CHAPTER 6

Advances in Virus ResearchISSN 0065-3527, DOI: 10.1

* Department of Molecula{ Laboratory of MolecularBiophysics, Polish Acade

Phage l—New Insights intoRegulatory Circuits

Grzegorz Wegrzyn,* Katarzyna Licznerska,* and

Alicja Wegrzyn†

Contents I. Introduction: The Bacteriophage l Paradigm 156

, Volum016/B9

r BiologBiologymy of S

e 82 # 201278-0-12-394621-8.00016-9 All righ

y, University of Gda�nsk, Gda�nsk, Poland(affiliated with the University of Gda�nsk), Institute of Biochemciences, Gda�nsk, Poland

Elsts

ist

II. E

jection of l DNA from Virion into the Host Cell 158

III. T

he Lysis-Versus-Lysogenization Decision and

l DNA Integration into Host Chromosome

158

A

. T he developmental decision 158

B

. A ntiphage response of the host cell 160

C

. In tegration of l DNA into host chromosome 160

IV. P

rophage Maintenance and Induction 162

A

. M odels of the genetic switch 162

B

. A role for Cro in l prophage induction 163

C

. S tructure and function of the cI repressor 164

D

. F actors causing prophage induction 164

E

. E xcision of the prophage 165

V. P

hage l DNA Replication 165

VI. G

eneral Recombination System Encoded by l 169

VII. T

ranscription Antitermination 170

VIII. F

ormation of Mature Progeny Virions 171

IX. H

ost Cell Lysis 171

X. C

oncluding Remarks 172

Ackn

owledgment 173

Refer

ences 173

evier Inc.reserved.

ry and

155

156 Grzegorz Wegrzyn et al.

Abstract Bacteriophage l, rediscovered in the early 1950s, has served as a

model in molecular biology studies for decades. Although currently

more complex organisms and more complicated biological systems

can be studied, this phage is still an excellent model to investigate

principles of biological processes occurring at the molecular level.

In fact, very few other biological models provide possibilities to

examine regulations of biological mechanisms as detailed as

performed with l. In this chapter, recent advances in our under-

standing of mechanisms of bacteriophage l development are

summarized and discussed. Particularly, studies on (i) phage DNA

injection, (ii) molecular bases of the lysis-versus-lysogenization

decision and the lysogenization process itself, (iii) prophage main-

tenance and induction, (iv), l DNA replication, (v) phage-encoded

recombination systems, (vi) transcription antitermination, (vii) for-

mation of the virion structure, and (viii) lysis of the host cell, as

published during several past years, will be presented.

I. INTRODUCTION: THE BACTERIOPHAGE l PARADIGM

Bacteriophage l has been used in studies on the molecular mechanism ofbiological processes for over half a century. In fact, it is now recognized asa paradigm and reference in many regulatory mechanisms occurring inboth prokaryotic and eukaryotic systems. Therefore, this model virus, andprocesses crucial for its development, has been described in many reviewarticles; this introductory section presents only a very concise overviewon the phage life styles and basic mechanisms. For more detailed reviewson both l-based advantages in basic research and phage-dependent bio-technological approaches, readers are advised to find various availablearticles, published in recent years, and exemplified by Gottesman andWeisberg (2004), Dodd et al. (2005), Ptashne (2005), Wegrzyn andWegrzyn (2005), Van Duyne (2005), Garufi et al. (2005), Oppenheim et al.(2005), Court et al. (2007a), Thomason et al. (2007), Krupovic and Bamford(2008), and Valdez-Cruz et al. (2010). We do not refer here to 20th-centuryreferences describing the actual discovery of phage l by Ester Lederberg;recognition of l genome peculiarities by Al Hershey; genetic characteri-zation of l genes by Allan Campbell; characterization of the basic mecha-nism of phage-dependent cell lysis by Alina Taylor and Ry Young; firstunraveling of the transcriptional patterns and discovery that both DNAstrands can be transcribed by Karol Taylor and Waclaw Szybalski;and identification of crucial events in l DNA replication by Ross Inman,Maria Schnos, Costa Georgopoulos, Roger McMacken, Karol Taylor, andMaciej Zylicz, to mention a few. We also do not refer here to the role of lin the development of biotechnology, particularly genetic engineering,

Bacteriophage l 157

although we must remember that this bacteriophage, after suitablemodifications, provided first cloning vectors and, employing l geneticregulatory elements, allowed researchers to construct very useful tools forcontrolled expression of cloned genes (works by Waclaw Szybalski, AnnaJ, Podhajska, and Marian S. Sektas must be acknowledged here). Further-more, the first transcriptional and physical (by electronmicroscopy) mapsof l, including nomenclature and definition of regulatory elements (oL , oR ,pL, pR, pR’, pI , pM, oop, ori, nutL, nutR, qut and others) were developed byWaclaw Szybalski and his collaborators.

Phage l virions adsorb on their host, Escherichia coli, cells employingthe bacterial LamB receptor, located in external membrane, and playingthe role of maltose porin in E. coli. Phage genome, the double-strandedDNA molecule composed of 48,502bp (with 12 nucleotide single-stranded, complementary ends), enters the host cell cytoplasm throughthe phage noncontractile tail, due to action of the E. coli enzymatic system,composed of PtsP and PtsM proteins, located in the internal membraneand used normally for the transportation of mannose. Following DNAcircularization, the decision whether to lysogenize the host cell or toproceed to lytic development is made on the basis of the specific phageregulatory system, in which many phage- and host-encoded factors areemployed. If the lysogenic pathway is chosen, phage DNA is incorporatedinto the host chromosome and persists in the formof a prophage. This statecan be kept formany cell generations. Lytic development starts either afterprophage induction (resulting in excision of the l genome from the hostchromosome) or when the lysis-versus-lysogenization decision is made infavor of the former option. At the early stage of lytic development, phageDNA is replicating predominantly according to the circle-to-circle (or y)mode, which is switched later to the rolling circle (or s) mode. Thelatter replication mechanism results in the appearance of long concate-meric DNAmolecules encompassing several l genomes. Simultaneously,general recombination of phageDNA takes place, and expression of phagegenes occurs (which is controlled precisely by various mechanisms,including activation, repression, and antitermination of transcription,as well as RNA stability and efficiency of translation initiation), leadingto the production of capsid proteins. During the process of the assem-bly of progeny virions, concatemeric lDNAmolecules are cut into mono-mers in the process called packaging, and maturation of capsidsleads to the formation of progeny virions. Phage progeny is liberatedfrom the host cell after its lysis, caused by products of phagegenes. Importantly, all the stages of bacteriophage l development arecontrolled precisely by mechanisms whose molecular details can beunderstood in more and more detail. These mechanisms also providemodels for studies on biological processes in both bacterial cells andeukaryotic organisms.

158 Grzegorz Wegrzyn et al.

This chapter does not refer further to basic information about particu-lar processes. Rather, it focuses on novel findings, which provided newinsights into the regulation of certain mechanisms operating during thedevelopment of bacteriophage l.

II. EJECTION OF l DNA FROM VIRION INTO THE HOST CELL

Findings on the mechanism of l DNA ejection focused on physical andchemical factors that influence this process. Koster et al. (2009) demon-strated that an internal capsid pressure influences efficiency of the ejec-tion process. A decrease in the capsid pressure correlated with a decreasein the efficiency of cell lysis caused by phage infection. Another studyindicated that the ejection process is independent of ionic concentrations,which might seem quite surprising (Wu et al., 2010). These studiesprovided important information about mechanistic processes occurringat the very first stage of phage l development.

III. THE LYSIS-VERSUS-LYSOGENIZATION DECISION ANDl DNA INTEGRATION INTO HOST CHROMOSOME

A. The developmental decision

Shortly after phage l DNA is ejected and enters the host cell, the majordevelopmental decision must be done, namely whether to lysogenize thehost or to perform lytic development. Several proteins and other factorsplay roles in the process of making the decision, and this process iscomplicated enough to make it impossible to predict precisely at thecurrent state of knowledge. Although we can estimate what conditionssupport lysogenization and what is required for lytic development, it isimpossible to calculate precisely what percentage of phages enters one oranother pathway under certain conditions. Therefore, various methods ofthe simulation of the decision process are being developed. Building suchmodels of the regulatory network is a usual step in attempts to under-stand the system in more detail (Avlund et al., 2009a; Wegrzyn andWegrzyn, 2005).

A very interesting quantitative kinetic analysis of such a network hasbeen performed that suggested that the Cro protein may be the key playerin sensing whether the host cell is infected by one or more phages (Kobileret al., 2005). In fact, a number of phages infecting a single host cell is one ofthe factors influencing the developmental decision. Recent simulation ofthe ‘‘counting’’ process suggested that apart from the already knownimportance of the ratio of Cro and cII proteins, additional regulatory

Bacteriophage l 159

mechanisms may exist (Avlund et al., 2009b). Another study suggestedthat the presence of the restriction–modification system can influencephage l development even if the phage has already been ‘‘adapted’’ tothis particular system (Gregory et al., 2010). The simulation used by theauthors of that study showed that adaptation and readaptation to aparticular restriction and modification system result in lower efficiencyof phage propagation and delayed lysis of bacterial cells relative to non-restricting host bacteria.

Among proteins involved in the lysis-versus-lysogenization decision,Cro, cI, cII, and cIII appear to be crucial. Therefore, understanding thedetails of structures and functions of these proteins is necessary to buildmore solid models of phage developmental regulation. The Cro proteinfunctions as a dimer, thus the efficiency of formation of such a structuredetermines its activity. Jia et al. (2005) demonstrated that native Cromonomers have compact structures, which are hard to unfold. However,unfolded or partially folded monomers appear to be preferred substratesfor Cro dimer formation. This implicates that the assembly of Cro into adimer is a slow process. Thus, in vivo Cro binding may be under kineticrather than thermodynamic control.

Several papers on cII structure and function have been published.Datta et al. (2005b) demonstrated that 15C-terminal amino acid residuesof cII are flexible and may act as a target for proteolysis in the host cell. Inthis 97 amino acid long protein, residues 70–82 were found to be neces-sary for tetramer formation, DNA binding, and transcription activation.The same research group (Datta et al., 2005a) solved the three-dimensionalstructure of cII at 2.6A resolution. The tetrameric structure of cII is ratherunusual, as it is a dimer of dimers, however, without a closed symmetry.Such a structure may be necessary for efficient binding of cII to majorgrooves of DNA, from one face of this molecule. Further studies by Paruaet al. (2010a) indicated that residues 70, 74, and 78 are crucial for main-taining the tetrameric structure of cII. The model of binding of cII to oneface of DNA was used to propose a mechanism for cII-mediated activa-tion of transcription with involvement of the RNA polymerase a subunit(Kedzierska et al., 2004). This can explain how cII stimulates promoteractivity when it is bound at the site overlapping the �35 box and when itinteracts with both s70 and a subunits of RNA polymerase. Interestingly,one of the cII-activated promoters, paQ, has been found to be stimulateddirectly by guanosine tetraphosphate (ppGpp), the stringent control alar-mone (Potrykus et al., 2004).

The cII protein is degraded in E. coli cell by the host-encoded HflBprotease (also called FtsH). Another host factor, the HflD protein, inter-acts with cII and facilitates its degradation. Parua et al. (2010b) demon-strated that HflD also negatively influences binding of cII to DNA,inhibiting transcription activation by this factor. Two other Hfl proteins,

160 Grzegorz Wegrzyn et al.

HflC and HflK, make the HflB-mediated proteolysis of cII less efficient.Bandyopadhyay et al. (2010) have purified HflC and HflK separately(which was not achieved previously) and found that each of theseproteins binds to HflB and inhibits cII proteolysis.

The best characterized inhibitor of HflB is l cIII protein. Kobiler et al.(2007) showed that cIII functions as a competitive inhibitor of HflB, pre-venting binding of the cII protein by this protease. cIII–HflB interactions,but not those between cII and cIII, were found to be responsible forinhibition of the protease (Halder et al., 2008). Kobiler et al. (2007) alsosuggested that oligomerization of cIII is required for its function as theHflB inhibitor. However, Halder et al. (2007) proposed that the cIII proteinexists as a dimer under native conditions.

Apart from the regulatory factors influencing the lysis-versus-lysogenization decision known for many years, perhaps surprisingly, itis still possible to discover novel players in this game. One of them is theEa8.5 protein, encoded by a l gene located in the b (nonessential) region ofthe phage genome. Overproduction of this protein resulted in a decreasein the efficiency of lysogenization, most probably due to impairment incII-mediated transcription activation (Łos et al., 2008).

Current knowledge about the regulatory network responsible for thecontrolofphageldevelopmentat thestageof the lysis-versus-lysogenizationdecision is presented schematically in Figure 1. This scheme is based onthe previous picture by Wegrzyn and Wegrzyn (2005) but contains anupdate on regulations discovered during the last several years.

B. Antiphage response of the host cell

Recent discoveries informed us about the existence of the prokaryoticantiviral (antiphage) system. This system, abbreviated as CRISPR, con-sists of clusters of regularly interspaced short palindromic repeats inbacterial genomes (Brouns et al., 2008). Because this topic is discussed inmore detail in another chapter of this book, this chapter only signals itsinfluence on lysogenization or lytic development. Namely, it appears thatCRISPR affects lysogenization by phage l, maintenance of the lysogenicstate, and prophage induction (Edgar and Qimron, 2010). Interestingly,the function of CRISPR appears to be regulated by H-NS and LeuOproteins (Westra et al., 2010).

C. Integration of l DNA into host chromosome

If the lysogenic pathway of phage l development is chosen, the crucialstep to form the prophage is integration of the viral genome into the hostchromosome. This reaction is catalyzed by the phage-encoded Int protein,also called integrase. Production of this enzyme is dependent on the

sib int cIII N cI cro cII O P Q S R Rz

pR

pL pM

pE

pO paQ

pR’/tR’

Int cIII N cI

Cro cII O P Q

pI

Low temp.

High temp.

HflB (FtsH)ATP

cAMP + PPi

Glucosetransport

ppGpp

Amino acidstarvation

DnaA

SeqA

ClpP/ClpX

DnaB

oop RNA AAAAAAAA

RNases

High growth rate

Low growth rate

PAP I

RNase III

RecA*

Low temp.High temp.

Ea8.5

HflC/HflK

HflD

DksA

IHF

OxyR

AC

FIGURE 1 A regulatory network at the ‘‘lysis-versus-lysogenization’’ decision of bacte-

riophage l. The crucial l regulatory genes are presented between two thick horizontal

lines that symbolize a fragment of the phage genome. Promoters are marked in thin

boxes, and transcripts are shown as thick arrows, with arrowheads indicating direction-

ality of transcription (oop RNA is an exception, see later). Phage l gene products

(proteins and one nontranslatable transcript, oop RNA) are marked in thick boxes. Host

(Escherichia coli) proteins and specific conditions are presented without boxes. Regu-

latory processes are indicated as thin arrows and thin blunt-ended lines (positive

regulations are represented by arrows and negative regulations are represented by blunt-

ended lines). AC, adenylate cyclase; ATP, adenosine triphosphate; cAMP, cyclic AMP, High

temp., high temperature; Low temp., low temperature; PAP I, poly(A) polymerase I (the

pcnB gene product); ppGpp, guanosine tetraphosphate; PPi, pyrophosphate. This figure is

an updated version of the scheme published by Wegrzyn and Wegrzyn (2005).

162 Grzegorz Wegrzyn et al.

function of the cII-dependent pI promoter and the tI terminator. Althoughtranscription of the int gene is also possible from the pL promoter,transcripts derived from this promoter do not terminate at tI due toN-dependent antitermination, thus int mRNA is highly unstable due tothe retroregulation mechanism (degradation of mRNA initiated at thespecific secondary RNA structure located downstream of the codingsequence). Therefore, only transcripts that stop at tI may give functionalintmRNA. Detailedmapping of the tI terminator has been performed onlyrecently by Martinez-Trujillo et al. (2010), who identified sequencesrequired for transcription termination at this site and mapped the alterna-tive ends of the transcript. Another published paper focused on the inte-grase activity itself. In fact, novel integrase variants were also selected,which helped not only to understand biochemical function of this enzyme,but also provided interesting biotechnological tools (Tay et al., 2010).

IV. PROPHAGE MAINTENANCE AND INDUCTION

Once the host cell is lysogenized by phage l, the prophage state isstabilized by the action of the phage-encoded cI protein, which is a strongrepressor of the main lytic promoters pR and pL and an activator of its ownpromoter, pM. The prophage is maintained as long as cI is active. Thisrepressor is sensitive to autoproteolysis, taking place upon interaction ofcI with the activated form of the RecA protein, called RecA*, whichchanges its conformation after binding to single-stranded DNA frag-ments, appearing as a result of the damage of the genetic material(which is a signal triggering the specific cellular response, called theSOS response). Cleavage of the cI repressor results in derepression of pRand pL and subsequent excision of the prophage from the host chromo-some, followed by phage lytic development. This drastic change in thebacteriophage life style is known as l prophage induction and is a para-digm of the genetic switch—a process of the drastic changes in the patternof expression of various genes leading to a major change in the life style.

A. Models of the genetic switch

A large body of data indicated that the prophage state is stable, showingthat it is a specific type of cellular memory (Oppenheim et al., 2005). Infact, studies indicated that this state is exceptionally stable, as the fre-quency of prophage induction events is lower than 10-8 per cell generationin absence of the SOS response (Cao et al., 2008; Little and Michalowski,2010; Morelli et al., 2009; Zong et al., 2010).

Despite this high stability, under natural conditions, prophage induc-tion occurs relatively often due to frequent stress conditions met by

Bacteriophage l 163

lysogenic bacteria in their habitats. Therefore, modeling of the regulationof the switch is important in understanding this crucial process in phagelife. Novel models of the l genetic switch have been published in recentyears. These include mathematical modeling, which described the switchas a piecewise, quadratic second-order differential equation (Laschov andMargaliot, 2009), and the experimental evolution approach, showing howlambdoid prophages can change their regulatory elements in response toselection (Refardt and Rainey, 2010). Other experimental and theoreticalanalyses suggested that some elements of the regulatory system maybe dispensable, playing a role in modulating the response rather thandetermining the main event (Little, 2010), that a constant level of the cIrepressor can be maintained over a wide range of its degradation rate,ensuring heritability of the lysogenic state (Cao et al., 2010), and thatthis state is in a monostable regime rather than in a bistable regime (Louet al., 2007).

B. A role for Cro in l prophage induction

An interesting question is the role of the Cro protein in l prophageinduction. Cro is known as a strong repressor of the pM promoter(required for the cI gene expression) and a weak repressor of pR and pL,playing a role as a facilitator of lytic development. However, there werealso reports indicating that Cro may have a role in the l genetic switch.Recent works indicated that Cro is not crucial in the switch, but maymoderate this process by a weak repression of early lytic promoters(Svenningsen et al., 2005). Atsumi and Little (2006) replaced Cro withthe Lac repressor and showed that a phage bearing the lacI gene and lacoperators instead of cro is viable, but some details of the phage develop-ment are changed. This underlined once again the complexity of theregulatory system and its sensitivity to subtle changes in the regulatoryprocesses (Ptashne, 2006). Further changes in the l genome, namelyreplacement of the cI gene with the gene coding for Tet repressor, resultedin construction of a still viable phage, able to proceed either lytic orlysogenic development depending on levels of factors controlling partic-ular promoters (Atsumi and Little, 2006). These experiments also sug-gested that we might be quite close to learning how to control phage ldevelopment. However, Schubert et al. (2007) postulated that the role ofCro is critical, rather than modulatory, in prophage induction. Theycreated mutants that strongly impaired Cro-mediated repression of pMwithout significant changes in cI-mediated regulation of this promoterand demonstrated that inhibition of transcription from pM is crucialfor efficient prophage induction, as synthesis of new cI molecules cansignificantly impede phage lytic development.

164 Grzegorz Wegrzyn et al.

C. Structure and function of the cI repressor

It is clear that prophage maintenance depends mainly on activity of the cIrepressor. However, new insights into both structure and function of thisprotein have appeared recently. A role for the formation of cI oligomersbound to both oR and oL regions has been demonstrated previously, andtheir structures have been determined at high resolution by experimentsemploying an atomic force microscope (Wang et al., 2009). The cI repres-sor binds to specific DNA sequences cooperatively; however, results byBabic and Little (2007) suggest that this cooperativity is not essential butrather is a refinement to a more basic circuit. It seems that the cooperativebinding of cI may increase stability of the prophage under variousenvironmental conditions. This could be supported by analyses basedon a validated mathematical model (Gedeon et al., 2008).

The cI-mediated activation of the pM promoter has been studied by em-ploying advanced biophysical (Bakk, 2005) and biochemical (Kedzierskaet al., 2007)methods. Results of such studies strongly suggest that cI contactsboths70 anda subunits of theRNApolymerase atpM(Kedzierska et al., 2007)and that activation of this promoter can be two- to fourfold higher whenDNA is looped (Anderson and Yang, 2008).

Determination of the crystal structure of the intact l cI repressor dimerbound to DNA (Stayrook et al., 2008) provided an excellent tool in under-standing the function of this protein in more detail. In fact, this structureindicates that the two subunits of the cI dimer adopt different conforma-tions (Hochschild and Lewis, 2009; Stayrook et al., 2008).

D. Factors causing prophage induction

Because the cI repressor is sensitive to its autocleavage triggered by theRecA* protein, which also triggers the SOS response, l prophage inductionwas usually studied in ultraviolet-irradiated cells. However, it is clearthat other factors occurring in natural habitats of E. coli must also causethe genetic switch. Identification of such factors is important in understand-ing the physiology of l and relative phages. Interestingly, it was demon-strated that significant differences exist in the mechanism of l prophageinduction and further lytic development between twoexperimental systemsthat are used commonly to study this phenomenon, namely the SOS-mediated induction of wild-type prophage and heat-mediated inductionof a prophage bearing a mutation in the gene coding for a temperature-sensitive cI repressor (Rokney et al., 2008). This finding strongly supports theprediction that our knowledgeof prophage inductionmechanisms,which isbased on a very limited number of experimental systems, may be highlyincomplete.

Bacteriophage l 165

Shkilnyj and Koudelka (2007) found that a concentration of salt (NaCl)as high as 200mM can cause efficient induction of a lambdoid prophage.This was an important discovery because it indicated that some condi-tions found in a natural environment may trigger the l genetic switch.Another work demonstrated that l prophage may be induced by acylhomoserine lactones (Ghosh et al., 2009). These compounds are known assignaling molecules of quorum sensing in Gram-negative bacteria. There-fore, one may speculate that prophage induction may be a response to ahigh concentration of bacterial cells. Another factor triggering the induc-tion of lambdoid prophages is hydrogen peroxide. This oxidative stress-mediating factor was shown to be an efficient inductor of not only l butalso other lambdoid phages, including Shiga toxin-converting phages(Łos et al., 2009, 2010). The mechanism of H2O2-caused prophageinduction may involve action of the host-encoded OxyR protein, whosefunction in regulation of the activity of the l pM promoter has beendescribed elsewhere (Glinkowska et al., 2010).

E. Excision of the prophage

Following inactivation of the cI repressor, which is always the first step ofl prophage induction, excision of the phage DNA from the host chromo-some is necessary to start lytic development. This excision is mediated bytwo phage-encoded proteins, Int and Xis, but is facilitated by at least fourhost proteins. The architecture of the nucleoprotein complex, containingsix proteins and a l DNA fragment encompassing the att site (the regionof the site-specific recombination that occurs during prophage excision),has been studied using the fluorescence resonance energy transfer tech-nique and employing a metric matrix distance-geometry algorithm (Sunet al., 2006). These studies provided important data, which helped inunderstanding the actual structure of this complex in more detail.Another work underlined the important role of the Fis protein, a hostDNA-binding protein, in l DNA excision from the E. coli chromosome(Papagiannis et al., 2007). In fact, upon prophage induction, a very highlevel of synthesis of the l Xis protein has been observed; however, thisprotein was ineffective in the absence of Fis. It is also worth mentioningthat some other lambdoid phages, such as the Shiga toxin-convertingphage F24B, may encode additional factors controlling the excisionevents (Fogg et al., 2011).

V. PHAGE l DNA REPLICATION

Bacteriophage l encodes only two proteins involved directly in replica-tion of its genome, O and P proteins. The O protein binds to the orilregion, and the P protein delivers the host-encoded DnaB helicase to this

166 Grzegorz Wegrzyn et al.

site. Then, a replication complex containing other replication proteinsfrom the host cell (including DNA polymerase III holoenzyme, DnaGprimase, and gyrase) is formed. However, contrary to host replicationmachinery, initiation of l DNA bidirectional replication requires theactivities of DnaJ, DnaK, and GrpE chaperones. Interestingly, this initia-tion is not effective in the absence of transcription proceeding near theoril region. This transcription, called transcriptional activation of theorigin, somehow activates the replication complex to start bidirectionalDNA replication. After several rounds of such a replication, a switchfrom circle-to-circle (or y) to rolling-circle (or s) replication modeoccurs, which leads to the production of long concatemeric l DNAmolecules, used for packaging of the phage genomes into newly assem-bled capsids.

During recent years, work on the regulation of l DNA replicationfocused on the process of transcriptional activation of oril. Thiswas becausethis process, rather than the assembly of the replication complex per se,appears to trigger the replication event. Moreover, because only a unidirec-tional replication can start from oril in the absence of transcriptional activa-tion, it was proposed that the switch from y to s replication mode is due toan inhibition of transcription; after one round of unidirectional replication,the 50 end of the replicating DNA strand can be displaced by the growing 30

end, which can result in initiation of rolling-circle replication (Wegrzyn andWegrzyn, 2005). As the transcription that activates DNA replication fromoril starts from the pR promoter, any factors that influence its strength mayalso influence l genome replication. Thus, studies on the function andregulation of this promoter have a high impact on our understanding ofthe regulation of l DNA replication.

A novel technique enabling calibration of biochemical parametersin vivo was tested to characterize the pR promoter (Rosenfeld et al.,2005). This provided important details on the function of this promoter,particularly suggesting that its activity fluctuates during the cell cycle.Then, the isomerization step of transcription initiation from pR was char-acterized in more detail (Kontur et al., 2006). It is worth mentioning thatthis promoter is controlled by many factors, including negative (cI, Cro,ppGpp) and positive (DnaA, SeqA) regulators. Interestingly, both DnaAand SeqA proteins stimulate the transcription from pR and bind down-stream of the transcription start point, which is very rare in prokaryoticsystems (Łyze�n et al., 2006). SeqA was found to activate pR at the stage ofpromoter clearance (Łyze�n et al., 2006). This protein influences DNAtopology, and it was demonstrated that another such protein, IHF, canalso stimulate pR-initiated transcription (Łyze�n et al., 2008). Interestingly,the SeqA protein may also control l DNA replication indirectly by influ-encing the stability of the replication complex (this complex, under stan-dard laboratory conditions of cultivation of bacteria, is stable and can be

Bacteriophage l 167

inherited by one of two daughter DNA molecules for many cell genera-tions if bound to a plasmid derived from bacteriophage l)(Narajczyket al., 2007a). Another stimulator of the pR promoter has been identified,namely the DksA protein (Łyze�n et al., 2009). Interestingly, this proteinoften acts together and synergistically with guanosine tetraphosphate(ppGpp), the stringent control alarmone, whereas their actions are inde-pendent and antagonistic at pR (ppGpp inhibits and DksA stimulates pRactivity)(Łyze�n et al., 2009). Interestingly, the phage-encoded P proteinwas found to inhibit biochemical activities of DnaA, which normallyfunctions as a stimulator of pR-initiated transcription (Datta et al., 2005c,d). All these new discoveries were bases for the updated proposal of themechanism of the switch from y to s replication mode during the lyticdevelopment of bacteriophage l, published by Narajczyk et al. (2007b)and presented schematically in Figure 2.

Szambowska et al. (2011) provided another input into the puzzle of theregulatory mechanism of l DNA replication regulation. It has beendemonstrated that RNA polymerase interacts directly with the l O pro-tein. This may indicate that RNA polymerase can play an additional rolein l DNA replication initiation apart from transcribing the oril region.Moreover, it was found that binding of the l O protein to tandem copiesof specific DNA sequences affects DNA topology more significantly thansuggested earlier (Chen et al., 2010). Perhaps a large nucleoprotein struc-ture is built whose activity is controlled in a more complex manner thanpredicted previously. This speculation may be supported by other recentdiscoveries that some lambdoid bacteriophages bear six O-bindingsequences at the ori region instead of four (like in l)(Nejman et al., 2009)and that single amino acid substitutions in O and P proteins may drasti-cally change requirements for factors influencing the transcriptional acti-vation of oril (such as DnaA, ppGpp, and DksA)(Nejman et al., 2011). Inaddition, activity of the CbpA protein, which can replace DnaJ in l DNAreplication, is controlled at multiple levels (Chenoweth and Wickner,2008). Perhaps newly developed methods, such as allowing either directobservation of enzymes replicating DNA molecules (Kulczyk et al., 2010)or dynamic analyses of phage–host interactions at the protein level(Maynard et al., 2010), should help in understanding the complicatedregulation of l DNA replication initiation in more detail.

At the late stage of l DNA replication, when long concatemers of thephage genome are produced as a result of rolling-circle replication, it isnecessary to protect these linear ‘‘tails’’ from host nucleases. The RecBCDnuclease is one of them, and l encodes an inhibitor of this enzyme, calledGam. The crystal structure of Gam has been resolved, which helped toshow that this protein inhibits RecBCD by preventing it from bindingDNA (Court et al., 2007b). Interestingly, it seems that l encodes its owninhibitor of DNA replication. It was known that the cII protein is toxic

pR

DnaA

5 – 6 rounds

~ 50 copies ofλ DNA

oriλ

Cro

P

SeqA

FIGURE 2 Model for regulation of the switch from circle-to-circle (y) to rolling-circle

(s) replication of bacteriophage l DNA. Shortly after infection, there are a few copies (or

even just one copy) of phage DNA (thick circles, representing double-stranded DNA

molecules) and a certain number of free DnaA (small filled circles) and SeqA (small open

circles) molecules, which can potentially bind toweak DnaA or GATC boxes, respectively,

at the pR promoter (a thick slash) region. This binding is required to stimulate transcrip-

tion from pR, which activates the origin region (rectangle). Such an activation facilitates

formation of the preprimosomal complex, consisting of the l O protein (the replication

initiator protein that binds to four iterone sequences located in oril), the l P protein (a

protein that delivers the host-encoded DNA helicase to oril), and the DnaB protein (the

DNA helicase). DnaA- and SeqA-mediated stimulation of transcriptional activation of

oril allows for initiation of bidirectional y replication of l DNA. After five to six rounds

of such a replication, about 50 copies of l genome appear. Due to the presence of many

168 Grzegorz Wegrzyn et al.

Bacteriophage l 169

when overproduced in E. coli cells; however, the mechanism of its toxicityremained unknown. Kedzierska et al. (2003) found that DNA replicationis impaired in cells overexpressing the cII gene efficiently, which suggeststhat the replication machinery may be a target for cII toxic activity.

VI. GENERAL RECOMBINATION SYSTEM ENCODED BY l

As an alternative to the hypothesis that the switch from y to s replicationmode is the result of a change from bidirectional to unidirectional repli-cation (Narajczyk et al., 2007b), it has been suggested that this switchdepends on formation of the recombination intermediate (reviewed byWeigel and Seitz, 2006). This could be possible, as bacteriophage lencodes its own system for general genetic recombination, called Red,and is composed of two proteins: Exo and Bet. This system is known asone of the most efficient recombination pathways identified to date.Interestingly, recent works indicated another possible link between repli-cation and recombination.

Although mechanisms of Red recombination based on strand anneal-ing or strand invasion have been proposed previously, neither of themcould explain the results of experiments in which crosses between anonreplicating linear l genome and a replicating plasmid bearing acloned fragment of l DNA were studied (Poteete, 2008). Therefore,another mechanism, in which the replisome invasion is considered, hasbeen proposed. This mechanism implies that replication is directlyinvolved in Red recombination (Poteete, 2008). Further work byMaresca et al. (2010) demonstrated that Red recombination indeedrequires replication of the target molecule. They proposed a model inwhich the formation of single-stranded DNA heteroduplexes at the repli-cation fork is required. The third work implicating the requirement of

DnaA- and SeqA-binding sequences in l DNA, cellular DnaA and SeqA proteins are

titrated out. Moreover, continuous production of the l P protein (small open oval)

results in its accumulation and interaction with DnaA, causing inhibition of activities of

the latter protein. In addition, the high amount of the Cro repressor (small gray oval),

which blocks transcription from pR when occurring at elevated concentrations in cell, is

also produced. Therefore, transcriptional activation of oril becomes impaired. This leads

to the initiation of unidirectional y replication, which, after one round, switches to the smode because a round of unidirectional y replication initiated at oril is followed by

displacement of the 50 end of the newly synthesized leading strand by its growing 30 end(arrow). For simplification of the diagram, at the switch from y to s replication, only the

fate of the parental strand (thin lines) directing the synthesis of the leading strand is

shown. This figure is an updated version of the scheme published by Wegrzyn and

Wegrzyn (2005).

170 Grzegorz Wegrzyn et al.

DNA replication in Red recombination was published by Mosberg et al.(2010). They suggested that the Exo protein degrades one l DNA strandand leaves the other strand intact as single-stranded DNA, which isannealed at the replication fork in the reaction catalyzed by the Betprotein. Altogether, these studies indicate that DNA replication isrequired for efficient recombination by the Red system.

VII. TRANSCRIPTION ANTITERMINATION

Transcription antitermination is a specific mechanism of regulation of theefficiency of RNA production. It is based on the formation of complexesbetween RNA polymerase and regulatory proteins, which prevent tran-scription termination at otherwise functional terminator regions. Bacteri-ophage l encodes two antitermination proteins, N and Q. In N-dependentantitermination, a large nucleoprotein complex is formed, which containsRNA polymerase, the l N protein, and a set of host-encoded regulatoryproteins, including NusA, NusB, NusE, andNusG. The N antiterminationcomplex, assembled on the RNA strand formed just after transcription ofthe corresponding DNA fragment, is one of the largest prokaryotictranscription complexes. Therefore, recent works have focused on moredetailed description of the structure of this complex and interactionsbetween its elements to better understand its function.

Conant et al. (2005) described quantitatively the binding states of the Nprotein, while Horiya et al. (2009) concluded about similar issues on thebasis of replacement of boxB RNA (a part of the transcript that, togetherwith boxA RNA, forms a structure recognized by the antiterminationcomplex) and N with the heterologous system composed of interactingRNA and peptide molecules. That work led to a determination of spatialrequirements for RNA–protein interactions. A role for RNA looping wastested by Conant et al. (2008), who proposed that such a looping facilitatesinteraction of RNAwith other elements of the N antitermination complex.Interestingly, it appears that pR promoter activity can influence assemblyof the N antitermination complex significantly (Zhou et al., 2006). Finally,interactions of the NusA protein with the l N protein and the specificRNA sequence were studied by Prasch et al. (2006 and 2009, respectively),and affinity of the NusB–NusE complex to boxA RNA was determined byBurmann et al. (2010). All these studies provided very important informa-tion, which now can be used for building more detailed models on thefunction of the large nucleoprotein complex enabling N-dependenttranscription antitermination. It appears significant, that one of the firstexamples of the new field of Synthetic Biology was the organic chemicalsynthesis of the antitermination nut sites and its mutants (Brown andSzybalski, 1985).

Bacteriophage l 171

In the Q-dependent antitermination system, a significantly less com-plicated complex is formed, which includes only RNA polymerase, the Qprotein, and NusA. Contrary to the N protein, the Q gene product inter-acts with DNA rather than with RNA. However, Nickels et al. (2006)demonstrated that RNA-mediated destabilization of the interactionbetween s70 and b subunits is required for binding of the Q protein tothe RNA polymerase holoenzyme. In fact, it was subsequently demon-strated that Q is a stable component of the transcription elongation com-plex (Deighan andHochschild, 2007) and that this antitermination proteincontacts the b-flap domain of RNA polymerase (Deighan et al., 2008).

VIII. FORMATION OF MATURE PROGENY VIRIONS

Assembly of mature virions is a complicated process in whichmany kindsof proteins, which build phage head and tail, are involved. Although allplayers of this reaction are perhaps already identified, details of particularsteps of this process and detailed roles of particular proteins remainedpoorly understood. For example, it was known that protein cross-linkingand proteolyticmaturation events are necessary for phage head formation,but the specific protease remained unknown. Medina et al. (2010) demon-strated that the phage-encoded C protein is the specific protease responsi-ble for l procapsidmaturation. Interestingly, it was found that major headand tail proteins, E and V, respectively, as well as a capsid decorationprotein D, are associated with transition metals (Zhang et al., 2011).

l DNA packaging into preformed heads, together with addition of thepreformed tail, finalizes the virion assembly. The packaging reaction iscatalyzed by the complex of the phage-encoded A and Nu1 proteins, calledthe terminase complex, and the specificities of terminases from differentlambdoid phages have been studied, including phylogenetic analyses ofthese enzymes (Feiss et al., 2010).An interestingworkhas been published byYang et al. (2009), who determined that phage lDNA is packaged at the rateof about 120bpper secondat 4�Cand at 1mMATP.Although thepackagingreaction usually occurs at higher temperatures in a natural environment,these results provide important information facilitating estimation of theactual rate of this reaction proceeding inside the host cell.

IX. HOST CELL LYSIS

The final step of bacteriophage l development is lysis of the host cell. Thisreaction is performed by two major phage-encoded proteins, S holin andR transglycosylase, and two accessory proteins, Rz and Rz1. All theseproteins are products of phage genes transcribed from the l late promoter

172 Grzegorz Wegrzyn et al.

pR’. Work by Shao and Wang (2009) led to a description of the model forprediction of the effects of pR’ activity on lysis time.

The specific roles of Rz and Rz1 proteins were poorly understood for arelatively long time. It is worth mentioning here that the existence andfunctionality of Rz and Rz1 genes have been discovered by Alina Taylorand co-workers (Hanych et al., 1993; Taylor et al., 1983) relatively late, wellafter determining the whole sequence of the phage l genome. Now weknow that Rz and Rz1, classified as spanins, are involved in disruptingthe outer membrane of the bacterial cell. Structures of these proteins weredetermined in more detail recently (Berry et al., 2010), which was the basisfor speculations on the model of Rz-Rz1-dependent disruption of theouter membrane.

The protein making holes in the cytoplasmic membrane is encoded bythe phage l S gene. Using cryoelectron microscopy, Dewey et al. (2010)demonstrated that holes made by this protein are surprisingly large,having an average diameter of 340nm, while some holes were even aslarge as 1mm in diameter. Interestingly, most cells had only one large hole.

An old and intriguing question was why cell lysis occurs at the veryend of phage development, while genes coding for S, R, Rz, and Rz1proteins are located at the beginning of the pR’ operon. It was knownthat the S cistron encodes two proteins, the holin (called S105) and theantiholin (S107), due to the presence of two alternative translation startsites. However, it remained unclear what actually triggers lysis, initiatedby formation of the hole in the cytoplamic membrane. White et al. (2011)suggest that lysis occurs when the holin reaches a critical concentrationand nucleates to form rafts. Therefore, a significant amount of time isneeded for the production of sufficient amounts of the S105 protein, andthe presence of S107 prevents premature formation of S105 rafts.

X. CONCLUDING REMARKS

In 2001, David Friedman and Don Court published an article entitled‘‘Bacteriophage l: Alive and Well and Still Doing Its Thing’’ to indicatethat this classical model, used for years in biological research, could stillserve as an excellent tool in studies on molecular mechanisms of basiccellular processes. It appears obvious that after 10years, the statement byFriedman and Court (2001) is still valid and that the use of bacteriophagel (as well as other lambdoid phages) in basic and applied research may berequired to solve some still difficult problems in understandingmolecularmechanisms of biological processes. This statement is supported by thenumber of excellent papers describing studies on l, published in previousseveral years and cited in this article, that concerned various aspects ofphage biology, particularly phage DNA injection, molecular bases of the

Bacteriophage l 173

lysis-versus-lysogenization decision and the lysogenization process itself,prophage maintenance and induction, DNA replication, genetic recombi-nation, transcription antitermination, formation of the virion structure,and lysis of the host cell.

ACKNOWLEDGMENT

This work was supported by Ministry of Science and Higher Education, Poland (Grant NN301 192439 to AW).

REFERENCES

Anderson, L. M., and Yang, H. (2008). DNA looping can enhance lysogenic CI transcriptionin phage lambda. Proc. Natl. Acad. Sci. USA 105:5827–5832.

Atsumi, S., and Little, J. W. (2006). A synthetic phage lambda regulatory circuit. Proc. Natl.

Acad. Sci. USA 103:19045–19050.Avlund, M., Dodd, I. B., Semsey, S., Sneppen, K., and Krishna, S. (2009a). Why do phage play

dice? J. Virol. 83:11416–11420.Avlund, M., Dodd, I. B., Sneppen, K., and Krishna, S. (2009b). Minimal gene regulatory

circuits that can count like bacteriophage lambda. J. Mol. Biol. 394:681–693.Babic, A. C., and Little, J. W. (2007). Cooperative DNA binding by CI repressor is dispensable

in a phage lambda variant. Proc. Natl. Acad. Sci. USA 104:17741–17746.Bakk, A. (2005). Transcriptional activation mechanisms of the PRM promoter of lambda

phage. Biophys. Chem. 114:229–234.Bandyopadhyay, K., Parua, P. K., Datta, A. B., and Parrack, P. (2010). Escherichia coliHflK and

HflC can individually inhibit the HflB (FtsH)-mediated proteolysis of lambdaCII in vitro.Arch. Biochem. Biophys. 501:239–243.

Berry, J., Savva, C., Holzenburg, A., and Young, R. (2010). The lambda spanin componentsRz and Rz1 undergo tertiary and quaternary rearrangements upon complex formation.Protein Sci. 19:1967–1977.

Brown, A. L., and Szybalski, W. (1985). Transcriptional antitermination activity of thesynthetic nut elements of coliphage lambda. I. Assembly of the nutR recognition sitefrom boxA and nut core elements. Gene 39:121–127.

Brouns, S. J., Jore, M. M., Lundgren, M., Westra, E. R., Slijkhuis, R. J., Snijders, A. P.,Dickman, M. J., Makarova, K. S., Koonin, E. V., and van der Oost, J. (2008). Small CRISPRRNAs guide antiviral defense in prokaryotes. Science 321:960–964.

Burmann, B. M., Luo, X., Rosch, P., Wahl, M. C., and Gottesman, M. E. (2010). Fine tuning ofthe E. coli NusB:NusE complex affinity to BoxA RNA is required for processiveantitermination. Nucleic Acids Res 38:314–326.

Cao, Y., Lu, H. M., and Liang, J. (2008). Stochastic probability landscape model for switchingefficiency, robustness, and differential threshold for induction of genetic circuit in phage.Conf. Proc. IEEE Eng. Med. Biol. Soc. pp. 611–614.

Cao, Y., Lu, H. M., and Liang, J. (2010). Probability landscape of heritable and robustepigenetic state of lysogeny in phage lambda. Proc. Natal. Acad. Sci. USA 107:18445–18450.

Chen, B., Xiao, Y., Liu, C., Li, C., and Leng, F. (2010). DNA linking number change inducedby sequence-specific DNA-binding proteins. Nucleic Acids Res. 38:3643–3654.

Chenoweth, M. R., and Wickner, S. (2008). Complex regulation of the DnaJ homolog CbpAby the global regulators sigmaS and Lrp, by the specific inhibitor CbpM, and by theproteolytic degradation of CbpM. J. Bacteriol. 190:5153–5161.

174 Grzegorz Wegrzyn et al.

Conant, C. R., Goodarzi, J. P., Weitzel, S. E., and vonHippel, P. H. (2008). The antiterminationactivity of bacteriophage lambda N protein is controlled by the kinetics of an RNA-looping-facilitated interaction with the transcription complex. J. Mol. Biol. 384:87–108.

Conant, C. R., Van Gilst, M. R., Weitzel, S. E., Rees, W. A., and von Hippel, P. H. (2005). Aquantitative description of the binding states and in vitro function of antiterminationprotein N of bacteriophage lambda. J. Mol. Biol. 348:1039–1057.

Court, D. L., Oppenheim, A. B., and Adhya, S. L. (2007a). A new look at bacteriophagelambda genetic networks. J. Bacteriol. 189:298–304.

Court, R., Cook, N., Saikrishnan, K., andWigley, D. (2007b). The crystal structure of lambda-Gam protein suggests a model for RecBCD inhibition. J. Mol. Biol. 371:25–33.

Datta, A. B., Panjikar, S., Weiss, M. S., Chakrabarti, P., and Parrack, P. (2005a). Structure oflambda CII: Implications for recognition of direct-repeat DNA by an unusual tetramericorganization. Proc. Natl. Acad. Sci. USA 102:11242–11247.

Datta, A. B., Roy, S., and Parrack, P. (2005b). Role of C-terminal residues in oligomerizationand stability of lambda CII: Implications for lysis-lysogeny decision of the phage. J. Mol.

Biol. 345:315–324.Datta, I., Banik-Maiti, S., Adhakari, L., Sau, S., Das, N., and Mandal, N. C. (2005c). The

mutation that makes Escherichia coli resistant to lambda P gene-mediated host lethality islocated within the DNA initiator Gene dnaA of the bacterium. J. Biochem. Mol. Biol.

38:89–96.Datta, I., Sau, S., Sil, A. K., and Mandal, N. C. (2005d). The bacteriophage lambda DNA

replication protein P inhibits the oriC DNA- and ATP-binding functions of the DNAreplication initiator protein DnaA of Eshcerichia coli. J. Biochem. Mol. Biol. 38:97–103.

Deighan, P., and Hochschild, A. (2007). The bacteriophage lambdaQ anti-terminator proteinregulates late gene expression as a stable component of the transcription elongationcomplex. Mol. Microbiol. 63:911–920.

Deighan, P., Diez, C. M., Leibman, M., Hochschild, A., and Nickels, B. E. (2008). Thebacteriophage lambda Q antiterminator protein contacts the beta-flap domain of RNApolymerase. Proc. Natl. Acad. Sci. USA 105:15305–15310.

Dewey, J. S., Savva, C. G., White, R. L., Vitha, S., Holzenburg, A., and Young, R. (2010).Micron-scale holes terminate the phage infection cycle. Proc. Natl. Acad. Sci. USA

107:2219–2223.Dodd, I. B., Shearwin, K. E., and Egan, J. B. (2005). Revisited gene regulation in bacteriophage

lambda. Curr. Opin. Genet. Dev. 15:145–152.Edgar, R., and Qimron, U. (2010). The Escherichia coli CRISPR system protects from

l lysogenization, lysogens, and prophage induction. J. Bacteriol. 192:6291–6294.Feiss, M., Reynolds, E., Schrock, M., and Sippy, J. (2010). DNA packaging by lambda-like

bacteriophages: Mutations broadening the packaging specificity of terminase, thelambda-packaging enzyme. Genetics 184:43–52.

Fogg, P. C., Rigden, D. J., Saunders, J. R., McCarthy, A. J., and Allison, H. E. (2011).Characterization of the relationship between integrase, excisionase and antirepressoractivities associated with a superinfecting Shiga toxin encoding bacteriophage. Nucleic

Acids Res. 39:2116–2129.Friedman, D. I., and Court, D. L. (2001). Bacteriophage lambda: Alive andwell and still doing

its thing. Curr. Opin. Microbiol. 4:201–207.Garufi, G., Minenkova, O., Lo Passo, C., Pernice, I., and Felici, F. (2005). Display libraries on

bacteriophage lambda capsid. Biotechnol. Annu. Rev. 11:153–190.Gedeon, T., Mischaikow, K., Patterson, K., and Traldi, E. (2008). Binding cooperativity in

phage lambda is not sufficient to produce an effective switch. Biophys. J. 94:3384–3392.Ghosh, D., Roy, K., Williamson, K. E., Srinivasiah, S., Wommack, K. E., and Radosevich, M.

(2009). Acyl-homoserine lactones can induce virus production in lysogenic bacteria:An alternative paradigm for prophage induction. Appl. Environ. Microbiol. 75:7142–7152.

Bacteriophage l 175

Glinkowska, M., Łos, J. M., Szambowska, A., Czyz, A., Całkiewicz, J., Herman-Antosiewicz, A., Wrobel, B., Wegrzyn, G., Wegrzyn, A., and Łos, M. (2010). Influenceof the Escherichia coli oxyR gene function on lambda prophage maintenance. Arch.

Microbiol. 192:673–683.Gottesman, M. E., and Weisberg, R. A. (2004). Little lambda, who made thee? Microbiol.

Mol. Biol. Rev. 68:796–813.Gregory, R., Saunders, V. A., and Saunders, J. R. (2010). Rule-based simulation of temperate

bacteriophage infection: Restriction-modification as a limiter to infection in bacterialpopulations. Biosystems 100:166–177.

Halder, S., Banerjee, S., and Parrack, P. (2008). Direct CIII-HflB interaction is responsible forthe inhibition of the HflB (FtsH)-mediated proteolysis of Escherichia coli sigma(32) bylambda CIII. FEBS J. 275:4767–4772.

Halder, S., Datta, A. B., and Parrack, P. (2007). Probing the antiprotease activity of lambda-CIII, an inhibitor of the Escherichia coli metalloprotease HflB (FtsH). J. Bacteriol.

189:8130–8138.Hanych, B., Kedzierska, S., Walderich, B., Uzna�nski, B., and Taylor, A. (1993). Expression of

the Rz gene and the overlapping Rz1 reading frame present at the right end of thebacteriophage lambda genome. Gene 129:1–8.

Hochschild, A., and Lewis, M. (2009). The bacteriophage lambda CI protein finds an asym-metric solution. Curr. Opin. Struct. Biol. 19:79–86.

Horiya, S., Inaba, M., Koh, C. S., Uehara, H., Masui, N., Ishibashi, M., Matsufuji, S., andHarada, K. (2009). Analysis of the spacial requirements for RNA-protein interactionswithin the N antitermination complex of bacteriophage lambda. Nucleic Acids Symp. Ser.

53:91–92.Jia, H., Satumba, W. J., Bidwell, G. L., 3rd, and Mossing, M. C. (2005). Slow assembly and

disassembly of lambda Cro repressor dimers. J. Mol. Biol. 350:919–929.Kedzierska, B., Glinkowska, M., Iwanicki, A., Obuchowski, M., Sojka, P., Thomas, M. S., and

Wegrzyn, G. (2003). Toxicity of the bacteriophage lambda cII gene product to Escherichiacoli arises from inhibition of host cell DNA replication. Virology 313:622–628.

Kedzierska, B., Lee, D. J., Wegrzyn, G., Busby, S. J., and Thomas, M. S. (2004). Role of theRNA polymerase alpha subunits in CII-dependent activation of the bacteriophagelambda pE promoter: Identification of important residues and positioning of the alphaC-terminal domains. Nucleic Acids Res. 32:834–841.

Kedzierska, B., Szambowska, A., Herman-Antosiewicz, A., Lee, D. J., Busby, S. J.,Wegrzyn, G., and Thomas, M. S. (2007). The C-terminal domain of the Escherichia coliRNA polymerase alpha subunit plays a role in the CI-dependent activation of thebacteriophage lambda pM promoter. Nucleic Acids Res. 35:2311–2320.

Kobiler, O., Rokney, A., Friedman, N., Court, D. L., Stavans, J., and Oppenheim, A. B. (2005).Quantitative kinetic analysis of the bacteriophage lambda genetic network. Proc. Natl.Acad. Sci. USA 102:4470–4775.

Kobiler, O., Rokney, A., and Oppenheim, A. B. (2007). Phage lambda CIII: A proteaseinhibitor regulating the lysis-lysogeny decision. PLoS One 2:e363.

Kontur, W. S., Saecker, R. M., Davis, C. A., Capp, M. W., and Record, M. T., Jr. (2006). Soluteprobes of conformational changes in open complex (RPo) formation by Escherichia coli

RNA polymerase at the lambdaPR promoter: Evidence for unmasking of the active site inthe isomerization step and for large-scale coupled folding in the subsequent conversionto RPo. Biochemistry 45:2161–2177.

Koster, S., Evilevitch, A., Jeembaeva, M., andWeitz, D. A. (2009). Influence of internal capsidpressure on viral infection by phage lambda. Biophys. J. 97:1525–1529.

Krupovic, M., and Bamford, D. H. (2008). Holin of bacteriophage lambda: Structural insightsinto a membrane lesion. Mol. Microbiol. 69:781–783.

176 Grzegorz Wegrzyn et al.

Kulczyk, A. W., Tanner, N. A., Loparo, J. J., Richardson, C. C., and van Oijen, A. M. (2010).Direct observation of enzymes replicating DNA using a single-molecule DNA stretchingassay. J. Vis. Exp. 37:1689.

Little, J. W. (2010). Evolution of complex gene regulatory circuits by addition of refinements.Curr. Biol. 20:R724–R734.

Little, J. W., and Michalowski, C. B. (2010). Stability and instability in the lysogenic state ofphage lambda. J. Bacteriol. 192:6064–6076.

Lou, C., Yang, X., Liu, X., He, B., and Ouyang, Q. (2007). A quantitative study of lambda-phage SWITCH and its components. Biophys. J. 92:2685–2693.

Łos, J. M., Łos, M., Wegrzyn, A., and Wegrzyn, G. (2008). Role of the bacteriophage lambdaexo-xis region in the virus development. Folia Microbiol. 53:443–450.

Łos, J. M., Łos, M., Wegrzyn, G., and Wegrzyn, A. (2009). Differential efficiency of inductionof various lambdoid prophages responsible for production of Shiga toxins in response todifferent induction agents. Microb. Pathog. 47:289–298.

Łos, J. M., Łos, M., Wegrzyn, A., and Wegrzyn, G. (2010). Hydrogen peroxide-mediatedinduction of the Shiga toxin-converting lambdoid prophage ST2-8624 in Escherichia coli

O157:H7. FEMS Immunol. Med. Microbiol. 58:322–329.Łyze�n, R., Kochanowska, M., Wegrzyn, G., and Szalewska-Pałasz, A. (2008). IHF- and SeqA-

binding sites, present in plasmid cloning vectors, may significantly influence activities ofpromoters. Plasmid 60:125–130.

Łyze�n, R., Kochanowska, M., Wegrzyn, G., and Szalewska-Palasz, A. (2009). Transcriptionfrom bacteriophage lambda pR promoter is regulated independently and antagonisti-cally by DksA and ppGpp. Nucleic Acids Res. 37:6655–6664.

Łyze�n, R., Wegrzyn, G., Wegrzyn, A., and Szalewska-Pałasz, A. (2006). Stimulation of thelambda pR promoter by Escherichia coli SeqA protein requires downstream GATCsequences and involves late stages of transcription initiation.Microbiology 152:2985–2992.

Maresca, M., Erler, A., Fu, J., Friedrich, A., Zhang, Y., and Stewart, A. F. (2010). Single-stranded heteroduplex intermediates in lambda Red homologous recombination. BMCMol. Biol. 11:54.

Martınez-Trujillo, M., Sanchez-Trujillo, A., Ceja, V., Avila-Moreno, F., Bermudez-Cruz, R. M., Court, D., and Montanez, C. (2010). Sequences required for transcriptiontermination at the intrinsic lambda t1 terminator. Can. J. Microbiol. 56:168–177.

Maynard, N. D., Birch, E. W., Sanghvi, J. C., Chen, L., Gutschow, M. V., and Covert, M. W.(2010). A forward-genetic screen and dynamic analysis of lambda phage host-dependen-cies reveals an extensive interaction network and a new anti-viral strategy. PLoS Genet. 6:e1001017.

Medina, E., Wieczorek, D., Medina, E. M., Yang, Q., Feiss, M., and Catalano, C. E. (2010).Assembly and maturation of the bacteriophage lambda procapsid: gpC is the viralprotease. J. Mol. Biol. 401:813–830.

Morelli, M. J., Ten Wolde, P. R., and Allen, R. J. (2009). DNA looping provides stability androbustness to the bacteriophage lambda switch. Proc. Natl. Acad. Sci. USA 106:8101–8106.

Mosberg, J. A., Lajoie, M. J., and Church, G. M. (2010). Lambda Red recombineering inEscherichia coli occurs through a fully single-stranded intermediate. Genetics 186:791–799.

Narajczyk, M., Bara�nska, S., Szambowska, A., Glinkowska, M., Wegrzyn, A., andWegrzyn, G. (2007a). Modulation of lambda plasmid and phage DNA replication byEscherichia coli SeqA protein. Microbiology 153:1653–1663.

Narajczyk, M., Bara�nska, S., Wegrzyn, A., and Wegrzyn, G. (2007b). Switch from theta tosigma replication of bacteriophage lambda DNA: Factors involved in process and amodel for its regulation. Mol. Genet. Genomics 278:65–74.

Nejman, B., Łos, J. M., Łos, M., Wegrzyn, G., andWegrzyn, A. (2009). Plasmids derived fromlambdoid bacteriophages as models for studying replication of mobile genetic elements

Bacteriophage l 177

responsible for the production of Shiga toxins by pathogenic Escherichia coli strains. J. Mol.

Microbiol. Biotechnol. 17:211–220.Nejman, B., Nadratowska-Wesołowska, B., Szalewska-Pałasz, A., Wegrzyn, A., and

Wegrzyn, G. (2011). Replication of plasmids derived from Shiga toxin-converting bacter-iophages in starved Escherichia coli. Microbiology 157:220–233.

Nickels, B. E., Roberts, C. W., Roberts, J. W., and Hochschild, A. (2006). RNA-mediateddestabilization of the sigma(70) region 4/beta flap interaction facilitates engagement ofRNA polymerase by the Q antiterminator. Mol. Cell 24:457–468.

Oppenheim, A. B., Kobiler, O., Stavans, J., Court, D. L., and Adhya, S. (2005). Switches inbacteriophage lambda development. Annu. Rev. Genet. 39:409–429.

Papagiannis, C. V., Sam, M. D., Abbani, M. A., Yoo, D., Cascio, D., Clubb, R. T., andJohnson, R. C. (2007). Fis targets assembly of the Xis nucleoprotein filament to promoteexcisive recombination by phage lambda. J. Mol. Biol. 367:328–343.

Parua, P. K., Datta, A. B., and Parrack, P. (2010a). Specific hydrophobic residues in the alpha4helix of lambda CII are crucial for maintaining its tetrameric structure and directing thelysogenic choice. J. Gen. Virol. 91:306–312.

Parua, P. K., Mondal, A., and Parrack, P. (2010b). HflD, an Escherichia coli protein involved inthe lambda lysis-lysogeny switch, impairs transcription activation by lambda CII. Arch.Biochem. Biophys. 493:175–183.

Poteete, A. R. (2008). Involvement of DNA replication in phage lambda Red-mediatedhomologous recombination. Mol. Microbiol. 68:66–74.

Potrykus, K., Wegrzyn, G., and Hernandez, V. J. (2004). Direct stimulation of the lambdapaQpromoter by the transcription effector guanosine-30,50-(bis)pyrophosphate in a definedin vitro system. J. Biol. Chem. 279:19860–19866.

Prasch, S., Jurk, M., Washburn, R. S., Gottesman, M. E., Wohrl, B. M., and Rosch, P. (2009).RNA-binding specificity of E. coli NusA. Nucleic Acids Res 37:4736–4742.

Prasch, S., Schwarz, S., Eisenmann, A., Wohrl, B. M., Schweimer, K., and Rosch, P. (2006).Interaction of the intrinsically unstructured phage lambda N protein with Escherichia coliNusA. Biochemistry 45:4542–4549.

Ptashne, M. (2005). Regulation of transcription: From lambda to eukaryotes. Trends Biochem.

Sci. 30:275–279.Ptashne, M. (2006). Lambda’s switch: Lessons from amodule swap. Curr. Biol. 16:R459–R462.Refardt, D., and Rainey, P. B. (2010). Tuning a genetic switch: Experimental evolution and

natural variation of prophage induction. Evolution 64:1086–1097.Rokney, A., Kobiler, O., Amir, A., Court, D. L., Stavans, J., Adhya, S., and Oppenheim, A. B.

(2008). Host responses influence on the induction of lambda prophage. Mol. Microbiol.

68:29–36.Rosenfeld, N., Young, J. W., Alon, U., Swain, P. S., and Elowitz, M. B. (2005). Gene regulation

at the single-cell level. Science 307:1962–1965.Schubert, R. A., Dodd, I. B., Egan, J. B., and Shearwin, K. E. (2007). Cro’s role in the CI Cro

bistable switch is critical for l’s transition from lysogeny to lytic development. Genes Dev.

21:2461–2472.Shao, Y., and Wang, I. N. (2009). Effect of late promoter activity on bacteriophage lambda

fitness. Genetics 181:1467–7145.Shkilnyj, P., and Koudelka, G. B. (2007). Effect of salt shock on stability of limm434 lysogens.

J. Bacteriol. 189:3115–3123.Stayrook, S., Jaru-Ampornpan, P., Ni, J., Hochschild, A., and Lewis, M. (2008). Crystal

structure of the lambda repressor and a model for pairwise cooperative operator binding.Nature 452:1022–1025.

Sun, X., Mierke, D. F., Biswas, T., Lee, S. Y., Landy, A., and Radman-Livaja, M. (2006).Architecture of the 99 bp DNA-six-protein regulatory complex of the lambda att site.Mol. Cell. 24:569–580.

178 Grzegorz Wegrzyn et al.

Svenningsen, S. L., Costantino, N., Court, D. L., and Adhya, S. (2005). On the role of Cro inlambda prophage induction. Proc. Natl. Acad. Sci. USA 102:4465–4469.

Szambowska, A., Pierechod, M., Wegrzyn, G., and Glinkowska, M. (2011). Coupling oftranscription and replication machineries in l DNA replication initiation: Evidence fordirect interaction of Escherichia coli RNA polymerase and lO protein. Nucleic Acids Res.

39:168–177.Tay, Y., Ho, C., Droge, P., and Ghadessy, F. J. (2010). Selection of bacteriophage lambda

integrases with altered recombination specificity by in vitro compartmentalization.Nucleic Acids Res. 38:e25.

Taylor, A., Benedik, M., and Campbell, A. (1983). Location of the Rz gene in bacteriophagelambda. Gene 26:159–163.

Thomason, L., Court, D. L., Bubunenko, M., Costantino, N., Wilson, H., Datta, S., andOppenheim, A. (2007). Recombineering: Genetic engineering in bacteria using homolo-gous recombination. Curr. Protoc. Mol. Biol. Chapt. 1, 1.16.

Valdez-Cruz, N. A., Caspeta, L., Perez, N. O., Ramirez, O. T., and Trujillo-Roldan, M. A.(2010). Production of recombinant proteins in E. coli by the heat inducible expressionsystem based on the phage lambda pL and/or pR promoters. Microb. Cell Fact 9:18.

Van Duyne, G. D. (2005). Lambda integrase: Armed for recombination. Curr. Biol. 15:

R658–R660.Wang, H., Finzi, L., Lewis, D. E., and Dunlap, D. (2009). AFM studies of lambda repressor

oligomers securing DNA loops. Curr. Pharm. Biotechnol. 10:494–501.Weigel, C., and Seitz, H. (2006). Bacteriophage replication modules. FEMS Microbiol. Rev.

30:321–381.Wegrzyn, G., and Wegrzyn, A. (2005). Genetic switches during bacteriophage lambda

development. Prog. Nucleic Acid Res. Mol. Biol. 79:1–48.Westra, E. R., Pul, U., Heidrich, N., Jore, M. M., Lundgren, M., Stratmann, T., Wurm, R.,

Raine, A., Mescher, M., Van Heereveld, L., Mastop, M., Wagner, E. G., Schnetz, K., VanDer Oost, J., Wagner, R., and Brouns, S. J. (2010). H-NS-mediated repression of CRISPR-based immunity in Escherichia coli K12 can be relieved by the transcription activatorLeuO. Mol. Microbiol. 77:1380–1393.

White, R., Chiba, S., Pang, T., Dewey, J. S., Savva, C. G., Holzenburg, A., Pogliano, K., andYoung, R. (2011). Holin triggering in real time. Proc. Natl. Acad. Sci. USA 108:798–803.

Wu, D., Van Valen, D., Hu, Q., and Phillips, R. (2010). Ion-dependent dynamics of DNAejections for bacteriophage lambda. Biophys. J. 99:1101–1109.

Yang, Q., Catalano, C. E., and Maluf, N. K. (2009). Kinetic analysis of the genome packagingreaction in bacteriophage lambda. Biochemistry 48:10705–10715.

Zhang, Y., Thompson, R., and Caruso, J. (2011). Probing the viral metallome: Searching formetalloproteins in bacteriophage l: The hunt begins. Metallomics 3:472–481.

Zhou, Y., Shi, T., Mozola, M. A., Olson, E. R., Henthorn, K., Brown, S., Gussin, G. N., andFriedman, D. I. (2006). Evidence that the promoter can influence assembly of antitermi-nation complexes at downstream RNA sites. J. Bacteriol. 188:2222–2232.

Zong, C., So, L. H., Sepulveda, L. A., Skinner, S. O., and Golding, I. (2010). Lysogen stability isdetermined by the frequency of activity bursts from the fate-determining gene.Mol. Syst.Biol. 6:440.