Pseudomonas aeruginosa plasmids as suicide vectors in Escherichia coli: resolution of genomic cointegrates through short regions of homology

Abstract

Pseudomonas aeruginosa plasmids which cannot replicate in Escherichia coli have been used to introduce specific modifications into the E. coli chromosome by homologous recombination (‘gene targeting’). The E. coli gene ( gpt) encoding guanine-xanthine phosphoribosyltransferase (Gpt) was used for initial targeting studies owing to the availability of a powerful positive selection for loss of the Gpt + phenotype (6-thioguanine resistance or 6TG R or Gpt −). P. aeruginosa plasmids containing selectable markers flanked by gpt sequences were introduced as supercoiled DNA into an E. coli strain which contained a normal gpt locus. Primary cointegration of such plasmids into the E. coli genome results in a gene duplication event which maintains Gpt function; a secondary recombinational event which resolves the cointegrate either reverses the primary event or results in replacement of the original gpt copy with the modified version. A 316-bp region of homology was sufficient for cointegrate formation, and resolution of the cointegrates through a shorter (92 bp) homologous flank was selectable through loss of Gpt function. The frequency of cointegrate resolution under these conditions was significantly above the spontaneous gpt mutational loss rate.