THE CYCHJKL GENES OF RHIZOBIUM-MELILOTI INVOLVED IN CYTOCHROME-C BIOGENESIS ARE REQUIRED FOR RESPIRATORY NITRATE REDUCTION EX PLANTA AND FOR NITROGEN-FIXATION DURING SYMBIOSIS

We report the genetic and biochemical analysis of Rlzizol?ilkm meliloti mutants defective in symbiotic nitrogen fixation (Fix(-)) and ''respiratory'' nitrate reduction (Rnr(-)). The mutations were mapped close to the ade-1 and cys-46 chromosomal markers and the mutated locus proved to be identical to the previously described fix-14 locus. 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 cycH exhibited a high degree of similarity to the Bradyrhizobium japonicum counterpart, while CycK and CycL shared more than 50% amino acid sequence identity with the Rhodobacter capsulatus Ccl1 and Ccl2 proteins, respectively. cycJ encodes a novel membrane anchored protein of 150 amino acids. We suggest that this gene cluster codes for (parts of) a multisubunit cytochrome c haem 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.