A coelectroporation method for the isolation of cryptic plasmids from Lactococcus lactis.

Abstract

A coelectroporation method using a marker plasmid for indirect selection of lactococcal plasmids with unassigned functions was evaluated. Cryptic plasmids were mixed with an erythromycin resistance (Eryr) marker plasmid and introduced into a recipient strain by electroporation, followed by plasmid extraction of erythromycin-resistant transformants. By optimizing the ratio between the marker plasmid and the cryptic plasmids, an average of 20% cotransformants was obtained, including combinations of more than one cryptic plasmid. The marker plasmid pSA3 was easily eliminated from the cotransformed cells by subculture without selective pressure. This cotransformation approach reduces the number of colonies that must be screened to find transformants harbouring cryptic plasmids. The method facilitates the isolation of cryptic plasmids, helps in assigning functions to unknown plasmids and allows construction of food-grade lactococcal strains with new combinations of wild-type plasmids.