VEX-capture: a new technique that allows in vivo excision, cloning, and broad-host-range transfer of large bacterial genomic DNA segments

We have developed a novel and easily performed procedure for the targeted excision, cloning, and broad-host-range transfer of large bacterial genomic DNA segments. This procedure, called Vector-mediated excision and Capture (VEX-Capture), represents a new molecular tool for the convenient manipulation and exchange of large (20-40+ kb) bacterial genomic fragments. VEX-Capture utilizes lox/Cre-mediated site-specific recombination for excision of the targeted genomic segment and homologous recombination for cloning of the excised DNA section onto a self-transmissible, broad-host-range IncP plasmid. The captured genomic DNA segment can then be transferred to a wide variety of Gram-negative hosts for basic research and bioengineering purposes. To demonstrate the utility and function of VEX-Capture, we have excised and cloned three separate genomic islands from the Salmonella typhimurium chromosome ranging in size from 26.7 to 40.0 kb. To test the ability of these islands to be established in different bacterial hosts, we transferred them to six other Gram-negative species and monitored their establishment via phenotypic and molecular analysis. RT-PCR was used to assay the expression of selected S. typhimurium island genes in the different species. This analysis led to the discovery that an island-encoded master regulator of S. typhimurium virulence functions is expressed in a species-specific manner. Our results demonstrate the potential for VEX-Capture to be used as a convenient genetic technique for fundamental biological applications in a wide variety of bacterial species. (C) 2004 Elsevier B.V. All rights reserved.