Lipopolysaccharide Transport Involves Long-Range Coupling between Cytoplasmic and Periplasmic Domains of the LptB₂FGC Extractor

ABSTRACT

The cell surface of the Gram-negative cell envelope contains lipopolysaccharide (LPS) molecules, which form a permeability barrier against hydrophobic antibiotics. The LPS transport (Lpt) machine composed of LptB₂FGCADE forms a proteinaceous transenvelope bridge that allows for the rapid and specific transport of newly synthesized LPS from the inner membrane (IM) to the outer membrane (OM). This transport is powered from the IM by the ATP-binding cassette transporter LptB₂FGC. The ATP-driven cycling between closed- and open-dimer states of the ATPase LptB₂ is coupled to the extraction of LPS by the transmembrane domains LptFG. However, the mechanism by which LPS moves from a substrate-binding cavity formed by LptFG at the IM to the first component of the periplasmic bridge, the periplasmic β-jellyroll domain of LptF, is poorly understood. To better understand how LptB₂FGC functions in Escherichia coli, we searched for suppressors of a defective LptB variant. We found that defects in LptB₂ can be suppressed by both structural modifications to the core oligosaccharide of LPS and changes in various regions of LptFG, including a periplasmic loop in LptF that connects the substrate-binding cavity in LptFG to the periplasmic β-jellyroll domain of LptF. These novel suppressors suggest that interactions between the core oligosaccharide of LPS and periplasmic regions in the transporter influence the rate of LPS extraction by LptB₂FGC. Together, our genetic data reveal a path for bidirectional coupling between LptB₂ and LptFG that extends from the cytoplasm to the entrance to the periplasmic bridge of the transporter.

IMPORTANCE Gram-negative bacteria are intrinsically resistant to many antibiotics due to the presence of lipopolysaccharide (LPS) at their cell surface. LPS is transported from its site of synthesis at the inner membrane to the outer membrane by the Lpt machine. Lpt proteins form a transporter that spans the entire envelope and is thought to function similarly to a Pez candy dispenser. This transenvelope machine is powered by the cytoplasmic LptB ATPase through a poorly understood mechanism. Using genetic analyses in Escherichia coli, we found that LPS transport involves long-ranging bidirectional coupling across cellular compartments between cytoplasmic LptB and periplasmic regions of the Lpt transporter. This knowledge could be exploited in developing antimicrobials that overcome the permeability barrier imposed by LPS.