The trans-envelope Tol-Pal complex is part of the cell division machinery and required for proper outer-membrane invagination during cell constriction in E. coli - PubMed (original) (raw)

The trans-envelope Tol-Pal complex is part of the cell division machinery and required for proper outer-membrane invagination during cell constriction in E. coli

Matthew A Gerding et al. Mol Microbiol. 2007 Feb.

Abstract

Fission of bacterial cells involves the co-ordinated invagination of the envelope layers. Invagination of the cytoplasmic membrane (IM) and peptidoglycan (PG) layer is likely driven by the septal ring organelle. Invagination of the outer membrane (OM) in Gram-negative species is thought to occur passively via its tethering to the underlying PG layer with generally distributed PG-binding OM (lipo)proteins. The Tol-Pal system is energized by proton motive force and is well conserved in Gram-negative bacteria. It consists of five proteins that can connect the OM to both the PG and IM layers via protein-PG and protein-protein interactions. Although the system is needed to maintain full OM integrity, and for class A colicins and filamentous phages to enter cells, its precise role has remained unclear. We show that all five components accumulate at constriction sites in Escherichia coli and that mutants lacking an intact system suffer delayed OM invagination and contain large OM blebs at constriction sites and cell poles. We propose that Tol-Pal constitutes a dynamic subcomplex of the division apparatus in Gram-negative bacteria that consumes energy to establish transient trans-envelope connections at/near the septal ring to draw the OM onto the invaginating PG and IM layers during constriction.

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Figures

Fig. 1

Map of chromosomal tol-pal cluster, mutations and constructs. The E. coli tol-pal gene cluster is arranged in two operons (ybgC-tolA and tolB-ybgF) the transcription of which proceeds from left to right (Vianney et al., 1996). Indicated are the site of EZTn insertion in strain FB20229 [_tolA_], the deletions in strains MG4 [_tolQ-pal_] and MG5 [_pal_], and the inserts present in the plasmids or phage encoding GFP or RFP fusions. Numbers refer to base pairs counting from the start codon of tolQ. GFP refers to GFPmut2 (Cormack et al., 1996), and RFP to the cherry variant of monomeric RFP (Shaner et al., 2004). The immature form of the TTGFP–TolB fusion encoded by λNP7 lacks the native signal peptide of TolB but contains the N-terminal signal sequence of TorA such that it is routed to the periplasm via the Tat system. The other four fusions contain complete native polypeptide appended to GFP (TolQ, R, A) or RFP (Pal), as indicated.

Fig. 2

Functionality of GFP–TolA and Pal–RFP. Panels A–D show suppression of cell chaining in tolA cells by GFP–TolA, and in pal cells by Pal–RFP. Strains FB20229/pMLB1113ΔH (A), FB20229/pNP4 (B), MG5/pMLB1113ΔH (C) and MG5/pMG36 (D) were grown in LB (0% NaCl) medium supplemented with 5 μM (A–C) or 100 μM (D) IPTG. Plasmid pMLB1113ΔH is an appropriate vector control for both pNP4 and pMG36. Panels A and C show DIC images, and panels B and D show both DIC and corresponding fluorescence images. The insert in A shows part of a tolA chain at higher magnification. Bar equals 2.5 (insert in A) or 5 μm. Panel E shows suppression of hypersensitivity to SDS by GFP–TolA in tolA cells, and by Pal–RFP in pal cells. Overnight cultures of wt and mutant strains carrying pNP4 [Plac∷_gfp-tolA_] or pMG36 [Plac∷_pal-rfp_] were diluted to optical densities (600 nm) of 2 × 10−3, 2 × 10−4 and 2 × 10−5 (left to right), and 5 μl aliquots were spotted on LB agar containing SDS and/or IPTG as indicated. Plates were incubated at 30°C for 24 h and then at 20°C for 72 h. Strains used were TB28 [wt], MG4 [_tolQ-pal_], MG5 [_pal_] and FB20229 [_tolA_].

Fig. 3

Increased periplasmic volume at the constriction site of tol-pal mutants, and release of OM vesicles. Periplasmic, Tat-targeted, GFP (TTGFP) in wt (A), tolA (B), pal (C) and tolQ-pal (D) cells. Note: (i) the squatness of mutant versus wt cells, (ii) the pronounced accumulation of TTGFP around constriction sites (some are indicated by arrowheads) in mutant cells versus the even peripheral distribution of TTGFP in wt cells, and (iii) the presence of fluorescence-filled blebs at the poles of mutant cells, and numerous free vesicles in the culture media. Arrows point to some of the cell-associated and free vesicles. The inset in D shows a prominent bleb associated with the constriction site. Strains used were TB28 (A), FB20229 (B), MG5 (C) and MG4 (D), each harbouring pTB6 [Plac∷TT_gfp_]. Cells were grown at 30°C in M9-glucose medium supplemented with 5 μM IPTG. Bar equals 2 μm. DIC images in this and following figures are shown immediately to the right, or below, the corresponding fluorescence images.

Fig. 4

Localization of TolQ–GFP to the division site in wt and mutant cells. TolQ–GFP in wt (A), tolA (B), pal (C) and tolQ-pal (D) cells. Strains used were TB28 (A), FB20229 (B), MG5 (C) and MG4 (D), each harbouring pNP2 [Plac∷_tolQ-gfp_]. Cells were grown at 30°C in M9-glucose medium supplemented with 25 μM IPTG. Bar equals 2 μm.

Fig. 5

Localization of GFP–TolR in wt and mutant cells. GFP–TolR in wt (A), tolA (B), pal (C) and tolQ-pal (D) cells. Note the failure of the fusion to accumulate at constriction sites in cells that lack the other four Tol–Pal proteins (D). Strains used were TB28 (A), FB20229 (B), MG5 (C) and MG4 (D), each harbouring pNP3 [Plac∷_gfp-tolR_]. Cells were grown at 30°C in M9-glucose medium supplemented with 5 μM IPTG. Bar equals 2 μm.

Fig. 6

Localization of GFP–TolA to the division site in wt and mutant cells. GFP–TolA in wt (A), tolA (B), pal (C) and tolQ-pal (D) cells. Note the normal morphology of cells in B. Strains used were TB28 (A), FB20229 (B), MG5 (C) and MG4 (D), each harbouring pNP4 [Plac∷_gfp-tolA_]. Cells were grown at 30°C in M9-glucose medium supplemented with 5 μM IPTG. Bar equals 2 μm.

Fig. 7

Localization of Pal–RFP in wt and mutant cells. Pal–RFP in wt (A), tolA (B), pal (C) and tolQ-pal (D) cells. Note the normal morphology of cells in C, and vesicle formation in D. The inset in D shows the formation of a large vesicle at the constriction site of a cell, and the absence of Pal–RFP in the lumen of the vesicle. Strains used were TB28 (A), FB20229 (B), MG5 (C) and MG4 (D), each harbouring pMG36 [Plac∷_pal-rfp_]. Cells were grown at 30°C in M9-glucose medium supplemented with 50 μM IPTG. Bar equals 2 μm.

Fig. 8

Localization of TTGFP–TolB to division sites. Panel A shows the accumulation of TTGFP–TolB at sites of constriction in wt cells. Cells from a single field were rearranged to highlight the localization pattern of the fusion during the division cycle. Panel B shows the localization of unfused TTGFP as a control. Note the absence of accumulation at constriction sites of TTGFP. Strain TB28 lysogenic for λNP7 [Plac∷TT_gfp-tolB_] (A) or λTB6 [Plac∷TT_gfp_] (B) was grown at 30°C in M9-glucose medium supplemented with 50 μM IPTG. Bar equals 2 μm.

Fig. 9

Recruitment of Tol–Pal to division sites requires FtsN. Panels A and B show the dispersal of GFP–TolA in SfiA(SulA)-induced filaments of strain TB28/pJE80/pNP4 [wt/PBAD∷sfiA/Plac∷gfp-tolA_]. Cells were grown in LB supplemented with 25 μM IPTG and either 0.1% glucose (A), or 0.005% arabinose (B). Panels C–G illustrate the localization of GFP–TolA (C and D), TolQ–GFP (E and F) and ZipA–GFP (G) in FtsN+ cells (C and E) and FtsN-depleted filaments (D, F and G). Note the sharp rings in C, E and G versus the peripherally dispersed signals in D and F. Fusions to TolR, TolB and Pal similarly failed to localize to rings in FtsN− filaments (not shown). Strain CH34/pMG20 [Δ_ftsN/PBAD∷TT_bfp_-ED_ftsN_] carrying pNP4 [Plac∷_gfp-tolA_] (C and D), pNP2 [Plac∷_tolQ-gfp_] (E and F), or lysogenic for λCH151 [Plac∷_zipA-gfp_] (G), was grown in M9-maltose (0.2%) supplemented with 5 (C and D), 25 (G), or 50 (E and F) μM IPTG, and with either 0.1% arabinose (C and E) or 0.1% glucose (D, F and G). Bar equals 2 μm.

Fig. 10

Model of Tol–Pal action in OM invagination during cell constriction. Panels represent a longitudinal section through the SR apparatus at the site of constriction. Indicated are the core machinery (core SR), representing all essential division proteins in the SR (dark-blue oval), the periplasmic murein hydrolases responsible for splitting septal murein in separate PG layers (green triangle), and general OM (lipo)proteins, such as Lpp and OmpA, with affinity for PG (yellow ovals). Superimposed are the IM-associated TolQ (purple ovals), TolR (green ovals) and TolA (straight or kinked light-blue ovals) proteins, the periplasmic TolB protein (blue squares), and the Pal OM lipoprotein (red ovals). The results of this study indicate that Pal–PG and Pal–TolA interactions must be readily reversible in vivo. Panels A–C represent a TolA–Pal engagement–disengagement cycle near the start of OM invagination, and D–F represent a cycle when constriction has progressed further. Ion potential over the IM is converted by TolQ and R to allow TolA to engage Pal (Cascales et al., 2000; 2001; Germon et al., 2001). It is proposed that TolA acts as a grabbing device that in its energized form extends through meshes of newly split murein to reach for free Pal in the OM (B and E). Upon catching a Pal partner, TolA snaps back, drawing Pal inwards, and then disengages, allowing Pal to engage the PG layer (C and F). TolA repeats this action as the TolQRA subcomplexes in the IM move along with the constricting core machinery (D–F). The Pal–PG interaction is also transient, and TolB may play a role in dislodging Pal to be reused in a subsequent TolA–Pal interaction event. The resulting waves of Tol–Pal mediated OM–PG and OM–IM connections at/near the core SR allow other OM (lipo)proteins to secure the OM to the PG in a more permanent manner. See the text for additional discussion.

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The trans-envelope Tol-Pal complex is part of the cell division machinery and required for proper outer-membrane invagination during cell constriction in E. coli - PubMed (original) (raw)