REVIEWS
CELL-CYCLE PROGRESSION AND
THE GENERATION OF ASYMMETRY
IN CAULOBACTER CRESCENTUS
Jeffrey M. Skerker and Michael T. Laub
Microorganisms make tractable model systems and Caulobacter crescentus has emerged as the
main model for understanding the regulation of the bacterial cell cycle. Mechanisms that mediate
the generation and maintenance of spatial asymmetry are being uncovered using this model
bacterium. Now, the advent of genomic technologies together with the completion of the
Caulobacter crescentus genome sequence is enabling global analyses that have revolutionized the
pace of research into the genetic networks that control the bacterial life cycle.
G1 PHASE
The period of time in the cell
cycle before DNA replication
starts and during which the
cell contains only one copy of
its genome.
Bauer Center for Genomics
Research, 7 Divinity Avenue,
Cambridge, Massachusetts
02138, USA.
Correspondence to M.T.L.
e-mail:
laub@cgr.harvard.edu
doi:10.1038/nrmicro864
Progression through the cell cycle, in all organisms,
requires the precise coordination of four main processes:
DNA replication, chromosome segregation, cell division and cell growth. The aquatic, non-pathogenic
bacterium Caulobacter crescentus (which will be
referred to as Caulobacter in this review) has emerged
as the main model system for analysis of the prokaryotic
cell cycle. This experimentally tractable organism
can be investigated using a wide range of genetic, biochemical and cell biological techniques. The recent
sequencing of the Caulobacter genome1 has now
enabled genomic approaches, such as DNA microarraybased expression profiling2,3, and a variety of proteomic
approaches4–6. Significantly, Caulobacter cells are easily
synchronized, thereby allowing precise temporal
analysis of the cell cycle. Caulobacter has also emerged
as a model system for understanding how bacteria
establish and maintain cellular asymmetry. Each cell
division in this organism produces two daughter cells
that are morphologically and physiologically distinct
(FIG. 1). Asymmetric cell division is a common feature
in the life cycle of many bacteria7. Perhaps the bestknown example is Bacillus subtilis, which, under certain
starvation conditions, can divide asymmetrically as
part of a developmental pathway that produces a
spore. In addition, many bacteria, such as Escherichia
coli, Pseudomonas aeruginosa, Shigella flexneri and
Listeria monocytogenes, might appear symmetric but
NATURE REVIEWS | MICROBIOLOGY
have, on closer inspection, asymmetric poles. This
article will review the progress that has been made in
elucidating the molecular mechanisms underlying
both cell-cycle progression and the establishment of
asymmetry in Caulobacter.
Recent studies have shown that many of the mechanisms discovered in Caulobacter are evolutionarily
conserved among other members of the α-subdivision of
proteobacteria. These species, including Sinorhizobium
meliloti, Agrobacterium tumefaciens, Rickettsia prowazekii
and Brucella abortus, have important roles in a wide
range of environmental, medical and biowarfare protection applications8–14; therefore, the fundamental
research in Caulobacter has far-reaching implications.
In particular, the identification of genes that are
essential to cell-cycle progression in Caulobacter
could help to identify new targets for antibacterial
drug discovery.
The cell cycle of Caulobacter crescentus:
Progression through the Caulobacter life cycle (FIG. 1)
requires the precise coordination of morphological,
metabolic and cell-cycle events. The life cycle starts
with a motile, chemotactic ‘swarmer’ cell. This cell type
has a single polar flagellum that is used for motility,
and polar type IV pili that mediate adhesion to biotic
and abiotic surfaces. The swarmer cell cannot initiate
DNA replication and remains in G1 PHASE with a single
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a
Stalked cell
Swarmer cell
0
15
30
45
G1
Early
predivisional
cell
60
75
Late
predivisional
cell
90
105
120
S
135
150
G2
DNA replication
initiation
Flagellum biogenesis
Pilus biogenesis
DNA replication, nucleotide
metabolism, recombination/repair
Chemotaxis machinery
DNA methylation
Chromosome
segregation
Growth: cell envelope, ribosomes,
oxidative respiration
Cell division
c
b
Figure 1 | Cell-cycle progression in Caulobacter crescentus. a | Schematic of the cell cycle.
Motile, piliated swarmer cells differentiate into stalked cells at the G1–S transition by shedding their
polar flagellum, growing a stalk at that site, losing the polar pili and initiating DNA replication. Circles
and ‘θ’ structures in the cell represent quiescent and replicating chromosomes, respectively.
An asymmetric predivisional cell yields two different progeny after division — a swarmer and a
stalked cell. The coloured bars indicate timing of gene transcription for functionally related sets of
genes. Each set of genes shows ‘just-in-time’ transcription: the genes are transcribed immediately
before, or coincident with, the execution of the event for which they are required. Electron
micrographs of a Caulobacter swarmer cell (b) and predivisional cell (c) prepared by negative
staining with uranyl acetate. Scale bars equal 0.5 µm.
S PHASE
The period of time in the cell
cycle in which a cell is actively
synthesizing/replicating its
genome.
G2 PHASE
The period of time in the cell
cycle after DNA replication has
been completed, but before cell
division.
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| APRIL 2004 | VOLUME 2
chromosome. In response to cues that are not understood, the swarmer cell differentiates into a ‘stalked’ cell
(FIG. 1). During this differentiation, the polar flagellum
is released and the polar pili are retracted. A stalk,
which is a narrow elongation of the cell wall and
membranes, is constructed at the same location as
the discarded flagellum. Coincident with this morphological transition, the cell enters S PHASE and initiates DNA replication. Like most eukaryotes, but
unlike many other prokaryotes such as E. coli,
Caulobacter cells exhibit strict once-and-only-once
replication behaviour; so, S phase produces exactly
two daughter chromosomes. By contrast, rapidly
growing E. coli cells can initiate DNA replication as
many as four or more times between intervening cell
divisions, such that daughter cells inherit a fully
replicated chromosome that has already initiated several rounds of DNA replication. DNA replication and
segregation of daughter chromosomes to opposite
ends of the growing predivisional cell occurs during
S phase and a brief, but distinct, G2 PHASE. Before dividing, the predivisional cell also builds a new flagellum
and starts to construct new pili at the pole opposite
the stalk. Once the flagellum is complete, cell division
proceeds, yielding two daughter cells that are physiologically and morphologically different. One daughter cell
is a stalked cell that immediately reinitiates another round
of S phase and the other daughter cell is a swarmer cell
that cannot start DNA replication until after the obligate
swarmer-to-stalked cell differentiation step.
Global approaches to cell-cycle study
The elaborate spatial and temporal process of cellcycle progression in Caulobacter has been dissected at
a molecular level using various genetic, biochemical
and cell biological tools. More recently, the sequencing
of the 4-Mb Caulobacter genome1, encoding 3,767
genes, has provided a resource that has facilitated
genomic approaches.
Early work on Caulobacter led to the identification
of more than 70 genes, many involved in DNA replication or flagellar biogenesis, that are transcriptionally
regulated during the cell cycle. Whereas these genes were
characterized one at a time, the completion of the
genome sequence allowed the development and use of
DNA microarrays for studying global patterns of gene
expression2,3. In the first global study3, DNA microarrays
were used to analyse changes in gene expression during
cell-cycle progression. A population of Caulobacter
cells were synchronized in the G1/swarmer phase and
then allowed to proceed through the cell cycle, with
RNA collected every 15 minutes and analysed on
whole-genome DNA microarrays. The resulting
expression profiles revealed a set of 553 genes — more
than 15% of the genome — that are transcribed in a
cell-cycle-dependent manner. The cell-cycle-dependent
expression patterns of more than 70 genes that had
been studied previously were confirmed by the DNA
microarray data.
Functional classification of these cell-cycle-regulated
genes showed that Caulobacter couples gene expression to cell-cycle events with extraordinary precision;
genes, the products of which are involved in a specific
cell-cycle event, have a peak in expression immediately before, or coincident with, the timing of the
event (FIG. 1). For example, transcription of the genes
that are involved in DNA metabolism and DNA replication is induced and reaches a maximal level at the
G1–S transition, whereas transcription of the genes
that are involved in chromosome segregation is maximal in late S phase. This theme of ‘just-in-time’ transcription applies to nearly all of the known cell-cycle
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REVIEWS
a
b
Order of assembly
Hook
Class III
Outer membrane
Order of gene expression
Class IV
Filament
Cell wall
Basal
body
Periplasm
Class II
Inner membrane
Class I
Figure 2 | Transcriptional control of flagellar assembly. a | The order of assembly of the
Caulobacter flagellum proceeds from the inside to the outside of the cell, with class II components
assembled first. Class II components include the inner-membrane secretion apparatus and the
inner rings. This is followed by assembly of the class III components, which include two rings, the
proximal and distal rod and the hook. The final step in the process is assembly of the filament, which
is encoded by the class IV genes. b | Clustered expression profiles of genes required for flagellar
assembly3, with cell-cycle progression running from left to right. Expression levels are colour-coded,
with yellow indicating high mRNA levels and blue representing low mRNA levels. The order of gene
transcription parallels the order of assembly.
PULSE–CHASE STUDY
A technique in which a cell, or
cell extract, is briefly treated
with a radioactive compound
(the ‘pulse’). This allows
incorporation of the radiolabel
into cellular constituents. The
pulse is followed by addition of
excess, non-radioactive
compound (the ‘chase’).
Monitoring the radiolabelled
compound over time then
allows its location or stability to
be tracked.
events (FIG. 1). Precise transcriptional control might
allow more efficient use of cellular resources and
help to ensure the correct timing of events during
the cell cycle.
Close inspection of a subset of functionally related
genes uncovered even more elaborate patterns of cellcycle transcription (FIG. 2). The genes that are required
for flagellum assembly can be organized into four
discrete classes (I–IV) on the basis of their expression
profiles. The order of expression of these four classes of
genes was known from earlier work to parallel the order
in which the gene products are assembled into a functioning flagellum, and the comprehensive DNA
microarray analysis confirmed this temporal pattern.
For a comprehensive review of flagellar assembly in
Caulobacter, see REF. 15. Class I, which is expressed earliest, is composed of a single transcription factor, called
CtrA (BOX 1), that sets the cascade of flagellar gene
expression in motion and is the master regulator. This
transcription factor activates expression of the class II
genes, which encode components of the flagellum that
are assembled in the inner membrane. Correct assembly
of these components triggers the expression of the
class III genes, which encode flagellar components that
are assembled in the periplasm and outer membrane.
Finally, after a short delay, the flagellar filament subunits (class IV genes), which comprise the flagellar
filament, are expressed. The striking co-linearity of
gene expression and organelle assembly occurs not
NATURE REVIEWS | MICROBIOLOGY
only with flagellum organelle assembly, but is also
found for pilus biogenesis: transcription of the genes
that encode components of the pilus temporally mirrors
their order of assembly3,16.
An alternative global analysis of cell-cycle expression
came from a large-scale study of protein synthesis rates
using two-dimensional (2D)-gel electrophoresis and
mass spectrometry4. A strong correlation between the
DNA microarray results and the 2D-gel data was found.
However, there was a small group of genes for which the
mRNA levels did not correlate with protein synthesis,
indicating that post-transcriptional regulation of these
genes occurs. Finally, a large-scale PULSE–CHASE STUDY4
identified 48 proteins that have extremely short
half-lives. Twenty-six of these proteins also showed
cell-cycle-regulated expression, indicating that the
abundance, and presumably the activity, of these proteins is precisely controlled during the cell cycle. This
set of 26 proteins includes several essential proteins
that had previously been shown to be degraded at specific times during the cell cycle17–19. Caulobacter, like
eukaryotic cells, seems to use regulated proteolysis as a
mechanism for regulating the cell cycle and ensuring
irreversible transitions during its development.
CtrA regulation. How are the intricate gene-expression
patterns that are observed during the Caulobacter cell
cycle coordinated and regulated? Previous work has
identified a key master regulator, CtrA, which is essential
for viability and controls a large number of cell-cycle
events20 (BOX 1). CtrA is a member of the highly conserved, ubiquitous two-component signal-transduction
family, comprised of histidine kinases and their substrates, response regulators (BOX 2). CtrA, a response
regulator, contains a DNA-binding domain, and phosphorylation of CtrA at a single site increases its affinity
for target promoters21. Phosphorylated CtrA (CtrA~P)
functions as a transcriptional activator and repressor
during the cell cycle.
Like master regulators in many organisms, CtrA is
itself subject to multiple levels of regulation (FIG. 3),
which help to ensure the fidelity of cell-cycle progression17,20,22–27. CtrA~P is present at high concentrations
in swarmer/G1 cells. Rapid proteolysis — a ClpXPdependent process — eliminates CtrA at the swarmerto-stalked cell transition17,22. The proteolysis of CtrA,
which binds with high affinity to five sites in the origin
of replication to repress chromosome replication,
allows DNA replication to begin28. By mechanisms that
are not fully understood, the commencement of
S phase leads to an initiation in transcription of ctrA
from the promoter P1. As the concentration of CtrA
increases, CtrA binds to, and represses, transcription
from P1, but strongly activates transcription from promoter P2 (REF. 29). This positive feedback results in a
rapid accumulation of newly synthesized CtrA, with
estimates of production of ~22,000 molecules per
cell30. Newly synthesized CtrA is phosphorylated,
which enables it to regulate the expression of its 95 target genes. In the stalked half of the late predivisional
cell, CtrA is again degraded in a ClpXP-dependent
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Box 1 | The master regulator CtrA
CtrA (cell-cycle transcription regulator)
A genetic screen for essential genes that activate the flagellar hierarchy identified CtrA as a transcription factor
controlling multiple cell-cycle processes. The master regulator CtrA coordinates the timing of cellular events with
chromosome replication. Important features of CtrA include:
• it is essential for viability
• it is a member of the two-component signal-transduction family of proteins — functions as a response regulator (BOX 2)
• it is conserved in many species of the α-subdivision of proteobacteria
• it binds to the chromosomal origin of replication to repress DNA replication initiation
• it functions as a transcription factor to directly regulate 95 genes involved in polar morphogenesis, cell-cycle
progression and regulatory processes
• its activity during the cell cycle is controlled on at least three partially redundant levels: transcription, proteolysis and
phosphorylation.
manner. Cell division results in a swarmer cell that
contains high concentrations of phosphorylated CtrA,
and a stalked cell that contains little or no CtrA. The
asymmetric distribution of phosphorylated CtrA is a
crucial step in establishing daughter cells that have
distinct fates in Caulobacter.
The regulation of CtrA is controlled at the level of
transcription, proteolysis and phosphorylation. Below,
we review the recent progress that has been made in
revealing the details of each of these control mechanisms,
as well as initial evidence that subcellular localization
might also have a role in regulating CtrA function.
Transcription of ctrA. DNA methylation has recently
been shown to have an important role in the activation
of the ctrA P1 promoter in stalked cells. The P1 promoter, which contains a DNA methylation site
between the –10 and –35 regions, is sensitive to methylation, such that it is more active in a hemi-methylated
or unmethylated state23. At the onset of S phase, the
chromosome, including the ctrA locus, is fully methylated
and the P1 promoter is inactive. During chromosome
replication, the replication fork passes the ctrA locus and,
because newly synthesized DNA is not immediately
methylated, the ctrA loci in the new chromosomes are
hemi-methylated. At this point, P1 becomes transcriptionally active. Consistent with this model, moving the
ctrA locus closer to the chromosome replication terminus delays transcription from P1. Once CtrA accumulates at high concentrations in the predivisional cell, it
activates transcription of ccrM, which encodes a DNA
methyltransferase that fully methylates the daughter
chromosomes. This renders the P1 promoter of both
new copies of the ctrA locus less transcriptionally
active. The precise mechanism by which the change in
methylation status of P1 affects transcription is unclear.
One likely possibility is that the transition from a fully
methylated to a hemi-methylated state increases the
affinity of the promoter for an as-yet-uncharacterized
transcription factor.
Box 2 | Two-component signal transduction.
DNA METHYLATION
The addition of a methyl (CH3)
group to adenine or cytosine
bases in DNA.
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Two-component signal transduction systems are
widespread in bacteria, fungi and plants, and are used by
these organisms to respond to various environmental
and intracellular stimuli75,76. The two components are
sensor histidine kinases and response regulators. As
shown in the figure (part a), activation of a histidine
kinase leads to autophosphorylation on a conserved
histidine residue. The phosphoryl group is then typically
transferred to an aspartate residue that is located on the
receiver domain of a response regulator, thereby
activating it; response regulators are frequently
transcription factors, and phosphorylation renders them
competent to bind DNA and regulate the transcriptional
output of target genes. Multicomponent phosphorelays
also exist (see figure part b), in which the phosphoryl
group is shuttled from a histidine kinase, to a response
regulator, to an intermediate protein known as a
histidine phosphotransferase and then to a final response
regulator. An important subclass of the histidine kinases
are the ‘hybrid kinases’, which are histidine kinases with a
carboxy-terminal response-regulator domain.
a
Input
signal
Input
signal
b
H
Histidine
kinase
Histidine
kinase
H~ P
P ~H
H
P ~D
Response
regulator
P ~H
D~ P
Histidine
phosphotransferase
Response
regulator
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REVIEWS
CtrA~P
CtrA proteolysis
ctrA P1 transcription
ctrA P2 transcription
CtrA phosphorylation
P1
P2
CtrA
ctrA
CtrA ~ P
Gene
expression
Figure 3 | Regulation of the master regulator CtrA. Coloured bars below a diagram of the
Caulobacter cell cycle indicate the timing of CtrA proteolysis, transcription and phosphorylation.
Active, phosphorylated CtrA protein (pink shading) is present in the swarmer cell and the early
predivisional cell. CtrA is specifically degraded at the swarmer-to-stalked cell transition, and in the
late predivisional cell after compartmentalization22. ctrA transcription is initiated at two promoters29,
P1 and P2, shown at the bottom of the figure. The weak P1 promoter is activated first in the early
predivisional cell. The initial accumulation of active CtrA leads to negative feedback on promoter
P1, and leads to strong positive feedback at the P2 promoter. The net result is the rapid
accumulation in the predivisional cell of CtrA~P, which is required for a number of essential cellcycle events. Even if CtrA is present throughout the cell cycle, regulated phosphorylation ensures
cell-cycle control of CtrA activity22.
HOMOLOGY MODELLING
A procedure in which an
unknown protein structure is
modelled by matching — fitting
— to the known structure of a
closely related protein by
matching conserved amino
acids. Allows an approximation
of the 3D shape and
organization of the protein to be
obtained.
Proteolysis and compartmentalization of CtrA. Precise
temporal and spatial control of CtrA proteolysis helps
to regulate cell-cycle progression and the production
of asymmetric daughter cells. Cell-cycle-regulated
proteolysis of CtrA is dependent on the ClpXP
protease17. Depletion of either of the two Clp subunits
(ClpX or ClpP) in vivo leads to stabilization of CtrA,
which in turn results in a defect in the G1–S transition,
presumably because CtrA remains bound to the origin
of replication. A second essential response regulator,
DivK also contributes to CtrA proteolysis. Cells that have
a cold-sensitive mutant allele of divK do not degrade
CtrA at the G1–S transition at the non-permissive
temperature25. CtrA is not degraded in vitro by purified,
active ClpXP and phosphorylated DivK25, which might
indicate either that DivK control of CtrA proteolysis is
indirect, or that another necessary proteolysis factor
remains to be found. In E. coli, ClpXP-mediated proteolysis often requires substrate-specificity factors that
target particular substrates for degradation31,32, which
supports the hypothesis that another factor is required.
The ClpXP protease typically recognizes hydrophobic
residues at the carboxyl terminus of substrate proteins.
Deletion of the C-terminal three residues of CtrA, or
modification of the last two residues from Ala–Ala to
Asp–Asp, results in a proteolytically stable form of CtrA22.
Although the C-terminal portion of CtrA is necessary for
proteolysis, it does not seem to be sufficient to trigger
degradation. However, the amino-terminal 56 residues of
NATURE REVIEWS | MICROBIOLOGY
CtrA — also known as the receiver domain (RD) —
together with the 15 C-terminal residues (RD+15) were
shown to be sufficient for cell-cycle-regulated proteolysis
of CtrA24. To delineate those residues that are necessary
for ClpXP-mediated proteolysis of CtrA, Ryan et al.24
fused the RDs from five separate CtrA homologues
(from closely related species in the α-subdivision of proteobacteria) to the last 15 C-terminal residues of the
Caulobacter CtrA. Four out of the five hybrid proteins,
when expressed in Caulobacter, were degraded temporally with a pattern that nearly matches that of native
CtrA, indicating that the RDs of these four proteins
contain a common proteolytic signal. By contrast, the
hybrid protein that contained the RD of CzcR, the CtrA
homologue from Rickettsia prowazekii, was stable
throughout the cell cycle. A sequence alignment of the
five CtrA RDs identified 10 amino acids that are conserved in all except CzcR, which makes these sites good
candidates for forming part of the proteolytic signal.
HOMOLOGY MODELLING of the Caulobacter CtrA RD using a
solved structure of the RD of B. subtilis Spo0F predicts
that 9 of these 10 residues are on one surface of the
protein and could form a binding surface for a factor that
regulates the temporal pattern of CtrA proteolysis. This
factor is unlikely to be a subunit of the ClpXP protease,
because clpX and clpP are constitutively expressed and are
present throughout the cell cycle17.
The Caulobacter CtrA RD+15 is sufficient to target
heterologous proteins that are unrelated to CtrA for
degradation, presumably by ClpXP. For example, a
fusion of yellow fluorescent protein (YFP) to the
CtrA RD+15 (YFP–RD+15), was sufficient to confer
a cell-cycle-regulated degradation pattern on YFP24.
This YFP construct allows direct visualization, by fluorescence microscopy, of protein turnover in vivo. As
expected, the YFP–RD+15 construct was degraded
during the swarmer-to-stalked cell transition and was
degraded in the portion of the predivisional cell that
was destined to become the stalked cell. Surprisingly, a
significant percentage of cells localized the fluorescent
fusion to the flagellar pole of the swarmer cell immediately prior to degradation at the swarmer-to-stalked
transition. The functional significance of this remains to
be shown, but it seems likely that subcellular localization
is coupled to degradation.
How is the proteolysis of CtrA confined to only one
portion of the predivisional cell? Using the YFP–RD+15
construct and fluorescence microscopy, Judd et al.30
showed that CtrA proteolysis occurs in the stalked half
of the predivisional cell only after septum formation,
which presumably prevents free diffusion of CtrA
between the two nascent daughter cells. This indicates
that septum formation is coupled to the initiation of
proteolysis, but the mechanism of this coupling remains
uncharacterized.
Phosphorylation of CtrA. Phosphorylation is perhaps
the most important, but least well-understood, aspect
of CtrA regulation. CtrA only becomes competent to
activate transcription after phosphorylation of the amino
acid residue Asp51 (REF. 20). Several two-component
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Box 3 | A beginner’s guide to Caulobacter gene nomenclature
Ple (pleiotropic phenotype)
A screen for mutants with defects in multiple processes, including motility, phage sensitivity
and stalk formation, yielded the two-component signalling genes pleC and pleD.
Div (division phenotype)
A screen for suppressors of the pleC pleiotropic phenotype yielded three two-component
signalling genes, divJ, divK and divL; mutations in each individually yield a cell-division
phenotype.
Cck (cell-cycle kinase)
A screen for temperature-sensitive, lethal, pleiotropic mutants identified the kinase CckA.
A yeast two-hybrid screen for proteins that interact with DivK identified two other
histidine kinases that might be involved in the cell cycle, CckN and CckO.
systems have been identified that have roles in controlling the phosphorylation state of CtrA (FIG. 4). One of
the proteins that regulates CtrA, the histidine kinase
CckA, is essential for viability, and the phenotype of a
cckA mutant is similar to that of a ctrA mutant33 (BOX 3).
Also, DNA-microarray analysis has shown that ctrA
and cckA mutants have almost identical expression
profiles34. Perhaps the strongest evidence for the role of
CckA in the regulation of CtrA is that a temperaturesensitive cckA mutant contains little to no phosphorylated CtrA33. CckA is also phosphorylated in a temporal
cell-cycle pattern that closely matches that of CtrA34.
These data indicate that an important role of CckA is
to phosphorylate CtrA, but it is not known whether
the effect is direct or indirect. CckA is a hybrid histidine kinase, which is a fusion between a kinase and a
response regulator (BOX 2). Hybrid histidine kinases
often participate in MULTI-COMPONENT PHOSPHORELAYS, in
which a histidine phosphotransferase (Hpt) protein
Histidine kinase
Response regulator
DivJ
PleC
CckN
CckO
DivL
CckA
DivK
Hpt
Histidine phosphotransferase
Response regulator
Hpt
CtrA
Figure 4 | Phosphorylation of the essential response regulators CtrA and DivK. The figure
shows a model for the signal transduction pathways that affect DivK and CtrA phosphorylation.
Solid arrows indicate pathways that are supported by direct experiments and dashed arrows
show proposed or hypothetical pathways. There is evidence for at least five histidine kinases that
bind to and/or influence the phosphorylation state of the essential response regulator DivK. DivJ
has been shown to phosphorylate DivK in vitro37. PleC seems to function as a phosphatase for
DivK~P37,65. CckN, CckO and DivL have been found to interact with DivK in a two-hybrid screen36.
Although DivJ and DivL can both phosphorylate CtrA in vitro27, only CckA has been shown to be
required for CtrA phosphorylation in vivo 33. An allele of ctrA was identified that can suppress
mutations in the three div genes and pleC 26, suggesting that all five components lie in the same
pathway. This might involve an unidentified histidine phosphotransferase (Hpt), which shuttles a
phosphoryl group from DivK to CtrA, both response regulators. CckA is a hybrid kinase (BOX 2),
which also raises the possibility of a Hpt-mediated pathway that controls CtrA phosphorylation.
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transfers a phosphoryl group from the hybrid kinase to
a second response regulator. This raises the possibility
that a Hpt protein functions in Caulobacter to mediate
signal transduction between CckA and CtrA.
The kinases PleC, DivJ and DivL, and the response
regulator DivK, are also thought to help mediate the
phosphorylation state of CtrA26,27. DivJ, DivK and DivL
were each identified as genetic suppressors of pleC 35, and
an allele of ctrA was subsequently found that was a suppressor of the three mutant div alleles26, which suggests
that all of these genes function in the same pathway. More
recently, a yeast two-hybrid analysis, which used DivK as
the bait, showed that DivK interacts not only with DivJ,
PleC and DivL, but also with two other uncharacterized
kinases, CckN and CckO36. Using purified proteins, direct
biochemical links have been established in vitro for
some of these two-component proteins26,27,37, but the
in vivo significance of these interactions remains unclear
(FIG. 4). A complete understanding of the complex pathways controlling CtrA phosphorylation will ultimately
require more detailed genetic and biochemical analysis,
similar to that needed for elucidating other complex
two-component signalling pathways, such as the phosphorelay pathway that leads to activation of Spo0A, the
master regulator of sporulation in B. subtilis38.
Mapping the CtrA regulon
The complex regulation of CtrA activity, along with
the essential nature of the ctrA gene, indicates that this
transcription factor has an important role in controlling cell-cycle patterns of gene expression. Early work
on CtrA showed that it transcriptionally controls
about a dozen genes, the products of which participate
in cell-cycle events16,18,20,39,40.
More recently, whole-genome DNA microarray
analyses have provided a complete description of the
CtrA regulon2,3 (FIG. 5). Global expression profiles of a
Caulobacter strain with a temperature-sensitive allele
of ctrA were obtained using microarrays. In total,
144, or 26% of all 553 cell-cycle-regulated genes, were
significantly affected by shifting to the restrictive
temperature and the subsequent loss of CtrA function 3; this included 84 genes that are activated by
CtrA and 60 genes that are repressed by CtrA.
Expression profiling alone is insufficient to determine
whether these regulatory effects are direct, but a technique known as location analysis (BOX 4) allows mapping of the DNA binding sites of a protein in vivo on
a genome-wide scale41,42. This technique was used to
identify the 95 genes in 55 operons that have regulatory regions bound by CtrA, and that also showed
significant changes in transcript levels in the ctrA
mutant strain. These 95 directly regulated genes comprise the CtrA regulon2. Twenty-nine of these genes
are repressed by CtrA — most of these genes show
maximal expression during the G1–S transition,
which is precisely when CtrA has been degraded and
therefore depleted from the cell. Sixty-six genes are
activated by CtrA. Although most of these genes
are expressed after accumulation of CtrA during
S phase, their peak induction times span a 60-minute
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P1
P2
ctrA
CtrA
CtrA ~ P
CtrA binding site
and promoter
Regulatory
pathways
Cell-cycle
regulation
8 regulatory genes
PilA
Pilus
biogenesis
Che
Chemotaxis
machinery
pilA
che (14 genes)
Cell-cycle
regulation
3 regulatory genes
DivK
divK
Fla
Flagellum
biosynthesis
CcrM
DNA
methylation
fla (16 genes)
ClpP
CtrA
proteolysis
HfaAB
Holdfast
synthesis
clpP
ccrM
hfaAB
FtsZ
?
ftsZ
39 unknown genes
Fts
Cell division,
cell-wall synthesis
fts (ftsQ, ftsW, murG)
Chromosome
replication
Origin of replication
Figure 5 | CtrA regulon. The master regulator CtrA controls many cell-cycle events. Phosphorylated CtrA (CtrA~P) directly
activates or represses the transcription of 95 genes2, which constitute the CtrA regulon. These include genes that are required for
essential cell-cycle processes, such as cell division (ftsZ) and DNA methylation (ccrM). CtrA~P also controls polar morphogenesis
by activating the flagellar biogenesis cascade, activating the transcription of the pilA gene, which encodes the pilin subunit, and
activating the transcription of genes that are needed for holdfast synthesis (hfaAB). CtrA~P blocks chromosome replication in the
swarmer cell by binding to five sites in the origin of replication28. CtrA~P also regulates a number of other regulatory genes,
including two (divK and clpP) that are involved in cell-cycle-regulated proteolysis of CtrA.
MULTI-COMPONENT
PHOSPHORELAY
A signalling pathway involving
two-component signal
transduction molecules, in
which a phosphoryl group from
ATP is transferred to more than
two components. Usually
involves transfer from a histidine
kinase to a response regulator to
a histidine phosphotransferase
to another response regulator.
CLOSED-LOOP SET
A set of regulatory factors that
regulate each other such that the
overall topology produces a
circle, or loop, of interactions.
period. This raises the question of how the timing of
expression of CtrA-activated genes can vary so much.
One possibility is that the affinities of different promoters
for CtrA vary significantly, affecting the timing of gene
expression39. Alternatively, additional transcription
factors, or alternative sigma factors, could ‘fine-tune’
the timing of different CtrA-dependent genes.
The functions of CtrA-regulated genes can be
divided into three main classes2: first, genes that are
required for polar morphogenesis, including flagellum
and pilus biogenesis; second, genes that are essential
for cell-cycle processes, such as cell division and DNA
methylation; and third, regulatory genes (FIG. 5). CtrA
directly controls expression of 11 regulatory genes,
which indicates that the Caulobacter cell cycle might be
controlled, at least in part, by a cascaded, CLOSED-LOOP
SET of regulatory genes, similar to that proposed for the
yeast cell cycle43. It is of particular interest to note that
two members of the CtrA regulon2, divK and clpP, are
involved in the cell-cycle-regulated proteolysis of
CtrA. Positive transcriptional activation of divK and
NATURE REVIEWS | MICROBIOLOGY
clpP by CtrA, followed by negative feedback in the
form of DivK/ClpXP-mediated CtrA proteolysis,
might form the basis of an oscillator that underlies
cell-cycle progression in Caulobacter.
Cell cycle: dependencies and checkpoints
Cell-cycle progression in Caulobacter requires the precise
temporal coordination of cellular and morphological
events, and seems to be accomplished largely by the organization of cell-cycle events into a series of dependent
relationships. In particular, cell division is known to be
dependent on successful DNA replication44 and the completion of chromosome segregation. But what is the
molecular basis for these critical dependencies? Hartwell
and Weinert45 proposed that similar ‘dependent events’
in the eukaryotic cell cycle could be coupled by either of
two distinct mechanisms: substrate–product relationships and checkpoints. We will briefly review each of
these mechanisms and then discuss what is known about
their role in coupling dependent processes during the
cell cycle and during morphogenesis in Caulobacter.
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Box 4 | DNA binding site analysis by immunoprecipitation and DNA microarrays
The figure shows a schematic
CtrA
CtrA-bound
promotor
diagram of location analysis, a
procedure that is used to identify
in vivo binding sites for
transcription factors on a genomewide scale2,41,42. Cells are treated
with formaldehyde to crosslink
CtrA to its in vivo binding sites2.
Crosslink CtrA to DNA,
Genomic DNA is extracted,
extract and shear DNA
fragmented and then subjected to
immunoprecipitation (IP) with an
anti-CtrA antibody.
IP without
IP with
A mock IP is performed without
antibody
CtrA-specific
(mock IP)
antibody
antibody as a control. The
crosslinks are reversed, and the
immunoprecipitated DNA is
Reverse crosslinks,
amplified by ligation-mediated
amplify and label by PCR
PCR. The DNA amplified from the
IP is labelled with the fluorophore
Cy5 (shown in red) and, using a
DNA microarray, is compared with
DNA amplified from the mock IP
that is labelled with the
fluorophore Cy3 (shown in green). DNA targets bound in vivo by CtrA should be enriched in the CtrA–IP sample and
the corresponding spots will appear more red (high Cy5:Cy3 ratio) on the array. Location analysis can be done with
any protein for which an antibody is available, and has been used to study binding of transcription factors in a number
of organisms41,43,77. Figure reproduced with permission from REF. 2 © (2002) National Academy of Sciences, USA.
The substrate–product mechanism is exemplified
by bacteriophage T4 baseplate morphogenesis46. The
T4 baseplate is composed of six identical ‘wedges’, each
containing seven different proteins. The assembly of the
wedge occurs in a precisely ordered pathway (FIG. 6a).
Each step in the assembly process forms an essential
substrate for the addition of the next subunit. The order
of assembly is controlled by the physical chemistry of
protein–protein interactions, and by the conformational
changes that occur with proper assembly46. This
example shows that a complex process can be precisely
ordered without the need for any extrinsic factors; the
components of the process itself have an intrinsic set of
dependent interactions. Alternatively, a checkpoint
mechanism could exist to ensure the proper execution of
cell-cycle events. Hartwell and Weinert45 originally
defined a cell-cycle checkpoint as a dedicated surveillance
mechanism that, in response to a failure to complete
event A, negatively regulates event B. Such a system is
extrinsic to the events themselves, because the components of the checkpoint system are not involved in the
execution of either event. The key to distinguishing a
checkpoint mechanism from a substrate–product
relationship is the identification of a mutation that
gives ‘relief of inhibition’45. A mutation in an extrinsic,
checkpoint system would relieve the inhibition that
is imposed on event B by the failure of event A. For
substrate–product relationships, no such mutation can
be found, because the dependency is caused entirely by
factors intrinsic to events A and B.
The SOS response is one example of a cell-cycle
checkpoint in a bacterial system47 (FIG. 6b). After exposure
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to ultraviolet (UV) irradiation, or other DNA damaging agents, E. coli cells are blocked for cell division until
the damage is repaired, which ensures that any progeny cells do not inherit a damaged chromosome; cell
division is therefore considered to be dependent on
successful DNA replication. In response to DNA damage, E. coli rapidly synthesizes the protein SulA, which
functions to specifically block cell division by binding
to FtsZ, an essential component of the cell division
machinery. By contrast, a sulA mutant does not properly arrest the cell cycle when DNA damage occurs,
because without SulA, FtsZ, and therefore cell division,
cannot be inhibited. The sulA mutation provides ‘relief
of inhibition’48. SulA is not normally required for DNA
replication or cell division. It acts extrinsically to both
processes, and ensures that their dependent relationship
is maintained in perturbed conditions; it constitutes a
true checkpoint system.
Are there cell-cycle checkpoints in Caulobacter? Although
Caulobacter does not have a sulA homologue, it does
couple DNA replication and cell division49,50. Treatment
of Caulobacter cells with DNA damaging agents transiently blocks cell division. In response to DNA damage,
transcription from the P2 promoter (FIG. 3) of ctrA is
rapidly reduced50. This leads to a decrease in the concentration of active, phosphorylated CtrA in the predivisional cell, which in turn prevents transcription of
two CtrA-dependent genes, ftsQ and ftsA, which are
required for the final stages of cell division. This establishes a mechanistic basis for coupling DNA damage to
the inhibition of cell division. However, no mutations
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a T4 baseplate wedge assembly
gp11
10
10
10
10
7
gp7
10
10
11
11
gp8
8 8
7
10
10
11
gp6
53
6
6
7
8 8
6
gp53
7
10
10
10
11
53
6
8 8
gp25
6
7
10
10
11
25
6
8 8
10
11
b Escherichia coli DNA damage response
DNA damage checkpoint
SOS response
DNA damage
sensed
SulA
DNA replication
Cell division
Cell division cycle
Figure 6 | Strategies for coupling-dependent
processes. Dependent processes can be controlled by
either a substrate–product or checkpoint mechanism.
a | Morphogenesis of the T4 baseplate wedge is a classic
example of a substrate–product relationship46. Assembly of
the wedge occurs in an invariant order by the successive
addition of subunits. For example, gp7 can bind only to the
gp10 dimer, and gp8 cannot bind to gp7 unless the latter is
preassembled into a gp10–gp7 complex. These
dependencies ensure that wedge assembly follows an
invariant pathway (gp10, gp7, gp8, gp6, gp53 and gp25).
If any of the subunits are missing, baseplate assembly is
blocked. Addition of gp11 can occur at any time, so it is not
part of the dependency pathway. b | The SOS response in
Escherichia coli is an example of a bacterial cell-cycle
checkpoint47, as described in the text. During an unperturbed
cell cycle (blue box), cell division is dependent on DNA
replication and undamaged DNA. In response to DNA
damage, the checkpoint system is activated (orange box).
The checkpoint surveillance mechanism is extrinsic to the
process it controls and is dispensable in a normal cell cycle.
Detection of DNA damage activates the SOS response,
resulting in production of the SulA protein. SulA inhibits the
polymerization of FtsZ, which is an essential cell division
protein, and cells arrest until the DNA damage is repaired.
Mutations in sulA relieve this inhibition.
NATURE REVIEWS | MICROBIOLOGY
have yet been found that give ‘relief of inhibition’, which
is the gold standard for identifying a checkpoint.
Hypothetically, the identification of relief-of-inhibition
mutations would reveal genes that function in a dedicated
surveillance system that monitors DNA damage, and
transmits an inhibitory signal to regulators of, or essential
components of, the cell-cycle machinery, such as CtrA.
Another dependency relationship in the Caulobacter
cell cycle that could be governed by a checkpoint
mechanism is the requirement for completion of
chromosome segregation before cell division. Several
highly conserved genes, including parA, parB, parC,
parE and smc in Caulobacter, have been shown to have
essential roles in chromosome segregation51–54.
Depletion of any one of these proteins prevents chromosome segregation, which leads to a block of cell
division. This dependency ensures that each daughter
cell inherits a full complement of the Caulobacter
genome, but no mutation has yet been described that
gives ‘relief of inhibition’, leading to an attempt at cell
division without the completion of chromosome segregation. Such a mutation would help to identify a
dedicated, extrinsic surveillance system that monitors
the status of chromosome segregation, if one exists.
Alternatively, the coupling of chromosome segregation
to cell division might be driven by a mechanism that is
more similar to the substrate–product mechanism
described above. In fact, in E. coli, the interruption of
chromosome segregation and the physical presence
of DNA at the mid-cell site, prevents the cytokinesis
machinery from executing cell division, a mechanism
called nucleoid occlusion55. Whether Caulobacter
operates in a similar manner or uses a dedicated
checkpoint system remains to be shown.
Coupling morphological events to gene expression.
There are several well-studied examples in Caulobacter
that show that gene expression is dependent on the
completion of a morphological event, most of which
function to ensure the proper assembly of the polar flagellum. This complex organelle is composed of more
than 30 gene products, and as described before, these
gene products are arranged in a four-tiered cascade
(class I–IV), in which the order of gene expression parallels the order of flagellum assembly (FIG. 2). The expression of the genes in each class depends on the expression
and assembly of the preceding class. These so-called
‘morphological couplings’56 seem to involve dedicated
surveillance machinery, but are intrinsic to the system,
and therefore incorporate aspects of both of the coupling
mechanisms described by Hartwell and Weinert.
A defect in the expression or assembly of a class II
flagellar component prevents the transcription of class
III and class IV genes. Mutants were identified that
restore class III gene expression in a class II mutant57.
These ‘bypass of flagellar assembly’ (bfa) mutants are
dominant mutations in two regulatory genes, flbD, and
fliX58–60. FliX is not a structural component of the flagellum, but instead seems to transmit the state of flagellar
assembly to FlbD, which is a transcription factor. FliX,
by an unknown mechanism, keeps FlbD inactive until
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class II gene products have properly assembled. FlbD
is then activated, class III gene expression, and therefore flagellar assembly, continues. Neither fliX nor flbD
are part of the flagellum — they seem to be dedicated
to surveillance of flagellar assembly and so are reminiscent of a checkpoint system. But both FliX and FlbD are
required every time a flagellum is built in Caulobacter,
so they are intrinsic to the assembly process, in contrast
to canonical cell-cycle checkpoint systems that are
required when a process malfunctions, but are otherwise
dispensable.
Morphological coupling also occurs between class
III and class IV flagellar genes: mutations in class III
genes prevent the production of flagellar filament
monomers that are encoded by the class IV genes. The
FlbT protein binds to a 5′ untranslated region of class IV
mRNAs and facilitates their degradation61–63. By a
mechanism that is not yet understood, the proper
assembly of flagellar subunits encoded by class III
genes prevents FlbT-mediated degradation of class IV
flagellin mRNAs. This, in turn, leads to synthesis of the
flagellin monomers and, ultimately, to their assembly
into a functioning flagellar filament. In common with
fliX and flbD, flbT seems to be dedicated to the surveillance of proper flagellar assembly, but it is intrinsic
to the process because it is required each time the
organism assembles a flagellum. The FlbT-based coupling of class III and class IV flagellar genes therefore
has elements of both a classical checkpoint system and
a substrate–product mechanism.
Subcellular protein localization
NASCENT SWARMER POLE
The pole opposite the stalked
pole in a Caulobacter
predivisional cell, where the
flagellum and pilus secretion
apparatus must be assembled
before cell division takes place.
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In addition to the elaborate circuitry that controls
temporal progression through the cell cycle, every
round of cell division requires the spatial organization
of an asymmetric predivisional cell to produce two different daughter cells. From work with in vivo fluorescent
reporters, such as green fluorescent protein (GFP), it
has become clear in recent years that in many bacteria,
subcellular localization of regulatory proteins can contribute to these spatial patterning processes — bacteria
can no longer be perceived as simple ‘bags of
enzymes’7,64.
The subcellular localization of three Caulobacter
histidine kinases (CckA, DivJ and PleC) is important for
the production of daughter cells that have distinct fates
(FIG. 7). In the predivisional cell, CckA and PleC localize
predominantly at the NASCENT SWARMER POLE, whereas DivJ
localizes almost entirely at the stalked pole33,65. CckA is
necessary for the phosphorylation and activation of the
master regulator CtrA. The localization of CckA to the
swarmer cell compartment is consistent with the observation that swarmer cells contain increased concentrations of CtrA~P compared with stalked cells. Therefore,
it seems that the localization and asymmetric distribution of the CckA kinase helps to establish an important
difference in daughter cell fate: new swarmer cells cannot
initiate DNA replication because high concentrations of
CtrA~P repress the origin of replication, whereas new
stalked cells, with little to no CtrA~P, are free to initiate
S phase. DivJ and PleC also help to establish different
cell fates in the two daughter cells. DivJ is a kinase that
phosphorylates two response regulators, DivK and PleD.
By contrast, PleC has demonstrated phosphatase activity
for DivK~P, and has recently been shown in vivo to
directly or indirectly inhibit the formation of PleD~P,
possibly as a phosphatase26,27,37,66. Taken together, these
data indicate that the localization of DivJ and PleC to
opposite poles65 — stalked and swarmer, respectively —
of the late predivisional cell helps to ensure high levels of
phosphorylated DivK (DivK~P) and phosphorylated
PleD (PleD~P) only in the new stalked cell. The asymmetric distribution of DivJ and PleC might therefore
help to determine daughter cell fates: active DivK is
required for CtrA degradation25, so its presence in the
stalked portion of septated predivisional cells presumably targets CtrA for degradation and thereby triggers
initiation of DNA replication in this daughter cell.
Conversely, without active DivK in the nascent
swarmer cell, CtrA will not be degraded and will
repress the initiation of DNA replication. By a mechanism
that is not yet understood, phosphorylated PleD triggers
flagellar release at the swarmer-to-stalked-cell transition67,68, so the inheritance of PleC to the swarmer cell
probably helps to prevent flagellar release in that cell type
as PleC inhibits the formation of PleD~P. Differentiation
into a stalked cell, and the localization of DivJ to the
stalked pole, could therefore initiate PleD-mediated
flagellar release. Localization of PleC to the swarmer pole
is also critical for the proper assembly of pili only in
swarmer cells69,70. PleC is required for accumulation of
the pilin subunit PilA, which polymerizes into the pilus
filament. Interestingly, cells containing an active, but
delocalized, PleC fail to accumulate PilA, which provides
the first evidence in Caulobacter that precise subcellular
localization of a kinase in the membrane is required for
kinase activity71.
PodJ is at the top of a localization hierarchy. How are
these kinases targeted to, and retained at, a specific cell
pole? Little is known about the localization of CckA or
DivJ, but a localization determinant for the kinase
PleC has recently been identified71. This factor, which
is encoded by the podJ gene, is a large, membraneassociated protein that is localized to the nascent
swarmer pole in predivisional cells. PodJ is required for
the correct localization of PleC to the swarmer pole,
and also specifies the localization of CpaC and CpaE —
two membrane-bound proteins that are involved in
pilus biogenesis. After localizing to the nascent swarmer
pole, and directing the localization of PleC, CpaC and
CpaE, PodJ is processed by an unknown protease to a
shorter form that is retained at the swarmer pole,
although the functional consequences of this processing
are not understood71,72. Full-length PodJ is predicted to
contain a long, α-helical region at the N-terminus73,
and has motifs that are a signature of coiled-coils — a
structural motif that is often involved in protein–protein
interactions. PodJ could interact with itself through
this region, potentially providing a structural scaffold
for the assembly of other proteins, such as PleC. PodJ
might also localize other proteins through its predicted
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PleC
PleC
CckA
CckA
CtrA ~ P
CckA
PleD
CtrA ~ P
CtrA ~ P
PleC
PleD
DivK
DivK
DivK ~ P
CtrA ~ P
DivK ~ P
PleD
DivK
DivK ~ P
PleD ~ P
PleD ~ P
PleD ~ P
PleC
CckA
DivK ~ P
DivJ
CckA
DivJ
PleD ~ P
CckA
DivJ
DivJ
Figure 7 | Subcellular localization and cellular asymmetry. Asymmetry in the Caulobacter predivisional cell is determined largely
by the differential localization of three histidine kinases (PleC, CckA and DivJ) and the response regulators that they control (CtrA,
DivK and PleD). PleC and CckA localize predominantly to the nascent flagellar pole33,65, whereas DivJ is found mostly at the stalked
pole65. PleC has activity as a DivK~P phosphatase and acts to inhibit, perhaps as a phosphatase, the formation of phosphorylated
PleD (PleD~P) in vivo37,65,66. By contrast, DivJ acts as a DivK and PleD kinase37,66. The combination of these factors leads to higher
concentrations of PleD~P and DivK~P in stalked cells than in swarmer cells. PleD~P is required for loss of flagellar motility and
DivK~P is required for CtrA degradation; therefore, higher concentrations of these two active response regulators in stalked cells
helps determine key aspects of stalked cell fate. CckA is required for CtrA phosphorylation and is predominantly localized to the
swarmer pole. This presumably helps establish active CtrA only in the swarmer cell where it can inhibit DNA replication.
tetratricopeptide repeat motifs (TPR), which are motifs
that are known to mediate protein–protein interactions
in a number of other systems74.
How is PodJ localized to the cell pole? Intriguingly,
the extreme C-terminus of PodJ has weak homology to
peptidoglycan-binding domains. So, one possibility is
that PodJ interacts with a cell wall structure that is itself
specifically located at the nascent swarmer pole, which, as
the site of cell division in the preceding cell cycle, might
contain a peptidoglycan structure that was formerly associated with septum assembly. Although little is known
about how PodJ functions, this protein clearly has an
important role in the spatial patterning processes in
Caulobacter, and it will prove fascinating as the mechanisms of localization are unravelled. Homologues of
PodJ are found throughout the α-subdivision of proteobacteria, so PodJ could have an important role in
establishing cell polarity in many bacteria.
Future directions
Caulobacter has become the predominant model system
for understanding the regulation of bacterial cellcycle progression at a molecular level. Studying the
Caulobacter cell-division cycle has begun to reveal the
mechanisms by which bacterial cells establish and maintain spatial asymmetry. Specifically, two-component signal transduction proteins have emerged as factors that
are of prime importance in the control of the cell cycle
and asymmetric cell division. The advent and application of genomic technologies has revolutionized the pace
NATURE REVIEWS | MICROBIOLOGY
of progress in mapping and understanding the intricate
genetic network controlling the Caulobacter life cycle.
Global analyses have so far focused on transcriptional
regulation, because DNA microarrays, the most mature
of genomic technologies, are designed to assay gene
expression and RNA levels. Global analyses of other
modes of regulation, including post-translational modifications, protein–protein interactions and subcellular
localization, will be important in the development of a
more complete, molecular-level picture of cell-cycle progression in Caulobacter. Better computational tools will
be needed to assemble, store, analyse and mine these new
and complex data sets.
In this era of global analyses, new challenges also arise
beyond understanding the role of individual components
in the cell-cycle regulatory network. For example, how
does a single Caulobacter cell coordinate the activities of
more than 100 two-component signalling proteins?
Given that these proteins are very closely related to each
other, how does the cell ensure specificity of cognate pairs
and prevent unwanted cross-talk? In the case of response
regulators such as DivK and CtrA, which are controlled
by numerous kinases, is there signal integration? Or are
these pathways responding to different signals at different
times? Systematic analysis of the two-component systems
in Caulobacter will be needed to begin addressing these
‘systems-level’ questions. We anticipate that such analyses
will illuminate general design principles and regulatory
schemes that are used to ensure the fidelity of cell-cycle
progression and to generate cellular asymmetry.
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Acknowledgements
We apologize to our colleagues whose work was not cited owing
to space constraints. We thank L. Garwin and A. Greenwood for
helpful comments on the manuscript. Work in the Laub laboratory
is supported by the Office of Science (BER), US Department of
Energy, the National Institutes of Health and the Defense Advanced
Research Projects Agency.
Competing interests statement
The authors declare that they have no competing financial interests.
Online links
DATABASES
The following terms in this article are linked online to:
Entrez: http://www.ncbi.nlm.nih.gov/Entrez/
Agrobacterium tumefaciens | Caulobacter crescentus |
Escherichia coli | Pseudomonas aeruginosa | Rickettsii prowazekii |
Sinorhizobium meliloti
SwissProt: http://www.ca.expasy.org/sprot/
CckA | CpaC | CpaE | CtrA | DivJ | DivK | DivL | FlbD | FliX | FtsZ |
PleC | PodJ | Spo0A | Spo0F
Access to this interactive links box is free online.
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