A complex of ExbB, ExbD, andTonB couples cytoplasmic membrane (CM) proton motive force (pmf) to the active transport of large, scarce, or important nutrients across the outer membrane (OM). TonB interacts with OM transporters to enable ligand transport. Several mechanical models and a shuttle model explain how TonB might work. In the mechanical models, TonB remains attached to the CM during energy transduction, while in the shuttle model theTonB N terminus leaves the CM to deliver conformationally stored potential energy to OM transporters. Previous studies suggested thatTonB did not shuttle based on the activity of a GFP-TonB fusion that was anchored in the CM by the GFP moiety. When we recreated the GFP-TonB fusion to extend those studies, in our hands it was proteolytically unstable, giving rise to potentially shuttleable degradation products. Recently, we discovered that a fusion of the Vibrio cholerae ToxR cytoplasmic domain to the N terminus of TonB was proteolytically stable. ToxR-TonB was able to be completely converted into a pro-teinase K-resistant conformation in response to loss of pmf in spheroplasts and exhibited an ability to form a pmf-dependent formaldehyde crosslink to ExbD, both indicators of its location in the CM. Most importantly, ToxR-TonB had the same relative specific activity as wild-type TonB. Taken together, these results provide conclusive evidence thatTonB does not shuttle during energy transduction. We had previously concluded thatTonB shuttles based on the use of an Oregon Green® 488 maleimide probe to assess periplasmic accessibility of N-terminalTonB. Here we show that the probe was permeant to the CM, thus permitting the labeling of theTonB N-terminus. These former results are reinterpreted in the context thatTonB does not shuttle, and suggest the existence of a signal transduction pathway from OM to cytoplasm. © 2011 Gresock, Savenkova, Larsen, Ollis and Postle.
Larsen, Ray A.; Gresock, Michael G.; Savenkova, Marina I.; Ollis, Anne A.; and Postle, Kathleen, "Death of the TonB Shuttle Hypothesis" (2011). Biological Sciences Faculty Publications. 6.
Frontiers in Microbiology
Frontiers Research Foundation