Lpt-protein dynamics reveal that Lipopolysaccharide promotes trans-envelope bridge formation

Abstract

Gram-negative bacteria have two lipid bilayers, the inner membrane and the outer membrane as part of their cell envelope structure.1 A special feature of the outer layer is its asymmetric membrane structure, comprised of phospholipids in the inner leaflet and lipopolysaccharide (LPS) in the outer leaflet. The presence of LPS in the outer leaflet of the outer membrane provides a robust physical barrier, protecting Gram-negative bacteria from external toxins, making them especially difficult to kill with e. g. antibiotics.2-3 LPS is a large amphipathic molecule comprised of fatty acyl chains attached to polysaccharides, whose biosynthesis is completed in the inner membrane, and which then needs to be transported from its synthesis site to the cell surface.4 LPS must be extracted from the inner membrane, moved across the aqueous periplasm that separates the two membranes, then translocate through the outer membrane and assembled on the cell surface.5 LPS transport and assembly requires seven conserved lipopolysaccharide transport components (LptA-G), which are proposed to form a continuous protein bridge spanning the periplasm that provides a path for LPS to reach the cell surface.6-7 However, this model has not been validated in living cells. It is also unknown how stable the proposed bridges are, what influences their formation and breakage and what is their order of assembly and disassembly. This thesis presents live cell single molecule tracking results to address these questions. In chapter 2 we show that Lpt protein dynamics are consistent with the bridge model and that half of the inner membrane Lpt proteins exist in a bridge state. In chapter 3 we describe the life time measurement of the bridge in cells. We found that bridges persist for 5-10 seconds, showing that their organization is highly dynamic. In chapter 4 we present evidence that Lpt-bridge formation is facilitated by LPS, providing a mechanism by which the production of LPS can be directly coupled to its transport. Finally, bridge decay kinetics suggest that two different kinds of bridges may exist, whose stability differs according to the presence (long lived) or absence (short lived) of LPS. Taken together, the data of this thesis support a model in which LPS is both a substrate and structural component of highly dynamic Lpt bridges that promote outer membrane assembly.