A chimeric vector for efficient chromosomal modification in Enterococcus faecalis and other lactic acid bacteria.

ABSTRACT

AIM: To construct a chimeric vector named pBVGh for quickly generating gene modifications in Enterococcus faecalis. METHODS AND RESULTS: 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. CONCLUSIONS: The pBVGh vector allows a straightforward inactivation or modification of target genes as well as a fast selection of enterococcal mutant strains. SIGNIFICANCE AND IMPACT OF THE STUDY 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.