FtsZ Ring Research Articles

The Ethanologenic Bacterium Zymomonas mobilis Divides Asymmetrically and Exhibits Heterogeneity in DNA Content.

The alphaproteobacterium Zymomonas mobilis exhibits extreme ethanologenic physiology, making this species a promising biofuel producer. Numerous studies have investigated its biology relevant to industrial applications and mostly at the population level. However, the organization of single cells in this industrially important polyploid species has been largely uncharacterized. In the present study, we characterized basic cellular behavior of Z. mobilis strain Zm6 under anaerobic conditions at the single-cell level. We observed that growing Z. mobilis cells often divided at a nonmidcell position, which contributed to variant cell size at birth. However, the cell size variance was regulated by a modulation of cell cycle span, mediated by a correlation of bacterial tubulin homologue FtsZ ring accumulation with cell growth. The Z. mobilis culture also exhibited heterogeneous cellular DNA content among individual cells, which might have been caused by asynchronous replication of chromosome that was not coordinated with cell growth. Furthermore, slightly angled divisions might have resulted in temporary curvatures of attached Z. mobilis cells. Overall, the present study uncovers a novel bacterial cell organization in Z. mobilisIMPORTANCE With increasing environmental concerns about the use of fossil fuels, development of a sustainable biofuel production platform has been attracting significant public attention. Ethanologenic Z. mobilis species are endowed with an efficient ethanol fermentation capacity that surpasses, in several respects, that of baker's yeast (Saccharomyces cerevisiae), the most-used microorganism for ethanol production. For development of a Z. mobilis culture-based biorefinery, an investigation of its uncharacterized cell biology is important, because bacterial cellular organization and metabolism are closely associated with each other in a single cell compartment. In addition, the current work demonstrates that the polyploid bacterium Z. mobilis exhibits a distinctive mode of bacterial cell organization, likely reflecting its unique metabolism that does not prioritize incorporation of nutrients for cell growth. Thus, another significant result of this work is to advance our general understanding in the diversity of bacterial cell architecture.

ZapG (YhcB/DUF1043), a novel cell division protein in gamma-proteobacteria linking the Z-ring to septal peptidoglycan synthesis

YhcB, a poorly understood protein conserved across gamma-proteobacteria, contains a domain of unknown function (DUF1043) and an N-terminal transmembrane domain. Here, we used an integrated approach including X-ray crystallography, genetics, and molecular biology to investigate the function and structure of YhcB. The Escherichia coli yhcB KO strain does not grow at 45 °C and is hypersensitive to cell wall–acting antibiotics, even in the stationary phase. The deletion of yhcB leads to filamentation, abnormal FtsZ ring formation, and aberrant septum development. The Z-ring is essential for the positioning of the septa and the initiation of cell division. We found that YhcB interacts with proteins of the divisome (e.g., FtsI, FtsQ) and elongasome (e.g., RodZ, RodA). Seven of these interactions are also conserved in Yersinia pestis and/or Vibrio cholerae. Furthermore, we mapped the amino acid residues likely involved in the interactions of YhcB with FtsI and RodZ. The 2.8 Å crystal structure of the cytosolic domain of Haemophilus ducreyi YhcB shows a unique tetrameric α-helical coiled-coil structure likely to be involved in linking the Z-ring to the septal peptidoglycan-synthesizing complexes. In summary, YhcB is a conserved and conditionally essential protein that plays a role in cell division and consequently affects envelope biogenesis. Based on these findings, we propose to rename YhcB to ZapG (Z-ring-associated protein G). This study will serve as a starting point for future studies on this protein family and on how cells transit from exponential to stationary survival.

From Fuzziness to Clarity: Regulation of DRP5B Ring Dynamics at the Chloroplast Division Site.

Plant cells harbor a varying number of chloroplasts, and chloroplasts multiply by division to maintain the continuity of chloroplasts. Chloroplast division is performed by the constriction of two self-assembling GTPase rings in the middle of the chloroplast: the FtsZ ring on the stromal side and the

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.

Bacterial Division: Journey to the Center of the Cell.

Most bacteria divide by corralling the tubulin-like FtsZ protein to mid-cell, where it assembles into a ring of treadmilling membrane-tethered oligomers. A study in this issue reveals new details about how FtsZ finds its way to the ring.

A gradient-forming MipZ protein mediating the control of cell division in the magnetotactic bacterium Magnetospirillumgryphiswaldense.

Cell division needs to be tightly regulated and closely coordinated with other cellular processes to ensure the generation of fully viable offspring. Here, we investigate division site placement by the cell division regulator MipZ in the alphaproteobacterium Magnetospirillum gryphiswaldense, a species that forms linear chains of magnetosomes to navigate within the geomagnetic field. We show that M.gryphiswaldense contains two MipZ homologs, termed MipZ1 and MipZ2. MipZ2 localizes to the division site, but its absence does not cause any obvious phenotype. MipZ1, by contrast, forms a dynamic bipolar gradient, and its deletion or overproduction cause cell filamentation, suggesting an important role in cell division. The monomeric form of MipZ1 interacts with the chromosome partitioning protein ParB, whereas its ATP-dependent dimeric form shows non-specific DNA-binding activity. Notably, both the dimeric and, to a lesser extent, the monomeric form inhibit FtsZ polymerization in vitro. MipZ1 thus represents a canonical gradient-forming MipZ homolog that critically contributes to the spatiotemporal control of FtsZ ring formation. Collectively, our findings add to the view that the regulatory role of MipZ proteins in cell division is conserved among many alphaproteobacteria. However, their number and biochemical properties may have adapted to the specific needs of the host organism.

Quantification of Predictive Uncertainty in Models of FtsZ ring assembly in Escherichia coli

Quantitative predictions of FtsZ protein polymerization are essential for understanding the self-regulating mechanisms in biochemical systems. Due to structural complexity and parametric uncertainty, existing kinetic models remain incomplete and their predictions error-prone. To address such challenges, we perform probabilistic uncertainty quantification and global sensitivity analysis of the concentrations of various protein species predicted with a recent FtsZ protein polymerization model. Our results yield a ranked list of modeling shortcomings that can be improved in order to develop more accurate predictions and more realistic representations of key mechanisms of such biochemical systems and their response to changes in internal or external conditions. Our conclusions and improvement recommendations can be extended to other kinetics models.

Allelic Variation in the Chloroplast Division Gene FtsZ2-2 Leads to Natural Variation in Chloroplast Size.

Chloroplast size varies considerably in nature, but the underlying mechanisms are unknown. By exploiting a near-isogenic line population derived from a cross between the Arabidopsis (Arabidopsis thaliana) accessions Cape Verde Islands (Cvi-1), which has larger chloroplasts, and Landsberg erecta (Ler-0), with smaller chloroplasts, we determined that the large-chloroplast phenotype in Cvi-1 is associated with allelic variation in the gene encoding the chloroplast-division protein FtsZ2-2, a tubulin-related cytoskeletal component of the contractile FtsZ ring inside chloroplasts. Sequencing revealed that the Cvi-1 FtsZ2-2 allele encodes a C-terminally truncated protein lacking a region required for FtsZ2-2 interaction with inner-envelope proteins, and functional complementation experiments in a Columbia-0 ftsZ2-2 null mutant confirmed this allele as causal for the increased chloroplast size in Cvi-1. Comparison of FtsZ2-2 coding sequences in the 1001 Genomes database showed that the Cvi-1 allele is rare and identified additional rare loss-of-function alleles, including a natural null allele, in three other accessions, all of which had enlarged-chloroplast phenotypes. The ratio of nonsynonymous to synonymous substitutions was higher among the FtsZ2-2 genes than among the two other FtsZ family members in Arabidopsis, FtsZ2-1, a close paralog of FtsZ2-2, and the functionally distinct FtsZ1-1, indicating more relaxed constraint on the FtsZ2-2 coding sequence than on those of FtsZ2-1 or FtsZ1-1 Our results establish that allelic variation in FtsZ2-2 contributes to natural variation in chloroplast size in Arabidopsis, and they also demonstrate that natural variation in Arabidopsis can be used to decipher the genetic basis of differences in fundamental cell biological traits, such as organelle size.

Regulation of Streptococcus pneumoniae FtsZ assembly by divalent cations: paradoxical effects of Ca2+ on the nucleation and bundling of FtsZ polymers.

The assembly and disassembly of the FtsZ ring drives the division of bacteria cells, including Streptococcus pneumoniae, which causes pneumonia and meningitis. In contrast to FtsZ from other bacterial species, Streptococcus pneumoniae (Spn) FtsZ contains two tryptophan residues. Here, we demonstrate that the assembly and disassembly of Streptococcus pneumoniae FtsZ (SpnFtsZ) monomers can be monitored by the intrinsic tryptophan fluorescence of FtsZ. We found that the assembly of SpnFtsZ is closely associated with its GTPase activity. Guanosine 5'-[β,γ-imido]triphosphate, a nonhydrolyzable analog of GTP, stabilized the FtsZ filaments without inducing their bundling. Using intrinsic tryptophan fluorescence, light scattering, and electron microscopy, we could differentiate the effects of divalent calcium and magnesium on the assembly of FtsZ. Though Mg2+ increased the stability of the FtsZ filaments, it could not prevent the disassembly of the filaments under conditions where GTP was limiting. Thus, our results indicate that Mg2+ primarily enhances the longitudinal assembly of FtsZ. Low concentrations of Ca2+ strongly promoted the bundling of FtsZ filaments and inhibited the disassembly of the filaments, suggesting that low concentrations of Ca2+ enhance the lateral interactions between the FtsZ filaments. Interestingly, Ca2+ delayed the nucleation process of FtsZ assembly, indicating that Ca2+ exerts paradoxical effects on the assembly of FtsZ. However, higher concentrations of Ca2+ did not enhance the bundling of FtsZ filaments. In addition, Ca2+ altered the secondary structure of FtsZ and increased the fluorescence of the FtsZ-1-anilinonaphthalene-8-sulfonic acid complex, indicating that Ca2+ induces conformational changes in FtsZ. The study provides an interesting insight into the assembly of SpnFtsZ and its regulation by divalent cations.

Hsp90 of E. coli modulates assembly of FtsZ, the bacterial tubulin homolog

Heat shock protein 90 (Hsp90) is a highly conserved molecular chaperone involved in ATP-dependent client protein remodeling and activation. It also functions as a protein holdase, binding and stabilizing clients in an ATP-independent process. Hsp90 remodels over 300 client proteins and is essential for cell survival in eukaryotes. In bacteria, Hsp90 is a highly abundant protein, although very few clients have been identified and it is not essential for growth in many bacterial species. We previously demonstrated that in Escherichia coli, Hsp90 causes cell filamentation when expressed at high levels. Here, we have explored the cause of filamentation and identified a potentially important client of E. coli Hsp90 (Hsp90Ec), FtsZ. We observed that FtsZ, a bacterial tubulin homolog essential for cell division, fails to assemble into FtsZ rings (divisomes) in cells overexpressing Hsp90Ec Additionally, Hsp90Ec interacts with FtsZ and inhibits polymerization of FtsZ in vitro, in an ATP-independent holding reaction. The FtsZ-Hsp90Ec interaction involves residues in the client-binding region of Hsp90Ec and in the C-terminal tail of FtsZ, where many cell-division proteins and regulators interact. We observed that E. coli deleted for the Hsp90Ec gene htpG turn over FtsZ more rapidly than wild-type cells. Additionally, the length of ΔhtpG cells is reduced compared to wild-type cells. Altogether, these results suggest that Hsp90Ec is a modulator of cell division, and imply that the polypeptide-holding function of Hsp90 may be a biologically important chaperone activity.

Localization of the key cell division determinant FtsZ in Francisella tularensis.

Francisella tularensis, the causative agent of tularemia, is a highly infectious gram-negative bacterium capable of replicating within macrophages, leukocytes, dendritic cells and epithelial cells. In addition, F. tularensis also invades erythrocytes. FtsZ, a homologue of eukaryotic tubulin, is an essential cell division protein in almost all bacteria. Polymerization of FtsZ into a contractile ring is a critical first step in division and the FtsZ ring acts in recruiting the cell wall synthesis machinery to the nascent septum. We have generated a recombinant fusion of ftsZftto emgfp (Emerald Green Fluorescent Protein) which can be expressed, in trans, under its presumed native promotor or a strong Francisella promoter (pGRP). As expected, FtsZft-Emgfp can be seen localizing as rings in F. tularensis. Our aim is to utilize fluorescently tagged FtsZ to investigate replication of F. tularensis in a range of in vitro host cell invasion assays and as a marker for viability to study the morphological and physiological differentiation of F. tularensis as it transitions into a viable but non-culturable (VBNC) state. (This research was made possible by NASA West Virginia Space Grant Consortium Training Grant #NNX15A101H and by NIH Grant P20GM103434 to the West Virginia IDeA Network for Biomedical Research Excellence).

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.

Z-ring membrane anchors associate with cell wall synthases to initiate bacterial cell division

During the transition from elongation to septation, Escherichia coli establishes a ring-like peptidoglycan growth zone at the future division site. This preseptal peptidoglycan synthesis does not require the cell division-specific peptidoglycan transpeptidase PBP3 or most of the other cell division proteins, but it does require FtsZ, its membrane-anchor ZipA and at least one of the bi-functional transglycosylase-transpeptidases, PBP1A or PBP1B. Here we show that PBP1A and PBP1B interact with ZipA and localise to preseptal sites in cells with inhibited PBP3. ZipA stimulates the glycosyltransferase activity of PBP1A. The membrane-anchored cell division protein FtsN localises at preseptal sites and stimulates both activities of PBP1B. Genes zipA and ftsN can be individually deleted in ftsA* mutant cells, but the simultaneous depletion of both proteins is lethal and cells do not establish preseptal sites. Our data support a model according to which ZipA and FtsN-FtsA have semi-redundant roles in connecting the cytosolic FtsZ ring with the membrane-anchored peptidoglycan synthases during the preseptal phase of envelope growth.

Photobleaching Enables Super-resolution Imaging of the FtsZ Ring in the Cyanobacterium Prochlorococcus.

Super-resolution microscopy has been widely used to study protein interactions and subcellular structures in many organisms. In photosynthetic organisms, however, the lateral resolution of super-resolution imaging is only ~100 nm. The low resolution is mainly due to the high autofluorescence background of photosynthetic cells caused by high-intensity lasers that are required for super-resolution imaging, such as stochastic optical reconstruction microscopy (STORM). Here, we describe a photobleaching-assisted STORM method which was developed recently for imaging the marine picocyanobacterium Prochlorococcus. After photobleaching, the autofluorescence of Prochlorococcus is effectively reduced so that STORM can be performed with a lateral resolution of ~10 nm. Using this method, we acquire the in vivo three-dimensional (3-D) organization of the FtsZ protein and characterize four different FtsZ ring morphologies during the cell cycle of Prochlorococcus. The method we describe here might be adopted for the super-resolution imaging of other photosynthetic organisms.

MapZ Forms a Stable Ring Structure That Acts As a Nanotrack for FtsZ Treadmilling in Streptococcus mutans.

Bacterial binary cell division requires accurate placement of division machinery. FtsZ, a vital component of the division machinery, can assemble into filaments and self-organize into a ring structure (Z ring) at the appropriate site for cell division. MapZ, a recently identified FtsZ regulator in Streptococcaceae, has been found to localize at the midcell where it helps to properly position the FtsZ ring. However, its mechanism is still unclear. Here, by using total internal reflection fluorescence microscopy, super-resolution imaging, and single molecule tracking, we investigated the mechanism by which MapZ controls the position of the FtsZ ring. Our results show that FtsZ exhibits a dynamic treadmilling motion in S. mutans. Importantly, depletion of MapZ leads to the unconstrained movement of treadmilling FtsZ filaments and a shorter lifetime of the constricting FtsZ ring, which is frequently misplaced. Furthermore, by revealing that MapZ forms an immobile ring-like nanostructure at the division site, our study suggests that MapZ forms a stable ring that acts as a nanotrack to guide and restrict treadmilling FtsZ filaments in S. mutans.

Treadmilling analysis reveals new insights into dynamic FtsZ ring architecture.

FtsZ, the primary protein of the bacterial Z ring guiding cell division, has been recently shown to engage in intriguing treadmilling dynamics along the circumference of the division plane. When coreconstituted in vitro with FtsA, one of its natural membrane anchors, on flat supported membranes, these proteins assemble into dynamic chiral vortices compatible with treadmilling of curved polar filaments. Replacing FtsA by a membrane-targeting sequence (mts) to FtsZ, we have discovered conditions for the formation of dynamic rings, showing that the phenomenon is intrinsic to FtsZ. Ring formation is only observed for a narrow range of protein concentrations at the bilayer, which is highly modulated by free Mg2+ and depends upon guanosine triphosphate (GTP) hydrolysis. Interestingly, the direction of rotation can be reversed by switching the mts from the C-terminus to the N-terminus of the protein, implying that the filament attachment must have a perpendicular component to both curvature and polarity. Remarkably, this chirality switch concurs with previously shown inward or outward membrane deformations by the respective FtsZ mutants. Our results lead us to suggest an intrinsic helicity of FtsZ filaments with more than one direction of curvature, supporting earlier hypotheses and experimental evidence.

Efficient models of polymerization applied to FtsZ ring assembly in Escherichia coli

High protein concentrations complicate modeling of polymer assembly kinetics by introducing structural complexity and a large variety of protein forms. We present a modeling approach that achieves orders of magnitude speed-up by replacing distributions of lengths and widths with their average counterparts and by introducing a hierarchical classification of species and reactions into sets. We have used this model to study FtsZ ring assembly in Escherichia coli The model's prediction of key features of the ring formation, such as time to reach the steady state, total concentration of FtsZ species in the ring, total concentration of monomers, and average dimensions of filaments and bundles, are all in agreement with the experimentally observed values. Besides validating our model against the in vivo observations, this study fills some knowledge gaps by proposing a specific structure of the ring, describing the influence of the total concentration in short and long kinetics processes, determining some characteristic mechanisms in polymer assembly regulation, and providing insights about the role of ZapA proteins, critical components for both positioning and stability of the ring.

Absence of the Min System Does Not Cause Major Cell Division Defects in Agrobacterium tumefaciens

In A. tumefaciens, the essential FtsZ protein is located at the growth pole before shifting to the mid-cell right before division. Loss of FtsZ causes a halt in cell separation and lysis of cells. To understand how FtsZ polymerization is regulated to properly localize the FtsZ ring at the mid-cell, we have conducted a systematic characterization of the Min system in A. tumefaciens. Our findings indicate that the Min system is not required for cell survival. Yet, we find that the deletion of either minE or minCDE results in a broad cell size distribution, including an increase in the proportion of short and long cells. We observe that the site of constriction is misplaced in the minE or minCDE deletion strains allowing for short cells to arise from sites of constriction near the cell poles. Remarkably, the short cells are viable and contain DNA. In order to observe chromosome replication and segregation in these strains, YFP-ParB is used as a proxy to track the origin of replication as cells elongate and divide. In the absence of the Min proteins, duplication and segregation of the origin of replication is frequently delayed. Taken together, our data suggest that the Min system contributes to the proper regulation of FtsZ placement and subsequent cell division. Furthermore, the failure to precisely place FtsZ rings at mid-cell in the min mutants impacts other cell cycle features including chromosome segregation.

Phosphatidylglycerol is implicated in divisome formation and metabolic processes of cyanobacteria

Phosphatidylglycerol is an essential phospholipid for photosynthesis and other cellular processes. We investigated the role of phosphatidylglycerol in cell division and metabolism in a phophatidylglycerol-auxotrophic strain of Synechococcus PCC7942. Here we show that phosphatidylglycerol is essential for the photosynthetic electron transfer and for the oligomerisation of the photosynthetic complexes, notably, we revealed that this lipid is important for non-linear electron transport. Furthermore, we demonstrate that phosphatidylglycerol starvation elevated the expressions of proteins of nitrogen and carbon metabolism. Moreover, we show that phosphatidylglycerol-deficient cells changed the morphology, became elongated, the FtsZ ring did not assemble correctly, and subsequently the division was hindered. However, supplementation with phosphatidylglycerol restored the ring-like structure at the mid-cell region and the normal cell size, demonstrating the phosphatidylglycerol is needed for normal septum formation. Taken together, central roles of phosphatidylglycerol were revealed; it is implicated in the photosynthetic activity, the metabolism and the fission of bacteria.