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New Details About Bacteriophage T7-Host Interactions 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 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 Escherichia coli Host Phage T7 depends on its bacterial host Summary Escherichia coli to propagate. A great deal • Because bacteriophages 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 genes of T7 and the Udi Qimron is a • Phage gene 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 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- 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 pathogens 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 viruses, specif- every aspect, and I am full of grat- dents the knowledge I have ically bacteriophage T7. “Re- itude for them.” gained,” he says.
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