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.     

Glutamine synthetase and arginine inhibition of nitrate reductase activity in Anabaena cycadeae

Wild-type Anabaena cycadeae with normal glutamine synthetase (GS) activity utilized arginine as sole N source whereas a mutant strain lacking GS activity did not. Nitrate reductase (NR) activity, higher in the mutant strain than the wild-type strain, was inhibited by arginine though arginine-dependent NH ₄ ⁺ generation was higher in the mutant strain than in the wild-type. This suggests that (1) NR activity is NO _inf3 ^sup- -inducible and arginine-repressible; and (2) while GS activity is required for the assimilation of arginine as sole N-source, it is not required for arginine inhibition of NR activity.S. Singh was with the Department of Biochemistry, North-Eastern Hill University, Shillong-793014, India, and is now with P.S. Bisen at the Department of Microbiology, Barkatullah University, Bhopal-462026, India     

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.     

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.     

Blue-light-control of the uptake of amino acids and of ammonia in Chlorella mutants

In non-photosynthetic, yellow or colourless mutant cells of Chlorella kessleri , grown with nitrate as sole nitrogen source, blue light inhibited the uptake of the amino acids glycine, proline and arginine and of ammonia in growing cells, while it enhanced the uptake of these amino acids in resting cells. On the other hand, in cells grown with ammonia as the only nitrogen source without nitrate reductase activity, blue light did not influence the uptake of amino acids and of ammonia in growing cells, while it enhanced the uptake of amino acids in resting cells. Addition of methionine sulphoximine, a potent inhibitor of glutamine synthetase, to growing cells, resulted in intracellular ammonia-accumulation and inhibition of uptake of glycine and of ammonia. For the colourless mutant, blue light was shown to activate purified nitrate reductase. These results indicate that in the mutant cells of Chlorella examined, uptake of ammonia seems to be influenced by nitrate reductase and the uptake of amino acids was influenced by both nitrate reductase and an unknown blue-light-receptor(s). The uptake of urea in mutant cells is not influenced by the irradiation with blue light. Uptake of glycine was also increased after addition of glucose (hexose) in the dark. Because blue light is known to enhance the breakdown of starch, a reaction producing glucose for oxidative degradation in the algae used, the role of glucose (hexose) in the blue light-affected uptake of amino acids is discussed.     

Glutathione formation in Penicillium chrysogenum: stimulatory effect of ammonium

Penicillium chrysogenum produced glutathione after growth in a defined medium containing 10 mM-NH4Cl as the sole source of nitrogen. The use of higher ammonium concentrations (100 mM) resulted in stimulation of growth and glutathione formation. In addition, increases in the intracellular pools of glutamate, alanine and glutamine, proportional to the amount of ammonium present in the medium were observed. Resting cell systems, prepared from cells previously grown with ammonium, were able to produce glutathione when incubated with ammonium or the amino acids glutamate, alanine and glutamine. A mutant lacking NADP-dependent glutamate dehydrogenase activity (which has a leaky phenotype on ammonium as sole nitrogen source) required glutamate to synthesize glutathione. Resting cell systems of this mutant, prepared from cells previously grown with ammonium, did not produce glutathione even when incubated with glutamate or glutamine. On the other hand, resting cell systems of this mutant produced glutathione if prepared from cells previously grown with glutamate. The addition of glutamate to resting cell systems of the wild-type strain stimulated the synthesis of gamma-glutamylcysteine synthetase, the first enzyme of glutathione biosynthesis.     

The pathways of ammonium assimilation in Rhizobium meliloti

Two pathways of ammonium assimilation are known in bacteria, one mediated by glutamate dehydrogenase, the other by glutamine synthetase and glutamate synthase. The activities of these three enzymes were measured in crude extracts from four Rhizobium meliloti wild-type strains, 2011, M15S, 444 and 12. All the strains had active glutamine synthetase and NADP-linked glutamate synthase. Assimilatory glutamate dehydrogenase activity was present in strains 2011, M15S, 444, but not in strain 12. Three glutamate synthase deficient mutants were isolated from strain 2011. They were unable to use 1 mM ammonium as a sole nitrogen source. However, increased ammonium concentration allowed these mutants to assimilate ammonium via glutamate dehydrogenase. It was found that the sole mode of ammonium assimilation in strain 12 is the glutamine synthetase-glutamate synthase route; whereas the two pathways are functional in strain 2011.Abbreviations GS glutamine synthetase - GOGAT glutamate synthase - GDH glutamate dehydrogenase     

Immunochemical characterization of glutamine synthetase from Neurospora crassa glutamine auxotrophs.

Glutamine synthetase derived from two Neurospora crassa glutamine auxotrophs was characterized. Previous genetic studies indicated that the mutations responsible for the glutamine auxotrophy are allelic and map in chromosome V. When measured in crude extracts, both mutant strains had lower glutamine synthetase specific activity than that found in the wild-type strain. The enzyme from both auxotrophs and the wild-type strain was partially purified from cultures grown on glutamine as the sole nitrogen source, and immunochemical studies were performed in crude extracts and purified fractions. Quantitative rocket immunoelectrophoresis indicated that the activity per enzyme molecule is lower in the mutants than in the wild-type strain; immunoelectrophoresis and immunochemical titration of enzyme activity demonstrated structural differences between the enzymes from both auxotrophs. On the other hand, the monomer of glutamine synthetase of both mutants was found to be of a molecular weight similar to that of the wild-type strain. These data indicate that the mutations are located in the structural gene of N. crassa glutamine synthetase.     

Glutamine assimilation pathways in Neurospora crassa growing on glutamine as sole nitrogen and carbon source

Neurospora crassa wild-type is almost unable to grow on glutamine as sole nitrogen and carbon source but a GDH-; GS +/- double mutant strain, lacking NADP-dependent glutamate dehydrogenase and partially lacking glutamine synthetase did grow. Under these conditions, the double mutant had a higher chemical energy content than the wild-type. Enzyme assays and labelling experiments with glutamine indicated that in the double mutant glutamine was degraded to ammonium and to carbon skeletons by glutamate synthase, the catabolic (NADH-dependent) glutamate dehydrogenase and the glutamine transaminase-omega-amidase pathway.     

Glutathione is required by Rhizobium etli for glutamine utilization and symbiotic effectiveness

Here, we provide genetic and biochemical evidence indicating that the ability of Rhizobium etli bacteria to efficiently catabolize glutamine depends on its ability to produce reduced glutathione (l-γ-glutamyl-l-cysteinylglycine [GSH]). We find that GSH-deficient strains, namely a gshB (GSH synthetase) and a gor (GSH reductase) mutant, can use different amino acids, including histidine, alanine, and asparagine but not glutamine, as sole source of carbon, energy, and nitrogen. Moreover, l-buthionine(S,R)-sulfoximine, a GSH synthesis inhibitor, or diamide that oxidizes GSH, induced the same phenotype in the wild-type strain. Among the steps required for its utilization, glutamine uptake, occurring through the two well-characterized carriers (Aap and Bra systems) but not glutamine degradation or respiration, was largely reduced in GSH-deficient strains. Furthermore, GSH-deficient mutants of R. etli showed a reduced symbiotic efficiency. Exogenous GSH was sufficient to rescue glutamine uptake or degradation ability, as well as the symbiotic effectiveness of GSH mutants. Our results suggest a previously unknown GSH-glutamine metabolic relationship in bacteria.     

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.     

Salmonella typhimurium LT-2 mutants with altered glutamine synthetase levels and amino acid uptake activities.

To determine whether Salmonella typhimurium has a nitrogen control response, we have examined the regulation of nitrogen utilization in two mutants with fivefold and threefold elevations in their glutamine synthetase activities. The mutants do not require glutamine for growth on glucose--ammonia medium but do have altered growth on other nitrogen sources. They grow better than an isogenic control on media containing arginine or asparate, but more slowly with proline or alanine as nitrogen sources. This unusual growth pattern is not due to altered regulation of the ammonia assimilatory enzymes, glutamate dehydrogenase and glutamate synthase, or to changes in the enzymes for aspartate degradation. However, transport for several amino acids may be affected. Measurement of amino acid uptake show that the mutants with high glutamine synthetase levels have increased rates for glutamine, arginine, aspartate, and lysine, but a decreased rate for proline. The relationship between glutamine synthetase levels and uptake was examined in two mutants with reduced, rather than increased, glutamine synthetase production. The uptake rates for glutamine and lysine were lower in these two glutamine auxotrophs than in the Gln+ controls. These results show a correlation between the glutamine synthetase levels and the uptake rates for several amino acids. In addition, the pleiotropic growth of the mutants with elevated glutamine synthetase activities suggests that a nitrogen control response exists for S. typhimurium and that it can be altered by mutations affecting glutamine synthetase regulation.     

gltB gene and regulation of nitrogen metabolism by glutamine synthetase in Escherichia coli.

A mutant (gltB) of Escherichia coli lacking glutamate synthase (GOGAT) was unable to utilize a wide variety of compounds as sole nitrogen source (e.g., arginine, proline, gamma-aminobutyrate, and glycine). Among revertants of these Asm- strains selected on one of these compounds (e.g., arginine, proline, or gamma-aminobutyrate) were those that produce glutamine synthetase (GS) constitutively (GlnC phenotype). These revertants had a pleiotropically restored ability to grow on compounds that are metabolized to glutamate. This suggested that the expression of the genes responsible for the metabolism of these nitrogen sources was regulated by GS. An examination of the regulation of proline oxidase confirmed this hypothesis. The differential sensitivities of GlnC and wild-type strains to low concentrations (0.1 mM) of the glutamine analog L-methionine-DL-sulfoximine supported the conclusion that the synthesis of a glutamine permease was also positively controlled by GS. During the course of this study we found that the reported position of the locus (gltB) for glutamate synthase is incorrect. We have relocated this gene to be 44% linked to the argG locus by P1 transduction. Further mapping has shown that the locus previously called aspB is in reality the gltB locus and that the "suppressor" of the aspB mutation (A. M. Reiner, J. Bacteriol. 97:1431-1436, 1969) is the locus for glutamate dehydrogenase (gdhA).     

Nitrite metabolism in the cyanobacterium Anabaena cycadeae: Regulation of nitrite uptake and nitrite reductase by ammonia

Abstract Nitrogen regulation of nitrite uptake and nitrite reductase was studied in the cyanobacterium Anabaena cycadeae and its glutamine-auxotrophic mutant. The development of the nitrite-uptake system preceded, and was independent of, the development of nitrate reductase. The levels of both of the systems were higher in the glutamine auxotroph lacking glutamine synthetase (GS) than in the wild-type strain having normal GS activity. The nitrite-uptake system was found to be constitutive and ammonia-repressible whereas the nitrite-reductase system was ammonia-repressible and nitrite-inducible. Ammonia did not inhibit the nitrite-uptake and nitrite reductase activities in the glutamine auxotroph whereas glutamine did so, suggesting that repression of nitrite-uptake and nitrite reductase systems by ammonia requires the operation of GS and probably involves the participation of some organic nitrogen metabolites like glutamine.     

Regulation of glutamine synthesis by glycine and serine in Neurospora crassa.

The biosynthetic activities of the polypeptide subunits alpha and beta of glutamine synthetase (GS) were inhibited in vitro by glycine and serine. These amino acids inhibited the growth of a mutant strain with partial GS activity when grown on glutamate as the nitrogen source and also blocked the synthesis of the glutamine in vivo, thus demonstrating the inhibitory effect on GS activity in vivo. Glycine and serine lowered the intracellular glutamine pool and regulated GS beta synthesis. A preferential induction of synthesis of the GS beta polypeptide was observed when either of these amino acids was present in the medium. On this basis, we obtained a glycine-sensitive mutant which showed a structural alteration of the GS beta polypeptide. The double regulatory effect of either glycine or serine on glutamine synthesis may be considered an example of the regulation of glutamine synthesis by alpha-amino nitrogen. It may be a mechanism that regulates the assimilation of ammonium into glutamate versus glutamine.     

Cloning, sequencing and analysis of a gene encoding Escherichia coli proline dehydrogenase

Abstract Using a genomic subtraction technique, we cloned a DNA sequence that is present in wild-type Escherichia coli strain CSH4 but is missing in a presumptive proline dehydrogenase deletion mutant RM2. Experimental evidence indicated that the cloned fragment codes for proline dehydrogenase (EC 1.5.99.8) since RM2 cells transformed with a plasmid containing this sequence was able to survive on minimal medium supplemented with proline as the sole nitrogen and carbon sources. The cloned DNA fragment has an open reading frame of 3942 bp and encodes a protein of 1313 amino acids with a calculated M _r of 143 808. The deduced amino acid sequence of the E. colli proline dehydrogenase has an 84.9% homology to the previously reported Salmonella typhimurium putA gene but it is 111 amino acids longer at the C-terminal than the latter.     

Improvement of Nitrogen Assimilation and Fermentation Kinetics under Enological Conditions by Derepression of Alternative Nitrogen-Assimilatory Pathways in an Industrial Saccharomyces cerevisiae Strain

Metabolism of nitrogen compounds by yeasts affects the efficiency of wine fermentation. Ammonium ions, normally present in grape musts, reduce catabolic enzyme levels and transport activities for nonpreferred nitrogen sources. This nitrogen catabolite repression severely impairs the utilization of proline and arginine, both common nitrogen sources in grape juice that require the proline utilization pathway for their assimilation. We attempted to improve fermentation performance by genetic alteration of the regulation of nitrogen-assimilatory pathways in Saccharomyces cerevisiae. One mutant carrying a recessive allele of ure2 was isolated from an industrial S. cerevisiae strain. This mutation strongly deregulated the proline utilization pathway. Fermentation kinetics of this mutant were studied under enological conditions on simulated standard grape juices with various nitrogen levels. Mutant strains produced more biomass and exhibited a higher maximum CO₂ production rate than the wild type. These differences were primarily due to the derepression of amino acid utilization pathways. When low amounts of dissolved oxygen were added, the mutants could assimilate proline. Biomass yield and fermentation rate were consequently increased, and the duration of the fermentation was substantially shortened. S. cerevisiae strains lacking URE2 function could improve alcoholic fermentation of natural media where proline and other poorly assimilated amino acids are the major potential nitrogen source, as is the case for most fruit juices and grape musts.     

Effect of applied benzyladenine on endogenous cytokinin content during the early stages of bud development of kiwifruit

The filamentous non-N₂-fixing cyanobacterium Phormidium laminosum (strain OH-1-p.Cl₁) was able to utilize glutamine as the sole nitrogen source. The addition to ammonium-grown cultures of the irreversible inhibitor of glutamine synthetase activity L-methionine-D, L-sulfoximine (MSX) inhibited cell growth. Supplying glutamine to the culture restored cell growth. This re-established growth was not due to interference by glutamine of MSX uptake by the cells, since glutamine synthetase (GS, EC 6.3.1.2) activity remained completely inhibited by MSX even when glutamine was simultaneously present. Both glutamine and ammonium exerted a negative effect on nitrate reductase (NR. EC 1.7.7.2) and nitrite reductase (NiR, EC 1.7.7.1) in vivo. This negative effect was reversed by MSX. When glutamine was added to MSX-treated cells, intracellular glutamine level was high, but the activity of both reductases remained at a high level. These results suggest that the presence of the active form of glutamine synthetase is required for the in vivo prevention of nitrate assimilation caused by ammonium and glutamine.