Improvement of pCVD442, a suicide plasmid for gene allele exchange in bacteria.

Allelic exchange experiments allow investigation of the functions of many unknown genes identified during the sequencing of entire genomes. Isogenic strains differing by only specific mutations can be constructed. Among other tools, suicide plasmids are widely used for this task. They present many advantages because they leave no scars on the chromosome, and therefore allow combining several mutations in the same genetic background. While using the previously described pCVD442 suicide plasmid [Infect. Immun. 59 (1991) 4310], we found untargeted recombination events due to the presence of an IS1 element on this plasmid. The plasmid was therefore improved by removal of the IS1 element. We also replaced the bla gene of pCVD442, conferring ampicillin resistance, by the cat gene conferring chloramphenicol resistance, leading to the new suicide plasmid pDS132. The plasmid was entirely sequenced. We demonstrate that this new vector can be easily used to introduce various types of mutations into different genetics backgrounds: removal of IS elements, introduction of point mutations or deletions. It can be introduced into bacterial strains by either transformation or conjugation. Copyright 2004 Elsevier Inc.