Targeted mutagenesis of enterococcal genes

A series of methods was developed for targeted mutagenesis in enterococci. First, a transposon mutagenesis system, miniγδ-200 (mγδ), which was used previously to make insertion mutants in streptococci, was shown to be useful for generation of mutants in enterococci. After mutagenesis of cosmid clones carrying enterococcal DNA inserts in Escherichia coli with mγδ, we were able to isolate the mutants by phenotype or to screen for them by immunoblotting or comparison of restriction digestion patterns. Clones with mγδ insertions in targeted enterococcal genes were then introduced into enterococci by electroporation to generate targeted disruption mutations. Allelic replacement en masse, that is, electroporation of enterococci with DNA from a pool of mutagenized cosmid clones, was shown to be an efficient method to obtain mutations in genes with detectable phenotypes in enterococci. We next constructed a vector for mutagenesis using small intragenic fragments of enterococcal genes to disrupt targeted genes. This vector was modified from pBluescript SK (-) by cloning the kanamycin resistance determinant from mγδ into the ScaI site internal to the ampicillin resistance gene. It was then used to generate insertion mutants in Enterococcus faecalis with an intragenic fragment as small as about 500 bp. A third method, based on the conjugation system reported by Trieu-Cuot et al., was developed in order to circumvent difficulties in the electroporation of some enterococcal strains and to improve the efficiency with which targeted mutations can be generated in enterococci. This system was capable of mobilizing both small plasmids and large cosmids into enterococci by conjugation, and produced disruption mutations by homologous recombination.