Identification of Residues in the Lipopolysaccharide ABC Transporter That Coordinate ATPase Activity with Extractor Function.

Brent W Simpson,Rebecca M Davis,Tristan W Owens,Sunia A Trauger,Matthew J Orabella,Natividad Ruiz,Janine M May,Daniel Kahne,Susan Gottesman

doi:10.1128/mbio.01729-16

Brent W Simpson, Rebecca M Davis + Show 7 more

Open Access

https://doi.org/10.1128/mbio.01729-16

Copy DOI

Journal: mBio	Publication Date: Oct 18, 2016
Citations: 32	License type: CC BY 4.0

Affiliation: The Ohio State University, Harvard University

Abstract

ABSTRACTThe surface of most Gram-negative bacteria is covered with lipopolysaccharide (LPS), creating a permeability barrier against toxic molecules, including many antimicrobials. To assemble LPS on their surface, Gram-negative bacteria must extract newly synthesized LPS from the inner membrane, transport it across the aqueous periplasm, and translocate it across the outer membrane. The LptA to -G proteins assemble into a transenvelope complex that transports LPS from the inner membrane to the cell surface. The Lpt system powers LPS transport from the inner membrane by using a poorly characterized ATP-binding cassette system composed of the ATPase LptB and the transmembrane domains LptFG. Here, we characterize a cluster of residues in the groove region of LptB that is important for controlling LPS transport. We also provide the first functional characterization of LptFG and identify their coupling helices that interact with the LptB groove. Substitutions at conserved residues in these coupling helices compromise both the assembly and function of the LptB2FG complex. Defects in LPS transport conferred by alterations in the LptFG coupling helices can be rescued by changing a residue in LptB that is adjacent to functionally important residues in the groove region. This suppression is achieved by increasing the ATPase activity of the LptB2FG complex. Taken together, these data identify a specific binding site in LptB for the coupling helices of LptFG that is responsible for coupling of ATP hydrolysis by LptB with LptFG function to achieve LPS extraction.

Highlights

The surface of most Gram-negative bacteria is covered with lipopolysaccharide (LPS), creating a permeability barrier against toxic molecules, including many antimicrobials
Residue F90 in the LptB groove is essential for the assembly of the LptB2FGC complex in Escherichia coli [10], suggesting that the groove of LptB forms part of an interacting surface with LptFG
To further characterize this groove region, we generated plasmid-carried mutant alleles of lptB that alter side chains flanking this groove and assessed their abilities to complement the loss of the wild-type (WT) chromosomal lptB allele in rich (LB) and minimal media since slow-growth conditions can suppress severe LPS transport defects [27]

Summary

Introduction

The surface of most Gram-negative bacteria is covered with lipopolysaccharide (LPS), creating a permeability barrier against toxic molecules, including many antimicrobials. To assemble LPS on their surface, Gram-negative bacteria must extract newly synthesized LPS from the inner membrane, transport it across the aqueous periplasm, and translocate it across the outer membrane. Defects in LPS transport conferred by alterations in the LptFG coupling helices can be rescued by changing a residue in LptB that is adjacent to functionally important residues in the groove region This suppression is achieved by increasing the ATPase activity of the LptB2FG complex. We still lack important details of the architecture and mechanism of function of the Lpt system, a model, known as the PEZ model, has been proposed to explain how Lpt transports LPS from the IM to the cell surface [5] In this model, ATP hydrolysis by LptB is used to extract newly synthesized LPS molecules from the IM and to load them onto the Lpt complex. Identification of functionally relevant contacts between LptB and LptFG is crucial to understanding LPS transport

Methods

Results

Conclusion