Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutamicum.

Abstract

Here we describe small mobilizable vectors based on the Escherichia coli plasmids pK18 and pK19. We combined the useful properties of the pK plasmids (e.g., multiple cloning site, lacZ alpha fragment, sequencing with M13 primers) with the broad-host-range transfer machinery of plasmid RP4 and a modified sacB gene from Bacillus subtilis. The new pK derivatives can be transferred by RP4-mediated conjugation into a wide range of Gram- and Gram+ bacteria, and should facilitate gene disruption and allelic exchange by homologous recombination. As an application example, the generation of a defined deletion of the hom-thrB genes in the chromosome of the Gram+ bacterium Corynebacterium glutamicum is presented.