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PubMed1981

Nitrate Utilization by Nitrate Reductase-deficient Barley Mutants.

Two nitrate reductase-deficient barley mutants were studied for growth on nitrate and ammonium sources of nitrogen and for resistance to chlorate. Although nitrate reductase-deficient mutants in some species are chlorate-resistant (unable to reduce chlorate to chlorite), the barley mutants used in these studies when grown on nitrate and treated with chlorate were only slightly more resistant to chlorate than the control. When grown to maturity on vermiculite supplemented with either nitrate or ammonium nutrient solutions, the mutants produced as much dry weight and reduced nitrogen per plant as the control. The in vivo and in vitro nitrate reductase activities in the roots and shoots of the mutants grown on nitrate were consistently less than 10% of the control. To avoid the possibility that the mutants received reduced nitrogen from microbial sources, excised embryos were cultured under sterile conditions. Again the mutants were capable of growth and reduced nitrogen accumulation with nitrate as the sole source of nitrogen. In spite of the low apparent nitrate reductase activity, the nitrate reductase-deficient mutants are capable of substantial nitrate reduction.

Journal Article↗
PubMed1981

Purification and Characterization of NAD(P)H:Nitrate Reductase and NADH:Nitrate Reductase from Corn Roots.

The nitrate reductase activity of 5-day-old whole corn roots was isolated using phosphate buffer. The relatively stable nitrate reductase extract can be separated into three fractions using affinity chromatography on blue-Sepharose. The first fraction, eluted with NADPH, reduces nearly equal amounts of nitrate with either NADPH or NADH. A subsequent elution with NADH yields a nitrate reductase which is more active with NADH as electron donor. Further elution with salt gives a nitrate reductase fraction which is active with both NADH and NADPH, but is more active with NADH. All three nitrate reductase fractions have pH optima of 7.5 and Stokes radii of about 6.0 nanometers. The NADPH-eluted enzyme has a nitrate K(m) of 0.3 millimolar in the presence of NADPH, whereas the NADH-eluted enzyme has a nitrate K(m) of 0.07 millimolar in the presence of NADH. The NADPH-eluted fraction appears to be similar to the NAD(P)H:nitrate reductase isolated from corn scutellum and the NADH-eluted fraction is similar to the NADH:nitrate reductases isolated from corn leaf and scutellum. The salt-eluted fraction appears to be a mixture of NAD(P)H: and NADH:nitrate reductases.

Journal Article↗
PubMed1993

Insertion of transposon Tn5 into a structural gene of the membrane-bound nitrate reductase of Thiosphaera pantotropha results in anaerobic overexpression of periplasmic nitrate reductase activity.

Chlorate-resistant mutants of the denitrifying bacterium Thiosphaera pantotropha were generated by transposon Tn5 mutagenesis. One class was deficient in membrane-bound nitrate reductase activity but retained a periplasmic nitrate reductase activity. Using transposon marker rescue it was shown that in one such mutant, M-6, the transposon was inserted in the membrane-bound nitrate reductase beta subunit structural gene (termed narH in order to be consistent with the nomenclature of the Escherichia coli major nitrate reductase operon). The translated sequence (total of 106 amino acids) from around the point of transposon insertion showed approximately 90% amino acid identity with the beta subunits of the E. coli nitrate reductases. Under anaerobic growth conditions M-6 overproduced the periplasmic nitrate reductase activity allowing anaerobic growth with nitrate as electron acceptor. A regulatory link was inferred between the presence of the membrane-bound nitrate reductase and expression of the periplasmic nitrate reductase. This is the first demonstration of full denitrification in an organism possessing only a periplasmic nitrate reductase.

Amino Acid Sequence↗
PubMed1999

Nitrate and nitrite control of respiratory nitrate reduction in denitrifying Pseudomonas stutzeri by a two-component regulatory system homologous to NarXL of Escherichia coli.

Bacterial denitrification is expressed in response to the concurrent exogenous signals of low-oxygen tension and nitrate or one of its reduction products. The mechanism by which nitrate-dependent gene activation is effected was investigated in the denitrifying bacterium Pseudomonas stutzeri ATCC 14405. We have identified and isolated from this organism the chromosomal region encoding the two-component sensor-regulator pair NarXL and found that it is linked with the narG operon for respiratory nitrate reductase. The same region encodes two putative nitrate or nitrite translocases, NarK and NarC (the latter shows the highest similarity to yeast [Pichia] and plant [Nicotiana] nitrate transporters), and the nitrate-regulated transcription factor, DnrE, of the FNR family. The roles of NarX and NarL in nitrate respiration were studied with deletion mutants. NarL activated the transcription of narG, narK, and dnrE but did not affect the denitrification regulons for the respiratory substrates nitrite, nitric oxide, and nitrous oxide. The promoters of narG, narK, and dnrE carry sequence motifs, TACYYMT, which correspond to the NarL recognition sequence established for Escherichia coli. The cellular response toward nitrate and nitrite was mediated by the sensor protein NarX, which discriminated weakly between these oxyanions. Our data show that the NarXL two-component regulatory system has been incorporated into the bacterial denitrification process of P. stutzeri for selective regulation of nitrate respiration.

Amino Acid Sequence↗
PubMed1995

Plasma nitrate clearance in mice: modeling of the systemic production of nitrate following the induction of nitric oxide synthesis.

Nitric oxide (NO) is produced in mammals by the enzyme NO synthase (NOS) in response to a number of agents, including the experimental antitumour agent flavone acetic acid (FAA) and the cytokine tumour necrosis factor-alpha (TNF). NO is converted rapidly in the presence of oxygen, water and haemoglobin to oxidation products, largely nitrate. To quantitate the production of nitric oxide it is necessary to know the clearance of nitrate. The concentration of nitrite and nitrate ion in the plasma of C3H and BDF1 (C57BL6 x DBA2) mice was assessed before and after injection of sodium nitrate and sodium nitrite. Nitrite was covered rapidly to nitrate and the kinetics of elimination of nitrate were determined. There was no significant difference between results obtained with different mouse strains, between levels of nitrite and nitrate, or between i.p. and i.v. administration, and the observations were therefore combined. The volume of distribution of nitrate was 0.71 +/- 0.04 l/kg and the clearance was 0.32 +/- 0.02 l/h-1/kg-1 (plasma half-life, 1.54 h). Using previously published data, we developed a pharmacokinetic-pharmacodynamic model that relates the production of TNF in response to administration of FAA, the enhancement of NOS activity in response to TNF, and the elevation of plasma nitrate in response to NO production. This information permits the prediction from observed plasma nitrate values of the amount of NOS induced in vivo.

Amino Acid Oxidoreductases↗
PubMed2002

Accumulations of nitrite and nitrate in the tissues of Penaeus monodon exposed to a combined environment of elevated nitrite and nitrate.

Penaeus monodon (11.86 +/- 0.63 g) exposed individually to six different nitrite and nitrate regimes [nitrite at 0.002 (control), 0.360, and 1.455 mM combined with nitrate at 0.005 (control) and 7.275 mM] in 25 ppt sea water were examined for the nitrite and nitrate concentrations in tissues and nitrite uptake and nitrate uptake after 24 h in 25.3 degrees C. In P. monodon following exposure to 1.455 mM nitrite only, nitrite levels increased by factors of 0.33, 1.05, 1.36, 1.79, 2.10, 2.16, 2.21, and 3.58 in muscle, hepatopancreas, gill, foregut, hemolymph, heart, eyestalk, and midgut, respectively, over the ambient nitrite level. In P. monodon, following exposure to combined solutions of 1.455 mM nitrite and 7.275 mM nitrate, nitrite levels were factors of 0.34, 1.05, 1.44, 1.86, 2.21, 2.30, 2.67, and 3.83 in muscle, hepatopancreas, gill, foregut, heart, hemolymph, eyestalk, and midgut, respectively, over the ambient nitrite levels, whereas nitrate levels were factors of 0.06, 0.11, 0.27, 0.29, 0.45, 0.51, 0.57, and 0.61 in muscle, hepatopancreas, foregut, heart, hemolymph, gill, midgut, and eyestalk, respectively, over the ambient nitrate levels. It is concluded that incorporation of nitrite is converted to nitrate in midgut, heart, hemolymph, foregut, and eyestalk, whereas incorporation of nitrate in tissues is converted to nitrite and accumulated in eyestalk when P. monodon are exposed to combined nitrite and nitrate environments.

Animals↗
PubMed2006

Removal of added nitrate in cotton burr compost, mulch compost, and peat: mechanisms and potential use for groundwater nitrate remediation.

We conducted batch tests on the nature of removal of added nitrate in cotton burr compost, mulch compost, and sphagnum peat that may be potentially used in a permeable reactive barrier (PRB) for groundwater nitrate remediation. A rigorous steam autoclaving protocol (121 degrees C for 2h each day for three consecutive days) for the cotton burr compost and autoclaving of all labware and the nitrate working solutions resulted in drastically different results compared to the non-autoclaved treatment. In the non-autoclaved cotton burr compost, added nitrate at 20 mg N l(-1) decreased rapidly and was not detected after 3d; whereas, the autoclaved cotton burr compost showed persistent nitrate above 15.5 mg N l(-1) even after 10d, which is comparable with nitrate concentrations above 17.6 mg N l(-1) in a treatment using NaN(3) at 1000 mg l(-1). Dewaxed cotton burr compost showed decreased nitrate reduction compared to the pristine cotton burr compost. No nitrate reduction was detected in the dewaxed sphagnum peat. It is concluded that nitrate removal in the organic media is controlled by microbiologically mediated processes. The use of readily available cotton burr and mulch composts may offer a cost-effective method of nitrate removal from contaminated groundwater.

Gossypium↗
PubMed2002

Accumulation of nitrate in the tissues of Penaeus monodon following elevated ambient nitrate exposure after different time periods.

Penaeus monodon (10.56 +/- 0.71 g) exposed individually to 0.026 (control), 0.774, 3.646, 7.004, 21.234 and 36.079 mM nitrate in 25 ppt seawater were examined for nitrate accumulation in hemolymph, gill, eyestalk, heart, foregut, midgut, hepatopancreas and muscle after 1, 3, 6, 12 and 24 h, respectively. Concentration of nitrate in the tissues increased directly with ambient nitrate concentration and exposure time except for muscle. Following 24-h exposure to 3.646 mM nitrate, nitrate concentrations measured in the muscle (0.202 micromol g(-1)), hepatopancreas (0.330 micromol g(-1)), heart (0.527 micromol g(-1)), foregut (0.632 micromol g(-1)), gill (0.927 micromol g(-1)), hemolymph (0.946 micromol ml(-1)), eyestalk (1.214 micromol g(-1)), and midgut (1.529 micromol g(-1)), respectively. The concentration of muscle nitrate was the lowest, and the concentration of midgut nitrate was the highest among the tissues tested. P. monodon following exposure to ambient nitrate at 21.234 and 36.079 mM, nitrate accumulation measured in the muscle (0.854 and 0.980 micromol g(-1)), hepatopancreas (1.139 and 1.552 micromol g(-1)), heart (1.468 and 1.879 micromol g(-1)), foregut (2.195 and 3.341 micromol g(-1)), gill (2.398 and 3.325 micromol g(-1)), hemolymph (3.327 and 3.948 micromol ml(-1)), eyestalk (3.461 and 4.264 micromol g(-1)) and midgut (3.343 and 5.239 micromol g(-1)), and never reached a plateau after 24 h.

Animals↗
PubMed1990

The effect of dietary nitrate on nitrate and nitrite excretion in man.

Dietary nitrate and nitrite may affect colonic pathophysiology. These anions influence fermentation, and nitrite has been shown to augment sodium absorption by the colon and participate in the formation of N-nitroso compounds. There is, however, no general agreement as to how much dietary nitrate and nitrite reaches the colon. To help resolve this question, balance studies were performed on six healthy ileostomy subjects who were given diets that varied in nitrate content from 0.83 to 5.20 mmol/d. Nitrate and nitrite excretion in ileal effluent and urine were measured by anion-exchange chromatography with conductivity detection. There was no significant nitrite in the diets, urine or ileal effluent. Dietary nitrate was largely excreted in urine (1.31-4.25 mmol/d). The urinary excretion findings indicated net synthesis of nitrate at low dietary intakes and net catabolism of nitrate at high intakes. Nitrate losses in ileal effluent were very low (0.03-0.05 mmol/d, 0.03-0.06 mmol/kg) and unrelated to intake for all the diets. It is concluded that dietary nitrate and nitrite do not enter the colon from the small intestine in amounts that would affect fermentation and mucosal metabolism in man. The possibility of significant amounts of nitrate reaching the colon via blood in normal subjects has not been excluded.

Aged↗
PubMed1986

Nitrate, nitrite balance, and de novo synthesis of nitrate in humans consuming cured meats.

Nitrate balance was measured in nine men consuming a fixed daily diet with constant nitrate (570 mumol/day), varying nitrite (18 to 150 mumol/day), and varying erythorbate levels. Nitrite and erythorbate were added to meat that was then cured and frozen until consumption. All diets were consumed by each subject for 17 days each. Average daily urinary nitrate excretion ranged from 959 to 2382 mumol/day. Subjects excreted significantly more nitrate in urine when fed nitrite cured meats with or without erythorbate than when fed uncured meat (1617 and 1577 versus 1430 mumol nitrate/day, respectively). The amount of nitrate excreted in urine consistently exceeded intakes of nitrate and nitrite by an average 870 mumol/day. This excess represented endogenous synthesis by subjects and was not due to unmeasured nitrate in the diet. The commonly used Greiss and xylenol procedures were unable to measure all nitrate in urine and in diets when compared to high performance liquid chromatographic analyses. The Greiss and xylenol analyses underestimated nitrate synthesis by 220 and 150 mumol/day, respectively when results were not adjusted by determining recovery of added nitrate.

Adult↗
PubMed2004

Mutation of a nitrate transporter, AtNRT1:4, results in a reduced petiole nitrate content and altered leaf development.

Unlike nitrate uptake of plant roots, less is known at the molecular level about how nitrate is distributed in various plant tissues. In the present study, characterization of the nitrate transporter, AtNRT1:4, revealed a special role of petiole in nitrate homeostasis. Electrophysiological studies using Xenopus oocytes showed that AtNRT1:4 was a low-affinity nitrate transporter. Whole-mount in situ hybridization and RT-PCR demonstrated that AtNRT1:4 was expressed in the leaf petiole. In the wild type, the leaf petiole had low nitrate reductase activity, but a high nitrate content, indicating that it is the storage site for nitrate, whereas, in the atnrt1:4 mutant, the petiole nitrate content was reduced to 50-64% of the wild-type level. Moreover, atnrt1:4 mutant leaves were wider than wild-type leaves. This study revealed a critical role of AtNRT1:4 in regulating leaf nitrate homeostasis, and the deficiency of AtNRT1:4 can alter leaf development.

Anion Transport Proteins↗
PubMed2002

Corn leaf nitrate reductase--a nontoxic alternative to cadmium for photometric nitrate determinations in water samples by air-segmented continuous-flow analysis.

Development, characterization, and operational details of an enzymatic, air-segmented continuous-flow analytical method for colorimetric determination of nitrate + nitrite in natural-water samples is described. This method is similar to U.S. Environmental Protection Agency method 353.2 and U.S. Geological Survey method 1-2545-90 except that nitrate is reduced to nitrite by soluble nitrate reductase (NaR, EC 1.6.6.1) purified from corn leaves rather than a packed-bed cadmium reactor. A three-channel, air-segmented continuous-flow analyzer-configured for simultaneous determination of nitrite (0.020-1.000 mg-N/L) and nitrate + nitrite (0.05-5.00 mg-N/L) by the nitrate reductase and cadmium reduction methods-was used to characterize analytical performance of the enzymatic reduction method. At a sampling rate of 90 h(-1), sample interaction was less than 1% for all three methods. Method detection limits were 0.001 mg of NO2- -N/L for nitrite, 0.003 mg of NO3-+ NO2- -N/L for nitrate + nitrite by the cadmium-reduction method, and 0.006 mg of NO3- + NO2- -N/L for nitrate + nitrite bythe enzymatic-reduction method. Reduction of nitrate to nitrite by both methods was greater than 95% complete overthe entire calibration range. The difference between the means of nitrate + nitrite concentrations in 124 natural-water samples determined simultaneously bythe two methods was not significantly different from zero at the p = 0.05 level.

Cadmium↗
PubMed2003

Monomeric NarB is a dual-affinity nitrate reductase, and its activity is regulated differently from that of nitrate uptake in the unicellular diazotrophic cyanobacterium Synechococcus sp. strain RF-1.

Synechococcus sp. strain RF-1 is a unicellular freshwater cyanobacterium that fixes N(2) aerobically and exhibits a circadian rhythm for nitrogenase activity under a light-dark regimen. Synechococcus sp. strain RF-1 also utilizes nitrate, nitrite, or ammonium for growth. Under the diazotrophic growth, the nitrate uptake in Synechococcus sp. strain RF-1 was induced by nitrate or nitrite but repressed by ammonium. In contrast, a prominent nitrate reductase (NR) activity was detected in diazotrophically grown cells using the reduced methyl viologen assay. The NR activity was not inhibited by ammonium and only slightly enhanced by nitrate. The different expression patterns of nitrate uptake and NR in Synechococcus sp. strain RF-1 were reflected in general at the transcript level determined by reverse transcriptase PCR. Under both nitrate-induced and uninduced conditions, the in situ NR activity exhibited similar biphasic kinetics for nitrate. The recombinant NR encoded by the narB gene of Synechococcus sp. strain RF-1, expressed in E. coli, also showed the biphasic kinetics with similar pH and temperature profiles. By in-gel NR activity assay, the recombinant NarB was found to exist as a single form. Both the high- and low-affinity NR activities of the recombinant NarB showed the same thermostability. When modified at the N terminus by a polyhistidine tag, the recombinant NR activity was shifted from biphasic to hyperbolic kinetics and showed only a single K(m) for nitrate, indicating the functional importance of the NarB N-terminal structure in NR kinetics.

Bacterial Proteins↗
PubMedDate not supplied

Enzymatic nitrate assay by a kinetic method employing Escherichia coli nitrate reductase.

An enzymatic assay system for nitrate employing the membrane-bound nitrate reductase (EC 1.7.99.4) of E. coli is described. Contrary to previous enzymatic assay systems, the present method is a kinetic one, i.e. the substrate, nitrate, is assayed by measuring the reaction rate of the nitrate reductase-catalyzed reaction. Based on the observation that the nitrate reductase-catalyzed reaction obeys pseudo-first order kinetics, a test system is described allowing the assay of nitrate at a concentration as low as 1 ppm. The relatively high Michaelis-Menten constant for nitrate (0.3 mM) of the E. coli nitrate reductase favours nitrate assay by the kinetic method.

Catalysis↗
PubMed1989

cDNA Clones for Corn Leaf NADH:Nitrate Reductase and Chloroplast NAD(P):Glyceraldehyde-3-Phosphate Dehydrogenase : Characterization of the Clones and Analysis of the Expression of the Genes in Leaves as Influenced by Nitrate in the Light and Dark.

cDNA clones were selected from a corn (Zea mays L.) leaf lambda gt11 expression library using polyclonal antibodies for corn leaf NADH:nitrate reductase. One clone, Zmnrl, had a 2.1 kilobase insert, which hybridized to a 3.2 kilobase mRNA. The deduced amino acid sequence of Zmnrl was nearly identical to peptide sequences of corn leaf NADH:nitrate reductase. Another clone, Zm6, had an insert of 1.4 kilobase, which hybridized to a 1.4 kilobase mRNA, and its sequence coded for chloroplastic NAD(P)(+):glyceraldehyde-3-phosphate dehydrogenase based on comparisons to sequences of this enzyme from tobacco and corn. When nitrate was supplied to N-starved, etiolated corn plants, nitrate reductase, and glyceraldehyde-3-phosphate dehydrogenase mRNA levels in leaves increased in parallel. When green leaves were treated with nitrate, only nitrate reductase mRNA levels were increased. Nitrate is a specific inducer of nitrate reductase in green leaves, but appears to have a more general effect in etiolated leaves. In the dark, nitrate induced nitrate reductase expression in both etiolated and green leaves, indicating light and functional chloroplast were not required for enzyme expression.

Journal Article↗
PubMed1997

Effects of Nitrate Availability and the Presence of Glyceria maxima on the Composition and Activity of the Dissimilatory Nitrate-Reducing Bacterial Community.

The effects of nitrate availability and the presence of Glyceria maxima on the composition and activity of the dissimilatory nitrate-reducing bacterial community were studied in the laboratory. Four different concentrations of NO(inf3)(sup-), 0, 533, 1434, and 2,905 (mu)g of NO(inf3)(sup-)-N g of dry sediment(sup-1), were added to pots containing freshwater sediment, and the pots were then incubated for a period of 69 days. Upon harvest, NH(inf4)(sup+) was not detectable in sediment that received 0 or 533 (mu)g of NO(inf3)(sup-)-N g of dry sediment(sup-1). Nitrate concentrations in these pots ranged from 0 to 8 (mu)g of NO(inf3)(sup-)-N g of dry sediment(sup-1) at harvest. In pots that received 1,434 or 2,905 (mu)g of NO(inf3)(sup-)-N g of dry sediment(sup-1), final concentrations varied between 10 and 48 (mu)g of NH(inf4)(sup+)-N g of dry sediment(sup-1) and between 200 and 1,600 (mu)g of NO(inf3)(sup-)-N g of dry sediment(sup-1), respectively. Higher input levels of NO(inf3)(sup-) resulted in increased numbers of potential nitrate-reducing bacteria and higher potential nitrate-reducing activity in the rhizosphere. In sediment samples from the rhizosphere, the contribution of denitrification to the potential nitrate-reducing capacity varied from 8% under NO(inf3)(sup-)-limiting conditions to 58% when NO(inf3)(sup-) was in ample supply. In bulk sediment with excess NO(inf3)(sup-), this percentage was 44%. The nitrate-reducing community consisted almost entirely of NO(inf2)(sup-)-accumulating or NH(inf4)(sup+)-producing gram-positive species when NO(inf3)(sup-) was not added to the sediment. The addition of NO(inf3)(sup-) resulted in an increase of denitrifying Pseudomonas and Moraxella strains. The factor controlling the composition of the nitrate-reducing community when NO(inf3)(sup-) is limited is the presence of G. maxima. In sediment with excess NO(inf3)(sup-), nitrate availability determines the composition of the nitrate-reducing community.

Journal Article↗
PubMed2002

Isolation and characterization of a chlorate-resistant mutant (Clo- R) of the symbiotic cyanobacterium Nostoc ANTH: heterocyst formation and N(2)-fixation in the presence of nitrate, and evidence for separate nitrate and nitrite transport systems.

Nostoc ANTH is a filamentous, heterocystous cyanobacterium capable of N(2)-fixation in the absence of combined nitrogen. A chlorate-resistant mutant (Clo- R) of Nostoc ANTH was isolated that differentiates heterocysts and fixes N(2) in the presence of nitrate, but not in the presence of nitrite or ammonium. The mutant lacks nitrate uptake and thereby also lacks induction of nitrate reductase activity by nitrate. However, this mutant is able to transport and assimilate nitrite, indicating that there is a transport system for nitrite that is distinct from that for the nitrate. The lack of inhibitory effect of nitrate on N(2)-fixation was owing to lack of nitrate uptake and not to lack of enzymes for its assimilation (nitrate reductase and glutamine synthetase) or the lack of an ammonium transport system for retention of ammonia. The mutant has potential for use as a biofertilizer supplementing chemical nitrate fertilizer in rice fields, without N(2)-fixation being adversely affected.

Biological Transport↗
PubMed2006

Bacterial nitrate reductases: Molecular and biological aspects of nitrate reduction.

Nitrogen is a vital component in living organisms as it participates in the making of essential biomolecules such as proteins, nucleic acids, etc. In the biosphere, nitrogen cycles between the oxidation states +V and -III producing many species that constitute the biogeochemical cycle of nitrogen. All reductive branches of this cycle involve the conversion of nitrate to nitrite, which is catalyzed by the enzyme nitrate reductase. The characterization of nitrate reductases from prokaryotic organisms has allowed us to gain considerable information on the molecular basis of nitrate reduction. Prokaryotic nitrate reductases are mononuclear Mo-containing enzymes sub-grouped as respiratory nitrate reductases, periplasmic nitrate reductases and assimilatory nitrate reductases. We review here the biological and molecular properties of these three enzymes along with their gene organization and expression, which are necessary to understand the biological processes involved in nitrate reduction.

Bacteria↗