Deriving Phage-Insensitive Lactococci Using a Food-Grade Vector Encoding Phage and Nisin Resistance

Abstract

Plasmid pFK012 is a potential food-grade vector comprising only lactococcal DNA, and it encodes bacteriophage insensitivity and nisin resistance. Efforts were made to transform several lactococcal strains with pFK012 via electroporation. Nisin-resistant transformants were selected on M17 agar containing .5% glucose and 300 IU of nisin/ml. Southern hybridization analysis of plasmid pools of the transformants using a nisin resistance probe confirmed the presence of pFK012. Two plasmid-free strains (Lactococcus lactis ssp. lactis LM0230 and MG1363) were successfully transformed and evaluated for phage sensitivity. Wild-type lactococcal strains were more difficult to transform, although two acquired pFK0l2 (L. lactis ssp. lactis C2 and SK2). These transformants acquired an abortive phage resistance mechanism, were stably maintained at 21 and 37°C after 100 generations, and retained the same rate of acid production as the parental strains. The difficulty in transforming parental strains could be related to incompatibility of plasmids or to restriction of incoming DNA. In this study, L. lactis ssp. lactis H1 was found to be naturally resistant to nisin but did not produce nisin. The plasmid pool from H1 was found to contain a plasmid that had strong homology with a nisin resistance probe.