FtsZ Dynamics Research Articles

GTPase activity regulates FtsZ ring positioning in Caulobacter crescentus.

Bacterial cell division is crucial for replication and requires careful coordination via proteins collectively called the divisome. The tubulin-like GTPase FtsZ is the master regulator of this process and serves to recruit downstream divisome proteins and regulate their activities. Upon assembling at mid-cell, FtsZ exhibits treadmilling motion driven by GTP binding and hydrolysis. Treadmilling is proposed to play roles in Z-ring condensation and in distribution and regulation of peptidoglycan (PG) cell wall enzymes. FtsZ polymer superstructure and dynamics are central to its function, yet their regulation is incompletely understood. We addressed these gaps in knowledge by evaluating the contribution of GTPase activity to FtsZ's function in vitro and in Caulobacter crescentus cells. We observed that a lethal mutation that abrogates FtsZ GTP hydrolysis impacts FtsZ dynamics and Z-ring positioning, but not constriction. Aberrant Z-ring positioning was due to insensitivity to the FtsZ regulator MipZ when GTPase activity is reduced. Z-ring mislocalization resulted in DNA damage, likely due to constriction over the nucleoid. Collectively, our results indicate that GTP hydrolysis serves primarily to position the Z-ring at mid-cell in Caulobacter. Proper Z-ring localization is required for effective coordination with chromosome segregation to prevent DNA damage and ensure successful cell division.

Vertical Immobilization Method for Time-Lapse Microscopy Analysis in Filamentous Cyanobacteria.

A main event in bacterial cell division is the septation process, where the protein FtsZ is the key element. FtsZ polymerizes forming a ring-like structure (Z-ring) in the middle of the cell that serves as a scaffold for other division proteins. Super-resolution microscopy in bacterial models Escherichia coli and Bacillus subtilis showed that the Z-ring is discontinuous, while live cell imaging studies demonstrated that FtsZ moves along the ring by a mechanism known as treadmilling. To study the dynamics of FtsZ in vivo, a special cell placement in a vertical position is necessary for imaging the complete structure of the ring in the XY plane. In the case of FtsZ imaging in multicellular cyanobacteria, such as Anabaena sp. PCC7120, maintaining the filaments in a vertical position is challenging because of the size of the cells and the filaments' length. In this article, we describe a method that allows the vertical immobilization of Anabaena sp. PCC 7120 filaments using low melting point agarose and syringes, to record the Z-ring in a mutant that expresses a FtsZ-sfGFP fusion protein. This method is a rapid and inexpensive way to register protein dynamics at the division site using confocal microscopy.

Bacterial cell-size changes resulting from altering the relative expression of Min proteins

The timing of cell division, and thus cell size in bacteria, is determined in part by the accumulation dynamics of the protein FtsZ, which forms the septal ring. FtsZ localization depends on membrane-associated Min proteins, which inhibit FtsZ binding to the cell pole membrane. Changes in the relative concentrations of Min proteins can disrupt FtsZ binding to the membrane, which in turn can delay cell division until a certain cell size is reached, in which the dynamics of Min proteins frees the cell membrane long enough to allow FtsZ ring formation. Here, we study the effect of Min proteins relative expression on the dynamics of FtsZ ring formation and cell size in individual Escherichia coli bacteria. Upon inducing overexpression of minE, cell size increases gradually to a new steady-state value. Concurrently, the time required to initiate FtsZ ring formation grows as the size approaches the new steady-state, at which point the ring formation initiates as early as before induction. These results highlight the contribution of Min proteins to cell size control, which may be partially responsible for the size fluctuations observed in bacterial populations, and may clarify how the size difference acquired during asymmetric cell division is offset.

Dynamics of the Synechococcus elongatus cytoskeletal GTPase FtsZ yields mechanistic and evolutionary insight into cyanobacterial and chloroplast FtsZs

The division of cyanobacteria and their chloroplast descendants is orchestrated by filamenting temperature-sensitive Z (FtsZ), a cytoskeletal GTPase that polymerizes into protofilaments that form a "Z ring" at the division site. The Z ring has both a scaffolding function for division-complex assembly and a GTPase-dependent contractile function that drives cell or organelle constriction. A single FtsZ performs these functions in bacteria, whereas in chloroplasts, they are performed by two copolymerizing FtsZs, called AtFtsZ2 and AtFtsZ1 in Arabidopsis thaliana, which promote protofilament stability and dynamics, respectively. To probe the differences between cyanobacterial and chloroplast FtsZs, we used light scattering to characterize the invitro protofilament dynamics of FtsZ from the cyanobacterium Synechococcus elongatus PCC 7942 (SeFtsZ) and investigate how coassembly of AtFtsZ2 or AtFtsZ1 with SeFtsZ influences overall dynamics. SeFtsZ protofilaments assembled rapidly and began disassembling before GTP depletion, whereas AtFtsZ2 protofilaments were far more stable, persisting beyond GTP depletion. Coassembled SeFtsZ-AtFtsZ2 protofilaments began disassembling before GTP depletion, similar to SeFtsZ. In contrast, AtFtsZ1 did not alter disassembly onset when coassembled with SeFtsZ, but fluorescence recovery after photobleaching analysis showed it increased the turnover of SeFtsZ subunits from SeFtsZ-AtFtsZ1 protofilaments, mirroring its effect upon coassembly with AtFtsZ2. Comparisons of our findings with previous work revealed consistent differences between cyanobacterial and chloroplast FtsZ dynamics and suggest that the scaffolding and dynamics-promoting functions were partially separated during evolution of two chloroplast FtsZs from their cyanobacterial predecessor. They also suggest that chloroplasts may have evolved a mechanism distinct from that in cyanobacteria for promoting FtsZ protofilament dynamics.

FtsZ phosphorylation brings about growth arrest upon DNA damage in Deinococcus radiodurans.

The polymerization/depolymerization dynamics of FtsZ play a pivotal role in cell division in the majority of the bacteria. Deinococcus radiodurans, a radiation-resistant bacterium, shows an arrest of growth in response to DNA damage with no change in the level of FtsZ. This bacterium does not deploy LexA/RecA type of DNA damage response and cell cycle regulation, and its genome does not encode SulA homologues of Escherichia coli, which attenuate FtsZ functions in response to DNA damage in other bacteria. A radiation-responsive Ser/Thr quinoprotein kinase (RqkA), characterized for its role in radiation resistance in this bacterium, could phosphorylate several cognate proteins, including FtsZ (drFtsZ) at Serine 235 (S235) and Serine 335 (S335) residues. Here, we reported the detailed characterization of S235 and S335 phosphorylation effects in the regulation of drFtsZ functions and demonstrated that the phospho-mimetic replacements of these residues in drFtsZ had grossly affected its functions that could result in cell cycle arrest in response to DNA damage in D. radiodurans. Interestingly, the phospho-ablative replacements were found to be nearly similar to drFtsZ, whereas the phospho-mimetic mutant lost the wild-type protein's signature characteristics, including its dynamics under normal conditions. The kinetics of post-bleaching recovery for drFtsZ and phospho-mimetic mutant were nearly similar at 2h post-irradiation recovery but were found to be different under normal conditions. These results highlighted the role of S/T phosphorylation in the regulation of drFtsZ functions and cell cycle arrest in response to DNA damage, which is demonstrated for the first time, in any bacteria.

Halogenation of tyrosine perturbs large-scale protein self-organization

Protein halogenation is a common non-enzymatic post-translational modification contributing to aging, oxidative stress-related diseases and cancer. Here, we report a genetically encodable halogenation of tyrosine residues in a reconstituted prokaryotic filamentous cell-division protein (FtsZ) as a platform to elucidate the implications of halogenation that can be extrapolated to living systems of much higher complexity. We show how single halogenations can fine-tune protein structures and dynamics of FtsZ with subtle perturbations collectively amplified by the process of FtsZ self-organization. Based on experiments and theories, we have gained valuable insights into the mechanism of halogen influence. The bending of FtsZ structures occurs by affecting surface charges and internal domain distances and is reflected in the decline of GTPase activities by reducing GTP binding energy during polymerization. Our results point to a better understanding of the physiological and pathological effects of protein halogenation and may contribute to the development of potential diagnostic tools.

Assembly dynamics of FtsZ and DamX during infection-related filamentation and division in uropathogenic E. coli

During infection of bladder epithelial cells, uropathogenic Escherichia coli (UPEC) can stop dividing and grow into highly filamentous forms. Here, we find that some filaments of E. coli UTI89 released from infected cells grow very rapidly and by more than 100 μm before initiating division, whereas others do not survive, suggesting that infection-related filamentation (IRF) is a stress response that promotes bacterial dispersal. IRF is accompanied by unstable, dynamic repositioning of FtsZ division rings. In contrast, DamX, which is associated with normal cell division and is also essential for IRF, is distributed uniformly around the cell envelope during filamentation. When filaments initiate division to regenerate rod cells, DamX condenses into stable rings prior to division. The DamX rings maintain consistent thickness during constriction and remain at the septum until after membrane fusion. Deletion of damX affects vegetative cell division in UTI89 (but not in the model E. coli K-12), and, during infection, blocks filamentation and reduces bacterial cell integrity. IRF therefore involves DamX distribution throughout the membrane and prevention of FtsZ ring stabilization, leading to cell division arrest. DamX then reassembles into stable division rings for filament division, promoting dispersal and survival during infection.

A conserved cell division protein directly regulates FtsZ dynamics in filamentous and unicellular actinobacteria.

Bacterial cell division is driven by the polymerization of the GTPase FtsZ into a contractile structure, the so-called Z-ring. This essential process involves proteins that modulate FtsZ dynamics and hence the overall Z-ring architecture. Actinobacteria like Streptomyces and Mycobacterium lack known key FtsZ-regulators. Here we report the identification of SepH, a conserved actinobacterial protein that directly regulates FtsZ dynamics. We show that SepH is crucially involved in cell division in Streptomyces venezuelae and that it binds FtsZ via a conserved helix-turn-helix motif, stimulating the assembly of FtsZ protofilaments. Comparative in vitro studies using the SepH homolog from Mycobacterium smegmatis further reveal that SepH can also bundle FtsZ protofilaments, indicating an additional Z-ring stabilizing function in vivo. We propose that SepH plays a crucial role at the onset of cytokinesis in actinobacteria by promoting the assembly of FtsZ filaments into division-competent Z-rings that can go on to mediate septum synthesis.

Artificial modulation of cell width significantly affects the division time of Escherichia coli

Bacterial cells have characteristic spatial and temporal scales. For instance, Escherichia coli, the typical rod-shaped bacteria, always maintains a relatively constant cell width and cell division time. However, whether the external physical perturbation of cell width has an impact on cell division time remains largely unexplored. In this work, we developed two microchannel chips, namely straight channels and ‘necked’ channels, to precisely regulate the width of E. coli cells and to investigate the correlation between cell width and division time of the cells. Our results show that, in the straight channels, the wide cells divide much slower than narrow cells. In the ‘necked’ channels, the cell division is remarkably promoted compared to that in straight channels with the same width. Besides, fluorescence time-lapse microscopy imaging of FtsZ dynamics shows that the cell pre-constriction time is more sensitive to cell width perturbation than cell constriction time. Finally, we revealed a significant anticorrelation between the death rate and the division rate of cell populations with different widths. Our work provides new insights into the correlation between the geometrical property and division time of E. coli cells and sheds new light on the future study of spatial–temporal correlation in cell physiology.

A Unified Model for Treadmilling and Nucleation of Single-Stranded FtsZ Protofilaments

Bacterial cell division is tightly coupled to the dynamic behavior of FtsZ, a tubulin homolog. Recent experimental work in vitro and in vivo has attributed FtsZ’s assembly dynamics to treadmilling, in which subunits add to the bottom and dissociate from the top of protofilaments. However, the molecular mechanisms producing treadmilling have yet to be characterized and quantified. We have developed a Monte Carlo model for FtsZ assembly that explains treadmilling, and also explains assembly nucleation by the same mechanisms. A key element of the model is a conformational change from R (relaxed), which is highly favored for monomers, to T (tense), which is favored for subunits in a protofilament. This model was created in MATLAB. Kinetic parameters were converted to probabilities of execution during a single, small time step. These were used to stochastically determine FtsZ dynamics. Our model is able to accurately describe the results of several in vitro and in vivo studies for a variety of FtsZ flavors. With standard conditions, the model FtsZ polymerized and produced protofilaments that treadmilled at 23 nm/s, hydrolyzed GTP at 3.6–3.7 GTP min−1 FtsZ−1, and had an average length of 30–40 subunits, all similar to experimental results. Adding a bottom capper resulted in shorter protofilaments and higher GTPase, similar to the effect of the known bottom capper protein MciZ. The model could match nucleation kinetics of several flavors of FtsZ using the same parameters as treadmilling and varying only the R to T transition of monomers.

Bacterial cell division at a glance

Bacterial cell division is initiated by the midcell assembly of polymers of the tubulin-like GTPase FtsZ. The FtsZ ring (Z-ring) is a discontinuous structure made of dynamic patches of FtsZ that undergo treadmilling motion. Roughly a dozen additional essential proteins are recruited to the division site by the dynamic Z-ring scaffold and subsequently activate cell wall synthesis to drive cell envelope constriction during division. In this Cell Science at a Glance article and the accompanying poster, we summarize our understanding of the assembly and activation of the bacterial cell division machinery. We introduce polymerization properties of FtsZ and discuss our current knowledge of divisome assembly and activation. We further highlight the intimate relationship between the structure and dynamics of FtsZ and the movement and activity of cell wall synthases at the division site, before concluding with a perspective on the most important open questions on bacterial cell division.

The Min System Disassembles FtsZ Foci and Inhibits Polar Peptidoglycan Remodeling in Bacillus subtilis.

A microfluidic system coupled with fluorescence microscopy is a powerful approach for quantitative analysis of bacterial growth. Here, we measure parameters of growth and dynamic localization of the cell division initiation protein FtsZ in Bacillus subtilis Consistent with previous reports, we found that after division, FtsZ rings remain at the cell poles, and polar FtsZ ring disassembly coincides with rapid Z-ring accumulation at the midcell. In cells mutated for minD, however, the polar FtsZ rings persist indefinitely, suggesting that the primary function of the Min system is in Z-ring disassembly. The inability to recycle FtsZ monomers in the minD mutant results in the simultaneous maintenance of multiple Z-rings that are restricted by competition for newly synthesized FtsZ. Although the parameters of FtsZ dynamics change in the minD mutant, the overall cell division time remains the same, albeit with elongated cells necessary to accumulate a critical threshold amount of FtsZ for promoting medial division. Finally, the minD mutant characteristically produces minicells composed of polar peptidoglycan shown to be inert for remodeling in the wild type. Polar peptidoglycan, however, loses its inert character in the minD mutant, suggesting that the Min system not only is important for recycling FtsZ but also may have a secondary role in the spatiotemporal regulation of peptidoglycan remodeling.IMPORTANCE Many bacteria grow and divide by binary fission in which a mother cell divides into two identical daughter cells. To produce two equally sized daughters, the division machinery, guided by FtsZ, must dynamically localize to the midcell each cell cycle. Here, we quantitatively analyzed FtsZ dynamics during growth and found that the Min system of Bacillus subtilis is essential to disassemble FtsZ rings after division. Moreover, a failure to efficiently recycle FtsZ results in an increase in cell size. Finally, we show that the Min system has an additional role in inhibiting cell wall turnover and contributes to the "inert" property of cell walls at the poles.

Mechanistic insight into the effect of BT-benzo-29 on the Z-ring in Bacillus subtilis.

The assembly and disassembly of FtsZ play an essential role in bacterial cell division. Using single-cell imaging, we report that short exposure to BT-benzo-29 inhibits Z-ring formation in live Bacillus subtilis cells. Fluorescence recovery after photobleaching of the Z-ring in live bacteria demonstrated that BT-benzo-29 strongly suppressed the assembly dynamics of FtsZ in the Z-ring. Furthermore, B. subtilis cells expressing V275A-FtsZ resisted the antibacterial activity of BT-benzo-29 providing evidence that BT-benzo-29 inhibits bacterial proliferation by targeting FtsZ. In addition, a brief (8 min) exposure of BT-benzo-29 destroyed the Z-ring without perturbing the localization of a late cell division protein, DivIVA, the nucleoid segregation, and membrane permeability. BT-benzo-29, when used in combination with vancomycin and polymyxin B (PMB), produced a much stronger inhibitory effect on Bacillus subtilis and Escherichia coli cells, respectively. The combination index of BT-benzo-29 with vancomycin and PMB was determined to be <1, suggesting that BT-benzo-29 exhibits synergistic inhibitory effects on bacterial proliferation when used along with these antibiotics.

Movement dynamics of divisome proteins and PBP2x:FtsW in cells of Streptococcus pneumoniae

Bacterial cell division and peptidoglycan (PG) synthesis are orchestrated by the coordinated dynamic movement of essential protein complexes. Recent studies show that bidirectional treadmilling of FtsZ filaments/bundles is tightly coupled to and limiting for both septal PG synthesis and septum closure in some bacteria, but not in others. Here we report the dynamics of FtsZ movement leading to septal and equatorial ring formation in the ovoid-shaped pathogen, Streptococcus pneumoniae Conventional and single-molecule total internal reflection fluorescence microscopy (TIRFm) showed that nascent rings of FtsZ and its anchoring and stabilizing proteins FtsA and EzrA move out from mature septal rings coincident with MapZ rings early in cell division. This mode of continuous nascent ring movement contrasts with a failsafe streaming mechanism of FtsZ/FtsA/EzrA observed in a ΔmapZ mutant and another Streptococcus species. This analysis also provides several parameters of FtsZ treadmilling in nascent and mature rings, including treadmilling velocity in wild-type cells and ftsZ(GTPase) mutants, lifetimes of FtsZ subunits in filaments and of entire FtsZ filaments/bundles, and the processivity length of treadmilling of FtsZ filament/bundles. In addition, we delineated the motion of the septal PBP2x transpeptidase and its FtsW glycosyl transferase-binding partner relative to FtsZ treadmilling in S. pneumoniae cells. Five lines of evidence support the conclusion that movement of the bPBP2x:FtsW complex in septa depends on PG synthesis and not on FtsZ treadmilling. Together, these results support a model in which FtsZ dynamics and associations organize and distribute septal PG synthesis, but do not control its rate in S. pneumoniae.

Molecular dynamics analysis of the effects of GTP, GDP and benzimidazole derivative on structural dynamics of a cell division protein FtsZ from Mycobacterium tuberculosis

The prevailing multi-drug resistance in Mycobacterium tuberculosis continues to remain one of the main challenges to combat tuberculosis. Hence, it becomes imperative to focus on novel drug targets. Filamenting temperature-sensitive mutant Z (FtsZ) is an essential cell division protein, a eukaryotic tubulin homologue and a promising drug target. During cytokinesis, FtsZ polymerises in the presence of GTP to form Z-ring and recruits other proteins at this site that eventually lead to the formation of daughter cells. Benzimidazoles were experimentally shown to inhibit Mtb-FtsZ, with one of the benzimidazole derivatives, M1, being reported to have the minimum inhibitory concentration (MIC) value of 3.13 µg/mL. In the present study, mechanism of destabilisation of FtsZ in the presence of M1 was computationally investigated in the presence of its substrate GTP/GDP employing molecular dynamics (MD) simulation analysis, principal component analysis (PCA), molecular mechanics combined with the generalised Born and surface area continuum salvation (MM-GBSA) and density functional theory (DFT). From the analyses, it is proposed that binding of M1 in the inter-domain cleft induces structural changes in the GTP-binding region that affect GTP binding, thus switching the preference of this protein towards depolymerised state and eventually inhibiting the cell division. Hence, this study provides mechanistic insights into the design of novel benzimidazole inhibitors against Mtb-FtsZ.Communicated by Ramaswamy H. Sarma

Cell shape-independent FtsZ dynamics in synthetically remodeled bacterial cells

FtsZ is the main regulator of bacterial cell division. It has been implicated in acting as a scaffolding protein for other division proteins, a force generator during constriction, and more recently, as an active regulator of septal cell wall production. FtsZ assembles into a heterogeneous structure coined the Z-ring due to its resemblance to a ring confined by the midcell geometry. Here, to establish a framework for examining geometrical influences on proper Z-ring assembly and dynamics, we sculpted Escherichia coli cells into unnatural shapes using division- and cell wall-specific inhibitors in a micro-fabrication scheme. This approach allowed us to examine FtsZ behavior in engineered Z-squares and Z-hearts. We use stimulated emission depletion (STED) nanoscopy to show that FtsZ clusters in sculpted cells maintain the same dimensions as their wild-type counterparts. Based on our results, we propose that the underlying membrane geometry is not a deciding factor for FtsZ cluster maintenance and dynamics in vivo.

Mutation of G51 in SepF impairs FtsZ assembly promoting ability of SepF and retards the division of Mycobacterium smegmatis cells.

The role of FtsZ-associated proteins in the regulation of the assembly dynamics of Mycobacterium smegmatis FtsZ is not clear. In this work, we examined the effect of M. smegmatis SepF on the assembly and stability of M. smegmatis FtsZ polymers. We discovered a single dominant point mutation in SepF (G51D or G51R) that renders the protein inactive. SepF promoted the polymerization of FtsZ, induced the bundling of FtsZ filaments, stabilized FtsZ filaments and reduced the GTPase activity of FtsZ. Surprisingly, both G51D-SepF and G51R-SepF neither stabilized FtsZ filaments nor showed a discernable effect on the GTPase activity of FtsZ. The binding affinity of SepF to FtsZ was found to be stronger than the binding affinity of G51R/D-SepF to FtsZ. Interestingly, the binding affinity of SepF to G51R-SepF was determined to be 45 times stronger than FtsZ. In addition, the interaction of SepF with G51R-SepF was found to be 2.6 times stronger than SepF-SepF interaction. Furthermore, G51R-SepF impaired the ability of SepF to promote the assembly of FtsZ. In addition, the overexpression of G51R-SepF in M. smegmatis mc2 155 cells retarded the proliferation of these cells and increased the average length of the cells. The results indicated that SepF positively regulates the assembly of M. smegmatis FtsZ and the G51 residue has an important role in the functioning of SepF.

Escherichia coli ZipA Organizes FtsZ Polymers into Dynamic Ring-Like Protofilament Structures.

ABSTRACTZipA is an essential cell division protein in Escherichia coli. Together with FtsA, ZipA tethers dynamic polymers of FtsZ to the cytoplasmic membrane, and these polymers are required to guide synthesis of the cell division septum. This dynamic behavior of FtsZ has been reconstituted on planar lipid surfaces in vitro, visible as GTP-dependent chiral vortices several hundred nanometers in diameter, when anchored by FtsA or when fused to an artificial membrane binding domain. However, these dynamics largely vanish when ZipA is used to tether FtsZ polymers to lipids at high surface densities. This, along with some in vitro studies in solution, has led to the prevailing notion that ZipA reduces FtsZ dynamics by enhancing bundling of FtsZ filaments. Here, we show that this is not the case. When lower, more physiological levels of the soluble, cytoplasmic domain of ZipA (sZipA) were attached to lipids, FtsZ assembled into highly dynamic vortices similar to those assembled with FtsA or other membrane anchors. Notably, at either high or low surface densities, ZipA did not stimulate lateral interactions between FtsZ protofilaments. We also used E. coli mutants that are either deficient or proficient in FtsZ bundling to provide evidence that ZipA does not directly promote bundling of FtsZ filaments in vivo. Together, our results suggest that ZipA does not dampen FtsZ dynamics as previously thought, and instead may act as a passive membrane attachment for FtsZ filaments as they treadmill.

Effect of Non Antibiotic Antimicrobial Curcuma Longa on Helicobacter Pylori Infection

Helicobacter pylori [H. pylori] induced gastric infection and inflammation, which is potentially carcinogenic, is common in India. Despite the availability of triple drug therapy, its failure is quite common due to the high cost factor, adverse drug reactions and emerging resistance to antibiotics. This necessitates the need for discovering new drugs for treating H. pylori infection. Curcumin [diferuloylmethane], an active ingredient of turmeric [curcumin longa], has been extensively studied and proved to be effective in eradicating H. pylori infection, correcting gastric mucosal damage and to prevent or regress gastric carcinogenesis. Curcumin has strong free radical scavenging and anti oxidant property. It bears anti inflammatory and antimicrobial potential against H. pylori. Mechanisms of its antimicrobial actions are l) The inhibition of shikimate pathway by inhibiting enzyme shikimate dehydrogenase [SDH] resulting in to the failure of synthesizing aromatic amino acids [AAA]. folates and ubiquinone. 2) By inhibiting the assembly dynamics of bacterial profilament FtsZ. Curcumin has anti tumerogenesis potential through it’s anti inflammatory, antioxidant and free radical scavenging actions. Thus curcumin provides potential alternative or an adjunct therapy to treat H. pylori infection and prevent it’s complications.

A conserved coiled-coil protein pair focuses the cytokinetic Z-ring in Caulobacter crescentus.

The cytoskeletal GTPase FtsZ assembles at midcell, recruits the division machinery and directs envelope invagination for bacterial cytokinesis. ZapA, a conserved FtsZ-binding protein, promotes Z-ring stability and efficient division through a mechanism that is not fully understood. Here, we investigated the function of ZapA in Caulobacter crescentus. We found that ZapA is encoded in an operon with a small coiled-coil protein we named ZauP. ZapA and ZauP co-localized at the division site and were each required for efficient division. ZapA interacted directly with both FtsZ and ZauP. Neither ZapA nor ZauP influenced FtsZ dynamics or bundling, in vitro, however. Z-rings were diffuse in cells lacking zapA or zauP and, conversely, FtsZ was enriched at midcell in cells overproducing ZapA and ZauP. Additionally, FtsZ persisted at the poles longer when ZapA and ZauP were overproduced, and frequently colocalized with MipZ, a negative regulator of FtsZ polymerization. We propose that ZapA and ZauP promote efficient cytokinesis by stabilizing the midcell Z-ring through a bundling-independent mechanism. The zauPzapA operon is present in diverse Gram-negative bacteria, indicating a common mechanism for Z-ring assembly.