Transport of ferric siderophores across the outer membrane derives energy from the inner membrane protonmotive force. This requires an energy-transducing TonB complex in the inner membrane (blue), consisting of TonB, ExbB and ExbD proteins. TonB interacts with outer membrane transporters (TBDT) at the TonB-box motif. Transport of ferric siderophores across the inner membrane requires a periplasmic binding protein and an ABC transporter. Once the ferric siderophore enters the cytoplasm, ferric ion (Fe³⁺) is reduced to ferrous ion (Fe²⁺), which is destined for storage or incorporation into enzymes. Excess Fe²⁺ (which could induce the formation of radicals harmful to the cell) binds to the repressor protein Fur, which in turn binds target promoters (P_fur) and inhibits transcription of siderophore transport genes. Some TBDTs, such as E. coli FecA are additionally regulated by σ/anti-σ factor systems. In addition to transporting diferric dicitrate, FecA regulates the expression of fecABCDE transport genes initiated by the binding of ferric citrate to FecA. This involves the N-terminal extension of FecA (green), the inner membrane regulator FecR (σ regulator, pink), and the cytoplasmic sigma factor FecI (ECF σ factor, pink). Both transport and induction require energy transduction from the TonB-ExbB-ExbD complex in the inner membrane.

TBDTs have an N-terminal plug domain that sits inside a C-terminal 22-stranded β-barrel domain. The conserved TonB box is found near the N-terminus of the plug domain facing the periplasm and is generally thought to remain sequestered inside the β-barrel domain in the absence of ligand. Upon binding ligand, a conformational change leads to exposure of the TonB box and subsequent interaction with TonB and siderophore transport. Panel a represents the FhuA-ferrichrome crystal structure (1BY5) with FhuA shown in ribbon and ferrichrome in spacefill model, panel b represents only the beta-barrel domain, panel c represents only the plug domain, and panel d shows the FhuA apo structure (1BY3) with those residues with at least 50% conservation highlighted in blue. Top view represents the extracellular view, side view represents the membrane view, and bottom represents the periplasmic view. The TonB box was found disordered in both structures and is represented by dashed lines.

It is generally accepted that the plug domain of TBDTs must undergo some form of conformational change to facilitate sideophore transport. In panel a, studies with FepA have shown that engineered cysteines (S46C/G54C) within the plug domain (indicated by blue spheres) become labeled by periplasmic thio-reactive reagents only during transport of ligand. Other studies have shown that engineered disulfides which tether the plug domain to the inner face of the barrel domain (indicated by yellow spheres) in both FepA (I14C/G300C) and in FhuA (panel b, C27/C533, L109C/S356C and Q112C/M383C) significantly reduce or eliminate sideophore transport. Together, these studies provide evidence that partial or full ejection of the plug domain from the β-barrel may be required for siderophore and/or colicin transport.