1887

Abstract

The periplasmic nitrate reductase of is important during anaerobic growth in low-nitrate environments. The operon encoding this nitrate reductase comprises seven genes including a gene, , that encodes a putative cytoplasmic iron–sulphur protein of uncertain subcellular location and function. In this study, N-terminal sequence analysis, cell fractionation coupled with immunoblotting and construction of LacZ and PhoA fusion proteins were used together to establish that NapF is located in the cytoplasm. A bacterial two-hybrid protein–protein interaction system was used to demonstrate that NapF interacted in the cytoplasm with the terminal oxidoreductase NapA, but that it did not self-associate or interact with other electron-transport components of the Nap system, NapC, NapG or NapH, or with another cytoplasmic component, NapD. NapF, purified as a His-tagged protein, exhibited spectral properties characteristic of an iron–sulphur protein. This protein was able to pull down NapA from soluble extracts of . A growth-based assay for NapF function in intact cell cultures was developed and applied to assess the effect of mutation of a number of conserved amino acids. It emerged that neither a highly conserved N-terminal double-arginine motif, nor a conserved proline motif, is essential for NapF-dependent growth. The combined data indicate that NapF plays one or more currently unidentified roles in the post-translational modification of NapA prior to the export of folded NapA via the twin-arginine translocation pathway into the periplasm.

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2006-11-01
2019-11-20
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References

  1. Berks, B. C., Ferguson, S. J., Moir, J. W. & Richardson, D. J. ( 1995a; ). Enzymes and associated electron transport systems that catalyse the respiratory reduction of nitrogen oxides and oxyanions. Biochim Biophys Acta 1232, 97–173.[CrossRef]
    [Google Scholar]
  2. Berks, B. C., Richardson, D. J., Reilly, A. R., Willis, A. & Ferguson, S. J. ( 1995b; ). The napEDABC gene cluster encoding the periplasmic nitrate reductase system of Thiosphaera pantotropha. Biochem J 309, 983–992.
    [Google Scholar]
  3. Berks, B. C., Sargent, F. & Palmer, T. ( 2000; ). The Tat protein export pathway. Mol Microbiol 35, 260–274.[CrossRef]
    [Google Scholar]
  4. Blasco, F., Iobbi, C., Ratouchniak, J., Bonnefoy, V. & Chippaux, M. ( 1990; ). Nitrate reductases of Escherichia coli: sequence of the second nitrate reductase and comparison with that encoded by the narGHJI operon. Mol Gen Genet 222, 104–111.
    [Google Scholar]
  5. Bolivar, F., Rodriguez, R. L., Greene, P. J., Betlach, M. C., Heyneke, N. L., Boyer, H. W., Crosa, J. H. & Falkow, S. ( 1977; ). Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene 2, 95–113.[CrossRef]
    [Google Scholar]
  6. Brondijk, T. H., Fiegen, D., Richardson, D. J. & Cole, J. A. ( 2002; ). Roles of NapF, NapG and NapH, subunits of the Escherichia coli periplasmic nitrate reductase, in ubiquinol oxidation. Mol Microbiol 44, 245–255.[CrossRef]
    [Google Scholar]
  7. Brondijk, T. H. C., Nilavongse, A., Filenko, N., Richardson, D. J. & Cole, J. A. ( 2004; ). The NapGH components of the periplasmic nitrate reductase of Escherichia coli K-12: location, topology, and physiological roles in quinol oxidation and redox balancing. Biochem J 379, 47–55.[CrossRef]
    [Google Scholar]
  8. Clegg, S. J., Jia, W. & Cole, J. A. ( 2006; ). Role of the Escherichia coli nitrate transport protein, NarU, during severe nutrient starvation and slow growth. Microbiology 152, 2091–2100.[CrossRef]
    [Google Scholar]
  9. Grove, J., Tanapongpipat, S., Thomas, G., Griffiths, L., Crooke, H. & Cole, J. ( 1996; ). Escherichia coli K-12 genes essential for the synthesis of c-type cytochromes and a third nitrate reductase located in the periplasm. Mol Microbiol 19, 476–481.
    [Google Scholar]
  10. Iobbi, C., Santini, C. L., Bonnefoy, V. & Giordano, G. ( 1987; ). Biochemical and immunological evidence for a second nitrate reductase in Escherichia coli K12. Eur J Biochem 168, 451–459.[CrossRef]
    [Google Scholar]
  11. Iobbi-Nivol, C., Santini, C. L., Blasco, F. & Giordano, G. ( 1990; ). Purification and further characterization of the second nitrate reductase of Escherichia coli K12. Eur J Biochem 188, 679–687.[CrossRef]
    [Google Scholar]
  12. Jayaraman, P.-S., Peakman, T. C., Busby, S. J. W., Quincey, R. V. & Cole, J. A. ( 1987; ). 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 196, 781–788.[CrossRef]
    [Google Scholar]
  13. Karimova, G., Pidoux, J., Ullmann, A. & Ladant, D. ( 1998; ). A bacterial two-hybrid system based on a reconstituted signal transduction pathway. Proc Natl Acad Sci U S A 95, 5752–5756.[CrossRef]
    [Google Scholar]
  14. Karimova, G., Ullmann, A. & Ladant, D. ( 2000; ). A bacterial two-hybrid system that exploits a cAMP signaling cascade in Escherichia coli. Methods Enzymol 328, 59–73.
    [Google Scholar]
  15. Kim, H. R., Lee, Y. C., Won, J. S. & Choe, M. H. ( 2003; ). AAS and ICP-AES analysis of the iron–sulfur cluster in YojG (NapF) protein of aeg-46.5 operon in Escherichia coli. Bull Korean Chem Soc 24, 1849–1852.[CrossRef]
    [Google Scholar]
  16. Ladant, D. & Ullmann, A. ( 1999; ). Bordetella pertussis adenylate cyclase: a toxin with multiple talents. Trends Microbiol 7, 172–176.[CrossRef]
    [Google Scholar]
  17. Manoil, C. ( 1991; ). Analysis of membrane protein topology using alkaline phosphatase and beta-galactosidase gene fusions. Methods Cell Biol 34, 61–75.
    [Google Scholar]
  18. Minton, N. P. ( 1984; ). Improved plasmid vectors for the isolation of translational lac gene fusions. Gene 31, 269–273.[CrossRef]
    [Google Scholar]
  19. Olmo-Mira, F., Richardson, D. J., Castillo, F., Moreno-Vivian, C. & Roldan, D. ( 2004; ). NapF is a cytoplasmic iron-sulfur protein required for Fe-S cluster assembly in the periplasmic nitrate reductase. J Biol Chem 279, 49727–49735.[CrossRef]
    [Google Scholar]
  20. Paulsen, I. T., Brown, M. H., Dunstan, S. J. & Skurray, R. A. ( 1995; ). Molecular characterization of the staphylococcal multidrug resistance export protein QacC. J Bacteriol 177, 2827–2833.
    [Google Scholar]
  21. Pope, N. R. & Cole, J. A. ( 1984; ). Pyruvate and ethanol as electron donors for nitrite reduction by Escherichia coli K12. J Gen Microbiol 130, 1279–1284.
    [Google Scholar]
  22. Potter, L. C. & Cole, J. A. ( 1999; ). Essential roles for the products of the napABCD genes, but not napFGH, in periplasmic nitrate reduction by Escherichia coli K-12. Biochem J 344, 69–76.[CrossRef]
    [Google Scholar]
  23. Potter, L. C., Millington, P., Griffiths, L., Thomas, G. H. & Cole, J. A. ( 1999; ). 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 344, 77–84.[CrossRef]
    [Google Scholar]
  24. Reyes, F., Roldan, M. D., Klipp, W., Castillo, F. & Moreno-Vivian, C. ( 1996; ). Isolation of periplasmic nitrate reductase genes from Rhodobacter sphaeroides DSM 158: structural and functional differences among prokaryotic nitrate reductases. Mol Microbiol 19, 1307–1318.[CrossRef]
    [Google Scholar]
  25. Reyes, F., Gavira, M., Castillo, F. & Moreno-Vivian, C. ( 1998; ). Periplasmic nitrate-reducing system of the phototrophic bacterium Rhodobacter sphaeroides DSM 158: transcriptional and mutational analysis of the napKEFDABC gene cluster. Biochem J 331, 897–904.
    [Google Scholar]
  26. Richardson, D. J. & Ferguson, S. J. ( 1992; ). The influence of carbon substrate on the activity of the periplasmic nitrate reductase in aerobically grown Thiospaera pantotropha. Arch Microbiol 157, 535–537.
    [Google Scholar]
  27. Santini, C. L., Ize, B., Chanal, A., Muller, M., Giordano, G. & Wu, L. F. ( 1998; ). A novel Sec-independent periplasmic protein translocation pathway in Escherichia coli. EMBO J 17, 101–112.[CrossRef]
    [Google Scholar]
  28. Sears, H. J., Sawers, G., Berks, B. C., Ferguson, S. J. & Richardson, D. J. ( 2000; ). Control of periplasmic nitrate reductase gene expression (napEDABC) from Paracoccus pantotrophus in response to oxygen and carbon substrates. Microbiology 146, 2977–2985.
    [Google Scholar]
  29. Simon, J., Sanger, M., Schuster, S. C. & Cross, R. ( 2003; ). Electron transport to periplasmic nitrate reductase (NapA) of Wolinella succinogenes is independent of a NapC protein. Mol Microbiol 49, 69–75.[CrossRef]
    [Google Scholar]
  30. Soballe, B. & Poole, R. K. ( 1999; ). Microbial ubiquinones: multiple roles in respiration, gene regulation and oxidative stress management. Microbiology 145, 1817–1830.[CrossRef]
    [Google Scholar]
  31. Stanley, N. R., Sargent, F., Buchanan, G., Shi, J., Stewart, V., Palmer, T. & Berks, B. C. ( 2002; ). Behaviour of topological marker proteins targeted to the Tat protein transport pathway. Mol Microbiol 43, 1005–1021.[CrossRef]
    [Google Scholar]
  32. Stewart, V. ( 1988; ). Nitrate respiration in relation to facultative metabolism in enterobacteria. Microbiol Rev 52, 190–232.
    [Google Scholar]
  33. Stewart, V., Lu, Y. & Darwin, A. J. ( 2002; ). Periplasmic nitrate reductase (NapABC enzyme) supports anaerobic respiration in Escherichia coli K-12. J Bacteriol 184, 1314–1323.[CrossRef]
    [Google Scholar]
  34. Unden, G. ( 1988; ). Differential roles for menaquinone and demethylmenaquinone in anaerobic electron transport of E. coli and their fnr-independent expression. Arch Microbiol 150, 499–503.[CrossRef]
    [Google Scholar]
  35. Wang, H., Tseng, C. P. & Gunsalus, R. P. ( 1999; ). The napF and narG nitrate reductase operons in Escherichia coli are differentially expressed in response to submicromolar concentrations of nitrate but not nitrite. J Bacteriol 184, 5303–5308.
    [Google Scholar]
  36. Weiner, J. H., Bilous, P. T., Shaw, G. M., Lubitz, S. P., Frost, L., Thomas, G. H., Cole, J. A. & Turner, R. J. ( 1998; ). A novel and ubiquitous system for membrane targeting and secretion of cofactor-containing proteins. Cell 93, 93–101.[CrossRef]
    [Google Scholar]
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