Detection of two forms of glutamine synthetase in Bacillus brevis (A. G. 4)

A laboratory isolate of $\text{Bacillus}\text{brevis}$ could grow and sporulate on an amino acid, viz., alanine or glutamate or aspartate as single source of carbon and nitrogen. It failed to sporulate if the amino acid was replaced by the corresponding keto acid and ammonium sulphate in the medium, although, normal growth was observed. One of the key enzymes in nitrogen assimilation, the glutamine synthetase, has been purified by DE-52 and affinity column chromatography from both alanine and pyruvate grown cells. The kinetic and other properties of both of these enzymes were studied. The enzyme isolated from alanine grown cells differed significantly from that isolated from pyruvate grown cells (viz.,pH optima, response to Mg⁺⁺ and other effectors). A possible role of glutamine synthetase in the initiation of bacterial sporulation is discussed.     

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.     

Glutamine-requiring mutants of Bacillus subtilis.

Two glutamine-requiring (Gln^?) mutants of $\text{Bacillus subtilis}$ SMY were deficient in glutamine synthetase activity $\text{in vitro}$. The Gln^? mutants sporulated poorly unless glutamine was provided at high concentrations. The differential rate of histidase synthesis following induction was 4- to 6-fold higher in the Gln^? mutants than in wild-type cells. In addition, glucose repression of utilization of alternative carbohydrates appeared to be partially relieved in the Gln^? mutants.     

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.     

Ammonium (methylammonium) transport by Klebsiella pneumoniae

Klebsiella pneumoniae can accumulate methylammonium up to 80-fold by means of a transport system as indicated by the energy requirement, saturation kinetics and a narrow pH profile around pH 6.8. Methylammonium transport (apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}\text{= 100 μ}\text{M}$, $\text{V = 40 μ}\text{mol/min}$ per g dry weight at 15°C) is competitively inhibited by ammonium (apparent $\text{K}{}_{\mathrm{<mtext>i</mtext>}}\text{= 7 μ}\text{M}$). The low $\text{K}{}_{\mathrm{<mtext>i</mtext>}}$ value and the finding that methylammonium cannot serve as a nitrogen source indicate that ammonium rather than methylammonium is the natural substrate. Uphill transport is driven by a component of the protonmotive force, probably the membrane potential. The transport system is under genetic control; it is partially repressed by amino acids and completely by ammonium. Analysis of mutants suggest that the synthesis of the ammonium transport system is subject to the same ‘nitrogen control’ as nitrogenase and glutamine synthetase.     

Effect of ammonia and glutamine on macromolecule synthesis and breakdown during sporulation of Saccharomyces cerevisiae.

The effect of two known inhibitors of sporulation in yeast, ammonia and glutamine, on certain biochemical events during sporogenesis have been studied using sporulating $\text{a}\text{α}$ and non sporulating $\text{α}\text{α}$ cells. Both strains gave similar results on the increase in dry cell weight, protein and RNA breakdown and the suppression of the intensive RNA and protein syntheses occurring after 4 hours. The inhibitory effect of ammonia and glutamine on RNA and protein syntheses is reversible under the same conditions which do so for sporulation.     

The role of O2-limitation in control of nitrogenase in continuous cultures of Rhizobrium sp.

Oxygen-limited continuous cultures of the cowpea $\text{Rhizobium}$ sp. strain CB756, had high levels of nitrogenase activity, which were not significantly affected by excess ammonium ions or glutamine. When the growth-restricting O₂-limitation was partially relieved, nitrogenase was repressed and this was accompanied by increased adenylylation of glutamine synthetase. It is suggested that the restricted supply of ATP interferes with adenylylation of glutamine synthetase during O₂-limited growth, thus preventing repression of nitrogenase in the presence of excess ammonium ions.     

Utilization of ammonia for tryptophan synthesis

A strain of $\text{Escherichia coli}$ in which the glutamine amidotransferase function (anthranilate synthetase component II) of anthranilate synthetase has been deleted synthesizes tryptophan using NH₃-dependent anthranilate synthetase component I (AS-I). In NH₃-limited media this strain is a tryptophan auxotroph. Mutants that acquired the capacity to grow in NH₃-limited media were isolated. Growth of mutant strains in NH₃-limited media correlates with increased AS-I activity. Glutamine-dependent AS activity was not found in any of the mutant strains indicating that another glutamine amidotransferase had not been recruited to function with AS-I.     

Porphyrin mutants of Saccharomyces cerevisiae: Correlated lesions in sterol and fatty acid biosynthesis

The $\text{ole2}$, $\text{3}$ and $\text{4}$ mutants of yeast require an unsaturated fatty acid and methionine for growth and do not synthesise ergosterol. They have very similar sterol compositions and all accumulate lanosterol. The mutants lack cytochrome pigments and have negligible respiratory activity. Porphyrin intermediates alleviate the lipid requirement of $\text{ole2}$ and $\text{ole3}$ and restore respiratory competence. It is concluded that the primary defects in these mutants are lesions in porphyrin biosynthesis.     

The occurrence of glutamate synthase in algae.

The presence of glutamate synthase in the green algae $\text{Chlorella fusca}$ var. $\text{vacuolata}$ has been demonstrated using a whole cell assay as well as cell free extracts. The assay is complicated by the presence of glutamine (amino): α-oxoglutarate transaminase, but this enzyme can be inhibited by amino oxyacetate. The rates of glutamate synthase activity are sufficient to account for the known rates of nitrate assimilation to occur via the glutamine synthetase/glutamate synthase pathway.     

Evidence for methionine sulfoximine as a transition-state analog for glutamine synthetase from NMR and EPR data.

Manganese(II)bound at the “tight” metal ion site of unadenylylated glutamine synthetase (E. coli W) has two rapidly exchanging first coordination sphere water molecules. The solvation number was evaluated from a study of the frequency dependence of $\text{1}\text{pT}{}_{\mathrm{<mtext>1p</mtext>}}$, the paramagnetic contribution to the longitudinal relaxation rate of solvent protons. The number of rapidly exchanging water molecules is reduced to one in the presence of saturating L-glutamate and to ～0.2 when L-methionine SR-sulfoximine (MSOX) is present. MSOX is a linear competitive inhibitor (K_I=3μM) of glutamine synthetase when L-Glu is the substrate. The dissociation constant of MSOX measured by following the 18 fold decrease in $\text{1}\text{pT}{}_{\mathrm{1p}}$ (at 48 MHz) is 30μM and is lowered to ～9μM in the presence of ADP. The high affinity of MSOX for the enzyme suggests that this compound mimicks the “transition-state” for the glutamine synthetase reaction. Further evidence for this postulate is found from the dramatic sharpening of the epr spectrum of enzyme-bound Mn(II) in the presence of MSOX and MSOX plus ADP. The intense change in the epr spectrum arises from reduced solvent accessibility to bound Mn(II) and conformational changes produced by binding MSOX and ADP. The suggestion is made from these data that L-Glu and MSOX bind near or directly to the Mn(II) at the “tight” metal ion site in glutamine synthetase isolated from E. coli W.     

Glutatmine synthetase activity in the chick brain during postnatal growth. Effect of MSO

Glutamine synthetase activity was estimated in the chick cerebral hemispheres, optic lobes and cerebellum between the 1st and the 30th day of postnatal growth. Glutamine synthetase activity is higher in the cerebellum than in the cerebral hemispheres and lowest in the optic lobes at 1 day after hatching; at 30 days after hatching, it is the same in the optic lobes and in the cerebellum and lowest in the cerebral hemispheres. The great increase of glutamine synthetase activity between the 1st and the 4th day after hatching corresponds to the appearance of the heterogeneity of the chick brain glutamate metabolism. The glutamine synthetase activity is inhibited by MSO $\text{in vivo}$ at a concentration of 100 mg kg ^?1 at values of 87, 90 and 89 % in cerebral hemispheres, optic lobes and cerebellum of 1, 2 and 4-day-old chicks. The enzyme inhibition is less pronounced $\text{in vitro}$ and reaches values of about 25 and 75 % for 1 and 10 mM MSO concentrations respectively in the three brain areas of the 1 to 4-day-old chick and values slightly lower in the 30-day-old chick brain.     

Preferential utilization of glutamine for amination of xanthosine 5'-phosphate to guanosine 5'-phosphate by purified enzymes from Escherichia coli

GMP synthetase was purified 180-fold from $\text{E. coli}$ B and 18-fold from the derepressed purine auxotroph, $\text{E. coli}$ B-96. The enzymes from both sources show the same preference for glutamine over ammonia as amino donor. Each is dimeric, consisting of subunits of molecular weight about 60,000. Thus the two are apparently identical. The similarities between GMP synthetase and xanthosine 5′-phosphate aminase of $\text{E. coli}$ B-96 (N. Sakamoto, G.W. Hatfield, and H.S. Moyed, J. Biol. Chem. (1972) $\text{247}$, 5880–5887) in respect to structure, state of derepression, and behavior during purification, lead us to the conclusion that the synthetase and the aminase are a single entity. We observe no loss or separation of glutamine-dependent activity upon purification of GMP synthetase and we suggest that such loss, reported by other workers, results artifactually by inactivation of an intrinsic glutamine-binding site. GMP synthetase appears not to contain a glutamine-binding subunit which is separable from the xanthosine 5′-phosphate-aminating component.     

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.     

Crystals of glutamine synthetase from Escherichia coli.

Further details are given of crystals of glutamine synthetase prepared from Escherichia coli. Crystals of two kinds have been observed: (1) rhombic dodecahedra which correspond to the morphology of the crystals studied by Eisenberg et al. (1971) (and which were found by them to contain dodecamers), and (2) rhombohedra, reported here. Cell dimensions and packing considerations led to the consideration of two possible structures for the rhombohedral crystals. These we have called the “T = 7 structure” and the “B.C.C. structure”. The T = 7 structure would be related to that derived by Eisenberg and would contain dodecamers, but is inconsistent with our X-ray intensity data. The B.C.C. structure is considered more probable. It is built of cubic octomers or square tetramers. Electron micrographs of our glutamine synthetase preparations show a wide variety of aggregates, including dodecamers and tetramers. The unit cell dimensions of our crystals are $\text{a = 140 ± 2}\text{A}\text{̊}$, and $\text{c = 148 ± 2}\text{A}\text{̊}$. The Laue symmetry group is $\text{3}\text{̄}$m P3₁.     

Two forms of glutamine synthetase in free-living root-nodule bacteria.

Cell-free extracts of $\text{Rhizobium japonicum}$ 61A76 contain two forms of glutamine synthetase (EC 6.3.1.2) which can be easily separated by isoelectric focusing. The more acid form (pI = 5.4), like the enzyme from $\text{E. coli}$, is stable at 50° and catalyses an ADP-dependent transferase reaction, whose inhibition by excess Mg⁺⁺ can be relieved by snake venom phosphodiesterase. The more alkaline form (pI = 6.1) is labile at 50°, and catalyses and ADP-dependent transferase reaction which is strongly inhibited by Mg⁺⁺ regardless of phosphodiesterase treatment. We have also observed the two forms of the enzyme in a nitrogenaseless mutant of 61A76, and in other strains of rhizobia, but only the acid form in $\text{E. coli}$ W, $\text{A. vinelandii}$ OP, and $\text{K. pneumoniae}$ M 51A.     

Role of glutamine synthetase in the initiation of sporulation in Bacillus polymyxa

The activity of glutamine synthetase (GS) was investigated during culture development of Bacillus polymyxa CN 2219 and its asporogenous mutant deficient in protease production. At 28°C, temperature permissive for sporulation, the glutamine synthetase activity was found to decline in the wild type cells which acquire the competence for sporulation. This decline was not observed in the asporogenous mutant. Incubation at 37°C (temperature non permissive) suppressed sporulation in the wild type and maintained glutamine synthetase activity. The involvement of glutamine synthetase in the repression of sporulation was further confirmied by the action of l-methionine sulfoximine a specific inhibitor of glutamine synthetase, which overcomes the catabolite repression by ammonium and induces sporulation. Intracellular proteases were measured as early markers of the initiation of sporulation and were found to be induced during sporulation.Abbreviations GS glutamine synthetase - MSO l-methionine sulfoximine - GYS glucose-yeast extract-salts - GT -glutamyltransferase - PMSF phenylmethylsulfonylfluoride