Regulation of the Nitric Oxide Reduction Operon (norRVW) in Escherichia coli
2003; Elsevier BV; Volume: 278; Issue: 12 Linguagem: Inglês
10.1074/jbc.m212462200
ISSN1083-351X
AutoresAnne M. Gardner, Christopher R. Gessner, Paul R. Gardner,
Tópico(s)Wastewater Treatment and Nitrogen Removal
ResumoNitric oxide (NO) induces NO-detoxifying enzymes in Escherichia coli suggesting sensitive mechanisms for coordinate control of NO defense genes in response to NO stress. Exposure of E. coli to sub-micromolar NO levels under anaerobic conditions rapidly induced transcription of the NO reductase (NOR) structural genes, norV and norW, as monitored by lac gene fusions. Disruption ofrpoN (ς54) impaired the NO-mediated induction of norV and norW transcription and NOR expression, whereas disruption of the upstream regulatory gene,norR, completely ablated NOR induction. NOR inducibility was restored to NorR null mutants by expressing NorR intrans. Furthermore, an internal deletion of the N-terminal domain of NorR activated NOR expression independent of NO exposure. Neither NorR nor ς54 was essential for NO-mediated induction of the NO dioxygenase (flavohemoglobin) encoded byhmp. However, elevated NOR activity inhibited NO dioxygenase induction, and, in the presence of dioxygen, NO dioxygenase inhibited norV induction by NO. The results demonstrate the role of NorR as a ς54-dependent regulator ofnorVW expression. A role for the NorR N-terminal domain as a transducer or sensor for NO is suggested. Nitric oxide (NO) induces NO-detoxifying enzymes in Escherichia coli suggesting sensitive mechanisms for coordinate control of NO defense genes in response to NO stress. Exposure of E. coli to sub-micromolar NO levels under anaerobic conditions rapidly induced transcription of the NO reductase (NOR) structural genes, norV and norW, as monitored by lac gene fusions. Disruption ofrpoN (ς54) impaired the NO-mediated induction of norV and norW transcription and NOR expression, whereas disruption of the upstream regulatory gene,norR, completely ablated NOR induction. NOR inducibility was restored to NorR null mutants by expressing NorR intrans. Furthermore, an internal deletion of the N-terminal domain of NorR activated NOR expression independent of NO exposure. Neither NorR nor ς54 was essential for NO-mediated induction of the NO dioxygenase (flavohemoglobin) encoded byhmp. However, elevated NOR activity inhibited NO dioxygenase induction, and, in the presence of dioxygen, NO dioxygenase inhibited norV induction by NO. The results demonstrate the role of NorR as a ς54-dependent regulator ofnorVW expression. A role for the NorR N-terminal domain as a transducer or sensor for NO is suggested. nitric oxide NO reductase NO dioxygenase Luria-Bertani chloramphenicol resistance, Apr, ampicillin resistance tetracycline resistance kanamycin resistance Nitric oxide (NO)1 is a free radical with multiple and diverse biological functions (reviewed in Ref. 1Ignarro L.J. Kidney Int. Suppl. 1996; 55: S2-S5PubMed Google Scholar). NO serves as an intermediate in microbial denitrification (2Zumft W. Microbiol. Mol. Biol. 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Microbes also express NO dioxygenases (NODs) that utilize O2 to convert NO to nitrate (12Gardner P.R. Gardner A.M. Martin L.A. Salzman A.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10378-10383Crossref PubMed Scopus (497) Google Scholar, 13Gardner A.M. Martin L.A. Gardner P.R. Dou Y. Olson J.S. J. Biol. Chem. 2000; 275: 12581-12589Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 14Gardner P.R. Gardner A.M. Martin L.A. Dou Y. Li T. Olson J.S. Zhu H. Riggs A.F. J. Biol. Chem. 2000; 275: 31581-31587Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 15Hausladen A. Gow A.J. Stamler J.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14100-14105Crossref PubMed Scopus (255) Google Scholar, 16Liu L. Zeng M. Hausladen A. Heitman J. Stamler J.S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4672-4676Crossref PubMed Scopus (166) Google Scholar, 17Ouellet H. Ouellet Y. Richard C. Labarre M. Wittenberg B. Wittenberg J. Guertin M. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5902-5907Crossref PubMed Scopus (237) Google Scholar, 18Pathania R. Navani N.K. Gardner A.M. Gardner P.R. Dikshit K.L. Mol. Microbiol. 2002; 45: 1303-1314Crossref PubMed Scopus (119) Google Scholar). Escherichia coli employs both of these enzymes. An inducible NOD (flavohemoglobin), encoded by the genehmp (19Vasudevan S.G. Armarego W.L.F. Shaw D.C. Lilley P.E. Dixon N.E. Poole R.K. Mol. Gen. Genet. 1991; 226: 49-58Crossref PubMed Scopus (184) Google Scholar), detoxifies NO under aerobic growth conditions (12Gardner P.R. Gardner A.M. Martin L.A. Salzman A.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10378-10383Crossref PubMed Scopus (497) Google Scholar,15Hausladen A. Gow A.J. Stamler J.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14100-14105Crossref PubMed Scopus (255) Google Scholar, 20Gardner A.M. Gardner P.R. J. Biol. Chem. 2002; 277: 8166-8171Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). An inducible O2-sensitive NOR activity encoded by the norRVW operon detoxifies NO under anaerobic and 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 (256) Google Scholar, 20Gardner A.M. Gardner P.R. J. Biol. Chem. 2002; 277: 8166-8171Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). NorV is a di-iron center-containing flavorubredoxin-type NOR with orthologues in the Archeae, strict anaerobes, and facultative anaerobes (21Gomes C.M. Vicente J.B. Wasserfallen A. Teixeira M. Biochemistry. 2000; 39: 16230-16237Crossref PubMed Scopus (60) Google Scholar, 22Wasserfallen A. Ragettli S. Jouanneau J. Leisinger T. Eur. J. Biochem. 1998; 254: 325-332Crossref PubMed Scopus (79) Google Scholar, 23Frazão C. Silva G. Gomes C.M. Matias P. Coelho R. Sieker L. Macedo S. Liu M.-Y. Oliveira S. Teixeira M. Xavier A.V. Rodrigues-Pousada C. Carrondo M.A. LeGall J. Nat. Struct. Biol. 2000; 7: 1041-1045Crossref PubMed Scopus (198) Google Scholar, 24Das A. Coulter E.D. Kurtz Jr., D.M. Ljungdahl L.G. J. Bacteriol. 2001; 183: 1560-1567Crossref PubMed Scopus (68) Google Scholar). It is distinct from the bacterial heme/nonheme iron-containing cytochrome bc-type NORs and the fungal P450-type NOR (2Zumft W. Microbiol. Mol. Biol. Rev. 1997; 61: 533-616Crossref PubMed Scopus (2897) Google Scholar). NorW functions as an NADH:flavorubredoxin oxidoreductase (21Gomes C.M. Vicente J.B. Wasserfallen A. Teixeira M. Biochemistry. 2000; 39: 16230-16237Crossref PubMed Scopus (60) Google Scholar) and is required for maximal flavorubredoxin-catalyzed NO reduction in cells (8Gardner A.M. Helmick R.A. Gardner P.R. J. Biol. Chem. 2002; 277: 8172-8177Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar) and in vitro (25Gomes C.M. Giuffrè 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 (177) Google Scholar). Together, the O2-dependent NOD and the O2-sensitive NOR (NorVW) detoxify NO throughout the physiological [O2] range (7Gardner P.R. Costantino G. Salzman A.L. J. Biol. Chem. 1998; 273: 26528-26533Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 8Gardner A.M. Helmick R.A. Gardner P.R. J. Biol. Chem. 2002; 277: 8172-8177Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar, 20Gardner A.M. Gardner P.R. J. Biol. Chem. 2002; 277: 8166-8171Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). NORs and NODs are induced by NO or NO-generating agents suggesting fine-tuned mechanisms for the coordination of microbial NO defenses to NO stress levels. In denitrifying Pseudomonas andRhodobacter, cytochrome bc-type NORs are up-regulated by the Fnr-like DnrD/NnrR transcription regulators in response to nanomolar NO (26Vollack K.-U. Zumft W.G. J. Bacteriol. 2001; 183: 2516-2526Crossref PubMed Scopus (102) Google Scholar, 27Kwiatkowski A.V. Shapleigh J.P. J. Biol. Chem. 1996; 271: 24382-24388Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 28Hutchings M.I. Shearer N. Wastell S. van Spanning R.J. Spiro S. J. Bacteriol. 2000; 182: 6434-6439Crossref PubMed Scopus (46) Google Scholar). However, unlike Fnr (29Cruz-Ramos H. Crack J. Wu G. Hughes M.N. Scott C. Thomson A.J. Green J. Poole R.K. EMBO J. 2002; 21: 3235-3244Crossref PubMed Scopus (253) Google Scholar), DnrD/NnrR do not bear NO-reactive [4Fe-4S] centers, and the NO sensing mechanism is currently unknown (26Vollack K.-U. Zumft W.G. J. Bacteriol. 2001; 183: 2516-2526Crossref PubMed Scopus (102) Google Scholar, 27Kwiatkowski A.V. Shapleigh J.P. J. Biol. Chem. 1996; 271: 24382-24388Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 28Hutchings M.I. Shearer N. Wastell S. van Spanning R.J. Spiro S. J. Bacteriol. 2000; 182: 6434-6439Crossref PubMed Scopus (46) Google Scholar, 30Zumft W.G. J. Mol. Microbiol. Biotechnol. 2002; 4: 277-286PubMed Google Scholar). In the denitrifierRalstonia eutropha, the tripartite transcription factor NorR regulates denitrification, norA1B1 transcription, and NOR activity expression in a ς54-dependent mechanism in response to exposures to sodium nitroprusside, the NO donor compound NOC18, or during growth with nitrite or nitrate (31Pohlmann A. Cramm R. Schmelz K. Friedrich B. Mol. Microbiol. 2000; 38: 626-638Crossref PubMed Scopus (98) Google Scholar).E. coli and related microbes contain norRorthologues suggesting a global regulatory role for NorR in controlling defenses (i.e. norVW, norBC, andhmp) against the incipient toxicity of NO and secondarily derived reactive nitrogen species (8Gardner A.M. Helmick R.A. Gardner P.R. J. Biol. Chem. 2002; 277: 8172-8177Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar, 31Pohlmann A. Cramm R. Schmelz K. Friedrich B. Mol. Microbiol. 2000; 38: 626-638Crossref PubMed Scopus (98) Google Scholar, 32Hutchings M.I. Mandhana N. Spiro S. J. Bacteriol. 2002; 184: 4640-4643Crossref PubMed Scopus (104) Google Scholar). Recently, Hutchings et al. (32Hutchings M.I. Mandhana N. Spiro S. J. Bacteriol. 2002; 184: 4640-4643Crossref PubMed Scopus (104) Google Scholar) reported NorR-dependent activation of norV transcription by the NO+ donor and NO-generating compound nitroprusside in support of the proposed regulatory function. Interestingly, nitroprusside-elicited norV transcription was increased >5-fold by normoxic O2 suggesting mechanisms for NorR activation involving O2-derived reactive nitrogen intermediates rather than NO per se. The large oxygen enhancement of norV transcription observed with or without nitroprusside exposure has also supported proposals for aerobic functions for the norRVW operon, including O2reduction and the detoxification of O2-derived reactive nitrogen intermediates (25Gomes C.M. Giuffrè 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 (177) Google Scholar, 32Hutchings M.I. Mandhana N. Spiro S. J. Bacteriol. 2002; 184: 4640-4643Crossref PubMed Scopus (104) Google Scholar). We now report the rapid and robust induction of norVand norW transcription and NorVW activity by sub-micromolar NO via a NorR- and ς54-dependent mechanism inE. coli. We also show that a deletion within the conserved NorR N-terminal domain activates NorVW expression independent of NO exposure, thus demonstrating the role of the N terminus in NO sensing and signaling. Contrary to the results obtained with nitroprusside (32Hutchings M.I. Mandhana N. Spiro S. J. Bacteriol. 2002; 184: 4640-4643Crossref PubMed Scopus (104) Google Scholar), O2 greatly diminished norV andnorW induction by NO. The results are discussed in light of the proposed NO reduction and detoxification function of thenorRVW operon within the NO defense network. Bovine liver catalase (260,000 units/ml) was purchased from Roche Molecular Biochemicals. Glucose oxidase (4,000 units/ml) and β-galactosidase (1190 units/ml) were obtained from Sigma. Saturated NO stocks (2 mm) were prepared in water as previously described (7Gardner P.R. Costantino G. Salzman A.L. J. Biol. Chem. 1998; 273: 26528-26533Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Compressed gas cylinders containing 1200 ppm (±5%) of NO in ultrapure N2, 99.999% N2, 99.993% O2, and 1.05% O2 in ultrapure N2 were obtained from Praxair (Bethlehem, PA). Strains and plasmids are described in Table I. ChromosomallacZ gene fusions of norR, norV, andnorW were created as previously described (8Gardner A.M. Helmick R.A. Gardner P.R. J. Biol. Chem. 2002; 277: 8172-8177Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar). A Tn10 mutation in the rpoN locus was transduced with P1 phage and mutants were selected for tetracycline resistance. The pUC19NorR construct is a 1.9-kb SalI-PstI fragment cloned in pUC19 containing the intragenic region betweennorR and norV in addition to the norRcoding region from the lambda phage 9G10 (36Rudd K.E. Nucleic Acids Res. 2000; 28: 60-64Crossref PubMed Scopus (211) Google Scholar). To construct pUC19NorRΔ30–164, a 1.1-kb fragment encoding the C terminus was PCR-amplified using pUC19NorR as the template and using the oligonucleotide primers 5′-GTTGCGGATCCAACAACTGGAAAGCCAGAATATGC-3′ and 5′-CATGCCTGCAGGATTTCTATCAGGCCG-3′ containingBamHI and PstI sites (underlined), respectively. pUC19NorR was digested withBcl1 and PstI, and the 1.1-kb PCR fragment was subcloned. This procedure generated an in-frame fusion of NorR that deleted amino acids 30–164 and added a glutamate residue at the junction. A second in-frame deletion of amino acids 30–214 was created by digesting pUC19NorR with Bcl1 and religating.Table IE. coli strains and plasmids used in this studyStrain or plasmidCharacteristic or descriptionReferenceStrainsAB1157F−,thr-1, leu-6, proA2, his-4, thi-1, argE3, lacY1, galK2, ara14, xyl-5, mtl-1, tsx-33, strA31, sup-37,λ−(33Greenberg J.T. Demple B. J. Bacteriol. 1986; 168: 1026-1029Crossref PubMed Google Scholar)YMC18YMC10rpoN(glnF208)::Tn10; Tcr(34Ueno-Nishio S. Backman K.C. Magasanik B. J. Bacteriol. 1983; 153: 1247-1251Crossref PubMed Google Scholar)AG500rpoN::Tn10 P1 transduction from YMC18 into AB1157This workAG200AB1157 φ(norR-lacZ)286; Cmr(8Gardner A.M. Helmick R.A. Gardner P.R. J. Biol. Chem. 2002; 277: 8172-8177Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar)AG300AB1157 φ(norV-lacZ)232; Cmr(8Gardner A.M. Helmick R.A. Gardner P.R. J. Biol. Chem. 2002; 277: 8172-8177Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar)AG400AB1157 φ(norW-lacZ)11; Cmr(8Gardner A.M. Helmick R.A. Gardner P.R. J. Biol. Chem. 2002; 277: 8172-8177Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar)AG305AG500rpoN::Tn10φ(norV-lacZ)232; CmrTcrThis workAG301AG300 hmp::Tn5Knr(20Gardner A.M. Gardner P.R. J. Biol. Chem. 2002; 277: 8166-8171Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar)PlasmidspUC19Vector; Apr(35Yanisch-Perron C. Vieira C. Messing J. Gene (Amst.). 1985; 33: 103-119Crossref PubMed Scopus (11465) Google Scholar)pUC19NorRThe norR regulatory region and the norR structural gene on a 1.9-kbSal1 to Pst1 fragment in pUC19This workpUC19NorRΔ30–164NorR gene with internal deletion of amino acids 30–164 in pUC19This work Open table in a new tab Anerobic starter cultures were grown static overnight at 37 °C in 15-ml tubes containing 10 ml of phosphate-buffered LB medium supplemented with 20 mm glucose (7Gardner P.R. Costantino G. Salzman A.L. J. Biol. Chem. 1998; 273: 26528-26533Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Aerobic and microaerobic starter cultures were grown overnight in 5 ml of phosphate-buffered LB medium in 15-ml tubes shaking at 200 rpm at 37 °C. Chloramphenicol and ampicillin were added as indicated at 30 and 50 μg/ml, respectively. Culture growth was monitored by following the turbidity at 550 nm (A 550) and by plating and counting. AnA 550 value of 1.0 in a 1-cm cuvette was equivalent to 3 × 108 bacteria per milliliter for cultures grown in phosphate-buffered LB media. Gases were mixed and delivered to sealed 50-ml growth flasks as previously described (20Gardner A.M. Gardner P.R. J. Biol. Chem. 2002; 277: 8166-8171Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). Whole cell NO consumption rates were measured at 37 °C with a 2-mm ISO-NOP NO electrode (World Precision Instruments, Sarasota, FL) in the presence or absence of O2 as previously described (12Gardner P.R. Gardner A.M. Martin L.A. Salzman A.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10378-10383Crossref PubMed Scopus (497) Google Scholar, 20Gardner A.M. Gardner P.R. J. Biol. Chem. 2002; 277: 8166-8171Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). Cells were harvested by centrifugation and washed in 100 mm sodium phosphate buffer, pH 7.0. β-Galactosidase activity was measured according to the method of Miller (37Miller J.H. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1972Google Scholar) with the following modifications. Frozen cell pellets were suspended at ∼1 × 1010 cells per milliliter in 100 mm sodium phosphate buffer, pH 7.0, and sonicated on ice. Cell-free extracts were prepared by centrifuging lysates at 12,000 × g for 5 min. Assays were incubated for 15 min at room temperature in a 0.1-ml volume with 1–15 μg of extract protein in a 96-well plate. Extract activities were determined using a standard curve generated with 0–3.5 milliunits of β-galactosidase. Activity is reported in milliunits per milligram of extract protein where one milliunit cleaves 1 nmol ofo-nitrophenyl-β-d-galactopyranoside per minute at room temperature. Background β-galactosidase activity in parental AB1157 cells was 3.3 ± 0.6 milliunits/mg extract protein in anaerobic cells, 3.5 ± 0.7 in low aerobic cells, and 8.3 ± 1.0 in aerobic cultures. β-Galactosidase activity remained constant in AB1157 cells irrespective of NO exposure and was subtracted from the activities measured in lacZ fusion strains under similar growth conditions. Soluble protein was measured using Bio-Rad dye reagent with bovine serum albumin as the standard (38Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216377) Google Scholar). Statistical significance (p < 0.05) was determined using the Tukey Kramer honestly significantly different method in the JMP program (SAS Institute). norV and norW transcriptional units are arranged in a head-to-tail fashion with the start methionine of NorW located within the coding region of norV suggesting coordinate transcription and translation in response to NO stress (8Gardner A.M. Helmick R.A. Gardner P.R. J. Biol. Chem. 2002; 277: 8172-8177Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar). In contrast, norR is divergently transcribed fromnorVW (8Gardner A.M. Helmick R.A. Gardner P.R. J. Biol. Chem. 2002; 277: 8172-8177Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar) and is autogenously regulated (32Hutchings M.I. Mandhana N. Spiro S. J. Bacteriol. 2002; 184: 4640-4643Crossref PubMed Scopus (104) Google Scholar). Strain AG300 carrying a norV-lacZ fusion within thenorV genomic locus and lacking inducible anaerobic NOR activity (8Gardner A.M. Helmick R.A. Gardner P.R. J. Biol. Chem. 2002; 277: 8172-8177Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar) was used to measure the responsiveness of norVtranscription to authentic NO. Exposure of anaerobic AG300 to 960 ppm gaseous NO (≤2 μm in solution) induced β-galactosidase activity by ∼50-fold within 5 min. β-Galactosidase expression peaked after 30–45 min of exposure resulting in ≥1000-fold induction (Fig. 1 A). A 30-fold increase in norV transcription was observed with 120 ppm NO (≤0.25 μm in solution) (Fig. 1 B). Maximal induction of β-galactosidase activity was observed with 480 ppm NO (≤1 μm in solution). Expression was blunted with 960 ppm NO exposure suggesting toxicity of NO under these conditions. NO similarly induced norW-lacZ in strain AG400 under anaerobic conditions (Table II). However, the norW-lac fusion was induced to a 10-fold lower extent than that observed for the norV-lac fusion following a similar NO exposure.Table IIExpression of norV-, norW-, and norR-lacZ fusionsβ-Galactosidase activityStrainGenotypewith N2 onlywith 0.5% O2with 21% O2ControlNOControlNOControlNOmilliunits/mgAG300Φ(norV-lacZ)2321 ± 12591 ± 3141 ± 1828 ± 2731 ± 1120 ± 7AG400Φ(norW-lacZ)113 ± 1538 ± 913 ± 1294 ± 994 ± 138 ± 15AG200Φ(norR-lacZ)28619 ± 213 ± 324 ± 122 ± 426 ± 135 ± 1 Open table in a new tab The dampened response of norW-lac to NO may be explained by the production of significant NOR activity from norVexpression within strain AG400 (8Gardner A.M. Helmick R.A. Gardner P.R. J. Biol. Chem. 2002; 277: 8172-8177Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar), thus resulting in a lower steady-state NO level. It is also possible that higher steady-state NO levels are required to activate maximal norW transcription. Nevertheless, the results clearly demonstrate a rapid, robust, and coordinate up-regulation of norV and norWtranscription in response to low levels of NO. The results in Table IIalso demonstrate a low non-inducible level of transcription fromnorR consistent with previous results obtained using nitroprusside as the potential inducer (32Hutchings M.I. Mandhana N. Spiro S. J. Bacteriol. 2002; 184: 4640-4643Crossref PubMed Scopus (104) Google Scholar). NO-mediated induction of norV-lac andnorW-lac fusions was significantly inhibited by O2. Under fully aerated conditions (∼200 μmO2), induction ratios for norV-lacZ andnorW-lacZ fusions were reduced 200- and 20-fold, respectively, with no change in the basal expression levels (Table II). At a lower O2 concentration (∼5 μm),norV-lac and norW-lac induction ratios were reduced 3.1- and 1.8-fold, respectively. Lower norV andnorW induction in the presence of O2 can be explained by the decrease in cellular NO levels achieved by the inducible NOD. Indeed, NOD expression decreased norV-lacexpression by ∼96% in cells exposed to an atmosphere containing 960 ppm NO in 21% O2 for 45 min. NOD-deficient strain AG301 and control strain AG300 produced 3453 ± 493 and 149 ± 23 milliunits/mg β-galactosidase (n = 4, ±S.E.), respectively. Thus, norV and norW are maximally induced under conditions in which the O2-sensitive NOR functions most effectively (8Gardner A.M. Helmick R.A. Gardner P.R. J. Biol. Chem. 2002; 277: 8172-8177Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar, 20Gardner A.M. Gardner P.R. J. Biol. Chem. 2002; 277: 8166-8171Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar), and NOD indirectly regulates NOR expression. The central domain of the tripartite NorR protein is highly homologous with ς54-dependent response regulators (31Pohlmann A. Cramm R. Schmelz K. Friedrich B. Mol. Microbiol. 2000; 38: 626-638Crossref PubMed Scopus (98) Google Scholar, 39Ramseier T.M. Figge R.M. Saier Jr., M.H. DNA Sequence. 1994; 5: 17-24Crossref PubMed Scopus (8) Google Scholar) thus suggesting an important role for ς54 in the NO response. Furthermore, the region upstream of the norVWgenes contains the respective −12 and −24 elements TTGCA and TGGCA characteristic of ς54-dependent promoters (40Reitzer L. Schneider B.L. Microbiol. Mol. Biol. Rev. 2001; 65: 422-444Crossref PubMed Scopus (220) Google Scholar, 41Barrios H. Valderrama B. Morett E. Nucleic Acids Res. 1999; 27: 4305-4313Crossref PubMed Scopus (303) Google Scholar). We used rpoN mutants to test the role of ς54 in NO-induced norV transcription and NOR activity expression. β-Galactosidase activity was measured in AG300 and ς54-deficient strain AG305 following a 45-min exposure to 600 ppm gaseous NO under microaerobic conditions. In the absence of ς54, norV-lacZ expression was substantially impaired (Fig.2 A). The ς54-deficient strain AG500 and parental AB1157 were similarly exposed to NO under low O2 and tested for anaerobic NOR and aerobic NOD activity. NOR activity was significantly reduced in strain AG500 (Fig. 2 B). There was no significant effect of ς54 on NOD (hmp) expression under these conditions (Fig. 2 C). The results clearly demonstrate a role for ς54 in norV transcription. The residual induction of norV transcription and NOR activity in the absence of ς54 suggests ancillary roles for other ς factors in norV transcription or mechanisms for post-transcriptional regulation. Expression of NorR from a multicopy plasmid rescued the NO inducibility of NOR activity in the norR deletion strain AG200 (Fig.3 A) thus confirming thetrans-acting regulatory role of NorR in the activation ofnorVW transcription and NOR activity expression (8Gardner A.M. Helmick R.A. Gardner P.R. J. Biol. Chem. 2002; 277: 8172-8177Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar, 32Hutchings M.I. Mandhana N. Spiro S. J. Bacteriol. 2002; 184: 4640-4643Crossref PubMed Scopus (104) Google Scholar). In the absence of NO, there was no measurable NOR activity expressed (Fig.3 A, open bars) indicating that overexpression of wild-type NorR does not by itself increase NorVW expression. However, internal deletion of NorR, eliminating amino acids 30–164 containing the putative signaling domain (31Pohlmann A. Cramm R. Schmelz K. Friedrich B. Mol. Microbiol. 2000; 38: 626-638Crossref PubMed Scopus (98) Google Scholar, 39Ramseier T.M. Figge R.M. Saier Jr., M.H. DNA Sequence. 1994; 5: 17-24Crossref PubMed Scopus (8) Google Scholar), but retaining the entire central ς54-interacting ATPase domain and the C-terminal DNA binding domain, induced NOR activity in the absence of NO (Fig.3 A). Further deletion of NorR to amino acid 214, eliminating part of the ς54-interacting ATPase domain, did not induce β-galactosidase activity (data not shown) thus further delineating the requirement for ς54 interaction with NorR for transcriptional activation. Interestingly, the NO-mediated induction of NOD activity was significantly (p < 0.05) reduced in strains expressing NorR and elevated NOR activity (Fig. 3 B) thus suggesting an indirect role for NorR and NOR in regulating NOD expression by reducing NO levels. These results demonstrate the signaling function of the N-terminal domain of the E. coli norVW transcription regulator NorR similar to that described for other tripartite regulators (31Pohlmann A. Cramm R. Schmelz K. Friedrich B. Mol. Microbiol. 2000; 38: 626-638Crossref PubMed Scopus (98) Google Scholar, 42Stock A.M. Robinson V.L. Goudreau P.N. Ann. Rev. Biochem. 2000; 69: 183-215Crossref PubMed Scopus (2456) Google Scholar). In addition, the results demonstrate that neither NorR nor ς54 is directly involved in the NO-mediated up-regulation of the E. coli NOD (hmp). Our data demonstrate that the exposure of E. coli to NO induces transcription of the norV and norW genes via a NorR and ς54-dependent mechanism. The data extend the results of Hutchings et al. (32Hutchings M.I. Mandhana N. Spiro S. J. Bacteriol. 2002; 184: 4640-4643Crossref PubMed Scopus (104) Google Scholar) demonstrating activation of norV transcription by the NO+ donor nitroprusside, nitrite, or nitrate in a NorR-dependent fashion. Given the relatively low concentration of NO required for anaerobic norV induction (Fig. 1 B), NO is the most probable physiological signal modulating NorR and norVW transcription. Our results differ from those of Hutchings et al. (32Hutchings M.I. Mandhana N. Spiro S. J. Bacteriol. 2002; 184: 4640-4643Crossref PubMed Scopus (104) Google Scholar) who reported that constitutive and induced norV transcription was greater in the presence of O2. One likely explanation for the discrepancy is that we used NO gas and Hutchings et al. (32Hutchings M.I. Mandhana N. Spiro S. J. Bacteriol. 2002; 184: 4640-4643Crossref PubMed Scopus (104) Google Scholar) used nitroprusside as a NO+ donor and potential NO-generating agent. Nitroprusside may have deleterious effects on transcription or, alternatively, may generate NO at higher levels in aerobic cells. Pure NO gas is readily available and is clearly preferred for investigations of the effects of NO on NO defense gene regulation. The use of pure NO gas for the quantitative evaluation of gene expression responses also presents challenges because of the existence of multiple pathways for rapid and inducible NO metabolism and because of the incipient toxicity of NO. Nevertheless, the demonstration that the NO levels required for norV induction correspond with levels shown to exert cellular damage strongly supports the proposed role of the norRVW operon in NO reduction and detoxification. Thus, 240 ppm gaseous NO (≤0.5 μm in solution) inactivated E. coli aconitase and 6-phosphogluconate dehydratase and inhibited growth in the absence of the induced NorVW activity (6Gardner P.R. Costantino G. Szabó C. Salzman A.L. J. Biol. Chem. 1997; 272: 25071-25076Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar, 8Gardner A.M. Helmick R.A. Gardner P.R. J. Biol. Chem. 2002; 277: 8172-8177Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar). Furthermore, the level of NO inducing half-maximal norV transcription (≤0.7 μm) approximates the apparent K m (NO) value of ∼0.4 μm determined for NorVW-catalyzed NO reduction (8Gardner A.M. Helmick R.A. Gardner P.R. J. Biol. Chem. 2002; 277: 8172-8177Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar). These results diminish the likelihood of a significant function of the operon in O2 detoxification or in the detoxification of unspecified reactive nitrogen intermediates generated from nitroprusside or NO exposure as previously suggested (25Gomes C.M. Giuffrè 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 (177) Google Scholar, 32Hutchings M.I. Mandhana N. Spiro S. J. Bacteriol. 2002; 184: 4640-4643Crossref PubMed Scopus (104) Google Scholar). A search of GenBankTM (NCBI) with the N-terminal 182 amino acids of NorR identifies several NorR orthologues (Fig.4). As in E. coli, NorR orthologues in Salmonella typhimurium, Klebsiella pnuemoniae, Shigella flexnerei (AAN44223), andVibrio vulnificus CMCP6 (NP_763239) are positioned upstream of norVW orthologues. Interestingly, NorR orthologues in thePseudomonas aeruginosa, Vibrio cholera,Azetobacter vinelandii (ZP_00091183),Burkholderia sp. strain TH2 (BAC16772), andBurkholderia fungorum (ZP00028693) genomes are found divergently transcribed from flavohemoglobin (hmp) genes suggesting a potential role for NorR in regulating NODs in response to NO. In this regard it is noteworthy that NorR was not required for the induction of NOD activity in response to NO in E. coli (Fig.3 B), thus demonstrating the existence of one or more separate NO-responsive regulator(s) of hmp in E. coli. NorR belongs to the family of two-component response regulators (42Stock A.M. Robinson V.L. Goudreau P.N. Ann. Rev. Biochem. 2000; 69: 183-215Crossref PubMed Scopus (2456) Google Scholar). Similar to other tripartite regulators in this family, deletion of the N-terminal signaling or inhibitory domain of NorR activated NorVW expression independent of NO (Fig. 3 A). Furthermore, conserved aspartate residues in the NorR N-terminal signaling domain suggest the potential for phosphorylation by a sensor histidine-kinase similar to that described for the NtrB/NtrC pair (43Weiss V. Magasanik B. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 8919-8923Crossref PubMed Scopus (186) Google Scholar). In particular, aspartates 57 and 62 are in position to accept phosphate, and the conserved acidic residue at position 14 may serve to optimize phosphorylation (Fig. 4) (44Stock J.B. Ninfa A.J. Stock A.M. Microbiol. Rev. 1989; 53: 450-490Crossref PubMed Google Scholar). Alternatively, the NorR N-terminal domain could activate transcription by interacting with a signal transducing protein as described for NifL/NifA (45Henderson N. Austin S.A. Dixon R.A. Mol. Gen. Genet. 1989; 216: 484-491Crossref Scopus (63) Google Scholar) or by binding NO directly as the formate-sensing transcription regulator FhlA binds formate (46Hopper S. Bock A. J. Bacteriol. 1995; 177: 2798-2803Crossref PubMed Google Scholar). The NorR N-terminal domain contains potential metal-liganding histidine and cysteine residues that could form the NO sensor module. For example, NorR contains an His111-X-Cys113 site reminiscent of the Cys75-X-His77heme iron ligand-switch motif in the carbon monoxide-sensing CooA of Rhodospirillum rubrum (47Lanzilotta W.N. Schuller D.J. Thorsteinsson M.V. Kerby R.L. Roberts G.P. Poulos T.L. Nat. Struct. Biol. 2000; 7: 876-880Crossref PubMed Scopus (234) Google Scholar). Fig. 5 summarizes our current view of the NO defense network in E. coli. NO exposure elicits the synthesis of two major NO-metabolizing enzymes, NOD and NOR (NorVW) by activating transcription of their corresponding genes, hmpand norVW. The respective contribution of each enzyme to NO detoxification depends primarily on the availability of O2. NOD is effective under aerobic and microaerobic conditions (K m (O2) = 60–100 μm) (13Gardner A.M. Martin L.A. Gardner P.R. Dou Y. Olson J.S. J. Biol. Chem. 2000; 275: 12581-12589Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 14Gardner P.R. Gardner A.M. Martin L.A. Dou Y. Li T. Olson J.S. Zhu H. Riggs A.F. J. Biol. Chem. 2000; 275: 31581-31587Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). The NOR activity of NorVW is unique in that its exquisite sensitivity to O2 restricts its NO scavenging function to 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 (256) Google Scholar, 20Gardner A.M. Gardner P.R. J. Biol. Chem. 2002; 277: 8166-8171Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). The results also support a model in which norVW and hmptranscription are indirectly influenced by O2 availability, because O2 levels affect NorVW and NOD activities, which ultimately determine NO steady-state levels and the activity of transcription regulators such as NorR and Fnr (29Cruz-Ramos H. Crack J. Wu G. Hughes M.N. Scott C. Thomson A.J. Green J. Poole R.K. EMBO J. 2002; 21: 3235-3244Crossref PubMed Scopus (253) Google Scholar, 48Poole R.K. Anjum M.F. Membrillo-Hernández J. Kim S.O. Hughes M.N. Stewart V. J. Bacteriol. 1996; 178: 5487-5492Crossref PubMed Scopus (208) Google Scholar). Interestingly, neither SoxRS nor OxyR, which have been persistently proposed to be critical NO stress response sensor-regulators (49Demple B. Mol. Cell. Biochem. 2002; 234: 11-18Crossref PubMed Scopus (45) Google Scholar, 50Kim S.O. Merchant K. Nudelman R. Beyer Jr., W.F. Keng T. DeAngelo J. Hausladen A. Stamler J.S. Cell. 2002; 109: 383-396Abstract Full Text Full Text PDF PubMed Scopus (390) Google Scholar) appear to be involved in the regulation of either hmp ornorVW in E. coli (Fig. 5) (48Poole R.K. Anjum M.F. Membrillo-Hernández J. Kim S.O. Hughes M.N. Stewart V. J. Bacteriol. 1996; 178: 5487-5492Crossref PubMed Scopus (208) Google Scholar). 2A. M. Gardner and P. R. Gardner, unpublished results. Future investigations will aim to further clarify the diverse roles and mechanisms of NO defense genes, enzymes, and regulators in microbial adaptations to NOin vitro and in various models of infection. We thank Drs. Alex Ninfa and Kenn Rudd for supplying strains and phage used in these investigations.
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