Isolation and Characterization of Glutamate Synthase Mutants of Azospirillum brasilense

Isolation of a glutamate synthase (GOGAT)-negative, pleiotropically N utilization-defective mutant of Azospirillum brasilense: cloning and partial characterization of GOGAT structural gene

“…The specific activity of GOGAT in RG was maximal when grown with 0.1% NH4Cl; lower activities were observed when it was grown with 0.01% NH4Cl, glutamate, or glutamine as the sole nitrogen source (Table 1). Compared with activities in the wild type, some reductions of the GS and cold-labile GDH activities were observed in N12; lower activities of these two enzymes were also noticed with other Asm -mutants (2).…”

“…It is not yet very clear why GOGAT deficiency would lead to pleiotropic N-assimilation defects (Asm -) in the mutants. An abnormally high concentration of glutamine may cause adenylylation of GS, and repression of sensitive operons in GOGAT -mutants has been suggested (2). The GOGAT structural gene of Escherichia coli has been cloned (5) and sequenced (23), and it showed that the cx-and (-subunits of GOGAT are determined by the gltB and gltD genes of the gltBDF operon (5).…”

“…The importance of the GS-GOGAT pathway was first demonstrated by Nagatani et al (19), who observed that GOGAT-deficient mutants of Klebsiella pneumoniae showed an ammonia assimilation (Asm-) defect; they failed to fix N2 (Nif-) and utilize a number of amino acids as N sources. GOGAT-deficient mutants of N2-fixing bacteria showing the Asm-phenotype have been described for A. brasilense (2), Bradyrhizobium japonicum (21), Rhizobium meliloti (13,15,24), and Azorhizobium sesbaniae ORS571 (11). It is not yet very clear why GOGAT deficiency would lead to pleiotropic N-assimilation defects (Asm -) in the mutants.…”

“…In Azospirillum brasilense, a nitrogen-fixing aerobic soil bacterium well known for its ability to colonize roots of grass plants of economic importance and to increase plant productivity (8,22), two enzymatic pathways exist for ammonia assimilation: the glutamate dehydrogenase (GDH; EC 1.4.1.3) pathway in which ammonia is available at a high level (16,35) and the glutamine synthetase (GS; EC 6.3.1.2)-glutamate synthase (GOGAT [glutamine 2-oxoglutarate amidotransferase]; EC 1.4.1.13) pathway in which ammonia availability is low, for example, during N2 fixation (2,4,7,8,35). The GS-GOGAT pathway is particularly important for all freeliving N2-fixing bacteria for growth under ammonia-deficient conditions (3,25), but for symbiotic bacteria, e.g., Rhizobium spp.…”

“…The glt (GOGAT) locus of A. brasilense was identified by isolating a broad-host-range pLAFR1 cosmid clone from a gene library of the bacterium that rectified Asm -and GOGAT -defects (full recovery of activities of the nitrogenase, the assimilatory nitrate and nitrite reductases, and the glutamate synthase). A 7.5-kb (2,4,7,8,35). The GS-GOGAT pathway is particularly important for all freeliving N2-fixing bacteria for growth under ammonia-deficient conditions (3,25), but for symbiotic bacteria, e.g., Rhizobium spp.…”