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From K. Kruse, Bacterial Organization in Space and Time. In: Edward H. Egelman, editor: Comprehensive
Biophysics, Vol 7, Cell Biophysics, Denis Wirtz. Oxford: Academic Press, 2012. pp. 208-221.
ISBN: 978-0-12-374920-8
© Copyright 2012 Elsevier B.V.
Academic Press.
Author's personal copy
7.13 Bacterial Organization in Space and Time
K Kruse, Theoretical Physics, Saarland University, Germany
r 2012 Elsevier B.V. All rights reserved.
7.13.1
7.13.2
7.13.2.1
7.13.3
7.13.3.1
7.13.3.2
7.13.3.3
7.13.3.4
7.13.4
7.13.4.1
7.13.4.1.1
7.13.4.1.2
7.13.4.1.3
7.13.4.2
7.13.5
7.13.6
7.13.6.1
7.13.6.2
7.13.7
7.13.7.1
7.13.7.2
7.13.8
References
Introduction
Organization Principles
Mechanisms vs. Models
The Bacterial Cytoskeleton
Prokaryotic Cytoskeletal Proteins
Helices and Rings
Induced Assembly and Disassembly of Cytoskeletal Filaments
Spatial Distribution of Magnetosomes and Carboxysomes
Positioning The Z-Ring
The Min Oscillations
Biochemistry of the proteins MinD and MinE
Min oscillations result from MinD-MinE self-organization in presence of a membrane
Cooperativity is needed for spontaneous emergence of Min oscillations
Polar Localization of MinD/DivIVA in B. subtilis
Polar Protein Localization
Chromosome segregation and protein distributions
Entropic chromosome segregation
Active Chromosome Segregation
Temporal Organization
Division Entry
A three-protein Circadian clock
Concluding remarks
Glossary
Divisome The molecular assembly that generates
formation of a new cell wall to divide a bacterial
cell.
Meanfield models A variant of computational models
used to describe the evolution of protein distributions that
takes the form of partial differential equations.
7.13.1
Introduction
Bacteria were long considered to be mere bags of proteins
and DNA with the macromolecules they contained homogenously distributed. This view was based on several experimental observations: Diffusion constants for cytosolic
macromolecules in bacteria have been measured to be on
the order of 1–10 mm2s1,1–3 such that for cells of typical
bacterial sizes of a few micrometers, diffusion homogenizes
these molecules on time scales of a second. Furthermore, early
optical and electron microscopy did not reveal clearly discernable compartments that would separate different parts
within bacteria as is the case for eukaryotes. Finally, based on
sequence comparisons, bacteria were thought to possess no
cytoskeleton, which in eukaryotes is to a large extent responsible for spatial and temporal organization.
This view of unstructured bacteria has been shattered in
the last 20 years or so, by the use of modern electron and
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Nucleoid A region of the bacterial cell that contains the
chromosomal DNA.
Origin of replication A site on the chromosomal DNA
where DNA replication starts.
Walker ATPase An ATPase for which the nucleotidebinding pocket contains two conserved sequence motifs,
namely the Walker A and Walker B motif.
fluorescence microscopy as well as by the discovery of the
bacterial cytoskeleton.4 Even prior to this time, it was, of
course, clear that bacteria are to some extent internally organized: cell division does not occur randomly in space and the
duplicated chromosome is evenly divided between the two
daughter cells. Furthermore, some bacteria like Caulobacter
crescentus, Bacillus subtilis, or Myxococcus xanthus were known
to be at least temporarily polarized. The degree of bacterial
organization we are aware of today, however, came as a
complete surprise. We now know that bacteria can have a
nose, they possess organelles, which are non-randomly distributed in the cells, and regular filamentous structures exist,
either in form of rings or helices.
In addition, a number of bacterial cytoskeletal proteins
have been discovered and by now it is clear that bacteria
contain homologs of the eukaryotic cytoskeletal proteins actin
and tubulin. Also intermediate filaments have found prokaryotic counterparts.
Comprehensive Biophysics, Volume 7
doi:10.1016/B978-0-12-374920-8.00717-7
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Bacterial Organization in Space and Time
Biophysics has played an important role in discovering
bacterial organization, and in revealing the mechanisms
underlying their formation. Think of the major advances in
optical microscopy that not only allow us to locate proteins
within bacterial cells with an extension of only a few microns,
but also to track individual proteins and quantify their
mobility. Theoretical analysis is indispensible if one aims at
a quantitative understanding of spatiotemporal protein patterns, and this can often be used to distinguish between
qualitatively different mechanisms. Furthermore, the reconstitution of bacterial systems in vitro plays an increasingly
important role.5
In the following chapter, a number of prominent bacterial
structures will be discussed. Instead of trying to be comprehensive, the systems are chosen to illustrate various general
organizational principles. A major part is devoted to the bacterial cytoskeleton, because similar to its eukaryotic counterpart it plays an essential role in bacterial organization. Before
discussing specific examples of bacterial organization, some
general principles underlying the generation of spatiotemporal order will first be presented.
7.13.2
Organization Principles
The mechanisms underlying the spatiotemporal organization
of proteins in cells can generally be separated into two classes,
depending on whether or not they exploit external cues.6 In this
context, an external cue does not necessarily refer to a signal
coming from outside of the bacterium, but rather to any entity
that determines the localization of the protein under investigation. Such entities could be other proteins. For example, a
ring built from the protein FtsZ, called the Z-ring, forms the
scaffold for all proteins that will assemble into the divisome,
the structure that drives cell division by generating the formation of a new cell wall, which ultimately leads to two daughter
cells. As another example, the dynamic distribution of MinC in
Escherichia coli is imposed by the distribution of MinD. As a
different kind of cue, membrane curvature has long been
thought to play a role in bacterial organization. Membrane
curvature could be used to localize proteins, for example, to the
poles of rod-shaped cells. Indeed, there is now compelling
evidence that some proteins assemble preferentially on membranes with a specific curvature.7 Also the lipid composition of
the cytoplasmic membrane, which in turn might depend on
membrane curvature,8,9 could be exploited as an external cue
to localize proteins. As a final example, let us mention active
directed transport, which might play a minor role in prokaryotes, but is heavily used in eukaryotes, where it relies on
external cues provided by the microtubule network (see Chapter
4.15).
With exception of the last example, the protein distributions result in the presence of external cues from a diffuse-andcapture mechanism; proteins move randomly in the cytoplasm or on the membrane until they hit an appropriate target
to which they adhere. Due to the comparatively large diffusion
constants of molecules in the bacterial cytoplasm,2,3 which
makes them explore the whole cell volume within seconds or
faster, this mechanism is very efficient. However, it relies on
pre-existing structures and the obvious following question is:
209
How do these structures themselves form in the right places at
the right time?
Mechanisms underlying the emergence of structures in
absence of external cues can again be divided into two classes.
Self-assembled structures are equilibrium structures, while
self-organized structures require a constant flux of energy
and/or matter to persist. As a non-biological example of
self-assembly, consider the formation of salt crystals. As a
contrasting example, the spontaneous emergence of convection rolls in water heated from below is due to self-organization; the rolls vanish if the heating is switched off and no
energy is put into the system.10 Both self-assembly and selforganization are also present in bacteria. For example, receptor
clusters on membranes can result from self-assembly (In spite
of the article’s title, the mechanism underlying formation
of membrane clusters of chemotactic receptors proposed in
Greenfield1 is really based on self-assembly of the receptors:
No energy or matter flux is required for maintaining the
clusters.), whereas the Min oscillations (see below) are clearly
self-organized patterns.11 Physics has developed an impressive
armada of tools and concepts to analyze self-assembled
and self-organized patterns,10 which can all be employed to
understand bacterial organization.
The distinction between self-organization and -assembly
on one hand, and organization due to external cues on the
other may not always be as clear cut as the previous paragraphs might suggest. This is due to numerous feedbacks
that exist between various components. We consider selforganization to be a powerful and adequate concept if the
structure at hand results from the interaction of a small
number of components. This is the case for the Min oscillations in E. coli. Other examples exist, and for eukaryotes this
concept has also been successfully applied to explain cellular
structures.12,13 As soon as the number of components
becomes too large, though, the insight gained by classifying a
pattern as self-organized might be limited, even if correct.
Think, for example, of the Z-ring that is influenced by a rather
large number of regulatory proteins, which themselves are
localized to the Z-ring by diffusion and capture.14 In such a
case, one can either focus on a subsystem consisting of few
different proteins, or it might pay to ‘‘coarse grain’’ the system
and to consider interactions between a small number functional entities rather than between individual proteins. Such
entities could capture essential features of single proteins or
of protein complexes. Depending on the context, it is thus
sometimes useful to emphasize the aspect of self-organization
or -assembly, and sometimes to point out external cues.
There are a few common structural ‘‘motifs’’ that result
from these mechanisms. Protein gradients are widely employed to spatially structure multicellular organisms15 and
eukaryotic cells, for example, during cell division,16 but are
also present in bacteria. In developing organisms, they often
emerge from localized protein synthesis, diffusion away from
their source, and degradation. In single cells, gradients are
rather formed by proteins in a specific phosphorylation state.
Gradients provide a nice example of structures that can form
by external cues (although they do not have to) and that
themselves serve in turn as external cues in the formation of
other structures. In rod-shaped bacteria, such gradients can
take a special form with the proteins being accumulated at the
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cell poles. Another common motif is given by filamentous
structures. These can either be straight, helical, or they can
form a closed ring around the cytoplasm. The spectrum of
temporal structural motives is much smaller and in essence
limited to oscillations.
In the following, these general motifs will be illustrated by
particular examples. As mentioned before, the focus will be on
protein structures and how they organize bacterial DNA,
organelles, and other proteins. However, also the distribution
and organization of the chromosomal DNA contributes to
the internal organization of bacteria. By presenting specific
binding sites and possibly through a process termed ‘‘transertion’’17,18 it can strongly influence the distribution of proteins. On several occasions, we will briefly touch on this
subject. The mechanical aspects of bacterial organization
will only play a minor role in the following discussion, even
though they might be very important. They are discussed in
more detail in (see Chapter 7.6) on bacterial morphology.
Before discussing these examples, a few comments on the
role of theory in our endeavor to reveal the mechanisms of
bacterial structure formation seem in order.
7.13.2.1
Mechanisms vs. Models
Patterns resulting from the presence of external cues are relatively easy to grasp and their understanding does usually not
require an elaborate conceptual physical basis. While experimental biophysical methods (FRET, PALM etc.) might make
essential contributions to elucidate such mechanisms, theoretical insights are expected to be minor in such a case. This
is utterly different for mechanisms involving self-assembly
or self-organization! These mechanisms rely on interactions
between a large number of proteins.12,13 Consequently, a
quantitative understanding cannot be achieved without the
concepts of Statistical Mechanics and Non-linear Dynamics.
Even on a qualitative level it is often difficult to see whether a
proposed mechanism can result in the desired structure
without invoking some kind of formal analysis.
In this context, one should not confuse the computational
models that are used to investigate mechanisms with the physical basis of these mechanisms. The models can take various
appearances, depending on the aspects of the problem they
are emphasizing. Sometimes so-called meanfield models in the
form of partial differential equations will provide an appropriate
framework, on other occasions stochastic models might be
preferable.19 As a consequence it often makes no sense to quarrel
about different models that represent the same mechanism.
In the following, the focus will be on those mechanisms
that are interesting from a biophysical point of view, although
other mechanisms will be mentioned, too.
7.13.3
The Bacterial Cytoskeleton
While the cytoskeleton was once thought to be a defining
feature of eukaryotic cells, we now know that all eukaryotic
cytoskeletal proteins have one or more bacterial analogs,4 see
Figure 1. Similar to its role in the eukaryotic cell, the
cytoskeleton is an important factor for determining bacterial
organization.
7.13.3.1
Prokaryotic Cytoskeletal Proteins
The first prokaryotic cytoskeletal protein to be identified was
FtsZ,20,21 a homolog of the eukaryotic tubulin. In spite of their
weak sequence similarity, FtsZ and tubulin bear a high structural similarity.21,22 Another, more recently discovered, similar
protein is TubZ.23 Prokaryotic homologs of eukaryotic actin
include MreB, FtsA, ParM, and MamK.4 Crescentin was the
first prokaryotic intermediate filament to be discovered.24 It is
a coiled coil rich protein and can form filaments. Other filament forming coiled coil rich proteins are FilP and AglZ. A
further family of bacterial cytoskeletal proteins is formed by
the Walker Box ATPases ParA and ParF, and the deviant
Walker-type ATPase MinD.25
All the above proteins are capable of forming filamentous
structures. These filaments can be straight like MamK,26 wind
as helices around the cytoplasm like MreB,27–29 or form rings
like FtsZ.20 These forms are not mutually exclusive. For
example, FtsZ can switch between an arrangement in a ring
and in a helix.30,31 With the exception of the intermediate
filament-like proteins, all other prokaryotic cytoskeletal proteins are ATPases or GTPases. In a cellular environment, these
filaments are thus kept out of equilibrium. Often they present
a high turnover with rates up to 0.1/s. Some of these filaments
(e.g., TubZ and MreB), have been reported to show treadmilling,23,32 that is, assembly at one and disassembly at the
other end. This dynamic behavior requires a structural polarity
of the filament. Other filaments show a behavior similar to the
dynamic instability of microtubules as they present alternating
phases of shrinkage and of growth at both ends.33
The bacterial cytoskeleton is employed in various processes. First of all, it determines cell morphology. For example,
the rod shape of E. coli or of C. crescentus depends on the
presence of MreB. Destruction of MreB filaments by the drug
A22 results in aberrantly large round cells.34 In C. crescentus,
MreB, furthermore, determines the polarity,35 while Cresentin
accounts for the curved (crescent) shape of the bacterium.24
A second major function lies in the proper distribution
and localization of cellular components: Magnetosomes that
endow magnetotactic bacteria with the ability to sense external
magnetic fields are aligned by MamK filaments,26 low copy
number plasmids are distributed evenly on the daughter cells
by ParA or ParM,36 and a ring formed by FtsZ recruits the
division inery to the future site of cell division14 In Myxococcus
xanthus, AglZ is involved in cell motility.37
Cytoskeletal proteins are thus of broad importance for
bacteria, in determining their structural and dynamical properties. In contrast to eukaryotic tubulin or actin, most prokaryotic cytoskeletal proteins can assemble spontaneously
into filaments.38 Still, their dynamics can be controlled by
auxiliary proteins. In the following, the formation of cytoskeletal structures in bacteria will be discussed.
7.13.3.2
Helices and Rings
As mentioned above, cytoskeletal filaments can form rings or
helices; see Figure 2. In Bacillus subtilis, but also in Escherichia coli,
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Bacterial Organization in Space and Time
-tubulin
GDP
GTP
+
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GDP
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FtsZ
GTP
GTP
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-tubulin
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ParM
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Figure 1 Structure of bacterial cytoskeletal proteins and their eukaryotic homologs. (a) Structure of the a/b tubulin heterodimer (left) and the
FtsZ dimer (right). (b) Structure of actin filaments (F-actin), MreB- and ParM-filaments. The arrow indicates the direction of the filament
orientations. From Michie, K. A.; Lowe. J. Annu. Rev. Biochem. 2006, 75, 467–492. Copyright by Annual Review.
FtsZ is observed to switch between annular and helical
arrangements; helices can grow out from existing rings.30,31 In
B. subtilis, under starvation conditions, the helix will collapse
at another location and there induce spore formation. In C.
crescentus, MreB forms a helix that determines cell polarity.35
Prior to cell division, the helix collapses into a ring and then
reappears in the two future daughter cells. In E. coli, MreB
rings and helices can coexist.39
A mechanism based on the growth of filaments states that
the configuration of a filament is completely determined by
the orientation of a filament nucleus from which it grows
straight.40 However, this approach neglects any mechanical
contributions, but turns the question into what determines the
orientation of the nucleus and can thus not provide a completely satisfying answer.
The arrangements of the filaments can be understood
purely on mechanical grounds, apart from their assembly and
disassembly dynamics. Accounting for a possible spontaneous
curvature of the filaments, their bending stiffness, and possible
anisotropies in the interaction between the filaments and the
membrane, the filaments can form rings and helices on curved
membranes.41 Which form is then adopted depends on the
values of the spontaneous curvatures and the spontaneous
twist. These values can be affected by the state of the nucleotide-binding site. For example, FtsZ curls when bound to GDP
and is straight when bound to GTP.
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2
1
3
4
(a)
1
2
3
4
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(b)
Figure 2 Examples of rings and helices formed by cytoskeletal
proteins in rod-shaped bacteria. (a) FtsZ rings and helices in B.
subtilis. From Ben-Yehuda, S.; Losick, R. Asymmetric cell division in
B-subtilis involves a spiral-like intermediate of the cytokinetic protein
FtsZ. Cell 2002, 109, 257–266. (b) MreB rings and helices in E. coli.
From Shih, Y.; Le, T.; Rothfield, L. Division site selection in
Escherichia coli involves dynamic redistribution of Min proteins
within coiled structures that extend between the two cell poles. Proc.
Natl. Acad. Sci. USA 2003, 100, 7865–7870. Copyright by PNAS.
An alternative mechanism, motivated by the apparent lack
of spontaneous curvature of MreB, is based on constrain forces
resulting from filament-membrane interactions.42 Such forces
could result from filament polymerization or from other forcegenerating entities. If these forces are located at the filament
ends, rings and straight lines can become unstable and result
in a helix. Which of these mechanisms – if any – is responsible
for helix formation in rod-shaped bacteria is currently
unknown. Further experimental input is certainly necessary to
improve our understanding.
on the actin-like protein ParM, a centromere-like sequence on
the plasmid denoted parS, and the protein ParR binding to
parS.36 ParM forms a filament extending from the parS locus
on one copy of one plasmid to the corresponding locus on the
other plasmid. ParR then induces hydrolysis of the ATP bound
to ParM, which results in unbinding of the filament from the
plasmid, thereby allowing a new ATP-bound ParM molecule
to attach to the filament and the plasmid. In this way, ParR
stimulates growth of ParM filaments such that the two plasmids are pushed to opposite ends of the cell. This mechanism
has been shown to work by an in vitro reconstruction containing ParM, ParR, and parS containing plasmids.43
There is an alternative mechanism that assures an equal
distribution of low copy number plasmids in the daughter
cells: Throughout the whole cell cycle, some plasmids are
evenly distributed along rod-shaped bacteria in a manner that
depends on ParA.44 The Walker Box ATPase ParA is a bacterial
cytoskeletal protein without known homolog in eukaryotes. It
is contained in the type I partitioning system, which in addition encodes for ParB, a DNA binding protein, and parC, the
binding site of ParB on the DNA. ParA dimers bind cooperatively and nonspecifically to DNA. ParA filaments polymerize
until they contact ParB, which itself may be bound or not to
parC, stimulating hydrolysis of the ATP bound to ParA.44 In
striking contrast to ParM, the ParA filament will not transiently detach from the plasmid and grow further. Instead it
will shrink and thereby pull on the plasmid. A theoretical
analysis has shown that this process will lead to an even distribution of plasmids if the rate at which the plasmid detaches
from the ParA filament depends on the current filament length
such that it is smaller for longer than for shorter filaments.45
An alternative mechanism has been proposed that assigns an
important role to the nucleoid in distributing ParA and thus
the plasmids,46 see below.
7.13.3.4
7.13.3.3
Induced Assembly and Disassembly of
Cytoskeletal Filaments
It has already been mentioned that, in contrast to tubulin or
actin, there is no kinetic barrier for forming bacterial cytoskeletal filaments. They can thus form anywhere in the cell.
As in eukaryotes and as mentioned above, the assembly and
disassembly of prokaryotic cytoskeletal filaments can nevertheless be regulated by other proteins. Such interactions can
be used to position cytoskeletal structures or to control their
effects on other cellular components. Some of these proteins
are themselves distributed in structures independent of the
cytoskeleton and act as proper external cues. Examples are the
proteins MinC or SlmA that affect the position of the Z-ring
(see below). In other cases, however, there is a feedback
between their distribution and the cytoskeleton, opening the
possibility for self-organization. The positioning of plasmids
mediated by the bacterial cytoskeleton will serve to illustrate
such processes.
Plasmids are short circular strands of DNA. Some of them
exist in very low copy numbers and active processes have
evolved to guarantee an even distribution of such plasmids
onto the daughter cells.36 The type II partitioning system relies
Spatial Distribution of Magnetosomes and
Carboxysomes
Similar to the plasmids carrying the type I partition complex
there are other macromolecular complexes that are evenly
distributed along the long axis of rod-shaped bacteria.
Two striking examples are provided by carboxysomes and
magnetosomes; see Figure 3. Carboxysomes are organelles of
cyanobacteria with a proteinaceous shell that sequester
enzymes involved in carbon fixation.47 Within the rod-shaped
bacterium Synechococcus elongatus, carboxysomes are evenly
spaced in a ParA-dependent manner.48 Whether the underlying mechanism is the same as for the even distribution
of plasmids carrying the type I partitioning systems is as
yet unknown. Magnetososmes are membraneous organelles
of magnetotactic bacteria.49 They contain magnetite, and are
surrounded by a membrane that is continuous with the
cytoplasmic membrane. In one cell, there are about 15–20
magnetosomes each containing a magnetite crystal of about
50 nm in size. Similar to carboxysomes, magnetosomes
form chains that are necessary for efficient detection of
an external magnetic field. Chain formation depends on
another actin-like cytoskeletal protein, MamK, a homolog of
MreB.26 As for carboxysomes, the mechanism behind the
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RbcL-CFP
(a)
(b)
Figure 3 Alignment of bacterial organelles. (a) Linear arrangement of carboxysomes. From Savage, D. F.; Afonso, B.; Chen, A. H.; Silver, P. A.
Spatially ordered dynamics of the bacterial carbon fixation machinery. Science 2010, 327, 1258–1261. Copyright by AAAS. (b) Localization of
magnetosomes (A, B) and schematic representation of MamK filaments (C). From Komeili, A.; Li, Z.; Newman, D.; Jensen, G. Magnetosomes are
cell membrane invaginations organized by the actin-like protein MamK. Science 2006, 311, 242–245. Copyright by AAAS.
cytoskeleton-dependent regular distributions of magnetosomes is still unidentified.
7.13.4
Positioning The Z-Ring
It was mentioned before that the Z-ring, a structure formed by
FtsZ filaments, provides the scaffold for the proteins that make
up the divisome. Correct positioning of the Z-ring is a vital
task for bacteria, because it assures the even distribution of
one copy of the chromosome onto each daughter cell. In
contrast to animal cells, where the cytoskeleton plays an active
part in positioning the contractile actin ring that will eventually cleave the cell, the position of the Z-ring is essentially
determined by external cues. In many rod-shaped bacteria,
two mechanisms cooperate to this end.14 One mechanism is
called nucleoid occlusion and prevents formation of the Z-ring
and thereby division over the nucleoids. A nucleoid is the
bacterial equivalent of a eukaryote’s nucleus, that is, an
intracellular region occupied by the bacterial chromosome
(unlike the nucleus of eukaryotes, it is not delimited by a
membrane). The physiological benefit is obvious; nucleoid
occlusion prevents the chromosome being cut into pieces by
the growing septum.
A second mechanism is then necessary to select the region
between the two segregated copies of the chromosome as the
site of cell division. Otherwise, one daughter cell would
remain without a chromosome. Different mechanisms have
evolved to this end, however, all those that we know of act
through inhibition of Z-ring formation at unwanted sites
rather than promoting it at the future division site. In E. coli
and Bacillus subtilis, the negative regulator of Z-ring assembly is
MinC. In B. subtilis it is localized in a stationary manner to the
cell poles, while in E. coli its localization alternates between
the two poles. In both species, though, the localization
mechanism relies on the protein MinD. A rather different
mechanism seems to operate in C. crescentus that apparently
lacks nucleoid occlusion. There, the protein MipZ forms a
static gradient with maxima at the two cell poles. MipZ directly
inhibits Z-ring formation and thus directs formation of the
divisome to the cell center.
From a biophysical perspective, the mechanisms underlying the appropriate distribution of MinC are probably the
most interesting ones. They will be discussed in detail below
as they exemplify protein self-organization as well as the role
of geometrical cues for bacterial organization. Before this,
however, some words on nucleoid occlusion and the distribution of MipZ are in order.
In E. coli and B. subtilis, nucleoid occlusion depends on
DNA-binding proteins that are able to destabilize FtsZ filaments.50,51 Interestingly, these proteins, SlmA in E. coli and
Noc in B. subtilis, seem to be unrelated although they ultimately act in very similar ways. SlmA interacts with specific
binding sites on the bacterial chromosome and was suggested
to sever FtsZ filaments by inducing hydrolysis of GTP bound
to FtsZ.52 Its effect on FtsZ is enhanced many times by binding
to DNA, such that SlmA floating freely in the cytoplasm does
not destroy FtsZ structures. Similarly, for Noc, specific binding
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sites on the chromosome have been identified.53 How
Noc mediates Z-ring disassembly is currently not known. Both
SlmA and Noc have been suggested as playing a role in
coordinating chromosome segregation and cell division
(see below).
Similarly to ParA and MinD, MipZ is a Walker-type ATPase
which associates via ParB to the chromosome of C. crescentus.54 Binding occurs only in the vicinity of the replication
origin of the chromosome. During segregation, the duplicated
origin is localized in an MreB-dependent way to the cell poles.
There, it is possible that MipZ filaments originate and thus
establish a gradient.
7.13.4.1
The Min Oscillations
In E. coli the Min system consists of three poteins, namely
MinC, an inhibitor of FtsZ filament assembly, MinD, a deviant
Walker-type ATPase, and MinE, a protein capable of enhancing
the ATPase activity of MinD.55 Microscopy of fluorescently
labeled Min proteins in living cells has revealed a remarkable
spatiotemporal pattern of the Min-protein distribution: The
proteins localize for about half a minute in one cell half, then
switch rapidly to the opposite cell half, where they reside
again for about half a minute, switch back and so forth, 56,57
see Figure 4(a). In this way, the Min system directs Z-ring
assembly with an accuracy of a few percent to the middle of
the bacterial long axis resulting in two equally sized daughter
cells after division.58 Several lines of evidence suggest that
these so-called Min oscillations emerge spontaneously from
interactions of MinD and MinE with the cytoplasmic membrane and do not require any additional cue.59 Also, MinC is
not involved in the generation of the oscillations.
7.13.4.1.1
Biochemistry of the proteins MinD and MinE
Let us start by presenting some biochemical properties of
the Min proteins,55 as illustrated in Figure 4(b). In absence
of ATP, MinD is cytoplasmic. After binding ATP, it forms
an amphipathic helix through which it can associate with
the cytoplasmic membrane. Due to its low ATPase activity, in
cells lacking MinE, most MinD will locate to the cytoplasmic
membrane. On membranes, MinD can form aggregates,
which take the form of tight helices on lipid vesicles.60 These
helices have a pitch of about 6 nm (comparable to the long
axis of a MinD molecule) and induce the formation of
lipid tubes with a diameter of 50–100 nm. Their appearance
requires the amount of MinD to exceed a critical concentration and most probably a conformational change of membrane-bound MinD that depends on ATP hydrolysis: In
the presence of ATPgS, a non-hydrolysable analog of ATP,
MinD still binds to lipid vesicles, but does not form helices.
MinD has also been observed to form filaments in vitro,
however, under unphysiological salt concentrations. The
structure of membrane-bound MinD aggregates in vivo is
currently not known.
MinE can bind to membrane-bound MinD, where it
competes with MinC for overlapping binding sites, and significantly increases its ATPase activity. Thereby, MinE can
induce detachment of MinD from the membrane. It was
shown in vitro that MinE can act processively, that is, it can
remove several MinD molecules from the membrane before
detaching itself.61 While the molecular mechanism for processive detachment is not yet understood, it likely involves
transient MinE binding to the cytoplasmic membrane.62
In vivo it forms a marked structure at the boundary of a MinD
aggregate, which is known as the MinE ring.
7.13.4.1.2
Min oscillations result from MinD-MinE selforganization in presence of a membrane
A number of mechanisms have been proposed to explain
the dynamic behavior of the Min-protein distribution
in vivo as a consequence of Min-protein self-organization
in the absence of external cues.11 Compelling evidence supporting the ideas have been provided by an in vitro assay
consisting of a supported lipid bilayer and a buffer essentially
containing MinD, MinE, and ATP,63 shown in Figure 4(c). In
this setup, the Min proteins have been found to self-organize
into traveling planar and spiral waves (see Figure 4(d)).
Theoretical analysis has shown that the same mechanism
underlying the patterns observed in vitro can also explain the
patterns observed in vivo if one assumes a reduced diffusion
constant of membrane-bound MinD in vivo compared to
in vitro.
While this indicates the possibility of generating Min
oscillations by protein self-organization, other cues might still
be important for producing the pattern in vivo. Several
observations argue otherwise, though. First of all, the Minprotein pattern displays an intrinsic characteristic length: in
filamentous cells the oscillating Min pattern has several nodes
regularly separated from each other by a distance of about
3 mm.56 No structural unit has so far been associated with
these nodes. Indeed, while the cells grow, transitions between
standing waves as described above and traveling waves can be
observed as the number of nodes is increased. Furthermore, in
sufficiently short cells, the Min-protein distribution does not
oscillate.64 Instead, the proteins accumulate in one cell half
and switch only stochastically to the other half. Only after a
certain critical length has been exceeded will the Min oscillations start. The critical length depends on the expression level
of the Min proteins such that this regime does presumably not
exist in wild-type bacteria.65
While the oscillatory behavior might be unique to the
Min proteins, there is a system in B. subtilis that shows
similar stochastic switching. It consists of the proteins Spo0J
and Soj, which are involved in chromosome segregation
and transcriptional regulation. The Soj proteins switch
stochastically between the two cell poles66 or jump from
nucleoid to nucleoid.67 Similarly to the Min oscillations,
this process might result from a dynamic instability where
Soj plays a role similar to MinD while Spo0J takes the part
analogous to MinE.68 However, rather than binding to Soj,
Spo0J switches between a condensed and an uncondensed
form, where condensation is triggered by the presence of
Soj and where the inverse process occurs spontaneously.
Condensed Spo0J stimulates Soj’s ATPase activity. Theoretical
analysis of this mechanism has revealed stochastic switching,
but also the possibility of regular oscillations.68 A thorough
experimental test of this prediction has not been attempted
so far.
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Bacterial Organization in Space and Time
215
MinE ring
MinD
MinE
+ATP/−ADP
x
2
Polar cap of
MinD and MinC
t
1
3
4b
5
–P
4a
MinD-ADP
MinC
MinD-ATP
MinE
(c)
FtsZ ring
1 min
Intensity (a.u.)
2 µm
0
MinC concentration gradient
Nucleoid occlusion
1
(b)
Intensity (a.u.)
(a)
0.5
Cell length
MinD
MinE
0
(e)
20
40
60
80
Distance (µm)
50 µm
(d)
Figure 4 Min oscillations in E. coli. (a) Kymograph of fluorescently labeled MinD and time-averaged distribution. (b) Schematic illustration of
Z-ring positioning by the Min proteins and nucleoid occlusion. (c) Illustration of the reaction kinetics of MinD and MinE. (d) Fluorescenc profiles
of MinD (green) and MinE (red) on a supported lipid bilayer. The structure moves in the direction indicated by the arrow. (e) Fluorescence
intensity profiles of MinD and MinE in a wave. From Loose, M.; Kruse, K.; Schwille, P. Protein self-organization: lessons from the min system.
Annu. Rev. Biophys. 2011, 40, 315–336. Copyright by Annual Review.
7.13.4.1.3
Cooperativity is needed for spontaneous
emergence of Min oscillations
A crucial ingredient to all mechanisms that have been proposed to explain the Min oscillations without reference to
external cues is a reduced diffusion of membrane bound
proteins compared to cytoplasmic proteins. Different diffusion constants are indeed essential for pattern formation in
chemical systems, as proposed by A. Turing in his seminal
paper on the chemical basis of morphogenesis.69 The introduction of membranes that on one hand reduce the diffusion
constant and on the other hand reduce the dimensionality of
the space the molecules reside in presents a new feature for
reaction-diffusion systems. Their consequences are by far not
yet fully explored.
Another essential ingredient for all proposed mechanisms
underlying the Min oscillations is the existence of some sort of
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Bacterial Organization in Space and Time
cooperativity among the MinD molecules. It could manifest
itself in cooperative binding of MinD to the membrane, or in
the formation of MinD aggregates on the membrane by a
mechanism reminiscent of nucleation; small clusters grow by
incorporating further molecules diffusing on the membrane.11
In spite of in vitro experiments suggesting a two-step
mechanism for forming membrane tubes induced by MinD,
whereby by first MinD attaches to the membrane and then
aggregates,60 the former seems to have more explanatory
power than the latter. A careful analysis of the wave profiles
on supported lipid bilayers suggests further sources of cooperativity,61 see Figures 4(d) and 4(e). Attachment of MinE to
membrane-bound MinD occurs initially at a constant rate – as
is revealed by the linear increase of the MinE concentration as
a function of time. As a critical concentration is passed,
however, the MinE profile becomes highly non-linear with a
sharp maximum at a wave’s trailing edge, which is probably
the in vitro analogue of the MinE ring in vivo. This sharp
increase could be due to cooperative binding of MinE. However, as mentioned before, MinE presumably removes MinD
processively from the membrane. It has been argued that such
an effect can explain the formation of the MinE ring.70,71 This
is similar in spirit to the accumulation of the depolymerizing,
microtubule-associated, molecular motor Kin-13.72
To end this section let us give an intuitive explanation of
one possible mechanism underlying the spontaneous emergence of the Min oscillations. Assume that all MinD reside in
one cell half. Therefore, MinE will also be recruited to this half
and start to remove MinD from the membrane. Due to the
overwhelming presence of MinD in that half, most of the
detached MinD will quickly rebind here. There will be a leak,
though, through which some MinD will escape to the adjacent
cell half. At some point, the system will topple over; due to the
presence of MinE in the original cell half, more MinD will be
removed from the membrane than added, while it can freely
attach in the adjacent half. At some point also MinE will
detach and after some time bind to the MinD, which is now
bound to the membrane in the cell half originally devoid of
MinD. At this point the process starts anew. Let us emphasize
again, that this intuitive explanation can only be stated after
the fact. Without a detailed computational analysis it cannot
be clear whether this mechanism will really bring the desired
effect or not.
7.13.4.2
Polar Localization of MinD/DivIVA in B. subtilis
As already mentioned above, the distribution of MinD and
hence of MinC is stationary in B. subtilis. There, the concentration of MinD is maximal at both poles simultaneously
and decays towards the cell center. Furthermore, MinD accumulates at the in-growing septum. The localization of MinD
depends on the protein DivIVA, which, similarly to MinE,
affects the stability of MinD on the membrane. These two
proteins are unrelated, though, and the effect of DivIVA is
opposite to that of MinE, since it stabilizes membrane-bound
MinD. In principle, the distribution of MinD could again
result from a dynamic instability of the homogenous distribution induced by DivIVA. In this case one would expect a
characteristic wavelength to be associated with the MinD
distribution. The existence of a characteristic wavelength is,
however, incompatible with the accumulation of MinD at
nascent cell poles and its absence from the cell center in filamentous cells.73
An early theoretical work explaining polar localization of
MinD invoked geometrical cues in terms of membrane curvature.74 The idea was that MinD has a reduced affinity for
binding to curved membranes and is thus depleted at the cell
poles. According to this proposal, DivIVA furthermore binds
preferentially to the rim of MinD regions, such that it accumulates at the cell poles or mature septa. Consequently, MinD
would be stabilized and accumulate in these regions in spite of
its low affinity for curved membranes. More recent observations, however, indicate that DivIVA itself senses membrane
curvature; simplifying the picture considerably.75,76
7.13.5
Polar Protein Localization
It is common in rod-shaped cells that proteins accumulate at
the cell poles, as illustrated in Figure 5. As we have just seen,
one example is DivIVA in B. subtilis, but other examples are
legion. The mechanism underlying DivIVA localization is
particularly simple, in that this protein directly reads out a
geometrical cue, namely, (negative) membrane curvature. Also
SpoVM, a small protein that recruits other proteins to newly
forming spores in B. subtilis, has been found to locate preferentially to regions of a specific curvature.77 In contrast to
DivIVA, though, SpoVM recognizes positive curvature.
A different but conceptually similarly simple mechanism
relies on cues associated with remnants from previous divisomes that are left behind at newly created poles after division. An example of such a protein is provided by TipN in C.
crescentus.78,79 Understanding these two mechanisms does not
require biophysical analysis, but rather a detailed knowledge
of the structural elements of these proteins that mediate curvature recognition. There is, however, a third mechanism for
polar localization that requires cooperative effects.
In E. coli, chemoreceptors cluster at the cell poles.80 These
clusters help to increase the bacterial sensitivity to changes in
chemo-attractant or -repellant concentrations. It is known that
chemoreceptors can form clusters anywhere on the membrane. High-resolution fluorescence microscopy has shown
that the spatial distribution of such clusters correlates with
their size; the largest clusters are found at the poles, and their
size decreases as their location becomes closer to the cell
center.1 The generation of this distribution does not need
external cues, but can result from a simple nucleation process:
Proteins attach to the membrane on which they diffuse81. As
receptors meet, attractive interactions glue them together. As
soon as a cluster exceeds a critical size, it becomes stable and
will grow by incorporation of further receptor proteins. In an
unbound system, this mechanism can lead to a periodic
arrangement of clusters. In the confined geometry of an E. coli
cell of wild-type length, it can lead to the observed localization
of the largest clusters at the cell poles. As the reader
might have noticed, such a nucleation mechanism operating
on MinD is also at the heart of one of the proposed
mechanisms explaining the spontaneous emergence of the
Min oscillations.70
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Bacterial Organization in Space and Time
Finally, the distribution of DNA is able to mediate protein
localization at the poles. We will now briefly touch upon some
aspects of chromosomal organization in bacterial cells and
then discuss this possibility.
7.13.6
Chromosome segregation and protein
distributions
As has already become clear, the distribution of certain binding
sites on the bacterial chromosome can serve as an external cue
to organize the distribution of proteins. The mechanism behind
A
217
the segregation of the two copies of a chromosome can have a
strong influence on the internal organization of the chromosome. In this section, we will discuss mechanism behind the
segregation of the two copies of a chromosome can have a
strong influence on the internal organization of the cell.82 At
the time when the existence of a bacterial cytoskeleton was
ignored, Jacob and coworkers proposed that for rod-shape
bacteria, the replication origins of the duplicating chromosome
attach to the cell poles.83 Elongation of the cell would then lead
to forces on the chromosomes that would pull them apart.
Experimentally this view has been challenged using fluorescence microscopy, revealing that the replication origins in B.
subtilis and C. crescentus separate 10 times faster than these cells
elongate.34 Alternative mechanisms invoke entropic effects,
ascribe an important role to cytoskeletal filaments, or postulate
that the chromosome is indeed linked to the growing cell wall.
7.13.6.1
Entropic chromosome segregation
A purely passive mechanism was proposed by Jun and Mulder,
based on the observation that highly confined polymers segregate due to entropy.84 In fact, the free energy of two overlapping polymers consisting of N monomers confined to a
tube of diameter D scales as FBD1/nNkBT, where kBT is the
thermal energy and n ¼ 3/5 is the Flory constant defined
through RgBNn, where Rg is the radius of gyration. This
indicates a very strong repulsion between the two polymers
that increases linearly with chain length. Monte Carlo simulations of two overlapping circular polymers in a bacterial
geometry confirm the entropic segregation suggested by the
scaling analysis. Adding the dynamics of DNA replication, the
positions of the replication origins can be tracked. The simulations are in qualitative agreement with experimental data.
B
(a)
Time
DIC
7.13.6.2
Active Chromosome Segregation
TipN-GFP
Similar to plasmid segregation, the cytoskeleton has also been
proposed to be involved in chromosome segregation. Similar
to the suggestion by Jacob et al. mentioned above, the
majority of the mechanisms involve site-specific sequestration
of DNA. In C. crescentus, segregation of the chromosomal
region close to the replication origin involves MreB.34 The
Old
New
TipN-GFP
New
Old
(b)
Old
epi-PALM
(c)
Figure 5 Polar localization of proteins in rod-shaped bacteria. (a)
Distribution of DivIVA in wild-type (A) and mutant (B) E. coli. The
mutant is unable to fission daughter cells after septum closure. In
addition to the poles, DivIVA localizes to the curved regions of the
newly formed septa. From Lenarcic, R.; Halbedel, S.; Visser, L.;
Shaw, M.; Wu, L. J.; Errington, J.; Marenduzzo, D.; Hamoen, L. W.
Localisation of DivIVA by targeting to negatively curved membranes.
Embo. J. 2009, 28, 2272–2282. Copyright by Nature. (b) Localization
of TlpN in dividing C. crescentus. After initial localization to the
(younger) pole opposite to the stalk, it shifts to the nascent pole.
From Lam, H.; Schofield, W. B.; Jacobs-Wagner, C. Landmark protein
essential for establishing and perpetuating the polarity of a bacterial
cell. Cell 2006, 124, 1011–1023. (c) Distribution of chemotactic
receptors in E. coli. From Greenfield, D.; McEvoy, A. L.; Shroff, H.;
Crooks, G. E.; Wingreen, N. S.; Betzig, E.; Liphardt, J. Selforganization of the Escherichia coli chemotaxis network imaged with
super-resolution light microscopy. Plos. Biol. 2009, 7, e1000137.
Copyright by PLoS One.
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Bacterial Organization in Space and Time
molecular details of this mechanism remain to be elucidated.
A possibility is that treadmilling MreB filaments that are
associated with the cytoplasmic membrane push the chromosomes apart.85
An interesting variant of this mechanism is the following:
When genes of membrane proteins are transcribed, the nascent mRNA can immediately be translated into the corresponding amino acid sequence, which in turn inserts itself
immediately into the cytoplasmic membrane/cell wall and
thereby links the DNA mechanically to the wall.17,18 This
process is called transertion. The compacted nucleoid is elastically linked along its whole extension to the elongating cell
wall, and thus a force is generated on the replicating nucleoid
that might pull the two copies apart. On the other hand, it
20
24
28
32
36
generates stress in the cell wall. This stress has a minimum in
the cell center. It has been speculated that this stress distribution might be read by mechanosensitive proteins, and
help to select the cell center as the site of Z-ring assembly.86
One consequence of active segregation that relies on specific DNA binding sites is a particular distribution of these
binding sites. This distribution can be employed as an external
cue to organize the bacterium.6
7.13.7
Temporal Organization
Many of the spatial patterns discussed above appear in connection with cell division. For a cell, it is a vital point to decide
min
80
Total
T-KaiC
ST-KaiC
S-KaiC
ori
% KaiC
60
MipZ-YFP
40
20
0
Overlay
0
20
(b)
40
60
Time (h)
80
(a)
B
U
KaiA
80
ST
60
% KaiC
T
KaiB
A
Total
T-KaiC
ST-KaiC
S-KaiC
40
S
0
kXY (S) = kXY
+
A
A(S)
kXY
k 1/2 + A(S)
A(S) = max {0, [KaiA] − 2S}
(c)
20
0
0
20
40
60
Time (h)
80
Figure 6 Examples of temporal regulation in bacteria. (a) Entry into cell division in C. crescentus mediated by MipZ. MipZ co-localizes with the
replication origins. The Z-ring can form and induce division as soon as the regions of high MipZ-concentration have sufficiently departed from
each other. From Thanbichler, M.; Shapiro, L. Getting organized – how bacterial cells move proteins and DNA. Nat. Rev. Microbiol. 2008, 6,
28–40. Copyright by Nature. (b) Periodic change in the phosphorylation level of the protein KaiA from the circadian clock formed by KaiA-C. (c)
Wiring diagram indicating the mutual effects of the Kai proteins on each other (A) and numerical solution of a corresponding computational
model (B). (b) and (c) from Rust, M. J.; Markson, J. S.; Lane, W. S.; Fisher, D. S.; O’Shea, E. K. Ordered phosphorylation governs oscillation of
a three-protein circadian clock. Science 2007, 318, 809–812. Copyright by AAAS.
Author's personal copy
Bacterial Organization in Space and Time
on the time she enters into cytokinesis. Hardly surprisingly, a
number of propositions have been made which link the spatial patterns to check points for entering cytokinesis.54 In
addition to the cell cycle, some bacteria are also temporally
organized in order to match the day-night cycle on Earth. Such
a circadian oscillator can be encoded in gene expression networks – as is the case for eukaryotes. A few years ago the
proteins KaiA, KaiB, and KaiC of the cyanobacterium Synechococcus elongatus were shown to constitute a biochemical
oscillator in vitro. In contrast to genetic oscillators it does not
require protein degradation and synthesis, but rather periodically changes the phosphorylation status of KaiC.
7.13.7.1
Division Entry
The evolving distribution of FtsZ-regulating proteins allows
for straightforward ways to determine the onset of cell division. An early theoretical suggestion was based on a putative
role of FtsZ-disassembly for the initiation of cytokinesis:87 As
E. coli cells grow longer, the oscillation pattern of the Min
proteins changes. Instead of pole-to-pole oscillations, the Min
proteins will organize into a pattern with high concentrations
simultaneously present at both poles followed by a phase
where the Min proteins accumulate in the cell center. At this
time the Z-ring is positioned correctly in the cell center and
the divisome might have assembled. The presence of MinC
could then activate the divisome through FtsZ disassembly.
More recent studies on the dynamics of the Min proteins in
dividing cells, however, show that division sets in before the
pattern changes.64,88
Experimental evidence has been obtained, however, for a
mechanism of cell division entry that works similar in spirit.54
In C. crescentus, the assembly of FtsZ is regulated by MipZ,
which in turn is coupled to the distribution of the replication
origins on the chromosome. As mentioned above, MipZ binds
to specific DNA sites that are enriched close to the replication
origin. For this reason, MipZ accumulates at these regions. As
the chromosomes segregate, these regions depart from each
other and a region depleted of MipZ emerges between them.
At some points, the MipZ maxima will be sufficiently far apart
that the MipZ concentration in between will be low enough as
to allow for Z-ring formation. In this way, assembly of the
divisome occurs only after chromosome segregation and can
be followed immediately by cell division. In essentially
the same way, the distribution of ParA was proposed to be
regulated by the structure of the nucleoid, such that low
copy number plasmids would be segregated faithfully46
(Figure 6(a)).
7.13.7.2
A three-protein Circadian clock
A fair number of different mechanisms have been proposed to
explain the periodic change in the amount of phosphorylated
KaiC.89 KaiC is a hexameric molecule that possesses two sites
which it can autophosphorylate and autodephosphorylate.
Rather than giving a comprehensive account of all the proposed mechanisms, we will focus on one that accounts for
the differences in the two phosphorylation sites as indicated
by experiment (see Figures 6(b) and 6(c)).90 Let T-KaiC and
219
S-KaiC denote the two respective species of singly phosphorylated KaiC, ST-KaiC doubly phosphorylated KaiC, and
U-KaiC unphosphorylated KaiC. During a complete cycle
starting with U-KaiC, first T-KaiC forms, then ST-KaiC followed by S-KaiC, and finally the protein returns to U-KaiC.
While all steps of this linear reaction chain are reversible, the
presence of KaiA promotes the transitions from U-KaiC to TKaiC and from T-KaiC to ST-KaiC. Furthermore it inhibits the
transition from ST-KaiC to S-KaiC, while promoting the
inverse reaction. In turn, S-KaiC inactivates KaiA with the help
of KaiB. That is, starting from U-KaiC, the system will arrive in
a state that is dominated by ST-KaiC. If the latter concentration
is high enough, the concentration of S-KaiC will exceed
a critical value, which in turn inactivates KaiA such that the
system relaxes through S-KaiC into a state dominated by
U-KaiC and the cycle starts over again.
The KaiABC system of S. elongatus thus presents an example
of spontaneous oscillations.
7.13.8
Concluding remarks
The modern view of bacteria is that of cells that are highly
organized in space and time. Concepts have emerged that trace
this organization back to a few key processes: Target structures
emerge from protein self-organization or protein and lipid
self-assembly, as well as from the distribution of specific sites
on the chromosomal DNA. Other proteins locate to these
target sites by a diffusion-and-capture mechanism. Biophysical
techniques have contributed to our understanding of all these
mechanisms, by providing the experimental tools necessary
for a quantitative analysis of protein dynamics in space, and
also by providing appropriate concepts and theoretical tools
for their study. In some cases, our understanding has advanced
so much that essential aspects of vital systems can be reconstituted in vitro. In spite of this success story, a lot remains to
be done. There are many systems that we have only just started
to understand. It is almost definite that bacteria still have
some surprises for us up their sleeves.
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