Identification of New In Vivo TonB-FepA Rendezvous Sites

The TonB system of Gram-negative bacteria uses the protonmotive force of the cytoplasmic membrane to energize active transport of large or scarce nutrients across the outer membrane by means of customized beta-barrels known as TonB-dependent transporters (TBDTs). The lumen of each TBDT is occluded by an amino-terminal domain, called the cork, which must be displaced for transport of nutrients or translocation of the large protein toxins that parasitize the system. A complex of cytoplasmic membrane proteins consisting of TonB, ExbB and ExbD harnesses the protonmotive force that TonB transmits to the TBDT. The specifics of this energy transformation are a source of continuing interest. The amino terminal domain of a TBDT contains a region called the TonB box, that is essential for the reception of energy from TonB. This domain is the only identified site of in vivo interaction between the TBDT and TonB, occurring through a non-essential region centered on TonB residue Q160. Because TonB binds to TBDTs whether or not it is active or even intact, the mechanism and extent of cork movement in vivo has been challenging to discover. In this study, we used in vivo disulfide crosslinking between eight engineered Cys residues in Escherichia coli TonB and 42 Cys substitutions in the TBDT FepA, including the TonB box, to identify novel sites of interaction in vivo. The TonB Cys substitutions in the core of an essential carboxy terminal amphipathic helix (residues 199-216) were compared to TonB Q160C interactions. Functionality of the in vivo interactions was established when the presence of the inactive TonB H20A mutation inhibited them. A previously unknown functional interaction between the hydrophilic face of the amphipathic helix and the FepA TonB box was identified. Interaction of Q160C with the FepA TonB box appeared to be less functionally important. The two different parts of TonB also differed in their interactions with the FepA cork and barrel turns. While the TonB amphipathic helix Cys residues interacted only with Cys residues on the periplasmic face of the FepA cork, TonB Q160C interacted with buried Cys substitutions within the FepA cork, the first such interactions seen with any TBDT. Both sets of interactions required active TonB. Taken together, these data suggest a model where the amphipathic helix binds to the TonB box, causing the mechanically weak domain of the FepA cork to dip sufficiently into the periplasmic space for interaction with the TonB Q160 region, which is an interaction that does not occur if the TonB box is deleted. The TonB amphipathic helix also interacted with periplasmic turns between FepA β-strands in vivo supporting a surveillance mechanism where TonB searched for TBDTs on the periplasmic face of the outer membrane.

The TonB system in Aeromonas hydrophila NJ-35 is essential for MacA2B2 efflux pump-mediated macrolide resistance

The TonB system is generally considered as an energy transporting device for the absorption of nutrients. Our recent study showed that deletion of this system caused a significantly increased sensitivity of Aeromonas hydrophila to the macrolides erythromycin and roxithromycin, but had no effect on other classes of antibiotics. In this study, we found the sensitivity of ΔtonB123 to all macrolides tested revealed a 8- to 16-fold increase compared with the wild-type (WT) strain, but this increase was not related with iron deprivation caused by tonB123 deletion. Further study demonstrated that the deletion of tonB123 did not damage the integrity of the bacterial membrane but did hinder the function of macrolide efflux. Compared with the WT strain, deletion of macA2B2, one of two ATP-binding cassette (ABC) types of the macrolide efflux pump, enhanced the sensitivity to the same levels as those of ΔtonB123. Interestingly, the deletion of macA2B2 in the ΔtonB123 mutant did not cause further increase in sensitivity to macrolide resistance, indicating that the macrolide resistance afforded by the MacA2B2 pump was completely abrogated by tonB123 deletion. In addition, macA2B2 expression was not altered in the ΔtonB123 mutant, indicating that any influence of TonB on MacA2B2-mediated macrolide resistance was at the pump activity level. In conclusion, inactivation of the TonB system significantly compromises the resistance of A. hydrophila to macrolides, and the mechanism of action is related to the function of MacA2B2-mediated macrolide efflux.

Characterization of Escherichia coli ExbD protein modification in vivo

ABSTRACTThe TonB system of Escherichia coli couples the protonmotive force of the cytoplasmic membrane to active transport of nutrients across the outer membrane. In the cytoplasmic membrane, this system consists of three known proteins, TonB, ExbB, and ExbD. ExbB and ExbD appear to harvest the protonmotive force and transmit it to TonB, which then makes direct physical contact with TonB-dependent active transport proteins in the outer membrane. Using two-dimensional gel electrophoresis, we found that ExbD exists as two different species with the same apparent molecular mass but with different pIs. The more basic ExbD species was consistently present, while the more acidic species arose when cells were starved for iron by the addition of iron chelators. The cause of the modification was, however, more complex than simple iron starvation. Absence of either TonB or ExbB protein also gave rise to modified ExbD under iron-replete conditions where the wild-type strain exhibited no ExbD modification. The effect of the tonB or exbB mutations were not entirely due to iron limitation since an equally iron-limited aroB mutation did not replicate the ExbD modification. This constitutes the first report of in vivo modification for any of the TonB system proteins.

The Intrinsically Disordered Region of ExbD Is Required for Signal Transduction

ABSTRACT The TonB system actively transports vital nutrients across the unenergized outer membranes of the majority of Gram-negative bacteria. In this system, integral membrane proteins ExbB, ExbD, and TonB work together to transduce the proton motive force (PMF) of the inner membrane to customized active transporters in the outer membrane by direct and cyclic binding of TonB to the transporters. A PMF-dependent TonB-ExbD interaction is prevented by 10-residue deletions within a periplasmic disordered domain of ExbD adjacent to the cytoplasmic membrane. Here, we explored the function of the ExbD disordered domain in more detail. In vivo photo-cross-linking through sequential pBpa substitutions in the ExbD disordered domain captured five different ExbD complexes, some of which had been previously detected using in vivo formaldehyde cross-linking, a technique that lacks the residue-specific information that can be achieved through photo-cross-linking: two ExbB-ExbD heterodimers (one of which had not been detected previously), previously detected ExbD homodimers, previously detected PMF-dependent ExbD-TonB heterodimers, and for the first time, a predicted, ExbD-TonB PMF-independent interaction. The fact that multiple complexes were captured by the same pBpa substitution indicated the dynamic nature of ExbD interactions as the energy transduction cycle proceeded in vivo. In this study, we also discovered that a conserved motif—V45, V47, L49, and P50—within the disordered domain was required for signal transduction to TonB and to the C-terminal domain of ExbD and was the source of motif essentiality. IMPORTANCE The TonB system is a virulence factor for Gram-negative pathogens. The mechanism by which cytoplasmic membrane proteins of the TonB system transduce an electrochemical gradient into mechanical energy is a long-standing mystery. TonB, ExbB, and ExbD primary amino acid sequences are characterized by regions of predicted intrinsic disorder, consistent with a proposed multiplicity of protein-protein contacts as TonB proceeds through an energy transduction cycle, a complex process that has yet to be recapitulated in vitro. This study validates a region of intrinsic disorder near the ExbD transmembrane domain and identifies an essential conserved motif embedded within it that transduces signals to distal regions of ExbD suggested to configure TonB for productive interaction with outer membrane transporters.

An ExbD Disordered Domain Peptide Inhibits TonB System Activity

ABSTRACTThe TonB system energizes transport of essential nutrients, such as iron siderophores, across unenergized outer membranes of Gram-negative bacteria. The integral cytoplasmic membrane proteins of the TonB system--ExbB, ExbD, and TonB--transduce the protonmotive force of the cytoplasmic membrane to TonB-dependent outer membrane transporters for active transport. ExbD protein is anchored in the cytoplasmic membrane, with the majority of it occupying the periplasm. We previously identified a conserved motif within a periplasmic disordered domain that is essential for TonB system function. Here we demonstrated that export of a peptide derived from that motif into the periplasm prevented TonB system function and inhibited all known ExbD interactions in vivo. Formaldehyde crosslinking captured the ExbD peptide in multiple ExbD and TonB complexes. Furthermore, peptides with mutations in the conserved motif not only had significantly reduced ability to inhibit TonB system activity, but they also altered interactions with ExbD and TonB, indicating the specificity of the interaction. Conserved motif peptide interactions with ExbD and TonB mostly occurred between Stage II and Stage III of the TonB energy transduction cycle, a transition that is characterized by the use of protonmotive force. Taken together, the data suggest that the ExbD disordered domain motif has multiple interactions with TonB and ExbD during between Stage II and III of the TonB energization cycle. Because of the essentiality of the motif, it may be a potential template for design of novel antibiotics that target the TonB system.IMPORTANCEGram-negative bacteria are intrinsically antibiotic-resistant due to the diffusion barrier posed by their outer membranes. The TonB system allows them to circumvent this barrier for their own nutritional needs, including iron. The ability of bacteria to acquire iron is a virulence factor for many Gram-negative pathogens. However, no antibiotics currently target the TonB system. Because TonB and ExbD must interact productively in the periplasm for transport across the outer membrane, they constitute attractive targets for potential antibiotic development where chemical characteristics need not accommodate the need to cross the hydrophobic cytoplasmic membrane. Here we show that a small ExbD-derived peptide can interfere with the TonB-ExbD interaction to inhibit the TonB system in vivo.

A TonB-dependent receptor constitutes the outer membrane transport system for a lignin-derived aromatic compound

TonB-dependent receptors (TBDRs) mediate substrate-specific transport across the outer membrane, utilizing energy derived from the proton motive force transmitted from the TonB−ExbB−ExbD complex located in the inner membrane (TonB system). Although a number of TonB systems involved in the uptake of siderophores, vitamin B12 and saccharides have been identified, their involvement in the uptake and catabolism of aromatic compounds was previously unknown. Here, we show that the outer membrane transport of a biphenyl compound derived from lignin is mediated by the TonB system in a Gram-negative bacterium capable of degrading lignin-derived aromatic compounds, Sphingobium sp. strain SYK-6. Furthermore, we found that overexpression of the corresponding TBDR gene enhanced the uptake of this biphenyl compound, contributing to the improved rate of platform chemical production. Our results will provide an important basis for establishing engineered strains optimized for use in lignin valorisation.

The Intrinsically Disordered Region of ExbD is Required for Signal Transduction

ABSTRACTThe TonB system actively transports vital nutrients across the unenergized outer membranes of the majority of Gram-negative bacteria. In this system, integral membrane proteins ExbB, ExbD, and TonB work together to transduce the protonmotive force (PMF) of the inner membrane to customized active transporters in the outer membrane by direct and cyclic binding of TonB to the transporters. A PMF-dependent TonB-ExbD interaction is prevented by 10-residue deletions within a periplasmic disordered domain of ExbD adjacent to the cytoplasmic membrane. Here we explored the function of the ExbD disordered domain in more detail. In vivo photo-cross-linking through sequential pBpa substitutions in the ExbD disordered domain captured five different ExbD complexes, some of which had been previously detected using in vivo formaldehyde crosslinking, a technique that lacks the residue-specific information that can be achieved through photo-cross-linking: 2 ExbB-ExbD heterodimers (one of which had not been detected previously), previously detected ExbD homodimers, previously detected PMF-dependent ExbD-TonB heterodimers, and for the first time, a predicted, ExbD-TonB PMF-independent interaction. The fact that multiple complexes were captured by the same pBpa substitution indicated the dynamic nature of ExbD interactions as the energy transduction cycle proceeded in vivo. In this study, we also discovered that a conserved motif, (V45, V47, L49, P50), within the disordered domain was required for signal transduction to TonB and to the C-terminal domain of ExbD and was the source of its essentiality.ImportanceThe TonB system is a virulence factor for many Gram-negative pathogens including E-S-K-A-P-E pathogenic species Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa. Because the majority of protein-protein interactions in the TonB system occur in the periplasm, it is an appealing target for novel antibiotics. Understanding the molecular mechanism of the TonB system will provide valuable information for design of potential inhibitors targeting the system.

From Homodimer to Heterodimer and Back: Elucidating the TonB Energy Transduction Cycle

ABSTRACTThe TonB system actively transports large, scarce, and important nutrients through outer membrane (OM) transporters of Gram-negative bacteria using the proton gradient of the cytoplasmic membrane (CM). InEscherichia coli, the CM proteins ExbB and ExbD harness and transfer proton motive force energy to the CM protein TonB, which spans the periplasmic space and cyclically binds OM transporters. TonB has two activity domains: the amino-terminal transmembrane domain with residue H20 and the periplasmic carboxy terminus, through which it binds to OM transporters. TonB is inactivated by all substitutions at residue H20 except H20N. Here, we show that while TonB trapped as a homodimer through its amino-terminal domain retained full activity, trapping TonB through its carboxy terminus inactivated it by preventing conformational changes needed for interaction with OM transporters. Surprisingly, inactive TonB H20A had little effect on homodimerization through the amino terminus and instead decreased TonB carboxy-terminal homodimer formation prior to reinitiation of an energy transduction cycle. That result suggested that the TonB carboxy terminus ultimately interacts with OM transporters as a monomer. Our findings also suggested the existence of a separate equimolar pool of ExbD homodimers that are not in contact with TonB. A model is proposed where interaction of TonB homodimers with ExbD homodimers initiates the energy transduction cycle, and, ultimately, the ExbD carboxy terminus modulates interactions of a monomeric TonB carboxy terminus with OM transporters. After TonB exchanges its interaction with ExbD for interaction with a transporter, ExbD homodimers undergo a separate cycle needed to re-energize them.IMPORTANCECanonical mechanisms of active transport across cytoplasmic membranes employ ion gradients or hydrolysis of ATP for energy. Gram-negative bacterial outer membranes lack these resources. The TonB system embodies a novel means of active transport across the outer membrane for nutrients that are too large, too scarce, or too important for diffusion-limited transport. A proton gradient across the cytoplasmic membrane is converted by a multiprotein complex into mechanical energy that drives high-affinity active transport across the outer membrane. This system is also of interest since one of its uses in pathogenic bacteria is for competition with the host for the essential element iron. Understanding the mechanism of the TonB system will allow design of antibiotics targeting iron acquisition.

Catechol Siderophore Transport by Vibrio cholerae

ABSTRACTSiderophores, small iron-binding molecules secreted by many microbial species, capture environmental iron for transport back into the cell.Vibrio choleraesynthesizes and uses the catechol siderophore vibriobactin and also uses siderophores secreted by other species, including enterobactin produced byEscherichia coli.E. colisecretes both canonical cyclic enterobactin and linear enterobactin derivatives likely derived from its cleavage by the enterobactin esterase Fes. We show here thatV. choleraedoes not use cyclic enterobactin but instead uses its linear derivatives.V. choleraelacked both a receptor for efficient transport of cyclic enterobactin and enterobactin esterase to promote removal of iron from the ferrisiderophore complex. To further characterize the transport of catechol siderophores, we show that the linear enterobactin derivatives were transported intoV. choleraeby either of the catechol siderophore receptors IrgA and VctA, which also transported the synthetic siderophore MECAM [1,3,5-N,N′,N″-tris-(2,3-dihydroxybenzoyl)-triaminomethylbenzene]. Vibriobactin is transported via the additional catechol siderophore receptor ViuA, while theVibrio fluvialissiderophore fluvibactin was transported by all three catechol receptors. ViuB, a putativeV. choleraesiderophore-interacting protein (SIP), functionally substituted for theE. coliferric reductase YqjH, which promotes the release of iron from the siderophore in the bacterial cytoplasm. InV. cholerae, ViuB was required for the use of vibriobactin but was not required for the use of MECAM, fluvibactin, ferrichrome, or the linear derivatives of enterobactin. This suggests the presence of another protein inV. choleraecapable of promoting the release of iron from these siderophores.IMPORTANCEVibrio choleraeis a major human pathogen and also serves as a model for theVibrionaceae, which include other serious human and fish pathogens. The ability of these species to persist and acquire essential nutrients, including iron, in the environment is epidemiologically important but not well understood. In this work, we characterize the ability ofV. choleraeto acquire iron by using siderophores produced by other organisms. We resolve confusion in the literature regarding its ability to use theEscherichia colisiderophore enterobactin and identify the receptor and TonB system used for the transport of several siderophores. The use of some siderophores did not require the ferric reductase ViuB, suggesting that an uncharacterized ferric reductase is present inV. cholerae.