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

Aims: A coelectroporation method using a marker plasmid for indirect selection of lactococcal plasmids with unassigned functions was evaluated. Methods and Results: Cryptic plasmids were mixed with an erythromycin resistance (Ery(r)) 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. Conclusions: This cotransformation approach reduces the number of colonies that must be screened to find transformants harbouring cryptic plasmids. Significance and Impact of the Study: 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.