Development of tools for the genetic manipulation of Pseudomonas aeruginosa

Abstract

To facilitate study of the opportunistic bacterial pathogen Pseudomonas aeruginosa, several genetic tools were developed. These tools include a series of cassettes carrying (a) the minimal sequence for the origin of transfer (oriT) of RP4 plasmid for introducing plasmid into P. aeruginosa via conjugation, (b) a minimal sequence for P. aeruginosa replicon (stabilizing fragment or SF) for maintenance of plasmids in P. aeruginosa, and (c) the transcriptionally non-polar tetracycline resistance gene (TcR) for insertional mutagenesis. Additional genetic constructs include (d) two conjugative and suicide lacZ reporter fusion plasmids for studying gene expression at the transcriptional or translational level, (e) a gentamicin resistant promoter-probing mini-Tn5 lacZ, and (f) a tightly regulated T7 promoter/repressor system to control gene expression in P. aeruginosa.