Bacterial cell division involves the invagination of the cellular membrane(s) and peptidoglycan (PG) cell wall to split the cell into two progeny cells. In most bacteria, cell division is initiated when the tubulin homologue and GTPase, FtsZ, polymerizes to form a ring-like structure, called the Z-ring, at the division site (Bi and Lutkenhaus, 1991; de Boer et al., 1992; Mukherjee and Lutkenhaus, 1994). The Z-ring serves as a scaffold onto which about a dozen other proteins assemble to form a mature cell division machinery called ‘divisome’ (Haeusser and Margolin, 2016). In the Gram-positive bacterium Bacillus subtilis, divisome assembly proceeds in two steps. First, proteins that interact directly with FtsZ and promote its association with the membrane are recruited to the divisome (Gamba et al., 2009). One essential protein recruited at this stage is FtsA, an actin homologue which tethers FtsZ to the membrane via its conserved C-terminal amphipathic helix (Pichoff and Lutkenhaus, 2005; Szwedziak et al., 2014). FtsA can also form protofilaments via polymerization in an ATP-dependent manner (Szwedziak et al., 2012). Second, proteins involved in septal PG synthesis and remodeling are recruited to the divisome (Gamba et al., 2009). Recent studies suggest that treadmilling by FtsZ filaments serves as a platform to drive the circumferential motion of septal PG synthases in both B. subtilis and Escherichia coli, suggesting that this dynamic property of FtsZ filaments can regulate cell wall remodeling in the septal disc (Bisson-Filho et al., 2017; Yang et al., 2017).

B. subtilis is a unique model system to study cell division as it can divide via two modes (Figure 1). During vegetative growth, the septum is formed at the midcell, producing two daughter cells of equal sizes (Wang and Lutkenhaus, 1993). However, during sporulation, the division septum is formed closer to one pole to produce a smaller forespore and a larger mother cell. Subsequently, the forespore is engulfed by the mother cell, producing an internal endospore, that upon maturation, is released by mother cell lysis (Khanna et al., 2020; Riley et al., 2021b; Tan and Ramamurthi, 2014). Vegetative and sporulating cells differ not only in the positioning of the division site but also in the thickness of their septa, with the medial vegetative septum being almost four times thicker than the polar sporulating septum (~80 nm vs. ~25 nm) (Illing and Errington, 1991; Tocheva et al., 2013). Another distinction is the presence of a membrane protein SpoIIE, which colocalizes with and directly interacts with FtsZ in sporulating cells (Arigoni et al., 1995; Levin et al., 1997; Lucet et al., 2000), but is absent in vegetative cells. Recent evidence suggests that SpoIIE localizes only on the forespore side of the sporulation septum at the onset of membrane constriction before being released into the forespore membrane upon septum formation (Carniol et al., 2005; Errington, 2003; Eswaramoorthy et al., 2014; Guberman et al., 2008; Wu et al., 1998). Certain spoIIE mutants also form thicker polar septa compared to wild-type sporangia (Barák and Youngman, 1996; Illing and Errington, 1991). These findings posit that there may be differences in the divisome organization between the vegetative and the sporulation septum that play a role in regulating septal thickness. However, a mechanistic understanding of how cell division is regulated during B. subtilis sporulation remains elusive.

Figure 1

Download asset Open asset

In this study, we used cryo-focused ion beam milling coupled with cryo-electron tomography (cryo-FIB-ET) to reveal the molecular architecture of the cell division machinery in B. subtilis during vegetative growth and sporulation. Our results demonstrate that FtsAZ filaments have distinct spatial organization during the two modes of division and that SpoIIE regulates the positioning of FtsAZ filaments during sporulation. We further provide evidence that this distinct organization leads to a thinner polar septum during sporulation. Our results pave the way for future studies in the field of bacterial cell division, giving rise to testable hypothesis on how bacteria regulate protein localization in space and time to carry out the critical process of cytokinesis under different gene expression programs.

In this study, we elucidate the differences in the architecture of the cell division machinery in B. subtilis during vegetative growth and sporulation and their impact on septal PG thickness. First, we show that during vegetative growth, two cytoskeletal rings localize uniformly around the leading edge of the invaginating medial septum to orchestrate cell division, a membrane-proximal ring of FtsA filaments that tethers the membrane-distal ring of FtsZ filaments to the membrane (Figures 3–6). Second, we demonstrate that during the asymmetrically positioned division event in the cell at the onset of sporulation, FtsAZ filaments are assembled only on the mother cell side of the invaginating polar septum (Figure 7A–D), demonstrating a surprising degree of asymmetry even in the localization of the division machinery. Third, our data suggest that during septal biogenesis the number of FtsAZ filaments tracking the sporulation septum is approximately half of that in the vegetative septum, which likely gives rise to a thinner sporulation septum by mediating the circumferential motion of fewer PG synthases around the division plane during sporulation (Figure 7E, F). Finally, we show that a sporulation-specific protein SpoIIE, which is essential for cell-specific gene expression after polar septation, is also essential for the asymmetric assembly of FtsAZ filaments and septal thickness at the onset of sporulation (Figures 8 and 9). These results demonstrate that the sporulation septum is both asymmetrically positioned in the cell and that it is assembled by an asymmetrically localized division machinery.

Most of our understanding of bacterial cytokinesis comes from studies in model Gram-positive and Gram-negative bacteria, namely B. subtilis and E. coli, respectively. Although the overall mechanism of cytokinesis is conserved in that a Z-ring that recruits other components of the divisome is formed at the division site, there are subtle differences with regards to the spatial and the temporal regulation of the process and the composition of the divisome for the two types of bacteria. Previously, only a single cytoskeleton ring of FtsZ was observed using cryo-ET in Gram-negative bacteria including E. coli and C. crescentus (Li et al., 2007; Szwedziak et al., 2014). However, we observed two rings corresponding to FtsA and FtsZ filaments in cryo-FIB-ET images of B. subtilis during both vegetative growth and sporulation, even though the ratio of FtsZ to FtsA is same (5:1) in B. subtilis and E. coli and there are enough molecules of FtsA to form a circumferential A-ring in both species (Feucht et al., 2001; Rueda et al., 2003). But an A-ring is apparent only in B. subtilis, although it appears more patchy as compared to the mostly continuous Z-ring. One possible explanation for this difference between species is that cell division in B. subtilis involves the constriction of a septal disc composed of thicker septal PG (~25 nm during sporulation and ~50 nm during vegetative growth) than in E. coli or C. crescentus, where the septal disc consists of only ~4-nm-thick PG. Hence, cytokinesis in B. subtilis may require more FtsA filaments in order to tether the increased number of PG synthetases that are required to build a thicker septum during constriction. Other differences between our data and that from Gram-negative bacteria are the decreased lateral spacing between FtsZ filaments (~5.5 nm in B. subtilis vs. 6.5–8 nm in E. coli and C. crescentus), and the shorter distances of the Z-ring and the A-ring from the membrane (~14 nm and ~6.5 nm, respectively, in B. subtilis vs. ~16 nm and ~8 nm, respectively, in E. coli). It is possible that a tighter bundling of FtsAZ filaments with each other and with the membrane in B. subtilis may be necessary for the synthesis of the thick septal PG. Further studies will determine if these mechanisms of Z-ring and A-ring formation are also conserved in other sporulating and non-sporulating Gram-positive bacteria.

Our result that the Z-ring is mostly continuous during vegetative growth is similar to a previous cryo-ET study that also revealed the continuity of Z-rings in both E. coli and C. crescentus (Szwedziak et al., 2014). However, this contrasts with super-resolution microscopy in which the Z-ring in both B. subtilis and E. coli appeared patchy with regions of low fluorescence intensity that may indicate gaps in the ring (Holden et al., 2014; Strauss et al., 2012). We note that cryo-ET has better spatial resolution than super-resolution microscopy and enables visualization of cellular structures in a near-native state. It is possible that we are unable to detect gaps in our cryo-FIB-ET data because they are either present in the cellular region that was ablated by cryo-FIB milling or not sampled due to missing wedge associated with cryo-ET data collection (Figure 2—figure supplement 1). However, these scenarios seem less likely because different tomograms capture different parts of the septal disc during constriction, and the ring appeared continuous in each of them. Thus, it seems likely that at least under the conditions used here the Z-ring is largely contiguous during constriction, although further studies are required to determine if this varies under other experimental conditions or at specific stages of FtsZ ring assembly, constriction or disassembly. During sporulation, we notice that the septal membranes are not as sharply defined as during vegetative growth, probably due to molecular crowding by membrane proteins that participate in cell division as well as those that are produced specifically during sporulation. Also, since FtsAZ are only present on the mother cell side and the distance spanned by the bundle around the invaginating septum is much shorter during sporulation, the filaments are detected in only a few slices in cross-sectional views as opposed to vegetative growth. These limitations make it difficult to conclude with confidence whether the Z-ring is also continuous during sporulation. However, the Z-ring is continuous during sporulation in SpoIIE sporangia in the tomograms we acquired, suggesting their continuous nature in at least under this condition during sporulation.

Our data demonstrate that during sporulation not only is there asymmetry associated with the positioning of the division septum within the cell, but also with the positioning of the division machinery at the septum, since FtsAZ filaments are only present on the mother cell side of the invaginating septum. This is an unanticipated observation. Further, we noted that only half the number of filaments assemble at the septum and track the division plane during sporulation than during vegetative growth, and that the septum is also half as thick in sporulating cells. These results suggest that the number of FtsAZ filaments could directly dictate the number of complexes involved in PG synthesis.

A thinner septum could be beneficial during sporulation in two ways. First, several protein complexes are proposed to form channels across both septal membranes during sporulation, including SpoIIQ-SpoIIIAH and SpoIIIE (Blaylock et al., 2004; Fleming et al., 2010; Meisner et al., 2008; Riley et al., 2021a; Yen Shin et al., 2015). A thinner septum could facilitate interaction between the subunits located in the forespore and those in the mother cell. Second, a thinner septum may facilitate the phagocytosis-like process of engulfment, during which migration of the mother cell membrane is driven by the remodeling of the junction between the septal PG and the lateral PG via the coordinated synthesis and degradation of the septal PG (Khanna et al., 2019; Ojkic et al., 2016). This remodeling event may be more straightforward if the sporulation septum contained relatively few or potentially only one layer of PG.

In addition to facilitating the formation of a thinner septum, asymmetric distribution of FtsAZ to the mother cell side of the septum may also be beneficial for other downstream events during sporulation. First, it is possible that such a strategy allows enough FtsAZ complexes to remain in the mother cell to support a second division site at the opposite cell pole as a backup should the first septum be misassembled or fail to support the onset of cell-specific gene expression. This second polar division site is dissolved after the onset of cell-specific gene expression by either the PG degradation complex SpoIIDMP (Abanes-De Mello et al., 2002; Eichenberger et al., 2001; Pogliano et al., 1999) or MciZ (Bisson-Filho et al., 2015; Handler et al., 2008) in the mother cell. Retaining FtsAZ complexes in the mother cell would ensure their availability for the second division event at the distal pole.

Second, if FtsAZ complexes were present on the forespore side of the septum after septation, they may induce aberrant cell division events in the small forespore if their activity was not efficiently inhibited by the Min and the nucleoid occlusion proteins (Marston et al., 1998; Wu and Errington, 2004). Thus, ensuring that the forespore does not harbor active cytoskeletal filaments upon septation could provide an additional safety mechanism. Time-lapse microscopy of FtsA-mNeonGreen and FtsZ-mNeonGreen indicated that just after polar septation there was some FtsAZ signal in the forespore that gradually diminished as engulfment proceeded in these cells (Figure 9—figure supplement 1). It is possible that there are protein(s) in the forespore that mediate degradation of FtsAZ polymers that do not participate in cell division yet remain in the forespore, ensuring an extra layer of safety mechanism to prevent any aberrant cell division events in the forespore. A recent study from the lab showed that certain proteins involved in central carbon metabolism and metabolic precursor synthesis were depleted specifically in the forespore after polar septation (Riley et al., 2021b). Further studies will address if FtsAZ are depleted from the forespore via a similar mechanism.

Finally, we have previously shown that PG synthases, including Pbp2B, a component of the divisome, preferentially localize to the forespore side of the leading edge of the engulfing membrane (Khanna et al., 2019; Ojkic et al., 2016), where they act together with the mother cell PG degradation complex SpoIIDMP to mediate the coordinated synthesis and degradation of the cell wall to drive engulfment membrane migration (Ojkic et al., 2016). The mechanism by which these proteins localize to the region of the forespore membrane that is adjacent to the migrating mother cell membrane remains unclear, but our fluorescence microscopy data indicate that at the onset of membrane constriction, a punctum of FtsA-mNeonGreen remains at the corner formed by the septum and the outer envelope likely in the forespore. It is possible that other cell division proteins such as those involved in cell wall synthesis also remain at this site, where they would be poised to participate in engulfment after the completion of cell division. Development of cryo-ET compatible electron-dense tags to track the localization of individual divisome proteins in conjunction with single-molecule studies will aid in testing these hypotheses in the future.

Asymmetric cell division is a common developmental theme in eukaryotes to generate progeny with diverse cellular fates (Li, 2013). In these cases of asymmetric cell division, the general theme is the establishment of cell division machinery along an axis of polarity and distribution of cell fate determinants in a polarized manner along this axis. In case of B. subtilis sporulation, an axis of polarity is likely established when the division plane shifts from medial to polar position and cell fate determinants are differentially distributed between the future forespore and the mother cell compartments at the onset of membrane constriction. In this study, we have identified SpoIIE as a cell fate determinant that also restricts FtsAZ filaments to the mother cell side of the asymmetrically positioned septum, likely by its preferential localization to the forespore side of the invaginating septum (Eswaramoorthy et al., 2014; Guberman et al., 2008; Wu et al., 1998). We show that regions I and II of SpoIIE corresponding to the transmembrane domain and the FtsZ-binding domain, respectively, regulate the septal thickness and the distribution of FtsAZ at the sporulation septum. Region III, C-terminal part of SpoIIE, consists of a phosphatase domain that is essential to activate σ^F in the forespore by dephosphorylating SpoIIAA after septum closure. It was previously shown that a SpoIIE mutant that is enzymatically inactive in dephosphorylating SpoIIA (D746A substitution) behaved similar to wild type with respect to polar septation during sporulation (Carniol et al., 2005). In another study, missense mutations spoIIE64 and spoIIE71 that map to region III of SpoIIE and affect its phosphatase activity were shown to have thin polar septa in thin-section electron microscopy images (Barák and Youngman, 1996), suggesting that the phosphatase activity of SpoIIE likely does not affect the septal thickness. However, we cannot exclude the possibility that the deletion of the entire region III or some other mutations in region III may play a role in regulating the septal thickness and/or FtsAZ localization at the septum. Also, investigating the physiological relevance of septal thickness during sporulation is complicated by the pleiotropic nature of SpoIIE mutations. SpoIIE directly participates in polar septation, but it is also required for the activation of σ^F in the forespore, and consequently, the rest of the sporulation-specific σ factors. Although the two roles seem to depend on different parts of the protein, separating them is not straightforward, as making mutations that affect polar septation may also affect the localization of SpoIIE and, therefore, the protein might not be released to the forespore to activate σ^F. Future studies to examine more mutations in different regions of SpoIIE and their overexpression will provide better insights into the mechanism of regulation of septal thickness by SpoIIE. Further biochemical and genetic studies are required to elucidate the cellular mechanism by which the nascent sporulation septum is assembled asymmetrically, so that SpoIIE, which is required for forespore-specific gene expression, assembles on the forespore side of the nascent septum, while FtsAZ rings assembles on the mother cell face of the nascent septum.

Our data provide a significant advancement in the understanding of the organization of the divisome, its regulators and its role in septal PG synthesis in B. subtilis, providing, to the best of our knowledge, the first evidence for the asymmetric assembly of the divisome in bacteria and showing that this depends on the SpoIIE protein that is required for the onset of cell-specific gene expression. Future efforts to identify the molecular arrangement of FtsZ and its regulators inside the cell such as SpoIIE will aid in the development of new antimicrobials targeting the cell division machinery in important pathogens.

All data generated or analysed during this study are included in the manuscript and supporting files. Additionally, we have deposited representative tomograms for each growth condition in the Electron Microscopy Data Bank (EMDB) accession codes EMD-23963, EMD-23964, EMD-23965, EMD-23966, EMD-23967 and EMD-23968, and tilt series in the Electron Microscopy Public Image Archive (EMPIAR) database accession code EMPIAR-10710.

1. 1. Khanna K
   2. Lopez-Garrido J
   3. Sugie J
   4. Pogliano K
   5. Villa E

   (2021) Electron Microscopy Data Bank

   ID EMD-23963. Tomogram of a dividing vegetative cell of Bacillus subtilis.

   https://www.ebi.ac.uk/pdbe/entry/emdb/EMD-23963
2. 1. Khanna K
   2. Lopez-Garrido J
   3. Sugie J
   4. Pogliano K
   5. Villa E

   (2021) Electron Microscopy Data Bank

   ID EMD-23964. Tomogram of a dividing vegetative cell of Bacillus subtilis.

   https://www.ebi.ac.uk/pdbe/entry/emdb/EMD-23964
3. 1. Khanna K
   2. Lopez-Garrido J
   3. Sugie J
   4. Pogliano K
   5. Villa E

   (2021) Electron Microscopy Data Bank

   ID EMD-23965. Tomogram of a dividing vegetative cell of Bacillus subtilis FtsZ-linker(Q-rich) strain.

   https://www.ebi.ac.uk/pdbe/entry/emdb/EMD-23965
4. 1. Khanna K
   2. Lopez-Garrido J
   3. Sugie J
   4. Pogliano K
   5. Villa E

   (2021) Electron Microscopy Data Bank

   ID EMD-23966. Tomogram of a dividing sporulating cell of Bacillus subtilis.

   https://www.ebi.ac.uk/pdbe/entry/emdb/EMD-23966
5. 1. Khanna K
   2. Lopez-Garrido J
   3. Sugie J
   4. Pogliano K
   5. Villa E

   (2021) Electron Microscopy Data Bank

   ID EMD-23967. Tomogram of a dividing sporulating cell of Bacillus subtilis.

   https://www.ebi.ac.uk/pdbe/entry/emdb/EMD-23967
6. 1. Khanna K
   2. Lopez-Garrido J
   3. Sugie J
   4. Pogliano K
   5. Villa E

   (2021) Electron Microscopy Data Bank

   ID EMD-23968. Tomogram of a dividing sporulating cell of Bacillus subtilis SpoIIE null mutant.

   https://www.ebi.ac.uk/pdbe/entry/emdb/EMD-23968
7. 1. Khanna K
   2. Lopez-Garrido J
   3. Sugie J
   4. Pogliano K
   5. Villa E

   (2021) Electron Microscopy Public Image Archive

   ID EMPIAR-10710. Tilt series of dividing vegetative and sporulating cells of Bacillus subtilis.

   https://www.ebi.ac.uk/pdbe/emdb/empiar/EMPIAR-10710

1. 1. Abanes-De Mello A
   2. Sun YL
   3. Aung S
   4. Pogliano K

   (2002) A cytoskeleton-like role for the bacterial cell wall during engulfment of the Bacillus subtilis forespore

   Genes & Development 16:3253–3264.

   https://doi.org/10.1101/gad.1039902

   • PubMed
   • Google Scholar
2. 1. Arigoni F
   2. Pogliano K
   3. Webb CD
   4. Stragier P
   5. Losick R

   (1995) Localization of protein implicated in establishment of cell type to sites of asymmetric division

   Science 270:637–640.

   https://doi.org/10.1126/science.270.5236.637

   • PubMed
   • Google Scholar
3. 1. Barák I
   2. Youngman P

   (1996) SpoIIE mutants of Bacillus subtilis comprise two distinct phenotypic classes consistent with a dual functional role for the SpoIIE protein

   Journal of Bacteriology 178:4984–4989.

   https://doi.org/10.1128/JB.178.16.4984-4989.1996

   • PubMed
   • Google Scholar
4. 1. Beall B
   2. Lutkenhaus J

   (1992) Impaired cell division and sporulation of a Bacillus subtilis strain with the ftsA gene deleted

   Journal of Bacteriology 174:2398–2403.

   https://doi.org/10.1128/JB.174.7.2398-2403.1992

   • PubMed
   • Google Scholar
5. 1. Bi EF
   2. Lutkenhaus J

   (1991) FtsZ ring structure associated with division in Escherichia coli

   Nature 354:161–164.

   https://doi.org/10.1038/354161a0

   • PubMed
   • Google Scholar
6. 1. Bisson-Filho AW
   2. Discola KF
   3. Castellen P
   4. Blasios V
   5. Martins A
   6. Sforça ML
   7. Garcia W
   8. Zeri AC
   9. Erickson HP
   10. Dessen A
   11. Gueiros-Filho FJ

   (2015) FtsZ filament capping by MciZ, a developmental regulator of bacterial division

   PNAS 112:E2130–E2138.

   https://doi.org/10.1073/pnas.1414242112

   • PubMed
   • Google Scholar
7. 1. Bisson-Filho AW
   2. Hsu YP
   3. Squyres GR
   4. Kuru E
   5. Wu F
   6. Jukes C
   7. Sun Y
   8. Dekker C
   9. Holden S
   10. VanNieuwenhze MS
   11. Brun YV
   12. Garner EC

   (2017) Treadmilling by FtsZ filaments drives peptidoglycan synthesis and bacterial cell division

   Science 355:739–743.

   https://doi.org/10.1126/science.aak9973

   • PubMed
   • Google Scholar
8. 1. Blaylock B
   2. Jiang X
   3. Rubio A
   4. Moran CP
   5. Pogliano K

   (2004) Zipper-like interaction between proteins in adjacent daughter cells mediates protein localization

   Genes & Development 18:2916–2928.

   https://doi.org/10.1101/gad.1252704

   • PubMed
   • Google Scholar
9. 1. Carniol K
   2. Ben-Yehuda S
   3. King N
   4. Losick R

   (2005) Genetic dissection of the sporulation protein SpoIIE and its role in asymmetric division in Bacillus subtilis

   Journal of Bacteriology 187:3511–3520.

   https://doi.org/10.1128/JB.187.10.3511-3520.2005

   • PubMed
   • Google Scholar
10. 1. de Boer P
    2. Crossley R
    3. Rothfield L

    (1992) The essential bacterial cell-division protein FtsZ is a GTPase

    Nature 359:254–256.

    https://doi.org/10.1038/359254a0

    • PubMed
    • Google Scholar
11. 1. Duman R
    2. Ishikawa S
    3. Celik I
    4. Strahl H
    5. Ogasawara N
    6. Troc P
    7. Löwe J
    8. Hamoen LW

    (2013) Structural and genetic analyses reveal the protein SepF as a new membrane anchor for the Z ring

    PNAS 110:E4601–E4610.

    https://doi.org/10.1073/pnas.1313978110

    • PubMed
    • Google Scholar
12. 1. Eichenberger P
    2. Fawcett P
    3. Losick R

    (2001) A three-protein inhibitor of polar septation during sporulation in Bacillus subtilis

    Molecular Microbiology 42:1147–1162.

    https://doi.org/10.1046/j.1365-2958.2001.02660.x

    • PubMed
    • Google Scholar
13. 1. Errington J

    (2003) Regulation of endospore formation in Bacillus subtilis

    Nature Reviews Microbiology 1:117–126.

    https://doi.org/10.1038/nrmicro750

    • PubMed
    • Google Scholar
14. 1. Eswaramoorthy P
    2. Winter PW
    3. Wawrzusin P
    4. York AG
    5. Shroff H
    6. Ramamurthi KS

    (2014) Asymmetric division and differential gene expression during a bacterial developmental program requires DivIVA

    PLOS Genetics 10:e1004526.

    https://doi.org/10.1371/journal.pgen.1004526

    • PubMed
    • Google Scholar
15. 1. Feucht A
    2. Lucet I
    3. Yudkin MD
    4. Errington J

    (2001) Cytological and biochemical characterization of the FtsA cell division protein of Bacillus subtilis

    Molecular Microbiology 40:115–125.

    https://doi.org/10.1046/j.1365-2958.2001.02356.x

    • PubMed
    • Google Scholar
16. 1. Fleming TC
    2. Shin JY
    3. Lee SH
    4. Becker E
    5. Huang KC
    6. Bustamante C
    7. Pogliano K

    (2010) Dynamic SpoIIIE assembly mediates septal membrane fission during Bacillus subtilis sporulation

    Genes & Development 24:1160–1172.

    https://doi.org/10.1101/gad.1925210

    • PubMed
    • Google Scholar
17. 1. Gamba P
    2. Veening JW
    3. Saunders NJ
    4. Hamoen LW
    5. Daniel RA

    (2009) Two-step assembly dynamics of the Bacillus subtilis divisome

    Journal of Bacteriology 191:4186–4194.

    https://doi.org/10.1128/JB.01758-08

    • PubMed
    • Google Scholar
18. 1. Guberman JM
    2. Fay A
    3. Dworkin J
    4. Wingreen NS
    5. Gitai Z

    (2008) PSICIC: noise and asymmetry in bacterial division revealed by computational image analysis at sub-pixel resolution

    PLOS Computational Biology 4:e1000233.

    https://doi.org/10.1371/journal.pcbi.1000233

    • PubMed
    • Google Scholar
19. 1. Gueiros-Filho FJ
    2. Losick R

    (2002) A widely conserved bacterial cell division protein that promotes assembly of the tubulin-like protein FtsZ

    Genes & Development 16:2544–2556.

    https://doi.org/10.1101/gad.1014102

    • PubMed
    • Google Scholar
20. 1. Haeusser DP
    2. Margolin W

    (2016) Splitsville: structural and functional insights into the dynamic bacterial Z ring

    Nature Reviews Microbiology 14:305–319.

    https://doi.org/10.1038/nrmicro.2016.26

    • PubMed
    • Google Scholar
21. 1. Handler AA
    2. Lim JE
    3. Losick R

    (2008) Peptide inhibitor of cytokinesis during sporulation in Bacillus subtilis

    Molecular Microbiology 68:588–599.

    https://doi.org/10.1111/j.1365-2958.2008.06173.x

    • PubMed
    • Google Scholar
22. 1. Holden SJ
    2. Pengo T
    3. Meibom KL
    4. Fernandez Fernandez C
    5. Collier J
    6. Manley S

    (2014) High throughput 3D super-resolution microscopy reveals Caulobacter crescentus in vivo Z-ring organization

    PNAS 111:4566–4571.

    https://doi.org/10.1073/pnas.1313368111

    • PubMed
    • Google Scholar
23. 1. Huang KC
    2. Mukhopadhyay R
    3. Wen B
    4. Gitai Z
    5. Wingreen NS

    (2008) Cell shape and cell-wall organization in Gram-negative Bacteria

    PNAS 105:19282–19287.

    https://doi.org/10.1073/pnas.0805309105

    • PubMed
    • Google Scholar
24. 1. Illing N
    2. Errington J

    (1991) Genetic regulation of morphogenesis in Bacillus subtilis: roles of sigma E and sigma F in prespore engulfment

    Journal of Bacteriology 173:3159–3169.

    https://doi.org/10.1128/JB.173.10.3159-3169.1991

    • PubMed
    • Google Scholar
25. 1. Khanna K
    2. Lopez-Garrido J
    3. Zhao Z
    4. Watanabe R
    5. Yuan Y
    6. Sugie J
    7. Pogliano K
    8. Villa E

    (2019) The molecular architecture of engulfment during Bacillus subtilis sporulation

    eLife 8:e45257.

    https://doi.org/10.7554/eLife.45257

    • PubMed
    • Google Scholar
26. 1. Khanna K
    2. Lopez-Garrido J
    3. Pogliano K

    (2020) Shaping an endospore: architectural transformations during Bacillus subtilis Sporulation

    Annual Review of Microbiology 74:361–386.

    https://doi.org/10.1146/annurev-micro-022520-074650

    • PubMed
    • Google Scholar
27. 1. Kremer JR
    2. Mastronarde DN
    3. McIntosh JR

    (1996) Computer visualization of three-dimensional image data using IMOD

    Journal of Structural Biology 116:71–76.

    https://doi.org/10.1006/jsbi.1996.0013

    • PubMed
    • Google Scholar
28. 1. Lee TK
    2. Huang KC

    (2013) The role of hydrolases in bacterial cell-wall growth

    Current Opinion in Microbiology 16:760–766.

    https://doi.org/10.1016/j.mib.2013.08.005

    • PubMed
    • Google Scholar
29. 1. Levin PA
    2. Losick R
    3. Stragier P
    4. Arigoni F

    (1997) Localization of the sporulation protein SpoIIE in Bacillus subtilis is dependent upon the cell division protein FtsZ

    Molecular Microbiology 25:839–846.

    https://doi.org/10.1111/j.1365-2958.1997.mmi505.x

    • PubMed
    • Google Scholar
30. 1. Li Z
    2. Trimble MJ
    3. Brun YV
    4. Jensen GJ

    (2007) The structure of FtsZ filaments in vivo suggests a force-generating role in cell division

    The EMBO Journal 26:4694–4708.

    https://doi.org/10.1038/sj.emboj.7601895

    • PubMed
    • Google Scholar
31. 1. Li R

    (2013) The art of choreographing asymmetric cell division

    Developmental Cell 25:439–450.

    https://doi.org/10.1016/j.devcel.2013.05.003

    • PubMed
    • Google Scholar
32. 1. Lopez-Garrido J
    2. Ojkic N
    3. Khanna K
    4. Wagner FR
    5. Villa E
    6. Endres RG
    7. Pogliano K

    (2018) Chromosome translocation inflates Bacillus forespores and impacts cellular morphology

    Cell 172:758–770.

    https://doi.org/10.1016/j.cell.2018.01.027

    • PubMed
    • Google Scholar
33. 1. Lucet I
    2. Feucht A
    3. Yudkin MD
    4. Errington J

    (2000) Direct interaction between the cell division protein FtsZ and the cell differentiation protein SpoIIE

    The EMBO Journal 19:1467–1475.

    https://doi.org/10.1093/emboj/19.7.1467

    • PubMed
    • Google Scholar
34. 1. Marston AL
    2. Thomaides HB
    3. Edwards DH
    4. Sharpe ME
    5. Errington J

    (1998) Polar localization of the MinD protein of Bacillus subtilis and its role in selection of the mid-cell division site

    Genes & Development 12:3419–3430.

    https://doi.org/10.1101/gad.12.21.3419

    • PubMed
    • Google Scholar
35. 1. Martinez-Sanchez A
    2. Garcia I
    3. Asano S
    4. Lucic V
    5. Fernandez JJ

    (2014) Robust membrane detection based on tensor voting for electron tomography

    Journal of Structural Biology 186:49–61.

    https://doi.org/10.1016/j.jsb.2014.02.015

    • PubMed
    • Google Scholar
36. 1. Mastronarde DN

    (1997) Dual-axis tomography: an approach with alignment methods that preserve resolution

    Journal of Structural Biology 120:343–352.

    https://doi.org/10.1006/jsbi.1997.3919

    • PubMed
    • Google Scholar
37. 1. Mastronarde DN

    (2005) Automated electron microscope tomography using robust prediction of specimen movements

    Journal of Structural Biology 152:36–51.

    https://doi.org/10.1016/j.jsb.2005.07.007

    • PubMed
    • Google Scholar
38. 1. Meisner J
    2. Wang X
    3. Serrano M
    4. Henriques AO
    5. Moran CP

    (2008) A channel connecting the mother cell and forespore during bacterial endospore formation

    PNAS 105:15100–15105.

    https://doi.org/10.1073/pnas.0806301105

    • PubMed
    • Google Scholar
39. 1. Mukherjee A
    2. Lutkenhaus J

    (1994) Guanine nucleotide-dependent assembly of FtsZ into filaments

    Journal of Bacteriology 176:2754–2758.

    https://doi.org/10.1128/JB.176.9.2754-2758.1994

    • PubMed
    • Google Scholar
40. 1. Ojkic N
    2. López-Garrido J
    3. Pogliano K
    4. Endres RG

    (2016) Cell-wall remodeling drives engulfment during Bacillus subtilis sporulation

    eLife 5:e18657.

    https://doi.org/10.7554/eLife.18657

    • PubMed
    • Google Scholar
41. 1. Pichoff S
    2. Lutkenhaus J

    (2005) Tethering the Z ring to the membrane through a conserved membrane targeting sequence in FtsA

    Molecular Microbiology 55:1722–1734.

    https://doi.org/10.1111/j.1365-2958.2005.04522.x

    • PubMed
    • Google Scholar
42. 1. Pogliano J
    2. Osborne N
    3. Sharp MD
    4. Abanes-De Mello A
    5. Perez A
    6. Sun YL
    7. Pogliano K

    (1999) A vital stain for studying membrane dynamics in Bacteria: a novel mechanism controlling septation during Bacillus subtilis sporulation

    Molecular Microbiology 31:1149–1159.

    https://doi.org/10.1046/j.1365-2958.1999.01255.x

    • PubMed
    • Google Scholar
43. 1. Riley EP
    2. Schwarz C
    3. Derman AI
    4. Lopez-Garrido J

    (2021a) Milestones in Bacillus subtilis sporulation research

    Microbial Cell 8:1–16.

    https://doi.org/10.15698/mic2021.01.739

    • Google Scholar
44. 1. Riley EP
    2. Lopez-Garrido J
    3. Sugie J
    4. Liu RB
    5. Pogliano K

    (2021b) Metabolic differentiation and intercellular nurturing underpin bacterial endospore formation

    Science Advances 7:eabd6385.

    https://doi.org/10.1126/sciadv.abd6385

    • PubMed
    • Google Scholar
45. 1. Rueda S
    2. Vicente M
    3. Mingorance J

    (2003) Concentration and assembly of the division ring proteins FtsZ, FtsA, and ZipA during the Escherichia coli cell cycle

    Journal of Bacteriology 185:3344–3351.

    https://doi.org/10.1128/JB.185.11.3344-3351.2003

    • PubMed
    • Google Scholar
46. 1. Sandman K
    2. Losick R
    3. Youngman P

    (1987) Genetic analysis of Bacillus subtilis spo mutations generated by Tn917-mediated insertional mutagenesis

    Genetics 117:603–617.

    https://doi.org/10.1093/genetics/117.4.603

    • PubMed
    • Google Scholar
47. 1. Singh JK
    2. Makde RD
    3. Kumar V
    4. Panda D

    (2007) A membrane protein, EzrA, regulates assembly dynamics of FtsZ by interacting with the C-terminal tail of FtsZ

    Biochemistry 46:11013–11022.

    https://doi.org/10.1021/bi700710j

    • PubMed
    • Google Scholar
48. 1. Squyres GR
    2. Holmes MJ
    3. Barger SR
    4. Pennycook BR
    5. Ryan J
    6. Yan VT
    7. Garner EC

    (2020) Dynamics of bacterial cell division: z ring condensation is essential for cytokinesis

    Nature Microbiology 6:553–562.

    https://doi.org/10.1038/s41564-021-00878-z

    • Google Scholar
49. 1. Stachowiak JC
    2. Schmid EM
    3. Ryan CJ
    4. Ann HS
    5. Sasaki DY
    6. Sherman MB
    7. Geissler PL
    8. Fletcher DA
    9. Hayden CC

    (2012) Membrane bending by protein-protein crowding

    Nature Cell Biology 14:944–949.

    https://doi.org/10.1038/ncb2561

    • PubMed
    • Google Scholar
50. 1. Strauss MP
    2. Liew AT
    3. Turnbull L
    4. Whitchurch CB
    5. Monahan LG
    6. Harry EJ

    (2012) 3D-SIM super resolution microscopy reveals a bead-like arrangement for FtsZ and the division machinery: implications for triggering cytokinesis

    PLOS Biology 10:e1001389.

    https://doi.org/10.1371/journal.pbio.1001389

    • PubMed
    • Google Scholar
51. 1. Szwedziak P
    2. Wang Q
    3. Freund SM
    4. Löwe J

    (2012) FtsA forms actin-like protofilaments

    The EMBO Journal 31:2249–2260.

    https://doi.org/10.1038/emboj.2012.76

    • PubMed
    • Google Scholar
52. 1. Szwedziak P
    2. Wang Q
    3. Bharat TA
    4. Tsim M
    5. Löwe J

    (2014) Architecture of the ring formed by the tubulin homologue FtsZ in bacterial cell division

    eLife 3:e04601.

    https://doi.org/10.7554/eLife.04601

    • PubMed
    • Google Scholar
53. 1. Tan IS
    2. Ramamurthi KS

    (2014) Spore formation in Bacillus subtilis

    Environmental Microbiology Reports 6:212–225.

    https://doi.org/10.1111/1758-2229.12130

    • PubMed
    • Google Scholar
54. 1. Tocheva EI
    2. López-Garrido J
    3. Hughes HV
    4. Fredlund J
    5. Kuru E
    6. Vannieuwenhze MS
    7. Brun YV
    8. Pogliano K
    9. Jensen GJ

    (2013) Peptidoglycan transformations during Bacillus subtilis sporulation

    Molecular Microbiology 88:673–686.

    https://doi.org/10.1111/mmi.12201

    • PubMed
    • Google Scholar
55. 1. Wang X
    2. Lutkenhaus J

    (1993) The FtsZ protein of Bacillus subtilis is localized at the division site and has GTPase activity that is dependent upon FtsZ concentration

    Molecular Microbiology 9:435–442.

    https://doi.org/10.1111/j.1365-2958.1993.tb01705.x

    • PubMed
    • Google Scholar
56. 1. Wu LJ
    2. Feucht A
    3. Errington J

    (1998) Prespore-specific gene expression in Bacillus subtilis is driven by sequestration of SpoIIE phosphatase to the prespore side of the asymmetric septum

    Genes & Development 12:1371–1380.

    https://doi.org/10.1101/gad.12.9.1371

    • PubMed
    • Google Scholar
57. 1. Wu LJ
    2. Errington J

    (2004) Coordination of cell division and chromosome segregation by a nucleoid occlusion protein in Bacillus subtilis

    Cell 117:915–925.

    https://doi.org/10.1016/j.cell.2004.06.002

    • PubMed
    • Google Scholar
58. 1. Yang X
    2. Lyu Z
    3. Miguel A
    4. McQuillen R
    5. Huang KC
    6. Xiao J

    (2017) GTPase activity-coupled treadmilling of the bacterial tubulin FtsZ organizes septal cell wall synthesis

    Science 355:744–747.

    https://doi.org/10.1126/science.aak9995

    • PubMed
    • Google Scholar
59. 1. Yen Shin J
    2. Lopez-Garrido J
    3. Lee SH
    4. Diaz-Celis C
    5. Fleming T
    6. Bustamante C
    7. Pogliano K

    (2015) Visualization and functional dissection of coaxial paired SpoIIIE channels across the sporulation septum

    eLife 4:e06474.

    https://doi.org/10.7554/eLife.06474

    • PubMed
    • Google Scholar
60. 1. Youngman P
    2. Perkins JB
    3. Losick R

    (1984) A novel method for the rapid cloning in Escherichia coli of Bacillus subtilis chromosomal DNA adjacent to Tn917 insertions

    Molecular and General Genetics MGG 195:424–433.

    https://doi.org/10.1007/BF00341443

    • PubMed
    • Google Scholar
61. 1. Zheng SQ
    2. Palovcak E
    3. Armache JP
    4. Verba KA
    5. Cheng Y
    6. Agard DA

    (2017) MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy

    Nature Methods 14:331–332.

    https://doi.org/10.1038/nmeth.4193

    • PubMed
    • Google Scholar

Author details

1. Kanika Khanna

   Division of Biological Sciences, University of California San Diego, La Jolla, United States

   Present address

   Department of Bioengineering and ChemH, Stanford University, Stanford, United States

   Contribution

   Conceptualization, Formal analysis, Investigation, Methodology, Writing - original draft

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-7150-0350

2. Javier Lopez-Garrido

   Division of Biological Sciences, University of California San Diego, La Jolla, United States

   Present address

   Max Planck Institute for Evolutionary Biology, Plön, Germany

   Contribution

   Conceptualization, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-8907-502X

3. Joseph Sugie

   Division of Biological Sciences, University of California San Diego, La Jolla, United States

   Contribution

   Formal analysis, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-2911-1807

4. Kit Pogliano

   Division of Biological Sciences, University of California San Diego, La Jolla, United States

   Contribution

   Conceptualization, Supervision, Funding acquisition, Writing - review and editing

   For correspondence

   kpogliano@ucsd.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-7868-3345

5. Elizabeth Villa

   Division of Biological Sciences, University of California San Diego, La Jolla, United States

   Contribution

   Conceptualization, Supervision, Funding acquisition, Writing - review and editing

   For correspondence

   evilla@ucsd.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-4677-9809

• 3,646

  views

• 500

  downloads

• 35

  citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.