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

## TL;DR

This study identifies ORF70 from the cyanophage MaMV-DC as a holin-like protein that causes bacterial cell death by disrupting membranes.

## Contribution

The study provides the first characterization of ORF70 as a holin-like protein from a cyanophage, revealing its membrane-disrupting activity and potential for phage-based therapies.

## Key findings

- ORF70 exhibits membrane-associated localization and features characteristic of class III holins.
- Overexpression of ORF70 in E. coli leads to cell death, membrane disruption, and β-galactosidase leakage.
- ORF70's activity is sensitive to 2,4-dinitrophenol, indicating membrane depolarization and holin-like behavior.

## Abstract

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 E. coli 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 E. coli 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.