Research Article

Oscillating Global Regulators Control the Genetic Circuit Driving a Bacterial Cell Cycle

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Science  14 May 2004:
Vol. 304, Issue 5673, pp. 983-987
DOI: 10.1126/science.1095191

Abstract

A newly identified cell-cycle master regulator protein, GcrA, together with the CtrA master regulator, are key components of a genetic circuit that drives cell-cycle progression and asymmetric polar morphogenesis in Caulobacter crescentus. The circuit drives out-of-phase temporal and spatial oscillation of GcrA and CtrA concentrations, producing time- and space-dependent transcriptional regulation of modular functions that implement cell-cycle processes. The CtrA/GcrA regulatory circuit controls expression of polar differentiation factors and the timing of DNA replication. CtrA functions as a silencer of the replication origin and GcrA as an activator of components of the replisome and the segregation machinery.

Progress of the bacterial cell cycle involves an intricately coordinated succession of genetic events that control cell growth, genome replication and segregation, and cell division. For bacteria with asymmetric cell cycles (i.e., with progeny having different characteristics), additional cell position–specific processes have to be integrated and coordinated. Caulobacter crescentus divides asymmetrically in each cell cycle, producing swarmer and stalked progeny cells with different morphological features and different cell fates. The expression of about 15% of Caulobacter's genes (∼550 out of 3760) vary over the cell cycle (1).

The previously known master regulator protein CtrA is one of several temporally and spatially regulated two-component signal-transduction proteins that play a key role in coordinating chromosome replication and multiple other cell-cycle events (25). CtrA directly controls the transcription of 95 cell cycle–regulated genes (6) by binding to a conserved motif (7) in the promoters of 55 operons. Although CtrA had been shown to control multiple functional modules, such as polar organelle development, cell division, and DNA methylation, other cell cycle–regulated events, such as formation of the replisome and chromosome segregation, remained unaccounted for. We therefore predicted the existence of additional master regulator proteins that together with CtrA would form a regulatory circuit to provide top-down control of Caulobacter cell-cycle progression (3). Here, we identify an essential protein, GcrA, and we show that the genetic circuit involving GcrA and CtrA provides the motive force for cell-cycle progression.

The tight temporal and spatial control of CtrA expression, activation, and degradation mediates differential gene expression and protein accumulation during the cell cycle (6). CtrA is present in swarmer cells, where it prevents initiation of DNA replication by binding to five conserved sites within the origin of replication (8). At the swarmer-to-stalked cell transition, CtrA is proteolyzed by the ClpXP protease (9), relieving the repression of the origin of replication. DNA synthesis subsequently ties the start of de novo accumulation of CtrA to the progression of chromosome replication by a mechanism involving the methylation status of the ctrA P1 promoter (3, 10). The P1 promoter is repressed when fully methylated and activated only after the replication fork has passed. Rapid production of CtrA and its activation by the CckA histidine kinase (11, 12) controls the expression of many cell cycle–regulated genes (1, 7), including the ccrM methyltransferase. The ctrA P1 promoter is then turned off first by autorepression by phosphorylated CtrA (CtrA∼P) (13) and then through the action of CcrM, which converts the active hemimethylated promoter into the inactive fully methylated state (10). Considerably before cell division, the predivisional cell is compartmentalized (14) and CtrA∼P in the stalked-cell compartment is inactivated by dephosphorylation and targeted proteolysis. The disappearance of CtrA∼P then permits re-initiation of DNA replication (13) and allows expression of the new global regulator, GcrA.

gcrA is an essential gene. The same temperature-sensitive genetic screen used to identify CtrA (7) and the CckA histidine kinase (11) was used to isolate gcrA (CC2245) (15). gcrA is predicted to encode a 25-kD protein that is conserved in several α-proteobacteria and that lacks known functional motifs. To analyze the function of this protein, the endogenous gcrA gene was disrupted and the gcrA coding sequence under the control of a xylose-inducible promoter (PxylX) (16) was integrated into the chromosome in single copy at the xyl locus. In this LS3707 strain, the majority of the original wild-type gcrA coding region was replaced with a spectinomycin/streptomycin resistance gene cassette (ΔgcrA::Ω) flanked by transcriptional and translational stop signals (17). Two hours after shifting LS3707 from peptone-yeast extract (PYE) + 0.3% xylose (PYEX) into PYE + 0.2% glucose (PYEG) to inactivate PxylX, GcrA could no longer be detected by immunoblots (Fig. 1A). LS3707 is viable when grown in PYEX, but GcrA depletion is lethal 6 hours after shifting to PYEG (Fig. 1B), indicating that GcrA is an essential protein.

Fig. 1.

gcrA is an essential gene. (A) Immunoblot analysis with GcrA antibodies of LS3707 cells depleted of GcrA after a shift from PYEX (xylose) into PYEG (glucose) medium. The GcrA protein is not detectable after 2 hours of growth in PYEG. wt, wild-type control. (B) Viability of strain LS3707 after shift from PYEX to PYEG medium.

gcrAis negatively regulated by CtrA. The transcript initiation site of gcrA, determined by primer extension (Fig. 2A), was mapped to an adenosine residue 92 nucleotides (nt) upstream of the ATG start codon (Fig. 2C). A CtrA binding motif overlaps the –10 region of the gcrA transcript initiation site (Fig. 2C); DNase I protection assays showed that CtrA∼P binds at this predicted CtrA motif (Fig. 2, B and C).

Fig. 2.

The gcrA promoter is negatively controlled by the CtrA response regulator. (A) Primer extension mapped the gcrA transcript start site to 92 nt upstream from the ATG start codon. (B) DNase I protection assays (footprints) of the gcrA promoter by purified His6-CtrA∼P using a 100-bp PCR-amplified fragment of the gcrA promoter region. Concentrations of CtrA∼P (μg/ml) are indicated above each lane. – indicates negative control without the addition of CtrA∼P. (C) The gcrA promoter region showing the transcription start site (arrow) and the position of the DNase I–protected region (black bar). The CtrA binding motif is underlined. (D) gcrA promoter activity measured as Miller units of β-galactosidase activity in the ctrA401ts strain LS2195 carrying the lacZ gene under the control of the gcrA promoter on the plasmid pLacZ290-gcrAP at 0, 2, and 4 hours after the shift from 28°C to 37°C (left). As a control, β-galactosidase activity was also measured using the same reporter construct in wild-type cells upon shift from 28°C to 37°C (right). (E) Immunoblot analysis, with GcrA antibody, of GcrA levels after a shift to restrictive temperature of a ctrA401ts mutant (LS2195), a cckAts mutant (LS3309), and wild-type cells.

We determined that CtrA∼P represses transcription of gcrA by using a reporter construct to assess the gcrA promoter activity in the ctrA401ts mutant upon shift to the restrictive temperature (Fig. 2D). A DNA fragment from –507 to +92 relative to the gcrA transcript initiation site was used to construct a transcriptional fusion to lacZ on the low-copy plasmid, pLacZ290. The resulting plasmid, pLacZ290-gcrAP, was introduced into the ctrA401ts mutant (Fig. 2D). Measurement of the relative levels of β-galactosidase activity showed that gcrA promoter activity increased about 50% 2 hours after the shift from 28°C to 37°C and increased approximately 100% 4 hours after the shift. β-galactosidase activity from the gcrA promoter in a wild-type background remained constant 2 hours after a shift from 28°C to 37°C and increased by only 25% at 4 hours after the shift. These data indicate that CtrA is a negative regulator of the gcrA promoter. Previous microarray assays of global transcript levels in the ctrA401ts mutant and a ts mutant of the gene encoding CtrA's cognate CckA histidine kinase, cckAts, also showed elevated gcrA transcript levels at the restrictive temperature (12).

Immunoblot analysis of GcrA protein levels in the CtrA401ts and CckAts strains showed approximately a doubling of GcrA levels in both mutant strains at 2 and 4 hours after shifting to the restrictive temperature (Fig. 2E). In contrast, in wild-type cells GcrA protein levels increased only about 10% 4 hours after shifting from 28°C to 37°C (Fig. 2E). Thus, the levels of active CtrA response regulator and of the CckA histidine kinase that activates CtrA (12) contribute to the repression of gcrA expression.

gcrAexpression is cell-cycle dependent. Immunoblots of synchronized cell populations show that the GcrA level is cell-cycle dependent and both temporally and spatially out of phase with CtrA (Fig. 3, A to C). Isolated swarmer cells were grown synchronously, and samples collected every 20 min were assayed by immunoblots with antibodies to GcrA and CtrA (18). GcrA was not detected in swarmer cells, but accumulated in stalked cells during the G1/S transition and in the stalked progeny after cell division. CtrA, on the other hand, is present in swarmer cells but is cleared from stalked cells by directed proteolysis at the swarmer-to-stalked cell transition and in the stalked compartment of the predivisional cell after barrier formation (Fig. 3, A and B) (14, 18). Consistent with the changes in GcrA levels from the immunoblot assays, previous microarray analyses of global transcript levels during the cell cycle showed that gcrA messenger RNA (mRNA) levels are maximal in stalked cells and decrease in predivisional cells when CtrA levels increase (1) (Fig. 3C). Thus, gcrA transcription is activated upon proteolysis of CtrA and decreases after de novo synthesis of CtrA, again consistent with the hypothesis that the rise in GcrA levels upon proteolysis of CtrA is due to negative regulation of gcrA transcription by CtrA (Fig. 2, D and E). Uncoupling gcrA transcription from regulation by CtrA by expressing GcrA from PxylX provided further support for this model.

Fig. 3.

GcrA is cell cycle–regulated, accumulating as CtrA is proteolyzed at the G1/S transition. (A) Schematic of the Caulobacter cell cycle. Red indicates CtrA and blue indicates GcrA. Theta structures indicate replicating DNA. SW, swarmer cell; ST, stalked cell; PD, predivisional cell. (B) Immunoblots of cell extracts from a synchronized culture using GcrA and CtrA antibodies at the indicated times in the cell cycle. Also shown are immunoblots of cell extracts from newly divided stalked and swarmer cells harvested at the end of a synchronized cell-division cycle. A low amount of CtrA is detectable in the stalked-cell fraction because of the presence of a small proportion of predivisional cells. (C) GcrA and CtrA protein levels from densitometry graphs of the CtrA and GcrA immunoblots. gcrA mRNA levels obtained from microarray assays of global transcription patterns as a function of the cell cycle (1). The dashed vertical lines bound the time of initiation of gcrA transcription upon CtrA proteolysis.

GcrA activates the ctrA promoter. The ctrA gene is regulated by two promoters. The weaker P1 promoter is active only in the late stalked cell, and the stronger P2 promoter is active in the predivisional cell. CtrA expressed from the P1 promoter enables transcription from the positively autoregulated P2 promoter (Fig. 4C). The resulting rapid increase in CtrA∼P represses the P1 promoter in parallel with performing CtrA's numerous other regulatory functions (12, 13) (Fig. 5B).

Fig. 4.

A feedback circuit controls the sequential expression of the gcrA and ctrA global regulators. (A) Differences in transcript levels from the individual ctrA P1 and P2 promoters driving expression of the lacZ reporter gene on plasmids pAR164 and pAR165, respectively, in strain LS3707 upon depletion of GcrA. Schematics of the lacZ reporter constructs are shown above the graphs of P1 and P2 activity. The graphs show relative β-galactosidase activities from the P1 or P2 promoter activity in strain LS3707 upon depletion of GcrA 2 hours and 4 hours after a shift from PYEX to PYEG. (B) In vivo association of GcrA and CtrA with promoter DNA using chromatin immunoprecipitation assays. GcrA and CtrA were immunoprecipitated from cell extracts after formaldehyde cross-linking (3). After reversing the cross-links, DNA in the immunoprecipitates was amplified by PCR and primer pairs to the ctrA, podJ, dnaA, and fixL promoters. The templates used were total chromosomal DNA (lane 1), DNA from the anti-GcrA immunoprecipitate (lane 2), and DNA from the anti-CtrA immunoprecipitate (lane 3). The PCR products were separated on 8% polyacrylamide gels. (C) Model of the GcrA/CtrA genetic circuit.

Fig. 5.

(A) Activation or repression of proteins regulated by GcrA. Immunoblot of LS3707 cells depleted of GcrA 2 hours and 4 hours after shift from PYEX to PYEG using antibodies directed against ParE, CtrA, DnaA, PodJL, and PodJS. (B) Schematic of the regulatory functions of CtrA and GcrA. GcrA regulatory pathways are shown in red, and CtrA regulatory pathways are shown in green.

Individual fusions of the P1 and P2 ctrA promoters to the lacZ gene on plasmids pAR164 and pAR165, respectively, were assayed for β-galactosidase activity in strain LS3707 (Fig. 4A). Four hours after these strains were shifted from PYEX to PYEG to deplete GcrA, the activity of P1 ctrA decreased 58%, whereas the activity of the P2 promoter decreased only 18%. In wild-type cells, the shift from PYEX to PYEG medium had no effect on the P1 and P2 promoter activity. Thus, GcrA is specifically involved in the activation of the ctrA P1 promoter.

GcrA is a transcriptional regulatory protein. We used a chromatin immunoprecipitation assay to determine whether GcrA interacts with promoter sequences in vivo (19). Briefly, formaldehyde was added to log-phase cultures to cross-link protein and DNA, and the cells were sonicated to shear the DNA to an average size of 500 to 1000 base pairs (bp). Protein-DNA complexes were then immunoprecipitated with antibodies against GcrA and CtrA, the cross-links were reversed, and the precipitated DNA was analyzed by polymerase chain reaction (PCR) to test for the presence of different promoters (19). As shown in Fig. 4B, the ctrA and podJ promoter regions were immunoprecipitated by both antibodies. In the present study, we show that the transcription of both ctrA and podJ changes significantly after GcrA depletion. In addition, both genes are directly regulated by CtrA (6), and CtrA footprints its own P1 and P2 promoters (13). GcrA binds the promoter of dnaA, but CtrA does not, in confirmation of microarray analyses showing that dnaA RNA levels change upon GcrA depletion (table S1) (20, 21) but do not change in the ctrA401ts strain at the restrictive temperature (1, 12). As a negative control, we tested the binding of GcrA and CtrA to the promoters of three metabolic genes: the histidine kinase gene fixL (CC0759), cytochrome D ubiquinol oxidase subunit I gene (CC0762), and the gene encoding ubiquinol oxidase subunit II (CC1773). These genes are not regulated by either GcrA or CtrA, and their promoters were not immunoprecipitated by antibodies to GcrA or CtrA (only the data for the fixL promoter is shown). Thus, our data suggest that GcrA binds either directly to promoter DNA or to another protein that binds to these promoters.

CtrA and GcrA are oscillating global regulators. We propose that the genetic circuit shown in Fig. 4C causes the spatially and temporally alternating expression of the GcrA and the CtrA global regulators. We can trace the action of the circuit from the time in the cell cycle when the replication fork passes the position of the ctrA gene on the chromosome (Fig. 5B, Switch A). Just before that point in the cell cycle, there is no CtrA in the cell and GcrA has accumulated [Fig. 3, A and C (∼50 min)]. GcrA then initiates CtrA expression at the hemimethylated P1 promoter of the newly replicated ctrA gene to seed the subsequent rapid production of CtrA from the P2 promoter [Fig. 3C (∼60 min) and Fig. 4C]. Thereafter, in the early predivisional cell, newly synthesized and activated CtrA∼P binds to and represses the gcrA and ctrA P1 promoters [Fig. 3C (∼80 min) and Fig. 4C] while activating transcription of the ctrA P2 promoter and ccrM. The newly expressed CcrM DNA methyltransferase remethylates and silences the ctrA P1 promoter (10) (Fig. 4C). Proteolysis of CtrA in the stalked compartment of the predivisional cell relieves repression of gcrA (Fig. 5B, Switch B), and GcrA accumulates in the progeny stalked cell. In contrast, in the swarmer compartment of the predivisional cell CtrA remains at high levels, continuing repression of initiation of chromosome replication as well as gcrA expression, thus resulting in the exclusion of GcrA from the progeny swarmer cells (Fig. 3B). This process may further be aided by either the targeted proteolysis of GcrA in the swarmer-cell compartment of the predivisional cell or an inherently short half-life of GcrA throughout the cell cycle. At the swarmer-to-stalked cell transition in the ensuing cell cycle (Fig. 3A), CtrA is also removed by proteolysis triggered by an unknown factor mediated by the DivK response regulator (22), so that GcrA is expressed and the cycle continues (Fig. 5B, Switch B).

The two events labeled Switch A and Switch B in Fig. 5B tie progression of this CtrA/GcrA–fueled cycle to successful completion of milestone events in the cell cycle. Switch A ties timing of CtrA expression to the progress of the replication fork past ctrA. This prevents premature expression of CtrA that could disrupt formation of the replisome and assures that there are two copies of ctrA in the cell to facilitate fast and reliable CtrA production thereafter. Switch B, which controls CtrA proteolysis, is triggered in the stalked compartment of the late predivisional cell (14) and during the swarmer-to-stalked cell transition (by an unknown DivK-dependent mechanism in both cases). In both instances, accelerated proteolysis (and dephosphorylation) of CtrA releases repression of gcrA, relieves silencing of the chromosome origin of replication, and, importantly, breaks the autoregulatory feedback loop maintaining CtrA production from the ctrA P2 promoter (Fig. 4C).

Using oligo microarrays with probes for 3761 predicted Caulobacter genes, we found that depletion of GcrA directly or indirectly altered expression levels of 125 genes. In these experiments, RNA extracted from LS3707 and wild-type cells was compared 2 hours after the cells were shifted from PYEX to PYEG. These 125 genes exhibited comparable changes in RNA levels 4 hours after the shift to PYEG. Among the 125 genes regulated by GcrA, 49 are cell-cycle regulated and eight of these 49 are also regulated by CtrA. Genes that both exhibit a decrease in mRNA levels upon depletion of GcrA and are activated by CtrA in wild-type cells probably reflect a secondary effect due to reduced ctrA P1 promoter activity and lower CtrA protein levels caused by GcrA depletion (Fig. 5A). Genes that in the wild type are repressed by CtrA and upon GcrA depletion show reduced mRNA levels are predicted to be regulated by both, negatively by CtrA and positively, directly or indirectly, by GcrA.

The 125 GcrA-dependent genes belong to multiple COG (clusters of orthologous groups) (23) categories, which suggests that GcrA is involved in the regulation of multiple aspects of the cell cycle. These clusters compromise 15 genes involved in DNA replication, recombination, and repair; 25 regulatory genes; 8 in cell motility and polar development; 5 in cell-wall biogenesis; and 5 in amino acid transport and metabolism. There are 41 genes of unknown function in the 125 GcrA-dependent genes.

The regulatory genes encode, among others, the PleC histidine kinase (24, 25), the response regulators CtrA and DivK (22, 26), and the sigma-54 factor activator TacA (27). Since tacA and divK have been reported to be regulated by CtrA (6, 27), changes in their mRNA levels upon GcrA depletion may be a secondary effect due to reduced CtrA levels. The CtrA and GcrA proteins together control only about 30% of the ∼550 (1) genes whose expression levels vary over the cell cycle. While some of the remainder are indirectly controlled by one of these proteins (e.g., several late flagellar genes), there are probably additional global cell-cycle regulatory proteins that are integrated into the cell-cycle network.

Protein levels of GcrA-regulated genes. The level of selected proteins in GcrA-depleted cells was assayed directly by immunoblot analysis (Fig. 5A). In the case of the topoisomerase IV subunit ParE, CtrA, and the two isoforms of the PodJ localization factor (PodJL and PodJS) (28), protein levels decreased upon GcrA depletion, indicating that their expression is activated by GcrA (Fig. 5A). In contrast, the levels of DnaA increased after depletion of GcrA, suggesting that it is negatively controlled by GcrA, perhaps to prevent the re-initiation of replication. In each of these cases, with the exception of ctrA, the mRNA changes observed in microarray assays of GcrA-depleted strains were consistent with the changes in protein levels. In the case of CtrA, the change in mRNA levels was below the statistical cutoff, probably because only the ctrA P1 promoter, and not the ctrA P2 promoter, is normally activated by GcrA.

Coregulation by GcrA and CtrA. The GcrA protein is essential, and depletion of GcrA affects multiple aspects of the cell cycle. The GcrA regulator works in conjunction with CtrA to control the timing and execution of chromosome replication (Fig. 5B). In swarmer cells, CtrA∼P binds to and silences the origin of replication. Upon proteolysis of CtrA at the swarmer-to-stalked cell transition, the origin is freed, enabling the formation of an active replisome (29). Previous work has shown that persistence of an active CtrA derivative freezes the cell cycle in G1 (18). Critically, upon proteolysis of CtrA, the gcrA promoter is freed from repression. The ensuing accumulation of GcrA controls the expression of multiple components of the replication machinery (Fig. 5B), allowing replisome synthesis and the initiation of chromosome replication. Several additional replication factors are released from repression at this time in the cell cycle by an unknown mechanism (Fig. 5B).

GcrA and CtrA also cooperate in directing polar morphogenesis and asymmetry. The podJ gene, encoding a required factor for the polar positioning of the PleC histidine kinase and the pilus secretion apparatus, is an example of sequential regulation of the same gene by CtrA and GcrA. Transcription of podJ is directly repressed by CtrA in swarmer cells and activated in the early stalked cell upon proteolysis of CtrA (6, 30). In a synchronized culture of cells lacking the CtrA binding site in the podJ promoter, the amplitude of the podJ promoter activity was altered but the timing during the cell cycle was not, which indicates that a factor other than CtrA is responsible for cell cycle–dependent activation of the podJ promoter (30). Upon GcrA depletion, a strong decrease of podJ mRNA and protein levels was observed, suggesting that in wild-type cells GcrA functions, directly or indirectly, to positively activate the podJ promoter in the early stalked cell. The function of this coregulation is to assure the proper timing of polar organelle development at the incipient swarmer pole of the predivisional cell, yielding asymmetry before cell division. PodJ localization at this pole has been shown to be essential for the subsequent localization of the PleC histidine kinase at that pole (28).

CtrA and GcrA are key elements of the genetic circuit (Fig. 4C) that causes the spatially and temporally alternating expression of these global regulators. The CtrA/GcrA circuit provides the motive force propelling the advancement of Caulobacter cell-cycle events. However, progression is paced by ties to successful completion of important cell-cycle events such as progress of the replication fork to the ctrA gene, which initiates CtrA production; predivisional cell compartmentalization, which initiates CtrA degradation in the stalked cell compartment; and a currently unknown mechanism that inactivates CtrA∼P either by dephosphorylation or by targeted proteolysis when the swarmer cell differentiates into a stalked cell. These are the Switch A and Switch B events in Fig. 5B. An apt comparison is to the spring and escapement component of a mechanical clock where the spring (or a weight) provides the motive impetus but the escapement paces progress. In the cell, the GcrA/CtrA genetic circuit provides the motive force analogous to the pressure of the clock's spring. In the clock, the progress is paced by the regular oscillations of a pendulum, whereas in the cell, progress is paced by the rate of progression or completion of many other complex biochemical and genetic cell-cycle events. Although the timing of the completion of these events is uncertain because of the random nature of chemical reactions, the orderly progression of the whole system is maintained by assuring that events occur in the proper order.

Every organism contains, within its regulatory system, self-sustaining pacemaker circuitry that coordinates progression of the cell cycle. At a high level, all these cell-cycle control circuits exhibit parallels in mechanism and function. For example, phosphorylation pathways are interconnected with transcriptional regulatory networks, critical positive and negative feedback pathways, and targeted proteolysis. These mechanisms lead to the cyclical presence and/or activation state of key regulatory proteins that in turn control the modular functions that implement the cell cycle. The specific cell-cycle regulatory proteins in bacteria and eukaryotic cells are not homologous; rather, the homologies are at the level of system design.

Supporting Online Material

www.sciencemag.org/cgi/content/full/1095191/DC1

Materials and Methods

SOM Text

Tables S1 and S2

References and Notes

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