Assimilatory nitrate uptake in Pseudomonas fluorescens studied using nitrogen-13

The mechanism of nitrate uptake for assimilation in procaryotes is not known. We used the radioactive isotope, ¹³N as NO₃ ^-, to study this process in a prevalent soil bacterium, Pseudomonas fluorescens. Cultures grown on ammonium sulfate or ammonium nitrate failed to take up labeled nitrate, indicating ammonium repressed synthesis of the assimilatory enzymes. Cultures grown on nitrite or under ammonium limitation had measurable nitrate reductase activity, indicating that the assimilatory enzymes need not be induced by nitrate. In cultures with an active nitrate reductase, the form of ¹³N internally was ammonium and amino acids; the amino acid labeling pattern indicated that ¹³NO₃ ^- was assimilated via glutamine synthetase and glutamate synthase. Cultures grown on tungstate to inactivate the reductase concentrated NO₃ ^- at least sixfold. Chlorate had no effect on nitrate transport or assimilation, nor on reduction in cell-free extracts. Ammonium inhibited nitrate uptake in cells with and without active nitrate reductases, but had no effect on cell-free nitrate reduction, indicating the site of inhibition was nitrate transport into the cytoplasm. Nitrate assimilation in cells grown on nitrate and nitrate uptake into cells grown with tungstate on nitrite both followed Michaelis-Menten kinetics with similar K _mvalues, 7 M. Both azide and cyanide inhibited nitrate assimilation. Our findings suggest that Pseudomonas fluorescens can take up nitrate via active transport and that nitrate assimilation is both inhibited and repressed by ammonium.     

Transformations of inorganic nitrogen by Azospirillum spp.

Azospirillum spp. participate in all steps of the nitrogen cycle except nitrification. They can fix molecular nitrogen and perform assimilatory nitrate reduction and nitrate respiration. Culture conditions have been defined under which nitrate is used both as terminal respiratory electron acceptor and as nitrogen source for growth. Nitrate and, possibly to a very limited extent, nitrite, but not sulfate, iron or fumarate support anaerobic respiration. Under anaerobic conditions, nitrate can also supply energy for nitrogen fixation but without supporting growth. Nitrate-dependent nitrogenase activity lasts only for 3–4 h until the enzymes of assimilatory nitrate reduction are synthesized. Nitrite accumulates during this period and inhibits nitrogenase activity at concentrations of about 1 mM.     

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.     

Regulation of Nitrate Assimilation and Nitrate Respiration in Aerobacter aerogenes

The influence of growth conditions on assimilatory and respiratory nitrate reduction in Aerobacter aerogenes was studied. The level of nitrate reductase activity in cells, growing in minimal medium with nitrate as the sole nitrogen source, was much lower under aerobic than anaerobic conditions. Further, the enzyme of the aerobic cultures was very sensitive to sonic disintegration, as distinct from the enzyme of anaerobic cultures. When a culture of A. aerogenes was shifted from anaerobic growth in minimal medium with nitrate and NH(4) (+) to aerobiosis in the same medium, but without NH(4) (+), the production of nitrite stopped instantaneously and the total activity of nitrate reductase decreased sharply. Moreover, there was a lag in growth of about 3 hr after such a shift. After resumption of growth, the total enzymatic activity increased again slowly and simultaneously became gradually sensitive to sonic disintegration. These findings show that oxygen inactivates the anaerobic nitrate reductase and represses its further formation; only after a de novo synthesis of nitrate reductase with an assimilatory function will growth be resumed. The enzyme in aerobic cultures was not significantly inactivated by air, only by pure oxygen. The formation of the assimilatory enzyme complex was repressed, however, by NH(4) (+), under both aerobic and anaerobic conditions. The results indicate that the formation of the assimilatory enzyme complex and that of the respiratory enzyme complex are regulated differently. We suggest that both complexes have a different composition, but that the nitrate reductase in both cases is the same protein.     

Inhibition of Assimilatory Nitrate Uptake by Ammonium Ions in Azorhizobium caulinodans IRBG 46

Addition of ammonium sulphate at low concentrations to Azorhizobium caulinodans IRBG 46 cells caused an immediate cessation of nitrate uptake activity, which was restored when the added ammonium ions were exhausted from the medium. Blockage of ammonium assimilation by L-methionine sulfoximine did not prevent the negative effect of ammonium on the assimilatory nitrate uptake, thus indicating that ammonium ions per se and not its assimilatory product(s) are actual regulators of assimilatory nitrate uptake.     

A novel class of ammonium assimilation mutants of the photosynthetic bacterium Rhodobacter capsulatus

Nitrogen assimilation in Rhodobacter capsulatus has been shown to proceed via the coupled action of glutamine synthetase (GS) and glutamate synthase (GOGAT) with no measurable glutamate dehydrogenase (GDH) present. We have recently isolated a novel class of mutants of R. capsulatus strain B100 that lacks a detectable GOGAT activity but is able to grow at wild type rates under nitrogen-fixing conditions. While NH ₄ ⁺ -supported growth in the mutants was normal under anaerobic/photosynthetic conditions, the growth rate was decreased under aerobic conditions. Ammonium and methylammonium uptake experiments indicated that there was a clear difference in the ammonium assimilatory capabilities in these mutants under aerobic versus anaerobic growth. Regulation of expression of a nifH : : lacZ fusion in these mutants was not impaired. The possible existence of alternative ammonium assimilatory pathways is discussed.     

Nitrate Reduction and the Growth of Veillonella alcalescens

Veillonella alcalescens, a strict anaerobe, was found to possess a nitrate reductase system which has characteristics of both assimilatory and respiratory nitrate reduction. The nitrate reductase has been identified tentatively as a particulate enzyme which utilizes a variety of electron donors for the reduction of nitrate. By use of ¹⁵N-labeled nitrate, it was shown that under appropriate conditions nitrate nitrogen is incorporated into cell material. V. alcalescens grown on pyruvate and nitrate has a greater growth rate than cells grown on pyruvate alone. Growth can occur in a medium with hydrogen and nitrate as the sole energy source. Ammonium chloride decreases the rate of nitrate reduction but does not completely inhibit reduction or incorporation. The results suggest that nitrate assimilation and respiration are not as distinct as in some other organisms.     

Effect of nitrogen concentration on the activity of manganese peroxidase ofPhanerochœte chrysosporium

Manganese peroxidase as an extracellular enzyme is produced by the white rot fungusPhanerochœte chrysosporium under nutrient nitrogen or carbon limitation. The effect of nitrogen concentration on the activity of manganese peroxidase was studied using ammonium nitrate andl-asparagine as nitrogen sources. The highest activity of the enzyme was observed in cultures grown in a medium containing 75 mg/L ammonium nitrate and 0.15 g/Ll-asparagine. Manganese peroxidase was not detectable in cultures grown in the presence 0.5 g/L ammonium nitrate and 1 g/Ll-asparagine.     

Microbial nitrate respiration--genes, enzymes and environmental distribution

Nitrate is a key node in the network of the assimilatory and respiratory nitrogen pathways. As one of the ‘fixed’ forms of nitrogen, nitrate plays an essential role in both nature and industry. For bacteria, it is both a nitrogen source and an electron acceptor. In agriculture and wastewater treatment, nitrate respiration by microorganisms is an important issue with respect to economics, greenhouse gas emission and public health. Several microbial processes compete for nitrate: denitrification, dissimilatory nitrate reduction to ammonium and anaerobic ammonium oxidation. In this review we provide an up to date overview of the organisms, genes and enzymes involved in nitrate respiration. We also address the molecular detection of these processes in nature. We show that despite rapid progress in the experimental and genomic analyses of pure cultures, knowledge on the mechanism of nitrate reduction in natural ecosystems is still largely lacking.     

Influence of nitrogen source and concentration on nitrogen isotopic discrimination in two barley genotypes (Hordeum vulgare L.)

The occurrence of nitrogen isotope discrimination with absorption and assimilation of nitrate (NO₃^–) and ammonium (NH₄⁺) was investigated using two genotypes of barley, Hordeum vulgare L. cv. Steptoe and Az12 : Az70, the latter of which lacks the characterized nitrate reductase isozymes. Plants were grown under two situations: a closed system with limited nitrogen or an open system with unlimited nitrogen, to elucidate the conditions and processes that influence discrimination. There was no discrimination observed for Az12 : Az70 when supplied with limited nitrogen. Discrimination was observed for Steptoe seedlings at high external NO₃^– concentrations, but not with low NO₃^– when assimilation is probably rapid and complete. The same pattern was observed for Steptoe when NH₄⁺ was supplied; indicating that for both nitrogen forms discrimination is dependent upon the presence of the assimilatory enzyme and the external concentration. The implications of this study are that both internal (assimilatory enzyme distribution) and external (source concentration) factors may have a larger impact on tissue δ ¹⁵N than the form of nitrogen utilized. This suggests that tissue δ ¹⁵N may not always be a reliable indicator of a plant's integrated nitrogen nutrition.     

NITROGENOUS NUTRITION OF ALEXANDRIUM CATENELLA (DINOPHYCEAE) IN CULTURES AND IN THAU LAGOON,SOUTHERN FRANCE1

Alexandrium catenella (Whedon et Kofoid) Balech was isolated from Thau lagoon (northern Mediterranean) and its growth and uptake characteristics measured for nitrate, ammonium, and urea. Although affinity constants did not indicate a preference for ammonium over nitrate, there was a strong inhibition of nitrate uptake by ammonium when both nitrogen (N) sources were present. Nitrogen budgets during growth in cultures revealed major imbalances between decreases in dissolved N and increases in particulate N, indicating excretion of dissolved organic N during the early part of the growth phase and uptake during the later part. A quasi‐unialgal bloom in November 2001 (4×10⁶ cells·L^?1) allowed measurements of uptake of nitrate, nitrite, ammonium, and urea; net and gross growth rate of A. catenella; and grazing rates on this organism. The affinity constants indicate that it is not a strong competitor for the N nutrients tested when these are in low concentrations (<10 μgat N·L^?1), compared with other members of the phytoplankton community. Indirect evidence from cultures indicate that dissolved organic N compounds could be important in triggering those blooms. Finally, the strongly unbalanced growth observed in the field indicates that A. catenella exhibits a storage rather than a growth response to a nutrient pulse and is adapted to low frequency events such as the passage of frontal disturbances. The disappearance of A. catenella was due to grazing that balanced growth at the peak of the bloom.     

Effects of non-steady-state iron limitation on nitrogen assimilatory enzymes in the marine diatom thalassiosira weissflogii (BACILLARIOPHYCEAE)

Since the recognition of iron‐limited high nitrate (or nutrient) low chlorophyll (HNLC) regions of the ocean, low iron availability has been hypothesized to limit the assimilation of nitrate by diatoms. To determine the influence of non‐steady‐state iron availability on nitrogen assimilatory enzymes, cultures of Thalassiosira weissflogii (Grunow) Fryxell et Hasle were grown under iron‐limited and iron‐replete conditions using artificial seawater medium. Iron‐limited cultures suffered from decreased efficiency of PSII as indicated by the DCMU‐induced variable fluorescence signal (F_v/F_m). Under iron‐replete conditions, in vitro nitrate reductase (NR) activity was rate limiting to nitrogen assimilation and in vitro nitrite reductase (NiR) activity was 50‐fold higher. Under iron limitation, cultures excreted up to 100 fmol NO₂^?·cell^?1·d^?1 (about 10% of incorporated N) and NiR activities declined by 50‐fold while internal NO₂^? pools remained relatively constant. Activities of both NR and NiR remained in excess of nitrogen incorporation rates throughout iron‐limited growth. One possible explanation is that the supply of photosynthetically derived reductant to NiR may be responsible for the limitation of nitrogen assimilation at the NO₂^? reduction step. Urease activity showed no response to iron limitation. Carbon:nitrogen ratios were equivalent in both iron conditions, indicating that, relative to carbon, nitrogen was assimilated at similar rates whether iron was limiting growth or not. We hypothesize that, diatoms in HNLC regions are not deficient in their ability to assimilate nitrate when they are iron limited. Rather, it appears that diatoms are limited in their ability to process photons within the photosynthetic electron transport chain which results in nitrite reduction becoming the rate‐limiting step in nitrogenassimilation.     

The activities of two pathways of nitrate reduction in Rhodopseudomonas capsulata

1. The disappearance of nitrate from suspensions of intact, washed cells of Rhodopseudomonas capsulata strain N22DNAR⁺ was measured with an ion selective electrode. In samples taken from phototrophic cultures grown to late exponential phase, nitrate disappearance was partially inhibited by light but was not affected by the presence of ammonium. Nitrate disappearance from samples from low density cultures in the early exponential phase of growth was first inhibited and later stimulated by light. In these cells ammonium ions inhibited the light-dependent but not the dark disappearance of nitrate. It is concluded that cells in the early exponential phase of growth possess both an ammonium-sensitive, assimilatory pathway for nitrate reduction (NRI) and an ammonium-insensitive pathway for nitrate reduction (NRII) which is linked to respiratory electron flow and energy conservation. In cells harvested in late exponential phase only the respiratory pathway for pitrate reduction is detectable.
2. Nitrate reduction, as judged by the oxidation of reduced methyl viologen by anaerobic cell suspensions, was measured at high rates in those strains of R. capsulata (AD2, BK5, N22DNAR⁺) which are believed to possess NRII activity but not in those strains (Kbl, R3, N22) which only manifest the ammonium-sensitive NRI pathway. On this basis we have used nitrate-dependent oxidation of reduced methyl viologen as a diagnostic test for the nitrate reductase of NRII in cells harvested from cultures of R. capsulata strain AD2. The activity was readily detectable in cells from cultures grown aerobically in the dark with ammonium nitrate as source of nitrogen. When the oxygen supply to the culture was withdrawn, the level of methyl viologen-dependent nitrate reductase increased considerably and nitrite accumulated in the culture medium. Upon reconnecting the oxygen supply, methyl viologen-dependent nitrate reductase activity decreased and the reduction of nitrate to nitrite in the culture was inhibited. It is concluded that the respiratory nitrate reductase activity is regulated by the availability of electron transport pathways that are linked to the generation of a proton electrochemical gradient.

     

Nitrate assimilation in Candida nitratophila and other yeasts

Nitrate assimilation has been studied in four species of yeasts; Candida nitratophila, Candida utilis, Hansenula anomala and Rhodotorula glutinis. Ammonium-grown cultures of these organisms did not assimilate nitrate but acquired the capacity to do so after a 3 h period of nitrogenstarvation. Ammonium inhibited nitrate assimilation completely in nitrate-grown cultures of R. glutinis. With Candida spp. ammonium and nitrate were assimilated simultaneously but each was assimilated at a lower rate than when either was supplied alone. Nitrogen-starved cultures of C. nitratophila contained enough nitrate reductase activity to sustain high rates of nitrate assimilation. Results indicate that the high levels of nitrate reductase in nitrate-grown cultures of C. nitratophila do not limit nitrate assimilation. Nitrate assimilation appears to be limited by nitrate uptake and/or the supply of reducing equivalents for nitrate reduction in these cultures.     

Mutants of Azotobacter vinelandii altered in the regulation of nitrate assimilation

The two enzymes involved in the assimilatory pathway of nitrate in Azotobacter vinelandii are corregulated. Nitrate reductase and nitrite reductase are inducible by nitrate and nitrite. Ammonium represses induction by nitrate of both reductases. Repression by ammonium is higher in media containing 2-oxo-glutarate as carbon source than in media containing sucrose. Mutants in the gene ntrC lost nitrate and nitrite reductase simultaneously. Ten chlorate-resistant mutants with a new phenotype were isolated. In media without ammonium they had a normal phenotype, being sensitive to the toxic effect of chlorate. In media containing low ammonium concentrations they were resistant to chlorate. These mutants seem to be affected in the repression of nitrate and nitrite reductases by ammonium.     

Methylammonium-resistant mutants ofNicotiana plumbaginifolia are affected in nitrate transport

This work reports the isolation and preliminary characterization ofNicotiana plumbaginifolia mutants resistant to methylammonium.Nicotiana plumbaginifolia plants cannot grow on low levels of nitrate in the presence of methylammonium. Methylammonium is not used as a nitrogen source, although it can be efficiently taken up byNicotiana plumbaginifolia cells and converted into methylglutamine, an analog of glutamine. Glutamine is known to repress the expression of the enzymes that mediate the first two steps in the nitrate assimilatory pathway, nitrate reductase (NR) and nitrite reductase (NiR). Methylammonium has therefore been used, in combination with low concentrations of nitrate, as a selective agent in order to screen for mutants in which the nitrate pathway is de-repressed. Eleven semi-dominant mutants, all belonging to the same complementation group, were identified. The mutant showing the highest resistance to methylammonium was not affected either in the utilization of ammonium, accumulation of methylammonium or in glutamine synthase activity. A series of experiments showed that utilization of nitrite by the wild-type and the mutant was comparable, in the presence or the absence of methylammonium, thus suggesting that the mutation specifically affected nitrate transport or reduction. Although NR mRNA levels were less repressed by methylammonium treatment of the wild-type than the mutant, NR activities of the mutant remained comparable with or without methylammonium, leading to the hypothesis that modified expression of NR is probably not responsible for resistance to methylammonium. Methylammonium inhibited nitrate uptake in the wild-type but had only a limited effect in the mutant. The implications of these results are discussed.     

Nitrate reductase and glutamate dehydrogenase activities in Skeletonema costatum as measures of nitrogen assimilation rates

Nitrate reductase (NADH-NR) and glutamate dehydrogenase (NADPH-GDH)activities were measured in Skeletonema costatum (Grev.) Clevein ammonium and nitrate limited continuous cultures before andafter additions of nitrate and/or ammonium. Comparisons of enzymicactivity with nitrogen uptake and assimilation rates, externaland internal nitrate concentrations, and external ammonium concentrationswere made in order to assess the roles of NR and GDH in nitrogenassimilation and to determine their suitability as measuresof nitrogen assimilation rates. NR activity appeared to be inducedby internal rather than external nitrate concentrations. Ammoniumin the medium reduced NR activity under some environmental conditions,but not others. However, ammonium acted indirectly, perhapsby causing the accumulation of an internal pool of an intermediateof ammonium assimilation. NR activity was found to approximatenitrate assimilation rates during growth limited by the nitratesupply and undeT some conditions in the presence of high nitrateand ammonium concentrations in the medium. Under other environmentalconditions, NR activity did not agree with nitrate assimilationrates; a second nitrate reducing mechanism may operate whenthese conditions prevail. GDH activities were consistently low,representing less than 5% of the ammonium uptake and assimilationrates. Consequently, it is proposed that ODH is not the primaryammonium assimilating enzyme under most environmental conditionsand cannot be used as a measure of ammonium assimilation. ¹ Contribution number 1095 from the Department of Oceanography,University of Washington