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Cytoskeletal Elements
in Bacteria
Peter L. Graumann
Institute of Microbiology, Faculty for Biology, University of Freiburg, 179104
Freiburg, Germany; email: [email protected]
Annu. Rev. Microbiol. 2007. 61:589–618
Key Words
The Annual Review of Microbiology is online at
micro.annualreviews.org
cytoskeleton, MreB, FtsZ, intermediate filaments, cell
morphology, cell cycle
This article’s doi:
10.1146/annurev.micro.61.080706.093236
c 2007 by Annual Reviews.
Copyright All rights reserved
0066-4227/07/1013-0589$20.00
Abstract
All cytoskeletal elements known from eukaryotic cells are also
present in bacteria, where they perform vital tasks in many aspects
of the physiology of the cell. Bacterial tubulin (FtsZ), actin (MreB),
and intermediate filament (IF) proteins are key elements in cell division, chromosome and plasmid segregation, and maintenance of
proper cell shape, as well as in maintenance of cell polarity and assembly of intracellular organelle-like structures. Although similar
tasks are performed by eukaryotic cytoskeletal elements, the individual functions of FtsZ, MreBs, and IFs are different from those
performed by their eukaryotic orthologs, revealing a striking evolutional plasticity of cytoskeletal proteins. However, similar to the
functions of their eukaryotic counterparts, the functions conferred
by bacterial cytoskeletal proteins are driven by their ability to form
dynamic filamentous structures. Therefore, the cytoskeleton was a
prokaryotic invention, and additional bacteria-specific cytoskeletal
elements, such as fibril and MinD-type ATPases, that confer various
functions in cell morphology and during the cell cycle have been
observed in prokaryotes. The investigation of these elements will
give fundamental information for all types of cells and can reveal the
molecular mode of action of cytoskeletal, filament-forming proteins.
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Contents
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INTRODUCTION . . . . . . . . . . . . . . . . .
TUBULIN-LIKE PROTEINS . . . . . .
FtsZ Forms a Ring that Initiates
Cell Division . . . . . . . . . . . . . . . . . .
A Switch in the Localization of
FtsZ During the Developmental
Process of Sporulation . . . . . . . . .
Proteins Regulating the Function
of FtsZ . . . . . . . . . . . . . . . . . . . . . . .
Positioning of the FtsZ Ring:
MinD Filaments, Nucleoid
Occlusion, and MipZ . . . . . . . . . .
Bacterial Tubulins BtubA and
BtubB . . . . . . . . . . . . . . . . . . . . . . . .
ACTIN-LIKE PROTEINS . . . . . . . . .
FtsA: A Crucial Component of the
Division Machinery . . . . . . . . . . .
MreB: A Filamentous Cytoskeletal
Structure Performing an
Essential Function in Many
Bacteria with a Complex Cell
Shape . . . . . . . . . . . . . . . . . . . . . . . . .
Biochemical Properties of MreB
Proteins . . . . . . . . . . . . . . . . . . . . . .
Dynamic Localization of MreB
Proteins . . . . . . . . . . . . . . . . . . . . . .
Gene Multiplicity . . . . . . . . . . . . . . . .
A Function in Control of Cell
Morphology . . . . . . . . . . . . . . . . . .
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IFs: intermediate
filaments
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Most if not all types of cells require factors
that stabilize their cell membrane and provide some rigidity to the cell. Such factors
are essential for the maintenance of cell morphology (the shape of an organism). Cellular
life is also dependent on several dynamic processes such as physical segregation of chromosomes and division of the cell into two
daughter cells, as well as movement of subcellular structures within the cell. Nature has
evolved a class of proteins called cytoskeletal elements that are key factors in both asGraumann
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INTRODUCTION
Cell morphology:
outward appearance
(shape, structure,
color, pattern) of an
organism
A Function During Bacterial
Development . . . . . . . . . . . . . . . . .
A Function in Chromosome
Segregation . . . . . . . . . . . . . . . . . . .
ParM and AlfA: A Function in
Plasmid Partitioning . . . . . . . . . . .
Cytoskeletal Elements in
Magnetotactic Bacteria . . . . . . . .
An Archaeal Actin-Like Protein . . .
INTERMEDIATE
FILAMENT-LIKE PROTEINS. .
Crescentin Mediates Cell
Curvature in C. crescentus . . . . . . .
Cytoplasmic Filaments in
Spirochetes: CfpA and Scc
Proteins . . . . . . . . . . . . . . . . . . . . . .
AglZ from Myxococcus xanthus . . . . .
OTHER TYPES OF
CYTOSKELETAL ELEMENTS
Cytoskeletal Elements in Cell
Wall–Less Bacteria: Fibril
Protein and MreB-Like
Filaments . . . . . . . . . . . . . . . . . . . . .
ParA-Type Proteins form
Plasmid-Segregating Filaments.
Bacterial Dynamin . . . . . . . . . . . . . . .
Cytoskeletal Elements of
Unknown Composition . . . . . . . .
pects, cell stability/morphology and cell dynamics. Cytoskeletal elements generally form
filamentous structures that can be highly dynamic or static, depending on which task they
confer.
Three types of cytoskeletal elements
in eukaryotic cells, tubulins, actins, and
intermediate filaments (IFs), have been
described and characterized. Tubulin forms
microtubules consisting of straight protofilaments that assemble into hollow tubules
through lateral contacts to both sides of the
protofilaments (30) (Figure 1). Microtubules
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extend through association of GTP-bound
α- and β-tubulin heterodimers onto the plus
end, while the dimers are released from the
minus end after hydrolysis, which destabilizes
the intrafilament contacts. However, microtubules can rapidly release dimers from both
ends and thus shrink, a stochastic event called
catastrophic collapse, giving rise to dynamic
instability of the filaments. Thus, tubulin
filaments are highly dynamic elements within
the cells. These tubules set up the tracks of
the mitotic spindle apparatus that are used to
segregate chromosomes during mitosis and
meiosis through dedicated motor proteins.
Tubulin filaments also serve as tracks for the
transport of intracellular vesicles and other
cargo, which are moved through cells by
motor proteins that have a defined direction
of movement on microtubules.
Actin forms a two-stranded, right-handed
helical filament with an axial rise of 5.4 nm
per monomer that has a plus-/minus-end polarity and is also dynamic (44, 80) (Figure 1).
Owing to filament asymmetry, ATP-bound
actin adds to the plus end (also called barbed
end) much faster than it does to the minus
end (pointed end) when actin concentrations
are high. ATPase activity leads to conversion to ADP-actin within the filament, and
ADP-actin is released at the minus end. This
process is called treadmilling and leads to a net
polymerization at one end and depolymerization at the other end. Thus, while the center
of the filament (and each subunit) remains stationary, the tip of the filament can push objects by limiting them in their diffusion such
that they can diffuse only in the direction of
the extended filament. Actin polymerization
is required for cell movement via extension
of pseudopods, in which additional proteins
induce branching of actin filaments, generating a network that pushes against the leading
edge of the membrane in a brush-like manner
(44). Actin is also required for the movement
of some types of vesicles through cells (44,
115).
IFs are composed of extended coiledcoil proteins that assemble into rigid sheets
GTP
Tubulin
GTP
α-/β-tubulin
-
+
GDP GDP
GDP
GTP
-
+
FtsZ
GTP
GDP
ATP
ATP
Actin
ParM
ADP
ATP
Strand 1
- ADP
+
ATP
Strand 2
ATP
MreB
-
+
ADP
IFs
Figure 1
Schematic drawing of cytoskeletal elements in eukaryotes (tubulin, actin,
and IFs) and in bacteria (FtsZ, with putative protofilament structure, ParA,
MreB, and IFs). IFs, intermediate filaments. Both actin and MreB filaments
(green and gray) are composed of identical subunits.
(Figure 1). IFs form 8- to 10-nm-thick cytoskeletal elements that provide internal mechanical support for the cell and position different organelles, e.g., Golgi apparatus and
mitochondria (107). For example, keratin provides mechanical strength in skin cells, even
after the cells have died, as does vimentin in
endothelial cells. IFs are much less conserved
in sequence than are tubulins or actin, because
different sequences can make up coiled-coil
motifs.
It has taken much longer to realize that
similar cytoskeletal elements are also present
in prokaryotic cells (43). Filamentous structures in bacteria have been described in
many reports, in which electron microscopy
www.annualreviews.org • Cytoskeletal Elements in Bacteria
Dynamic
instability: rapid
depolymerization
and shrinkage of
filaments
Filament: long
chain of proteins
Treadmilling:
constant removal of
the protein subunits
from these filaments
at one end while
protein subunits are
constantly added at
the other end
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TUBULIN-LIKE PROTEINS
b
a
FtsZ Forms a Ring that Initiates Cell
Division
FtsZ-CFP
FtsZ-CFP
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c
d
YFP-MreB
Crescetin
Figure 2
Fluorescence microscopy of cytoskeletal elements in bacteria. (a) FtsZ
forms a ring at the middle of the cell (Bacillus subtilis cells expressing
FtsZ-CFP), initiating division. (b) FtsZ switches its position during
differentiation; B. subtilis cells express FtsZ-CFP at the onset of
sporulation. White arrowheads indicate two polar Z rings. Note the spiral
forms of FtsZ in several cells. (c) MreB forms helical filaments underneath
the cell membrane (B. subtilis cells expressing YFP-MreB). (d ) Crescentin
localizes to the concave side of the bent Caulobacter crescentus cells
(immunofluorescence with anti-crescentin antibodies; cells are stained
with the blue DNA stain DAPI). Panel d courtesy of C. Jacobs-Wagner,
Yale University. White lines in panels a–c indicate septa between cells that
grow in chains. White bars in panels a–c and white line in panel d are
2 μm.
Polymerization:
bonding of
monomers or “single
units” together from
longer chains called
polymers
Differentiation:
acquisition of a (new)
cell type
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was used. The first cytoskeletal element in
a prokaryote was discovered in 1980 by
Lutkenhaus et al. (72), and the second element was discovered in 1988 by Matsuhashi
and coworkers (23). In spite of the power
of bacterial genetics, the true function of
FtsZ and MreB only became apparent with
the investigation of the localization of the
proteins within cells. Adaptation of cytological techniques developed for eukaryotic
cells for the study of cellular organization
of bacteria has shown that bacteria contain
a multitude of specifically subcellularly localized proteins and possess proteins that
confer highly dynamic and directional processes. Among these proteins, cytoskeletal elements play several crucial roles in bacterial
physiology.
Graumann
FtsZ was the first protein described to form
a cytoskeletal structure in prokaryotic cells.
Using screens selecting for temperaturesensitive mutants that form filaments at high
temperature (filamentation temperature sensitive), researchers have identified many genes
involved in cell division, and ftsZ was first described in 1980 (72). Using immunoelectron
microscopy, Bi & Lutkenhaus (9) showed that
FtsZ is present primarily at the invaginating
edge of the septum. The use of immunofluorescence microscopy revealed that FtsZ forms
a ring (Z ring) at the middle of the cell, even
if the membrane does not yet show any detectable invagination (2) (Figure 2a). Depletion of FtsZ leads to the formation of long
aseptate cells, in which chromosomes are normally segregated and which ultimately lyse.
FtsZ is the first protein of the division machinery that visibly localizes at midcell, and
the localization of all division proteins is dependent on FtsZ (71). Formation of new FtsZ
rings occurs rapidly (within 1 min) and can
occur at future division sites even in the predivisional cell, before the midcell Z rings have
almost completely closed (104). These experiments suggest that FtsZ is first to establish
the division machinery, and in support of this,
it is the most highly regulated and conserved
division protein.
Several reviews have summarized knowledge on FtsZ and cell division (3, 73); therefore, this review contains only a condensed
account of the nature and function of FtsZ.
An ftsZ gene is present in almost all bacterial and archaeal cells (round-, rod-, or more
complex-shaped species) (Figure 3), except
for Sulfolobus species, other Crenarchaeota,
and some cell wall-less bacteria (belonging to
the Mollicutes). Its essential function in cell
division notwithstanding, FtsZ is dispensable
for growth in Streptomyces coelicolor. Curiously,
this organism can grow in the absence of
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FtsZ, during which one giant cell is formed
that can produce a whole colony (78). In fact,
Streptomyces cells divide rather irregularly and
distribute their chromosomes sloppily. Thus,
there is no apparent need to divide during
growth in these organisms. However, S. coelicolor is unable to sporulate in the absence of
FtsZ, because cell division is required to pinch
off spores at the tips of aerial hyphae.
FtsZ has two domains, one of which is
a GTPase domain related to the GTPase
domain of EF-Tu. The structure of FtsZ is
highly similar to α- and β-tubulin (which
are highly similar to each other) (70). GTP
is bound between the two domains, and hydrolysis is thought to occur through contacts
between the bottom of one FtsZ and the
GTPase pocket of the following molecule
within the filament, analogous to tubulin.
During polymerization of tubulin, GTP in
β-tubulin is immediately hydrolyzed, whereas
hydrolysis in α- and β-tubulin is much slower.
Conversely, GTP is hydrolyzed much more
rapidly in FtsZ filaments (94), and hydrolysis may occur within each FtsZ monomer
during polymerization. Because GDP-bound
tubulin filaments are more unstable than the
GDP/GTP-bound structures, FtsZ filaments
are more unstable in vitro than the tubulin assemblies. The two domains of FtsZ
can interact and polymerize when they are
Pbps:
penicillin-binding
proteins
−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−→
Figure 3
Scheme of cytoskeletal elements in bacteria.
(a) FtsZ (and frequently also FtsA) forms a ring in
the middle of coccal cells. In many cocci, division
planes alternate in two or even three dimensions,
giving rise to growth as tetrads or packets of cells,
respectively. (b) FtsZ forms a midcell ring in
rod-shaped cells and recruits cytosolic division
proteins and (c) membrane-bound division
proteins, such that the division septum is
synthesized by penicillin-binding proteins (Pbps).
MinD forms spiral structures that are enriched at
the cell poles, preventing assembly of Z rings.
Nucleoids (which contain the chromosomes)
prevent formation of Z rings, such that only the
middle of the cell is competent for FtsZ
polymerization after nucleoids have separated.
MreB forms dynamic helical filaments that move
underneath the cell membrane and affect
chromosome segregation and maintenance of cell
morphology. (c) MreB proteins interact with
membrane proteins (MreC) that affect cell
morphology and in turn interact with Pbps.
Cytoskeletal structures in (d ) C. crescentus and
(e) in spiral formed bacteria. Spirochetes contain
cytoskeletal filaments along the long side of the
cells, and fibril forms a ribbon-like structure along
the short axis of cell wall–less Spiroplasma cells.
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FRAP: fluorescence
recovery after
photobleaching
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noncovalently linked, suggesting that polymerization of the ancestor evolved through
fusion of the two domains (85). FtsZ protein forms different kinds of polymers in
vitro, such as single protofilaments, sheets,
and tubule-like structures, that are dependent on the presence of GTP (31). However, because FtsZ lacks loops that make lateral contacts between tubulin protofilaments,
it is unlikely to form hollow microtubules like
tubulin in vivo (Figure 1). Additionally, αand β-tubulin contain several inserts in their
sequence that are absent in FtsZ and that are
important for binding of additional factors
that affect the formation of microtubules (3).
However, FtsZ interacts with many other proteins that appear to affect its polymerization
(see below).
Using fluorescence recovery after photobleaching (FRAP), the Erickson group (102)
has shown that the Z ring is highly dynamic
in vivo. FRAP exploits the property of green
fluorescent protein (GFP) to require an extensive time (in the range of up to 60 min) to
regain excitability after an excitation/emission
event. Thus, if a rigid structure of a GFPtagged protein is bleached within a subcellular
area of the cell, recovery of fluorescence will
depend on the time it takes for the bleached
GFP molecules to regain fluorescence. However, if a structure is dynamic, exchange with
molecules from a nonbleached area in the cell
will speed up recovery of fluorescence. Using
this technique, the Erickson group showed
that upon bleaching of one half of an FtsZ
ring, fluorescence is regained within a few seconds. Fluorescence recovery was markedly reduced in a mutant that possesses much slower
GTPase activity, revealing that exchange of
FtsZ monomers (and thus filament disassembly) depends on GTP hydrolysis. It has been
proposed that the Z ring consists of many FtsZ
filaments that constantly assemble and disassemble or that extend at one end and disassemble at the other end (called ratchet mechanism). This idea is supported by findings from
the Margolin group (105), who found that in
a subset of growing Escherichia coli cells, FtsZ
Graumann
forms spirals that dynamically extend away
from the central Z ring or move underneath
the cell membrane in young cells lacking a
Z ring within a time frame of a few seconds.
Oscillatory waves of FtsZ spirals were observed in elongated cells and were independent of MreB spirals (see below). Indeed, FtsZ
can be captured in a spiral form as a specific
mutant version that is shifted to nonpermissive temperature and resumes formation of
normal rings and cell division after temperature shift-back (79). These data suggest that
FtsZ forms long dynamic spirals that are reorganized into a ring structure at the onset
of division, which possibly consists of shortpitched tightly stacked helices.
A Switch in the Localization of FtsZ
During the Developmental Process
of Sporulation
When B. subtilis cells are starved, or enter
stationary phase, cells can differentiate into
a spore that is dormant for a long period
(spores can survive for 100 years in distilled
water) and highly resistant to high temperature or a variety of chemical insults. The
hallmark of sporulation is the generation of
a septum close to one cell pole, rather than
at midcell. This asymmetric division generates a small cell that differentiates into the
spore and a large cell (called the mother cell)
that lyses. Polar division is achieved owing
to a change in the pattern of localization of
FtsZ, which forms spiral structures at the onset of sporulation (Figure 2b) rather than a
ring structure at midcell and finally localizes
as two polar rings, both of which initiate divisome assembly (7). One septum matures faster
than the other, and upon completion of one
septum, a genetic program is initiated that
leads to the activation of proteins that prevent the completion of the second (slower)
polar septum and mediate disassembly of the
second Z ring (29). The switch between midcell and bipolar assembly of Z rings is driven
by overproduction of ftsZ transcription relative to exponentially growing cells (due to the
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induction of an additional promoter driven
by a sporulation-specific sigma factor) and by
the sporulation-specific synthesis of a membrane protein, SpoIIE, that stabilizes polar
FtsZ rings (7). Artificial overproduction of
FtsZ in growing cells indeed leads to the formation of spiral structures and, if accompanied by induction of SpoIIE synthesis, to the
formation of stable bipolar Z rings. Therefore, inhibition of polar ring formation by the
Min system is apparently overridden by high
FtsZ levels and induction of SpoIIE, and formation of a Z ring at midcell may be prevented
by a sporulation-specific structure of the nucleoid, called axial filaments, in which the nucleoid now extends from pole to pole but lacks
a visible constriction at the cell center.
Proteins Regulating the Function
of FtsZ
Just as tubulin assembly is regulated by a large
array of proteins, several proteins that interact
with FtsZ influence the stability of the Z ring.
MinC inhibits polymerization of FtsZ close to
the cell poles (see below), whereas FtsA and
ZipA appear to stabilize Z rings in E. coli cells.
Both FtsA and ZipA are essential for cell division but not for the formation of Z rings,
and they interact with FtsZ independently
(Figure 3c). However, in the absence of both
FtsA and ZipA, formation of Z rings is abolished (86). That a single mutation in FtsA can
bypass the requirement for ZipA during division supports the idea that FtsA and ZipA have
overlapping functions in the establishment
and/or maintenance of Z rings (38). Instead of
possessing ZipA, B. subtilis possesses the unrelated ZapA, EzrA, and SepF/YlmA proteins,
all of which bind to FtsZ, and of which ZapA
and SepF/YlmA appear to stabilize Z rings and
EzrA appears to destabilize FtsZ polymers in
vivo (45, 47, 54). Consistent with these ideas,
EzrA prevents FtsZ polymerization in vitro
(46), and purified ZapA promotes bundling of
FtsZ filaments (45). In addition, induction of
DNA damage elicits the SOS response, causing transcription of genes that encode DNA
repair, as well as of sulA in E. coli (or yneA in
B. subtilis). SulA binds to FtsZ and prevents
polymerization, effectively inhibiting division
until DNA damage has been repaired (83).
The battery of regulatory proteins shows that
regulation of FtsZ polymerization is required
to prevent unwanted and premature division and to elicit productive formation of a
ring structure at the appropriate time in the
cell cycle.
Cell cycle: the
series of events that
take place in a cell
between its
formation and the
moment it has
duplicated itself
Positioning of the FtsZ Ring: MinD
Filaments, Nucleoid Occlusion,
and MipZ
FtsZ forms a ring relatively precisely at midcell in E. coli and B. subtilis, but not at any other
position within the cell and only late during
the cell cycle. Positioning of the Z ring is regulated by two additive systems. Noc (nucleoid
occlusion; B. subtilis) and SlmA (synthetically
lethal with a defective Min system) (E. coli)
bind to the nucleoid and prevent the formation of a Z ring—SlmA does this directly by interfering with polymerization of FtsZ (8, 118).
Thus, the nucleoid prevents the formation of
the Z ring in its vicinity (occlusion), such that
nonseparated chromosomes are not bisected
by premature cell division. This leaves the
cell center (as well as the cell poles, in which
there is no DNA) competent for Z ring formation after nucleoids have separated (thus
ensuring that Z ring formation follows late in
the cell cycle after segregation of the chromosomes). Here, MinC prevents FtsZ polymerization (53). MinC is positioned at both
cell poles in B. subtilis, or oscillates between
the cell poles in E. coli, by MinD, a Walkertype ATPase with which MinC forms a complex (73, 90). MinD in turn is recruited to the
poles in B. subtilis by DivIVa, a late component
of the cell division machinery whose localization to the septum (which later becomes the
new pole) depends on the FtsZ protein (74).
In E. coli, MinD forms filamentous spirals as
it moves from one pole to the other and back
(Figure 3b) while MinC is carried piggyback
(98).
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It is possible that the MinD filaments assemble at one pole and extend to the other
pole by addition of MinD to the growing tip
of the filament(s), in a manner analogous to
actin. MinD also forms short double-stranded
filaments in vitro in the presence of ATP
(103). The addition of phospholipid vesicles
greatly enhances the formation of MinD filaments, which increase in length and selfassociate into bundles, supporting the notion
that MinD self-assembles along the cell membrane (51). Membrane association is mediated
by a short C-terminal sequence and requires
ATP or a nonhydrolyzable analog. MinE disassembles MinD filaments in vitro, because it
stimulates MinD ATPase activity (52, 103),
and also localizes in an oscillatory manner
(48). MinE forms a ring structure close to
midcell that moves toward the MinD assembly at one pole. After MinD has disassembled
from the pole, the MinE ring moves toward
the other pole after MinD has assembled at
this site, apparently “pushing” MinD away
from one pole to the other and back. The net
results of both systems in B. subtilis and in
E. coli are a higher concentration of MinC
close to the poles, masking these for FtsZ
polymerization, and a low concentration at
midcell.
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ANRV322-MI61-27
Bacterial Tubulins BtubA and BtubB
Verrucomicrobia are a phylum of bacteria of
uncertain lineage that contains species that
live in close association or as ectosymbionts
of eukaryotic ciliates. Microtubule-like structures that serve as protection against predators
have been observed in several species. Close
tubulin homologs, termed BtubA and BtubB,
have been found encoded in the genome
of a free-living member of Verrucomicrobia,
Prosthecobacter dejongeii, whereas ftsZ is absent from the genome (58). BtubA and BtubB
form heterodimers that rapidly polymerize
into double protofilaments or bundles of double filaments in the presence of GTP (101).
Slow GTP hydrolysis results in depolymerization of filaments. The 3D structure of the
596
Graumann
BtubA/B heterodimer closely resembles that
of α-/β-tubulin, including the C-terminal domain of tubulin that forms the outside of microtubules, which is absent in FtsZ (96). The
close resemblance of BtubA/BtubB to tubulin
and their absence in any other known bacterial genome suggest that the genes have
been transferred to P. dejongeii (and possibly also other Verrucomicrobia) via horizontal gene transfer to replace FtsZ. In any
event, BtubA/B represent an intermediate
step between FtsZ and tubulin, in terms of
both structure and folding, because like FtsZ,
BtubA/B do not require chaperones for folding, as tubulin does. It will be interesting to
determine the distribution of BtubA/B in Verrucomicrobia and to elucidate their function
in vivo.
ACTIN-LIKE PROTEINS
FtsA: A Crucial Component of the
Division Machinery
FtsA belongs to the family of actin-fold proteins (111). FtsA interacts directly with the extreme C terminus of FtsZ and localizes to the
FtsZ ring (Figure 3c) invariably dependent
on FtsZ but not on any other component of
the division machinery, whereas most division
proteins other than FtsZ are recruited to the
FtsZ ring dependent on FtsA (32) (Figure 3c).
These data show that FtsA is recruited to the
FtsZ ring at an early time point. FtsA is essential in E. coli and many other bacteria including
cocci (Streptococcus pneumoniae), whereas deletion of ftsA (which is usually upstream of ftsZ)
in B. subtilis leads to a severe defect in division but allows for slow growth. The ration of
FtsZ to FtsA is 5:1 in E. coli and B. subtilis and
must be maintained to ensure proper division
(16). In vitro, S. pneumoniae FtsA assembles
into long curved polymers, apparently consisting of a 2+2 pair of paired filaments, in the
presence of various nucleotide diphosphates
and triphosphates (66). FtsA generally binds
to ATP; however, ATPase activity could be
detected only in B. subtilis FtsA but not in the
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S. pneumoniae ortholog. In the absence of
FtsA, FtsZ forms abnormal rings in B. subtilis
cells, and loss of FtsA and ZipA abolishes FtsZ
ring-formation in E. coli, showing that FtsA
is important for FtsZ polymerization (possibly in counteracting inhibition with MinC)
or stability of FtsZ polymers at midcell. FtsA
interacts directly with later-recruited division proteins (32) and thus plays an additional role in the assembly of the divisome.
The exact function of FtsA remains to be
uncovered.
MreB: A Filamentous Cytoskeletal
Structure Performing an Essential
Function in Many Bacteria with a
Complex Cell Shape
In the late 1960s, screens designed to find
genes whose product affects cell morphology
in rod-shaped cells identified several loci in
which mutations led to the formation of osmotically stable, round cells. One of these loci
was the Mre (murein cluster E) operon, which
contains three genes in E. coli, and in many
other bacteria, called mreB, mreC, and mreD
(23). Mutations in all three genes lead to formation of irregularly bulged or oval to round
cells. MreB was first described in 1988, but
its similarity to actin became apparent only
in 1992. MreB contains five sequence motifs,
called phosphate 1, phosphate 2, adenosine,
connect 1, and connect 2, that are conserved
in actin and other seemingly unrelated proteins. It was proposed in 1992 that MreB, together with several sugar kinases, FtsA, and
plasmid-encoded StbA, belongs to the actinfold protein family (11). Actin consists of two
domains of similar fold, domains I and II, and
ATP is bound in the cleft between the two
domains, triggering movement of the two domains. Domain movement in turn may mediate formation of filaments, but this is still
under debate. Both domains are composed
of four subdomains (two each), called Ia, Ib,
IIa, and IIB. Subdomains Ia and IIa connect
the two major domains and have a similar
fold with identical topology, which most likely
arose through gene duplication. Phosphate 1
and phosphate 2 motifs are situated on two
β-hairpins on subdomains Ia and IIa and bind
to the phosphate tail of ATP, critically influencing ATP binding and ATP hydrolysis (3).
Compared with MreB, eukaryotic actin has
several sequence insertions, all of which mediate either allosteric interactions within the
actin monomer or binding to chaperones and
cofactors.
MreBCD genes are usually absent in round
(coccoid) bacteria, although several round
cyanobacteria and planctomycetes species
contain an mreB ortholog. Mutation or deletion of mreB in E. coli results in formation
of round slowly growing cells that are hypersensitive to antibiotics targeting cell wall synthesis enzymes (114). A similar phenotype is
observed upon mutation of the downstream
mreC and mreD genes (113). These experiments suggest that MreB is involved in cell
wall synthesis. Because isolated peptidoglycan (PG) sacculi (called ghosts) retain a rod
cell shape, or bent cell shape for sacculi from
vibrios, it is clear that the structure of the
cell wall determines the cell shape. MreB proteins may dictate the shape of the sacculus (see
below). E. coli cells with a deletion of mreB,
mreC, mreD, or all three genes are suppressor mutants, showing that cells are not viable
in the absence of MreB/C/D but that their
function can somehow be compensated for by
extragenic mutations (63). MreB is also essential in B. subtilis, Caulobacter crescentus, and
Rhodobacter sphaeroides (35, 59, 100), but not in
S. coelicolor or Azospirillum brasilense (10, 77),
showing that mreB is not essential in all bacteria with complex cell morphology.
R. sphaeroides is an interesting case because the bacterium changes shape, from a rod
to a coccobacillus, and undergoes extensive
cytoplasmic membrane invagination when it
switches from aerobic to anaerobic photosynthetic growth. MreB is essential for viability in both cell forms and was found only in
the cytosolic fraction of lysed cells, indicating
that it is only weakly membrane associated or
not associated at all (100). Deletion of mreB
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PG: peptidoglycan
597
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Cytoskeleton:
cellular scaffolding,
dynamic structure
that maintains cell
shape and drives
cellular division,
enables some cell
motion, and plays
important roles in
intracellular
transport
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in the nitrogen-fixing α-Proteobacterium
A. brasilense results in a highly pleiotrophic
phenotype in which cells are large and round,
secrete modified exopolysaccharides, have an
altered and larger capsule, aggregate more
strongly, and are more resistant to osmotic
stress than are wild-type cells (10). These phenotypes are similar to many changes observed
in A. brasilense cells that undergo physiological conversion to cyst-like structures when
cells colonize plant roots or other plant tissue. Apparently, the function of MreB in this
bacterium is complex and includes control of
cell permeability, cell surface structures, and,
possibly, cell differentiation.
Many proteins specifically localize to the
cell poles in several bacteria. Deletion of mreB
in C. crescentus and in E. coli leads to a loss of
polar localization of several proteins (40, 84),
showing that MreB is also involved in the establishment or maintenance of cell polarity. A
second MreB paralog, Mbl (MreB-like), was
found in B. subtilis and in B. cereus. Deletion of
mbl is not lethal in B. subtilis but leads to the
formation of highly irregularly shaped cells
that grow much slower than wild-type cells
(1). Using immunofluorescence microscopy,
Errington and coworkers (59) showed that
MreB and Mbl form distinct structures that
had never before been observed in bacteria.
Both proteins form dots and helical filamentous threads underneath the cell membrane
(Figure 2c). Mbl filaments extend from pole
to pole, and MreB filaments were predominantly found towards the cell center. Mbl filaments appeared to have a pitch (the distance
from one point of a helix to the next point one
helical turn away) different from that of MreB
filaments and therefore to form independent
structures. These experiments showed that a
cytoskeletal structure that controls cell morphology exists in bacteria. On the basis of the
different phenotypes of the depletion of MreB
or deletion of mbl, which generates round or
elongated and twisted cells, respectively, it was
proposed that MreB controls the width of the
cell and that Mbl controls longitudinal growth
(59).
Graumann
A helical arrangement of MreB filaments
was also found in E. coli and in the curved
C. crescentus cells (35, 64, 98) (Figure 3d ).
In the C. crescentus cells, helical MreB bands
switch to a ring-like central localization
around the time of cell division, which depends on the presence of FtsZ, showing that
MreB has a cell cycle–dependent localization
during C. crescentus growth and may be involved in cell division. However, in contrast
to most cell cycle proteins, whose intracellular
level changes dramatically during the C. crescentus cell cycle, MreB levels remain constant
in synchronized cells (35). These findings suggest that the MreB cytoskeletal element is
important to mediate complex cell morphology by providing an intracellular scaffold for
cell wall synthesis and/or membrane structure. Curiously, loss of MreB can be suppressed by overproduction of FtsQ, FtsA, and
FtsZ in E. coli (63). Round MreB-depleted
cells have a much larger diameter than normal rod-shaped cells and thus require more
FtsZ to form complete Z rings. Indeed, round
MreB-depleted B. subtilis cells frequently have
incomplete Z rings (59), and overproduction of FtsZ may therefore allow round cells
to keep up cell division, allowing for their
(slow) propagation in spite of an abnormal cell
shape.
Biochemical Properties of MreB
Proteins
When Löwe and coworkers (110) showed that
the structure of MreB is similar to that of
actin, and that MreB is thus the bacterial homolog of actin, it became clear that bacteria contain an actin-like cytoskeleton. The
3D structure of MreB from Thermotoga maritima can be superimposed onto actin over
almost its entire length with only minor deviations. MreB is also similar to Hsp70; however, the Hsp70 contains a large insert within
one subdomain and a large C-terminal domain, absent in both actin and MreB. MreB
is less similar to hexokinase or FtsA, and thus
most closely related to actin (110). With the
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use of electron microscopy (EM), Löwe and
colleagues showed that MreB assembled into
long filamentous structures, dependent on the
presence of ATP or GTP (but not of magnesium), in which two MreB strands form a
straight protofilament (110). In contrast to eukaryotic actin, this two-stranded filament is
straight rather than helical. In vitro, polymerized MreB structures rarely consist of twostranded filaments; rather, they consist mostly
of sheets or bundles of filaments that can be
rather straight or highly curved. Further biochemical analysis of T. maritima MreB has
shown that polymerization of MreB is influenced by Ca2+ and Mg2+ concentrations and
that end-to-end annealing of filaments plays
no detectable role in polymerization (33).
ATP hydrolysis of MreB has kinetics similar
to that of actin, but it occurs at a much lower
critical concentration (about 2 orders of magnitude), as does polymerization of MreB. This
suggests that the affinity of MreB monomers
to each other is much higher than the affinity
between actin monomers. Given that about
8000 molecules of MreB were determined to
be present in B. subtilis, which corresponds to a
concentration of 5.6 μM, a lower critical concentration for polymerization is required for
the formation of filaments in vivo, in contrast
to actin, which is much more abundant in eukaryotic cells (about 550 μM).
Unlike actin, which favors ATP over GTP,
MreB can use ATP and GTP equally well for
the formation of filaments, which are much
more rigid than those formed by actin (34).
Further, MreB assembles into bundles of filaments, whereas actin assembles into single
protofilaments. These experiments show that
MreB filaments have the potential to provide
mechanical rigidity to bacterial cells, which
may contribute substantially to the maintenance of the rod shape or other complex types
of shapes.
Ultracentrifugation experiments have
shown that MreB is largely present as a
polymer in C. crescentus, and a high proportion of MreB is present in membrane
preparations, in parallel with cytoplasmic
MreB. Although MreB does not contain
any motifs typical for membrane association or membrane integration, the protein
remained in the membrane fraction after
removal of peripheral membrane proteins by
sodium carbonate, revealing a close association with the C. crescentus cell membrane
(35).
EM: electron
microscopy
Dynamic Localization of MreB
Proteins
MreB and Mbl filaments underneath the cell
membrane were shown to be highly dynamic
in vivo when functional GFP fusions became
available. Time-lapse microscopy showed that
GFP-Mbl filaments change their pitch and
even orientation throughout the cell cycle (14). Using FRAP, Carballido-Lopez &
Errington (14) further showed that GFP-Mbl
filaments undergo rapid turnover. In these experiments, a longitudinal half-side of a cell (a
half-cell cut along the long axis) was bleached
with a laser light. After GFP is excited, it takes
an extended period (up to 60 min) until the
molecule can be excited again. Therefore, if
a static structure containing GFP is bleached,
fluorescence will be lost for a long time. GFPMbl fluorescence reappears after about 5 min
in the bleached side of the cell, revealing that
the structures must exchange GFP-Mbl from
the nonbleached section of the cell (14). Importantly, concomitant with the recovery of
fluorescence, the nonbleached GFP-Mbl filaments in the other cell part lost fluorescence,
indicating that GFP-Mbl filaments turn over
by movement of nonbleached GFP from one
lateral side of the cell to the other. Rapid
time-lapse microscopy studies confirmed that
MreB-like filaments are highly dynamic structures. GFP-MreB and GFP-Mbl filaments
change their localization within 10-s intervals, and filaments apparently move underneath the membrane along a helical patch with
a speed of about 0.1 μm s−1 (19). This speed
of movement corresponds to the lower end of
extension of actin filaments measured in vivo
(0.1–1 μm s−1 for actin).
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In contrast to the structures of MreB in
B. subtilis, time-lapse microscopy of E. coli
MreB has suggested that these structures are
static (105). However, experiments visualizing
single MreB-YFP molecules in C. crescentus
have recently shown that MreB also localizes
dynamically in this bacterium. Using continuous illumination and capturing 65 frames s−1 ,
Kim et al. (60) observed fast-moving populations (most likely corresponding to unpolymerized MreB) and slowly moving—almost
static—populations of MreB-YFP molecules.
The slowly moving populations are expected
to be polymerized MreB molecules within the
helical filaments, suggesting that MreB treadmills by adding to the growing tip of a filament
(and thus becoming static) and dissociating
from the shrinking end. Because the slow population did show some slow movement, the
authors argue that MreB forms many short
(∼400 nm) filaments, i.e., treadmills within
many short filaments. The fast-moving MreB
population did not move as slowly as expected
for freely diffusing 64-kDa (MreB + GFP)
proteins. Rather, the polymerization rate was
comparable to that of membrane proteins,
suggesting that MreB may be membrane associated and thus diffuse along the membrane
until it hits the tip of a growing filament
(60).
The dynamic localization of MreB in B.
subtilis is highly important for the function
of the protein. A mutation in the phosphate
2 motif is dominant-negative in E. coli and
in B. subtilis, in that it leads to aberrant cell
shape (21, 64). This MreB mutant forms helical elements; however, they are abnormal and
static in B. subtilis, i.e., there is no extension
of filaments in time-lapse experiments and
no recovery of fluorescence in FRAP experiments (21). Although this mutant does form
filaments, B. subtilis cells expressing the mutant version but not wild-type MreB grow
poorly and show a strong cell shape and segregation defect (21). Thus, MreB dynamics are not essential for viability, but they
are important for the proper function of
MreB.
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Gene Multiplicity
In contrast to E. coli and relatives, which
contain only one MreB ortholog, several
other bacteria contain two (e.g., T. maritima,
Fusobacterium nucleatum) or three orthologs
(bacilli, many gram-positive bacteria, and
some cyanobacteria, e.g., Gloeobacter violaceus). B. subtilis contains three mreB-like
genes: mreB, mbl, and mreBH. The depletion of MreB, Mbl, or MreBH has different
consequences for the morphology of the cell
(15, 18), suggesting that the proteins perform
different functions. However, it has recently
become clear that the three MreB orthologs
form one cytoskeletal element, because all
three proteins extensively colocalize within
the helical filaments, as revealed by functional
CFP/YFP fusions (15, 21), and because they
interact with each other, as shown by fluorescence resonance energy transfer (FRET)
and bimolecular fluorescence complementation (BiFC) (21). Moreover, a dominantnegative allele of MreB that affects the localization of wild-type MreB also affects the
dynamic localization of Mbl, whose filaments
become static (21). Thus, the interaction of
MreB and Mbl is physiologically relevant, and
a defect in one MreB ortholog will translate
into a defect in the other orthologs. MreB,
Mbl, and MreBH may form mixed polymers,
and/or MreB orthologs may be important for
the structural integrity of each of the helical
filaments. MreB filaments were highly abnormal in the absence of Mbl but showed only
a mildly aberrant localization in the absence
of MreBH (20). These data suggest that Mbl
may be more important than MreBH for the
putative mixed filaments. The idea of mixed
polymers must be biochemically verified; nevertheless, MreB, Mbl, and MreBH can no
longer be viewed as independent helical filamentous structures.
A Function in Control of Cell
Morphology
How might an intracellular helical structure
affect the formation of a cylindrical PG cell
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envelope across the cell membrane? Although
this point is still unclear, a possible concept
is the interaction of MreB with membranebound cell wall–synthesizing enzymes (called
Pbps, penicillin-binding proteins) (Figure 3c)
and/or with membrane proteins that would
mediate a directed localization of Pbps. Indeed, Daniel & Errington (17) have shown by
using the fluorescently labeled antibiotic vancomycin (Van-FL) that binds to the d-Alad-Ala moieties of cell wall precursors that the
incorporation of cell wall precursors occurs in
a helical arrangement within the cell wall in
many rod-shaped cells. Helical arrangement
of cell wall synthesis was supported by the
use of another drug that binds to the reducing
end of PG precursors (106). During cell extension, new cell wall material is thus inserted
in a helical pattern along the lateral cell wall,
while during cell division, a major proportion
of Van-FL localizes as a ring to the site of division. Daniel & Errington (17) further showed
that rod-shaped cells that lack MreB, such
as gram-positive Corynebacterium and Streptomyces species, grow by inserting new cell wall
material into polar zones, but not along the
lateral wall, and at the division site, and that
cocci synthesize new cell wall layers only at
the division site. These experiments show that
different patterns of cell wall growth exist in
bacteria, and that the MreB cytoskeleton may
direct the insertion of lateral cell wall material. Helical insertion of Van-FL was reported
to be absent in mbl mutant cells (17), but this
finding has recently been disputed (106).
In support of the idea that MreB orthologs
may influence the 3D organization of cell wall
synthesis, C. crescentus Pbps localize in a bandlike pattern along the cell membrane, which
are lost in the absence of MreB (35). Several
Pbps were coimmunoprecipitated from membrane preparations, suggesting that they form
a complex in vivo whose position may be directed via MreB (35). B. subtilis Pbps also appear to be localized in a helical arrangement
within the cell membrane (95). Furthermore,
the MreC protein localizes at discrete areas
in a seemingly helical pattern in C. crescen-
tus (where it is a periplasmic protein) (22, 26)
and in B. subtilis (where MreC has one membrane span and thus is likely a membrane protein) (67). Depletion of MreC leads to loss of
distinct localization of Pbps (22, 26), as well
as loss of additional outer membrane proteins
that also localize in helical band-like patterns
(22). In E. coli, MreB interacts with MreC
(membrane anchored), which in turn interacts with MreD (63) (Figure 3c). Likewise,
Mbl and MreC colocalize and interact in B.
subtilis (21), suggesting that the spatial information of MreB/Mbl filaments is transferred
across the membrane via MreC, which has a
small cytosolic domain and a large periplasmic domain. Indeed, depletion of MreC interferes with the formation of MreB filaments
in both organisms (20, 63). Finally, C. crescentus MreC interacts with Pbps (22), suggesting that the link MreB-MreC-Pbps may direct
cell wall synthesis. However, now the puzzle
begins. B. subtilis Pbps maintain their specific
(probably helical) localization in the absence
of MreB or Mbl (95). Although the helical
incorporation of cell wall precursors was reported to be lost in the absence of Mbl, this
was recently disputed, and helical localization
of Van-FL was maintained in mbl-null cells
and MreB-depleted cells (106). Moreover,
C. crescentus MreB and MreC do not colocalize
(26), and disruption of MreB filaments by the
specific inhibitor A22 retains the helical localization of MreC and of Pbps (in this case, cells
stop to divide and grow but retain their proper
cell morphology). Only in cells that lose their
curved morphology during depletion of MreB
do Pbps lose their specific localization pattern
(22). Thus, MreB and MreC form two distinct
subcellular structures in C. crescentus, both of
which are required for proper localization of
Pbps, whereas MreB and MreC in E. coli and
Mbl and MreC in B. subtilis physically interact.
R. sphaeroides MreB localizes at midcell
in a transverse ring-like structure, but also
in a helical pattern away from midcell, indicating that it may be involved in some aspect of cell division and/or control of cell
wall extension (100). MreC and Pbps also
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Van-FL: fluorescent
vancomycin
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localize in a similar helical band-like pattern in
R. sphaeroides and colocalize throughout the
cell cycle (99). MreB colocalizes with Pbp2
only during the elongation phase of cell
growth, and during septation, MreB remains
at the septation site, whereas Pbp2 relocalizes to the one-quarter and three-quarter positions. Thus, Pbp2 and MreC are involved
in PG synthesis during cell elongation, and
MreB appears to be more restricted to cell
wall synthesis occurring during cell division.
Thus, although the concept of helical arrangement of cell wall synthesis through helical arrangement of the MreB cytoskeletal
structures appears attractive, it is still unclear
if and how the helical pattern of MreB orthologs affects helical incorporation of cell
wall material. It is also unclear how MreBs
obtain their helical arrangement within the
cytosol.
A variation of the MreB/cell wall concept
appears to apply to the third MreB ortholog of
B. subtilis, MreBH. Depletion of MreBH leads
to the formation of bent and bulged cells (15,
18). MreBH interacts with LytE, a cell wall
hydrolase. Because the murein sacculus is enlarged inside gram-positive bacteria and additional layers are laid down, the outer layers
need to be loosened and ultimately released to
make space for the enlargement of the inner
rings. This crucial activity is mediated by hydrolases, and in fact, B. subtilis cells constantly
give off cell wall material into the medium.
LytE also localizes in a helical pattern within
the cell wall and interacts with MreBH, as
shown by yeast two-hybrid experiments (15).
Loss of MreBH abolishes the specific localization of LytE (15), suggesting the MreBH
directs the pattern of LytE localization and
action, possibly by directing the secretion of
LytE. Indeed, the Sec secretion system localizes in a spot-like pattern following a helical
path within the membrane in B. subtilis (13).
However, the puzzle comes back again: SecY
maintains its helical pattern in the absence
of MreB or of Mbl, so the complete story of
MreBH-directed localization of LytE still remains to be unraveled.
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A Function During Bacterial
Development
As outlined above, MreB might play a role
during development in Azospirillum. In Streptomyces coelicolor, MreB is dispensable for
growth (77), in agreement with the idea that
incorporation of new cell wall material occurs at the tip of growing S. coelicolor chains
of cells, but not at the lateral sides (17).
S. coelicolor mreB mutants show a strong defect in the formation of spores (77), which
are produced from aerial hyphae that extend
away from the surface into the air. MreB
mutant spores are highly enlarged, distorted,
and strongly sensitive to stress conditions in
which wild-type spores easily survive. MreB
localizes to the septa of sporulating aerial hyphae (77). Thus, in Streptomyces the mreB gene
is used for a differentiation process, influencing the formation of proper cell walls in
spores, rather than for rod-shape maintenance
during growth. In agreement with this notion, MreB (and a second mreB paralogous
gene) is present only in actinomycetes that
form spores (such as streptomycetes), but not
in nonsporulating species, such as Corynebacterium glutamicum cells, which also grow by
polar extension (17). Deletion of mreC results
in slowly growing cells, suggesting that, in
contrast to MreB, MreC plays an important
role in vegetative S. coelicolor cells (77).
A Function in Chromosome
Segregation
Three independent approaches have shown
that MreB proteins also play an important role
in chromosome segregation. After initiation
of replication, and long before its termination,
duplicated regions of the chromosome are
separated toward opposite cell poles in rodshaped bacteria such as B. subtilis and E. coli.
Origin regions of the chromosome are separated with an average speed of ∼0.2 μm min−1
over a distance of about 1.5 μm, even during inhibition of cell wall growth, followed
by all other regions soon after these have
been duplicated by the replication machinery
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located in the center of the cell (117). These
data show that an active intracellular machinery must exist that pushes or pulls duplicated
chromosome regions, one into each cell half.
What drives chromosome segregation is
still unclear, but the action of MreB is important or essential for this process. Depletion of MreB in B. subtilis leads to rapid decondensation of the nucleoid that contains the
chromosome(s), and to the formation of anucleate cells, i.e., cells that lack DNA after cell
division has occurred, even during continued
synthesis of MreC and MreD (whose genes
are driven by the same promoter as mreB) (18,
20). This defect in chromosome segregation
arises even before a defect in cell morphology is apparent, ruling out a secondary effect
via cell shape abnormalities. The segregation
defect occurs at the stage of separation of origin regions; during depletion of MreB (and
to a lesser extent of Mbl), origin regions frequently remain in the cell center or are moved
into the same cell half. In E. coli, induction
of a dominant-negative ATPase mutant allele
of MreB leads to similar phenotypes and, although origin regions can still be separated (in
contrast to terminus regions), one sister cell
frequently receives both chromosomes (64).
The most direct evidence for the involvement
of MreB orthologs in chromosome partitioning comes from experiments in C. crescentus,
in which the addition of the drug A22 rapidly
and specifically interferes with the formation
of MreB helical filaments and leads to disintegration of the cytoskeletal structures. Loss of
MreB filaments is accompanied by the inability to separate origin regions, although termini separate when A22 is added after origins
have moved apart (41). MreB is associated
with DNA of the origin regions, but not of
the terminus regions, showing that MreB is
a crucial component in the process of early
chromosome segregation (41).
It has been speculated that extension of
MreB filaments toward the poles could provide the force to separate origin regions, in
agreement with an observed potential poleward movement of 0.24 μm min−1 for MreB
(accounting for the pitch of MreB helices)
(44). Because actin can propel whole bacteria
(Listeria) through the cytosol in macrophages
and can push beads through a viscous solution
(80), it is possible that MreB pushes duplicated chromosome regions toward opposite
cell poles. Indeed, such a mechanism operates
during segregation of plasmids (see below), in
which an MreB homolog forms a filament that
drives plasmids toward opposite cell poles. If
this scenario is true, an adaptor (or several)
must exist that links extending MreB filaments
to those regions on the chromosome that have
been replicated. Alternatively, as proposed by
Gerdes and coworkers (62), MreB could serve
as an anchor for RNA polymerase (RNAP),
on the basis of the finding that E. coli MreB
interacts with RNAP in vivo. Intriguingly, it
had been proposed before that RNAP may
be the driving force for chromosome segregation, owing to the finding that inhibition
of transcription prevents separation of origin
regions, even of those that have been duplicated (inhibition of transcription also interferes with replication) (25). Most genes are
oriented away from replication origins and
may be moved toward opposite cell poles via
tracking through many directionally MreBanchored RNAP molecules.
An mreB in frame deletion can be generated in a special medium containing high sucrose and magnesium concentrations, which
stabilize the cell wall and membrane (36).
This finding reinforces the idea that MreB
has an important influence on the composition and/or structure of the cell envelope.
Exponentially growing mreB-null cells had a
normal cell shape in the special medium and
did not show any chromosome segregation
defect, which suggests that MreB is not involved in chromosome segregation. An explanation for these puzzling findings might be
that in the absence of MreB, Mbl is essential
for viability, even in the special medium (21).
Depletion of Mbl in mreB cells leads to cessation of growth, a complete loss of regular
cell shape, and a defect in chromosome segregation. This observation suggests that Mbl
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RNAP: RNA
polymerase
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can partially assume MreB functions under
special conditions, and it reinforces the idea
that loss of one paralog weakens the mixed
MreB/Mbl/MreBH polymer, which may be
compensated for through stabilization by special media conditions.
ParM and AlfA: A Function in
Plasmid Partitioning
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Beyond dispute, a bacterial actin homolog has
been shown to be a critical element in the
separation of a low-copy-number plasmid in
E. coli (high-copy-number plasmids are separated randomly and do not need an active
mechanism that ensures their propagation).
R1 plasmid harbors the ParRMC, which consists of parR and parM genes, and a cis-acting
partitioning locus ( parC ), which contains several ParR binding repeats. ParR binds cooperatively to these repeats and may pair parC sites
of two replicated plasmids (81) (Figure 4c).
ParM is an actin-like protein and forms Factin-like structures in vitro in the presence
of ATP (112). In contrast to MreB filaments,
and similar to actin, the two ParM strands coil
a
c
around each other in a right-handed helical
path. The crystal structure of ParM is similar
to that of MreB and of actin. Upon binding of
ADP (compared with the crystal structure of
nucleotide-free ParM), the interdomain cleft
closes as domains I and II approach each other,
and the two domains of ParM undergo a large
(25◦ ) movement. It is unclear if this movement
is the basis for induction of polymerization.
In vivo, ParM forms one or two helical filaments in E. coli cells carrying a ParRMC plasmid (and only if ParR and parC are present)
(82). However, ParM filaments were found
only in a subset of cells in a growing culture
that contained two separated plasmids. Filaments were not present in cells containing
one or two plasmids at the middle of the cell
(82). Most importantly, plasmids were always
present at the tip of the ParM filaments (81),
suggesting that the filaments push plasmids
away from the middle of the cell toward opposite cell poles (Figure 4a). A simple and elegant mechanism appears to regulate directed
pushing of plasmid through ParM filaments.
ParM strongly interacts with ParR bound to
the parS site, which is dependent on ATP
ATP-ParM
ADP-ParM
ParR bound
to parS site
ParM/plasmid
d
b
e
AlfA-GFP
Figure 4
Plasmid-segregating actin homologs ParM and AlfA, which form filaments that probably push plasmids
toward opposite cell poles. (a) Immunofluorescence microscopy of E. coli cells carrying
LacI-GFP-labeled plasmid (red ) and ParM stained with antibodies ( green). Outlines of cells are captured
by phase contrast. Image reproduced with permission of the publisher. (b) B. subtilis cells expressing
AlfA-GFP; membranes are stained in red. Image courtesy of J. Pogliano. (c–e) Cartoon showing
segregation of plasmids toward opposite cell poles through growing ParM and AlfA filaments.
604
Graumann
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binding to ParM. ParR in turn enhances the
ATPase activity of ParM, and ATP hydrolysis leads to dissociation of the ParM/ParR interaction (81). ParR appears to pair two plasmids via their parC sites, which biorients the
plasmids (Figure 4d ) and further triggers filament formation of ParM (Figure 4e ). ParM
in turn interacts with ParR, ensuring a close
but transient contact between motor and target. Thus, ATP-bound ParM molecules can
add to the tip of the filament (or rather, at the
tips of the bundles of filaments), in between
the tip and the (ParR-bound) parC site, generating a treadmilling process that prevents
backward diffusion of plasmids and drives
replicated plasmids apart into both cell halves
(Figure 4e). Transport appears to be rapid because filaments of intermediate length could
not be detected, in which the plasmids would
be halfway between midcell and cell pole (82).
This mechanism is a simple but efficient way
to separate two pieces of DNA by using (a) an
attachment site and (b) an adaptor that interacts with (c) a polymer motor and that affects
the state of nucleotide binding of the motor
subunits.
Strikingly, polymerization of ParM could
be observed in vitro. Fluorescently labeled
purified ParM polymerizes in an ATPdependent manner through addition of ATPbound ParM to both sides of the filament,
supporting the finding that bidirectional symmetrical polymerization can push plasmids
towards the cell poles (37). Like actin, a critical minimal concentration of ParM is required to induce polymerization and, similar to barbed ends of actin filaments, ParM
filaments extended ∼2 μm s−1 at each end
(5 μM−1 s−1 ). However, a recent report shows
that ParM forms randomly oriented filament
bundles in the presence of crowding agents
(87); therefore the observed single filaments
in vitro and in vivo could consist of bidirectionally oriented bundles of filaments. A nonhydrolyzable ATP analog stabilized ParM filaments, whereas ATP-bound filaments showed
dynamic instability, that is, at some variable,
critical point, growing ParM filaments rapidly
disintegrated from one end and less often
from both ends. Thus, ATP hydrolysis can
trigger disassembly of the dynamic filaments.
Dynamic instability is a hallmark of microtubules, and that tubulin and ParM bear no
similarity at the protein level suggests that dynamic instability has arisen through convergent evolution, as has their similar role in the
segregation of DNA molecules.
AlfA has recently been identified as a
plasmid-segregating actin-like protein in B.
subtilis. It is encoded on a plasmid and, together with its downstream gene alfB, it is
necessary and sufficient for plasmid segregation during growth and sporulation (6). AlfA
forms long filaments that extend from pole
to pole in cells (Figure 4b) and probably
push the duplicated plasmids toward opposite poles, analogous to ParM. AlfA filaments
have a high turnover rate, and a mutant version of AlfA that displays reduced turnover
within the filaments is impaired in plasmid
segregation, showing that also in this case filament dynamics are important for function
in vivo (6). ParM and ActA form two phylogenetically discrete groups of actin-like proteins, each representing an evolutionary class
distinct from MreB, actin, and FtsA. AlfA
has so far been found in gram-positive organisms, whereas ParM has been detected
in gram-negative bacteria. The evolution of
these classes of actin-like proteins shows that
early during evolution the actin-like precursor evolved into different classes that perform
distinct functions, all of which are based on
the ability to form polymers that extend and
shrink in a highly dynamic manner.
Cytoskeletal Elements in
Magnetotactic Bacteria
Magnetotactic species are a group of bacteria
that have a specialized machinery (usually encoded on a chromosomal island) that sets up
a linear array of 15 to 20 magnetosomes in a
straight line along the short axis of the vibrio (or spiral-formed cell) (97) (Figure 5b).
Magnetosomes are membranous structures
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a
b
Magnetosome
MamK/MamJ filament
MamJ-GFP
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c
Basal bodies
Cytoplasmic filament
Periplasmic flagellar filament
Figure 5
MamK (actin homolog) and MamJ form a cytoskeletal element that positions magnetosomes in a straight
line. (a) Schematic drawing of the MamK/MamJ structure that assembles at the short axis of the cell, with
MamK forming cytoskeletal filaments with which MamJ probably interacts. (b) MamJ-GFP fusion
expressed in Magnetospirillum gryphiswaldense cells (image by A. Scheffel, H.J. Defeu Soufo & D. Schüler).
(c) Electron micrograph of a spirochete cell, from which the outer membrane has been removed to reveal
the periplasmic flagellar filament, basal bodies (where flagella are attached), and cytoplasmic filaments,
which run along the long axis of the cells. Image from Reference 56 with permission from the publisher.
Magnetotaxis:
ability of certain
motile, aquatic
bacteria to sense a
magnetic field and
coordinate their
movement in
response
606
(usually 50 nm in diameter) filled with magnetite (Fe3 O4 ) that arise through invagination of the inner cell membrane and are used
for orientation according to the magnetic
field. Because most magnetotactic bacteria are
microaerophilic and thus prefer to dwell in
the rather small interface between oxic and
anoxic zones within soil, it is believed that
magnetotaxis provides spatial information for
up and down movement within soil to find the
optimal conditions for growth.
Until recently, it had been unclear how
magnetosomes are aligned in a straight line
along the cell’s short axis. With the use of
cryo-electron tomography, a network of up to
seven filaments ∼4 nm thick that run parallel
to the line of magnetosomes close to the cell
Graumann
membrane has been visualized in Magnetospirillum magneticum and in M. gryphiswaldense.
This cytoskeletal structure could consist of
two factors that have recently been identified to be essential for alignment of magnetosomes: an actin ortholog called MamK and
a novel type of protein called MamJ. In the absence of MamK or MamJ, both of which are
encoded from genes situated in the magnetosome island, magnetosomes still form but
are mislocalized and frequently clustered in
the middle of the cells (61, 93). Both proteins
fused to GFP, showing filamentous structures
along the short axis of the cell (Figure 5a),
corresponding to the aligned magnetosome
structure. However, although magnetosomes
are aligned along the center of the helical
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cells, filamentous MamK-GFP and MamJGFP structures extend from pole to pole.
In addition to MamK, M. magneticum contains an MreB paralog encoded on the chromosome, adjacent to genes encoding for other
morphology factors. MamK, ParM, and MreB
form three distinct phylogenetic groups, each
of which arose early during evolution, a testament to their different functions. The filamentous structures detected in cryo-EM are
∼6 nm thick, indicating that they may derive from actin-like protofilaments composed
of MamK (61). MamK forms a straight filamentous structure from pole to pole when
expressed in E. coli cells (88), suggesting that
it self-assembles into the cytoskeletal structure as seen in Magnetospirillum. Guiding the
arrangement of intracellular organelle-like
structures in a bacterium is a novel function
for bacterial actin orthologs. After induction
in E. coli MamK filaments slowly formed and
became visible after ∼75 min, apparently nucleating at one of the few sites in the cell.
When a MamK-GFP fusion and a MamKmCherry fusion were expressed sequentially,
a mostly mosaic pattern with generally separated green and red sections arose within the
filament (88). These experiments support the
idea of nucleation and extension events and
of extension of preexisting filaments. MamK
filaments are dynamic structures, similar to
those formed by MreB and ParM, but as evidenced by their diverse phylogenetic evolution, MamK and ParM form straight filaments
rather than helical filaments, as formed by
MreB orthologs.
MamJ is a highly acidic 456-amino-acid
protein and has a unique repetitive domain
structure (93). In the absence of all other
magnetosome genes, MamJ-GFP localizes
throughout the cytosol, suggesting that MamJ
does not form a cytoskeletal structure itself
but is associated with such a structure and
possibly connects magnetosomes with a cytoskeletal element, possibly MamK. Timecourse experiments showed that magnetosomes form at many different positions within
the cell. In wild-type cells they assemble at
the cell center in a straight line at a later time
point, and in the absence of MamJ, they collapse into an agglomeration after their assembly (93). These experiments suggest that magnetosomes gather into a filament after they
have formed through an invagination of the
cell membrane around a magnetite crystal at
many positions within the cell, through the
combined action of MamK and MamJ.
An Archaeal Actin-Like Protein
A fifth phylogenetic type of prokaryotic actinlike proteins is present in several species
within the Euryarchaeota. Notably, three
strains of the order Thermoplasmatales contain a highly conserved copy of Ta0583, whose
structure was determined from Thermoplasma
acidophilum. Ta0583 is most similar to ParM
(rather than to MreB or actin) in its overall
structure (92), has ATPase activity, forms helices with a filament width of 5.5 nm and an
axial repeating unit of 5.5 nm, both of which
are comparable to eukaryotic actin, and forms
crystalline sheets in vitro (49, 92). On the
basis of the packaging of monomers within
the crystal, Ta0583 can indeed form actin-like
protofilaments that interact with each other
to form sheet-like structures seen in EM images in vitro. Ta0583 is closer to actin in
terms of its polymerization kinetics and phylogeny (49). In vivo, Ta0583 appears to be a
low-abundance protein unlikely to form an
extended cytoskeletal structure, although it
may play an important role in cell morphology in Thermoplasmatales especially because
these bacteria lack a rigid cell wall structure.
The true function of Ta0583 remains to be
elucidated.
INTERMEDIATE
FILAMENT-LIKE PROTEINS
Crescentin Mediates Cell Curvature
in C. crescentus
IF-type proteins assemble into 8- to 10-nmthick reversible filamentous structures and
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Motility: ability to
move spontaneously
and independently,
usually directed by
chemotaxis
18:23
covalently cross-linked meshworks in many
eukaryotic cells (Figure 1), wherein they frequently provide mechanical strength. The
first bacterial IF-type protein was identified by
Jacobs-Wagner and coworkers (4) in a screen
for straight C. crescentus cells. A gene, creS, was
identified whose deletion resulted in growing
but completely straight cells. The encoded
crescentin protein is predicted to contain a
high content of coiled coils as well as characteristic stutters, which are positions at which
coiled coils are clearly disrupted. Extended
coiled coils containing stutters are characteristic of eukaryotic IF proteins. Through the
use of immunofluorescence and GFP fusion
it was vizualized that crescentin forms a filamentous structure along the short axis of
the cell (Figure 2d ). Strikingly, in stationaryphase C. crescentus cells that are elongated
and highly spiraled, crescentin localized at the
short axis throughout the large cells (4). Thus,
the curved cytoskeletal crescentin structure
is essential for cell curvature by an unknown
mechanism.
Like eukaryotic IF proteins, crescentin
assembles into 10-nm-thick filaments and
sheets in vitro without any need for cofactors or energy (4). Owing to their high
degree of coiled coils, IFs usually have a
highly elongated structure. Because many different sequences can form coiled coils, sequence conservation between IF proteins is
low, making it difficult to identify them solely
in silico. However, IF-type proteins are predicted to be present in many other bacterial
species. With the identification of crescentin,
it has now been established that none of the
three cytoskeletal elements within eukaryotic
cells was a eukaryal invention. It will be interesting to investigate the detailed role and
function of IF-type proteins in Caulobacter and
other bacteria.
Cytoplasmic Filaments in
Spirochetes: CfpA and Scc Proteins
Spirochaeta are a phylum (major genetic lineage) within the kingdom of Bacteria and in608
Graumann
clude many human pathogens. Spirochetes
are unusual in that they have a helical or
flat-plane wave shape, are motile via flagella
that run along the length of the cells within
the periplasmic space, and are attached to
both ends of the cells. These features enable spirochetes to move through viscous and
dense media and to pass through cell layers during infection. EM and cryo-EM studies have revealed the presence of cytoplasmic filaments 5 to 7 nm in diameter, four
to six of which run the length of the cells
underneath the cell membrane (50, 55, 56).
In contrast to crescentin, the filaments run
in a helical path along the long axis of the
cells (Figure 3e). Cross-bridging structures
appear to cross-link filaments with each other
on the cytosolic side of the filaments, which on
the opposite side are connected to the membrane by different proteins (55). Apparently,
the filaments are located underneath the corresponding group of periplasmic flagella and
were originally suggested to be involved in
motility. Gentle lysis of the cells preserves
the cytoplasmic filaments that still exhibit the
same helical periodicity of the formerly intact
cell.
The major constituent of the filaments
was identified to be CfpA, a 79-kDa polypeptide unique to spirochetes that is predicted to
contain several extended coiled-coil regions
(120). This property suggests that CfpA may
belong to the family of IF proteins. When
expressed in E. coli, CfpA induces the formation of extended filamentous structures,
indicating that CfpA may indeed form filaments by itself. Deletion of the cfpA gene has
a pleiotrophic phenotype, including formation of chains of cells, reduction in motility,
and, most strikingly, a defect in the intracellular arrangement of DNA (57). In contrast to wild-type cells in which the DNA
is distributed throughout the cytoplasm, cfpA
mutants contain compacted DNA at various
subcellular positions and form anucleate cells
(2% of the population). Thus, CfpA may affect the positioning of DNA within the cells
(and/or cell division) and motility. Periplasmic
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flagella localize normally in the absence of
CfpA, and vice versa, suggesting that both systems assemble independently of each other
(56). Because lower mobility of the cfpA mutants could be due to the chaining morphology, it is unclear if CfpA has a direct or indirect
effect on the function of flagella. Spirochetes
have a highly unusual genome structure, including one large chromosome that is frequently linear and more than 20 additional
minichromosomes and plasmids, each with
different replicons, that are also frequently
linear and have terminal hairpin telomeres.
Possibly, this unusual genome requires a specialized IF-like filamentous structure for its
propagation into daughter cells, which arise
through FtsZ-dependent medial cell division
(24) (Figure 3e).
An additional protein containing an extended N-terminal coiled-coil region is
present in Leptospira species. Scc protein
forms helical structures 2 to 3 μm in length
and also single filamentous structures 6 to
10 nm in diameter in vitro, independent of
any cofactor, that are stable for days (76).
When expressed in E. coli cells, Scc-GFP
forms a single extended filament through the
cell from pole to pole (highly reminiscent of
ParM and AlfA) or underneath the cell membrane. Purified Scc binds to RNA and to DNA
and forms striking rod-shaped nucleoprotein
structures. Deletion of the Scc-encoding gene
altered colony morphology, but not cell morphology or motility, indicating that Scc may
be involved in chemotaxis during spreading
of cells (76). It will be interesting to elucidate
further the function of cytoplasmic filaments,
CfpA, and Scc, as well as the mode of motility
in spirochetes.
AglZ from Myxococcus xanthus
AglZ in involved in social motility in the
bacterium Myxococcus xanthus and interacts
with a small GTPase that is also involved in
this process (119). The N terminus of AglZ
shows similarity to the receiver domain of
two-component response regulators, and the
C terminus contains coiled-coil motifs. The
purified coiled-coil part of AglZ forms filamentous structures in vitro. It will be interesting to determine the function of the
protein, which may belong to the IF protein
family.
OTHER TYPES OF
CYTOSKELETAL ELEMENTS
Cytoskeletal Elements in Cell
Wall–Less Bacteria: Fibril Protein
and MreB-Like Filaments
Mollicutes are a class of bacteria (e.g.,
Mycoplasma, Acholeplasma, and Spiroplasma)
that do not contain a cell wall, although
they phylogenetically belong to the grampositive phylum of bacteria. However, Mollicutes can have intricate cell morphologies,
such as the long corkscrew-like Spiroplasma
species. These bacteria can be as long as
10 μm but are only ∼300 nm in diameter. It
has been unclear how Mollicutes can have a
defined cell shape in the absence of a rigid
wall. Thin specimens are ideal for a new technology called cryo-electron tomography. This
technique uses subtle density differences in
frozen cells, in which the formation of ice
crystals has been suppressed through sudden
freezing to –10◦ C (liquid methane) in milliseconds. Several images are taken from different angles, and 3D image reconstruction is
computationally achieved from calculating actual density differences from all images. The
Baumeister group (65) has resolved intracellular cytoskeletal elements in Spiroplasma melliferum that were previously unknown. Two
types of sheets of filamentous structures lie
underneath the membrane along the short
axis of the cell (Figure 3e). A central sheet
of nine thin filaments spaced ∼4 nm apart is
flanked on both sides by a sheet of five thick
filaments spaced 11 nm apart (65). The dimensions of the inner filaments are highly
similar to those formed by actin or MreB and
could thus be composed of an MreB ortholog.
Indeed, Spiroplasma contains five mreB genes
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in its genome. The outer sheets are composed of fibril, a protein unique to Spiroplasma.
Fibril is a 59-kDa protein that is thought to
form a dimer; these proteins appear to form
tetramers that polymerize into extended helical structures with an axial repeat of 9 nm (108,
116). Given that each monomer is ∼5 nm
in diameter, the 11-nm-wide filaments observed are most likely composed of fibril
tetramers.
Computer models predict that Spiroplasma
can locally change its handedness, from rightto left-handed or vice versa (65, 109). Shortening one of the outer sheets relative to the
other would generate such a change in handedness, and if such a change moved through
the cell from one end to the other (like
a wave), this would generate a propelling
force suitable to propel Spiroplasma through
a liquid. It remains to be seen if this is indeed a mechanism for swimming without a
flagellum.
Bacteria from the species Mycoplasma
can have intricate cell shapes, ranging from
pear-shaped cells to cells having stalk-like
extension and bulged cell bodies. Using
conventional EM, researchers (42, 75) have
observed distinct subcellular structures in
Mycoplasma genitalium that appeared to
be fibrils or helically arranged blade-like
structures. When membranes and cytosolic
proteins are removed with Triton X100,
an intriguing rod-like structure (apparently
consisting of many parallel filaments) with a
rounded tip that has the rough dimensions of
300 × 40 nm can be visualized (42). Although
the composition of this Triton-insoluble
fraction of Mycoplasma cells has been investigated (91), the defined components of the
rod-like structure are still unclear, and the
function of these structures remains to be
elucidated.
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Plasmid-Segregating Filaments
In addition to the ParR/ParM system (also
called the type II segregation system), a sec610
Graumann
ond type of segregation system exists (type I)
that distributes low-copy-number plasmids to
future bacterial daughter cells. The plasmidencoded system consists of a defined partitioning site ( par) and two genes in a bicistronic
operon that encode for a Walker-box ATPase
(ParA, which is similar to MinD) and for a
DNA binding protein (ParB). ParB-type proteins bind to the par site (or in some cases sites)
situated close to the par operon and interact
with their ParA counterparts (39). ParA-type
proteins form helical filaments that rapidly oscillate along the nucleoids (27). In vitro, ParA
forms ATP-dependent filaments and bundles
of filaments, in which the filaments appear to
twist around each other (5, 28, 68). Consistent
with the formation of higher-order filamentous structures, ParA ATPase activity is cooperative. Thus, formation of dynamic filaments
appears to be a conserved function of MinDtype ATPases. The interaction between ParA
and ParB suggests that ParA dynamics distribute plasmids along the nucleoids and thus
ensure that sufficient copies are positioned
within each cell half before cell division occurs. It is also clear that a helical pattern is a
recurring scheme for the localization of many
bacterial proteins that form highly dynamic
filaments.
Bacterial Dynamin
Dynamins are a family of multidomain
GTPases that perform a mechanochemical
function in endocytosis, vesicle trafficking,
and mitochondrial division and morphology
in eukaryotic cells (89). For example, dynamin
I forms a helical collar around the neck of
clathrin-coated vesicles and pinches them off
from the membrane. In vitro, dynamins form
oligomeric rings and long helical structures
that constrict upon binding of GTP. The pitch
of dynamin spirals assembled on lipid vesicles
increases upon GTP hydrolysis, suggesting
that dynamins pinch off vesicles in a springlike manner (89).
Recently, the structure of a cyanobacterial
dynamin-like protein has been resolved,
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revealing striking conservation of the GTPase domain structure (69). The Nostoc
punctiforme bacterial dynamin-like protein
(BDLP) is a low-affinity GTPase that forms
long tubules in the presence of GMP-PNP
(a nonhydrolyzable analog of GTP) and of
vesicles in vitro, and it tubulates vesicles
in a compact helical coating with a 6-nm
interfilament pitch repeat and a diameter
of ∼45 nm. The helix has a pattern of a
cartwheel with a 17-fold symmetry, when
looked at from top or bottom, similar to that
of eukaryotic dynamins. Lipids may be bound
at the inside of the tubules, via the hydrophilic
paddle region. N. punctiforme BDLP localizes
to the cell membrane in the filamentous
cyanobacterium in a punctate pattern (69).
The B. subtilis BDLP ortholog also localizes
in a similar pattern (M. Krishnamurthy & P.L.
Graumann, unpublished results), indicating
that BDLPs may play a role at the membrane
in many bacterial species, because genes encoding dynamin-like proteins are also present
in many other bacterial genomes. It will be
exciting to determine which function BDLPs
confer.
Cytoskeletal Elements of Unknown
Composition
Using cryo-EM, Briegel et al. (12) observed
apparently novel as yet uncharacterized
cytoskeletal elements in C. crescentus cells.
About one-fourth of the cells contained bundles of filaments adjacent to the membrane
on the concave side of the cells, which were
observed even in creS-deleted cells and in
cells in which MreB filament formation was
inhibited by A22. Thus, these elements that
were much shorter than the long axis of
the cell may present novel filament-forming
proteins. Similarly, structures called cytoplasmic ribbons (bundles of three to five
ribbons without an apparent connection to
the membrane) were detected in about half
of all cells analyzed, even in the absence of
CreS and MreB. Two additional structures,
polar ribbons and a ring-like structure, were
detectable at low frequency, but nothing is
known about their nature. Thus, at least four
independent filamentous structures appear
to exist in C. crescentus, suggesting that the
bacterial cytoskeleton is even more intricate
than recently believed (12).
BDLP: bacterial
dynamin-like protein
SUMMARY POINTS
1. Cytoskeletal elements also exist in bacteria and thus arose early during evolution.
2. Bacterial tubulin-like protein FtsZ and actin-like MreB proteins form highly dynamic
filaments and interact with proteins involved in cell division or cell shape maintenance,
respectively.
3. FtsZ is the first protein known to form a ring at midcell and recruits further cell
division proteins to form the divisome that ultimately drives formation of the division
septum.
4. MreB proteins form helical filaments underneath the cell membrane and are involved
in maintenance of cell shape, possibly directing locations of cell wall synthesis; MreB
may also play a role in chromosome segregation.
5. Actin-like ParM, AlfA, and MamK proteins form straight filaments. ParM and AlfA
drive plasmids toward opposite cell poles, and MamK positions magnetosomes (special
organelles) in a straight line underneath the cell membrane.
6. ParA- and MinD-like Walker box ATPases form filaments within cells and mediate
partitioning of certain plasmids or positioning of the FtsZ ring, respectively.
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7. Crescentin, an intermediate filament-like protein, is essential for cell curvature in
C. crescentus and forms a filamentous structure at the short axis of the cells.
8. Spirochetes and Spiroplasma cells have additional distinct filamentous cytoskeletal
elements, the function of which is still unclear.
DISCLOSURE STATEMENT
Annu. Rev. Microbiol. 2007.61:589-618. Downloaded from arjournals.annualreviews.org
by Universidad de Chile on 06/02/08. For personal use only.
The author is not aware of any biases that might be perceived as affecting the objectivity of
this review.
ACKNOWLEDGMENTS
I would like to thank Christine Jacobs-Wagner and Matt Cabeen (Yale University), Herve
Joel Defeu Soufo (University of Freiburg), Joe Pogliano (UCSD), Andre Scheffel (Max Planck
Institute, Bremen, Germany) and Dirk Schüler (University of Munich) for providing images.
Work in my laboratory is supported by grants from the Deutsche Forschungsgemeinschaft.
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Annual Review of
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Contents
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Volume 61, 2007
Frontispiece
Margarita Salas p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p xiv
40 Years with Bacteriophage Ø29
Margarita Salas p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 1
The Last Word: Books as a Statistical Metaphor for Microbial Communities
Patrick D. Schloss and Jo Handelsman p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 23
The Mechanism of Isoniazid Killing: Clarity Through the Scope
of Genetics
Catherine Vilchèze and William R. Jacobs, Jr. p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 35
Development of a Combined Biological and Chemical Process for
Production of Industrial Aromatics from Renewable Resources
F. Sima Sariaslani p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 51
The RNA Degradosome of Escherichia coli: An mRNA-Degrading
Machine Assembled on RNase E
Agamemnon J. Carpousis p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 71
Protein Secretion in Gram-Negative Bacteria via the Autotransporter
Pathway
Nathalie Dautin and Harris D. Bernstein p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 89
Chlorophyll Biosynthesis in Bacteria: The Origins of Structural and
Functional Diversity
Aline Gomez Maqueo Chew and Donald A. Bryant p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p113
Roles of Cyclic Diguanylate in the Regulation of Bacterial Pathogenesis
Rita Tamayo, Jason T. Pratt, and Andrew Camilli p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p131
Aggresomes and Pericentriolar Sites of Virus Assembly:
Cellular Defense or Viral Design?
Thomas Wileman p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p149
As the Worm Turns: The Earthworm Gut as a Transient Habitat for
Soil Microbial Biomes
Harold L. Drake and Marcus A. Horn p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p169
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Biogenesis of the Gram-Negative Bacterial Outer Membrane
Martine P. Bos, Viviane Robert, and Jan Tommassen p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p191
SigB-Dependent General Stress Response in Bacillus subtilis and
Related Gram-Positive Bacteria
Michael Hecker, Jan Pané-Farré, and Uwe Völker p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p215
Annu. Rev. Microbiol. 2007.61:589-618. Downloaded from arjournals.annualreviews.org
by Universidad de Chile on 06/02/08. For personal use only.
Ecology and Biotechnology of the Genus Shewanella
Heidi H. Hau and Jeffrey A. Gralnick p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p237
Nonhomologous End-Joining in Bacteria: A Microbial Perspective
Robert S. Pitcher, Nigel C. Brissett, and Aidan J. Doherty p p p p p p p p p p p p p p p p p p p p p p p p p p p p259
Postgenomic Adventures with Rhodobacter sphaeroides
Chris Mackenzie, Jesus M. Eraso, Madhusudan Choudhary, Jung Hyeob Roh,
Xiaohua Zeng, Patrice Bruscella, Ágnes Puskás, and Samuel Kaplan p p p p p p p p p p p p p p p p p283
Toward a Hyperstructure Taxonomy
Vic Norris, Tanneke den Blaauwen, Roy H. Doi, Rasika M. Harshey,
Laurent Janniere, Alfonso Jiménez-Sánchez, Ding Jun Jin,
Petra Anne Levin, Eugenia Mileykovskaya, Abraham Minsky,
Gradimir Misevic, Camille Ripoll, Milton Saier, Jr., Kirsten Skarstad,
and Michel Thellier p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p309
Endolithic Microbial Ecosystems
Jeffrey J. Walker and Norman R. Pace p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p331
Nitrogen Regulation in Bacteria and Archaea
John A. Leigh and Jeremy A. Dodsworth p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p349
Microbial Metabolism of Reduced Phosphorus Compounds
Andrea K. White and William W. Metcalf p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p379
Biofilm Formation by Plant-Associated Bacteria
Thomas Danhorn and Clay Fuqua p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p401
Heterotrimeric G Protein Signaling in Filamentous Fungi
Liande Li, Sara J. Wright, Svetlana Krystofova, Gyungsoon Park,
and Katherine A. Borkovich p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p423
Comparative Genomics of Protists: New Insights into the Evolution
of Eukaryotic Signal Transduction and Gene Regulation
Vivek Anantharaman, Lakshminarayan M. Iyer, and L. Aravind p p p p p p p p p p p p p p p p p p p p453
Lantibiotics: Peptides of Diverse Structure and Function
Joanne M. Willey and Wilfred A. van der Donk p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p477
The Impact of Genome Analyses on Our Understanding of
Ammonia-Oxidizing Bacteria
Daniel J. Arp, Patrick S.G. Chain, and Martin G. Klotz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p503
Contents
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Morphogenesis in Candida albicans
Malcolm Whiteway and Catherine Bachewich p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p529
Structure, Assembly, and Function of the Spore Surface Layers
Adriano O. Henriques and Charles P. Moran, Jr. p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p555
Cytoskeletal Elements in Bacteria
Peter L. Graumann p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p589
Indexes
Annu. Rev. Microbiol. 2007.61:589-618. Downloaded from arjournals.annualreviews.org
by Universidad de Chile on 06/02/08. For personal use only.
Cumulative Index of Contributing Authors, Volumes 57–61 p p p p p p p p p p p p p p p p p p p p p p p p619
Cumulative Index of Chapter Titles, Volumes 57–61 p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p622
Errata
An online log of corrections to Annual Review of Microbiology articles may be found
at http://micro.annualreviews.org/
viii
Contents