The long term goal of this project is to elucidate the mechanism of broad host range conjugation mediated by the IncP plasmids of gram negative bacteria. The P plasmids carry antibiotic resistance genes and are capable of transferring DNA to most gram negative bacteria as well as gram positives and yeast. Recent evidence also suggests a relationship between IncP conjugation and Agrobacterium mediated DNA transfer to plants. Study of IncP conjugation will enhance the understanding of gene exchange mechanisms and facilitate use of P plasmids as genetic tools in a wide variety of medically important bacteria. One broad goal of this current proposal is to define the structural interactions between essential transfer proteins and binding sites within the origin of transfer that lead to initiation of the transfer process. A 250 basepair(bp) sequence is required for oriT function, and two plasmid specific proteins, TraJ and TraI, are required for the site specific nicking that initiates transfer at oriT. Specific aims for this project are to use site directed mutagenesis to define the role of the 19 bp inverted repeat and the sequence adjacent to the nick site, and to correlate these structural changes with protein-DNA interactions between the essential TraJ and TraI proteins of the nicking complex and their binding sites in oriT. A second broad goal is to define transfer genes and gene products responsible for the broad host range property. Specific aims are to analyze the role of the two DNA primase proteins in host range by constructing clones that produce one or the other exclusively, and using these to complement a primase deletion mutant in transfer to a variety of gram negative bacteria, to determine the roles in conjugation of the two genes that are encoded by different reading frames in traI.