<p> 1 Supplementary Methods</p><p>2</p><p>3 Bacterial strains and growth condition</p><p>4 B. cereus ATCC 10876 was used to isolate and propagate the bacteriophages BPS10C </p><p>5 and BPS13. The bacteria strains used for the determination of the antibacterial spectra of these </p><p>6 bacteriophages are described in Table 1. All of the bacteria were cultivated in Luria-Bertani (LB)</p><p>7 broth (Difco, Detroit, MI, USA) at 37°C with vigorous shaking (Lab Companion SI-600R </p><p>8 Benchtop Shaker, JEIO TECH, Korea/ RPM: 220/ platform size: 16.1" x 16.1" / Motion type: </p><p>9 orbital motion), and the agar plate was prepared with a final agar (Difco) concentration of 1.5%.</p><p>10</p><p>11 Bacteriophage isolation and purification </p><p>12 Food waste samples were collected from Mok-dong, Seoul in South Korea and used for </p><p>13 the isolation of B. cereus-infecting bacteriophages. To isolate the bacteriophages, 25 g of each </p><p>14 sample was mixed with 225 ml of Butterfield’s phosphate-buffered dilution water (0.25 M </p><p>15 KH2PO4, pH 7.2) in sterile bags. After homogenization, 25 ml of each diluted sample was mixed </p><p>16 with 25 ml of 2X LB broth medium, and the mixture was incubated with shaking at 37°C for 12 </p><p>17 h. Then, 0.5 ml of chloroform was added to the mixture, and the mixture incubated for 5 min at </p><p>18 room temperature. The supernatant of the culture was collected by centrifugation at 6,000 × g for</p><p>19 10 min and filtered using 0.22-µm-pore-size filters (Millipore, Billerica, MA). Forty-five </p><p>20 milliliters of each filtrate was mixed with an equal volume of LB broth containing 107 CFU/ml </p><p>21 B. cereus ATCC 10876, and the mixture was then incubated at 37°C for 12 h with shaking. After </p><p>22 the incubation, the mixed culture was centrifuged at 6,000 × g for 10 min, and the supernatant </p><p>23 was filtered through 0.22-µm pore-size filters to remove the B. cereus cells. The filtered 1 supernatant was used for plaque formation in molten 0.4% LB soft agar containing 107 CFU/ml </p><p>2 B. cereus ATCC 10876. Each plaque was picked with a sterile tip and eluted with 1 ml of sodium</p><p>3 chloride-magnesium sulfate (SM) buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, and 10 mM </p><p>4 MgSO4·7H2O). This phage purification step was repeated at least five times.</p><p>5 For phage propagation, either BPS10C or BPS13 was added to a culture of B. cereus </p><p>6 ATCC 10876 at a multiplicity of infection (MOI) of 1 when the optical density (OD) of the </p><p>7 culture at 600 nm reached 1.0. The mixture was incubated at 37°C for 4 h with shaking. After the</p><p>8 incubation of the mixture, the phage particles were recovered from the B. cereus cell debris by </p><p>9 centrifugation at 6,000 × g for 10 min and filtration using 0.22-µm-pore-size filters. The phage </p><p>10 particles were then purified by precipitation with polyethylene glycol (PEG) 6,000 (Sigma, St. </p><p>11 Louis, MO, USA) and CsCl density gradient ultracentrifugation (Himac CP 100β, Hitachi, </p><p>12 Japan) with different CsCl steps (step density = 1.3, 1.45, 1.5, and 1.7 g/ml) at 78,500 × g and </p><p>13 4°C for 2 h. The purified phage particles were recovered, dialyzed using standard dialysis buffer </p><p>14 (5 M NaCl, 1 M MgCl2, and 1 M Tris·HCl at pH 8.0), and stored at 4°C until further analysis.</p><p>15</p><p>16 Transmission electron microscopy</p><p>17 The morphology of phages BPS10C and BPS13 were observed using Energy-Filtered </p><p>18 Transmission Electron Microscope (EF-TEM). The phage samples were diluted with SM buffer, </p><p>19 and 5 μl of each phage sample was applied to the surface of carbon-coated copper grids. The </p><p>20 negatively stained samples with 2% uranyl acetate were allowed to absorb for 2 min. The </p><p>21 prepared samples were observed using an EF-TEM (JEM-1010, JEOL, Tokyo, Japan) at 80 kV. </p><p>22 The BPS10C and BPS13 phages were identified based on their morphology and classified into </p><p>23 their relative family according to the guidelines of the International Committee on Taxonomy of 1 Viruses [6].</p><p>2</p><p>3 Bacteriophage host range test</p><p>4 Five milliliters of molten 0.4% LB top agar containing 100 µl of each test bacterial </p><p>5 culture was overlaid on 1.5% LB base agar plates. Then, 10 µl of serially diluted phage solutions </p><p>6 (102 to 1011) were spotted on the overlaid agar plate and incubated at 37°C. The sensitivity of the </p><p>7 test bacteria to each of the phages was determined based on whether a phage plaque formed. The </p><p>8 efficiency of plating (EOP) was determined by a comparison of the titers between each selected </p><p>9 test bacterium and the propagation strain B. cereus ATCC 10876.</p><p>10</p><p>11 Stability test under various pH or temperature conditions</p><p>12 To test the phage stability under various pH conditions, each phage (final concentration </p><p>13 109 PFU/ml) was added to SM buffer that was pH adjusted using HCl or NaOH to a pH range of </p><p>14 2.0 to 10.5 and the phage suspensions were incubated at 37°C for 24 h. Then, phage titers were </p><p>15 calculated using the overlaid agar plate as host range test with a reference strain, B. cereus ATCC</p><p>16 10876. The phage suspensions at pH 6.0 were used as controls. And for stability test under </p><p>17 various temperature conditions, each phage (final concentration 109 PFU/ml) was added to SM </p><p>18 buffer and the phage suspensions were incubated for 1 h at 25, 42, 50, 60, and 70°C, respectively.</p><p>19 And then, the phage titers were calculated using the overlaid agar plate with the same reference </p><p>20 strain. The phage suspensions at 25°C were used as controls.</p><p>21</p><p>22 Bacterial challenge test</p><p>23 To confirm the host lysis activity of the phages, a B. cereus ATCC 10876 culture at 1 OD600 nm of 1.0 was infected with the corresponding phage (BPS10C or BPS13) at an MOI of 1.0.</p><p>2 The optical density of the mixture was monitored at 600 nm at 1-h intervals. A B. cereus culture </p><p>3 without phage infection was used as a control. This test was performed in triplicate.</p><p>4</p><p>5 Bacteriophage DNA purification </p><p>6 The genomic DNAs of phages BPS10C and BPS13 were isolated as previously </p><p>7 described by Wilcox et al. [11]. Before the isolation of the phage genomic DNA, the phage </p><p>8 particles were treated with DNase I and RNase A at 37°C for 1 h to remove the bacterial DNA </p><p>9 and RNA, respectively. To isolate the phage genomic DNA, the phage particles were lysed with </p><p>10 lysis buffer (1% sodium dodecyl sulfate (SDS), 0.5 mol/l EDTA, and 10 mg/ml proteinase K) for</p><p>11 2 h at 56°C. A standard phenol-chloroform DNA purification and ethanol precipitation was </p><p>12 performed [9].</p><p>13</p><p>14 Bacteriophage genome sequencing and bioinformatics analysis</p><p>15 The purified phage genomic DNAs were sequenced using a Genome Sequencer FLX </p><p>16 (GS-FLX) instrument (Roche, Mannheim, Germany), and the filtered sequence reads were </p><p>17 assembled with Newbler 2.3 (Roche) at Macrogen Inc. (Seoul, South Korea). The prediction of </p><p>18 all of the open reading frames (ORFs) was conducted using Glimmer v3.02 [4], GeneMarkS [2], </p><p>19 and FgenesB (Softberry, Inc. Mount Kisco, NY, USA) and confirmed by RBSFinder (J. Craig </p><p>20 Venter Institute, Rockville, MD, USA). The annotation and functional analysis of the predicted </p><p>21 ORFs were performed using the BLASTP [1] and InterProScan [12] programs. The comparative </p><p>22 genome analysis of these phages was conducted using the BLASTN [1] and Easyfig [10] </p><p>23 programs. The phylogenetic analysis was conducted using MEGA5 with the neighbor-joining 1 method [7].</p><p>2 1 Supplementary Results</p><p>2</p><p>3 Phylogenetic analysis and phage lifestyle prediction. </p><p>4 The phylogenetic analysis of the isolated phages using the major capsid proteins of </p><p>5 several phages suggested that these two phages belong to the Spounavirinae subfamily. However,</p><p>6 these two phages did not belong to any known genera (Spo1-like virus and Twort-like virus), </p><p>7 similarly to the previously reported phage Bc431v3 (Fig. S2) [5]. To predict the lifestyle of the </p><p>8 BPS10C and BPS13 phages, the amino acid sequences of predicted ORFs were analyzed using </p><p>9 the Phage Classification Tool Set (PHACTS) program [8]. However, this program was unable to </p><p>10 predict whether the lifestyle of these two phages was virulent or temperate, which suggests that </p><p>11 these phages have genomes that are significantly different compared with the other phage </p><p>12 genomes in the GenBank database. The prediction of the packaging type [3] showed that the </p><p>13 packaging strategies of these two phages were not belong to any known packaging strategies of </p><p>14 other phages (Fig. S3).</p><p>15 1 Supplementary Tables and Figures</p><p>2</p><p>3 Table S1. Host range of B. cereus bacteriophages BPS10C and BPS13.</p><p>Plaque formationa Plaque formation Bacterial isolate Sourceb of BPS10C of BPS13 Bacillus cereus ATCC 10876 CC CC ATCC Bacillus cereus ATCC 13061 C C ATCC Bacillus cereus ATCC 14579 C C ATCC Bacillus thuringiensis ATCC 29730 CC CC ATCC Bacillus thuringiensis subsp. kurstaki ATCC 35866 CC CC ATCC Bacillus mycoides ATCC 6462 T T ATCC Listeria monocytogenes ATCC 19115 - - ATCC Staphylococcus aureus ATCC 29213 - - ATCC Staphylococcus epidermis ATCC 35983 - - ATCC 4 a CC, the efficiency of plating (EOP) ranged from 1 to 0.1 with clear plaque; C, the EOP ranged from 0.1 to 0.001 5 with clear plaque; T, turbid plaque; -, not susceptible to the indicated phage. 6 b ATCC, American Type Culture Collection. 7</p><p>8</p><p>9 1 Fig. S1. Electron microscopy images of phages BPS10C (A) and BPS13 (B).</p><p>2</p><p>3</p><p>4 1</p><p>2 Fig. S2. Phylogenetic analysis of major capsid proteins in the Spounavirinae subfamily of </p><p>3 bacteriophages. The major capsid proteins were compared using the ClustalW program, and the </p><p>4 phylogenetic tree was generated through the neighbor-joining method with P distance values </p><p>5 using the MEGA5 program.</p><p>6 1 1 Fig. S3. Phylogenetic analysis of the terminase large subunits in several bacteriophages. </p><p>2 The terminase large subunits were compared using the ClustalW program, and the phylogenetic </p><p>3 tree was generated through the neighbor-joining method with P distance values using the </p><p>4 MEGA5 program.</p><p>5</p><p>6 References</p><p>7 1. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment 8 search tool. J Mol Biol 215:403-410 9 2. Besemer J, Lomsadze A, Borodovsky M (2001) GeneMarkS: a self-training method for 10 prediction of gene starts in microbial genomes. Implications for finding sequence motifs 11 in regulatory regions. Nucleic Acids Res 29:2607-2618 12 3. Casjens SR, Gilcrease EB (2009) Determining DNA packaging strategy by analysis of 13 the termini of the chromosomes in tailed-bacteriophage virions. Methods Mol Biol 14 502:91-111 15 4. Delcher AL, Bratke KA, Powers EC, Salzberg SL (2007) Identifying bacterial genes and 16 endosymbiont DNA with Glimmer. Bioinformatics 23:673-679 17 5. El-Arabi T, Griffiths M, She Y-M, Villegas A, Lingohr E, Kropinski A (2013) Genome 18 sequence and analysis of a broad-host range lytic bacteriophage that infects the Bacillus 19 cereus group. Virol J 10:48 20 6. King AMQ, Lefkowitz E, Adams MJ, Carstens EB (2011) Virus taxonomy: ninth report 21 of the international committee on taxonomy of viruses. Elsevier Science 22 7. Kumar S, Nei M, Dudley J, Tamura K (2008) MEGA: a biologist-centric software for 23 evolutionary analysis of DNA and protein sequences. Brief Bioinform 9:299-306 24 8. McNair K, Bailey BA, Edwards RA (2012) PHACTS, a computational approach to 25 classifying the lifestyle of phages. Bioinformatics 28:614-618 26 9. Sambrook J, Russell D (2001) Molecular cloning: a laboratory manual, 3rd ed. Cold 27 Spring Harbor Laboratory Press, NY 28 10. Sullivan MJ, Petty NK, Beatson SA (2011) Easyfig: a genome comparison visualiser. 29 Bioinformatics 27:1009-1010 30 11. Wilcox SA, Toder R, Foster JW (1996) Rapid isolation of recombinant lambda phage 31 DNA for use in fluorescence in situ hybridization. Chromosome Res 4:397-398 32 12. Zdobnov EM, Apweiler R (2001) InterProScan--an integration platform for the signature- 33 recognition methods in InterPro. Bioinformatics 17:847-848</p><p>34</p><p>35</p><p>36 1</p><p>2</p>