噬菌体形态特征分析
Description
Using C. indologenes MA9 as the host, a phage was isolated from a mixed sample of soil eluate and hospital wastewater using the double-agar plate method. This phage was named MA9V-3. The results showed that phage MA9V-3 formed uniform plaques on the double-agar plate, with a diameter of approximately 1-3 mm. The centers of the plaques were clear and transparent, surrounded by a distinct halo (Figure 1A).Transmission electron microscopy (Figure 1B) revealed that MA9V-3 has an icosahedral protein "head" with a 20-sided structure, and a contractile tail. The phage tail undergoes conformational changes during the adsorption process, forming a channel through which the viral genome is injected into the host. The overall length of the phage is 248.48 nm, with a head diameter of approximately 84.85 nm and a tail length of 163.63 nm.As shown in （Figure 2A）, the titer of phage MA9V-3 was 1.6 × 10¹⁰ PFU/mL, with the highest titer observed at an optimal MOI of 1. This indicates that the highest number of progeny phages, approximately 10¹⁰ PFU/mL, was produced when the phage and host bacteria were mixed at this ratio.The phage adsorption curve reflects the ability of the phage to bind with the host. As shown in（Figure 2B）, after 20 minutes, only 40% of the free phages remained, while approximately 60% of the phages had adsorbed onto the host. This indicates that phage MA9V-3 has a high adsorption efficiency. The high adsorption rate and rapid adsorption time are beneficial for phage MA9V-3 to efficiently recognize and attach to the host.Given the host specificity of the phage, genetically engineered phages, utilizing reporter genes or fluorescent/luminescent probe-based sensors, can be used for rapid detection of pathogenic bacteria in food (Figure 2C).The one-step growth curve of phage MA9V-3, as shown in（Figure 2D）, reflects its lytic capacity. The latent period of phage MA9V-3 is approximately 40 minutes, during which the phage population grows slowly. The lytic phase lasts for 50 minutes, followed by a stationary phase after 90 minutes, where the number of released progeny phages peaks at 160 PFU/cell.The effect of temperature and pH on phage activity was evaluated by counting plaques on double-agar plates and determining the phage titer under various conditions. As shown in Figure 2E, phage MA9V-3 exhibited optimal activity between 20°C and 50°C, with the highest titer at 40°C. However, the optimal growth temperature for the host bacterium was 28°C, and the phage was completely inactivated at temperatures above 60°C. Regarding pH stability, phage MA9V-3 showed no activity at pH ≤ 3 or pH ≥ 12, with a titer of zero. The phage remained active within the pH range of 4.0 to 11.0, with the highest titer observed at pH 6.0, which is considered the optimal pH for phage MA9V-3.