Res. Microbiol. 152 (2001) 523529 2001 ditions scientiques et mdicales Elsevier SAS.

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Mini-review

The Tol-Pal proteins of the Escherichia coli cell envelope: an energized system required for outer membrane integrity?
Roland Lloubs , 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 and larger molecules unable to diffuse freely through the porins. However, outer membrane proteins together with the Tol-Pal and TonB systems have been exploited for the entry of macromolecules such as bacteriocins and phage DNA through the Escherichia 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 dened than its requirement for cell envelope integrity. These two systems, containing an energized inner membrane protein interacting with outer membrane proteins, share similarities. 2001 ditions scientiques et mdicales Elsevier SAS Tol-Pal and TonB systems / outer membrane integrity / proton motive force

1. Introduction Outer membrane proteins (OMPs) together with two cell envelope protein systems have been parasitized for the import of bacterial toxins and phage DNA. In Escherichia coli, these toxins have been called colicins and two groups have been dened according to their dependence on the Tol or TonB system for their translocation. The Tol system has been shown to be required for the import of group A colicins (i.e.: A, E1 to E9, N) and of lamentous bacteriophage (Ff) DNA (M13, fd and ) through the cell envelope [47]. The TonB system allows the entry of group B colicins (i.e.: B, D, Ia, Ib, M) and of Tl and 80 phage DNA (for colicin import review see [31]). The tol genes have been mapped and sequenced in E. coli and were shown to be organized in a cluster transcribed from two promoters corresponding to ybgC-tolQ-tolR-tolA-tolB-pal-ybgF and tolBpal-ybgF. Each of the tol and pal mutants has been found to exhibit outer membrane defects characterized by hypersensitivity to harmful compounds, the release of periplasmic proteins in the medium and the formation of outer membrane vesicles [33]. These ef Correspondence and reprints.

E-mail address: lloubes@ibsm.cnrs-mrs.fr (R. Lloubs).

fects are absent with mutations in the ybgC and ybgF genes [44, 46]. In addition, tol mutations have been shown to produce a temperature-dependent mucoid effect in E. coli which has been related to their genetic regulation by the RcsC sensor of colanic acid synthesis [14]. Alongside this effect, a negative regulatory effect produced by iron has been detected in P. aeruginosa [27] while in both species, varying the temperature of growth was shown to modify the level of expression of tol genes. The TonB system is encoded by three genes mapping two loci on E. coli chromosome. These correspond to the tonB gene and the exbB-exbD operon. Mutations in each of these genes affect the active transport of iron siderophore and cobalamin [9]. The analysis of the genetic organization of the tol-pal and tonB-exb genes in bacterial genomes has shown that tol-pal genes always form a cluster while tonB-exb may form a cluster, and that these genes are found throughout the Eubacteria but are absent in Gram-positive bacteria [43]. At present in E. coli, the function of the TonB system has been well characterized, while extensive studies have been conducted with the Tol-Pal system to further understand the mechanism of macromolecule import. In this review, the import mechanism will not be analyzed; rather we have focused our attention on the possible roles of the Tol-Pal proteins in the cell envelope stability and maturation.

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2. Tol-Pal proteins of the E. coli cell envelope The Tol-Pal system of the E. coli cell envelope is composed of seven proteins. Three of them are located in the inner membrane. TolA and TolR have one transmembrane (TM) domain and the rest of the molecule protrudes into the periplasm while the integral membrane TolQ protein contains three TM domains. TolB and YbgF are periplasmic proteins, Pal is a peptidoglycan-associated lipoprotein anchored in the outer membrane and YbgC is found in the cytoplasm. Previous genetic and biochemical analyses have indicated that the Tol-Pal proteins form two complexes. One is located in the cytoplasmic membrane and contains the TolA, TolQ and TolR proteins interacting with each other through their transmembrane segments (gure 1A) [18, 32]. However, it remains to be determined whether the TolQ-R-A proteins interact simultaneously. The second complex is associated with the outer membrane and contains TolB and Pal [6]. In vivo, Pal forms a complex with OmpA and its presence is required for the interaction of TolB with Lpp and OmpA [15]. Thus, the TolBPal complex is tightly associated with structural components of the cell envelope. Recent data have indicated that the inner and outer membrane complexes are linked via TolA C-terminal domain binding with Pal [13] and with TolB (A. Walburger, C. Lazdunski, Y. Corda: unpublished results). Moreover, TolA was found to be an inner membrane energized protein, and the TolA-Pal interaction, linking the two membranes, was shown to depend on the proton motive force (pmf) [13]. These interactions clearly demonstrate the presence of a trans-envelope protein complex that was previously suspected (gure 1B) [22]. The analyses of these interactions using puried TolB, Pal and soluble domains of TolA will further indicate their afnity constants and whether a ternary complex is formed between TolA, TolB and Pal. Biophysical analyses of some of these proteins have been performed. Crystals of TolB have been obtained and the 3-D structure has been determined [1, 12]. TolB is a two-domain protein with a Cterminal six-bladed -propeller and an N-terminal / domain. Preliminary crystallographic studies have begun with the periplasmic domain of TolR [2] and with a recombinant unacylated Pal protein [3]. While the TolA C-terminal domain structure has been solved in a cocrystal with the g3p phage capsid protein [38], its NMR spectrum attribution has

Figure 1. Schematic representation of the Tol-Pal and TonB systems of the E. coli cell envelope. A) Interactions of the TM segments (circle) of the inner membrane TolA-Q-R proteins; the C-terminal domain of TolR (box indicated by III), which possesses membrane afnity, is also represented. B) The C-terminal domain of TolA (TolAIII); interactions with TolB and Pal, together with TolB interactions with Lpp and OmpA, are indicated. C) The TonBExbB-D proteins and the interactions of the C-terminal region of TonB with outer membrane receptors are shown.

been solved [17] and information concerning its interactions with other Tol-Pal proteins should be expected. 3. Outer membrane integrity depends on the Tol-Pal proteins The impairment of E. coli outer membrane integrity has been observed with tol and pal mutants which are leaky for periplasmic proteins, hypersensitive to drugs and detergents [33], and release outer membrane vesicles [5]. Mutations affecting the cell barrier such as tolC (TolC being an outer membrane channel-tunnel protein, required for type I secretion and for drug efux systems, [4]) and rfaD (a mutation which removes the core oligosaccharide of the LPS

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molecule) have been reported to induce hypersensitivity to drugs and detergents. However, outer membrane perturbations similar to those found in tol-pal strains have only been observed with the lpp mutation [45]. Outer membrane vesicle (OMV) formation is a general feature found in Gram-negative bacteria [36], and its prevalance could be linked to turgor pressure of the cell envelope during bacterial growth [48]. Like E. coli tol-pal mutants, some Gram-negative intracellular species (i.e., Porphyromonas and Neisseria) have been shown to form large numbers of OMVs, and these bacteria have been postulated to lack a functional Tol-Pal system [43]. The OMVs of E. coli tol-pal strains, which are very abundant, contain outer membrane porins and periplasmic proteins such as TolB [5]. In these vesicles, OMPs were found correctly assembled as deduced from the immunodetection of the external L9 loop of LamB (gure 2). OM stabilization was observed in lpp strain when TolA was overexpressed, while its overexpression in a pal strain had no effect [13]. According to the low abundance of TolA within the cell and the severe OM defects found with tolA mutations, these results together with the discovery of the TolA-Pal interaction may indicate that this interaction has an important effect on the outer membrane stability. However, the relationship between the Tol complex and the different phenotypes observed in the tol-pal cells remains to be elucidated: i) how can the hypersensitivity to hydrophobic compounds be explained, i.e. in what way are the efux systems affected, since we observed similar amounts of TolC in tol and parent cells [13]; ii) does the periplasmic leakage result from OMV formation? And iii) why are so many OMVs obtained in the tolA strain and how are they formed? E. coli K12 strains have also been shown to present tol phenotypes when such cells are transformed with plasmids coding for exported soluble periplasmic domains of either TolA (the C-terminal domain of TolA [35]) or TolR [25]. Similarly, the periplasmic targeting of the colicin A translocation domain (a domain which interacts with Tol components) also induces a tol phenotype [7], characterized by colicin tolerance, hypersensitivity to hydrophobic compounds and RNaseI periplasmic release. Moreover, OMV formation was detected as well when the periplasmic domains of TolA and TolR or the colicin translocation domain were exported into the periplasm (our unpublished 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 mM Tris-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 that tol-pal mutations as well as the periplasmic targeting of soluble TolR, TolA and colicin translocation domains which may affect the Tol-Pal complex formation result in the impairment of the OM integrity. The knowledge of the stoechiometry of these proteins at present indicates that TolA and TolR (which is presumably a dimer [25]), are minor proteins (400800 and 20003000 copies per cell, [34, 41]) while TolQ is about three times as abundant as TolR ([22]; our unpublished results). The outer membrane Pal protein was estimated in the range of 10,000 to 30,000 copies/cell (our unpublished results). Besides their translational regulation,

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the expression of these proteins may also be related to their genetic organization in two operons in which the tolB-pal-ybgF are transcribed from two promoters. However, the Tol-Pal protein expression levels remain to be determined in a given strain in order to give their precise stoechiometry. 4. The energized TolA protein shares similarities with TonB The Tol-Pal and TonB systems of E. coli form two inner membrane complexes; however, mutations in the exbB, exbD or tonB genes do not result in outer membrane perturbations. While the TonB system is devoid of the additional TolB, YbgF, Pal and YbgC components, the ExbB-TolQ and ExbD-TolR proteins share homologies in their amino acid sequences and topologies [31]. The TonB-ExbB-ExbD proteins act for the active transport of vitamin B12 and iron siderophores through specic receptors of the outer membrane (gure 1C) [9, 40]. The gated receptors are opened upon ligand binding in a process dependent on the TonB protein and on the pmf provided by the inner membrane. The in vivo interactions of TonB with FepA [28] and FhuA in the presence of ligand [40] and BtuB [11] have been demonstrated, and TonB has been shown to be the energized protein which undergoes conformational changes dependent on the pmf, ExbB and ligand binding to FepA [29]. In a similar transmembrane link, TolA was found to interact with the OM Pal lipoprotein. This interaction was found to be coupled with the pmf of the inner membrane since, in the presence of ATP but in the absence of a pmf, the interaction was not detected. Moreover, TolA was shown to be an energized protein which might adopt conformational change under energization [33] to permit its interaction with Pal [13]. However, the role of the TolA-Pal interaction remains unclear but may be involved in transducing energy from the cytoplasmic membrane to (or via) Pal for an unknown event. The role of the TolQ and TolR proteins in this process should be elucidated since their overexpression was found to enhance the TolAPal interaction [13] and, as for ExbB and ExbD [29], these proteins could be essential for explaining the mechanism of the energization of TolA. While the tol-pal gene clusters were found to be widespread in Gram-negative bacteria, the TolA sequence was found to be very poorly conserved compared to the TolQ, TolR and Pal amino acid sequences

[43]. E. coli TolA is divided into three predicted domains separated by glycine-rich regions [34]. The rst domain contains the N-terminal membrane anchor, the second is an -helix-rich central domain, and the third corresponds to the C-terminal domain whose / structure has been solved in a cocrystal with g3p [38]. While TolQ and TolR are highly homologous to ExbB and ExbD, and can function with TonB [10], only the membrane anchors of TolA and TonB were shown to contain a four-conservedresidue motif (gure 3A) suspected to be functionally signicant [26]. The essential role of the Ser and His residues within the TM motif of TolA and TonB was demonstrated and found to be implicated in their respective interactions with the rst TM segment of TolQ [21] and ExbB [30]. However, the comparison of TolA and TonB sequences from different species of the subdivision (E. coli, Haemophilus inuenzae, Pseudomonas aeruginosa, Pseudomonas putida, and Vibrio cholerae which have been analyzed for their tol genes) indicate few points of similarity between TolA and TonB. While only TonB contains an elevated amount of Pro residues, both TonB and TolA are found with a high content of Lys residues (between 9 to 14% and 14 to 20% for TonB and TolA respectively), conferring a theoretical pI of 8.8 to 9.8 for TonB and 9.0 to 9.6 for TolA. Thus, TolA and TonB are very basic proteins (in a similar range to histone and Skp proteins), and this could explain their numerous interactions. A homology between TolA of E. coli and TonB of Haemophilus ducreyi C-terminal sequences has previously been reported [38]. Similarly, homology searches of the SwissProt database using the BLAST program with the C-terminal sequences of TolA of P. aeruginosa and P. putida gave a hit with TonB of H. inuenzae. Alignment using the CLUSTALW program of the C-terminal TolA and TonB sequences from the ve different species of the subdivision showed only weak conservation; however, the C-terminal TolA sequence comparisons with that of TonB of the same species gave identity values between 18 and 26%. The identities found in E. coli C-terminal regions of the TolA and TonB proteins are shown in gure 3B. It is noteworthy that in E. coli, these C-terminal sequences of TolA and TonB were both implicated in numerous interactions. The C-terminal region of TolA and TonB interacted, respectively, with Pal and TolB, and with BtuB (Q-QY residue 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 Cterminal 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 -helix, -strand and 3/10 structures [38]), are indicated on the top sequences.

(the 48 last residues) [28]. The knowledge of the folding structure of TonB and the comparison with that of TolA in E. coli, both interacting with outer membrane components, together with new data on these proteins from other species, would further indicate the structural or functional relevance of the homologies found in their C-terminal domains. 5. Possible function of the Tol-Pal complexes Aside from its implications in outer membrane integrity, the specic function of the Tol-Pal system remains unknown, although the tol-pal gene cluster has been found in many Gram-negative bacteria. A hypothetical function has been suggested in which TolA would drive newly synthesized outer membrane components across the periplasm [35]. This hypothesis is in accordance with the observation of in vitro interactions of TolB and the central domain of TolA with outer membrane trimeric porins [19, 42]. Thus, the Tol proteins might function in the dynamic assembly of outer membrane porins that require de novo LPS synthesis. Furthermore, a recent observation indicated that TolA was found to be required for surface expression of O-lipopolysaccharide, and to a lesser extent, for the synthesis of the LPS core [20]. In relation to OMV formation during cell growth, we suspect that any defect of the Tol-Pal system could indirectly increase the OMV amount since the defect may interfere with the dynamics of outer membrane component assembly. Recently, tol mutants have been described in genetic contexts different from E. coli K12. The tol-

pal mutants of P. putida were found to induce outer membrane defects similar to those described in E. coli K12, and also extensive lamentation [37]. E. coli O7 tolQ and V. cholerae tol mutants were found to be hypersensitive to deleterious agents and formed cell laments [20, 23]. Similarly, but only under low and high osmolarities, the E. coli K12 tolA mutant formed laments with impaired septation [39]. These morphological changes indicate that tol mutations may affect the formation of the cell wall peptidoglycan and normal septation [20]. The tolB mutation of Salmonella typhimurium was also obtained and TolB was found to be implicated in its virulence [8], but the tolB phenotype was not characterized. However, tolA mutants were never obtained in P. aeruginosa and E. coli O7, suggesting that this mutation is lethal [16, 20], with the death resulting from the possible toxicity of the accumulated O antigen. Thus, the TolA-QR protein complex, previously recovered at a density corresponding to the contact sites between inner and outer membranes [22], may participate in a functional site of export of cell envelope components through the periplasm. The in vivo cellular localization of the TolQ-R-A proteins still remains to be analyzed by microscopy, since the proposed contact sites between the two membranes remain a predicted region for cell growth [24]. 6. Conclusion Studies on the interactions between Tol-Pal components have shown six protein complexes present in the E. coli cell envelope which are localized in the

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[3] Abergel C., Walburger A., Chenivesse S., Lazdunski C., Crystallization and preliminary crystallographic study of the peptidoglycan-associated lipoprotein from E. coli , Acta cryst. 57 (2001) 317319. [4] Andersen C., Hugues C., Koronakis V., Chunnel vision: export and efux through bacterial channel-tunnels, EMBO Reports 1 (2000) 313318. [5] Bernadac A., Gavioli M., Lazzaroni J.C., Raina S., Lloubs R., Escherichia coli tol-pal mutants form outer membrane vesicles, J. Bacteriol. 180 (1998) 48724878. [6] Bouveret E., Derouiche R., Rigal A., Lloubs R., Lazdunski C., Bndetti H., Peptidoglycan-associated lipoproteinTolB interaction, J. Biol. Chem. 270 (1995) 1107111077. [7] Bouveret E., Rigal A., Lazdunski C., Bndetti H., Distinct regions of the colicin A translocation domain are involved in the interaction with TolA and TolB proteins upon import into Escherichia coli , Mol. Microbiol. 1 (1998) 143157. [8] Bowe F., Lipps C.J., Tsolis R.M., Groisman E., Heffron F., Kusters J.G., At least four percent of the Salmonella typhimurium genome is required for fatal infection of mice, Infect. Immun. 66 (1998) 33723377. [9] Braun V., Energy-coupled transport and signal transduction through the gram-negative outer membrane via TonBExbB-ExbD-dependent receptor proteins, FEMS Microbiol. Rev. 16 (1995) 295307. [10] Braun V., Herrmann C., Evolutionary relationship of uptake systems for biopolymers in Escherichia coli : crosscomplementation between the TonB-ExbB-ExbD and the TolA-TolQ-TolR proteins, Mol. Microbiol. 8 (1993) 261268. [11] Cadieux N., Kadner R.J., Site-directed disulde bonding reveals an interaction site between energy-coupling protein TonB and BtuB, the outer membrane cobalamin transporter, Proc. Natl. Acad. Sci. USA 96 (1999) 1067310678. [12] Carr S., Penfold C., Bamford V., James R., Hemmings A., The structure of TolB, an essential component of the Tol-dependent translocation system, and its protein-protein interaction with the translocation domain of colicin E9, Structure 8 (1999) 5766. [13] Cascales E., Gavioli M., Sturgis J., Lloubs R., Proton motive force drives the interaction of the inner membrane TolA and outer membrane Pal proteins in E. coli , Mol. Microbiol. 38 (2000) 904915. [14] Clavel T., Lazzaroni J.C., Vianney A., Portalier R., Expression of the tolQRA genes of Escherichia coli K-12 is controlled by the RcsC sensor protein involved in capsule synthesis, Mol. Microbiol. 19 (1996) 1925. [15] Clavel T., Germon P., Vianney A., Portalier R., Lazzaroni J.C., TolB protein of Escherichia coli K-12 interacts with the outer membrane peptidoglycan-associated proteins Pal, Lpp and OmpA, Mol. Microbiol. 29 (1998) 359367. [16] Dennis J.J., Lafontaine E.R., Sokol P.A., Identication and characterization of the tolQRA genes of Pseudomonas aeruginosa, J. Bacteriol. 178 (1996) 70597068. [17] Deprez C., Blanchard L., Simorre J.P., Gavioli M., Guerlesquin F., Lazdunski C., Lloubs R., Marion D., Assignment of the 1 H, 15 N and 13 C resonances of the C-terminal domain of the TolA protein of Escherichia coli, involved in cell envelope integrity, J. Biomol. NMR 18 (2000) 179180. [18] Derouiche R., Bndetti H., Lazzaroni J.C., Lazdunski C., Lloubs R., Protein complex within Escherichia coli inner

inner and outer membranes, linking the two membranes. Knowledge of simultaneous or transient interactions between these different complexes (TolQTolR, TolQ-TolA, TolR-TolA, TolA-Pal, TolA-TolB and TolB-Pal) would further indicate the oligomeric form of the Tol-Pal complex in the presence or absence of a pmf. The TolQ-R-A complex, sharing homologies with the TonB system, may be involved in energy transduction to Pal for an unknown event or in driving outer membrane components through the periplasm in an energy-dependent process. The effects of tol-pal mutations together with the physiological analyses indicate that the whole system is involved in OM integrity, which is consistent with the TolB-Pal complex interacting with the structural network linking OM to the peptidoglycan. Sequence similarities found between the C-terminal domain of TolB and the active site of class B -lactamases have suggested a role in peptidoglycan metabolism [1]. Preliminary experiments have also shown that vancomycin susceptibility (an antibiotic inhibiting the transpeptidation step in peptidoglycan synthesis) was increased in tol-pal strains, indicating a difference from the lpp strain [13]. Together with the possible N-acetyl-muramoyl-L-alanine amidase activity of the YbgF proteins found in Chlamydia species [43], these results tend to indicate that the Tol-Pal system is associated with cell wall metabolism. Acknowledgements The authors thank M. Gavioli for technical assistance, J. Sturgis for helpful discussions and careful reading of the manuscript, A. Charbit for the gift of monoclonal antibody E302, and A. Rigal and H. Bndetti for their contribution to the Tol system. Computations were performed at the SIB using the BLAST network service. This work was in part supported by a grant from PCV, MENRT, and the E.E.C. References
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