New Details about Bacteriophage
T7-Host Interactions
Researchers are showing renewed interest in learning how phages
interact with bacterial hosts, adapting to and overcoming their defenses
Udi Qimron, Stanley Tabor, and Charles C. Richardson
he abundance of phages and their
T importance to evolution and to ecol-
ogy provide an incentive to study
them. The golden era for studying
phages stretched from the 1920s
through the late 1980s, when the relative sim-
plicity of their replication cycle proved critical
for learning fundamental biology, including
identifying the hereditary role of DNA and un-
covering the nature of the genetic code.
During the 1990s and until recently, phage
biology fell into relative neglect. However,
bioinformatics and resources such as bacterial
knockout collections and open reading frame
(ORF) libraries are reviving the field, and it
again is bringing important payoffs. For exam-
ple, researchers recently elucidated the CRISPR
Summary
• Because bacteriophages provide insights into
complex phenomena, they are attractive sub-
jects for research.
• Phage gene products that prevent or eliminate
bacterial infections could provide alternative
strategies for fighting antibiotic-resistant patho-
gens.
• Bacteriophage T7 overcomes host obstacles at
every step of its lytic cycle, adapting to changes
in host receptors, evading restriction enzymes,
generating needed nucleotides, and inactivating
host enzymes that interfere with its propaga-
tion.
• Studying how bacteriophage T7 interacts with
its host expands our understanding of how vi-
ruses or other microorganisms interact with
even more complex host organisms.
system as a bacterial defense mechanism against
phages (Microbe, May 2009, p. 224).
Moreover, there is renewed interest in phages
as therapeutic agents against bacterial infec-
tions, reflecting, in part, frustrations over the
emerging resistance of bacteria to conventional
antibiotics. Thus, for example, in the country of
Georgia, physicians are using phages to treat
infections. Although phage-based treatments of
patients are not authorized in the United States,
the Food and Drug Administration recently ap-
proved the use of a phage mixture to use on
particular foods to prevent them becoming con-
taminated with Listeria.
Bacteriophage T7 and Its
Escherichia coli Host
Phage T7 depends on its bacterial host
Escherichia coli to propagate. A great deal
is known about this host-viral partnership,
including the tentative roles of more than
half of each of the 56 genes of T7 and the Udi Qimron is a
4,453 genes of E. coli. senior lecturer in
T7, an obligatory lytic phage, unleashes the Department of
more than 100 progeny phage per host in Clinical Immunology
less than 25 min under optimal conditions. and Microbiology at
The 39,937-bp, double-stranded DNA ge- the Tel Aviv Univer-
nome of T7 is transcribed from left to right, sity Sackler Medical
with each gene on the physical map sequen- School, Tel Aviv,
tially numbered (Fig. 1). The essential genes Israel, and Stanley
are assigned integral numbers, while the Tabor is a lecturer
nonessential genes are assigned noninteger and Charles C. Ri-
numbers reflecting their relative positions chardson is a Pro-
between essential genes. Genes 2.5, 6.7, and fessor at Harvard
7.3 are essential gene exceptions to that Medical School,
naming practice, as is gene 7, which is now Boston, Mass.
Volume 5, Number 3, 2010 / Microbe Y 117
 FIGURE 1 considered nonessential. Class I genes,
expressed early in infection, establish
favorable conditions for phage growth,
class II genes are expressed later and
mainly encode DNA replication pro-
teins, and class III genes are expressed
during the late stages of phage growth
and mainly encode structural gene
products.

Overcoming Host Obstacles to

Bacteriophage T7 Propagation

Genetic map of bacteriophage T7. T7 DNA is depicted as a black line. Boxes represent The lytic cycle of T7 phage is divided
genes or promoters. Genes and elements relevant to this review are indicated in the into several steps, including adsorption,
map. DNA penetration, DNA replication,
and DNA packaging (Fig. 2). During
adsorption, T7 attaches its tail fiber
FIGURE 2 proteins to lipopolysaccharide mole-
cules (LPS) on the outer membrane of a
host cell. Loss of function of any of the
nine nonessential genes in the LPS core-
biosynthesis pathway of the host con-
fers resistance to T7 infection, and the
frequency of these mutations in the lab-
oratory is about 10-5. However, T7
phage can overcome this resistance by
acquiring mutations in genes encoding
its tail proteins. In fact, by selecting for
T7 phages that infect LPS mutants, we
isolated T7 phages that adsorb to the
host in a LPS-independent manner.
These mutants extend their host range
about 200-fold compared to wild-type
T7.
The mainstay of resistance to phage
is the bacterial restriction system, which
recognizes and cleaves specific DNA se-
quences. By having underrepresented
recognition sequences, T7 phage evades
restriction enzymes, particularly the
type II restriction systems. The phage
genome also encodes gene product (gp)
0.3, a protein that mimics the structure
of a DNA molecule and specifically
binds to and inactivates the type I re-
striction enzyme. As an additional pro-
tective measure, the DNA sequence en-
Obstacles arising in the T7 lytic cycle. Modification to the host LPS may eliminate
recognition by the phage receptor in the adsorption step. Host restriction systems, coding gene 0.3 lacks any sequence
nucleases, as well as the CRISPR system cleave the newly incoming DNA in the recognized by the type I restriction sys-
penetration step. The phage has to generate high enough nucleotide pool and to maintain tem. Further, the sequences located to-
it during the DNA replication step. Finally, the phage has to inactivate the host RNA
polymerase in order to maintain DNA integrity during the packaging step. See text for ward the middle and end of the T7
details. genome enter the cell only after gene 0.3

118 Y Microbe / Volume 5, Number 3, 2010

Qimron: Addressing Thorny Questions Regarding Bacteriophage T7
Udi Qimron traces his Sabra na-
ture to three years in the Israeli
army, an experience that “sculp-
tures the personality and facili-
tates maturity,” he says. “The Is-
raeli culture is very influenced by
the army and its roughness. . .re-
flected in the nickname of the na-
tive born Israelis as ‘Sabra,’ a
thorny desert plant with a thick
hide that conceals a sweet, softer
interior.” Qimron rediscovered
the roots to some of his personal-
ity quirks after returning to his
homeland late last year following
five years of postdoctoral research
in Boston, where the atmosphere
was more relaxed, even if the cli-
mate was not.
“Here and there I suffer from
some thorns from the Sabra men-
tality, although I am readjusting
quite fast,” he says. “I definitely
can’t complain about the weather.
Compared to Boston, the week-
ends here are heavenly nice. Rain
is considered here—in the semi-
desert place—a blessing and, as
such, comes only in small, rare
portions.”
Qimron, 35, is a senior lecturer
in the Department of Clinical Im-
munology and Microbiology at
the Tel Aviv University Sackler
Medical School. His scientific fo-
cus is on bacterial viruses, specif-
ically bacteriophage T7. “Re-
markably, despite its isolation
over 60 years ago, followed by
numerous thorough studies, the
functions of almost half of its en-
coded genes are still not known,”
he says. “We try to identify and
characterize the specific functions
of all its genetic elements.”
Qimron also is studying a
newly identified bacterial defense
mechanism against bacterio-
phages, CRISPR, with the goal of
discovering phage products to
counteract it, as well as for genes
that participate and regulate this
system. “Once identified, we will
characterize these genes and add
another brick to the understand-
ing of this fascinating system,” he
says. “Lastly, we are also looking
for novel ways to reverse antibi-
otic resistance of pathogens using
bacteriophages.” His interest in
bacteria and bacteriophages arose
long ago. “Even though they have
very complicated regulatory path-
ways, most of their behavior is
possible to interpret using basic
principles,” he says. “This relative
simplicity, compared to higher eu-
karyotes, is what attracts me the
most to studying them.”
Qimron was born in Jerusalem.
His father, Elisha, is a leading ac-
ademic in the study of ancient He-
brew and is regarded as an expert
on the language used in the Dead
Sea Scrolls. “Even though he was
a bit disappointed that I did not
walk in his exact footsteps, he was
glad that at least I have the pas-
sion and curiosity for following a
different kind of science,” Qim-
ron says. “He and my mother sup-
port me in every step of my life, in
every aspect, and I am full of grat-
itude for them.”
His family and friends encour-
aged him to study medicine at Tel
Aviv University— he was admit-
ted to the medical school there—
but he decided to study microbi-
ology instead. “My passion for
research prevailed,” he says, al-
though today his family and
friends “are extremely happy”
that he joined the medical school
there, even if he is not a clinician.
Earlier, he earned his B.Sc. degree
in 2000 from Ben Gurion Univer-
sity, majoring in biochemistry and
microbiology. He continued at
that institution, completing his
Ph.D. in microbiology and immu-
nology in 2004. “I did my Ph.D.
in the lab of Angel Porgador. As in
all Ph.D. studies, a side project is
always essential for backup,” he
says. “My side project was a girl
named Noga, who did her M.Sc.
at that time in the same lab, and
since I was also her side project,
we mutually agreed to marry.”
He spent 2004 –2009 at Har-
vard Medical School as a postdoc-
toral fellow. “Coming to Boston
was the greatest experience of my
life,” he says. “I encountered sev-
eral giants of science, but more
importantly, I encountered my
postdoc advisor, Charles Rich-
ardson, who is also an amazingly
kind, smart, and warm person.
He financially supported my fel-
lowship throughout the years,
and gave me all the scientific tools
that are required to prosper.” The
decision to go to Boston “was
planned with the ultimate goal of
returning to my home country,”
to help “prepare the next genera-
tion of scientists by teaching stu-
dents the knowledge I have
gained,” he says.
He and Noga have two daugh-
ters, both born in Boston, Aya, 2,
and Stav, six months. “In my free
time I like to play with my daugh-
ters,” he says. “When they get
tired of me, I go read books or
watch TV.”
Marlene Cimons
Marlene Cimons lives and writes in
Bethesda, Md.
Volume 5, Number 3, 2010 / Microbe Y 119
 FIGURE 3
Synthesis pathways and maintenance of deoxynucleotides used by T7 phage. The major
source of dNMP is from degradation products of the host DNA. The dNMPs are
phosphorylated by the indicated gene products into their respective dNDPs. The host ndk
gene product further phosphorylates the dNDPs into dNTPs that are now available for
DNA synthesis. Gp1.2 protects the elevated pool of dGTP from being degraded by the
host dgt product. See text for details.
is expressed at a sufficient level to protect them.
Meanwhile, how T7 avoids the host RecBCD
nuclease complex that degrades linear DNA re-
mains a mystery. The phage encodes gp5.9 that
specifically binds and inhibits the nuclease activ-
ity of RecBCD nuclease. However, gene 5.9 is
not expressed until after T7 DNA enters a host
cell.
The clustered, regularly interspaced short
palindromic repeats (CRISPR) system is an-
other way in which host cells defend against
bacteriophages and other extrachromosomal
elements. No phage gene product is known to
inhibit the CRISPR system. However, we de-
termined that the T7 protein kinase phosphor-
ylates threonine and serine residues of one of
the components of the CRISPR system, CasB.
Moreover, when this T7 protein kinase phos-
phorylates several enzymes, including the host
RNA polymerase and RNaseE, it inhibits
them, whereas it enhances the activity of other
enzymes, including E. coli RNase III. We are
testing whether gp0.7 phosphorylation of
120 Y Microbe / Volume 5, Number 3, 2010
CasB is a phage counter-defense
against CRISPR.
The rapid synthesis of T7 DNA to yield
100 progeny phage necessitates access to
a large pool of nucleoside 5⬘-tripho-
phates, which is a complex synthetic
pathway within host cells (Fig. 3). More
than 80% of nucleotides used in synthe-
sizing the DNA of T7 progeny derive
from the host chromosome. T7 achieves
this efficient utilization of nucleosides by
encoding an endonuclease, gp3, and an
exonuclease, gp6, that together degrade
the host chromosome into nucleoside 5⬘-
monophosphates. These nucleoside
monophosphates must be phosphory-
lated to yield nucleoside 5⬘-triphosphate
substrates for T7 DNA polymerase.
These phosphorylations are carried out
by both host and phage enzymes. For
example, T7 gp1.7 is a nucleotide kinase
that phosphorylates both dGMP and
dTMP. Its activity was identified through
disruptive mutations in a genetic screen
to isolate T7 DNA polymerase mutants
that are protected against the chain-ter-
minating dideoxthymidine triphosphate
(ddTTP).
In another screen, we found that the
host cmk gene product, dCMP/CMP
kinase, is essential for T7— but not host cell—
growth. Thus, while T7 phage encodes a dGMP/
dTMP kinase, it does not encode a dCMP ki-
nase. Because dCTP can be synthesized from
UTP via another pathway, the cmk product is
dispensable for the host but not T7, presumably
due to its faster metabolism. Another explana-
tion for the growth deficiency of T7 phage in the
absence of the cmk gene product is the reduced
pool of rCTP. Indeed, we find that a specific
mutation in T7 primase partially overcomes this
growth deficiency on cmk cells. T7 primase
makes primers that are rich in cytosine, and
ongoing experiments suggest that a mutant pri-
mase is less restricted to using CTP for primers
than is the wild-type enzyme.
The dNDP molecules are phosphorylated to
the corresponding dNTPs by the abundant ndk
gene product, a broad-acting nucleoside diphos-
phate kinase. The expanded pool of nucleoside
triphosphates in a cell following T7 phage infec-
tion is unusual for the host. In fact, dgt of E. coli
encodes a dGTPase that hydrolyzes dGTP to
guanosine and tripolyphosphate, thus FIGURE 4
maintaining a balanced pool of dGTP.
An absence of this dGTPase yields a
mutator phenotype. T7 phage, which
requires high levels of dGTP, encodes
gp1.2, which specifically inhibits this
host enzyme. However, gp1.2 is essen-
tial for phage growth only when dgt is
overexpressed. For example, a dgt pro-
moter mutation results in increased dgt
expression, restricting phage growth in
the absence of gp1.2. Even though
gp1.2 activity is dispensable for phage
growth when dgt is expressed at normal
levels, its presence presumably helps
maintain levels of dGTP in the host cell
that are sufficient for phage propaga-
tion.

Packaging of Progeny T7 DNA

Is a Complicated Process
Packaging T7 DNA is a complicated
process during which a concatemeric Requirement for inhibition of host RNA polymerase during T7 DNA packaging. The left
T7 DNA molecule is converted into panels depict the processes occurring during normal processing of T7 DNA. T7 RNA
polymerase transcribes from the ⌽OR promoter (a), and pauses at a unique site
unit lengths, each of which is then in- immediately downstream to the concatemer junction (b). This pause, enhanced by gp3.5,
serted into an individual capsid. The T7 recruits the prohead and the terminase complex to nick the DNA and thus produce a T7
RNA polymerase (RNAP) recognizes end for packaging (c). The right panels depict the processes in the absence of sufficient
inhibition of host RNA polymerase. Transcription by T7 RNA initiates either from the ⌽OR
the genomic end of each separate unit of or from the ⌽OL promoters (d,e). Transcription by the slower host RNA polymerase from
the concatemer, presumably pausing the strong E. coli promoters on the left end of the T7 DNA forms a “roadblock” to T7 RNA
the elongation complex at a unique site polymerase. This blockage causes the T7 RNA polymerase to pause at a pseudo-pause
site, and thus recruits the terminase and prohead complex, resulting in truncated left
located immediately after the concate- ends (f).
meric junction. The role of T7 RNAP as
a signaling molecule marking the
genomic end upon pause in transcrip- We find that when the host gene, udk, is overex-
tion provides several advantages to the phage. pressed, T7 gp2 no longer inactivates the host
First, the T7 RNAP elongation complex selects RNAP to a sufficient extent, leading to prema-
only T7 DNA, excluding remnants of host ture breakage of T7 DNA and lack of phage
DNA. Second, the requirement for elongation growth.
complex prevents stochastic packaging at differ- According to our model (Fig. 4), T7 DNA is
ent T7 promoters. Third, pausing dictates spec- cleaved due to the host RNAP causing a road-
ificity for the packaging site of the T7 genome. block to the faster T7 RNAP. This accidental
Because the host RNAP could cleave T7 DNA pause recruits the DNA packaging machinery,
near its early host promoters, T7 inactivates that which begins to cleave the DNA. Ordinarily,
host enzyme during the late stages of the T7 lytic these steps occur only at the unique pause site of
cycle. The T7 gene 2 product, gp2, which is the T7 RNAP, a site that is immediately down-
produced early during infection, binds to the ␤⬘ stream of the concatemer junction where it gen-
subunit of the host RNAP and prevents its load- erates genomic ends. The accidental pauses fol-
ing onto RNA polymerase promoters near the lowed by recruitment of the packaging
left end of the T7 genome. Furthermore, T7 machinery, however, damage the phage DNA,
gp0.7 phosphorylates the ␤⬘ subunit of the host especially near the host promoter sites.
RNAP, increasing its transcription termination. Several lines of evidence support this model.

Volume 5, Number 3, 2010 / Microbe Y 121

 First, the host RNAP destroys T7 DNA, while
deleting the early host promoters alleviates the
requirement to inactivate the host RNAP. Sec-
ond, in the absence of T7 DNA packaging pro-
teins such as gp19 and gp10, the phage DNA
remains intact despite host RNAP activity, indi-
cating that packaging proteins can mediate
DNA cleavage. Third, mutations in gp3.5 that
reduce the pausing of T7 RNAP also alleviate
the requirement to inactivate the host RNAP.
Thus, pausing leads to cleavage of the T7 DNA
in the absence of sufficient host RNAP inactiva-
tion.
Outlook for T7 Phage Research
Despite a wealth of data on T7 phage structure,
genetic organization, timing of gene expression,
and each step of its lytic cycle, there is still much
to learn about this relatively simple virus. For
example, we understand the function of only
half the genes of T7 phage. Protein-protein in-
teractions with the host and with other phage
proteins also remain obscure. Identifying these
functions and interactions may reveal new
mechanisms of gene regulation and of host re-
sponse against viral attacks.
In addition, its fast growth rate and rapid
adaptivity make T7 phage well suited for ex-
ploring evolutionary principles. Not only do
such studies complement biochemical and ge-
netic research on this phage, they also could lead
to a better understanding of viral resistance to
inhibitors and to new approaches for developing
tools to fight antibiotic-resistant pathogens.

SUGGESTED READING
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encoding a dGTP triphosphohydrolase. J. Bacteriol. 190:6931– 6939.
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Microbiol. 11:186 –193.
Qimron, U., A. W. Kulczyk, S. M. Hamdan, S. Tabor, and C. C. Richardson. 2008. Inadequate inhibition of host RNA
polymerase restricts T7 bacteriophage growth on hosts overexpressing udk. Mol. Microbiol. 67:448 – 457.
Qimron, U., B. Marintcheva, S. Tabor, and C. C. Richardson. 2006. Genomewide screens for Escherichia coli genes affecting
growth of T7 bacteriophage. Proc. Natl. Acad. Sci. USA 103:19039 –19044.
Zhang, X., and F. W. Studier. 2004. Multiple roles of T7 RNA polymerase and T7 lysozyme during bacteriophage T7
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122 Y Microbe / Volume 5, Number 3, 2010