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.     

Energy-Dependent Conformational Change in the TolA Protein of Escherichia coli Involves Its N-Terminal Domain, TolQ, and TolR

TolQ, TolR, and TolA inner membrane proteins of Escherichia coli are involved in maintaining the stability of the outer membrane. They share homology with the ExbB, ExbD, and TonB proteins, respectively. The last is involved in energy transduction between the inner and the outer membrane, and its conformation has been shown to depend on the presence of the proton motive force (PMF), ExbB, and ExbD. Using limited proteolysis experiments, we investigated whether the conformation of TolA was also affected by the PMF. We found that dissipation of the PMF by uncouplers led to the formation of a proteinase K digestion fragment of TolA not seen when uncouplers are omitted. This fragment was also detected in Delta tolQ, Delta tolR, and tolA(H22P) mutants but, in contrast to the parental strain, was also seen in the absence of uncouplers. We repeated those experiments in outer membrane mutants such as lpp, pal, and Delta rfa mutants: the behavior of TolA in lpp mutants was similar to that observed with the parental strain. However, the proteinase K-resistant fragment was never detected in the Delta rfa mutant. Altogether, these results suggest that TolA is able to undergo a PMF-dependent change of conformation. This change requires TolQ, TolR, and a functional TolA N-terminal domain. The potential role of this energy-dependent process in the stability of the outer membrane is discussed.     

Isolation and characterization of extragenic suppressor mutants of the tolA-876 periplasmic-leaky allele in Escherichia coli K-12

tolA mutants of Escherichia coli K-12 release periplasmic proteins into the extracellular medium; they are sensitive to growth inhibitors such as cholic acid and tolerant to group A colicins and filamentous bacteriophage. Suppressor mutants of the tolA-876 allele were isolated by selecting for cholic acid resistant clones that did not release periplasmic ribonuclease I. One class of tolA suppressor strains carried mutations in the staA gene (for suppressor of tolA) located a 41 min. tolA-876 staA strains partially recovered a wild-type phenotype: they exported alkaline phosphatase and beta-lactamase into the periplasm and only released very low amounts of periplasmic proteins; moreover, they were sensitive to E1 and A colicins and more resistant than tolA-876 staA+ strains to various growth inhibitors. Furthermore, tolA-876 staA-2 and tolA+staA-2 mutants were 10- to 2700-times more resistant than staA+ strains to bacteriophages TuIa, TuIb and T4, and TuII whose receptors are major outer membrane proteins OmpF, OmpC and OmpA, respectively. SDS-PAGE analysis suggested that cell envelopes of staA or staA+ strains contained similar amounts of these proteins but characterization of strains carrying ompF (or C or A)-phoA gene fusions showed that mutation stA-2 reduced ompF gene expression by a factor of two. Analysis of double mutants strains carrying mutation staA-2 and a tolA, tolB, excC or excD periplasmic-leaky mutation showed that staA suppression was allele specific which suggested that proteins TolA and StaA might directly interact.     

Mutational analysis of the TolA C-terminal domain of Escherichia coli and genetic evidence for an interaction between TolA and TolB

The Tol proteins are involved in the outer membrane stability of gram-negative bacteria. The C-terminal domain of TolA was mutagenized to identify residues important for its functions. The isolation of suppressor mutants of tolA mutations in the tolB gene confirmed an interaction between TolAIII and the N-terminal domain of TolB.     

Filamentous phage infection: required interactions with the TolA protein.

Infection of Escherichia coli by the filamentous phage f1 is initiated by binding of the phage to the tip of the F conjugative pilus via the gene III protein. Subsequent translocation of phage DNA requires the chromosomally encoded TolQ, TolR, and TolA proteins, after the pilus presumably has withdrawn, bringing the phage to the bacterial surface. Of these three proteins, TolA is proposed to span the periplasm, since it contains a long helical domain (domain II), which connects a cytoplasmic membrane anchor domain (domain I) to the carboxyl-terminal domain (domain III). By using a transducing phage, the requirement for TolA in an F+ strain was found to be absolute. The role of TolA domains II and III in the infective process was examined by analyzing the ability of various deletion mutants of tolA to facilitate infection. The C-terminal domain III was shown to be essential, whereas the polyglycine region separating domains I and II could be deleted with no effect. Deletion of helical domain II reduced the efficiency of infection, which could be restored to normal by retaining the C-terminal half of domain II. Soluble domain III, expressed in the periplasm but not in the cytoplasm or in the medium, interfered with infection of a tolA+ strain. The essential interaction of TolA domain III with phage via gene III protein appears to require interaction with a third component, either the pilus tip or a periplasmic entity.     

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.     

Escherichia coli TolA tolerates multiple amino-acid substitutions as revealed by screening randomized variants for membrane integrity and phage receptor function

Escherichia coli TolA is a cytoplasmic membrane protein required for outer membrane integrity and the translocation of F-specific filamentous (Ff) bacteriophage DNA. Both phage infection and membrane integrity depend on several TolA interactions, e.g. those of the TolA C-terminal domain (TolAIII). Membrane integrity involves interaction with two host proteins and phage translocation requires direct interaction with the N-terminal domain (N1) of Ff phage protein g3p. Although cocrystallization of TolAIII and N1g3p has identified several contact points, it is still uncertain which residues are selectively involved in the different TolA functions. Thus, four different limited substitution libraries of TolA were created, targeting contacts at positions 415-420. These libraries were introduced into the tolA strain K17DE3tolA/F(+) and several variants, containing complementing, multiple amino-acid substitutions, were identified. However, most randomized variants did not complement the tolA strain K17DE3tolA/F(+). The TolA variants that restored sensitivity to phage infection displayed a considerable sequence variation, while the few variants that restored tolerance to detergent were from the same library. A comparison of the generated residue variation and natural variation, suggests that structural dependence overrides contact residue dependence. Thus, library screening can be efficient in identifying TolA variants with different functionally associated characteristics.     

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.     

Identification by genetic suppression of Escherichia coli TolB residues important for TolB-Pal interaction

The Tol-Pal system of Escherichia coli is involved in maintaining outer membrane stability. Mutations in tolQ, tolR, tolA, tolB, or pal genes result in sensitivity to bile salts and the leakage of periplasmic proteins. Moreover, some of the tol genes are necessary for the entry of group A colicins and the DNA of filamentous bacteriophages. TolQ, TolR, and TolA are located in the cytoplasmic membrane where they interact with each other via their transmembrane domains. TolB and Pal form a periplasmic complex near the outer membrane. We used suppressor genetics to identify the regions important for the interaction between TolB and Pal. Intragenic suppressor mutations were characterized in a domain of Pal that was shown to be involved in interactions with TolB and peptidoglycan. Extragenic suppressor mutations were located in tolB gene. The C-terminal region of TolB predicted to adopt a beta-propeller structure was shown to be responsible for the interaction of the protein with Pal. Unexpectedly, none of the suppressor mutations was able to restore a correct association between Pal and peptidoglycan, suggesting that interactions between Pal and other components such as TolB may also be important for outer membrane stability.     

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.     

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.     

Pal Lipoprotein of Escherichia coli Plays a Major Role in Outer Membrane Integrity

The Tol-Pal system of gram-negative bacteria is composed of five proteins. TolA, TolQ, and TolR are inner membrane proteins, TolB is a periplasmic protein, and Pal, the peptidoglycan-associated lipoprotein, is anchored to the outer membrane. In this study, the roles of Pal and major lipoprotein Lpp were compared in Escherichia coli. lpp and tol-pal mutations have previously been found to perturb the outer membrane permeability barrier and to cause the release of periplasmic proteins and the formation of outer membrane vesicles. In this study, we showed that the overproduction of Pal is able to restore the outer membrane integrity of an lpp strain but that overproduced Lpp has no effect in a pal strain. Together with the previously reported observation that overproduced TolA complements an lpp but not a pal strain, these results indicate that the cell envelope integrity is efficiently stabilized by an epistatic Tol-Pal system linking inner and outer membranes. The density of Pal was measured and found to be lower than that of Lpp. However, Pal was present in larger amounts compared to TolA and TolR proteins. The oligomeric state of Pal was determined and a new interaction between Pal and Lpp was demonstrated.     

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 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.     

Nucleotide sequences of the tolA and tolB genes and localization of their products, components of a multistep translocation system in Escherichia coli.

Various mutations in the tolQRAB gene cluster of Escherichia coli render the bacteria tolerant to high concentrations of the E, A, or K colicins as well as tolerant to infection by the single-stranded filamentous bacteriophage. The nucleotide sequence of a 2.8-kilobase fragment containing the tolA and tolB genes was determined. This sequence predicts TolA to be a 421-amino-acid protein of molecular mass 44,190 daltons. Studies using minicells show it to be associated with the inner membrane, presumably via a 21-amino-acid hydrophobic sequence between residues 13 and 35. The remaining 387 residues on the carboxyl side of this region are located in the periplasm. Within this region of TolA is a 230-residue portion that is predicted to form a very long helical segment. This region is rich in alanine, lysine, and glutamic and aspartic acids. The TolB protein is predicted to contain 431 amino acids. Localization studies using minicells show two proteins encoded by this open reading frame. The larger protein of 47.5 kilodaltons appears to be associated with the membrane fractions. The smaller protein is 43 kilodaltons in size and is found with the periplasmic components of the cell.     

Movements of the TolR C-terminal domain depend on TolQR ionizable key residues and regulate activity of the Tol complex

The TolQRA proteins of Escherichia coli form an inner membrane complex involved in the maintenance of the outer membrane stability and in the late stages of cell division. The TolQR complex uses the proton-motive force to regulate TolA conformation and its interaction with the outer membrane Pal lipoprotein. It has been proposed that an ion channel forms at the TolQR transmembrane helix interface. This complex assembles with a minimal TolQ/TolR ratio of 4:2, therefore involving at least 14 transmembrane helices, which may form the ion pathway. The C-terminal periplasmic domain of TolR protein interacts with TolQ and has been proposed to control the TolQR channel activity. Here, we constructed unique cysteine substitutions in the last 27 residues of TolR. Each of the substitutions results in a functional TolR protein. Disulfide cross-linking demonstrates that the TolQR complex is dynamic, involving conformational modifications of TolR C-terminal domain. We monitored these structural changes by cysteine accessibility experiments and showed that the conformation of this domain is responsive to the proton-motive force and on the presence of critical residues of the ion pathway.     

Deletion analyses of the peptidoglycan-associated lipoprotein Pal reveals three independent binding sequences including a TolA box

The Tol-Pal system of the Escherichia coli cell envelope is composed of five proteins. TolQ, TolR and TolA form a complex in the inner membrane, whereas TolB is a periplasmic protein interacting with Pal, the peptidoglycan-associated lipoprotein anchored to the outer membrane. This system is required for outer membrane integrity and has been shown to form a trans-envelope bridge linking inner and outer membranes. The TolA-Pal interaction plays an important role in the function of this system and has been found to depend on the proton motive force and the TolQ and TolR proteins. The Pal lipoprotein interacts with many components, such as TolA, TolB, OmpA, the major lipoprotein and the murein layer. In this study, six pal deletions were constructed. The analyses of the resulting Pal protein functions and interactions defined an N-terminal region of 40 residues, which can be deleted without any cell-damaging effect, and three independent regions required for its interaction with TolA, OmpA and TolB or the peptidoglycan. The analyses of the integrity of the cells producing the various Pal lipoproteins revealed strong outer membrane destabilization only when binding regions were deleted. Furthermore, a conserved polypeptide sequence located downstream of the peptidoglycan binding motif of Pal was required for the TolA-Pal interaction and for the maintenance of outer membrane stability.     

Signal sequence processing is required for the assembly of LamB trimers in the outer membrane of Escherichia coli.

Proteins destined for either the periplasm or the outer membrane of Escherichia coli are translocated from the cytoplasm by a common mechanism. It is generally assumed that outer membrane proteins, such as LamB (maltoporin or lambda receptor), which are rich in beta-structure, contain additional targeting information that directs proper membrane insertion. During transit to the outer membrane, these proteins may pass, in soluble form, through the periplasm or remain membrane associated and reach their final destination via sites of inner membrane-outer membrane contact (zones of adhesion). We report lamB mutations that slow signal sequence cleavage, delay release of the protein from the inner membrane, and interfere with maltoporin biogenesis. This result is most easily explained by proposing a soluble, periplasmic LamB assembly intermediate. Additionally, we found that such lamB mutations confer several novel phenotypes consistent with an abortive attempt by the cell to target these tethered LamB molecules. These phenotypes may allow isolation of mutants in which the process of outer membrane protein targeting is altered.     

Mutational Analysis of the Escherichia coli K-12 TolA N-Terminal Region and Characterization of Its TolQ-Interacting Domain by Genetic Suppression

The Tol-Pal proteins of Escherichia coli are involved in maintaining outer membrane integrity. They form two complexes in the cell envelope. Transmembrane domains of TolQ, TolR, and TolA interact in the cytoplasmic membrane, while TolB and Pal form a complex near the outer membrane. The N-terminal transmembrane domain of TolA anchors the protein to the cytoplasmic membrane and interacts with TolQ and TolR. Extensive mutagenesis of the N-terminal part of TolA was carried out to characterize the residues involved in such processes. Mutations affecting the function of TolA resulted in a lack or an alteration in TolA-TolQ or TolR-TolA interactions but did not affect the formation of TolQ-TolR complexes. Our results confirmed the importance of residues serine 18 and histidine 22, which are part of an SHLS motif highly conserved in the TolA and the related TonB proteins from different organisms. Genetic suppression experiments were performed to restore the functional activity of some tolA mutants. The suppressor mutations all affected the first transmembrane helix of TolQ. These results confirmed the essential role of the transmembrane domain of TolA in triggering interactions with TolQ and TolR.     

Crystal Structures of a CTXφ pIII Domain Unbound and in Complex with a Vibrio cholerae TolA Domain Reveal Novel Interaction Interfaces

Vibrio cholerae colonize the small intestine where they secrete cholera toxin, an ADP-ribosylating enzyme that is responsible for the voluminous diarrhea characteristic of cholera disease. The genes encoding cholera toxin are located on the genome of the filamentous bacteriophage, CTXφ, that integrates as a prophage into the V. cholerae chromosome. CTXφ infection of V. cholerae requires the toxin-coregulated pilus and the periplasmic protein TolA. This infection process parallels that of Escherichia coli infection by the Ff family of filamentous coliphage. Here we demonstrate a direct interaction between the N-terminal domain of the CTXφ minor coat protein pIII (pIII-N1) and the C-terminal domain of TolA (TolA-C) and present x-ray crystal structures of pIII-N1 alone and in complex with TolA-C. The structures of CTXφ pIII-N1 and V. cholerae TolA-C are similar to coliphage pIII-N1 and E. coli TolA-C, respectively, yet these proteins bind via a distinct interface that in E. coli TolA corresponds to a colicin binding site. Our data suggest that the TolA binding site on pIII-N1 of CTXφ is accessible in the native pIII protein. This contrasts with the Ff family phage, where the TolA binding site on pIII is blocked and requires a pilus-induced unfolding event to become exposed. We propose that CTXφ pIII accesses the periplasmic TolA through retraction of toxin-coregulated pilus, which brings the phage through the outer membrane pilus secretin channel. These data help to explain the process by which CTXφ converts a harmless marine microbe into a deadly human pathogen.