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SUPPLEMENTARY INFORMATION Table S1. List of strains Strains Genotype Source B. subtilis PY79 Prototrophic derivative of B. subtilis 168 (60) RL1070 spovid::kan (12) HS176 cotz phs2 (cotz-gfp spc) (22) KW363 spovid::kan amye::spovid-cfp cat coteωppm19 (cote-yfp spc) (58) PE793 yaah pkw10 (yaah-gfp spc) (58) PE1496 coto ppm4 (coto-yfp spc) (58) PE1547 spoiva spoiva-yfp spc (58) PE2091 spovid::kan amye::spovid 125-136 -cfp cat (58) PE2094 spovid::kan amye::spovid 125-136 -cfp cat cote cote-yfp spc (58) PE2269 This work PE2273 PE2274 PE2275 PE2276 PE2277 PE2283 PE2608 PE2611 PE2612 PE2621 PE2616 PE2619 spovid::kan amye::spovid L125A -cfp cat spovid::kan amye::spovid I127A -cfp cat spovid::kan amye::spovid D130A -cfp cat spovid::kan amye::spovid I133A -cfp cat spoiva spoiva-yfp spc This work spovid::kan amye::spovid 125-136 -cfp cat coto coto-yfp spc This work spovid::kan amye::spovid 125-136 -cfp cat cotz cotz-gfp spc This work spovid::kan amye::spovid 125-136 -cfp cat yaah yaah-gfp spc This work spovid::kan amye::spovid 125-136 -cfp cat spoiva spoiva-yfp spc This work coto coto-yfp spc This work cotz cotz-gfp spc This work 1

PE2620 yaah yaah-gfp spc This work E. coli PE2622 spovid-his in pet21b (DH5 ) This work PE2623 spovid-his in pet21b (BL21) This work PE2624 cote-stop in pet21b (BL21) This work PE2625 spovid L125A -his in pet21b (BL21) This work PE2626 spovid I127A -his in pet21b (BL21) This work PE2627 spovid D130A -his in pet21b (BL21) This work PE2628 spovid L131A -his in pet21b (BL21) This work PE2629 spovid 125-136 -his in pet21b (BL21) This work PE2630 spovid I133A -his in pet21b (BL21) This work Yeast Y8800 Y8930 MATa leu2-3,112 trp1-901 his3δ200 ura3-52 gal4δ gal80δ cyh2 R GAL2::ADE2, GAL1::HIS3-@LYS2, GAL7::lacZ@met2 (2) MAT leu2-3,112 trp1-901 his3δ200 ura3-52 gal4δ gal80δ cyh2 R GAL2::ADE2, GAL1::HIS3-@LYS2, GAL7::lacZ@met2 (2) YPE324 Y8930 + pcote(1-158)-ad This work YPE343 Y8800 + pspovid(1-144)-bd This work YPE511 Y8800 + pspovid(1-144 L131A)-BD This work YPE513 Y8800 + pspovid(1-144 I133A)-BD This work YPE518 Y8930 + pdest-ad This work YPE519 Y8800 + pdest-bd This work YPE600 Y8800/Y8930 + pfos-ad + pjun-bd (55) Table S2. Primers Primer Nucleotide Sequence MHC001 MHC002 MHC003 MHC004 MHC007 MHC008 MHC009 CAATTGACGGATTCGCGCATTGCTACAATTCAAGCTGATTTAGCG CGCTAAATCAGCTTGAATTGTAGCAATGCGCGAATCCGTCAATTG CGGATTCGCGCATTTTAACAGCTCAAGCTGATTTAGCGATCG CGATCGCTAAATCAGCTTGAGCTGTTAAAATGCGCGAATCCG CGCATTTTAACAATTCAAGCTGCTTTAGCGATCGAAGGGCTTTTG CAAAAGCCCTTCGATCGCTAAAGCAGCTTGAATTGTTAAAATGCG GCATTTTAACAATTCAAGCTGATGCTGCGATCGAAGGGCTTTTGG 2

MHC010 MHC011 MHC012 MAD37 MAD38 CCAAAAGCCCTTCGATCGCAGCATCAGCTTGAATTGTTAAAATGC CAATTCAAGCTGATTTAGCGGCTGAAGGGCTTTTGGACGATACG CGTATCGTCCAAAAGCCCTTCAGCCGCTAAATCAGCTTGAATTC GCGCCATATGCCGCAAAATCATGCATTGCAATTTTC GCGCCTCGAGCGCATGGCTATTTTTATATTGAGGAATATAG AGAATCTGTCCTGC MAD39 MAD96 KW206 KW207 CotEF1 CotER1 SpoVIDF1 SpoVIDR1 GCGCCATATGTCTGAATACAGGGAAATTATTACGAAGGC GCGCCTCGAGTTATTATTCTTCAGGATCTCCCACTAAAAACTCCG TTGACGGATTCGCGCATTTTGGACGATACGCAAGACAAAG CTTTGTCTTGCGTATCGTCCAAAATGCGCGAATCCGTCAA GGGGACAACTTTGTACAAAAAAGTTGGCTCTGAATACAGGGAAATT ATTACGAAG GGGGACAACTTTGTACAAGAAAGTTAATCTTCCTCGTCATCCTCTTC GGGGACAACTTTGTACAAAAAAGTTGGCCCGCAAAATCATCGATTG GGGGACAACTTTGTACAAGAAAGTTACTCTTTGTCTTGCGTATCGTCC Table S3. List of Plasmids Number Primers or source Cloning vector pkw50 (58) amy-spovid L125A -cfp MHC001 and MHC002 pkw50 amy-spovid I127A -cfp MHC003 and MHC004 pkw50 amy-spovid D130A -cfp MHC007 and MHC008 pkw50 amy-spovid L131A -cfp MHC009 and MHC010 pkw50 amy-spovid I133A -cfp MHC011 and MHC012 pkw50 pet21b-spovid-his MAD37 and MAD38 pet21b pet21b-cote-stop MAD39 and MAD96 pet21b pet21b-spovid 125-136 -his KW206 and KW207 pet21b-spovid-his 3

pet21b-spovid L125A -his MHC001 and MHC002 pet21b-spovid-his pet21b-spovid I127A -his MHC003 and MHC004 pet21b-spovid-his pet21b-spovid D130A -his MHC007 and MHC008 pet21b-spovid-his pet21b-spovid L131A -his MHC009 and MHC010 pet21b-spovid-his pet21b-spovid I133A -his MHC011 and MHC012 pet21b-spovid-his pdest-ad (56) pdest-bd (56) pdonr223 Invitrogen pcote 1-158 -AD CotE F1 and CotE R1 pdest-ad pspovid 1-144 -BD pspovid 1-144 (L131A) -BD pspovid 1-144 (I133A) -BD SpoVID F1 and SpoVID R1 pdest-bd SpoVID F1 and SpoVID R1 pdest-bd SpoVID F1 and SpoVID R1 pdest-bd 4

5

Figure S1: Specific residues in the N-terminal region of SpoVID are essential for encasement. Cells were sporulated at 37 C and imaged 3 (top panel) and 6 hours (lower panel) after resuspension in SM medium. Samples were stained with membrane stain (FM4-64) and analyzed by fluorescence microscopy. The spovid point mutations are listed to the left of the images. SpoVID-CFP fluorescence is shown in the first 6

column. The second column shows the fluorescence of the membranes. CotE-YFP fluorescence in the same cells is reported in the third column. The fourth column is an overlay of the CotE-YFP fluorescence (yellow) on the membrane fluorescence (red). The fifth column shows the presence of phase bright forespores by phase contrast microscopy (brightfield). A. KW363 (spovid::kan amye::spovid-cfp cat cote cote-yfp spc) B. PE2094 (spovid::kan amye::spovid 125-136 -cfp cat cote cote-yfp spc). C. PE2273 (spovid::kan amye::spovid L125A -cfp cat cote cote-yfp spc). D. PE2274 (spovid::kan amye::spovid I127A -cfp cat cote cote-yfp spc). E. PE2275 (spovid::kan amye::spovid D130A -cfp cat cote cote-yfp spc). F. PE2276 ( cote cote-yfp spc). G. PE2277 (spovid::kan amye::spovid I133A -cfp cat cote cote-yfp spc). 7

Figure S2. Subcellular localization of representative coat protein-fluorescence protein fusions in spovid mutant strains Cells were resuspended at 37 C by resuspension in SM medium, stained with membrane stain (FM4-64) and analyzed by fluoresecence microscopy at hour 3 (A- C) or hour 6 (D) of sporulation. One representative field of cells is shown for each condition. First column: GFP or YFP fluorescence. Second column: fluorescence of membranes. Third column: overlay of coat protein fusion (green or yellow) and membranes (red). A. SpoIVA-YFP. First row: SpoIVA-YFP in an otherwise wild type 8

strain (PE1547). Second row: SpoIVA-YFP in a spovid 125-136 -cfp expressing strain (PE2621). Third row: SpoIVA-YFP in a spovid L -cfp expressing strain (PE2283). B. YaaH-GFP. First row: YaaH-GFP in an otherwise wild type strain (PE793). Second row: YaaH-GFP in a spovid 125-136 -cfp expressing strain (PE2612). Third row: YaaH- GFP in a spovid L -cfp expressing strain (PE2620). C. CotO-YFP. First row: CotO- YFP in an otherwise wild type strain (PE1496). Second row: CotO-YFP in a spovid 125-136 -cfp expressing strain (PE2608). Third row: CotO-YFP in a spovid L - cfp expressing strain (PE2616). D. CotZ-GFP. First row: CotZ-GFP in an otherwise wild type strain (HS176). Second row: CotZ-GFP in a spovid 125-136 -cfp expressing strain (PE2611). Third row: CotZ-GFP in a spovid L -cfp expressing strain (PE2619). 9

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