1 Supplementary Methods
2
3 Bacterial strains and growth condition
4 B. cereus ATCC 10876 was used to isolate and propagate the bacteriophages BPS10C
5 and BPS13. The bacteria strains used for the determination of the antibacterial spectra of these
6 bacteriophages are described in Table 1. All of the bacteria were cultivated in Luria-Bertani (LB)
7 broth (Difco, Detroit, MI, USA) at 37°C with vigorous shaking (Lab Companion SI-600R
8 Benchtop Shaker, JEIO TECH, Korea/ RPM: 220/ platform size: 16.1" x 16.1" / Motion type:
9 orbital motion), and the agar plate was prepared with a final agar (Difco) concentration of 1.5%.
10
11 Bacteriophage isolation and purification
12 Food waste samples were collected from Mok-dong, Seoul in South Korea and used for
13 the isolation of B. cereus-infecting bacteriophages. To isolate the bacteriophages, 25 g of each
14 sample was mixed with 225 ml of Butterfield’s phosphate-buffered dilution water (0.25 M
15 KH2PO4, pH 7.2) in sterile bags. After homogenization, 25 ml of each diluted sample was mixed
16 with 25 ml of 2X LB broth medium, and the mixture was incubated with shaking at 37°C for 12
17 h. Then, 0.5 ml of chloroform was added to the mixture, and the mixture incubated for 5 min at
18 room temperature. The supernatant of the culture was collected by centrifugation at 6,000 × g for
19 10 min and filtered using 0.22-µm-pore-size filters (Millipore, Billerica, MA). Forty-five
20 milliliters of each filtrate was mixed with an equal volume of LB broth containing 107 CFU/ml
21 B. cereus ATCC 10876, and the mixture was then incubated at 37°C for 12 h with shaking. After
22 the incubation, the mixed culture was centrifuged at 6,000 × g for 10 min, and the supernatant
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
2 B. cereus ATCC 10876. Each plaque was picked with a sterile tip and eluted with 1 ml of sodium
3 chloride-magnesium sulfate (SM) buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, and 10 mM
4 MgSO4·7H2O). This phage purification step was repeated at least five times.
5 For phage propagation, either BPS10C or BPS13 was added to a culture of B. cereus
6 ATCC 10876 at a multiplicity of infection (MOI) of 1 when the optical density (OD) of the
7 culture at 600 nm reached 1.0. The mixture was incubated at 37°C for 4 h with shaking. After the
8 incubation of the mixture, the phage particles were recovered from the B. cereus cell debris by
9 centrifugation at 6,000 × g for 10 min and filtration using 0.22-µm-pore-size filters. The phage
10 particles were then purified by precipitation with polyethylene glycol (PEG) 6,000 (Sigma, St.
11 Louis, MO, USA) and CsCl density gradient ultracentrifugation (Himac CP 100β, Hitachi,
12 Japan) with different CsCl steps (step density = 1.3, 1.45, 1.5, and 1.7 g/ml) at 78,500 × g and
13 4°C for 2 h. The purified phage particles were recovered, dialyzed using standard dialysis buffer
14 (5 M NaCl, 1 M MgCl2, and 1 M Tris·HCl at pH 8.0), and stored at 4°C until further analysis.
15
16 Transmission electron microscopy
17 The morphology of phages BPS10C and BPS13 were observed using Energy-Filtered
18 Transmission Electron Microscope (EF-TEM). The phage samples were diluted with SM buffer,
19 and 5 μl of each phage sample was applied to the surface of carbon-coated copper grids. The
20 negatively stained samples with 2% uranyl acetate were allowed to absorb for 2 min. The
21 prepared samples were observed using an EF-TEM (JEM-1010, JEOL, Tokyo, Japan) at 80 kV.
22 The BPS10C and BPS13 phages were identified based on their morphology and classified into
23 their relative family according to the guidelines of the International Committee on Taxonomy of 1 Viruses [6].
2
3 Bacteriophage host range test
4 Five milliliters of molten 0.4% LB top agar containing 100 µl of each test bacterial
5 culture was overlaid on 1.5% LB base agar plates. Then, 10 µl of serially diluted phage solutions
6 (102 to 1011) were spotted on the overlaid agar plate and incubated at 37°C. The sensitivity of the
7 test bacteria to each of the phages was determined based on whether a phage plaque formed. The
8 efficiency of plating (EOP) was determined by a comparison of the titers between each selected
9 test bacterium and the propagation strain B. cereus ATCC 10876.
10
11 Stability test under various pH or temperature conditions
12 To test the phage stability under various pH conditions, each phage (final concentration
13 109 PFU/ml) was added to SM buffer that was pH adjusted using HCl or NaOH to a pH range of
14 2.0 to 10.5 and the phage suspensions were incubated at 37°C for 24 h. Then, phage titers were
15 calculated using the overlaid agar plate as host range test with a reference strain, B. cereus ATCC
16 10876. The phage suspensions at pH 6.0 were used as controls. And for stability test under
17 various temperature conditions, each phage (final concentration 109 PFU/ml) was added to SM
18 buffer and the phage suspensions were incubated for 1 h at 25, 42, 50, 60, and 70°C, respectively.
19 And then, the phage titers were calculated using the overlaid agar plate with the same reference
20 strain. The phage suspensions at 25°C were used as controls.
21
22 Bacterial challenge test
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.
2 The optical density of the mixture was monitored at 600 nm at 1-h intervals. A B. cereus culture
3 without phage infection was used as a control. This test was performed in triplicate.
4
5 Bacteriophage DNA purification
6 The genomic DNAs of phages BPS10C and BPS13 were isolated as previously
7 described by Wilcox et al. [11]. Before the isolation of the phage genomic DNA, the phage
8 particles were treated with DNase I and RNase A at 37°C for 1 h to remove the bacterial DNA
9 and RNA, respectively. To isolate the phage genomic DNA, the phage particles were lysed with
10 lysis buffer (1% sodium dodecyl sulfate (SDS), 0.5 mol/l EDTA, and 10 mg/ml proteinase K) for
11 2 h at 56°C. A standard phenol-chloroform DNA purification and ethanol precipitation was
12 performed [9].
13
14 Bacteriophage genome sequencing and bioinformatics analysis
15 The purified phage genomic DNAs were sequenced using a Genome Sequencer FLX
16 (GS-FLX) instrument (Roche, Mannheim, Germany), and the filtered sequence reads were
17 assembled with Newbler 2.3 (Roche) at Macrogen Inc. (Seoul, South Korea). The prediction of
18 all of the open reading frames (ORFs) was conducted using Glimmer v3.02 [4], GeneMarkS [2],
19 and FgenesB (Softberry, Inc. Mount Kisco, NY, USA) and confirmed by RBSFinder (J. Craig
20 Venter Institute, Rockville, MD, USA). The annotation and functional analysis of the predicted
21 ORFs were performed using the BLASTP [1] and InterProScan [12] programs. The comparative
22 genome analysis of these phages was conducted using the BLASTN [1] and Easyfig [10]
23 programs. The phylogenetic analysis was conducted using MEGA5 with the neighbor-joining 1 method [7].
2 1 Supplementary Results
2
3 Phylogenetic analysis and phage lifestyle prediction.
4 The phylogenetic analysis of the isolated phages using the major capsid proteins of
5 several phages suggested that these two phages belong to the Spounavirinae subfamily. However,
6 these two phages did not belong to any known genera (Spo1-like virus and Twort-like virus),
7 similarly to the previously reported phage Bc431v3 (Fig. S2) [5]. To predict the lifestyle of the
8 BPS10C and BPS13 phages, the amino acid sequences of predicted ORFs were analyzed using
9 the Phage Classification Tool Set (PHACTS) program [8]. However, this program was unable to
10 predict whether the lifestyle of these two phages was virulent or temperate, which suggests that
11 these phages have genomes that are significantly different compared with the other phage
12 genomes in the GenBank database. The prediction of the packaging type [3] showed that the
13 packaging strategies of these two phages were not belong to any known packaging strategies of
14 other phages (Fig. S3).
15 1 Supplementary Tables and Figures
2
3 Table S1. Host range of B. cereus bacteriophages BPS10C and BPS13.
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
8
9 1 Fig. S1. Electron microscopy images of phages BPS10C (A) and BPS13 (B).
2
3
4 1
2 Fig. S2. Phylogenetic analysis of major capsid proteins in the Spounavirinae subfamily of
3 bacteriophages. The major capsid proteins were compared using the ClustalW program, and the
4 phylogenetic tree was generated through the neighbor-joining method with P distance values
5 using the MEGA5 program.
6 1 1 Fig. S3. Phylogenetic analysis of the terminase large subunits in several bacteriophages.
2 The terminase large subunits were compared using the ClustalW program, and the phylogenetic
3 tree was generated through the neighbor-joining method with P distance values using the
4 MEGA5 program.
5
6 References
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
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