PERSPECTIVES molecular biology research. In this Timeline, TIMELINE we highlight the impact of phages in the first 100 years since their discovery in terms of A century of the phage: past, present the origins of molecular biology, our knowl- edge of ecology and evolution, and their and future biotechnological exploitation. We encourage readers to try to imagine what the modern world would look like if phages did not exist; George P. C. Salmond and Peter C. Fineran we are clearly indebted to the most abundant Abstract | Viruses that infect bacteria (bacteriophages; also known as phages) were biological entities on Earth. discovered 100 years ago. Since then, phage research has transformed The origins of molecular biology fundamental and translational biosciences. For example, phages were crucial in Key questions in biology addressed. In establishing the central dogma of molecular biology — information is sequentially the early twentieth century, the nature of passed from DNA to RNA to proteins — and they have been shown to have major the gene was a central biological question. roles in ecosystems, and help drive bacterial evolution and virulence. Furthermore, Physicists, including Leo Szilard, Salvador Luria and Max Delbrück as well as other phage research has provided many techniques and reagents that underpin modern researchers (the ‘phage group’), began tack- biology — from sequencing and genome engineering to the recent discovery and ling this and other fundamental biological exploitation of CRISPR–Cas phage resistance systems. In this Timeline, we discuss questions by working with phages as biologi- a century of phage research and its impact on basic and applied biology. cal models5. Delbrück urged researchers to use select ‘authorized phages’, the T‑phages, in their studies to facilitate comparability of Bacteriophages were discovered indepen- led to a decline in interest in phage therapy, results between laboratories. The T-phages dently in 1915 by Frederick Twort, a British although research continued in the former were able to infect Escherichia coli, which pathologist1, and in 1917 by Félix d’Hérelle, Soviet Union and other Eastern European was rapidly becoming the model Gram- a French–Canadian microbiologist2 (FIG. 1). countries. During this period, insights into negative bacterium. In 1939, Emory Ellis Twort described the “glassy transformation” fundamental phage biology were limited, and Delbrück characterized phage growth of micrococci colonies, whereas d’Hérelle and up until the 1940s the viral nature of in the ‘one-step growth experiment’, which isolated an “anti-microbe” of Shigella and phages was still disputed. Visualization of revealed key phage-related concepts, such devised the term ‘bacteriophage’ — literally phages by electron microscopy in the early as adsorption, the latent period and viral meaning bacteria-eater. 1940s proved their particulate nature4. burst6. A few years later, Luria and Delbrück Phages are obligate intracellular para- Since their discovery, phages have had demonstrated that mutations pre-existed sites of bacteria and have diverse life cycles an immense and unforeseen impact on our in the absence of selection — a prediction (BOX 1). The ability of phages to infect bacte- understanding of the wider biological world. of Darwinian theory7. This ‘fluctuation ria led d’Hérelle to examine their therapeutic Their ‘simplicity’ enabled our understanding test’ involved growing independent E. coli potential against bacterial infection. Even of core biological processes that are relevant cultures without selection and then plating in his first paper, he noted that the presence to all biology. Phages provided tractable the bacteria to determine both the total and of phages correlated with disease clearance model systems that gave rise to molecular phage T1‑resistant cell numbers. Consistent in patients with dysentery, and he carried biology and provided many biotechnologi- with a model of pre-existing mutations, the out a rabbit study, in which phages provided cally useful reagents, including restriction number of mutants varied markedly — a fac- protection from infection with Shigella. enzymes, en route. In addition, their influ- tor influenced by when the mutant arose in Most early phage research conducted in the ence on nutrient cycles, pathogenicity and each culture. Retrospectively, we now inter- 1920s and 1930s focused on the develop- bacterial evolution further underlines their pret these experiments in terms of mutations ment of phage therapy for the treatment of central role in global ecology and evolution. in DNA, but the nature of the gene was not bacterial infections, and companies began Furthermore, the inexorable rise of antibi- known at that time. to market phage preparations3. However, otic resistance has provided added impetus By using phages, evidence that genes were in the late 1930s, the Council on Pharmacy for ‘back to the future’ phage-based solutions composed of DNA was provided by Alfred and Chemistry of the American Medical to bacterial infection. We are also currently Hershey and Martha Chase in their ‘Waring Association concluded that the efficacy of witnessing incredible advances in the bio- blender experiment’ (REF. 8). Phages provided phage therapy was ambiguous and that fur- technological exploitation of CRISPR–Cas an ideal model system, as they are composed ther research was required3. These concerns, phage defence systems, which are revolu- of a protein coat and internal DNA — the and the success of emerging antibacterials, tionizing both prokaryotic and eukaryotic two leading genetic contenders at that NATURE REVIEWS | MICROBIOLOGY VOLUME 13 | DECEMBER 2015 | 777 © 2015 Macmillan Publishers Limited. All rights reserved PERSPECTIVES Twort observed “acute infectious apparent activation of enzymes only in 1915 disease of micrococci” (REF. 1) the presence of the substrate11. Using the d’Hérelle identified 1917 lac system in E. coli, they demonstrated ‘bacteriophages’ (REF. 2) co‑transcription of genes that were regulated Phage therapy experiments 1919 conducted by d’Hérelle by a common repressor. Although these experiments exploited some phage-based 1939 Ellis and Delbrück: one-step growth experiment6 constructions, Jacob had also been working Phage visualization by EM4 and on λ prophage induction, which shared simi- beginnings of the ‘phage group’ 1940 lar principles. Further studies on the genetic 1943 Luria and Delbrück: mutations Hershey and Chase: arise without selection7 circuits of phage λ provided numerous DNA is genetic material8 paradigms for gene regulation, including the 1952 Zinder and Lederberg: identification of DNA-binding repressor and transduction40 1955 Benzer: fine structure of phage T4 rII9 activator proteins, and transcriptional anti- terminators. The Nobel Prize in Physiology Lwoff, Horne and Tournier: 1962 virus classification91 or Medicine in 1965 was shared by Jacob, Monod and André Lwoff “for their discover- 1970 Type II restriction enzyme isolated15 ies concerning genetic control of enzyme Fiers: phage MS2 ssRNA and virus synthesis”. These pioneering phage 22 1976 genome sequenced studies defined what we now call ‘genetic Sanger: phage ΦX174 1977 ssDNA genome sequenced23 engineering’ by combining analytical power with simple and elegant in vivo experiments. Smith: phage display developed64 1985 Phages shown to be The ‘birth’ of molecular biology. An unex- 1989 25 abundant in water pected translational benefit of this early Role of phages in microbial turnover demonstrated26–28 1990 research was the discovery of molecular biology reagents, which was facilitated by 1996 Lysogenic conversion in Vibrio cholerae demonstrated37 the increased molecular understanding of Phage relatedness and phages. Restriction–modification (R–M) was 32 1999 mosaicism proposed first observed in the early 1950s as a non- Phage diversity apparent 2002 (ecology and viromics)31 heritable variation following phage passage through particular E. coli hosts12,13. However, 75 First synthetic genome (ΦX174) 2003 Prophage abundance in it was in the late 1960s and early 1970s that genomes appreciated36 CRISPR–Cas adaptive phage 2007 R–M systems were shown to modify DNA immunity demonstrated81 (often by methylation) to protect it from Cas9 RNA-guided nuclease 2012 for genome editing82 cleavage by the partner restriction endonu- clease14. The sequence-specific nature of a PhagoBurn: phage therapy 2013 Phase I/II clinical trial initiated class of restriction enzymes (type II) encour- aged their biotechnological development for cutting discrete DNA fragments15, and phage T4 DNA ligase could be used to join the molecules together16. The ability to use these Figure 1 | Some major events in the 100 years of phage research. EM, electron microscopy; ssDNA, Nature Reviews | Microbiology new reagents to clone genes was a major step single-stranded DNA; ssRNA, single-stranded RNA. in the development of molecular biology and biotechnology industries14. For their work on “the discovery of restriction enzymes and time. They specifically radiolabelled either Shortly after the work of Hershey and Chase, their application to problems of molecular phage proteins (with 35S) or DNA (with 32P). the fine structure of genes was analysed by genetics”, Werner Arber, Daniel Nathans and Unlabelled cells were separately infected Seymour Benzer by investigating the rII Hamilton Smith were awarded the Nobel with the two differentially labelled phage T2 region of phage T4 (REF. 9). He calculated Prize