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REVIEW
A Bacterial Cell-Cycle Regulatory Network
Operating in Time and Space
Harley H. McAdams* and Lucy Shapiro
Transcriptional regulatory circuits provide only a fraction of the signaling pathways
and regulatory mechanisms that control the bacterial cell cycle. The CtrA regulatory
network, important in control of the Caulobacter cell cycle, illustrates the critical role
of nontranscriptional pathways and temporally and spatially localized regulatory
proteins. The system architecture of Caulobacter cell-cycle control involves topdown control of modular functions by a small number of master regulatory proteins
with cross-module signaling coordinating the overall process. Modeling the cell cycle
probably requires a top-down modeling approach and a hybrid control system
modeling paradigm to treat its combined discrete and continuous characteristics.
In the past few years, microarrays and other
high-throughput experimental methods have
supported cellwide or “global” assays of gene
expression activity in cells. We can realistically expect these methods to yield virtually
complete descriptions of transcriptional wiring diagrams of some yeast and bacterial
species, perhaps within the next decade.
However, the transcriptional regulatory circuitry provides only a fraction of the signaling pathways and regulatory mechanisms that
control the cell. Rates of gene expression are
modulated through posttranscriptional mechanisms that affect mRNA half-lives and
translation initiation and progression, as well
as DNA structural or chemical state modifications that affect transcription initiation
rates. Phosphotransfer cascades provide fast
point-to-point signaling and conditional signaling mechanisms to integrate internal and
external status signals, activate regulatory
molecules, and coordinate the progress of
diverse asynchronous pathways. As if this
were not complex enough, we are now finding that the interior of bacterial cells is highly
spatially structured, with the cellular position
Department of Developmental Biology, Stanford University School of Medicine, B300 Beckman Center,
Stanford, CA 94305, USA.
*To whom correspondence should be addressed. Email: [email protected]
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of many regulatory proteins as tightly controlled at each time in the cell cycle as are
their concentrations.
Architecture of Bacterial Cell-Cycle
Control System
A few top-level master regulatory proteins organize and coordinate multiple processive modular
functions that do much of the work to execute
the cell cycle (1, 2). Herein, “modules” refers to
groups of proteins that work together to execute
a function (3). Bacterial genes for module proteins are frequently organized in one or several
operons. Proteins in a module generally interact
strongly with each other, either by forming multiprotein machines that accomplish complex
functions (e.g., gene transcription, mRNA translation, and chromosome replication) or by creating signaling or reaction pathways. Production
of the proteins constituting these modules is
tightly controlled so that they are expressed just
at the time needed and in the order required for
assembly (1, 4, 5) and destroyed when they are
not needed. Stoichiometry and, frequently, the
place of expression in the cell also are often
controlled (5). Once launched, modules act autonomously (e.g., modular metabolic pathways)
or processively (e.g., the replisome, RNA polymerase, and the construction of structures such
as flagella and pili) to complete their job. Because of inherent stochastic differences in the
time required to execute any reaction cascade
(6), intermodule regulatory linkages are required
for synchronization when maintenance of relative timing is important.
Top-level master regulators work together to
switch modular functions on and off in orderly
succession (1) as needed to progress through the
cell cycle. Many of the genes controlled by
master regulator proteins are themselves regulatory molecules, so that coordinated activation of
a complex mix of activities can be achieved. For
example, in the yeast Saccharomyces cerevisiae,
12 cell-cycle regulatory proteins control a subset
of regulators forming an interconnected loop of
activators that contribute to cell-cycle progression (7). Other master regulator proteins control
complex cell responses such as the Escherichia
coli general stress response (8) and the activation of the Bacillus subtilis sporulation pathway
(9). The advantages of a regulatory circuit architecture involving hierarchical top-down control of modular functions by master regulators
may relate to simplification of control signal
communication pathways, as well as to facilitation of both short-term (i.e., immediate within
the resources of the current cell design) and
long-term [i.e., evolutionary evolvability (10)]
adaptation to changes in environmental conditions. Disadvantages include the creation of lethal failure points when regulation of large numbers of critical functions depends on a limited
number of master regulatory proteins. Failures
can also occur because of stochastic risk of
failure in any given pathway (6). This risk is
alleviated in cells by redundant control of critical regulatory proteins, as we see in the case of
CtrA in Caulobacter.
Caulobacter crescentus is a productive model
system for the study of bacterial cell-cycle regulation and the mechanisms of asymmetric cell
division. Caulobacter always divides asymmetrically, producing daughter cells with differing polar structures, different cell fates, and asymmetric
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Synchronization of Cell Functions and
Control of CtrA Timing
During the course of the cell cycle, functions such
as flagella construction, chromosome replication,
and cell division must be both started and completed in proper order. The time required for the
modules executing these complex multistage actions to complete their work will vary depending
on (i) environmental considerations, such as variations in the cell’s nutritional state; (ii) the inherently stochastic temporal pattern of protein production from activated genes (6); and (iii) natural
stochastic differences in the time to complete a
chemical reaction cascade. Reaction cascades can
be quite complex, and multiple cascades may
have to be completed before a given reaction can
proceed. The time to complete any reaction cascade is never precisely predictable. However, by
adding regulatory complexity, the timing can be
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regulation of the initiation of chromosome replication (Fig. 1A). The role of master regulatory
proteins in top-down control of functional modules is well illustrated by Caulobacter’s CtrA
regulatory network (Fig. 1B), which is parallel in
many ways to the Spo0A master regulator of
sporulation in B. subtilis (11). Examples of CtrAregulated modular functions include flagella biogenesis, the chemotaxis control subsystem, the
divisiome (FtsZ), the pili structure, and the replisome. Both CtrA and Spo0A are response regulator members of two-component signal-transduction pathways that control large numbers of genes
involved in multiple modular functions. CtrA controls, directly or indirectly, 26% of the Caulobacter cell-cycle–regulated genes and directly
controls 95 genes in 55 different operons (1, 2).
There are presumably additional as-yet-unidentified master regulators of comparable scope that in
coordination with CtrA control the remaining
74% of the cell-cycle–regulated genes.
The “just-in-time” principle for expression of
genes supporting modular functions can be seen in
the early derepression of metabolic and ribosomal
genes as swarmer cells differentiate into stalked
cells, in the timing of ftsZ (the earliest expressed
gene in cell division) expression, and in the timing
of flagella biogenesis and the associated chemotaxis control subsystem (Fig. 1B). Another example is the timing of production of CtrA. In a
narrow window of the cell cycle, Caulobacter
produces ⬃22,000 CtrA molecules (12). Why so
many CtrA molecules are needed so quickly is
unclear. Perhaps the purpose is to minimize stochastic variations in start times of important cellcycle functions that must complete before cell
division. In any case, the Caulobacter cell is designed to produce the CtrA molecules at just the
right time relative to the progress of chromosome
replication, and then, a little later, to remove them
with equal alacrity by activating an as-yet-unidentified cell-cycle–regulated recognition factor that
delivers CtrA to the ClpP/ClpX proteolysis system (Fig. 1).
Fig. 1. (A) Schematic of the Caulobacter cell cycle. The motile swarmer cell has a single polar
flagellum, polar chemotaxis receptors, and multiple polar pili. Shaded cells contain CtrA.
Chromosome replication is repressed in swarmer cells by binding of CtrA to the origin of
replication. After about a third of its cell cycle, the swarmer discards the flagellum, chemoreceptors, and pili as it differentiates into a stalked cell. At the swarmer-to-stalked cell
transition, CtrA is cleared from the cell, allowing the initiation of DNA replication (shown as
the ␪-like structure in the cell). Upon completion of separation of the predivisional cell into two
compartments before cell division, CtrA is cleared just from the nascent stalked cell compartment, allowing replisome formation while maintaining repression of replication initiation in the
swarmer compartment (12). (B) Genetic network controlled by the CtrA response regulator.
The transcriptional network directly controlled by CtrA (green lines) is integrated with
nontranscriptional regulatory pathways (red lines). A negative feedback loop through the CcrM
DNA methylation pathway ties the timing of CtrA synthesis to a specific point in the progress
of chromosome replication. Activation by one or more phosphorelays involving the membranelocalized CckA histidine kinase (15), and probably the DivL tyrosine kinase (32) and the DivK
response regulator (33), are required and thought to integrate other currently unknown signals
into the control of timing of CtrA activation. Red indicates regulatory genes. Timing in the cell
cycle of transcriptional regulation (as assayed by gene expression microarrays and lacZ
transcriptional fusions) is indicated by the bars on the right. Numbers over each bar indicate
the numbers of genes regulated in that time period. Redundant control of the timing of
chromosome replication involves both repression of the origin of replication at five CtrA
binding sites (34) and transcriptional repression of components of the replication machinery by
an unknown factor (35). The metabolic and ribosomal proteins controlled by CtrA are
derepressed in the swarmer cell at about the time of the swarmer-to-stalked differentiation,
perhaps to support accelerated growth in the stalked cell.
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made more predictable, as in the case of the
regulatory network controlling circadian rhythms
(13). Even when different functions are started at
exactly the proper times in the cell cycle, maintenance of synchronization requires regulatory signaling between modules at intermediate points
(checkpoints) to coordinate the overall process
(14). This cross-module signaling involving
mechanisms such as two-component systems or
phosphorelays, either blocks cell-cycle progress
until necessary precursor functions are completed
or initiates coordinated responses to emergencies
such as nutrient shortages or DNA damage.
Improper timing of some events in the cell
cycle will have fatal consequences; thus, highly
reliable synchronization mechanisms are required.
An obvious example is the need to complete
separation of the two chromosomes before completing cell division. Other critical timing events in
Caulobacter include: (i) somewhat surprisingly,
the timing of CtrA expression versus the state of
progression of chromosome replication noted
above, and (ii) the timing of initiation of CtrA
degradation in the stalked compartment of the late
predivisional cell. In the swarmer cell, CtrA binds
to and silences the chromosomal origin of replication; later, CtrA has to be cleared from the
stalked cell for initiation of replication. Because
chromosome replication is differentially regulated
in the swarmer and stalked progeny, Caulobacter
faces a tricky problem in the predivisional cell: It
has to quickly and reliably get rid of CtrA in the
Fig. 2. Spatial and temporal dynamics of selected regulatory and structural proteins. (A) Scaled cell cross
sections starting with the newly divided swarmer cell (0 min) and proceeding to cell division (240 min).
Notable features include the diverse dynamics of histidine kinases and response regulators that are, for
example, variously synthesized and degraded (CtrA), localized to the membrane and delocalized to the
cytoplasm (DivK), localized to the pole and delocalized in the membrane (DivJ), and transiently present and
localized at a pole (PleC); the controlled positioning of the chromosome origin and terminus of replication as
well as the replisome; the continuously changing complement of regulatory and structural proteins at the cell
poles; and the bifurcating cytoplasmic chemistry at the time of cell compartmentalization (⬃220 min). (B)
Regulatory connections between localized features in (A). CckA is delocalized in the membrane of the
swarmer cell and early stalked cell and accumulates at the predivisional cell pole, concomitant with transfer
of its phosphate moiety to newly synthesized CtrA (140 min). The membrane-bound PleC histidine kinase is
required for signaling polar pili assembly, biogenesis of holdfast material at the tip of the stalk, and chemotaxis
functions. PleC is localized at the flagellated pole (⬃140 min) and delocalized at the swarmer-to-stalked cell
transition time. Positioning of the PodJ localization factor at the pole opposite the stalk (90 min) is necessary
for subsequent positioning of PleC and the components of the pili secretion apparatus. As the swarmer cell
changes into a stalked cell, the replisome assembles on the polar chromosome origin of replication and PleC
is released from the pole and replaced by the DivJ histidine kinase. In the late predivisional cell (⬃220 min),
CtrA proteolysis commences in the stalked compartment, the CckA histidine kinase is released from the cell
poles, and DivK is released from the pole of the swarmer compartment.
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nascent stalked cell while reliably keeping the
concentration high in the nascent swarmer cell.
Failure to maintain a high concentration of CtrA
in the swarmer compartment risks premature activation of chromosome replication and disruption
of the swarmer cell’s distinctive behavior and
morphology. Caulobacter has a robust mechanism for avoiding this problem, involving the
compartmentalization of the predivisional cell.
Partitioning the cell apparently enables activation
of a CtrA degradation signal, perhaps involving a
phosphorylation pathway, which accelerates the
CtrA degradation rate (12, 15). CtrA proteolysis is
only initiated in the stalked half of the predivisional cell after cytoplasmic compartmentalization
has occurred, about 15 to 18 min before cell
separation. This ensures that DNA replication begins only in the progeny stalked cell (12, 16)
Another novel synchronization mechanism is
a resettable switch that ties the timing of CtrA
expression to the progress of chromosome replication. The hemimethylated state of newly replicated DNA containing the ctrA gene is used as a
signal for ctrA promoter activation (15). Before
the initiation of replication, the chromosome is
fully methylated at sites recognized by the CcrM
methyltransferase. As the replication fork passes
through the ctrA gene, the promoter region goes
from the fully methylated to the hemimethylated
state. CtrA is transcribed from two promoters: the
P1 promoter, which can only be activated when it
is hemimethylated, and the P2 promoter, which is
autoactivated when the CtrA produced from P1
accumulates to threshold levels and is activated by
phosphorylation. This system ensures that two
gene copies are present before beginning CtrA
expression, thereby increasing the rate of CtrA
production and reducing the stochastic variations
in the rate of its production. The increasing CtrA
level represses P1 and activates many genes, including those that express the CcrM methyltransferase (Fig. 1B). CcrM methylates a site at the P1
promoter. Finally, both CtrA and CcrM are degraded to reset the switch for the next cell cycle.
When the ctrA gene is moved to a site on the
chromosome near the termination of DNA replication, the cells lose size control, probably as a
result of defective coordination of timing (17).
Subcellular Localization of Proteins
and Regions of the Genome
In the Caulobacter cell, and presumably in any
cell undergoing asymmetric division, asymmetries in the intracellular environment, particularly
differentially localized regulatory molecules, are
essential for the implementation of spatially differentiated cell morphogenesis. The design principles underlying these spatially distributed circuits are poorly understood. Also, the role of the
physical geometry of the cellular reaction chamber (the cell interior and the membrane) in which
the circuitry operates has not been well characterized in bacterial cells. During B. subtilis sporulation, differing chemistry on the two sides of the
septum separating the compartments (9) as cell
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of structures in the bacterial cell. Integration of
these imaging technologies with global gene expression studies of wild-type and mutant cells
will enable new advances in genomics by integrating the spatial dimension of regulation.
Implications for Modeling the Cell
Success with subsystem models has inspired ambitions to develop “whole-cell” models to put the
individual parts together to create so-called “in
silico” models (27, 28). However, major, perhaps
insurmountable, difficulties must be overcome before whole-cell models based on extensions of the
current “low-level” modeling and simulation
methodologies, which emphasize kinetics of coupled reaction systems, will be feasible. Problems
include lack of quantitative data on molecular
concentrations and kinetic parameters, as well as
only piecemeal characterization of the cell’s regulatory circuitry. Although great progress is being
made in identifying global regulatory pathways by
using high-throughput assays and sophisticated
algorithms, it is still a difficult and labor-intensive
biochemical and genetic job to verify details of
any pathway. This suggests that it will be a long
time before the data to support bottom-up models
of the whole cell will be feasible.
On the other hand, the hierarchical, top-down
regulatory architecture and the modular structure
of the functional elements that implement the cell
cycle suggest that it may be useful to consider a
top-down modeling paradigm. This approach
would focus on the network of global regulators and
the orderly initiation and coordinated execution of
modular functions. Details of the wiring diagrams
of global regulatory networks controlling the cell
cycle, responses to environmental signals, and stress
responses will have to be identified. With these
results, along with characterization of the top-level
regulation of modular functions and intermodule
signaling pathways, realistic modeling of overall
cell behavior and responses to environmental events
should be possible.
With all the switches and checkpoint mechanisms controlling cell-cycle progress, the resulting behavior of the cell is very machinelike, with
the cell cycle progressing in a ratchetlike manner
as conditional gates controlling forward progression from one state to another are successively
opened. This discontinuous progression of the
cell through the cell cycle is not well suited to
modeling with differential equation-based cellcycle models with their implicit assumptions of
continuity. Alternative modeling paradigms may
prove necessary, probably involving modeling
the cell cycle as a hybrid control system, i.e., a
system with both discrete and continuous dynamics (29). Hybrid control models reflect the
combination of discrete and continuous control
mechanisms found in the cell (29). Biological
applications of hybrid system models approximate transcriptional regulation with piecewiselinear approximations of sigmoidal responses
(29–31) and map modular biological functions
into corresponding software objects (29). This
approach, although promising, is less developed
than the differential equation-based modeling of
systems of coupled chemical reactions, and, at
least for the Caulobacter cell, the three-dimensional nature of regulation with polar-localized
regulatory molecules and bifurcating cytoplasmic chemistry adds complications. At a minimum, this leads to complex boundary conditions
for the models. If reaction-diffusion models have
to be incorporated (for example, to properly treat
the function of localized components of phospho-cascades), the mathematics will be even
more complicated. These will be exciting challenges for the new breed of “systems biologists”
to address in the next decade.
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division nears completion is critical to differential
development of the mother cell and prespore.
There is no evidence for such asymmetric chemistry at the division plane in Caulobacter. Rather,
in Caulobacter differential polar chemistry seems
to be central to establishing the distinctive identities of the daughter cells. There is a complex
interaction between the regulatory mechanisms
operative at transcription initiation sites, the dynamically changing portfolio of regulatory proteins, the subset of those proteins localized to the
cell poles (18, 19), and the instantaneous physical
deployment of the replicating chromosome (19,
20) within the cell. As a result of these spatially
distributed interactions, the dynamic deployment
of regulatory proteins throughout the cell has to be
considered when analyzing how individual components of the cell-cycle control circuitry work.
Figure 2 provides a schematic view of the
Caulobacter cell’s geometry and spatial deployment of proteins over the course of a cell cycle.
Multiple two-component signal-transduction proteins, localization proteins, replication proteins,
membrane-associated secretion and export proteins, and pili and flagellar structural proteins are
dynamically created and destroyed at the poles as
the cell cycle progresses and the polar organelles
mature. The Caulobacter property of having only
one flagellum made at a precise time in the cell
cycle and strictly located at the pole is one result
of precise temporal and spatial regulatory controls.
Although the regulatory mechanisms for construction of a flagellum are well characterized (5),
the mechanism that establishes its polar location is
unknown (21). Some proteins that control or affect polar positioning of structures have been identified, but mechanisms of action are not known. In
the case of polar pili biogenesis, a localization
factor, PodJ (which is present in both long, PodJL,
and short, PodJS, forms), has been identified that
functions to place both the PleC histidine kinase
required for pilus assembly and the pilus secretory
protein at the cell pole (22, 23). PleC, in turn,
signals the accumulation of the pilin subunit that
assembles into the polar pilus; if active PleC is
mislocalized, pili are not assembled (24). Mutations in polar-localized regulatory proteins can
cause aberrant cell morphologies, defective or
missing polar structures, failures in progression of
the cell cycle, or cell death. Thus, it now appears
that the transcriptional circuitry that implements
the temporal control of cell-cycle progression is an
integrated function of the spatial deployment of
structural and regulatory proteins, so that the
changing transcriptome both affects, and is affected by, polar developmental progress.
Solving the puzzle of the connection between
spatial positioning of regulatory proteins, cellcycle progression, and asymmetry in cell division and polar organelle development is a current
challenge. Recent advances in protein labeling
for electron microscope imaging (25) and in
biological imaging using soft x-ray microscopy
(26) offer prospects for unprecedented visibility
into the dynamic three-dimensional positioning
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