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Constitutive and Adaptive Detoxification of Nitric Oxide in Escherichia coli

1998; Elsevier BV; Volume: 273; Issue: 41 Linguagem: Inglês

10.1074/jbc.273.41.26528

1083-351X

Paul R. Gardner, Giuseppina Costantino, Andrew L. Salzman,

Neutrophil, Myeloperoxidase and Oxidative Mechanisms

Nitric oxide (NO⋅) is a naturally occurring toxin that some organisms adaptively resist. In aerobic or anaerobic Escherichia coli, low levels of NO⋅exposure inactivated the NO⋅-sensitive citric acid cycle enzyme aconitase, and inactivation was more effective when the adaptive synthesis of NO⋅-defensive proteins was blocked with chloramphenicol. Protection of aconitase in aerobically grown E. coli was dependent upon O2, was potently inhibited by cyanide, and was correlated with an induced rate of cellular NO⋅consumption. Constitutive and adaptive cellular NO⋅ consumption in aerobic cells was also dependent upon O2 and inhibited by cyanide. Exposure of aerobic cells to NO⋅ accordingly elevated the activity of the O2-dependent and cyanide-sensitive NO⋅ dioxygenase (NOD). Anaerobic E. coli exposed to NO⋅ or nitrate induced a modest O2-independent and cyanide-resistant NO⋅-metabolizing activity and a more robust O2-stimulated cyanide-sensitive activity. The latter activity was attributed to NOD. The results support a role for NOD in the aerobic detoxification of NO⋅ and suggest functions for NOD and a cyanide-resistant NO⋅ scavenging activity in anaerobic cells. Nitric oxide (NO⋅) is a naturally occurring toxin that some organisms adaptively resist. In aerobic or anaerobic Escherichia coli, low levels of NO⋅exposure inactivated the NO⋅-sensitive citric acid cycle enzyme aconitase, and inactivation was more effective when the adaptive synthesis of NO⋅-defensive proteins was blocked with chloramphenicol. Protection of aconitase in aerobically grown E. coli was dependent upon O2, was potently inhibited by cyanide, and was correlated with an induced rate of cellular NO⋅consumption. Constitutive and adaptive cellular NO⋅ consumption in aerobic cells was also dependent upon O2 and inhibited by cyanide. Exposure of aerobic cells to NO⋅ accordingly elevated the activity of the O2-dependent and cyanide-sensitive NO⋅ dioxygenase (NOD). Anaerobic E. coli exposed to NO⋅ or nitrate induced a modest O2-independent and cyanide-resistant NO⋅-metabolizing activity and a more robust O2-stimulated cyanide-sensitive activity. The latter activity was attributed to NOD. The results support a role for NOD in the aerobic detoxification of NO⋅ and suggest functions for NOD and a cyanide-resistant NO⋅ scavenging activity in anaerobic cells. nitric oxide nitric-oxide reductase nitric-oxide dioxygenase Luria-Bertani. Nitric oxide (NO⋅)1 is released by leukocytes and functions as an antibiotic (1MacMicking J.D. North R.J. LaCourse R. Mudgett J.S. Shah S.K. Nathan C.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5243-5248Crossref PubMed Scopus (913) Google Scholar, 2Stenger S.N. Donhauser H. Thüring M. Röllinghoff M. Bogdan C. J. Exp. Med. 1996; 183: 1501-1514Crossref PubMed Scopus (284) Google Scholar, 3Fang F.C. J. Clin. Invest. 1997; 99: 2818-2825Crossref PubMed Google Scholar). It may also be produced endogenously by bacteria during the reduction of NO2− by nitrate reductase (4Ji X. Hollocher T.C. Biochem. Biophys. Res. Commun. 1988; 157: 106-108Crossref PubMed Scopus (75) Google Scholar). In addition, bacteria may encounter NO⋅ released by competing microorganisms (5Morita H. Yoshikawa H. Sakata R. Nagata Y. Tanaka H. J. Bacteriol. 1997; 179: 7812-7815Crossref PubMed Scopus (83) Google Scholar). Regardless of the source, NO⋅ produced at sufficient levels directly or indirectly damages critical cell processes (3Fang F.C. J. Clin. Invest. 1997; 99: 2818-2825Crossref PubMed Google Scholar). Indeed, NO⋅ is bacteristatic toward some bacteria (6Mancinelli R. McKay C.P. Appl. Environ. Microbiol. 1983; 46: 198-202Crossref PubMed Google Scholar), and NO⋅ or NO⋅-derived species may display bactericidal activities in vitro and in infected animals (1MacMicking J.D. North R.J. LaCourse R. Mudgett J.S. Shah S.K. Nathan C.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5243-5248Crossref PubMed Scopus (913) Google Scholar, 2Stenger S.N. Donhauser H. Thüring M. Röllinghoff M. Bogdan C. J. Exp. Med. 1996; 183: 1501-1514Crossref PubMed Scopus (284) Google Scholar, 3Fang F.C. J. Clin. Invest. 1997; 99: 2818-2825Crossref PubMed Google Scholar, 7Hurst J.K. Lymar S.V. Chem. Res. Toxicol. 1997; 10: 802-810Crossref PubMed Scopus (60) Google Scholar, 8Shank J.L. Silliker J.H. Harper R.H. Appl. Microbiol. 1962; 10: 185-189Crossref PubMed Google Scholar, 9Brunelli L. Crow J.P. Beckman J.S. Arch. Biochem. Biophys. 1995; 316: 327-334Crossref PubMed Scopus (266) Google Scholar, 10Pacelli R. Wink D.A. Cook J.A. Krishna M.C. DeGraff W. Friedman N. Tsokos M. Samuni A. Mitchell J.B. J. Exp. Med. 1995; 182: 1469-1479Crossref PubMed Scopus (224) Google Scholar). Various organisms may benefit from adaptive mechanisms for NO⋅detoxification. Denitrifying bacteria (11Dermastia M. Turk T. Hollocher T.C. J. Biol. Chem. 1991; 266: 10899-10905Abstract Full Text PDF PubMed Google Scholar, 12Fujiwara T. Fukumori Y. J. Bacteriol. 1996; 178: 1866-1871Crossref PubMed Google Scholar) and fungi (13Nakahara K. Tanimoto T. Hatano K. Usuda K. Shoun H. J. Biol. Chem. 1993; 268: 8350-8355Abstract Full Text PDF PubMed Google Scholar) are known to produce NO⋅-inducible (14Kwiatkowski A.V. Shapleigh J.P. J. Biol. Chem. 1996; 271: 24382-24388Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar) NO⋅-detoxifying NORs. NORs catalytically reduce NO⋅ to produce nitrous oxide (N2O). NORs also increase the anaerobic energy production capacity of denitrifiers by catalyzing an essential step in the reduction of nitrate (NO3−) and nitrite (NO2−) to N2 (15Carr G.J. Page M.D. Ferguson S.J. Eur. J. Biochem. 1989; 179: 683-692Crossref PubMed Scopus (70) Google Scholar). Considerably less is known of the adaptations of nondenitrifying bacteria to NO⋅ or of their normal exposures to NO⋅. An adaptation of Escherichia coli to NO⋅ under the transcriptional control of the antioxidant regulators SoxRS and OxyR has been suggested, since these global antioxidant regulators provide some survival and growth benefits against NO⋅ (16Nunoshiba T. Derojas-Walker T. Tannenbaum S.R. Demple B. Infect. Immun. 1995; 63: 794-798Crossref PubMed Google Scholar, 17Nunoshiba T. Derojas-Walker T. Wishnok J.S. Tannenbaum S.R. Demple B. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9993-9997Crossref PubMed Scopus (280) Google Scholar) or nitrosothiols (18Hausladen A. Privalle C.T. Keng T. DeAngelo J. Stamler J.S. Cell. 1996; 86: 719-729Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar), respectively. Yet, it remains unclear how these regulators protect bacteria. E. coli does not appear to produce a typical NOR activity, but it does produce a multiheme nitrite reductase with NO⋅-reducing capacity (19Costa C. Macedo A. Moura I. Moura J.J.G. Le Gall J. Berlier Y. Liu M.-Y. Payne W.J. FEBS Lett. 1990; 276: 67-70Crossref PubMed Scopus (58) Google Scholar) and a nitric oxide dioxygenase (NOD) that has been proposed to function in NO⋅detoxification (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 (500) Google Scholar). We have observed, and now report, an increased susceptibility to NO⋅ of the NO⋅-sensitive citric acid cycle enzyme aconitase (21Gardner 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, 22Kennedy M.C. Antholine W.E. Beinert H. J. Biol. Chem. 1997; 272: 20340-20347Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar) in aerobic or anaerobic E. coli inhibited for de novo protein synthesis. Protection of aconitase activity correlated with an increased rate of cellular NO⋅consumption and correlated with an increased NOD activity in cell-free extracts. The results support the proposed function of NOD in the constitutive and adaptive detoxification of NO⋅ in aerobicE. coli. The possible role of the NO⋅-induced NOD activity in anaerobic E. coli is discussed. E. coli strain DH5α was from Life Technologies, Inc. Mutants deficient in the terminal oxidases ECL936 (Δcyo), ECL937 (Δcyd), and the parent ECL933 were kindly provided by E. C. C. Lin (23Iucchi S. Chepuri V. Fu H.-A. Gennis R.B. Lin E.C.C. J. Bacteriol. 1990; 172: 6020-6025Crossref PubMed Google Scholar). Compressed gas cylinders containing 1200 ppm (± 5%) NO⋅ in ultrapure N2, 99.998% N2, and 99.993% O2 were obtained from Praxair (Bethlehem, PA). NO⋅-saturated water was prepared by stirring N2-equilibrated water under 98.5% NO⋅ gas (Aldrich), which was first bubbled through 1 n NaOH. Saturated NO⋅ was stored at 4 °C in a rubber septum-sealed glass tube. Sodium cyanide, NADP+, FAD, chloramphenicol, glucose oxidase from Aspergillus niger, glucose-6-phosphate dehydrogenase from bakers' yeast, sodium nitrite, sodium nitrate, succinic acid, d-glucose, glucose 6-phosphate,l-arginine-HCl, thiamine HCl, Tris, and MnCl2were from Sigma. Nitrate reductase from Aspergillus nigerand bovine liver catalase were purchased from Boehringer Mannheim. Tryptone and yeast extract were obtained from Difco. The minimal salts medium was made up with tap water and contained 60 mm K2HPO4, 33 mmKH2PO4, 7.6 mm(NH4)2SO4, 1.7 mmsodium citrate, 1 mm MgSO4, 10 μmMnCl2, 10 μg/ml thiamine Cl, 40 μg/mll-arginine, and 10 mm sodium succinate or 10 mm glucose as indicated. The phosphate-buffered LB medium was prepared with 10 g of tryptone, 5 g of yeast extract, and 10 g of NaCl per liter of 66 mmK2HPO4, 33 mmKH2PO4, and 10 μmMnCl2. The pH of the phosphate-buffered LB and minimal salts media were adjusted to 7.0 with HCl or NaOH. MnCl2was routinely added to media to ensure full expression of the inducible O⨪2-scavenging manganese-containing superoxide dismutase, maximal activity of the O⨪2-sensitive aconitase, and thus optimal growth on citric acid cycle-dependent substrates (32Gardner P.R. Fridovich I. J. Biol. Chem. 1992; 267: 8757-8763Abstract Full Text PDF PubMed Google Scholar). To achieve maximal gas exchange, cultures were routinely grown in a gyrorotary water bath shaking at >200 rpm at 37 °C with a medium:flask volume ratio of at most 1:5, and growth was monitored by following the turbidity at 550 nm. Bacterial densities were determined by dilution, plating, and colony counting. An absorbance of 1.0 at 550 nm corresponded to 7 × 108 bacteria/ml when bacteria were grown in the minimal medium. Anoxic growth of cultures was achieved by incubating cultures at 37 °C in static stopper-sealed 50-ml Erlenmeyer flasks filled with 50 ml of medium. To minimize the disturbance of head space gases, culture aliquots were removed from gas-equilibrated culture flasks using a 1-ml tuberculin syringe connected via small tubing. Culture aliquots were immediately transferred to 1.5-ml Eppendorf tubes and were quickly centrifuged at 20,000 × g for 25 s, the supernatant was aspirated, and the cell pellet was frozen on dry ice. Cell pellets were resuspended and lysed by sonicating in 0.1 ml of buffer containing 50 mm Tris-Cl, pH 7.4, 0.6 mm MnCl2, and 20 μm barium dl-fluorocitrate, and the lysate was frozen on dry ice. Cell lysates were stored at −70 °C for up to 2 weeks without noticeable loss of aconitase activity. Lysates were thawed in a 25 °C water bath and clarified by centrifugation for 25 s at 20,000 × g immediately prior to the assay of aconitase activity. Cell lysates were prepared for the assay of NOD activity essentially as described for the assay of aconitase except that the lysis buffer contained 50 mm potassium phosphate, pH 7.8, and 0.1 mm EDTA. A three-way gas proportioner (Cole-Parmer Instrument Co.) was used to produce various mixtures of O2, N2, and NO⋅ at a constant flow rate of 30 ml/min, and gas mixtures were passed through a trap containing NaOH pellets to remove higher oxides of nitrogen. Aconitase activity and protein were assayed as described previously (21Gardner 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). For the measurement of media NO2− and NO3−, cultures were clarified by centrifugation, and supernatants were incubated at 37 °C for 2 h in a 0.1-ml reaction mixture containing 7 milliunits of nitrate reductase and 40 μm NADPH in 100 mm Tris-Cl, pH 7.5. Samples were assayed for NO2−with the Griess reagent (24Green L.C. Wagner D.A. Glogowski J. Skipper P.L. Wishnok J.S. Tannenbaum S.R. Anal. Biochem. 1982; 126: 131-138Crossref PubMed Scopus (10881) Google Scholar) using sodium nitrite as a standard. NO⋅ consumption was measured at 37 °C with an NO⋅ microelectrode (Diamond General Inc.) fitted in a water-jacketed glass-stoppered 2-ml capacity cell (Gilson Inc.) equipped with a magnetic stirrer. One microliter of NO⋅ was delivered to the cell with a Hamilton syringe from a saturated solution (2 mm) prepared in water. Rates of NO⋅ consumption by bacteria were measured in minimal medium salts containing chloramphenicol and glucose or succinate as indicated. Rates of NO⋅ consumption were determined from initial rates and were corrected for the background rate of NO⋅ decomposition. O2 consumption was measured at 37 °C with a Clark-type O2 electrode (Yellow Springs Instrument Co.) in a water-jacketed cell in a total volume of 2.0 ml of minimal succinate medium. The O2 concentration for media saturated with air at normal atmospheric pressure and 37 °C was taken to be 200 μm (25Chappell J.B. Biochem. J. 1964; 90: 225-237Crossref PubMed Scopus (221) Google Scholar). Cell-free extracts were assayed for NOD activity at 37 °C in a 2-ml reaction mixture containing 50 mm potassium phosphate buffer, pH 7.8, 0.1 mmEDTA, 1 μm FAD, 0.2 mm NADP+, 0.5 units/ml glucose-6-phosphate dehydrogenase, 2.5 mm glucose 6-phosphate, and 1 μm NO⋅. Initial rates of NO⋅ disappearance from reaction mixtures were followed amperometrically with an NO⋅ electrode and were corrected for the background rate of NO⋅ decomposition. Where indicated, O2 was removed by incubating the mixture with 10 mm glucose, 2 units/ml glucose oxidase, and 130 units/ml catalase for 5 min prior to the addition of NO⋅ and extract. O2 removal was followed amperometrically with an O2 electrode. Results are representative of two or more independent experiments. We measured the effects of NO⋅ on the growth of E. coli in order to gauge the capacity of cells to adapt to NO⋅ under various growth conditions. Exposure of log phase cultures to an atmosphere containing 960 ppm NO⋅ had little effect on the aerobic growth of E. coli in either the minimal succinate or the rich LB medium (Fig. 1, A and B, comparelines 1 and 2). Similarly, 480 ppm NO⋅exerted no discernible effect on the anaerobic growth of E. coli in a minimal glucose medium supplemented with 10 mm nitrate (data not shown). Interestingly, however, NO⋅ exposure caused a small, but significant, decrease in the aerobic growth rate and the yield of E. coli in the LB medium at the end of the log phase (Fig. 1 B, comparelines 1 and 2). The effect of growth phase on NO⋅ inhibition was explored further. Exposure of late stationary phase E. coli to 960 ppm NO⋅ strongly inhibited growth (Fig. 1, C and D, compare lines 1 and 2), and the growth-inhibitory effects of NO⋅ were more pronounced in the LB medium (Fig. 1 D). Growth inhibition was readily reversible, as indicated by the ability of NO⋅-treated cells to resume normal growth following NO⋅ removal (Fig. 1, C and D, comparelines 2 and 3). The susceptibility of stationary phase cultures to NO⋅-mediated growth inhibition suggests a requirement for nutritional resources, protein synthesis, or growth competency for NO⋅ resistance. Moreover, the ability of cells to grow normally at NO⋅ levels that were previously shown to potently inactivate the citric acid cycle enzyme aconitase (21Gardner 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) suggests that NO⋅ resistance is due to the presence of adaptive mechanisms. The NO⋅-sensitive aconitase (21Gardner 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, 22Kennedy M.C. Antholine W.E. Beinert H. J. Biol. Chem. 1997; 272: 20340-20347Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar) was used to explore the mechanism of adaptation of E. coli to NO⋅. Thus, we supposed that adaptation to NO⋅ should correlate with the protection of aconitase from inactivation. Indeed, aconitase activity was more sensitive to NO⋅ in the presence of the protein synthesis inhibitor chloramphenicol than in its absence (Fig. 2, compare lines 1 and 2). Further, NO⋅ was a more effective inactivator of aconitase in the absence of O2 than in its presence (Fig. 2, compare lines 2 and 4), and, interestingly, chloramphenicol had no apparent effect on the susceptibility of aconitase to NO⋅-mediated inactivation in the absence of O2 (Fig. 2, compare lines 3 and 4). The data clearly indicate inducible protective mechanisms for aconitase and demonstrate a role of O2 in the adaptive mechanism. We also measured the effects of chloramphenicol on NO⋅-mediated aconitase inactivation in anaerobic E. coli, since aerobic succinate-adapted E. coli are dependent upon O2for growth, respiration, and ATP production, which may have affected the ability of cells to adapt. Exposure of anaerobic glucose-adaptedE. coli to 240 ppm NO⋅ in N2 caused a greater decline of aconitase activity in the presence of chloramphenicol than in its absence (Fig. 3, compare lines 1 and 2). It is noteworthy that the inducible protection of aconitase was relatively less effective under anaerobic than aerobic conditions. Thus, a ∼20% loss of aconitase was observed following 60 min of 240 ppm NO⋅ exposure in anaerobic cultures (Fig. 3,line 1), whereas 480 ppm NO⋅ did not affect the aconitase activity in aerobic cultures (Fig. 2, line 1). Thus, anaerobic as well as aerobic E. coli protect aconitase from NO⋅-mediated inactivation. Moreover, while O2was not absolutely essential for adaptive protection, it did increase the apparent NO⋅ detoxification capacity of cells. We supposed that the decreased sensitivity of aconitase to NO⋅ in chloramphenicol-treated E. coliin the presence of O2 might be due, at least in part, to a lower exposure to NO⋅, since the O2-mediated oxidation of NO⋅ to form NO2·and N2O3 would be expected to decrease the steady-state NO⋅ levels and increase NO2− and NO3− formation (26Ignarro L.J. Fukuto J.M. Griscavage J.M. Rogers N.E. Byrns R.E. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8103-8107Crossref PubMed Scopus (759) Google Scholar). Alternatively, the O2-dependent protection may be due to an NO⋅ metabolic pathway, which was directly or indirectly dependent upon O2. For example, the protective effect of O2 may be linked to the respiration of E. coli.Thus, the homologous mitochondrial terminal oxidase, cytochromec oxidase, is thought to metabolize NO⋅ (27Borutaité V. Brown G.C. Biochem. J. 1996; 315: 295-299Crossref PubMed Scopus (232) Google Scholar,28Cooper C.E. Torres J. Sharpe M.A. Wilson M.T. FEBS Lett. 1997; 414: 281-284Crossref PubMed Scopus (107) Google Scholar). To evaluate the contribution of O2-mediated NO⋅oxidation to the protection, we measured the concentration of O2 required for aconitase protection in E. coliexposed to NO⋅ and compared it with that required for NO⋅oxidation as detected by the formation of NO2− and NO3− in the culture medium. Surprisingly, aconitase was near maximally protected from the inactivating effect of 240 ppm NO⋅ by the lowest O2concentration tested (∼17 μm O2) (Fig. 4, closed circles). However, at this O2 level, NO2− and NO3− formation was only a fraction of that achievable via NO⋅ autoxidation (Fig. 4, open circles). Thus, the O2-mediated decomposition of NO⋅ does not appear to account for the protective effects of O2. The role of respiration and the terminal respiratory oxidases in the O2-dependent protection of aconitase was assessed by measuring the effects of the inhibitor cyanide. The addition of cyanide (25 μm) to aerobic cultures completely blocked the protection of aconitase by O2 (Fig. 5 A, compare lines 1and 2), while cyanide was without effect in the absence of O2 (compare lines 3 and 4). Importantly, cyanide was effective at decreasing aconitase protection at much lower concentrations than those required for the inhibition of respiration. Thus, while ∼5 μm NaCN was saturating in its effect on the O2-dependent protection of aconitase (Fig. 5 B), half-maximal inhibition of respiration required >50 μm NaCN (Fig. 5 B, inset). The results clearly demonstrate a cyanide-sensitive mechanism of aconitase protection; however, the difference in cyanide sensitivities indicates a mechanism of inhibition independent of cell respiration and the terminal respiratory oxidases. We also investigated the effects of O2 and cyanide on the NO⋅ sensitivity of aconitase in naive aerobic cultures and compared these effects with those in NO⋅-treated cultures to determine whether the adaptive protection displayed a similar O2 dependence and cyanide sensitivity. Indeed, the induced protection of aconitase was O2-dependent; however, this protection appeared less sensitive to cyanide than the constitutive activity (Fig. 6). We were unable to test the effects of higher cyanide concentrations, because aconitase activity was sensitive to cyanide at >25 μm. This may be understandable in light of the ability of cyanide to stimulate O⨪2 production by respiring E. colimembranes (29Imlay J.A. J. Biol. Chem. 1995; 270: 19767-19777Abstract Full Text Full Text PDF PubMed Google Scholar) and the ability of O⨪2 to inactivate aconitase (30Gardner P.R. Biosci. Rep. 1997; 17: 33-42Crossref PubMed Scopus (153) Google Scholar). To determine whether aconitase protection in E. coli was associated with an increased rate of NO⋅metabolism, we measured the rate of NO⋅ consumption by aerobic and anaerobic cells. We also measured the effects of O2 and cyanide on these rates. As shown by the data in Fig. 7 A, aerobic E. coliconsumed NO⋅. Moreover, NO⋅ consumption was sensitive to cyanide and dependent upon O2. Further, NO⋅consumption was induced approximately 13-fold in aerobic cultures exposed to 480 ppm NO⋅ for 60 min (Fig. 7 B). The induced rate of NO⋅ consumption was also sensitive to cyanide and dependent upon O2. Control anaerobic cultures did not express an NO⋅ consumption activity, whereas anaerobic cells exposed to 960 ppm NO⋅ for 60 min produced an NO⋅-consuming activity that did not require O2 and that was insensitive to cyanide (Table I, Experiment A). Interestingly, however, the presence of O2in the assay revealed a cryptic NO⋅ consumption activity that was cyanide-sensitive and was also induced ∼12-fold by the NO⋅exposure. NO3− (10 mm) also induced an NO⋅-consuming activity in anoxic cultures with similar properties as that induced by gaseous NO⋅ (Table I, Experiment B).Table INO consumption by anaerobically grown E. coliTreatmentFinal growthWithout O2With O2Basal+ NaCNBasal+ NaCNA550nmol NO/min/10 cellsExpt. A N2 control0.720.0 ± 0.00.0 ± 0.02.0 ± 0.10.0 ± 0.0 + 960 ppm NO·0.632.2 ± 0.32.4 ± 0.324.4 ± 1.50.0 ± 0.0Expt. B Anoxic control0.560.0 ± 0.00.0 ± 0.01.1 ± 0.10.0 ± 0.0 + 10 mM NaNO30.690.5 ± 0.00.6 ± 0.115.9 ± 0.50.0 ± 0.0Anaerobic cultures of DH5α (Experiment A) were grown overnight in stoppered Erlenmeyer flasks filled to capacity with minimal glucose medium supplemented with amino acids. Cells were washed and resuspended at their original density (A 550 = ∼0.5) in fresh medium and were incubated under an atmosphere of N2 for 30 min prior to a 60-min exposure to 960 ppm NO· in N2 or to N2 alone. For Experiment B, static overnight anoxic cultures of DH5α were grown under similar conditions except that amino acids were omitted and 10 mM NaNO3 was added as indicated. Cells were harvested and washed, and NO· consumption was measured in minimal glucose medium as described under "Materials and Methods." Growth was initiated with 2% inocula from aerobic log phase cultures grown in phosphate-buffered LB medium. Results represent the average ± S.D. of three measurements.Expt., experiment. Open table in a new tab Anaerobic cultures of DH5α (Experiment A) were grown overnight in stoppered Erlenmeyer flasks filled to capacity with minimal glucose medium supplemented with amino acids. Cells were washed and resuspended at their original density (A 550 = ∼0.5) in fresh medium and were incubated under an atmosphere of N2 for 30 min prior to a 60-min exposure to 960 ppm NO· in N2 or to N2 alone. For Experiment B, static overnight anoxic cultures of DH5α were grown under similar conditions except that amino acids were omitted and 10 mM NaNO3 was added as indicated. Cells were harvested and washed, and NO· consumption was measured in minimal glucose medium as described under "Materials and Methods." Growth was initiated with 2% inocula from aerobic log phase cultures grown in phosphate-buffered LB medium. Results represent the average ± S.D. of three measurements. Expt., experiment. Prompted by the aforementioned results, we identified an O2-dependent cyanide-sensitive NO⋅-converting activity in extracts of E. coli that was NOD/flavohemoglobin and has been described elsewhere (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 (500) Google Scholar). Further, we supposed that this NOD activity might account for the NO⋅inducible, cyanide-sensitive, and O2-dependent protection of aconitase and NO⋅ consumption by cells. As shown by the data in Table II, NOD activity in DH5α cells was induced ∼36-fold following an exposure to 960 ppm NO⋅. Extract NOD activity was dependent upon O2, was potently inhibited by cyanide, and displayed cofactor requirements consistent with the flavohemoglobin/NOD activity (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 (500) Google Scholar). NOD activity was also measured in anaerobic cultures exposed to NO⋅ or NO3− as described in the legend to Table I. NOD activity was increased 33-fold (588 milliunits/mgversus 18 milliunits/mg) following exposure to 960 ppm NO⋅ and 40-fold (644 milliunits/mg versus 16 milliunits/mg) during anoxic growth with NO3−. Thus, NO⋅-induced NOD activity levels correlate with the NO⋅-induced aconitase protection and NO⋅ consumption in both aerobic and anaerobic cells.Table IINO consumption by cell-free extracts of E. coliSampleAssaySpecific activityActivitynmol/min/mg%Air controlComplete5.8100NO·-exposedComplete209.5100− FAD2.3<2− NADP+2.4<2− G-6-P1.1<1− G-6-PD0.00− O27.1<4+ NaCN27.3<15NO· consumption activity was measured in extracts prepared from strain DH5α grown aerobically in minimal salts succinate medium to an A550 = ∼0.5. Cells were exposed either to air or to 960 ppm NO· in 21% O2 balanced with N2 for 60 min prior to harvest. The effects of omission of individual components of the reaction mixture, removal of O2, or the addition of 0.25 mm NaCN on the induced activity were measured. Cells were grown, extracts were prepared, and NO· consumption and protein were assayed as described under "Materials and Methods."Glucose 6-phosphate.Glucose-6-phosphate dehydrogenase. Open table in a new tab NO· consumption activity was measured in extracts prepared from strain DH5α grown aerobically in minimal salts succinate medium to an A550 = ∼0.5. Cells were exposed either to air or to 960 ppm NO· in 21% O2 balanced with N2 for 60 min prior to harvest. The effects of omission of individual components of the reaction mixture, removal of O2, or the addition of 0.25 mm NaCN on the induced activity were measured. Cells were grown, extracts were prepared, and NO· consumption and protein were assayed as described under "Materials and Methods." Glucose 6-phosphate. Glucose-6-phosphate dehydrogenase. Our results demonstrate aerobic and anaerobic pathways for NO⋅ detoxification that appear to differ from the NORs described in denitrifiers (11Dermastia M. Turk T. Hollocher T.C. J. Biol. Chem. 1991; 266: 10899-10905Abstract Full Text PDF PubMed Google Scholar, 12Fujiwara T. Fukumori Y. J. Bacteriol. 1996; 178: 1866-1871Crossref PubMed Google Scholar, 13Nakahara K. Tanimoto T. Hatano K. Usuda K. Shoun H. J. Biol. Chem. 1993; 268: 8350-8355Abstract Full Text PDF PubMed Google Scholar, 14Kwiatkowski A.V. Shapleigh J.P. J. Biol. Chem. 1996; 271: 24382-24388Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 15Carr G.J. Page M.D. Ferguson S.J. Eur. J. Biochem. 1989; 179: 683-692Crossref PubMed Scopus (70) Google Scholar). Foremost among these differences was the requirement for O2. Moreover, the similar NO⋅inducibility, O2 dependence, and cyanide sensitivity between the NOD activity in extracts (Table II) and the inducible and constitutive aconitase protection and NO⋅ consumption by intactE. coli strongly supports a role for the recently described NOD (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 (500) Google Scholar) in the observed aerobic NO⋅ removal and detoxification pathway. We can estimate that the NOD activity measured in cell extracts is within range of the NO⋅ consumption activity of intact aerobic E. coli. Thus, assuming roughly 10−13 g of soluble protein per E. coli, we can calculate the NOD activity in extracts from control and NO⋅-exposed E. coli to equal 0.1 and 2.1 nmol of NO⋅/min/108 cells, respectively. These values are comparable with the respective values of 0.2 and 3.2 measured for intact aerobic DH5α grown under similar conditions (Fig. 7). Furthermore, NO⋅ consumption by cells does not appear to involve the terminal respiratory oxidases as proposed for mitochondria (27Borutaité V. Brown G.C. Biochem. J. 1996; 315: 295-299Crossref PubMed Scopus (232) Google Scholar,28Cooper C.E. Torres J. Sharpe M.A. Wilson M.T. FEBS Lett. 1997; 414: 281-284Crossref PubMed Scopus (107) Google Scholar), since cyanide was without effect on the respiration of E. coli at levels that inhibited NO⋅ detoxification (Figs. 5and 7). Moreover, E. coli strains deficient in either of the terminal respiratory oxidases (23Iucchi S. Chepuri V. Fu H.-A. Gennis R.B. Lin E.C.C. J. Bacteriol. 1990; 172: 6020-6025Crossref PubMed Google Scholar) expressed normal levels of the constitutive and inducible aerobic NO⋅ consumption activity (data not shown), whereas NOD/flavohemoglobin-deficient E. coli express essentially no constitutive or inducible aerobic NO⋅ consumption activity (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 (500) Google Scholar). The identity of the inducible anaerobic pathway for NO⋅metabolism and detoxification is less clear, since there is no evidence for a typical NOR in E. coli. The demonstration of NOD induction and a cryptic O2-stimulated and cyanide-sensitive NO⋅ consumption activity in anaerobically grown E. coli exposed to NO⋅ or NO3−suggests an additional role of NOD in anaerobic NO⋅detoxification. However, both the failure of cyanide to inhibit the anaerobic activity in vivo (Table I) and the failure of the induced NOD to metabolize NO⋅ in the absence of O2(Table II) suggests a limited role of NOD/flavohemoglobin in anaerobic NO⋅ metabolism and detoxification. Nevertheless, NOD may have other anaerobic protective functions or may prepare the cell for more efficient NO⋅ detoxification upon exposure to O2. It will now be important to assess the overall contributions of NOD and other potential NO⋅-metabolizing systems, such as the NO⋅-reducing multiheme nitrite reductase (19Costa C. Macedo A. Moura I. Moura J.J.G. Le Gall J. Berlier Y. Liu M.-Y. Payne W.J. FEBS Lett. 1990; 276: 67-70Crossref PubMed Scopus (58) Google Scholar), to NO⋅detoxification in E. coli using deletion strains and overexpressors under various growth conditions. Finally, it is hoped that greater knowledge of the function and regulation of NO⋅ detoxification systems, including the E. coli NOD, may reveal the true antibiotic potential of NO⋅toward a variety of organisms. For example, the greater ability of NO⋅ to inhibit the growth of stationary phase E. coli(Fig. 1) suggests that latent or dormant pathogens will be more susceptible to the antibiotic action of NO⋅ than healthy growing microorganisms simply because they are unable to induce their NO⋅ detoxification systems. Indeed, this may account for the activation of latent Mycobacterium tuberculosis and Leishmania spp. infections in nitric-oxide synthase-deficient mice (1MacMicking J.D. North R.J. LaCourse R. Mudgett J.S. Shah S.K. Nathan C.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5243-5248Crossref PubMed Scopus (913) Google Scholar, 2Stenger S.N. Donhauser H. Thüring M. Röllinghoff M. Bogdan C. J. Exp. Med. 1996; 183: 1501-1514Crossref PubMed Scopus (284) Google Scholar). The ability of O2 to increase the NO⋅ detoxification capacity of cells through the action of an NOD activity, like that of the (flavo)hemoglobin (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 (500) Google Scholar), may also explain the sensitivity of intraerythrocytic malaria parasites to NO⋅ at low O2 tensions (31Taylor-Robinson A.W. Looker M. Lancet. 1998; 351: 1630Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Furthermore, greater knowledge of the NODs, NORs, and other NO⋅ detoxification systems may facilitate the design of new drugs that target these systems and allow the expression of the full potential of NO⋅ as a natural broad spectrum antibiotic (3Fang F.C. J. Clin. Invest. 1997; 99: 2818-2825Crossref PubMed Google Scholar). We are grateful to Dr. Edmund Lin for kindly supplying bacterial strains. We thank Dr. Irwin Fridovich for comments on the manuscript.