The ongoing evolutionary battle between bacteria and bacteriophages has led to the emergence of diverse prokaryotic antiviral defense systems. Historically, research focused on well-known defense mechanisms like restriction-modification (RM), abortive infection (Abi) systems and the widely-recognized CRISPR-Cas system. However, recent advancements in exploring the vast bacterial pangenome, the collective genetic pool of a species, are revealing a hidden arsenal of previously unknown defense systems clustered in "defense islands" [1, 2].
In this study, we explore the molecular mechanisms and functional optimization of four novel defense systems: PD-T4-1, PD-T7-1, Azaca and Mokosh I. Our research starts by testing whether the defence systems are able to protect against bacteriophages. We have tested the defence systems against bacteriophages from the BASEL collection and have shown that they are active and able to defend against some of the bacteriophages in the collection. We were also able to isolate several bacteriophage mutants (escapers). In future studies, the genomic DNA information isolated from these bacteriophage mutants will provide a more detailed understanding of the defence systems we are studying. Subsequently, we purified wild-type and mutant proteins from the Azaca, PD-T4-1, and PD-T7-1 systems and conducted nuclease activity assays using various DNA substrates. These experiments revealed the specificities and efficiencies of the nucleases, highlighting key functional domains and residues. Finally, we performed optimization studies to determine the optimal conditions for the nuclease activity of the wild-type proteins, enhancing our understanding of their biochemical properties and potential applications.
This work not only advances our knowledge of prokaryotic antiviral defense mechanisms but also lays the groundwork for the development of novel biotechnological tools and therapeutic strategies.