1 Supplemental Materials and Methods, supplementary Tables S1S5, and supplementary Figures S1S9 Insights into the evolutionary relationships of LytA autolysin and Ply pneumolysin- like genes in Streptococcus pneumoniae and related streptococci María Morales, Antonio J. Martín-Galiano, Mirian Domenech and Ernesto García* 2 Materials and Methods Bacterial strains, growth conditions and transformation Pneumococcal strains were grown in C medium (Lacks and Hotchkiss 1960) either supplemented (C + Y) or not supplemented with 0.08% yeast extract. Escherichia coli strains were grown in Luria-Bertani medium (Sambrook and Russell 2001). For in vitro biofilms assays, we used the non-encapsulated pneumococcal strain R6 and its derivatives: R924 (lytA::kan) (Moscoso and Claverys 2004), P206 (plyA), and P259 (plyA lytA::kan). The following E. coli strains were used: DH5 (supE44 ΔlacU169 (80 lacZΔM15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1) (Hanahan 1983), and BL21 (DE3) (hsdS gal (cIts857 ind1 Sam7 nin5 lacUV5-T7 gene 1) (Novagen). Transformation of E. coli strains was performed by the rubidium chloride method (Sambrook and Russell 2001). Biofilm formation assay and quantification The optimal conditions for biofilm formation by pneumococcal cells have been previously described (Moscoso, et al. 2006). Cells were grown in C medium to an optical density at 550 nm (OD550) of 0.5–0.6, sedimented by centrifugation, resuspended in an equal volume of C medium, and diluted 1/10. Aliquots of 200 l were dispensed into each well of polystyrene microtiter plates (Corning, New York, USA). After 6 h of incubation at 34C, the biofilm formed was stained with 0.2% crystal violet and rinsed three times with distilled water to remove non-adherent bacteria. After solubilizing the biofilm in 95% ethanol, the OD595 was determined using an Anthos 2020 microplate absorbance reader (Anthos Labtec Instruments, Wals, Austria). 3 PCR amplification and cloning of lytA alleles Routine DNA manipulations were performed essentially as described elsewhere (Sambrook and Russell 2001). Restriction endonucleases and T4 DNA ligase were purchased from Takara Bio and used according to the recommendations of the suppliers. DNA fragments were purified using the Geneclean II kit (Bio 101). For PCR reactions, recombinant DNA polymerase from Thermus thermophilus HB27 was used (Biotools B&M Laboratories, Spain). For cloning, lytASpn alleles coding for the NAM- amidase alleles 1, 2, 4, 5, or 7 (Supplemental Table S4) were PCR amplified using a mixture of Taq and Pfu DNA polymerases (Biotools B&M Laboratories, Spain) and oligonucleotides LytA-ATG and LytA-STOP (Moscoso, et al. 2010). The products amplified by PCR were purified using a High Pure PCR product purification kit (Roche). The resultant DNA fragments (1,012 bp each) were digested with NdeI and BamHI and cloned into plasmid pT7-7 (Tabor 1990). The ligation mixtures were used to transform E. coli DH5. Next, the recombinant plasmids were introduced into E. coli BL21 (DE3). The accuracy of the different constructs was confirmed by completely sequencing the insert of the corresponding recombinant plasmid. All primers for PCR amplification and nucleotide sequencing were purchased from Sigma-Aldrich. Overproduction and purification of LytA alleles, and assay of cell wall lytic activity For overproduction of LytA NAM-amidases, E. coli BL21 (DE3) cells harboring the appropriate plasmid were incubated in Luria-Bertani medium with ampicillin (100 g ml1) up to an optical density at 600 nm of 0.4. At this time point, isopropyl--D- thiogalactopyranoside (0.1 mM) was added, and incubation continued for 4 h at 37C with shaking. The cultures were centrifuged, and the bacteria were resuspended in 20 mM sodium phosphate buffer (pH 6.9) and disrupted by two 10 s periods of sonication. After centrifugation (10,000 × g, 10 min, 4C), the supernatant was loaded onto a DEAE-cellulose column and NAM-amidases were purified as previously described (Sánchez-Puelles, et al. 1992). LytA concentration was determined 4 spectrophotometrically using a molar absorption coefficient at 280 nm of 113,750 M1 cm1 (Usobiaga, et al. 1996). Pneumococcal cell walls were radioactively labeled with [methyl-3H]choline and assays for cell wall lytic (NAM-amidase) activity were conducted according to standard procedures described elsewhere (Mosser and Tomasz 1970; Romero, et al. 2004). One unit of amidase activity was defined as the amount of enzyme needed to catalyze the hydrolysis (solubilization) of 1 g of cell wall material in 10 min at 37C. Statistical analysis The data for NAM-amidase activity include the mean ± standard error of at least three independent experiments, each performed in triplicate. Statistical significance was examined using the Student t test. For multiples comparisons, one-way analyses of variance (ANOVA) were performed, followed by Dunnet’s post hoc test when the ANOVA rejected the null hypothesis. Differences were considered statistically significant when P <0.05. The SAS 9.3 statistical package (SAS Institute, Cary, NC) was used for all analyses. 5 Table S1. Complete and draft genomic sequences of SMG and other relevant streptococcia. Species Complete genomes Draft genomes Referenceb SMGc S. australis 0 2 (Willcox, et al. 2001) S. cristatus 0 2 (Handley, et al. 1991) S. dentisani 0 2 (Camelo-Castillo, et al. 2014) S. gordonii 1 0 (Kilian, et al. 1989) S. infantis 0 6 (Kawamura, et al. 1998) S. lactarius 0 0 (Martín, et al. 2011) S. mitis 1 30 (Andrewes and Horder 1906) S. oligofermentans 1 0 (Tong, et al. 2003) S. oralis 1 12 (Bridge and Sneath 1982) S. parasanguinis 2 7 (Whiley, et al. 1990) S. peroris 0 1 (Kawamura, et al. 1998) S. pneumoniae 27 275 (Chester 1901) S. pseudopneumoniae 1 6 (Arbique, et al. 2004) S. rubneri 0 0 (Huch, et al. 2013) S. sanguinis 1 21 (White and Niven 1946) S. sinensis 0 1 (Woo, et al. 2002) S. tigurinus 0 4 (Zbinden, et al. 2012) S. troglodytidis 0 0 (Zhang, et al. 2013) Other streptococci S. agalactiae 18 295 S. anginosus 4 10 S. mutans 4 161 S. pyogenes 30 209 S. suis 20 87 S. thermophilus 7 13 Other speciesd 37 167 a. Genomic sequences available at the National Center for Biotechnology Information (NCBI) (last date accessed, 25 October 2014). b. Publication corresponds only to SMG. c. Streptococcus massiliensis has also been proposed to belong to SMG but its phylogenetic position is still unclear (Glazunova, et al. 2006). d. S. anginosus, S. caballi, S. canis, S. castoreus, S. constellatus, S. cricetus, D. devriesei, S. didelphis, S. downei, S. dysgalactiae, S. equi, S. equinus, S. ferus, S. gallolyticus, S. henryi, S. hyovaginalis, S. ictaluri, S. infantarius, S. iniae, S. intermedius, S. lutetiensis, S. macacae, S. macedonicus,S. marimammalium, S. massiliensis, S. merionis, S. minor, S. orisratti, S. ovis, S. parauberis, S. pasteurianus, S. plurextorum, S. porci, S. porcinus, S. pseudoporcinus, S. ratti, S. salivarius, S. thoraltensis, S. uberis, S. urinalis S. vestibularis,and Streptococcus sp. 6 Table S2. See separate file Table S3. See separate file 7 Table S4. Correspondence between lytA and LytA alleles of S. pneumoniae lytASpn allele (Fam_)a (Acc. No.) LytASpn Alleleb Specific activity (Units mg1)c Significant (P <0.05)d 1 (B) (AE007317) 1 1.3 × 106 ± 0.97 × 105 allele 1 vs. 7: Yes 2 (B) (AE005672) 2 1.3 × 106 ± 2.2 × 105 allele 2 vs. 7: Yes 3 (A) (CP002176) 3 4 (B) (FQ312030) 4 1.2 × 106 ± 1.5 × 105 allele 4 vs. 7: Yes 5 (A) (AGPA01000015) 5 1.6 × 106 ± 1.9 × 105 allele 5 vs. 7: No 6 (A) (AWTX01000004) 5 7 (A) (FM211187) 5 8 (A) (FQ312027) 5 9 (A) (ALAZ01000017) 6 10 (A) (FQ312045) 2 11 (A) (CP002121) 7 1.9 × 106 ± 2.1 × 105 12 (B) (AGID01000009) 2 13 (A) (CP001845) 2 14 (B) (AIKV01000007) 2 15 (A) (CP000920) 5 16 (B) (CP000918) 2 17 (B) (ALCR01000016) 2 18 (B) (AFAX01000007) 8 19 (B) (ALCV01000008) 5 20 (B) (ALBF01000021) 2 21 (B) (AQTO01000004) 9 22 (A) (AKQY01000003) 10 23 (A) (ALBM01000008) 11 24 (A) (AILM01000015) 2 25 (A) (AILB01000008) 12 26 (A) (AIKT01000015) 5 27 (A) (ALBP01000011) 13 28 (A) (ALBK01000014) 6 29 (A) (ABAC01000008) 7 30 (A) (ABAB01000008)  a Fam_A and Fam_B of lytASpn alleles have been described elsewhere (Morales, et al. 2010). b The lytA30-Spn allele was not translated into protein since it may contain a sequencing error. c Expressed as the mean ± the standard error of the mean. d For multiples comparisons, one-way analyses of variance (ANOVA) were performed, followed by Dunnet’s post hoc test when the ANOVA rejected the null hypothesis. Differences were considered statistically significant when P <0.05. 8 References Morales M, et al. 2010. Evidence of localized prophage-host recombination in the lytA gene encoding the major pneumococcal autolysin. J Bacteriol 192:26242632. 9 Nucleotide sequences of the lytASpn alleles analyzed in this study >1_SPN ATGGAAATTAATGTGAGTAAATTAAGAACAGATTTGCCTCAAGTCGGCGTGCAACCATATAGGCAAGTACACGCACACTCAACTGGGA ATCCGCATTCAACCGTACAGAATGAAGCGGATTATCACTGGCGGAAAGACCCAGAATTAGGTTTTTTCTCGCACATTGTTGGGAACGG TTGCATCATGCAGGTAGGACCTGTTGATAATGGTGCCTGGGACGTTGGGGGCGGTTGGAATGCTGAGACCTATGCAGCGGTTGAACTG ATTGAAAGCCATTCAACCAAAGAAGAGTTCATGACGGACTACCGCCTTTATATCGAACTCTTACGCAATCTAGCAGATGAAGCAGGTT TGCCGAAAACGCTTGATACAGGGAGTTTAGCTGGAATTAAAACGCACGAGTATTGCACGAATAACCAACCAAACAACCACTCAGACCA CGTTGACCCTTATCCATATCTTGCTAAATGGGGCATTAGCCGTGAGCAGTTTAAGCATGATATTGAGAACGGCTTGACGATTGAAACA GGCTGGCAGAAGAATGACACTGGCTACTGGTACGTACATTCAGACGGCTCTTATCCAAAAGACAAGTTTGAGAAAATCAATGGCACTT GGTACTACTTTGACAGTTCAGGCTATATGCTTGCAGACCGCTGGAGGAAGCACACAGACGGCAACTGGTACTGGTTCGACAACTCAGG CGAAATGGCTACAGGCTGGAAGAAAATCGCTGATAAGTGGTACTATTTCAACGAAGAAGGTGCCATGAAGACAGGCTGGGTCAAGTAC AAGGACACTTGGTACTACTTAGACGCTAAAGAAGGCGCCATGGTATCAAATGCCTTTATCCAGTCAGCGGACGGAACAGGCTGGTACT ACCTCAAACCAGACGGAACACTGGCAGACAGGCCAGAATTCACAGTAGAGCCAGATGGCTTGATTACAGTAAAATAA >2_SPN ATGGAAATTAATGTGAGTAAATTAAGAACAGATTTGCCTCAAGTCGGCGTGCAACCATATAGGCAAGTACACGCACACTCAACTGGGA ATCCGCATTCAACCGTACAGAATGAAGCGGATTATCACTGGCGGAAAGACCCAGAATTAGGTTTTTTCTCGCACATTGTTGGGAACGG TTGCATCATGCAGGTAGGACCTGTTGATAATGGTGCCTGGGACGTTGGGGGCGGTTGGAATGCTGAGACCTATGCAGCGGTTGAACTG ATTGAAAGCCATTCAACCAAAGAAGAGTTCATGACGGACTACCGCCTTTATATCGAACTCTTACGCAATCTAGCAGATGAAGCAGGTT TGCCGAAAACGCTTGATACAGGGAGTTTAGCTGGAATTAAAACGCACGAGTATTGCACGAATAACCAACCAAACAACCACTCAGACCA CGTTGACCCTTATCCATATCTTGCTAAATGGGGCATTAGCCGTGAGCAGTTTAAGCATGATATTGAGAACGGCTTGACGATTGAAACA GGCTGGCAGAAGAATGACACTGGCTACTGGTACGTACATTCAGACGGCTCTTATCCAAAAGACAAGTTTGAGAAAATCAATGGCACTT GGTACTACTTTGACAGTTCAGGCTATATGCTTGCAGACCGCTGGAGGAAGCACACAGACGGCAACTGGTACTGGTTCGACAACTCAGG CGAAATGGCTACAGGCTGGAAGAAAATCGCTGATAAGTGGTACTATTTCAACGAAGAAGGTGCCATGAAGACAGGCTGGGTCAAGTAC AAGGACACTTGGTACTACTTAGACGCTAAAGAAGGCGCCATGGTATCAAATGCCTTTATCCAGTCAGCGGACGGAACAGGCTGGTACT ACCTCAAACCAGACGGAACACTGGCAGACAAGCCAGAATTCACAGTAGAGCCAGATGGCTTGATTACAGTAAAATAA >3_SPN ATGGAAATTAATGTGAGTAAATTAAGAACAGATTTGCCTCAAGTCGGCGTGCAACCATATAGGCAAGTACACGCACACTCAACTGGGA ATCCGCATTCAACCGTACAGAATGAAGCGGATTATCACTGGCGGAAAGACCCAGAATTAGGTTTTTTCTCGCACATTGTTGGGAACGG TTGCATCATGCAGGTAGGACCTGTTGATAATGGTGCCTGGGACGTTGGGGGCGGTTGGAATGCTGAGACCTATGCAGCGGTTGAACTG ATTGAAAGCCATTCAACTAAAGAAGAGTTCATGACGGACTACCGCCTTTATATCGAACTCTTACGCAATCTAGCAGATGAAGCAGGTT TGCCGAAAACGCTTGATACAGGGAGTTTAGCTGGAATTAAAACGCACGAGTATTGCACGAATAACCAACCAAACAACCACTCAGACCA TGTGGATCCATACCCTTACTTGGCAAAATGGGGCATTAGCCGTGAGCAGTTTAAGTATGATATTGAGAACGGCTTGACGATTGAAACA GGCTGGCAGAAGAATGACACTGGCTACTGGTACGTACATTCAGACGGCTCTTATCCAAAAGACAAGTTTGAGAAAATCAATGGCACTT GGTACTACTTTGACAGTTCAGGCTATATGCTTGCAGACCGCTGGAGGAAGCACACAGACGGCAACTGGTACTGGTTCGACAACTCAGG CGAAATGGCTACAGGCTGGAAGAAAATCGCTGAGAAGTGGTACTATTTCAACGAAGAAGGTGCCATGAAGACAGGCTGGGTCAAGTAC AAGGACACTTGGTACTACTTAGACGCTAAAGAAGGCGCCATGGTATCAAATGCCTTTATCCAGTCAGCGGACGGAACAGGCTGGTACT ACCTCAAACCAGACGGAACACTGGCAGACAAGCCAGAATTCACAGTAGAGCCAGATGGCTTGATTACAGTTAAATAA >4_SPN ATGGAAATTAATGTGAGTAAATTAAGAACAGATTTGCCTCAAGTCGGCGTGCAACCATATAGGCAAGTACACGCACACTCAACTGGGA ATCCGCATTCAACCGTACAGAATGAAGCGGATTATCACTGGCGGAAAGACCCAGAATTAGGTTTTTTCTCGCACATTGTTGGGAACGG TTGCATCATGCAGGTAGGACCTGTTGATAATGGTGCCTGGGACGTTGGGGGCGGTTGGAATGCTGAGACCTATGCAGCGGTTGAACTG ATTGAAAGCCATTCAACCAAAGAAGAGTTCATGACGGACTACCGCCTTTATATCGAACTCTTACGCAATCTAGCAGATGAAGCAGGTT TGCCGAAAACGCTTGATACAGGGAGTTTAGCTGGAATTAAAACGCACGAGTATTGCACGAATAACCAACCAAACAACCACTCAGACCA CGTTGACCCTTATCCATATCTTGCTAAATGGGGCATTAGCCGTGAGCAGTTTAAGCATGATATTGAGAACGGCTTGACGATTGAAACA GGCTGGCAGAAGAATGACACTGGCTACTGGTACGTACATTCAGACGGCTCTTATCCAAAAGACAAGTTTGAGAAAATCAATGGCACTT GGTACTACTTTGACAGTTCAGGCTATATGCTTGCAGACCGCTGGAGGAAGCACACAGACGGCAACTGGTACTGGTTCGACAACTCAGG CGAAATGGCTACAGGCTGGAAGAAAATCGCTGATAAGTGGTACTATTTCAACGAAGAAGGTGCCATGAAGACAGGCTGGGTCAAGTAC AAGGACACTTGGTACTACTTAGACGCTAAAGAAGGCGCCATGGTATCAAATGCCTTTATCCAGTCAGCGGACGGAACAGGCTGGTACT ACCTCAAACCAGACGGAACACTGGCAGACAAGCCAGACTTCACAGTAGAGCCTGAAGGCTTGATTACAGTAAAATAA >5_SPN ATGGAAATTAATGTGAGTAAATTAAGAACAGATTTGCCTCAAGTCGGCGTGCAACCATATAGGCAAGTACACGCACACTCAACTGGGA ATCCGCATTCAACCGTACAGAATGAAGCGGATTATCACTGGCGGAAAGACCCAGAATTAGGTTTTTTCTCGCACATTGTTGGGAACGG TTGCATCATGCAGGTAGGACCTGTTGATAATGGTGCCTGGGACGTTGGGGGCGGTTGGAATGCTGAGACCTATGCAGCGGTTGAACTG ATTGAAAGCCATTCAACTAAAGAAGAGTTCATGACGGACTACCGTCTTTATATCGAACTCTTACGCAATCTAGCAGATGAAGCAGGTT TGCCGAAAACGCTTGATACAGGGAGTTTAGCTGGAATTAAAACGCACGAGTATTGCACGAATAACCAACCAAACAACCACTCAGACCA TGTGGATCCATACCCTTACTTGGCAAAATGGGGCATTAGCCGTGAGCAGTTTAAGTATGATATTGAGAACGGCTTGACGATTGAAACA GGCTGGCAGAAGAATGACACTGGCTACTGGTACGTACATTCAGACGGCTCTTATCCAAAAGACAAGTTTGAGAAAATCAATGGCACTT GGTACTACTTTGACAGTTCAGGCTATATGCTTGCAGACCGCTGGAGGAAGCACACAGACGGCAACTGGTACTGGTTCGACAACTCAGG TGAAATGGCTACAGGCTGGAAGAAAATCGCTGATAAGTGGTACTATTTCAACGAAGAAGGTGCCATGAAGACAGGCTGGGTCAAGTAC AAGGACACTTGGTACTACTTAGACGCTAAAGAAGGCGCCATGGTATCAAACGCCTTTATCCAGTCAGCGGACGGAACAGGCTGGTACT ACCTCAAACCAGACGGAACACTGGCAGACAAGCCAGAATTCACAGTAGAGCCAGATGGCTTGATTACAGTAAAATAA >6_SPN ATGGAAATTAATGTGAGTAAATTAAGAACAGATTTGCCTCAAGTCGGCGTGCAACCATATAGGCAAGTACACGCACACTCAACTGGGA ATCCGCATTCAACCGTACAGAATGAAGCGGATTATCACTGGCGGAAAGACCCAGAATTAGGTTTTTTCTCGCACATTGTTGGGAACGG TTGCATCATGCAGGTAGGACCTGTTGATAATGGTGCCTGGGACGTTGGGGGCGGTTGGAATGCTGAGACCTATGCAGCGGTTGAACTG ATTGAAAGCCATTCAACTAAAGAAGAGTTCATGACGGACTACCGCCTTTATATCGAACTCTTACGCAATCTAGCAGATGAAGCAGGTT TGCCGAAAACGCTTGATACAGGGAGTTTAGCTGGAATTAAAACGCACGAGTATTGCACGAATAACCAACCAAACAACCACTCAGACCA TGTGGATCCATACCCTTACTTGGCAAAATGGGGCATTAGCCGTGAGCAGTTTAAGTATGATATTGAGAACGGCTTGACGATTGAAACA GGCTGGCAGAAGAATGACACTGGCTACTGGTACGTACATTCAGACGGCTCTTATCCAAAAGACAAGTTTGAGAAAATCAATGGCACTT GGTACTACTTTGACAGTTCAGGCTATATGCTTGCAGACCGCTGGAGGAAGCACACAGACGGCAACTGGTACTGGTTCGACAACTCAGG CGAAATGGCTACAGGCTGGAAGAAAATCGCTGATAAGTGGTACTATTTCAACGAAGAAGGTGCAATGAAGACAGGCTGGGTCAAGTAC AAGGACACTTGGTACTACTTAGACGCTAAAGAAGGCGCCATGGTATCAAATGCCTTTATCCAGTCAGCGGACGGAACAGGCTGGTACT ACCTCAAACCAGACGGAACACTGGCAGACAAGCCAGAATTCACAGTAGAGCCAGATGGCTTGATTACAGTAAAATAA >7_SPN ATGGAAATTAATGTGAGTAAATTAAGAACAGATTTGCCTCAAGTCGGCGTGCAACCATATAGGCAAGTACACGCACACTCAACTGGGA ATCCGCATTCAACCGTACAGAATGAAGCGGATTATCACTGGCGGAAAGACCCAGAATTAGGTTTTTTCTCGCACATTGTTGGGAACGG 10 TTGCATCATGCAGGTAGGACCTGTTGATAATGGTGCCTGGGACGTTGGGGGCGGTTGGAATGCTGAGACCTATGCAGCGGTTGAACTG ATTGAAAGCCATTCAACTAAAGAAGAGTTCATGACGGACTACCGCCTTTATATCGAACTCTTACGCAATCTAGCAGATGAAGCAGGTT TGCCGAAAACGCTTGATACAGGGAGTTTAGCTGGAATTAAAACGCACGAGTATTGCACGAATAACCAACCAAACAACCACTCAGACCA TGTGGATCCATACCCTTACTTGGCAAAATGGGGCATTAGCCGTGAGCAGTTTAAGTATGATATTGAGAACGGCTTGACGATTGAAACA GGCTGGCAGAAGAATGACACTGGCTACTGGTACGTACATTCAGACGGCTCTTATCCAAAAGACAAGTTTGAGAAAATCAATGGCACTT GGTACTACTTTGACAGTTCAGGCTATATGCTTGCAGACCGCTGGAGGAAGCACACAGACGGCAACTGGTACTGGTTCGACAACTCAGG CGAAATGGCTACAGGCTGGAAGAAAATCGCTGATAAGTGGTACTATTTCAACGAAGAAGGTGCCATGAAGACAGGCTGGGTCAAGTAC AAGGACACTTGGTACTACTTAGACGCTAAAGAAGGCGCCATGGTATCAAATGCCTTTATCCAGTCAGCGGACGGAACAGGCTGGTACT ACCTCAAACCAGACGGAACACTGGCAGACAAGCCAGAATTCACAGTAGAGCCAGATGGCTTGATTACAGTAAAATAA >8_SPN ATGGAAATTAATGTGAGTAAATTAAGAACAGATTTGCCTCAAGTCGGCGTGCAACCATATAGGCAAGTACACGCACACTCAACTGGGA ATCCGCATTCAACCGTACAGAATGAAGCGGATTATCACTGGCGGAAAGACCCAGAATTAGGTTTTTTCTCGCACATTGTTGGGAACGG TTGCATCATGCAGGTAGGACCTGTTGATAATGGTGCCTGGGACGTTGGGGGCGGTTGGAATGCTGAGACCTATGCAGCGGTTGAACTG ATTGAAAGCCATTCAACTAAAGAAGAGTTCATGACGGACTACCGCCTTTATATCGAACTCTTACGCAATCTAGCAGATGAAGCAGGTT TGCCGAAAACGCTTGATACAGGGAGTTTAGCTGGAATTAAAACGCACGAGTATTGCACGAATAACCAACCAAACAACCACTCAGACCA TGTGGATCCATACCCTTACTTGGCAAAATGGGGCATTAGCCGTGAGCAGTTTAAGTATGATATTGAGAACGGCTTGACGATTGAAACA GGCTGGCAGAAGAATGACACTGGCTACTGGTACGTACATTCAGACGGCTCTTATCCAAAAGACAAGTTTGAGAAAATCAATGGCACTT GGTACTACTTTGACAGTTCAGGCTATATGCTTGCAGACCGCTGGAGGAAGCACACAGACGGCAACTGGTACTGGTTCGACAACTCAGG CGAAATGGCTACAGGCTGGAAGAAAATCGCTGATAAGTGGTACTATTTCAACGAAGAAGGTGCCATGAAGACAGGCTGGGTCAAGTAC AAGGACACTTGGTACTACTTAGACGCTAAAGAAGGCGCCATGGTATCAAATGCTTTTATCCAGTCAGCGGACGGAACAGGCTGGTACT ACCTCAAACCAGACGGAACACTGGCAGACAAGCCAGAATTCACAGTAGAGCCAGATGGCTTGATTACAGTAAAATAA >9_SPN ATGGAAATTAATGTGAGTAAATTAAGAACAGATTTGCCTCAAGTCGGCGTGCAACCATATAGGCAAGTACACGCACACTCAACTGGGA ATCCGCATTCAACCGTACAGAATGAAGCGGATTATCACTGGCGGAAAGACCCAGAATTAGGTTTTTTCTCGCACATTGTTGGGAACGG TTGCATCATGCAGGTAGGACCTGTTGATAATGGTGCCTGGGACGTTGGGGGCGGTTGGAATGCTGAGACCTATGCAGCGGTTGAACTG ATTGAAAGCCATTCAACTAAAGAAGAGTTCATGACGGACTACCGCCTTTATATCGAACTCTTACGCAATCTAGCAGATGAAGCAGGTT TGCCGAAAACGCTTGATACAGGGAGTTTAGCTGGAATTAAAACGCACGAGTATTGCACGAATAACCAACCAAACAACCACTCAGACCA TGTGGATCCATACCCTTACTTGGCAAAATGGGGCATTAGCCGTGAGCAGTTTAAGCATGATATTGAGAACGGCTTGACGATTGAAACA GGCTGGCAGAAGAATGACACTGGCTACTGGTACGTACATTCAGACGGCTCTTATCCAAAAGACAAGTTTGAGAAAATCAATGGCACTT GGTACTACTTTGACAGTTCAGGCTATATGCTTGCAGACCGCTGGAGGAAGCACACAGACGGCAACTGGTACTGGTTCGACAACTCAGG CGAAATGGCTACAGGCTGGAAGAAAATCGCTGAGAAGTGGTACTATTTTGATGTAGAAGGTGCCATGAAGACAGGCTGGGTCAAGTAC AAGGACACTTGGTACTACTTAGACGCTAAAGAAGGCGCCATGGTATCAAATGCCTTTATCCAGTCAGCGGACGGAACAGGCTGGTACT ACCTCAAACCAGACGGAACACTGGCAGATAAACCAGAGTTCACAGTAGAGCCAGATGGCTTGATTACAGTAAAATAA >10_SPN ATGGAAATTAATGTGAGTAAATTAAGAACAGATTTGCCTCAAGTCGGCGTGCAACCATATAGGCAAGTACACGCACACTCAACTGGGA ATCCGCATTCAACCGTACAGAATGAAGCGGATTATCACTGGCGGAAAGACCCAGAATTAGGTTTTTTCTCGCACATTGTTGGGAACGG TTGCATCATGCAGGTAGGACCTGTTGATAATGGTGCCTGGGACGTTGGGGGCGGTTGGAATGCTGAGACCTATGCAGCGGTTGAACTG ATTGAAAGCCATTCAACTAAAGAAGAGTTCATGACGGACTACCGCCTTTATATCGAACTCTTACGCAATCTAGCAGATGAAGCAGGTT TGCCGAAAACGCTTGATACAGGGAGTTTAGCTGGAATTAAAACGCACGAGTATTGCACGAATAACCAACCAAACAACCACTCAGACCA TGTGGATCCATACCCTTACTTGGCAAAATGGGGCATTAGCCGTGAGCAGTTTAAGCATGATATTGAGAACGGCTTGACGATTGAAACA GGCTGGCAGAAGAATGACACTGGCTACTGGTACGTACACTCAGACGGCTCTTATCCAAAAGACAAGTTTGAGAAAATCAATGGCACTT GGTACTACTTTGACAGTTCAGGCTATATGCTTGCAGACCGCTGGAGGAAGCACACAGACGGCAACTGGTACTGGTTCGACAACTCAGG CGAAATGGCTACAGGCTGGAAGAAAATCGCTGATAAGTGGTACTATTTCAACGAAGAAGGTGCCATGAAGACAGGCTGGGTCAAGTAC AAGGACACTTGGTACTACTTAGACGCTAAAGAAGGCGCCATGGTATCAAATGCCTTTATCCAGTCAGCGGACGGAACAGGCTGGTACT ACCTCAAACCAGACGGAACACTGGCAGACAAGCCAGAATTCACAGTAGAGCCAGATGGCTTGATTACAGTAAAATAA >11_SPN ATGGAAATTAATGTGAGTAAATTAAGAACAGATTTGCCTCAAGTCGGCGTGCAACCATATAGGCAAGTACACGCACACTCAACTGGGA ATCCGCATTCAACCGTACAGAATGAAGCGGATTATCACTGGCGGAAAGACCCAGAATTAGGTTTTTTCTCGCACATTGTTGGGAACGG TTGCATCATGCAGGTAGGACCTGTTGATAATGGTGCCTGGGACGTTGGGGGCGGTTGGAATGCTGAGACCTATGCAGCGGTTGAACTG ATTGAAAGCCATTCAACTAAAGAAGAGTTCATGACGGACTACCGCCTTTATATCGAACTCTTACGCAATCTAGCAGATGAAGCAGGTT TGCCGAAAACGCTTGATACAGGGAGTTTAGCTGGAATTAAAACGCACGAGTATTGCACGAATAACCAACCAAACAACCACTCAGACCA TGTGGATCCATACCCTTACTTGGCAAAATGGGGCATTAGCCGTGAGCAGTTTAAGTATGATATTGAGAACGGCTTGACGATTGAAACA GGCTGGCAGAAGAATGACACTGGCTACTGGTACGTACATTCAGACGGCTCTTATCCAAAAGACAAGTTTGAGAAAATCAATGGCACTT GGTACTACTTTGACAGTTCAGGCTATATGCTTGCAGACCGCTGGAGGAAGCACACAGACGGCAACTGGTACTGGTTCGACAACTCAGG CGAAATGGCTACAGGCTGGAAGAAAATCGCTGAGAAGTGGTACTATTTTGATGTAGAAGGTGCCATGAAGACAGGCTGGGTCAAGTAC AAGGACACTTGGTACTACTTAGACGCTAAAGAAGGCGCCATGGTATCAAATGCCTTTATCCAGTCAGCGGACGGAACAGGCTGGTACT ACCTCAAACCAGACGGAACACTGGCAGACAAGCCAGAATTCACAGTAGAGCCAGATGGCTTGATTACAGTAAAATAA >12_SPN ATGGAAATTAATGTGAGTAAATTAAGAACAGATTTGCCTCAAGTCGGCGTGCAACCATATAGGCAAGTACACGCACACTCAACTGGGA ATCCGCATTCAACCGTACAGAATGAAGCGGATTATCACTGGCGGAAAGACCCAGAATTAGGTTTTTTCTCGCACATTGTTGGGAACGG TTGCATCATGCAGGTAGGACCTGTTGATAATGGTGCCTGGGACGTTGGGGGCGGTTGGAATGCTGAGACCTATGCAGCGGTTGAACTT ATTGAAAGCCATTCAACCAAAGAAGAGTTCATGACGGACTACCGCCTTTATATCGAACTCTTACGCAATCTAGCAGATGAAGCAGGTT TGCCGAAAACGCTTGATACAGGGAGTTTAGCTGGAATTAAAACGCACGAGTATTGCACGAATAACCAACCAAACAACCACTCAGACCA CGTTGACCCTTATCCATATCTTGCTAAATGGGGCATTAGCCGTGAGCAGTTTAAGCATGATATTGAGAACGGCTTGACGATTGAAACA GGCTGGCAGAAGAATGACACTGGCTACTGGTACGTACATTCAGACGGCTCTTATCCAAAAGACAAGTTTGAGAAAATCAATGGCACTT GGTACTACTTTGACAGTTCAGGCTATATGCTTGCAGACCGCTGGAGGAAGCACACAGACGGCAACTGGTACTGGTTCGACAACTCAGG CGAAATGGCTACAGGCTGGAAGAAAATCGCTGATAAGTGGTACTATTTCAACGAAGAAGGTGCCATGAAGACAGGCTGGGTCAAGTAC AAGGACACTTGGTACTACTTAGACGCTAAAGAAGGCGCCATGGTATCAAATGCCTTTATCCAGTCAGCGGACGGAACAGGCTGGTACT ACCTCAAACCAGACGGAACACTGGCAGACAAGCCAGAATTCACAGTAGAGCCAGATGGCTTGATTACAGTAAAATAA >13_SPN ATGGAAATTAATGTGAGTAAATTAAGAACAGATTTGCCTCAAGTCGGCGTGCAACCATATAGGCAAGTACACGCACACTCAACTGGGA ATCCGCATTCAACCGTACAGAATGAAGCGGATTATCACTGGCGGAAAGACCCAGAATTAGGTTTTTTCTCGCACATTGTTGGGAACGG TTGCATCATGCAGGTAGGACCTGTTGATAATGGTGCCTGGGACGTTGGGGGCGGTTGGAATGCTGAGACCTATGCAGCGGTTGAACTG ATTGAAAGCCATTCAACTAAAGAAGAGTTCATGACGGACTACCGCCTTTATATCGAACTCTTACGCAATCTAGCAGATGAAGCAGGTT TGCCGAAAACGCTTGATACAGGGAGTTTAGCTGGAATTAAAACGCACGAGTATTGCACGAATAACCAACCAAACAACCACTCAGACCA TGTGGATCCATACCCTTACTTGGCAAAATGGGGCATTAGCCGTGAGCAGTTTAAGCATGATATTGAGAACGGCTTGACGATTGAAACA GGCTGGCAGAAGAATGACACTGGCTACTGGTACGTACATTCAGACGGCTCTTATCCAAAAGACAAGTTTGAGAAAATCAATGGCACTT GGTACTACTTTGACAGTTCAGGCTATATGCTTGCAGACCGCTGGAGGAAGCACACAGACGGCAACTGGTACTGGTTCGACAACTCAGG 11 CGAAATGGCTACAGGCTGGAAGAAAATCGCTGATAAGTGGTACTATTTCAACGAAGAAGGTGCCATGAAGACAGGCTGGGTCAAGTAC AAGGACACTTGGTACTACTTAGACGCTAAAGAAGGCGCCATGGTATCAAATGCCTTTATCCAGTCAGCGGACGGAACAGGCTGGTACT ACCTCAAACCAGACGGAACACTGGCAGACAAGCCAGAATTCACAGTAGAGCCAGATGGCTTGATTACAGTAAAATAA >14_SPN ATGGAAATTAATGTGAGTAAATTAAGAACAGATTTGCCTCAAGTCGGCGTGCAACCATATAGGCAAGTACACGCACACTCAACTGGGA ATCCGCATTCAACCGTACAGAATGAAGCGGACTATCACTGGCGGAAAGACCCAGAATTAGGTTTTTTCTCGCACATTGTTGGGAACGG TTGCATCATGCAGGTAGGACCTGTTGATAATGGTGCCTGGGACGTTGGGGGCGGTTGGAATGCTGAGACCTATGCAGCGGTTGAACTG ATTGAAAGCCATTCAACCAAAGAAGAGTTCATGACGGACTACCGCCTTTATATCGAACTCTTACGCAATCTAGCAGATGAAGCAGGTT TGCCGAAAACGCTTGATACAGGGAGTTTAGCTGGAATTAAAACGCACGAGTATTGCACGAATAACCAACCAAACAACCACTCAGACCA CGTTGACCCTTATCCATATCTTGCTAAATGGGGCATTAGCCGTGAGCAGTTTAAGCATGATATTGAGAACGGCTTGACGATTGAAACA GGCTGGCAGAAGAATGACACTGGCTACTGGTACGTACATTCAGACGGCTCTTATCCAAAAGACAAGTTTGAGAAAATCAATGGCACTT GGTACTACTTTGACAGTTCAGGCTATATGCTTGCAGACCGCTGGAGGAAGCACACAGACGGCAACTGGTACTGGTTCGACAACTCAGG CGAAATGGCTACAGGCTGGAAGAAAATCGCTGATAAGTGGTACTATTTCAACGAAGAAGGTGCCATGAAGACAGGCTGGGTCAAGTAC AAGGACACTTGGTACTACTTAGACGCTAAAGAAGGCGCCATGGTATCAAATGCCTTTATCCAGTCAGCGGACGGAACAGGCTGGTACT ACCTCAAACCAGACGGAACACTGGCAGACAAGCCAGAATTCACAGTAGAGCCAGATGGCTTGATTACAGTAAAATAA >15_SPN ATGGAAATTAATGTGAGTAAATTAAGAACAGATTTGCCTCAAGTCGGCGTGCAACCATATAGGCAAGTACACGCACACTCAACTGGGA ATCCGCATTCAACCGTACAGAATGAAGCGGATTATCACTGGCGGAAAGACCCAGAATTAGGTTTTTTCTCGCACATTGTTGGGAACGG TTGCATCATGCAGGTAGGACCTGTTGATAATGGTGCCTGGGACGTTGGGGGCGGTTGGAATGCTGAGACCTATGCAGCGGTTGAACTG ATTGAAAGCCATTCAACTAAAGAAGAGTTCATGACGGATTACCGCCTTTATATCGAACTCTTACGCAATCTAGCAGATGAAGCAGGTT TGCCGAAAACGCTTGATACAGGGAGTTTAGCTGGAATTAAAACGCACGAGTATTGCACGAATAACCAACCAAACAACCACTCAGACCA TGTGGATCCATACCCTTACTTGGCAAAATGGGGCATTAGCCGTGAGCAGTTTAAGTATGATATTGAGAACGGCTTGACGATTGAAACA GGCTGGCAGAAGAATGACACTGGCTACTGGTACGTACATTCAGACGGCTCTTATCCAAAAGACAAGTTTGAGAAAATCAATGGCACTT GGTACTACTTTGACAGTTCAGGCTATATGCTTGCAGACCGCTGGAGGAAGCACACAGACGGCAACTGGTACTGGTTCGACAACTCAGG CGAAATGGCTACAGGCTGGAAGAAAATCGCTGATAAGTGGTACTATTTCAACGAAGAAGGTGCCATGAAGACAGGCTGGGTCAAGTAC AAGGACACTTGGTACTACTTAGACGCTAAAGAAGGCGCCATGGTATCAAATGCCTTTATCCAGTCAGCGGACGGAACAGGCTGGTACT ACCTCAAACCAGACGGAACACTGGCAGACAAGCCAGAATTCACAGTAGAGCCAGATGGCTTGATTACAGTAAAATAA >16_SPN ATGGAAATTAATGTGAGTAAATTAAGAACAGATTTGCCTCAAGTCGGCGTGCAACCATATAGGCAAGTACACGCACACTCAACTGGGA ATCCGCATTCAACCGTACAGAATGAAGCGGATTATCACTGGCGGAAAGACCCAGAATTAGGTTTTTTCTCGCACATTGTTGGGAACGG TTGCATCATGCAGGTAGGACCTGTTGATAATGGTGCCTGGGACGTTGGGGGCGGTTGGAATGCTGAGACCTATGCAGCGGTTGAACTG ATTGAAAGCCATTCAACTAAAGAAGAGTTCATGACGGACTACCGCCTTTATATCGAACTCTTACGCAATCTAGCAGATGAAGCAGGTT TGCCGAAAACGCTTGATACAGGGAGTTTAGCTGGAATTAAAACGCACGAGTATTGCACGAATAACCAACCAAACAACCACTCAGACCA CGTTGACCCTTATCCATATCTTGCTAAATGGGGCATTAGCCGTGAGCAGTTTAAGCATGATATTGAGAACGGCTTGACGATTGAAACA GGCTGGCAGAAGAATGACACTGGCTACTGGTACGTACATTCAGACGGCTCTTATCCAAAAGACAAGTTTGAGAAAATCAATGGCACTT GGTACTACTTTGACAGTTCAGGCTATATGCTTGCAGACCGCTGGAGGAAGCACACAGACGGCAACTGGTACTGGTTCGACAACTCAGG CGAAATGGCTACAGGCTGGAAGAAAATTGCTGATAAGTGGTACTATTTCAACGAAGAAGGTGCCATGAAGACAGGCTGGGTCAAGTAC AAGGACACTTGGTACTACTTAGACGCTAAAGAAGGCGCCATGGTATCAAATGCCTTTATCCAGTCAGCGGACGGAACAGGCTGGTACT ACCTCAAACCAGACGGAACACTGGCAGACAAGCCAGAATTCACAGTAGAGCCAGATGGCTTGATTACAGTAAAATAA >17_SPN ATGGAAATTAATGTGAGTAAATTAAGAACAGATTTGCCTCAAGTCGGCGTGCAACCATATAGGCAAGTACACGCACACTCAACTGGGA ATCCGCATTCAACCGTACAGAATGAAGCGGATTATCACTGGCGGAAAGACCCAGAATTAGGTTTTTTCTCGCACATTGTTGGGAACGG TTGCATCATGCAGGTAGGACCTGTTGATAATGGTGCCTGGGACGTTGGGGGCGGTTGGAATGCTGAGACCTATGCAGCGGTTGAACTG ATTGAAAGCCATTCAACCAAAGAAGAGTTCATGACGGACTACCGCCTTTATATCGAACTCTTACGCAATCTAGCAGATGAAGCAGGTT TGCCGAAAACGCTTGATACAGGGAGTTTAGCTGGAATTAAAACGCACGAGTATTGCACGAATAACCAACCAAACAACCACTCAGACCA CGTTGACCCTTATCCATATCTTGCTAAATGGGGCATTAGCCGTGAGCAGTTTAAGCATGATATTGAGAACGGCTTGACGATTGAAACA GGCTGGCAGAAGAATGACACTGGCTACTGGTACGTACATTCAGACGGCTCTTATCCAAAAGACAAGTTTGAGAAAATCAATGGCACTT GGTACTACTTTGACAGTTCAGGCTATATGCTTGCAGACCGCTGGAGGAAGCACACAGACGGCAACTGGTACTGGTTCGACAACTCAGG CGAAATGGCTACAGGCTGGAAGAAAATTGCTGATAAGTGGTACTATTTCAACGAAGAAGGTGCCATGAAGACAGGCTGGGTCAAGTAC AAGGACACTTGGTACTACTTAGACGCTAAAGAAGGCGCCATGGTATCAAATGCCTTTATCCAGTCAGCGGACGGAACAGGCTGGTACT ACCTCAAACCAGACGGAACACTGGCAGACAAGCCAGAATTCACAGTAGAGCCAGATGGCTTGATTACAGTAAAATAA >18_SPN ATGGAAATTAATGTGAGTAAATTAAGAACAGATTTGCCTCAAGTCGGCGTGCAACCATATAGGCAAGTACACGCACACTCAACTGGGA ATTCGCATTCAACCGTACAGAATGAAGCGGACTATCACTGGCGGAAAGACCCAGAATTAGGTTTTTTCTCGCACATTGTTGGGAACGG TTGCATCATGCAGGTAGGACCTGTTGATAATGGTGCCTGGGACGTTGGGGGCGGTTGGAATGCTGAGACCTATGCAGCGGTTGAACTG ATTGAAAGCCATTCAACCAAAGAAGAGTTCATGACGGACTACCGCCTTTATATCGAACTCTTACGCAATCTAGCAGATGAAGCAGGTT TGCCGAAAACGCTTGATACAGGGAGTTTAGCTGGAATTAAAACGCACGAGTATTGCACGAATAACCAACCAAACAACCACTCAGACCA CGTTGACCCTTATCCATATCTTGCTAAATGGGGCATTAGCCGTGAGCAGTTTAAGCATGATATTGAGAACGGCTTGACGATTGAAACA GGCTGGCAGAAGAATGACACTGGCTACTGGTACGTACATTCAGACGGCTCTTATCCAAAAGACAAGTTTGAGAAAATCAATGGCACTT GGTACTACTTTGACAGTTCAGGCTATATGCTTGCAGACCGCTGGAGGAAGCACACAGACGGCAACTGGTACTGGTTCGACAACTCAGG CGAAATGGCTACAGGCTGGAAGAAAATCGCTGATAAGTGGTACTATTTCAACGAAGAAGGTGCCATGAAGACAGGCTGGGTCAAGTAC AAGGACACTTGGTACTACTTAGACGCTAAAGAAGGCGCCATGGTATCAAATGCCTTTATCCAGTCAGCGGACGGAACAGGCTGGTACT ACCTCAAACCAGACGGAACACTGGCAGACAAGCCAGAATTCACAGTAGAGCCAGATGGCTTGATTACAGTAAAATAA >19_SPN ATGGAAATTAATGTGAGTAAATTAAGAACAGATTTGCCTCAAGTCGGCGTGCAACCATATAGGCAAGTACACGCACACTCAACTGGGA ATCCGCATTCAACCGTACAGAATGAAGCGGATTATCACTGGCGGAAAGACCCAGAATTAGGTTTTTTCTCGCACATTGTTGGGAACGG TTGCATCATGCAGGTAGGACCTGTTGATAATGGTGCCTGGGACGTTGGGGGCGGTTGGAATGCTGAGACCTATGCAGCGGTTGAACTG ATTGAAAGCCATTCAACCAAAGAAGAGTTCATGACGGACTACCGCCTTTATATCGAACTCTTACGCAATCTAGCAGATGAAGCAGGTT TGCCGAAAACGCTTGATACAGGGAGTTTAGCTGGAATTAAAACGCACGAGTATTGCACGAATAACCAACCAAACAACCACTCAGACCA CGTTGACCCTTATCCATATCTTGCTAAATGGGGCATTAGCCGTGAGCAGTTTAAGTATGATATTGAGAACGGCTTGACGATTGAAACA GGCTGGCAGAAGAATGACACTGGCTACTGGTACGTACATTCAGACGGCTCTTATCCAAAAGACAAGTTTGAGAAAATCAATGGCACTT GGTATTACTTTGACAGTTCAGGCTATATGCTTGCAGACCGCTGGAGGAAGCACACAGACGGCAACTGGTACTGGTTCGACAACTCAGG CGAAATGGCTACAGGCTGGAAGAAAATCGCTGATAAGTGGTACTATTTCAACGAAGAAGGTGCCATGAAGACAGGCTGGGTCAAGTAC AAGGACACTTGGTACTACTTAGACGCTAAAGAAGGCGCCATGGTATCAAATGCCTTTATCCAGTCAGCGGACGGAACAGGCTGGTACT ACCTCAAACCAGACGGAACACTGGCAGACAAGCCAGAATTCACAGTAGAGCCAGATGGCTTGATTACAGTAAAATAA >20_SPN ATGGAAATTAATGTGAGTAAATTAAGAACAGATTTGCCTCAAGTCGGCGTGCAACCATATAGGCAAGTACACGCACACTCAACTGGGA ATCCGCATTCAACCGTACAGAATGAAGCGGATTATCACTGGCGGAAAGACCCAGAATTAGGTTTTTTCTCGCACATTGTTGGGAACGG 12 TTGCATCATGCAGGTAGGACCTGTTGATAATGGTGCCTGGGACGTTGGGGGCGGTTGGAATGCTGAGACCTATGCAGCGGTTGAACTG ATTGAAAGCCATTCAACCAAAGAAGAGTTCATGACGGACTACCGCCTTTATATCGAACTCTTACGCAATCTAGCAGATGAAGCAGGTT TGCCGAAAACGCTTGATACAGGGAGTTTAGCTGGAATTAAAACGCACGAGTATTGCACGAATAACCAACCAAACAACCACTCAGACCA CGTTGACCCTTATCCATATCTTGCTAAATGGGGCATTAGCCGTGAGCAGTTTAAGCATGATATTGAGAACGGCTTGACGATTGAAACA GGCTGGCAGAAGAATGACACTGGCTACTGGTACGTACATTCAGACGGCTCTTATCCAAAAGACAAGTTTGAGAAAATCAATGGCACTT GGTACTACTTTGACAGTTCAGGCTATATGCTTGCAGACCGCTGGAGGAAGCACACAGACGGCAATTGGTACTGGTTCGACAACTCAGG CGAAATGGCTACAGGCTGGAAGAAAATCGCTGATAAGTGGTACTATTTCAACGAAGAAGGTGCCATGAAGACAGGCTGGGTCAAGTAC AAGGACACTTGGTACTACTTAGACGCTAAAGAAGGCGCCATGGTATCAAATGCCTTTATCCAGTCAGCGGACGGAACAGGCTGGTACT ACCTCAAACCAGACGGAACACTGGCAGACAAGCCAGAATTCACAGTAGAGCCAGATGGCTTGATTACAGTAAAATAA >21_SPN ATGGAAATTAATGTGAGTAAATTAAGAACAGATTTGCCTCAAGTCGGCGTGCAACCATATAGGCAAGTACACGCACACTCAACTGGGA ATCCGCATTCAACCGTACAGAATGAAGCGGACTATCACTGGCGGAAAGACCCAGAATTAGGTTTTTTCTCGCACATTGTTGGGAACGG TTGCATCATGCAGGTAGGACCTGTTGATAATGGTGCCTGGGACGTTGGGGGCGGTTGGAATGCTGAGACCTATGCAGCGGTTGAACTG ATTGAAAGCCATTCAACCAAAGAAGAGTTCATGACGGACTACCGCCTTTATATCGAACTCTTACGCAATCTAGCAGATGAAGCAGGTT TGCCGAAAACGCTTGATACAGGGAGTTTAGCTGGAATTAAAACGCACGAGTATTGCACGAATAACCAACCAAACAACCACTCAGACCA CGTTGACCCTTATCCATATCTTGCTAAATGGGGCATTAGCCGTGAGCAGTTTAAGCATGATATTGAGAACGGCTTGACGATTGAAACA GGCTGGCAGAAGAATGACACTGGCTACTGGTACGTACATTCAGACGGCTCTTATCCAAAAGACAAGTTTGAGAAAATCAATGGCACTT GGTACTACTTTGACAGTTCAGGCTATATGCTTGCAGACCGCTGGAGGAAGCACACAGACGGCAACTGGTACTGGTTCGACAACTCAGG CGAAATGGCTACAGGCTGGAAGAAAATCGCTGATAAGTGGTACTATTTCAACGAAGAAGGTGCCATGAAGACAGGCTGGGTCAAGTAC AAGGACACTTGGTACTACTTAGACGCTAAAGAAGGCGCCATGGTATCAAATGCCTTTATCCAGTCAGCGGACGGAACAGGCTGGTACT ACCTCAAACCAGACGGAACACTGGCAGACAAGCCAGACTTCACAGTAGAGCCAGATGGCTTGATTACAGTAAAATAA >22_SPN ATGGAAATTAATGTGAGTAAATTAAGAACAGATTTGCCTCAAGTCGGCGTGCAACCATATAGGCAAGTACACGCACACTCAACTGGGA ATCCGCATTCAACCGTACAGAATGAAGCGGATTATCACTGGCGGAAAGACCCAGAATTAGGTTTTTTCTCGCACATTGTTGGGAACGG TTGCATCATGCAGGTAGGACCTGTTGATAATGGTGCCTGGGACGTTGGGGGCGGTTGGAATGCTGAGAGTTATGCAGCGGTTGAACTG ATTGAAAGCCATTCAACTAAAGAAGAGTTCATGACGGACTACCGCCTTTATATCGAACTCTTACGCAATCTAGCAGATGAAGCAGGTT TGCCGAAAACGCTTGATACAGGGAGTTTAGCTGGAATTAAAACGCACGAGTATTGCACGAATAACCAACCAAACAACCACTCAGACCA TGTGGATCCATACCCTTACTTGGCAAAATGGGGCATTAGCCGTGAGCAGTTTAAGCATGATATTGAGAACGGCTTGACGATTGAAACA GGCTGGCAGAAGAATGACACTGGCTACTGGTACGTACATTCAGACGGCTCTTATCCAAAAGACAAGTTTGAGAAAATCAATGGCACTT GGTACTACTTTGACAGTTCAGGCTATATGCTTGCAGACCGCTGGAGGAAGCACACAGACGGCAACTGGTACTGGTTCGACAACTCAGG CGAAATGGCTACAGGCTGGAAGAAAATCGCTGATAAGTGGTACTATTTCAACGAAGAAGGTGCCATGAAGACAGGCTGGGTCAAGTAC AAGGACACTTGGTACTACTTAGACGCTAAAGAAGGCGCCATGGTATCAAATGCCTTTATCCAGTCAGCGGACGGAACAGGCTGGTACT ACCTCAAACCAGACGGAACACTGGCAGACAAGCCAGAATTCACAGTAGAGCCAGATGGCTTGATTACAGTAAAATAA >23_SPN ATGGAAATTAATGTGAGTAAATTAAGAACAGATTTGCCTCAAGTCGGCGTGCAACCATATAGGCAAGTACACGCACACTCAACTGGGA ATCCGCATTCAACCGTACAGAATGAAGCGGATTATCACTGGCGGAAAGACCCAGAATTAGGTTTTTTCTCGCACATTGTTGGGAACGG TTGCATCATGCAGGTAGGACCTGTTGATAATGGTGCCTGGGACGTTGGGGGCGGTTGGAATGCTGAGACCTATGCAGCGGTTGAACTG ATTGAAAGCCATTCAACTAAAGAAGAGTTCATGACGGACTACCGCCTTTATATCGAACTCTTACGCAATCTAGCAGATGAAGCAGGTT TGCCGAAAACGCTTGATACAGGGAGTTTAGCTGGAATTAAAACGCACGAGTATTGCACGAATAACCAACCAAACAACCACTCAGACCA TGTGGATCCATACCCTTACTTGGCAAAATGGGGCATTAGCCGTGAGCAGTTTAAGTATGATATTGAGAACGACTTGACGATTGAAACA GGCTGGCAGAAGAATGACACTGGCTACTGGTACGTACATTCAGACGGCTCTTATCCAAAAGACAAGTTTGAGAAAATCAATGGCACTT GGTACTACTTTGACAGTTCAGGCTATATGCTTGCAGACCGCTGGAGGAAGCACACAGACGGCAACTGGTACTGGTTCGACAACTCAGG CGAAATGGCTACAGGCTGGAAGAAAATCGCTGATAAGTGGTACTATTTCAACGAAGAAGGTGCCATGAAGACAGGCTGGGTCAAGTAC AAGGACACTTGGTACTACTTAGACGCTAAAGAAGGCGCCATGGTATCAAATGCCTTTATCCAGTCAGCGGACGGAACAGGCTGGTACT ACCTCAAACCAGACGGAACACTGGCAGACAAGCCAGAATTCACAGTAGAGCCAGATGGCTTGATTACAGTAAAATAA >24_SPN ATGGAAATTAATGTGAGTAAATTAAGAACAGATTTGCCTCAAGTCGGCGTGCAACCATATAGGCAAGTACACGCACACTCAACTGGGA ATCCGCATTCAACCGTACAGAATGAAGCGGATTATCACTGGCGGAAAGACCCAGAATTAGGTTTTTTCTCGCACATTGTGGGGAACGG TTGCATCATGCAGGTAGGACCTGTTGATAATGGTGCCTGGGACGTTGGGGGCGGTTGGAATGCTGAGACCTATGCAGCGGTTGAACTG ATTGAAAGCCATTCAACTAAAGAAGAGTTCATGACGGACTACCGCCTTTATATCGAACTCTTACGCAATCTAGCAGATGAAGCAGGTT TGCCGAAAACGCTTGATACAGGGAGTTTAGCTGGAATTAAAACGCACGAGTATTGCACGAATAACCAACCAAACAACCACTCAGACCA TGTGGATCCATACCCTTACTTGGCAAAATGGGGCATTAGCCGTGAGCAGTTTAAGCATGATATTGAGAACGGCTTGACGATTGAAACA GGCTGGCAGAAGAATGACACTGGCTACTGGTACGTACACTCAGACGGCTCTTATCCAAAAGACAAGTTTGAGAAAATCAATGGCACTT GGTACTACTTTGACAGTTCAGGCTATATGCTTGCAGACCGCTGGAGGAAGCACACAGACGGCAACTGGTACTGGTTCGACAACTCAGG CGAAATGGCTACAGGCTGGAAGAAAATCGCTGATAAGTGGTACTATTTCAACGAAGAAGGTGCCATGAAGACAGGCTGGGTCAAGTAC AAGGACACTTGGTACTACTTAGACGCTAAAGAAGGCGCCATGGTATCAAATGCCTTTATCCAGTCAGCGGACGGAACAGGCTGGTACT ACCTCAAACCAGACGGAACACTGGCAGACAAGCCAGAATTCACAGTAGAGCCAGATGGCTTGATTACAGTAAAATAA >25_SPN ATGGAAATTAATGTGAGTAAATTAAGAACAGATTTGCCTCAAGTCGGCGTGCAACCATATAGGCAAGTACACGCACACTCAACTGGGA ATCCGCATTCAACCGTACAGAATGAAGCGGATTATCACTGTCGGAAAGACCCAGAATTAGGTTTTTTCTCGCACATTGTTGGGAACGG TTGCATCATGCAGGTAGGACCTGTTGATAATGGTGCCTGGGACGTTGGGGGCGGTTGGAATGCTGAGACCTATGCAGCGGTTGAACTG ATTGAAAGCCATTCAACTAAAGAAGAGTTCATGACGGACTACCGCCTTTATATCGAACTCTTACGCAATCTAGCAGATGAAGCAGGTT TGCCGAAAACGCTTGATACAGGGAGTTTAGCTGGAATTAAAACGCACGAGTATTGCACGAATAACCAACCAAACAACCACTCAGACCA TGTGGATCCATACCCTTACTTGGCAAAATGGGGCATTAGCCGTGAGCAGTTTAAGCATGATATTGAGAACGGCTTGACGATTGAAACA GGCTGGCAGAAGAATGACACTGGCTACTGGTACGTACACTCAGACGGCTCTTATCCAAAAGACAAGTTTGAGAAAATCAATGGCACTT GGTACTACTTTGACAGTTCAGGCTATATGCTTGCAGACCGCTGGAGGAAGCACACAGACGGCAACTGGTACTGGTTCGACAACTCAGG CGAAATGGCTACAGGCTGGAAGAAAATCGCTGATAAGTGGTACTATTTCAACGAAGAAGGTGCCATGAAGACAGGCTGGGTCAAGTAC AAGGACACTTGGTACTACTTAGACGCTAAAGAAGGCGCCATGGTATCAAATGCCTTTATCCAGTCAGCGGACGGAACAGGCTGGTACT ACCTCAAACCAGACGGAACACTGGCAGACAAGCCAGAATTCACAGTAGAGCCAGATGGCTTGATTACAGTAAAATAA >26_SPN ATGGAAATTAATGTGAGTAAATTAAGAACAGATTTGCCTCAAGTCGGCGTGCAACCATATAGGCAAGTACACGCACACTCAACTGGGA ATCCGCATTCAACCGTACAGAATGAAGCGGATTATCACTGGCGGAAAGACCCAGAATTAGGTTTTTTCTCGCACATTGTTGGGAACGG TTGCATCATGCAGGTAGGACCTGTTGATAATGGTGCCTGGGACGTTGGGGGCGGTTGGAATGCTGAGACCTATGCAGCGGTTGAACTG ATTGAAAGCCATTCAACTAAAGAAGAGTTCATGACGGACTACCGCCTTTATATCGAACTCTTACGCAATCTAGCAGATGAAGCAGGTT TGCCGAAAACGCTTGATACAGGGAGTTTAGCTGGAATTAAAACGCACGAGTATTGCACGAATAACCAACCAAACAACCACTCAGACCA TGTGGATCCATACCCTTACTTGGCAAAATGGGGCATTAGCCGTGAGCAGTTTAAGTATGATATTGAGAACGGCTTGACGATTGAAACA GGCTGGCAGAAGAATGACACTGGCTACTGGTACGTACATTCAGACGGCTCTTATCCAAAAGACAAGTTTGAGAAAATCAATGGCACTT GGTACTACTTTGACAGTTCAGGCTATATGCTTGCAGACCGCTGGAGGAAGCACACAGACGGCAACTGGTACTGGTTCGACAACTCAGG 13 TGAAATGGCTACAGGCTGGAAGAAAATCGCTGATAAGTGGTACTATTTCAACGAAGAAGGTGCCATGAAGACAGGCTGGGTCAAGTAC AAGGACACTTGGTACTACTTAGACGCTAAAGAAGGCGCCATGGTATCAAACGCCTTTATCCAGTCAGCGGACGGAACAGGCTGGTACT ACCTCAAACCAGACGGAACACTGGCAGACAAGCCAGAATTCACAGTAGAGCCAGATGGCTTGATTACAGTAAAATAA >27_SPN ATGGAAATTAATGTGAGTAAATTAAGAACAGATTTGCCTCAAGTCGGCGTGCAACCATATAGGCAAGTACACGCACACTCAACTGGGA ATCCGCATTCAACCGTACAGAATGAAGCGGATTATCACTGGCGGAAAGACCCAGAATTAGGTTTTTTCTCGCACATTGTTGGGAACGG TTGCATCATGCAGGTAGGACCTGTTGATAATGGTGCCTGGGACGTTGGGGGCGGTTGGAATGCTGAGACCTATGCAGCGGTTGAACTG ATTGAAAGCCATTCAACTAAAGAAGAGTTCATGACGGACTACCGCCTTTATATCGAACTCTTACGCAATCTAGCAGATGAAGCAGGTT TGCCGAAAACGCTTGATACAGGGAGTTTAGCTGGAATTAAAACGCACGAGTATTGCACGAATAACCAACCAAACAACCACTCAGACCA TGTGGATCCATACCCTTACTTGGCAAAATGGGGCATTAGCCGTGAGCAGTTTAAGTATGATATTGAGAACGGCTTGACGATTGAAACA GGCTGGCAGAAGAATGACACTGGCTACTGGTACGTACATTCAGACGGCTCTTATCCAAAAGACAAGTTTGAGAAAATCAATGGCACTT GGTACTACTTTGACAGTTCAGGCTATATGCTTGCAGACCGCTGGAGGAAGCACACAGACGGCAACTGGTACTGGTTCGACAACTCAGG CGAAATGGCTACAGGCTGGAAGAAAATCGCTGATAAGTGGTACTATTTTGATGTAGAAGGTGCCATGAAGACAGGCTGGGTCAAGTAC AAGGACACTTGGTACTACTTAGACGCTAAAGAAGGCGCCATGGTATCAAATGCCTTTATCCAGTCAGCGGACGGAACAGGCTGGTACT ACCTCAAACCAGACGGAACACTGGCAGATAAACCAGAGTTCACAGTAGAGCCAGATGGCTTGATTACAGTAAAATAA >28_SPN ATGGAAATTAATGTGAGTAAATTAAGAACAGATTTGCCTCAAGTCGGCGTGCAACCATATAGGCAAGTACACGCACACTCAACTGGGA ATCCGCATTCAACCGTACAGAATGAAGCGGATTATCACTGGCGGAAAGACCCAGAATTAGGTTTTTTCTCGCACATTGTTGGGAACGG TTGCATCATGCAGGTAGGACCTGTTGATAATGGTGCCTGGGACGTTGGGGGCGGTTGGAATGCTGAGACCTATGCAGCGGTTGAACTG ATTGAAAGCCATTCAACTAAAGAAGAGTTCATGACGGACTACCGCCTTTATATCGAACTCTTACGCAATCTAGCAGATGAAGCAGGTT TGCCGAAAACGCTTGATACAGGGAGCTTAGCTGGAATTAAAACGCACGAGTATTGCACGAATAACCAACCAAACAACCACTCAGACCA TGTGGATCCATACCCTTACTTGGCAAAATGGGGCATTAGCCGTGAGCAGTTTAAGCATGATATTGAGAACGGCTTGACGATTGAAACA GGCTGGCAGAAGAATGACACTGGCTACTGGTACGTACATTCAGACGGCTCTTATCCAAAAGACAAGTTTGAGAAAATCAATGGCACTT GGTACTACTTTGACAGTTCAGGCTATATGCTTGCAGACCGCTGGAGGAAGCACACAGACGGCAACTGGTACTGGTTCGACAACTCAGG CGAAATGGCTACAGGCTGGAAGAAAATCGCTGAGAAGTGGTACTATTTTGATGTAGAAGGTGCCATGAAGACAGGCTGGGTCAAGTAC AAGGACACTTGGTACTACTTAGACGCTAAAGAAGGCGCCATGGTATCAAATGCCTTTATCCAGTCAGCGGACGGAACAGGCTGGTACT ACCTCAAACCAGACGGAACACTGGCAGATAAACCAGAGTTCACAGTAGAGCCAGATGGCTTGATTACAGTAAAATAA >29_SPN ATGGAAATTAATGTGAGTAAATTAAGAACAGATTTGCCTCAAGTCGGCGTGCAACCATATAGGCAAGTACACGCACACTCAACTGGGA ATCCGCATTCAACCGTACAGAATGAAGCGGATTATCACTGGCGGAAAGACCCAGAATTAGGTTTTTTCTCGCACATTGTTGGGAACGG TTGCATCATGCAGGTAGGACCTGTTGATAATGGTGCCTGGGACGTTGGGGGCGGTTGGAATGCTGAGACCTATGCAGCGGTTGAACTG ATTGAAAGCCATTCAACTAAAGAAGAGTTCATGACGGACTACCGCCTTTATATCGAACTCTTACGCAATCTAGCAGATGAAGCAGGTT TGCCGAAAACGCTTGATACAGGGAGTTTAGCTGGAATTAAAACGCACGAGTATTGCACGAATAACCAACCAAACAACCACTCAGACCA TGTGGATCCATACCCTTACTTGGCAAAATGGGGCATTAGCCGTGAGCAGTTTAAGTATGATATTGAGAACGGCTTGACGATTGAAACA GGCTGGCAGAAGAATGACACTGGCTACTGGTACGTACATTCAGACGGCTCTTATCCAAAAGACAAGTTTGAGAAAATCAATGGCACTT GGTACTACTTTGACAGTTCAGGCTATATGCTTGCAGACCGCTGGAGGAAGCACACAGACGGCAACTGGTACTGGTTCGACAACTCAGG CGAAATGGCTACAGGCTGGAAGAAAATCGCTGAGAAGTGGTACTATTTTGATGTAGAAGGTGCCATGAAGACAGGCTGGGTCAAGTAC AAGGACACTTGGTACTACTTAGACGCTAAAGAAGGCGCCATGGTATCAAATGCCTTTATCCAGTCAGCGGACGGAACAGGCTGGTACT ACCTCAAACCAGACGGAACACTGGCAGACAAGCCAGAGTTCACAGTAGAGCCAGATGGCTTGATTACAGTAAAATAA >30_SPN ATGGAAATTAATGTGAGTAAATTAAGAACAGATTTGCCTCAAGTCGGCGTGCAACCATATAGGCAAGTACACGCACACTCAACTGGGA ATCCGCATTCAACCGTACAGAATGAAGCGGATTATCACTGGCGGAAAGACCCAGAATTAGGTTTTTTCTCGCACATTGTTGGGAACGG TTGCATCATGCAGGTAGGACCTGTTGATAATGGTGCCTGGGACGTTGGGGGCGGTTGGAATGCTGAGACCTATGCAGCGGTTGAACTG ATTGAAAGCCATTCAACTAAAGAAGAGTTCATGACGGACTACCGCCTTTATATCGAACTCTTACGCAATCTAGCAGATGAAGCAGGTT TGCCGAAAACGCTTGATACAGGGAGTTTAGCTGGAATTAAAACGCACGAGTATTGCACGAATAACCAACCAAACAACCACTCAGACCA TGTGGATCCATACCCTTACTTGGCAAAATGGGGCATTAGCCGTGAGCAGTTTAAGCATGATATTGAGAACGGCTTGACGATTGAAACA GGCTGGCAGAAGAATGACACTGGCTACTGGTACGTACACTCAGACGGCTCTTATCCAAAAGACAAGTTTGAGAAAATCAATGGCACTT GGTACTACTTTGACAGTTCAGGCTATATGCTTGCAGACCGCTGGAGGAAGCACACAGACGGCAACTGGTACTGGTTCGACAACTCAGG CGAAATGGCTACAGGCTGGAAGAAAATCGCTGATAAGTGGTACTATTTCAACGAAGAAGGTGCCATGAAGACTTGAAGGGTCAAGTAC AAGGACACTTGGTACTACTTAGACGCTAAAGAAGGCGCCATGGTATCAAATGCCTTTATCCAGTCAGCGGACGGAACAGGCTGGTACT ACCTCAAACCAGACGGAACACTGGCAGACAAGCCAGAATTCACAGTAGAGCCAGATGGCTTGATTACAGTAAAATAA 14 Amino acid sequences of the LytASpn alleles analyzed in this study >1_SPN MEINVSKLRTDLPQVGVQPYRQVHAHSTGNPHSTVQNEADYHWRKDPELGFFSHIVGNGCIMQVGPVDNGAWDVGGGWNAETYAAVEL IESHSTKEEFMTDYRLYIELLRNLADEAGLPKTLDTGSLAGIKTHEYCTNNQPNNHSDHVDPYPYLAKWGISREQFKHDIENGLTIET GWQKNDTGYWYVHSDGSYPKDKFEKINGTWYYFDSSGYMLADRWRKHTDGNWYWFDNSGEMATGWKKIADKWYYFNEEGAMKTGWVKY KDTWYYLDAKEGAMVSNAFIQSADGTGWYYLKPDGTLADRPEFTVEPDGLITVK >2_SPN MEINVSKLRTDLPQVGVQPYRQVHAHSTGNPHSTVQNEADYHWRKDPELGFFSHIVGNGCIMQVGPVDNGAWDVGGGWNAETYAAVEL IESHSTKEEFMTDYRLYIELLRNLADEAGLPKTLDTGSLAGIKTHEYCTNNQPNNHSDHVDPYPYLAKWGISREQFKHDIENGLTIET GWQKNDTGYWYVHSDGSYPKDKFEKINGTWYYFDSSGYMLADRWRKHTDGNWYWFDNSGEMATGWKKIADKWYYFNEEGAMKTGWVKY KDTWYYLDAKEGAMVSNAFIQSADGTGWYYLKPDGTLADKPEFTVEPDGLITVK >3_SPN MEINVSKLRTDLPQVGVQPYRQVHAHSTGNPHSTVQNEADYHWRKDPELGFFSHIVGNGCIMQVGPVDNGAWDVGGGWNAETYAAVEL IESHSTKEEFMTDYRLYIELLRNLADEAGLPKTLDTGSLAGIKTHEYCTNNQPNNHSDHVDPYPYLAKWGISREQFKYDIENGLTIET GWQKNDTGYWYVHSDGSYPKDKFEKINGTWYYFDSSGYMLADRWRKHTDGNWYWFDNSGEMATGWKKIAEKWYYFNEEGAMKTGWVKY KDTWYYLDAKEGAMVSNAFIQSADGTGWYYLKPDGTLADKPEFTVEPDGLITVK >4_SPN MEINVSKLRTDLPQVGVQPYRQVHAHSTGNPHSTVQNEADYHWRKDPELGFFSHIVGNGCIMQVGPVDNGAWDVGGGWNAETYAAVEL IESHSTKEEFMTDYRLYIELLRNLADEAGLPKTLDTGSLAGIKTHEYCTNNQPNNHSDHVDPYPYLAKWGISREQFKHDIENGLTIET GWQKNDTGYWYVHSDGSYPKDKFEKINGTWYYFDSSGYMLADRWRKHTDGNWYWFDNSGEMATGWKKIADKWYYFNEEGAMKTGWVKY KDTWYYLDAKEGAMVSNAFIQSADGTGWYYLKPDGTLADKPDFTVEPEGLITVK >5_SPN MEINVSKLRTDLPQVGVQPYRQVHAHSTGNPHSTVQNEADYHWRKDPELGFFSHIVGNGCIMQVGPVDNGAWDVGGGWNAETYAAVEL IESHSTKEEFMTDYRLYIELLRNLADEAGLPKTLDTGSLAGIKTHEYCTNNQPNNHSDHVDPYPYLAKWGISREQFKYDIENGLTIET GWQKNDTGYWYVHSDGSYPKDKFEKINGTWYYFDSSGYMLADRWRKHTDGNWYWFDNSGEMATGWKKIADKWYYFNEEGAMKTGWVKY KDTWYYLDAKEGAMVSNAFIQSADGTGWYYLKPDGTLADKPEFTVEPDGLITVK >6_SPN MEINVSKLRTDLPQVGVQPYRQVHAHSTGNPHSTVQNEADYHWRKDPELGFFSHIVGNGCIMQVGPVDNGAWDVGGGWNAETYAAVEL IESHSTKEEFMTDYRLYIELLRNLADEAGLPKTLDTGSLAGIKTHEYCTNNQPNNHSDHVDPYPYLAKWGISREQFKHDIENGLTIET GWQKNDTGYWYVHSDGSYPKDKFEKINGTWYYFDSSGYMLADRWRKHTDGNWYWFDNSGEMATGWKKIAEKWYYFDVEGAMKTGWVKY KDTWYYLDAKEGAMVSNAFIQSADGTGWYYLKPDGTLADKPEFTVEPDGLITVK >7_SPN MEINVSKLRTDLPQVGVQPYRQVHAHSTGNPHSTVQNEADYHWRKDPELGFFSHIVGNGCIMQVGPVDNGAWDVGGGWNAETYAAVEL IESHSTKEEFMTDYRLYIELLRNLADEAGLPKTLDTGSLAGIKTHEYCTNNQPNNHSDHVDPYPYLAKWGISREQFKYDIENGLTIET GWQKNDTGYWYVHSDGSYPKDKFEKINGTWYYFDSSGYMLADRWRKHTDGNWYWFDNSGEMATGWKKIAEKWYYFDVEGAMKTGWVKY KDTWYYLDAKEGAMVSNAFIQSADGTGWYYLKPDGTLADKPEFTVEPDGLITVK >8_SPN GA13499 MEINVSKLRTDLPQVGVQPYRQVHAHSTGNSHSTVQNEADYHWRKDPELGFFSHIVGNGCIMQVGPVDNGAWDVGGGWNAETYAAVEL IESHSTKEEFMTDYRLYIELLRNLADEAGLPKTLDTGSLAGIKTHEYCTNNQPNNHSDHVDPYPYLAKWGISREQFKHDIENGLTIET GWQKNDTGYWYVHSDGSYPKDKFEKINGTWYYFDSSGYMLADRWRKHTDGNWYWFDNSGEMATGWKKIADKWYYFNEEGAMKTGWVKY KDTWYYLDAKEGAMVSNAFIQSADGTGWYYLKPDGTLADKPEFTVEPDGLITVK >9_SPN 801 MEINVSKLRTDLPQVGVQPYRQVHAHSTGNPHSTVQNEADYHWRKDPELGFFSHIVGNGCIMQVGPVDNGAWDVGGGWNAETYAAVEL IESHSTKEEFMTDYRLYIELLRNLADEAGLPKTLDTGSLAGIKTHEYCTNNQPNNHSDHVDPYPYLAKWGISREQFKHDIENGLTIET GWQKNDTGYWYVHSDGSYPKDKFEKINGTWYYFDSSGYMLADRWRKHTDGNWYWFDNSGEMATGWKKIADKWYYFNEEGAMKTGWVKY KDTWYYLDAKEGAMVSNAFIQSADGTGWYYLKPDGTLADKPDFTVEPDGLITVK >10_SPN PCS81218 MEINVSKLRTDLPQVGVQPYRQVHAHSTGNPHSTVQNEADYHWRKDPELGFFSHIVGNGCIMQVGPVDNGAWDVGGGWNAESYAAVEL IESHSTKEEFMTDYRLYIELLRNLADEAGLPKTLDTGSLAGIKTHEYCTNNQPNNHSDHVDPYPYLAKWGISREQFKHDIENGLTIET GWQKNDTGYWYVHSDGSYPKDKFEKINGTWYYFDSSGYMLADRWRKHTDGNWYWFDNSGEMATGWKKIADKWYYFNEEGAMKTGWVKY KDTWYYLDAKEGAMVSNAFIQSADGTGWYYLKPDGTLADKPEFTVEPDGLITVK >11_SPN 2081074 MEINVSKLRTDLPQVGVQPYRQVHAHSTGNPHSTVQNEADYHWRKDPELGFFSHIVGNGCIMQVGPVDNGAWDVGGGWNAETYAAVEL IESHSTKEEFMTDYRLYIELLRNLADEAGLPKTLDTGSLAGIKTHEYCTNNQPNNHSDHVDPYPYLAKWGISREQFKYDIENDLTIET GWQKNDTGYWYVHSDGSYPKDKFEKINGTWYYFDSSGYMLADRWRKHTDGNWYWFDNSGEMATGWKKIADKWYYFNEEGAMKTGWVKY KDTWYYLDAKEGAMVSNAFIQSADGTGWYYLKPDGTLADKPEFTVEPDGLITVK >12_SPN GA47597 MEINVSKLRTDLPQVGVQPYRQVHAHSTGNPHSTVQNEADYHCRKDPELGFFSHIVGNGCIMQVGPVDNGAWDVGGGWNAETYAAVEL IESHSTKEEFMTDYRLYIELLRNLADEAGLPKTLDTGSLAGIKTHEYCTNNQPNNHSDHVDPYPYLAKWGISREQFKHDIENGLTIET GWQKNDTGYWYVHSDGSYPKDKFEKINGTWYYFDSSGYMLADRWRKHTDGNWYWFDNSGEMATGWKKIADKWYYFNEEGAMKTGWVKY KDTWYYLDAKEGAMVSNAFIQSADGTGWYYLKPDGTLADKPEFTVEPDGLITVK >13_SPN 2082239 MEINVSKLRTDLPQVGVQPYRQVHAHSTGNPHSTVQNEADYHWRKDPELGFFSHIVGNGCIMQVGPVDNGAWDVGGGWNAETYAAVEL IESHSTKEEFMTDYRLYIELLRNLADEAGLPKTLDTGSLAGIKTHEYCTNNQPNNHSDHVDPYPYLAKWGISREQFKYDIENGLTIET GWQKNDTGYWYVHSDGSYPKDKFEKINGTWYYFDSSGYMLADRWRKHTDGNWYWFDNSGEMATGWKKIADKWYYFDVEGAMKTGWVKY KDTWYYLDAKEGAMVSNAFIQSADGTGWYYLKPDGTLADKPEFTVEPDGLITVK 15 Table S5. Correspondence between plyA and PlyA alleles of S. pneumoniaea plyA allele (Fam_)b PlyA allelec Acc. No. of plyA alleles in databases References Specific activity (HU mg1)d 1 (1) 2 AE005672 (Tettelin, et al. 2001) 4.4 × 105 2 (1) 2 GU968237 (Jefferies, et al. 2010) 3 (1) 2 GU968219 (Jefferies, et al. 2010) 4 (1) 2 FQ312045 (Donati, et al. 2010) 5 (1) 2 GU968217 (Jefferies, et al. 2010) 6 (1) 2 EF413941 (Jefferies, et al. 2007) 7 (1) 2 CP000918 Unpublished 8 (1) 2 DQ251179 (Kirkham, et al. 2006) 9 (1) 2 GU968385 (Jefferies, et al. 2010) 10 (1) 1 GU968223 (Jefferies, et al. 2010) 4.1 × 105 11 (1) 1 EF413947 (Jefferies, et al. 2007) 12 (1) 1 GU968317 (Jefferies, et al. 2010) 13 (1) 1 CP002176 Unpublished 14 (2) 5 EF413960 (Jefferies, et al. 2007) Non-hemolytic 15 (1) 1 GU968247 (Jefferies, et al. 2010) 16 (1) 1   17 (1) 1   18 (1) 1 CP000936 Unpublished 19 (1) 2   20 (2) 5 EF368014 (Marks, et al. 2007) 21 (2) 10 EF413934 (Jefferies, et al. 2007) 1.1 × 105 22 (1) 11 EF413933 (Jefferies, et al. 2007) 5.9 × 105 23 (1) 2   24 (1) 9 GU968397 (Jefferies, et al. 2010) 3.1 × 105 25 (1) 16 GU968252 (Jefferies, et al. 2010) Unknown 26 (1) 2 GU968225 (Jefferies, et al. 2010) 27 (1) 2 GU968238 (Jefferies, et al. 2010) 28 (1) 2 GU968396 (Jefferies, et al. 2010) 29 (2) 3 EF413957 (Jefferies, et al. 2007) 6.9 × 103 30 (1) 20e   Unknown 31 (1) 1   32 (1) 2   33 (1) 8 GU968401 (Jefferies, et al. 2010) 9.1 × 104 34 (2) 3   35 (1) 1 GU968221 (Jefferies, et al. 2010) a Novel plyA or PlyA alleles are shaded in a light or dark gray background respectively. b Fam_1 and Fam_2 designate 1416- and 1410-bp-long plyASpn alleles respectively. c Numbering of PlyA alleles correspond to those previously proposed (Jefferies, et al. 2010). 16 d The specific activities of the PlyA alleles [in hemolytic units (HU) per miligram of protein] were adapted from Jefferies et al. (2007). e Since up to 19 different PlyA alleles has been reported to date (Jefferies, et al. 2007; Jefferies, et al. 2010; Harvey, et al. 2011), this new allele has preliminarily named as alelle 20. References Donati C, et al. 2010. Structure and dynamics of the pan-genome of Streptococcus pneumoniae and closely related species. Genome Biol. 11:R107. Harvey RM, Ogunniyi AD, Chen AY, Paton JC. 2011. Pneumolysin with low hemolytic activity confers an early growth advantage to Streptococcus pneumoniae in the blood. Infect Immun. 79:41224130. Jefferies JMC, et al. 2007. Presence of nonhemolytic pneumolysin in serotypes of Streptococcus pneumoniae associated with disease outbreaks. J Infect Dis. 196:936944. Jefferies JMC, et al. 2010. Identification of novel pneumolysin alleles from paediatric carriage isolates of Streptococcus pneumoniae. J Med Microbiol. 59:808814. Kirkham L-AS, et al. 2006. Identification of invasive serotype 1 pneumococcal isolates that express nonhemolytic pneumolysin. J Clin Microbiol. 44:151159. Marks M, et al. 2007. Influence of neutropenia on the course of serotype 8 pneumococcal pneumonia in mice. Infect Immun. 75:15861597. Tettelin H, et al. 2001. Complete genome sequence of a virulent isolate of Streptococcus pneumoniae. Science 293:498506. 17 Nucleotide sequences of novel plyA alleles >plyA16 ATGGCAAATAAAGCAGTAAATGACTTTATACTAGCTATGAATTACGATAAAAAGAAACTCTTGACCCATCAGGGAGAAAGTATTGAAA ATCGTTTCATCAAAGAGGGTAATCAGCTACCCGATGAGTTTGTTGTTATCGAAAGAAAGAAGCGGAGCTTGTCGACAAATACAAGTGA TATTTCTGTAACAGCTACCAACGACAGTCGCCTCTATCCTGGAGCACTTCTCGTAGTGGATGAGACCTTGTTAGAGAATAATCCCACT CTTCTTGCGGTCGATCGTGCTCCGATGACTTATAGTATTGATTTGCCTGGTTTGGCAAGTAGCGATAGCTTTCTCCAAGTGGAAGACC CCAGCAATTCAAGTGTTCGCGGAGCGGTAAACGATTTGTTGGCTAAGTGGCATCAAGATTATGGTCAGGTCAATAATGTCCCAGCTAG AATGCAGTATGAAAAAATCACGGCTCACAGCATGGAACAACTCAAGGTCAAGTTTGGTTCTGACTTTGAAAAGACAGGGAATTCTCTT GATATTGATTTTAACTCTGTCCATTCAGGCGAAAAGCAGATTCAGATTGTTAATTTTAAGCAGATTTATTATACAGTCAGCGTAGACG CTGTTAAAAATCCAGGAGATGTGTTTCAAGATACTGTAACGGTAGAGGATTTAAAACAGAGAGGAATTTCTGCAGAGCGTCCTTTGGT CTATATTTCGAGTGTTGCTTATGGGCGCCAAGTCTATCTCAAGTTGGAAACCACGAGTAAGAGTGATGAAGTAGAGGCTGCTTTTGAA GCTTTGATAAAAGGAGTCAAGGTAGCTCCTCAGACAGAGTGGAAGCAGATTTTGGACAATACAGAAGTGAAGGCGGTTATTTTAGGGG GCGACCCAAGTTCGGGTGCCCGAGTTGTAACAGGCAAGGTGGATATGGTAGAGGACTTGATTCAAGAAGGCAGTCGCTTTACAGCAGA TCATCCAGGCTTGCCGATTTCCTATACAACTTCTTTTTTACGTGACAATGTAGTTGCGACCTTTCAAAACAGTACAGACTATGTTGAG ACTAAGGTTACAGCTTACAGAAACGGAGATTTACTGCTGGATCATAGTGGTGCCTATGTTGCCCAATATTATATTACTTGGGATGAAT TATCCTATGATCATCAAGGTAAGGAAGTCTTGACTCCTAAGGCTTGGGACAGAAATGGGCAGGATTTGACGGCTCACTTTACCACTAG TATTCCTTTAAAAGGGAATGTTCGTAATCTCTCTGTCAAAATTAGAGAGTGTACCGGGCTTGCCTGGGAATGGTGGCGTACGGTTTAT GAAAAAACCGATTTGCCACTAGTGCGTAAGCGGACGATTTCTATTTGGGGAACAACTCTCTATCCTCAGGTAGAGGATAAGGTAGAAA ATGATTAG >plyA17 ATGGCAAATAAAGCAGTAAATGACTTTATACTAGCTATGAATTACGATAAAAAGAAACTCTTGACCCATCAGGGAGAAAGTATTGAAA ATCGTTTCATCAAAGAGGGTAATCAGCTACCCGATGAGTTTGTTGTTATCGAAAGAAAGAAGCGGAGCTTGTCGACAAATACAAGTGA TATTTCTGTAACAGCTACCAACGACAGTCGCCTCTATCCTGGAGCACTTCTCGTAGTGGATGAGACCTTGTTAGAGAATAATCCCACT CTTCTTGCGGTCGATCGTGCTCCGATGACTTATAGTATTGATTTGCCTGGTTTGGCAAGTAGCGATAGCTTTCTCCAAGTGGAAGACC CCAGCAATTCAAGTGTTCGCGGAGCGGTAAACGATTTGTTGGCTAAGTGGCATCAAGATTATGGTCAGGTCAATAATGTCCCAGCTAG AATGCAGTATGAAAAAATCACGGCTCACAGCATGGAACAACTCAAGGTCAAGTTTGGTTCTGACTTTGAAAAGACAGGGAATTCTCTT GATATTGATTTTAACTCTGTCCATTCAGGCGAAAAGCAGATTCAGATTGTTAATTTTAAGCAGATTTATTATACAGTCAGCGTAGATG CTGTTAAAAATCCAGGAGATGTGTTTCAAGATACTGTAACGGTAGAGGATTTAAAACAGAGAGGAATTTCTGCAGAGCGTCCTTTGGT CTATATTTCGAGTGTTGCTTATGGGCGCCAAGTCTATCTCAAGTTGGAAACCACGAGTAAGAGTGATGAAGTAGAGGCTGCTTTTGAA GCTTTGATAAAAGGAGTCAAGGTAGCTCCTCAGACAGAGTGGAAGCAGATTTTGGACAATACAGAAGTGAAGGCGGTTATTTTAGGGG GCGATCCAAGTTCGGGTGCCCGAGTTGTAACAGGCAAGGTGGATATGGTAGAGGACTTGATTCAAGAAGGCAGTCGCTTTACAGCAGA TCATCCAGGCTTGCCGATTTCCTATACAACTTCTTTTTTACGTGACAATGTAGTTGCGACCTTTCAAAACAGTACAGACTATGTTGAG ACTAAGGTTACAGCTTACAGAAACGGAGATTTACTGCTGGATCATAGTGGTGCCTATGTTGCCCAATATTATATTACTTGGGATGAAT TATCCTATGATCATCAAGGTAAGGAAGTCTTGACTCCTAAGGCTTGGGACAGAAATGGGCAGGATTTGACGGCTCACTTTACCACTAG TATTCCTTTAAAAGGGAATGTTCGTAATCTCTCTGTCAAAATTAGAGAGTGTACCGGGCTTGCCTGGGAATGGTGGCGTACGGTTTAT GAAAAAACCGATTTGCCACTAGTGCGTAAGCGGACGATTTCTATTTGGGGAACAACTCTCTATCCTCAGGTAGAAGATAAGGTAGAAA ATGACTAG >plyA19 ATGGCAAATAAAGCAGTAAATGACTTTATACTAGCTATGAATTACGATAAAAAGAAACTCTTGACCCATCAGGGAGAAAGTATTGAAA ATCGTTTCATCAAAGAGGGTAATCAGCTACCCGATGAGTTTGTTGTTATCGAAAGAAAGAAGCGGAGCTTGTCGACAAATACAAGTGA TATTTCTGTAACAGCTACCAACGACAGTCGCCTCTATCCTGGAGCACTTCTCGTAGTGGATGAGACCTTGTTAGAGAATAATCCCACT CTTCTTGCGGTCGATCGTGCTCCGATGACTTATAGTATTGATTTGCCTGGTTTGGCAAGTAGCGATAGCTTTCTCCAAGTGGAAGACC CCAGCAATTCAAGTGTTCGCGGAGCGGTAAACGATTTGTTGGCTAAGTGGCATCAAGATTATGGTCAGGTCAATAATGTCCCAGCTAG AATGCAGTATGAAAAAATCACGGCTCACAGCATGGAACAACTCAAGGTCAAGTTTGGTTCTGACTTTGAAAAGACAGGGAATTCTCTT GATATTGATTTTAACTCTGTCCATTCAGGCGAAAAGCAGATTCAGATTGTTAATTTTAAGCAGATTTATTATACAGTCAGCGTAGACG CTGTTAAAAATCCAGGAGATGTGTTTCAAGATACTGTAACGGTAGAGGATTTAAAACAGAGAGGGATTTCTGCAGAGCGTCCTTTGGT CTATATTTCGAGTGTTGCTTATGGGCGCCAAGTCTATCTCAAGTTGGAAACCACGAGTAAGAGTGATGAAGTAGAGGCTGCTTTTGAA GCTTTGATAAAAGGAGTCAAGGTAGCTCCTCAGACAGAGTGGAAGCAGATTTTGGACAATACAGAAGTGAAGGCGGTTATTTTAGGGG GCGACCCAAGTTCGGGTGCCCGAGTTGTAACAGGCAAGGTGGATATGGTAGAGGACTTGATTCAAGAAGGCAGTCGCTTTACAGCAGA TCATCCAGGCTTGCCGATTTCCTATACAACTTCTTTTTTACGTGACAATGTAGTTGCGACCTTTCAAAATAGTACAGACTATGTTGAG ACTAAGGTTACAGCTTACAGAAACGGAGATTTACTGCTGGATCATAGTGGTGCCTATGTTGCCCAATATTATATTACTTGGAATGAAT TATCCTATGATCATCAAGGTAAGGAAGTCTTGACTCCTAAGGCTTGGGACAGAAATGGGCAGGATTTAACGGCTCACTTTACCACTAG TATTCCTTTAAAAGGGAATGTTCGTAATCTCTCTGTCAAAATTAGAGAGTGTACCGGGCTTGCCTGGGAATGGTGGCGTACGGTTTAT GAAAAAACCGATTTGCCACTAGTGCGTAAGCGGACGATTTCTATTTGGGGAACAACTCTCTATCCTCAGGTAGAAGATAAGGTAGAAA ATGACTAG >plyA23 ATGGCAAATAAAGCAGTAAATGACTTTATACTAGCTATGAATTACGATAAAAAGAAACTCTTGACCCATCAGGGAGAAAGTATTGAAA ATCGTTTCATCAAAGAGGGTAATCAGCTACCCGATGAGTTTGTTGTTATCGAAAGAAAGAAGCGGAGCTTGTCGACAAATACAAGTGA TATTTCTGTAACAGCTACCAACGACAGTCGCCTCTATCCTGGAGCACTTCTCGTAGTGGATGAGACCTTGTTAGAGAATAATCCCACT CTTCTTGCGGTCGATCGTGCTCCGATGACTTATAGTATTGATTTGCCTGGTTTGGCAAGTAGCGATAGCTTTCTCCAAGTGGAAGACC CCAGCAATTCAAGTGTTCGCGGAGCGGTAAACGATTTGTTGGCTAAGTGGCATCAAGATTATGGTCAGGTCAATAATGTCCCAGCTAG AATGCAGTATGAAAAAATCACGGCTCACAGCATGGAACAACTCAAGGTCAAGTTTGGTTCTGACTTTGAAAAGACAGGGAATTCTCTT GATATTGATTTTAACTCTGTCCATTCAGGCGAAAAGCAGATTCAGATTGTTAATTTTAAGCAGATTTATTATACAGTCAGCGTAGACG CTGTTAAAAATCCAGGAGATGTGTTTCAAGATACTGTAACGGTAGAGGATTTAAAACAGAGAGGAATTTCTGCAGAGCGTCCTTTGGT CTATATTTCGAGTGTTGCTTATGGGCGTCAAGTCTATCTCAAGTTGGAAACCACGAGTAAGAGTGATGAAGTAGAGGCTGCTTTTGAA GCTTTGATAAAAGGAGTCAAGGTAGCTCCTCAGACAGAGTGGAAGCAGATTTTGGACAATACAGAAGTGAAGGCGGTTATTTTAGGGG GCGACCCAAGTTCGGGTGCCCGAGTTGTAACAGGCAAGGTGGATATGGTAGAGGACTTGATTCAAGAAGGCAGTCGCTTTACAGCAGA TCATCCAGGCTTGCCGATTTCCTATACAACTTCTTTTTTACGTGACAATGTAGTTGCGACCTTTCAAAATAGTACAGACTATGTTGAG ACTAAGGTTACAGCTTACAGAAACGGAGATTTACTGCTGGATCATAGTGGTGCCTATGTTGCCCAATATTATATTACTTGGAATGAAT TATCCTATGATCATCAAGGTAAGGAAGTCTTGACTCCTAAGGCTTGGGACAGAAATGGGCAGGATTTAACGGCTCACTTTACCACTAG TATTCCTTTAAAAGGGAATGTTCGTAATCTCTCTGTCAAAATTAGAGAGTGTACCGGGCTTGCCTGGGAATGGTGGCGTACGGTTTAT GAAAAAACCGATTTGCCACTAGTGCGTAAGCGGACGATTTCTATTTGGGGAACAACTCTCTATCCTCAGGTAGAAGATAAGGTAGAAA ATGACTAG >plyA30 ATGGCAAATAAAGCAGTAAATGACTTTATACTAGCTATGAATTACGATAAAAAGAAACTCTTGACCCATCAGGGAGAAAGTATTGAAA ATCGTTTCATCAAAGAGGGTAATCAGCTACCCGATGAGTTTGTTGTTATCGAAAGAAAGAAGCGGAGCTTGTCGACAAATACAAGTGA TATTTCTGTAACAGCTACCAACGACAGTCGCCTCTATCCTGGAGCACTTCTCGTAGTGGATGAGACCTTGTTAGAGAATAATCCCACT 18 CTTCTTGCGGTCGATCGTGCTCCGATGACTTATAGTATTGATTTGCCTGGTTTGGCAAGTAGCGATAGCTTTCTCCAAGTGGAAGACC CCAGCAATTCAAGTGTTCGCGGAGCGGTAAACGATTTGTTGGCTAAGTGGCATCAAGATTATGGTCAGGTCAATAATGTCCCAGCTAG AATGCAGTATGAAAAAATCACGGCTCACAGCATGGAACAACTCAAGGTCAAGTTTGGTTCTGACTTTGAAAAGACAGGGAATTCTCTT GATATTGATTTTAACTCTGTCCATTCAGGCGAAAAGCAGATTCAGATTGTTAATTTTAAGCAGATTTATTATACAGTCAGCGTAGACG CTGTTAAAAATCCAGGAGATGTGTTTCAAGATACTGTAACGGTAGAGGATTTAAAACAGAGAGGAATTTCTGCAGAGCGTCCTTTGGT CTATATTGCGAGTGTTGCTTATGGGCGCCAAGTCTATCTCAAGTTGGAAACCACGAGTAAGAGTGATGAAGTAGAGGCTGCTTTTGAA GCTTTGATAAAAGGAGTCAAGGTAGCTCCTCAGACAGAGTGGAAGCAGATTTTGGACAATACAGAAGTGAAGGCGGTTATTTTAGGGG GCGACCCAAGTTCGGGTGCCCGAGTTGTAACAGGCAAGGTGGATATGGTAGAGGACTTGATTCAAGAAGGCAGTCGCTTTACAGCAGA TCATCCAGGCTTGCCGATTTCCTATACAACTTCTTTTTTACGTGACAATGTAGTTGCGACCTTTCAAAACAGTACAGACTATGTTGAG ACTAAGGTTACAGCTTACAGAAACGGAGATTTACTGCTGGATCATAGTGGTGCCTATGTTGCCCAATATTATATTACTTGGGATGAAT TATCCTATGATCATCAAGGTAAGGAAGTCTTGACTCCTAAGGCTTGGGACAGAAATGGGCAGGATTTGACGGCTCACTTTACCACTAG TATTCCTTTAAAAGGGAATGTTCGTAATCTCTCTGTCAAAATTAGAGAGTGTACCGGGCTTGCCTGGGAATGGTGGCGTACGGTTTAT GAAAAAACCGATTTGCCACTAGTGCGTAAGCGGACGATTTCTATTTGGGGAACAACTCTCTATCCTCAGGTAGAGGATAAGGTAGAAA ATGATTAG >plyA31 ATGGCAAATAAAGCAGTAAATGACTTTATACTAGCTATGAATTACGATAAAAAGAAACTCTTGACCCATCAGGGAGAAAGTATTGAAA ATCGTTTCATCAAAGAGGGTAATCAGCTACCCGATGAGTTTGTTGTTATCGAAAGAAAGAAGCGGAGCTTGTCGACAAATACAAGTGA TATTTCTGTAACAGCTACCAACGACAGTCGCCTCTATCCTGGAGCACTTCTCGTAGTGGATGAGACCTTGTTAGAGAATAATCCCACT CTTCTTGCGGTCGATCGTGCTCCGATGACTTATAGTATTGATTTGCCTGGTTTGGCAAGTAGCGATAGCTTTCTCCAAGTGGAAGACC CCAGCAATTCAAGTGTTCGCGGAGCGGTAAACGATTTGTTGGCTAAGTGGCATCAAGATTATGGTCAGGTCAATAATGTCCCAGCTAG AATGCAGTATGAAAAAATCACGGCTCACAGCATGGAACAACTCAAGGTCAAGTTTGGTTCTGACTTTGAAAAGACAGGGAATTCTCTT GATATTGATTTTAACTCTGTCCATTCAGGCGAAAAGCAGATTCAGATTGTTAATTTTAAACAGATTTATTATACAGTCAGCGTAGACG CTGTTAAAAATCCAGGAGATGTGTTTCAAGATACTGTAACGGTAGAGGATTTAAAACAGAGAGGAATTTCTGCAGAGCGTCCTTTGGT CTATATTTCGAGTGTTGCTTATGGGCGCCAAGTCTATCTCAAGTTGGAAACCACGAGTAAGAGTGATGAAGTAGAGGCTGCTTTTGAA GCTTTGATAAAAGGAGTCAAGGTAGCTCCTCAGACAGAGTGGAAACAGATTTTGGACAATACAGAAGTGAAGGCGGTTATTTTAGGGG GCGACCCAAGTTCGGGTGCCCGAGTTGTAACAGGCAAGGTGGATATGGTAGAGGACTTGATTCAAGAAGGCAGTCGCTTTACAGCAGA TCATCCAGGCTTGCCGATTTCCTATACAACTTCTTTTTTACGTGACAATGTAGTTGCGACCTTTCAAAACAGTACAGACTATGTTGAG ACTAAGGTTACAGCTTACAGAAACGGAGATTTACTGCTGGATCATAGTGGTGCCTATGTTGCCCAATATTATATTACTTGGGATGAAT TATCCTATGATCATCAAGGTAAGGAAGTCTTGACTCCTAAGGCTTGGGACAGAAATGGGCAGGATTTGACGGCTCACTTTACCACTAG TATTCCTTTAAAAGGGAATGTTCGTAATCTCTCTGTCAAAATTAGAGAGTGTACCGGGCTTGCCTGGGAATGGTGGCGTACGGTTTAT GAAAAAACCGATTTGCCACTAGTGCGTAAGCGGACGATTTCTATTTGGGGAACAACTCTCTATCCTCAGGTAGAAGATAAGGTAGAAA ATGACTAG >plyA32 ATGGCAAATAAAGCAGTAAATGACTTTATACTAGCTATGAATTACGATAAAAAGAAACTCTTGACCCATCAGGGAGAAAGTATTGAAA ATCGTTTCATCAAAGAGGGTAATCAGCTACCCGATGAGTTTGTTGTTATCGAAAGAAAGAAGCGGAGCTTGTCGACAAATACAAGTGA TATTTCTGTAACAGCTACCAACGACAGTCGCCTCTATCCTGGAGCACTTCTCGTAGTGGATGAGACCTTGTTAGAGAATAATCCTACT CTTCTTGCGGTCGATCGTGCTCCGATGACTTATAGTATTGATTTGCCTGGTTTGGCAAGTAGCGATAGCTTTCTCCAAGTGGAAGACC CCAGCAATTCAAGTGTTCGCGGAGCGGTAAACGATTTGTTGGCTAAGTGGCATCAAGATTATGGTCAGGTCAATAATGTCCCAGCTAG AATGCAGTATGAAAAAATCACGGCTCACAGCATGGAACAACTCAAGGTCAAGTTTGGTTCTGACTTTGAAAAGACAGGGAATTCTCTT GATATTGATTTTAACTCTGTCCATTCAGGCGAAAAGCAGATTCAGATTGTTAATTTTAAGCAGATTTATTATACAGTCAGTGTAGATG CTGTTAAAAATCCAGGAGATGTGTTTCAAGATACTGTAACGGTAGAGGATTTAAAACAGAGAGGAATTTCTGCAGAGCGTCCTTTGGT CTATATTTCGAGTGTTGCTTATGGGCGCCAAGTCTATCTCAAGTTGGAAACCACGAGTAAGAGTGATGAAGTAGAGGCTGCTTTTGAA GCTTTGATAAAAGGAGTCAAGGTAGCTCCTCAGACAGAGTGGAAGCAGATTTTGGACAATACAGAAGTGAAGGCGGTTATTTTAGGGG GCGACCCAAGTTCGGGTGCCCGAGTTGTAACAGGCAAGGTGGATATGGTAGAGGACTTGATTCAAGAAGGCAGTCGCTTTACAGCAGA TCATCCAGGCTTGCCGATTTCCTATACAACTTCTTTTTTACGTGACAATGTAGTTGCGACCTTTCAAAATAGTACAGACTATGTTGAA ACTAAGGTTACAGCTTACAGAAACGGAGATTTACTGCTGGATCATAGTGGTGCCTATGTTGCCCAATATTATATTACTTGGAATGAAT TATCCTATGATCATCAAGGTAAGGAAGTCTTGACTCCTAAGGCTTGGGACAGAAATGGGCAGGATTTAACGGCTCACTTTACCACTAG TATTCCTTTAAAAGGGAATGTTCGTAATCTCTCTGTCAAGATTAGAGAGTGTACCGGGCTTGCCTGGGAATGGTGGCGTACGGTTTAT GAAAAAACCGATTTGCCACTAGTGCGTAAGCGGACGATTTCTATTTGGGGAACAACTCTCTATCCTCAGGTAGAAGATAAGGTAGAAA ATGACTAG >plyA34 (1410 nt) ATGGCAAATAAAGCAGTAAATGACTTTATACTAGCTATGAATTACGATAAAAAGAAACTCTTGACCCATCAGGGAGAAAGTATTGAAA ATCGTTTCATCAAAGAGGGTAATCAGCTACCCGATGAGTTTGTTGTTATCGAAAGAAAGAAGCGGAGCTTGTCGACAAATACAAGTGA TATTTCTGTAACAGCTACCAACGACAGTCGCCTCTATCCTGGAGCACTTCTCGTAGTGGATGAGACCTTGTTAGAGAATAATCCCACT CTTCTTGCGGTCGATCGTGCTCCGATGACTTATAGTATTGATTTGCCTGGTTTGGCAAGTAGCGATAGCTTTCTCCAAGTGGAAGACC CCAGCAATTCAAGTGTTCGCGGAGCGGTAAACGATTTGTTGGCTAAGTGGCATCAAGATTATGGTCAGGTCAATAATGTCCCAGCTAG AATGCAGTATGAAAAAATCACGGCTCACAGCATGGAACAACTCAAGGTCAAGTTTGGTTCTGACTTTGAAAAGATAGGGAATTCTCTT GATATTGATTTTAACTCTGTCCATTCAGGAGAAAAGCAGATTCAGATTGTTAATTTTAAGCAGATTTATTATACAGTCAGCGTAGATG CTGTTAAAAATCCAGGAGATGTGTTTCAAGATACTGTAACGGTAGAGGATTTAAGGCAGAGAGGAATTTCTGCAGAGCGTCCTTTGGT CTATATTTCGAGTGTTGCTTATGGGCGCCAAGTCTATCTCAAGTTGGAAACCACGAGTAAGAGTGATGAAGTAGAGGCTGCTTTTGAA TCTTTGATAAAAGGAGTAGCTCCTCAGACAGAGTGGAAGCAGATTTTGGACAATACAGAAGTGAAGGCGGTTATTTTAGGGGGCGACC CAAGTTCGGGTGCCCGAGTTGTAACAGGCAAGGTGGATATGGTAGAGGACTTGATTCAAGAAGGCAGTCGCTTTACAGCCGATCATCC AGGCTTGCCGATTTCCTATACAACTTCTTTTTTACGTGACAATGTAGTTGCGACCTTTCAAAACAGTACAGACTATGTTGAGACTAAG GTTACAGCTTACAGAAACGGAGATTTACTGCTGGATCATAGTGGTGCCTATGTTGCTCAATATTATATTACTTGGGATGAATTATCCT ATGATCATCAAGGCAAGGAAGTCTTGACTCCTAAGGCTTGGGACAGAAATGGGCAGGATTTAACGGCTCACTTTACCACTAGTATTCC TTTAAAAGGGAATGTTCGTAATCTCTCTGTCAAAATTAGAGAGTGTACCGGGCTTGCCTGGGAATGGTGGCGTACGGTTTATGAAAAA ACCGATTTGCCACTAGTGCGTAAGCGGACGATTTCTATTTGGGGAACAACTCTCTATCCTCAGGTAGAAGATAAGGTAGAAAATGACT AG 19 Amino acid sequence of the novel PlyASpn-20 allele >PlyA20 MANKAVNDFILAMNYDKKKLLTHQGESIENRFIKEGNQLPDEFVVIERKKRSLSTNTSDISVTATNDSRLYPGALLVVDETLLENNPT LLAVDRAPMTYSIDLPGLASSDSFLQVEDPSNSSVRGAVNDLLAKWHQDYGQVNNVPARMQYEKITAHSMEQLKVKFGSDFEKTGNSL DIDFNSVHSGEKQIQIVNFKQIYYTVSVDAVKNPGDVFQDTVTVEDLKQRGISAERPLVYIASVAYGRQVYLKLETTSKSDEVEAAFE ALIKGVKVAPQTEWKQILDNTEVKAVILGGDPSSGARVVTGKVDMVEDLIQEGSRFTADHPGLPISYTTSFLRDNVVATFQNSTDYVE TKVTAYRNGDLLLDHSGAYVAQYYITWDELSYDHQGKEVLTPKAWDRNGQDLTAHFTTSIPLKGNVRNLSVKIRECTGLAWEWWRTVY EKTDLPLVRKRTISIWGTTLYPQVEDKVEND This novel allele differs from PlyA1 by a S238A conserved substitution. 20 Fig. S1. Diagram of SMG genes homologous to S. pseudopneumoniae SPPN_02090 (llyA1 and llyA2 subfamilies). A. With the exception of this gene, S. pneumoniae and S. pseudopneumoniae genomes are syntenic in this region, whereas disparities are characteristic of S. mitis and S. oralis genomes. A SPPN_02090 ortholog is also present upstream of pulA in the S. mitis strains SK597 and SK1080. B. A different subfamily of SPPN_02090 orthologous genes (llyA2) is located downstream of parC in many different S. mitis strains (also including SK597). Regions showing ≥90% sequence identity are represented by identical color and shading. S. oralis Uo5 S. mitis B6 02090 02105 02080 02100 02110 rpsL polCrpsG fusApulA fus l 02085 S. mitis SK597_1 pulArpoC rpoB S. mitis SK1080 S. pneumoniae D39 02530249 0251 0254 rpsL polCrpsG fusA 02520250 pulA fusAl polC 1714 1723 pulA 1681 1683 rpsLrpsGfusApolC 0209 fusA pulAl 18480261 0263 0264 rpsL polCrpsG fusA pulApolC 18491846 1847 rpoC rpoB 1712 1713 rpoC rpoB S. pseudopneumoniae IS7493 A 0 1 2 3 4 5 6 7 8 9 10 11 kb parE parC S. mitis SK564PF04439 parE parCPF04439 S. mitis SK597_2 cppA S. mitis SK608 parE parCPF04439 04295 04340 0434504330 04335 S. pseudopneumoniae IS7493 parE parC S. mitis SK616 PF04439 cppA S. mitis SK575 S. mitis SK569 parE parCPF04439 S. mitis SK1073 ilvE S. mitis SK629 parE parCPF04439 S. mitis SK667IS1167 parE parC S. mitis SK642PF04439 ilvE ilvE parE parC S. mitis 21/39PF04439 cppA Streptococcus sp. M334 parE parC 0746 0748 0749 S. pneumoniae D39ilvE parE parC 0897 0905 09060904 S. mitis B6ilvE Signal peptide F5_F8_type_C domain Thiol_cytolysin domain B 21 Fig. S2. Diagram of SMG genes (and gene products) homologous to S. pseudopneumoniae SPPN_04220 (llyB subfamily). Thin arrows represent interrupted genes (pseudogenes). Regions showing ≥90% sequence identity are represented by identical color and/or shading. 0 1 2 3 4 5 6 7 8 9 10 11 kb 04230 rpiA deoB punA deoD rpsT 0422004200 04225 04235 S. pseudopneumoniae IS7493 0879 rpiA deoB punA rpsT 0873 0874 0876 S. mitis B6deoD 0881 rpiA deoB deoD rpsT S. mitis SK564punA rpiA deoB deoD rpsT S. mitis SK1080 punA S. mitis SK597 rpiA deoB punA deoD rpsT S. pseudopneumoniae 1321 S. mitis SK575 S. mitis SK608 S. mitis 21/39 Signal peptide Lipase_3 domain F5_F8_type_C domain Thiol_cytolysin domain 0730 rpiA deoB punA deoD rpsT 0723 0724 S. pneumoniae D39 07270729 01474 rpiAdeoBpunAdeoDrpsT 0148201476 01480 S. pneumoniae 110.58 0147701479 01483 22 S. mitis B6 CATTAGGAATCTCCTTTTTTCACATTTTAATCTTTCTTATTATAACACAAGTTTTTTCGA NCTC 12261 CATTAGGAATCTCCTTTTTTCACATTTTAATCTTTCTTATTATAACACAAGTTTTTTCGA 11/5 CATTAGGAATCTCCTTTTTTCACATTTTAATCTTTCTTATTATAACACAAGTTTTTTCGA 13/39 CATTAGGAATCTCCTTTTTTCACATTTTAATCTTTCTTATTATAACACAAGTTTTTTCGA 17/34 CATTAGGAATCTCCTTTTTTCACATTTTAATCTTTCTTATTATAACACAAGTTTTTTCGA 18/56 CATTAGGAATCTCCTTTTTTCACATTTTAATCTTTCTTATTATAACACAAGTTTTTTCTA 29/42 CATTAGGAATCTCCTTTTTTCACATTTTAATCTTTCTTATTATAACACAAGTTTTTTCGA F0392 CATTAGGAATCTCCTTTTTTCACATTTTAATCTTTCTTATTATAACACAAGTTTTTTCGA SK95 CATTAGGAATCTCCTTTTTTCACATTTTAATCTTTCTTATTATAACACAAGTTTTTTCGA SK321 CATTAGGAATCTCCTTTTTTCACATTTTAATCTTTCTTATTATAACACAAGTTTTTTCGA SK569 CATTAGGAATCTCCTTTTTTCACATTTTAATCTTTCTTATTATAACACAAGTTTTTTCGA SK575 CATTAGGAATCTCCTTTTTTCACATTTTAATCTTTCTTATTATAACACAAGTTTTTTCGA SK579 CATTAGGAATCTCCTTTTTTCACATTTTAATCTTTCTTATTATAACACAAGTTTTTTCGA SK616 CATTAGGAATCTCCTTTTTTCACATTTTAATCTTTCTTATTATAACACAAGTTTTTTCGA S. oralis Uo5 CATTAGGAATCTCCTTTTTTCACATTTTAATCTTTCTTATTATAACACAAGTTTTTTCGA ATCC 35037 CATTAGGAATCTCCTTTTTTCACATTTTAATCTTTCTTATTATAACACAAGTTTTTTCGA ATCC 49296 CATTAGGAATCTCCTTTTTTCACATTTTAATCTTTCTTATTATAACACAAGTTTTTTCGA SK10 CATTAGGAATCTCCTTTTTTCACATTTTAATCTTTCTTATTATAACACAAGTTTTTTCGA SK100 CATTAGGAATCTCCTTTTTTCACATTTTAATCTTTCTTATTATAACACAAGTTTTTTCGA SK313 CATTAGGAATCTCCTTTTTTCACATTTTAATCTTTCTTATTATAACACAAGTTTTTTCGA S. infantis ATCC 700779 CATTAGGAATCTCCTTTTTTCACATTTTAATCTTTCTTATTATAACACAAGTCTAGTCGA X CATTAGGAATCTCCTTTTTTCACATTTTAATCTTTCTTATTATAACACAAGTCTAGTCGA S. tigurinus AZ_3A CATTAGGAATCTCCTTTTTTCACATTTTAATCTTTCTTATTATAACACAAGTCTAGCTGA 1366 CATTAGGAATCTCCTTTTTTCACATTTTAATCTTTCTTATTATAACACAAGTTTTTTCGA 2425 CATTAGGAATCTCCTTTTTTCACATTTTAATCTTTCTTATTATAACACAAGTTTTTTCGA 2426 CATTAGGAATCTCCTTTTTTCACATTTTAATCTTTCTTATTATAACACAAGTTTTTTCGA Streptococcus sp. F0407 CATTAGGAATCTCCTTTTTTCACATTTTAATCTTTCTTATTATAACACAAGTTTTTTCGA GMD1S CATTAGGAATCTCCTTTTTTCACATTTTAATCTTTCTTATTATAACACAAGTTTTTTCGA GMD2S CATTAGGAATCTCCTTTTTTCACATTTTAATCTTTCTTATTATAACACAAGTTTTTTCGA GMD4S CATTAGGAATCTCCTTTTTTCACATTTTAATCTTTCTTATTATAACACAAGTTTTTTCGA GMD6S CATTAGGAATCTCCTTTTTTCACATTTTAATCTTTCTTATTATAACACAAGTTTTTTCGA SK643 CATTAGGAATCTCCTTTTTTCACATTTTAATCTTTCTTATTATAACACAAGTTTTTTCGA SPAR10 CATTAGGAATCTCCTTTTTTCACATTTTAATCTTTCTTATTATAACACAAGTCTAGTCGA **************************************************** * * B6 TTTTCACTAGAGGAAATGGATTTTATT--------------------------------- NCTC 12261 TTTTCACTAGAGGAAATGGATTTTATT--------------------------------- 11/5 TTTTCACTAGAGGAAATGGATTTTATT--------------------------------- 13/39 TTTTCACTAGAGGAAATGGATTTTATT--------------------------------- 17/34 TTTTCACTAGAGGAAATAGATTTTATT--------------------------------- 18/56 TTTTCACTAGAGGAAATGGATTTTATT--------------------------------- 29/42 TTTTCACTAGAGGAAATGGATTTTATT--------------------------------- F0392 TTTTCACTAGAGGAAATGGATTTTGTT--------------------------------- SK95 TTTTCACTAGAGGAAATGGATTTTCTT--------------------------------- SK321 TTTTCACTAGAGGAAATAGATTTTATT--------------------------------- SK569 TTTTCACTAGAGGAAATGGATTTTATT--------------------------------- SK575 TTTTCACTAGAGGAAATAGATTTTATT--------------------------------- SK579 TTTTCACTAGAGGAAATAGATTTTATT--------------------------------- SK616 TTTTCACTAGAGGAAATGGATTTTAT---------------------------------- Uo5 TTTTCACTAGAGGAAAGGGATTTTATTTTATACTCAATGAAAATCAAAGAGCAAACTAGG ATCC 35037 TTTTCACTAGAGGAAATGGATTTTGTT--------------------------------- ATCC 49296 TTTTCACTAGAGGAAATGGATTTTATTTTATACTCAATGAAAATCAAAGAGCAAACTAGG SK10 TTTTCACTAGAGGAAATGGATTTTATT--------------------------------- SK100 TTTTCACTAGAGGAAATGGATTTTATT--------------------------------- SK313 TTTTCACTAGAGGAAATGGATTTTCTT--------------------------------- ATCC 700779 TTTTCACTAGAGGAAATGGATTTTTCTT-------------------------------- X TTTTCACTAGAGGAAATGGATTTTTCTT-------------------------------- AZ_3A TTTTCACTAGAGGAAATGGATTTTCTT--------------------------------- 1366 TTTTCACTAGAGGAAATGGATTTTCTT--------------------------------- 2425 TTTTCACTAGAGGAAATGGATTTTCTT--------------------------------- 2426 TTTTCACTAGAGGAAATGGATTTTCTT--------------------------------- F0407 TTTTCACTAGAGGAAATGGATTTTATT--------------------------------- GMD1S TTTTCACTAGAGGAAATGGATTTTATT--------------------------------- GMD2S TTTTCACTAGAGGAAATGGATTTTATT--------------------------------- GMD4S TTTTCACTAGAGGAAATGGATTTTATT--------------------------------- GMD6S TTTTCACTAGAGGAAATGGATTTTATT--------------------------------- SK643 TTTTCACTAGAGGAAACGGATTTTATT--------------------------------- SPAR10 TTTTCACTAGAGGAAATGGATTTTTCTT-------------------------------- **************** ****** 23 B6 ------------------------------------------------------------ NCTC 12261 ------------------------------------------------------------ 11/5 ------------------------------------------------------------ 13/39 ------------------------------------------------------------ 17/34 ------------------------------------------------------------ 18/56 ------------------------------------------------------------ 29/42 ------------------------------------------------------------ F0392 ------------------------------------------------------------ SK95 ------------------------------------------------------------ SK321 ------------------------------------------------------------ SK569 ------------------------------------------------------------ SK575 ------------------------------------------------------------ SK579 ------------------------------------------------------------ SK616 ------------------------------------------------------------ Uo5 AAGCTAGCCGCAGGCTGTACTTGAGTACGGCAAGACTAAGCTGACACGGTTTGAATTTGA ATCC 35037 ------------------------------------------------------------ ATCC 49296 AAGCTAGCCGCAGGCTGTACTTGAGTACGGCAAGGCTAAGCTGACACGGTTTGAATTTGA SK10 ------------------------------------------------------------ SK100 ------------------------------------------------------------ SK313 ------------------------------------------------------------ ATCC 700779 ------------------------------------------------------------ X ------------------------------------------------------------ AZ_3A ------------------------------------------------------------ 1366 ------------------------------------------------------------ 2425 ------------------------------------------------------------ 2426 ------------------------------------------------------------ F0407 ------------------------------------------------------------ GMD1S ------------------------------------------------------------ GMD2S ------------------------------------------------------------ GMD4S ------------------------------------------------------------ GMD6S ------------------------------------------------------------ SPAR10 ------------------------------------------------------------ SK643 ------------------------------------------------------------ B6 --------------TAGCAAATACAGCTAGGATTGCACTTTTGTTGCCAAGATAGCCTTG NCTC 12261 --------------CAGTAAATACAACTAGGATAGCACTTTAGTTGCTAAAATTTCCTTG 11/5 --------------CAGTAAATACAACTAGGATAGCACTTTAGTTGCTAAAATTTCCTTG 13/39 --------------TAGCAAATACAGCTAGGAGAGCACTTTAATTGCCAAGATGGACTTG 17/34 --------------CAGTAAATCAAGCTAGGATGACACTTTTGTTGCCAAGATGGCCTTG 18/56 --------------TAGCAAATCAAGCTAGGATGGTACTTTTGCTACCAAGATTTCCTTG 29/42 --------------CAGTAAATACAACTAGGATAGCACTTTAGTTGCTAAAATTTCCTTG F0392 --------------CAGCAAATCCAGCTAGGATTGTCCTTTGGCTTCCAAGATTTCCTTG SK95 --------------TCTTAAATCCAGCTAGGATTATCCTTTAGCTGCCAAGATTGCCTTG SK321 --------------CAGTAAATCCAACTAGGATAGCACTTTGGTTGCCAAGATGACCTTA SK569 --------------CAGTAAATCAAGCTAGGATGACACTTTTGTTGCCAAGATGGTCTTG SK575 --------------TAGTAAATACAGCTAGGATTGCACTTTGGTTACCAAGATTGCCTTG SK579 --------------CAGCAAATCGAGCTATGATGACAGTTTTGTTGCCAAGATGGCCTTG SK616 --------------TAGCAAATACAGCTAGGATATCACTTTGGTTGCCAAGATTTCCTTG Uo5 TTTTCGAAGAGTATTAGCAAATCCAGCTAGGAGTGTCCTTTAGCAGCCAAGACTGCCTTG ATCC 35037 --------------CAGCAAACCCAGCTAGTATTGTCCTTTGGCTGCCAAGATTGCCTTG ATCC 49296 TTTTCGAAGAGTATTAGCAAATCCAGCTAGGATTGTCCTTTGGCTGCCAAGATTGCCTTG SK10 --------------CAGCAAATCCAGCTAGGATTGTCCTTTGGCTGCCAAGATTGCCTTT SK100 --------------CAGCAAATCCAGCTAGGATTGTCCTTTTGTTGCCAAGATTGCCTTG SK313 --------------TTGCAAAGCAAGCTAGGCTTTGCCTCCGACTGCCACGATTTTCTTG ATCC 700779 --------------TTATAAATGAAGCTAGGATCGTCCGCCGACAGCCAAGATTTTCTTG X --------------TTATAAATGAAGCTAGGATTGTCCGCCGACAGCCAAGATTTTCTTG AZ_3A --------------TTGCAAAGCAAGCTAGAATTTCCCTCCGACTGCCACGATTTTCTTG 1366 --------------TTGCAAAGCAAGCTAGGATTTCCCTCCGACTGCCACGATTTTCTTG 2425 --------------TTGCAAAGCAAGCTAGGATTTCCCTCCGACTGCCACGATTTTCTTG 2426 --------------TTGCAAAGCAAGCTAGGATTTCCCTCCGACTGCCACGATTTTCTTG F0407 --------------CAGCAAATCCAGCTAGGATTGTCCTTTGGCTGCCAAGATTGCCTTG GMD1S --------------CAGCAAATCCAGCTAGGATTGTCCTTTGGCTGCCAAGATTGCCTTG GMD2S --------------CAGCAAATCCAGCTAGGATTGTCCTTTGGCTGCCAAGATTGCCTTG GMD4S --------------CAGCAAATCCAGCTAGGATTGTCCTTTGGCTGCCAAGATTGCCTTG GMD6S --------------CAGCAAATCCAGCTAGGATTGTCCTTTGGCTGCCAAGATTGCCTTG SPAR10 --------------TTATAAATGAAGCTAGGATTGTCCGCCGACAGCCAAGATTTTCTTG SK643 --------------CAGCAAATCGAGCTATGATGACACTTTTGTTGCCAAGATGGCCTTG *** * *** * * * *** 24 B6 CCTTCTTTTATCAAAGGGTGACGAAACAGTGAGAAATACAGTTGGATAGTCATGGCAACC NCTC 12261 CCTTCTTTTATCAAGGGGTGACGAAACAGTGAGAAATACAGTTGAATGGTCATGGCAACC 11/5 CCTTCTTTTATCAAGGGGTGACGAAACAGTGAGAAATACAGTTGAATGGTCATGGCAACC 13/39 CCTTCTTTTATCAAGGGGTGACGGAAAAGCGAAAAGTACAGTTGAATGGTCATGGCAACC 17/34 CCATCTTTTATCAAAGGATGACGGAAGAGTGAGAAGTACAATTGAATGGTCATGGCAACC 18/56 CCTTCTTTTATCAAAGGGTGACGAAACAGTGAAAAATAAAGTTGTATGGTCATGGCAACC 29/42 CCTTCTTTTATCAAGGGGTGACGAAACAGTGAGAAATACAGTTGAATGGTCATGGCAACC F0392 CCTTCTTTTATCAGGGGATGGCGGAAAAGTGAAAAGTACAGTTGGATAGTCATGGCAACC SK95 CCTTCTTTTATCAGGGGATGGCGGAAAAGTGAAAAATACAGTTGGGTGGTCATGGCAACC SK321 CCTTCTTTTATCAAGGGGTGACGAAACAGTGAGAAATACAGTTGGATGGTCATGGCAACC SK569 CCTTCTTTTATCAAGGGGTGGCGAAACAGTGAGAAGTACAGCTGAATAGTCATGGCAACC SK575 CCTTCTTTTATCAAGGGGTGACGGAAGAGTGAGAAGTAGAGTTGAATGGTCATGGCAACC SK579 CCTTCTTTTATCAAGGGATGACGGAACAGTGAAAAGTACAGTTGAATGGTCATGGCAACC SK616 CCTTCTTTTATCAAGGGGTGGCGGAAAAGCGAAAAGTACAGTTGGATGGTCATGGCAACC Uo5 CCTTCTTTTATCAGGGGATGGCGGAAAAGTGAGAAGTACAGTTGGGTGGTCATGGCAACC ATCC 35037 CCTTCTTTTATCAGGGGATGGCGGAAAAGTGAAAAGTACAGTTGTGTAGTCATGGCAACC ATCC 49296 CCTTCTTTTATCAGGGGGTGACGGAAAAGTGAGAAGTACAGTTGGGTGGTCATGGCGACC SK10 CCTTCTTTTATCAGGGGATGGCGGAAAAGTGAAAAGTACAGTTGGGTAGTCATGGCAACC SK100 CCTTCTTTTATCAGGGGATGGCGGAAAAGTGAAAAGTACAGTTGGGTAGTCATGGCAACC SK313 CCTTCTTTTATCCAAGGGTGTCGGGAAAGGGAGAAGTAAAGTTGGGCAGTCATCACAACC ATCC 700779 CCTTCTTTTATCCAAGGGTGTTGAGAGAGTGAGATATAAAGCTGTGCAGTCATAACAAGC X CCTTCTTTTATCCAAGGGTGCTGAGAGAGTGAGATATAAAGCTGTGCAGTCATAACAAGC AZ_3A CCTTCTTTTATCCAAGGGTGTCGGGAAAGGGAGAAGTAAAGTTGGACAGTCATCACAACC 1366 CCTTCTTTTATCCAAGGGTGTCGGGAAAGGGAGAAGTAAAGTTGGACAGTCATCACAACC 2425 CCTTCTTTTATCCAAGGGTGTCGGGAAAGGGAGAAGTAAAGTTGGACAGTCATCACAACC 2426 CCTTCTTTTATCCAAGGGTGTCGGGAAAGGGAGAAGTAAAGTTGGACAGTCATCACAACC F0407 CCTTCTTTTATCAGGGGATGACGGAAAAGTGAAAAGTACAGTTGGGTGGTCATGGCAACC GMD1S CCTTCTTTTATCAGGGGATGGCGGAAAAGTGAAAAGTACAGTTGGGTGGTCATGGCAACC GMD2S CCTTCTTTTATCAGGGGATGGCGGAAAAGTGAAAAGTACAGTTGGGTGGTCATGGCAACC GMD4S CCTTCTTTTATCAGGGGATGGCGGAAAAGTGAAAAGTACAGTTGGGTGGTCATGGCAACC GMD6S CCTTCTTTTATCAGGGGATGGCGGAAAAGTGAAAAGTACAGTTGGGTGGTCATGGCAACC SK643 CCTTCTTTTATCAAGGGATGACGGAACAGTGAGAAATAAAGTTGGATGGTCATGGCAACC SPAR10 CCTTCTTTTATCCAAGGATGCTGAGAGAGTGAGATATAAAGCTGTGCAGTCATAACAAGC ** ********* ** ** * * ** ** * ** * ** ***** * * * Fig. S3. Multiple alignment of the DNA region located between the 3’ end of dinF and the gene located immediately downstream in selected SMG lacking a lytASMG gene. The orientation of the sequences corresponds to that in the genome of the S. pneumoniae D39 strain. The nucleotides that match those of the plREP copy overlapping the 3’ end of dinF are shown in a black background. Asterisks indicate identical nucleotides in all the sequences. Hyphens indicate gaps introduced to maximize similarity. The termination codon of dinF is marked in a red background whereas the initiation codon of the downstream gene is highlighted in a green background. In S. oralis strains Uo5 and ATCC 49296, an intervening BOX element is present (boxC is labeled in blue; boxA is boldfaced and labeled in gray). 25 Fig. S4. Diagram of the approximate location and orientation of genes cinA, recA, and dinF in the chromosomes of different streptococci. Only complete genomes were analyzed. The first gene of each genome is dnaA. The positions were calculated as the percentage of the respective genome. Interestingly, S. thermophilus strains lack a functional dinF gene. Note that in S. salivarius, the location and orientation of the three genes appear to be strain-specific. Ssa, S. salivarius; Sgl, S. gallolyticus; Sth, S. thermophilus; Ssu, S. suis; Smu, S. mutans. Ssa C C H SS3 Ssa 5 7 .I Ssa JIM 8 7 7 7 Sg lU C N 3 4 Sth Ssu Sm u cinA recA dinF 100% 0% 25%75% 50% 26 Fig. S5. Chromosomal rearrangement in the serotype 3 equine S. pneumoniae A45 strain. The genome of the D39 strain is shown for comparison. The plyA gene and the capsular cluster (cap/cps) are shown in light and dark blue respectively. Several genes encoding choline-binding proteins are shown as red arrows that indicate the direction of transcription. Genes are identified as ordered in D39 (prefix SPD_)/A45 (SPNA45_) and correspond to the following: 0126/01912 (pspA); 0345/01670 (cbpC); 0357/01659 (cbpF); 0821/01231 (cbpE); 0853/01263 (lytB); 1403/00649 (lytC); 1726/ (plyA); 1737/ (lytASpn); 1965/00072 (pcpA); 2017/02044 (cbpA); 2028/02054 (cbpD). The open reading frames SPNA45_01659 and SPNA45_01670 appear to correspond to pseudogenes. The chromosomal fragments of strain A45 that are inverted with respect to D39 are labeled in red. The chromosomal fragment that has been translocated (but not inverted) is marked in yellow. Hatched and blue boxes correspond to complete or defective prophages respectively. 27 Fig. S6. Alignment of the 8.6 kb DNA fragment found in the plyAlytA island of various pneumococcal isolates with the sequences deposited in the databases. For complete genomes, the accession no. and nucleotide positions are indicated in parentheses. The inset shows the percentages of nucleotide similarity compared with the fragment of strain GA43257. Predicted proteins were compared with the Pfam database: PF03009, GDPD (glycerophosphoryl diester phosphodiesterase family); PF13343, SBP_bac_6 (bacterial extracellular solute-binding protein); PF00005, ABC_tran (ATP-binding domain of ABC transporters); PF08402, TOBE_2 (TOBE domain); PF00528, BPD_transp_1 (binding-protein-dependent transport system inner membrane component); PF02308, MgtC (MgtC family); PF13419, HAD_2 (haloacid dehalogenase-like hydrolase); PF00356, LacI (bacterial regulatory proteins, lacI family); PF13416, SBP_bac_8 (bacterial extracellular solute-binding protein). S. suis D9 (CP002641; 11193441111658) S. pseudopneumoniae IS7493 (CP002925; 16593041654816) Clostridium pasteurianum BC1 (CP003261; 38400273845911) S. pneumoniae D39 (CP000410; 16255841621096) S. mitis SK597 S. oralis SK304 Granulicatella elegans ATCC 700633 S. pneumoniae GA43257 (AIKU01000006; 2409332700) Roseburia intestinalis M50/1 (FP929049; 617269619935) 0 2 4 6 8 10 12 14 16 18 20 22 kb S. pneumoniae D39  10/20 kb S. mitis B6 S. oralis Uo5 S. mitis SK597  5 kb S. pseudopneumoniae IS7493  10 kb kb 6070%70% RUP 28 Fig. S7. Alignment of the putative, defective prophage found in the plyAlytA island of various pneumococcal isolates with the sequences deposited in the databases. Potential phage genes are also shown as arrows indicating the direction of transcription. Blackened arrows account for genes encoding hypothetical proteins. The inset shows the percentages of nucleotide similarity compared with the defective prophage of strain GA02270. Predicted proteins were compared with the Pfam database: PF01695, IstB_IS21 (IstB-like ATP binding protein); PF09524, Phg_2220_C (Conserved phage C-terminus); PF09681, Phage_rep_org_N (N-terminal phage replisome organiser); PF09669, Phage_pRha (Phage regulatory protein Rha); PF01381, HTH_3 (Helix-turn-helix); PF00717, Peptidase_S24 (Peptidase S24-like); PF13443, HTH_26 (Helix-turn-helix); PF00589, Phage_integrase (Phage integrase family); PF14659, Phage_int_SAM_3 (Phage integrase, N-terminal SAM-like domain). S. pneumoniae SPNA45 (19029801887193) S. mitis B6 (20559332067453) S. pneumoniae GA02270 S. pseudopneumoniae IS7493 (202692190016) S. pneumoniae 670-6B (320518860) S. suis D39 (21559622162200) Prophage EJ-1 (40621829) >90% 8090% 7080% 0 2 4 6 8 10 12 14 16 18 20 22 kb S. pneumoniae D39  10/20 kb S. mitis B6 S. oralis Uo5 S. mitis SK597  5 kb S. pseudopneumoniae IS7493  10 kb kb 29 Fig. S8. Biofilm formation capacity of lytA, plyA, and plyA lytA mutants of the S. pneumoniae strain R6. The non-encapsulated R6 strain was used as a control. Cells were grown in C medium for 6 h at 34C. Open and filled bars indicate growth and biofilm formation, respectively. In all panels, the results represent the means ± standard errors from at least four independent experiments, each performed in triplicate. *, P <0.05 (compared to the R6 or R924 strains; ANOVA with post-hoc tests). 3 2 1 O D 5 9 5 * * 30 Fig. S9. Dendrogram of S. pneumoniae genomes showing the arrangement distribution of the plyA—lytA island. The number of additional genomes represented in each branch is shown in parentheses. Red diamonds indicate the location of the 44 S. pneumoniae genomes analyzed by Donati et al. (2010). Group II non-Ec-Spn are indicated in a gray background. The figure was slightly modified from http://www.ncbi.nlm.nih.gov/genome/?term=CP000410 (Tatusova, et al. 2014). The names of the different arrangements (A—H) are as in Fig. 6. A D B A A D D D A A A A A D A A AD C A A H A A A A A 1% D GA17971 (+1) 70585 SPN13637 (+1) R6 (+6) P1031 +(22) 845 801 SPNA45 MNZ37 (+1) MNZ14 (+11) MNZ41 (+10) CGSP14 (+8) Hungary19A-6 JJA (+12) TIGR4 (+7) North Carolina6A-23/GA17328/GA02270 (+14) MLV-016 SP14-BS292 (+5) CDC3059-06 (+23) GA43380/WL677 2090008 (+3) SP19-BS75 (+1) CDC0288-04 (+1) TCH8431/19A/GA11856 (+8) SP195 (+17) SPN061370 (+7) 2072047/2061617 (+3) SP23-BS72/208913 (+5) GA60080 GA41317 (+10) SP18-BS74 (+4) GA47502 GA40563 CDC1087-00 (+5) SPN072838/SP3-BS71 (+11) GA41538/670-6B (+9) 1488 (+1) GA17484 (+1) GA17227 (2) BHN237 (+3) GA47210 (+2) 3051 G54/GA47179 (+4) Taiwan19F-14 (+25) GA47033 (+6) DAR831 (+3) SP6-BS73 CCRI 1974 (+5) A G F A A A D DA D D E 31 References Andrewes FW, Horder TJ 1906. A study of the streptococci pathogenic for man. Lancet. 168:708713. Arbique JC, et al. 2004. 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