Characterization of the Azorhizobium sesbaniae ORS571 genomic locus encoding NADPH-glutamate synthase.
Sixteen independent Azorhizobium sesbaniae ORS571 vector insertion (Vi) mutants defective in ammonium assimilation (Asm-) were selected; genomic DNA sequences flanking the insertion endpoints were cloned directly. Resulting recombinant plasmids were used to identify, by hybridization, corresponding wild-type DNA sequences from an A. sesbaniae lambda EMBL3 genomic library (lambda Asm phages). All 16 Asm- Vi mutants physically mapped to a single genomic locus. Plasmid subclones of recombinant phage lambda Asm152 were able to complement both Escherichia coli gltB and A. sesbaniae Asm- Vi mutants; NADPH-glutamate synthase activity was detected in all such strains complemented to Asm+. Heterologous and homologous complementations required both A. sesbaniae gltA+ and (inferred) gltB+ genes. Eleven A. sesbaniae Asm- Vi mutants mapped to a 4-kilobase-pair (kbp) DNA region that exhibited homology with Bacillus subtilis gltA+. In E. coli maxicell labeling experiments, this 4-kbp DNA region encoded a 165-kilodalton polypeptide that was inferred to be the product of the A. sesbaniae gltA+ gene (glutaminase NADPH-dependent L-glutamate synthase subunit). Site-directed Tn5-lacZ mutagenesis of a glt plasmid subclone identified a region that bisected this locus into (at least) two cistrons. Because the remaining five A. sesbaniae Asm- mutants mapped to a 1.5-kbp region adjacent to gltA+, these mutants probably define a single gltB+ gene (glutamate dehydrogenase NADPH-dependent L-glutamate synthase subunit); this region did not exhibit homology with the B. subtilis gltB+ gene.
Donald
R G
RG
Department of Biology, Thimann Laboratories, University of California, Santa Cruz 95064.
Lapointe
J
J
Ludwig
R A
RA
D000641:Ammonia / Q000378:metabolism; D003001:Cloning, Molecular; D004251:DNA Transposable Elements; D004269:DNA, Bacterial / Q000235:genetics; D005796:Genes; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D005970:Glutamate Synthase / Q000235:genetics*; D007763:Lac Operon; D009154:Mutation; D012230:Rhizobiaceae / Q000235:genetics*; D000637:Transaminases / Q000235:genetics*
D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D000641:Ammonia; D005970:Glutamate Synthase; D000637:Transaminases