Alielic exchange in Pseudomonas aeruginosa using novel ColE1‐type vectors and a family of cassettes containing a portable oriT and the counter‐selectable Bacillus subtilis sacB marker

An improved method for allele replacement in Pseudomonas aeruginosa was developed. The two main ingredients of the method are: (i) novel ColE1‐type cloning vectors derived from pBR322 and pUC19; and (ii) a family of cassettes containing a portable oriT, the sacB gene from Bacillus subtilis as a counter‐selectable marker, and a chloramphenicol‐resistance gene allowing positive selection of both oriT and sacB. Introduction of plasmid‐borne DNA into the chromosome was achieved in several steps. The DNA to be exchanged was first cloned into the new ColE1‐type vectors. After insertion of the oriT and sacB sequences, these plasmid were conjugally transferred into P. aeruginosa and plasmid integrants were selected. Plating on sucrose‐containing medium allowed positive selection for both plasmid excision and curing since Pseudomonas aeruginosa strains containing the sacB gene in single‐ or multiple copy were highly sensitive to 5% sucrose in rich medium. This procedure was successfully used to introduce an agmR mutation into P. aeruginosa wild‐type strain PA01 and should allow the exchange of any DNA segment into any non‐essential regions of the P. aeruginosa chromosome.