Res. Microbiol. 152 (2001) 523–529 2001 Éditions scientifiques et médicales Elsevier SAS. All rights reservedS0923-2508(01)01226-8/REV

Mini-review

The Tol-Pal proteins of the Escherichia coli cell envelope: an energized systemrequired for outer membrane integrity?

Roland Lloubès∗, Eric Cascales, Anne Walburger, Emmanuelle Bouveret, Claude Lazdunski,Alain Bernadac, Laure Journet

Institut de Biologie Structurale et Microbiologie (CNRS), UPR 9027, 31 chemin Joseph Aiguier, 13402 Marseille cedex 20, France

Received 31 January 2001; accepted 27 February 2001

Abstract – The outer membrane of Gram-negative bacteria acts as a barrier against harmful lipophilic compounds andlarger molecules unable to diffuse freely through the porins. However, outer membrane proteins together with the Tol-Paland TonB systems have been exploited for the entry of macromolecules such as bacteriocins and phage DNA through theEscherichia coli cell envelope. The TonB system is involved in the active transport of iron siderophores and vitamin B12,while no more precise physiological role of the Tol-Pal system has yet been defined than its requirement for cell envelopeintegrity. These two systems, containing an energized inner membrane protein interacting with outer membrane proteins,share similarities. 2001 Éditions scientifiques et médicales Elsevier SAS

Tol-Pal and TonB systems / outer membrane integrity / proton motive force

1. Introduction

Outer membrane proteins (OMPs) together withtwo cell envelope protein systems have been para-sitized for the import of bacterial toxins and phageDNA. In Escherichia coli, these toxins have beencalled colicins and two groups have been defined ac-cording to their dependence on the Tol or TonB sys-tem for their translocation. The Tol system has beenshown to be required for the import of group A col-icins (i.e.: A, E1 to E9, N) and of filamentous bac-teriophage (Ff) DNA (M13, fd and fl) through thecell envelope [47]. The TonB system allows the en-try of group B colicins (i.e.: B, D, Ia, Ib, M) and ofTl and φ80 phage DNA (for colicin import reviewsee [31]). Thetol genes have been mapped and se-quenced inE. coli and were shown to be organized ina cluster transcribed from two promoters correspond-ing to ybgC-tolQ-tolR-tolA-tolB-pal-ybgF and tolB-pal-ybgF. Each of thetol and pal mutants has beenfound to exhibit outer membrane defects character-ized by hypersensitivity to harmful compounds, therelease of periplasmic proteins in the medium and theformation of outer membrane vesicles [33]. These ef-

∗ Correspondence and reprints.E-mail address: lloubes@ibsm.cnrs-mrs.fr (R. Lloubès).

fects are absent with mutations in theybgC andybgFgenes [44, 46]. In addition,tol mutations have beenshown to produce a temperature-dependent mucoideffect in E. coli which has been related to their ge-netic regulation by the RcsC sensor of colanic acidsynthesis [14]. Alongside this effect, a negative reg-ulatory effect produced by iron has been detected inP. aeruginosa [27] while in both species, varying thetemperature of growth was shown to modify the levelof expression oftol genes. The TonB system is en-coded by three genes mapping two loci onE. colichromosome. These correspond to thetonB gene andthe exbB-exbD operon. Mutations in each of thesegenes affect the active transport of iron siderophoreand cobalamin [9]. The analysis of the genetic orga-nization of thetol-pal andtonB-exb genes in bacterialgenomes has shown thattol-pal genes always form acluster whiletonB-exb may form a cluster, and thatthese genes are found throughout the Eubacteria butare absent in Gram-positive bacteria [43].

At present inE. coli, the function of the TonBsystem has been well characterized, while extensivestudies have been conducted with the Tol-Pal systemto further understand the mechanism of macromole-cule import. In this review, the import mechanism willnot be analyzed; rather we have focused our attentionon the possible roles of the Tol-Pal proteins in the cellenvelope stability and maturation.

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2. Tol-Pal proteins of the E. coli cell envelope

The Tol-Pal system of theE. coli cell envelope iscomposed of seven proteins. Three of them are lo-cated in the inner membrane. TolA and TolR haveone transmembrane (TM) domain and the rest of themolecule protrudes into the periplasm while the inte-gral membrane TolQ protein contains three TM do-mains. TolB and YbgF are periplasmic proteins, Palis a peptidoglycan-associated lipoprotein anchored inthe outer membrane and YbgC is found in the cy-toplasm. Previous genetic and biochemical analyseshave indicated that the Tol-Pal proteins form twocomplexes. One is located in the cytoplasmic mem-brane and contains the TolA, TolQ and TolR pro-teins interacting with each other through their trans-membrane segments (figure 1A) [18, 32]. However,it remains to be determined whether the TolQ-R-Aproteins interact simultaneously. The second complexis associated with the outer membrane and containsTolB and Pal [6]. In vivo, Pal forms a complex withOmpA and its presence is required for the interactionof TolB with Lpp and OmpA [15]. Thus, the TolB-Pal complex is tightly associated with structural com-ponents of the cell envelope. Recent data have indi-cated that the inner and outer membrane complexesare linked via TolA C-terminal domain binding withPal [13] and with TolB (A. Walburger, C. Lazdunski,Y. Corda: unpublished results). Moreover, TolA wasfound to be an inner membrane energized protein, andthe TolA-Pal interaction, linking the two membranes,was shown to depend on the proton motive force(pmf) [13]. These interactions clearly demonstrate thepresence of a trans-envelope protein complex that waspreviously suspected (figure 1B) [22]. The analyses ofthese interactions using purified TolB, Pal and solu-ble domains of TolA will further indicate their affin-ity constants and whether a ternary complex is formedbetween TolA, TolB and Pal.

Biophysical analyses of some of these proteinshave been performed. Crystals of TolB have beenobtained and the 3-D structure has been determined[1, 12]. TolB is a two-domain protein with a C-terminal six-bladedβ-propeller and an N-terminalα/β domain. Preliminary crystallographic studieshave begun with the periplasmic domain of TolR [2]and with a recombinant unacylated Pal protein [3].While the TolA C-terminal domain structure hasbeen solved in a cocrystal with the g3p phage cap-sid protein [38], its NMR spectrum attribution has

Figure 1. Schematic representation of the Tol-Pal and TonBsystems of the E. coli cell envelope. A) Interactions of the TMsegments (circle) of the inner membrane TolA-Q-R proteins; theC-terminal domain of TolR (box indicated by III), which possessesmembrane affinity, is also represented. B) The C-terminal domainof TolA (TolAIII); interactions with TolB and Pal, together with TolBinteractions with Lpp and OmpA, are indicated. C) The TonB-ExbB-D proteins and the interactions of the C-terminal region ofTonB with outer membrane receptors are shown.

been solved [17] and information concerning its in-teractions with other Tol-Pal proteins should be ex-pected.

3. Outer membrane integritydepends on the Tol-Pal proteins

The impairment ofE. coli outer membrane in-tegrity has been observed withtol and pal mutantswhich are leaky for periplasmic proteins, hypersen-sitive to drugs and detergents [33], and release outermembrane vesicles [5]. Mutations affecting the cellbarrier such astolC (TolC being an outer membranechannel-tunnel protein, required for type I secretionand for drug efflux systems, [4]) andrfaD (a mutationwhich removes the core oligosaccharide of the LPS

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molecule) have been reported to induce hypersensi-tivity to drugs and detergents. However, outer mem-brane perturbations similar to those found intol-palstrains have only been observed with thelpp muta-tion [45]. Outer membrane vesicle (OMV) formationis a general feature found in Gram-negative bacteria[36], and its prevalance could be linked to turgor pres-sure of the cell envelope during bacterial growth [48].Like E. coli tol-pal mutants, some Gram-negativeintracellular species (i.e.,Porphyromonas and Neis-seria) have been shown to form large numbers ofOMVs, and these bacteria have been postulated tolack a functional Tol-Pal system [43]. The OMVs ofE. coli tol-pal strains, which are very abundant, con-tain outer membrane porins and periplasmic proteinssuch as TolB [5]. In these vesicles, OMPs were foundcorrectly assembled as deduced from the immunode-tection of the external L9 loop of LamB (figure 2).OM stabilization was observed inlpp strain whenTolA was overexpressed, while its overexpression ina pal strain had no effect [13]. According to the lowabundance of TolA within the cell and the severe OMdefects found withtolA mutations, these results to-gether with the discovery of the TolA-Pal interactionmay indicate that this interaction has an important ef-fect on the outer membrane stability. However, the re-lationship between the Tol complex and the differentphenotypes observed in thetol-pal cells remains tobe elucidated: i) how can the hypersensitivity to hy-drophobic compounds be explained, i.e. in what wayare the efflux systems affected, since we observedsimilar amounts of TolC intol and parent cells [13];ii) does the periplasmic leakage result from OMV for-mation? And iii) why are so many OMVs obtained inthe tolA strain and how are they formed?

E. coli K12 strains have also been shown to presenttol phenotypes when such cells are transformed withplasmids coding for exported soluble periplasmic do-mains of either TolA (the C-terminal domain of TolA[35]) or TolR [25]. Similarly, the periplasmic target-ing of the colicin A translocation domain (a domainwhich interacts with Tol components) also inducesa tol phenotype [7], characterized by colicin toler-ance, hypersensitivity to hydrophobic compounds andRNaseI periplasmic release. Moreover, OMV forma-tion was detected as well when the periplasmic do-mains of TolA and TolR or the colicin translocationdomain were exported into the periplasm (our unpub-lished results).

Figure 2. Immunolabeling of LamB from tolA cells. LG10 cells(5) grown on solid support containing minimal medium with 0.4%maltose (a) or in its absence (b), were suspended in 20 mMTris-HCl pH 7.0, 150 mM NaCl and adsorbed on coated grids.Immunogold labeling with anti-LamB monoclonal antibody (E302)was performed.

These results indicate thattol-pal mutations as wellas the periplasmic targeting of soluble TolR, TolAand colicin translocation domains which may affectthe Tol-Pal complex formation result in the impair-ment of the OM integrity. The knowledge of the stoe-chiometry of these proteins at present indicates thatTolA and TolR (which is presumably a dimer [25]),are minor proteins (400–800 and 2000–3000 copiesper cell, [34, 41]) while TolQ is about three timesas abundant as TolR ([22]; our unpublished results).The outer membrane Pal protein was estimated inthe range of 10,000 to 30,000 copies/cell (our unpub-lished results). Besides their translational regulation,

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the expression of these proteins may also be relatedto their genetic organization in two operons in whichthe tolB-pal-ybgF are transcribed from two promot-ers. However, the Tol-Pal protein expression levelsremain to be determined in a given strain in order togive their precise stoechiometry.

4. The energized TolA proteinshares similarities with TonB

The Tol-Pal and TonB systems ofE. coli form twoinner membrane complexes; however, mutations inthe exbB, exbD or tonB genes do not result in outermembrane perturbations. While the TonB system isdevoid of the additional TolB, YbgF, Pal and YbgCcomponents, the ExbB-TolQ and ExbD-TolR proteinsshare homologies in their amino acid sequences andtopologies [31]. The TonB-ExbB-ExbD proteins actfor the active transport of vitamin B12 and ironsiderophores through specific receptors of the outermembrane (figure 1C) [9, 40]. The gated receptors areopened upon ligand binding in a process dependenton the TonB protein and on the pmf provided by theinner membrane. The in vivo interactions of TonBwith FepA [28] and FhuA in the presence of ligand[40] and BtuB [11] have been demonstrated, andTonB has been shown to be the energized proteinwhich undergoes conformational changes dependenton the pmf, ExbB and ligand binding to FepA [29].In a similar transmembrane link, TolA was found tointeract with the OM Pal lipoprotein. This interactionwas found to be coupled with the pmf of the innermembrane since, in the presence of ATP but in theabsence of a pmf, the interaction was not detected.Moreover, TolA was shown to be an energized proteinwhich might adopt conformational change underenergization [33] to permit its interaction with Pal[13]. However, the role of the TolA-Pal interactionremains unclear but may be involved in transducingenergy from the cytoplasmic membrane to (or via) Palfor an unknown event. The role of the TolQ and TolRproteins in this process should be elucidated sincetheir overexpression was found to enhance the TolA-Pal interaction [13] and, as for ExbB and ExbD [29],these proteins could be essential for explaining themechanism of the energization of TolA.

While the tol-pal gene clusters were found to bewidespread in Gram-negative bacteria, the TolA se-quence was found to be very poorly conserved com-pared to the TolQ, TolR and Pal amino acid sequences

[43]. E. coli TolA is divided into three predicted do-mains separated by glycine-rich regions [34]. Thefirst domain contains the N-terminal membrane an-chor, the second is anα-helix-rich central domain,and the third corresponds to the C-terminal domainwhoseα/β structure has been solved in a cocrys-tal with g3p [38]. While TolQ and TolR are highlyhomologous to ExbB and ExbD, and can functionwith TonB [10], only the membrane anchors of TolAand TonB were shown to contain a four-conserved-residue motif (figure 3A) suspected to be function-ally significant [26]. The essential role of the Ser andHis residues within the TM motif of TolA and TonBwas demonstrated and found to be implicated in theirrespective interactions with the first TM segment ofTolQ [21] and ExbB [30]. However, the comparisonof TolA and TonB sequences from different speciesof theγ subdivision (E. coli, Haemophilus influenzae,Pseudomonas aeruginosa, Pseudomonas putida, andVibrio cholerae which have been analyzed for theirtol genes) indicate few points of similarity betweenTolA and TonB. While only TonB contains an ele-vated amount of Pro residues, both TonB and TolA arefound with a high content of Lys residues (between 9to 14% and 14 to 20% for TonB and TolA respec-tively), conferring a theoretical pI of 8.8 to 9.8 forTonB and 9.0 to 9.6 for TolA. Thus, TolA and TonBare very basic proteins (in a similar range to histoneand Skp proteins), and this could explain their numer-ous interactions.

A homology between TolA ofE. coli and TonBof Haemophilus ducreyi C-terminal sequences haspreviously been reported [38]. Similarly, homologysearches of the SwissProt database using the BLASTprogram with the C-terminal sequences of TolA ofP. aeruginosa and P. putida gave a hit with TonBof H. influenzae. Alignment using the CLUSTALWprogram of the C-terminal TolA and TonB sequencesfrom the five different species of theγ subdivi-sion showed only weak conservation; however, theC-terminal TolA sequence comparisons with that ofTonB of the same species gave identity values be-tween 18 and 26%. The identities found inE. coliC-terminal regions of the TolA and TonB proteinsare shown infigure 3B. It is noteworthy that inE.coli, these C-terminal sequences of TolA and TonBwere both implicated in numerous interactions. TheC-terminal region of TolA and TonB interacted, re-spectively, with Pal and TolB, and with BtuB (Q-QYresidue of TonB at position 160 to 163) [11] and FepA

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Figure 3. Comparisons between the N and C-terminal regions of E. coli TolA and TonB proteins. Identities (*) found in the N and C-terminal regions of TolA and TonB. (A) The SHLS motif of the TM segments of TolA and TonB is indicated. (B) The residues of TonB (+)involved in the interaction with BtuB (Q-QY) and with FepA (the 48 th C-terminal residues), and the secondary structure of TolA (with a,b and 3 corresponding, respectively, to α-helix, β-strand and 3/10 structures [38]), are indicated on the top sequences.

(the 48 last residues) [28]. The knowledge of the fold-ing structure of TonB and the comparison with that ofTolA in E. coli, both interacting with outer membranecomponents, together with new data on these proteinsfrom other species, would further indicate the struc-tural or functional relevance of the homologies foundin their C-terminal domains.

5. Possible function of the Tol-Pal complexes

Aside from its implications in outer membrane in-tegrity, the specific function of the Tol-Pal system re-mains unknown, although thetol-pal gene cluster hasbeen found in many Gram-negative bacteria. A hypo-thetical function has been suggested in which TolAwould drive newly synthesized outer membrane com-ponents across the periplasm [35]. This hypothesis isin accordance with the observation of in vitro inter-actions of TolB and the central domain of TolA withouter membrane trimeric porins [19, 42]. Thus, theTol proteins might function in the dynamic assem-bly of outer membrane porins that require de novoLPS synthesis. Furthermore, a recent observation in-dicated that TolA was found to be required for surfaceexpression of O-lipopolysaccharide, and to a lesserextent, for the synthesis of the LPS core [20]. In re-lation to OMV formation during cell growth, we sus-pect that any defect of the Tol-Pal system could indi-rectly increase the OMV amount since the defect mayinterfere with the dynamics of outer membrane com-ponent assembly.

Recently,tol mutants have been described in ge-netic contexts different fromE. coli K12. The tol-

pal mutants ofP. putida were found to induce outermembrane defects similar to those described inE. coliK12, and also extensive filamentation [37].E. coli O7tolQ andV. cholerae tol mutants were found to be hy-persensitive to deleterious agents and formed cell fila-ments [20, 23]. Similarly, but only under low and highosmolarities, theE. coli K12 tolA mutant formed fil-aments with impaired septation [39]. These morpho-logical changes indicate thattol mutations may affectthe formation of the cell wall peptidoglycan and nor-mal septation [20]. ThetolB mutation ofSalmonellatyphimurium was also obtained and TolB was foundto be implicated in its virulence [8], but thetolBphenotype was not characterized. However,tolA mu-tants were never obtained inP. aeruginosa and E.coli O7, suggesting that this mutation is lethal [16,20], with the death resulting from the possible toxic-ity of the accumulated O antigen. Thus, the TolA-Q-R protein complex, previously recovered at a densitycorresponding to the contact sites between inner andouter membranes [22], may participate in a functionalsite of export of cell envelope components throughthe periplasm. The in vivo cellular localization of theTolQ-R-A proteins still remains to be analyzed by mi-croscopy, since the proposed contact sites betweenthe two membranes remain a predicted region for cellgrowth [24].

6. Conclusion

Studies on the interactions between Tol-Pal com-ponents have shown six protein complexes present inthe E. coli cell envelope which are localized in the

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inner and outer membranes, linking the two mem-branes. Knowledge of simultaneous or transient in-teractions between these different complexes (TolQ-TolR, TolQ-TolA, TolR-TolA, TolA-Pal, TolA-TolBand TolB-Pal) would further indicate the oligomericform of the Tol-Pal complex in the presence or ab-sence of a pmf. The TolQ-R-A complex, sharing ho-mologies with the TonB system, may be involved inenergy transduction to Pal for an unknown event orin driving outer membrane components through theperiplasm in an energy-dependent process. The ef-fects of tol-pal mutations together with the physi-ological analyses indicate that the whole system isinvolved in OM integrity, which is consistent withthe TolB-Pal complex interacting with the structuralnetwork linking OM to the peptidoglycan. Sequencesimilarities found between the C-terminal domain ofTolB and the active site of class Bβ-lactamases havesuggested a role in peptidoglycan metabolism [1].Preliminary experiments have also shown that van-comycin susceptibility (an antibiotic inhibiting thetranspeptidation step in peptidoglycan synthesis) wasincreased intol-pal strains, indicating a differencefrom the lpp strain [13]. Together with the possibleN-acetyl-muramoyl-L-alanine amidase activity of theYbgF proteins found inChlamydia species [43], theseresults tend to indicate that the Tol-Pal system is as-sociated with cell wall metabolism.

Acknowledgements

The authors thank M. Gavioli for technical assis-tance, J. Sturgis for helpful discussions and care-ful reading of the manuscript, A. Charbit for thegift of monoclonal antibody E302, and A. Rigal andH. Bénédetti for their contribution to the Tol system.Computations were performed at the SIB using theBLAST network service. This work was in part sup-ported by a grant from PCV, MENRT, and the E.E.C.

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