SITE-DIRECTED MUTATIONS IN THE RELAXASE OPERON OF RP4

Mutations were constructed by site-directed mutagenesis in the relaxas e operon of the broad-host-range plasmid RP4. The mutations were const ructed in smaller plasmids, recombined into the 60-kb RP4 plasmid, and tested for their ability to transfer. The relaxase operon contains th e transfer genes traJ, traH, and traI, which are involved in nicking a t the transfer origin to generate the single strand destined to be tra nsferred to the recipient cell. In the first mutant, the C terminus of TraI was truncated, leaving TraH intact. This mutant decreased transf er by approximately 500-fold in Escherichia coli, and the traI mutatio n could be complemented by a wild-type copy of tral in trans in the do nor. The traI mutation similarly decreased transfer between a variety of gram-negative bacteria. A site-specific mutation was made by the po lymerase chain reaction-based unique-site mutagenesis procedure to alt er the start site of traH. This mutation had no effect on intraspecifi c E. coli transfer but reduced transfer by up to sevenfold for some gr am-negative bacteria. The traH mutation had no effect on plasmid stabi lity. Thus, neither TraH nor the C terminus of TraI is required for co njugative transfer, but both increase mating efficiency in some hosts.