SUPPLEMENTAL DATA

Figure S1. Multiple sequence alignment of SpoIIQ (Q) orthologs. Amino acid residues displaying >50% identity or similarity among the Q orthologs are shaded black or gray, respectively. The amino-terminal transmembrane domain of the Q proteins (TMD; residues 22-

42 of Bacillus subtilis Q) is indicated, as is the highly conserved tyrosine within the TMD that was switched to an alanine in the B. subtilis QY28A mutant protein. Also shown is the region of homology to the lysostaphin-like M23 peptidase family (residues 117-222 of B. subtilis Q; Pfam

“Peptidase_M23” family, E-value = 3.4x10-38) (Finn et al. 2008). For comparison, a multiple sequence alignment of the region of homology from four representative members of the M23 peptidase family is given below. Amino acid residues displaying >50% identity or similarity among the M23 peptidases are shaded black or gray, respectively. Residues that have been conserved among and between the Q orthologs and M23 peptidases are indicated with solid or dashed connector lines. Residues that coordinate a zinc ion (Zn2+) in M23 peptidases are labeled

(^) (corresponding to LytM His210, Asp214, and His293); also labeled is the presumed catalytic histidine residue (*) (corresponding to LytM His291) (Gustin et al. 1996; Odintsov et al. 2004;

Firczuk et al. 2005; Ragumani et al. 2008; Rawlings et al. 2008). The corresponding residues in the B. subtilis Q protein (Ser119, Asp123, His204, and His202, respectively) that were switched to alanine in the QH202A and QS119A,D123A,H202A,H204A mutants are shown. Finally, the residues deleted in the B. subtilis Q∆202-216 and Q∆C50 mutant proteins are labeled. Q protein sequences from the following bacteria were included in this analysis: B. subtilis (Bsu; Accession

NP_391536), Bacillus licheniformis (Bli; Accession YP_080970), Geobacillus kaustophilus

(Gka; Accession YP_149192), Bacillus anthracis (Ban; Accession NP_847683), Bacillus

1 halodurans (Bha; Accession NP_244613), Bacillus clausii (Bcl; Accession YP_177337), and

Oceanobacillus iheyensis (Oih; Accession NP_693883). Fragments from the following M23 peptidase family members were included in this analysis: Staphylococcus aureus LytM

(Accession NP_645067), Staphylococcus simulans Lysostaphin (Accession AAB53783), B. subtilis LytH (Accession NP_391114), and Pseudomonas aeruginosa LasA (Accession

NP_250562). The ClustalW program (Thompson et al. 1994) was utilized to generate the multiple sequence alignment.

2 Figure S1.

3 SUPPLEMENTAL MATERIALS AND METHODS

Strain construction

Unless otherwise noted, strains used in this study were derived by transformation of the prototrophic laboratory strain PY79 (Youngman et al. 1984) or derivatives thereof with chromosomal or plasmid DNA. The genes utilized to confer resistance to antibiotics are referred to as follows: cat (chloramphenicol), erm (erythromycin plus lincomycin), spc (spectinomycin), kan (kanamycin), and phleo (phleomycin). Competent Bacillus subtilis cells were prepared as previously described (Wilson and Bott 1968). Full genotypes of strains used in this study are given in Supplemental Table S1. Plasmids used for strain construction are listed in

Supplemental Table S2. Details of plasmid design and construction are provided in the “Plasmid construction” section below. Finally, Supplemental Table S3 describes primers used in this work.

Deletion mutants. The ∆sigF::erm and ∆spoIIIA::erm deletions (gifts of P. Stragier) were obtained from the laboratory stock strains RL1275 and RL2765, respectively. The unmarked sigG deletion allele spoIIIG∆1 has been previously described (Karmazyn-Campelli et al. 1989) and was obtained from laboratory stock strain RL214 (Kunkel et al. 1988). The unmarked, in- frame deletion of spoIIIAH was moved from strain MO1429 (gift of P. Stragier) into PY78

(Sandman et al. 1988) by congression, yielding strain AHB770. The ∆sigG::kan and

∆spoIIQ::erm deletion strains (AHB98 and AHB141, respectively) have been previously described (Camp and Losick 2008). Transformation of RL2765 and AHB141 with pEr::Pm

(erm::phleo) (Steinmetz and Richter 1994) yielded the erythromycin-sensitive, phleomycin-

4 resistant strains AHB1225 (∆spoIIIAA-AH::erm::phleo) and AHB1227 (∆spoIIQ::erm::phleo), respectively.

Construction of strains with insertions at ylnF and ywrK. Strains AHB271

(ylnF::Tn917::amyE::cat), AHB262 (ywrK::Tn917), AHB282 (ywrK::Tn917::amyE::cat) and

AHB375 (ywrK::Tn917::amyE::kan), or strains derived thereof, served as recipient strains that permitted plasmids with homology to Tn917 and/or amyE to insert at the ylnF and ywrK loci.

Strains AHB262, AHB282 and AHB375 have been previously described (Camp and Losick

2008), but their construction will be described again here for completeness. Tn917 transposons inserted into the B. subtilis chromosome at map positions 140˚ (zdi-82::Tn917) and 317˚ (zii-

83::Tn917) (Vandeyar and Zahler 1986) were moved by transformation into PY79 (selecting for the erythromycin resistance conferred by Tn917) from Bacillus Genetic Stock Center (BGSC) strains 1A633 and 1A644, yielding strains AHB260 and AHB262, respectively. The precise sites of Tn917 insertion were determined to be immediately downstream of the ylnF gene (in the case of zdi-82::Tn917) and in the non-essential ywrK gene (in the case of zii-83::Tn917). These genetic positions are consistent with the calculated map positions of the integrated transposons

(Vandeyar and Zahler 1986). For clarity, we henceforth refer to these transposon insertions as ylnF::Tn917 and ywrK::Tn917. Next, AHB260 and AHB262 were transformed with pAH120

(Tn917::amyE::cat) (Camp and Losick 2008), which replaced the majority of the Tn917 sequence (including the erythromycin resistance gene) with the cat gene flanked by amyE sequences, yielding the chloramphenicol resistant, erythromycin-sensitive strains AHB271

(ylnF::Tn917::amyE::cat) and AHB282 (ywrK::Tn917::amyE::cat). AHB282 was then transformed with pER82 (amyE::kan) (Driks et al. 1994), yielding strain AHB375

(ywrK::Tn917::amyE::kan). To confirm insertion of plasmids containing Tn917 or amyE

5 homology into these modified ylnF and ywrK loci, transformants were routinely screened for loss of the antibiotic resistance originally associated with the ylnF or ywrK locus, and, when appropriate, were tested for α-amylase activity to confirm that the endogenous amyE locus was intact.

Plasmid construction

Supplemental Table S2 provides a list of plasmids used in this study. The sequences of primers used in plasmid construction are given in Supplemental Table S3. Chromosomal DNA from PY79 served as a template for PCR, unless otherwise noted. Plasmid mutagenesis was performed in all cases with the QuikChange II XL site-directed mutagenesis kit (Stratagene).

Plasmids were cloned and propagated in the Escherichia coli strain DH5α.

pAH122 (amyE::PsigG-sigG [spc]) was constructed by ligating an EcoRI/BglII PCR fragment spanning the sigG promoter, ribosome binding site (RBS), and open reading frame

(ORF) (amplified with AH6 and AH7) into EcoRI/BamHI-digested pDG1730 (amyE::spc)

(Guerout-Fleury et al. 1996). Low plasmid yield of pAH122, likely due to sigG toxicity in E. coli (Sun et al. 1991; Camp and Losick 2008), prohibited clone confirmation by restriction digest or direct sequencing; instead, candidate clones were transformed directly into a B. subtilis sigG deletion strain, screened for functionality, and sequenced following PCR amplification from the chromosome.

pAH124 (amyE::lacZ [cat]) was generated by ligating a HindIII/BamHI PCR product containing the lacZ ORF (amplified from pDG268 [Karmazyn-Campelli et al. 1989] with primers AH72 and AH73) into HindIII/BamHI-digested pDG1662 (amyE::cat) (Guerout-Fleury et al. 1996).

6 pAH129 (Tn917::amyE::PsspB-lacZ [cat]) was constructed in two steps. First, a

HindIII/HindIII PCR product containing the sspB promoter, RBS, and start codon (amplified with AH60 and AH96) was ligated into HindIII-digested pAH124 (amyE::lacZ [cat]), yielding pAH125. Second, the EcoRI/BamHI fragment harboring the PsspB-lacZ fusion was subcloned from pAH125 into EcoRI/BamHI-digested pAH120 (Tn917::amyE::cat) (Camp and Losick

2008), yielding pAH129.

pAH136 (amyE::PspoIIQ-lacZ [cat]) was generated by ligating an EcoRI/HindIII PCR product harboring the spoIIQ promoter, RBS, and start codon (amplified with AH1 and AH70) into EcoRI/HindIII-digested pAH124 (amyE::lacZ [cat]).

pAH306 (amyE::PspoIIQ-T7RNAP [spc]) harbors the spoIIQ promoter, RBS, and start codon (amplified as an EcoRI/HindIII fragment with AH1 and AH70) fused to the gene encoding the phage T7 RNA polymerase (amplified from E. coli strain BL21 chromosomal DNA as a

HindIII/BglII fragment with AH278 and AH279), ligated between the EcoRI and BamHI sites of pDG1730 (amyE::spc) (Guerout-Fleury et al. 1996).

pAH294 (amyE::PT7-lacZ [cat]) was constructed by ligating a DNA linker harboring an optimal T7 RNA polymerase-recognized promoter, optimal RBS, and an ATG start codon

(generated by annealing oligonucleotides AH280 and AH281) into EcoRI/HindIII-digested pAH124 (amyE::lacZ [cat]).

pAH357 (sacA::PspoIIQ-spoIIQ [kan]) was constructed by ligating an EcoRI/BamHI PCR fragment spanning the spoIIQ promoter, RBS, and ORF (amplified with AH1 and AH342) into

EcoRI/BamHI-digested pSac-Kan (sacA::kan) (Middleton and Hofmeister 2004).

∆C50 ∆C100 pAH363 (sacA::PspoIIQ-spoIIQ [kan]), pAH365 (sacA::PspoIIQ-spoIIQ [kan]), and

∆C230 pAH383 (sacA::PspoIIQ-spoIIQ [kan]) were constructed by ligating EcoRI/BamHI PCR

7 fragments spanning the spoIIQ promoter, RBS, and various truncations of the spoIIQ ORF

(lacking the coding sequence for the C-terminal 50, 100, or 230 amino acids, respectively) into

EcoRI/BamHI-digested pSac-Kan (sacA::kan) (Middleton and Hofmeister 2004). The primer

∆C50 pairs used for PCR amplification were as follows: AH1/AH352 (PspoIIQ-spoIIQ ),

∆C100 ∆C230 AH1/AH353 (PspoIIQ-spoIIQ ), and AH1/AH368 (PspoIIQ-spoIIQ ).

Y28A H202A pAH372 (sacA::PspoIIQ-spoIIQ [kan]), pAH377 (sacA::PspoIIQ-spoIIQ [kan]),

∆202-216 ∆2-43 pAH388 (sacA::PspoIIQ-spoIIQ [kan]), and pAH386 (sacA::PspoIIQ-spoIIQ [kan]) were generated by targeted mutagenesis of pAH357 (sacA::PspoIIQ-spoIIQ [kan]) with the complementary primer pairs AH344/AH345 (Y28A), AH350/AH351 (H202A), AH366/AH367

(∆202-216), and AH364/AH365 (∆2-43).

Y28A,∆202-216 pAH400 (sacA::PspoIIQ-spoIIQ [kan]) and pAH433 (sacA::PspoIIQ-

Y28A,∆C230 ∆202-216 spoIIQ [kan]) were generated by mutagenizing pAH388 (sacA::PspoIIQ-spoIIQ

∆C230 [kan]) and pAH383 (sacA::PspoIIQ-spoIIQ [kan]), respectively, with primer pair

AH344/AH345 (Y28A).

TMD pAH401 (sacA::PspoIIQ-malF -spoIIQ [kan]) was constructed in two steps. First, a three-way ligation was performed with an EcoRI/HindIII PCR product containing the spoIIQ promoter, RBS, and ATG start codon (amplified with AH1 and AH70), a HindIII/BamHI PCR product harboring codons 2-38 of the E. coli malF gene followed by an in-frame XhoI site

(amplified from pLMG130 [Guzman et al. 1997] with AH375 and AH376), and EcoRI/BamHI- digested pSac-Kan (sacA::kan) (Middleton and Hofmeister 2004), yielding pAH398. Next, pAH398 was digested with XhoI and BamHI and ligated with a XhoI/BamHI PCR product containing codons 44-283 of spoIIQ (amplified with AH377 and AH342), yielding pAH401.

8 TMD ∆202-216 pAH416 (sacA::PspoIIQ-malF -spoIIQ [kan]) was generated by mutagenizing

TMD plasmid pAH401 (sacA::PspoIIQ-malF -spoIIQ [kan]) with primer pair AH366/AH367 (∆202-

216).

pAH422 (amyE::P2spoIIIA-gfp-spoIIIAH [cat]) was constructed in three steps. First, pAH315 (sacA::P2spoIIIA-spoIIIAH [kan]) (Camp and Losick 2008) was mutagenized with primer pair AH385/AH386 to insert an in-frame NheI restriction site downstream of the second spoIIIAH codon, yielding pAH418. Second, an NheI/NheI PCR fragment containing the gfp

ORF (amplified from pAC172 [Chastanet and Losick 2007] with primers AH387 and AH388) was ligated into NheI-digested pAH418, yielding pAH419. Finally, the BglII/EcoRI fragment containing P2spoIIIA-spoIIIAH was subcloned from pAH419 into BamHI/EcoRI-digested pDG1662 (amyE::cat) (Guerout-Fleury et al. 1996), yielding pAH422.

pAH420 (lacA::erm) was constructed in two steps. First, pAX01 (lacA::PxylA, xylR [erm])

(Hartl et al. 2001) was digested with SacI to excise the cassette containing PxylA and xylR; the plasmid backbone was then gel purified and re-ligated, yielding pAH152. Next, a DNA linker harboring the restriction sites NheI, BamHI, and EcoRI (generated by annealing oligonucleotides

AH389 and AH390) was ligated into SacII/SacI-digested pAH152, yielding pAH420.

TMD pAH429 (lacA::PspoIIQ-malF -spoIIQ [erm]) was constructed by subcloning an

TMD EcoRI/BamHI DNA fragment harboring PspoIIQ-malF -spoIIQ from pAH401 (sacA::PspoIIQ- malFTMD-spoIIQ [kan]) into EcoRI/BamHI-digested pAH420 (lacA::erm).

9 SUPPLEMENTAL TABLES

Table S1. Strains used in this study.

Strain Genotype Source or reference PY79a Prototrophic wild type Youngman et al. 1984 PY78 glnA100 Sandman et al. 1988 AHB260 ylnF::Tn917 (formerly zdi-82::Tn917) This study; Vandeyar and Zahler 1986; BGSCb strain 1A633 → PY79 AHB271 ylnF::Tn917::amyE::cat This study AHB262 ywrK::Tn917 (formerly zii-83::Tn917) Vandeyar and Zahler 1986; Camp and Losick 2008; BGSC strain 1A644 → PY79 AHB282 ywrK::Tn917::amyE::cat Camp and Losick 2008 AHB375 ywrK::Tn917::amyE::kan Camp and Losick 2008 RL1275 ∆sigF::erm Laboratory stock; MO173 (gift of P. Stagier) → PY79 RL214 spoIIIG∆1 Kunkel et al. 1988; Karmazyn-Campelli et al. 1989 AHB98 ∆sigG::kan Camp and Losick 2008 RL2765 ∆spoIIIAA-AH::erm Laboratory stock; MO1433 (gift of P. Stragier) → PY79 AHB1225 ∆spoIIIAA-AH::erm::phleo This study AHB770 ∆spoIIIAH This study; MO1429 (gift of P. Stragier) → PY78 AHB141 ∆spoIIQ::erm Camp and Losick 2008 AHB1227 ∆spoIIQ::erm::phleo This study

AHB324 ywrK::Tn917::amyE::PsspB-lacZ (cat) This study

AHB1361 ∆spoIIQ::erm sacA::PspoIIQ-spoIIQ (kan) This study ∆C50 AHB1362 ∆spoIIQ::erm sacA::PspoIIQ-spoIIQ (kan) This study ∆C100 AHB1363 ∆spoIIQ::erm sacA::PspoIIQ-spoIIQ (kan) This study ∆C230 AHB1372 ∆spoIIQ::erm sacA::PspoIIQ-spoIIQ (kan) This study ∆202-216 AHB1375 ∆spoIIQ::erm sacA::PspoIIQ-spoIIQ (kan) This study H202A AHB1369 ∆spoIIQ::erm sacA::PspoIIQ-spoIIQ (kan) This study ∆2-43 AHB1374 ∆spoIIQ::erm sacA::PspoIIQ-spoIIQ (kan) This study TMD AHB1418 ∆spoIIQ::erm sacA::PspoIIQ-malF -spoIIQ (kan) This study TMD ∆202-216 AHB1533 ∆spoIIQ::erm sacA::PspoIIQ-malF -spoIIQ (kan) This study

10 AHB1399 ywrK::Tn917::amyE::PsspB-lacZ (cat) ∆spoIIQ::erm This study

AHB1401 ywrK::Tn917::amyE::PsspB-lacZ (cat) ∆spoIIQ::erm sacA::PspoIIQ- This study spoIIQ (kan)

AHB1408 ywrK::Tn917::amyE::PsspB-lacZ (cat) ∆spoIIQ::erm sacA::PspoIIQ- This study spoIIQ∆202-216 (kan)

AHB1405 ywrK::Tn917::amyE::PsspB-lacZ (cat) ∆spoIIQ::erm sacA::PspoIIQ- This study spoIIQH202A (kan)

AHB1537 ywrK::Tn917::amyE::PsspB-lacZ (cat) ∆spoIIQ::erm sacA::PspoIIQ- This study malFTMD-spoIIQ (kan)

AHB1538 ywrK::Tn917::amyE::PsspB-lacZ (cat) ∆spoIIQ::erm sacA::PspoIIQ- This study malFTMD-spoIIQ∆202-216 (kan)

AHB1508 ∆spoIIIAH amyE::P2spoIIIA-gfp-spoIIIAH (cat) This study

AHB1516 ∆spoIIIAH amyE::P2spoIIIA-gfp-spoIIIAH (cat) ∆spoIIQ::erm This study

AHB1527 ∆spoIIIAH amyE::P2spoIIIA-gfp-spoIIIAH (cat) ∆spoIIQ::erm This study sacA::PspoIIQ-spoIIQ (kan)

AHB1531 ∆spoIIIAH amyE::P2spoIIIA-gfp-spoIIIAH (cat) ∆spoIIQ::erm This study ∆202-216 sacA::PspoIIQ-spoIIQ (kan) AHB1552 This study ∆spoIIIAH amyE::P2spoIIIA-gfp-spoIIIAH (cat) ∆spoIIQ::erm H202A sacA::PspoIIQ-spoIIQ (kan)

AHB1532 ∆spoIIIAH amyE::P2spoIIIA-gfp-spoIIIAH (cat) ∆spoIIQ::erm This study TMD sacA::PspoIIQ-malF -spoIIQ (kan)

AHB1555 ∆spoIIIAH amyE::P2spoIIIA-gfp-spoIIIAH (cat) ∆spoIIQ::erm This study TMD ∆202-216 sacA::PspoIIQ-malF -spoIIQ (kan)

AHB881 amyE::PspoIIQ-lacZ (cat) This study

AHB938 amyE::PspoIIQ-lacZ (cat) ∆sigF::erm This study

AHB882 amyE::PspoIIQ-lacZ (cat) ∆sigG::kan This study

AHB1305 amyE::PspoIIQ-lacZ (cat) ∆sigG::kan ywrK::Tn917::amyE::PsigG- This study sigG (spc)

AHB1134 amyE::PspoIIQ-lacZ (cat) ∆spoIIIAA-AH::erm This study

AHB939 amyE::PspoIIQ-lacZ (cat) ∆spoIIQ::erm This study

AHB915 amyE::PspoIIQ-lacZ (cat) ∆sigG::kan ∆sigF::erm This study

AHB1017 amyE::PspoIIQ-lacZ (cat) ∆sigG::kan ∆spoIIIAA-AH::erm This study

AHB916 amyE::PspoIIQ-lacZ (cat) ∆sigG::kan ∆spoIIQ::erm This study

AHB1453 amyE::PspoIIQ-lacZ (cat) spoIIIG∆1 ∆spoIIQ::erm This study

AHB1463 amyE::PspoIIQ-lacZ (cat) spoIIIG∆1 ∆spoIIQ::erm This study sacA::PspoIIQ-spoIIQ (kan)

AHB1470 amyE::PspoIIQ-lacZ (cat) spoIIIG∆1 ∆spoIIQ::erm This study ∆202-216 sacA::PspoIIQ-spoIIQ (kan)

AHB1125 ywrK::Tn917::amyE::PT7-lacZ (cat) amyE::PspoIIQ-T7RNAP (spc) This study

AHB1131 ywrK::Tn917::amyE::PT7-lacZ (cat) amyE::PspoIIQ-T7RNAP (spc) This study ∆sigF::erm

11 AHB1132 ywrK::Tn917::amyE::PT7-lacZ (cat) amyE::PspoIIQ-T7RNAP (spc) This study ∆spoIIIAA-AH::erm

AHB1382 ywrK::Tn917::amyE::PT7-lacZ (cat) amyE::PspoIIQ-T7RNAP (spc) This study ∆202-216 ∆spoIIQ::erm sacA::PspoIIQ-spoIIQ (kan)

AHB1449 ywrK::Tn917::amyE::PT7-lacZ (cat) ylnF::Tn917::amyE::PspoIIQ- This study T7RNAP (spc)

AHB1474 ywrK::Tn917::amyE::PT7-lacZ (cat) ylnF::Tn917::amyE::PspoIIQ- This study T7RNAP (spc) ∆sigF::erm

AHB1475 ywrK::Tn917::amyE::PT7-lacZ (cat) ylnF::Tn917::amyE::PspoIIQ- This study T7RNAP (spc) ∆spoIIIAA-AH::erm

AHB1545 ywrK::Tn917::amyE::PT7-lacZ (cat) ylnF::Tn917::amyE::PspoIIQ- This study ∆202-216 T7RNAP (spc) ∆spoIIQ::erm sacA::PspoIIQ-spoIIQ (kan)

AHB1542 ywrK::Tn917::amyE::PT7-lacZ (cat) ylnF::Tn917::amyE::PspoIIQ- This study T7RNAP (spc) ∆spoIIQ::erm sacA::PspoIIQ-spoIIQ (kan)

AHB1547 ywrK::Tn917::amyE::PT7-lacZ (cat) ylnF::Tn917::amyE::PspoIIQ- This study TMD ∆202-216 T7RNAP (spc) ∆spoIIQ::erm sacA::PspoIIQ-malF -spoIIQ (kan)

AHB1543 ywrK::Tn917::amyE::PT7-lacZ (cat) ylnF::Tn917::amyE::PspoIIQ- This study Y28A T7RNAP (spc) ∆spoIIQ::erm sacA::PspoIIQ-spoIIQ (kan)

AHB1544 ywrK::Tn917::amyE::PT7-lacZ (cat) ylnF::Tn917::amyE::PspoIIQ- This study ∆C230 T7RNAP (spc) ∆spoIIQ::erm sacA::PspoIIQ-spoIIQ (kan)

AHB1567 ywrK::Tn917::amyE::PT7-lacZ (cat) ylnF::Tn917::amyE::PspoIIQ- This study TMD T7RNAP (spc) ∆spoIIQ::erm::phleo lacA::PspoIIQ-malF -spoIIQ (erm)

AHB1571 ywrK::Tn917::amyE::PT7-lacZ (cat) ylnF::Tn917::amyE::PspoIIQ- This study TMD T7RNAP (spc) ∆spoIIQ::erm::phleo lacA::PspoIIQ-malF -spoIIQ ∆C230 (erm) sacA::PspoIIQ-spoIIQ (kan)

AHB1579 ywrK::Tn917::amyE::PT7-lacZ (cat) ylnF::Tn917::amyE::PspoIIQ- This study TMD T7RNAP (spc) ∆spoIIQ::erm::phleo lacA::PspoIIQ-malF -spoIIQ Y28A,∆C230 (erm) sacA::PspoIIQ-spoIIQ (kan)

AHB1562 ywrK::Tn917::amyE::PT7-lacZ (cat) ylnF::Tn917::amyE::PspoIIQ- This study Y28A T7RNAP (spc) ∆spoIIQ::erm sacA::PspoIIQ-spoIIQ (kan) ∆spoIIIAA-AH::erm::phleo

AHB1546 ywrK::Tn917::amyE::PT7-lacZ (cat) ylnF::Tn917::amyE::PspoIIQ- This study Y28A,∆202-216 T7RNAP (spc) ∆spoIIQ::erm sacA::PspoIIQ-spoIIQ (kan)

AHB1476 ywrK::Tn917::amyE::PT7-lacZ (cat) ylnF::Tn917::amyE::PspoIIQ- This study T7RNAP (spc) ∆spoIIQ::erm aAll strains are isogenic with PY79; bBGSC, Bacillus Genetic Stock Center

12 Table S2. Plasmids used in this study.

Plasmid Description Source or reference pDG1662 amyE::cat (directs double crossover insertion into amyE) Guerout-Fleury et al. 1996 pDG1730 amyE::spc (directs double crossover insertion into amyE) Guerout-Fleury et al. 1996 pER82 amyE::kan (directs double crossover insertion into amyE) Driks et al. 1994 pEr::Pm erm::phleo (inserts phleo antibiotic resistance cassette by double Steinmetz and Richter 1994 crossover into erm antibiotic resistance cassette) pSac-Kan sacA::kan (directs double crossover insertion into sacA) Middleton and Hofmeister 2004 pAH120 Tn917::amyE::cat (inserts amyE and cat sequences into Tn917) Camp and Losick 2008 pAH122 amyE::PsigG-sigG (spc) This study pAH124 amyE::lacZ (cat) (used for construction of lacZ fusions) This study pAH129 Tn917::amyE::PsspB-lacZ (cat) This study pAH136 amyE::PspoIIQ-lacZ (cat) This study pAH294 amyE::PT7-lacZ (cat) This study pAH306 amyE::PspoIIQ-T7RNAP (spc) This study pAH357 sacA::PspoIIQ-spoIIQ (kan) This study ∆C50 pAH363 sacA::PspoIIQ-spoIIQ (kan) This study ∆C100 pAH365 sacA::PspoIIQ-spoIIQ (kan) This study Y28A pAH372 sacA::PspoIIQ-spoIIQ (kan) This study H202A pAH377 sacA::PspoIIQ-spoIIQ (kan) This study ∆C230 pAH383 sacA::PspoIIQ-spoIIQ (kan) This study ∆2-43 pAH386 sacA::PspoIIQ-spoIIQ (kan) This study ∆202-216 pAH388 sacA::PspoIIQ-spoIIQ (kan) This study Y28A,∆202-216 pAH400 sacA::PspoIIQ-spoIIQ (kan) This study TMD pAH401 sacA::PspoIIQ-malF -spoIIQ (kan) This study TMD ∆202-216 pAH416 sacA::PspoIIQ-malF -spoIIQ (kan) This study pAH422 amyE::P2spoIIIA-gfp-spoIIIAH (cat) This study Y28A,C230 pAH433 sacA::PspoIIQ-spoIIQ (kan) This study pAH420 lacA::erm (directs double crossover insertion into lacA) This study; derived from pAX01 (Hartl et al. 2001) TMD pAH429 lacA:: PspoIIQ-malF -spoIIQ (erm) This study

13 Table S3. Primers used in this study.

Primer Sequence (5’→3’) Description

AH1 gatcgaattccggtatcggctgttaccatt Forward primer, PspoIIQ upstream (EcoRI)

AH6 gatcgaattcagcggatatgatggggatttc Forward primer, PsigG upstream (EcoRI) AH7 gacagatctcgtaaaccatccataatcaatag Reverse primer, sigG downstream (BglII)

AH60 gatcgaattcacgagatacatgaactgatgc Forward primer, PsspB upstream (EcoRI)

AH70 gatcaagcttcattgtttcatcacctcagcaac Reverse primer, PspoIIQ downstream including start codon (HindIII) AH72 gatcaagcttgtggaagttactgacgtaagattacg Forward primer, lacZ upstream (HindIII) AH73 gatcggatccagtacataatggatttccttacgcg Reverse primer, lacZ downstream (BamHI)

AH96 gatcaagcttcatgtgtaaaatctcctttttatttagtatgg Reverse primer, PsspB downstream including start codon (HindIII) AH278 gatcaagcttaacacgattaacatcgctaagaacgacttc Forward primer, T7RNAP upstream (HindIII) AH279 gatcagatctttacgcgaacgcgaagtccgac Reverse primer, T7RNAP downstream (BglII) AH280 aattcttaatacgactcactatagggaataaggaggcatgcctatga Oligonucleotide containing T7 RNAP promoter, optimal RBS, ATG start codon, top strand AH281 agcttcataggcatgcctccttattccctatagtgagtcgtattaag Oligonucleotide containing T7 RNAP promoter, optimal RBS, ATG start codon, bottom strand AH342 gatcggatccttaagactgttcagtgtcttctgttgtgc Reverse primer, spoIIQ downstream (BamHI) AH344 ggtgttccctgcaattGCcttagtcagtgcggccg Mutagenesis primer to switch spoIIQ Tyr28 → Ala, top strand AH345 cggccgcactgactaagGCaattgcagggaacacc Mutagenesis primer to switch spoIIQ Tyr28 → Ala, bottom strand AH350 cctttacagtgaagacagcggaaacGCcgtgcactttgaaatcc Mutagenesis primer to switch spoIIQ His202 → Ala, top strand AH351 ggatttcaaagtgcacgGCgtttccgctgtcttcactgtaaagg Mutagenesis primer to switch spoIIQ His202 → Ala, bottom strand AH352 gatcggatccttattgggtggcagctttttcaatagaag Reverse primer spoIIQ internal (omitting C-terminal 50 codons), includes a stop codon (BamHI) AH353 gatcggatccttattgttttactttgtcaccttgctctacg Reverse primer spoIIQ internal (omitting C-terminal 100 codons) includes a stop codon (BamHI) AH364 gctgaggtgatgaaacaatg//tcagtatcaaatgatgaggtaaagg Mutagenesis primer to delete spoIIQ codons 2-43, top strand AH365 cctttacctcatcatttgatactga//cattgtttcatcacctcagc Mutagenesis primer to delete spoIIQ codons 2-43, bottom strand

14 AH366 cctttacagtgaagacagcggaaac//ttaaactttatggacaaacc Mutagenesis primer to delete spoIIQ codons 202-216, top strand AH367 ggtttgtccataaagtttaa//gtttccgctgtcttcactgtaaagg Mutagenesis primer to delete spoIIQ codons 202-216, bottom strand AH368 gatcggatccttactgatcctttacctcatcatttgatactg Reverse primer spoIIQ internal (omitting C-terminal 230 codons) includes a stop codon (BamHI) AH375 gatcaagcttgatgtcattaaaaagaaacattggtggc Forward primer malF upstream (starting at at codon 2) (HindIII) AH376 gatcggatccacgtctcgagcccttgtgcgtacattaaaacaacaagg Reverse primer malF internal (through codon 38) (XhoI, BamHI) AH377 gatcctcgagtcagtatcaaatgatgaggtaaaggatcag Forward primer spoIIQ internal (starting with codon 44) (XhoI) AH385 ggaggattcataaatgcttGCTAGCaaaaaacaaaccgtttggc Mutagenesis primer to insert NheI site at 5’ end of spoIIIAH ORF, top strand AH386 gccaaacggtttgttttttGCTAGCaagcatttatgaatcctcc Mutagenesis primer to insert NheI site at 5’ end of spoIIIAH ORF, bottom strand AH387 gatcgctagcagtaaaggagaagaacttttcactggag Forward primer gfp upstream (starting with codon 2) (NheI) AH388 gatcgctagctttgtatagttcatccatgccatgtg Reverse primer gfp downstream (omitting stop codon) (NheI) AH389 ggatagctagcattggatccgatcgaatccagcgagct Oligonucleotide containing NheI, BamHI, and EcoRI sites, top strand AH390 cgctgaattcgatcggatccaatgctagctatccgc Oligonucleotide containing NheI, BamHI, and EcoRI sites, bottom strand Restriction endonuclease recognition sites are underlined. Substitutions or insertions in mutagenesis primers are indicated in uppercase. “//” designates sites of deleted nucleotides in mutagenesis primers.

15 SUPPLEMENTAL REFERENCES

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