The Tol/Pal system has been extensively studied; however, the normal cellular function of the tol system in E. coli is less clear. It is thought that the Tol proteins play a role in maintaining cell envelope integrity through a network of interactions spanning the cytoplasmic membrane, periplasm, and outer membrane (Lloubes, Cascales et al. 2001). Moreover, the Tol system is coupled to the proton motive force across the CM (Lloubes, Cascales et al. 2001) and thus transfers energy between CM and OM. The tol dependent translocation system consists of the proteins TolA, TolB, TolQ, TolR and Pal (James, Penfold et al. 2002). Strains containing a mutation in the tol genes are insensitive to group A colicins. Pal is a lipoprotein associated with the peptidoglycan (Mizuno 1979) and TolB is a periplasmic protein (Isnard, Rigal et al. 1994). TolB is associated with the OM through the interaction of its C- terminal -propeller domain with Pal, while its N-terminal domain interacts with the C terminal domain of the TolA protein, TolAIII (Bouveret, Derouiche et al. 1995; Dubuisson, Vianney et al. 2002). The TolA protein is anchored in
the cytoplasmic membrane, via its N-terminal TolAI region which facilitates its interaction with TolQ and TolR (Germon, Ray et al. 2001) forming an inner membrane complex.
The production levels of the Tol-Pal have not yet been simultaneously measured in a given strain, although, it is estimated by Western blotting that TolA is present at approximately 800 copies per cell and there are 2000-3000 copies of TolR (Levengood, Beyer et al. 1991; Muller, Vianney et al. 1993). This is consistent with the results of radioactive labelling studies which suggest that the TolA:TolR:TolQ ratio is 1:2:6 (Guihard, Boulanger et al. 1994). Pal has been found to be present in great excess over the TolA protein and it is predicted that there are 10,000 to 30,000 Pal molecules per cell (Cascales, Gavioli et al. 2000). It is not yet clear if the Tol system is dependent on the formation of stable complexes of defined stoichiometry or on more subtle transient interactions between different protein partners.
The tol mutations induce sensitivity to dyes and detergents and their tendency to release periplasmic proteins into the medium indicates that the membrane permeability of tol mutants is affected. Consequently, it has been suggested that the Tol system maintains outer membrane integrity (Lazzaroni, Fognini- Lefebvre et al. 1989). Identification of the TolA-Pal and TolB-Pal interactions and the interaction of TolB and Pal with OmpA and Lpp (Clavel, Germon et al. 1998; Cascales, Bernadac et al. 2002), suggested that the Tol system could link the OM and CM to peptidoglycan. The observed interaction of TolA and TolB with porin trimers (Derouiche, Gavioli et al. 1996; Rigal, Bouveret et al. 1997; Clavel, Germon et al. 1998; Dover, Evans et al. 2000) suggested that these
proteins could be involved in porin assembly. This is supported by the decrease in the quantity of OmpF and LamB in tol mutants (Lazdunski, Bouveret et al. 1998). LPS translocation or assembly could also be regulated by the Tol proteins as tol mutants can have reduced LPS content and TolA is required for the surface expression of O-antigen and LPS (Lazzaroni, Germon et al. 1999). The Tol-Pal proteins might assist porin trimer-LPS complexes with a molecular weight greater than 50 kDa, the cut-off point for translocation across the peptidoglycan layer, to cross the peptidoglycan layer (Clavel, Germon et al. 1998). The Tol-Pal system contributes in the transport of newly synthesized OM components by bringing the CM and OM into close proximity via the TolA-Pal or TolA-TolB interactions (Cascales and Lloubes 2004).
Studies that demonstrated that the CM Tol complex, TolA, TolQ and TolR, can transduce energy dependent on the proton motive force, suggest that TolQRA could act as an ion potential-driven molecular motor (Cascales, Gavioli et al. 2000). This is supported by the recent findings that TolQ and TolR transduce energy to TolA that eventually lead to release of immunity protein as a requirement of ColE9 to transport across the periplasm (Vankemmelbeke, Zhang et al. 2009).
Several studies have now revealed that interaction of group A colicins with Tol proteins is required for translocation. Three major classes of colicin-tolerant (tol) mutants of E. coli K-12, which adsorbed colicins but were not killed by them, were isolated and studied genetically and physiologically (Nagel de Zwaig and Luria 1967). The Tol system was suggested to contribute to the cell
envelop integrity, this was based on the pleotropic phenotypes exhibited by tol and pal mutants including the hypersensitivity to drugs and detergents and the release of periplasmic contents into the extracellular medium (Nagel de Zwaig and Luria 1967; Fognini-Lefebvre, Lazzaroni et al. 1987). Tol mutants have shown a defect in the O-antigen insertion into the OM, so Tol-pal proteins interfere with the assembly of OM components (Meury and Devilliers 1999; Cascales, Buchanan et al. 2007). The tolABQR and pal mutants form OM vesicles that contain periplasmic proteins. The OM blebbing in tol mutants possibly reflects an incorrect assembly of the OM structure during cell growth.
E. coli strains with tonB mutations are tolerant to all group B colicins, although, exbB or exbD mutants retain some slight sensitivity to colicins due to cross interaction with the Tol system (Davies and Reeves 1975a; Braun and Herrmann 1993). Strains with both exbB/D and tolQ/R mutations are tolerant to group B colicins (Braun 1989). Mutations in the TonB transmembrane domain that inactivates TonB were found to prevent PMF-dependant conformational changes (Larsen and Postle 2001). Colicin D shares 96 % homology with ColB yet mutants in TonB Arg158Ser and Pro161Leu are resistant to ColD and sensitive to B, Ia, M and bacteriophage (Mora, Diaz et al. 2005). It was shown that point mutation within TolQR abolish the PMF- dependent TolA interaction.