Rhizobium etli cycHJKL gene locus involved in c-type cytochrome biogenesis: sequence analysis and characterization of two cycH mutants

Abstract

The cycHJKL gene locus was cloned from Rhizobium etli by the rescue of a Tn 5mob insertion of a mutant (IFC01) which was affected in the production of c-type cytochromes. The cycH, cycJ, cycK and cycL genes are proposed to code for different subunits of a haem lyase complex involved in the attachment of haem to cytochrome c apoproteins. CycH of 365 aa shared 27, 36, 47 and 63% identity with CycH from Paracoccus denitrificans, Bradyrhizobium japonicum, R. meliloti , and R. leguminosarum, respectively. CycJ of 153 aa shared 52, 71, and 85% identity to the cycJ gene product of B. japonicum, R. meliloti, R. leguminosarum, respectively. CycK of 666 aa shared 62, 73, and 90% homology with CycK from B. japonicum, R. meliloti , and R. leguminosarum, respectively, while CycL of 151 aa shared 57, 67 and 86% homology with CycL from the abovementioned species. The Tn 5mob insertion present in the IFC01 strain was located in the cycH gene. This strain was able to infect bean plants, but unable to fix nitrogen during symbiosis. A previously described R. etli cytochrome c-deficient MuD1 lac-induced mutant (CFN4202) that induced empty nodules on Phaseolus vulgaris, also have lesions in cycH. Complementation analysis suggested that the MuD1 lac insertion of the CFN4202 strain was polar on expression of genes downstream of cycH in contrast with the Tn 5mob insertion present in IFC01, which showed no polarity on cycJKL. Our data suggest that CycH may not be essential for the infection process, but is necessary for nitrogen fixation.