Identification and Characterization of Holin-like Protein ORF70 from Cyanophage MaMV-DC

In this study, we characterized the holin-like protein ORF70 from the cyanophage MaMV-DC, offering valuable insights into its role in phage-mediated host cell lysis. ORF70 shares key features with class III holins, such as a hydrophobic transmembrane domain and membrane-associated localization, which are crucial for its bacteriolytic activity. Subcellular localization studies suggested its association with the membrane, supporting its classification as a holin-like protein. Overexpression of ORF70 in <i>E. coli</i> resulted in significant growth inhibition, increased β-galactosidase leakage, and visual confirmation of cell death through live/dead staining. Additionally, ORF70's sensitivity to the energy toxin 2,4-dinitrophenol (DNP) further indicated its holin-like activity by promoting membrane depolarization. Transmission electron microscopy and Gram staining revealed characteristic morphological changes in <i>E. coli</i> cells, including membrane disruption, consistent with damage caused by holins. These results suggest that ORF70 acts as a holin-like protein that disrupts the host membrane, leading to bacterial cell death. Our study provides evidence supporting the holin-like activity of ORF70 from cyanophage MaMV-DC. This research significantly enhances our understanding of phage-host interactions and opens new avenues for developing phage-based therapies, offering promising alternatives to traditional antibiotics amidst the growing challenge of antibiotic resistance.