Endogenous nitrate loss as an assay for nitrate reduction in vivo

An in vivo assay method for nitrate reduction is proposed, based on the use of endogenous nitrate rather than on the accumulation of nitrite. Loss of endogenous nitrate and accumulation of nitrite were studied in barley (Hordeum vulgare L. cv. Gars Clipper ex Napier) leaves. Leaf sections were incubated in the dark in a gaseous environment of air or N₂. Nitrate disappeared under both conditions, the highest loss being observed in tissue under anaerobiosis. Nitrite accumulated only in leaf sections under anaerobiosis, but the amount of nitrite accumulated was much lower than the amount of nitrate lost. A comparative study of the capacity of barley leaf sections to use endogenous nitrate and accumulate nitrite showed that both activities were dependent on temperature in a manner characteristic of enzymatic reactions. Disappearance of endogenous nitrate increased with increasing levels of nitrate in the tissue.     

Effects of inorganic nitrogen compounds on the activity and synthesis of nitrogenase in Gloeothece (Nägeli) sp. ATCC 27152

Addition of 2 mM nitrite or ammonium to aerobically incubated cultures of Gloeothece rapidly inhibited N₂ fixation (measured as acetylene reduction). In contrast, 2 mM nitrate inhibited N₂ fixation less rapidly and less extensively, and often temporarily stimulated nitrogenase activity. The inhibitory effects of both nitrate and ammonium could be prevented by addition of 3 mM L-methionine-DL-sulphoximine, suggesting that the true inhibitor of N₂ fixation was an assimilatory product of ammonium rather than either ammonium or nitrate itself. The inhibition of N₂ fixation by nitrite could not, however, be prevented by addition of L-methionine-DL- sulphoximine. On the other hand, nitrite (unlike nitrate and ammonium) did not inhibit N₂ fixation in cultures incubated under a gas phase lacking oxygen. These findings suggest that the mechanism whereby nitrite inhibits N₂ fixation in Gloeothece differs from that of either nitrate or ammonium. The inhibitory effect of nitrite on N₂ fixation did not involve reduction of nitrite to nitric oxide, though nitric oxide was a potent inhibitor of nitrogenase activity in Gloeothece . Nitrate and nitrite inhibited the synthesis of nitrogenase in Gloeothece , while ammonium not only inhibited nitrogenase synthesis but also stimulated degradation of the enzyme. In addition, all three compounds favoured the appearance of the Fe-protein of nitrogenase in its larger, presumed inactive, form.     

Measurement of denitrification in estuarine sediment using membrane inlet mass spectrometry

Abstract Denitrification was measured in intact sediment cores and in homogenised slurries using membrane inlet mass spectrometry. Dissolved concentrations of O₂, N₂, N₂O and CO₂ were simultaneously monitored. Using a 0.8 mm diameter needle probe, a comparison was made of the gas profiles of intact cores obtained under different conditions, i.e. with air or argon as the headspace gas and after the addition of nitrate and/or a carbon source to the sediment surface. O₂ was detectable to a depth of 1 cm under a headspace of air and the depth at which the maxima of denitrification products occurred was 1.5–2 cm. Denitrification products (N₂O, N₂) occurred in the surface layers where O₂ was above the minimum level of detectability (> 0.25 μM): diffusion of N₂ and N₂O upwards from the anoxic zone, local anaerobic microenvironments or aerobic denitrification are alternative explanations for this observation. The addition of nitrate and/or acetate increased the concentrations of N₂, N₂O and CO₂ in the sediment core. In sediment slurries, the pH, nitrate concentration, carbon source and the depth from which the sample was taken affected the rate of denitrification. Nitrogen was the sole detectable end product. Maximum denitrification occurred at pH 7.5 and at 20 mM nitrate. Denitrification was at a maximum in those slurries prepared from sections of core at 1–2 cm depth.     

Role of nitrate and nitrite for production and consumption of nitric oxide during denitrification in soil

Abstract Anaerobic production and consumption of NO was measured in a calcic cambisol (KBE; pH 7.3) and a forest luvisol (PBE; pH 4.4) which were incubated at 80% water-holding capacity and continuously flushed with N₂. Both NO production and NO consumption were negligibly low when nitrate and nitrite concentrations in the soil were exhausted. Addition of glucose alone had no effect, but addition of nitrate ± glucose greatly stimulated both NO production and NO consumption. NO consumption followed an apparent first-order reaction at low NO mixing ratios (1–3 ppmv), but a higher NO mixing ratios it followed Michaelis-Menten kinetics. In PBE the apparent K _m was 980 ppbv NO (1.92 nM in soil water). During reduction of nitrate, nitrite intermediately accumulated and simultaneously, production rates of NO and N₂O were at the maximum. Production rates of NO plus N₂O amounted to 20% and 34% of the nitrate reduction rate in KBE and PBE, respectively. NO production was hyperbolically related to the nitrite concentration, indicating an apparent K_m of 1.6 μg nitrite-N g⁻¹ d.w. soil (equivalent to 172 μM nitrite in soil solution) for the reduction of nitrite to NO in KBE. Under nitrate and nitrite-limiting conditions, 62–76% and 93–97% of the consumed NO-N were recovered as N₂O-N in KBE and PBE, respectively. Gassing of nitrate plus nitrite-depretsu KBE with increasing mixing ratios of NO₂ resulted in increasing rates of NO₂ uptake and presumably in the formation of low concentrations of nitrite and nitrate. This NO₂ uptake resulted in increasing rates of both NO production and NO consumption indicating that nitrite or nitrate was limiting for both reactions.     

A SALT-TOLERANT DENITRIFYING BACILLUS STRAIN WHICH 'BLOWS' CANNED BACON

SUMMARY: A Bacillus strain, isolated from a 'blown' can of bacon, is described: it tolerates 15% NaCl and reduces nitrate and nitrite to N₂O and N₂. The pH-salt relationship, the tolerance to nitrite under aerobic and anaerobic conditions, the heat resistance of the spores, and the composition of the gas produced, have been investigated. The characters of the organism are such that, should its spores survive the processing of a cured meat, their subsequent development and the spoilage of the product seem almost certain.     

Regulation of nitrogen fixation by nitrite and glutamine in Derxia gummosa

Abstract Nitrogenase activity of cells of Derxia gummosa (30 h growth in cultures without combined nitrogen) was not inhibited on adding nitrate. However, on adding either azaserine or methionine sulfoximine (MSX) with nitrate to these cells, nitrogenase (C₂H₂ reduction) was inhibited because nitrite accumulated in the reaction mixtures. Nitrite inhibition of the in vivo C₂H₂ reduction had a K _i value of 16 μM. Both ammonia and glutamine inhibited N₂ fixation (C₂H₂ reduction) in intact cells and in those treated with toluene. This inhibition by ammonia was relieved by methionine sulfoximine but not by glutamine. Azaserine enhanced the inhibition of nitrogenase produced by either ammonia or glutamine, since these treatments resulted in an accumulation of glutamine.     

The metabolism of sugar and malic acid by Leuconostoc oenos: effect of malic acid, pH and aeration conditions

The co-metabolism of sugars by Leuconostoc oenos was studied under different environmental conditions. Under aerobic conditions, growth and sugar metabolism were poorer than under CO₂ or N₂ atmosphere and acetic acid accumulated to a larger extent. Glycerol was found in the aerobic cultures while erythritol was detected under N₂ or CO₂. When medium conditions make growth difficult (low pH, aerobic conditions, low nutrients), sugars were only slightly metabolized and growth was very slow while malic acid was rapidly and completely degraded, leading to an increase in the y _ATP. Aeration effects on the malic acid degradation rate depended on the nutrients and carbon source in the medium. Malic acid clearly stimulated bacterial growth, allowing an increase in the molar growth yields and ATP production. The results suggest that under adverse conditions cells are not able to grow and malic degradation supplies additional energy production.     

Co-denitrification by the denitrifying system of the fungus Fusarium oxysporum

Abstract Nitrogen compounds such as azide, salicylhydroxamic acid, and possibly ammonium ions were converted to nitrous oxide (N₂O) or dinitrogen (N₂) by Fusarium oxysporum under denitrifying conditions. Nitrogen atoms in these compounds were combined with another nitrogen atom from nitrite to form a hybrid N₂O species. The fungus exhibited much higher converting activities as compared with similar reactions catalyzed by bacterial denitrifiers. We thus propose the phenomenon be called co-denitrification, which means that such nitrogen compounds are denitrified by the system induced by nitrite (or nitrate) but are incapable by themselves of inducing the denitrifying system.     

Effect of nitrate on nitrogen fixation and nodule carbohydrate and organic acid concentrations in pea mutants deficient in nitrate reductase

Nitrate inhibits symbiotic N₂ fixation and a number of hypotheses concerned with NO₃⁻ assimilation have been suggested to explain this inhibition. These hypotheses were tested using a pea ( Pisum sativum L. cv. Juneau) with normal nitrate reductase NR; (EC 1,6,6,4) activity and two mutants of cv. Juneau, A317 and A334, with impaired NR activity. The plants were inoculated with three strains of Rhizobium leguminosarum and grown for 3 weeks in N-free medium, followed by 1 week in medium supplemented with 0, 5 or 10 m M KNO₃ before harvesting. NO₃ was taken up at comparable rates by the parent and the mutants and accumulated in leaf and stem tissue of the latter. Acetylene reduction rates were inhibited similarly in both the parent and mutants in the presence of KNO₃ but there were differences among rhizobial strains. Starch concentration of the nodules decreased by 46% in the presence of KNO₃ and there were differences among rhizobial strains but not among pea genotypes. Malate and succinate accumulated in nodules in the presence of KNO₃. These data are not consistent with the photosynthate deprivation hypothesis as a primary mechanism for NO₃ inhibition of N₂ fixation since NO₃ affected the nodule carbohydrate composition of all three pea genotypes in a similar manner. The lack of correlation between NR activity and NO₃ inhibition of N₂ fixation suggests that NO₃ assimilation may be only indirectly involved in the inhibition phenomenon.     

Studies of diurnal variations of nitrate reductase activity in barley leaves using various assay methods

The in vitro and various modifications of the in vivo assay for nitrate reductase have been compared in order to elucidate their usefulness in studies of diurnal variations of enzyme activity in barley leaves ( Hordeum vulgare L. cv. Herta). Generally, activity was low in the morning and increased rapidly during the first hours of the photoperiod. In the in vivo assay the leaf tissue was vacuum-infiltrated, whereafter either N₂ was bubbled through the assay buffer (anaerobic assay), or no N₂ was used (aerobic assay). Activity was 2–25 times higher in the anaerobic than in the aerobic assay. Anaerobiosis enhanced activity most during the dark period when the nitrate reductase level was low. Aerobic in vivo activity usually showed a more rapid decrease towards the end of the light period than did anaerobic activity. Addition of glucose and/or nitrate to the in vivo assay buffer usually stimulated activity more in the aerobic than in the anaerobic assay. In the morning, at the end of the dark period, these additives stimulated activity by 20–400% depending on growth and assay conditions. Later in the day stimulation was usually less, and even a slight inhibition was observed when only nitrate (0.1 M ) was added. The effect of these additives on the activity patterns determined was to dampen the oscillations. The additives were therefore not advantageous when testing diurnal variations. However, when the plants were grown under relatively poor light conditions it was necessary to add nitrate and glucose to the aerobic in vivo assay buffer since activity was otherwise too low to be measured. The in vitro assay gave about 5 times higher activity than the anaerobic in vivo assay. During the last part of the dark period in vivo activity (without glucose and KNO₃ in the assay buffer) decreased while in vitro activity remained constant.     

Metabolism of nitric oxide in soil and denitrifying bacteria

Abstract Production and consumption of NO was measured under anaerobic conditions in a slightly alkaline and an acidic soil as well as in pure cultures of denitrifying Pseudomonas aeruginosa, P. stutzeri, P. fluorescens, Paracoccus denitrificans, Azospirillum brasilense , and A. lipoferum . Growing bacterial cultures reduced nitrate and intermediately accumulated nitrite, NO, N₂O, but not NO₂. Addition of formaldehyde inhibited NO production and NO consumption. In the presence of acetylene NO was reduced to N₂O. Net NO release rates in denitrifying bacterial suspensions and in soil samples decreased hyperbolically with increasing NO up to mixing ratios of about 5 ppmv NO. This behaviour could be modelled by assuming a constant rate of NO production simultaneously with a NO consumption activity that increased with NO until V _max was reached. The data allowed calculation of the gross rates ( P ) of NO production, of the rate constants ( k ), V _max and K _m of NO consumption, and of the NO compensation mixing ratio ( m _c). In soil, P was larger than V _max resulting in net NO release even at high NO mixing ratios unless P was selectively inhibited by chlorate + chlorite or by aerobic incubation conditions. In bacteria, V _max was somewhat larger than P resulting in net NO uptake at high NO mixing ratios. Both P and V _max were dependent on the supply of electron donor (e.g. glucose). Both in soil (aerobic or anaerobic) and in pure culture, the K _m values of NO consumption were in a similar low range of about 0.5–6.0 nM. Anaerobic soil and denitrifying bacteria exhibited m _c values of 1.6–2.1 ppmv NO and 0.2–4.0 ppmv NO, respectively.     

Metabolism of nitric oxide and nitrous oxide during nitrification and denitrification in soil at different incubation conditions

Abstract NO production and consumption rates as well as N₂O accumulation rates were measured in a loamy cambisol which was incubated under different conditions (i.e. soil moisture content, addition of nitrogen fertilizer and/or glucose, aerobic or anaerobic gas phase). Inhibition of nitrification with acetylene allowed us to distinguish between nitrification and denitrification as sources of NO and N₂O. Under aerobic conditions untreated soil showed very low release of NO and N₂O but high consumption of NO. Fertilization with NH₄⁺ or urea stimulated both NO and N₂O production by nitrification. Addition of glucose at high soil moisture contents led to increased N₂ and N₂O production by denitrification, but not to increased NO production rates. Anaerobic conditions, however, stimulated both NO and N₂O production by denitrification. The production of NO and N₂O was further stimulated at low moisture contents and after addition of glucose or NO₃⁻. Anaerobic consumption of NO by denitrification followed Michaelis-Menten kinetics and was stimulated by addition of glucose and NO₃⁻. Aerobic consumption of NO followed first-order kinetics up to mixing ratios of at least 14 ppmv NO, was inhibited by autoclaving but not by acetylene, and decreased with increasing soil moisture content. The high NO-consumption activity and the effects of soil moisture on the apparent rates of anaerobic and aerobic production and consumption of NO suggest that diffusional constraints have an important influence on the release of NO, and may be a reason for the different behaviour of NO release vs N₂O release.     

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.     

Oxygen-induced recovery from short-term nitrate inhibition of N2 fixation in white clover plants from spaced and dense stands

Nitrogenase (N₂ase; EC 1.18.6.1) activity (H₂ evolution) and root respiration (CO₂ evolution) were measured under either N₂:O₂ or Ar:O₂ gas mixtures in intact nodulated roots from white clover ( Trifolium repens L.) plants grown either as spaced or as dense stands. The short-term nitrate (5 m M ) inhibition of N₂-fixation was promoted by competition for light between clover shoots, which reduced CO₂ net assimilation rate. Oxygen-diffusion permeability of the nodule declined during nitrate treatment but after nitrate removal from the liquid medium its recovery parallelled that of nitrogenase activity. Rhizosphere pO₂ was increased from 20 to 80 kPa under N₂:O₂. A simple mono-exponential model, fitted to the nodule permeability response to pO₂, indicated NO⁻₃ induced changes in minimum and maximum nodule O₂-diffusion permeability. Peak H₂ production rates at 80 kPa O₂ and in Ar:O₂ were close to the pre-decline rates at 20 kPa O₂. At the end of the nitrate treatment, this O₂-induced recovery in nitrogenase activity reached 71 and 82%; for clover plants from spaced and dense stands, respectively. The respective roles of oxygen diffusion and phloem supply for the short-term inhibition of nitrogenase activity in nitrate-treated clovers are discussed.     

Metabolism of nitric oxide and nitrous oxide during nitrification and denitrification in soil at different incubation conditions

Abstract NO production and consumption rates as well as N₂O accumulation rates were measured in a loamy cambisol which was incubated under different conditions (i.e. soil moisture content, addition of nitrogen fertilizer and/or glucose, aerobic or anaerobic gas phase). Inhibition of nitrification with acetylene allowed us to distinguish between nitrification and denitrification as sources of NO and N₂O. Under aerobic conditions untreated soil showed very low release of NO and N₂O but high consumption of NO. Fertilization with NH₄⁺ or urea stimulated both NO and N₂O production by nitrification. Addition of glucose at high soil moisture contents led to increased N₂ and N₂O production by denitrification, but not to increased NO production rates. Anaerobic conditions, however, stimulated both NO and N₂O production by denitrification. The production of NO and N₂O was further stimulated at low moisture contents and after addition of glucose or NO₃⁻. Anaerobic consumption of NO by denitrification followed Michaelis-Menten kinetics and was stimulated by addition of glucose and NO₃⁻. Aerobic consumption of NO followed first-order kinetics up to mixing ratios of at least 14 ppmv NO, was inhibited by autoclaving but not by acetylene, and decreased with increasing soil moisture content. The high NO-consumption activity and the effects of soil moisture on the apparent rates of anaerobic and aerobic production and consumption of NO suggest that diffusional constraints have an important influence on the release of NO, and may be a reason for the different behaviour of NO release vs N₂O release.     

Thermophilic denitrifying bacteria: A survey of hot springs in Southwestern Iceland

Abstract Samples of water, sediment and bacterial mat from hot springs in Grændalur and Hveragerdi areas in southwestern Iceland were screened at 70°C and 80°C for thermophilic denitrifying bacteria by culturing in anaerobic media containing nitrate or N₂O as the terminal oxidant. The springs ranged in temperature from 65–100°C and included both neutral (pH 7–8.5) and acidic (pH 2.5–4) types. Nitrate reducing bacteria (nitrate → nitrite) and denitrifiers (nitrate → N₂) were found that grew at 70°C but not at 80°C in nutrient media at pH 8. Samples from neutral springs that were cultured at pH 8 failed to yield a chemolithotrophic, sulfur-oxidizing and nitrate-reducing bacterium, and samples from acidic springs that were cultured at pH 3.5 seemed entirely to lack dissimilatory, nitrate-utilizing bacteria. No sample yielded an organism capable of growth solely by N₂O respiration. The denitrifiers appeared to be Bacillus . Two such Bacillus strains were examined in pure culture and found to exhibit the unusual denitrification phenotype described previously for the mesophile, Pseudomonas aeruginosa , and one other strain of thermophilic Bacillus . The phenotype is characterized by the ability to grow by reduction of nitrate to N₂ with N₂O as an intermediate but a virtual inability to reduce N₂O when N₂O was the sole oxidant.     

Thermophilic denitrifying bacteria: A survey of hot springs in Southwestern Iceland

Abstract Samples of water, sediment and bacterial mat from hot springs in Grændalur and Hveragerdi areas in southwestern Iceland were screened at 70°C and 80°C for thermophilic denitrifying bacteria by culturing in anaerobic media containing nitrate or N₂O as the terminal oxidant. The s springs ranged in temperature from 65–100°C and included both neutral (pH 7–8.5) and acidic (pH 2.5–4) types. Nitrate reducing bacteria (nitrate → nitrite) and denitrifiers (nitrate → N₂) were found that grew at 70°C but not at 80°C in nutrient media at pH 8. Samples from neutral springs that were cultured at pH 8 failed to yield a chemolithotrophic, sulfur-oxidizing and nitrate-reducing bacterium, and samples from acidic springs that were cultured at pH 3.5 seemed entirely to lack dissimilatory, nitrate-utilizing bacteria. No sample yielded an organism capable of growth solely by N₂O respiration. The denitrifiers appeared to be Bacillus . Two such Bacillus strains were examined in pure culture and found to exhibit the unusual denitrification phenotype described previously for the mesophile, Pseudomonas aeruginosa , and one other strain of thermophilic Bacillus . The phenotype is characterized by the ability to grow by reduction of nitrate to N₂ with N₂O as an intermediate but a virtual inability to reduce N₂O when N₂O was the sole oxidant.     

Effects of oxygen, pH and nitrate concentration on denitrification by Pseudomonas species

Abstract The production of nitrogen-containing gases by denitrification in three organisms was examined using membrane inlet mass spectrometry. The effects of O₂ (during both growth and maintenance) and of pH, nitrate concentration and carbon source were tested in non-proliferating cell suspensions. Two strains of Pseudomonas aeruginosa were capable of co-respiration of NO₃⁻ and O₂ and, under controlled O₂ supply, gave oscillatory denitrification. Variations in culture and assay conditions affected both the rate of denitrification and the ratio of end products (N₂O:N₂). Higher rates were seen following anaerobic growth. Optimum values of pH and nitrate concentration for denitrification are given. Generally, the optimum pH was 7.0–7.5, approximately that of the growth medium. Optimum nitrate concentration was generally 20 mM.     

Membrane inlet mass spectrometric analysis of N-isotope labelling for aquatic denitrification studies

Abstract: Techniques are described for measuring the isotope distribution in dissolved nitrate and N₂ using membrane inlet mass spectrometry, which allows several gases to be measured in a water sample without the need for any separation steps. The isotope distribution in dissolved nitrate was measured using denitrifying Pseudomonas nautica to reduce the nitrate to N₂ which was then measured by mass spectrometry. Pseudomonas nautica NCIMB 1967 was easily grown in nitrate-limited continuous culture minimising intra- or extracellular nitrate or nitrite pools, and the bioassay was tolerant of a range of salinities. The precision of the bioassay when measuring samples with high ¹⁵NO₃⁻ contents (0.5 μmol) was 0.05 atom%; with 0.1 μmol ¹⁵NO₃⁻, the precision was around 0.2 atom%. Differences in labelling of N₂ in preserved samples obtained from ¹⁵NO₃⁻ incubations of water-covered sediment cores were measured on parallel samples with membrane inlet MS and GC-MS. The membrane inlet technique was accurate but the precision on ratio measurements was lower than by GC-MS.     

Nitrate metabolism in roots and nodules of Vicia faba in response to exogenous nitrate

Activities of nitrate reduction enzymes, nitrate reductase activity (NRA) and nitrite reductase activity (NiRA) from roots and nodules of 5 mutant genotypes and one commercial cultivar (Alameda) of faba bean ( Vicia faba L. var. minor) grown in the presence of N₂ alone or with additional NO⁻₃ in the medium have been studied. A naturally occurring mutant (VFM109) with impaired ability to reduce nitrate in its nodules is described. All the other cultivars of V. faba showed nodule NRA, although the range was very wide, from almost negligible (VFM72) up to 2 μmol h⁻¹ (g FW)⁻¹. This activity was entirely of plant origin. Root NRA also ranged widely accross cultivars. However, the level of activity expressed as well as the response of NRA to nitrate followed a pattern opposite to that observed in nodules. Roots and nodules of all cultivars showed very high rates of NiRA, respectively 50 and 150-fold higher than NRA, thus precluding accumulation of nitrite in these tissues. Root enzymes were significantly stimulated by nitrate while negative (NRA) or little effect (NiRA) was found for nodules. Nitrate and nitrite reduction are carried out by inducible enzymes in roots of V. faba and by constitutive enzymes in nodules, indicating that there may be different forms of these enzymes in each tissue. Differences in the plant genotype were a major cause of the variability in nitrate and nitrite reduction by nodulated root systems of V. faba .