REVIEW ARTICLE
Cytoskeleton, October 2012 69:778–790 (doi: 10.1002/cm.21054)
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V
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 cytoskeletal families, tubulin and actin, play critical roles in
cytokinesis of bacterial cells. FtsZ is the ancestral
homologue of tubulin and assembles into the Z ring
that determines the division plane. FtsA, a member of
the actin family, is involved in coordinating cell wall
synthesis during cytokinesis. FtsA assists in the formation of the Z ring and also has a critical role in
recruiting downstream division proteins to the Z ring
to generate the divisome that divides the cell. Spatial
regulation of cytokinesis occurs at the stage of Z ring
assembly and regulation of cell size occurs at this stage
or during Z ring maturation. V 2012 Wiley Periodicals, Inc
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Key Words:
cytokinesis, FtsZ/tubulin, FtsA/actin
Introduction
B
acterial cytokinesis has been studied primarily in E.
coli and B. subtilis, two bacteria with a similar rod
shape. A comparison between these two organisms, which
are separated in evolutionary time longer than yeast and
humans, has revealed the basic components of the cytokinetic machinery [Errington et al., 2003]. Despite some
differences, defining the machinery in these two bacteria
has demonstrated a core of essential components that are
used by many bacteria. Furthermore, these investigations
have facilitated the study of cytokinesis in other bacteria,
by allowing investigators to hone in on differences. Chloroplasts and many Archaea use FtsZ for division but this
will not be discussed here [Wang and Lutkenhaus, 1996;
Miyagishima, 2011]. In contrast, some Archaea and most
mitochondria use Escrt or dynamin based cell division
machineries, respectively [Osteryoung, 2001; Bernander
and Ettema, 2010].
*Address correspondence to: Joe Lutkenhaus, Department of
Microbiology, Molecular Genetics and Immunology, University of
Kansas Medical Center, Kansas City, Kansas 66160.
E-mail: [email protected]
Published online 30 August 2012 in Wiley Online Library
(wileyonlinelibrary.com).
n 778
Cytokinesis in bacteria can be split into at least three
steps [de Boer, 2010]. First, is the assembly of the Z ring
on the cytoplasmic membrane with the aid of membrane
tethering proteins [Pichoff and Lutkenhaus, 2002]. This
step is under spatial and temporal control to ensure that
the Z ring is assembled between segregated chromosomes
[Lutkenhaus, 2007]. In the second step, which usually
occurs after a considerable lag, the remaining cell division
proteins are added to the Z ring to form the complete
divisome [Aarsman et al., 2005; Gamba et al., 2009;
Goley et al., 2011]. Formation of this complex machine
involves the addition of many essential proteins and an
increasing number of nonessential proteins that have partially overlapping functions [Goehring and Beckwith,
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
[Gerding et al., 2009]. This third step is under complex
topological control so that cell wall degrading enzymes are
only activated at the correct place after septal cell wall
synthesis has initiated [Uehara and Bernhardt, 2011].
FtsZ and Assembly of the Z Ring
FtsZ
FtsZ is considered the ancestral homologue of eukaryotic
tubulins [Nogales et al., 1998]. Overall the amino acid
identity is only on the order of 10% with the highest
degree of conservation involving residues required for
GTP binding and hydrolysis [Nogales et al., 1998; Erickson, 2007] (Fig. 1). GTP is bound on one end of an
FtsZ/tubulin (the þ end) subunit with the aid of the signature FtsZ/tubulin loop (GGGTG[S/T]G) which binds
the phosphates. The GTPase catalytic site is formed during 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
of the monomers and filaments, as well a similar mechanism of the GTPase, argue that these two proteins are evolutionary related filament forming proteins [Michie and
L€owe, 2006]. Furthermore, the recent isolation of an inhibitor of FtsZ that stabilizes FtsZ filaments and the
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
binding site in tubulin, which stabilizes microtubules, further highlights the similarity [Haydon et al., 2008;
Andreu et al., 2010; Elsen et al., 2012].
FtsZ forms dynamic filaments in the presence of GTP
that are structurally similar to a protofilament present in a
microtubule [Mukherjee and Lutkenhaus, 1998; L€owe and
Amos, 1999]. The filaments readily bundle depending
upon the in vitro conditions, however, the basic unit of
assembly is a filament that is a single subunit thick
[Mukherjee and Lutkenhaus, 1994; Chen et al., 2005].
Under conditions that favor maximal GTPase activity the
average filament contains about 30 subunits, however,
many in vitro conditions lead to lateral bundling which
slows down the GTPase and results in longer filaments
[Chen and Erickson, 2009]. However, close inspection of
bundled filaments did not reveal an orderly arrangement
and it was argued that lateral bonds do not exist [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
[Romberg et al., 2001], it is now clear that FtsZ displays
a critical concentration around 1 lM, remarkably similar
to tubulin [Mukherjee and Lutkenhaus, 1998; Chen
et al., 2005]. The nucleated assembly of a linear filament
CYTOSKELETON
is accounted for by having an inactive monomer undergo
an izomerization reaction to an active form before association with the next monomer [Dajkovic et al., 2008; Huecas et al., 2008; Miraldi et al., 2008]. The existence of
two forms of FtsZ is supported by the recent structure of
FtsZ in the presence of an inhibitor [Elsen et al., 2012].
FtsZ filaments display different degrees of curvature
depending upon the nucleotide. Initially, straight filaments
were associated with bound GTP and highly curved filaments with GDP [Lu et al., 2000]. However, bound GTP
has also been associated with a gently curved form by
both atomic force microscopy (AFM) and electron microscopy [Mingorance et al., 2005]. The possibility that GTP
containing filaments adopt a fixed curvature, which can
deform membranes in vitro, has been used as a basis for a
proposal that FtsZ provides the force for constriction
[Erickson et al., 2010]. The reader is referred to a comprehensive review on the in vitro properties and behavior
of FtsZ [Erickson et al., 2010].
Z Ring
The Z ring was first visualized as an entity by immunoelectron microscopy [Bi and Lutkenhaus, 1991], then by
immunofluorescence microscopy [Addinall et al., 1996;
Levin and Losick, 1996], but it is now readily visualized
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 components that are recruited to the Z ring with green fluorescent protein (GFP) [Ma et al., 1996]. So far all studies
have been done with GFP tagged FtsZ, which can not
substitute for FtsZ and is toxic. However, the expression
of GFP-FtsZ does not appear to interfere with division if
the expression level is less than 25% of the native FtsZ.
The use of GFP-FtsZ revealed that the Z ring is
assembled gradually with FtsZ being initially loosely
organized at midcell before eventually coalescing into a
ring. This transition correlates with the segregation of the
nucleoids [Inoue et al., 2009] and is promoted by various
nonessential Z associated proteins (Zap). Since some of
these Zap proteins have the ability to bundle FtsZ filaments in vitro [Gueiros-Filho and Losick, 2002; Monahan
et al., 2009; Dajkovic et al., 2010; Hale et al., 2011;
Durand-Heredia et al., 2012], it suggests that some degree
of bundling of FtsZ filaments occurs in the Z ring.
Efforts to visualize the Z ring by cryoelectron microscopy in Caulobacter, which is amenable to this technique
due to its small diameter, yielded only a few scattered
short filaments in the septal region perpendicular to the
long axis of the cell [Li et al., 2007]. However, quantitative fluorescence measurements indicate that in E. coli and
B. subtilis about 30–40% of the FtsZ in the cell is in the
Z ring, which is enough FtsZ to form two to three fila-
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Lutkenhaus et al.
ments completely encircling the septum [Anderson et al.,
2004; Geissler et al., 2007]. This suggests not all FtsZ filaments are captured by cryoelectron microscopy. FRAP
(fluorescent recovery after photobleaching) studies have
shown that the subunits in the Z ring are rapidly turning
over (T1/2 of 8–10 s) [Stricker et al., 2002; Erickson
et al., 2010]. To account for the amount of FtsZ in the Z
ring and the rapid turnover, it was proposed that the Z
ring consists of short overlapping filaments. One of the
major questions in the field is the substructure of the Z
ring. Overproduction of FtsZ or removal of spatial regulators leads to Z rings at midcell and the poles of the cell,
or doublets between nucleoids in long cells, rather than a
thickening of the existing ring suggesting that Z rings
have a defined structure [Bi and Lutkenhaus, 1990; Yu
and Margolin, 1999; Quardokus et al., 2001; Weart and
Levin, 2003].
The first known step in bacterial cytokinesis is the assembly of the Z ring at the future division site [Bi and
Lutkenhaus, 1991]. In E. coli formation of the Z ring
requires the presence of one of two proteins (ZipA or
FtsA) that can tether FtsZ to the membrane [Pichoff and
Lutkenhaus, 2002] (Fig. 2). Other nonessential FtsZ interacting proteins (ZapA-D), at least two of which are highly
conserved (ZapA and ZapD), are also present and have a
partially overlapping function in promoting the integrity
CYTOSKELETON
of the Z ring [Gueiros-Filho and Losick, 2002; de Boer,
2010; Hale et al., 2011; Durand-Heredia et al., 2012].
ZipA has a transmembrane domain attached by a long
flexible linker to the FtsZ binding domain and FtsA binds
the membrane through a C-terminal amphipathic helix
that is attached to the main body of FtsA by a long flexible linker [Hale and de Boer, 1997; Pichoff and Lutkenhaus, 2005]. Both proteins bind to a short highly
conserved tail of FtsZ (the C-terminal 12 amino acids)
that is connected to the main body of FtsZ by a flexible
linker [Ma and Margolin, 1999; Haney et al., 2001].
Even though these conserved residues are important for
binding to both FtsA and ZipA, crystal structures of ZipA
and FtsA complexed with the conserved tail reveal that
the tail is in different conformations in the two complexes
and the respective binding sites in ZipA and FtsA have no
similarity [Mosyak et al., 2000; Szwedziak et al., 2012]
(Fig. 2). This tail of FtsZ is extremely conserved in evolution even in bacteria that lack ZipA and FtsA. However,
proteins with features similar to ZipA, but lacking significant amino acid homology, have been found in other bacteria. One such protein from Neissera gonorrheae can
substitute for ZipA in E. coli suggesting there is little
restraint in the evolution of ZipA [Du and Arvidson,
2003]. In B. subtilis the interaction between FtsZ and
both SepF [Singh et al., 2008] and EzrA [Singh et al.,
2007], which also has features similar to ZipA, depends
upon the FtsZ tail sequence. The FtsZ tail also binds to
at least one antagonist of FtsZ assembly, MinC/MinD
and to one of the Z interacting proteins, ZapD [Shen and
Lutkenhaus, 2009; Durand-Heredia et al., 2012].
The fact that either of two unrelated proteins can promote Z ring formation suggests that the self organization
leading to Z ring formation is largely a property of FtsZ
assembly and membrane attachment. This concept was reinforced by the experiments from the Erickson lab that
demonstrated that the addition of a membrane targeting
sequence (MTS) to FtsZ (also tagged with GFP for visualization) leads to formation of dynamic Z rings inside artificial phospholipid tubules that coalesce and partially
constrict the tubule [Osawa et al., 2008]. Placing the
MTS at the other end of FtsZ (so it would be on the
other side of the filament) leads to formation of Z rings
on the outside of the lipid tubule that coalesce to cause a
constriction [Osawa and Erickson, 2011]. This assembly
of Z rings on the inside versus outside of the tubule based
on the position of the membrane tether suggests that
intrinsic filament curvature is an important feature in
establishment of the Z ring. In the absence of GTP hydrolysis Z rings assemble and constriction occurs although
not as deep. This suggests that the intrinsic curvature of
GTP bound FtsZ filaments along with their lateral association can deform a vesicle surface. It was suggested that
the remodeling of the filaments driven by GTP hydrolysis
is required to get deeper constrictions.
CYTOSKELETON
Mutations in ftsZ that prevent GTP hydrolysis are lethal,
however, at least one hydrolysis deficient mutant can support growth in the presence of an unknown suppressor
mutation [Bi and Lutkenhaus, 1992; Mukherjee et al.,
2001; Dajkovic and Lutkenhaus, 2006; Osawa and Erickson, 2006]. This mutant, FtsZ2 (D212G), has a substitution in the synergy loop required for GTP hydrolysis (Fig.
1). The most notable phenotype of this mutant is a large
fraction of cells displaying a contorted septum suggesting
that GTP hydrolysis by FtsZ is not essential for cytokinesis
but remodeling of FtsZ at the Z ring is required for symmetrical invagination of the septum [Addinall and Lutkenhaus, 1996]. One possibility is that constant remodeling of
FtsZ at the leading edge of the septum (and adapting to the
constriction of the cell) acts as a guide to septal peptidoglycan synthesis (which would be the primary motor for invagination). This possibility is suggested by mutants of FtsZ
that form spiral shaped structures and spiraled shaped septa,
and also by the fact that cells inhibited for septal peptidoglycan synthesis do not invaginate the inner membrane
[Addinall and Lutkenhaus, 1996].
Spatial Regulation of the Z Ring
The spatial regulation of Z ring assembly has been extensively studied in three widely divergent rod shaped bacteria, E. coli, B. subtilis and Caulobacter crescentus, but is
beginning to be studied in others [Lutkenhaus, 2012].
The theme that emerges from these studies is that antagonists of FtsZ assembly are positioned away from midcell
to prevent Z ring formation in their vicinity [Lutkenhaus,
2007]. For at least three of these negative regulators that
have been studied in vitro, MinC, SlmA and MipZ, the
mechanism appears similar, when activated, they break
FtsZ filaments [Hu et al., 1999; Thanbichler and Shapiro,
2006; Cho et al., 2011; Tonthat et al., 2011]. By doing
so in vivo they would prevent FtsZ filaments from obtaining the necessary length to coalesce into a Z ring.
The filament breaking mechanism used by spatial regulators is in contrast to the best studied FtsZ inhibitor, SulA,
which prevents assembly by sequestering FtsZ monomers
[Cordell et al., 2003; Dajkovic et al., 2008; Chen et al.,
2012]. This inhibitor is produced in E. coli as part of the
SOS (DNA damage) response [Huisman and D’Ari, 1981].
When produced it also leads to the rapid dissolution of
dynamic Z rings as monomers released by the dynamic
turnover are sequestered [Dajkovic et al., 2008].
E. coli and B. subtilis employ two negative regulatory
systems, Min (minicell) and NO (nucleoid occlusion),
named for their effect on positioning of the septum (Z
ring). Eliminating the Min system leads to Z ring assembly at the poles and minicell formation. In contrast, eliminating the known effectors of the NO system has little
phenotype in exponential cells but leads to Z ring assembly occurring over the nucleoid under conditions that
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;
Bernhardt and de Boer, 2005]. Elimination of both systems leads to filamentous cell death as cells are unable to
divide, presumably due to FtsZ being scattered among
many immature FtsZ assemblies throughout the cell
[Bernhardt and de Boer, 2005]. By preventing these spurious assemblies Min and NO ensure that there is sufficient
FtsZ available to construct a complete Z ring at midcell.
Consistent with this, overproduction of FtsZ is able to
rescue a double mutant lacking both systems.
Min System
The Min system prevents Z ring assembly near the poles
of the cell through the spatial regulation of the FtsZ antagonist MinC [Lutkenhaus, 2007] (Fig. 3). MinC is tethered to the membrane by binding MinD, which not only
concentrates MinC at the membrane but also enhances
MinC’s ability to bind to the conserved tail of FtsZ
[Johnson et al., 2002; Shen and Lutkenhaus, 2009]. This
binding to the tail of FtsZ, which requires the C-terminal
domain of MinC, positions the N-terminus of MinC near
the FtsZ filament. Genetic and biochemical evidence indicates the N-terminal domain attacks FtsZ filaments at the
junction of two subunits following GTP hydrolysis [Shen
and Lutkenhaus, 2010].
MinC and MinD are positioned by one of two regulators
[Rothfield et al., 2005]. In E. coli, and most Gram negative
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Lutkenhaus et al.
bacteria, the regulator is MinE, which undergoes a coupled
oscillation along with MinC/MinD between the poles of
the cell [Hu and Lutkenhaus, 1999; Raskin and de Boer,
1999; Fu et al., 2001; Hale et al., 2001] (Fig. 3). MinD, an
ATPase that dimerizes and binds the membrane in the presence of ATP, and MinE, an activator of the MinD ATPase,
constitute the oscillator whereas MinC is a passenger. Many
of the details of this oscillatory mechanism have been elaborated and it has been modeled extensively [Meinhardt and
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
able to dynamically self organize in vitro [Loose et al.,
2008; Ivanov and Mizuuchi, 2010]. They form traveling
waves on a lipid bilayer fueled by the ATP hydrolysis of
MinD, which is stimulated by MinE. The in vitro waves
have characteristics that mimic the in vivo oscillation,
including a maximum concentration of MinE at the trailing
edge of the wave, which is estimated to be in a 1:1 ratio
with MinD [Loose et al., 2011]. MinD promotes conformational changes in MinE resulting in an active form that
binds MinD and the membrane which allows MinE to
swing from one membrane bound MinD to the next despite knocking MinD off the membrane [Park et al., 2011].
In B. subtilis, and most Gram positive bacteria, the regulator of MinC/MinD is a coiled-coil protein designated
DivIVA [Marston et al., 1998]. This protein is recruited
to the site of membrane curvature generated by the
CYTOSKELETON
initiation of cytokinesis [Eswaramoorthy et al., 2011]
(Fig. 3). DivIVA recruits MinC and MinD through an
intermediary designated MinJ [Bramkamp et al., 2008;
Patrick and Kearns, 2008]. Thus, a DivIVA ring, decorated with the Min proteins, is formed on either side of
the incipient septum and prevents FtsZ released from the
ongoing cytokinesis from reforming a ring at the newly
forming poles [Gregory et al., 2008].
the double mutant can be suppressed by increasing FtsZ
(also by a slower growth rate). Also, growth of E. coli in
chambers smaller than the diameter of the cell selects for
flattened cells. In these deformed cells a Z ring forms
between segregated nucleoids even in the absence of SlmA
and Min suggesting that NO is the primary determinant
of Z ring placement and that SlmA is not the only component of the NO system [Mannik et al., 2012].
Another Spatial Regulator
NO System
Remarkably, the NO systems of B. subtilis and E. coli
employ two unrelated DNA binding proteins, Noc and
SlmA, respectively, to perform the same task [Wu and
Errington, 2004; Bernhardt and de Boer, 2005]. Both
proteins bind to their specific DNA binding sites, which
are scattered in the origin proximal 2/3 of the circular
chromosome [Wu et al., 2009]. For SlmA, it has been
shown that it is activated to attack FtsZ filaments upon
binding to its cognate sequence [Cho et al., 2011; Tonthat
et al., 2011]. Therefore, as the duplicating chromosomes
segregate the tethered regulators are moved away from the
cell center making it permissive for Z ring assembly (Fig.
3). Thus, the NO systems seem ideally suited to couple
DNA segregation to formation of the Z ring, but as
pointed out above, elimination of the known effectors of
NO has little phenotype in exponentially growing cells,
suggesting their primary role is to prevent guillotining of
the chromosome under conditions of replication/segregation stress [Wu and Errington, 2004; Bernhardt and de
Boer, 2005].
B. subtilis offers an advantage in the study of cytokinesis
since examination of the Z ring in germinating spores
offers a chance to look at formation of a Z ring in the absence of influences of the previous division [Migocki
et al., 2002]. Spores of a mutant lacking Min and Noc
(generated under permissive growth conditions for the
double mutant) were germinated and followed for 1–2
generations of growth. Z ring formation, although
delayed, still occurred primarily at midcell [Rodrigues and
Harry, 2012]. This result indicates that an additional factor, other than Min or Noc, positions the Z ring and
remains to be identified. Also, it is clear that the proteins
identified to mediate NO, SlmA and Noc, represent only
part of NO and that additional factor (s) are involved.
Together the results favor a model in which there is a
limited amount of FtsZ available in the cell but FtsZ assembly is promiscuous and is actively countered by localized antagonists, Min and SlmA (Noc). By limiting this
promiscuous behavior away from midcell, they ensure
there is sufficient FtsZ available to assemble a complete Z
ring at the desired location. In their absence, FtsZ assembly between nucleoids is favored, however, the free FtsZ is
insufficient to form a complete ring due to too much
FtsZ tied up in spurious assemblies. Consistent with this,
CYTOSKELETON
Although Min and SlmA or Noc are widely distributed,
especially among rod shaped bacteria, they are not present
in all bacteria. Caulobacter lacks Min and both NO
homologues and spatial regulation is due to MipZ, a protein related to MinD [Thanbichler and Shapiro, 2006].
Monomers of MipZ are recruited to the pole of the cell
by interaction with ParB, which is bound near the origin
of replication, and along with ParA, is involved in chromosome segregation (Fig. 3). ParB promotes MipZ dimerization, the form that antagonizes FtsZ assembly, and the
dimers diffuse away and bind nonspecifically to the chromosome [Kiekebusch et al., 2012]. This binding is transient as the intrinsic ATPase of MipZ causes release from
the DNA. Repeated cycling of MipZ between the bound
ParB and the DNA leads to a gradient of MipZ on the
chromosome that is highest near ParB. When the origin
duplicates, one segregates to the other pole, resulting in a
bipolar gradient of MipZ with the low point near midcell
where the Z ring forms.
Z Ring Assembly During Sporulation
Although spatial regulation of Z ring assembly is focused
mostly on exponentially growing cells, sporulation offers
unique opportunities to examine spatial regulation. In
sporulating cells of B. subtilis the Z ring formed at midcell
spirals away to form a Z ring at each of the cell’s poles,
one of which leads to asymmetric septation and the other
is dismantled [Ben-Yehuda and Losick, 2002]. Although
the switch involves an increase in FtsZ expression and the
production of two sporulation specific proteins, SpoIIE,
membrane protein that binds FtsZ, and RefZ, a protein
related to SlmA, a detailed mechanism is lacking [Wagner-Herman et al., 2012]. Streptomyces species also sporulate and have a life style similar to filamentous fungi with
a mycelial growth containing few septa and the formation
of aerial hyphae that eventually contain a ladder like structure of Z rings that form simultaneously [Grantcharova
et al., 2005]. An interesting aspect of this system is the
identification of a protein (SsgB) that precedes FtsZ localization [Willemse et al., 2011]. How it is localized is still
not known but it represents the first case where a protein
precedes FtsZ to the division site. This protein is not
widespread among bacteria, however, it is possible that
analogues exist in other bacteria.
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
length. Levin’s lab found that a mutant in B. subtilis, deficient in the synthesis of a nonessential cell wall polymer,
was unable to increase its cell length upon shift to a faster
growth rate [Weart et al., 2007]. This result indicated that
this pathway is used to communicate information about
the growth rate to the division apparatus to delay division
at faster growth rates. The effector of this response is the
last enzyme in this pathway, UgtP, which interacts directly
with FtsZ. Although the mechanism is not clear, it is possible that the metabolic flux through UgtP regulates its
interaction with FtsZ. At fast growth rates UgtP may
decrease the amount of active FtsZ so that cell division is
delayed until more FtsZ accumulates [Hill et al., 2012].
An E. coli mutant (pgm) corresponding to the original B.
subtilis mutant (pgcA) also displays a defect in adjusting
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
gain of function allele of ftsA (ftsA*) raising the possibility
that the ftsA* mutant is resistant to this regulation [Hill
et al., 2012]. A potential effector protein in E. coli is not
clear since it lacks a homologue of UgtP.
Formation of the Divisome
The assembly of the complete divisome occurs in two
temporally distinct steps in E. coli, B. subtilis and Caulobacter [Aarsman et al., 2005; Gamba et al., 2009; Goley
et al., 2011]. Following assembly of the Z ring there is a
considerable gap before the subsequent accrual of additional proteins to constitute the complete divisome. In
E. coli at least nine additional essential division proteins
are added almost simultaneously to the Z ring [Schmidt
et al., 2004; Goehring and Beckwith, 2005] (Fig. 4).
How these additional proteins are connected to the Z ring
is not clear but FtsA plays a key role. Dependency studies
indicate that there is a linear order to the assembly process
even though several of the proteins have been shown to
exist in complexes even when they are not associated with
the Z ring [Goehring et al., 2006]. The roles of these
additional essential division proteins in E. coli are
described below.
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Lutkenhaus et al.
Fig. 4. Diagram of assembly of the Z ring and maturation
to the divisome in E. coli. FtsZ polymers (FtsZ[n]) are tethered to the membrane by FtsA and ZipA, which leads to formation of the Z ring. However, FtsA is not immediately available
to recruit downstream proteins (FtsA[i]). Although not essential,
several FtsZ interacting proteins (ZapA-D) localize to the ring
and promote the integrity of the Z ring. Antagonists of FtsZ assembly, MinC/D and SlmA, are positioned away from midcell
so that it is permissive for Z ring formation. When enough
monomeric FtsA (active [a]) is present at the Z ring the remaining Fts proteins and PBP1b are recruited. The arrival of FtsN
signals the divisome is complete and activation of septal PG
(peptidoglycan) synthesis occurs (divisome [a]). The septal cross
wall is split by AmiB, which is activated by EnvC (recruited earlier), and AmiC, which is activated by NlpD. Although EnvC is
recruited early, the arrival of AmiC, AmiB and NlpD depend
upon the start of septal PG synthesis induced by the arrival of
FtsN. The Tol-Pal complex is needed for efficient invagination
of the outer membrane (i), inactive; (a), active.
FtsE and FtsX encode an ABC transporter with the
most homology to the Lol system, which extracts lipoproteins out of the cytoplasmic membrane [Schmidt et al.,
2004]. FtsX encodes the membrane component and FtsE
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
[Reddy, 2007]. However, there is a slight division defect
under suppressing conditions and recently it was shown
that FtsE/FtsX recruit an activator, EnvC, of a septal peptidoglycan amidase (AmiB) to the Z ring [Yang et al.,
2011]. EnvC is recruited directly by FtsX but the ATPase
activity of FtsE is required for EnvC to activate AmiB. In
the absence of FtsE/FtsX this activity is missing and splitting of the septum is due to other amidases with partially
redundant activity.
FtsK is a DNA translocase with a membrane domain
containing four transmembrane spanning segments fused
to the DNA translocase domain by a long linker [Begg
et al., 1995]. When located at the septum this protein is
able to translocate DNA away from the septum due to
specific sequences (KOPS) located throughout the chromosome, which give directionality to the movement of
the DNA [Bigot et al., 2005]. Although this protein can
rescue DNA trapped at the septum this only comes into
play during stress and is not an essential function [Steiner
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
and have no role in septation. 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].
FtsN is the last essential division protein to arrive at the
ring and may signal that the divisome complex is complete and septation should be initiated, basically acting as
a trigger for septation [Goehring and Beckwith, 2005;
Gerding et al., 2009]. FtsN is a bitopic protein with a
short cytoplasmic region connected to a larger periplasmic
region by a single transmembrane domain [Dai et al.,
1993]. The most conserved region of FtsN lies at the Cterminus (SPOR domain) and binds a form of peptidoglycan that is only present at the septum. However, this domain is not essential [Gerding et al., 2009]. FtsN,
however, is required for the recruitment of a host of
downstream proteins whose activities are partially redundant. It is unlikely that this recruitment is direct but may
rely upon FtsN’s ability to trigger septation [Bernard
et al., 2007]. At least one allele of ftsA can bypass the
requirement for FtsN suggesting that septation can be
triggered another way [Bernard et al., 2007].
Role of FtsA in Formation of the Divisome
FtsA plays two critical roles in cytokinesis. First, along
with ZipA, it tethers FtsZ filaments to the membrane
[Pichoff and Lutkenhaus, 2002]. Second, along with
ZipA, it is required for recruitment of all the downstream
division proteins [Hale and de Boer, 2002]. Importantly,
Margolin’s lab isolated an allele of ftsA, ftsA*, that was
able to bypass the requirement for ZipA [Geissler et al.,
2007]. This, along with evidence that FtsA is much more
conserved in evolution than ZipA, and interacts with a
number of downstream division proteins, indicate that
FtsA has a more direct role in their recruitment [Corbin
et al., 2004]. In addition to bypassing ZipA, FtsA* has a
number of unusual properties, including resistance to various treatments that destabilize the Z ring, including excess
MinC, and allowing cells to divide at a smaller cell size
CYTOSKELETON
Fig. 5. Structures of FtsA and MreB. Two molecules of FtsA
are shown as arranged in a filament (PDB 4A2A). The structure
of MreB (PDB 1JCE) is similar to conventional actin. FtsA
lacks domain 1B but has a new domain 1C (colored red in one
FtsA) that interacts with other division proteins.
[Bernard et al., 2007]. Bacterial two hybrid tests indicated
FtsA* interacted more strongly with itself leading to the
suggestion that increased interaction between FtsA molecules stabilized the Z ring to destabilizing conditions
[Shiomi and Margolin, 2007].
Although FtsA is a member of the actin family, it is
missing one of actin’s four subdomains but has an additional subdomain (1C) located elsewhere in the structure
[Szwedziak et al., 2012] (Fig. 5). Many bacteria possess
another actin homologue (MreB) that has the same domain structure as actin [van den Ent et al., 2001] (Fig. 5),
but in E. coli and B. subtilis it is required for the maintenance of the rod shape by organizing peptidoglycan synthesis 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
reliable ATPase activity accompanied assembly and the filaments were not dynamic [Lara et al., 2005; Krupka
et al., 2012]. Other reports indicated FtsA without its
membrane binding domain assembles in vivo, forming
cytoplasmic filaments when overexpressed [Pichoff and
Lutkenhaus, 2005]. Recently, L€owe’s lab solved the structure of FtsA revealing actin-like protofilaments [Szwedziak
et al., 2012]. They also observed FtsA polymers on a lipid
monolayer and in the cytoplasm of cell (without the
membrane binding domain) with the same repeat distance
as observed in the crystal structure. Moreover, mutations
that would be expected to interfere with polymer contacts
were less efficient at division.
In a separate study Pichoff et al. [Pichoff et al., 2012]
isolated mutations that interfered with the ability of FtsA
to form cytoplasmic filaments. Surprisingly, ftsA* was
Bacterial Cytokinesis: From Z Ring to Divisome
785 n
among them and a decrease in self-interaction was confirmed by independent tests. In addition, selection of
many additional mutations that bypassed ZipA led to the
inescapable conclusion that such mutations decrease FtsA’s
self interaction. Consistent with this, the altered residues
mapped to the four major contact points between subunits in the FtsA filament. Whether some low level of FtsA
self interaction is essential is not clear. Nonetheless, this
finding led to a new model for how FtsA recruits downstream division proteins. In this model, FtsA switches
between a form that is unable to interact with downstream proteins (polymeric) and a form that is active in
recruitment (monomeric). Somehow this switch is regulated by the dynamic interaction of proteins with the tail
of FtsZ.
Model for FtsA Recruitment of
Downstream Division Proteins
Two complementary lines of evidence indicate FtsA, and
in particular domain 1C, plays a critical role by in assembly of the divisome. Artificially targeting domain 1C to
the poles of the cell leads to polar localization of several
late division proteins (FtsN and FtsI) [Corbin et al.,
2004]. On the other hand, FtsA deleted for the 1C domain localizes efficiently to the Z ring but is unable to
recruit the late cell division proteins [Rico et al., 2004].
Although the best evidence is for interaction between domain 1C and FtsN [Busiek et al., 2012], some evidence,
although indirect, also suggests interaction of this domain
with FtsI and FtsQ. These proteins are all single pass
bitopic membrane proteins with short cytoplasmic N-termini and large extracytoplasmic domains. A direct interaction with the cytoplasmic FtsA has to be with the short
N-termini of one or more of these proteins.
Since ftsA mutations that bypass ZipA result in less self
interaction, it was proposed that the essential function of
ZipA is to antagonize FtsA self assembly [Pichoff et al.,
2012]. Furthermore, FtsA mutants that no longer require
ZipA self-interact less well, suggesting that it is the monomer form of FtsA that recruits one or more of the downstream division proteins, presumably because domain 1C
becomes available. In this model, the Z ring is formed as
FtsA and ZipA interact with polymers of FtsZ and tether
them to the membrane. In shorter cells, other proteins
(such as MinC/MinD) that interact with the FtsZ tail,
compete with FtsA and reduce the amount of FtsA’s domain IC available at the Z ring. However, a combination
of a build up of FtsA at the Z ring along with conditions
favoring monomers increase the availability of domain 1C
and the recruitment of downstream proteins commences.
During constriction the presence of the downstream division proteins at the septum perpetuates the monomeric
form of FtsA through interaction with the 1C domain.
n 786
Lutkenhaus et al.
Together the information suggests the following more
explicit model. In the polymer form the 1C domain is
not available for interaction with a protein such as FtsN,
since it is occupied in the filament. In the monomer
form, however, the 1C domain is free and the connection
to the body of the protein by a hinge allows movement
and the acceptance of the N-terminus of FtsN. The structure of proteins from the Type IV pilus from Pseudomonas
suggests a likely scenario [Busiek et al., 2012]. PilM,
which is similar in structure to FtsA, binds to the N-terminus of the bitopic PilN protein [Karuppiah and Derrick, 2011]. Comparison of the structures of FtsA with
the PilM-PilN complex is consistent with the necessary
membrane orientation. The tail of FtsA, which attaches to
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.
Triggering Septation and Cell
Separation
Upon completion of the divisome septation is triggered
leading to synthesis of peptidoglycan and its eventual
splitting. In E. coli the triggering event coincides with the
arrival of FtsN at the divisome [Gerding et al., 2009].
FtsN recruitment has at least two requirements: (1) it
requires the immediate preceding protein, FtsI [Wissel
and Weiss, 2004]; and (2) it requires FtsA [Goehring
et al., 2006]. Thus, FtsN arrival signals completion of
divisome assembly and by activating FtsI, which along
with PBP1b synthesizes septal specific peptidoglycan,
results in septation. The activation of FtsI leads to the
recruitment of additional proteins that metabolize the
peptidoglycan and invaginate the outer membrane [Gerding et al., 2007]. In E. coli four proteins are recruited to
the septum by a conserved SPOR domain that binds septal specific peptidoglycan, presumably glycan chains that
have been metabolized by amidases [Gerding et al., 2009;
Arends et al., 2010]. These SPOR containing proteins
make good markers revealing the onset of septation.
E. coli contains three amidases, enzymes that remove
the peptide side chains attached to the glycan chains that
constitute peptidoglycan. Removal of these peptide side
chains, which crosslink the glycan chains, allows cells to
be separated [Bernhardt and de Boer, 2003]. The activity
of these amidases overlap and the presence of any one
ensures survival. Two of them, AmiB and AmiC, are localized to the septum [Peters et al., 2011]. AmiB requires
EnvC and thus, FtsX for recruitment and also FtsE for
activation. EnvC localizes early whereas its partner, AmiB
requires FtsN for localization and so is localized later.
AmiC and its activator (a lipoprotein called NlpD), both
require FtsN for localization. The requirement for FtsN
means that the amidases won’t be specifically recruited
CYTOSKELETON
and activated until the switch for peptidoglycan synthesis
is thrown. However, in the absence of AmiB and AmiC,
AmiA (which does not localize specifically to the septum)
is still able to carry out the splitting of the septum
through activation by the localized EnvC [Peters et al.,
2011].
In a Gram negative bacterium like E coli invagination
of the outer membrane follows septal peptidoglycan synthesis. A large set of proteins including a cytoplasmic
complex of Tol proteins (TolQAR) interacts with an outer
membrane lipoprotein (Pal) to ensure timely and efficient
invagination of this layer [Gerding et al., 2007]. These
proteins are not essential though and in their absence
invagination occurs.
Summary and Prospective
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DedD, and RlpA. J Bacteriol 192:242–255.
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over chromosomes in E. coli. Mol Cell 18:555–564.
The study of cytokinesis in bacteria has lagged behind
studies in eukaryotes due to the small size of bacteria and
the lack of prominent intracellular structures. However,
the availability of GFP, improving microscopic techniques
and the relative simplicity of the bacterial systems has
resulted in rapid gains in understanding this process. One
outcome of the study of cytokinesis in bacteria is the realization that the eukaryotic cytoskeleton had its origins in
bacteria. Another is that components of the bacterial system are relatively small in number making in vitro reconstitution experiments feasible. Many important questions
remain, however, including the structure of the Z ring,
how Z filament dynamics are regulated, how the Z ring is
activated to constrict and how the Z ring is connected to
cell wall synthesis.
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in Escherichia coli. J Bacteriol 172:2765–2768.
Acknowledgment
Chen Y, Erickson HP. 2009. FtsZ filament dynamics at steady state:
subunit exchange with and without nucleotide hydrolysis. Biochemistry 48:6664–6673.
Work in the author’s laboratory is supported by NIH
grant GM29764.
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