The cycHJKLgenes of Rhizobium melilotiinvolved in cytochrome cbiogenesis are required for “respiratory” nitrate reduction ex plantaand for nitrogen fixation during symbiosis

We report the genetic and biochemical analysis of Rhizobium melilotimutants defective in symbiotic nitrogen fixation (Fix−) and “respiratory” nitrate reduction (Rnr−). The mutations were mapped close to the ade-1and cys-46chromosomal markers and the mutated locus proved to be identical to the previously described fix-14locus. By directed Tn5 mutagenesis, a 4.5 kb segment of the chromosome was delimited in which all mutations resulted in Rnr−and Fix−phenotypes. Nucleotide sequence analysis of this region revealed the presence of four open reading frames coding for integral membrane and membrane-anchored proteins. Biochemical analysis of the mutants showed that the four proteins were necessary for the biogenesis of all cellular c-type cytochromes. In agreement with the nomenclature proposed for rhizobial genes involved in the formation of c-type cytochromes, the four genes were designated cycH, cycJ, cycK, and cycL, respectively. The predicted protein product of cycHexhibited a high degree of similarity to the Bradyrhizobium japonicumcounterpart, while CycK and CycL shared more than 50% amino acid sequence identity with the Rhodobacter capsulatusCcll and Cc12 proteins, respectively. cycJencodes a novel membrane anchored protein of 150 amino acids. We suggest that this gene cluster codes for (parts of) a multi-subunit cytochrome chaem lyase. Moreover, our results indicate that in R. meliloti c-type cytochromes are required for respiratory nitrate reduction ex planta, as well as for symbiotic nitrogen fixation in root nodules.