Yue, J.; Manne, A.; Guo, W.; Yu, H.; Wang, J.; Rahman, M. K.; Lees, K.; Xu, H.; Botting, J. M.; MOTALEB, M. A.; Liu, J.

The transmembrane export apparatus is a conserved core of bacterial type III secretion systems, shared by the flagellum and injectisome. Powered by the proton-motive force (PMF), this machinery translocates protein substrates across the bacterial envelope to support motility and virulence. However, its assembly and operation within native membranes remain poorly understood. Here, using in-situ single-particle cryo-electron microscopy, we resolve near-atomic-resolution structures of the flagellar export apparatus within the periplasmic flagellar motor of the Lyme disease spirochete Borrelia burgdorferi. Structural and genetic analyses suggest that coordinated, stepwise assembly of the flagellar export apparatus and MS-ring drives local deformation of the cytoplasmic membrane through extensive protein-lipid-protein interactions, constructing a funnel-shaped conduit optimal for substrate translocation. In addition, a conserved hydrophilic pathway lies within core export protein FlhA, with key acidic residues essential for export activity and supporting PMF-driven substrate translocation. Together, these findings establish a mechanistic framework for export apparatus assembly and energy transduction, defining core principles of type III secretion.