DnaA coordinates replication initiation and cell cycle transcription in Caulobacter crescentus


E-mail hmcadams@stanford.edu; Tel. (+1) 650 858 1864; Fax (+1) 650 858 1886.


The level of DnaA, a key bacterial DNA replication initiation factor, increases during the Caulobacter swarmer-to-stalked transition just before the G1/S transition. We show that DnaA coordinates DNA replication initiation with cell cycle progression by acting as a global transcription factor. Using DnaA depletion and induction in synchronized cell populations, we have analysed global transcription patterns to identify the differential regulation of normally co-expressed genes. The DnaA regulon includes genes encoding several replisome components, the GcrA global cell cycle regulator, the PodJ polar localization protein, the FtsZ cell division protein, and nucleotide biosynthesis enzymes. In cells depleted of DnaA, the G1/S transition is temporally separated from the swarmer-to-stalked cell differentiation, which is normally coincident. In the absence of DnaA, the CtrA master regulator is cleared by proteolysis during the swarmer-to-stalked cell transition as usual, but DNA replication initiation is blocked. In this case, expression of gcrA, which is directly repressed by CtrA, does not increase in conjunction with the disappearance of CtrA until DnaA is subsequently induced, showing that gcrA expression requires DnaA. DnaA boxes are present upstream of many genes whose expression requires DnaA, and His6-DnaA binds to the promoters of gcrA, ftsZ and podJ in vitro. This redundant control of gcrA transcription by DnaA (activation) and CtrA (repression) forms a robust switch controlling the decision to proceed through the cell cycle or to remain in the G1 stage.


Two master regulators, CtrA and GcrA, have been shown to drive Caulobacter crescentus cell cycle progression (Holtzendorff et al., 2004). CtrA and GcrA, however, influence the transcription of only about 30% of the over 500 genes that are expressed at specific times in the cell cycle (Laub et al., 2000; Holtzendorff et al., 2004), suggesting that one or more additional factors also act as global cell cycle regulators. Here, we show that DnaA, a key replication initiation factor found in most Eubacteria (Gil et al., 2003), functions as a critical transcription factor required for the expression of the gene encoding the GcrA master regulator whose levels oscillate with CtrA during the cell cycle. Other genes in the DnaA regulon include those encoding nucleotide biosynthesis enzymes and components of the DNA replication machinery. DnaA has also been shown to act as a transcription factor in Escherichia coli (Messer and Weigel, 1997) and Bacillus subtilis (Burkholder et al., 2001).

Synchronized populations of swarmer-stage Caulobacter cells can easily be obtained and followed through the cell cycle (reviewed in Ryan and Shapiro, 2003). The motile swarmer cells have a single polar flagellum and several pili at one pole of the cell (Fig. 1A). CtrA, a DNA-binding response regulator that directly controls transcription of at least 95 genes in 55 operons (Laub et al., 2002), is present in swarmer cells where it binds to the Caulobacter origin of replication and blocks replication initiation (Quon et al., 1998). Swarmer cells undergo a swarmer-to-stalked cell differentiation during which the polar pili, flagellum and chemotaxis apparatus are lost and are replaced with a stalk, which grows at the pole previously occupied by the flagellum. Concurrent with the swarmer/stalked transition, Caulobacter cells degrade CtrA, initiate DNA replication, and progress from the G1 (pre-replication) to the S (DNA synthesis) phase of the cell cycle. Following the disappearance of CtrA, gcrA, whose promoter is directly repressed by CtrA, is transcribed. GcrA levels rise and affect the transcription of over 125 genes, including genes encoding components of the DNA replication and chromosome segregation machinery (Holtzendorff et al., 2004). Additionally, GcrA activates the P1 promoter of ctrA, the first of the two ctrA promoters to be consecutively activated during the cell cycle (Holtzendorff et al., 2004). During DNA replication, stalked cells elongate and develop into predivisional cells, which constrict at the future division plane and assemble a new flagellum at the pole opposite the stalk. Each predivisional cell ultimately divides asymmetrically into a sessile, replication competent stalked cell and a DNA replication quiescent swarmer cell.

Figure 1.

Location of the swarmer/stalked and G1/S transitions in the normal Caulobacter cell cycle and the DnaA depletion/induction cell cycle.
A and B. In the schematics of the (A) wild-type (CB15N) and (B) DnaA depletion/induction cell cycles, a theta structure represents a replicating chromosome and a ring structure indicates a non-replicating chromosome. The DnaA depletion/induction cell cycle uses strain GM2471 where the only copy of dnaA is under the control of a xylose-inducible promoter (Gorbatyuk and Marczynski, 2001). In the DnaA depletion/induction cell cycle, GM2471 swarmer cells are isolated from a culture grown in M2GX and are placed in M2G, which turns off transcription of dnaA, leading to DnaA depletion. After 75 min of DnaA depletion in M2G, xylose is added, making M2GX and turning on dnaA transcription. If dnaA transcription is not induced, GM2471 cells continue to elongate as stalked cells with a single chromosome.
C. GM2471 swarmer cells isolated from a M2GX culture were released into either M2G or M2GX and allowed to proceed synchronously through the cell cycle. Arrows indicate typical stalks, which were present in both the M2G and M2GX cultures after 60 min. After 120 min, the M2GX population had developed into pinched predivisional cells while the M2G population had elongated as unpinched, stalked cells.
D. GM2471 swarmers from a M2GX culture were released in M2G, and after 75 min, xylose was added to part of the culture to induce DnaA. At the indicated times, aliquots were taken and incubated at 30°C for 3 h with rifampicin (15 µg ml−1), which allowed cells that had initiated DNA replication prior to rifampicin addition to finish chromosome duplication, but blocked new initiations. Cells were then fixed and analysed by flow cytometry as in (Winzeler et al., 1997). Data were collected with a Becton Dickinson FACStar Plus machine and analysed using FLOJO software. The numbers below each panel indicate chromosome content. Cells that had not initiated replication at the time of a sample have a DNA content of one chromosome, while cells that had initiated replication have a DNA content of two chromosomes.

DnaA is essential for replication initiation (Gorbatyuk and Marczynski, 2001), and DnaA binds specifically to DnaA boxes, nine base pair asymmetric DNA motifs found in the origin of replication of most Eubacteria (Mackiewicz et al., 2004). DnaA bound to the origin of replication causes the two strands of DNA to unwind and separate, allowing replication factors to enter and form an initiation complex (reviewed in Messer et al., 2001). The cellular level of DnaA is under cell cycle control, peaking at the G1/S transition (Gorbatyuk and Marczynski, 2005). To separate the effects of DnaA from other regulatory mechanisms active at the time of the G1/S transition, we used a dnaA-inducible strain (Gorbatyuk and Marczynski, 2001) to modulate dnaA expression and DnaA accumulation. Delaying dnaA expression delays processes that are largely DnaA-independent, such those involved in swarmer-to-stalked cell differentiation (Gorbatyuk and Marczynski, 2001), much less than it delays events dependent on DnaA, such as replication initiation and the transcription of some genes. Using this technique, we separated normally concurrent events in order to identify separate but overlapping regulatory pathways in complex cellular circuitry.


Delaying dnaA transcription separates the G1/S transition from the swarmer-to-stalked cell differentiation

At the time DnaA initiates DNA replication and causes Caulobacter cells to undergo the G1/S transition, many other cell cycle processes, including the swarmer/stalked morphological transition, are also happening (Fig. 1A). To distinguish DnaA-dependent events from other events that occur during the G1/S transition, we developed a DnaA depletion/induction cell cycle protocol that delays transcription of dnaA until after the swarmer/stalked transition and (presumably) other DnaA-independent events, that normally happen concurrently with the G1/S transition, have occurred (Fig. 1B). The protocol uses GM2471, a strain of C. crescentus where the only copy of the monocistronic dnaA gene is on the chromosome under the control of a xylose-inducible promoter (Gorbatyuk and Marczynski, 2001).

In the DnaA depletion/induction protocol, GM2471 swarmer cells are isolated from a culture grown in M2GX (minimal salts media with glucose and xylose) where dnaA is transcribed and placed in M2G (minimal salts media with glucose as the only carbon source) where dnaA is off. After 60 min in M2G, most GM2471 cells develop into stalked cells, and only about 10% of the cells, as judged by light microscopy, are still motile (Fig. 1C). During continued DnaA depletion in M2G, GM2471 cells fail to initiate DNA replication (Fig. 1D) or develop into pinched predivisional cells (Fig. 1C). In the DnaA depletion/induction protocol, xylose is added, inducing dnaA, after 75 min of DnaA depletion in M2G. Most cells then initiate DNA replication 25–45 min later (Fig. 1D), finish the cell cycle, and divide. The number of viable cells doubles within 3 h of DnaA induction (Fig. S1). By depleting DnaA for 75 min, we obtained the maximum separation between DnaA-independent and DnaA-dependent processes without decreasing the synchronicity of the subsequent recovery (data not shown).

Delaying dnaA transcription delays cell cycle-regulated transcription

Using gene expression microarrays, we obtained time courses of transcription during both the DnaA depletion/induction cell cycle and the wild-type cell cycle (Fig. 2A). Some genes previously identified as cell cycle-regulated (Laub et al., 2000) are not included in Fig. 2A because their expression profiles were too noisy, had insufficient variation during the cell cycle, or were affected by xylose, which was used to induce dnaA. We found that 267 genes had robust, reproducible, time-varying behaviour during both the wild-type and the DnaA depletion/induction cell cycles.

Figure 2.

Transcription during the wild-type and DnaA depletion/induction cell cycles.
A. Microarray expression profiles of the wild-type and DnaA depletion/induction cell cycles are shown with approximate cell cycle stages. RNA samples were collected at the times indicated and were compared with a common reference RNA sample. Each row shows the expression profiles for a different gene. For both the wild-type and DnaA depletion/induction cell cycles, expression profiles for each gene, which are shown on a colour-coded, base two logarithmic scale, were independently centred about zero. Yellow indicates times of above average expression. Blue indicates times when a gene was expressed at a below average value. The vertical pink line indicates the time of DnaA induction. Black boxes indicate genes in the DnaA regulon. Genes with decreased (increased) expression after a 2 h GcrA depletion (Holtzendorff et al., 2004) are indicated by green (red) boxes in the GcrA column. A green (red) box in the CtrA column indicates that the gene had decreased (increased) expression in the ctrA401ts strain at the restrictive temperature compared with the permissive temperature and is in a transcription unit that is a direct target of CtrA (Crymes et al., 1999; Hung and Shapiro, 2002; Laub et al., 2002; Holtzendorff et al., 2004). CC2234 appears alone at the bottom. The data (Table S2) and a larger version of the figure (Fig. S2), including gene annotations, are available online.
B. The transition interval (period when a profile climbs from 25% to 75% of its maximum) and the transition midpoint (time when an expression profile has undergone 50% of its increase) of an example expression profile are indicated. The profile is normalized so the minimum and maximum of the main upward transition are zero and one respectively.
C. A wild-type cell cycle transition interval and a DnaA depletion/induction cell cycle transition interval for an example gene are plotted versus each other. The x and y coordinates of the black circle correspond to the transition midpoints for the wild-type and DnaA depletion/induction time courses respectively. The dotted horizontal and vertical lines indicate the transition intervals for the wild-type and DnaA depletion/induction cell cycles respectively. The wild-type transition interval shown is the same as the one used in B.
D. Transition intervals for the expression profiles in the Pre-DnaA, Post-DnaA and Late sets are shown using the style illustrated in C. Transition midpoints are indicated by red circles (Pre-DnaA set), green squares (Post-DnaA set), or blue triangles (Late set). The black horizontal line at 75 min shows the time of DnaA induction.

To facilitate comparisons between expression profiles from the wild-type cell cycle and the DnaA depletion/induction cell cycle, we defined a transition interval as the time during which a gene's expression profile increased from 25% to 75% of its maximum (Fig. 2B). We also defined the transition midpoint as the time when a profile had completed half of its increase (Fig. 2B). Transition times during the wild-type and the DnaA depletion/induction cell cycles were compared graphically (Fig. 2C). Based on their transition midpoint times, genes that ‘turned-on’ (went from low expression to high expression) during both the wild-type and DnaA depletion/induction cell cycles were partitioned into three groups (Fig. 2D). Genes in both the (i) Pre-DnaA and (ii) Post-DnaA sets are co-expressed early (before ctrA transcription) in the wild-type cell cycle. The Pre-DnaA genes, however, turn on before DnaA induction in the DnaA depletion/induction cell cycle, indicating that their expression does not require DnaA. In contrast, Post-DnaA genes turn on only after DnaA induction indicating that their expression requires DnaA and is not induced by the swarmer/stalked transition alone. The (iii) Late set contains genes transcribed with or later than ctrA in both the wild-type and the DnaA depletion/induction cell cycles. Exceptions are discussed in Supplementary material.

Additionally, we identified an Early set which contains genes whose observed expression variations are likely due to the stress of the synchrony process (carbon starvation and cold) rather than the normal cell cycle. These Early genes were expressed at the beginning of both the wild-type and DnaA depletion/induction cell cycles, but, unlike genes expressed in swarmer cells, were not expressed at the end of the wild-type or DnaA depletion/induction cell cycle experiments.

The 59 Pre-DnaA genes in Table 1 and the 40 Post-DnaA genes in Table 2 belong to multiple functional classes, with the Post-DnaA set being particularly enriched for genes encoding nucleotide biosynthesis enzymes and components of the DNA replication, recombination and repair machinery. The Post-DnaA set contains genes encoding PodJ, a polarity determinant (Viollier et al., 2002); PleC, a dynamically localized histidine kinase required for multiple polar events (Wheeler and Shapiro, 1999); and FtsZ, a GTPase that forms a ring at the division plane and is required for cell division (Quardokus et al., 2001). Of particular interest, the expression of gcrA, which encodes one of the two oscillating global cell cycle master regulators, waits for DnaA induction. Furthermore, 10 of the 40 genes in the Post-DnaA set are GcrA-induced (Holtzendorff et al., 2004).

Table 1.  Early cell cycle genes whose transcription does not require DnaA (Pre-DnaA set).
Chromosome partitioning and cell division
 CC3678intracellular septation protein A, putative
DNA replication, recombination and repair
 CC1283ATPase related to the helicase subunit of the Holliday junction resolvase
 CC1711DnaA-related protein
Nucleotide biosynthesis
 CC1889ribonucleotide reductase-related protein
Other metabolism
 CC0360ornithine decarboxylase, putative
 CC1090cytochrome c family protein
 CC1482sulphate adenylate transferase, subunit 1/adenylylsulphate kinase cysN/C
 CC1724enolase eno
 CC21385-methyltetrahydrofolate – homocysteine methyltransferase
 CC2253lysophospholipase L2 pldB
 CC3085alcohol dehydrogenase adhA
 CC3403cytochrome c oxidase assembly protein, putative
 CC3763uroporphyrinogen decarboxylase hemE
 CC3679signal recognition particle-docking protein ftsY
Transcriptional regulators
 CC1213, CC1280, CC1356, CC2883 (sigU), CC3475 (sigT)
Translation (ribosomal proteins)
 CC1249 (rplD), CC1252 (rpsS), CC1253 (rplV), CC1254 (rpsC), CC1256 (rpmC), CC1257 (rpsQ), CC1263 (rplF), CC1264 (rplR), CC1266 (rpmD)
 CC0322biopolymer transport protein exbD
 CC0362phosphonates ABC transporter, periplasmic phosphonates-binding protein phnD
 CC1666TonB-dependent receptor
 CC2954permeases of the major facilitator superfamily
 CC3229OmpA family protein
 CC3373ABC transporter, ATP-binding protein
Unknown function
 CC0568, CC0600, CC0622, CC1178, CC1290, CC1475, CC1532, CC1988, CC2039, CC2047, CC2178, CC2365, CC2441, CC2476, CC2477, CC2574, CC2987, CC3218, CC3257, CC3467, CC3476, CC3483, CC3613, CC3676
Table 2.  Early cell cycle genes whose transcription requires DnaA (Post-DnaA set).
Cell envelope
 CC1581lipopolysaccharide core biosynthesis protein kdtB
 CC2384exopolysaccharide production protein pss
 CC2557phospho-N-acetylmuramoyl-pentapeptide-transferase mraY
 CC3692VacJ-like lipoprotein
Cell polarity
 CC2045polar organelle development protein podJ
 CC2482non-motile and phage-resistance protein pleC
Chromosome partitioning and cell division
 CC2165similar to ATPases involved in chromosome partitioning
 CC2540cell division protein ftsZ
DNA replication, recombination and repair
 CC0005DNA polymerase III, ɛ subunit dnaQ
 CC0008chromosomal replication initiator protein dnaA
 CC0012DNA mismatch repair protein mutS
 CC0379ribonuclease HII rnhB
 CC1468single-strand binding protein ssb
 CC1665replicative DNA helicase dnaB
 CC1983DNA repair protein recN
 CC2038similar to helicases
 CC2246exodeoxyribonuclease large subunit xseA
 CC2331DNA-binding protein HU
 CC1464flagellin modification protein, putative
 CC1077flagellar biosynthesis protein flhB
Nucleotide biosynthesis
 CC0260ribonucleoside-diphosphate reductase, β subunit nrdB
 CC3492ribonucleoside-diphosphate reductase, α subunit nrdA
 CC3713deoxyuridine 5′-triphosphate nucleotidohydrolase dut
Other metabolism
 CC14954-hydroxy-2-oxoglutarate aldolase/2-deydro-3-deoxyphosphogluconate aldolase eda
 CC1642arsenate reductase and related proteins, glutaredoxin family
 CC2622NADP-dependent malic enzyme maeB
Transcriptional regulators
 CC0949, CC2245 (gcrA)
 CC0286sulphate-binding protein sbp
 CC3298similar to ABC-type Na + efflux pump, permease component
 CC3694ABC transporter, substrate-binding protein, putative
Unknown function
 CC0048, CC0527, CC0710, CC1415, CC2280, CC2377, CC2593

Although DnaA depletion does not prevent the expression of the 59 Pre-DnaA genes, DnaA depletion does delay the expression of 23 Pre-DnaA genes by at least 20 min, based on the start time of the corresponding transition intervals. Additionally, transition intervals for 30 Pre-DnaA genes are at least 20 min longer for the DnaA depletion/induction cell cycle than for the wild-type cell cycle. The slow turn-on of many Pre-DnaA genes during DnaA depletion suggests that DnaA directly or indirectly affects their expression.

The Pre-DnaA set includes the hemE gene, adjacent to the Caulobacter origin of replication. The hemE gene has two promoters: the weak Pw promoter, whose transcripts are translated into HemE protein, and the CtrA-repressed, strong Ps promoter, whose transcription during the stalked stage may help unwind the origin of replication, promoting DNA replication initiation (Marczynski et al., 1995). Consistent with the results of Gorbatyuk and Marczynski (Gorbatyuk and Marczynski, 2001), we find that hemE is transcribed during DnaA depletion after the swarmer/stalked transition, presumably in response to the absence of CtrA (see below), but that hemE transcription is insufficient for DNA replication initiation.

Using transition midpoint times, we found that while the 139 Late genes turn on in essentially wild-type order during the DnaA depletion/induction cell cycle, transcription of Late genes in the DnaA depletion/induction cell cycle is delayed ∼60 min compared with the wild-type cell cycle. In both the wild-type and DnaA depletion/induction cell cycles, ctrA is one of the first genes in the Late set to be expressed. Many CtrA-activated genes are in the Late set, including genes encoding the DivK and PleD response regulators, the CcrM methyltransferase, and flagellum, pili, holdfast and chemotaxis proteins. The comparable activation order of genes in the Late class during both the wild-type and the DnaA depletion/induction cell cycle suggests that once ctrA transcription has been turned on, the cells have largely recovered from the DnaA depletion. The widespread dependence on CtrA of transcription that occurs after ctrA activation appears to buffer the timing of expression of genes needed near end of the cell cycle from perturbations that happen earlier in the cell cycle.

The expression profiles for CC2234 (bottom of Fig. 2A), which is expressed early in the DnaA depletion/induction cell cycle and late in the wild-type cell cycle, do not fit into any of the four main classes. CC2234 encodes a protein of unknown function, and the cause of CC2234's anomalous behaviour is unknown.

GcrA, FtsZ and PodJ accumulation requires DnaA

Using immunoblots, we investigated the accumulation of GcrA, FtsZ and PodJ, which are encoded by genes in the Post-DnaA set, during the DnaA depletion/induction cell cycle. We also tracked the levels of CtrA, which disappears during the swarmer/stalked transition in both wild-type cells (Fig. 1A) and cells undergoing DnaA depletion (Fig. 3A). In the wild-type cell cycle, depicted in Fig. 1A, GcrA appears concurrent with CtrA degradation (Holtzendorff et al., 2004). In contrast, during DnaA depletion, GcrA levels remain low following CtrA degradation, leading to a situation where neither CtrA nor GcrA is present (Figs 1B and 3A). Subsequent induction of DnaA results in the accumulation of GcrA, consistent with the observed increase in gcrA mRNA levels following DnaA induction. These results suggest that the gcrA promoter, in addition to being repressed by CtrA (Holtzendorff et al., 2004), is activated by a DnaA-dependent factor or by DnaA itself.

Figure 3.

GcrA, FtsZ and PodJ accumulation requires DnaA. Using immunoblots, CtrA (A), GcrA (A), FtsZ (B) and PodJ (C) levels were followed in a synchronized population of GM2471 swarmer cells undergoing DnaA depletion. After 75 min, DnaA was induced in part of each culture (A–C, top) while DnaA depletion continued in the rest of the culture (A–C, middle). Each lane contains protein from equal amounts of culture. Sample times (min) are shown above each blot. After 200 min, the induced culture was 1.3 times denser than the uninduced culture (as measured by optical density). For each protein, values were quantified with ImageQuant and are shown as a fraction of the maximum attained by the culture where DnaA was induced (A–C, bottom).

Like the gcrA promoter, the ftsZ and podJ promoters are directly repressed by CtrA (Kelly et al., 1998; Crymes et al., 1999; Holtzendorff et al., 2004). During the DnaA depletion/induction cell cycle, ftsZ and podJ mRNA levels, like gcrA mRNA levels, increase little in response to CtrA degradation and instead wait for DnaA induction. During DnaA depletion, FtsZ levels increase slightly following the swarmer/stalked transition and CtrA degradation, and induction of DnaA causes an additional increase in FtsZ levels (Fig. 3B).

The podJ gene is specifically transcribed following the swarmer/stalked cell transition and PodJ in its long form, PodJL, is positioned at the incipient swarmer cell pole of predivisional cells (Viollier et al., 2002). At cell division, PodJL is processed to a short form, PodJS, which remains at the pole of the progeny swarmer cell until it is cleared by a regulated proteolytic event during the swarmer/stalked transition (Chen et al., 2005). While the clearance of PodJS at the swarmer/stalked transition does not require DnaA, PodJL accumulates only after DnaA induction (Fig. 3C). The failure of cells to accumulate GcrA, FtsZ and PodJ while the G1/S transition is blocked by DnaA depletion demonstrates the high degree of coordination between DNA replication, polar development and cell division in Caulobacter.

DnaA boxes are present upstream of some DnaA-inducible genes

Of the 40 genes in the Post-DnaA set, 21 contain at least one match (seven of nine or better) to TTATNCACA, the E. coli consensus DnaA box (Messer and Weigel, 1997), in the 200 bases upstream of their translation start site (either strand). Neither random sets of genes nor the Pre-DnaA, Early, or Late sets contain similar enrichments of upstream DnaA boxes. Thus, the Post-DnaA set possesses a unique feature, and the DnaA boxes upstream of Post-DnaA genes are most likely functional rather than a chance occurrence, suggesting that Caulobacter DnaA acts as a transcription factor.

We created an improved, more specific model for Caulobacter DnaA boxes from the candidate DnaA boxes upstream of the most strongly induced genes in the Post-DnaA set and the four best matches to the E. coli DnaA box present in the Caulobacter origin of replication (Marczynski and Shapiro, 1992) using MEME (Bailey and Elkan, 1994). Thirteen genes that belong to the Post-DnaA set and contain a DnaA box (MEME model) in the 200 bases upstream of the translational start site were identified as candidates for the DnaA regulon (Table 3). Expression of all 13 genes increases following DnaA induction. Of these 13 genes, six have DnaA boxes upstream of the −10 to −35 promoter region and two have DnaA boxes downstream of their +1 sites. The +1 sites for five of the 13 genes have not been identified. The DnaA boxes upstream of podJ and gcrA both overlap DNA sites recognized by the CcrM methyltransferase, so the methylation state of the DNA, which reflects the replication state of the chromosome (Marczynski, 1999), may affect DnaA binding.

Table 3. Caulobacter DnaA regulon identified by delaying dnaA transcription.
GeneDistance from
translational start sitea
from +1a,b
  • a

    . Distances refer to the base in the motif closest to the start codon or +1 site. Translational start sites came from (Nierman et al., 2001).

  • b

    . The podJ+1 site came from (Crymes et al., 1999). The gcrA+1 site came from (Holtzendorff et al., 2004). The ftsZ+1 site came from (Kelly et al., 1998). Others are from P. McGrath (unpubl. data). +1 site information is not available (NA) for all genes. For CC3692 the +1 site data indicate the actual translational start site is downstream of the (Nierman et al., 2001) prediction.

  • c

    .‘+’ indicates the motif is on the same strand as the gene; ‘–’ indicates the motif is on the opposite strand as the gene.

CC0005 DNA polymerase III, ɛ subunit dnaQ 49NATTCTCAACA
CC0260 ribonucleoside-diphosphate reductase, β subunit nrdB114 72+TTATCCACA
CC1415 hypothetical protein113NATGATCCTCA
CC1468 single-strand binding protein ssb103 51+TTGCCCACA
CC1665 replicative DNA helicase dnaB 71NA+ACATACACA
CC2045 polar organelle development protein podJ 98 80+TCCTCCACA
CC2165 similar to ATPases involved in chromosome partitioning 87NACAATCCACA
CC2245 global cell cycle regulator gcrA 92 51CTGTCCACA
CC2280 hypothetical protein 91NACGATCCACA
CC2331 DNA-binding protein HU 18−22+TGATTCACA
CC2540 cell division protein ftsZ172

CC3492 ribonucleoside-diphosphate reductase, α subunit nrdA 84−11+TCAGCCACA
CC3692 VacJ-like lipoprotein122130TGATCCACC
Caulobacter Multi-level consensus   TTATCCACA

His6-DnaA binds to the gcrA, ftsZ and podJ promoters in vitro

We used an in vitro gel-shift assay to test the binding of His6-DnaA to the gcrA, ftsZ and podJ promoters. γ[32P]ATP-labelled, 29 base pair, double-stranded DNA probes containing the DnaA boxes centred 64 (ftsZ-1) and 47 (ftsZ-2) bases upstream of the ftsZ+1 site (Kelly et al., 1998), 55 bases upstream of the gcrA+1 site (Holtzendorff et al., 2004), and 84 bases upstream of the podJ+1 site (Crymes et al., 1999) formed concentration-dependent, specific complexes with His6-DnaA (Fig. 4A). The gcrA probe had the strongest binding while the ftsZ-2 probe had the weakest. The close proximity of two of the three DnaA boxes in the ftsZ promoter suggests that DnaA may bind cooperatively to the full-length region. We used a region of the origin of replication containing a DnaA box as a positive control. As a negative control, we used the same region with a two base pair mutation in the DnaA box known to be incompatible with replication initiation (Marczynski and Shapiro, 1992). The wild-type origin region formed a complex with His6-DnaA while the mutated origin did not. An excess of unlabelled probe in the reactions disrupted the binding of the [32P]-labelled probe, but excesses of either of two arbitrary competitors did not (Fig. 4B). The presence of DnaA boxes upstream of genes that are induced rapidly following DnaA induction (the Post-DnaA set) coupled with the binding of His6-DnaA to promoters of selected DnaA-inducible genes suggests that DnaA acts as a transcription factor in Caulobacter.

Figure 4.

His6-DnaA binds to the gcrA, podJ and ftsZ promoters.
A. Electrophoretic mobility shift assays of His6-DnaA and DNA probes of a DnaA box from the origin, a mutated version of the origin probe with a two-base change in the DnaA box, and the gcrA, podJ and ftsZ promoters were performed with the indicated protein concentrations and a probe concentration of ∼1 nM.
B. A 500-fold excess of unlabelled probe (S) or of one of two arbitrary DNA sequences (A1 or A2) was added to the reaction. The DnaA concentration was 1.2 µM and the probe concentration was ∼1 nM.


The essential DnaA replication initiation factor is present at peak concentrations during the Caulobacter G1/S transition coincident with swarmer-to-stalked cell differentiation (Gorbatyuk and Marczynski, 2001; 2005). Furthermore, in both E. coli (reviewed in Messer and Weigel, 1997) and B. subtilis (Burkholder et al., 2001), DnaA has been shown to function as a transcription factor. We have shown here that DnaA plays a central role in the top-level genetic circuit that controls Caulobacter cell cycle progression.

In the normal Caulobacter cell cycle, many regulatory events occur coincidently during the swarmer/stalked cell and G1/S transitions, obscuring the role of DnaA. By delaying dnaA expression, we separated DnaA-dependent events including the G1/S transition from the swarmer/stalked cell transition and other early cell cycle pathways that normally execute concurrently (Fig. 5). Of 99 genes expressed during the swarmer/stalked cell transition, we identified 59 (Table 1) whose expression does not require DnaA and 40 whose expression is DnaA-dependent (Table 2). Thirteen of the DnaA-dependent genes have a DnaA box in their promoter regions and likely belong to the DnaA regulon (Table 3). Because delaying dnaA expression delays DNA replication, some genes whose expression appears to be indirectly dependent on DnaA may actually be dependent on DNA replication (Humphery-Smith, 1999). For example, expression of some genes may depend on the methylation state of the DNA, which reflects the replication state of the chromosome (Marczynski, 1999).

Figure 5.

Delaying dnaA transcription delays expression of some, but not all, genes expressed early in the cell cycle.
A. During the normal cell cycle, Caulobacter expresses both DnaA-independent and DnaA-dependent genes early in the cell cycle at overlapping times.
B. Delaying dnaA expression delays expression of some early cell cycle genes and expression of all genes expressed in late stalked and predivisional cells, including ctrA. Approximate cell cycle stages and DnaA protein levels are shown for reference.

The DnaA regulon includes genes encoding nucleotide biosynthesis enzymes, components of the DNA replication machinery, and GcrA, a global regulator of the Caulobacter cell cycle. The DnaA regulon also contains podJ, which encodes a polarity determinant that is asymmetrically localized in predivisional cells (Viollier et al., 2002), and ftsZ, which encodes an essential cell division protein (Quardokus et al., 2001). His6-DnaA directly binds the promoters of gcrA, ftsZ and podJ.

Multiple regulatory factors control a subset of the DnaA-regulated genes. CtrA directly regulates four genes (Laub et al., 2002) and GcrA directly or indirectly regulates seven genes in the DnaA regulon (Holtzendorff et al., 2004). For four genes in the DnaA regulon, DnaA is the only known regulator. While DnaA boxes are upstream of the −10 to −35 promoter regions of most genes in the DnaA regulon, DnaA boxes are downstream of the +1 sites for hu and nrdA, similar to the case in E. coli where DnaA positively regulates the bacteriophage λpR promoter by binding downstream of the +1 site (Glinkowska et al., 2003).

The Caulobacter DnaA regulon likely contains more than the 13 genes conservatively identified in this work. For example, genes whose expression is strongly affected by the carbon source used to turn on and off the inducible promoter employed to delay dnaA expression (Meisenzahl et al., 1997; Gorbatyuk and Marczynski, 2001) were not considered for membership in the DnaA regulon. In addition, genes induced by both DnaA and other factors in a redundant fashion may not have been identified as DnaA-dependent. Somewhat surprisingly, repression by DnaA, which can be caused by DnaA binding within a promoter region or DnaA binding within a transcribed region leading to transcriptional termination (reviewed in Messer and Weigel, 1997), was not definitively observed, suggesting that any DnaA-repressed genes are also affected by other factors. Additionally, if DnaA regulates genes at times in the cell cycle other than the G1/S transition, these genes would not have been identified.

DnaA is a master regulator of the Caulobacter cell cycle

As an activator of gcrA transcription, DnaA, along with CtrA and GcrA, is an integral component of the top-level genetic circuit controlling Caulobacter cell cycle progression (Fig. 6). In swarmer cells, CtrA actively inhibits replication initiation by binding to the chromosome origin of replication (Quon et al., 1998). At the same time, CtrA directly represses transcription of gcrA (Holtzendorff et al., 2004), ftsZ (Kelly et al., 1998) and podJ (Crymes et al., 1999), blocking the early steps in cell division and polar development. The concurrent proteolysis of DnaA in swarmer cells (Gorbatyuk and Marczynski, 2005) removes DnaA's activating effect on the same components. During the swarmer/stalked transition, CtrA is degraded (Quon et al., 1996) while DnaA levels increase (Gorbatyuk and Marczynski, 2005). The presence of DnaA, and not just the absence of CtrA, is required to trigger an increase in GcrA levels and start the next wave of cell cycle transcription, which includes the expression of genes encoding nucleotide biosynthesis and DNA replication enzymes. Thus, DnaA not only initiates DNA replication but also promotes the transcription of the components necessary for successful chromosome duplication. DnaA also activates transcription of ftsZ and podJ, starting the cell division and polar organelle development processes that, in addition to DNA replication, will prepare the cell for asymmetric division. Ultimately, the DnaA-induced increase in GcrA levels coupled with DNA replication turns ctrA transcription back on (Holtzendorff et al., 2004).

Figure 6.

DnaA coordinates cell cycle processes by acting as a replication initiation factor and a transcription factor. GcrA, CtrA and DnaA regulated pathways are shown in light grey, medium grey and black respectively. To emphasize the role of DnaA, some GcrA and CtrA pathways have been omitted. Possible regulators of dnaA which include GcrA (Holtzendorff et al., 2004), DnaA itself (Zweiger and Shapiro, 1994), and the unknown protein that binds to the RRF (repression of replication factors) motif in the dnaA promoter (Winzeler and Shapiro, 1996; Keiler and Shapiro, 2001) are not shown.

CtrA and DnaA levels exhibit inverse behaviour throughout the cell cycle (Gorbatyuk and Marczynski, 2005). For example, swarmer cells contain CtrA and only low levels of DnaA while stalked cells contain DnaA but not CtrA. Caulobacter cells use the reciprocal behaviour of CtrA and DnaA in a regulatory logic motif involving (i) CtrA negative regulation and (ii) DnaA positive regulation to form a robust switch to implement the decision to stay in the G1 stage or to undertake a round of cell division.

We surmise that this reliable, redundant control of entry into the cell division pathway would be advantageous for an organism like Caulobacter that normally lives in an environment that permits only infrequent divisions (Poindexter et al., 2000). Having to change the level of two master regulators, CtrA and DnaA, rather than just one, in order to cause key cell cycle events like replication initiation or gcrA, ftsZ and podJ transcription reduces the chance of erroneously triggering cell cycle progression.

Many bacteria use DnaA as a transcription factor, but the DnaA regulon varies widely among organisms (Wang and Kaguni, 1989; Tesfa-Selase and Drabble, 1992; 1996; Augustin et al., 1994; Quinones et al., 1997; Burkholder et al., 2001). In each organism, the genes in the regulon must be selected to provide functions needed at the time of DnaA accumulation that are important to that organism's fitness strategy. B. subtilis, for example, uses DnaA to ensure that DNA replication precedes sporulation (Burkholder et al., 2001), while Caulobacter uses DnaA as a master regulator to coordinate DNA replication with cell cycle-regulated transcription, cell division and polar organelle development.

Experimental procedures

Bacterial strains and growth conditions

Liquid cultures of C. crescentus CB15N (wild-type) and GM2471 (Gorbatyuk and Marczynski, 2001) were grown at 30°C in peptone yeast extract (PYE) or M2 minimal media containing either 0.2% glucose (M2G) or 0.2% glucose and 0.3% xylose (M2GX) (Ely, 1991). Cell cultures were synchronized using Ludox density centrifugation (Quon et al., 1996) or Percoll density centrifugation (Tsai and Alley, 2001).

Oligo microarrays

Samples for microarray analysis were collected by centrifugation and were frozen in liquid nitrogen. RNA was isolated using Trizol (Invitrogen) (Holtzendorff et al., 2004).

For one wild-type cell cycle data set, a synchronized culture of CB15N swarmer cells grown in M2G was sampled every 15 min during the 150 min cell cycle. For a second wild-type cell cycle data set, a synchronized culture of CB15N swarmer cells grown in PYE (rich media) was sampled every 10 min during the faster 100 min cell cycle. We also used the wild-type cell cycle data from (Laub et al., 2000), which provided a 15 min sampling of the CB15N cell cycle in M2G. All times shown for the wild-type cell cycle refer to the 150 min M2G cell cycle, not the faster PYE cell cycle.

For each of two DnaA depletion/induction cell cycles, swarmer cells were isolated from GM2471 cultures grown in M2GX and placed in M2G, which turned off dnaA transcription. After 74 min in M2G, xylose was added to induce dnaA transcription. Samples were taken every 15 min from 0 to 195 min. The delay in dnaA transcription causes the DnaA depletion/induction cell cycle to take longer than the M2G CB15N cell cycle even though both experiments use similar growth conditions.

Microarrays containing a 50mer oligo for each Caulobacter gene were printed, hybridized, and washed as in Holtzendorff et al. (2004), except that arrays for the wild-type M2G cell cycle were hybridized at 45°C; all other microarrays were hybridized at 44°C. Each microarray compared the relative abundance of mRNA species from one time during the cell cycle to a common reference of RNA from a log-phase CB15N culture that contained all cell cycle stages. For example, the M2G wild-type cell cycle data set consists of arrays comparing the 0 min sample to the reference, the 15 min sample to the reference, the 30 min sample to the reference, . . . , and the 150 min sample to the reference. All samples from each cell cycle experiment used the same reference RNA. Reference RNA for the PYE, M2G and DnaA depletion/induction cell cycles was isolated from cells grown in PYE, M2G and M2GX respectively. Reference RNA for the DnaA depletion/induction series was additionally treated with DNase I (Hottes et al., 2004).

Arrays were scanned using a GenePix 4000B scanner (Axon Instruments), and spots were located and quantified using GenePix Pro 4.0 (Axon Instruments) software. Ratios were filtered and normalized as in (Hottes et al., 2004). Microarray data were visualized using Treeview (Eisen et al., 1998).

Identifying the effect of delaying dnaA expression on cell cycle transcription

Based on pair-wise correlation coefficients between expression profiles from different repetitions and the fold change in expression during each experiment, we identified 1482 genes that changed reproducibly during the DnaA depletion/induction cell cycle and 921 genes that behaved reproducibly during the wild-type cell cycle. Overall, 569 genes belonged to both sets. Using simulations with randomized profiles, the false discovery rate [(false positives)/(total discovered)] was estimated to be 1% (5.6/569) (Benjamini and Hochberg, 1995). The number of repeatable DnaA depletion/induction profiles is larger than the number of reproducible wild-type profiles because the preliminary selection did not discriminate between reproducible responses to the addition of xylose and reproducible responses to cell cycle progression. Expression profiles for genes that are neither cell cycle-regulated nor xylose-sensitive do not contain large, reproducible variations and were removed.

To characterize the transcriptional response to the addition of xylose, mixed cultures of wild-type (CB15N) cells grown in M2GX were subjected to a mock synchrony, which did not separate out the different cell types, and then were placed in M2G. The mock synchrony was similar to a Ludox density centrifugation synchrony (Quon et al., 1996) except that ice-cold M2 was used instead of Ludox. One RNA sample was taken after 70 min in M2G. After 75 min in M2G, xylose was added, making M2GX. Thirty minutes later (105 min from the beginning) a second RNA sample was taken. Four paired samples from independent cultures were compared using oligo microarrays.

A DnaA depletion/induction cell cycle expression profile's xylose response component was considered negligible compared with its cell cycle response component if one of two conditions were met. First, a profile was considered mainly cell cycle-responsive if the gene's expression changes during a part of the DnaA depletion/induction cell cycle when the cells were in a single media (i.e. M2G or M2GX) exceeded the gene's expression change in response to xylose addition. Second, a profile was considered cell cycle-responsive if the gene's immediate response to DnaA induction (xylose addition) was greater than its response to xylose addition alone. Genes with a rapid response to the synchronization process (i.e. a large expression change between 0 and 15 min) and no additional variation over the course of the experiment, are neither xylose nor cell cycle-responsive and did not meet the above criteria. Overall 267 genes had reproducible wild-type and reproducible, non-xylose DnaA depletion/induction expression profiles. Microarray data (Table S1) and additional details about data processing are available in Supplementary material and in the NCBI's GEO database under Accession number GSE3171.

Locating DnaA boxes

In searching for DnaA boxes using the MEME-generated position-specific scoring matrix (Bailey and Elkan, 1994), the likelihood that each sequence contained a DnaA box was compared with the likelihood that it was a typical Caulobacter intergenic sequence. Caulobacter intergenic sequence was represented using a third-order Markov model (Liu et al., 2001). Only high scoring motifs that were at least a 7/9 match to TTATNCACA, the E. coli consensus DnaA box (Messer and Weigel, 1997) were accepted. The middle base, which often is cytosine in E. coli, was not constrained because DnaA does not interact with it in a base-specific manner (Fujikawa et al., 2003). The cut-off for the presence of a DnaA box was set by trading off between the number of DnaA boxes found in random sequences and the number of DnaA boxes found upstream of the most strongly induced genes in the Post-DnaA set. The cut-off used yielded 7.7 false positives for every 100 promoters of random sequence (200 base pairs) generated using the background model. Similarly, the motif is present in the 200 bases upstream of the translational start sites of 6.7% of Caulobacter genes. As 40 genes in the Post-DnaA set were searched for DnaA boxes, we estimate that the 13 gene DnaA regulon identified contains ∼3 false positives. In addition to the one DnaA box upstream of ftsZ that met the cut-off, two other nearby possible DnaA boxes are also shown. Sequences were manipulated using Genome-tools (Lee and Chen, 2002).

We used an E. coli-derived model for DnaA boxes (seven of nine or better match to TTATNCACA) to determine that DnaA boxes are enriched upstream of the genes in the Post-DnaA set to avoid biasing the analysis by training the model on the same Caulobacter data that would later be tested. As the E. coli model lacks specificity (∼35% of Caulobacter genes have a seven of nine or better match in their upstream 200 bases), we used MEME and the best Caulobacter candidate DnaA boxes to refine the model before predicting the Caulobacter DnaA regulon.

Western blots

Immunoblotting was carried out as described in (Chen et al., 2005). Each pair of blots (induced and non-induced) in Fig. 3 includes the same samples for 0, 20, 40 and 60 min. Data between corresponding blots were normalized using a linear model (Weisberg, 1985) based on the data for the shared time points. Experiments were performed at least twice.


Cells were fixed in 2.5% formaldehyde and 30 mM sodium phosphate buffer, pH 7.5 at the indicated times. Cells were either immobilized on agarose pads or placed directly on glass slides. Nomarski differential interference contrast (DIC) images were taken with a 100 × DIC objective on a Nikon E800 microscope with a 5 MHz Micromax 5600 cooled CCD camera controlled through Metamorph (Universal Imaging). Images were prepared using Adobe Photoshop and Metamorph version 4.5.

His6-DnaA gel shift

The complete coding sequence of the dnaA gene from the CB15 wild-type Caulobacter strain was amplified using primers 5′-CGTTAGCCCCGCAGCTTGCGCG-3′ and 5′-GGCG GTGGACATATGACCATGAAGGGC-3′. The polymerase chain reaction (PCR) product was digested with NdeI and HindIII and cloned into NdeI-HindIII-digested pET-28a(+) (Novagen). The resulting plasmid, pHis6DnaA, expresses an N-terminal His6-tagged DnaA protein. Plasmid preparation, DNA cloning and PCR amplifications were carried out according to manufacturers’ instructions.

His6-DnaA was over-expressed in the BL21 E. coli strain by inducing for 4 h at 30°C with isopropyl-β-D-thiogalactopyranosid and was purified using native conditions (Qiagen, 2003). Gel shift reactions were carried out as in (Ausubel et al., 2001). Details and oligos used are in Supplementary material.


We thank Swain Chen for constructing the His6-DnaA over-expression strain, Greg Marczynski (McGill University) for providing strain GM2471, Michael Laub and Melanie Prasol for advice on oligo microarray protocols, Maliwan Meewan for microarray printing, Justine Collier for critical reading of the manuscript, and Ji Park for computer support. This work was supported by ONR Grant N00014-02-1-053 to H.M., DOE Grants DE-FG03-01ER63219-A001 and DE-FG02-01ER63219 to H.M., and NIH Grants GM51426 and GM32506 to L.S.