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 β-lactamase-like domain. Here, we provide in-depth characterisation of the nuclease activity of the β-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 β-lactamase-like domain is enhanced when the OB domain is encoded on the same polypeptide chain. Additionally, we identify a pin loop within the β-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.