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Notes: The TonB-dependent energy transduction system couples cytoplasmic membrane proton motive force to active transport of iron–siderophore complexes across the outer membrane in Gram-negative bacteria. In Escherichia coli, the primary players known in this process to date are: FepA, the TonB-gated transporter for the siderophore enterochelin; TonB, the energy-transducing protein; and two cytoplasmic membrane proteins with less defined roles, ExbB and ExbD. In this study, we report the per cell numbers of TonB, ExbB, ExbD and FepA for cells grown under iron-replete and iron-limited conditions. Under iron-replete conditions, TonB and FepA were present at 335 ± 78 and 504 ± 165 copies per cell respectively. ExbB and ExbD, despite being encoded from the same operon, were not equimolar, being present at 2463 ± 522 and 741 ± 105 copies respectively. The ratio of these proteins was calculated at one TonB:two ExbD:seven ExbB under all four growth conditions tested. In contrast, the TonB:FepA ratio varied with iron status and according to the method used for iron limitation. Differences in the method of iron limitation also resulted in significant differences in cell size, skewing the per cell copy numbers for all proteins.

Notes: TonB protein functions as an energy transducer, coupling cytoplasmic membrane electrochemical potential to the active transport of vitamin B12 and Fe(III)–siderophore complexes across the outer membrane of Escherichia coli and other Gram-negative bacteria. Accumulated evidence indicates that TonB is anchored in the cytoplasm, but spans the periplasmic space to interact physically with outer membrane receptors. It has been presumed that this ability is caused by a conserved (Glu–Pro)n–(Lys–Pro)m repeat motif, predicted to assume a rigid, linear conformation of sufficient length to reach the outer membrane. Based on in vitro studies with synthetic peptides and purified FhuA outer membrane receptor, it has been suggested that this region contains a site that directly binds outer membrane receptors and is essential for energy transduction. We have found a TonB lacking the (Glu–Pro)n–(Lys–Pro)m, repeat motif (TonBΔ(66–100)). TonBΔ(66–100) is fully capable of irreversible 80 adsorption, except under physiological circumstances where the periplasmic space is expanded. Based on the ability of TonBΔ(66–100) to interact with outer membrane receptors and components of the energy transduction apparatus under normal physiological conditions, it is evident that the TonB proline-rich region has no role in energy transduction other than to provide a physical extension sufficient to reach the outer membrane.

Notes: Active transport of vitamin B12 and Fe(III)-siderophore complexes across the outer membrane of Escherichia coli appears to be dependent upon the ability of the TonB protein to couple cytoplasmic membrane-generated protonmotive force to outer membrane receptors. TonB is supported in this role by an auxiliary protein, ExbB, which, in addition to stabilizing TonB against the activities of endogenous envelope proteases, directly contributes to the energy transduction process. The topological partitioning of TonB and ExbB to either side of the cytoplasmic membrane restricts the sites of interaction between these proteins primarily to their transmembrane domains. In this study, deletion of valine 17 within the amino-terminal transmembrane anchor of TonB resulted in complete loss of TonB activity, as well as loss of detectable in vivo crosslinking into a 59 kDa complex believed to contain ExbB. The ΔV17 mutation had no effect on TonB export. The loss of crosslinking appeared to reflect conformational changes in the TonB/ExbB pair rather than loss of interaction since ExbB was still required for some stabilization of TonBΔV17. Molecular modeling suggested that the ΔV17 mutation caused a significant change in the predicted conserved face of the TonB amino-terminal membrane anchor. TonBΔV17 was unable to achieve the 23 kDa proteinase K-resistant form in lysed sphaeroplasts that is characteristic of active TonB. Wild-type TonB also failed to achieve the proteinase K-resistant configuration when ExbB was absent. Taken together these results suggested that the ΔV17 mutation interrupted productive TonB–ExbB interactions. The apparent ability to crosslink to ExbB as well as a limited ability to transduce energy were restored by a second mutation (A39E) in or near the first predicted transmembrane domain of the ExbB protein. Consistent with the weak suppression, a 23 kDa proteinase K-resistant form of TonBΔV17 was not observed in the presence of ExbBA39E. Neither the ExbBA39E allele nor the absence of ExbB affected TonB or TonBΔV17 export. Unlike the tonBΔV17 mutation, the exbBA39E mutation did not greatly alter a modelled ExbB transmembrane domain structure. Furthermore, the suppressor ExbBA39E functioned normally with wild-type TonB, suggesting that the suppressor was not allele specific. Contrary to expectations, the TonBδV17, ExbBA39E pair resulted in a TonB with a greatly reduced half-life (≅ 10 min). These results together with protease susceptibility studies suggest that ExbB functions by modulating the conformation of TonB.

Notes: TonB couples the cytoplasmic membrane protonmotive force (pmf) to active transport across the outer membrane, potentially through a series of conformational changes. Previous studies of a TonB transmembrane domain mutant (TonB-ΔV17) and its phenotypical suppressor (ExbB-A39E) suggested that TonB is conformationally sensitive. Here, two new mutations of the conserved TonB transmembrane domain SHLS motif were isolated, TonB-S16L and -H20Y, as were two new suppressors, ExbB-V35E and -V36D. Each suppressor ExbB restored at least partial function to the TonB mutants, although TonB-ΔV17, for which both the conserved motif and the register of the predicted transmembrane domain α-helix are affected, was the most refractory. As demonstrated previously, TonB can undergo at least one conformational change, provided both ExbB and a functional TonB transmembrane domain are present. Here, we show that this conformational change reflects the ability of TonB to respond to the cytoplasmic membrane proton gradient, and occurs in proportion to the level of TonB activity attained by mutant–suppressor pairs. The phenotype of TonB-ΔV17 was more complex than the -S16L and -H20Y mutations, in that, beyond the inability to be energized efficiently, it was also conditionally unstable. This second defect was evident only after suppression by the ExbB mutants, which allow transmembrane domain mutants to be energized, and presented as the rapid turnover of TonB-ΔV17. Importantly, this degradation was dependent upon the presence of a TonB-dependent ligand, suggesting that TonB conformation also changes following the energy transduction event. Together, these observations support a dynamic model of energy transduction in which TonB cycles through a set of conformations that differ in potential energy, with a transition to a higher energy state driven by pmf and a transition to a lower energy state accompanying release of stored potential energy to an outer membrane receptor.

Notes: Gram-negative bacteria are able to convert potential energy inherent in the proton gradient of the cytoplasmic membrane into active nutrient transport across the outer membrane. The transduction of energy is mediated by TonB protein. Previous studies suggest a model in which TonB makes sequential and cyclic contact with proteins in each membrane, a process called shuttling. A key feature of shuttling is that the amino-terminal signal anchor must quit its association with the cytoplasmic membrane, and TonB becomes associated solely with the outer membrane. However, the initial studies did not exclude the possibility that TonB was artifactually pulled from the cytoplasmic membrane by the fractionation process. To resolve this ambiguity, we devised a method to test whether the extreme TonB amino-terminus, located in the cytoplasm, ever became accessible to the cys-specific, cytoplasmic membrane-impermeant molecule, Oregon Green® 488 maleimide (OGM) in vivo. A full-length TonB and a truncated TonB were modified to carry a sole cysteine at position 3. Both full-length TonB and truncated TonB (consisting of the amino-terminal two-thirds) achieved identical conformations in the cytoplasmic membrane, as determined by their abilities to cross-link to the cytoplasmic membrane protein ExbB and their abilities to respond conformationally to the presence or absence of proton motive force. Full-length TonB could be amino-terminally labelled in vivo, suggesting that it was periplasmically exposed. In contrast, truncated TonB, which did not associate with the outer membrane, was not specifically labelled in vivo. The truncated TonB also acted as a control for leakage of OGM across the cytoplasmic membrane. Further, the extent of labelling for full-length TonB correlated roughly with the proportion of TonB found at the outer membrane. These findings suggest that TonB does indeed disengage from the cytoplasmic membrane during energy transduction and shuttle to the outer membrane.