© 2004 Landes Bioscience. Do Not Distribute
replication. The stalked cell immediately initiates and new round of DNA replication,
while the swarmer cell must first differentiate into a stalked cell as described above to reini-
tiate the cell cycle. Global transcriptional analysis of CtrA mutants10has shown that CtrA
not only acts as an origin inhibitor, but also regulates genes required for cell division, DNA
methylation, polar flagellar and pili biogenesis, and chemotaxis.
[Cell Cycle 3:10, e55-e57, EPUB Ahead of Print: http://www.landesbioscience.com/journals/cc/abstract.php?id=1181; October 2004]; ©2004 Landes Bioscience
2004; Vol. 3 Issue 10
Department of Developmental Biology; Stanford University School of Medicine;
Stanford, California USA
*Correspondence to: Lucy Shapiro; Department of Developmental Biology;
Stanford University School of Medicine; Beckman Center B300; Stanford, California
94305 USA; Tel.: 650.725.7678; Fax: 650.725.7739; Email: email@example.com-
Received 08/13/04; Accepted 08/17/04
This manuscript has been published online, prior to printing for CellCycle, Volume
3, Issue 10. Definitive page numbers have not been assigned. The current citation
is: Cell Cycle 2004; 3(10):
Once the issue is complete and page numbers have been assigned, the citation will
cell cycle, bacteria, Caulobacter, genetic circuit,
A Genetic Oscillator and the Regulation of Cell Cycle Progression in
Analyses of cell polarity, division, and differentiation in prokaryotes have identified
several regulatory proteins that exhibit dramatic changes in expression and spatial local-
ization over the course of a cell cycle. The dynamic behavior of these proteins is often
intrinsically linked to their function as polarity determinants.1-3In the α-proteobacterium,
Caulobacter crescentus, the CtrA global transcriptional regulator exhibits a spatially and
temporally dynamic expression pattern across the cell cycle. CtrA plays key roles in
asymmetric cell division and in the timing of chromosome replication.3,4An additional
global regulator, GcrA, has recently been discovered that both regulates and is regulated
by CtrA.5Together, these regulatory proteins create a genetic circuit in which the cellular
concentrations of CtrA and GcrA oscillate spatially and temporally to control daughter
cell differentiation and cell cycle progression.
CELL CYCLE PROGRESSION AND DIMORPHISM IN CAULOBACTER CRESCENTUS
In C. crescentus, a tightly orchestrated signaling network controls asymmetric cell division
yielding two developmentally-distinct daughter cell types: (1) swarmer cells that possess a
single polar flagellum and pili and whose chromosome replication is inhibited, and (2)
stalked cells that possess a thin polar stalk capped with an adhesive holdfast that actively
replicate their chromosome (Fig. 1A). The dimorphic nature of Caulobacter permits isolation
of a homogenous population of swarmer cells by means of density gradient centrifugation.6
These synchronized swarmer cells can then be tracked through the cell cycle as they first
shed their polar flagellum and pili and then grow a stalk at the same pole. The newly-
formed stalked cells subsequently create chemotaxis control circuitry and build a flagellum
and pili secretion complex at the pole opposite the stalk, which becomes a pole of the
nascent daughter swarmer cell (Fig. 1A). Thus the complement of polar organelles at the
Caulobacter cell poles is radically changed during the course of a cell cycle, with each pole
fated to first grow a flagellum and then a stalk.
The timing of chromosome replication is also tightly controlled during the cell cycle.
The phosphorylated form of the CtrA response regulator (CtrA~P) binds to the chromo-
somal origin of replication in the swarmer cell, repressing initiation of replication (Fig. 1A).
Shortly after the swarmer cell’s flagellum and pili are shed, CtrA~P is proteolyzed7by the
ClpXP protease8permitting initiation of chromosome replication during the stalked and
predivisional phases of the cell cycle. A restrictive barrier forms between the incipient
daughter cells following the completion of chromosome partitioning to the two halves of
the predivisional cell, but well before cell separation.9As soon as two distinct compartments
are formed, CtrA~P is proteolyzed in the daughter stalked cell7but not in the daughter
swarmer cell where the continued presence of CtrA~P represses initiation of chromosome
CTRA AND GCRA ARE OSCILLATING COREGULATORS OF THE
CAULOBACTER CELL CYCLE
CtrA is removed from early stalked cells by proteolysis, then is resynthesized to high
levels in late-predivisional and swarmer cells7(Figs. 1A and B). This changing level of
© 2004 Landes Bioscience. Do Not Distribute
activates transcription of ctrA from its hemi-methylated
P1 promoter, which is created by the passage of the repli-
cation fork (Fig. 2). This ties activation of ctrAtranscription
by GcrA to a specific time in the progression of DNA replication. A
chromatin immunoprecipitation assay revealed an interaction
between GcrA and the promoter regions of several genes including
the replication initiation factor dnaA, the polarity regulator podJ,
and ctrA. It is not known if these promoter interactions are direct or
are mediated by another protein. Notably, alignment of GcrA
against the Conserved Domain Database20reveals no homology to
known DNA-binding domains. Thus, if the interaction with DNA
CtrA is key to its role as a silencer of the origin of DNA
replication in swarmer cells and as an activator of the gene
encoding the CcrM DNA methyltransferase and flagellar
and chemotaxis genes in predivisional cells.10-12The strik-
ing temporal and spatial oscillatory pattern of CtrA
concentration during the cell cycle raises the question of
what regulatory factors control CtrA expression and activity.
It is known that regulated proteolysis by ClpXP8plays an
important role by rapidly decreasing CtrA concentration
at the swarmer-to-stalked cell transition. Another layer of
CtrA regulation is provided by the essential histidine
kinase CckA, which directly phosphorylates CtrA to the
CtrA~P form,13thereby serving as a post-translational
regulator of its DNA-binding activity14(Fig. 2).
Additional regulation occurs at the P1 and P2 promoters
of ctrA itself, with CtrA serving as an auto-activator of its
P2 promoter and a repressor of its P1 promoter15(Fig. 2).
Transcription of ctrA in stalked cells begins from the P1
promoter shortly after DNA replication initiates. The
positive feedback loop between CtrA and its P2 promoter
upregulates ctrAtranscription in predivisional cells15(Fig. 2).
The methylation state of the chromosome introduces yet
another layer of regulation. Specifically, the ctrA P1 promoter
is repressed when the chromosome is fully methylated,
and accessible for activation only when hemi-methylated.16
Replication initiates on a fully methylated chromosome,
and as the replication fork passes the ctrA gene two hemi-
methylated copies are generated, thus priming ctrA for
transcriptional activation. The essential methyltransferase
CcrM,17which is under positive transcriptional control
by CtrA, subsequently silences the P1 promoter as it
remethylates the chromosome in the late predivisional cell.
While the mechanisms described above partially
explain how the concentration of CtrA is controlled
throughout the cell cycle, until recently no factor had
been attributed to the upregulation of ctrA transcription
in stalked and predivisional cells. Now studies of a temper-
ature sensitive Caulobacter mutant by Holtzendorff and
colleagues5have identified another essential gene, gcrA,
which acts as a positive regulator of ctrA and that is itself
repressed by CtrA. gcrA encodes a 190 amino acid protein
that is conserved among the α-proteobacteria but is not
found in other families of sequenced prokaryotes or
eukaryotes. GcrA is most highly expressed in stalked and
early predivisional cells. GcrA positively regulates transcrip-
tion of genes required for maintenance of cellular asym-
metry such as the localization factor, podJ18and the histi-
dine kinase pleC19as well as genes required for formation
of the replication machinery. Importantly, GcrA also
is direct, GcrA almost certainly binds DNA in a novel way.
The oscillatory behavior of the CtrA/GcrA genetic circuit arises
because GcrA acts as a positive regulator of CtrA, while CtrA acts as
a negative regulator of GcrA. The CtrA/GcrA genetic circuit shown
in Figure 2, results in spatial and temporal separation of expression
of these global regulators over the course of a cell cycle.5These
out-of-phase oscillations of the CtrA and GcrA master regulators
(Fig. 1B) drive the progression of the Caulobacter cell cycle. In
A GENETIC OSCILLATOR IN CAULOBACTER
Figure 1. (A) Cartoon of the Caulobacter crescentus cell cycle. CtrA is shown in red and
GcrA in blue. The chromosome is shown as a circle inside the cell with the theta-like
structure representing replicating DNA. (B) Protein levels of CtrA (solid line) and GcrA
(dashed line) over the course of the cell cycle.
Figure 2. Model of the CtrA/GcrA genetic circuit.
© 2004 Landes Bioscience. Do Not Distribute
1. Margolin W. Spatial regulation of cytokinesis in bacteria. Curr Opin Microbiol 2001;
2. Shapiro L, McAdams HH, Losick R. Generating and exploiting polarity in bacteria.
Science 2002; 298:1942-6.
3. Ausmees N, Jacobs-Wagner C. Spatial and temporal control of differentiation and cell cycle
progression in Caulobacter crescentus. Annu Rev Microbiol 2003; 57:225-47.
4. Skerker JM, Laub MT. Cell-cycle progression and the generation of asymmetry in
Caulobacter crescentus. Nat Rev Microbiol 2004; 2:325-37.
5. Holtzendorff J, Hung D, Brende P, Reisenauer A, Viollier PH, McAdams HH, Shapiro L.
Oscillating global regulators control the genetic circuit driving a bacterial cell cycle. Science
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e57Cell Cycle 2004; Vol. 3 Issue 10
particular, the presence of CtrA~P in the swarmer cell inactivates
DNA replication by blocking the origin of replication and repressing
GcrA expression. At the swarmer-to-stalked cell transition, CtrA~P
is proteolyzed by ClpXP in response to an unknown signal. Upon
degradation of CtrA, gcrA is derepressed allowing its gene product to
activate transcription of genes required for DNA replication and
polar morphogenesis as well as transcription of ctrA when the
hemi-methylated promoter appears. The subsequent increase in
CtrA concentration renews repression of gcrA in predivisional and
ADDITIONAL REGULATORY PATHWAYS COUPLED TO THE
The oscillatory CtrA/GcrA feedback circuit described above is a
central element of the regulation of the Caulobacter cell cycle and
asymmetric cell division. Yet, even though these two master regulatory
proteins have been shown to directly control some 145 genes, it is
clear there must be one or more additional top-level master regulators.
One indicator of these missing regulatory proteins is the presence of
numerous other cell cycle-regulated genes that do not appear to be
regulated by either CtrA or GcrA (as one example, several components
of the DNA replication complex). In addition, there are numerous
status reporting signals from various cell cycle processes to the top-level
CtrA/GcrA circuit. Examples include signals indicating progress of
cell compartmentalization21and progress in construction of polar
organelles. When cells encounter environmental stress conditions or
sustain DNA damage, signals to halt or slow cell cycle progression
Sensory/signaling pathways that tie the intra- and extracellular
environment into cell cycle regulation remain largely unexplored. In
its natural dilute freshwater habitat, Caulobacter normally experiences
a wide range of physical and chemical conditions to which it must
adapt to survive. With sixty-one sensor histidine kinases annotated
in its genome and a total of 105 two-component regulatory proteins,22
Caulobacter appears to be well equipped to respond to its environment.
However, function has only been assigned to a handful of these sensory
proteins and the environmental signals affecting Caulobacter kinase
activity remain uncharacterized. Environmental signals (e.g., oxygen
binding, light detection, redox change, small molecule binding, etc.)
may feed into the pathway controlling CckA kinase activity to affect
the phosphorylation state and DNA-binding activity of CtrA.
Analysis of wild-type and two-component mutant strains of
Caulobacter grown under a variety of physiological conditions will
provide insight into how cell cycle progression is modulated by
changes in the nutrient availability, oxygen concentration, or the
presence of other physical and chemical signals. Certainly, we have
only begun to understand the regulatory systems underlying cell
cycle progression in this “simple” bacterial cell.
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transcriptional regulator controls the G1-to-S transition in a bacterial cell cycle. Cell 1997;
8. Jenal U, Fuchs T. An essential protease involved in bacterial cell-cycle control. EMBO J
9. Judd EM, Ryan KR, Moerner WE, Shapiro L, McAdams HH. Fluorescence bleaching
reveals asymmetric compartment formation prior to cell division in Caulobacter. Proc Natl
Acad Sci USA 2003; 100:8235-40.
10. Laub MT, Chen SL, Shapiro L, McAdams HH. Genes directly controlled by CtrA, a mas-
ter regulator of the Caulobacter cell cycle. Proc Natl Acad Sci USA 2002; 99:4632-7.
11. Quon KC, Marczynski GT, Shapiro L. Cell cycle control by an essential bacterial two-com-
ponent signal transduction protein. Cell 1996; 84:83-93.
12. Laub MT, McAdams TH, Feldblyum T, Fraser CM, Shapiro L. Global analysis of the
genetic network controlling a bacterial cell cycle. Science 2000; 290:2144-8.
13. Jacobs C, Domian IJ, Maddock JR, Shapiro L. Cell cycle-dependent polar localization of
an essential bacterial histidine kinase that controls DNA replication and cell division. Cell
14. Jacobs C, Ausmees N, Cordwell SJ, Shapiro L, Laub MT. Functions of the CckA histidine
kinase in Caulobacter cell cycle control. Mol Microbiol 2003; 47:1279-90.
15. Domian IJ, Reisenauer A, Shapiro L. Feedback control of a master bacterial cell-cycle reg-
ulator. Proc Natl Acad Sci USA 1999; 96:6648-53.
16. Reisenauer A, Shapiro L. DNA methylation affects the cell cycle transcription of the CtrA
global regulator in Caulobacter. EMBO J 2002; 21:4969-77.
17. Stephens C, Reisenauer A, Wright R, Shapiro L. A cell cycle-regulated bacterial DNA
methyltransferase is essential for viability. Proc Natl Acad Sci USA 1996; 93:1210-4.
18. Viollier PH, Sternheim N, Shapiro L. Identification of a localization factor for the polar
positioning of bacterial structural and regulatory proteins. Proc Natl Acad Sci USA 2002;
19. Viollier PH, Sternheim N, Shapiro L. A dynamically localized histidine kinase controls the
asymmetric distribution of polar pili proteins. EMBO J 2002; 21:4420-8.
20. Marchler-Bauer A, Anderson JB, DeWeese-Scott C, Fedorova ND, Geer LY, He S, Hurwitz
DI, Jackson JD, Jacobs AR, Lanczycki CJ, Liebert CA, Liu C, Madej T, Marchler GH,
Mazumder R, Nikolskaya AN, Panchenko AR, Rao BS, Shoemaker BA, Simonyan V, Song
JS, Thiessen PA, Vasudevan S, Wang Y, Yamashita RA, Yin JJ, Bryant SH. CDD: A curat-
ed Entrez database of conserved domain alignments. Nucleic Acids Res 2003; 31:383-7.
21. McGrath PT, Viollier P, McAdams HH. Setting the pace: Mechanisms tying Caulobacter
cell-cycle progression to macroscopic cellular events. Curr Opin Microbiol 2004; 7:192-7.
22. Nierman WC, Feldblyum TV, Laub MT, Paulsen IT, Nelson KE, Eisen JA, Heidelberg JF,
Alley MR, Ohta N, Maddock JR, Potocka I, Nelson WC, Newton A, Stephens C, Phadke
ND, Ely B, DeBoy RT, Dodson RJ, Durkin AS, Gwinn ML, Haft DH, Kolonay JF, Smit
J, Craven MB, Khouri H, Shetty J, Berry K, Utterback T, Tran K, Wolf A, Vamathevan J,
Ermolaeva M, White O, Salzberg SL, Venter JC, Shapiro L, Fraser CM, Eisen J. Complete
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A GENETIC OSCILLATOR IN CAULOBACTER