Direct interaction between fd phage pilot protein pIII and the TolQ-TolR proton-dependent motor provides new insights into the import of filamentous phages.

Filamentous phages are one of the simplest examples of viruses with a protein capsid that protects a circular single-stranded DNA genome. The infection is very specific, nonlytic, and can strongly affect the physiology or provide new pathogenic factors to its bacterial host. The infection process is proposed to rely on a pore-forming mechanism similar to that of certain nonenveloped eukaryotic viruses. The Ff coliphages (including M13, fd, and f1) have been intensively studied and were used to establish the sequence of events taking place for efficient crossing of the host envelope structure. However, the mechanism involved in the penetration of the cell inner membrane is not well understood. Here, we identify new host players involved in the phage translocation mechanism. Interaction studies by a combination of in vivo biochemical methods demonstrate that the adhesion protein pIII located at the tip of the phage binds to TolQ and TolR, two proteins that form a conserved proton-dependent molecular motor in the inner membrane of the host cell. Moreover, in vivo cysteine cross-linking studies reveal that the interactions between the pIII and TolQ or TolR occur between their transmembrane helix domains and may be responding to the proton motive force status of the cell. These results allow us to propose a model for the late stage of filamentous phage translocation mediated by multiple interactions with each individual component of the host TolQRA complex.

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.

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.

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.

Timing of TolA and TolQ Recruitment at the Septum Depends on the Functionality of the Tol-Pal System.

Efficient cell division of Gram-negative bacteria requires the presence of the Tol-Pal system to coordinate outer membrane (OM) invagination with inner membrane invagination (IM) and peptidoglycan (PG) remodeling. The Tol-Pal system is a trans-envelope complex that connects the three layers of the cell envelope through an energy-dependent process. It is composed of the three IM proteins, TolA, TolQ and TolR, the periplasmic protein TolB and the OM lipoprotein Pal. The proteins of the Tol-Pal system are dynamically recruited to the cell septum during cell division. TolA, the central hub of the Tol-Pal system, has three domains: a transmembrane helix (TolA1), a long second helical periplasmic domain (TolA2) and a C-terminal globular domain (TolA3). The TolQR complex uses the PMF to energize TolA, allowing its cyclic interaction via TolA3 with the OM TolB-Pal complex. Here, we confirm that TolA2 is sufficient to address TolA to the site of constriction, whereas TolA1 is recruited by TolQ. Analysis of the protein localization as function of the bacterial cell age revealed that TolA and TolQ localize earlier at midcell in the absence of the other Tol-Pal proteins. These data suggest that TolA and TolQ are delayed from their septal recruitment by the multiple interactions of TolA with TolB-Pal in the cell envelope providing a new example of temporal regulation of proteins recruitment at the septum.

The Tol-Pal system of Escherichia coli plays an unexpected role in the import of the oxyanions chromate and phosphate.

Chromate is a toxic metal that enters bacteria by using oxyanion importers. Here, we show that each mutant of the Tol-Pal system of Escherichia coli exhibited increased chromate resistance. This system, which spans the cell envelope, plays a major role in envelope integrity and septation. The ΔtolQR mutant accumulated three-fold less chromate than the wild-type. Addition of phosphate but not sulfate to rich medium drastically reduced chromate toxicity and import in the wild-type strain. Furthermore, the intracellular concentration of free inorganic phosphate was significantly reduced for the ΔtolR mutant in comparison to the wild-type strain. Moreover, extracellular labeled phosphate was significantly less incorporated into the ΔtolR mutant. Finally, two distinct TolQR mutant complexes, specifically affected in Tol-Pal energization without affecting the TolQRA complex structure, did not complement the ΔtolQR mutant for inorganic phosphate accumulation. We thus propose that, while the Pst system is well known to import inorganic phosphate, the Tol-Pal system participates to phosphate uptake in particular at medium to high extracellular phosphate concentrations. Since mutations disabling the Tol-Pal system lead to pleiotropic effects, chromate resistance and reduced inorganic phosphate import could occur from an indirect effect of mutations in components of the Tol-Pal system.

Colicin biology.

Colicins are proteins produced by and toxic for some strains of Escherichia coli. They are produced by strains of E. coli carrying a colicinogenic plasmid that bears the genetic determinants for colicin synthesis, immunity, and release. Insights gained into each fundamental aspect of their biology are presented: their synthesis, which is under SOS regulation; their release into the extracellular medium, which involves the colicin lysis protein; and their uptake mechanisms and modes of action. Colicins are organized into three domains, each one involved in a different step of the process of killing sensitive bacteria. The structures of some colicins are known at the atomic level and are discussed. Colicins exert their lethal action by first binding to specific receptors, which are outer membrane proteins used for the entry of specific nutrients. They are then translocated through the outer membrane and transit through the periplasm by either the Tol or the TonB system. The components of each system are known, and their implication in the functioning of the system is described. Colicins then reach their lethal target and act either by forming a voltage-dependent channel into the inner membrane or by using their endonuclease activity on DNA, rRNA, or tRNA. The mechanisms of inhibition by specific and cognate immunity proteins are presented. Finally, the use of colicins as laboratory or biotechnological tools and their mode of evolution are discussed.

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.

Deciphering the catalytic domain of colicin M, a peptidoglycan lipid II-degrading enzyme.

Colicin M inhibits Escherichia coli peptidoglycan synthesis through cleavage of its lipid-linked precursors. It has a compact structure, whereas other related toxins are organized in three independent domains, each devoted to a particular function: translocation through the outer membrane, receptor binding, and toxicity, from the N to the C termini, respectively. To establish whether colicin M displays such an organization despite its structural characteristics, protein dissection experiments were performed, which allowed us to delineate an independent toxicity domain encompassing exactly the C-terminal region conserved among colicin M-like proteins and covering about half of colicin M (residues 124-271). Surprisingly, the in vitro activity of the isolated domain was 45-fold higher than that of the full-length protein, suggesting a mechanism by which the toxicity of this domain is revealed following primary protein maturation. In vivo, the isolated toxicity domain appeared as toxic as the full-length protein under conditions where the reception and translocation steps were by-passed. Contrary to the full-length colicin M, the isolated domain did not require the presence of the periplasmic FkpA protein to be toxic under these conditions, demonstrating that FkpA is involved in the maturation process. Mutational analysis further identified five residues that are essential for cytotoxicity as well as in vitro lipid II-degrading activity: Asp-229, His-235, Asp-226, Tyr-228, and Arg-236. Most of these residues are surface-exposed and located relatively close to each other, hence suggesting they belong to the colicin M active site.

Toxicity of the colicin M catalytic domain exported to the periplasm is FkpA independent.

Colicin M (ColM) is a bactericidal protein that kills sensitive cells by hydrolyzing lipid II, involved in the biosynthesis of cell wall peptidoglycan. It recognizes FhuA on the outer leaflet, and its translocation through the outer membrane depends on the energized Ton complex in the inner membrane. To be active in the periplasm, ColM must be translocated through the outer membrane and then interact with FkpA, a periplasmic protein that exhibits both cis- and trans-peptidylprolyl isomerase (PPiase) and chaperon activities. In an attempt to directly target ColM to the periplasm of the producing bacteria, we fused the presequence of OmpA to ColM (sp-ColM). We found that expression of this hybrid protein in an Escherichia coli strain devoid of ColM immunity protein (Cmi) was bactericidal. We showed that sp-ColM was correctly expressed, processed, and associated with the inner membrane. sp-ColM toxicity was related to its enzymatic activity and did not rely on the TonB import proteins or the FhuA receptor. The presence of both activity domains of FkpA was still required for sp-ColM activity. Analyses of deletion mutants of sp-ColM show that the domain required for toxicity corresponds to the C-terminal last 153 amino acids of ColM. Like the full-length protein, this domain is not active in the presence of the immunity protein Cmi. On the other hand, it does not require FkpA for toxic activity.

Determinants of the proton selectivity of the colicin A channel.

The channel formed by colicin A in planar lipid bilayers has an outsized selectivity for protons compared to any other ion, even though it allows large ions, such as tetraethylammonium, to permeate readily. A mechanism to account for this discrepancy remains obscure. We considered that protons may traverse a separate pathway but were unable to find any evidence for one. Manipulations that interfere with ionic conduction, such as replacing some of the water in the pore with a nonelectrolyte, reduce the proton current along with the ionic current. Lipids have been proposed to play a structural role in the channel, but we found that the proton selectivity was unaffected by various gross changes in the lipid composition of the bilayer, effectively ruling out any specific effect of lipids in the selectivity and offering no support for their role in structure. The 10-helix channel-forming domains of colicins Ia and E1 are structurally homologous to that of colicin A but do not select so remarkably for protons; thus we were able to use them to probe for the regions responsible for the high selectivity. Using hybrids made by helix swapping among these proteins, we found that the anomalous selectivity could be localized to the five C-terminal helices of colicin A.

Similarities and Differences between Colicin and Filamentous Phage Uptake by Bacterial Cells.

Gram-negative bacteria have evolved a complex envelope to adapt and survive in a broad range of ecological niches. This physical barrier is the first line of defense against noxious compounds and viral particles called bacteriophages. Colicins are a family of bactericidal proteins produced by and toxic to Escherichia coli and closely related bacteria. Filamentous phages have a complex structure, composed of at least five capsid proteins assembled in a long thread-shaped particle, that protects the viral DNA. Despite their difference in size and complexity, group A colicins and filamentous phages both parasitize multiprotein complexes of their sensitive host for entry. They first bind to a receptor located at the surface of the target bacteria before specifically recruiting components of the Tol system to cross the outer membrane and find their way through the periplasm. The Tol system is thought to use the proton motive force of the inner membrane to maintain outer membrane integrity during the life cycle of the cell. This review describes the sequential docking mechanisms of group A colicins and filamentous phages during their uptake by their bacterial host, with a specific focus on the translocation step, promoted by interactions with the Tol system.

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.

Cell Fractionation.

Protein function is generally dependent on its subcellular localisation. In Gram-negative bacteria such as Escherichia coli, a protein can be targeted to five different compartments: the cytoplasm, the inner membrane, the periplasm, the outer membrane and the extracellular medium. Different approaches can be used to determine the protein localisation within a cell such as in silico identification of protein signal sequences and motifs, electron microscopy and immunogold labelling, optical fluorescence microscopy, and biochemical technics. In this chapter, we describe a simple and efficient method to isolate the different compartments of Escherichia coli by a fractionation method and to determine the presence of the protein of interest. For inner membrane proteins we propose a method to discriminate between integral and peripheral membrane proteins.

The pore-forming domain of colicin A fused to a signal peptide: a tool for studying pore-formation and inhibition.

Pore-forming colicins are plasmid-encoded bacteriocins that kill Escherichia coli and closely related bacteria. They bind to receptors in the outer membrane and are translocated across the cell envelope to the inner membrane where they form voltage-dependent ion-channels. Colicins are composed of three domains, with the C-terminal domain responsible for pore-formation. Isolated C-terminal pore-forming domains produced in the cytoplasm of E. coli are inactive due to the polarity of the transmembrane electrochemical potential, which is the opposite of that required. However, the pore-forming domain of colicin A (pfColA) fused to a prokaryotic signal peptide (sp-pfColA) is transported across and inserts into the inner membrane of E. coli from the periplasmic side, forming a functional channel. Sp-pfColA is specifically inhibited by the colicin A immunity protein (Cai). This construct has been used to investigate colicin A channel formation in vivo and to characterise the interaction of pfColA with Cai within the inner membrane. These points will be developed further in this review.

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.

Immunity protein protects colicin E2 from OmpT protease.

The endonuclease colicin E2 (ColE2), a bacteriocidal protein, and the associated cognate immunity protein (Im2) are released from producing Escherichia coli cells. ColE2 interaction with the target cell outer membrane BtuB protein and Tol import machinery allows the dissociation of Im2 from its colicin at the outer membrane surface. Here, we use in vivo approaches to show that a small amount of ColE2-Im2 protein complex bound to sensitive cells is susceptible to proteolytic cleavage by the outer membrane protease, OmpT. The presence of BtuB is required for ColE-Im2 cleavage by OmpT. The amount of colicin cleaved by OmpT is greatly enhanced when ColE2 is dissociated from Im2. We further demonstrate that OmpT cleaves the C-terminal DNase domain of the toxin. As expected, strains that over-produce OmpT are less susceptible to infection by ColE2 than by ColE2-Im2. Our findings reveal an additional function for the immunity protein beside protection of producing cells against their own colicin in the cytoplasm. Im2 protects ColE2 against OmpT-mediated proteolytic attack.

Colicin E2 is still in contact with its receptor and import machinery when its nuclease domain enters the cytoplasm.

Colicins reach their targets in susceptible Escherichia coli strains through two envelope protein systems: the Tol system is used by group A colicins and the TonB system by group B colicins. Colicin E2 (ColE2) is a cytotoxic protein that recognizes the outer membrane receptor BtuB. After gaining access to the cytoplasmic membrane of sensitive Escherichia coli cells, ColE2 enters the cytoplasm to cleave DNA. After binding to BtuB, ColE2 interacts with the Tol system to reach its target. However, it is not known if the entire colicin or only the nuclease domain of ColE2 enters the cell. Here I show that preincubation of ColE2 with Escherichia coli cells prevents binding and translocation of pore-forming colicins of group A but not of group B. This inhibition persisted even when cells were incubated with ColE2 for 30 min before the addition of pore-forming colicins, indicating that ColE2 releases neither its receptor nor its translocation machinery when its nuclease domain enters the cells. These competition experiments enabled me to estimate the time required for ColE2 binding to its receptor and translocation.