Cell Division
Frederico Gueiros-Filho
4
Abstract
Cell division in rod-shaped bacteria like Bacillus subtilis is carried out by a
contractile protein ring, known as the divisome or septalsome, which is made
up of about a dozen different polypeptides. This sophisticated macromolecular
machine, which is centered around the tubulin-like protein FtsZ, is capable of
promoting the coordinated invagination of the cell membrane and cell wall to
create the so-called division septum. The goal of this chapter is to provide an
overview of the mechanism of septum formation in B. subtilis. Emphasis will be
placed on describing the properties of the individual division proteins and how
they assemble into the divisome complex, and on a discussion of the regulatory
mechanisms that ensure that septum formation will happen with great spatial
and temporal precision at every cell cycle. In addition, the peculiar asymmetric
division that happens during B. subtilis sporulation will be described.
Introduction
Cell division, in its most encompassing sense, is the process of generating two
viable descendants from a progenitor cell. This involves two main coordinated
events: the replication and segregation of the bacterial chromosome and the
splitting of the progenitor cell by cytokinesis, which in bacteria is also known
as septum formation. This chapter focuses on the mechanism of septum formation. For a detailed description of chromosome replication and segregation, see
the Chapters 1 to 3 in this volume.
Not surprisingly, given the importance of efficient reproduction for evolutionary success, bacterial cells have developed a remarkably sophisticated protein
machinery capable of precisely splitting a progenitor cell at the right place and
time in every cell cycle. This machine, which is often referred to as divisome
or septalsome (septosome) (Nanninga, 1998), is based on a contractile protein
ring, much as in the case of their more complex eukaryotic relatives. In contrast
with eukaryotic cells, however, which frequently use actin and myosin as the
base of their contractile protein ring, in bacteria the contractile machinery is
based on the tubulin-like protein FtsZ.
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Understanding the composition and functioning of the divisome started
almost 40 years ago when the first mutants impaired on division were isolated
(Hirota et al., 1968; Nukushina and Ikeda, 1969). These mutants provided a first
handle on the components of the division pathway. It was not until the pioneering work of Bi and Lutkenhaus in the early 90s, which used electron microscopy
to determine the distribution of FtsZ during the cell cycle and discovered the Z
ring (Bi and Lutkenhaus, 1991), that the function of these components began
to be understood. Since then, the field has blossomed, advanced by the powerful
genetic tools available for E. coli and B. subtilis, as well as the intensive use of cell
biological approaches. In this regard, it is noteworthy that the study of cytokinesis, by popularizing the use of protein localization methods, has helped to
establish a new view of cellular microbiology, centered on the idea that bacterial
cells possess bona fide cytoskeletal elements and discretely localized proteins and
thus are far more architecturally complex than previously anticipated.
The goal of this chapter is to review the mechanism of cytokinesis in B.
subtilis. This mechanism seems to be generally conserved between B. subtilis and
E. coli and often E. coli information will be used to fill gaps in the B. subtilis
literature. Whenever appropriate, the distinctions between B. subtilis and E. coli
will be highlighted.
This chapter starts with a brief description of the anatomy of the division
site and an overview of cytokinesis. Next, it will present information on the
components of the B. subtilis divisome, followed by a discussion of the mechanisms involved in regulating the timing and placement of the division septum.
Finally, the peculiar asymmetric division that happens during sporulation will
be discussed.
Anatomy of B. subtilis and E. coli envelopes:
implications for cytokinesis
Cytokinesis requires the coordinated remodeling of the different layers that
make up the bacterial envelope. Even though the overall mechanism of septum
formation is highly conserved in B. subtilis and E. coli, there are some differences in the architecture of their envelopes and the way in which the septum
is constructed that are worth highlighting. The envelope of B. subtilis, typical
of Gram-positive bacteria, consists of the cytoplasmic membrane plus a thick
cell wall made of peptidoglycan and associated anionic polymers such as teichoic acid (Fig. 4.1A) (see Chapter 10 by Scheffers). The situation in E. coli is
more complex, since it has an LPS-containing outer membrane besides the cell
wall and the cytoplasmic membrane (Fig. 4.1C). The cell wall in E. coli is also
much thinner than in B. subtilis, probably a single peptidoglycan molecule thick.
The presence of an outer membrane does not seem to create a need for special
division mechanisms, since it seems to passively track with the cell wall. The
difference in cell wall, on the other hand, may be related to a marked difference
in the anatomy of the B. subtilis and E. coli developing division sites. In B. subtilis, division happens by the synthesis of a complete cross wall of peptidoglycan
sandwiched by membrane bilayers. Initially, there is no obvious invagination or
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Figure 4.1 Anatomy of septation in B. subtilis and E. coli. (A) Schematic representation
of the B. subtilis envelope and (B) fluorescence microscopy image of a septating B.
subtilis cell. (C) Schematic representation of the E. coli envelope and (D) fluorescence
microscopy image of a septating E. coli cell. Images B and D represent live cells that
were treated with a fluorescent membrane dye. Note the absence of constriction at
the B. subtilis septum. (E) Electron micrograph of a growing septum in B. subtilis,
highlighting the simultaneous invagination of the two envelope layers (CW and PM). (F)
Typical chain of cells in an exponentially growing culture of B. subtilis. Chain formation
is due to the delay that exists between septation and cell separation. Membrane stains
are often used to assess septation in B. subtilis cultures since septa that have not yet
undergone constriction are difficult to detect by phase contrast or DIC methods. CW,
cell wall; PM, plasma membrane; OM, outer membrane.
constriction at the division site (Fig. 4.1A, B, E, F). Invagination and maturation
of the septa into new hemispherical poles will eventually occur by the degradation of the inner part of the septal peptidoglycan. In contrast, in E. coli synthesis
of the division septum is concomitant with constriction (Fig. 4.1C, D). This is
probably due to the thinner peptidoglycan septal wall in E. coli, which gets split
in the middle as it is synthesized (Holtje, 1998). The thick cell wall of B. subtilis
is probably also to blame for the time it takes for the wall in between cells to
get degraded and the poles to mature. A consequence of this delay between cell
division and cell separation is that fast growing cultures of this bacterium reveal
mostly cells associated into long chains (Fig. 4.1F). Each cell in these chains is
fully separated from the next by a complete septum.
Overview of cytokinesis
Cytokinesis in B. subtilis, and probably for any other rod-shaped bacterium, can
be summarized in the following series of events. First, FtsZ, which is normally
a cytoplasmic protein (Fig. 4.2A), assembles into a ring like structure associated
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Figure 4.2 The steps of cytokinesis. (A) In a newborn cell, FtsZ (gray circles) and the
other divisome proteins, here represented as a membrane associated complex (black
“paddle”), are dispersed throughout the cell. (B) Upon the decision to divide, FtsZ selfassociates into the Z ring at the cell center. (C) The Z ring acts as a scaffold and recruits
the other division proteins to the division site, forming the divisome. (D) The assembly
of a complete divisome triggers the ingrowth of the peptidoglycan cell wall and the
invagination of the plasma membrane required for the synthesis of the new septum.
Septum formation is followed by constriction of the divisome and disassembly of its
components. (E) The ingrowth of the cell wall and membrane will eventually produce a
complete septum separating the progenitor cell into two independent daughter cells.
Finishing of the septum requires a membrane fusion event to separate the membranes
of the daughter cells. This step has been omitted from the figure for simplicity. (F)
Separation of the daughter cells is accomplished by the degradation of the inner part of
the septal peptidoglycan through the action of murein hydrolases.
with the inner face of the cytoplasmic membrane (Fig. 4.2B). This structure,
known as the Z ring, is a cytoskeletal element that serves as a scaffold onto
which the divisome complex will assemble. Z ring formation is the target of
careful regulatory mechanisms, described in detail below, that ensure that division will happen in coordination with other cell cycle events and at the proper
place along the rod-shaped cell. Next, the Z ring nucleates the assembly of
the divisome by attracting about ten other proteins to the division site. These
components of the divisome are mostly integral membrane proteins and their
recruitment to the Z ring occurs through both direct and indirect interactions
with FtsZ (Fig. 4.2C). Once a mature divisome has been formed, it will promote
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Cell Division | 97
the coordinated synthesis of a transversal cell wall and the invagination of the
cytoplasmic membrane (Fig. 4.2D). The ingrowth of the cell wall and membrane
will eventually produce a complete septum separating the progenitor cell into
two independent daughter cells. Completion of the septum requires a membrane fusion event to separate the membranes of the daughter cells (Fig. 4.2E).
Septum formation is accompanied by the constriction and disassembly of the
divisome into its elementary parts (Fig. 4.2D) such that division proteins will
have been redistributed back to the cytoplasm and cell membrane at the end
of cytokinesis. No divisome proteins remain associated with the newly formed
septum (Fig. 4.2E). The last step of cytokinesis is separation of the daughter
cells by the action of murein hydrolases that degrade the inner part of the septal
peptidoglycan cross wall (Fig. 4.2F).
B. subtilis division proteins
There are currently 18 proteins known to be associated with division in B. subtilis: ClpX, DivIB, DivIC, DivIVA, EzrA, FtsA, FtsL, FtsW, FtsZ, MinC, MinD,
Noc, PBP2B, SepF, SpoIIE, SpoIIIE, YneA and ZapA. These can be divided in
two main groups: proteins that make up the divisome and are directly involved in
the construction of the division septum (DivIB, DivIC, FtsA, FtsL, FtsW, FtsZ,
PBP2B, SepF) and proteins that regulate the assembly of the divisome (ClpX,
DivIVA, EzrA, MinC, MinD, Noc, YneA, and ZapA). Some of the regulatory
proteins, like EzrA and ZapA, are tightly associated with the divisome and thus
can be considered as part of this complex. SpoIIIE, a protein whose function
is to coordinate septum assembly with chromosome segregation, represents a
third functional group. A summary of the features of these division proteins
can be found in Table 4.1. The table also points to the correspondence between
the B. subtilis and E. coli proteins, since the nomenclature is not unified in both
species. Most of the components of the B. subtilis divisome have counterparts
in E. coli and many other bacteria. These genes seem to make up a core division
machinery that was probably already present in the last common ancestor of
all bacteria (Robson F. Souza, Sandro de Souza and Frederico Gueiros-Filho,
unpublished observations).
The genes for many cell division proteins are often found next to each other
as well as to some cell wall biosynthetic genes in a conserved region that has been
called the dcw (for “divison and cell wall”) cluster. The remarkably conserved
gene order of this region in diverse bacterial genomes has led some authors to
suggest that there are functional consequences of this arrangement for division
and the maintenance of a rod shape (Mingorance et al., 2004). The dcw cluster
of B. subtilis is located at around 135 degrees in the chromosome. The ftsA, ftsZ,
divIB (ftsQ) pbpB (ftsI), ftsL and ftsW genes are some of the genes commonly
present at the dcw cluster. In B. subtilis and several other Gram-positives, the dcw
cluster seems to extend further on one side to include also the gene for the division site selection protein DivIVA and several conserved “ylm” genes (Massidda
et al., 1998), which, based on recent evidence, are also likely to participate in
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E. coli ortholog
ClpX
FtsQ
FtsB
–
–
FtsA
FtsL
FtsW
FtsZ
MinC
B. subtilis ortholog
ClpX
DivIB
DivIC
DivIVA
EzrA
FtsA
FtsL
FtsW
FtsZ
MinC
25 kDa cytoplasmic protein. Site-specific FtsZ inhibitor. Prevents polar Z ring formation (with MinD)
40 kDa cytoplasmic protein. Structurally and functionally related to tubulin. Self-assembles to forms
cytoskeletal scaffold (Z ring) for divisome assembly
44 kDa multipass transmembrane protein. Putative peptidoglycan precursor transporter
13 kDa membrane protein with extracytoplasmic domain. Part of multiprotein complex connecting the
peptidoglycan synthesizing proteins to the early divisome
48 kDa membrane associated protein. Structurally similar to actin. Stabilizes and tethers Z ring to
membrane. Recruits downstream divisome proteins
65 kDa membrane protein with cytoplasmic domain. Negative regulator of FtsZ polymerization. Prevents
formation of multiple Z rings
19 kDa cytoplasmic protein. Topological determinant of Min system. Localizes MinCD to cell poles thus
restricting division inhibition to this location. Analogous function to MinE in E. coli
15 kDa membrane protein with extracytoplasmic domain. Part of multiprotein complex connecting the
peptidoglycan synthesizing proteins to the early divisome
30 kDa membrane protein with extracytoplasmic domain. Part of multiprotein complex connecting the
peptidoglycan synthesizing proteins to the early divisome
46 kDa cytoplasmic protein. Substrate recognition subunit of the ClpXP protease complex. Negative
modulator of Z-ring formation
Features and function
Table 4.1 Summary of B. subtilis division genes and their E. coli counterparts
?1
FtsI or PBP3
(ftsI or pbpB)
–
–
FtsK
–
ZapA (ygfE)
Noc
PBP2b
(pbpB)
SepF or YlmF
SpoIIE
SpoIIIE
YneA
ZapA
10 kDa cytoplasmic protein. Positive modulator of FtsZ. Binds to FtsZ and promotes polymer bundling in
vitro. Stabilizes Z ring in vivo. Dispensable for septum formation
12 kDa predicted membrane protein. SOS-induced septation inhibitor. Analogous function to SulA in E. coli
87 kDa protein with four transmembrane segments and a large cytoplasmic domain. DNA pump involved in
chromosome segregation and dimer resolution. Dispensable for septum formation in B. subtilis but required
in E. coli
92 kDa transmembrane protein with cytoplasmic domain. Sporulation protein needed for efficient polar
septum formation. Binds to FtsZ. Exhibits also phosphatase activity required for the activation of foresporespecific gene expression
17 kDa cytoplasmic FtsZ-binding protein. Absence reduces division frequency and affects septum structure.
Overexpression can compensate for the absence of FtsA
79 kDa membrane protein with large extracytoplasmic domain. Transpeptidase involved in septal
peptidoglycan biosynthesis. Likely to be associated with DivIB, DivIC and FtsL in a multiprotein complex
comprising a subassembly of the divisome
33 kDa DNA-associated FtsZ inhibitor. Effector of nucleoid occlusion. Analogous function to SlmA in E. coli
29 kDa membrane associated ATPase. Interacts with MinC and brings it to membrane. Prevents polar Z-ring
formation
Gene names that differ from the corresponding protein name are presented in parentheses.
1Noc is a member of the ParB family of DNA binding proteins that have several orthologs in E. coli. It is unclear which of the E. coli family members is
the true ortholog of Noc.
MinD
MinD
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division (Fadda et al., 2003; Hamoen et al., 2006). The remaining genes are
scattered throughout the genome. Curiously, in B. subtilis, the genes for the division inhibitor MinCD are in the same operon as the mre genes, which are the
components of the actin-like cytoskeleton responsible for maintaining cell shape
and chromosome segregation. It is unclear whether this organization implies a
functional relationship between the division and morphogenesis systems.
In the next sections, the current knowledge about the best-studied B. subtilis
division proteins will be summarized. This will be presented in the order they
assemble in the divisome.
FtsZ and the Z ring
The tubulin-like FtsZ is the earliest-acting division protein and a master coordinator of septum formation. This characterization is based on several observations. First, inactivation of FtsZ produces a block in division that seems to
occur earlier than a block caused by inactivation of any other division protein
(Taschner et al., 1988). Second, all known division proteins depend on FtsZ
for their proper localization to the division site, while, conversely, localization of
FtsZ to the division site is independent of any other division protein (Addinall
et al., 1996; Errington et al., 2003). Third, cellular FtsZ levels seem to be the
main determinant of division frequency (Ward and Lutkenhaus, 1985; Dai and
Lutkenhaus, 1991). Fourth, FtsZ is the most widely distributed and conserved
division protein. It is present in essentially all the bacterial genomes that have
been sequenced to date, with a few notable exceptions, such as the obligate intracellular Chlamydia, the planctomycete Pirellula and the mollicutes Ureaplasma
(Vaughan et al., 2004).
Despite a low primary sequence identity with tubulin, FtsZ has been shown
to be very similar to tubulin both at the structural and at the functional level. FtsZ
is a 40 kDa globular protein that folds into two independent domains (Oliva
et al., 2004) (Fig. 4.3A). Self-assembly of FtsZ involves interactions between
the C-terminal domain of one subunit with the N-terminal domain of another
subunit (Oliva et al., 2004) (Fig. 4.3B). Like tubulin, FtsZ polymer formation
is regulated by guanine nucleotides. Binding of GTP to a pocket located in the
N-terminal domain induces the assembly of polymers (Erickson et al., 1996;
Mukherjee and Lutkenhaus, 1998) (Fig. 4.3C). An important consequence of
FtsZ assembly is to activate its GTPase activity, which is essentially absent when
the protein is monomeric. Structurally, activation of GTP hydrolysis is due to
the positioning of a catalytic aspartate residue present in a C-terminal domain
loop (the T7 loop) of one FtsZ monomer next to the GTP sitting in the binding pocket of another monomer. Hydrolysis leads to instability of the polymers
and to disassembly. Thus, FtsZ polymers are highly dynamic assemblies in vitro
(Mukherjee and Lutkenhaus, 1998), as would be expected of a cytoskeletal element that needs to undergo recurring assembly and disassembly during every
cell cycle. Another similarity between the FtsZ and the tubulin cytoskeleton is
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Figure 4.3 FtsZ and the Z ring. (A) Crystal structure of FtsZ highlighting its main
features. Note that the protein can be divided by the helix H7 into two independently
folding domains (boundaries marked by dashed line): the N- and C-terminal domains.
The nucleotide binding pocket is in the N-terminal domain whereas the T7 loop involved
in catalyzing nucleotide hydrolysis is located in the C-terminal domain. (B) Model of an
FtsZ protofilament showing the contacts involved in the self-association of monomers.
The spacing between subunits is 42 Å, very similar to the spacing between subunits in
a tubulin protofilament (40 Å). Molecules of GDP can be seen occupying the binding
interface in between the monomers. Plus and minus signs indicate the polarity of the
polymer. The plus end corresponds to the nucleotide binding surface of the protein.
(C) Electron micrograph of a negatively stained sample of B. subtilis FtsZ polymerized
by GTP. This sample exhibits predominantly straight protofilaments. (D) Fluorescence
image of a Z ring in a dividing B. subtilis cell expressing GFP-tagged FtsZ. (E) A Z
ring in three dimensions. 3D reconstruction of the Z ring was carried out from optical
sections of a cell expressing FtsZ-GFP like the one in D. The dotted white line shows
the boundaries of the imaged cell. Images in panels A and B adapted from Lowe et al.,
Annu. Rev. Biophys. Biomol. Struct. 2004;33:177–98 and reprinted with permission.
Microscopy images in panels D and E adapted from Ben-Yehuda and Losick (2002) and
reprinted with permission.
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that FtsZ assembly seems to be a cooperative process (Wang and Lutkenhaus,
1993; Huecas and Andreu, 2003; Chen et al., 2005).
Structural studies of FtsZ polymerization using electron microscopy under
different conditions have shown that FtsZ is capable of forming various types of
polymers (Romberg and Levin, 2003). The simplest kind of polymer formed in
the presence of GTP is the protofilament, a one-subunit thick head to tail string
of FtsZ monomers (Fig. 4.3C). Protofilaments induced by GTP are straight
polymers, indicating the absence of a tilt or angle in the bond between subunits.
Straight protofilaments can also associate into two dimensional sheets or 3D
bundles through lateral interactions (Gonzalez et al., 2003). The tendency of
FtsZ polymers to form lateral interactions is greatly increased by the presence
of cations such as Ca2+ (Mukherjee and Lutkenhaus, 1999), DEAE-dextran,
or cationic phospholipids (Erickson et al., 1996) and by FtsZ binding proteins,
such as ZapA discussed below. Polymerization can be achieved also in the presence of GDP as long as stabilizing factors such as DEAE-dextran are present
in the reaction. In contrast with GTP-FtsZ, polymers formed by GDP-bound
FtsZ are mostly curved. Helical tubes, mini rings and curved protofilaments
have been detected in different preparations (Erickson et al., 1996; Lu et al.,
1998). Both GTP-induced and GDP-induced FtsZ polymers are highly similar
to tubulin polymers produced under equivalent in vitro conditions, reinforcing
the notion that the two proteins are related. The main difference between FtsZ
and tubulin is that FtsZ has never been seen to assemble as hollow microtubules
like those formed by tubulin.
Much less is known about the structure of the Z ring in live cells. The Z ring
has never been detected using standard transmission electron microscopy. This
is either due to inappropriate fixation conditions or because the FtsZ polymer
that makes up the ring is not bulky enough to be distinguished above the high
density of the surrounding bacterial cytoplasm. Based on the in vitro studies
and the fact that in vivo FtsZ should be mostly bound to GTP, it is currently
assumed that the physiologically relevant FtsZ polymer is some form of protofilament sheet. This sheet could contain between 3 and 12 protofilaments, based
on estimates made in B. subtilis and E. coli that there are 5000 to 20 000 FtsZ
molecules in a single cell (Feucht et al., 2001; Romberg and Levin, 2003). The
actual number of protofilaments in a Z ring could be as low as one, if recent
experiments indicating that the Z ring seems to contain only one third of the
cellular FtsZ are taken into account (Stricker et al., 2002). Another important
question that remains unanswered is whether the Z ring is made up of long
continuous protofilaments or by an association of short protofilament segments.
Advances, such as the use of cryoelectron tomography, which has already been
used successfully for high resolution studies of other structural proteins in bacteria (Kurner et al., 2005), should facilitate the emergence of a more detailed
description of the FtsZ polymer structure in vivo.
Meanwhile, valuable information about the assembly and the dynamic
behavior of the Z ring has been gathered by fluorescence microscopy. This
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Cell Division | 103
approach revealed that Z ring assembly and disassembly are fast processes,
occurring in a timescale of minutes (Addinall et al., 1997; Sun and Margolin,
1998). FtsZ assembly in vivo has been proposed to start from a discrete point
on the cytoplasmic membrane and extend bidirectionally until a complete ring
is formed (Addinall and Lutkenhaus, 1996). This has suggested the existence
of a nucleation site at the membrane. The nature of this nucleation site is still
elusive (see below). At any given time during cytokinesis, only about a third
of the FtsZ in a cell is associated with the Z ring. The other two thirds are
present in the cytoplasm. Photobleaching experiments with bacteria expressing
GFP-tagged FtsZ have revealed that both in E. coli and in B. subtilis, FtsZ in the
ring is constantly being exchanged with the cytoplasmic FtsZ pool (Stricker et
al., 2002; Anderson et al., 2004). The rate of exchange is very fast (an average
ring will turn over with a half life of between 8 and 30 seconds) and seems to
correlate with the GTPase activity of the protein. The rate of exchange of ZipA,
a membrane-associated FtsZ binding protein of E. coli, was as fast as that of
FtsZ, indicating that not only the Z ring, but probably the whole divisome is
a dynamic structure that is constantly recycling itself. The dynamic behavior
of the Z ring and associated proteins has been proposed to be important to
generate the mechanical force required for divisome constriction (Stricker et al.,
2002).
Z ring formation in vivo is affected by a number of proteins that interact
with FtsZ and modulate its polymerization properties. These modulatory factors, which are capable of either inhibiting or stimulating the assembly of FtsZ,
play key roles in the spatiotemporal regulation of Z ring formation (see section
below on regulation). This is analogous to the regulation of the microtubule
cytoskeleton by tubulin binding proteins such as the MAPs.
FtsZ binding proteins—early divisome components
In B. subtilis, there are four divisome proteins, FtsA, EzrA, ZapA and SepF
(YlmF), which interact directly with FtsZ (Fig. 4.4A). Of the four, FtsA and
ZapA are quite conserved, being present in diverse bacterial groups including E.
coli. SepF, on the other hand, is present only in Gram-positive and cyanobacteria
and EzrA is restricted to a subset of Gram-positive bacteria. A fifth well-studied
FtsZ binding protein, ZipA, is absent from B. subtilis but present in E. coli and
closely related enterobacteria.
The FtsZ binding proteins of B. subtilis play different roles in division. FtsA
seems to be important to connect the Z ring to the membrane and is required
for the assembly of the later components of the divisome and septum formation.
On the other hand, ZapA and EzrA affect the polymerization of FtsZ and are
likely to play regulatory roles. A summary of the properties and function of FtsA
and SepF are presented below. A detailed description of ZapA and EzrA can be
found in the section on regulation of septum formation.
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Figure 4.4 Divisome components and assembly. (A) The B. subtilis divisome. This
scheme represents a hypothetical divisome complex as it would be viewed in a cross
section of the bacterium at the division site. Protein topologies and the interactions
between FtsZ and the proteins that interact directly with it are accurately represented.
Some of the other interactions are only meant to illustrate the existence of a complex
and do not necessarily reflect reality (for example, there is no evidence for a direct
interaction between FtsA and DivIB). (B) Pathway of divisome assembly in B. subtilis
and E. coli. In each of these pathways, assembly of a given protein is dependent on all
the proteins to its left and independent of the proteins to its right. Proteins that associate
early with the divisome but that are not necessary for the recruitment of later proteins are
shown in gray. Question marks represent placements for which experimental evidence
is lacking. SepF has been omitted from the figure because there is little information on
its interaction with other divisome components. CW, cell wall; CYT, cytoplasm.
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FtsA
FtsA is a 45 kD cytoplasmic protein that belongs to the superfamily of ATPbinding proteins that includes actin, Hsp70 and hexokinase. Indeed, determination of the crystal structure of FtsA revealed that it is quite similar to actin (van
den Ent and Lowe, 2000). Both proteins have a similar two-domain fold and
nucleotide binding cleft. The main structural difference between actin and FtsA
is in subdomain 1B, which in FtsA adopts an alternative topology and therefore
has been named 1C (van den Ent and Lowe, 2000). In contrast with the other
prokaryotic actin-like proteins MreB and ParM, FtsA does not seem to self-associate and form filaments under normal conditions.
Studies in E. coli have shown that FtsA binds to a conserved 15 amino acid
sequence present at the extreme C-terminus of FtsZ (Ma and Margolin, 1999;
Wang et al., 1997). This is probably the case for B. subtilis as well given that
several of the residues important for the FtsA-FtsZ interaction are present in B.
subtilis FtsZ (Erickson, 2001). The C-terminal peptide of FtsZ has been shown
to be also the binding site for the E. coli protein ZipA (Haney et al., 2001). The
region of FtsA involved in binding to the C-terminus of FtsZ is still not known.
It is also unknown whether the binding of FtsA to FtsZ is capable of promoting
the assembly of FtsZ polymers, like ZapA and ZipA do.
FtsA is required for septum formation in both B. subtilis and E. coli. Division
inhibition due to the absence of FtsA is lethal in E. coli. In contrast, ftsA null
mutants have been obtained in B. subtilis, although these are highly filamentous
and unhealthy (Beall and Lutkenhaus, 1992). Several studies, predominantly
in E. coli, have determined that FtsA seems to have two distinct roles in septum formation. The first role of FtsA is to promote the assembly of the Z ring.
Assembly of the Z ring in E. coli requires both FtsA and ZipA, since mutants
lacking either of these proteins still form Z rings (Addinall et al., 1996; Hale
and de Boer, 1999; Liu et al., 1999). However, in an ftsA zipA double mutant,
assembly of Z rings is hampered (Pichoff and Lutkenhaus, 2002). In B. subtilis,
a mutation in ftsA alone seems to prevent Z ring formation ( Jensen et al., 2005).
This is consistent with the fact that B. subtilis lacks ZipA. The primary role
played by FtsA in B. subtilis is likely to be the rule rather than the exception,
given that most bacteria have FtsA but lack a recognizable homolog of ZipA.
FtsA promotes Z ring formation by bringing FtsZ polymers to the cytoplasmic membrane (Pichoff and Lutkenhaus, 2005). Although FtsA lacks a
transmembrane segment, it associates with the membrane through a conserved
C-terminal amphipathic alpha-helix (Pichoff and Lutkenhaus, 2005). Tethering
of FtsZ to the membrane could facilitate Z ring formation by increasing the
probability with which FtsZ polymers would associate into stable multifilament
structures. Besides connecting FtsZ polymers to the membrane, binding of FtsA
to FtsZ seems to stabilize FtsZ polymers. This is suggested by the observation
that FtsA overexpression is capable of overcoming the division block mediated
by the Min inhibitor (see below) both in E. coli ( Justice et al., 2000) and in B.
subtilis (Alessandra Pancetti and Frederico Gueiros-Filho, unpublished).
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The second role of FtsA during septation is to recruit downstream divisome
proteins to the Z ring. In the absence of FtsA none of the late division proteins
localize properly to the division site (Errington et al., 2003). Recently, it has been
shown that FtsA is indeed able to interact with late divisome proteins independently of FtsZ (Corbin et al., 2004). This same study implicated subdomain 1C
as the important region for divisome protein recruitment (Corbin et al., 2004).
The role of subdomain 1C was further supported by the observation that deletion of this region did not affect FtsA association with the Z ring but prevented
the recruitment of several late proteins to the divisome (Rico et al., 2004). It is
likely that FtsA also serves to connect the Z ring to the late division proteins
in B. subtilis. Testing this will require finding specific mutants or using an assay
that separates the two roles role of FtsA, since this protein seems to be strictly
required for Z ring formation in B. subtilis ( Jensen et al., 2005).
SepF (YlmF)
SepF is a newly discovered component of the B. subtilis divisome (Hamoen et
al., 2006; Ishikawa et al., 2006). The gene for SepF lies close to the gene for
the division site selection protein DivIVA in an extension of the dcw cluster
that is conserved among Gram-positive bacteria (see above). SepF is a 17 kDa
protein that was shown to interact directly with FtsZ by two-hybrid analysis.
Inactivation of the gene for SepF leads to mild division inhibition and to alterations in septum morphology. SepF is likely to complement the role of FtsA in
promoting the assembly of the Z ring, since deletion of sepF exacerbates the
division inhibition of an ftsA mutant, whereas SepF overexpression rescues
the division block of the same mutant (Ishikawa et al., 2006). The alteration in
septum morphology in a sepF mutant suggests that SepF may also play a role
at a late stage of division, during the synthesis of the peptidoglycan crosswall
(Hamoen et al., 2006).
Late divisome components and the synthesis of septal
peptidoglycan
In B. subtilis, six integral membrane proteins are recruited to the divisome late in
its assembly (Fig. 4.4A, B). None of these appear to bind directly to FtsZ. Thus,
their recruitment must depend on interactions with FtsZ-binding proteins
such as FtsA, although this has not been demonstrated experimentally in B.
subtilis. One of these proteins, SpoIIIE, is involved in coordinating chromosome
partitioning and septation. Most of the other late divisome proteins have large
extracytoplasmic domains, suggesting that they function outside the cell in the
construction of the septal peptidoglycan wall. Despite rather intense efforts by
several laboratories, the actual functions of these proteins remain unknown. The
only exception is PBP2B, which is a member of the penicillin binding protein
family involved in the late stages of peptidoglycan assembly. In the following sections, an overview of what is known about each of these proteins is presented.
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SpoIIIE
SpoIIIE is a member of a family of DNA translocases involved in chromosome segregation and conjugation of mobile elements (Iyer et al., 2004). The
counterpart of SpoIIIE in E. coli is FtsK. These proteins generally have four
transmembrane segments followed by a large cytoplasmic domain that functions
as an ATP-driven DNA pump (Aussel et al., 2002; Bath et al., 2000) (see also
Chapter 3). SpoIIIE localizes to the division complex through its membrane
domain. Once constriction begins, SpoIIIE becomes organized as a focus at the
leading edge of the closing septum. The SpoIIIE focus is presumed to represent
a DNA translocation pore responsible for the correct distribution of chromosomes that occasionally fail to completely segregate and get trapped by the constricting septum (see Fig. 3.7) (Britton and Grossman, 1999). Thus, SpoIIIE
represents functional link between the cytokinesis and the segregation systems
in B. subtilis. The chromosome pumping role of SpoIIIE is crucial during sporulation, hence, the Spo name of the protein. This is because during sporulation,
segregation of the chromosome destined for the developing spore always occurs
after formation of the asymmetric septum (see Fig. 3.8, for a more detailed
description of chromosome segregation during sporulation see Chapters 3 and
11). Besides pumping misaligned chromosomes, there is clear evidence for a role
for FtsK in promoting chromosome dimer resolution by the Xer recombinase
system in E. coli (Recchia et al., 1999; Steiner et al., 1999; Aussel et al., 2002). It
is controversial whether SpoIIIE plays an equivalent role in B. subtilis (Lemon et
al., 2001; Sciochetti et al., 2001).
In E. coli, FtsK is required for septum formation, in addition to its role in
chromosome segregation (Begg et al., 1995). Mutations in FtsK block divisome
assembly by preventing the recruitment of other late division proteins such as
FtsI, FtsL and FtsQ (Chen and Beckwith, 2001). The division-promoting activity of FtsK resides in the N-terminal membrane part of the protein and can be
clearly separated from its DNA translocation activity (Draper et al., 1998; Liu et
al., 1998; Wang and Lutkenhaus, 1998). In contrast, in B. subtilis, SpoIIIE is not
required for divisome assembly. This is reflected in the fact that the N-terminal
regions of the two proteins are quite divergent.
DivIB, DivIC, and FtsL
DivIB, DivIC, and FtsL are small bitopic membrane proteins, with a short
N-terminal cytoplasmic region, a single transmembrane segment and a larger
C-terminal extracytoplasmic region (Harry and Wake, 1989; Levin and Losick,
1994; Daniel et al., 1998). DivIB is the largest of the three (30 kDa), with a
210 amino acid globular extracytoplasmic region. DivIC and FtsL, on the other
hand, have significantly shorter external regions (67 and 63 amino acids, respectively) that are predicted to adopt a coiled coil configuration. The E. coli
counterparts of DivIB and DivIC are FtsQ and FtsB, respectively. FtsL follows
the same nomenclature in both species.
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The three proteins localize to the division site at a late stage in cytokinesis
(Harry and Wake, 1997; Katis et al., 1997; Daniel and Errington, 2000; Sievers
and Errington, 2000b) and, with the exception of DivIB, are essential for septum formation (Levin and Losick, 1994; Daniel et al., 1998). DivIB mutants
are also impaired in septation but this defect is mild when cells are grown at
low temperatures (Beall and Lutkenhaus, 1989). Domain swap experiments
demonstrated that only the extracytoplasmic domain of DivIB is required for
localization and functioning in division (Chen et al., 1999; Katis and Wake,
1999). This seems also to be the case for DivIC (Katis and Wake, 1999). The
function of FtsL, on the other hand, seems to require both its extracytoplasmic
domain as well as its transmembrane segment (Ghigo and Beckwith, 2000;
Sievers and Errington, 2000a).
DivIB, DivIC, and FtsL are likely to interact to form a ternary complex, as
recruitment of any one of these proteins to the divisome is dependent on the
other two. Moreover, the intracellular levels of DivIC and FtsL, both intrinsically
unstable proteins, are affected by the presence or absence of DivIB (Robson et al.,
2002). Recent evidence obtained in E. coli suggests that the equivalent complex
(FtsB-FtsL-FtsQ) is preassembled in the membrane before being recruited to the
cytokinetic ring, thus representing a subcomplex of the divisome (Buddelmeijer
and Beckwith, 2004). Interaction among the proteins in this complex is likely
to involve the coiled coil domains of DivIC and FtsL. The interacting region in
DivIB is not known. The DivIB-DivIC-FtsL subcomplex in B. subtilis might also
include PBP2B, since this protein requires DivIB, DivIC, FtsL to localize to the
divisome and, reciprocally, PBP2b is required to localize DivIB, DivIC, FtsL to
the divisome (Daniel et al., 2000). Thus, the DivIB-DivIC-FtsL ternary complex
seems to connect the early divisome proteins to the peptidoglycan synthesizing
machinery. The instability of some of the proteins in this complex has led to the
hypothesis that this may be a regulatory step in the maturation of the divisome
(Errington et al., 2003). The association of DivIB-DivIC-FtsL with the early
divisome is presumed to involve an interaction with FtsA, although no evidence
is available to support this hypothesis. Finally, the relatively large size of the extracytoplasmic domain of DivIB suggests that this protein may carry out some
other function in septum formation besides just promoting the recruitment of
other divisome components. Interestingly, a recent report showed that DivIB
affects the Spo0J/Soj proteins involved in the organization and segregation of
chromosomal origins (Real et al., 2005). Thus, DivIB may provide another connection between the cytokinesis and segregation systems in B. subtilis, besides
the one described above for SpoIIIE.
PBP2B
The assembly of peptidoglycan from its precursors requires the polymerization
of GlucNAc-MurNAc sugar subunits into glycan strands and the connection of
glycan strands through peptide chain cross links. The penicillin binding proteins
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Cell Division | 109
(PBPs) are the family of enzymes that execute these reactions. For a more detailed account of the role of PBPs in peptidoglycan synthesis, see Chapter 10.
There are over 10 different PBPs in B. subtilis and E. coli (see Table 10.1).
High molecular weight PBPs are divided in two classes. Class A contains enzymes that possess both transglycosylase and transpeptidase activities. Class B,
on the other hand, possess only transpeptidase activity (Popham and Young,
2003). There are two possible modes of peptidoglycan synthesis for a rod-shaped
bacterium. During growth, elongation of the cell wall is required whereas during division, a transverse wall will be synthesized. Genetic evidence indicates
that different class B PBPs are involved in the different kinds of peptidoglycan
synthesis. Thus, in B. subtilis PBP2B, encoded by the pbpB gene, is responsible
for septal wall synthesis. Mutation in pbpB blocks septum formation without
affecting elongation (Daniel et al., 2000; Yanouri et al., 1993). In contrast, mutations in the genes encoding the other class 2 enzymes, PBP2a and PBPH, block
elongation without affecting septum formation, and thus lead to the production
of spherical cells (Wei et al., 2003). An equivalent situation exists in E. coli where
PBP3 (encoded by ftsI) is essential for septum formation while PBP2 is required
for elongation (Popham and Young, 2003).
PBP2B is an 80 kDa protein with one transmembrane segment and two
extracytoplasmic domains. One of these is the transpeptidase domain. The
function of the other domain is unknown. In accordance with its role in septation, PBP2B and its E. coli counterpart localize to the division septum (see
Fig. 10.4B) (Daniel et al., 2000; Weiss et al., 1999). As discussed above, localization of PBP2B to the divisome depends on DivIB, DivIC and FtsL, suggesting
that these proteins associate cooperatively in a complex. How PBP2B interacts
with its partner proteins in B. subtilis remains unclear, however. In E. coli, interaction with the divisome appears to involve the protein’s transmembrane segment (Weiss et al., 1999). Besides protein–protein interactions with divisome
components, localization of PBP2B could also be promoted by the recognition
of a specific substrate, such as a modified cell wall precursor, at the division
site. A precedent for such a mechanism exists in the Gram-positive bacterium
Staphylococcus aureus, where localization of the counterpart of PBP2B to the
division site depends on the availability its transpeptidation substrates (Pinho
and Errington, 2005).
Other members of the PBP family besides PBP2B were recently found to
localize preferentially, or at least partially, to the septum (Scheffers et al., 2004).
One of these is PBP1, a class A enzyme, which is strongly targeted to the division complex (Pedersen et al., 1999; Scheffers et al., 2004). This suggests that
these enzymes are also involved in septal synthesis, perhaps by being part of a
peptidoglycan “factory” or holoenzyme complex (Holtje, 1998). Inactivation of
PBP1 seems to cause only a slight inhibition of septation (Pedersen et al., 1999;
Scheffers and Errington, 2004). The non-essential nature of PBPs other than
PBP2B for septal peptidoglycan synthesis could be explained by the existence of
extensive redundancy of activities.
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FtsW
FtsW is a member of the SEDS (shape, elongation, division and sporulation)
protein family. Proteins of this family are generally found associated with a
cognate class B PBP. Thus, in E. coli, FtsW is a partner of the septation-specific
PBP3, whereas the elongation-specific PBP2 is functionally associated with
RodA, another SEDS protein in that organism. B. subtilis possesses three SEDS
proteins: FtsW, RodA and SpoVE. FtsW is the partner of PBP2B, although
they are not in the same operon, as are the orthologous genes in E. coli. RodA is
a likely partner for the elongation PBPs, PBP2A and PBPH whereas SpoVE is
a protein required for the synthesis of the spore peptidoglycan cortex.
In E. coli, FtsW is required to localize PBP3 to the divisome (Mercer and
Weiss, 2002). The localization of FtsW itself depends on FtsL and on FtsQ
(DivIB in B. subtilis) (Mercer and Weiss, 2002). There are no published observations on either the essentiality or the requirements for FtsW localization in
B. subtilis. Given that it seems to interact with PBP3 in E. coli, it is likely that
FtsW will be part of a large complex with PBP2B, DivIB, DivIC and FtsL in
B. subtilis. FtsW has 10 transmembrane domains (Gerard et al., 2002), leading
to speculation that it will function as a transporter of peptidoglycan precursors
that will be acted upon by PBP2B on the outside of the cell. FtsW could also
provide a link between the Z ring and the membrane, since it has been reported
that, in Mycobacteria, FtsW is capable of binding directly to FtsZ (Datta et al.,
2002). This role of FtsW in Mycobacteria could have evolved to compensate for
the absence of FtsA in these organisms.
Assembly of the divisome
An important issue in bacterial division is to define how the multiprotein
complex of the divisome is formed. One of the approaches to this question has
been to test how the elimination of one protein affects the recruitment of the
remaining proteins to the complex. This type of experiment has produced a pattern of localization interdependency among the individual divisome proteins,
which was discussed in the sections above, and that can be summarized in the
form of a putative assembly pathway for divisome formation in both B. subtilis
and E. coli (Fig. 4.4B) (Errington et al., 2003). These pathways revealed that
the late proteins in B. subtilis seem to behave as a multiprotein complex—a
subcomplex of the divisome—as shown by the fact that their recruitment is
fully interdependent. The situation in E. coli is somewhat different, since the late
proteins assemble following a predominantly linear dependency (Errington et
al., 2003; Goehring and Beckwith, 2005). This could mean that the late proteins
do not form a single subcomplex in this organism. The early proteins (FtsA,
ZapA, EzrA in B. subtilis and FtsA, ZipA in E. coli), on the other hand, seem
to assemble independently of each other, in line with their ability to interact
directly with FtsZ.
It is important to note that the pathways in Fig. 4.4B do not necessarily
reflect a temporal sequence of events from Z ring to mature divisome. To deter-
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Cell Division | 111
mine the order of events, experiments that address the timing of arrival of each
of the proteins at the complex must be carried out. The data from the single
report that has been done towards that end suggest that, in E. coli, the divisome
assembles in two steps (Aarsman et al., 2005). In the first step, proteins up to
FtsK will arrive at the complex. After a ~15 minute delay, the remaining proteins
will assemble. The interval between the two waves of assembly could mean that
the division site has to be modified by the early assembling proteins in some way,
perhaps at the level of the local peptidoglycan structure, before the late proteins
can recognize it.
Finally, an important approach to understand divisome assembly will be to
characterize the protein–protein interactions that hold the complex together.
Several two-hybrid screens have identified potential interactions between divisome components (Di Lallo et al., 2003; Karimova et al., 2005). These screens
reported a large number of potential interactions between the division proteins,
several of them not anticipated by the dependency pathways. There are two
potential technical caveats that may explain the surprisingly high number of interactions found. First, the screens were carried out in homologous systems (i.e.
the interaction between a pair of E. coli proteins was measured in E. coli, in the
presence of all the other cognate divisome proteins) such that a positive signal
could be due to an indirect association between the tested proteins. Second, they
often relied on overexpression of the proteins to be tested, a situation that may
promote physiologically irrelevant interactions.
The final steps of septum formation: divisome
constriction, membrane fusion, and cell separation
Once the divisome is fully assembled, it will trigger the synthesis of the septal
peptidoglycan and subsequent invagination of the cell envelope. The signal that
induces peptidoglycan synthesis and constriction remains unknown. Another
important mystery is the force that drives constriction of the divisome and the
invagination of the envelope. There are two main views about this problem. One
view argues that constriction is an indirect consequence of the ingrowth of a
rigid peptidoglycan wall. The other view is that the force for constriction results
from the dynamic behavior of the FtsZ cytoskeleton. This view is supported by
three types of observations. First, there are bacteria like Mycoplasma, which are
devoid of cell walls and whose division machinery seems to be made up solely of
FtsZ (Vaughan et al., 2004). Second, wall-less forms of bacteria that normally
contain peptidoglycan, such as E. coli’s L-forms, are still capable of dividing (Siddiqui et al., 2006). And third, membrane invagination can be uncoupled from
septal wall growth by mutations such as the inactivation of PBP2B (Daniel et
al., 2000). How the FtsZ cytoskeleton would produce force is still a matter of
debate. An actomyosin-like mechanism seems unlikely, given that there are no
known motor proteins in bacteria that could power constriction by sliding FtsZ
filaments against each other. Other potential mechanisms would include the
regulated disassembly of the FtsZ polymer while maintaining the ring associ-
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ated with the membrane, or alterations in filament curvature induced by the
hydrolysis of GTP to GDP in the polymer (Romberg and Levin, 2003). Even
though constriction can occur in the absence of a cell wall, it cannot be ruled out
that under normal conditions constriction will be powered by both FtsZ and
cell wall ingrowth.
Constriction of the cell envelope will eventually promote the formation of a
complete septum. A required step in the completion of the septum is the fusion
of the invaginating membranes. How septal membrane fusion is accomplished
is not known. There are no obvious homologs of eukaryotic proteins specialized in membrane fusion such as dynamins and SNAREs in bacterial genomes.
Recent findings, however, suggest that the DNA-translocation protein SpoIIIE
could play a role in membrane fusion during the engulfment step of sporulation
(see Fig. 11.1, and further details in Chapter 11) (Sharp and Pogliano, 2003).
Sporulation membrane fusion promoted by SpoIIIE seems to be carried out by
its N-terminal membrane domain. Since a deletion of SpoIIIE has no effect in
septum formation, it remains unclear what is the contribution of this protein for
septal membrane fusion during growth.
Following completion of the septum, hydrolysis of the inner portion of the
peptidoglycan cross-wall allows the final separation of the daughter cells. As
discussed before, daughter cell separation in B. subtilis does not happen simultaneously to septum formation. Hydrolysis of septal peptidoglycan is carried
out by murein hydrolases, a large family of enzymes involved in several aspects
of peptidoglycan turnover and recycling necessary for growth and division. At
least five of these enzymes have been implicated in cell separation in B. subtilis,
since mutations in their genes lead to an increase in the average size of the chains
of cells in the mutants (Smith et al., 2000). One way to restrict the hydrolase
activity to the desired location during cell separation would be to target the enzymes to the septum. This seems to be the case for the LytF and LytE proteins
(Yamamoto et al., 2003). In E. coli, the AmiC murein hydrolase was also shown
to localize specifically to the periplasmic space around the septum (Bernhardt
and de Boer, 2003). Localization of AmiC to the septum was dependent on
recruitment by the late divisome protein FtsN. There is no information on how
LytF and LytE are recruited to the B. subtilis septum.
Regulation of septum formation
As discussed above, several lines of evidence have demonstrated that Z ring
formation is the earliest detectable event and the rate-limiting step in septum
formation. Thus, any regulatory mechanism operating to ensure that cytokinesis
will happen at the right place and time during the cell cycle must be affecting
either FtsZ availability or its propensity to assemble into the Z ring.
Timing of Z-ring formation during the cell cycle
Restricting FtsZ expression to a period of the cell cycle would be the simplest
explanation for the timing of Z-ring formation. This hypothesis has been tested
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Cell Division | 113
using synchronized populations of E. coli with conflicting results. Even though
there are at least two studies that showed fluctuations in the amount of ftsZ
mRNA (Garrido et al., 1993; Zhou and Helmstetter, 1994), levels of FtsZ or of
the FtsZ-binding proteins FtsA and ZipA do not seem to change during the E.
coli cell cycle (Rueda et al., 2003). Because B. subtilis is not as amenable to synchronization as E. coli, studies of B. subtilis ftsZ expression have not emphasized
cell cycle control and focused instead on the developmental regulation of this gene
during sporulation. In B. subtilis, ftsZ forms an operon with the neighboring ftsA
and both genes are expressed from three promoters located upstream of ftsA.
Two of these promoters (P1 and P3) are controlled by the housekeeping sigma A
factor and are “on” during vegetative growth. The third promoter (P2) is turned
on by a stationary phase/sporulation specific sigma factor and is responsible for
an increase in FtsZ concentration as cells enter sporulation (Gholamhoseinian
et al., 1992; Gonzy-Treboul et al., 1992). This burst in FtsZ expression seems
to be required for the switch in Z ring position that occurs during sporulation,
as will be discussed below. Despite the absence of any evidence for fluctuation
of ftsZ mRNA or protein levels during the cell cycle, the observation that the
natural ftsZ promoters can be substituted by an IPTG-controlled promoter
without a noticeable alteration in the pattern of division (Beall and Lutkenhaus,
1991; Weart and Levin, 2003) suggests that transcriptional regulation is not the
main component of Z ring control in B. subtilis.
The only bacterium for which there is clear evidence for cell cycle control
of FtsZ levels is Caulobacter crescentus. In this bacterium, every division cycle
produces two different types of progeny: the sessile stalked cell, which is competent for chromosome replication and cell division; and the flagellated swarmer
cell, which has to differentiate back to a stalked cell before it can reproduce.
Remarkably, FtsZ is absent from swarmer cells as a consequence of targeted
proteolysis combined with gene repression by CtrA, the master cell cycle regulatory protein of C. crescentus (Kelly et al., 1998). As swarmer cells differentiate
into replication-competent stalked cells, FtsZ levels increase concomitantly.
However, the fluctuation of FtsZ levels during the C. crescentus cell cycle is
likely to be an exceptional situation, given that it is associated with the peculiar
differentiation that occurs at every C. crescentus cell cycle, which is absent from
most bacteria. Thus, the current view is that the timing of Z ring formation in
B. subtilis and most bacteria is not primarily determined by fluctuating FtsZ
levels. Instead, the timing may depend on mechanisms operating at the level of
the activity of preexisting FtsZ, such as factors that inhibit or stimulate FtsZ
polymerization.
Another aspect of the timing of cytokinesis has been to define whether Z
ring formation is dependent on other cell cycle events, in particular DNA replication. Studies carried out with germinating spores as a means to obtain synchronous B. subtilis populations have suggested that Z ring formation depends
on both replication initiation and the cells being capable of replicating at least the
origin proximal region of their chromosome (Harry, 2001b). In contrast, Z ring
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114 | Gueiros-Filho
formation in E. coli was shown to coincide with termination of DNA replication
(Den Blaauwen et al., 1999). Despite these observations, the actual connection,
if any, between DNA replication and Z ring formation remains unclear. In B.
subtilis, it has been suggested that during the initial phases of DNA replication
the replisome would be located at midcell where it would mask a putative FtsZ
nucleation site. Upon replication progression, the replisome would move away
from midcell, freeing the nucleation site for Z ring assembly. This model has been
recently disfavored by the finding that replisome localization during early DNA
replication is not restricted to the cell center and seems too dynamic to account
for the masking of a Z nucleation site (Migocki et al., 2004). An alternative model
to explain the coordination between DNA replication and cytokinesis invokes
the relieving of nucleoid occlusion by chromosome segregation, as described in
more detail below (Fig. 4.5A, B). Finally, it must be emphasized that, in contrast
to eukaryotes, there is no strict interdependence or checkpoint tying cytokinesis
to DNA replication in bacteria, because both in B. subtilis and in E. coli Z-ring
formation is only delayed but will eventually occur even in the absence of DNA
replication (Harry, 2001b). The converse is also true, as inhibition of septation
will generate long filaments containing multiple, evenly spaced, nucleoids (that
contain the chromosomes, see Fig. 3.2A), indicating that septum formation is
not required for DNA replication or segregation to occur.
Spatial control of septum formation
One of the most intriguing questions in bacterial cytology is how can a bacterium
determine where its middle is so precisely? Measurements of the positioning of
Z rings in B. subtilis have shown that the division site is placed to within 6% of
the cell middle, i.e. with very high precision (Migocki et al., 2002). Initial models
to explain this exquisite site preference proposed that only a few sites along the
rod-shaped cell (more specifically, the midcell and the cell poles) would be recognized as potential division sites. These potential division sites would be defined
by the existence of a positive signal, also referred to as a nucleation site, at the
place where the Z ring was supposed to form. These models have gradually lost
their appeal because no one could demonstrate the existence, let alone reveal the
nature, of the putative positive signal triggering Z-ring formation. Moreover,
there has been mounting evidence that Z ring formation can potentially happen
anywhere along the bacterial cell cylinder (Harry, 2001a; Margolin, 2001). Thus,
the current view is that the division site is determined by two complementary
inhibitory systems that prevent Z ring formation everywhere but at midcell
(Fig. 4.5A). These two systems are the Min proteins and nucleoid occlusion.
The Min system
Initial insight into the control of septum position in bacteria came from the
isolation of mutants in which division frequently occurred asymmetrically at
one of the cell poles, generating a larger cell with two nucleoids and a small
DNA-less minicell (Adler et al., 1967; Reeve et al., 1973). These mutants defined
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Cell Division | 115
Figure 4.5 Spatio-temporal control of cytokinesis. (A) Positioning of the division
septum at midcell by the combined action of two inhibitory systems. The MinCD
proteins (gray crescents at the cell poles, top panel) prevent the use of polar sites for
division. Nucleoid occlusion (N.O.) mediated by proteins such as Noc (middle panel)
prevents Z-ring formation in the vicinity of the nucleoids, and thus in the region between
the cell poles and the midcell; the combined action of both systems restricts Z ring
formation to the middle of the cell (bottom panel). (B) A model for the cell cycle timing
of Z ring formation. At birth, the combination of polarly localized MinCD and the central
location of an unreplicated nucleoid prevent Z ring formation throughout the cell. As the
cell progresses through the cell cycle, replication and concomitant segregation of the
nucleoid will eventually create a zone free of inhibitor at the cell middle.
the so-called min genes, encoding a set of proteins responsible for suppressing
division specifically at the cell poles. In B. subtilis, the Min system is composed
of the proteins MinC, MinD and DivIVA (Cha and Stewart, 1997; Edwards
and Errington, 1997; Levin et al., 1992). MinC and MinD interact to form a
membrane-associated complex capable of inhibiting Z ring formation in a sitespecific manner. Inhibition of Z ring formation by the MinCD complex has
been shown to be carried out by MinC, a 25 kDa protein that is organized in
two functional domains: the N-terminal domain interacts with FtsZ while the
C-terminal domain has been implicated in MinC dimerization and interaction
with MinD (Hu et al., 1999). MinD, on the other hand, is a 29 kDa ATPase of
the ParA family, which associates with the membrane through an amphipathic
alpha helix at its C-terminus (Szeto et al., 2003). Binding of MinC to MinD is
necessary to localize and concentrate the inhibitory complex at the inner face
of the cytoplasmic membrane, where it ultimately has to act. Recent evidence
suggests that the interaction between MinC and MinD is necessary to activate
the inhibitory action of MinC beyond simply increasing its local concentration
near the membrane.
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In B. subtilis, DivIVA is the protein that imparts site selectivity to the Min
inhibitor (Cha and Stewart, 1997; Edwards and Errington, 1997). Thus, this
protein, and its functional analog MinE in E. coli, are commonly known as the
topological specificity factors of the Min system. DivIVA is a small protein with
a long tropomyosin-like coiled-coil region and that is conserved in several Grampositive species. How does DivIVA restrict the action of the MinCD inhibitor
to the poles? Insight into this question came from studies using GFP fusions to
determine the subcellular distribution of the Min and DivIVA proteins in B.
subtilis (Marston et al., 1998). These fusions revealed that the MinCD complex
is predominantly associated with the cell poles, exhibiting a crescent like staining pattern at the old pole and strong staining at the recently formed septum,
which will become the new pole once cell separation happens. This localization
was dependent on DivIVA, since in its absence MinCD appeared evenly distributed throughout the cell membrane. DivIVA itself was also shown to localize to
the septum and the poles. Initial experiments suggested that polar localization
of DivIVA was dependent on the assembly of the divisome, as it was blocked
in the absence of FtsZ or other cell division proteins. This led to a model in
which the anchoring of the MinCD complex to the cell poles would occur by a
DivIVA-mediated recruitment of this complex to the divisome at a late stage in
its assembly. Upon completion of division, the DivIVA-MinCD complex would
be retained at the newly formed poles (Marston et al., 1998). Using the divisome
to recruit a division inhibitor to its proper location may sound paradoxical. It
must be remembered, however, that MinCD inhibits the first step of divisome
assembly such that its association with a mature divisome would be unlikely to
affect its stability. The recent observation that DivIVA and MinD localize to
the poles of outgrowing spores even before the assembly of the division complex
challenges the notion that recruitment of DivIVA, and consequently of MinCD,
requires a direct interaction between DivIVA and the division complex (Harry
and Lewis, 2003). Although this does not rule out the existence of affinity between DivIVA and the division complex, it suggests that DivIVA would be also
attracted by some physical property of the division site and cell poles, such as
membrane curvature. This view is supported by the fact that DivIVA is capable
of associating with the cell poles when expressed heterologously in E. coli and
even in S. pombe, a eukaryote whose division machinery is completely unrelated
to the bacterial one (Edwards et al., 2000).
Besides piloting MinCD to the cell poles, DivIVA is also involved in the
anchoring of the origin regions of chromosomes to the cell poles during sporulation (Thomaides et al., 2001; Ben-Yehuda et al., 2003). This is important to
ensure correct segregation of a chromosome copy to the developing spore and
will be discussed in more detail below. Thus, the ability of DivIVA to recognize
and mark the poles of a bacterial cell has been co-opted during evolution to play
two distinct roles in the reproductive cycle of B. subtilis.
In E. coli, the MinCD complex is also restricted to the cell poles. However,
the way in which the polar localization of MinCD is achieved is remarkably
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different. In contrast to the static localization of MinCD in B. subtilis, MinCD
in E. coli exhibit a dynamic oscillation in which the proteins move from one pole
of the cell to the other approximately every minute (Raskin and de Boer, 1999;
Rothfield et al., 2005). During this oscillation, MinD (and the associated MinC)
initially forms a polar zone, a MinD-rich region on the membrane periphery
extending from one of the poles toward the cell middle. MinD in the polar zone
exists as a helical polymer created by the self-assembly of ATP-bound MinD
subunits. Once the polar zone has grown enough, the topological specificity protein MinE will associate with it, creating the so-called MinE ring at the leading
edge of the MinD polymers. The MinE ring will then promote the disassembly
of the polar zone by inducing the ATPase activity of MinD. Next, the disassembled MinD, which now is bound to ADP, will diffuse away from the pole,
exchange the bound ADP for ATP and then reassemble into a new polar zone
at the opposite cell pole, until the next round of MinE-mediated disassembly
occurs (Rothfield et al., 2005). The net effect of this fast alternation between cell
poles is that the average occupancy of these sites by the MinCD proteins during
the division cycle will be higher than that of the cell middle. Thus, over time, this
dynamic system promotes the same effect as the static anchoring of MinCD in
B. subtilis. An intriguing evolutionary question is why do two mechanistically
quite different solutions for the problem of targeting proteins to the cell poles
exist?
Despite its well-established capacity to block polar Z-ring formation, the
Min system cannot explain by itself the use of the midcell site with high precision during bacterial division. This is supported by work in B. subtilis and E.
coli showing that the midcell site is still favored even in the absence of the Min
proteins under certain conditions, and by the fact that several bacteria, like C.
crescentus, lack min homologs in their genomes (Levin et al., 1998; Margolin,
2001; Migocki et al., 2002). The residual spatial preference in the absence of the
Min proteins is likely to be a consequence of the nucleoid occlusion effect, as
described below.
Nucleoid occlusion
In the absence of the Min system cells divide only at midcell and at the poles,
even though any position along the length of a rod-shaped cell can sustain Z
ring formation. So, there must be a second inhibitory system preventing the use
of the space between the cell middle and the poles. Since this space ordinarily
corresponds to the position occupied by the nucleoid in the cell, an attractive
possibility is that the nucleoid would play a role in defining the site of division.
A precedent for such a mechanism exists in the case of eukaryotic cells like S.
pombe where the nucleus defines the correct plane of division, and recent evidence
described below has shown that the bacterial “nucleus” is indeed important for
determining where cells will divide.
The recognition that bacteria avoid forming a septum over their nucleoid
under conditions that perturb DNA replication and segregation led to the for-
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mulation of the hypothesis that nucleoids were capable of exerting a “veto effect”
on septum formation in their vicinity (Woldringh et al., 1991). This veto by the
nucleoid has been called nucleoid occlusion (NO) and was shown to happen at
the level of Z ring formation by subsequent studies (Harry, 2001a; Margolin,
2001). These studies produced two other important observations regarding
NO. First, blocking of Z rings by the nucleoid seemed to be relieved as soon as
the nucleoid had undergone partial replication and segregation and assumed a
bilobed shape. And second, mutations that reduced the general organization or
condensation state of nucleoids, such as those in the smc or mukB genes, greatly
reduced the efficacy of NO. Therefore, NO seems to be affected by the topology
and/or local DNA concentration in the vicinity of the cytoplasmic membrane.
The molecular basis of NO remained mysterious until recent reports
showed that in both B. subtilis and E. coli NO could be exerted by the DNAbinding proteins Noc (in B. subtilis) and SlmA (in E. coli) (Bernhardt and de
Boer, 2005; Wu and Errington, 2004). These proteins were implicated in NO
because mutations in their genes promoted a severe division block and synthetic
lethality when combined with a min mutation. Microscopic observation of conditional noc min or slmA min double mutants revealed that instead of forming
the typical rings, FtsZ assembled in a haphazard pattern of arcs, foci and other
irregular structures often present across the nucleoids. The explanation for this
initially surprising phenotype is that in the absence of its two main negative
regulatory systems, there are too many sites available for FtsZ to polymerize and
this prevents formation of a productive Z ring.
In line with the idea that Noc and SlmA are nucleoid-associated inhibitors
of Z ring formation, both proteins localize diffusely on the nucleoids and promote division inhibition when overexpressed. Moreover, deletion of noc or slmA
led to frequent Z ring formation over nucleoids, and eventually nucleoid bisecting by a septum in situations in which chromosome replication or segregation
were perturbed. Interestingly, however, Z ring formation and division continued
to occur preferentially between segregated nucleoids in noc and slmA mutants
when replication was unperturbed. This observation implies that nucleoids still
exhibit some NO activity even in the absence of Noc and SlmA and suggests
that there may be other nucleoid-associated protein inhibitors of Z ring formation awaiting discovery. Alternatively, there could be a protein-independent
component to the NO effect, as originally predicted in the “transertion” model.
As stated by this model, the NO effect would be a consequence of the molecular
crowding effects arising from a physical connection between the nucleoid and
the adjacent membrane promoted by the simultaneous transcription, translation and insertion (hence, “transertion”) of integral membrane proteins into
the cytoplasmic membrane (Rabinovitch et al., 2003). Determining the precise
contributions of Min and NO to the spatial control of septum formation will
require a better understanding of the molecular mechanism of NO.
Besides regulating the position of the septum, nucleoid occlusion could also
provide a mechanism for temporal coordination between chromosome replica-
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tion and segregation, and septum formation. According to this hypothesis, a
newborn cell will not form a Z ring immediately because the combined distribution of Min and NO would inhibit Z ring formation throughout the cell. Thus,
at the beginning of the cell cycle there would be no place available for a Z ring to
form. As cells progress through the division cycle, chromosome replication and
segregation would occur, relieving nucleoid occlusion at midcell and creating an
inhibitor-free region in the cell for the first time. Thus, the timing of Z ring formation would be determined by a change in the pattern of NO promoted by the
concomitant replication and segregation of the bacterial nucleoid (Fig. 4.5B).
Other regulators of Z-ring formation
Even though the Min system and nucleoid occlusion seem to account for most
of the spatial control of Z-ring formation, there are at least four other proteins
in B. subtilis that affect the general propensity of FtsZ to assemble and thus contribute to the regulation of division. Three of these modulators (EzrA, ZapA,
ClpX) play roles in normal cell division whereas YneA is an SOS-induced
protein important to prevent division in cells that suffered DNA damage.
ZapA
ZapA is a positive modulator of FtsZ, which was first identified in B. subtilis
as a protein capable of overcoming the division block produced by overexpression of the MinD inhibitor (Gueiros-Filho and Losick, 2002). ZapA is a small
cytoplasmic protein (10 kDa) widely conserved in bacteria and that is recruited
to the divisome at an early stage in its assembly through a direct interaction with
FtsZ. Binding of FtsZ by ZapA in vitro promotes the formation of higher order
assemblies of FtsZ (Gueiros-Filho and Losick, 2002; Low et al., 2004). If GTP
is present in the reactions, bundles of straight protofilaments are formed. ZapA
can also promote the assembly of FtsZ structures in the presence of GDP. In
this case, curved FtsZ assemblies, such as minirings, are formed.
The crystal structure of Pseudomonas aeruginosa ZapA was recently solved
and revealed a two-domain protein with a globular head and an elongated stalk
corresponding to a long C-terminal alpha helix (Low et al., 2004). The crystal
structure also showed that the protein oligomerizes, predominantly through a
coiled coil association between the C-terminal alpha helices of monomers. P.
aeruginosa ZapA is a tetramer in the crystal but biochemical experiments in P.
aeruginosa and B. subtilis suggest that the protein is likely a dimer in solution
(Low et al., 2004) (Aaron Handler, Glenn King and Frederico Gueiros-Filho,
unpublished). Despite the availability of 3D structures for both proteins, the
details of the interaction between ZapA and FtsZ remain unclear at this point.
In contrast to FtsA (and ZipA in E. coli), ZapA is not essential for Z ring or
septum formation under normal laboratory conditions. The frequency and pattern of division of zapA mutants is essentially identical to wild type cells. ZapA
becomes required, however, if secondary mutations are used to weaken the
tendency of Z rings to form, either by reducing the available pool of cytoplasmic
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120 | Gueiros-Filho
FtsZ or by deregulating a division inhibitor (Gueiros-Filho and Losick, 2002).
This suggests that ZapA facilitates the assembly of the Z ring, perhaps through
its ability to bundle FtsZ protofilaments. Whatever its exact contribution, the
dispensability of ZapA indicates that it plays a redundant role in the assembly
of the Z-ring. Likely candidates to overlap with ZapA are the FtsZ binding
proteins FtsA and SepF described above. It remains a possibility that ZapA
becomes required under specific physiological conditions. At present, there is no
hint as to what these conditions could be.
EzrA
EzrA is a negative regulator of Z-ring formation that was identified in a genetic
screen for suppressors of an ftsZ temperature-sensitive allele (Levin et al., 1999).
It is a 65 kDa single-span membrane protein with a short N-terminal transmembrane segment and a large cytoplasmic domain. EzrA is distributed homogenously throughout the cytoplasmic membrane, but localizes preferentially
to the Z-ring once it is formed (Levin et al., 1999). In vitro, EzrA binds to FtsZ
and prevents the growth of polymers from unassembled protein (Haeusser et al.,
2004). However, EzrA is incapable of disassembling preformed polymers. This
inability of EzrA to disassemble FtsZ polymers might explain why EzrA can
localize to the Z ring without disturbing it.
The precise role of EzrA during division remains unclear. In contrast to the
site-specific action of MinCD, EzrA seems to generally decrease the propensity
of FtsZ to polymerize inside the cell. Thus, EzrA could be in charge of controlling that Z rings would not form prematurely or too frequently during the cell
cycle. This is supported by the observation that inactivation of ezrA leads to
cells that have more than one Z ring (Levin et al., 1999). Interestingly, the extra
rings are frequently present at one of the cell poles, presumably because in the
absence of EzrA, FtsZ polymers become hyperstable and therefore less sensitive
to inhibitors, such as MinCD, which prevent their assembly at aberrant places
in the cell. An alternative view of the function of EzrA is that it is necessary for
the complete disassembly of the Z ring at the end of division. According to this
view, the extra Z rings present at cell poles would be derived from incompletely
disassembled polymer from the previous round of division. The idea that EzrA
is involved in the disassembly of FtsZ polymers is also supported by its tendency
to associate with the Z ring.
Another interesting feature of EzrA is that it seems to functionally interact
with several of the other regulators of FtsZ polymerization. Two well-documented examples are the observation that a mutation in ezrA is capable of suppressing the overexpression of MinCD (Levin et al., 2001), and the observation
that a mutation in ezrA exhibits a synergistic effect and severely blocks division
when combined with a mutation in zapA (Gueiros-Filho and Losick, 2002).
The first observation is easy to rationalize—the elimination of one destabilizing
factor compensating for the overexpression of another destabilizing factor—and
has led to the idea that the overall balance of stabilizing versus destabilizing
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activities is key for the proper functioning of the FtsZ polymer during division
(Romberg and Levin, 2003). The second case, however, seems paradoxical since
the simultaneous elimination of a negative (EzrA) and a positive (ZapA) regulator would be expected to have compensatory effects on FtsZ polymer assembly
and be benign for division. The division inhibition of the ezrA zapA double
mutant suggests that the interplay between positive and negative regulators of
FtsZ may be more complex than the simple addition or subtraction of opposite
activities.
ClpX
ClpX is member of the HSP100 family of AAA+ ATPases, which play a role
in protein degradation by serving as the substrate recognition portion of the
ClpXP protease (see Fig. 7.1, and see Chapter 7 for more details). In addition,
ClpX can function as a molecular chaperone in the absence of ClpP, preventing
protein aggregation and promoting the remodeling and disassembly of multiprotein complexes (Burton and Baker, 2005). Recent observations have implicated
ClpX as a negative regulator of Z ring formation in B. subtilis (Weart et al.,
2005). Inactivation of clpX suppresses the division defect of a temperature sensitive ftsZts mutant. Conversely, ClpX overexpression inhibits Z-ring formation.
The effect of ClpX on Z ring formation is independent of ClpP and does not
involve proteolysis of FtsZ. Moreover, purified ClpX inhibits FtsZ polymerization in vitro. Thus, ClpX must be preventing the assembly or promoting the
disassembly of FtsZ polymers through its chaperone activity. In E. coli, ClpX
was shown to bind FtsZ suggesting that the modulatory role of this chaperone
in cell division may be generally conserved among bacteria (Burton and Baker,
2005).
YneA
Induction of the SOS response by DNA damage in bacteria (see also Chapter
2 by Alonso) often leads to a block in division. This block in division offers cells
time to repair all damage and resume replication of their genome, and prevents
the formation of anucleate cells or the fragmentation of nucleoids by the formation of a septum over unreplicated chromosomes. Suppression of division by the
SOS response in E. coli is caused by the induction of SulA, a protein that binds
tightly to the T7 loop surface of FtsZ and inhibits its polymerization (Huisman
et al., 1984; Cordell et al., 2003). In B. subtilis, suppression of division during
the SOS response is caused by YneA (Kawai et al., 2003). The gene encoding
YneA is located next to the gene for DinR, the repressor that controls the SOS
response in B. subtilis. Induction of YneA causes filamentation and a reduction
in the frequency of Z rings. No direct interaction between YneA and FtsZ
could be demonstrated and no evidence is available on the effect of YneA on
FtsZ polymerization (Kawai et al., 2003). Thus, it is still unclear whether YneA
blocks division at the level of Z ring formation or at a later step. The absence of
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sequence similarity between YneA and SulA is consistent with the possibility
that these proteins function by different mechanisms.
Asymmetric division during sporulation
When faced with nutrient deprivation, B. subtilis and related bacteria undergo
a developmental process that leads to the formation of a spore, a highly specialized cell capable of resisting a number of environmental stresses and staying
dormant for long periods of time. Once conditions become favorable again,
spores germinate and give rise to vegetative cells capable of resuming growth.
Spore formation starts with the asymmetric division of a progenitor cell into
two unequal cellular compartments by the formation of a polar septum. The
smaller cell will become the resistant spore and is called the forespore, whereas
the larger compartment is called the mother cell. Establishment of morphological asymmetry by polar septation is a key event in triggering distinct programs
of gene expression in each of the compartments and, thus, to promote spore
differentiation. For a detailed description of sporulation see Chapter 11.
The polar septum made during sporulation uses essentially the same division machinery responsible for septum formation in growing cells. Mutations
that block division in vegetative cells also block formation of the sporulation
septum (Beall and Lutkenhaus, 1991; Beall and Lutkenhaus, 1992). Thus, the
alteration in septum position during sporulation must be due to the interaction
of the division machinery with sporulation-specific proteins. In addition to the
change in positioning, vegetative and sporulation septa also differ in septal peptidoglycan content. Sporulation septa have a thinner peptidoglycan layer than
vegetative septa, a likely adaptation to the requirement that septal peptidoglycan
be completely removed to permit the engulfment of the forespore by the mother
cell (see Chapter 11). The only sporulation protein with a well-established role
in polar septation is SpoIIE. This protein seems to play a role both in controlling
the thickness of the peptidoglycan in the polar septum as well as in the switching
of the location of the division site (Barak and Youngman, 1996; Feucht et al.,
1996). How SpoIIE affects the septal peptidoglycan is not known. The role of
SpoIIE in the change of division site is discussed below.
The mechanism of the switch of division site
The switch from medial to asymmetric division during sporulation is a consequence of reprogramming the sites allowed for Z ring formation in the cell (Levin
and Losick, 1996). In fact, the pattern of Z ring formation during sporulation
is exactly the opposite of that during growth: whereas in vegetative cells the
midcell site is always used and the cell poles are forbidden, in sporulating cells,
Z rings avoid the midcell site and instead form close to each of the cell poles
generating a typical bipolar pattern. Subsequently, only one of these polar rings
will develop into a mature division complex to generate the asymmetric septum
(Fig. 4.6).
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Figure 4.6 Asymmetric division during sporulation. Left: During sporulation, cells will
deliberately divide at the pole as a consequence of switching the position of Z rings.
Repositioning of the Z ring from midcell to the cell poles involves a transient FtsZ spiral
intermediate, which will eventually produce a cell with two polar Z rings. Only one of
these Z rings will become a septum. Right: Representative fluorescence microscopy
images of the different stages of asymmetric division. The top three panels show two
cells with medial Z rings, a cell with a Z spiral, and a cell with bipolar Z rings, respectively,
as detected by immunofluorescence with anti-FtsZ antibodies. The bottom panel shows
the fluorescently labeled membranes of a typical sporulating cell after polar septation.
At this later stage, the second FtsZ ring will have dissipated, due to inhibition of septum
formation at the slower-forming Z ring. Microscopy images adapted from Ben-Yehuda
and Losick (2002) and reprinted with permission.
Through careful cytological analysis, it is now recognized that the switch of
Z rings to the cell poles involves the dynamic redeployment of FtsZ structures
across the cell (Ben-Yehuda and Losick, 2002) according to the sequence of
events depicted in Fig. 4.6. Initially, a cell that is ready to sporulate will construct a medial Z ring, which, in contrast to the rings in vegetative cells, will not
mature into a complete divisome. Instead, this “metastable” Z ring seems to be
converted into spiral-like structures that emanate from the midcell towards the
cell poles. Eventually, the FtsZ spirals reach the poles where they are converted
back to stable bipolar Z rings (Ben-Yehuda and Losick, 2002). FtsZ spirals had
been previously detected in E. coli when FtsZ was overexpressed or in strains
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124 | Gueiros-Filho
bearing certain mutant ftsZ alleles, but the detection of a spiral intermediate in
sporulating B. subtilis was the first time in which these structures were shown to
be physiologically relevant. In fact, these observations have led to the suggestion
that the Z ring is actually a tightly wound helical polymer whose interconversion
between ring and spiral structures could be controlled by changing the pitch of
the FtsZ helices. Concomitant with the above-described FtsZ choreography, at
the onset of sporulation, there is also an important change in nucleoid organization. The pair of nucleoids present in the sporulating cell fails to fully segregate
and adopt an extended structure that stretches from pole-to-pole of the cell,
and has been named the “axial filament” (see Fig. 3.8B). This change in nucleoid
organization is key to allow the efficient trapping of a complete genome in the
future spore through a specialized form of chromosome segregation (Errington,
2003). The axial filament is also likely to be an important factor in the Z ring
switch, as discussed below.
Even though a rather refined description of the events from commitment
to sporulation to polar septation exists, a clear understanding of the molecular
mechanism responsible for the change in preference for the division site is still
lacking. Asymmetric division is controlled by two transcriptional regulators that
orchestrate the first steps of sporulation: the response regulator Spo0A and the
alternative sigma factor σH (see Fig. 11.2). Mutants lacking each of these fail to
switch the position of Z rings in sporulation (Ben-Yehuda and Losick, 2002;
Levin and Losick, 1996). Despite the fact that Spo0A and σH control about
one hundred genes at the onset of sporulation (Britton et al., 2002; Molle et al.,
2003), genetic experiments suggest that there are only two genes whose activity
seem to be required for the Z ring switch. One of these is ftsZ itself, whose
expression increases about two-fold during sporulation due to the activation of
a supplementary σH-responsive (P2) promoter (Gholamhoseinian et al., 1992;
Gonzy-Treboul et al., 1992). The other one is spoIIE, which encodes a multifunctional protein expressed exclusively during sporulation. SpoIIE is a membrane-bound protein phosphatase (see Fig. 11.5A) whose activity is required
for triggering the gene expression program in the developing forespore compartment (Duncan et al., 1995; Errington, 2003). SpoIIE is also capable of binding
directly to FtsZ, and this interaction has been hypothesized to contribute to
the stability and the anchoring of FtsZ polymers to the cytoplasmic membrane
(Lucet et al., 2000). Mutations inactivating either the ftsZ P2 promoter or spoIIE
lead to a mild inhibition of the Z ring switch. The combination of both mutations, however, practically abolishes polar septation, indicating that these are
the main factors required for the change in Z ring position (Ben-Yehuda and
Losick, 2002). Further support for this hypothesis came from experiments in
which simultaneously boosting the expression of ftsZ and artificially expressing
SpoIIE in vegetative cells led to polar septum formation during growth (BenYehuda and Losick, 2002).
How do the increase in FtsZ levels and the expression of SpoIIE promote
the switch? In order to answer this question we should take into account the
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known division site selection mechanisms operating in growing cells. As discussed above, asymmetric division in vegetative cells is prevented by the presence of the MinCD inhibitor at the cell poles. The localization of DivIVA, the
protein that guides MinCD to the cell poles, does not change significantly as
cells enter sporulation (Thomaides et al., 2001). Although similar experimental
evidence is lacking for MinCD, it seems reasonable to assume that at the onset
of sporulation the Min proteins will be present and active at the cell poles. How,
than, is the inhibitory action of MinCD at the poles overridden? One possibility
is that the SpoIIE interaction with FtsZ makes polymer formation refractory to
inhibition. Alternatively, MinCD would move away from the cell poles during
sporulation. According to this model, displacement of MinCD from the cell
poles would be promoted by RacA, a DNA binding protein induced early on
during sporulation that promotes the anchoring of the replication origin region
of nucleoids to the cell poles (Ben-Yehuda et al., 2003). Anchoring of the nucleoids to the cell poles depends on an interaction between RacA and DivIVA.
Thus, the RacA-DivIVA interaction could alter the properties of DivIVA such
that it would no longer be capable of maintaining MinCD at the cell poles (BenYehuda et al., 2003). Even though there is a compelling logic behind this model,
further experiments will be necessary to elucidate how MinCD is neutralized
during sporulation.
Another important unanswered question about the mechanism of the Z
ring switch is what is the role, if any, of nucleoid occlusion? During vegetative
growth, nucleoid occlusion delays Z ring formation at the midcell position until
the replicating nucleoids have undergone at least partial segregation. Given that
the sister nucleoids remain unsegregated and instead form the “axial filament”
structure at the onset of sporulation, nucleoid occlusion may explain why the
cell stops using the midcell site. The formation of polar Z rings over the nucleoid
in sporulation would seem like an argument against nucleoid occlusion being
active at the time. It is possible, however, that some special property of the edges
of the nucleoids, such as a special chromatin organization (Pogliano et al., 2002)
or lowered local DNA concentration would reduce the effectiveness of nucleoid
occlusion at the ends of the nucleoid without affecting it at midcell. It would be
interesting to determine if inactivation of noc, the only currently known effector
of nucleoid occlusion, would have any effect on the position of the sporulation
septum.
Why does only one of the bipolar Z rings becomes a septum?
Another striking feature of division during sporulation is that only one of the
two polar Z rings matures into a polar septum (Levin and Losick, 1996). Formation of a single polar septum is important to ensure that a cell pair consisting
of a large mother cell and a small forespore is always formed, thus maintaining
the morphological asymmetry required for proper spore development. The use
of a single Z ring seems to result from the inability of the cell to simultaneously
assemble two complete division complexes. This is suggested by observations
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that division proteins like FtsA and PBP2b localize to a single polar ring in
sporulating cells (Daniel et al., 2000; Feucht et al., 2001). The choice of which
of the two Z rings to use is probably stochastic. Thus, in contrast with vegetative cells, where Z-ring formation seems to be the limiting step for division, in
sporulating cells septum formation seems to be limited by the availability of
division proteins other than FtsZ.
The Z ring that does not normally become a septum is also competent to assemble a mature divisome, as shown by the identification of mutants that divide
at both poles, generating “disporic” cells. Formation of the two polar septa in a
disporic mutant is sequential, in line with the idea that one or more divisome
proteins are limiting (Pogliano et al., 1999). Maturation of the second septum
probably depends on divisome components being made available by the disassembly of the complex involved in the synthesis of the first septum. Time-lapse
microscopic observation of septum formation during early sporulation revealed
that even normal cells tend to start the synthesis of the second septum, although
these septa seem to regress and are rarely completed (Pogliano et al., 1999).
These observations suggest the existence of a mechanism to actively suppress the
formation of a second polar septum. Suppression of the second polar septum
depends on the activation of three proteins, SpoIID, SpoIIM and SpoIIP, in
the mother cell (Pogliano et al., 1999; Eichenberger et al., 2001). These three
proteins have features of peptidoglycan hydrolases and are likely to inhibit the
second polar septum by preventing the growth of the peptidoglycan crosswall.
Perspectives
During the past 15 years remarkable progress has been made in understanding
the molecular mechanism of septum formation. In this period researchers went
from a list of loci which affected division to our current picture of the divisome as
a intricate multiprotein complex capable of promoting the ingrowth of the bacterial envelope with high spatial precision and in perfect synchrony with DNA
replication and segregation. Furthermore, it has been clearly demonstrated that
the assembly of the division machinery is coordinated by a bacterial cytoskeleton
based on the tubulin-like protein FtsZ. Despite these advances there are many
discoveries still to be made and throughout the chapter I have tried to highlight
specific gaps in our knowledge. Among the things we still do not know, I have
singled out a few themes there are likely to pose the greatest challenges and to
generate the most exciting discoveries in this field in the near future.
First, it will be necessary to refine our understanding of the mechanisms
controlling Z-ring and divisome assembly. Even though we know a number of
proteins that affect Z-ring formation, the details of how they function remain
largely unknown. Another remaining mystery is how the timing of Z-ring formation is achieved. Current proposals invoking the relieving of nucleoid occlusion by chromosome segregation, although attractive, need to be experimentally
validated. Furthermore, the recent discovery that DnaA plays a broader role
than anticipated as a transcriptional regulator during the cell cycle (Goranov et
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Cell Division | 127
al., 2005; Hottes et al., 2005) suggests that oscillations in the cellular concentration of division proteins might play a role in the timing of division after all.
Perhaps the biggest challenge for the future will be to improve our understanding of the late events of septum formation, including how the synthesis
of the septal peptidoglycan is controlled, the specific role of the late divisome
proteins in this process and the source of the driving force for divisome constriction. Dealing with these questions will benefit from the recent development of
reagents, such as a fluorescent derivative of vancomycin (Daniel and Errington,
2003), capable of probing the local structure of the peptidoglycan in intact cells.
It will probably also require the reconstitution in vitro of at least some of the
steps of septal peptidoglycan synthesis, a daunting task because the synthesis of
peptidoglycan involves large membrane-embedded protein complexes acting on
an insoluble polymeric template.
Finally, if we are to understand the process of bacterial division in all its
complexity and detail it will be important to eventually identify the full complement of cell division proteins in model organisms like B. subtilis or E. coli. The
steady reporting of novel cell division proteins every year and the substantial
number of predicted proteins still with unknown function in the B. subtilis
genome suggests that there is no end in sight for this quest. Novel cell division
proteins will likely also include previously known proteins with an unforeseen
role in division, such as ClpX. If we take into account that the recently discovered division proteins were not required for septum formation or had very
mild loss-of-function phenotypes it becomes evident that the comprehensive
identification of new cell division proteins will not be trivial and shall require
the combination of approaches such as clever genetic screens (gain-of-function,
synthetic lethal) and large scale protein interaction and localization studies.
Acknowledgments
I would like to thank Jonathan Dworkin for critical reading of the manuscript,
and Sigal Ben-Yehuda, Ulysses Lins and Fernanda Abreu for images.
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