Article Figures & Data
Figures
- 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
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
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
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
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
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
- Table 1
Previously published lambda-host PPIsa
Function/role and PPI λ Hostb Description Source or reference Transcription 1 CI RecA RecA degrades CIc 26 2 CI RpoA 27 3 CI RpoD 28, 29 4 CII ClpYQ ClpYQ degrades CII in vitro 26 5 CII ClpAP ClpAP degrades CII in vitro 26 6 CII HflD HflD makes CII more vulnerable to FtsH (hfl = high-frequency lysogenization) 30 7 CII HflB* HflB (= FtsH) protease degrades CII 31, 32 8 CII RpoA 33, 34 9 CII RpoD 33 10 CIII HflB CIII inhibits HflB; HflB degrades CIII 26, 35 11 gpN NusA Transcriptional regulation 36 12 gpN Lon Lon degrades gpN 26, 37 13 gpQ σ70 gpQ makes RNAP insensitive to cis termin 29, 34, 38, 39 Head 14 gpB GroE Genetic interaction; E. coli GroE 40–42 15 gpE GroE Genetic interaction 41 Tail 16 gpJ LamB LamB is the E. coli receptor 43–47 17 Stf OmpC OmpC is a secondary E. coli receptor 48 Recombination 18 Xis Lon Xis is degraded by E. coli Lon protease 49 19 Xis FtsH Xis is degraded by E. coli FtsH protease 49 20 Xis Fis Both required for excision 50–52 21 Int IHF Both catalyze recombination at attP/attB 53, 54 22 Gam SbcC SbcCD is a dsDNA exonuclease + ssDNA endonuclease 55 23 Gam RecB Gam inhibits RecBCD 56 24 NinB SSB NinB also binds ssDNA 57 25 Ral Hsd Ral inhibits restriction enzyme complex HsdMSR (by binding to HsdM or S) 58 Replication 26 gpO ClpXP ClpXP degrades gpO 26 27 gpO DnaK 59 28 gpO RpoB 60 29 gpP DnaA 61 30 gpP DnaB 62 31 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 λ ORF/pGBKT7g/pGADT7g High copy E. coli W3110 Arrayed pool λ ORF/pGBKT7g/pGADT7g High copy E. coli W3110 Prey pool λ ORF/pDEST32/pDEST22 Low copy E. coli W3110 Prey pool - Table 3
Numeric results of phage lambda-host pool screens
Screen type Parameter No. E. coli prey proteins interacting with lambda Total 294 Promiscuous 25 Unique bait-prey pairs Total 631 Single hits 334 Multiple hits 144 With promiscuous prey 218 Unique bait-prey pairs, pDEST32/22 only Total 56 Single hits 38 High-confidence hits 20 Unique bait-prey pairs, pGBKT7g/pGADT7g only Total 573 Single hits 296 High-confidence hits 122 Unique bait-prey pairs found in both pDEST32/22 and pGBKT7g/pGADT7g 2 Retested individually 162 Total High-confidence hits 62 - Table 4
High-confidence interactions in this studya
λ locus no. λ locus E. coli protein Annotation lambdap01 nu1 DcrB Conserved protein involved in bacteriophage adsorption lambdap02 A NohA Bacteriophage DNA packaging protein lambdap02 A NohB Bacteriophage DNA packaging protein lambdap09 Fi YdgH Conserved hypothetical protein lambdap09 Fi FixB Probable flavoprotein subunit required for carnitine metabolism lambdap09 Fi MinE Cell division topological specificity factor MinE lambdap09 Fi CchB Ethanolamine utilization EutN lambdap14 G ClpP ATP-dependent Clp protease, proteolytic subunit ClpP lambdap14 G FhuF Ferric iron reductase protein FhuF lambdap14 G FdoH Formate dehydrogenase, beta subunit lambdap14 G YohN Conserved hypothetical protein lambdap14 G ChaC Cation transport protein ChaC lambdap14 G ProQ ProP effector lambdap14 G YliL Hypothetical protein lambdap16 H YliL Hypothetical protein lambdap16 H YeiW Proteinase inhibitor lambdap16 H YfcQ Conserved hypothetical protein lambdap16 H YohH Predicted membrane protein lambdap16 H YehD Fimbrial protein lambdap33 int NohB Bacteriophage DNA packaging protein lambdap36 ea8.5 YjdI Conserved hypothetical protein lambdap36 ea8.5 MinE Cell division topological specificity factor MinE lambdap36 ea8.5 YeiW Proteinase inhibitor lambdap38 orf63 YqhC Putative HTH-type transcriptional regulator YqhC lambdap45 ea10 Rmf b0953 ribosome modulation factor (rmf) lambdap45 ea10 RpmA Ribosomal protein L27 lambdap45 ea10 YliL Hypothetical protein lambdap45 ea10 RpsG Ribosomal protein S7 lambdap45 ea10 PriC Primosomal replication N lambdap45 ea10 CobB NAD-dependent deacetylase lambdap45 ea10 SoxS Regulatory protein SoxS lambdap45 ea10 YcbG Cell division protein MatP lambdap49 N Hcr NADH oxidoreductase Hcr lambdap49 N NuoG NADH-quinone oxidoreductase, chain G lambdap49 N YebR Free methionine-(R)-sulfoxide reductase lambdap49 N NusA Transcription elongation NusA lambdap49 N EnvR Probable acrEF/envCD operon repressor lambdap49 N MinC Septum site-determining protein MinC lambdap49 N RpoS RNA polymerase sigma factor RpoS lambdap49 N YcbG Cell division protein MatP lambdap61 P AtpC ATP synthase F1, epsilon subunit lambdap61 P EutC Ethanolamine ammonia-lyase, light chain lambdap61 P YcbG Cell division protein MatP lambdap61 P YqhC Putative HTH-type transcriptional regulator YqhC lambdap65 NinD PyrF Orotidine 5′-phosphate decarboxylase lambdap65 NinD YhdW General l-amino acid-binding periplasmic protein AapJ lambdap65 NinD MinE Cell division topological specificity factor MinE lambdap65 NinD Slp Outer membrane protein Slp lambdap65 NinD SmpA Small protein A lambdap65 NinD YjdI Conserved hypothetical protein lambdap65 NinD CsrA Carbon storage regulator lambdap65 NinD HycG Hydrogenase-4 component I lambdap65 NinD SdiA Regulatory protein SdiA lambdap65 NinD SoxS Regulatory protein SoxS lambdap65 NinD YebR Free methionine-(R)-sulfoxide reductase lambdap71 Q GlyQ Glycyl-tRNA synthetase, alpha subunit lambdap71 Q YqhC Putative HTH-type transcriptional regulator YqhC lambdap71 Q PaaC Phenylacetate-CoA oxygenase, PaaI subunit lambdap71 Q RfaD ADP-l-glycero-d-manno-heptose-6-epimerase lambdap75 R FhuF Ferric iron reductase protein FhuF lambdap75 R CaiF Transcriptional activatory protein CaiF lambdap79 YbcW Conserved 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 bait λ homolog E. coli prey Description of E. coli prey gpA Nu1 NohA Bacteriophage DNA packaging protein gpN Nu1 NohA Bacteriophage DNA packaging protein Int Nu1 NohB DLP12 prophage, DNA packaging protein gpA Nu1 NohB DLP12 prophage, DNA packaging protein Orf401 Orf314 StfR Rac prophage; predicted tail fiber protein gpO Orf194 TfaQ Qin prophage; predicted tail fiber assembly protein Orf314 Orf194 TfaQ Qin prophage; predicted tail fiber assembly protein Orf314 Orf194 TfaR Rac prophage; predicted tail fiber protein Orf79 Orf79 YbcW DLP12 prophage, predicted protein gpP YdaG Rac 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.
Supplemental material
Files in this Data Supplement:
- Supplemental file 1
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Table S1 (All interactions (bait-prey pairs) and promiscous preys.)
XLS, 90K
- Supplemental file 1
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