Highlights d The CBASS-associated nuclease NucC kills phage-infected cells by genome destruction d Binding of a cyclic triadenylate second messenger causes NucC homohexamer assembly d NucC hexamer assembly juxtaposes pairs of active sites to activate DNA cleavage d NucC is found in both CBASS systems and type III CRISPR/ Cas systems

Bacteria are continually challenged by foreign invaders, including bacteriophages, and have evolved a variety of defenses against these invaders. Here, we describe the structural and biochemical mechanisms of a bacteriophage immunity pathway found in a broad array of bacteria, including E. coli and Pseudomonas aeruginosa. This pathway uses eukaryoticlike HORMA domain proteins that recognize specific peptides, then bind and activate a cGAS/DncV-like nucleotidyltransferase (CD-NTase) to generate a cyclic triadenylate (cAAA) second messenger; cAAA in turn activates an endonuclease effector, NucC. Signaling is attenuated by a homolog of the AAA+ ATPase Pch2/TRIP13, which binds and disassembles the active HORMA-CD-NTase complex. When expressed in non-pathogenic E. coli, this pathway confers immunity against bacteriophage l through an abortive infection mechanism. Our findings reveal the molecular mechanisms of a bacterial defense pathway integrating a cGAS-like nucleotidyltransferase with HORMA domain proteins for threat sensing through protein detection and negative regulation by a Trip13 ATPase.

Bacteria encode myriad defences that target the genomes of infecting bacteriophage, including restriction–modification and CRISPR–Cas systems1. In response, one family of large bacteriophages uses a nucleus-like compartment to protect its replicating genomes by excluding host defence factors2–4. However, the principal composition and structure of this compartment remain unknown. Here we find that the bacteriophage nuclear shell assembles primarily from one protein, which we name chimallin (ChmA). Combining cryo-electron tomography of nuclear shells in bacteriophage-infected cells and cryo-electron microscopy of a minimal chimallin compartment in vitro, we show that chimallin self-assembles as a flexible sheet into closed micrometre-scale compartments. The architecture and assembly dynamics of the chimallin shell suggest mechanisms for its nucleation and growth, and its role as a scaffold for phage-encoded factors mediating macromolecular transport, cytoskeletal interactions, and viral maturation.

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Understanding how biological species arise is critical for understanding the evolution of life on Earth. Bioinformatic analyses have recently revealed that viruses, like multicellular life, form reproductively isolated biological species. Viruses are known to share high rates of genetic exchange, so how do they evolve genetic isolation? Here, we evaluate two related bacteriophages and describe three factors that limit genetic exchange between them: 1) A nucleus-like compartment that physically separates replicating phage genomes, thereby limiting inter-phage recombination during co-infection; 2) A tubulin-based spindle that orchestrates phage replication and forms nonfunctional hybrid polymers; and 3) A nuclear incompatibility factor that reduces phage fitness. Together, these traits maintain species differences through Subcellular Genetic Isolation where viral genomes are physically separated during co-infection, and Virogenesis Incompatibility in which the interaction of cross-species components interferes with viral production.