1887

Abstract

The enteric bacterium serovar Typhimurium is a pathogen that is highly adapted for both intracellular and extracellular survival in a range of oxic and anoxic environments. The cytotoxic radical nitric oxide (NO) is encountered in many of these environments. Protection against NO may involve reductive detoxification in low-oxygen environments, and three enzymes, flavorubredoxin (NorV), flavohaemoglobin (HmpA) and cytochrome nitrite reductase (NrfA), have been shown to reduce NO . In this work we determined the role of these three enzymes in NO detoxification by by assessing the effects of all eight possible combinations of , and single, double and triple mutations. The mutant strains were cultured and exposed to NO following either glucose fermentation (when nitrite reductase activity is low), or anaerobic respiration (when nitrite reductase activity is high). Wild-type cultures were more sensitive to the addition of a pulse of NO when grown under fermentative conditions compared with anaerobic respiratory conditions. Analysis of the mutant strains suggested an important additive role for both NorV and NrfA in both environments, since the mutant could not grow after NO addition. The results also suggested a minor role for HmpA in anaerobic detoxification of NO under the two growth conditions, and a larger role for HmpA in aerobic NO detoxification was confirmed. Activity assays and measurements of NO consumption showed that increased nitrite reductase activity correlates with an elevated capacity for NO reduction by intact cells. Taken together, the results reveal a combined role for NorV and NrfA in NO detoxification under anaerobic conditions, and highlight the influence that growth conditions have on the sensitivity to NO of this pathogenic bacterium.

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2008-04-01
2019-11-14
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References

  1. Bang, I. S., Liu, L., Vazquez-Torres, A., Crouch, M. L., Stamler, J. & Fang, F. C. ( 2006; ). Maintenance of nitric oxide and redox homeostasis by the Salmonella flavohemoglobin Hmp. J Biol Chem 281, 28039–28047.[CrossRef]
    [Google Scholar]
  2. Bodenmiller, D. M. & Spiro, S. ( 2006; ). The yjeB (nsrR) gene of Escherichia coli encodes a nitric oxide-sensitive transcriptional regulator. J Bacteriol 188, 874–881.[CrossRef]
    [Google Scholar]
  3. Contreras, I., Toro, C., Troncoso, G. & Mora, G. ( 1997; ). Salmonella typhi mutants defective in anaerobic respiration are impaired in their ability to replicate within epithelial cells. Microbiology 143, 2665–2672.[CrossRef]
    [Google Scholar]
  4. Costa, C., Macedo, A., Moura, I., Moura, J., LeGall, J., Berlier, Y., Liu, M.-Y. & Payne, W. ( 1990; ). Regulation of the hexaheme nitrite/nitric oxide reductase of Desulfovibrio desulfuricans, Wolinella succinogenes and Escherichia coli. A mass spectrometric study. FEBS Lett 276, 67–70.[CrossRef]
    [Google Scholar]
  5. Crawford, M. J. & Goldberg, D. E. ( 1998; ). Role for the Salmonella flavohemoglobin in protection from nitric oxide. J Biol Chem 273, 12543–12547.[CrossRef]
    [Google Scholar]
  6. da Costa, P. N., Teixeira, M. & Saraiva, L. M. ( 2003; ). Regulation of the flavorubredoxin nitric oxide reductase gene in Escherichia coli: nitrate repression, nitrite induction, and possible post-transcription control. FEMS Microbiol Lett 218, 385–393.[CrossRef]
    [Google Scholar]
  7. Darwin, A., Hussain, H., Griffiths, L., Grove, J., Sambongi, Y., Busby, S. & Cole, J. ( 1993; ). Regulation and sequence of the structural gene for cytochrome c 552 from Escherichia coli: not a hexahaem but a 50 kDa tetrahaem nitrite reductase. Mol Microbiol 9, 1255–1265.[CrossRef]
    [Google Scholar]
  8. Datsenko, K. A. & Wanner, B. L. ( 2000; ). One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A 97, 6640–6645.[CrossRef]
    [Google Scholar]
  9. Eriksson, S., Lucchini, S., Thompson, A., Rhen, M. & Hinton, J. C. ( 2003; ). Unravelling the biology of macrophage infection by gene expression profiling of intracellular Salmonella enterica. Mol Microbiol 47, 103–118.
    [Google Scholar]
  10. Farr, S. B. & Kogoma, T. ( 1991; ). Oxidative stress responses in Escherichia coli and Salmonella typhimurium. Microbiol Rev 55, 561–585.
    [Google Scholar]
  11. Field, S. J., Thorndycroft, F. H., Matorin, A. D., Richardson, D. J. & Watmough, N. J. ( 2008; ). The respiratory nitric oxide reductase (NorBC) from Paracoccus denitrificans. Methods Enzymol 437, in press
    [Google Scholar]
  12. Filenko, N., Spiro, S., Browning, D. F., Squire, D., Overton, T. W., Cole, J. & Constantinidou, C. ( 2007; ). The NsrR regulon of Escherichia coli K-12 includes genes encoding the hybrid cluster protein and the periplasmic, respiratory nitrite reductase. J Bacteriol 189, 4410–4417.[CrossRef]
    [Google Scholar]
  13. Flatley, J., Barrett, J., Pullan, S., Hughes, M., Green, J. & Poole, R. ( 2005; ). Transcriptional responses of Escherichia coli to S-nitrosoglutathione under defined chemostat conditions reveal major changes in methionine biosynthesis. J Biol Chem 280, 10065–10072.[CrossRef]
    [Google Scholar]
  14. Gardner, A. M. & Gardner, P. R. ( 2002; ). Flavohemoglobin detoxifies nitric oxide in aerobic, but not anaerobic, Escherichia coli. Evidence for a novel inducible anaerobic nitric oxide-scavenging activity. J Biol Chem 277, 8166–8171.[CrossRef]
    [Google Scholar]
  15. Gardner, P. R., Gardner, A. M., Martin, L. A. & Salzman, A. L. ( 1998; ). Nitric oxide dioxygenase: an enzymic function for flavohemoglobin. Proc Natl Acad Sci U S A 95, 10378–10383.[CrossRef]
    [Google Scholar]
  16. Gardner, A. M., Helmick, R. A. & Gardner, P. R. ( 2002; ). Flavorubredoxin, an inducible catalyst for nitric oxide reduction and detoxification in Escherichia coli. J Biol Chem 277, 8172–8177.[CrossRef]
    [Google Scholar]
  17. Gilberthorpe, N. J., Lee, M. E., Stevanin, T. M., Read, R. C. & Poole, R. K. ( 2007; ). NsrR: a key regulator circumventing Salmonella enterica serovar Typhimurium oxidative and nitrosative stress in vitro and in IFN-γ-stimulated J774.2 macrophages. Microbiology 153, 1756–1771.[CrossRef]
    [Google Scholar]
  18. Gomes, C. M., Giuffre, A., Forte, E., Vicente, J. B., Saraiva, L. M., Brunori, M. & Teixeira, M. ( 2002; ). A novel type of nitric-oxide reductase. Escherichia coli flavorubredoxin. J Biol Chem 277, 25273–25276.[CrossRef]
    [Google Scholar]
  19. Hausladen, A., Gow, A. J. & Stamler, J. S. ( 1998; ). Nitrosative stress: metabolic pathway involving the flavohemoglobin. Proc Natl Acad Sci U S A 95, 14100–14105.[CrossRef]
    [Google Scholar]
  20. Hoiseth, S. K. & Stocker, B. A. ( 1981; ). Aromatic-dependent Salmonella typhimurium are non-virulent and effective as live vaccines. Nature 291, 238–239.[CrossRef]
    [Google Scholar]
  21. Hutchings, M. I., Mandhana, N. & Spiro, S. ( 2002; ). The NorR protein of Escherichia coli activates expression of the flavorubredoxin gene norV in response to reactive nitrogen species. J Bacteriol 184, 4640–4643.[CrossRef]
    [Google Scholar]
  22. Justino, M. C., Vicente, J. B., Teixeira, M. & Saraiva, L. M. ( 2005; ). New genes implicated in the protection of anaerobically grown Escherichia coli against nitric oxide. J Biol Chem 280, 2636–2643.[CrossRef]
    [Google Scholar]
  23. Kim, S. O., Orii, Y., Lloyd, D., Hughes, M. N. & Poole, R. K. ( 1999; ). Anoxic function for the Escherichia coli flavohaemoglobin (Hmp): reversible binding of nitric oxide and reduction to nitrous oxide. FEBS Lett 445, 389–394.[CrossRef]
    [Google Scholar]
  24. Membrillo-Hernandez, J., Coopamah, M., Anjum, M., Stevanin, T., Kelly, A., Hughes, M. & Poole, R. ( 1999; ). The flavohemoglobin of Escherichia coli confers resistance to a nitrosating agent, a “nitric oxide releaser”, and paraquat and is essential for transcriptional responses to oxidative stress. J Biol Chem 274, 748–754.[CrossRef]
    [Google Scholar]
  25. Mills, C. E., Sedelnikova, S., Soballe, B., Hughes, M. N. & Poole, R. K. ( 2001; ). Escherichia coli flavohaemoglobin (Hmp) with equistoichiometric FAD and haem contents has a low affinity for dioxygen in the absence or presence of nitric oxide. Biochem J 353, 207–213.[CrossRef]
    [Google Scholar]
  26. Mukhopadhyay, P., Zheng, M., Bedzyk, L. A., LaRossa, R. A. & Storz, G. ( 2004; ). Prominent roles of the NorR and Fur regulators in the Escherichia coli transcriptional response to reactive nitrogen species. Proc Natl Acad Sci U S A 101, 745–750.[CrossRef]
    [Google Scholar]
  27. Poock, S. R., Leach, E. R., Moir, J. W., Cole, J. A. & Richardson, D. J. ( 2002; ). Respiratory detoxification of nitric oxide by the cytochrome c nitrite reductase of Escherichia coli. J Biol Chem 277, 23664–23669.[CrossRef]
    [Google Scholar]
  28. 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]
  29. Pullan, S. T., Gidley, M. D., Jones, R. A., Barrett, J., Stevanin, T. M., Read, R. C., Green, J. & Poole, R. K. ( 2007; ). Nitric oxide in chemostat-cultured Escherichia coli is sensed by Fnr and other global regulators: unaltered methionine biosynthesis indicates lack of S nitrosation. J Bacteriol 189, 1845–1855.[CrossRef]
    [Google Scholar]
  30. Shiloh, M. U., MacMicking, J. D., Nicholson, S., Brause, J. E., Potter, S., Marino, M., Fang, F., Dinauer, M. & Nathan, C. ( 1999; ). Phenotype of mice and macrophages deficient in both phagocyte oxidase and inducible nitric oxide synthase. Immunity 10, 29–38.[CrossRef]
    [Google Scholar]
  31. Stach, P., Einsle, O., Schumacher, W., Kurun, E. & Kroneck, P. M. ( 2000; ). Bacterial cytochrome c nitrite reductase: new structural and functional aspects. J Inorg Biochem 79, 381–385.[CrossRef]
    [Google Scholar]
  32. Stevanin, T. M., Poole, R. K., Demoncheaux, E. A. & Read, R. C. ( 2002; ). Flavohemoglobin Hmp protects Salmonella enterica serovar Typhimurium from nitric oxide-related killing by human macrophages. Infect Immun 70, 4399–4405.[CrossRef]
    [Google Scholar]
  33. Stevanin, T. M., Read, R. C. & Poole, R. K. ( 2007; ). The hmp gene encoding the NO-inducible flavohaemoglobin in Escherichia coli confers a protective advantage in resisting killing within macrophages, but not in vitro: links with swarming motility. Gene 398, 62–68.[CrossRef]
    [Google Scholar]
  34. Vazquez-Torres, A. & Fang, F. C. ( 2001; ). Salmonella evasion of the NADPH phagocyte oxidase. Microbes Infect 3, 1313–1320.[CrossRef]
    [Google Scholar]
  35. Weinberg, J. ( 1999; ). Human mononuclear phagocyte nitric oxide production and inducible nitric oxide synthase expression. In Nitric Oxide and Infection, pp. 95–150. Edited by F. Fang. New York: Kluwer Academic/Plenum Press.
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