Antonie van Leeuwenhoek (2010) 98:39–50 DOI 10.1007/s10482-010-9426-4
ORIGINAL PAPER
Analyses of bifidobacterial prophage-like sequences Marco Ventura • Francesca Turroni • Elena Foroni • Sabrina Duranti • Vanessa Giubellini • Francesca Bottacini Douwe van Sinderen
•
Received: 2 February 2010 / Accepted: 3 March 2010 / Published online: 15 March 2010 Ó Springer Science+Business Media B.V. 2010
Abstract The genomes of 22 putative prophages (bifidoprophages), previously identified in bifidobacterial genomes, were analyzed to detect the presence and organization of functional modules. Bifidoprophages were shown to display a classical modular genomic organization in which the DNA lysogeny module and the DNA packaging regions are the most highly conserved. Furthermore, single phage gene as well as multiple phage gene-based phylogenetic analyses clearly revealed the chimeric make-up of the genomes of bifidoprophages. Keywords Prophages Bifidobacterium Genomes Modular organization
Electronic supplementary material The online version of this article (doi:10.1007/s10482-010-9426-4) contains supplementary material, which is available to authorized users. M. Ventura (&) F. Turroni E. Foroni S. Duranti V. Giubellini F. Bottacini Department of Genetics, Biology of Microorganisms, Anthropology and Evolution, University of Parma, Parco Area delle Scienze 11a, 43100 Parma, Italy e-mail:
[email protected] F. Bottacini D. van Sinderen Alimentary Pharmabiotic Centre and Department of Microbiology, Bioscience Institute, National University of Ireland, Western Road, Cork, Ireland
Introduction Due to their commercial importance as health-promoting microorganisms, bifidobacteria have become the focus of substantial research efforts which made them one of the most intensely investigated bacterial groups that are present in the human gastro intestinal tract (GIT) (Ventura et al. 2009a). Bifidobacterial genome-based analyses have revealed the presence of prophage sequences, which constitute up to 3% of the total pangenome of bifidobacteria (Ventura et al. 2005, 2009b). Such findings have profoundly changed our knowledge on bacteriophage distribution in these bacteria, which for a long time were considered to be free from phage attacks (Canchaya et al. 2003). Bifidobacteria belong to the Actinobacteria phylum, within which they form the deepest taxonomic branch (Ventura et al. 2007). The natural ecological niche for many bifidobacterial species is represented by the large intestine, where, particularly in infants, they reach high cell densities and are thus considered to be a very important bacterial commensal (Turroni et al. 2009a, b). Although prophage-like elements appear to be rare in actinobacterial genomes, a relatively large number of such bacteriophages, also termed actinophages, are known to infect various Actinobacteria, including Streptomyces bacteriophage phi31 (Smith et al. 1999) and Mycobacterium phage L5 or Bxb1 (Hatfull et al. 2006; Mediavilla et al. 2000; Pena et al. 1997). The proportion of the mobilome that corresponds to prophages sequences is under-represented in
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actinobacterial genomes; in fact only the genome of Corynebacterium diphtheriae, Propionibacterium acnes and Mycobacterium tuberculosis contain prophage-like elements, whereas the other so far sequenced genomes of Streptomyces ssp., Nocardia ssp., Leifsonia, Frankia, Thermobifida and Tropheryma do not contain any prophage like-elements (Ventura et al. 2007). Interestingly, the genome sequences of actinophages are clearly mosaic in nature (Pedulla et al. 2003), with regions of obvious sequence similarity interspersed with segments that appear to be unrelated, suggesting that extensive horizontal genetic exchange (or shuffling) among bacteriophages is common (Hendrix et al. 1999). So far, no complete phage particles infecting Bifidobacterium species have been identified. However, prophages have previously been described in several bifidobacterial genomes (Ventura et al. 2005, 2009b), which represents a clear sign of the existence of bifidobacteriophages. Only four out of the 22 so far identified so-called bifidoprophages (Blj-1, Bent-2, Binf-4 and Ban-1) were shown to be inducible (Ventura et al. 2005, 2009b), although this may be due to technical difficulties (e.g., specific growth condition requirements of the host) that so far prevented the isolation of phage particles from bifidobacteria. In the present report, we have expanded our genomic knowledge on bifidobacterial prophages through the genomic analyses of 22 bifidoprophages, which clearly revealed a modular organization and a highly mosaic genome structure, suggesting that bifidoprophages are chimeric in nature.
Results Identification of prophage sequences in bifidobacterial genomes As previously described (Ventura et al. 2009b), the screening of bifidobacterial genome sequences for prophages, using genes encoding integrases and/or cI-type repressors as identification markers, allowed the identification of nineteen prophages. These presumptive prophages, together with three previously described prophages (Ventura et al. 2005), represent our database of presumed bifidoprophages. The genomic features of the identified bifidoprophages are described in Table 1 and in the Supplementary
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data. Notably, many of the investigated bifidobacterial genomes were shown to be polylysogenic, i.e. B. dentium Bd1, B. dentium ATCC 27678, B. longum subsp. infantis ATCC 15697, B. longum subsp. infantis CCUG 52486, B. bifidum 317B, B. bifidum NCIMB 41171 and B. adolescentis L2-32 (Ventura et al. 2009b). Only the genome of B. breve UCC2003, B. longum subsp. longum DJO10A, B. longum subsp. longum NCC2705 and B. animalis subsp. lactis AD011 genomes contain a single prophage (Ventura et al. 2005). In addition, certain bifidobacterial genomes do not appear to harbour any prophage-like sequences, i.e., B. adolescentis ATCC15703, Bifidobacterium angulatum ATCC 27535, Bifidobacterium gallicum LMG 11596 and Bifidobacterium asteroides LMG 10735 (NCBI source and unpublished data). Genomic structure of bifidoprophages Database matches allowed a tentative subdivision of these prophage-like elements into functional modules, including genetic modules that encode functions involved in lysogeny, DNA replication, DNA packaging, head-to tail joining, tail morphogenesis, and host lysis (Botstein 1980). Based on these analyses it appears that nine prophage sequences are genomically complete or at least near-complete (i.e. Binf-1, Binf-4, Binf-5, Bbif-1, Bbif-2, Bcat-1, Blj-1, Bdent-1, and Bdent-3), that ten bifidoprophages genomes are partially complete (Bdent-2, Bdent-4, Binf-2, Binf-3, Binf-6, Binf-7, Bado-1, Bado-2, Bado-3 and Bl-1), while four bifidoprophages are very clearly incomplete and thus considered to represent phage remnants (Bbr-1, Binf-remn-3, Binf-3, and Ban-1). Genomic based analyses of the most complete prophages clearly indicated similarities to many Siphoviridae phages; however, in order to assign these prophages to a specific phage family, it will be necessary to determine the morphology of the corresponding phage particles. Previous bioinformatic analyses of the prophage genomes allowed the identification of genomic modules based on similarities at protein level with corresponding modules of phages infecting both Actinobacteria (e.g., mycobacteriophages) as well as Firmicutes (e.g., phages infecting Lactococcus lactis and other lactic acid bacteria) (Ventura et al. 2009b). Based on the identified genomic modules, a consensus bifidoprophage genome was defined
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Table 1 Genomic features of bifidoprophages Prophages
Host
Size (bp)
Genome organization
Lysogenymodule
DNA replicationmodule
DNA packagingmodule
Tail module
Host lysismodule
Bdent-1
B. dentium Bd1
29558
Complete
Yes
Yes
Yes
Yes
Yes
16503
Incomplete
Yes
No
Yes
No
No
29498
Complete
Yes
Yes
Yes
Yes
Yes
16858
Incomplete
Yes
No
Yes
No
No
42372
Complete
Yes
Yes
Yes
Yes
Yes
30701
Incomplete
Yes
Yes
No
No
No
28089
Incomplete
No
No
Yes
Yes
Yes
Bdent-2 Bdent-3
B. dentium ATCC 27678
Bdent-4 Binf-1 Binf-2
B. longum subsp. infantis ATCC 15697
Binf-3 Binf-4
39276
Complete
Yes
Yes
Yes
Yes
Yes
Binf-remn-3
4185
Incomplete
No
No
Yes
No
Yes
B. longum subsp. infantis CCUG 52486
35459
Complete
Yes
Yes
Yes
Yes
Yes
32913 12508
Incomplete Incomplete
Yes Yes
Yes No
Yes Yes
Yes Yes
No Yes
Bbif-1
B. bifidum 317B
45252
Complete
Yes
Yes
Yes
Yes
Yes
Bbif-2
B. bifidum NCIMB 41171
40136
Complete
Yes
Yes
Yes
Yes
Yes
Bado-1
B. adolescentis L2-32
Binf-5 Binf-6 Binf-7
41551
Incomplete
Yes
No
Yes
Yes
Yes
Bado-2
25889
Incomplete
Yes
No
Yes
Yes
Yes
Bado-3
29951
Incomplete
Yes
No
Yes
Yes
Yes
12415
Incomplete
Yes
Yes
Yes
No
No
Ban-1
B. animalis subsp. lactis AD011
Bcat-1
B. catenulatum DSM 16992
40265
Complete
Yes
Yes
Yes
Yes
Yes
Bbr-1
B. breve UCC2003
28153
Incomplete
Yes
No
Yes
No
Yes
Blj-1
B. longum subsp. longum DJO10A
37284
Complete
Yes
Yes
Yes
Yes
Yes
Bl-1
B. longum subsp. longum NCC2705
17463
Incomplete
Yes
No
Yes
Yes
Yes
(Fig. 1a). Notably, the most conserved modules are represented by the lysogeny and the DNA packaging-module, in which a consistent level of conservation with respect to gene composition was noticed. In contrast, the most variable genome module in bifidoprophages is represented by the DNA replication region (Fig. 1a). In the following sections we will discuss the identified module contents of the investigated bifidoprophages in more detail. Lysogeny module In temperate phages the genes necessary for the establishment and maintenance of the lysogenic state are generally organized into a compact lysogeny module such as in streptococcal phages (Lucchini et al. 1999b) and BPs mycobacteriophage (Sampson
et al. 2009), whereas in other temperate phages [e.g., lambda or in L5 mycobacteriophage (Hatfull and Sarkis 1993)], the integrase-encoding gene does not cluster with the genes that specify the lysogenic switch. The lysogeny module generally encompasses genes encoding an integrases, a superinfection exclusion protein, a cI-type repressor, a Cro-type protein, and an anti-repressor (Lucchini et al. 1999a; McGrath et al. 2002), and is arranged as two oppositely oriented sets of genes located at one end of the integrated prophage genome (Canchaya et al. 2003). However, except for the Binf-5 genome, whose lysogeny module does indeed resemble this expected composition and organization (Fig. 2), none of the other analyzed bifidobacterial phages display the genetic features of this classical lysogeny module, although they do exhibit certain characteristics typical of this module, such as a gene encoding an integrase belonging to the
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Fig. 1 Schematic representation of the genome organization of bifidoprophages (a) and their integrase relatedness (b). In a, the different colour shadings represent the level of conservation of
the different genes within each module as described in the figure’s inset. In b, the different bifidoprophages groups are indicated (Color figure online)
tyrosine integrase family (Groth and Calos 2004). Notably, the alignment of all identified bifidoprophage-encoded integrases revealed the existence of
three integrase groups, designated I, II and III. Phylogenetic tree analyses showed that group I and Cyanobacteria phage integrases are each others
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2407
2434
43
2447
2467
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Lysin Holin
1099
Tail
91%
1116
Protease
Capsid
Ter.
Portal
83% 1128
1149
83%
Protease
88% 1140
77%
Primase
Rus A
Ant.
Int.
cI Cro
Binf-5
Tail
Ter.
1528
Major head
Portal
Ter L
Rus A
1497
1490
Cro
Ant.
Binf-1
Antitoxin
Int.
44% 1572
1567
Ant.
Ant.
Methyl trans.
1558
Transcr. reg.
SSB
1548
cI cro
RecE
Binf-2
47%
87%
75%
100%
44% 99%
51%
1832
Tail Fiber
Tail
Major Tail
Tail
66%
1811
1780
50%
1791
41%
Head prot.
Portal
Ter L
Binf-3
Int.
Minor tail
1656
51%
Binf-rmn3 Endonuclease HNH
Ter.
capsid
Major
Minor tail
Lysin
Int.
Holin
Binf-7
Minor tail
Major capsid 577
Scaffold prot.
Ter. 581
Primase Reductase
Module killer Transcr. reg. Endonuclease
Repl. prot.
Oligori.nuclease
599
597
Sie
Binf-6
34%
TerS TerL
1073
Transcr. reg.
Minor tail
Major tail
1090
1078
80%
Major head
Portal
Ter L
t-RNA Trp 95%
1097
93%
Oligori.nuclease
79%
1120
1133
93%
1137
96%
Par B
97%
Repl. prot.
SSB
Binf-4
Fig. 2 Comparative genome maps of the B. longum subsp. infantis ATCC15697 and B. longum subsp. infantis CCUG52486 prophage-like elements Binf-1, Binf-2, Binf-3, Binf-4, Binf-5, Binf-6, Binf-7 and Binf-rmn-3. Similar genes are linked by shading with the amino acid similarity given in percentage. Predicted functions of encoded proteins identified are indicated. The modular structure of the genomes is indicated by different
patterns, which indicates their predicted function (black: lysogeny module; dark grey: DNA replication; pale grey: DNA packaging and head; diagonal stripes: tail and tail fiber; vertical stripes: lysis module; open arrows: hypothetical protein; open arrows with thick lines: similar to bacterial protein; vertical lines: tRNA genes) (Color figure online)
closest relatives, while bifidoprophage integrases from group II and group III share similarities with many phages infecting Myxococcus, Firmicutes and Actinobacteria (Fig. 1b). Notably, group II and III bifidoprophage integrases represent two clearly distinguishable subclusters from their closest related phage integrase (e.g., derived from phages that infect Myxococcus, Firmicutes and Actinobacteria). Furthermore, such a cluster distribution provides evidence that, from an evolutionary perspective, the group II and III bifidobacterial integrases seem to have only recently diverged from a common ancestor.
The other genes identified in the lysogeny module of bifidobacteriophages (where present, e.g., cI, cro, sie and ant) display limited or no similarity among each other. These findings suggest that the lysogeny module of bifidobacteriophages, except for the integrase gene, has been subject to a very high level of genetic exchange with other bacteriophages during their evolutionary development. Notably, for seven bifidoprophages (e.g., Binf-7, Bado-1, Bado-2, Bado3, bbr-1, Bl-1, Blj-1), the integrase gene is placed next to the holin gene, being completely separated from the cI-type-encoding repressor gene, thereby
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normally found at either genome end of prophages infecting Firmicutes (near the attL and attR sites) (Canchaya et al. 2003). 0919
0922
0959
0979
representing an unusual and unique genetic organization of the lysogeny module in these bifidophages. In fact, phage integrase and lysis cassette are
Holin Lysin Transc. reg.
Tail fiber
Tail tape
Major head Head fiber
Portal
Primase Ter S
Trans. reg.
Oligori.nuclease
98% 100%
1273
100%
1254
ParB
SSB 98%
1326
97%
1304
100%
Ant
Cro
Int
Bbif-1
Bbif-2
20000
30000
are indicated by colours (see legend of Fig. 2 for an explanation). The degree of amino acid identity is indicated by colour shading (Color figure online)
1153
40000
1174
1207 Primosomal protein
30000
1191
1233 Int
20000
Lysin Holin
10000
1165
Fig. 3 Alignment of the B. bifidum 317B and B. bifidum NCIMB 41171 prophage-like elements Bbif-1 and Bbif-2 prophages. The modular structure and function of the genomes
40000
Tail
10000
10000
5280
8000
6000
5290
5315
Major tail
DNA helicase
4000
5350
2000
Phage major capsid
Endonuclease
TerL
Bcat-1
Fig. 4 Genome maps of the B. catenulatum DSM16992 prophage-like element Bcat-1 and B. animalis subsp. lactis AD011 prophage-like elements Ban-1. The modular structure
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Prohead-protease
DNA helicase
cI
Int
Ban-1
and function of the genomes is indicated by colours (see legend of Fig. 2 for an explanation) (Color figure online)
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917
916
Ter
t-RNA Ser t-RNA Trp t-RNA Leu
956
The putative head morphogenesis module in bifidoprophages encompasses a variable number of genes. For example, the bifidoprophage Bado-2 displays an apparently complete head morphogenesis region, which encompasses, among others, a set of genes showing strong sequence similarity to a ClpP protease, a portal protein, a large subunit terminase protein and the major head protein (Figs. 2 and 5). The attribution of the ClpP protease to the serine proteases family is also supported by multiple alignments. When compared with Clp protease family members of bacteria, plant and animal origin, the bifidoprophage-derived ClpP proteins (e.g., Bado-2, Binf-5, Ban-1, Bdent-2 and Bdent-4) share extensive sequence similarities. Phage-encoded ClpP proteins have previously been described in several phages infecting Firmicutes such as streptococcal and lactococcal phages (Desiere et al. 1999). Notably,
Holin Lysin
The lysogeny module of a number of bifidoprophages (Binf-1, Binf-2, Binf-4, Binf-5, Binf-6, Bbif-1, Bbif-2, Bcat-1, Ban-1, Bdent-1, Bdent-3, Bbr-1, Bl-1, Blj-1) is flanked by a disparate set of genes specifying proteins that show bioinformatic links to predicted DNA replication functions including a helicase, a single-stranded DNA binding protein, a primase, an oligoribonuclease and a RusA protein (DNA structure-specific endonuclease that resolves Holliday junction intermediates formed during DNA replication). Apparently, all three bifidoprophages identified in the genome of B. adolescentis L32-22 (Bado-1 to Bado-3), as well as the B. dentium Bdent-1 and Bent3 phages lack such a DNA replication module (Figs. 3 and 4). In contrast, bifidoprophages Binf-6 and the identified B. bifidum prophages contain the most complete DNA replication module with respect to their predicted functional content (Figs. 2 and 3).
958
Head morphogenesis genome module
Int
DNA replication genome module
21%
Portal
Minor head
Major head
Major Tail
Tail fiber
RM/ M
RM/ M
Bado-1
62%
55%
42%
1214
1197
1188
1182
55%
HNH endonuclease
Ter
Portal
Major head
1404
1370
52%
Prohead protease
Clp Protease
Lysin
Bado-2
10000
Fig. 5 Comparative genome maps of the B. adolescentis L232 prophage-like elements Bado-1, Bado-2 and Bado-3. Genes sharing similarity are linked by shading. Probable functions of
20000
30000
Ter
t-RNA-Ser
Portal
Major head
Trasc. reg.
Rev transcriptase
t-RNA-Met Int cI Holin Lysin
Bado-3
40000
encoded proteins identified by bioinformatic analysis are noted. The degree of amino acid identity is indicated by shading as described in the legend of Fig. 2
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relatedness for phage classification purposes (Desiere et al. 1999). When we applied such an approach to the bifidoprophages’ terminase proteins, three clusters were identified, which are different to those identified by dendrogram-based integrase proteins (data not shown).
The genome organization of the putative head-to-tail joining and tail genes in bifidoprophages is well conserved in temperate streptococcal pac-site as well as cos-site and resembles that of phage k (Ackermann 1998). The genomes of almost all bifidoprophages contained two adjacent genes encoding a putative tail-tape measure protein and a tail fiber protein (Figs. 2, 3, 4, 5, 6). Sequence comparison of the tail fiber proteins revealed the existence of two highly conserved domains separated by a variable C-terminal domain, with the first domain containing collagen-like repeats, which may be involved in host recognition (Pfam protein domain family code
BDP 1477
BDP 1461 Portal
Tail morphogenesis region
BDP 1472
BDP 1460 TerL
phylogenetic ClpP-based analyses show close evolutionary relationships between the Bado-2-encoded ClpP protease and those specified by Burkholderia phages (data not shown). The gene organization of the head morphogenesis module also provides information regarding the DNA packaging strategy used by the phage (Casjens 2005). Thus, the finding that the Bado-2, Binf-5, Ban-1, Bdent-2 and Bdent-4 prophages contain a ClpP protease-enoding gene in the putative head morphogenesis module suggests that these bifidoprophages belong to the pac-site phages, which package their DNA using a terminal redundancy mechanism (Sternberg and Coulby 1987). In phage lambda DNA packaging is performed by the phage terminase (Brussow 2001; Hendrix et al. 1984). The terminase consists of a small and a large subunit that mediate the interaction between the preformed prohead of the phage and the bacteriophage DNA during genome packaging (Brussow 2001). A phylogenetic tree, based on the terminase and portal proteins, is commonly used to define patterns of
Xis
Methylase
Lysin
Tail-host specificity protein
BDG 20
BDG 30
BDG 38
BDG 42
BDG 27
100%
BDG 50
99% BDG 52
Tape mesaure prot.
Major tail
Major head
Int
DNA helicase
Bdent1
BDP 576
BDP 579
BDP 589
BDP 601
BDP 602
Bdent3
cI
TerS Xis
BDG 556
99% BDG 549
BDG 547
BDG 540
TerL
Portal
99%
100%
100% BDG 534
Prohead protease
Minor head
Int
Met-tRNA Met-tRNA
Bdent2
Bdent4
Fig. 6 Genetic maps of the B. dentium Bd1 and B. dentium ATCC 27678 prophage-like elements Bdent-1, Bdent-2, Bdent3 and Bdent-4. Genes sharing similarity are linked by shading.
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Probable functions of encoded proteins identified by bioinformatics analysis are noted. The degree of amino acid similarity is indicated by shading as described in the legend of Fig. 2
Antonie van Leeuwenhoek (2010) 98:39–50
PF05737). Interestingly, the receptor-recognizing protein in T4 phages also consists of hypervariable regions separated by conserved domains, containing oligoglycine stretches, which are considered to be involved in host-recognition in T4 phages (Tetart et al. 1998).
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Lysis cassette Many different types of lytic enzymes have been described in phages, i.e. muramidases, for example those identified in Lactococcus phages (e.g., Tuc2009 and LC-3), amidases, such as those identified in
Fig. 7 Supertree computed from the concatenation of integrase and portal protein sequences encoded by the genomes of bifidoprophages plus other phages belonging to representative groups. Bifidoprophages are highlighted
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Streptococcus (Gasson 1996), and mycolylarabinogalactan esterases, found in mycobacteriophages (Payne et al. 2009). The alignment of the lysin-encoding gene, as identified on the genomes of various bifidophages, revealed a typical two-domain structure, represented by an N-terminal domain that contains the cell walldegrading activity and a C-terminal domain specifying the substrate-binding activity. Furthermore, in a large proportion of the investigated bifidoprophages (Figs. 2, 3, 4, 5, 6), the lysin gene is preceded by a gene encoding a putative holin, which contains two predicted transmembrane domains. Chimeric prophages Tree analyses that were performed for selected phage proteins clearly suggest that bifidoprophage genomes are genetic chimers. For example, an integrase-based tree shows a different evolutionary pattern of bifidoprophage as compared to a phylogenetic tree based on particular proteins of the head morphogenesis module (e.g., portal protein, major head protein and ClpP protease). Choosing the phage head morphogenesis module to analyse the evolutionary development of bifidoprophages was inspired by the presumption that this module is the most ancient phage module and thus a valuable candidate for an evolutionary analysis (Desiere et al. 1999). In order to investigate bifidoprophage phylogeny a multigene approach was also considered. The concatenation of genes has previously been shown to be very useful in order to infer phylogeny in bacteria (Teichmann and Mitchison 1999). In the current study the phylogenetic tree that was generated using the concatenated sequences of the various integrase and portal protein sequences is presented in Fig. 7. Notably, two phylogenetic groups were detected; the larger is well resolved and separated from all other clusters, which include the concatenated sequences from so far classified/isolated bacteriophages. Such findings suggest that the bifidoprophages described here represent a novel phage lineage. However, phylogenetic tree analyses of complete phage proteins or protein sets must be interpreted with extreme caution. For example, in multi-domain phage proteins an individual protein may harbour domains that belong to different evolutionary lineages, as described for the anti-repressor proteins in Streptococcus phages (Lucchini et al. 1999a).
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Conclusions This study provides an extensive analysis of the genome organization of prophage-like sequences in bifidobacteria. Bifidobacteria were previously considered to be free from phage infection (Canchaya et al. 2003). However, this and previous genome analyses (Ventura et al. 2005, 2009a) have clearly demonstrated the presence of prophage-like sequences that display a classical modular organization of their genomes. Sequence matches as well as phylogenetic investigations have suggested that bifidoprophage genomes display similarities with other phages, which represent a broad phylogenetic range of host bacteria (Ventura et al. 2005, 2009a). Here, we confirmed these results through phylogeny-based analyses involving genes that are considered to be canonical molecular markers to investigate bacteriophage phylogeny (Bruttin et al. 1997; Desiere et al. 2001). Furthermore, genome analyses of bifidoprophages, including those that display an incomplete genome organization, revealed the existence of putative lysogenic conversion genes, which do not encompass virulence factors but genes whose products might enhance the ecological fitness of the lysogens (e.g. by allowing a more efficient colonization of the human intestine by the bacterial host) (Ventura et al. 2009b). Additional Bifidobacterium genome sequencing projects are currently in progress, and undoubtedly these efforts will identify new prophage sequences. This increased resource of bifidobacterial phage sequences will expand our ability to provide answers to questions such as those concerning horizontal versus vertical DNA transfer within different species of Bifidobacterium. Altogether, our results reveal an extensive interaction between bifidobacteria and phages, substantiate the notion that (pro)phages drive the co-evolution of both phage and host genomes and furthermore emphasize that the bifidobacterial population and diversity, as an important representative of the gut microbiota, may fluctuate due to bacteriophage-mediated extermination. Acknowledgments This material is based upon works supported by a Principal Investigator Grant (to DvS, grant number 08/IN.1/B1909) and the Alimentary Pharmabiotic Centre, a Centre for Science and Technology, both funded by Science Foundation Ireland (SFI) through the Irish Government’s National Development Plan, by the Italian
Antonie van Leeuwenhoek (2010) 98:39–50 Award for Outstanding Young Researcher scheme ‘‘Incentivazione alla mobilita’ di studiosi stranieri e italiani residenti all’estero’’ and to the Marie Curie Reintegration Grant (MERG-CT-2005-03080) to MV and by an IRCSET Embark postgraduate fellowship to F.B. The project described was partially supported by NIH-NIGMS T32-GM08799 (DAS). Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIGMS or NIH.
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