Classification of Sir2-HerA systems reveals a multilayered regulatory cascade gating the type III antiphage activity.

Sir2-HerA systems are abortive infection defenses that integrate multiple enzymatic activities to induce growth arrest, thereby limiting phage propagation. However, the molecular logic that couples phage sensing to a precisely gated antiviral response, thereby ensuring infection-specific activation, remains unclear. Through genomic mining of Escherichia coli, we show that E. coli Sir2-HerA immunity diversifies into functional subtypes and identify three types (I-III) with distinct protection profiles. Focusing on the most potent type III system, we identified its phage activators, Gp2.5 and Gp5.9, through an unbiased T7 proteome screen. Mechanistically, type III Sir2-HerA follows a multilayered gating logic. Phage proteins trigger both the HerA nickase and Sir2 NADase; however, the activated NADase remains dormant due to an ATP-mediated checkpoint. The HerA nickase introduces DNA nicks, leading to the accumulation of end-exposed DNA intermediates that engage the complex-associated ATPase to drive ATP consumption. This process relieves the checkpoint, thereby unleashing the full trigger-dependent NADase activity and enabling robust NAD+ depletion. Together, our findings reveal a sophisticated molecular logic that integrates diverse enzymatic activities into a tiered gating architecture, ensuring high-fidelity phage defense while preventing inadvertent activation.