
==== Front
Mol Biol Cell
Mol Biol Cell
molbiolcell
mboc
Molecular Biology of the Cell
1059-1524
1939-4586
The American Society for Cell Biology

38758654
E23-09-0365
10.1091/mbc.E23-09-0365
Special Issue on Cell Biology of Bacteria and Archaea
GTPase activity regulates FtsZ ring positioning in Caulobacter crescentus
Barrows Jordan M. a †
Talavera-Figueroa Barbara K. a
Payne Isaac P. a
Smith Erika L. a
Goley Erin D. * a
a Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205
Garner Ethan Monitoring Editor
Harvard University
Author contributions: J.M.B. and E.D.G. designed and performed experiments, conceptualized and contributed to writing and editing the manuscript. B.K.T.F. performed phase contrast imaging for constriction rate analysis. I.P.P. constructed strains and performed imaging for DNA damage quantification. E.L.S. performed control western blots to characterize FtsZ depletion. J.M.B. carried out quantitative and statistical analysis.

†Present address: Department of Integrative Structural and Computational Biology, Scripps Research Institute, La Jolla, CA 92037.

National Institutes of Health (grant numbers R35GM136221 and T32GM007445

ORCID ID: Erin D. Goley, 0000-0002-8518-2303

*Address correspondence to: Erin D Goley (egoley1@jhmi.edu).
01 7 2024
01 7 2024
35 7 ar9713 9 2023
06 5 2024
08 5 2024
© 2024 Barrows et al. “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society for Cell Biology.
2024
https://creativecommons.org/licenses/by-nc-sa/4.0/ This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 4.0 Unported Creative Commons License.

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.

Bacterial division is orchestrated by a tubulin-like GTPase called FtsZ that recruits downstream proteins and regulates constriction. How FtsZ function is regulated is incompletely understood.

The authors leverage Caulobacter crescentus to investigate the effects of FtsZ’s GTPase activity on FtsZ assembly, dynamics, and function in vitro and in cells. They found that GTPase activity is critical for spatial regulation of FtsZ in coordination with chromosome segregation.

As FtsZ is an essential and broadly conserved division regulator, deciphering its regulation is central to understanding bacterial replication and identifying novel antibacterial targets.
==== Body
pmcINTRODUCTION

Cell division in bacteria is a complex process that is essential for replication. In most bacteria, cell division occurs through active remodeling of the peptidoglycan (PG) cell wall, which drives constriction of the bacterial envelope and eventually results in cell separation. In rod-shaped organisms, failure to properly regulate this process leads to filamentation and eventual lysis. Cell division is carried out by a multiprotein complex known as the “divisome,” which comprises over 20 proteins, including PG synthases that are responsible for building the cell wall. While the identities of most players in the divisome have been known for some time, there are still fundamental questions regarding the interactions between and regulation of members of the divisome (Mahone and Goley, 2020).

The tubulin homolog FtsZ is the master regulator of the divisome and is responsible for several functions before and throughout cell division: i) localization to mid-cell to form a “Z-ring” and mark the future site of division, ii) recruitment of downstream members of the divisome, and iii) regulation of divisome activity and distribution of PG synthases (Barrows and Goley, 2021). In Caulobacter crescentus (hereafter Caulobacter), Z-ring formation is mediated by FtsZ self-assembly and its interaction with positive (ZapA/ZauP; Woldemeskel et al., 2017) and negative (MipZ; Thanbichler and Shapiro, 2006) regulators of polymerization. While ZapA/ZauP primarily affect Z-ring morphology, MipZ is responsible for Z-ring positioning, depolymerizing FtsZ at the cell poles and allowing formation of a stable Z-ring only at mid-cell.

In numerous bacteria, FtsZ has been shown to exhibit treadmilling dynamics, polymerizing and depolymerizing in a polarized manner to result in net movement of polymers along the inner circumference of the cell (Bisson-Filho et al., 2017; Yang et al., 2017). Treadmilling dynamics are linked to the rate of GTP hydrolysis, as mutants with decreased hydrolytic activity have decreased treadmilling rates (Bisson-Filho et al., 2017; Yang et al., 2017). In Escherichia coli, FtsZ treadmilling helps to distribute PG synthases about the division plane, and mutations that reduce treadmilling velocity also affect the movement rates of these enzymes but not the rate of septum closure (Yang et al., 2017, 2021).

Caulobacter is a Gram-negative alphaproteobacterium that has long served as a model for bacterial morphogenesis and cell-cycle progression (Barrows and Goley, 2023). While the majority of divisome components are conserved from Caulobacter to other well-studied Gram-negative bacterial models, Caulobacter lacks the membrane anchor ZipA, which is specific to gammaproteobacteria (Hale and De Boer, 1997), and possesses FzlC and FzlA, which are FtsZ binding partners specific to alphaproteobacteria (Goley et al., 2010; Meier et al., 2016; Lariviere et al., 2018). Additionally, Z-ring localization is regulated by the MipZ system in Caulobacter (Thanbichler and Shapiro, 2006), which is not present in other organisms in which FtsZ dynamics have been studied (Barrows and Goley, 2021). Caulobacter cultures can also be synchronized at the swarmer stage of the cell cycle (Schrader and Shapiro, 2015), allowing for precise analysis of cell-cycle dependent events (e.g., Z-ring formation and constriction). This ability to synchronize Caulobacter makes it a desirable model system in which to evaluate FtsZ’s role in cell division. Although Caulobacter has been leveraged as a model for understanding cell division, a thorough study of the regulation of Caulobacter FtsZ structure and dynamics is necessary to further our mechanistic understanding of cell division.

Here, we explored the roles of GTPase activity in Caulobacter FtsZ function. By examining a GTPase-deficient variant of FtsZ, we delineated the contribution(s) of GTP turnover to Z-ring assembly and subsequent constriction. We found that GTP hydrolysis is primarily responsible for proper Z-ring placement. We also observed that GTPase-deficient FtsZ stabilizes polymers both in vitro and in vivo, but that the kinetics of constriction remain unaffected by GTPase rate. Finally, we demonstrate that Z-ring misplacement is due to attenuated regulation of FtsZ by MipZ. Aberrant constriction localization results in increased incidence of DNA damage, providing a mechanism for the associated loss of viability. Overall, our results support a model wherein GTP turnover guides FtsZ localization in tandem with MipZ to ensure proper spatial coordination with chromosome segregation before constriction and ensure successful cell division.

RESULTS

A GTPase variant of FtsZ is deficient for GTP turnover but not polymerization

To begin assessing the role of GTPase activity in the function of Caulobacter FtsZ, we made two mutations based on previously studied variants: G109S and D216A (G105S and D212A in E. coli, respectively). Each of these substitutions resulted in decreased in GTP turnover in E. coli. However, the G105S mutant can complement loss of wild-type ftsZ while the D212A mutant cannot (Stricker and Erickson, 2003), suggesting that they have differing effects on FtsZ dynamics and function. We began our investigation by evaluating polymerization of wild-type and GTPase variants of Caulobacter FtsZ. Wild-type FtsZ formed polymers upon addition of GTP as observed by transmission electron microscopy (TEM), like previous observations (Figure 1A; Sundararajan and Goley, 2017; Barrows et al., 2020). FtsZD216A formed polymers morphologically similar to those formed by wild-type FtsZ (Figure 1A), suggesting that its ability to bind GTP and polymerize is relatively unimpaired. To confirm that this variant is deficient for GTPase activity, we quantified GTP turnover rates and found that wild-type FtsZ exhibited a hydrolysis rate of ∼5 GTP min–1, comparable to what was observed previously (Sundararajan and Goley, 2017), while the D216A variant had no detectable GTPase activity (Figure 1B). Finally, we employed right-angle light scattering to observe the relative stability of FtsZD216A filaments compared with those formed by wild-type FtsZ. Wild-type FtsZ exhibited an increase in light scattering upon addition of a limiting concentration of GTP, indicative of polymer formation, followed by a return to baseline as GTP was depleted (Figure 1C, dark blue solid line). FtsZD216A formed polymers as well, but these were maintained through the duration of the experiment even after wild-type FtsZ had depolymerized (Figure 1C, cyan dashed line).

FIGURE 1: The D216A variant of FtsZ exhibits decreased GTP turnover and dynamics. (A) Representative TEM micrographs for 2 μM of each indicated protein incubated in indicated polymerization buffers with 2 mM GTP. (B) GTP turnover rates for indicated FtsZ variants (4 µM) in same buffers as in (A). (C) Right-angle light scattering at 350 nm over time for 4 μM of each indicated protein upon addition of 0.5 mM GTP, indicated by the black arrow. Representative lines of two independent replicates are shown.

We also found that the G109S full-length variant is deficient for GTP turnover (Supplemental Figure 1A). However, in contrast to the D216A variant, we observed no light scattering by FtsZG109S following addition of GTP (Supplemental Figure 1B), suggesting that it fails to form polymers. This finding is supported by observations via TEM, wherein we were unable to detect polymers (Supplemental Figure 1C). This is consistent with previous reports of the corresponding variant in E. coli FtsZ failing to form polymers or even bind to GTP in vitro (RayChaudhuri and Park, 1992). Thus, we are unable to recapitulate the polymer-forming activity of the G109S variant in vitro, precluding our ability to draw conclusions about its effect on FtsZ function. It is unlikely that this mutation confers a loss of polymerization in vivo, however, as this mutant can fully complement loss of wild-type ftsZ in E. coli (Stricker and Erickson, 2003).

GTPase activity is required for growth and viability in Caulobacter

Previous work with the G109S mutant indicated that production of this species in Caulobacter is dominant lethal with drastic morphological defects (Wang et al., 2001). However, in that and related studies (Li et al., 2007; Goley et al., 2010), the G109S mutant was overexpressed from a high copy replicating plasmid. Upon expression of ftsZG109S from an integrating plasmid, either in a merodiploid or depletion strain, cells failed to form the expected “dumbbell” morphology exhibited in the overexpression strain (Supplemental Figure 2), suggesting that the dominant lethality observed previously is dose dependent. However, we were also unable to complement loss of native ftsZ with the G109S mutant, suggesting that unlike in E. coli, the G109S mutant is lethal in the absence of wild-type FtsZ. As a result of this finding and the fact that we were unable to observe polymerization of the G109S variant in vitro, we elected to continue this study with only the D216A mutant.

To observe the effects of GTPase deficiency in vivo, we constructed strains for the simultaneous depletion of wild-type FtsZ (expression driven by a vanillate-inducible promoter) and production of a desired variant under the control of a xylose-inducible promoter (Supplemental Figure 3A). This approach allowed us to cultivate these strains in the presence of wild-type FtsZ and observe the effects of substituting it with a desired variant over time. As a control for this experiment and later ones performed using cells depleted of FtsZ, we depleted wild-type FtsZ over the course of 5 h following removal of vanillate (in the absence of xylose) and probed FtsZ levels by immunoblotting. We found that FtsZ levels decrease 50–70% in this time frame (Supplemental Figure 3, B and C). Following removal of vanillate from and addition of xylose to the growth medium, FtsZ- and FtsZD216A-producing strains yielded similar levels of protein at the expected size for FtsZ (Supplemental Figure 3, D and E), indicating that the variants were produced to similar levels and are similarly stable. While some wild-type FtsZ remains in the absence of vanillate, these data indicate that the majority of FtsZ in the cell is derived from the xylose-induced copy under these conditions, allowing us to observe effects of FtsZD216A when it is the predominant form in the cell. Depletion of wild-type FtsZ in liquid culture resulted in continued increase in cell density (Supplemental Figure 4A, left), likely as a result of filamentation, until eventual growth arrest and lysis (Supplemental Figure 4B). Production of FtsZD216A resulted in a reduced growth rate and final density in liquid culture (Supplemental Figure 4A, right) and complete lethality on solid media (Supplemental Figure 4B), suggesting that GTPase deficiency hampers growth and is ultimately lethal.

FtsZ GTPase activity is required for proper Z-ring placement and constriction localization

To understand the function of FtsZ GTPase activity in vivo, we next investigated cells depleted of FtsZ and expressing either ftsZ or ftsZD216A from a xylose-inducible integrated plasmid (same strains used for results in Supplemental Figures 3 and 4). Phase contrast imaging indicated that cells of both strains continued to divide, suggesting that the divisome is functional, but the D216A mutant exhibited constriction localization defects over time (Figure 2A, aberrant constriction localization indicated by red arrowhead). This defect resulted in an increase in mean cell length (Figure 2B) and no discernable change in maximum width (Figure 2C) after 3 h of expression. Notably, while the mean length did increase, there was also a significant population of short cells (Figure 2, A and B, 3-h time point) suggesting an overall defect in length regulation. To further characterize this defect, we plotted constriction location relative to the length of the cell (Figure 2, D and E), defining the pole closest to the constriction as the right-hand pole in Figure 2D. Cells producing FtsZ (navy blue) exhibited consistent constriction localization centered at ∼5% away from mid-cell. However, cells producing FtsZD216A had constrictions over a much greater range of the cell length, formed multiple constrictions in some cells (Figure 2D, cells with localizations with negative x-values contain at least two constrictions), and had constrictions farther from mid-cell on average (Figure 2E). We hypothesized that the mislocalization of constriction sites in the ftsZD216A strain was a result of Z-ring mislocalization, which we tested by observing localization of a C-terminal fusion of mNeonGreen (mNG) to ZapA (ZapA-mNG; fusion contains a 10-amino acid flexible linker), which binds directly to FtsZ and reports on its localization (Woldemeskel et al., 2017). While ZapA-mNG consistently localized at mid-cell with a single focus in cells producing FtsZ, ZapA-mNG exhibited multiple bands and non-mid-cell localization in the majority of cells producing FtsZD216A (Supplemental Figure 5A). ZapA-mNG consistently localized to rings ∼5% away from mid-cell in the wild-type strain, but ring localization was more widely distributed along the length of the cell in the D216A mutant (Supplemental Figure 5, B and C), reminiscent of the constriction localization defect we observed (Figure 2, A, D, and E). Taken together, our observations support a critical role for GTPase activity in placement of the Z-ring and, subsequently, the constriction site.

FIGURE 2: FtsZD216A production results in aberrant constriction localization. (A) Representative phase contrast micrographs of indicated strains at given time points following simultaneous removal of vanillate and induction of xylose-driven expression of indicated ftsZ with 0.3% xylose. Red arrowhead indicates constriction located near the pole. Blue arrowhead indicates a minicell resulting from mislocalized constriction. Scale bar, 2 μm. (B and C) Dot plots of cell length (B) or maximum width (C) for indicated strains at 1 or 3 h postdepletion/induction. Circles represent individual cell measurements from three independent replicates (orange, cyan, and magenta; 100 cells per replicate) and outlined triangles represent mean values for each replicate. Line indicates mean of replicate means and error bars are SD. Unpaired t tests were performed using population mean values (N = 3) to determine indicated p values (n.s., not significant; **, p ≤ 0.01). (D) Scatter plot of constriction position in cells from (A) at 3 h postdepletion/induction sorted by length. The right-hand pole is defined as the pole closest to the constriction site (swarmer pole). Cells expressing ftsZ or ftsZD216A are shown in dark blue and cyan, respectively. Data was acquired in three independent replicates, represented for each strain by circles, triangles, and squares. (E) Dot plots of relative distance of constrictions from midcell in cells from (D). Circles represent individual cell measurements from three independent replicates (orange, cyan, and magenta; 50 cells per replicate) and outlined triangles represent median values for each replicate. Line indicates mean of replicate medians and error bars are SD. An unpaired t test was performed using population median values (N = 3) to determine indicated p value (****, p ≤ 0.0001).

GTPase activity decreases FtsZ polymer dynamics in vivo

Given our observations that GTPase deficiency resulted in decreased polymer dynamics in vitro (Figure 1) and Z-rings with aberrant localization in vivo (Figure 2; Supplemental Figure 5), we hypothesized that polymer dynamics are likely disrupted in vivo as well. Previous reports in E. coli have demonstrated that FtsZ variants with attenuated GTPase activity exhibit decreased FtsZ polymer dynamics (Yang et al., 2017). To evaluate polymer dynamics in vivo, we first attempted to measure mNG-FtsZ cluster velocity via fluorescence total internal reflection fluorescence (TIRF) microscopy, as had been done previously in E. coli (Yang et al., 2017) and Bacillus subtilis (Bisson-Filho et al., 2017). Due to the relatively small size of Caulobacter, however, we were unable to resolve individual clusters sufficiently to confidently report on velocity.

Instead, we investigated polymer dynamics by measuring FtsZ monomer lifetime – that is, the length of time that a given FtsZ monomer is present in a polymer. A previous study in B. subtilis demonstrated that FtsZ monomer lifetime is affected by perturbations to FtsZ treadmilling dynamics (Squyres et al., 2021), so we reasoned that the variants investigated here would affect monomer lifetime as well. Thus, we sought to measure monomer lifetime using a fusion of HaloTag to FtsZ (Halo-FtsZ; fusion contains a 27-amino acid flexible linker). Fluorescent fusions to FtsZ are typically not viable, and that remains the case for the Halo-FtsZ variant. However, these fusions have been demonstrated to localize correctly, recruit downstream members of the divisome, and facilitate constriction for several generations (Sundararajan et al., 2015; Meier et al., 2016, 2017; Woldemeskel et al., 2017; Barrows et al., 2020), suggesting that Halo-FtsZ and other FtsZ fusions serve as useful proxies for FtsZ activity in vivo.

Imaging single molecules of Halo-FtsZ (Figure 3A) and tracking the fluorescence intensity over time yielded a step-like pattern indicative of molecules that are cycling between diffusive (low intensity; monomeric) and immobile (high intensity; polymeric) states (Figure 3B), allowing us to measure the duration of each period within the polymer. Relative to wild-type FtsZ (12.2 ± 0.3 s), the D216A variant (16.2 ± 0.5 s) resulted in increased monomer lifetime (Figure 3C), indicating an increase in polymer stability and/or length, consistent with our prediction and previous data in other bacteria that GTP turnover is correlated with polymer dynamics. The difference observed in monomer lifetimes was smaller than expected based on measurements in B. subtilis (Squyres et al., 2021), but this could be a result of residual wild-type FtsZ still being present at the imaging time (Figure 3D). Overall, our results are consistent with a model wherein GTPase activity is necessary for and modulates bulk Z-ring placement as well as individual polymer dynamics in vivo.

FIGURE 3: GTPase activity modulates FtsZ polymer dynamics in vivo. (A) Representative phase contrast and maximum projection of JF646 fluorescence stack acquired for single molecule tracking of FtsZ. The molecule of interest in this stack is within the red circle. Scale bar, 1 µm. (B) Representative intensity traces (black) and approximate fits (cyan) demonstrating step-like pattern. The length of each step was used to calculate the monomer lifetime for each trace. (C) Frequency distribution of monomer lifetime measurements for indicated strains. (D) Immunoblots of indicated strains bearing inducible Halo-tagged full-length FtsZ variants before synchrony (presync), postsynchrony (postsync), and 1 h postsynchrony (postsync 1 h) to evaluate degradation of wild-type FtsZ. Blots are probed with α-FtsZ (top) and α-CdnL (bottom, loading control) primary antibodies.

FtsZ GTPase activity does not affect constriction rate but is necessary to prevent DNA damage during constriction

Given that we observed effects on polymer dynamics both in vitro (Figure 1) and in vivo (Figure 3), we next asked whether there was a connection between FtsZ dynamics and cell growth/constriction. The relationship between FtsZ dynamics and PG synthesis has been explored in E. coli (Yang et al., 2017), B. subtilis (Bisson-Filho et al., 2017), Streptococcus pneumoniae (Perez et al., 2019), and Staphylococcus aureus (Monteiro et al., 2018), with differing degrees of coupling – or not – between the two depending on the species. Therefore, we sought to investigate whether a loss of GTPase activity in Caulobacter affects the kinetics of constriction, which is dependent on PG synthesis.

Using the same strains as in Figure 2, we predepleted cultures of wild-type FtsZ for 30 min then synchronized to isolate swarmer cells, spotted them on agarose pads with growth media and xylose, and observed their morphology over the course of a cell cycle via timelapse imaging. We used these data to measure the rates of elongation and constriction, as well as the time before constriction and constriction duration. As before, cells producing FtsZD216A frequently exhibited mislocalized constriction sites (Figure 4A). However, none of the parameters we measured (elongation rate, constriction rate, elongation/constriction rate ratio, preconstriction time, or constriction duration) were different between strains producing FtsZ or FtsZD216A (Figure 4, B–F). We conclude that while division site placement depends on FtsZ GTPase activity, constriction initiation, rate, and duration do not.

FIGURE 4: Loss of GTPase activity does not affect constriction kinetics. (A) Montage of representative phase micrographs of a cells from indicated strains at indicated time points following synchrony. Constriction start (Ti) and constriction end (Tf) times are indicated below images. Scale bar, 2 µm. (B–F) Dot plots of constriction rate (B), elongation rate (C), the ratio of constriction rate to elongation rate (D), preconstriction time (E), or constriction duration (F) for strains inducible for the indicate FtsZ mutant. Circles represent individual cell measurements from three independent replicates (orange [WT: 226 cells; D216A: 182 cells], cyan [WT: 100 cells; D216A: 149 cells], and magenta [WT: 125 cells; D216A: 52 cells]) and outlined triangles represent mean values for each replicate. Line indicates mean of replicate means and error bars are SD. A parametric student’s t test was performed using population mean values (N = 3) to compare values for each measurement, none of which were determined to be significantly different.

MipZ regulation of FtsZ depends on GTPase activity

Thus far, our findings suggest that GTP hydrolysis by FtsZ is crucial for positioning the Z-ring at mid-cell. This finding is consistent the model proposed by (Thanbichler and Shapiro, 2006), in which the ATPase MipZ actively depolymerizes FtsZ by stimulating its GTPase activity. As MipZ concentration is highest at the cell poles, stable polymer condensation only occurs at mid-cell where MipZ is absent. Thus, we hypothesized, therefore, that GTPase-deficient FtsZ might be insensitive to MipZ’s positioning signals. To investigate further, we expressed mipZ labeled with mCherry (MipZ-mChy; fusion contains a 20-amino acid flexible linker) in cells producing mNG fusions of FtsZ and FtsZD216A (mNG-FtsZ and mNG-FtsZD216A; fusions contain a 13-amino acid flexible linker). We observed that MipZ-mChy localized to the poles in most cells and in periodic foci corresponding to multiple origins of replication in filamentous cells regardless of the FtsZ variant present (Supplemental Figure 6), suggesting that its localization is not affected by the FtsZ variants. However, we observed multiple instances of mNG-FtsZD216A rings that appeared close to MipZ foci or at the poles (see white arrowheads in 3-h time point), suggesting that MipZ has decreased influence on the in vivo localization of these variants.

Next, we performed an FtsZ pelleting assay to determine whether the depolymerizing effect of MipZ in vitro (Thanbichler and Shapiro, 2006) would be mitigated by the loss of FtsZ GTPase activity (Figure 5). As expected, the addition of MipZ resulted in a decrease in FtsZ in the pellet, indicative of depolymerization activity (Figure 5A). However, there was no difference in the fraction of FtsZD216A in the pellet in the presence or absence of MipZ (Figure 5A), while MipZ itself remained mostly soluble regardless of the species of FtsZ (Figure 5B). Next, we tested whether we could see a decrease in polymers via TEM under the same conditions. While MipZ did affect FtsZ polymers in both cases, FtsZD216A polymers appear to be more robust upon addition of MipZ compared with wild-type FtsZ polymers, which were shorter and more highly curved than those of FtsZD216A with MipZ (Figure 5C). Taken together, our results indicate that while FtsZD216A is not completely blind to regulation by MipZ, the loss of GTPase activity prevents depolymerization by MipZ enough to result in failure to localize to midcell, yielding the mislocalized constrictions and Z-rings observed (Figure 2; Supplemental Figure 5).

FIGURE 5: FtsZ GTPase activity is required for coordination with chromosome segregation (A and B) Quantification of fraction of FtsZ (3 µM; A) or MipZ (6 µM; B) in pellet upon centrifugation following polymerization with the indicated protein components with 2 mM GTP/2 mM ATP. Experiments were completed in triplicate (N = 3) and parametric student’s t tests were performed on indicated pairs of columns to determine indicated p values (n.s., not significant; **, p ≤ 0.01). (C) Representative TEM micrographs for 3 μM indicated FtsZ variant incubated with 2 mM GTP/2 mM ATP without or with MipZ (6 µM). Scale bar, 100 nm. (D) Percent of cells with high GFP signal from indicated strains with indicated inducers (van, 0.5 mM vanillate; xyl, 0.3% xylose) after indicated hours of induction (3, 5, or 7 h). High GFP signal was determined as the mean signal for the top 1% of cells producing FtsZ with vanillate at 3 h (left-most column). One-way ANOVA with Šídák’s multiple comparisons test was performed to determine indicated p values (N = 3 biological replicates, all of which included >200 cells; *, p ≤ 0.05; ***, p ≤ 0. 001).

The regulation of Z-ring formation and positioning by MipZ is a primary mechanism by which cell division is linked to chromosome replication and segregation. Constriction must occur near midcell to ensure that daughter cells are properly sized, but also to coordinate clearing of the replicated chromosomes from the future constriction site, and failure to do so has been shown to lead to DNA damage in other organisms (Wang and Lutkenhaus, 1998; Wang et al., 2006). Thus, we predicted that the toxicity we observed following production of the GTPase-deficient FtsZ is at least partially a result of DNA damage. To test this hypothesis, we employed the use of a fluorescence-based reporter for activation of the PsidA promoter, an indicator of DNA damage (Modell et al., 2011; Mahone et al., 2024). Neither the presence of wild-type FtsZ protein produced by vanillate induction nor the production of xylose-induced wild-type FtsZ resulted in an increase in fluorescence of the reporter, indicating little to no DNA damage (Figure 5D). The production of FtsZD216A, however, resulted in a substantial increase in fluorescence after 7 h (Figure 3G, FtsZD216A xyl), suggesting that cells experience DNA damage within four to five generations contributing to the observed toxicity.

DISCUSSION

FtsZ is a crucial factor for initiating and regulating cell division in PG-bearing bacteria as it is responsible for demarcation of the future site of division, recruitment of downstream divisome factors, and modulation of their activity. Disruption of any of these functions has profound effects on division and morphogenesis, inspiring the need to understand them in detail. In the present study, we sought to dissect the contributions of the GTPase activity of FtsZ in regulating FtsZ dynamics and function in Caulobacter. Our findings support a model wherein GTPase activity is primarily responsible for Z-ring and divisome positioning and dynamics (Figure 6A).

FIGURE 6: GTP hydrolysis directs FtsZ localization to determine constriction site placement and coordinate with chromosome segregation. (A) FtsZ condenses at mid-cell before constriction to form a Z-ring (cyan), localization of which primarily depends on regulation by MipZ (magenta) and GTP hydrolysis by FtsZ. After Z-ring formation, FtsZ recruits and regulates the activity of downstream divisome components, including PG enzymes (red and green ellipses), to drive constriction of the cell envelope. Z-ring and constriction positioning are coordinated with chromosome (beige and white striped ellipse outlines) segregation to avoid DNA damage. (B) GTPase-deficient FtsZ (navy blue) can polymerize independent of spatial regulation by MipZ, allowing formation of Z-rings even in regions where MipZ concentrations are higher (magenta shading), resulting in mislocalized and occasionally multiple constriction sites. Constriction over nucleoid regions (i.e., where the bulk of the chromosome is located) results in DNA damage (red lightning bolts).

Based on studies in E. coli, we constructed FtsZ bearing the D216A substitution, resulting in polymer stabilization through complete abrogation of GTP turnover (Figure 1). Expression of this mutant in vivo caused mislocalization of Z-rings (Supplemental Figure 5), resulting in a distribution of constriction sites along the cell length as opposed to near mid-cell (Figure 2). Although Halo-FtsZD216A exhibited an increased dwell time in polymers in vivo (Figure 3), the results from our constriction rate analysis (Figure 4) fail to indicate any alteration in either constriction initiation or constriction rate. This is consistent with observations in E. coli (Coltharp et al., 2016), Streptococcus pneumoniae (Perez et al., 2019), and Staphylococcus aureus (Monteiro et al., 2018). Although FtsZ treadmilling can impact constriction rate in B. subtilis, it is dispensable for constriction after Z-ring condensation in that organism (Whitley et al., 2021). Collectively, these findings suggest that while GTPase activity is necessary for proper Z-ring positioning and dynamics (Figure 6), it does not play a major role in regulating constriction rate in most bacteria. Interestingly, although movement of an inactive subpopulation of the PG synthase FtsW is driven by FtsZ dynamics in Caulobacter (Mahone et al., 2024), cells producing GTPase-deficient FtsZ were able to divide several times. However, these cells were not viable (Supplemental Figure 4), likely due to the accumulation of DNA damage (Figure 5D) resulting from guillotining of nucleoids due to improper coordination of constriction and chromosome segregation (Figure 6B).

The mislocalization defect brought on by GTPase deficiency is at least in part due to reduced responsiveness to MipZ. We observed overlapping mNG-FtsZD216A and MipZ foci in cells (Supplemental Figure 6), and MipZ had a decreased propensity to depolymerize FtsZD216A in vitro compared with FtsZ (Figure 5). As MipZ has been demonstrated to enhance GTP hydrolysis by FtsZ, it follows that an FtsZ variant incapable of hydrolysis might be blind to regulation by MipZ, resulting in Z-ring formation even in regions containing higher MipZ levels. A more recent study suggested that MipZ disrupts FtsZ polymer formation not only through enhancement of GTP hydrolysis, but also by capping FtsZ polymers and perhaps sequestering FtsZ monomers near the poles where MipZ concentrations are highest (Corrales-Guerrero et al., 2022). The fact that there is inhibitory activity of MipZ independent of FtsZ GTPase activity concurs with our observations that FtsZD216A exhibits modest but apparent resistance to depolymerization by MipZ. Our results indicate that enhancement of GTPase activity remains a critical mechanism of FtsZ regulation by MipZ in cells.

Overall, our findings suggest that GTPase activity is primarily responsible for Z-ring positioning and is dispensable for constriction (Figure 6). Further investigation is required to understand the specific mechanism(s) by which FtsZ performs its functions and how they are influenced by both GTP hydrolysis activity and other regulators of division.

MATERIALS AND METHODS

Request a protocol through Bio-protocol.

Caulobacter crescentus and Escherichia coli growth media and conditions

Caulobacter NA1000 (Evinger and Agabian, 1977) cells were grown at 30°C in peptone-yeast extract (PYE) medium. Antibiotic concentrations used in liquid (solid) media for Caulobacter were as follows: gentamicin, 1 (5) µg ml–1; kanamycin, 5 (25) µg ml–1; spectinomycin, 25 (100) µg ml–1; streptomycin, (5) µg ml–1. For experiments involving inducible gene expression, inducer concentrations were as follows: glucose, 0.2% (wt/vol); xylose, 0.3% (wt/vol); vanillate, 0.5 mM. E. coli Rosetta(DE3)/pLysS cells were grown at 37°C in Luria-Bertani (LB) medium. Antibiotic concentrations used in liquid (solid) media for E. coli were as follows: ampicillin, 50 (100) µg ml–1. Strains used in this study are listed in Supplemental Table 1. Plasmids used in this study are listed in Supplemental Table 2.

FtsZ protein purification

Wild-type FtsZ, as well as GTPase variants, from Caulobacter were purified using the protocol described for CcFtsZ (Sundararajan and Goley, 2017). ftsZ (or variant) expression was induced in E. coli Rosetta(DE3)/pLysS cells bearing ftsZ (or variant) on a pET21a vector. The culture was grown at 37°C to an OD600 of 1.0, at which point protein expression was induced using 0.5 mM isopropyl-β-d-thiogalactopyranoside (IPTG) at 37°C for 3 h. Cells were pelleted and resuspended in lysis buffer (50 mM Tris-HCl [pH 8.0], 50 mM KCl, 1 mM EDTA, 10% glycerol, DNase I, 1 mM β-mercaptoethanol, 2 mM phenylmethylsulfonyl fluoride [PMSF], 1 complete mini, EDTA-free protease inhibitor tablet [Roche]) and incubated with 1 mg ml–1 lysozyme for 1 h at 25°C for lysis, followed by sonication. Protein was purified using an anion exchange chromatography column (HiTrap Q HP, 5 ml; GE Life Sciences) followed by ammonium sulfate precipitation (20 to 30% ammonium sulfate saturation, depending on the variant). The precipitated pellet was resuspended in FtsZ storage buffer (50 mM HEPES-KOH [pH 7.2], 50 mM KCl, 0.1 mM EDTA, 1 mM β-mercaptoethanol, 10% glycerol) and was further purified by size exclusion chromatography (Superdex 200 10/300 GL column; GE Life Sciences/Cytiva). Peak fractions were pooled, aliquoted, and snap frozen in liquid nitrogen for long-term storage at –80°C in FtsZ storage buffer.

MipZ protein purification

His6-MipZ was purified as previously described (Sundararajan et al., 2015). Rosetta(DE3) pLysS E. coli cells carrying pMT183 were grown at 37°C to OD600 of 0.6 then induced with 0.5 mM IPTG for 3 h. Cells were harvested by centrifugation at 6000 × g for 10 min at 4°C then washed in PBS and frozen in liquid nitrogen for storage at –80°C until purification. The cell pellet was resuspended in Ni lysis buffer (50 mM Tris-HCl [pH 8.0], 300 mM NaCl, 10 mM imidazole, 1 mM EDTA, 10% glycerol) with protease inhibitors (1 mini complete protease inhibitor table per 30 ml [Roche] and 2 mM PMSF and 2 U/ml DNAse I. Cells were lysed by passage through a French Press at 15,000 psi. Lysates were clarified by centrifugation at 15,000 × g for 30 min at 4°C. His6-MipZ was purified by binding to Ni-NTA agarose (Qiagen), washed in Ni wash buffer (Ni lysis buffer with 20 mM imidazole) and eluted in Ni elution buffer (Ni lysis buffer with 300 mM imidazole). Protein was applied to a Superdex 200 10/300 GL column (Cytiva) equilibrated in 50 mM HEPES-NaOH [pH 7.2], 50 mM NaCl, 0.1 mM EDTA, 1 mM β-mercaptoethanol, and 10% glycerol. Fractions containing His6-MipZ were pooled, concentrated, frozen in liquid nitrogen and stored at –80°C.

Phosphate release assay for FtsZ GTPase activity

Phosphate release by GTP hydrolysis was observed using an assay similar to that described in (Sundararajan and Goley, 2017). Thawed protein was diluted to 4 µM in polymerization buffer (50 mM HEPES-KOH [pH 7.2], 50 mM KCl, 0.1 mM EDTA) with 2.5 mM MgCl2. 2 mM GTP was added, and reactions were run for 0 to 30 min in 5-min intervals, stopping each reaction by adding to quench buffer (50 mM HEPES-KOH [pH 7.2], 50 mM KCl, 21.3 mM EDTA). Malachite green reagent (SensoLyte MG Phosphate Assay Kit [AnaSpec]) was added to each reaction and incubated for 30 min before measuring absorbance at 660 nm. Values were compared with a standard curve and plotted to determine GTPase rate. The rate for each protein was measured in triplicate.

FtsZ polymerization kinetic assay

FtsZ polymerization kinetics were observed using an assay similar to that described in (Sundararajan and Goley, 2017). Thawed protein was diluted to 4 µM in polymerization buffer (50 mM HEPES-KOH [pH 7.2], 50 mM KCl, 0.1 mM EDTA) with 2.5 mM MgCl2 in cuvettes. Following addition of a limiting concentration of GTP (0.5 mM), polymerization was measured using a Fluoromax-3 spectrofluorometer (Jobin Yvon) to measure right-angle light scattering (excitation and emission at 350 nm, 2-nm slits) every 10 s for 40 min.

Transmission electron microscopy (TEM)

TEM to visualize FtsZ polymers was performed as described in (Sundararajan et al., 2015). Thawed protein was diluted to 2 µM in polymerization buffer (50 mM HEPES-KOH [pH 7.2], 50 mM KCl, 0.1 mM EDTA) with 2.5 mM MgCl2. Reactions to test interactions with MipZ included 6 µM MipZ protein and 2 mM ATP with 5 mM MgCl2. Polymerization was induced with addition of 2 mM GTP, and reactions were incubated for 15 min before spotting on glow-discharged carbon-coated copper grids (Electron Microscopy Sciences, Hatfield, PA). Grids were blotted and stained twice with 0.75% uranyl formate for 2 min. Grids were then dried and imaged at 100,000 × magnification using a Hitachi 7600 TEM (operated at 80 kV) with an AMT XR80 8-megapixel CCD camera (AMT Imaging).

High-speed pelleting assay to measure FtsZ polymerization

The fraction of steady-state polymerized FtsZ was measured similar to as described in (Goley et al., 2010; Sundararajan and Goley, 2017; Sundararajan et al., 2018). Frozen aliquots of FtsZ or FtsZD216A in storage buffer were thawed and spun at 250,000 × g for 15 min at 25°C to remove nonspecific aggregates. Clarified FtsZ was diluted to 2 µM in polymerization buffer (50 mM HEPES-KOH [pH 7.2], 50 mM KCl, 0.1 mM EDTA) with 5 mM MgCl2 and 2 mM ATP with or without 6 µM MipZ. Polymerization was induced with addition of 2 mM GTP, and reactions were incubated for 15 min before pelleting by ultracentrifugation at 250,000 × g for 15 min at 25°C. Pellet and supernatant fractions were visualized via SDS–PAGE and Coommassie staining followed by analysis using ImageLab (Bio-Rad).

Phase-contrast and epifluorescence microscopy and image analysis

Logphase cultures of cells were spotted onto 1% agarose pads and were imaged using a Nikon Eclipse Ti inverted microscope through a Nikon Plan Fluor 100x (numeric aperture, 1.30) oil Ph3 objective with a Photometrics CoolSNAP HQ2 cooled CCD camera. Background intensity was subtracted from all raw fluorescence images before analysis/figure preparation using the background subtraction function in FIJI (Schindelin et al., 2012; rolling ball radius = 25 pixels). Images were prepared for presentation in Photoshop (Adobe) by adjusting fluorescence signal to the same levels (unless otherwise indicated) across samples in each experiment without oversaturating pixels. Length, maximum width, and constriction/Z-ring location analyses were performed using the MicrobeJ plugin for FIJI (Ducret et al., 2016). For the position measurements, cells were oriented using the Feature function, defining pole 1 as the pole closest to the constriction/Z-ring. Relative distance from midcell values were calculated by subtracting 0.5 from the arbitrary relative value generated by MicrobeJ and taking the absolute value. Each analysis was performed in biological triplicate for each strain, and comparisons were evaluated using either a student’s t test or one-way ANOVA with Šídák’s multiple comparisons test as indicated in corresponding figure legends.

Cell preparation for FtsZ single molecule tracking

Log-phase cultures containing vanillate were synchronized (Schrader and Shapiro, 2015), induced with 0.3% xylose for expression of the indicated Halo-tagged mutant, and incubated at 30°C for 30 min. Cells were then incubated with 0.7 nM Janelia fluor 646 dye conjugated to Halo-tag ligand (JF646; Promega) in the dark at 30°C for 15 min and washed with plain PYE to remove excess dye. Cells were then mounted on a 1% agarose pad made with plain PYE and dried before imaging.

Microscope and imaging for FtsZ single molecule tracking

Labeled strains were imaged on an Olympus IX-71 microscope with a 100x/1.30 NA Oil Ph3 objective and an Andor iXon 897 Ultra EM-CCD camera with EM-gain set to 300 using Metamorph software. A region of 250 × 250 pixels (160 nm/pixel) was selected, and a 300-frame image stack was acquired (0.5 s/frame) with constant illumination with a 647-nm laser set to 5 W/cm2. Phase contrast images were acquired before and after acquisition of fluorescence images to detect cell drift.

Analyzing single molecule trajectories and extracting monomer lifetime values

Image stacks were analyzed in a manner similar to that described in (McCausland et al., 2021). Briefly, fluorescence image stacks were processed using the ThunderSTORM plugin (Ovesný et al., 2014) for FIJI to detect and localize particle spots. Processed data was then analyzed using custom scripts (McCausland et al., 2021) in Matlab R2022b to link particles to trajectories, manually unwrap cells, segment trajectories and classify trajectories, and output monomer lifetime values. Final data are the results of three biological replicates pooled into a single population and then presented as frequency distribution curves.

Time-lapse imaging and constriction rate analysis

Imaging and analysis of constriction rate were performed similar to that described previously (Lariviere et al., 2018). Log-phase cultures of cells grown with vanillate for FtsZ production were washed with plain PYE and incubated for 30 min to begin FtsZ depletion. Cells were then synchronized (Schrader and Shapiro, 2015) and spotted onto 1% agarose pads containing PYE and xylose to induce production of the indicated FtsZ variant. Cells were then imaged by phase contrast microscopy as described above every 5 min until cells had completed division. Constriction/elongation rate analysis was undertaken using MicrobeJ, tracking each cell throughout the timelapse and recording the presence/absence of a constriction as well as the length and width for each frame. These values were then used to calculate the following parameters: preconstriction/constriction time (number of frames before/during which a constriction is present multiplied by 5 min/frame), constriction rate (cell width at constriction initiation divided by the constriction time), and elongation rate (difference of initial and final cell lengths divided by the sum of preconstriction and constriction time).

DNA damage characterization

Log phase cultures of cells with a plasmid bearing an eGFP reporter under the transcriptional control of the sidA promoter were treated with either vanillate (wild-type FtsZ control) or xylose (experimental) for indicated amounts of time before imaging. The mean fluorescence intensity was measured for each cell, and mean intensity values representing the 99th percentile for each replicate of the wild-type strain treated with vanillate were averaged. This value was used as a benchmark to calculate the “% with high GFP” at each time point, which is defined as the percentage of cells with a mean intensity greater than the benchmark. These values were plotted and used to compare the numbers of cells with high intensity in each strain via one-way ANOVA with Šídák’s multiple comparisons tests.

Growth rate measurement

Log phase cultures of cells grown without inducer were diluted to an OD600 of 0.05 and grown in 96-well plates for 24 h at 30°C with constant shaking on a BioTek Cytation 1 plate reader (Agilent), measuring the absorbance at 600 nm every 30 min for three technical replicates for each strain. Shaded region represents SD for each strain.

Spot dilution assay

Log phase cultures of cells grown without inducer were diluted to an OD600 of 0.05 and serially diluted up to 10–5 before spotting onto solid media with indicated inducer concentrations. Plates were incubated for 48 h at 30°C and imaged with a GE Healthcare Amersham Imager 600.

Immunoblots to measure FtsZ levels

Indicated strains were grown to log phase in the presence of 0.5 mM vanillate, depleted of native FtsZ by washing cells in PYE, and incubated with either 0.2% glucose (control) or 0.3% xylose to induce expression of the indicated ftsZ mutant. Samples were collected at the indicated time points after induction and resuspended in 1x SDS loading dye (0.2 M Tris-HCl [pH 6.8], 2% SDS, 10% glycerol, 0.18 M β-mercaptoethanol, 1.5 mM bromophenol blue). Samples were run on 12% SDS–PAGE gels and transferred to nitrocellulose membranes, at which point they were blocked overnight at 4°C with 5% milk in Tris-Buffered Saline Tween-20 (TBST). Blocked blots were probed with 1°C antibodies (α-FtsZ: rabbit, 1:20,000 [Sundararajan et al., 2015]; α-CdnL: rabbit, 1:5000 [Woldemeskel et al., 2020]) for 1 h at room temperature, washed three times for 5 min with TBST, probed with 2°C α-rabbit-HRP antibody (1:10,000) for 1 h at room temperature, washed three more times for 5 min with TBST, and developed with Clarity Western ECL Substrate (Bio Rad) for subsequent imaging on a GE Healthcare Amersham 600 imager. Quantification of bands was performed in Image Lab, version 6.0 (Bio Rad), via manual band detection and automatic integration of volume. FtsZ values were adjusted with CdnL loading controls and normalized to the 0 h time point in each replicate before being plotted using Prism (GraphPad).

Supplementary Material

We would like to thank members of the Goley lab for helpful discussions throughout this work and feedback on the manuscript. We would also like to thank members of the Xiao lab, particularly Martin Yepes and Dr. Josh McCausland for their guidance with planning, carrying out, and performing analysis for FtsZ monomer lifetime measurements. Work in the Goley laboratory on Caulobacter was supported by the National Institutes of Health (grant numbers R35GM136221 [to E.D.G.] and T32GM007445 [training grant support of and J.M.B., B.K.T.F., I.P.P., and E.S.]).

Abbreviations used:

eGFP enhanced green fluorescent protein

mChy mCherry

mNG mNeonGreen

PG peptidoglycan

TEM transmission electron microscopy

TIRF total internal reflection fluorescence

Reviewer Report

This article was published online ahead of print in MBoC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E23-09-0365) on May 17, 2024.
==== Refs
References

Barrows JMGoley ED (2021). FtsZ dynamics in bacterial division: What, how, and why? Curr Opin Cell Biol 68 , 163–172.33220539
Barrows JMGoley ED (2023). Synchronized swarmers and sticky stalks: Caulobacter crescentus as a model for bacterial cell biology. J Bacteriol 205 , e0038422.36715542
Barrows JMSundararajan KBhargava AGoley ED (2020). FtsA regulates Z-ring morphology and cell wall metabolism in an FtsZ C-terminal linker-dependent manner in Caulobacter crescentus. J Bacteriol 202 , 1–20.
Bisson-Filho AWHsu YPSquyres GRKuru EWu FJukes CSun YDekker CHolden SVanNieuwenhze MS, et al. (2017). Treadmilling by FtsZ filaments drives peptidoglycan synthesis and bacterial cell division. Science (1979) 355 , 739–743.28209898
Coltharp CBuss JPlumer TMXiao J (2016). Defining the rate-limiting processes of bacterial cytokinesis. Proc Natl Acad Sci USA 113 , E1044–E1053.26831086
Corrales-Guerrero LSteinchen WRamm BMücksch JRosum JRefes YHeimerl TBange GSchwille PThanbichler M (2022). MipZ caps the plus-end of FtsZ polymers to promote their rapid disassembly. Proc Natl Acad Sci USA 119 .
Ducret AQuardokus EMBrun YV (2016). MicrobeJ, a tool for high throughput bacterial cell detection and quantitative analysis. Nat Microbiol 1 , 16077.27572972
Evinger MAgabian N (1977). Envelope-associated nucleoid from Caulobacter crescentus stalked and swarmer cells. J Bacteriol 132 , 294–301.334726
Goley EDDye NAWerner JNGitai ZShapiro L (2010). Imaging-based identification of a critical regulator of FtsZ protofilament curvature in Caulobacter. Mol Cell 39 , 975–987.20864042
Hale CADe Boer PAJ (1997). Direct binding of FtsZ to ZipA, an essential component of the septal ring structure that mediates cell division in E. coli. Cell 88 , 175–185.9008158
Lariviere PJSzwedziak PMahone CRLöwe JGoley ED (2018). FzlA, an essential regulator of FtsZ filament curvature, controls constriction rate during Caulobacter division. Mol Microbiol 107 , 180–197.29119622
Li ZTrimble MJBrun YVJensen GJ (2007). The structure of FtsZ filaments in vivo suggests a force-generating role in cell division. EMBO J 26 , 4694–4708.17948052
Mahone CRGoley ED (2020). Bacterial cell division at a glance. J Cell Sci 133 , 1–7.
Mahone CRPayne IPLyu ZMcCausland JWBarrows JMXiao JYang XGoley ED (2024). Integration of cell wall synthesis and chromosome segregation during cell division in Caulobacter. J Cell Biol 223 , e202211026.38015166
McCausland JWYang XSquyres GRLyu ZBruce KELamanna MMSöderström BGarner ECWinkler MEXiao J, et al. (2021). Treadmilling FtsZ polymers drive the directional movement of sPG-synthesis enzymes via a Brownian ratchet mechanism. Nat Commun 12 , 609.33504807
Meier ELRazavi SInoue TGoley ED (2016). A novel membrane anchor for FtsZ is linked to cell wall hydrolysis in Caulobacter crescentus. Mol Microbiol 101 , 265–280.27028265
Meier ELDaitch AKYao QBhargava AJensen GJGoley ED (2017). FtsEX-mediated regulation of the final stages of cell division reveals morphogenetic plasticity in Caulobacter crescentus. PLoS Genet 13 , e1006999.28886022
Modell JWHopkins ACLaub MT (2011). A DNA damage checkpoint in Caulobacter crescentus inhibits cell division through a direct interaction with FtsW. Genes Dev 25 , 1328–1343.21685367
Monteiro JMPereira ARReichmann NTSaraiva BMFernandes PBVeiga HTavares ACSantos MFerreira MTMacário V, et al. (2018). Peptidoglycan synthesis drives an FtsZ-treadmilling-independent step of cytokinesis. Nature 554 , 528–532.29443967
Ovesný MKřížek PBorkovec JŠvindrych ZHagen GM (2014). ThunderSTORM: A comprehensive ImageJ plug-in for PALM and STORM data analysis and super-resolution imaging. Bioinformatics 30 , 2389–2390.24771516
Perez AJCesbron YShaw SLVillicana JBTsui H-CTBoersma MJYe ZATovpeko YDekker CHolden S, et al. (2019). Movement dynamics of divisome proteins and PBP2x:FtsW in cells of Streptococcus pneumoniae. Proc Natl Acad Sci USA 116 , 3211–3220.30718427
RayChaudhuri DPark JT (1992). Escherichia coli cell-division gene ftsZ encodes a novel GTP-binding protein. Nature 359 , 251–254.1528267
Schindelin JArganda-Carreras IFrise EKaynig VLongair MPietzsch TPreibisch SRueden CSaalfeld SSchmid B, et al. (2012). Fiji: An open-source platform for biological-image analysis. Nat Methods 9 , 676–682.22743772
Schrader JMShapiro L (2015). Synchronization of Caulobacter crescentus for Investigation of the bacterial cell cycle. J Vis Exp 2015 , 52633.
Squyres GRHolmes MJBarger SRPennycook BRRyan JYan VTGarner EC (2021). Single-molecule imaging reveals that Z-ring condensation is essential for cell division in Bacillus subtilis. Nat Microbiol 6 , 553–562.33737746
Stricker JErickson HP (2003). In vivo characterization of Escherichia coli ftsZ mutants: Effects on Z-ring structure and function. J Bacteriol 185 , 4796–4805.12896999
Sundararajan KGoley ED (2017). The intrinsically disordered C-terminal linker of FtsZ regulates protofilament dynamics and superstructure in vitro. J Biol Chem 292 , 20509–20527.29089389
Sundararajan KMiguel ADesmarais SMMeier ELHuang KCGoley ED (2015). The bacterial tubulin FtsZ requires its intrinsically disordered linker to direct robust cell wall construction. Nat Commun 6 , 7281.26099469
Sundararajan KVecchiarelli AMizuuchi KGoley ED (2018). Species- and C-terminal linker-dependent variations in the dynamic behavior of FtsZ on membranes in vitro. Mol Microbiol 110 , 47–63.30010220
Thanbichler MShapiro L (2006). MipZ, a spatial regulator coordinating chromosome segregation with cell division in Caulobacter. Cell 126 , 147–162.16839883
Wang LLutkenhaus J (1998). FtsK is an essential cell division protein that is localized to the septum and induced as part of the SOS response. Mol Microbiol 29 , 731–740.9723913
Wang SCEWest LShapiro L (2006). The bifunctional FtsK protein mediates chromosome partitioning and cell division in Caulobacter. J Bacteriol 188 , 1497–1508.16452433
Wang YJones BDBrun YV (2001). A set of ftsZ mutants blocked at different stages of cell division in Caulobacter. Mol Microbiol 40 , 347–360.11309118
Whitley KDJukes CTregidgo NKarinou EAlmada PCesbron YHenriques RDekker CHolden S (2021). FtsZ treadmilling is essential for Z-ring condensation and septal constriction initiation in Bacillus subtilis cell division. Nat Commun 12 , 2448.33907196
Woldemeskel SADaitch AKAlvarez LPanis GZeinert RGonzalez DSmith ECollier JChien PCava F, et al. (2020). The conserved transcriptional regulator CdnL is required for metabolic homeostasis and morphogenesis in Caulobacter. PLoS Genet 16 , e1008591.31961855
Woldemeskel SAMcQuillen RHessel AMXiao JGoley ED (2017). A conserved coiled-coil protein pair focuses the cytokinetic Z-ring in Caulobacter crescentus. Mol Microbiol 105 , 721–740.28613431
Yang XLyu ZMiguel AMcQuillen RHuang KCXiao J (2017). GTPase activity-coupled treadmilling of the bacterial tubulin FtsZ organizes septal cell wall synthesis. Science (1979) 355 , 744–747.28209899
Yang XMcQuillen RLyu ZPhillips-Mason PDe La Cruz AMcCausland JWLiang HDeMeester KESantiago CCGrimes CL, et al. (2021). A two-track model for the spatiotemporal coordination of bacterial septal cell wall synthesis revealed by single-molecule imaging of FtsW. Nat Microbiol 6 , 584–593.33495624
