TONB PROTEIN APPEARS TO TRANSDUCE ENERGY BY SHUTTLING BETWEEN THE CYTOPLASMIC MEMBRANE AND THE OUTER-MEMBRANE IN ESCHERICHIA-COLI

The energy source for active transport of iron-siderophore complexes a nd vitamin B12 across the outer membrane in Gram-negative bacteria is the cytoplasmic membrane proton-motive force (pmf). Tone protein is re quired in this process to transduce cytoplasmic membrane energy to the outer membrane, In this study, Escherichia coli Tone was found to be distributed in sucrose density gradients approximately equally between the cytoplasmic membrane and the outer membrane fractions, while two proteins with which it is known to interact, ExbB and ExbD, as well as the NADH oxidase activity characteristic of the cytoplasmic membrane, were localized in the cytoplasmic membrane fraction. Neither the N-te rminus of TonB nor the cytoplasmic membrane pmf, both of which are ess ential for Tone activity, were required for Tone to associate with the outer membrane, When the Tone C-terminus was absent, TonB was found a ssociated with the cytoplasmic membrane, suggesting that the C-terminu s was required for outer membrane association. When ExbB and ExbD, as well as their cross-talk-competent homologues TolQ and TolR, were abse nt, Tone was found associated with the outer membrane. TetA-TonB prote in, which cannot interact with ExbB/D, was likewise found associated w ith the outer membrane. These results indicated that the role of ExbB/ D in energy transduction is to bring Tone that has reached the outer m embrane back to associate with the cytoplasmic membrane. Two possible explanations exist for the observations presented in this study. One p ossibility is that TonB transduces energy by shuttling between membran es, and, at some stages in the energy-transduction cycle, is associate d with either the cytoplasmic membrane or the outer membrane, but not with both at the same time. This hypothesis, together with the alterna tive interpretation that TonB remains localized in the cytoplasmic mem brane and changes its affinity for the outer and cytoplasmic membrane during energy transduction, are incorporated with previous observation s into two new models, consistent with the novel aspects of this syste m, that describe a mechanism for TonB-dependent energy transduction.