Zorya is a recently identified and widely distributed bacterial immune system that protects bacteria from viral (phage) infections. Three Zorya subtypes have been discovered, each containing predicted membrane-embedded ZorAB complexes paired with soluble subunits that differ among Zorya subtypes, notably ZorC and ZorD in type I Zorya systems^1,2. Here, we investigate the molecular basis of Zorya defense using cryo-electron microscopy, mutagenesis, fluorescence microscopy, proteomics, and functional studies. We present cryo-EM structures of ZorAB and show that it shares stoichiometry and features of other 5:2 inner membrane ion-driven rotary motors. The ZorA₅B₂ complex contains a dimeric ZorB peptidoglycan binding domain and a pentameric α-helical coiled-coil tail made of ZorA that projects approximately 70 nm into the cytoplasm. We also characterize the structure and function of the soluble Zorya components, ZorC and ZorD, finding that they harbour DNA binding and nuclease activity, respectively. Comprehensive functional and mutational analyses demonstrate that all Zorya components work in concert to protect bacterial cells against invading phages. We provide evidence that ZorAB operates as a proton-driven motor that becomes activated upon sensing of phage invasion. Subsequently, ZorAB transfers the phage invasion signal through the ZorA cytoplasmic tail to recruit and activate the soluble ZorC and ZorD effectors, which facilitate degradation of the phage DNA. In summary, our study elucidates the foundational mechanisms of Zorya function as an anti-phage defense system.

Zorya 是一种最近发现且分布广泛的细菌免疫系统，可保护细菌免受病毒（噬菌体）感染。已发现三种 Zorya 亚型，每种亚型都包含预测的膜包埋 ZorAB 复合物，这些复合体与不同 Zorya 亚型不同的可溶性亚基配对，特别是 I 型 Zorya 系统中的 ZorC 和 ZorD^1,2。在这里，我们使用冷冻电子显微镜、诱变、荧光显微镜、蛋白质组学和功能研究来研究 Zorya 防御的分子基础。我们介绍了 ZorAB 的冷冻电镜结构，并表明它与其他 5：2 内膜离子驱动旋转电机具有相同的化学计量和特征。ZorA₅B₂ 复合物包含一个二聚体 ZorB 肽聚糖结合结构域和一个由 ZorA 制成的五聚体 α 螺旋卷曲螺旋尾部，该尾部投射约 70 nm 到细胞质中。我们还表征了可溶性 Zorya 成分 ZorC 和 ZorD 的结构和功能，发现它们分别具有 DNA 结合和核酸酶活性。全面的功能和突变分析表明，所有 Zorya 成分协同工作，以保护细菌细胞免受噬菌体入侵。我们提供的证据表明 ZorAB 作为质子驱动的马达运行，在感应到噬菌体侵袭时被激活。随后，ZorAB 通过 ZorA 细胞质尾部转移噬菌体侵袭信号，以募集并激活可溶性 ZorC 和 ZorD 效应子，从而促进噬菌体 DNA 的降解。总之，我们的研究阐明了 Zorya 作为抗噬菌体防御系统功能的基本机制。