Application and evaluation of the phage resistance- and bacteriocin-encoding plasmid pMRC01 for the improvement of dairy starter cultures

Abstract

The conjugative 63-kb lactococcal plasmid pMRC01 encodes bacteriophage resistance and production of and immunity to a novel broad-spectrum bacteriocin, designated lacticin 3147 (M.P. Ryan, M.C. Rea, C. Hill, and R.P. Ross, Appl. Environ. Microbiol. 62:612-619, 1996). The phage resistance is an abortive infection mechanism which targets the phage-lytic cycle at a point after phage DNA replication. By using the genetic determinants for bacteriocin immunity encoded on the plasmid as a selectable marker, pMRC01 was transferred into a variety of lactococcal starter cultures to improve their phage resistance properties. Selection of resulting transconjugants was performed directly on solid media containing the bacteriocin. Since the starters exhibited no spontaneous resistance to the bacteriocin as a selective agent, this allowed the assessment of the transfer of the naturally occurring plasmid into a range of dairy starter cultures. Results demonstrate that efficient transfer of the plasmid was dependent on the particular recipient strain chosen, and while high-frequency transfer (10(-3) per donor) of the entire plasmid to some strains was observed, the plasmid could not be conjugated into a number of starters. In this study, transconjugants for a number of lactococcal starter cultures which are phage resistant and bacteriocin producing have been generated. This bacteriocin-producing phenotype allows for control of nonstarter flora in food fermentations, and the phage resistance property protects the starter cultures in industry. The 63-kb plasmid was also successfully transferred into Lactococcus lactis MG1614 cells via electroporation.