DnaA couples DNA replication and the expression of two cell cycle master regulators
2006; Springer Nature; Volume: 25; Issue: 2 Linguagem: Inglês
10.1038/sj.emboj.7600927
ISSN1460-2075
AutoresJustine Collier, Sean R. Murray, Lucy Shapiro,
Tópico(s)CRISPR and Genetic Engineering
ResumoArticle5 January 2006free access DnaA couples DNA replication and the expression of two cell cycle master regulators Justine Collier Justine Collier Search for more papers by this author Sean Richard Murray Sean Richard Murray Search for more papers by this author Lucy Shapiro Corresponding Author Lucy Shapiro Department of Developmental Biology, School of Medicine, Stanford University Medical Center, Beckman Center, Stanford, CA, USA Search for more papers by this author Justine Collier Justine Collier Search for more papers by this author Sean Richard Murray Sean Richard Murray Search for more papers by this author Lucy Shapiro Corresponding Author Lucy Shapiro Department of Developmental Biology, School of Medicine, Stanford University Medical Center, Beckman Center, Stanford, CA, USA Search for more papers by this author Author Information Justine Collier, Sean Richard Murray and Lucy Shapiro 1 1Department of Developmental Biology, School of Medicine, Stanford University Medical Center, Beckman Center, Stanford, CA, USA *Corresponding author. Department of Developmental Biology, School of Medicine, Stanford University Medical Center, Beckman Center, Stanford B351, CA 94305, USA. Tel.: +1 650 725 7678; Fax: +1 650 725 7739; E-mail: [email protected] The EMBO Journal (2006)25:346-356https://doi.org/10.1038/sj.emboj.7600927 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Cell cycle progression in Caulobacter is driven by the master transcriptional regulators CtrA and GcrA. The cellular levels of CtrA and GcrA are temporally and spatially out-of-phase during the cell cycle, with CtrA repressing gcrA transcription and GcrA activating ctrA transcription. Here, we show that DnaA, a protein required for the initiation of DNA replication, also functions as a transcriptional activator of gcrA, which in turn activates multiple genes, notably those involved in chromosome replication and segregation. The cellular concentration of DnaA is cell cycle-controlled, peaking at the time of replication initiation and gcrA induction. Regulated proteolysis of GcrA contributes to the cell cycle variations in GcrA abundance. We propose that DnaA couples DNA replication initiation with the expression of the two oscillating regulators GcrA and CtrA and that the DnaA/GcrA/CtrA regulatory cascade drives the forward progression of the Caulobacter cell cycle. Introduction Caulobacter crescentus divides asymmetrically in each cell cycle, giving rise to two different progeny cells with distinct morphological features and cell fates: a stalked cell that can immediately initiate DNA replication and a swarmer cell that cannot initiate DNA replication until it differentiates into a stalked cell (Figure 1A). Caulobacter, therefore, must not only coordinate DNA replication with cell division, but also with morphogenetic events. Multiple cell cycle events, such as the G1/S transition, flagellar and pili biogenesis, DNA methylation, chromosome segregation and cytokinesis are mediated by the GcrA and CtrA master regulators, which oscillate out of phase during the cell cycle (Holtzendorff et al, 2004). These events are coordinated, in part, by an interdependent feedback loop between GcrA and CtrA and by the differential methylation state of the chromosome during the cell cycle (Reisenauer and Shapiro, 2002; Holtzendorff et al, 2004). Here, we show that DnaA is a critical coordination factor, functioning to allow replication initiation while activating the expression of gcrA, with the consequent expression of ctrA and genes involved in chromosome replication and segregation. Figure 1.Cell cycle expression of gcrA. (A) Schematic of the Caulobacter cell cycle. Gray indicates accumulation of GcrA. Theta structures indicate replicating DNA. SW, swarmer cell; ST, stalked cell; PD, predivisional cell. (B) From the same synchronized LS4220 (gcrA+ gcrAP(−507…+92)-lacZ) strain culture, aliquots were taken at the times indicated and were either pulse-labelled with [35S]methionine to follow β-galactosidase synthesis or GcrA synthesis using either anti-β-galactosidase or anti-GcrA immunoprecipitation, or immunobloted with anti-GcrA. Autoradiograms from the immunoprecipitation of labelled β-galactosidase ([35S]β-Gal) and GcrA ([35S]GcrA), and immunoblots with anti-GcrA (GcrA) are shown. Also shown are autoradiograms and immunoblots from cell extracts from newly divided stalked and swarmer cells harvested at the end of the cell cycle. (C) Histograms of the relative rates of gcrA transcription (β-galactosidase synthesis), GcrA synthesis and GcrA accumulation as measured by phosphorimaging or densitometric scanning of immunoblots. Values were normalized to the maximum value of each experiment (at 40 min from 0 to 140 min and in stalked progenies). Download figure Download PowerPoint CtrA is an essential transcription factor and a selective chromosome replication inhibitor in Caulobacter (Quon et al, 1996, 1998). CtrA accumulates in swarmer cells, where it directly binds to five sites within the Caulobacter origin of DNA replication (Cori) and thereby blocks replication initiation. CtrA is cleared from the cell during the swarmer-to-stalked cell differentiation, which is functionally analogous to the eukaryotic G1-to-S transition of the cell cycle. Concurrently, as CtrA is proteolyzed, the dnaA and the himA genes, which encode the DNA replication initiator protein DnaA and the α-subunit of the ‘histone-like’ integration host factor (IHF), are transcribed (Zweiger and Shapiro, 1994; Laub et al, 2000). The DnaA protein, conserved in most eubacteria, directly binds to DNA at DnaA boxes (Fuller et al, 1984). Five DnaA boxes and an exceptional AT-rich region are found in the Caulobacter Cori (Marczynski and Shapiro, 1992). DnaA binding to Cori has been proposed to unwind the AT-rich region and provide an entry site for protein components of the replisome that then initiates chromosome replication (Bramhill and Kornberg, 1988; Gille and Messer, 1991). An IHF-binding site overlaps one of the CtrA-binding sites in Cori. IHF binding to the Cori may help displace CtrA from the Cori sequence and bend DNA, promoting the initiation of replication (Siam et al, 2003). Many functions needed for normal progression of the Caulobacter cell cycle are regulated by discrete transcription patterns: genes are activated at the time when their function is needed. Whole-genome analysis of messenger RNA (mRNA) accumulation during the Caulobacter cell cycle has revealed that the expression of about 19% of the Caulobacter genes (∼550 out of 3760) are regulated in a cell-cycle-dependent manner (Laub et al, 2000). More than 15% of these cell-cycle-regulated genes are directly regulated by CtrA, which binds to a conserved motif in the promoters of 55 operons (Quon et al, 1996; Laub et al, 2002). CtrA's targets include genetic modules for cell division, DNA methylation, flagellum and pili biosynthesis, chemotaxis and metabolism (Laub et al, 2002). GcrA is another essential regulatory protein controlling 49 cell-cycle-regulated genes, of which eight are also regulated by CtrA (Holtzendorff et al, 2004). GcrA notably controls polar morphogenesis and multiple components of the chromosome replication and segregation machinery. The cellular levels of GcrA are cell-cycle-dependent, both temporally and spatially out-of-phase with CtrA ((Holtzendorff et al, 2004) and Figure 5). Functional complementarities of the conserved CtrA and GcrA proteins allow time- and cell type-specific transcriptional regulation of key cell cycle events, thus providing a motive force for cell cycle progression. The expression and the activity of the CtrA and GcrA master regulators must therefore be tightly regulated. The cellular accumulation of CtrA is controlled by at least two mechanisms: transcriptional regulation and targeted proteolysis. The ctrA gene is transcribed from two promoters: P1 and P2. The ctrA P1 promoter can be methylated by the CcrM DNA methyltransferase at a GANTC site and a fully methylated ctrA P1 promoter cannot be transcribed (Reisenauer and Shapiro, 2002). At the initiation of replication, the ctrA P1 promoter is in the fully methylated state. The ctrA P1 promoter can only be transcribed after the passage of the replication fork that generates two hemi-methylated copies of the gene. Thus, ctrA transcription is coordinated with the progression of chromosomal replication. Once in the hemi-methylated state, transcription from the ctrA P1 promoter is activated by GcrA (Holtzendorff et al, 2004). As CtrA accumulates and is activated by phosphorylation, it represses the ctrA P1 promoter and activates the strong ctrA P2 promoter, leading to a burst of CtrA synthesis in late stalked cells and early predivisional cells (Domian et al, 1999), simultaneously repressing the gcrA promoter (Holtzendorff et al, 2004). Once the division plane establishes two cellular compartments prior to cell division, CtrA is rapidly proteolyzed by the ClpXP protease (Jenal and Fuchs, 1998) in the stalked compartment of the late predivisional cell and then at the swarmer-to-stalked cell transition, when the cell is preparing to initiate DNA replication (Domian et al, 1997; Judd et al, 2003). Even though the cell cycle regulation of ctrA expression has been extensively studied, the mechanisms governing the oscillation of CtrA and GcrA concentrations during the cell cycle are not well understood, notably because little is known about the regulation of gcrA expression or GcrA stability. Previous microarray analyses showed that gcrA mRNA levels are maximal in stalked cells and decrease in predivisional cells (Laub et al, 2000), suggesting that gcrA transcription may be an important level of regulation of gcrA cell cycle expression. It was also shown that CtrA represses transcription of gcrA by directly binding to a CtrA-binding site within the gcrA promoter (Holtzendorff et al, 2004). In this study, we demonstrate that as DnaA accumulates during the swarmer-to-stalked cell transition, it directly activates gcrA transcription as the CtrA repressor is being cleared from the cell. Temporally regulated proteolysis further modulates GcrA cellular concentration during the cell cycle. The ‘dual-use’ DnaA protein functions to both enable replisome formation at the origin and to control the transcription of gcrA. The accumulation of GcrA, in turn, activates the transcription of genes involved in DNA replication and chromosome segregation, and directly activates ctrA transcription. Thus, DnaA serves as a central coordinator of multiple cell cycle events. Results Cell cycle control of gcrA expression To determine how GcrA protein levels are temporally regulated, each step of GcrA synthesis was analyzed during the Caulobacter cell cycle. To measure the transcription of gcrA, a gcrAP-lacZ transcriptional fusion (Holtzendorff et al, 2004) was integrated into the Caulobacter chromosome, at a site close to the natural gcrA locus (LS4220). Transcription and translation from gcrAP results in production of β-galactosidase. We measured newly synthesized β-galactosidase at different time points during the cell cycle. Synchronous cells were pulse-labelled with [35S]methionine and β-galactosidase was immunoprecipitated to assay the activity of the gcrA promoter (Figure 1). Transcription increased more than two-fold during the swarmer-to-stalked cell transition. The maximum level of transcription was reached in stalked cells and then decreased in the predivisional cells. The cell-cycle-dependent transcription of gcrA is comparable to the changes in the steady-state levels of gcrA mRNA during the cell cycle (Laub et al, 2000), suggesting that the increase in gcrA mRNA levels during the swarmer-to-stalked cell transition is the result of increased transcription, rather than stabilization of gcrA mRNA. The rate of gcrA transcription in separated populations of swarmer and stalked cells was also measured immediately after cell division (at ∼150 min) and was found to be significantly higher in stalked than swarmer progeny cells (Figure 1). Overall, these results demonstrate that the transcriptional regulation of gcrA contributes to the control of the cell-cycle-dependent accumulation of GcrA. To measure the temporal synthesis of GcrA, we immunoprecipitated GcrA from the pulse-labelled cell samples from the same synchronous culture used to measure the temporal transcription of gcrA. We could thereby determine how much new GcrA protein was synthesized from the natural gcrA gene during the pulse (Figure 1). Synthesis of the GcrA protein increased during the swarmer-to-stalked cell transition, reached a maximum in stalked cells, decreased in predivisional cells and was much higher in the stalked than the swarmer progenies after cell division. Since the cell cycle variations of GcrA synthesis are comparable to the cell cycle variations of gcrA transcription, we conclude that gcrA mRNA translation and stability are not significantly cell cycle regulated, even though the 5′ untranslated region of the gcrA mRNA is surprisingly long (92 nucleotides long (Holtzendorff et al, 2004)). The proteolysis of GcrA is cell cycle regulated The accumulation of the GcrA protein was compared with the efficiency of its synthesis during the cell cycle by immunoblots using anti-GcrA antibodies (Figure 1). Cell-cycle-regulated synthesis of GcrA can approximately account for the increase of GcrA steady-state levels in the stalked cells. Nevertheless, the rapid decrease in GcrA protein levels in predivisional cells, approximately concurrent with the decrease in GcrA synthesis, indicates that GcrA is an unstable protein. To measure GcrA half-life in a mixed cell population, we performed pulse-chase experiments in the wild-type strain grown in exponential phase. We found that the half-life of GcrA is about 24 min (data not shown), which is substantially shorter that the 150 min cell cycle of wild-type Caulobacter cells. The absence of detectable GcrA proteins in swarmer cells (at time=0 min and after isolation of the swarmer progeny at time=140 min), despite a basal level of GcrA synthesis (Figure 1), suggests that GcrA is particularly unstable in swarmer cells. If GcrA proteolysis changes during the cell cycle, accumulated levels of GcrA should vary even when GcrA is constitutively synthesized throughout the cell cycle. To test this hypothesis, we used a strain (LS3707) in which the sole copy of gcrA is under the control of the chromosomal xylose inducible promoter PxylX, yielding constitutive expression of gcrA in the presence of xylose (Holtzendorff et al, 2004). Swarmer cells from LS3707 grown in minimal media supplemented with xylose were isolated, and protein samples were collected at different time points throughout the cell cycle in the same medium for immunoblot analysis using anti-GcrA antibodies (Figure 2A). We observed that GcrA accumulation changed during the cell cycle, even under conditions when gcrA is transcribed constitutively throughout the cell cycle, with predominant accumulation in stalked and predivisional cells. This result suggests that GcrA is significantly more unstable in swarmer cells than other Caulobacter cell types. To ascertain that the weaker accumulation of GcrA in swarmer cells than in stalked cells was due to more rapid proteolysis of GcrA, we determined the half-life of GcrA in independent isolated populations of LS3707 swarmer and stalked cells (Figure 2B). We found that the half-life of GcrA is ∼10.5 min in swarmer cells, as compared to ∼43.7 min in stalked cells. Since the rate of synthesis of GcrA in wild-type swarmer cells is very low, we were not able to assess accurately the half-life of GcrA in swarmer cells from a wild-type strain. Nevertheless, we assessed the half-life of GcrA in stalked cells from a wild-type strain and found it was also about 42 min (data not shown), which confirms that proteolysis of GcrA in the LS3707 strain is comparable to proteolysis of GcrA in wild-type cells. Overall, these results show that GcrA proteolysis is cell cycle regulated, with GcrA being degraded approximately four-fold faster in swarmer than stalked cells. Figure 2.Proteolysis of GcrA is cell cycle regulated. (A) GcrA accumulation when gcrA is transcribed constitutively. Immunoblots of cell extracts from a synchronized NA1000 and LS3707 (ΔgcrA PxylX∷gcrA) culture using GcrA antibodies at the indicated times of the cell cycle. In the schematic of the Caulobacter cell cycle, gray indicates accumulation of GcrA in the NA1000 cells. (B) Stability of GcrA synthesized in swarmer and stalked LS3707 cells. A synchronized population of LS3707 swarmer cells was allowed to proceed through the cell cycle. At 5 (swarmer cells) and 30 min (swarmer-to-stalked cell transition), aliquots of the culture were pulse-labelled with [35S]methionine and chased for increasing amounts of time. Aliquots were taken at the times indicated and the remaining radiolabelled GcrA was determined by immunoprecipitation using GcrA antibodies, followed by SDS–PAGE and phosphorimaging. The t1/2 corresponds to the calculated half-life of GcrA in each cell type, along with the calculated standard deviation from three independent LS3707 populations. Download figure Download PowerPoint Characterization of the gcrA promoter A DNA fragment from −507 to +92 relative to the gcrA transcript initiation site (Holtzendorff et al, 2004) was used to construct the transcriptional fusion to lacZ that we used to study the temporal transcription of gcrA shown in Figure 1. We show (in Supplementary data) that all significant regulatory sequences should be included in the −78 to +1 region of the gcrA promoter. This sequence notably contains the previously characterized CtrA-binding site (Holtzendorff et al, 2004), in addition to a putative DnaA box and two putative DNA methylation sites (Figure 3A). Figure 3.Opposite effects of CtrA and DnaA on gcrA transcription. (A) The nucleotide sequence of the gcrA promoter is shown. The transcriptional +1 start site (Holtzendorff et al, 2004), the CtrA-binding site (underlined) (Holtzendorff et al, 2004), the two putative DNA methylation sites (boxes), the putative DnaA box (underlined) and a putative hairpin structure (two arrows) are indicated. (B) The sequence of the CtrA-binding site and the DnaA box within the gcrA promoter (gcrAP) are shown and compared to the consensus CtrA-binding site and DnaA box in Caulobacter. Mutations introduced in the gcrAP are underlined and in capital letters. The names of the pLacZ290 derivatives carrying the unmodified and the mutated gcrAP fused to lacZ are also shown. (C) The graph shows the relative β-galactosidase activities from the six plasmids in (B) in an unsynchronized NA1000 strain. (D) The graph shows the relative β-galactosidase activities from pLacZ290-gcrAP(WT) and pLacZ290-gcrAP(DnaA) in an unsynchronized GM2471 (ΔdnaA∷Ω PxylX∷dnaA) strain, upon depletion of DnaA 4 h after a shift from PYE+xylose (PYEX) to PYE+glucose (PYEG). Activities in Miller units were normalized so that the activity of the gcrAP(WT) equals 100% in PYE (NA1000) or PYEX (GM2471), to facilitate comparison. Errors bars indicate the standard deviations (when they were more than 1%). Download figure Download PowerPoint CtrA is a negative regulator of gcrA transcription A previous study from our laboratory, assessing the activity of the gcrA promoter in a ctrA401ts mutant shifted to restrictive temperature, showed that CtrA represses gcrA transcription (Holtzendorff et al, 2004). Even though CtrA directly binds to the gcrA promoter, the activation of gcrA transcription at restrictive temperature in the ctrA401ts strain could partially be an indirect effect of the cell cycle arrest caused by the CtrA depletion. To quantitatively assess the effect of CtrA on gcrA transcription in a wild-type strain, we analyzed the activity of three mutant gcrA promoters, carrying mutations in the CtrA-binding site in the gcrA promoter fused to lacZ carried on the low copy number plasmid pLacZ290 (Figure 3B). The consensus DNA sequence for CtrA binding in Caulobacter is TTAA-N7-TTAA (Marczynski et al, 1995) and both the CtrA-binding half-sites and the N7 spacing are critical for CtrA-mediated transcriptional regulation (Ouimet and Marczynski, 2000). The first mutant gcrA promoter we constructed (gcrAP(CtrAL)) carries a 4 base-pair mutation in the left portion of the CtrA-binding site, the second (gcrAP(CtrAM)) carries a double base-pair mutation in the middle part of the CtrA-binding site, and the third (gcrAP(CtrAR)) carries a single base-pair mutation in the right portion of the CtrA-binding site. All mutated promoters were cloned in pLacZ290 and introduced into a wild-type Caulobacter strain to compare their activities with the wild-type promoter (gcrAP(WT)) by β-galactosidase assays. In all three cases, the activities of the mutated promoters were higher than that of the wild-type gcrA promoter, with a 25–50% difference in the activity (Figure 3C). To check whether the mutations we created in the gcrA promoter render the gcrA promoter independent of CtrA protein levels in the cell, we also introduced these constructs in a ctrA401ts strain. After a shift from permissive temperature to restrictive temperature, the activity of the gcrAP(CtrAL) did not change significantly, unlike the activity of the wild-type gcrA promoter, which increased significantly (data not shown). We conclude that the mutation we introduced in the left portion of the CtrA-binding site of the gcrA promoter (gcrAP(CtrAL)) renders it independent of CtrA protein levels in the cell. The activities of the gcrAP(CtrAR) and the gcrAP(CtrAM) in a ctrA401ts strain at restrictive temperature slightly increased, but more moderately than the wild-type promoter (data not shown), showing that these two mutated promoters are still slightly sensitive to CtrA protein levels in the cell, which probably explains why the effect of these two mutations on gcrA transcription are more moderate than the mutation in the left portion of the CtrA-binding site (Figure 3C). Overall, these results confirm that CtrA is a significant repressor of gcrA transcription. Yet, β-galactosidase assays using lacZ transcriptional fusions measure transcriptional levels in log phase cultures, but not temporal control. In order to understand the role of CtrA in the regulation of gcrA transcription during the cell cycle, we integrated the apparently CtrA-independent gcrAP(CtrAL)-lacZ transcriptional fusion into the Caulobacter chromosome. As was done for the wild-type promoter (Figure 1), transcription and translation from the gcrAP(CtrAL) at different time points of the cell cycle were compared by pulse-labelling cell samples from a synchronous culture with [35S]methionine and immunoprecipitating the β-galactosidase (Figure 4A). Surprisingly, we found that transcription from the mutated gcrAP(CtrAL) promoter is still cell cycle regulated. We also immunoprecipitated GcrA from the same pulse-labelled samples, which allowed us to accurately compare GcrA synthesis from the wild-type gcrA promoter to β-galactosidase synthesis from the mutated promoter, during the same synchronization experiment. This experiment shows that the level of transcription from the mutated gcrAP(CtrAL) is still cell cycle controlled. We conclude that the degradation of the CtrA repressor is not solely responsible for the increase in gcrA transcription during the swarmer-to-stalked cell transition, suggesting that other regulatory elements are also involved in the temporal control of gcrA transcription. Figure 4.Effects of CtrA and DnaA on gcrA cell cycle transcription. Cell cycle activities of the mutated gcrA promoters were measured by immunoprecipitation of β-galactosidase from pulse-labelled samples of synchronized Caulobacter cultures. To compare with the activity of the gcrAP(WT), radiolabelled GcrA was also immunoprecipitated from the same synchrony. Relative rates of gcrA transcription from the mutated gcrA promoter and GcrA synthesis from the wild-type gcrA promoter at the indicated times of the cell cycle were measured by phosphorimaging and were normalized so that the maximum value of each experiment equals 1. The averages of the results of multiple independent experiments are shown. In the schematic of the Caulobacter cell cycle, gray indicates accumulation of GcrA in the NA1000 cells. (A) The LS4224 (gcrA+ gcrAP(CtrAL)-lacZ) strain was used to compare the cell cycle activity of the gcrAP(CtrAL) with the cell cycle activity of the gcrAP(WT). (B) The NA1000 (gcrA+) strain carrying the pLacZ290-gcrAP(DnaA) plasmid was used to compare the cell cycle activity of the gcrAP(DnaA) with the cell cycle activity of the gcrAP(WT). (C) The NA1000 (gcrA+) strain carrying the pLacZ290-gcrAP(DnaA+CtrAL) plasmid was used to compare the cell cycle activity of the gcrAP(DnaA+CtrAL) with the cell cycle activity of the gcrAP(WT). Download figure Download PowerPoint DnaA is critical for the induction of gcrA transcription The gcrA promoter has a putative DnaA box about 50 base pairs upstream of its +1 transcriptional start site (Figure 3A), which matches seven of the nine base-pair Caulobacter consensus DnaA box sequence (Marczynski and Shapiro, 1992). DnaA is a dual function protein: it is essential for the initiation of DNA replication and it acts as a transcription factor in Escherichia coli (for a review, see Messer and Weigel, 1997), Bacillus subtilis (Goranov et al, 2005) and Caulobacter (Hottes et al, 2005). To determine whether DnaA is a regulator of gcrA transcription, we used a strain (GM2471) in which the sole copy of dnaA is under the control of the chromosomal xylose-inducible promoter PxylX. The gcrAP-lacZ fusion on plasmid pLacZ290-gcrAP(WT) was assayed in strain GM2471 (Figure 3D). At 4 h after this strain was shifted from PYEX to PYEG to deplete DnaA, the activity of the gcrA promoter decreased by more than 50%. Thus, DnaA is directly or indirectly involved in the activation of the gcrA promoter. To test whether the activation of gcrA transcription by DnaA requires the presence of the DnaA box in the gcrA promoter, we analyzed the transcription of a mutant gcrA promoter (gcrAP(DnaA)), in which the DnaA box has been disrupted (Figure 3B). The gcrAP(DnaA) was cloned in the pLacZ290 and introduced in a wild-type Caulobacter strain to compare its activity with the wild-type promoter (gcrAP(WT)) by β-galactosidase assays. We observed a striking 80% loss of activity of the gcrAP(DnaA) as compared to the wild-type promoter (Figure 3C). Similarly, when the construct was introduced in the GM2471 strain grown in PYEX, the activity of the gcrAP(DnaA) was also five-fold lower than the activity of the wild-type promoter (Figure 3D). Since the mutated gcrAP(DnaA) has the same activity in GM2471 grown for 4 h in either xylose or glucose containing media, we conclude that the mutations we introduced in the DnaA box renders the mutated gcrAP(DnaA) independent of the DnaA protein levels in the cell. The dependence of gcrA transcription on DnaA and on the presence of an intact DnaA box in the gcrA promoter provides evidence that the induction of gcrA transcription by DnaA is direct and requires binding of DnaA to the gcrA promoter. To understand the effect of DnaA on the temporal control of gcrA transcription, we analyzed the transcription and translation from the gcrAP(DnaA) at different time points of the cell cycle by pulse labelling cell samples from a synchronous culture and immunoprecipitating the β-galactosidase (Figure 4B). We then compared GcrA synthesis from the wild-type gcrA promoter to β-galactosidase synthesis from the DnaA-independent gcrAP(DnaA) promoter during the same synchronization experiments. This experiment shows that the low-level activity from the DnaA-independent promoter is still cell cycle controlled, parallel to our results with the CtrA-independent promoter and with the wild-type gcrA promoter. Caulobacter integrates the negative control by CtrA and the positive control by DnaA to regulate gcrA cell cycle transcription We observed that the temporal regulation of the mutated gcrAP(CtrAL) and gcrAP(DnaA) promoters are equivalent to the wild-type gcrA promoter. This result suggests that CtrA and DnaA both contribute to the control of the temporal transcription of gcrA. Another possibility is that a third regulator is responsible for gcrA temporal regulation. To discriminate between these two possibilities, we constructed a gcrA promoter double mutant (gcrAP(DnaA+CtrAL)) that is mutated in both the left portion of the CtrA-binding site and in the DnaA box (Figure 3B). This mutated promoter was cloned in pLacZ290 and introduced into a wild-type Caulobacter strain and its activity was compared to strains with single promoter mutants: β-galactosidase assays were carried out in all three strains (Figure 3C). Like the gcrAP(DnaA), we observed that the activity of the gcrAP(DnaA+CtrAL) is much lower than the activity of the wild-type promoter when introduced in a wild-type Caulobacter strain (Figure 3C), showing that DnaA can activate the CtrA-independent gcrAP(CtrAL). Yet, the activity of the gcrAP(DnaA+CtrAL) is about 26% higher than the activity of the gcrAP(DnaA) (Figure 3C), showing that CtrA can repress the basal transcription from the DnaA-independent gcrAP(DnaA). These observations suggest that CtrA and DnaA independently contribute to the level of expression of the gcrA promoter but that their activities may function together to provide correct tempora
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