The multifarious roles of Tol-Pal in Gram-negative bacteria

Abstract

In the 1960s several groups reported the isolation and preliminary genetic mapping of Escherichia coli strains tolerant towards the action of colicins. These pioneering studies kick-started two new fields in bacteriology; one centred on how bacteriocins like colicins exploit the Tol (or more commonly Tol-Pal) system to kill bacteria, the other on the physiological role of this cell envelope-spanning assembly. The following half century has seen significant advances in the first of these fields whereas the second has remained elusive, until recently. Here, we review work that begins to shed light on Tol-Pal function in Gram-negative bacteria. What emerges from these studies is that Tol-Pal is an energised system with fundamental, interlinked roles in cell division - coordinating the re-structuring of peptidoglycan at division sites and stabilising the connection between the outer membrane and underlying cell wall. This latter role is achieved by Tol-Pal exploiting the proton motive force to catalyse the accumulation of the outer membrane peptidoglycan associated lipoprotein Pal at division sites while simultaneously mobilising Pal molecules from around the cell. These studies begin to explain the diverse phenotypic outcomes of tol-pal mutations, point to other cell envelope roles Tol-Pal may have and raise many new questions.

Keywords: Ton; cell envelope; divisome; outer membrane; peptidoglycan; proton motive force.

Figure 1.

Structures of Tol-Pal proteins. The figure presents all currently known structures in the PDB for soluble domains and/or complexes of Tol-Pal proteins. A, The solution-state structure of the TolR periplasmic domain dimer in its ‘open’ PG-binding conformation (PDB code: 2JWK); the groove running between the two monomers is thought to be the PG binding site. The structure is that of H. influenzae TolR (residues 59–130) (Parsons, Grishaev and Bax 2008). See text for details. B, Crystal structure of the strand-swapped TolR periplasmic domain dimer, the ‘closed’ state (PDB code: 5BY4). This is the E. coli TolR structure (residues 36–142) in which the additional N- and C-terminal sequences occlude the deep groove between the monomers and block binding to PG (Wojdyla et al. 2015). In both a and b, the position of Tyr117 is shown. A Tyr117Cys substitution forms a spontaneous disulphide bond between TolR monomers that inactivates the Tol-Pal system in vivo (Goemaere et al. 2007b). These residues are only close enough to form a disulphide in b suggesting inactivation comes from stabilising the closed state of the stator complex (Wojdyla et al. 2015). C, Crystal structure of P. aeruginosa TolA^III (PDB code: 1LR0) (Witty et al. 2002). D, Solution state structure of the P. aeruginosa TolA^III-TolB^22–33 complex (PDB code: 6S3W). TolB binds through a β-strand augmentation mechanism in which the C-terminal α-helix (α4) of TolA is displaced by the N-terminus of TolB (Glu22-Ser33, in orange) (Szczepaniak et al. 2020). E, Crystal structure of E. coli TolB (PDB code: 1CRZ). TolB is comprised of an N-terminal α/β domain and a six-bladed β-propeller domain (Abergel et al. 1999). F, Crystal structure of the E. coli TolB-Pal complex (PDB code:2W8B) (Bonsor et al. 2009). The structure is rotated 90° relative to TolB in e. Shown in orange (Glu22-Ser33) is the N-terminus of TolB that becomes ordered in the Pal-bound state (Bonsor et al. 2009). G, Crystal structure of E. coli Pal (1OAP). H, Solution state structure of H. influenzae Pal bound to the peptidoglycan precursor UDP-N-acetylmuramyl-L-Ala-α-D-Glu-m-Dap-D-Ala-D-Ala (PDB code: 2AIZ) (Parsons, Lin and Orban 2006). The figure shows how the m-DAP residue of PG reaches into the binding pocket of Pal.

Figure 2.

Model of the TolQ-TolR stator. A, Alignment of the trans-pore helix regions of E. coli TolR, ExbD and MotB. Asp23 and Phe32 (TolR numbering) are conserved across all three proteins. The alignment was generated using MUSCLE ClustalW. B, Model of the TolQ-TolR complex based upon the 5:2 structure of ExbB-ExbD (Celia et al. 2019). Horizontal lines represent approximate position of the inner membrane. The model was generated using SWISS-MODEL (Waterhouse et al. 2018) (https://swissmodel.expasy.org/). C, Model of each TolQ monomer. D, Co-localization of functionally important TolQ and TolR residues in the model. The TPH of TolR and three transmembrane helices of TolQ (residues 19–37, 138–156 and 169–187) are shown. The figure highlights the proximity of residues TolQ Thr145, Thr178 and TolR Asp23 within the model, all of which have been identified previously as functionally important (Goemaere et al. 2007b). The conserved residue Phe32 is also shown. E, Comparison of the electrostatic surfaces for the cytoplasmic chambers of the TolQ model with that of the ExbB structure (PDB code: 6TYI) (Celia et al. 2019). Figures were generated using chimera (Jurrus et al. 2018). Upper panels are cut-throughs of each stator protein while the lower panels are 90° rotations showing the cytoplasmic constriction. The TolQ chamber is predominantly electronegatively charged whereas ExbB has bands of positive and negative charge. The transmembrane region of both proteins is a predominantly neutral pore in which the TPHs of the TolR dimer reside (not shown in this figure).

Figure 3.

Structural basis for Tol-Pal parasitism by bacteriocins and filamentous bacteriophages. See text Box 1 for details. A, Phage g3p N1 domain binds E. coli TolA^III through a β-strand augmentation mechanism at the same site as TolB (Fig. 1D) but in the opposite orientation (PDB code: 1TOL) (Lubkowski et al. 1999). B, Colicin A (residues 53–107) also binds TolA^III through β-strand augmentation, but on the opposing side of the β-sheet targeted by phage g3p N1 and TolB (PDB code: 3QDR) (Li et al. 2012). C, Crystal structure of the colicin E9 translocation (T-) domain (residues 32–47) bound to TolB (PDB code: 2IVZ) (Loftus et al. 2006). Colicin E9 binds at the same β-propeller site on TolB as used by Pal but does not induce the conformational changes in TolB that sequester its N-terminus, as in Fig. 1F. The N-terminus of TolB in this complex (not shown) is disordered thereby promoting binding to TolA^III (Bonsor et al. 2009).

Figure 4.

PMF-driven mobilisation-and-capture of Pal by Tol proteins drives Pal accumulation at division sites. Figure adapted from (Szczepaniak et al. 2020). See text for details. The following model assumes that TolB-Pal complexes are actively dissociated by PMF-linked TolQ-TolR-TolA, and that dissociated TolB molecules are translocated through holes in the PG layer by TolA. Top panel—Elongating cell. A, The stator complex TolQ-TolR (depicted as a 5:2 complex based on the modelling presented in Fig. 2) and TolA are free to diffuse in the inner membrane (IM). The periplasmic domain of TolR is shown as a strand-swapped dimer, consistent with available structural data (Fig. 1). Pal is bound to the mDAP moiety of peptidoglycan (PG; white line against grey Pal) unless in complex with TolB, which blocks PG binding and increases Pal diffusion in the outer membrane (OM). B, TolA associates with TolQ-TolR. It is not known if the complex is a stable TolQ-TolR-TolA complex or if the association is transient. C, Proton flux through the residues of the TolQ pentamer and the transpore helices of the TolR dimer, coupled to possible rotatory motions of the stator subunits, cause unravelling of the strand-swapped periplasmic domain of TolR allowing it to extend and bind the cell wall. Consequent with these changes, TolA extends through a hole in the PG layer, possibly aided by interactions with the TolR-PG complex. At the outer membrane, TolA binds the N-terminus of TolB which is in complex with Pal. D, Loss of protonation causes the whole assembly to relax back to its starting position, providing the driving force to bring TolB down through the PG layer into the lower periplasmic compartment. E, TolB now dissociates from TolA—presumably because TolA is no longer exerting a force and the complex has a weak affinity—and diffuses until it encounters a hole in the PG through which it can reach the outer membrane and rebind Pal to repeat the process. Bottom panel—Dividing cell. The TolQ-TolR-TolA complex is recruited to the divisome which confines its TolB capturing activity. As TolB-Pal complexes diffuse past the septum they are actively dissociated, releasing Pal and recycling TolB, as described above. Thus, Pal located at the divisome is kept free of TolB by localised TolQ-TolR-TolA. Recycled TolB diffuses away and mobilises non-septal Pal molecules. Because TolQ-TolR-TolA is not freely circulating this leads to a greater number of TolB molecules being located in the outer periplasmic compartment (i.e. TolB-Pal complexes are longer lived than in an elongating cell) and as a result Pal mobility increases throughout the cell except at the septum. More and more Pal molecules now accumulate at the septum where they stabilise the link between the outer membrane and the underlying cell wall in daughter cells.