Identification of int and attP on the genome of lactococcal bacteriophage Tuc2009 and their use for site-specific plasmid integration in the chromosome of Tuc2009-resistant Lactococcus lactis MG1363.

The DNA sequence of the int-attP region of the small-isometric-headed lactococcal bacteriophage Tuc2009 is presented. In this region, an open reading frame, int, which potentially encodes a protein of 374 amino acids, representing the Tuc2009 integrase, was identified. The nucleotide sequence of the bacteriophage attachment site, attP, and the sequences of attB, attL, and attR in the lysogenic host Lactococcus lactis subsp. cremoris UC509 were determined. A sequence almost identical to the UC509 attB sequence was found to be present in the plasmid-free Tuc2009-resistant L. lactis subsp. cremoris MG1363. This site could be used for the site-specific integration of a plasmid carrying the Tuc2009 int-attP region in the chromosome of MG1363, thereby demonstrating that the application of chromosomal insertion vectors based on bacteriophage integration functions is not limited to the prophage-cured original host strain of the phage.