Summary
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.
Cell division in E. coli is carried out by the divisome, a large complex of proteins that is organized and propelled by treadmilling FtsZ filaments. Here, we summarize the latest findings on divisome assembly and activation as well as provide our perspective on how these two processes might be regulated.
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.
The Z ring is discontinuous and consists of patches of FtsZ assemblies that treadmill circumferentially at the division site.The mechanism of FtsZ treadmilling was revealed by determination of the conformational changes that accompany assembly and the kinetic polarity of FtsZ filaments.FtsZ employs an intrinsically disordered linker and a molecular recognition motif (MoRF) to achieve flexibility and avidity in tethering FtsZ filaments to the membrane.Evidence exists that crosslinking and bundling of FtsZ filaments contribute to assembly of the Z ring, but how filaments are organized in patches awaits further investigation.Treadmilling FtsZ filaments are coupled to the septal PG machinery through membrane anchors.
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.
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
, 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 (
ctivation of Fts
) 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.
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.
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.
In
E. 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.
ABSTRACT
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.
In
E. 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.
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.
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.
Bacillus coagulans is a probiotic agent widely used in various industries. In this study, we isolated a novel strain of B. coagulans, X26, from soil and characterized its properties. X26 exhibited ...superior enzyme, acid, and biomass yields when compared with other bacterial probiotics and an antibiotic. Moreover, X26 significantly improved the body weight of rats, highlighting its potential for industrial development as a supplement for animals. To optimize the fermentation process of this bacterium, we adopted the response surface design. When X26 was cultured in a medium with 16.5 g/L maltose, 25.00 g/L yeast extract, and 3.5 g/L K.sub.2 HPO.sub.4, the optimal yield was predicted to be 5.1 x 10.sup.9 CFU/mL. Consistent with the prediction, the yield of X26 in a 500-mL flask culture was (5.12 ± 0.01) x 10.sup.9 CFU/mL, and in a 30-L fermenter was (5.11 ± 0.02) x 10.sup.9 CFU/mL, accounting for a 9.9-fold higher field than that with a basal medium before optimization. We further optimized the fermentation process in the 30-L and a 10-T fermenter, generating yields of (7.8 ± 0.2) x 10.sup.9 CFU/mL (spore rate: 96.54%) and (8.7 ± 0.1) x 10.sup.9 CFU/mL (spore rate: 97.93%), respectively. These yields and spore rates were achieved at 45-55°C, the typical fermentation temperature of B. coagulans. Our findings indicate that B. coagulans X26 is a promising probiotic with considerable potential for cost-effective industrial fermentation.
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.
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