Nitrogen assimilation in Rhodopseudomonas acidophila

Rhodopseudomonas acidophila strain 7050 assimilated ammonia via a constitutive glutamine synthetase/glutamate synthase enzyme system.Glutamine synthetase had a K _m for NH ₄ ⁺ of 0.38 mM whilst the nicotinamide adenine dinucleotide linked glutamate synthase had a K _m for glutamine of 0.55 mM. R. acidophila utilized only a limited range of amino acids as sole nitrogen sources: l-alanine, glutamine and asparagine. The bacterium did not grow on glutamate as sole nitrogen source and lacked glutamate dehydrogenase. When R. acidophila was grown on l-alanine as the sole nitrogen source in the absence of N₂ low levels of a nicotinamide adenine dinucleotide linked l-alanine dehydrogenase were produced. It is concluded, therefore, that this reaction was not a significant route of ammonia assimilation in this bacterium except when glutamine synthetase was inhibited by methionine sulphoximine. In l-alanine grown cells the presence of an active alanine-glyoxylate aminotransferase and, on occasions, low levels of an alanine-oxaloacetate aminotransferase were detected. Alanine-2-oxo-glutarate aminotransferase could not be demonstrated in this bacterium.Abreviations ADH alanine dehydrogenase - GDH glutamate dehydrogenase - GS glutamine synthetase - GOGAT glutamate synthase - MSO methionine sulphoximine     

Nitrogen metabolism inRhodopseudomonas globiformis

Rhodopseudomonas globiformis strain 7950 grew with a variety of amino acids, urea, or N₂ as sole nitrogen sources. Cultures grown on N₂ reduced acetylene to ethylene; this activity was absent from cells grown on nonlimiting NH ₄ ⁺ . Glutamate dehydrogenase could not be detected in extracts of cells of strain 7950, although low levels of an alanine dehydrogenase were present. Growth ofR. globiformis on NH ₄ ⁺ was severely inhibited by the glutamate analogue and glutamine synthetase inhibitor, methionine sulfoximine. High levels of glutamine synthetase (as measured in the -glutamyl transferase assay) were observed in cell extracts of strain 7950 regardless of the nitrogen source, although N₂ and amino acid grown cells contained somewhat higher glutamine synthetase contents than cells grown on excess NH ₄ ⁺ . Levels of glutamate synthase inR. globiformis were consistent with that reported from other phototrophic bacteria. Both glutamate synthase and alanine dehydrogenase were linked to NADH as coenzyme. We conclude thatR. globiformis is capable of fixing N₂, and assimilates NH ₄ ⁺ primarily via the glutamine synthetase/glutamate synthase pathway.Abbreviations GS glutamine synthetase - GOGAT Glutamineoxoglutarate aminotransferase - GDH Glutamate dehydrogenase - ADH Alanine dehydrogenase - MSO Methionine sulfoximine     

The enzymes of the ammonia assimilation in Pseudomonas aeruginosa

Glutamine synthetase from Pseudomonas aeruginosa is regulated by repression/derepression of enzyme synthesis and by adenylylation/deadenylylation control. High levels of deadenylylated biosynthetically active glutamine synthetase were observed in cultures growing with limiting amounts of nitrogen while synthesis of the enzyme was repressed and that present was adenylylated in cultures with excess nitrogen.NADP-and NAD-dependent glutamate dehydrogenase could be separated by column chromatography and showed molecular weights of 110,000 and 220,000, respectively. Synthesis of the NADP-dependent glutamate dehydrogenase is repressed under nitrogen limitation and by growth on glutamate. In contrast, NAD-dependent glutamate dehydrogenase is derepressed by glutamate. Glutamate synthase is repressed by glutamate but not by excess nitrogen.     

Growth of Bacillus fastidiosus on glycerol and the enzymes of ammonia assimilation

Bacillus fastidiosus was able to grow on glycerol as a carbon source when allantoin or urate was used as nitrogen source. The primary assimilatory enzyme for glycerol was glycerol kinase; glycerol dehydrogenase could not be detected. The glycerol kinase activity was increased 30-fold in allantoin/glycerol-grown cells as compared to alantoin-grown cells. Under both growth conditions high levels of glutamate dehydrogenase were found. Glutamine synthetase and glutamate synthase activities could not be demonstrated, while low levels of alanine dehydrogenase were present. It is concluded that B. fastidiosus assimilates ammonia by the NADP-dependent glutamate dehydrogenase.Abbreviations GS glutamine synthetase - GOGAT glutamate synthase - GDH glutamate dehydrogenase - ADH alanine dehydrogenase     

Ammonium assimilation in an Arthrobacter sp. “fluorescens”

Ammonium assimilation was studied in a nitrogenfixing Arthrobacter strain grown in both batch and ammonium-limited continuous cultures. Arthrobacter sp. fluorescens grown in nitrogen-free medium or at low ammonium levels assimilated NH ₄ ⁺ via the glutamine synthetase/glutamate synthase pathway. When ammonium was in excess it was assimilated via the alanine dehydrogenase pathway. Very low levels of glutamate dehydrogenase were found, irrespective of growth conditions.Abbreviations GS glutamine synthetase - GOGAT glutamine oxoglutarate aminotransferase - GDH glutamate dehydrogenase - ADH alanine dehydrogenase - GOT glutamate oxaloacetate transaminase - GPT glutamate pyruvate transaminase     

NAD and NADP-dependent glutamate dehydrogenase in Hydrogenomonas H 16

Summary Hydrogenomonas H 16 synthetized two chromatographically distinct forms of glutamate dehydrogenase which differed in their thermolability. One glutamate dehydrogenase utilized NAD, the other NADP as a coenzyme.Low specific activity of NAD-dependent glutamate dehydrogenase was found in cells grown with glutamate as sole nitrogen source or in cells grown with a high concentration of ammonium ions. In the presence of a low concentration of ammonium ions or in a nitrogen free medium, the specific activity of the NAD-dependent enzyme increased. Corresponding to the formation of the NAD-dependent glutamate dehydrogenase the enzyme glutamine synthetase was synthesized. The ratio of NAD-dependent glutamate dehydrogenase to glutamine synthetase activity differed only slightly in cells grown with different nitrogen and carbon sources.The NADP-dependent glutamate dehydrogenase was found in high specific activity in cells grown with an excess of ammonium ions. Under nitrogen starvation the formation of the NADP-dependent glutamate dehydrogenase ceased and the enzyme activity decreased.     

Nitrogen control in Pseudomonas aeruginosa: A role for glutamine in the regulation of the synthesis of NADP-dependent glutamate dehydrogenase,urease and histidase

In Pseudomonas aeruginosa the formation of urease, histidase and some other enzymes involved in nitrogen assimilation is repressed by ammonia in the growth medium. The key metabolite in this process appears to be glutamine or a product derived from it, since ammonia and glutamate did not repress urease and histidase synthesis in a mutant lacking glutamine synthetase activity when growth was limited for glutamine. The synthesis of these enzymes was repressed in cells growing in the presence of excess glutamine. High levels of glutamine were also required for the derepression of NADP-dependent glutamate dehydrogenase formation in the glutamine synthetase-negative mutant.     

The effect of various culture conditions on the levels of ammonia assimilatory enzymes of Corynebacterium callunae

Corynebacterium callunae (NCIB 10338) grows faster on glutamate than ammonia when used as sole nitrogen sources. The levels of glutamine synthetase (GS; EC 6.3.1.2) and glutamate synthase (GOGAT; EC 1.4.1.13) of C. callunae were found to be influenced by the nitrogen source. Accordingly, the levels of GS and GOGAT activities were decreased markedly under conditions of ammonia excess and increased under low nitrogen conditions. In contrast, glutamate dehydrogenase (GDH; EC 1.4.1.4) activities were not significantly affected by the type or the concentration of the nitrogen source supplied. The carbon source in the growth medium could also affect GDH, GS and GOGAT levels. Of the carbon sources tested in the presence of 2 mM or 10 mM ammonium chloride as the nitrogen source pyruvate, acetate, fumarate and malate caused a decrease in the levels of all three enzymes as compared with glucose. GDH, GS and GOGAT levels were slightly influenced by aeration. Also, the enzyme levels varied with the growth phase. Methionine sulfoximine, an analogue of glutamine, markedly inhibited both the growth of C. callunae cells and the transferase activity of GS. The apparent K _m values of GDH for ammonia and glutamate were 17.2 mM and 69.1 mM, respectively. In the NADPH-dependent reaction of GOGAT, the apparent K _m values were 0.1 mM for -ketoglutarate and 0.22 mM for glutamine.Abbreviations GDH glutamate dehydrogenase - GS glutamine synthetase - GOGAT glutamate synthase     

Ammonia assimilation and glutamate incorporation in coenzyme F420 derivatives ofMethanosarcina barkeri

Methanosarcina barkeri was able to grow on L-alanine and L-glutamate as sole nitrogen sources. Cell yields were 0.5 g/l and 0.7 g/l (wet wt), respectively. The mechanism of ammonia assimilation inMethanosarcina barkeri strain MS was studied by analysis of enzyme activities. Activity levels of nitrogen-assimilating enzymes in extracts of cells grown on different nitrogen sources (ammonia, 0.05–100 mM; L-alanine, 10 mM; L-glutamate, 10 mM) were compared. Activities of glutamate dehydrogenase, glutamate synthase, glutamine synthetase, glutamate oxaloacetate transaminase and glutamate pyruvate transaminase could be measured in cells grown on these three nitrogen sources. Alanine dehydrogenase was not detected under the growth conditions used. None of the measured enzyme activities varied significantly in response to the NH₄ ⁺ concentration. The length of the poly--glutamyl side chain of F₄₂₀ derivatives turned out to be independent of the concentration of ammonia in the culture medium.Abbreviations ADH alanine dehydrogenase - FO 7,8-didemethyl-8-hydroxy-5-deazariboflavin - GDH glutamate dehydrogenase - GOGAT glutamate synthase - GOT glutamate oxaloacetate transaminase - GPT glutamate pyruvate transaminase - GS glutamine synthetase - H₄MPT tetrahydromethanopterin     

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.     

Ammonium uptake and metabolism by nitrogen fixing bacteria

The primary steps of N₂, ammonia and nitrate metabolism in Klebsiella pneumoniae grown in a continuous culture are regulated by the kind and supply of the nitrogenous compound. Cultures growing on N₂ as the only nitrogen source have high activities of nitrogenase, unadenylated glutamine synthetase and glutamate synthase and low levels of glutamate dehydrogenase. If small amounts of ammonium salts are added continuously, initially only part of it is absorbed by the organisms. After 2–3 h complete absorption of ammonia against an ammonium gradient coinciding with an increased growth rate of the bacteria is observed. The change in the extracellular ammonium level is paralleled by the intracellular glutamine concentration which in turn regulates the glutamine synthetase activity. An increase in the degree of adenylation correlates with a repression of nitrogenase synthesis and an induction of glutamate dehydrogenase synthesis. Upon deadenylation these events are reversed.—After addition of nitrate ammonia appears in the medium, probably due to the action of a membrane bound dissimilatory nitrate reductase.—Addition of dinitrophenol causes transient leakage of intracellular ammonium into the medium.     

Nitrogen nutrition in the estuarine zone: the case of Suaeda maritima var. macrocarpa

Suaeda maritima L. var. macrocarpa is a halophytic species distributed in the lower parts of salt marshes of the French coasts. The influence of salinity on nitrogen nutrition and on levels of the key enzymes involved in nitrogen assimilation is analyzed by growing Suaeda under experimental conditions. Use of ¹⁵N-labelled NO₃ ^- and NH₄ ⁺ shows that both ions are effective sources of inorganic nitrogen for Suaeda. The plant is found to use NH₄ ⁺ ions with a good yield, chiefly at high salinities (up to 130 mM). Nitrate reduction and ammonium assimilation by the glutamine synthetase/glutamate synthase pathway occurs mainly in leaves when Suaeda is grown at optimal saline conditions (130 mM NaCl). Absence of NaCl creates less favourable conditions and lowers the activity of nitrate reductase and glutamine synthetase but leads to an important activity of glutamate dehydrogenase in roots. This enzyme could play a major role under suboptimal environmental conditions (i.e., absence of NaCl for Suaeda maritima).Part of this paper is taken from a thesis that was submitted by J. P. Billard in fulfillment of the Doctorat d'Etat degree at the University of Caen, France.     

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     

N2 fixation and NH 4 + assimilation in the thermophilic anaerobes Clostridium thermosaccharolyticum and Clostridium thermoautotrophicum

Inorganic nitrogen metabolism in the obligate anaerobic thermophiles Chlostridium thermosaccharolyticum and Clostridium thermoautotrophicum differs in several respects. C. thermosaccharolyticum contains a nitrogenase as inferred from NH ₄ ⁺ repressible C₂H₂ reduction, a glutamine synthetase which is partially repressed by ammonium, very labile glutamate synthase activities with both NADH and NADPH, NADPH-dependent glutamate dehydrogenase, and NH ₄ ⁺ -dependent asparagine synthetase. C. thermoautotrophicum contains no nitrogenase, but glutamine synthetase, no glutamate synthase, no glutamate dehydrogenase, but a NADH-dependent alanine dehydrogenase and a NH ₄ ⁺ -dependent asparagine synthetase.Abbreviation GOGAT glutamine-oxoglutarate amidotransferase amidotransferase (glutamate synthase)     

Utilization of nitrogen compounds and ammonia assimilation by chromatiaceae

Chromatium vinosum strain D, Thiocapsa roseopersicina strain 6311 and Ectothiorhodospira mobilis strain 8112 were grown anaerobically in the light with various single nitrogen sources. When substituted for NH₄Cl only glutamine and casamino acids supported good growth of all strains tested. Peptone and urea were utilized by C. vinosum and T. roseopersicina, glutamate, asparagine and nitrate only by C. vinosum. The strains were able to grow with molecular nitrogen; complete inhibition of this growth was observed in the presence of alanine with E. mobilis, and of alanine or asparagine with T. roseopersicina.Glutamate dehydrogenase, requiring either NADH or NADPH, NADH-linked glutamate synthase, and glutamine synthetase were demonstrate in the above organisms grown on NH₄Cl.     

Ammonium assimilation inNocardia asteroides

Specific enzymes of ammonium assimilation were measured in cell-free extracts ofNocardia asteroides grown in a synthetic medium with glutamate as the nitrogen source. Cell-free extracts had active glutamine synthetase (GS) and glutamate synthase (GOGAT) and alanine dehydrogenase (ADH) but glutamate dehydrogenase (GDH) could not be detected in the enzyme preparation. This shows that GS/GOGAT is the major pathway of ammonium assimilation inN. asteroides.     

Carbon and nitrogen metabolism in barley (Hordeum vulgare L.) mutants lacking ferredoxin-dependent glutamate synthase

Five mutant lines of barley (Hordeum vulgare L.), which are only able to grow at elevated levels of CO₂, contain less than 5% of the wild-type activity of ferredoxin-dependent glutamate synthase (EC 1.4.7.1). Two of these lines (RPr 82/1 and RPr 82/9) have been studied in detail. Leaves and roots of both lines contain normal activities of NADH-dependent glutamate synthase (EC 1.4.1.14) and the other enzymes of ammonia assimilation. Under conditions that minimise photorespiration, both mutants fix CO₂ at normal rates; on transfer to air, the rates drop rapidly to 15% of the wild-type. Incorporation of ¹⁴CO₂ into sugar phosphates and glycollate is increased under such conditions, whilst incorporation of radioactivity into serine, glycine, glycerate and sucrose is decreased; continuous exposure to air leads to an accumulation of ¹⁴C in malate. The concentrations of malate, glutamine, asparagine and ammonia are all high in air, whilst aspartate, alanine, glutamate, glycine and serine are low, by comparison with the wild-type parent line (cv. Maris Mink), under the same conditions. The metabolism of [¹⁴C]glutamate and [¹⁴C]glutamine by leaves of the mutants indicates a very much reduced ability to convert glutamine to glutamate. Genetic analysis has shown that the mutation in RPr 82/9 segregates as a single recessive nuclear gene.Abbreviations GDH glutamate dehydrogenase (EC 1.4.1.2) - GS glutamine synthetase (EC 6.3.1.2) - RuBP ribulose 1,5-bisphosphate     

Inhibition of Astrocyte Glutamine Production by α-Ketoisocaproic Acid

Abstract: We have evaluated the effect of α-ketoisocaproic acid (KIC), the ketoacid of leucine, on the production of glutamine by cultured astrocytes. We used ¹⁵NH₄Cl as a metabolic tracer to measure the production of both [5-¹⁵N]glutamine, reflecting amidation of glutamate via glutamine synthetase, and [2-¹⁵N]glutamine, representing the reductive amination of 2-oxoglutarate via glutamate dehydrogenase and subsequent conversion of [¹⁵N]-glutamate to [2-¹⁵N]glutamine. Addition of KIC (1 mM) to the medium diminished the production of [5-¹⁵N]glutamine and stimulated the formation of [2-¹⁵N]glutamine with the overall result being a significant inhibition of net glutamine synthesis. An external KIC concentration as low as 0.06 mM inhibited synthesis of [5-¹⁵N]glutamine and a level as low as 0.13 mM enhanced labeling (atom% excess) of [2-¹⁵N]glutamine. Higher concentrations of KIC in the medium had correspondingly larger effects. The presence of KIC in the medium did not affect flux through glutaminase, which was measured using [2-¹⁵N]glutamine as a tracer. Nor did KIC inhibit the activity of glutamine synthetase that was purified from sheep brain. Addition of KIC to the medium caused no increased release of lactate dehydrogenase from the astrocytes, suggesting that the ketoacid was not toxic to the cells. KIC treatment was associated with an approximately twofold increase in the formation of ¹⁴CO₂ from [U-¹⁴C]glutamate, indicating that transamination of glutamate with KIC increases intraastrocytic α-ketoglutarate, which is oxidized in the tricarboxylic acid cycle. KIC inhibited glutamine synthesis more than any other ketoacid tested, with the exception of hydroxypyruvate. The data indicate that KIC diminishes flux through glutamine synthetase by lowering the intraastrocytic glutamate concentration below the K_m of glutamine synthetase for glutamate, which we determined to be ～7 mM.     

Nitrogen metabolism of Frankia strain,Cc01, Mg+, At4 and Hr18

The composition and levels of amino acids in four Frankia strains isolated from different actinorhizal plants, were determined. Minor differences in the amino acid profiles were noted with GLN (GLU) being the major amino acid in all four strains. Enzyme actives of ammonia metabolism, GS (glutamine synthetase), GOGAT (glutamate synthetase), and GDH (glutamate dehydrogenase), were also measured. In strains At4 and Hr18, GS and GOGAT activity levels were elevated in N₂-grown cells but significant amounts of GDH activity were present in ammonia-grown cells. No GDH was detected in strain Cc01 and Mg⁺. The characters of heat-stable and heat-labile GSs were described. In N₂-fixing cells, the ATP and amino acid content was much lower, but ammonia content was higher than in NH _inf4 ^sup+ -grown cells.     

Regulation by ammonium and glucose of Arthrobacter fluorescens alanine dehydrogenase

Alanine dehydrogenase in Arthrobacter fluorescens exhibited an allosteric behaviour and two K _m values for ammonium were estimated. In batch cultures at different ammonium concentrations and in continuous culture following an NH₄ ⁺ pulse, the level of ADH activity seems to be regulated by the ammonium concentration, high activities being observed when extracellular ammonium was in excess. The response to the growth rate of an ammonium-limited chemostat culture of A. fluorescens seems to indicate that alanine dehydrogenase and glutamine synthetase activities were inversely related. High activities of glutamate oxaloacetate transaminase and glutamate pyruvate transaminase have been found in crude extract of ammonium-limited cultures. From the results obtained in batch cultures grown at different glucose concentrations and in carbon-limited chemostat culture it appeared that the limitation by glucose influenced alanine dehydrogenase activity negatively. No glutamate dehydrogenase activity and no glutamate synthase activity could be detected with either NADH or NADPH as coenzymes.Abbreviations ADH alanine dehydrogenase - GS glutamine synthetase - GDH glutamate dehydrogenase - GOGAT glutamine oxoglutarate aminotransferase - GOT glutamate oxaloacetate transaminase - GPT glutamate pyruvate transaminase