An in vitro gram-negative double bilayer envelope mimetic

The gram-negative bacterial envelope is a complex structure composed of two membranes and a peptidoglycan layer that together maintain the cell's integrity and shape. Despite extensive knowledge of its composition, replicating this system in vitro remains a challenge, particularly for trans-envelope studies, as existing models focus on either the outer or inner membrane alone. The study of transport processes between these membranes, especially biogenic transport responsible for outer membrane (OM) formation, is therefore limited. One key system, the PqiABC complex, is believed to facilitate glycerophospholipid (GPL) transport by forming a trans-envelope tunnel, tethering the two membranes. Previous work by us has shown that PqiABC can be reconstituted and used to construct a double bilayer architecture on a planar surface. In this proposal, we aim to utilise a non-functional variant of PqiABC as a scaffold to study other trans-envelope biogenic processes. As a proof of concept, by integrating the Maintenance of Outer Membrane Lipid Asymmetry (Mla) pathway, which regulates GPL distribution between the membranes, we will investigate its activity in this controlled mimetic system. This approach has the potential to significantly advance the accuracy of in vitro studies of gram-negative envelope biology, enabling more precise investigations into membrane dynamics and transport processes.