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Lactococcus lactis phage P335 is a virulent type phage for the species that bears its name and belongs to the Siphoviridae family. Morphologically, P335 resembled the L. lactis phages TP901-1 and Tuc2009, except for a shorter tail and a different collar/whisker structure. Its 33,613-bp double-stranded DNA genome had 50 open reading frames. Putative functions were assigned to 29 of them. Unlike other sequenced genomes from lactococcal phages belonging to this species, P335 did not have a lysogeny module. However, it did carry a dUTPase gene, the most conserved gene among this phage species. Comparative genomic analyses revealed a high level of identity between the morphogenesis modules of the phages P335, ul36, TP901-1, and Tuc2009 and two putative prophages of L. lactis SK11. Differences were noted in genes coding for receptor-binding proteins, in agreement with their distinct host ranges. Sixteen structural proteins of phage P335 were identified by liquid chromatography-tandem mass spectrometry. A 2.8-kb insertion was recognized between the putative genes coding for the activator of late transcription (Alt) and the small terminase subunit (TerS). Four genes within this region were autonomously late transcribed and possibly under the control of Alt. Three of the four deduced proteins had similarities with proteins from Streptococcus pyogenes prophages, suggesting that P335 acquired this module from another phage genome. The genetic diversity of the P335 species indicates that they are exceptional models for studying the modular theory of phage evolution.

The type II restriction-modification (R-M) system LlaDII isolated from Lactococcus lactis contains two tandemly arranged genes, llaDIIR and llaDIIM , encoding a restriction endonuclease (REase) and a methyltransferase (MTase), respectively. Interestingly, two LlaDII recognition sites are present in the llaDIIM promoter region, suggesting that they may influence the activity of the promoter through methylation status. In this study, separate promoters for llaDIIR and llaDIIM were identified, and the regulation of the two genes at the transcriptional level was investigated. DNA fragments containing the putative promoters were cloned in a promoter probe vector and tested for activity in the presence and absence of the active MTase. The level of expression of the MTase was 5- to 10-fold higher than the level of expression of the REase. The results also showed that the presence of M.LlaDII reduced the in vivo expression of the llaDIIM promoter (P llaDIIM ) up to 1,000-fold, whereas the activity of the llaDIIR promoter (P llaDIIR ) was not affected. Based on site-specific mutations it was shown that both of the LlaDII recognition sites within P llaDIIM are required to obtain complete repression of transcriptional activity. No regulation was found for llaDIIR , which appears to be constitutively expressed.

The Lla DII restriction/modification (R/M) system was found on the naturally occurring 8.9-kb plasmid pHW393 in Lactococcus lactis subsp. cremoris W39. A 2.4-kb Pst I- Eco RI fragment inserted into the Escherichia coli-L. lactis shuttle vector pCI3340 conferred to L. lactis LM2301 and L. lactis SMQ86 resistance against representatives of the three most common lactococcal phage species: 936, P335, and c2. The Lla DII endonuclease was partially purified and found to recognize and cleave the sequence 5′-GC↓NGC-3′, where the arrow indicates the cleavage site. It is thus an isoschizomer of the commercially available restriction endonuclease Fnu 4HI. Sequencing of the 2.4-kb Pst I- Eco RI fragment revealed two open reading frames arranged tandemly and separated by a 105-bp intergenic region. The endonuclease gene of 543 bp preceded the methylase gene of 954 bp. The deduced amino acid sequence of the Lla DII R/M system showed high homology to that of its only sequenced isoschizomer, Bsp 6I from Bacillus sp. strain RFL6, with 41% identity between the endonucleases and 60% identity between the methylases. The genetic organizations of the Lla DII and Bsp 6I R/M systems are identical. Both methylases have two recognition sites (5′-GCGGC-3′ and 5′-GCCGC-3′) forming a putative stem-loop structure spanning part of the presumed −35 sequence and part of the intervening region between the −35 and −10 sequences. Alignment of the Lla DII and Bsp 6I methylases with other m 5 C methylases showed that the protein primary structures possessed the same organization.

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.