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. 2025 Jan 2;36(1):ar10. doi: 10.1091/mbc.E24-07-0302

GpsB interacts with FtsZ in multiple species and may serve as an accessory Z-ring anchor

Dipanwita Bhattacharya a, Asher King a, Lily McKnight a, Pilar Horigian a, Prahathees J Eswara a,b,*
Editor: Erin Goleyc
PMCID: PMC11742113  PMID: 39602291

Abstract

Bacterial cytokinesis commences when a tubulin-like GTPase, FtsZ, forms a Z-ring to mark the division site. Synchronized movement of Z-ring filaments and peptidoglycan synthesis along the axis of division generates a division septum to separate the daughter cells. Thus, FtsZ needs to be linked to the peptidoglycan synthesis machinery. GpsB is a highly conserved protein among species of the Firmicutes phylum known to regulate peptidoglycan synthesis. Previously, we showed that Staphylococcus aureus GpsB directly binds to FtsZ by recognizing a signature sequence in its C-terminal tail (CTT) region. As the GpsB recognition sequence is also present in Bacillus subtilis, we speculated that GpsB may interact with FtsZ in this organism. Earlier reports revealed that disruption of gpsB and ftsA or gpsB and ezrA is deleterious. Given that both FtsA and EzrA also target the CTT of FtsZ for interaction, we hypothesized that in the absence of other FtsZ partners, GpsB-FtsZ interaction may become apparent. Our data confirm that is the case, and reveal that GpsB interacts with FtsZ in multiple species and stimulates the GTPase activity of the latter. Moreover, it appears that GpsB may serve as an accessory Z-ring anchor such as when FtsA, one of the main anchors, is absent.

  • The arrival of FtsZ and activation of cell wall synthesis are needed to build a division septum. GpsB directly interacts with FtsZ and regulates its polymerization kinetics in Staphylococcus aureus. However, whether this role of GpsB is conserved in other organisms is unknown.

  • In this report, the authors reveal that GpsB interacts with FtsZ in multiple species and could serve as a surrogate FtsZ anchor.

  • As the rapid rise in antibiotic resistance is threatening public health globally, knowledge of unique important cell division factors could be harnessed to develop new antibacterial therapeutics.

INTRODUCTION

Most bacteria divide by a process called binary fission in which the division of parental cell produces two nearly identical daughter cells (Adams and Errington, 2009; Eswara and Ramamurthi, 2017; Naha et al., 2023; Cameron and Margolin, 2024). Although various studies conducted in the past decades elucidated the mechanism of bacterial cell division in great detail, many factors remain elusive (Goley, 2013; Pinho et al., 2013; Mahone and Goley, 2020). One of the most studied cell division proteins is FtsZ, a tubulin-like GTPase that marks the site of cell division (Haeusser and Margolin, 2016; Du and Lutkenhaus, 2019; Barrows and Goley, 2021). FtsZ is conserved in almost all bacteria and archaea (Du and Lutkenhaus, 2019; Ithurbide et al., 2022), and in eukaryotic organelles mitochondria and chloroplasts (Miyagishima et al., 2004; Kraus et al., 2021). The assembly of FtsZ into FtsZ ring (Z-ring) is spatiotemporally regulated by certain proteins that help tether FtsZ to the cell membrane (Naha et al., 2023). These Z-ring anchors play a vital role in facilitating the cell division process.

GpsB is a Firmicutes-specific protein that has been shown to interact with a variety of proteins involved in cell wall synthesis (Halbedel and Lewis, 2019; Hammond et al., 2019). Our group previously showed that Staphylococcus aureus GpsB is capable of directly interacting with FtsZ and stimulating its GTPase activity (Eswara et al., 2018). We also showed that GpsB interacts with FtsZ through a conserved R-X-X-R recognition sequence (S-R-R-T-R-R) located in the C-terminal tail (CTT) region of FtsZ (Sacco et al., 2024). A positive interaction between FtsZ and GpsB has not been reported in other organisms (Halbedel and Lewis, 2019). However, we observed that R-X-X-R motif is present in the CTT region of other FtsZ homologues in Bacillus subtilis, Enterococcus faecalis, Listeria monocytogenes, but is absent in Streptococcus pneumoniae (Figure 1A). Based on this, we speculated that GpsB–FtsZ interaction may be conserved beyond S. aureus. The CTT region is a known hot spot for Z-ring anchors FtsA and SepF (Huang et al., 2013), as well as FtsZ regulator EzrA which becomes essential in the absence of SepF (Hamoen et al., 2006) (note: the role of EzrA in cell division is more complex, please see supplementary discussion in the Squyres et al., 2021 article). Thus, we wondered whether GpsB–FtsZ interaction or GpsB-related division phenotype is masked by the presence of other Z-ring anchors/regulators. Interestingly, it has been reported that deletion or depletion of either gpsB and ezrA or gpsB and ftsA is synthetic sick/lethal in B. subtilis (Claessen et al., 2008; Tavares et al., 2008). Therefore, it appears that GpsB is able to compensate for the lack of EzrA or FtsA and possibly serve as a surrogate Z-ring anchor and/or regulator.

FIGURE 1:

FIGURE 1:

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GpsB stimulates the GTPase activity of FtsZ in multiple species. (A) Cartoon representation of FtsZ domains. FtsZ anchoring proteins FtsA, SepF, EzrA, and GpsB (protein of interest in this study) interact with the C-terminal tail of FtsZ. The disordered linker connecting N-terminal GTP-binding domain and C-terminal tail region are shown in dotted lines. Multiple sequence alignment of the C-terminal end of FtsZ from representative species of Firmicutes phylum is provided. The GpsB recognition motif (R-X-X-R) is highlighted. (B–E) GTPase activity analyses of full-length and C-terminal truncated Bs FtsZ (B), Ef FtsZ (C), Lm FtsZ (D), and Sp FtsZ (E) in the absence and presence of GpsB. Error bars indicate SD and n = 4. ****, ** and * indicate p < 0.0001, 0.001, and 0.01, respectively.

In this study, we sought to investigate GpsB–FtsZ interaction in B. subtilis and other species. Biochemically, we were able to show that B. subtilis GpsB (Bs GpsB) induces the GTPase activity of B. subtilis FtsZ (Bs FtsZ) in a CTT-dependent manner, as shown previously for S. aureus GpsB/FtsZ pair (Eswara et al., 2018; Sacco et al., 2024). This was the case for E. faecalis and L. monocytogenes GpsB and FtsZ, however, the latter was not dependent on the R-X-X-R motif. To our surprise, we also detected a modest but reproducible stimulation of S. pneumoniae FtsZ GTPase activity in the presence of GpsB, even though the R-X-X-R sequence is absent in this organism. Thus, it is possible another interaction surface (or noncanonical interaction motif) may be present to facilitate GpsB–FtsZ interaction in L. monocytogenes and S. pneumoniae. Other partners of GpsB, such as B. subtilis EzrA and MreC (Claessen et al., 2008; Hammond et al., 2019; Halbedel and Lewis, 2019), do not have a R-X-X-R motif at their C-termini so the presence of this motif may not be strictly required for recognition.

It has been shown that deletion of gpsB in B. subtilis does not exhibit any cell division phenotype under normal growth conditions except in the absence of EzrA or FtsA (Claessen et al., 2008; Tavares et al., 2008). We posited that GpsB-dependent cell division phenotypes would become more prominent in the absence of other FtsZ partners such as EzrA, SepF, or FtsA (Naha et al., 2023); as well as in the absence of GpsB's known interaction partner PBP1 (Claessen et al., 2008; Cleverley et al., 2019). PBP1 is a major bifunctional (class A) transglycosylase-transpeptidase enzyme that is involved in cell division and cell elongation (Scheffers and Errington, 2004; Dion et al., 2019). Using fluorescence microscopy, we noticed that increased production of GpsB in otherwise wild-type (WT) cells does not lead to cell division inhibition. In contrast, cell elongation was observed in strains lacking EzrA or SepF. Using a temperature-sensitive ftsA (ftsA*) mutant (Karmazyn-Campelli et al., 1992), we show that cell elongation at nonpermissive temperature is suppressed by ectopic expression of gpsB. Furthermore, we provide evidence that in a ponA (which encodes PBP1) deletion background, increased production of GpsB leads to cell division inhibition suggesting PBP1 is an efficient inhibitor of excess GpsB. We also show that GpsB facilitates cell division, as increased cell lengths of ΔezrA ΔponA, ΔsepF ΔponA, and ftsA* ΔponA are greatly reduced upon overexpression of gpsB. Recent reports in S. aureus suggest that FtsZ treadmilling kinetics is unaffected in the absence of GpsB (Costa et al., 2024) and that GpsB plays a preferential role in peptidoglycan synthesis (Sutton et al., 2023; Costa et al., 2024). Our data show that the GpsB phenotype is more severe in the absence of another FtsZ anchor, EzrA, in S. aureus similar to our observation in B. subtilis. Therefore, we believe the role of GpsB as a FtsZ regulator is masked by the presence of other FtsZ partners. Thus, taken together, our data establish GpsB as a FtsZ partner in multiple organisms and possibly as an accessory Z-ring anchor in certain conditions.

RESULTS

GpsB stimulates the GTPase activity of FtsZ in multiple species

The CTT of FtsZ is the main interaction target for its partners. The mechanistic details of the interaction between FtsA (Yan et al., 2000; Szwedziak et al., 2012; Huang et al., 2013), EzrA (Haeusser et al., 2007; Singh et al., 2007), and SepF (Duman et al., 2013; Zhang et al., 2022) with CTT of FtsZ have been uncovered (Figure 1A). We recently showed that S. aureus GpsB also targets CTT of FtsZ for interaction by recognizing the R-X-X-R motif (Sacco et al., 2024). We also showed that S. aureus GpsB stimulates the GTPase activity of FtsZ (Eswara et al., 2018), in a manner dependent on the terminal R-X-X-R motif (Sacco et al., 2024). Furthermore, we provided evidence that the binding affinity between GpsB and FtsZ is lost upon truncation of the C-terminal 6 residues in FtsZ (Sacco et al., 2024). Upon generating a multiple sequence alignment of the CTT residues of different FtsZ homologues of representative bacterial species of Firmicutes phylum, we noticed the presence of R-X-X-R sequence in B. subtilis, E. faecalis, L. monocytogenes, but not in S. pneumoniae (Figure 1A).

To test whether GpsB-mediated stimulation of GTPase activity of FtsZ occurs in other species, we purified recombinant GpsB/FtsZ pairs of other organisms. We also tested the truncated FtsZ versions lacking the RXXR sequence. As shown in Figure 1B, coincubation of B. subtilis GpsB (Bs GpsB) and B. subtilis FtsZ (Bs FtsZ) stimulated the GTPase activity by nearly 4-fold. This GpsB-dependent enhancement was absent upon removal of the C-terminal 6 residues in FtsZ (Bs FtsZΔC6), similar to what we reported for S. aureus GpsB/FtsZ pair (Sacco et al., 2024). GpsB alone did not hydrolyze GTP (Supplemental Figure S1A). Next, we tested E. faecalis GpsB/FtsZ proteins (Ef GpsB/Ef FtsZ). Our results reveal that Ef GpsB stimulates the GTPase activity of Ef FtsZ ∼ 3-fold, but not in the absence of R-X-X-R (Figure 1C). L. monocytogenes GpsB (Lm GpsB) also stimulated the L. monocytogenes FtsZ (Lm FtsZ) GTPase activity by nearly 2-fold (Figure 1D). However, this was not R-X-X-R dependent, as truncated FtsZ (Lm FtsZΔC5) by itself displayed a higher GTP hydrolysis rate and the addition of Lm GpsB increased the rate further by ∼1.5-fold. These data suggest that Lm GpsB interaction with Lm FtsZ is not solely reliant on the R-X-X-R motif. Finally, we tested the GTPase activity stimulation of S. pneumoniae FtsZ (Sp FtsZ), which naturally lacks R-X-X-R sequence, by S. pneumoniae GpsB (Sp GpsB). We observed a modest but statistically significant and reproducible increase in GTPase activity in the presence of Sp GpsB (Figure 1E). This result indicates that Sp FtsZ and Sp GpsB may also be partners but do not require R-X-X-R for interaction. Overall, this set of results indicates that the GpsB–FtsZ interaction we initially observed in S. aureus is also conserved in B. subtilis, E. faecalis, L. monocytogenes, and S. pneumoniae. However, the GpsB–FtsZ interaction in the latter two appears to be independent of the R-X-X-R recognition motif.

Binding of GpsB and FtsZ is widely conserved

Using the purified recombinant GpsB/FtsZ protein pairs, we conducted a fluorescence-based binding assay. Simply, we labeled FtsZ with fluorescein isothiocyanate (FITC) and monitored the change in fluorescence in the presence of equimolar GpsB. It has been previously shown that a change in fluorescence represents binding (Kuchibhatla et al., 2011). First, we monitored the background fluorescence spectra of FITC-labeled Bs FtsZ by itself (Figure 2, A and F). Next, we measured the fluorescence spectra of Bs FtsZ mixed with Bs GpsB and noted more than double the peak fluorescence intensity. On the contrary, fluorescence spectra of Bs FtsZΔC6 and Bs FtsZΔC6 + Bs GpsB did not show any measurable difference in the fluorescence intensity, suggesting R-X-X-R motif in Bs FtsZ is needed for binding. This trend is also seen in Sa FtsZ/Sa GpsB and Sa FtsZΔC6/Sa GpsB pairs (Figure 2, B and F), consistent with what we observed previously (Sacco et al., 2024). Further, to determine the binding efficiency, a fixed concentration of FITC-Bs FtsZ was incubated with increasing concentrations of GpsB. The fluorescence intensity of FITC labeled Bs FtsZ increased significantly upon incubating with increasing concentrations of Bs GpsB, indicating that Bs GpsB specifically interacts with Bs FtsZ (Supplemental Figure S2A). However, deletion of C-terminal 6 amino acid residues from Bs FtsZ abolished its interaction with Bs GpsB as no change in the fluorescence intensity of FITC-Bs FtsZΔC6 could be observed upon incubation with Bs GpsB (Supplemental Figure S2C). Using the change in the fluorescence of FITC-Bs FtsZ in the presence of Bs GpsB, the Kd for the binding of Bs GpsB with Bs FtsZ was determined to be 48 ± 2 µM (Supplemental Figure S2B). After similar analysis of Sa FtsZ/Sa GpsB pair, we established the Kd for their interaction to be 53.81 ± 8 µM (Supplemental Figure S2, D and E), which is comparable to the Kd value obtained by surface plasmon resonance previously (40 ± 2 µM) (Sacco et al., 2024), and is in comparable range to other Z-anchors (Mosyak et al., 2000; Szwedziak et al., 2012; Sogues et al., 2020). Again, we failed to see a robust concentration-dependent increase in fluorescence signal between Sa FtsZΔC6 and Sa GpsB (Supplemental Figure S2F). To ensure the specificity of this binding assay, we tested the interaction of FtsZ and an unrelated protein (bovine serum albumin; BSA), which did not result in an increase in fluorescence signal for any of the FtsZ versions tested (Supplemental Figure S2G).

FIGURE 2:

FIGURE 2:

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Binding of GpsB and FtsZ is widely conserved. The binding of GpsB to either full-length or C-terminal truncated FtsZ of Bs (A), Sa (B), Ef (C), Lm (D), and Sp (E) was monitored using fluorescence spectroscopy. The representative fluorescence spectra of full-length FITC-FtsZ (1 µM) in the absence (●) and presence (○) of GpsB (1 µM) were plotted against the wavelength. Similarly, the truncated version of FITC-FtsZ (1 µM) was incubated without (■) or with (□) GpsB, and the fluorescence spectra were monitored. (F) The average of maximum fluorescence intensities at 520 nm; n = 4 and **, *** and **** indicate p ∼ 0.0025, 0.0006, and < 0.0001, respectively.

We also investigated the interaction between the FtsZ/GpsB pairs of other species. We noticed a significant increase in fluorescence intensity between FITC-Ef FtsZ and Ef GpsB to a similar magnitude observed for B. subtilis and S. aureus proteins (Figure 2, C and F). This enhancement in fluorescence intensity was absent when Ef GpsB was incubated with FITC-Ef FtsZΔC4. This result is consistent with the GTPase assay results (Figure 1C). This suggests that the interaction between Ef FtsZ and Ef GpsB is mediated by the 4 C-terminal amino acids of Ef FtsZ. Next, we monitored the fluorescence intensity of FITC-Lm FtsZ and FITC-Lm FtsZΔC5 with or without Lm GpsB (Figure 2, D and F). We observed an increase in fluorescence intensity for FITC-Lm FtsZ and Lm GpsB consistent with other pairs. However, in striking contrast, we also noticed an increase in fluorescence intensity for FITC-Lm FtsZΔC5 and Lm GpsB. We noted previously that stimulation of GTPase activity was also seen when Lm FtsZΔC5 was incubated with Lm GpsB (Figure 1D), which is not the case for other species. This set of data reveals that in L. monocytogenes, the interaction between FtsZ and GpsB is conserved but independent of the signature GpsB recognition (R-X-X-R) motif located within the 5 C-terminal residues of Lm FtsZ. Finally, we investigated the fluorescence spectra of Sp GpsB incubated with FITC-Sp FtsZ, which lacks the R-X-X-R motif in the C-terminus, and noted a statistically significant increase in peak fluorescence intensity (Figure 2, E and F). This is consistent with the stimulation of GTPase activity of Sp FtsZ by Sp GpsB (Figure 1E). It is possible the terminal residues in Sp FtsZ (KNR) that weakly resemble R-X-X-R may mediate this interaction.

Taken together, the binding of GpsB to FtsZ through the R-X-X-R motif was confirmed in B. subtilis and E. faecalis using fluorescence assay. However, GpsB–FtsZ interaction in L. monocytogenes and S. pneumoniae, although present, appears to be independent of the R-X-X-R motif. Thus, the interaction between GpsB and FtsZ that we first observed in S. aureus (Sacco et al., 2024) is also present in multiple species.

The effects of GpsB are only apparent in the absence of known FtsZ partners

It is known that under standard growth conditions, there are no discernible gpsB phenotypes in B. subtilis (Claessen et al., 2008; Tavares et al., 2008). Based on our results confirming that GpsB interacts with FtsZ, we speculated that potential gpsB phenotypes are masked by the presence of redundant FtsZ anchors/partners such as SepF, FtsA, and EzrA that all target the same binding site - the CTT region on FtsZ (Figure 1A). Therefore, we hypothesized that gpsB phenotypes will become more apparent in the absence of other FtsZ targeting proteins. To test this, we constructed a B. subtilis strain harboring an inducible copy of gpsB at an ectopic locus and investigated the gpsB overexpression phenotype in ΔsepF and ΔezrA strains. We also tested this in a temperature-sensitive ftsA mutant (ftsA*; FtsAS9N) (Karmazyn-Campelli et al., 1992) background grown at nonpermissive temperature, as deletion of ftsA in B. subtilis leads to severe/lethal filamentation (Beall and Lutkenhaus, 1992; Jensen et al., 2005; Duman et al., 2013). Overexpression of gpsB by itself did not have any noticeable effect on cell length (Figure 3, A and B; compare the cell lengths in the absence and presence of inducer). Although ΔsepF or ΔezrA cells are longer in general, overexpression of gpsB in these strain backgrounds leads to a further increase in cell length suggesting that excess GpsB is negatively affecting FtsZ ring assembly. Similar cell elongation was observed when ezrA or sepF was overexpressed (Haeusser et al., 2004; Gao et al., 2017; Squyres et al., 2021). In contrast, we see that the ectopic expression of gpsB rescues cell division when ftsA* cells are grown at a nonpermissive temperature which suggests that GpsB can step in as a Z-anchor when FtsA is nonfunctional (Supplemental Figure S3, A and B). We also tested whether GpsB overproduction would have any effect on cells lacking ZapA, which does not interact with the C-terminal tail region of FtsZ (Gueiros-Filho and Losick, 2002; Gao et al., 2017), and thus not compete with GpsB. As shown in Supplemental Figure S3C, overexpression of gpsB in ΔzapA strain background resulted in a modest but statistically significant cell length increase. In addition, we investigated the gpsB phenotypes in ΔfacZ background, as FacZ also has a role in proper Z-ring assembly (Bartlett et al., 2024). Although ΔfacZ cells are longer as previously reported, our results reveal that deletion of gpsB or overexpression of gpsB does not affect cell length further (Supplemental Figure S3D). These data underscore a possible regulatory role of GpsB in B. subtilis cell division in the absence of the other known FtsZ partners.

FIGURE 3:

FIGURE 3:

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GpsB overproduction worsens the cell elongation phenotypes of strains lacking SepF or EzrA. (A) The effect of gpsB overexpression in B. subtilis in otherwise WT (GG18), ΔsepF (BDB12), and ΔezrA (BDB1) backgrounds. Micrographs of cells grown in the absence (left) or presence (right) of the inducer are shown. The cell membrane was stained with synapto-red. The scale bar is 1 µm. (B) Quantification of the cell lengths of B. subtilis strains shown in A. **** indicates p < 0.0001; n = 100.

GpsB serves as a FtsZ regulator in the absence of its partner PBP1

GpsB is known to interact with penicillin-binding proteins (PBPs) in B. subtilis, S. aureus, and other organisms by recognizing the R-X-X-R motif (Cleverley et al., 2019; Hammond et al., 2019; Halbedel and Lewis, 2019; Sacco et al., 2024). Therefore, we hypothesized that in the absence of ponA (which encodes GpsB interaction partner, PBP1), GpsB will be more likely to interact with FtsZ as both PBP1 and FtsZ interact with GpsB through their R-X-X-R motifs. In affirmation of our speculation, overexpression of gpsB in ΔponA background leads to cell length increase (Figure 4, A and B), which is not the case in the absence of an inducer. Next, we wanted to probe whether GpsB can serve as a FtsZ regulator in the absence of PBP1. To test this, we investigated the effects of gpsB overexpression in the absence of PBP1 and one of the known FtsZ partners. In the absence of gpsB induction, deletion of ponA and either sepF or ezrA lead to increased cell lengths. Interestingly, in the presence of inducer, the cell lengths of ΔponA ΔsepF and ΔponA ΔezrA were greatly reduced (Figure 4, A and B). Similarly, cell division inhibition in ΔponA ftsA* grown at nonpermissive temperature was also abrogated by overexpression of gpsB (Supplemental Figure S4, A and B). Overall, these results suggest that GpsB can in fact facilitate cell division by possibly serving as FtsZ regulator in the absence of other partners of GpsB or FtsZ.

FIGURE 4:

FIGURE 4:

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GpsB overproduction phenotypes in ΔponA and upon additional deletion of ΔsepF or ΔezrA. (A) Micrographs of gpsB overexpression strain (GG18), and gpsB overexpression in ΔponA (BDB20), ΔponA ΔsepF (BDB18), or ΔponA ΔezrA (BDB22) strain backgrounds. Cells were grown in the absence (left) or presence (right) of the inducer. The cell membrane was stained with synapto-red. The scale bar is 1 µm. (B) Cell length quantification of strains shown in A. ** and **** indicate p < 0.001 and p < 0.0001, respectively; n = 100. (C) Simplified interaction network of GpsB and Z-anchors. (D) Tug-of-war model of GpsB roles. A well-characterized role of GpsB is in regulating cell wall synthesis (thick arrow) which is executed through partners such as PBP1, MreC, and RodZ. The function of GpsB in facilitating Z-ring assembly is minor due to the presence of redundant FtsZ anchors and binding site competition for the FtsZ C-terminal tail. However, the ability of GpsB to anchor FtsZ becomes apparent in the absence of one of the major Z-ring anchors or PBP1. Other GpsB binding partners that may be involved in these processes are also listed. Proteins that are not determined to be direct partners are listed within parentheses. The proteins shown in green are involved in sporulation.

Based on our results, we wondered whether GpsB facilitated cell division in ftsA*, ΔsepF, and ΔponA strains. To test this, we deleted gpsB in these strain backgrounds. We noticed that the cells lengths of ΔsepF ΔgpsB and ΔponA ΔgpsB were significantly longer than single deletions (Supplemental Figure S4C). This suggested that indeed GpsB is capable of promoting cell division in these backgrounds. However, we did not notice any difference in cell lengths between ftsA* and ftsA* ΔgpsB strains. We suspect that in ftsA*, although nonfunctional, FtsA is still present and thus could occupy and occlude the binding site needed for GpsB to help anchor FtsZ. It has been reported that GpsB becomes important when FtsA is depleted (Tavares et al., 2008), we confirmed this result independently by showing that ΔftsA ΔgpsB cells are nonviable (Supplemental Figure S4E). We were unable to generate a ΔezrA ΔgpsB strain without an inducible copy of gpsB, consistent with previously reported synthetic sick/lethal phenotype (Claessen et al., 2008). Therefore, we analyzed the cell lengths of this strain in the absence or presence of inducer. In the absence of inducer, the culture grew poorly, and the cells were long (Supplemental Figure S4D). However, in the presence of inducer, cell length decreased. Again, this result confirmed that in cells lacking ezrA, presence of GpsB is critically needed to facilitate cell division. Overall, our results reveal that GpsB is capable of mediating efficient cell division in the absence of other FtsZ anchors/regulators or the well-characterized interaction partner of GpsB, PBP1 (Cleverley et al., 2019).

S. aureus cells lacking ezrA display severe GpsB-dependent cell division phenotype

It has been shown that the division site localization of GpsB depends on EzrA in S. aureus (Steele et al., 2011). Evidence of direct interaction between GpsB and septum-localized peptidoglycan (PG) synthesis machinery proteins such as PBP2 and PBP4 are also available (Barbuti et al., 2023; Costa et al., 2024; Sacco et al., 2024). Recent reports suggest that the predominant role of GpsB is in maintaining normal S. aureus cell morphology by spatially restricting PBP2 and PBP4 enzymes at the division site for septal PG synthesis (Sutton et al., 2023; Costa et al., 2024). Therefore, we speculated that in the absence of EzrA, excess GpsB will lead to increased peripheral PG synthesis. As predicted, we see that cells overexpressing gpsB in a ΔezrA strain background were significantly enlarged in comparison to the cells overexpressing gpsB in an otherwise WT background (Figure 5, A and B). We infer our results to mean that in the absence of EzrA, GpsB is unable to remain at the division site and promote septum-specific PG synthesis. Thus, EzrA is a spatial regulator of GpsB. Alternatively, it is possible the GpsB overexpression phenotype in ΔezrA background is due to the lack of EzrA-mediated regulation of FtsZ. Recently, Bartlett and colleagues showed that ezrA and a new cell division gene facZ are synthetically essential, but ezrA and gpsB are not (Bartlett et al., 2024). They further showed that ΔfacZ cells are enlarged only in the presence of GpsB and that FacZ is needed for proper localization of GpsB. Thus, it appears that both EzrA and FacZ are redundant GpsB positioning factors. Based on our results and recent reports from other groups (Sutton et al., 2023; Costa et al., 2024; Bartlett et al., 2024), it appears that the major role of GpsB in S. aureus is to restrict PBP2 and PBP4-mediated PG synthesis to division septa. As our data clearly show Sa FtsZ–Sa GpsB interaction (Figures 1 and 2) (Eswara et al., 2018; Sacco et al., 2024), it is possible that GpsB's regulatory role becomes important in the absence of other FtsZ anchors such as FtsA/SepF and/or PG synthesis enzymes PBP2/PBP4 or under nonstandard growth/stress conditions. However, this remains to be tested.

FIGURE 5:

FIGURE 5:

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GpsB overexpression in S. aureus cells lacking ezrA leads to increased cell diameter typical of cell division inhibition. (A) The micrographs of S. aureus WT cells harboring empty vector (EV; PE355), vector with IPTG-inducible gpsB (PE356), and ΔezrA cells harboring EV (LM121) or inducible gpsB (LM122) are shown. The cells were grown in the absence (left) and presence (right) of the inducer. (B) Cell diameter quantification of the strains shown in A. **** indicates p < 0.0001; n = 100. (C) Simplified interaction network of Z-anchors in S. aureus.

DISCUSSION

In this report, we provide evidence showing that GpsB-FtsZ pairs of B. subtilis and E. faecalis require the presence of C-terminal R-X-X-R recognition sequence in FtsZ for interaction and stimulation of GTPase activity, which is in line with what we previously reported for S. aureus GpsB–FtsZ interaction (Sacco et al., 2024). In contrast, we find that the interaction between GpsB and FtsZ is independent of the terminal R-X-X-R motif in L. monocytogenes. This is the case for S. pneumoniae which naturally lacks the terminal GpsB recognition motif. However, we cannot rule out the possibility that interaction is mediated by a motif similar to R-X-X-R located in other parts of FtsZ. Overall, our data suggest that GpsB–FtsZ interaction is likely broadly conserved in Firmicutes.

To further validate the physiological relevance of our in vitro results, we focused on FtsZ and GpsB in B. subtilis. In this bacterium, it was previously reported that simultaneous deletion/depletion of gpsB with either ezrA or ftsA is synthetically sick/lethal (Claessen et al., 2008; Tavares et al., 2008). In addition, the enrichment of GpsB at division sites has been noted previously (Claessen et al., 2008; Tavares et al., 2008; Hammond et al., 2022). We find that although overexpression of gpsB by itself does not reveal any noticeable phenotype, we see increased cell lengths (cell division inhibition) when gpsB is overexpressed in the absence of a functional copy of known FtsZ anchors/regulators SepF or EzrA. In contrast, we see that GpsB facilitated division in cells lacking functional FtsA. These results suggested that robust GpsB–FtsZ interaction likely happens only in the absence of other FtsZ partners due to competition for the same binding site. We also observed that the overproduction of GpsB in cells lacking its other interaction partner, PBP1 (which competes with FtsZ using its own R-X-X-R motif [Cleverley et al., 2019]), resulted in cell elongation. This suggests that PBP1 is capable of sequestering excess GpsB away from FtsZ. Upon inspecting the cells lacking ponA and one of the known FtsZ anchors/regulators, we noticed that these cells are longer in the absence of an inducer. However, upon GpsB overproduction cell division was restored, and cell elongation was significantly reduced. These data suggested that GpsB is capable of serving as FtsZ anchor in the absence of its partner PBP1 and any one of the known FtsZ interaction partners. It has been shown that SepF, a peripheral membrane protein, can anchor FtsZ (solely in certain genetic conditions [Gulsoy et al., 2024]), and cells lacking SepF and either FtsA or EzrA are nonviable (Duman et al., 2013; White and Eswara, 2021). Overproduction of SepF rescues the cell division inhibition seen in cells lacking FtsA (Ishikawa et al., 2006). Thus, it is conceivable that in the absence of other factors perhaps GpsB also aids in anchoring FtsZ, as overexpression of gpsB restores cell division in the absence of functional FtsA. In support of this notion, we find that cells lacking ezrA, sepF, or ponA are longer and absence of GpsB in these strain backgrounds lead to severe cell division inhibition in the case of ΔezrA and mild but significant cell elongation in the latter two. Consistent with the published finding (Tavares et al., 2008), we note that GpsB becomes essential in the absence of FtsA. Therefore, we believe that GpsB is an accessory but a bona fide FtsZ anchor. However, further experiments are needed to confirm this.

PBP1 plays a major role in peripheral PG synthesis, as deleting ponA restores some of the abnormal cell width phenotypes (Kawai et al., 2009; Dion et al., 2019; Sassine et al., 2020). Besides PBP1 and EzrA, other interaction partners of GpsB include MreC, RodZ, YpbE, and PatA (DapX) (Claessen et al., 2008; Cleverley et al., 2019; O'Reilly et al., 2023). While the first two have clear roles in peripheral cell wall synthesis (Sun et al., 2023), the functions of the latter two are less clear. After FtsZ protofilaments arrive at the potential division site, the ring-like structure is further condensed by FtsZ bundling proteins to initiate septal PG synthesis (Squyres et al., 2021; Whitley et al., 2021). We identified that another divisome protein, FtsL, is also critical for FtsZ condensation (White et al., 2022). FtsL controls the functionality of main septal PG synthase machinery made of FtsW and PBP2B (transglycosylase and transpeptidase pair) (Daniel et al., 2006; Morales Angeles et al., 2020; White et al., 2022). It was shown that the effects of ezrA deletion can be countered by ftsL overexpression (Kawai and Ogasawara, 2006). In fact, FtsL is less stable in cells lacking EzrA (Gamba et al., 2015). It is known that EzrA and GpsB directly interact with bifunctional PBP1 (Claessen et al., 2008; Gamba et al., 2009; Cleverley et al., 2019). PBP1 is also a part of the division machinery and is enriched at division sites (Scheffers and Errington, 2004; Scheffers et al., 2004). It appears that B. subtilis cells grown in different media conditions may require PBP1 for proper cell division (Pedersen et al., 1999). It is also to be noted that cell envelopes of Gram-positive monoderm organisms such as B. subtilis are composed of two distinct layers of PG (Matias and Beveridge, 2005; Pasquina-Lemonche et al., 2020; Straume et al., 2021). Thus, cell division in B. subtilis utilizes both FtsW/PBP2B as well as PBP1 for efficient cell division. Interestingly, in another member of the Firmicutes phylum Clostridioides difficile bifunctional class A PBP (PBP1) is the main driver of septal PG synthesis during vegetative cell division (Shrestha et al., 2023). In C. difficile, which does not encode gpsB, Z-ring anchors must therefore connect FtsZ to class A PBP1. Bacterial species of other phyla, such as Escherichia coli and Acinetobacter baumannii, also utilize class A PBP for efficient cell division (Gray et al., 2015; Kang et al., 2021).

It has been reported that PBP1 localizes to asymmetric cell division sites during sporulation and is required for efficient septation (Scheffers and Errington, 2004). Evidence showing that GpsB localizes to asymmetric division sites and interacts with another sporulation-specific FtsZ interaction partner, SpoIIE, exists (Muchova et al., 2020; Tavares et al., 2008). SpoIIE also interacts with PBP1 and EzrA among others. Thus, it is possible GpsB help anchor/regulate FtsZ for efficient asymmetric cell division during sporulation. It has been reported that GpsB also interacts with YrrS and PBP4B (yrrR/pbpI) which are produced during sporulation (Cleverley et al., 2019). During vegetative growth, a paralog of GpsB, DivIVA, that controls an FtsZ positioning (Min) system in B. subtilis (Eswaramoorthy et al., 2011; Eswara and Ramamurthi, 2017), is also an interaction partner of GpsB (Pompeo et al., 2015). A newly identified factor FacZ, which is found to interact with GpsB in S. aureus, appears to also play a role in Z-ring positioning in B. subtilis (Bartlett et al., 2024). Taking all of this into account, we propose a tug-of-war model where the role of GpsB in promoting cell wall synthesis and FtsZ ring assembly is influenced by its interaction partners or other factors that compete with GpsB for binding its substrate proteins (Figure 4D).

Previously, we showed that the overproduction of S. aureus GpsB in B. subtilis is lethal due to cell division inhibition (Eswara et al., 2018; Sacco et al., 2024). Furthermore, spontaneous suppressor mutations that tolerated lethal Sa GpsB overproduction mapped to tagG or tagH genes that encode wall teichoic acids exporter complex (Hammond et al., 2022). Here we show that Sa GpsB also utilizes R-X-X-R of B. subtilis FtsZ for recognition (Supplemental Figure S1). We believe that the major source of Sa GpsB toxicity is due to direct interaction with Bs FtsZ, that TagG/TagH mutants circumvent by an unknown mechanism. We suspect that we did not isolate any suppressors harboring mutations in Bs FtsZ as the interaction surface is vital for other FtsZ interaction partners.

Recent reports indicate that S. aureus GpsB plays a predominant role in the spatiotemporal regulation of peptidoglycan synthesis (Sutton et al., 2023; Costa et al., 2024) and that there is no effect on the kinetics of FtsZ ring assembly and constriction (Costa et al., 2024). As EzrA and FacZ form a synthetic-lethal pair and are known to affect GpsB localization (Steele et al., 2011; Bartlett et al., 2024), it could be argued that EzrA and FacZ play a redundant role in restricting GpsB to division sites. Additionally, other regulatory interaction partners of EzrA such as CozEa and CozEb are also present in S. aureus (Stamsas et al., 2018). The PBPs, PBP2 and PBP4 are interaction partners of GpsB (Costa et al., 2024; Sacco et al., 2024), and are normally enriched at sites of cell division (Barbuti et al., 2023). However, it appears that in the absence of GpsB, PBP2, and PBP4 are no longer enriched at division sites, and they synthesize PG throughout the cell periphery instead (Costa et al., 2024). Conversely, overproduction of GpsB leads to increased cell diameter (Eswara et al., 2018). In this report, we find that this phenotype is exacerbated in the absence of EzrA. Using super-resolution microscopy, we previously showed that GpsB localizes to the leading edge of the invaginating membrane during cell division (Eswara et al., 2018), almost identical to what would be expected of FtsZ during Z-ring constriction. We also uncovered that GpsB is highly capable of directly interacting with FtsZ and stimulating its GTPase activity (Eswara et al., 2018; Sacco et al., 2024). Thus in S. aureus, GpsB is capable of both (i) stimulating FtsZ ring remodeling and (ii) promoting septal peptidoglycan synthesis needed for the construction of division septum. However, due to redundant well-established Z-ring regulators/anchors such as EzrA, SepF, FtsA, and newly uncovered cell division factors such as FacZ (Bartlett et al., 2024) and PcdA (Ramos-Leon et al., 2024), the roles of GpsB in cell division is eclipsed at least under standard laboratory conditions.

Similar to its homologue in other organisms, L. monocytogenes GpsB is also targeted to division sites (Rismondo et al., 2016). GpsB of this species interacts with FtsZ anchors SepF and EzrA, as well as several other divisome proteins (Cleverley et al., 2019). L. monocytogenes GpsB is uniquely essential when grown at higher temperature (42°C), and suppressors have been mapped to pathways that enhance the PG precursor pool (Rismondo et al., 2017). As GpsB interacts with class A PBP A1 (Rismondo et al., 2016; Cleverley et al., 2019), it is speculated that in the absence of GpsB, PBP A1 consumes PG precursors for PG synthesis and that increase in PG precursors therefore restores cell division (Rismondo et al., 2017). However, as PBP A1 is required for septal cell wall synthesis (Rismondo et al., 2017), it can be hypothesized that in the absence of GpsB at 42°C, PBP A1 is dysregulated and functions outside of the divisome complex. Therefore, perhaps GpsB directly (Figures 1 and 2) and/or indirectly via interaction with SepF and EzrA coordinates Z-ring remodeling with the action of PBP A1.

The role of GpsB in septal peptidoglycan synthesis has also been established in S. pneumoniae, where it is essential in certain strain backgrounds (Fleurie et al., 2014; Rued et al., 2017). It was shown that GpsB localizes to division sites, and lack of GpsB leads to increased cell length due to aberrant Z-ring assembly (Fleurie et al., 2014). It was proposed that the main role of GpsB is to facilitate septal PG synthesis and restrict peripheral PG synthesis of the elongasome machinery (Fleurie et al., 2014; Rued et al., 2017). Thus, it would not be surprising if GpsB was able to interact with FtsZ to execute its role in enabling septal PG synthesis. However, although GpsB and FtsZ are found to be in a complex via co-immunoprecipitation assay (Rued et al., 2017), the interaction was absent when tested by bacterial two-hybrid analysis (Fleurie et al., 2014; Rued et al., 2017). In this organism, GpsB partner and FtsZ regulator, EzrA, is essential (Perez et al., 2021). Nevertheless, given that EzrA localization depends on GpsB (Fleurie et al., 2014), it is possible that GpsB–FtsZ interaction may be needed for the efficient recruitment of EzrA to the division site and to facilitate septal PG synthesis. FtsA is also essential in S. pneumoniae, and the aberrantly long cell morphology of cells lacking SepF or GpsB can be rescued by increased levels of FtsA (Mura et al., 2017). Therefore, it is conceivable there may be a role for GpsB, albeit minor, in anchoring FtsZ if our biochemical data shown in Figures 1 and 2 were to hold true when tested by other methods.

Although the precise function of enterococcal GpsB remains to be understood, it has been shown that cells lacking gpsB display cell separation defects and are highly susceptible to cell walls targeting antibiotics (Minton et al., 2022). Interestingly, deletion of ser/thr kinase IreK also leads to increased sensitivity to antibiotics, and it was shown that the phosphoablative mutant of GpsB is unable to fully complement ΔgpsB phenotype. Thus, it appears the posttranslational regulation of GpsB is important for antibiotic resistance. In fact, GpsB of other species are phosphorylated and/or GpsB stimulates the kinase activity of the corresponding ser/thr kinase (Junker et al., 2018; Halbedel and Lewis, 2019; Hammond et al., 2019; Wamp et al., 2020; Kelliher et al., 2021; Ulrych et al., 2021; Minton et al., 2022; Mikkat et al., 2024). Thus, it would be interesting to study whether the binding of GpsB and its interaction partners is affected by phosphorylation.

In summary, we show that the interaction between FtsZ and GpsB is conserved in other organisms and may not strictly require the R-X-X-R sequence for recognition. As cell division is an essential process, multiple redundant mechanisms are in place to ensure the faithful generation of progenies. Our data reveal that GpsB is a widely conserved FtsZ interaction partner which may also serve as the latter's membrane anchor. However, its significance is only apparent in the absence of one of the other FtsZ anchors/regulators or PBP1. The importance of FtsZ–GpsB interaction in E. faecalis, L. monocytogenes, S. pneumoniae, and possibly other organisms remains to be elucidated.

MATERIALS AND METHODS

Strain construction

All B. subtilis strains used in this study are derivatives of PY79 (Youngman et al., 1984). The strain details are provided in Supplemental Table S1. To generate B. subtilis strains BDB1, BDB12, BDB13, plasmid pGG5 (Eswara et al., 2018) harboring gpsB under IPTG-inducible promoter (gpsB+) was used to clone into the amyE locus of the parent strains GG17, RB73, and MW393 lacking ezrA, sepF, and harboring temperature-sensitive ftsAS9N, respectively. The recombination was confirmed via amyE screening. To generate strains lacking ponA in the above-mentioned strain backgrounds, the genomic DNA was isolated from strain PE719 and was transformed into BDB1, BDB12, and BDB13 resulting in strains BDB22, BDB18, and BDB24 respectively. In addition, ΔponA gpsB+ strain was constructed by transforming GG18 with the chromosomal DNA from PE482 resulting in strain BDB20. The genomic DNA of GG13 was used to transform into GG18 resulting in strain BDB38. To generate ΔzapA strain, the PY79 strain was transformed with the chromosomal DNA of RL2647 (Gueiros-Filho and Losick, 2002) to generate strain BDB53. The resultant strain was transformed with plasmid pGG5 to construct BDB55. To generate double deletion strains, the chromosomal DNA of GG13 was used to transform strains RB73 and MW393 resulting in strains BDB31 and BDB34, respectively. As ΔponA strain is noncompetent, we used PE482 chromosomal DNA to transform GG13 to generate BDB41. We were unable to generate ΔezrA ΔgpsB strain without the inducible copy of gpsB present, to generate BDB36 we used the chromosomal DNA of GG17 to transform BDB38. To create ΔfacZ strains, the chromosomal DNA of BKE29780 (Bacillus Genetic Stock Center) was transformed into PY79, GG13, and GG18 to create strains LM138, LM139, and LM140, respectively.

For recombinant protein expression and purification, pET28a plasmid was used to clone the ftsZ and gpsB genes. The chromosomal DNA from PY79, L. monocytogenes EGDe (ATCC strain), E. faecalis 29212 (ATCC strain), and S. pneumoniae D39 (chromosomal DNA; gift – Nicholas De Lay lab) were used as templates. The amplified products were ligated to the pET28a vector between NdeI and XhoI restriction sites for ftsZ and XbaI and BamHI sites for gpsB. The final cloned plasmids (from DH5a) were transformed into BL21-DE3 cells for recombinant protein purification. The strains generated and the oligonucleotides used in this study are provided in Supplemental Tables S1 and S2. Purified FtsZ and GpsB proteins cloned from different organisms mentioned were used for the biochemical experiments performed in this study.

Media and growth condition for microscopy

B. subtilis cultures were grown overnight at 30°C in LB medium. Then the cultures were diluted to 1:10 in fresh LB and allowed to grow at 37°C until the mid-log phase (OD600 ∼ 0.6–0.8) following which, the cultures were standardized to OD600 ∼ 0.1 in fresh LB medium. IPTG was used at 1 mM final concentration at OD600 ∼ 0.1 to induce the expression of genes under IPTG-inducible promoter. The cultures were grown for an additional 2 h at 37°C.

Recombinant protein purification

BL21-DE3 overexpressing recombinant full-length and truncated ftsZ were grown until the OD600 reached 0.8. Then the cells were induced using IPTG (1 mM for ftsZ constructs and 0.5 mM for gpsB constructs) and were further allowed to grow at 30°C for an additional 6 h. Subsequently, the cells were harvested by centrifuging the culture at 8000 rpm for 10 min. The cell pellet was dissolved, homogenized, and lysed in ice-cold lysis buffer A (20 mM HEPES, pH 7.4; 50 mM KCl; 5 mM MgCl2; and 10% glycerol) containing 1 mM PMSF and 1 mg/mL lysozyme. The cell suspension was sonicated, and the lysate was cleared by centrifugation at 35,000 rpm for 1 h in an ultracentrifuge. Imidazole (10 mM) was added to the supernatant, and it was loaded to the Ni-NTA column. The column was washed extensively with lysis buffer containing 25 mM and subsequently, 50 mM imidazole to elute loosely bound proteins. Both FtsZ and truncated FtsZ were then eluted with lysis buffer containing 250 mM imidazole. The eluted fractions were analyzed via SDS–PAGE and those containing FtsZ were pooled and loaded onto a gravity column containing Sephadex G-25 fine resin (Cytiva) for salt removal. Proteins were eluted in a storage buffer containing (20 mM HEPES and 10% glycerol, pH 7.4). FtsZ constructs were then concentrated using Amicon 30 kDa MWCO (Sigma) centrifugal filter and stored at −80°C.

BL21-DE3 cells overexpressing gpsB were allowed to grow until OD600 0.6 and then induced with 0.5 mM IPTG and was grown for a further 6 h at 37°C. The cells were harvested, and the protein was purified as described in (Bhattacharya et al., 2017).

GTPase assay

The effect of GpsB on the inorganic phosphate released by FtsZ and truncated FtsZ was examined using a malachite green phosphate assay kit (Sigma). Briefly, either FtsZ or truncated FtsZ (30 µM) was incubated with GpsB (10 µM) in 25 mM HEPES buffer containing 140 mM KCl, 5 mM MgCl2, and 2 mM GTP at 37°C for 15 min. Then, the reaction mixtures were incubated with the working reagent provided with the kit for a further 30 min in the dark and the absorbance of the reaction milieu were taken at 650 nm (Bhattacharya et al., 2017; Eswara et al., 2018).

Fluorescence microscopy

Fluorescence microscopy was performed as described previously (Brzozowski et al., 2019). Briefly, 500 µl aliquots of B. subtilis cultures to be imaged were pelleted and washed with 1X PBS. The pellets were resuspended in 100 µl PBS and 1 µg/ml Synapto-Red was added to the cell suspension to stain the cell membrane. A small volume (5 µl) of the suspension was spotted on the glass bottom dish (MatTek) and 1% agarose pad was gently placed on top. The images were taken using DeltaVision Elite deconvolution fluorescence microscope with a Photometrics CoolSnap HQ2 camera. Images were deconvoluted by SoftWorx imaging software provided by the manufacturer. The cell lengths were analyzed by ImageJ software and the statistical analysis was carried out using GraphPad Prism.

Labeling of FtsZ

Both full-length and truncated versions of FtsZ (50 µM) was incubated with FITC (250 µM) in 50 mM phosphate buffer (pH 8.0) for 3 h on ice. The reaction was terminated by adding 5 mM Tris-HCl (pH 8.0). The free FITC was separated from the labeled protein by passing the reaction mixture through the Sephadex G25 fine column (Cytiva). The concentration of FITC-bound FtsZ was determined by taking the absorbance at 495 nm. The concentration of the unlabeled protein was measured using a Bradford reagent (Bradford, 1976). The stoichiometry of labeling of FITC per FtsZ was determined to be ∼0.7 (Bhattacharya et al., 2017).

Fluorescence spectroscopy

To check the specificity of binding of GpsB to FtsZ, either FITC-FtsZ (100 nM) or FITC-FtsZΔC6 (100 nM) was incubated with increasing concentrations of GpsB (10–80 µM) in 25 mM HEPES (pH 6.7) for 10 min at 25°C. The fluorescence spectra of FITC-FtsZ (510–650 nm) were monitored by exciting the reaction mixtures at 495 nm. The changes in the fluorescence intensities of FITC-FtsZ in the presence of different concentrations of GpsB were plotted. The dissociation constants of binding were determined by fitting the values in the following quadratic binding equation (Bhattacharya et al., 2017).

graphic file with name mbc-36-ar10-e001.jpg

Where ΔF and ΔFmax are the change and maximum change in the fluorescence intensity of FITC-FtsZ upon binding to GpsB respectively. P0 and L0 are the concentrations of FITC-FtsZ and GpsB used, respectively. ΔF was calculated by subtracting the fluorescence intensity of FITC-FtsZ in the presence of GpsB from the intensity of the same in the absence of GpsB. The data were analyzed using Graph Pad Prism software.

Supplementary Material

mbc-36-ar10-s001.pdf (8MB, pdf)

Abbreviations used:

ATCC

American Type Culture Collection

Bs

Bacillus subtilis

BSA

bovine serum albumin

CTT

FtsZ C-terminal tail region

Ef

Enterococcus faecalis

FITC

Fluorescein isothiocyanate

IPTG

Isopropyl β-D-1-thiogalactopyranoside

Lm

Listeria monocytogenes

PBPs

penicillin binding proteins

PG

peptidoglycan

Sa

Staphylococcus aureus

Sp

Streptococcus pneumoniae

WT

Wild type

Z-ring

a ring-like structure made of FtsZ protofilaments.

Footnotes

This article was published online ahead of print in MBoC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E24-07-0302) on November 27, 2024.

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