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 melilot i mutants defective in symbiotic nitrogen fixation (Fix(-)) and ''resp iratory'' nitrate reduction (Rnr(-)). The mutations were mapped close to the ade-1 and cys-46 chromosomal markers and the mutated locus prov ed to be identical to the previously described fix-14 locus. By direct ed Tn5 mutagenesis, a 4.5 kb segment of the chromosome was delimited i n which all mutations resulted in Rnr(-) and Fix(-) phenotypes. Nucleo tide sequence analysis of this region revealed the presence of four op en reading frames coding for integral membrane and membrane-anchored p roteins. Biochemical analysis of the mutants showed that the four prot eins were necessary for the biogenesis of all cellular c-type cytochro mes. In agreement with the nomenclature proposed for rhizobial genes i nvolved in the formation of c-type cytochromes, the four genes were de signated cycH, cycJ, cycK, and cycL, respectively. The predicted prote in product of cycH exhibited a high degree of similarity to the Bradyr hizobium japonicum counterpart, while CycK and CycL shared more than 5 0% amino acid sequence identity with the Rhodobacter capsulatus Ccl1 a nd Ccl2 proteins, respectively. cycJ encodes a novel membrane anchored protein of 150 amino acids. We suggest that this gene cluster codes f or (parts of) a multisubunit cytochrome c haem lyase. Moreover, our re sults indicate that in R. meliloti c-type cytochromes are required for respiratory nitrate reduction ex planta, as well as for symbiotic nit rogen fixation in root nodules.