Relation between Glutamine Synthetase and Nitrogenase Activities in the Symbiotic Association between Rhizobium japonicum and Glycine max

The activity and extent of adenylylation of glutamine synthetase was examined in both free-living and bacteroid forms of Rhizobium japonicum in the presence of excess ammonia. Ammonia caused an apparent repression of glutamine synthetase in free-living R. japonicum and adenylylation of the enzyme was also increased. In contrast, neither the activity nor the extent of adenylylation of the bacteroid enzyme was consistently affected by ammonium treatment of bacteroid suspensions. Similar results were obtained after ammonium treatment of soybean plants even though nitrogenase activity was reduced markedly. We have been unable to demonstrate ammonium repression of nitrogenase activity in R. japonicum-Glycine max symbiotic association that is mediated through bacteroid glutamine synthetase. This result is in contrast to the situation in nitrogen-fixing strains of Klebsiella where a role of glutamine synthetase in the regulation of nitrogenase has been reported.     

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.     

Effect of oxygen tension on nitrogenase and on glutamine synthetases I and II in Rhizobium jaonicum 61A76.

When grown under aerobic conditions, $\text{Rhizobium japonicum}$ 61A76 contains two forms of glutamine synthetase, GSI and GSII, as previously described. In contrast, cells grown under the low O₂ tensions required for nitrogenase synthesis contain only GSI. GSII activity disappears completely at O₂ levels below 0.4%. GSI activity decreases by only 50%, but the enzyme appears to become highly adenylylated under the low O₂ tensions required for nitrogenase synthesis.     

The relationship between the state of adenylylation of glutamine synthetase I and the export of ammonium in free-living,N2-fixing Rhizobium

The relationship between ammonium assimilation and ammonium export has been studied in free-living, N₂-fixing Rhizobium sp. 32H1. After 55 to 67 h of microaerobic growth under a gas phase of 0.2% O₂ – 1.0% CO₂ – 98.8% Ar high levels of nitrogenase were observed concomitant with a slightly adenylylated glutamine synthetase (GSI) and some glutamine synthetase (GSII) activity. However, after growth of 89 h, or longer, GSI became adenylylated and the level of GSII had decreased. When the gas phase was shifted to 0.2% O₂ – 1.0% CO₂ – 98.8% N₂, a lag was observed before ammonium export could be detected in the 55 to 67 h cultures. No lag in ammonium export was observed in the cultures previously grown for 89 h. The onset of ammonium export in the 55 to 67 h cultures was found to correlate with the adenylylation state of GSI. There appeared to be no correlation between the level of GSII and the export of ammonium. Neither an increase in the adenylylation level of GSI nor ammonium export was observed when the 55 to 67 h cultures were maintained under the Ar gas mixture.Abbreviations GOGAT Glutamate synthase - GS glutamine synthetase - BES [N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid] - CTAB cetyltrimethylammonium bromide - MES [2-(N-morpholino)-ethane sulfonic acid]     

Regulation of glutamine synthetase in fed-batch cultures of Neurospora crassa.

The effect of the nitrogen and carbon sources in the regulation of g$\text{l}$u tamine synthetase has been studied in fed-batch cultures of $\text{Neurospora crassa}$. The limitation of ammonium in an excess of the carbon source, leads to an accumulation of α-ketoglutarate and elevation of glutamine sy$\text{n}$ thetase. The limitation of sucrose in an excess of ammonium results in a decrease in glutamine synthetase activity. These results indicate that the carbon source exerts a positive control in the regulation of glutamine synthetase.     

Relationships between nitrogenase,glutamine synthetase,glutamine, and energy charge in Azotobacter vinelandii

When continuous cultures of Azotobacter vinelandii were supplied with ammonium or nitrate in amounts, which just repressed nitrogenase synthesis completely, both the intracellular glutamine level and the degree of adenylylation of the glutamine synthetase (GS) increased only slightly (from 0.45–0.50 mM and from 2 to 3 respectively), while the total GS level remained unaffected. Higher amounts of ammonium additionally inhibited the nitrogenase activity, caused a strong rise in the intracellular glutamine concentration and adenylylation of the GS, but caused no change in the ATP/ADP ratio. These results are considered as evidence that in A. vinelandii the regulation of nitrogenase synthesis is not linked to the adenylylation state of the GS and to the intracellular glutamine level, and that the inhibition of the nitrogenase activity as a consequence of a high extracellular ammonium level is not mediated via a change in the energy charge.Abbreviations GS glutamine synthetase - GS-S(Mg) Mg²⁺ dependent synthetic activity of GS - GS-T(Mn) Mn²⁺ dependent transferase activity of GS     

Nitrogen fixation in cultured cowpea Rhizobia: inhibition and regulation of nitrogenase activity.

Nitrogenase activity in agar cultures of cowpea rhizobia, strain 32H1, was rapidly inhibited by $\text{NH}{}_4{}^{\mathrm{+}}$ but this was relieved by increased O₂ tension. Inhibition was more rapid than that caused by inhibitors of protein synthesis and was not relieved by methionine sulfoximine or methionine sulfone. Under conditions were nitrogenase activity was inhibited by $\text{NH}{}_4{}^{\mathrm{+}}$, glutamine synthetase and glutamate synthase were substantially unaffected. Glutamate dehydrogenase was undetected in either nitrogenase active or $\text{NH}{}_4{}^{\mathrm{+}}$ inhibited cultures. These results indicate that $\text{NH}{}_4{}^{\mathrm{+}}$ inhibition of nitrogenase activity in strain 32H1 is not effected through glutamine synthetase regulation of nitrogenase synthesis.     

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 NH+4 is in equiblibrium 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 of glutamine synthetase in cell free extracts. Also, activities in biosynthetic assays were positively correlated with activities in gamma-glutamyl transferase assays containing 60 mM Mg2+. Relative adenylylation of glutamine synthetase was conveniently measured in cell free extracts as the ratio of gamma-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 NH+4, were masked when oxygen strongly limited culture yield. Partial relief of the limitation in cultures supplied with 10 mM NH+4 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.     

The glutamine synthetase-glutamate synthase system in Rhodopseudomonas sphaeroides

The phototrophic purple bacterium Rhodopseudomonas sphaeroides, strain 2R, can assimilate ammonium by means of glutamine synthetase and glutamate synthase. A higher activity of glutamine synthetase is displayed by cells grown in the medium with glutamate or in the atmosphere of molecular nitrogen. The activity of glutamate synthase also rises when cells grow in the atmosphere of N2. However, in contrast to glutamine synthetase, the activity of glutamate synthase does not decrease in the presence of considerable NH4+ amounts. The glutamine synthetase of R. sphaeroides is modified by adenylylation/deadenylylation. In the presence of nitrogenase in R. sphaeroides, the glutamine synthetase is found mainly in the deadenylylation state. Methionine sulfone, an inhibitor of glutamine synthetase, partly restores the activity of nitrogenase in the presence of ammonium, and prevents adenylylation of glutamine synthetase.     

Ammonia and light effect on nitrogenase activity in nitrogen-limited continuous cultures of Rhodopseudomonas capsulata. Role of glutamine synthetase

Nitrogen-limited continuous cultures of Rhodopseudomonas capsulata were used to investigate some aspects of the regulation of nitrogenase activity. The role of glutamine synthetase (GS) in this regulation was examined by measuring changes of its adenylylation state when the light intensity and the nitrogen source were varied. Maximal nitrogenase activity was observed at a dilution rate corresponding to about one third of the maximum specific growth rate (_max), both in ammonia- and in glutamate-limited cultures. At higher dilution rates, both GS and nitrogenase were inactivated by ammonia. Determination of the kinetics of inhibition of both enzymes indicated that the degree of inactivation of nitrogenase and the adenylylation state of GS were not closely related. Increase of light intensity stimulated nitrogenase activity dramatically. Conversely, a shift-down in light intensity to a limiting value resulted in a decrease of nitrogenase activity suggesting that synthesis was inhibited. On the other hand, the adenylylation state of glutamine synthetase appeared to be unaffected by changes in light intensity, indicating that GS is probably not involved in the regulation of nitrogenase expression by light.Abbreviations GS glutamine synthetase - R Rhodopseudomonas - Rs. Rhodospirillum - CTAB cetyltrimethylammonium bromide Dedicated to Prof. Dr. H. G. Schlegel on the occasion of his 60th birthday     

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     

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.     

Nitrogenase switch-off by ammonia in Rhodopseudomonas palustris: Loss under nitrogen deficiency and independence from the adenylylation state of glutamine synthetase

Nitrogenase activity in Rhodopseudomonas palustris is subject to a rapid switch-off in response to exogenous ammonia. When cells were grown on limiting nitrogen and eventually became nitrogen deficient, nitrogenase synthesis was fully derepressed but the enzyme was insensitive to ammonia. The transformation of ammonia-sensitive to ammonia-insensitive cells was a slow, but fully reversible process. The switch-off effect in ammonia-sensitive cells paralleled changes in the adenylylation state of glutamine synthetase. Ammonia-insensitive cells, however, showed similar changes in glutamine synthetase activity although nitrogenase activity was unaffected. We conclude that nitrogenase regulation and adenylylation of glutamine synthetase are independent processes, at least under conditions of nitrogen deficiency.     

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.     

Regulation of ammonium uptake and metabolism by nitrogen fixing bacteria. III. Clostridium pasteurianum

Addition of ammonium salts to N₂ fixing continuous cultures of Clostridium pasteurianum caused immediate stop of nitrogenase synthesis, while the levels of glutamine synthetase, glutamate dehydrogenase and asparagine synthetase remained constant. No evidence for an interconversion of the glutamine synthetase was found. The activities of glutamate synthase in crude extracts were inversely related to the nitrogenase levels. The intracellular glutamine pool rapidly expanded during nitrogenase repression and decreased as fast during derepression while the pool sizes of all other amino acids were not strongly related to the rate of nitrogenase formation. These investigations suggest glutamine as corepressor of nitrogenase synthesis.     

Metabolite regulation of the state of adenylylation of glutamine synthetase

When glutamine synthetase is incubated in a mixture containing adenylyltrans-ferase, the regulatory protein (P_II) and several effectors, including ATP, UTP, P_i, α-ketoglutarate, glutamine, and Mg²⁺ and/or Mn²⁺, it ultimately assumes a constant state of adenylylation. The final state of adenylylation (i.e., the number of adenylylated subunits per mole of enzyme) can vary from 0 to 12 and is specified by the concentrations and ratios of the various effectors and by the extent of uridylylation of P_II (i.e., the P_IIA:P_IID ratio). Under otherwise identical conditions, increasing the concentrations of either UTP, P_i, α-ketoglutarate, Mn²⁺, or P_IID decreases the state of adenylylation finally reached, whereas increasing the concentrations of either glutamine, ATP, or Pua increases the final state of adenylylation. The final state of adenylylation is independent of the concentrations of glutamine synthetase, adenylyltransferase, and P_II (but not of the P_IIA:P_IIDratio), and also of the initial average state of adenylylation of glutamine synthetase. Various lines of evidence show that the final state of adenylylation represents a dynamic steady state in which the rates of adenylylation and deadenylylation of glutamine synthetase are equal. It is concluded that the regulation of glutamine synthetase activity by the adenylylation mechanism utilizes a significant amount of ATP energy, but this amount is less than 0.1% that utilized directly by the glutamine synthetase in the synthesis of glutamine.     

NADH-dependent glutamate synthase and nitrogen metabolism in Neurospora crassa

The GDH (NADPH) mutant strain $\text{am-1}$ of $\text{N. crassa}$ has sizable pools of glutamine and glutamate under ammonium-limited conditions for which requires an elevated glutamine synthetase activity. Glutamine in the pre$\text{s}$ ence of 2-oxoglutarate, stimulated nicotinamide nucleotide oxidation by crude and purified extracts of the $\text{am-1}$ strain and led to a reductant dependent formation of two molecules of glutamate. Aminooxyacetate did not have any effect on the reaction, whereas azaserine inhibited it completely. It is concluded that in $\text{N. crassa}$ glutamine synthetase and glutamate synthase are responsible for the assimilation of low ammonium concentrations.     

Effects of L-methionine-DL-sulfoximine and beta-N-oxalyl-L-alpha, beta-diaminopropionic acid on nitrogenase biosynthesis and activity in Rhodopseudomonas capsulata.

Inhibitors of glutamine synthetase cause derepression of nitrogenase biosynthesis in the presence of $\text{NH}{}_4{}^{\mathrm{+}}$ in the photosynthetic bacterium Rhodopseudomonas capsulata. A new derepressor of nitrogenase biosynthesis, β-N-oxalyl-L-α,β-diaminopropionic acid (ODAP), is here compared with the widely used L-methionine-DL-sulfoximine (MSX). With both compounds, a quantitative correlation has been observed between inhibition of glutamine synthetase and derepression of nitrogenase biosynthesis. We also find that both MSX and ODAP inhibit nitrogenase activity in vivo in R. capsulata. The latter effect seems to be indirect and related to the previously reported reversible inhibition of nitrogenase activity in vivo by $\text{NH}{}_4{}^{\mathrm{+}}$. As a control it was observed that neither $\text{NH}{}_4{}^{\mathrm{+}}$ nor MSX nor ODAP inhibit nitrogenase activity in vivo in Clostridium pasteurianum.     

Effect of Ammonium Chloride and Methionine Sulfoximine on the Acetylene Reduction of Detached Root Nodules of Peas (Pisum sativum)

Acetylene-reducing activity of detached pea nodules was determined by submerging the nodules in buffer solution [tris(hydroxymethyl)aminomethane-hydrochloride, pH 7.4] containing 100 mM sodium succinate and incubating under a gas phase of 90% O₂ and 10% C₂H₂. The nitrogenase activity was 4 to 8 μmol of C₂H₄ formed per g of nodule fresh weight per h and remained constant for at least 4 h. Addition of NH₄Cl to the buffer solution (at a concentration of 10 mM or more) resulted in a significant decrease of nitrogenase activity, which was more pronounced at higher concentrations of ammonium chloride. The inhibition of nitrogenase activity by NH₄Cl was reversible; when the NH₄Cl-containing buffer solution was replaced by buffer without NH₄Cl, the original activity was partly restored. Treatment of the nodules with NH₄Cl had almost no effect on the amount of nitrogenase, as measured by the acetylene-reducing activity of ethyl-enediaminetetraacetate-toluene-treated bacteroid suspensions. The effect of NH₄Cl was largely eliminated by simultaneous addition of 10 mM methionine sulfoximine to the assay solution. This suggests that the assimilation of ammonium ions by glutamine synthetase controls the functioning of nitrogenase activity in the nodules. However, no effect of glutamine, glutamate, or aspartate on the acetylene reduction by detached nodules could be detected.     

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.