Four Escherichia coli O157:H7 Phages: A New Bacteriophage Genus and Taxonomic Classification of T1-Like Phages

Abstract

The T1-like bacteriophages vB_EcoS_AHP24, AHS24, AHP42 and AKS96 of the family Siphoviridae were shown to lyse common phage types of Shiga toxin-producing Escherichia coli O157:H7 (STEC O157:H7), but not non-O157 E. coli. All contained circularly permuted genomes of 45.7-46.8 kb (43.8-44 mol% G+C) encoding 74-81 open reading frames and 1 arginyl-tRNA. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the structural proteins were identical among the four phages. Further proteomic analysis identified seven structural proteins responsible for tail fiber, tail tape measure protein, major capsid, portal protein as well as major and minor tail proteins. Bioinformatic analyses on the proteins revealed that genomes of AHP24, AHS24, AHP42 and AKS96 did not encode for bacterial virulence factors, integration-related proteins or antibiotic resistance determinants. All four phages were highly lytic to STEC O157:H7 with considerable potential as biocontrol agents. Comparative genomic, proteomic and phylogenetic analysis suggested that the four phages along with 17 T1-like phage genomes from database of National Center for Biotechnology Information (NCBI) can be assigned into a proposed subfamily "Tunavirinae" with further classification into five genera, namely "Tlslikevirus" (TLS, FSL SP-126), "Kp36likevirus" (KP36, F20), Tunalikevirus (T1, ADB-2 and Shf1), "Rtplikevirus" (RTP, vB_EcoS_ACG-M12) and "Jk06likevirus" (JK06, vB_EcoS_Rogue1, AHP24, AHS24, AHP42, AKS96, phiJLA23, phiKP26, phiEB49). The fact that the viruses related to JK06 have been isolated independently in Israel (JK06) (GenBank Assession #, NC_007291), Canada (vB_EcoS_Rogue1, AHP24, AHS24, AHP42, AKS96) and Mexico (phiKP26, phiJLA23) (between 2005 and 2011) indicates that these similar phages are widely distributed, and that horizontal gene transfer does not always prevent the characterization of bacteriophage evolution. With this new scheme, any new discovered phages with same type can be more properly identified. Genomic- and proteomic- based taxonomic classification of phages would facilitate better understanding phages diversity and genetic traits involved in phage evolution.

Full text

Four
Escherichia coli
O157:H7 Phages: A New
Bacteriophage Genus and Taxonomic Classification of
T1-Like Phages
Yan D. Niu
1
*, Tim A. McAllister
2
, John H. E. Nash
3,4
, Andrew M. Kropinski
3,5
, Kim Stanford
1
*
1Alberta Agriculture and Rural Development, Agriculture Centre, Lethbridge, Alberta, Canada, 2Lethbridge Research Centre, Agriculture and Agri-Food Canada,
Lethbridge, Alberta, Canada, 3Public Health Agency of Canada, Laboratory for Foodborne Zoonoses, Guelph, Ontario, Canada, 4Department of Pathobiology, Ontario
Veterinary College, University of Guelph, Guelph, Ontario, Canada, 5Department of Cellular and Molecular Biology, University of Guelph, Guelph, Ontario, Canada
Abstract
The T1-like bacteriophages vB_EcoS_AHP24, AHS24, AHP42 and AKS96 of the family Siphoviridae were shown to lyse
common phage types of Shiga toxin-producing Escherichia coli O157:H7 (STEC O157:H7), but not non-O157 E. coli. All
contained circularly permuted genomes of 45.7–46.8 kb (43.8–44 mol% G+C) encoding 74–81 open reading frames and 1
arginyl-tRNA. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the structural proteins were identical
among the four phages. Further proteomic analysis identified seven structural proteins responsible for tail fiber, tail tape
measure protein, major capsid, portal protein as well as major and minor tail proteins. Bioinformatic analyses on the
proteins revealed that genomes of AHP24, AHS24, AHP42 and AKS96 did not encode for bacterial virulence factors,
integration-related proteins or antibiotic resistance determinants. All four phages were highly lytic to STEC O157:H7 with
considerable potential as biocontrol agents. Comparative genomic, proteomic and phylogenetic analysis suggested that the
four phages along with 17 T1-like phage genomes from database of National Center for Biotechnology Information (NCBI)
can be assigned into a proposed subfamily ‘‘Tunavirinae’’ with further classification into five genera, namely ‘‘Tlslikevirus’’
(TLS, FSL SP-126), ‘‘Kp36likevirus’’ (KP36, F20), Tunalikevirus (T1, ADB-2 and Shf1), ‘‘Rtplikevirus’’ (RTP, vB_EcoS_ACG-M12)
and ‘‘Jk06likevirus’’ (JK06, vB_EcoS_Rogue1, AHP24, AHS24, AHP42, AKS96, phiJLA23, phiKP26, phiEB49). The fact that the
viruses related to JK06 have been isolated independently in Israel (JK06) (GenBank Assession #, NC_007291), Canada
(vB_EcoS_Rogue1, AHP24, AHS24, AHP42, AKS96) and Mexico (phiKP26, phiJLA23) (between 2005 and 2011) indicates that
these similar phages are widely distributed, and that horizontal gene transfer does not always prevent the characterization
of bacteriophage evolution. With this new scheme, any new discovered phages with same type can be more properly
identified. Genomic- and proteomic- based taxonomic classification of phages would facilitate better understanding phages
diversity and genetic traits involved in phage evolution.
Citation: Niu YD, McAllister TA, Nash JHE, Kropinski AM, Stanford K (2014) Four Escherichia coli O157:H7 Phages: A New Bacteriophage Genus and Taxonomic
Classification of T1-Like Phages. PLoS ONE 9(6): e100426. doi:10.1371/journal.pone.0100426
Editor: Mark J van Raaij, Centro Nacional de Biotecnologia – CSIC, Spain
Received January 9, 2014; Accepted May 23, 2014; Published June 25, 2014
Copyright: ß2014 Niu 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: This work was supported by the Alberta Livestock and Meat Agency, Agriculture and Agri-Food Canada Peer-Reviewed Project Program. 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.
* Email: dongyan.niu@gov.ab.ca (YDN); kim.stanford@gov.ab.ca (KS)
Introduction
Tailed bacteriophages (phages) with double-strand DNA
genomes belonging to the order Caudovirales are the most abundant
viruses on earth, accounting for 96% of all the phages observed
[1]. Based on tail morphology, these viruses are classified by the
International Committee on Taxonomy of Viruses (ICTV), into
three families2Myoviridae (long contractile tail), Siphoviridae (long
non-contractile tail) and Podoviridae (short non-contractile tail).
Recent advances in sequencing technologies has led to a
proliferation in the sequencing of phage genomes [2,3], enabling
comparative genomics and proteomics to better define phage
taxonomy. The family Myoviridae now contains three subfamilies,
Peduovirinae,Spounavirinae and Tevenvirinae [4], and 18 genera. The
family Podoviridae has been further divided into the Autographivirinae
and Picovirinae subfamilies [2], with a total of eleven genera. The
Siphoviridae account for .61% of described phages [1] and this
family also represents the largest group of fully sequenced phages,
but no subfamilies, and only nine bacterial-specific phage genera
have been described. Classification of the Siphoviridae is currently
under review by ICTV (Adriaenssens, personal communication).
T1-like phages possess terminally redundant and circularly
permuted genomes of ,50 kb, and are currently classified as
members of one genus (Tunalikevirus) within Siphoviridae [5].
Morphologically, they have a polyhedral head 60 nm in diameter
with an extremely flexible non-contractile tail 151 nm in length
and 8 nm in diameter [6]. At present, ICTV only recognizes nine
species of phages within this genus with 1, 1, 6 and 1 infecting
Cronobacter, Enterobacter, Escherichia coli, and Shigella, respectively.
Shiga-toxin producing E. coli O157:H7 (STEC O157:H7)
remains one of leading causes of foodborne illnesses in North
America [7,8]. Although the food production continuum has
introduced control measures to prevent the pathogen from
entering food chain, outbreaks of STEC O157:H7 linked to fresh
produce and beef products continue (http://www.cdc.gov/ecoli/
outbreaks.html and http://www.phac-aspc.gc.ca/fs-sa/fs-fi/ecoli-
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eng.php). Lytic phages offer promise in the prevention and therapy
of bacterial infections in humans [9], livestock [10,11] and plants
[12] and have been employed to decontaminate processed foods
and agricultural products [9,10]. However, the use of phage
therapy to target bacterial pathogens such as STEC O157:H7
[13,14] and Salmonella [15,16] in the digestive tract of livestock
remains challenging. Factors such as the development of phage
resistance, the complexity of predator-prey relationships between
phages and hosts, the diversity and abundance of microflora in the
gastro-intestinal tract all may undermine the effectiveness of phage
therapy. Recently, in-vitro experiments in our laboratory have
indicated that competitive interference between different phage
types may be another factor impacting effectiveness of phage
cocktails [17], even though this approach is often advocated as a
means of avoiding resistance. An improved understanding of
phage taxonomy, proteomics and target receptors could lead to
the formulation of more effective phage cocktails that overcome
resistance development while remaining efficacious.
Previously, four STEC O157:H7-infecting bacteriophages
(vB_EcoS_AHP24, AHS24, AHP42 and AKS96) originally
isolated from cattle feedlots in southern Alberta, Canada were
classified as T1-like Siphoviridae by electron microscopy, but
exhibited divergent genotypes based on EcoRI- or HindIII-
digestion profiles [18]. This study aimed to further define their
genomic and proteomic characteristics as well as infectivity against
STEC O157:H7 and non-pathogenic E. coli (ECOR) strains. We
also conducted comparative genomic, proteomic and phylogenetic
analysis among known T1-like phages in an effort to determine
how these viruses could be optimally classified.
Materials and Methods
Bacteriophage, bacteria and media
Four phages infecting STEC O157:H7 strain R508 (phage type,
PT14) were isolated from the feces of commercial feedlot cattle in
2007 in Alberta, Canada [19] with AHP24 (Pen 10), AHS24 (Pen
10) and AHP42 (Pen 6) from Feedlot B and AKS97 (Pen 2) from
Feedlot A [19] with permission. A single discrete plaque from each
phage was purified three times by the soft agar (0.6%) overlay
method [20] and propagated as previously discribed [18]. Titers of
phages in the stock filtrates were then determined by the soft agar
overlay technique [20]. STEC O157:H7 strain R508 was used as a
host for plaque purification, propagation and titration of the phage
stocks. Other standard laboratory strains of STEC O157:H7
(n = 24) and non-O157 E. coli (n = 73) [21] used to evaluate host
range of four T1-like phages are listed in Table 1. Unless otherwise
indicated the bacterial strains were grown in tryptic soy broth
and/or tryptic soy agar.
Host range and lytic capability
Host range and lytic capability of the phages for STEC
O157:H7 and non-O157 E. coli was assessed using a microplate
phage virulence assay [22]. To estimate multiplicity of infection
(MOI), high titre phage stocks (10
9
210
10
PFU/ml) were serially
diluted and incubated at 37uC for 5 h with 10-fold diluted
overnight cultures of STEC O157:H7 in a 96-well microplate.
After incubation, wells were examined visually for turbidity and
the highest dilution that resulted in complete lysis (no discernable
turbidity) of bacteria was recorded. The MOI for each phage-host
assay was calculated by dividing the initial number of phages in the
highest-dilution wells by the initial number of bacteria added, as
determined by plate counts of serially diluted bacterial cultures.
CsCl density gradient centrifugation
Bacterial nucleic acids were removed from filtered phage lysates
(,10
9
PFU/ml) using DNase1 (Sigma-Aldrich, Oakville, ON,
Canada) and RNaseA (Sigma-Aldrich), and the phage lysates were
concentrated in polyethylene glycol (PEG) 8000 and purified
through two rounds of CsCl density gradient centrifugation [20].
Genome sequencing and annotation
Phage DNA was extracted from the CsCl-purified phage lysates
using the SDS-proteinase K protocol of Sambrook and Russell
[20]. Purified phage DNA was submitted to the Plate-forme
Table 1. Host range and lytic capability of phages AHP24, AHS24, AHP42 and AKS96.
Bacteria Strains
a
Sensitivity
b
of T1-like phages
AHP24 AHS24 AHP42 AKS96
STEC O157:H7 PT8, 33, 38 ++++
PT10, 14a, 28, 32, 34, 46248, 54, 68, 80, 88 +++ +++ +++ +++
PT24 +++ +++ +++ +
PT31 ++ ++ ++ ++
PT45 +++ +++ ++ +++
PT49 ++ ++ ++ ++
PT50, 67 +++ +++ ++ ++
PT51 +++ +++ + +
PT63 ++ ++ + +
PT74 ++ ++ +++ +++
non-O157 E. coli ECOR collection
c
2222
a
PT represents phage type of STEC O157:H7 strains
b
Sensitivities are grouped on the basis of multiplicity of infection (MOI: the lowest ratio of phage to bacteria that resulted in complete lysis of an overnight bacterial
culture during 5 h of incubation with serial dilutions of the phage). +++: extremely susceptible (MOI ,0.01); ++: highly susceptible (0.01 #MOI ,1); +: moderately
susceptible (1 #MOI ,10);
–: non-susceptible (i.e., no lysis observed).
c
ECOR collection represents standard reference strains of Escherichia coli [21].
doi:10.1371/journal.pone.0100426.t001
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Table 2. Features of the T1-like phages from NCBI database.
Phages Phylogenic Cluster Host Head Dimension (nm) Tail Dimension (nm) Genome size (bp) Mole% G
+
C Reference Accession #
TLS A E. coli,Shigella NA NA 49,902 42.7 51 NC_009540
FSL SP-126 A Salmonella NA NA 51,092 42.9 53 KC139513
vB_KpnS_KP36 B Klebsiella NA NA 49,818 50.7 55 NC_019781
F20 B Enterobacter 50 15067 51,543 47.9 54 JN672684
T1 C E. coli,Shigella 60 15168 48,836 45.6 51 NC_005833
Shfl1 C Shigella NA NA 50,661 45.4 NA NC_015456
ADB-2 C E. coli NA NA 50,552 46 52 NC_019725
RTP D E. coli NA NA 46,219 44.3 57 NC_007603
vB_EcoS_ACG-M12 D E. coli 57 17267 46,054 44 56 NC_019404
phiEB49 E E. coli 50 NA 47,180 44 58 JF770475
vB_EcoS_Rogue1 E E. coli 53 15268 45,805 44.2 40 NC_019718
phiJLA23 E E. coli NA NA 43,017 44.2 49 KC333879
phiKP26 E E. coli,Salmonella NA NA 47,285 44.3 50 KC579452
JK06 E E. coli NA NA 46,072 44 NA NC_007291
pSf-1 NA Shigella 73 103613 51,821 44 59 NC_021331
ESP2949-1 NA Cronobacter NA NA 49,116 50.1 60 NC_019509
vB_XveM_DIBBI NA Xanthomonas NA NA 49,981 52.4 NA NC_017981
a
NA: Not applicable.
doi:10.1371/journal.pone.0100426.t002
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d’Analyses Ge´nomiques of the Institut de Biologie Inte´grative et
des Syste`mes (Laval University, Que´bec, QC, Canada) for
sequencing. For each sample, a tagged GS-FLX rapid library
was made according to the manufacturer’s instructions (Roche/
454 sequencing, Brandford, USA). Phage libraries were pooled for
sequencing on a GS-FLX+instrument using titanium chemistry
according to the manufacturer’s instructions (Roche/454). Se-
quencing reads were assembled with the gsAssembler module of
Newbler v. 2.5.3.
Initial genome annotation was completed using myRAST [23].
Geneious v5.4 program (Biomatters Ltd., Auckland, New Zealand)
was used to visually scan the sequence for potential genes. All
predicted proteins were scanned for homologues using BLASTP
and PSI-BLAST [24]. Rho-independent terminators were identi-
fied using WebGeSTer at http://pallab.serc.iisc.ernet.in/gester/
rungester.html [25] and TransTermHP [26]. Promoters were
identified by neural network promoter prediction [27] with visual
inspection. Transfer RNA (tRNA) genes were screened using
Aragorn [28] at http://130.235.46.10/ARAGORN/and tRNAS-
can at http://lowelab.ucsc.edu/tRNAscan-SE/[29]. Transmem-
brane domains were described using TMHMM 2.0 at http://
www.cbs.dtu.dk/services/TMHMM/[30], Phobius at http://
phobius.sbc.su.se/[31] and SPLIT 4.0 at http://split.pmfst.hr/
split/4/[32]. The phage genomes were rendered syntenic by
opening at the initiation codon for the small subunit terminase
gene prior to dotplot alignment, ClustalW alignment and
EMBOSS Stretcher analysis. Whole genome sequences of the
four phages studied, as well as another 17 T1-like phages (Table 2)
from the database of National Center for Biotechnology Infor-
mation (NCBI) were analyzed by local ClustalW algorithm [33,34]
using default parameters and phylogenetic trees were visualized by
FigTree program (available from http://tree.bio.ed.ac.uk/
software/figtree/). CLUSTAL omega [35] was used to align
amino acid sequences of T1-like phages. The GenBank accession
numbers for AHP24, AHS24, AHP42 and AKS96 sequence are
KF771236, KF771238, KF771237 and KF771239, respectively.
Analysis of structural proteins
The in-gel digest and mass spectrometry experiments were
performed by the Proteomics platform of the Eastern Quebec
Genomics Center (Quebec, Canada). CsCl-purified phage parti-
cles were analyzed for structural proteins by standard Tris-glycine
12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE). Samples were mixed with sample loading buffer and
boiled for 5 min before loading. Proteins were stained with
Coomassie brilliant blue R250 (Bio-Rad Laboratories, Missis-
sauga, ON, Canada) and subsequently characterized using
Bionumerics 6.6 software (Applied Maths, Austin, TX, USA).
Bands of interest were excised and de-stained with water. Tryptic
digestion was performed on a MassPrep liquid handling robot
(Waters, Milford, USA) according to the manufacturer’s specifi-
cations and to the modified protocol [36,37]. Briefly, following
reduction with 10 mM dithiothreitol (DTT) and alkylation with
55 mM iodoacetamide, the protein was digested by 126 nM of
modified porcine trypsin (Sequencing grade, Promega, Madison,
WI, USA) at 58uC for 1 h. The proteolytic peptides were then
extracted using 1% formic acid, 2% acetonitrile followed by 1%
formic acid and 50% acetonitrile. The recovered extracts were
pooled, dried by vacuum centrifuge and resuspended into 7 mlof
0.1% formic acid for mass spectrometry. Peptide resuspensions
(2 ml) were separated by online reversed-phase (RP) nanoscale
capillary liquid chromatography (nanoLC) and analyzed by
electrospray mass spectrometry (ES MS/MS). The experiments
were performed with a Agilent 1200 nano pump connected to a
triple time-of-flight mass spectrometer (AB Sciex 5600, Framing-
ham, MA,USA) equipped with a nanoelectrospray ion source (AB
Sciex 5600). Briefly, 2 ml of the peptide resuspension was injected
onto a 15 cm675 mm (internal diameter) PicoFrit column (New
Objective, Woburn, MA), packed with reversed phase C18
particles (5 mm diameter; 300 A
˚pore size; Jupiter 300, Phenom-
enex, Torrance, CA, USA) and eluted in a linear gradient from 2–
50% buffer B (0.1% formic acid in acetonitrile) at flow rate of
300 nl/min for 30 min. Mass spectra were acquired using a data-
dependent acquisition mode using Analyst software version 1.6
(AB Sciex 5600). Each full scan mass spectrum (400 to 1250 m/z)
was followed by collision-induced dissociation of the twenty most
intense ions. Dynamic exclusion was set for a period of 3 sec and a
tolerance of 100 ppm.
All MS/MS peak lists were generated with ProteinPilot Version
4.5 (AB Sciex, Framingham, MA, USA) and analyzed using
Table 3. General genome features of AHP24, AHS24, AHP42 and AKS96.
Phages
Feature AHP24 AHS24 AHP42 AKS96
Size (bp) 46,719 46,440 46,847 45,746
Sequence coverage 31.6 74.6 23.4 47.5
G+C content (%) 43.8 43.8 44 43.9
Total ORFs 78 81 76 74
Average ORF size (bp) 552 531 560 569
% of genome coding for proteins 92.2 92.8 90.9 92.2
No. of gene products similar to known proteins, total 73 71 72 69
No. of gene products similar to known T1, total 72 70 71 68
No. of conserved hypothetical proteins with unknown function 43 42 44 42
No. of hypothetical proteins 5 10 5 4
No. of tRNAs 1111
No. of s
70
promoters 56107
No. of rho-independent terminators 18 18 21 21
doi:10.1371/journal.pone.0100426.t003
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T1-Like Bacteriophages
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Mascot (Matrix Science, London, UK; version 2.3.02). Proteins
were identified by searching against the Uniref100-SiphoViridea
database (release 12-05) as well as an in-house protein database
derived from genome sequences of AHP24, AHS24, AHP42 and
AKS96. Parameters for Mascot used a fragment ion mass
tolerance of 0.10 Da and a parent ion tolerance of 0.10 Da.
Iodoacetamide derivatives of cysteine were specified as a fixed
modification and oxidation products of methionine were specified
as a variable modification with up to two missed cleavages
allowed. Scaffold version 4.0.1 (Proteome Software Inc., Portland,
OR, USA) was used to validate MS/MS based peptide and
protein identifications. The protein identification cut off was set at
a confidence level of 95% (MASCOT score .33) with a
requirement for at least two peptides to match to a protein.
Proteins with similar peptides that could not be differentiated
based on MS/MS analysis alone were grouped to satisfy the
principles of parsimony.
Results
Phages were able to lyse all 24 STEC O157:H7 strains tested,
but displayed no activity against any of the 73 non-O157 E. coli
strains (Table 1). Phages AHP24 and AHS24 exhibited the same
infective pattern, with 17 strains extremely susceptible, 4 strains
highly susceptible and 3 strains moderately susceptible. On the
basis of MOI value, the lytic capability of these two phages was
slightly higher than AHP42 or AKS96.
General genomic feature
All four phages contained circularly permuted genomes of 45.7–
46.8 kb (43.8–44 mol% G+C) encoding 74–81 open reading
frames (ORFs) and 1 arginyl-tRNA (Tables 3 and S12S4).
Furthermore, 18221 rho-dependent terminators and 5210
promoters recognized by host RNA polymerase were identified.
The majority (68–72 ORFs, 86–94%) of the proteins displayed
homology to proteins of other T1-like phages with 32239 of DNA
replication, morphogenesis, genome packing and lysis (Tables 3
and S12S4). Based on functional comparison of the ORFs to the
NCBI database of non-redundant protein sequences, none of the
genes encoded for proteins associated with pathogenesis, integra-
tion or antibiotic resistance.
Comparative analysis
Comparative computational genomic analysis revealed that the
four T1-like phages were collinear (.90.8% pairwise similarity),
with AHP24 and AHS24 having the highest sequence identity
(99.2%). All four phages were 80.8–86.5% identical to phages
JK06, vB_EcoS_Rogue1, phiJLA23 and phiKP26 and 65%
similar to phiEB49 in Cluster E and ,56% related to other
known T1-like phages (Fig. 1A, Table 4). At the protein level,
computational analysis of CoreGenes demonstrated that com-
pared to phage vB_EcoS_Rogue1, phages AHP24 and AHS24
shared greatest number of proteins (67–68, Avg. 91.2% similarity)
in common, followed by AHP42, AKS96, phiKP26 and phiJLA23
(65, 87.8%), phiJLA23, JK06 and phiEB49 (56–58,
Avg.77%)(Table 5).
To obtain a global phylogenetic overview of the relationships
between the T1-like phages, we employed genomic dot-plots of
these genome sequences against each other (Fig. 1A). Clearly,
nucleotide sequence aligned well within each cluster. Phylogenic
analysis of whole genome also demonstrated that phages within
each cluster shared close relatedness at nucleotide level (Fig. 1B).
Nucleotide similarity of phages within each cluster was 82.6%
(Cluster A), 72.8% (Cluster B), 77.6–81.5% (Cluster C), 65.1%
(Cluster D) and 64.2–99.2% (Cluster E), whereas nucleotide
identity shared between each cluster was 48.6–55.7% (Fig. 1C,
Tables 2 and 4). Phages pSf-1 and ESP2949-1 demonstrated lower
nucleotide similarity to phages from each genus (Table 4).
Computational analysis of CoreGenes showed that phages within
same cluster had greater number of homologues (75.7–91.2%)
than those among different clusters (43.7–68%; Table 5). Orphan
phage species pSf-1 and ESP2949-1 did not have over 55 gene
products (,64%) in common as compared with other phages in
each cluster. Considering the close relatedness at both nucleotide
and protein level exhibited by the phages within each cluster, we
propose the establishment of a new subfamily ‘‘Tunavirinae’’
which can be divided into five genera, i.e. ‘‘Tlslikevirus’’ (Cluster
A), ‘‘Kp36likevirus’’ (Cluster B), Tunalikevirus (Cluster C), ‘‘Rtpli-
kevirus’’ (Cluster D) and ‘‘Jk06likevirus’’ (Cluster E) (Fig. 1,
Tables 4 and 5), each of which is named after the first isolated
phage of its type.
Phylogenetic trees were constructed to further investigate
common proteomic features for the large subunit of terminase
(TerL), portal protein (PorT), tail fiber (FibA) and major capsid
proteins (CapS) (Fig. 2). Overall, these analyses substantiated the
establishment of the proposed genera. Interestingly, PorT and
CapS of phage phiEB49 were more closely related to those from
the ‘‘Rtplikevirus’’ (84.4–93% aa identity, ID) than those from the
‘‘Jk06likevirus’’ (70.4–75.2% aa ID). Within the ‘‘Jk06likevirus’’,
CapS from AHP24, AHS24, AHP42 and AKS96 (100% aa ID)
was found to be 70.4–72.3% (aa) related to that from phages JK06,
Rogue1, phiKP26 and phiJLA23 (97.8–99.7% aa ID). Likewise
high diversity of the whole genome presented by orphan phages
pSf-1 and ESP2949-1, low amino acid sequence similarities (,
73.5%) were identified for each of the proteins studied, as
compared to those of other members of the T1-like family.
Proteomics
SDS-PAGE revealed that the structural proteins generated
identical banding patterns among the four phages (Fig. 3). Further
shotgun proteomics by liquid chromatography-tandem mass
spectrometry identified up to 52% of the amino acids in seven
structural proteins including tail fiber, tail tape measure protein,
major capsid, portal protein as well as major and minor tail
proteins (Table 6 and Fig. 3). A major capsid protein (Fig. 3, band
D) was observed to have a molecular mass of 29.7 kDa, similar to
the 33 kDa of the major head subunit P7 protein previously
identified from T1 phage [38]. Also, a conserved hypothetical
protein with a molecular mass of 14.2 kDa (Table 6; Fig. 3, band
Figure 1. Comparative genomic analysis of the 21 known T1-like phages. A, Dot plot alignment of nucleotide identity of the 21 known T1-
like phages using Gepard [61]. The vertical axis shows the phage IDs and horizontal axis indicates phage clusters (highlighted in red box). The
apparent black diagonal lines indicate high degrees of nucleotide sequence identity; while each phage shows 100% identity to itself (displayed as
diagonal line). B, Phylogenetic analysis of whole genomes of the 21 known T1-like phages by ClustalW algorithm. Scale bar represents 0.1
substitutions. C, Whole genome comparisons of phages AHP24 (A), KP36 (B), Rogue1 (C), RTP (D), TLS (E) and T1 (F) using a progressive MAUVE
alignment [62]. The degree of sequence similarity is indicated by the intensity of the colored region. The contiguous black boxes under the colored
region represent the position of the genes; red, large subunit of terminase; green, tail tape measure protein; blue, tail fiber protein I; black, tail fiber
protein II; orange, helicase; pink, common hypothetical protein.
doi:10.1371/journal.pone.0100426.g001
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Table 4. Pairwise nucleotide sequence identity of T1-like collinearized genomes as calculated by EMBOSS Stretcher[44,45].
Cluster Genus DNA sequence similarity (%)
Within
genus between genus and/or species
a
‘‘Tlslikevirus’’ ‘‘Kp36likevirus’’
Tunalikevirus
‘‘Rtplikevirus’’ ‘‘Jk06likevirus’’ Putative orphan species
pSf-1 ESP2949-1
A ‘‘Tlslikevirus’’ 82.6 100 53.4 54.9 48.9 50.3 57.4 56.4
B ‘‘Kp36likevirus’’ 72.8 100 53.3 48.6 49.3 52.6 53.3
CTunalikevirus 77.6–81.5 100 50.6 51.3 54.7 54.6
D ‘‘Rtplikevirus’’ 65.1 100 55.7 49.6 49
E ‘‘Jk06likevirus’’ 64.2–99.2 100 50.5 49.9
a
DNA sequence similarity between different genera/or species was calculated using phage TLS a reference genome for the ‘‘Tlslikevirus’’, phage KP36 for the ‘‘Kp36likevirus’’, phage T1 for the Tunalikevirus, phage RTP for the
‘‘Rtplikevirus’’ and phage JK06 for the ‘‘Jk06likevirus’’.
doi:10.1371/journal.pone.0100426.t004
Table 5. Proteomic analysis of T1-like collinearized genomes using CoreGenes 3.0 program [46–48].
Cluster Genus No. of common proteins shared (%)
a
Within
genus between genus and/or species
‘‘Tlslikevirus’’ ‘‘Kp36likevirus’’
Tunalikevirus
‘‘Rtplikevirus’’ ‘‘Jk06likevirus’’ Putative orphan species
pSf-1 ESP2949-1
A ‘‘Tlslikevirus’’ 72 (82.8) (100) 45 (51.7) 46 (52.9) 42 (48.3) 38 (43.7) 55 (63.2) 35 (46.7)
B ‘‘Kp36likevirus’’ 67 (83.8) (100) 41 (51.3) 38 (47.5) 36 (45) 42 (52.5) 38 (47.5)
CTunalikevirus 61–66 (78.1–
84.6)
(100) 38 (48.7) 37 (47.4) 47 (60.3) 36 (46.2)
D ‘‘Rtplikevirus’’ 58 (77.3) (100) 51 (68) 42 (52.5) 38 (47.5)
E ‘‘Jk06likevirus’’ 56–68 (75.7–
91.2)
(100) 41 (55.4) 33 (44.6)
a
Percentage value in each row is the ratio of homologs shared to total genes which was calculated using phage TLS a reference genome for the ‘‘Tlslikevirus’’, phage KP36 for the ‘‘Kp36likevirus’’, phage T1 for the Tunalikevirus,
phage RTP for the ‘‘Rtplikevirus’’ and phage Rogue1 for the ‘‘Jk06likevirus’’.
doi:10.1371/journal.pone.0100426.t005
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G) was similar to P11 (16 kDa) from phage T1, which has been
proposed to be a second major head component that stabilizes the
later stages of head assembly [39]. A major 23.6 kDa tail protein
(Table 6; Fig. 3, band F) was consistent with a major tail protein
from phage Rogue1 (gp29, 25.9kDa) [40] and from phage T1
(P10, 26 kDa) [39].
Discussion
This study revealed that phages AHP24, AHS24, AHP42 and
AKS96 are closely related members of new proposed genus–
‘‘Jk06likevirus’’. Not surprisingly, the highest degree of nucleotide
identity was shared between AHP24 and AHS24, as they were
isolated simultaneously from fecal pats and manure slurry from the
same feedlot pen [19]. AHP42 and AKS96 originated from
different feedlots, but displayed the second highest degree of
nucleotide sequence similarity, a result that confirms our previous
findings of genomic relatedness of these two isolates based on
restriction enzyme profiles [18]. Our ongoing work has also
characterized a number of additional STEC O157:H7-infecting
phages with TEM morphology, genome size and restriction
enzyme profiles (Niu et al. unpublished data) that are similar to the
four phages in this study, possibly because they were also obtained
from the same commercial feedlots in 2007 [19]. This may suggest
that ‘‘Jk06likevirus’’ are widespread in Alberta feedlots. All four
phages were active against a broad range of STEC O157:H7
reference strains, but did not target non-O157 E. coli, suggesting
that they could be used to control STEC O157:H7 without
harming generic commensal E. coli. Also, the four phages exhibited
strong lytic capability against vast majority of PT strains of STEC
Figure 2. Evolutionary relationships of major proteins. The evolutionary history was inferred using the Neighbor-Joining method [63]. The
optimal tree with the sum of branch length for A, large subunit of terminase ( = 2.05), B, portal protein (= 2.2), C, tail fiber ( =1.61), D, major capsid
( = 2.05), is shown; The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (500 replicates) are shown
next to the branches [64]. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the
phylogenetic tree. The evolutionary distances were computed using the JTT matrix-based method [65] and are in the units of the number of amino
acid substitutions per site. The analysis involved 20 amino acid sequences. All positions containing gaps and missing data were eliminated. There
were a total of 429 positions (A), 244 positions (B), 807 positions (C), 305 positions (D), in each final dataset. Evolutionary analyses were conductedin
MEGA5 [66]. Scale bar represents 0.1 substitutions. The asterisk represents phages of which evolutionary relationships of major proteins differed from
those of whole genome.
doi:10.1371/journal.pone.0100426.g002
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O157:H7, although lytic capability may vary with propagation
hosts. This would make them effective biocontrol agents and
possible low dosage required for therapeutic application.
A total of 21 phages with genome sizes ranging from 43 to
52 kb, similar genomic structure and TEM morphology have been
described. Based on current taxonomic classification of ICTV,
these phages were classified as T1-like phage (Tunalikevirus) within
Siphoviridae. Undoubtedly, more T1-like phages will be identified in
the future and there is a need to establish a more defined
taxonomic system in order to explore the evolutionary relation-
ships and genetic linkages in these types of phage. In the present
study, we aligned whole genome sequences from all 21 T1-like
phages using the ClustalW algorithm, which has been widely used
for nucleotide sequence alignment of viruses [41-43]. The
phylogenetic analysis showed that the T1-related phages fall into
five clusters. Moreover, computational EMBOSS Stretcher
[44,45] and CoreGenes programs [46–48] showed that phages
within each proposed genus were more closely related than those
among genera at both the nucleotide and protein level. This was
also confirmed by the phylogenetic analysis of four key functional
phage proteins. The fact that the viruses related to JK06 have been
isolated independently in Israel (JK06) (GenBank Assession #,
NC_007291), Canada (vB_EcoS_Rogue1, AHP24, AHS24,
AHP42, AKS96) [40] and Mexico (phiKP26, phiJLA23) [49,50]
between 2005 and 2011 indicates that these similar phages are
widely distributed, and that horizontal gene transfer does not
always prevent the characterization of bacteriophage evolution.
Similar finding have been noted as part of the Phage Hunters
Integrating Research and Education (PHIRE) program (http://
phagesdb.org/) and for the global distribution of viruses related to
Listeria phage A511. The results indicate that a new subfamily, the
Figure 3. T1-like structural proteins (Lane 2-5) alongside the
standard marker (Lane1) separated on 12% SDS-PAGE gel and
visualized by Coomassie brilliant blue R250 stain. A, tail fiber
protein; B, tail tape measure protein; C, portal protein; D, major capsid
protein; E, minor tail protein; F, major tail protein; G, conserved
hypothetical protein.
doi:10.1371/journal.pone.0100426.g003
Table 6. Structural proteins of phages AHP24, AHS24, AHP42 and AKS96 identified by mass spectrometry.
Gel band ORF in T1-like phages Theoretical mass (kDa) Observed mass (kDa) Putative function No. of peptides
Sequence coverage
(%)
AHP24 AHS24 AHP42 AKS96
A 24 25 25 23 124.4 128 Tail fiber protein 11–23 11–24
B 18 19 19 18 108.9 94.8 Tail tape measure protein 43–55 43–50
C 04 04 04 04 46.3 43.9 Portal protein 14–25 29–47
D 09 10 10 09 34.7 29.7 Major capsid protein 3–7 9–27
E 21 22 21 20 28.1 27.7 Minor tail protein 3–10 10–50
F 15 16 16 15 23.5 23.6 Major tail protein 5–9 31–52
G 12 13 13 12 13.5 14.2 Conserved hypothetical
protein
3–6 27–42
doi:10.1371/journal.pone.0100426.t006
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‘‘Tunavirinae’’ created within the family Siphoviridae containing the
following genera: a modified Tunalikevirus (T1, ADB-2, Shfl1)
[51,52], ‘‘Tlslikevirus’’ (TLS, FSL SP-126) [51,53], ‘‘Kp36like-
virus’’ (KP36, F20) [54,55], ‘‘Rtplikevirus’’ (RTP, vB_EcoS_ACG-
M12) [56,57]; and ‘‘Jk06likevirus’’ (JK06, vB_EcoS_Rogue1,
AHP24, AHS24, AHP42, AKS96, phiJLA23, phiKP26, phiEB49)
[40,49,50,58]. This would leave two putative orphan species: pSf-1
[59] and ESP2949-1 [60] to be further classified as more phages
are characterized. There is a move within ICTV to eliminate the
order Caudovirales, and its three families (Myoviridae, Siphoviridae and
Podoviridae) as they are not compatible with emerging genomic and
proteomic information on phage phylogeny.
Mitigation of STEC O157:H7 has been a challenge in feedlot
cattle. The newly discovered four members of ‘‘Jk06likevirus’’
exhibited broad host range and strong lytic capability against
STEC O157:H7, emphasizing efficacy and suitability for phage-
based biocontrol of this zoonotic pathogen. In this study, we also
proposed further classification of the 21 known T1-like phages into
one subfamily with five genera, constructing a basis for proper
identification of new phages within the same type. Genomic- and
proteomic- based taxonomic classification of phages would
facilitate a better understanding of phage diversity and genetic
traits involved in phage evolution.
Supporting Information
Table S1 Feature of phage AHP24 gene products and their
functional assignments.
(XLSX)
Table S2 Feature of phage AHS24 gene products and their
functional assignments.
(XLSX)
Table S3 Feature of phage AHP42 gene products and their
functional assignments.
(XLSX)
Table S4 Feature of phage AKS96 gene products and their
functional assignments.
(XLSX)
Acknowledgments
We gratefully acknowledge J. Peters and H. Zahiroddini for technical
assistance and support, and R. Johnson (Public Health Agency of Canada,
Guelph, ON, Canada) for kind provision of bacterial strains.
Author Contributions
Conceived and designed the experiments: YDN TAM KS. Performed the
experiments: YDN. Analyzed the data: YDN JHEN AMK. Contributed
reagents/materials/analysis tools: TAM KS. Wrote the paper: YDN TAM
JHEN AMK KS.
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