Bacterial strains and culture media
Bacillus cereus strain LJH431 used in this study was obtained from the Canadian Research Institute for Food Safety culture collection (CRIFS, University of Guelph, Guelph, ON, Canada). Another fifteen strains of B. cereus were provided generously by T. Abee (Laboratory of Food Microbiology, Wageningen University, Netherlands). Strains A2 and D2 of B. cereus were kindly provided by A. Hudson (Christchurch Science Centre, Institute of Environmental Science and Research, New Zealand). Strains of B. weihenstephanensis were kindly provided by S. Scherer (Department of Biosciences, Wissenschaftszentrum Weihenstephan, Germany). Finally B. anthracis ΔSterne was obtained from K. Amoako (Canadian Food Inspection Agency, Ottawa, ON).
B. cereus strain LJH431 was grown in half-strength media of Tryptic Soy Broth (TSB; Difco Laboratories, Detroit, MI), half-strength Tryptic Soy Agar (TSA: TSB plus 1.5% agar) and half-strength Tryptic Soy Soft Agar (TSB + 0.5% agar) at 30°C for 24 h.
Transmission electron microscopy
The morphology of the phage Bc431v3 was examined by transmission electron microscopy as described
. Briefly, 1 ml of each phage (~ 9 log10 PFU ml-1) was centrifuged at 16,000 × g for 1 h at 4°C (Beckman J-20 centrifuge, Beckman Coulter Inc., Mississauga, ON), pellets were washed once using CM buffer (2.5 g/L MgSO4.7H2O; 0.05 g/L gelatin; 6 ml/L 1 M Tris buffer; 0.735 g/L CaCl2; pH 7.5) and then resuspended in 300 μl of CM buffer. Five microliters of the phage suspension were applied onto 300-mesh copper grids coated with formvar and allowed to stand for 1 min. The excess liquid was removed by filter paper and grids were stained with 2% uranyl acetate for 30 s then carefully blotted with filter paper to remove excess stain solution. Negatively stained phages were examined with a LEO 912AB transmission electron microscope (Energy filtered TEM [EFTEM], LEO 912AB model at 100kv, Zeiss, Germany).
Phage isolation and purification and, extraction of DNA
Phage vB_BceM_Bc431v3 was isolated from sludge samples collected from a local wastewater management plant, and plaque purified. Lysates were prepared using the solid propagation method as described elsewhere
, clarified from bacterial cell debris by centrifugation (5,400× g, 15 min) and filtered through 0.45 μm mixed cellulose ester (MCE) Fisher brand syringe filters (Fisher Scientific Company, Ottawa, ON, Canada). DNase I and RNase A (Sigma-Aldrich, Oakville, ON, Canada) were added to 10 μg/ml and held at 37°C for 30 min. Subsequent DNA purification steps were carried on according to the handbook of QIAGEN® Lambda midi Kit (Qiagen Inc., Mississauga, ON, Canada). DNA concentration was determined by absorbance at 260 nm using a Nanodrop™ 1000 spectrophotometer (Thermo Scientific, Ottawa, ON, Canada).
Determination of host specificity using Bioscreen C technology
The host range of the phage Bc431v3 on the selected strains of Bacillus spp. was determined using the spot test technique and also by measuring the optical density (OD) in liquid medium of the tested bacterium in the presence of phage using the Bioscreen C Microbiology Plate Reader (Labsystems, Helsinki, Finland) as described elsewhere
. The following settings were used: wide band (wb) wavelength; 25°C incubation temperature; 10 min preheating time; kinetic measurement; measurement time 24 h at time intervals of 30 min with medium intensity shaking for 10 s before and after each measurement. Fifty microliters of each phage lysate were transferred to each of the 100 wells of the sterilized honeycomb plates of the Bioscreen C reader (Fischer Scientific), and then each of the wells was inoculated with 125 μl of an overnight bacterial culture at final concentrations of 3 log10 CFU ml-1. The multiplicity of infection (M.O.I) used to determine the host range of the phage was around 103. In this experimental design, three types of control samples were used: phage only in broth, bacteria only and sterile medium. The absorbance data were analyzed using the Bioscreen C data processing software version 5.26 (Labsystems) to determine the detection time (time required for each test well to increase by 0.3 OD units). Detection times (h:min) were converted to decimal values, averaged and the mean control detection time was subtracted from all test data for each isolate tested and expressed as detection time difference (DT diff.). Based on the host specificity, phages were divided into three groups: (++), when phages completely inhibited the growth of the host bacterium; (+), indicating that phages delayed the growth of the host bacterium; and (−), indicating that the phage had no effect on bacterial growth.
One-step growth curve
Burst sizes and latent periods of the selected phages were determined by a one-step growth experiment as described by Anany
. Phages were added to its host bacterium at an MOI of around 0.1 and incubated in a water bath at 30°C for 5 min. One ml was removed and added to 100 μl of chloroform and mixed well. One hundred microliters of this mixture were added to 100 μl of an overnight culture of the host bacterium and mixed with 4 ml of overlay media and poured onto TSA agar plates to determine the degree of adsorption of the phage to bacterial cells. After an additional 30 s at 30°C, 100 μl were transferred to a tube containing 9.9 ml of fresh TSB and then diluted 10 times in fresh TSB (0.1 log10 ml-1). One ml of the 10-1 dilution tube was additionally diluted 10 times in fresh TSB (0.01 log10 ml-1) and all three tubes (the original plus 10-1 and 10-2 dilutions) were incubated in a water bath at 30°C. After 6 min, samples were collected every 5 min for 3 h and phages were titrated in each respective sample as previously described. The relative burst size was determined according to the equation:
Relative burst size = [(Final titre – Initial titre)/ Initial titre]
The relative burst size at different times was plotted against time to determine the latent period.
Determination of phage genome size using PFGE
Phage genome size was determined by pulsed-field gel electrophoresis (PFGE) as described elsewhere
. Phage particles were embedded in 1% Seakem Gold agarose (Mandel Scientific, Guelph, ON) and subjected to electrophoresis in 0.5X TBE buffer (5X: 20 ml of 0.5 M EDTA [pH 8.0], 53 g/l Tris base, and 27.5 g/l boric acid) at 14°C for 18 h, using a CHEF DR-III Mapper electrophoresis system (Bio-Rad, Mississauga, ON) with pulse times of 2.2-54.2 s, at 6 V/cm. Low range DNA marker and phage lambda DNA concatemers (New England Biolabs) were used as size standards. The gels stained with ethidium bromide and DNA bands were visualized under UV transillumination. PFGE results were analyzed using BioNumerics software (Applied Maths Inc., Austin, TX).
Genome sequencing and annotation
The sequencing of phage Bc431v3 DNA was carried out at the McGill University Genome Quebec Innovation Centre (Montreal, QC, Canada) using pyrosequencing (454 technology). AutoFACT automated annotation software
 was initially used for genome annotation and then all open reading frames (ORFs) were confirmed using Kodon version 2.0 (Applied Maths). The individual proteins were analyzed using BLASTP against the protein databases at NCBI (http://www.ncbi.nlm.nih.gov). Protein motifs structures were identified using Pfam 24 (http://pfam.sanger.ac.uk/)
. In the case of the phage sigma factors a motif analysis was carried out using MEME v.4.5.0
. Phage-encoded tRNA genes were identified with Aragorn
 and tRNAscan-SE v.1.21
 using the default parameters. DNAMAN (Lynnon Corp., Vaudreuil-Dorion, QC, Canada) was used to determine the codon usage information of both Bc431v3 and its bacterial host Bacillus cereus, which also provided information on the GC content and presence of direct repeats in Bc431v3 genome. Putative promoters were identified by visual inspection for sequence similarity to TTGACA-N15-18-TATAAT, and by MEME analysis on the 5′ sequences extracted using extractUpStreamDNA (http://lfz.corefacility.ca/extractUpStreamDNA/). Rho-independent terminators were determined by ARNold and TransTerm software
[60, 61] and verified by examining the secondary structure of the DNA adjacent to polyT sequences using Mfold
. The physicochemical parameters of the gene products were determined using Molecule Weight and Isoelectric Point Finder (http://greengene.uml.edu/programs/FindMW.html). Transmembrane domains were predicted using TMHMM v2.0 and Phobius or Split 4.0
[63–65]. Sequences of bacterial sigma factors and sigma-factor binding sites were identified through DBTBS database (http://dbtbs.hgc.jp)
Genomic comparisons at the proteomic level were made using CoreGenes
[47, 48]. For alignments of multiple genomes and defining sequence homology percentage with related phages, progressive Mauve was used
. For genomic map visualization and annotation pipelines, the CGview software was used (http://wishart.biology.ualberta.ca/cgview/)
GenBank accession number
The sequence of genome of this phage has been deposited with GenBank under accession number JX094431.
Phage Bc431v3 purified through CsCl gradients, was reduced with 10 mM dithiothreitol (56°C, 1 hr) and alkylated by 55 mM iodoacetamide (room temperature, dark, 1 hr), and then dialyzed against 10 mM NH4HCO3, and dried by SpeedVac concentrator (Savant, Fisher scientific, Nepean, Ontario). Enzymatic digestions were performed on ~20 μg of the purified protein using either sequencing grade trypsin or chymotrypsin (100 ng, Roche Diagnostics GmbH, Indianapolis, IN) for 4 hours. The digests were subsequently diluted by 0.2% formic acid and analyzed by online nanoAcquity ultra-performance liquid chromatography (UPLC, Waters, Milford, MA ) coupled with linear ion-trap Fourier transform ion cyclotron resonance (LTQ-FT ICR, Thermo Fisher, San Jose, CA) mass spectrometry. Peptides were trapped by a RP Symmetry C18 column (180 μm i.d. × 20 mm length, 5 μm) at 5 μl/min, and subsequently separated on a C18 analytical column (100 μm i.d. × 100 mm, 1.7 μm, BEH 130) at 400 nl/min. Peptide elution was achieved using mobile phases consisting of solvent A (0.1% FA) and solvent B (acetonitrile/0.1% FA) at a linear gradient from 5% to 30%, and then 85% of solvent B (65 min run). FT-MS scans were acquired with high resolution (100,000) at the mass range of m/z 300 to 2000, and low resolution MS/MS measurements in linear ion-trap mode were obtained by data-dependent scans of the top eight most intense precursor ions at multiply charged states of 2+, 3+, and 4+. Dynamic exclusion was enabled for a period of 180 S.
Protein identification was performed using an in-house Mascot Server (version 2.3.0, Matrix Science, London, UK), and the raw data were searched against the Bc431v3 protein database. The parameter settings allowed specific trypsin digestion for maximum 2 missed cleavage sites, and non-specific digestion of chymotrypsin. Cystein carbamidomethylation was designated as a fixed modification of peptides, and deamidation of asparagine and glutamine, methionine oxidation, pyro-Glu of Gln conversion at the N-terminus were considered as variable modifications. Mass tolerances were set up to 10 ppm for the FT MS ions and 1 Da for ion trap MS/MS fragment ions. Peptide assignments were filtered by an ion score cut off of 20, and the significance threshold was adjusted to 0.001 to achieve a false discovery rate (FDR) of less than 3%