Supporting InformationStrahl and Hamoen 10.1073/pnas.1005485107SI Materials and MethodsConstruction of Plasmids and Strains. The strains used in this studyare listed in Table S1. For the localization of the different pro-teins we made use of previously described GFP fusion proteins,except for the following proteins.The strains encoding fusions between GFP and TlpA, KinA, KinB,

and ComKwere constructed by amplification of the correspondinggenes tlpA, kinA, kinB, and comK using oligos (tlpA-for/-rev, kinA-for/-rev, kinB-for/-rev, and comK-for/-rev), followed by cloning intopSG1154 (kinA, kinB, and tlpA) or pSG1729 (comK), using In-Fusion Dry-Down PCR Cloning Kit (Clontech). The plasmidspSG1154 and pSG1729 were linearized using primers pSG1154-for/-rev andpSG1729-for/-rev. Finally, the plasmidswere integratedintoB. subtilis 168 resulting in strainsB. subtilisHS27-30. The strainsencoding SecA and Hbs GFP fusion proteins were constructed byamplification of secA using oligos secA-for/rev and hbs using oligoshbs-for/rev followed by cloning into pSG1154 (secA) and pJWV017(hbs) using restriction sites Asp718/SpeI (secA) and EcoRI/NheI(hbs), and transformation into B. subtilis 168. The strain B. subtilisHM160 was constructed by double crossover integration ofpHM110 into HM31 (1). pHM110 encodes a neomycin resistancecassette and the soj-spo0J operon with spo0J fused to gfp. The polarlocalization of the chemoreceptor TlpA, and the nucleoid locali-zation of the transcription factor ComK fusion proteins were aspredicted indicating the ability of the fusion protein to localizecorrectly. The functionality of KinA and KinB fusion proteins wasverified by the ability to induce sporulation upon overexpression.A B. subtilis strain allowing the use of immunofluorescence mi-

croscopyofFtsAwas constructedby introducing aFlag-epitope inNterminus of FtsA. The strain B. subtilisHS21 encodes an induciblecopy of ftsA with a N-terminal Flag-epitope (MDYKDDDDK-GS-FtsA), and itwas constructedusing primersflag-ftsA-for and ftsA-revfollowedby cloning intopSG1729 linearizedwith primerspSG1729-for and pSG1154-rev using In-Fusion Dry-Down PCR Cloning Kit(Clontech) thereby replacing the gfp with flag-ftsA. The activity ofthe fusion was verified by complementation of theΔftsA phenotypein B. subtilis YK206 strain background.To uncouple the dissipation of pmf from the decrease in cellular

ATP levels, a strain B. subtilis HS13 harboring a disruption of theatpIBEFHAGDC-operon encoding the F1Fo ATP synthase wasconstructed by homologous integration of pMutin4. The corre-sponding plasmid was constructed by amplifying an atpB-fragmentusing primers atpB-for and atpB-rev and cloning into pMutin4 inreverse complementary direction using restriction sites HindIIIand BamHI.A GFP fusion protein allowing the analysis of the localization

of E. coli FtsA was constructed. The plasmid pHJS101 coding fora fusion protein between superfolderGFP (sfGFP) and E. coliFtsA with a flexible SGSGSG-linker was constructed by two-stepPCR using primers sfgfp-for/rev and ftsA-for/rev followed by li-gation into pBAD322/sfGFP using restrictions sites EcoRI andNotI thereby replacing the sfgfp coding sequence with sfgfp-ftsA.The activity of the fusion protein was verified by the ability to lo-calize correctly and to complement both growth and cell morphol-ogy in a temperature sensitive FtsA strain (E. coli PS236) undernonpermissive conditions.Several amino acid exchanges allowing a more detailed analysis

of the pmf-dependency of GFP-MinD were constructed. Thestrain B. subtilis HS15 encoding a K16A-point mutation in GFP-MinD was constructed by incorporation of the correspondingmutation in plasmid pSG1730 using primers minDK16A-for andminDK16A-rev and QuikChange Site-Directed Mutagenesis Kit

(Stratagen) followed by integration into B. subtilis 1901 strain.The strains B. subtilis HS16 and HS17 encoding I260E, E250Q,and E251Q amino acid exchanges in GFP-MinD were constru-cted accordingly using primers minDI260E-for, minDI260E-rev,minDEE250-251QQ-for, minDEE250-251QQ-rev.To analyze the membrane binding of the amphipathic helix of

B. subtilis MinD in E. coli, the plasmid pHJS100 encoding anarabinose inducible fusion between GFP, JunLZ, and B. subtilisMinD-membrane targeting amphipathic helix (MinD248–268, MTS)was constructed. The E. coliMinD-MTS coding region on plasmidpTS37 was replaced with B. subtilis MinD-MTS by amplificationof the corresponding sequence using primers BsMTS-for andBsMTS-rev followed by cloning into pTS37 using restriction sitesPtsI and HindIII.

Cellular ATP Measurements. For cellular ATP level measurements,B. subtilis wild type and F1Fo ATP synthase-deficient strainswere grown at 30 °C in LB supplemented with 0.1% glucoseto OD600 = 0.5. ATP levels after addition of 100 μM CCCP dis-solved in DMSO or 0.1% DMSO were measured using ATP bio-luminescence Assay Kit HSII (Roche Applied Science) and BMGFluostar Optima luminometer/fluorimeter following manu-facturers’ instructions. The absence of cell lysis during the timeperiod of sample collectionwas verified bymeasurement of opticaldensity and microscopy. The different samples were equalizedusing quantification of whole cell protein content using Bio-RadProtein Assay Kit (Bio-Rad). All measurements were carried outin triplicate.

Fluorescence Spectroscopy. For fluorimetric measurement of ΔΨ,the cells were grown in LB supplemented with 50mMHepes-HCl,pH 7.5, 300 mMKCl and 0.1% glucose. In midlog phase, the cellswere harvested and twofold concentratedwith 50mMHepes-HCl,pH 7.5, 300 mM KCl and 0.1% glucose. ΔΨ was measured quali-tatively by following the electrophoretic accumulation of themembrane permeable cationic potential sensitive dye DiSC3(5)(1 μM) in glucose energized cells, and the release of the dye afterdissipation of ΔΨ by 30 μM valinomycin using BGM FluostarGalaxy fluorimeter, excitation 544–10 nm, emission 660–10 nm(Fig. S7A). The level ofΔΨ induced in liposomes, the ability of theliposomes to maintain ΔΨ, and the dissipation of ΔΨ by nigericinwasmeasured accordingly (Fig. S7C, seeMaterials andMethods fordetails). For fluorimetric measurement of the cytoplasmic pH, thecells were grown in LB supplemented with 50mMHepes-HCl, pH7.5, 300mMKCl and 0.1%glucose. Inmidlog phase, the cells wereharvested and resuspended in LB supplemented with 300mMKCland 0.1% glucose and buffered with 50mM Hepes-HCl pH 7.5–7or 50mM Mes-NaOH pH 6.5–6. The pH sensitive dye BCECF-AM (16 μM) was internalized during 30 min incubation undershaking and the relative pH-dependent cytoplasmic fluorescenceof BCECFwas measured in triplicate using BGMFluostar Galaxyfluorimeter, excitation 485–12 nm, emission 520–10 nm. The acid-ification of the cytoplasm in low medium pH as a result of the H+

transport activity of nigericin wasmeasured after 2min incubationwith 5 μM nigericin (dissolved in DMSO). As a control, the ab-sence of acidification by DMSO (0.1%) was measured. The valuesare presented relative to the untreated sample (100%).

Immunofluorescence Microscopy. For immunolocalization of FtsA,cells expressing a single copy of FtsA with an N-terminal Flagepitope (B. subtilisHS21) were grown and the pmf dissipated withCCCP as described for GFP fusion proteins. Cells were fixed and

Strahl and Hamoen www.pnas.org/cgi/content/short/1005485107 1 of 11

permeabilized essentially as described by Pogliano et al. (2). Theused antibodies were monoclonal mouse anti-FlagM2 (1:3,000,Sigma-Aldrich) and donkey anti-mouse Alexa488 (1:10,000, Mo-lecular Probes). The specificity of used antibodies and protocol

were verified by using wild-type B. subtilis as a control. The cellsamples were mounted in Vector Shield (Vector Laboratories)and visualized immediately using epifluorescence and phase-contrast microscopy.

1. Murray H, Errington J (2008) Dynamic control of the DNA replication initiation proteinDnaA by Soj/ParA. Cell 135:74–84.

2. Pogliano K, Harry E, Losick R (1995) Visualization of the subcellular location ofsporulation proteins in Bacillus subtilis using immunofluorescence microscopy. MolMicrobiol 18:459–470.

A

B

Fig. S1. Influence of different glass slide supports for microscopy imaging on membrane potential and MinD localization. (A) Visualization of membranepotential using potential sensitive fluorescent dye DiSC3(5) in B. subtilis cells immobilized on agarose-pad (Left) or with poly-l-lysine (Right). DiSC3(5) accu-mulates in cells with high membrane potential. (B) Cellular localization of GFP-MinD in cells (B. subtilis 1981) immobilized on agarose-pad (Left) or with poly-l-lysine (Right).

Strahl and Hamoen www.pnas.org/cgi/content/short/1005485107 2 of 11

Mbl

Mbl/CCCP

MreBH

MreBH/CCCP

MreD

MreD/CCCP

MinC

MinC/CCCP Soj/CCCP

Soj FtsZ

FtsZ/CCCP

MreC

MreC/CCCP

FtsA (IFM) FtsA (IFM)/CCCP

A

B MreB MreC MreD

MreB/CCCP MreC/CCCP MreD/CCCP

Fig. S2. Pmf-dependent localization of proteins. (A) Cellular localization of B. subtilis MinC, Soj, FtsZ, Mbl, MreBH, MreD, and MreC GFP fusion proteins andimmunofluorescence microscopy (IFM) of FtsA in the absence and presence of CCCP (100 μM). Strains used: B. subtilis 1999 (GFP-MinC), B. subtilis HM4 (GFP-Soj),B. subtilis 2020 (GFP-FtsZ), B. subtilis HS21 (Flag-FtsA), B. subtilis 3751 (YFP-Mbl), B. subtilis 3750 (YFP-MreBH), B. subtilis 3416 (GFP-MreD), and B. subtilis 3417(GFP-MreC). (B) Cellular localization of E. coli MreB, MreC and MreD YFP-fusion proteins (deconvolved images) in the absence or presence of CCCP (100 μM).Strains used: E. coli MC1000/pLE7 (YFP-MreB), E. coli MC1000/pVP1 (YFP-MreC) and E. coli MC1000/pVP2 (YFP-MreC).

Strahl and Hamoen www.pnas.org/cgi/content/short/1005485107 3 of 11

FtsA FtsZZap

ASep

FEzrA

Pbp2B

0

10

20

30

40no CCCP2 min CCCP6 min CCCP

% a

t sep

tum

whole cell fluorescence

septal

non septal

% at septum=septal-non septal

whole cell 100*

FtsA

FtsZ

ZapA

SepF

EzrA

Pbp2B

no CCCP 2 min CCCP 6 min CCCP

A

B

C

Fig. S3. Quantification of septal localization of cell division proteins after dissipation of the pmf. (A) Schematic representation of septal localization mea-surement from fluorescence micrographs. (B) Quantification of the percentage of the fluorescence signal localized at septa for YFP-FtsA, GFP-FtsZ, YFP-ZapA,SepF-GFP, EzrA-GFP, and GFP-Pbp2B in untreated cells, and after 2 and 6 min of dissipation of the pmf with CCCP (n = 30 cells). (C) Representative images ofcells used to quantify the effect of CCCP (100 μM) on septal localization. Strains used: B. subtilis PG62 (YFP-FtsA), B. subtilis 2020 (GFP-FtsZ), B. subtilis PG67 (YFP-ZapA), B. subtilis 4181 (SepF-GFP), B. subtilis 3312 (EzrA-GFP), and B. subtilis 3122 (GFP-Pbp2B).

Strahl and Hamoen www.pnas.org/cgi/content/short/1005485107 4 of 11

FtsA

MinDMbl

MreBH

MinC

Soj

FtsZ

F1Fo ATP-synthasedeficient B. subtilis strain -CCCP +CCCP

F1Fo ATP-synthasedeficient E. coli strain

-CCCP +CCCP

A B

Fig. S4. Pmf-dependent localization of proteins is independent of ATP levels. (A) Localization of YFP-Mbl, YFP-MreBH, GFP-MinC, GFP-Soj, and GFP-FtsZproteins in the F1Fo ATP synthase-deficient B. subtilis strain in the presence (Right) or absence (Left) of CCCP (100 μM). Strains used: B. subtilis HS24 (YFP-Mbl), B.subtilis HS25 (YFP-MreBH), B. subtilis HS25 (GFP-MinC), B. subtilis HS26 (GFP-Soj), and B. subtilis HS19 (GFP-FtsZ). (B) Localization of GFP-MinD and GFP-FtsA inthe F1Fo ATP synthase-deficient E. coli strain in the presence (Right) or absence (Left) of CCCP (100 μM). Strains used: E. coli DK8/pFX9 (GFP-MinD), and E. coliDK8/pHJS101 (GFP-FtsA).

A

untreated colicin N

MinD

FtsA

MreB

DiSC3(5)

FtsA

MinD

BB. subtilis E. coliuntreated nisin

Fig. S5. Nisin and colicin N-dependent localization MinD, FtsA, and MreB. (A) Localization of B. subtilis GFP-MinD, YFP-FtsA and GFP-MreB in the presence(Right) or absence (Left) of the channel forming peptide antibiotic nisin (375 nM). Strains used: B. subtilis 1981 (GFP-MinD), B. subtilis PG62 (YFP-FtsA), B. subtilisYK405 (GFP-MreB), and B. subtilis 168 [for visualization of membrane potential using ΔΨ-dependent dye DiSC3(5)]. (B) Localization of E. coli GFP-MinD and GFP-FtsA in the presence (Right) or absence (Left) of the bacteriocin colicin N (1000 molecules/cell). Strains used: E. coli RC1/pFX9 (GFP-MinD) and E. coli MC1000/pHJS101 (GFP-FtsA).

Strahl and Hamoen www.pnas.org/cgi/content/short/1005485107 5 of 11

+nigericin +valinomycin valinomycin valinomycin hyperpolarization depolarization

MinD

FtsA

MreB

DiSC3(5)

A B

Mbl

MreBH

MinC

Soj

FtsZ

Fig. S6. Pmf-dependent localization is based on the membrane potential (ΔΨ). (A) Cellular localization of YFP-Mbl, YFP-MreBH, GFP-MinC, GFP-Soj, and GFP-FtsZ in B. subtilis cells in the presence of the ΔΨ-dissipating ionophore valinomycin (30 μM) or in the presence of the ΔpH-dissipating ionophore nigericin(5 μM). All images were taken within 2 min after addition of the ionophores. Strains used: B. subtilis 3751 (YFP-Mbl), B. subtilis 3750 (YFP-MreBH), B. subtilis1999 (GFP-MinC), B. subtilis HM4 (GFP-Soj), and B. subtilis 2020 (GFP-FtsZ). (B) Cellular localization of B. subtilis GFP-MinD, YFP-FtsA and GFP-MreB in thepresence of valinomycin in medium supplemented with 300 mM NaCl (hyperpolarization of ΔΨ resulting from diffusion of K+ out of the cell), and 300 mM KCl(depolarization of ΔΨ resulting from diffusion of K+ into the cell). The level of membrane potential is visualized using the membrane potential sensitive dyeDiSC3(5). Strains used: B. subtilis 1981 (GFP-MinD), B. subtilis PG62 (YFP-FtsA), B. subtilis YK405 (GFP-MreB), and B. subtilis 168 [for visualization of membranepotential using ΔΨ-dependent dye DiSC3(5)].

Strahl and Hamoen www.pnas.org/cgi/content/short/1005485107 6 of 11

0 2 4 60

2,000

4,000

6,000

8,000

10,000 Δψ-dependent dye uptake Dye release due to low Δψ

+DISC3(5) +valinomycin

A

0 2 4 6 8 100

500

1,000

1,500

2,000

2,500

time [min]

+DiSC3(5)+valinomycin +nigericin

DIS

C3(

5) -fl

uore

scen

ce

time [min]

C

B

6.0 6.5 7.0 7.50

25

50

75

100

D MS Oniger ic in

medium pH

% B

CEC

F-flu

ores

cenc

eD

ISC

3(5)

-fluo

resc

ence

Fig. S7. Fluorimetric measurement of the activity of valinomycin and nigericin. (A) The membrane potential in B. subtilis cells is measured as the cytoplasmicaccumulation of the ΔΨ-sensitive fluorescent dye DiSC3(5), which results in fluorescence quenching. Dissipation of ΔΨ by valinomycin (30 μM) is measured asrelease of the dye to the medium resulting in an increase in fluorescence. (B) The activity of nigericin (5 μM) is measured as nigericin-dependent acidification ofthe cytoplasm in the presence of low medium pH using the pH sensitive dye BCECF-AM. As a control, the absence of the acidification is measured for DMSOthat is used as the solvent for nigericin. (C) The generation of an artificial membrane potential in liposomes using valinomycin (0.2 μM) is measured as theΔΨ-dependent accumulation and subsequent fluorescence quenching of the dye DiSC3(5). The dissipation of the ΔΨ by nigericin (1 μM) is measured as thereduction of the fluorescence quenching due to the release of the dye into the buffer.

Strahl and Hamoen www.pnas.org/cgi/content/short/1005485107 7 of 11

MinD

MinD+CCCP

MinDI260E

MinDI260E

+CCCP

apolar charged polar

KG

M

M

A

K

I

K

S

FF

wild type

KG

M

M

A

I

K

S

F

KG

M

M

A

KK

S

F

K

FF

E

I260EE250QE251Q

G G G

MinDE250-251Q

MinDE250-251Q

+CCCP

Fig. S8. Localization of GFP-MinD carrying the amino acid exchanges I260E and E250Q/E251Q in B. subtilis cells. Sequence of the wild-type amphipathic helixand of variants carrying I260E and E250Q/E251Q-substitutions, and the predicted amphipathical regions are underlined and presented as helical wheel dia-grams (Upper). The cellular localization of GFP-MinD, GFP-MinDI260E and GFP-MinDE250Q, E251Q in the presence or absence of 100 μM CCCP is shown (Lower).Strains used: B. subtilis 1981 (GFP-MinD), B. subtilis HS16 (GFP-MinDI260E), and B. subtilis HS17 (GFP-MinDE250Q, E251Q).

before

30min

60min

30minrecovery

MinD FtsA FtsZ MreB DiSC3(5)

MinD

FtsA

FtsZ

MreB

DiSC3(5)

MinD FtsA FtsZ MreB DiSC3(5)

MinD FtsA FtsZ MreB DiSC3(5)

Fig. S9. Oxygen dependent localization of B. subtilis MinD, FtsA, and MreB. Depletion for oxygen (30 min) results in dissipation of membrane potential asvisualized by the membrane potential sensitive dye DiSC3(5) (Right). Delocalization of GFP-MinD, YFP-FtsA, and GFP-MreB after 30 and 60 min is shown (Left).GFP-FtsZ is visualized as a control for the presence of septa. Reaeration (30 min) is sufficient to regenerate the membrane potential, and to restore proteinlocalization. Strains used: B. subtilis 1981 (GFP-MinD), B. subtilis PG62 (YFP-FtsA), B. subtilis 2020 (GFP-FtsZ), B. subtilis YK405 (GFP-MreB), and B. subtilis 168 [forvisualization of membrane potential using the ΔΨ-dependent dye DiSC3(5)].

Strahl and Hamoen www.pnas.org/cgi/content/short/1005485107 8 of 11

Table S1. Strains and plasmids

Relevant genotype/properties

Usedinduction

Source/construction

StrainsB. subtilis 168 trpC2 wild type − lab collectionB. subtilis HS13 erm atpB::pMutin4 − this workB. subtilis 1901 erm minD::ermC − 1B. subtilis RD021 tet minJ::tet − 2B. subtilis 1981 erm spc minD::ermC

amyE::Pxyl-gfp-minD0.1% xylose 1

B. subtilis HS14 erm spc atpB::pMutin4amyE::Pxyl-gfp-minD

0.1% xylose this work

B. subtilis HS15 erm spc minD::ermCamyE::Pxyl-gfp-minDK16A

0.1% xylose this work

B. subtilis HS16 spc amyE::Pxyl-gfp-minDI260E 0.1% xylose this workB. subtilis HS17 erm spc minD::ermC

amyE::Pxyl-gfp-minDE250Q, E251Q

0.1% xylose this work

B. subtilis 1803 cat divIVA-gfp − 3B. subtilis MB002 spc amyE::Pxyl-minJ-gfp 0.1% xylose 2B. subtilis HS18 tet spc minJ::tet

amyE::Pxyl-gfp-minD0.1% xylose this work

B. subtilis 2020 spc amyE::Pxyl-gfp-ftsZ 0.1% xylose 4B. subtilis HS19 erm spc atpB::pMutin4

amyE::Pxyl-gfp-ftsZ0.1% xylose this work

B. subtilis 3122 cat pbpB::pSG5061(Pxyl-gfp-pbpB)

0.1% xylose 5

B. subtilis PG62 spc aprE::Pspac-yfp-ftsA 0.1 mM IPTG 6B. subtilis HS20 erm spc atpB::pMutin4

aprE::Pspac-yfp-ftsA0.1 mM IPTG this work

B. subtilis YK206 ftsA::erm Pspac-ftsZ − 7B. subtilis HS21 erm spc ftsA::erm Pspac-FtsZ

amyE::Pxyl-flag-ftsA0.1% xylose this work

B. subtilis PG67 spc aprE::Pspac-yfp-zapA 0.1 mM IPTG 6B. subtilis 3312 cat ezrA-gfp − 6B. subtilis 4181 spc amy::Pxyl-sepF-gfp 0.1% xylose 8B. subtilis 1999 spc amyE::Pxyl-gfp-minC minD 0.1% xylose 9B. subtilis HS22 erm spc atpB::pMutin4

amyE::Pxyl-gfp-minC minD0.1% xylose this work

B. subtilis YK405 spc amyE::Pxyl-gfp-mreB 0.3% xylose 10B. subtilis HS23 erm spc atpB::pMutin4

amyE::Pxyl-gfp-mreB0.3% xylose this work

B. subtilis 3751 spc amyE::Pxyl-yfp-mbl 0.3% xylose 11B. subtilis HS24 erm spc atpB::pMutin4

amyE::Pxyl-yfp-mbl0.3% xylose this work

B. subtilis 3750 spc amyE::Pxyl-yfp-mreBH 0.3% xylose 11B. subtilis HS25 erm spc atpB::pMutin4

amyE::Pxyl-yfp-mreBH0.3% xylose this work

B. subtilis 3416 cat mreD::Pxyl-gfp-mreD 0.3% xylose 12B. subtilis 3417 cat mreC::Pxyl-gfp-mreC 0.3% xylose 12B. subtilis HM160 neo spo0J-gfp − this workB. subtilis HM4 neo gfp-soj − 13B. subtilis HS26 erm neo atpB::pMutin4 gfp-soj − this workB. subtilis

PolC-GFPspc amyE::Pxyl-polC-gfp 0.1% xylose 14

B. subtilis JK03 cat clpP–gfp − 15B. subtilis JK05 cat clpC–yfp − 15B. subtilis JK06 cat clpX–yfp − 15B. subtilis BS23 cat atpA–gfp − 16B. subtilis HS27 spc amyE::Pxyl-tlpA-gfp 0.1% xylose this workB. subtilis HS28 spc amyE::Pxyl-kinA-gfp 0.01% xylose this workB. subtilis HS29 spc amyE::Pxyl-kinB-gfp 0.01% xylose this workB. subtilis HS30 spc amyE::Pxyl-gfp-comK 0.1% xylose this workB. subtilis

JWV042cat amyE::Phbs-hbs-gfp − this work

Strahl and Hamoen www.pnas.org/cgi/content/short/1005485107 9 of 11

Table S1. Cont.

Relevant genotype/properties

Usedinduction

Source/construction

B. subtilisBSN103

spc amyE::Pxyl-secA-gfp 0.1% xylose this work

E. coli MC1000 Δlac Δara − lab collectionE. coli RC1 Δlac Δara ΔminCDE − 17E. coli PB114 ΔminB (minCDE) − 18E. coli DK8 tet ilv::Tn10 ΔatpBEFHAGDC − 19E. coli PS236 W3110 ftsA12 leu::Tn10 − 20C. crescentus

LS3814neo xylX::Pxyl-gfp-mreB 0.03% xylose 21

PlasmidspSG1730 bla Pxyl-gfp-minD 0.1% xylose 1pFX9 bla Plac-gfp-minD minE 10 μM IPTG 22pTS37 cat Paragfp-junLZ-EcMTS256–270 0–0.1% arabinose 23pHJS100 cat Paragfp-junLZ-BsMTS248–268 0–0.1% arabinose this workpVP1 bla Plac-yfp-mreC 20 μM IPTG 24pVP2 bla Plac-yfp-mreD 20 μM IPTG 24pBAD322 bla rop araC Para − 25pHJS101 bla rop araC Parasfgfp-ftsA − this workpLE7 bla Plac-yfp-mreB 20 μM IPTG 24pMutin4 bla ery lacI Pspac lacZ − 26pSG1154 bla spc amyE3′ Pxyl-’gfpmut1 amyE5′ − 27pSG1729 bla spc amyE3′ Pxyl-gfpmut1’ amyE5′ − 27pJWV017 cat spec amyE3′ gfp amyE5′ − 28

1. Marston AL, Thomaides HB, Edwards DH, Sharpe ME, Errington J (1998) Polar localization of the MinD protein of Bacillus subtilis and its role in selection of the mid-cell division site.Genes Dev 12:3419–3430.

2. Bramkamp M, et al. (2008) A novel component of the division-site selection system of Bacillus subtilis and a new mode of action for the division inhibitor MinCD. Mol Microbiol 70:1556–1569.

3. Edwards DH, Thomaides HB, Errington J (2000) Promiscuous targeting of Bacillus subtilis cell division protein DivIVA to division sites in Escherichia coli and fission yeast. EMBO J 19:2719–2727.

4. Stokes NR, et al. (2005) Novel inhibitors of bacterial cytokinesis identified by a cell-based antibiotic screening assay. J Biol Chem 280:39709–15.5. Scheffers DJ, Jones LJ, Errington J (2004) Several distinct localization patterns for penicillin-binding proteins in Bacillus subtilis. Mol Microbiol 51:749–764.6. Gamba P, Veening JW, Saunders NJ, Hamoen LW, Daniel RA (2009) Two-step assembly dynamics of the Bacillus subtilis divisome. J Bacteriol 191:4186–4194.7. Ishikawa S, Kawai Y, Hiramatsu K, Kuwano M, Ogasawara N (2006) A new FtsZ-interacting protein, YlmF, complements the activity of FtsA during progression of cell division in Bacillus

subtilis. Mol Microbiol 60:1364–1380.8. Hamoen LW, Meile JC, de Jong W, Noirot P, Errington J (2006) SepF, a novel FtsZ-interacting protein required for a late step in cell division. Mol Microbiol 59:989–999.9. Marston AL, Errington J (1999) Selection of the midcell division site in Bacillus subtilis through MinD-dependent polar localization and activation of MinC. Mol Microbiol 33:84–96.10. Kawai Y, Daniel RA, Errington J (2009) Regulation of cell wall morphogenesis in Bacillus subtilis by recruitment of PBP1 to the MreB helix. Mol Microbiol 71:1131–1144.11. Carballido-López R, et al. (2006) Actin homolog MreBH governs cell morphogenesis by localization of the cell wall hydrolase LytE. Dev Cell 11:399–409.12. Leaver M, Errington J (2005) Roles for MreC and MreD proteins in helical growth of the cylindrical cell wall in Bacillus subtilis. Mol Microbiol 57:1196–1209.13. Murray H, Errington J (2008) Dynamic control of the DNA replication initiation protein DnaA by Soj/ParA. Cell 135:74–84.14. Migocki MD, Lewis PJ, Wake RG, Harry EJ (2005) The midcell replication factory in Bacillus subtilis is highly mobile: Implications for coordinating chromosome replication with other

cell cycle events. Mol Microbiol 54:452–463.15. Kirstein J, Strahl H, Molière N, Hamoen LW, Turgay K (2008) Localization of general and regulatory proteolysis in Bacillus subtilis cells. Mol Microbiol 70:682–694.16. Johnson AS, van Horck S, Lewis PJ (2004) Dynamic localization of membrane proteins in Bacillus subtilis. Microbiology 150:2815–2824.17. Rowland SL, et al. (2000) Membrane redistribution of the Escherichia coli MinD protein induced by MinE. J Bacteriol 182:613–619.18. de Boer PAJ, Crossley RE, Rothfield LI (1989) A division inhibitor and a topological specificity factor coded for by the minicell locus determine proper placement of the division septum

in E. coli. Cell 56:641–649.19. Klionsky DJ, Brusilow WS, Simoni RD (1984) In vivo evidence for the role of the ε subunit as an inhibitor of the proton-translocating ATPase of Escherichia coli. J Bacteriol 160:

1055–1060.20. Pichoff S, Lutkenhaus J (2002) Unique and overlapping roles for ZipA and FtsA in septal ring assembly in Escherichia coli. EMBO J 21:685–693.21. Gitai Z, Dye N, Shapiro L (2004) An actin-like gene can determine cell polarity in bacteria. Proc Natl Acad Sci USA 101:8643–8648.22. Shih YL, Le T, Rothfield L (2003) Division site selection in Escherichia coli involves dynamic redistribution of Min proteins within coiled structures that extend between the two cell

poles. Proc Natl Acad Sci USA 100:7865–7870.23. Szeto TH, Rowland SL, Habrukowich CL, King GF (2003) The MinD membrane targeting sequence is a transplantable lipid-binding helix. J Biol Chem 278:40050–40056.24. Vats P, Shih YL, Rothfield L (2009) Assembly of the MreB-associated cytoskeletal ring of Escherichia coli. Mol Microbiol 72:170–182.25. Cronan JE (2006) A family of arabinose-inducible Escherichia coli expression vectors having pBR322 copy control. Plasmid 55:152–157.26. Vagner V, Dervyn E, Ehrlich SD (1998) A vector for systematic gene inactivation in Bacillus subtilis. Microbiology 144:3097–3104.27. Lewis PJ, Marston AL (1999) GFP vectors for controlled expression and dual labelling of protein fusions in Bacillus subtilis. Gene 227:101–110.28. Veening JW, Murray H, Errington J (2009) A mechanism for cell cycle regulation of sporulation initiation in Bacillus subtilis. Genes Dev 23:1959–1970.

Strahl and Hamoen www.pnas.org/cgi/content/short/1005485107 10 of 11

Table S2. Oligos/peptides

Name Sequence

atpB-for GCGCGAAGCTTGATGATTACTGTGGCGAGTGatpB-rev GCGCGGGATCCCGGCGAAGATGTTACCATACminDK16A-for GCGGAGTAGGTGCGACAACAACATCTGminDK16A-rev CAGATGTTGTTGTCGCACCTACTCCGCminDEE250-251QQ-for CCTTTACAGGTGCTTCAACAGCAAAACAAAGGAATGATGminDEE250-251QQ-rev CATCATTCCTTTGTTTTGCTGTTGAAGCACCTGTAAAGGBsMTS-for GCGCGCTGCAGGTGCTTGAAGAGCAAAACAAAGGBsMTS-rev GCGCGAAGCTTAAGATCTTACTCCGAAAAATGACtlpA-for GATGAACTATACAAAATGAAAAAAACACTCACCACTATTCtlpA-rev GGGCCCGTGGATCCGTTATTTGTCTACTTTAAATTGTTTTGTCAGkinA-for GAGATTCCTAGGATGGAACAGGATACGCAGCATGkinA-rev TTCTCCTTTACTCATTTTTTTTGGAAATGAAATTTTAAACGCkinB-for GAGATTCCTAGGATGGAAATTCTAAAAGACTATCTTCkinB-rev TTCTCCTTTACTCATGTGAGGAAGATCAGCGGGAAGcomK-for GATGAACTATACAAAATGAGTCAGAAAACAGACGCACcomK-rev GGGCCCGTGGATCCGCTAATACCGTTCCCCGAGCTCACsecA-for ATGCTTGGTACCTTAAATAAAATGTTTGATCCAACsecA-rev ATATGTCGACTTCAGTACGGCCGCAGChbs-for GCGCGAATTCGGCTTAATCGCCATCATCChbs-rev GCTCGCTAGCTCCAGCTTTAGCTGCAGCTTCTCCACCAGATCC

TTTTCCGGCAACTGCGTCTTTAAGCGCTTTACCpSG1154-for CATCCTAGGAATCTCCTTTCTAGpSG1154-rev ATGAGTAAAGGAGAAGAACTTTTCACpSG1729-for TTTGTATAGTTCATCCATGCCATGTGpSG1729-rev CGGATCCACGGGCCCCCCCTCsfgfp-for GGAGGAATTCATGAGCAAAGGsfgfp-rev CCTGAGCCGCTTCCTGATTTGTAGAGCTCATCCATGCCftsA-for CAGGAAGCGGCTCAGGAATGATCAAGGCGACGGACAGftsA-rev GCGCGGCGGCCGCGGTTAAAACTCTTTTCGCAGCCAACTATTGflag-ftsA-for GAGATTCCTAGGATGGACTATAAAGACGATGACGATA

AAGGATCAATGACAACAATGAACTTTACGTCftsA-rev GGGCCCGTGGATCCGCTATTCCCAAAACATGCTTAATAGMTS1 5-FAM-VLEEQNKGMMAKIKSFFGVRS*MTS2 5-FAM-VLQQQNKGMMAKIKSFFGVRS*MTS3 5-FAM-VLEEQNKGMMAKEKSFFGVRS*

*5-carboxyfluorescein-labeled synthetic peptides corresponding to B. subtilis MinD248–268.

Movie S1. Pmf-dependent pole to pole-oscillation of E. coli GFP-MinD. Time lapse of the GFP-MinD pole-to-pole oscillation in untreated E. coli cells (Left), andin the presence of 100 μM CCCP (Center) or 250 molecules/cell of colicin N (Right). The oscillation was recorded in MinCDE-deficient (Upper), and F1Fo ATPsynthase deficient (Lower) strain backgrounds. Strains used: E. coli RC1/pFX9 (ΔminCDE) and E. coli DK8/pFX9 (ΔatpB-C).

Movie S1

Strahl and Hamoen www.pnas.org/cgi/content/short/1005485107 11 of 11