Glutamine metabolism during aerial mycelium growth of Neurospora crassa

During vegetative growth, glutamine is accumulated in the mycelium of Neurospora crassa. This high pool of glutamine seems to be required for aerial mycelium growth. Enzymes responsible for the synthesis and catabolism of glutamine were measured before and during the partial transformation of a mycelial mat into aerial mycelium. In the transforming mycelial mat,considerable activities of the biosynthetic NADP-glutamate dehydrogenase and glutamine synthetase (predominantly β polypeptide) and also some activity of glutamate synthase were observed. In the aerial mycelium, glutamine synthetase (predominantly β polypeptide) was detected, but very low activities of NADP-glutamate dehydrogenase and glutamate mycelium could derive from glutamine. No glutaminase activity could be detected. It is suggested that glutamate is formed through the activities of the glutamine transaminase-ω -amidase pathway and another transaminase. High activities of glutamine and alanine transaminases were observed in the aerial mycelium. These results are discussed in terms of the possible role of glutamine as a nitrogen carrier from the mycelium to the growing aerial hyphae.     

Amino acid pool composition of the basidiomycete Coprinus cinereus

The leaf-litter fungus Coprinus cinereus maintains a pool of free amino acid in its mycelium. When the organism is grown under conditions of high nitrogen availability with 13.2 mmol.L-1 L-asparagine as the nitrogen source, the primary constituents of this pool are glutamine, alanine, and glutamic acid. Together these 3 amino acids comprise approximately 70% of the pool. Nitrogen deprivation reduces the size of the free amino acid pool by 75%, and neither a high concentration of ammonium nor a protein nitrogen source support a similar pool size as L-asparagine. Nitrogen deprivation also reduces the concentration of glutamine to the pool while increasing glutamate. Concomitant with this shift is a marked increase in mycelial ammonium.     

Free amino acid pools of Trichoderma aureoviride during conditions of glucose-limited growth and glucose starvation

Glucose-limited and glucose-starved cultures of Trichoderma aureoviride were analyzed for the size and composition of the mycelial free amino acid pool. In glucoselimited mycelia the pool size increased as a function of the specific growth rate above a value of ca. 0.08 h^-1 and this was due principally to increasing concentrations of alanine and glutamic acid. During glucose starvation, the net pool size decreased only by ca 20% although a transient elevation of free amino acids was observed, the latter being attributed to the turnover of mycelial proteins. The amino acid pool compositions were categorized according to their ionic nature and, although no particular group varied significantly in its percentage contribution to the total pool size of growing mycelia, the observed variations during starvation were mostly attributable to the basic and acidic amino acids. Comparisons are made of the results with those obtained for other species of filamentous fungi and some possible explanations for the observed variations are discussed.     

Effect of 24-hour starvation on amino acid pool composition and enzyme activities of rat brown adipose tissue

The effect of 24-hr starvation on the amino acid pool composition and its concentration ratios with respect to blood and plasma as well as the activities of alanine, aspartate and branched chain amino acid transaminases, glutamate dehydrogenase, glutamine synthetase and adenylate deaminase have been studied in rat brown adipose tissue. Starvation induced a considerable decrease of pool amino acid concentration. Alanine and taurine were the amino acids in which the decrease was more marked. Small changes were observed in the activities of the enzymes studied, with decreases only in glutamate dehydrogenase and adenylate deaminase. These changes agree with a decrease in amino acid utilization in this tissue induced by starvation.     

Characterization of amino acid pools in the vacuolar compartment of Saccharomyces cerevisiae

Intact vacuoles are released from spheroplasts of Saccharomyces cerevisiae by means of a gentle mechanical disintegration method. They are purified by centrifugation in isotonic density gradients (flotation and subsequent sedimentation), and analyzed for their soluble amino acid content. The results indicate that about 60% of the total amino acid pool of spheroplasts is contained in the vacuoles. This may be an underestimate, as it presupposes no loss of amino acids from the vacuoles during the purification procedure. The amino acid concentration in the vecuoles is calculated to be approximately 5 times that in the cytoplasm if the total volumes of the two compartments are used for the calculation. The vacuolar amino acid pool is rich in basic amino acids, and in citrulline and glutamine, but contains a remarkably small amount of glutamate. Radioactive labeling experiments with spheroplasts indicate that the vacuolar amino acids are separated from the metabolically active pools located in the cytoplasm. This is particularly evident for the basic amino acids and glutamine; in contrast, the neutral amino acids and glutamate appear to exchange more rapidly between the cytoplasmic and the vacuolar compartments of the cells.     

Morphogenesis and free amino acid composition of the ascomycete Sphaerostilbe repens as influenced by nitrogen and calcium

Abstract Sphaerostilbe repens utilizes nitrate and ammonium as nitrogen sources. Differentiation of mycelium into rhizomorphs and coremia was reduced in the presence of nitrate and completely inhibited in the absence of calcium. The most abundant free amino acids were, in decreasing order: alanine, glutamine, glutatomic acid, serine, aspartic acid, γ-aminobutyric acid, arginine and threonine. These compounds represented 90% of the total amino acid pool.
The free amino acid composition did not vary with cultural conditions although concentrations of individual amino acids differed. In ammonium-grown cells, γ-aminobutyric acid increased in concentration and glutamate, aspartate and alanine decreased. Calcium-deficient media reduced amino acid concentrations, especially of arginine and ornithine. Amino acid contents increased during the growth period and were higher in rhizomorphs than in vegetative mycelia.     

Environmental factors affecting basic nitrogen metabolism and seasonal levels of various nitrogen fractions in tissues of bilberry, Vaccinium myrtillus

Bilberry tissues accumulated nitrogen for the winter in the form of reduced low-molecular weight amino compounds. The storage organ was principally the underground stem and the oldest parts of the aerial shoot. Most of the nitrogen was stored in arginine and ammonium compounds, and less in glutamine and other amino acids. Proteins did not accumulate during the winter. The soluble nitrogenous compounds were discharged from storage in May, when nitrogen was translocated from the lower parts of the stem to the growing leaves and buds. Amino acid compositions and concentrations in winter were almost identical under the snow and in snowless areas, only the concentration of glutamine being lower and that of glutathione higher in the snowless area. The level of total protein, particularly in the leaves and buds was much higher in a nitrogen-polluted industrial area than in unpolluted urban forests. The same difference was observed in total amino compounds, but among individual substances it only appeared in ammonium compounds. Certain species differences in the amino acid pool were recorded between V. myrtillus and V. vitis-idaea.     

Changes in Amino Acid Content and the Metabolism of γ-Aminobutyrate in Cucurbita moschata Seedlings

Ungerminated pumpkin (Cucurbita moschata Poir.) cotyledons contained 30 % of their dry weight as lipid and 26 % as protein, of which 93 % was globulin. There was a rapid degradation of these reserves 4 to 8 days after planting when the cotyledons had their maximum metabolic activity. About half of the mole percent of amino acids found in the globulin reserve was in arginine, glutamate, aspartate, and their amides. The cotyledons had a large soluble pool of arginine, glutamine, glutamate, and leucine. Most amino acids increased steadily in amount in the cotyledons during germination, except glutamine, ornithine, alanine, serine, glycine, and γ-aminobutyrate and these appeared in large amounts in the translocation stream to the axis tissue. Little arginine or proline was translocated. By 10 days, when translocation had decreased, amino acids accumulated. Ornithine, γ-aminobutyrate, and aspartate were rapidly utilized in the hypocotyl, while glutamine, glycine, and alanine accumulated there. Cysteine and methionine levels were low in the reserve, trans-location stream and soluble fractions. γ-Aminobutyrate-U^?14C injected into cotyledons or incubated with hypocotyls was utilized in a similar fashion. The label appeared in citric acid cycle acids and in the amino acids closely related to this cycle, but the bulk of the label appeared in CO₂. The labeling pattern suggests that γ-aminobutyrate was utilized via succinate, and thus entered the citric acid cycle. A close relationship between arginine, ornithine, glutamate, and γ-aminobutyrate exists in the cotyledon with all but arginine being translocated rapidly to the axis tissue where these amino acids are rapidly metabolized.     

Effect of low temperature on amino Acid metabolism in wintering poplar: arginine-glutamine relationships

Analyses of free amino acids in poplar (Populus gelrica) were carried out throughout a year to see the effect of low temperature on a system regulating amino acid metabolism in the tree. The results indicated that during the wintering phase arginine was the major amino acid both in bark and xylem, particularly in xylem, and that at the time of budding and growing glutamine and glutamate became dominant. Changes in the relative levels of glutamine (plus glutamate) and arginine to the total amino acids of the α-ketoglutarate family indicated the presence of a regulatory system annually controlling the synthesis between glutamine (plus glutamate) and arginine. The system appeared to be governed and sensitized by low temperatures. Neither a transition of the synthesis from arginine to glutamine (plus glutamate) nor budding occurred in the poplars which spent the winter months in a greenhouse.     

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.     

Influence of Carbon or Nitrogen Starvation on Amino Acid Transport in Pseudomonas aeruginosa

Pseudomonas aeruginosa was shown to utilize the majority of commonly occurring amino acids for growth as either the sole carbon or the sole nitrogen source. During carbon or nitrogen deprivation, the rates of transport of most of the amino acids remained unchanged; however, the transport rates for glutamate, alanine, and glycine increased under these conditions and the transport rates for leucine and valine decreased. Normal transport rates for these amino acids were resumed immediately upon the addition of the required nutrient. In the absence of an external source of carbon or of nitrogen, pool amino acids underwent rapid degradation. (14)C-Amino acid pulse experiments indicated that the constitutive amino acid catabolic enzymes, normally present in the organism during growth with glucose as the carbon source, were responsible for rapid pool losses. Nutrient starvation in the presence of chloramphenicol did not prevent amino acid catabolism. This enzymic activity is interpreted as providing P. aeruginosa with a selective advantage for survival during conditions of carbon or nitrogen starvation.     

Regulative influence of o-aminobenzoic acid on the biosynthesis of nourseothricin in cultures of Streptomyces noursei JA 3890b. III. Change of redox state of nicotinamide-adenine-dinucleotides in the presence of aminobenzoic acids.

o-Aminobenzoic acid (OABA, anthranilic acid) and related compounds which are known to stimulate the biosynthesis of streptothricin-type antibiotic nourseothricin by Streptomyces noursei JA 3890b were found to increase strongly the NADH/NAD+ ratio in growing mycelium of this strain suggesting that these effectors are capable of interfering with the function of the respiratory chain. In parallel, a complex shift of metabolism was induced shown by simultaneous alteration of mycelial activities of alanine dehydrogenase, glutamine synthetase, and glutamate dehydrogenase. These changes may be responsible for the observed delay of amino acid catabolism and may improve the precursor supply of the secondary metabolism.     

Aerial growth inNeurospora crassa: Characterization of an experimental model system

Liquid-grownNeurospora crassa mycelia, when filtered and exposed to the air, rapidly formed aerial hyphae that conidiated in a synchronous manner. The air-exposed mycelial mat could be separated into two different layers: an upper layer of adherent mycelia and a lower layer of loose mycelia. Adhesion of the hyphae occurred in less than 0.5 h and the hyphae were characteristically arranged in interconnecting bundles. This upper layer produced the aerial hyphae that could be seen after 3 h exposure to air. Aerial hyphae grew during the next 9 h, conidiophores were formed after about 9 h exposure to air, and at 12 h loose conidia were obtained. In both layers, profuse septation occurred during the first hours of incubation, vacuolation increased, and the number of nuclei diminished with time. Aerial hyphae were also septated and vacuolation was observed at their bases; numerous nuclei were seen in the aerial hyphae and conidiophores. The lower layer also contributed to the formation of aerial hyphae in an indirect manner. The lower layer was metabolically much less active than the upper layer with regard to incorporation of labeled amino acids into both hyphae and macromolecules. Translocation of label from the lower layer to the upper layer and to the aerial hyphae was detected. Ammonium, nitrate, and glutamine inhibited, whereas sucrose stimulated aerial growth.     

Regulation of ammonium uptake and metabolism by nitrogen fixing bacteria. III. Clostridium pasteurianum

Addition of ammonium salts to N₂ fixing continuous cultures of Clostridium pasteurianum caused immediate stop of nitrogenase synthesis, while the levels of glutamine synthetase, glutamate dehydrogenase and asparagine synthetase remained constant. No evidence for an interconversion of the glutamine synthetase was found. The activities of glutamate synthase in crude extracts were inversely related to the nitrogenase levels. The intracellular glutamine pool rapidly expanded during nitrogenase repression and decreased as fast during derepression while the pool sizes of all other amino acids were not strongly related to the rate of nitrogenase formation. These investigations suggest glutamine as corepressor of nitrogenase synthesis.     

EFFECT OF MALTOSE RELEASE ON UPTAKE AND ASSIMILATION OF AMMONIUM BY SYMBIOTIC CHLORELLA (CHLOROPHYTA)1

When incubated at pH 4–5, Chlorella freshly isolated from symbiosis with Hydra viridissima PALLAS 1766 (green hydra) release large amounts of photosynthetically fixed carbon in the form of maltose, and assimilation of inorganic N is inhibited. Physiological responses to N starvation of the cultured 3N813A strain of maltose-releasing Chlorella differed from those caused by 48 h of maltose release induced by low pH. N starvation increased rates of ammonium assimilation at pH 7.0 in light or darkness, and ammonium assimilation in darkness stimulated cell respiration. In contrast, cells pretreated at pH 5.0 to induce maltose release were unable to take up ammonium at pH 7.0 unless supplied with an external carbon source such as bicarbonate, acetate, or succinate, and rates of uptake were similar to control cells. Freshly isolated symbionts displayed a similar dependency. Rates of ammonium uptake by cells pretreated at pH 5.0 were reduced in darkness and did not stimulate cell respiration. N-starved cells supplied with ammonium also showed a large short-term increase in glutamine pools at the expense of glutamate, as might be expected if large amounts of ammonium were rapidly assimilated via glutamine synthetase/glutamate synthase, whereas after long-term maltose release cells showed only a small increase in glutamine when supplied with ammonium. Furthermore, maltose release caused a fall in pool sizes of a number of amino acids, including glutamine and glutamate, and also caused a decrease in pool sizes of 2-oxoglutarate and phospho-enol-pyruvate, which are required for ammonium assimilation into amino acids. Cells stimulated to synthesize and release maltose may be unable to assimilate ammonium and synthesize amino acids because of diversion of fixed carbon from N metabolism. We estimate that 40–50% affixed C is required for maximal maltose synthesis, whereas up to 30% fixed C is required for ammonium assimilation. These results are discussed in the context of host regulation of symbiotic algal growth.     

Biosynthesis of penicillin N and cephalosporin C: Antibiotic production and other features of the metabolism of a Cephalosporium sp

1. The production of penicillin N and cephalosporin C by two mutants of a Cephalosporium sp. has been studied with cultures grown in a chemically defined medium and with suspensions of washed mycelium in water or a buffered salt solution. 2. Antibiotic synthesis began at an early stage of growth and its rate per unit weight of mycelium appeared to pass its maximum as morphological changes were occurring in young hyphae. This rate subsequently declined, but rapid production could continue after net growth had ceased. 3. In a series of shake-flask fermentations in the growth medium, increases in the yield of penicillin N above the mean were correlated with much smaller increases in the yield of cephalosporin C and vice versa. 4. In suspensions of washed mycelium, moderate decreases in the efficiency of aeration increased the yield of penicillin N and decreased that of cephalosporin C. A similar result normally followed the addition of methionine to the suspension fluid, and in both cases there was usually an increase in the yield of the two antibiotics combined. 5. The apparent intracellular concentrations of the antibiotics were much lower than those attained extracellularly and also much lower than those of most of the amino acids in the intracellular pool. No detectable amount of [(14)C]penicillin N added to the extracellular fluid was found to enter the mycelium. 6. Very small amounts of peptide material whose behaviour was similar to that of the sulphonic acid of delta-(alpha-amino-adipoyl)cysteinylvaline on paper electrophoresis at pH1.8 were found in extracts of the mycelium that had been oxidized with performic acid. 6-Aminopenicillanic acid and 7-aminocephalosporanic acid were not detected. 7. Ultrasonic treatment of the mycelium resulted in rapid fragmentation of mycelial chains, rupture of many individual cells, and the liberation of amino acids and other substances into the medium. 8. Ultrasonically treated preparations synthesized penicillin N and cephalosporin C rapidly after a lag of 12hr. Antibiotic synthesis was accompanied by the growth of hyphae from swollen mycelial fragments and by the re-establishment of permeability barriers resulting in the uptake of amino acids from the medium.     

Amino Acid Pools in Herbaceous Plants at the Wintering Stage and at the Beginning of Growth

Free amino acids in 40 herbaceous perennial plants were analyzedunder natural conditions. From the major amino acid contentat the wintering stage, the pools were separated into the followingfive types: 1) a group which accumulated arginine (20 plantsout of 40); 2) a group which accumulated arginine and proline(9 plants); 3) a group which accumulated glutamate and glutamine(3 plants); 4) a group which accumulated asparagine (4 plants);and 5) a group which accumulated proline (4 plants). Changesin the amino acid pools in the plants occurred under snow duringwintering for about five months. Particularly, asparagine wasno longer the major amino acid in the group which had accumulatedit in fall. There was a tendency for the glutamine content toincrease, suggesting that NH₃ is utilized for the synthesisof the amide. Also, the relative concentrations of almost allthe free amino acids increased several-fold, which was indicativeof the occurrence of biosynthetic processes of general aminoacids during wintering. As the mobile fractions of stored nitrogen,the amino acids appeared to contribute to the initial stageof rapid growth in early spring. (Received August 4, 1986; Accepted November 17, 1986)     

Mobilization of vacuolar arginine in Neurospora crassa. Mechanism and role of glutamine

Nitrogen starvation has been shown to increase the cytosolic arginine concentration and to accelerate protein turnover in mycelia of Neurospora crassa. The cytosolic arginine is derived from a metabolically inactive vacuolar pool. Redistribution of arginine between cytosolic and vacuolar compartments is the result of mobilization of this metabolite in response to nitrogen starvation. Mobilization of arginine (and purines) also occurred in response to glutamine limitation, but arginine accumulated upon proline starvation. These observations indicate that mobilization is a consequence of glutamine limitation rather than a general response to amino acid starvation (or limitation). Analysis of the amino acid pools in mycelia subjected to starvation or limitation suggests that glutamine (or a metabolite derived from glutamine) provides a signal which determines the metabolic fate of vacuolar arginine. The results are consistent with the hypothesis that vacuolar compartmentation provides a readily available store of nitrogen-rich compounds to be utilized during differentiation or under conditions of nutritional stress.     

Anaerobic accumulation of amino acids in rice roots: role of the glutamine synthetase/glutamate synthase cycle

Summary. Accumulation of amino acids was studied in rice roots of 3-day-old seedlings subjected for 48 h to anaerobic conditions. Alanine and Gaba were the main amino acids accumulated under anoxia. Their synthesis was strongly inhibited by MSX and AZA, inhibitors of glutamine synthetase and glutamate synthase. These activities increased after 8 h of anaerobic treatment and, by immunoprecipitation of ³⁵S-labeled proteins, it was shown that glutamine synthetase and ferredoxin-dependent glutamate synthase were synthesized during the treatment. These findings indicate that the glutamine synthetase/glutamate synthase cycle play an important role in anaerobic amino acid accumulation. Received April 5, 1999     

Regulation of the ammonia assimilatory enzymes in Salmonella typhimurium.

The regulation of glutamate dehydrogenase (EC 1.4.1.4), glutamine synthetase (EC 6.3.1.2), and glutamate synthase (EC 2.6.1.53) was examined for cultures of Salmonella typhimurium grown with various nitrogen and amino acid sources. In contrast to the regulatory pattern observed in Klebsiella aerogenes, the glutamate dehydrogenase levels of S. typhimurium do not decrease when glutamine synthetase is derepressed during growth with limiting ammonia. Thus, it appears that the S. typhimurium glutamine synthetase does not regulate the synthesis of glutamate dehydrogenase as reported for K. aerogenes. The glutamate dehydrogenase activity does increase, however, during growth of a glutamate auxotroph with glutamate as a limiting amino acid source. The regulation of glutamate synthase levels is complex with the enzyme activity decreasing during growth with glutamate as a nitrogen source, and during growth of auxotrophs with either glutamine or glutamate as limiting amino acids.