Ammonia assimilation and glutamate formation in the anaerobe Selenomonas ruminantium.

Selenomonas ruminantium was found to possess two pathways for NH4+ assimilation that resulted in net glutamate synthesis. One pathway fixed NH4+ through the action of an NADPH-linked glutamate dehydrogenase (GDH). Maximal GDH activity required KCl (about 0.48 M), but a variety of monovalent salts could replace KCl. Complete substrate saturation of the enzyme by NH4+ did not occur, and apparent Km values of 6.7 and 23 mM were estimated. Also, an NADH-linked GDH activity was observed but was not stimulated by KCl. Cells grown in media containing non-growth-rate-limiting concentrations of NH4+ had the highest levels of GDH activity. The second pathway fixed NH4+ into the amide of glutamine by an ATP-dependent glutamine synthetase (GS). The GS did not display gamma-glutamyl transferase activity, and no evidence for an adenylylation/deadenylylation control mechanism was detected. GS activity was highest in cells grown under nitrogen limitation. Net glutamate synthesis from glutamine was effected by glutamate synthase activity (GOGAT). The GOGAT activity was reductant dependent, and maximal activity occurred with dithionite-reduced methyl viologen as the source of electrons, although NADPH or NADH could partially replace this artificial donor system. Flavin adenine dinucleotide, flavin mononucleotide, or ferredoxin could not replace methyl viologen. GOGAT activity was maximal in cells grown with NH4+ as sole nitrogen source and decreased in media containing Casamino Acids.     

浑球红假单胞菌氨同化途径的研究

本文测定了浑球红假单胞菌(Rhodobacter sphaeroides)菌株601谷氨酰胺合成酶(GS)、谷氨酸合酶(GOGAT)、谷氨酸脱氢酶(GDH)和丙氨酸脱氢酶(ADH)的活性。低氨时,GS/GOGAT活力高,GDH活力低,高氨时,GS/GOGAT活力低,GDH活力高。在以分子氮或低浓度氨为氮源的培养条件下,加入GS抑制刑MSX(L—methionine—DL—sulphoximine),细菌生长受到抑制。但是,生长在以谷氨酸为氮源的细菌则不受影响。上述结果表明,浑球红假单胞菌菌株601氨同化是通过GS/GOGAT途径和GDH途径。     

Evidence of ammonium assimilation via the glutamine synthetase-glutamate synthase system in rice seedling roots

When rice seedling roots were fed ¹⁵N-ammonium for 1 hr, theamide nitrogen of glutamine showed the highest ¹⁵N abundance.Moreover, glutamine amino, glutamic acid, aspartic acid andalanine showed higher ¹⁵N abundance than ammonium did. In roots whose GS activity was inhibited with MS, both the amountof ammonium and its ¹⁵N abundance were increased. In contrast,both the amount of all examined amino acids containing glutamicacid and their ¹⁵N abundance decreased in roots whose GS activitywas inhibited. From these results, it could be concluded thatthe first step of ammonium assimilation in rice seedling rootswas mainly glutamine synthesis by GS and the second was glutamicacid formation by the GOGAT system. The results of an experiment using ¹⁵N glutamine also supportedthis conclusion. (Received February 23, 1977; )     

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     

Glutamate synthesis in Streptomyces coelicolor.

Both glutamate synthase (GOGAT) and glutamate dehydrogenase (GDH) are involved in glutamate synthesis in Streptomyces coelicolor. The highest levels of GDH were seen in extracts of cells grown with high levels of ammonium as the nitrogen source. GOGAT activity was reduced two- to threefold in extracts of cells grown with good sources of glutamate. S. coelicolor mutants deficient in GOGAT (Glt-) required glutamate for growth with L-alanine, asparagine, arginine, or histidine as the nitrogen source but grew like wild-type cells when ammonium, glutamine, or aspartate was the nitrogen source. The glt mutations were tightly linked to hisA1. Mutants deficient in both GOGAT and GDH (Gdh-) required glutamate for growth in all media. The gdh-5 mutation was mapped to the left region of the S. coelicolor chromosomal map, between proA1 and uraA1.     

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.     

氮素水平对花生氮素代谢及相关酶活性的影响

 在大田高产条件下研究了氮素水平对花生(Arachis hypogaea)可溶性蛋白质、游离氨基酸含量及氮代谢相关酶活性的影响, 结果表明, 适当提高氮素水平既能增加花生各器官中可溶性蛋白质和游离氨基酸的含量, 又能提高硝酸还原酶、谷氨酰胺合成酶和谷氨酸脱氢酶等氮素同化酶的活性, 使其达到同步增加; 氮素水平过高虽能提高硝酸还原酶和籽仁蛋白质含量, 但谷氨酰胺合成酶(GS)和谷氨酸脱氢酶(GDH)的活性下降; N素施肥水平不改变花生植株各器官中可溶性蛋白质、游离氨基酸含量以及硝酸还原酶(NR)、谷氨酰胺合成酶、谷氨酸脱氢酶活性的变化趋势, 但适量施N (A2和A3处理)使花生各营养器官中GS、GDH活性提高; 氮素水平对花生各叶片和籽仁中GS、GDH活性的高低影响较大, 但对茎和根中GDH活性大小的影响较小。     

The effect of nitrogen addition on N2-fixation and on glutamate dehydrogenase and glutamate synthase activities. in nodules and roots of soybeans inoculated with various strains of Rhizobium japonicum

The addition of nitrogen in the form of urea decreased the activitiesof glutamate dehydrogenase (GDH) and glutamate synthase (GOGAT)in root nodules of Glycine max, whereas the same addition greatlyenhanced root GDH activity. Division of nodules into a mitochondrialand bacteroid fraction indicated that the addition of nitrogenas urea, ammonia, or nitrate most greatly inhibits GDH activityin the mitochondrial fraction. Studies with plants having floralprimordia indicated that added nitrate inhibits nodular GDHmore than either ammonia or urea, while plants inoculated withan ineffective strain (non-nitrogen fixing) of Rhizobium japonicumshowed an increase in nodular GDH activity with nitrogen addition.GOGAT activity was greatly reduced after floral initiation.GDH, GOGAT, and nitrogenase activities in root nodules appearedto vary with the strain of Rhizobium japonicum used as inoculum.In general, strains which produced nodules with high GDH activityproduced bacteroids with low GOGAT activity and the strain whichproduced nodules with the lowest GDH activity produced bacteroidswith the highest GOGAT activity. (Received May 24, 1976; )     

Disturbed ammonium assimilation is associated with growth inhibition of roots in rice seedlings caused by NaCl

The effects of NaCl on changes in ammonium level and enzyme activities of ammonium assimilation in roots growth of rice (Oryza sativa L.) seedlings were investigated. NaCl was effective in inhibiting root growth and stimulated the accumulation of ammonium in roots. Accumulation of ammonium in roots preceded inhibition of root growth caused by NaCl. Both effects caused by NaCl are reversible. Exogenous ammonium chloride and methionine sulfoximine (MSO), which caused ammonium accumulation in roots, inhibited root growth of rice seedlings. NaCl decreased glutamine synthetase and glutamate synthase activities in roots, but increased glutamate dehydrogenase activity. The growth inhibition of roots by NaCl or MSO could be reversed by the addition of L-glutamic acid or L-glutamine. The current results suggest that disturbance of ammonium assimilation in roots may be involved in regulating root growth reduction caused by NaCl.Abbreviations GDH glutamate dehydrogenase - GOGAT glutamate synthase - GS glutamine synthetase - MSO methionine sulfoximine     

Ammonium metabolism in the symbiotic sea anemone Anemonia viridis

The temperate sea anemone Anemonia viridis (Forskål) forms an endosymbiotic association with dinoflagellate algae commonly referred to as zooxanthellae. It is now well established that under appropriate environmental conditions, these associations can be autotrophic for carbon. Under such conditions, many of these symbioses, including A. viridis, not only retain excretory ammonium, but can take up ammonium added to the surrounding seawater. The flux from inorganic to organic nitrogen will be via the free amino acid pools and in A. viridis these were found to be markedly different between zooxanthellae and host with glycine and taurine dominant in the latter. When anemones were maintained with 20 M ammonium, the concentration of free amino groups increased in the zooxanthellae but appeared not to change in the host. There was no evidence that the ratio of glutamine – glutamate in zooxanthellae changed when anemones were maintained with 20 M ammonium for 47 days. These ratios imply that zooxanthellae from this temperate symbiosis may not be nitrogen-limited. GDH was detected in both zooxanthellae and host where it was most active with the coenzyme NADPH. In addition, GDH showed activity when glutamine replaced ammonium as the substrate, indicating that the host may have alternative means to assimilate ammonium. Zooxanthellae were shown to possess GOGAT activity in the presence of a ferredoxin analogue. This suggests that in vivo zooxanthellae could assimilate ammonium via the activity of GS linked with ferredoxin-dependent GOGAT. Given evidence from other studies of rapid ammonium assimilation and essential amino acid synthesis in symbiotic host tissue, it appears that the capacity of cnidarians to metabolise nitrogen may at present be underestimated.     

Influence of nitrogen and phosphorus fertilizers on ammonia-assimilating enzymes of Azolla

Summary A field experiment was conducted and studied the effect of nitrogen and phosphorus on ammonia assimilating enzymes of Azolla. Nitrogen and phosphorus at 30 and 60 kg/ha respectively were tested andAzolla pinnata was inoculated at 200 g/m². The Azolla samples were drawn on 24th hr, 7th day and 14th day and the ammonia assimilating enzymes glutamine synthetase (GS), glutamate synthase (GOGAT) and glutamine dehydrogenase (GDH) were estimated. Nitrogen and phosphorus have markedly suppressed the GDH activity but fertilizer nitrogen has no significant influence in inhibiting the enzyme activity of GOGAT and GS. In general phosphorus application also has stimulated the GS activity significantly during the first sampling period of 24th hour.     

NaCl对水稻谷氨酸合酶和谷氨酸脱氢酶的胁迫作用

在ＮａＣｌ的胁迫下,水稻幼苗根和叶的谷氨酸合酶和谷氨酸脱氢酶的活性随着营养液中的ＮａＣｌ浓度的升高而降低;游离ＮＨ４＾＋在叶中积累,在根中未见明显变化。与根相比,叶对ＮａＣｌ的胁迫作用更为敏感。叶的ＮＡＤＨ－ＧＯＧＡＴ和ＮＡＤＨ－ＧＤＨ活性在ＮａＣｌ胁迫降低的程度明显大于根。无论是否有ＮａＣｌ存在,根的ＮＡＤＨ－ＧＤＨ活性明显高于叶。ＧＳ／ＧＤＨ比值分析提示,对对照下,根中的ＮＨ４＾存在,根的ＮＡ     

Glutamine synthetase/glutamate synthase ammonium-assimilating pathway in Schizosaccharomyces pombe

Kinetic parameters of glutamine synthetase (GS) and glutamate synthase (glutamineoxoglutarate aminotransferase) (GOGAT) activities, including initial velocity, pH, and temperature optima, as well as K _m values, were estimated in Schizosaccharomyces pombe crude cell-free extracts. Five glutamine auxotrophic mutants of S. pombe were isolated following MNNG treatment. These were designated gln1-1,2,3,4,5, and their growth could be repaired only by glutamine. Mutants gln1-1,2,3,4,5 were found to lack GS activity, but retained wild-type levels of NADP-glutamate dehydrogenase (GDH), NAD-GDH, and GOGAT. One further glutamine auxotrophic mutant, gln1-6, was isolated and found to lack both GS and GOGAT but retained wild-type levels of NADP-GDH and NAD-GDH activities. Fortuitously, this isolate was found to harbor an unlinked second mutation (designated gog1-1), which resulted in complete loss of GOGAT activity but retained wild-type GS activity. The growth phenotype of mutant gog1-1 (in the absence of the gln1-6 mutation) was found to be indistinguishable from the wild type on various nitrogen sources, including ammonium as a sole nitrogen source. Double-mutant strains containing gog1-1 and gdh1-1 or gdh2-1 (mutations that result specifically in the abolition of NADP-GDH activity) result in a complete lack of growth on ammonium as sole nitrogen source in contrast to gdh or gog mutants alone.     

Changes in the activity of enzymes involved with primary nitrogen metabolism due to ectomycorrhizal symbiosis on jack pine seedlings

Jack pine (Pinus banksiana Lamb.) seedlings were inoculated with either one of the ectomycorrhizal (ECM) fungi, Laccaria bicolor (Maire) Orton or Pisolithus tinctorius (Pers.) Coker and Couch, and grown for 16 weeks in a growth chamber along with non-ECM controls. Five enzymes involved with the assimilation of nitrogen or the synthesis of amino acids were measured in the 3 jack pine root systems as well as in the pure fungal cultures. Pisolithus tinctorius in pure culture had no detectable activity of nitrate reductase (NR. EC 1.6.6.1), glutamate dehydrogenase (GDH. EC 1.4.1.2), glutamate decarboxylase (GDCO. EC 4.1.1.15) or glutamate oxoglutarate aminotransferase (GOGAT, EC 1.4.1.13) but did have some glutamine synthetase (GS, EC 6.3.1.2) activity. Laccaria bicolor in pure culture had no NR activity, small levels of GDCO activity, and high GS, GDH and GOGAT activity. The high levels of enzymatic activity present in L. bicolor indicate that it may play a greater role in the nitrogen metabolism of its host plant than P. tinctorius. ECM infection clearly altered the enzymatic activity in jack pine roots but the nature of these changes depended on the fungal associate. Non-ECM root systems had higher specific activities than ECM root systems for NR, GS, GDH and GDCO but GOGAT activites were the same for both the ECM and non-ECM roots. Root systems infected with L. bicolor had significantly greater NR and GDCO activity than those infected with P. tinctorius. Differences in the GS activity of the two fungi in pure culture corresponded to the GS activity of jack pine roots in symbiotic association with these fungi. While the free amino acid profiles in roots were significantly affected by ECM infection, the profile of free amino acids exported to the stem was the same for all treatments. High asparagine and low glutamine in roots infected with P. tinctorius indicates that asparagine synthetase (EC x.x.x.x) activity should be higher within this symbiotic association than in the L. bicolor association or in the non-mycorrhizal roots.     

Heme oxygenase up-regulation under salt stress protects nitrogen metabolism in nodules of soybean plants

The behaviour of enzymes involved in nitrogen metabolism, as well as oxidative stress generation and heme oxygenase gene and protein expression and activity, were analysed in soybean (Glycine max L.) nodules exposed to 50, 100 and 200 mM NaCl concentrations. A significant increase in lipid peroxidation was found with 100 and 200 mM salt treatments. Moreover, superoxide dismutase, catalase and peroxidase activities were decreased under 100 and 200 mM salt. Nitrogenase activity and leghemeoglobin content were diminished and ammonium content increased only under 200 mM NaCl. At 100 mM NaCl, glutamine synthetase (GS) and NADH-glutamate dehydrogenase (GDH) activities were similar to controls, whereas a significant increase (64%) in NADH-glutamate synthase (GOGAT) activity was observed. GS activity did not change at 200 mM salt treatment, but GOGAT and GDH significantly decreased (40 and 50%, respectively). When gene and protein expression of GS and GOGAT were analysed, it was found that they were positively correlated with enzyme activities. In addition, heme oxygenase (HO) activity, protein synthesis and gene expression were significantly increased under 100 mM salt treatment. Our data demonstrated that the up-regulation of HO, as part of antioxidant defence system, could be protecting the soybean nodule nitrogen fixation and assimilation under saline stress conditions.     

Assimilation of 13NH4+ by Azospirillum brasilense grown under nitrogen limitation and excess.

The specific activities of glutamine synthetase (GS) and glutamate synthase (GOGAT) were 4.2- and 2.2-fold higher, respectively, in cells of Azospirillum brasilense grown with N2 than with 43 mM NH4+ as the source of nitrogen. Conversely, the specific activity of glutamate dehydrogenase (GDH) was 2.7-fold higher in 43 mM NH4+-grown cells than in N2-grown cells. These results indicate that NH4+ could be assimilated and that glutamate could be formed by either the GS-GOGAT or GDH pathway or both, depending on the cellular concentration of NH4+. The routes of in vivo synthesis of glutamate were identified by using 13N as a metabolic tracer. The products of assimilation of 13NH4+ were, in order of decreasing radioactivity, glutamine, glutamate, and alanine. The formation of [13N]glutamine and [13N]glutamate by NH4+-grown cells was inhibited in the additional presence of methionine sulfoximine (an inhibitor of GS) and diazooxonorleucine (an inhibitor of GOGAT). Incorporation of 13N into glutamine, glutamate, and alanine decreased in parallel in the presence of carrier NH4+. These results imply that the GS-GOGAT pathway is the primary route of NH4+ assimilation by A. brasilense grown with excess or limiting nitrogen and that GDH has, at best, a minor role in the synthesis of glutamate.     

Kinetic flux profiling dissects nitrogen utilization pathways in the oleaginous green alga Chlorella protothecoides

As a promising candidate for biodiesel production, the green alga Chlorella protothecoides can efficiently produce oleaginous biomass and the lipid biosynthesis is greatly influenced by the availability of nitrogen source and corresponding nitrogen assimilation pathways. Based on isotope‐assisted kinetic flux profiling (KFP), the fluxes through the nitrogen utilization pathway were quantitatively analyzed. We found that autotrophic C. protothecoides cells absorbed ammonium mainly through glutamate dehydrogenase (GDH), and partially through glutamine synthetase (GS), which was the rate‐limiting enzyme of nitrogen assimilation process with rare metabolic activity of glutamine oxoglutarate aminotransferase (GOGAT, also known as glutamate synthase); whereas under heterotrophic conditions, the cells adapted to GS‐GOGAT cycle for nitrogen assimilation in which GS reaction rate was associated with GOGAT activity. The fact that C. protothecoides chooses the adenosine triphosphate‐free and less ammonium‐affinity GDH pathway, or alternatively the energy‐consuming GS‐GOGAT cycle with high ammonium affinity for nitrogen assimilation, highlights the metabolic adaptability of C. protothecoides exposed to altered nitrogen conditions.     

Biochar improved nodulation and nitrogen metabolism of soybean under salt stress

To investigate salt stress and biochar application effects on nodulation and nitrogen metabolism of soybeans (Glycine max cv. M7), an experiment was conducted under the control condition. The treatments comprised three biochar rates (non, 50 and 100 g kg^?1 soil) and three salinities (0, 5 and 10 dS m^?1 NaCl), with four replications of treatments. Salt stress diminished the number of nodules and their weights in the soybean roots. Nitrogen content and metabolism decreased in nodules, roots and shoots, while reducing the activity of glutamate dehydrogenase (GDH), glutamine synthetase (GS), glutamine oxoglutarate aminotransferase (GOGAT) and nitrate reductase (NR). Also, salinity brought down root and shoot weight, total plant biomass, chlorophyll content, leaf area (LA) and rubisco activity in the soybean. On the other hand, application of biochar improved nodulation, nitrogen content, rubisco activity, GDH, GS, GOGAT and NR activities in different parts of the soybean and nodules under salt stress, and consequently improved chlorophyll content, LA, root and shoot weight. Both the 50 and 100 g kg^?1 biochar rates showed similar effects in improving nitrogen metabolism and plant performance under salt stress. Generally, biochar increased nodulation and nitrogen metabolism of the soybean under saline conditions.     

Root and Nodule Enzymes of Ammonia Assimilation in Two Plant-Conditioned Symbiotically Ineffective Genotypes of Alfalfa (Medicago sativa L.)

Biochemical and physiological parameters associated with nitrogen metabolism were measured in nodules and roots of glasshouse-grown clones of two symbiotically ineffective alfalfa (Medicago sativa L.) genotypes supplied with either NO₃⁻ or NH₄⁺. Significant differences were observed between genotypes for nodule soluble protein concentrations and glutamine synthetase (GS) and glutamate synthase (GOGAT) specific activities, both in untreated controls and in response to applied N. Nodule soluble protein of both genotypes declined in response to applied N, while nodule GS, GOGAT, and glutamate dehydrogenase (GDH) specific activities either decreased or remained relatively constant. In contrast, no genotype differences were observed in roots for soluble protein concentrations and GS, GOGAT, and GDH specific activities, either in untreated controls or in response to applied N. Root soluble protein levels and GS and GOGAT specific activities of N-treated plants increased 2- to 4-fold within 4 days and then decreased between days 13 and 24. Root GDH specific activity of NH₄⁺-treated plants increased steadily throughout the experiment and was 50 times greater than root GS or GOGAT specific activities by day 24.     

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