TonB, a cytoplasmic membrane protein, couples cytoplasmic membrane protonmotive force to active transport across the outer membrane of Escherichia coli. In vivo cross-linking studies were initiated to analyze TonB interactions with other cell envelope proteins. Four TonB-specific cross-linked complexes were detected with apparent molecular masses of 195, 77, 59, and 43.5 kDa. The 195-kDa complex was shown to contain both TonB and FepA, the outer membrane receptor for the siderophore enterochelin. The 195-kDa complex is absent in strains missing either TonB or FepA and can be detected by either TonB-specific or FepA-specific monoclonal antibodies. This is the first direct in vivo evidence that TonB can span the periplasmic space to interact physically with outer membrane receptors. Consistent with that observation, the outer membrane protease OmpT was shown to play a role in TonB turnover, both in the presence and absence of ExbB results in the rapid degradation of TonB. The absence of OmpT could be used to stabilize TonB in an exbB::Tn10 strain such that steady state levels of TonB protein are identical to a wild-type strain. Under those conditions, the absence of ExbB results in greatly reduced TonB activity, indicating that ExbB plays a direct role in energy transduction and probably secondarily protects TonB protein from proteolysis. The 59-kDa complex was absent in an exbB::Tn10 strain, suggesting either that ExbB is in the complex with TonB or that ExbB is required to form the 59-kDa complex. A tolQ nonsense mutation had no effect on the cross-linking profile observed, confirming that its participation in TonB-dependent phenomena is minor and most likely the result of evolutionary cross-talk.
Summary 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 β‐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.
ABSTRACT The TonB system of Escherichia coli resolves the dilemma posed by its outer membrane that protects it from a variety of external threats, but also constitutes a diffusion barrier to nutrient uptake. Our working model involves interactions among a set of cytoplasmic membrane-bound proteins: tetrameric ExbB that serves as a scaffold for a dimeric TonB complex (ExbB 4 -TonB 2 ), and also engages dimeric ExbD (ExbB 4 -ExbD 2 ). Through a set of synchronized conformational changes and movements these complexes are proposed to cyclically transduce cytoplasmic membrane protonmotive force to energize active transport of nutrients through TonB-dependent transporters in the outer membrane (described in Gresock et al. , J. Bacteriol. 197:3433). In this work, we provide experimental validation of three important aspects of the model. The majority of ExbB is exposed to the cytoplasm, with an ∼90-residue cytoplasmic loop and an ∼50 residue carboxy terminal tail. Here we found for the first time, that the cytoplasmic regions of ExbB served as in vivo contacts for three heretofore undiscovered proteins, candidates to move ExbB complexes within the membrane. Support for the model also came from visualization of in vivo PMF-dependent conformational transitions in ExbD. Finally, we also show that TonB forms homodimers and heterodimers with ExbD through its transmembrane domain in vivo . This trio of in vivo observations suggest how and why solved in vitro structures of ExbB and ExbD differ significantly from the in vivo results and submit that future inclusion of the unknown ExbB-binding proteins may bring solved structures into congruence with proposed in vivo energy transduction cycle intermediates.
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.
The TonB system of Gram-negative bacteria uses the proton motive force (PMF) of the cytoplasmic membrane to energize active transport of nutrients across the outer membrane. The single transmembrane domain (TMD) anchor of TonB, the energy transducer, is essential. Within that TMD, His20 is the only TMD residue that is unable to withstand alanine replacement without a loss of activity. H20 is required for a PMF-dependent conformational change, suggesting that the importance of H20 lies in its ability to be reversibly protonated and deprotonated. Here all possible residues were substituted at position 20 (H20X substitutions). The His residue was also relocated throughout the TonB TMD. Surprisingly, Asn, a structurally similar but nonprotonatable residue, supported full activity at position 20; H20S was very weakly active. All the remaining substitutions, including H20K, H20R, H20E, and H20D, the obvious candidates to mimic a protonated state or support proton translocation, were inactive. A second-site suppressor, ExbB(A39E), indiscriminately reactivated the majority of H20 substitutions and relocations, including H20V, which cannot be made protonatable. These results suggested that the TonB TMD was not on a proton conductance pathway and thus only indirectly responds to PMF, probably via ExbD.
Summary Although iron is an essential nutrient, its toxicity at high levels necessitates regulated transport. In Gram‐negative bacteria a central target for regulation is the TonB protein, an energy transducer that couples the cytoplasmic membrane proton motive force to active transport of (Fe III )‐siderophore complexes across the outer membrane. We have previously demonstrated the threefold repression of tonB transcription by excess iron in the presence of Fur repressor protein under aerobic conditions. In this report, we examine tonB regulation under anaerobic conditions where the solubility of iron is not a limiting factor and, presumably, siderophore‐mediated transport is not required. Under these conditions, tonB transcription is repressed at least 10‐foid by excess iron in the presence of Fur, but can be fully derepressed in the absence of Fur. Based on several lines of evidence, this anaerobic repression is not due to increased negative supercoiling as previously postulated. Our results rule out both supercoiling mediated decreased promoter function and increased Fur binding as mediators of anaerobic repression. Under iron‐limiting anaerobic conditions tonB expression is as high or higher than under iron‐limiting aerobic conditions, suggesting that promoter function has not decreased anaerobically. Furthermore, under anaerobic conditions in tonB + strains, tonB promoter function is insensitive to the gyrase inhibitor novobiocin and to changes in medium osmolarity and temperature, three conditions known to change levels of supercoiling. We also rule out effects of mutations in arcA or fnr as mediators of anaerobic repression. Results from in vivo dimethyl sulphate protection foot‐printing indicate that Fur binds to an operator site between the ‐10 and ‐35 regions of the promoter but not to a less homologous operator site centered at +26. The binding is, if anything, weaker under anaerobic conditions, indicating that anaerobic repression is not mediated through Fur. Additional changes in the in vivo footprint upstream from the promoter implicate a second factor in tonB anaerobic repression. Together, these results suggest that the mechanism responsible for this regulation (and, by analogy, that of other anaerobically repressed, iron‐regulated genes such as cir, exbB, and fhuA ) is a novel one.
ABSTRACT The TonB system of Escherichia coli uses the cytoplasmic membrane protonmotive force (PMF) to energize active transport of nutrients across the otherwise unenergized outer membrane. Because it overcomes limitations for nutrient diffusion through outer membrane size-limiting porins, it provides a growth advantage and is widespread among Gram-negative bacteria. It consists of three known cytoplasmic membrane proteins, TonB, ExbB and ExbD that energize a variety of customized TonB-dependent transporters in the outer membrane. The sole ExbD transmembrane domain is proposed to consist of residues 23-43 (Kampfenkel and Braun, 1992, J. Bacteriol. 174:5485-7). Here we showed that the charge and location of residue Asp25 were essential for activity of the TonB system, thus identifying it as the only PMF-responsive element in the TonB system. The proposed boundaries of the transmembrane domain α-helix were revised to consist of residues 23-39, with residues 40-43 initiating the subsequent disordered region required for signal transduction (Kopp and Postle, 2020, J. Bacteriol. 202, e00687-19). Trapping of disulfide-linked ExbD homodimers through T42C or V43C prevented TonB system activity that was restored by addition of the reducing agent dithiothreitol, indicating a requirement for motion. In vivo photo-cross-linking experiments suggested that motion was rotation of ExbD transmembrane domains. Inactivity of ExbD L132Q, the first ExbD mutant identified, was likely due to steric hindrance. A conserved and defined site of in vivo ExbD interaction with TonB was identified. Exogenous addition of a cyclic peptide based on that site inhibited ExbD-TonB interaction while concomitantly decreasing iron transport efficiency. This suggested that a novel antimicrobial strategy against ESKAPE and other Gram-negative pathogens could be developed by targeting ExbD protein-protein interactions.
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.
