Molecular cloning and functional characterization of Rhizobium leguminosarum structural nif-genes by site-directed transposon mutagenesis and expression in Escherichia coli minicells.

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.