Fluoresceination of FepA during colicin B killing: effects of temperature, toxin and TonB

Smallwood, Chuck R.; Marco, Amparo Gala; Xiao, Qiaobin; Trinh, Vy; Newton, S. A.; Klebba, Phillip E.

doi:10.1111/j.1365-2958.2009.06715.x

Cited by 22 publications

(27 citation statements)

References 59 publications

(92 reference statements)

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“…(7) and pGT (36) was grown in MOPS minimal medium to late log, which derepressed the synthesis of FepAS271C and GFP-TonB, respectively. We pelleted the bacteria by centrifugation and exposed them to 5 μM A 680 M (Upper) or A 555 M (Lower) in PBS at pH 6.5, which restricts the labeling reaction to the genetically engineered Cys sulfhydryl in FepA (46,48). raised the possibility that the anisotropy of GFP-TonB may change in response to inhibition of energy metabolism.…”

Section: Resultsmentioning

confidence: 99%

“…Bacteria were grown overnight in LB broth with appropriate antibiotics, subcultured into 3-(N-morpholino)propanesulfonic acid (MOPS) minimal medium with the same antibiotics, and grown for 5.5 h to late exponential phase (46,48). The cells were washed with Tris-buffered saline (TBS), adsorbed to slides coated with poly-L-lysine hydrobromide (8.33 mg/mL) for 15 min, and observed by a Nikon confocal microscope.…”

Section: Methodsmentioning

confidence: 99%

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Energy-dependent motion of TonB in the Gram-negative bacterial inner membrane

Jordan

Zhou

Smallwood

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Gram-negative bacteria acquire iron with TonB-dependent uptake systems. The TonB-ExbBD inner membrane complex is hypothesized to transfer energy to outer membrane (OM) iron transporters. Fluorescence microscopic characterization of green fluorescent protein (GFP)-TonB hybrid proteins revealed an unexpected, restricted localization of TonB in the cell envelope. Fluorescence polarization measurements demonstrated motion of TonB in living cells, which likely was rotation. By determining the anisotropy of GFP-TonB in the absence and presence of inhibitors, we saw the dependence of its motion on electrochemical force and on the actions of ExbBD. We observed higher anisotropy for GFP-TonB in energy-depleted cells and lower values in bacteria lacking ExbBD. However, the metabolic inhibitors did not change the anisotropy of GFP-TonB in ΔexbBD cells. These findings demonstrate that TonB undergoes energized motion in the bacterial cell envelope and that ExbBD couples this activity to the electrochemical gradient. The results portray TonB as an energized entity in a regular array underlying the OM bilayer, which promotes metal uptake through OM transporters by a rotational mechanism.bioenergetics | membrane transport | FepA | iron transport F rom its importance in aerobic metabolism, iron is essential to most pro-and eukaryotes and therefore is a determinant of bacterial disease. Its sequestration by transferrin, lactoferrin, ferritin, heme compounds, and lipocalins defends animal cells, fluids, and tissues by "nutritional immunity" (1). However, efficient pathogens overcome this barrier and capture Fe 3+ either by producing siderophores (2) or by directly removing the metal from eukaryotic proteins (3). The trilaminar cell envelope of Gram-negative bacteria, composed of inner membrane (IM), outer membrane (OM), and the periplasm between them, contains protein components that confer the uptake of metabolic solutes, including sugars, amino acids, nucleotides, vitamins, and metals such as iron (4, 5). Enigmatic OM active transporters acquire metal complexes (ferric siderophores, heme, vitamin B 12 ) from the environment (6). The OM protein ferric enterobactin permease A (FepA), for example, internalizes the siderophore ferric enterobactin (FeEnt) (7). It is typical of many homologous metal transporters in commensal and pathogenic organisms. These uptake reactions also require TonB (8), a cell envelope protein that long ago was proposed to transduce energy (9-12). However, many questions exist about TonB's mediation of iron uptake, including its physical mechanism and its relationship to bioenergetics. Proton motive force (PMF) may drive OM active transport (6-8), but the mode of energy transmission to the OM and TonB's potential role in it are unknown. We addressed these topics by characterizing the localization of TonB in the cell membranes, by monitoring its physical motion, and by determining the dependence of its movements on metabolic energy and the additional IM proteins ExbBD.Iron chelates bind to their OM transporter...

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Energy-dependent motion of TonB in the Gram-negative bacterial inner membrane

Jordan

Zhou

Smallwood

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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“…S3); FepB did not detectably bind to FepA under any conditions. Specific sites within FepA are chemically modifiable during binding and transport of FeEnt (32,43), and chemical modification of the substitution G54C occurs during active FeEnt uptake. Located in the N-domain of FepA, G54C is susceptible to fluoresceination during FeEnt transport (32) but not labeled in tonB or energy-poisoned cells.…”

Section: Pub Determinations Of Om Transportmentioning

confidence: 99%

Direct Measurements of the Outer Membrane Stage of Ferric Enterobactin Transport

Newton

Trinh

et al. 2010

Journal of Biological Chemistry

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When Gram-negative bacteria acquire iron, the metal crosses both the outer membrane (OM) and the inner membrane, but existing radioisotopic uptake assays only measure its passage through the latter bilayer, as the accumulation of the radionuclide in the cytoplasm. We devised a methodology that exclusively observes OM transport and used it to study the uptake of ferric enterobactin (FeEnt) by Escherichia coli FepA. This technique, called postuptake binding, revealed previously unknown aspects of TonB-dependent transport reactions. The experiments showed, for the first time, that despite the discrepancy in cell envelope concentrations of FepA and TonB (ϳ35:1), all FepA proteins were active and equivalent in FeEnt uptake, with a maximum turnover number of ϳ5/min. FepA-mediated transport of FeEnt progressed through three distinct phases with successively decreasing rates, and from its temperature dependence, the activation energy of the OM stage was 33-35 kcal/mol.

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“…Furthermore, both in vivo and in vitro studies have detected significant movement of only roughly the amino-terminal one-third of the cork residues that are accessible from the periplasm. While removing one-third of the cork residues from the barrel would leave an opening sufficient for passage of transport ligands such as siderophores and vitamin B 12 , it may not be sufficient for larger ligands such as the 55-kDa protein toxin colicin B (24,(29)(30)(31)(32).…”

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Going Outside the TonB Box: Identification of Novel FepA-TonB Interactions In Vivo

Gresock

Postle

2017

J Bacteriol

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In Gram-negative bacteria, the cytoplasmic membrane protein TonB transmits energy derived from proton motive force to energize transport of important nutrients through TonB-dependent transporters in the outer membrane. Each transporter consists of a beta barrel domain and a lumen-occluding cork domain containing an essential sequence called the TonB box. To date, the only identified site of transporter-TonB interaction is between the TonB box and residues ϳ158 to 162 of TonB. While the mechanism of ligand transport is a mystery, a current model based on site-directed spin labeling and molecular dynamics simulations is that, following ligand binding, the otherwise-sequestered TonB box extends into the periplasm for recognition by TonB, which mediates transport by pulling or twisting the cork. In this study, we tested that hypothesis with the outer membrane transporter FepA using in vivo photo-cross-linking to explore interactions of its TonB box and determine whether additional FepA-TonB interaction sites exist. We found numerous specific sites of FepA interaction with TonB on the periplasmic face of the FepA cork in addition to the TonB box. Two residues, T32 and A33, might constitute a ligandsensitive conformational switch. The facts that some interactions were enhanced in the absence of ligand and that other interactions did not require the TonB box argued against the current model and suggested that the transport process is more complex than originally conceived, with subtleties that might provide a mechanism for discrimination among ligand-loaded transporters. These results constitute the first study on the dynamics of TonB-gated transporter interaction with TonB in vivo. IMPORTANCE The TonB system of Gram-negative bacteria has a noncanonical active transport mechanism involving signal transduction and proteins integral to both membranes. To achieve transport, the cytoplasmic membrane protein TonB physically contacts outer membrane transporters such as FepA. Only one contact between TonB and outer membrane transporters has been identified to date: the TonB box at the transporter amino terminus. The TonB box has low information content, raising the question of how TonB can discriminate among multiple different TonBdependent transporters present in the bacterium if it is the only means of contact. Here we identified several additional sites through which FepA contacts TonB in vivo, including two neighboring residues that may explain how FepA signals to TonB that ligand has bound.KEYWORDS TonB, FepA, cork domain, p-benzoyl-L-phenylalanine, enterochelin, TonB box, energy transduction I ron is essential for growth of Gram-negative bacteria but can be difficult to acquire because of its insolubility under aerobic conditions, its scarcity in hosts, and the protective nature of the Gram-negative bacterial outer membrane (OM) (1). The majority of Gram-negative bacteria synthesize and secrete siderophores, which are high-

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Fluoresceination of FepA during colicin B killing: effects of temperature, toxin and TonB

Cited by 22 publications

References 59 publications

Energy-dependent motion of TonB in the Gram-negative bacterial inner membrane

Energy-dependent motion of TonB in the Gram-negative bacterial inner membrane

Direct Measurements of the Outer Membrane Stage of Ferric Enterobactin Transport

Going Outside the TonB Box: Identification of Novel FepA-TonB Interactions In Vivo

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