Shishen Du

Bacterial cytokinesis: From Z ring to divisome †

Ancestral homologues of the major eukaryotic cytoskeletal families, tubulin and actin, play critical roles in cytokinesis of bacterial cells. FtsZ is the ancestral homologue of tubulin and assembles into the Z ring that determines the division plane. FtsA, a member of the actin family, is involved in coordinating cell wall synthesis during cytokinesis. FtsA assists in the formation of the Z ring and also has a critical role in recruiting downstream division proteins to the Z ring to generate the divisome that divides the cell. Spatial regulation of cytokinesis occurs at the stage of Z ring assembly and regulation of cell size occurs at this stage or during Z ring maturation.

SlmA antagonism of FtsZ assembly employs a two-pronged mechanism like MinCD.

Assembly of the Z-ring over unsegregated nucleoids is prevented by a process called nucleoid occlusion (NO), which in Escherichia coli is partially mediated by SlmA. SlmA is a Z ring antagonist that is spatially regulated and activated by binding to specific DNA sequences (SlmA binding sites, SBSs) more abundant in the origin proximal region of the chromosome. However, the mechanism by which SBS bound SlmA (activated form) antagonizes Z ring assembly is controversial. Here, we report the isolation and characterization of two FtsZ mutants, FtsZ-K190V and FtsZ-D86N that confer resistance to activated SlmA. In trying to understand the basis of resistance of these mutants, we confirmed that activated SlmA antagonizes FtsZ polymerization and determined these mutants were resistant, even though they still bind SlmA. Investigation of SlmA binding to FtsZ revealed activated SlmA binds to the conserved C-terminal tail of FtsZ and that the ability of activated SlmA to antagonize FtsZ assembly required the presence of the tail. Together, these results lead to a model in which SlmA binding to an SBS is activated to bind the tail of FtsZ resulting in further interaction with FtsZ leading to depolymerization of FtsZ polymers. This model is strikingly similar to the model for the inhibitory mechanism of the spatial inhibitor MinCD.

The bypass of ZipA by overexpression of FtsN requires a previously unknown conserved FtsN motif essential for FtsA-FtsN interaction supporting a model in which FtsA monomers recruit late cell division proteins to the Z ring

Assembly of the divisome in Escherichia coli occurs in two temporally distinct steps. First, FtsZ filaments attached to the membrane through interaction with FtsA and ZipA coalesce into a Z ring at midcell. Then, additional proteins are recruited to the Z ring in a hierarchical manner to form a complete divisome, activated by the arrival of FtsN. Recently, we proposed that the interaction of FtsA with itself competes with its ability to recruit downstream division proteins (both require the IC domain of FtsA) and ZipAs essential function is to promote the formation of FtsA monomers. Here, we tested whether overexpression of a downstream division protein could make ZipA dispensable, presumably by shifting the FtsA equilibrium to monomers. Only overexpression of FtsN bypassed ZipA and a conserved motif in the cytoplasmic domain of FtsN was required for both the bypass and interaction with FtsA. Also, this cytoplasmic motif had to be linked to the periplasmic E domain of FtsN to bypass ZipA, indicating that linkage of FtsA to periplasmic components of the divisome through FtsN was essential under these conditions. These results are used to further elaborate our model for the role of FtsA in recruiting downstream division proteins.

FtsEX acts on FtsA to regulate divisome assembly and activity

Significance Understanding divisome assembly and activation has become the focus of research on bacterial cytokinesis. However, very little is known about how this process is regulated. Here, we find that FtsEX (an ATP-binding cassette transporter-like complex) acts on the bacterial actin homolog FtsA to regulate divisome assembly and function in Escherichia coli. Our results suggest that FtsEX antagonizes FtsA polymerization to promote divisome assembly and continual ATP hydrolysis by FtsEX is needed for the divisome to synthesize septal peptidoglycan. Because FtsEX is also required for cell wall hydrolysis at the septum, our study indicates that FtsEX couples cell wall synthesis and hydrolysis at the septum by acting through FtsA. Our study also implies that unpolymerized FtsA is favored for division and FtsW plays a critical role in divisome activation. Bacterial cell division is driven by the divisome, a ring-shaped protein complex organized by the bacterial tubulin homolog FtsZ. Although most of the division proteins in Escherichia coli have been identified, how they assemble into the divisome and synthesize the septum remains poorly understood. Recent studies suggest that the bacterial actin homolog FtsA plays a critical role in divisome assembly and acts synergistically with the FtsQLB complex to regulate the activity of the divisome. FtsEX, an ATP-binding cassette transporter-like complex, is also necessary for divisome assembly and inhibits division when its ATPase activity is inactivated. However, its role in division is not clear. Here, we find that FtsEX acts on FtsA to regulate both divisome assembly and activity. FtsX interacts with FtsA and this interaction is required for divisome assembly and inhibition of divisome function by ATPase mutants of FtsEX. Our results suggest that FtsEX antagonizes FtsA polymerization to promote divisome assembly and the ATPase mutants of FtsEX block divisome activity by locking FtsA in the inactive form or preventing FtsA from communicating with other divisome proteins. Because FtsEX is known to govern cell wall hydrolysis at the septum, our findings indicate that FtsEX acts on FtsA to promote divisome assembly and to coordinate cell wall synthesis and hydrolysis at the septum. Furthermore, our study provides evidence that FtsA mutants impaired for self-interaction are favored for division, and FtsW plays a critical role in divisome activation in addition to the FtsQLB complex.

Oligomerization of FtsZ converts the FtsZ tail motif (conserved carboxy‐terminal peptide) into a multivalent ligand with high avidity for partners ZipA and SlmA

A short conserved motif located at the carboxy terminus of FtsZ, referred to here as the CCTP (conserved carboxy‐terminal peptide), is required for the interaction of FtsZ with many of its partners. In Escherichia coli interaction of FtsZ with its membrane anchors, ZipA and FtsA, as well as the spatial regulators of Z‐ring formation, MinC and SlmA, requires the CCTP. ZipA interacts with FtsZ with high affinity and interacts with the CCTP with low affinity, but the reason for this difference is not clear. In this study, we show that this difference is due to the oligomerization of FtsZ converting the CCTP to a multivalent ligand that binds multiple ZipAs bound to a surface with high avidity. Artificial dimerization of the CCTP is sufficient to increase the affinity for ZipA in vitro. Similar principles apply to the interaction of FtsZ with SlmA. Although done in vitro, these results have implications for the recruitment of FtsZ to the membrane in vivo, the interaction of FtsZ with spatial regulators and the reconstitution of FtsZ systems in vitro.

Assembly and activation of the Escherichia coli divisome

Cell division in Escherichia coli is mediated by a large protein complex called the divisome. Most of the divisome proteins have been identified, but how they assemble onto the Z ring scaffold to form the divisome and work together to synthesize the septum is not well understood. In this review, we summarize the latest findings on divisome assembly and activation as well as provide our perspective on how these two processes might be regulated.

MinC/MinD copolymers are not required for Min function

In Escherichia coli, precise placement of the cytokinetic Z ring at midcell requires the concerted action of the three Min proteins. MinD activates MinC, an inhibitor of FtsZ, at least in part, by recruiting it to the membrane and targeting it to the Z ring, while MinE stimulates the MinD ATPase inducing an oscillation that directs MinC/MinD activity away from midcell. Recently, MinC and MinD were shown to form copolymers of alternating dimers of MinC and MinD, and it was suggested that these copolymers are the active form of MinC/MinD. Here, we use MinD mutants defective in binding MinC to generate heterodimers with wild‐type MinD that are unable to form MinC/MinD copolymers. Similarly, MinC mutants defective in binding to MinD were used to generate heterodimers with wild‐type MinC that are unable to form copolymers. Such heterodimers are active and in the case of MinC were shown to mediate spatial regulation of the Z ring demonstrating that MinC/MinD copolymer formation is not required. Our results are consistent with a model in which a membrane anchored MinC/MinD complex is targeted to the Z ring through the conserved carboxy tail of FtsZ leading to breakage of FtsZ filaments.

E. coli Cell Cycle Machinery

Cytokinesis in E. coli is organized by a cytoskeletal element designated the Z ring. The Z ring is formed at midcell by the coalescence of FtsZ filaments tethered to the membrane by interaction of FtsZs conserved C-terminal peptide (CCTP) with two membrane-associated proteins, FtsA and ZipA. Although interaction between an FtsZ monomer and either of these proteins is of low affinity, high affinity is achieved through avidity - polymerization linked CCTPs interacting with the membrane tethers. The placement of the Z ring at midcell is ensured by antagonists of FtsZ polymerization that are positioned within the cell and target FtsZ filaments through the CCTP. The placement of the ring is reinforced by a protein network that extends from the terminus (Ter) region of the chromosome to the Z ring. Once the Z ring is established, additional proteins are recruited through interaction with FtsA, to form the divisome. The assembled divisome is then activated by FtsN to carry out septal peptidoglycan synthesis, with a dynamic Z ring serving as a guide for septum formation. As the septum forms, the cell wall is split by spatially regulated hydrolases and the outer membrane invaginates in step with the aid of a transenvelope complex to yield progeny cells.

MipZ: One for the Pole, Two for the DNA

In this issue of Molecular Cell,Kiekebusch et al. (2012) show that a novel regulatory mechanism of the ATPase cycle of MipZ, together with nonspecific DNA binding, generates the MipZ gradient that spatially regulates Z ring formation in Caulobacter crescentus.

Oligomerization of FtsZ converts the FtsZ tail motif (CCTP) into a multivalent ligand with high avidity for partners ZipA and SlmA

A short conserved motif located at the carboxy terminus of FtsZ, referred to here as the CCTP (conserved carboxy‐terminal peptide), is required for the interaction of FtsZ with many of its partners. In Escherichia coli interaction of FtsZ with its membrane anchors, ZipA and FtsA, as well as the spatial regulators of Z‐ring formation, MinC and SlmA, requires the CCTP. ZipA interacts with FtsZ with high affinity and interacts with the CCTP with low affinity, but the reason for this difference is not clear. In this study, we show that this difference is due to the oligomerization of FtsZ converting the CCTP to a multivalent ligand that binds multiple ZipAs bound to a surface with high avidity. Artificial dimerization of the CCTP is sufficient to increase the affinity for ZipA in vitro. Similar principles apply to the interaction of FtsZ with SlmA. Although done in vitro, these results have implications for the recruitment of FtsZ to the membrane in vivo, the interaction of FtsZ with spatial regulators and the reconstitution of FtsZ systems in vitro.