Skip to main content
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1997 Jun;179(11):3534–3540. doi: 10.1128/jb.179.11.3534-3540.1997

Characterization of the nitric oxide reductase-encoding region in Rhodobacter sphaeroides 2.4.3.

T B Bartnikas 1, I E Tosques 1, W P Laratta 1, J Shi 1, J P Shapleigh 1
PMCID: PMC179145  PMID: 9171397

Abstract

A gene cluster which includes genes required for the expression of nitric oxide reductase in Rhodobacter sphaeroides 2.4.3 has been isolated and characterized. Sequence analysis indicates that the two proximal genes in the cluster are the Nor structural genes. These two genes and four distal genes apparently constitute an operon. Mutational analysis indicates that the two structural genes, norC and norB, and the genes immediately downstream, norQ and norD, are required for expression of an active Nor complex. The remaining two genes, nnrT and nnrU, are required for expression of both Nir and Nor. The products of norCBQD have significant identity with products from other denitrifiers, whereas the predicted nnrT and nnrU gene products have no similarity with products corresponding to other sequences in the database. Mutational analysis and functional complementation studies indicate that the nnrT and nnrU genes can be expressed from an internal promoter. Deletion analysis of the regulatory region upstream of norC indicated that a sequence motif which has identity to a motif in the gene encoding nitrite reductase in strain 2.4.3 is critical for nor operon expression. Regulatory studies demonstrated that the first four genes, norCBQD, are expressed only when the oxygen concentration is low and nitrate is present but that the two distal genes, nnrTU, are expressed constitutively.

Full Text

The Full Text of this article is available as a PDF (276.4 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. Arai H., Igarashi Y., Kodama T. Expression of the nir and nor genes for denitrification of Pseudomonas aeruginosa requires a novel CRP/FNR-related transcriptional regulator, DNR, in addition to ANR. FEBS Lett. 1995 Aug 28;371(1):73–76. doi: 10.1016/0014-5793(95)00885-d. [DOI] [PubMed] [Google Scholar]
  3. Arai H., Igarashi Y., Kodama T. Structure and ANR-dependent transcription of the nir genes for denitrification from Pseudomonas aeruginosa. Biosci Biotechnol Biochem. 1994 Jul;58(7):1286–1291. doi: 10.1271/bbb.58.1286. [DOI] [PubMed] [Google Scholar]
  4. Arai H., Igarashi Y., Kodama T. The structural genes for nitric oxide reductase from Pseudomonas aeruginosa. Biochim Biophys Acta. 1995 Apr 4;1261(2):279–284. doi: 10.1016/0167-4781(95)00018-c. [DOI] [PubMed] [Google Scholar]
  5. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chung C. T., Niemela S. L., Miller R. H. One-step preparation of competent Escherichia coli: transformation and storage of bacterial cells in the same solution. Proc Natl Acad Sci U S A. 1989 Apr;86(7):2172–2175. doi: 10.1073/pnas.86.7.2172. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dermastia M., Turk T., Hollocher T. C. Nitric oxide reductase. Purification from Paracoccus denitrificans with use of a single column and some characteristics. J Biol Chem. 1991 Jun 15;266(17):10899–10905. [PubMed] [Google Scholar]
  8. Gennis R. B., Casey R. P., Azzi A., Ludwig B. Purification and characterization of the cytochrome c oxidase from Rhodopseudomonas sphaeroides. Eur J Biochem. 1982 Jun 15;125(1):189–195. doi: 10.1111/j.1432-1033.1982.tb06667.x. [DOI] [PubMed] [Google Scholar]
  9. Goretski J., Zafiriou O. C., Hollocher T. C. Steady-state nitric oxide concentrations during denitrification. J Biol Chem. 1990 Jul 15;265(20):11535–11538. [PubMed] [Google Scholar]
  10. Hill J., Goswitz V. C., Calhoun M., Garcia-Horsman J. A., Lemieux L., Alben J. O., Gennis R. B. Demonstration by FTIR that the bo-type ubiquinol oxidase of Escherichia coli contains a heme-copper binuclear center similar to that in cytochrome c oxidase and that proper assembly of the binuclear center requires the cyoE gene product. Biochemistry. 1992 Nov 24;31(46):11435–11440. doi: 10.1021/bi00161a023. [DOI] [PubMed] [Google Scholar]
  11. Iwata S., Ostermeier C., Ludwig B., Michel H. Structure at 2.8 A resolution of cytochrome c oxidase from Paracoccus denitrificans. Nature. 1995 Aug 24;376(6542):660–669. doi: 10.1038/376660a0. [DOI] [PubMed] [Google Scholar]
  12. Jüngst A., Zumft W. G. Interdependence of respiratory NO reduction and nitrite reduction revealed by mutagenesis of nirQ, a novel gene in the denitrification gene cluster of Pseudomonas stutzeri. FEBS Lett. 1992 Dec 21;314(3):308–314. doi: 10.1016/0014-5793(92)81495-8. [DOI] [PubMed] [Google Scholar]
  13. Kastrau D. H., Heiss B., Kroneck P. M., Zumft W. G. Nitric oxide reductase from Pseudomonas stutzeri, a novel cytochrome bc complex. Phospholipid requirement, electron paramagnetic resonance and redox properties. Eur J Biochem. 1994 Jun 1;222(2):293–303. doi: 10.1111/j.1432-1033.1994.tb18868.x. [DOI] [PubMed] [Google Scholar]
  14. Keen N. T., Tamaki S., Kobayashi D., Trollinger D. Improved broad-host-range plasmids for DNA cloning in gram-negative bacteria. Gene. 1988 Oct 15;70(1):191–197. doi: 10.1016/0378-1119(88)90117-5. [DOI] [PubMed] [Google Scholar]
  15. Koike I., Hattori A. Energy yield of denitrification: an estimate from growth yield in continuous cultures of Pseudomonas denitrificans under nitrate-, nitrite- and oxide-limited conditions. J Gen Microbiol. 1975 May;88(1):11–19. doi: 10.1099/00221287-88-1-11. [DOI] [PubMed] [Google Scholar]
  16. Kokotek W., Lotz W. Construction of a lacZ-kanamycin-resistance cassette, useful for site-directed mutagenesis and as a promoter probe. Gene. 1989 Dec 14;84(2):467–471. doi: 10.1016/0378-1119(89)90522-2. [DOI] [PubMed] [Google Scholar]
  17. Kwiatkowski A. V., Shapleigh J. P. Requirement of nitric oxide for induction of genes whose products are involved in nitric oxide metabolism in Rhodobacter sphaeroides 2.4.3. J Biol Chem. 1996 Oct 4;271(40):24382–24388. doi: 10.1074/jbc.271.40.24382. [DOI] [PubMed] [Google Scholar]
  18. Maier T., Jacobi A., Sauter M., Böck A. The product of the hypB gene, which is required for nickel incorporation into hydrogenases, is a novel guanine nucleotide-binding protein. J Bacteriol. 1993 Feb;175(3):630–635. doi: 10.1128/jb.175.3.630-635.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Manoil C. Analysis of protein localization by use of gene fusions with complementary properties. J Bacteriol. 1990 Feb;172(2):1035–1042. doi: 10.1128/jb.172.2.1035-1042.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Maskus M., Pariente F., Wu Q., Toffanin A., Shapleigh J. P., Abruña H. D. Electrocatalytic reduction of nitric oxide at electrodes modified with electropolymerized films of [Cr(v-tpy)2]3+ and their application to cellular NO determinations. Anal Chem. 1996 Sep 15;68(18):3128–3134. doi: 10.1021/ac951063g. [DOI] [PubMed] [Google Scholar]
  21. Penfold R. J., Pemberton J. M. An improved suicide vector for construction of chromosomal insertion mutations in bacteria. Gene. 1992 Sep 1;118(1):145–146. doi: 10.1016/0378-1119(92)90263-o. [DOI] [PubMed] [Google Scholar]
  22. Saraste M., Castresana J. Cytochrome oxidase evolved by tinkering with denitrification enzymes. FEBS Lett. 1994 Mar 14;341(1):1–4. doi: 10.1016/0014-5793(94)80228-9. [DOI] [PubMed] [Google Scholar]
  23. Shapleigh J. P., Davies K. J., Payne W. J. Detergent inhibition of nitric-oxide reductase activity. Biochim Biophys Acta. 1987 Feb 25;911(3):334–340. doi: 10.1016/0167-4838(87)90074-4. [DOI] [PubMed] [Google Scholar]
  24. Shapleigh J. P., Gennis R. B. Cloning, sequencing and deletion from the chromosome of the gene encoding subunit I of the aa3-type cytochrome c oxidase of Rhodobacter sphaeroides. Mol Microbiol. 1992 Mar;6(5):635–642. doi: 10.1111/j.1365-2958.1992.tb01511.x. [DOI] [PubMed] [Google Scholar]
  25. Shapleigh J. P., Payne W. J. Nitric oxide-dependent proton translocation in various denitrifiers. J Bacteriol. 1985 Sep;163(3):837–840. doi: 10.1128/jb.163.3.837-840.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Solioz M., Carafoli E., Ludwig B. The cytochrome c oxidase of Paracoccus denitrificans pumps protons in a reconstituted system. J Biol Chem. 1982 Feb 25;257(4):1579–1582. [PubMed] [Google Scholar]
  27. Spiro S. The FNR family of transcriptional regulators. Antonie Van Leeuwenhoek. 1994;66(1-3):23–36. doi: 10.1007/BF00871630. [DOI] [PubMed] [Google Scholar]
  28. Toffanin A., Wu Q., Maskus M., Caselia S., Abruña H. D., Shapleigh J. P. Characterization of the gene encoding nitrite reductase and the physiological consequences of its expression in the nondenitrifying Rhizobium "hedysari" strain HCNT1. Appl Environ Microbiol. 1996 Nov;62(11):4019–4025. doi: 10.1128/aem.62.11.4019-4025.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Tosques I. E., Kwiatkowski A. V., Shi J., Shapleigh J. P. Characterization and regulation of the gene encoding nitrite reductase in Rhodobacter sphaeroides 2.4.3. J Bacteriol. 1997 Feb;179(4):1090–1095. doi: 10.1128/jb.179.4.1090-1095.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Tosques I. E., Shi J., Shapleigh J. P. Cloning and characterization of nnrR, whose product is required for the expression of proteins involved in nitric oxide metabolism in Rhodobacter sphaeroides 2.4.3. J Bacteriol. 1996 Aug;178(16):4958–4964. doi: 10.1128/jb.178.16.4958-4964.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Van Spanning R. J., De Boer A. P., Reijnders W. N., Spiro S., Westerhoff H. V., Stouthamer A. H., Van der Oost J. Nitrite and nitric oxide reduction in Paracoccus denitrificans is under the control of NNR, a regulatory protein that belongs to the FNR family of transcriptional activators. FEBS Lett. 1995 Feb 27;360(2):151–154. doi: 10.1016/0014-5793(95)00091-m. [DOI] [PubMed] [Google Scholar]
  32. Wing H. J., Williams S. M., Busby S. J. Spacing requirements for transcription activation by Escherichia coli FNR protein. J Bacteriol. 1995 Dec;177(23):6704–6710. doi: 10.1128/jb.177.23.6704-6710.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. 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]
  34. Yokoyama K., Hayashi N. R., Arai H., Chung S. Y., Igarashi Y., Kodama T. Genes encoding RubisCO in Pseudomonas hydrogenothermophila are followed by a novel cbbQ gene similar to nirQ of the denitrification gene cluster from Pseudomonas species. Gene. 1995 Feb 3;153(1):75–79. doi: 10.1016/0378-1119(94)00808-6. [DOI] [PubMed] [Google Scholar]
  35. Zumft W. G., Braun C., Cuypers H. Nitric oxide reductase from Pseudomonas stutzeri. Primary structure and gene organization of a novel bacterial cytochrome bc complex. Eur J Biochem. 1994 Jan 15;219(1-2):481–490. doi: 10.1111/j.1432-1033.1994.tb19962.x. [DOI] [PubMed] [Google Scholar]
  36. Zumft W. G. The biological role of nitric oxide in bacteria. Arch Microbiol. 1993;160(4):253–264. doi: 10.1007/BF00292074. [DOI] [PubMed] [Google Scholar]
  37. de Boer A. P., van der Oost J., Reijnders W. N., Westerhoff H. V., Stouthamer A. H., van Spanning R. J. Mutational analysis of the nor gene cluster which encodes nitric-oxide reductase from Paracoccus denitrificans. Eur J Biochem. 1996 Dec 15;242(3):592–600. doi: 10.1111/j.1432-1033.1996.0592r.x. [DOI] [PubMed] [Google Scholar]
  38. van der Oost J., de Boer A. P., de Gier J. W., Zumft W. G., Stouthamer A. H., van Spanning R. J. The heme-copper oxidase family consists of three distinct types of terminal oxidases and is related to nitric oxide reductase. FEMS Microbiol Lett. 1994 Aug 1;121(1):1–9. doi: 10.1111/j.1574-6968.1994.tb07067.x. [DOI] [PubMed] [Google Scholar]

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

RESOURCES