Probing phospholipid transport by the Pqi pathway

In Gram-negative bacteria, the cell envelope is a complex tripartite structure composed of an inner membrane (IM), outer membrane (OM), and an intervening peptidoglycan layer. These components collectively maintain cell integrity and shape. While the envelope’s structure is well characterized, replicating it in vitro remains challenging. Current models typically isolate either the OM or IM, limiting study of processes spanning the entire envelope.

This is particularly problematic for studying trans-envelope transport systems—multi-protein machineries essential for processes such as OM biogenesis. These systems often include components embedded in both membranes and may physically tether the IM and OM. One example is the PqiABC complex, implicated in phospholipid transport, which spans the periplasm and links the membranes.

We have developed a method to reconstitute PqiABC on a planar surface, enabling structural analysis. Using differentially deuterated inner and outer membranes, we observed lipid mixing between bilayers. However, whether this mixing is mediated by PqiABC or is an artefact of membrane deposition remains unclear.

This proposal will address this via a deuterated lipid pulse-chase experiment. By comparing lipid transport in wild-type PqiABC and a non-functional mutant, we will determine whether phospholipid transfer is protein-mediated or incidental—clarifying PqiABC’s role and supporting future study of trans-envelope systems.