A lactococcal pWV01-based integration toolbox for bacteria

A conditionally replicating lactococcal vector system is described, based on pWV01, that is used for chromosomal integration in Lactococcus lactis. The system consists of plasmids that are all based on the broad-host-range lactococcal replicon pWV01 which has been deprived of its gene encoding the replication initiation protein RepA. These so-called pORI plasmids can only replicate if RepA is provided in trans. Special Escherichia coli, Bacillus subtilis and L. lactis helper strains, producing RepA in trans (RepA+), are used as intermediate hosts for the construction of pORI integration plasmids. The presence of a lactococcal chromosomal DNA fragment in a pORI plasmid enables its chromosomal integration by homologous recombination in an L. lactis strain that does not produce RepA (RepA–). A set of special purpose pORI vectors are available: (i) pORI280, designed to mutate or delete genes or to insert new genes in the chromosome in such a way that no heterologous DNA or antibiotic resistance markers are left in the recombinant strain, here designated as silent gene replacement; (ii) pORI19, suitable for random mutagenesis of the chromosome, and (iii) pORI13, constructed to make random single copy transcriptional fusions in the chromosome to search for (environmentally regulated) promoters. Some of these vectors have also been succesfully applied for integration in E. coli and B. subtilis. We strongly believe that the systems described here can be used in various other bacterial species