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.     

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.     

Ammonium and amino acids as regulators of nitrate reductase in corn roots

When amino acids or ammonia are added to plant systems, the effects on the development of nitrate-dependent nitrate reductase activity are variable. In addition, amino acids added singly or as casein hydrolysate may not support a normal growth. A physiologically correct mixture of amino acids, one similar in composition to amino acids released by the endosperm, has been shown to support normal growth and protein synthesis in corn (Zea mays) embryos. In this investigation, we have used the mixture of corn amino acids to determine whether amino acids have an effect on the appearance or disappearance of nitrate reductase activity. The results show that these amino acids partially inhibit the induction of nitrate reductase in corn roots. The effect is more pronounced in mature root than in root tip sections. When glutamine and asparagine are included along with the "corn amino acid mixture," the inhibition is more severe. Amino acids or amino acid analogues added singly to the induction medium have a similar effect: i.e. when the induction of nitrate reductase is inhibited in the root tips (lysine, canavanine, azaserine, azetidine-2-carboxylic acid, dl-4-azaleucine, asparagine, and glutamine), that inhibition is more severe in mature root sections. Arginine enhanced the recovery of nitrate reductase in root tips but inhibited it in mature root sections. The effect of the amino acids is apparently on some phase of the induction processes (i.e. the uptake or distribution of nitrate or a direct effect on the synthesis of the enzyme) and not on the turnover of the enzyme.     

Amino acid limited growth of starter cultures in milk

The specific growth rates of several Streptococcus cremoris strains were 10–40% lower in milk than in other growth in media. The growth rates in milk increased when an amino acid mixture or casein was added, whereas, when milk was diluted, the specific growth rate of the streptococci decreased. This decrease could be overcome by bringing the casein concentration in the diluted milk back to the normal value (3%). This indicates that casein-hydrolysis proceeded at a rate too low for the streptococci to reach their potential maximum specific growth rates in milk so that growth in milk is essentially amino acid-limited. This was subsequently demonstrated for S. cremoris by continuous cultivation in media with low casein concentrations. At a low dilution rate casein hydrolysis was fast enough to supply the cells with enough amino acids and lactose was growth-limiting, whereas at higher dilution rates amino acids became growth-limiting. In cultures exponentially growing in milk the concentration of free amino acids was measured to determine which amino acid(s) was(were) absent and could possibly limit growth. A number of essential amino acids (leucine, methionine, glutamate and in some cases phenylalanine) were not detected and addition of these, together, stimulated the growth of S. cremoris in milk. The amino acids leucine and phenylalanine appeared to play a particularly important role in this stimulation. These two are, supposedly, the first amino acids that become limiting during growth in milk. The effect of competition for casein and amino acids by different organisms was studied in continuous cultures. At different dilution rates different strains became dominant in these mixed cultures, suggesting that differences in apparent affinity constants (K_S) for casein, leucine and glutamate existed between the strains.     

Nitrogen metabolite repression of nitrate reductase in Neurospora crassa.

The effect of different nitrogen compounds on the induction of reduced nicotinamide adenine dinucleotide phosphate-nitrate reductase was examined in Neurospora crassa. Whereas in the wild-type strain several amino acids and ammonia inhibit the formation of nitrate reductase, only glutamine, cysteine, and histidine are shown to inhibit the synthesis of nitrate reductase in a glutamine-requiring auxotroph. None of the amino acids inhibited nitrate reductase activity in vitro. The effects of cysteine and histidine are nonspecific, these amino acids being inhibitory of the growth of the organism. The effect of glutamine on the induction of nitrate reductase is not due to an inhibition of the uptake of the inducer nitrate. By the use of histidine-, pyrimidine-, and arginine-requiring auxotrophs, it was shown that glutamine appears to act per se and does not seem to be converted to another product in order to be effective in repression. The repression of nitrate reductase by ammonia appears, from the results described herein, to be indirect; ammonia has to be converted first to glutamine in order to be effective in repression.     

Nitrogen fixation byAlnus viridis (Chaix) DC.

Summary InAlnus viridis nodule growth relative to plant growth was inversely related to the quantity of nitrate added to nutrient solutions. Nodulated plants showed maximum growth when grown independently of supplied nitrogen and made better growth in its absence than unnodulated plants at any level of added nitrogen. Low levels of nitrate caused a depression of growth of nodulated plants, apparently by suppressing both nitrogen fixation and nodule growth. Nodules in nitrogen-free sand culture fixed atmospheric nitrogen at a rate of 6.6 mg/day/g nodule. Phosphorus deficiency was induced by low levels of phosphate and resulted in small plants with dark-green foliage. Root and nodule growth as a percentage of total plant growth and the percentage of total accumulated plant nitrogen below ground were greater at a root temperature of 11°C than 21°C. Thus at low root temperature processes other than nitrogen fixation were limiting to plant growth. Excised nodules were exposed to an N ₂ ¹⁵ -enriched atmosphere. A positive correlation between rate of nitrogen fixation and temperature was obtained, with optimum fixation occurring at about 20°C. It was shown that in spite of decreasing mean temperatures with increase in altitude, rate of nitrogen fixation by nodules of plants growing in the field increased with increase in altitude. This latter trend was deduced to be a reflection of the extent to which the field sites were nitrogen deficient in relation to climatically possible growth.     

The capability of the algaeChlorella vulgaris andScenedesmus obliquus of utilizing amino acids as the sole nitrogen source

The capability of utilizing 20 amino acids and 2 amides as the sole nitrogen source for growth was studied in two green algae (Chlorophyceae). A comparison was made of the growth rate of algae in a mineral nutrient solution containing nitrate as the nitrogen source, with that in the same solution in which nitrogen in the form of nitrate was substituted by an equivalent nitrogen amount in the form of various amino acids. In addition to this, another series of experiments was carried out in whioh both culture media were supplied with glucose. The results show that both algae utilize a series of amino acids in dependence of their structure (mostly 3-carbon amino acids). The growth rate ofChlorella in the presence of these sources is the same as in nitrate, that ofScenedesmus even much higher. In the cultures containing glucose both algal species exhibit a higher growth rate in the media with the nitrate nitrogen source than in those with amino acids (with the exception of glycine inScenedesmus).     

Assimilation of Nitrogen in Mutants Lacking Enzymes of the Glutamate Synthase Cycle

¹⁵N labelling was used to investigate the pathway of nitrogenassimilation in photorespiratory mutants of barley (Hordeumvulgare cv. Maris Mink), in which the leaves have low levelsof glutamine synthetase (GS) or glutamate synthase, key enzymesof ammonia assimilation. These plants grew normally when maintainedin high CO₂, but the deletions were lethal when photorespirationwas initiated by transfer to air. Enzyme levels in roots weremuch less affected, compared to leaves, and assimilation oflabelled nitrate into amino acids of the root showed very littledifference between wild type and mutants. Organic nitrogen wasexported from roots in the xylem sap mainly as glutamine, levelsof which were somewhat reduced in the GS-deficient mutant andenhanced in the glutamate synthase deficient mutant. In theleaf, the major effect was seen in the glutamatesynthase mutant,which had an extremely limited capacity to utilize the importedglutamine and amino acid synthesis was greatlyrestricted. Thiswas confirmed by the supply of [¹⁵N]-glutamine directly to leaves.Leaves of the GS-deficient mutant assimilatedammonia at about75% the rate found for the wild type, and this was almost completelyeliminated by addition of the inhibitormethionine sulphoximine.Root enzymes, together with residual levels of the deleted enzymesin the leaves, have sufficient capacityfor ammonia assimilation,through the glutamate synthase cycle, to provide adequate inputof nitrogen for normal growth of themutants, if photorespiratoryammonia production is suppressed. Key words: Hordeum vulgare, ¹⁵N, glutamine synthetase, glutamate synthase, ammonia assimilation     

Studies on utilization of 2-ketoglutarate,glutamate and other amino acids by the unicellular alga Cyanidium caldarium

Two strains of Cyanidium caldarium which possess different biochemical and nutritional characteristics were examined with respect to their ability to utilize amino acids or 2-ketoglutarate as substrates.One strain utilizes alanine, glutamate or aspartate as nitrogen sources, and glutamate, alanine or 2-ketoglutarate as carbon and energy sources for growth in the dark. The growth rate in the dark on 2-ketoglutarate is almost twice as high or higher than that on glutamate or alanine. During growth or incubation of this alga on amino acids, large amounts of ammonia are formed; however, ammonia formation is strongly inhibited by 2-ketoglutarate. The capacity of the alga to form ammonia from amino acids is inducible and develops fully only when the cells are grown or incubated in the presence of glutamate.By contrast, the other strain of Cyanidium caldarium cannot utilize alanine or aspartate as nitrogen sources. It utilizes glutamate only very poorly and does not excrete ammonia into the external medium. This strain is unable to utilize amino acids or 2-ketoglutarate as carbon and energy sources for heterotrophic growth.Cell-free extracts were tested for the occurrence of enzymes which could account for amino acid metabolism and ammonia formation.     

Nitrogen nutrition and regulation of cephalosporin production in Streptomyces clavuligerus.

When used as sole nitrogen source, certain amino acids (e.g., proline, asparagine) supported both growth and sporulation by Streptomyces clavuligerus streaked onto solid defined medium. Ammonium supported growth but suppressed sporulation. Amino nitrogen was best for cephalosporin production in liquid defined medium, although urea was almost as useful. A comparison of amino acids showed asparagine and glutamine to be the best nitrogen sources and arginine to be almost as good. Ammonium salts supported a somewhat lower growth rate than asparagine, but antibiotic production was very poor on these inorganic nitrogen sources. Addition of ammonium to asparagine did not affect growth rate but increased mycelial mass; cephalosporin production was reduced by about 75%. Antibiotic production was more closely associated with growth in the absence of ammonium than in its presence, indicating a strong inhibitory and (or) repressive effect of NH4+ on antibiotic production. Ammonium exerted its negative effect when added at 24h or earlier, i.e. before antibiotic formation began.     

Nitrogen utilization in bacterial isolates from the equine cecum.

A total of 114 bacterial isolates were obtained from the cecal contents of two mature cecally fistulated horses on a habitat-simulating medium containing 40% energy-depleted cecal fluid. Of these isolates, 108 were maintained in pure cultures and were tentatively grouped on the basis of cell morphology and physiological characteristics. Gram-negative rods (50.9%), gram-positive rods (22.8%), and gram-positive cocci (21.9%) represented the largest groups isolated from these animals. Fifty isolates were tested for their ability to grow in media containing urea, ammonia, peptones, or amino acids as sole nitrogen sources. None of the isolates had a unique requirement for urea or ammonia since nitrogen derived from peptones, amino acids, or both supported growth as well as did ammonia or urea in a low nitrogen medium. Of the cecal isolates, 18% were able to use urea for growth, and 20.5% were able to grow with ammonia as the sole nitrogen source. All organisms grew in the experimental media containing peptones as the sole nitrogen source. Urease activity was detected in only 2 of 114 isolates tested. The inability of isolates to use urea or ammonia as nitrogen sources may have been a reflection of growth conditions in the habitat-stimulating medium used for isolation, but it could also suggest that many cecal bacteria require nitrogen sources other then ammonia or urea for growth.     

Investigations on Growth and Metabolism of Plant Cells: VII. Sources of Nitrogen for Tissue Cultures under Optimal Conditions for Their Growth

Tissue cultures from explants of carrot root and potato tuber,stimulated into rapid growth by the addition of coconut milkorAesculus liquid endosperm to the medium, become to a certaindegree heterotrophic for nitrogen. Maximum growth-rates areattained only when nitrate is supplemented with various reducednitrogen compounds. The effects of casein hydrolysate, amino-acidmixtures, ammonia, tryptophan, urea, and allantoin have beeninvestigated, and their possible biochemical roles are discussed.     

Inhibition of nitrate influx by glutamine in Lolium perenne depends upon the contribution of the HATS to the total influx

Plants of Lolium perenne L. were grown in sterile solution culture supplied with 2 mol m(-3) nitrogen as either nitrate or ammonium. Glutamine at 5 mol m(-3) was added to the nutrient solution of half the plants for 24 h. Root nitrate influx (at external nitrate concentrations 0-2000 mmol m(-3)) and amino acid concentrations were determined. In a second experiment the concentration of the added glutamine was varied from 0-5 mol m(-3) and nitrate influx determined at 250 and 2000 mmol m(-3). The maximum rate of influx attributed to the high affinity transport system (HATS) was reduced by 66% by the presence of glutamine achieved through an 84% reduction in its constitutive component and a 59% reduction in its inducible component. Influx attributed to LATS was unaffected by the addition of glutamine. The inhibition of total nitrate influx by glutamine was positively related to the contribution of HATS to the total influx. In both nitrate- and ammonium-grown plants, the concentration of glutamine required to inhibit nitrate influx significantly was lower when influx was determined at 250 mmol m(-3) compared with 2000 mmol m(-3) nitrate. The addition of glutamine increased its concentrations in root tissue. However, the results cannot be attributed to changes in glutamine alone as its addition also resulted in increased concentrations of other amino acids. Implications for plants growing under field conditions are discussed.     

The utilization of nitrogen for growth in mice fed blends of purified proteins

Reasons for differences in growth of weanling mice fed different blends of purified proteins in diets apparently adequate in indispensable amino acids were investigated. Dietary nitrogen provided at increasing levels from a mixture of casein, gelatin, and zein, or from these plus gliadin and lactalbumin resulted in similar weight gains and efficiency of feed utilization in weanling mice. These responses were always lower (P less than 0.05) than for mice fed the control diet. When diets were based on true digestible nitrogen and amino acids, weight gains and feed efficiency decreased (P less than 0.05) as the dietary nitrogen level increased. High plasma glycine concentrations and high phenylalanine to tyrosine ratios were associated with high dietary glycine levels provided by gelatin. When a mixture of constant proportions of protein sources, predominantly casein, supplied most of the dietary nitrogen, small increases in weight gains and feed efficiency were observed as the nitrogen level in the diet increased. Varying the proportions of sources, which increased the percentage of gelatin, resulted in decreased gains in response to more dietary nitrogen. Replacing the amino acids found in gelatin by crystalline L-amino acids and replacing glycine by glutamic acid improved gains and feed efficiency (P less than 0.05) compared with gelatin-containing diets. These studies demonstrated that glycine at dietary concentrations of approximately 1-2%, either from gelatin or as the free amino acid, inhibited growth and feed utilization of growing mice.     

Derepression of asparaginase II during exponential growth of Saccharomyces cerevisiae on ammonium ion

The biosynthesis of asparaginase II in Saccharomyces cerevisiae is sensitive to nitrogen catabolite repression. In cell cultures growing in complete ammonia medium, asparaginase II synthesis is repressed in the early exponential phase but becomes derepressed in the midexponential phase. When amino acids such as glutamine or asparagine replace ammonium ion in the growth medium, the enzyme remains repressed into the late exponential phase. The three nitrogen compounds permit a similar rate of cell growth and are assimilated at nearly the same rate. In the early exponential phase the internal amino acid pool is larger in cells growing with glutamine or asparagine than in cells growing with ammonium sulfate as the sole source of nitrogen.     

Effect of specific amino acids on growth and aflatoxin production by Aspergillus parasiticus and Aspergillus flavus in defined media.

Four amino acids were used as sole nitrogen sources or as supplements to ammonium sulfate, and casein and ammonium sulfate were used as sole nitrogen sources to examine their effects on aflatoxin production by Aspergillus parasiticus NRRL 2999 and Aspergillus flavus 3357 grown on synthetic liquid media. In general, when proline, asparagine, casein, and ammonium sulfate were used as sole nitrogen sources, they supported more growth and toxin production than tryptophan or methionine. However, proline stimulated more toxin production per gram of mycelium in stationary cultures than the other nitrogen sources, including the amino acid asparagine, which is generally recognized as supporting good aflatoxin production. The exact responses to individual nitrogen sources were influenced by the species of fungus and whether cultures were stationary or shaken. In shake cultures, but not in stationary cultures, increased growth was generally associated with increased toxin production.     

Nitrogen utilization in bacterial isolates from the equine cecum

A total of 114 bacterial isolates were obtained from the cecal contents of two mature cecally fistulated horses on a habitat-simulating medium containing 40% energy-depleted cecal fluid. Of these isolates, 108 were maintained in pure cultures and were tentatively grouped on the basis of cell morphology and physiological characteristics. Gram-negative rods (50.9%), gram-positive rods (22.8%), and gram-positive cocci (21.9%) represented the largest groups isolated from these animals. Fifty isolates were tested for their ability to grow in media containing urea, ammonia, peptones, or amino acids as sole nitrogen sources. None of the isolates had a unique requirement for urea or ammonia since nitrogen derived from peptones, amino acids, or both supported growth as well as did ammonia or urea in a low nitrogen medium. Of the cecal isolates, 18% were able to use urea for growth, and 20.5% were able to grow with ammonia as the sole nitrogen source. All organisms grew in the experimental media containing peptones as the sole nitrogen source. Urease activity was detected in only 2 of 114 isolates tested. The inability of isolates to use urea or ammonia as nitrogen sources may have been a reflection of growth conditions in the habitat-stimulating medium used for isolation, but it could also suggest that many cecal bacteria require nitrogen sources other then ammonia or urea for growth.     

De Novo Synthesis of Amino Acids by the Ruminal Bacteria Prevotella bryantii B14, Selenomonas ruminantium HD4, and Streptococcus bovis ES1

The influence of peptides and amino acids on ammonia assimilation and de novo synthesis of amino acids by three predominant noncellulolytic species of ruminal bacteria, Prevotella bryantii B₁4, Selenomonas ruminantium HD4, and Streptococcus bovis ES1, was determined by growing these bacteria in media containing ¹⁵NH₄Cl and various additions of pancreatic hydrolysates of casein (peptides) or amino acids. The proportion of cell N and amino acids formed de novo decreased as the concentration of peptides increased. At high concentrations of peptides (10 and 30 g/liter), the incorporation of ammonia accounted for less than 0.16 of bacterial amino acid N and less than 0.30 of total N. At 1 g/liter, which is more similar to peptide concentrations found in the rumen, 0.68, 0.87, and 0.46 of bacterial amino acid N and 0.83, 0.89, and 0.64 of total N were derived from ammonia by P. bryantii, S. ruminantium, and S. bovis, respectively. Concentration-dependent responses were also obtained with amino acids. No individual amino acid was exhausted in any incubation medium. For cultures of P. bryantii, peptides were incorporated and stimulated growth more effectively than amino acids, while cultures of the other species showed no preference for peptides or amino acids. Apparent growth yields increased by between 8 and 57%, depending on the species, when 1 g of peptides or amino acids per liter was added to the medium. Proline synthesis was greatly decreased when peptides or amino acids were added to the medium, while glutamate and aspartate were enriched to a greater extent than other amino acids under all conditions. Thus, the proportion of bacterial protein formed de novo in noncellulolytic ruminal bacteria varies according to species and the form and identity of the amino acid and in a concentration-dependent manner.     

Analysis of regulatory factors for urea synthesis by isolated perfused rat liver. II. Comparison of urea synthesis in livers of rats subjected to different dietary conditions.

Capacities for urea synthesis and amino acid patterns in the perfused livers isolated from rats fed low and high-protein diets were compared. Urea formation with amjonium chlorode as the nitrogen source in perfused livers isolated from rats fed on a 70% casein diet was rapid and the efficiency of conversion of ammonia to urea was 97.9%. However, that in livers isolated from rats fed on a 5% casein diet was much slower and the efficiency of conversion of ammonia to urea was only 36.1%. The ratios of the rate of urea formation from ammonium chloride to activity of ornithine transcarbamylase [EC 2.1.3.3.] in the perfused livers of rats fed on 5 and 70% casein diets were calculated. The ratio of the former condition was much lower than that of the latter. The ratios reached nearly the same level by the addition of ornithine and N-acetylglutamate, the addition of which to the perfusate caused marked elevation of the ratios in both cases. In the perfused livers from rats fed on a 5% casein diet a considerable portion of the ammonia added to the perfusate was fixed into an amino ro an amide group of amino acids such as alamin, aspartate, and glutamine. On the other hand, in the perfused livers from rats fed on a 70% casein diet most of the ammonia added was converted to urea. The regulation of urea synthesis and the relation between anabolism and catabolism of amino acids in rat livers subjected to different dietary conditions were compared.     

Effect of growth conditions on accumulation of internal nitrate,ammonium, amino acids,and protein in three marine diatoms

The capacity of marine phytoplankton to change their cellular content of nitrate, ammonium, amino acids, and protein in response to different growth conditions was systematically investigated. Cellular concentrations of these compounds were measured in N-starved, N-deficient, and N-sufficient Skeletonema costatum (Grev.) Cleve and in N-deficient Chaetoceros debilis Cleve and Thalassiosira gravida Cleve, both before and after the addition of a pulse of nitrogen.N-sufficient Skeletonema costatum contains high concentrations of protein, large persistent pools of amino acids, and, if it is growing on nitrate, sizeable amounts of nitrate. As it becomes N-starved, the total cellular nitrogen decreases, the internal nitrate and amino acids become entirely depleted, and the protein content is drastically reduced. After nitrogen additions to N-deficient and N-starved cultures, transient pools of unassimilated nitrogen form which can account for a large fraction of newly taken up nitrogen. The size and kind of pool which accumulates is determined by the preconditioning of the cells, the nitrogen compound which is added, and the species identity. The pools which form in S. costatum indicate that nitrate reduction is the slowest step in nitrogen assimilation, the synthesis of protein from amino acids is the next slowest, and the incorporation of ammonium into amino acid is the fastest. However, the rate limiting steps may vary between diatom species.For the first time, measurements of the variation in cellular nitrogen compounds over a wide range of environmental conditions reveal the ability of some phytoplankton to buffer the effects of a changing, and sometimes growth-limiting, nitrogen supply. They accomplish this by utilizing stored internal nitrogen for growth when the external supply is low and by quickly storing unassimilated nitrogen when the external supply is suddenly increased beyond their ability to immediately assimilate it. The accumulation of large pools of unassimilated nitrogen compounds can explain the often observed difference between nitrogen uptake rates and growth rates.