ROSET Model of TonB Action in Gram-Negative Bacterial Iron Acquisition

Therotationalsurveillance andenergytransfer (ROSET) model of TonB action suggests a mechanism by which the electrochemical proton gradient across the Gram-negative bacterial inner membrane (IM) promotes the transport of iron through ligand-gated porins (LGP) in the outer membrane (OM). TonB associates with the IM by an N-terminal hydrophobic helix that forms a complex with ExbBD. It also contains a central extended length of rigid polypeptide that spans the periplasm and a dimericC-terminal-ββαβ-domain (CTD) with LysM motifs that binds the peptidoglycan (PG) layer beneath the OM bilayer. The TonB CTD forms a dimer with affinity for both PG- and TonB-independent OM proteins (e.g., OmpA), localizing it near the periplasmic interface of the OM bilayer. Porins and other OM proteins associate with PG, and this general affinity allows the TonB CTD dimer to survey the periplasmic surface of the OM bilayer. Energized rotational motion of the TonB N terminus in the fluid IM bilayer promotes the lateral movement of the TonB-ExbBD complex in the IM and of the TonB CTD dimer across the inner surface of the OM. When it encounters an accessible TonB box of a (ligand-bound) LGP, the monomeric form of the CTD binds and recruits it into a 4-stranded β-sheet. Because the CTD is rotating, this binding reaction transfers kinetic energy, created by the electrochemical proton gradient across the IM, through the periplasm to the OM protein. The equilibration of the TonB C terminus between the dimeric and monomeric forms that engage in different binding reactions allows the identification of iron-loaded LGP and then the internalization of iron through their trans-outer membrane β-barrels. Hence, the ROSET model postulates a mechanism for the transfer of energy from the IM to the OM, triggering iron uptake.

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High-Throughput Screening Assay for Inhibitors of TonB-Dependent Iron Transport

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057115613788 ◽

2015 ◽

Vol 21 (3) ◽

pp. 316-322 ◽

Cited By ~ 4

Author(s):

Mathew Hanson ◽

Lorne D. Jordan ◽

Yan Shipelskiy ◽

Salete M. Newton ◽

Phillip E. Klebba

Keyword(s):

Outer Membrane ◽

High Throughput ◽

High Throughput Screening ◽

Iron Transport ◽

Iron Acquisition ◽

Metabolic Inhibitors ◽

Screening Assay ◽

Gram Negative ◽

Microtiter Plates ◽

High Throughput Screening Assay

The TonB-dependent Gram-negative bacterial outer membrane protein FepA actively transports the siderophore ferric enterobactin (FeEnt) into the periplasm. We developed a high-throughput screening (HTS) assay that observes FeEnt uptake through FepA in living Escherichia coli, by monitoring fluorescence quenching that occurs upon binding of FeEnt, and then unquenching as the bacteria deplete it from solution by transport. We optimized the labeling and spectroscopic methods to screen for inhibitors of TonB-dependent iron uptake through the outer membrane. The assay works like a molecular switch that is on in the presence of TonB activity and off in its absence. It functions in 96-well microtiter plates, in a variety of conditions, with Z factors of 0.8–1.0. TonB-dependent iron transport is energy dependent, and the inhibitory effects of the metabolic inhibitors carbonyl cyanide m-chlorophenylhydrazone, 2,4-dinitrophenol, azide, cyanide, and arsenate on FeEnt uptake were readily detected by the assay. Because iron acquisition is a determinant of bacterial pathogenesis, HTS with this method may identify inhibitors that block TonB function and constitute novel therapeutics against infectious disease caused by Gram-negative bacteria.

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Evidence for phospholipid export from the gram-negative inner membrane: time to rethink the Mla pathway?

10.1101/388546 ◽

2018 ◽

Cited By ~ 1

Author(s):

Gareth W. Hughes ◽

Stephen C. L. Hall ◽

Claire S. Laxton ◽

Pooja Sridhar ◽

Amirul H. Mahadi ◽

...

Keyword(s):

Crystal Structure ◽

Outer Membrane ◽

Binding Pocket ◽

Periplasmic Protein ◽

Inner Membrane ◽

Gram Negative ◽

Membrane Atpase ◽

Phospholipid Transport ◽

First Time ◽

Β Sheet

AbstractThe Mla pathway is believed to be involved in maintaining the asymmetrical Gram-negative outer membrane via retrograde phospholipid transport. The pathway is composed of 3 components: the outer membrane MlaA-OmpC/F complex, a soluble periplasmic protein, MlaC, and the inner membrane ATPase, MlaFEDB complex. Here we solve the crystal structure of MlaC in its phospholipid free closed apo conformation, revealing a novel pivoting β-sheet mechanism which functions to open and close the phospholipid-binding pocket. Using the apo form of MlaC we provide evidence that the Mla pathway functions in an anterograde rather than a retrograde direction by showing the inner membrane MlaFEDB machinery exports phospholipids and transfers them to MlaC in the periplasm. We confirm that the lipid export process occurs through the MlaD component of the MlaFEDB complex. This lipid export process is shown to be independent of ATP. Our data provides, for the first time, evidence of an apparatus for lipid export to the outer membrane.

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Formation of a β-barrel membrane protein is catalyzed by the interior surface of the assembly machine protein BamA

eLife ◽

10.7554/elife.49787 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 8

Author(s):

James Lee ◽

David Tomasek ◽

Thiago MA Santos ◽

Mary D May ◽

Ina Meuskens ◽

...

Keyword(s):

Membrane Protein ◽

Outer Membrane ◽

Molecular Mechanisms ◽

Gram Negative Bacteria ◽

Site Specific ◽

Gram Negative ◽

Interior Surface ◽

Essential Component ◽

Bam Complex ◽

Β Sheet

The β-barrel assembly machine (Bam) complex in Gram-negative bacteria and its counterparts in mitochondria and chloroplasts fold and insert outer membrane β-barrel proteins. BamA, an essential component of the complex, is itself a β-barrel and is proposed to play a central role in assembling other barrel substrates. Here, we map the path of substrate insertion by the Bam complex using site-specific crosslinking to understand the molecular mechanisms that control β-barrel folding and release. We find that the C-terminal strand of the substrate is stably held by BamA and that the N-terminal strands of the substrate are assembled inside the BamA β-barrel. Importantly, we identify contacts between the assembling β-sheet and the BamA interior surface that determine the rate of substrate folding. Our results support a model in which the interior wall of BamA acts as a chaperone to catalyze β-barrel assembly.

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Structure and Stoichiometry of the Ton Molecular Motor

International Journal of Molecular Sciences ◽

10.3390/ijms21020375 ◽

2020 ◽

Vol 21 (2) ◽

pp. 375 ◽

Cited By ~ 8

Author(s):

Herve Celia ◽

Nicholas Noinaj ◽

Susan K Buchanan

Keyword(s):

Outer Membrane ◽

Mode Of Action ◽

Molecular Motor ◽

Proton Gradient ◽

Gram Negative Bacteria ◽

Periplasmic Space ◽

Inner Membrane ◽

Gram Negative ◽

X Ray ◽

X Ray Crystallography

The Ton complex is a molecular motor that uses the proton gradient at the inner membrane of Gram-negative bacteria to generate force and movement, which are transmitted to transporters at the outer membrane, allowing the entry of nutrients into the periplasmic space. Despite decades of investigation and the recent flurry of structures being reported by X-ray crystallography and cryoEM, the mode of action of the Ton molecular motor has remained elusive, and the precise stoichiometry of its subunits is still a matter of debate. This review summarizes the latest findings on the Ton system by presenting the recently reported structures and related reports on the stoichiometry of the fully assembled complex.

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From Homodimer to Heterodimer and Back: Elucidating the TonB Energy Transduction Cycle

Journal of Bacteriology ◽

10.1128/jb.00484-15 ◽

2015 ◽

Vol 197 (21) ◽

pp. 3433-3445 ◽

Cited By ~ 17

Author(s):

Michael G. Gresock ◽

Kyle A. Kastead ◽

Kathleen Postle

Keyword(s):

Outer Membrane ◽

Active Transport ◽

Cytoplasmic Membrane ◽

Transmembrane Domain ◽

Energy Transduction ◽

Proton Gradient ◽

Carboxy Terminus ◽

Gram Negative ◽

Amino Terminal ◽

Tonb System

ABSTRACTThe TonB system actively transports large, scarce, and important nutrients through outer membrane (OM) transporters of Gram-negative bacteria using the proton gradient of the cytoplasmic membrane (CM). InEscherichia coli, the CM proteins ExbB and ExbD harness and transfer proton motive force energy to the CM protein TonB, which spans the periplasmic space and cyclically binds OM transporters. TonB has two activity domains: the amino-terminal transmembrane domain with residue H20 and the periplasmic carboxy terminus, through which it binds to OM transporters. TonB is inactivated by all substitutions at residue H20 except H20N. Here, we show that while TonB trapped as a homodimer through its amino-terminal domain retained full activity, trapping TonB through its carboxy terminus inactivated it by preventing conformational changes needed for interaction with OM transporters. Surprisingly, inactive TonB H20A had little effect on homodimerization through the amino terminus and instead decreased TonB carboxy-terminal homodimer formation prior to reinitiation of an energy transduction cycle. That result suggested that the TonB carboxy terminus ultimately interacts with OM transporters as a monomer. Our findings also suggested the existence of a separate equimolar pool of ExbD homodimers that are not in contact with TonB. A model is proposed where interaction of TonB homodimers with ExbD homodimers initiates the energy transduction cycle, and, ultimately, the ExbD carboxy terminus modulates interactions of a monomeric TonB carboxy terminus with OM transporters. After TonB exchanges its interaction with ExbD for interaction with a transporter, ExbD homodimers undergo a separate cycle needed to re-energize them.IMPORTANCECanonical mechanisms of active transport across cytoplasmic membranes employ ion gradients or hydrolysis of ATP for energy. Gram-negative bacterial outer membranes lack these resources. The TonB system embodies a novel means of active transport across the outer membrane for nutrients that are too large, too scarce, or too important for diffusion-limited transport. A proton gradient across the cytoplasmic membrane is converted by a multiprotein complex into mechanical energy that drives high-affinity active transport across the outer membrane. This system is also of interest since one of its uses in pathogenic bacteria is for competition with the host for the essential element iron. Understanding the mechanism of the TonB system will allow design of antibiotics targeting iron acquisition.

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Electrophysiological Characterization of Transport Across Outer Membrane Channels from Gram-Negative Bacteria in Their Native Environment

10.26434/chemrxiv.8282204.v1 ◽

2019 ◽

Author(s):

Jiajun Wang ◽

Rémi Terrasse ◽

Jayesh Arun Bafna ◽

Lorraine Benier ◽

Mathias Winterhalter

Keyword(s):

Outer Membrane ◽

Lipid Membrane ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

Multi Drug Resistance ◽

Gram Negative Bacteria ◽

Membrane Channels ◽

Gram Negative ◽

Electrophysiological Characterization

Multi-drug resistance in Gram-negative bacteria is often associated with low permeability of the outer membrane. To investigate the role of membrane channels in the uptake of antibiotics, we extract, purify and reconstitute them into artificial planar membranes. To avoid this time-consuming procedure, here we show a robust approach using fusion of native outer membrane vesicles (OMV) into planar lipid bilayer which moreover allows also to some extend the characterization of membrane protein channels in their native environment. Two major membrane channels from Escherichia coli, OmpF and OmpC, were overexpressed from the host and the corresponding OMVs were collected. Each OMV fusion revealed surprisingly single or only few channel activities. The asymmetry of the OMV´s translates after fusion into the lipid membrane with the LPS dominantly present at the side of OMV addition. Compared to conventional reconstitution methods, the channels fused from OMVs containing LPS have similar conductance but a much broader distribution. The addition of Enrofloxacin on the LPS side yields somewhat higher association (kon) and lower dissociation (koff) rates compared to LPS-free reconstitution. We conclude that using outer membrane vesicles is a fast and easy approach for functional and structural studies of membrane channels in the native membrane.

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