Structural bases for F plasmid conjugation and F pilus biogenesis in Escherichia coli

Abstract

Bacterial conjugation systems are members of the large type IV secretion system (T4SS) superfamily. Conjugative transfer of F plasmids residing in theEnterobacteriaceaewas first reported in the 1940s, yet the architecture of F plasmid-encoded transfer channel and its physical relationship with the F pilus remain unknown. We visualized F-encoded structures in the native bacterial cell envelope by in situ cryoelectron tomography (CryoET). Remarkably, F plasmids encode four distinct structures, not just the translocation channel or channel-pilus complex predicted by prevailing models. The F1 structure is composed of distinct outer and inner membrane complexes and a connecting cylinder that together house the envelope-spanning translocation channel. The F2 structure is essentially the F1 complex with the F pilus attached at the outer membrane (OM). Remarkably, the F3 structure consists of the F pilus attached to a thin, cell envelope-spanning stalk, whereas the F4 structure consists of the pilus docked to the OM without an associated periplasmic density. The traffic ATPase TraC is configured as a hexamer of dimers at the cytoplasmic faces of the F1 and F2 structures, where it respectively regulates substrate transfer and F pilus biogenesis. Together, our findings present architectural renderings of the DNA conjugation or “mating” channel, the channel–pilus connection, and unprecedented pilus basal structures. These structural snapshots support a model for biogenesis of the F transfer system and allow for detailed comparisons with other structurally characterized T4SSs.

Type IV Coupling Proteins as Potential Targets to Control the Dissemination of Antibiotic Resistance

“…Finally, the conjugative pilus, a structure essential for the direct contact between bacterial cells (Lawley et al, 2003), is composed of VirB2 and VirB5 proteins. Recent structural studies on the F-plasmid have provided new insights into the different types of channel structures and conjugative pili that can be present in donor bacteria (Hu et al, 2019a).…”

Analysis of Dot/Icm Type IVB Secretion System Subassemblies by Cryoelectron Tomography Reveals Conformational Changes Induced by DotB Binding

“…The DotB (VirB11-related) ATPase is dynamic, and, presumably, one important stage that is necessary for translocation of substrates by the Dot/Icm machine is the recruitment of a DotB hexamer from the cytosol to the type IVB complex through a direct interaction with the DotO complex (15). Recently, in situ cryo-ET structures of the Cag secretion system in H. pylori and the F plasmid conjugation system in E. coli (19,20) provided evidence that the VirB4-related ATPases in these systems assemble a structure that is similar to that of the DotO complex.…”

Bacterial injection machines: Evolutionary diverse but functionally convergent

“…Craig Roy reported a DotB‐DotO hexameric complex, involved in substrate recruitment, which creates a cytoplasmic channel whose assembly opens up the T4SS, thus directing the translocation of substrates through the T4SS (Chetrit et al, ). A very similar structure is revealed in the case of the system associated with the conjugative plasmid F, with a 13‐fold symmetry OMC linked to a sixfold symmetry IMCC (Hu et al, a). In addition, Bo Hu from University of Houston reported a refined cryo‐ET structure of the Cag T4SS confirming the previously reported 14‐fold symmetry at the OMC but showing a clear sixfold symmetry in the cytoplasmic complex (Hu et al, b).…”

“…Comparison of the 3D structure of T4SS from plasmid R388 (Low et al, ), plasmid F (Hu et al, a), Legionella pneumophila Dot‐Icm (Chetrit et al, ), and Helicobacter pylori Cag (Hu et al, b). Below each T4SS, the nature of the translocated substrate (protein or protein–DNA complexes) and the target cell (bacteria or human) is indicated.…”