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

Abstract

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...

Structural Insight into the Role of the Ton Complex in Energy Transduction

“…The Tol complex is required for cell envelope integrity 23,24 and to maintain cellular structure during cell division 25 . And similar to TonB for the Ton system, energy dependent conformational changes have been demonstrated for TolA 20,26,27 . The Ton complex is also evolutionarily related to the Mot complex, which drives bacterial flagellar motion 22,[28][29][30] To better understand the role of the Ton complex in energy transduction to the OM, we solved crystal structures of the E. coli Ton subcomplex.…”

“…The exact stoichiometry of components of the Ton complex has been a matter of debate for years [16][17][18][19] . Evidence favoring a dynamic mechanism has been reported in which fluorescence anisotropy studies showed that the presence of TonB within the Ton complex sustains a rotational motion dependent on the pmf at the IM 20 .…”

“…The Ton complex relies on the pmf for its function 27,33 and it has been proposed that the Ton complex acts as a proton-conducting channel that shuttles protons from the periplasm to the cytoplasm and this powers a mechanical motion within the complex 20 . Mutagenesis studies have previously identified a number of residues that are necessary for harnessing the pmf, including D25 of ExbD and T148 and T181 of ExbB 37,38 .…”

New insights into iron acquisition by cyanobacteria: an essential role for ExbB-ExbD complex in inorganic iron uptake

“…Over the past three decades, many aspects of this TonB-ExbB-ExbD-dependent transport system have been revealed. The crystal structures of several OM transporters and their complexes with TonB are now known (Ferguson et al, 1998(Ferguson et al, , 2002Buchanan et al, 1999;Ferguson and Deisenhofer, 2004;Pawelek et al, 2006;Shultis et al, 2006;Krieg et al, 2009), the signal transduction of OM transporters by interaction with TonB has been elucidated (Ferguson et al, 2007;Kim et al, 2007) and the rotational mechanism of TonB motion has been reported (Jordan et al, 2013). However, with regard to the substrates of the transport system, we are probably only seeing the 'tip of the iceberg' (Schauer et al, 2008).…”

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

“…3 The detailed mechanism of FeEnt transport through FepA remains uncertain, but the process is controlled by TonB. 4 Active transport across the OM poses an energetic challenge: How does accumulation occur in a membrane containing open porin channels (e.g., OmpF) that preclude an electrochemical gradient? TonB presumably overcomes this problem by transferring energy from the inner membrane (IM) to the OM, which facilitates FeEnt passage through FepA.…”