TPW22, a Lactococcal Temperate Phage with a Site-Specific Integrase Closely Related to Streptococcus thermophilus Phage Integrases

The interactions of lactococcal phages with bacterial hosts have been subjected to studies not only because of the ability of bacteriophages to disturb fermentation of industrial products but also because of the potential use of phage genetic elements in the study and genetic modification of lactococcal bacteria. Much of this work has been inspired by the findings and knowledge achieved from work with bacteriophage λ and its host, Escherichia coli. A model of the insertion of the bacteriophage λ genome into the chromosome of the host bacterium through site-specific recombination between the phage attachment site (attP) and the bacterial attachment site (attB) has been suggested (19). It has been shown that the insertion is mediated by an integrase protein (Int) encoded by bacteriophage λ (29, 67) as well as an integration host factor encoded by the host bacterium (41, 51). The mechanism of this insertion, as well as the excision of bacteriophage λ, is complicated but well characterized (for a review, see reference 45). Both the integrase-encoding genes (int) and the phage attachment sites have been identified and sequenced in five separate temperate lactococcal phages. The sequence information on the int gene of the φLC3 phage was the first published and revealed relationship with the Int family of site-specific recombinases (5, 47). Later, the int genes of Tuc2009, r1t, and BK5-T were also reported (11, 63, 64). The integrase proteins deduced from these sequences are found to be almost identical with φLC3 Int and could be called the φLC3 type of integrases. In addition, the cores of the attachment sites in these phages are identical. The Int protein of the lactococcal phage TP901-1 shows features relating to the resolvase-integrase family of site-specific recombinases (23, 58). Botstein (10) has proposed that the evolution of lambdoid phages in particular happens by exchange of genes organized in functional modules with homologous areas interspersed between the genes as recombination sites. Examples supporting this kind of recombination in lambdoid phages have been summarized by Campbell (20). In temperate lactococcal phages, the application of this model has been validated by the observation of different codon usage in lysin and holin genes of the phages φLC3 and Tuc2009 (4, 9); by the presence of moderate homology among genes of temperate Lactococcus lactis, Streptococcus thermophilus, and Lactobacillus phages (17, 24, 26, 42, 43, 52, 60); and by the proposal of a conserved integration cassette of the phages BK5-T, φLC3, Tuc2009, and r1t (11, 12, 64). Additional examples supporting the Botstein evolutionary theory have not been presented for temperate lactococcal phages, although it is well established that lactococcal phages are able to evolve by acquisition of host chromosomal DNA (31, 50). In this paper we report on the characterization of a temperate phage, TPW22, from a bacterial isolate, Lactococcus lactis subsp. cremoris W22, isolated from the mixed starter culture TK5 (39). The integrase of this phage is identified and characterized.