Cyclic dinucleotides (CDNs) play central roles in bacterial homeostasis and virulence as nucleotide second messengers. Bacterial CDNs also elicit immune responses during infection when they are detected by pattern recognition receptors in animal cells. Here, we performed a systematic biochemical screen for bacterial signaling nucleotides and discovered a broad family of cGAS / DncV-like nucleotidyltransferases (CD-NTases) that use both purine and pyrimidine nucleotides to synthesize an exceptionally diverse range of CDNs. A series of crystal structures establish CD-NTases as a structurally conserved family and reveal key contacts in the active-site lid that direct purine or pyrimidine selection. CD-NTase products are not restricted to CDNs and also include an unexpected class of cyclic trinucleotide compounds. Biochemical and cellular analysis of novel signaling nucleotides demonstrate that these molecules activate distinct host receptors and thus may modulate the interaction of both pathogens and commensal microbiota with their animal and plant hosts.

Stimulator of interferon genes (STING) is a receptor in human cells that senses foreign cyclic dinucleotides released during bacterial infection and endogenous cyclic GMP–AMP signaling during viral infection and antitumor immunity 1 – 5 . STING shares no structural homology with other known signaling proteins 6 – 9 , limiting functional analysis and preventing explanation for the origin of cyclic dinucleotide signaling in mammalian innate immunity. Here we discover functional STING homologues encoded within prokaryotic defense islands and reveal a conserved mechanism of signal activation. Crystal structures of bacterial STING define a minimal homodimeric scaffold that selectively responds to c-di-GMP synthesized by a neighboring cGAS/DncV-like nucleotidyltransferase (CD-NTase) enzyme. Bacterial STING domains couple cyclic dinucleotide recognition with protein filament formation to drive TIR effector domain oligomerization and rapid NAD + cleavage. We reconstruct the evolutionary events following acquisition of STING into metazoan innate immunity and determine the structure of a full-length TIR-STING fusion from the Pacific oyster C. gigas . Comparative structural analysis demonstrates how metazoan-specific additions to the core STING scaffold enabled a switch from direct effector function to regulation of antiviral transcription. Together, our results explain the mechanism of STING-dependent signaling and reveal conservation of a functional cGAS-STING pathway in prokaryotic bacteriophage defense.