Identification and phenotypical characterization of a cluster of fix genes, including a nif regulatory gene, from Rhizobium leguminosarum PRE

Summary A nif regulatory gene in R. leguminosarum PRE was identified by interspecies DNA hybridization and site-directed Tn5 mutagenesis. Significant homology was found with the K. pneumoniae nifA locus, a R. meliloti symbiotic regulatory gene and E. coli ntrC; Tn5 insertions within this nifA gene inhibit the expression of the nifHDK operon, encoding synthesis of the nitrogenase polypeptides.Specific DNA hybridization also was detected between a downstream adjacent part of the PRE sym plasmid and the R. leguminosarum 248 fixZ gene, a homologue of the K. pneumoniae nifB locus. To detect further fix genes we investigated a region of the sym plasmid which is localized within a short distance upstream from the nifA gene and is transcribed selectively at a high rate during symbiosis. This approach revealed the existence of a fix cluster in which Tn5-mutations cause a Fix^- phenotype although wild-type levels of nitrogenase synthesis were detectable. In a sym plasmid fragment, which is immediately upstream adjacent to the nifA locus and only moderately expressed in Rhizobium bacteroids, a second fix gene conferring the same symbiotic phenotype was detected.