Sampling, Isolation and Purification of Escherichia Phage IME392
Phage IME392 and its host cell, Escherichia coli E2, were isolated from sewage samples taken from a chicken farm in Qingdao, China. For the purification of phage particles, the sewage samples were firstly centrifuged at 12,000×g for 5 min and then filtered through a 0.22-μm membrane. After that, an equal amount of the filtrate was added to the 3×LB medium containing the log-phase host bacteria E2 (OD600 = 0.4), and cultured overnight at 37 °C,180 rpm. The culture was centrifuged at 12,000×g for 5 minutes and precipitation was discarded while the supernatant was filtrated by passing through a 0.22-μm filter to remove host cells. The filtrate was serially diluted with sterile PBS in a 10-fold dilution method, and then 100 μL of the dilution was mixed with 200 μL of the log-phase host bacteria culture, followed by incubation at room temperature for 5 minutes. The mixture was added to 5 mL of preheated 0.75% LB soft agar, and poured on the surface of 1.5% hard agar plates. After solidification, the plates were incubated at 37 °C overnight. Single plaques were isolated from the plates and again incubated over night with a liquid culture of E2 with shaking at 37 °C. Cultures were re-centrifuged, sterile filtrated and the filtrates were subjected to another round of plaque assays. This process is repeated three times to obtain pure phage stocks.
Multilocus sequence typing
According to the previous reports , primer pairs for eight housekeeping genes including dinB, icdA, pabB, polB, putP, trpA, trpB and uidA were designed for PCR amplification. All PCR products were purified by gel extraction and then sequenced by Beijing Ruiboxingke Biotechnology Co., Ltd. using universal sequencing primer OF and/or OR. Further details on this MLST scheme can be found at http://www.pasteur.fr/mlst.
DNA Extraction, Gene Sequencing and Bioinformatic Analysis
According to a modified standard phenol-chloroform extraction protocol , DNase I and RNase A (Thermo Scientific, USA) with a final concentration of 1 μg/mL were added to the purified phage IME392 stock solution and incubated overnight at 37 °C. After incubating at 80 °C for 15 minutes to inactivate DNase I and cooling down to room temperature, the lysis buffer with a final concentration of 0.5% SDS, 50 μg/ml protease K and 20 mM EDTA was added. The solution was incubated for 1 hour at 56 °C, and added an equal volume of Tris-saturated phenol. The mixture was vortexed to form a uniform emulsion. After 10,000×g centrifugation at 4 °C for 5 minutes, the upper aqueous phase was collected and transferred to a new tube, while also adding an equal volume of extraction agent (phenol: chloroform: isoamyl alcohol= 25:24:1). The mixture was centrifuged again (10,000×g, 4 °C, 5 min), and the aqueous phase was collected and added to an equal volume of isopropanol. The mixture was incubated at -20 °C for more than 1 hour, followed by 10,000×g centrifugation at 4 °C for 20 minutes, precipitating phage DNA. The precipitation was collected and washed twice with 1 mL of 75% cold ethanol, then resuspended in 30 μL of deionized water and stored at -20 °C.
According to the manufacturer's instructions, a 2×300 nt paired-end DNA library was prepared using NEBNext® UltraTM II DNA Library Prep Kit for Illumina®. The Bioruptor UCD-200TS ultrasound system is used to fragment a total volume of 50 μl DNA (about 100 ng) into 300-600 bp fragments. The resulting fragmented DNA was end repaired and ligated to NEBNext adaptor. Cleanup of Adaptor-ligated DNA was used AMPure XP Beads. Finally, the cleaned DNA was amplified by PCR for 4 to 5 cycles, and the PCR product was purified again using AMPure XP Beads. Agilent 2100 Bioanalyzer system was used to measure the size distribution of the constructed library fragments, and the library was quantified using the KAPA Library Quantification Kits. Whole genome sequencing was performed on Illumina Miseq sequencing platform (San Diego, CA, United States) with a 600-cycle MiSeq v3 Reagent kit to generate 2×300 bp paired-end reads. Overall, 555,564 raw reads were generated.
The raw sequencing data quality was analyzed using the quality control software FastQC v0.11.5 and filtered for low quality reads and adapter regions using Trimmomatic 0.36 with default parameters . The generated high-quality reads were assembled by SPAdes v3.13.0 with default parameters and approximately 4,872 contigs were generated . For the assembled contigs, Bandage v0.8.1 , which is a tool for visualizing assembly graphs with connections, was used to display the connections between those contigs. Among them, only 3 contigs are circularity with lengths of 116,460, 39,440 and 4,888 bp respectively and coverage of 352×, 7× and 18× respectively. By BLASTn analysis, it is confirmed that the two shorter contigs are lysogenic phage and plasmid respectively. The mapping was carried out with CLC Genomics Workbench 12.0.2 (Length fraction = 0.95; Similarity fraction = 0.95) which was also used to adjust the sequences and for result checking. A consensus genome was generated that spanned 100% of the reference genome and the 425,959 mapped reads had an 884.1 mean read coverage. Nucleic acid sequence similarity search was performed by BLASTn (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Gene annotation was first run on RAST  (http://rast.nmpdr.org/), then refined by amino acid sequence comparisons on BLASTp. The genome function map was generated by the laboratory's self-built script and optimized using Inkscape 0.92.1.
The amino acid sequence of the major capsid protein and the terminase large subunit of the bacteriophage IME392 was used to construct a neighbor-joining phylogenetic tree via MEGA 7.0 with 1000 bootstrap replicates and optimized by online website tool EvolView (https://www.evolgenius.info/evolview/).
Transmission Electron Microscopy
After centrifuging the co-culture of the phage and its host at 12,000×g and filtering with a 0.22-μm filter, the phage particles were purified by sucrose density gradient centrifugation . Approximately 20 μL purified, enriched phage sample were deposited on carbon-coated copper grids for a 15 minutes absorption and then dried using filter paper. The phage particles were negatively stained by 2% (w/v) phosphotungstic acid (pH 7.0) for 2 min, and examined using a JEM-1200EX transmission electron microscope (Jeol Ltd., Tokyo, Japan) at the acceleration voltage of 80 kV.
Host Range Determination
The host range of phage IME392 was determined by spot assay and was confirmed with the plaque assays. Suspected hosts were cultured at 37 °C to reach the optical density of 1.0. 300 mL of bacterial cultures was added to 5 mL of preheated 0.7% LB agar, and poured on 1.5% agar plates respectively. After solidification, each plate was examined by pipetting 5 μL phage suspension onto the bacterial lawn. Eligible hosts are detected by plaque formation after incubating overnight at 37 °C. The plaque assay procedure is as described in the phage purification section.
Determination of Optimal Multiplicity of Infection
Multiplicity of infection (MOI), or the ratio of phage particles to bacteria cells prior to culture, affects the final production of bacteriophages. At optimal MOI, the cultured product contains the largest amount of phage particles after reaching stationary phase. In order to determine the optimal MOI, firstly, the colony forming unit of the log-phase (OD600 = 0.6) host bacteria E2 culture and the plaque forming unit of the bacteriophage IME392 stock solution were calculated separately. The bacteria-phage mixture was added to 5mL LB medium according to different ratios to achieve a MOI of 10, 1, 0.1, 0.01, 0.001, or 0.0001, which were subsequently incubated at 37 °C, 220 rpm for 4 hours. After centrifuging at 12,000×g for 1min and filtering the culture through a 0.22-μm filter, the phage titer was calculated using the double-layer plate method after serial dilution. Three replicates were conducted for determination and the MOI produced the highest phage titer was considered the best MOI of the phage.
One-step Growth Curve
The one-step growth curve was examined by the following method. A mixture of phage and bacteria at optimal MOI (0.1) was allowed to incubate in 37 °C for 10 minutes for absorption. After centrifugation at 12,000×g for 1 min, the supernatant which contained the unabsorbed phage particles were discarded, while the precipitation was then washed twice with LB medium and re-suspended in 20 mL of LB medium. The moment when the precipitation was re-suspended in medium was defined as time zero. The obtained culture was subsequently transferred to a shaker and cultured at 37 °C, 220 rpm for 140 min. The 200 μL samples were collected every 10 minutes (every 5 minutes in the first 30 minutes) and then centrifuged and plated on double agar plates to determine the phage titer. Each sample were plated on 3 separate plates respectively. Finally, the one-step growth curve was plotted by Graphpad Prism 8.0.
Determination of pH and Temperature Tolerance
To determine the tolerance of phage particles to different pH, LB medium were adjusted to a variety of pH (2.0, 3.0, 4.0 … 13.0) with 5 M HCl or NaOH solution and then filtered through a 0.22-μm filter. 100 μl purified phage suspension were added into the 900 μl LB medium of different pHs and incubated at 37 °C for 1 hour. To investigate the thermal stability of the phage, 100 μl purified phage suspension were added to 900 μl LB medium and incubated at a variety of temperatures (30 °C, 37 °C, 40 °C, 50 °C, 60 °C, 70 °C, 80 °C) for 1 hour. The titer of the remaining viable phage particles was determined using the double-layer agar method. All assays were performed in triplicate.
The phage particles were concentrated by PEG and then purified by sucrose density gradient centrifugation . Purified phages were mixed with 6× protein loading buffer (TransGen Biotech Co., LTD) and then boiled for 10 minutes, followed by concentrating at 12,000×g, 4 °C for 3 min. The proteins were separated by 12% SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis) and the bands were visualized by staining the gels with Coomassie brilliant blue. Gel slices were then excised and trypsinized. Briefly, 2.5μg Trypsin enzyme was added to 100 μg protein solution according the ratio of protein:enzyme = 40:1, and enzymatically digested at 37 °C for 4 hours. Add Trypsin one more time according to the above ratio, and continue enzymatic digested at 37 °C for 8 hours. The enzymatically digested peptides are desalted by Strata X column and vacuumed to dry. The dried peptide sample was analyzed by using Liquid chromatography–mass spectrometry (LC-MS). The full spectrum identification of protein is mainly based on experimental tandem mass spectrometry data, matching with theoretical mass spectrometry data obtained by database simulation, so as to obtain protein identification results. First the original mass spectrum data was converted into a mass spectrum peak file, and then searched and matched the sequence in the database using Mascot v2.3.02 (parameter: Enzyme Trypsin, Fragment Mass Tolerance 0.05 Da, Fixed modifications Carbamidomethyl (C), Variable modifications Oxidation (M); Gln->pyro-Glu (N-term Q); Deamidated (NQ), Max Missed Cleavages 1, Instrument type ESI-FTICR, Database Bacteriophage_392_nr.fasta) and performed some filtering and quality control (Mascot evalue <= 0.05) on the search results to give reliable protein identification results.