The Protein Interaction Network of Bacteriophage Lambda with ItsHost, Escherichia coli

Sonja Blasche,a Stefan Wuchty,b* Seesandra V. Rajagopala,c Peter Uetzd

Genomics and Proteomics Core Facilities, German Cancer Research Center, Heidelberg, Germanya; National Center of Biotechnology Information, National Institutes ofHealth, Bethesda, Maryland, USAb; J. Craig Venter Institute, Rockville, Maryland, USAc; Center for the Study of Biological Complexity, Virginia Commonwealth University,Richmond, Virginia, USAd

Although most of the 73 open reading frames (ORFs) in bacteriophage � have been investigated intensively, the function ofmany genes in host-phage interactions remains poorly understood. Using yeast two-hybrid screens of all lambda ORFs forinteractions with its host Escherichia coli, we determined a raw data set of 631 host-phage interactions resulting in a set of62 high-confidence interactions after multiple rounds of retesting. These links suggest novel regulatory interactions be-tween the E. coli transcriptional network and lambda proteins. Targeted host proteins and genes required for lambda in-fection are enriched among highly connected proteins, suggesting that bacteriophages resemble interaction patterns ofhuman viruses. Lambda tail proteins interact with both bacterial fimbrial proteins and E. coli proteins homologous toother phage proteins. Lambda appears to dramatically differ from other phages, such as T7, because of its unusually largenumber of modified and processed proteins, which reduces the number of host-virus interactions detectable by yeast two-hybrid screens.

More than 60 years ago, Esther Lederberg discovered phagelambda (1). Since this seminal discovery, lambda has be-

come a model organism, contributing to our current understand-ing of the ways genes work and viruses take control of their hosts.However, phage lambda is still far from being completely under-stood given that the function of several genes in its 48.5-kb ge-nome remain only vaguely defined. For instance, 14 of 73 pre-dicted lambda proteins are still largely uncharacterized (2).

Some of the best-characterized aspects of lambda biology arethe genetic switch that determines whether a phage lyses the cell orintegrates into its host genome as a prophage and the mechanismof transcriptional antitermination (3). Also, lambda continues toprovide new insights into its gene regulatory circuits (4, 5), withrecent studies of its DNA packaging motor as the vanguard ofnanomotor research (6).

Key to lambda biology is a detailed understanding of thephage’s proteins, including their interactions. We recently ana-lyzed 95 protein-protein interactions (PPIs) between �70 lambdaproteins, capturing 16 of 33 previously published interactions (2).Although protein interactions are reasonably well characterizedwithin the virus particle, host-phage interactions remain incom-pletely known during the replication and recombination stages ofthe lambda life cycle in the host cell. In addition, the moleculardetails of virion assembly are still largely mysterious. Altogether,about 30 host-lambda interactions have been discovered in thepast 50 years (Table 1).

To improve our understanding of lambda PPIs, we have cloned68 lambda open reading frames (ORFs) (2) and performed screensagainst a pooled Escherichia coli library using a multivector yeasttwo-hybrid (Y2H) strategy. Among a total of 631 raw interactions,we found 244 reproducible interactions. Furthermore, we con-firmed 162 interactions in an additional round of retesting, allow-ing us to obtain an overlapping set of 62 high-confidence interac-tions. Based on our analysis of this set of phage-host interactions,we show that lambda preferably targets highly connected E. coliproteins, indicating that the phage affects central host proteins.

Utilizing a set of genes that are necessary for phage infection in E.coli, we elucidated a regulatory subnetwork that suggests new waysthe phage interferes with host transcription factors to take controlof the cell.

MATERIALS AND METHODSYeast two-hybrid screens. For the yeast two-hybrid screens, 68 phagelambda ORFs were cloned into the bait vectors pGBKT7g and pDEST32(2) and screened against the E. coli W3110 library (7) in the prey vectorspGADT7g and pDEST22 (8). As a result, we obtained two bait-prey com-binations, pGBKT7g-pGADT7g and pDEST32-pDEST22. The screenswere performed in three runs, each including the lambda baits once ormultiple times against one library using one screening method (Table 2and Fig. 1).

In the first run, arrayed pool screens were performed using the wholeE. coli pGADT7g library arranged on two plates, where each well con-tained a different prey pool of 24 clones. Each bait was mated with eachprey pool, and the diploids were arrayed on selective agarose plates. A flowchart of the procedure is shown in Fig. 1. In the second and third runs thelambda bait proteins were screened against pooled E. coli pGADT7g andpDEST22 libraries, respectively. The diploid yeasts were plated on selec-tive agarose plates, and prey plasmids were identified by colony PCR andsequencing.

The pGBKT7g/pGADT7g and pDEST32/22 vector systems differmainly by copy number and, as a consequence, in the amount of pro-

Received 29 August 2013 Accepted 10 September 2013

Published ahead of print 18 September 2013

Address correspondence to Peter Uetz, peter@uetz.us.

* Present address: Stefan Wuchty, Department of Computer Science, University ofMiami, Coral Gables, Florida, USA.

S.B. and S.W. contributed equally to this article.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/JVI.02495-13.

Copyright © 2013, American Society for Microbiology. All Rights Reserved.

doi:10.1128/JVI.02495-13

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tein expressed in the yeast cell. As previously shown (9), the number ofobtained screening hits varies clearly between the vector systems, andthe number of common pairs detected in both systems is usually low.However, the two vector systems complement each other, suggestingthat using both led to an increase in identified protein-protein inter-actions.

Promiscuous baits. Potentially promiscuous baits were identified byusing two different approaches: (i) Binary tests of all baits against emptyprey vector and (ii) generation of a “bait promiscuity factor” using pair-

wise yeast Y2H tests of phage baits against 184 preys that were found inour initial screen. These preys were retested to make sure that they arereproducible. Some of the baits turned out to show nearly random inter-actions and were therefore discarded.

In the first approach, the lambda baits in pGBKT7g and pDEST32 baitvectors were tested against empty pGADT7g and pDEST22 prey vectors(i.e., without prey protein), respectively. This step resulted in the identi-fication of four random activators (lambdap18 � gpL, lambdap29 � Tfa,lambdap47 � Orf28, and lambdap67 � NinF) in the pGBKT7g/pGADT7g vector system and five random activators (lambdap05 � gpC,lambdap06 � Nu3, lambdap29 � Tfa, lambdap47 � Orf28, and lamb-dap67 � NinF) in the pDEST32/22 system. Note that lambdap29 (Tfa),lambdap47 (Orf28), and lambdap67 (NinF) yielded positive results(“pseudo-interactions”) with empty vectors in both systems. All baits pro-ducing “interactions” with empty prey vector were deemed nonscreenableand were thus excluded.

In the second approach, pairwise tests of phage lambda bait pro-teins against different prey proteins suggested that lambdap16 (gpH),

TABLE 1 Previously published lambda-host PPIsa

Function/role and PPI � Hostb Description Source or reference

Transcription1 CI RecA RecA degrades CIc 262 CI RpoA 273 CI RpoD 28, 294 CII ClpYQ ClpYQ degrades CII in vitro 265 CII ClpAP ClpAP degrades CII in vitro 266 CII HflD HflD makes CII more vulnerable to FtsH (hfl � high-frequency lysogenization) 307 CII HflB* HflB (� FtsH) protease degrades CII 31, 328 CII RpoA 33, 349 CII RpoD 3310 CIII HflB CIII inhibits HflB; HflB degrades CIII 26, 3511 gpN NusA Transcriptional regulation 3612 gpN Lon Lon degrades gpN 26, 3713 gpQ �70 gpQ makes RNAP insensitive to cis termin 29, 34, 38, 39

Head14 gpB GroE Genetic interaction; E. coli GroE 40–4215 gpE GroE Genetic interaction 41

Tail16 gpJ LamB LamB is the E. coli receptor 43–4717 Stf OmpC OmpC is a secondary E. coli receptor 48

Recombination18 Xis Lon Xis is degraded by E. coli Lon protease 4919 Xis FtsH Xis is degraded by E. coli FtsH protease 4920 Xis Fis Both required for excision 50–5221 Int IHF Both catalyze recombination at attP/attB 53, 5422 Gam SbcC SbcCD is a dsDNA exonuclease � ssDNA endonuclease 5523 Gam RecB Gam inhibits RecBCD 5624 NinB SSB NinB also binds ssDNA 5725 Ral Hsd Ral inhibits restriction enzyme complex HsdMSR (by binding to HsdM or S) 58

Replication26 gpO ClpXP ClpXP degrades gpO 2627 gpO DnaK 5928 gpO RpoB 6029 gpP DnaA 6130 gpP DnaB 6231 gpP DnaK 59

a Modified from the study by Häuser et al. (15). CbpA (63) may interact with DnaA and SeqA (64–66).b Boldfacing indicates hosts required for infection (12). Underlining indicates host proteins found in our screen. *, HflB � FtsH.c After DNA damage, RecA bound to single-stranded DNA (ssDNA) stimulates autocleavage of the CI repressor (26). dsDNA, double-stranded DNA.

TABLE 2 Summary of screening procedures for lambda bait against E.coli prey libraries

Bait/bait vector/prey vector Property Prey library Screen type

� ORF/pGBKT7g/pGADT7g High copy E. coli W3110 Arrayed pool� ORF/pGBKT7g/pGADT7g High copy E. coli W3110 Prey pool� ORF/pDEST32/pDEST22 Low copy E. coli W3110 Prey pool

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lambdap36 (Ea8.5), lambdap45 (Ea10), lambdap65 (NinD), andlambdap83 (Ea22) were putative random activators. A bait protein wasdefined as a random activator if it bound to more than 30 of 184 preysthat were not identified previously in the phage/host pool screens.

According to their tendency to interact randomly, a promiscuity scoreranging from 1 (nonpromiscuous) to 3 was assigned to all screenable baitproteins. Baits of categories 2 (lambdap16 � gpH, lambdap36 � Ea8.5,and lambdap83 � Ea22) and 3 (lambdap45 � Ea10 and lambdap65 �NinD) had �45 and �60 host interaction partners, respectively. No pro-tein had between 45 and 60 partners.

Promiscuous preys. A prey protein was tagged as potentially promis-cuous or “sticky” if it interacted with five or more different lambda baitsreproducibly and/or if it interacted randomly in pairwise yeast two-hybridtests. A list of potential promiscuous preys and the number of phagelambda baits they interacted with is provided in Table S1 in the supple-mental material.

Most promiscuous preys were found by the pGBKT7g/pGADT7g vec-tor system. The pDEST system revealed only interactions with two stickyproteins, LeuB and YajI, both of which were found as single hits. Weacknowledge that the pGBKT7g/pGADT7g system is less stringent thanthe pDEST system, since it also detected most of the interactions reportedin reference 10. Prey proteins identified as promiscuous were excludedfrom further evaluation.

Verification of single hits by pairwise Y2H assays. 144 interactinglambda-host pairs were found as multiple hits in our initial screens. Thisset and selected additional interactions from the initial screen were re-tested using binary Y2H tests. Out of these 162 were tested positive, yield-ing an overlap of 62 “high-confidence” interactions (see Table S1 in thesupplemental material). The retests were performed as previously de-scribed by Chen et al. (8). A summary of all screens is provided in Table 3.

Molecular interactions data of E. coli. We utilized 3,559 experimen-tally determined, physical interactions between 1,987 proteins of E. coli(S. V. Rajagopala et al., unpublished data). As for regulatory interactions,we utilized data from the RegulonDB database (11). In particular, we used

FIG 1 Screening strategy. (A) Arrayed pool screening. (B) Typical array screen with positive yeast colonies indicated by arrows. The plates are in 384 format, andeach prey pool is pinned as quadruplicate. (C) We cloned 68 lambda ORFs into two Y2H bait vectors and screened them against two Y2H prey libraries (one inpDEST22 and one in pGADT7g). All screening results were pooled, yielding 631 raw interactions. After the removal of 487 “low-confidence” interactions thatinvolved promiscuous baits and preys and another round of retesting, we obtained 244 interactions. Furthermore, we retested the initial raw set of interactions,allowing us to confirm 162 interactions, yielding a set of 62 “high-confidence” interactions.

TABLE 3 Numeric results of phage lambda-host pool screens

Screen type Parameter No.

E. coli prey proteins interacting with lambda Total 294Promiscuous 25

Unique bait-prey pairs Total 631Single hits 334Multiple hits 144With promiscuous prey 218

Unique bait-prey pairs, pDEST32/22 only Total 56Single hits 38High-confidence hits 20

Unique bait-prey pairs,pGBKT7g/pGADT7g only

Total 573

Single hits 296High-confidence hits 122

Unique bait-prey pairs found in bothpDEST32/22 and pGBKT7g/pGADT7g

2

Retested individually 162

Total High-confidence hits 62

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4,442 regulatory interactions between 187 transcription factors and 1,638genes in E. coli that RegulonDB collected from the literature.

Functional groups. All lambda-phage proteins and corresponding E.coli interactors were assigned to 9 and 14 different functional groups,respectively. As for lambda, these classes included virion head, virion tail,superinfection exclusion, transcription, replication, recombination, lysis,inhibition of host replication, and proteins of unknown function. Thecategories for E. coli involved biosynthesis of cofactors prosthetic groupsand carriers, cell envelope, phage origin, cellular processes, DNA metab-olism, energy metabolism, fimbrial proteins, protein fate, protein synthe-sis, regulatory functions, transcription, transport and binding proteins,unknown/uncharacterized/hypothetical proteins, and others with knownfunction. Functional annotations of phage lambda were obtained fromthe reference proteome set in Uniprot (http//www.uniprot.org). Func-tional groups of E. coli were obtained from EcoGene 3.0 (http://ecogene.org).

Phage-host dependencies and essential genes. We used a set of 57genes of E. coli that are required for phage infection as assessed by mea-suring their effect on � replication when knocked out in E. coli (12). A totalof 705 essential E. coli genes were obtained from the Database of EssentialGenes (DEG) (13).

Enrichment analysis. We grouped proteins of E. coli according totheir number of interaction partners in an underlying protein interactionsnetwork. We represented each group by N�k proteins that had at least kinteractions. In each group we calculated the number of proteins with acertain feature i such as being targeted by the phage or required for theinfection process Ni,�k. Randomly assigning feature i to proteins we de-

fined Ei,�k �Ni,�k

Ni,�kr as the enrichment of proteins with feature i, where

Ni,�kr is the corresponding random number of proteins with feature i

among all Ni,�k proteins. After averaging Ei over 10,000 randomizations,Ei � 1 pointed to an enrichment and vice versa, whereas Ei � 1 indicateda random process (14).

RESULTSInteractions of phage lambda with its host: overview. Over thepast several decades, about 30 interactions between proteins ofphage lambda and its host have been identified by using a widerange of approaches and studies (15) (Table 1). After our success-ful study of lambda-lambda interactions (2), we decided to use asimilar approach to screen all lambda ORFs against a library of E.coli clones using the yeast two-hybrid system. In short, wescreened each lambda ORF as Y2H bait against an array of E. colipreys, as well as a pooled library of prey clones (Fig. 1 and Table 2;see Materials and Methods for details). These screens provided araw data set of 631 unique bait-prey pairs, involving 294 distinct E.coli proteins. A total of 244 of these interactions were found inmultiple hits and, after removal of promiscuous proteins and re-peated retesting, we finally obtained a set of 62 “high-confidence”interactions (Table 4; see Table S1 in the supplemental material).

As expected (10), different Y2H vectors resulted in very differ-ent interactions: 573 raw pairs were found using the pGBKT7g/pGADT7g vector system, while the pDEST32/22 system yieldedonly 56 interactions. Two were found in both systems (lambdagpG-E. coli ClpP and lambda gpA-E. coli NohB), and their signif-icance is discussed below. The results of all phage lambda-hostscreens are summarized in Table 4 and Table S1 in the supplemen-tal material.

Phage-phage versus phage-host interactions and genome or-ganization. In an interaction network of phage proteins, we previ-ously observed that structural proteins of the virion were involved inmost interactions (2). In contrast, patterns of interactions of phageproteins with their corresponding host proteins are less clear

(Fig. 2A). Most structural proteins do not appear to interact with hostproteins, except for gpH and gpG, the tape measure and the tail as-sembly chaperone, respectively. Furthermore, the proteins encodedby the early left operon and early right operon significantly interactwhile the structural proteins of the late operon have only few interac-tions with the host.

Interactions among functional groups. The 62 high-confi-dence phage lambda-host interactions were combined in acomplete network along with 31 previously known interac-tions. A comparison of these two sets indicated an overlap ofone interaction. Furthermore, we annotated each host andphage protein with its corresponding function (Fig. 2B). Un-expectedly, we found few clear patterns, indicating that only afew phage proteins interacted with very specific groups of tar-get proteins. Most notably, the E. coli group “transcription”(NusA, RpoABD, RpoS, Fis, and ihfAB) was targeted by phagelambda proteins involved in transcription (gpN, CI), replica-tion (Xis, gpO, and gpP), recombination (Int) and lysis (CII),whereas lambda transcription factors (gpN and gpQ) targetedmost functional groups of the host. Similarly, many proteinsinvolved in phage transcription interacted with host proteinsinvolved in energy metabolism. Interestingly, an E. coli fimbrialprotein, YehD, was exclusively linked to phage lambda proteinsthat were involved in virion tail formation such as the tapemeasure protein gpH. Even though host fimbrial proteins donot have any homologs in lambda, the tape measure proteinappears to be capable of selectively targeting host fimbrial pro-teins. We also found that the E. coli group “protein synthesis”was targeted mainly by the putative single strand binding pro-tein Ea10, a protein that we observed to bind the ribosomalproteins RpmA and RpsG, as well as the ribosome modulationfactor Rmf. “Transport and binding” host proteins interactedmainly with virion tail proteins. Given that transport proteinsare expected to be transmembrane proteins, these proteins mayhelp the phage to attach to the host cells. Interestingly, gpHappears to be injected with the DNA and thus may interact withintracellular proteins. Finally, proteins of unknown function(or hypothetical proteins) of both host and phage were in-volved in interactions with several functional categories. Still,most functionally unknown proteins of the host interactedwith unknown phage proteins.

Protein-protein interactions and host factors required forlambda infection. Maynard et al. (12) screened the E. coli KeioCollection (16) and found 57 host proteins that are required forlambda infection and reproduction. However, only four ofthese have been previously known to physically interact withlambda proteins, namely, LamB, the lambda receptor, IhfAB,the integration host factor, and HflD, which interacts with theCII protein, catalyzing its degradation and DNA bindingability.

In the combined network of high-confidence and previouslyknown phage lambda-host interactions, we found that lambdasignificantly targeted a total of five genes that are necessary forphage infection (P � 1.4 � 103, Fisher’s exact test). However, thesignificance of these interactions is not clear. For instance, HldD isinvolved in the synthesis of the lipopolysaccharide (LPS) and isrequired for lambda to infect its host (12). In our set of 62 high-confidence host-phage interactions, HldD (� RfaD) interactswith gpQ, a phage protein involved in transcription antitermina-tion (Fig. 3).

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Phage lambda targets essential E. coli genes and genes in-volved in transcription. It has been suggested that both bacterialand viral proteins tend to interact preferentially with highly con-nected host proteins, so-called hubs (17). Similarly, such hubshave been proposed to be more likely to be essential for a cell (18).In the combined network we observed a similar significant enrich-

ment among the 22 essential genes in E. coli targeted by lambda(P � 4.7 � 104, Fisher’s exact test). Ten E. coli genes that werepreviously known to interact with eight lambda proteins areessential, whereas twelve essential host genes were found tointeract with six phage proteins in our screens (Fig. 3). Further-more, we observed that lambda targeted nine transcription fac-

TABLE 4 High-confidence interactions in this studya

� locus no. � locus E. coli protein Annotation

lambdap01 nu1 DcrB Conserved protein involved in bacteriophage adsorptionlambdap02 A NohA Bacteriophage DNA packaging proteinlambdap02 A NohB Bacteriophage DNA packaging proteinlambdap09 Fi YdgH Conserved hypothetical proteinlambdap09 Fi FixB Probable flavoprotein subunit required for carnitine metabolismlambdap09 Fi MinE Cell division topological specificity factor MinElambdap09 Fi CchB Ethanolamine utilization EutNlambdap14 G ClpP ATP-dependent Clp protease, proteolytic subunit ClpPlambdap14 G FhuF Ferric iron reductase protein FhuFlambdap14 G FdoH Formate dehydrogenase, beta subunitlambdap14 G YohN Conserved hypothetical proteinlambdap14 G ChaC Cation transport protein ChaClambdap14 G ProQ ProP effectorlambdap14 G YliL Hypothetical proteinlambdap16 H YliL Hypothetical proteinlambdap16 H YeiW Proteinase inhibitorlambdap16 H YfcQ Conserved hypothetical proteinlambdap16 H YohH Predicted membrane proteinlambdap16 H YehD Fimbrial proteinlambdap33 int NohB Bacteriophage DNA packaging proteinlambdap36 ea8.5 YjdI Conserved hypothetical proteinlambdap36 ea8.5 MinE Cell division topological specificity factor MinElambdap36 ea8.5 YeiW Proteinase inhibitorlambdap38 orf63 YqhC Putative HTH-type transcriptional regulator YqhClambdap45 ea10 Rmf b0953 ribosome modulation factor (rmf)lambdap45 ea10 RpmA Ribosomal protein L27lambdap45 ea10 YliL Hypothetical proteinlambdap45 ea10 RpsG Ribosomal protein S7lambdap45 ea10 PriC Primosomal replication Nlambdap45 ea10 CobB NAD-dependent deacetylaselambdap45 ea10 SoxS Regulatory protein SoxSlambdap45 ea10 YcbG Cell division protein MatPlambdap49 N Hcr NADH oxidoreductase Hcrlambdap49 N NuoG NADH-quinone oxidoreductase, chain Glambdap49 N YebR Free methionine-(R)-sulfoxide reductaselambdap49 N NusA Transcription elongation NusAlambdap49 N EnvR Probable acrEF/envCD operon repressorlambdap49 N MinC Septum site-determining protein MinClambdap49 N RpoS RNA polymerase sigma factor RpoSlambdap49 N YcbG Cell division protein MatPlambdap61 P AtpC ATP synthase F1, epsilon subunitlambdap61 P EutC Ethanolamine ammonia-lyase, light chainlambdap61 P YcbG Cell division protein MatPlambdap61 P YqhC Putative HTH-type transcriptional regulator YqhClambdap65 NinD PyrF Orotidine 5=-phosphate decarboxylaselambdap65 NinD YhdW General L-amino acid-binding periplasmic protein AapJlambdap65 NinD MinE Cell division topological specificity factor MinElambdap65 NinD Slp Outer membrane protein Slplambdap65 NinD SmpA Small protein Alambdap65 NinD YjdI Conserved hypothetical proteinlambdap65 NinD CsrA Carbon storage regulatorlambdap65 NinD HycG Hydrogenase-4 component Ilambdap65 NinD SdiA Regulatory protein SdiAlambdap65 NinD SoxS Regulatory protein SoxSlambdap65 NinD YebR Free methionine-(R)-sulfoxide reductaselambdap71 Q GlyQ Glycyl-tRNA synthetase, alpha subunitlambdap71 Q YqhC Putative HTH-type transcriptional regulator YqhClambdap71 Q PaaC Phenylacetate-CoA oxygenase, PaaI subunitlambdap71 Q RfaD ADP-L-glycero-D-manno-heptose-6-epimeraselambdap75 R FhuF Ferric iron reductase protein FhuFlambdap75 R CaiF Transcriptional activatory protein CaiFlambdap79 YbcW Conserved hypothetical proteina The protein interactions from this publication have been submitted to the IMEx (http://www.imexconsortium.org) consortium through IntAct (67) and assigned the identifierIM-21394.

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tors (1.7 � 103, Fisher’s exact test). In particular, four transcrip-tion factors interacting with three lambda proteins were found inpreviously published interactions. In turn, our screens detectedfive transcription factors that were targeted by six lambda proteins(Fig. 3).

Interaction of lambda with lambda homologs in the E. coligenome. Many E. coli proteins derive from prophages and, whilemany of these proteins have mutated or become pseudogenes,some still seem to be active (19). Utilizing all obtained host-phageinteractions, we found seven E. coli proteins of phage origin thatwere involved in 10 phage-host interactions (Table 5). Six of theseseven proteins were highly similar to phage lambda proteins witha sequence similarity of up to 100%. Previously, lambda gpA wasfound to interact with lambda Nu1 and Orf79 interacted withitself (2). Since all three proteins have E. coli homologs of phageorigin, it was not surprising that the lambda proteins also interactwith the cellular homologues of Nu1 (E. coli NohA and NohB) andOrf79 (E. coli YbcW), respectively (Table 5). The Nu1 homologueNohA was also found to interact with the phage antiterminatorgpN. Even though the biological relevance of this interaction re-mains unclear, binding of gpN to lambda Nu1 has been previouslyreported (2).

Interactions of the phage lambda defective tail fiber proteinsOrf314 and Orf401 with the host-encoded tail fiber assembly pro-teins TfaR and TfaQ and the tail fiber protein StfR, respectively,appear plausible. In turn, the biological relevance of other inter-actions remain obscure, such as lambda integrase-NohB, lambda

gpO-TfaQ, and gpP-YdaG, given that phage lambda has no ho-mologues of YdaG, a protein of Rac prophage origin.

Topological properties of the phage-host interaction net-work. To investigate the location of targeted proteins, we analyzeda network of protein-protein interactions of E. coli. Utilizing thecombined network of high-confidence and previously knownhost-phage interactions, we calculated the enrichment of targetedproteins as a function of their degree (Fig. 4A), suggesting thathost proteins with an increased number of interaction partners areprime targets for the phage. Similarly, genes that are required forthe phage infection are enriched among higher connected hostproteins as well.

As a corollary to the observed phage’s preference to target cen-tral positions in the protein interaction network of E. coli, wehypothesized that targeted proteins allow the pathogen to reachother proteins efficiently. In particular, we calculated shortestpaths from reference proteins to remaining proteins in the under-lying network of interaction between E. coli proteins. Figure 4Bsuggests that the lengths of paths from targeted proteins to allother proteins are significantly shorter than the correspondingpaths from required (P 1010, Student’s t test) and nontargetedand nonrequired proteins (P 1010), respectively. As for therelative placement of targeted proteins and genes that lambda re-quires for its infection and proliferation, we calculated shortestpaths between such sets of proteins. In Fig. 4C, we show that thedistribution of path lengths between targeted and required geneswas significantly shifted to smaller values than paths from targeted

FIG 2 Core E. coli-lambda interactome organized by function. (A) Protein interactions mapped onto the lambda genome. All previously published (green bars),244 final (orange bars), and 62 “high-confidence” interactions (red bars) between proteins of E. coli and the lambda phage were plotted along the lambda genomefor each protein. Furthermore, we labeled the functional groups of genes (red, structural proteins; green, regulatory proteins; blue, DNA replication; yellow, lysis)and indicated the interactions between lambda proteins. (B) We augmented the network of 62 “high-confidence” protein interactions with more than 30previously published interactions (green arrows). All proteins are colored according to their functional annotations.

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proteins to nonrequired, other proteins (P � 2.9 � 104, Studentt test).

Interaction of protein-protein and gene regulatory net-works. We utilized PPIs and regulatory interactions between tran-scription factors and their target genes in E. coli to identify pathsfrom targeted genes to genes that are required for the infection ofthe phage. Specifically, we observed five required host genes thatare directly targeted by phage PPIs (hflD, hldD, ihfAB, and lamB).To control their expression, we identified the shortest path fromrequired genes to targeted genes that started with a transcriptionfactor-gene interaction, prompting us to focus on 27 requiredgenes involving 78 shortest paths. In Fig. 5, we show that all re-quired genes involved in LamB regulation were either directly

targeted (lamB) or were under the control of transcription factorCrp (malT, malI, and cyaA). As for other genes that play a role inthe ability of the phage to penetrate the cell, Crp controls theexpression of manZ, a required gene and encoding a subunit of the

FIG 3 Host-lambda regulatory network. In the core E. coli-lambda interac-tome, we augmented the network of 62 “high-confidence” protein interactionswith more than 30 previously published interactions. Among the targets of thephage, we highlighted 9 transcription factors, 22 essential proteins, and 5 genesthat are required for the lambda infection.

TABLE 5 High- and low-confidence interactions between lambdaproteins and their E. coli homologsa

� bait � homolog E. coli prey Description of E. coli prey

gpA Nu1 NohA Bacteriophage DNA packaging proteingpN Nu1 NohA Bacteriophage DNA packaging proteinInt Nu1 NohB DLP12 prophage, DNA packaging proteingpA Nu1 NohB DLP12 prophage, DNA packaging proteinOrf401 Orf314 StfR Rac prophage; predicted tail fiber proteingpO Orf194 TfaQ Qin prophage; predicted tail fiber

assembly proteinOrf314 Orf194 TfaQ Qin prophage; predicted tail fiber

assembly proteinOrf314 Orf194 TfaR Rac prophage; predicted tail fiber proteinOrf79 Orf79 YbcW DLP12 prophage, predicted proteingpP YdaG Rac prophage; predicted proteina The lambda phage targets E. coli proteins of phage origin. Proteins in the “� homolog”column are lambda proteins that are homologous to the E. coli prey. High-confidencebait-prey interactions are indicated in boldface.

FIG 4 Topological analysis of host-phage interactions. (A) Utilizing ourcombined set of previously published and “high-confidence” interactions,we observed that highly connected host proteins are increasingly targetedby the phage. Focusing on genes that are required for the lambda phageinfection, we observed a similar, albeit weaker trend. (B) We calculated theshortest paths from reference proteins to all other host proteins. The short-est paths from targeted proteins are significantly shorter than the corre-sponding paths from genes that are necessary for phage infection (Studentt test, P 1010). (C) Upon determining the shortest paths from targetedproteins, we found that the distances to genes that are necessary for phageinfection were significantly shorter than for the remaining proteins (P �2.9 � 104).

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ManXYZ mannose permease. Similarly, genes that are involved inCII degradation were directly targeted by the phage (hflD) or werecontrolled by Crp (hflK and hflC).

DISCUSSIONOverlap with previously known interactions. Given our limitedknowledge about lambda-host interactions, we identified 62mostly new high confidence interactions (15). Although we wereinitially disappointed by finding few known PPIs, we can explainmany of the false negatives in retrospect. Nine of the more than 30previously published interactions involve host proteases and theirphage substrates. Such interactions are hard to detect with a high-throughput approach since they are probably weak, short, andtransient, although we did find one protease, ClpP, to interactwith gpO. The other detected, known interaction appears betweengpN and NusA, a well-known interaction involved in the antiter-mination of lambda transcription (15). We did not find the fivepreviously known interactions involving transcription factors,namely, those among CI, CII, gpQ, and RNA polymerase or sig-ma-70 (15). Multiple proteins may be required to stabilize suchproteins in a quaternary structure, a condition our Y2H assay didnot capture. Of the remaining known interactions, two appear

between phage tail proteins and the phage receptors LamB andOmpC. Since we did not clone and screen gpJ in our Y2H assay, wecannot assess whether these interactions are amenable to ourassay.

Although we found an interaction between replication pro-teins gpO and gpP in our previous study (2), we were not able todetect interactions among gpP and DnaA or DnaB. The latterinteraction may require the hexameric structure of DnaB. Fur-thermore, we did not find an interaction between gpP and DnaA,possibly because this interaction may necessitate DnaA to bindDNA. The remaining four undetected interactions are all involvedin recombination (Xis-Fis, Int-IHF, Gam-RecB, and NinB-SSB).Xis is one of the few proteins that we were not able to clone andscreen. Since IHF is a heterodimer, its interaction with Int mayrequire three proteins and therefore may have eluded our experi-mental approach. As for the remaining two missed interactions(Gam-RecB and NinB-SSB), we also surmise that they may re-quire DNA binding to be stabilized.

In summary, results of our screens are clearly impaired by falsenegatives (i.e., known but undetected interactions). The mostplausible explanations for this finding are 3-fold. First, many host-phage interactions appear to require additional factors such asother proteins or DNA. Second, lambda appears to have an un-usually high fraction of proteins whose interactions with the hostrequire posttranslational modification such as proteolytic cleav-age. This observation is in stark contrast to our lambda-lambdaprotein interaction screen: lambda proteins do not appear to in-volve many modified proteins, and thus we found about half of allpreviously reported interactions (2). Finally, many intraviral in-teractions are structural, making them more stable than interac-tions involved in transcription, translation, and regulatory pro-cesses. Thus, they may be easier to detect by Y2H screens than PPIsinvolved in the virus-host interplay.

Differences between phages. Our results may suggest that theY2H system works well for phage-phage interactions but not forphage-host interactions, given the sophisticated interactionmechanisms that involve protein processing or other posttransla-tional mechanisms inside the host. However, this conclusion isnot supported by results from other phages. For instance, whereaslambda has nine interactions involving proteases, no such inter-actions appear to occur in phage T7 (15). Similarly, whereas agenetic screen of E. coli mutants identified 57 host genes requiredfor lambda infection, a similar screen with T7 identified only 11, 9(82%) of which are involved in LPS biosynthesis (20). In thelambda screen, only 8 (14%) of 57 genes were involved in LPSbiosynthesis (12). Clearly, different phages have evolved manydifferent ways to conquer the same host, suggesting that there maybe even more infection mechanisms that are currently unknown(Fig. 6).

Interactions with required genes. In our screens and amongpreviously known interactions, we found only five targeted hostgenes that are required for the infection process of lambda(namely, the genes encoding HflD, LamB, IhfAB, and RfaD; Ta-bles 1 and 4). Although small, this number is not entirely surpris-ing. In fact, almost half of the E. coli proteins that interact withlambda are essential proteins and are therefore not present in theKeio collection (i.e., Maynard et al. could not find them). Unex-pectedly, however, we found only one required gene in our high-confidence Y2H data (namely, RfaD � HldD) that interacted withlambda gpQ. Although small, the size of the overlap between tar-

FIG 5 Gene regulatory interactions targeted by lambda. Utilizing a network ofprotein-protein and transcription factor-gene interactions in E. coli, we calcu-lated the shortest paths from a set of genes that are required for lambda infec-tion to targeted host genes. Specifically, we demanded that a shortest path fromrequired to targeted proteins started with a transcription factor-gene interac-tion. We determined 78 shortest paths from 27 required genes to proteins thatwere targeted by the lambda phage.

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geted and required genes is comparable to previously publishedinteractions: only three of the genes found by Maynard et al. (theHflD, LamB, and IHF genes) have been reported in previous stud-ies to directly interact with lambda proteins.

Another reason why we may have not found many requiredgenes is the fact that roughly half of them are involved in metab-olism such as LPS synthesis or tRNA thiolation (possibly affectingthe translation of lambda transcripts). That is, while our screensare biased toward structural or signaling proteins, genetic screensas in Maynard et al. (12) are biased toward genes that are involvedin metabolism. However, metabolic enzymes are much morerarely involved in PPIs (except for homo- and heterodimeriza-tion) but rather in enzyme-substrate binding, resulting in largelynonoverlapping hits.

Topological characteristics of host-phage interactions. Ana-lyzing their network topology, we observed that proteins with anincreasing number of interaction partners were preferably tar-geted by the phage. We observed a similar and yet weaker trend forgenes required for infection (Fig. 4A), observations that have beenreported for human host-virus interactions (17, 21), as well as forhost-parasite interactions (22, 23). Another topological measurethat reflects the central placement of targeted proteins is the short-est path between proteins in a protein interaction network. Wefound that targeted proteins have shorter paths to other proteins,suggesting that these targets have a more central role in the cellthan others (24). Furthermore, paths from targeted proteins torequired genes were significantly shorter compared to remainingproteins, further indicating that required genes are more closelyplaced to targeted host proteins. These results suggest that host-phage interactions allow the phage to invade the host cell effec-tively on different levels of cellular organization, similar to humanviruses (21).

Phage interference with regulatory interactions. The avail-ability of a large set of host-phage interactions enabled us to iden-tify potential paths the phage uses to exert control on a small set ofrequired genes. Determining connections from the phage to re-quired host genes we identified transcription factors that are cen-tral to the regulation of required genes (such as Crp or Fis) and areat least indirectly targeted by the phage. Although our results arebased on topological considerations only, our data make several

predictions, e.g., that transcriptions factors such as Crp and Fisshould have a significant effect on lambda or vice versa (Fig. 5).This prediction has been confirmed in recent experiments (25).

ACKNOWLEDGMENTS

We thank Sherwood Casjens for constructive comments on the manu-script.

This project was partly funded by National Institutes of Health grantR01GM79710 and the European Union (grant HEALTH-F3-2009-223101, AntipathoGN). Research at the National Center for Biotechnol-ogy Information was supported by the National Institutes of Health/De-partment of Health and Human Services as part of the IntramuralResearch program of the National Library of Medicine.

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