Supporting InformationMunoz-Espín et al. 10.1073/pnas.0906465106SI TextBacterial Strains, Phages, Growth Conditions, and Protein Levels. E.coli strain DH5�, used for cloning, was grown in Luria Bertani(LB) medium. When appropriate, ampicillin was added tocultures and plates at a final concentration of 100 �g/mL. E. colistrains were grown in LB medium containing 5 mM MgSO4 at37 °C and supplemented with appropriate antibiotics: strepto-mycin (50 �g/mL), kanamycin (50 �g/mL), and/or chloramphen-icol (50 �g/mL). B. subtilis strains were grown in LB mediumcontaining 5 mM MgSO4 at 37 °C and supplemented withappropriate antibiotics: kanamycin (5 �g/mL), chloramphenicol(5 �g/mL), erythromycin (1 �g/mL), lincomycin (25 �g/mL),and/or spectinomycin (50 �g/mL). When cytoskeleton mutantswere used, MgSO4 concentrations were increased to 25 mM inall of the cultures. Expression of GFP fusions was induced byaddition of 0.5% xylose at the time of phage infection. Generally,overnight cultures were diluted 1/50 in fresh medium andincubated for 2–3 h to re-establish exponential growth beforemanipulation. For induction of the hyperspank promoter, theculture media were supplemented with 1 mM isopropyl-�-D-thiogalactopyranoside (IPTG) at the time of infection.

It has been previously determined that the �29 DNA poly-merase increases to about 1,000 molecules per cell during thephage replication cycle (1). It has also been shown that in B.subtilis Mbl is present in about 12,000 to 14,000 molecules per celland MreB in about 8,000 molecules per cell (2). In the case ofp16.7, the amount of protein present during the infection cyclewas estimated as approximately 65,000 molecules per cell (3).Whereas these levels are about 5–8-fold higher than those of thecytoskeletal proteins, it is worth mentioning that 1 functionalDNA-binding unit is composed of 3 p16.7 dimers, forming acavity that interacts with the phage DNA in a non-specific way(4).

Bacterial Transformation, Conjugation, and DNA Labeling. Selectionfor B. subtilis transformants was carried out on nutrient agar(Oxoid) plates, supplemented with appropriate antibiotics and0.5% xylose and/or 25 mM MgSO4 when necessary. In DNAlabeling experiments, the thymine analogue BrdU (Sigma) wasadded to the growth medium at a final concentration of 150 �M.Incorporation of BrdU into chromosomal DNA of B. subtilis wasinhibited by adding 75 �M 6-(p-hydroxyphenylazo)-uracil(HpUra) (5) to the growth medium 2 min before BrdU addition.

Plasmid Construction. Construction of the 16.7-gfpmut1 fusion(pSGW1) has been described (3). The N- and C-terminal gfpfusions of �29 gene 2, encoding the DNA polymerase, wereconstructed as follows. Gene 2 was amplified by PCR from �29DNA using primer sets P2GFP�U and P2GFP�L (C-terminalGFP fusion) or GFPP2�U and GFPP2�L (N-terminal GFPfusion)(see Table S4). The PCR products obtained were digestedwith KpnI/HindIII or SalI/EcoRI, respectively, and cloned intoB. subtilis amyE integration vectors pSG1154 and pSG1729 (6)digested with the same enzymes, respectively. As a result, gene2 fused in frame to the gfpmut1 gene (7) at its N terminus(pSGDM1) or C terminus (pSGDM2) were located behind thexylose-inducible promoter Pxyl. The C-terminal yfp fusion of �29gene 2 present on plasmid pSGDM3 was constructed by isolatingthe �29 gene 2-containing a BamHI-EcoRI fragment frompSGDM2 and cloning it into pSG5472 digested with the sameenzymes. Plasmid pSG5472, containing the yfpmut2 gene (8), isa derivative of pSG1729. pSGDM1, pSGDM2, and pSGDM3

were used to transform competent B. subtilis cells. Spectinomy-cin resistant transformants were tested for their ability todegrade starch to select for double-crossover transformants.

Immunofluorescence Microscopy. Blocking buffer contained 0.5%(wt/vol) casein (Sigma). Affinity-purified rat and rabbit poly-clonal antibodies against p16.7 were used at 1:1,000 dilution andincubations were carried out overnight at 4 °C. Polyclonal anti-bodies were centrifuged for 10 min at 14,000 � g at 4 °C beforeuse to precipitate possible antibody aggregates. The N-terminalc-Myc-tagged MreB protein (c-Myc-MreB) was previouslyshown to be functional and determination of the MreB IFdistribution pattern gave clearer results using monoclonal anti-bodies against c-Myc than detecting native MreB using poly-clonal anti-MreB antibodies (2). Monoclonal antibodies mouseanti-c-Myc (Sigma) and mouse anti-BrdU (Caltag) were used at1/50 and 1/100 dilutions, respectively, and incubated at 4 °Covernight. FITC-conjugated anti-rabbit, anti-rat, and anti-mouseantibodies (Sigma) were used at a dilution of 1:1,000 andincubated for 2–4 h at room temperature. C�3-conjugatedanti-rabbit and anti-rat antibodies (Sigma) were used at adilution of 1:2,000 and incubated for 2 h at room temperature.These and all subsequent steps were performed with minimalexposure of the samples to light. For the immunodetection ofBrdU, cells were incubated with 4 M HCl for 15 min at roomtemperature, and then washed 6 times with PBS before incuba-tion with anti-BrdU antibodies. To detect only �29 dsDNA,ssDNA was removed by treating the samples with S1 nuclease(Sigma) at a final dilution of 1/75 for 30 min at 37 °C before theHCl denaturation step. All samples were mounted for epif luo-rescence microscopy in multispot microscope slides (C.A. Hend-ley, Essex, LTD) supplemented with 0.2 �g/mL DAPI.

Epifluorescence Microscopy. For fluorescence microscopy, over-night cultures were diluted in LB medium containing 5 or 25 mMMgSO4 and grown to early exponential phase at 37 °C. At anOD600 � 0.3–0.6, cells were infected at a MOI � 5 with theindicated �29 phage (Table S2) and supplemented with 0.5%xylose.

Image Acquisition and Image Analysis. Imaging acquisition wasperformed as described (9) using a Sony CoolSnap HQ cooledCCD camera (Roper Scientific) attached to a Zeiss Axiovert200M microscope. The digital images were acquired and ana-lyzed with METAMORPH version 6 software. Images of fluo-rescent samples were deconvolved within METAMORPH andassembled in Adobe PHOTOSHOP version 7. Image manipu-lation was kept to a minimum. For general purposes images werescaled and then saved as 8-bit images.

Real-Time PCR. The following primer sets were used to amplifyregions of the genome of phage �29, SPP1, and PRD1, respec-tively: R-OUT-SUPER and R-25; oriR-A and oriR-B; andR-PRD1-A and R-PRD1-B (see Table S4). The data obtainedfor samples were interpolated to standard curves constructedwith known amounts of phage DNA. The results were expressedas �g DNA per mL culture.

Analysis of Viral DNA by Gel Electrophoresis. The method used toanalyze synthesis of viral DNA in vivo was carried out asdescribed (10). Basically, total intracellular DNA was isolated at

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different times after infection, and analyzed in 0.5% agarosegels.

Bacterial 2-Hybrid Assay. In this system positive interactions giverise to the formation of an active adenyl cyclase enzyme,resulting in the relaxation of catabolite repression in the host cell,rather than an active transcriptional activator. This has theadvantage that the association of proteins does not need to

involve also association with the DNA. Colonies were analyzedon nutrient agar plates supplemented with X-gal. The appear-ance of blue pigment within colonies indicates a positive inter-action. Plasmids used are listed in Table S3 and obtained fromEuromedex. A 10-�L aliquot from each transformation reactionwas spotted onto a nutrient agar plate containing 100 �g/mLampicillin, 50 �g/mL kanamycin, and 0.008% X-gal. Plates wereincubated at 30 °C for 48 h.

1. Bravo A, Salas M (1997) Initiation of bacteriophage �29 DNA replication in vivo:assembly of a membrane-associated multiprotein complex. J Mol Biol 269:102–112.

2. Jones LJ, Carballido-Lopez R, Errington J (2001) Control of cell shape in bacteria:Helical, actin-like filaments in Bacillus subtilis. Cell 104:913–922.

3. Meijer WJ, Serna-Rico A, Salas M (2001) Characterization of the bacteriophage �29-encoded protein p16.7: A membrane protein involved in phage DNA replication. MolMicrobiol 39:731–746.

4. Albert A, et al. (2005) Structural basis for membrane anchorage of viral �29 DNA duringreplication. J Biol Chem 280:42486–42488.

5. Brown NC (1970) 6-(-p-Hydroxyphenylazo)-uracil: A selective inhibitor of host DNAreplication in phage-infected Bacillus subtilis. Proc Natl Acad Sci USA 67:1454–1461.

6. Lewis PJ, Marston AL (1999) GFP vectors for controlled expression and dual labelling ofprotein fusions in Bacillus subtilis Gene 227:101–110.

7. Cormack BP, Valdivia RH, Falkow S (1996) FACS-optimized mutants of the greenfluorescent protein (GFP). Gene 173:33–38.

8. Lemon KP, Grossman AD (2000) Movement of replicating DNA through a stationaryreplisome. Mol Cell 6:1321–1330.

9. Lewis PJ, Errington J (1997) Direct evidence for active segregation of oriC regions of theBacillus subtilis chromosome and co-localization with the Spo0J partitioning protein.Mol Microbiol 25:945–954.

10. Bravo A, Hermoso JM, Salas M (1994) A genetic approach to the identification offunctional amino acids in protein p6 of Bacillus subtilis phage �29. Mol Gen Genet245:529–536.

11. Meijer WJ, et al. (2005) Molecular basis for the exploitation of spore formation assurvival mechanism by virulent phage �29. EMBO J 24:3647–3657.

12. Castilla-Llorente V, Munoz-Espín D, Villar L, Salas M, Meijer WJ (2006) Spo0A, the keytranscriptional regulator for entrance into sporulation, is an inhibitor of DNA replica-tion. EMBO J 25:3890–3899.

13. Moreno F, Camacho A, Vinuela E, Salas M (1974) Suppressor-sensitive mutants andgenetic map of Bacillus subtilis bacteriophage �29. Virology 62:1–16.

14. Kruse T, Moller-Jensen J, Lobner-Olesen A, Gerdes K (2003) Dysfunctional MreB inhibitschromosome segregation in Escherichia coli. EMBO J 22:5283–5292.

15. Carballido-Lopez R, Errington J (2003) The bacterial cytoskeleton: In vivo dynamics ofthe actin-like protein Mbl of Bacillus subtilis. Dev Cell 4:19–28.

16. Carballido-Lopez R (2006) Orchestrating bacterial cell morphogenesis. Mol Microbiol60:815–819.

17. Formstone A, Errington J (2005) A magnesium-dependent mreB null mutant: Implica-tions for the role of mreB in Bacillus subtilis. Mol Microbiol 55:1646–1657.

18. Sandman K, Losick R, Youngman P (1987) Genetic analysis of Bacillus subtilis spomutations generated by Tn917-mediated insertional mutagenesis. Genetics 117:603–617.

19. Mellado RP, Vinuela E, Salas M (1976) Isolation of a strong suppressor of nonsensemutations in Bacillus subtilis. Eur J Biochem 65:213–223.

20. Xu K, Strauch MA (1996) Identification, sequence, and expression of the gene encodinggamma-glutamyltranspeptidase in Bacillus subtilis. J Bacteriol 178:4319–4322.

21. Jimenez F, Camacho A, de la Torre J, Vinuela E, Salas M (1977) Assembly of Bacillussubtilis phage �29. 2. Mutants in the cistrons coding for the non-structural proteins.Eur. J Biochem 73:57–72.

22. Karimova G, Pidoux J, Ullmann A, Ladant D (1998) A bacterial two-hybrid system basedon a reconstituted signal transduction pathway. Proc Natl Acad Sci USA 95:5752–5756.

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Fig. S1. Genetic and transcriptional map of the �29 genome and mechanism of in vitro �29 DNA replication. (A) Map of the �29 genome. The direction oftranscription and length of the transcripts are indicated by arrows and the positions of genes are indicated with numbers. TD1 corresponds to the bidirectionaltranscriptional terminator located in between the convergently transcribed late and right-side early operons. Black circles represent the terminal proteincovalently linked to the 5� DNA ends. Adapted from Meijer et al. (11). (B) Overview of in vitro �29 DNA replication mechanism. Replication starts by recognitionof the p6-nucleoprotein complexed origins of replication by a TP/DNA polymerase heterodimer. The DNA polymerase then catalyses the addition of the firstdAMP to the TP present in the heterodimer complex. Next, after a transition step, these 2 proteins dissociate and the DNA polymerase continues processiveelongation until replication of the nascent DNA strand is completed. Replication is coupled to strand displacement. The �29-encoded SSB protein p5 binds tothe displaced ssDNA strands and is removed by the DNA polymerase during later stages in the replication process. Continuous polymerization results in thegeneration of 2 fully replicated �29 genomes. Circles, TP; triangles, DNA polymerase; ovals, replication initiator protein p6; diamonds, SSB protein p5; de novosynthesized DNA is shown as beads on a string. Adapted from Castilla-Llorente et al. (12).

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Fig. S2. The amount of accumulated viral DNA is severely affected in mreB mutants. Agarose gel electrophoresis analysis showing the amount of viral DNAaccumulated at different times after infection in wild-type and mreB-like mutants of B. subtilis (A and B) and E. coli (C) cells infected with phage �29 (A), SPP1(B), or PRD1 (C). Cells were infected at a MOI of 5 (A and B) or at a MOI of 25 (C), and aliquots were harvested and processed at the indicated postinfection times.

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Fig. S3. Efficient �29 DNA replication is restored upon MreB induction in a conditional mreB strain. B. subtilis strain 2060, which contains a disruption of mreBat its natural position and a xylose inducible copy of c-myc-mreB at an ectopic locus (amyE), was grown in LB medium supplemented with 25 mM MgSO4 and2% glucose at 37 °C. Cultures containing 0.5% xylose (black bars) or in the absence of xylose (striped bars) were infected with �29 phage sus14(1242) at a MOIof 1. At 30 min postinfection a fraction of the cell culture grown without xylose was separated and supplemented with 0.5% xylose (gray bars). Cells wereharvested at the indicated postinfection times and processed for quantitative real-time PCR analysis as described in the Materials and Methods. The amountsof accumulated phage DNA (ng viral DNA per mL culture) are expressed as a function of time after infection. In the absence of xylose (striped bars) the amountof replicated DNA was slightly higher compared to that obtained using the knock out mreB deletion strain (Fig. 1, Left), most likely because the promoter is notcompletely inactive in the absence of xylose. To avoid possible pleoitropic effects in the absence of MreB, all media were supplemented with 25 mM MgSO4.

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Fig. S4. The constructed xylose-inducible N- and C-terminal fusions of GFP to the �29 DNA polymerase p2 are functional in vivo. Exponentially growing B. subtiliscells of strains DM-010 (GFP-p2) and DM-015 (p2-GFP) were infected with �29 mutant phage sus2(513) containing a suppressible stop codon in the DNApolymerase-encoding gene 2 (13). Next, samples were mixed with liquid top agar containing or not 0.5% xylose, spread on LB agar plates and incubated overnightat 37 °C.

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Fig. S5. Phage �29 dsDNA localizes in a helix-like pattern at the periphery of infected cells. B. subtilis cells (168 �spo0A) were grown in LB medium supplementedwith 5 mM MgSO4 at 37 °C. At an OD600 of 0.4, the culture was split and half of it was infected with sus14(1242) phage at a MOI of 5. Samples were harvestedand processed 20 min later. (A and B) phase contrast and lack of IF signals in non-infected cells. (C and D) Phase contrast and unprocessed image of theimmunofluorescence signal of BrdU in S1-nuclease and HCl-treated cells. (E) Same cell as shown in (D) after deconvolution of an image stack, as a‘‘max-projection.’’

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Fig. S6. Subcellular localization of GFP-p2 in cells infected with a sus16.7 mutant phage. GFP fluorescence and phase contrast of typical cells expressing axylose-inducible GFP-p2 fusion (strain DM-010) infected with a sus14(1242) (A) or a sus16.7(48)/sus14(1242) (B) mutant phage. Fluorescence images were obtainedat 25 min postinfection and correspond to ‘‘max projections’’ of a deconvolved stack of optical sections. DM-010 cells were grown at 37 °C in LB mediumsupplemented with 25 mM MgSO4 to an OD600 of 0.4. Next, xylose was added to a final concentration of 0.5% and the culture was infected at a multiplicity of5. Arrows indicate mislocalization of GFP-p2.

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Table S1. Strains used

Strain Relevant genotype* Construction, source or reference†

E. coliDH5 � F� �80dlacZ�M15 �(lacZYA-argF) U169 recA1 endA1 hsdR17(rk

�, mk�) Laboratory stock

phoA supE44 �� thi-1 gyrA96 relA1?K529 Strain bearing virulence plasmid pLM2 �tet(Am), ampR(Am), kan)� C.G.S.C‡

MC1000 �araD139 �(ara, leu)7697 �lacX74 galU galK atrA� Kruse et al. (14)MC1000�mreB �araD139 �(ara, leu)7697 �lacX74 galU galK atrA� (mreB::cat) Kruse et al. (14)DM-040 MC1000 containing plasmid pLM2 K5293MC1000 (Km, Str)DM-041 MC1000�mreB containing plasmid pLM2 K5293MC1000�mreB (Km, Str)

B. subtilis110NA trpC2 spo0A3 su� Moreno et al. (13)110WA trpC2 spo0A3 su� (amyE::Pxyl-16.7-gfp spc) Meijer et al. (3)DM-000 trpC2 (amyE::Pxyl-16.7-gfp spc) pSGW13 168 (Sp)168 trpC2, considered wild type strain B.G.S.C.§

168�Mbl trpC2 (mbl::neo) Jeff Errington Lab.2060 trpC2 (amyE::Pxyl-c-myc-mreBCD spc) (mreB::neo) Jones et al. (2)2501 trpC2 (mbl::pMUTIN4, erm) Carballido-Lopez et al. (15)2505 trpC2 (mbl::spc) Jones et al. (2)2536 trpC2 (mreBH::cat) Carballido-Lopez et al. (16)3725 trpC2 (neo3427)�mreB Formstone et al. (17)3748 trpC2 (mreB-Pxyl cfp-mreB cat) Carballido-Lopez et al. (16)DM-001 trpC2 (neo3427)�mreB (spo0A::erm) KS2893 3725 (Em)DM-002 trpC2 (mbl::spc) (spo0A::erm) KS2893 2505 (Sp)DM-003 trpC2 (mreBH::cat) (spo0A::erm) KS2893 2536 (Cm)DM-004 trpC2 (amyE::Pxyl-16.7-gfp spc) (spo0A::Tn917�289, erm) KS2893 DM-000 (Em)DM-009 trpC2 (amyE::Pxyl-gfp-p2 spc) pSGDM23 168 (Sp)DM-010 trpC2 (amyE::Pxyl-gfp-p2 spc) (spo0A::kan) SWV2153 DM-009 (Km)DM-011 trpC2 (amyE::Pxyl-gfp-p2 spc) (neo3427)�mreB (spo0A::Tn917�289, erm) KS289, 37253 DM-009 (Em, Km)DM-012 trpC2 (amyE::Pxyl-gfp-p2 spc) (mbl::neo) (spo0A::Tn917�289, erm) KS289, 168�Mbl3 DM-009 (Em, Km)DM-013 trpC2 (amyE::Pxyl-gfp-p2 spc)) (mreBH::cat) (spo0A::Tn917�289, erm) KS289, 25363 DM-009 (Em, Cm)DM-014 trpC2 (amyE::Pxyl-p2-gfp spc) pSGDM13 168 (Sp)DM-015 trpC2 (amyE::Pxyl-p2-gfp spc) (spo0A::kan) SWV2153 DM-014 (Km)DM-016 trpC2 (amyE::Pxyl-p2-gfp spc) (neo3427)�mreB (spo0A::Tn917�289, erm) KS289, 37253 DM-014 (Em, Km)DM-017 trpC2 (amyE::Pxyl-p2-gfp spc) (mbl::neo) (spo0A::Tn917�289, erm) KS289, 168�Mbl3 DM-014 (Em, Km)DM-018 trpC2 (amyE::Pxyl-p2-gfp spc) (mreBH::cat) (spo0A::Tn917�289, erm) KS289, 25363 DM-014 (Em, Cm)DM-019 trpC2 (mreB-Pxyl cfp-mreB cat) (amyE::Pxyl-yfp-p2 spc) (spo0A::Km) pSG5472, 3748, SWV2153 168 (Sp, Cm, Km)KS289 trpC2 (spo0A::Tn917�289, erm) Sandman et al. (18)MO-101-P thr� spo0A� su� Mellado et al. (19)SWV215 trpC2 pheA1 (spo0A::kan) Xu and Strauch (20)3738 trpC2 (neo3427)�mreB (amyE::Pxyl-mreB-his) Y. Kawai (unpublished)YK827 trpC2 (amyE::Pxyl-mreB-his) (mreB::neo) (spo0A::kan) SWV2153 3738

*Antibiotic resistance gene abbreviations are as follows: neo, neomycin; spc, spectinomycin; cat, chloramphenicol; kan, kanamycin; erm, erythromycin andlincomycin.

†Bacillus Genetic Stock Center‡X3 Y indicates that strain Y was transformed with DNA from source X, with selected marker in parentheses. Cm, chloramphenicol; Sp, spectinomycin; Em,erythromycin; Km, kanamycin.

§Coli Genetic Stock Center

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Table S2. �29 phages used

Phage Source or reference

�29 wild type Laboratory stocksus14(1242) Jiménez et al. (21)sus16.7(48)/sus14(1242) Meijer et al. (3)sus2(513) Moreno et al. (13)SPP1 wild type Juan Carlos Alonso Lab.PRD1 wild type Dennis Bamford Lab.

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Table S3. Plasmids used

Plasmids Relevant features Reference

pSG1154 bla amyE3� spc Pxyl-gfpmut1 amyE5� , C-terminal GFP fusion vector Lewis and Marston (6)pSG1729 bla amyE3� spc Pxyl-gfpmut1� amyE5�, N-terminal GFP fusion vector Lewis and Marston (6)pSG5472 pSG1729 derivative containing yfpmut2 gene instead of gfpmut1 A. Formstone (unpublished)pSGW1 pSG1154 containing 16.7::gfp fusion Meijer et al. (3)pSGDM1 pSG1154 containing p2::gfp fusion This workpSGDM2 pSG1729 containing gfp::p2 fusion This workpSGDM3 pSG5472 containing yfp::p2 fusion This workpUT18 C-terminal T18 fusion vector Karimova et al. (22)pUT18C N-terminal T18 fusion vector Karimova et al. (22)p25N C-terminal T25 fusion vector R. Emmins (unpublished)pKT25 N-terminal T25 fusion vector Karimova et al. (22)pUT18–16.7 pUT18 containing 16.7::T18 This workpUT18C-mreB pUT18C containing T18::mreB This workp25N-16.7 p25N containing 16.7::T25 This workpKT25-mreB pKT25 containing T25::mreB This work

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Table S4. Oligos used

Name Sequence

P2GFP�U (5�-tga agg gta ccc cga gaa aga tgt ata gtt gtg ac-3�)P2GFP�L (5�-aca aaa agc ttt ttg att gtg aat gtg tca tca ac-3�)GFPP2�U (5�-gga tgg tcg aca tgc cga gaa aga tgt ata gtt gt-3�)GFPP2�L (5�-taa acg aat tct tat ttg att gtg aat gtg tca tc-3�)R-OUT-SUPER (5�-aaa tag att ttc ttt ctt ggc tac-3�)R-25 (5�-aaa gta ggg tac agc gac aac ata c-3�)oriR-A (5�-aaa gaa gca gag cca ttc c-3�)oriR-B (5�-cag cct cgc cta gat cag-3�)R-PRD1-A (5�-cgc tat gtg gct tgg gtt gag cgc c-3�)R-PRD1-B (5�-ggg gat acg tgc ccc tcc cc acct a-3�)B2H-16.7-F (5�-atg gta ccc att tca atg acc ccc gat ata gtt ttt tct g-3�)B2H-16.7-R (5�-atg gat cca atg gaa gct att tta atg atc ggt gta ctt gc-3�)B2H-mreB-F (5�-gtc gtc gac tat gtt tgg aat tgg tgc t-3�)B2H-mreB-R (5�-gaa gaa ttc tta tct agt ttt ccc ttt g-3�)

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Movie S1

Movie S1. Protein p16.7 localizes as helix-like structures at the membrane of infected B. subtilis cells. Cells (168 �spo0A) were grown in LB medium containing5 mM MgSO4 at 37 °C. At an OD600 of 0.4 the culture was infected with sus14(1242) mutant �29 phage at a MOI � 5; samples were harvested 20 min later andprocessed for p16.7 immunodetection. Deconvolved stacks of z-sections were assembled in a movie using METAMORPH Version 6 software.

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