Effects of D-Glutamate on Enzymes of Ammonia Assimilation in Bacillus megaterium NCIB 7581

Abstract

Enzymes possibly concerned in the assimilation of ammonia were assayed in Bacillus megaterium NCIB 7581 after growth with ammonium sulphate as sole source of nitrogen. Alanine dehydrogenase and aspartase were not detected, but both NADPH-dependent glutamate dehydrogenase and glutamate synthase (NADH-or NADPH-dependent) were found. Glutamine synthetase was also present, though differing methods of assay gave different values for its activity.Glutamate dehydrogenase was competitively inhibited by D-glutamate, whereas glutamate synthase was insensitive to D-glutamate or D-glutamine (singly or together). D-Glutamate was a substrate for glutamine synthetase, and the use of L-glutamate by this enzyme was partly inhibited by a 10-fold molar excess of D-glutamate. L-Glutamine strongly inhibited glutamine synthetase, but D-glutamine had no effect.A D-glutamate-resistant substrain (grown with ammonium sulphate as nitrogen source) contained 10 times more glutamate dehydrogenase activity than did the wild-type under the same conditions, and the enzyme showed altered affinity for 2-oxoglutarate and for Dglutamate. Activities of glutamate synthase and glutamine synthetase in the substrain were similar to those in the wild-type.Glutamate dehydrogenase was absent from organisms grown with nitrate as sole nitrogen source, yet growth under these conditions was still inhibited by D-glutamate. I N T R O D U C T I O NGrowth of Bacillus megaterium NCIB 7581 in a simple chemically defined medium was strongly inhibited by D-glutamate when ammonium sulphate was the nitrogen source (White, 1979). D-Glutamate was not inhibitory in the presence of L-glutamate or other L-amino acids that, like L-glutamate, could act as sole nitrogen sources for growth. DGlutamate might therefore inhibit the assimilation of ammonia by B. megaterium 758 1 and this paper describes a study of the enzymes (glutamate dehydrogenase, glutamate synthase, glutamine synthetase, aspartase and alanine dehydrogenase) that might take part in this process. The effect of D-glutamate on transaminases that use L-glutamate as substrate was also examined. METHODSOrganisms. BuciZlus megaterium NCIB 7581 and 758 1 GRA-1 (a D-glutamate-resistant substrain) were described by White (1979); 7581 GRN-1 (another D-glutamate-resistant substrain) is described in this paper. Escherichiu coli W (ATCC 9637) was described by White (1976). This organism was used to provide an extract with high enzymic activity to check the proper functioning of the assays for glutamine synthetase (four methods) and glutaminase.Media. Medium A1 (supplemented with biotin and citrate) was described by White (1979). When NaNO, (3 g 1-l) was used as the sole nitrogen source, (NH4),S04 was omitted from medium A1 and Na2S04. 10HzO 0022-1287/79/oooO-8123 $02.00 0 1979 SGM I1M I C 114

Minimal Nutritional Requirements of Bacillus sphaericus NCTC9602 and 26 other Strains of this Species: the Majority Grow and Sporulate with Acetate as Sole Major Source of Carbon

“…Enzymic activities in the extracts were measured at 30 "C. Pyruvate dehydrogenase complex (overall reaction) was assayed by recording the anaerobic (see White, 1979) pyruvate-dependent reduction of NAD+ in a reaction mixture similar to that of Guest & Creaghan (1973); 2-oxoglutarate dehydrogenase complex (overall reaction) was assayed similarly. Pyruvate dehydrogenase El (EC 1 .2.4.1) was assayed spectrophotometrically at 420 nm and pH 6.3 or 7-0 with ferricyanide as electron acceptor (Hager & Kornberg, 1961), and 2-oxoglutarate dehydrogenase El (EC 1.2.4.2) was assayed similarly.…”

“…Isocitrate dehydrogenase (EC 1.1.1.41) was assayed by the method of Plaut (1962). Glutamate dehydrogenase (EC 1 .4.1.4) and glutamate synthase (EC 1 .4.1.13) were assayed anaerobically at pH 7.6 as described by Meers et al (1970) and White (1979) with NADPH as cofactor.…”

Enhancement of methionine production by methionine analogue ethionine resistant mutants of Brevibacterium heali

“…The lyophilized culture of the methionine producer Breuibacterium heali [6], was revived in 25 ml of a synthetic broth medium of ALFOLDIS [7] with 1% glucose. Prior to mutagenic treatment for obtaining the analogue (ethionine) resistant mutants, the sensitivity of the parent strain to this analogue was tested.…”

Bacillus thuringiensis growth and toxicity

“…The pathways for ammonia assimilation are catabolized by alanine dehydrogenase and glutamate dehydrogenase (26,30). Although glutamate synthase and glutamine synthetase have been found in Bacillus megaterium (31,32), Bacillus licheniformis (33), and Bacillus subtilis (34), as far as the authors are aware, no report about the presence of this system in BT has been made. Meers et al (35) found that the expression of these enzymes was very high for B. megaterium (among other bacteria) when the growth was ammonium limited.…”