Proteinase k

Cephradine capsules (batch number: 151101) were purchased from Suzhou Chung-Hwa Chemical & Pharmaceutical Industrial Co. Ltd., and gentamicin sulfate (batch number: 5150307) was purchased from Yichang Pharmaceutical Industrial Co. Ltd. The two antibiotics were then prepared into a mixture at a concentration of 62.5 g·L^-1[26 (link)]. The DNA extraction reagents (such as Proteinase K, lysozyme, Tris-saturated phenol-chloroform-isoamyl alcohol (25:24:1) and TE buffer) were purchased from Beijing Dingguo Changsheng Biotechnology Co. Ltd., and prepared in the lab (such as 10% SDS, chloroform-isoamyl alcohol (24:1), 5 mol·L^-1 NaCl, 0.1 mol·L^-1 PBS and CTAB/NaCl). D. hansenii, which was provided by the laboratory and shaken at 28°C for 36 hours after being inoculated into liquid Potato Sucrose medium in a 300 mL erlenmeyer flask. The cells were then gathered by centrifugation at 2000×g for 4 minutes after washed 1∼2 times repeatedly with sterile stroke-physiological saline solution. The above cells were diluted to 10¹⁰ mL^-1 with sterile storke-physiological saline solution eventually after being counted by hemocytometer, and stored at 4°C for subsequent experiments[27 (link)].

Overnight-cultured A. hydrophila was adjusted to a concentration of 5 × 10⁸ CFU/mL and transferred into 20 mL of LB media at a dilution of 1:100. Bacteriophage G65 was applied to the bacterial culture to yield a final concentration of 5 × 10⁴ PFU/mL, and the cocultures were incubated at 28°C for 24 h with 180 rpm shaking. Phage cultured alone served as a control. Phage enumeration was determined at a regular interval of 2 h between this 24-h incubation period by counting the plaques using a double-layer plaque assay.
To determine whether bacterial surface proteins or polysaccharides were involved in phage-bacterial host recognition, A. hydrophila strains were cultured under the same conditions as shown above except that either 0.1 mg/mL proteinase K (Dingguo, Nanjing, China) or 2.14 mg/mL sodium meta-periodate (NaIO₄; Dingguo) was added to the medium prior to coculture with phage G65. The samples were treated with proteinase K or NaIO₄ at 37°C for 2 h. The assay was performed in three independent biological experiments.

Tris-saturated phenol–chloroform-isoamyl alcohol (25:24:1), lysozyme, proteinase K, chloroform, isoamyl alcohol, acetone, and TE buffer were purchased from Beijing Dingguo Biotechnology Co. Ltd. 10% sodium dodecyl sulfate (SDS), 0.1 mol/L phosphaTE buffer solution (PBS) buffer, 5 mol/L NaCl, chloroform-isoamyl alcohol (24:1), cetyl trimethyl ammonium bromide (CTAB)/NaCl, 3 mol/L sodium acetate and 70% anhydrous ethanol, were prepared in the laboratory.