Replication initiator DnaA binds at the Caulobacter centromere and enables chromosome segregation

Significance

DnaA is an essential and conserved bacterial protein that enables the initiation of DNA replication. Although it is commonly held that the onset of bacterial chromosome segregation depends on the initiation of DNA replication, we have found that in Caulobacter crescentus, chromosome segregation can be induced in a DnaA-dependent, yet replication-independent manner. The chromosome replication origin, containing essential DnaA binding motifs, resides 8 kb from the centromere parS region that also contains DnaA binding motifs. The centromere parS region bound to the ParB partition protein initiates movement across the cell followed by the origin region. Mutations in a centromere DnaA motif that alter DnaA–centromere interaction exhibit aberrant patterns of ParB/parS translocation, implicating DnaA in the process of chromosome segregation.

Abstract

During cell division, multiple processes are highly coordinated to faithfully generate genetically equivalent daughter cells. In bacteria, the mechanisms that underlie the coordination of chromosome replication and segregation are poorly understood. Here, we report that the conserved replication initiator, DnaA, can mediate chromosome segregation independent of replication initiation. It does so by binding directly to the parS centromere region of the chromosome, and mutations that alter this interaction result in cells that display aberrant centromere translocation and cell division. We propose that DnaA serves to coordinate bacterial DNA replication with the onset of chromosome segregation.

Cell division requires the faithful transmission of genetic information to each daughter cell. Thus, in all forms of life, multiple mechanisms cooperate to ensure that DNA synthesis and chromosome segregation are temporally controlled and coordinated. Unlike eukaryotes, in which chromosomes are fully replicated and organized into higher order structures before segregation (1), most bacteria segregate their chromosomes progressively during replication (2). DnaA is a conserved bacterial protein responsible for the initiation of DNA synthesis at the chromosomal origin of replication (ori) (3, 4). The mechanism by which chromosome segregation is initiated in bacteria is less well understood.

Although the factors responsible for DNA replication are highly conserved among bacterial species, multiple mechanisms have been proposed to account for chromosome segregation (5). In the G₁ phase of the Caulobacter crescentus cell cycle, the centromeric region of the chromosome (parS) is tethered to one pole of the cell (Fig. 1A). Upon the swarmer to stalked cell transition, replication initiates with replisome assembly at the origin of replication. The Par system in Caulobacter includes parS and two partitioning proteins, ParA and ParB. The ParB protein binds to parS (6, 7), which, in turn, interacts with the nucleoid-associated ParA ATPase to effect centromere movement (8–11).

Fig. 1.

DnaA-dependent chromosome translocation. (A) Dynamics of Caulobacter chromosome segregation. (B–F) Time-lapse fluorescence micrographs of CFP-ParB in synchronized cells grown on M2G agarose pads (Left). Plots in Right depict fraction of synchronized cells with CFP-ParB translocated to the new pole (reported as % cells) grown in liquid media. Samples were taken every 30 min and imaged over a 3-h time-course. Data are represented as mean ± SD from two independent experiments with n equals on average 200 cells per time point per experiment. Caulobacter cells have a faster generation time in liquid media than on agarose pads. (B) Wild-type cells (parB::cfp-parB; MT190). Cells with dnaA expression regulated by P_xylX (parB::cfp-parB, dnaA::Ω, xylX::dnaA; LS5368) grown on M2G + 0.3% xylose (C) or M2G only (D). Cells with dnaA expression regulated by P_vanA (parB::cfp-parB, dnaA::Ω, vanA:dnaA; LS5369) grown on M2G + 250 μM vanillate (E) or M2G only (F); arrows indicate segregation of CFP-ParB/parS. (G) Vanillate promoter has leaky expression of DnaA. Relative levels of DnaA were followed by using Western blots with anti-DnaA antibodies (1 in 10,000 dilution) in mixed population of LS101 (Top), LS5368 (Middle), and LS5369 (Bottom) undergoing DnaA depletion. Cells were washed three times and grown on liquid M2G. Exposures of all samples were done simultaneously. (H) DnaA-dependent centromere translocation. Plotted are the percentages of cells (parB::cfp-parB, dnaA::Ω, xylX::dnaA; LS5368) with a single CFP-ParB focus translocated to the distant pole as a function of levels of xylose inducer added (i.e., varying subphysiological levels of DnaA). Localization of CFP-ParB was determined by fluorescence microscopy of cells grown in M2G liquid media with the respective xylose concentration.

In Vibrio cholerae and Bacillus subtilis, the chromosome partitioning protein ParA (Soj) has been reported to regulate replication initiation by directly interacting with the DnaA replication initiator protein, suggesting a connection between segregation and the initiation of replication (12–14). However, the signals that trigger the Par system to initiate chromosome segregation are not known. By generating Caulobacter strains that express limited concentrations of DnaA, we sought to determine whether replication initiation is a prerequisite for the translocation of the centromere complex. Under these conditions, we were able to detect translocation of the chromosome in the absence of replication. We show that DnaA binds directly within the parS region and that altering binding of DnaA to parS leads to compromised chromosome segregation. These results suggest that, in Caulobacter, DnaA plays a direct role in the initiation of chromosome segregation.

Results

DnaA-Dependent Chromosome Translocation.

In Caulobacter, the ParB partitioning protein directly occupies the parS site, found 8 kb from ori (7). In cells expressing a functional CFP-ParB fusion protein, the number of fluorescent foci reflects the copy number of ori (Fig. 1A). In a wild-type background, initiation of DNA replication results in the migration of one copy of the CFP-ParB/parS complex toward the opposite end of the cell, culminating in the establishment of a second fluorescent ParB focus at the distal pole (7, 11) (Fig. 1 A and B). To investigate the dependency of chromosome segregation on DNA replication, we imaged the translocation of CFP-ParB/parS foci in cells expressing subphysiological DnaA concentrations. We constructed a Caulobacter strain in which transcription of the sole copy of dnaA was controlled by the tightly regulated, xylose-inducible P_xylX promoter (15) and in which parB was replaced with a translational fusion of the gene encoding CFP to parB. When xylose was present in the growth media in this dnaA depletion strain, DnaA was expressed and fluorescent imaging revealed CFP-ParB localized to both cell poles, indicating replication initiation had occurred (Fig. 1C). When these cells were shifted to media lacking xylose, a single CFP-ParB focus remained at the stalked pole (Fig. 1D), consistent with the requirement of DnaA to initiate replication (16).

To characterize the effect of limited DnaA levels on chromosome segregation, we constructed a dnaA depletion strain in which dnaA expression was driven by the leaky, vanillate-inducible P_vanA promoter. Growth of this strain in the absence of vanillate led to an 80% reduction in relative levels of DnaA after 1 h, whereas DnaA levels decreased below the detection threshold in a P_xylX-dnaA strain within 30 min following removal of inducer (Fig. 1G and Fig. S1). In the presence of vanillate (250 μM), the dynamic localization of CFP-ParB during the cell cycle was indistinguishable from that observed in a wild-type strain (Fig. 1E). Growth in the absence of vanillate produced cells with a single CFP-ParB focus. Notably, in this background, we found that this single focus translocated to the opposite pole in more than 60% of the cells (Fig. 1F).

These observations raised the possibility that DnaA is competent to promote the initiation of chromosome segregation when levels are depleted below a concentration threshold that is critical for replication initiation. To determine whether DnaA exhibits this behavior at steady state, we constitutively expressed dnaA from the P_xylX-dnaA promoter at a range of subphysiological DnaA levels (Fig. 1H). We titrated the levels of the xylose inducer in the dnaA depletion strain and imaged cells expressing CFP-ParB for the translocation of a single CFP-ParB/parS centromere. DnaA-dependent translocation of only a single CFP-ParB focus was observed in the presence of very low (≤0.007%) xylose concentrations. Titration of the xylose inducer over a narrow range of concentrations revealed an approximately linear relationship between xylose concentration and the proportion of cells in a population exhibiting translocation of a single CFP-ParB focus (Fig. 1H). A mixed population of cells (containing either one or two CFP-ParB foci) was observed when the P_xylX-dnaA depletion strain was grown in the presence of greater than 0.007% xylose, implying that dnaA expression is sufficient to initiate replication in at least a subset of cells above this level of induction. In sum, these results support the possibility that subphysiological levels of DnaA are sufficient to induce CFP-ParB/parS translocation in the apparent absence of replication initiation.

Limited Levels of DnaA Are Sufficient To Initiate Chromosome Segregation but Not Replication.

The appearance of two ParB foci at opposite poles in wild-type cells reflects replication of the centromeric region of the chromosome and the translocation of a replicated ParB/parS to the opposite pole. The translocation of an apparent solitary CFP-ParB focus could represent either the translocation of an unreplicated centromere or the segregation of a duplicated but closely apposed CFP-ParB/parS whose fluorescent foci could not be resolved by using diffraction-limited epifluorescence imaging (Fig. 1F). To determine whether the translocation of the apparently single CFP-ParB focus observed in the presence of limited DnaA levels occurs in the absence of DNA replication, we measured the total chromosomal content in DnaA-limited cells by using fluorescence-activated cell sorting (FACS). The P_vanA-dnaA depletion strain was treated with chloramphenicol under conditions in which reinitiation of DNA replication was prevented while allowing the completion of replication (17). In the presence of vanillate (dnaA expression induced), cells contained either one (1n) or two (2n) copies of the chromosome (Fig. 2A, dashed curve), indicating that replication proceeds normally when DnaA is abundant. In contrast, the absence of the vanillate inducer resulted in an accumulation of mononucleate (1n) cells (Fig. 2A, black curve), suggesting that limited levels of DnaA (Fig. 1G), which are sufficient to instigate translocation (Fig. 1F), did not support chromosome duplication.

Fig. 2.

Translocation of CFP-ParB/parS is independent of DNA replication. (A) Chromosome content. Strain LS5369 (parB::cfp-parB, dnaA:: Ω, vanA:dnaA) was grown in the presence or absence of the vanillate inducer for 3 h in M2G media and then treated with chloramphenicol. Cells were fixed and stained with Vibrant DyeCycle orange before analyzing their DNA content by flow cytometry. The x axis represents the number of complete chromosomal copies (n). (B) Relative ori:ter ratio obtained from qPCR analyses on extracted genomic DNA. Wild-type LS101 swarmer cells (nonreplicating cells, 1:1 ratio) were used to normalize values. For control of 2:1 ratio, mixed population of LS101 were treated with the ribonucleotide reductase inhibitor hydroxyurea (HU) to prevent completion of replication. For the “no DnaA” control, LS5368 (parB::cfp-parB, dnaA:: Ω, xylX::dnaA) was grown in the absence of xylose inducer. For limited DnaA, LS5369 was grown in the absence of vanillate inducer.

Although the FACS assay provides a useful metric for determining ploidy in a population of cells, it does not address the possibility that replication initiated properly in cells with limited DnaA, but arrested after a short elongation period, yielding apposed CFP-ParB/parS foci. To test for partial replication, we measured the ratio of ori (which is replicated early) to ter (which is replicated late) in a population of cells expressing limited levels of DnaA. We determined the copy number per cell of ori relative to ter by using quantitative PCR (qPCR). In hydroxyurea-treated wild-type cells (in which replication stalls as a consequence of nucleotide depletion), replication initiation led to an increase in the ori:ter ratio compared to cells lacking DnaA (Fig. 2B). We observed a near 1:1 ratio of ori to ter in swarmer cells in which DNA replication does not occur and in cells with undetectable levels of DnaA. Notably, cells with limited levels of DnaA (Fig. 1G) exhibited a 1:1 ratio of ori to ter (Fig. 2B), arguing that DNA replication is not initiated under these conditions. Complementary to these results, we showed that the replisome does not assemble at the origin of replication at any time point during the observed movement of the ParB/parS complex in the DnaA-limited strain (Fig. S2 and SI Text). Cumulatively, these results support the notion that DnaA at low concentrations can stimulate chromosome segregation in a replication-independent manner.

Replication-Independent Translocation of the Centromere Requires ParA.

The ParA partition protein interacts with ParB to mediate the movement of the Caulobacter ParB/parS centromere to the opposite cell pole (8–10). To determine whether the observed replication-independent translocation of parS/ParB requires ParA activity, we analyzed the frequency of centromere translocation in cells expressing a nonfunctional ParA mutant. We used a parA merodiploid strain that contains the wild-type allele of parA at the native locus and a xylose-inducible, dominant negative mutant parA allele encoding a missense mutation in the ATPase domain (ParA^K20R) that inhibits chromosome segregation even in the presence of wild-type ParA (7). We assessed the localization pattern of CFP-ParB following removal of vanillate (to limit the levels of DnaA) in either the presence or absence of parAK20R expression. We observed that ∼60% of cells containing wild-type ParA and limited DnaA levels exhibited single centromere translocation as evidenced by shuttling of CFP-ParB away from the stalked pole (Fig. 3A). However, when expression of the dominant negative ParA^K20R variant was induced in this background, centromere translocation failed to occur; rather, these cells displayed a single, arrested CFP-ParB focus, as was observed in a P_xylX-dnaA depletion strain grown in the absence of xylose (Fig. 3A). These results suggest that a centromere that is translocated in a replication-independent fashion retains the requirement for a functional ParA segregation complex.

Fig. 3.

Effects of DnaA-dependent replication-independent centromeric translocation. (A) Centromeric translocation requires a functioning segregation apparatus. Frequency of centromere translocation was determined by analyzing fluorescence micrographs of CFP-ParB in cells grown for 3 h in liquid M2G media. No DnaA: LS5368 (parB::cfp-parB, dnaA:: Ω, xylX::dnaA). Limited DnaA: LS5369 (parB::cfp-parB, dnaA:: Ω, vanA:dnaA). Limited DnaA with expression of ParA^K20R (M2G + xylose): LS5371 (parB::cfp-parB, dnaA:: Ω vanA:dnaA xylX::parA(K20R)). Limited DnaA^K195I (M2G): LS5372 (parB::cfp-parB, dnaA:: Ω, xylX::dnaA vanA:dnaA(K195I)). (B) Localization of anchoring protein PopZ. Overlay of phase contrast images of CFP-ParB (green) or mCherry-PopZ (purple). LS5374 (parB::cfp-parB, dnaA:: Ω, vanA::dnaA, xylX::mcherry-popZ) were grown for 3 h in M2G + vanillate to induce dnaA expression (Top) and M2G only to allow centromere translocation in cells with limited DnaA levels (Bottom; cells with centromere translocated shown only). Xylose was added for 1 h to allow for mCherry-PopZ expression. (C) MipZ localization. Overlay of phase-contrast images of MipZ-CFP (yellow). LS5375 (dnaA:: Ω, vanA::dnaA mipZ::mipZ-cfp) cells were grown in the presence of vanillate to allow dnaA expression (Left) or absence of vanillate to allow centromere translocation (Right; cells with centromere translocated shown only) for 4 h. (D) The graph shows the relative β-galactosidase activities from P_ftsZ (LS5377; Top) and P_gcrA (LS5376; Bottom). Data are represented as mean ± SD. Cells were washed three times and resuspended in M2G with vanillate, xylose, or no inducer.

Translocation of the Single ParB/parS Centromere Is Sufficient for Relocalization of the Polar Proteins MipZ and PopZ.

In Caulobacter, polar factors have been identified to interact with the parS/ParB complex to enable chromosome segregation and cytokinesis. One such protein is the polar scaffold PopZ, which interacts with the ParB/parS complex to enable directional chromosome segregation (18, 19). PopZ is positioned at the stalked cell pole early in the cell cycle and forms a second focus at the opposite pole upon the initiation of replication and segregation of the chromosome to anchor the translocated centromere to the new cell pole (18–21). To determine whether replication-independent translocation of ParB/parS is accompanied by the assembly of a second PopZ focus at the new cell pole, we constructed a popZ merodiploid derived from the P_vanA-dnaA depletion strain containing CFP-ParB and expressing a xylose-inducible allele of popZ fused to mCherry. When grown under DnaA-limited conditions, cells that had their single CFP-ParB focus translocated to the opposite cell pole also exhibited the concurrent localization of PopZ to the new pole (Fig. 3B). These results indicate that replication-independent centromere translocation signals the assembly of a second PopZ polar focus, as is observed in wild-type cells. These results are consistent with the recent report that bipolar localization of PopZ can occur when replication is blocked by novobiocin (21).

Another regulatory factor, the MipZ inhibitor of divisome Z-ring polymerization, assembles on the Caulobacter centromere via a direct interaction with ParB (22). Upon the initiation of chromosome replication and the bipolar positioning of the ParB/parS complex, MipZ is localized to both cell poles and consequently restricts the formation of the division ring to midcell by inhibiting the polymerization of the essential divisome component FtsZ near the cell poles. To assess the pattern of MipZ localization in cells with limited DnaA levels, we replaced the native mipZ allele with a mipZ-cfp translational fusion and tracked MipZ-CFP in the P_vanA-dnaA depletion strain in the presence or absence of inducer. We found that the localization of MipZ-CFP correlates precisely with that of CFP-ParB; MipZ is present at both poles following segregation in DnaA-replete cells, but exhibits a unipolar distribution when DnaA is limited (Fig. 3C). Notably, the restriction of MipZ to the new pole in cells limited for DnaA explains the occasionally observed aberrant formation of division septa near the stalked pole (Fig. 3 B and C). These findings indicate that limited amounts of DnaA initiate a cascade of events including MipZ and PopZ accumulation at the new cell pole and chromosome segregation in the absence of replication.

A DnaA Variant Capable of Activating Transcription Does Not Promote Centromere Translocation.

In addition to its role in enabling the initiation of DNA replication, DnaA functions as a transcription factor that regulates the expression of a large number of genes (23–25). To address the possibility that translocation of the centromere is stimulated indirectly through a target of the DnaA transcriptional regulon, we constructed a strain expressing DnaA^K195I, a variant encoding a mutation in DnaA’s Walker A box, which affects its ability to bind ATP and, therefore, is not competent to initiate replication (26). We demonstrated that Caulobacter cells exclusively expressing this mutant dnaA(K195I) failed to generate two resolvable foci of CFP-ParB/parS, suggesting that this mutant is unable to initiate replication (Fig. S3). We then asked whether DnaA^K195I retains the ability to activate transcription by generating transcriptional fusions of two DnaA-dependent promoters (P_ftsZ and P_gcrA) to lacZ and measuring the activity of each promoter in a strain that was depleted for wild-type DnaA but expressed DnaA^K195I. We found that the ftsZ and gcrA promoters remained active in the presence of DnaA^K195I, demonstrating that this variant is stable and able to mediate transcription of these DnaA-target genes (Fig. 3D).

Having established the ability of DnaA^K195I to activate transcription of DnaA-dependent promoters, we asked whether chromosome segregation occurred in cells exclusively expressing limited levels of DnaA^K195I. To mirror the conditions of limited wild-type DnaA, we constructed a dnaA depletion strain in which dnaA(K195I) transcription was driven by the same vanillate-inducible P_vanA promoter. We found that expression of limited levels of DnaA^K195I did not result in chromosome segregation (Fig. 3A); indeed, with respect to DNA replication and translocation, DnaA^K195I behaves as a null-like variant in our imaging assay. Although we cannot conclusively rule out the existence of an unknown segregation factor that specifically requires DnaA-ATP for transcriptional activation, the apparent ability of DnaA^K195I to act as a transcription factor and its inability to promote ParB/parS translocation argues that DnaA-dependent stimulation of chromosome segregation is unlikely to be the indirect result of a DnaA-regulated gene.

DnaA Binds Directly to the Centromere.

A chromosomal parS sequence of ∼100 bp containing two ParB binding sites has been identified as the Caulobacter centromere (7) (Fig. 4A, referred here as parS⁺). This chromosomal region serves as a protein hub where the segregation machinery and additional interacting factors contribute to the temporal and spatial control of chromosome segregation (8, 9, 19, 20, 22, 27). Because DnaA is known to bind ori to enable replication initiation (28), we considered the possibility that DnaA binds directly to the parS centromere region to activate ParB/parS translocation. Inspection of the parS sequence revealed three Caulobacter DnaA binding sites (23, 29), including one that closely resembles a site (TTATCCACA) shown to have high affinity for DnaA (29) (Fig. 4A). Using in vivo chromatin-immunoprecipitation assays followed by quantitative PCR (ChIP-qPCR), we identified parS among DNA sequences that copurify with DnaA (Fig. 4B). To confirm that the DnaA-parS interaction revealed by ChIP-qPCR is direct, we conducted an in vitro DNA footprinting assay with purified DnaA and radiolabeled native parS (parS⁺). We observed that within the 42-bp sequence between the two ParB binding sites, DnaA binding protects DNA from nuclease digestion (Fig. 4C, parS⁺).

Fig. 4.

DnaA binds directly on the centromere. (A) Sequence of wild-type parS (parS⁺) and a site-directed mutagenized parS (parS*). Asterisks represent specific nucleotide changes made on parS⁺ to generate parS*. (B) ChIP of DnaA quantified by qPCR. Three chromosomal regions were tested in wild-type cells (LS101): parS⁺, ori (used to normalize the data), and ter (negative control). Values presented are ratios of DNA fold enrichment of parS⁺ (or ter) over levels of ori. (C) DNaseI footprinting by purified native DnaA (0, 250, and 750 nM) of parS (parS⁺) and the modified sequence (parS*). Dashed boxes depict the region of the wild-type and mutated DnaA-binding site. (D) Reduced frequency of replication-independent centromere translocation in parS* cells. Frequency of single ParB focus at the new pole was determined by analyzing fluorescence micrographs of CFP-ParB in cells grown for 3 h in liquid minimal (M2G) and rich (PYE) media. Number of cells (n) are on average 800 cells per time point, and data are represented as mean ± SD. Strain parS⁺ (LS5369, parB::cfp-parB, dnaA:: Ω, vanA::dnaA) and parS* (LS5378, parB::cfp-parB, dnaA:: Ω, vanA::dnaA, parS⁺::parS*). (E) Morphological defects of parS* cells grown in rich media expressing dnaA. LS5369 and LS5378 were grown in PYE + vanillate (dnaA expression induced). Images were taken at OD_600nm ∼0.3.

To determine whether DnaA’s interaction with parS mediated the observed replication-independent ParB/parS translocation, we constructed a DnaA-limited strain with the native parS (parS⁺) replaced by a variant (parS*) bearing mutations at the high affinity DnaA binding site (29). We modified this DnaA-binding site (TTGTCCACA) to a variant sequence (gcGaCCcgt) previously shown to significantly diminish the binding affinity of Caulobacter DnaA (29) (Fig. 4A). Using footprinting assays, we confirmed that these mutations affect DnaA’s ability to bind that site (Fig. 4C, parS*). Having established an altered interaction between DnaA and parS* in vitro, we assessed the effects of the mutant DnaA binding motif on chromosome segregation in vivo. When the parS* mutant was grown with limited levels of DnaA, the frequency of replication-independent centromere translocation decreased (by 30% in minimal medium, and by 50% in rich medium) compared to cells carrying parS⁺ (Fig. 4D). Furthermore, the parS* mutations caused aberrant localization of ParB and cell division defects in cells grown in rich media even when the expression of dnaA was fully induced (Fig. 4E). Taken together, these results suggest that DnaA plays a role in chromosome segregation by assembling directly onto the Caulobacter centromere.

Discussion

Upon the initiation of DNA replication, the parS centromere, bound to the essential ParB partition factor, is located at the stalked pole of the cell (Fig. 1A). DnaA bound to the origin, positioned 8 kb from parS, enables replisome formation and the initiation of DNA replication (28). Once replication is initiated, one of the two ParB/parS complexes moves rapidly to the opposite cell pole. Under conditions of limited DnaA levels, we observed a single polar CFP-ParB/parS focus translocate across the cell in the absence of DNA replication. We showed that this translocation closely mirrors chromosome segregation in wild-type cells in that it requires ParA ATPase activity and promotes downstream, segregation-dependent events, including the dynamic localization of the polar factors PopZ and MipZ.

Because DnaA is a multifunctional protein, we addressed the possibility that DnaA indirectly mediates chromosome segregation via transcriptional activation of downstream target genes by using a DnaA variant that cannot bind ATP and, therefore, does not function as a replication initiator. We showed that this DnaA variant retains its ability to activate transcription yet cannot promote translocation of the centromere, suggesting that the ability of DnaA to mediate chromosome segregation requires ATP-binding, reminiscent of its role in replication initiation.

Examination of the Caulobacter parS sequence revealed several putative DnaA binding sites between the two ParB binding motifs (Fig. 4A). In vitro footprinting of the parS region with DnaA showed protection of parS DNA in the absence of any additional factors. Site-directed mutagenesis of one of these DnaA binding sites (Fig. 4C) resulted in loss of protection by DnaA at that site, but did not eliminate protection by DnaA at other sites within the parS region. Notably, this modification in the DnaA–parS interaction observed in the mutant parS background resulted in significantly lower frequency of replication-independent centromere translocation. Cumulatively, these results support the hypothesis that DnaA binding to parS mediates chromosome segregation.

In light of these findings, it is conceivable that the DnaA binding affinity for parS is higher than that for ori. Our observed enrichment of parS over ori in a DnaA ChIP-qPCR assay supports this hypothesis (Fig. 4B). If true, a preferential binding to parS offers a tentative explanation for the uncoupling of replication and segregation in DnaA-limited cells. This supposition is further corroborated by the presence of the Escherichia coli-like DnaA binding motif in parS (TTgTCCACA), and recent in vitro analyses revealing that Caulobacter DnaA has greater affinity for the DnaA box found in the E. coli origin of replication (TTaTCCACA) than it does for known DnaA binding sites within the Caulobacter ori (29).

Based on results presented here, we propose that Caulobacter DnaA is independently involved in both chromosome segregation and replication initiation. Several potential mechanisms by which DnaA could mediate the onset of both segregation and initiation can be envisioned. For example, it is possible that the proximity between ori and parS in Caulobacter (∼8 kb; ref. 7) enables the assembly of DnaA into a polymeric structure that links both chromosomal loci to integrate the processes of replication and segregation. However, any model positing a DnaA-directed interaction between ori and parS must account for the observation that parS can be artificially relocated hundreds of kilobases away from ori and segregation is still maintained (7). Alternatively, it is conceivable that the DNA bending ability of DnaA (30) is coopted to modify the structure of parS so as to influence the structure and/or composition of the multicomponent, centromeric protein/DNA complex. A similar mechanism has been proposed for the parS/ParAB partitioning system of P1 plasmid in E. coli in which the DNA-bending protein, IHF (integration host factor), binds between ParB-binding sites and bends P1 parS, resulting in a high affinity protein–DNA complex (31, 32).

The results presented here apply to the replication and segregation of the Caulobacter chromosome. However, the use of replication initiators to coordinate replication with segregation could be a strategy used by other bacterial species. For example, in the opportunistic pathogen Burkholderia cenocepacia, DnaA binding sites close to parS sites have been reported in at least two of its four chromosomes (33). Although demonstration of DnaA binding sites coincident with verified parS sites are required for other bacterial species, a similar strategy to coordinate replication with segregation has been observed with the eukaryotic replication initiator complex (ORC). Independent of DNA replication, components of ORC directly affect chromosome segregation by properly localizing cohesion complexes of sister chromatids and by regulating the formation of protein complexes required for microtubule organization (34). In fact, the human Orc2 protein localizes to the centrosome, centromeres, and heterochromatin, suggesting that replication initiator proteins function to coordinate the entire chromosome inheritance cycle (35). These findings demonstrate that even when replication and segregation are separated in time and space, cells maintain factors that coordinate these two events. These observations, along with the results presented here, posit a conserved function of replication initiators as key coordinators of DNA synthesis and chromosome segregation in both eukaryotes and bacteria.

Methods

All Caulobacter strains used were derived from the wild-type strain CB15N (NA1000) and grown at 28 °C in either minimal or rich medium. Swarmer cells were isolated from mixed population cultures using percoll density centrifugation protocol (36). Fluorescence microscopy followed by image processing with Adobe Photoshop and manual cell counting was used to determine intracellular location of labeled loci. Details regarding experimental protocols, reagents, and data analyses are included in SI Methods.