Contemporary Phage Biology: From Classic Models to New Insights

Bacteriophages, discovered about a century ago, have been pivotal as models for understanding the fundamental principles of molecular biology. While interest in phage biology declined after the phage “golden era,” key recent developments, including advances in phage genomics, microscopy, and the discovery of the CRISPR-Cas anti-phage defense system, have sparked a renaissance in phage research in the past decade. This review highlights recently discovered unexpected complexities in phage biology, describes a new arsenal of phage genes that help them overcome bacterial defenses, and discusses advances toward documentation of the phage biodiversity on a global scale. Bacteriophages, discovered about a century ago, have been pivotal as models for understanding the fundamental principles of molecular biology. While interest in phage biology declined after the phage “golden era,” key recent developments, including advances in phage genomics, microscopy, and the discovery of the CRISPR-Cas anti-phage defense system, have sparked a renaissance in phage research in the past decade. This review highlights recently discovered unexpected complexities in phage biology, describes a new arsenal of phage genes that help them overcome bacterial defenses, and discusses advances toward documentation of the phage biodiversity on a global scale. Bacteriophages (or phages for short) are viruses that infect bacteria. Like any virus, they are obligatory parasites, requiring the host cellular machinery to reproduce. Infection begins by attachment of the phage particle to its host cell through specific recognition of a receptor on the host surface, followed by delivery of the phage nucleic acids into the infected cell. Once inside the bacterium, the phage takes over the bacterial cell, hijacks its cellular components and shuts down its defense mechanisms. Phage genes are expressed, and the phage genome is replicated and eventually packed into self-assembled phage particles. At the end of the lytic infection cycle, progeny phage particles emerge from the cell in a process that usually involves cell lysis by phage proteins (Calendar and Abedon, 2006Calendar R. Abedon S.T. The bacteriophages. Oxford University Press, 2006Google Scholar) (Figure 1A). Most isolated phages (>95%) discovered to date have linear, double-stranded DNA (dsDNA) genomes packed into a tailed proteinaceous capsid (Ackermann, 2007Ackermann H.-W. 5500 Phages examined in the electron microscope.Arch. Virol. 2007; 152: 227-243Crossref PubMed Scopus (315) Google Scholar). Other groups of phages can have non-tailed capsids with dsDNA genome or non-tailed capsids with single-stranded DNA (ssDNA) or RNA genomes (Ackermann, 2006Ackermann H.-W. Classification of Bacteriophages.in: Calendar R. Abedon S.T. The Bacteriophages. Oxford University Press, 2006: 8-16Google Scholar) (Figure 1B). Phages were independently discovered twice: by Twort in 1915 (Twort, 1915Twort F.W. An investigation on the nature of ultra-microscopic viruses.Lancet. 1915; 186: 1241-1243Abstract Google Scholar) and d’Hérelle in 1917 (D’Herelle, 1917D’Herelle F. Sur un microbe invisible antagoniste des bacilles dysentériques.C.R. Acad. Sci. Paris. 1917; 165: 373-375Google Scholar). They were initially studied as anti-bacterials and were later widely used in the clinic, mainly in the former Soviet Union (Abedon et al., 2011Abedon S.T. Kuhl S.J. Blasdel B.G. Kutter E.M. Phage treatment of human infections.Bacteriophage. 2011; 1: 66-85Crossref PubMed Google Scholar). Starting the 1940s, phages became model organisms that facilitated the birth of molecular biology and were utilized to derive the most basic discoveries on the nature of life at the molecular level, including the random nature of mutation (Luria and Delbrück, 1943Luria S.E. Delbrück M. Mutations of bacteria from virus sensitivity to virus resistance.Genetics. 1943; 28: 491-511Crossref PubMed Google Scholar), the discovery that DNA is the genetic material (Hershey and Chase, 1952Hershey A.D. Chase M. Independent functions of viral protein and nucleic acid in growth of bacteriophage.J. Gen. Physiol. 1952; 36: 39-56Crossref PubMed Google Scholar), and the understanding of gene-expression control (Jacob and Monod, 1961Jacob F. Monod J. Genetic regulatory mechanisms in the synthesis of proteins.J. Mol. Biol. 1961; 3: 318-356Crossref PubMed Google Scholar). The history of phage research and its contributions to molecular biology has been recently reviewed (Salmond and Fineran, 2015Salmond G.P.C. Fineran P.C. A century of the phage: past, present and future.Nat. Rev. Microbiol. 2015; 13: 777-786Crossref PubMed Scopus (91) Google Scholar). The deep understanding of phage biology that stemmed from these early studies led to the development of fundamental molecular tools. Such tools are very widely used to date and include gene-expression systems based on the phage T7 RNA polymerase (Studier and Moffatt, 1986Studier F.W. Moffatt B.A. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes.J. Mol. Biol. 1986; 189: 113-130Crossref PubMed Scopus (4419) Google Scholar), the phi29 DNA polymerase that allows single-cell genomics and is an essential component of the PacBio SMRT sequencing technology (Eid et al., 2009Eid J. Fehr A. Gray J. Luong K. Lyle J. Otto G. Peluso P. Rank D. Baybayan P. Bettman B. et al.Real-time DNA sequencing from single polymerase molecules.Science. 2009; 323: 133-138Crossref PubMed Scopus (1671) Google Scholar), and the phage P1-derived Cre-Lox system that is used for site-specific recombination in numerous applications (Sauer, 1987Sauer B. Functional expression of the cre-lox site-specific recombination system in the yeast Saccharomyces cerevisiae.Mol. Cell. Biol. 1987; 7: 2087-2096Crossref PubMed Scopus (303) Google Scholar). The phage display technology, where filamentous phages are used as carriers of peptide libraries displayed on the virion surface, is commonly applied for antibody development and protein interaction studies (Pande et al., 2010Pande J. Szewczyk M.M. Grover A.K. Phage display: concept, innovations, applications and future.Biotechnol. Adv. 2010; 28: 849-858Crossref PubMed Scopus (231) Google Scholar). In addition, phage-centered research led to the discovery of restriction-modification systems, which opened the door to genetic engineering, and more recently the CRISPR-Cas system that became the basis for the genome-editing revolution. Both of these systems are primarily naturally used by bacteria as anti-phage defense systems (Roberts, 2005Roberts R.J. How restriction enzymes became the workhorses of molecular biology.Proc. Natl. Acad. Sci. USA. 2005; 102: 5905-5908Crossref PubMed Scopus (72) Google Scholar, Sorek et al., 2013Sorek R. Lawrence C.M. Wiedenheft B. CRISPR-mediated adaptive immune systems in bacteria and archaea.Annu. Rev. Biochem. 2013; 82: 237-266Crossref PubMed Scopus (273) Google Scholar). Together, these molecular tools are estimated to sell at many millions of dollars per year, exemplifying the huge impact that phage-derived biology had and still has on modern biotechnology. The early decades of phage research led to very deep understanding of phage biology, but the overwhelming majority of knowledge was derived from a very small set of model phages, primarily those infecting Escherichia coli. Between the 1980s and the early 2000s, phages received much less attention as a research subject, presumably because the topic was perceived as largely understood (Young, 2006Young R. Foreword.in: Calendar R. The Bacteriophages. Oxford University Press, New York2006: v-xCrossref Google Scholar). However, advances in genomics and molecular ecology, as well as the discovery that bacteria have an adaptive immune system against phages—CRISPR-Cas—have revived interest in phage research and sparked a renaissance in the field. The purpose of this review is to describe major new knowledge from the past decade or so in phage biology. We note that, as the phage literature is vast, no single review can cover all aspects of phage biology; rather, we focus on novel emerging concepts that stem from studying new, non-model phages or from applying new techniques to study established phage models. Specifically, we highlight discoveries in three areas. First, we discuss the massive expansion of known phage sequence space that occurred in recent years. Second, we describe the complexity of molecular circuits in phages' lysis-lysogeny decisions that was recently revealed to deviate from the known paradigm of the well-studied lambda phage and also discuss an expanding set of cases where lysogenized phages became themselves decision-making switches in bacteria. Third, we review new discoveries regarding the molecular mechanisms employed by phages in their arms race against bacterial defenses and specifically the ways they interact with CRISPR-Cas systems. We conclude with new insights into phage biology revealed by advances in high-resolution microscopy. Through the advances described below, our aim is to highlight the major impact that the recent revitalization of phage research had on our understating of their biology. Phages are known to be highly abundant in multiple environments, and in most environments they outnumber their bacterial hosts (Parikka et al., 2017Parikka K.J. Le Romancer M. Wauters N. Jacquet S. Deciphering the virus-to-prokaryote ratio (VPR): insights into virus-host relationships in a variety of ecosystems.Biol. Rev. Camb. Philos. Soc. 2017; 92: 1081-1100Crossref PubMed Scopus (6) Google Scholar). In seawater, where environmental phages were intensively studied, they are consistently documented to exist in 106–107 particles per milliliter (Parikka et al., 2017Parikka K.J. Le Romancer M. Wauters N. Jacquet S. Deciphering the virus-to-prokaryote ratio (VPR): insights into virus-host relationships in a variety of ecosystems.Biol. Rev. Camb. Philos. Soc. 2017; 92: 1081-1100Crossref PubMed Scopus (6) Google Scholar) and are known to have major ecological roles (Weinbauer, 2004Weinbauer M.G. Ecology of prokaryotic viruses.FEMS Microbiol. Rev. 2004; 28: 127-181Crossref PubMed Scopus (858) Google Scholar). For example, apart from directly shaping the bacterial communities through killing of bacteria, phages rewire bacterial metabolism through phage-encoded metabolic genes (Hurwitz and U’Ren, 2016Hurwitz B.L. U’Ren J.M. Viral metabolic reprogramming in marine ecosystems.Curr. Opin. Microbiol. 2016; 31: 161-168Crossref PubMed Scopus (25) Google Scholar) and can terminate bacterial blooms through the induction of lysogens (Brum et al., 2016Brum J.R. Hurwitz B.L. Schofield O. Ducklow H.W. Sullivan M.B. Seasonal time bombs: dominant temperate viruses affect Southern Ocean microbial dynamics.ISME J. 2016; 10: 437-449Crossref PubMed Scopus (53) Google Scholar). A major development in phage ecology research, which was derived from the genomic and metagenomic revolution, is our ability to begin appreciating the diversity and abundance of phage species in global scales. Prior to this revolution, there was no good way to really assess the extent of this diversity, although hints for the existence of a huge “dark matter” of unexplored phages was available from early studies on phage isolates (Rohwer, 2003Rohwer F. Global phage diversity.Cell. 2003; 113: 141Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar) and from analyses of CRISPR spacers, revealing that only 2% of spacers had hits to known phage sequences and suggesting that 98% of the world “phagome” was unknown (Mojica et al., 2005Mojica F.J.M. Díez-Villaseñor C. García-Martínez J. Soria E. Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements.J. Mol. Evol. 2005; 60: 174-182Crossref PubMed Scopus (701) Google Scholar). Recent applications of the metagenomic approach, in which DNA extracted from environmental samples is directly sequenced and studied, indeed revealed that the majority of phage genomes discovered through this approach are new to science, exposing an overwhelming diversity of phage genomes that were not encountered before (Andersson and Banfield, 2008Andersson A.F. Banfield J.F. Virus population dynamics and acquired virus resistance in natural microbial communities.Science. 2008; 320: 1047-1050Crossref PubMed Scopus (296) Google Scholar, Brum and Sullivan, 2015Brum J.R. Sullivan M.B. Rising to the challenge: accelerated pace of discovery transforms marine virology.Nat. Rev. Microbiol. 2015; 13: 147-159Crossref PubMed Scopus (97) Google Scholar, Brum et al., 2015Brum J.R. Ignacio-Espinoza J.C. Roux S. Doulcier G. 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Sunagawa S. Duhaime M.B. Loy A. Poulos B.T. Solonenko N. Lara E. Poulain J. et al.Tara Oceans CoordinatorsEcogenomics and potential biogeochemical impacts of globally abundant ocean viruses.Nature. 2016; 537: 689-693Crossref PubMed Scopus (104) Google Scholar). Despite the huge diversity of phage genomes revealed via metagenomics, deep sampling of certain habitats brings us closer to documenting a significant portion of their biodiversity. For example, in the surface photic ocean, the most extensively sampled habitat for phage genomes, the discovery rate of new phage gene clusters in metagenomic samples is approaching saturation (Roux et al., 2016Roux S. Brum J.R. Dutilh B.E. Sunagawa S. Duhaime M.B. Loy A. Poulos B.T. Solonenko N. Lara E. Poulain J. et al.Tara Oceans CoordinatorsEcogenomics and potential biogeochemical impacts of globally abundant ocean viruses.Nature. 2016; 537: 689-693Crossref PubMed Scopus (104) Google Scholar). In more sparsely sampled habitats, such as the deeper ocean, discovery rates of new genes and new phages still seem far from saturation (Mizuno et al., 2016Mizuno C.M. Ghai R. Saghaï A. López-García P. Rodriguez-Valera F. Genomes of abundant and widespread viruses from the deep ocean.MBio. 2016; 7: e00805-e00816Crossref PubMed Scopus (11) Google Scholar, Roux et al., 2014Roux S. Hawley A.K. Torres Beltran M. Scofield M. Schwientek P. Stepanauskas R. Woyke T. Hallam S.J. Sullivan M.B. Ecology and evolution of viruses infecting uncultivated SUP05 bacteria as revealed by single-cell- and meta-genomics.eLife. 2014; 3: e03125Crossref PubMed Scopus (50) Google Scholar, Roux et al., 2016Roux S. Brum J.R. Dutilh B.E. Sunagawa S. Duhaime M.B. Loy A. Poulos B.T. Solonenko N. Lara E. Poulain J. et al.Tara Oceans CoordinatorsEcogenomics and potential biogeochemical impacts of globally abundant ocean viruses.Nature. 2016; 537: 689-693Crossref PubMed Scopus (104) Google Scholar). Although the most deeply studied habitat for phage diversity is the marine environment, a new frontier is the study of phages in the human microbiome (Mirzaei and Maurice, 2017Mirzaei M.K. Maurice C.F. Ménage à trois in the human gut: interactions between host, bacteria and phages.Nat. Rev. Microbiol. 2017; 15: 397-408Crossref PubMed Scopus (47) Google Scholar), where saturation in new phage gene discovery also seems to be within reach. Phages are highly abundant in the human gut microbiome and were suggested to be involved in shaping the healthy gut microbiome as well as having a role in pathogenic conditions (Mirzaei and Maurice, 2017Mirzaei M.K. Maurice C.F. Ménage à trois in the human gut: interactions between host, bacteria and phages.Nat. Rev. Microbiol. 2017; 15: 397-408Crossref PubMed Scopus (47) Google Scholar). Although gut phages are very diverse, high-intensity, deep sampling of gut microbiomes revealed a core set of phages that are common among healthy individuals (Manrique et al., 2016Manrique P. Bolduc B. Walk S.T. van der Oost J. de Vos W.M. Young M.J. Healthy human gut phageome.Proc. Natl. Acad. Sci. USA. 2016; 113: 10400-10405Crossref PubMed Scopus (175) Google Scholar, Stern et al., 2012Stern A. Mick E. Tirosh I. Sagy O. Sorek R. CRISPR targeting reveals a reservoir of common phages associated with the human gut microbiome.Genome Res. 2012; 22: 1985-1994Crossref PubMed Scopus (94) Google Scholar). One of the most abundant phages in the human gut microbiome is called crAssPhage, probably infecting a Bacteroides host (Dutilh et al., 2014Dutilh B.E. Cassman N. McNair K. Sanchez S.E. Silva G.G.Z. Boling L. Barr J.J. Speth D.R. Seguritan V. Aziz R.K. et al.A highly abundant bacteriophage discovered in the unknown sequences of human faecal metagenomes.Nat. Commun. 2014; 5: 222-227Crossref Scopus (104) Google Scholar). Although this phage is responsible for up to 90% of viral reads in some gut metagenomic samples (Manrique et al., 2016Manrique P. Bolduc B. Walk S.T. van der Oost J. de Vos W.M. Young M.J. Healthy human gut phageome.Proc. Natl. Acad. Sci. USA. 2016; 113: 10400-10405Crossref PubMed Scopus (175) Google Scholar), it was only recently discovered (Dutilh et al., 2014Dutilh B.E. Cassman N. McNair K. Sanchez S.E. Silva G.G.Z. Boling L. Barr J.J. Speth D.R. Seguritan V. Aziz R.K. et al.A highly abundant bacteriophage discovered in the unknown sequences of human faecal metagenomes.Nat. Commun. 2014; 5: 222-227Crossref Scopus (104) Google Scholar). With the intensive sampling of gut phageomes, discovery of new phage genes in gut microbiome samples now seems to approach saturation (Paez-Espino et al., 2016Paez-Espino D. Eloe-Fadrosh E.A. Pavlopoulos G.A. Thomas A.D. Huntemann M. Mikhailova N. Rubin E. Ivanova N.N. Kyrpides N.C. Uncovering Earth’s virome.Nature. 2016; 536: 425-430Crossref PubMed Scopus (155) Google Scholar). Apart from sampling efforts targeted at viral communities, the repository of phage genomes has recently been significantly expanded by applying new analysis tools to existing sequence databases. Specifically, new tools that detect prophages in bacterial genomes identified close to 13,000 phage genomes integrated within their host genomes, identifying for the first time phages infecting important phyla of bacteria with no previously known phage (Roux et al., 2015Roux S. Hallam S.J. Woyke T. Sullivan M.B. Viral dark matter and virus-host interactions resolved from publicly available microbial genomes.eLife. 2015; 4: 1-20Crossref Google Scholar). In another study, a massive search for virus-associated genes in metagenomic data from over 3,000 geographically diverse samples documented more than 125,000 new viral contigs (large pieces of viral genomes) (Paez-Espino et al., 2016Paez-Espino D. Eloe-Fadrosh E.A. Pavlopoulos G.A. Thomas A.D. Huntemann M. Mikhailova N. Rubin E. Ivanova N.N. Kyrpides N.C. Uncovering Earth’s virome.Nature. 2016; 536: 425-430Crossref PubMed Scopus (155) Google Scholar). Such analyses of metagenomic data revealed novel strategies in phage biology, including extensive utilization of non-canonical codons and stop codons in phage genomes (Ivanova et al., 2014Ivanova N.N. Schwientek P. Tripp H.J. Rinke C. Pati A. Huntemann M. Visel A. Woyke T. Kyrpides N.C. Rubin E.M. Stop codon reassignments in the wild.Science. 2014; 344: 909-913Crossref PubMed Scopus (50) Google Scholar). While metagenomics is the most rapid approach for massive discovery of new phage genomes, deep functional phage research usually necessitates isolation of cultivable phages and their hosts. A recent endeavor, called SEA-PHAGES, attempted to characterize the entire repertoire of phages infecting a single host using a parallel effort of phage isolation, performed by high-school and undergraduate students as part of their early scientific training (Jordan et al., 2014Jordan T.C. Burnett S.H. Carson S. Caruso S.M. Clase K. DeJong R.J. Dennehy J.J. Denver D.R. Dunbar D. Elgin S.C.R. et al.A broadly implementable research course in phage discovery and genomics for first-year undergraduate students.MBio. 2014; 5 (e01051–e13)Crossref PubMed Scopus (86) Google Scholar). This resulted in the largest collection of phages infecting a single host—currently containing ∼8,500 isolated phages infecting Mycobacterium smegmatis, over 1,300 of them fully sequenced and annotated (Pope et al., 2015Pope W.H. Bowman C.A. Russell D.A. Jacobs-Sera D. Asai D.J. Cresawn S.G. Jacobs W.R. Hendrix R.W. Lawrence J.G. Hatfull G.F. Science Education Alliance Phage Hunters Advancing Genomics and Evolutionary SciencePhage Hunters Integrating Research and EducationMycobacterial Genetics CourseWhole genome comparison of a large collection of mycobacteriophages reveals a continuum of phage genetic diversity.eLife. 2015; 4: e06416Crossref PubMed Scopus (70) Google Scholar). Comparison of phage genomes within this large dataset revealed extensive genome mosaicism among these phages, suggesting extremely frequent genetic exchange between phages infecting the same host, even to a point of a “continuum of diversity” (Pope et al., 2015Pope W.H. Bowman C.A. Russell D.A. Jacobs-Sera D. Asai D.J. Cresawn S.G. Jacobs W.R. Hendrix R.W. Lawrence J.G. Hatfull G.F. Science Education Alliance Phage Hunters Advancing Genomics and Evolutionary SciencePhage Hunters Integrating Research and EducationMycobacterial Genetics CourseWhole genome comparison of a large collection of mycobacteriophages reveals a continuum of phage genetic diversity.eLife. 2015; 4: e06416Crossref PubMed Scopus (70) Google Scholar). Studying phages from this set revealed new modes of lysogeny regulation (Broussard et al., 2013Broussard G.W. Oldfield L.M. Villanueva V.M. Lunt B.L. Shine E.E. Hatfull G.F. Integration-dependent bacteriophage immunity provides insights into the evolution of genetic switches.Mol. Cell. 2013; 49: 237-248Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar) and exposed an abundant phenomenon of prophage-mediated protection against attack by other phages (Dedrick et al., 2017Dedrick R.M. Jacobs-Sera D. Bustamante C.A.G. Garlena R.A. Mavrich T.N. Pope W.H. Reyes J.C.C. Russell D.A. Adair T. Alvey R. et al.Prophage-mediated defence against viral attack and viral counter-defence.Nat. Microbiol. 2017; 2: 16251Crossref PubMed Scopus (14) Google Scholar). The expanding set of known phage genome sequences has implications on our understanding of their evolution—for example, a recent study, which analyzed 1,440 genomes of dsDNA viruses, showed evolutionary relatedness between phages and metazoan Herpesvirales based on shared capsid proteins and packaging components (Iranzo et al., 2016Iranzo J. Krupovic M. Koonin E.V. The double-stranded DNA virosphere as a modular hierarchical network of gene sharing.MBio. 2016; 7 (e00978–e16)Crossref PubMed Scopus (24) Google Scholar). Analysis of over 2,000 complete dsDNA phage genomes also revealed two distinct evolutionary strategies of high versus low horizontal gene transfer flux among phage genomes, which are influenced by both the phage ecology and genetic modules (Mavrich and Hatfull, 2017Mavrich T.N. Hatfull G.F. Bacteriophage evolution differs by host, lifestyle and genome.Nat. Microbiol. 2017; 2: 17112Crossref PubMed Scopus (1) Google Scholar). A more complete record of the phage sequence space should enable a more detailed and fine-grained understanding of their evolutionary paths. While we are still very far from documenting the entire space of phage groups in the world, the studies described above demonstrate that very deep sampling can yield near-saturated documentation of the phages that exist in specific habitats or infecting specific hosts, a task considered largely unachievable until recently. While this holds true for the abundant tailed phages, recent discoveries suggest that non-tailed dsDNA, ssDNA and RNA phages are underrepresented in conventional metagenomic studies (Brum et al., 2013Brum J.R. Schenck R.O. Sullivan M.B. Global morphological analysis of marine viruses shows minimal regional variation and dominance of non-tailed viruses.ISME J. 2013; 7: 1738-1751Crossref PubMed Scopus (50) Google Scholar, Kauffman et al., 2018Kauffman K.M. Hussain F.A. Yang J. Arevalo P. Brown J.M. Chang W.K. VanInsberghe D. Elsherbini J. Sharma R.S. Cutler M.B. et al.A major lineage of non-tailed dsDNA viruses as unrecognized killers of marine bacteria.Nature. 2018; 554: 118-122Crossref PubMed Scopus (0) Google Scholar)—a challenge that needs to be addressed. With the exponential growth of available genomic and metagenomic data, we can expect extensive phage documentation in more and more habitats in the foreseeable future, which should ultimately enable true ecology-scale characterization of phage global abundance and diversity. Another field in which key insights have been recently obtained from application of new techniques is phage lysogeny. Lysogeny is a very common alternative life cycle that temperate phages can employ, where instead of replicating and lysing their host, they become latent, either by integrating into their host genome or by forming an episome within the host cell. Once lysogenized, these phage genomes are replicated together with the host genome and can eventually initiate their lytic cycle upon specific cues that usually involve host stress (Howard-Varona et al., 2017Howard-Varona C. Hargreaves K.R. Abedon S.T. Sullivan M.B. Lysogeny in nature: mechanisms, impact and ecology of temperate phages.ISME J. 2017; 11: 1511-1520Crossref PubMed Scopus (5) Google Scholar). Temperate phages affect bacterial communities on multiple levels—they transfer new genes to their hosts, alter the expression of host genes, provide protection against infection by other phages, and kill host populations upon induction. These phenomena were recently reviewed (Howard-Varona et al., 2017Howard-Varona C. Hargreaves K.R. Abedon S.T. Sullivan M.B. Lysogeny in nature: mechanisms, impact and ecology of temperate phages.ISME J. 2017; 11: 1511-1520Crossref PubMed Scopus (5) Google Scholar). A temperate phage needs to make a decision every time it infects a bacterial cell—either to execute the lytic cycle or to become a prophage. The lysis-lysogeny decision (or “molecular switch”) has been a paradigm for molecular decision-making processes since the very early days of molecular biology (Golding, 2011Golding I. Decision making in living cells: lessons from a simple system.Annu. Rev. Biophys. 2011; 40: 63-80Crossref PubMed Scopus (0) Google Scholar, Herskowitz and Hagen, 1980Herskowitz I. Hagen D. The lysis-lysogeny decision of phage λ: explicit programming and responsiveness.Annu. Rev. Genet. 1980; 14: 399-445Crossref PubMed Google Scholar). This topic was thoroughly investigated in the E. coli phage lambda, where it was found to be a complex process involving an intricate network that includes transcriptional repressors and transcriptional activators, as well as RNA degradation, transcription antitermination, and proteolysis. The network integrates information on the metabolic state of the cell and the phage multiplicity of infection to make the eventual decision (Oppenheim et al., 2005Oppenheim A.B. Kobiler O. Stavans J. Court D.L. Adhya S. Switches in bacteriophage lambda development.Annu. Rev. Genet. 2005; 39: 409-429Crossref PubMed Scopus (221) Google Scholar). The basic understanding of the lambda lysis-lysogeny decision began by studying phage mutants in the 50s (Kaiser, 1957Kaiser A.D. Mutations in a temperate bacteriophage affecting its ability to lysogenize Escherichia coli.Virology. 1957; 3: 42-61Crossref PubMed Google Scholar) and was considered largely solved in the 1980s (Herskowitz and Hagen, 1980Herskowitz I. Hagen D. The lysis-lysogeny decision of phage λ: explicit programming and responsiveness.Annu. Rev. Genet. 1980; 14: 399-445Crossref PubMed Google Scholar). However, this decision was thought to be strongly influenced by stochasticity (or “noise”) (Oppenheim et al., 2005Oppenheim A.B. Kobiler O. Stavans J. Court D.L. Adhya S. Switches in bacteriophage lambda development.Annu. Rev. Genet. 2005; 39: 409-429Crossref PubMed Scopus (221) Google Scholar). New studies that examined this process using single-cell techniques now showed that much of the stochasticity ca