S. thermophilus phage 7201 shows graded relatedness with Sfi21-like streptococcal and lactococcal phages.

S. thermophilus phage 7201 is not inhibited by the Sfi21-type origin of phage replication (51). Since the DNA replication module is highly conserved in S. thermophilus phages (19), a distinct genome organization was suspected for phage 7201. The sequencing of the 35,466-bp-long phage 7201 genome revealed a distinct replication module in an otherwise standard Sfi21-like phage genome. The predicted genetic map is depicted in Fig. ​Fig.22 and is compared to that of cos site virulent S. thermophilus phage Sfi19, an established member of the Sfi21 phage group (41). Note that the two virulent streptococcal phages are depicted in a hypothetical prophage configuration to facilitate the comparison with the L. lactis prophage bIL286 (top of Fig. ​Fig.2).2). The database matches with phage DT1 and an S. thermophilus prophage remnant (54) identified the first three 7201 genes (ORFs 1, 2, and 3) as the lysogeny replacement module (Table ​(Table2).2). Over the next eight predicted genes (ORF 4 to 10), phage 7201 shared no similarity with any described S. thermophilus phages. However, with the exception of ORF 6a and 6b, the products of a DNA duplication event, all predicted proteins matched likely DNA replication proteins identified on plasmids or phages and prophages from pathogenic streptococci (51).

Open in a separate window

FIG. 2.

Alignment of the genetic maps from the L. lactis prophage bIL286 with the virulent S. thermophilus phages 7201 and SfiI9. The open reading frames are color coded according to their predicted function as in Fig. ​Fig.1.1. Selected genes or genomic features are denoted. Genes encoding proteins that showed significant amino acid sequence similarity are linked by red shading, and the percentage of amino acid identity is indicated. The degree of amino acid identity (>90, >80, >70, and <70%) is reflected in the color intensity of the red shading. Note that the genomes of the virulent phages were rearranged to allow an easier comparison with the prophage bIL286. The natural ends of the streptococcal phage DNA flank the indicated cos sites.

For the remainder of the phage 7201 genome, covering ORF 11 to ORF 44, closely related genes were found at corresponding genome positions in the cos site S. thermophilus phages Sfi19. One exception is represented by ORFs 13 and 14, which showed a close match with a putative DNA methylase from L. lactis phage Tuc2009 and a putative transcriptional regulator from mycobacteriophage L5, respectively. The only other exception is provided by ORFs 36 and 37, which are closely related to corresponding genes in bIL286.

Sequence identity between S. thermophilus phages 7201 and Sfi19 was high and exceeded 80% at the amino acid level for proteins encoded by the head, tail, and part of the tail fiber genes (Fig. ​(Fig.2).2). At the DNA level, both phages could be aligned with few interruptions over three-quarters of the respective genomes (Fig. ​(Fig.3A).3A). Proteins specified by the entire structural gene cluster of phage 7201 also displayed significant amino acid sequence similarity to the proteins encoded by the equivalent region of the Sfi21-like L. lactis prophage bIL286. However, the level of protein sequence identity between 7201 and bIL286 was lower than that between any two S. thermophilus phages belonging to the Sfi21 group. DNA sequence similarity between 7201 and bIL286 was limited to one DNA replication gene, several head genes, the genes involved in DNA packaging, the major tail gene, and two tail fiber genes (Fig. ​(Fig.3B).3B). Due to the high level of sequence identity (85 and 80% base pair and amino acid identity, respectively, between 7201 ORF 36 and bIL286 ORF 55), this tail fiber gene is a candidate for lateral gene transfer between streptococcal and lactococcal phages.

Open in a separate window

FIG. 3.

Dot plot analysis for the S. thermophilus phage 7201 genome versus the S. thermophilus phage Sfi19 genome (A) and the S. thermophilus phage 7201 genome versus the L. lactis prophage bIL286 genome (B). The comparison window was 50 bp, and the stringency was 30 bp. Genes sharing nucleotide sequence similarity are highlighted in black on the gene maps (see Fig. ​Fig.22 for an expanded view of the maps) of the phages depicted at the sides of the dot plot.

The 7201 sequence confirms the widespread distribution of Sfi21-like phages in S. thermophilus, and the sequence also confirmed the close link between streptococcal and lactococcal phages as already deduced from the Sfi21/L. lactis phage BK5-T phage comparison (20).

Dot plot matrix for lactococcal phages.

In apparent contradiction to the widespread distribution of Sfi21-like phages for S. thermophilus, Sfi21-/BK5-T-like L. lactis phages have only rarely been isolated from the cheese factory. However, Sfi21-like lactococcal phages were detected as prophages during the sequencing of the L. lactis strain IL1403 (16), increasing the number of complete lactococcal phage genomes to 11. It is, therefore, appropriate to study the DNA sequence relationships between all currently available L. lactis phages in the dot plot matrix displayed in Fig. ​Fig.4.4. This analysis allows investigation into the genetic differences underlying the lactococcal phage species and genus classification established by phage taxonomists (7). Several observations are noteworthy. The virulent lactococcal phages represent two clearly separated groups with respect to DNA sequence relationships: c2-like phages (an International Committee on Taxonomy in Virology (ICTV)-accepted genus in Siphoviridae [43]) and sk1-like phages. Each group is represented by two sequenced members that share close sequence relationships with each other (c2 and bIL67 on one side and sk1 and bIL170 on the other side) but practically no DNA sequence relationships with any other group of lactococcal phages (Fig. ​(Fig.4).4). Both c2- and sk1-like phages are abundant for L. lactis, while phages with similar genome organization have, until now, not been isolated from any bacterial species other than L. lactis. Temperate lactococcal phages like r1t (53), Tuc2009 (J. F. M. L. Seegers, M. van de Guchte, M. Creaven, G. F. Fitzgerald, and D. van Sinderen, unpublished data), and TP901-1 (9) (currently classified as a P335 species of lactococcal phages) are also prominent factory isolates in contrast to temperate phages from the lactococcal BK5-T species (42). All temperate lactococcal phages are linked by DNA sequence relationships. This is most evident for the lysogeny, DNA replication, and putative transcriptional regulation genes, which are shared to a large extent by all seven temperate lactococcal phages (Fig. ​(Fig.4,4, diagonal line in top seven squares of leftmost column). However, with respect to DNA sequence homology over the structural genes, several groups could be differentiated within the temperate lactococcal phages. The first group is represented by BK5-T-like phages. This group contains three sequenced members: phages BK5-T, bIL286, and bIL309. They are clearly members of the proposed Sfi21-like genus of Siphoviridae. The second group is represented by phages Tuc2009 and TP901-1, while a third group is represented by a single phage isolate, r1t (Fig. ​(Fig.4).4). In the current taxonomic system of lactococcal phages, the latter two phage groups are classified together as P335 species. This classification leads to problems, however. Tuc 2009 and TP901-1 are pac site phages which are members of the recently proposed Sfi11-like genus of Siphoviridae (10, 11), which are as widely distributed in low-GC-content gram-positive bacteria as phages from the proposed Sfi21-like genus. In contrast, phage r1t is a cos site phage with a clearly distinct DNA sequence and genome organization of the structural gene module. Closely and distantly related phages were found in S. pyogenes and mycobacteria, respectively (20, 21), indicating that we might have here a further widely distributed structural gene module. Prophage bIL285 constitutes a fourth DNA homology group for the structural genes in temperate lactococcal phages. Therefore, at least six distinct structural modules can be defined for Siphoviridae from L. lactis phages, and more are likely to exist (e.g., in the 949 lactococcal phage species). What might be the reason for this much broader genetic diversity for phages from L. lactis than for those from S. thermophilus, which showed only two distinct structural modules for both virulent and temperate phages? Since L. lactis phages have been investigated for a longer time and by more laboratories, it might represent an observational bias. We judge this unlikely, since more than 200 S. thermophilus phage isolates have been investigated in several laboratories, revealing just two major phage types (12, 35). We prefer an alternative interpretation. Numerous plasmid-encoded phage inhibition systems have been described for L. lactis, while plasmids are rare for S. thermophilus, and even rarer are S. thermophilus plasmids that encode phage resistance mechanisms (26). Interestingly, one of the rare S. thermophilus plasmids (30) showed sequence similarity to the origin of replication of S. thermophilus phages (25). However, in contrast to the situation for E. coli, where phages can replicate as extrachromosomal elements (phage P1), no good evidence for prophage-like plasmids exists for lactic acid bacteria (15). L. lactis phages are therefore under a much greater selection pressure to diversify within the species or to accept “foreign” phage modules from the environment than S. thermophilus phages. We speculate that the latter process might be favored by the broader ecological range of L. lactis compared to S. thermophilus, which appears to be restricted to milk.

Open in a separate window

FIG. 4.

Dot plot matrix calculated for the complete genomes of the L. lactis prophages bIL285, bIL286, and bIL309, all from the genome sequence of L. lactis strain IL1403 (13), of the temperate L. lactis phages Tuc2009 (P335 lactococcal species, proposed Sfi11-like genus of pac site Siphoviridae), BK5-T (BK5-T lactococcal species, proposed Sfi21-like genus of cos site Siphoviridae), TP901-1 (P335 lactococcal species, proposed Sfi11-like genus of pac site Siphoviridae), r1t (P335 lactococcal species, new r1t/LC3-like genus (?) of cos site Siphoviridae) and of the virulent L. lactis phages bIL67 and c2 (both c2 lactococcal species, c2-like genus of Siphoviridae) and sk1 and bIL170 (936 species of lactococcal phages, new sk1-like genus (?) of Siphoviridae). The prophages and temperate phages are aligned so that the phage integrase is at the utmost left (top) of each indicated square, followed by the lysogeny and DNA replication modules; in the center are the structural genes (DNA packaging, head, and tail genes); at the right (bottom) of each indicated square are tail fiber and lysis genes. The virulent phages are given with the late structural genes at the right and the early nonstructural genes at the left. The BK5-T and c2 sequences were rearranged or inversed, respectively, with respect to the database entry. The left y axis provides a scale in kilobases; the right y axis and the bottom x axis identify the phage genomes that were compared in the corresponding square. The dot matrix was calculated using Dotter (50). The comparison window was 50 bp, and the stringency was 30 bp.

To explore the genetic diversity demonstrated by lactococcal phages, we performed a comparative genomics analysis for the L. lactis phages with an Sfi21-like gene map.

Prophages bIL286 and bIL309 are further examples of Sfi21-like L. lactis phages.

Over the entire late gene cluster, the L. lactis prophage bIL286 exhibited a high degree of protein sequence identity with phage BK5-T, an established member of the proposed Sfi21-like Siphoviridae genus (Fig. ​(Fig.5).5). In particular, the proteins encoded by the DNA packaging, major head and tail, and lysis genes were nearly identical between these two phages. Major differences between the late genes were limited to three regions. The first is the additional ORF 14 in BK5-T, located in a region where phage lambda shows frameshifting between the tail genes G and T. The putative tail fiber genes ORF 54 and 55 from bIL286 have no counterpart in BK5-T, while a gene specifying a closely related protein was detected in the L. lactis prophage bIL309. This is actually the second example where a single ORF in one phage has similarity to multiple ORFs in another phage (see also ORF 35 and 38 from 7201 compared to ORF 1291 in Sfi19) (Fig. ​(Fig.2).2). The large gene in phage Sfi19 encodes the likely receptor-recognizing tail fiber gene (39) and represents a multidomain protein, which is due to internal collagen-like repeats that are the target of recombination events (23, 39). It cannot be decided whether the fused or split genes represent the ancient gene constellation. A third difference was observed in the lysis module: bIL286 possesses a relatively large anonymous gene upstream of the holin gene (ORF 59), while a similarly sized gene was located downstream of the lysin gene in BK5-T (ORF 30). The latter gene is one of the few transcribed genes of the BK5-T prophage (6) and thus a candidate for a lysogenic conversion gene (54). Over the nonstructural gene region, only isolated genes shared sequence similarity between bIL286 and BK5-T.

Open in a separate window

FIG. 5.

Alignment of the genetic maps from the L. lactis prophage bIL309 with the L. lactis prophage bIL286 and temperate L. lactis phage BK5-T. The open reading frames are color coded according to their predicted function as in Fig. ​Fig.1.1. Selected genes or genomic features are denoted. Genes encoding proteins that showed significant amino acid sequence are linked by red shading, and the percentage of amino acid identity is indicated. The degree of amino acid identity (>90, >80, >70, or <70%) is reflected in the color intensity of the red shading.

A qualitatively similar pattern of relatedness was observed when the L. lactis prophages bIL309 and bIL286 were compared (Fig. ​(Fig.5).5). However, important quantitative differences were observed. The degree of sequence similarity between bIL286 and bIL309 was, with only a few exceptions, lower than that between bIL286 and BK5-T. At the DNA level, sequence homologies were restricted to small genome segments, the longest covering two adjacent putative tail fiber genes (Fig. ​(Fig.4).4). In contrast, BK5-T and bIL286 phages shared DNA sequence similarity essentially over the entire structural gene cluster (Fig. ​(Fig.44).

Prophages bIL285: a distant member of the Sfi21-like phage group.

An interesting case is presented by L. lactis prophage bIL285. Despite its lack of any DNA or protein sequence similarity with the structural gene cluster from any other sequenced lactococcal phage, the gene map suggested an Sfi21-like phage (Fig. ​(Fig.6).6). Diagnostic for this group is the protease/major head gene constellation presented by ORF 43 and ORF 44 in bIL285. Notably, the L. lactis prophage bIL285 and the Listeria innocua prophage 5 are closely related at the protein level for most of the predicted structural proteins. Exceptions were the bIL285 ORF 45 insertion/deletion and ORF 55, a probable tail fiber gene which we predict to be involved in host cell recognition since it is closely related to a corresponding gene in phage Tuc2009 (Fig. ​(Fig.6).6). The similarity between bIL285 and the Listeria prophage extended to the DNA sequence level (Fig. ​(Fig.7).7). This is unusual, since up to now DNA sequence similarity between phages from low-GC-content gram-positive bacteria have been limited to phages infecting the two closely related bacterial genera Streptococcus and Lactococcus (20, 21). Listeria, in contrast, is a more distant member of this branch of bacteria. Notably, lactococcal Sfi21-like phages cover genetic relationships ranging from close similarity at the DNA sequence level over relatedness at the protein sequence level to the sharing of a comparable gene map in the absence of any sequence similarity. This observation confirms the previously described graded relationships between phages in low-GC-content gram-positive bacteria and thus provides evidence for shared evolution of these phages that may be deciphered by comparative genomics. Apparently, at some point the Lactococcus, Listeria, and Streptococcus phages have been in the same host (which may be none of the above bacteria) and have shared their DNA. Since sequence relatives of the structural modules identified in all temperate lactococcal phages have also been found in Streptococcus and Listeria phages, it is unlikely that these structural modules have specifically evolved in the L. lactis species.

Open in a separate window

FIG. 6.

Alignment of the genetic maps from the temperate L. lactis phage Tuc2009 with L. lactis prophage bIL285 and L. innocua prophage 5. The open reading frames are color coded according to their predicted function as in Fig. ​Fig.1.1. Selected genes from bIL285 are annotated. Genes encoding proteins that showed significant amino acid sequence similarity are linked by red shading, and the percentage of amino acid identity is indicated. The degree of amino acid identity (>90, >80, >70, or <70%) is reflected in the color intensity of the red shading. The brackets under the bIL285 gene map demarcate the patchwise sequence sharing of bIL285 genes with other lactococcal phages (B, BK5-T, T, TP901-1; 286, bIL286), possibly identifying genes whose proteins have to interact in a common function (see the text).

Open in a separate window

FIG. 7.

Dot plot analysis for the L. innocua prophage 5 (29) genome versus the L. lactis prophage bIL285 genome. The comparison window was 50 bp, and the stringency was 30 bp. Genes sharing nucleotide sequence similarity are highlighted in black on the gene maps (see Fig. ​Fig.66 for an expanded view of the prophage maps) of the phages depicted at the sides of the dot plot.

The nonstructural gene module in temperate lactococcal phages: association of genes by comparative genomics—the case of bIL285.

All temperate lactococcal phages share substantial DNA sequence similarity over the nonstructural gene cluster. This sharing can be quite substantial as indicated in Fig. ​Fig.66 for phages bIL285 and Tuc2009. However, the dot plot matrix demonstrated mainly a patchwise pattern of DNA sequence sharing for most phage comparisons. On the basis of the modular theory of phage evolution (5), one predicts involvement in related functions for those adjacent genes that share a high level of DNA sequence conservation in a context of sequence-unrelated genes. This rationale allows then a tentative subdivision of parts of the phage genome. The principle is illustrated for the lactococcal prophage bIL285 in Fig. ​Fig.6.6. The comparison with L. lactis phage TP901-1 associated bIL285 ORFs 2, 3, 4, 5, and 6 (predicting lysogeny-related functions) and ORFs 14 and 15 (predicting related DNA replication functions), respectively, (Fig. ​(Fig.6,6, brackets T below bIL285 gene map) as partners in two distinct genetic functions. The comparison with BK5-T associated a different, but complementary, set of bIL285 ORFs (Fig. ​(Fig.6,6, brackets B below bIL285 gene map). From the latter comparison, ORFs 7, 8, and 9 are predicted to fulfill a common function (excisionase function to cooperate with the BK5-T like integrase?). Further groups of genes associated by prophage bIL286 comparisons are annotated in Fig. ​Fig.66 (brackets 286 below bIL285 gene map in figure; unknown replication and transcription control functions?). While still conjectural, these associations provide a subdivision of the prophage genome organization that can serve as a basis for future genetic and biochemical experiments exploring the function of the predicted genes.

We are grateful for PB-EXP01-23 funding from FICYT (principado de Asturias) and the project BIO2201-3621 from CICYT (Spanish Ministry of Science and Technology).