A new family of mobilizable suicide plasmids based on broad host range R388 plasmid (IncW) and RP4 plasmid (IncPα) conjugative machineries and their cognate Escherichia coli host strains

We describe the construction of the pSW family of conditionally replicating plasmids which are based on the IncX oriV origin (oriV(R6K gamma)) of replication that is dependent on the pir-encoded protein. We constructed several Escherichia coli derivatives expressing pir from different chromosomal loci, and the pir gene could be transduced by phage P I to any E. coli strain. These chromosomal constructions generate dapA and thyA knockouts, which lead to diaminopimelate or thymidine auxotrophies, respectively, and they serve to provide absolute counterselection even in rich media. These strains can be easily counterselected if used in plasmid transfer experiments into markerless recipients, and they have been demonstrated to work efficiently in E. coli x Vibrio or E. coli x Bartonella matings. We constructed different pSW plasmids carrying either the oriT(RP4) or the oriT(R388), and we demonstrated that these derivatives can be efficiently transferred using RP4 and R388 conjugation machineries, respectively. We also constructed two plasmids expressing the R388 conjugation machinery, but lacking the oriT(R388). We demonstrated that these plasmids enabled efficient and exclusive transfer of a pSW-oriT(R388) derivative from E. coli to V cholerae. and we offer an alternative to the popular RN-based delivery system. (c) 2004 Elsevier SAS. All rights reserved.