Regulation of nitrogen fixation. Nitrogenase-derepressed mutants of Klebsiella pneumoniae.

1. A new procedure is described for selecting nitrogenase-derepressed mutants based on the method of Brenchley et al. (Brenchley, J.E., Prival, M.J. and Magasanik, B. (1973) J. Biol. Chem. 248, 6122-6128) for isolating histidase-constitutive mutants of a non-N2-fixing bacterium. 2. Nitrogenase levels of the new mutants in the presence of NH4+ were as high as 100% of the nitrogenase activity detected in the absence of NH4+. 3. Biochemical characterization of these nitrogen fixation (nif) derepressed mutants reveals that they fall into three classes. Three mutants (strains SK-24, 28 and 29), requiring glutamate for growth, synthesize nitrogenase and glutamine synthetase constitutively (in the presence of NH4+). A second class of mutants (strains SK-27 and 37) requiring glutamine for growth produces derepressed levels of nitrogenase activity and synthesized catalytically inactive glutamine synthetase protein, as determined immunologically. A third class of glutamine-requiring, nitrogenase-derepressed mutants (strain SK-25 and 26) synthesizes neither a catalytically active glutamine synthetase enzyme nor an immunologically cross-reactive glutamine synthetase protein. 4. F-prime complementation analysis reveals that the mutant strains SK-25, 26, 27, 37 map in a segment of the Klebsiella chromosome corresponding to the region coding for glutamine synthetase. Since the mutant strains SK-27 and SK-37 produce inactive glutamine synthetase protein, it is concluded that these mutations map within the glutamine synthetase structural gene.     

Amino acids as repressors of nitrogenase biosynthesis in Klebsiella pneumoniae

Nitrogenase biosynthesis in Klebsiella pneumoniae including mutant strains, which produce nitrogenase in the presence of NH₄⁺ (Shanmugam, K.T., Chan, Irene, and Morandi, C. (1975) Biochim. Biophys. Acta 408, 101–111) is repressed by a mixture of L-amino acids. Biochemical analysis shows that glutamine synthetase activity in strains SK-24, SK-28, and SK-29 is also repressed by amino acids, with no detectable effect on glutamate dehydrogenase. Among the various amino acids, L-glutamine in combination with L-aspartate was found to repress nitrogenase biosynthesis completely. In the presence of high concentrations of glutamine (1 mg/ml) even NH₄⁺ repressed nitrogenase biosynthesis in the strains SK-27, SK-37, SK-55 and SK-56. Under these conditions, increased glutamate dehydrogenase activity was also detected. Physiological studies show that nitrogenase derepressed strains are unable to utilize NH₄⁺ as sole source of nitrogen for biosynthesis of glutamate, whereas back mutations leading to NH₄⁺ utilization results in sensitivity to repression by NH₄⁺. These findings suggest that amino acids play an important role as regulators of nitrogen fixation.     

Activity of nitrogenase and glutamine synthetase in relation to availability of oxygen in continuous cultures of a strain of cowpea Rhizobium sp. supplied with excess ammonium

In samples from nitrogen-fixing continuous cultures of strain CB756 of the cowpea type rhizobia (Rhizobium sp.), newly fixed NH₄⁺ is in equilibrium with the medium, from where it is assimilated by the glutamine synthetase/glutamate synthase pathway. In samples from steady state cultures with different degrees of oxygen-limitation, nitrogenase activity was positively correlated with the biosynthetic activity of glutamine synthetase in cell free extracts. Also, activities in biosynthetic assays were positively correlated with activities in γ-glutamyl transferase assays containing 60 mM Mg²⁺. Relative adenylylation of glutamine synthetase was conveniently measured in cell free extracts as the ratio of γ-glutamyl transferase activities without and with addition of 60 mM Mg²⁺.Automatic control of oxygen supply was used to facilitate the study of transitions between steady-state continuous cultures with high and low nitrogenase activities. Adenylylation of glutamine synthetase and repression of nitrogenase activity in the presence of excess NH₄⁺, were masked when oxygen strongly limited culture yield. Partial relief of the limitation in cultures supplied with 10 mM NH₄⁺ produced early decline in nitrogenase activity and increase in relative adenylylation of glutamine synthetase. Decreased oxygen supply produced a rapid decline in relative adenylylation, followed by increased nitrogenase activity, supporting the concept that control of nitrogenase synthesis is modulated by glutamine synthetase adenylylation in these bacteria.     

Inhibition of the adenylylation of glutamine synthetase by methionine sulfone during nitrogenase derepression

Adenylylation of glutamine synthetase was suppressed during derepression of nitrogenase synthesis in the presence of methionine sulfone and an excess of NH₄⁺. Deadenylylation of glutamine synthetase was also promoted during nitrogenase derepression under the same conditions. These results are consistent with the hypothesis that the unadenylylated form of glutamine synthetase is required for derepression of nitrogenase.     

Amino acids as repressors of nitrogenase biosynthesis in Klebsiella pneumoniae.

Nitrogenase biosynthesis in Klebsiella pneumoniae including mutant strains, which produce nitrogenase in the presence of NH+4 (Shanmugam, K.T., Chan, Irene, and Morandi, C. (1975) Biochim. Biophys. Acta 408, 101--111) is repressed by a mixture of L-amino acids. Biochemical analysis shows that glutamine synthetase activity in strains SK-24, SK-28, and SK-29 is also repressed by amino acids, with no detectable effect on glutamate dehydrogenase. Among the various amino acids, L-glutamine in combination with L-aspartate was found to repress nitrogenase biosynthesis completely. In the presence of high concentrations of glutamine (1 mg/ml) even NH+4 repressed nitrogenase biosynthesis in the strains SK-27, SK-37, SK-55 and SK-56. Under these conditions, increased glutamate dehydrogenase activity was also detected. Physiological studies show that nitrogenase derepressed strains are unable to utilize NH+4 as sole source of nitrogen for biosynthesis of glutamate for biosynthesis of glutamate, whereas back mutations leading to NH+4 utilization results in sensitivity to repression by NH+4. These findings suggest that amino acids play an important role as regulators of nitrogen fixation.     

Ammonium uptake by nitrogen fixing bacteria

Both the changes in the activities of nitrogenase, glutamine synthetase and glutamate dehydrogenase and in the extracellular and intracellular NH₄ ⁺ concentrations were investigated during the transition from an NH₄ ⁺ free medium to one containing NH₄ ⁺ ions for a continuous culture of Azotobacter vinelandii. If added in amounts causing 80–100% repression of nitrogenase, ammonium acetate, lactate and phosphate are absorbed completely, whereas chloride, sulfate and citrate are only taken up to about 80%. After about 1–2 hrs the NH₄ ⁺ remaining in the medium is absorbed too, indicating the induction or activation of a new NH₄ ⁺ transport system. One of the new permeases allows the uptake of citrate in the presence of sucrose. Addition of inorganic NH₄ ⁺ salts leads to acidification of the culture. Anaerobiosis suppresses NH₄ ⁺ transport. A rise in the extracellular NH₄ ⁺ level leads to a reversible rise in the glutamine synthetase activity, which is not prevented by chloramphenicol, and to a reversible decrease in nitrogenase activity. During these measurements glutamate dehydrogenase activity remains close to zero. The intracellular NH₄ ⁺ level of about 0.6 mM does not change when extracellular NH₄ ⁺ is taken up and repression of nitrogenase starts.     

The photoproduction of H2 and NH+4 fixed from N2 by a derepressed mutant of Rhodospirillum rubrum

A mutant of Rhodospirillum rubrum has been isolated, after mutagenesis with nitrosoguanidine, which is characterized by its inability to grow in the light on malate-minimal media with exogenous ammonia or alanine, poor growth on glutamine and vigorous growth on glutamate. This mutant produces low levels of a key NH⁺₄ assimilation enzyme, glutamate synthase (NADPH-dependent). It also exhibits significant derepression of nitrogenase biosynthesis in the presence of ammonia or alanine, being 15% derepressed for the former and about 70% derepressed for the latter.Some of this mutant's fixed N₂ is excreted into the medium as NH⁺₄ (1 μmol NH⁺₄ per mg cell protein in 50 h). Nitrogenase-mediated H₂ production by this strain is considerable (42 μmol H₂ per mg cell protein in 50 h), approximately twice that of the wild type assayed under similar conditions.These results demonstrate that genetic alteration of the photosynthetic N₂-fixer's NH⁺₄ assimilation system disrupts the tight coupling of N₂ fixation and NH⁺₄ assimilation normally observed in these organisms, enabling photochemical conversion steps to be utilized for the photoproduction of NH⁺₄ and H₂.     

Energy status,growth and nitrogenase activity in continuous cultures of Rhizobium sp. strain CB756 supplied with NH+4 and various rates of aeration

Continuous cultures of the cowpea-type Rhizobium sp., strain CB756, were grown in the presence of NH⁺₄ at automatically controlled concentrations of dissolved O₂ and rates of aeration. Nitrogenase activity of steady-state cultures was only detected under microaeration conditions (dissolved O₂ typically <0.03 μM; aeration rate typically 0.6 μmol O₂/ml per h), when the cellular ATP pool size was 0.8–1.8 nmol/mg dry wt., (optimum 1.1) and the energy charge 0.6–0.7. At twice this aeration rate and dissolved O₂ concentration of about 0.15 μM, the yield of bacteria doubled, the ATP pool increased and energy charge increased to 0.8. With similar rates of O₂ supply but high concentration of dissolved O₂ (approx. 150 μM), cultures were NH⁺₄-limited and the ATP pool and energy charge were slightly reduced. Amongst all of these O₂ supply conditions the total pool of adenosine phosphates was not significantly different (2.6 S.D. 0.7 nmol/mg dry wt.). In steady-state, O₂-limited cultures, concentrations of cyclic GMP were higher when nitrogenase was present. When rates of O₂ supply to steady-state cultures were changed, oscillations in bacterial energy status and growth rate were induced decreasing in amplitude until a new steady state was reached. This made it difficult to discern precisely the energy status in which nitrogenase activity was derepressed or repressed. However, generally, increases in nitrogenase activity followed decreases in ATP and energy charge and decreased nitrogenase activity accompanied increases in these energy parameters. These results are discussed in relation to the possible involvement of adenylation or deadenylation of glutamine synthetase and to the control of nitrogenase synthesis in the presence of NH⁺₄. It is concluded that the small ATP pool size is responsible for failure of adenylylation of glutamine synthetase and is related to nitrogenase synthesis at microaeration rates.     

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     

Effects of L-methionine-DL-sulphoximine on the assimilation of newly fixed NH3, acetylene reduction and heterocyst production in Anabaena cylindrica.

The addition of exogenous L-methionine-DL-sulphoximine (MSO) to N₂-fixing cultures of the blue-green alga Anabaena cylindrica results in over half of the newly fixed NH₃ being released into the medium. MSO also inhibits glutamine synthetase (GS) activity, has negligible effect on alanine dehydrogenase activity, and glutamate dehydrogenase activity under N₂-fixing conditions is negligible. In the presence of MSO, intracellular pools of glutamate and glutamine decrease, those of aspartate and alanine + glycine show little change, and the NH₃ pool increases. MSO alleviates the inhibitory effect of exogenous NH₄⁺ on nitrogenase synthesis and heterocyst production. The results suggest that in N₂-fixing cultures of A. cylindrica the primary NH₃ assimilating pathway involves GS, and probably glutamate synthase (GOGAT), and that the repressor of nitrogenase synthesis and heterocyst production is not NH₄⁺ but is GS, GOGAT, or a product of their reactions.     

Regulation of nitrogen fixation in Rhizobium spp. Isolation of mutants of Rhizobium trifulii which induce nitrogenase activity

This communication describes the isolation and characterization of mutants of Rhizobium trifolii which can induce nitrogenase activity in defined liquid medium. Two procedures were used for the isolation of these mutants from R. trifolii strain DT-6: (1) following chemical mutagenesis, slow growin mutants were selected which were unable to utilize NH₄⁺ as sole source of nitrogen; (2) as spontaneous mutants resistant to the glutamate analogue L-methionine-DL-sulfoximine.Mutants (DT-71, DT-125) isolated by these procedures induced nitrogenase activity in the free-living state, whereas the parent strain lacked this property. Induction of nitrogenase activity in these mutants occurred during the late exponential phase of growth when the rate of protein synthesis was decreasing. The addition of NH₄⁺ to a medium containing glutamate as the nitrogen-source resulted in a 50–70% reduction (repression?) of nitrogenase activity; in contrast, the rate of protein synthesis or the rate of respiration was not influenced by exogenous NH₄⁺.Biochemistry analysis showed that these mutants (strains DT-71 and DT-125) have defects in both nitrogen and carbon metabolism. The levels of glutamate synthase (both NADP⁺-and NAD⁺-dependent activities) and glutamate dehydrogenase (NAD⁺-dependent activity) were markedly lower. In addition, the mutants were found to have no detectable ribitol dehydrogenase or β-galactosidase activity. These findings are discussed in relation to a mechanism of regulation of symbiotic nitrogen fixation.     

Mutants of Azospirillum brasilense resistant to methylammonium

One hundred and twenty-nine mutants of Azospirillum brasilense strain Sp6, resistant to methylammonium, were isolated. Three of the mutants were found to be able to reduce acetylene in the presence of 4 mM ammonium or 120mM methylammonium, concentrations which strongly reduced the nitrogenase activity of the parental strain. Under N₂-fixing conditions, two mutants failed to switch off nitrogenase when NH₄Cl was added. Moreover, the three mutants showed a reduced capacity to incorporate [¹⁴C]methylammonium. The level of glutamine synthetase activity found in the mutants was not reduced as compared to that of the parental strain. All of the data indicate an impairement in the mechanism of ammonium uptake by the bacterial cell.Abbreviations MEA Methylammonium - MSP minimal medium (ammonium free) - PY complete medium - GS glutamine synthetase     

ColE1 hybrid plasmids containing Escherichia coli genes involved in the biosynthesis of glutamate and glutamine

The Clarke-Carbon bank of Escherichia coli strains carrying ColE1 hybrid plasmids was screened for complementation of gdh, gltB, and glnA mutations affecting nitrogen metabolism in E. coli. Plasmids which complemented each one of these mutations were isolated. In every case, the plasmids conferred to otherwise mutant cells the capacity to synthesize the corresponding wild-type enzymes: glutamate dehydrogenase, glutamate synthase, and glutamine synthetase (GS), respectively. For three representative plasmids, endonuclease restriction maps were constructed. One of the plasmids, pACR1, which complemented glnA mutations, including the glnA21::Tn5 insertion, was deemed to carry the glnA⁺ allele. GS synthesis by pACR1 $\text{glnA}{}^{\mathrm{+}}\text{glnA20}$ heterozygous merodiploids was subjected to repression by growth on 15 mm NH₄⁺ and had a twofold high derepressed level than wild-type (glnA⁺) haploid cells when grown on 0.5 mm NH₄⁺ or on glutamate as only nitrogen sources. The presence of glutamine as sole nitrogen source promoted repressed GS synthesis in the $\text{glnA}{}^{\mathrm{+}}\text{glnA20}$ merodiploids. By contrast, glutamine allowed almost fully derepressed synthesis of GS in glnA⁺ haploid cells.     

MSX-resistant mutants of Anabaena 7120 with derepressed heterocyst development and nitrogen fixation

To investigate the role of ammonium-assimilating enzyme in heterocyst differentiation, pattern formation and nitrogen fixation, MSX-resistant and GS-impaired mutants of Anabaena 7120 were isolated using transposon (Tn5-1063) mutagenesis. Mutant Gs1 and Gs2 (impaired in GS activity) exhibited a similar rate of nitrogenase activity compared to that of the wild type under dinitrogen aerobic conditions in the presence and absence of MSX. Filaments of Gs1 and Gs2 produced heterocysts with an evenly spaced pattern in N₂-grown conditions, while addition of MSX altered the interheterocyst spacing pattern in wild type as well as in mutant strains. The wild type showed complete repression of heterocyst development and nitrogen fixation in the presence of NO₃ ^– or NH₄ ⁺, whereas the mutants Gs1 and Gs2 formed heterocysts and fixed nitrogen in the presence of NO₃ ^– and NH₄ ⁺. Addition of MSX caused complete inhibition of glutamine synthetase activity in wild type but Gs1 and Gs2 remained unaffected. These results suggest that glutamine but not ammonium is directly involved in regulation of heterocyst differentiation, interheterocyst spacing pattern and nitrogen fixation in Anabaena.     

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     

Correlation between nitrogenase and glutamine synthetase expression in the cyanobacterium Anabaena cylindrical

Distribution pattern and levels of nitrogenase (EC 1.7.99.2) and glutamine synthetase (GS, EC 6.3.1.2) were studied in N₂-, NO₃^? and NH₄⁺ grown Anabaena cylindrica (CCAP 1403/2a) using immunogold electron microscopy. In N₂- and NO₃^? grown cultures, heterocysts were formed and nitrogenase activity was present. The nitrogenase antigen appeared within the heterocysts only and showed an even distribution. The level of nitrogenase protein in the heterocysts was identical with both nitrogen sources. In NO₃^? grown cells the 30% reduction in the nitrogenase activity was due to a corresponding decrease in the heterocyst frequency and not to a repressed nitrogenase synthesis. In NH₄^? grown cells, the nitrogenase activity was almost zero and new heterocysts were formed to a very low extent. The heterocysts found showed practically no nitrogenase protein throughout the cytoplasm, although some label occurred at the periphery of the heterocyst. This demonstrates that heterocyst differentiation and nitrogenase expression are not necessarily correlated and that while NH₄⁺ caused repression of both heterocyst and nitrogenase synthesis, NO₃^? caused inhibition of heterocyst differentiation only. The glutamine synthetase protein label was found throughout the vegetative cells and the heterocysts of all three cultures. The relative level of the GS antigen varied in the heterocysts depending on the nitrogen source, whereas the GS level was similar in all vegetative cells. In N₂- and NO₃⁺ grown cells, where nitrogenase was expressed, the GS level was ca 100% higher in the heterocysts compared to vegetative cells. In NH₄⁺ grown cells, where nitrogenase was repressed, the GS level was similar in the two cell types. The enhanced level of GS expressed in heterocysts of N₂ and NO₃^? grown cultures apparently is related to nitrogenase expression and has a role in assimilation of N₂derived ammonia.     

NH4+-Excreting Azospirillum brasilense Mutants Enhance the Nitrogen Supply of a Wheat Host

Spontaneous ethylenediamine-resistant mutants of Azospirillum brasilense were selected on the basis of their excretion of NH₄⁺. Two mutants exhibited no repression of their nitrogenase enzyme systems in the presence of high (20 mM) concentrations of NH₄⁺. The nitrogenase activities of these mutants on nitrogen-free minimal medium were two to three times higher than the nitrogenase activity of the wild type. The mutants excreted substantial amounts of ammonia when they were grown either under oxygen-limiting conditions (1 kPa of O₂) or aerobically on nitrate or glutamate. The mutants grew well on glutamate as a sole nitrogen source but only poorly on NH₄Cl. Both mutants failed to incorporate [¹⁴C]methylamine. We demonstrated that nitrite ammonification occurs in the mutants. Wild-type A. brasilense, as well as the mutants, became established in the rhizospheres of axenically grown wheat plants at levels of > 10⁷ cells per g of root. The rhizosphere acetylene reduction activity was highest in the preparations containing the mutants. When plants were grown on a nitrogen-free nutritional medium, both mutants were responsible for significant increases in root and shoot dry matter compared with wild-type-treated plants or with noninoculated controls. Total plant nitrogen accumulation increased as well. When they were exposed to a ¹⁵N₂-enriched atmosphere, both A. brasilense mutants incorporated significantly higher amounts of ¹⁵N inside root and shoot material than the wild type did. The results of our nitrogen balance and ¹⁵N enrichment studies indicated that NH₄⁺-excreting A. brasilense strains potentially support the nitrogen supply of the host plants.     

Photoproduction of ammonium ion from N2 inRhodospirillum rubrum

NH ₄ ⁺ excretion was undetectable in N₂-fixing cultures ofRhodospirillum 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 ⁺ (>40 moles NH ₄ ⁺ /mg cell protein in 6 days) orl-glutamate (>60 moles NH ₄ ⁺ /mg cell protein in 6 days) was added to the cultures together with MSX.Biochemical analysis revealed thatR. 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.     

Regulation of non-autotrophic carbon dioxide assimilation by ammonia in cultured cells of Acer pseudoplatanus L

The assimilation of ¹⁴CO₂ by Acer pseudoplatanus cells in the dark was stimulated by the addition of either NH₄Cl or methylamine. Results were obtained demonstrating that methylamine was not metabolised to any appreciable extent by Acer cells. This suggests that the mechanism of stimulation of dark fixation by methylamine does not involve metabolism via the glutamine synthetase reaction. NH₄⁺ stimulation of CO₂ fixation also occurred in cells pretreated with the glutamine-synthetase inhibitor methionine sulfoximine. This further supports the conclusion that neither NH₄⁺ nor methylamine exerts its effect on CO₂-assimilation via a mechanism that depends upon the assimilation of NH₃ by glutamine synthetase.     

In vivo regulation of glutamine synthetase by ammonium in the cyanobacterium Anabaena L-31

In cell-free preparations of NH₄⁺-grown cultures of the cyanobacterium Anabaena L-31 the glutamine synthetase activity is only half as much as in N₂-grown cultures. Using a procedure which enables quantitative purification of the enzyme to homogeneity it has been shown that the decrease in the enzyme activity is caused by NH₄⁺-mediated repression. Glutamine synthetase activity in both N₂-grown and NH₄⁺-grown Anabaena remains stable for more than 24 h in the presence of chloramphenicol suggesting low enzyme turnover and an enzyme half-life greater than the generation time (16–18 h) of the cyanobacterium. In N₂-grown cultures, a drastic decrease in the enzyme activity by exogenous NH₄⁺ can be discerned when fresh protein synthesis is prevented by chloramphenicol. The enzyme purified from such cultures has K_m values for NH₄⁺, glutamate Mg²⁺, and ATP similar to those observed for the enzyme from N₂- and NH₄⁺-grown Anabaena, but shows depression in V for all the substrates, leading to drastic decrease in specific activity. The modified enzyme also shows a sharper thermal denaturation profile. These results indicate that NH₄⁺-mediated modification to a less active form may be a means of regulation of glutamine synthetase in N₂-fixing cultures of Anabaena.