Regulation of nitrogen utilization of hisT mutants of Salmonella typhimurium.

Mutations in the hisT gene of Salmonella typhimurium alter pseudouridine synthetase I, the enzyme that modifies two uridines in the anticodon loop of numerous transfer ribonucleic acid species. We have examined two strains carrying different hisT mutations for their ability to grow on a variety of nitrogen sources. The hisT mutants grew more rapidly than did hisT+ strains with either arginine or proline as the nitrogen source and glucose as the carbon source. The hisT mutations were transduced into new strains to show that these growth properties were due to the hisT mutations. The hisT mutations did not influence the growth of mutants having altered glutamine synthetase regulation. Assays of the three primary ammonia-assimilatory enzymes, glutamate dehydrogenase, glutamine synthetase, and glutamate synthase, showed that glutamate synthase activities were lower in hisT mutants than in isogenic hisT+ controls; however, the glutamate dehydrogenase activity was about threefold higher in the hisT strains grown in glucose-arginine medium. The results suggest that the controls for enzyme synthesis for nitrogen utilization respond either directly or indirectly to transfer ribonucleic acid species affected by the hisT mutation.     

Comparison of hup trait and intrinsic antibiotic resistance for assessing rhizobial competitiveness axenically and in soil

A defined medium for growth of 24 strains of Moraxella (Branhamella) catarrhalis was devised. This medium (medium B4) contains sodium lactate as a partial carbon source, proline as both a partial carbon source and a partial nitrogen source, aspartate as a partial nitrogen source, and the growth factors arginine, glycine, and methionine. Either aspartate, glutamate, or proline could serve as sole nitrogen source, but growth occurred at a significantly better rate if proline was present together with either aspartate or glutamate, or with both aspartate and glutamate. With the exception of strain ATCC 23246, all the strains had an absolute requirement for arginine and either a partial or absolute requirement for glycine. The concentration of glycine required for optimal growth was found to be relatively high for an amino acid growth factor. Heart infusion broth was found to be growth inhibitory for spontaneous mutants of one strain able to grow in the absence of arginine, and such mutants reverted readily to arginine dependence accompanied by the ability to grow faster on the complex medium. Growth rates in the defined medium B4 were enhanced by the simultaneous addition of asparagine, glutamate, glutamine, leucine, lysine, histidine, and phenylalanine.     

Comparison of Hup Trait and Intrinsic Antibiotic Resistance for Assessing Rhizobial Competitiveness Axenically and in Soil

A defined medium for growth of 24 strains of Moraxella (Branhamella) catarrhalis was devised. This medium (medium B4) contains sodium lactate as a partial carbon source, proline as both a partial carbon source and a partial nitrogen source, aspartate as a partial nitrogen source, and the growth factors arginine, glycine, and methionine. Either aspartate, glutamate, or proline could serve as sole nitrogen source, but growth occurred at a significantly better rate if proline was present together with either aspartate or glutamate, or with both aspartate and glutamate. With the exception of strain ATCC 23246, all the strains had an absolute requirement for arginine and either a partial or absolute requirement for glycine. The concentration of glycine required for optimal growth was found to be relatively high for an amino acid growth factor. Heart infusion broth was found to be growth inhibitory for spontaneous mutants of one strain able to grow in the absence of arginine, and such mutants reverted readily to arginine dependence accompanied by the ability to grow faster on the complex medium. Growth rates in the defined medium B4 were enhanced by the simultaneous addition of asparagine, glutamate, glutamine, leucine, lysine, histidine, and phenylalanine.     

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.     

Role of glutamine synthetase activity in the uptake and metabolism of arginine and proline in the cyanobacterium Anabaena cycadeae

Abstract The uptake of arginine and proline and their assimilation as nitrogen source have been studied in the cyanobacterium Anabaena cycadeae and its glutamine auxotropic mutant lacking glutamine synthetase activity. The uptake pattern of arginine and proline was found to be biphasic in both wild-type and mutant strains, consisting of an initial fast phase lasting up to 60 s followed by a slower second phase. The uptake activities of both the amino acids were also found to be similar in both the strains. The wild-type strain, having normal glutamine synthetase activity, utilized arginine and proline as sole nitrogen source, whereas the mutant strain lacking glutamine synthetase activity could not do so. These results suggest that: (1) glutamine synthetase activity is necessarily required for the assimilation of arginine and proline as nitrogen source, but it is not required for the uptake of these amino acids; and (2) glutamine synthetase serves as the sole ammonia-assimilating enzyme as well as glutamine-forming route in heterocystous cyanobacteria.     

Evidence for a role of glutamine synthetase in assimilation of amino acids as nitrogen source in the cyanobacterium Nostoc muscorum.

Methylammonium/ammonium ion, glutamine, glutamate, arginine and proline uptake, and their assimilation as nitrogen sources, was studied in Nostoc muscorum and its glutamine synthetase-deficient mutant. Glutamine served as nitrogen source independent of glutamine synthetase activity. Glutamate was not metabolised as a nitrogen source but still inhibited nitrogenase activity and diazotrophic growth. Glutamine synthetase activity was essential for the assimilation of N2, ammonia, arginine and proline as nitrogen sources but not for the control of their transport, heterocyst formation, and production of ammonia or aminoacid dependent repressor signal for N2-fixing heterocysts. These results also suggest that glutamine synthetase serves as the sole route of ammonia assimilation and glutamine synthesis, and ammonia per se as the repressor signal for N2-fixing heterocysts and methylammonium (ammonium) transport.     

Mutant strains (nit) of Salmonella typhimurium with a pleiotropic defect in nitrogen metabolism.

We have isolated mutant strains (nit) of Salmonella typhimurium that are defective in nitrogen metabolism. They have a reduced ability to use a variety of compounds including glutamate, proline, arginine, N-acetyl-glucosamine, alanine, and adenosine as sole nitrogen source. In addition, although they grow normally on high concentrations of ammonium chloride (greater than 1 mM) as nitrogen source, they grow substantially more slowly than wild type at low concentrations (less than 1 mM). We postulated that the inability of these strains to utilize low concentrations of ammonium chloride accounts for their poor growth on other nitrogen sources. The specific biochemical lesion in strains with a nit mutation is not known; however, mutant strains have no detectable alteration in the activities of glutamine synthetase, glutamate synthetase, or glutamate dehydrogenase, the enzymes known to be involved in assimilation of ammonia. A nit mutation is suppressed by second-site mutations in the structural gene for glutamine synthetase (glnA) that decrease glutamine synthetase activity.     

Evidence for the role of intracellular glutamine level in the regulation of glutamine uptake in the cyanobacteriumAnabaena 7120

The role of intracellular glutamine concentration in the regulation of¹⁴C-glutamine uptake was studied in a diazotrophic cyanobacteriumAnabaena 7120. The uptake pattern was found to be biphasic, consisting of a rapid first phase lasting up to 60 s followed by a slower second phase. Azaserine, which could not inhibit in vitro and in vivo glutamine synthetase (GS) activity effectively, inhibited the¹⁴C-glutamine uptake. Glutamine uptake was also not significantly affected when glutamate, methylglutamate, aspartate, arginine, lysine, hydroxylysine, ornithine, and GS inhibitor,L-methionine-DL-sulfoximine (MSX) were simultaneously available during uptake assay, suggesting that glutamine uptake takes place via a general amino acid permease which does not, however, transport basic and acidic amino acids. The azaserine-treated cells had increased and decreased levels of glutamine and glutamate, respectively, suggesting that the increased intracellular glutamine level is responsible for the inhibition of¹⁴C-glutamine uptake and provides evidence here for the role of an intracellular glutamine pool in the regulation of¹⁴C-glutamine uptake inAnabaena 7120.     

L-Methionine SR-sulfoximine-resistant glutamine synthetase from mutants of Salmonella typhimurium

Two mutants of Salmonella typhimurium resistant to growth inhibition by the glutamine synthetase transition state analog, L-methionine SR-sulfoximine, were isolated and characterized. These mutants are glutamine bradytrophs and cannot use growth rate-limiting nitrogen sources. Although this phenotype resembles that of mutants with lesions in the regulatory gene for glutamine synthetase, glnG, these mutations do not lie in the glnG gene. Purification and characterization of the glutamine synthetase from one of the mutants and a control strain demonstrated that the mutant enzyme is defective in the reverse gamma-glutamyltransferase activity but has biosynthetic activity that is resistant to inhibition by L-methionine SR-sulfoximine. The mutant enzyme also has a 4.4-fold higher apparent Km for glutamate (0.2 mM versus 2.1 mM, respectively) and a 13.8-fold higher Km for NH3 (6.4 mM versus 0.46 mM) than the enzyme from the control. These data show that the glutamine synthetase protein has been altered by this mutation, designated as glnA982, and suggest that the L-methionine SR-sulfoximine resistance is conferred by a change in the NH3 binding domain of the enzyme.     

Lack of a regulatory function for glutamine synthetase protein in the synthesis of glutamate dehydrogenase and nitrite reductase in Escherichia coli K12.

Synthesis of glutamine synthetase (GS) in anaerobic batch cultures of Escherichia coli was repressed when excess NH4+ was available, but derepressed during growth with a poor nitrogen source. In wild-type bacteria there was only a weak inverse correlation between the activities of GS and glutamate dehydrogenase (GDH) during growth in various media. No positive correlations were found between the activities of GS and nitrite reductase, or between GS and cytochrome c552: both of these proteins were synthesized normally by mutants that contained no active GS. Although activities of GS and GDH were low in two mutants that are unable to synthesize cytochrome c552 or reduce nitrite because of defects in the nirA gene, the nirA defect was separated from the GS and GDH defects by transduction with bacteriophage P1. Attempts to show that catabolite repression of proline oxidase synthesis could be relieved during NH4+ starvation also failed. It is, therefore, unlikely that nitrite reduction or proline oxidation by E. coli are under positive control by GS protein. The regulation of the synthesis of enzymes for the utilization of secondary nitrogen sources in E. coli, therefore, different from that in Klebsiella aerogenes, but is similar to that in Salmonella typhimurium.     

Characterization of Salmonella typhimurium Strains Sensitive and Resistant to Methionine Sulfoximine

Methionine sulfoximine inhibits the growth of Salmonella typhimurium at a concentration of 50 muM, and the addition of glutamine, but not glutamate, is sufficient to overcome this inhibition. The analogue causes 50% inhibition of glutamine synthetase activity at 2 to 4 muM and of glutamate synthase at 2 to 3 mM when these enzymes are assayed in vitro. No inhibition of glutamate dehydrogenase activity is observed at analogue concentrations as high as 50 mM. Two mutants selected for their resistance to methionine sulfoximine inhibition have a partial growth requirement for glutamine and a reduction in the glutamine synthetase and glutamate synthase activities. The sensitivity of the remaining glutamine synthetase activity in these mutants to methionine sulfoximine inhibition appears unaltered, and the lesions conferring the analogue resistance may not affect glutamine synthetase directly.     

Amino Acid transport and metabolism in relation to the nitrogen economy of a legume leaf

Net balances of amino acids were constructed for stages of development of a leaf of white lupin (Lupinus albus L.) using data on the N economy of the leaf, its exchanges of amino acids through xylem and phloem, and net changes in its soluble and protein-bound amino acids. Asparagine, aspartate, and γ-aminobutyrate were delivered to the leaf in excess of amounts consumed in growth and/or phloem export. Glutamine was supplied in excess until full leaf expansion (20 days) but was later synthesized in large amounts in association with mobilization of N from the leaf. Net requirements for glutamate, threonine, serine, proline, glycine, alanine, valine, isoleucine, leucine, tyrosine, phenylalanine, histidine, lysine, and arginine were met mainly or entirely by synthesis within the leaf. Amides furnished the bulk of the N for amino acid synthesis, asparagine providing from 24 to 68%. In vitro activity of asparaginase (EC 3.5.1.1) exceeded that of asparagine:pyruvate aminotransferase (EC 2.6.1.14) during early leaf expansion, when in vivo estimates of asparagine metabolism were highest. Thereafter, aminotransferase activity greatly exceeded that of asparaginase. Rates of activity of one or both asparagine-utilizing enzymes exceeded estimated rates of asparagine catabolism throughout leaf development. In vitro activities of glutamine synthetase (EC 6.3.1.2) and glutamate synthase (EC 1.4.7.1) were consistently much higher than that of glutamate dehydrogenase (EC 1.4.1.3), and activities of the former two enzymes more than accounted for estimated rates of ammonia release in photorespiration and deamidation of asparagine.     

Regulation of nitrogen catabolic enzymes in Streptomyces clavuligerus

The levels of several enzymes involved in assimilation of different nitrogen compounds were investigated in Streptomyces clavuligerus in relation to the nitrogen source supplied to the cultures. Threonine dehydratase, serine dehydratase, proline dehydrogenase, histidase and urocanase were not decreased in the presence of ammonium. The latter two enzymes were induced by histidine in the culture medium, while proline dehydrogenase was induced by proline. Glutamine synthetase, urease and ornithine aminotransferase levels were higher with poor nitrogen sources and were repressed by ammonium. Arginase was induced by arginine and repressed by ammonium. Glutamine synthetase was rapidly inactivated upon addition of ammonium to the culture, and could be reactivated in vitro by treatment with snake venom phosphodiesterase, which suggested that adenylylation is involved in the inactivation. Three previously isolated mutants with abnormal glutamine synthetase activities showed pleiotropic effects on urease formation. All these data point to a mechanism controlling preferential utilization of some nitrogen sources in this species.     

The influence of peptides on the uptake of amino acids inFusobacterium; predicted interactions withPorphyromonas gingivalis

A study of the uptake of amino acids and its influence by a peptide source was carried out withFusobacterium varium as a convenient representative of the genus. Reference strains and a clinical isolate had similar amino acid uptake profiles, but most amino acids were incorporated at lower concentrations by the latter. In general, high levels of serine, asparagine, glutamate, cysteine, and arginine were incorporated by all species. Histidine, lysine, threonine, and aspartate were taken up at lower levels, whereas the nonpolar neutral amino acids such as alanine, valine, leucine, isoleucine, glycine, proline, phenylalanine, and methionine were poorly metabolized. Yeast extract, as a source of peptides, stimulated the uptake of several amino acids such as histidine and glutamate, whereas others such as methionine, threonine, and asparagine were repressed. The incorporation of some amino acids such as aspartate, ornithine, lysine, and arginine was unaffected by the presence of peptides. Equimolar nitrogen concentrations of amino acids or ammonia could not replace the peptide requirement, emphasizing the importance of peptides as an energy source. The limited capacity ofFusobacterium spp. to hydrolyze proteins increased approximately 30% in the presence of the proteolytic species,Porphyromonas gingivalis, and may represent one bacterial interaction in which peptides may become available toFusobacterium species in vivo.     

Isolation and characterization of nitrogen-deregulated mutants of Streptomyces clavuligerus

Two screening methods for isolation of mutants of Streptomyces clavuligerus with altered control of nitrogen metabolism enzymes are described. Thirty-eight prototrophic mutants with simultaneous deregulation of urease and glutamine synthetase were isolated. Nine mutants were examined in more detail and they also showed deregulated formation of arginase and ornithine aminotransferase. Different patterns of altered control of all four enzymes were observed. Inactivation of glutamine synthetase after ammonium shock took place to different extents in these nine strains, and seven of them had a thermosensitive glutamine synthetase activity. It is concluded that a system of nitrogen control, in which glutamine synthetase has a key role, is present in S. clavuligerus. Cephalosporin production was depressed by ammonium in all the mutants, irrespective of the alterations in nitrogen control of primary metabolism.     

Regulation of gamma-aminobutyric acid degradation in Escherichia coli by nitrogen metabolism enzymes.

The possible role of glutamate dehydrogenase, glutamate synthase, and glutamine synthetase in the regulation of enzyme formation in the gamma-aminobutyric acid (GABA) catabolic pathway of Escherichia coli K-12 was investigated. Evidence is presented indicating that glutamine synthetase acts as a positive regulator in the E. coli GABA control system. Mutations impairing glutamate synthase activity prevent the depression of the enzymes of the GABA pathway in ammonia-limited glucose media. However, mutations resulting in constitutive synthesis of glutamine synthetase (GlnC) restore the ability of the glutamate synthase-less mutants to grow in glucose-GABA media and result in depressed synthesis of the GABA enzymes. It is suggested that the loss of glutamate synthesis activity affects the GABA control system indirectly by lowering glutamine synthetase levels.     

Rhodopseudomonas sphaeroides mutants defective in nitrogen fixation

Mutants of phototrophic bacterium Rhodopseudomonas sphaeroides deficient in nitrogen fixation and unable to utilize alanine, proline, arganine and glutamic acid as nitrogen sources have been obtained as a result of nitrosomethylurea mutagenesis. The majority of the nif-mutants have no nitrogenase activity and aminotransferase activity of glutamine synthetase during their growth in glutamine containing medium is sharply lowered. The specific activity of glutamate synthase and alanine dehydrogenase in the mutants does not differ from that of the wild type strain. One of the mutants (NF-42) has higher glutamine synthetase activity in comparison with the wild type strain. The pleiotropic character of the changes obtained in the nif-mutants shows that the loss of nitrogen fixation ability is due to defects in regulation system of nitrogen metabolism.     

The regulation of ammonia assimilating enzymes in Lemma minor

Summary Lemna minor has the potential to assimilate ammonia via either the glutamine or glutamate pathways. A 3-4 fold variation in the level of ferredoxindependent glutamate synthase may occur, when plants are grown on different nitrogen sources, but these changes show no simple relationship to changes in the endogenous pool of glutamate. High activities of glutamate synthase and glutamine synthetase at low ammonia availability suggests that these two enzymes function in the assimilation of low ammonia concentrations. Increasing ammonia availability leads to a reduction in level of glutamate synthase and glutamine synthetase and an increase in the level of glutamate dehydrogenase. Glutamine synthetase and glutamate dehydrogenase are subject to concurrent regulation, with glutamine rather than ammonia, exerting negative control on glutamine synthetase and positive control on glutamate dehydrogenase. The changes in the ratio of these two enzymes in response to the internal pool of glutamine could regulate the direction of the flow of ammonia into amino acids via the two alternative routes of assimilation.Abbreviations GS Glutamine synthetase - GDH Glutamate dehydrogenase - GOGAT Glutamate synthase     

Analysis of glutamate homeostasis by overexpression of Fd-GOGAT gene in Arabidopsis thaliana

Glutamate plays a central role in nitrogen flow and serves as a nitrogen donor for the production of amino acids. In plants, some amino acids work as buffers: during photorespiration, ammonium derived from the conversion of glycine to serine is promptly reassimilated into glutamate by the glutamine synthetase (GS-2)/ferredoxin-dependent glutamate synthase (Fd-GOGAT) cycle. The glutamate concentration is relatively stable compared with those of other amino acids under environmental changes. The few studies dealing with glutamate homeostasis have but all used knockouts or mutants of these enzymes. Here, we generated Fd-GOGAT (GLU1)-overexpressing Arabidopsis plants to analyze changes in the amino acid pool caused by glutamate overproduction under different ammonium conditions controlled by CO₂ concentration, light intensity and nitrate concentration. Under photorespiratory conditions with sufficient ammonium supply, aspartate increased and glutamine and glycine decreased, but glutamate barely changed. Under non-photorespiratory conditions, however, glutamate and most other amino acids increased. These results suggest that the synthesized glutamate is promptly converted into other amino acids, especially aspartate. In addition, ammonium supply by photorespiration does not limit glutamate biosynthesis, but glutamine and glycine are important. This study will contribute to the understanding of glutamate homeostasis in plants.