Structural and biochemical insights reveal substrate-modulated nuclease activity of ComEC during DNA processing

Natural transformation enables bacteria to internalise extracellular DNA, driving adaptation and the spread of antibiotic resistance. The membrane protein ComEC mediates translocation of single-stranded DNA across the cytoplasmic membrane while degrading the complementary strand, yet the structural basis of its activity remains incompletely defined. Here, we report a cryo-electron microscopy structure of full-length ComEC from Neomoorella carbonis in a pre-translocation state, revealing a three-domain architecture and a conserved transmembrane channel captured in a closed conformation. Structural analysis indicates that conformational rearrangements of channel-lining helices are required to accommodate single-stranded DNA. Biochemical assays show that, relative to the isolated {beta}-lactamase-like domain, full-length ComEC degrades DNA more efficiently. Importantly, coating of the DNA by the periplasmic DNA receptor ComEA suppresses endonucleolytic cleavage, thereby modulating nuclease activity. Together, these findings provide the first characterisation of the nuclease activity of full-length ComEC and show how ComEA-mediated protection of the substrate directs ComECs nuclease activity to ensure high fidelity during the natural transformation process.