New Details about 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 system as a bacterial defense mechanism against importance to evolution and to ecol- phages (Microbe, May 2009, p. 224). ogy provide an incentive to study Moreover, there is renewed interest in phages T them. The golden era for studying as therapeutic agents against bacterial infec- phages stretched from the 1920s tions, reflecting, in part, frustrations over the through the late 1980s, when the relative sim- emerging resistance of bacteria to conventional plicity of their replication cycle proved critical antibiotics. Thus, for example, in the country of for learning fundamental biology, including Georgia, physicians are using phages to treat identifying the hereditary role of DNA and un- infections. Although phage-based treatments of covering the nature of the genetic code. patients are not authorized in the United States, During the 1990s and until recently, phage the Food and Drug Administration recently ap- biology fell into relative neglect. However, proved the use of a phage mixture to use on bioinformatics and resources such as bacterial particular foods to prevent them becoming con- knockout collections and open reading frame taminated with Listeria. (ORF) libraries are reviving the field, and it again is bringing important payoffs. For exam- ple, researchers recently elucidated the CRISPR Bacteriophage T7 and Its Host

Phage T7 depends on its bacterial host Summary Escherichia coli to propagate. A great deal • Because provide insights into is known about this host-viral partnership, complex phenomena, they are attractive sub- including the tentative roles of more than jects for research. half of each of the 56 of T7 and the Udi Qimron is a • Phage products that prevent or eliminate 4,453 genes of E. coli. senior lecturer in bacterial infections could provide alternative T7, an obligatory lytic phage, unleashes the Department of strategies for fighting antibiotic-resistant patho- more than 100 progeny phage per host in Clinical Immunology gens. less than 25 min under optimal conditions. and Microbiology at • Bacteriophage T7 overcomes host obstacles at The 39,937-bp, double-stranded DNA ge- the Tel Aviv Univer- every step of its lytic cycle, adapting to changes nome of T7 is transcribed from left to right, sity Sackler Medical in host receptors, evading restriction enzymes, generating needed nucleotides, and inactivating with each gene on the physical map sequen- School, Tel Aviv, host enzymes that interfere with its propaga- tially numbered (Fig. 1). The essential genes Israel, and Stanley tion. are assigned integral numbers, while the Tabor is a lecturer • Studying how bacteriophage T7 interacts with nonessential genes are assigned noninteger and Charles C. Ri- its host expands our understanding of how vi- numbers reflecting their relative positions chardson is a Pro- ruses or other microorganisms interact with between essential genes. Genes 2.5, 6.7, and fessor at Harvard even more complex host organisms. 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 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 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- mechanism against bacterio- there, even if he is not a clinician. ture to three years in the Israeli phages, CRISPR, with the goal of Earlier, he earned his B.Sc. degree army, an experience that “sculp- discovering phage products to in 2000 from Ben Gurion Univer- tures the personality and facili- counteract it, as well as for genes sity, majoring in biochemistry and tates maturity,” he says. “The Is- that participate and regulate this microbiology. He continued at raeli culture is very influenced by system. “Once identified, we will that institution, completing his the army and its roughness. . .re- characterize these genes and add Ph.D. in microbiology and immu- flected in the nickname of the na- another brick to the understand- nology in 2004. “I did my Ph.D. tive born Israelis as ‘Sabra,’ a ing of this fascinating system,” he in the lab of Angel Porgador. As in thorny desert plant with a thick says. “Lastly, we are also looking all Ph.D. studies, a side project is hide that conceals a sweet, softer for novel ways to reverse antibi- always essential for backup,” he interior.” Qimron rediscovered otic resistance of using says. “My side project was a girl the roots to some of his personal- bacteriophages.” His interest in named Noga, who did her M.Sc. ity quirks after returning to his bacteria and bacteriophages arose at that time in the same lab, and homeland late last year following long ago. “Even though they have since I was also her side project, five years of postdoctoral research very complicated regulatory path- we mutually agreed to marry.” in Boston, where the atmosphere ways, most of their behavior is He spent 2004–2009 at Har- was more relaxed, even if the cli- possible to interpret using basic vard Medical School as a postdoc- mate was not. principles,” he says. “This relative toral fellow. “Coming to Boston “Here and there I suffer from simplicity, compared to higher eu- was the greatest experience of my some thorns from the Sabra men- karyotes, is what attracts me the life,” he says. “I encountered sev- tality, although I am readjusting most to studying them.” eral giants of science, but more quite fast,” he says. “I definitely Qimron was born in Jerusalem. importantly, I encountered my can’t complain about the weather. His father, Elisha, is a leading ac- postdoc advisor, Charles Rich- Compared to Boston, the week- ademic in the study of ancient He- ardson, who is also an amazingly ends here are heavenly nice. Rain brew and is regarded as an expert kind, smart, and warm person. is considered here—in the semi- on the language used in the Dead He financially supported my fel- desert place—a blessing and, as Sea Scrolls. “Even though he was lowship throughout the years, such, comes only in small, rare a bit disappointed that I did not and gave me all the scientific tools portions.” walk in his exact footsteps, he was that are required to prosper.” The Qimron, 35, is a senior lecturer glad that at least I have the pas- decision to go to Boston “was in the Department of Clinical Im- sion and curiosity for following a planned with the ultimate goal of munology and Microbiology at different kind of science,” Qim- returning to my home country,” the Tel Aviv University Sackler ron says. “He and my mother sup- to help “prepare the next genera- Medical School. His scientific fo- port me in every step of my life, in tion of scientists by teaching stu- cus is on bacterial , specif- every aspect, and I am full of grat- dents the knowledge I have ically bacteriophage T7. “Re- itude for them.” gained,” he says. markably, despite its isolation His family and friends encour- He and Noga have two daugh- over 60 years ago, followed by aged him to study medicine at Tel ters, both born in Boston, Aya, 2, numerous thorough studies, the Aviv University—he was admit- and Stav, six months. “In my free functions of almost half of its en- ted to the medical school there— time I like to play with my daugh- coded genes are still not known,” but he decided to study microbi- ters,” he says. “When they get he says. “We try to identify and ology instead. “My passion for tired of me, I go read books or characterize the specific functions research prevailed,” he says, al- watch TV.” of all its genetic elements.” though today his family and Marlene Cimons Qimron also is studying a friends “are extremely happy” Marlene Cimons lives and writes in newly identified bacterial defense that he joined the medical school Bethesda, Md.

Volume 5, Number 3, 2010 / Microbe Y 119 FIGURE 3 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 5Ј-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 5Ј- monophosphates. These nucleoside monophosphates must be phosphory- lated to yield nucleoside 5Ј-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 Synthesis pathways and maintenance of deoxynucleotides used by T7 phage. The major disruptive mutations in a genetic screen source of dNMP is from degradation products of the host DNA. The dNMPs are to isolate T7 DNA polymerase mutants phosphorylated by the indicated gene products into their respective dNDPs. The host ndk that are protected against the chain-ter- 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 minating dideoxthymidine triphosphate host dgt product. See text for details. (ddTTP). In another screen, we found that the host cmk gene product, dCMP/CMP is expressed at a sufficient level to protect them. kinase, is essential for T7—but not host cell— Meanwhile, how T7 avoids the host RecBCD growth. Thus, while T7 phage encodes a dGMP/ nuclease complex that degrades linear DNA re- dTMP kinase, it does not encode a dCMP ki- mains a mystery. The phage encodes gp5.9 that nase. Because dCTP can be synthesized from specifically binds and inhibits the nuclease activ- UTP via another pathway, the cmk product is ity of RecBCD nuclease. However, gene 5.9 is dispensable for the host but not T7, presumably not expressed until after T7 DNA enters a host due to its faster metabolism. Another explana- cell. tion for the growth deficiency of T7 phage in the The clustered, regularly interspaced short absence of the cmk gene product is the reduced palindromic repeats (CRISPR) system is an- pool of rCTP. Indeed, we find that a specific other way in which host cells defend against mutation in T7 partially overcomes this bacteriophages and other extrachromosomal growth deficiency on cmk cells. T7 primase elements. No phage gene product is known to makes primers that are rich in cytosine, and inhibit the CRISPR system. However, we de- ongoing experiments suggest that a mutant pri- termined that the T7 protein kinase phosphor- mase is less restricted to using CTP for primers ylates threonine and serine residues of one of than is the wild-type enzyme. the components of the CRISPR system, CasB. The dNDP molecules are phosphorylated to Moreover, when this T7 protein kinase phos- the corresponding dNTPs by the abundant ndk phorylates several enzymes, including the host gene product, a broad-acting nucleoside diphos- RNA polymerase and RNaseE, it inhibits phate kinase. The expanded pool of nucleoside them, whereas it enhances the activity of other triphosphates in a cell following T7 phage infec- enzymes, including E. coli RNase III. We are tion is unusual for the host. In fact, dgt of E. coli testing whether gp0.7 phosphorylation of encodes a dGTPase that hydrolyzes dGTP to

120 Y Microbe / Volume 5, Number 3, 2010 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 . 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 ␤Ј 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 ␤Ј 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 and each step of its lytic cycle, there is still much deleting the early host promoters alleviates the to learn about this relatively simple . For requirement to inactivate the host RNAP. Sec- example, we understand the function of only ond, in the absence of T7 DNA packaging pro- half the genes of T7 phage. Protein-protein in- teins such as gp19 and gp10, the phage DNA teractions with the host and with other phage remains intact despite host RNAP activity, indi- proteins also remain obscure. Identifying these cating that packaging proteins can mediate functions and interactions may reveal new DNA cleavage. Third, mutations in gp3.5 that mechanisms of gene regulation and of host re- reduce the pausing of T7 RNAP also alleviate sponse against viral attacks. the requirement to inactivate the host RNAP. In addition, its fast growth rate and rapid Thus, pausing leads to cleavage of the T7 DNA adaptivity make T7 phage well suited for ex- in the absence of sufficient host RNAP inactiva- ploring evolutionary principles. Not only do tion. such studies complement biochemical and ge- netic research on this phage, they also could lead Outlook for T7 Phage Research to a better understanding of viral resistance to Despite a wealth of data on T7 phage structure, inhibitors and to new approaches for developing genetic organization, timing of gene expression, tools to fight antibiotic-resistant pathogens.

SUGGESTED READING Beauchamp, B. B., and C. C. Richardson. 1988. A unique deoxyguanosine triphosphatase is responsible for the optA1 phenotype of Escherichia coli. Proc. Natl. Acad. Sci. USA 85:2563–2567. Dunn, J. J., and F. W. Studier. 1983. Complete nucleotide sequence of bacteriophage T7 DNA and the locations of T7 genetic elements. J. Mol. Biol. 166:477–535. Gawel, D., M. D. Hamilton, and R. M. Schaaper. 2008. A novel mutator of Escherichia coli carrying a defect in the dgt gene, encoding a dGTP triphosphohydrolase. J. Bacteriol. 190:6931–6939. Hamdan, S. M., and C. C. Richardson. 2009. Motors, switches, and contacts in the . Annu. Rev. Biochem. 78:205–243. Molineux, I. J. 2005. The T7 group, p. 275–299. In S. T. Abedon and R. L. Calendar (ed.), The bacteriophages. Oxford University Press, Oxford. Nechaev, S., and K. Severinov. 2008. The elusive object of desire—interactions of bacteriophages and their hosts. Curr. Opin. 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 during bacteriophage T7 infection. J. Mol. Biol. 340:707–730.

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