浑球红细菌谷氨酸合酶基因(glt)的克隆和图谱分析

利用转座子Tn5随机插入诱变筛选得到12株浑球红细菌(Rhodobacter sphaeroides)氨同化缺陷突变株(Asm~-)。这些突变株胞内均无GOGAT活性,同时它们均无固氮酶活性(Nif~-),并且具有氮代谢多效性缺失表型(Ntr~-)。将含有Azorhizobium sesbaniae ORS571的完整glt基因的质粒pHB10转入突变株中能互补上述表型。通过筛选携带Tn5的R-prime质粒克隆了glt::Tn5片段。Southern杂交证明所克隆glt::Tn5片段与E. coli的gltBD基因有同源性。用此片段与以pLAFR3为载体所构建的R. sphaeroides 601基因文库进行菌落原位杂交筛选到了携带glt基因的cosmid pLT27。pLT27能互补所有12株R.sphaeroides氨同化缺陷突变株。酶切分析表明在该cosmid中插人的染色体DNA片段大小约为26.5kb。以pRK415为载体亚克隆了4.0kb与10.5kh的pLT27的Hindlll酶切片段,分别命名为pLTRK271与pLTRK272。pLTRK272能互补变种GT6、GT10、GT11,pLTRK…

CLONING AND PHYSICAL MAPPING OF THE glt GENE IN RHODOBACTER SPHAERUIDES

Twelve Rhodobacter sphaeroides mutants disturbed in ammonia assimilation ( Asm-) were independently isolated by employing transposon Tn5-generated mutation. Biochemical and physiological analysis revealed that these mutants had no activity of glutamate synthase (GOGAT-) and showed a defective pleiotropic phenotype in nitrogen metabolism ( Ntr-) and a Nlf- phenotype in RCVBN medium. The glt gene locus of Azorhizobium sesbaniae ORS571 is able to complement the R. sphaeroides mutants containing Tn5 insertions which produced characteristic Asm-phenotypes. The git:: Tn5 region of the mutants was cloned and shown to have a homological sequence with the Escherichia coli gltBD genes using Southern hybridization analysis. A cosmid named pLT27 containing glt gene was identified using git:: Tn5 fragment as a probe from R. sphaeroides 601 genomic library constructed by using cosmid pLAFR3. pLT27 was able to complement the 12 GOGAT-mutants. After physical mapping of the 26.5kb of R. sphaeroides insert DNA in pLT 4kb and 10.5kb HindIII fragments of pLT27 were subcloned in pRK415. The resulted plasmid pLTRK272 could complement mutants GT6, GT10 and GT11, and failed tocomplement other 9 mutants. But pLTRK271 only complemented the 9 mutants. The cloned 10.5kb Hindlll fragment of pLTRK272 had homology with E. coli gltBD genes. Southern hybridization indicated that the specific positions of transposon insertions of the mutants GT6, GT10 and GT11 mapped within a 7kb EcoRI-Hind111 fragment of pLT27, while those of other 9 mutants located at a 4kb HindIII fragment of pLT27. The physical maps of pLTRK271 and pLTRK272 were constructed. A 11kb EcoRI-HindIII fragment of R. sphaeroides genome DNA contains genetic information corresponding to some structrual or regulatory loci for GOGAT biosynthesis.