2.1 Bacteriophage isolation
To isolate bacteriophages, 50 mL sea water sample and 1–2 mL of overnight V. alginolyticus ATCC 33787 culture were added to 50 mL of double concentrated LB broth, shaking at 200 rpm overnight at 30°C. The broth mixture was then centrifuged at 10,000 ×g for 10 min and the supernatant was filtered through a 0.22 µm filter. The bacteriophage supernatant was diluted with a 10-fold gradient using SM buffer (100 mM NaCl, 8 mM MgSO4, 50 mM Tris-HCl pH 7.5, 0.01% (w/v) gelatin). 100 microliters of each dilution were mixed with 1 mL of V. alginolyticus solution, and stood at 30 ℃ for 20 minutes. Then 9 mL of warm semi-solid top agar (culture medium with 0.6% agar) were added into the solution and immediately mix to prepare a double-layer plate. The double-layer agar plate was cultured at 30°C for 12 h, and then phage plaques were visualized. The isolation on double-layer agar plates was repeated at least three times to purify the phages.
2.2 Bacteriophage characterization
The bacteriophages were morphologically characterized by electron microscopy. Purified phage (> 1010 PFU/ml) were deposited onto carbon-coated copper grids and allowed to adsorb for 5 min. The grids were allowed to dry, stained with 2% phosphotungstic acid (pH 7.0) for 5 min, and washed with PBS. The morphology was examined under a Hitachi TEM System HC-1 at 80 kV.
The host range of phage was also determined by the double-layer agar plate method as mentioned above. Specifically, 1 mL of each freshly grown potential host cultures (107-108 cfu/ml) was mixed with 100 µL of phage and 3 ml of warm top agar, then poured on bottom agar plates. After incubation, plaque formation on the plates was observed to determine the sensitivity of the host bacteria to phage.
Thermal stability was assessed by exposing aliquots of the phage at various temperatures (40, 50, 60, 70 and 80°C) for 30 minutes or 1 h. After incubation, samples were diluted with SM buffer, and titers were evaluated by the double-layer agar method in triplicates.
For the determination of the stability of the phages at different pH values, phage lysates were added to SM buffer at different pH levels (pH 3–11), which was adjusted by using HCl or NaOH. After incubation of the mixtures at 30°C for 1 h, the titers were evaluated by the double-layer method.
To determine the optimal multiplicity of infection (MOI), different titers of phage were co-cultured with V. alginolytics at MOI ranging from 0.001 to 100 for 10 h at 30 ℃ with shaking. The titer of the phage was then determined by the double-layer agar method. The optimal MOI was the MOI of the phage sample with the highest titer.
2.3 Genome sequencing and bioinformatic analysis
Genomic DNA was extracted from the cell pellets with a Bacteria DNA Kit (OMEGA) according to the manufacturer’s instructions, and high qualified DNA sample (OD260/280 = 1.8 ~ 2.0, >6ug) is utilized to construct fragment library. Library construction and Illumina HiSeq sequencing were performed by Biozeron Biotechnology Co., Ltd. (Shanghai, China.). Paired-end libraries with insert sizes of ~ 400bp were prepared following Illumina’s standard genomic DNA library preparation procedure. The qualified Illumina pair-end library would be used for Illumina NovaSeq 6000 sequencing.
ABySS (Jackman et al. 2017) was used to do genome assembly with multiple-Kmer parameters and got the optimal results of the assembly. The complete genome of phage was sequenced and assembled by Biozeron Biotechnology Company. Gene models were identified using GeneMark (Besemer et al. 2005). Then all gene models were blastp against non-redundant (NR in NCBI) database, SwissProt (http://uniprot.org), KEGG (http://www.genome.jp/kegg/), and COG (http://www.ncbi.nlm.nih.gov/COG) to do functional annotation. In addition, tRNA were identified using the tRNAscan-SE (v1.23, http://lowelab.ucsc.edu/tRNAscan-SE) and rRNA were determined using the RNAmmer (v1.2, http://www.cbs.dtu.dk/services/RNAmmer/).
Phylogenetic and molecular evolutionary analyses were conducted using MEGA version 11 (Tamura et al. 2021). Phylogenetic trees were constructed in MEGA using the neighbor-joining algorithm.
2.4 Antibacterial effect in vitro
V. alginolyticus ATCC 33787 was cultured to OD600 0.1, and then phage (4×107 PFU) was added to the culture to achieve the optimal MOI (0.01). Host bacteria with chloramphenicol (finally 20 µg/mL) were used as positive controls, and host bacteria with PBS served as negative controls. The mixtures were shaken for 12 h at 30°C, and the optical density at 600 nm was monitored every 1 h. Each treatment was performed in triplicate.
2.5 Therapeutic efficacy of VaPW against V. alginolyticus infection in shrimp
Phage therapy was performed using 10 randomly selected shrimp in a PE water tank with seawater for each experimental group. the V. alginolyticus was added into tank to a final concentration of 1.59 × 108 CFU/mL, with a total volume of 3 L. And then bacteriophage was added with MOI of 1, 0.01, and 0.0001, respectively. Three independent experiments were performed for each condition. A group adding PBS of the same volume was used as the negative control group. A control group containing only bacteriophages (~ 1.59 × 108 CFU/mL) was set up to test whether VaPW itself has toxic effects on shrimp health. Any dead shrimp was observed and recorded every 12 hours for a total of 7 days, and a survival curve was drawn.
2.6 Bactericidal activity of recombinant lysozyme and holin of VaPW
The complete holin (ORF 26) gene, lysozame (ORF 27) gene, and holin-lysozyme (ORF 26–27) fragment were amplified using PrimSTAR Max DNA Polymerase (Takara, China). Plasmids and primers used in this study are listed in Table S1 & S2. The vector fragment was amplified using pSCT32 vector (Zhang et al. 2017) as template and then recombinated with the amplified lysozame, holin, and lysozame-holin gene fragments, respectively, using One Step Cloning Kit (Vazyme, China) according to the manufacturer’s instructions. The reaction mixtures were transformed into competent E. coli DH5α cells. The resulting pSCT-lysozyme, pSCT-holin, and pSCT-holin-lysozame plasmids were transferred into E. coli BL21 cells for expression.
The BL21 cells containing the above-mentioned plasmids were cultured in LB medium with 50 µg/mL kanamycin until the OD600 nm reached at about 1.0. The cultures were then supplemented with IPTG with the final concentration of 0.1 mM, and incubated for 8 hours at 30°C. The OD values of cultures were monitored and the expression of recombination proteins, which were tagged with a hexahistidine epitope tag, was analyzed by previous published SDS-PAGE and Western blotting methods (Zhang et al. 2018).
2.7 Statistical analysis
The statistical study of the obtained results was performed with GraphPad Prism 7.0. Results are expressed as means ± the standard deviations (SD). One-way analysis of variance (ANOVA) was used to determine the statistical significance. Significant differences were considered present at *P < 0.05 and **P < 0.01.
2.8 Data availability
The sequence data for the phage VaPW were deposited at GenBank under accession no. 2649232.