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The Tol proteins of Escherichia coli and their involvement in the uptake of biomolecules and outer membrane stability

Jean Claude Lazzaroni, Pierre Germon, Marie-Céline Ray, Anne Vianney
DOI: http://dx.doi.org/10.1111/j.1574-6968.1999.tb13731.x 191-197 First published online: 1 August 1999

Abstract

The Tol proteins of Escherichia coli are involved in outer membrane stability. They are also required for the uptake of the group A colicins and the translocation of filamentous phage DNA into the cytoplasm. The tol-pal genes constitute two operons in the E. coli genome, orf1tolQRA and tolBpalorf2. The TolQ TolR TolA proteins form a complex in the cytoplasmic membrane, while TolB and Pal interact near the outer membrane. Most of the amino acid residues of TolA, TolB, TolR and Pal are localized in the periplasm. Recent advances in the knowledge of interactions of Tol-Pal proteins with other envelope components, or with group A colicins, are presented, together with current hypotheses about the role of the Tol proteins in outer membrane stability.

Keywords
  • TolQ
  • TolR
  • TolA
  • TolB
  • Pal
  • Outer membrane stability
  • Periplasm
  • Uptake of macromolecule

1 Introduction

The outer membrane of Gram-negative bacteria acts as a permeability barrier protecting the cell against most antimicrobial agents. With the increasing threat of drug-resistant strains of bacteria to human health, a better understanding of the molecular basis of outer membrane stability should permit new strategies to be developed for antibacterial agents. Although most of the components of the outer membrane have been well characterized, we still have only a limited understanding of how they are functionally integrated and interact in the outer membrane. These components are synthesized in the cytoplasm and cross the cytoplasmic membrane and the periplasm to reach their final localization. While the mechanisms of the export of macromolecules across the cytoplasmic membrane are well documented, the translocation of macromolecules from the cytoplasmic to the outer membrane is still poorly understood [1]. The periplasm is the key compartment in this process. Although the periplasm has long been considered an aqueous compartment, it is now recognized to be highly viscous, because of the high content of proteins and unpolymerized peptidoglycan [2]. The peptidoglycan network is highly hydrated and acts as a molecular sieve to limit the diffusion of proteins and other macromolecules [3]. In addition, the strongly anionic nature of most osmotically regulated periplasmic glucans, also called MDO in Escherichia coli, gives rise to a Donnan potential across the outer membrane.

The outer membrane is linked to the peptidoglycan network via many outer membrane proteins. One third of Lpp, the major outer membrane lipoprotein, is found covalently associated with the peptidoglycan, and two thirds of Lpp, as well as Pal, OmpA and porins, are non-covalently associated with this network.

Two models have been proposed for the translocation of outer membrane components across the periplasm. Adhesion sites between the cytoplasmic and outer membranes have been visualized by electron microscopy and have been postulated to be implicated in the export of lipopolysaccharide, phospholipids and some outer membrane proteins [4]. However, their in vivo existence is still a matter of debate. Periplasmic transporters provide an alternative mechanism and are necessary for at least some hydrophobic components to cross the periplasm. Such a pathway is illustrated by the transport of outer membrane lipoproteins which use the LolA-LolB route to reach their final location [5]. The two pathways are not mutually exclusive and indeed one can imagine that, rather than true adhesion sites involving proteins and lipids, some protein complexes might transiently link the two membranes, or bring them into close proximity to help the translocation of some outer membrane components. Such complexes exist for the type I secretion pathway of proteins and for the efflux pumps generating drug resistance in Gram-negative bacteria. The protein components of these transport mechanisms include an integral cytoplasmic membrane protein which provides energy to the system, and a cytoplasmic membrane lipoprotein, also called membrane fusion protein, which interacts with an outer membrane transporter [6,7].

To maintain outer membrane stability, the bacterium must not only synthesize and transport its components, but also synchronize their integration while the cells grow and divide. The isolation of mutants altered in outer membrane stability has assisted in characterizing the elements involved in this process. In addition to its role as a barrier against external antibacterial agents, the outer membrane also prevents the release of periplasmic components. Therefore mutants which are hypersensitive to antibacterial agents and/or are able to release periplasmic components represent interesting candidates for the study of outer membrane stability. One class of mutants hypersensitive to drugs is impaired in various components of efflux pumps, such as the TolC/AcrAB system [7]. Other mutants are altered in outer membrane components, such as rfa, lpp and pal mutants. rfa mutants altered in their LPS composition release periplasmic contents and are hypersensitive to many antibacterial agents. lpp mutants, lacking the major outer membrane lipoprotein, Lpp, release periplasmic components, while pal mutants release periplasmic proteins and are sensitive to antibacterial agents [8]. The tolQRAB mutants represent another class of mutants with a similar phenotype. The level of the release of periplasmic components, as well as the degree of sensitivity to antibacterial agents of these mutants are quite different, with the tolQRAB mutants sharing the most severe effect on the integrity of the outer membrane (Table 1).

View this table:
Table 1

Outer membrane stability and drug sensitivity of various cell envelope mutants of E. coli K-12

MutationRelease of alkaline phosphatase (%)Sensitivity to
exponential phasestationary phaseNa cholateSDS
Wild-type2560>100
ΔtolA861007.50.3
ΔtolQRA76100100.3
pal7193100.3
tolB98100100.3
Δrfa1122930<0.1
tolC::Tn10294010
Δlpp740601
  • Strains were all isogenic derivatives of JC188 (Hfr P4X metB pstS lacI). The release of periplasmic alkaline phosphatase was expressed as the percent of total activity recovered in the extracellular medium in mid-exponential or stationary phase of growth in the absence of cell lysis. The sensitivity to drugs was expressed as the highest concentration (in mg ml−1) allowing growth of colonies on agar plates.

In this review we will present a description of the TolQRAB system and discuss the progress in our understanding of its potential role in the translocation of macromolecules and in outer membrane stability.

2 The Tol proteins

E. coli K-12 has been used for most studies on the TolQRAB proteins. However, the DNA sequence data from microbial genomes indicate that these proteins are likely to be ubiquitous and well conserved in Gram-negative bacteria. The tolQRAB and pal genes are located at 17 min on the E. coli chromosome. They constitute two operons orf1tolQRA and tolBpalorf2 [9]. Orf1 is a cytoplasmic protein of unknown function, Orf2 encodes a periplasmic protein. Mutations in any of the tolQRAB genes of E. coli make the bacterium hypersensitive to bile salts and other deleterious agents, and result in the release of periplasmic proteins into the medium [10]. The tolQRAB and pal mutants form outer membrane vesicles which contain periplasmic components that are released into the medium [11]. Like rfa and pal mutants, tolQRAB mutants show a temperature-dependent mucoid phenotype, due to the induction of colanic acid synthesis which is believed to protect the cell against desiccation. This is consistent with the observation that expression of the orf1tolQRA operon is also regulated by RcsC, the cytoplasmic membrane sensor protein of the RcsB-RcsC two-component system involved in the regulation of capsule synthesis [12]. The TolQRA proteins are required for the translocation of the filamentous bacteriophage DNA into the cytoplasm during the process of infection [13,14], and also participate in the translocation of group A colicins to their respective sites of action [15].

2.1 The TolQ-TolR-TolA complex

The TolQ, TolR, TolA and TolB proteins are localized in the cell envelope. TolQ spans the inner membrane three times, with the bulk of the protein in the cytoplasm. Both TolA and TolR are anchored in the inner membrane via a single membrane spanning region near the amino terminus, leaving most of the protein exposed to the periplasm. TolA has a three-domain structure. In addition to the N-terminal anchoring region, it contains a large central domain with a high degree of α-helical content and a C-terminal domain [16]. Each of the three domains is separated by a stretch of glycine residues. TolR has a N-terminal anchor and most of its residues are localized in the periplasm. However, the C-terminal residues may form an amphipathic helix interacting with the cytoplasmic membrane. TolB is a periplasmic protein with the C-terminal residues organized in a β-propeller structure.

A combination of biochemical and genetic studies has been used to show that the TolQ, TolR and TolA proteins form a complex in the cytoplasmic membrane [17,18]. The N-terminal transmembrane domain of TolA interacts with the transmembrane domain of TolR and the first transmembrane domain of TolQ (Fig. 1). The third transmembrane domain of TolQ interacts with the N- and C-termini of TolR and the first transmembrane domain of TolQ. The stoichiometry of the proteins in the complex has not been entirely elucidated, but at least TolR has been shown to form dimers (Lazdunski et al., personal communication).

Figure 1

Model of Tol transmembrane interacting domains. Only the transmembrane domains of TolA, TolQ, TolR and the C-terminal part of TolR are shown.

2.2 The TolB-Pal complex

Pal and TolB also form a complex near the outer membrane [19]. The two proteins can be co-immunoprecipitated and can be cross-linked in vivo with formaldehyde. The C-terminal part of Pal interacts with TolB and the peptidoglycan [20]. The C-terminal domain of TolB (residues 165–420) has been proposed to form a β-propeller structure. Using a genetic suppressor approach, we have recently shown that this part of TolB is responsible for interaction with Pal (our unpublished results).

2.3 Relation between the TolQ-TolR-TolA and TolB-Pal complexes

There is no direct experimental support for an interaction between the TolQ-TolR-TolA and TolB-Pal complexes. However, several lines of evidence suggest that they might interact at least transiently. In membrane fractionation experiments, the Tol proteins are localized mainly in an intermediate fraction supposed to contain contact sites between the cytoplasmic and outer membranes [15]. When cells are treated with colicin A, the quantity of Tol protein doubles. TolA and TolB are both involved in interactions with porin trimers and group A colicins [21,22].

2.4 Interactions with other membrane components

Several outer membrane components interact with some of the Tol proteins. TolB can be cross-linked to OmpA and Lpp [20]. The central domain of TolA and TolB interact with porin trimers in vitro in the presence of SDS but not with OmpA [21,22]. The TolA C-terminal domain interferes with the wild-type protein, when it is overproduced in the periplasm [23]. This result is consistent with interaction of the C-terminal domain of TolA with components of the periplasm or the inner surface of the outer membrane. However, unlike the related Ton system, where it has been clearly demonstrated that TonB interacts with outer membrane proteins, no such in vivo interaction has yet been shown for the Tol system.

2.5 Comparison of the TonB and Tol systems

The TolQ-TolR-TolA complex presents some similarities with the related TonB-ExbB-ExbD complex, which is involved in the uptake of iron-siderophore complexes and vitamin B12[24]. TolQ and TolR are structurally and functionally homologous to ExbB and ExbD, respectively [25]. TolA and TonB have a similar elongated conformation but are homologous only in their N-terminal transmembrane domains, where they both have a well conserved S-X(3)-H-X(6)-L-X(3)-S motif [17,26]. The TolQ-TolR-TolA-N-terminus complex can partially replace ExbB-ExbD-TonB-N-terminus for energy transduction ([26], our unpublished results). However, the TolA and TonB proteins span the periplasm by quite different mechanisms, since the TolA central domain has an α-helical structure whereas that of TonB has a stretch of X-Pro repeats. Interestingly, the TolA protein of Brucella abortus has a central domain organized in X-Pro structure, very similar to TonB, again showing a link between the two systems (A. Tibor, personal communication). In addition, the tolQRA genes of Pseudomonas aeruginosa are controlled by Fur, a global regulator protein involved in iron regulation of the components of the Ton system in Gram-negative bacteria.

TonB is thought to open outer membrane channels via an energy-dependent conformational change, either while located in the cytoplasmic membrane or by shuttling between the two membranes [27]. The ExbB-ExbD-TonB complex mediates the energy transfer of the electrochemical potential from the cytoplasmic membrane to the outer membrane. We have recently shown that TolA could undergo a change of conformation which depends on TolQ, TolR and the proton-motive force (our unpublished results). In addition, hybrid proteins consisting of the N-terminal transmembrane domain of TolA fused to the TonB protein deleted from its own transmembrane part are able to transduce energy to the outer membrane ([26], our unpublished results). Therefore the TolQ-TolR-TolA proteins, like the TonB-ExbB-ExbD proteins, might also be able to transduce electrochemical potential from the cytoplasm to the periplasm or the outer membrane.

3 Function of the Tol proteins

3.1 Colicin import

The involvement of the Tol proteins in colicin import has been recently reviewed [15]. Therefore only the most significant results will be summarized in this review. To enter cells, group A colicins use outer membrane proteins as receptors. These include BtuB, and Tsx which are vitamin B12 and nucleoside receptors, respectively, or the OmpF porin. Many studies suggest that colicins interact with the Tol proteins upon translocation. The N-terminal domain of group A colicins interacts with TolB and the C-terminal domain of TolA, and colicin A N-terminal domain can form a trimeric complex with TolA and TolB [28]. The Tol proteins appear to maintain the colicins in an unfolded conformation which helps them to cross the periplasm and reach their final target in the cytoplasmic membrane or in the cytoplasm [15].

3.2 Uptake of filamentous phage DNA

Infection of E. coli by filamentous phages is initiated when the end of the particle containing the pIII protein interacts with the F conjugative pilus. TolA domain III is essential for infection, interacting with the amino-terminal portion of pIII protein [13,14]. For this reason, this TolA domain is referred to as the coreceptor for filamentous phage infection [14]. The TolQ, TolR and TolA proteins are also required for insertion of the pVIII protein into the cytoplasmic membrane, suggesting a role of these proteins as a channel for the phage DNA to cross the cytoplasmic membrane [13].

3.3 Outer membrane stability

The only known physiological role of the Tol proteins is to maintain outer membrane integrity. Therefore these proteins are likely to be involved in the biogenesis of some outer membrane components. One potential role of the Tol proteins could be to participate in the transport of an outer membrane component crucial for its stability, such as LPS, Lpp or Pal. The fact that no outer membrane component is lacking does not support a role of the Tol proteins in the transport of molecules, although the possibility that the Tol proteins help transport some outer membrane components cannot be ruled out.

There is some evidence that the Tol proteins could play a role in porin and/or LPS translocation or assembly. Some variations in the porin and in the LPS content of tol mutants have been described by different authors. The amount of OmpF and LamB is lowered in tol mutants while that of OmpC is increased [15]. A periplasmic ‘leaky’ mutant, probably located in the tol-pal locus, has a diminution in the neutral sugar content in its core LPS [29]. In enteropathogenic strains of E. coli, tol mutants are unable to secrete smooth LPS containing O-antigen residues, although rough LPS is synthesized and localized normally [30].

The interaction of TolA and TolB with porin trimers also suggests that the Tol proteins are involved in some steps of the assembly of these proteins which is thought to involve the association with LPS and periplasmic intermediates [31,32]. The protein-to-LPS ratio is probably crucial for correct assembly of the outer membrane.

Another important step in porin trimer assembly is the translocation across the peptidoglycan network. The cut-off value for the passage of proteins through the peptidoglycan is about 50 kDa [3]. The Tol-Pal proteins might help high-molecular-mass molecules, such as porin trimer-LPS complexes, to cross the peptidoglycan [20].

Outer membrane blebbing in tol mutants probably reflects an incorrect assembly of this structure during cell growth. It is surprising that tolQRA mutants produce more vesicles than do tolB or pal mutants. The formation of vesicles by tolB and pal mutants can be interpreted as some peeling off of the outer membrane, when it is more loosely attached to the peptidoglycan. Alternatively, the Tol-Pal proteins may be involved in the formation of naturally produced membrane vesicles by most Gram-negative bacteria [33]. This natural and common phenomenon may give certain Gram-negative bacteria an advantage to survive in some environments. These outer membrane vesicles are able to concentrate components such as virulence factors or cell-wall degradative enzymes able to lyse surrounding dissimilar bacteria thereby releasing inorganic compounds for growth.

Whereas we have now a good picture of the interactions between the Tol proteins, many aspects of Tol function in outer membrane stabilization remain unclear. As the Ton and Tol systems show some functional similarities, the Tol system may be involved in an energy-dependent process necessary for the translocation of some outer membrane components. The TolA and TolB proteins have been proposed to play a chaperone-like role in the translocation of colicins [15]. The Tol proteins may function bidirectionally either in the entry of colicins and filamentous phage DNA or in the translocation of outer membrane components from the cytoplasmic membrane.

Recent advances in the understanding of interactions between the Tol-Pal proteins, as well as the availability of numerous mutants in the system, should help elaborate a molecular modelling of the Tol proteins and understand their role in the translocation of biomolecules across the periplasm.

Acknowledgements

We thank Robert Webster for reading the manuscript. Work in the authors’ laboratory was supported by the Life Science Department of the CNRS and the Ministère de l'Enseignement Supérieur et de la Recherche.

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View Abstract