The long ongoing fight between bacteria and bacteriophages has led to bacteria evolving and/or acquiring diverse defense mechanisms. Many of these have become irreplaceable tools in molecular biology and biochemistry. CRISPR-Cas systems for genome editing and restriction-modification systems (RM) for manipulating nucleic acids are all derived from prokaryotic antiviral defense systems[1]. However, there is always a need for new tools to be discovered. Fortunately, recent bioinformatics approaches have identified “defense islands” (DIs), genomic regions enriched in defense-related genes, leading to the identification of numerous novel and uncharacterized defense systems[2, 3].
This study aims to characterize the novel prokaryotic antiviral defense system - Olokun, discovered in the E.coli UMB0934 strain. To better understand the underlying mechanism of Olokun, site-specific mutagenesis targeting the predicted active centers has been carried out. Following mutagenesis, Western blotting was used to confirm the expression of the defense system mutants. At the same time, phage plaque assays were employed to determine the effect of these mutations on the system’s ability to protect bacteria from bacteriophage infection in vivo. The purified mutant protein was also subjected to biochemical assays to investigate its nucleolytic activity in vitro.
This work contributes to a deeper understanding of prokaryotic antiphage defense and its potential applications in biotechnology.