FtsA acts through FtsW to promote cell wall synthesis during cell division in Escherichia coli.

In Escherichia coli, FtsQLB is required to recruit the essential septal peptidoglycan (sPG) synthase FtsWI to FtsA, which tethers FtsZ filaments to the membrane. The arrival of FtsN switches FtsQLB in the periplasm and FtsA in the cytoplasm from a recruitment role to active forms that synergize to activate FtsWI. Genetic evidence indicates that the active form of FtsQLB has an altered conformation with an exposed domain of FtsL that acts on FtsI to activate FtsW. However, how FtsA contributes to the activation of FtsW is not clear, as it could promote the conformational change in FtsQLB or act directly on FtsW. Here, we show that the overexpression of an activated FtsA (FtsA*) bypasses FtsQ, indicating it can compensate for FtsQ's recruitment function. Consistent with this, FtsA* also rescued FtsL and FtsB mutants deficient in FtsW recruitment. FtsA* also rescued an FtsL mutant unable to deliver the periplasmic signal from FtsN, consistent with FtsA* acting on FtsW. In support of this, an FtsW mutant was isolated that was rescued by an activated FtsQLB but not by FtsA*, indicating it was specifically defective in activation by FtsA. Our results suggest that in response to FtsN, the active form of FtsA acts on FtsW in the cytoplasm and synergizes with the active form of FtsQLB acting on FtsI in the periplasm to activate FtsWI to carry out sPG synthesis.

Disruption of divisome assembly rescued by FtsN-FtsA interaction in Escherichia coli.

Cell division requires the assembly of a protein complex called the divisome. The divisome assembles in a hierarchical manner, with FtsA functioning as a hub to connect the Z-ring with the rest of the divisome and FtsN arriving last to activate the machine to synthesize peptidoglycan. FtsEX arrives as the Z-ring forms and acts on FtsA to initiate recruitment of the other divisome components. In the absence of FtsEX, recruitment is blocked; however, a multitude of conditions allow FtsEX to be bypassed. Here, we find that all such FtsEX bypass conditions, as well as the bypass of FtsK, depend upon the interaction of FtsN with FtsA, which promotes the back-recruitment of the late components of the divisome. Furthermore, our results suggest that these bypass conditions enhance the weak interaction of FtsN with FtsA and its periplasmic partners so that the divisome proteins are brought to the Z-ring when the normal hierarchical pathway is disrupted.

FtsZ filaments have the opposite kinetic polarity of microtubules.

FtsZ is the ancestral homolog of tubulin and assembles into the Z ring that organizes the division machinery to drive cell division in most bacteria. In contrast to tubulin that assembles into 13 stranded microtubules that undergo dynamic instability, FtsZ assembles into single-stranded filaments that treadmill to distribute the peptidoglycan synthetic machinery at the septum. Here, using longitudinal interface mutants of FtsZ, we demonstrate that the kinetic polarity of FtsZ filaments is opposite to that of microtubules. A conformational switch accompanying the assembly of FtsZ generates the kinetic polarity of FtsZ filaments, which explains the toxicity of interface mutants that function as a capper and reveals the mechanism of cooperative assembly. This approach can also be employed to determine the kinetic polarity of other filament-forming proteins.

How FtsEX localizes to the Z ring and interacts with FtsA to regulate cell division.

In Escherichia coli, FtsEX, a member of the ABC transporter superfamily, is involved in regulating the assembly and activation of the divisome to couple cell wall synthesis to cell wall hydrolysis at the septum. Genetic studies indicate FtsEX acts on FtsA to begin the recruitment of the downstream division proteins but blocks septal PG synthesis until a signal is received that divisome assembly is complete. However, the details of how FtsEX localizes to the Z ring and how it interacts with FtsA are not clear. Our results show that recruitment of FtsE and FtsX is codependent and suggest that the FtsEX complex is recruited through FtsE interacting with the conserved tail of FtsZ (CCTP), thus adding FtsEX to a growing list of proteins that interacts with the CCTP of FtsZ. Furthermore, we find that the N-terminus of FtsX is not required for FtsEX localization to the Z ring but is required for its functions in cell division indicating that it interacts with FtsA. Taken together, these results suggest that FtsEX first interacts with FtsZ to localize to the Z ring and then interacts with FtsA to promote divisome assembly and regulate septal PG synthesis.

FtsEX acts on FtsA to regulate divisome assembly and activity.

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.

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 ZipA's 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.

FtsA mutants impaired for self-interaction bypass ZipA suggesting a model in which FtsA's self-interaction competes with its ability to recruit downstream division proteins.

Z-ring assembly requires polymers of the tubulin homologue FtsZ to be tethered to the membrane. Although either ZipA or FtsA is sufficient to do this, both of these are required for recruitment of downstream proteins to form a functional cytokinetic ring. Gain of function mutations in ftsA, such as ftsA* (ftsA-R286W), bypass the requirement for ZipA suggesting that this atypical, well-conserved, actin homologue has a more critical role in Z-ring function. FtsA forms multimers both in vitro and in vivo, but little is known about the role of FtsA polymerization. In this study we identify FtsA mutants impaired for self-interaction. Such mutants are able to support Z-ring assembly and are also able to bypass the requirement for ZipA. These mutants, including FtsA*, have reduced ability to self-interact but interact normally with FtsZ and are less toxic if overexpressed. These results do not support a model in which FtsA monomers antagonize FtsZ polymers. Instead, we propose a new model in which FtsA self-interaction competes with its ability to recruit downstream proteins. In this model FtsA self-interaction at the Z ring is antagonized by ZipA, allowing unpolymerized FtsA to recruit downstream proteins such as FtsN.

Cell Cycle-Dependent Recruitment of FtsN to the Divisome in Escherichia coli.

Cell division in Escherichia coli starts with the formation of an FtsZ protofilament network at midcell, the Z ring. However, only after a considerable lag period does the cell start to form a midcell constriction. The onset of constriction depends upon the arrival of so-called late divisome proteins, among which, FtsN is the last essential one. The timing and dependency of FtsN arrival to the divisome, along with genetic evidence, suggests it triggers cell division. In this study, we used high-throughput fluorescence microscopy to determine the arrival of FtsN and the early divisome protein ZapA to midcell at a single-cell level during the cell cycle. Our data show while the recruitment of ZapA/FtsZ is gradual in the cell cycle, recruitment of FtsN is rapid and begins at about the onset of constriction. At this time, the fraction of ZapA/FtsZ in the Z ring approaches its peak value. We also find a second increase in FtsN recruitment to the divisome, which begins once the amount of ZapA/FtsZ at midcell starts decreasing. Increasing hypermorphic FtsA* (FtsA R286W), but not FtsA, accelerates FtsN recruitment but not constriction. This finding is consistent with FtsA* recruiting FtsN with some other divisome component being rate-limiting for constriction under these conditions. Finally, our data support the recently proposed idea that ZapA/FtsZ and FtsN are part of physically separate complexes in midcell throughout the whole septation process. IMPORTANCE Cell division in most bacteria starts with the formation of an FtsZ protofilament network at midcell, the Z ring. However, cells only start to constrict after a considerable lag. A factor thought to trigger the onset of constriction in Escherichia coli is FtsN, which is the last essential protein to be recruited to the Z ring. Using a high-throughput quantitative fluorescence microscopy, we determine the cell cycle-dependent recruitment of FtsN to the Z ring. Our data show rapid accumulation of FtsN to the Z ring about a quarter of the cell cycle after the formation of the Z ring. This initial wave is followed by another increase in FtsN recruitment once the FtsZ protofilament network starts to disassemble. The presence of FtsA* accelerates FtsN recruitment to the Z ring but does not lead to earlier constrictions. Our data furthermore suggest FtsZ and FtsN are part of physically separate complexes throughout the division process.

Cross-linking FtsZ polymers into coherent Z rings.

A key event in bacterial cytokinesis is the formation of the Z ring, which serves as a mechanical scaffold that recruits other cytokinetic proteins to establish functional divisomes. This scaffolding function of Z rings is essential throughout cytokinesis, but the underlying molecular interactions are poorly understood. Here we report that a widely conserved FtsZ binding protein, ZapA, has cytological, biochemical and biophysical properties that argue for the importance of cross-linking interactions between FtsZ polymers in the coherence of Z rings. Escherichia coli zapA null mutant cells have Z rings that are structurally looser and many helical precursors of Z rings fail to coalesce into coherent rings. Biophysical behaviour of FtsZ in the presence of ZapA reveals that ZapA not only bundles, but also cross-links FtsZ polymers, which makes it the first cross-linking protein of the bacterial cytoskeleton. Cross-linking in vitro occurs at the stoichiometry of FtsZ-ZapA interaction at the Z rings in vivo, where nearly all intracellular ZapA is dynamically associated. ZapA also stabilizes longitudinal bonds between FtsZ monomers since it promotes the polymerization of FtsZ mutants with lesions at the polymerization interface and since it reverses the inhibitory effects of SulA, a known antagonist of FtsZ longitudinal interactions.

Roles of ATP Hydrolysis by FtsEX and Interaction with FtsA in Regulation of Septal Peptidoglycan Synthesis and Hydrolysis.

In Escherichia coli, FtsEX coordinates peptidoglycan (PG) synthesis and hydrolysis at the septum. It acts on FtsA in the cytoplasm to promote recruitment of septal PG synthetases and recruits EnvC, an activator of septal PG hydrolases, in the periplasm. Following recruitment, ATP hydrolysis by FtsEX is thought to regulate both PG synthesis and hydrolysis, but how it does this is not well understood. Here, we show that an ATPase mutant of FtsEX blocks septal PG synthesis similarly to cephalexin, suggesting that ATP hydrolysis by FtsEX is required throughout septation. Using mutants that uncouple the roles of FtsEX in septal PG synthesis and hydrolysis, we find that recruitment of EnvC to the septum by FtsEX, but not ATP hydrolysis, is required to promote cell separation when the NlpD-mediated cell separation system is present. However, ATP hydrolysis by FtsEX becomes necessary for efficient cell separation when the NlpD system is inactivated, suggesting that the ATPase activity of FtsEX is required for optimal activity of EnvC. Importantly, under conditions that suppress the role of FtsEX in cell division, disruption of the FtsEX-FtsA interaction delays cell separation, highlighting the importance of this interaction in coupling the cell separation system with the septal PG synthetic complex.IMPORTANCE Cytokinesis in Gram-negative bacteria requires coordinated invagination of the three layers of the cell envelope; otherwise, cells become sensitive to hydrophobic antibiotics and can even undergo cell lysis. InE. coli, the ABC transporter FtsEX couples the synthesis and hydrolysis of the stress-bearing peptidoglycan layer at the septum by interacting with FtsA and EnvC, respectively. ATP hydrolysis by FtsEX is critical for its function, but the reason why is not clear. Here, we find that in the absence of ATP hydrolysis, FtsEX blocks septal PG synthesis similarly to cephalexin. However, an FtsEX ATPase mutant, under conditions where it cannot block division, rescues ftsEX phenotypes as long as a partially redundant cell separation system is present. Furthermore, we find that the FtsEX-FtsA interaction is important for efficient cell separation.

Overview of cell shape: cytoskeletons shape bacterial cells.

An evolving hypothesis is that bacterial cell shape is determined by cytoskeletal elements that localize peptidoglycan synthetic machineries. In most bacteria FtsZ assembles into the Z ring which recruits the machinery necessary for cytokinesis. Most rod shaped cells require MreB which assembles into cables that run between the poles of the cell and distribute various components of peptidoglycan metabolism along the cell length. Cells with other shapes have additional cytoskeletal elements that either localize synthetic machineries or possibly influence their activity.

Roles of FtsEX in cell division.

FtsEX is a member of a small subclass of ABC transporters that uses mechano-transmission to perform work in the periplasm. FtsEX controls periplasmic peptidoglycan (PG) hydrolase activities in many Gram negative and positive organisms to ensure the safe separation of daughter cells during division. In these organisms FtsEX localizes to the Z ring and uses its ATPase activity to regulate its periplasmic effectors. In Escherichia coli, FtsEX also participates in building the divisome and coordinates PG synthesis with PG hydrolysis. This review discusses studies that are beginning to elucidate the mechanisms of FtsEX's various roles in cell division.

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.

Identification of a region of FtsA required for interaction with FtsZ.

The assembly of the Z ring is the earliest step in bacterial cell division. In Escherichia coli this assembly requires either FtsA or ZipA which bind to a conserved, C-terminal 17 amino acid motif in FtsZ and to the membrane. The FtsZ-ZipA interaction is well characterized; however, nothing is known about the region of FtsA involved in the interaction with FtsZ even though the FtsA-FtsZ interaction is nearly ubiquitous in Eubacteria. FtsA is proposed to bind to the membrane through its conserved C-terminal amphiphatic helix before efficiently interacting with FtsZ. Based upon this model we designed a genetic screen to identify mutants specifically impaired for the FtsA-FtsZ interaction. The mutants obtained retain the ability to be targeted to the membrane but fail to be recruited to the Z ring or interact with FtsZ in the yeast two-hybrid system. These mutants do not complement an ftsA-depletion strain. Through this approach we have identified a region of FtsA containing some invariant residues which is required for binding to FtsZ. The results support our model that FtsA is targeted to the membrane before it interacts with FtsZ and demonstrates that this interaction plays an essential role in E. coli cell division.

Tethering the Z ring to the membrane through a conserved membrane targeting sequence in FtsA.

The cytokinetic Z ring is required for bacterial cell division. It consists of polymers of FtsZ, the bacterial ancestor of eukaryotic tubulin, linked to the cytoplasmic membrane. Formation of a Z ring in Escherichia coli occurs as long as one of two proteins, ZipA or FtsA, is present. Both of these proteins bind FtsZ suggesting that they might function to tether FtsZ filaments to the membrane. Although ZipA has a transmembrane domain and therefore can function as a membrane anchor, interaction of FtsA with the membrane has not been explored. In this study we demonstrate that FtsA, which is structurally related to eukaryotic actin, has a conserved C-terminal amphipathic helix that is essential for FtsA function. It is required to target FtsA to the membrane and subsequently to the Z ring. As FtsA is much more widely conserved in bacteria than ZipA, it is likely that FtsA serves as the principal membrane anchor for the Z ring.

Unique and overlapping roles for ZipA and FtsA in septal ring assembly in Escherichia coli.

ZipA and FtsA are essential division proteins in Escherichia coli that are recruited to the division site by interaction with FtsZ. Utilizing a newly isolated temperature-sensitive mutation in zipA we have more fully characterized the role of ZipA. We confirmed that ZipA is not required for Z ring formation; however, we found that ZipA, like FtsA, is required for recruitment of FtsK and therefore all downstream division proteins. In the absence of FtsA or ZipA Z rings formed; however, in the absence of both, new Z rings were unable to form and preformed Z rings were destabilized. Consistent with this, we found that an FtsZ mutant unable to interact with both ZipA and FtsA was unable to assemble into Z rings. These results demonstrate that ZipA and FtsA are both required for recruitment of additional division proteins to the Z ring, but either one is capable of supporting formation and stabilization of Z rings.