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, USAb Biology Department, University of Utah, Salt Lake City, UT 84112, USAc Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260, USA

a r t i c l e i n f o

Article history:Received 9 December 2014Returned to author for revisions13 January 2015Accepted 5 February 2015Available online 3 March 2015

Keywords:BacteriophageLambdaLambdoid phages

a b s t r a c t

Molecular genetic research on bacteriophage lambda carried out during its golden age from the mid-1950s to mid-1980s was critically important in the attainment of our current understanding of thesophisticated and complex mechanisms by which the expression of genes is controlled, of DNA virusassembly and of the molecular nature of lysogeny. The development of molecular cloning techniques,ironically instigated largely by phage lambda researchers, allowed many phage workers to switch theirefforts to other biological systems. Nonetheless, since that time the ongoing study of lambda and itsrelatives has continued to give important new insights. In this review we give some relevant earlyhistory and describe recent developments in understanding the molecular biology of lambda's life cycle.

& 2015 Elsevier Inc. All rights reserved.

Contents

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 310Historical importance and recent progress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 311

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

The future . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323

Introduction

From the earliest studies on lambda genetics by Jean Weigle(1953), Francois Jacob and Elie Wollman (1953, 1954) and Dale Kaiser(1955 in volume number 1 of VIROLOGY) in the 1950s until it helpedusher in the age of genetic engineering in the late 1970s and early1980s, phage lambda was in its golden age at the center of the

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Virology

http://dx.doi.org/10.1016/j.virol.2015.02.0100042-6822/& 2015 Elsevier Inc. All rights reserved.

n Corresponding author at: Division of Microbiology and Immunology, PathologyDepartment, University of Utah School of Medicine, Emma Eccles Jones MedicalResearch Building, 15 North Medical Drive East, Salt Lake City, UT 84112, USA.Tel.: þ1 801 581 5980.

E-mail address: sherwood.casjens@path.utah.edu (S.R. Casjens).

Virology 479-480 (2015) 310–330

molecular genetic universe. During those decades lambda was one ofthe few experimental “organisms” that was sufficiently experimen-tally accessible to be viewed as potentially completely understand-able. Its genome was small enough not to be overwhelming, yet itwas capable of making a decision of whether to lysogenize or growlytically and was clearly complex enough to be interesting andinformative on many fronts. In particular it was unique in that agenetic and molecular understanding of the control of gene expres-sion seemed to be within almost immediate reach. Those of us luckyenough to be involved in the study of phage lambda during this timewere tremendously excited about future possibilities and threwourselves into the work with all the energy we had.

Lambda was originally discovered in 1951 by Esther Lederberg(1951) at the University of Wisconsin (Madison), when she seren-dipitously found it was released from the laboratory Escherichia colistrain K-12 after ultraviolet irradiation. Since that time all aspects oflambda's lytic and lysogenic lifestyles have been studied. Lambdabecame a more important and approachable experimental systemwhen Allan Campbell (1961) isolated a set of suppressible nonsensemutants of lambda (or amber mutations, originally called suppres-sor sensitive or sus mutations) that identified genes essential forits lytic growth. Additional amber mutations isolated by SandyParkinson (1968) and Goldberg and Howe (1969) and prophagemutations isolated by Clarence Fuerst (Mount et al., 1968) that werelethal for lambda lytic growth identified a few more essential genes.Genetic complementation tests of these mutations succeeded indefining all but one of lambda's 29 genes that are essential forplaque formation under laboratory conditions (this includes theframeshifted G and G–T gene pair and the alternate starts of theS105 and S107 genes as well as the C and Nu3 genes as two geneseach; see below). We note that two additional genes, Rz and Rz1should probably also be considered essential genes even thoughplaques often form in their absence. They are absolutely requiredfor disruption of the outer membrane and lysis unless externalphysical solution forces that are often present during laboratorygrowth help to destabilize the outer membrane of infected cells(Berry et al., 2012). Only the small cro gene was not found by theabove random mutant hunts. It was discovered during the geneticanalysis of the control of lysogeny (Eisen et al., 1970), and itsabsence leads to the lysogenic state and hence no lytic growth.Recombination frequency and deletion mapping of these andvarious promoter and operator mutations isolated by others duringthis time allowed the construction of a detailed genetic map.Analysis of the phage DNA molecule led to the most detailedphysical map of any organism's genome at the time. Detailed studyof the phenotypes of phage mutants defective in known, mappedgenes gave an early overall picture of the lambda life cycle. Indeed,the careers of both authors here started at about the same timewith the analysis of lambda virion morphogenesis by utilizing thevarious amber mutants to identify the proteins expressed from thelambda morphogenetic genes and determining the nature of thedefects in their absence – S.R.C. in the laboratory of Dale Kaiser atStanford University and R.W.H. in the laboratory of Jim Watson atHarvard University (Happily, rather than generating animosity, ourearly competition in this arena led to mutual respect and a lifelongfriendship). We believe that the clever use of forward geneticselections and screens to isolate informative mutations and combi-nations of mutations and the use of these molecular geneticexperiments to deduce the underlying molecular mechanismsreached its zenith in the study of phage lambda during this period.Those were heady days with lambda running at the front of thescientific pack.

We discuss here early experiments that led to our currentunderstanding of phage lambda and molecular biological pro-cesses in general. During this period VIROLOGY published many ofthese important findings. We also mention recent developments

to show how far the field has come in the past 60 years (weassume a basic knowledge of the phage lambda life cycle; seeHendrix et al., 1983; Hendrix and Casjens, 2006). We includediscussion of phages related to lambda, which are commonly refe-rred to as “lambdoid” phages. This ill-defined (and often incor-rectly used) term refers phages with very similar lifestyles tolambda and genomes that are mosaically related to lambda. Thisdefinition included the notion that a lambdoid phage is capable ofrecombination with lambda itself to produce a functional hybridphage, as was first shown with phage 434 by Kaiser and Jacob(1957). Recent phage genome sequences have caused an expansionof this term to mean a phage with the same functional gene orderas lambda and that carries patches of nucleotide sequence homol-ogy with lambda or another lambdoid phage. Thus, in theory asingle recombination event between lambdoid phages could giverise to a fully functional phage that has all the necessary genes(Hendrix, 2002; Casjens, 2005; Hendrix and Casjens, 2006; Groseand Casjens, 2014). In this discussion we use lambdoid to includefor example, the three phages HK97, P22 and N15, which typifythree of the best-studied lambdoid groups that have significantdifferences from lambda. Citations are in general not meant tobestow credit for the original discoveries but to allow the readeraccess to the literature. Other more detailed historical treatmentsof some of the topics covered below can be found in the booksBacteriophage lambda (Hershey, 1971) and Lambda II (Hendrixet al., 1983) (and in Gottesman and Weisberg, 2004; Stahl, 2005;Georgopoulos, 2006; Campbell, 2007; Court et al., 2007a).

Historical importance and recent progress

The lambda genome

Phage lambda DNA was one of the earliest model systems forstudying the physical nature of DNA and genes, and a substantialamount of early research examined the hydrodynamic propertiesof the lambda chromosome with the goal of, among other things,determining its absolute molecular weight. At the same timemethods (now considered to be primitive) were devised forseparating the two halves of the lambda chromosome and formapping genes identified genetically to these halves and tosmaller physical intervals (reviewed by Davidson and Szyblaski,1971). Soon thereafter electron microscopic analysis of absolutelength and of lambda DNA in heteroduplex with various alteredlambda DNAs gave rise to a major step forward, the construction ofa detailed physical map of lambda DNA – a gene map in base pairs(bps) rather than recombination frequency units (Fiandt et al.,1971; Simon et al., 1971). The lambda virion chromosome was alsoamong the first natural linear DNAs whose end structure wasunderstood in detail; it was found not to have blunt ends, but tohave complementary 12 bp protruding 50-ends called cohesive (orsticky) ends, since the two single-strand ends can pair and thusbind to each other (Hershey et al., 1963; Hershey and Burgi, 1965).Meanwhile, lambda contributed greatly to the ultimate mappingof DNA, the complete nucleotide sequence. It is not generallyappreciated that the 12 bp lambda cohesive ends were the subjectof the first direct nucleotide sequencing of a biological DNA. RayWu devised methodology for using DNA polymerase to fill in thecohesive ends with specifically labeled nucleotides, followed byanalysis of the product to determine this exact 12 bp nucleotidesequence (Wu and Kaiser, 1968; Onaga, 2014). This sequencing wasnot a trivial undertaking, as determining this 0.012 kbp of sequ-ence required several years of painstaking work. Fourteen yearslater, Fred Sanger et al. (1982) used lambda as the subject for thefirst determination of the complete genome sequence of a dsDNAvirus – 48,502 bp – using his dideoxynucleotide chain termination

S.R. Casjens, R.W. Hendrix / Virology 479-480 (2015) 310–330 311

method. This, along with anecdotal sequencing of various lambdagenes and mutants of them by the laborious and more error-pronechemical sequencing method (Maxam and Gilbert, 1977) in theprevious few years, unleashed a rapid avalanche of studies relatingthe many extraordinary genetic studies on lambda from the pre-vious decades to the genome sequence. This in turn gave rise to anunderstanding of phage lambda genes and their expression thatwas unprecedented at the time (reviewed in Hendrix et al., 1983).Fig. 1 shows a map of the lambda chromosome; more detailedannotated maps of lambda, HK97, P22 and N15 can be found in thesupplementary material of Hendrix and Casjens (2006).

In a sense the complete nucleotide sequence of the lambdagenome and publication of the Lambda II book (Hendrix et al.,1983) heralded the demise of lambda's golden age and its exaltedposition in molecular genetic research. The complete sequencegave the (false) impression to researchers outside this field thateverything important about lambda was known. This impression,along with the approximately simultaneous development of sim-ple molecular cloning techniques that could be used in virtuallyany molecular biology laboratory, allowed new and powerfulmolecular genetic access to essentially any biological system aresearcher wished to study. Ironically, lambda played a major partin the development of these techniques (below).

Control of transcription during lytic growth

Lambda was one of the first biological entities whose transcrip-tional regulation was studied and understood in detail. It was

found early on, like other phages under study at the time, to have atemporally controlled pattern of transcription and gene expres-sion, called in this case immediate early, early (sometimes called“delayed early”) and late transcription (reviewed by Echols, 1971;Friedman and Gottesman, 1983). Lambda's major transcriptionunits are shown in Fig. 1. When Jeffrey Roberts (1969), then astudent in the Watson/Gilbert lab at Harvard, used purified RNApolymerase to transcribe lambda DNA in vitro, it initiated RNAsynthesis at the immediate early promoters PL and PR, but thetranscripts made were longer than those observed in vivo. He thenused this system to discover a host protein (that he named Rho)that caused shorter transcripts to be made that were the samelength as those observed in vivo. Rho was later shown to causetranscription termination at the normal lambda sites in vivo, andto be required for a subset of transcription termination events in E.coli. Thus lambda was directly responsible for the discovery of thisfirst transcription controlling “factor” (if the sigma subunit isconsidered to be part of the basic RNA polymerase enzyme). Lyticinfection by lambda phage carrying a conditional lethal mutationin its N gene drastically lowered protein and transcript synthesisfrom nearly all lambda genes compared to a wild type infection(e.g., Radding, 1964; Echols, 1971). These findings were rapidlyfollowed by many ingenious molecular genetic studies that con-verged on the idea that N protein causes termination of the PL andPR immediate early transcripts to fail. The only immediate earlygenes, N and cro, lie upstream of these terminators, in the PL andPR transcripts, respectively. Further work showed that transcrip-tion is antiterminated at terminators tL1 and tR1 (and other

non-essential

8 9 10 11 12 13 14 15 16

tail tip

0 1 2 3 4 5 6 7

Z U V G–T H M L K Inu1 A W B C D E FI FIInu3

terminase procapsids (heads)

PR'

PaQ

39 40 41 42 43 46 47 48 kbp

ori

45

RQPO Rzren 290 57 56 rap68 221 64

lysisnon-essential non-essential

S107 small 26 60orf borsieBralkil

cIIIea10

34 36 37 38

N rexB rexA cI cro cII

conversion replication

recombination

23 24 25 26 27

ea47 ea31 ea59

non-essential (b2 region)

17 18 19 20 21 22

stf tfa

side tail fibers

Ea22

28 30 31 32

int ea8.5 exo bet gamsmall non-essential

33

recombination

lom xisJ

tail shaft

PR

PsieBPRM

t'I

tI

tR'tR2 tR3 tR4

tL3tL4

tL2

PO

tR1

sib

PRE

attP

29

44

tL1 PL

PI

t aQ

lysogenic DNA

homologous site-specific

Rz1 orfs?

orfsea22

tO

35

esc

55

S105

G

nutLnutR

cos

♦ ♦ ♦ ♦♦ ♦

∗∗ ∗

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 andfunctional 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 (♦) markregulatory genes. Important DNA sites (e.g., P, promoters; t, terminators) are indicated below the genes. Thick horizontal arrows below indicate mRNAs: black, transcriptsmade in a lysogen; orange, immediate–early transcripts; green, early transcripts; red, late transcripts; blue, transcripts made in response to high CII levels. Asterisks (n) markRNAs with regulatory activity (the detailed role of PRE-initiated cro antisense message remains unclear (Spiegelman et al., 1972)). The chromosome is circularized in the cellduring infection, so the PR0-initiated late transcript is continuous from the lower right to the upper left in the figure.

S.R. Casjens, R.W. Hendrix / Virology 479-480 (2015) 310–330312

downstream terminators; Fig. 1) by N protein to allow expressionof the downstream (delayed) early genes (summerized in Echols,1971; Franklin, 1974; Friedman and Gottesman, 1983 and refer-ences therein). N protein does not, however, simply inactivate Rho.Instead N somehow makes the transcribing RNA polymerase thathas passed over a nut (N utilization) site in its presence insensitive todownstream termination at Rho-dependent and Rho-independentterminators.

Since its identification as a transcription antiterminator, N proteinhas been the subject of many studies aimed at elucidating themechanism by which it accomplishes this feat. Jack Greenblatt firstsucceeded in accomplishing N action in vitro (Greenblatt, 1972), andmany subsequent studies have learned much about the detailed rolesof the several host proteins that participate in N-mediated antiter-mination (see section “Interaction between phage lambda and its host”below for a more detailed discussion of these factors). N protein, anatively unfolded protein (Van Gilst and von Hippel, 1997;Goldenberg and Argyle, 2014), binds to the boxB sequence of thenut site in the nascent PL and PR mRNAs, as well as to the host NusAprotein (Mogridge et al., 1998; Mishra et al., 2013) and RNApolymerase. It probably binds RNA polymerase on its β subunit nearthe RNA exit channel (Cheeran et al., 2005). Exactly how N proteinblocks termination is not yet known, but the recent observation thatit reduces transcriptional slippage may provide a clue if slippagecontributes to termination (Parks et al., 2014).

All characterized lambdoid phages possess an N protein-mediated transcription antitermination mechanism, with theexception of phage HK022. In lambda antitermination, as outlinedabove, the nascent PL or PR transcript interacts through its nutsites with N protein, RNA polymerase and other host proteins tocreate a transcription elongation complex that is insensitive totermination signals. HK022 accomplishes the same end without Nand without the known host factors. We now know that nascentPL and PR transcripts of HK022 interact directly with the β0

subunit of the transcribing RNA polymerase through secondarystructure formed at the put sites in the nascent RNA (whichcorrespond in position to the nut sites on the lambda transcripts)(Oberto et al., 1993; Sen et al., 2002; Komissarova et al., 2008). Theresulting transcription elongation complex, as with lambda, isinsensitive to termination signals. Thus, HK022 early transcriptantitermination is apparently mediated by RNA alone, rather thanby RNA in conjunction with N and host transcription factors.

HK022 has not finished its dalliance with non-canonical mechan-isms of regulating transcription with its unusual mechanism ofaccomplishing antitermination. It also has a mechanism to promoteinappropriate termination in lambda-like phages during co-infectionof the same bacterium. While HK022 does not have an N gene, itdoes have a heterologous gene, called nun, at the same position inthe gene order as N in lambda. The Nun protein interacts with the nutsites of lambda as well as with the other host factors and RNApolymerase (Burova et al., 1999; Watnick and Gottesman, 1998;Vitiello et al., 2014). This resembles what happens with N protein,but the resulting transcription elongation complex is biased towardtermination. For a phage like lambda such inappropriate terminationof transcription would be a lethal event. These stories and othersabout how HK022 deviates in informative ways from the lambdaparadigm, have largely come out of the laboratories of Max Gottes-man and the late BobWeisberg (Weisberg et al., 1999). Ongoing workon these topics provides a good illustration of how phage investiga-tions can still give new information on how RNA polymerase receivesand responds to signals.

Following N-mediated antitermination of PR transcription, gene Qis transcribed at the distal end of the early right operon. The Q proteinin turn acts as a transcriptional antiterminator that allows read-through of the tR0 terminator (Fig. 1) to allow expression of the lateoperon (Roberts et al., 1998 and references therein). However,

Q protein works by a very different mechanism from that of N protein.Studies with phage lambda and the lambdoid phage 82 have shownthat it is a DNA-binding protein that joins the transcription complex atthe Q binding element (QBE) between the -35 and -10 parts of the lateoperon promoter PR0 and stays with the transcription elongationcomplex as it traverses the late operon. In contrast to N-mediatedantitermination, there are no known host proteins that participate inits action. The recent structure of the DNA-binding domain of lambdaQ protein sheds considerable light on its contacts with the QBE DNAsequence, the R4 region of sigma factor and the tip of the RNApolymerase β flap (Deighan and Hochschild, 2007; Shankar et al.,2007; Muteeb et al., 2012; Vorobiev et al., 2014).

Control of lysogeny

In 1961 Jacob and Monod put forward their operon hypothesis,deduced entirely from their genetic work on the lac operon andphage lambda model systems (Jacob and Monod, 1961). The earlyobservations by Kaiser that CI is required for maintenance oflambda lysogeny and that it is responsible for immunity to super-infecting lambda phages strongly suggested, but did not absolutelyprove, that cI encoded a protein that directly turns off all the lambdalytic genes in the prophage state (Kaiser, 1957; Kaiser and Jacob,1957). The subsequent isolation of suppressible amber nonsensemutants in the cI gene proved that this function is mediated by theprotein product of the cI gene (Jacob et al., 1962; Thomas andLambert, 1962). By the mid 1960s experiments had demonstratedquite clearly that the lack of lytic gene expression from the lambdaprophage was explained by blockage of mRNA synthesis by the cIgene product (Jacob and Campbell, 1959; Thomas, 1971; Ptashne,1971 and references therein). In 1965–1966 a competition devel-oped between Mark Ptashne (with the lambda CI repressor) andWally Gilbert (with the E. coli LacI repressor) to be the first to isolatea transcriptional repressor and thus absolutely prove the existenceof repressor proteins that regulate gene expression. They worked inlaboratories on different floors of the same building at Harvard, andthis race for the repressor caught the attention of ABC television,which filmed a documentary, “The Scientist,” giving CI and LacI abrief moment of popular fame (and somewhat amusing andirritating the other scientists in the building who experienced thefilming). Gilbert and Muller-Hill (1966) attained their goal first, butPtashne (1967) was not far behind, and today both repressorsremain important model systems for the study of sequence-spe-cific DNA binding by proteins.

While maintenance of lambda lysogeny only requires onephage protein, the process of establishment of the lysogenic stateis more complex. Dale Kaiser's original genetic studies identifiedtwo additional genes, cII and cIII, in which defects lower thefrequency of establishment by several orders of magnitude (Kaiser,1957). These two genes lie in the early right and early left operons,respectively. After the discovery of Cro it was first thought that thelysis/lysogeny decision might simply be explained by a directcompetition for operator binding between CI and Cro (a bistablegenetic switch), but more recent experiments have shown that it isnot this simple. We now know that the CII protein is a transcrip-tional activator that stimulates strong cI gene transcription fromthe PRE promoter, and that high CII protein levels are critical inensuring the high CI concentrations required to establish, but notmaintain, the lysogenic state (Court et al., 2007a and referencestherein). The cIII gene encodes a protein that controls the stabilityof the CII protein by inhibiting the action of the host-encodedHflB(FtsH)-HflC-HflK protease complex (Kobiler et al., 2007;Bandyopadhyay et al., 2010). In addition to this host proteasecontrol of CII stability, the host physiological state impacts thelysis/lysogeny decision through the following: (1) cAMP andppGpp levels may control HflBCK levels (Hong et al., 1971; Rao

S.R. Casjens, R.W. Hendrix / Virology 479-480 (2015) 310–330 313

and Raj, 1973; Joung et al., 1994; Slominska et al., 1999). (2) RNaseIII is under strong growth rate regulation (Wilson et al., 2002), andits action controls cIII mRNA stability (Altuvia et al., 1991).(3) Integration host factor (IHF) modulates CII and CIII levels(Mahajna et al., 1986; Krinke and Wulff, 1990; Giladi et al., 1998)and N gene translation (Kameyama et al., 1991; Wilson et al.,2002). (4) RecA is activated by DNA damage to cause CI inactiva-tion (see below). (5) HflD may directly inhibit DNA binding by CIIprotein (Parua et al., 2010). (6) It has also been suggested that hostproteins DnaA and SeqA may affect PL transcription and thus thelysis/lysogeny decision (Wegrzyn et al., 2012). Finally, (7) the OOPRNA made from the lambda Po promoter is antisense to the 30

portion of cII gene mRNA and acts to destabilize that message withthe help of RNase III, and the OOP promoter is repressed by thehost LexA protein (Krinke and Wulff, 1990; Krinke et al., 1991;Lewis et al., 1994). Thus, the current consensus is that CII is thecentral player in establishing lysogeny, and Fig. 2 summarizes thiscomplex regulatory circuitry.

When a decision is made to establish lysogeny, lambda ensuresthat the proteins that favor the lytic growth pathway disappearquickly, thereby minimizing stuttering between the lytic andlysogenic life cycle directions and ensuring that a decision isdefinitive. The O, N, CII, CIII and Xis proteins are all extremelymetabolically unstable and are destroyed within a few minutes ofbeing made. Thus, when their synthesis stops, their DNA replica-tion initiation, delayed early gene expression and lysogeny pro-moting activities are quickly lost (Kobiler et al., 2004). The hostproteases utilized for this purpose are ClpX/ClpP for O (Gonciarz-Swiatek et al., 1999; Thibault and Houry, 2012), Lon for N(Gottesman et al., 1981; Maurizi, 1987; Mikita et al., 2013) andFtsH for CII and CIII (Shotland et al., 2000; Kobiler et al., 2002,2007), and Lon for Xis (Leffers and Gottesman, 1998).

In addition to preventing the lytic gene transcription cascade,the CI protein has two other known regulatory functions, turningdown expression of the host pckA gene and autoregulating its ownsynthesis. Array analysis of mRNAs showed that the E. coli pckAgene (which encodes phosphoenolpyruvate carboxykinase) is the

most highly down-regulated host gene in a lambda lysogen (Chenet al., 2005). This gene is required for growth on succinate andsome other carbon sources. It is not known why lowering itsexpression might be beneficial, but the fact that the pckA promoteralso overlaps lambdoid phage 21, P22, 434 and H-19B repressorbinding targets (all different from lambda CI operator specificity),suggests that this regulation is evolutionarily important to thelambdoid phages. Louis Reichardt, a student in Kaiser's lab in theearly 1970s, devised a quantitative assay for CI repressor proteinand performed an early set of intricate and elegant experimentsthat showed that CI activates its own synthesis at low CI levels andrepresses at high levels (Reichardt and Kaiser, 1971). This was oneof the first demonstrations of autoregulation of a gene. Otherexperiments illuminated the amazing intricacies of CI and Crobinding to the three tandem operators at PL to regulate thedivergent PRM and PL promoters (Ptashne, 1992 and referencestherein). More recent study of the lambda prophage repressor hasshown that it forms multimers that bind simultaneously tooperators OL and OR at PL and PR, respectively, thus looping theDNA between these two promoters, and that this looping isimportant in the autoregulation that keeps the CI concentrationwithin narrow limits in lysogens (Lewis et al., 2011; Cui et al.,2013a, 2013b; Hensel et al., 2013; Michalowski and Little, 2013;Priest et al., 2014). Host supercoil density impacts this looping, butits role in vivo is not known (Ding et al., 2014).

The CI–CII–CIII circuitry is nearly universally present in thelambdoid phages, but some of these phages have additionalcontrols on lysogeny. The first of these to be discovered was theP22 antirepressor (Ant), which binds to repressor and causes it torelease repression (Susskind and Botstein, 1975). Transcription ofthe ant gene in a prophage is kept off by two additional phage-encoded repressors, Mnt and Arc, and a small antisense RNA, Sar,that controls the translation of ant mRNA (Prell and Harvey, 1983;Liao et al., 1987; Wu et al., 1987; Schaefer and McClure, 1997).Salmonella lambdoid phage gifsy-1 encodes an antirepressor GfoRthat is repressed by the host LexA repressor (Lemire et al., 2011).Other phages such as N15 encode a different type of antirepressor

HflB/C/K

CI

Int

Q

N

CIII

IHF

glucose

cAMPRNaseIII

Ant

ArcMnt

*

Early gene expressioninluding N, cII, cIII and Q

PaQ*

? Cro

Late gene expression

Prophageintegration

NusA/B/E(S1)/G

CII Proteolysis

PR

PLPR PL

PR’

PL

tR1/tR2PRM

PRMPRE

PI

CII outputInput on CII

CII

PL

Rho

PL

PRM PR PL

Sar RNA

Fis

HexRecA

CI AutoproteolysisUV

HflDPOLexA

#

#†

Poop RNA†

†

Fig. 2. Lambda regulatory circuits. The figure shows phage lambda-encoded functions (yellow ovals) and host encoded functions (blue ovals) that impact the lysis-lysogenydecision 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 ovalsrespectively. 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 FISphysical participation in the integrase complex that catalyzes the integration of prophage DNA.

S.R. Casjens, R.W. Hendrix / Virology 479-480 (2015) 310–330314

control system. Here, transcription of AntA (an antirepressorwhose mode of action is not known) is negatively regulated bythe N15 prophage repressor and by premature transcriptionaltermination that is mediated by processing of a portion of theleader of the antirepressor operon mRNA (Ravin et al., 1999;Mardanov and Ravin, 2007). The exact biological role(s) of anti-repressors remains unclear; they could be prophage inductionmodules (see below) but could also impact the lytic/lysogenydecision.

Integration and excision

An aspect of the establishment of lysogeny not discussed aboveis the physical integration of the phage chromosome into the hostchromosome. The phenomenon of lysogeny had been known butpoorly understood for several decades before lambda was dis-covered (Lwoff, 1953). The quiescent phage genome (prophage) ina lysogen appeared to be attached to the host chromosome insome way that could be mapped in bacterial conjugation experi-ments (Lederberg and Lederberg, 1953; Wollman, 1953), but thenature of such an attachment was not known. Our current under-standing of prophage insertion derives from the idea of site-specific recombination between a circular phage lambda genomeand the host chromosome put forward by Allan Campbell (1962).Numerous subsequent genetic and physical tests of this idea byCampbell and others soon validated this model (see Gottesmanand Weisbeg, 1971). The existence of a lambda encoded function,integrase (Int), that was necessary for prophage insertion wassimultaneously demonstrated by the isolation of int defectivemutants by Zissler (1967), Zissler et al. (1971), Gingery andEchols (1967) and Gottesman and Yarmolinsky (1968). HowardNash and co-workers accomplished in vitro integration (reviewedby Weisberg and Landy, 1983), and many subsequent ingeniousgenetic and biochemical studies elucidated the complex mechan-ism of the integration reaction. The host integration host factor(IHF; named for this reaction but now known to have manypleiotropic functions in E. coli and Fis protein were found to beinvolved (Ball and Johnson, 1991). Genetic studies by Ethan Signer,Robert Weisberg and others combined with biochemical studiesfrom the Art Landy lab have resulted in a marvelously detailedpicture of the integration protein DNA complex bound to itstarget (the attachment site, att) and its actions that result in attsite-specific recombination and lambda prophage insertion (Seahet al., 2014; Tong et al., 2014 and references therein).

As Esther Lederberg originally discovered (above), lambdaprophages are induced by DNA damaging agents such as UV lightor mitomycin C to enter a cycle of lytic phage growth. Release of CIrepression causes the lytic gene expression cascade to initiate, andthe prophage is excised from the host chromosome to allow circle-to-circle DNA replication (below). Interestingly, the CI repressorcontains a cryptic autoprotease activity that is activated by bindingto the host RecA recombination protein, but only when RecA isactivated by ssDNA (Little, 1984; Kim and Little, 1993; Mustard andLittle, 2000). Integration and excision are directional; while Int(with its host factors) is sufficient to integrate lambda DNA,another phage-encoded protein Xis is required for the reverseexcision reaction. Control of the relative amounts of Int and Xis arenecessary to ensure that integration occurs during establishmentof lysogeny and excision occurs during induction of a lysogen. Thisis accomplished in two ways. First, when lysogeny is beingestablished by high CII levels (above) the Int/Xis ratio needs tobe high to ensure that integration occurs. To achieve this, CIIprotein activates the promoter PI, and its transcript encodes Intbut not Xis. Second, a process dubbed retroregulation lowers thelevel of Int expression during lytic infection. Here, a site sib in theRNA downstream of int and the PI transcript causes more rapid

decay of PL initiated int mRNA early after infection, ensuring thatInt is made in large amounts only when a high CII level commitsthe cell to lysogeny. In addition, the sib site is separated from PL inthe integrated prophage, so after induction moderate levels of bothInt and Xis made from PL ensure excision (Schindler and Echols,1981; Gottesman et al., 1982; Guarneros et al., 1982). CII alsoactivates a third promoter, PaQ, whose product is a small antisenseRNA of the Q gene, which presumably helps keep the late operonoff while a decision is being whether to lysogenize (Ho andRosenberg, 1985; Hoopes and McClure, 1985). In vivo visualizationexperiments have recently shown that, after injection, lambdaDNA is confined near its point of entry and replication-drivenmovement of the host DNA allows proper juxtaposition of the hostand phage attachment sites. These experiments also demonstratethat even bacterial attachment site location in the chromosomeaffects the frequency of lysogenization (Tal et al., 2014).

In an interesting variation of lysogeny, lambdoid phages N15,øKO2 and PY54 do not integrate into their host's genome, but existas linear plasmid prophages (Ravin and Shulga, 1970; Hertwiget al., 2003; Casjens et al., 2004). In these phages the int gene isreplaced by a ptl gene that encodes the enzyme, called protelo-merase, that creates the ends of these plasmids. These ends arecovalently closed hairpins in which one DNA strand turns aroundand becomes the other strand with no break in the phosphodiesterbackbone (Rybchin and Svarchevsky, 1999). The mechanism ofaction of protelomerase has been examined, and it recognizes aspecific inverted repeat sequence, makes staggered nicks 6 bpapart in the two DNA strands, separates the strands between thenicks, and after strand swapping ligates the ends to form twohairpins (Huang et al., 2004; Aihara et al., 2007). These prophagesencode their own plasmid segregation and replication proteins(Ravin, 2003; Ravin et al., 2003) and so do not turn off their earlygenes as completely as integrated prophages (Ravin et al., 2000).

DNA replication and homologous recombination

Lambda DNA replication initiates at a single origin and pro-ceeds bidirectionally from that point to generate circle-to-circlereplication until several tens of circles accumulate in the infectedcell. At that point replication switches (by a still poorly understoodmechanism) to rolling circle mode to generate the concatemericDNA molecules that are the substrate for DNA packaging intovirions (below) (Skalka et al., 1972; Wake et al., 1972; Narajczyket al., 2007). The lambda genome carries two essential DNAreplication genes, O and P. Four dimers of O protein bind at foursequence repeats in the origin, which lies inside the O gene, toform an “O-some” complex. The other essential replication protein,P, binds to the host DnaB helicase, and this binary complex bindsthe O-some to form the ori:O:P:DnaB preprimosomal complex(Zylicz et al., 1998 and references therein). Host chaperones DnaK-DnaJ-GrpE (see below) are involved in activating the preprimoso-mal complex to recruit the rest of the host replication machinery(Learn et al., 1997). Thus, the early study of lambda replicationcontributed greatly to an understanding of the detailed stepsrequired to accomplish controlled initiation. In addition, the originis activated by rightward transcription from PR, but this phenom-enon remains poorly understood (Leng and McMacken, 2002;Hayes et al., 2012; Olszewski et al., 2014). In some other lambdoidphages like P22, the P gene is replaced by a gene that encodes anactual DnaB type helicase (Wickner, 1984a, 1984b), and phagesAPSE-1 and N15 encode a putative DNA polymerase (van der Wilket al., 1999) or a bifunctional helicase-primase (Ravin et al., 2000;Mardanov and Ravin, 2006), respectively.

Genetic recombination was first demonstrated for lambda byJacob and Wollman (1954), and mutants of lambda that recombineat reduced frequency were isolated by Franklin (1967), Echols and

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Gingery (1968) and Signer and Weil (1968). These mutations werefound to affect genes exo and bet that lie in the early PL transcript.Exo is an exonuclease (Zhang et al., 2011 and references therein)and Bet is a strand-annealing protein (Matsubara et al., 2013 andreferences therein). These two proteins can substitute for RecA inhomologous recombination. In addition, a host RecBCD nucleaseinhibitor is encoded by the gam gene, which is located immedi-ately upstream of exo and bet (Court et al., 2007b and referencestherein). Lambda also carries two genes in its nin region that affectrecombination, orf and rap. Orf protein is a recombination med-iator that binds to host single-strand binding protein and ssDNA(Maxwell et al., 2005; Curtis et al., 2011, 2014), and Rap protein is aDNA structure-specific endonuclease that cleaves Holliday junc-tion branch points (Sharples et al., 1998; Poteete et al., 2002).Homologous recombination occurs by several different pathways,and these phage-encoded proteins participate with host proteinsin various ways (see for example Poteete, 2013). We will notreview these in detail here, but these lambdoid proteins continueto be important in ongoing studies of the systems that catalyzehomologous recombination.

Early work on lambda recombination also led directly to ourcurrent understanding of host RecBCD action. In about 1969mutants were isolated by David Henderson in Ken and NoreenMurray's laboratory that increased the growth of lambda that wasmissing its recombination genes. These were studied further byFrank Stahl, Gerry Smith and coworkers (Stahl, 2005 and refer-ences therein; Smith et al., 1981a, 1981b). These mutations turnedout to have created a unique asymmetric 8 bp nucleotide sequence(now called the Chi site, for crossover hotspot instigator), and theirstudy gave rise to our current understanding that the RecBCDnuclease enters DNA at double-strand breaks and degrades bothstrands (differentially) from there until it hits a properly orientedChi site. Passing over a Chi site “civilizes” the RecBCD exonuclease(to use Stahl's words) so that it continues along the DNA stands asa helicase, separating but not degrading them. Finally the hostRecA protein is recruited to initiate a recombination event. Wildtype lambda has no native Chi site, but its E. coli host has one every5 kbp on average, and they are highly preferentially orientedaccording to the direction replication passes over them. It is nowbelieved that Chi plays a major role in RecBCD-mediated recom-binational repair of collapsed E. coli replication forks (Kuzminov,2001). The lack of Chi sites in most invading foreign DNAs may alsoallow the RecBCD nuclease to help protect E. coli from such“invaders” (Stahl, 2005).

Other lambdoid phages carry different recombination genes inthe exo/bet/gam chromosomal location. The best studied of theseare genes abc1, abc2, erf and arf of phage P22. The two Abcproteins inhibit RecBCD nuclease, Erf is a strand annealing protein,and Arf is a poorly understood recombination accessory function(Botstein and Matz, 1970; Poteete and Fenton, 1983; Poteete et al.,1988, 1991). Other lambdoid phages carry a third type of recom-bination genes, recE and recT, that encode an exonuclease (ExoVIII)and a strand annealing protein, respectively (Kushner et al., 1974;Hall et al., 1993; Zhang et al., 2009). The latter two genes wereoriginally identified as E. coli genes, but were later found to lie inthe defective lambdoid prophage Rac (Clark et al., 1993); variousfully functional lambdoid phages such as gifsy-1 carry recE andrecT genes (Lemire et al., 2008). In addition, some other lambdoidphage nin regions carry a rusA gene which encodes a Hollidayjunction resolvase that is different from lambda Rap (Sharpleset al., 1994).

Recently the lambda exo and bet recombination functionshave been put to valuable technological use in accomplishingvery efficient homologous recombination in vivo. Don Court andcoworkers devised and optimized systems in which short 30–50 bphomologous tails on a selectable gene (generated by amplification

by PCR primers with such tails) are inserted by homologousrecombination into any desired site in a bacterial genome accordingto the sequence of the tails. Then, since the inserted gene is chosento be counter-selectable, homologous recombination can replacethe inserted gene with any DNA that has the proper homologies(summarized in Thomason et al., 2014). This now widely usedstrategy, called “recombineering,” is dependent upon high levels ofectopically expressed Exo and Bet and allows rapid, efficient andscarless engineering of bacterial genomes. Very recently, Farzadfardand Lu (2014) have used lambda bet-mediated ssDNA recombineer-ing to develop a genomic memory system that “writes” (recom-bines) a chosen sequence into a second site in the genome of E. coliin response to endogenous expression of ssDNA containing thesequence. The system thus genomically encodes a memory of pastexpression of the promoter engineered to express the ssDNA.

Interactions between phage lambda and its host

In the early 1970s Costa Georgopoulos, David Friedman and NatSternberg independently devised methods of isolating mutationsin the E. coli host that allowed lambda virions to adsorb and DNAto be injected, but which block phage infection at some later stage.These mutations affect lambda's head assembly, DNA replicationand early transcription. They alter specific host proteins that arerequired for successful transcription and for proper protein fold-ing/unfolding dynamics.

The host mutations that affect head assembly identified thehost GroEL-GroES protein folding chaperone (also now known asHSP60-HSP10) (Georgopoulos et al., 1973; Sternberg, 1973). Thefunctions defined by these host mutations were named “gro” for totheir inability of grow lambda (Georgopoulos and Herskowitz,1971), and the “E” was added to reflect the fact that mutations inlambda gene E (major head protein) overcome this gro block(Georgopoulos et al., 1973). However, in actual fact, mutations ineither gene E or B (portal protein) have this property, and althoughthis particular reaction has still not been studied in biophysicaldetail, subsequent work suggests that it is probably folding of thegene B protein that actually requires this host chaperone (Murialdoand Becker, 1978; Kochan and Murialdo, 1983; Georgopoulos, 2006).GroEL was originally known simply as GroE, but it acquired theL when its smaller partner, GroES, was discovered (Tilly et al., 1981).In agreement with the genetic evidence that GroEL-GroES interactswith lambda head proteins, examination of the results of lambdainfection of a groEL mutant host showed that head production wasseriously aberrant as seen in a B or C minus infection (Georgopouloset al., 1973). The mechanism of GroEL-GroES protein folding chaper-one action has now been studied in detail with other more tractablesubstrates (Horwich and Fenton, 2009).

The groE gene was one of the first bacterial genes to be cloned.Its cloning relied on the confluence of microbial genetics in theform of the groE mutants isolated by Costa Georgopoulos, and oneof the earliest recombinant DNA techniques in the form of ashotgun (random) library of E. coli DNA fragments in a phagelambda vector. One of us (RWH) packed some of Costa's groEmutants in a suitcase and went to visit Ron Davis at Stanford. Ronhad constructed one of the first libraries of E. coli DNA cloned intophage lambda genomic DNA and packaged in vitro into lambdavirions (below), and when this phage library was plated on a groEmutant bacterial strain it contained plaque-forming phages at afrequency of 10�4 relative to wild type E. coli. The rare plaque-forming phages carried an 8 kbp insert of E. coli DNA. (As anindication of how new the recombinant DNA technology was, thesize of the insert was measured not with restriction enzymes andagarose gels but by the much better established technique ofmeasuring the density of phage carrying the insert in an equili-brium CsCl gradient.) The 8 kbp insert was subsequently shown to

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contain both the groEL and groES genes and led to the identifica-tion of the GroEL protein as a 60 kDa protein with ATPase activitythat assembles into a cylindrical 14 subunit structure (two stackedrings of seven subunits) (Hendrix, 1979). Sequencing of this clonedDNA led to the realization, which was very surprising at the time,that GroEL had homologs in the chloroplasts of green plants in theform of “rubisco large subunit binding protein” (Hemmingsenet al., 1988). Rubisco is the photosynthesis enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase, and the plant GroEL homo-logue has a role in folding its large subunit (Gatenby, 1996). Whilewe were cloning and characterizing GroE, very similar experi-ments were done by Barbara and Tom Hohn in Basel, Switzerland,using another early lambda library of E. coli DNA that wasconstructed by Ken and Noreen Murray (Georgopoulos andHohn, 1978; Hohn et al., 1979).

The host mutants that affect DNA replication identified the DnaK-DnaJ-GrpE protein folding/unfolding chaperone (DnaK is also calledHSP70) (Georgopoulos and Herskowitz, 1971; Georgopoulos andWelch, 1993 and references therein), which is now known to berequired for disassembly/loosening of the protein complex that formsat the origin of lambda (above), thus enabling the replication fork tomove away from the origin (Hoffmann et al., 1992; Osipiuk et al., 1993;Wyman et al., 1993). In addition to pointing out that protein folding,assembly and disassembly can be catalyzed by other proteins, it wasnoticed early that transcription of the genes encoding the GroE andDnaK protein chaperone systems is enhanced at high temperatures.Further examination of this latter phenomenon by many workers hasled to our current understanding of the mechanisms of stress controlof gene expression (Tilly et al., 1983; Georgopoulos and Welch, 1993).Thus, lambda work was seminal in the nucleation of the idea ofcatalyzed protein folding and in the discovery and understanding oftwo important chaperone systems. Before this, all protein folding wasthought to be spontaneous.

The host mutants in which lambda fails to express (delayed)early genes affect the ability of the lambda N protein to cause RNApolymerase to bypass terminators and thus transcribe the down-stream essential early genes (above). These mutations alter severaltranscription factors now called NusA, NusB, NusE and NusG(originally for N under supplied but more recently N utilizationsubstance) that are all required for successful N antitermination(Georgopoulos, 1971; Friedman, 1971; Friedman and Baron, 1974;Friedman and Gottesman, 1983; Friedman and Court, 1995). NusAis an RNA-binding protein that interacts with the α subunit of RNApolymerase and modulates transcriptional pausing (Prasch et al.,2009; Yang et al., 2009; Yang and Lewis, 2010; Schweimer et al.,2011; Zhou et al., 2011; Muteeb et al., 2012; Kolb et al., 2014), aswell as interacting with lambda N protein (Mishra et al., 2013). TheNusB–NusE complex binds the BoxA RNA sequence of the nut sitesand binds RNA polymerase (Burmann et al., 2010; Stagno et al.,2011). NusG protein homologs are present in all three domains oflife, and in E. coli NusG is a transcriptional pause-suppressingfactor that might also be involved in suppressing aberrant anti-sense transcription in E. coli (Herbert et al., 2010; Peters et al.,2012; Yakhnin and Babitzke, 2014). It has two domains, an N-terminal domain that binds RNA polymerase (Belogurov et al.,2009) and a C-terminal domain that binds NusE and Rho(Burmann et al., 2010). NusE, which is now known to be ribosomalsmall subunit protein S10, binds NusG and as such is thought to beinvolved in ensuring that a ribosome closely follows the transcrib-ing RNA polymerase, thus coupling translation to transcription(Burmann et al., 2010; McGary and Nudler, 2013). In addition,NusA interacts with translesion DNA polymerases to affect DNArepair (Cohen et al., 2009), and with NusB it may even be involvedin intracellular RNA folding and processing (Bubunenko et al.,2013). Thus, these early lambda–host interaction genetic studiesrevealed previously unsuspected complexities of transcription and

led directly to the discovery and characterization of factors thatcontrol RNA polymerase elongation and termination. The study ofthe mechanisms of action of these factors continues to the present.

Virion assembly

The study of the assembly of phage lambda virions began in the1960s when Jean Weigle (1966, 1968) showed that lambda headsand tails assemble independently and spontaneously join in vitroto form infectious virions and with the deduction in the EduardKellenbeger, Louis Siminovitch and Dale Kaiser laboratories thatgene E encodes its major capsid protein (MCP—also called coatprotein) (Karamata et al., 1962; Kemp et al., 1968; Casjens et al.,1970). Fig. 3A shows an electron micrograph of the phage lambdavirion (with the side fibers that are not present in laboratorystrains, see below).

At that time, the study of viral structural proteins and theirassembly was a non-trivial enterprise. For example, one of us(S.R.C.) began his Ph.D. thesis project in 1968 by simply attemptingto determine how many different proteins are present in thelambda virion. I had been toiling over various strategies forseparating lambda virion proteins solubilized with urea, aceticacid or guanidine hydrochloride for nearly a year without notice-able success, when Bill Studier (who was studying phage T7 at theBrookhaven Laboratory) gave a seminar at Stanford describing thesodium dodecyl-sulfate (SDS)-polyacrylamide gel electrophoresistechnique that he and Jake Maizel had developed (Studier andMaizel, 1969; Maizel, 2000). The combined facts that boiling in0.1% SDS disassembles virtually all protein complexes and thatelectrophoresis could be performed in SDS solutions instantlymade the prospects for my thesis very bright. This, along withwhat was probably the first published use of a slab gel apparatusthat allowed accurate comparison of adjacent lanes, allowed me toget my thesis started with a definitive proof that gene E encodesthe major head protein of lambda (Casjens et al., 1970). I note herethat it was the ability to do the rigorous controls necessary toreally prove (rather than surmise or strongly suggest) our conclu-sions that first attracted me to phage work and has kept me herefor nearly 50 years. Uli Laemmli (a phage T4 worker) and JakeMaizel soon modified this technique to give considerably higherresolution (called discontinuous SDS gel electrophoresis). In acurious twist of fate this now universally used and extremelyhighly cited method was published only in a figure legend in Uli'spaper on T4 morphogenetic protein cleavage (Laemmli, 1970; cited215,930 times at the time of this writing).

With the advent of discontinuous SDS gel electrophoresis andmethods to tamp down host protein synthesis relative to lambdaprotein synthesis in infected cells (Ptashne, 1967; Hendrix, 1971)came the rapid identification of the rest of the morphogeneticgene products as specific bands in such gels (Buchwald et al., 1970;Hendrix, 1971; Georgopoulos et al., 1973). This in turn led toanalysis of the structures made by infections with various mutantphages and the gene products in them. These studies also illuminatedthe common protein cleavages that accompany lambda and manyother virus assembly processes and make them irreversible and/orallow access to final conformations not available to the full-lengthproteins (Murialdo and Siminovitch, 1972; Georgopoulos et al., 1973;Hendrix and Casjens, 1974, 1975). This information allowed thededuction of the basic assembly pathway of lambda virions (the orderof action and general role of each of the morphogenetic proteins;summarized in Georgopoulos et al., 1983; Hendrix and Casjens, 2006),but it did not elucidate the detailed functions of each of the 21 phage-encoded proteins required for virion assembly. Work continues in thisdirection to this day.

The fact that lambda procapsids (precursor head structures) areassembled first and DNA is subsequently pumped into them by a

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DNA translocase was initially suggested by analysis of head-likestructures made in vivo (Hendrix and Casjens, 1975) and wasrigorously proven by Kaiser et al. (1975). Two proteins, the productsof the Nu1 and A genes are required for inserting DNA into theprocapsid. The Nu1 protein recognizes the DNA to be packaged(Shinder and Gold, 1988; de Beer et al., 2002). The A protein cleaveslambda DNA at the cos site during packaging to create the cohesiveends (Wang and Kaiser, 1973; Feiss et al., 1983; Davidson et al.,1991) and is also the ATP cleavage-driven translocase that movesthe DNA into the procapsid (Duffy and Feiss, 2002; Tsay et al., 2010).Neither of these proteins is present in the completed virion. MikeFeiss and co-workers performed extensive and informative geneticanalyses of lambda DNA packaging (Sippy et al., 2014; Feiss and Rao,2012 and references therein), and Carlos Catalano's laboratory hasstudied its biochemistry (e.g., Andrews and Catalano, 2013; Singhet al., 2013). Most viruses do not depend on the host to help buildtheir virions (except for chaperones, above), but lambda DNApackaging initiation requires the host IHF protein, which binds toa sequence near the Nu1 protein recognition sites and bends theDNA. This bend allows formation of the DNA:Nu1:A nucleoproteincomplex that initiates cos site cleavage and DNA packaging (Xin etal., 1993; Sanyal et al., 2014). Laser tweezer measurements of theforce generated by this molecular motor in vitro by Douglas Smithand collaborators have shown it to be among the most powerfulnanomotors known (Tsay et al., 2009, 2010).

Lambda tail assembly work has focused largely on how its shaft isassembled. Isao Katsura and Roger Hendrix found that the length ofthe gene H-encoded tape measure protein determines the length ofthe shaft. It acts as a measuring device, most likely by serving as a

template for shaft assembly (Katsura and Hendrix, 1984; Katsura,1987). Curiously, the H protein is proteolytically cleaved before tailattachment to heads, but the role of this cleavage and the proteasethat causes it remain unknown (Hendrix and Casjens, 1974; Tsui andHendrix, 1983). In addition, Levin et al. (1993) found that a pro-grammed translational frameshift is required for expression of theessential T reading frame as a C-terminal extension of the gene Gprotein (called G–T protein). The frameshift ensures the proper ratio(�30:1) of G and G–T proteins. This was one of the first demonstra-tions of programmed frameshifting in prokaryotes, and it is nowknown to be an almost universal feature of the assembly of longphage tails, with the curious apparent exception of phage T4 and itsallies (Xu et al., 2004a). This phenomenon has only been studied inmore detail in lambda, where both G and G–T proteins are requiredfor tail assembly, but neither is present in completed tails. The Gand G–T proteins act as a chaperone in which the G domain bindsthe tape measure protein, and the T domain of G–T binds the majortail subunit (the gene V protein) (Xu et al., 2013, 2014). An under-standing of the detailed dynamics of this assembly process awaitsfuture study.

The different lambdoid phages have quite diverse virion struc-tures (below) and as such have proven to be very fertile ground instudies of virion assembly – in particular, studies of phages P22 andHK97 have been very informative. Salmonella phage P22 was ofinterest early because it was the first phage found to carry outgeneralized transduction (Zinder and Lederberg, 1952). HorstSchmieger isolated P22 mutants with greatly increased frequenciesof transduction (Schmieger, 1972; Casjens et al., 1992b), which madeP22 the easiest transducing phage to use in the laboratory. As such it

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 reproducedwith 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 thelocations 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 rainbowcolor depiction by MacPyMOL copyright 2009–2010 Schrodinger, LLC). (C) Subnanometer-resolution 3-dimensional cyroelectron microscopic reconstruction of the phagelambda 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) andreproduced with permission of publisher.

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contributed significantly to the rise of Salmonella enterica as one ofthe important bacterial model organisms (Neidhardt, 1996). MyronLevine, David Botstein and Jonathan King, studied P22 on its ownmerits with the (prescient) view that phages related to lambdawould benefit from the early lambda studies but would be differentenough from lambda to be interesting and informative. Transductionis the result of errors in phage DNA packaging, and the study of P22has led to a much better understanding of headful DNA packaging(Tye et al., 1974; Jackson et al., 1978; Casjens and Hayden, 1988;Casjens et al., 1992c; Wu et al., 2002; Leavitt et al., 2013). P22 wasalso the first virus found to require a scaffolding protein for properassembly of its MCP into a capsid shell. Scaffolding protein is presentin large numbers inside of procapsids, but it exits the structure beforeDNA enters to form complete virions. This was one of the firstdiscoveries of a protein that catalyzes the assembly of other proteins(King and Casjens, 1974; Earnshaw et al., 1976; Weigele et al., 2005;Chen et al., 2011; Padilla-Meier et al., 2012), and scaffolding proteinshave since been found to be a general feature of the assembly of largevirus particles.

The study of lambdoid phage HK97 began in the Hendrix labbecause its tail has a slightly different length from that of lambda,but it was soon noticed that its MCP behaved as a smear near thetop of an SDS electrophoresis gel of virion proteins (Popa et al.,1991). Analysis of this phenomenon showed that its head proteinsare very different from those of lambda and that its virion MCP isauto-catalytically cross-linked into inter-locked rings as “molecu-lar chain mail” (Duda, 1998; Wikoff et al., 2000; Helgstrand et al.,2003; Dierkes et al., 2009). We now know that such a chain mailarrangement is not unique to HK97 and may not be uncommon inphage capsids (Duda, 1998; Gilakjan and Kropinski, 1999). Inaddition, structural studies in the Alasdair Steven and Jack Johnsonlaboratories led to a uniquely detailed picture of HK97 procapsidmaturation (MCP shell expansion) into virion heads (Duda et al.,2006; Hendrix and Johnson, 2012; Cardone et al., 2014). The studyof P22 and epsilon15 (a remote lambda relative that is not quitelambdoid) gave rise to the first asymmetric cryo-electron micro-scopic (EM) reconstructions of virions that show the tail as well asthe MCP at subnanometer resolution (Jiang et al., 2006, 2008;Lander et al., 2006; Chang et al., 2006; Tang et al., 2011). Finally,and perhaps most importantly, the HK97 MCP shell crystallizesand remains today as the only large phage MCP that has beensolved to atomic resolution by x-ray crystallography. The fold ofthe HK97 MCP was novel at the time of its determination (Wikoffet al., 2000) (Fig. 3B). Subsequent high resolution cryoEM struc-tures have shown that this protein fold (with minor variations) ispresent in all tailed phage MCPs examined, including those oflambdoid phages lambda, P22, Sf6 and CUS-3 (Lander et al., 2008;Parent et al., 2012a, 2014a; Singh et al., 2013; Rizzo et al., 2014), aswell as herpesvirus MCP (Baker et al., 2005), the MCP of anarchaea virus with tailed phage morphology (Pietila et al., 2013),and a cellular nanocompartment (McHugh et al., 2014). In addi-tion, Pell et al. (2009) determined the structure of the lambda tailtube subunit (gene V protein). This fold is also present in theprotein that makes up the tail tubes of both noncontractile andcontractile tails of other phages, as well as in the shaft of thebacterial type VI secretion apparatus (reviewed by Davidson et al.,2012). Thus, the lambdoid phages gave us the structures – nowcalled the “HK97 fold” and the “tail tube fold” – of what are surelytwo of the most abundant proteins on Earth.

Cell lysis

Five lambda genes have been identified that are important in lysisof infected cells, S105, S107, R, Rz and Rz1. The enzyme responsible forcleaving the cell wall peptidoglycan is encoded by gene R (Campbell,1961; Harris et al., 1967). This endolysin is a true lysozyme in some

phages like P22 and a transglycosidase in lambda (Bienkowska-Szewczyk et al., 1981; Weaver et al., 1985; Evrard et al., 1998;Mooers and Matthews, 2006). Ryland Young and colleagues havedescribed themechanism(s) by which the lambda R protein is releasedinto the periplasm. Release is controlled by the S105 and S107 holinproteins, which are expressed from different start codons of the samereading frame, resulting in one protein, S107 that is two amino acidslonger than the other, S105. The shorter one, S105, causes lysis andS107 inhibits lysis; thus, the ratio of the two determines the exacttiming of lysis (Wang et al., 2000a). Lambda S105 protein is a smallmembrane protein that forms large physical holes in the membranethat allow endolysin escape across the cytoplasmic membrane. Theseholes are irregular, average 4300 nm in diameter and involvehundreds of S105 molecules (Savva et al., 2008; To and Young,2014). Exactly how S105 molecules aggregate to form such large holesafter accumulating in the membrane in a non-hole-forming stateremains mysterious. More recent study of the lambdoid phage 21 hasshown that its mechanism of endolysin release is different from that oflambda. Its endolysin is secreted by the host Sec translocon machineryinto the periplasm in a catalytically inactive form, tethered to the innermembrane by an N-terminal transmembrane domain. When thephage 21 “pinholin” forms small holes that depolarize the innermembrane, the endolysin is released from the membrane and isactivated to cleave the peptidoglycan (Pang et al., 2009, 2013; Xu et al.,2004b). In addition to holin disruption of the inner membrane and cellwall breakdown, the outer membrane must also be disrupted forcomplete cell lysis to occur. The two lambda proteins that perform thisstill poorly understood process are Rz and Rz1, which are an innermembrane protein and an outer membrane lipoprotein, respectively.Together they form the “spanin” complex (so named because it spansthe entire periplasm) (Berry et al., 2008, 2012; Young, 2014). Spaninsare not always essential in laboratory phage infections becausebuffeting of cells suspended in solution can physically disrupt theouter membrane in their absence; however, their nearly universalpresence in tailed phages suggests a critical evolutionary importance.Curiously, in lambdoid and many other phages the Rz1 gene lieswholly within the Rz gene in its þ1 reading frame, where it is aspectacular example of complete gene overlap. These three features ofcell lysis, a peptidoglycan degrading enzyme, a holin or pinholin and aspanin, are essentially universal among the tailed phages, and theiractions were unraveled through study of the lambdoid phages.

DNA delivery from the virion into target cells

Most mutations that protect bacteria from killing by bacteriophageinfection are due to the loss of the primary surface receptors thatvirions bind to on the cell surface, and studies with lambda identifiedthe LamB protein (an outer membrane porin involved in maltoseuptake) as its primary receptor (Thirion and Hofnung, 1972; Randall-Hazelbauer and Schwartz, 1973; Roessner et al., 1983). Other lambdoidphages utilize different surface receptors. For example ø80 binds theFhuA outer membrane protein (Vostrov et al., 1996; Endriss and Braun,2004), P22 binds (and cleaves) the O-antigen polysaccharide (Wrightand Kanegasaki, 1971), and Sf6 utilizes O-antigen and OmpA protein(Parent et al., 2014b). In all phages where it has been studied in detail,fibers or spikes at the distal tip of the tail make the first interactionwith the host. In lambda the J protein that resides at the tip of thelambda tail (R. Duda and R. Hendrix, unpublished) and its C-terminal149 AAs (of 1132 AAs) are sufficient to interact with LamB (Charbitet al., 1994; Wang et al., 2000b; Berkane et al., 2006; Meyer et al.,2012). In addition to receptor alterations, mutations in the host innermembrane protein ManY (mannose phosphotransferase) were foundthat block injection of lambda DNA. Mutations in the phage V or Hgenes overcome this block, and this role of ManY does not depend onits enzymatic activity (Emmons et al., 1975; Scandella and Arber, 1976;Elliott and Arber, 1978; Esquinas-Rychen and Emi, 2001). Phage HK97,

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which has a tail that is quite similar to that of lambda, requires thehost's inner membrane glucose transporter protein PstG and periplas-mic chaperone FkpA for successful DNA entry, and these requirementsare determined by the tape measure protein (Cumby et al., 2015). Theactual role of host proteins in DNA injection remains a mystery.

A reconstruction of the early laboratory history of lambdarevealed some unexpected additional information about lambda'stail fibers. When Dale Kaiser began his work on lambda as a graduatestudent at Caltech he saw that the phage made very small plaques,and he isolated a mutant that made larger, more robust plaques. Thislarge plaque size made his classic experiments defining the cI, cII andcIII genes (above) possible, since plaque turbidity is much morevisible in larger plaques. Subsequently, as a postdoc at the PasteurInstitute in Paris, he crossed the large plaque mutant he had broughtfrom Pasadena with the version of lambda in use in Paris at that timeto make what came to be called lambda Pasadena/Paris or lambdaPaPa. This version of lambda is the one used in virtually all laboratoryexperiments thereafter, and it soon came to be thought of as lambdawild type. Finally, although lambda PaPa has only one short J proteinfiber at its tail tip, and does not have long tail fibers, its genomesequence contains a side tail fiber gene stf and a tail fiber additiongene tfa downstream of J. The STF protein has substantial sequencesimilarity to the phage T4 gene 37 tail fiber protein but contains aframeshift mutation (Hendrix and Duda, 1992). When it wasexamined, the original lambda prophage in E. coli K-12 (Lederberg,1951), which has been called Ur-lambda, was found to lack theframeshift mutation in the stf gene and to produce virions that havelong thin side tail fibers (Fig. 3A); the two halves of stf generated bythe frameshift in lambda PaPa were originally called orf401 andorf314 by Sanger et al. (1982). These long fibers cause lambda tomake smaller plaques (they probably splay out and slow diffusion inagar), so the stf frameshift mutationwas present in the plaque pickedby Kaiser. The side fibers apparently bind weakly to E. coli outermembrane protein OmpC, and, although they are not essential, theyspeed up adsorption (Hendrix and Duda, 1992).

The mechanism of DNA release from tailed phage virions andpassage into cells remains poorly understood. The right end of theDNA in lambda virions extends from the head about one-third ofthe way into the tail, so it is primed for directional release duringinjection (Thomas, 1974). The great majority of the DNA is packedvery tightly into phage heads with no DNA-binding proteinsholding it in place (Casjens and Hendrix, 1974; Earnshaw et al.,1979; Chang et al., 2006; Lander et al., 2006, 2008). Phosphatecharge repulsion, DNA bending, partial dehydration and lowentropy state must be overcome to package DNA, and its compres-sion in the phage head creates a kind of DNA pressure inside thecapsid. DNA is spontaneously released from lambda virions whenthey bind purified LamB receptor (Roessner et al., 1983; Roessnerand Ihler, 1987); however, Molineux (2006), Molineux and Panja(2013) and Panja and Molineux (2010) have argued that cellularturgor pressure acts against phage DNA injection into cells andhave reviewed the ongoing discussion of the exact nature of theforces that drive DNA delivery into cells from virions. WilliamGelbart, Alex Evilevitch and co-workers have studied the ener-getics of lambda DNA condensation and release (e.g., Grayson et al.,2006; Qiu et al., 2011; Liu et al., 2014). Rob Phillips and co-workershave found that lambda DNA release on pure LamB in vitro iscomplete in about 10 s, while injection into cells appears toaverage about 5 min (Van Valen et al., 2012). Unless the DNA-bound dye confounds the latter experiments, this suggests thatforces inside the cell do affect the rate of DNA entry into cells fromlambda virions.

Very little is known about the role of the other lambda tailproteins in DNA injection beyond the fact that the H protein isinjected into the host, probably into the inner membrane andahead of the DNA (Roessner et al., 1983; Roessner and Ihler, 1987),

and that there must be an as yet uncharacterized rearrangement ofthe tail tip that allows release of the DNA and H protein from thevirion. Curiously, one of the minor tail tip proteins, the L protein,contains a (4Fe–4S)2þ iron–sulfur cluster (Tam et al., 2013). Whilethe function of the cluster is not known, the ability of the L proteinto form the complex with this cluster is essential for both tailassembly and DNA injection (Dai, 2009). While lambda appears torelease only a few molecules of one protein from the virion duringDNA injection, the short-tailed lambdoid phage P22 releases 6–20molecules each of three different proteins with its DNA (Botsteinet al., 1973; Casjens and King, 1974; Israel, 1977). The roles of theseproteins also remain unclear. It is not known how the DNAtraverses the periplasm and penetrates the inner membrane. Inthe short tailed phages the injected proteins may form a tempor-ary channel through the periplasm (Perez et al., 2009; reviewed byCasjens and Molineux, 2012; see also Hu et al., 2013). Indeed oneof the earliest observations in this area, that bacteria infected withlambda can take up exogenously added lambda DNA only if it hasat least one cohesive end is still not understood (Kaiser andHogness, 1960; Kaiser, 1962).

Non-essential accessory genes

In addition to lambda's 29 essential lytic genes, and cI, cII, cIIIand int, which are essential for efficient lysogeny, 38 additionalopen reading frames (ORFs) whose protein products are notabsolutely essential to lambda lytic growth or lysogeny in thelaboratory were identified in the original genome sequence (asdelineated in Hendrix et al., 1983; Hendrix and Casjens, 2006).Many of these have been shown to be functional genes thatproduce proteins. Recent ribosome profiling analysis of lambdaby Jeff Roberts and co-workers (Liu et al., 2013) confirmed thatessentially all of these ORFs are translated. This observation, likethe determination of the lambda genome sequence before it,provides striking confirmation of the accuracy of the catalog oflambda genes with identifiable functions, which was determinedlargely by genetic methods, decades before development of manyof the sophisticated techniques that contemporary students mayassume have always been available. Surprisingly, the ribosomeprofiling also identified 55 possibly translated ORFs in addition tothe 71 previously known genes. These “new” ORFs are all quitesmall (all but one are r76 codons in length), and none completelyoverlaps a previously identified gene. It is not known if the shortputative proteins they might encode (or perhaps the act of theirtranslation?) have functions. The profiling technique used isrelatively new, and the authors do not rule out the possibility thatsome or all of the putative new genes could be the result ofartifacts of the method. On the other hand, if ongoing workconfirms the reality of even a fraction these new potential genes,lambda will once again have shown that the descriptions found intextbooks fall short, even for a “simple” little virus like lambda.

Each of the major lambda transcription units containsaccessory genes whose functions are known. The late regionincludes the lom and bor genes that are expressed from theprophage and appear to help the host bind to eukaryotic cellsand confer blood serum resistance on the host, respectively(Barondess and Beckwith, 1995; Vaca Pacheco et al., 1997). Ea47,Ea31 and Ea59 (the latter encodes an ATP-dependent endonu-clease; Benchimol et al., 1982) are non-essential genes that liebetween the tail region and int whose biological roles are notknown. The early left operon contains a number of useful butnonessential genes. For example, the exo, bet and gam proteinscatalyze homologous recombination (see section “DNA replicationand homologous recombination” above), the kil gene product dis-rupts host division septum formation (Greer, 1975; Haeusser et al.,2014), and the less well studied bin and ral functions block host

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replication initiation (Sergueev et al., 2001) and modulate hostrestriction-modification systems (Loenen and Murray, 1986),respectively. Nonessential genes rexA and rexB are co-transcribedwith the cI repressor gene and are discussed below with lysogenicconversion genes. Near the 30-end of lambda's early right operon lieten contiguous nonessential nin genes, so named because they areabsent in large deletions that remove tR2 and make transcription ofthe essential gene Q (and thus the late genes) high enough to allowthe phage to be N independent (Court and Sato, 1969). The ningenes include the ren, orf, rap and orf221, genes that encodeprotection from rexAB exclusion (Toothman and Herskowitz,1980b), a homologous recombination function (Tarkowski et al.,2002; Curtis et al., 2014), a Holliday junction resolvase active inhomologous recombination (Barik, 1993; Sharples et al., 2004) anda serine/threonine/tyrosine protein phosphatase (Voegtli et al.,2000) whose role is unknown, respectively.

Lysogenic conversion

Accessory genes that are expressed from the prophage andalter the host in some way are called lysogenic conversion genes,e.g. the lambda bor and lom genes (above). Many temperate phagescarry genes, called superinfection exclusion genes, that protect theprophage-carrying host from attack by other phages, and lambdais no exception. Its rexAB (above) and sieB genes block infection bycertain other phages (e.g., Parma et al., 1992; Engelberg-Kulkaet al., 1998; Ranade and Poteete, 1993). In both these cases thedetailed mechanism of exclusion remains unclear, but it occursafter DNA from the superinfecting phage has entered the cell andin both cases a second protein, ren and esc, respectively, protectlambda from self-exclusion (Toothman and Herskowitz, 1980a;Ranade and Poteete, 1993). Other lambdoid phages encode exclu-sion proteins that operate through other mechanisms. Severalexamples are as follows: The P22 sieA gene blocks injection of DNAby some phages (Susskind et al., 1974), phage 933W encodes atyrosine kinase that excludes HK97 (Friedman et al., 2011), and thephage ø80 and N15 cor genes block adsorption of superinfectingphages to the outer membrane FhuA protein (Vostrov et al., 1996;Uc-Mass et al., 2004).

Many lambdoid phages encode proteins that modify the bacterialO-antigen surface polysaccharide. These modifications change thespectrum of phages that can adsorb to the O-antigens of suchlysogens and also alter the antigenicity and thus the pathogenicityof the host bacterium (Allison and Verma, 2000; Broadbent et al.,2010; Kintz et al., 2015). O-antigen modifying genes are present intwo common forms in lambdoid phages. Some phages, for exampleShigella phages Sf6 and Sf101, encode O-acetyl transferases that areexpressed from the prophage and acetylate sugar moieties in theO-antigen repeat (Verma et al., 1991; Allison and Verma, 2000;Jakhetia et al., 2014). Other lambdoid phages, for example P22, SfXand epsilon34, carry a three gene gtrABC operon; the first two genesin such a cluster, gtrA and gtrB, are rather highly conserved and arethought to encode an undecaprenol phosphate-glucose flippase thatmoves the sugar part of its substrate from the cytoplasmic toperiplasmic side of the inner membrane (Liu et al., 1996) and abactoprenol transferase that transfers the glucose from UDP-glucoseto form undecaprenol phosphate-glucose (Guan et al., 1999), respec-tively. The third gene, gtrC, is much more variable and different gtrC'sencode different glycosyltransferases that add various sugars as sidechains to the O-antigen backbone (Markine-Goriaynoff et al., 2004;Thanweer et al., 2008; Villafane et al., 2008; Davies et al., 2013).Interestingly, the phage P22 prophage gtr operon is subject to on/offphase variation that is epigenetically controlled by the host Dammethylase and OxyR regulatory protein (Broadbent et al., 2010).

A variation on lysogenic conversion in the lambdoid phages is thepresence of genes that affect the host during lytic infection. It has

been known for over half a century that prophages often encodevirulence factors that are important in bacterial diseases, e.g.,diphtheria toxin (Freeman, 1951; Uchida et al., 1971; PappenheimerandMurphy, 1983), but it was assumed that these are expressed fromuninduced prophages. A surprising more recent observation is thatthe Shiga-like toxins do not follow this paradigm. The Shiga-liketoxins are important in the human disease E. coli hemorrhagic colitis,and the stxA and stxB genes that encode the toxin are carried mostlyby lambdoid phage 933Wand its close relatives (Plunkett et al., 1999;Smith et al., 2012), where they lie within the late operon between thepromoter and lysis genes (Neely and Friedman, 1998). David Fried-man, Matthew Waldor and colleagues have studied two of thesephages, 933W and H-19B, and they find that high level expression ofstxAB is largely dependent on prophage induction and replication ofthe prophage to increase the DNA copy number. In addition, phagemediated cell lysis is required to release the toxin from the cell(Wagner et al., 2002; Tyler et al., 2004). Thus, it appears that“spontaneous” induction, lysis and death of a small fraction of thesebacteria produces the toxin that is presumably advantageous to thebacterium during disease. This seems to be an example of sibselection and/or altruism in which lysis of a few members of abacterial clone give an advantage to their clonal siblings (Livny andFriedman, 2004).

Molecular cloning of DNA

In 1968 Peter Lobban, a Ph.D. student in Dale Kaiser's labora-tory in the Biochemistry Department of Stanford University wasthe first person to realize that the tools to clone DNA existed andthat it would be a useful thing to do. He presented this proposal ata Kaiser lab group meeting, defended the idea in his Ph.D.preliminary exam, and proceeded to do his thesis work on cloningmethodology (Lobban, 1972; Lobban and Kaiser, 1973). This idea,which in part sprang from the fact that Kaiser's laboratory wasvery near those of Arthur Kornberg and Robert Lehman whereDNA polymerase and DNA ligase were being characterized at thattime (e.g., Olivera and Lehman, 1967; Englund et al., 1968), was sopowerful that others devised more rapidly attainable cloning goalsand were able to publish before Lobban (Jackson et al., 1972;Cohen et al., 1973). Nonetheless, the idea of molecular cloningoriginated with him (Kornberg, 1991; Berg, 1993; Casjens, 1994;Yi, 2008; for more extensive historical treatments of molecularcloning and genetic engineering see Lear, 1978; Judson, 1979;McElheny, 2014).

In addition, other important parts of cloning technology werealso developed with phage lambda. Werner Arber's discovery andcharacterization of bacterial restriction-modification systems wasdone using phage lambda (Arber, 1971 and references therein).Ron Davis at Stanford developed phage lambda into a powerfulcloning vector by showing that some restriction enzymes leavesticky ends and then utilizing these enzymes for DNA cloning(Mertz and Davis, 1972; Cameron et al., 1975), Kaiser and Masuda(1973) devised a procedure for packaging exogenously addedlambda DNA into lambda virions, and lambda-based cosmidvectors were developed for cloning relatively large DNA secti-ons up to about 40 kbp (Hohn and Murray, 1977; Collins andHohn, 1978).

Thus, lambda contributed greatly to the development of DNAcloning technology, but these advances contributed to the demiseof the golden age of lambda, since molecular cloning became thefirst step in being able to understand cellular (both prokaryoticand eukaryotic) DNA function at a molecular level. Because lambdawas no longer uniquely experimentally accessible, and because thebasic aspects of its life cycle were by then fairly well understood,the 1980s and 1990s saw fewer and fewer laboratories dedicatedsolely to the study of phage lambda. Nonetheless, a handful of

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laboratories persisted in delving deeper into the details of the lifecycles of lambda and its relatives. In addition, the well-understoodlambda system has often been used for proof-of-principle demon-strations for new technologies. Thus, even though they are the soleobjects of study in relatively few laboratories at present, thelambdoid phages continue to give new insights into the amazingdetails of gene expression, gene function and virus life cycles tothis day.

Bacteriophage diversity and the evolution of modular viral genomes

The early decision to study a small number of chosen bacterio-phages (including the most highly studied phages lambda, T4 and T7)for highly focused hypothesis-driven research certainly sped up theattainment of our current overall knowledge of gene expressionmechanisms and numerous viral functions immensely. However, atthe same time it restricted our view of the diversity of life on Earthand in particular the diversity and natural abundance of bacterio-phages. Even in the early days of the study of phages several lambdarelatives (e.g., phages 21, 434, P22 and ø80) had been isolated andwere known to form viable hybrids with lambda (Kaiser and Jacob,1957). Because of their different repressor-operator specificities, theseother phages were of seminal use in developing an understanding ofthe control of early gene expression and the reasons why phagelambda cannot infect a lambda lysogen (e.g., Bode and Kaiser (1965)showed that the lambda cII and cIII genes cannot complement from aheteroimmune repressed hybrid prophage). Analysis of heteroduplexDNA pairs with lambda showed early on that these phages havemosaically related genomes in which patches of highly relatedsequence are interspersed with patches of less related or unrelatedsequences (Fiandt et al., 1971; Simon et al., 1971; Botstein, 1980;Campbell and Botstein, 1983; Casjens et al., 1992a; Campbell, 1994).Phage genome sequences have greatly increased our understanding ofthis genome mosaicism in recent years (Hendrix et al., 2000; Juhalaet al., 2000; Hendrix, 2002; Casjens, 2005; Casjens and Thuman-Commike, 2011; Grose and Casjens, 2014). Fig. 4 shows some of thebetter-characterized variations among lambdoid phages.

Many relatives of phage lambda have now been isolated, and atpresent over 80 such phage genomes have been completelysequenced (compiled in Grose and Casjens, 2014), along with

hundreds of related prophages in bacterial genome sequences(see, for example, Casjens and Thuman-Commike (2011) for the 45such P22-like prophages known at that time). All these phagesinfect members of the Enterobacteriaceae bacterial family, but theirdiversity is nonetheless huge. These 80 phages fall into 17 different“types” or “clusters” that all have lambda-like gene (functionalmodule) orders and apparently similar gene expression cascades(Grose and Casjens, 2014). (We note that there are a very smallnumber of notable exceptions to this synteny; some phage N15and ASPE-1 early genes have an order that is different from that oflambda; van der Wilk et al., 1999; Ravin et al., 2000.) The 17lambdoid phage clusters were defined in terms of overall sequencesimilarity, in which a dot matrix plot comparison of members ofdifferent clusters at relatively low stringency shows recognizablenucleic acid similarity over more than 50% of the genome withinbut not between clusters (Grose and Casjens, 2014). A very preli-minary survey of prophages present in the hundreds of reportedenterobacterial genome sequences has revealed at least fouradditional lambdoid phage clusters (S. Casjens, unpublished).Clearly, the lambda life-style has been extremely successful andhas diverged to fill many presumed niches.

All these lambdoid phages are transitively related by patches ofsimilar sequence that can potentially undergo homologous recom-bination. The “unrelated” patches that lie in parallel chromosomallocations can be either homologous sequence that has diverged tothe point that it is no longer significantly related or entirelyunrelated sequence from a different source that performs thesame function. An example of the former is the lambdoid proph-age repressor. The repressors all perform the same role, shuttingoff the lytic genes in the prophage, yet they are extremely variableand often barely recognizable or even not recognizable as beinghomologous. The lambdoid repressors that have been examinedhave similarities in their DNA-binding domain folds, and so arealmost certainly ancient homologs (Pabo et al., 1990; Watkinset al., 2008). We are not aware of a recent analysis, but in 1992eleven different repressor operator-binding specificities wereknown (Casjens et al., 1992a), and the currently known sequencediversity of these proteins suggests a very much larger repertoire.Another example of very divergent homologs is the procapsidassembly gene cluster (including the MCP and portal protein) of

DNA pack

aging

/Term

inase

Regula

tion (

N,cIII)

Lysis

Propha

ge re

press

or (cI

)

Acces

sory

(nin r

egion

)

Homolo

gous

reco

mbinati

on

Propha

ge en

ds (in

t, ptl)

Acces

sory/

Lyso

genic

conv

ersion

Tail f

ibers/

Tails

pikes

TailsProc

apsid

asse

mbly/H

eads

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sory

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tion (

Q)

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on

λHK97P22

λP22SfV

λN15

λP22Gifsy-2

λP22

λP22L434ø80etc.

λP22N15SfVøP27Fels1

ManyMany λP22933WSf6

λ21P22øKO2

λP22HK97SfVES18Gifsy-2933WøES15ZF40

P22Sf6N15øKO2

λ933WSf6St64BN15

Regula

tion (

cro, c

II)

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 genesin 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 definedin 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 longcontractile, 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 areextremely divergent homologs (e.g., portal and MCPs in the procapsid assembly section and probably all the different prophage repressors with different operator bindingspecificities). 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 ninregion 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 genesbetween 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 andbetween tail and tailspike genes in P22, respectively).

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which there are 14 very different types among the lambdoidphages (Grose and Casjens, 2014). Examples of apparently non-homologous proteins that perform analogous functions in variouslambdoid phages are the three largely unrelated tail types – short,long contractile and long noncontractile (e.g., P22, SfV and lambda,respectively), the integrases of integrating phages like lambda andP22 and the protelomerases of phages N15, PY54 and øKO2(above), and the multiple types of homologous recombinationmodules (above). We have previously discussed the differencesand similarities among the four best-studied lambdoid phages,lambda, HK97, P22 and N15 in more depth, and the reader isreferred to Hendrix and Casjens (2006) for more examples of suchmosaic relationships.

We believe that extant lambdoid phage diversity has been attainedin several ways. These phages are likely extremely old, and naturaldivergence has had sufficient time to make even some of theirhomologous protein sequences no longer significantly related. Newmosaic boundaries/novel sequence joints can form in two ways. First,highly divergent lambdoid phages could recombine through illegiti-mate recombination events to generate a new boundary. Second,illegitimate recombination could incorporate novel sequences into aphage by insertion between or replacement of resident genes. Theseforeign inserts between pre-existing lambdoid phage genes have beencalled “morons” since there is more DNA when they are present(Hendrix et al., 2000; Juhala et al., 2000), and the known moronsusually carry their own promoters for independent expression. Thesources of such inserts and replacements may be divergent lambdoidphages, other phages or genes with no phage relationship, since theirsources are currently unknown. Thus, new mosaic boundaries likelyform very rarely, but the probability of such a fortuitous eventbetween two phages giving rise to a functional progeny phage isgreatly increased if the two participating phages have the same geneorder (as all lambdoid phages have). Given the enormous numbers ofphages on Earth (Whitman et al., 1998; Wommack and Colwell, 2000;Hendrix, 2002), we imagine that this type of novel joint formationmay well be random, and that all possible single-recombination noveljoints between divergent lambdoid phage genomes might be formed.However, only the extremely small fraction of such recombinants thathas a functional, advantageous phage genome would be found as thephages present today. Once a new functional mosaic boundary isformed in a lambdoid phage genome, homologous recombinationbetween similar sequences in other mosaically related lambdoidphages could rapidly cause reassortment to form new combinationsof such boundaries (and sequence patches). Interestingly, althoughboth of these processes, formation and reassortment of mosaicpatches, will tend to homogenize the genetic makeup of the partici-pating phages, neither process is fast enough to mask the presence ofclear clustering of different types within the lambdoid phage group(Grose and Casjens, 2014).

The future

Lambdoid phage research was seminal in the attainment of ourcurrent understanding on such varied fronts as gene and operonstructure and function, the mechanisms of control of gene expres-sion by regulatory proteins and small RNAs, protein chaperones,prophage integration, protein and RNA turnover, virion assemblyand modular virus genome evolution. Although from outsidelambdoid phages may be seen as “fully understood,” we believethat there is still much that these phages can teach us. In additionto their roles as model systems, the amazing abundance of tailedphages on our planet means that they are extremely important inall ecological processes that involve bacteria such as global carbonand nitrogen cycling (Suttle, 2007; Ankrah et al., 2014). Phages areinvolved through both their ability to kill bacteria and so affect

their population size as well as modification of bacteria throughlysogenic conversion and transduction. Thus, it is important tounderstand phages in their own right.

Recent work has also found the lambdoid phages to havetechnological applications and human health importance. Lambdaand P22 have been developed into powerful and versatile proteindisplay agents, where both the head decoration proteins lambda D(Sternberg and Hoess, 1995; Mikawa et al., 1996; Nicastro et al.,2013; Chang et al., 2014) and P22 Dec (Parent et al., 2012b), as wellas the lambda major tail V protein (Maruyama et al., 1994; Zanghiet al., 2007) and P22 MCP head protein (Kang et al., 2008; Servidet al., 2013) have been used successfully to display foreignpeptides and proteins. Lambda virions have also been used asDNA or protein vaccine delivery vehicles in mammals (Jepson andMarch, 2004; Lankes et al., 2007; Clark et al., 2011; Mattiacio et al.,2011; Saeedi et al., 2014). Trevor Douglas and Peter Prevelige aredeveloping P22 into a flexible multi-use nanoparticle platform,where foreign molecular cargo can be placed on the surface orwithin the interior of the particle (O'Neil et al., 2011, 2012; Luconet al., 2012; Uchida et al., 2012) to make, for example, magneticresonance contrast enhancing agents (Qazi et al., 2013, 2014) orenzyme-containing nanoreactors (Patterson et al., 2012, 2014).Finally, the stability of cross-linked HK97 capsids may give themstability advantages, and HK97 nanoparticles are under develop-ment (Huang et al., 2011). Human health relevance comes from thefact that these phages carry bacterial disease virulence factorgenes such as the lom and bor genes of lambda and the Shiga-like toxin stx genes (above).

It is now nearly 65 years since Esther Lederberg discovered andnamed bacteriophage lambda, but we still have not reached themythical goal of a complete understanding of its molecular lifecycle. If nothing else, work on these phages should have taught usthat there are many levels of intricacy and sophistication in allaspects of phage life cycles, and each time a level is “understood” itopens doors to yet deeper levels of understanding. Each time wethink we have learned something new and important aboutlambda, we have really learned something new and importantabout biology more generally, and the lambdoid phages have notyet run out of interesting biology to illuminate. Despite thechanging fashions in research, the vicissitudes of NIH grant funding,and the changing cast of characters studying them, these phageshave been a remarkably consistent and productive source of newbiological insight that remains an important experimental modelsystem in the vanguard of biological science. This seems to usunlikely to change any time soon.

Acknowledgments

The authors' research is supported by NIH Grants GM47795 andGM114817 from the U.S. Public Health Service to R.W.H. and S.R.C.,respectively. We thank our colleagues in the phage research fieldfor many years of gratifyingly open and stimulating discussions.We especially thank Costa Georgopoulos, Peter Lobban and twoanonymous reviewers for their welcome suggestions and correc-tions of our less than perfectly accurate memories.

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