Author Correction: Cryo-EM structure of the bacterial Ton motor subcomplex ExbB-ExbD provides information on structure and stoichiometry.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Decoupling Filamentous Phage Uptake and Energy of the TolQRA Motor in Escherichia coli.

Filamentous phages are nonlytic viruses that specifically infect bacteria, establishing a persistent association with their host. The phage particle has no machinery for generating energy and parasitizes its host's existing structures in order to cross the bacterial envelope and deliver its genetic material. The import of filamentous phages across the bacterial periplasmic space requires some of the components of a macrocomplex of the envelope known as the Tol system. This complex uses the energy provided by the proton motive force (pmf) of the inner membrane to perform essential and highly energy-consuming functions of the cell, such as envelope integrity maintenance and cell division. It has been suggested that phages take advantage of pmf-driven conformational changes in the Tol system to transit across the periplasm. However, this hypothesis has not been formally tested. In order to decouple the role of the Tol system in cell physiology and during phage parasitism, we used mutations on conserved essential residues known for inactivating pmf-dependent functions of the Tol system. We identified impaired Tol complexes that remain fully efficient for filamentous phage uptake. We further demonstrate that the TolQ-TolR homologous motor ExbB-ExbD, normally operating with the TonB protein, is able to promote phage infection along with full-length TolA.IMPORTANCE Filamentous phages are widely distributed symbionts of Gram-negative bacteria, with some of them being linked to genome evolution and virulence of their host. However, the precise mechanism that permits their uptake across the cell envelope is poorly understood. The canonical phage model Fd requires the TolQRA protein complex in the host envelope, which is suspected to translocate protons across the inner membrane. In this study, we show that phage uptake proceeds in the presence of the assembled but nonfunctional TolQRA complex. Moreover, our results unravel an alternative route for phage import that relies on the ExbB-ExbD proteins. This work provides new insights into the fundamental mechanisms of phage infection and might be generalized to other filamentous phages responsible for pathogen emergence.

Cryo-EM structure of the bacterial Ton motor subcomplex ExbB-ExbD 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.

Tol Energy-Driven Localization of Pal and Anchoring to the Peptidoglycan Promote Outer-Membrane Constriction.

During cell division, gram-negative bacteria must coordinate inner-membrane invagination, peptidoglycan synthesis and cleavage and outer-membrane (OM) constriction. The OM constriction remains largely enigmatic, and the nature of this process, passive or active, is under debate. The proton-motive force-dependent Tol-Pal system performs a network of interactions within these three compartments. Here we confirm that the trans-envelope Tol-Pal complex accumulates at constriction site in Escherichia coli. We show that the inner-membrane complex composed of TolA, TolQ and TolR recruits the OM complex TolB-Pal to the septum, in an energy-dependent process. Pal recruitment then allows its binding to peptidoglycan and subsequently OM constriction. Our results provide evidence that the constriction of the OM is an energized process.

Large Complexes: Cloning Strategy, Production, and Purification.

Membrane proteins can assemble and form complexes in the cell envelope. In Gram-negative bacteria, a number of multiprotein complexes, including secretion systems, efflux pumps, molecular motors, and pilus assembly machines, comprise proteins from the inner and outer membranes. Besides the structures of isolated soluble domains, only a few atomic structures of these assembled molecular machines have been elucidated. To better understand the function and to solve the structure of protein complexes, it is thus necessary to design dedicated production and purification processes. Here we present cloning procedures to overproduce membrane proteins into Escherichia coli cells and describe the cloning and purification strategy for the Type VI secretion TssJLM membrane complex.

Electrostatic interactions between the CTX phage minor coat protein and the bacterial host receptor TolA drive the pathogenic conversion of Vibrio cholerae.

Vibrio cholerae is a natural inhabitant of aquatic environments and converts to a pathogen upon infection by a filamentous phage, CTXΦ, that transmits the cholera toxin-encoding genes. This toxigenic conversion of V. cholerae has evident implication in both genome plasticity and epidemic risk, but the early stages of the infection have not been thoroughly studied. CTXΦ transit across the bacterial periplasm requires binding between the minor coat protein named pIII and a bacterial inner-membrane receptor, TolA, which is part of the conserved Tol-Pal molecular motor. To gain insight into the TolA-pIII complex, we developed a bacterial two-hybrid approach, named Oxi-BTH, suited for studying the interactions between disulfide bond-folded proteins in the bacterial cytoplasm of an Escherichia coli reporter strain. We found that two of the four disulfide bonds of pIII are required for its interaction with TolA. By combining Oxi-BTH assays, NMR, and genetic studies, we also demonstrate that two intermolecular salt bridges between TolA and pIII provide the driving forces of the complex interaction. Moreover, we show that TolA residue Arg-325 involved in one of the two salt bridges is critical for proper functioning of the Tol-Pal system. Our results imply that to prevent host evasion, CTXΦ uses an infection strategy that targets a highly conserved protein of Gram-negative bacteria essential for the fitness of V. cholerae in its natural environment.

Host cell interactions of outer membrane vesicle-associated virulence factors of enterohemorrhagic Escherichia coli O157: Intracellular delivery, trafficking and mechanisms of cell injury.

Outer membrane vesicles (OMVs) are important tools in bacterial virulence but their role in the pathogenesis of infections caused by enterohemorrhagic Escherichia coli (EHEC) O157, the leading cause of life-threatening hemolytic uremic syndrome, is poorly understood. Using proteomics, electron and confocal laser scanning microscopy, immunoblotting, and bioassays, we investigated OMVs secreted by EHEC O157 clinical isolates for virulence factors cargoes, interactions with pathogenetically relevant human cells, and mechanisms of cell injury. We demonstrate that O157 OMVs carry a cocktail of key virulence factors of EHEC O157 including Shiga toxin 2a (Stx2a), cytolethal distending toxin V (CdtV), EHEC hemolysin, and flagellin. The toxins are internalized by cells via dynamin-dependent endocytosis of OMVs and differentially separate from vesicles during intracellular trafficking. Stx2a and CdtV-B, the DNase-like CdtV subunit, separate from OMVs in early endosomes. Stx2a is trafficked, in association with its receptor globotriaosylceramide within detergent-resistant membranes, to the Golgi complex and the endoplasmic reticulum from where the catalytic Stx2a A1 fragment is translocated to the cytosol. CdtV-B is, after its retrograde transport to the endoplasmic reticulum, translocated to the nucleus to reach DNA. CdtV-A and CdtV-C subunits remain OMV-associated and are sorted with OMVs to lysosomes. EHEC hemolysin separates from OMVs in lysosomes and targets mitochondria. The OMV-delivered CdtV-B causes cellular DNA damage, which activates DNA damage responses leading to G2 cell cycle arrest. The arrested cells ultimately die of apoptosis induced by Stx2a and CdtV via caspase-9 activation. By demonstrating that naturally secreted EHEC O157 OMVs carry and deliver into cells a cocktail of biologically active virulence factors, thereby causing cell death, and by performing first comprehensive analysis of intracellular trafficking of OMVs and OMV-delivered virulence factors, we provide new insights into the pathogenesis of EHEC O157 infections. Our data have implications for considering O157 OMVs as vaccine candidates.

Structural insight into the role of the Ton complex in energy transduction.

In Gram-negative bacteria, outer membrane transporters import nutrients by coupling to an inner membrane protein complex called the Ton complex. The Ton complex consists of TonB, ExbB, and ExbD, and uses the proton motive force at the inner membrane to transduce energy to the outer membrane via TonB. Here, we structurally characterize the Ton complex from Escherichia coli using X-ray crystallography, electron microscopy, double electron-electron resonance (DEER) spectroscopy, and crosslinking. Our results reveal a stoichiometry consisting of a pentamer of ExbB, a dimer of ExbD, and at least one TonB. Electrophysiology studies show that the Ton subcomplex forms pH-sensitive cation-selective channels and provide insight into the mechanism by which it may harness the proton motive force to produce energy.

(1)H, (15)N and (13)C resonance assignments of the C-terminal domain of Vibrio cholerae TolA protein.

Vibrio cholerae is the bacterial causative agent of the human disease cholera. Non-pathogenic bacterium can be converted to pathogenic following infection by a filamentous phage, CTXΦ, that carries the cholera toxin encoding genes. A crucial step during phage infection requires a direct interaction between the CTXΦ minor coat protein (pIII(CTX)) and the C-terminal domain of V. cholerae TolA protein (TolAIIIvc). In order to get a better understanding of TolA function during the infection process, we have initiated a study of the V. cholerae TolAIII domain by 2D and 3D heteronuclear NMR. With the exception of the His-tag (H123-H128), 97 % of backbone (1)H, (15)N and (13)C resonances were assigned and the side chain assignments for 92 % of the protein were obtained (BMRB deposit with accession number 25689).

Virulence from vesicles: Novel mechanisms of host cell injury by Escherichia coli O104:H4 outbreak strain.

The highly virulent Escherichia coli O104:H4 that caused the large 2011 outbreak of diarrhoea and haemolytic uraemic syndrome secretes blended virulence factors of enterohaemorrhagic and enteroaggregative E. coli, but their secretion pathways are unknown. We demonstrate that the outbreak strain releases a cocktail of virulence factors via outer membrane vesicles (OMVs) shed during growth. The OMVs contain Shiga toxin (Stx) 2a, the major virulence factor of the strain, Shigella enterotoxin 1, H4 flagellin, and O104 lipopolysaccharide. The OMVs bind to and are internalised by human intestinal epithelial cells via dynamin-dependent and Stx2a-independent endocytosis, deliver the OMV-associated virulence factors intracellularly and induce caspase-9-mediated apoptosis and interleukin-8 secretion. Stx2a is the key OMV component responsible for the cytotoxicity, whereas flagellin and lipopolysaccharide are the major interleukin-8 inducers. The OMVs represent novel ways for the E. coli O104:H4 outbreak strain to deliver pathogenic cargoes and injure host cells.

Non classical secretion systems.

Bacteria use molecular machines or weapons to colonize, invade or fight other bacteria and eukaryotic cells. In addition to these various secretion systems, two different systems that release bacterial compounds have also been described. The first one corresponds to membrane vesicle formation and to long distance delivery of membrane or soluble components. The second system is dependent of the expression of the colicin lysis genes known for releasing cytoplasmic colicins as well as other soluble proteins. Both systems will be described thereafter.

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.

Colicin M, a peptidoglycan lipid-II-degrading enzyme: potential use for antibacterial means?

Colicins are proteins produced by some strains of Escherichia coli to kill competitors belonging to the same species. Among them, ColM (colicin M) is the only one that blocks the biosynthesis of peptidoglycan, a specific bacterial cell-wall polymer essential for cell integrity. ColM acts in the periplasm by hydrolysing the phosphoester bond of the peptidoglycan lipid intermediate (lipid II). ColM cytotoxicity is dependent on FkpA of the targeted cell, a chaperone with peptidylprolyl cis-trans isomerase activity. Dissection of ColM was used to delineate the catalytic domain and to identify the active-site residues. The in vitro activity of the isolated catalytic domain towards lipid II was 50-fold higher than that of the full-length bacteriocin. Moreover, this domain was bactericidal in the absence of FkpA under conditions that bypass the import mechanism (FhuA-TonB machinery). Thus ColM undergoes a maturation process driven by FkpA that is not required for the activity of the isolated catalytic domain. Genes encoding proteins with similarity to the catalytic domain of ColM were identified in pathogenic strains of Pseudomonas and other genera. ColM acts on several structures of lipid II representative of the diversity of peptidoglycan chemotypes. All together, these data open the way to the potential use of ColM-related bacteriocins as broad spectrum antibacterial agents.

The C-tail anchored TssL subunit, an essential protein of the enteroaggregative Escherichia coli Sci-1 Type VI secretion system, is inserted by YidC.

Type VI secretion systems (T6SS) are macromolecular complexes present in Gram-negative bacteria. T6SS are structurally similar to the bacteriophage cell-puncturing device and have been shown to mediate bacteria-host or bacteria-bacteria interactions. T6SS assemble from 13 to 20 proteins. In enteroaggregative Escherichia coli (EAEC), one of the subassemblies is composed of four proteins that form a trans-envelope complex: the TssJ outer membrane lipoprotein, the peptidoglycan-anchored inner membrane TagL protein, and two putative inner membrane proteins, TssL and TssM. In this study, we characterized the TssL protein of the EAEC Sci-1 T6SS in terms of localization, topology, and function. TssL is a critical component of the T6SS, anchored to the inner membrane through a single transmembrane segment located at the extreme C-terminus of the protein. We further show that this transmembrane segment is essential for the function of the protein and its proper insertion in the inner membrane is dependent upon YidC and modulated by the Hsp70 homologue DnaK.

Characterization of colicin M and its orthologs targeting bacterial cell wall peptidoglycan biosynthesis.

For a long time, colicin M was known for killing susceptible Escherichia coli cells by interfering with cell wall peptidoglycan biosynthesis, but its precise mode of action was only recently elucidated: this bacterial toxin was demonstrated to be an enzyme that catalyzes the specific degradation of peptidoglycan lipid intermediate II, thereby provoking the arrest of peptidoglycan synthesis and cell lysis. The discovery of this activity renewed the interest in this colicin and opened the way for biochemical and structural analyses of this new class of enzyme (phosphoesterase). The identification of a few orthologs produced by pathogenic strains of Pseudomonas further enlarged the field of investigation. The present article aims at reviewing recently acquired knowledge on the biology of this small family of bacteriocins.

An epigenetic switch involving overlapping fur and DNA methylation optimizes expression of a type VI secretion gene cluster.

Type VI secretion systems (T6SS) are macromolecular machines of the cell envelope of Gram-negative bacteria responsible for bacterial killing and/or virulence towards different host cells. Here, we characterized the regulatory mechanism underlying expression of the enteroagregative Escherichia coli sci1 T6SS gene cluster. We identified Fur as the main regulator of the sci1 cluster. A detailed analysis of the promoter region showed the presence of three GATC motifs, which are target of the DNA adenine methylase Dam. Using a combination of reporter fusion, gel shift, and in vivo and in vitro Dam methylation assays, we dissected the regulatory role of Fur and Dam-dependent methylation. We showed that the sci1 gene cluster expression is under the control of an epigenetic switch depending on methylation: fur binding prevents methylation of a GATC motif, whereas methylation at this specific site decreases the affinity of Fur for its binding box. A model is proposed in which the sci1 promoter is regulated by iron availability, adenine methylation, and DNA replication.

Regulation of type VI secretion gene clusters by sigma54 and cognate enhancer binding proteins.

Type VI secretion systems (T6SS) are bacteriophage-derived macromolecular machines responsible for the release of at least two proteins in the milieu, which are thought to form an extracellular appendage. Although several T6SS have been shown to be involved in the virulence of animal and plant pathogens, clusters encoding these machines are found in the genomes of most species of gram-negative bacteria, including soil, marine, and environmental isolates. T6SS have been associated with several phenotypes, ranging from virulence to biofilm formation or stress sensing. Their various environmental niches and large diversity of functions are correlated with their broad variety of regulatory mechanisms. Using a bioinformatic approach, we identified several clusters, including those of Vibrio cholerae, Aeromonas hydrophila, Pectobacterium atrosepticum, Pseudomonas aeruginosa, Pseudomonas syringae pv. tomato, and a Marinomonas sp., which possess typical -24/-12 sequences, recognized by the alternate sigma factor sigma 54 (σ(54) or σ(N)). σ(54), which directs the RNA polymerase to these promoters, requires the action of a bacterial enhancer binding protein (bEBP), which binds to cis-acting upstream activating sequences. Putative bEBPs are encoded within the T6SS gene clusters possessing σ(54) boxes. Using in vitro binding experiments and in vivo reporter fusion assays, we showed that the expression of these clusters is dependent on both σ(54) and bEBPs.

Mapping the interactions between Escherichia coli TolQ transmembrane segments.

The tolQRAB-pal operon is conserved in Gram-negative genomes. The TolQRA proteins of Escherichia coli form an inner membrane complex in which TolQR uses the proton-motive force to regulate TolA conformation and the in vivo interaction of TolA C-terminal region with the outer membrane Pal lipoprotein. The stoichiometry of the TolQ, TolR, and TolA has been estimated and suggests that 4-6 TolQ molecules are associated in the complex, thus involving interactions between the transmembrane helices (TMHs) of TolQ, TolR, and TolA. It has been proposed that an ion channel forms at the interface between two TolQ and one TolR TMHs involving the TolR-Asp(23), TolQ-Thr(145), and TolQ-Thr(178) residues. To define the organization of the three TMHs of TolQ, we constructed epitope-tagged versions of TolQ. Immunodetection of in vivo and in vitro chemically cross-linked TolQ proteins showed that TolQ exists as multimers in the complex. To understand how TolQ multimerizes, we initiated a cysteine-scanning study. Results of single and tandem cysteine substitution suggest a dynamic model of helix interactions in which the hairpin formed by the two last TMHs of TolQ change conformation, whereas the first TMH of TolQ forms intramolecular interactions.

Channel domain of colicin A modifies the dimeric organization of its immunity protein.

Proteins conferring immunity against pore-forming colicins are localized in the Escherichia coli inner membrane. Their protective effects are mediated by direct interaction with the C-terminal domain of their cognate colicins. Cai, the immunity protein protecting E. coli against colicin A, contains four cysteine residues. We report cysteine cross-linking experiments showing that Cai forms homodimers. Cai contains four transmembrane segments (TMSs), and dimerization occurs via the third TMS. Furthermore, we observe the formation of intramolecular disulfide bonds that connect TMS2 with either TMS1 or TMS3. Co-expression of Cai with its target, the colicin A pore-forming domain (pfColA), in the inner membrane prevents the formation of intermolecular and intramolecular disulfide bonds, indicating that pfColA interacts with the dimer of Cai and modifies its conformation. Finally, we show that when Cai is locked by disulfide bonds, it is no longer able to protect cells against exogenous added colicin A.