Bacterial Cytokinesis: from Z Ring to Divisome

REVIEW ARTICLE Cytoskeleton, October 2012 69:778–790 (doi: 10.1002/cm.21054) VC 2012 Wiley Periodicals, Inc.

Bacterial Cytokinesis: From Z Ring to Divisome

Joe Lutkenhaus, Sebastien Pichoff, and Shishen Du Department of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center, Kansas City, Kansas Received 21 June 2012; Revised 18 July 2012; Accepted 20 July 2012 Monitoring Editor: Joseph Sanger

Ancestral homologues of the major eukaryotic cytos- Cytokinesis in bacteria can be split into at least three keletal families, tubulin and actin, play critical roles in steps [de Boer, 2010]. First, is the assembly of the Z ring cytokinesis of bacterial cells. FtsZ is the ancestral on the cytoplasmic membrane with the aid of membrane homologue of tubulin and assembles into the Z ring tethering proteins [Pichoff and Lutkenhaus, 2002]. This that determines the division plane. FtsA, a member of step is under spatial and temporal control to ensure that the actin family, is involved in coordinating cell wall the Z ring is assembled between segregated chromosomes synthesis during cytokinesis. FtsA assists in the forma- [Lutkenhaus, 2007]. In the second step, which usually tion of the Z ring and also has a critical role in occurs after a considerable lag, the remaining cell division recruiting downstream division proteins to the Z ring proteins are added to the Z ring to form the complete to generate the divisome that divides the cell. Spatial divisome [Aarsman et al., 2005; Gamba et al., 2009; regulation of cytokinesis occurs at the stage of Z ring Goley et al., 2011]. Formation of this complex machine assembly and regulation of cell size occurs at this stage involves the addition of many essential proteins and an or during Z ring maturation. VC 2012 Wiley Periodicals, Inc increasing number of nonessential proteins that have partially overlapping functions [Goehring and Beckwith, Key Words: cytokinesis, FtsZ/tubulin, FtsA/actin 2005; Vicente and Rico, 2006; de Boer, 2010]. Third, the divisome is activated to synthesize septal peptidoglycan, which has to be split so that the progeny cells can separate Introduction [Gerding et al., 2009]. This third step is under complex topological control so that cell wall degrading enzymes are acterial cytokinesis has been studied primarily in E. only activated at the correct place after septal cell wall Bcoli and B. subtilis, two bacteria with a similar rod synthesis has initiated [Uehara and Bernhardt, 2011]. shape. A comparison between these two organisms, which are separated in evolutionary time longer than yeast and FtsZ and Assembly of the Z Ring humans, has revealed the basic components of the cytokinetic machinery [Errington et al., 2003]. Despite some FtsZ differences, defining the machinery in these two bacteria has demonstrated a core of essential components that are FtsZ is considered the ancestral homologue of eukaryotic used by many bacteria. Furthermore, these investigations tubulins [Nogales et al., 1998]. Overall the amino acid have facilitated the study of cytokinesis in other bacteria, identity is only on the order of 10% with the highest by allowing investigators to hone in on differences. Chlor- degree of conservation involving residues required for oplasts and many Archaea use FtsZ for division but this GTP binding and hydrolysis [Nogales et al., 1998; Erick- will not be discussed here [Wang and Lutkenhaus, 1996; son, 2007] (Fig. 1). GTP is bound on one end of an Miyagishima, 2011]. In contrast, some Archaea and most FtsZ/tubulin (the þ end) subunit with the aid of the sig- mitochondria use Escrt or dynamin based cell division nature FtsZ/tubulin loop (GGGTG[S/T]G) which binds machineries, respectively [Osteryoung, 2001; Bernander the phosphates. The GTPase catalytic site is formed dur- and Ettema, 2010]. ing filament assembly by the addition of the synergy loop (NxDxx[D/E]) from the incoming subunit. Despite this limited sequence identity, however, the similar structures *Address correspondence to: Joe Lutkenhaus, Department of of the monomers and filaments, as well a similar mecha- Microbiology, Molecular Genetics and Immunology, University of nism of the GTPase, argue that these two proteins are ev- Kansas Medical Center, Kansas City, Kansas 66160. E-mail: [email protected] olutionary related filament forming proteins [Michie and Published online 30 August 2012 in Wiley Online Library L€owe, 2006]. Furthermore, the recent isolation of an in- (wileyonlinelibrary.com). hibitor of FtsZ that stabilizes FtsZ filaments and the

n 778 Fig. 1. Structures of FtsZ and tubulin. The residues that are most conserved in FtsZ (PDB 2VAW) and tubulin (PDB 1TUB) are involved in the binding and hydrolyzing of GTP (colored cyan) and include the synergy loop (NxDxx[D/E] (important residues in caps and colored magenta in the structure) and the signature loop (GGGTG[T/S]G, colored blue) that binds the phosphates. The synergy loop in b-tubulin does not induce GTP hydrolysis due to a positive charged residue (K) substituted for an acidic residue (E) in the loop. A substitution of the acidic residue in FtsZ with G (FtsZ2) results in loss of GTPase activity.

realization that its binding site is analogous to the taxol is accounted for by having an inactive monomer undergo binding site in tubulin, which stabilizes microtubules, fur- an izomerization reaction to an active form before associa- ther highlights the similarity [Haydon et al., 2008; tion with the next monomer [Dajkovic et al., 2008; Hue- Andreu et al., 2010; Elsen et al., 2012]. cas et al., 2008; Miraldi et al., 2008]. The existence of FtsZ forms dynamic filaments in the presence of GTP two forms of FtsZ is supported by the recent structure of that are structurally similar to a protofilament present in a FtsZ in the presence of an inhibitor [Elsen et al., 2012]. microtubule [Mukherjee and Lutkenhaus, 1998; L€owe and FtsZ filaments display different degrees of curvature Amos, 1999]. The filaments readily bundle depending depending upon the nucleotide. Initially, straight filaments upon the in vitro conditions, however, the basic unit of were associated with bound GTP and highly curved fila- assembly is a filament that is a single subunit thick ments with GDP [Lu et al., 2000]. However, bound GTP [Mukherjee and Lutkenhaus, 1994; Chen et al., 2005]. has also been associated with a gently curved form by Under conditions that favor maximal GTPase activity the both atomic force microscopy (AFM) and electron micros- average filament contains about 30 subunits, however, copy [Mingorance et al., 2005]. The possibility that GTP many in vitro conditions lead to lateral bundling which containing filaments adopt a fixed curvature, which can slows down the GTPase and results in longer filaments deform membranes in vitro, has been used as a basis for a [Chen and Erickson, 2009]. However, close inspection of proposal that FtsZ provides the force for constriction bundled filaments did not reveal an orderly arrangement [Erickson et al., 2010]. The reader is referred to a com- and it was argued that lateral bonds do not exist [Erickson prehensive review on the in vitro properties and behavior et al., 2010]. of FtsZ [Erickson et al., 2010]. Although there was some concern whether assembly of a filament a single molecule thick would undergo nucleated assembly and display a critical concentration Z Ring [Romberg et al., 2001], it is now clear that FtsZ displays The Z ring was first visualized as an entity by immunoe- a critical concentration around 1 lM, remarkably similar lectron microscopy [Bi and Lutkenhaus, 1991], then by to tubulin [Mukherjee and Lutkenhaus, 1998; Chen immunofluorescence microscopy [Addinall et al., 1996; et al., 2005]. The nucleated assembly of a linear filament Levin and Losick, 1996], but it is now readily visualized

CYTOSKELETON Bacterial Cytokinesis: From Z Ring to Divisome 779 n Fig. 2. FtsZ filaments are tethered to the membrane by FtsA and ZipA. ZipA and FtsA bind to the highly conserved C-tail of FtsZ, which is connected to main body of FtsZ by a flexible linker. Both ZipA and FtsA are tethered to the membrane by a flexible linker that connects the membrane binding domains of these proteins to the main body of the protein.

in live cells by tagging FtsZ or any of a number of com- ments completely encircling the septum [Anderson et al., ponents that are recruited to the Z ring with green fluo- 2004; Geissler et al., 2007]. This suggests not all FtsZ fil- rescent protein (GFP) [Ma et al., 1996]. So far all studies aments are captured by cryoelectron microscopy. FRAP have been done with GFP tagged FtsZ, which can not (fluorescent recovery after photobleaching) studies have substitute for FtsZ and is toxic. However, the expression shown that the subunits in the Z ring are rapidly turning of GFP-FtsZ does not appear to interfere with division if over (T1/2 of 8–10 s) [Stricker et al., 2002; Erickson the expression level is less than 25% of the native FtsZ. et al., 2010]. To account for the amount of FtsZ in the Z The use of GFP-FtsZ revealed that the Z ring is ring and the rapid turnover, it was proposed that the Z assembled gradually with FtsZ being initially loosely ring consists of short overlapping filaments. One of the organized at midcell before eventually coalescing into a major questions in the field is the substructure of the Z ring. This transition correlates with the segregation of the ring. Overproduction of FtsZ or removal of spatial regula- nucleoids [Inoue et al., 2009] and is promoted by various tors leads to Z rings at midcell and the poles of the cell, nonessential Z associated proteins (Zap). Since some of or doublets between nucleoids in long cells, rather than a these Zap proteins have the ability to bundle FtsZ fila- thickening of the existing ring suggesting that Z rings ments in vitro [Gueiros-Filho and Losick, 2002; Monahan have a defined structure [Bi and Lutkenhaus, 1990; Yu et al., 2009; Dajkovic et al., 2010; Hale et al., 2011; and Margolin, 1999; Quardokus et al., 2001; Weart and Durand-Heredia et al., 2012], it suggests that some degree Levin, 2003]. of bundling of FtsZ filaments occurs in the Z ring. The first known step in bacterial cytokinesis is the as- Efforts to visualize the Z ring by cryoelectron micros- sembly of the Z ring at the future division site [Bi and copy in Caulobacter, which is amenable to this technique Lutkenhaus, 1991]. In E. coli formation of the Z ring due to its small diameter, yielded only a few scattered requires the presence of one of two proteins (ZipA or short filaments in the septal region perpendicular to the FtsA) that can tether FtsZ to the membrane [Pichoff and long axis of the cell [Li et al., 2007]. However, quantita- Lutkenhaus, 2002] (Fig. 2). Other nonessential FtsZ inter- tive fluorescence measurements indicate that in E. coli and acting proteins (ZapA-D), at least two of which are highly B. subtilis about 30–40% of the FtsZ in the cell is in the conserved (ZapA and ZapD), are also present and have a Z ring, which is enough FtsZ to form two to three fila- partially overlapping function in promoting the integrity

n 780 Lutkenhaus et al. CYTOSKELETON of the Z ring [Gueiros-Filho and Losick, 2002; de Boer, Mutations in ftsZ that prevent GTP hydrolysis are lethal, 2010; Hale et al., 2011; Durand-Heredia et al., 2012]. however, at least one hydrolysis deficient mutant can sup- ZipA has a transmembrane domain attached by a long port growth in the presence of an unknown suppressor flexible linker to the FtsZ binding domain and FtsA binds mutation [Bi and Lutkenhaus, 1992; Mukherjee et al., the membrane through a C-terminal amphipathic helix 2001; Dajkovic and Lutkenhaus, 2006; Osawa and Erick- that is attached to the main body of FtsA by a long flexi- son, 2006]. This mutant, FtsZ2 (D212G), has a substitu- ble linker [Hale and de Boer, 1997; Pichoff and Lutken- tion in the synergy loop required for GTP hydrolysis (Fig. haus, 2005]. Both proteins bind to a short highly 1). The most notable phenotype of this mutant is a large conserved tail of FtsZ (the C-terminal 12 amino acids) fraction of cells displaying a contorted septum suggesting that is connected to the main body of FtsZ by a flexible that GTP hydrolysis by FtsZ is not essential for cytokinesis linker [Ma and Margolin, 1999; Haney et al., 2001]. but remodeling of FtsZ at the Z ring is required for sym- Even though these conserved residues are important for metrical invagination of the septum [Addinall and Lutken- binding to both FtsA and ZipA, crystal structures of ZipA haus, 1996]. One possibility is that constant remodeling of and FtsA complexed with the conserved tail reveal that FtsZ at the leading edge of the septum (and adapting to the the tail is in different conformations in the two complexes constriction of the cell) acts as a guide to septal peptidogly- and the respective binding sites in ZipA and FtsA have no can synthesis (which would be the primary motor for inva- similarity [Mosyak et al., 2000; Szwedziak et al., 2012] gination). This possibility is suggested by mutants of FtsZ (Fig. 2). This tail of FtsZ is extremely conserved in evolu- that form spiral shaped structures and spiraled shaped septa, tion even in bacteria that lack ZipA and FtsA. However, and also by the fact that cells inhibited for septal peptido- proteins with features similar to ZipA, but lacking signifi- glycan synthesis do not invaginate the inner membrane cant amino acid homology, have been found in other bac- [Addinall and Lutkenhaus, 1996]. teria. One such protein from Neissera gonorrheae can substitute for ZipA in E. coli suggesting there is little Spatial Regulation of the Z Ring restraint in the evolution of ZipA [Du and Arvidson, 2003]. In B. subtilis the interaction between FtsZ and The spatial regulation of Z ring assembly has been exten- both SepF [Singh et al., 2008] and EzrA [Singh et al., sively studied in three widely divergent rod shaped bacte- 2007], which also has features similar to ZipA, depends ria, E. coli, B. subtilis and Caulobacter crescentus, but is upon the FtsZ tail sequence. The FtsZ tail also binds to beginning to be studied in others [Lutkenhaus, 2012]. at least one antagonist of FtsZ assembly, MinC/MinD The theme that emerges from these studies is that antago- and to one of the Z interacting proteins, ZapD [Shen and nists of FtsZ assembly are positioned away from midcell Lutkenhaus, 2009; Durand-Heredia et al., 2012]. to prevent Z ring formation in their vicinity [Lutkenhaus, The fact that either of two unrelated proteins can pro- 2007]. For at least three of these negative regulators that mote Z ring formation suggests that the self organization have been studied in vitro, MinC, SlmA and MipZ, the leading to Z ring formation is largely a property of FtsZ mechanism appears similar, when activated, they break assembly and membrane attachment. This concept was re- FtsZ filaments [Hu et al., 1999; Thanbichler and Shapiro, inforced by the experiments from the Erickson lab that 2006; Cho et al., 2011; Tonthat et al., 2011]. By doing demonstrated that the addition of a membrane targeting so in vivo they would prevent FtsZ filaments from obtain- sequence (MTS) to FtsZ (also tagged with GFP for visual- ing the necessary length to coalesce into a Z ring. ization) leads to formation of dynamic Z rings inside arti- The filament breaking mechanism used by spatial regula- ficial phospholipid tubules that coalesce and partially tors is in contrast to the best studied FtsZ inhibitor, SulA, constrict the tubule [Osawa et al., 2008]. Placing the which prevents assembly by sequestering FtsZ monomers MTS at the other end of FtsZ (so it would be on the [Cordell et al., 2003; Dajkovic et al., 2008; Chen et al., other side of the filament) leads to formation of Z rings 2012]. This inhibitor is produced in E. coli as part of the on the outside of the lipid tubule that coalesce to cause a SOS (DNA damage) response [Huisman and D’Ari, 1981]. constriction [Osawa and Erickson, 2011]. This assembly When produced it also leads to the rapid dissolution of of Z rings on the inside versus outside of the tubule based dynamic Z rings as monomers released by the dynamic on the position of the membrane tether suggests that turnover are sequestered [Dajkovic et al., 2008]. intrinsic filament curvature is an important feature in E. coli and B. subtilis employ two negative regulatory establishment of the Z ring. In the absence of GTP hy- systems, Min (minicell) and NO (nucleoid occlusion), drolysis Z rings assemble and constriction occurs although named for their effect on positioning of the septum (Z not as deep. This suggests that the intrinsic curvature of ring). Eliminating the Min system leads to Z ring assem- GTP bound FtsZ filaments along with their lateral associ- bly at the poles and minicell formation. In contrast, elimi- ation can deform a vesicle surface. It was suggested that nating the known effectors of the NO system has little the remodeling of the filaments driven by GTP hydrolysis phenotype in exponential cells but leads to Z ring assem- is required to get deeper constrictions. bly occurring over the nucleoid under conditions that

CYTOSKELETON Bacterial Cytokinesis: From Z Ring to Divisome 781 n Fig. 3. Antagonists of FtsZ assembly are positioned in the cell to spatially regulate Z ring assembly. NO is mediated by SlmA in E. coli (Noc in B. subtilis) and prevents Z ring formation over the nucleoid. SlmA and Noc are localized by binding to sequences in the origin proximal region of the chromosome. The Min system prevents Z ring assembly near the poles. MinC is activated by being recruited to the membrane by MinD and oscillates between the poles of the cell under the control of MinD and MinE. In B. subtilis MinC/MinD are recruited to incipient septa by MinJ (not shown) and DivIVA. In Caulobacter crescentus MipZ forms a gradient on the nucleoid. The gradient emanates from its partner ParB, which is bound near the origin and anchored to the pole by interaction with PopZ. Following initiation of replication one origin segregates to the opposite pole where it dislodges FtsZ left over from the previous division. This FtsZ, along with newly synthesized FtsZ, assembles at the lowpoint of the bipolar MipZ gradient.

delay replication or segregation [Wu and Errington, 2004; bacteria, the regulator is MinE, which undergoes a coupled Bernhardt and de Boer, 2005]. Elimination of both sys- oscillation along with MinC/MinD between the poles of tems leads to filamentous cell death as cells are unable to the cell [Hu and Lutkenhaus, 1999; Raskin and de Boer, divide, presumably due to FtsZ being scattered among 1999; Fu et al., 2001; Hale et al., 2001] (Fig. 3). MinD, an many immature FtsZ assemblies throughout the cell ATPase that dimerizes and binds the membrane in the pres- [Bernhardt and de Boer, 2005]. By preventing these spuri- ence of ATP, and MinE, an activator of the MinD ATPase, ous assemblies Min and NO ensure that there is sufficient constitute the oscillator whereas MinC is a passenger. Many FtsZ available to construct a complete Z ring at midcell. of the details of this oscillatory mechanism have been elabo- Consistent with this, overproduction of FtsZ is able to rated and it has been modeled extensively [Meinhardt and rescue a double mutant lacking both systems. de Boer, 2001; Howard and Kruse, 2005; Lutkenhaus, 2007]. Importantly, only MinD and MinE are needed for the oscillation. Consistent with this, MinD and MinE are Min System able to dynamically self organize in vitro [Loose et al., The Min system prevents Z ring assembly near the poles 2008; Ivanov and Mizuuchi, 2010]. They form traveling of the cell through the spatial regulation of the FtsZ an- waves on a lipid bilayer fueled by the ATP hydrolysis of tagonist MinC [Lutkenhaus, 2007] (Fig. 3). MinC is teth- MinD, which is stimulated by MinE. The in vitro waves ered to the membrane by binding MinD, which not only have characteristics that mimic the in vivo oscillation, concentrates MinC at the membrane but also enhances including a maximum concentration of MinE at the trailing MinC’s ability to bind to the conserved tail of FtsZ edge of the wave, which is estimated to be in a 1:1 ratio [Johnson et al., 2002; Shen and Lutkenhaus, 2009]. This with MinD [Loose et al., 2011]. MinD promotes confor- binding to the tail of FtsZ, which requires the C-terminal mational changes in MinE resulting in an active form that domain of MinC, positions the N-terminus of MinC near binds MinD and the membrane which allows MinE to the FtsZ filament. Genetic and biochemical evidence indi- swing from one membrane bound MinD to the next de- cates the N-terminal domain attacks FtsZ filaments at the spite knocking MinD off the membrane [Park et al., 2011]. junction of two subunits following GTP hydrolysis [Shen In B. subtilis, and most Gram positive bacteria, the reg- and Lutkenhaus, 2010]. ulator of MinC/MinD is a coiled-coil protein designated MinC and MinD are positioned by one of two regulators DivIVA [Marston et al., 1998]. This protein is recruited [Rothfield et al., 2005]. In E. coli, and most Gram negative to the site of membrane curvature generated by the

n 782 Lutkenhaus et al. CYTOSKELETON initiation of cytokinesis [Eswaramoorthy et al., 2011] the double mutant can be suppressed by increasing FtsZ (Fig. 3). DivIVA recruits MinC and MinD through an (also by a slower growth rate). Also, growth of E. coli in intermediary designated MinJ [Bramkamp et al., 2008; chambers smaller than the diameter of the cell selects for Patrick and Kearns, 2008]. Thus, a DivIVA ring, deco- ﬂattened cells. In these deformed cells a Z ring forms rated with the Min proteins, is formed on either side of between segregated nucleoids even in the absence of SlmA the incipient septum and prevents FtsZ released from the and Min suggesting that NO is the primary determinant ongoing cytokinesis from reforming a ring at the newly of Z ring placement and that SlmA is not the only com- forming poles [Gregory et al., 2008]. ponent of the NO system [Mannik et al., 2012].

Another Spatial Regulator NO System Although Min and SlmA or Noc are widely distributed, Remarkably, the NO systems of B. subtilis and E. coli especially among rod shaped bacteria, they are not present employ two unrelated DNA binding proteins, Noc and in all bacteria. Caulobacter lacks Min and both NO SlmA, respectively, to perform the same task [Wu and homologues and spatial regulation is due to MipZ, a pro- Errington, 2004; Bernhardt and de Boer, 2005]. Both tein related to MinD [Thanbichler and Shapiro, 2006]. proteins bind to their specific DNA binding sites, which Monomers of MipZ are recruited to the pole of the cell are scattered in the origin proximal 2/3 of the circular by interaction with ParB, which is bound near the origin chromosome [Wu et al., 2009]. For SlmA, it has been of replication, and along with ParA, is involved in chro- shown that it is activated to attack FtsZ filaments upon mosome segregation (Fig. 3). ParB promotes MipZ dime- binding to its cognate sequence [Cho et al., 2011; Tonthat rization, the form that antagonizes FtsZ assembly, and the et al., 2011]. Therefore, as the duplicating chromosomes dimers diffuse away and bind nonspecifically to the chro- segregate the tethered regulators are moved away from the mosome [Kiekebusch et al., 2012]. This binding is tran- cell center making it permissive for Z ring assembly (Fig. sient as the intrinsic ATPase of MipZ causes release from 3). Thus, the NO systems seem ideally suited to couple the DNA. Repeated cycling of MipZ between the bound DNA segregation to formation of the Z ring, but as ParB and the DNA leads to a gradient of MipZ on the pointed out above, elimination of the known effectors of chromosome that is highest near ParB. When the origin NO has little phenotype in exponentially growing cells, duplicates, one segregates to the other pole, resulting in a suggesting their primary role is to prevent guillotining of bipolar gradient of MipZ with the low point near midcell the chromosome under conditions of replication/segrega- where the Z ring forms. tion stress [Wu and Errington, 2004; Bernhardt and de Boer, 2005]. Z Ring Assembly During Sporulation B. subtilis offers an advantage in the study of cytokinesis since examination of the Z ring in germinating spores Although spatial regulation of Z ring assembly is focused offers a chance to look at formation of a Z ring in the ab- mostly on exponentially growing cells, sporulation offers sence of influences of the previous division [Migocki unique opportunities to examine spatial regulation. In et al., 2002]. Spores of a mutant lacking Min and Noc sporulating cells of B. subtilis the Z ring formed at midcell (generated under permissive growth conditions for the spirals away to form a Z ring at each of the cell’s poles, double mutant) were germinated and followed for 1–2 one of which leads to asymmetric septation and the other generations of growth. Z ring formation, although is dismantled [Ben-Yehuda and Losick, 2002]. Although delayed, still occurred primarily at midcell [Rodrigues and the switch involves an increase in FtsZ expression and the Harry, 2012]. This result indicates that an additional fac- production of two sporulation specific proteins, SpoIIE, tor, other than Min or Noc, positions the Z ring and membrane protein that binds FtsZ, and RefZ, a protein remains to be identified. Also, it is clear that the proteins related to SlmA, a detailed mechanism is lacking [Wag- identified to mediate NO, SlmA and Noc, represent only ner-Herman et al., 2012]. Streptomyces species also sporu- part of NO and that additional factor (s) are involved. late and have a life style similar to filamentous fungi with Together the results favor a model in which there is a a mycelial growth containing few septa and the formation limited amount of FtsZ available in the cell but FtsZ as- of aerial hyphae that eventually contain a ladder like struc- sembly is promiscuous and is actively countered by local- ture of Z rings that form simultaneously [Grantcharova ized antagonists, Min and SlmA (Noc). By limiting this et al., 2005]. An interesting aspect of this system is the promiscuous behavior away from midcell, they ensure identification of a protein (SsgB) that precedes FtsZ local- there is sufficient FtsZ available to assemble a complete Z ization [Willemse et al., 2011]. How it is localized is still ring at the desired location. In their absence, FtsZ assem- not known but it represents the first case where a protein bly between nucleoids is favored, however, the free FtsZ is precedes FtsZ to the division site. This protein is not insufficient to form a complete ring due to too much widespread among bacteria, however, it is possible that FtsZ tied up in spurious assemblies. Consistent with this, analogues exist in other bacteria.

CYTOSKELETON Bacterial Cytokinesis: From Z Ring to Divisome 783 n Growth Rate Regulation of Z Ring Function

Bacteria have a remarkable ability to adjust their cell size with growth rate. Fast growing cells (s ¼ 20 min) can be up to eight times the volume of slow growing cells (s > 60 min). This increase in cell volume accommodates the increased DNA content of the cell, which results from initiation of DNA replication occurring before the previous round of replication is completed. In B. subtilis the increased size is due to an increase in cell length while in E. coli the increase in size is due to increases in width and Fig. 4. Diagram of assembly of the Z ring and maturation to the divisome in E. coli. FtsZ polymers (FtsZ[n]) are teth- length. Levin’s lab found that a mutant in B. subtilis, defi- ered to the membrane by FtsA and ZipA, which leads to forma- cient in the synthesis of a nonessential cell wall polymer, tion of the Z ring. However, FtsA is not immediately available was unable to increase its cell length upon shift to a faster to recruit downstream proteins (FtsA[i]). Although not essential, growth rate [Weart et al., 2007]. This result indicated that several FtsZ interacting proteins (ZapA-D) localize to the ring this pathway is used to communicate information about and promote the integrity of the Z ring. Antagonists of FtsZ as- the growth rate to the division apparatus to delay division sembly, MinC/D and SlmA, are positioned away from midcell so that it is permissive for Z ring formation. When enough at faster growth rates. The effector of this response is the monomeric FtsA (active [a]) is present at the Z ring the remain- last enzyme in this pathway, UgtP, which interacts directly ing Fts proteins and PBP1b are recruited. The arrival of FtsN with FtsZ. Although the mechanism is not clear, it is pos- signals the divisome is complete and activation of septal PG sible that the metabolic flux through UgtP regulates its (peptidoglycan) synthesis occurs (divisome [a]). The septal cross interaction with FtsZ. At fast growth rates UgtP may wall is split by AmiB, which is activated by EnvC (recruited ear- lier), and AmiC, which is activated by NlpD. Although EnvC is decrease the amount of active FtsZ so that cell division is recruited early, the arrival of AmiC, AmiB and NlpD depend delayed until more FtsZ accumulates [Hill et al., 2012]. upon the start of septal PG synthesis induced by the arrival of An E. coli mutant (pgm) corresponding to the original B. FtsN. The Tol-Pal complex is needed for efficient invagination subtilis mutant (pgcA) also displays a defect in adjusting of the outer membrane (i), inactive; (a), active. cell size to growth rate suggesting that this is a conserved regulatory feature. Furthermore, the small cell size at fast growth rate observed in the pgm mutant is mimicked by a FtsE and FtsX encode an ABC transporter with the gain of function allele of ftsA (ftsA*) raising the possibility most homology to the Lol system, which extracts lipopro- that the ftsA* mutant is resistant to this regulation [Hill teins out of the cytoplasmic membrane [Schmidt et al., et al., 2012]. A potential effector protein in E. coli is not 2004]. FtsX encodes the membrane component and FtsE clear since it lacks a homologue of UgtP. encodes the ATPase. Since these genes can be deleted under conditions of high osmolarity without a dramatic effect on cell division, they have been given less attention Formation of the Divisome [Reddy, 2007]. However, there is a slight division defect under suppressing conditions and recently it was shown The assembly of the complete divisome occurs in two that FtsE/FtsX recruit an activator, EnvC, of a septal pep- temporally distinct steps in E. coli, B. subtilis and Caulo- tidoglycan amidase (AmiB) to the Z ring [Yang et al., bacter [Aarsman et al., 2005; Gamba et al., 2009; Goley 2011]. EnvC is recruited directly by FtsX but the ATPase et al., 2011]. Following assembly of the Z ring there is a activity of FtsE is required for EnvC to activate AmiB. In considerable gap before the subsequent accrual of addi- the absence of FtsE/FtsX this activity is missing and split- tional proteins to constitute the complete divisome. In ting of the septum is due to other amidases with partially E. coli at least nine additional essential division proteins redundant activity. are added almost simultaneously to the Z ring [Schmidt FtsK is a DNA translocase with a membrane domain et al., 2004; Goehring and Beckwith, 2005] (Fig. 4). containing four transmembrane spanning segments fused How these additional proteins are connected to the Z ring to the DNA translocase domain by a long linker [Begg is not clear but FtsA plays a key role. Dependency studies et al., 1995]. When located at the septum this protein is indicate that there is a linear order to the assembly process able to translocate DNA away from the septum due to even though several of the proteins have been shown to specific sequences (KOPS) located throughout the chro- exist in complexes even when they are not associated with mosome, which give directionality to the movement of the Z ring [Goehring et al., 2006]. The roles of these the DNA [Bigot et al., 2005]. Although this protein can additional essential division proteins in E. coli are rescue DNA trapped at the septum this only comes into described below. play during stress and is not an essential function [Steiner

n 784 Lutkenhaus et al. CYTOSKELETON et al., 1999]. The essential function of FtsK lies in the four transmembrane segments which may play a role in fusion of the invaginating membrane to complete cytokinesis [Fleming et al., 2010]. FtsQ, FtsL and FtsB have no known enzymatic activity and appear to function as a link between the Z ring and the peptidoglycan biosynthetic machinery [Goehring and Beckwith, 2005]. FtsI and FtsW are part of the peptidoglycan machinery dedicated to septation [Typas et al., 2012]. Their orthologues, PBP2 and RodA, respectively, are dedicated to peptidoglycan synthesis during cell elongation andhavenoroleinseptation.These proteins alone cannot synthesize peptidoglycan, which requires a transglycoslyase. E. coli has several proteins with this activity and it appears that the majority of peptidoglycan synthesis is carried out by PBP1A and PBP1B. Even though these two synthetases are thought to primarily be involved in cell elongation and division, respectively, their function overlaps, as only one is necessary for cells to survive [Typas et al., 2012]. Fig. 5. Structures of FtsA and MreB. Two molecules of FtsA are shown as arranged in a filament (PDB 4A2A). The structure FtsN is the last essential division protein to arrive at the of MreB (PDB 1JCE) is similar to conventional actin. FtsA ring and may signal that the divisome complex is com- lacks domain 1B but has a new domain 1C (colored red in one plete and septation should be initiated, basically acting as FtsA) that interacts with other division proteins. a trigger for septation [Goehring and Beckwith, 2005; Gerding et al., 2009]. FtsN is a bitopic protein with a [Bernard et al., 2007]. Bacterial two hybrid tests indicated short cytoplasmic region connected to a larger periplasmic FtsA* interacted more strongly with itself leading to the region by a single transmembrane domain [Dai et al., suggestion that increased interaction between FtsA mole- 1993]. The most conserved region of FtsN lies at the C- cules stabilized the Z ring to destabilizing conditions terminus (SPOR domain) and binds a form of peptidogly- [Shiomi and Margolin, 2007]. can that is only present at the septum. However, this do- Although FtsA is a member of the actin family, it is main is not essential [Gerding et al., 2009]. FtsN, missing one of actin’s four subdomains but has an addi- however, is required for the recruitment of a host of tional subdomain (1C) located elsewhere in the structure downstream proteins whose activities are partially redun- [Szwedziak et al., 2012] (Fig. 5). Many bacteria possess dant. It is unlikely that this recruitment is direct but may another actin homologue (MreB) that has the same do- rely upon FtsN’s ability to trigger septation [Bernard main structure as actin [van den Ent et al., 2001] (Fig. 5), et al., 2007]. At least one allele of ftsA can bypass the but in E. coli and B. subtilis it is required for the mainte- requirement for FtsN suggesting that septation can be nance of the rod shape by organizing peptidoglycan syn- triggered another way [Bernard et al., 2007]. thesis for lateral wall synthesis [Jones et al., 2001; Typas et al., 2012]. Nonetheless, various reports demonstrated that FtsA assembles into filaments in vitro although no Role of FtsA in Formation of the Divisome reliable ATPase activity accompanied assembly and the fil- FtsA plays two critical roles in cytokinesis. First, along aments were not dynamic [Lara et al., 2005; Krupka with ZipA, it tethers FtsZ filaments to the membrane et al., 2012]. Other reports indicated FtsA without its [Pichoff and Lutkenhaus, 2002]. Second, along with membrane binding domain assembles in vivo, forming ZipA, it is required for recruitment of all the downstream cytoplasmic filaments when overexpressed [Pichoff and division proteins [Hale and de Boer, 2002]. Importantly, Lutkenhaus, 2005]. Recently, L€owe’s lab solved the struc- Margolin’s lab isolated an allele of ftsA, ftsA*, that was ture of FtsA revealing actin-like protofilaments [Szwedziak able to bypass the requirement for ZipA [Geissler et al., et al., 2012]. They also observed FtsA polymers on a lipid 2007]. This, along with evidence that FtsA is much more monolayer and in the cytoplasm of cell (without the conserved in evolution than ZipA, and interacts with a membrane binding domain) with the same repeat distance number of downstream division proteins, indicate that as observed in the crystal structure. Moreover, mutations FtsA has a more direct role in their recruitment [Corbin that would be expected to interfere with polymer contacts et al., 2004]. In addition to bypassing ZipA, FtsA* has a were less efficient at division. number of unusual properties, including resistance to vari- In a separate study Pichoff et al. [Pichoff et al., 2012] ous treatments that destabilize the Z ring, including excess isolated mutations that interfered with the ability of FtsA MinC, and allowing cells to divide at a smaller cell size to form cytoplasmic filaments. Surprisingly, ftsA*was

CYTOSKELETON Bacterial Cytokinesis: From Z Ring to Divisome 785 n among them and a decrease in self-interaction was con- Together the information suggests the following more firmed by independent tests. In addition, selection of explicit model. In the polymer form the 1C domain is many additional mutations that bypassed ZipA led to the not available for interaction with a protein such as FtsN, inescapable conclusion that such mutations decrease FtsA’s since it is occupied in the filament. In the monomer self interaction. Consistent with this, the altered residues form, however, the 1C domain is free and the connection mapped to the four major contact points between subu- to the body of the protein by a hinge allows movement nits in the FtsA filament. Whether some low level of FtsA and the acceptance of the N-terminus of FtsN. The struc- self interaction is essential is not clear. Nonetheless, this ture of proteins from the Type IV pilus from Pseudomonas finding led to a new model for how FtsA recruits down- suggests a likely scenario [Busiek et al., 2012]. PilM, stream division proteins. In this model, FtsA switches which is similar in structure to FtsA, binds to the N-ter- between a form that is unable to interact with down- minus of the bitopic PilN protein [Karuppiah and Der- stream proteins (polymeric) and a form that is active in rick, 2011]. Comparison of the structures of FtsA with recruitment (monomeric). Somehow this switch is regu- the PilM-PilN complex is consistent with the necessary lated by the dynamic interaction of proteins with the tail membrane orientation. The tail of FtsA, which attaches to of FtsZ. the membrane, positions FtsA to interact with the FtsN protein. Furthermore, FtsZ is attached to the opposite side of FtsA so that it would be on the cytoplasmic side. Model for FtsA Recruitment of Downstream Division Proteins Triggering Septation and Cell Two complementary lines of evidence indicate FtsA, and Separation in particular domain 1C, plays a critical role by in assembly of the divisome. Artificially targeting domain 1C to Upon completion of the divisome septation is triggered the poles of the cell leads to polar localization of several leading to synthesis of peptidoglycan and its eventual late division proteins (FtsN and FtsI) [Corbin et al., splitting. In E. coli the triggering event coincides with the 2004]. On the other hand, FtsA deleted for the 1C do- arrival of FtsN at the divisome [Gerding et al., 2009]. main localizes efficiently to the Z ring but is unable to FtsN recruitment has at least two requirements: (1) it recruit the late cell division proteins [Rico et al., 2004]. requires the immediate preceding protein, FtsI [Wissel Although the best evidence is for interaction between do- and Weiss, 2004]; and (2) it requires FtsA [Goehring main 1C and FtsN [Busiek et al., 2012], some evidence, et al., 2006]. Thus, FtsN arrival signals completion of although indirect, also suggests interaction of this domain divisome assembly and by activating FtsI, which along with FtsI and FtsQ. These proteins are all single pass with PBP1b synthesizes septal specific peptidoglycan, bitopic membrane proteins with short cytoplasmic N-ter- results in septation. The activation of FtsI leads to the mini and large extracytoplasmic domains. A direct interac- recruitment of additional proteins that metabolize the tion with the cytoplasmic FtsA has to be with the short peptidoglycan and invaginate the outer membrane [Gerd- N-termini of one or more of these proteins. ing et al., 2007]. In E. coli four proteins are recruited to Since ftsA mutations that bypass ZipA result in less self the septum by a conserved SPOR domain that binds sep- interaction, it was proposed that the essential function of tal specific peptidoglycan, presumably glycan chains that ZipA is to antagonize FtsA self assembly [Pichoff et al., have been metabolized by amidases [Gerding et al., 2009; 2012]. Furthermore, FtsA mutants that no longer require Arends et al., 2010]. These SPOR containing proteins ZipA self-interact less well, suggesting that it is the mono- make good markers revealing the onset of septation. mer form of FtsA that recruits one or more of the down- E. coli contains three amidases, enzymes that remove stream division proteins, presumably because domain 1C the peptide side chains attached to the glycan chains that becomes available. In this model, the Z ring is formed as constitute peptidoglycan. Removal of these peptide side FtsA and ZipA interact with polymers of FtsZ and tether chains, which crosslink the glycan chains, allows cells to them to the membrane. In shorter cells, other proteins be separated [Bernhardt and de Boer, 2003]. The activity (such as MinC/MinD) that interact with the FtsZ tail, of these amidases overlap and the presence of any one compete with FtsA and reduce the amount of FtsA’s do- ensures survival. Two of them, AmiB and AmiC, are local- main IC available at the Z ring. However, a combination ized to the septum [Peters et al., 2011]. AmiB requires of a build up of FtsA at the Z ring along with conditions EnvC and thus, FtsX for recruitment and also FtsE for favoring monomers increase the availability of domain 1C activation. EnvC localizes early whereas its partner, AmiB and the recruitment of downstream proteins commences. requires FtsN for localization and so is localized later. During constriction the presence of the downstream divi- AmiC and its activator (a lipoprotein called NlpD), both sion proteins at the septum perpetuates the monomeric require FtsN for localization. The requirement for FtsN form of FtsA through interaction with the 1C domain. means that the amidases won’t be specifically recruited

n 786 Lutkenhaus et al. CYTOSKELETON and activated until the switch for peptidoglycan synthesis coli septal ring proteins that contain a SPOR domain: DamX, is thrown. However, in the absence of AmiB and AmiC, DedD, and RlpA. J Bacteriol 192:242–255. AmiA (which does not localize specifically to the septum) Begg KJ, Dewar SJ, Donachie WD. 1995. A new Escherichia coli is still able to carry out the splitting of the septum cell division gene, ftsK. J Bacteriol 177:6211–6222. through activation by the localized EnvC [Peters et al., Ben-Yehuda S, Losick R. 2002. Asymmetric cell division in B. subti- 2011]. lis involves a spiral-like intermediate of the cytokinetic protein FtsZ. Cell 109:257–266. In a Gram negative bacterium like E coli invagination Bernander R, Ettema TJ. 2010. FtsZ-less cell division in archaea of the outer membrane follows septal peptidoglycan syn- and bacteria. Curr Opin Microbiol 13:747–752. thesis. A large set of proteins including a cytoplasmic Bernard CS, Sadasivam M, Shiomi D, Margolin W. 2007. An complex of Tol proteins (TolQAR) interacts with an outer altered FtsA can compensate for the loss of essential cell division membrane lipoprotein (Pal) to ensure timely and efficient protein FtsN in Escherichia coli. Mol Microbiol 64:1289–1305. invagination of this layer [Gerding et al., 2007]. These Bernhardt TG, de Boer PA. 2003. The Escherichia coli amidase proteins are not essential though and in their absence AmiC is a periplasmic septal ring component exported via the invagination occurs. twin-arginine transport pathway. Mol Microbiol 48:1171–1182. Bernhardt TG, de Boer PA. 2005. SlmA, a nucleoid-associated, Summary and Prospective FtsZ binding protein required for blocking septal ring assembly over chromosomes in E. coli. Mol Cell 18:555–564. The study of cytokinesis in bacteria has lagged behind Bi E, Lutkenhaus J. 1990. FtsZ regulates frequency of cell division studies in eukaryotes due to the small size of bacteria and in Escherichia coli. J Bacteriol 172:2765–2768. the lack of prominent intracellular structures. However, Bi E, Lutkenhaus J. 1992. Isolation and characterization of ftsZ al- the availability of GFP, improving microscopic techniques leles that affect septal morphology. J Bacteriol 174:5414–5423. and the relative simplicity of the bacterial systems has Bi EF, Lutkenhaus J. 1991. FtsZ ring structure associated with divi- resulted in rapid gains in understanding this process. One sion in Escherichia coli. Nature 354:161–164. outcome of the study of cytokinesis in bacteria is the real- Bigot S, Saleh OA, Lesterlin C, Pages C, El Karoui M, Dennis C, Grigoriev M, Allemand JF, Barre FX, Cornet F. 2005. KOPS: DNA ization that the eukaryotic cytoskeleton had its origins in motifs that control E. coli chromosome segregation by orienting the bacteria. Another is that components of the bacterial sys- FtsK translocase. EMBO J 24:3770–3780. tem are relatively small in number making in vitro recon- Bramkamp M, Emmins R, Weston L, Donovan C, Daniel RA, stitution experiments feasible. Many important questions Errington J. 2008. A novel component of the division-site selection remain, however, including the structure of the Z ring, system of Bacillus subtilis and a new mode of action for the division how Z filament dynamics are regulated, how the Z ring is inhibitor MinCD. Mol Microbiol 70:1556–1569. activated to constrict and how the Z ring is connected to Busiek KK, Eraso JM, Wang Y, Margolin W. 2012. The early divi- cell wall synthesis. some protein FtsA interacts directly through its 1c subdomain with the cytoplasmic domain of the late divisome protein FtsN. J Bacter- iol 194:1989–2000. Acknowledgment Chen Y, Erickson HP. 2009. 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