TraG from RP4 and TraG and VirD4 from Ti plasmids confer relaxosome specificity to the conjugal transfer system of pTiC58

Plasmid conjugation systems are composed of two components, the DNA transfe r and replication system, or Dtr, and the mating pair formation system, or Mpf. During conjugal transfer an essential factor, called the coupling prot ein, is thought to interface the Dtr, in the form of the relaxosome, with t he Mpf, in the form of the mating bridge. These proteins, such as TraG from the IncP1 plasmid RP4 (TraG(RP4)) and TraG and VirD4 from the conjugal tra nsfer and T-DNA transfer systems of Ti plasmids, are believed to dictate sp ecificity of the interactions that can occur between different Dtr and Mpf components. The Ti plasmids of Agrobacterium tumefaciens do not mobilize ve ctors containing the oriT of RP4, but these IncP1 plasmid derivatives lack the trans-acting Dtr functions and TraG(RP4). A. tumefaciens donors transfe rred a chimeric plasmid that contains the oriT and Dtr genes of RP4 and the Mpf genes of pTiC58, indicating that the Ti plasmid mating bridge can inte ract with the RP4 relaxosome. However, the Ti plasmid did not mobilize tran sfer from an IncQ relaxosome. The Ti plasmid did mobilize such plasmids if TraG(RP4) was expressed in the donors. Mutations in traG(RP4) with defined effects on the RP4 transfer system exhibited similar phenotypes for Ti plas mid-mediated mobilization of the IncQ vector. When provided with VirD4, the tra system of pTiC58 mobilized plasmids from the IncQ relaxosome. However, neither TraG(RP4) nor VirD4 restored transfer to a traG mutant of the Ti p lasmid. VirD4 also failed to complement a traG(RP4) mutant for transfer fro m the RP4 relaxosome or for RP4-mediated mobilization from the IncQ relaxos ome. TraG(RP4)-mediated mobilization of the IncQ plasmid by pTiC58 did not inhibit Ti plasmid transfer, suggesting that the relaxosomes of the two pla smids do not compete for the same mating bridge. We conclude that TraG(RP4) and VirD4 couples the IncQ but not the Ti plasmid relaxosome to the Ti pla smid mating bridge. However, VirD4 cannot couple the IncP1 or the IncQ rela xosome to the RP4 mating bridge. These results support a model in which the coupling proteins specify the interactions between Dtr and Mpf components of mating systems.