Inorganic nitrogen assimilation in the non-N2-fixing cyanobacterium Phormidium laminosum. I. Cellular levels of glutamine synthetase and NADPH-dependent glutamate dehydrogenase

Cell-free extracts of nitrate-grown as well as of ammonium-grown cells of the filamentous non-nitrogen-fixing cyanobacterium Phormidium laminosum (strain OH-1-p.Cl₁) showed detectable levels of both glutamine synthetase (GS, EC 6.3.1.2) and NADPH-dependent glutamate dehydrogenase (GDH, EC 1.4.1.4) activities. The GS level of nitrate-grown cells was higher than that of ammonium-grown cells, whereas the GDH level was higher in ammonium-grown cells and depended on the external ammonium concentration. When nitrate-grown cells were transferred to an ammonium-containing medium, a decrease of GS and an increase of GDH specific activities occurred, even in the presence of nitrate. Conversely, when ammonia-grown cells were transferred to a nitrate-containing medium, an increase of GS and a decrease of GDH-specific activities took place. Both these effects were inhibited by chloramphenicol and were probably mediated by de novo protein synthesis. When either cell type was transferred to a medium without nitrogen source, the specific activities of both enzymes increased. When nitrate-grown cells were transferred to nitrate medium with L-methionine-DL-sulphoximine (MSX) added, the specific activity of GDH also increased. Here we present some evidence that, under certain conditions of nitrogen availability, GDH would play a minor role in ammonium assimilation.     

Immunoelectrophoretic approach to the metabolic regulation of glutamine synthetase in Rhodopseudomonas capsulata E1F1: role of glutamine

Anti-glutamine synthetase serum was raised in rabbits by injecting purified glutamine synthetase (GS) of the phototrophic bacterium Rhodopseudomonas capsulata E1F1. The antibodies were purified to monospecificity by immunoaffinity chromatography in GS-sepharose gel. These anti-GS antibodies were used to measure the antigen levels in crude extracts from bacteria, grown phototrophically with dinitrogen, nitrate, nitrite, ammonia, glutamate, glutamine or alanine as nitrogen sources. The amount of GS detected by rocket immunoelectrophoresis was proportional to Mn²⁺-dependent transferase activity measured in the crude extracts. Addition of GS inhibitor l-methionine-d,l-sulfoximine (MSX) to the actively growing cells promoted increased antigen levels, that were not found in the presence of glutamine or chloramphenicol. The ammonia-induced decrease in GS relative levels was reverted by MSX. GS levels remained constant when phototrophically growing cells were kept in the dark.Abbreviations GS glutamine synthetase - MOPS 2-(N-morpholine) propane sulfonate - MSX l-methionine-d,l-sulfoximine     

Transport and Assimilation of Nitrogen by Stichococcus bacillaris Grown in the Presence of Methionine Sulfoximine

Stichococcus bacillaris Naeg., a green soil alga, can grow in the presence of methionine sulfoximine (MSX), an inhibitor of glutamine synthetase, by maintaining a high level of NADPH-glutamate dehydrogenase activity. MSX-grown cells can utilize both NH₄⁺ and NO₃⁻ as nitrogen source for growth. [¹⁴C]Methylammonium is not metabolized by S. bacillaris, and is transported by a carrier system that obeys Michaelis Menten kinetics, and is insensitive to MSX.     

CONTRASTING EFFECTS OF METHIONINE SULFOXIMINE ON UPTAKE AND ASSIMILATION OF AMMONIUM IN ULVA INTESTINALIS (CHLOROPHYCEAE)1

Ammonium is assimilated in algae by the glutamine synthetase (GS)–glutamine:2‐oxoglutarate aminotransferase pathway. In addition to the assimilation of external ammonium taken up across the cell membrane, an alga may have to reassimilate ammonium derived from endogenous sources (i.e. nitrate reduction, photorespiration, and amino acid degradation). Methionine sulfoximine (MSX), an irreversible inhibitor of GS, completely inhibited GS activity in Ulva intestinalis L. after 12 h. However, assimilation of externally derived ammonium was completely inhibited after only 1–2 h in the presence of MSX and was followed by production of endogenous ammonium. However, endogenous ammonium production in U. intestinalis represented only a mean of 4% of total assimilation attributable to GS. The internally controlled rate of ammonium uptake (V_i) was almost completely inhibited in the presence of MSX, suggesting that V_i is a measure of the maximum rate of ammonium assimilation. After complete inhibition of ammonium assimilation in the presence of MSX, the initial or surge (V_s) rate of ammonium uptake in the presence of 400 μM ammonium chloride decreased by only 17%. However, the amount that the rate of ammonium uptake decreased by was very similar to the uninhibited rate of ammonium assimilation. In addition, the decrease in the rate of ammonium uptake in darkness (in the absence of MSX) in the presence of 400 μM ammonium chloride matched the decrease in the rate of ammonium assimilation. However, in the presence of 10 μM ammonium chloride, MSX completely inhibited ammonium assimilation but had no effect on the rate of uptake.     

Effcts of Blue Light and Ammonia on Nitrogen Metabolism in a Colorless Mutant of Chlorella

Illumination of a colorless mutant of Chlorella vulgaris 1lh(M125) with blue light enhanced both the uptake of nitrate andthe release of ammonia. These effects were not observed underillumination with red light. The release of ammonia was alsoenhanced by the addition of methionine sulphoximine (MSX), aninhibitor of glutamine synthetase (GS). Addition of MSX to culturesin the dark increased the rate of breakdown of starch. Algal cells grown in nitrate-containing medium did not showthe aminating activity of glutamate dehydrogenase (GDH). Additionof large (millimolar) amounts of ammonia in the dark resultedin the induction of NADPH-GDH activity and, in addition, a decreasein GS activity. From these results it appears that GS catalyzesthe primary step in the assimilation of ammonia in algal cellsgrown in nitrate-containing medium. Two isoforms (GS1 and GS2)of GS have been separated by ion exchange chromatography. Theactivities of both isoforms were decreased upon the additionof ammonia. Illumination of the alga with blue light at intensities up to10,000 mW m^–2 enhanced the measurable activity of GS invitro, while higher intensities were ineffective. In red lightno such effect was observed. The effects of blue light and ammonia on nitrogen metabolismin algal cells are discussed. (Received November 25, 1988; Accepted March 6, 1989)     

Regulation of Pseudomonas aeruginosa chemotaxis by the nitrogen source.

The regulation of amino acid chemotaxis by nitrogen was investigated in the gram-negative bacterium Pseudomonas aeruginosa. The quantitative capillary tube technique was used to measure chemotactic responses of bacteria to spatial gradients of amino acids and other attractants. Chemotaxis toward serine, arginine, and alpha-aminoisobutyrate was sharply dependent on the form in which nitrogen was presented to the bacteria. Bacteria grown on mineral salts-succinate with potassium nitrate gave responses to amino acids that were 2 to 3 times those of cells grown on ammonium sulfate and 10 to 20 times those of cells grown in mineral salts-succinate with Casamino Acids as the nitrogen source. A combination of ammonium sulfate and glutamate was as effective as Casamino Acids in depressing serine taxis. The threshold concentration for alpha-aminoisobutyrate taxis was consistently lower in nitrate-grown bacteria than in ammonia-grown bacteria. Responsiveness to sodium succinate, however, was not subject to regulation by nitrogen, and glucose chemotaxis was inhibited, rather than enhanced, in nitrate-grown bacteria. These results indicate that chemotaxis of P. aeruginosa toward amino acids is subject to regulation by nitrogen and that this regulation probably is expressed at the level of the chemoreceptors or transducers.     

Impact of nitrogen assimilation on regulation of antibiotic production in Streptomyces hygroscopicus 155

The enzymes of the assimilation pathways in cultures of S. hygroscopicus grown in the presence of various nitrogen sources were investigated. No assimilation activity of glutamate dehydrogenase (GDH) was observed. Activities of alanine dehydrogenase (ADH), GDH, glutamine: 2-oxoglutarate aminotransferase (GOGAT) and glutamate synthetase (GS) were studied. High concentrations of ammonium and alanine induced ADH formation. The levels of GS remained low in media with NH4Cl. Various nitrogen sources had no impact on the activity of GOGAT which suggested the involvement of constitutive synthesis. ADH was likely to play an alternative role. Determination of the quantitative and qualitative composition of the free amino acids confirmed the involvement of the GS-GOGAT pathway in nitrogen assimilation. The concentration of ammonium ions in the media with one amino acid or in the presence of several amino acids lowered the antibiotic activity while in the media with alanine and the other nitrogen compounds it increased the antibiotic activity.     

Expression of the bacterial gdhA gene encoding a NADPH glutamate dehydrogenase in tobacco affects plant growth and development

We investigated the effects of genetic modification of nitrogen metabolism via the bacterial glutamate dehydrogenase (GDH) on plant growth and metabolism. The gdhA gene from Escherichia coli encoding a NADPH-GDH was expressed in tobacco plants under the control of the 35 S promoter. The specific activity of GDH in gdhA plants was 8-fold of that in E. coli. Damage caused by spray application of 1.35 mM of phosphinothricin (PPT) herbicide, a glutamine synthetase (GS) inhibitor, was less pronounced in gdhA plants as compared with the control plants which suggests that the introduced GDH can assimilate some of the excess ammonium, at least during GS inhibition. However, gdhA plants were susceptible to 2.7 mM PPT. Biomass production was consistently increased in gdhA transgenic plants grown under controlled conditions and in the field. Total free amino acids and total carbohydrates were increased in gdhA plants grown in the greenhouse suggesting that both nitrogen and carbon metabolism were altered. We conclude that the modifications in transgenic plants may result from both increased nitrogen efficiency and altered gene expression and metabolism. This revised version was published online in June 2006 with corrections to the Cover Date.     

Regulation of asparaginase, glutamine synthetase, and glutamate dehydrogenase in response to medium nitrogen concentrations in a euryhaline chlamydomonas species

The ammonium assimilatory enzymes glutamine synthetase (EC 6.3.1.2) and glutamate dehydrogenase (EC 1.4.1.3) were investigated for a possible role in the regulation of asparaginase (EC 3.5.1.1) in a Chlamydomonas species isolated from a marine environment. Cells grown under nitrogen limitation (0.1 millimolar NH(4) (+), NO(3) (-), or l-asparagine) possessed 6 times the asparaginase activity and approximately one-half the protein of cells grown at high nitrogen levels (1.5 to 2.5 millimolar). Biosynthetic glutamine synthetase activity was 1.5 to 1.8 times greater in nitrogen-limited cells than cells grown at high levels of the three nitrogen sources.Conversely, glutamate dehydrogenase (both NADH- and NADPH-dependent activities) was greatest in cells grown at high levels of asparagine or ammonium, while nitrate-grown cells possessed little activity at all concentrations employed. For all three nitrogen sources, glutamate dehydrogenase activity was correlated to the residual ammonium concentration of the media after growth (r = 0.88 and 0.94 for NADH- and NADPH-dependent activities, respectively).These results suggest that glutamate dehydrogenase is regulated in response to ambient ammonium levels via a mechanism distinct from asparaginase or glutamine synthetase. Glutamine synthetase and asparaginase, apparently repressed by high levels of all three nitrogen sources, are perhaps regulated by a common mechanism responding to intracellular nitrogen depletion, as evidenced by low cellular protein content.     

Effect of glutamine on glutamine-synthetase regulation in the green alga Monoraphidium braunii

Glutamine-synthetase (GS; EC 6.3.1.2) activity and protein levels were measured in crude extracts from Monoraphidium braunii Näegeli, strain 202-7d, cultures grown under different nitrogen sources. Only ammonium and l-glutamine promoted a partial enzyme inactivation, which, in the case of l-glutamine, was accompanied by a significant repression of GS. Methionine sulfoximine (MSX), a strong inhibitor of GS, produced a drastic inactivation of GS which was concomitant with a marked increase in GS protein as measured by rocket immunoelectrophoresis. Such an increase was prevented in the presence of cycloheximide. The effect of the l-glutamine analog on GS activity and protein was partially inhibited if l-glutamine was also added to cell cultures, possibly indicating competition in the transport of these two substances. In addition, the effects of MSX were reversed after longer times when cultures were treated with smaller concentrations of inhibitor. Treatment of cell cultures with azaserine, a specific inhibitor of glutamate synthase, the second enzyme acting in the ammonium assimilation pathway, promoted a strong GS inactivation and a partial repression of this enzyme, which paralleled a specific increase in the intracellular pools of glutamine High-performance liquid chromatography measurements of intracellular amino-acid concentrations showed that glutamine levels correlated negatively with GS concentration. A role for glutamine as a negative effector of GS synthesis is proposed.Abbreviations GS l-glutamine synthetase - GOGAT l-glu-tamine:2-oxoglutarate amidotransferase - MSX methionine sulfoximine During the course of this work, J.A. was supported by a fellowship from Junta de Andalucía, and J.M. G-F. by a fellowship from the Spanish Ministerio de Educatión y Ciencia. This work was supported by the Junta de Andalucía.     

Glutamine synthetase of Streptomyces cattleya: purification and regulation of synthesis

Glutamine synthetase (GS; EC 6.3.1.2) from Streptomyces cattleya was purified using a single affinity-gel chromatography step, and some of its properties were determined. Levels of GS in S. cattleya cells varied by a factor of 8 depending upon the source of nitrogen in the growth medium. Of 24 nitrogen sources examined only glutamine or NH4Cl utilization resulted in very low GS activity. Addition of NH4Cl to a culture with high GS levels appeared to stop further synthesis and resulted in a progressive decrease in the specific activity of the enzyme. The GS inhibitor methionine sulphoximine (MSX) inhibited GS activity but had no effect on exponentially growing cells. The presence of MSX either lengthened or shortened the period between spore inoculation and initiation of exponential growth, depending on the source of nitrogen. In glutamine minimal medium MSX produced earlier and more efficient spore germination while in glutamate or nitrate minimal medium germination was delayed by its presence.     

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.     

Nitrogen Metabolism of the Marine Microalga Chlorella autotrophica

The levels of glutamine synthetase (GS) and glutamate dehydrogenase (GDH) in Chlorella autotrophica (clone 580) are strongly regulated by the nitrogen source and salt concentration of the medium. GS is present at high levels in NO₃⁻-grown cells, and at maximum levels in nitrogen-starved cells. However, the levels of GS in these cells are somewhat decreased by increasing salinity. Cells growing on NH₄⁺ have high NADPH-GDH activity, the levels of which increase with increasing NH₄⁺ supply, while GS decreases to a very low level under these conditions. Salinity intensifies the induction of NADPH-GDH activity in NH₄⁺-grown cells. The levels of NADH-GDH are low in this alga, but present under all growth conditions. Methionine sulfoximine (MSX) has little effect on growth and nitrogen assimilation of the alga in the presence of NH₄⁺.     

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.     

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.     

The Relationship between Inorganic Nitrogen Metabolism and Proline Accumulation in Osmoregulatory Responses of Two Euryhaline Microalgae

Chlorella autotrophica, a euryhaline marine alga, and Stichococcus bacillaris, a salt-tolerant soil alga, grow in the presence of methionine sulfoximine (MSX), an inhibitor of glutamine synthetase, by maintaining high levels of NADPH-glutamate dehydrogenase. Nitrate reductase showed no change in MSX-adapted cells. For both species, MSX-adapted cells retained their capacity to accumulate proline in response to salinity, and in S. bacillaris no major shift was observed in the presence of MSX toward the accumulation of sorbitol. Following transfer from 33 to 150% artificial seawater (ASW), both algae exhibited increases in organic solute levels without a lag. Within 6 h of this sudden increase in salinity, the levels of proline in C. autotrophica and of proline and sorbitol in S. bacillaris were similar to those found in steady state 150% ASW cultures. Following transfer from 33 to 150% ASW, S. bacillaris continued [¹⁴C] bicarbonate photoassimilation at a normal rate and maintained active enzymes of nitrogen assimilation. The incorporation of [¹⁴C]phenylalanine into proteins was inhibited for about 30 minutes in MSX-free cells and 90 minutes in MSX-adapted cells following transfer from 33 to 150% ASW; the recovery after these lag periods was almost complete.     

Regulation of nitrate assimilation in the cyanobacterium Phormidium laminosum

The intracellular ratio of 2-oxoglutarate to glutamine has been analyzed under nutritional conditions leading to different activity levels of nitrate-assimilating enzymes in Phormidium laminosum (Agardh) Gom. This non-N₂-fixing cyanobacterium adapted to the available nitrogen source by modifying its nitrate reductase (NR; EC 1.7.7.2), nitrite reductase (NiR; EC 1.7.7.1) and glutamine synthetase (GS; EC 6.3.1.2) activities. The 2-oxoglutarate/glutamine ratio was similar in cells adapted to grow with nitrate or ammonium. However, metabolic conditions that increased this ratio [i.e., nitrogen starvation or l-methionine-d,l-sulfoximine (MSX) treatment] corresponded to high activity levels of NR, NiR, GS (except in MSX-treated cells) and glutamate synthase (GOGAT; EC 1.4.7.1). By contrast, metabolic conditions that diminished this ratio (i.e., addition of ammonium to nitrate-growing cells or addition of nitrate or ammonium to nitrogen-starved cells) resulted in low activity levels. The variation in the 2-oxoglutarate/glutamine ratio preceded the changes in enzyme activities. These results suggest that changes in the 2-oxoglutarate/glutamine ratio could be the signal that triggers the adaptation of P. laminosum cells to variations in the available nitrogen source, as occurs in enterobacteria.Abbreviations Chl chlorophyll - GOGAT ferredoxin-dependent glutamate synthase (EC 1.4.7.1) - GS glutamine synthetase (EC 6.3.1.2) - MSX l-methionine-d,l-sulfoximine - NiR nitrite reductase (EC 1.7.7.1) - NR nitrate reductase (EC 1.7.7.2) - TP total protein This work has been partially supported by grants from the Spanish Ministry of Education and Science (DGICYT PB88-0300 and PB92-0464) and the University of the Basque Country (042.310-EC203/94). M.I.T. was the recipient of a fellowship from the Basque Government.     

Characterization of a Mutant of Chlamydomonas reinhardtii That Uses L-Methionine-S-Sulfoximine and Phosphinothricin as Nitrogen Sources for Growth

A strain of Chlamydomonas reinhardtii, named ARF-1, which grows with the glutamine synthetase (GS) inhibitor L-methionine-S-sulfoximine (MSX), has been isolated and characterized. Mutant ARF-1 is affected at a single and dominant gene, tentatively assigned to the allele msr-1-2. Neither the uptake of ammonia nor the two GS isoenzyme activities of the mutant were affected by MSX in vivo. GS activities, however, were fully abolished in vitro, thus suggesting that neither GS isoform was an altered enzyme resistant to the inhibitor. Resistance to MSX does not seem to be due to either a defect in a permease responsible for the transport of MSX or over-expression of GS activity, nor did we find an alternative enzymatic pathway for the assimilation of ammonium. Resistance was independent of the nitrogen source used and was strongly enhanced by the addition of acetate. Unlike the parental strain, mutant ARF-1 can degrade and utilize MSX as the sole nitrogen source for growth, which could account for the observed resistance. Thus, this mutant can be classified as a novel type of MSX-resistant mutant. This mutant can also use phosphinothricin, methionine sulfone, or methionine sulfoxide as the sole sources of nitrogen. This capability cosegregated in the genetic crosses and was also observed in all the diploids isolated. An MSX/[alpha]-ketoglutarate aminotransferase activity, not present in the parental strain 305, was detected in mutant ARF-1 cells. Therefore, we propose that the locus msr-1-2 either codes for this transaminase activity or its product gene is necessary to express this transaminase activity.     

A common transport system for methionine, L-methionine-DL-sulfoximine (MSX), and phosphinothricin (PPT) in the diazotrophic cyanobacterium Nostoc muscorum

We present evidence, for the first time, of the occurrence of a transport system common for amino acid methionine, and methionine/glutamate analogues l-methionine-dl-sulfoximine (MSX) and phosphinothricin (PPT) in cyanobacterium Nostoc muscorum. Methionine, which is toxic to cyanobacterium, enhanced its nitrogenase activity at lower concentrations. The cyanobacterium showed a biphasic pattern of methionine uptake activity that was competitively inhibited by the amino acids alanine, isoleucine, leucine, phenylalanine, proline, valine, glutamine, and asparagine. The methionine/glutamate analogue-resistant N. muscorum strains (MSX-R and PPT-R strains) also showed methionine-resistant phenotype accompanied by a drastic decrease in ³⁵S methionine uptake activity. Treatment of protein extracts from these mutant strains with MSX and PPT reduced biosynthetic glutamine synthetase (GS) activity only in vitro and not in vivo. This finding implicated that MSX- and PPT-R phenotypes may have arisen due to a defect in their MSX and PPT transport activity. The simultaneous decrease in methionine uptake activity and in vitro sensitivity toward MSX and PPT of GS protein in MSX- and PPT-R strains indicated that methionine, MSX, and PPT have a common transport system that is shared by other amino acids as well in N. muscorum. Such information can become useful for isolation of methionine-producing cyanobacterial strains.     

Glutamine metabolism regulation in Chlorella pyrenoidosa. Regulation of Chlorella glutamine synthetase activity by amino acids]

Effect of glutamine and its metabolites (amino acids) on Chlorella glutamine synthetase (GS) (E.C.6.3.1.2) in the presence of Mg or Mn was studied. Purified GS preparation was used, isolated from Chlorella grown in the presence of NH as a sole nitrogen source. Glutamate, aspartate, alanine and glycine inhibit GS activity in the presence of both Mg and Mn. Tryptophane and valine (up to 15 mM) activate GS in the presence of Mn. Tryptophane inhibits GS in the system with Mg. Sinergistic inhibition was observed under the combined effect of amino acids on GS in the presence of Mn and aspartate or alanine. The change of GS activity observed is supposed to be due to the inhibitory effect of glutamine and amino acids studied, since the glutamine content is increased (in 2.5 times for 5 min) and that of alanine and dicarbonic amino acids (for the following 15 min) under NH assimilation in Chlorella cells.