Evidence for an involvement of glutamine synthetase in regulation of nitrogenase activity in Rhodopseudomonas capsulata

In the presnet studies with whole cells and extracts of the photosynthetic bacterium Rhodopseudomonas capsulata the rapid inhibition of nitrogenase dependent activities (i.e. N₂-fixation acetylene reduction, or photoproduction of H₂) by ammonia was investigated. The results suggest, that the regulation of the nitrogenase activity by NH ₄ ⁺ in R. capsulata is mediated by glutamine synthetase (GS). (i) The glutamate analogue methionine sulfoximine (MSX) inhibited GS in situ and in vitro, and simultaneously prevented nitrogenase activity in vivo. (ii) When added to growing cultures ammonia caused rapid adenylylation of GS whereas MSX abolished the activity of both the adenylylated and unadenylylated form of the enzyme. (iii) Recommencement of H₂ production due to an exhaustion of ammonia coincided with the deadenylylation of GS. (iv) In extracts, the nitrogenase was found to be inactive only when NH ₄ ⁺ or MSX were added to intact cells. Subsequently the cells had to be treated with cetyltrimethylammonium bromide (CTAB). (v) In extracts the nitrogenase activity declined linearily with an increase of the ration of adenylylated vs. deadenylylated GS. A mechanism for inhibition of nitrogenase activity by ammonia and MSX is discussed.Abbreviations BSA bovin serum albumine - CTAB cetyltrimethylammonium bromide - GOGAT l-glutamine: 2-oxoglutarate amino transferase - GS glutamine synthetase - HEPES N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid - MSX l-methionine-d,l-sulfoximine     

Fixation of [13N]N2 and transfer of fixed nitrogen in the Anthoceros-Nostoc symbiotic association

The initial product of fixation of [¹³N]N₂ by pure cultures of the reconstituted symbiotic association between Anthoceros punctatus L. and Nostoc sp. strain ac 7801 was ammonium; it accounted for 75% of the total radioactivity recovered in methanolic extracts after 0.5 min and 14% after 10 min of incubation. Glutamine and glutamate were the primary organic products synthesized from [¹³N]N₂ after incubation times of 0.5–10 min. The kinetics of labeling of these two amino acids were characteristic of a precursor (glutamine) and product (glutamate) relationship. Results of inhibition experiments with methionine sulfoximine (MSX) and diazo-oxonorleucine were also consistent with the assimilation of N₂-derived NH ₄ ⁺ by Anthoceros-Nostoc through the sequential activities of glutamine synthetase (EC 6.3.1.2) and glutamate synthase (EC 1.4.7.1), with little or no assimilation by glutamate dehydrogenase (EC 1.3.1.3). Isolated symbiotic Nostoc assimilated exogenous ¹³NH ₄ ⁺ into glutamine and glutamate and their formation was inhibited by MSX, indicating operation of the glutamine synthetase-glutamate synthase (GS-GOGAT) pathway: However, relative to free-living cultures, isolated symbiotic Nostoc assimilated 80% less exogenous ammonium into glutamine and glutamate, implying that symbiotic Nostoc could assimilate only a fraction of N₂-derived NH ₄ ⁺ . This implication was tested by using Anthoceros associations reconstituted with wild-type or MSX-resistant strains of Nostoc incubated with [¹³N]N₂ in the presence of MSX. The results of these experiments indicated that, in situ, symbiotic Nostoc assimilated about 10% of the N₂-derived NH ₄ ⁺ and that NH ₄ ⁺ was made available to Anthoceros tissue where it was apparently assimilated by the GS-GOGAT pathway. Since less than 1% of the fixed N₂ was lost to the suspension medium, it appears that transfer of NH ₄ ⁺ from symbiont to host tissue was very efficient in this extracellular symbiotic association.Abbreviations DON 6-diazo-5-oxo-l-norleucine - GDH glutamate dehydrogenase - GOGAT glutamate synthase - GS glutamine synthetase - MSX l-methionine-dl-sulfoximine     

Photoproduction of ammonium ion from N2 in Rhodospirillum rubrum.

NH+4 excretion was undetectable in N2-fixing cultures of Rhodospirillum rubrum (S-1) and nitrogenase activity in these cultures was repressed by the addition of 10 mM NH+4 to the medium. The glutamate analog, L-methionine-DL-sulfoximine (MSX), derepressed N2 fixation even in the presence of 10 mM extracellular NH+4. When 10 mg MSX/ml was added to cultures just prior to nitrogenase induction they developed nitrogenase activity (20% of the control activities) and excreted most of their fixed N2 as NH+4. Nitrogenase activities and NH+4 production from fixed N2 were increased considerably when a combined nitrogen source, NH+4 (greater than 40 mumoles NH+4/mg cell protein in 6 days) or L-glutamate (greater than 60 mumoles NH+4/ mg cell protein in 6 days) was added to the cultures together with MSX. Biochemical analysis revealed that R. rubrum produced glutamine synthetase and glutamate synthase (NADP-dependent) but no detectable NADP-dependent glutamate dehydrogenase. The specific activity of glutamine synthetase was observed to be maximal when nitrogenase activity was also maximal. Nitrogenase and glutamine synthetase activities were repressed by NH+4 as well as by glutamate. The results demonstrate that utilization of solar energy to photoproduce large quantities of NH+4 from N2 is possible with photosynthetic bacteria by interfering with their regulatory control of N2 fixation.     

Assimilation of exogenous and dinitrogen-derived13NH 4 + byAnabaena azollae separated fromAzolla caroliniana Willd

Anabaena azollae was isolated fromAzolla caroliniana by the gentle roller method and differential centrifugation. Incubation of suchAnabaena preparations for 10 min with [¹³N]N₂ resulted in the formation of four radioactive compounds; ammonium, glutamine, glutamate and alanine. Ammonium accounted for 66% of the total radioactivity recovered and 58% of the ammonium was in an extracellular fraction. Since essentially no extracellular¹³N-labeled organic compounds were found, it appears that ammonium is the compound most probably made available toAzolla during dinitrogen-dependent growth of the association.The kinetics of incorporation of exogenous¹³NH ₄ ⁺ into glutamine and glutamate were characteristic of a precursor (glutamine)-product (glutamate) relationship and consistent with assimilation by the glutamine synthetase-glutamate synthase pathway. The results of experiments using the glutamine synthetase inhibitor, methionine sulfoximine, the glutamate synthase inhibitor, diazo-oxonorleucine, and increasing the ammonium concentration to greater than 1 mM, provided evidence for assimilation primarily by the glutamine synthetase-glutamate synthase pathway with little or no contribution from biosynthetic glutamate dehydrogenase.While showing that N₂ fixation and NH ₄ ⁺ assimilation were not tightly coupled metabolic processes in symbioticAnabaena, these results reflect a composite picture and do not indicate the extent to which ammonium assimilatory enzymes might be regulated in filaments associated with specific stages in theAzolla-Anabaena developmental profile.Non-standard abbreviations DON 6-Diazo-5-oxo-l-norleucine - GDH glutamate dehydrogenase - GOGAT glutamate synthase - GS glutamine synthetase - MSX l-methionine-Dl-sulfoximine     

Interactions between cyanobacterium and fungus during 15N2-incorporation and metabolism in the lichen Peltigera canina

On following N₂-incorporation and subsequent metabolism in the lichen Peltigera canina using ¹⁵N as tracer, it was found, over a 30 min period, that greatest initial labelling was into NH ₄ ⁺ followed by glutamate and the amide-N of glutamine. Labelling of the amino-N of glutamine, aspartate and alanine increased slowly. Pulse-chase experiments using ¹⁵N confirmed this pattern. On inhibiting the GS-GOGAT pathway using l-methionine-dl-sulphoximine and azaserine, ¹⁵N enrichment of glutamate, alanine and aspartate continued although labelling of glutamine was undetectable. From this and enzymic data, NH ₄ ⁺ assimilation in the P. canina thallus appears to proceed via GS-GOGAT in the cyanobacterium and via GDH in the fungus; aminotransferases were present in both partners. The cyanobacterium assimilated 44% of the ¹⁵N₂ fixed; the remainder was liberated almost exclusively as NH ₄ ⁺ and then assimilated by fungal GDH.Abbreviations ADH alanine dehydrogenase - APT aspartate-pyruvate aminotransferase - AOA aminooxyacetate - GDH glutamate dehydrogenase - GOT glutamate-oxaloacetate aminotransferase - GOGAT glutamate synthase - GPT glutamate-pyruvate aminotransferase - GS glutamine synthetase - HEPES 4-(2-hydroxyethyl)-1-piperazine ethanesulphonic acid - MSX l-methionine-dl-sulphoximine     

Enzymes of ammonia assimilation and their regulation inAzospirillum lipoferum strain D-2

Azospirillum lipoferum strain D-2 possesses the following enzymes for the assimilation of N₂ and NH ₄ ⁺ : nitrogenase, glutamine synthetase, NADPH-dependent glutamate synthase, NADH-/NADPH-dependent glutamate dehydrogenase, and NADH-dependent alanine dehydrogenase. Nitrogenase and glutamine synthetase are repressed, whereas glutamate dehydrogenase and alanine dehydrogenase are induced by NH ₄ ⁺ . Glutamine synthetase activity is modulated by both repression and depression and also by adenylylation.     

Alteration of cyanobacterial glutamine synthetase activity in vivo in response to light and NH 4 +

Extractable glutamine synthetase activity of the cyanobacterium Anabaena cylindrica was reduced by approximately 50% when N₂-fixing cultures were treated with 10 mM NH ₄ ⁺ or were placed in darkness. The deactivated enzyme could be rapidly reactivated (within 5 min) by adding 40 mM 2-mercaptoethanol to the biosynthetic reaction mixture. The enzyme could also be reactivated in vivo by replacing the culture in light or by removing NH ₄ ⁺ . When the enzyme was deactivated by simultaneously adding NH ₄ ⁺ and placing the culture in darkness, reactivation occurred on reillumination and removal of NH ₄ ⁺ . The removal of NH ₄ ⁺ in darkness did not result in reactivation. On in vitro reactivation of glutamine synthetase from dark or NH ₄ ⁺ -treated cultures the maximum glutamine synthetase activity observed frequently exceeded that of glutamine synthetase extracted from untreated cultures. Anacystis nidulans showed a similar type of reversible dark deactivation to A. cylindrica but Plectonema boryanum and a Nostoc did not. With A. cylindrica, a direct positive correlation between the size of the intracellular pool of glutamate and biosynthetic glutamine synthetase activity occurred during light/dark shifts, and on treatment with NH ₄ ⁺ . The changes in activity of glutamine synthetase in A. cylindrica in response to light resemble in some respects the light modulation of enzymes of the oxidative and reductive pentose phosphate pathways noted in cyanobacteria by others.     

Different ammonium-ion uptake,metabolism and detoxification efficiencies in two Lemnaceae

¹⁵N-Nuclear magnetic resonance spectroscopy was used to follow nitrogen assimilation and amino-acid production in Wolffia arrhiza (L.) Hork. ex. Wimmer, clone Golan exposed to 4.0 mM ¹⁵NH₄Cl solutions for 24 h. The main ¹⁵N-labelled metabolites were asparagine and glutamine, as well as substantial amounts of unreacted, intracellular NH ₄ ⁺ . These results were compared with those of a previous study on Lemna gibba L. clone Hurfeish (Monselise et al., 1987, New Phytol. 10, 341–345) with regard to NH ₄ ⁺ uptake, assimilation and detoxification efficiencies. Both species, grown under continuous white light, were capable of preferential uptake of NH ₄ ⁺ in the presence of nitrate. Relative growth rates indicate that both species tolerate increased levels of NH ₄ ⁺ , up to 10^–2 mol · 1^–1, with L. gibba showing a slightly greater tolerance. No ¹⁵N-labelled free NH ₄ ⁺ was detectable in L. gibba, while in W. arrhiza excess NH ₄ ⁺ was found within the cells. This fact indicates that L. gibba is more efficient in detoxification than W. arrhiza, presumably because of inability of W. arrhiza to regenerate the NH ₄ ⁺ traps, glutamate and aspartate, rapidly enough. This is also evident from the observation that addition of -ketoglutarate to the medium caused nearly complete assimilation of intracellular NH ₄ ⁺ in W. arrhiza. In both plants, addition of -ketoglutarate increased both NH ₄ ⁺ uptake and assimilation. Addition of l-methionine dl-sulfoximine, an inhibitor of glutamine synthetase inhibited NH ₄ ⁺ assimilation, while addition of azaserine, an inhibitor of glutamate synthase, resulted in ¹⁵N incorporation into the glutamine-amide position only. These results are consistent with the glutamine synthetase-glutamate synthase pathway being the major route of NH ₄ ⁺ assimilation in the two plants under the conditions used.Abbreviations AZA azaserine (O-diazoacetyl-l-serine) - GOGAT glutamine oxoglutarate amine transferase=]glutamate synthase E.C. 1.4.7. and E.C. 1.4.1.13. - GS glutamine synthetase E.C. 6.3.1.2. - -KG -ketoglutarate=2-oxoglutarate - MSO l-methionine dl-sulphoximine - NMR nuclear magnetic resonance - RGR relative growth rate This article is dedicated to Professor Bernhard Schrader on the occasion of his 60th birthdayWe wish to thank Professor Robert Glaser for helpful discussions, and Mrs. Aliza Levkoviz and Mr. Gideon Raziel for skillful assistance.     

Ethylenediamine uptake and metabolism in the cyanobacterium Anabaena variabilis

The cyanobacterium Anabaena variabilis showed a pH dependent uptake of ethylenediamine. No uptake of ethylenediamine was detected at pH 7.0. At higher pH values (e.g. pH 8.0 and pH 9.0) accumulation did occur and was attributed to diffusion of uncharged ethylenediamine in response to a pH gradient. A biphasic pattern of uptake was observed at these higher pH values. Treatment with l-methionine-d,l-sulphoximine (MSX) to inactivate glutamine synthetase (GS) inhibited the second slower phase of uptake without any significant alteration of the initial uptake. Therefore for sustained uptake, metabolism of ethylenediamine via GS was required. NH ₄ ⁺ did not alter the uptake of ethylenediamine. Ethylenediamine was converted in the second phase of uptake to an analogue of glutamine which could not be detected in uptake experiments at pH 7.0 or in uptake experiments at pH 9.0 following pretreatment of cells with MSX. Ethylenediamine treatment inhibited nitrogenase activity and this inhibition was greatest at high pH values.Abbreviations EDA 1,2-diaminoethane (ethylenediamine) - GS glutamine synthetase - HEPES 4-(2-hydroxyethyl)-1 piperazine ethanesulphonic acid - MSX l-methionine-dl-sulphoximine - membrane potential - Tricine N-tris(hydroxymethyl) methylglycine     

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     

Absence of the glutamine-synthetase-linked methylammonium (ammonium)-transport system in the cyanobiont of Cycas-cyanobacterial symbiosis

Using the ammonium analogue ¹⁴CH₃NH ₃ ⁺ , ammonium transport was studied in the cyanobiont cells freshly isolated from the root nodules of Cycas revoluta. An L-methionine-dl-sulphoximine (MSX)-insensitive ammonium-transport system, which was dependent on membrane potential (), was found in the cyanobiont. However, the cyanobiont was incapable of metabolizing exogenous ¹⁴CH₃NH ₃ ⁺ or NH ₄ ⁺ because of the absence of another ammonium-transport system responsible for the uptake of ammonium for assimilation via glutamine synthetase (EC 6.3.1.2). Such a modification seems to be the result of symbiosis because the free-living cultured isolate, Anabaena cycadeae, has been shown to possess both the ammonium-transport systems.Abbreviations and symbol ATS/ATSs ammonium transport system/systems - Chl chlorophyll - GS glutamine synthetase - MSX L-methionine-dl-sulphoximine - membrane potential     

Evidence for an ammonium transport system in free-living and symbiotic cyanobacteria

The free-living cyanobacterium Anabaena variabilis showed a biphasic pattern of ¹⁴CH₃NH ₃ ⁺ uptake. Initial accumulation (up to 60 s) was independent of CH₃NH ₃ ⁺ metabolism, but long-term uptake was dependent on its metabolism via glutamine synthetase (GS). The CH₃NH ₃ ⁺ was converted into methylglutamine which was not further metabolised. The addition of l-methionine-dl-sulphoximine (MSX), to inhibit GS, inhibited CH₃NH ₃ ⁺ metabolism, but did not affect the CH₃NH ₃ ⁺ transport system.NH ₄ ⁺ , when added after the addition of ¹⁴CH₃NH ₃ ⁺ , caused the efflux of free CH₃NH ₃ ⁺ ; when added before ¹⁴CH₃NH ₃ ⁺ , NH ₄ ⁺ inhibited its uptake indicating that both NH ₄ ⁺ and CH₃NH ₃ ⁺ share a common transport system. Carbonylcyanide m-chlorophenylhydrazone and triphenyl-methylphosphonium both inhibited CH₃NH ₃ ⁺ accumulation indicating that the transport system was -dependent. At pH 7 and at an external CH₃NH ₃ ⁺ concentration of 30 mol dm^-3, A. variabilis showed a 40-fold intracellular accumulation of CH₃NH ₃ ⁺ (internal concentration 1.4 mmol dm^-3). Packets of the symbiotic cyanobacterium Anabaena azollae, directly isolated from the water fern Azolla caroliniana, also showed a -dependent NH ₄ ⁺ transport system suggesting that the reduced inhibitory effect of NH ₄ ⁺ on nitrogenase cannot be attributed to the absence of an NH ₄ ⁺ transport system but is probably related to the reduced GS activity of the cyanobiont.Abbreviations CCCP carbonylcyanide m-chlorophenylhydrazone - GS glutamine synthetase - HEPES 4-(2-hydroxyethyl)-1-piperazine ethanesulphonic acid - MSX l-methionine-dl-sulphoximine - membrane potential - pH transmembrane pH difference - TPMP⁺ triphenylmethylphosphonium     

Ammonium uptake in Rhodopseudomonas capsulata

Investigations of the uptake of ammonium (NH ₄ ⁺ ) by Rhodopseudomonas capsulata B100 supported the presence of an NH ₄ ⁺ transport system. Experimentally NH ₄ ⁺ was determined by electrode or indophenol assay and saturation kinetics were observed with two apparent K _m's of 1.7 M and 11.1 M (pH 6.8, 30°) and a V _max at saturation of 50–60 nmol/min·mg protein. The optimum pH and temperature were 7.0 and 33° C, respectively. The Q₁₀ quotient was calculated to be 1.9 at 100 M NH ₄ ⁺ , indicating enzymatic involvement. In contrast to the wild type, B100, excretion of NH ₄ ⁺ , not uptake, was observed in a glutamine auxotroph, R. capsulata G29, which is derepressed for nitrogenase and lacks glutamine synthetase activity. G29R1, a revertant of G29, also took up NH ₄ ⁺ at the same rate as wild type and had fully restored glutamine synthetase activity. Partially restored derivatives, G29R5 and G29R6, grew more slowly than wild type on NH ₄ ⁺ as the nitrogen source, remained derepressed for nitrogenase in the presence of NH ₄ ⁺ , and displayed rates of NH ₄ ⁺ uptake in proportion to their glutamine synthetase activity. Ammonium uptake and glutamine synthetase activity were also restored in R. capsulata G29 exconjugants which had received the plasmid pPS25, containing the R. capsulata glutamine synthetase structural gene. These data suggest that NH ₄ ⁺ transport is tightly coupled to assimilation.Abbreviations used CHES cyclohexylaminoethanesulfonic acid - GS glutamine synthetase - SDS sodium dodecylsulfate     

Photoproduction of ammonium by immobilized mutant strains of Anabaena variabilis

Summary Ethylenediamine (EDA) is toxic to the cyanobacterium Anabaena variabilis and inhibits nitrogenase activity. The inhibition of nitrogenase was prevented by pretreatment of cells with l-methionine-d,l-sulphoximine (MSX). Mutant strains of Anabaena variabilis (ED81, ED92), resistant to EDA, had low levels of glutamine synthetase (GS) biosynthetic activity compared with the wild type strain. ED92 had a low level of GS protein whereas ED81 had a similar level to that of the parent strain as estimated using antibodies against GS. Both strains fixed N₂ and liberated NH₄ ⁺ into the media. Following immobilization of the mutant strains, sustained photoproduction of NH₄ ⁺ was obtained in air-lift reactors at rates of up to 50 mol NH₄ ⁺ mg chl a^–1 h^–1, which were comparable to the rates obtained when immobilized cyanobacteria were treated with MSX.Abbreviations EDA 1,2-diaminoethane (ethylenediamine) - GS glutamine synthetase - MSX l-methionine-d,l-sulphoximine     

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)     

Photorespiratory ammonium release by the cyanobacterium Anabaena cylindrica in the presence of methionine sulfoximine

A release of ammonium by non-nitrogen-fixing Anabaena cylindrica (grown on NH₄Cl) in the presence of MSX (methionine sulfoximine) and absence of any external nitrogen source was found. In the light the release was maximal at 0.2 mM MSX, a concentration which did not affect net CO₂ fixation nor the glycollate excretion, but inhibited the glutamine synthetase activity and the reassimilation of ammonium. It is suggested that the major source of the ammonium released is the photorespiratory conversion of glycine to serine as (1) the release was stimulated by increase in light intensity, (2) high CO₂ (3%) lowered the release, if not given as a longer pretreatment (as CO₂ or HCO ₃ ^- ) when a stimulation was observed, (3) glyoxylate and glutamate stimulated the release, the latter compound particularly under nitrogen-deficient conditions and (4) isonicotinic acid hydrazide caused a reduced release of ammonium. Furthermore, a substantial part of the ammonium released by N₂-fixing A. cylindrica in presence of MSX may thus originate from the glycollate pathway. The data show that in the light the glycine to serine conversion is active in cyanobacteria with a concomitant production of ammonium which is assimilated by glutamine synthetase.Abbreviations MSX L-methionine-Dl-sulfoximine - INH isonicotinic acid hydrazide - RuDP ribulose 1,5-diphosphate - Hepes N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - GS glutamine synthetase - GOGAT glutamate synthase - DTT Dl-dithiothreitol     

15N2 Incorporation and metabolism in the lichen Peltigera aphthosa Willd

The Nostoc in the cephalodia of the lichen Peltigera aphthosa Willd. fixed ¹⁵N₂ and the bulk of the nitrogen fixed was continuously transferred from it to its eukaryotic partners (a fungus and a green alga, Coccomyxa sp.). Kinetic studies carried out over the first 30 min, after exposure of isolated cephalodia to ¹⁵N₂, showed that highest initial ¹⁵N₂-labelling was into NH ₄ ⁺ . After 12 min little further increase in the NH ₄ ⁺ label occurred while that in the amide group of glutamine and in glutamate continued to increase. The ¹⁵N-labelling of the amino group of glutamine and of aspartate increased more slowly, followed by an increase in the labelling of alanine. When total incorporation of ¹⁵N-label was calculated, the overall pattern was found to be rather similar except that, throughout the experiment, the total ¹⁵N incorporated into glutamate was about six times greater than that into the amide group of glutamine. Pulse chase experiments, in which ¹⁴N₂ was added to cephalodia previously exposed to ¹⁵N₂, showed that the NH ₄ ⁺ pool rapidly became depleted of ¹⁵N-label, followed by decreases in the labelling of glutamate, the amide group of glutamine and aspartate. The ¹⁵N-labelling of alanine, however, continued to increase for a period. When isolated cephalodia were treated with L-methionine-SR-sulphoximine, an inhibitor of glutamine synthetase (EC 6.3.1.2), and azaserine, an inhibitor of glutamate synthase (EC 2.6.1.53), there was no detectable labelling in glutamine although the ¹⁵N-labelling of glutamate increased unimpaired. On treating the cephalodia with amino-oxyacetate, an inhibitor of aminotransferase activity, the alanine pool decreased. Evidence was obtained that glutamine synthetase and glutamate synthase were located in the Nostoc, and that glutamate dehydrogenase (EC 1.4.1.4) and various amino-transferases were located in the cephalodial fungus. Possible implications of these findings are discussed.Abbreviations MSX L-methionine-SR-sulphoximine - AOA amino-oxyacetate - HEPES N-2-hydroxymethylpiperazine-N-2-ethane sulphonic acid - Tris tris-(hydroxymethyl) methylamine - GS glutamine synthetase - GOGAT glutamate synthase - GDH glutamate dehydrogenase - GPT glutamate-pyruvate aminotransferase - APT aspartate-pyruvate aminotransferase - ADH alanine dehydrogenase - GOT glutamate-oxaloacetate aminotransferase     

Short-term ammonium inhibition of nitrate uptake by Azotobacter chroococcum

Addition of NH₄Cl at low concentrations to Azotobacter chroococcum cells caused an immediate cessation of nitrate uptake activity, which was restored when the added NH ₄ ⁺ was exhausted from the medium or by adding an NH ₄ ⁺ assimilation inhibitor, l-methionine-dl-sulfoximine (MSX) or l-methionine sulfone (MSF). In the presence of such inhibitors the newly-reduced nitrate was released into the medium as NH ₄ ⁺ . When the artificial electron donor system ascorbate/N-methylphenazinium methylsulfate (PMS), which is a respiratory substrate that was known to support nitrate uptake by A. chroococcum while inhibiting glutamine synthetase activity, was the energy source, externally added NH ₄ ⁺ had no effect on nitrate uptake. It is concluded that, in A. chroococcum cells, NH ₄ ⁺ must be assimilated to exert its short-term inhibitory effect on nitrate uptake. A similar proposal was previously made to explain the short-term ammonium inhibition of N₂ fixation in this bacterium.Abbreviations MOPS morpholinopropanesulfonic acid - MSX l-methionine-dl-sulfoximine - PMS N-methylphenazinium methylsulfate - MSF l-methionine sulfone     

The role of glutamine synthetase in the regulation of nitrogenase activity (“switch off” effect) in Rhodospirillum rubrum

We have studied the changes in the activities of both nitrogenase (switch off) and glutamine synthetase in Rhodospirillum rubrum upon addition of ammonium ions or glutamine to nitrogen fixing cultures. Both activities decrease drastically and return in a parallel manner when added ammonia is metabolized. The decrease in glutamine synthetase activity does not seem to be primarily due to adenylylation of the enzyme. Addition of glutamine to cells starved for nitrogen results in inactivation of glutamine synthetase but nitrogenase is only partially switched off.Abbreviations CeMe₃NBr Cetyltrimethylammonium bromide - Hepes N-2-hydroxyethyl-piperazine-N-2 sulfonic acid - MSO methionine-D,L-sulfoximine - Tea-Dmg triethanol amine-3,3-dimethylglutaric acid     

Genetic transformation of glutamine auxotrophy to prototrophy in the cyanobacterium Nostoc muscorum

Glutamine auxotrophic (Gln ^-) and l-methionine d,l-sulfoximine (MSX) resistant (MSX ^r) mutants of N. muscorum were isolated and characterized for nitrogen nutrition, nitrogenase activity, glutamine synthetase (GS) activity and glutamine amide, -keto-glutarate amido transferase (GOGAT) activity. The glutamine auxotroph was found to the GOGAT-containing GS-defective, incapable of growth with N₂ or NH ₄ ⁺ but capable of growth with glutamine as nitrogen source, thus, suggesting GS to be the primary enzyme of both ammonia assimilation and glutamine formation in the cyanobacterium. The results of transformation and reversion studies suggests that glutamine auxotrophy is the result of a mutation in the gln A gene and that gln A gene can be transferred from one strain to another by transformation.