Although most plasmids in Gram

Although most plasmids in Gram-negative bacteria have a restricted host-range, a few groups are widely distributed among unrelated species. These plasmids are disseminated primarily by conjugation, a process that can result in the transfer of plasmids not only between bacterial species, but also into eucaryotic cells. In our laboratory, we are focussing on how plasmid DNA is processed for conjugal transfer. This problem has practical medical significance, since broad host-range plasmids generally encode resistance to one or more antibiotics, and are probably contributing to the increasing frequency of multiple drug-resistance in bacteria by providing a mechanism for interspecific gene transfer and maintenance.

We have been investigating the conjugal transfer of the broad host-range plasmid R1162. Our objectives are to map the genes involved and to characterize their regulation, and also to identify the gene products and the sites at which they act. Ultimately, we wish to describe in detail the molecular steps in transfer, and to identify those properties which free the plasmid from the constraints imposed by different cytoplasms. We have identified the R1162 genes required for transfer, and have mapped oriT, the origin of transfer, to a 38 base-pair segment of the plasmid. We are in the process of determining how the R1162-encoded proteins interact at oriT to carry out the DNA processing steps required for the initiation and termination of transfer. To this end, several specific questions now are being investigated in our laboratory:

During conjugation, a single, linear DNA strand is transferred. This strand is generated by nicking at oriT, and occurs within the relaxosome, a complex of oriT DNA and several plasmid proteins. How does this nicking occur, and what determines its strand- and site-specificity?

The nicked strand is transferred with 5' to 3' polarity. Is there rolling circle replication of the plasmid DNA during conjugation, so that the transferred strand is replaced in the donor cell by synthesis primed at the 3' end?

Only one strand is transferred during conjugation, but R1162 consists of duplex DNA. How is the complementary strand synthesized in the recipient cell after transfer?

In the relaxosome, nicking and religation of oriT DNA are probably occurring continuously. What directs the nicked strand into the conjugation pathway?

R1162 is not self-transmissible, but depends on other plasmids for many of the functions required for conjugation. However, different plasmids are parasitized with distinct and characteristic efficiencies. How does R1162 communicate with its mobilizing vector during conjugation, and what determines the efficiency of transfer?