An extra repABC locus in the incRh2 Ti plasmid pTiBo542 exerts incompatibility toward an incRh1 plasmid

Ti/Ri plasmids in pathogenic Agrobacterium species are repABC replicons that are stably maintained by the function of repABC genes. Two Ti plasmids, pTiBo542 and pTiS4, belonging to incRh2 and incRh4 incompatibility groups, respectively, were reported to carry two repABC loci. In the present study, to reveal the roles of the two repABC loci in the two plasmids, we constructed mini-replicons carrying any one or both of the repABC loci (referred to as repABC1 and repABC2 here) and examined their replication and incompatibility properties. The introduction of mini-replicons into A. tumefaciens C58C1 strains suggested that repABC1 functions as replicator genes but repABC2 does not in both the Ti plasmids. Because the components of repABC2 of pTiBo542 have highly similar amino acid and nucleotide sequences to those of the incRh1-type repABC replicon, we introduced repABC2-containing replicons into cells harboring an incRh1 plasmid in order to check their incompatibility traits. As a result, the repABC2-containing replicon expelled the resident incRh1 plasmid, indicating that the extra repABC locus is dispensable for replication and could work as an incompatibility determinant against incRh1 group plasmids. We suggest that the locus contributes to plasmid retention by eliminating the burden of co-existing competitive plasmids in host cells through its incompatibility.