Article No. jmbi.1999.3137 available online at http://www.idealibrary.com on J. Mol. Biol. (1999) 293, 177±180

Bacteriophage lll: The Untold Story

Max Gottesman

E-mail address of the corresponding author:gottesma@cuccfa.ccc.columbia.edu

0022-2836/99/420177±4 $30.00/0

controls. Yarmolinsky was temporarily involved astudy of the biphasic induction curve of a lamb-doid prophage. The project was designed to selectmutants affecting site-speci®c recombination. Asmuch as I could understand of it, it seemed elegantand meticulously planned. However, the phenom-enon could never be explained (nor could West-moreland's, for that matter). On the other hand,we did isolate l mutants unable to perform site-speci®c recombination, and I soon becamewrapped up in the l integration and excision path-way, which offered the joys of pure genetics andeliminated the requirement to wear a coat in thesummer.

From the beginning the study of bacteriophageattracted scientists with a logical bent of mindaccompanied, frequently, by a lack of technicalskills. The latter probably forced them to concen-trate on the intellectual aspects of the ®eld, sincethe development of sophisticated technology wasbeyond their means. What could be more reward-ing, and less technically demanding, than to con-®rm or disprove de®nitively an elegantly craftedhypothesis, conceived in the morning, simply bycounting plaques in the afternoon? And, oftenenough, counting plaques was not even required.The most elegant hypotheses were tested simplyby noting the presence or absence of plaques.Some phageologists learned at the very beginningof their careers how to perform a single simpleassay, such as distinguishing a clear plaque from aturbid one, or how to band DNA in a cesiumchloride gradient, and have repeated this assayover the decades, thousands of times, to the pre-sent day and, perhaps, even into the next millen-ium. Blissfully in Hershey Heaven. One of my

Institute of CancerResearchCollege of Physiciansand Surgeons of ColumbiaUniversity, 701 West 168thStreet, New York, NY10032, USA

The study of bacteriophage lambda has provided key insights into funda-mental biological processes. This review recalls some highlights in thehistory of lambda research, and relates how simple (but elegant) experi-ments yielded major scienti®c breakthroughs. What we know aboutrecombination, gene regulation, and protein folding, for example, derivesin large part from bacteriophage lambda genetics. Lambda not only rep-resents a model system of scienti®c logic in a technology-driven age, butcontinues to reveal new principles of molecular biology.

# 1999 Academic Press

Keywords: bacteriophage lambda; scienti®c logic; gene regulation;recombination; protein folding

Introduction

That this is only the 40th anniversary of the Jour-nal of Molecular Biology reminds me of the remark-ably short history of our discipline, and of themajor role that bacteriophage has played in itsdevelopment. While the contribution of phage gen-etics to molecular biology is not exactly a secret,and far from an untold story, it does bear repeat-ing. Nothing is more astonishing to me as the lackof appreciation of students for the history ofscience, even of a science with such a short past,and of the contribution of particular scientists tothe ®eld. There is something disconcerting to seeour work ®lter down into the textbooks as unat-tributed dry facts, without a hint of the juicy char-acters of the scientists involved, or the intellectualframework that led to their discovery. On the otherhand, we did not choose to be writers or artists,®elds in which the author and the work are foreverinseparable. On second thoughts, some of the pio-neers in bacteriophage genetics were also excellentwriters, who described the beginnings and growthof the ®eld as well as their own eventful lives. Irefer the reader to the writings of Jacob, DelbruÈ cckand Luria, for example, for an insight into how itall started.

There was a time when I was entirely unawareof phage l. In 1966, after several years in a cold-room at Rockefeller University, I was offeredpositions by W. Westmoreland in Saigon andM. Yarmolinsky in Bethesda. Westmoreland'sproject, the effect of dioxin on fetal development inman, although lavishly funded, lacked proper

# 1999 Academic Press

178 Bacteriophage �

colleagues, who is partially colorblind, has chosento rely on an assay that distinguishes red coloredplaques (or colonies) from colorless. It doesn'tseem to matter how technically challenged he is;his intellect suf®ces and his contributions to the®eld have been seminal.

It was also the case that even if the l geneticistswere gifted at the bench, the equipment availableleft much to be desired. At the Pasteur Institute,for example, where much of the early genetic stu-dies of l were carried out, the glass pipettes wereso scored that their markings could not be distin-guished. One knew, of course, that, one was usinga ten milliliter pipette and not a one milliliter pip-ette, but ®ner distinctions were not possible. Incu-bator temperatures varied over several degreescentigrade (although the incubators were beauti-fully crafted of some exotic wood). These handi-caps provided a powerful incentive to designexperiments with all-or-none predictions.

I should also stress that the study of bacterio-phage was aided greatly by the spirit ofcooperation among the various laboratories.Strains, information, preprints all were freelyexchanged. For the most part, we all knew (know)and respected each other. Alas, this model of scien-ti®c interaction has become rather the exceptiontoday.

I shall con®ne this article to the temperate bac-teriophage l and its relatives. The two lifestyles ofl, lytic and lysogenic, and the subtle ways inwhich it subverts its host, Escherichia coli, havemade it a paradigm for a vast number of biologicalpathways. In particular, our understanding of posi-tive and negative gene regulation, site-speci®c andgeneralized recombination, assisted protein fold-ing, DNA replication, and other reactions (includ-ing molecular cloning) grew from studies of lbiology. And continues to grow.

Lambda, with its two lifestyles, is an ideal sub-ject for genetics. Mutant phage that cannot be pro-pagated lytically can be maintained as prophageand vice versa. Also, l, like other phages, makesspecial and taxing demands on its host. Thus it isrelatively easy to isolate viable bacterial mutantsthat fail to support phage growth. The mutationsoften reside in essential bacterial genes, but onlylower the activity of the gene product below thethreshold necessary for phage replication. Forexample, the chaperones were detected as E. colimutations that aborted lambda growth. The dnaK/J/grpE mutations de®ne the hsp70 family chaper-ones required for l DNA synthesis. The groEL/Smutations partially inactivate the hsp60 chaperonesneeded for l head formation. Null mutations inthese genes severely inhibit bacterial growth andwould have been a challenge to recover.

The genetic analysis of l was so sophisticatedthat when the DNA sequence of the phage wassolved, there were remarkably few surprises. Theessential features of the phage attachment site andoperators were predicted from the behaviour ofmutants, and the sequence provided a stunning

con®rmation of these predictions. On the otherhand, with the sequence in hand, we can ask gen-etic questions of even greater relevance thanbefore. Site-directed mutations can test veryspeci®c hypotheses. Of course, there is still valuein selecting mutations without preconceivednotions as to where they should lie. Surprises canbe informative.

The complexities of gene regulation wereapproached early in l. For example, the inability toform lysogens does not interfere with l lyticgrowth. Hence it was simple to detect and propa-gate phage that failed to make repressor. Wild-type phage make plaques with turbid centers thatcontain lysogenic survivors bearing l prophage.The lc mutants make clear plaques, i.e. they kill allthe bacteria they infect. The c mutants fall into twocategories: (1) those which never lysogenize (l cI);and (2) others which rarely lysogenize (l cII and lcIII). The latter class can be complemented bywild-type phage, or by phage mutant in a differentcomplementation group. The cI gene is the struc-tural gene for l repressor, and is required torepress the prophage early promoters, pL and pR.The cII and cIII products are proteins required onlyto initiate repressor synthesis. This suggested atonce, at least to certain prepared minds, thatrepressor positively regulates its own synthesis.The autoregulatory hypothesis was proven by aremarkably simple experiment that entailed merelyheating and cooling bacteria carrying a lysis-defec-tive l cIts prophage, and infecting with l. Theinactivation of the temperature-sensitive repressorresulted in the subsequent failure to synthesizerepressor upon return to the permissive tempera-ture. Thus, the cells became sensitive to superinfec-tion by l. The sequence of the l regulatory regionrevealed two promoters that transcribed cI. Thepre promoter, responsible for the initial burst ofrepressor, is positively controlled by CII. The prmpromoter is positively controlled by repressor, andis the promoter responsible for cI expression in alysogen. Prm is repressed by l Cro, which isexpressed very early in the lytic cycle. The on/offnature of this promoter represents an epigeneticswitch that decides whether l goes lysogenic orlytic.

Dominant phage mutants resistant to repressor,l vir, which were able to propagate on lambdalysogens, were also isolated, although less easilythan the lc mutants. The responsible mutationsde®ne the site of action of repressor i.e. the opera-tor sequences. Several mutations were required toobtain virulence, suggesting that the operatorswere, in some way, duplicated. The full signi®-cance of this ®nding awaited the biochemistry andthe DNA sequence of the operator region. Theoperators were shown, in fact, to be imperfectrepeats with dyad symmetry, three on each side ofthe cI gene. DNA binding studies with puri®edrepressor were highly informative, clarifying aregulatory aspect of l prophage induction. Unlikeinduction of the lac operon with IPTG, induction

Bacteriophage � 179

of l prophage by DNA-damaging agents is essen-tially all-or-nothing. Operator binding as a functionof repressor concentration yields a sigmoidalcurve, representing cooperative binding of repres-sor dimers. This cooperativity explains the steepinduction curve; it was also a major insight intohow DNA-binding proteins work. Interactionbetween DNA-binding proteins at their sites ofaction has proved true not only for l, but for pro-moter regulation in general. One can claim withsome justi®cation, in fact, that our understandingof transcription factors has its intellectual origins inthe analysis of the l repression system and is, inmany ways, only a re®nement of it.

Interactions between proteins that bind the lorigin of replication were also ®rst deduced fromgenetics, e.g. isolation of allele-speci®c suppressorsof phage replication gene mutations and sub-sequently veri®ed in vitro.

The story of the l integration/excision system isof particular interest in terms of the spin-offs itgenerated. Key genetic experiments revealed bio-chemical pathways of universal importance. Thusthe observation that the l virion and prophagegenetic maps were not identical, but representedcircular permutations of each other, led to the sug-gestion that l DNA, which is linear in the virion,formed a ring prior to recombining with the hostchromosome. The physical demonstration of lDNA rings occurred rapidly. The investigation ofthe biochemical pathway that converted linearDNA to supercoiled intracellular rings turned uptwo host enzymes, DNA ligase and topoisomerase.Both enzymes are quite remarkable and, of course,the isolation of the ligase directly led to the abilityto clone DNA and, for better or worse, the birth ofthe biotechnology industry.

It was believed, based on what was knownabout recombination at the time, that l integrationinvolved a cross-over between homologous DNAsegments on the phage and host chromosomes.However, the peculiar integrative properties of ltransducing particles, l derivatives in which bac-terial DNA fragments replace phage sequences,were incompatible with such a simple model. This``Guerrini Paradox'' could be best explained ifl integration involved a recombination eventbetween non-homologous DNA sequences. Theobservation that l integration and excision couldoccur in bacterial recA hosts defective in recombi-nation between homologous DNA segments sup-ported this, at the time, heretical idea. The isolationof l int mutants, which failed to integrate or exciseeven in recA� hosts, was consistent with thisnotion, and indicated the phage function respon-sible for recombining l into the host chromosome.Int speci®cally catalyzed recombination betweentwo non-identical DNA sequences, the l and hostattachment sites (attP and att B, respectively). Theproperties of the l site-speci®c recombinationsystem provided the theoretical and biochemicalframework for analogous systems, such as trans-poson hopping and retroviral integration.

The study of l site-speci®c recombination contin-ued to turn up surprises. Mutations in att thatcould be crossed between the bacterium and thephage indicated that the two must share, albeitsmall, homology. Sequence analysis shows a 15 bpregion of homology, too small to support homolo-gous recombination, but quite essential for recom-bination. Two clever selections yielded hostmutants that blocked l integration. The responsiblemutations inactivated the IHF protein, a small,basic, heterodimeric protein. The sole role of IHFin l site-speci®c recombination is to bend att DNAby inserting into the minor groove. Contactsbetween IHF and Int or other proteins bound at attare not involved. The critical role of the bend is toallow the two distinct DNA binding domains ofInt to contact their unique DNA sites. IHF mutantsalso displayed speci®c transcriptional defects. Posi-tive regulation of promoters by IHF probablyentails both architectural alterations and protein-protein interactions. The subsequent discovery ofan analogous protein, HMG I/Y that alters thearchitecture of certain eukaryotic promoters, didnot, therefore, come as a shock.

One feature of l gene regulation, antitermina-tion, was initially thought to be an odd mechanismcon®ned to a single bacteriophage. The l chromo-some has the following basic structure. Two earlydivergent promoters, pL and pR, ¯ank the cI geneand are repressed by CI in a lysogen. When thesepromoters are released from repression, however,only short transcripts are synthesized. Viral devel-opment requires the synthesis of a phage-speci®cprotein. The pL transcript encodes a protein, N,that suppresses l terminators, allowing the pL andpR transcripts to elongate into the early region.Later in development, a second antiterminator pro-tein, Q, enhances late gene expression by suppres-sing tR0, a terminator just downstream to aconstitutive promoter, pR0. The fact that N and Qwere regulatory proteins was evidenced by thepleiotropic defects of mutants, i.e. the absence ofsets of phage proteins in N or Q nulls. Transcriptsfrom promoters other than pL and pR were notantiterminated, indicating a speci®city element inthe pL and pR operons. These sites of N action,nutL and nutR, were de®ned genetically as cisdominant pleotropic mutations, and were thenidenti®ed in the l DNA sequence as near repeatsin the two operons. The nut sites could be dividedinto boxA and an inverted hyphenated repeat,boxB. The nutL element lies just downstream topL, whereas nutR lies distal to cro, an immediate-early gene in the pR operon. Neither nut site islocated in a translated portion of the transcript; theimportance of this is described below. The nutelements are both necessary and suf®cient for Naction, as demonstrated by the N-dependent anti-termination activity of a nutR fragment clonedbetween a non-viral promoter and terminator.

In addition to mutations in boxA and boxB, fra-meshift mutations in cro that extend the cro readingframe over nutR block antitermination in the pR

180 Bacteriophage �

operon. This experiment suggested that the activeform of nut was RNA, rather than DNA, and thatN was an RNA-binding regulatory protein. Thebiochemistry of the N reaction supported this gen-etic analysis. N binds to boxB RNA (referred to asBOXB), which forms a 15 nt stem-loop structure.Mutational analysis of one of the ®ve nucleotideloop sequences, GAAGA, indicated that the 50 Gresidue was essential for antitermination and Nbinding. Recently, the structure of aminoterminalRNA binding domain (the arginine-rich motive,ARM), bound to BOXB, was solved by NMR. BothN and BOXB change structure when they associate.N, which has little structure alone, adopts a bentalpha-helical con®guration. The BOXB loop under-goes a most peculiar change, swinging out thefourth residue (G), to generate a GNRA tetraloop.The importance of the 50 G residue now becomesclear.

N shares structural and functional homologywith the HIV TAT protein, another member of theARM family. TAT, like N, binds to a stem-loopsequence in an HIV transcript, and promotes themodi®cation of PolII. The modi®ed polymerase isin an elongation favorable form, permitting the fulltranscription of the HIV chromosome.

The speci®city of N for lambda transcription isdue to the absence of a BOXB sequence in theE. coli chromosome. However, gene regulation byantitermination is found in the E. coli ribosomalRNA operons. The promoters of each of theseoperons transcribes a BOXA sequence that is bothnecessary and suf®cient to suppress Rho-depen-dent terminators within the operons. A stem-loopsequence reminiscent of BOXB is also found, butthe relevance of this structure to antitermination isnot clear.

The mechanism of Q antitermination is quite dis-tinct from N. Q binds l DNA at the pR0 promoterand then interacts with RNA polymerase pausedat �16. The details of this reaction are still beingelucidated, but it is already known that the poly-merase must retain the s subunit for the reactionto occur. Prior to this ®nding, it was thought thats was rapidly lost after promoter clearance.

Consistent with the notion that if you dream of aregulatory mechanism, it probably exists in l orone of its cohorts, the temperate phage HK022exercises an antitermination mechanism that doesnot involve a phage-speci®c antitermination pro-tein. Instead, HK022 promotes antitermination ofearly phage transcription through the direct actionof transcribed sequences called put (for polymeraseutilization) sites. These sites, which have a complex

secondary structure, appear to interact with RNApolymerase, possibly through the b0 subunit, tomodify its elongation properties. The various anti-termination systems, which appear so different inmechanism, do have in common the ability toaccelerate the rate of transcription elongation, inaddition to suppressing terminators.

I have not even mentioned that key l regulatoryand recombination proteins have short half-lives,and that the relevant proteases may themselves besubject to regulation. Thus, proteolysis can act as acontrol mechanism in bacteriophage development.And that the l packaging system remains the beststudied of any virus.

As for the future, count on l to continue to pro-vide cutting edge information. The mechanisms ofaction or even the host targets of many l functionsare unknown, and the biochemical and structuralanalysis of those functions will continue to provideparadigms for the rest of the biological world.

Acknowledgments

My thanks to M. Yarmolinsky for his suggestions onthis piece, and in general.

References

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Eisen, H., Brachet, P., Pereira da Silva, L. & Jacob, F.(1970). Regulation of repressor expression inlambda. Proc. Natl Acad. Sci. USA, 66, 855-862.

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Luria, S. E., Gould, S. J. & Singer, S. (1981). A View ofLife, Benjamin/Cummings Publishers, Menlo Park,CA.

Ptashne, M. (1992). A Genetic Switch, Blackwell Scienti®cPublications and Cell Press, Cambridge, MA.