EFFECT OF NITROGEN SOURCE ON PLATYMONAS (CHLOROPHYTA) CELL COMPOSITION AND AMINO ACID UPTAKE RATES1

The effect of nitrogen source (nitrate, ammonia and/or amino acids) on cell composition and amino acid uptake rates was examined. Substantial levels of free amino acids accumulated intracellularly with all nitrogen sources used. Ammonia accumulated only when provided in the medium. The presence of ammonia in the medium decreased the intracellular accumulation of free amino acids, especially arginine. Amino acid uptake rates were suppressed by the presence of excess nitrogen, especially ammonia. However, the suppression of uptake did not show any particular relation to the nitrogenous cell composition.     

PERIODIC CHANGES IN RATE OF AMINO ACID UPTAKE DURING YEAST CELL CYCLE

Uptake of amino acids is a complex process but in cells growing with ammonia as sole nitrogen source the initial uptake rate of amino acids is a measure of the transport capacity of the uptake system (permease). In synchronous cultures of Saccharomyces cerevisiae amino acids were transported at all stages of the cell cycle. However, for any one amino acid the initial uptake rate was constant for most of the cycle and doubled during a discrete part of the cycle. Thus, for a variety of amino acids the functioning amino acid transport capacity of the membrane doubles once per cycle at a characteristic stage of the cycle. Arginine, valine, and phenylalanine exhibit periodic doubling of uptake rate at different stages of the cell cycle indicating that the transport of these amino acids is mediated by three different systems. Serine, phenylalanine, and leucine exhibit periodic doubling of the uptake rate at the same stage of the cycle. However, it is unlikely that serine and phenylalanine share the same transport system since the uptake of one is not inhibited by the other amino acid. This phenomenon is analogous to the periodic synthesis of soluble enzymes observed in S. cerevisiae.     

Formation of the intermediate products of the tricarboxylic acid cycle and ammonia from free amino acids in anoxic heart muscle

Glutamate and aspartate showed the highest rate of catabolism in oxygenated isolated rat heart with the formation of glutamine, asparagine and alanine. Under anoxia, the catabolism of branch chained amino acids and that of lysine, proline, arginine and methionine was inhibited. However, glutamate and aspartate catabolized at a higher rate as compared with oxygenation. Alanine was the product of their excessive degradation. During oxygenation, 70% of ammonia were produced via deamination of amino acids. Under anaerobic conditions the participation of amino acids in ammoniagenesis decreased to 4%; the principal source of ammonia was the adenine nucleotide pool. The total pool of the tricarboxylic acid cycle intermediates increased 2.5-fold due to accumulation of succinate. The data obtained suggest that the constant influx of intermediates into the cycle from amino acids is supported by coupled transamination of glutamate and aspartate. This leads to the formation of ATP and GTP in the tricarboxylic acid cycle during blocking of aerobic energy production.     

Regulation of exocellular protease in Neurospora crassa: induction and repression under conditions of nitrogen starvation.

Exponential-phase cells of Neurospora crassa require the continued presence of a protein inducer and nitrogen starvation to induce exocellular protease under conditions where protein is the sole nitrogen source. The nature of the protein inducer appears relatively unimportant, since both soluble proteins (e.g., myoglobin) and insoluble proteins (e.g., corn zein) will effect induction. Nonstarved cells of N. crassa appear to have small nitrogen pools, since nitrogen starvation of exponential cells prior to transfer into a medium where protein is the sole nitrogen source effects starvation-time-dependent decreases in protease biosynthesis. Ammonium ion represses protease synthesis, with apparent specificity at low concentrations. The amino acids arginine, tryptophan, and threonine effect repression of protease biosynthesis under conditions of nitrogen starvation. Under conditions of sulfur starvation, the amino acids cysteine, methionine, and cystine repress protease biosynthesis. In carbon-starved cells, all of the above amino acids, plus histidine, isoleucine, leucine, lysine, phenylalanine, and valine, effect repression. Examination of amino acid pools formed when cells are grown on protein as the sole nitrogen source demonstrated that the amino acids which repress protease biosynthesis under conditions where protein is the sole carbon source accumulate in significant amounts during the course of protease induction, with kinetics consonant with the induction process.     

Nickel deficiency disrupts metabolism of ureides, amino acids, and organic acids of young pecan foliage

The existence of nickel (Ni) deficiency is becoming increasingly apparent in crops, especially for ureide-transporting woody perennials, but its physiological role is poorly understood. We evaluated the concentrations of ureides, amino acids, and organic acids in photosynthetic foliar tissue from Ni-sufficient (Ni-S) versus Ni-deficient (Ni-D) pecan (Carya illinoinensis [Wangenh.] K. Koch). Foliage of Ni-D pecan seedlings exhibited metabolic disruption of nitrogen metabolism via ureide catabolism, amino acid metabolism, and ornithine cycle intermediates. Disruption of ureide catabolism in Ni-D foliage resulted in accumulation of xanthine, allantoic acid, ureidoglycolate, and citrulline, but total ureides, urea concentration, and urease activity were reduced. Disruption of amino acid metabolism in Ni-D foliage resulted in accumulation of glycine, valine, isoleucine, tyrosine, tryptophan, arginine, and total free amino acids, and lower concentrations of histidine and glutamic acid. Ni deficiency also disrupted the citric acid cycle, the second stage of respiration, where Ni-D foliage contained very low levels of citrate compared to Ni-S foliage. Disruption of carbon metabolism was also via accumulation of lactic and oxalic acids. The results indicate that mouse-ear, a key morphological symptom, is likely linked to the toxic accumulation of oxalic and lactic acids in the rapidly growing tips and margins of leaflets. Our results support the role of Ni as an essential plant nutrient element. The magnitude of metabolic disruption exhibited in Ni-D pecan is evidence of the existence of unidentified physiological roles for Ni in pecan.     

Nitrogen limitation and amino-acid metabolism of Chlorella symbiotic with green hydra

Chlorella algae symbiotic in the digestive cells of Hydra viridissima Pallas (green hydra) were found to contain less amino-N and smaller pools of free amino acids than their cultured counterparts, indicating that growth in symbiosis was nitrogen-limiting. This difference was reflected in uptake of amino acids and subsequent incorporation into protein; symbiotic algae incorporated a greater proportion of sequestered radioactivity, supplied as ¹⁴C-labelled alanine, glycine or arginine, than algae from nitrogen-sufficient culture, presumably because smaller internal pools diluted sequestered amino acids to a lesser extent. Further experiments with symbiotic algae showed that metabolism of the neutral amino acid alanine differed from that of the basic amino acid arginine. Alanine but not arginine continued to be incorporated into protein after uptake ceased, and while internal pools of alanine were exchangeable with alanine in the medium, those of arginine were not exchangeable with external arginine. Thin-layer chromatography of ethanol-soluble extracts of algae incubated with [¹⁴C]alanine or [¹⁴C]arginine showed that both were precursors of other amino acids. The significance of nitrogen-limiting growth of symbiotic algae is discussed in terms of host-cell regulation of algal cell growth and division.     

Growth reduction of<Emphasis Type="Italic"> Sphagnum magellanicum</Emphasis> subjected to high nitrogen deposition: the role of amino acid nitrogen concentration

We tested the relationship between Sphagnum growth and the amount of nitrogen stored in free amino acids in a fertilisation experiment with intact peat monoliths in an open greenhouse in The Netherlands. Three nitrogen deposition scenarios were used: no nitrogen deposition, field conditions and a doubling of the latter, corresponding to 0, 40 and 80 kg N ha(-1 )year(-1). Growth of Sphagnum as expressed by height increment was reduced in the 80 kg N treatment, but showed no correlation with the total nitrogen tissue concentration or with the concentration of individual or pooled free amino acids. The amount of nitrogen stored in free amino acids increased concomitantly with deposition, although it lagged more and more behind the total nitrogen concentration, the latter pointing to the accumulation of unmeasured nitrogen compounds. Asparagine clearly acted as the major storage compound for nitrogen in Sphagnum stem tissue, whereas arginine fulfilled this function to a lesser extent in the capitulum. It appears that nitrogen-induced growth inhibition of Sphagnum is related to acclimation rather than to certain threshold concentrations of amino nitrogen or total nitrogen. We propose that when Sphagnum is exposed to a step increase of nitrogen, its nitrogen metabolism does not adapt fast enough to keep up with the enhanced uptake rate. This imbalance between nitrogen uptake and assimilation may lead to an accumulation of toxic NH(4)(+ )in the cell and a subsequent reduction in growth.     

Amino acids as a nitrogen source for Chlorella symbiotic with green hydra

Supply of amino acids may be important in controlling cell division of Chlorella symbiotic with green hydra. Freshly isolated symbionts display characteristics of N-limited algae, and low pH in perialgal vacuoles and high levels of host glutamine synthetase (GS) limit uptake of ammonium. Movement of tritiated amino acids from host to algal pools suggests that symbiotic algae utilize amino acids derived from host digestion of prey. Amounts are significant in relation to host and algal amino acids pools. During host starvation, glutamine produced by host GS may be important as a nitrogen supply to the algae, which take up this amino acid at high rates at low pH.     

Free intracellular amino acid pools during autonomous oscillations in Saccharomyces cerevisiae

In the present work dynamic changes of free intracellular amino acid pools during autonomous oscillations of Saccharomyces cerevisiae were quantified in glucose-limited continuous cultivations. At a dilution rate of D = 0.22 h(-1) cyclic changes with a period of 120 min were found for many variables such as carbon dioxide production rate, dissolved oxygen, pH, biomass content, and various metabolite concentrations. On the basis of the observed dynamic patterns, free intracellular amino acids were classified to show oscillatory, stationary, or chaotic behavior. Amino acid pools such as serine, alanine, valine, leucine, or lysine were subjected to clear oscillations with a frequency of 120 min, identical to that of other described cultivation variables, indicating that there is a direct correlation between the periodic changes of amino acid concentrations and the metabolic oscillations on the cellular level. The oscillations of these amino acids were unequally phase-delayed and had different amplitudes of oscillation. Accordingly, they exhibited different patterns in phase plane plots vs. intracellular trehalose. Despite the complex and marked metabolic changes during oscillation, selected intracellular amino acids such as histidine, threonine, isoleucine, or arginine remained about constant. Concentrations of glutamate and glutamine showed a chaotic behavior. However, the ratio of glutamate to glutamine concentration was found to be oscillatory, with a period of 60 min and a corresponding figure eight-shaped pattern in a plot vs. trehalose concentration. Considering the described diversity, it can be concluded that the observed periodic changes are neither just the consequence of low or high rates of protein biosynthesis/degradation nor correlated to changing cell volumes during oscillation. The ratio between doubling time (189 min) and period of oscillation of intracellular amino acids (120 min) was 1:6. The fact that there is a close relationship between doubling time and period of oscillation underlines that the described autonomous oscillations are cell-cycle-associated.     

Nitrogen nutrition in the cyanobacterium Nostoc ANTH, a symbiotic isolate from Anthoceros: uptake and assimilation of inorganic-n and amino acids

Amino acid uptake and utilization of various nitrogen sources (amino acids, nitrite, nitrate and ammonia) were studied in Nostoc ANTH and i ts mu tant (Het(-)Nif(-)) isolate defective in heterocyst formation and N2-fixation. Both parent and its mutant grew at the expense of glutamine, asparagine and arginine as a source of fixed-nitrogen. Growth was better in glutamine-and asparagine-media as compared to that in arginine media. Glutamine and asparagine repressed heterocyst formation, N2-fixation and nitrate reduction in Nostoc ANTH, but arginine did so only partially. The poor growth in arginine-medium was not due to poor uptake rates, since the uptake rates were not significantly different from those for glutamine or asparagine. The glutamine synthetase activity remained unaffected during cultivation in media containing any one of the three amino acids tested. The uptake of amino acids was substrate-inducible, energy-dependent and required de novo protein synthesis. Nitrate and ammonium repressed ammonium uptake, but did not repress uptake of amino acids. In N2-medium (BG-11(0)), the uptake of ammonium and amino acids in the mutant was significantly higher than its parent strain. This was apparently due to nitrogen limitation since the mutant was unable to fix N2 and the growth medium lacked combined-N.     

Potassium Uptake in Synchronous and Synchronized Cultures of Escherichia coli

Criteria are presented for distinguishing between synchronous and synchronized cultures (natural vs. forced synchrony) on the basis of characteristics of growth and division during a single generation. These criteria were applied in an examination of the uptake of potassium during the cell growth and division cycle in synchronous cultures and in a synchronized culture of Escherichia coli. In the synchronous cultures the uptake of ⁴²K doubled synchronously with cell number, corresponding to a constant rate of uptake per cell throughout the cell cycle. In the synchronized culture, uptake rates also remained constant during most of the cycle, but rates doubled abruptly well within the cycle. This constancy of ⁴²K uptake per cell supports an earlier interpretation for steady-state cultures that uptake is limited in each cell by a constant number of functional sites for binding, transport, or accumulation of compounds from the growth medium, and that the average number of such sites doubles late in each cell cycle. The abrupt doubling of the rate of uptake of potassium per cell in the synchronized culture appears because of partial uncoupling of cell division from activation or synthesis of these uptake sites.     

Transgenic manipulation of a single polyamine in poplar cells affects the accumulation of all amino acids

The polyamine metabolic pathway is intricately connected to metabolism of several amino acids. While ornithine and arginine are direct precursors of putrescine, they themselves are synthesized from glutamate in multiple steps involving several enzymes. Additionally, glutamate is an amino group donor for several other amino acids and acts as a substrate for biosynthesis of proline and γ-aminobutyric acid, metabolites that play important roles in plant development and stress response. Suspension cultures of poplar (Populus nigra × maximowiczii), transformed with a constitutively expressing mouse ornithine decarboxylase gene, were used to study the effect of up-regulation of putrescine biosynthesis (and concomitantly its enhanced catabolism) on cellular contents of various protein and non-protein amino acids. It was observed that up-regulation of putrescine metabolism affected the steady state concentrations of most amino acids in the cells. While there was a decrease in the cellular contents of glutamine, glutamate, ornithine, arginine, histidine, serine, glycine, cysteine, phenylalanine, tryptophan, aspartate, lysine, leucine and methionine, an increase was seen in the contents of alanine, threonine, valine, isoleucine and γ-aminobutyric acid. An overall increase in percent cellular nitrogen and carbon content was also observed in high putrescine metabolizing cells compared to control cells. It is concluded that genetic manipulation of putrescine biosynthesis affecting ornithine consumption caused a major change in the entire ornithine biosynthetic pathway and had pleiotropic effects on other amino acids and total cellular carbon and nitrogen, as well. We suggest that ornithine plays a key role in regulating this pathway.     

Free amino acids and total nitrogen during shoot development in Scots pine seedlings

The concentration of free amino acids and total nitrogen was studied in needles, stems and roots of seedlings of Pinus sylvestris L. for five weeks during the second growth period ("summer"). In one group of seedlings the source/sink relation was disturbed through removal of the terminal buds. The seedlings were cultivated in artificial year-cycles in a climate chamber.
Total nitrogen increased in needles and sterns of intact seedlings in the beginning of the "summer" and decreased during shoot growth. In seedlings, from which the buds had been removed, nitrogen remained at high levels in the primary needles and accumulated in steins and roots. The results are consistent with utilization of nitrogen in older needles and in the stem during shoot elongation.
The pool of free amino acids increased in the beginning of the "summer" and decreased after bud break in primary needles, stems and roots. Arginine and glutamine, in the roots also asparagine, were the dominating amino acids (amides included). Together, these compounds (plus glutamate and aspartate) contributed about 90% of the nitrogen in the amino acid pool in all organs. In primary needles and in the stem, arginine predominated at the end of hardening (75–85% of the amino acid nitrogen). Free amino acids contributed at most ca 10% of the total nitrogen in primary needles, where the ratio of free amino acid nitrogen: total nitrogen was highest at the end of dormancy and in the early "summer". Free amino acids accumulated after bud removal in primary needles and especially in stems and roots. Glutamine became relatively more dominant than arginine in the different organs.
The observations are consistent with the role of arginine and glutamine for storage and transport of nitrogen in conifers. Because of the low concentrations of amino acid nitrogen in the primary needles, arginine is not considered a major nitrogen reserve in needles of Scots pine seedlings.     

Stable isotope peptide mass spectrometry to decipher amino acid metabolism in Dehalococcoides strain CBDB1

Dehalococcoides species are key players in the anaerobic transformation of halogenated solvents at contaminated sites. Here, we analyze isotopologue distributions in amino acid pools from peptides of Dehalococcoides strain CBDB1 after incubation with (13)C-labeled acetate or bicarbonate as a carbon source. The resulting data were interpreted with regard to genome annotations to identify amino acid biosynthesis pathways. In addition to using gas chromatography-mass spectrometry (GC-MS) for analyzing derivatized amino acids after protein hydrolysis, we introduce a second, much milder method, in which we directly analyze peptide masses after tryptic digest and peptide fragments by nano-liquid chromatography-electrospray ionization-tandem mass spectrometry (nano-LC-ESI-MS/MS). With this method, we identify isotope incorporation patterns for 17 proteinaceous amino acids, including proline, cysteine, lysine, and arginine, which escaped previous analyses in Dehalococcoides. Our results confirmed lysine biosynthesis via the α-aminoadipate pathway, precluding lysine formation from aspartate. Similarly, the isotopologue pattern obtained for arginine provided biochemical evidence of its synthesis from glutamate. Direct peptide MS/MS analysis of the labeling patterns of glutamine and asparagine, which were converted to glutamate and aspartate during protein hydrolysis, gave biochemical evidence of their precursors and confirmed glutamate biosynthesis via a Re-specific citrate synthase. By addition of unlabeled free amino acids to labeled cells, we show that in strain CBDB1 none of the 17 tested amino acids was incorporated into cell mass, indicating that they are all synthesized de novo. Our approach is widely applicable and provides a means to analyze amino acid metabolism by studying specific proteins even in mixed consortia.     

Nitrogen transport in the xylem sap of Quercus ilex: the role of ornithine

The storage and remobilization of nitrogen in deciduous and evergreen species is a major source of N, supporting the seasonal growth of trees. In evergreens, in addition to wood and roots, older leaves are important reservoirs of N used in the growth of new foliage. Just before bud burst, when transpiration is inactive or low, and when uptake of nitrogen by the roots may be restricted due to low temperatures, levels of organic N in the xylem are high. Amino acids usually comprise the bulk of this organic N. Changes in amino acid concentrations in early spring are thought to result mainly from hydrolysis of N reserves, and not from current N uptake. The seasonal profiles of amino acids in the xylem sap of Quercus ilex, an evergreen Mediterranean tree, were investigated. The first amino acid detected in the xylem sap before spring was ornithine, which may result from the breakdown of arginine present in storage proteins. Arginine is one of the main amino acids present in storage proteins because each arginine molecule has four nitrogen atoms. When protein degradation increases the free arginine pool, the arginase activity is enhanced and, consequently, the conversion of arginine to ornithine. It seems that ornithine has an important role in N transport early in the growth season of Q. ilex.     

Prediction of effects of amino acid supplementation on growth of E. coli B/r

A mathematical model for the growth of a single cell of E. coli on medium containing amino acid is presented. A mixture of purified amino acids (glutamate, aspartate, serine, tyrosine, and leucine) combined in the ratios found in a natural digest (casein) were employed as the nitrogen source. Each of these amino acids is the representative of a different family of amino acids. The transport mechanisms and assimilation routes for each amino acid were inserted into the prototype model. The enzyme activities and saturation constants used in the model were based on literature data. The maximum velocities for uptake systems were calculated from experimental data. The formation and homeostasis of amino acid pools were regulated through cross-control of the activities of biosynthetic enzymes and of membrane transport of exogenous nutrients. The size of each amino acid pool was determined with mass balance equations that included terms for a transport system, a biosynthesis system, a transaminase enzyme system for interchange between the amino acid families, and a consumption system. The predictions of the extended model with regard to nutrient concentrations and growth rates compared well with the experimental data.     

Neurospora crassa mutant impaired in glutamine regulation.

The final products of the catabolism of arginine that can be utilized as nitrogen sources by Neurospora crassa are ammonium, glutamic acid, and glutamine. Of these compounds, only glutamine represses arginase and glutamine synthetase. We report here the isolation and characterization of a mutant of N. crassa whose arginase, glutamine synthetase, and amino acid accumulations are resistant to glutamine repression (glnI). This mutant has a greater capacity than the wild type (glns) to accumulate most of the arginine and some of the glutamine in osmotically sensitive compartments while growing exponentially. Nonetheless, the major part of the glutamine remains soluble and metabolically available for repression. We propose that the lower repression of glutamine synthetase by glutamine in this mutant could be a necessary condition for sustaining the higher flow of nitrogen for the accumulation of amino acids observed in ammonium excess and that, if glutamine is the nitrogen signal that regulates the arginine accumulation of the vesicle, the glnr mutant has also escaped this control. Finally, in the glnr mutant, some glutamine resynthesis is necessary for arginine biosynthesis and accumulation.