Rhizobium leguminosarum bv viciae contains a second fnr/fixK-like gene and an unusual fixL homologue

Genes of Rhizobium legominosarum bv. viciae VF39 coding for the regulatory elements NifA, FixL and FixK were isolated, sequenced and genetically analysed. The fixK-fixL region is located upstream of the fixNOQP operon on the non-nodulation plasmid pRIeVF39c. The deduced amino acid sequence of FixL revealed an unusual structure in that it contains a receiver module (homologous to the N-terminal domain of response regulators) fused to its transmitter domain. An oxygen-sensing haem-binding domain, found in other FixL proteins, is conserved in R. leguminosarum bv. viciae FixL. R. leguminosarum bv. viciae possesses a second fnr-like gene, designated fixK, whose encoded gene product is very similar to Rhizobium meliloti and Azorhizobium caulinodans FixK. Individual R. leguminosarum bv. viciae fixK and fixL insertion mutants displayed a Fix(+) phenotype. A reduced nitrogen-fixation activity was found for a R. leguminosarum bv. viciae fnrN-deletion mutant, whereas no nitrogen-fixation activity was detectable for a fixK/fnrN double mutant. The R. leguminosarum bv. viciae nifA gene is expressed independently of FixL and FixK under aerobic and microaerobic conditions, whereas fixL gene expression is induced under microaerobiosis. Another orf was identified downstream of fixK-fixL and encodes a product which has homology to pseudoazurins from different species. Mutation of this azu gene showed that it is dispensable for nitrogen fixation.