PqiABC forms a membrane-bridging conduit and mediates bidirectional phospholipid transport across the Gram-negative bacterial envelope

The mechanism by which glycerophospholipids are transported between the inner and outer membranes in Gram-negative bacteria remains poorly understood. In Escherichia coli, the paraquat-inducible (Pqi) pathway, comprising the inner membrane protein PqiA, the periplasm-spanning MCE-family protein PqiB, and the outer membrane lipoprotein PqiC, has been implicated in this process. These components are proposed to assemble into a quaternary complex that forms a continuous channel bridging the inner and outer membranes. Here, using neutron reflectometry and quartz crystal microbalance with dissipation monitoring, we perform a dynamic structural analysis of PqiABC within a planar double bilayer membrane-mimetic system. This approach reveals that PqiABC assembles into a stable, envelope-spanning complex anchored to both membranes, consistent with its proposed conduit architecture. Furthermore, using neutron reflectometry in combination with complementary fluorescence-based assays, we demonstrate that PqiABC mediates passive glycerophospholipid transport, supporting bidirectional lipid exchange between membranes. Together, these findings establish PqiABC as a membrane-bridging lipid transport system and provide direct evidence for a mechanism of passive glycerophospholipid equilibration across the bacterial envelope.