Deselaers, S. | Search bioRxiv

bioRxiv2026

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.

biochemistry↗

bioRxiv2025

Molecular interplay between ComEC domains leads to efficient DNA translocation during natural transformation

Naturally competent bacteria can take up and incorporate free DNA from their environment using complex machinery that is endogenously encoded. This process is called natural transformation and is a key mechanism in the spread of antibiotic resistance amongst bacteria, including many human pathogens. All competent bacteria require ComEC to transport the transforming DNA across the cytoplasmic membrane. In addition to the transmembrane competence domain predicted to form the DNA channel, most ComEC orthologues additionally contain an oligonucleotide binding (OB) domain and {beta}-lactamase-like domain. Here, we provide in-depth characterisation of the nuclease activity of the {beta}-lactamase-like domain and the DNA binding activity of the OB domain, and present high-resolution structures of both domains. We show that the in vitro nuclease activity of the {beta}-lactamase-like domain is enhanced when the OB domain is encoded on the same polypeptide chain. Additionally, we identify a pin loop within the {beta}-lactamase-like domain responsible for melting the DNA duplex prior to cleavage of the non-translocating strand, and a DNA channel lined with aromatic residues that guide the uncleaved translocating strand through ComEC. On the basis of our biochemical, structural and functional characterisation, we provide a mechanistic model for how ComEC achieves the simultaneous tasks of DNA degradation and translocation, central to the natural transformation process.

microbiology↗