S. Peter Howard

In Vivo Evidence for TonB Dimerization

TonB, in complex with ExbB and ExbD, is required for the energy-dependent transport of ferric siderophores across the outer membrane of Escherichia coli, the killing of cells by group B colicins, and infection by phages T1 and phi80. To gain insights into the protein complex, TonB dimerization was studied by constructing hybrid proteins from complete TonB (containing amino acids 1 to 239) [TonB(1-239)] and the cytoplasmic fragment of ToxR which, when dimerized, activates the transcription of the cholera toxin gene ctx. ToxR(1-182)-TonB(1-239) activated the transcription of lacZ under the control of the ctx promoter (P(ctx)::lacZ). Replacement of the TonB transmembrane region by the ToxR transmembrane region resulted in the hybrid proteins ToxR(1-210)-TonB(33-239) and ToxR(1-210)-TonB(164-239), of which only the latter activated P(ctx)::lacZ transcription. Dimer formation was reduced but not abolished in a mutant lacking ExbB and ExbD, suggesting that these complex components may influence dimerization but are not strictly required and that the N-terminal cytoplasmic membrane anchor and the C-terminal region are important for dimer formation. The periplasmic TonB fragment, TonB(33-239), inhibits ferrichrome and ferric citrate transport and induction of the ferric citrate transport system. This competition provided a means to positively screen for TonB(33-239) mutants which displayed no inhibition. Single point mutations of inactive fragments selected in this manner were introduced into complete TonB, and the phenotypes of the TonB mutant strains were determined. The mutations located in the C-terminal half of TonB, three of which (Y163C, V188E, and R204C) were obtained separately by site-directed mutagenesis, as was the isolated F230V mutation, were studied in more detail. They displayed different activity levels for various TonB-dependent functions, suggesting function-related specificities which reflect differences in the interactions of TonB with various transporters and receptors.

A TonB‐like protein and a novel membrane protein containing an ATP‐binding cassette function together in exotoxin secretion

Protein secretion by many Gram‐negative bacteria occurs via the type II pathway involving translocation across the cytoplasmic and outer membranes in separate steps. The mechanism by which metabolic energy is supplied to the translocation across the outer membrane is unknown. Here we show that two Aeromonas hydrophila inner membrane proteins, ExeA and ExeB, are required for this process. ExeB bears sequence as well as topological similarity to TonB, a protein which opens gated ports for the inward translocation of ligands across the outer membrane. ExeA is a novel membrane protein which contains a consensus ATP‐binding site. Mutations in this site dramatically decreased the rate of secretion of the toxin aerolysin from the cell. ExeB was stable when overproduced in the presence of ExeA, but was degraded when synthesized in its absence, indicating that the two proteins form a complex. These results suggest that ExeA and ExeB may act together to transduce metabolic energy to the opening of a secretion port in the outer membrane.

Expression of the ExeAB complex of Aeromonas hydrophila is required for the localization and assembly of the ExeD secretion port multimer

Aeromonas hydrophila secretes protein toxins via the type II pathway, involving the products of at least two operons, exeAB (gspAB) and exeC‐N (gspC‐N). In the studies reported here, aerolysin secretion was restored to C5.84, an exeA::Tn5–751 mutant, by overexpression of exeD alone in trans. Expression studies indicated that these results did not reflect a role of ExeAB in the regulation of the exeC‐N operon. Instead, immunoblot analysis showed that ExeD did not multimerize in C5.84, and fractionation of the membranes showed that the monomeric ExeD remained in the inner membrane. Expression of ExeAB, but not either protein alone, from a plasmid in C5.84 resulted in increases in the amount of multimeric ExeD, which correlated with increases in aerolysin secretion. Pulse‐chase analysis also sug‐gested that the induction of ExeAB allowed multimerization of previously accumulated monomer ExeD. In C5.84 cells overproducing ExeD, it multimerized even in the absence of ExeAB and, although most remained in the inner membrane, an amount similar to that in wild‐type outer membranes fractionated with the outer membrane of the overproducing cells. These results indicate that the secretion defect of exeAB mutants is a result of an inability to assemble the ExeD secretin in the outer membrane. The localization and multimerization of overproduced ExeD in these mutants further suggests that the ExeAB complex plays either a direct or indirect role in the transport of ExeD into the outer membrane.

In Vivo Synthesis of the Periplasmic Domain of TonB Inhibits Transport through the FecA and FhuA Iron Siderophore Transporters of Escherichia coli

The siderophore transport activities of the two outer membrane proteins FhuA and FecA of Escherichia coli require the proton motive force of the cytoplasmic membrane. The energy of the proton motive force is postulated to be transduced to the transport proteins by a protein complex that consists of the TonB, ExbB, and ExbD proteins. In the present study, TonB fragments lacking the cytoplasmic membrane anchor were exported to the periplasm by fusing them to the cleavable signal sequence of FecA. Overexpressed TonB(33-239), TonB(103-239), and TonB(122-239) fragments inhibited transport of ferrichrome by FhuA and of ferric citrate by FecA, transcriptional induction of the fecABCDE transport genes by FecA, infection by phage phi80, and killing of cells by colicin M via FhuA. Transport of ferrichrome by FhuADelta5-160 was also inhibited by TonB(33-239), although FhuADelta5-160 lacks the TonB box which is involved in TonB binding. The results show that TonB fragments as small as the last 118 amino acids of the protein interfere with the function of wild-type TonB, presumably by competing for binding sites at the transporters or by forming mixed dimers with TonB that are nonfunctional. In addition, the interactions that are inhibited by the TonB fragments must include more than the TonB box, since transport through corkless FhuA was also inhibited. Since the periplasmic TonB fragments cannot assume an energized conformation, these in vivo studies also agree with previous cross-linking and in vitro results, suggesting that neither recognition nor binding to loaded siderophore receptors is the energy-requiring step in the TonB-receptor interactions.

An ExeAB complex in the type II secretion pathway of Aeromonas hydrophila: effect of ATP-binding cassette mutations on complex formation and function.

The energy‐dependent secretion of aerolysin by Aeromonas hydrophila requires the ExeA and ExeB proteins. An 85 kDa complex containing the two proteins was identified in wild‐type cells but not in cells producing either protein alone. Radiolabelling followed by cross‐linking, immunoprecipitation and then reduction of the cross‐links confirmed the presence of the two proteins in the same complex. The complex could also be extracted intact from cell membranes with non‐ionic detergents. A G229D substitution in the kinase‐3a motif of ExeA strongly reduced the level of aerolysin secretion, as did the replacement of the invariant Lys of the kinase‐1a motif (K56) with Arg. The G229D mutant contained very little of the ExeA–ExeB complex, but overexpression of the mutant complex until wild‐type levels were achieved allowed normal secretion. In contrast, the K56R mutation had no effect on complex formation, but normal secretion levels occurred only when there was a far greater amount of the complex present. These results are consistent with a model in which binding of ATP by ExeA is required for ExeA–ExeB complex formation, while hydrolysis is required for its function in secretion once established.

The solution structure of the periplasmic domain of the TonB system ExbD protein reveals an unexpected structural homology with siderophore‐binding proteins

The transport of iron complexes through outer membrane transporters from Gram‐negative bacteria is highly dependent on the TonB system. Together, the three components of the system, TonB, ExbB and ExbD, energize the transport of iron complexes through the outer membrane by utilizing the proton motive force across the cytoplasmic membrane. The three‐dimensional (3D) structure of the periplasmic domain of TonB has previously been determined. However, no detailed structural information for the other two components of the TonB system is currently available and their role in the iron‐uptake process is not yet clearly understood. ExbD from Escherichia coli contains 141 residues distributed in three domains: a small N‐terminal cytoplasmic region, a single transmembrane helix and a C‐terminal periplasmic domain. Here we describe the first well‐defined solution structure of the periplasmic domain of ExbD (residues 44–141) solved by multidimensional nuclear magnetic resonance (NMR) spectroscopy. The monomeric structure presents three clearly distinct regions: an N‐terminal flexible tail (residues 44–63), a well‐defined folded region (residues 64–133) followed by a small C‐terminal flexible region (residues 134–141). The folded region is formed by two α‐helices that are located on one side of a single β‐sheet. The central β‐sheet is composed of five β‐strands, with a mixed parallel and antiparallel arrangement. Unexpectedly, this fold closely resembles that found in the C‐terminal lobe of the siderophore‐binding proteins FhuD and CeuE. The ExbD periplasmic domain has a strong tendency to aggregate in vitro and 3D‐TROSY (transverse relaxation optimized spectroscopy) NMR experiments of the deuterated protein indicate that the multimeric protein has nearly identical secondary structure to that of the monomeric form. Chemical shift perturbation studies suggest that the Glu‐Pro region (residues 70–83) of TonB can bind weakly to the surface and the flexible C‐terminal region of ExbD. At the same time the Lys‐Pro region (residues 84–102) and the folded C‐terminal domain (residues 150–239) of TonB do not show significant binding to ExbD, suggesting that the main interactions forming the TonB complex occur in the cytoplasmic membrane.

The proline-rich domain of TonB possesses an extended polyproline II-like conformation of sufficient length to span the periplasm of Gram-negative bacteria.

TonB from Escherichia coli and its homologues are critical for the uptake of siderophores through the outer membrane of Gram‐negative bacteria using chemiosmotic energy. When different models for the mechanism of TonB mediated energy transfer from the inner to the outer membrane are discussed, one of the key questions is whether TonB spans the periplasm. In this article, we use long range distance measurements by spin‐label pulsed EPR (Double Electron–Electron Resonance, DEER) and CD spectroscopy to show that the proline‐rich segment of TonB exists in a PPII‐like conformation. The result implies that the proline‐rich segment of TonB possesses a length of more than 15 nm, sufficient to span the periplasm of Gram‐negative bacteria.

Secretion and properties of the large and small lobes of the channel-forming toxin aerolysin

Aerolysin is a dimeric protein secreted by Aeromonas spp. that binds to glycosylphosphatidylinositol‐anchored receptors on target cells and becomes insertion competent by oligomerizing. The protein comprises two lobes joined by a short arm. The large lobe is thought to be responsible for channel formation, whereas the small lobe is believed to stabilize the dimer, and it may also contain the receptor binding site. We cloned and expressed the DNA for both lobes of the toxin separately and together in A. salmonicida. The large lobe produced alone was secreted, although more poorly than native protein. The small lobe with the arm produced by itself was not secreted. When the large lobe without the arm was co‐produced with the small lobe with the arm, both were secreted, and they co‐purified as a stoichiometric complex. Analytical ultracentrifugation showed that they form a heterotetramer corresponding to the native dimer. The purified product was nearly as active as aerolysin, but lost activity and became trypsin sensitive above 25°C. The large lobe with the arm was also purified. It was shown to be monomeric, confirming that the small lobe is responsible for dimer stabilization. The large lobe had very low channel‐forming activity, although it was correctly processed by trypsin, and it could form stable oligomers. Surprisingly, the large lobe was found to bind to several glycosylphosphatidylinositol‐anchored proteins, indicating that it contains at least part of the receptor‐binding domain.

Involvement of the GspAB Complex in Assembly of the Type II Secretion System Secretin of Aeromonas and Vibrio Species

The type II secretion system (T2SS) functions as a transport mechanism to translocate proteins from the periplasm to the extracellular environment. The ExeA homologue in Aeromonas hydrophila, GspA(Ah), is an ATPase that interacts with peptidoglycan and forms an inner membrane complex with the ExeB homologue (GspB(Ah)). The complex may be required to generate space in the peptidoglycan mesh that is necessary for the transport and assembly of the megadalton-sized ExeD homologue (GspD(Ah)) secretin multimer in the outer membrane. In this study, the requirement for GspAB in the assembly of the T2SS secretin in Aeromonas and Vibrio species was investigated. We have demonstrated a requirement for GspAB in T2SS assembly in Aeromonas salmonicida, similar to that previously observed in A. hydrophila. In the Vibrionaceae species Vibrio cholerae, Vibrio vulnificus, and Vibrio parahaemolyticus, gspA mutations significantly decreased assembly of the secretin multimer but had minimal effects on the secretion of T2SS substrates. The lack of effect on secretion of the mutant of gspA of V. cholerae (gspA(Vc)) was explained by the finding that native secretin expression greatly exceeds the level needed for efficient secretion in V. cholerae. In cross-complementation experiments, secretin assembly and secretion in an A. hydrophila gspA mutant were partially restored by the expression of GspAB from V. cholerae in trans, further suggesting that GspAB(Vc) performs the same role in Vibrio species as GspAB(Ah) does in the aeromonads. These results indicate that the GspAB complex is functional in the assembly of the secretin in Vibrio species but that a redundancy of GspAB function may exist in this genus.

ExeA binds to peptidoglycan and forms a multimer for assembly of the type II secretion apparatus in Aeromonas hydrophila.

Aeromonas hydrophila uses the type II secretion system (T2SS) to transport protein toxins across the outer membrane. The inner membrane complex ExeAB is required for assembly of the ExeD secretion channel multimer, called the secretin, into the outer membrane. A putative peptidoglycan‐binding domain (Pfam number PF01471) conserved in many peptidoglycan‐related proteins is present in the periplasmic region of ExeA (P‐ExeA). In this study, co‐sedimentation analysis revealed that P‐ExeA was able to bind to highly pure peptidoglycan. The protein assembled into large multimers in the presence of peptidoglycan fragments, as shown in native PAGE, gel filtration and cross‐linking experiments. The requirement of peptidoglycan for multimerization was abrogated when the protein was incubated at 30°C and above. These results provide evidence that the putative peptidoglycan‐binding domain of ExeA is involved in physical contact with peptidoglycan. The interactions facilitate the multimerization of ExeA, favouring a model in which the protein forms a multimeric structure on the peptidoglycan during the ExeAB‐dependent assembly of the secretin multimer in the outer membrane.