Maintenance of Nitric Oxide and Redox Homeostasis by the Salmonella Flavohemoglobin Hmp

Intracellular pathogens must resist the antimicrobial actions of nitric oxide (NO·) produced by host cells. To this end pathogens possess several NO·-metabolizing enzymes. Here we show that the flavohemoglobin Hmp is the principal enzyme responsible for aerobic NO· metabolism by Salmonella enterica serovar typhimurium. We further show that Hmp is required for Salmonella virulence in mice, in contrast to S-nitrosoglutathione reductase, flavorubredoxin, or cytochrome c nitrite reductase. Abrogation of murine-inducible NO· synthase restores virulence to hmp mutant bacteria. In the presence of nitrosative stress, Hmp-deficient Salmonella exhibits reduced NO· consumption, impaired growth, increased protein S-nitrosylation, and filamentous morphology. However, under aerobic conditions in the absence of nitrosative stress, elevated hmp expression increases S. typhimurium susceptibility to hydrogen peroxide. Both the heme binding and flavoreductase domains are required for resistance to NO·, whereas the flavoreductase domain is responsible for iron-dependent susceptibility to oxidative stress. This provides a rationale for the regulation of hmp expression by the transcriptional repressor NsrR in response to both nitrosative stress and intracellular free iron concentration. The Hmp flavohemoglobin plays a central role in the response of Salmonella to nitrosative stress but requires precise regulation to avoid the exacerbation of oxidative stress that can result if electrons are shuttled to extraneous iron. Intracellular pathogens must resist the antimicrobial actions of nitric oxide (NO·) produced by host cells. To this end pathogens possess several NO·-metabolizing enzymes. Here we show that the flavohemoglobin Hmp is the principal enzyme responsible for aerobic NO· metabolism by Salmonella enterica serovar typhimurium. We further show that Hmp is required for Salmonella virulence in mice, in contrast to S-nitrosoglutathione reductase, flavorubredoxin, or cytochrome c nitrite reductase. Abrogation of murine-inducible NO· synthase restores virulence to hmp mutant bacteria. In the presence of nitrosative stress, Hmp-deficient Salmonella exhibits reduced NO· consumption, impaired growth, increased protein S-nitrosylation, and filamentous morphology. However, under aerobic conditions in the absence of nitrosative stress, elevated hmp expression increases S. typhimurium susceptibility to hydrogen peroxide. Both the heme binding and flavoreductase domains are required for resistance to NO·, whereas the flavoreductase domain is responsible for iron-dependent susceptibility to oxidative stress. This provides a rationale for the regulation of hmp expression by the transcriptional repressor NsrR in response to both nitrosative stress and intracellular free iron concentration. The Hmp flavohemoglobin plays a central role in the response of Salmonella to nitrosative stress but requires precise regulation to avoid the exacerbation of oxidative stress that can result if electrons are shuttled to extraneous iron. Host phagocytic cells use the NADPH phagocyte oxidase (Phox) and inducible nitric-oxide synthase (iNOS) 3The abbreviations used are: iNOS, inducible nitric-oxide synthase; GSNO, S-nitrosoglutathione; RNS, reactive nitrogen species; PBS, phosphate-buffered saline; cfu, colony-forming unit; NONOate, diazenium diolate.3The abbreviations used are: iNOS, inducible nitric-oxide synthase; GSNO, S-nitrosoglutathione; RNS, reactive nitrogen species; PBS, phosphate-buffered saline; cfu, colony-forming unit; NONOate, diazenium diolate. to generate the antimicrobial radicals superoxide anion and nitric oxide, respectively (1Fang F.C. Nat. Rev. Microbiol. 2004; 2: 820-832Crossref PubMed Scopus (1228) Google Scholar). Superoxide (O2·¯) and nitric oxide (NO·) in turn can be converted to other reactive oxygen species or reactive nitrogen species (RNS) such as hydrogen peroxide (H2O2), hydroxyl radical (·OH), nitrogen dioxide (NO2·), peroxynitrite (ONOO–), dinitrogen trioxide (N2O3), and nitrosothiols (RSNO). Enzymes responsible for the metabolism and detoxification of reactive oxygen species, including catalases, superoxide dismutases, and peroxidases, have been extensively studied and shown to contribute to bacterial virulence in experimental infections (2Seyler Jr., R.W. Olson J.W. Maier R.J. Infect. Immun. 2001; 69: 4034-4040Crossref PubMed Scopus (131) Google Scholar, 3Fang F.C. DeGroote M.A. Foster J.W. Baumler A.J. Ochsner U. Testerman T. Bearson S. Giard J.C. Xu Y. Campbell G. et al.Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7502-7507Crossref PubMed Scopus (181) Google Scholar, 4Wilson T.M. de Lisle G.W. Collins D.M. Mol. Microbiol. 1995; 15: 1009-1015Crossref PubMed Scopus (137) Google Scholar). However, although some bacterial enzymes capable of RNS detoxification have been characterized, their importance in pathogenesis has not been directly demonstrated. The enteric pathogens such as Salmonella enterica serovar typhimurium possess a number of enzymes with the ability to metabolize RNS. The flavohemoglobin Hmp detoxifies NO· by an O2-dependent denitrosylase mechanism, producing NO3- under aerobic or microaerobic conditions or by the slower O2-independent reduction of NO· to N2O (5Poole R.K. Biochem. Soc. Trans. 2005; 33: 176-180Crossref PubMed Scopus (207) Google Scholar, 6Hausladen A. Gow A.J. Stamler J.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14100-14105Crossref PubMed Scopus (254) Google Scholar, 7Hausladen A. Gow A. Stamler J.S. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10108-10112Crossref PubMed Scopus (140) Google Scholar). The flavorubredoxin NorV can reduce NO· to N2O under anaerobic or microaerobic conditions (8Gardner A.M. Helmick R.A. Gardner P.R. J. Biol. Chem. 2002; 277: 8172-8177Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar, 9Gomes C.M. Giuffre A. Forte E. Vicente J.B. Saraiva L.M. Brunori M. Teixeira M. J. Biol. Chem. 2002; 277: 25273-25276Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar) and is induced during experimental infection of macrophages (10Eriksson S. Lucchini S. Thompson A. Rhen M. Hinton J.C. Mol. Microbiol. 2003; 47: 103-118Crossref PubMed Scopus (707) Google Scholar). The GSH-dependent formaldehyde dehydrogenase AdhC has S-nitrosoglutathione (GSNO) reductase activity (11Liu L. Hausladen A. Zeng M. Que L. Heitman J. Stamler J.S. Nature. 2001; 410: 490-494Crossref PubMed Scopus (750) Google Scholar), which can limit levels of S-nitrosoglutathione formed during nitrosative stress. Last, the periplasmic cytochrome c nitrite reductase NrfA, which reduces NO2- to NH3, may also be able to directly reduce NO· (12Poock S.R. Leach E.R. Moir J.W. Cole J.A. Richardson D.J. J. Biol. Chem. 2002; 277: 23664-23669Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). The biochemistry of Hmp has been extensively characterized. As one of bacterial globins, Hmp binds NO· at its heme ligand. Structural analysis of flavohemoglobins has also revealed binding domains for FAD and NAD(P) in the C-terminal portion of the molecule (13Ilari A. Bonamore A. Farina A. Johnson K.A. Boffi A. J. Biol. Chem. 2002; 277: 23725-23732Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). This reductase domain is believed to transfer electrons from NAD(P)H to the ferric heme iron ligand via FAD, ultimately resulting in reduction of the liganded NO· to form a heme-bound nitroxyl anion (NO–) equivalent (7Hausladen A. Gow A. Stamler J.S. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10108-10112Crossref PubMed Scopus (140) Google Scholar, 14Poole R.K. Ioannidis N. Orii Y. Proc. Biol. Sci. 1994; 255: 251-258Crossref PubMed Scopus (56) Google Scholar, 15Kim S.O. Orii Y. Lloyd D. Hughes M.N. Poole R.K. FEBS Lett. 1999; 445: 389-394Crossref PubMed Scopus (140) Google Scholar). NO–/HNO is alternatively converted to NO3- or N2O in the presence or absence of O2, respectively. Biochemical studies of the Escherichia coli Hmp enzyme have revealed some evidence that Hmp might exacerbate oxidative stress under selected circumstances. In the absence of NO, Hmp binds O2. NADH oxidase activity of Hmp can then generate O2·¯ at the heme, and further dismutation or reduction could produce H2O2 (16Membrillo-Hernandez J. Ioannidis N. Poole R.K. FEBS Lett. 1996; 382: 141-144Crossref PubMed Scopus (61) Google Scholar, 17Mills C.E. Sedelnikova S. Soballe B. Hughes M.N. Poole R.K. Biochem. J. 2001; 353: 207-213Crossref PubMed Scopus (59) Google Scholar). Moreover, by consuming NADH and reducing free flavins, Hmp has the ability to reduce external electron acceptors, including ferric iron (Fe3+) (18Andrews S.C. Shipley D. Keen J.N. Findlay J.B. Harrison P.M. Guest J.R. FEBS Lett. 1992; 302: 247-252Crossref PubMed Scopus (90) Google Scholar, 19Eschenbrenner M. Coves J. Fontecave M. Biochem. Biophys. Res. Commun. 1994; 198: 127-131Crossref PubMed Scopus (20) Google Scholar, 20Poole R.K. Rogers N.J. D'Mello R.A. Hughes M.N. Orii Y. Microbiology. 1997; 143: 1557-1565Crossref PubMed Scopus (31) Google Scholar). Woodmansee and Imlay (21Woodmansee A.N. Imlay J.A. J. Biol. Chem. 2002; 277: 34055-34066Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar) have demonstrated the ability of reduced flavins generated by the NADPH-dependent flavin oxidoreductase Fre to promote oxidative damage by reducing intracellular free iron. Homology between Fre and the C-terminal portion of Hmp (22Frey A.D. Kallio P.T. FEMS Microbiol. Rev. 2003; 27: 525-545Crossref PubMed Scopus (173) Google Scholar) as well as the ability of Hmp to act as a ferrisiderophore reductase (18Andrews S.C. Shipley D. Keen J.N. Findlay J.B. Harrison P.M. Guest J.R. FEBS Lett. 1992; 302: 247-252Crossref PubMed Scopus (90) Google Scholar) supports a possible role of Hmp in reducing intracellular iron under physiological conditions. To examine the importance of RNS metabolism to Salmonella virulence in mice, we have constructed Salmonella mutant or of with show that the flavohemoglobin Hmp plays the role of enzymes in RNS produced during Salmonella We have also used to the of the heme and flavin binding domains of Hmp to NO· denitrosylase activity and the of oxidative stress. we have the by which hmp is in response to nitrosative stress and intracellular iron concentration. and and used for the of bacterial cells D.M. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The from from the between and nitrite as M.A. D. Xu Y. Campbell G. F.C. 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The cells in with and from selected by to a and for at in a of macrophages with with S. typhimurium with at a of infection of and to the for by with The macrophages and the are as by macrophages in response to infection by the A. J. F.C. J. PubMed Scopus Google Scholar). of NO· S. hmp mutant S. and S. typhimurium to for in the of NO· NO· in of with an NO· of levels in bacterial cells as L. Zeng M. Stamler J.S. PubMed Scopus Google Scholar). Salmonella in with for by by at for and or not with to of in the by J.S. J. J. J. D. J. Proc. Natl. Acad. Sci. U. S. A. 1992; PubMed Scopus Google Scholar). and to protein of by the at at with a in in to NO· and of the S. typhimurium hmp hmp from S. typhimurium with and The with enzyme and of the by and in hmp mutant use as the for to the of E. B. PubMed Scopus Google Scholar) with and used to from the The with enzyme to and cells by used for as and and cells in and cells with H2O2 at a of in at for H2O2 to not bacterial the by in and at by by and by of heme of cells and mutant Hmp by of protein in and H2O2 at S. B. Biochem. PubMed Scopus Google Scholar). The at of an the of the heme of mutant Hmp by with that of the Hmp and of hmp under conditions including or iron and NO· with or and for by a of the and the as M. J. F.C. Mol. Microbiol. 2005; PubMed Scopus Google Scholar). used and and and and and in during in hmp but the or Salmonella in the of reductase, flavorubredoxin, and cytochrome c nitrite reductase to Salmonella the virulence of and mutant and S. typhimurium in virulence in not However, in mice, the hmp mutant of whereas or virulence to that of of the hmp virulence in not hmp is the that the hmp a Moreover, expression of hmp from a to have a of with the R.K. J. Chem. 1994; PubMed Scopus Google Scholar) virulence to hmp mutant Salmonella that the flavohemoglobin Hmp Salmonella virulence by RNS generated by host cells. Moreover, in with hmp to by macrophages to macrophages with hmp produced of nitrite with macrophages with Salmonella although be this of NO· by Hmp or of NO· in macrophages with that are to Salmonella infection can be with S. typhimurium in D.M. D.M. S. J. 2004; PubMed Scopus Google Scholar). To Hmp is required for Salmonella we with of and hmp mutant bacteria. and by bacterial in The hmp mutant not in at but of infection that the flavohemoglobin Hmp Salmonella virulence during infection as well as during infection of hmp S. typhimurium to to of and of NO· by hmp mutant and in an NO·, whereas hmp NO· as as that Hmp is responsible for NO· in Salmonella under aerobic experimental conditions the of hmp mutant impaired in the presence of D. C.M. M. J. PubMed Scopus Google Scholar), D. C.M. M. J. PubMed Scopus Google Scholar), and M.A. D. Xu Y. Campbell G. F.C. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar) hmp mutant cells with as with has been shown to in with the that nitrosative stress F.C. Proc. Natl. Acad. Sci. U. S. A. 2003; PubMed Scopus Google Scholar). revealed that hmp mutant in to of cells that Hmp is responsible for the of NO· detoxification in Salmonella and can promote bacterial during nitrosative stress. of the or of Hmp Salmonella to that expression of the E. coli Hmp heme and flavin domains in resistance to nitrosative stress E. C.E. E. J. Poole R.K. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). To the importance of domain in the activity of Hmp of the Salmonella Hmp heme and flavoreductase domains and hmp from an in an hmp mutant analysis of the E. coli Hmp protein (13Ilari A. Bonamore A. Farina A. Johnson K.A. Boffi A. J. Biol. 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Microbiology. 1997; 143: 1557-1565Crossref PubMed Scopus (31) Google Scholar). of Hmp in the of Salmonella to by a and from a number the NO· resistance of an hmp mutant but bacterial under aerobic conditions in the absence of nitrosative stress not in with that increased Hmp expression might have The A. Res. 2004; PubMed Scopus Google Scholar) revealed between the C-terminal portion of Hmp and the flavin reductase ferric iron reductase activity of Hmp and Fre (18Andrews S.C. Shipley D. Keen J.N. Findlay J.B. Harrison P.M. Guest J.R. FEBS Lett. 1992; 302: 247-252Crossref PubMed Scopus (90) Google Scholar) also conditions of NADH the Fre flavin reductase of E. coli reduces FAD to which can in turn act as a ferric iron and the (21Woodmansee A.N. Imlay J.A. J. Biol. Chem. 2002; 277: 34055-34066Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). in a cells from iron-dependent oxidative and susceptibility to hydrogen peroxide (21Woodmansee A.N. Imlay J.A. J. Biol. 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Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar), but to the that the studies an an J. Biol. Chem. Full Text Full Text PDF Google Scholar). the hmp expression in Salmonella are To hmp expression is by NO·, iron and the used to hmp As shown in hmp is induced by iron with or to The of iron to cells of hmp However, iron-dependent regulation of hmp not The well J. C.M. J. 2003; PubMed Scopus Google Scholar) a for analysis of transcriptional that a hmp in species Biol. 2005; PubMed Scopus Google Scholar). an constructed in S. typhimurium. of hmp to be by the in with levels in cells by also by the as for an of the a hmp in mutant but this by cells with that iron and NO· hmp expression by of the NsrR repressor in S. typhimurium. of NO· by plays an role in to including Salmonella M.A. Ochsner A. Xu Y. F.C. Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google Scholar, Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). However, as a S. typhimurium to the antimicrobial actions of RNS. This has the of enzymes to Salmonella in studies metabolism of RNS by the flavorubredoxin, reductase, and cytochrome c nitrite reductase the flavohemoglobin Hmp to be for Salmonella virulence in an infection This that aerobic detoxification of NO· produced by the host enzyme is of importance to Salmonella during We have that AdhC can limit nitrosative stress during to NO· in not but may be between NorV and may have from a in Moreover, is that anaerobic detoxification of generated NO· or the of N. and Scholar, J. FEMS Microbiol. Rev. 2002; PubMed Google Scholar) may be in not demonstrated under experimental conditions. demonstrated that the flavohemoglobin is required for virulence of M. L. J.C. Stamler J.S. Heitman J. Biol. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar), that the detoxification of NO· is a flavohemoglobins produced by an of Hmp to Salmonella we have been able to an that Hmp Salmonella in macrophages T.M. Poole R.K. Infect. Immun. 2002; PubMed Scopus Google Scholar). Moreover, that Hmp is required for infection of Salmonella in that RNS are during as well as domain are produced by a number of bacterial species such as and A.D. J. Kallio P.T. Microbiol. 2002; PubMed Scopus Google Scholar, Y. M. B. J. M. Proc. Natl. Acad. Sci. U. S. A. 2002; PubMed Scopus Google Scholar). the role of is bacterial an reductase domain at the which flavin reductase or reductase with binding for FAD and The has demonstrated that of the or ligand of heme the of the Salmonella flavohemoglobin The and the and the of the FAD U. R.A. B. J. 1995; PubMed Scopus Google Scholar), which the reduction of heme-bound NO· to the flavin reductase domain is for electron from FAD to the heme ligand NO· is converted to NO3- under aerobic or microaerobic conditions or to N2O under anaerobic conditions (7Hausladen A. Gow A. Stamler J.S. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10108-10112Crossref PubMed Scopus (140) Google Scholar). the of Hmp has been to a to NO· reduction in to the flavorubredoxin NorV (8Gardner A.M. Helmick R.A. Gardner P.R. J. Biol. Chem. 2002; 277: 8172-8177Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar). However, a of the NO· susceptibility of hmp and mutant E. coli that both are under anaerobic conditions Vicente J.B. Teixeira M. Saraiva L.M. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar), whereas the Hmp is under the microaerobic conditions that in (7Hausladen A. Gow A. Stamler J.S. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10108-10112Crossref PubMed Scopus (140) Google Scholar). In the absence of NO·, Hmp can transfer electrons from NAD(P)H to external electron such as O2, ferric and cytochrome c (18Andrews S.C. Shipley D. Keen J.N. Findlay J.B. Harrison P.M. Guest J.R. FEBS Lett. 1992; 302: 247-252Crossref PubMed Scopus (90) Google Scholar, 19Eschenbrenner M. Coves J. Fontecave M. Biochem. Biophys. Res. Commun. 1994; 198: 127-131Crossref PubMed Scopus (20) Google Scholar, 20Poole R.K. Rogers N.J. D'Mello R.A. Hughes M.N. Orii Y. Microbiology. 1997; 143: 1557-1565Crossref PubMed Scopus (31) Google Scholar, N. C.E. Poole R.K. Biochem. J. 1992; PubMed Scopus Google Scholar, Poole R.K. FEMS Microbiol. Lett. 1995; PubMed Google Scholar). E. coli Hmp can generate superoxide and hydrogen peroxide by a (16Membrillo-Hernandez J. Ioannidis N. Poole R.K. FEBS Lett. 1996; 382: 141-144Crossref PubMed Scopus (61) Google Scholar, 17Mills C.E. Sedelnikova S. Soballe B. Hughes M.N. Poole R.K. Biochem. J. 2001; 353: 207-213Crossref PubMed Scopus (59) Google Scholar), one possible by which Hmp might contribute to oxidative stress. However, the an that of ferric iron reduction by reduced reductase activity of Hmp in an analysis of ferrisiderophore (18Andrews S.C. Shipley D. Keen J.N. Findlay J.B. Harrison P.M. Guest J.R. FEBS Lett. 1992; 302: 247-252Crossref PubMed Scopus (90) Google Scholar) and is to the reduction of flavins M. Coves J. Fontecave M. Biochem. Biophys. Res. Commun. 1994; 198: 127-131Crossref PubMed Scopus (20) Google Scholar) that can reduce ferric iron and (21Woodmansee A.N. Imlay J.A. J. Biol. Chem. 2002; 277: 34055-34066Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). Hmp might oxidative stress both by producing superoxide at the heme ligand and by reducing FAD at the flavoreductase Structural studies have shown that heme ligand in Hmp is by and (13Ilari A. Bonamore A. Farina A. Johnson K.A. Boffi A. J. Biol. Chem. 2002; 277: 23725-23732Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). The in Salmonella in the heme to be in an of and and shown to be for heme binding in the of S. J.A. J. G. M.A. M. Proc. Natl. Acad. Sci. U. S. A. 2004; PubMed Scopus Google Scholar). In this an to NO· detoxification and heme binding by Salmonella Hmp but not the of susceptibility to hydrogen whereas a in the FAD binding domain heme binding but the of Hmp that the increased susceptibility of cells to hydrogen peroxide under conditions from the reduction of flavins at the flavoreductase electrons are from NADH to free In the presence of NO·, of bacterial T.M. Ioannidis N. C.E. S.O. Hughes M.N. Poole R.K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) can intracellular of NAD(P)H (21Woodmansee A.N. Imlay J.A. J. Biol. Chem. 2002; 277: 34055-34066Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar), which might oxidative damage electrons are to Hmp (7Hausladen A. Gow A. Stamler J.S. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10108-10112Crossref PubMed Scopus (140) Google Scholar) to promote of heme-bound NO· with In this Hmp can oxidative stress. However, bacterial can also be in the absence of NO·, by antimicrobial produced by host cells T. Nat. Rev. 2003; PubMed Scopus Google Scholar). such elevated levels of NAD(P)H can to reduction of transfer of reducing could promote oxidative expression of Hmp in the absence of NO· may and iron-dependent damage by hydrogen peroxide and Salmonella this by hmp in the presence of NO· and expression intracellular free iron are have been some the responsible for of hmp expression in enteric bacteria. of the E. coli hmp in response to NO· has been to the transcriptional which to levels during nitrosative stress J. A. Hughes M.N. Poole R.K. Mol. Microbiol. 1998; PubMed Scopus Google Scholar). However, other not to be required for NO· of hmp expression in E. coli M. R.A. G. Proc. Natl. Acad. Sci. U. S. A. 2004; PubMed Scopus Google Scholar). the for binding are well between the E. coli and Salmonella hmp we not an of a Salmonella hmp under the experimental conditions used in this not has regulation of hmp by the transcriptional repressor NsrR Biol. 2005; PubMed Scopus Google Scholar), and this has been in E. coli D.M. S. J. PubMed Scopus Google Scholar). In Salmonella hmp expression to be induced by iron or NO· via a the protein J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). In the we show that expression of the Salmonella hmp is in induced by NO· and by intracellular free iron. However, we hmp regulation not to be by by NsrR This provides a by which hmp can be produced during nitrosative stress but under conditions in the absence of regulation the flavohemoglobin Hmp to a central role in NO· detoxification to oxidative stress is to that an between iron and NO· is also in the regulation of in which are by NO· and by iron Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar). regulation is in an such as S. typhimurium that must to both reactive oxygen species and RNS during its with host A. J. F.C. J. PubMed Scopus Google Scholar). with