Biological characteristics and genome sequences of two bacteriophage strains infecting Stenotrophomonas indicatrix

1.1 Source of Host Bacteria and Bacteriophages Indicator oligotrophic bacterium (S. indicatrix) EB12 was tested by the College of Life Science and Technology at Heilongjiang Bayi Agricultural Reclamation University in the field of agricultural and environmental microbiology The laboratory separates and preserves blue-green algae during the enrichment process. Bacteriophages Ste-X and Ste-D were isolated from the artificial lake of Heilongjiang Bayi Agricultural Reclamation University (N46 ° 35 ′, E Water samples collected at 125 ° 10 ′. 1.2 Test medium and buffer solution NA liquid medium, SM buffer, and 1 mol/L KCl solution. 1.3 Separation and Purification of Bacteriophages Separate and purify bacteriophages according to the method of Zhao Xinzhuo et al. [16]. Firstly, centrifuge the collected water sample and filter it through a 0.22 µ m membrane to remove impurities Except for some biological and non biological factors, this filtrate is used as a virus suspension. Take 0.3 mL of virus suspension and 0.2 mL in logarithmic growth phase (OD600 value) The S. indicatrix EB12 bacterial solution, with an OD600 value of 0.8 for all experiments, was left to stand in a test tube for 1 hour using a double-layer design Separate bacteriophages using the agar plate method, select individual plaques of different sizes and place them in EP tubes containing NA liquid culture medium. Let them stand in a refrigerator at 4 ℃ Extract the virus solution for 24 hours and purify the bacteriophage using a double-layer plate method. Repeat the purification process at least 5 times until uniform plaque size is observed Purified bacteriophages. 1.4 Concentration and Electron Microscopy Observation of Bacteriophages According to the method of Zhao Xinzhuo et al. [16], a virus concentrate was obtained, and Chengdu Century Meiyang Technology Co., Ltd. used transmission electron microscopy to analyze the bacteriophages Observe the morphology of the body. 1.5 Optimal infection multiplicity of bacteriophages Mix the EB12 bacterial liquid and bacteriophage according to the multiplicity of infection (MOI) values of 10000, 1000, 100, 10, respectively 1. Mix 0.1 and 0.01, refer to the method of Zhao Xinzhuo et al. [16] for specific operation. The potency was determined using a double-layer plate method with sterile water as the blank control, All experiments were repeated three times to determine the optimal number of infections. 1.6 Phage one-step growth curve, acid-base, temperature, and UV stability According to the method proposed by Zhao Xinzhuo et al. [16], the one-step growth curve of bacteriophages was determined. Starting from 0 min, take out 100 μ L of the mixture every 10 min, Use the double-layer plate method to determine its potency and draw a one-step growth curve, as shown in formula (1). Lysis=Phage titer at the end of lysis/Host bacterial concentration at the beginning of infection (1) Take 10 μ L of phage raw material and add it to 0.99 mL of NA liquid culture medium with different pH values ranging from 3.0 to 13.0. Use the double-layer plate method to determine its concentration Valence; Take 2 mL of phage raw material and incubate it in a constant temperature water bath at 20-80 ℃ for 1 hour, then measure its potency; Take 10 mL of bacteriophage The original solution was placed in three sterile 90 mm petri dishes and irradiated at a distance of 30 cm under a UV lamp (30 W). Samples were taken every 10 minutes for measurement Its potency. All the above experiments were repeated three times.