INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

Lactococcus lactis is used extensively as starter culture in the dairy industry, where the main function is to convert lactose to lactic acid. One of the predominant reasons for fermentation failures is infection of the starter culture by bacteriophages, resulting in slow acid formation and a product of inferior value. The sources of phage infection for the predominant c2 and 936 species are environmental and include raw milk and in-plant contamination. However, the use of lysogenic starter cultures has recently been implicated in the appearance of virulent P335 phages in the industry (1, 4, 19, 49). To overcome this problem, a variety of different strategies have been implemented, including improved sanitation, rotation of starter cultures (18, 61), use of multiple-strain cultures (64), and the use of phage-resistant starter cultures (for a review, see reference 22).

Lactococcal phages are classified into 12 different species based on morphology, DNA homology, and protein profiles (35), but only three phage species, the prolate-headed c2 species and the isometric-headed 936 and P335 species represent the major virulent types responsible for problems in dairy plants. There are no known temperate phages in the group of the c2 and 936 species, while the P335 species contains both temperate and lytic phages (35). Historically, it was believed that temperate phages did not contribute to the emergence of lytic phages in L. lactis (33). However, the increasing appearance of new lytic phages belonging to the P335 species, supported by DNA homology studies showing extensive homology between lytic and temperate P335 phage species, indicates that temperate phages or a phage remnant constitutes an important source for the development of new lytic bacteriophages (1, 19, 49, 69). Recent studies have shown that introduction of plasmids encoding phage resistance mechanisms selects for new virulent P335 phage species via acquisition of plasmid sequences or chromosomal regions of L. lactis (4, 19, 30, 49). The new recombinant phages are resistant to the original anti-phage system and, in some cases, have acquired new origins of replication (4, 19). Since the first complete phage genome sequence of bIL67 (c2 species) was published (60), several complete genome sequences of both temperate and lytic phages from Lactococcus, Lactobacillus, and Streptococcus thermophilus have become available (2, 22, 44, 67). The sequence information on LAB phages provided important insights into the genome organization, evolution, and relationships between lytic and temperate bacteriophages, which is essential for the future development of new phage-resistant starter culture strains. Genome analysis has shown that obligately lytic phages such as c2 (c2 species) and sk1 (936 species) contain genes required for lytic growth organized in differently oriented clusters (12, 42, 43), while the lytic genes of temperate phages belonging to the P335 species are found in one large cluster (6, 66). To our knowledge, no extensive sequence data are available on lytic members of the P335 species. Temperate phages of the P335 species and other temperate LAB phages are highly modular, organized with genes similar in function clustered together. Typically, genes needed for lysogeny are transcribed divergently and located adjacent to the genes required for lytic propagation. The lysogenic module usually encodes a repressor needed for maintenance of the prophage state and an integrase, which is essential for bacteriophage integration into the bacterial genome. The lytic module is comprised of another repressor, which acts by repressing the lysogeny module during lytic propagation. Following this repressor and in the same transcriptional orientation are gene clusters involved in phage replication, packaging, and lysis of the host cell (for a review, see reference 67).

Previous studies of the lytic phage 31 (P335 species) have shown that two distinct DNA regions exhibit high sequence identity to temperate members of the BK5-T and P335 species (16, 55, 70). However, no sequence data were available to explain the obligately lytic lifestyle of 31 and other lytic members of the P335 species. This study was conducted to understand the basis of the obligate lytic behavior of phage 31. A 10.8-kb DNA region covering the putative replication module and a genetic switch directing lytic or lysogenic components was identified in 31. The genetic components required to establish lysogeny, however, were incomplete in this virulent member of the P335 species.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Bacterial strains, plasmids, bacteriophage, and growth conditions. The bacterial strains, plasmids, and bacteriophage used in this study are listed in Table 1. L. lactis strains were grown in M17 broth with 0.5% (wt/vol) glucose at 30°C. For selection of plasmids in L. lactis, 1 µg of erythromycin per ml and/or 5 µg of chloramphenicol per ml was added to the medium when appropriate. Escherichia coli strain DH10B was cultivated in Luria-Bertani or brain heart infusion medium, supplemented with 200 µg of erythromycin per ml or 10 µg of chloramphenicol per ml when appropriate, at 37°C. Phage 31, a small, lytic, isometric phage with a DNA genome of 31.9 kb, was propagated on L. lactis NCK203 in GM17 medium supplemented with 10 mM CaCl₂. Determination of the efficiency of plaquing (EOP) was performed as previously described (63).

Measurement of -galactosidase activity. -Galactosidase assays were performed on exponentially growing cultures (optical density at 600 nm 0.75) as previously described (32). Measurements were averages of at least three independent experiments.

Cloning, plasmid and bacteriophage DNA isolation, and transformation. Standard procedures were used for DNA manipulations (59). Large-scale plasmid preparations for sequencing and cloning were isolated from E. coli using Jet Prep columns (Genomed) according to the manufacturer's directions. L. lactis small-scale plasmid preparations were performed as described previously (53). Genomic phage DNA was isolated as described elsewhere (19). E. coli competent cells (ElectroMAX DH10B) were electroporated as described by the manufacturer. L. lactis MG1363 was transformed as described by Holo and Nes (31), and L. lactis NCK203 was transformed as described by Walker and Klaenhammer (69).

PCR and DNA sequencing. Plasmid DNA sequencing was performed on both strands using an ABI model 377 automated gene sequencer (Perkin-Elmer) or an ALFexpress DNA sequencer (Pharmacia Biotech). DNA and deduced amino acid sequences were analyzed using the BlastN and BlastP programs available at www.ncbi.nlm.nih.gov/BLAST. PCR amplifications were performed using the Taq DNA polymerase from Gibco BRL as recommended by the manufacturer.

Plasmid constructions. pTRK627 was constructed by insertion of a 1.8-kb PvuII-EcoRV genomic fragment from 31 into the EcoRV site of pBluescript KS II (Stratagene). pTRK627 contains the early promoter region of 31. To study the regulation of the two promoters located in the early region, a collection of promoter fragments were fused to the lacLM reporter gene in the promoter probe vector pAK80 (32). The different promoter fragments were all amplified by PCR using genomic 31 DNA as the template. A 909-bp XhoI-BamHI fragment obtained by using primers cI-XhoI-P1 (5'GGC CGC TCG AGC CTG TTC CGT CTG CCG 3') and cI-BamHI-P2 (5' TAG TAG GAT CCT TTT GGG AGA GAT AAA GCG CC 3') was cloned into pAK80 digested with XhoI and BamHI, resulting in pSMBI 1. Plasmids pSMBI4 and pSMBI8 were constructed by cloning of a 1,240-bp BamHI fragment, obtained by using primers cro-BamHI-P1 (5' TAG TAG GAT CCC CTT TCT TTT TAT AAA GTT CAA ATT TTT TGG AC 3') and cI-BamHI-P2, into pAK80 digested with BamHI. The inserts in pSMBI4 and pSMBI8 are in opposite directions with respect to the lacLM reporter gene in pAK80.

RNA extraction, oligonucleotide labeling, and primer extension. Total RNA was isolated from L. lactis NCK203 at various time points during the 31 infection cycle (multiplicity of infection  5) using the TRIzol reagent from Gibco BRL as described previously (16). Primer extension analysis was performed as described by Kullen and Klaenhammer (38), except for the following modifications. Primers (750 ng) Phi31 Rev3 (5' CCG TAC TGA ATG CTC CAT GAT TGT TCG C 3') and BamHI-Prom1 (5' TAG TAG GAT CCT GTC TTA ATC CTC GGT CGT TC 3') were end labeled with 30 µCi of [-³²P]ATP using 10 U of T4 polynucleotide kinase. Approximately 50 µg of total RNA from each time point was mixed with 150 ng of each labeled primer. The subsequent treatments were done as previously described (38). The primer extension products were analyzed on a denaturing 6% polyacrylamide gel next to a sequence ladder of the same primer and plasmid pTRK627 as the template.

Nucleotide sequence accession number. The nucleotide sequence described in this study has been deposited in the EMBL database under accession number AJ292531.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Mapping of the 31 origin of replication. Previous studies have shown that a 4.5 kb SalI-BamHI fragment of 31 (ori31) is able to protect against 31 proliferation when carried on a high-copy-number plasmid (52). It was suggested that the plasmid-borne ori31 fragment interfered with phage replication by titrating out one or more of the replication factors needed for phage DNA replication (29, 52). To determine the precise location of the putative origin of replication, the 4.5-kb SalI-BamHI fragment located in pTRK361 was sequenced. Analysis of the sequence obtained showed a DNA region scattered with several open reading frames (ORFs), including a putative primase and a short noncoding region (Fig. 1). Most replication origins of phages from lactic acid bacteria are located within noncoding DNA regions which have a high AT content and are rich in direct repeats, as well as inverted repeats (2, 12, 21, 29). The identification of a 230-bp noncoding AT-rich region located just downstream of a gene encoding a putative primase indicates that this region encodes the replication origin of 31 (Fig. 2). Several direct and inverted repeats were identified within this area. A BlastN search with the noncoding DNA sequence did not reveal any homology to other DNA sequences in the databases. However, by using the Bestfit program, we obtained 70% identity between the 230-bp putative origin of 31 and a 253-bp region containing the putative origin of S. thermophilus phage Sfi21 (13) and, importantly, most of the repeats were similarly organized (data not shown). Additionally, the structural gene organization in this particular region of 31 is very similar to the organization found in several S. thermophilus phages, such as Sfi19, Sfi21, O1205, and DT1 (10, 13, 62, 65), and in Lactobacillus gasseri phage adh (2). In these phages, noncoding DNA regions were identified immediately downstream of the putative primase gene. Additionally, in some cases, a phage-encoded resistance (per) phenotype against the particular phage was also demonstrated, which is typical of a phage origin of replication (21, 29, 47). The failure of the 4.5-kb SalI-BamHI fragment to support DNA replication of a nonreplicating vector in L. lactis (data not shown) suggests that host factors alone are not sufficient to carry out DNA replication, but additional phage-encoded factors are required.

View larger version (15K): [in this window] [in a new window]   FIG. 1.   (A) Structural organization of the sequenced 10,814-bp region of the 31 genome. The arrowhead indicates the orientation of each ORF, which is named according to its length in amino acids. Bent arrows indicate the two early promoters, P1 and P2. Vertical arrows indicate the positions of inverted repeats, which may be involved in the termination of transcription. The origin of replication of 31 (ori31) is indicated. Unique restriction sites within the region are marked below the ORFs. The inserts of the three plasmids (pTRK627, pTRK402, and pTRK361) used for DNA sequencing are indicated by black lines. The figure is drawn to scale. (B) Restriction enzyme map of the 31 (31.9-kb) genome adapted from Alatossava and Klaenhammer (1). Only restriction sites relevant for this study are indicated. The map is linearized at the cos site. Black bars indicate previous 31 sequences which have been deposited in databases by Walker et al. (70) (AF022773) and Dinsmore and Klaenhammer (16) (U51128). The figure is drawn to scale.

View larger version (43K): [in this window] [in a new window]   FIG. 2.   Structure and sequence of the origin of replication of phage 31. The illustrated 439-bp sequence is contained in pSMBI10 and confers the per phenotype. Solid arrows indicate direct repeats, numbered DR1 to DR4, and dashed arrows indicate an inverted repeat (IR). The deduced amino acid sequences of the C terminus of ORF 492 (primase gene) and the N terminus of ORF 169a are shown below the nucleotide sequence. The asterisk represents the stop codon of ORF 492.

6

Identification of an early expressed promoter. The inability of the original 4.5-kb ori31 fragment to mediate autonomous DNA replication in L. lactis suggests that phage-encoded factors are essential for DNA replication. Phage genes encoding DNA replication factors are likely expressed from an early promoter that is recognized by the host transcription machinery without the need for additional phage-encoded transcription factors. Therefore, a search was initiated for phage promoters that are recognized immediately after phage 31 infection. To map early expressed promoters, we employed an RNA transcript-capping method used previously to pinpoint a phage-inducible promoter (17). Total RNA was isolated 3, 10, and 20 min after the onset of 31 infection and 5' labeled with vaccinia virus guanylyltransferase and [-³²P]GTP. Phage 31 genomic DNA was digested with EcoRV, PvuII, and EcoRV plus PvuII and subsequently hybridized with the labeled RNA probes. Based on the hybridization patterns obtained with the RNA probes generated 3 min after infection, a single minimal 1.8-kb PvuII-EcoRV fragment containing an early expressed promoter was identified (data not shown). According to the published restriction map of 31 (1), this fragment is located immediately upstream of the EcoRV-SalI fragment contained in pTRK402 (16) (Fig. 1). Only one early hybridizing signal was observed in each restriction enzyme digest, indicating that only a single early promoter region is located on the phage genome. The 1.8-kb PvuII-EcoRV fragment was inserted into pBluescript, resulting in pTRK627, and the DNA sequence was determined.

Nucleotide sequence of a 10.8-kb PvuII-BamHI fragment from 31. The identification of a region containing an early expressed promoter, the origin of replication, and a gene encoding a putative primase indicated that this region of the phage genome is involved in DNA replication. The DNA sequence and the gene organization of the putative 31 replication module were determined by assembling previous sequences and closing gaps as needed.

View this table: [in this window] [in a new window]   TABLE 2.   General features of the putative ORFs within the 10,814-bp region of the 31 genome

The incomplete lysin gene. Database searches with the deduced amino acid sequence of incomplete ORF 171' revealed a high degree of identity (98%) to ORF 259, encoding a cell wall-lytic enzyme from the temperate L. lactis phage BK5-T (6). Despite the lack of the 5' end of the gene, we propose that ORF 171' encodes the lysin of phage 31.

Identification of a regulatory region encoding two repressor homologues, cI and Cro. Located 368 bp downstream of the putative lysin gene, ORF 180 was identified in the opposite orientation. ORF 180 shows significant similarity to a group of phage proteins that function as repressors of lytic gene expression in temperate bacteriophages. The highest level of identity found (68%) was to ORF 4 (180 amino acids) of the temperate L. lactis phage TP901-1 (45). Similar to ORF 4 of TP901-1 and some other repressor proteins, ORF 180 showed no helix-turn-helix (HTH) DNA-binding motif or RecA-mediated cleavage site. The potential ribosome-binding site (5' AAGAA 3') located upstream of the AUG start codon shows weak complementarity to the L. lactis 3' 16S rRNA, indicating weak translation of cI. Alternatively, two other AUG start codons were located in the vicinity that might also serve as translation initiation codons. These would specify proteins of 174 and 187 amino acids, respectively. However, neither was preceded by ribosome-binding sites that were better than the ribosome-binding site located upstream of ORF 180. In the case of the 187-amino-acid alternative, only 6 bp separates the 10 promoter region from the AUG start codon. This is very analogous to the situation seen for the rro gene of the temperate L. lactis phage r1t (50) and for the cI repressor gene of E. coli phage  (57), where the adenine of the start codon also acts as the transcriptional start site. Despite the high diversity between ORF 180 (alternatively, ORF 174 or ORF 187) and typical cI proteins, we suggest that this ORF specifies a protein with a cI-like repressor function and therefore it was renamed cI.

View larger version (32K): [in this window] [in a new window]   FIG. 3.   Multiple-sequence alignment of the putative Cro sequence of 31; ORF69 (accession no. AF158600, AF158601, and AF115102) of S. thermophilus phages Sfi11, Sfi18, and Sfi19; ORF5 (accession no. U88974) of S. thermophilus phage O1205; and ORF 5 (accession no. Y14232) of L. lactis phage TP901-1. Identical amino acids are boxed. The putative HTH motif involved in DNA binding is shown above the sequence.

The region between cI and Cro encodes two divergent promoters. Previous studies with lambdoid and LAB phages have revealed an intergenic region located between cI and Cro that contains two oppositely oriented promoters and an operator site (5, 10, 20, 37, 40, 45, 50, 51). Two putative divergently oriented consensus promoters, designated P1 and P2, were found within the 213-bp intergenic region of 31. The P1 promoter reading toward the cro gene contained a perfect consensus sequence with an extended 10 motif (15), while P2 reading toward the cI gene deviated slightly from the consensus sequence in the 35 region (Fig. 4). The activity of the two putative promoters during temporal phage development was analyzed by primer extension using a cro-specific primer and a cI-specific primer. RNA was extracted at various time points during the 31 infection cycle and used for primer extension analysis. With the cro-specific primer, a cDNA product was identified 5 min after infection and it was retained for at least 40 min (Fig. 5). No cDNA product was obtained using RNA isolated from uninfected cells (time zero), demonstrating that the primer extension product obtained 5 min after infection was phage specific. The identified transcription start site is located 8 bp downstream of the extended 10 region of the P1 promoter. In contrast, no transcript initiated from the P2 promoter, suggesting that P2 is repressed during phage 31 infection. A 164-bp nontranslated mRNA leader region preceded the cro-specific transcript. The distance from the 10 region of the putative P2 promoter to the start codon of the cI gene is between 6 and 45 bp, depending on which initiation codon is used for cI translation. Analysis of the sequences intervening between cI and cro revealed two inverted repeats which may serve as repressor-binding sites for the cI or Cro protein. Binding to this inverted repeat could repress transcription of the cI gene, since one of the inverted repeats overlapped the putative 35 region of the P2 promoter. The other inverted repeat was located in the 5' end of the untranslated cro mRNA leader and could also act to stabilize mRNA or as a repressor-binding site. Site-directed mutagenesis will be required to deduce the exact role and importance of these inverted repeats for promoter regulation.

View larger version (23K): [in this window] [in a new window]   FIG. 4.   Nucleotide sequence of the divergent promoter region located between the oppositely directed cI and Cro homologues. The 10 and 35 sequences of promoters P1 and P2 are underlined. A bent arrow indicates the transcriptional start site located downstream of P1. Inverted repeats are indicated by solid arrows, and stippled arrows indicate a direct repeat. The possible translational initiation codons of the two genes are in boldface uppercase letters, while their respective ribosome-bindings sites (RBS) are in boldface lowercase letters. Alternative translational initiation codons of the cI protein are stippled.

View larger version (74K): [in this window] [in a new window]   FIG. 5.   Primer extension analysis of the early promoter region of phage 31. Total RNA was extracted at various time points after 31 infection, indicated above the panels (minutes), and used for determination of transcriptional start sites. The nucleotide sequence ladders obtained with the corresponding primers are also shown (GATC). The relevant nucleotide sequence of P1 is indicated to the right. The extended 10 sequence of P1 is boxed, and the transcriptional start site is marked with an asterisk. The putative 10 sequence of P2 is indicated to the left.

Identification of putative antirepressor ORF 238. ORF 238 was identified downstream of the Cro homologue and was preceded by a strong ribosome-binding site. The C-terminal half of ORF 238 exhibited 94% amino acid identity to ORF 6 from temperate L. lactis phage TP901-1 (45), while the N-terminal half of ORF 238 showed homology (54% amino acid identity) to a putative antirepressor homologue ORF 238 gene product from temperate S. thermophilus phage TP-J34 (51). No biological function has yet been proven for ORF 6, the putative TP-J34 antirepressor homologue, or related ORF 287 from temperate S. thermophilus phage Sfi21 (9, 10). A bipartite immunity system for control of lysogeny was suggested for Sfi21 (10), but a spontaneous Sfi21 deletion mutant lacking the C terminus of ORF 287 has been isolated (9), demonstrating that the putative antirepressor (ORF 287) is nonessential for both the lytic and lysogenic life cycles.

Identification of a putative helicase and primase. The deduced protein sequence of ORF 448 (or ORF 449, depending on which start codon is used for translation initiation) shows strong similarity (57 to 58% identity) to a group of helicase-like proteins encoded by several S. thermophilus phages (Fig. 6). Similar to the helicase homologues identified in the S. thermophilus phages, several motifs were found within the coding sequence of ORF 448. In the N terminus, a nucleotide-binding site and a DEAH box was identified, which is typical in a subgroup of helicases (26, 68). A second nucleotide-binding site was found in the middle of the putative helicase protein, and a zinc finger motif (CX₂CX₁₁CX₂C) was located in the C terminus of ORF 448 (Fig. 6). Based on the homology and the sequence features, it appears likely that ORF 448 is a helicase and plays a role in unwinding the DNA template during 31 replication.

View larger version (72K): [in this window] [in a new window]   FIG. 6.   Multiple alignment of the 31 helicase homologue (ORF 448) with putative helicases from S. thermophilus phages DT1 (accession no. AF085222), O1205 (accession no. U88974), and Sfi19 (accession no. AF115102). Identical amino acids are boxed. Two nucleotide-binding sites (Walker A motifs), located in the N terminus and in the middle of the protein, are marked in boldface letters. The conserved DEAH box is indicated in boldface letters and marked by asterisks above the sequence. The four conserved cysteine residues within the zinc finger motif are shaded.

ORF 169a is homologous to intron-encoded endonucleases. ORF 169a exhibits 36 to 41% identity to several bacteriophage-encoded site-specific endonucleases that all are located within self-splicing group I introns in different B. subtilis phages, such as SPO1, phi-E, and SP82 (24, 25). Whether ORF 169a actually is located within an intron in 31 is still unclear. All of the bacteriophage introns identified so far interrupt genes with different functions, but no obvious gene seems to be interrupted by ORF 169a in 31. Furthermore, we were not able to identify the conserved sequence elements common to group I introns (11; Robin Gutell, personal communication). Additionally, attempts to identify a spliced mRNA product using reverse transcription-PCR over this region were unsuccessful.

Cro is an efficient repressor of P1 and P2 promoter expression. To study the regulation of the divergent promoters P1 and P2, located between the cI and Cro repressor homologues, we made a collection of transcriptional gene fusions to the promoterless reporter genes lacLM of pAK80 (32) (Fig. 7). The region encompassing the two promoters and the cognate repressor genes were amplified by PCR and cloned in both orientations with respect to the lacLM reporter gene, allowing analysis of each promoter in the presence of one or both repressor proteins. Measurement of -galactosidase activity in L. lactis MG1363 in constructs with cI and Cro showed that the P2 promoter (pSMBI4) was completely repressed (<0.1 Miller unit), while the P1 promoter (pSMBI8) was marginally active (~3.5 Miller units). The closed state of P2 agrees with the primer extension analysis described above showing that no extension product was obtained using the cI gene-specific primer.

View larger version (21K): [in this window] [in a new window]   FIG. 7.   Analysis of P1 and P2 promoter regulation. The indicated individual fragments were cloned into L. lactis promoter probe vector pAK80. The right endpoint of each fragment was transcriptionally fused to the lacLM reporter gene of pAK80. The plasmids were introduced into L. lactis MG1363, and -galactosidase (-gal.) assays were performed as described in Materials and Methods. The figure is drawn to scale. Spec. act., specific activity.

trans-complementation studies of cro and cI. To confirm the strong negative regulation of both promoters by the Cro protein and the moderate repression of P2 by the cI protein, the two repressor genes were cloned individually into another compatible plasmid, pCI372, for trans-complementation studies. When Cro was provided in trans, a substantial decrease in -galactosidase activity was observed for both pSMBI25 and pSMBI 27 (Table 3). These data demonstrate that Cro has a trans-acting ability to repress the expression of both promoters. In contrast, the donation of cI in trans did not significantly repress either P1 or P2. These data suggest that the impact of cI, in cis, on P2 expression is likely minor.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

In this study, we have established the sequence of a 10.8-kb region from lytic Lactococcus phage 31 that encodes remnants from a genetic switch and the DNA replication region. Analysis of the nucleotide sequence revealed the presence of one incomplete ORF and 17 full-length ORFs longer than 40 amino acids. Among these were three ORFs described previously that were involved in sensitivity to the abortive phage infection system AbiA (16). The deduced amino acid sequence of the remaining ORFs was compared to the contents of the available protein databases, and several ORFs showing significant homology to ORFs from other LAB phages were identified. The identified ORFs were clustered in DNA modules that directed key functions responsible for lysis, DNA replication, and control of lifestyle (lytic versus lysogenic).

The 31 origin of replication (ori31) was originally located on a 4.5-kb fragment (52) by demonstration of its ability to retard phage replication when presented in trans on a high-copy-number plasmid. First demonstrated by Hill et al. (29) using phage 50, this phenomenon has been shown in other Lactococcus and S. thermophilus phages (21, 47). In this study, we mapped ori31 to a 439-bp noncoding DNA sequence located just downstream of a gene encoding the putative 31 primase. This was based on (i) the ability of the 439-bp fragment to reduce the EOP approximately 10⁶-fold (per phenotype), (ii) a typical structure with several repeats, and (iii) the conserved location for replication origins within a group of related phages from S. thermophilus and L. gasseri. The ori31 sequence was distinct from P335 phage 50 (29), consistent with our previous functional observation that the presence of ori50 does not interfere with phage 31 replication and that ori31 does not interfere in trans with phage 50 replication.

Flanked by the origin of replication and an early P1 promoter, a group of genes were identified that are likely to constitute the replication module of 31. This is based on the identification of two ORFs with several motifs that show strong similarity to helicases and primases. Five of the deduced proteins (ORF 245, helicase, ORF 150, ORF 268, and primase) located within the putative replication module of 31 showed strong similarity to a conserved DNA replication module from a group of virulent and temperate S. thermophilus phages, such as Sfi19, Sfi21, O1205, and DT1. Besides the high degree of amino acid identity (38 to 58%) to the S. thermophilus homologues, the sizes of the proteins and the topological organization of the corresponding genes were similar, suggesting that S. thermophilus phages and lactococcal phages belonging to the P335 species are closely related. Finally, we observed significant sequence identity and structural similarity between the origin of replication of 31 and the origin of replication of Sfi21. This is a very significant observation, given that both Lactococcus and S. thermophilus are used extensively in common dairy fermentation environments and one could anticipate opportunities for coevolution between phages infecting these two bacterial species (8).

Efforts to construct an autonomously replicating plasmid were based on cloning of the entire sequence extending from the P1 promoter to a point just downstream of the origin of replication. These efforts were unsuccessful, suggesting that one or more factors are missing from this region that are essential for phage replication. In a recent study, the complete DNA sequence of temperate L. gasseri phage adh was determined. A 10-kb fragment covering the putative replication module of adh was sufficient to mediate autonomously replication in both L. gasseri and L. lactis (2). Interestingly, the topological organization within the replication module of adh was similar to that of 31. Homologues of ORF 243, helicase, ORF 150, and the primase were identified in the same order in adh.

Another interesting feature from the promoter and sequence analysis of 31 was the identification of a typical genetic switch including two divergently oriented promoters and two cognate repressor genes that regulate promoters P1 and P2. Genetic switches are found in temperate phages, where they control the entrance into either the lytic or the lysogenic life cycle. Identification of a cI repressor homologue in the lytic bacteriophage 31 was unexpected, as the natural function of this protein is to repress the expression of genes required for lytic proliferation. In this regard, it is interesting that gene fusion experiments with promoter constructs expressing cI failed to significantly retard expression of P1, the promoter directed toward cro and the lytic gene functions of phage 31 (Fig. 7, construct pSMBI30). Whether or not this is a function of the effectiveness of the cI protein or its ability to bind the operator is unknown. To our knowledge, this is the first example of a lytic Lactococcus phage which contains a gene implicated in the establishment of the lysogenic state. Spontaneous deletion mutants of different temperate LAB phages (L. lactis temperate phage BK5-T, S. thermophilus temperate phage Sfi21, and Lactobacillus delbrueckii temperate phage LL-H) that have lost the capacity to lysogenize host cells have been described in the literature (7, 9, 48). These mutants have incurred deletions in a region covering the phage attachment site (attP) and the integrase gene, which both are required to establish lysogeny. The genome organization in temperate bacteriophages is highly conserved, and the genetic requirements (the attP and int genes) for integration are typically located between the lysin gene and the divergently oriented cI gene. In this study, we identified the 3' end of the lysin gene, but no attachment site or int gene was identified within the 368-bp sequence located between the lysin gene and the cI gene. These data indicate that 31 evolved from a temperate phage to an obligately lytic phage by deletion of a specific region that abolishes the ability of 31 to form stable lysogens. As a direct result of this close relationship to temperate phages within the P335 species, the obligately lytic phage 31 is further capable of acquiring prophage-encoded sequences essential for virulent replication and development (19).

In contrast to the normal situation in temperate phages, where the genetic switch is controlled by a sophisticated regulation mechanism involving differential interactions between the repressor proteins and the operator-binding sites (20, 41, 46, 50), the situation in 31 seems to be simpler. An example of this high complexity appears in the regulation of the genetic switch from temperate L. lactis phage TP901-1 (46). Despite the high overall similarity between the two repressor proteins of 31 and the corresponding proteins of TP901-1, the two genetic switches are quite differently regulated. The cI homologue (ORF 4) of TP901-1 was an efficient repressor of both early promoters, whereas the Cro homologue (ORF 5) alone had no effect on either of the two early promoters in TP901-1. In contrast, the cI homologue of 31 was not an efficient repressor of either P1 or P2 and, interestingly, allowed efficient expression of P1 reading into cro and the DNA replication module. In contrast to TP901-1, the Cro homologue of 31 efficiently repressed both P1 and P2. Comparison of the DNA sequences of the two early divergent promoter regions of 31 and TP901-1 showed identity inside the region flanked by the two 35 sequences in the two phages (Fig. 8). While an inverted repeat overlapped both 35 sequences in TP901-1, the equivalent inverted repeat of 31 only overlapped the 35 sequence of the P2 promoter (cI gene expression). These differences at the DNA level, in addition to the diversity between the repressor proteins, might contribute to the obvious difference in regulation between the two genetic switches of 31 and TP901-1 and their corresponding virulent and temperate lifestyles.

View larger version (13K): [in this window] [in a new window]   FIG. 8.   Comparison of the early promoter regions of lytic phage 31 and temperate phage TP901-1. The 10 and 35 regions of all four promoters are shaded. Vertical lines indicate identical base pairs. Inverted repeats are indicated by horizontal arrows. PL, lytic promoter of TP901-1; PR, lysogenic promoter of TP901-1. The depicted sequence is shown as 5' to 3'. The TP901-1 sequence included extends from position 3219 to position 3307 in EMBL entry Y14232.

Using the BlastN search tool, we identified several regions within the established DNA sequence that showed very high identity to DNA sequences from the temperate L. lactis phage species P335 and BK5-T. The most interesting identities are located around the lysin and cI genes, further supporting the idea that temperate phages contribute to the appearance of lytic phages in these species (19, 34, 49, 58). The presently available DNA sequence of the 31 lysin gene exhibits 94% identity to 3' end of the lysin gene of BK5-T. Three other regions of homology around the lysogenic modules of several temperate lactococcal phages and 31 were identified. (i) Just downstream of the lysin gene, a 22-bp DNA sequence, which is located in corresponding positions on the BK5-T, r1t, Tuc2009, and LC3 phage genomes, was identified. This sequence is 100% conserved among the three phages and contains, besides a putative transcription terminator, part of the BK5-T core sequence, which was involved in the formation of two spontaneous BK5-T.H28 and BK5-T.H210 deletion mutants (7). (ii) A DNA region with extensive homology to the region located downstream of the attP site and the int gene of BK5-T (93% identity; nucleotides 10518 to 10706 of the published sequence [6]) and r1t (97% identity; nucleotides 33069 to 33183 of the published sequence [66]) was identified adjacent to the 3' end of the cI gene of 31. (iii) Finally, a region that spans most of the untranslated cro leader was 97 and 98% identical to DNA sequences located in similar positions of r1t and BK5-T. The homologous DNA sequences identified that flank the lysogenic modules of r1t, BK5-T, and the cI gene of 31 might, therefore, represent recombination hot spots promoting exchange of lysogenic modules among the phages, consistent with the modular theory of phage evolution proposed by Botstein (3).

More evidence of a close evolutionary relationship between 31 and temperate phages was provided by the identification of a 952-bp region within the 31 sequence showing 96% sequence identity to temperate phage r1t. The 952-bp sequence is located in the region encoding ORFs 59 and 188 of 31, which is adjacent to the region containing the late phage inducible promoter that also exhibited high sequence identity to phage r1t (70)

The accumulating sequencing data and molecular studies of bacteriophages from lactic acid bacteria will certainly lead to a better understanding of phage evolution and provide more insight into the emergence of new virulent phages. This investigation adds to the mounting evidence that virulent members of the P335 species have arisen from temperate ancestors and freely exchange prophage-based sequences under the dynamics of the dairy fermentation environment. This perspective should fuel future efforts to identify P335 sequences in the genomic complement of Lactococcus starter cultures and eliminate them in an effort to the minimize genetic routes that support the appearance and adaptation of new virulent phages.