J. Embryol. exp. Morph. 83, Supplement, 75-88 (1984) 7 5Printed in Great Britain © The Company of Biologists Limited 1984

A programme for the construction of a lambdaphage

By W. J. BRAMMAR AND C. HADFIELDDepartment of Biochemistry and The Leicester Biocentre, Leicester University,

University Road, Leicester LEI 7RH, U. K.

TABLE OF CONTENTS

SummaryIntroductionThe lambda genomeThe expression of lambda genes on infection

(a) The uncommitted phase(b) Lytic development

Lysogenic developmentThe decision between lysis and lysogenyThe lysogenic stateSummary and conclusionsReferences

SUMMARY

Infection of a sensitive host by the lambdoid coliphages can cause death of the bacterial cellby lysis or can lead to a lysogenic cell, with the viral DNA stably integrated into the hostchromosome. These alternative responses both require the coordination of several host andphage functions, and lambda infection follows a well controlled developmental plan. The lyticand lysogenic pathways of lambdoid infection are reviewed, with emphasis on the variety ofcontrol mechanisms involved in the commitment to a particular pathway.

INTRODUCTION

The lambdoid bacteriophages are a family of viruses which multiply inEscherichia coli. Infection by a lambdoid phage can either cause death of the hostcell by lysis or can lead to the stable integration of the viral DNA as a prophageinto the host chromosome, with survival of a lysogenic cell.

The lytic and lysogenic responses both require the co-ordinated activities ofseveral functions, encoded by both viral and host genes. In lytic infection, DNAreplication, genome packaging and cell lysis proceed sequentially. In thelysogenic mode, the synthesis of the phage integration system and the repressorprotein must be synchronized. In both cases, functions that might interfere withthe chosen pathway must be excluded. Thus the lambdoid phage infection

76 W. J. BRAMMAR AND C. HADFIELD

follows a simple, properly co-ordinated developmental plan.While no one would claim that phage infection provides a valid model system

for the study of development, the present, detailed understanding of the lambdadevelopmental programme might have some valuable lessons for developmentalbiologists working on more complex systems. This review will present a simpleaccount of the key steps in the lytic and lysogenic pathways, with emphasis onthe control mechanisms governing co-ordination and commitment to a particularpathway.

THE LAMBDA GENOME

The genome of bacteriophage lambda contains approximately fifty genes,about half of which are essential for lytic growth. The DNA is packaged into thehead of the mature phage particle as a non-permuted, linear, duplex moleculewith single-stranded, 5' projections of 12 nucleotides at each end. These mutuallycohesive termini assure rapid circularization of the genome following infection.

The genetic and physical map of the lambda chromosome (Fig. 1) showsmarked clustering of genes of related function. The essential genes concernedwith head (A-F) and tail (Z-7) formation and assembly are contiguous within theleft-hand third of the genome. The region of the map between J and att, thephage attachment site, contains no essential genes (Hendrix, 1971). Genes to theright of att govern site-specific (int and xis) and generalized (red) recombinationof phage DNA. None of the genes between att and N is essential for lytic growthof lambda on normal hosts, though they may affect growth on certain mutanthost strains. The product of gene iVis the early regulatory protein that is normallynecessary to activate transcription of most other phage genes. The cl gene codesfor the lambda repressor, the regulatory protein that switches off transcriptionof prophage genes in the lysogenic state. The presence of the lambda repressormakes a A-lysogenic cell immune to superinfection by another lambda phage andis responsible for the characteristic turbidity of lambda plaques. Lambda carriesan additional regulatory gene, cro, that encodes a repressor-like, DNA-bindingprotein. The cro gene-product functions as an antagonist to the c/-product,acting to prevent lysogenic development and to promote lytic growth (Gussin etal. 1983). The O and P genes are required for replication of ADNA (Brooks,1965; Joyner, Isaacs, Echols & Sly, 1966), the Q gene product for activation oflate transcription (Dove, 1966; Couturier, Dambly & Thomas, 1973) and the S,R and Rz gene products for lysis of host cells (Harris, Mount, Fuerst &Siminovitch, 1967; Young etal. 1979).

THE EXPRESSION OF LAMBDA GENES ON INFECTION

(a) The uncommitted phase

The infection of an E. coli cell by lambda is initiated when a phage particleadsorbs, via its tail fibre, to a specific receptor on the cell surface (Schwartz, 1976;

A programme for the construction of a lambda phage 11

earlycontrol

head tail recombination

P

DNAreplication

immunitylysis

repliesmmunity /

pE latecontrol

att \ cro |

ABC EFZV H LK J Vmf red._ N_ cIlOP 9 SR." " ' '• • • " ' i I I II i II i * II i i IUI I I i IIU i i i i m i i

PR PkI 10 20 30 40 R

! ! ! ! ! 1 ! ! ! kb

Fig. 1. Genetic and physical map of bacteriophage lambda. Gene clusters with relatedfunctions are indicated above the brackets. There are ten known genes at the left endof the map concerned with head formation, and eleven genes from Z to/inclusive thatare required for formation of the tail. The region between / and att, the b region (forbuoyant density changes in CsCl gradients), codes for several proteins that are notessential for vegetative growth (Hendrix, 1971). The att (attachment) site is the site ofintegrative recombination between phage and bacterial DNA, catalysed by theproduct of the int gene. The red genes, red A (= exo) and redB (= bet), encode func-tions that catalyse general phage recombination. The N and Q genes encode theproteins that activate early and late transcription respectively. The phage repressor isencoded by the cl gene, while the product of the cro gene is a DN A-binding protein thatacts as an anti-repressor and promotes lytic growth. Promoters are indicated hyp witha subscript to indicate their role: pi is the promoter for int gene expression (= pmt); PLthe major leftward and p& the major rightward promoter; p^ is the promoter forestablishment of repressor synthesis, often called /?RE , and pu is the promoter formaintenance of repressor synthesis (=/?RM); P'R is the late promoter. Horizontalarrows indicate the extents and directions of specific transcripts, with — indicatingsignificant readthrough beyond a termination signal. The scale is in kilobase-pairs.

Thirion & Hofnung, 1972). The linear phage genome is injected through the cellmembrane into the cytoplasm (Mackay & Bode, 1976), where it rapidly cir-cularizes via its cohesive termini and is covalently sealed by host DNA ligase.

Transcription of lambda genes by the host's RNA polymerase proceeds left-wards from promoter ph through gene N and rightwards from promoter /?Rthrough the cro gene (Fig. 2). Most of these initial transcripts terminate at *LI andtei, immediately beyond genes N and cro respectively. The leftward transcriptis translated to yield the N protein and the early rightward transcript producesthe cro protein. Both of these gene products have important regulatory functionsthat influence subsequent A development.

The N protein exerts its controlling effect by influencing RNA polymerase toignore transcription-termination signals (Adhya, Gottesman & de Crom-brugghe, 1974; Franklin, 1974; Segawa & Imamoto, 1974). In the presence of Nprotein, transcription initiated at/?L and/?R is elongated through the terminatorstu and JRI and through a series of 'delayed early' genes whose products regulate

78 W. J. BRAMMAR AND C. HADFIELD

Fig. 2. Transcription of lambda genes during lytic growth. I: Transcription im-mediately after infection or induction. In the absence of lambda's Nprotein, leftwardtranscription from p^ terminates largely at tu, while rightward transcription ter:minates inefficiently at fo\, beyond the cro gene. About 20 % of the rightwardtranscripts (thin arrow) avoid termination at fax and proceed through the replicationgenes 0 and P to terminate at fe. II: The product of the TV gene influencessubsequent transcription to override termination sites tu, fei and tja.. Leftwardtranscription now expresses the recombination (red) genes, while rightward tran-scription proceeds through the O, P and Q genes, leading to DNA replication andthe activation of late transcription. Ill: The product of the Q gene elongates tran-scription from/?'R through genes 5, R (and Rz), governing cell-lysis, and throughgenes A to J, coding for head and tail proteins.

both the lytic and the lysogenic responses. The infecting phage genome shortlythereafter becomes committed to one pathway or the other.

The apparent specificity of the N protein for transcripts initiating at /?L andPR is not a consequence of the promoters themselves, but of nucleotidesequences, termed ^/-utilization ('nuf) sites, located between the promoters and

A programme for the construction of a lambda phage 79the first transcription termination signals (Salstrom & Szybalski, 1978; de Crom-brugghe, Mudryj, Di Lauro & Gottesman, 1979). The two nut sites, which have16 out of 17 identical nucleotides, show hyphenated dyad symmetry, consistentwith the formation of a stable stem-loop structure in the RNA or the DNA(Rosenberg et al. 1978). Though the nut sequences have been shown to besufficient to allow the Af-protein to influence RNA polymerase to ignoresubsequent termination sequences (de Crombrugghe et al. 1979), the precisemechanism by which this is achieved has not been elucidated.

During the phase of uncommitted growth, phage DNA replication is activatedby transcription in the vicinity of the unique origin within the O gene (Dove etal. 1969; Dove, Inokuhi & Stevens, 1971) and by the products of the genes O andP (Ogawa & Tomizawa, 1968). The early phase of replication, giving monomericcircular molecules, ceases after a few minutes. At this stage the genome eitherstops replicating and enters the lysogenic phase or switches to rolling-circlereplication as it begins the lytic cycle.

(b) Lytic development

During lytic growth, the production of mature phage particles requires thesynthesis of concatemeric phage DNA, composed of covalently joined, tandem-ly repeated unit copies of the lambda chromosome (Szpirer & Brachet, 1970;Stahl etal. 1972; Feiss & Margulies, 1973). The rolling-circle mode of replicationis believed to provide the mechanism for the production of the concatemericmolecules (Eisen, Pereira de Silva & Jacob, 1969; Gilbert & Dressier, 1969),which are the ideal substrate for DNA encapsidation (Skalka, 1977). Thepackaging of lambda DNA into phage heads requires the unit chromosome to bebounded by cos sites (Emmons, 1974; Feiss & Campbell, 1974); a monomericcircular lambda chromosome cannot be packaged in vivo (Szpirer & Brachet,1970).

The onset of rolling-circle replication is facilitated by lambda's gam geneproduct, an inhibitor of the host's recB, C-encoded exonuclease V (linger &Clark, 1972; Unger, Echols & Clark, 1972; Enquist & Skalka, 1973). In theabsence of the gam-product this nuclease attacks both the rolling circles them-selves and an intermediate required for their formation, thus inhibiting thesynthesis of maturable phage DNA.

An alternative route to packageable DNA can be provided by recombinationbetween two monomeric circular molecules to produce a circular dimer contain-ing two cos sites. Both the red systems of the phage and the rec system of the hostcan catalyse such recombinational dimerization (Stahl et al. 1972; Enquist &Skalka, 1973), but the latter does so very inefficiently with wild-type lambdaDNA (Stahl, Crasemann & Stahl, 1975).

While 'delayed early' leftward transcription gives rise to gam gene expression,similar transcription rightwards facilitates expression of the Q gene. Note thatthe expression of the Q gene will necessarily be delayed by some 2-5 min, the

80 W. J. BRAMMAR AND C. HADFIELD

time required for RNA polymerase to reach the gene from its promoter /?R , 7000base-pairs away. The product of Q activates transcription initiated at P'R totraverse the late genes, whose products are responsible for DNA encapsidationand cell lysis. The Q protein acts as an antiterminator, employing a <2-utilization('qut') site in a manner formally analogous to the N protein, allowing transcriptsinitiated atp'R to proceed through the termination sequence, J'R into the largelate gene region (Forbes & Herskowitz, 1982).

Delayed early transcription begins to diminish after about 5 min of infection,dropping to a very low level by 10 min (Szybalski etal. 1970). This diminution iscaused by the binding of the cro gene-product to the OL and OR operatorsequences, interfering with the initiation of transcription at/?L and/?R (Fig. 3).The kinetics of action of the croproduct are explained by the observation that theactive form of the DNA-binding protein is a dimer, and that relatively high con-centrations are needed to block the RNA polymerase-binding sites at/?L and/?R.

From about 10 min until the end of infection, transcription from/?'R activatedby the Q protein is predominant and expresses all the late genes. The productsof the A and Nul genes aggregate to form the terminase, which binds to theconcatemeric substrate DNA at the cos sites. Several other late gene productsare assembled into a capsid prohead, while others are independently assembledto form the tail fibre. The prohead attaches to a DNA-terminase complex, anda unit length of the phage chromosome is taken up until the next terminasecomplex is reached. The terminase then cleaves the DNA to yield a mature,DNA-filled head. The final stage of capsid assembly is the addition of a tail fibreto the mature phage head.

Coincident with the production of mature virions is the continuing expressionof the lysis genes. About 60min after infection initiated, the accumulatedproducts of the S, R and Rz genes are sufficient to lyse the host cell, releasing aburst of progeny phage.

LYSOGENIC DEVELOPMENT

The establishment of the lysogenic state involves the coordinated expressionof two key genes, the cl gene that encodes the phage repressor and the int gene,whose product catalyses the site-specific integration event. Transcription of cland int is synchronized by the action of a positive effector, the ell gene-product,which stimulates the binding of RNA polymerase to the separate promoters, preand /?int (Ho & Rosenberg, 1982) (Fig. 4). The c//-product is metabolicallyunstable, due to the action of the host's HflA protein, but is protected by thelambda clll product (Hoyt et al. 1982). The controlling ell and cIII genes areboth expressed mainly by delayed early transcription from/?R and /?L during theuncommitted phase of phage development.

The c/-encoded repressor is the effector of the switch into lysogenic develop-ment to the exclusion of lytic growth. The protein binds to the phage DNA at the

A programme for the construction of a lambda phage 81OL and OR operators, with initial preference for regions OLI and ORI . Theseoperator sub-sites are located within the RNA polymerase-binding sites and thebound repressor prevents further transcription from initiating atpL andpR. Therepressor, in blocking transcription of the N, gam and red genes from /?L and theO, P and Q genes frompR, effectively stops phage DNA replication, late proteinsynthesis and host cell lysis.

The repressed, circular phage DNA is integrated into the host chromosome bya site-specific recombination event catalysed by the int-pvotein (integrase). Theint gene is transcribed from pint, under the influence of ell protein, and from /?L ,driven by the action of Af-protein. However, an interesting post-transcriptionalmechanism, 'retroregulation', ensures that the latter transcription is non-productive during phage infection (Fig. 5). This phenomenon depends on a ex-acting regulatory element, sib, located downstream from int, beyond the phageattachment site (Mascarenhas, Kelley & Campbell, 1981; Guarneros, Mon-tanez, Hernandez & Court, 1982). The sib sequence displays extensive dyadsymmetry, enabling the elongated RNA transcript to form a hairpin structurethat is believed to be a preferential substrate for RNAse III. The action of thisenzyme on the RNA could create a 3' end, processive 3' to 5' degradation fromwhich destroys the adjacent int region of the transcript (Guarneros et al. 1982).

Transcription from the c//-dependent promoter, pint, terminates at a rho-dependent terminator, tmx, located within the sib region (Schmeissner, Court,McKenney & Rosenberg, 1981), and fails to complete the RNAse Ill-sensitive

(pcro\

A / att N PL cl cro OP Q SR

Fig. 3. The control of lambda transcription in the lytic phase. The product of the crogene represses transcription from /?L and /?R , moderating expression of the early anddelayed-early (Af-dependent) genes, and from p™, blocking synthesis of therepressor. The Q gene product causes transcription fromp'R to avoid terminationand thus to express the late genes.

Jatt int Pint N cl cro pre ell OP QSR

Fig. 4. Coordinate expression of int and cl leading to lysogeny. The two promotersPint and PRE , governing transcription of the int and cl genes respectively, are bothsites for activation of transcription by the ell gene-product, pell.

82 W. J. BRAMMAR AND C. HADFIELD

product+ 1 pell £

. . . . sib att int xis D,wlysogenic infection = - F —

Pi

RNAselll

sib att int xis n v ; A • .lytic infection = = = = = = = = = = = = = = = = = = = = = = = = = G££> PJ2I

pN

prophage induction — — • " •—~ rms+p_m£

Fig. 5. Retro-regulation of int gene expression. During infection, the cll gene-product activates promoter p\ (-pmt), within the xis gene, to give expression of int.The transcript terminates beyond att, and is translated to give the m/-product. Duringa lytic infection, transcription from/?L is activated by TV-protein, proceeding throughtermination signals to transcribe both xis and int. The transcript proceeds beyond thesib site, where RNAse III initiates a nucleolytic attack that leads to processiveelimination of the int gene transcript. Potentially deleterious integration of achromosome committed to the lytic cycle is avoided. On prophage induction, tran-scription from/?L can express both xis and int, since the sib site is no longer adjacentto the int gene. The site-specific recombination event at the att site that gives theintegrated prophage places the sib site at the opposite end of the prophage map. Theproducts of both int and xis are required for efficient excision of phage DNA fromthe prophage state.

structure. This regulatory mechanism ensures that only those int transcriptsinitiated at/?jnt are effective as integrase messenger, and the requirement for cllprotein ensures coordination with repressor synthesis.

In the prophage of a A-lysogenic cell the sib site has been removed fromproximity to the int gene by the integrative recombination event involving thephage attachment site. On induction of a A-lysogenic cell the adjacent int and xisgenes, whose products are both required to bring about excision of the prophageDNA, are coordinately expressed via transcription from/?L-

The alternative modes of expressing int during infection and induction thusensures that the appropriate protein is produced to drive the recombinationevent in the required direction.

Host proteins are also important in modifying the lysogenic response. The E.coli HflA protein decreases the amount of active c//-product in the infected cell,but is antagonized by the phage c///-product. The synthesis of the HflA proteinitself is repressed by the host cyclic AMP-activated catabolite repression system(Belfort & Wulff, 1974). The integrative host factor (IHF), a dimeric proteincomprised of the products of the E. coli him A and himD genes (Miller & Nash,

A programme for the construction of a lambda phage 83

1981; Nash & Robertson, 1981) has a dual function in lysogenic development.It is a DNA-binding protein that is essential for integrative recombination (Nash& Robertson, 1981) and promotes the synthesis of the phage ell product (Hoyutetal. 1982).

Overall, a single regulatory protein, the ell gene-product, coordinates thelysogenic response. Not only does the ell protein synchronize the expression ofthe cl and int genes, but it also acts as the receptor protein for transmission ofenvironmental signals via the host IHF and HflA control proteins.

THE DECISION BETWEEN LYSIS AND LYSOGENY

Within the phage-infected cell, an individual lambda genome can enter eitherthe lytic or the lysogenic pathway. Although these two pathways have their initialsteps in common, a decision between them is made at an early stage of phagedevelopment.

The particular pathway that ensues depends upon whether the cro-protein orthe repressor wins the competition for the operator sites, particularly OR . Thecro-product is able to lock the genome into the lytic pathway, inhibiting theexpression of ell and clll and ultimately preventing repressor synthesis. Therepressor ensures the lysogenic response by preventing expression of cro itself,and also of the O, P, and Q genes whose products are required for DNA replica-tion and late gene expression.

The delicately balanced competition between repressor and cro protein isinfluenced by several factors, the level of ell protein being a crucial one. The ellproduct is required to activate transcription of the cl gene from /?RE , and itsactivity is influenced by several host- and phage-coded products. When theactivity of the c//-product is high, the lysogenic response is favoured; when it islow, the lytic response is enhanced. Thus, for example, carbon-starved cells,which have reduced levels of HflA protein, favour activity of the ell protein andshow elevated frequencies of lysogenization (Belfort & Wulff, 1974).

THE LYSOGENIC STATE

In the lysogenic cell, the prophage state is maintained by the binding ofrepressor to the operator sites, OL and OR , blocking transcription from the twomajor early promoters, /?L and /?R .

Each of the operators OL and OR contains three repressor-binding sites(Ptashne et al. 1976) (Fig. 6). These are similar in sequence and contain axes oftwo-fold hyphenated symmetry. The three sites in OR overlap both/?R, the majorearly rightward promoter, and /?RM , the promoter from which the cl gene istranscribed in the prophage. At low concentrations, repressor binds preferenti-ally, as a dimer, at ORI: such binding potentiates subsequent binding of repressor

84 W. J. BRAMMAR AND C. HADFIELD

to OR2 (Johnson, Meyer &Ptashne, 1979). Binding to 0R3 shows reduced affinityand is only achieved at high concentrations of repressor.

The binding of repressor dimers at om and ORJ. stimulates the binding of theE. coli RNA polymerase to/?™, by providing extra contact sites at this position,thereby enhancing transcription of the cl gene (Reichardt & Kaiser, 1971;Meyer, Maurer & Ptashne, 1980). The repressor, in blocking transcription fromPR , is also acting to stimulate its own synthesis (Fig. 7). At high intracellularconcentrations repressor will bind to OR3 and inhibit its own synthesis, thusproviding an effective homeostatic mechanism.

When the DNA of a A-lysogenic cell is damaged, for example by ultravioletirradiation, thymine starvation or a chemical carcinogen, the prophage is in-duced into the lytic cycle. The induction process is triggered by the proteolyticcleavage of the repressor, catalysed by the activated product of the cellular recAgene (Roberts, Roberts & Craig, 1978; Craig & Roberts, 1981). Destruction ofthe repressor leads first to transcription from/?R , with production of the cro geneproduct, and slightly later to transcription from pL. (Repressor binds moretightly at OL than at OR .) The cro-protein, also active as a dimer, has high affinityfor the sites within OR , though in the order ORS > OR2 = ORI . Binding of cro-protein to OR3 blocks access of RNA polymerase to /?RM , eliminating the main-tenance mode of repressor synthesis (Fig. 7). This action of the era-producteffectively prevents recovery of repressor synthesis and locks the inducedprophage into the lytic cycle.

PL PRMf 4 «. , ^

4 ,att int xis red cIII NOL rex cl OR cro ell 0 P Q

I PRM 1 I PR J

Fig. 6. The sites controlling early lambda transcription. Transcription of lambda'searly genes is initiated at p^ and /?R and is controlled by the interaction of repressorwith the operator-sites, OL and OR . Transcription of cl can be initiated at pie, theestablishment promoter, or p^, the maintenance promoter. Transcripts are in-dicated by dashed lines. The expanded diagrams show the three repressor-bindingsites within OL and OR and their spatial relationships with /?L , p™ and pR. The crogene-product also binds at OL and OR to reduce transcription from /?L , PRM and pn.

A programme for the construction of a lambda phage 85repressor

-M

induction

pcro

cl

Fig. 7. The lysogenic state and prophage induction. In the lysogenic state therepressor dimers bind cooperatively at ORI and OR2 , blocking /?R and activating /?RMby providing extra contact'sites for the interaction of RNA polymerase with /?RM .Following induction, involving proteolytic inactivation of repressor by the host'srecA gene-product, polymerase gains access topR to transcribe the cro gene. The crogene-product binds as a dimer to om and prevents expression of cl from /?RM •

SUMMARY AND CONCLUSIONS

Lambda has evolved an effective system for monitoring the environment in theinfected cell and using this information in the simple decision between lysis andlysogeny. The alternative responses branch from a common pathway, theultimate direction being governed by the products of the cro and cl genes. Thesetwo DNA-binding proteins compete for the same region of the phage DNA andrepress each other's synthesis. The balance between the cl and cro products ismodulated by the ell protein, whose activity is sensitive to the cell's metabolicstate via its interaction with the host HflA gene-product.

Lambda's relatively simple control circuits reveal great variety in the mechan-isms by which gene expression is controlled. Genes can have alternative modesof expression; control proteins can affect either initiation or termination of

86 W. J. BRAMMAR AND C. HADFIELD

transcription and can act at multiple sites to coordinate the expression ofseparate genes, and the same protein can be both an activator and a repressorof transcription. It is generally the case that regulatory interactions betweencompeting pathways are reinforced, to prevent possible interference, and thatmany regulatory proteins are metabolically unstable, allowing rapid switchingshould circumstances change. It will be interesting to see how many of thesethemes are rediscovered in examining the mechanisms of determination andcommitment in more complex developmental systems.

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DE CROMBRUGGHE, B., MUDRYJ, M., DI LAURO, R. & GOTTESMAN, M. (1979). Specificity of thebacteriophage lambda N gene product (N): Nut sequences are necessary and sufficient forthe antitermination by N. Cell 18, 1145-1151.

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