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Flavorubredoxin, an Inducible Catalyst for Nitric Oxide Reduction and Detoxification in Escherichia coli

2002; Elsevier BV; Volume: 277; Issue: 10 Linguagem: Inglês

10.1074/jbc.m110471200

1083-351X

Anne M. Gardner, Ryan A. Helmick, Paul R. Gardner,

Wastewater Treatment and Nitrogen Removal

Nitric oxide (NO) is a poison, and organisms employ diverse systems to protect against its harmful effects. InEscherichia coli, ygaA encodes a transcription regulator (b2709) controlling anaerobic NO reduction and detoxification. Adjacent to ygaA and oppositely transcribed are ygaK (encoding a flavorubredoxin (flavoRb) (b2710) with a NO-binding non-heme diiron center) and ygbD (encoding a NADH:(flavo)Rb oxidoreductase (b2711)), which function in NO reduction and detoxification. Mutation of either ygaA orygaK eliminated inducible anaerobic NO metabolism, whereasygbD disruption partly impaired the activity. NO-sensitive [4Fe-4S] (de)hydratases, including the Krebs cycle aconitase and the Entner-Doudoroff pathway 6-phosphogluconate dehydratase, were more susceptible to inactivation in ygaK orygaA mutants than in the parental strain, and these metabolic poisonings were associated with conditional growth inhibitions. flavoRb (NO reductase) and flavohemoglobin (NO dioxygenase) maximally metabolized and detoxified NO in anaerobic and aerobic E. coli, respectively, whereas both enzymes scavenged NO under microaerobic conditions. We suggest designation of the ygaA-ygaK-ygbD gene cluster as the norRVWmodulon for NO reduction and detoxification. Nitric oxide (NO) is a poison, and organisms employ diverse systems to protect against its harmful effects. InEscherichia coli, ygaA encodes a transcription regulator (b2709) controlling anaerobic NO reduction and detoxification. Adjacent to ygaA and oppositely transcribed are ygaK (encoding a flavorubredoxin (flavoRb) (b2710) with a NO-binding non-heme diiron center) and ygbD (encoding a NADH:(flavo)Rb oxidoreductase (b2711)), which function in NO reduction and detoxification. Mutation of either ygaA orygaK eliminated inducible anaerobic NO metabolism, whereasygbD disruption partly impaired the activity. NO-sensitive [4Fe-4S] (de)hydratases, including the Krebs cycle aconitase and the Entner-Doudoroff pathway 6-phosphogluconate dehydratase, were more susceptible to inactivation in ygaK orygaA mutants than in the parental strain, and these metabolic poisonings were associated with conditional growth inhibitions. flavoRb (NO reductase) and flavohemoglobin (NO dioxygenase) maximally metabolized and detoxified NO in anaerobic and aerobic E. coli, respectively, whereas both enzymes scavenged NO under microaerobic conditions. We suggest designation of the ygaA-ygaK-ygbD gene cluster as the norRVWmodulon for NO reduction and detoxification. nitric-oxide reductases nitric-oxide dioxygenases flavohemoglobin flavorubredoxin rubredoxin:O2oxidoreductase Nitric oxide (NO) is present throughout the biosphere (1Zumft W. Microbiol. Mol. Biol. Rev. 1997; 61: 533-616Crossref PubMed Scopus (2799) Google Scholar, 2Payne W.J. Liu M.-Y. Bursakov S.A. LeGall J. Biofactors. 1997; 6: 47-52Crossref PubMed Scopus (10) Google Scholar, 3Watmough N.J. Butland G. Cheesman M.R. Moir J.W.B. Richardson D.J. Spiro S. Biochim. Biophys. Acta. 1999; 1411: 456-474Crossref PubMed Scopus (118) Google Scholar). In humans, tightly regulated NO synthases produce sufficient NO to poison pathogens, opportunistic organisms, and neoplastic tissue (4Fang F.C. J. Clin. Invest. 1997; 99: 2818-2825Crossref PubMed Google Scholar, 5Nathan C. FASEB J. 1992; 6: 3051-3064Crossref PubMed Scopus (4129) Google Scholar). Nanomolar NO potently inhibits terminal oxidases and aerobic respiration (6Brown G.C. Biochim. Biophys. Acta. 1999; 1411: 351-369Crossref PubMed Scopus (607) Google Scholar, 7Gardner P.R. Martin L.A. Hall D. Gardner A.M. Free Radic. Biol. Med. 2001; 31: 191-204Crossref PubMed Scopus (84) Google Scholar) and alters the amphibolic and regulatory reactions of the citric acid cycle enzyme aconitase by destroying its labile [4Fe-4S] center (7Gardner P.R. Martin L.A. Hall D. Gardner A.M. Free Radic. Biol. Med. 2001; 31: 191-204Crossref PubMed Scopus (84) Google Scholar, 8Gardner P.R. Costantino G. Szabó C. Salzman A.L. J. Biol. Chem. 1997; 272: 25071-25076Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 9Drapier J.-C. Hibbs Jr., J.B. Methods Enzymol. 1996; 269: 26-36Crossref PubMed Google Scholar, 10Kennedy M.C. Antholine W.E. Beinert H. J. Biol. Chem. 1997; 272: 20340-20347Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar). In addition, significant secondary toxicity of NO can occur via reactions of NO2, ONOO−, NO−, dinitrosyl iron, and nitrosothiols (11Beckman J.S. Beckman T.W. Chen J. Marshall P.A. Freeman B.A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1620-1624Crossref PubMed Scopus (6647) Google Scholar, 12Miranda K.M. Espey M.G. Yamada K. Krishna M. Ludwick N. Kim S. Jourd'heuil G.M.B. Feelisch M. Fukuto J.M. Wink D.A. J. Biol. Chem. 2001; 276: 1720-1727Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 13Boese M. Keese M.A. Becker K. Busse R. Mülsch A. J. Biol. Chem. 1997; 272: 21767-21773Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 14Boese M. Mordvintcev P.I. Vanin A.F. Busse R. Mülsch A. J. Biol. Chem. 1995; 270: 29244-29249Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar). It has become increasingly evident that most organisms metabolize and detoxify NO. Enzymes decompose NO in microorganisms (1Zumft W. Microbiol. Mol. Biol. Rev. 1997; 61: 533-616Crossref PubMed Scopus (2799) Google Scholar, 3Watmough N.J. Butland G. Cheesman M.R. Moir J.W.B. Richardson D.J. Spiro S. Biochim. Biophys. Acta. 1999; 1411: 456-474Crossref PubMed Scopus (118) Google Scholar, 15Carr G.J. Ferguson S.J. Biochim. Biophys. Acta. 1990; 1017: 57-62Crossref PubMed Scopus (80) Google Scholar, 16Gardner P.R. Costantino G. Salzman A.L. J. Biol. Chem. 1998; 273: 26528-26533Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 17Gardner A.M. Gardner P.R. J. Biol. Chem. 2002; 277: 8166-8171Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 18Stevanin T.M. Ioannidis N. Mills C.E. Kim S.O. Hughes M.N. Poole R.K. J. Biol. Chem. 2000; 275: 35868-35875Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar) and humans (7Gardner P.R. Martin L.A. Hall D. Gardner A.M. Free Radic. Biol. Med. 2001; 31: 191-204Crossref PubMed Scopus (84) Google Scholar) and prevent the accumulation of toxic NO levels. Nitric-oxide reductases (NORs)1 metabolize NO to N2O in anaerobic denitrifying bacteria and fungi and likely serve an additional role in minimizing NO toxicity (1Zumft W. Microbiol. Mol. Biol. Rev. 1997; 61: 533-616Crossref PubMed Scopus (2799) Google Scholar, 3Watmough N.J. Butland G. Cheesman M.R. Moir J.W.B. Richardson D.J. Spiro S. Biochim. Biophys. Acta. 1999; 1411: 456-474Crossref PubMed Scopus (118) Google Scholar, 19Householder T.C. Fozo E.M. Cardinale J.A. Clark V.L. Infect. Immun. 2000; 68: 5241-5246Crossref PubMed Scopus (83) Google Scholar). Nitric-oxide dioxygenases (NODs) convert NO to NO3− in organisms as diverse as bacteria and mammals and have been shown to protect aerobic cells from NO damage (7Gardner P.R. Martin L.A. Hall D. Gardner A.M. Free Radic. Biol. Med. 2001; 31: 191-204Crossref PubMed Scopus (84) Google Scholar, 20Gardner P.R. Gardner A.M. Martin L.A. Salzman A.L. Proc. Natl. Acad. Sci. U. S. 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In microorganisms, (flavo)hemoglobins catalyze NO dioxygenation (20Gardner P.R. Gardner A.M. Martin L.A. Salzman A.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10378-10383Crossref PubMed Scopus (488) Google Scholar, 21Gardner 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, 22Gardner 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 (131) Google Scholar, 23Liu L. Zeng M. Hausladen A. Heitman J. Stamler J.S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4672-4676Crossref PubMed Scopus (165) Google Scholar, 24Mills C.E. Sedelnikova S. Søballe B. Hughes M.N. Poole R.K. Biochem. J. 2001; 353: 207-213Crossref PubMed Scopus (59) Google Scholar, 27Kim S.O. Orii Y. Lloyd D. Hughes M.N. Poole R.K. FEBS Lett. 1999; 445: 389-394Crossref PubMed Scopus (139) Google Scholar, 28Hausladen A. Gow A.J. Stamler J.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14100-14105Crossref PubMed Scopus (250) Google Scholar). In the accompanying article (17Gardner A.M. Gardner P.R. J. Biol. Chem. 2002; 277: 8166-8171Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar), we provide evidence for an inducible and robust NO-metabolizing and -detoxifying activity in anaerobicEscherichia coli. Attempts to biochemically identify the NO reduction system have been complicated by its instability. Moreover, the E. coli genome lacks a NOR belonging to either the cytochrome bc complex or cytochrome P450 families (1Zumft W. Microbiol. Mol. Biol. Rev. 1997; 61: 533-616Crossref PubMed Scopus (2799) Google Scholar). The list of proteins displaying a reductase activity for NO in vitro with potential for function in E. coli is long and includes flavohemoglobin (flavoHb) (27Kim S.O. Orii Y. Lloyd D. Hughes M.N. Poole R.K. FEBS Lett. 1999; 445: 389-394Crossref PubMed Scopus (139) Google Scholar, 28Hausladen A. Gow A.J. Stamler J.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14100-14105Crossref PubMed Scopus (250) Google Scholar), cytochromec or c′ (29Cross R. Lloyd D. Poole R.K. Moir J.W.B. J. Bacteriol. 2001; 183: 3050-3054Crossref PubMed Scopus (53) Google Scholar), multi-heme nitrite reductase (2Payne W.J. Liu M.-Y. Bursakov S.A. LeGall J. Biofactors. 1997; 6: 47-52Crossref PubMed Scopus (10) Google Scholar,30Costa C. Macedo A. Moura I. Moura J.J.G. LeGall J. Berlier Y. Liu M.-Y. Payne W.J. FEBS Lett. 1990; 276: 67-70Crossref PubMed Scopus (58) Google Scholar), copper-nitrite reductase (31Jackson M.A. Tiedje J.M. Averill B.A. FEBS Lett. 1991; 291: 41-44Crossref PubMed Scopus (67) Google Scholar), bacterioferritin (32Le Brun N.E. Andrews S.C. Moore G.R. Thomson A.J. Biochem. J. 1997; 326: 173-179Crossref PubMed Scopus (26) Google Scholar), ribonucleotide reductase (33Haskin C.J. Ravi N. Lynch J.B. Munck E. Que Jr., L. Biochemistry. 1995; 34: 11090-11098Crossref PubMed Scopus (71) Google Scholar), Cu,Zn-superoxide dismutase (34Liochev S.I. Fridovich I. J. Biol. Chem. 2001; 276: 35253-35257Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar), and terminal respiratory oxidases (35Zhao X.-J. Sampath V. Caughey W.S. Biochem. Biophys. Res. Commun. 1995; 212: 1054-1060Crossref PubMed Scopus (71) Google Scholar). However, none of these candidate systems, including NO-inducible flavoHb (17Gardner A.M. Gardner P.R. J. Biol. Chem. 2002; 277: 8166-8171Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar), have a demonstrated NO reduction function in cells. Moreover, unlike the inducible NOR inE. coli (17Gardner A.M. Gardner P.R. J. Biol. Chem. 2002; 277: 8166-8171Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar), none of these “NO reductases” are sensitive to inactivation by O2. Genomic data and bioinformatics tools (NCBI Protein Database and BLAST) provided a strategy for identifying the E. coli system.E. coli ygaA encodes a protein bearing ∼42% identity to the NO-modulated Ralstonia eutrophus transcription regulators NorR1 and NorR2 (36Pohlmann A. Cramm R. Schmelz K. Friedrich B. Mol. Microbiol. 2000; 38: 626-638Crossref PubMed Scopus (96) Google Scholar). Intriguingly, NorR homologs are also located adjacent to the flavoHb gene (hmp) in bothVibrio cholera (37Heidelberg J.F. Eisen J.A. Nelson W.C. Clayton R.A. Gwinn M.L. Dodson R.J. Haft D.H. Hickey E.K. Peterson J.D. Umayam L.A. Gill S.R. Nelson K.E. Read T.D. Tettelin H. Richardson D. Ermolaeva M.D. Vamathevan J. Bass S. Qin H. Dragoi I. Sellers P. McDonald L. Utterback T. Fleishman R.D. Nierman W.C. White O. Salzberg S.L. Smith H.O. Colwell R.R. Mekalanos J.J. Venter J.C. Fraser C.M. Nature. 2000; 406: 477-483Crossref PubMed Scopus (1414) Google Scholar) and Pseudomonas aeruginosa(38Stover C.K. Pham X.-Q.T. Erwin A.L. Mizoguchi S.D. Warrener P. Hickey M.J. Brinkman F.S.L. Hufnagle W.O. Kowalik D.J. Lagrou M. Garber R.L. Goltry L. Tolentino E. Westbrook-Wadman S. Yuan Y. Brody L.L. Coulter S.N. Folger K.R. Kas A. Larbig K. Lim R.M. Smith K.A. Spencer D.H. Wong G.K.-S. Wu Z. Paulsen I.T. Reizer J. Saier M.H. Hancock R.E.W. Lory S. Olson M.V. Nature. 2000; 406: 959-964Crossref PubMed Scopus (3325) Google Scholar), suggesting a common control for NO detoxification systems in various organisms. Adjacent to ygaA and transcribed in the opposite direction with specific promoters are the genesygaK encoding a flavorubredoxin (flavoRb) with a NO-binding diiron center and ygbD encoding a flavoRb reductase (39Wasserfallen A. Ragettli S. Jouanneau J. Leisinger T. Eur. J. Biochem. 1998; 254: 325-332Crossref PubMed Scopus (79) Google Scholar, 40Gomes C.M. Vicente J.B. Wasserfallen A. Teixeira M. Biochemistry. 2000; 39: 16230-16237Crossref PubMed Scopus (59) Google Scholar) with potential for a NOR function (see Fig. 1). We demonstrate here the role of the E. coli NorR homolog YgaA in controlling NOR expression. We also elucidate the role of flavoRb (YgaK) and its reductase partner (YgbD) in the NO-induced anaerobic NOR activity in E. coli. We further demonstrate that NOR and NOD protect NO-sensitive [4Fe-4S]-containing (de)hydratases in critical anabolic and catabolic pathways and thus explain conditional growth defects observed with NO poisoning inE. coli. A mechanism for flavoRb-catalyzed NO reduction is envisioned. We suggest renaming the ygaA-ygaK-ygbD gene cluster as norRVW and annotating similar genes for a possible NO detoxification function. DNA restriction and modifying enzymes were obtained from New England Biolabs Inc. Porcine heart isocitrate dehydrogenase, Aspergillus niger glucose oxidase, lactate dehydrogenase, casamino acids, HEPES, citrate, 6-phosphogluconate, NADP+, lactose, tetrazolium red, and antibiotics were obtained from Sigma. NADH and bovine liver catalase (260,000 units/ml) were purchased from Roche Molecular Biochemicals. Yeast extract, Bacto-agar, and Bacto-Tryptone were purchased from Fisher. Mixtures of 1200 ppm NO balanced with ultrapure N2, 1.05% O2 balanced with N2, and 99.998% N2 and 99.999% O2 were from Praxair (Bethlehem, PA). NO (98.5%) and CO (99.999%) were obtained from Aldrich. CO (1 mm) and NO (2 mm) stock solutions were prepared as previously described (22Gardner 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 (131) Google Scholar). E. colistrains AB1157 (F− thr-1 ara-C14 leuB6 DE(gpt-proA)62 lacY1 tsx-33 gsr′-0 glnV44 galK2 Rac-0 hisG4 rfbD1 mgl-51 rpoS396 rpsL31(strR) kdgK51 xyl-A5 mtl-1 argE3 thi-1) and JTG10 (AB1157gshA::Tn10kanR) were obtained from Dr. Bruce Demple (Harvard University). AG103 (AB1157hmp::Tn5kanR) was prepared as previously described (17Gardner A.M. Gardner P.R. J. Biol. Chem. 2002; 277: 8166-8171Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). Strain AG301 was created by transducinghmp::Tn5 from AG103 to AG300. Strain JC7623 (F− thr-1 ara-C14 leuB6 DE(gpt-proA)62 lacY1 sbcC201 tsx-33 gsr′-0 glnV44 galK2 Rac-0 sbcB15 hisG4 rfbD1 rpoS396 recB21 recC22 rpsL31(strR) kdgK51 xyl-A5 mtl-1 argE3 thi-1) was obtained from the E. coli Genetic Stock Center of Yale University. QC719 and QC720 were obtained from Dr. Danièlle Touati (CNRS, Paris, France) (41Carlioz A. Touati D. EMBO J. 1986; 5: 623-630Crossref PubMed Scopus (608) Google Scholar). P1vir phage was obtained from Dr. Jim Imlay (University of Illinois). The E. coli miniset clone 9G10 (no. 447) of Kohara et al.(42Kohara Y. Akiyama K. Isono K. Cell. 1987; 50: 495-508Abstract Full Text PDF PubMed Scopus (1070) Google Scholar) in λ phage, encompassing thesrlA-ascF chromosome segment, was generously provided by Dr. Kenn Rudd (University of Miami). A 13.355-kbKpnI-BamHI fragment containing theygaA, ygaK, and ygbD genes and the corresponding open reading frames (b2709, b2710, and b2711, respectively) was subcloned from 9G10 into pAlter (Promega Corp., Madison, WI) that was modified to express ampicillin resistance. Disruptions in the ygaA, ygaK, andygbD genes were created by Mu transposon insertion. MudIIPR13 (camR), a generous gift of Dr. Danièlle Touati, was randomly transposed to pAlter9G10 by the method of Castilhoet al. (43Castilho B.A. Olfson P. Casadaban M.J. J. Bacteriol. 1984; 158: 488-495Crossref PubMed Google Scholar) as modified by Carlioz and Touati (41Carlioz A. Touati D. EMBO J. 1986; 5: 623-630Crossref PubMed Scopus (608) Google Scholar). Briefly, strain QC720 carrying a Mucts prophage and MudIIPR13 was transformed at 30 °C with pAlter9G10. Mu transposition, phage growth, and cell lysis were induced at 44 °C in LB medium prepared with 10 g of Tryptone, 5 g of yeast extract, and 10 g of NaCl prepared in 1 liter of deionized water and supplemented with 20 mm glucose. Phage lysates were used to transduce strain QC719 to ampicillin and chloramphenicol resistance, thus selecting for plasmids carrying a Mudlactransposon. Ampicillin- and chloramphenicol-resistant clones were further scored for β-galactosidase fusion phenotype on tetrazolium red-lactose agar (44Miller J.H. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1972Google Scholar). White β-galactosidase-positive colonies were selected and screened for Mudlac insertions inygaA, ygaK, and ygbD by restriction analysis. To enrich for NO-inducible, anaerobically expressed β-galactosidase gene fusions, ampicillin- and chloramphenicol-resistant colonies were replica-plated onto tetrazolium red-lactose agar with antibiotics and grown overnight under an atmosphere containing 180 ppm NO balanced with N2. Following an additional 24 h of growth in air, colonies that were originally red, but turned pink upon exposure to NO, were chosen for restriction analysis. Insertion sites were determined by sequencing from the 5′-end of the Mudlac insertions using primer 5′-AATACATCTGTTTCATTTG-3′. Plasmids with insertions at amino acids 311, 232, and 11 in the respective YgaA, YgaK, and YgbD open reading frames were isolated. Plasmids were linearized with XbaI, and the DNA was used to transform strain JC7623 to chloramphenicol resistance (45Kushner S.R. Nagaishi H. Temple A. Clark A.J. Proc. Natl. Acad. Sci. U. S. A. 1971; 68: 824-827Crossref PubMed Scopus (227) Google Scholar). Several chloramphenicol-resistant, ampicillin-sensitive colonies were selected for analysis. Site-specific insertion in theygaA, ygaK, and ygbD genes was confirmed by linkage to gshA using strain JTG10 as the recipient in P1 transduction analyses. Mutations were subsequently transduced to strain AB1157. Cultures were grown in phosphate-buffered LB medium (8Gardner P.R. Costantino G. Szabó C. Salzman A.L. J. Biol. Chem. 1997; 272: 25071-25076Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar) unless otherwise indicated. Minimal salts medium (pH 7.0) contained 60 mmK2HPO4, 33 mmKH2PO4, 7.6 mm(NH4)2SO4, 1.7 mmsodium citrate, 1 mm MgSO4, 10 μmMnCl2, and 10 μg/ml thiamin HCl and was supplemented with either 20 mm glucose or 2% potassium gluconate. Minimal medium was supplemented with 40 μg/ml l-arginine,l-histidine, l-leucine, l-proline, and l-threonine or with 0.25% casamino acids as indicated. Overnight 5-ml aerobic cultures were grown at 37 °C in 15-ml culture tubes with vigorous shaking. Overnight 10-ml anaerobic cultures were grown static at 37 °C in 15-ml culture tubes. For gas exposures, cultures were grown at 37 °C in rubber stopper-sealed 50-ml Erlenmeyer flasks continuously flushed with gas mixtures at 30 ml/min at a culture/flask volume ratio of at most 1:5 with vigorous shaking at 275 rpm. To minimize the disturbance of gases, culture aliquots were removed from flasks using a 1-ml tuberculin syringe connected via narrow tubing to the culture medium. Cell density was determined from culture absorbance at 550 nm and by plating and counting. An absorbance of 1.0 was taken to equal 3 × 108 and 7 × 108 bacteria/ml for cells grown in phosphate-buffered LB medium and minimal salts medium, respectively. Aerobic and anaerobic NO consumption activities were measured amperometrically using a 2-mm ISO-NOP NO electrode (World Precision Instruments, Sarasota, FL) as previously described (16Gardner P.R. Costantino G. Salzman A.L. J. Biol. Chem. 1998; 273: 26528-26533Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 17Gardner A.M. Gardner P.R. J. Biol. Chem. 2002; 277: 8166-8171Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). Aerobic and anaerobic activities were measured at 37 °C and at 1.0 and 1.5 μm NO, respectively, unless otherwise indicated. Cells were harvested; extracts were prepared; and aconitase, 6-phosphogluconate dehydratase, and protein were assayed as previously described (16Gardner P.R. Costantino G. Salzman A.L. J. Biol. Chem. 1998; 273: 26528-26533Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 46Gardner P.R. Methods Enzymol. 2002; 349: 9-23Crossref PubMed Scopus (244) Google Scholar, 47Gardner P.R. Fridovich I. J. Biol. Chem. 1991; 266: 1478-1483Abstract Full Text PDF PubMed Google Scholar, 48Gardner P.R. Fridovich I. J. Biol. Chem. 1992; 267: 8757-8763Abstract Full Text PDF PubMed Google Scholar). Significance of differences between data (p < 0.05) were determined using Student'st test. To test the individual roles of theygaA, ygaK, and ygbD gene products in the anaerobic NO consumption activity, we constructed strains carrying insertion mutations using random Mudlac transposition (Fig.1). The anaerobic and aerobic NO consumption activities of strains AG200, AG300, and AG400, with Mudlac insertions in ygaA, ygaK, andygbD, respectively, were measured and compared with those of the parental strain, AB1157. Strains bearing mutations inygaA or ygaK showed no anaerobic NO consumption activity following exposure to 960 ppm NO under anaerobic growth conditions, whereas the ygbD mutant produced a rate ∼40% lower than that of its parental strain (Fig.2 A). None of the strains showed significant anaerobic NO consumption activity in the absence of NO exposure. We also measured the aerobic NO consumption activity of anaerobically induced cells. Each strain showed the normal basal level of aerobic NO consumption activity (Fig. 2 B, comparewhite bars), and this activity was induced to high levels by NO (black bars). The absence of ygaA,ygaK, or ygbD resulted in small increases in the induction of the aerobic NO consumption activity (compare black bars). This aerobic NO consumption activity is fully attributable to flavoHb (20Gardner P.R. Gardner A.M. Martin L.A. Salzman A.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10378-10383Crossref PubMed Scopus (488) Google Scholar). These results demonstrate that YgaA (NorR) and YgaK (NorV, flavoRb) are essential for anaerobic NO consumption. Thus, YgaA and YgaK constitute a novel modulon for NO reduction and detoxification in E. coli, with YgbD (NorW, flavoRb reductase) acting as an accessory for NO reduction. YgaA, YgaK, and YgbD may decrease aerobic NO consumption activities by decreasing steady-state NO levels and flavoHb/NOD expression in anaerobic cells. We examined the efficiency of the anaerobic NO consumption activity for NO scavenging. NOR showed an apparentK m(NO) value of 400 nm (Fig.3, ●) and was CO-resistant (○) and cyanide-resistant (17Gardner A.M. Gardner P.R. J. Biol. Chem. 2002; 277: 8166-8171Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). The K m(NO) value is identical to the values estimated for the flavoHb-type NOD inE. coli (21Gardner 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) and for the cytochrome bc-type NOR in Pseudomonas perfectomarina (49Zafiriou O.C. Hanley Q.S. Snyder G. J. Biol. Chem. 1989; 264: 5694-5699Abstract Full Text PDF PubMed Google Scholar). The results demonstrate the efficiency of the anaerobic system for NO scavenging. The results also indicate low affinities of the NO scavenger flavoRb (YgaK) for CO and cyanide. To define the NO detoxification function of ygaA, ygaK, and ygbD in cells, it is necessary to understand the protective role(s) of the system for critical target(s) of NO poisoning under physiological conditions. Thus, we tested the role of ygaA, ygaK, andygbD in the inducible anaerobic protection of the NO-sensitive Krebs cycle enzyme aconitase (16Gardner P.R. Costantino G. Salzman A.L. J. Biol. Chem. 1998; 273: 26528-26533Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 17Gardner A.M. Gardner P.R. J. Biol. Chem. 2002; 277: 8166-8171Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). E. coli(AB1157) exposed to 480 ppm NO gas (<1 μm) lost ∼45% of the aconitase activity after 30 min (Fig.4). Aconitase inactivation increased significantly (p < 0.05) in the presence of chloramphenicol, thus demonstrating the protective role for newly synthesized protein(s). Moreover, loss of aconitase activity in theygaA (AG200) and ygaK (AG300) mutants was greater than in the parental strain, thus demonstrating critical roles forygaA and ygaK in the adaptive protection. In contrast, ygbD (AG400) was not essential for aconitase protection under these conditions. To further define the functional importance of ygaA, ygaK, andygbD for E. coli, we investigated the effects of specific insertion mutations in each of these genes on the anaerobic growth of E. coli exposed to a NO stress. Surprisingly, there was little growth inhibition with 240 ppm NO gas (≤0.5 μm) for cells growing on a rich phosphate-buffered LB medium (Fig. 5 A). Higher NO exposure levels caused comparable growth inhibition of the mutants and the parental strain (data not shown). These results suggest a limited role for ygaA, ygaK, and ygbD and anaerobic NO metabolism in growth protection. Nevertheless, the exquisite sensitivity of aconitase to NO-mediated inactivation (Fig. 4) (8Gardner P.R. Costantino G. Szabó C. Salzman A.L. J. Biol. Chem. 1997; 272: 25071-25076Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 16Gardner P.R. Costantino G. Salzman A.L. J. Biol. Chem. 1998; 273: 26528-26533Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar) strongly suggested that anaerobic growth protection may be better observed under conditions requiring aconitase function, the citric acid cycle, or other NO-sensitive metabolic pathways. Aconitase expression is relatively low under these conditions; and moreover, aconitase function is not expected to be limiting for E. coli growth with glucose supplied as the substrate for energy production (50Gardner P.R. Fridovich I. J. Biol. Chem. 1991; 266: 19328-19333Abstract Full Text PDF PubMed Google Scholar). The effect of NO on anaerobic growth of E. coli mutants was investigated under growth conditions demanding the function of putative NO-sensitive enzymes. The [4Fe-4S]-containing 6-phosphogluconate dehydratase of the gluconate-metabolizing Entner-Doudoroff pathway and the [4Fe-4S]-containing α,β-dihydroxyacid dehydratase of the branched-chain amino acid biosynthesis pathway are two enzymes that are predicted to be NO-sensitive (8Gardner P.R. Costantino G. Szabó C. Salzman A.L. J. Biol. Chem. 1997; 272: 25071-25076Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 51Gardner P.R. Biosci. Rep. 1997; 17: 33-42Crossref PubMed Scopus (148) Google Scholar). Anaerobic growth of strains AG200 and AG300 was significantly impaired by exposure to 240 ppm NO gas under growth conditions requiring gluconate metabolism (Fig.5 B) or amino acid biosynthesis (Fig. 5 C). These results demonstrate important, albeit conditional, roles for YgaA (NorR) and YgaK (NorV) in NO detoxification. Measurements of 6-phosphogluconate dehydratase activity following a 60-min exposure of parental or flavoRb-deficient cells to NO gas (240 ppm) demonstrated the protection that flavoRb (YgaK) and NO reduction afforded to 6-phosphogluconate dehydratase (Fig.6, compare white andblack bars). The results establish the sensitivity of this [4Fe-4S] enzyme to NO and further demonstrate the protection NO metabolism affords against metabolic NO poisoning. The anaerobic NO consumption activity in E. coli is O2-sensitive, decaying with a half-life of ∼5 min in air (17Gardner A.M. Gardner P.R. J. Biol. Chem. 2002; 277: 8166-8171Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar), suggesting that flavoRb may function poorly, if at all, in the presence of O2. On the other hand, the aerobic NOD activity of flavoHb shows a rather highK m value for O2 (60–100 μm) and is potently inhibited