Cryo-EM structure of a conjugative type IV secretion system suggests a molecular switch regulating pilus biogenesis

Conjugative type IV secretion systems (T4SS) mediate bacterial conjugation, a process that enables the unidirectional exchange of genetic materials between a donor and a recipient bacterial cell. Bacterial conjugation is the primary means by which antibiotic resistance genes spread among bacterial populations (Barlow 2009; Virolle et al, 2020). Conjugative T4SSs form pili: long extracellular filaments that connect with recipient cells. Previously, we solved the cryo-electron microscopy (cryo-EM) structure of a conjugative T4SS. In this article, based on additional data, we present a more complete T4SS cryo-EM structure than that published earlier. Novel structural features include details of the mismatch symmetry within the OMCC, the presence of a fourth VirB8 subunit in the asymmetric unit of both the arches and the inner membrane complex (IMC), and a hydrophobic VirB5 tip in the distal end of the stalk. Additionally, we provide previously undescribed structural insights into the protein VirB10 and identify a novel regulation mechanism of T4SS-mediated pilus biogenesis by this protein, that we believe is a key checkpoint for this process. Conjugative type IV secretion systems (T4SS) mediate transfer of genetic information between bacterial cells. This study presents a cryo-EM structure of a conjugative type IV secretion that provides insights into the various sub-complexes that form its machinery.New structural information provides insights into outer membrane core complex (OMCC) dynamics and mismatch symmetry. The complete structure of VirB8 is determined and a fourth VirB8 subunit is characterised within the arches and the inner membrane complex (IMC). The VirB10 protein structure from its N-terminus in the cytoplasm to its C-terminus in the OMCC is elucidated. Obstruction of the VirB2 pilin recruitment site on VirB6 by a trans-membrane region of VirB10 suggests a mechanism of pilus biogenesis regulation. New structural information provides insights into the four major compartments of a conjugative T4SS: the outer membrane core complex (OMCC), the stalk, the arches, and the inner membrane complex (IMC).