A chimeric vector for efficient chromosomal modification inEnterococcus faecalisand other lactic acid bacteria

Abstract

To construct a chimeric vector named pBVGh for quickly generating gene modifications in Enterococcus faecalis. The constructed plasmid pBVGh carries the pG(+)host replicon (a thermosensitive (TS) derivative of pWV01), allowing a simple generation of mutants by growing colonies first at the permissive temperature and then switching the culture to the nonpermissive temperature. Additionally, this vector facilitates the screening of mutants by a rapid colorimetric blue-white discrimination of plasmid-free bacteria. The pBVGh vector allows a straightforward inactivation or modification of target genes as well as a fast selection of enterococcal mutant strains. The broad range of the TS replicon utilized in this plasmid permits the easy establishment and the efficient generation of food-grade mutant strains in Ent. faecalis and several other gram-positive bacteria.