Protein import into Escherichia coli: colicins A and E1 interact with a component of their translocation system.

Colicins are antibiotic proteins that kill sensitive Escherichia coli cells. Their mode of action involves three steps: binding to specific receptors located in the outer membrane, translocation across this membrane, and action on their targets. A specific colicin domain can be assigned to each of these steps. Colicins have been subdivided into two groups (A and B) depending on the proteins required for them to cross the external membrane. Plasmids were constructed which led to an overproduction of the Tol proteins involved in the import of group A colicins. In vitro binding of overexpressed Tol proteins to either Tol-dependent (group A) or TonB-dependent (group B) colicins was analyzed. The Tol dependent colicins A and E1 were able to interact with TolA but the TonB dependent colicin B was not. The C-terminal region of TolA, which is necessary for colicin uptake, was also found to be necessary for colicin A and E1 binding to occur. Furthermore, only the isolated N-terminal domain of colicin A, which is involved in the translocation step, was found to bind to TolA. These results demonstrate the existence of a correlation between the ability of group A colicins to translocate and their in vitro binding to TolA protein, suggesting that these interactions might be part of the colicin import process.     

Tol-dependent macromolecule import through the Escherichia coli cell envelope requires the presence of an exposed TolA binding motif

The Tol-Pal proteins of the cell envelope of Escherichia coli are required for maintaining outer membrane integrity. This system forms protein complexes in which TolA plays a central role by providing a bridge between the inner and outer membranes via its interaction with the Pal lipoprotein. The Tol proteins are parasitized by filamentous bacteriophages and group A colicins. The N-terminal domain of the Ff phage g3p protein and the translocation domains of colicins interact directly with TolA during the processes of import through the cell envelope. Recently, a four-amino-acid sequence in Pal has been shown to be involved in Pal's interaction with TolA. A similar motif is also present in the sequence of two TolA partners, g3p and colicin A. Here, a mutational study was conducted to define the function of these motifs in the binding activity and import process of TolA. The various domains were produced and exported to the bacterial periplasm, and their cellular effects were analyzed. Cells producing the g3p domain were tolerant to colicins and filamentous phages and had destabilized outer membranes, while g3p deleted of three residues in the motif was affected in TolA binding and had no effect on cell integrity or colicin or phage import. A conserved Tyr residue in the colicin A translocation domain was involved in TolA binding and colicin A import. Furthermore, in vivo and in vitro coprecipitation analyses demonstrated that colicin A and g3p N-terminal domains compete for binding to TolA.     

The TolA-recognition site of colicin N. ITC, SPR and stopped-flow fluorescence define a crucial 27-residue segment

Colicins translocate across the Escherichia coli outer membrane and periplasm by interacting with several receptors. After first binding to the outer membrane surface receptors via their central region, they interact with TolA or TonB proteins via their N-terminal region. Colicin N residues critical to TolA binding have been discovered, but the full extent of any colicin TolA site is unknown. We present, for the first time, a fully mapped TolA binding site for a colicin. It was determined through the use of alanine-scanning mutants, glutathione S-transferase fusion peptides and Biacore/fluorescence binding studies. The minimal TolA binding region is 27 residues and of similar size to the TolA binding region of bacteriophage g3p-D1 protein. Stopped-flow kinetic studies show that the binding to TolA follows slow association kinetics. The role of other E. coli Tol proteins in colicin translocation was also investigated. Isothermal titration microcalorimetry (ITC) and in vivo studies conclusively show that colicin N translocation does not require the presence of TolB. ITC also demonstrated colicin A interaction with TolB, and that colicin A in its native state does not interact with TolAII-III. Colicin N does not bind TolR-II. The TolA protein is shown to be unsuitable for direct immobilisation in Biacore analysis.     

TolA central domain interacts with Escherichia coli porins.

TolA is an inner membrane protein with three domains: a transmembrane N-terminus and periplasmic central and C-terminal domains. The interaction of TolA with outer membrane porins of Escherichia coli was investigated. Western blot analyses of cell extracts with anti-TolA antibodies indicated that TolA forms high molecular weight complexes specifically with trimeric OmpF, OmpC, PhoE and LamB, but not with OmpA. The interaction of purified TolA domains with purified porins was also studied. TolA interacted with OmpF, PhoE and LamB porins via its central domain, but not with either their denatured monomeric forms or OmpA. Moreover, the presence or absence of lipopolysaccharides associated with trimeric porins did not modify the interactions. These results suggest that the specific interaction of TolA with outer membrane porins might be relevant to the function of Tol proteins.     

Allosteric β‐propeller signalling in TolB and its manipulation by translocating colicins

The Tol system is a five‐protein assembly parasitized by colicins and bacteriophages that helps stabilize the Gram‐negative outer membrane (OM). We show that allosteric signalling through the six‐bladed β‐propeller protein TolB is central to Tol function in Escherichia coli and that this is subverted by colicins such as ColE9 to initiate their OM translocation. Protein–protein interactions with the TolB β‐propeller govern two conformational states that are adopted by the distal N‐terminal 12 residues of TolB that bind TolA in the inner membrane. ColE9 promotes disorder of this ‘TolA box’ and recruitment of TolA. In contrast to ColE9, binding of the OM lipoprotein Pal to the same site induces conformational changes that sequester the TolA box to the TolB surface in which it exhibits little or no TolA binding. Our data suggest that Pal is an OFF switch for the Tol assembly, whereas colicins promote an ON state even though mimicking Pal. Comparison of the TolB mechanism to that of vertebrate guanine nucleotide exchange factor RCC1 suggests that allosteric signalling may be more prevalent in β‐propeller proteins than currently realized.     

The Tol/Pal system function requires an interaction between the C-terminal domain of TolA and the N-terminal domain of TolB

The Tol/Pal system of Escherichia coli is composed of the YbgC, TolQ, TolA, TolR, TolB, Pal and YbgF proteins. It is involved in maintaining the integrity of the outer membrane, and is required for the uptake of group A colicins and DNA of filamentous bacteriophages. To identify new interactions between the components of the Tol/Pal system and gain insight into the mechanism of colicin import, we performed a yeast two-hybrid screen using the different components of the Tol/Pal system and colicin A. Using this system, we confirmed the already known interactions and identified several new interactions. TolB dimerizes and the periplasmic domain of TolA interacts with YbgF and TolB. Our results indicate that the central domain of TolA (TolAII) is sufficient to interact with YbgF, that the C-terminal domain of TolA (TolAIII) is sufficient to interact with TolB, and that the amino terminal domain of TolB (D1) is sufficient to bind TolAIII. The TolA/TolB interaction was confirmed by cross-linking experiments on purified proteins. Moreover, we show that the interaction between TolA and TolB is required for the uptake of colicin A and for the membrane integrity. These results demonstrate that the TolA/TolB interaction allows the formation of a trans-envelope complex that brings the inner and outer membranes in close proximity.     

The TolB protein interacts with the porins of Escherichia coli.

TolB is a periplasmic protein of the cell envelope Tol complex. It is partially membrane associated through an interaction with the outer membrane lipoprotein PAL (peptidoglycan-associated lipoprotein), which also belongs to the Tol system. The interaction of TolB with outer membrane porins of Escherichia coli was investigated with a purified TolB derivative harboring a six-histidine tag. TolB interacted with the trimeric porins OmpF, OmpC, PhoE, and LamB but not with their denatured monomeric forms or OmpA. These interactions took place both in the presence and in the absence of lipopolysaccharide. TolA, an inner membrane component of the Tol system, also interacts with the trimeric porins via its central periplasmic domain (R. Dérouiche, M. Gavioli, H. Bénédetti, A. Prilipov, C. Lazdunski, and R. Lloubès, EMBO J. 15:6408-6415, 1996). In the presence of the purified central domain of TolA (TolAIIHis), the TolB-porin complexes disappeared to form TolAIIHis-porin complexes. These results suggest that the interactions of TolA and TolB with porins might take place in vivo and might be concomitant events participating in porin assembly. They also suggest that the Tol system as a whole may be involved in porin assembly in the outer membrane.     

Programmed cell death and the pathogenesis of tissue injury induced by type A Francisella tularensis

The protein Pal (peptidoglycan-associated lipoprotein) is anchored in the outer membrane (OM) of Gram-negative bacteria and interacts with Tol proteins. Tol–Pal proteins form two complexes: the first is composed of three inner membrane Tol proteins (TolA, TolQ and TolR); the second consists of the TolB and Pal proteins linked to the cell's OM. These complexes interact with one another forming a multiprotein membrane-spanning system. It has recently been demonstrated that Pal is essential for bacterial survival and pathogenesis, although its role in virulence has not been clearly defined. This review summarizes the available data concerning the structure and function of Pal and its role in pathogenesis.     

Import of colicins across the outer membrane of Escherichia coli involves multiple protein interactions in the periplasm

Several proteins of the Tol/Pal system are required for group A colicin import into Escherichia coli. Colicin A interacts with TolA and TolB via distinct regions of its N-terminal domain. Both interactions are required for colicin translocation. Using in vivo and in vitro approaches, we show in this study that colicin A also interacts with a third component of the Tol/Pal system required for colicin import, TolR. This interaction is specific to colicins dependent on TolR for their translocation, strongly suggesting a direct involvement of the interaction in the colicin translocation step. TolR is anchored to the inner membrane by a single transmembrane segment and protrudes into the periplasm. The interaction involves part of the periplasmic domain of TolR and a small region of the colicin A N-terminal domain. This region and the other regions responsible for the interaction with TolA and TolB have been mapped precisely within the colicin A N-terminal domain and appear to be arranged linearly in the colicin sequence. Multiple contacts with periplasmic-exposed Tol proteins are therefore a general principle required for group A colicin translocation.     

TolA Modulates the Oligomeric Status of YbgF in the Bacterial Periplasm

The trans-envelope Tol complex of Gram-negative bacteria is recruited to the septation apparatus during cell division where it is involved in stabilizing the outer membrane. The last gene in the tol operon, ybgF, is highly conserved, yet does not seem to be required for Tol function. We have addressed this anomaly by characterizing YbgF from Escherichia coli and its interaction with TolA, which, based on previous yeast two-hybrid data, is the only known physical link between YbgF and the Tol system. We show that the stable YbgF trimer undergoes a marked change in oligomeric state on binding TolA, forming a one-to-one complex with the Tol protein. Through a combination of pull-down assays, deletion analysis, and isothermal titration calorimetry, we map the TolA-YbgF interface to the C-terminal tetratricopeptide repeat domain of YbgF and 31 residues at the C-terminal end of TolA domain II (TolA^280-313). We show that TolB, which binds TolA domain III close to the YbgF binding site, has no impact on the YbgF-TolA association. We also report the crystal structures of the two component domains of YbgF, the N-terminal coiled coil from E. coli YbgF, which forms a stable trimer and controls the oligomeric status of YbgF, and the monomeric tetratricopeptide repeat domain from Xanthomonas campestris YbgF, which is also able to trimerize. Although the coiled coil is not directly involved in TolA binding, we demonstrate that the regular hydrophilic patterning of its otherwise hydrophobic core is a prerequisite for the TolA-induced oligomeric-state transition of YbgF. We postulate that rather than YbgF affecting Tol function, it is the change in YbgF oligomeric status (with an accompanying change in its function) that likely explains the necessity for tight co-regulation of the ybgF and tol genes in Gram-negative bacteria.     

Thermoregulation-dependent expression of Yersinia enterocolitica protein 1 imparts serum resistance to Escherichia coli K-12.

Resistance to the bactericidal action of normal human serum is one of the characteristics of virulent Yersinia enterocolitica. This property is attributable to the virulence plasmid harbored by pathogenic strains of the species. Serum resistance in Y. enterocolitica is thermoregulated, and its expression correlates well with the presence of virulence plasmid-encoded outer membrane proteins. To further examine the biochemical basis underlying resistance, we cloned a large segment (ca. 30 kilobases) of virulence plasmid DNA and studied the expression of plasmid-encoded outer membrane proteins in a serum-sensitive strain of Escherichia coli. The presence of the 160-kilodalton Y. enterocolitica-derived outer membrane protein 1 on E. coli transformants conferred a high degree of hydrophobicity, autoagglutinability, and resistance to serum killing. All of these properties were thermoregulated in E. coli with fidelity, suggesting that a functional thermoregulatory element was present in the cloned DNA. Elimination of protein 1 from the outer membrane of E. coli transformants by insertional inactivation of the structural gene with a Kanr gene cassette abrogated all of these properties and returned the serum-sensitive phenotype.     

Role of the carboxyl-terminal domain of TolA in protein import and integrity of the outer membrane.

The TolA protein is involved in maintaining the integrity of the outer membrane of Escherichia coli, as mutations in tolA cause the bacteria to become hypersensitive to detergents and certain antibiotics and to leak periplasmic proteins into the medium. This protein also is required for the group A colicins to exert their effects and for many of the filamentous single-stranded bacteriophage to infect the bacterial cell. TolA is a three-domain protein, with the amino-terminal domain anchoring it to the inner membrane. The helical second domain is proposed to span the periplasmic space to allow the carboxyl-terminal third domain to interact with the outer membrane. A plasmid that allowed the synthesis and transport of the carboxyl-terminal third domain into the periplasmic space was constructed. The presence of an excess of this domain in the periplasm of a wild-type cell resulted in an increased sensitivity to deoxycholate, the release of periplasmic alkaline phosphatase and RNase into the medium, and an increased tolerance to colicins E1, E2, E3, and A. There was no effect on the cells' response to colicin D, which depends on TonB instead of TolA for its action. The presence of the free carboxyl-terminal domain of TolA in the periplasm in a tolA null mutation did not restore the wild-type phenotype, suggesting that this domain must be part of the intact TolA molecule to perform its function. Our results are consistent with a model in which the carboxyl-terminal domain of TolA interacts with components in the periplasm or on the inner surface of the outer membrane to function in maintaining the integrity of this membrane.     

The Tol proteins of Escherichia coli and their involvement in the translocation of group A colicins

The Tol proteins are involved in outer membrane stability of Gram-negative bacteria. The TolQRA proteins form a complex in the inner membrane while TolB and Pal interact near the outer membrane. These two complexes are transiently connected by an energy-dependent interaction between Pal and TolA. The Tol proteins have been parasitized by group A colicins for their translocation through the cell envelope. Recent advances in the structure and energetics of the Tol system, as well as the interactions between the N-terminal translocation domain of colicins and the Tol proteins are presented.     

Distinct regions of the colicin A translocation domain are involved in the interaction with TolA and TolB proteins upon import into Escherichia coli

Group A colicins need proteins of the Escherichia coli envelope Tol complex (TolA, TolB, TolQ and TolR) to reach their cellular target. The N-terminal domain of colicins is involved in the import process. The N-terminal domains of colicins A and E1 have been shown to interact with TolA, and the N-terminal domain of colicin E3 has been shown to interact with TolB. We found that a pentapeptide conserved in the N-terminal domain of all group A colicins, the 'TolA box', was important for colicin A import but was not involved in the colicin A–TolA interaction. It was, however, involved in the colicin A–TolB interaction. The interactions of colicin A N-terminal domain deletion mutants with TolA and TolB were investigated. Random mutagenesis was performed on a construct allowing the colicin A N-terminal domain to be exported in the bacteria periplasm. This enabled us to select mutant protein domains unable to compete with the wild-type domain of the entire colicin A for import into the cells. Our results demonstrate that different regions of the colicin A N-terminal domain interact with TolA and TolB. The colicin A N-terminal domain was also shown to form a trimeric complex with TolA and TolB.     

Structural evidence that colicin A protein binds to a novel binding site of TolA protein in Escherichia coli periplasm

The Tol assembly of proteins is an interacting network of proteins located in the Escherichia coli cell envelope that transduces energy and contributes to cell integrity. TolA is central to this network linking the inner and outer membranes by interactions with TolQ, TolR, TolB, and Pal. Group A colicins, such as ColA, parasitize the Tol network through interactions with TolA and/or TolB to facilitate translocation through the cell envelope to reach their cytotoxic site of action. We have determined the first structure of the C-terminal domain of TolA (TolAIII) bound to an N-terminal ColA polypeptide (TA(53-107)). The interface region of the TA(53-107)-TolAIII complex consists of polar contacts linking residues Arg-92 to Arg-96 of ColA with residues Leu-375-Pro-380 of TolA, which constitutes a β-strand addition commonly seen in more promiscuous protein-protein contacts. The interface region also includes three cation-π interactions (Tyr-58-Lys-368, Tyr-90-Lys-379, Phe-94-Lys-396), which have not been observed in any other colicin-Tol protein complex. Mutagenesis of the interface residues of ColA or TolA revealed that the effect on the interaction was cumulative; single mutations of either partner had no effect on ColA activity, whereas mutations of three or more residues significantly reduced ColA activity. Mutagenesis of the aromatic ring component of the cation-π interacting residues showed Tyr-58 of ColA to be essential for the stability of complex formation. TA(53-107) binds on the opposite side of TolAIII to that used by g3p, ColN, or TolB, illustrating the flexible nature of TolA as a periplasmic hub protein.     

Role of TolR N-Terminal, Central, and C-Terminal Domains in Dimerization and Interaction with TolA and TolQ

The Tol-PAL system of Escherichia coli is a multiprotein system involved in maintaining the cell envelope integrity and is necessary for the import of some colicins and phage DNA into the bacterium. It is organized into two complexes, one near the outer membrane between TolB and PAL and one in the cytoplasmic membrane between TolA, TolQ, and TolR. In the cytoplasmic membrane, all of the Tol proteins have been shown to interact with each other. Cross-linking experiments have shown that the TolA transmembrane domain interacts with TolQ and TolR. Suppressor mutant analyses have localized the TolQ-TolA interaction to the first transmembrane domain of TolQ and have shown that the third transmembrane domain of TolQ interacts with the transmembrane domain of TolR. To get insights on the composition of the cytoplasmic membrane complex and its possible contacts with the outer membrane complex, we focused our attention on TolR. Cross-linking and immunoprecipitation experiments allowed the identification of Tol proteins interacting with TolR. The interactions of TolR with TolA and TolQ were confirmed, TolR was shown to dimerize, and the resulting dimer was shown to interact with TolQ. Deletion mutants of TolR were constructed, and they allowed us to determine the TolR domains involved in each interaction. The TolR transmembrane domain was shown to be involved in the TolA-TolR and TolQ-TolR interactions, while TolR central and C-terminal domains appeared to be involved in TolR dimerization. The role of the TolR C-terminal domain in the TolA-TolR interaction and its association with the membranes was also demonstrated. Furthermore, phenotypic studies clearly showed that the three TolR domains (N terminal, central, and C terminal) and the level of TolR production are important for colicin A import and for the maintenance of cell envelope integrity.     

The TolA protein interacts with colicin E1 differently than with other group A colicins.

The 421-residue protein TolA is required for the translocation of group A colicins (colicins E1, E2, E3, A, K, and N) across the cell envelope of Escherichia coli. Mutations in TolA can render cells tolerant to these colicins and cause hypersensitivity to detergents and certain antibiotics, as well as a tendency to leak periplasmic proteins. TolA contains a long alpha-helical domain which connects a membrane anchor to the C-terminal domain, which is required for colicin sensitivity. The functional role of the alpha-helical domain was tested by deletion of residues 56 to 169 (TolA delta1), 166 to 287 (TolA delta2), or 54 to 287 (TolA delta3) of the alpha-helical domain of TolA, which removed the N-terminal half, the C-terminal half, or nearly the entire alpha-helical domain of TolA, respectively. TolA and TolA deletion mutants were expressed from a plasmid in an E. coli strain producing no chromosomally encoded TolA. Cellular sensitivity to the detergent deoxycholate was increased for each deletion mutant, implying that more than half of the TolA alpha-helical domain is necessary for cell envelope stability. Removal of either the N- or C-terminal half of the alpha-helical domain resulted in a slight (ca. 5-fold) decrease in cytotoxicity of the TolA-dependent colicins A, E1, E3, and N compared to cells producing wild-type TolA when these mutants were expressed alone or with TolQ, -R, and -B. In cells containing TolA delta3, the cytotoxicity of colicins A and E3 was decreased by a factor of >3,000, and K+ efflux induced by colicins A and N was not detectable. In contrast, for colicin E1 action on TolA delta3 cells, there was little decrease in the cytotoxic activity (<5-fold) or the rate of K+ efflux, which was similar to that from wild-type cells. It was concluded that the mechanism(s) by which cellular uptake of colicin E1 is mediated by the TolA protein differs from that for colicins A, E3, and N. Possible explanations for the distinct interaction and unique translocation mechanism of colicin E1 are discussed.     

Mutational analyses define helix organization and key residues of a bacterial membrane energy-transducing complex

In Gram-negative bacteria, many biological processes are coupled to inner membrane ion gradients. Ions transit at the interface of helices of integral membrane proteins, generating mechanical energy to drive energetic processes. To better understand how ions transit through these channels, we used a model system involved in two different processes, one of which depends on inner membrane energy. The Tol machinery of the Escherichia coli cell envelope is dedicated to maintaining outer membrane stability, a process driven by the proton-motive force. The Tol system is parasitized by bacterial toxins called colicins, which are imported through the outer membrane using an energy-independent process. Herein, we mutated TolQ and TolR transmembrane residues, and we analyzed the mutants for outer membrane stability, colicin import and protein complex formation. We identified residues involved in the assembly of the complex, and a new class of discriminative mutations that conferred outer membrane destabilization identical to a tol deletion mutant, but which remained fully sensitive to colicins. Further genetic approaches revealed transmembrane helix interactions and organization in the bilayer, and suggested that most of the discriminative residues are located in a putative aqueous ion channel. We discuss a model for the function of related bacterial molecular motors.     

Proton motive force drives the interaction of the inner membrane TolA and outer membrane pal proteins in Escherichia coli

The Tol-Pal system of the Escherichia coli envelope is formed from the inner membrane TolQ, TolR and TolA proteins, the periplasmic TolB protein and the outer membrane Pal lipoprotein. Any defect in the Tol-Pal proteins or in the major lipoprotein (Lpp) results in the loss of outer membrane integrity giving hypersensitivity to drugs and detergents, periplasmic leakage and outer membrane vesicle formation. We found that multicopy plasmid overproduction of TolA was able to complement the membrane defects of an lpp strain but not those of a pal strain. This result indicated that overproduced TolA has an envelope-stabilizing effect when Pal is present. We demonstrate that Pal and TolA formed a complex using in vivo cross-linking and immunoprecipitation experiments. These results, together with in vitro experiments with purified Pal and TolA derivatives, allowed us to show that Pal interacts with the TolA C-terminal domain. We also demonstrate using protonophore, K+ carrier valinomycin, nigericin, arsenate and fermentative conditions that the proton motive force was coupled to this interaction.