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 with the use of deletion derivatives of the integration vector pBC143 (B. Christiansen, M. G. Johnsen, E. Stenby, F. K. Vogensen, and K. Ham mer, J. Bacteriol. 176:1069-1076, 1994). The phage-encoded elements ne cessary for integration were localized on a 2.8-kb NsiI-EcoRI fragment including the phage attachment site, attP. This fragment was DNA sequ enced, 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 wa s necessary for the integration process. Furthermore, it was found tha t Orf1, attP, and a 425-bp region upstream of the orf1 gene are suffic ient for integration. Orf1 contains 485 amino acids and is located jus t upstream of attP. The N-terminal 150 to 180 amino acids of Orf1 show ed 38 to 44% similarity to the resolvase group of site-specific integr ases, while no similarity to known proteins was found in the C-termina l end. Bacteriophage TP901-1 therefore contains a unique integration s ystem that does not resemble the Int class of site-specific integrases usually found in temperate bacteriophages. The constructed integratio n vector, pBC170, integrates into the chromosomal attachment site very efficiently and forms stable transformants with a frequency correspon ding to 20% of the transformation efficiency.