Abstract: Bacterial conjugation is the main mechanism for the dissemination of antibiotic resistance genes. A single DNA strand of the conjugative plasmid is transferred across bacterial membranes covalently bound to a large multi-domain protein, named relaxase, which must be unfolded to traverse the secretion channel. Two tyrosine residues of the relaxase (Y18 and Y26 in relaxase TrwC) play an important role in the processing of conjugative DNA. We have used nanopore technology to uncover the unfolding states that take place during translocation of the relaxase-DNA complex. We observed that the relaxase unfolding pathway depends on the tyrosine residue involved in conjugative DNA binding. Transfer of the nucleoprotein complex is faster when DNA is bound to residue Y18. This is the first time in which a protein-DNA complex that is naturally translocated through bacterial membranes has been analyzed by nanopore sensing, opening new horizons to apply this technology to study protein secretion.

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