exbB exbD Research Articles

Energization of Outer Membrane Transport by the ExbB ExbD Molecular Motor.

The outer membranes (OM) of Gram-negative bacteria contain a class of proteins (TBDTs) that require energy for the import of nutrients and to serve as receptors for phages and protein toxins. Energy is derived from the proton motif force (pmf) of the cytoplasmic membrane (CM) through the action of three proteins, namely, TonB, ExbB, and ExbD, which are located in the CM and extend into the periplasm. The leaky phenotype of exbB exbD mutants is caused by partial complementation by homologous tolQ tolR. TonB, ExbB, and ExbD are genuine components of an energy transmission system from the CM into the OM. Mutant analyses, cross-linking experiments, and most recently X-ray and cryo-EM determinations were undertaken to arrive at a model that describes the energy transfer from the CM into the OM. These results are discussed in this paper. ExbB forms a pentamer with a pore inside, in which an ExbD dimer resides. This complex harvests the energy of the pmf and transmits it to TonB. TonB interacts with the TBDT at the TonB box, which triggers a conformational change in the TBDT that releases bound nutrients and opens the pore, through which nutrients pass into the periplasm. The structurally altered TBDT also changes the interactions of its periplasmic signaling domain with anti-sigma factors, with the consequence being that the sigma factors initiate transcription.

The Ton Motor.

The Ton complex is a molecular motor at the inner membrane of Gram-negative bacteria that uses a proton gradient to apply forces on outer membrane (OM) proteins to permit active transport of nutrients into the periplasmic space. Recently, the structure of the ExbB–ExbD subcomplex was determined in several bacterial species, but the complete structure and stoichiometry of TonB have yet to be determined. The C-terminal end of TonB is known to cross the periplasm and interact with TonB-dependent outer membrane transport proteins with high affinity. Yet despite having significant knowledge of these transport proteins, it is not clear how the Ton motor opens a pathway across the outer membrane for nutrient import. Additionally, the mechanism by which energy is harnessed from the inner membrane subcomplex and transduced to the outer membrane via TonB is not well understood. In this review, we will discuss the gaps in the knowledge about the complete structure of the Ton motor complex and the relationship between ion flow used to generate mechanical work at the outer membrane and the nutrient transport process.

Targeted Killing of Pseudomonas aeruginosa by Pyocin G Occurs via the Hemin Transporter Hur

Pseudomonas aeruginosa is a priority pathogen for the development of new antibiotics, particularly because multi-drug-resistant strains of this bacterium cause serious nosocomial infections and are the leading cause of death in cystic fibrosis patients. Pyocins, bacteriocins of P. aeruginosa, are potent and diverse protein antibiotics that are deployed during bacterial competition. Pyocins are produced by more than 90% of P. aeruginosa strains and may have utility as last resort antibiotics against this bacterium. In this study, we explore the antimicrobial activity of a newly discovered pyocin called pyocin G (PyoG). We demonstrate that PyoG has broad killing activity against a collection of clinical P. aeruginosa isolates and is active in a Galleria mellonella infection model. We go on to identify cell envelope proteins that are necessary for the import of PyoG and its killing activity. PyoG recognizes bacterial cells by binding to Hur, an outer-membrane TonB-dependent transporter. Both pyocin and Hur interact with TonB1, which in complex with ExbB–ExbD links the proton motive force generated across the inner membrane with energy-dependent pyocin translocation across the outer membrane. Inner-membrane translocation of PyoG is dependent on the conserved inner-membrane AAA+ ATPase/protease, FtsH. We also report a functional exploration of the PyoG receptor. We demonstrate that Hur can bind to hemin in vitro and that this interaction is blocked by PyoG, confirming the role of Hur in hemin acquisition.

Cryo-EM structure of the bacterial Ton motor subcomplex ExbB\u2013ExbD provides information on structure and stoichiometry

The TonB–ExbB–ExbD molecular motor harnesses the proton motive force across the bacterial inner membrane to couple energy to transporters at the outer membrane, facilitating uptake of essential nutrients such as iron and cobalamine. TonB physically interacts with the nutrient-loaded transporter to exert a force that opens an import pathway across the outer membrane. Until recently, no high-resolution structural information was available for this unique molecular motor. We published the first crystal structure of ExbB–ExbD in 2016 and showed that five copies of ExbB are arranged as a pentamer around a single copy of ExbD. However, our spectroscopic experiments clearly indicated that two copies of ExbD are present in the complex. To resolve this ambiguity, we used single-particle cryo-electron microscopy to show that the ExbB pentamer encloses a dimer of ExbD in its transmembrane pore, and not a monomer as previously reported. The revised stoichiometry has implications for motor function.

Elucidating the origin of the ExbBD components of the TonB system through Bayesian inference and maximum-likelihood phylogenies

Uptake of ferric siderophores, vitamin B12, and other molecules in gram-negative bacteria is mediated by a multi-protein complex known as the TonB system. The ExbB and ExbD protein components of the TonB system play key energizing roles and are homologous with the flagellar motor proteins MotA and MotB. Here, the phylogenetic relationships of ExbBD and MotAB were investigated using Bayesian inference and the maximum-likelihood method. Phylogenetic trees of these proteins suggest that they are separated into distinct monophyletic groups and have originated from a common ancestral system. Several horizontal gene transfer events for ExbB–ExbD are also inferred, and a model for the evolution of the TonB system is proposed.

Energetics of colicin import revealed by genetic cross-complementation between the Tol and Ton systems

Colicins are bacterial toxins that parasitize OM (outer membrane) receptors to bind to the target cells, use an import system to translocate through the cell envelope and then kill sensitive cells. Colicins classified as group A (colicins A, E1-E9, K and N) use the Tol system (TolA, TolB, TolQ and TolR), whereas group B colicins (colicins B, D, Ia, M and 5) use the ExbB-ExbD-TonB system. Genetic evidence has suggested that TolQ and ExbB, as well as TolR and ExbD, are interchangeable, whereas this is not possible with TolA and TonB. Early reports indicated that group B colicin uptake requires energy input, whereas no energy was necessary for the uptake of the pore-forming colicin A. Furthermore, energy is required to dissociate the complex formed with colicin E9 and its cognate immunity protein during the import process. In the present paper, we detail the functional phenotypes and colicin-sensitivity results obtained in tolQ and exbB mutants and cross-complementation data of amino acid substitutions that lie within ExbB or TolQ TMHs (transmembrane helices). We also discuss on a specific phenotype that corresponds to group A colicin-sensitivity associated with a non-functional Tol system.

Oligomeric Structure of ExbB and ExbB-ExbD Isolated from Escherichia coli As Revealed by LILBID Mass Spectrometry

Energy-coupled transporters in the outer membrane of Escherichia coli and other Gram-negative bacteria allow the entry of scarce substrates, toxic proteins, and bacterial viruses (phages) into the cells. The required energy is derived from the proton-motive force of the cytoplasmic membrane, which is coupled to the outer membrane via the ExbB-ExbD-TonB protein complex. Knowledge of the structure of this complex is required to elucidate the mechanisms of energy harvesting in the cytoplasmic membrane and energy transfer to the outer membrane transporters. Here we solubilized an ExbB oligomer and an ExbB-ExbD subcomplex from the cytoplasmic membrane with the detergent undecyl maltoside. Using laser-induced liquid bead ion desorption mass spectrometry (LILBID-MS), we determined at moderate desorption laser energies the oligomeric structure of ExbB to be mainly hexameric (ExbB(6)), with minor amounts of trimeric (ExbB(3)), dimeric (ExbB(2)), and monomeric (ExbB(1)) oligomers. Under the same conditions ExbB-ExbD formed a subcomplex consisting of ExbB(6)ExbD(1), with a minor amount of ExbB(5)ExbD(1). At higher desorption laser intensities, ExbB(1) and ExbD(1) and traces of ExbB(3)ExbD(1), ExbB(2)ExbD(1), ExbB(1)ExbD(1), ExbB(3), and ExbB(2) were observed. Since the ExbB(6) complex and the ExbB(6)ExbD(1) complex remained stable during solubilization and subsequent chromatographic purification on nickel-nitrilotriacetate agarose, Strep-Tactin, and Superdex 200, and during native blue gel electrophoresis, we concluded that ExbB(6) and ExbB(6)ExbD(1) are subcomplexes on which the final complex including TonB is assembled.

TonB-dependent maltose transport by Caulobacter crescentus

We have shown previously that Caulobacter crescentus grows on maltodextrins which are actively transported across the outer membrane by the MalA protein. Evidence for energy-coupled transport was obtained by deletion of the exbB exbD genes which abolished transport. However, removal of the TonB protein, which together with the ExbB ExbD proteins is predicted to form an energy-coupling device between the cytoplasmic membrane and the outer membrane, left transport unaffected. Here we identify an additional tonB gene encoded by the cc2334a ORF, which when deleted abolished maltose transport. MalA contains a TonB box that reads EEVVIT and is predicted to interact with TonB. Replacement of valine number 15 in the TonB box by proline abolished maltose transport. Maltose was transported across the cytoplasmic membrane by the MalY protein (CC2283). Maltose transport was induced by maltose and repressed by the MalI protein (CC2284). In addition to MalA, MalY and MalI, the mal locus encodes two predicted cytoplasmic alpha-amylases (CC2285 and CC2286) and a periplasmic glucoamylase (CC2282). The TonB dependence together with the previously described ExbB ExbD dependence demonstrates energy-coupled maltose transport across the outer membrane. MalY is involved in maltose transport across the cytoplasmic membrane by a presumably ion-coupled mechanism.

From the Periplasmic Signaling Domain to the Extracellular Face of an Outer Membrane Signal Transducer of Pseudomonas aeruginosa: Crystal Structure of the Ferric Pyoverdine Outer Membrane Receptor

The pyoverdine outer membrane receptor, FpvA, from Pseudomonas aeruginosa translocates ferric pyoverdine across the outer membrane through an energy consuming mechanism using the proton motive force and the TonB–ExbB–ExbD energy transducing complex from the inner membrane. We solved the crystal structure of the full-length FpvA bound to iron-pyoverdine at 2.7 Å resolution. Signal transduction to an anti-sigma protein of the inner membrane and to TonB–ExbB–ExbD involves the periplasmic domain, which displays a β-α-β fold composed of two α-helices sandwiched by two β-sheets. One iron-pyoverdine conformer is bound at the extracellular face of FpvA, revealing the conformer selectivity of the binding site. The loop that contains the TonB box, involved in interactions with TonB, and connects the signaling domain to the plug domain of FpvA is not defined in the electron density following the binding of ferric pyoverdine. The high flexibility of this loop is probably necessary for signal transduction through the outer membrane.

Crystal Structure at High Resolution of Ferric-pyochelin and its Membrane Receptor FptA from Pseudomonas aeruginosa

Pyochelin is a siderophore and virulence factor common to Burkholderia cepacia and several Pseudomonas strains. We describe at 2.0 A resolution the crystal structure of the pyochelin outer membrane receptor FptA bound to the iron-pyochelin isolated from Pseudomonas aeruginosa. One pyochelin molecule bound to iron is found in the protein structure, providing the first three-dimensional structure at the atomic level of this siderophore. The pyochelin molecule provides a tetra-dentate coordination of iron, while the remaining bi-dentate coordination is ensured by another molecule not specifically recognized by the protein. The overall structure of the pyochelin receptor is typical of the TonB-dependent transporter superfamily, which uses the proton motive force from the cytoplasmic membrane through the TonB-ExbB-ExbD energy transducing complex to transport ferric ions across the bacterial outer membrane: a transmembrane 22 beta-stranded barrel occluded by a N-terminal domain that contains a mixed four-stranded beta-sheet. The N-terminal TonB box is disordered in two crystal forms, and loop L8 is found to point towards the iron-pyochelin complex, suggesting that the receptor is in a transport-competent conformation.

The iron–siderophore transporter FhuA is the receptor for the antimicrobial peptide microcin J25: role of the microcin Val11–Pro16 β-hairpin region in the recognition mechanism

The role of the outer-membrane iron transporter FhuA as a potential receptor for the antimicrobial peptide MccJ25 (microcin J25) was studied through a series of in vivo and in vitro experiments. The requirement for both FhuA and the inner-membrane TonB-ExbB-ExbD complex was demonstrated by antibacterial assays using complementation of an fhuA(-) strain and by using isogenic strains mutated in genes encoding the protein complex respectively. In addition, MccJ25 was shown to block phage T5 infection of Escherichia coli, in vivo, by inhibiting phage adhesion, which suggested that MccJ25 prevents the interaction between the phage and its receptor FhuA. This in vivo activity was confirmed in vitro, as MccJ25 inhibited phage T5 DNA ejection triggered by purified FhuA. Direct interaction of MccJ25 with FhuA was demonstrated for the first time by size-exclusion chromatography and isothermal titration calorimetry. MccJ25 bound to FhuA with a 2:1 stoichiometry and a K(d) of 1.2 microM. Taken together, our results demonstrate that FhuA is the receptor for MccJ25 and that the ligand-receptor interaction may occur in the absence of other components of the bacterial membrane. Finally, both differential scanning calorimetry and antimicrobial assays showed that MccJ25 binding involves external loops of FhuA. Unlike native MccJ25, a thermolysin-cleaved MccJ25 variant was unable to bind to FhuA and failed to prevent phage T5 infection of E. coli. Therefore the Val11-Pro16 beta-hairpin region of MccJ25, which is disrupted upon cleavage by thermolysin, is required for microcin recognition.

Identification of Fur regulated genes in the bacterial fish pathogen Photobacterium damselae ssp. piscicida using the Fur titration assay

Bacteria have developed a series of iron-scavenging and transport systems. The expression of many of the iron utilization genes is tightly regulated by the Fe2+ loaded Fur repressor protein. In this study, the Fur titration assay (FURTA) was used to screen for DNA fragments from a genomic DNA library of Photobacterium damselae ssp. piscicida containing potential Fe2+ Fur binding sites or iron binding-proteins which withdraw iron from Fur. One of the clones encoded a tonB gene and adjacent a functionally related exbB gene. An additional and complete tonB exbB exbD gene cluster was identified and sequenced. A gene homologous to the ferritin gene was found whose FURTA-positive phenotype may be explained by its iron-binding ability. Genes encoding a putative complete iron-regulated outer membrane transport protein and a pseudogene of a transport protein were found. The FURTA assay also revealed iron regulation of the AraC type transcriptional regulation.

Pathogenesis of enteric Campylobacter infection.

Campylobacter jejuni and related species are important human pathogens, causing acute human enterocolitis, and they are the most common cause of food-borne diarrhoea in many industrialized countries. Previous infection with certain strains of C. jejuni is also linked with the development of the neurological disorder Guillain-Barre syndrome (GBS). Despite its importance as a human pathogen, relatively little is understood of the mechanisms of C. jejuni-associated disease. The recent release of the complete genome sequence of C. jejuni strain NCTC 11168, together with new strategies for random and directed mutagenesis, have allowed a better insight into some of the genetic determinants of C. jejuni virulence. In this review current knowledge on the pathogenesis of C. jejuni infection is summarized, and possible directions of future research indicated.

Bordetella pertussis TonB, a Bvg-independent virulence determinant.

In gram-negative bacteria, high-affinity iron uptake requires the TonB/ExbB/ExbD envelope complex to release iron chelates from their specific outer membrane receptors into the periplasm. Based on sequence similarities, the Bordetella pertussis tonB exbB exbD locus was identified on a cloned DNA fragment. The tight organization of the three genes suggests that they are cotranscribed. A putative Fur-binding sequence located upstream from tonB was detected in a Fur titration assay, indicating that the tonB exbB exbD operon may be Fur-repressed in high-iron growth conditions. Putative structural genes of the beta-subunit of the histone-like protein HU and of a new two-component regulatory system were identified upstream from tonB and downstream from exbD, respectively. A B. pertussis DeltatonB exbB::Km(r) mutant was constructed by allelic exchange and characterized. The mutant was impaired for growth in low-iron medium in vitro and could not use ferrichrome, desferal, or hemin as iron sources. Levels of production of the major bacterial toxins and adhesins were similar in the TonB(+)/TonB(-) pair. The DeltatonB exbB mutant was still responsive to chemical modulators of virulence; thus, the BvgA/BvgS two-component system is not TonB dependent. Nevertheless, in vivo in the mouse respiratory infection model, the colonization ability of the mutant was reduced compared to the parental strain.

Role of the Haemophilus ducreyi Ton system in internalization of heme from hemoglobin.

By cloning into Escherichia coli and construction of isogenic mutants of Haemophilus ducreyi, we showed that the hemoglobin receptor (HgbA) is TonB dependent. An E. coli hemA tonB mutant expressing H. ducreyi hgbA grew on low levels of hemoglobin as a source of heme only when an intact H. ducreyi Ton system plasmid was present. In contrast, growth on heme by the E. coli hemA tonB mutant expressing hgbA was observed only at high concentrations of heme, was TonB independent, and demonstrated that H. ducreyi HgbA was not sufficient to function as a typical TonB-dependent heme receptor in E. coli. Allelic replacement of the wild-type H. ducreyi exbB, exbD, and tonB loci with the exbB, exbD, and tonB deletion resulted in an H. ducreyi isogenic mutant unable to utilize hemoglobin but able to utilize hemin at the same levels as the parent strain to fulfill its heme requirement. This finding confirms the TonB dependence of HgbA-mediated hemoglobin utilization and suggests that uptake of hemin in H. ducreyi is TonB independent. Additionally, the H. ducreyi Ton system mutant synthesized increased amounts of HgbA and other heme-regulated outer membrane proteins, consistent with derepression of these proteins due to lower intracellular heme and/or iron concentrations in the mutant. Sequencing of the Ton system genes revealed that the arrangement of the genes was exbB exbD tonB. The proximity and structure of these genes suggested that they are transcribed as an operon. This arrangement, as well as the DNA and deduced amino acid sequences of these H. ducreyi genes, was most similar to those from other pasteurellae.

Characterization of the exbBD operon of Escherichia coli and the role of ExbB and ExbD in TonB function and stability

TonB protein appears to couple the electrochemical potential of the cytoplasmic membrane to active transport across the essentially unenergized outer membrane of gram-negative bacteria. ExbB protein has been identified as an auxiliary protein in this process. In this paper we show that ExbD protein, encoded by an adjacent gene in the exb cluster at 65', was also required for TonB-dependent energy transduction and, like ExbB, was required for the stability of TonB. The phenotypes of exbB exbD+ strains were essentially indistinguishable from the phenotypes of exbB+ exbD strains. Mutations in either gene resulted in the degradation of TonB protein and in decreased, but not entirely absent, sensitivities to colicins B and Ia and to bacteriophage phi 80. Evidence that the absence of ExbB or ExbD differentially affected the half-lives of newly synthesized and steady-state TonB was obtained. In the absence of ExbB or ExbD, newly synthesized TonB was degraded with a half-life of 5 to 10 min, while the half-life of TonB under steady-state conditions was significantly longer, approximately 30 min. These results were consistent with the idea that ExbB and ExbD play roles in the assembly of TonB into an energy-transducing complex. While interaction between TonB and ExbD was suggested by the effect of ExbD on TonB stability, interaction of ExbD with TonB was detected by neither in vivo cross-linking assays nor genetic tests for competition. Assays of a chromosomally encoded exbD::phoA fusion showed that exbB and exbD were transcribed as an operon, such that ExbD-PhoA levels in an exbB::Tn10 strain were reduced to 4% of the levels observed in an exbB+ strain under iron-limiting conditions. Residual ExbD-PhoA expression in an exbB::Tn10 strain was not iron regulated and may have originated from within the Tn10 element in exbB.

Energy-coupled transport through the outer membrane of Escherichia coli small deletions in the gating loop convert the FhuA transport protein into a diffusion channel

Active transport of Fe 3+ as ferrichrome complex through the outer membrane of Escherichia coli is mediated by the FhuA outer membrane protein and the TonB—ExbB—ExbD protein complex in the cytoplasmic membrane. The required energy is provided by the electrochemical potential of the cytoplasmic membrane which is assumed to induce a conformation of the TonB protein that causes a conformational change in FhuA so that bound ferrichrome is released into the periplasmic space located between the outer and the cytoplasmic membrane. Excision of segments as small as 12 amino acids in the largest surface loop of FhuA converted FhuA into an open channel through which ferrichrome and antibiotics diffused independent of TonB—ExbB—ExbD. It is proposed that FhuA forms a closed channel which is opened by movement of the gating loop through a kind of allosteric interaction with TonB. The gating loop is also involved in binding of all FhuA ligands which in addition to ferrichrome are the phages T1, T5, φ80, colicin M and the antibiotic albomycin.