Mutations in the Escherichia coli receptor FepA reveal residues involved in ligand binding and transport

FepA is the Escherichia coli outer membrane receptor for ferric enterobactin, colicin D and colicin B. The transport processes through FepA are energy-dependent, relying on the periplasmic protein TonB to interact with FepA. Through this interaction, TonB tranduces energy derived from the cytoplasmic membrane across the periplasmic space to FepA. In this study, random mutagenesis strategies were used to define residues of FepA important for its function. Both polymerase chain reaction (PCR)-generated random mutations in the N-terminal 180 amino acids of FepA and spontaneous chromosomal fepA mutations were selected by resistance to colicin B. The PCR mutagenesis strategy targeted the N-terminus because it forms a plug inside the FepA barrel that is expected to be involved in ligand binding, ligand transport, and interaction with TonB. We report the characterization of 15 fepA missense mutations that were localized to three regions of the FepA receptor. The first region was a stretch of eight amino acids referred to as the TonB box. The second region included extracellular loops of both the barrel and the plug. A third region formed a cluster near the barrel wall around positions 75 and 126 of the plug. These mutations provide initial insight into the mechanisms of ligand binding and transport through the FepA receptor.     

Studies on colicin B translocation: FepA is gated by TonB

Colicin B is a 55 kDa dumbbell-shaped protein toxin that uses the TonB system (outer membrane transporter, FepA, and three cytoplasmic membrane proteins TonB/ExbB/ExbD) to enter and kill Escherichia coli. FepA is a 22-stranded beta-barrel with its lumen filled by an amino-terminal globular domain containing an N-terminal semiconserved region, known as the TonB box, to which TonB binds. To investigate the mechanism of colicin B translocation across the outer membrane, we engineered cysteine (Cys) substitutions in the globular domain of FepA. Colicin B caused increased exposure to biotin maleimide labelling of all Cys substitutions, but to different degrees, with TonB as well as the FepA TonB box required for all increases. Because of the large increases in exposure for Cys residues from T13 to T51, we conclude that colicin B is translocated through the lumen of FepA, rather than along the lipid-barrel interface or through another protein. Part of the FepA globular domain (residues V91-V142) proved relatively refractory to labelling, indicating either that the relevant Cys residues were sequestered by an unknown protein or that a significant portion of the FepA globular domain remained inside the barrel, requiring concomitant conformational rearrangement of colicin B during its translocation. Unexpectedly, TonB was also required for colicin-induced exposure of the FepA TonB box, suggesting that TonB binds FepA at a different site prior to interaction with the TonB box.     

Phage display reveals multiple contact sites between FhuA, an outer membrane receptor of Escherichia coli, and TonB

The ferric hydroxamate uptake receptor FhuA from Escherichia coli transports siderophores across the outer membrane (OM). TonB-ExbB-ExbD transduces energy from the cytoplasmic membrane to the OM by contacts between TonB and OM receptors that contain the Ton box, a consensus sequence near the N terminus. Although the Ton box is a region of known contact between OM receptors and TonB, our biophysical studies established that TonB binds to FhuA through multiple regions of interaction. Panning of phage-displayed random peptide libraries (Ph.D.-12, Ph.D.-C7C) against TonB identified peptide sequences that specifically interact with TonB. Analyses of these sequences using the Receptor Ligand Contacts (RELIC) suite of programs revealed clusters of multiply aligned peptides that mapped to FhuA. These clusters localized to a continuous periplasm-accessible surface: Ton box/switch helix; cork domain/beta1 strand; and periplasmic turn 8. Guided by such matches, synthetic oligonucleotides corresponding to DNA sequences identical to fhuA were fused to malE; peptides corresponding to the above regions were displayed at the N terminus of E.coli maltose-binding protein (MBP). Purified FhuA peptides fused to MBP bound specifically to TonB by ELISA. Furthermore, they competed with ligand-loaded FhuA for binding to TonB. RELIC also identified clusters of multiply aligned peptides corresponding to the Ton box regions in BtuB, FepA, and FecA; to periplasmic turn 8 in BtuB and FecA; and to periplasmic turns 1 and 2 in FepA. These experimental outcomes identify specific molecular contacts made between TonB and OM receptors that extend beyond the well-characterized Ton box.     

A C-terminal truncation results in high-level expression of the functional photoreceptor sensory rhodopsin I in the archaeon Halobacterium salinarium

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.     

TonB interacts with nonreceptor proteins in the outer membrane of Escherichia coli

The Escherichia coli TonB protein serves to couple the cytoplasmic membrane proton motive force to active transport of iron-siderophore complexes and vitamin B(12) across the outer membrane. Consistent with this role, TonB has been demonstrated to participate in strong interactions with both the cytoplasmic and outer membranes. The cytoplasmic membrane determinants for that interaction have been previously characterized in some detail. Here we begin to examine the nature of TonB interactions with the outer membrane. Although the presence of the siderophore enterochelin (also known as enterobactin) greatly enhanced detectable cross-linking between TonB and the outer membrane receptor, FepA, the absence of enterochelin did not prevent the localization of TonB to the outer membrane. Furthermore, the absence of FepA or indeed of all the iron-responsive outer membrane receptors did not alter this association of TonB with the outer membrane. This suggested that TonB interactions with the outer membrane were not limited to the TonB-dependent outer membrane receptors. Hydrolysis of the murein layer with lysozyme did not alter the distribution of TonB, suggesting that peptidoglycan was not responsible for the outer membrane association of TonB. Conversely, the interaction of TonB with the outer membrane was disrupted by the addition of 4 M NaCl, suggesting that these interactions were proteinaceous. Subsequently, two additional contacts of TonB with the outer membrane proteins Lpp and, putatively, OmpA were identified by in vivo cross-linking. These contacts corresponded to the 43-kDa and part of the 77-kDa TonB-specific complexes described previously. Surprisingly, mutations in these proteins individually did not appear to affect TonB phenotypes. These results suggest that there may be multiple redundant sites where TonB can interact with the outer membrane prior to transducing energy to the outer membrane receptors.     

Point mutations in a conserved region (TonB box) of Escherichia coli outer membrane protein BtuB affect vitamin B12 transport.

Uptake of cobalamins and iron chelates in Escherichia coli K-12 is dependent on specific outer membrane transport proteins and the energy-coupling function provided by the TonB protein. The btuB product is the outer membrane receptor for cobalamins, bacteriophage BF23, and the E colicins. A short sequence near the amino terminus of mature BtuB, previously called the TonB box, is conserved in all tonB-dependent receptors and colicins and is the site of the btuB451 mutation (Leu-8----Pro), which prevents energy-coupled cobalamin uptake. This phenotype is partially suppressed by certain mutations in tonB. To examine the role of individual amino acids in the TonB box of BtuB, more than 30 amino acid substitutions in residues 6 to 13 were generated by doped oligonucleotide-directed mutagenesis. Many of the mutations affecting each amino acid did not impair transport activity, although some substitutions reduced cobalamin uptake and the Leu-8----Pro and Val-10----Gly alleles were completely inactive. To test whether the btuB451 mutation affects only cobalamin transport, a hybrid gene was constructed which encodes the signal sequence and first 39 residues of BtuB fused to the bulk of the ferrienterobactin receptor FepA (residues 26 to 723). This hybrid protein conferred all FepA functions but no BtuB functions. The presence of the btuB451 mutation in this fusion gene eliminated all of its tonB-coupled reactions, showing that the TonB box of FepA could be replaced by that from BtuB. These results suggest that the TonB-box region of BtuB is involved in active transport in a manner dependent not on the identity of specific side chains but on the local secondary structure.     

TonB protein and energy transduction between membranes

TonB protein couples cytoplasmic membrane electrochemical potential to active transport of iron-siderophore complexes and vitamin B12 through high-affinity outer membrane receptors of Gram-negative bacteria. The mechanism of energy transduction remains to be determined, but important concepts have already begun to emerge. Consistent with its function, TonB is anchored in the cytoplasmic membrane by its uncleaved amino terminus while largely occupying the periplasm. Both the connection to the cytoplasmic membrane and the amino acid sequences of the anchor are essential for activity. TonB directly associates with a number of envelope proteins, among them the outer membrane receptors and cytoplasmic membrane protein ExbB. ExbB and TonB interact through their respective transmembrane domains. ExbB is proposed to recycle TonB to an active conformation following energy transduction to the outer membrane. TonB most likely associates with the outer membrane receptors through its carboxy terminus, which is required for function. In contrast, the novel prolinerich region of TonB can be deleted without affecting function. A model that incorporates this information, as well as tempered speculation, is presented.     

Identification of functionally important TonB-ExbD periplasmic domain interactions in vivo

In gram-negative bacteria, the cytoplasmic membrane proton-motive force energizes the active transport of TonB-dependent ligands through outer membrane TonB-gated transporters. In Escherichia coli, cytoplasmic membrane proteins ExbB and ExbD couple the proton-motive force to conformational changes in TonB, which are hypothesized to form the basis of energy transduction through direct contact with the transporters. While the role of ExbB is not well understood, contact between periplasmic domains of TonB and ExbD is required, with the conformational response of TonB to presence or absence of proton motive force being modulated through ExbD. A region (residues 92 to 121) within the ExbD periplasmic domain was previously identified as being important for TonB interaction. Here, the specific sites of periplasmic domain interactions between that region and the TonB carboxy terminus were identified by examining 270 combinations of 45 TonB and 6 ExbD individual cysteine substitutions for disulfide-linked heterodimer formation. ExbD residues A92C, K97C, and T109C interacted with multiple TonB substitutions in four regions of the TonB carboxy terminus. Two regions were on each side of the TonB residues known to interact with the TonB box of TonB-gated transporters, suggesting that ExbD positions TonB for correct interaction at that site. A third region contained a functionally important glycine residue, and the fourth region involved a highly conserved predicted amphipathic helix. Three ExbD substitutions, F103C, L115C, and T121C, were nonreactive with any TonB cysteine substitutions. ExbD D25, a candidate to be on a proton translocation pathway, was important to support efficient TonB-ExbD heterodimerization at these specific regions.     

Characterization of TonB Interactions with the FepA Cork Domain and FecA N-terminal Signaling Domain

The mechanism of TonB dependent siderophore uptake through outer membrane transporters in Gram-negative bacteria is poorly understood. In an effort to expand our knowledge of the interaction between TonB and the outer membrane transporters, we have cloned and expressed the FepA cork domain (11–154) from Salmonella typhimurium and characterized its interaction with the periplasmic C-terminal domain of TonB (103–239) by isotope assisted FTIR and NMR spectroscopy. For comparison we also performed similar experiments using the FecA N-terminal domain (1–96) from Escherichia coli which includes the conserved TonB box. The FepA cork domain was completely unfolded in solution, as observed for the E. coli cork domain previously [Usher et al. (2001) Proc Natl Acad Sci USA 98, 10676–10681]. The FepA cork domain was found to bind to TonB, eliciting essentially the same chemical shift changes in TonB C-terminal domain as was observed in the presence of TonB box peptides. The FecA construct did not cause this same structural change in TonB. The binding of the FepA cork domain to TonB-CTD was found to decrease the amount of ordered secondary structure in TonB-CTD. It is likely that the FecA N-terminal domain interferes with TonB-CTD binding to the TonB box. Binding of the FepA cork domain induces a loss of secondary structure in TonB, possibly exposing TonB surface area for additional intermolecular interactions such as potential homodimerization or additional interactions with the barrel of the outer membrane transporter.     

Role of the TonB amino terminus in energy transduction between membranes.

Escherichia coli TonB protein is an energy transducer, coupling cytoplasmic membrane energy to active transport of vitamin B12 and iron-siderophores across the outer membrane. TonB is anchored in the cytoplasmic membrane by its hydrophobic amino terminus, with the remainder occupying the periplasmic space. In this report we establish several functions for the hydrophobic amino terminus of TonB. A G-26-->D substitution in the amino terminus prevents export of TonB, suggesting that the amino terminus contains an export signal for proper localization of TonB within the cell envelope. Substitution of the first membrane-spanning domain of the cytoplasmic membrane protein TetA for the TonB amino terminus eliminates TonB activity without altering TonB export, suggesting that the amino terminus contains sequence-specific information. Detectable TonB cross-linking to ExbB is also prevented, suggesting that the two proteins interact primarily through their transmembrane domains. In vivo cleavage of the amino terminus of TonB carrying an engineered leader peptidase cleavage site eliminates (i) TonB activity, (ii) detectable interaction with a membrane fraction having a density intermediate to those of the cytoplasmic and outer membranes, and (iii) cross-linking to ExbB. In contrast, the amino terminus is not required for cross-linking to other proteins with which TonB can form complexes, including FepA. Additionally, although the amino terminus clearly is a membrane anchor, it is not the only means by which TonB associates with the cytoplasmic membrane. TonB lacking its amino-terminal membrane anchor still remains largely associated with the cytoplasmic membrane.     

Quantification of known components of the Escherichia coli TonB energy transduction system: TonB, ExbB, ExbD and FepA

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.     

In vivo evidence of TonB shuttling between the cytoplasmic and outer membrane in Escherichia coli

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(R) 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.     

Nucleotide sequence of the gene for the ferrienterochelin receptor FepA in Escherichia coli. Homology among outer membrane receptors that interact with TonB

We have determined the nucleotide sequence of the Escherichia coli fepA gene, which codes for the outer membrane receptor for ferrienterochelin and colicins B and D. The predicted FepA polypeptide has a molecular weight of 79,908 and consists of 723 amino acids. A 22-amino acid leader or signal peptide preceded the mature protein. With respect to overall composition, hydropathy, net charge and distribution of nonpolar segments, the FepA polypeptide was typical of other E. coli outer membrane proteins, except that FepA contained 2 cysteine residues. Comparison of the deduced amino acid sequence of FepA with that of three other TonB-dependent receptors (BtuB, FhuA, and IutA) revealed only a few regions of sequence homology; one of these included the amino-termini. An amino acid substitution within the conserved amino-terminal region of BtuB resulted in production of a receptor that had normal binding functions but was incapable of energy-dependent transport of vitamin B12. This result suggests that the amino-terminal end of these four polypeptides is involved in interaction with the TonB protein or another step of energy transduction. Three other regions of homology were shared among the four proteins: one near residues 50 to 70, another at about residue 100 to 140, and the last between 20 and 40 amino acid residues from the carboxyl terminus. The function of these three regions remains speculative.     

A beta strand lock exchange for signal transduction in TonB-dependent transducers on the basis of a common structural motif

Transport of molecules larger than 600 Da across the outer membrane involves TonB-dependent receptors and TonB-ExbB-ExbD of the inner membrane. The transport is energy consuming, and involves direct interactions between a short N-terminal sequence of receptor, called the TonB box, and TonB. We solved the structure of the ferric pyoverdine (Pvd-Fe) outer membrane receptor FpvA from Pseudomonas aeruginosa in its apo form. Structure analyses show that residues of the TonB box are in a beta strand which interacts through a mixed four-stranded beta sheet with the periplasmic signaling domain involved in interactions with an inner membrane sigma regulator. In this conformation, the TonB box cannot form a four-stranded beta sheet with TonB. The FhuA-TonB or BtuB-TonB structures show that the TonB-FpvA interactions require a conformational change which involves a beta strand lock-exchange mechanism. This mechanism is compatible with movements of the periplasmic domain deduced from crystallographic analyses of FpvA, FpvA-Pvd, and FpvA-Pvd-Fe.     

Transport of iron across the outer membrane

Summary The TonB protein is involved in energy-coupled receptor-dependent transport processes across the outer membrane. The TonB protein is anchored in the cytoplasmic membrane but exposed to the periplasmic space. To fulfill its function, it has to couple the energy-providing metabolism in the cytoplasmic membrane with regulation of outer membrane receptor activity. Ferrichrome and albomycin transport, uptake of colicin M, and infection by the phages T1 and80 occur via the same receptor, the FhuA protein in the outer membrane. Therefore, this receptor is particularly suitable for the study of energy-coupled TonB-dependent transport across the outer membrane. Ferrichrome, albomycin and colicin M bind to the FhuA receptor but are not released into the periplasmic space of unenergized cells, ortonB mutants. In vivo interaction between FhuA and TonB is suggested by the restoration of activity of inactive FhuA proteins, bearing amino acid replacements in the TonB box, by TonB derivatives with single amino acid substitutions. Point mutations in thefhuA gene are suppressed by point mutations in thetonB gene. In addition, naturally occurring degradation of the TonB protein and its derivatives is preferentially prevented in vivo by FhuA and FhuA derivatives where functional interaction takes place. It is proposed that in the energized state, TonB induces a conformation in FhuA which leads to the release of the FhuA-bound compounds into the periplasmic space. Activation of FhuA by TonB depends on the ExbBD proteins in the cytoplasmic membrane. They can be partially replaced by the TolQR proteins which show strong sequence similarity to the ExbBD proteins. A physical interaction of these proteins with the TonB protein is suggested by TonB stabilization through ExbB and TolQR. We propose a permanent or reversible complex in the cytoplasmic membrane composed of the TonB protein and the ExbBD/TolQR proteins through which TonB is energized.     

Energy-dependent changes in the gonococcal transferrin receptor

The pathogenic Neisseria spp. are capable of iron utilization from host iron-binding proteins including transferrin and lactoferrin. Transferrin iron utilization is an energy-dependent, receptor-mediated event in which two identified transferrin-binding proteins participate. One of these proteins, TbpA, is homologous to the TonB-dependent family of outer membrane receptors that are required for high-affinity uptake of vitamin B₁₂ and ferric siderophores. The 'TonB box' is a conserved domain near the amino-terminus of these proteins that has been implicated in interaction with TonB. Interaction between a periplasmic domain of TonB and the TonB box allows energy transduction to occur from the cytoplasmic membrane to the energy-dependent receptor in the outer membrane. We created a TonB box mutant of gonococcal TbpA and demonstrated that its binding and protease accessibility characteristics were indistinguishable from those of gonococcal Ton system mutants. The protease exposure of the second transferrin-binding protein, TbpB, was affected by the energization of TbpA, consistent with an interaction between these proteins. TbpB expressed by the de-energized mutants was readily accessible to protease, similar to TbpB expressed in the absence of TbpA. The de-energized mutants exhibited a marked decrease in transferrin diffusion rate, suggesting that receptor energization was necessary for ligand release. We propose a model to explain the observed Ton-dependent changes in the binding parameters and exposures of TbpA and TbpB.     

Import of the transfer RNase colicin D requires site-specific interaction with the energy-transducing protein TonB

The transfer RNase colicin D and ionophoric colicin B appropriate the outer membrane iron siderophore receptor FepA and share a common translocation requirement for the TonB pathway to cross the outer membrane. Despite the almost identical sequences of the N-terminal domains required for the translocation of colicins D and B, two spontaneous tonB mutations (Arg158Ser and Pro161Leu) completely abolished colicin D toxicity but did not affect either the sensitivity to other colicins or the FepA-dependent siderophore uptake capacity. The sensitivity to colicin D of both tonB mutants was fully restored by specific suppressor mutations in the TonB box of colicin D, at Ser18(Thr) and Met19(Ile), respectively. This demonstrates that the interaction of colicin D with TonB is critically dependent on certain residues close to position 160 in TonB and on the side chains of certain residues in the TonB box of colicin D. The effect of introducing the TonB boxes from other TonB-dependent receptors and colicins into colicins D and B was studied. The results of these and other changes in the two TonB boxes show that the role of residues at positions 18 and 19 in colicin D is strongly modulated by other nearby and/or distant residues and that the overall function of colicin D is much more dependent on the interaction with TonB involving the TonB box than is the function of colicin B.     

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 and vitamin B12 across the outer membrane in Gram-negative bacteria is the cytoplasmic membrane proton-motive force (pmf). TonB protein is required in this process to transduce cytoplasmic membrane energy to the outer membrane. In this study, Escherichia coli TonB 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-terminus of TonB nor the cytoplasmic membrane pmf, both of which are essential for TonB activity, were required for TonB to associate with the outer membrane. When the TonB C-terminus was absent, TonB was found associated with the cytoplasmic membrane, suggesting that the C-terminus was required for outer membrane association. When ExbB and ExbD, as well as their cross-talk-competent homologues TolQ and TolR, were absent, TonB was found associated with the outer membrane. TetA–TonB protein, which cannot interact with ExbB/D, was likewise found associated with the outer membrane. These results indicated that the role of ExbB/D in energy transduction is to bring TonB that has reached the outer membrane back to associate with the cytoplasmic membrane. Two possible explanations exist for the observations presented in this study. One possibility is that TonB transduces energy by shuttling between membranes, and, at some stages in the energy-transduction cycle, is associated with either the cytoplasmic membrane or the outer membrane, but not with both at the same time. This hypothesis, together with the alternative interpretation that TonB remains localized in the cytoplasmic membrane and changes its affinity for the outer and cytoplasmic membrane during energy transduction, are incorporated with previous observations into two new models, consistent with the novel aspects of this system, that describe a mechanism for TonB-dependent energy transduction.