Genetic and biochemical characterization of TraA, the relaxase of symbiotic plasmid pRetCFN42d from Rhizobium etli, is described. After purifying the relaxase domain (N265TraA), we demonstrated nic binding and cleavage activity in vitro and thus characterized for the first time the nick site (nic) of a plasmid in the family Rhizobiaceae. We studied the range of N265TraA relaxase specificity in vitro by testing different oligonucleotides in binding and nicking assays. In addition, the ability of pRetCFN42d to mobilize different Rhizobiaceae plasmid origins of transfer (oriT) was examined. Data obtained with these approaches allowed us to establish functional and phylogenetic relationships between different plasmids of this family. Our results suggest novel characteristics of the R. etli pSym relaxase for previously described conjugative systems, with emphasis on the oriT cis-acting preference of this enzyme and its possible biological relevance.This work was supported by grant BFU2005-03477/BMC to F.C., by grants BIO99-0904 and AGR258 to J.S., and by the PAI-Plan Andaluz de Investigación (Spain). D.P.M. was supported by a PFPI fellowship from the Ministerio de Educación y Ciencia (Spain) and by a postdoctoral fellowship from Fundación Marqués de Valdecilla (IFIMAV). M.L. was supported by a fellowship from IFIMAV. J.A.H.C. was supported by a Ramón y Cajal contract from the Ministerio de Ciencia y Tecnología (Spain).Peer reviewed