The cell envelope of bacteria mediates their interaction with the outside world and determines what can enter the cell. Gram-negative bacteria have a cell envelope defined by two membranes: an inner membrane, which surrounds the cytoplasm, and an outer membrane, which together with the inner membrane delimits an additional cellular compartment termed the periplasm. The inner membrane of Gram-negative bacteria is primarily composed of a phospholipid bilayer. The outer membrane, in contrast, contains phospholipids in its inner leaflet, and the essential glycolipid lipopolysaccharide (LPS) in the outer leaflet. The presence of LPS in the outer leaflet renders the outer membrane relatively impermeable, and therefore grants the cell resistance to noxious compounds in the environment, such as antibiotics. LPS is synthesized in the cytoplasmic face of the inner membrane, though it can also undergo non-stoichiometric modifications in the periplasmic face, and must thereafter be transported across the rest of the cell envelope. Transversal of the inner membrane by LPS is mediated by the ATP-binding cassette transporter MsbA. LPS extraction from the inner membrane, and subsequent transport across the rest of the cell envelope, is mediated by the LPS transport, or Lpt, complex. The Lpt complex is composed of eight proteins: a dimer of LptB in the cytoplasm that binds and hydrolyzes ATP to drive LPS transport; two transmembrane domains, LptF and LptG, which form a cavity in the inner membrane that accepts LPS and extracts it; LptC, LptA, and LptD, which form a bridge across the periplasm to allow the hydrophobic portion of LPS to traverse the aqueous periplasm; and LptE, which in conjunction with LptD facilitates the transport of LPS across the outer membrane.
Here, we describe work in which we dissect the molecular mechanisms by which the Lpt system’s inner membrane complex, LptB2FGC, interacts with LPS. In chapter two, we describe the identification of a residue within LptG, K34, which is critical for LPS transport. Through structure-function and suppressor analyses, we show K34 of LptG mediates early contact with unmodified LPS as it enters the cavity formed by LptF and LptG. Further, our results imply that modified LPS interacts with the transporter distinctly from unmodified LPS.
Chapter three describes the structure of the LptB2FGC inner membrane complex, as determined by X-ray crystallography, and defines the path LPS takes to the periplasmic bridge of the Lpt system through site-specific crosslinking. We show that the transmembrane anchor of LptC intercalates between transmembrane domains LptF and LptG and is ergo likely to be functionally involved in transport. Further, the periplasmic bridge is preferentially coupled to LptF, rather than LptG.
In chapter four, we utilized a mutant producing LptG altered at residue K34 to perform suppressor analysis and further elucidate how the LptB2FGC complex interacts with LPS. We found that increased LPS production could suppress the defects produced by this variant, but not those produced by other altered lpt alleles. We therefore postulate that this suppression could be used as a probe of what stages of LPS transport are altered by a given lpt allele, or Lpt system inhibitor. Finally, we found that partial-loss-of-function lpt alleles suppressed otherwise lethal overproduction of LPS, which we believe supports a model in which LPS is essential to balance the rate at which material is added to the leaflets of the outer membrane.