Integration-proficient plasmids for Pseudomonas aeruginosa: Site-specific integration and use for engineering of reporter and expression strains

An improved method for integration of exogenous DNA fragments at a defined site within the genome of Pseudomonas aeruginosa was developed. The method relies on two integration-proficient vectors, mini-CTX1 and mini-CTX2. Thes e two vectors contain (1) a tetracycline (tet) selectable marker, (2) an or iT for conjugation-mediated plasmid transfer, (3) the pMB1-derived origin o f replication, (4) a modified phi CTX integrase (int) gene, (5) a versatile multiple cloning site (MCS) flanked by T4 transcriptional termination sequ ences (Omega elements), and (6) the phi CTX attachment site. The MCS and Om ega elements are flanked by yeast Flp recombinase target sites that allow i n vivo excision of unwanted plasmid backbone sequences, including tet and i nt, from the genome of integrants by Flp recombinase. In the mini-CTX2 vect or int transcription is driven from the strong trc promoter, which is regul ated by the Lac repressor that is encoded by lacl(q) also contained on the plasmid. Upon conjugal transfer,mini-CTX1 and mini-CTX2 integrated at frequ encies of 10(-8) and 10(-7), respectively. The usefulness of the integratio n vectors for gene fusion analyses was demonstrated by chromosomal insertio n of autoinducer (AI)-regulated lasB-lacZ and rhlA-lacZ fusions into wild-t ype and AI synthase mutants. In wild-type, the fusions responded in a cell density-dependent manner and expression of both fusions was either greatly reduced or abolished in AI synthase mutants. Finally, an expression cassett e containing the T7 polymerase gene under Lac repressor control was constru cted, integrated into the P. aeruginosa chromosome, and used to express the hexahistidine-tagged P. aeruginosa AI synthase RhlI. (C) 2000 Academic Pre ss.