SINGLE-STEP CONJUGATIVE CLONING OF BACTERIAL GENE FUSIONS INVOLVED INMICROBE-HOST INTERACTIONS

In vivo expression technology (IVET) is a genetic strategy for isolati ng genes expressed in vivo. In order to fully exploit this technology, it is necessary to analyse large numbers of IVET-generated gene fusio ns, which must be recovered from the chromosome of host bacteria. In b acteria for which transductional methods are not available, the recove ry of integrated fusion plasmids is problematic and currently limits b road application of IVET. We describe a rapid, single-step, triparenta l conjugative approach for recovering chromosomally integrated fusion plasmids from both Pseudomonas fluorescens and Salmonella typhimurium. This simple and broadly applicable conjugative cloning system extends the utility of the IVET approach to clinically and agronomically rele vant microbes and may be employed to recover non-replicating and integ rated plasmids in other systems.