The temperate lactococcal phage TP901-1, induced by UV light from Lactococcus lactis subsp. cremoris 901-1, was characterized. The restriction map was found to be circular, and the packaging of TP901-1 DNA was concluded to occur by a headful mechanism. The pac region was localized on the 38.4-kb phage genome. TP901-1 belongs to the class of P335 phages (V. Braun, S. Hertwig, H. Neve, A. Geis, and M. Teuber, J. Gen. Microbiol. 135:2551-2560, 1989. Evidence is presented that the phages TP936-1 (V. Braun, S. Hertwig, H. Neve, A. Geis, and M. Teuber, J. Gen. Microbiol. 135:2551-2560, 1989) and C3-T1 (A. W. Jarvis, V. R. Parker, and M. B. Bianchin, Can. J. Microbiol. 38:398404, 1992) are very closely related to or are identical to TP901-1. The lytically propagated TP901-1 phages were able to lysogenize both indicator strains Lactococcus cremoris 3107 and Wg2. Lysogenization resulted in site-specific integration of the phage genome into the bacterial chromosome. Only one chromosomal attB site was found in 20 independent lysogens. The attP region of TP901-1 and the attL and attR regions were cloned and sequenced. The results showed a core region of only 5 bp, in which the recombination occurs, followed after a 1-bp mismatch by a 7-bp identical region, TCAAT(T/C)AAGGTAA. This result was further verified by sequencing of the attB region obtained by PCR. An integration vector was constructed with the 6.5-kb EcoRl fragment from TP901-1 containing attP. This vector also functions in the plasmid-free strains MG1363 and LM0230 with only one specific attB site, strongly indicating a more general use of the TP901-1-based integration vector in lactococci. In this report, we present the restriction map of phage TP901-1, including the location of the attP andpac regions. We have been able to monitor the lysogenic life cycle of the phage and demonstrate the presence of only one major attachment site. An integration vector based on TP901-1 DNA sequences was constructed, and the attP, attL, attR, and attB regions were identified, cloned, and sequenced. The integration system from TP901-1 was also shown to be functional and site specific in the laboratory strains often used for genetic studies of lactococci, namely Lactococcus lactis subsp. lactis MG1363 and LM0230. This strongly indicates that the integration system of TP901-1 may be of general use as a genetic tool with lactococci. MATERIALS AND METHODSBacteria, plasmids, and phages. Bacterial strains and plasmids used in this work are listed in Table 1. Phage DNA from phages C3-T1 (13) and XLC3 (22) Temperate phages were induced from their hosts by UV irradiation. M17 broth was inoculated with 1% (vol/vol) of an overnight culture of the lysogenic strain. The culture was at an optical density at 600 nm of 0.15, harvested at 5,000 x g for 10 min, and resuspended in 0.5 volume of NC (0.5% NaCl [wt/vol], 5 mM CaCl2). The suspension was pumped through a quartz flow cuvette placed at the bottom of a UV field (254 1069

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. Hammer, J. Bacteriol. 176:1069-1076, 1994). 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-bp region upstream of the orf1 gene are sufficient for integration. Orf1 contains 485 amino acids and is located just upstream of attP. The N-terminal 150 to 180 amino acids of Orf1 showed 38 to 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 that does not resemble 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.Most temperate bacteriophages from gram-negative and gram-positive bacteria, including the well-known bacteriophage, integrate their DNA into the host chromosome when they enter the lysogenic cycle. The integration often occurs site specifically and has, in these cases, been found to be mediated by an integrase (Int) belonging to the Int class of site-specific recombinases (1, 24).TP901-1 is a pac-type, lactococcal temperate bacteriophage with a small isometric head and a long noncontractile tail. It belongs to a group of temperate bacteriophages which show a high degree of homology to a group of virulent bacteriophages, represented by the type bacteriophage P335 (3, 15). It is likely that TP901-1 is identical to TP936-1 and C3-T1 on the basis of DNA restriction patterns and hybridizations (5, 16). We have previously described the site-specific integration of the temperate lactococcal bacteriophage TP901-1 and the construction of a TP901-1-based integration vector (5). The phage attachment site (attP) and the chromosomal attachment site (attB) are unrelated to those found in other phages of lactic acid bacteria and their host strains, e.g., adh, mv4, LC3, Tuc2009, and BK5-T (2,8,10,21,36).This communication contains a further characterization of the integration system from TP901-1 by deletion analysis, DNA sequencing, and mutational analysis. The data presented show that the integration system from TP901-1 is very different from the bacteriophage integration systems reported so far, since Orf1, which is identified to be necess...

A method for the construction and isolation of specifically designed mutations of the temperate lactococcal phage TP901-1 has been developed. Two different erm-labeled mutants were isolated. One was shown to be defective in lysogenization and excision. The other, showing normal lysogenization, was used for host range studies.