Reconstitution of bacterial outer membrane TonB-dependent transporters in planar lipid bilayer membranes

Abstract

Micronutrients such as siderophore-bound iron and vitamin B12 cross the outer membrane of Gram-negative bacteria through a group of 22-stranded ␤-barrel proteins. They share the unusual feature that their N-terminal end inserts from the periplasmic side into the ␤-barrel and plugs the lumen. Transport results from energy-driven movement of TonB protein, which either pulls the plug out of the barrel or causes it to rearrange within the barrel. Attempts to reconstitute native plugged channels in an ionconducting state in lipid bilayer membranes have so far been unsuccessful. We, however, have discovered that if the cis solution contained 4 M urea, then, with the periplasmic side of the channel facing that solution, macroscopic conductances and single channel events could be observed. These results were obtained with FhuA, Cir, and BtuB; for the former two, the channels were closed by removing the 4 M urea. Channels generated by 4 M urea exposure were not a consequence of general protein denaturation, as their ligand-binding properties were preserved. Thus, with FhuA, addition of ferrichrome (its siderophore) to the trans, extracellularfacing side reversibly inhibited 4 M urea-induced channel opening and blocked the channels. With Cir, addition of colicin Ia (the microbial toxin that targets Cir) to the trans, extracellular-facing side prevented 4 M urea-induced channel opening. We hypothesize that 4 M urea reversibly unfolds the FhuA and Cir plugs, thereby opening an ion-conducting pathway through these channels, and that this mimics to some extent the in vivo action of TonB on these plugs.BtuB ͉ Cir ͉ FhuA ͉ urea-denaturation

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

“…The amino-terminal residues from position 28 to 51 proved to be the most impervious to pBpa substitution and supported 55 Fe-enterochelin transport at between 65 and 100% of the pFepA (pKP1693) initial uptake rate, indicating that they retained essentially wild-type function ( Fig. 2A).…”

“…However, several substitutions within FepA residues 63 to 145 (Fig. 2B) supported significantly decreased 55 Fe-enterochelin uptake rates. The region of the FepA cork from S63 to I145 was fairly sensitive to pBpa substitutions, with over half unable to support transport of 55 Fe-enterochelin at greater than ϳ35% of wild-type rates.…”

“…The region of the FepA cork from S63 to I145 was fairly sensitive to pBpa substitutions, with over half unable to support transport of 55 Fe-enterochelin at greater than ϳ35% of wild-type rates. In fact, R75pBpa, T82pBpa, and V142pBpa were completely unable to transport 55 Fe-enterochelin (Fig. 2B).…”

“…As is commonly seen at the fifth position in the TonB box, FepA V16pBpa was largely inactive. Wild-type, His-tagged FepA expressed from pKP1693 supported full 55 Fe-enterochelin uptake compared to chromosomal FepA and served as the standard for 100% uptake.…”

“…Strain KP1270 (W3110 aroB) transformed with pEVOL-BpF (cFepA/pEVOL) and ΔFepA/pEVOL served as uptake controls. Initial rates of 55 Fe-enterochelin transport were determined as described in Materials and Methods. At an A 550 of 0.20 (early exponential phase), all samples were treated with 0.4 mM pBpa, and expression of pBpa substitutions was induced by the addition of sodium propionate.…”