Broad-host-range plasmid and M13 bacteriophage-derived vectors for promoter analysis in Escherichia coli and Pseudomonas aeruginosa

Abstract

A set of bacteriophage and plasmid vectors containing xylE as a reporter gene was constructed for the analysis of promoters functional in Escherichia coli and in other Gram-negative bacteria. Two M13 bacterio-phage derivatives, M13mVDX18 and M 13mMK010, were designed for rapid cloning, screening and sequencing of DNA fragments promoting transcription in E. coli. To demonstrate their utility, total cellular DNA from a variety of bacterial species including Pseudomonas aeruginosa strain PAO was shotgun cloned in M13 vectors and clones displaying promoter activity in E. coli were isolated. These randomly cloned promoters from P. aeruginosa Borrelia burgdorferi Streptococcus pneumoniae and other bacterial species were sequenced without a need for further subcloning manipulation. The promoter activity of P. aeruginosa clones was verified by subcloning inserts on a broad-host-range promoter probe vector pVDXIS and assaying the xylE transcription from these promoters in P. aeruginosa. The pVDXIS vector was also used for initial characterization of the algD promoter controlling mucoidy in P. aeruginosa. The activities of the wild-type and deletion clones of the algD promoter were compared. Results indicated that the region containing direct and inverted repeats at −55 to −110 bp upstream of the mRNA 5' end was important for the activation of the algD transcription in mucoid P. aeruginosa infecting cystic fibrosis patients.