TonB-dependent Outer Membrane Research Articles

A Novel Extracytoplasmic Function (ECF) Sigma Factor Regulates Virulence in Pseudomonas aeruginosa

Next to the two-component and quorum sensing systems, cell-surface signaling (CSS) has been recently identified as an important regulatory system in Pseudomonas aeruginosa. CSS systems sense signals from outside the cell and transmit them into the cytoplasm. They generally consist of a TonB-dependent outer membrane receptor, a sigma factor regulator (or anti-sigma factor) in the cytoplasmic membrane, and an extracytoplasmic function (ECF) sigma factor. Upon perception of the extracellular signal by the receptor the ECF sigma factor is activated and promotes the transcription of a specific set of gene(s). Although most P. aeruginosa CSS systems are involved in the regulation of iron uptake, we have identified a novel system involved in the regulation of virulence. This CSS system, which has been designated PUMA3, has a number of unusual characteristics. The most obvious difference is the receptor component which is considerably smaller than that of other CSS outer membrane receptors and lacks a β-barrel domain. Homology modeling of PA0674 shows that this receptor is predicted to be a bilobal protein, with an N-terminal domain that resembles the N-terminal periplasmic signaling domain of CSS receptors, and a C-terminal domain that resembles the periplasmic C-terminal domains of the TolA/TonB proteins. Furthermore, the sigma factor regulator both inhibits the function of the ECF sigma factor and is required for its activity. By microarray analysis we show that PUMA3 regulates the expression of a number of genes encoding potential virulence factors, including a two-partner secretion (TPS) system. Using zebrafish (Danio rerio) embryos as a host we have demonstrated that the P. aeruginosa PUMA3-induced strain is more virulent than the wild-type. PUMA3 represents the first CSS system dedicated to the transcriptional activation of virulence functions in a human pathogen.

A survey of TonB‐dependent receptors in fluorescent pseudomonads

For bacteria with an aerobic lifestyle, iron is in the oxidized Fe(3+) form, hence poorly soluble. The solution is the synthesis and excretion of siderophores with a high affinity for iron. These ferrisiderophores are recognized by TonB-dependent outer membrane receptors in Gram-negative bacteria. Haem is also a source of iron and is captured via TonB-dependent receptors as well. In many cases bacterial genomes encode genes for receptors for siderophores produced by other microorganisms (xenosiderophores). Pseudomonads are known for their high adaptive capacity and it is therefore not surprising to find a relatively large number of genes encoding these receptors. In this study we analysed the genomes of three fluorescent pseudomonads available in the Pseudomonas genome database (http://www.pseudomonas.com; P. aeruginosa, P. putida, P. syringae) in order to extract the genes coding for TonB-dependent receptors. As expected we observed differences between species for the number of receptors. We also report differences within species, suggesting the acquisition of some genes via horizontal gene transfer, including those coding for the ferripyoverdine receptors. We also report cases where duplications of receptor genes are observed and the presence of 'receptor islands'. Our study strongly supports the notion of 'core' and 'accessory' TonB-dependent receptors within each species, with the ferripyoverdine receptors belonging to the last category.

Proteomics of Porphyromonas gingivalis within a model oral microbial community

BackgroundPorphyromonas gingivalis is a periodontal pathogen that resides in a complex multispecies microbial biofilm community known as dental plaque. Confocal laser scanning microscopy showed that P. gingivalis can assemble into communities in vitro with Streptococcus gordonii and Fusobacterium nucleatum, common constituents of dental plaque. Whole cell quantitative proteomics, along with mutant construction and analysis, were conducted to investigate how P. gingivalis adapts to this three species community.Results1156 P. gingivalis proteins were detected qualitatively during comparison of the three species model community with P. gingivalis incubated alone under the same conditions. Integration of spectral counting and summed signal intensity analyses of the dataset showed that 403 proteins were down-regulated and 89 proteins up-regulated. The proteomics results were inspected manually and an ontology analysis conducted using DAVID. Significant decreases were seen in proteins involved in cell shape and the formation of the cell envelope, as well as thiamine, cobalamin, and pyrimidine synthesis and DNA repair. An overall increase was seen in proteins involved in protein synthesis. HmuR, a TonB dependent outer membrane receptor, was up-regulated in the community and an hmuR deficient mutant was deficient in three species community formation, but was unimpaired in its ability to form mono- or dual-species biofilms.ConclusionCollectively, these results indicate that P. gingivalis can assemble into a heterotypic community with F. nucleatum and S. gordonii, and that a community lifestyle provides physiologic support for P. gingivalis. Proteins such as HmuR, that are up-regulated, can be necessary for community structure.

A TonB-dependent outer membrane protein as aBacteroides fragilisfibronectin-binding molecule

The binding of Bacteroides fragilis to plasmatic fibronectin was investigated using strains isolated from healthy subjects and from patients with bacteremia. They were cultivated in a synthetic media in which variations in cysteine concentrations determined alterations in the oxidation-reduction potential (Eh). All the strains assayed were capable of adhering to plasmatic fibronectin when cultivated under oxidizing and reducing conditions. Bacteroides fragilis 1405 showed the greatest difference when the results under these conditions were compared and it was selected for further investigations. Chemical treatments suggested the involvement of a protein in the interaction between B. fragilis and plasmatic fibronectin. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis of outer membrane proteins (OMPs) revealed differences between the extracts obtained from cultures grown under the two conditions. Protein bands of c. 102, 100, 77, 73, 50 and 40 kDa were more highly expressed under oxidizing than reducing conditions. Dot blot analysis showed a stronger recognition of plasmatic fibronectin by OMPs obtained from cultures grown under higher Eh, and Western blot assays confirmed a band of c. 102 kDa as fibronectin-binding protein. This protein was sequenced and revealed to be a putative TonB-dependent OMPs. PCR analysis confirmed the presence of this gene in all the studied strains.

Expression, purification, crystallization and preliminary X-ray diffraction analysis of the TonB-dependent haem outer membrane transporter ShuA fromShigella dysenteriae

As part of efforts towards understanding the crystallization of membrane proteins and membrane transport across the outer membrane of Gram-negative bacteria, the TonB-dependent haem outer membrane transporter ShuA of Shigella dysenteriae bound to heavy atoms was crystallized in several crystallization conditions using detergents. The insertion of a His(6) tag into an extracellular loop of ShuA, instead of downstream of the Escherichia coli peptide signal, allowed efficient targeting to the outer membrane and the rapid preparation of crystallizable protein. Crystals diffracting X-rays beyond 3.5 A resolution were obtained by co-crystallizing ShuA with useful heavy atoms for phasing (Eu, Tb, Pb) by the MAD method at the synchrotron, and the SAD or SIRAS method at the Cu wavelength. The authors collected X-ray diffraction data at 2.3 A resolution using one crystal of ShuA-Pb, and at 3.2 A resolution at an energy remote from the Pb M absorption edges for phasing on PROXIMA-1 at SOLEIL.

Use of an in-house approach to study the three-dimensional structures of various outer membrane proteins: structure of the alcaligin outer membrane transporter FauA fromBordetella pertussis

Bordetella pertussis is the bacterial agent of whooping cough in humans. Under iron-limiting conditions, it produces the siderophore alcaligin. Released to the extracellular environment, alcaligin chelates iron, which is then taken up as a ferric alcaligin complex via the FauA outer membrane transporter. FauA belongs to a family of TonB-dependent outer membrane transporters that function using energy derived from the proton motive force. Using an in-house protocol for membrane-protein expression, purification and crystallization, FauA was crystallized in its apo form together with three other TonB-dependent transporters from different organisms. Here, the protocol used to study FauA is described and its three-dimensional structure determined at 2.3 A resolution is discussed.

Protein-Protein Interactions And The Energy Coupling Mechanism In TonB-Dependent Transport

BtuB is a TonB-dependent outer membrane transporter of vitamin B12 in E. coli. In this work, we investigated the interaction between BtuB and the inner membrane protein TonB using site-directed spin labeling (SDSL). In CHAPS/POPC mixed micelles, the Ton box of BtuB undergoes an order-to-disorder transition upon addition of vitamin B12 which appears to be identical to that seen in POPC bilayers. Under these conditions, addition of a C-terminal fragment of TonB broadens the EPR lineshapes, indicating that there is an ordering of the Ton box and an interaction between the transporter Ton box and this C-terminal fragment. Residues N-terminal to the Ton box do not appear to interact with TonB. These changes appear to be independent of the addition of the substrate, vitamin B12. The EPR data obtained are generally consistent with the crystal structure that has been obtained for this complex (Shultis et al. Science 312, (2006)); however, preliminary distance measurements using DEER indicate that there may be multiple states of TonB when it is bound to BtuB. Spin labels incorporated into TonB also become ordered upon interaction with BtuB, and the EPR lineshapes indicate that there is a decrease in backbone dynamics of TonB upon association with BtuB. An EPR based equilibrium binding assay was carried out to determine the affinity between this C-terminal TonB fragment and BtuB, and was performed using either labels on BtuB or labels on TonB. Both labels indicate that there is an affinity of approximately 50 μM between BtuB and TonB, which is, unexpectedly, independent of substrate addition.This work was supported by GM035215.

Overproduction, purification and preliminary X-ray diffraction analysis of YncE, an iron-regulated Sec-dependent periplasmic protein from Escherichia coli.

The yncE gene of Escherichia coli encodes a predicted periplasmic protein of unknown function. The gene is de-repressed under iron restriction through the action of the global iron regulator Fur. This suggests a role in iron acquisition, which is supported by the presence of the adjacent yncD gene encoding a potential TonB-dependent outer-membrane transporter. Here, the preliminary crystallographic structure of YncE is reported, revealing that it consists of a seven-bladed beta-propeller which resembles the corresponding domain of the ;surface-layer protein' of Methanosarcina mazei. A full structure determination is under way in order to provide insight into the function of this protein.

Alr0397 Is an Outer Membrane Transporter for the Siderophore Schizokinen inAnabaenasp. Strain PCC 7120

Iron uptake in proteobacteria by TonB-dependent outer membrane transporters represents a well-explored subject. In contrast, the same process has been scarcely investigated in cyanobacteria. The heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120 is known to secrete the siderophore schizokinen, but its transport system has remained unidentified. Inspection of the genome of strain PCC 7120 shows that only one gene encoding a putative TonB-dependent iron transporter, namely alr0397, is positioned close to genes encoding enzymes involved in the biosynthesis of a hydroxamate siderophore. The expression of alr0397, which encodes an outer membrane protein, was elevated under iron-limited conditions. Inactivation of this gene caused a moderate phenotype of iron starvation in the mutant cells. The characterization of the mutant strain showed that Alr0397 is a TonB-dependent schizokinen transporter (SchT) of the outer membrane and that alr0397 expression and schizokinen production are regulated by the iron homeostasis of the cell.

Vibrio choleraeVciB Promotes Iron Uptake via Ferrous Iron Transporters

Vibrio cholerae uses a variety of strategies for obtaining iron in its diverse environments. In this study we report the identification of a novel iron utilization protein in V. cholerae, VciB. The vciB gene and its linked gene, vciA, were isolated in a screen for V. cholerae genes that permitted growth of an Escherichia coli siderophore mutant in low-iron medium. The vciAB operon encodes a predicted TonB-dependent outer membrane receptor, VciA, and a putative inner membrane protein, VciB. VciB, but not VciA, was required for growth stimulation of E. coli and Shigella flexneri strains in low-iron medium. Consistent with these findings, TonB was not needed for VciB-mediated growth. No growth enhancement was seen when vciB was expressed in an E. coli or S. flexneri strain defective for the ferrous iron transporter Feo. Supplying the E. coli feo mutant with a plasmid encoding either E. coli or V. cholerae Feo, or the S. flexneri ferrous iron transport system Sit, restored VciB-mediated growth; however, no stimulation was seen when either of the ferric uptake systems V. cholerae Fbp and Haemophilus influenzae Hit was expressed. These data indicate that VciB functions by promoting iron uptake via a ferrous, but not ferric, iron transport system. VciB-dependent iron accumulation via Feo was demonstrated directly in iron transport assays using radiolabeled iron. A V. cholerae vciB mutant did not exhibit any growth defects in either in vitro or in vivo assays, possibly due to the presence of other systems with overlapping functions in this pathogen.

NagA-dependent uptake of N-acetyl-glucosamine and N-acetyl-chitin oligosaccharides across the outer membrane of Caulobacter crescentus.

Among the 67 predicted TonB-dependent outer membrane transporters of Caulobacter crescentus, NagA was found to be essential for growth on N-acetyl-beta-D-glucosamine (GlcNAc) and larger chitin oligosaccharides. NagA (93 kDa) has a predicted typical domain structure of an outer membrane transport protein: a signal sequence, the TonB box EQVVIT, a hatch domain of 147 residues, and a beta-barrel composed of 22 antiparallel beta-strands linked by large surface loops and very short periplasmic turns. Mutations in tonB1 and exbBD, known to be required for maltose transport via MalA in C. crescentus, and in two additional predicted tonB genes (open reading frames cc2327 and cc3508) did not affect NagA-mediated GlcNAc uptake. nagA is located in a gene cluster that encodes a predicted PTS sugar transport system and two enzymes that convert GlcNAc-6-P to fructose-6-P. Since a nagA insertion mutant did not grow on and transport GlcNAc, diffusion of GlcNAc through unspecific porins in the outer membrane is excluded. Uptake of GlcNAc into tonB and exbBD mutants and reduction but not abolishment of GlcNAc transport by agents which dissipate the electrochemical potential of the cytoplasmic membrane (0.1 mM carbonyl cyanide 3-chlorophenylhydrazone and 1 mM 2,4-dinitrophenol) suggest diffusion of GlcNAc through a permanently open pore of NagA. Growth on (GlcNAc)(3) and (GlcNAc)(5) requires ExbB and ExbD, indicating energy-coupled transport by NagA. We propose that NagA forms a small pore through which GlcNAc specifically diffuses into the periplasm and functions as an energy-coupled transporter for the larger chitin oligosaccharides.

Dual Roles of the Central Domain of Colicin D tRNase in TonB-mediated Import and in Immunity

Colicin D import into Escherichia coli requires an interaction via its TonB box with the energy transducer TonB. Colicin D cytotoxicity is inhibited by specific tonB mutations, but it is restored by suppressor mutations in the TonB box. Here we report that there is a second site of interaction between TonB and colicin D, which is dependent upon a 45-amino acid region, within the uncharacterized central domain of colicin D. In addition, the 8th amino acids of colicin D (a glycine) and colicin B (a valine), adjacent to their TonB boxes, are also required for TonB recognition, suggesting that high affinity complex formation involves multiple interactions between these colicins and TonB. The central domain also contributes to the formation of the immunity complex, as well as being essential for uptake and thus killing. Colicin D is normally secreted in association with the immunity protein, and this complex involves the following two interactions: a major interaction with the C-terminal tRNase domain and a second interaction involving the central domain of colicin D and, most probably, the alpha4 helix of ImmD, which is on the opposite side of ImmD compared with the major interface. In contrast, formation of the immunity complex with the processed cytotoxic domain, the form expected to be found in the cytoplasm after colicin D uptake, requires only the major interaction. Klebicin D has, like colicin D, a ribonuclease activity toward tRNAArg and a central domain, which can form a complex with ImmD but which does not function in TonB-mediated transport.

A β 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.

Impact of alcaligin siderophore utilization on in vivo growth of Bordetella pertussis.

Bordetella pertussis, the causative agent of human whooping cough, or pertussis, is an obligate human pathogen with diverse high-affinity transport systems for the assimilation of iron, a biometal that is essential for growth. Under iron starvation stress conditions, B. pertussis produces the siderophore alcaligin. The alcaligin siderophore gene cluster, consisting of the alcABCDERS and fauA genes, encodes activities required for alcaligin biosynthesis, the export of the siderophore from the cell, the uptake of the ferric alcaligin complex across the outer membrane, and the transcriptional activation of alcaligin system genes by an autogenous mechanism involving alcaligin sensing. The fauA gene encodes a 79-kDa TonB-dependent outer membrane receptor protein required for the uptake and utilization of ferric alcaligin as an iron source. In this study, using mixed-infection competition experiments in a mouse respiratory model, inactivation of the B. pertussis ferric alcaligin receptor protein was found to have a profound impact on in vivo growth and survival of a fauA mutant compared with a coinfecting wild-type strain. The attenuating effect of fauA inactivation was evident early in the course of the infection, suggesting that the contribution of ferric alcaligin transport to the ecological fitness of B. pertussis may be important for adaptation to iron-restricted host conditions that exist at the initial stages of infection. Alcaligin-mediated iron acquisition by B. pertussis may be critical for successful host colonization and establishment of infection.

Mechanics of Force Propagation in TonB-Dependent Outer Membrane Transport

For the uptake of scarce yet essential organometallic compounds, outer membrane transporters of Gram-negative bacteria work in concert with an energy-generating inner membrane complex, thus spanning the periplasmic space to drive active transport. Here, we examine the interaction of TonB, an inner membrane protein, with an outer membrane transporter based upon a recent crystal structure of a TonB-transporter complex to characterize two largely unknown steps of the transport cycle: how energy is transmitted from TonB to the transporter and how energy transduction initiates transport. Simulations of TonB in complex with BtuB reveal that force applied to TonB is transmitted to BtuB without disruption of the very small connection between the two, supporting a mechanical mode of coupling. Based on the results of different pulling simulations, we propose that the force transduction instigates a partial unfolding of the pore-occluding luminal domain of the transporter, a potential step in the transport cycle. Furthermore, analysis of the electrostatic potentials and salt bridge interactions between the two proteins during the simulations hints at involvement of electrostatic forces in long-range interaction and binding of TonB and BtuB.

Expression and Characterization of an Iron-Regulated Hemin-Binding Protein, HbpA, fromLeptospira interrogansSerovar Lai

In an earlier study, based on the ferric enterobactin receptor FepA of Escherichia coli, we identified and modeled a TonB-dependent outer membrane receptor protein (LB191) from the genome of Leptospira interrogans serovar Lai. Based on in silico analysis, we hypothesized that this protein was an iron-dependent hemin-binding protein. In this study, we provide experimental evidence to prove that this protein, termed HbpA (hemin-binding protein A), is indeed an iron-regulated hemin-binding protein. We cloned and expressed the full-length 81-kDa recombinant rHbpA protein and a truncated 55-kDa protein from L. interrogans serovar Lai, both of which bind hemin-agarose. Assay of hemin-associated peroxidase activity and spectrofluorimetric analysis provided confirmatory evidence of hemin binding by HbpA. Immunofluorescence studies by confocal microscopy and the microscopic agglutination test demonstrated the surface localization and the iron-regulated expression of HbpA in L. interrogans. Southern blot analysis confirmed our earlier observation that the hbpA gene was present only in some of the pathogenic serovars and was absent in Leptospira biflexa. Hemin-agarose affinity studies showed another hemin-binding protein with a molecular mass of approximately 44 kDa, whose expression was independent of iron levels. This protein was seen in several serovars, including nonpathogenic L. biflexa. Sequence analysis and immunoreactivity with specific antibodies showed this protein to be LipL41.

Mutagenesis and Molecular Modeling Reveal Three Key Extracellular Loops of the Membrane Receptor HasR That Are Involved in Hemophore HasA Binding

On the basis of the three-dimensional model of the heme/hemophore TonB-dependent outer membrane receptor HasR, mutants with six-residue deletions in the 11 putative extracellular loops were generated. Although all mutants continued to be active TonB-dependent heme transporters, mutations in three loops abolished hemophore HasA binding both in vivo and in vitro.

Characterization of the Outer Membrane Receptor ShuA from the Heme Uptake System of Shigella dysenteriae: SUBSTRATE SPECIFICITY AND IDENTIFICATION OF THE HEME PROTEIN LIGANDS

Shigella dysenteriae, like many bacterial pathogens, has evolved outer membrane receptor-mediated pathways for the uptake and utilization of heme as an iron source. As a first step toward understanding the mechanism of heme uptake we have undertaken a site-directed mutagenesis, spectroscopic, and kinetic analysis of the outer membrane receptor ShuA of S. dysenteriae. Purification of the outer membrane receptor gave a single band of molecular mass 73 kDa on SDS-PAGE. Initial spectroscopic analysis of the protein in either detergent micelles or lipid bicelles revealed residual heme bound to the receptor, with a Soret maximum at 413 nm. Titration of the protein with exogenous heme gave a Soret peak at 437 nm in detergent micelles, and 402 nm in lipid bicelles. However, transfer of heme from hemoglobin yields a Soret maximum at 413 nm identical to that of the isolated protein. Further spectroscopic and kinetic analysis revealed that hemoglobin in the oxidized state is the most likely physiological substrate for ShuA. In addition, mutation of the conserved histidines, H86A or H420A, resulted in a loss of the ability of the receptor to efficiently extract heme from hemoglobin. In contrast the double mutant H86A/H420A was unable to extract heme from hemoglobin. These findings taken together confirm that both His-86 and His-420 are essential for substrate recognition, heme coordination, and transfer. Furthermore, the full-length TonB was shown to form a 1:1 complex with either apo-ShuA H86A/H420A or the wild-type ShuA. These observations provide a basis for future studies on the coordination and transport of heme by the TonB-dependent outer membrane receptors.

Identification of Transferrin-Binding Domains in TbpB Expressed by Neisseria gonorrhoeae

The transferrin iron acquisition system of Neisseria gonorrhoeae is necessary for iron uptake from transferrin in the human host and requires the participation of two distinct proteins: TbpA and TbpB. TbpA is a TonB-dependent outer membrane transporter responsible for the transport of iron into the cell. TbpB is a lipid-modified protein, for which a precise role in receptor function has not yet been elucidated. These receptor complex proteins show promise as vaccine candidates; therefore, it is important to identify surface-exposed regions of the proteins required for wild-type functions. In this study we examined TbpB, which has been reported to be surface exposed in its entirety; however, this hypothesis has never been tested experimentally. We placed the hemagglutinin (HA) epitope into TbpB with the dual purpose of examining the surface exposure of particular epitopes as well as their impact on receptor function. Nine insertion mutants were created, placing the epitope downstream of the signal peptidase II cleavage site. We report that the HA epitope is surface accessible in all mutants, indicating that the full-length TbpB is completely surface exposed. By expressing the TbpB-HA fusion proteins in N. gonorrhoeae, we were able to examine the impact of each insertion on the function of TbpB and the transferrin acquisition process. We propose that TbpB is comprised of two transferrin-binding-competent lobes, both of which are critical for efficient iron uptake from human transferrin.

The rag Locus of Porphyromonas gingivalis Contributes to Virulence in a Murine Model of Soft Tissue Destruction

The rag locus of Porphyromonas gingivalis encodes a putative TonB-dependent outer membrane receptor, RagA, and a 55-kDa immunodominant antigen, RagB. Inactivation of either ragA or ragB prevented expression of both RagA and RagB. Both the ragA and ragB mutants were significantly less virulent than wild-type strains in a murine model of infection.