wild-type ∆spoIIIE

Figure S1A Doan et al.

T2

T2.5

wild-type ∆spoIIIE

Figure S1B Doan et al.

T2

T2.5

wt ΔfisB

T 2

T 2.

5 T

3 T

3.5

T 4

T 4.

5 T

1.5

phase membranes CFP membranes phase CFP

Figure S2 Doan et al.

FM4-64 TMA-DPH TMA-DPH + CFP

Figure S3 Doan et al.

Figure S4 Doan et al.

0  

50  

100  His-FisBECD His-FisBFL

A28

0nm

(mA

U)

A28

0nm

(AU

)

monomer

Elution volume (mL)

0  

50  

100  

150  

200 66

13.6

Elution volume (mL)

monomer

oligomer oligomer

YFP-FisB membranes merge

Figure S5 Doan et al.

T2

T2.5

T3

Figure S6 Doan et al.

-IPTG

+IP

TG

S10

0

FT

P10

0

Frx

1

Frx

2

Frx

3

Frx

4

His-FisB

M -IPTG

+IP

TG

S10

0

FT

P10

0

Frx

1

Frx

2

Frx

3

Frx

4

mock

M

anti-HIS

Frx

1 Fr

x 2

Frx

3 Fr

x 4

Frx

1 Fr

x 2

Frx

3

Frx

4

mock His-FisB

Frx

1

Frx

2

Frx

3

Frx

4

Frx

1

Frx

2

Frx

3

Frx

4

mock His-FisB B

A

Figure S7 Doan et al.

30

25

20

15

10

5

0 10 30 50 70 90 110

time (min)

% m

axim

um fl

uore

scen

ce

Figure S8 Doan et al.

YFP

-Fis

B

mem

bran

es

mer

ge

wt spoIIIE36 ΔspoIIIE B

FisB

σF

wt

2 2.5

3 3.5

4

spoIIIE36 ΔspoIIIE

2 2.5

3 3.5

4 2 2.5

3 3.5

4 Δfis

B

1.5

1.5

1.5

A

Figure S9 Doan et al.

Figure S10 Doan et al.

mem

bran

es

mer

ge

cfp σG

CFP

wt ΔfisB

σG

σG

Figure S11 Doan et al.

wt ∆fisB ∆sigG ∆fisB ∆sigG ∆fisB ∆sigK

mem

bran

es

phas

e m

erge

3.8% 30.9% 4.3% 4.2% 27.8% % lysis:

1  

SUPPLEMENTAL MATERIAL

SUPPLEMENTAL FIGURE LEGENDS

Figure S1. Cells lacking SpoIIIE have defects in membrane migration. Membranes from

a wild-type (PY79) and a SpoIIIE null mutant (BDR1066) were visualized with TMA-

DPH at hour 2 (top) and 2.5 (bottom) of sporulation. Examples of sporulating cells in

which membrane migration is impaired are highlighted (yellow carets). The images are

from cells mounted on polylysine-treated coverslips (A) or on agarose pads containing

resuspension medium (B).

Figure S2. Cells lacking FisB are specifically impaired in membrane fission. Membrane

migration and fission were monitored by fluorescence microscopy during a sporulation

time course. Cells were imaged every 30 min from hour 1.5 to 4.5 in wild-type (BKM15)

and a FisB mutant (BTD3200). Both strains contained a fluorescent forespore reporter

(PspoIIQ-cfp) to detect all forespores. The membranes (false-colored red) were visualized

with FM4-64 and merged with the CFP signal (false-colored green). Yellow carets

Examples of sporulating cells that had undergone membrane fission are highlighted

(yellow carets). Examples of cell lysis are indicated (red carets).

Figure S3. Comparison of the membrane dyes FM4-64 and TMA-DPH. Wild-type

sporulating cells (BKM15) at hour 3 of sporulation were stained with FM4-64 and TMA-

DPH. This strain contained a fluorescent forespore reporter (PspoIIQ-cfp) to detect all

forespores. Examples of sporulating cells that had undergone membrane fission are

highlighted (yellow carets). In these cells the forespore membranes are not detectable by

FM4-64 but are weakly stained by TMA-DPH.

Figure S4. FisB forms high molecular weight oligomers in vitro. Detergent-solubilized

full-length His-tagged FisB (His-FisBFL) and soluble His-tagged FisB extracellular

domain (His-FisBECD) were purified by affinity chromatography on Ni2+-NTA agarose

and then analyzed by size-exclusion chromatography on a Superdex 200 10/300 GL

column. All fractions were analyzed by immunoblot (not shown). Monomer and

2  

oligomers are indicated. In both cases, the proteins were soluble after centrifugation at

100,000Xg. For His-FisBECD, the elution volumes for three standard proteins (amylase,

200 kDa; BSA, 66 kDa; cytochrome C, 13.6 kDa) are indicated (red carets).

Figure S5. Low-level expression of YFP-FisB (strain BTD3124) reveals specific

localization at the cell pole at the time of fission. Larger fields of cells showing the

localization of YFP-FisB by fluorescence microscopy during a time course of sporulation

from hour 2, 2.5 and 3. The membranes from the same fields were visualized with TMA-

DPH (false-colored red) and merged with the YFP-FisB signal (false-colored green).

White carets (at hour 2 of sporulation) highlight examples of the punctate YFP-FisB

localization pattern prior to fission. Yellow carets show examples of sporulating cells

discrete YFP-FisB foci at or near the cell pole.

Figure S6. Purified full-length His-FisB (His-FisB) and mock-purified proteins.

Purifications were from E. coli cells harboring the His6-FisBfull-length expression plasmid

(pDT355) and the empty expression plasmid (pET28a). A complete description of the

purifications can be found in Text S1. (A) Fractions from the protein purification were

analyzed by SDS-PAGE and coomassie blue staining. The red arrow indicates the

monomeric form of His-FisB and the red bracket highlights SDS-dependent His-FisB

multimers. (B) The eluted fractions from the Ni-NTA column (from separate

purifications) were analyzed by immunoblot using anti-His antibodies. The red arrow

indicates the monomeric form of His-FisB and the red bracket highlights SDS-dependent

His-FisB multimers. The nature of the SDS-induced aggregation of His-FisB is not

known but is reminiscent of SDS-induced aggregation of neuronal SNARES (Hayashi et

al. 1994).

Figure S7. SNARE proteins were purified and reconstituted into liposomes as described

in (Weber et al. 1998) except lipid-to-protein ratios were decreased to 1:200 for Vamp2

and 1:400 for t-SNAREs. The liposome fusion assay was performed as described in

Figure 4, except the temperature was increased to 37˚C. As a control, t-SNARE

liposomes were incubated with the cytoplasmic domain of Vamp2 (cdv) in excess to

3  

block fusion. After 2 hours, detergent was added to determine the maximum

fluorescence. The data was normalized and plotted as a percent of maximum fluorescence

versus time in minutes.

Figure S8. Mutations in spoIIIE affect FisB levels and/or localization. (A) Immunoblot

analysis of whole cell lysates from sporulating cells. FisB levels were analyzed in

sporulating cells from wild-type (strain PY79), a FisB mutant (BDR1083), spoIIIE36

(BDR1050) and a SpoIIIE null mutant (BDR1066) using anti-FisB antibodies. σF levels

were monitored to control for efficiency of sporulation and loading. Time (in hours) after

the initiation of sporulation is indicated. (B) YFP-FisB localization was monitored by

fluorescence microscopy at hour 3 of sporulation in wild-type (BTD3124), a spoIIIE36

mutant (BTD3143), and a SpoIIIE null mutant (BTD3142). The membranes were

visualized using TMA-DPH (false-colored red) and merged with the YFP-FisB signal

(false-colored green). Examples of sporulating cells in which YFP-FisB forms foci at the

cell pole are highlighted (yellow carets).

Figure S9. Engulfment membrane fission and septal membrane fission (cytokinesis) have

opposite membrane topologies. Schematic representations showing membrane fission

during asymmetric division (top) and at the end of engulfment (bottom). A single-pass

membrane protein (with a green ball in the cytoplasm) is shown to highlight the opposite

topologies.

Figure S10. σG is activated in the absence of membrane fission. σG activity was

monitored in single cells using a σG-responsive promoter fusion to cfp (PsspB-cfp) in wild-

type (BTD1609) and a FisB mutant (BTD2045). Cells were visualized at hour 3.5 of

sporulation. The membranes (false-colored red) were visualized with FM4-64 and

merged with the CFP signal (false-colored green). Examples of sporulating cells in which

the forespores have σG activity in the absence of membrane fission (yellow carets) or

after membrane fission has occurred (blue carets) are indicated. White carets highlight

sporulating cells that have not completed membrane fission in which σG activity is not

detected in the forespore.

4  

Figure S11. Lysis in the FisB mutant is suppressed in the absence of σG. Membrane

fission (monitored with FM4-64 and PspoIIQ-cfp) and cell lysis (assessed by phase

contrast) were analyzed 5 hours after the onset of sporulation in wild-type (BKM15), a

FisB mutant (BTD3200), a sigmaG mutant (ΔsigG, BTD3218), a FisB, sigG double

mutant (BTD3219), and a FisB, sigmaK double mutant (ΔfisB, ΔsigK, BTD3217). For

each strain, the percentage of lysed cells compared to total sporulating cells (including

lysed cells) is indicated below the image. A subset of cells lacking FisB lyse at this time

point. Lysis is suppressed in the absence of the forespore transcription factor σG but not

in the absence of mother cell transcription factor σK. This result suggests that σG-

dependent gene expression in cells that have not undergone membrane fission causes cell

lysis. Yellow carets highlight examples of lysed cells.

Movie M1A and B. GFP-FisB forms dynamic foci in the mother-cell membranes. GFP-

FisB (strain, BAM003) localization was monitored during sporulation at hour 2.5 by

time-lapse fluorescence microscopy. The focal plane was at the bottom surface of the

cell. Images were acquired every 5 seconds.

Movie M2. GFP-FisB foci are immobile at the cell pole at the time of membrane fission.

GFP-FisB (strain, BAM003) localization was monitored during sporulation at hour 2.5 by

time-lapse fluorescence microscopy. The focal plane was at mid-cell. Images were

acquired every 30 seconds.

SUPPLEMENTAL TEXT

Protein purification

His6-FisBFL and His6-FisBECD fusion proteins were expressed in E. coli BL21 DE3 pLysS

and purified by affinity chromatography on Ni2+-NTA agarose (Qiagen). MBP-FisBECD

fusion protein was expressed in E. coli NB42 and purified by affinity chromatography on

an amylose resin (New England Biolabs).

5  

For His6-FisBECD, cells were grown in LB with 20 µg/ml kanamycin at 16˚C to an OD600

of 0.6 and induced overnight at 16˚C by the addition of IPTG to 1 mM. Cells were

harvested by centrifugation and resuspended in 1/50th volume buffer I (50 mM Tris-HCl

pH 8, 200 mM NaCl, 5 mM 2-Mercaptoethanol, 1 mM PMSF, and 10 mM imidazole)

and flash-frozen in N2(l). A crude extract was prepared by freeze-thawing the cells

followed by two passes through a French press. A soluble fraction from 100,000Xg spin

was loaded on a 1 mL Ni2+-NTA agarose (Qiagen) column equilibrated with buffer I.

Bound protein was washed with buffer II (20 mM Tris-HCl pH 8, 200 mM NaCl, 5 mM

2-Mercaptoethanol, 2% glycerol, 10 mM imidazole) and eluted in buffer III (20 mM Tris-

HCl pH 8, 200 mM NaCl, 5 mM 2-Mercaptoethanol, and 150 mM imidazole).

For His6-FisBFL (and mock-purified proteins) cells were grown in LB with 20 µg/ml

kanamycin at 30˚C to an OD600 of 0.6 and induced by the addition of IPTG to 1 mM.

After 3 h of induction, cells were harvested by centrifugation and resuspended in 1/50th

volume buffer I. A crude extract was prepared by freeze-thawing the cells followed by

two passes through a French press. The membrane fraction was isolated by 100,000Xg

spin and resuspended in 1/250th volume buffer B (20 mM Tris-HCl pH 8, 200 mM NaCl,

2% glycerol, 5 mM 2-Mercaptoethanol, 1 mM PMSF, and 10 mM imidazole). Membrane

proteins were solubilized by the addition of the nonionic lipid-like detergent foscholine-

12 (Anatrace) to a final concentration of 2%. The mixture was rotated at 4˚C for 2 hours.

Soluble and insoluble fractions were separated by centrifugation at 100,000Xg for 1 hour

at 4˚C. The soluble fraction was collected and loaded on a Ni2+-NTA agarose (Qiagen)

column equilibrated with buffer I containing 0.1% foscholine-12. Bound protein was

washed with buffer II containing 0.1% foscholine-12 and eluted in buffer III containing

0.1% foscholine-12.

For MBP-FisBECD, cells were grown in LB with 100 µg/ml ampicillin at 37˚C to an

OD600 of 0.6 and induced by the addition of IPTG to 1 mM. After 2 h of induction, cells

were harvested by centrifugation and resuspended in 1/50th volume buffer A (20 mM

Tris-HCl pH 8, 1 M NaCl, 1 mM EDTA, 5 mM 2-Mercaptoethanol, 1 mM PMSF) and

flash-frozen in N2(l). A crude extract was prepared by freeze-thawing the cells followed

6  

by two lysis cycles in a French press. A soluble fraction was made by 100,000Xg spin

and was loaded on 1ml of amylose resin (New England Biolabs) equilibrated with buffer

A. Bound protein was washed with buffer A and eluted in buffer A containing 10 mM

maltose.

Plasmid construction

pKM008 [amyE::PspoIIQ-cfp (spec)] was generated in a two-way ligation with a HindIII-

BamHI PCR fragment containing the cfp gene (codon optimized for B. subtilis; (Doan et

al. 2005)) with an optimized RBS and pKM001 cut with HindIII and BamHI. pKM001

[amyE::PspoIIQ (spec)] was built in a two-way ligation with an EcoRI-HindIII PCR

fragment containing the spoIIQ promoter (oligonucleotide primers oDR234 and oDR235

and template DNA pDR172 [amyE::PspoIIQ-gfp (kan)] (DZR, unpublished)) and pLD30

(Garsin et al. 1998) cut with EcoRI and HindIII.

pDT343 [ycgO::PspoIID-RBSfisB-yfp-fisB (erm)] was built in a three-way ligation with a

HindIII-XhoI PCR fragment containing the coding sequence of YFP with a modified fisB

RBS (oligonucleotide primers oTD269 and oDR108 and plasmid template pKL183

(Lemon and Grossman 2000), a XhoI-BamHI PCR product containing the FisB coding

sequence (oligonucleotide primers oDR328 and oDR329 and PY79 genomic DNA as

template) and pDT92 [ycgO::PspoIID-spoIVB (erm)] (TD and DZR, unpublished) cut with

HindIII and BamHI.

pDT357 [ycgO::fisB (erm)] was generated in a two-way ligation with a NheI-BamHI

PCR fragment containing the fisB locus (oligonucleotide primers oTD291 and oTD118

and PY79 genomic DNA as template) and pKM84 cut with NheI and BamHI. pKM84 [ycgO::erm] is an ectopic integration vector for double crossover insertions into the nonessential ycgO locus (KM and DZR, unpublished).

pDT240 [mbp-fisBECD] was generated in a two-way ligation with a BamHI-PstI PCR

product encoding the FisB extracellular domain (216 amino acids) (oligonucleotide

7  

primers oTD234 and oTD235 and PY79 genomic DNA as template) and pMAL-C2x

(New England Biolabs) cut with BamHI and PstI.

pDT355 [his6-fisBfull-length] was built in a two-way ligation with a NheI-XhoI PCR

fragment encoding full length FisB (oligonucleotide primers oTD279 and oDR406 and

PY79 genomic DNA as template) and pET28a (Novagen) cut with NheI and XhoI.

pDT389 [his6-fisBECD] was generated in a two-way ligation with a BamHI-XhoI PCR

product encoding the FisB extracellular domain (216 amino acids) (oligonucleotide

primers oTD306 and oTD307 and pDT355 as template) and pRsetA (Invitrogen) cut with

BamHI and XhoI.

pAM002 [ycgO::PfisB-gfp-fisB (cat)] was generated in a three-way ligation with a

HindIII-XhoI PCR product containing monomeric gfp preceded by the native fisB RBS

and the fisB TTG start codon (oligonucleotide primers oDR834 and oDR108), a XhoI-

BamHI PCR product containing the fisB coding sequence (oligonucleotide primers

oDR835 and oDR836), and pAM001 cut with HindIII and BamHI. pAM001 [ycgO::PfisB

(cat)] was built in a two-way ligation with an EcoRI-HindIII PCR product

consisting of 300 nucleotides preceding the fisB coding sequence (oligonucleotide

primers oDR832 and oDR833) and pKM077 (ycgO::cat) cut with EcoRI and

HindIII. pKM077 [ycgO::cat] is an ectopic integration vector for double crossover insertions into the nonessential ycgO locus (KM and DZR unpublished).

Supplemental Reference

Doan T, Marquis KA, Rudner DZ. 2005. Subcellular localization of a sporulation membrane protein is achieved through a network of interactions along and across the septum. Mol Microbiol 55: 1767-1781.

Garsin DA, Paskowitz DM, Duncan L, Losick R. 1998. Evidence for common sites of contact between the antisigma factor SpoIIAB and its partners SpoIIAA and the developmental transcription factor sigmaF in Bacillus subtilis. J Mol Biol 284: 557-568.

8  

Hayashi T, McMahon H, Yamasaki S, Binz T, Hata Y, Südhof TC, Niemann H. 1994. Synaptic vesicle membrane fusion complex: action of clostridial neurotoxins on assembly. EMBO J 13: 5051-5061.

Lemon KP, Grossman AD. 2000. Movement of replicating DNA through a stationary replisome. Mol Cell 6: 1321-1330. Weber T, Zemelman BV, McNew JA, Westermann B, Gmachl M, Parlati F, Söllner TH,

Rothman JE. 1998. SNAREpins: minimal machinery for membrane fusion. Cell 92: 759-772.

Table 1. Strains used in this study

Strain Genotype Source PY79 Prototrophic wild-type strain Youngman et al., 1983 BDR1050 spoIIIE36 Wu and Errington, 1994 BDR1066 spoIIIE::neo Wu and Errington, 1994 BDR1083 fisB::tet Eichenberger et al., 2002 BKM15 amyE::PspoIIQ-cfp (spec) This work BTD1609 yycR::PsspB-cfp (phleo) Doan et al., 2009 BTD2045 yycR::PsspB-cfp (phleo), fisB::tet This work BAM027 yycR::PspoIIQ-cfp (phleo), dynA::cat This work BTD3124 fisB::tet, ycgO::PspoIID-RBSfisB-yfp-fisB (erm) This work BTD3142 fisB::tet, ycgO::PspoIID-RBSfisB-yfp-fisB (erm), spoIIIE::neo This work BTD3143 fisB::tet, ycgO::PspoIID-RBSfisB-yfp-fisB (erm), spoIIIE36 This work BTD3200 amyE::PspoIIQ-cfp (spec), fisB::tet This work BTD3217 amyE::PspoIIQ-cfp (spec), fisB::tet, spoIVCB::erm This work BTD3218 amyE::PspoIIQ-cfp (spec), spoIIIG::neo This work BTD3219 amyE::PspoIIQ-cfp (spec), fisB::tet, spoIIIG::neo This work BAM003 fisB::tet ycgO::PfisB-gfp-fisB (cat) This work

Table 2. Plasmids used in this study

plasmid description source pKM008 amyE::PspoIIQ-cfp (spec) This work pDT343 ycgO::PspoIID-RBSfisB-yfp-fisB (erm) This work pDT357 ycgO::fisB (erm) This work pDT240 mbp-fisBECD This work pDT355 his6-fisBfull-length This work pDT389 his6-fisBECD This work pAM002 ycgO::PfisB-gfp-fisB (cat) This work

Table 3. Oligonucleotide primers used in this study

primer sequence oDR108 cggCTCGAGtttgtatagttcatccatgc oDR234 gccGAATTCcatgcttcgtcaatgtatatgctg oDR235 cggAAGCTTagcaacattctgaacacttttctg oDR328 cggCTCGAGttgccaagatatcgcggccc oDR329 gccGGATCCcgcatttgagggcgtgg oDR406 cggCTCGAGccgcctcttcaacaggcg oTD118 gccGGATCCttatttgctgcttttttctttttt oTD234 gttGGATCCggctcgatcaaacctgtttta oTD235 cggCTGCAGttatttgctgcttttttcttttt oTD269 gttAAGCTTatgtaaggggggattatgagtaaaggagaagaactt oTD279 gccGCTAGCttgccaagatatcgcggcc oTD291 ggcGCTAGCggccgtcgccaatgatcac oTD306 gtgCTCGAGttatttgctgcttttttctttttt oTD307 cgcGGATCCaatggctcgatcaaacctgt oDR832 gccGAATTCccaaaacgaattttatggcc oDR833 cgcAAGCTTcatatcacaatgtatgcttgtaag oDR834 gcgAAGCTTaaggggggattttcttgagtaaaggagaagaacttttc oDR835 cggCTCGAGttgccaagatatcgcggcc oDR836 cgcGGATCCcgcatttgagggcgtg oAM028 ctgtgcaggaggagccagcggac oAM029 agggtaactattgccgtatgctgtcataagtctgaacccc oAM030 ggggttcagacttatgacagcatacggcaatagttaccct oAM031 gccaactgaattttacattttggagctgtaatataaaaaccttc oAM032 gaaggtttttatattacagctccaaaatgtaaaattcagttggc oAM033 gcgttcagcttttcaagcatacgctc

capital letters indicate the endonuclease recognition sequences