Bacteriophage deploys a RecA-dependent nuclease to inhibit Staphylococcus aureus replication and promote phage propagation.

Bacteriophages have evolved diverse strategies to manipulate host processes, yet the molecular mechanisms employed by phage-encoded effector proteins remain poorly understood. Here, we identify Gp16, an early-expressed protein from Staphylococcus aureus phage ΦNM1, as a RecA-dependent nuclease that plays a dual role in host inhibition and phage propagation. Gp16 is rapidly expressed upon infection, and its overexpression alone is sufficient to inhibit bacterial growth where deletion of gp16 severely impairs phage DNA replication, progeny production, and host cell lysis, underscoring its essential role in the phage life cycle. Structural modeling predicts Gp16 is a nuclease, and its overexpression induces DNA condensation in vivo. Biochemical and cellular analyses show that Gp16 interacts with the host RecA protein to inhibit growth, and functions as a nickase in vitro, requiring the catalytic cysteine C181 for DNA cleavage. RecA further enhances its cleavage activity. During phage infection, RecA activation is required for efficient phage propagation, while Gp16 concurrently suppresses host DNA replication and promotes DNA condensation, thereby facilitating phage replication. Together, these findings reveal a previously unrecognized strategy in which a phage-encoded nuclease exploits the host RecA machinery to couple host suppression with productive phage propagation.