Phage Genomics as an Educational Platform
Graham F. Hatfull1,2*, Marisa L. Pedulla1,2¤a, Deborah Jacobs-Sera1,2, Pauline M. Cichon1,2, Amy Foley3,
Michael E. Ford1,2¤b, Rebecca M. Gonda1,2, Jennifer M. Houtz1,2, Andrew J. Hryckowian1,2, Vanessa A. Kelchner1,2¤c,
Swathi Namburi1,2, Kostandin V. Pajcini1,2¤d, Mark G. Popovich4, Donald T. Schleicher1,2, Brian Z. Simanek1,2¤e,
Alexis L. Smith1,2, Gina M. Zdanowicz1,2¤f, Vanaja Kumar5, Craig L. Peebles1,2, William R. Jacobs Jr.6,7,
Jeffrey G. Lawrence1,2, Roger W. Hendrix1,2
1 Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America, 2 Pittsburgh Bacteriophage Institute, University of
Pittsburgh, Pittsburgh, Pennsylvania, United States of America, 3 Hampton High School, Allison Park, Pennsylvania, United States of America, 4 Lincoln High School, Elwood
City, Pennsylvania, United States of America, 5 Tuberculosis Research Center, ICMR, Chetpet, Chennai, India, 6 Department of Microbiology and Immunology, Albert Einstein
College of Medicine, New York, New York, United States of America, 7 Howard Hughes Medical Institute, Albert Einstein College of Medicine, New York, New York, United
States of America
Bacteriophages are the most abundant forms of life in the biosphere and carry genomes characterized by high genetic
diversity and mosaic architectures. The complete sequences of 30 mycobacteriophage genomes show them collectively
to encode 101 tRNAs, three tmRNAs, and 3,357 proteins belonging to 1,536 ‘‘phamilies’’ of related sequences, and a
statistical analysis predicts that these represent approximately 50% of the total number of phamilies in the
mycobacteriophage population. These phamilies contain 2.19 proteins on average; more than half (774) of them
contain just a single protein sequence. Only six phamilies have representatives in more than half of the 30 genomes,
and only three—encoding tape-measure proteins, lysins, and minor tail proteins—are present in all 30 phages,
although these phamilies are themselves highly modular, such that no single amino acid sequence element is present
in all 30 mycobacteriophage genomes. Of the 1,536 phamilies, only 230 (15%) have amino acid sequence similarity to
previously reported proteins, reflecting the enormous genetic diversity of the entire phage population. The abundance
and diversity of phages, the simplicity of phage isolation, and the relatively small size of phage genomes support
bacteriophage isolation and comparative genomic analysis as a highly suitable platform for discovery-based education.
Citation: Hatfull GF, Pedulla ML, Jacobs-Sera D, Cichon PM, Foley A, et al. (2006) Exploring the mycobacteriophage metaproteome: Phage genomics as an educational
platform. PLoS Genet 2(6): e92. DOI: 10.1371/journal.pgen.0020092
Approximately 1031tailed bacteriophages are estimated to
be on planet Earth, representing the majority of all biological
entities in the biosphere [1,2]. Phages exert considerable
influence on the microbial community  and cohabit with
their bacterial hosts in highly dynamic relationships [3–5].
While phages show only somewhat limited morphological
variation, the genomes of the approximately 300 completely
sequenced double-strand DNA (dsDNA) phage genomes
reveal both a high level of genetic diversity and a high
proportion of genes that are dissimilar to any that have been
previously sequenced [6–10]. Furthermore, phage genomic
architecture is characterized by a high degree of mosaicism
that likely arises from extensive horizontal genetic exchange
occurring over perhaps as many as 3 billion years [9, 11–13].
dsDNA tailed phages are a substantial proportion of the
total phage population, and the number of completely
sequenced genomes has grown dramatically over the past 5
years to more than 150. These infect a wide variety of
bacterial hosts, but the phages of enteric bacteria, bacteria
relevant to the dairy industry, Mycobacterium spp., Pseudomomas
spp., Staphylococcus spp., and marine bacteria are prevalent
among the completely sequenced dsDNA tailed phage
genomes [6,14–17]. These genomes vary in size from 25 to
greater than 200 kilobase-pairs (kbp) and are harbored by
viruses with a variety of virion morphologies . The high
degree of phage genetic diversity is indicated not only by the
sequenced genomes of individual phages but also by viral
metagenomic approaches showing that the majority of genes
sequenced from uncultured viral libraries have no significant
similarity to known genes [19–23].
Editor: Claire Fraser-Liggett, The Institute for Genomic Research, United States of
Received February 24, 2006; Accepted May 4, 2006; Published June 9, 2006
Copyright: ? 2006 Hatfull 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.
Abbreviations: dsDNA, double-strand DNA; kbp, kilobase-pairs
* To whom correspondence should be addressed. E-mail: firstname.lastname@example.org
¤a Current address: Biology Department, Montana Tech of the University of
Montana, Butte, Montana, United States of America
¤b Current address: J. Weill Medical College of Cornell University, New York, New
York, United States of America
¤c Current address: Bristol-Myers Squibb, Pennington, New Jersey, United States of
¤d Current address: Department of Microbiology and Immunology, Stanford
University, Stanford, California, United States of America
¤e Current address: Department of Mathematics and Statistics, Williams College,
Bronfman Science Center, Williamstown, Massachusetts, United States of America
¤f Current address: Department of Pathology, University of Pittsburgh, Pittsburgh,
Pennsylvania, United States of America
PLoS Genetics | www.plosgenetics.orgJune 2006 | Volume 2 | Issue 6 | e920835
Mycobacteriophages—viruses of the mycobacteria—have
facilitated the development of mycobacterial genetic systems
[24–27] and provided insights into viral diversity and the
evolutionary mechanisms that generate them [6,28–31]. The
comparative genomic analysis of 14 mycobacteriophages
showed that they have relatively large genomes (average
length, 70 kbp), contain large numbers of previously
unidentified genes, and are highly diverse at both the
nucleotide and amino acid sequence levels . Moreover,
phage genome architectures are pervasively mosaic; each
genome apparently contains a unique combination of
individual modules, of which the majority correspond to
one or a small cluster of genes. Generation of these mosaic
genomes reflects a high level of horizontal genetic exchange
within the phage population, with illegitimate recombination
events underlying the generation of new module boundaries.
This model is supported by the identification of a small
number of exchange events that have occurred relatively
recently and for which there is no evidence of targeting to
specific locations, such as gene extremities [6,7].
Since the majority of newly sequenced phage genes,
including those from mycobacteriophages, are not closely
related to known gene sequences, their functions remain
largely unknown [15,19]. In many, but not all, dsDNA tailed
phages, the genes encoding the virion structure and assembly
functions are clustered into long operons with a well-defined
gene order, and these genes can thus be more easily identified
. In phages adorned with a long flexible tail, the length of
the tail is determined by the tmp gene, whose length
corresponds directly to the tail length [6,33]. Since it is
common for these phages to have tail lengths in excess of 100
nm, the tmp gene is frequently the longest open reading frame
in the genome . Outside of the structural operons, gene
functions are well understood only in those phages that have
been thoroughly dissected genetically and biochemically, and
many phages contain large numbers of relatively small open
reading frames of unknown function [6,34].
While the characteristic mosaic architecture of phage
genomes can be explained by abundant horizontal genetic
exchange events in their evolutionary history, little is known
about which genomes participate in these events or the rates
at which exchange occurs. Phages infecting the same host are
expected to be more likely to exchange genes, but phages can
readily switch or expand their host-range by a variety of
mechanisms [35,36]. Moreover, some phages have very broad
host-ranges . In addition, capsid packaging imposes
nonselective constraints on genome length that are quite
distinct from the functional advantages and disadvantages of
phage genes. Little is known about the rates of nonselective
acquisition, or the subsequent fates of such genes in the
phage population, although it is likely that gene acquisition
and loss contribute significantly to the evolution of bacterial
Bacteriophages exchange genes not only among themselves
but also with their host chromosomes, and it is common for
phages to carry genes that profoundly influence the physiol-
ogy of their hosts [41–44]; these include genes that confer
bacterial pathogenicity, such as the toxins responsible for
pathogenesis of cholera, diphtheria, and shigellosis [45,46]. If
bacteriophage mosaicism is largely generated by a process of
illegitimate recombination, then the acquisition of genes
from the host is not surprising . However, for host-like
genes identified by phage genomics, it is unclear what
advantage such genes may confer upon lysogenic hosts or
whether they remain in the phage population for long or
We have expanded our collection of complete mycobacter-
iophage genomes to a total of 30 and utilized the enlarged set
of gene sequences to explore their genetic diversity. The
organization of protein-encoding genes into ‘‘phamilies’’ of
related sequences provides insights into which genes are most
prevalent in these phages, whether there are signature
sequences that are characteristic of mycobacteriophages,
and how phamily size corresponds to phage and bacterial
homologs outside of the mycobacteriophage group. The
genetic diversity, abundance of novel genes, and technical
feasibility of phage isolation and genomic characterization
strongly support the utilization of phage discovery and
comparative genomics as an effective educational platform
for undergraduate and high school students.
Isolation and Characterization of Mycobacteriophages
We previously reported the genome sequences of myco-
bacteriophages L5 , D29 , TM4 , and Bxb1 ,
along with a comparative genomic analysis of these and ten
additional mycobacteriophages: Barnyard, Bxz1, Bxz2, Che8,
Che9c, Che9d, Cjw1, Corndog, Omega, and Rosebush . We
have isolated an additional 16 mycobacteriophages using the
methods described previously  from a variety of sources;
Che12 was isolated in Chennai, India; Bethlehem and U2 are
from Bethlehem, Pennsylvania, United States; 244 is from
Connecticut, United States; and Catera, Cooper, Halo, Llij,
Orion, PBI1, PG1, Pipefish, P-Lot, PMC, Qyrzula, and Wildcat
are from Pittsburgh, Pennsylvania, United States, and the
surrounding areas. Each of these 16 new phages was plaque-
purified, grown in quantity, banded through an equilibrium
CsCl density gradient, and used to isolate virion DNA. DNA
was hydrodynamically sheared, cloned into plasmid vectors,
sequenced, and assembled as described previously . Many
of these phages were isolated, named, and characterized by
undergraduate and high school students, taking advantage of
phage discovery and genomics as a platform for integrating
scientific research and education as described in detail below.
PLoS Genetics | www.plosgenetics.org June 2006 | Volume 2 | Issue 6 | e920836
Bacteriophages are viruses that infect bacterial hosts and are
estimated to be the most numerous biological entities in the
biosphere. Insights into the genetic diversity of the bacteriophage
population and the evolutionary mechanisms that give rise to it can
be obtained using comparative genomic analyses. The genomic
analysis of 30 complete mycobacteriophages—viruses that infect
mycobacterial hosts—reveals them to be genetically diverse and to
contain many previously unidentified genes. The high diversity and
relatively small genome sizes of these phages provide an ideal
platform for introducing high school and undergraduate students to
the research laboratory, isolating and naming novel viruses, and
determining their genomic sequences. The thrill of discovering new
viruses and previously unidentified genes, coupled with ownership
of individual phage projects, provides strong motivations for
students to engage in and pursue scientific research.
Our view of the general features of mycobacteriophage
genomes changes only modestly with a doubling of the
number of genome sequences available (Table 1). The total
sequence information is increased from 979,434 bp to
2,071,001 bp but the average genome length has not changed
significantly. The largest of the newer genomes is Catera
(153.7 kbp), slightly smaller than Bxz1 (156.1 kbp), the largest
previously sequenced mycobacteriophage. One of the newly
sequenced genomes, Halo, is only 42.3 kbp long, substantially
smaller than any other mycobacteriophage genome (Table 1).
Neither the base composition (average %GC content, 63.7%)
nor the average ORF length (600 bp) has changed significantly
(Table 1). These genomes also encode 101 tRNAs and three
tmRNAs. The distribution of these small translation-system
RNAs is quite uneven, with three phages (Bxz1, Wildcat, and
Catera) contributing 80% of the total tRNAs and all three
tmRNAs. Since the primary focus of this paper is to report on
the diversity of mycobacteriophage gene phamilies and the
use of phage genomics as a discovery-based educational
platform, further details of the individual phages and their
genomes will be reported elsewhere.
Nucleotide Sequence Diversity of Mycobacteriophages
Comparison of the 30 mycobacteriophages with each other
at the nucleotide level reveals considerable overall diversity,
with small groups having recognizable sequence similarity
(Figure 1). The most numerous phage genome cluster
contains seven that are more closely related to each other
than to other phages; these include L5, the first sequenced
mycobacteriophage genome , D29 , Bxb1 , Bxz2
, Che12, Bethlehem, and U2. The next most numerous
group contains six members, including the previously
described Rosebush , Orion, PG1, Cooper, Qyrzula, and
Pipefish. Phages PMC, Che8, and Llij form another cluster,
with parts of Che9d and Omega having similarity to these;
two-member groups are formed by phages P-Lot and PBI1,
Cjw1 and 244, and Bxz1 and Catera. Phages showing little or
no nucleotide sequence similarity to any of the others are
TM4, Halo, Che9c, Barnyard, Corndog, and Wildcat.
The profile of nucleotide sequence similarities differs in
several notable ways from the previously reported compar-
ison of only 14 genomes . While the overall diversity
remains very high, 11 of the newly sequenced genomes have
recognizable nucleotide sequence similarity to at least one of
the previously reported genomes. A particular surprise is the
finding that five of the newly sequenced genomes have
detectable nucleotide sequence similarity to the previously
sequenced genome of phage Rosebush (of which no closely
related genomes had been described), although, with the
exception of Qyrzula, the degree of similarity is low. In
contrast, the Bxz1/Catera and Cjw1/244 groups are more
closely related than any other pairs of mycobacteriophage
genomes. Both pairs exhibit greater than 90% nucleotide
sequence identity with a number of small insertions or
deletions accounting for much of the difference. In contrast
to other groups of phages that have been analyzed, the
diversity at the nucleotide sequence level of the mycobacter-
iophages appears to be greater than that of either dairy
phages  or staphylococcal phages , although it is not
yet clear whether this reflects underlying biological diversity
differences or just the relatively small numbers that have
been analyzed and the isolation approaches utilized.
There is no clear correlation between membership in these
clusters of similar phages and the phages’ geographic origins.
Although all the members of the Rosebush group came from
the environs of Pittsburgh, the seven members of the L5
cluster came from Japan, New York City, California, Chenai,
India, and Bethlehem, PA. The smaller groups are also mostly
geographically diverse. Our view at the current level of data
collection is that the clusters of similar phages are widely
Mycobacteriophage Genes and Gene Phamilies
The 30 mycobacteriophage genomes encode a total of 3,357
open reading frames (Table 1). As expected, the newly
sequenced genomes possess a mosaic architecture similar to
those described previously, with modules—frequently con-
taining just a single gene—shared by otherwise distantly
related phages. In order to better understand this genetic
diversity, we have assembled all 3,357 open reading frames
into gene phamilies, i.e., groups of related sequences, using
the criteria that an encoded protein must share amino acid
sequence similarity at an E-value of 0.001 or better or 25%
amino acid identity across its length with at least one other
Table 1. Features of Completely Sequenced Mycobacteriophage
Phage GC% Size (bp)Number
aNumber of tRNAs predicted by tRNA-Scan-SE with a COVE score ¼ 20.
bNumber of tmRNAs predicted by Aragorn v1.1.
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member of the phamily (Table S1). This generates a total of
1,536 phamilies, with a mean phamily size of 2.19 genes
(Figure 2). However, more than half of the phamilies (774,
50.3%) contain just a single constituent gene, and 88% of the
genes are contained within phamilies containing three or
fewer members (Figure 2).
Surprisingly, there are only three phamilies that contain
members in all 30 of the mycobacteriophage genomes. Since
these phamilies have the potential to contain genes or
domains that are present in all mycobacteriophage ge-
nomes—and may thus correspond to mycobacteriophage
signatures—we have examined these more closely. The first of
these, Pham7, contains Lysin A genes, one of two putative
lysins encoded by the mycobacteriophages . The second
lysin, Lysin B (Pham9), is also present in 27 of the 30 genomes
(Figure 2); however, both of these phams are highly diverse,
and they appear to be composed of subgenic modules with
reasonably defined boundaries (Figure 3A). While each of the
Pham7 members has sequence similarity to at least one other
member of the phamily, no single sequence element within
Pham7 is present in all 30 genomes (Figure 3A).
Both of the remaining 30-genome phamilies correspond to
tail proteins. One of these encodes the tape-measure protein
(Tmp) (Pham23) that plays a role in tail assembly and
determines the lengths of noncontractile tails [6,33]. This
phamily is also highly diverse, but the relationships among
the members are complicated, and no well-defined bounda-
ries between shared modules could be recognized; this
complexity is illustrated by the long branch lengths within a
phylogenetic analysis (Figures 3B). The second tail protein
phamily (Pham28) is equally complicated, with ill-defined
module boundaries (Figure 3C), and a total of 81 genes fall
into this phamily (Figure 2A). Nevertheless, as seen in Pham7,
no single sequence element in either of these tail protein
phamilies is present in all 30 genomes.
It is noteworthy that the six most abundant phamilies
(Phams 28, 23, 7, 9, 25, and 109; Figure 2A) appear to be
highly represented not just because of the conservation of
essential functions but also because of their highly divergent
and modular natures. These phamilies correspond to phage
functions—predominantly tail proteins and lysis proteins—
that are expected to be intimately involved in interacting
with their bacterial hosts, and high diversity among phage tail
proteins has been described previously [48,49]. Although the
Pham109 members are related to carboxypeptidases (Figure
2A), Pham109 genes are typically located among tail genes
[6,29]. Thus, we postulate that they are likely to be structural
components of tails.
Phamily-Based Clustering of Mycobacteriophage
The comparison of mycobacteriophage genomes at the
nucleotide level (Figure 1) reveals not only considerable
genetic diversity but also small groups of phages that appear
to be more closely related to each other than they are to other
mycobacteriophages. To explore this further, we examined
Figure 1. Nucleotide Sequence Comparison of 30 Mycobacteriophage Genomes as Illustrated in a Dotter Plot Using a Sliding Window of 25 bp 
The lower triangle represents the relationships at an elevated level of gray-scale relative to the upper triangle, revealing weaker sequence relationships.
PLoS Genetics | www.plosgenetics.orgJune 2006 | Volume 2 | Issue 6 | e920838
the relationships among these phages by asking whether or
not each genome contains a member of each gene phamily by
using the program Splitstree , which accommodates
alternative phylogenetic relationships, to express these data
(Figure 4A). This analysis reveals six clearly defined groups of
genomes that we have termed clusters A through F. Cluster A
contains the previously characterized phages L5, D29, Bxb1,
and Bxz2, plus the newly sequenced genomes of Bethlehem,
Che12, and U2. The second most numerous cluster (cluster B)
includes six genomes, of which only one (Rosebush) was
previously characterized ; the remaining four clusters are
closely related pairs of genomes (Figure 4A).
The relationships presented in Figure 4A help to organize
the discussion of the general features of these genomes, and
we note that this approach bears some resemblance to the
phage proteomic tree described previously , although the
Splitstree presentation enables at least partial inclusion of
phylogenetic ambiguities that arise from the comparison of
genomes that are pervasively mosaic in their architecture. But
while these representations reflect the global relationships of
the phages, it is important to note that they represent
aggregate representations of the evolutionary histories of
these viruses, and this ignores the numerous constituent
phamilies that have phylogenies that are distinct from that
shown in Figure 4A. The six phamilies (Phams 61, 137, 58,
1,072, 216, and 933) shown in Figure 4B illustrate this
problem, underscoring the important role of horizontal
genetic exchange in phage evolution.
An alternative representation of phage genome relation-
ships utilizes phamily circles to identify the participants and
indicate the strength of relationships within each phamily
(Figure 5). For example, Pham58 is present in eight genomes,
with the strongest relationships between the phamily mem-
bers in four of the seven components of cluster A and both
members of cluster E. In contrast, Pham61 members are also
in three of the same four members of cluster A as Pham58,
but these are all closely related to a Pham61 member in
Omega. This representation also illustrates the phylogeny of
the intein present in the Pham216 member in Omega, which
differs substantially from the remainder of the phamily
members. While the phamily circles shown in Figure 5
obviously represent only a subset of all of the possible genetic
relationships in the bacteriophages, a hypothetical extension
to all of the 762 phamilies that contain two or more members
would constitute a complete and accurate representation of
the phylogenies of the protein-encoding genes of these
genomes. The future development of automated circle
drawing software should greatly facilitate this.
Figure 2. Size and Distribution of Mycobacteriophage Phamilies
All 3,357 mycobacteriophage genes were assorted into 1,536 phamilies based on amino acid sequence similarity with a BLAST E value of 0.001 or better
to at least one other member of the phamily.
(A) The distribution of phamilies is shown ranked according to the number of mycobacteriophage genomes containing at least one phamily member.
Examples of specific phams and the total number of mycobacteriophage genes within that pham are shown.
(B) Pie-chart representation of the phamily-size distribution. Phamilies with eight or more members represent about 2% of the total.
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How Many Mycobacteriophage Phams Exist?
To estimate the total number of phams shared among
mycobacteriophages, we calculated the number of phams
found among randomly chosen subsets of the 30 phages
described above (Figure 6). A hyperbolic curve was fit to the
data, where PhamMax is the maximum number of phams in
the population being sampled, and K_Phage is the number
of phages that must be sampled to uncover one-half of the
phams. We found that a curve with PhamMax ¼ 3,064 and
K_Phage¼29 fit the data well (Figure 6). That is, this sample
of 30 phages contains representatives of only half of the
predicted total number of 3,064 phams to be found among
mycobacteriophages, and sequencing a further 100 mycobac-
teriophage genomes would probably uncover members of an
additional approximately 1,000 phams, assuming that phages
that are closely related to known phages are not excluded
from the sample. We note that on average the addition of a
thirtieth phage to a randomly selected group of 29 phages
leads to identifying approximately 25 new phams.
Genetic Novelty of Phage Phamilies
The organization of mycobacteriophage genes into phami-
lies simplifies the process of determining how these are
related to genomes of other phages and their bacterial hosts.
For example, of the 1,536 phamilies, only 230 (15%) have
recognizable sequence similarity (Blast E value of 0.001 or
better) to existing (nonmycobacteriophage) database entries
(Figure 7). Remarkably, over 85% of these mycobacterio-
Figure 3. Complex Relationships within Highly Abundant Mycobacteriophage Phamilies
(A) Complex relationships among members of the Pham7 (Lysin A) phamily. The output of a BLAST comparison of Wildcat gp49 against other
mycobacteriophage proteins shows that only 16 other mycobacteriophage proteins are matched and that these correspond to different parts of
Wildcat gp49. Colored bars represent the strength of the matches, with red being the strongest, followed by purple, blue, and black.
(B) Phylogenetic relationships between members of mycobacteriophage Pham23 (tape-measure protein; Tmp). Amino acid sequences for each of the
30 constituent members of Pham23 were aligned using ClustalW and the unrooted phylogenetic relationships represented using NJTree. Bootstrap
values from 1,000 reiterations are shown.
(C) Chimerism in Pham28 (minor tail) proteins. Llij gp18 is related to both gp18 and gp19 of phage Che8 at high levels of amino acid sequence identity,
and these proteins are related in turn to other members of Pham28 as shown.
PLoS Genetics | www.plosgenetics.orgJune 2006 | Volume 2 | Issue 6 | e920840
phage phamilies, a total of 1,306, represent previously
unidentified genetic sequences, consistent with the idea that
phages may represent the largest reservoir of unexplored
genes in the biosphere [6, 19]. Interestingly, only 126 (54.8%)
of the 230 phamilies with matches correspond to genes found
in other phages, whereas 104 (45.2%) are related to nonphage
(predominantly bacterial) genomes; 57 (24.8%) of the 230
phamilies are found in both phage and bacterial genomes
(Figure 7). Some the phams listed as exclusively matching
bacterial genomes may be identifying unrecognized or
It is clear from this analysis as well as that of other phage
genomes that there is little overlap between the metapro-
teome of phages and that of prokaryotes. Moreover, typically,
greater than 80% of genes within bacterial genomes can be
assigned to clusters of orthologous groups, and in some cases
(e.g., Buchnera) virtually every gene can [52, 53]; this is in
contrast to the high proportion of mycobacteriophage genes
(approximately 50%) that are unrelated to other genes (i.e.,
ORFans). Grouping of the phage genes into phamilies is
therefore justified similarly to the separate ordering of
eukaryotic genes into eukaryotic clusters of orthologous
genes . We also note that only a small proportion of
mycobacteriophage phamilies (approximately 20%) would
qualify as adding to or expanding the current cluster of
orthologous groups database.
Examination of the phams that match other phage or
nonphage genes, or both, reveals qualitative differences
among these groups, particularly in regard to the numbers
of mycobacteriophage genomes within each pham. For
example, the average pham size (defined as the number of
mycobacteriophage genomes containing a phamily member)
of all phams with matches to nonmycobacteriophage genes is
3.88, compared to 1.83 for those with no matches (Figure 7).
Thus, if a mycobacteriophage gene has sequence similarity to
any nonmycobacteriophage gene, it will have more relatives
within the group of mycobacteriophages than one that does
The average pham size is different for the groups of phams
that match phage and nonphage genes (Figure 7). However,
the average pham size for the 126 phams matching all other
phage genes is 4.67, substantially larger than the average size
for all phams (2.19) or those with matches (3.88) (Figure 7);
the subset of these that specifically matches both phage and
nonphage sequences is even larger, with an average pham size
of 5.12 (Figure 7). On the other hand, the average pham size
for the 104 phams that exclusively match nonphage genes is
What is the basis for different distributions of pham sizes?
While the dataset remains relatively small and the 30 genomes
are not all equally different from each other (Figures 1 and 4),
we postulate that the differences in average pham sizes reflect
the extent to which these phams provide functions of general
utility to the mycobacteriophages (i.e., high pham size) or
provide specialist functions to smaller numbers of mycobac-
teriophages that are required to infect specific hosts to or
survive within particular environmental or biological niches.
Examples of the pham group that match other phages include
both structure and assembly genes (e.g., capsids, portals,
terminases) as well as nonstructural genes (e.g., ruvC,
integrases, excises), but these phams presumably provide
important functions to both mycobacteriophages and other
phages. In contrast, the phams matching nonphage gene
products with smaller pham sizes include gene products such
as WhiB, Glutaredoxins, FtsK, Lsr2, DinD, DinG, Ro, and
PurA; these presumably provide more specialist functions. It
seems likely that these specialist functions have been acquired
directly from host genomes, and they are relatively rare
Figure 4. Representation of Mycobacteriophage Clusters Using Splitstree
(A) The relationships between 30 mycobacteriophages are represented by Splitstree representation of a dataset in which each of the 1,536 gene
phamilies is annotated as being either present or absent in each of the 30 genomes. Clusters A through F of genomes that are more closely-related to
each other than to other mycobacteriophages are shown by colored circles.
(B) The distribution of the members of six phamilies on the Splitstree representation in (A) illustrates that individual phamilies have notably different
evolutionary histories than the aggregate representation.
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Figure 5. Phamily Circle Representations of Phamily Relationships
All 30 genomes are shown around the circumference, and the phamily members are linked by a line with the width representing the degree of
similarity. Phamily circles of Pham58 (upper left), Pham61 and Pham1072 (upper right), and Pham137 and Pham993 (lower right) are shown using
different colors for different Phams. Pham216 (bottom left) is shown in turquoise, with the intein present within the Omega phamily member (which
has a different set of relationships) shown in purple.
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because of their restricted utility to a subset of phages, rather
than their lack of opportunity to spread more broadly among
this group of phages that infect at least one common host (i.e.,
Constraints on the Modular Construction of Phage
If there are approximately 3,000 different sequence
phamilies among phages that infect M. smegmatis, how many
different ways can these be combined to make a functional
phage genome? If we assume that an average genome contains
100 genes, then there are approximately 10350possible
ordered phamily combinations. Since there are estimated to
be 1031phage particles in the biosphere and mycobacter-
iophages represent only a fraction of these, it is clear that
only a miniscule fraction of the conceivable gene combina-
tions and organizations have been used. More important, we
expect that there are strong functional constraints imposed
on the generation of competent phage genomes. These
constraints can be grouped into four main categories. First,
each genome must encode all necessary functions not
provided by the host, mandating inclusion of virion structural
and assembly genes, lysis genes, and possibly some DNA
replication functions. Thus, a specific subset of phamilies
with required functions must be present in each genome.
Second, specific gene combinations are required when a gene
can provide functional benefits only in combination with
another gene. One example might be the joint action of holin
and lysin components of the lysis machinery. Third, some
phamilies may encode alternative functions, where the
inclusion of a member of one phamily (e.g., a virion capsid)
precludes the inclusion of any members from another
phamily (e.g., a nonhomologous capsid protein). Fourth, gene
organization is likely to be an important constraint, for
example, allowing cotranscription of genes that need to be
Clearly, the 30 mycobacteriophage genomes are not simply
the result of random assortment of phamilies. At both the
nucleotide (Figure 1) and protein (Figure 4) levels, we can
identify clusters of phages that exhibit greater levels of
similarity to each other than to the larger population; for
example, 21 of the 30 phages fall into six separate clusters at
the nucleotide sequence and the phamily inclusion levels used
here (Figure 4). While it is likely that this clustering reflects in
part the particular host and the isolation procedures used, it
also suggests that there are pools of related phages that enjoy
success within the current environment. This is reminiscent
of what has been seen for phages of other hosts, including
those of Escherichia coli, where separate clusters are repre-
sented by phages lambda, T4, T7, and P2. The types of phages
defined by the clusters of mycobacteriophages are not
obviously correlated with the types seen in the coliphages,
suggesting the possibility that each host type has a distinct set
of phage types associated with it. We suspect instead that each
of the clusters defined by the current collection of genome
sequences is part of a continuum of types extending over a
range of hosts, with no sharply definable boundaries.
However, more data will be required to clarify this question.
It seems likely that an additional constraint on the
assortment of phamilies is that their distribution within
genomes is not independent of each other. For example,
examination of the data set reveals that Pham56 is found in
diverse bacteriophages but only in those that carry the
Figure 6. Estimating the Number of Mycobacteriophage Phams
A subset of the 30 phages was randomly selected without replacement,
and the total number of Phams was determined; this was repeated
10,000 times with the mean shown as a blue circle. For each subset, an
additional phage was then randomly chosen, and the average number of
new Phams found in that phage was determined; these data are shown
as red squares. The total number of Phams was fit to a hyperbolic
function, with the best-fit equation determined by least-squares
Figure 7. Relationships between Mycobacteriophage Phams and
Previously Sequenced Proteins
The number and size of mycobacteriophage phamilies with sequence
similarity to nonmycobacteriophage genes are shown. The numbers of
Phams shared by mycobacteriophages, other phages, and nonphage
genomes are shown, along with the average pham size, defined as the
number of mycobacteriophage genomes containing at least one
member of that phamily. The red circle represents mycobacteriophage
genomes, the green circle represents all dsDNA phage genomes other
than the mycobacteriophages, and the blue circle represents all
nonphage genomes. The number of phams shared between these
groups and the mean mycobacteriophages pham size of those phams
are shown, with arrows indicating whether they are shared by
mycobacteriophages (red circle), nonmycobacteriophage phage ge-
nomes (green circle), or nonphage genomes (blue circle).
PLoS Genetics | www.plosgenetics.orgJune 2006 | Volume 2 | Issue 6 | e920843
Pham25 minor tail protein, suggesting both a role for
Pham56 in virion assembly and a lack of function in genomes
lacking Pham25. The nine members of Pham297 are not only
found in Pham25-bearing genomes, but they are adjacent to
the Pham25-encoding genes, suggesting an even more
intimate association between these proteins. The coassocia-
tion of genes among bacteriophage genomes can only be
assessed using a large collection of diverse genomes, and this
demonstrates how datasets such as this one can be used to
uncover plausible gene functions and interactions.
Phage Discovery as an Educational Tool
The newly discovered phages described here were isolated
and characterized by junior members of the laboratory,
including high school and undergraduate students (Table 2).
The educational benefits of performing scientific research
have been reported previously [54,55], although identifying
suitable research projects and laboratory environments
represents a significant challenge to the research community.
The Phagehunters Program developed at the University of
Pittsburgh—in which students discover and genomically
characterize their own bacteriophages—provides a partic-
ularly strong combination of attributes that maximize the
educational benefits within a research environment; we note
that a successful program with some common features also
was developed by R. Young and colleagues . While there
may be many others, we have identified seven key attributes
of this project that facilitate successful outcomes, and these
are described in Table 3. Identifying the key features of
projects that are well suited for student research should
facilitate the identification and development of other
Two particularly important features of this educational
platform are the strong emphasis on scientific discovery and
project ownership. As evident from the comparative genomic
analysis described above, there are three main discovery
elements. First, there is an excellent prospect of each student
isolating a phage from the environment that is different to all
previously described viruses. Second, within each genome
there is an opportunity to discover new genes that are distinct
to all previously identified genes. Third, many of these phage
genomes surprisingly contain homologs of known genes that
have not been previously found in phage genomes. The high
diversity of the mycobacteriophage population (Figure 1), the
preponderance of novel genes (Figure 7), and the mosaic
architecture of these genomes provide a high promise of
discovery for each participating student.
The opportunity for students to discover novel genes and
viruses is important since it is stimulating and highly
motivating, providing a strong impetus for students to
become engaged in scientific research and to maintain their
involvement even through the more challenging aspects of
their projects. The modest genome size of the phages is such
that a single student can reasonably manage an individual
phage, and project ownership adds motivation and commit-
ment. The prospect of naming their new phage generates
considerable interest among these novice scientists while also
offering opportunities to learn about the justification of a
nonsystematic nomenclature for viruses that contain ge-
nomes with highly individualized mosaic architectures. This
educational platform is clearly not restricted to mycobacter-
iophages and can be readily extended to the isolation and
characterization of environmental phages for other non-
pathogenic hosts; comparative genomic analyses of dairy,
Table 2. Isolation, Sequencing, and Annotation of Mycobacteriophage Genomes
Phage Isolated byStatus Sequencera
High school student
High school student
High school student
High school student
High school teacher
High school teacher
P-LotPBIGC MLP and CB group
aInitials indicate author primarily responsible for sequencing and annotation. PBIGC, Pittsburgh Bacteriophage Institute Genome Center; CB group, a group of ten University of Pittsburgh
undergraduate students taking a course in computational biology. Phage genome sequence determination and annotation were considered a contribution warranting authorship of this
communication. Other authors contributed to experimental design, data analysis, and manuscript preparation.
PLoS Genetics | www.plosgenetics.orgJune 2006 | Volume 2 | Issue 6 | e92 0844
staphylococcal, and pseudomonal phages show that these also
are genetically diverse and typically harbor a high proportion
of novel genes [14,16,17].
The development of multiple projects with parallel
structures as described here brings both advantages and
disadvantages. The main disadvantage is that since the
research path is well established, students generally have only
limited opportunities for experimental planning and design.
However, there are also considerable advantages to parallel
project structures. First, it provides opportunities for a
greater number of students to participate than if each were
independently structured. Second, it facilitates the training
of students through peer and near-peer mentoring systems,
and we find that mentorship of high school students by
undergraduates is a particularly effective combination. More-
over, undergraduate student mentors can be readily trained
using the Entering Mentoring program developed by Pfund
and colleagues . The parallel project structure, coupled
with this mentoring system and the technical simplicity of the
initial project stages, represents essential ingredients for
enabling students in the early stages of their educational
development to engage in scientific research.
Finally, this phage discovery educational platform requires
only modest prior comprehension of biological facts and
concepts. This simplifies access of young students to scientific
research and provides opportunities to students who do not
necessarily excel in more traditional classroom settings. The
platform offers numerous opportunities for students to learn
concepts in microbiology, ecology, genetics, computational
biology, and evolution within an inquiry-driven environment
and is fully inclusive of a diverse variety of learning styles.
Additionally, the significant bioinformatics component of the
program appeals to students with computer science and
engineering backgrounds, and in doing so, it creates a diverse
research group that offers advantages both to the partic-
ipants and the research agenda itself. Detailed protocols are
available at http://www.pitt.edu/;gfh.
The comparative genomic analysis of 30 mycobacterio-
phage genomes provides important new insights into the
diversity and architecture of phage genomes and offers
insights about gene exchange between phage genomes and
between phages and their hosts. It is likely that these general
features will be shared by most other phages, and the recent
comparative analysis of 27 Staphylococcus and 18 Pseudomonas
phages also shows relatively high genetic diversity [16,17].
Phage isolation and genomics is a powerful educational
platform that provides research opportunities to students
from diverse educational backgrounds. The high diversity of
the phage population offers the excitement that each student
can isolate a unique virus and uses genomic approaches to
understand the relationship of the newly discovered phage to
the broader biological world. The ability of students to
contribute successfully to achieving the key scientific goals of
understanding viral diversity, and the underlying evolu-
tionary mechanisms that give rise to it, suggests that phage
isolation and characterization can be used broadly for
Materials and Methods
Phage isolation. Phages were isolated from the following locations:
Cooper, Halo, Llij, Orion, PBI1, PG1, P-Lot, Pipefish, PMC, and
Qyrzula were from Pittsburgh; Bethlehem and U2, Bethlehem,
Pennsylvania, Unites States; Catera and Wildcat, Latrobe, Pennsylva-
nia, United States; 244, Connecticut; and Che12, Chennai, India.
Samples from various sources were extracted with phage buffer,
plated directly onto solid overlays containing 0.35% agar and
Mycobacterium smegmatis mc2155, and incubated at 37 8C for 24 h as
described previously, with the exception of Che12, which was isolated
using Mycobacterium tuberculosis as a host . Individual plaques were
picked and purified through several rounds and purified by CsCl
equilibrium density gradient centrifugation.
Genome sequencing and analysis. Approximately 10 lg of purified
phage DNA was sheared hydrodynamically and repaired, and 1- to 3-
kbp fragments inserted into plasmid pBluescript (Stratagene, La Jolla,
California, United States). Individual clones were sequenced using
ABI3730 or ABI3100 instruments (Applied Biosystems, Foster City,
California, United States), and the sequences were assembled into a
single or small number of contigs . At approximately 8-fold
redundancy, sequence data from oligonucleotide primers used with
phage template DNA generated a single contig and resolved sequence
ambiguities. Genome termini were sometimes identifiable as an
overabundance of clone ends, and a comparison of the sequences
generated using primers annealed to ligated and unligated phage
DNA allowed determination of the molecular ends, where possible.
Sequence assembly was performed using the Phred/Phrap/Consed
suite of programs and annotated using a variety of programs
including DNA Master (available from http://cobamide2.bio.pitt.edu),
Genemark , and Glimmer . tRNA and tmRNA genes were
identified using tRNA-Scan-SE and ARAGORN [61,62]. BLAST
analyses were performed either locally or remotely at the National
Center for Biotechnology Information and were used to assemble a
database of related sequences in Microsoft Excel.
Table 3. Seven Helpful Attributes for High School and Under-
graduate Research Projects
Attribute Description (Phage-Hunting Examples)
1Technical simplicity, especially at initial stages
The initial phage isolation procedures are technically
approachable, and while they become more sophisticated
with genomic characterization, they are within the grasp
of pre-college and undergraduate students working
within a pre-established genomics infrastructure.
Conceptual simplicity and minimal background requirements
To begin phage hunting, no deep conceptual understanding
beyond the high school curriculum is required, although
conceptual appreciation is acquired throughout the project.
Compatibility with flexible scheduling
The varied demands on students’ time requires scheduling
accommodations, and phage isolation and characterization
offers flexible timing.
Multiple achievement milestones
Significant research and educational accomplishments
accrue at distinct steps, such as phage isolation, DNA
purification, microscopy, sequence determination,
and annotation. Achieving each milestone is a success.
Parallel project structure enabling greater numbers of students
While sacrificing some student-initiated research planning,
parallel projects facilitate the involvement of multiple
students and are well suited to peer and near-peer mentoring.
Real research that is publishable and interesting to others
The value of the scientific findings is validated by
Project ownership provides strong motivation
Identifying a new biological entity is an exciting prospect,
and the discovery by students of their own new virus—virtually
guaranteed given the genetic diversity of bacteriophages
and their great abundance—is highly motivating.
Furthermore, naming new viruses using nonsystematic
nomenclature—justified by the nonhierarchical
taxonomic relationships—is fun!
PLoS Genetics | www.plosgenetics.orgJune 2006 | Volume 2 | Issue 6 | e92 0845
Table S1. The Pham Database
Each of the putative proteins encoded by 30 mycobacteriophage
genomes are tabulated according to sequence similarity and
assembled into phamilies. Putative pham functions are also listed.
Found at DOI: 10.1371/journal.pgen.0020092.st001 (250 KB XLS).
GenBank (http://www.ncbi.nlm.nih.gov/Genbank) accession numbers
for phages are L5 (Z18946), D29 (AF022214), Bxb1 (AF271693), TM4
(AF068845), Barnyard (AY129339), Bxz1 (AY129337), Bxz2
(AY129332), Che8 (AY129330), Che9c (AY129333), Che9d
(AY129336), Corndog (AY129335), Cjw1 (AY129331), Omega
(AY129338), Rosebush (AY129334), Catera (DQ398053), Halo
(DQ398042), Wildcat (DQ398052), Pipefish (DQ398049), 244
(DQ398041), Cooper (DQ398044), Llij (DQ398045), Orion
(DQ398046), PMC (DQ398050), Qyrzula (DQ398048), Bethlehem
(AY500153), U2 (AY500152), Che12 (DQ398043), PBI1 (DQ398047),
PG1 (AF547430), and P-Lot (DQ398051).
We thank Molly Scanlon for superb technical assistance and Steve
Cresawn for comments on the manuscript. We are grateful to Jo
Handelsman and colleagues for assistance with the Entering Mentoring
program developed by them. We would also thank Rajeswaru
Dandapani, Curt Wadsworth, Lori Bibb, John Lewis, Bill Brucker,
Vanaja Kumar, Joe Gross, and Jake Falbo for their contributions to
phage isolation. Eric Polinko provided invaluable assistance in the
bioinformatic analyses and coordinating the annotation efforts of ten
students in his Computational Biology class at the University of
Pittsburgh. The specific contributions of other students are listed in
Author contributions. GFH, WRJ, JGL, and RWH conceived and
SN, KVP, MGP, DTS, BZS, ALS, and GMZ performed the experiments.
GFH, MLP, DJS, PMC, AF, MEF, RMG, JMH, AJH, VAK, SN, KVP, MGP,
AF, RMG, JMH, AJH, VAK, SN, KVP, MGP, DTS, BZS, ALS, GMZ, VK,
and JGL contributed reagents/materials/analysis tools. GFH, MLP, DJS,
CLP, WRJ, JGL, and RWH wrote the paper.
Funding. This work was supported in part by a grant to the
University of Pittsburgh by the Howard Hughes Medical Institute
(HHMI) in support of GFH under HHMI’s Professors Program.
Support was also provided by grants from the National Institutes of
Health to RWH (GM51975), GFH (AI28927), WRJ (AI26170), and V.
Kumar (training grant AITTRP); from the David and Lucille Packard
Foundation (JGL); and from the Ellison Medical Foundation (WRJ).
Competing interests. The authors have declared that no competing
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