Nitrate Reduction to Nitrite,Nitric Oxide and Ammonia by Gut Bacteria under Physiological Conditions

The biological nitrogen cycle involves step-wise reduction of nitrogen oxides to ammonium salts and oxidation of ammonia back to nitrites and nitrates by plants and bacteria. Neither process has been thought to have relevance to mammalian physiology; however in recent years the salivary bacterial reduction of nitrate to nitrite has been recognized as an important metabolic conversion in humans. Several enteric bacteria have also shown the ability of catalytic reduction of nitrate to ammonia via nitrite during dissimilatory respiration; however, the importance of this pathway in bacterial species colonizing the human intestine has been little studied. We measured nitrite, nitric oxide (NO) and ammonia formation in cultures of Escherichia coli, Lactobacillus and Bifidobacterium species grown at different sodium nitrate concentrations and oxygen levels. We found that the presence of 5 mM nitrate provided a growth benefit and induced both nitrite and ammonia generation in E.coli and L.plantarum bacteria grown at oxygen concentrations compatible with the content in the gastrointestinal tract. Nitrite and ammonia accumulated in the growth medium when at least 2.5 mM nitrate was present. Time-course curves suggest that nitrate is first converted to nitrite and subsequently to ammonia. Strains of L.rhamnosus, L.acidophilus and B.longum infantis grown with nitrate produced minor changes in nitrite or ammonia levels in the cultures. However, when supplied with exogenous nitrite, NO gas was readily produced independently of added nitrate. Bacterial production of lactic acid causes medium acidification that in turn generates NO by non-enzymatic nitrite reduction. In contrast, nitrite was converted to NO by E.coli cultures even at neutral pH. We suggest that the bacterial nitrate reduction to ammonia, as well as the related NO formation in the gut, could be an important aspect of the overall mammalian nitrate/nitrite/NO metabolism and is yet another way in which the microbiome links diet and health.     

Effect of sodium in the nutrient medium on the incidence of potassium-deficiency symptoms in tomato plants

Summary Tomato plants (Lycopersicon esculentum cv. Amberley Cross) were grown in sand culture and were fed with four concentrations of potassium nitrate in combination with two levels of sodium nitrate. After six weeks the plants were scored for the presence and absence of a symptom of potassium deficiency, namely, marginal chlorosis and/or necrosis in the young, fully-expanded leaves. These leaves were also analysed for K and Na. Marginal chlorosis and/or necrosis occurred in plants given a nutrient solution containing 0.5 meq K/I or less and supplied with either of the sodium nitrate levels. However, the symptoms occurred more frequently in plants receiving the lower level of sodium nitrate. The laminae on plants receiving the lower concentration of sodium nitrate had a 50 per cent incidence of chlorisis and/or necrosis when the tissue potassium content was 0.74 per cent of the dry wt, while those laminae on plants receiving the higher level did not show a 50 per cent incidence until their potassium fell to 0.64 per cent of the dry wt.     

Dissimilatory nitrate reduction by a strain ofClostridium butyricum isolated from estuarine sediments

Nitrate dissimilation in chemostat grown cultures ofClostridium butyricum SS6 has been investigated. Sucrose limited cultures grown on nitrate produced nitrite as the principal end-product of nitrate reduction whilst under nitrate-limiting conditions ammonia accumulated in the spent media. Nitrate reduction was accompanied by the synthesis of a soluble nitrate reductase (123 nmol·NADH oxidised · min^-1 · mg protein^-1) and in addition, under N-limiting conditions, a soluble nitrite reductase (56 nmol NADH oxidised min^-1 · mg protein^-1). Corresponding ammonia grown cultures synthesised neither enzyme. Concurrent with the dissimilation of nitrate to nitrite and ammonia cell population densities increased by 18% (C-limitation) and 32% (N-limitation). Spent media analyses of the fermentation products from ammonia and nitrate grown cells showed the accumulation of acetate in nitrate dissimilating cultures. Molar ratios of acetate/butyrate increased by a factor of 5 (C-limitation) to 12 (N-limitation) upon adding nitrate to the growth medium. In C-limited cultures, grown on nitrate, hydrogenase activity was 340 nmol · min^-1 · mg protein^-1 and under N-limitation this increased to 906 nmol · min^-1 · mg protein^-1. Since N-limited cultures are electron acceptor limited, the increase in hydrogenase activity enables excess electrons to be spilled by this route.     

Variable Ammonia Production Among Smooth and Rough Strains of Pseudomonas pseudomallei: Resemblance to Bacteriocin Production

The colonial morphology of some strains of Pseudomonas pseudomallei was correlated with certain biochemical and physiological traits. After 3 days of growth on Wahba or heart infusion agars, smooth-colony strains generated toxic amounts of ammonia. Under the same conditions, the rough strains simultaneously produced oxalic acid which decreased the inhibitory concentration of ammonia. The ammonia-ammonium concentrations in smooth cultures exhibited certain bacteriocin-like characteristics. An unusually stable, smooth strain (strain 165) was chosen to compare and emphasize any differences with typical, rough strain 7815. Three-day-old smooth cultures grown on Wahba agar containing 3% (w/v) glycerol demonstrated ammonia toxicity. The substitution of glucose for glycerol completely obviated this toxicity. In highly aerated Wahba broth containing glucose, the amount of ammonia found in strain 165 smooth cultures and the amount of oxalic acid found in strain 7815 rough cultures were greatly reduced. In Difco nitrate broth smooth strain 165 did not form gas, and it reduced nitrate to nitrite only. Strain 7815 produced a gas and reduced both nitrate and nitrite.     

NarK enhances nitrate uptake and nitrite excretion in Escherichia coli.

narK mutants of Escherichia coli produce wild-type levels of nitrate reductase but, unlike the wild-type strain, do not accumulate nitrite when grown anaerobically on a glucose-nitrate medium. Comparison of the rates of nitrate and nitrite metabolism in cultures growing anaerobically on glucose-nitrate medium revealed that a narK mutant reduced nitrate at a rate only slightly slower than that in the NarK+ parental strain. Although the specific activities of nitrate reductase and nitrite reductase were similar in the two strains, the parental strain accumulated nitrite in the medium in almost stoichiometric amounts before it was further reduced, while the narK mutant did not accumulate nitrite in the medium but apparently reduced it as rapidly as it was formed. Under conditions in which nitrite reductase was not produced, the narK mutant excreted the nitrite formed from nitrate into the medium; however, the rate of reduction of nitrate to nitrite was significantly slower than that of the parental strain or that which occurred when nitrite reductase was present. These results demonstrate that E. coli is capable of taking up nitrate and excreting nitrite in the absence of a functional NarK protein; however, in growing cells, a functional NarK promotes a more rapid rate of anaerobic nitrate reduction and the continuous excretion of the nitrite formed. Based on the kinetics of nitrate reduction and of nitrite reduction and excretion in growing cultures and in washed cell suspensions, it is proposed that the narK gene encodes a nitrate/nitrite antiporter which facilitates anaerobic nitrate respiration by coupling the excretion of nitrite to nitrate uptake. The failure of nitrate to suppress the reduction of trimethylamine N-oxide in narK mutants was not due to a change in the level of trimethylamine N-oxide reductase but apparently resulted from a relative decrease in the rate of anaerobic nitrate reduction caused by the loss of the antiporter system.     

Les nitrate-réductases bactériennes

Résumé M. halodenitrificans possède la nitrate-réductase A. Cet enzyme se trouve sous la forme particulaire dans les extraits bruts. Il ne présente pas un caractère halophile: NaCl, KCl ou CsCl 1 ou 0,5 M ne l'activent pas. NaCl 1 M active cependant la réduction du nitrate en nitrite par les cellules en présence de lactate comme donneur d'électrons. Cet effet n'est pas dû à une action du sel sur la nitrate-réductase. Les cultures anaérobies avec nitrate synthétisent approximativement 7 fois plus d'enzyme que les cultures aérobies sans nitrate.

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Bacterial nitrate reductasesVIII. Preliminary Study of the enzyme of Micrococcus halodenitrificans

Summary M. halodenitrificans has nitrate reductase A. This enzyme appears to be in particulate form in crude extracts. It does not present a halophilic character: 1 or 0.5 M NaCl, KCl, or CsCl does not activate it. However, 1 M NaCl activates the reduction of nitrate to nitrite by cells in the presence of lactate as electron donor. This effect is not due to an action of the salt on nitrate reductase. Anaerobic cultures containing nitrate form approximately 7 times more enzyme than aerobic cultures not containing nitrate.

     

Influence of Carbon Source on Nitrate Removal by Nitrate-Tolerant Klebsiella oxytoca CECT 4460 in Batch and Chemostat Cultures

The nitrate-tolerant organism Klebsiella oxytoca CECT 4460 tolerates nitrate at concentrations up to 1 M and is used to treat wastewater with high nitrate loads in industrial wastewater treatment plants. We studied the influence of the C source (glycerol or sucrose or both) on the growth rate and the efficiency of nitrate removal under laboratory conditions. With sucrose as the sole C source the maximum specific growth rate was 0.3 h⁻¹, whereas with glycerol it was 0.45 h⁻¹. In batch cultures K. oxytoca cells grown on sucrose or glycerol were able to immediately use sucrose as a sole C source, suggesting that sucrose uptake and metabolism were constitutive. In contrast, glycerol uptake occurred preferentially in glycerol-grown cells. Independent of the preculture conditions, when sucrose and glycerol were added simultaneously to batch cultures, the sucrose was used first, and once the supply of sucrose was exhausted, the glycerol was consumed. Utilization of nitrate as an N source occurred without nitrite or ammonium accumulation when glycerol was used, but nitrite accumulated when sucrose was used. In chemostat cultures K. oxytoca CECT 4460 efficiently removed nitrate without accumulation of nitrate or ammonium when sucrose, glycerol, or mixtures of these two C sources were used. The growth yields and the efficiencies of C and N utilization were determined at different growth rates in chemostat cultures. Regardless of the C source, yield carbon (Y_C) ranged between 1.3 and 1.0 g (dry weight) per g of sucrose C or glycerol C consumed. Regardless of the specific growth rate and the C source, yield nitrogen (Y_N) ranged from 17.2 to 12.5 g (dry weight) per g of nitrate N consumed. In contrast to batch cultures, in continuous cultures glycerol and sucrose were utilized simultaneously, although the specific rate of sucrose consumption was higher than the specific rate of glycerol consumption. In continuous cultures double-nutrient-limited growth appeared with respect to the C/N ratio of the feed medium and the dilution rate, so that for a C/N ratio between 10 and 30 and a growth rate of 0.1 h⁻¹ the process led to simultaneous and efficient removal of the C and N sources used. At a growth rate of 0.2 h⁻¹ the zone of double limitation was between 8 and 11. This suggests that the regimen of double limitation is influenced by the C/N ratio and the growth rate. The results of these experiments were validated by pulse assays.     

Nitrate Reductase and Soluble Cytochrome c in Spirillum itersonii

The addition of nitrate to cultures of Spirillum itersonii incubated under low aeration produced a diauxic growth pattern in which the second exponential phase was preceded by the appearance of nitrite in the medium. The organism also grew anaerobically in the presence of nitrate. Nitrate reductase activity could be demonstrated in cell-free extracts by use of reduced methyl viologen as the electron donor. The enzyme was located in the supernatant fraction after centrifugation of extracts for 2 hr at 40,000 x g, and it sedimented as a single peak when centrifuged in a sucrose gradient. Nitrate reductase activity was found in cells grown with low aeration without nitrate, but was increased about twofold by addition of nitrate. Enzyme activity was negligible in cells grown with high aeration. The proportion of soluble cytochrome c was increased two- to threefold in cells grown with nitrate. The specific activities of nitrate reductase and soluble cytochrome c rose when nitrate or nitrite was added to cell suspensions incubated with low aeration; nitrite was more effective than nitrate during the early stages of incubation. A nitrate reductase-negative mutant synthesized increased amounts of soluble cytochrome c in response to nitrate or to nitrite in the cell suspension system. It is concluded that enhanced synthesis of soluble cytochrome c does not require the presence of a functional nitrate reductase.     

Effect of Medium Composition on the Denitrification of Nitrate by Paracoccus denitrificans

The course of denitrification of nitrate in static cultures of Paracoccus denitrificans was studied. Reduction of nitrate to gaseous nitrogen without accumulation of nitrite because of parallel and balanced activities of nitrate and nitrite reductases was observed in nutrient broth. In minimal liquid cultures supplemented with either methanol, acetate, or ethanol as a sole carbon source, substantial amounts of nitrite (up to 70%) accumulated. The reduction in nitrite concentration began just after the transformation of nitrate to nitrite was completed. The addition of some growth factors to minimal media shortened the bacterial biomass doubling time. A correlation coefficient of 0.71 between the doubling time and the amount of accumulated nitrite in cultures was found. My results indicated that the type of denitrification carried out by P. denitrificans is not stable and depends on the nutritional composition of the culture medium.     

Effect of CO2 and phosphate deprivation on the control of nitrate, nitrite and ammonium metabolism in Chlorella

Chlorella vulgaris Beijerinck, strain 211/12, uses nitrate, nitrite and ammonium at pH 8.2 but not at pH 6.4 when kept under conditions of CO₂-deprivation, as observed in cell suspensions aerated with CO₂-free air during a 20–30. h period Most of the nitrate absorbed at pH 8.2, however, was not assimilated but was released into the external medium as nitrite and ammonium. Cells of Chlorella previously grown in phosphate-limited continuous cultures were unable to absorb nitrate, nitrite or ammonium under conditions of phosphate starvation at either pH 6.4 or 8.2 in cell suspensions flushed with air containing 5% CO₂, However, in cell suspensions flushed with CO₂-free air, the capacity of the alga to absorb and reduce nitrate and to excrete nitrite and ammonium at pH 8.2 was restored.
It is hypothesized that in Chlorella the metabolism of nitrate, nitrite and ammonium is influenced by the availability of other nutrients and controlled by the cell's carbon status at the level of ion entry into the cell. With respect to nitrate this carbon-dependent control is distinct and works independently of that triggered by the cell's nitrogen status.     

Kinetic Explanation for Accumulation of Nitrite, Nitric Oxide, and Nitrous Oxide During Bacterial Denitrification

The kinetics of denitrification and the causes of nitrite and nitrous oxide accumulation were examined in resting cell suspensions of three denitrifiers. An Alcaligenes species and a Pseudomonas fluorescens isolate characteristically accumulated nitrite when reducing nitrate; a Flavobacterium isolate did not. Nitrate did not inhibit nitrite reduction in cultures grown with tungstate to prevent formation of an active nitrate reductase; rather, accumulation of nitrite seemed to depend on the relative rates of nitrate and nitrite reduction. Each isolate rapidly reduced nitrous oxide even when nitrate or nitrite had been included in the incubation mixture. Nitrate also did not inhibit nitrous oxide reduction in Alcaligenes odorans, an organism incapable of nitrate reduction. Thus, added nitrate or nitrite does not always cause nitrous oxide accumulation, as has often been reported for denitrifying soils. All strains produced small amounts of nitric oxide during denitrification in a pattern suggesting that nitric oxide was also under kinetic control similar to that of nitrite and nitrous oxide. Apparent K_m values for nitrate and nitrite reduction were 15 μM or less for each isolate. The K_m value for nitrous oxide reduction by Flavobacterium sp. was 0.5 μM. Numerical solutions to a mathematical model of denitrification based on Michaelis-Menten kinetics showed that differences in reduction rates of the nitrogenous compounds were sufficient to account for the observed patterns of nitrite, nitric oxide, and nitrous oxide accumulation. Addition of oxygen inhibited gas production from ¹³NO₃⁻ by Alcaligenes sp. and P. fluorescens, but it did not reduce gas production by Flavobacterium sp. However, all three isolates produced higher ratios of nitrous oxide to dinitrogen as the oxygen tension increased. Inclusion of oxygen in the model as a nonspecific inhibitor of each step in denitrification resulted in decreased gas production but increased ratios of nitrous oxide to dinitrogen, as observed experimentally. The simplicity of this kinetic model of denitrification and its ability to unify disparate observations should make the model a useful guide in research on the physiology of denitrifier response to environmental effectors.     

The Effect of Calcium Nutrition of Ethylene-induced Abscission

The influence of calcium nutrition on ethylene-induced abscission was studied by growing cotton (Gossypium hirsutum L. cv. Stoneville 213) and bean (Phaseolus vulgaris L. cv. Resistant Black Valentine) plants for several weeks in nutrient solutions containing 2, 10 (normal level), 15, or 20 meq/l of calcium, and then treating the plants with ethylene. Increasing the calcium level of cotton from 2 to 20 meq/l resulted in a 9-fold increase in the calcium content of the abscission zone and a maximum reduction of 25% in the amount of leaf abscission induced by ethylene (9 μl/l). Bean plants grown on 10, 15, or 20 meq/l calcium solutions showed corresponding increases in the calcium content of the abscission zone but showed no significant differences in the rate of ethyleneinduced abscission. Only at the lowest calcium level of 2 meq/l, where deficiency symptoms became apparent, was a significant effect observed. These results suggest that under normal cultural practices calcium nutrition has little influence on the rate of ethylene-induced abscission.     

Osmotic stress induced-capsaicin production in suspension cultures of Capsicum chinense Jacq.cv. Naga King Chili

The influence of osmotic stress on capsaicin production was investigated in cell suspension cultures of Capsicum chinense Jacq.cv. Naga King Chili, a chili species native to Northeastern India. The sterilized seeds were germinated in Murashige and Skoog medium. Two-week-old hypocotyls were excised from in vitro germinated seedlings and implanted in MS medium containing 2, 4-dichlorophenoxyacetic acid (2?mg/l), and Kinetin (0.5?mg/l) for callus induction. Capsaicin production in the suspension cultures was significantly affected using sucrose, mannitol, and NaCl in the medium. Stoichiometric analysis with different combinations of sucrose and non-sugar osmotic agent (NaCl) showed that osmotic stress was an important factor for enhancing capsaicin production in cell suspension cultures of C. chinense. The capsaicin content of 1,644.1???g?g^?1 f.wt was recorded on day 15 in cultures grown in MS medium containing 87.64?mM sucrose in combination with 40?mM NaCl. However, osmotic stress treatment at 160?mM NaCl with sucrose resulted in lowering capsaicin accumulation and separation of cell wall from their cytoplasm, under microscopic observation.     

Endonuclease V of Escherichia coli prevents mutations from nitrosative deamination during nitrate/nitrite respiration

Endonuclease V (Endo V) of Escherichia coli participates in the excision repair of hypoxanthine and xanthine (deaminated adenine and guanine) in DNA. It thereby reduces the mutagenic effects of nitrous acid by attacking lesions caused by nitrosative deamination. Nitrosating agents may be produced endogenously when E. coli is grown in oxygen-poor cultures, during which nitrate and nitrite replace oxygen as preferred electron acceptors. In this study, the protective effect of Endo V was observed under such conditions. During micro-aerobic growth, an nfi (Endo V) mutation enhanced the frequency of nitrate- and nitrite-induced A:T-->G:C and G:C-->A:T transition mutations, which are consistent with a defect in the removal of DNA hypoxanthine and xanthine, respectively. Similar effects were observed in saturated, aerobic cultures but not in well-aerated, logarithmically growing ones. A narG (nitrate reductase) mutation blocked the mutagenesis of the nfi mutant by nitrate but not by nitrite. These results differed from those of previous studies in which cell suspensions generated an exogenous nitrosating agent from nitrite, but not from nitrate, in a reaction that was narG-dependent. Nitrate/nitrite metabolism is also known to generate endogenous alkylating agents through N-nitrosation. However, an nfi mutation did not appreciably enhance mutagenesis by N-methyl-N-nitrosourea, suggesting that the mutator effect of nfi is not due to a defect in alkylation repair. The overall results indicate that Endo V functions during normal growth by helping to repair nitrosatively deaminated bases in DNA, which are by-products of anaerobic nitrate/nitrite respiration.     

Nitrite accumulation by Synechococcus 6301 as a consequence of carbon- or sulfur-deficiency

Abstract Air grown cultures of the cyanobacterium Synechococcus 6301, when incubated under continuous illumination with nitrate as the sole nitrogen source, started to liberate nitrite from the second day of inoculation. Nitrite accumulation depended on culture density and was caused by CO₂ deficiency since it could be prevented by addition of 5% CO₂ to the gas stream. Nitrite excreted during growth with air (0.035% CO₂) was taken up after an increase in CO₂ concentration to 5%.
In sulfur depleted cultures, nitrite excretion took place also with saturating CO₂ concentration. In this case nitrite accumulation could be reversed by addition of a suitable sulfur source.
Under both conditions for nitrite accumulation, carbon and sulfur deficiency, a significant decrease in nitrite reductase activity was observed which might account for nitrite liberation.     

Characterization of a Corynebacterium Strain That Can Grow in Medium Containing up to 2 M Nitrate

A gram-positive bacterium, identified as Corynebacterium K37, was isolated from the waste effluent of a dairy farm. The bacterium thrived and expressed nitrate-reducing activity at nitrate concentrations of up to 2 M, and reduced nitrate concentration from 0.4 M to 11.4 mM and also from 0.4 M to 23.4 mM in aerobic and anaerobic fed-batch cultures, respectively. Cells of K37 were able to utilize a variety of carbon sources for nitrate reduction with little or no accumulation of nitrite. In aerobic cultures, the residual nitrite was minimal and it was completely reduced after prolonged incubation. Growth on acetate or pyruvate in anaerobic cultures resulted in lower nitrite reductase activities and concomitant higher residual nitrite concentrations than did growth on ethanol or glucose, suggesting that diminished electron availability was a factor in the accumulation of residual nitrite. The bacterium also survived in 2 M concentrations of NaCl, KCl, and CaCl₂. Corynebacterium sp. K37 may be useful in bioremediation of high nitrate pollution in contaminated soils and water.     

Nitrate and Nitrite Reduction by Microorganisms Embedded in a Filter Paper Incubated Aerobically

Pseudomonas aeruginosa, grown to steric saturation between the cellulose fibers of a filter paper, reduced nitrate or nitrite or both when the cell-filled paper was washed, transferred to phosphate buffer, nitrate, or nitrite or both, and glucose agar plates, and incubated under aerobiosis as resting cells. The biological nature of the reduction was ascertained by the use of nitrate and nitrite reductaseless mutants. The mesh of cellulose fibers was necessary to create a sufficient barrier to oxygen diffusion, since denitrification was not obtained within large and thick colonies of P. aeruginosa. When a soil suspension was used to inoculate the filter paper, ammonium and nitrite accumulated. Concomitant to nitrate reduction, the total nonvolatile inorganic nitrogen decreased and then increased as if part of it was immobilized to be subsequently mineralized.     

Nitrogen transformation under different dissolved oxygen levels by the anoxygenic phototrophic bacterium <Emphasis Type="Italic">Marichromatium gracile</Emphasis>

Marichromatium gracile: YL28 (M. gracile YL28) is an anoxygenic phototrophic bacterial strain that utilizes ammonia, nitrate, or nitrite as its sole nitrogen source during growth. In this study, we investigated the removal and transformation of ammonium, nitrate, and nitrite by M. gracile YL28 grown in a combinatorial culture system of sodium acetate-ammonium, sodium acetate-nitrate and sodium acetate-nitrite in response to different initial dissolved oxygen (DO) levels. In the sodium acetate-ammonium system under aerobic conditions (initial DO?=?7.20–7.25 mg/L), we detected a continuous accumulation of nitrate and nitrite. However, under semi-anaerobic conditions (initial DO?=?4.08–4.26 mg/L), we observed a temporary accumulation of nitrate and nitrite. Interestingly, under anaerobic conditions (initial DO?=?0.36–0.67 mg/L), there was little accumulation of nitrate and nitrite, but an increase in nitrous oxide production. In the sodium acetate-nitrite system, nitrite levels declined slightly under aerobic conditions, and nitrite was completely removed under semi-anaerobic and anaerobic conditions. In addition, M. gracile YL28 was able to grow using nitrite as the sole nitrogen source in situations when nitrogen gas produced by denitrification was eliminated. Taken together, the data indicate that M. gracile YL28 performs simultaneous heterotrophic nitrification and denitrification at low-DO levels and uses nitrite as the sole nitrogen source for growth. Our study is the first to demonstrate that anoxygenic phototrophic bacteria perform heterotrophic ammonia-oxidization and denitrification under anaerobic conditions.     

Nitrite reduction in Rhizobium “hedysari” strain HCNT 1

Rhizobium hedysari strain HCNT 1 rapidly reduced nitrite to N₂O, only slowly reduced nitrate to nitrite and did not exhibit nitrous oxide reductase activity. Nitrite reduction in this rhizobium strain may be a detoxification mechanism for conversion of nitrite, which inhibits O₂ uptake, to non-toxic N₂O. Concentrations of nitrite as small as 3 M diminished O₂ uptake in whole cells. The bacterium did not couple energy conservation with nitrate or nitrite reduction. Cells neither grew anaerobically at the expense of these nitrogen oxides nor translocated protons during reduction of nitrite. Induction of nitrite reductase activity was not a response to the presence of nitrate or nitrite, but occurred instead when the O₂ concentration in culture atmospheres fell to <16.5% of air saturation. Sensitivity of cytochrome o, which is synthesized only in cells grown under O₂-limited conditions, may account for the toxicity of nitrite in strain HCNT 1.     

A new method to measure nitrate/nitrite with a NO-sensitive electrode

There are different methods to measure the unstable moleculenitric oxide (NO). We will describe a new sensitive method to measure NO by reconversion of nitrate/nitrite to NO, which will bedetermined with an amperometric Clark-type electrode. Nitrate andnitrite are the degradation products of NO. First, nitrate isenzymatically converted to nitrite with the use of the nitrate reductase. Second, nitrite is reduced to equimolar NO concentrations byan acidic iodide solution. The detection limit of the electrode in anaqueous solution was 2 nmol/l NO (meaning the threshold was dependingon the volume added: 500 µl of a 0.2 µmol/l nitrite solution addedto a 10-ml bath). This method provides the ability to assess basal andagonist-stimulated NO releases of different biological models. Wemeasured basal and carbachol-stimulated NO release of nativeendothelial cells from porcine coronary arteries and porcine aorticendothelial cell cultures. Moreover, it was possible to measure thenitrate/nitrite concentration in the coronary effluent of a guinea pigheart. In conclusion, we present a valid, highly sensitive new methodof measuring nitrite/NO in biological systems with a commerciallyavailable electrode.