Widespread photosynthesis reaction centre barrel proteins are necessary for haloarchaeal cell division.

Cell division is fundamental to all cellular life. Most archaea depend on either the prokaryotic tubulin homologue FtsZ or the endosomal sorting complex required for transport for division but neither system has been robustly characterized. Here, we show that three of the four photosynthesis reaction centre barrel domain proteins of Haloferax volcanii (renamed cell division proteins B1/2/3 (CdpB1/2/3)) play important roles in cell division. CdpB1 interacts directly with the FtsZ membrane anchor SepF and is essential for cell division, whereas deletion of cdpB2 and cdpB3 causes a major and a minor division defect, respectively. Orthologues of CdpB proteins are also involved in cell division in other haloarchaea, indicating a conserved function of these proteins. Phylogenetic analysis shows that photosynthetic reaction centre barrel proteins are widely distributed among archaea and appear to be central to cell division in most if not all archaea.

Widespread PRC barrel proteins play critical roles in archaeal cell division.

Cell division is fundamental to all cellular life. Most of the archaea employ one of two alternative division machineries, one centered around the prokaryotic tubulin homolog FtsZ and the other around the endosomal sorting complex required for transport (ESCRT). However, neither of these mechanisms has been thoroughly characterized in archaea. Here, we show that three of the four PRC (Photosynthetic Reaction Center) barrel domain proteins of Haloferax volcanii (renamed Cell division proteins B1/2/3 (CdpB1/2/3)), play important roles in division. CdpB1 interacts directly with the FtsZ membrane anchor SepF and is essential for division, whereas deletion of cdpB2 and cdpB3 causes a major and a minor division defect, respectively. Orthologs of CdpB proteins are also involved in cell division in other haloarchaea. Phylogenetic analysis shows that PRC barrel proteins are widely distributed among archaea, including the highly conserved CdvA protein of the crenarchaeal ESCRT-based division system. Thus, diverse PRC barrel proteins appear to be central to cell division in most if not all archaea. Further study of these proteins is expected to elucidate the division mechanisms in archaea and their evolution.

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.

Identification of the potential active site of the septal peptidoglycan polymerase FtsW.

SEDS (Shape, Elongation, Division and Sporulation) proteins are widely conserved peptidoglycan (PG) glycosyltransferases that form complexes with class B penicillin-binding proteins (bPBPs, with transpeptidase activity) to synthesize PG during bacterial cell growth and division. Because of their crucial roles in bacterial morphogenesis, SEDS proteins are one of the most promising targets for the development of new antibiotics. However, how SEDS proteins recognize their substrate lipid II, the building block of the PG layer, and polymerize it into glycan strands is still not clear. In this study, we isolated and characterized dominant-negative alleles of FtsW, a SEDS protein critical for septal PG synthesis during bacterial cytokinesis. Interestingly, most of the dominant-negative FtsW mutations reside in extracellular loops that are highly conserved in the SEDS family. Moreover, these mutations are scattered around a central cavity in a modeled FtsW structure, which has been proposed to be the active site of SEDS proteins. Consistent with this, we found that these mutations blocked septal PG synthesis but did not affect FtsW localization to the division site, interaction with its partners nor its substrate lipid II. Taken together, these results suggest that the residues corresponding to the dominant-negative mutations likely constitute the active site of FtsW, which may aid in the design of FtsW inhibitors.

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.

Genetic analysis of the septal peptidoglycan synthase FtsWI complex supports a conserved activation mechanism for SEDS-bPBP complexes.

SEDS family peptidoglycan (PG) glycosyltransferases, RodA and FtsW, require their cognate transpeptidases PBP2 and FtsI (class B penicillin binding proteins) to synthesize PG along the cell cylinder and at the septum, respectively. The activities of these SEDS-bPBPs complexes are tightly regulated to ensure proper cell elongation and division. In Escherichia coli FtsN switches FtsA and FtsQLB to the active forms that synergize to stimulate FtsWI, but the exact mechanism is not well understood. Previously, we isolated an activation mutation in ftsW (M269I) that allows cell division with reduced FtsN function. To try to understand the basis for activation we isolated additional substitutions at this position and found that only the original substitution produced an active mutant whereas drastic changes resulted in an inactive mutant. In another approach we isolated suppressors of an inactive FtsL mutant and obtained FtsWE289G and FtsIK211I and found they bypassed FtsN. Epistatic analysis of these mutations and others confirmed that the FtsN-triggered activation signal goes from FtsQLB to FtsI to FtsW. Mapping these mutations, as well as others affecting the activity of FtsWI, on the RodA-PBP2 structure revealed they are located at the interaction interface between the extracellular loop 4 (ECL4) of FtsW and the pedestal domain of FtsI (PBP3). This supports a model in which the interaction between the ECL4 of SEDS proteins and the pedestal domain of their cognate bPBPs plays a critical role in the activation mechanism.

Essential Role for FtsL in Activation of Septal Peptidoglycan Synthesis.

Spatiotemporal regulation of septal peptidoglycan (PG) synthesis is achieved by coupling assembly and activation of the synthetic enzymes (FtsWI) to the Z ring, a cytoskeletal element that is required for division in most bacteria. In Escherichia coli, the recruitment of the FtsWI complex is dependent upon the cytoplasmic domain of FtsL, a component of the conserved FtsQLB complex. Once assembled, FtsWI is activated by the arrival of FtsN, which acts through FtsQLB and FtsA, which are also essential for their recruitment. However, the mechanism of activation of FtsWI by FtsN is not clear. Here, we identify a region of FtsL that plays a key role in the activation of FtsWI which we designate AWI (activation of FtsWI) and present evidence that FtsL acts through FtsI. Our results suggest that FtsN switches FtsQLB from a recruitment complex to an activator with FtsL interacting with FtsI to activate FtsW. Since FtsQLB and FtsWI are widely conserved in bacteria, this mechanism is likely to be also widely conserved.IMPORTANCE A critical step in bacterial cytokinesis is the activation of septal peptidoglycan synthesis at the Z ring. Although FtsN is the trigger and acts through FtsQLB and FtsA to activate FtsWI the mechanism is unclear. Here, we find an essential role for FtsL in activating septal peptidoglycan (PG) synthesis and find that it acts on FtsI. Our results suggest a model where FtsWI is recruited in an inactive form by FtsQLB, and upon the arrival of FtsN, FtsQLB undergoes a conformational change so that a region of FtsL, which we designate the AWI domain, becomes available to interact with FtsI and activate the FtsWI complex. This mechanism for activation of the divisome has similarities to the activation of the elongasome and is likely to be widely conserved in bacteria.

Structural Characterization of the Essential Cell Division Protein FtsE and Its Interaction with FtsX in Streptococcus pneumoniae.

FtsEX is a membrane complex widely conserved across diverse bacterial genera and involved in critical processes such as recruitment of division proteins and in spatial and temporal regulation of muralytic activity during cell division or sporulation. FtsEX is a member of the ABC transporter superfamily. The component FtsX is an integral membrane protein, whereas FtsE is an ATPase and is required for the transmission of a conformational signal from the cytosol through the membrane to regulate the activity of cell wall hydrolases in the periplasm. Both proteins are essential in the major human respiratory pathogenic bacterium Streptococcus pneumoniae, and FtsX interacts with the modular peptidoglycan hydrolase PcsB at the septum. Here, we report high-resolution structures of pneumococcal FtsE bound to different nucleotides. Structural analysis revealed that FtsE contains all the conserved structural motifs associated with ATPase activity and afforded interpretation of the in vivo dimeric arrangement in both the ADP and ATP states. Interestingly, three specific FtsE regions with high structural plasticity were identified that shape the cavity in which the cytosolic region of FtsX would be inserted. The residues corresponding to the FtsX coupling helix, responsible for contacting FtsE, were identified and validated by in vivo mutagenesis studies showing that this interaction is essential for cell growth and proper morphology.IMPORTANCE Bacterial cell division is a central process that requires exquisite orchestration of both the cell wall biosynthetic and lytic machineries. The essential membrane complex FtsEX, widely conserved across bacteria, plays a central role by recruiting proteins to the divisome apparatus and by regulating periplasmic muralytic activity from the cytosol. FtsEX is a member of the type VII family of the ABC-superfamily, but instead of being a transporter, it couples the ATP hydrolysis catalyzed by FtsE to mechanically transduce a conformational signal that provokes the activation of peptidoglycan (PG) hydrolases. So far, no structural information is available for FtsE. Here, we provide the structural characterization of FtsE, confirming its ATPase nature and revealing regions with high structural plasticity which are key for FtsE binding to FtsX. The complementary binding region in FtsX has also been identified and validated in vivo Our results provide evidence on how the difference between the ATP/ADP-bound states in FtsE would dramatically alter the interaction of FtsEX with the PG hydrolase PcsB in pneumococcal division.

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.

"Asymmetry Is the Rhythmic Expression of Functional Design," a Quotation from Jan Tschichold.

The outer membranes of Gram-negative bacteria provide a permeability barrier to antibiotics and other harmful chemicals. The integrity of this barrier relies on the maintenance of the lipid asymmetry of the outer membrane, and studies of suppressors of a decades-old mutant reveal that YejM plays a key regulatory role and provide a model for the maintenance of this asymmetry.

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.

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.

At the Heart of Bacterial Cytokinesis: The Z Ring.

Bacterial cell division is mediated by the divisome which is organized by the Z ring, a cytoskeletal element formed by the polymerization of the tubulin homologue FtsZ. Despite billions of years of bacterial evolution, the Z ring is nearly universal among bacteria that have a cell wall and divide by binary fission. Recent studies have revealed the mechanism of cooperative assembly of FtsZ and that the Z ring consists of patches of FtsZ filaments tethered to the membrane that treadmill to distribute the septal biosynthetic machinery. Here, we summarize these advances and discuss questions raised by these new findings.

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.

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.

MinC and FtsZ mutant analysis provides insight into MinC/MinD-mediated Z ring disassembly.

The Min system negatively regulates the position of the Z ring, which serves as a scaffold for the divisome that mediates bacterial cytokinesis. In Escherichia coli, this system consists of MinC, which antagonizes assembly of the tubulin homologue FtsZ. MinC is recruited to the membrane by MinD and induced by MinE to oscillate between the cell poles. MinC is a dimer with each monomer consisting of functionally distinct MinCN and MinCC domains, both of which contact FtsZ. According to one model, MinCC/MinD binding to the FtsZ tail positions MinCN at the junction of two GDP-containing subunits in the filament, leading to filament breakage. Others posit that MinC sequesters FtsZ-GDP monomers or that MinCN caps the minus end of FtsZ polymers and that MinCC interferes with lateral interactions between FtsZ filaments. Here, we isolated minC mutations that impair MinCN function and analyzed FtsZ mutants resistant to MinC/MinD. Surprisingly, we found mutations in both minC and ftsZ that differentiate inhibition by MinC from inhibition by MinC/MinD. Analysis of these mutations suggests that inhibition of the Z ring by MinC alone is due to sequestration, whereas inhibition by MinC/MinD is not. In conclusion, our genetic and biochemical data support the model that MinC/MinD fragments FtsZ filaments.

MinE conformational dynamics regulate membrane binding, MinD interaction, and Min oscillation.

In Escherichia coli MinE induces MinC/MinD to oscillate between the ends of the cell, contributing to the precise placement of the Z ring at midcell. To do this, MinE undergoes a remarkable conformational change from a latent 6ß-stranded form that diffuses in the cytoplasm to an active 4ß-stranded form bound to the membrane and MinD. How this conformational switch occurs is not known. Here, using hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) we rule out a model in which the two forms are in rapid equilibrium. Furthermore, HDX-MS revealed that a MinE mutant (D45A/V49A), previously shown to produce an aberrant oscillation and unable to assemble a MinE ring, is more rigid than WT MinE. This mutant has a defect in interaction with MinD, suggesting it has difficulty in switching to the active form. Analysis of intragenic suppressors of this mutant suggests it has difficulty in releasing the N-terminal membrane targeting sequences (MTS). These results indicate that the dynamic association of the MTS with the ß-sheet is fine-tuned to balance MinE's need to sense MinD on the membrane with its need to diffuse in the cytoplasm, both of which are necessary for the oscillation. The results lead to models for MinE activation and MinE ring formation.

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 FtsZ's 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.

The N-succinyl-l,l-diaminopimelic acid desuccinylase DapE acts through ZapB to promote septum formation in Escherichia coli.

Spatial regulation of cell division in Escherichia coli occurs at the stage of Z ring formation. It consists of negative (the Min and NO systems) and positive (Ter signal mediated by MatP/ZapA/ZapB) regulators. Here, we find that N-succinyl-L,L-diaminopimelic acid desuccinylase (DapE) facilitates functional Z ring formation by strengthening the Ter signal via ZapB. DapE depends on ZapB to localize to the Z ring and its overproduction suppresses the division defect caused by loss of both the Min and NO systems. DapE shows a strong interaction with ZapB and requires the presence of ZapB to exert its function in division. Consistent with the idea that DapE strengthens the Ter signal, overproduction of DapE supports cell division with reduced FtsZ levels and provides some resistance to the FtsZ inhibitors MinCD and SulA, while deletion of dapE, like deletion of zapB, exacerbates the phenotypes of cells impaired in Z ring formation such as ftsZ84 or a min mutant. Taken together, our results report DapE as a new component of the divisome that promotes the integrity of the Z ring by acting through ZapB and raises the possibility of the existence of additional divisome proteins that also function in other cellular processes.