A HIGHLY EFFICIENT AND STABLE SYSTEM FOR SITE-SPECIFIC INTEGRATION OFGENES AND PLASMIDS INTO THE PHAGE PHI-LC3 ATTACHMENT SITE (ATTB) OF THE LACTOCOCCUS-LACTIS CHROMOSOME

An integration vector system based on the site-specific integration ap paratus of the temperate lactococcal bacteriophage phi LC3 was develop ed. A 1.6-kb recombinogenic DNA cassette, containing the phi LC3 integ rase gene (int) and the phage attachment site (attP), mediated site-sp ecific integration of a single marker-gene, as well as of a replicatio n-thermosensitive (-ts) plasmid (pINT2), into the phi LC3 attB site of Lactococcus lactis subsp. lactis LM0230 chromosome after introduction of the DNA into the cells by electroporation. Both the marker gene an d the pINT2 plasmid were stably inserted as single copies in an orient ation-specific and integrase dependent manner, the pINT2-ts replicon b eing stably maintained at temperatures both permissive and non-permiss ive for plasmid-directed replication. Essentially all transformants ob tained with the pINT2 plasmid appeared to be integrants, demonstrating the remarkably high efficiency of the system. This high efficiency re ndered possible the detection of transformation-plus-integration event s using DNA directly obtained from ligase reaction mixtures, thus avoi ding initial subcloning in a nonlactococcal strain and subsequent coin tegration of foreign replication functions into the chromosome of L. l actis. The above results, the observation that the phi LC3 attB site a ppear to be conserved in L. lactis, and the fact that the int-attP cas sette functions efficiently in a non-phi LC3-host strain, show that th e phi LC3 site-specific integration apparatus provides an efficient an d 'food grade' tool for stable integration of genetic elements into th e chromosome of L. lactis.