Insights into the assembly and regulation of the bacterial divisome

Abstract

The ability to split one cell into two is fundamental to all life, and many bacteria can accomplish this feat several times per hour with high accuracy. Most bacteria call on an ancient homologue of tubulin, called FtsZ, to localize and organize the cell division machinery, the divisome, into a ring-like structure at the cell midpoint. The divisome includes numerous other proteins, often including an actin homologue (FtsA), that interact with each other at the cytoplasmic membrane. Once assembled, the protein complexes that comprise the dynamic divisome coordinate membrane constriction with synthesis of a division septum, but only after overcoming checkpoints mediated by specialized protein-protein interactions. In this Review, we summarize the most recent evidence showing how the divisome proteins of Escherichia coli assemble at the cell midpoint, interact with each other and regulate activation of septum synthesis. We also briefly discuss the potential of divisome proteins as novel antibiotic targets.

Fig. 1.. Centering and organizing the divisome.

(A) Escherichia coli cell division cycle. Newborn cells (i) carry several spatial FtsZ positioning systems, including SlmA and MatP on the nucleoid and MinCD oscillating between the cell poles, that keep FtsZ from assembling into a Z ring at the cell poles or on top of the unsegregated nucleoid. As the replicated daughter nucleoids segregate, the Z ring assembles at midcell (ii) and recruits other septum-synthesis proteins (iii), which result in formation of the division septum. During septum synthesis, FtsZ leaves the division site prior to the other septation proteins (iv). The final step of cytokinesis results in two daughter cells (v), which begin the next cycle during growth conditions. Nutrient depletion or stress induces a quiescent state (vi) in which FtsZ localizes to putative biomolecular condensates that can revert back to polymer forms after growth restart. (B) Reconstitution of a centered proto-ring in lipid vesicles. After FtsZ, FtsA, and Min proteins are synthesized from a plasmid template or added as purified protein to lipid vesicles, they self-organize into membrane-associated polymers or gradients. Oscillation of MinCDE within the liposome helps to corral FtsA-tethered FtsZ polymers into a focused ring, mimicking in vivo behavior. (C) Theme and varations of Z ring positioning systems in diverse bacterial species, showing three examples of negative spatial regulators (orange gradients) and three examples of positive spatial regulators (blue gradients) and the proteins involved.

Fig. 2.. The E. coli divisome is built in stages.

FtsZ is tethered to the membrane by FtsA and ZipA, forming the proto-ring. This is accompanied by the recruitment of the ABC transporter-like FtsEX complex and FtsK, which contains an N-terminal membrane bound domain required for cell division connected to a cytoplasmic DNA motor domain via a long linker. The next complex to be recruited is FtsQLB, which is important for recruitment and activation of the septum-specific transpeptidase (FtsI) and glycosyltransferase (FtsW). FtsN binds to and activates FtsWI enzymatic activity and is present at high concentrations at later stages. FtsN’s four distinct domains, the short cytoplasmic tail (FtsN_cyto), transmembrane domain, essential periplasmic domain (FtsN^E), and SPOR domain, are highlighted in the cartoon. The recruitment stage for each protein is highlighted by more intense coloring.

Fig. 3.. FtsA oligomerization state regulates divisome activation.

(A) Distinct oligomeric states of FtsA and their effects. WT FtsA initially forms mini-rings on lipid membranes that tether and align separated FtsZ polymers. Although mini-rings have not yet been visualized in vivo, unlocking of FtsA mini-rings by ZipA, FtsX, FtsN (arrows), or by mutations, would be expected to form open FtsA oligomers (arcs) or DS antiparallel FtsA filaments. In vitro, both of these unlocked FtsA states seem to induce tethered FtsZ polymers to laterally associate into bundles, possibly by increasing membrane packing density. FtsA*-like variants bypass several divisome defects in vivo and bypass the mini-ring form in vitro, instead assembling mostly into arcs (e.g. R286W) or DS filaments (G50E). FtsZ polymer bundling may be another divisome activation signal, as the self-bundling FtsZ_L169R (FtsZ*) variant bypasses several divisome defects in vivo and may convert FtsA into an unlocked, active form, suggesting a positive feedback loop (red and black arrows). In addition to FtsN’s ability to unlock FtsA mini-rings, unlocked mini-rings may facilitate binding of FtsN (red and black arrows). (B) FtsA interactions, variants and activities. FtsA interacts with FtsN_cyto through subdomain 1C, FtsZ and ZipA through interactions in subdomain 2B, FtsX through subdomain 1A, another FtsA subunit through several interfaces, and the cytoplasmic membrane via a membrane-targeting amphipathic helix. FtsA*-like substitutions such as R286W likely weaken monomer-monomer interactions that result in unlocking of mini-rings in vitro. FtsN bypass alleles (I143L, E124A) map to subdomain 1C, potentially mimicking FtsN_cyto binding. (C) The changes in FtsA oligomeric state signal the later divisome proteins such as FtsW to switch to the activated form (upward arrow). In addition to the cytoplasmic activation signal from FtsA, conformational changes in FtsQLB in the periplasm, along with FtsN’s essential domain, are also crucial for activation of FtsWI (downward arrow).

Fig. 4.. Divisome proteins build the septum in two tracks.

(A) Cross section of a dividing E. coli cell depicting several processive, FtsZ treadmilling complexes that move rapidly along the cytoplasmic face of the inner membrane to organize septum synthesis, in coordination with several independently moving, slower track of proteins that actively engage in septal peptidoglycan (PG) synthesis in the periplasm. (B) Detail of complexes comprising the different speed modes during septum formation. A subset of FtsN proteins are transiently stationary, possibly because they are bound to denuded septal PG resulting from amidase activity. Another subset of FtsN proteins is on the slow track (~9 nm/s) when in complex with FtsQLB and FtsWI as they synthesize septal PG, activated by FtsN. On the right is a putative fast moving (~30 nm/s) complex containing FtsZ and FtsA that spatially guides FtsQLB and FtsWI so the latter are properly placed to be handed off to the slow track for septum synthesis.

Fig. 5.. Binding of a small molecule inhibitor to the FtsZ interdomain cleft.

A dimer of the core polymerizing domain of FtsZ is shown, highlighting the interdomain cleft that connects the N-terminal GTP binding domain with the C-terminal domain. Many small molecule inhibitors of FtsZ, including the benzamide PC190723 and its derivatives, insert into the interdomain cleft (inset) and inhibit conformational changes within the FtsZ subunit required for its ability to dynamically treadmill.