Phage-encoded NARP3 system rebuilds NAD ⁺ to subvert bacterial immunity

Phages deploy diverse countermeasures to evade bacterial nicotinamide adenine dinucleotide (NAD ⁺ )–directed immunity in a perpetual arms race. Known pathways include NAD ⁺ reconstitution pathway 1 (NARP1), which recycles adenosine 5′-diphosphate–ribose, and NARP2, which converts nicotinamide into NAD ⁺ via nicotinamide mononucleotide ligation. Whether additional NAD ⁺ -restoring strategies exist has remained unclear. Here, we identify NARP3, a conserved two-gene NAD ⁺ -restoring phage pathway widespread in Enterobacteriaceae-infecting phages. NARP3 encodes a pyridine nucleoside uptake system C (PnuC)-like nicotinamide riboside transporter (Bas30_87) and a bifunctional NAD ⁺ biosynthesis regulator (NadR)-like enzyme (Bas30_86). Bas30_87 imports nicotinamide riboside, and Bas30_86 converts it to NAD ⁺ through sequential phosphorylation and adenylation. NARP3 fully restores NAD ⁺ pools depleted by Sir2-HerA defenses, enabling robust phage replication. We solve x-ray crystal structures of Bas30_86 alone and bound to NAD ⁺ , revealing coordinated substrate capture, intermediate handling, and product formation. Mutational analyses confirm that both transport and enzymatic activities are essential. NARP3 functions as a metabolite-centered countermeasure, expands the phage arsenal, and underscores NAD ⁺ metabolism as a central battlefield in host-phage conflicts. Its discovery provides a blueprint for engineering phages to bypass NAD ⁺ -dependent bacterial immunity and offers a mechanistic framework to harness metabolite-guided viral strategies for biotechnological applications.