Confined Mobility of TonB and FepA in Escherichia coli Membranes.

Yoriko Lill,Ken Ritchie,Phillip E Klebba,Chuck R Smallwood,Lorne D Jordan,Markus A Lill,Salete M Newton

doi:10.1371/journal.pone.0160862

Yoriko Lill, Ken Ritchie + Show 5 more

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https://doi.org/10.1371/journal.pone.0160862

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Abstract

The important process of nutrient uptake in Escherichia coli, in many cases, involves transit of the nutrient through a class of beta-barrel proteins in the outer membrane known as TonB-dependent transporters (TBDTs) and requires interaction with the inner membrane protein TonB. Here we have imaged the mobility of the ferric enterobactin transporter FepA and TonB by tracking them in the membranes of live E. coli with single-molecule resolution at time-scales ranging from milliseconds to seconds. We employed simple simulations to model/analyze the lateral diffusion in the membranes of E.coli, to take into account both the highly curved geometry of the cell and artifactual effects expected due to finite exposure time imaging. We find that both molecules perform confined lateral diffusion in their respective membranes in the absence of ligand with FepA confined to a region μm in radius in the outer membrane and TonB confined to a region μm in radius in the inner membrane. The diffusion coefficient of these molecules on millisecond time-scales was estimated to be μm2/s and μm2/s for FepA and TonB, respectively, implying that each molecule is free to diffuse within its domain. Disruption of the inner membrane potential, deletion of ExbB/D from the inner membrane, presence of ligand or antibody to FepA and disruption of the MreB cytoskeleton was all found to further restrict the mobility of both molecules. Results are analyzed in terms of changes in confinement size and interactions between the two proteins.

Highlights

The important process of nutrient uptake in Escherichia coli requires transport across the lipopolysaccharide (LPS)-rich outer membrane (OM), passage through the periplasmic space that contains the peptidoglycan (PG), and transport across the cell’s inner membrane (IM), that surrounds the cytoplasm
TonB-dependent transporters (TBDTs) are energy-dependent gated channels that usually transport large metal complexes which cannot fit through porins, and are too scarce to enter by mass-action-driven transport
Site-directed alkylation experiments indicated that the N-terminal domain of FepA at least in part dislodges into the periplasm to allow passage of ferric enterobactin [10]

Summary

Introduction

The important process of nutrient uptake in Escherichia coli requires transport across the lipopolysaccharide (LPS)-rich outer membrane (OM), passage through the periplasmic space that contains the peptidoglycan (PG), and transport across the cell’s inner membrane (IM), that surrounds the cytoplasm. In E. coli TBDTs scavenge and bind micronutrients with high affinities, especially iron chelates (called siderophores), and vitamin B12, and they are parasitized by colicins, phages, and naturally occurring antibiotics [5]. In other bacteria they were reported to serve as receptors for nickel complexes and even carbohydrates [6]. Ferric enterobactin transport through FepA may involve dislodgement of the plug domain from the B-barrel (ball-and-chain model), or formation of a smaller diffusion channel (transient pore model) [9, 10]. Site-directed alkylation experiments indicated that the N-terminal domain of FepA at least in part dislodges into the periplasm to allow passage of ferric enterobactin [10]

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