Omega mutagenesis in gram-negative bacteria: a selectable interposon which is strongly polar in a wide range of bacterial species

Abstract

We have used the 2.0-kb DNA fragment omega [Prentki and Krisch, Gene 29 (1984) 303-313] to mutagenize in vitro a broad-host-range plasmid carrying the entire meta-cleavage pathway of the Pseudomonas putida TOL plasmid pWW0. The mutant plasmids were subsequently introduced by conjugal mobilization into a variety of Gram-negative bacteria. The omega fragment carries a selectable marker (aadA+; SpcR/SmR), which is expressed in all species tested, as well as flanking transcription and translation termination signals and synthetic polylinkers. Expression of the plasmid-borne catechol 2,3-dioxygenase (C23O) gene, situated downstream from the site of omega insertion, was substantially reduced in all strains tested. The transcription terminators originally cloned from bacteriophage T4 gene 32, are apparently functional in a wide range of hosts. Insertional mutagenesis with the omega 'interposon' can thus be used in a wide variety of species, with the advantages of a positive selection for the presence of the fragment, the termination of RNA and protein synthesis beyond the site of insertion, and genetic stability of the resulting mutation.