The mechanisms of nitrogen assimilation in Pseudomonads

Pseudomonas aeruginosa, Ps. fluorescens and 3 marine psychrophylic pseudomonads were grown in chemostat cultures with nitrate ammonia or glutamate as nitrogen source. In cultures grown on nitrate (either carbon- or nitrogen-limited) and in ammonia nitrogen-limited cultures ammonia was assimilated via the GS/GOGAT pathway. With a excess of ammonia in the culture however ammonia was assimilated via GDH and GS was either present only at low levels or absent. Two distinct GDH activities were detected in all 5 bacteria, one specific to NAD and one to NADP. The presence of these activities was determined by the environment in which cells were grown. These activities showed differences with respect to substrate affinity (Km values) for ammonia, incubation temperature and to a lesser extent pH and may involve separate GDH isoenzymes. GS from the marine bacterium PL₁ had a very high affinity for ammonia (Km of 0.3mm) but a low affinity for glutamate (Km of 19mm).     

AMMONIA PRODUCTION IN UREA-GROWN CULTURES OF CHLORELLA ELLIPSOIDEA12

Production of ammonia by urea-grown Chlorella ellipsoidea was investigated. Ammonia was produced during the stationary growth phase in cultures with urea as sole nitrogen source and glucose as supplementary carbon source. Ammonia was produced only in medium containing excess urea and limiting amounts of glucose. Ammonia production was accompanied by increase in pH. In cultures with nitrate as sole nitrogen source and glucose as supplementary carbon source, growth and pH changes were similar to those in urea-glucose medium, but no ammonia was detected. Cultures grown in urea-acetate medium were similar to those grown in urea medium without additional organic carbon source. No ammonia was produced under these circumstances and growth was significantly lower than that achieved in glucose-supplemented cultures. C. ellipsoidea evidently produces an enzyme or enzyme system which forms ammonia from urea. This organism was reportedly urease-free because previous workers did not detect ammonia formation from urea. Our findings indicate that special circumstances are required to produce detectable amounts of ammonia from urea. These findings are in agreement with a recent report of urea-splitting, cofactor-requiring enzyme in cell-free extracts of Chlorella.     

Nitrogen metabolism in plant cell suspension cultures: I. Effect of amino acids on growth

Certain amino acids inhibit growth of tobacco (Nicotiana tabacum L. var. xanthi), tomato (Lycopersicon esculentum) carrot (Daucus carota), and soybean (Glycerine max L. co. Mandarin) cell cultures when nitrate or urea are the nitrogen sources but not when ammonia is the nitrogen source. These amino acids also inhibit development of nitrate reductase activity (NADH:nitrate oxidoreductase EC 1.6.6.1) in tobacco and tomato cultures. Threonine, the most inhibitory amino acid, also inhibits nitrate uptake in tobacco cells. Arginine, and some other amino acids, abolish the inhibition effects caused by other amino acids. We suggest that amino acids inhibit assimilation of intracellular ammonium into amino acids in cells grown on nitrate or urea.     

A comparison of growth rate of algae as influenced by variation in nitrogen nutrition inChlorella pyrenoidosa andScenedesmus obliquus

Algae were cultivated in nutrient solutions containing nitrates or ammonium salts as the nitrogen source. We measured the culture density in the case of autotrophic and mixotrophic cultivation at different pH values, in the presence of nitrate also at different concentrations and in the absence of molybdenum. The dry weight and amount of nitrogen in the cells under these conditions were determined. According to results obtained we conclude thatChlorella, in contrast toScenedesmus, prefers ammonia as the nitrogen source in the first growth period, later both prefer nitrogen from nitrates. Nitrogen from ammonium salts has a positive effect only during short-time cultivation. If glucose was used as a mixotrophic energy source (or perhaps H-donor), the unsuitability of ammonia as a nitrogen source was more marked. We suppose that the lower growth intensity of older cultures (i.e., after seven days) in ammonia medium is the result of the use of endogenous energy sources. Both algae have adaptive systems making it possible to use nitrate nitrogen even in the absence of molybdate.     

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.     

Aspects of inorganic nitrogen assimilation in yeasts

Cultures of Candida utilis utilise glutamate in preference to ammonia and ammonia in preference to nitrate. The nitrate reductase of this organism is induced by nitrate and repressed in cultures grown on glutamate or ammonia. Nitrate-grown cultures of C. utilis, irrespective of the medium nitrate concentration, behave as though nitrogen-limited. In contrast to C. utilis, Saccharomyces cerevisiae utilises ammonia in preference to glutamate. In eight yeasts studied the highest cellular contents of biosynthetic NADP-linked glutamate dehydrogenase were found in batch cultures containing low concentrations of ammonia or in nitrogen-limited chemostat cultures. NAD-linked glutamate dehydrogenase activity was detected in extracts of cells grown in the presence of glutamate but not in those grown in the presence of ammonia.     

Nitrogen Starvation and the Regulation of Glutamine Synthetase in Agmenellum quadruplicatum

The level of glutamine synthetase activity in Agmenellum quadruplicatum strain PR-6 was dependent on the nitrogen source used for growth and on the nutritional status of the cells. During exponential growth, glutamine synthetase activity was low in cells grown on ammonia, urea, or nitrate. During the transition from nitrogen replete to nitrogen starved growth, glutamine synthetase activity began to rise. With ammonia as a nitrogen source, glutamine synthetase activity as determined in whole cells increased from 1 nanomole per minute per milliliter during exponential growth to 22 nanomoles per minute per milliliter during severe nitrogen starvation. In cells grown on nitrate the increase was from 5 to 39 nanomoles per minute per milliliter, and in cells grown on urea the increase was from 4 to 31 nanomoles per minute per milliliter.     

Nitrate reductase and glutamine synthetase activities,nitrate and ammonia assimilation,in the unicellular alga Cyanidium caldarium

Nitrogen-limited continuous cultures of Cyanidium caldarium contained induced levels of glutamine synthetase and nitrate reductase when either nitrate or ammonia was the sole nitrogen source. Nitrate reductase occurred in a catalytically active form. In the presence of excess ammonia, glutamine synthetase and nitrate reductase were repressed, the latter enzyme completely. In the presence of excess nitrate, intermediate levels of glutamine synthetase activity occurred. Nitrate reductase was derepressed but occurred up to 60% in a catalytically inactive form.Cell suspensions of C. caldarium from nitrate- or ammonialimited cultures assimilated either ammonia or nitrate immediately when provided with these nutrients. In these types of cells, as well as in cells grown with excess nitrate, the rate of ammonia assimilation was 2.5-fold higher than the rate of nitrate assimilation. It is proposed that the reduced rate at which nitrate was assimilated as compared to ammonia might be due to regulatory mechanisms which operate at the level of nitrate reductase activity.     

Bioconversion of dethiobiotin to biotin by a cell-free system of a bioYB transformant of Bacillus sphaericus

Abstract Potassium-limited cultures of Candida utilis grown with ammonium chloride as the sole nitrogen source attained a higher dry weight than similar cultures grown with sodium nitrate as the sole nitrogen source. This increase depended on the extracellular ammonia concentration and was not due to accumulation of storage polymers. We conclude that in this yeast ammonium ions can fulfill to some extent the physiological role of potassium ions and that the intracellular concentration of ammonium ions is predominantly determined by the ammonia concentration in the culture medium.     

Nitrogen Metabolism of Lemna minor. II. Enzymes of Nitrate Assimilation and Some Aspects of Their Regulation

In L. minor grown in sterile culture, the primary enzymes of nitrate assimilation, nitrate reductase (NR), nitrite reductase (NiR) and glutamate dehydrogenase (GDH) change in response to nitrogen source. NR and NiR levels are low when grown on amino acids (hydrolyzed casein) or ammonia; both enzymes are rapidly induced on addition of nitrate, while addition of nitrite induces NiR only. Ammonia represses the nitrate induced synthesis of both NR and NiR.NADH dependent GDH activity is low when grown on amino acids and high when grown on nitrate or ammonia, but the activities of NADPH dependent GDH and Alanine dehydro-genase (AIDH) are much less affected by nitrogen source. NADH-GDH and AIDH are induced by ammonia, and it is suggested that these enzymes are involved in primary nitrogen assimilation.     

Synthesis of Nitrate Reductase in Chlorella: II. EVIDENCE FOR SYNTHESIS IN AMMONIA-GROWN CELLS

Antiserum was prepared against nitrate reductase (EC 1.6.6.1) purified to homogeneity from Chlorella vulgaris Beijerinck. Both crude antiserum and anti-nitrate reductase antibodies prepared from it were used as re-agents to study the synthesis of nitrate reductase. Cell extracts from cultures which were grown with ammonia salts as the sole source of nitrogen contained almost no active enzyme. These extracts did contain material which binds to antibody and is thus immunologically related to purified nitrate reductase. The presence of this cross reacting material in cell extracts was detected by the ability of these extracts to (a) lower the titer of antisera; (b) form a biphasic precipitin curve with purified antibody; and (c) increase the peak height of a standard amount of purified nitrate reductase in rocket immunoelectrophoresis assay. These results suggest that ammonia-grown cells contain nitrate reductase precursor protein.     

Interaction of Mineral Deficiency and Nitrogen Source on Acid Phosphatase Activity in Leaf Extracts

Plants were grown in sand cultures to observe the effects ofmineral deficiencies, with different sources of nitrogen supply,on leaf acid phosphatase activity using phenolphthalein phosphateas substrate. Large increases ranging from 7 to 18 times normal (on freshweight or protein basis) occurred with deficiency of phosphoruswith nitrate, nitrite or ammonia nitrogen; increases from 4to 8 times normal occurred with deficiencies of zinc with nitriteor ammonia; and of copper with nitrite. Less pronounced increases,from 2 to 3 times normal on a protein basis, occurred with deficiencyof zinc with nitrate; of boron with all nitrogen sources; ofmanganese and of iron with nitrite. Activities on both proteinand fresh-weight basis were often greater with nitrite or ammoniumsulphate than with nitrate. Effects of phosphorus, zinc, copper,boron, and nitrogen sources were significant. Molybdenum deficiency caused approximately 2 times normal activityon a protein basis in tomato, tobacco, sunflower, cauliflower,lucerne, mustard, spinach beet, and lettuce when nitrate wasgiven. Effects with ammonia nitrogen were not consistent inmolybdenum-deficient plants but 50 per cent. increases occurredin the first 5 species listed. Activities varied widely betweenspecies. It was concluded that the increases in activity were causedby increases in amounts of the enzyme produced during growth,and that different combinations of deficiencies and nitrogensupplies produce different specific responses in terms of enzymesynthesis.     

The utilization of organic nitrogen for growth of algae: physiological aspects

The ability of 27 algae belonging to 11 taxonomic divisions to grow at the expense of organic nitrogen was tested in axenic culture. Experiments were carried out in buffered media: morpholine propanesulphonic acid (MOPS) was used successfully for all fresh water strains except diatoms. It was not used as a nitrogen source by any strain examined but served as a good source of sulphur for four strains. The range of substrates used was extensive and included amino acids, urea, acetamide, urate and some nucleosides. Growth rates varied widely but growth yields were generally comparable to those attained with nitrate or ammonia except for substrates containing more than one utilizable nitrogen atom. Limited experiments were carried out in the dark, and it was found that a given substrate was equally suitable for both dark heterotrophic and photolithotrophic growth. Levels of chlorophyll a were measured during growth of two algae with nitrate, glycine and urate. With nitrate, levels of specific chlorophyll a declined some ten-fold during growth: with poor substrates supporting only slow growth, levels were more uniform but were only 10–15% of those attained during growth with good substrates. One strain grown with MOPS as sole source of sulphur produced cultures with levels of chlorophyll a about half those found in sulphate-grown cultures. Tolerance of sodium chloride was examined in a few strains and even putatively fresh water strains were found to be appreciably tolerant.     

Effect of Nitrogen Source on Growth and Trichloroethylene Degradation by Methane-Oxidizing Bacteria

The effect of nitrogen source on methane-oxidizing bacteria with respect to cellular growth and trichloroethylene (TCE) degradation ability were examined. One mixed chemostat culture and two pure type II methane-oxidizing strains, Methylosinus trichosporium OB3b and strain CAC-2, which was isolated from the chemostat culture, were used in this study. All cultures were able to grow with each of three different nitrogen sources: ammonia, nitrate, and molecular nitrogen. Both M. trichosporium OB3b and strain CAC-2 showed slightly lower net cellular growth rates and cell yields but exhibited higher methane uptake rates, levels of poly-β-hydroxybutyrate (PHB) production, and naphthalene oxidation rates when grown under nitrogen-fixing conditions. The TCE-degrading ability of each culture was measured in terms of initial TCE oxidation rates and TCE transformation capacities (mass of TCE degraded/biomass inactivated), measured both with and without external energy sources. Higher initial TCE oxidation rates and TCE transformation capacities were observed in nitrogen-fixing mixed, M. trichosporium OB3b, and CAC-2 cultures than in nitrate- or ammonia-supplied cells. TCE transformation capacities were found to correlate with cellular PHB content in all three cultures. The results of this study suggest that the nitrogen-fixing capabilities of methane-oxidizing bacteria can be used to select for high-activity TCE degraders for the enhancement of bioremediation in fixed-nitrogen-limited environments.     

Glycolate metabolism is under nitrogen control in chlorella

The utilization of nitrate and ammonia as nitrogen sources had different effects on the metabolism of glycolate in Cholorella sorokiniana. During photolithotrophic growth with nitrate as nitrogen source, glycolate was metabolized via the glycine-serine pathway. Ammonia, produced as a result of glycolate metabolism, was reassimilated by glutamine synthetase. Two isoforms of this enzyme were present at different relative abundance in C. sorokiniana wild type and in a mutant with an increased capacity for the metabolism of glycolate (strain OR).

During photolithotrophic growth in the presence of ammonia as sole nitrogen source, several lines of evidence indicated that glycolate was metabolized to malate, pyruvate, tricarboxylic acid cycle intermediates and related amino acids in C. sorokiniana wild-type cells. Malate synthase was induced and glycine decarboxylase and serine-glyoxylate aminotransferase were repressed in cells grown with ammonia. An inverse correlation was observed between aminating NADPH-glutamate dehydrogenase and the in vivo glycine decarboxylation rate.

     

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.     

Regulation of nitrogen catabolic enzymes in Bacillus spp.

The levels of the inducible nitrogen catabolic enzymes arginase (L-arginine amidinohydrolase, EC 3.5.3.1) and alanine dehydrogenase (L-alanine:NAD+ oxidoreductase [deaminating], EC 1.4.1.1) from Bacillus licheniformis and histidase (L-histidine ammonia-lyase, EC 4.3.1.3) from Bacillus subtilis and the ammonia assimilatory enzymes from B. licheniformis were determined in cultures grown in the presence of different nitrogen sources. Although the levels of these enzymes were dependent upon the nitrogen source present, induction of the catabolic enzymes in response to the addition of inducer occurred even in the presence of preferred nitrogen sources. Intracellular pool sizes of ammonia, glutamate, glutamine, and alpha-ketoglutarate were measured in continuous cultures of b. licheniformis growing in the presence of different nitrogen sources. A comparison of the pool sizes of these metabolites with the ammonia assimilatory enzyme levels showed that the pools of the metabolites did not change in a manner consistent with their use as regulators of the synthesis of any of these enzymes.     

Altered nitrogenous pools induced by the azolla-anabaena azolla symbiosis

The free amino acid and ammonia pools of Azolla caroliniana were analyzed by quantitative column chromatography on columns capable of separating all of the nitrogenous constituents normally found in physiological fluids. Comparisons were made of plants containing symbiotic algae and grown on nitrogen-free media, plants grown on media containing nitrate, and algae-free plants also grown on nitrate media. The major feature of the data was a very high level of intracellular ammonia found in plants which contain N₂-fixing algal symbionts. In addition to the more usual amino acids, serine and cystathionine were found in the free amino acid pool.     

NITROGEN METABOLISM OF AQUATIC ORGANISMS. II. THE ASSIMILATION OF NITRATE,NITRITE, AND AMMONIA BY BIDDULPHIA AURITA1

Biddulphia aurita, a centric diatom, can grow on either nitrate, nitrite, or ammonia as its sole nitrogen, source. Cells remove ammonium nitrogen from the medium 2.3–2.4 times faster than either nitrate or nitrite nitrogen and, when grown for 24 hr in the ammonium medium, contain higher levels of non-protein nitrogen than cells grown in the nitrate or nitrite medium for the same period of time. The nitrogenous compounds in the nonprotein nitrogen fraction from cells grown in the nitrate, nitrite, or ammonium medium contain the same level of soluble-free amino nitrogen, combined amino nitrogen, and ammonium nitrogen. The high level of soluble nonprotein nitrogen in the medium of the cells grown in the ammonium medium is due to soluble amide nitrogen which represents 18% of the total soluble nitrogen present in these cells, whereas it represents only 2% in cells from the nitrite medium, and its level is negligible in cells from the nitrate medium. Cells grown in the nitrate medium have both nitrate- and nitrite-reductase activity. Cells grown in the nitrite medium have only nitrite-reductase activity in significant levels, while cells grown in the ammonium medium lack both enzymes.     

Linking phytoplankton community composition to seasonal changes in f-ratio

Seasonal changes in nitrogen assimilation have been studied in the western English Channel by sampling at approximately weekly intervals for 12 months. Nitrate concentrations showed strong seasonal variations. Available nitrogen in the winter was dominated by nitrate but this was close to limit of detection from May to September, after the spring phytoplankton bloom. The ¹⁵N uptake experiments showed that nitrate was the nitrogen source for the spring phytoplankton bloom but regenerated nitrogen supported phytoplankton productivity throughout the summer. The average annual f-ratio was 0.35, which demonstrated the importance of ammonia regeneration in this dynamic temperate region. Nitrogen uptake rate measurements were related to the phytoplankton responsible by assessing the relative abundance of nitrate reductase (NR) genes and the expression of NR among eukaryotic phytoplankton. Strong signals were detected from NR sequences that are not associated with known phylotypes or cultures. NR sequences from the diatom Phaeodactylum tricornutum were highly represented in gene abundance and expression, and were significantly correlated with f-ratio. The results demonstrate that analysis of functional genes provides additional information, and may be able to give better indications of which phytoplankton species are responsible for the observed seasonal changes in f-ratio than microscopic phytoplankton identification.