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,