In situ structure of the flagellar export apparatus reveals membrane remodeling and lipid-mediated coupling between...

The transmembrane export apparatus is a conserved core of bacterial type III secretion systems shared by flagella and injectisomes. Powered by the proton-motive force (PMF), this machinery translocates protein substrates across the cytoplasmic membrane to support bacterial motility and virulence. However, how the export apparatus assembles and functions within native membranes remains poorly understood. Here, using in situ single-particle cryo-electron microscopy and cryo-electron tomography, we resolve high-resolution structures of the flagellar export apparatus within intact cells of the Lyme disease spirochete Borrelia burgdorferi. Structural and genetic analyses suggest that coordinated assembly of the export apparatus and MS-ring drives extensive deformation of the cytoplasmic membrane, generating a funnel-shaped conduit for substrate translocation. The FlhA nonamer and FliPQR-FlhB export gate lack direct transmembrane contacts and are separated by a thin, bilayer-like density, revealing a lipid-mediated interface. The conserved FlhB hinge helix extends into the FlhA tunnel and may couple PMF-driven energy transduction to substrate translocation. We further identify a conserved hydrophilic pathway within FlhA and show that key acidic residues lining this pathway are essential for export activity. Together, these findings establish a mechanistic framework for export apparatus assembly and energy transduction, providing broader insights into type III secretion.