Plasmids determining antibiotic resistance have become widespread in gram-negative bacteria and account for the major portion of clinically important drug resistance. Conjugation is the major mechanism by which plasmids are transferred among gram-negative bacteria. Many fundamental aspects of conjugation are poorly understood, including the recipient cell surface structures needed for proper mating, the process of initiation of DNA transfer, and the mechanisms of DNA replication during conjugation. In addition, the role of conjugation systems in determining the host range of transmissible plasmids is known. Using the narrow host range E. coli F factor and the broad host range R plasmid RK2 as model conjugation systems, the proposed research will investigate: 1) the initiation events in RK2 DNA transfer, including the site and the DNA sequence of the origin of transfer and the function of the RK2 relaxation complex in initiating DNA transfer, 2) the biochemical mechanisms of transfer DNA replication, 3) the role of the conjugation system in determining the host range of F and RK2, and 4) the recipient surface structures important in the cell-cell interactions required for conjugation. These studies will provide a detailed knowledge of the mechanism of DNA transfer and a comparison of the conjugation systems of narrow and broad host range plasmids. This research is necessary to understand and control the spread of antibiotic resistance among diverse gram-negative bacteria.