Structural basis of unidirectional export of lipopolysaccharide to the cell surface.

Gram-negative bacteria are surrounded by an inner cytoplasmic membrane and by an outer membrane, which serves as a protective barrier to limit entry of many antibiotics. The distinctive properties of the outer membrane are due to the presence of lipopolysaccharide1. This large glycolipid, which contains numerous sugars, is made in the cytoplasm; a complex of proteins forms a membrane-to-membrane bridge that mediates transport of lipopolysaccharide from the inner membrane to the cell surface1. The inner-membrane components of the protein bridge comprise an ATP-binding cassette transporter that powers transport, but how this transporter ensures unidirectional lipopolysaccharide movement across the bridge to the outer membrane is unknown2. Here we describe two crystal structures of a five-component inner-membrane complex that contains all the proteins required to extract lipopolysaccharide from the membrane and pass it to the protein bridge. Analysis of these structures, combined with biochemical and genetic experiments, identifies the path of lipopolysaccharide entry into the cavity of the transporter and up to the bridge. We also identify a protein gate that must open to allow movement of substrate from the cavity onto the bridge. Lipopolysaccharide entry into the cavity is ATP-independent, but ATP is required for lipopolysaccharide movement past the gate and onto the bridge. Our findings explain how the inner-membrane transport complex controls efficient unidirectional transport of lipopolysaccharide against its concentration gradient.

A cluster of residues in the lipopolysaccharide exporter that selects substrate variants for transport to the outer membrane.

Most Gram-negative bacteria assemble lipopolysaccharides (LPS) on their surface to form a permeability barrier against many antimicrobials. LPS is synthesized at the inner membrane and then transported to the outer leaflet of the outer membrane. Although the overall LPS structure is conserved, LPS molecules can differ in composition at the species and strain level. Some bacteria also regulate when to modify phosphates on LPS at the inner membrane in order to become resistant to cationic antimicrobial peptides. The multi-protein Lpt trans-envelope machine, which transports LPS from the inner to the outer membrane, must therefore handle a variety of substrates. The most poorly understood step in LPS transport is how the ATP-binding cassette LptB2 FG transporter extracts LPS from the inner membrane. Here, we define residue K34 in LptG as a site within the structural cavity of the Escherichia coli LptB2 FG transporter that interacts electrostatically with phosphates on unmodified LPS. Alterations to this residue cause transport defects that are suppressed by the activation of the BasSR two-component signaling system, which results in modifications to the LPS phosphates. We also show this residue is part of a larger site in LptG that differentially contributes to the transport of unmodified and modified LPS.