A dynamic duo: Understanding the roles of FtsZ and FtsA for Escherichia coli cell division through in vitro approaches.

Bacteria divide by binary fission. The protein machine responsible for this process is the divisome, a transient assembly of more than 30 proteins in and on the surface of the cytoplasmic membrane. Together, they constrict the cell envelope and remodel the peptidoglycan layer to eventually split the cell into two. For Escherichia coli, most molecular players involved in this process have probably been identified, but obtaining the quantitative information needed for a mechanistic understanding can often not be achieved from experiments in vivo alone. Since the discovery of the Z-ring more than 30 years ago, in vitro reconstitution experiments have been crucial to shed light on molecular processes normally hidden in the complex environment of the living cell. In this review, we summarize how rebuilding the divisome from purified components - or at least parts of it - have been instrumental to obtain the detailed mechanistic understanding of the bacterial cell division machinery that we have today.

Structural insights into the FtsEX-EnvC complex regulation on septal peptidoglycan hydrolysis in Vibrio cholerae.

During bacterial cell division, hydrolysis of septal peptidoglycan (sPG) is crucial for cell separation. This sPG hydrolysis is performed by the enzyme amidases whose activity is regulated by the integral membrane protein complex FtsEX-EnvC. FtsEX is an ATP-binding cassette transporter, and EnvC is a long coiled-coil protein that interacts with and activates the amidases. The molecular mechanism by which the FtsEX-EnvC complex activates amidases remains largely unclear. We present the cryo-electron microscopy structure of the FtsEX-EnvC complex from the pathogenic bacteria V. cholerae (FtsEX-EnvCVC). FtsEX-EnvCVC in the presence of ADP adopts a distinct conformation where EnvC is "horizontally extended" rather than "vertically extended". Subsequent structural studies suggest that EnvC can swing between these conformations in space in a nucleotide-dependent manner. Our structural analysis and functional studies suggest that FtsEX-EnvCVC employs spatial control of EnvC for amidase activation, providing mechanistic insights into the FtsEX-EnvC regulation on septal peptidoglycan hydrolysis.

The C-terminal domain of MinC, a cell division regulation protein, is sufficient to form a copolymer with MinD.

Assembly of cell division protein FtsZ into the Z-ring at the division site is a key step in bacterial cell division. The Min proteins can restrict the Z-ring to the middle of the cell. MinC is the main protein that obstructs Z-ring formation by inhibiting FtsZ assembly. Its N-terminal domain (MinCN ) regulates the localization of the Z-ring by inhibiting FtsZ polymerization, while its C-terminal domain (MinCC ) binds to MinD as well as to FtsZ. Previous studies have shown that MinC and MinD form copolymers in vitro. This copolymer may greatly enhance the binding of MinC to FtsZ, and/or prevent FtsZ filaments from diffusing to the ends of the cell. Here, we investigated the assembly properties of MinCC -MinD of Pseudomonas aeruginosa. We found that MinCC is sufficient to form the copolymers. Although MinCC -MinD assembles into larger bundles, most likely because MinCC is spatially more readily bound to MinD, its copolymerization has similar dynamic properties: the concentration of MinD dominates their copolymerization. The critical concentration of MinD is around 3 µm and when MinD concentration is high enough, a low concentration MinCC could still be copolymerized. We also found that MinCC -MinD can still rapidly bind to FtsZ protofilaments, providing direct evidence that MinCC also interacts directly with FtsZ. However, although the presence of minCC can slightly improve the division defect of minC-knockout strains and shorten the cell length from an average of 12.2 ± 6.7 to 6.6 ± 3.6 µm, it is still insufficient for the normal growth and division of bacteria.

Activator-induced conformational changes regulate division-associated peptidoglycan amidases.

AmiA and AmiB are peptidoglycan-hydrolyzing enzymes from Escherichia coli that are required to break the peptidoglycan layer during bacterial cell division and maintain integrity of the cell envelope. In vivo, the activity of AmiA and AmiB is tightly controlled through their interactions with the membrane-bound FtsEX-EnvC complex. Activation of AmiA and AmiB requires access to a groove in the amidase-activating LytM domain of EnvC which is gated by ATP-driven conformational changes in FtsEX-EnvC complex. Here, we present a high-resolution structure of the isolated AmiA protein, confirming that it is autoinhibited in the same manner as AmiB and AmiC, and a complex of the AmiB enzymatic domain bound to the activating EnvC LytM domain. In isolation, the active site of AmiA is blocked by an autoinhibitory helix that binds directly to the catalytic zinc and fills the volume expected to accommodate peptidoglycan binding. In the complex, binding of the EnvC LytM domain induces a conformational change that displaces the amidase autoinhibitory helix and reorganizes the active site for activity. Our structures, together with complementary mutagenesis work, defines the conformational changes required to activate AmiA and/or AmiB through their interaction with their cognate activator EnvC.

An MltA-Like Lytic Transglycosylase Secreted by Bdellovibrio bacteriovorus Cleaves the Prey Septum during Predatory Invasion.

Lytic transglycosylases cut peptidoglycan backbones, facilitating a variety of functions within bacteria, including cell division, pathogenesis, and insertion of macromolecular machinery into the cell envelope. Here, we identify a novel role of a secreted lytic transglycosylase associated with the predatory lifestyle of Bdellovibrio bacteriovorus strain HD100. During wild-type B. bacteriovorus prey invasion, the predator rounds up rod-shaped prey into spherical prey bdelloplasts, forming a spacious niche within which the predator grows. Deleting the MltA-like lytic transglycosylase Bd3285 still permitted predation but resulted in three different, invaded prey cell shapes: spheres, rods, and "dumbbells." Amino acid D321 within the catalytic C-terminal 3D domain of Bd3285 was essential for wild-type complementation. Microscopic analyses revealed that dumbbell-shaped bdelloplasts are derived from Escherichia coli prey undergoing cell division at the moment of Δbd3285 predator invasion. Prelabeling of E. coli prey peptidoglycan prior to predation with the fluorescent D-amino acid HADA showed that the dumbbell bdelloplasts invaded by B. bacteriovorus Δbd3285 contained a septum. Fluorescently tagged Bd3285, expressed in E. coli, localized to the septum of dividing cells. Our data indicate that B. bacteriovorus secretes the lytic transglycosylase Bd3285 into the E. coli periplasm during prey invasion to cleave the septum of dividing prey, facilitating prey cell occupation. IMPORTANCE Antimicrobial resistance is a serious and rapidly growing threat to global health. Bdellovibrio bacteriovorus can prey upon an extensive range of Gram-negative bacterial pathogens and thus has promising potential as a novel antibacterial therapeutic and is a source of antibacterial enzymes. Here, we elucidate the role of a unique secreted lytic transglycosylase from B. bacteriovorus which acts on the septal peptidoglycan of its prey. This improves our understanding of mechanisms that underpin bacterial predation.

Models versus pathogens: how conserved is the FtsZ in bacteria?

Combating anti-microbial resistance by developing alternative strategies is the need of the hour. Cell division, particularly FtsZ, is being extensively studied for its potential as an alternative target for anti-bacterial therapy. Bacillus subtilis and Escherichia coli are the two well-studied models for research on FtsZ, the leader protein of the cell division machinery. As representatives of gram-positive and gram-negative bacteria, respectively, these organisms have provided an extensive outlook into the process of cell division in rod-shaped bacteria. However, research on other shapes of bacteria, like cocci and ovococci, lags behind that of model rods. Even though most regions of FtsZ show sequence and structural conservation throughout bacteria, the differences in FtsZ functioning and interacting partners establish several different modes of division in different bacteria. In this review, we compare the features of FtsZ and cell division in the model rods B. subtilis and E. coli and the four pathogens: Staphylococcus aureus, Streptococcus pneumoniae, Mycobacterium tuberculosis, and Pseudomonas aeruginosa. Reviewing several recent articles on these pathogenic bacteria, we have highlighted the functioning of FtsZ, the unique roles of FtsZ-associated proteins, and the cell division processes in them. Further, we provide a detailed look at the anti-FtsZ compounds discovered and their target bacteria, emphasizing the need for elucidation of the anti-FtsZ mechanism of action in different bacteria. Current challenges and opportunities in the ongoing journey of identifying potent anti-FtsZ drugs have also been described.

FtsEX-independent control of RipA-mediated cell separation in Corynebacteriales.

The bacterial cell wall is a multi-layered mesh, whose major component is peptidoglycan (PG), a sugar polymer cross-linked by short peptide stems. During cell division, a careful balance of PG synthesis and degradation, precisely coordinated both in time and space, is necessary to prevent uncontrolled destruction of the cell wall. In Corynebacteriales, the D,L endopeptidase RipA has emerged as a major PG hydrolase for cell separation, and RipA defaults have major implications for virulence of the human pathogens Mycobacterium tuberculosis and Corynebacterium diphtheriae. However, the precise mechanisms by which RipA mediates cell separation remain elusive. Here we report phylogenetic, biochemical, and structural analysis of the Corynebacterium glutamicum homologue of RipA, Cg1735. The crystal structures of full-length Cg1735 in two different crystal forms revealed the C-terminal NlpC/P60 catalytic domain obtruded by its N-terminal conserved coiled-coil domain, which locks the enzyme in an autoinhibited state. We show that this autoinhibition is relieved by the extracellular core domain of the transmembrane septal protein Cg1604. The crystal structure of Cg1604 revealed a (ß/α) protein with an overall topology similar to that of receiver domains from response regulator proteins. The atomic model of the Cg1735-Cg1604 complex, based on bioinformatical and mutational analysis, indicates that a conserved, distal-membrane helical insertion in Cg1604 is responsible for Cg1735 activation. The reported data provide important insights into how intracellular cell division signal(s), yet to be identified, control PG hydrolysis during RipA-mediated cell separation in Corynebacteriales.

MipZ caps the plus-end of FtsZ polymers to promote their rapid disassembly.

The spatiotemporal regulation of cell division is a fundamental issue in cell biology. Bacteria have evolved a variety of different systems to achieve proper division site placement. In many cases, the underlying molecular mechanisms are still incompletely understood. In this study, we investigate the function of the cell division regulator MipZ from Caulobacter crescentus, a P-loop ATPase that inhibits the polymerization of the treadmilling tubulin homolog FtsZ near the cell poles, thereby limiting the assembly of the cytokinetic Z ring to the midcell region. We show that MipZ interacts with FtsZ in both its monomeric and polymeric forms and induces the disassembly of FtsZ polymers in a manner that is not dependent but enhanced by the FtsZ GTPase activity. Using a combination of biochemical and genetic approaches, we then map the MipZ-FtsZ interaction interface. Our results reveal that MipZ employs a patch of surface-exposed hydrophobic residues to interact with the C-terminal region of the FtsZ core domain. In doing so, it sequesters FtsZ monomers and caps the (+)-end of FtsZ polymers, thereby promoting their rapid disassembly. We further show that MipZ influences the conformational dynamics of interacting FtsZ molecules, which could potentially contribute to modulating their assembly kinetics. Together, our findings show that MipZ uses a combination of mechanisms to control FtsZ polymerization, which may be required to robustly regulate the spatiotemporal dynamics of Z ring assembly within the cell.

FtsK, a DNA Motor Protein, Coordinates the Genome Segregation and Early Cell Division Processes in Deinococcus radiodurans.

Filament temperature-sensitive mutant K (FtsK)/SpoIIIE family proteins are DNA translocases known as the fastest DNA motor proteins that use ATP for their movement on DNA. Most of the studies in single chromosome-containing bacteria have established the role of FtsK in chromosome dimer resolution (CDR), connecting the bacterial chromosome segregation process with cell division. Only limited reports, however, are available on the interdependent regulation of genome segregation and cell division in multipartite genome harboring (MGH) bacteria. In this study, for the first time, we report the characterization of FtsK from the radioresistant MGH bacterium Deinococcus radiodurans R1 (drFtsK). drFtsK shows the activity characteristics of a typical FtsK/SpoIIIE/Tra family. It stimulates the site-specific recombination catalyzed by Escherichia coli tyrosine recombinases. drFtsK interacts with various cell division and genome segregation proteins of D. radiodurans. Microscopic examination of different domain deletion mutants of this protein reveals alterations in cellular membrane architecture and nucleoid morphology. In vivo localization studies of drFtsK-RFP show that it forms multiple foci on nucleoid as well as on the membrane with maximum density on the septum. drFtsK coordinates its movement with nucleoid separation. The alignment of its foci shifts from old to new septum indicating its cellular dynamics with the FtsZ ring during the cell division process. Nearly, similar positional dynamicity of FtsK was observed in cells recovering from gamma radiation exposure. These results suggest that FtsK forms a part of chromosome segregation, cell envelope, and cell division machinery in D. radiodurans. IMPORTANCE Deinococcus radiodurans show extraordinary resistance to gamma radiation. It is polyploid and harbors a multipartite genome comprised of 2 chromosomes and 2 plasmids, packaged in a doughnut-shaped toroidal nucleoid. Very little is known about how the tightly packed genome is accurately segregated and the next divisional plane is determined. Filament temperature-sensitive mutant K (FtsK), a multifunctional protein, helps in pumping the septum-trapped DNA in several bacteria. Here, we characterized FtsK of D. radiodurans R1 (drFtsK) for the first time and showed it to be an active protein. The absence of drFtsK causes many defects in morphology at both cellular and nucleoid levels. The compact packaging of the deinococcal genome and cell membrane formation is hindered in ftsK mutants. In vivo drFtsK is dynamic, forms foci on both nucleoid and septum, and coordinates with FtsZ for the next cell division. Thus, drFtsK role in maintaining the normal genome phenotype and cell division in D. radiodurans is suggested.

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.

Screening of plant-based natural compounds as an inhibitor of FtsZ from Salmonella Typhi using the computational, biochemical and in vitro cell-based studies.

Salmonella Typhi is emerging as a drug-resistant pathogen, particularly in developing countries. Hence, the progressive development of new antibiotics against novel drug targets is essential to prevent the spread of infections and mortality. The cell division protein FtsZ is an ideal drug target as the cell wall synthesis in bacteria is driven by the dynamic treadmilling nature of the FtsZ. The polymerization of the FtsZ provides the essential mechanical constricting force and flexibility to modulate the cell wall synthesis. Any alteration in FtsZ polymerization leads to the bactericidal or bacteriostatic effect. In this study, we have evaluated the secondary metabolites of natural compounds berberine chloride, cinnamaldehyde, scopoletin, quercetin and eugenol as potential inhibitors of FtsZ from Salmonella Typhi (stFtsZ) using computational, biochemical, and in vivo cell-based assays. Out of these five compounds, berberine chloride and cinnamaldehyde exhibited the best binding affinity of Kd = 7 µM and 10 µM, respectively and inhibit stFtsZ GTPase activity and polymerization by 70 %. The compound berberine chloride showed the best MIC of 500 µg/mL and 175 µg/mL against gram-negative and gram-positive bacterial strains. The findings support that these natural compounds can be used as a backbone structure to develop a broad spectrum of antibacterial agents.

The lipoprotein DolP affects cell separation in Escherichia coli, but not as an upstream regulator of NlpD.

Bacterial amidases are essential to split the shared envelope of adjunct daughter cells to allow cell separation. Their activity needs to be precisely controlled to prevent cell lysis. In Escherichia coli, amidase activity is controlled by three regulatory proteins NlpD, EnvC and ActS. However, recent studies linked the outer membrane lipoprotein DolP (formerly YraP) as a potential upstream regulator of NlpD. In this study we explored this link in further detail. To our surprise DolP did not modulate amidase activity in vitro and was unable to interact with NlpD in pull-down and MST (MicroScale Thermophoresis) assays. Next, we excluded the hypothesis that ΔdolP phenocopied ΔnlpD in a range of envelope stresses. However, morphological analysis of double deletion mutants of amidases (AmiA, AmiB AmiC) and amidase regulators with dolP revealed that ΔamiAΔdolP and ΔenvCΔdolP mutants display longer chain length compared to their parental strains indicating a role for DolP in cell division. Overall, we present evidence that DolP does not affect NlpD function in vitro, implying that DolP is not an upstream regulator of NlpD. However, DolP may impact daughter cell separation by interacting directly with AmiA or AmiC, or by a yet undiscovered mechanism.

Dendrimersome Synthetic Cells Harbor Cell Division Machinery of Bacteria.

The integration of active cell machinery with synthetic building blocks is the bridge toward developing synthetic cells with biological functions and beyond. Self-replication is one of the most important tasks of living systems, and various complex machineries exist to execute it. In Escherichia coli, a contractile division ring is positioned to mid-cell by concentration oscillations of self-organizing proteins (MinCDE), where it severs membrane and cell wall. So far, the reconstitution of any cell division machinery has exclusively been tied to liposomes. Here, the reconstitution of a rudimentary bacterial divisome in fully synthetic bicomponent dendrimersomes is shown. By tuning the membrane composition, the interaction of biological machinery with synthetic membranes can be tailored to reproduce its dynamic behavior. This constitutes an important breakthrough in the assembly of synthetic cells with biological elements, as tuning of membrane-divisome interactions is the key to engineering emergent biological behavior from the bottom-up.

How Do MinC-D Copolymers Act on Z-Ring Localization Regulation? A New Model of Bacillus subtilis Min System.

Division site selection in rod-shaped bacteria is strictly regulated spatially by the Min system. Although many sophisticated studies, including in vitro recombination, have tried to explain these regulations, the precise mechanisms are still unclear. A previous model suggested that the concentration gradient of MinC, an FtsZ inhibitor, regulates the position of the Z-ring in the cell. In Escherichia coli, the oscillation of MinCDE proteins leads to a gradient of Min proteins with the average concentration being lowest in the middle and highest near the poles. In contrast to the Min system of E. coli, the Min system of Bacillus subtilis lacks MinE and exhibits a stable concentration distribution, which is regulated by the binding of DivIVA to the negative curvature membrane. The Min proteins first accumulate at the poles of the cell and relocalize near the division site when the membrane invagination begins. It is inconsistent with the previous model of high concentrations of MinC inhibiting Z-ring formation. Our preliminary data here using electron microscopy and light scattering technology reported that B. subtilis MinC (BsMinC) and MinD (BsMinD) also assembled into large straight copolymers in the presence of ATP, similar to the Min proteins of E. coli. Their assembly is fast and dominated by MinD concentration. When BsMinD is 5 µM, a clear light scattering signal can be observed even at 0.3 µM BsMinC. Here, we propose a new model based on the MinC-D copolymers. In our hypothesis, it is not the concentration gradient of MinC, but the MinC-D copolymer assembled in the region of high concentration MinD that plays a key role in the regulation of Z-ring positioning. In B. subtilis, the regions with high MinD concentration are initially at both ends of the cell and then appear at midcell when cell division began. MinC-D copolymer will polymerize and form a complex with MinJ and DivIVA. These complexes capture FtsZ protofilaments to prevent their diffusion away from the midcell and narrow the Z-ring in the middle of the cell.

FtsZ-mediated fission of a cuboid bacterial symbiont.

Less than a handful of cuboid and squared cells have been described in nature, which makes them a rarity. Here, we show how Candidatus Thiosymbion cuboideus, a cube-like gammaproteobacterium, reproduces on the surface of marine free-living nematodes. Immunostaining of symbiont cells with an anti-fimbriae antibody revealed that they are host-polarized, as these appendages exclusively localized at the host-proximal (animal-attached) pole. Moreover, by applying a fluorescently labeled metabolic probe to track new cell wall insertion in vivo, we observed that the host-attached pole started septation before the distal one. Similarly, Ca. T. cuboideus cells immunostained with an anti-FtsZ antibody revealed a proximal-to-distal localization pattern of this tubulin homolog. Although FtsZ has been shown to arrange into squares in synthetically remodeled cuboid cells, here we show that FtsZ may also mediate the division of naturally occurring ones. This implies that, even in natural settings, membrane roundness is not required for FtsZ function.

The coiled-coil domain of Escherichia coli FtsLB is a structurally detuned element critical for modulating its activation in bacterial cell division.

The FtsLB complex is a key regulator of bacterial cell division, existing in either an off state or an on state, which supports the activation of septal peptidoglycan synthesis. In Escherichia coli, residues known to be critical for this activation are located in a region near the C-terminal end of the periplasmic coiled-coil domain of FtsLB, raising questions about the precise role of this conserved domain in the activation mechanism. Here, we investigate an unusual cluster of polar amino acids found within the core of the FtsLB coiled coil. We hypothesized that these amino acids likely reduce the structural stability of the domain and thus may be important for governing conformational changes. We found that mutating these positions to hydrophobic residues increased the thermal stability of FtsLB but caused cell division defects, suggesting that the coiled-coil domain is a "detuned" structural element. In addition, we identified suppressor mutations within the polar cluster, indicating that the precise identity of the polar amino acids is important for fine-tuning the structural balance between the off and on states. We propose a revised structural model of the tetrameric FtsLB (named the "Y-model") in which the periplasmic domain splits into a pair of coiled-coil branches. In this configuration, the hydrophilic terminal moieties of the polar amino acids remain more favorably exposed to water than in the original four-helix bundle model ("I-model"). We propose that a shift in this architecture, dependent on its marginal stability, is involved in activating the FtsLB complex and triggering septal cell wall reconstruction.

Gossypol acetate: A natural polyphenol derivative with antimicrobial activities against the essential cell division protein FtsZ.

Antimicrobial resistance has attracted worldwide attention and remains an urgent issue to resolve. Discovery of novel compounds is regarded as one way to circumvent the development of resistance and increase the available treatment options. Gossypol is a natural polyphenolic aldehyde, and it has attracted increasing attention as a possible antibacterial drug. In this paper, we studied the antimicrobial properties (minimum inhibitory concentrations) of gossypol acetate against both Gram-positive and Gram-negative bacteria strains and dig up targets of gossypol acetate using in vitro assays, including studying its effects on functions (GTPase activity and polymerization) of Filamenting temperature sensitive mutant Z (FtsZ) and its interactions with FtsZ using isothermal titration calorimetry (ITC), and in vivo assays, including visualization of cell morphologies and proteins localizations using a microscope. Lastly, Bacterial membrane permeability changes were studied, and the cytotoxicity of gossypol acetate was determined. We also estimated the interactions of gossypol acetate with the promising target. We found that gossypol acetate can inhibit the growth of Gram-positive bacteria such as the model organism Bacillus subtilis and the pathogen Staphylococcus aureus [both methicillin-sensitive (MSSA) and methicillin-resistant (MRSA)]. In addition, gossypol acetate can also inhibit the growth of Gram-negative bacteria when the outer membrane is permeabilized by Polymyxin B nonapeptide (PMBN). Using a cell biological approach, we show that gossypol acetate affects cell division in bacteria by interfering with the assembly of the cell division FtsZ ring. Biochemical analysis shows that the GTPase activity of FtsZ was inhibited and polymerization of FtsZ was enhanced in vitro, consistent with the block to cell division in the bacteria tested. The binding mode of gossypol acetate in FtsZ was modeled using molecular docking and provides an understanding of the compound mode of action. The results point to gossypol (S2303) as a promising antimicrobial compound that inhibits cell division by affecting FtsZ polymerization and has potential to be developed into an effective antimicrobial drug by chemical modification to minimize its cytotoxic effects in eukaryotic cells that were identified in this work.

Targeting the Achilles Heel of FtsZ: The Interdomain Cleft.

Widespread antimicrobial resistance among bacterial pathogens is a serious threat to public health. Thus, identification of new targets and development of new antibacterial agents are urgently needed. Although cell division is a major driver of bacterial colonization and pathogenesis, its targeting with antibacterial compounds is still in its infancy. FtsZ, a bacterial cytoskeletal homolog of eukaryotic tubulin, plays a highly conserved and foundational role in cell division and has been the primary focus of research on small molecule cell division inhibitors. FtsZ contains two drug-binding pockets: the GTP binding site situated at the interface between polymeric subunits, and the inter-domain cleft (IDC), located between the N-terminal and C-terminal segments of the core globular domain of FtsZ. The majority of anti-FtsZ molecules bind to the IDC. Compounds that bind instead to the GTP binding site are much less useful as potential antimicrobial therapeutics because they are often cytotoxic to mammalian cells, due to the high sequence similarity between the GTP binding sites of FtsZ and tubulin. Fortunately, the IDC has much less sequence and structural similarity with tubulin, making it a better potential target for drugs that are less toxic to humans. Over the last decade, a large number of natural and synthetic IDC inhibitors have been identified. Here we outline the molecular structure of IDC in detail and discuss how it has become a crucial target for broad spectrum and species-specific antibacterial agents. We also outline the drugs that bind to the IDC and their modes of action.

A Novel Z-Ring Associated Protein ZapA-Like Protein (PA5407) From Pseudomonas aeruginosa Promotes FtsZ to Form Double Filaments.

Bacterial cell division is initiated by the assembly of the contraction ring (Z-ring), which consists of the self-assembled FtsZ protofilaments and dozens of other associate proteins. ZapA, a regulatory protein found in almost all bacteria, stabilizes FtsZ protofilaments to form bundles and enhances the Z-ring condensation. Here, we reported that another small protein from Pseudomonas aeruginosa, ZapA-Like protein (ZapAL; PA5407), is a new FtsZ associated protein. ZapAL exists in many Pseudomonas species and shares only 20% sequence identity to ZapA. ZapAL interacts with FtsZ and induces FtsZ to form long straight double filaments; in comparison, ZapA promotes long bundles with multiple FtsZ filaments. ZapAL has only a mild effect on GTPase activity of FtsZ, which is reduced by around 26% when 10 µM ZapAL is added in the solution. However, to study their assembly dynamics using light-scattering assay, we found that FtsZ-ZapAL double filament is stable and no depolymerization process is observed, which is different from ZapA. Further research found that ZapA and ZapL are likely to form heterodimers. The bundles formed by the mixture of FtsZ-ZapA-ZapAL will depolymerize after GTP is hydrolyzed. Consistent with ZapAL interaction with FtsZ in vitro, the expression of ZapAL-GFP was observed as a narrow band or spots in the middle of the cells, suggesting that it is a component of bacterial division machinery. Similar to ZapA, ZapAL is also not essential for bacterial cell division. Little changes were observed when zapAL gene was deleted, or overexpressed under normal conditions; however, overexpression of ZapAL caused zapA-deficient cells to grow approximately two times longer, showing a mild bacterial division defect. Although we still do not know the exact physiological roles of ZapAL, our results suggest that ZapAL is a novel Z-ring associate protein, which may work together with ZapA to stabilize the FtsZ protofilament and Z-ring structure.

CTP regulates membrane-binding activity of the nucleoid occlusion protein Noc.

ATP- and GTP-dependent molecular switches are extensively used to control functions of proteins in a wide range of biological processes. However, CTP switches are rarely reported. Here, we report that a nucleoid occlusion protein Noc is a CTPase enzyme whose membrane-binding activity is directly regulated by a CTP switch. In Bacillus subtilis, Noc nucleates on 16 bp NBS sites before associating with neighboring non-specific DNA to form large membrane-associated nucleoprotein complexes to physically occlude assembly of the cell division machinery. By in vitro reconstitution, we show that (1) CTP is required for Noc to form the NBS-dependent nucleoprotein complex, and (2) CTP binding, but not hydrolysis, switches Noc to a membrane-active state. Overall, we suggest that CTP couples membrane-binding activity of Noc to nucleoprotein complex formation to ensure productive recruitment of DNA to the bacterial cell membrane for nucleoid occlusion activity.