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Virology 479-480 (2015) 310–330

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Virology

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Review Bacteriophage lambda: Early pioneer and still relevant

Sherwood R. Casjens a,b,n, Roger W. Hendrix c

a Division of Microbiology and Immunology, Pathology Department, University of Utah School of Medicine, Emma Eccles Jones Medical Research Building, 15 North Medical Drive East, Salt Lake City, UT 84112, USA b Biology Department, University of Utah, Salt Lake City, UT 84112, USA c Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260, USA

article info abstract

Article history: Molecular genetic research on bacteriophage lambda carried out during its golden age from the mid- Received 9 December 2014 1950s to mid-1980s was critically important in the attainment of our current understanding of the Returned to author for revisions sophisticated and complex mechanisms by which the expression of genes is controlled, of DNA virus 13 January 2015 assembly and of the molecular nature of lysogeny. The development of molecular cloning techniques, Accepted 5 February 2015 ironically instigated largely by phage lambda researchers, allowed many phage workers to switch their Available online 3 March 2015 efforts to other biological systems. Nonetheless, since that time the ongoing study of lambda and its Keywords: relatives has continued to give important new insights. In this review we give some relevant early Bacteriophage history and describe recent developments in understanding the molecular biology of lambda's life cycle. Lambda & 2015 Elsevier Inc. All rights reserved. Lambdoid phages

Contents

Introduction...... 310 Historical importance and recent progress ...... 311 The lambda genome ...... 311 Control of transcription during lytic growth ...... 312 Control of lysogeny ...... 313 Integration and excision ...... 315 DNA replication and homologous recombination ...... 315 Interactions between phage lambda and its host ...... 316 Virion assembly...... 317 Celllysis...... 319 DNA delivery from the virion into target cells...... 319 Non-essential accessory genes ...... 320 Lysogenic conversion ...... 321 Molecular cloning of DNA...... 321 Bacteriophage diversity and the evolution of modular viral genomes ...... 322 Thefuture...... 323 Acknowledgments...... 323 References...... 323

Introduction

From the earliest studies on lambda genetics by Jean Weigle n Corresponding author at: Division of Microbiology and Immunology, Pathology (1953),FrancoisJacob and Elie Wollman (1953, 1954) and Dale Kaiser Department, University of Utah School of Medicine, Emma Eccles Jones Medical (1955 in volume number 1 of VIROLOGY) in the 1950s until it helped Research Building, 15 North Medical Drive East, Salt Lake City, UT 84112, USA. Tel.: þ1 801 581 5980. usher in the age of genetic engineering in the late 1970s and early E-mail address: [email protected] (S.R. Casjens). 1980s, phage lambda was in its golden age at the center of the

http://dx.doi.org/10.1016/j.virol.2015.02.010 0042-6822/& 2015 Elsevier Inc. All rights reserved. S.R. Casjens, R.W. Hendrix / Virology 479-480 (2015) 310–330 311 molecular genetic universe. During those decades lambda was one of to show how far the field has come in the past 60 years (we the few experimental “organisms” that was sufficiently experimen- assume a basic knowledge of the phage lambda life cycle; see tally accessible to be viewed as potentially completely understand- Hendrix et al., 1983; Hendrix and Casjens, 2006). We include able. Its genome was small enough not to be overwhelming, yet it discussion of phages related to lambda, which are commonly refe- was capable of making a decision of whether to lysogenize or grow rred to as “lambdoid” phages. This ill-defined (and often incor- lytically and was clearly complex enough to be interesting and rectly used) term refers phages with very similar lifestyles to informative on many fronts. In particular it was unique in that a lambda and genomes that are mosaically related to lambda. This genetic and molecular understanding of the control of gene expres- definition included the notion that a lambdoid phage is capable of sion seemed to be within almost immediate reach. Those of us lucky recombination with lambda itself to produce a functional hybrid enough to be involved in the study of phage lambda during this time phage, as was first shown with phage 434 by Kaiser and Jacob were tremendously excited about future possibilities and threw (1957). Recent phage genome sequences have caused an expansion ourselves into the work with all the energy we had. of this term to mean a phage with the same functional gene order Lambda was originally discovered in 1951 by Esther Lederberg as lambda and that carries patches of nucleotide sequence homol- (1951) at the University of Wisconsin (Madison), when she seren- ogy with lambda or another lambdoid phage. Thus, in theory a dipitously found it was released from the laboratory Escherichia coli single recombination event between lambdoid phages could give strain K-12 after ultraviolet irradiation. Since that time all aspects of rise to a fully functional phage that has all the necessary genes lambda's lytic and lysogenic lifestyles have been studied. Lambda (Hendrix, 2002; Casjens, 2005; Hendrix and Casjens, 2006; Grose became a more important and approachable experimental system and Casjens, 2014). In this discussion we use lambdoid to include when Allan Campbell (1961) isolated a set of suppressible nonsense for example, the three phages HK97, P22 and N15, which typify mutants of lambda (or amber mutations, originally called suppres- three of the best-studied lambdoid groups that have significant sor sensitive or sus mutations) that identified genes essential for differences from lambda. Citations are in general not meant to its lytic growth. Additional amber mutations isolated by Sandy bestow credit for the original discoveries but to allow the reader Parkinson (1968) and Goldberg and Howe (1969) and prophage access to the literature. Other more detailed historical treatments mutations isolated by Clarence Fuerst (Mount et al., 1968) that were of some of the topics covered below can be found in the books lethal for lambda lytic growth identified a few more essential genes. Bacteriophage lambda (Hershey, 1971) and Lambda II (Hendrix Genetic complementation tests of these mutations succeeded in et al., 1983) (and in Gottesman and Weisberg, 2004; Stahl, 2005; defining all but one of lambda's 29 genes that are essential for Georgopoulos, 2006; Campbell, 2007; Court et al., 2007a). plaque formation under laboratory conditions (this includes the frameshifted G and G–T gene pair and the alternate starts of the S105 and S107 genes as well as the C and Nu3 genes as two genes Historical importance and recent progress each; see below). We note that two additional genes, Rz and Rz1 should probably also be considered essential genes even though The lambda genome plaques often form in their absence. They are absolutely required for disruption of the outer membrane and lysis unless external Phage lambda DNA was one of the earliest model systems for physical solution forces that are often present during laboratory studying the physical nature of DNA and genes, and a substantial growth help to destabilize the outer membrane of infected cells amount of early research examined the hydrodynamic properties (Berry et al., 2012). Only the small cro gene was not found by the of the lambda chromosome with the goal of, among other things, above random mutant hunts. It was discovered during the genetic determining its absolute molecular weight. At the same time analysis of the control of lysogeny (Eisen et al., 1970), and its methods (now considered to be primitive) were devised for absence leads to the lysogenic state and hence no lytic growth. separating the two halves of the lambda chromosome and for Recombination frequency and deletion mapping of these and mapping genes identified genetically to these halves and to various promoter and operator mutations isolated by others during smaller physical intervals (reviewed by Davidson and Szyblaski, this time allowed the construction of a detailed genetic map. 1971). Soon thereafter electron microscopic analysis of absolute Analysis of the phage DNA molecule led to the most detailed length and of lambda DNA in heteroduplex with various altered physical map of any organism's genome at the time. Detailed study lambda DNAs gave rise to a major step forward, the construction of of the phenotypes of phage mutants defective in known, mapped a detailed physical map of lambda DNA – a gene map in base pairs genes gave an early overall picture of the lambda life cycle. Indeed, (bps) rather than recombination frequency units (Fiandt et al., the careers of both authors here started at about the same time 1971; Simon et al., 1971). The lambda virion chromosome was also with the analysis of lambda virion morphogenesis by utilizing the among the first natural linear DNAs whose end structure was various amber mutants to identify the proteins expressed from the understood in detail; it was found not to have blunt ends, but to lambda morphogenetic genes and determining the nature of the have complementary 12 bp protruding 50-ends called cohesive (or defects in their absence – S.R.C. in the laboratory of Dale Kaiser at sticky) ends, since the two single-strand ends can pair and thus Stanford University and R.W.H. in the laboratory of Jim Watson at bind to each other (Hershey et al., 1963; Hershey and Burgi, 1965). Harvard University (Happily, rather than generating animosity, our Meanwhile, lambda contributed greatly to the ultimate mapping early competition in this arena led to mutual respect and a lifelong of DNA, the complete nucleotide sequence. It is not generally friendship). We believe that the clever use of forward genetic appreciated that the 12 bp lambda cohesive ends were the subject selections and screens to isolate informative mutations and combi- of the first direct nucleotide sequencing of a biological DNA. Ray nations of mutations and the use of these molecular genetic Wu devised methodology for using DNA polymerase to fill in the experiments to deduce the underlying molecular mechanisms cohesive ends with specifically labeled nucleotides, followed by reached its zenith in the study of phage lambda during this period. analysis of the product to determine this exact 12 bp nucleotide Those were heady days with lambda running at the front of the sequence (Wu and Kaiser, 1968; Onaga, 2014). This sequencing was scientificpack. not a trivial undertaking, as determining this 0.012 kbp of sequ- We discuss here early experiments that led to our current ence required several years of painstaking work. Fourteen years understanding of phage lambda and molecular biological pro- later, Fred Sanger et al. (1982) used lambda as the subject for the cesses in general. During this period VIROLOGY published many of first determination of the complete genome sequence of a dsDNA these important findings. We also mention recent developments virus – 48,502 bp – using his dideoxynucleotide chain termination 312 S.R. Casjens, R.W. Hendrix / Virology 479-480 (2015) 310–330

terminase procapsids (heads) tail shaft tail tip G nu1 A W B Cnu3 D E FI FII ZU V G–T HMLKI cos

0 12345678 9 10111213141516

site-specific homologous side tail fibers non-essential (b2 region) recombination non-essential recombination ea22 small J lom stf tfa ea47 ea31 ea59 int xis55ea8.5Ea22 orfs exo bet gam

t sib attP t'I tL4 L3

17 18 19 20 21 22 23 24 25 26 27 2829 30 31 32 33

tI PI lysogenic DNA non-essential conversion replication non-essential lysis non-essential 26 60 Rz1 kil ea10 sieB 68 S107 small cIII ral esc N rexB rexA cI cro cII O P ren orf 290 57 56 rap 221 Q 64 S105 R Rz bor orfs? ♦ ♦ ♦ ♦♦ ♦ t t P P L2 L1 L R tR1 ori tR2 tR3 tR4 PR' tR'

34 35 36 37 38 39 40 41 42 4344 45 46 47 48 kbp ∗ ∗ ∗ P nutL tO sieB PO t P nutR P aQ aQ PRM RE

Fig. 1. Map of the bacteriophage lambda chromosome. The linear virion chromosome is shown with a scale in kbp below. Rectangles indicate known genes with names and functional regions shown above; red genes are transcribed rightward and green leftward; vertically offset gene rectangles are expressed from reading frames that overlap (C-Nu3, S107-S105 and sieB-esc in the same reading frame, rz-rz1 in different frames) or by programmed frameshifting (G–T, small arrow in figure). Diamonds (♦) mark regulatory genes. Important DNA sites (e.g., P, promoters; t, terminators) are indicated below the genes. Thick horizontal arrows below indicate mRNAs: black, transcripts made in a lysogen; orange, immediate–early transcripts; green, early transcripts; red, late transcripts; blue, transcripts made in response to high CII levels. Asterisks (n) mark RNAs with regulatory activity (the detailed role of PRE-initiated cro antisense message remains unclear (Spiegelman et al., 1972)). The chromosome is circularized in the cell during infection, so the PR0-initiated late transcript is continuous from the lower right to the upper left in the figure. method. This, along with anecdotal sequencing of various lambda found early on, like other phages under study at the time, to have a genes and mutants of them by the laborious and more error-prone temporally controlled pattern of transcription and gene expres- chemical sequencing method (Maxam and Gilbert, 1977)inthe sion, called in this case immediate early, early (sometimes called previous few years, unleashed a rapid avalanche of studies relating “delayed early”) and late transcription (reviewed by Echols, 1971; the many extraordinary genetic studies on lambda from the pre- Friedman and Gottesman, 1983). Lambda's major transcription vious decades to the genome sequence. This in turn gave rise to an units are shown in Fig. 1. When Jeffrey Roberts (1969), then a understanding of phage lambda genes and their expression that student in the Watson/Gilbert lab at Harvard, used purified RNA was unprecedented at the time (reviewed in Hendrix et al., 1983). polymerase to transcribe lambda DNA in vitro, it initiated RNA Fig. 1 shows a map of the lambda chromosome; more detailed synthesis at the immediate early promoters PL and PR, but the annotated maps of lambda, HK97, P22 and N15 can be found in the transcripts made were longer than those observed in vivo. He then supplementary material of Hendrix and Casjens (2006). used this system to discover a host protein (that he named Rho) In a sense the complete nucleotide sequence of the lambda that caused shorter transcripts to be made that were the same genome and publication of the Lambda II book (Hendrix et al., length as those observed in vivo. Rho was later shown to cause 1983) heralded the demise of lambda's golden age and its exalted transcription termination at the normal lambda sites in vivo, and position in molecular genetic research. The complete sequence to be required for a subset of transcription termination events in E. gave the (false) impression to researchers outside this field that coli. Thus lambda was directly responsible for the discovery of this everything important about lambda was known. This impression, first transcription controlling “factor” (if the sigma subunit is along with the approximately simultaneous development of sim- considered to be part of the basic RNA polymerase enzyme). Lytic ple molecular cloning techniques that could be used in virtually infection by lambda phage carrying a conditional lethal mutation any molecular biology laboratory, allowed new and powerful in its N gene drastically lowered protein and transcript synthesis molecular genetic access to essentially any biological system a from nearly all lambda genes compared to a wild type infection researcher wished to study. Ironically, lambda played a major part (e.g., Radding, 1964; Echols, 1971). These findings were rapidly in the development of these techniques (below). followed by many ingenious molecular genetic studies that con- verged on the idea that N protein causes termination of the PL and Control of transcription during lytic growth PR immediate early transcripts to fail. The only immediate early genes, N and cro, lie upstream of these terminators, in the PL and Lambda was one of the first biological entities whose transcrip- PR transcripts, respectively. Further work showed that transcrip- tional regulation was studied and understood in detail. It was tion is antiterminated at terminators tL1 and tR1 (and other S.R. Casjens, R.W. Hendrix / Virology 479-480 (2015) 310–330 313 downstream terminators; Fig. 1) by N protein to allow expression Q protein works by a very different mechanism from that of N protein. of the downstream (delayed) early genes (summerized in Echols, Studies with phage lambda and the lambdoid phage 82 have shown 1971; Franklin, 1974; Friedman and Gottesman, 1983 and refer- that it is a DNA-binding protein that joins the transcription complex at ences therein). N protein does not, however, simply inactivate Rho. the Q binding element (QBE) between the -35 and -10 parts of the late Instead N somehow makes the transcribing RNA polymerase that operon promoter PR0 and stays with the transcription elongation has passed over a nut (N utilization) site in its presence insensitive to complexasittraversesthelateoperon.IncontrasttoN-mediated downstream termination at Rho-dependent and Rho-independent antitermination, there are no known host proteins that participate in terminators. its action. The recent structure of the DNA-binding domain of lambda Since its identification as a transcription antiterminator, N protein Q protein sheds considerable light on its contacts with the QBE DNA has been the subject of many studies aimed at elucidating the sequence, the R4 region of sigma factor and the tip of the RNA mechanism by which it accomplishes this feat. Jack Greenblatt first polymerase β flap (Deighan and Hochschild, 2007; Shankar et al., succeeded in accomplishing N action in vitro (Greenblatt, 1972), and 2007; Muteeb et al., 2012; Vorobiev et al., 2014). many subsequent studies have learned much about the detailed roles of the several host proteins that participate in N-mediated antiter- Control of lysogeny mination (see section “Interaction between phage lambda and its host” below for a more detailed discussion of these factors). N protein, a In 1961 Jacob and Monod put forward their operon hypothesis, natively unfolded protein (Van Gilst and von Hippel, 1997; deduced entirely from their genetic work on the lac operon and Goldenberg and Argyle, 2014), binds to the boxB sequence of the phage lambda model systems (Jacob and Monod, 1961). The early nut site in the nascent PL and PR mRNAs, as well as to the host NusA observations by Kaiser that CI is required for maintenance of protein (Mogridge et al., 1998; Mishra et al., 2013)andRNA lambda lysogeny and that it is responsible for immunity to super- polymerase. It probably binds RNA polymerase on its β subunit near infecting lambda phages strongly suggested, but did not absolutely the RNA exit channel (Cheeran et al., 2005). Exactly how N protein prove, that cI encoded a protein that directly turns off all the lambda blocks termination is not yet known, but the recent observation that lytic genes in the prophage state (Kaiser, 1957; Kaiser and Jacob, it reduces transcriptional slippage may provide a clue if slippage 1957). The subsequent isolation of suppressible amber nonsense contributes to termination (Parks et al., 2014). mutants in the cI gene proved that this function is mediated by the All characterized lambdoid phages possess an N protein- protein product of the cI gene (Jacob et al., 1962; Thomas and mediated transcription antitermination mechanism, with the Lambert, 1962). By the mid 1960s experiments had demonstrated exception of phage HK022. In lambda antitermination, as outlined quite clearly that the lack of lytic gene expression from the lambda above, the nascent PL or PR transcript interacts through its nut prophage was explained by blockage of mRNA synthesis by the cI sites with N protein, RNA polymerase and other host proteins to gene product (Jacob and Campbell, 1959; Thomas, 1971; Ptashne, create a transcription elongation complex that is insensitive to 1971 and references therein). In 1965–1966 a competition devel- termination signals. HK022 accomplishes the same end without N oped between Mark Ptashne (with the lambda CI repressor) and and without the known host factors. We now know that nascent Wally Gilbert (with the E. coli LacI repressor) to be the first to isolate PL and PR transcripts of HK022 interact directly with the β0 a transcriptional repressor and thus absolutely prove the existence subunit of the transcribing RNA polymerase through secondary of repressor proteins that regulate gene expression. They worked in structure formed at the put sites in the nascent RNA (which laboratories on different floors of the same building at Harvard, and correspond in position to the nut sites on the lambda transcripts) this race for the repressor caught the attention of ABC television, (Oberto et al., 1993; Sen et al., 2002; Komissarova et al., 2008). The which filmed a documentary, “The Scientist,” giving CI and LacI a resulting transcription elongation complex, as with lambda, is brief moment of popular fame (and somewhat amusing and insensitive to termination signals. Thus, HK022 early transcript irritating the other scientists in the building who experienced the antitermination is apparently mediated by RNA alone, rather than filming). Gilbert and Muller-Hill (1966) attained their goal first, but by RNA in conjunction with N and host transcription factors. Ptashne (1967) was not far behind, and today both repressors HK022 has not finished its dalliance with non-canonical mechan- remain important model systems for the study of sequence-spe- isms of regulating transcription with its unusual mechanism of cific DNA binding by proteins. accomplishing antitermination. It also has a mechanism to promote While maintenance of lambda lysogeny only requires one inappropriate termination in lambda-like phages during co-infection phage protein, the process of establishment of the lysogenic state of the same bacterium. While HK022 does not have an N gene, it is more complex. Dale Kaiser's original genetic studies identified does have a heterologous gene, called nun, at the same position in two additional genes, cII and cIII, in which defects lower the thegeneorderasN in lambda. The Nun protein interacts with the nut frequency of establishment by several orders of magnitude (Kaiser, sitesoflambdaaswellaswiththeotherhostfactorsandRNA 1957). These two genes lie in the early right and early left operons, polymerase (Burova et al., 1999; Watnick and Gottesman, 1998; respectively. After the discovery of Cro it was first thought that the Vitiello et al., 2014). This resembles what happens with N protein, lysis/lysogeny decision might simply be explained by a direct but the resulting transcription elongation complex is biased toward competition for operator binding between CI and Cro (a bistable termination. For a phage like lambda such inappropriate termination genetic switch), but more recent experiments have shown that it is of transcription would be a lethal event. These stories and others not this simple. We now know that the CII protein is a transcrip- about how HK022 deviates in informative ways from the lambda tional activator that stimulates strong cI gene transcription from paradigm, have largely come out of the laboratories of Max Gottes- the PRE promoter, and that high CII protein levels are critical in man and the late Bob Weisberg (Weisberg et al., 1999). Ongoing work ensuring the high CI concentrations required to establish, but not on these topics provides a good illustration of how phage investiga- maintain, the lysogenic state (Court et al., 2007a and references tions can still give new information on how RNA polymerase receives therein). The cIII gene encodes a protein that controls the stability and responds to signals. of the CII protein by inhibiting the action of the host-encoded Following N-mediated antitermination of PR transcription, gene Q HflB(FtsH)-HflC-HflK protease complex (Kobiler et al., 2007; is transcribed at the distal end of the early right operon. The Q protein Bandyopadhyay et al., 2010). In addition to this host protease in turn acts as a transcriptional antiterminator that allows read- control of CII stability, the host physiological state impacts the through of the tR0 terminator (Fig. 1) to allow expression of the late lysis/lysogeny decision through the following: (1) cAMP and operon (Roberts et al., 1998 and references therein). However, ppGpp levels may control HflBCK levels (Hong et al., 1971; Rao 314 S.R. Casjens, R.W. Hendrix / Virology 479-480 (2015) 310–330

Input on CII CII output LexA Fis PO HflD Poop RNA Int Prophage † PI integration

CII Proteolysis CII HflB/C/K PaQ* Late gene Q expression CIII PR’ † PL PRE PL # PR Cro ? PRM PL IHF PRM PR PL CI Autoproteolysis RecA CI Early gene expression UV N, II, cIII Q PR PL inluding c and RNaseIII Hex † PL cAMP PRM tR1/tR2 N Ant * Sar RNA Rho

glucose # Mnt Arc NusA/B/E(S1)/G

Fig. 2. Lambda regulatory circuits. The figure shows phage lambda-encoded functions (yellow ovals) and host encoded functions (blue ovals) that impact the lysis-lysogeny decision and control the lytic gene expression cascade. Phage P22 (Liao et al., 1987; Wu et al., 1987) and phage 434 (Shkilnyj et al., 2013) genes are pink and orange ovals respectively. Green arrows denote positive (activation) functions and red lines denote inhibitory (repression) functions. Asterisks (*) indicate antisense RNA interactions, hashes () indicate translational repression, and daggers (†) indicate mRNA stability controls; other regulatory events are at the level of transcription except IHF and FIS physical participation in the integrase complex that catalyzes the integration of prophage DNA. and Raj, 1973; Joung et al., 1994; Slominska et al., 1999). (2) RNase most highly down-regulated host gene in a lambda lysogen (Chen III is under strong growth rate regulation (Wilson et al., 2002), and et al., 2005). This gene is required for growth on succinate and its action controls cIII mRNA stability (Altuvia et al., 1991). some other carbon sources. It is not known why lowering its (3) Integration host factor (IHF) modulates CII and CIII levels expression might be beneficial, but the fact that the pckA promoter (Mahajna et al., 1986; Krinke and Wulff, 1990; Giladi et al., 1998) also overlaps lambdoid phage 21, P22, 434 and H-19B repressor and N gene translation (Kameyama et al., 1991; Wilson et al., binding targets (all different from lambda CI operator specificity), 2002). (4) RecA is activated by DNA damage to cause CI inactiva- suggests that this regulation is evolutionarily important to the tion (see below). (5) HflD may directly inhibit DNA binding by CII lambdoid phages. Louis Reichardt, a student in Kaiser's lab in the protein (Parua et al., 2010). (6) It has also been suggested that host early 1970s, devised a quantitative assay for CI repressor protein proteins DnaA and SeqA may affect PL transcription and thus the and performed an early set of intricate and elegant experiments lysis/lysogeny decision (Wegrzyn et al., 2012). Finally, (7) the OOP that showed that CI activates its own synthesis at low CI levels and RNA made from the lambda Po promoter is antisense to the 30 represses at high levels (Reichardt and Kaiser, 1971). This was one portion of cII gene mRNA and acts to destabilize that message with of the first demonstrations of autoregulation of a gene. Other the help of RNase III, and the OOP promoter is repressed by the experiments illuminated the amazing intricacies of CI and Cro host LexA protein (Krinke and Wulff, 1990; Krinke et al., 1991; binding to the three tandem operators at PL to regulate the Lewis et al., 1994). Thus, the current consensus is that CII is the divergent PRM and PL promoters (Ptashne, 1992 and references central player in establishing lysogeny, and Fig. 2 summarizes this therein). More recent study of the lambda prophage repressor has complex regulatory circuitry. shown that it forms multimers that bind simultaneously to When a decision is made to establish lysogeny, lambda ensures operators OL and OR at PL and PR, respectively, thus looping the that the proteins that favor the lytic growth pathway disappear DNA between these two promoters, and that this looping is quickly, thereby minimizing stuttering between the lytic and important in the autoregulation that keeps the CI concentration lysogenic life cycle directions and ensuring that a decision is within narrow limits in lysogens (Lewis et al., 2011; Cui et al., definitive. The O, N, CII, CIII and Xis proteins are all extremely 2013a, 2013b; Hensel et al., 2013; Michalowski and Little, 2013; metabolically unstable and are destroyed within a few minutes of Priest et al., 2014). Host supercoil density impacts this looping, but being made. Thus, when their synthesis stops, their DNA replica- its role in vivo is not known (Ding et al., 2014). tion initiation, delayed early gene expression and lysogeny pro- The CI–CII–CIII circuitry is nearly universally present in the moting activities are quickly lost (Kobiler et al., 2004). The host lambdoid phages, but some of these phages have additional proteases utilized for this purpose are ClpX/ClpP for O (Gonciarz- controls on lysogeny. The first of these to be discovered was the Swiatek et al., 1999; Thibault and Houry, 2012), Lon for N P22 antirepressor (Ant), which binds to repressor and causes it to (Gottesman et al., 1981; Maurizi, 1987; Mikita et al., 2013) and release repression (Susskind and Botstein, 1975). Transcription of FtsH for CII and CIII (Shotland et al., 2000; Kobiler et al., 2002, the ant gene in a prophage is kept off by two additional phage- 2007), and Lon for Xis (Leffers and Gottesman, 1998). encoded repressors, Mnt and Arc, and a small antisense RNA, Sar, In addition to preventing the lytic gene transcription cascade, that controls the translation of ant mRNA (Prell and Harvey, 1983; the CI protein has two other known regulatory functions, turning Liao et al., 1987; Wu et al., 1987; Schaefer and McClure, 1997). down expression of the host pckA gene and autoregulating its own Salmonella lambdoid phage gifsy-1 encodes an antirepressor GfoR synthesis. Array analysis of mRNAs showed that the E. coli pckA that is repressed by the host LexA repressor (Lemire et al., 2011). gene (which encodes phosphoenolpyruvate carboxykinase) is the Other phages such as N15 encode a different type of antirepressor S.R. Casjens, R.W. Hendrix / Virology 479-480 (2015) 310–330 315 control system. Here, transcription of AntA (an antirepressor decay of PL initiated int mRNA early after infection, ensuring that whose mode of action is not known) is negatively regulated by Int is made in large amounts only when a high CII level commits the N15 prophage repressor and by premature transcriptional the cell to lysogeny. In addition, the sib site is separated from PL in termination that is mediated by processing of a portion of the the integrated prophage, so after induction moderate levels of both leader of the antirepressor operon mRNA (Ravin et al., 1999; Int and Xis made from PL ensure excision (Schindler and Echols, Mardanov and Ravin, 2007). The exact biological role(s) of anti- 1981; Gottesman et al., 1982; Guarneros et al., 1982). CII also repressors remains unclear; they could be prophage induction activates a third promoter, PaQ, whose product is a small antisense modules (see below) but could also impact the lytic/lysogeny RNA of the Q gene, which presumably helps keep the late operon decision. off while a decision is being whether to lysogenize (Ho and Rosenberg, 1985; Hoopes and McClure, 1985). In vivo visualization Integration and excision experiments have recently shown that, after injection, lambda DNA is confined near its point of entry and replication-driven An aspect of the establishment of lysogeny not discussed above movement of the host DNA allows proper juxtaposition of the host is the physical integration of the phage chromosome into the host and phage attachment sites. These experiments also demonstrate chromosome. The phenomenon of lysogeny had been known but that even bacterial attachment site location in the chromosome poorly understood for several decades before lambda was dis- affects the frequency of lysogenization (Tal et al., 2014). covered (Lwoff, 1953). The quiescent phage genome (prophage) in In an interesting variation of lysogeny, lambdoid phages N15, a lysogen appeared to be attached to the host chromosome in øKO2 and PY54 do not integrate into their host's genome, but exist some way that could be mapped in bacterial conjugation experi- as linear plasmid prophages (Ravin and Shulga, 1970; Hertwig ments (Lederberg and Lederberg, 1953; Wollman, 1953), but the et al., 2003; Casjens et al., 2004). In these phages the int gene is nature of such an attachment was not known. Our current under- replaced by a ptl gene that encodes the enzyme, called protelo- standing of prophage insertion derives from the idea of site- merase, that creates the ends of these plasmids. These ends are specific recombination between a circular phage lambda genome covalently closed hairpins in which one DNA strand turns around and the host chromosome put forward by Allan Campbell (1962). and becomes the other strand with no break in the phosphodiester Numerous subsequent genetic and physical tests of this idea by backbone (Rybchin and Svarchevsky, 1999). The mechanism of Campbell and others soon validated this model (see Gottesman action of protelomerase has been examined, and it recognizes a and Weisbeg, 1971). The existence of a lambda encoded function, specific inverted repeat sequence, makes staggered nicks 6 bp integrase (Int), that was necessary for prophage insertion was apart in the two DNA strands, separates the strands between the simultaneously demonstrated by the isolation of int defective nicks, and after strand swapping ligates the ends to form two mutants by Zissler (1967), Zissler et al. (1971), Gingery and hairpins (Huang et al., 2004; Aihara et al., 2007). These prophages Echols (1967) and Gottesman and Yarmolinsky (1968). Howard encode their own plasmid segregation and replication proteins Nash and co-workers accomplished in vitro integration (reviewed (Ravin, 2003; Ravin et al., 2003) and so do not turn off their early by Weisberg and Landy, 1983), and many subsequent ingenious genes as completely as integrated prophages (Ravin et al., 2000). genetic and biochemical studies elucidated the complex mechan- ism of the integration reaction. The host integration host factor DNA replication and homologous recombination (IHF; named for this reaction but now known to have many pleiotropic functions in E. coli and Fis protein were found to be Lambda DNA replication initiates at a single origin and pro- involved (Ball and Johnson, 1991). Genetic studies by Ethan Signer, ceeds bidirectionally from that point to generate circle-to-circle Robert Weisberg and others combined with biochemical studies replication until several tens of circles accumulate in the infected from the Art Landy lab have resulted in a marvelously detailed cell. At that point replication switches (by a still poorly understood picture of the integration protein DNA complex bound to its mechanism) to rolling circle mode to generate the concatemeric target (the attachment site, att) and its actions that result in att DNA molecules that are the substrate for DNA packaging into site-specific recombination and lambda prophage insertion (Seah virions (below) (Skalka et al., 1972; Wake et al., 1972; Narajczyk et al., 2014; Tong et al., 2014 and references therein). et al., 2007). The lambda genome carries two essential DNA As Esther Lederberg originally discovered (above), lambda replication genes, O and P. Four dimers of O protein bind at four prophages are induced by DNA damaging agents such as UV light sequence repeats in the origin, which lies inside the O gene, to or mitomycin C to enter a cycle of lytic phage growth. Release of CI form an “O-some” complex. The other essential replication protein, repression causes the lytic gene expression cascade to initiate, and P, binds to the host DnaB helicase, and this binary complex binds the prophage is excised from the host chromosome to allow circle- the O-some to form the ori:O:P:DnaB preprimosomal complex to-circle DNA replication (below). Interestingly, the CI repressor (Zylicz et al., 1998 and references therein). Host chaperones DnaK- contains a cryptic autoprotease activity that is activated by binding DnaJ-GrpE (see below) are involved in activating the preprimoso- to the host RecA recombination protein, but only when RecA is mal complex to recruit the rest of the host replication machinery activated by ssDNA (Little, 1984; Kim and Little, 1993; Mustard and (Learn et al., 1997). Thus, the early study of lambda replication Little, 2000). Integration and excision are directional; while Int contributed greatly to an understanding of the detailed steps (with its host factors) is sufficient to integrate lambda DNA, required to accomplish controlled initiation. In addition, the origin another phage-encoded protein Xis is required for the reverse is activated by rightward transcription from PR, but this phenom- excision reaction. Control of the relative amounts of Int and Xis are enon remains poorly understood (Leng and McMacken, 2002; necessary to ensure that integration occurs during establishment Hayes et al., 2012; Olszewski et al., 2014). In some other lambdoid of lysogeny and excision occurs during induction of a lysogen. This phages like P22, the P gene is replaced by a gene that encodes an is accomplished in two ways. First, when lysogeny is being actual DnaB type helicase (Wickner, 1984a, 1984b), and phages established by high CII levels (above) the Int/Xis ratio needs to APSE-1 and N15 encode a putative DNA polymerase (van der Wilk be high to ensure that integration occurs. To achieve this, CII et al., 1999) or a bifunctional helicase-primase (Ravin et al., 2000; protein activates the promoter PI, and its transcript encodes Int Mardanov and Ravin, 2006), respectively. but not Xis. Second, a process dubbed retroregulation lowers the Genetic recombination was first demonstrated for lambda by level of Int expression during lytic infection. Here, a site sib in the Jacob and Wollman (1954), and mutants of lambda that recombine RNA downstream of int and the PI transcript causes more rapid at reduced frequency were isolated by Franklin (1967), Echols and 316 S.R. Casjens, R.W. Hendrix / Virology 479-480 (2015) 310–330

Gingery (1968) and Signer and Weil (1968). These mutations were by PCR primers with such tails) are inserted by homologous found to affect genes exo and bet that lie in the early PL transcript. recombination into any desired site in a bacterial genome according Exo is an exonuclease (Zhang et al., 2011 and references therein) to the sequence of the tails. Then, since the inserted gene is chosen and Bet is a strand-annealing protein (Matsubara et al., 2013 and to be counter-selectable, homologous recombination can replace references therein). These two proteins can substitute for RecA in the inserted gene with any DNA that has the proper homologies homologous recombination. In addition, a host RecBCD nuclease (summarized in Thomason et al., 2014). This now widely used inhibitor is encoded by the gam gene, which is located immedi- strategy, called “recombineering,” is dependent upon high levels of ately upstream of exo and bet (Court et al., 2007b and references ectopically expressed Exo and Bet and allows rapid, efficient and therein). Lambda also carries two genes in its nin region that affect scarless engineering of bacterial genomes. Very recently, Farzadfard recombination, orf and rap. Orf protein is a recombination med- and Lu (2014) have used lambda bet-mediated ssDNA recombineer- iator that binds to host single-strand binding protein and ssDNA ing to develop a genomic memory system that “writes” (recom- (Maxwell et al., 2005; Curtis et al., 2011, 2014), and Rap protein is a bines) a chosen sequence into a second site in the genome of E. coli DNA structure-specific endonuclease that cleaves Holliday junc- in response to endogenous expression of ssDNA containing the tion branch points (Sharples et al., 1998; Poteete et al., 2002). sequence. The system thus genomically encodes a memory of past Homologous recombination occurs by several different pathways, expression of the promoter engineered to express the ssDNA. and these phage-encoded proteins participate with host proteins in various ways (see for example Poteete, 2013). We will not Interactions between phage lambda and its host review these in detail here, but these lambdoid proteins continue to be important in ongoing studies of the systems that catalyze In the early 1970s Costa Georgopoulos, David Friedman and Nat homologous recombination. Sternberg independently devised methods of isolating mutations Early work on lambda recombination also led directly to our in the E. coli host that allowed lambda virions to adsorb and DNA current understanding of host RecBCD action. In about 1969 to be injected, but which block phage infection at some later stage. mutants were isolated by David Henderson in Ken and Noreen These mutations affect lambda's head assembly, DNA replication Murray's laboratory that increased the growth of lambda that was and early transcription. They alter specific host proteins that are missing its recombination genes. These were studied further by required for successful transcription and for proper protein fold- Frank Stahl, Gerry Smith and coworkers (Stahl, 2005 and refer- ing/unfolding dynamics. ences therein; Smith et al., 1981a, 1981b). These mutations turned The host mutations that affect head assembly identified the out to have created a unique asymmetric 8 bp nucleotide sequence host GroEL-GroES protein folding chaperone (also now known as (now called the Chi site, for crossover hotspot instigator), and their HSP60-HSP10) (Georgopoulos et al., 1973; Sternberg, 1973). The study gave rise to our current understanding that the RecBCD functions defined by these host mutations were named “gro” for to nuclease enters DNA at double-strand breaks and degrades both their inability of grow lambda (Georgopoulos and Herskowitz, strands (differentially) from there until it hits a properly oriented 1971), and the “E” was added to reflect the fact that mutations in Chi site. Passing over a Chi site “civilizes” the RecBCD exonuclease lambda gene E (major head protein) overcome this gro block (to use Stahl's words) so that it continues along the DNA stands as (Georgopoulos et al., 1973). However, in actual fact, mutations in a helicase, separating but not degrading them. Finally the host either gene E or B (portal protein) have this property, and although RecA protein is recruited to initiate a recombination event. Wild this particular reaction has still not been studied in biophysical type lambda has no native Chi site, but its E. coli host has one every detail, subsequent work suggests that it is probably folding of the 5 kbp on average, and they are highly preferentially oriented gene B protein that actually requires this host chaperone (Murialdo according to the direction replication passes over them. It is now and Becker, 1978; Kochan and Murialdo, 1983; Georgopoulos, 2006). believed that Chi plays a major role in RecBCD-mediated recom- GroEL was originally known simply as GroE, but it acquired the binational repair of collapsed E. coli replication forks (Kuzminov, L when its smaller partner, GroES, was discovered (Tilly et al., 1981). 2001). The lack of Chi sites in most invading foreign DNAs may also In agreement with the genetic evidence that GroEL-GroES interacts allow the RecBCD nuclease to help protect E. coli from such with lambda head proteins, examination of the results of lambda “invaders” (Stahl, 2005). infection of a groEL mutant host showed that head production was Other lambdoid phages carry different recombination genes in seriously aberrant as seen in a B or C minus infection (Georgopoulos the exo/bet/gam chromosomal location. The best studied of these et al., 1973). The mechanism of GroEL-GroES protein folding chaper- are genes abc1, abc2, erf and arf of phage P22. The two Abc one action has now been studied in detail with other more tractable proteins inhibit RecBCD nuclease, Erf is a strand annealing protein, substrates (Horwich and Fenton, 2009). and Arf is a poorly understood recombination accessory function The groE gene was one of the first bacterial genes to be cloned. (Botstein and Matz, 1970; Poteete and Fenton, 1983; Poteete et al., Its cloning relied on the confluence of microbial genetics in the 1988, 1991). Other lambdoid phages carry a third type of recom- form of the groE mutants isolated by Costa Georgopoulos, and one bination genes, recE and recT, that encode an exonuclease (ExoVIII) of the earliest recombinant DNA techniques in the form of a and a strand annealing protein, respectively (Kushner et al., 1974; shotgun (random) library of E. coli DNA fragments in a phage Hall et al., 1993; Zhang et al., 2009). The latter two genes were lambda vector. One of us (RWH) packed some of Costa's groE originally identified as E. coli genes, but were later found to lie in mutants in a suitcase and went to visit Ron Davis at Stanford. Ron the defective lambdoid prophage Rac (Clark et al., 1993); various had constructed one of the first libraries of E. coli DNA cloned into fully functional lambdoid phages such as gifsy-1 carry recE and phage lambda genomic DNA and packaged in vitro into lambda recT genes (Lemire et al., 2008). In addition, some other lambdoid virions (below), and when this phage library was plated on a groE phage nin regions carry a rusA gene which encodes a Holliday mutant bacterial strain it contained plaque-forming phages at a junction resolvase that is different from lambda Rap (Sharples frequency of 104 relative to wild type E. coli. The rare plaque- et al., 1994). forming phages carried an 8 kbp insert of E. coli DNA. (As an Recently the lambda exo and bet recombination functions indication of how new the recombinant DNA technology was, the have been put to valuable technological use in accomplishing size of the insert was measured not with restriction enzymes and very efficient homologous recombination in vivo. Don Court and agarose gels but by the much better established technique of coworkers devised and optimized systems in which short 30–50 bp measuring the density of phage carrying the insert in an equili- homologous tails on a selectable gene (generated by amplification brium CsCl gradient.) The 8 kbp insert was subsequently shown to S.R. Casjens, R.W. Hendrix / Virology 479-480 (2015) 310–330 317 contain both the groEL and groES genes and led to the identifica- led directly to the discovery and characterization of factors that tion of the GroEL protein as a 60 kDa protein with ATPase activity control RNA polymerase elongation and termination. The study of that assembles into a cylindrical 14 subunit structure (two stacked the mechanisms of action of these factors continues to the present. rings of seven subunits) (Hendrix, 1979). Sequencing of this cloned DNA led to the realization, which was very surprising at the time, Virion assembly that GroEL had homologs in the chloroplasts of green plants in the form of “rubisco large subunit binding protein” (Hemmingsen The study of the assembly of phage lambda virions began in the et al., 1988). Rubisco is the photosynthesis enzyme ribulose-1,5- 1960s when Jean Weigle (1966, 1968) showed that lambda heads bisphosphate carboxylase/oxygenase, and the plant GroEL homo- and tails assemble independently and spontaneously join in vitro logue has a role in folding its large subunit (Gatenby, 1996). While to form infectious virions and with the deduction in the Eduard we were cloning and characterizing GroE, very similar experi- Kellenbeger, Louis Siminovitch and Dale Kaiser laboratories that ments were done by Barbara and Tom Hohn in Basel, Switzerland, gene E encodes its major capsid protein (MCP—also called coat using another early lambda library of E. coli DNA that was protein) (Karamata et al., 1962; Kemp et al., 1968; Casjens et al., constructed by Ken and Noreen Murray (Georgopoulos and 1970). Fig. 3A shows an electron micrograph of the phage lambda Hohn, 1978; Hohn et al., 1979). virion (with the side fibers that are not present in laboratory The host mutants that affect DNA replication identified the DnaK- strains, see below). DnaJ-GrpE protein folding/unfolding chaperone (DnaK is also called At that time, the study of viral structural proteins and their HSP70) (Georgopoulos and Herskowitz, 1971; Georgopoulos and assembly was a non-trivial enterprise. For example, one of us Welch, 1993 and references therein), which is now known to be (S.R.C.) began his Ph.D. thesis project in 1968 by simply attempting required for disassembly/loosening of the protein complex that forms to determine how many different proteins are present in the at the origin of lambda (above), thus enabling the replication fork to lambda virion. I had been toiling over various strategies for move away from the origin (Hoffmann et al., 1992; Osipiuk et al., 1993; separating lambda virion proteins solubilized with urea, acetic Wyman et al., 1993). In addition to pointing out that protein folding, acid or guanidine hydrochloride for nearly a year without notice- assembly and disassembly can be catalyzed by other proteins, it was able success, when Bill Studier (who was studying phage T7 at the noticed early that transcription of the genes encoding the GroE and Brookhaven Laboratory) gave a seminar at Stanford describing the DnaK protein chaperone systems is enhanced at high temperatures. sodium dodecyl-sulfate (SDS)-polyacrylamide gel electrophoresis Further examination of this latter phenomenon by many workers has technique that he and Jake Maizel had developed (Studier and led to our current understanding of the mechanisms of stress control Maizel, 1969; Maizel, 2000). The combined facts that boiling in of gene expression (Tilly et al., 1983; Georgopoulos and Welch, 1993). 0.1% SDS disassembles virtually all protein complexes and that Thus, lambda work was seminal in the nucleation of the idea of electrophoresis could be performed in SDS solutions instantly catalyzed protein folding and in the discovery and understanding of made the prospects for my thesis very bright. This, along with two important chaperone systems. Before this, all protein folding was what was probably the first published use of a slab gel apparatus thought to be spontaneous. that allowed accurate comparison of adjacent lanes, allowed me to The host mutants in which lambda fails to express (delayed) get my thesis started with a definitive proof that gene E encodes early genes affect the ability of the lambda N protein to cause RNA the major head protein of lambda (Casjens et al., 1970). I note here polymerase to bypass terminators and thus transcribe the down- that it was the ability to do the rigorous controls necessary to stream essential early genes (above). These mutations alter several really prove (rather than surmise or strongly suggest) our conclu- transcription factors now called NusA, NusB, NusE and NusG sions that first attracted me to phage work and has kept me here (originally for N under supplied but more recently N utilization for nearly 50 years. Uli Laemmli (a phage T4 worker) and Jake substance) that are all required for successful N antitermination Maizel soon modified this technique to give considerably higher (Georgopoulos, 1971; Friedman, 1971; Friedman and Baron, 1974; resolution (called discontinuous SDS gel electrophoresis). In a Friedman and Gottesman, 1983; Friedman and Court, 1995). NusA curious twist of fate this now universally used and extremely is an RNA-binding protein that interacts with the α subunit of RNA highly cited method was published only in a figure legend in Uli's polymerase and modulates transcriptional pausing (Prasch et al., paper on T4 morphogenetic protein cleavage (Laemmli, 1970; cited 2009; Yang et al., 2009; Yang and Lewis, 2010; Schweimer et al., 215,930 times at the time of this writing). 2011; Zhou et al., 2011; Muteeb et al., 2012; Kolb et al., 2014), as With the advent of discontinuous SDS gel electrophoresis and well as interacting with lambda N protein (Mishra et al., 2013). The methods to tamp down host protein synthesis relative to lambda NusB–NusE complex binds the BoxA RNA sequence of the nut sites protein synthesis in infected cells (Ptashne, 1967; Hendrix, 1971) and binds RNA polymerase (Burmann et al., 2010; Stagno et al., came the rapid identification of the rest of the morphogenetic 2011). NusG protein homologs are present in all three domains of gene products as specific bands in such gels (Buchwald et al., 1970; life, and in E. coli NusG is a transcriptional pause-suppressing Hendrix, 1971; Georgopoulos et al., 1973). This in turn led to factor that might also be involved in suppressing aberrant anti- analysis of the structures made by infections with various mutant sense transcription in E. coli (Herbert et al., 2010; Peters et al., phages and the gene products in them. These studies also illuminated 2012; Yakhnin and Babitzke, 2014). It has two domains, an N- the common protein cleavages that accompany lambda and many terminal domain that binds RNA polymerase (Belogurov et al., other virus assembly processes and make them irreversible and/or 2009) and a C-terminal domain that binds NusE and Rho allow access to final conformations not available to the full-length (Burmann et al., 2010). NusE, which is now known to be ribosomal proteins (Murialdo and Siminovitch, 1972; Georgopoulos et al., 1973; small subunit protein S10, binds NusG and as such is thought to be Hendrix and Casjens, 1974, 1975). This information allowed the involved in ensuring that a ribosome closely follows the transcrib- deduction of the basic assembly pathway of lambda virions (the order ing RNA polymerase, thus coupling translation to transcription of action and general role of each of the morphogenetic proteins; (Burmann et al., 2010; McGary and Nudler, 2013). In addition, summarized in Georgopoulos et al., 1983; Hendrix and Casjens, 2006), NusA interacts with translesion DNA polymerases to affect DNA but it did not elucidate the detailed functions of each of the 21 phage- repair (Cohen et al., 2009), and with NusB it may even be involved encoded proteins required for virion assembly. Work continues in this in intracellular RNA folding and processing (Bubunenko et al., direction to this day. 2013). Thus, these early lambda–host interaction genetic studies The fact that lambda procapsids (precursor head structures) are revealed previously unsuspected complexities of transcription and assembled first and DNA is subsequently pumped into them by a 318 S.R. Casjens, R.W. Hendrix / Virology 479-480 (2015) 310–330

N

* *

Fig. 3. The phage lambda virion: (A) negatively stained electron micrograph of the Ur-lambda virion. Micrograph is modified from Hendrix and Duda (1992) and reproduced with permission of publisher. (B) Ribbon diagram of the phage HK97 virion MCP subunit. The N- and C-termini of the protein are indicated, and the asterisks mark the locations of K169 and N356 whose side chains are crosslinked to adjacent subunits in the virion (from Helgstrand et al. (2003); protein database code 1OHG with rainbow color depiction by MacPyMOL copyright 2009–2010 Schrodinger, LLC). (C) Subnanometer-resolution 3-dimensional cyroelectron microscopic reconstruction of the phage lambda head. Coloration ranges from red to blue with increasing radius, and the black arrowhead indicates the center of a coat protein hexamer as shown in panel D. (D) Close up view centered on a major capsid protein hexamer of the head shown in panel C. Six gene D protein trimers are indicated in orange and seven major coat protein (gene E protein) subunits are shown in other colors. These colored subunits form one shell asymmetric unit. Panels C and D are modified from Lander et al. (2008) and reproduced with permission of publisher.

DNA translocase was initially suggested by analysis of head-like template for shaft assembly (Katsura and Hendrix, 1984; Katsura, structures made in vivo (Hendrix and Casjens, 1975)andwas 1987). Curiously, the H protein is proteolytically cleaved before tail rigorously proven by Kaiser et al. (1975). Two proteins, the products attachment to heads, but the role of this cleavage and the protease of the Nu1 and A genes are required for inserting DNA into the that causes it remain unknown (Hendrix and Casjens, 1974; Tsui and procapsid. The Nu1 protein recognizes the DNA to be packaged Hendrix, 1983). In addition, Levin et al. (1993) found that a pro- (Shinder and Gold, 1988; de Beer et al., 2002). The A protein cleaves grammed translational frameshift is required for expression of the lambda DNA at the cos site during packaging to create the cohesive essential T reading frame as a C-terminal extension of the gene G ends (Wang and Kaiser, 1973; Feiss et al., 1983; Davidson et al., protein (called G–T protein). The frameshift ensures the proper ratio 1991) and is also the ATP cleavage-driven translocase that moves (30:1) of G and G–T proteins. This was one of the first demonstra- the DNA into the procapsid (Duffy and Feiss, 2002; Tsay et al., 2010). tions of programmed frameshifting in prokaryotes, and it is now Neither of these proteins is present in the completed virion. Mike known to be an almost universal feature of the assembly of long Feiss and co-workers performed extensive and informative genetic phage tails, with the curious apparent exception of phage T4 and its analyses of lambda DNA packaging (Sippy et al., 2014; Feiss and Rao, allies (Xu et al., 2004a). This phenomenon has only been studied in 2012 and references therein), and Carlos Catalano's laboratory has more detail in lambda, where both G and G–Tproteinsarerequired studied its biochemistry (e.g., Andrews and Catalano, 2013; Singh for tail assembly, but neither is present in completed tails. The G et al., 2013). Most viruses do not depend on the host to help build and G–T proteins act as a chaperone in which the G domain binds their virions (except for chaperones, above), but lambda DNA the tape measure protein, and the T domain of G–Tbindsthemajor packaging initiation requires the host IHF protein, which binds to tail subunit (the gene V protein) (Xu et al., 2013, 2014). An under- a sequence near the Nu1 protein recognition sites and bends the standing of the detailed dynamics of this assembly process awaits DNA. This bend allows formation of the DNA:Nu1:A nucleoprotein future study. complex that initiates cos site cleavage and DNA packaging (Xin et The different lambdoid phages have quite diverse virion struc- al., 1993; Sanyal et al., 2014). Laser tweezer measurements of the tures(below)andassuchhaveproventobeveryfertilegroundin force generated by this molecular motor in vitro by Douglas Smith studies of virion assembly – in particular, studies of phages P22 and and collaborators have shown it to be among the most powerful HK97 have been very informative. Salmonella phage P22 was of nanomotors known (Tsay et al., 2009, 2010). interest early because it was the first phage found to carry out Lambda tail assembly work has focused largely on how its shaft is generalized transduction (Zinder and Lederberg, 1952). Horst assembled. Isao Katsura and Roger Hendrix found that the length of Schmieger isolated P22 mutants with greatly increased frequencies the gene H-encoded tape measure protein determines the length of of transduction (Schmieger, 1972; Casjens et al., 1992b), which made theshaft.Itactsasameasuringdevice,mostlikelybyservingasa P22 the easiest transducing phage to use in the laboratory. As such it S.R. Casjens, R.W. Hendrix / Virology 479-480 (2015) 310–330 319 contributed significantly to the rise of Salmonella enterica as one of phages like P22 and a transglycosidase in lambda (Bienkowska- the important bacterial model organisms (Neidhardt, 1996). Myron Szewczyk et al., 1981; Weaver et al., 1985; Evrard et al., 1998; Levine, David Botstein and Jonathan King, studied P22 on its own Mooers and Matthews, 2006). Ryland Young and colleagues have merits with the (prescient) view that phages related to lambda described the mechanism(s) by which the lambda R protein is released would benefit from the early lambda studies but would be different into the periplasm. Release is controlled by the S105 and S107 holin enough from lambda to be interesting and informative. Transduction proteins, which are expressed from different start codons of the same is the result of errors in phage DNA packaging, and the study of P22 reading frame, resulting in one protein, S107 that is two amino acids has led to a much better understanding of headful DNA packaging longer than the other, S105. The shorter one, S105, causes lysis and (Tye et al., 1974; Jackson et al., 1978; Casjens and Hayden, 1988; S107 inhibits lysis; thus, the ratio of the two determines the exact Casjens et al., 1992c; Wu et al., 2002; Leavitt et al., 2013). P22 was timing of lysis (Wang et al., 2000a). Lambda S105 protein is a small also the first virus found to require a scaffolding protein for proper membrane protein that forms large physical holes in the membrane assembly of its MCP into a capsid shell. Scaffolding protein is present that allow endolysin escape across the cytoplasmic membrane. These in large numbers inside of procapsids, but it exits the structure before holes are irregular, average 4300 nm in diameter and involve DNA enters to form complete virions. This was one of the first hundreds of S105 molecules (Savva et al., 2008; To and Young, discoveries of a protein that catalyzes the assembly of other proteins 2014). Exactly how S105 molecules aggregate to form such large holes (King and Casjens, 1974; Earnshaw et al., 1976; Weigele et al., 2005; after accumulating in the membrane in a non-hole-forming state Chen et al., 2011; Padilla-Meier et al., 2012), and scaffolding proteins remains mysterious. More recent study of the lambdoid phage 21 has have since been found to be a general feature of the assembly of large shown that its mechanism of endolysin release is different from that of virus particles. lambda. Its endolysin is secreted by the host Sec translocon machinery The study of lambdoid phage HK97 began in the Hendrix lab into the periplasm in a catalytically inactive form, tethered to the inner because its tail has a slightly different length from that of lambda, membrane by an N-terminal transmembrane domain. When the but it was soon noticed that its MCP behaved as a smear near the phage 21 “pinholin” forms small holes that depolarize the inner top of an SDS electrophoresis gel of virion proteins (Popa et al., membrane, the endolysin is released from the membrane and is 1991). Analysis of this phenomenon showed that its head proteins activated to cleave the peptidoglycan (Pang et al., 2009, 2013; Xu et al., are very different from those of lambda and that its virion MCP is 2004b). In addition to holin disruption of the inner membrane and cell auto-catalytically cross-linked into inter-locked rings as “molecu- wall breakdown, the outer membrane must also be disrupted for lar chain mail” (Duda, 1998; Wikoff et al., 2000; Helgstrand et al., complete cell lysis to occur. The two lambda proteins that perform this 2003; Dierkes et al., 2009). We now know that such a chain mail still poorly understood process are Rz and Rz1, which are an inner arrangement is not unique to HK97 and may not be uncommon in membrane protein and an outer membrane lipoprotein, respectively. phage capsids (Duda, 1998; Gilakjan and Kropinski, 1999). In Together they form the “spanin” complex (so named because it spans addition, structural studies in the Alasdair Steven and Jack Johnson the entire periplasm) (Berry et al., 2008, 2012; Young, 2014). Spanins laboratories led to a uniquely detailed picture of HK97 procapsid are not always essential in laboratory phage infections because maturation (MCP shell expansion) into virion heads (Duda et al., buffeting of cells suspended in solution can physically disrupt the 2006; Hendrix and Johnson, 2012; Cardone et al., 2014). The study outer membrane in their absence; however, their nearly universal of P22 and epsilon15 (a remote lambda relative that is not quite presence in tailed phages suggests a critical evolutionary importance. lambdoid) gave rise to the first asymmetric cryo-electron micro- Curiously, in lambdoid and many other phages the Rz1 gene lies scopic (EM) reconstructions of virions that show the tail as well as wholly within the Rz gene in its þ1 reading frame, where it is a the MCP at subnanometer resolution (Jiang et al., 2006, 2008; spectacular example of complete gene overlap. These three features of Lander et al., 2006; Chang et al., 2006; Tang et al., 2011). Finally, cell lysis, a peptidoglycan degrading enzyme, a holin or pinholin and a and perhaps most importantly, the HK97 MCP shell crystallizes spanin, are essentially universal among the tailed phages, and their and remains today as the only large phage MCP that has been actions were unraveled through study of the lambdoid phages. solved to atomic resolution by x-ray crystallography. The fold of the HK97 MCP was novel at the time of its determination (Wikoff DNA delivery from the virion into target cells et al., 2000)(Fig. 3B). Subsequent high resolution cryoEM struc- tures have shown that this protein fold (with minor variations) is Most mutations that protect bacteria from killing by bacteriophage present in all tailed phage MCPs examined, including those of infection are due to the loss of the primary surface receptors that lambdoid phages lambda, P22, Sf6 and CUS-3 (Lander et al., 2008; virions bind to on the cell surface, and studies with lambda identified Parent et al., 2012a, 2014a; Singh et al., 2013; Rizzo et al., 2014), as theLamBprotein(anoutermembraneporininvolvedinmaltose well as herpesvirus MCP (Baker et al., 2005), the MCP of an uptake) as its primary receptor (Thirion and Hofnung, 1972; Randall- archaea virus with tailed phage morphology (Pietila et al., 2013), Hazelbauer and Schwartz, 1973; Roessner et al., 1983). Other lambdoid and a cellular nanocompartment (McHugh et al., 2014). In addi- phages utilize different surface receptors. For example ø80 binds the tion, Pell et al. (2009) determined the structure of the lambda tail FhuA outer membrane protein (Vostrov et al., 1996; Endriss and Braun, tube subunit (gene V protein). This fold is also present in the 2004), P22 binds (and cleaves) the O-antigen polysaccharide (Wright protein that makes up the tail tubes of both noncontractile and and Kanegasaki, 1971), and Sf6 utilizes O-antigen and OmpA protein contractile tails of other phages, as well as in the shaft of the (Parent et al., 2014b). In all phages where it has been studied in detail, bacterial type VI secretion apparatus (reviewed by Davidson et al., fibers or spikes at the distal tip of the tail make the first interaction 2012). Thus, the lambdoid phages gave us the structures – now with the host. In lambda the J protein that resides at the tip of the called the “HK97 fold” and the “tail tube fold”–of what are surely lambda tail (R. Duda and R. Hendrix, unpublished) and its C-terminal two of the most abundant proteins on Earth. 149 AAs (of 1132 AAs) are sufficienttointeractwithLamB(Charbit et al., 1994; Wang et al., 2000b; Berkane et al., 2006; Meyer et al., Cell lysis 2012). In addition to receptor alterations, mutations in the host inner membrane protein ManY (mannose phosphotransferase) were found Five lambda genes have been identified that are important in lysis that block injection of lambda DNA. Mutations in the phage V or H of infected cells, S105, S107, R, Rz and Rz1. The enzyme responsible for genes overcome this block, and this role of ManY does not depend on cleaving the cell wall peptidoglycan is encoded by gene R (Campbell, its enzymatic activity (Emmons et al., 1975; Scandella and Arber, 1976; 1961; Harris et al., 1967). This endolysin is a true lysozyme in some Elliott and Arber, 1978; Esquinas-Rychen and Emi, 2001). Phage HK97, 320 S.R. Casjens, R.W. Hendrix / Virology 479-480 (2015) 310–330 which has a tail that is quite similar to that of lambda, requires the and that there must be an as yet uncharacterized rearrangement of host's inner membrane glucose transporter protein PstG and periplas- the tail tip that allows release of the DNA and H protein from the mic chaperone FkpA for successful DNA entry, and these requirements virion. Curiously, one of the minor tail tip proteins, the L protein, are determined by the tape measure protein (Cumby et al., 2015). The contains a (4Fe–4S)2þ iron–sulfur cluster (Tam et al., 2013). While actual role of host proteins in DNA injection remains a mystery. the function of the cluster is not known, the ability of the L protein A reconstruction of the early laboratory history of lambda to form the complex with this cluster is essential for both tail revealed some unexpected additional information about lambda's assembly and DNA injection (Dai, 2009). While lambda appears to tail fibers. When Dale Kaiser began his work on lambda as a graduate release only a few molecules of one protein from the virion during student at Caltech he saw that the phage made very small plaques, DNA injection, the short-tailed lambdoid phage P22 releases 6–20 andheisolatedamutantthatmadelarger,morerobustplaques.This molecules each of three different proteins with its DNA (Botstein largeplaquesizemadehisclassicexperimentsdefining the cI, cII and et al., 1973; Casjens and King, 1974; Israel, 1977). The roles of these cIII genes (above) possible, since plaque turbidity is much more proteins also remain unclear. It is not known how the DNA visible in larger plaques. Subsequently, as a postdoc at the Pasteur traverses the periplasm and penetrates the inner membrane. In InstituteinParis,hecrossedthelargeplaquemutanthehadbrought the short tailed phages the injected proteins may form a tempor- from Pasadena with the version of lambda in use in Paris at that time ary channel through the periplasm (Perez et al., 2009; reviewed by to make what came to be called lambda Pasadena/Paris or lambda Casjens and Molineux, 2012; see also Hu et al., 2013). Indeed one PaPa. This version of lambda is the one used in virtually all laboratory of the earliest observations in this area, that bacteria infected with experiments thereafter, and it soon came to be thought of as lambda lambda can take up exogenously added lambda DNA only if it has wild type. Finally, although lambda PaPa has only one short J protein at least one cohesive end is still not understood (Kaiser and fiber at its tail tip, and does not have long tail fibers, its genome Hogness, 1960; Kaiser, 1962). sequence contains a side tail fiber gene stf and a tail fiber addition gene tfa downstream of J. The STF protein has substantial sequence Non-essential accessory genes similarity to the phage T4 gene 37 tail fiber protein but contains a frameshift mutation (Hendrix and Duda, 1992). When it was In addition to lambda's 29 essential lytic genes, and cI, cII, cIII examined, the original lambda prophage in E. coli K-12 (Lederberg, and int, which are essential for efficient lysogeny, 38 additional 1951), which has been called Ur-lambda, was found to lack the open reading frames (ORFs) whose protein products are not frameshift mutation in the stf gene and to produce virions that have absolutely essential to lambda lytic growth or lysogeny in the long thin side tail fibers (Fig. 3A); the two halves of stf generated by laboratory were identified in the original genome sequence (as the frameshift in lambda PaPa were originally called orf401 and delineated in Hendrix et al., 1983; Hendrix and Casjens, 2006). orf314 by Sanger et al. (1982).Theselongfibers cause lambda to Many of these have been shown to be functional genes that make smaller plaques (they probably splay out and slow diffusion in produce proteins. Recent ribosome profiling analysis of lambda agar), so the stf frameshift mutation was present in the plaque picked by Jeff Roberts and co-workers (Liu et al., 2013) confirmed that by Kaiser. The side fibers apparently bind weakly to E. coli outer essentially all of these ORFs are translated. This observation, like membrane protein OmpC, and, although they are not essential, they the determination of the lambda genome sequence before it, speed up adsorption (Hendrix and Duda, 1992). provides striking confirmation of the accuracy of the catalog of The mechanism of DNA release from tailed phage virions and lambda genes with identifiable functions, which was determined passage into cells remains poorly understood. The right end of the largely by genetic methods, decades before development of many DNA in lambda virions extends from the head about one-third of of the sophisticated techniques that contemporary students may the way into the tail, so it is primed for directional release during assume have always been available. Surprisingly, the ribosome injection (Thomas, 1974). The great majority of the DNA is packed profiling also identified 55 possibly translated ORFs in addition to very tightly into phage heads with no DNA-binding proteins the 71 previously known genes. These “new” ORFs are all quite holding it in place (Casjens and Hendrix, 1974; Earnshaw et al., small (all but one are r76 codons in length), and none completely 1979; Chang et al., 2006; Lander et al., 2006, 2008). Phosphate overlaps a previously identified gene. It is not known if the short charge repulsion, DNA bending, partial dehydration and low putative proteins they might encode (or perhaps the act of their entropy state must be overcome to package DNA, and its compres- translation?) have functions. The profiling technique used is sion in the phage head creates a kind of DNA pressure inside the relatively new, and the authors do not rule out the possibility that capsid. DNA is spontaneously released from lambda virions when some or all of the putative new genes could be the result of they bind purified LamB receptor (Roessner et al., 1983; Roessner artifacts of the method. On the other hand, if ongoing work and Ihler, 1987); however, Molineux (2006), Molineux and Panja confirms the reality of even a fraction these new potential genes, (2013) and Panja and Molineux (2010) have argued that cellular lambda will once again have shown that the descriptions found in turgor pressure acts against phage DNA injection into cells and textbooks fall short, even for a “simple” little virus like lambda. have reviewed the ongoing discussion of the exact nature of the Each of the major lambda transcription units contains forces that drive DNA delivery into cells from virions. William accessory genes whose functions are known. The late region Gelbart, Alex Evilevitch and co-workers have studied the ener- includes the lom and bor genes that are expressed from the getics of lambda DNA condensation and release (e.g., Grayson et al., prophage and appear to help the host bind to eukaryotic cells 2006; Qiu et al., 2011; Liu et al., 2014). Rob Phillips and co-workers and confer blood serum resistance on the host, respectively have found that lambda DNA release on pure LamB in vitro is (Barondess and Beckwith, 1995; Vaca Pacheco et al., 1997). Ea47, complete in about 10 s, while injection into cells appears to Ea31 and Ea59 (the latter encodes an ATP-dependent endonu- average about 5 min (Van Valen et al., 2012). Unless the DNA- clease; Benchimol et al., 1982) are non-essential genes that lie bound dye confounds the latter experiments, this suggests that between the tail region and int whose biological roles are not forces inside the cell do affect the rate of DNA entry into cells from known. The early left operon contains a number of useful but lambda virions. nonessential genes. For example, the exo, bet and gam proteins Very little is known about the role of the other lambda tail catalyze homologous recombination (see section “DNA replication proteins in DNA injection beyond the fact that the H protein is and homologous recombination” above), the kil gene product dis- injected into the host, probably into the inner membrane and rupts host division septum formation (Greer, 1975; Haeusser et al., ahead of the DNA (Roessner et al., 1983; Roessner and Ihler, 1987), 2014), and the less well studied bin and ral functions block host S.R. Casjens, R.W. Hendrix / Virology 479-480 (2015) 310–330 321 replication initiation (Sergueev et al., 2001) and modulate host been known for over half a century that prophages often encode restriction-modification systems (Loenen and Murray, 1986), virulence factors that are important in bacterial diseases, e.g., respectively. Nonessential genes rexA and rexB are co-transcribed diphtheria toxin (Freeman, 1951; Uchida et al., 1971; Pappenheimer with the cI repressor gene and are discussed below with lysogenic and Murphy, 1983), but it was assumed that these are expressed from conversion genes. Near the 30-end of lambda's early right operon lie uninduced prophages. A surprising more recent observation is that ten contiguous nonessential nin genes, so named because they are the Shiga-like toxins do not follow this paradigm. The Shiga-like absent in large deletions that remove tR2 and make transcription of toxins are important in the human disease E. coli hemorrhagic colitis, the essential gene Q (and thus the late genes) high enough to allow and the stxA and stxB genes that encode the toxin are carried mostly the phage to be N independent (Court and Sato, 1969). The nin by lambdoid phage 933W and its close relatives (Plunkett et al., 1999; genes include the ren, orf, rap and orf221, genes that encode Smith et al., 2012), where they lie within the late operon between the protection from rexAB exclusion (Toothman and Herskowitz, promoter and lysis genes (Neely and Friedman, 1998). David Fried- 1980b), a homologous recombination function (Tarkowski et al., man, Matthew Waldor and colleagues have studied two of these 2002; Curtis et al., 2014), a Holliday junction resolvase active in phages, 933W and H-19B, and they find that high level expression of homologous recombination (Barik, 1993; Sharples et al., 2004)and stxAB is largely dependent on prophage induction and replication of a serine/threonine/tyrosine protein phosphatase (Voegtli et al., the prophage to increase the DNA copy number. In addition, phage 2000) whose role is unknown, respectively. mediated cell lysis is required to release the toxin from the cell (Wagner et al., 2002; Tyler et al., 2004). Thus, it appears that Lysogenic conversion “spontaneous” induction, lysis and death of a small fraction of these bacteria produces the toxin that is presumably advantageous to the Accessory genes that are expressed from the prophage and bacterium during disease. This seems to be an example of sib alter the host in some way are called lysogenic conversion genes, selection and/or altruism in which lysis of a few members of a e.g. the lambda bor and lom genes (above). Many temperate phages bacterial clone give an advantage to their clonal siblings (Livny and carry genes, called superinfection exclusion genes, that protect the Friedman, 2004). prophage-carrying host from attack by other phages, and lambda is no exception. Its rexAB (above) and sieB genes block infection by Molecular cloning of DNA certain other phages (e.g., Parma et al., 1992; Engelberg-Kulka et al., 1998; Ranade and Poteete, 1993). In both these cases the In 1968 Peter Lobban, a Ph.D. student in Dale Kaiser's labora- detailed mechanism of exclusion remains unclear, but it occurs tory in the Biochemistry Department of Stanford University was after DNA from the superinfecting phage has entered the cell and the first person to realize that the tools to clone DNA existed and in both cases a second protein, ren and esc, respectively, protect that it would be a useful thing to do. He presented this proposal at lambda from self-exclusion (Toothman and Herskowitz, 1980a; a Kaiser lab group meeting, defended the idea in his Ph.D. Ranade and Poteete, 1993). Other lambdoid phages encode exclu- preliminary exam, and proceeded to do his thesis work on cloning sion proteins that operate through other mechanisms. Several methodology (Lobban, 1972; Lobban and Kaiser, 1973). This idea, examples are as follows: The P22 sieA gene blocks injection of DNA which in part sprang from the fact that Kaiser's laboratory was by some phages (Susskind et al., 1974), phage 933W encodes a very near those of Arthur Kornberg and Robert Lehman where tyrosine kinase that excludes HK97 (Friedman et al., 2011), and the DNA polymerase and DNA ligase were being characterized at that phage ø80 and N15 cor genes block adsorption of superinfecting time (e.g., Olivera and Lehman, 1967; Englund et al., 1968), was so phages to the outer membrane FhuA protein (Vostrov et al., 1996; powerful that others devised more rapidly attainable cloning goals Uc-Mass et al., 2004). and were able to publish before Lobban (Jackson et al., 1972; Many lambdoid phages encode proteins that modify the bacterial Cohen et al., 1973). Nonetheless, the idea of molecular cloning O-antigen surface polysaccharide. These modifications change the originated with him (Kornberg, 1991; Berg, 1993; Casjens, 1994; spectrum of phages that can adsorb to the O-antigens of such Yi, 2008; for more extensive historical treatments of molecular lysogens and also alter the antigenicity and thus the pathogenicity cloning and genetic engineering see Lear, 1978; Judson, 1979; of the host bacterium (Allison and Verma, 2000; Broadbent et al., McElheny, 2014). 2010; Kintz et al., 2015). O-antigen modifying genes are present in In addition, other important parts of cloning technology were two common forms in lambdoid phages. Some phages, for example also developed with phage lambda. Werner Arber's discovery and Shigella phages Sf6 and Sf101, encode O-acetyl transferases that are characterization of bacterial restriction-modification systems was expressed from the prophage and acetylate sugar moieties in the done using phage lambda (Arber, 1971 and references therein). O-antigen repeat (Verma et al., 1991; Allison and Verma, 2000; Ron Davis at Stanford developed phage lambda into a powerful Jakhetia et al., 2014). Other lambdoid phages, for example P22, SfX cloning vector by showing that some restriction enzymes leave and epsilon34, carry a three gene gtrABC operon; the first two genes sticky ends and then utilizing these enzymes for DNA cloning in such a cluster, gtrA and gtrB, are rather highly conserved and are (Mertz and Davis, 1972; Cameron et al., 1975), Kaiser and Masuda thought to encode an undecaprenol phosphate-glucose flippase that (1973) devised a procedure for packaging exogenously added moves the sugar part of its substrate from the cytoplasmic to lambda DNA into lambda virions, and lambda-based cosmid periplasmic side of the inner membrane (Liu et al., 1996)anda vectors were developed for cloning relatively large DNA secti- bactoprenol transferase that transfers the glucose from UDP-glucose ons up to about 40 kbp (Hohn and Murray, 1977; Collins and to form undecaprenol phosphate-glucose (Guan et al., 1999), respec- Hohn, 1978). tively. The third gene, gtrC,ismuchmorevariableanddifferentgtrC's Thus, lambda contributed greatly to the development of DNA encode different glycosyltransferases that add various sugars as side cloning technology, but these advances contributed to the demise chains to the O-antigen backbone (Markine-Goriaynoff et al., 2004; of the golden age of lambda, since molecular cloning became the Thanweer et al., 2008; Villafane et al., 2008; Davies et al., 2013). first step in being able to understand cellular (both prokaryotic Interestingly, the phage P22 prophage gtr operon is subject to on/off and eukaryotic) DNA function at a molecular level. Because lambda phase variation that is epigenetically controlled by the host Dam was no longer uniquely experimentally accessible, and because the methylase and OxyR regulatory protein (Broadbent et al., 2010). basic aspects of its life cycle were by then fairly well understood, A variation on lysogenic conversion in the lambdoid phages is the the 1980s and 1990s saw fewer and fewer laboratories dedicated presence of genes that affect the host during lytic infection. It has solely to the study of phage lambda. Nonetheless, a handful of 322 S.R. Casjens, R.W. Hendrix / Virology 479-480 (2015) 310–330

) ) cI ) int, ptl ) ) N,cIII cro, cII Q

Accessory DNA packaging/TerminaseProcapsid Tailsassembly/HeadsTail fibers/TailspikesAccessory/LysogenicProphage conversion Homologousends ( recombinationRegulationProphage ( Regulation repressorDNA ( ( ReplicationAccessory (ninRegulation region) (Accessory Lysis

λ λ λ λ P22 λ λ Many λ λ Many λ λ λ HK97 P22 P22 P22 Sf6 N15 P22 P22 P22 933W P22 21 P22 HK97 SfV N15 Gifsy-2 L N15 Sf6 933W P22 SfV øKO2 434 SfV St64B Sf6 øKO2 ES18 ø80 øP27 N15 Gifsy-2 etc. Fels1 933W øES15 ZF40

Fig. 4. The lambdoid phage diversity menu. A schematic map of the lambda virion chromosome and its major transcripts are shown above (colored as in Fig. 1). The “menus” of various functional sections are labeled above and listed below the map in colored rectangles. In each section0s menu a few lambdoid phages that have very different genes in this region and are best understood in that particular regard are indicated; however, the extant diversity of most sections is larger than shown and remains poorly defined in many cases. In some cases the different phages indicated within one menu section have clearly non-homologous genes that encode parallel functions (e.g., SfV long contractile, lambda long noncontractile and P22 short tails; lambda Exo, P22 Erf and gifsy-2 RecE type homologous recombination systems), and in some cases the genes are extremely divergent homologs (e.g., portal and MCPs in the procapsid assembly section and probably all the different prophage repressors with different operator binding specificities). Each of the functional sections shown is populated by at least one gene in most lambdoid phages, although there are a few exceptions (e.g., Fels-1 has no nin region genes and HK022 has no N gene). In addition, any given lambdoid phage may have additional scattered accessory genes (e.g., in lambda the lom and rexAB genes between the tail and tail fiber genes and immediately downstream of cI, respectively, and in P22 the rha and ant genes between the lysis and DNA packaging genes and between tail and tailspike genes in P22, respectively). laboratories persisted in delving deeper into the details of the life hundreds of related prophages in bacterial genome sequences cycles of lambda and its relatives. In addition, the well-understood (see, for example, Casjens and Thuman-Commike (2011) for the 45 lambda system has often been used for proof-of-principle demon- such P22-like prophages known at that time). All these phages strations for new technologies. Thus, even though they are the sole infect members of the Enterobacteriaceae bacterial family, but their objects of study in relatively few laboratories at present, the diversity is nonetheless huge. These 80 phages fall into 17 different lambdoid phages continue to give new insights into the amazing “types” or “clusters” that all have lambda-like gene (functional details of gene expression, gene function and virus life cycles to module) orders and apparently similar gene expression cascades this day. (Grose and Casjens, 2014). (We note that there are a very small number of notable exceptions to this synteny; some phage N15 Bacteriophage diversity and the evolution of modular viral genomes and ASPE-1 early genes have an order that is different from that of lambda; van der Wilk et al., 1999; Ravin et al., 2000.) The 17 The early decision to study a small number of chosen bacterio- lambdoid phage clusters were defined in terms of overall sequence phages (including the most highly studied phages lambda, T4 and T7) similarity, in which a dot matrix plot comparison of members of for highly focused hypothesis-driven research certainly sped up the different clusters at relatively low stringency shows recognizable attainment of our current overall knowledge of gene expression nucleic acid similarity over more than 50% of the genome within mechanisms and numerous viral functions immensely. However, at but not between clusters (Grose and Casjens, 2014). A very preli- the same time it restricted our view of the diversity of life on Earth minary survey of prophages present in the hundreds of reported and in particular the diversity and natural abundance of bacterio- enterobacterial genome sequences has revealed at least four phages. Even in the early days of the study of phages several lambda additional lambdoid phage clusters (S. Casjens, unpublished). relatives (e.g., phages 21, 434, P22 and ø80) had been isolated and Clearly, the lambda life-style has been extremely successful and were known to form viable hybrids with lambda (Kaiser and Jacob, has diverged to fill many presumed niches. 1957). Because of their different repressor-operator specificities, these All these lambdoid phages are transitively related by patches of other phages were of seminal use in developing an understanding of similar sequence that can potentially undergo homologous recom- the control of early gene expression and the reasons why phage bination. The “unrelated” patches that lie in parallel chromosomal lambda cannot infect a lambda lysogen (e.g., Bode and Kaiser (1965) locations can be either homologous sequence that has diverged to showed that the lambda cII and cIII genes cannot complement from a the point that it is no longer significantly related or entirely heteroimmune repressed hybrid prophage). Analysis of heteroduplex unrelated sequence from a different source that performs the DNA pairs with lambda showed early on that these phages have same function. An example of the former is the lambdoid proph- mosaically related genomes in which patches of highly related age repressor. The repressors all perform the same role, shutting sequence are interspersed with patches of less related or unrelated off the lytic genes in the prophage, yet they are extremely variable sequences (Fiandt et al., 1971; Simon et al., 1971; Botstein, 1980; and often barely recognizable or even not recognizable as being Campbell and Botstein, 1983; Casjens et al., 1992a; Campbell, 1994). homologous. The lambdoid repressors that have been examined Phage genome sequences have greatly increased our understanding of have similarities in their DNA-binding domain folds, and so are this genome mosaicism in recent years (Hendrix et al., 2000; Juhala almost certainly ancient homologs (Pabo et al., 1990; Watkins et al., 2000; Hendrix, 2002; Casjens, 2005; Casjens and Thuman- et al., 2008). We are not aware of a recent analysis, but in 1992 Commike, 2011; Grose and Casjens, 2014). Fig. 4 shows some of the eleven different repressor operator-binding specificities were better-characterized variations among lambdoid phages. known (Casjens et al., 1992a), and the currently known sequence Many relatives of phage lambda have now been isolated, and at diversity of these proteins suggests a very much larger repertoire. present over 80 such phage genomes have been completely Another example of very divergent homologs is the procapsid sequenced (compiled in Grose and Casjens, 2014), along with assembly gene cluster (including the MCP and portal protein) of S.R. Casjens, R.W. Hendrix / Virology 479-480 (2015) 310–330 323 which there are 14 very different types among the lambdoid their population size as well as modification of bacteria through phages (Grose and Casjens, 2014). Examples of apparently non- lysogenic conversion and transduction. Thus, it is important to homologous proteins that perform analogous functions in various understand phages in their own right. lambdoid phages are the three largely unrelated tail types – short, Recent work has also found the lambdoid phages to have long contractile and long noncontractile (e.g., P22, SfV and lambda, technological applications and human health importance. Lambda respectively), the integrases of integrating phages like lambda and and P22 have been developed into powerful and versatile protein P22 and the protelomerases of phages N15, PY54 and øKO2 display agents, where both the head decoration proteins lambda D (above), and the multiple types of homologous recombination (Sternberg and Hoess, 1995; Mikawa et al., 1996; Nicastro et al., modules (above). We have previously discussed the differences 2013; Chang et al., 2014) and P22 Dec (Parent et al., 2012b), as well and similarities among the four best-studied lambdoid phages, as the lambda major tail V protein (Maruyama et al., 1994; Zanghi lambda, HK97, P22 and N15 in more depth, and the reader is et al., 2007) and P22 MCP head protein (Kang et al., 2008; Servid referred to Hendrix and Casjens (2006) for more examples of such et al., 2013) have been used successfully to display foreign mosaic relationships. peptides and proteins. Lambda virions have also been used as We believe that extant lambdoid phage diversity has been attained DNA or protein vaccine delivery vehicles in mammals (Jepson and in several ways. These phages are likely extremely old, and natural March, 2004; Lankes et al., 2007; Clark et al., 2011; Mattiacio et al., divergence has had sufficienttimetomakeevensomeoftheir 2011; Saeedi et al., 2014). Trevor Douglas and Peter Prevelige are homologous protein sequences no longer significantly related. New developing P22 into a flexible multi-use nanoparticle platform, mosaic boundaries/novel sequence joints can form in two ways. First, where foreign molecular cargo can be placed on the surface or highly divergent lambdoid phages could recombine through illegiti- within the interior of the particle (O'Neil et al., 2011, 2012; Lucon mate recombination events to generate a new boundary. Second, et al., 2012; Uchida et al., 2012) to make, for example, magnetic illegitimate recombination could incorporate novel sequences into a resonance contrast enhancing agents (Qazi et al., 2013, 2014)or phage by insertion between or replacement of resident genes. These enzyme-containing nanoreactors (Patterson et al., 2012, 2014). foreign inserts between pre-existing lambdoid phage genes have been Finally, the stability of cross-linked HK97 capsids may give them called “morons” since there is more DNA when they are present stability advantages, and HK97 nanoparticles are under develop- (Hendrix et al., 2000; Juhala et al., 2000), and the known morons ment (Huang et al., 2011). Human health relevance comes from the usually carry their own promoters for independent expression. The fact that these phages carry bacterial disease virulence factor sources of such inserts and replacements may be divergent lambdoid genes such as the lom and bor genes of lambda and the Shiga- phages, other phages or genes with no phage relationship, since their like toxin stx genes (above). sources are currently unknown. Thus, new mosaic boundaries likely It is now nearly 65 years since Esther Lederberg discovered and form very rarely, but the probability of such a fortuitous event named bacteriophage lambda, but we still have not reached the between two phages giving rise to a functional progeny phage is mythical goal of a complete understanding of its molecular life greatly increased if the two participating phages have the same gene cycle. If nothing else, work on these phages should have taught us order (as all lambdoid phages have). Given the enormous numbers of that there are many levels of intricacy and sophistication in all phages on Earth (Whitman et al., 1998; Wommack and Colwell, 2000; aspects of phage life cycles, and each time a level is “understood” it Hendrix, 2002), we imagine that this type of novel joint formation opens doors to yet deeper levels of understanding. Each time we may well be random, and that all possible single-recombination novel think we have learned something new and important about joints between divergent lambdoid phage genomes might be formed. lambda, we have really learned something new and important However, only the extremely small fraction of such recombinants that about biology more generally, and the lambdoid phages have not has a functional, advantageous phage genome would be found as the yet run out of interesting biology to illuminate. Despite the phages present today. Once a new functional mosaic boundary is changing fashions in research, the vicissitudes of NIH grant funding, formed in a lambdoid phage genome, homologous recombination and the changing cast of characters studying them, these phages between similar sequences in other mosaically related lambdoid have been a remarkably consistent and productive source of new phages could rapidly cause reassortment to form new combinations biological insight that remains an important experimental model of such boundaries (and sequence patches). Interestingly, although system in the vanguard of biological science. This seems to us both of these processes, formation and reassortment of mosaic unlikely to change any time soon. patches, will tend to homogenize the genetic makeup of the partici- pating phages, neither process is fast enough to mask the presence of clear clustering of different types within the lambdoid phage group Acknowledgments (Grose and Casjens, 2014). The authors' research is supported by NIH Grants GM47795 and GM114817 from the U.S. Public Health Service to R.W.H. and S.R.C., The future respectively. We thank our colleagues in the phage research field for many years of gratifyingly open and stimulating discussions. Lambdoid phage research was seminal in the attainment of our We especially thank Costa Georgopoulos, Peter Lobban and two current understanding on such varied fronts as gene and operon anonymous reviewers for their welcome suggestions and correc- structure and function, the mechanisms of control of gene expres- tions of our less than perfectly accurate memories. sion by regulatory proteins and small RNAs, protein chaperones, prophage integration, protein and RNA turnover, virion assembly and modular virus genome evolution. Although from outside References lambdoid phages may be seen as “fully understood,” we believe that there is still much that these phages can teach us. 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