Open AccessCharacterization of the Azorhizobium sesbaniae ORS571 genomic locus encoding NADPH-glutamate synthase
Author(s) -
Robert G. K. Donald,
Jerome P. Lapointe,
Robert A. Ludwig
Publication year - 1988
Publication title -
journal of bacteriology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.652
H-Index - 246
eISSN - 1067-8832
pISSN - 0021-9193
DOI - 10.1128/jb.170.3.1197-1204.1988
Subject(s) - biology , microbiology and biotechnology , plasmid , mutant , locus (genetics) , genetics , homology (biology) , genomic dna , shuttle vector , gene , restriction map , genomic library , insertion , bacillus subtilis , recombinant dna , peptide sequence , bacteria , vector (molecular biology)
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.