Pettis 9604879 During their complex life cycle, bacteria of the Gram-positive genus, Streptomyces, are capable of the highly efficient transfer of both plasmid and chromosomal genes. Conjugation in Streptomyces appears to be unique among microbes in that Streptomyces plasmids encode remarkably little transfer-related genetic information, a characteristic that further implies that host functions also play critical roles in the conjugation process. The overall goal of this project is to define the potentially novel interactions that direct conjugation in Streptomyces using the S. lividans plasmid pIJ101 as part of the model system. Transfer of pIJ101 requires the plasmid-encoded Tra protein and an additional plasmid locus, clt, which acts in cis by an undetermined mechanism to promote efficient plasmid transfer. In contrast to its role in plasmid transfer, however, clt was found to be dispensible for tra-mediated mobilization of chromosomal genes, a result which implies that the Streptomyces chromosome may encode its own clt-like locus (or loci) that is required for host gene transfer. Interestingly, clt is also required in cis for expression of the plasmid-encoded Tra protein. To further elucidate the molecular events during conjugation in Streptomyces, the specific objectives of this project are: (1) To isolate transfer-related protein-DNA complexes involving clt+ plasmids formed in vivo and then examine these complexes for induction (by ethidium bromide treatment) of conjugation-associated DNA nicking, as well as characterize the proteins involved by SDS-PAGE and Western blotting; as an alternative approach, the pIJ101 Tra protein will be purified and tested in vitro for a variety of possible DNA processing functions including qpecific nicking of clt+ plasmids, helicase activity and specific binding to clt+ DNA; (2) to clone from the Streptomyces chromosome the putative functional equivalent of the pIJ101 clt locus based on the ability of such a locus (or loci) either to restore in ci s expression of the plasmid-encoded Tra protein (thereby producing a transfer-competent plasmid derivative), or to hybridize specifically to pIJ101 clt sequences. This research will provide important insights about a potentially novel form of communication that certain bacteria use to transfer genetic information from one cell to another. The ability of bacteria to transfer genes between cells ultimately promotes the survival of their populations under often hostile environmental conditions.
