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。     

Resistance to l-methionine-S-sulfoximine in Chlamydomonas reinhardtii is due to an alteration in a general amino acid transport system

It was suggested that the mutant ARF1 of Chlamydomonas reinhardtii is resistant to l-methionine-S-sulfoximine (MSX, an irreversible inhibitor of glutamine synthetase, EC 6.3.1.2) because this strain degraded and utilized this compound as a nitrogen source for growth (A.R. Franco et al., 1996, Plant Physiol 110: 1215–1222). Resistance to MSX has now been characterized in a double mutant of this alga, called MPA1, which is resistant to MSX and lacks l-amino acid oxidase (LAO activity, EC 1.4.3.2). Biochemical and genetic evidence indicate that the mutant MPA1 is altered in the same MSX-resistance locus as mutant ARF1. However, mutant MPA1 neither degraded nor utilized MSX as a nitrogen source. This led us to conclude that (i) resistance to MSX is not linked to its utilization, and (ii) that LAO activity accounts for the degradation of MSX in mutant ARF1. Data indicate that C. reinhardtii possesses a broad-specificity carrier system responsible for the transport of arginine and other amino acids, including MSX. We propose that the alteration of this carrier confers resistance to MSX in mutants ARF1 and MPA1. Received: 6 April 1998 / Accepted: 8 June 1998     

Ammonium-assimilating enzymes and their regulation in wild and NADP-glutamate dehydrogenase-deficient strains of the ectomycorrhizal fungus Hebeloma cylindrosporum

Hebeloma cylindrosporum strain h 17 was grown on media containing either glutamate or ammonium as nitrogen source. Growth tests and in vitro activity measurements revealed that both glutamine synthetase (GS. EC 6.3.1.2) and NADP-specific glutamate dehydrogenase (NADP-GDH, EC 1.4.1.4) are fully functional in wild type mycelia grown on glutamate or ammonium as sole nitrogen source. However, NADP-GDH appeared to be more active than GS in stationary growing mycelia. NADP-GDH is also able to sustain adequate ammonium assimilation in methionine sulfoximine (MSX)-treated mycelia since they grew as well as mycelia fed with ammonium alone. The NADP-GDH also appeared to be L-glutamate inducible whereas GS was repressed by ammonium. The NADP-GDH deficient strain, when transferred from a glutamate containing medium to an ammonium containing medium, exhibited a derepressed GS, although this enzyme did not fully substitute for the deficiency of NADP-GDH in ammonium assimilation. The low NADP-GDH activity of the mutant strain exhibited a reduced mobility on a 6% constant polyacrylamide gel. By contrast, the two enzymes had identical molecular weights, estimated to be ca 295 kDa on gradient polyacrylamide gel. The involvement of NADP-GDH and GS enzymes in nitrogen assimilation is discussed.     

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 carbon and nitrogen flow by glutamate synthase in Neurospora crassa

A glycine-resistant Neurospora crassa mutant (am-132;glyr), derived from the am-132 mutant, was isolated and characterized. [am-132 itself has a deletion in the structural gene for NADP-dependent glutamate dehydrogenase (GDH).] This new mutation also conferred resistance to serine and methionine sulphoximine (MS), which are inhibitors of glutamine synthetase (GS). In addition, the mutant obtained grew better on ammonium than the am-132 parental strain. Resistance to glycine was not due to increased synthesis of glutamine by an altered or induced GS, nor to increased glutamate synthesis by induction of the catabolic NAD-dependent GDH, nor to NADH-dependent glutamate synthase (GOGAT), which was as sensitive to inhibitors as the GOGAT from the parental strain. The glycine-resistance mutation lowered but did not abolish the carbon flow; this resulted in a lower content of tricarboxylic acid cycle intermediates. GOGAT activity was inhibited in vitro by several organic acids and methionine sulphone (MSF). The higher growth rate of the glycine-resistant mutant on ammonium or on ammonium plus glycine, serine or MS was explained by an increased capacity of GOGAT to synthesize glutamate in vivo due to a lower content of inhibitory tricarboxylic acid cycle intermediates; the higher glutamate content overcomes the effect of the GS inhibitors and explains the MSF resistance of the mutant.     

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.     

Regulation of urease and ammonia assimilatory enzymes in Selenomonas ruminantium.

Urease and glutamine synthetase activities in Selenomonas ruminantium strain D were highest in cells grown in ammonia-limited, linear-growth cultures or when certain compounds other than ammonia served as the nitrogen source and limited the growth rate in batch cultures. Glutamate dehydrogenase activity was highest during glucose (energy)-limited growth or when ammonia was not growth limiting. A positive correlation (R = 0.96) between glutamine synthetase and urease activities was observed for a variety of growth conditions, and both enzyme activities were simultaneously repressed when excess ammonia was added to ammonia-limited, linear-growth cultures. The glutamate analog methionine sulfoximine (MSX), inhibited glutamine synthetase activity in vitro, but glutamate dehydrogenase, glutamate synthase, and urease activities were not affected. The addition of MSX (0.1 to 100 mM) to cultures growing with 20 mM ammonia resulted in growth rate inhibition that was dependent upon the concentration of MSX and was overcome by glutamine addition. Urease activity in MSX-inhibited cultures was increased significantly, suggesting that ammonia was not the direct repressor of urease activity. In ammonia-limited, linear-growth cultures, MSX addition resulted in growth inhibition, a decrease in GS activity, and an increase in urease activity. These results are discussed with respect to the importance of glutamine synthetase and glutamate dehydrogenase for ammonia assimilation under different growth conditions and the relationship of these enzymes to urease.     

NITROGEN METABOLISM AND AMINO ACID NUTRITION IN THE SOIL ALGA STICHOCOCCUS BACILLARIS (CHLOROPHYCEAE)1

Nitrate-grown cells of Stichococcus bacillaris Naeg. (UTEX 314) contained much higher activities of glutamine synthetase (GS) and NADPH-glutamate dehydrogenase (GDH) than ammonium-grown cells. Methylamine, a non-metabolizable ammonium analog, caused a decrease in GS activity in nitrate-grown cells suggesting that GS is regulated by the size of the endogenous ammonium pool. The decrease in GS observed in methylammonium-loaded nitrate-grown cells was accompanied by an increase in NADPH-GDH activity. Stichococcus bacillaris can be grown in the presence of methionine sulfoximine (MSX), a potent inhibitor of GS. However, only a fraction of a control cell population showed a requirement for glutamine or arginine for growth following MSX addition. Fully adapted MSX-grown cells were indistinguishable from control cells in their ability to photosynthesize and utilize amino acids as nitrogen sources. Alanine, arginine, asparagine, glutamine, glycine and proline were good nitrogen sources, and maximum capacity for amino acid transport was developed in cells grown on these amino acids. Compared to nitrate-grown cells the activity of GS in ammo acid-grown cells was low, whereas NADPH-GDH was very active. The activity of NADH-GDH in amino acid-grown cells was highest under heterotrophic conditions.     

Effect of methionine sulfoximine on asparaginase activity and ammonium levels in pea leaves

In developing leaves of Pisum sativum the levels of ammonium did not change during the light-dark photoperiod even though asparaginase (EC 3.5.1.1) did; asparaginase activity in detached leaves doubled during the first 2.5 hours in the light. When these leaves were supplied with 1 millimolar methionine sulfoximine (MSX, an inhibitor of glutamine synthetase, GS, activity) at the beginning of the photoperiod, levels of ammonium increased 8-to 10-fold, GS activity was inhibited 95%, and the light-stimulated increase in asparaginase activity was completely prevented, and declined to less than initial levels. When high concentrations of ammonium were supplied to leaves, the light-stimulated increase of asparaginase was partially prevented. However, it was also possible to prevent asparaginase increase, in the absence of ammonium accumulation, by the addition of MSX together with aminooxyacetate (AOA, which inhibits transamination and some other reactions of photorespiratory nitrogen cycling). AOA alone did not prevent light-stimulated asparaginase increase; neither MSX, AOA, or elevated ammonium levels inhibited the activity of asparaginase in vitro. These results suggest that the effect of MSX on asparaginase increase is not due solely to interference with photorespiratory cycling (since AOA also prevents cycling, but has no effect alone), nor to the production of high ammonium concentration or its subsequent effect on photosynthetic mechanisms. MSX must have further inhibitory effects on metabolism. It is concluded that accumulation of ammonium in the presence of MSX may underestimate rates of ammonium turnover, since liberation of ammonium from systems such as asparaginase is reduced by the effects of MSX.     

酰胺态氮瞬间调节荚膜红假单孢菌光合固氮活性的GS传感机制

天门冬酰胺(Asn)和谷氨酰胺(Gln)对荚膜红假单孢菌固氮酶活性抑制,在表观上类似于氨关闭效应,这种抑制效应由GS参与,相似于氨抑的传感机制。中断Gln代谢的6-diazo-5-oxo-L-norleucine(DON)存在时,氨抑的持续时间延长,与此相类似,Gln抑制加剧,这可能归之于Gln的积累。但是,Gln抑制被methionine sulfoximine(MSX,GS的抑制剂)消除,消除时MSX对Gln的浓度比值约为0.2,与氨抑消除所需的MSX对氨的浓度比值相当。此外,MSX消除氨抑不为DON拮抗,表明Gln抑制固氮酶活性由GS传感。然而,不能抑制GS转谷酰基活性的methionine suffone(MSF,谷氨酸的类似物)却与MSX相同,能消除Gln和氨对固氮活性的抑制。上述观察结果也可延伸至Asn的关闭固氮酶活性效应。     

Rhizobium sp. strain ORS571 ammonium assimilation and nitrogen fixation.

Among rhizobia studied, Rhizobium sp. strain ORS571 alone grew unambiguously on N2 as sole N source. In ORS571 , only the glutamine synthetase (GS)-glutamate synthase ( GOGAT ) pathway assimilated ammonium. However, ORS571 exhibited two unique physiological aspects of this pathway: ORS571 had only GS I, whereas all other Rhizobiaceae studied had both GS I and GS II, and both NADPH- and NADH-dependent GOGAT activities were present. ORS571 GS-affected and NADPH- GOGAT -affected mutant strains were defective in both ammonium assimilation (Asm-) and N2 fixation (Nif-) in culture and in planta ; NADH- GOGAT mutants were Asm- but Nif+. "Bacteroid" GS activity was essentially nil, suggesting symbiotic ammonium export. Physiological studies on effects of glutamine, ammonium, methionine sulfoximine, and diazo-oxo-norleucine on nitrogenase induction in culture implied a regulatory role for the intracellular glutamine pool.     

Nitrogen control of atrazine utilization in Pseudomonas sp. strain ADP

Pseudomonas sp. strain ADP uses the herbicide atrazine as the sole nitrogen source. We have devised a simple atrazine degradation assay to determine the effect of other nitrogen sources on the atrazine degradation pathway. The atrazine degradation rate was greatly decreased in cells grown on nitrogen sources that support rapid growth of Pseudomonas sp. strain ADP compared to cells cultivated on growth-limiting nitrogen sources. The presence of atrazine in addition to the nitrogen sources did not stimulate degradation. High degradation rates obtained in the presence of ammonium plus the glutamine synthetase inhibitor MSX and also with an Nas(-) mutant derivative grown on nitrate suggest that nitrogen regulation operates by sensing intracellular levels of some key nitrogen-containing metabolite. Nitrate amendment in soil microcosms resulted in decreased atrazine mineralization by the wild-type strain but not by the Nas(-) mutant. This suggests that, although nitrogen repression of the atrazine catabolic pathway may have a strong impact on atrazine biodegradation in nitrogen-fertilized soils, the use of selected mutant variants may contribute to overcoming this limitation.     

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)     

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.     

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.     

Isolation and characterization of a dihydropyrimidine dehydrogenase mutant of Pseudomonas chlororaphis

A dihydropyrimidine dehydrogenase mutant of Pseudomonas chlororaphis ATCC 17414 was isolated and characterized in this study. Initially, reductive catabolism of uracil was confirmed to be active in ATCC 17414 cells. Following chemical mutagenesis and d-cycloserine counterselection, a mutant strain unable to utilize uracil as a nitrogen source was identified. It was also unable to utilize thymine as a nitrogen source but could use either dihydrouracil or dihydrothymine as a sole source of nitrogen. Subsequently, it was determined that the mutant strain was deficient for the initial enzyme in the reductive pathway dihydropyrimidine dehydrogenase. The lack of dehydrogenase activity did not seem to have an adverse effect upon the activity of the second reductive pathway enzyme dihydropyrimidinase activity. It was shown that both dihydropyrimidine dehydrogenase and dihydropyrimidinase levels were affected by the nitrogen source present in the growth medium. Dihydropyrimidine dehydrogenase and dihydropyrimidinase activities were elevated after growth on uracil, thymine, dihydrouracil or dihydrothymine as a source of nitrogen.     

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

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

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.     

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     

Isolation and characterization of a Neurospora crassa mutant altered in the alpha polypeptide of glutamine synthetase.

We report the isolation and characterization of a Neurospora crassa glutamine synthetase (GS) mutant altered in one of the two polypeptides (GS alpha) of this enzyme. We used the gln-1bR8 mutant strain that synthesizes only the GS alpha monomer and lacks the GS beta monomer and selected for growth in minimal medium in the presence of alpha-methyl-DL-methionine-SR-sulfoximine (alpha-me-MSO), an inhibitor of GS activity. The GS activity of the gln-1bR8;alpha-me-MSOR strain drastically reduced its transferase activity and only slightly reduced its synthetase activity, and it was resistant to inhibition by alpha-me-MSO and L-methionine-DL-sulfoximine. The mutation that overcame the inhibitory effect of alpha-me-MSO also altered the antigenic, kinetic, and physical properties of GS alpha. The low GS activity of the alpha-me-MSO-resistant strain was compensated for by a higher glutamate/glutamine ratio and a lower glutamate synthase activity, allowing this strain to grow as well as the parental strain. The mutation that conferred resistance to alpha-me-MSO was not linked to the gln-1bR8 mutation, providing direct evidence of the existence of two genes involved with the structure of the two polypeptides of N. crassa GS.