R. sphaeroides genes seem to be organized into a napKEFDABC transcriptional unit. In addition, a partial open reading frame similar to the Azorhizobium caulinodans yntC gene and the Escherichia coli yjcC and yhjK genes is present upstream from this nap gene cluster. The R. sphaeroides napK gene codes for a putative 6.3 kDa transmembrane protein which is not similar to known proteins and the napE gene codes for a 6.7 kDa transmembrane protein similar to the Thiosphaera pantotropha NapE. The R. sphaeroides napF gene product is a 16.4 kDa protein with four cysteine clusters that probably bind four [4Fe-4S] centres. This iron-sulphur protein shows similarity to the NapF and NapG proteins of E. coli and Haemophilus influenzae. Finally, the napD gene product is a 9.4 kDa soluble protein which is also found in E. coli and T. pantotropha. The 5' end of the nap transcript has been determined by primer extension, and a sigma70-like promoter has been identified upstream from the napK gene. The same transcriptional start site is found for cells growing aerobically or anaerobically with nitrate. Different mutant strains carrying defined polar and non-polar insertions in each nap gene were constructed. Characterization of these mutant strains demonstrates the participation of the nap gene products in the periplasmic nitrate reduction in R. sphaeroides.

Full Text

The Full Text of this article is available as a PDF (651.7 KB).

Selected References

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

  1. Arnold W., Pühler A. A family of high-copy-number plasmid vectors with single end-label sites for rapid nucleotide sequencing. Gene. 1988 Oct 15;70(1):171–179. doi: 10.1016/0378-1119(88)90115-1. [DOI] [PubMed] [Google Scholar]
  2. Berks B. C. A common export pathway for proteins binding complex redox cofactors? Mol Microbiol. 1996 Nov;22(3):393–404. doi: 10.1046/j.1365-2958.1996.00114.x. [DOI] [PubMed] [Google Scholar]
  3. Berks B. C., Ferguson S. J., Moir J. W., Richardson D. J. Enzymes and associated electron transport systems that catalyse the respiratory reduction of nitrogen oxides and oxyanions. Biochim Biophys Acta. 1995 Dec 12;1232(3):97–173. doi: 10.1016/0005-2728(95)00092-5. [DOI] [PubMed] [Google Scholar]
  4. Berks B. C., Richardson D. J., Reilly A., Willis A. C., Ferguson S. J. The napEDABC gene cluster encoding the periplasmic nitrate reductase system of Thiosphaera pantotropha. Biochem J. 1995 Aug 1;309(Pt 3):983–992. doi: 10.1042/bj3090983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Berks B. C., Richardson D. J., Robinson C., Reilly A., Aplin R. T., Ferguson S. J. Purification and characterization of the periplasmic nitrate reductase from Thiosphaera pantotropha. Eur J Biochem. 1994 Feb 15;220(1):117–124. doi: 10.1111/j.1432-1033.1994.tb18605.x. [DOI] [PubMed] [Google Scholar]
  6. Blattner F. R., Burland V., Plunkett G., 3rd, Sofia H. J., Daniels D. L. Analysis of the Escherichia coli genome. IV. DNA sequence of the region from 89.2 to 92.8 minutes. Nucleic Acids Res. 1993 Nov 25;21(23):5408–5417. doi: 10.1093/nar/21.23.5408. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Brown N. L., Misra T. K., Winnie J. N., Schmidt A., Seiff M., Silver S. The nucleotide sequence of the mercuric resistance operons of plasmid R100 and transposon Tn501: further evidence for mer genes which enhance the activity of the mercuric ion detoxification system. Mol Gen Genet. 1986 Jan;202(1):143–151. doi: 10.1007/BF00330531. [DOI] [PubMed] [Google Scholar]
  8. Castillo F, Dobao MM, Reyes F, Blasco R, Roldan MD, Gavira M, Caballero FJ, Moreno-Vivian C, Martinez-Luque M. Molecular and Regulatory Properties of the Nitrate Reducing Systems of Rhodobacter. Curr Microbiol. 1996 Dec;33(6):341–346. doi: 10.1007/s002849900125. [DOI] [PubMed] [Google Scholar]
  9. Colbeau A., Richaud P., Toussaint B., Caballero F. J., Elster C., Delphin C., Smith R. L., Chabert J., Vignais P. M. Organization of the genes necessary for hydrogenase expression in Rhodobacter capsulatus. Sequence analysis and identification of two hyp regulatory mutants. Mol Microbiol. 1993 Apr;8(1):15–29. doi: 10.1111/j.1365-2958.1993.tb01199.x. [DOI] [PubMed] [Google Scholar]
  10. Darwin A. J., Stewart V. Nitrate and nitrite regulation of the Fnr-dependent aeg-46.5 promoter of Escherichia coli K-12 is mediated by competition between homologous response regulators (NarL and NarP) for a common DNA-binding site. J Mol Biol. 1995 Aug 4;251(1):15–29. doi: 10.1006/jmbi.1995.0412. [DOI] [PubMed] [Google Scholar]
  11. Dreusch A., Bürgisser D. M., Heizmann C. W., Zumft W. G. Lack of copper insertion into unprocessed cytoplasmic nitrous oxide reductase generated by an R20D substitution in the arginine consensus motif of the signal peptide. Biochim Biophys Acta. 1997 Apr 11;1319(2-3):311–318. doi: 10.1016/s0005-2728(96)00174-0. [DOI] [PubMed] [Google Scholar]
  12. Eiglmeier K., Honoré N., Iuchi S., Lin E. C., Cole S. T. Molecular genetic analysis of FNR-dependent promoters. Mol Microbiol. 1989 Jul;3(7):869–878. doi: 10.1111/j.1365-2958.1989.tb00236.x. [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. Goldman B. S., Lin J. T., Stewart V. Identification and structure of the nasR gene encoding a nitrate- and nitrite-responsive positive regulator of nasFEDCBA (nitrate assimilation) operon expression in Klebsiella pneumoniae M5al. J Bacteriol. 1994 Aug;176(16):5077–5085. doi: 10.1128/jb.176.16.5077-5085.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Goodrich J. A., Schwartz M. L., McClure W. R. Searching for and predicting the activity of sites for DNA binding proteins: compilation and analysis of the binding sites for Escherichia coli integration host factor (IHF). Nucleic Acids Res. 1990 Sep 11;18(17):4993–5000. doi: 10.1093/nar/18.17.4993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Green J., Anjum M. F., Guest J. R. Regulation of the ndh gene of Escherichia coli by integration host factor and a novel regulator, Arr. Microbiology. 1997 Sep;143(Pt 9):2865–2875. doi: 10.1099/00221287-143-9-2865. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Kyte J., Doolittle R. F. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. doi: 10.1016/0022-2836(82)90515-0. [DOI] [PubMed] [Google Scholar]
  19. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  20. Lübben M., Warne A., Albracht S. P., Saraste M. The purified SoxABCD quinol oxidase complex of Sulfolobus acidocaldarius contains a novel haem. Mol Microbiol. 1994 Jul;13(2):327–335. doi: 10.1111/j.1365-2958.1994.tb00426.x. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. Moreno-Vivian C., Hennecke S., Pühler A., Klipp W. Open reading frame 5 (ORF5), encoding a ferredoxinlike protein, and nifQ are cotranscribed with nifE, nifN, nifX, and ORF4 in Rhodobacter capsulatus. J Bacteriol. 1989 May;171(5):2591–2598. doi: 10.1128/jb.171.5.2591-2598.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Moreno-Vivian C., Schmehl M., Masepohl B., Arnold W., Klipp W. DNA sequence and genetic analysis of the Rhodobacter capsulatus nifENX gene region: homology between NifX and NifB suggests involvement of NifX in processing of the iron-molybdenum cofactor. Mol Gen Genet. 1989 Apr;216(2-3):353–363. doi: 10.1007/BF00334376. [DOI] [PubMed] [Google Scholar]
  24. Muro-Pastor M. I., Reyes J. C., Florencio F. J. The NADP+-isocitrate dehydrogenase gene (icd) is nitrogen regulated in cyanobacteria. J Bacteriol. 1996 Jul;178(14):4070–4076. doi: 10.1128/jb.178.14.4070-4076.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Pawlowski K., Klosse U., de Bruijn F. J. Characterization of a novel Azorhizobium caulinodans ORS571 two-component regulatory system, NtrY/NtrX, involved in nitrogen fixation and metabolism. Mol Gen Genet. 1991 Dec;231(1):124–138. doi: 10.1007/BF00293830. [DOI] [PubMed] [Google Scholar]
  26. Reyes F., Roldán M. D., Klipp W., Castillo F., Moreno-Vivián C. Isolation of periplasmic nitrate reductase genes from Rhodobacter sphaeroides DSM 158: structural and functional differences among prokaryotic nitrate reductases. Mol Microbiol. 1996 Mar;19(6):1307–1318. doi: 10.1111/j.1365-2958.1996.tb02475.x. [DOI] [PubMed] [Google Scholar]
  27. Reyes J. C., Florencio F. J. Electron transport controls transcription of the glutamine synthetase gene (glnA) from the cyanobacterium Synechocystis sp. PCC 6803. Plant Mol Biol. 1995 Feb;27(4):789–799. doi: 10.1007/BF00020231. [DOI] [PubMed] [Google Scholar]
  28. Richardson D. J., McEwan A. G., Page M. D., Jackson J. B., Ferguson S. J. The identification of cytochromes involved in the transfer of electrons to the periplasmic NO3- reductase of Rhodobacter capsulatus and resolution of a soluble NO3(-)-reductase--cytochrome-c552 redox complex. Eur J Biochem. 1990 Nov 26;194(1):263–270. doi: 10.1111/j.1432-1033.1990.tb19452.x. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. Siddiqui R. A., Warnecke-Eberz U., Hengsberger A., Schneider B., Kostka S., Friedrich B. Structure and function of a periplasmic nitrate reductase in Alcaligenes eutrophus H16. J Bacteriol. 1993 Sep;175(18):5867–5876. doi: 10.1128/jb.175.18.5867-5876.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Simon R., Quandt J., Klipp W. New derivatives of transposon Tn5 suitable for mobilization of replicons, generation of operon fusions and induction of genes in gram-negative bacteria. Gene. 1989 Aug 1;80(1):161–169. doi: 10.1016/0378-1119(89)90262-x. [DOI] [PubMed] [Google Scholar]
  32. Sofia H. J., Burland V., Daniels D. L., Plunkett G., 3rd, Blattner F. R. Analysis of the Escherichia coli genome. V. DNA sequence of the region from 76.0 to 81.5 minutes. Nucleic Acids Res. 1994 Jul 11;22(13):2576–2586. doi: 10.1093/nar/22.13.2576. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Spiro S., Guest J. R. FNR and its role in oxygen-regulated gene expression in Escherichia coli. FEMS Microbiol Rev. 1990 Aug;6(4):399–428. doi: 10.1111/j.1574-6968.1990.tb04109.x. [DOI] [PubMed] [Google Scholar]
  34. 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]
  35. Weaver P. F., Wall J. D., Gest H. Characterization of Rhodopseudomonas capsulata. Arch Microbiol. 1975 Nov 7;105(3):207–216. doi: 10.1007/BF00447139. [DOI] [PubMed] [Google Scholar]
  36. Weiner J. H., Rothery R. A., Sambasivarao D., Trieber C. A. Molecular analysis of dimethylsulfoxide reductase: a complex iron-sulfur molybdoenzyme of Escherichia coli. Biochim Biophys Acta. 1992 Aug 28;1102(1):1–18. doi: 10.1016/0005-2728(92)90059-b. [DOI] [PubMed] [Google Scholar]
  37. Williamson M. P. The structure and function of proline-rich regions in proteins. Biochem J. 1994 Jan 15;297(Pt 2):249–260. doi: 10.1042/bj2970249. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Ye R. W., Fries M. R., Bezborodnikov S. G., Averill B. A., Tiedje J. M. Characterization of the structural gene encoding a copper-containing nitrite reductase and homology of this gene to DNA of other denitrifiers. Appl Environ Microbiol. 1993 Jan;59(1):250–254. doi: 10.1128/aem.59.1.250-254.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Zumft W. G. Cell biology and molecular basis of denitrification. Microbiol Mol Biol Rev. 1997 Dec;61(4):533–616. doi: 10.1128/mmbr.61.4.533-616.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. von Heijne G. Membrane protein structure prediction. Hydrophobicity analysis and the positive-inside rule. J Mol Biol. 1992 May 20;225(2):487–494. doi: 10.1016/0022-2836(92)90934-c. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES