Cloning and sequence analysis of the Rhizobium etli ccmA and ccmB genes involved in c-type cytochrome biogenesis

In this paper we describe the sequence analysis of two Rhizobium etli genes (ccmA and ccmB) which are believed to participate in the transport of the haem moiety to the periplasm in other bacterial species. The characterized DNA region was isolated by complementation of a R. etli Tn5mob induced mutant (CFN4201) which was affected in the production of c-type cytochromes. Sequence analysis of this region identified three open reading frames, two were identified as the ccmA and ccmB genes. The predicted protein sequence of ccmA showed significant homology with ATP binding proteins of the ABC-type transporter systems, while ccmB encodes for a hydrophobic protein probably associated with the ccmA gene product. The Tn5mob insertion in CFN4201 strain was located in the carboxy terminus of CcmB. Restriction mapping of the EcoRI fragment containing the Tn5mob insertion showed that it involved a deletion of approximately 1.5 kb. Mutagenesis of the wild-type region with a miniMu transposon and complementation analysis showed that the mutation in ccmB, and not the deletion, was responsible for the phenotype of CFN4201 strain and that ccmA and ccmB are independent transcription units. We found that a region located downstream of ccmB is reiterated twice, one near the chromosomal ccmA-ccmB locus while the second in plasmid e. Finally, CFN4201 membranes had detectable levels of c(1) apoprotein which did not contain bound haem. This data could suggest that haem binding to the apoprotein occurs after the translocation of the apo form of c(1) to the membrane.