Role of hypothetical protein PA1-LRP in antibacterial activity of endolysin from a new Pantoea phage PA1

Phage PA1 belonging to the <i>Chaseviridae</i> family exhibited a broad host range against <i>P. ananatis</i> strains, with a latent period of 40 minutes and a burst size of 17.17 phages per infected cell. PA1-Lys remained stable at pH 6-10 and temperatures of 20-50°C and showed lytic activity against various Gram-negative bacteria, while PA1-Lys alone could not directly lyse bacteria, its lytic activity was enhanced in the presence of EDTA. Surprisingly, PA1-LRP inhibited bacterial growth when expressed alone. After 24 h of incubation, the OD₆₀₀ value of pET28a-LRP decreased by 0.164 compared to pET28a. Furthermore, the lytic effect of co-expressed PA1-LRP and PA1-Lys was significantly stronger than each separately. After 24 h of incubation, compared to pET28a-LRP, the OD₆₀₀ value of pET28a-Lys-LRP decreased by 0.444, while the OD₄₂₀ value increased by 3.121. Live/dead cell staining, and flow cytometry experiments showed that the fusion expression of PA1-LRP and PA1-Lys resulted in 41.29% cell death, with bacterial morphology changing from rod-shaped to filamentous. Notably, PA1-LRP provided stronger support for endolysin-mediated cell lysis than exogenous transmembrane domains. Additionally, our results demonstrated that the HPP fused with PA1-Lys, led to 40.60% cell death, with bacteria changing from rod-shaped to spherical and exhibiting vacuolation. Taken together, this study provides insights into the lysis mechanisms of <i>Pantoea</i> phages and identifies a novel lysis-related protein, PA1-LRP, which could have potential applications in phage therapy and bacterial disease control.