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Virus-Cell Interactions

The Protein Interaction Network of Bacteriophage Lambda with Its Host, Escherichia coli

Sonja Blasche, Stefan Wuchty, Seesandra V. Rajagopala, Peter Uetz
Sonja Blasche
aGenomics and Proteomics Core Facilities, German Cancer Research Center, Heidelberg, Germany
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Stefan Wuchty
bNational Center of Biotechnology Information, National Institutes of Health, Bethesda, Maryland, USA
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Seesandra V. Rajagopala
cJ. Craig Venter Institute, Rockville, Maryland, USA
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Peter Uetz
dCenter for the Study of Biological Complexity, Virginia Commonwealth University, Richmond, Virginia, USA
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DOI: 10.1128/JVI.02495-13
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  • Fig 1
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    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, and each 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 in pDEST22 and one in pGADT7g). All screening results were pooled, yielding 631 raw interactions. After the removal of 487 “low-confidence” interactions that involved 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.

  • Fig 2
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    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 genome for 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 30 previously published interactions (green arrows). All proteins are colored according to their functional annotations.

  • Fig 3
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    Fig 3

    Host-lambda regulatory network. In the core E. coli-lambda interactome, we augmented the network of 62 “high-confidence” protein interactions with more than 30 previously published interactions. Among the targets of the phage, we highlighted 9 transcription factors, 22 essential proteins, and 5 genes that are required for the lambda infection.

  • Fig 4
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    Fig 4

    Topological analysis of host-phage interactions. (A) Utilizing our combined set of previously published and “high-confidence” interactions, we observed that highly connected host proteins are increasingly targeted by the phage. Focusing on genes that are required for the lambda phage infection, we observed a similar, albeit weaker trend. (B) We calculated the shortest paths from reference proteins to all other host proteins. The shortest paths from targeted proteins are significantly shorter than the corresponding paths from genes that are necessary for phage infection (Student t test, P < 10−10). (C) Upon determining the shortest paths from targeted proteins, we found that the distances to genes that are necessary for phage infection were significantly shorter than for the remaining proteins (P = 2.9 × 10−4).

  • Fig 5
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    Fig 5

    Gene regulatory interactions targeted by lambda. Utilizing a network of protein-protein and transcription factor-gene interactions in E. coli, we calculated the shortest paths from a set of genes that are required for lambda infection to targeted host genes. Specifically, we demanded that a shortest path from required to targeted proteins started with a transcription factor-gene interaction. We determined 78 shortest paths from 27 required genes to proteins that were targeted by the lambda phage.

  • Fig 6
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    Fig 6

    Comparison of host-phage interactions in lambda and T7. We collected previously published protein interactions between E. coli and the phages lambda and T7. We observed that only a small minority of host proteins is targeted by both phages (RecB and the HsdMS complex).

Tables

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  • Additional Files
  • Table 1

    Previously published lambda-host PPIsa

    Function/role and PPIλHostbDescriptionSource or reference
    Transcription
        1CIRecARecA degrades CIc26
        2CIRpoA27
        3CIRpoD28, 29
        4CIIClpYQClpYQ degrades CII in vitro26
        5CIIClpAPClpAP degrades CII in vitro26
        6CIIHflDHflD makes CII more vulnerable to FtsH (hfl = high-frequency lysogenization)30
        7CIIHflB*HflB (= FtsH) protease degrades CII31, 32
        8CIIRpoA33, 34
        9CIIRpoD33
        10CIIIHflBCIII inhibits HflB; HflB degrades CIII26, 35
        11gpNNusATranscriptional regulation36
        12gpNLonLon degrades gpN26, 37
        13gpQσ70gpQ makes RNAP insensitive to cis termin29, 34, 38, 39
    Head
        14gpBGroEGenetic interaction; E. coli GroE40–42
        15gpEGroEGenetic interaction41
    Tail
        16gpJLamBLamB is the E. coli receptor43–47
        17StfOmpCOmpC is a secondary E. coli receptor48
    Recombination
        18XisLonXis is degraded by E. coli Lon protease49
        19XisFtsHXis is degraded by E. coli FtsH protease49
        20XisFisBoth required for excision50–52
        21IntIHFBoth catalyze recombination at attP/attB53, 54
        22GamSbcCSbcCD is a dsDNA exonuclease + ssDNA endonuclease55
        23GamRecBGam inhibits RecBCD56
        24NinBSSBNinB also binds ssDNA57
        25RalHsdRal inhibits restriction enzyme complex HsdMSR (by binding to HsdM or S)58
    Replication
        26gpOClpXPClpXP degrades gpO26
        27gpODnaK59
        28gpORpoB60
        29gpPDnaA61
        30gpPDnaB62
        31gpPDnaK59
    • ↵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 vectorPropertyPrey libraryScreen type
    λ ORF/pGBKT7g/pGADT7gHigh copyE. coli W3110Arrayed pool
    λ ORF/pGBKT7g/pGADT7gHigh copyE. coli W3110Prey pool
    λ ORF/pDEST32/pDEST22Low copyE. coli W3110Prey pool
  • Table 3

    Numeric results of phage lambda-host pool screens

    Screen typeParameterNo.
    E. coli prey proteins interacting with lambdaTotal294
    Promiscuous25
    Unique bait-prey pairsTotal631
    Single hits334
    Multiple hits144
    With promiscuous prey218
    Unique bait-prey pairs, pDEST32/22 onlyTotal56
    Single hits38
    High-confidence hits20
    Unique bait-prey pairs, pGBKT7g/pGADT7g onlyTotal573
    Single hits296
    High-confidence hits122
    Unique bait-prey pairs found in both pDEST32/22 and pGBKT7g/pGADT7g2
    Retested individually162
    TotalHigh-confidence hits62
  • Table 4

    High-confidence interactions in this studya

    λ locus no.λ locusE. coli proteinAnnotation
    lambdap01nu1DcrBConserved protein involved in bacteriophage adsorption
    lambdap02ANohABacteriophage DNA packaging protein
    lambdap02ANohBBacteriophage DNA packaging protein
    lambdap09FiYdgHConserved hypothetical protein
    lambdap09FiFixBProbable flavoprotein subunit required for carnitine metabolism
    lambdap09FiMinECell division topological specificity factor MinE
    lambdap09FiCchBEthanolamine utilization EutN
    lambdap14GClpPATP-dependent Clp protease, proteolytic subunit ClpP
    lambdap14GFhuFFerric iron reductase protein FhuF
    lambdap14GFdoHFormate dehydrogenase, beta subunit
    lambdap14GYohNConserved hypothetical protein
    lambdap14GChaCCation transport protein ChaC
    lambdap14GProQProP effector
    lambdap14GYliLHypothetical protein
    lambdap16HYliLHypothetical protein
    lambdap16HYeiWProteinase inhibitor
    lambdap16HYfcQConserved hypothetical protein
    lambdap16HYohHPredicted membrane protein
    lambdap16HYehDFimbrial protein
    lambdap33intNohBBacteriophage DNA packaging protein
    lambdap36ea8.5YjdIConserved hypothetical protein
    lambdap36ea8.5MinECell division topological specificity factor MinE
    lambdap36ea8.5YeiWProteinase inhibitor
    lambdap38orf63YqhCPutative HTH-type transcriptional regulator YqhC
    lambdap45ea10Rmfb0953 ribosome modulation factor (rmf)
    lambdap45ea10RpmARibosomal protein L27
    lambdap45ea10YliLHypothetical protein
    lambdap45ea10RpsGRibosomal protein S7
    lambdap45ea10PriCPrimosomal replication N
    lambdap45ea10CobBNAD-dependent deacetylase
    lambdap45ea10SoxSRegulatory protein SoxS
    lambdap45ea10YcbGCell division protein MatP
    lambdap49NHcrNADH oxidoreductase Hcr
    lambdap49NNuoGNADH-quinone oxidoreductase, chain G
    lambdap49NYebRFree methionine-(R)-sulfoxide reductase
    lambdap49NNusATranscription elongation NusA
    lambdap49NEnvRProbable acrEF/envCD operon repressor
    lambdap49NMinCSeptum site-determining protein MinC
    lambdap49NRpoSRNA polymerase sigma factor RpoS
    lambdap49NYcbGCell division protein MatP
    lambdap61PAtpCATP synthase F1, epsilon subunit
    lambdap61PEutCEthanolamine ammonia-lyase, light chain
    lambdap61PYcbGCell division protein MatP
    lambdap61PYqhCPutative HTH-type transcriptional regulator YqhC
    lambdap65NinDPyrFOrotidine 5′-phosphate decarboxylase
    lambdap65NinDYhdWGeneral l-amino acid-binding periplasmic protein AapJ
    lambdap65NinDMinECell division topological specificity factor MinE
    lambdap65NinDSlpOuter membrane protein Slp
    lambdap65NinDSmpASmall protein A
    lambdap65NinDYjdIConserved hypothetical protein
    lambdap65NinDCsrACarbon storage regulator
    lambdap65NinDHycGHydrogenase-4 component I
    lambdap65NinDSdiARegulatory protein SdiA
    lambdap65NinDSoxSRegulatory protein SoxS
    lambdap65NinDYebRFree methionine-(R)-sulfoxide reductase
    lambdap71QGlyQGlycyl-tRNA synthetase, alpha subunit
    lambdap71QYqhCPutative HTH-type transcriptional regulator YqhC
    lambdap71QPaaCPhenylacetate-CoA oxygenase, PaaI subunit
    lambdap71QRfaDADP-l-glycero-d-manno-heptose-6-epimerase
    lambdap75RFhuFFerric iron reductase protein FhuF
    lambdap75RCaiFTranscriptional activatory protein CaiF
    lambdap79YbcWConserved hypothetical protein
    • ↵a The protein interactions from this publication have been submitted to the IMEx (http://www.imexconsortium.org) consortium through IntAct (67) and assigned the identifier IM-21394.

  • Table 5

    High- and low-confidence interactions between lambda proteins and their E. coli homologsa

    λ baitλ homologE. coli preyDescription of E. coli prey
    gpANu1NohABacteriophage DNA packaging protein
    gpNNu1NohABacteriophage DNA packaging protein
    IntNu1NohBDLP12 prophage, DNA packaging protein
    gpANu1NohBDLP12 prophage, DNA packaging protein
    Orf401Orf314StfRRac prophage; predicted tail fiber protein
    gpOOrf194TfaQQin prophage; predicted tail fiber assembly protein
    Orf314Orf194TfaQQin prophage; predicted tail fiber assembly protein
    Orf314Orf194TfaRRac prophage; predicted tail fiber protein
    Orf79Orf79YbcWDLP12 prophage, predicted protein
    gpPYdaGRac prophage; predicted protein
    • ↵a The lambda phage targets E. coli proteins of phage origin. Proteins in the “λ homolog” column are lambda proteins that are homologous to the E. coli prey. High-confidence bait-prey interactions are indicated in boldface.

Additional Files

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  • Supplemental material

    Files in this Data Supplement:

    • Supplemental file 1 -

      Table S1 (All interactions (bait-prey pairs) and promiscous preys.)

      XLS, 90K

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The Protein Interaction Network of Bacteriophage Lambda with Its Host, Escherichia coli
Sonja Blasche, Stefan Wuchty, Seesandra V. Rajagopala, Peter Uetz
Journal of Virology Nov 2013, 87 (23) 12745-12755; DOI: 10.1128/JVI.02495-13

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The Protein Interaction Network of Bacteriophage Lambda with Its Host, Escherichia coli
Sonja Blasche, Stefan Wuchty, Seesandra V. Rajagopala, Peter Uetz
Journal of Virology Nov 2013, 87 (23) 12745-12755; DOI: 10.1128/JVI.02495-13
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