A dual-action bacteriophage protein Ped1 degrades DNA and blocks replication in Pseudomonas aeruginosa.

To fully harness phage therapy against escalating antimicrobial resistance, it is imperative to elucidate the fundamental mechanisms by which viruses systematically dismantle host physiology. In this study, we identify Ped1, a protein from Pseudomonas aeruginosa phage PaoP5, which suppresses bacterial growth through two mechanisms: degradation of genomic DNA and direct inhibition of the replication machinery. Our 2.3-Å crystal structure and biochemical profiling reveal that Ped1 adopts a previously uncharacterized α/β-nuclease architecture, enabling the efficient cleavage of both double-stranded and single-stranded DNA. Furthermore, we demonstrate that Ped1 physically interacts with the host replicative helicase, DnaB, to suppress its activity during DNA replication. Reciprocally, DnaB binds Ped1 to neutralize its nuclease function, acting as an internal immunity partner. Evolutionary analysis indicates that Ped1 is a lineage-specific toxin strictly conserved among Pseudomonas phages. Collectively, this work identifies a novel, dual-function phage effector that coordinately disrupts genomic integrity and replication fidelity, thus advancing our understanding of the phage-host molecular arms race, and describing a promising scaffold for the development of next-generation antimicrobials against multidrug-resistant P. aeruginosa.