Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 2004 Apr 1;379(Pt 1):47–55. doi: 10.1042/BJ20031115

NapGH components of the periplasmic nitrate reductase of Escherichia coli K-12: location, topology and physiological roles in quinol oxidation and redox balancing.

T Harma C Brondijk 1, Arjaree Nilavongse 1, Nina Filenko 1, David J Richardson 1, Jeffrey A Cole 1
PMCID: PMC1224043  PMID: 14674886

Abstract

Nap (periplasmic nitrate reductase) operons of many bacteria include four common, essential components, napD, napA, napB and napC (or a homologue of napC ). In Escherichia coli there are three additional genes, napF, napG and napH, none of which are essential for Nap activity. We now show that deletion of either napG or napH almost abolished Nap-dependent nitrate reduction by strains defective in naphthoquinone synthesis. The residual rate of nitrate reduction (approx. 1% of that of napG+ H+ strains) is sufficient to replace fumarate reduction in a redox-balancing role during growth by glucose fermentation. Western blotting combined with beta-galactosidase and alkaline phosphatase fusion experiments established that NapH is an integral membrane protein with four transmembrane helices. Both the N- and C-termini as well as the two non-haem iron-sulphur centres are located in the cytoplasm. An N-terminal twin arginine motif was shown to be essential for NapG function, consistent with the expectation that NapG is secreted into the periplasm by the twin arginine translocation pathway. A bacterial two-hybrid system was used to show that NapH interacts, presumably on the cytoplasmic side of, or within, the membrane, with NapC. As expected for a periplasmic protein, no NapG interactions with NapC or NapH were detected in the cytoplasm. An in vitro quinol dehydrogenase assay was developed to show that both NapG and NapH are essential for rapid electron transfer from menadiol to the terminal NapAB complex. These new in vivo and in vitro results establish that NapG and NapH form a quinol dehydrogenase that couples electron transfer from the high midpoint redox potential ubiquinone-ubiquinol couple via NapC and NapB to NapA.

Full Text

The Full Text of this article is available as a PDF (269.2 KB).

Selected References

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

  1. 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]
  2. 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]
  3. Berks B. C., Sargent F., Palmer T. The Tat protein export pathway. Mol Microbiol. 2000 Jan;35(2):260–274. doi: 10.1046/j.1365-2958.2000.01719.x. [DOI] [PubMed] [Google Scholar]
  4. Blasco F., Iobbi C., Ratouchniak J., Bonnefoy V., Chippaux M. Nitrate reductases of Escherichia coli: sequence of the second nitrate reductase and comparison with that encoded by the narGHJI operon. Mol Gen Genet. 1990 Jun;222(1):104–111. doi: 10.1007/BF00283030. [DOI] [PubMed] [Google Scholar]
  5. Brondijk T. H. C., Fiegen D., Richardson D. J., Cole J. A. Roles of NapF, NapG and NapH, subunits of the Escherichia coli periplasmic nitrate reductase, in ubiquinol oxidation. Mol Microbiol. 2002 Apr;44(1):245–255. doi: 10.1046/j.1365-2958.2002.02875.x. [DOI] [PubMed] [Google Scholar]
  6. Elliott Sean J., Léger Christophe, Pershad Harsh R., Hirst Judy, Heffron Kerensa, Ginet Nicolas, Blasco Francis, Rothery Richard A., Weiner Joel H., Armstrong Fraser A. Detection and interpretation of redox potential optima in the catalytic activity of enzymes. Biochim Biophys Acta. 2002 Sep 10;1555(1-3):54–59. doi: 10.1016/s0005-2728(02)00254-2. [DOI] [PubMed] [Google Scholar]
  7. Iobbi-Nivol C., Santini C. L., Blasco F., Giordano G. Purification and further characterization of the second nitrate reductase of Escherichia coli K12. Eur J Biochem. 1990 Mar 30;188(3):679–687. doi: 10.1111/j.1432-1033.1990.tb15450.x. [DOI] [PubMed] [Google Scholar]
  8. Iobbi C., Santini C. L., Bonnefoy V., Giordano G. Biochemical and immunological evidence for a second nitrate reductase in Escherichia coli K12. Eur J Biochem. 1987 Oct 15;168(2):451–459. doi: 10.1111/j.1432-1033.1987.tb13438.x. [DOI] [PubMed] [Google Scholar]
  9. Jayaraman P. S., Peakman T. C., Busby S. J., Quincey R. V., Cole J. A. Location and sequence of the promoter of the gene for the NADH-dependent nitrite reductase of Escherichia coli and its regulation by oxygen, the Fnr protein and nitrite. J Mol Biol. 1987 Aug 20;196(4):781–788. doi: 10.1016/0022-2836(87)90404-9. [DOI] [PubMed] [Google Scholar]
  10. Karimova G., Pidoux J., Ullmann A., Ladant D. A bacterial two-hybrid system based on a reconstituted signal transduction pathway. Proc Natl Acad Sci U S A. 1998 May 12;95(10):5752–5756. doi: 10.1073/pnas.95.10.5752. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Karimova G., Ullmann A., Ladant D. A bacterial two-hybrid system that exploits a cAMP signaling cascade in Escherichia coli. Methods Enzymol. 2000;328:59–73. doi: 10.1016/s0076-6879(00)28390-0. [DOI] [PubMed] [Google Scholar]
  12. Kröger A. Fumarate as terminal acceptor of phosphorylative electron transport. Biochim Biophys Acta. 1978 Oct 23;505(2):129–145. doi: 10.1016/0304-4173(78)90010-1. [DOI] [PubMed] [Google Scholar]
  13. Ladant D., Ullmann A. Bordatella pertussis adenylate cyclase: a toxin with multiple talents. Trends Microbiol. 1999 Apr;7(4):172–176. doi: 10.1016/s0966-842x(99)01468-7. [DOI] [PubMed] [Google Scholar]
  14. Manoil C. Analysis of membrane protein topology using alkaline phosphatase and beta-galactosidase gene fusions. Methods Cell Biol. 1991;34:61–75. doi: 10.1016/s0091-679x(08)61676-3. [DOI] [PubMed] [Google Scholar]
  15. Minton N. P. Improved plasmid vectors for the isolation of translational lac gene fusions. Gene. 1984 Nov;31(1-3):269–273. doi: 10.1016/0378-1119(84)90220-8. [DOI] [PubMed] [Google Scholar]
  16. Paulsen I. T., Brown M. H., Dunstan S. J., Skurray R. A. Molecular characterization of the staphylococcal multidrug resistance export protein QacC. J Bacteriol. 1995 May;177(10):2827–2833. doi: 10.1128/jb.177.10.2827-2833.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Pope N. R., Cole J. A. Pyruvate and ethanol as electron donors for nitrite reduction by Escherichia coli K12. J Gen Microbiol. 1984 May;130(5):1279–1284. doi: 10.1099/00221287-130-5-1279. [DOI] [PubMed] [Google Scholar]
  18. Potter L. C., Cole J. A. Essential roles for the products of the napABCD genes, but not napFGH, in periplasmic nitrate reduction by Escherichia coli K-12. Biochem J. 1999 Nov 15;344(Pt 1):69–76. doi: 10.1042/0264-6021:3440069. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Potter L. C., Millington P., Griffiths L., Thomas G. H., Cole J. A. Competition between Escherichia coli strains expressing either a periplasmic or a membrane-bound nitrate reductase: does Nap confer a selective advantage during nitrate-limited growth? Biochem J. 1999 Nov 15;344(Pt 1):77–84. [PMC free article] [PubMed] [Google Scholar]
  20. Potter L., Angove H., Richardson D., Cole J. Nitrate reduction in the periplasm of gram-negative bacteria. Adv Microb Physiol. 2001;45:51–112. doi: 10.1016/s0065-2911(01)45002-8. [DOI] [PubMed] [Google Scholar]
  21. Reyes F., Gavira M., Castillo F., Moreno-Vivián C. Periplasmic nitrate-reducing system of the phototrophic bacterium Rhodobacter sphaeroides DSM 158: transcriptional and mutational analysis of the napKEFDABC gene cluster. Biochem J. 1998 May 1;331(Pt 3):897–904. doi: 10.1042/bj3310897. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. Santini C. L., Ize B., Chanal A., Müller M., Giordano G., Wu L. F. A novel sec-independent periplasmic protein translocation pathway in Escherichia coli. EMBO J. 1998 Jan 2;17(1):101–112. doi: 10.1093/emboj/17.1.101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Sears H. J., Sawers G., Berks B. C., Ferguson S. J., Richardson D. J. Control of periplasmic nitrate reductase gene expression (napEDABC) from Paracoccus pantotrophus in response to oxygen and carbon substrates. Microbiology. 2000 Nov;146(Pt 11):2977–2985. doi: 10.1099/00221287-146-11-2977. [DOI] [PubMed] [Google Scholar]
  25. Simon Jörg, Sänger Monica, Schuster Stephan C., Gross Roland. Electron transport to periplasmic nitrate reductase (NapA) of Wolinella succinogenes is independent of a NapC protein. Mol Microbiol. 2003 Jul;49(1):69–79. doi: 10.1046/j.1365-2958.2003.03544.x. [DOI] [PubMed] [Google Scholar]
  26. Stanley N. R., Palmer T., Berks B. C. The twin arginine consensus motif of Tat signal peptides is involved in Sec-independent protein targeting in Escherichia coli. J Biol Chem. 2000 Apr 21;275(16):11591–11596. doi: 10.1074/jbc.275.16.11591. [DOI] [PubMed] [Google Scholar]
  27. Stanley Nicola R., Sargent Frank, Buchanan Grant, Shi Jiarong, Stewart Valley, Palmer Tracy, Berks Ben C. Behaviour of topological marker proteins targeted to the Tat protein transport pathway. Mol Microbiol. 2002 Feb;43(4):1005–1021. doi: 10.1046/j.1365-2958.2002.02797.x. [DOI] [PubMed] [Google Scholar]
  28. Stewart V., MacGregor C. H. Nitrate reductase in Escherichia coli K-12: involvement of chlC, chlE, and chlG loci. J Bacteriol. 1982 Aug;151(2):788–799. doi: 10.1128/jb.151.2.788-799.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Stewart V. Nitrate respiration in relation to facultative metabolism in enterobacteria. Microbiol Rev. 1988 Jun;52(2):190–232. doi: 10.1128/mr.52.2.190-232.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Søballe B., Poole R. K. Microbial ubiquinones: multiple roles in respiration, gene regulation and oxidative stress management. Microbiology. 1999 Aug;145(Pt 8):1817–1830. doi: 10.1099/13500872-145-8-1817. [DOI] [PubMed] [Google Scholar]
  31. Tanapongpipat S., Reid E., Cole J. A., Crooke H. Transcriptional control and essential roles of the Escherichia coli ccm gene products in formate-dependent nitrite reduction and cytochrome c synthesis. Biochem J. 1998 Sep 1;334(Pt 2):355–365. doi: 10.1042/bj3340355. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Weiner J. H., Bilous P. T., Shaw G. M., Lubitz S. P., Frost L., Thomas G. H., Cole J. A., Turner R. J. A novel and ubiquitous system for membrane targeting and secretion of cofactor-containing proteins. Cell. 1998 Apr 3;93(1):93–101. doi: 10.1016/s0092-8674(00)81149-6. [DOI] [PubMed] [Google Scholar]

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

RESOURCES