Restricted Mobility Of Tonb And Fepa In E. Coli Membranes

Nutrient uptake in Escherichia coli requires transport across the cell's lipopolysaccharide-rich outer membrane, passage through the peptidoglycan layer containing periplasmic space and finally transport across the cell's inner cytoplasmic membrane. In many cases, the first step in this process involves transit through a class of beta-barrel proteins in the outer membrane known as TonB-dependent transporters (TBDTs). This uptake requires interaction between the TBDTs and the cytoplasmic membrane protein, TonB. TonB, in complex with ExbB and ExbD, is thought to derive energy from proton-motive force of the cytoplasmic membrane to be transduced to the outer membrane TBDTs for transport. Here, we have investigated the mobility of TonB in the inner membrane and FepA, a TBDT responsible for transport of the iron chelating siderophore enterobactin (FeEnt), in the outer membrane of E. coli at the single molecule level. We find that FepA is located throughout the outer membrane while TonB is found excluded from the inner membrane's polar regions. Both proteins are observed to undergo free diffusion in the absence of FeEnt within confining domains in their respective membranes (a domain radius of 130 nm for FepA and 240 nm for TonB). Effects of depolarization of the inner membrane, deletion of ExbB/D and presence of FeEnt will be discussed in term of interactions between the proteins.