title: Molecular cloning and functional characterization of Rhizobium leguminosarum structural nif-genes by site-directed transposon mutagenesis and expression in Escherichia coli minicells.
abstract: In order to study the structural organization and regulation of the expression of the nitrogenase gene cluster in Rhizobium leguminosarum PRE we selected relevant subfragments of the sym-plasmid from clone banks by homology with R. meliloti nif-genes. Site-directed Tn5 mutagenesis was applied to a nif DH-specific clone and subsequently the transposon insertions were transferred back into the wild-type rhizobial genome by homologous recombination. Phenotypic effects of Tn5 mutations in the region of the structural nif-genes were determined by measuring acetylene reduction in nodulated plants and by immunological analysis of bacteroid-specific proteins. The localization of Tn5 insertion sites was in accordance with observed consequences: two genotypically different Tn5-induced mutations within nif D caused repression of CI alpha and beta synthesis and a strong reduction of CII production, thus resulting in a Fix- phenotype. Expression of different cloned Rhizobium DNA inserts, bearing nif K, nif D, nif H, or nif DH, was achieved in Escherichia coli minicells dependent upon the presence of a strong upstream vector promoter sequence. Gene products were identified by immunoprecipitation with specific antisera. Endogenous rhizobial transcriptional start signals in one case (nif H) seemed to be recognized at a low rate by the E. coli system; in contrast, Rhizobium ribosome binding sites for all three structural nif-genes functioned normally in minicells. The approximate location of the coding regions for nif KDH genes was determined and found to be contiguous.
mesh_terms: D002874:Chromosome Mapping; D003001:Cloning, Molecular; D004251:DNA Transposable Elements; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D009154:Mutation; D009591:Nitrogenase / Q000235:genetics*; D009876:Operon; D010957:Plasmids; D012231:Rhizobium / Q000235:genetics*
chemical_list: D004251:DNA Transposable Elements; D009591:Nitrogenase