Consistent with this, we found that OptE robustly inhibited bacterial growth unless an OptS-derived nucleotide was present and that OptS constitutively synthesizes 2′,3′-c-di-AMP in vivo in the absence of phage infection.

These findings left us puzzled: How does the Acb2 nucleotide sponge protein activate the Panoptes system? Based on its ability to bind diverse nucleotide products, we proposed that Acb2 also binds the product of OptS (2′,3′-c-di-AMP), which is exactly what we found.

Intriguingly, the escaper phages we identified encoded loss-of-function mutations exclusively in the anti-CBASS 2 (acb2) gene. Consistent with this, we found that acb2 was necessary and sufficient to activate Panoptes defense.

We tested the activity of OptS by incubating it with a range of NTPs and found that it used ATP as a substrate to specifically produce 2′,3′-c-di-AMP, an isomer of c-di-AMP. We used a series of phosphodiesterases to show that the linkage included a 3′–5′ and 2′–5′ bond.

To better understand the mechanism of this system, we determined a crystal structure of OptS and found that it resembled the catalytic regions of both Cas10 and GGDEF enzymes and formed a stable tetrameric complex.

We first explored the antiphage activity of Panoptes and found that it robustly and specifically defended against Teven phages from the Straboviridae family.

Nucleotide-based signaling is a hallmark of many antiphage systems (CBASS, Pycsar, Thoeris, etc.). Here we investigated Panoptes, a two-gene operon (optSE) that we predicted would similarly use nucleotide second messengers to carry out an immune response.