Article

Comparative (Meta)genomic Analysis and Ecological Profiling of Human Gut-Specific Bacteriophage φB124-14

Centre for Biomedical and Health Science Research, School of Pharmacy and Biomolecular Sciences, University of Brighton, Brighton, United Kingdom.
PLoS ONE (Impact Factor: 3.23). 04/2012; 7(4):e35053. DOI: 10.1371/journal.pone.0035053
Source: PubMed

ABSTRACT

Bacteriophage associated with the human gut microbiome are likely to have an important impact on community structure and function, and provide a wealth of biotechnological opportunities. Despite this, knowledge of the ecology and composition of bacteriophage in the gut bacterial community remains poor, with few well characterized gut-associated phage genomes currently available. Here we describe the identification and in-depth (meta)genomic, proteomic, and ecological analysis of a human gut-specific bacteriophage (designated φB124-14). In doing so we illuminate a fraction of the biological dark matter extant in this ecosystem and its surrounding eco-genomic landscape, identifying a novel and uncharted bacteriophage gene-space in this community. φB124-14 infects only a subset of closely related gut-associated Bacteroides fragilis strains, and the circular genome encodes functions previously found to be rare in viral genomes and human gut viral metagenome sequences, including those which potentially confer advantages upon phage and/or host bacteria. Comparative genomic analyses revealed φB124-14 is most closely related to φB40-8, the only other publically available Bacteroides sp. phage genome, whilst comparative metagenomic analysis of both phage failed to identify any homologous sequences in 136 non-human gut metagenomic datasets searched, supporting the human gut-specific nature of this phage. Moreover, a potential geographic variation in the carriage of these and related phage was revealed by analysis of their distribution and prevalence within 151 human gut microbiomes and viromes from Europe, America and Japan. Finally, ecological profiling of φB124-14 and φB40-8, using both gene-centric alignment-driven phylogenetic analyses, as well as alignment-free gene-independent approaches was undertaken. This not only verified the human gut-specific nature of both phage, but also indicated that these phage populate a distinct and unexplored ecological landscape within the human gut microbiome.

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Available from: Jonathan L Caplin
Comparative (Meta)genomic Analysis and Ecological
Profiling of Human Gut-Specific Bacteriophage wB124-14
Lesley A. Ogilvie
1
, Jonathan Caplin
2
, Cinzia Dedi
1
, David Diston
, Elizabeth Cheek
3
, Lucas Bowler
4
,
Huw Taylor
2
, James Ebdon
2
, Brian V. Jones
1
*
1 Centre for Biomedical and Health Science Research, School of Pharmacy and Biomolecular Sciences, University of Brighton, Brighton, United Kingdom, 2 School of
Environment and Technology, University of Brighton, Brighton, United Kingdom, 3 School of Computing, Engineering and Mathematics, University of Brighton, Brighton,
United Kingdom, 4 Sussex Proteomics Centre, University of Sussex, Brighton, United Kingdom
Abstract
Bacteriophage associated with the human gut microbiome are likely to have an important impact on community structure
and function, and provide a wealth of biotechnological opportunities. Despite this, knowledge of the ecology and
composition of bacteriophage in the gut bacterial community remains poor, with few well characterized gut-associated
phage genomes currently available. Here we describe the identification and in-depth (meta)genomic, proteomic, and
ecological analysis of a human gut-specific bacteriophage (designated wB124-14). In doing so we illuminate a fraction of the
biological dark matter extant in this ecosystem and its surrounding eco-genomic landscape, identifying a novel and
uncharted bacteriophage gene-space in this community. wB124-14 infects only a subset of closely related gut-associated
Bacteroides fragilis strains, and the circular genome encodes functions previously found to be rare in viral genomes and
human gut viral metagenome sequences, including those which potentially confer advantages upon phage and/or host
bacteria. Comparative genomic analyses revealed wB124-14 is most closely related to wB40-8, the only other publically
available Bacteroides sp. phage genome, whilst comparative metagenomic analysis of both phage failed to identify any
homologous sequences in 136 non-human gut metagenomic datasets searched, supporting the human gut-specific nature
of this phage. Moreover, a potential geographic variation in the carriage of these and related phage was revealed by
analysis of their distribution and prevalence within 151 human gut microbiomes and viromes from Europe, America and
Japan. Finally, ecological profiling of wB124-14 and wB40-8, using both gene-centric alignment-driven phylogenetic
analyses, as well as alignment-free gene-independent approaches was undertaken. This not only verified the human gut-
specific nature of both phage, but also indicated that these phage populate a distinct and unexplored ecological landscape
within the human gut microbiome.
Citation: Ogilvie LA, Caplin J, Dedi C, Diston D, Cheek E, et al. (2012) Comparative (Meta)genomic Analysis and Ecological Profiling of Human Gut-Spec ific
Bacteriophage wB124-14. PLoS ONE 7(4): e35053. doi:10.1371/journal.pone.0035053
Editor: Jonathan H. Badger, J. Craig Venter Institute, United States of America
Received November 23, 2011; Accepted March 8, 2012; Published April 25, 2012
Copyright: ß 2012 Ogilvie et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: Dr. Lesley Ogilvie is supported by funding from the Medical Research Council (Grant ID number 93344/G0901553 awarded to Dr. B. V. Jones). Research
in the laboratory of Dr. B. V. Jones is also supported by funding from the Hospital Infection Society, The Royal Society, The Society for Applied Microbiology, The
University of Brighton, and the European Union (FP7 Marie Curie IAPP scheme). The funders had no role in study design, data collection and analysis, decision to
publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: B.V.Jones@Brighton.ac.uk
¤ Current address: Bundesamt fu
¨
r Gesundheit, Ko
¨
niz, Bern, Switzerland
Introduction
The human gut harbours a diverse microbial community which
in turn plays host to a variety of mobile genetic elements (MGE)
and bacteriophages, forming the gut mobile metagenome [1–4].
The role of this flexible gene pool in the development and
functioning of the gut microbial community remains largely
unexplored, yet there is emerging evidence that this mobile
metagenome reflects the co-evolution of host and microbe in this
community, and that some MGE may be unique to or enriched
within this ecosystem [1,4–8].
Identification and characterization of such elements will provide
much insight into fundamental aspects of development and
functioning of the gut microbiota, and provide the raw material
for the development of novel molecular tools. Furthermore, MGE
comprising the human gut mobile metagenome are also likely to
encode a range of functions of biotechnological or pharmaceutical
interest [9]. Bacteriophages in particular have the potential to
influence community structure and function [10–15], and are
regarded to be of considerable biotechnological value, exemplified
by the growing interest in their use as novel and highly selective
therapeutic agents (for review see [16]). Initial studies of the gut
virome have already provided evidence of distinct viral population
dynamics and gene content in this ecosystem, with a dominance of
apparently temperate phage and a relative lack of the predator-
prey phage-host relationship commonly observed in other
microbial communities [6].
Through selective elimination of species within the gut
microbiota, phage may alter community function, metabolic
output and subsequently impact on host health [17–19].
Furthermore, there is also scope for the direct interaction of
bacteriophage with the host immune system, which may be
important in the pathogenesis of some gut related disorders [17].
The observation that dense bacteriophage populations are
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associated with the gut mucosa, and numbers are elevated in
patients with Crohn’s disease, emphasizes the possible role of these
bacterial viruses in community function, interaction with the host,
and disease pathogenesis [19].
The characterisation of bacteriophage specific to the human gut
is also of considerable interest for the development of microbial
source tracking tools (MST), which permit determination of faecal
source in surface and ground waters [20–24]. Faecal contamina-
tion of surface waters poses a major risk to public health, and
bacteriophages specific to human faecal indicator bacteria (and the
human gut microbiome) have already been successfully employed
as water quality indicators that can specifically identify pollution
originating from human sources [20–22,25,26]. Bacteriophage
offer numerous advantages in these applications and are not only
thought to persist longer in the environment than host bacteria but
can often be found in higher numbers making them a more
sensitive source tracking tool [22]. In particular, the development
of rapid and sensitive culture-independent methods for detection
of human faecal indicator phage, directly in environmental
samples, offers significant advantages over classic culture-based
approaches, and there is presently much interest in developing and
implementing such strategies [27].
However, the development of culture-independent phage-based
MST tools, along with our improved understanding of bacterio-
phage in the gut community, is hindered by the lack of well-
characterized bacteriophage with defined host-ranges and avail-
able genome sequences, which infect prominent and important
species of human gut bacteria. A prime example is the availability
of only one complete Bacteroides spp. phage genome sequence in
public databases (as of Oct 2011), despite the prominence and
importance of this group of bacteria in the human gut microbiome
[28,29].
We have previously isolated bacteriophage infecting the human
faecal indicator bacteria Bacteroides sp. GB-124 from municipal
wastewaters, and found these to be present in human faecal
samples, but absent from faecal samples derived from a wide range
of domestic and wild animals, as well as from the general
environment, strongly suggesting these phage are human gut-
specific [20]. Because of the apparent gut-specific nature of these
phage, and the growing evidence of their usefulness as MST, in-
depth genomic characterization would not only begin to address
the current lack of knowledge regarding gut-associated bacterio-
phage (and Bacteroides phage in particular), but would also provide
the genetic information required for development of culture-
independent MST.
This motivated us to undertake an in-depth characterization of
one such phage designated wB124-14. This phage was selected as
it not only appears to be representative of a morphologically and
phenotypically homogenous group of human-specific phages, but
also displayed greater environmental stability than other phage
tested (particularly in terms of UV resistance), suggesting an
excellent environmental ‘‘half-life’’ (D. Diston Jan 2010, pers.
comm.). Here we have characterized the host range, complete
genome sequence and proteome of wB124-14. Using comparative
metagenomic analysis and genome signature-based approaches we
subsequently examined its ecological profile in relation to 611
other bacteriophage genomes available on GenBank, as well as
human gut-specific viral metagenomes [6].
Overall, these investigations support the human gut specific
nature of wB124-14 and indicate that this phage occupies a distinct
and largely unexplored ecological landscape within the human gut
microbiome. We also increase the available number of well-
characterized genomes of bacteriophage infecting prominent
members of the human gut microbiota. This will not only enhance
our fundamental understanding of this important microbial
ecosystem, but will facilitate the development of sensitive and
rapid culture-independent MST tools.
Results and Discussion
wB124-14 physical characteristics and host range
Transmission Electron Microscopy (TEM) shows wB124-14 has
a binary morphology with an icosahedral head and a non-
contractile tail (Figure 1A), placing it in the Caudoviriale order,
Siphoviridae family [30]. The phage produces small (0.7 mm 60.3),
clear plaques on a lawn of the original host Bacteroides sp. GB-124.
Structural dimensions are similar to the B. fragilis faecal pollution
indicator phage B40-8 (wB40-8; also referred to as phage ATCC
51477-B1; GenBank accession no. FJ008913.1) [31], with tail
length of 162 nm 621, tail diameter of 13.6 nm 61.6, and a
slightly smaller head diameter measurement of 49.8 nm 63.9
(versus wB40-8 measurements of 60 6 4.0 nm). The morphology
of the wB124-14 capsid is in keeping with metagenomic surveys of
human gut bacteriophage, in which the majority of identifiable
viruses were Siphophages [10].
Previous studies indicated that the host bacterium, Bacteroides sp.
GB-124, was most closely related to B. ovatus based on comparison
of 16S rRNA gene sequences (96% identity; [26]). However, since
97% identity between 16S genes is typically used as the cut off for
species level identification [32], and in light of the recent release of
many additional Bacteroides genome sequences from human gut
isolates (as part of the human microbiome project), we investigated
the identity of Bacteroides sp. GB-124 in more detail. This new
analysis revealed GB-124 to be a strain of B. fragilis (designated
here B. fragilis GB-124), with 16S rRNA gene sequences exhibiting
99% identity to a number of other B. fragilis strains, including those
isolated from the same municipal wastewater site as well as strains
HSP40, 683R, YCH46, JCM 17586 and JCM 17587 isolated
from various human body sites and human faeces (Table S1,
Figure 1B; accession numbers given on figure).
Investigation of the ability of wB124-14 to infect and lyse a
range of Bacteroides spp. commonly associated with the human gut
microbiota, demonstrated that this phage has a highly restricted
host range. wB124-14 was capable of infecting only a subset of B.
fragilis strains isolated from the same municipal wastewater site and
B. fragilis strain DSM 1396 (Figure 1B; Table S1), originally
isolated from human pleural fluid [33]. No activity was observed
against other Bacteroides spp. tested, or against strains of B. fragilis
isolated from geographically distinct municipal wastewaters,
namely, Galicia, Spain [20] and Hawaii, USA [34] (Figure 1B;
Table S1).
Overall, these observations indicate that Bacteroides spp. within
the human gut microbiota play host to bacteriophage with
extremely narrow host ranges, and in at least some cases these are
restricted to closely related strains. Such narrow host range may be
the result of the extreme niche specialization thought to occur at
short phylogenetic distances in gut bacteria [7], likely resulting in
strain-to-strain variation in surface proteins or other structures
exploited by phage as receptors. In this regard, it is notable that
horizontal gene transfer mediated by phage and other mobile
elements [35], as well as the selective pressure imposed on host
bacteria by phage themselves [6,36,37] can all promote modifi-
cation of surface structures and contribute to strain diversification.
In the case of surface structures, since these are often key to host-
microbe interactions, and may include those that are important to
nutrient acquisition and competition between strains, such
diversification also has the potential to influence the interaction
of bacteria with the human host [35]. Phage with such restricted
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host ranges are also unlikely to produce a significant impact on
overall microbial community structure and functioning due to
functional redundancy among members of the gut microbiome
[6,29].
Genome structure and sequence overvi. The dsDNA
genome of wB124-14 is 47,159 bp with an average G+C content
of 38.75%, and predicted to encode 68 open reading frames
(ORFs) with an average size of 212 aa. The genome exhibits the
high coding density typical of bacteriophage, with non-coding
sequence limited to 8.2% of the genome (Figure 2A). Restriction
fragment patterns are compatible with a circular genome
structure, and indicated that the wB124-14 is packaged as a
circular molecule (Figure 3), as has been described for
bacteriophages P2 and P4 [38,39].
Similar to the host bacterial species, B. fragilis, the majority of
putative ORFs detected are predicted to be initiated by an ATG
start codon, with one ORF (ORF10) presenting a CTG start
codon, and two initiated by GTG codons (ORF34, ORF44). A
number of ORF start and stop codons overlap (Table S2;
Figure 2A); a feature common to bacteriophage genomes, which
has been hypothesised to facilitate gene regulation and allow an
increased repertoire of proteins without a corresponding increase
in genome size [40,41]. Based on the protein BLAST algorithm
(BlastP; http://blast.ncbi.nlm.nih.gov/Blast.cgi) and proteomic
analysis, 18 of the predicted ORFs have an assignable function,
and 12 ORFs contain conserved domain signatures (Table S2;
Figure 2A). The majority of ORFs with an assigned function
encode proteins with predicted roles in DNA replication and
regulation, with the remainder predicted to encode functions
related to capsid structure, packaging, and host lysis (Table S2;
Figure 2A).
No function could be predicted for 50 ORFs which were all
designated as proteins of unknown function (Table S2;
Figure 2A,B). Of these, 29 were homologous to ORFs within the
wB40-8 genome or the genomes of Bacteroides spp., and a further 21
exhibited no significant homology to any sequences within the
public databases (Table S2; Figure 2A, B, C). This likely reflects
the general paucity of bacteriophage genome sequences within
public databases (only one other complete Bacteroides spp. phage
genome [31] is currently available in public databases, as of
October 2011), as well as the high level of uncharacterized
functions typically encoded by phage genomes [6,42].
Genome architecture and phage e ncoded functions
The clustering of functionally related genes and modular gene
architecture is a common feature of bacteriophage genomes.
Based on gene architecture, putative transcriptional coupling, and
the functional assignments of ORFs, the wB124-14 genome also
exhibits a modular organisation with functional gene clusters
related to packaging, capsid structure and assembly, as well as
DNA replication and regulation, and host lysis (Figure 2A,B;
Table S2). A comparable gene architecture and functional
clustering has also been described in wB40-8, which exhibits a
similar array of loosely defined gene modules containing high
levels of ORFs of unknown function [31]. However, many ORFs
assigned to particular modules in both wB40-8 and wB124-14
genomes cannot be assigned a specific function due to lack of
homology to any sequences in current databases, or any direct
experimental evidence.
Figure 1. Physical structure and host range of WB124-14. A. Transmission electron micrograph of WB124-14 showing phage capsid composed
of an icosahedral head and a non-contractile tail. Magnification 50,0006. Scale Bar, 20 nm. B. Phylogenetic characterisation of B. fragilis wB124-14
host strains. Consensus maximum likelihood trees were constructed from 16S rRNA gene sequences, with 1000 bootstrap resamplings using MEGA
v5. Bootstrap values of 40 or greater are shown adjacent to respective nodes. Accession numbers for bacterial 16S sequences are given in brackets
following species names on the tree. The ability of wB124-14 to replicate in a particular host species was tested in standard agar overlay assays, in
which replication of wB124-14 in a particular host was indicated by production of plaques in bacterial lawns. Species tested in host range assays are
denoted by open or filled circles. Filled red circles indicate strains which support wB124-14 replication, and open grey circles indicate strains in which
wB124 did not replicate.
doi:10.1371/journal.pone.0035053.g001
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Structure and packaging
The wB124-14 structure and packaging cluster potentially
comprises 24 ORFs, constituting 52% of the phage genome
(Figure 2A). Across this cluster seven ORFs could be assigned
putative functions based on sequence homologies and analysis of
the mature virion proteome (Table S2; Figures 2 and 4). ORFs 38,
41 and 42 are predicted to encode the main structural proteins
comprising the phage capsid, and exhibit high homology to
corresponding capsid proteins from wB40-8 at the amino acid level
(MP1 Major Capsid Protein 1, MP3 Major Capsid Protein 3,
and MP2 Major Protein 2, respectively; Table S2). ORF20
encodes a putative tail fibre protein, which is thought to be
involved in host recognition and phage attachment [43]. However,
with the exception of the predicted tail fibre protein, which
exhibits homology with other proteins annotated in Bacteroides spp.
genome sequences, capsid proteins of both phage (wB124-14
ORF38, 39, 41, 42 and corresponding wB40-8 ORFS; Table S2)
show no significant homology to any other sequences in BlastP
searches of the nr dataset (e = 0.03 or greater), and all lack
conserved domains found in other phage capsid proteins.
The presence of ORF38 (wB40-8 Major Capsid Protein 1
homologue), and ORF42 (wB40-8 Major Protein 2 homologue) in
the mature virion was confirmed by analysis of the phage
proteome by tandem mass spectroscopy (Figure 4). Proteomic
analyses also identified an additional protein within the structure
and packaging cluster (encoded by ORF39) as present within the
mature virion, confirming a role for this previously hypothetical
protein in capsid structure (Figures 2 and 4). In contrast, ORF41
and ORF20 (wB40-8 Major Capsid Protein 3 homologue and Tail
Fibre homologues, respectively) were not detected in mature
Figure 2. Architecture and characteristics of wB124-14 genome. A. Physical map of WB124-14 genome. Outer track: Position and
orientation of each predicted ORF. Block arrows represent individual ORFs and indicate direction of transcription. ORF colour indicates functional
assignment based on BlastP and conserved domain searches (minimum 20% identity, and an e-value of 1e
25
or lower), as well as analysis of the
phage proteome, as described in the figure legend. ORFs marked with purple triangles indicate ORF function has been confirmed through LC-MS/MS
analysis of the mature phage proteome (See Figure 4). Middle track: Bars represent location of proposed functional gene clusters and ORFs
belonging to each cluster. Colours of bars indicate putative role of each gene cluster in phage replication, based on predicted functions of member
ORFs. Inner track: G+C content: dark grey lines = above average genome G+C content; light grey lines = below average genome G+C content. B.
Percentage of ORFs assigned to each functional category, including unassigned ORFs. ORFs of unknown function are further broken down in a
secondary pie chart to illustrate those with homologues in the other available Bacteroides phage genome (wB40-8), and those with no significant
homology (nsh) to any sequences present in public databases encompassed by the nr dataset. C. Percentage of wB124-14 ORFs with highest
homology (based on top hits by bit score in BlastP searches) to sequences of various phylogenetic origin. Only hits generating e values of 1e
25
or
lower were considered significant in this analysis. nsh no significant homology. D. Illustrates the percentage of predicted ORFs assigned to each of
the three predicted functional modules in the wB124-14 genome.
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Figure 3. Physical structure of wB124-14 genome. Left and middle panels show in silico digest and electrophoresis to visualise restriction
fragment profiles of wB124-14 expected for each permutation of the genome (linear or circular) generated by pDRAW32. Right panel shows results
obtained from digestion of 1.5
mgofwB124-14 DNA (3 h at 37uC) with restriction enzymes used in in silico analysis. Restriction enzymes tested are
indicated above each lane. MW = 1 kb Molecular Weight marker (Promega). UC = uncut wB124-14 DNA.
doi:10.1371/journal.pone.0035053.g003
Figure 4. Analysis of the mature wB124-14 proteome. Spectra of wB124-14 proteins identified by tandem mass spectrometry. Example peptide
spectra for each of the three proteins identified are shown. Table provides protein coverage and associated number of unique peptides matched and
the sequence of the top four matches (ranked by by XCorr score).
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wB124-14 phage capsids, and their role in virus capsid structure
remains hypothetical.
The lack of identifiable products from ORF41 and ORF20 in
the proteome of mature wB124-14 virions may indicate that these
are present at relatively low levels that do not permit accurate
identification using the proteomic techniques employed here.
Although the theoretical sensitivity of the Orbitrap XL LC-MS
system utilised in this study implies proteins should be detected at
the low fmol range, subsets of comparatively rare proteins can be
‘‘masked’’ by the presence of highly abundant proteins in any
given sample [44]. Alternatively, ORF41 and ORF20 may be
non-functional in wB124-14 and could be dispensable for capsid
formation, or play only transient roles in capsid assembly,
potentially limited to aspects that occur in vivo in host cells.
In the case of ORF20, the assignment of this as a tail fibre
protein is based solely on its similarity to the homologous protein
annotated as a tail fibre in the wB40-8 genome sequence.
However, a closer examination of both wB124-14 and wB40-8
proteins revealed only low levels of homology to other tail fibre
proteins, with the highest homology observed with an Enterococcus
phage phiEF24C-P2 protein annotated as a tail fibre component
(32% identity, 10% query coverage, 6e
210
). The large size of
ORF20 is more typical of tail tape measure proteins, and given the
low homology to other tail fibre proteins, and the apparent
absence from the phage structural proteome, the possibility that
ORF20 encodes a tape measure protein rather than a tail fibre
should be noted. Nevertheless, in the absence of experimental
evidence demonstrating a specific function, the available genomic
data indicates wB124-14 ORF20 to be most closely related to the
wB40-8 putative ‘‘tail fibre’’ protein, and the wB124-14 genome
has been annotated to reflect this.
DNA replication and regulation. ORFs assigned to the
DNA replication and regulation cluster account for more than half
of all those encoded by wB124-14 but constitute only 35% of the
phage genome (Figure 2A,D). The devotion of a large number of
ORFs with roles in replication and DNA synthesis is concordant
with recent large-scale analyses of the human gut viral
metagenome in which genes involved in nucleotide replication
and synthesis were found to be enriched [6]. In addition, this is
also observed in the only other publically available complete
Bacteroides sp. phage genome, wB40-8 [31]. However, of the ORFs
affiliated to this putative cluster, as with phage encoded ORFs in
general, only a small proportion (25%) could be assigned a
putative function (Figure 2A, B, C).
Of particular interest in the regulation and replication cluster is
a predicted thymidylate synthase (TS; ORF8). TS is a ubiquitous
enzyme in bacteria that catalyzes the formation of deoxythymidine
59-monophosphate (dTMP) from deoxyuridine 59-monophosphate
(dUMP), which is essential for dTTP synthesis and DNA
replication [45]. Based on sequence homology and conserved
domain searches, the wB124-14 ORF8 appears to encode a ThyA
type enzyme which is predicted to be utilised by ,70% of
microorganisms [46], but seemingly rare in human gut viral
genomes and most likely acquired from host bacterial species [6].
However, conserved domain searches indicate that the wB124-14
TS may also exhibit dUMP hydroxymethylase activity and thus
constitute a bi-functional enzyme involved in the manufacture of
modified nucleotides (Table S2). This latter function is thought to
protect phage DNA from restriction-based host defence mecha-
nisms [47].
In addition, owing to the importance of TS activity for bacterial
survival, it has also been suggested that phage-encoded TS are of
benefit to host bacteria [48]. The provision of additional copies of
ThyA may enhance bacterial growth through gene dosing effects
as well as providing redundancy for a key activity and safeguarding
against its loss [48]. Furthermore, the efficiency of thymidylate
metabolism has also been implicated as a limiting factor in
prokaryote genome expansion and evolution, as well as cell
proliferation [46]. Overall, any enhancement in host survival
ability and replication rate is also of obvious benefit to
bacteriophage, since facilitating survival and replication of host
bacteria will contribute directly to phage survival.
The wB124-14 replication and regulation module also encodes
recombination proteins (ORF4) and phage antirepressors (ORF63
and 67) (Table S2; Figure 2). Phage encoded recombination
proteins are often involved in facilitating recombination between
the phage attP site and the attB site in the host chromosome
during formation of prophage insertions [49]. Phage antirepressors
are also often found in prophage elements [50], and these
regulators typically control the activation and de-repression of
genes required for re-entry into the lytic life cycle, often in
response to changes in the physiological status of the host cells
[50].
Host lysis. As with the previously characterised wB40-8 [31],
wB124-14 lacks a well-defined lytic module, and there is a relative
absence of ORFs encoding proteins with an obvious role in host
cell lysis. This lack of a well-defined lytic module is also a general
feature of other phages belonging to the Siphoviridae family [31,51].
Only one protein (ORF17), encoding a putative M15 type
metallopeptidase, could be assigned a clear function potentially
related to host lysis; phage-encoded peptidases are often involved
in disruption of the host cell envelope [52,53]. However, ORF17
appears to form part of a small gene cluster with several ORFs of
unknown function, which collectively constitute a putative lytic
module (Figure 2A). Two of these putative lytic module members
(ORFs 16 and 19) are predicted to encompass transmembrane
signal sequences and it is possible that these function to target the
encoded proteins to the cell wall or periplasm, as is often observed
with holin-endolysin systems [54].
Phage life cycle
Although the wB124-14 genome encodes some genes normally
related to temperate life cycles (recombinases, transcriptional
repressors and anti-repressors; Figure 2, Table S2), no evidence
for a lysogenic cycle was indicated in previous host range analyses.
The existence of wB124-14, or homologous elements, as prophage
was investigated within currently available Bacteroides genome
sequences using the nucleotide BLAST algorithm (Blastn).
Although lytic replication of wB124-14 appears to be confined to
only a few closely related strains of B. fragilis (Figure 1B), this
investigation encompassed 48 available complete and draft
genomes (Table S3), within the genus Bacteroides, including human
gut-specific species. A broad range of Bacteroides species was
included in this analysis to account for the possibility that an
alternate life cycle may occur in species other than B. fragilis, which
may not be detected under the laboratory conditions used to
elucidate host range in this study.
However, no evidence for a lysogenic life cycle or the existence
of wB124-14 as prophage was provided by this analysis, with all
chromosome sequences analysed devoid of any detectable wB124-
14-like prophage regions. Moreover, in addition to the production
of clear plaques and the absence of any identifiable integrase
genes, the large deviation between G+C content of the potential
bacterial host B. fragilis (G+ C,43.3%) and wB124-14 (G+C
37.5%) is also fitting with a lytic rather than lysogenic lifestyle [55].
Although deviation in G+C content may also be evident in
lysogenic prophage, a propensity for a larger reduction in the G+C
content of lytic phage, as well as the resulting increases in genome
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Page 6
signature differences (in terms of nucleotide repeat patterns), have
been used as indicators of a lytic lifestyle [55,56]. Thus, despite the
presence of genes often associated with a lysogenic cycle in other
phage, there is currently no evidence to indicate wB124-14
undergoes a temperate life cycle. Considering also the dynamic
and mosaic nature of phage genomes, in the case of wB124-14
genes such as anti-repressors could conceivably be remnants of
previous genomic incarnations, which no longer undertake their
original function.
Comparative genome analysis of available Bacteroides
phage sequences
Annotation and analysis of the wB124-14 genome sequence
indicated many ORFs were homologous to predicted proteins
from Bacteroides wB40-8 [31], also a member of the Siphoviridae
family and originally isolated from an urban sewage sample [57].
In order to examine the similarity between both phage in detail, a
comparative genomic analysis of the wB124-14 and wB40-8
complete genome sequences was undertaken.
Direct comparison of wB124-14 and wB40-8 complete genome
nucleotide sequences using the Artemis Comparison Tool (ACT;
[58]) (Figure 5A), as well as ORF-by-ORF comparison of
translated amino acid sequences (Figure 5B) revealed significant
homology over large areas of the phage genomes, encompassing
regions believed to be involved in structure and packaging, DNA
replication and regulation, and lysis, with a general conservation in
gene architecture and organisation evident (Figure 5). At the
nucleotide level, wB124-14 and wB40-8 are 57% identical across
the complete genome sequences, with the majority of ORFs in
each genome exhibiting homologues in the other (Figure 5).
Concordantly, Coregenes [59] analysis, which determines the core
set of genes common to two or more distinct genomes, showed that
39 of wB124-14 ORFs are shared with wB40-8 (BlastP identity of
75% or over), with structural genes displaying particularly high
levels of homology (96% identity or greater, Figure 5B).
Comparative metagenomic analysis of wB124-14 and
wB40-8
Due to the absence of phage infecting the host strain (B. fragilis
GB-124) from faecal samples derived from a wide range of
common domestic and wild animals, and from the general
environment, our previous work strongly suggested wB124-14 is
human gut specific [20]. w B40-8 has also been utilised as a marker
of human faecal pollution and is thought to be indicative of the
human gut microbiota [57]. To provide further insight into the
distribution of wB124-14 and wB40-8 in various microbiomes, and
evaluate their utility for the development of culture-independent
faecal source tracking methods, we undertook a comparative
metagenomic analysis using both complete bacteriophage genome
sequences.
The general distribution of sequences with homology to wB124-
14 and wB40-8 was investigated within all publically available
metagenomic datasets in the NCBI metagenome database (as of
June 2011, excluding those comprised solely of 16S rRNA gene
sequences), as well as the microbiomes of 124 individuals of
European origin which comprise the METAHIT dataset [28], 2
individuals of American origin [60], 13 Japanese individuals [8]
and the viromes of 12 individuals of American descent [6].
Searches using the full length phage nucleotide sequences failed
to identify metagenomic sequences with significant homology
(defined as a minimum of 80% identity over 100 nucleotides or
more, with an e-value of 1e
25
or lower) to either phage in any of
the available non-human gut metagenomes searched, or within the
available environmental metagenomes of aquatic and terrestrial
origin. Interestingly, sequences with high homology to wB124-14
and wB40-8 were detected within the termite gut metagenome
[61], but these were below the 80% identity threshold considered
to be significant for the purposes of this survey (#71% identity in
the termite metagenome). However, it should be noted that no
dataset currently provides complete coverage of representative
microbial communities and associated MGE.
The lack of homology to both phage within non-human gut
metagenomes will almost certainly reflect the distribution of
bacterial hosts in various microbial habitats. In the case of wB124-
14, the narrow host range observed for this phage supports
previous findings that the B. fragilis host strains it infects (Figure 1B)
are specific to the human gut [20].
Concordantly, sequences with homology to wB124-14 were
present in 104 of 124 (83.8%) human gut metagenomes within the
MetaHIT dataset (comprised of Danish and Spanish individuals),
3 out of 13 (23%) Japanese individuals and 2 out of 12 human gut
viromes (16.6%) (Figure 6A). By contrast, homologous sequences
to wB40-8 were detected in only 11.1% of individual MetaHIT
metagenomes, in only one gut metagenome of Japanese origin and
no homologous sequences were found in the 12 human gut
viromes searched (Figure 6A). Importantly, the observed incidence
of sequences homologous to both phage was only very weakly
positively correlated to the size of metagenomes (r
2
= 0.2;
P,0.0001; Figure 6B), indicating that observed differences in
incidence are unlikely to be an artifact of differing metagenome
size.
In contrast, homologous nucleotide sequences to both phages
were absent from the gut metagenomes of the two American
individuals [60] examined, with the search criteria employed. This
is perhaps unsurprising given the lack of sequences from Bacteroides
spp. within the metagenomic datasets generated by Gill and co-
workers [60]. However, a lack of homology to both phage was also
apparent in the American gut viral metagenomic dataset available
on GenBank at the time of study [62]. Given that this dataset
focuses on RNA viruses [62], a lack of homology to the DNA
viruses wB124-14 and wB40-80, was also not unexpected.
However, a general lack of homologous sequences was also
apparent in the gut viral metagenomes of American origin
generated by Reyes and colleagues (Figure 6A) [6].
Despite these caveats, the current observations indicate
potential geographic variation in the distribution of these phage,
and may also reflect inter-individual variation in actual levels of
bacteriophage resident in distinct human gut microbiomes. This
notion is congruent with observed differences in excretion of phage
amongst the human population; with Bacteroides HSP40 infecting
phages such as wB40-8 shown to be excreted by a lower number of
individuals than other B. fragilis phages [23,63]. These differences
are likely also indicative of the abundance of host strains within the
human gastrointestinal tract (GIT), as well as intra-individual
differences in gut viral community population structure.
wB124-14 ecological profiling
To further evaluate the potential utility of wB124-14 in broad-
scale MST applications, and the putative gut specific nature of this
phage (as indicated by previous studies [20,26,34] and our
comparative metagenomic analysis) the relationship of wB124-14
with the wider bacteriophage community was explored. This was
investigated using both conventional gene-centric alignment-
driven phylogenetic analysis, as well as gene-independent
alignment-free methodologies based on the pattern of tetranucle-
otide repeat frequencies encoded in the wB124-14 genome [51,64–
65]. In particular, the latter approach facilitates large-scale
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analyses of nucleotide sequence affiliation and relationships, which
permit a more expansive overview of wB124-14 ecology.
wB124-14 terminase based phylogeny. Since terminases
are thought to be the most highly conserved gene within phage
[66], conventional phylogenetic analysis was undertaken using the
putative wB124-14 terminase gene (ORF43) (Figure 7).
Homologous amino acid sequences from phage and bacterial
genomes (prophage), as well as from metagenomes of diverse
origin, were aligned with the predicted large subunit terminase of
wB124-14, and alignments used to construct phylogenetic trees.
This analysis further confirmed the close association of wB124-14
with wB40-8, and also revealed a strong association with predicted
terminase and terminase-like sequences originating from human
gut microbiomes [8,28] and viromes [6].
As expected, the closest association was observed with terminase
sequences originating from human gut-associated members of the
Bacteroidetes division, including B. fragilis (the host species of
wB124-14), and Alistipes spp. (Figure 7). In addition, the majority of
terminase sequences derived from human gut viral metagenomes
[6] represented in this tree appeared to be distinct from all other
sequences retrieved from other sources (Figure 7). This latter
observation suggests the existence of additional gut-specific
bacteriophage and hints at a close association between the human
host, its microbiome and components of the associated mobile
metagenome. However, this phylogenetic analysis was limited to
sequences possessing terminase genes closely related to that of
wB124-14, and also to those generating good alignments with the
wB124-14 sequence. By default this excludes the majority of
metagenomic virome sequences (due to the fragmentary nature of
such datasets), and provides only a limited view of wB124-14
ecology and evolution.
Gene-independent genome signature-based ecological
profiling..
In light of the narrow view offered by gene-centric
alignment-based phylogenetic methods for analysis of wB124-14,
and the problems associated with expanding such surveys when
analyzing bacteriophage genomes in general, we next explored the
broader ecological landscape occupied by wB124-14 using gene-
independent and alignment-free methods [51,64,65].
Since bacteriophage and other mobile genetic elements are
believed to reflect the genomic signatures of their host bacteria (in
terms of di-, tri-, and tetra-nucleotide repeat frequency (TRF)
patterns; [51,67]), it would be expected that bacteriophage with
Figure 5. Comparative genomic analysis of WB124-14 and WB40-8 (ATCC 51477-B1). A. Nucleotide sequences of wB124-14 and wB40-8
were compared using the Artemis Comparison Tool (ACT). Shaded areas between linear phage genome maps represent areas of high nucleotide
identity (90% or greater). Colour scale represents level of nucleotide identity at each region of homology. The ORF map for wB40-8 corresponds to the
annotations available in the GenBank submission (FJ008913.1). For the purposes of this analysis, the wB124-14 genome was linearised between ORFs
29 and 30 (Figure 2, Table S2), in order to compare the circular wB124-14 genome with that of wB40-8. Colours of ORFs correspond to functional
assignments as used in Figure 2. B. Comparison of amino acid sequences from wB124-14 ORFs with those annotated in the wB40-8 genome. Shading
between arrows indicates those sharing high amino acid sequence identity. Colour scale indicates level of amino acid identity between each
homologous ORF.
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Page 8
similar host ranges will exhibit comparable TRF signatures.
Therefore, comparison of these TRF genetic signatures may be
used to place wB124-14 in a wider ecological context with other
bacteriophage, bacterial host species, and sequences obtained from
metagenomic datasets.
To this end we compared the patterns of TRF in the genome of
wB124-14 to those encoded in the genomes of 611 other
bacteriophage, 48 chromosomal sequences from a range of
Bacteroides species, and all large fragments (.10 kb, n = 188
contigs) assembled from human gut meta-viromes generated by
Reyes et al. [6]. In light of the similarities observed between
wB124-14 and wB40-8 in other analyses undertaken here, TRF
scores for each bacteriophage were correlated to identify
ecological similarities or differences. This not only permitted the
evaluation of the effectiveness of this genetic signature-based
approach but also the exploration of the extent to which the
ecological landscapes populated by both phage overlap (Figure 8).
In general, results of this analysis were congruent with host
range studies, comparative genomic analyses, and trends observed
from construction of terminase phylogenetic trees (Figures 1, 5 and
7). As expected, wB40-8 was the most closely related bacterio-
phage to wB124-14 and sequences from B. fragilis strains were
found to be the most closely related chromosomal sequences
(Figure 8A; Figure S1). A high level of correlation was observed
between TRF scores derived from the comparison of each phage
(wB124-14 and wB40-8) against all other sequences analyzed
(r = 0.981 or above; Figure 8). This high level of correlation
indicates that both phage share closely related and highly similar
ecological niches, in keeping with known host ranges, and the close
phylogeny and evolutionary relationship observed in our other
investigations (Figure 8A).
The relationship of other complete bacteriophage genomes
(relative to wB124-14 and wB40-8) also exhibited a marked trend
based on the broad classification of bacterial host genera and its
association with the human gut microbiome (Figure 8A). The
genomes of phage infecting bacterial genera commonly found in
the human gut microbiome displayed a clear association relative to
Figure 6. Incidence of sequences homologous to WB124-14 and WB40-8 human gut metagenomes. Percentage of individual
metagenomes in which sequences homologous to wB124-14 or wB40-8 were identified ($80% identity over $100 nucleotides, 1e
25
or lower). The
microbial metagenomes examined were derived from individuals of European (MetaHit) [28], Japanese [8] and American [60] origin, alongside the
combined viromes from 12 individuals of American descent [6]. MH MetaHit All individuals represented in the MetaHit dataset; Jap All
individuals of Japanese origin; AM All individuals of American descent; Virome All viromes from individuals of American origin. B. Scatter plots
illustrating the relationship between size of individual metagenomes searched and detection of sequences homologous to wB124-14. r
2
= Pearson
correlation co-efficient. **P,0.0001.
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Figure 7. Phylogeny of WB124-14 large subunit terminase. Amino acid sequences homologous to wB124-14 terminase (ORF43), based on bit-
score, were retrieved from GenBank and metagenomic datasets, including human gut microbiomes and viromes [6,8,28] and marine microbial
metagenomes [92], and aligned using ClustalW. The unrooted concensus neighbour joining tree (1000 bootstrap resamplings) was produced using
MEGA v5. Bootstrap values $40 are shown adjacent to respective tree nodes. Scale indicates amino acid substitutions. Colours indicate phylum level
grouping or origin of metagenomic sequences. Black triangles indicate wB124-14 or wB40-8 terminase sequences; white triangles represent other
phage sequences; white circles represent sequences originating from human gut metagenomes.
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Page 10
the wB124-14 and wB40-8 genomes, forming a distinct grouping
centered around the majority of gut virome sequences (Figure 8A).
In contrast, those exhibiting the least similar TRF profiles were
phage infecting bacterial genera predominantly associated with
terrestrial, aquatic or marine environments and not members of
the normal human gut microbiota (Figure 8A). Identical analyses
utilizing the wB124-14 genome, and that of Burkholderia wKS10
[68] (which generated the lowest TRF correlation to wB124-14 of
all phage analyzed; Figure 8A) displayed none of the trends
observed between w B124-14 and wB40-8, and exhibited only
negative correlation coefficients (r = 20.409 or below) with
wB124-14 in relation to sequence categories or groups used
(Figure 8B). However, even in this analysis, gut-associated phage
genomes, bacterial genomes, and gut virome fragments were
Figure 8. Comparison of tetranucleotide repeat frequency patterns in bacteriophage genomes and ecological profiling of wB124-14
and wB40-8. The tetranucleotide repeat frequency (TRF) correlation scores for wB124-14, wB40-8 and Burkholderia wKS10, were compared using
scatter plots and correlation of data examined using the Pearson coefficient. A complete list of genomes and sequences utilised in this analysis is
provided in Table S3. A. Comparison of TRF scores for wB124-14 (x-axis) vs wB40-8 (y-axis). B. Comparison of TRF scores for wB124-14 (x-axis) vs
Burkholderia wKS10 (y-axis). A, B.
Upper charts plot scores for all phage genomes, viral metagenome fragments, and Bacteroides genomes. Phage
= TRF scores from comparisons to 611 phage and prophage genomes. Virome = TRF scores from comparisons to 188 large fragments (.10 Kb)
from human gut viral metagenomes [6]. Chromosome = TRF scores from comparison to 48 Bacteroides spp. genome sequences. Each sequence
type is represented by a different colour and symbol as indicated in the figure legends on each chart. The intensity of shading of data points reflects
the number of data points represented in a given area with a greater intensity indicating more overlapping data points. Values in parentheses
provide Pearson correlation scores for each sequence type.
Lower charts plot TRF scores for sequences assigned to one of three categories based
on their relation to the human gut microbiome: Gut = comprises bacteriophage infecting bacterial genera commonly forming part of the normal
human gut microbiota. Gut Associated = comprises bacteriophage genomes infecting bacterial genera whose member species are associated with
the gut but not generally considered to be members of the normal gut microbiota (such as primary invasive gut pathogens), or where member
species are more commonly associated with environmental habitats. Non-Gut = contains bacteriophage infecting bacterial genera with member
species not considered to be part of the human gut microbiota or typically associated with this community, and primarily encompasses
bacteriophage infecting genera of environmental origin. Virome = All large fragments (n = 188, .10 Kb) assembled using CAMERA workflows (per
individual) from human gut viral metagenomic libraries [6,91]. Each sequence category is represented by a different colour and symbol as indicated in
the figure legends on each chart. For the purposes of this analysis phage infecting a particular host bacterial genus were only utilised if four or more
representative phage genomes were available (540 complete phage genomes, representing 31 bacterial genera). The intensity of shading of data
points reflects the number of data points represented in a given area with a greater intensity indicating more overlapping data points. Values in
parentheses provide Pearson correlation scores for each sequence type.
doi:10.1371/journal.pone.0035053.g008
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observed to be much more closely affiliated with wB124-14,
displaying a distinct trend towards the wB124-14 axis (Figure 8B).
Collectively, these observations confirm the usefulness of the TRF
approach to investigate bacteriophage ecology (Figure 8).
Despite the observed trends and phage groupings, much overlap
was observed between bacteriophage assigned to different
categories, an observation that is not unexpected in light of the
constant ingress of ‘‘contaminants’’ into the gut ecosystem through
consumption of food, the malleable nature of phage genomes, and
the broad categories to which phage genomes were assigned in this
analysis. Nevertheless, the relationships indicated here suggest that
wB124-14 and wB40-8 have a strong association with the gut
microbiota and occupy a distinct and largely uncharacterized
ecological niche in this community.
As well as facilitating the development of novel MST tools,
genomic characterization of phage infecting prominent members
of the human gut community also provides fundamental insight
into a fraction of the mobile metagenome that constitutes an
immense and largely unexplored gene-space. This fraction of the
gut microbiome is likely to encode novel activities relevant to
development and functioning of the human GIT, and be of
pharmaceutical or biotechnological interest in its own right [1,9].
This is particularly relevant for phages infecting members of the
Bacteroidetes which constitute a major component of the human
GIT microbial community [28], and have been implicated in both
the onset of and protection against the development of gut-related
disorders [69–71]. Given the potential for phage to shape
microbial community structure and function [10–15], coupled
with their highly selective nature, the isolation and characteriza-
tion of gut-specific phage offer numerous possibilities for the
therapeutic manipulation of the human gut microbiota, and a
range of biotechnological applications including the development
of novel MST tools.
In this regard the genetic characterization of wB124-14 has
provided an essential first step in the development of culture-
independent microbial source tracking tools. In particular PCR-
based tools that will permit sensitive detection and quantification
of human gut-specific indicators (such as wB124-14 DNA), will be
made possible by the availability of this, and other, genome
sequences of human gut-specific bacteriophage. In this regard,
current efforts in developing portable, self-contained ‘‘chip’’ style
PCR systems, for accurate and rapid diagnosis of bacterial
infections at point-of-care [72–73], will translate well for microbial
source tracking applications. Ultimately, such methods will
eliminate the need for anaerobic culture, permitting rapid and
sensitive monitoring of faecal pollution in a range of samples from
surface water to shellfish.
Our analyses have also provided insight into a novel and
uncharted ecological landscape within the human gut microbiome.
Comparative metagenomic analysis, along with ecological profil-
ing confirmed the gut-specific nature of wB124-14, corroborating
our previous findings [20]. Intriguingly, this analysis also indicated
that wB124-14 and wB40-8 genomes are distinct from other phage
genomes and the meta-virome sequences examined here,
seemingly occupying an ecological sphere of the human gut
virome not represented in currently available human gut meta-
viromes, and by only two phage genomes (wB124-14 and wB40-8)
in public sequence databases.
In conjunction with the apparent broad geographical distribu-
tion of sequences homologous to wB124-14 in human gut
microbiomes (observed in our comparative metagenomic analysis),
this observation points to a long-term association with the human
gut microbiome. In keeping with this hypothesis is the observation
that both wB124-14 and wB40-8 encode functions (namely TS)
previously found to be absent from extensive viral datasets, but
present in gut-associated viral metagenomes [6], and which are
likely to play a role in wider metabolism and fitness of bacterial
hosts. If so, such phage may also contribute to more subtle
mechanisms influencing community structure and help shape this
ecosystem not only through selective elimination of host species,
but also through effects on host fitness and inter-strain or inter-
species competition [74–76].
However, the relative lack of homologous sequences to these
phage observed in comparative metagenomic analysis of American
datasets, suggests that phage complements may vary between
geographically distinct populations; for source tracking applica-
tions region or population specific phage may be required, a
picture that is also emerging from other studies [20,26,34]. In
addition, the large degree of inter-individual variation in the
human gut microbiome almost certainly extends to the mobile
metagenome, including the virome [6]. In this regard the goal of
developing a truly universal MST will most likely require the
utilization of multiple gut-specific elements, such as bacteriophage,
to construct a multivalent tool capable of detecting a range of
human faecal indicators.
Although much of the bacteriophage genetic landscape is
exceedingly poorly characterized in the majority of microbial
ecosystems investigated to date, including the human gut, here we
provide a glimpse of this biological dark matter and its
corresponding ecological context. Our findings suggest that the
gene-space and ecological neighborhood populated by wB124-14
and related
Bacteroides phage is even less well characterized than
other aspects of the gut virome, and may be almost entirely
uncharted at present. The availability of the complete genome
sequence of this and other such phage will now permit further
study of this aspect of the human gut mobile metagenome,
facilitate interpretation of metagenomic datasets, as well as the
development and application of novel, sensitive, and rapid culture-
independent MST tools.
Materials and Methods
Phage, host strains and growth conditions
wB124-14 was originally isolated from municipal wastewater
and is routinely propagated on Bacteroides sp. GB-124, as described
previously [20]. Phages were isolated by the double-agar protocol
(ISO 10705-4) [77] developed specifically for Bacteroides phages
using Bacteroides phage recovery medium (BPRM, per litre: meat
peptone, 10 g; casein peptone, 10 g; yeast extract, 2 g; NaCl, 5 g;
monohydrated l-cystein, 0.5 g; glucose, 1.8 g; MgSO
4
.7H
2
O,
0.12 g; CaCl
2
solution (0.05 g/ml), 1 ml; hemin, 10 ml of a 0.1%
(w/v) solution made up in NaOH 0.02%; 1M Na
2
CO
3
, 25 ml;
pH 6.860.5).
To ensure purity of wB124-14 isolates, agar plugs containing
single wB124-14 plaques (zones of lysis) were picked from plates
using a sterile Pasteur pipette and incubated at 4uC for 4 h in
400
ml phage isolation buffer (19.5 mM Na
2
HPO
4
,22mM
KH
2
PO
4
, 85.5 mM NaCl, 1 mM MgSO
4
, 0.1 mM CaCl
2
), and
phage presence was retested using the double-agar method above
to generate fresh plaques. This process was repeated three times
and the final purified phage suspension used to generate high titre
phage stocks for sequencing and other assays.
To generate high titre phage stocks, pure wB124-14 phage
suspensions were added to 27 ml of an exponential Bacteroides sp.
GB-124 culture (OD
620
0.33; cell density of approximately 2610
8
colony forming units; CFU) and incubated anaerobically (accord-
ing to [77]) overnight at 37uC to produce crude lysates. Phage
lysates producing plaques were subsequently added to 620 ml of
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Page 12
GB-124 (OD
620
0.33) and incubated overnight as before. Phage
suspensions were then purified and concentrated using polyethyl-
ene glycol 8000 [78] as follows: NaCl was added to a final
concentration of 1 M and phage suspensions were incubated for
2 h at 4uC, then centrifuged at 18006g for 10 min to remove
bacterial debris. Polyethylene glycol 8000 was added to a final
concentration of 10% (w/v), mixed for 30 min, and left overnight
at 4uC. Precipitated phage were collected by centrifuging at
11,0006g for 10 min at 4uC. Resulting supernatant was discarded
and 30 ml of phage isolation buffer (as above) was added.
Suspensions were stored at 4uC overnight, mixed gently to dissolve
pellet and centrifuged at 15006g for 10 min to remove remaining
debris. Phage suspensions were filtered through a 0.2
mM
polyvinylidene filter (Sartorius, UK). High titre stock suspensions
of 10
11
plaque forming units (pfu)/ml were stored in glass vials in
the dark at 4uC.
Transmission Electron Microscopy (TEM)
Purified phage particles (10
9
pfu/ml) were immobilised on a
200 mesh Formvar/Carbon copper electron microscope grids
(Agar Scientific, UK), and negatively stained with 1% uranyl
acetate. Phage were imaged by TEM using an Hitachi-7100 TEM
at 100 kV. Phage dimensions were estimated from positively
stained micrographs and values reported are the mean value 6
standard deviation (SD) of five virion measurements.
Analysis of wB124-14 host range
Purified phage particles (10
3
pfu/ml) were tested for their ability
to infect and replicate within a selection of host strains using the
double agar method as previously described [77]. Plates were
incubated for 24 h at 37uC, under anaerobic conditions and
presence of plaques was used to indicate ability to replicate in a
particular Bacteroides species. A number of strains previously
isolated from municipal wastewaters from a variety of geograph-
ical locations [26] as well as typed Bacteroides spp. were tested (see
Table S1 for full list of strains and species used). Novel strains were
identified further by 16S rRNA gene sequencing, from 16S PCR
products amplified with universal primers 27f and 1492r [79]
using standard conditions. Purified PCR amplicons were
sequenced directly by GATC Biotech AG (Konstanz, Germany)
using Sanger sequencing, and are deposited in the EMBL database
under the following accession numbers: HE608156, HE608157,
HE608158, HE608159 and HE608160.
Bacteroides host species phylogeny
The relationship between the wB124-14 Bacteroides fragilis host
strain GB-124 and other Bacteroides species was examined in closer
detail by construction of phylogenetic trees based on 16S rRNA
gene sequences. In addition to those 16S sequences generated in
this study, sequences homologous to the wB124-14 host species
16S rRNA were retrieved from GenBank based on best-hit Blast
analysis and aligned using ClustalW [80]. Evolutionary histories
were inferred by constructing consensus maximum likelihood
phylogenetic trees based on the Tamura-Nei model using MEGA
v5 [81]. The reliability of tree nodes was evaluated using %-age of
1,000 bootstrap resamplings, with bootstrap values $40% used to
define well-supported clusters of 16S rRNA gene sequences.
DNA extraction and sequencing
DNA was extracted from high titre phage stocks (10
10
pfu/ml),
as described previously [82], with minor modifications. Briefly,
each ml of phage stock was treated with DNAseI (1
mg/ml) and
RNAseA (100
mg/ml) to remove contaminating bacterial DNA,
before precipitating with 2M ZnCl
2
(20 ml/ml) for 5 min at 37uC.
Precipitate was centrifuged (1 min, 5,0006g) and resultant
supernatant discarded. The remaining pellet was gently resus-
pended in TES buffer (0.1 M Tris-HCl, pH 8; 0.1 M EDTA;
0.3% SDS) and incubated at 60uC for 15 min. Proteins and
polysaccharides were precipitated using 3 M potassium acetate
(pH 5.2) on ice for 15 min, then centrifuged for 1 min at 8,0006g.
DNA in the resultant supernatant was precipitated with isopro-
panol and centrifuged. The resulting DNA pellet was washed with
70% ethanol, air dried at room temperature and resuspended in
20
ml Tris-EDTA buffer (10 mM Tris-HCl, pH 8; 1 mM EDTA).
The complete genome sequence of wB124-14 was obtained by
pyrosequencing using a Roche GS FLX with Titanium chemistry.
A total of 16,952 reads with an average length of 355 nt were
generated and assembled using the GS De Novo Assembler. The
final assembly provided average sequence coverage of ,1276 for
the wB124-14 genome. All sequencing and genome assembly was
conducted by GATC Biotech AG (Konstanz, Germany). Genome
size was confirmed by restriction digest and agarose gel
electrophoresis, and fragment sizes calculated using Gene Tools
software (Syngene, UK). The complete wB124-14 genome has
been deposited in the EMBL database under the following
accession number: HE608841.
Annotation and bioinformatic analyses of wB124-14
genome
Open reading frames (ORFs) encoded by wB124-14 were
predicted using Glimmer (v3) [83], and annotated using Artemis
[84]. The putative function of predicted ORFs were assigned
based on homologies to proteins and protein conserved domains
identified in BlastP and tBlastn [85] searches against the NCBI-nr,
and Conserved Domains Database (CDD; encompassing all NCBI
entries plus protein models from Pfam, SMART, COG, PRK and
TIGRFAM, and ACLAME databases), respectively.
For BlastP and tBlastn searches only homologous sequences
generating e-values of lower than 1e
25
at $20% identity were
considered significant. For Conserved Domain searches, only hits
with an e-value of 0.01 or lower were considered significant.
Putative tRNA-encoding genes were searched for using tRNAs-
can-SE [86]. Transmembrane proteins and signal peptides were
predicted using the TMHMM v2 [87] and SignalP v3 [88] servers.
The presence of prophage with homology to wB124-14 and wB40-
8 in complete bacterial genome sequences were predicted using
Prophinder [89] and Blastn analysis of Bacteroides genomes
available within GenBank (See Table S3 for list of genomes).
Comparative analysis of bacteriophage genomes was carried out
using the Artemis Comparison Tool (ACT) [58]. Physical maps of
the annotated wB124-14 and wB40-8 genomes were generated
using Vector NTI Advance (v11.5).
Physical structure of phage genome
Phage genomic DNA was digested with HindIII, EcoRI
(Promega. UK), SwaI and SaII (NEB, UK), respectively, for 3 h
at 37uC and fragments resolved on a 0.8% Tris Acetate EDTA
(TAE) gel at 80 V for 3 h. Resulting restriction fragment profiles
were compared to in silico restriction profiles for linear or circular
permutations of the genome, which were generated by pDRAW32
(http://www.acaclone.com/).
Analysis of the wB124-14 proteome
wB124-14 lysate (10
11
pfu/ml) was filtered through a sterile
0.2
mM low protein binding filter (HT Tuffryn, Pall Corp.) to
remove cell debris. Resulting crude protein extract was diluted
Comparative Metagenomic Analyis of Human Gut Phage
PLoS ONE | www.plosone.org 13 April 2012 | Volume 7 | Issue 4 | e35053
Page 13
with an equal volume of 2,2,2-Trifluoroethanol (Fluka), 20 mM
DTT, and denatured and reduced at 60uC for 60 min, before
alkylation with 30 mM IAA at room temperature in the dark for
45 min. The sample was diluted 6-fold with 50 mM ammonium
bicarbonate and digested with sequencing grade trypsin (Promega,
UK) overnight at 37uC. Tryptic peptides were fractionated on a
250 mm 60.075 mm reverse phase column (Acclaim PepMap100,
C18, Dionex) using an Ultimate U3000 nano-LC system (Dionex)
and a 2 h linear gradient from 95% solvent A (0.1 % formic acid
in water) and 5% B (0.1% formic acid in 95% acetonitrile) to 50%
B at a flow rate of 250 nL/min. Eluting peptides were directly
analysed by tandem mass spectrometry using a LTQ Orbitrap XL
hybrid FTMS (ThermoScientific) and derived MS/MS data
searched against wB124-14 amino acid sequences using Sequest
version SRF v. 5 as implemented in Bioworks v 3.3.1 (Thermo
Fisher Scientific), assuming carboxyamidomethylation (Cys),
deamidation (Asn and Gln) and oxidation (Met) as variable
modifications. Filtering criteria used for positive protein identifi-
cations are Xcorr values greater than 1.9 for +1 spectra, 2.2 for +2
spectra and 3.75 for +3 spectra and a delta correlation (DCn) cut-
off of 0.1.
Comparative metagenomic analysis
Comparative metagenomic analysis were conducted as previ-
ously described [1,4,90]. The presence of wB124-14 and wB40-8-
like sequences among available metagenomes was investigated in
the first instance using the full set of microbial metagenomes of
diverse origin available within the NCBI database (158 metagen-
omes, June 2011). A more detailed investigation of the distribution
of wB124-14 and wB40-8-like sequences within the 124 human gut
microbial metagenomes from individuals of European descent
represented in the METAHIT dataset [28], 13 individuals of
Japanese origin [8], 2 individuals of American origin [60] and
within the viral metagenomes from 12 individuals of American
descent [6] was then carried out. To obtain assemblies of viral gut
metagenomes for these analyses, pyrosequencing reads for project
SRA012183 [6] were obtained from the NCBI Short Read
Archive and processed using CAMERA workflows [91]. Reads
were filtered to remove low quality reads and duplicates using the
454 QC and 454 Duplicate Clustering workflows, respectively,
with default parameters. The resulting high-quality, non-redun-
dant data sets were assembled using the CAMERA Meta-
Assembler which combines output from seven independent short
read assemblers run using pre-optimised parameters: Newbler,
Taipan, Celera, Velvet, SOAPdenovo, ABySS and SSAKE [91].
Individual metagenomes were processed separately. The com-
bined metagenomes from each dataset (MetaHIT, Japanese gut,
American gut and gut viral) were searched using Blastn for
nucleotide sequences with homology to wB124-14 and wB40-8.
Only sequences exhibiting an identity of 80% or greater over
100 bp or longer at 1e
25
or lower were considered significant and
used to calculate incidence of positive metagenomes as described
previously [1,4]. Correlation analysis (Scatter plots and Pearson
correlation co-efficient) was carried out using Microsoft Excel.
Ecological profiling of wB124-14
Alignment-driven phylogenetics was undertaken using the
wB124-14 terminase gene amino acid sequence. Homologous
sequences, based on top bit scores, were identified in metagenomic
datasets of human gut and marine origin [6,8,28,60,92], as well as
through BlastP searches of the nr dataset. Sequences were aligned
using ClustalW and the Neighbour-Joining method with the Jones-
Taylor-Thornton matrix model for protein distance, used to
construct phylogenetic trees using MEGA v5 [81]. Alignment-free
analysis, based on the TRF patterns encoded in microbial and
bacteriophage genomes, was used to investigate the broader
relationship of wB124-14 with the wider phage community, and
host bacterial species. Correlations between frequencies of all 256
possible tetranucleotide sequences in all phage genome sequences
available in GenBank (611 phage genome sequences as of October
2011), a wide range of Bacteroides spp. genomes (48 genome
sequences, obtained from GenBank, The Broad Institute http://
www.broadinstitute.org; and the Washington University Genome
Institute http://www.genome.wustl.edu), as well as all large
metagenomic fragments (.10 kb) assembled from the human gut
viral datasets generated by Reyes and colleagues [6], were
calculated according to the method of Teeling and colleagues,
using the standalone TETRA 1.0 program [65]. Draft Bacteroides
chromosomal sequences were also included in this analysis and for
each draft genome contigs were first concatenated before
processing using TETRA (concatenation was confirmed not to
obscure the inherent tetranucleotide genomes signature in draft
genomes processed this way; Figure S2). All sequences entered
into the TETRA standalone program were extended by their
reverse complement and used by the program to calculate
observed and expected TRFs [65]. The divergence between
observed and expected frequencies for each tetranucleotide
pattern were subsequently converted to Z-scores which were
compared pairwise between all sequences to generate a Pearson
similarity matrix of TRF patterns.
Supporting Information
Figure S1 Details of closest sequences to wB124-14 by
tetra score. For each sequence type represented (phage, virome,
chromosome), the top six closest sequences to wB124-14 by
tetranucleotide repeat frequency (TRF) score are indicated by
numerals on the scatter plot, and colours correspond to sequence
types (as detailed in chart legend). The table provides the names
and TRF correlation values against the wB124-14 genome for
each sequence indicated, arranged by sequence type. In the case of
complete phage genome sequences, the closest sequence to wB124-
14 is wB40-8 and vice versa.
(TIF)
Figure S2 Comparison of tetranucleotide correlation
scores for complete and draft concatenated genomes.
To verify that concatenation of draft genomes, and the unfinished
nature of these datasets did not corrupt the tetranucleotide genome
signatures of these genomes, complete and draft genomes for several
Bacteroides species were compared. It is expected that such strains
would exhibit a high level of correlation between tetranucleotide
genome signatures. Scatter plots indicate that concatenated draft
genomes retain their tetranucleotide signature, with perfect
correlation observed in all comparisons, in contrast to negative
control plots between the distantly related Bacteroides vulgatus and
Bifidobacterium longum genomes. A. B. thetaiotaomicron VPI-
5483 complete genome vs B. thetaiotaomicron 3330-1 draft
concatenated genome. B. B. vulgatus ATCC 8482 complete
genome vs B. vulgates 1_0 draft concatenated genome. C. B.
fragilis YCH46 complete genome vs B. fragilis 3_1_12_1 draft
concatenated genome. D. Negative control plot, B. fragilis YCH46
vs Bifidobacterium longum DJO10A. Corr = Correlation score.
(TIF)
Table S1 Origin of species and strains used in wB124-14
host range assays
1
.
1
highly related B. fragilis strains used for
tree construction (Figure 1B) also included. NT not tested.
(DOCX)
Comparative Metagenomic Analyis of Human Gut Phage
PLoS ONE | www.plosone.org 14 April 2012 | Volume 7 | Issue 4 | e35053
Page 14
Table S2. wB124-14 predicted ORFs and putative func-
tional assignments.
1
ORF numbers and functional assign-
ments correspond to those represent on genetic maps of the
FB124-14 genome presented in Figure 2.
2
ORFs were assigned
roles relating to broad functions based on results of BlastP and
conserved domain searches of translated ORF amino acid
sequences.
(DOCX)
Table S3 Bacterial chromosomes, phage genomes and
metagenomic fragments used in phage phylogenetic
analyses and ecological profiling (Figures 7 and 8).
1
Classification, refers to classification of genomes used for
ecological profiling in Figure 8B. Genomes from phage infecting
host bacteria belonging from a particular genus were assigned one
of three broad categories based on the relationship of bacterial
host genus with the human gut microbiota. For the purposes of this
analysis only bacteriophage with 4 or more representatives
infecting a particular genus of bacteria were included (540
complete phage genomes, representing 31 bacterial genera). G
= Gut, constitutes bacteriophage infecting genera commonly
forming part of the normal human gut microbiota as well as all
large fragments (.10 Kb) assembled using CAMERA workflows
from human gut viral metagenomic libraries (Reyes et al 2010,
Nature 466: 334–338 [6]). GA = Gut Associated, contains
bacteriophage genomes infecting genera with member species
associated with the gut but not considered to be members of the
normal microbiota (such as primary invasive gut pathogens), and/
or contain member species more commonly associated with
environmental habitats. NG = Non-Gut, contains bacteriophage
infecting genera with member species not considered to be
members of the human gut microbiota or typically associated with
this community. Primarily encompasses bacteriophage infecting
genera of environmental origin.
2 Source, indicates the source
of bacterial and bacteriophage genomes utilised in this study:
NCBI Complete bacteriophage genomes were obtained from
the NCBI Viruses home page (TaxID: 10239) and all genomes
present as of Oct 18th 2011 were downloaded using the Viral
homepage ftp. Complete finished Bacteroides genomes were
obtained from the NCBI Prokaryotes genome homepage and
downloaded individually. N NCBI Viral Homepage: http://www.
ncbi.nlm.nih.gov/genomes/GenomesHome.cgi?taxid = 10239; N
NCBI Viral FTP: ftp://ftp.ncbi.nih.gov/refseq/release/viral/; N
NCBI Prokaryote Homepage: http://www.ncbi.nlm.nih.gov/
genomes/lproks.cgi. NCBI SRA –Pyrosequencing reads generat-
ed from metagenomic libraries of virus-like particles by Reyes et al.
(2010) [6], were obtained from the NCBI Short read archive,
project SRA012183 (http://www.ncbi.nlm.nih.gov/sra). Reads
were subsequently processed for quality and assembled using
CAMERA workflows (https://portal.camera.calit2.net/
gridsphere/gridsphere). Broad Inst = Broad Institute. Draft
Bacteroides spp. genomes sequenced as part of the Human
Microbiome Project (Nelson et al 2010 Science 328 (5981):994–
999) at the Broad Institute were downloaded from the Bacteroides
group Sequencing project page: N Broad Institute homepage
(http://www.broadinstitute.org/); N Bacteroides Sequencing
Group Project Page (http://www.broadinstitute.org/annotation/
genome/bacteroides_group/MultiDownloads.html); N Human Mi-
crobiome Project Homepage (http://genome.wustl.edu/projects/
human_microbiome_project/human_gut_microbiome). WUGC
= Washington University Genome Centre. Draft Bacteroides
genomes sequenced as part of the Human Gut Microbiome Project
were also obtained from the Washington University Sequencing
Centre, Human Microbiome Project website. N HGM Home page:
http://genome.wustl.edu/projects/human_microbiome_project/
human_microbiome_project_description. N Genomes: http://
genome.wustl.edu/genomes/human_gut_microbiome_genomes.
(DOCX)
Acknowledgments
We thank Dr. Cormac Gahan and Dr. Caroline Jones for critical discussion
of the manuscript, and also wish to acknowledge and thank the research
groups whose data we have analysed as part of this study, for making this
publically available: Human gut viral metagenomes were generated by
Reyes and colleagues [6]; Draft genomes of Bacteroides species sequenced as
part of the Human Gut Microbiome project were obtained from the
‘‘Bacteroides group sequencing project’’ at the Broad Institute (http://www.
broadinstitute.org/), and the Washington University Genome Institute
(http://www.genome.wustl.edu).
Author Contributions
Conceived and designed the experiments: BVJ JC LO LB JE. Performed
the experiments: LO CD BVJ JE LB DD HT JC. Analyzed the data: LO
BVJ LB EC. Wrote the paper: LO BVJ.
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  • Source
    • "In contrast Reischer et al. (2013) have reported geographical stability for q-PCR targeted ruminant associated populations of Bacteroidetes. The reasons for this geographical instability are not known, though in the last decade reports have proliferated indicating that the community structure of the gut microbiota in general and that of Bacteroides in particular is deeply influenced by diet, host phylogeny and genetic factors of the host, and environmental factors (Sá nchez et al., 2001; Ley et al., 2008; Shanks et al., 2011; Ogilvie et al., 2012 ) and that consequently Bacteroides shows some signature of biogeography (Lee et al., 2011). Nevertheless, a feasible method to isolate Bacteroides discriminating hosts convenient for a given geographical area has been described (Payá n et al., 2005). "
    [Show abstract] [Hide abstract] ABSTRACT: Bacteriophages infecting certain strains of Bacteroides are amid the numerous procedures proposed for tracking the source of faecal pollution. These bacteriophages fulfil reasonably well most of the requirements identified as appropriate for a suitable marker of faecal sources. Thus, different host strains are available that detect bacteriophages preferably in water contaminated with faecal wastes corresponding to different animal species. For phages found preferably in human faecal wastes, which are the ones that have been more extensively studied, the amounts of phages found in waters contaminated with human fecal samples is reasonably high; these amounts are invariable through the time; their resistance to natural and anthropogenic stressors is comparable to that of other relatively resistant indicator of faecal pollution such us coliphages; the abundance ratios of somatic coliphages and bacteriophages infecting Bacteroides thetaiotaomicron GA17 are unvarying in recent and aged contamination; and standardised detection methods exist. These methods are easy, cost effective and provide data susceptible of numerical analysis. In contrast, there are some uncertainties regarding their geographical stability, and consequently suitable hosts need to be isolated for different geographical areas. However, a feasible method has been described to isolate suitable hosts in a given geographical area. In summary, phages infecting Bacteroides are a marker of faecal sources that in our opinion merits being included in the “toolbox” for microbial source tracking. However, further research is still needed in order to make clear some uncertainties regarding some of their characteristics and behaviour, to compare their suitability to the one of emerging methods such us targeting Bacteroidetes by qPCR assays; or settling molecular methods for their determination.
    Full-text · Article · May 2014 · Water Research
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    • "Recently, a strain of Bact. fragilis (GB-124) was identified as being susceptible to human-sourced phages in Europe (Ebdon et al. 2007; Nnane 2011; Nnane et al. 2011; Ebdon et al. 2012; Ogilvie et al. 2012). However , the geographic distribution of GB-124 and related phages is largely unreported outside of the EU, except for a recent method performance study conducted in California (Harwood et al. 2013). "
    [Show abstract] [Hide abstract] ABSTRACT: Unlabelled: Phages infecting human-associated Bacteroides fragilis (GB-124 phages) have been employed in the European Union (EU) to identify human faecal pollution, but their utility for the United States was unclear. Primary sewage samples were collected seasonally from seven wastewater treatment plants (WWTP) across the continental United States, and more time-intensive sampling was conducted at local WWTPs. All samples were assayed for plaque-forming units (PFU) of GB-124 phages, somatic and FRNA-specific coliphages, as well as adenoviruses (by quantitative PCR [qPCR]). Animal faecal samples (>250) from 14 different species were tested for the presence of the three phage groups. GB-124 phages were consistently detected in sewage (10-10(2) PFU ml(-1) ), but not in animal faeces. While density estimates of both coliphages in sewage were approximately one order of magnitude higher than GB-124 phages, they were both randomly detected in animal faecal samples (10(2) -10(5) g(-1) dry weight). Stability of all three phages was inversely proportional to temperature; persistence was greatest at 5°C compared to 20 and 35°C, where no phages were detectable after a week. In summary, GB-124 phages appear to be a feasible alternative indicator organism and benefit from being sewage associated, while providing an inexpensive detection technique for infectious virions. Significance and impact of the study: Bacteroides fragilis GB-124 phages appear to be restricted to human sewage sources in the United States, being absent from 264 animal faecal samples from 14 different species and present in approx. 90% (34/38) of primary sewage effluent samples collected across the country. Although somatic and F-specific coliphages were present in sewage samples at higher densities, unlike GB-124 phages, both coliphage types were also detected in animal faecal samples. Hence, GB-124 phages may prove to be a useful novel indicator group for human faecal pollution in the continental United States.
    Full-text · Article · Mar 2014 · Letters in Applied Microbiology
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    [Show abstract] [Hide abstract] ABSTRACT: Croceibacter atlanticus HTCC2559T, a marine bacterium isolated from the Sargasso Sea, is a phylogenetically unique member of the family Flavobacteriaceae. Strain HTCC2559T possesses genes related to interaction with primary producers, which makes studies on bacteriophages infecting the strain interesting. Here we report the genome sequence of bacteriophage P2559S, which was isolated off the coast of the Republic of Korea and lytically infects HTCC2559T. Many genes predicted in the P2559S genome had their homologs in Bacteroides phages.
    Full-text · Article · Aug 2012 · Journal of Virology
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