Assembly and Maintenance of Lipids at the Bacterial Outer Membrane

Abstract

The outer membrane of Gram-negative bacteria is essential for their survival in harsh environments and provides intrinsic resistance to many antibiotics. This membrane is remarkable; it is a highly asymmetric lipid bilayer. The inner leaflet of the outer membrane contains phospholipids, whereas the fatty acyl chains attached to lipopolysaccharide (LPS) comprise the hydrophobic portion of the outer leaflet. This lipid asymmetry, and in particular the exclusion of phospholipids from the outer leaflet, is key to creating an almost impenetrable barrier to hydrophobic molecules that can otherwise pass through phospholipid bilayers. It has long been known that these lipids are not made in the outer membrane. It is now believed that conserved multisubunit protein machines extract these lipids after their synthesis is completed at the inner membrane and transport them to the outer membrane. A longstanding question is how the cell builds and maintains this asymmetric lipid bilayer in coordination with the assembly of the other components of the cell envelope. This Review describes the trans-envelope lipid transport systems that have been identified to participate in outer-membrane biogenesis: LPS transport via the Lpt machine, and phospholipid transport via the Mla pathway and several recently proposed transporters.

Figure 1:. Major lipid components of the E. coli outer membrane.

A) Structure of the three types of phospholipids found in E. coli. The whole-cell phospholipid content of E. coli is 75% phosphatidylethanolamine, 20% phosphatidylglycerol, and 5% cardiolipin. Although fatty acid composition can change in response to signals, the major fatty acids in phospholipids under normal growth are 16:0, 16:1, and 18:1. B) Structure and main components of E. coli K-12 LPS. Potential modifications of Lipid A and the corresponding modifying enzymes are color coded. PagP cleaves an acyl chain from a phospholipid and ligates it (shown in red) onto Lipid A. PagL deacylates LPS by removing the R-3-hydroxymyristate shown in green. The C1 and C4′ phosphates can be modified with L-4-aminoarabinose (pink) by ArnT and phosphoethanolamine (blue) by EptA, respectively. The core oligosaccharide is composed of glucose (Glu), heptose (Hep), galactose (Gal), and 3-deoxy-d-manno-octulosonic acid (Kdo). Heptose residues are phosphorylated (P) and modified with phosphoethanolamine (PEtN). The numbers represent glycosidic linkage positions. The structure of the highly variable O antigen is not shown.

Figure 2:. Model for LPS transport by the Lpt system.

LPS is transported from the inner membrane (IM) to the outer membrane (OM) by the LptB₂FGCADE complex in an ATP-dependent manner. Each round of ATP binding and hydrolysis by the LptB₂FGC ABC transporter is thought to be used to extract one molecule of LPS from the inner membrane and place it onto the periplasmic Lpt bridge. Repeated rounds of hydrolysis extract LPS molecules that push others ahead on the bridge so that a stream of LPS travels towards the outer membrane through the Lpt bridge. LPS finally reaches the LptDE translocon and exits into the outer leaflet of the outer membrane. PG represents the peptidoglycan cell wall.

Figure 3:. Structures of Lpt factors.

Cartoon representations of the crystal structures of the components of the Lpt machine shown in their respective cellular compartments. Components were crystalized individually or as sub-complexes as follows: The LptB2FGC complex (PDB 6MJP), LptA (PDB 2R19), and LptDE complex (PDB 4Q35). To date, a structure of the entire trans-envelope complex is not available, but it is known that the β-jellyroll domains interact in a head-to-tail fashion (see main text for details). The number of LptA molecules in each bridge is unknown, but one could be sufficient to span the periplasm.

Figure 4:. Conformational states of the LptB2FG(C) complex.

Cartoon representations of structures of the LptB₂FG(C) transporter in different conformations thought to represent steps of the transport cycle. From left to right: crystal structure of apo LptB₂FGC (PDB 6MJP) representing the resting state, the cryo-EM structure of apo LptB₂FG bound to an LPS molecule (shown in orange) prior to extraction from the cavity (PDB 6MHU), and the cryo-EM structure of vanadate-trapped LptB₂FG (PDB 6MHZ) representing the post-extraction state (in this structure the periplasmic β-jellyroll domains of LptFG were not resolved). Panel (a) depicts a view of these structures from the membrane, panel (b) depicts a top-down view, cut away to show the substrate-binding cavity and the movement of the TM helices.

Figure 5:. Model for the mechanism of LPS extraction from the inner membrane by the LptB₂FGC ABC transporter.

For simplicity, the β-jellyroll domains of LptCFG are not shown. The LptB₂FGC transporter starts the cycle in an apo conformation. 1) LPS enters into the V-shaped cavity of the transporter in an ATP-independent manner. 2) The TM helix of LptC is thought to dissociate from LptFG through an unknown mechanism. This causes a partial closure of the LptFG cavity, resulting in the formation of more high-affinity contacts between LPS and LptFG. 3) ATP (yellow) binds to LptB, triggering the closure of the LptB dimer and the LptFG cavity. As the cavity closes, LPS is expelled out onto the periplasmic bridge (not shown). 4) Finally, ATP is hydrolyzed and ADP and Pi (red and black) are released, which leads to the reopening of the LptB dimer and LptF cavity and the resetting of the transporter. How and when the TM helix of LptC dissociates and re-associates with LptFG is unknown.

Figure 6:. Model for retrograde phospholipid transport by the Mla system.

Phospholipids that are mislocalized to the outer leaflet of the outer membrane first interact with the MlaA-OmpC complex and transverse the bilayer through a central pore in MlaA. Next, the phospholipid molecule is transferred to the periplasmic chaperone MlaC. MlaC moves across the periplasm and docks onto the IM MlaFEDB complex at the inner membrane. The phospholipid molecule is moved through this complex back into the inner membrane in an ATP-dependent fashion. It is unknown whether the phospholipid is placed in the inner or outer leaflet of the inner membrane. To avoid cluttering, the transmembrane domain of only one of the six MlaD monomers is depicted.

Figure 7:. Structures of Mla factors.

Cartoon representations of the structures of Mla proteins shown in their respective cellular compartments. Structures were obtained from individual components or sub-complexes as follows: The MlaD ₆E₂F₂B₂ complex (PBD 6IC4), MlaD bound to a PL molecule (PBD 5UWA), MlaA complexed with the OmpF trimer (PDB 5NUQ). MlaA can associate with OmpF and OmpC (not shown), but only OmpC has been shown to be functionally relevant.

Figure 8:. Potential mechanisms of anterograde phospholipid transport.

From left to right: i) Zones of hemi-fusion between the inner and outer membranes, termed Bayer junctions, would allow free bidirectional diffusion of phospholipids between the two membranes. ii) A chaperone-mediated mechanism would rely on a soluble protein that moves phospholipids through the periplasm. This chaperone would likely be loaded by an inner membrane component and eventually dock to an outer membrane protein that would incorporate the phospholipid molecule into the inner leaflet of the outer membrane iii) A protein bridge or tunnel between inner- and outer-membrane components would allow phospholipids to transverse the periplasm. The inner membrane component would load phospholipids on to the bridge, while the outer membrane portion would incorporate the PLs into the inner leaflet of the outer membrane. The outer membrane components in the protein-based models are depicted as lipoproteins that are embedded in the inner leaflet similarly to MlaA, but there is no evidence suggesting this type of factor.