A small derivative of the broad-host-range plasmid RK2 which can be switched from a replicating to a non-replicating state as a response to an externally added inducer

TrfA is the only plasmid-encoded protein required for RK2 replication. We r eport here the construction and characterization of an RK2-based vector in which trfA is expressed from the inducible promoter Pm. The resulting const ruct, pJBSDI, was found to replicate in Escherichia coli DH5a (recA(-)) onl y in the presence of a Pm inducer. In two tested E. coli recA(+) strains pJ BSD1 could replicate in the absence of inducer, but a replication inducer-d ependent phenotype was obtained in these strains by introducing a mutation known to reduce the trfA expression level. The plasmid construct could be u sed as a conditional suicide vector system for targeted chromosomal integra tion via homologous recombination. This feature may potentially be used for many types of studies in microbial molecular biology. (C) 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. A ll rights reserved.