Donald R G, Lapointe J, Ludwig R A
Department of Biology, Thimann Laboratories, University of California, Santa Cruz 95064.
J Bacteriol. 1988 Mar;170(3):1197-204. doi: 10.1128/jb.170.3.1197-1204.1988.
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.
筛选出16个独立的在铵同化方面有缺陷（Asm-）的 Sesbania rostrata根瘤菌ORS571载体插入（Vi）突变体；直接克隆插入端点两侧的基因组DNA序列。用所得重组质粒通过杂交从Sesbania rostrata根瘤菌λEMBL3基因组文库（λAsm噬菌体）中鉴定出相应的野生型DNA序列。所有16个Asm- Vi突变体在物理图谱上都定位到单个基因组位点。重组噬菌体λAsm152的质粒亚克隆能够互补大肠杆菌gltB和Sesbania rostrata根瘤菌Asm- Vi突变体；在所有互补为Asm+的此类菌株中都检测到了NADPH-谷氨酸合酶活性。异源和同源互补都需要Sesbania rostrata根瘤菌gltA+和（推测的）gltB+基因。11个Sesbania rostrata根瘤菌Asm- Vi突变体定位到一个与枯草芽孢杆菌gltA+具有同源性的4千碱基对（kbp）DNA区域。在大肠杆菌大细胞标记实验中，这个4-kbp DNA区域编码一个165千道尔顿的多肽，推测它是Sesbania rostrata根瘤菌gltA+基因（依赖NADPH的L-谷氨酸合酶亚基谷氨酰胺酶）的产物。对一个glt质粒亚克隆进行定点Tn5-lacZ诱变鉴定出一个区域，该区域将这个位点一分为二（至少）成两个顺反子。由于其余5个Sesbania rostrata根瘤菌Asm-突变体定位到与gltA+相邻的一个1.5-kbp区域，这些突变体可能定义了一个单一的gltB+基因（依赖NADPH的L-谷氨酸合酶亚基谷氨酸脱氢酶）；该区域与枯草芽孢杆菌gltB+基因没有同源性。
