Development of a P1 phagemid system for the delivery of DNA into Gram-negative bacteria

The inability to transform many clinically important Gram-negative bacteria has hampered genetic studies addressing the mechanism of bacterial pathogenesis. This report describes the development and construction of a delivery system utilizing the broad-host-range transducing bacteriophage P1. The phagemids used in this system contain a P1 pac initiation site to package the vector, a P1 lytic replicon to generate concatemeric DNA, a broad-host-range origin of replication and an antibiotic-resistance determinant to select bacterial clones containing the recircularized phagemid. Phagemid DNA was successfully introduced by infection and stably maintained in members of the families Enterobacteriaceae (Escherichia coli, Shigella flexneri, Shigella dysenteriae, Klebsiella pneumoniae and Citrobacter freundii) and Pseudomonadaceae (Pseudomonas aeruginosa). In addition to laboratory strains, these virions were used successfully to deliver phagemids to a number of strains isolated from patients. This ability to deliver genetic information to wild-type strains raises the potential for use in antimicrobial therapies and DNA vaccine development.