Themes and Variations in Prokaryotic Cell Division

FEMS Microbiology Reviews 24 (2000) 531^548 www.fems-microbiology.org

Themes and variations in prokaryotic cell division

William Margolin *

Department of Microbiology and Molecular Genetics, University of Texas-Houston Medical School, 6431 Fannin, Houston, Texas 77030, USA

Received 26 January 2000; received in revised form 6 June 2000; accepted 20 June 2000 Downloaded from https://academic.oup.com/femsre/article/24/4/531/510989 by guest on 04 October 2021

Abstract

Perhaps the biggest single task facing a bacterial cell is to divide into daughter cells that contain the normal complement of chromosomes. Recent technical and conceptual breakthroughs in bacterial cell biology, combined with the flood of genome sequence information and the excellent genetic tools in several model systems, have shed new light on the mechanism of prokaryotic cell division. There is good evidence that in most species, a molecular machine, organized by the tubulin-like FtsZ protein, assembles at the site of division and orchestrates the splitting of the cell. The determinants that target the machine to the right place at the right time are beginning to be understood in the model systems, but it is still a mystery how the machine actually generates the constrictive force necessary for cytokinesis. Moreover, although some cell division determinants such as FtsZ are present in a broad spectrum of prokaryotic species, the lack of FtsZ in some species and different profiles of cell division proteins in different families suggests that there are diverse mechanisms for regulating cell division. ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

Keywords: Bacterium; Cell division; Septation; Binary ¢ssion

Contents 1. Introduction ...... 532 2. Cell division: a major developmental event ...... 532 3. FtsZ, the keystone of the cell division apparatus ...... 532 4. FtsA and Z-interacting protein A (ZipA), FtsZ-interacting proteins ...... 533 5. FtsK and FtsW, polytopic integral membrane proteins ...... 536 6. The bitopic cell division proteins ...... 536 7. A conserved septum/cell wall synthesis cluster ...... 537 8. Other genes and factors that a¡ect cell division ...... 537 9. Cell division in other model systems: B. subtilis ...... 537 10. Cell division in other model systems: C. crescentus ...... 538 11. Cocci ...... 538 12. Chlamydia ...... 539 13. Mycoplasma and L-forms ...... 539 14. Archaea ...... 539 15. Organelles ...... 540 16. Factors involved in the speci¢cation of the division plane ...... 540 16.1. The Min system ...... 540 16.2. The nucleoid ...... 542 16.3. A new model for E. coli division site placement ...... 543 17. Perspectives ...... 543

Acknowledgements ...... 544

References ...... 544

* Tel.: +1 (713) 500-5452; Fax: +1 (713) 500-5499; E-mail: [email protected]

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1. Introduction been fully sequenced: Aeropyrum pernix, a crenarchaeon, and Ureaplasma urealyticum, a mycoplasma species. De- In order to proliferate vegetatively, all cells ¢rst dupli- spite these and probably other exceptions, FtsZ is present cate their chromosomes into separate subcellular compart- in lower and higher plants and appears to be important ments, then split by dividing their cytoplasms somewhere for chloroplast division (see Section 15). between the chromosomes to yield progeny cells. This ba- In E. coli, FtsZ appears to act at the earliest known step sic process of cell division is conceptually similar in eu- in cell division. Conditional mutants of ftsZ in E. coli fail karyotic and prokaryotic cells. There are several advan- to divide, yielding long ¢lamentous cells that replicate and tages of studying cell division in prokaryotes: the process segregate their chromosomes but have no sign of any di- is likely to be simpler, there are several outstanding model vision septa or cellular constrictions (thus the term `fts', systems for study such as Escherichia coli, Caulobacter for `¢lamentous temperature sensitive'). FtsZ is also the crescentus and Bacillus subtilis, and a greater understand- target of SulA protein, synthesis of which is induced ing of cell division in bacteria may lead to novel therapeu- upon DNA damage [10]. SulA transiently prevents FtsZ tic antimicrobial compounds. Over the last decade, advan- from functioning in cell division, thus inhibiting unwanted Downloaded from https://academic.oup.com/femsre/article/24/4/531/510989 by guest on 04 October 2021 ces in cytological and genomic technologies have greatly cell divisions until the damage to the chromosome can be increased our understanding of cell division in prokary- repaired [11^13]. otes, particularly in the model systems. It is likely, how- A structural role for FtsZ was initially suggested by its ever, that we will ¢nd a diversity of cell division mecha- abundance, about 15 000 monomers per average E. coli nisms that mirror the diversity of microbial life. The cell [14], and by its localization by immunogold labeling purpose of this review is to summarize the common to a ring structure at the future site of division [15]. Lo- themes of cell division as well as the likely variations cated at the cytoplasmic membrane, the Z ring, as it is among the vast prokaryotic world. called, appears to contract dynamically along with the membrane as it invaginates during formation of the division septum. The Z ring has also been detected by light 2. Cell division: a major developmental event microscopy in whole E. coli cells and in other bacteria and archaea by using either immuno£uorescence or green £uo- In many rod-shaped bacteria such as E. coli and B. rescent protein (GFP) fusions to FtsZ [16^20]. These subtilis, cell division involves the synthesis of a septum, methods con¢rmed the previous ¢ndings, demonstrated with some constriction also occurring in addition to sep- that Z rings occur in diverse prokaryotic species, and the tation in E. coli [1]. In others, such as C. crescentus, cell GFP studies directly demonstrated the dynamic properties division appears to occur exclusively by simultaneous con- of the Z ring during cell growth and division of E. coli striction of the entire cell envelope, resulting in tapered [21]. poles [2]. In these cases and in others, including cocci The localization of FtsZ to a ring structure at the divi- and in species such as cyanobacteria that form chains of sion site of E. coli (Figs. 1A and 2A) suggests that FtsZ cells, it is clear that cell division is a major developmental protein forms some type of cytoskeletal structure [5]. Un- event, arguably the predominant cellular event in the veg- fortunately, an actual FtsZ structure has not yet been vi- etative life cycle of a bacterial cell [3]. To redirect cell wall sualized in thin sections of E. coli cells. However, much growth in a new direction (formation of the septum) and/ has been learned about the properties of FtsZ protein that or to provide the constrictive force at a single location in can help formulate a model about its structural role in cell the cell, particularly against several atmospheres of turgor division. There is now overwhelming evidence in favor of pressure, it is easy to imagine that a fairly complex mo- the idea that FtsZ is a homolog of tubulin, the ubiquitous lecular machine must be required. Recent evidence indi- eukaryotic cytoskeletal protein involved in many essential cates that a protein machine dedicated to the process of cellular processes including mitosis [7]. Despite only lim- cell division is assembled between segregated chromo- ited primary sequence homology centered around a GTP somes at the proper time [4^6]. The key to this machine's binding motif termed the `tubulin signature sequence' [22^ assembly is FtsZ [7^9]. 25], the recently solved crystal structures of FtsZ and tubulin show extensive structural homology throughout the proteins [26]. In addition, FtsZ, like tubulin, binds and 3. FtsZ, the keystone of the cell division apparatus hydrolyzes GTP and assembles into proto¢laments that have structures similar to those within microtubules FtsZ is by far the most highly conserved of the known [27,28]. This assembly is GTP-dependent [29,30] and dis- cell division proteins. It is present in most species of pro- assembly occurs when the GTP is exhausted, suggesting karyotes examined to date (Table 1). Interestingly, it is not that FtsZ polymers, like microtubules, are dynamically present in the obligately intracellular chlamydiae that di- unstable [31]. FtsZ and tubulin also share similar re- vide by binary ¢ssion within a host inclusion. It is also not sponses to hydrophobic dyes: while bis-anilino-naphthale- present in two free-living species whose genomes have nesulfonate (bis-ANS) inhibits polymerization of both

FEMSRE 690 29-8-00 Cyaan Magenta Geel Zwart W. Margolin / FEMS Microbiology Reviews 24 (2000) 531^548 533 proteins, the related dye ANS has no e¡ect on either [32]. to the latter puzzle is Era, a highly conserved and essential Another link between FtsZ and tubulin in vivo is that they GTPase. Depletion of Era causes a cell cycle delay, result- can be made to coalign as polymers in mammalian cells in ing in cells containing four nucleoids and an unconstricted the presence of vinblastine, a microtubule-destabilizing Z ring [40]. Whether Era localizes to the Z ring and how it drug [33]. might be involved in regulating its activity awaits further FtsZ and the Z ring are essential for cell division [34]. investigation. Thermoinactivation of a mutant FtsZ or depletion of na- What is the function of the Z ring? Does it provide the tive FtsZ results in the rapid disappearance of rings and contractile force? Or does it lack motor properties itself the failure of cells to divide, yielding long ¢lamentous cells but instead serves as a cellular organizer that recruits other lacking Z rings or septa [35]. Because FtsZ is rate-limiting proteins into a molecular machine? To address this ques- for cell division [36,37], one possible trigger for assembly tion, it is necessary to review what is known about other of the ring is a critical concentration of FtsZ monomers in proteins essential for cell division in E. coli. the cell. In E. coli, the small variation in FtsZ levels during the cell cycle [38] is unlikely to be su¤cient to trigger Downloaded from https://academic.oup.com/femsre/article/24/4/531/510989 by guest on 04 October 2021 assembly. The average in vivo concentration is estimated 4. FtsA and Z-interacting protein A (ZipA), to be about 10 WM per average cell, about 5^10 times FtsZ-interacting proteins higher than the critical concentration for assembly in vitro [14,29,39]. Therefore, FtsZ may always be in excess in the FtsA was found by the original fts screen, except that cell and ring assembly may be subject to negative regula- ftsA mutant ¢laments exhibited regular constrictions in- tion by FtsZ assembly inhibitors (see Section 16). The stead of no constrictions observed in ftsZ mutant ¢la- speci¢c cell cycle signal that triggers Z ring assembly at ments. This suggested that FtsA acted later in the division the proper time remains elusive, as is the signal that trig- process [41,42]. FtsA is the only essential cell division pro- gers constriction of the ring once it is assembled. One clue tein other than FtsZ that is predicted to reside solely in the

Table 1 Cell division proteins across species Species Family FtsZ FtsA ZipA FtsK FtsW FtsQ FtsL FtsI FtsN MinC MinD MinE Eco Q-proteobacteria X X X X X XXXXXXX Hin Q-proteobacteria X X X X X XXXX Nme L-proteobacteria XX XXX X XXX Rpr K-proteobacteria XX X X x X Cje O-proteobacteria XX XX X X Hpy O-proteobacteria XX XX X x X X Ctr chlamydiae X X X Cpn chlamydiae X X X Tpa spirochaetes X X X X x X X Bbu spirochaetes X X X X x X X Mtu +high GC X X X x X X Bsu +low GC X X X X x X X X DivIVA Mga +low GC X Mpn +low GC X Uur +low GC X Ssp cyano/chlor X x X x X X X X Dra Deino/Thermus XX XXx X X Aae Aqui¢cales XXXXXX Tma Thermotogales XXXXX Mja euryarchaeota X X Mth euryarchaeota X X Afu euryarchaeota X X Pho euryarchaeota X X Pab euryarchaeota X X Ape crenarchaeota Bacteria are listed at the top and archaea at the bottom. All species shown are fully sequenced; all have been published except for Uur. Deino/thermus represents the Deinococcus/Thermus family. FtsW entries include the E. coli mrdE and B. subtilis spoVE families. FtsK entries include the SpoIIIE homologs. Large Xs represent strong similarity, and small xs re£ect weak similarity. Small xs in the FtsQ column represent weak sequence similarity with E. coli FtsQ but stronger similarity among other FtsQs. DivIVA has some functional similarity with MinE but no sequence similarity. Eco: E. coli; Hin: H. in£uenzae; Nme: Neisseria meningitidis; Rpr: Rickettsia prowazekii; Cje: Campylobacter jejuni;Hpy:Helicobacter pylori; Ctr: Chlamydia trachomatis; Cpn: Chlamydia pneumoniae; Tpa: Treponema pallidum; Bbu: Borrelia burgdorferi; Mtu: Mycobacterium tuberculosis; Bsu: B. subtilis; Mga: Mycoplasma genitalium;Mpn:Mycoplasma pneumoniae; Uur: U. urealyticum; Ssp: Synechocystis sp.; Dra: D. radiodurans; Aae: Aquifex aeolicus; Tma: Thermotoga maritima; Mja: Methanococcus jannaschii; Mth: Methanobacterium thermoautotrophicum; Afu: Archaeoglobus fulgidus; Pho: Pyrococcus horikoshii; Pab: Pyrococcus abyssi; Ape: A. pernix.

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Fig. 1. Models for the assembly of the cell division apparatus in E. coli. (A) A possible time course of events. Nucleoids are shown as red ovals, the cell envelope as large black ovals, and the Z ring and its associated components as colored rings. Di¡erent ring colors represent its putative di¡erent states of assembly. Early in the cell cycle, MinE forms a ring near the future division site, keeping the MinCD oscillating inhibitor away and clearing the way for Z ring assembly. DNA replication results in the duplication and rapid segregation of origins toward the poles [169]; this is followed by termination of replication, further nucleoid alignment and decatenation. Approximately when replication termination occurs, the Z ring assembles at the division site. The MinE ring may still persist but for simplicity is not shown after this point. Then the other essential cell division proteins are recruited in a linear order. This may occur all at once or may occur sequentially over a signi¢cant time period; for clarity, a sequential type of recruitment is shown. Finally, the ring contracts, the septum is laid down and the cells separate. (B) A possible cross-sectional view of a small section of the E. coli division machine at the cytoplasmic membrane. About 100 monomers of FtsZ, comprising one proto¢lament, are in the pictured part of the ring. In this speculative model, the other proteins are found in well-distributed clusters, with FtsA being central for the recruitment of the cluster. ZipA is shown in a separate location from the cluster, but there is no evidence for or against this idea other than FtsA and ZipA are recruited independently. cytoplasm (Fig. 2B). FtsA is a member of the ATPase and it is completely unknown how FtsA helps FtsZ to superfamily that includes actin and HSP70 [43]. It is divide the cell. Puri¢ed FtsA can be phosphorylated and well-conserved in many, though not all, bacteria, but it bind ATP, and the viability of a mutant that cannot bind is notably absent in mycobacteria, cyanobacteria and my- ATP suggests that ATP binding and possible hydrolysis coplasmas. It is also absent in archaea. The ftsA gene is may not be essential for FtsA function [46]. Additional usually present immediately upstream of ftsZ within the clues to FtsA function may emerge from mutants of cell division^cell wall biosynthesis gene cluster (dcw)ina FtsZ that can form Z rings but apparently can no longer variety of diverse species, suggesting that its product may recruit FtsA; these mutants have residue changes within a interact with FtsZ. Direct evidence for such an interaction short conserved region of the FtsZ C-terminus which may comes from yeast two-hybrid screens and colocalization represent a protein^protein interaction domain [47,48]. experiments with GFP-tagged FtsAs. Cytological experi- Another clue is the importance of the ratio between ments demonstrated that FtsA localizes to the Z ring and FtsA and FtsZ in the cell, which is estimated to be about its localization depends on FtsZ, whereas Z ring localiza- 1:100. Altering this ratio by overproducing FtsA, for ex- tion does not depend on FtsA [17,18,44,45]. This is con- ample, inactivates septation, but cell division can be re- sistent with the later cell division defect apparent in the stored under these conditions by proportionally increasing ftsA (ts) mutant. However, inactivation of FtsA also pre- levels of FtsZ [49,50]. FtsA and FtsZ appear to interact vents the recruitment of a number of other cell division directly in bacteria, as described above, and in the yeast proteins (see Sections 5 and 6). This indicates that FtsA two-hybrid system [48,51]. Taken together, the evidence may act relatively early in division, and that the numerous suggests that FtsA binds to FtsZ monomers within the ftsA (ts) mutants that have been isolated may all have ring, but because of the 1:100 ratio, FtsA must bind residual activity, allowing septum formation to initiate only to a small subset of these FtsZ monomers. Because but then abort. the other cell division proteins with periplasmic compo- Despite the genetic and cytological evidence, little is nents (see Sections 5 and 6) are probably less abundant known about the biochemical properties of FtsA protein, in the cell than FtsA, an important function of FtsA may

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Fig. 2. Micrographs of bacterial cells. A^C show immuno£uorescence images of Z rings in an E. coli ftsK mutant (A), wild-type B. subtilis (B) and an E. coli spherical (rodA) mutant (C). Nucleoids are stained with DAPI in (A) and (B) and appear red in pseudocolor. D^E show phase contrast images of E. coli cells before (D) and after (E) induction of synthesis of a C-terminally truncated FtsA, which causes the cells to bend dramatically [52].

be to limit the number of assembly sites for these proteins E. coli, ZipA is essential for cell division, requires the Z (Fig. 1B). If FtsA indeed distributes itself among a limited ring for recruitment, and is recruited to the Z ring inde- number of FtsZ monomers within the ring, it is not clear pendently of FtsA [55]. Z rings are present when ZipA is how this would be accomplished. depleted from the cell, although in some cases the number One unusual property of FtsA is that overproduction of of rings is signi¢cantly reduced. This ¢nding, as well as a C-terminally truncated FtsA causes E. coli cells to be- recent biochemical evidence [56], suggests that ZipA may come markedly curved [52] (Fig. 1D,E). This e¡ect does function to stabilize the Z ring immediately after it is not require functional FtsZ or FtsI [53]. One speculative assembled. The topology of ZipA implies that this stabili- explanation of this phenomenon is that high levels of the zation may be accomplished by ZipA-mediated anchoring truncated FtsA protein can assemble at a single site on the of the ring to the cytoplasmic membrane (Fig. 2B). Inter- cytoplasmic membrane, independent of the Z ring, and estingly, recent evidence indicates that the conserved ex- recruit but not properly orient a subset of cell division treme C-terminus of FtsZ may be involved in recruitment proteins. This would stimulate abnormal septum-like of ZipA as well as FtsA [47,57]. Assuming that this do- wall synthesis only on one side of the cell, resulting in a main is directly responsible for recruitment, it seems at curved cell morphology. ¢rst that it would not be possible for one small protein ZipA was not found in the screen for fts mutants. In- domain to recruit two di¡erent proteins. However, the stead, it was found in a biochemical screen for proteins predicted large excess of FtsZ relative to FtsA and ZipA that interacted with puri¢ed FtsZ [54]. ZipA is predicted might easily allow recruitment of both proteins to separate to be an integral membrane protein, with an unusual N- segments of the ring (Fig. 2B). It is notable that overpro- terminal membrane anchor and a C-terminal cytoplasmic duction of ZipA can inactivate cell division [54], which is domain. It is not particularly conserved, with homologs analogous to the e¡ect of FtsA overproduction. The only in other Q-proteobacteria (Table 1). Nevertheless, in mechanism of this inhibition is unknown. It may occur

FEMSRE 690 29-8-00 Cyaan Magenta Geel Zwart 536 W. Margolin / FEMS Microbiology Reviews 24 (2000) 531^548 in part because ZipA or FtsA, when overproduced, may vision complex so as to prevent chromosome scission. It compete for accessible FtsZ sites and therefore prevent does so presumably by actively transporting DNA away recruitment of the other. from the closing septum while simultaneously achieving the last step in septum closure. The viability of ftsK C- terminal deletions [70] indicates that not all chromosomes 5. FtsK and FtsW, polytopic integral membrane proteins get trapped and that backup systems exist. However, such deletion strains have defects in cell^cell separation, sta- FtsK and FtsW are both essential cell division proteins tionary phase survival and adaptation to stress [70]. More- that are predicted to span the membrane multiple times over, combining an ftsK C-terminal deletion with a null [58^60] (Fig. 2B). FtsK is recruited to the Z ring and mutation in mukB, which is probably involved in chromo- requires both FtsZ and FtsA for recruitment, but not some condensation, yields non-viable cells; this suggests FtsI or FtsQ [61,62]. FtsW may have a ZipA-like role, that decondensed chromosomes are more susceptible to as depletion of FtsW results in a decrease in Z rings; being trapped [66]. A similar synthetic phenotype between however, nothing else is known about how FtsW functions SMC (a probable functional homolog of mukB) and Downloaded from https://academic.oup.com/femsre/article/24/4/531/510989 by guest on 04 October 2021 in cell division or how it is recruited to the ring [63]. One spoIIIE was found in B. subtilis [71]. FtsK is also induced attractive idea is that FtsW serves to integrate signals be- upon SOS-mediated DNA damage, which is consistent tween the cytoplasmic components of the machine (FtsZ with its important role in DNA dynamics [62]. and FtsA) and the periplasmic components including FtsQ (see Section 6). FtsK is one of the largest proteins in E. coli and appears 6. The bitopic cell division proteins to have multiple functions. Its N-terminal 15% is su¤cient for its targeting to the Z ring and its function in cell Four known essential cell division proteins in E. coli, division [61,64]. Interestingly, the requirement for FtsK FtsQ, FtsL, FtsI and FtsN, possess a short N-terminal in cell division can be bypassed by deletion of the gene cytoplasmic anchor, a transmembrane segment and a rel- for penicillin binding protein 5 (PBP5), a carboxypeptidase atively large periplasmic domain [72^74]. FtsI, also known that removes a dipeptide from the pentapeptide side chain as PBP3, has been relatively well-characterized. There is of peptidoglycan to make a tripeptide. Overproduction of good evidence that it is a transpeptidase and functions in FtsN (see Section 6) can also bypass the FtsK requirement one of the key enzymatic steps in septal peptidoglycan [64]. The mechanism behind the bypass e¡ects is not clear. synthesis [75]. The L-lactam drugs cephalexin and aztreo- In the case of PBP5, shifting the balance toward precur- nam preferentially inhibit PBP3 activity without signi¢- sors with pentapeptide side chains, which are normally cantly inhibiting the activity of other PBPs that synthesize used in cell elongation, may compensate for the loss of lateral wall peptidoglycan, such as the PBP1 class. These FtsK. This suggests that FtsK-mediated septum closure drugs have been useful for speci¢cally blocking cell divi- may require di¡erent peptidoglycan precursors than the sion, and they are currently the only known drugs that tripeptide precursors normally used for septum synthesis directly target the cell division apparatus. [65]. Interestingly, overproduction of FtsK, as with ZipA All four of these proteins localize to the Z ring (Fig. and FtsA, results in inhibition of cell division; in the case 2A,B). By combining cytological techniques with fts mu- of FtsK, it appears that the major e¡ect is by inhibiting tants, enough evidence has accumulated to propose a the assembly of Z rings [64]. The mechanism of this inhi- model for their recruitment. For example, FtsQ tagged bition is unknown but may prove to be generally signi¢- at its cytoplasmic N-terminus with GFP localizes to the cant for our understanding of protein^protein interactions Z ring independently of FtsL and FtsI but requires FtsZ within the division complex. and FtsA [76]; a similar GFP^FtsI fusion protein fails to Whereas the N-terminal domain of FtsK is essential for localize in the absence of FtsZ, FtsA, FtsQ or FtsL [77], cell division, the C-terminal domain appears to have a role consistent with immuno£uorescence results with FtsI [63]. in coupling cell division with chromosome segregation FtsL requires FtsZ, FtsA and FtsQ but not FtsI to localize [66,67]. This domain is highly similar to the C-terminus [78]. FtsN is recruited last, and depends on FtsI [79]. In of SpoIIIE, which is required in B. subtilis to transport summary, these proteins appear to be recruited in a linear chromosomes through septa in which they have been series after FtsZ, FtsA, ZipA and FtsK (and perhaps trapped [68]. In E. coli, deletions of the C-terminal domain FtsW) are localized (Fig. 2A). of FtsK result in abnormal chromosome positioning and The functions of FtsQ, FtsL and FtsN are unknown, chromosome bisection by closing septa. These deletions but their tripartite domain structure has been exploited in also result in failure to resolve chromosome dimers at domain swapping studies. These studies indicate that re- the dif site, suggesting that many of the positioning defects placing domains becomes increasingly deleterious the later may be caused by dimer resolution problems [69]. The the protein is recruited. For example, all three domains of current working model for FtsK proposes that it helps FtsI, the periplasmic and cytoplasmic domains of FtsL, to keep chromosomes away from the contracting cell di- but only the periplasmic domain of FtsQ are essential

FEMSRE 690 29-8-00 Cyaan Magenta Geel Zwart W. Margolin / FEMS Microbiology Reviews 24 (2000) 531^548 537 for function and cannot be replaced [72,76,77,80]. The genes are present in Streptococcus pyogenes but are dis- periplasmic portion of FtsL has a coiled coil structure tributed around the genome in three non-contiguous clus- and is essential for its localization and function; recent ters [87]. In mycoplasmas that contain ftsZ, the gene order results suggest that this domain multimerizes and that is mraZ-mraW-MG223-ftsZ, indicating that the termini of this multimerization is important for function [80]. FtsQ the cluster in this `minimal genome' are still conserved but and FtsL are moderately conserved among bacteria, but that all of the other intervening dcw genes are replaced by FtsN is only found in E. coli and Haemophilus in£uenzae. a single gene of unknown function, MG223. In archaea, The role of FtsN is puzzling. Despite its late recruit- mraZ-mraW are missing, and ftsZ is the only recognizable ment, FtsN when overproduced can suppress mutations dcw representative. in ftsA and ftsK [64,81], implicating FtsN in some type of global process. Consistent with this, depletion of FtsN results in an `early' cell division defect, with ¢la- 8. Other genes and factors that a¡ect cell division ments lacking indentations. This suggests that most or all of the essential cell division proteins are already in The discussion so far has been limited to nine genes Downloaded from https://academic.oup.com/femsre/article/24/4/531/510989 by guest on 04 October 2021 place early during the septum formation process. One known to be essential for E. coli cell division. All but way that FtsN might in£uence the cell division machinery zipA were found in genetic screens for e¡ects on cell divi- globally is by regulating the availability of substrates or sion. The recent availability of genomic sequences of a modulating the ability of the cell envelope to invaginate. It variety of microbial species suggests that other highly con- is also possible that FtsN does not need to localize to the served genes may be required for cell division. The mraZ- ring in order to be active. mraW genes are examples of highly conserved genes that do not seem to have a cell division phenotype. However, yihA, which has GTPase motifs, has a cell division pheno- 7. A conserved septum/cell wall synthesis cluster type when inactivated but it is not clear if the e¡ect is direct or indirect [88]. It is likely that further functional Many of the essential cell division genes, including ftsI, genomic analysis of potential cell division genes will lead ftsW, ftsQ, ftsA and ftsZ, are found together with genes to additional essential cell division genes. for peptidoglycan synthesis within the dcw cluster. The In many cases, cell division can be inhibited by muta- dcw genes not exclusively involved in cell division include tions or overexpression of genes known to be involved in the mur (murein), ddl (alanine ligase) and mra gene fami- other cellular processes, indicating that there are many lies. Despite the omission or addition of speci¢c members, indirect ways to a¡ect cell division. One class of inhibitory this cluster and the gene order within it are highly con- e¡ects includes mutations that perturb chromosome repli- served in a number of bacteria. In E. coli, the transcrip- cation, structure or segregation; inhibition of cell division tional organization of the dcw cluster is complex, but all results from either SOS-mediated induction of SulA or a transcription proceeds in one direction [82]. Genes ftsZ topological veto by the nucleoid (see Section 16). Another and envA, a gene involved in a step in lipid biosynthesis class includes direct e¡ects on cell division proteins. For that is also important for cell separation [83], lie at the 3P example, altered synthesis of DnaK, trigger factor, and the end. It is likely that ftsZ is the most heavily transcribed of immunophilin homolog slyD results in ¢lament formation the genes, and there are additional upstream promoters [89^91]. In these and other cases, overproduction of FtsZ throughout the cluster that are responsible for high-level often can suppress the inhibition, suggesting that the in- ftsZ expression [84]. Regulation of ftsZ gene transcription hibitory e¡ects are caused by a decrease in the level of does not appear to play a signi¢cant role in activating cell FtsZ activity such as decreased ftsZ gene expression or division in E. coli, as cells in which ftsZ is expressed ec- improper FtsZ folding. De¢ciencies in factors as diverse topically from an inducible promoter in the absence of a as S-adenosyl methionine [92] or speci¢c membrane phos- functional native ftsZ grow and divide normally. How- pholipids [93] result in speci¢c cell division defects. Con- ever, several proteins regulate transcription of dcw genes, stitutive activation of the cpxA periplasmic stress pathway particularly ftsA and ftsZ, and it remains to be seen how does not a¡ect the cell's ability to divide, but division this regulation a¡ects and is a¡ected by cell physiology often occurs at abnormal sites [94]. Dissecting the mecha- [4,85,86]. nisms by which these and other proteins a¡ect cell division The genes at the termini of the cluster, including ftsZ at will be a challenge because of the multiple roles of many the downstream end and mraZ-mraW at the upstream of these proteins in cell physiology. end, appear to be the most conserved among bacteria, whereas the arrangement of internal genes within the cluster is more variable among di¡erent species. This variabil- 9. Cell division in other model systems: B. subtilis ity amidst the common core of dcw genes may correlate with the likely di¡erences in cell division mechanisms in Cell division in B. subtilis di¡ers from that of E. coli di¡erent species. For example, many of the usual dcw in several ways. First, cytokinesis in the Gram-positive

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B. subtilis is achieved by formation of a thick septum with- division is C. crescentus. One of the reasons C. crescentus out signi¢cant constriction of the outer cell envelope, is attractive for the study of cell division and the cell cycle whereas cytokinesis in E. coli appears to occur by simul- is that like B. subtilis, C. crescentus undergoes an asym- taneous formation of a septum and cell envelope constric- metric division that results in daughter cells having di¡er- tion. Second, septation in B. subtilis normally occurs at the ent developmental fates. The two distinct cell types are cell midpoint during vegetative growth. However, upon swarmer and stalked cells, which arise initially by the de- starvation, B. subtilis cells initiate a highly regulated devel- velopment of asymmetry within the mother cell prior to opmental program that culminates in the formation of a cell division. The cell cycle appears to be regulated much terminal endospore. One of the critical steps in this pro- more tightly than in the other bacterial model systems and gram is the switch from medial cell division to polar cell thus is reminiscent of an eukaryotic cell cycle, in which division, which results in the formation of a septum that each step is dependent on and regulated by a previous separates the mother cell from the prespore. The asymmet- step. For example, DNA replication initiates only in stalk ric compartments generated by this spore septum are im- cells and not in swarmer cells. Moreover, cell division is portant for the creation of two cells with complete ge- regulated in ways that appear to be distinct from those of Downloaded from https://academic.oup.com/femsre/article/24/4/531/510989 by guest on 04 October 2021 nomes but with di¡erent developmental fates [95,96], E. coli or B. subtilis. First, on a structural level, cell divi- although di¡erent-sized compartments are not absolutely sion appears to occur by constriction or a cleavage furrow required for this to occur [97]. The alteration of Z ring and not by formation of a septum [2]. As a result, the localization from midcell in vegetatively growing cells [98] poles are tapered, which may facilitate the biogenesis of (Fig. 1B) to the poles is controlled by the master response the polar stalk. Whether this re£ects the plasticity of the regulator Spo0A [16], but it is not yet known what factors cell envelope or a di¡erent mechanism of cell wall syn- directly cause the change in Z ring localization. Identi¢ca- thesis is not known. Second, unlike in E. coli, C. crescentus tion of factors involved more directly in the switch should FtsZ levels are tightly regulated by transcriptional and contribute important insights into the spatial control of posttranslational control [110,111]. The result is that the division plane. FtsZ is only present in cells during a short cell cycle win- As might be expected from their di¡erent cell envelopes, dow. Expression of ftsZ is repressed by the global regu- there are some di¡erences in the proteins involved in B. lator CtrA in swarmer cells, which do not divide, but is subtilis cell division with respect to those of E. coli. Normal derepressed in stalked cells [111]. In addition, FtsZ is pro- B. subtilis cell division requires FtsZ, FtsA, FtsL (a distant teolyzed speci¢cally in cells undergoing division, e¡ectively relative of E. coli FtsL), the FtsQ homolog DivIB and the removing FtsZ from both daughter cells. Interestingly, FtsI homolog PBP2b [99^103,176]. The B. subtilis homolog ftsZ is expressed ¢rst, followed by ftsA and ftsQ; this of FtsK, SpoIIIE, is essential for segregating the prespore timing of expression re£ects the timing of function of chromosome into the prespore and is involved in fusing the the gene products [112]. It will be instructive to ¢nd out inner and outer membranes of the spore upon its engulf- how the placement of the Z ring is controlled in this or- ment by the mother cell [104]. However, SpoIIIE is not ganism, given the apparent lack of some negative regula- required for vegetative division despite its recruitment to tory controls which are present in other species (see Sec- the septum [105], and detectable homologs of the other tion 16). proteins essential for E. coli division are not present or are not essential for division in B. subtilis. Conversely, Div- IC is an essential cell division protein in B. subtilis that is 11. Cocci not present in E. coli or most other species [106]. Like DivIB, DivIC is highly abundant, bitopic in structure and Very little is known about the molecular aspects of cell acts late, requiring FtsL for localization [99,107^109]. Cy- division in other bacteria. Nevertheless, one common tological evidence suggests a linear order of recruitment to thread likely to emerge is the role of FtsZ as a cellular the Z ring, with FtsZ and FtsA arriving ¢rst, followed by organizer. In most standard rod-shaped cells, FtsZ prob- FtsL, DivIB and PBP2b. This is somewhat di¡erent from ably forms a ring at the cell midpoint to divide the cell. E. coli in that FtsL recruitment in E. coli requires FtsQ(Di- Given the variable conservation of the other cell division vIB); however, B. subtilis FtsL shares little sequence ho- proteins and the examples of B. subtilis and E. coli,itis mology with E. coli FtsL and may also not be functionally likely that the Z ring recruits a di¡erent set of proteins to homologous. In addition, some factors involved in specify- the division complex in di¡erent species. Such plasticity ing the position of the Z ring are di¡erent and appear to act may re£ect di¡erent cell wall biosynthetic pathways, by a di¡erent mechanism (see Section 16). PBPs, lipid composition, and/or mechanisms of chromosome organization and partitioning. The problem becomes more complex in cocci, which 10. Cell division in other model systems: C. crescentus have an in¢nite number of theoretical cell division planes that can give rise to two equal daughter cells, as opposed The third well-characterized system for bacterial cell to rods, which have essentially one division site that ful¢lls

FEMSRE 690 29-8-00 Cyaan Magenta Geel Zwart W. Margolin / FEMS Microbiology Reviews 24 (2000) 531^548 539 this criterion. Moreover, the larger diameter of most cocci of dividing reticulate bodies that may be a candidate for a relative to rods means that the Z ring must be signi¢cantly cell division protein [121]. It is intriguing that despite the larger and therefore may need to regulate assembly of the lack of FtsZ, chlamydiae contain several other homologs division machine di¡erently. of essential cell division genes including ftsK, ftsW and Enterococci apparently deal with the problem of identi- ftsI. Assuming these genes have functional roles in divi- ¢cation of the division plane by zonal cell wall growth. sion, how do their gene products work if there is no FtsZ New wall growth begins at a structure called a wall band, to recruit them? which is represented by a circumferential hump-like structure on the outside of the cell. Wall material then grows outward bidirectionally, duplicating the band to form a 13. Mycoplasma and L-forms notch and forming a Y-shaped structure. The two bands push apart until they are located at the midcell point of Mycoplasma are bacteria that lack cell walls and cause the two daughter cells. A new notch is then formed at each a variety of infections. They also contain small genomes band by duplication, and the process repeats [113]. Thus, that are good models for a minimal set of genes. As with Downloaded from https://academic.oup.com/femsre/article/24/4/531/510989 by guest on 04 October 2021 the division site is di¡erentiated by duplication of the wall organelles and archaea (see below), the only recognizable band and division culminates in the synthesis of a septum essential cell division homolog in mycoplasmas is ftsZ at the base of the wall band. Just as FtsZ seems to be (Table 1). The absence of the other genes such as ftsA, responsible for the switch from elongation to septal syn- ftsQ, etc. suggests that FtsZ is su¤cient for cell division thesis mode in E. coli, it may also be responsible for the and that the other proteins serve to coordinate cell wall duplication of the wall band. In this case the division site and septum synthesis with FtsZ-mediated cytokinesis. It is seems to be an epigenetic structure. tempting to speculate, based on this genomic evidence, In other cocci such as Deinococcus, however, this model that FtsZ itself can provide the constrictive force necessary does not seem to hold. Deinococcus radiodurans and the to split cells. However, mycoplasmas also have unusual Gram-negative cocci represented by Neisseria, among cytoskeletal-like proteins that are involved in cytadher- others, appear to divide in alternating planes [114^116]. ence; interestingly, perturbing the gene encoding one Staphylococcus aureus mutants defective in cell separation such protein results in a morphological defect that may can also divide this way [117]. While the enterococcal sys- be a result of cell division inhibition [122]. More work tem provides an explanation for the selection of the divi- needs to be done to demonstrate if this or other large sion plane, it is not at all clear how alternating perpendic- attachment proteins are involved in cell division in myco- ular planes are speci¢ed and how the switch in direction is plasmas. Mycoplasmas appear to have divergent division made. It is reasonable to propose that in rods, a major patterns, with some species dividing mainly by binary ¢s- function of FtsZ is to switch the direction of cell wall sion and others by budding [123]. Most intriguing is the synthesis by 90³, and perhaps a similar directional switch lack of FtsZ in U. urealyticum, indicating that some other functions in spheres. Interestingly, spherical mutants of E. means must be used to divide these cells. coli divide in alternating planes [118], and studying Z ring Bacteria other than mycoplasmas can be stripped of assembly and placement in such mutants (Fig. 2C) may be their walls and cultivated. These wall-less variants, called able to address these issues in a more tractable system. It L-forms, can be made in many species including E. coli is tempting to speculate that the perpendicular switching [124]. The fact that E. coli L-forms can grow and divide by FtsZ may be evolutionarily related to the two perpen- indicates that the cell wall is not essential for the division dicular structures of the animal cell centriole, which plays machinery to function. The tantalizing question here is an important role in organizing the microtubule cytoskel- whether the cell division proteins other than FtsZ are nec- eton. essary for L-form division; if not, then E. coli L-forms may be a viable model system for studying the su¤ciency of FtsZ for cell division. 12. Chlamydia

Other systems pose additional challenging questions. 14. Archaea For example, chlamydia species lack FtsZ (Table 1), yet clearly undergo binary ¢ssion as reticulate bodies within Despite having many eukaryotic characteristics, such as the chlamydial inclusion [119]. Having minimal genomes, eukaryotic-based transcription and DNA replication sys- do chlamydiae co-opt host dynamin, which is involved in tems, archaea look like bacteria [125]. It was therefore pinching o¡ membranes during endocytosis, or microtu- exciting but not a complete surprise when it was found bules for their own cell division [120]? If so, how do they that archaea contain FtsZ homologs and that FtsZ forms regulate the placement of the division plane? If they use a a ring at the midcell division site [20,126,127]. However, chlamydial protein, what is it? Recently, a protein of un- several surprises have emerged from genomic studies of known function has been immunolocalized to the equator archaeal cell division genes. First, as with mycoplasmas,

FEMSRE 690 29-8-00 Cyaan Magenta Geel Zwart 540 W. Margolin / FEMS Microbiology Reviews 24 (2000) 531^548 no other known essential cell division homologs are found tric electron-dense rings at the site of chloroplast division in archaea despite the presence of cell walls. Second, many [136]. However, this raises the question of how targeting archaeal species harbor two distinct paralogs of FtsZ, and of two FtsZs on either side of the chloroplast might be a compilation of many archaeal genome sequences indi- coordinated. cates that these paralogs form two separate groups [128]. The role of FtsZ in mitochondrial division is less clear, Essentially nothing is known about archaeal cell division, but has been illuminated in some recent studies. Several including the di¡erent functions, if any, of the dual FtsZs. completely sequenced animal and fungal genomes, includ- It should be emphasized that additional copies of ftsZ are ing nematodes and budding yeast, lack obvious FtsZ ho- also found in plants (see Section 15), and there is one case mologs. This indicates that FtsZ is not universally re- in bacteria (Sinorhizobium meliloti) of two distinct ftsZ quired for mitochondrial division. Recently, however, a homologs [129]. This suggests that ftsZ has a tendency mitochondrial FtsZ homolog from a chromophyte alga to form gene families and potentially multiple isotypes, has been isolated [137]. This homolog is most related to as does tubulin. Whereas the FtsZ paralogs in plants the FtsZs of the K-proteobacteria, further supporting the may have distinct functions (see Section 15), the di¡eren- idea that mitochondria descended from this family of pro- Downloaded from https://academic.oup.com/femsre/article/24/4/531/510989 by guest on 04 October 2021 tial function of FtsZ paralogs in prokaryotes has yet to be karyotes. The absence of FtsZ homologs in animal and explored. fungal species so far suggests that mitochondria of most The third, and biggest, surprise revealed by archaeal species either do not need to divide regularly and can genomes is the complete absence of ftsZ to date in a subset segregate by other mechanisms, or that another cytoskel- of the archaea known as the crenarchaea. This group in- etal protein such as dynamin may act to divide organelles cludes the recently sequenced A. pernix [130]. Whereas the in these species [138]. absence of ftsZ in chlamydia can be rationalized because chlamydiae are obligately intracellular and depend on the host for many cellular functions, the absence of ftsZ in 16. Factors involved in the speci¢cation of the division plane free-living bacteria that divide is di¤cult to understand. Sulfolobus species, crenarchaea that divide by budding 16.1. The Min system and grow under fairly normal conditions, have the potential to be a good model system for cell cycle studies [131]. As stated previously, cell division is a major develop- In particular, this organism also may serve as a good mental event. The primary question that arises is how the FtsZ-free system in which to study FtsZ-based bacterial cell division plane is identi¢ed. Clearly, we now know that cell division and potentially to assemble the division appa- bacteria are not just bags of enzymes and DNA, and they ratus de novo inside a cell. have within them distinct subcellular addresses for protein assembly. In rod-shaped bacilli, the correct division site is usually the midpoint between the two poles. How does 15. Organelles FtsZ ¢nd this site? We have recently developed a model that invokes neg- It is currently accepted that organelles arose from bac- ative regulation. In this model, the two main determinants terial endosymbionts: chloroplasts came from cyanobac- of division site placement are (i) the Min system and (ii) teria while mitochondria came from the K-proteobacteria the nucleoid and its associated components [139]. The Min [132]. As organelles need to divide in order to proliferate system is composed of three proteins, MinC, D and E and be maintained within their dividing eukaryotic hosts, which are encoded by the minCDE operon of E. coli it is natural to ask whether bacterial division mechanisms [140]. In E. coli, MinC and D act together to negatively have been conserved. Recent evidence strongly indicates regulate the assembly of the Z ring, while MinE acts to that they have been [133]. Nuclear-encoded FtsZ homo- negatively regulate the action of MinC and D [141]. GFP logs have been found in a number of photosynthetic eu- fusions to the Min proteins have revealed much about karyotes, ranging from protists to monocotyledonous and how they might function as regulators [142]. MinE^GFP dicotyledonous plants. Not surprisingly, these FtsZs are localizes as a ring-like structure near the cell midpoint most highly related phylogenetically to cyanobacterial (Fig. 2A), although the ring is often o¡-center [143]. In FtsZs. In two di¡erent plant species, including the model contrast, the Z ring is precisely centered [139,144]. Local- system Arabidopsis thaliana, inhibition of FtsZ by anti- ization of MinE is independent of FtsZ, consistent with sense or by knockouts causes chloroplasts to enlarge and the role of MinE as a speci¢city determinant for FtsZ stop dividing [134,135]. Interestingly, A. thaliana contains placement [143]. GFP fusions to MinC and MinD, on at least two FtsZ homologs, one having a chloroplast im- the other hand, oscillate from pole to pole with a perio- port sequence and the other lacking it. This evidence sug- dicity on the order of 10^60 s [145^147]. The membrane gests the possibility that two Z rings may assemble, one localization and oscillation of MinC depend upon MinD, inside the organelle and the other outside. This would and both MinC and D appear to oscillate as a complex nicely ¢t with the microscopic observation of two concen- [146]. Interestingly, the oscillation of MinCD depends on

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Fig. 3. A model for negative regulation of division site placement in E. coli and B. subtilis. Cells at successive points in the cell cycle, from early in the replication cycle to initiation of septation, are shown from top to bottom for each species. Points above the dotted line represent intervals of less than a minute, in order to highlight the MinCD oscillation phenomenon. Blue ovals represent nucleoids, and yellow bands near midcell in E. coli represent MinE rings. MinE rings are shown to move about the center in order to explain why they are often found o¡-center, but there is no evidence that they actually move in this way. Dark shaded areas within cells represent the MinCD division inhibitor and the degree of shading illustrates the putative concentration gradient with respect to the poles. Red dots denote FtsZ molecules with potential to multimerize. Orange dots denote FtsZ molecules that have successfully nucleated and polymerized into a Z ring. The green squares denote nucleation sites at midcell that are revealed once chromosome segregation begins. Black lines above the cells indicate regions of the cell covered by the nucleoid that appear to inhibit multimerization of FtsZ.

MinE, and the midcell localization of MinE depends on often form closely packed double and triple rings [139]. MinD. The movement of MinCD may function to average This result indicates that Z rings require the presence of the distance between the two poles, while the MinE ring the Min system in order to be properly directed, and sug- may act as a molecular slingshot to repel MinCD during gests that MinCD inhibits Z ring assembly in all parts of its cellular traverse and to allow Z rings to assemble near the cell, not just at midcell and at the poles. the protection of MinE. Away from MinE, MinCD may In addition to the defect in division site placement, min act as a sweeper to keep Z rings from assembling at poles, mutants also have defects in chromosome partitioning one half-cell at a time (Fig. 3). Puri¢ed MinC is su¤cient [148,149]. This is of interest as MinD is homologous to to inhibit FtsZ polymer assembly in a dose-dependent the ParA family of plasmid and chromosomal partitioning manner [147]. MinD, therefore, probably enhances MinC proteins. One of these, Soj, oscillates from pole to pole in action by recruiting MinC to the membrane where its local B. subtilis and interacts with the centromere binding pro- concentration is signi¢cantly increased. tein Spo0J [150,151]. Soj is non-essential for growth, cell What happens in the absence of the Min system in E. division or chromosome segregation. It remains to be seen coli? Cells with inactivated minC or D divide either at their what speci¢c role MinD plays in E. coli chromosome seg- midpoint or at a pole, resulting in a mixture of short regation. ¢laments, nucleoid-free minicells and nucleoid-free rods The Min system of B. subtilis is also involved in pre- [139,148]. These mutations are not lethal because enough venting unwanted polar divisions [152], but there are im- cells still divide medially. Z rings in a vminCDE strain portant distinctions in both the players involved, their form promiscuously in all gaps between nucleoids and localization and their importance for division site selec-

FEMSRE 690 29-8-00 Cyaan Magenta Geel Zwart 542 W. Margolin / FEMS Microbiology Reviews 24 (2000) 531^548 tion. As in E. coli, MinCD acts as a division inhibitor, (ii) that some species may not use an analogous site place- preventing assembly of Z rings [153]. However, there is ment system. H. in£uenzae, for example, is highly related no MinE homolog in B. subtilis. Instead, a protein called to E. coli but lacks the Min proteins. So do many (but not DivIVA acts as the MinE-equivalent, antagonizing all) cocci. One rationale for this is that short rods, such as MinCD inhibition [154]. Another major di¡erence is that H. in£uenzae, do not need to prevent polar divisions be- MinC and D have not been observed to oscillate. Instead, cause there are no nucleoid-free areas at the poles. The in contrast to the E. coli system, MinCD and DivIVA in rationale for most cocci is that most of them have other B. subtilis are recruited to the Z ring after it forms, and mechanisms for identifying the division plane. Interest- stay bound to the ring until after cell division [153^155]. ingly, Neisseria and Deinococcus species both have MinD DivIVA helps to keep MinCD at the poles of the daughter homologs and Neisseria also has MinC and MinE; as cells, so that Z ring assembly is speci¢cally inhibited at the pointed out above, these species divide in alternating per- poles (Fig. 3). One of the conclusions to be made from pendicular planes. This may not be coincidental, and it is these observations is that MinCD prevents assembly of possible that this type of division system may use the new rings but not existing rings. Moreover, there must negative regulation by Min proteins as an extra level of Downloaded from https://academic.oup.com/femsre/article/24/4/531/510989 by guest on 04 October 2021 be factors other than MinCD that help to regulate local- topological control. It will be interesting to see if min ization of Z rings to the medial site in B. subtilis, because knockouts in these species result in randomized division MinCD is recruited by FtsZ and most cells lacking planes, and whether toporegulation by the Min system MinCD still contain properly placed medial Z rings conforms to one of the known models. [156]. A B. subtilis-speci¢c additional negative regulator of Z rings, EzrA, has recently been isolated. When ezrA 16.2. The nucleoid is inactivated, extra rings appear at the poles [157]. EzrA is recruited to the Z ring like MinCD and DivIVA, but EzrA The other regulatory element hypothesized to be in- is not retained later at the poles. This suggests that unlike volved in division site selection is the nucleoid. This makes MinCD, EzrA may serve to destabilize preassembled FtsZ good sense, as the proper replication, packaging and seg- polymers. regation of the chromosome should be a prerequisite for In some ways, the B. subtilis division site system seems cell division. However, in E. coli, evidence from several more streamlined than the E. coli system. By using the approaches suggests that chromosome replication or seg- existing ring to recruit the MinCD inhibitor and retain it regation is not an absolute requirement for cell division to at the incipient poles where it acts (Fig. 3), B. subtilis Min occur. For example, Z ring assembly and septum forma- proteins do not need to be able to identify the division site. tion can be triggered at cellular locations far from nucle- In E. coli, on the other hand, FtsZ is not able to identify oids and yet with some spatial precision [139,148,159]. Z speci¢c sites as readily and needs MinE to do this. It can rings and septa can also bisect abnormal nucleoids that be postulated that FtsZ of E. coli can assemble constitu- have not yet segregated [159^161]. tively and is negatively regulated, whereas FtsZ of B. sub- Nevertheless, wild-type E. coli cells do not septate on tilis either is capable of identifying the right site on its own top of or far away from nucleoids. In fact, there is some or uses other, yet unknown, cues. The factors that deter- evidence that the nucleoid acts negatively to inhibit Z ring mine FtsZ targeting to the proper subcellular sites in B. formation in its vicinity. This nucleoid veto is the basis of subtilis are still unknown. the `nucleoid occlusion' model proposed by Woldringh MinD is well-conserved among many bacteria and ar- and coworkers [162]. The model suggests that all positions chaea, and is also present in a number of eukaryotes that in the cell are potential division sites and that the nucleoid carry chloroplasts [158]. This is not as surprising as it prevents division from occurring at midcell until the veto seems, because recognizable Min homologs are present is released, presumably because of segregation. According in cyanobacteria. It will be interesting to see if MinD is to this model, minicells result from increased nucleoid-free required for proper Z ring placement in archaea and or- zones at poles in min mutants. The bisection of nucleoids ganelles, or if it acts in some other pathway. Judging in certain mutants can be rationalized by the idea that whether a species has MinD is complicated by (i) the pres- nucleoids with abnormal structure no longer have e¡ective ence of MinD-like proteins such as ParA and Soj that may veto power. However, some evidence clearly contradicts oscillate but probably do not in£uence division site place- the idea that nucleoid occlusion is su¤cient for division ment, and (ii) incorrect sequence annotations citing ho- site placement. First, Z rings are present at or near midcell mology to MinD when little similarity actually exists. in nucleoid-free cells [161]. Second, divisions occur within Nevertheless, obvious homologs of MinD as well as a ¢xed distance from a pole in DNA replication-defective MinC appear to be absent in many species. MinE is mutant ¢laments far from any nucleoids [159]. Third, the present in only a few characterized species. The B. subtilis Min system, which must have a role in division site selec- system and comparative genomic data suggest that (i) tion, is not included in the nucleoid occlusion model. many species may use other factors that interact in novel Therefore, this model is not su¤cient to explain division ways with MinD to regulate division site placement, and site placement.

FEMSRE 690 29-8-00 Cyaan Magenta Geel Zwart W. Margolin / FEMS Microbiology Reviews 24 (2000) 531^548 543

16.3. A new model for E. coli division site placement The other major model system, C. crescentus, may also o¡er some valuable insights into division site placement. We have formulated a new model that ¢ts the known Despite the tight control of protein levels and the impor- results and combines roles of both the Min system and the tance of chromosome integrity, the nucleoid veto e¡ect in nucleoid in negative regulation of Z ring placement in E. C. crescentus appears to be weak. For example, topoiso- coli (Fig. 3). Essentially, this model proposes that a com- merase IV mutants of C. crescentus are inhibited in chro- bination of negative regulatory signals from the nucleoid mosome segregation, but Z rings bisect the unsegregated and the Min system are necessary and perhaps su¤cient nucleoids, forming multiple constrictions throughout the for correct localization of the ring. Like the nucleoid oc- cell [168]. These constrictions fail to complete division, clusion model, our model suggests that all locations in the suggesting that C. crescentus has a cell cycle checkpoint cell are potential division sites, but these sites are masked that acts unusually late, although there are other chromo- by (i) the MinCD inhibitor, which sweeps the cell from some-related checkpoints that act earlier [169]. These ¢nd- pole to pole, and (ii) the nucleoid, which occupies much of ings, along with the lack of evidence so far for a Min the cell center. Z rings are kept from forming in polar system in C. crescentus or other K-proteobacteria, suggest Downloaded from https://academic.oup.com/femsre/article/24/4/531/510989 by guest on 04 October 2021 nucleoid-free regions by MinCD, while the nucleoid inhib- that some other system negatively regulates Z ring assem- its Z ring assembly in the remainder of the cell. It is not bly in these species. S. meliloti and its relatives such as known what the molecular mechanism of the proposed Agrobacterium species also share an unusual response nucleoid veto is, but as proposed in the original nucleoid to cell division inhibition: they form branched cells occlusion model, this veto is relieved at some point during [170,171]. The mechanism behind branching, whether it nucleoid segregation. This relief may occur early in segre- is in these species or in E. coli, where it occurs much gation of normal chromosomes, because Z ring assembly less frequently [172], is completely unknown but should occurs about the same time as replication termination eventually provide important insights into growth and [163] (Fig. 2A). MinCD would normally be able to still form [173]. prevent Z ring formation except for the presence of MinE at midcell, which protects FtsZ from MinCD. The promis- cuous assembly of Z rings in nucleoid-free regions of cells 17. Perspectives lacking the Min proteins (but not in cells with the Min proteins) prompted us to formulate this model. The bisec- Despite our vast knowledge of how gene expression is tion of nucleoids by septa under certain conditions, as regulated and how biosynthetic pathways are interrelated, discussed above, can be rationalized. In these cases, nucle- the challenging questions of how any cell grows and di- oid structure is perturbed, and intact nucleoid structure vides remain unanswered. The typical reductionist ap- may be required for the veto. proach used so successfully to tackle other problems, What could FtsZ ultimately be sensing? One possibility such as DNA replication, becomes much more challenging is that FtsZ is targeted to a membrane domain that also in light of the size, complexity, membrane association and targets replication proteins. For example, SeqA in E. coli topological constraints of the cellular division apparatus. and DNA polymerase in B. subtilis also localize to midcell The recent emergence of powerful genomic and cytological and quarter-cell regions [164,165]. Does an intact, properly tools for studying bacteria has signi¢cantly enhanced the positioned oriC complex in£uence FtsZ localization? Is ability to address the problem, but the hardest part will be the nucleoid veto e¡ect observed in other species? One bridging the gap between what is observable on the whole important postulate in our model is that the precise posi- cell level and what can be ascertained by biochemical ap- tioning of the cell division site may result from the pre- proaches. This gap is where macromolecular assemblies vious centering of the replication apparatus. It is un- reside. The only way that we can fully understand how known, however, how either replication proteins or bacterial cell division and other major cellular processes MinE ¢nd their midcell locations, and this is fertile ground work is by being able to characterize, build and quan- for future study. tify these assemblies. Future work in reconstituting the Studies of germinating spores of B. subtilis may address division apparatus will be di¤cult, but is really the the nucleoid veto mechanism. By being able to synchron- only way to understand and dissect how this machine ize initiation of DNA replication in this system, it may be works. Such studies may be enhanced by the compar- possible to dissect the e¡ects of oriC structure and nucle- ative genomics of a number of microorganisms and the oid dynamics on Z ring assembly [166]. It is interesting potential for the use of species that either do not require that the switch from medial to polar septation occurs at cell division for vegetative growth [174,175], or species the same time as the formation of a highly elongated nu- such as the crenarchaea that use FtsZ-independent mech- cleoid structure called the `axial ¢lament' [167]. Whether anisms. The future looks bright indeed for gaining a this ¢lamentous nucleoid structure results in a nucleoid better understanding of the fundamental problem of cell veto across most of the cell, pushing the Z rings to the division. poles, remains to be seen but is intriguing.

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