A Resolvase-like Protein is required for the Site-Specific Integration of the Temperate Lactococcal Bacteriophage TP901-1

The integration system of the temperate lactococcal phage TP901-1 was characterized in Lactococcus lactis subsp. cremoris LM0230 and MG1363 using deletion derivatives of the integration vector pBC143 (Christiansen et al. (1994), J. Bacteriol. 176, p. 1069-1076). The phage encoded elements necessary for integration were localized on a 2.8 kb NsiI-EcoRI fragment including the phage attachment site, attP. This fragment was DNA sequenced, and sequence analysis revealed three putatively expressed open reading frames Orf1, Orf2, and Orf3. By the introduction of mutations within the orf1, orf2, and orf3 genes, it was shown that only Orf1 was necessary for the integration process. Furthermore, it was found that Orf1, attP and a 425 base pair region upstream of the orf1 gene was sufficient for integration. Orf1 contains 485 amino acids and is located just upstream of attP. The N-terminal 150-180 amino acids of Orf1 showed 38-44 % similarity to the resolvase group of site-specific integrases, while no similarity to known proteins was found in the C-terminal end. Bacteriophage TP901-1 therefore contains a unique integration system, not resembling the Int-class of site-specific integrases usually found in temperate bacteriophages. The constructed integration vector pBC170, integrates into the chromosomal attachment site very efficiently and forms stable transformants with a frequency corresponding to 20 % of the transformation efficiency.