The widespread CBASS (cyclic oligonucleotide‐based anti‐phage signaling system) immune systems in bacteria protect their hosts from bacteriophage infection by triggering programmed cell death. CBASS systems all encode a cyclic oligonucleotide synthase related to eukaryotic cGAS but use diverse regulators and effector proteins including nucleases, phospholipases, and membrane‐disrupting proteins to effect cell death. Cap18 is a predicted 3′‐5′ exonuclease associated with hundreds of CBASS systems, whose structure, biochemical activities, and biological roles remain unknown. Here we show that Cap18 is a DEDDh‐family exonuclease related to the bacterial exonucleases RNase T and Orn and has nonspecific 3′‐5′ DNA exonuclease activity. Cap18 is commonly found in CBASS systems with associated CapW or CapH+CapP transcription factors, suggesting that it may coordinate with these proteins to regulate CBASS transcription in response to DNA damage. These data expand the repertoire of enzymatic activities associated with bacterial CBASS systems and provide new insights into the regulation of these important bacterial immune systems.
