A food-grade approach for functional analysis and modification of native plasmids in Lactococcus lactis

While plasmids from lactic acid bacteria possess many traits that are of industrial value, their exploitation is often frustrated by an inability to conduct food-grade engineering of native plasmids or to readily screen for their transfer. Here we describe a system that uses a RepA(+) temperature-sensitive helper plasmid and a RepA(-) cloning vector to overcome these problems while maintaining the food-grade status of the native plasmid. This strategy was used to precisely delete ltnA1 alone, or in conjunction with ItnA2 (encoding the structural proteins of the lantibiotic lacticin 3147), from the native 60.2-kb plasmid pMRC01 and to select for the transfer of pMRC01 between Lactococcus lactis strains.