Structure and activity of a bacterial defense-associated 3'-5' exonuclease.

Abstract

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. Cap18 is a predicted 3′-5′ exonuclease associated with hundreds of CBASS systems with diverse regulators and effectors. However, the structure, biochemical activities, and biological roles of Cap18 remain unknown. Here we determine a high-resolution X-ray crystal structure of E. coli upec-117 Cap18, and show that it adopts a homodimeric structure similar to the bacterial DEDDh-family nucleases RNase T and Orn. We show that the protein is an active exonuclease with a preference for ssDNA over ssRNA, presenting non-sequence specific activity on both single-stranded and 3’ overhang DNA substrates. It does not require a cGAS-derived second messenger molecule for activity. Since E. coli upec-117 CBASS system can likely respond to stress signals other than phage infection through its WYL-domain transcription factor CapW, we are currently exploring the biological roles of Cap18 in modulating CapW responses using biochemical and cell-based assays. Together, these findings provide structural and functional insights into a previously uncharacterized enzyme associated with bacterial CBASS immune systems.