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Center for Environmental Genomics, Department of Biology, McMaster University, 1280 Main St West, Hamilton, Ontario, Canada L8S 4K1
Correspondence
Turlough M. Finan
finan{at}mcmaster.ca
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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, 1984). oriC is the location of the initial strand opening event that precedes replication and the site of replisome assembly (Bramhill & Kornberg, 1988b). Many of the molecular events that occur at the Escherichia coli oriC have been well characterized, providing a general model of the essential biochemical process which results in the formation of two replication forks and duplication of oriC (Bramhill & Kornberg, 1988a). While E. coli oriC is the most extensively characterized chromosome origin, sequences capable of autonomous replication have been isolated from many bacterial chromosomes (Harding et al., 1982; Zyskind et al., 1983; Yee & Smith, 1990; Zakrzewska-Czerwinska & Schrempf, 1992; Fujita et al., 1990, 1992; Calcutt & Schmidt, 1992; Schaper et al., 2000; Moriya et al., 1992; Salazar et al., 1996; Jakimowicz et al., 1998). In most sequenced bacterial chromosomes, oriC has been predicted on the basis of sequence analysis where a switch in the asymmetry of the two strands of DNA coincides with the origin and terminus of the chromosome (Lobry, 1996). The architecture of the bacterial chromosome origin is nevertheless diverse with respect to the physical sequence as well as the number and organization of binding sites for proteins that control the timing of origin firing. Binding sites for the replication initiator DnaA (DnaA boxes) appear to be one of the hallmark features of bacterial chromosome origins and because of this the location of DnaA boxes in the bacterial genome has been used as a method for predicting oriCs (Mackiewicz et al., 2004). Another feature of many functional origins is that adjacent to the DnaA boxes is an exceptionally AT-rich region which is the site of initial strand separation. Bacterial chromosome origins are commonly located in close vicinity to the dnaA gene and attempts have been made to exploit this linkage in the cloning of oriCs (Smith et al., 1991; Fujita et al., 1990, 1992; Schaper et al., 2000; Moriya et al., 1992; Salazar et al., 1996; Jakimowicz et al., 1998). Examples do exist in which hallmarks of oriCs have not been found to be linked to the dnaA replication initiation gene, such as the chromosomes of Synechocystis spp. (Richter & Messer 1995), Prochlorococcus marinus (Richter et al., 1998) and Sinorhizobium meliloti (Margolin et al., 1995).
S. meliloti is a Gram-negative bacterium that forms root nodules on alfalfa. The colonization of alfalfa roots by S. meliloti provokes a complex differentiation program resulting in morphological changes in both organisms. Nitrogen-fixing bacteroids have been reported to contain more nucleic acid per cell than the free-living form and thus may undergo a process of endoreduplication within alfalfa (Paau et al., 1977). It is unclear what role DNA replication of the S. meliloti genome has during differentiation; however, the origins of replication in the S. meliloti genome may serve as important elements that coordinate this process within plant cells. The tripartite genome of S. meliloti (Galibert et al., 2001) is composed of a circular chromosome (3·6 Mb) and two megaplasmids, pSymA (1·35 Mb) and pSymB (1·68 Mb). Previously, the chromosomal origin was predicted to be located adjacent to the hemE gene (encoding uroporphyrinogen decarboxylase) on the basis of DNA strand asymmetry (Capela et al., 2001). Here we show that a DNA fragment encompassing this region can confer autonomous replication to a non-replicating plasmid and that these minichromosomes are maintained in S. meliloti at copy numbers of less than one per host chromosome. Using a combined bioinformatic, genetic and biochemical approach we have mapped essential DnaA-binding sites within oriC. We previously characterized the replication origins from both megaplasmids carried by S. meliloti (Chain et al., 2000; MacLellan et al., 2005) and this work completes our initial characterization of the third origin of replication carried by this bacterium.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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. E. coli was grown at 37 °C in LB broth and S. meliloti was grown at 30 °C in LB supplemented with 2·5 mM MgSO4 and 2·5 mM CaCl2 (LBmc) or on LB solidified with agar (16 g l–1).
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General molecular biology.
Plasmid DNA isolation, genomic DNA isolation, restriction analysis, PCR and site-directed mutagenesis were all performed according to standard protocols. PCR products were purified using QIAquick spin columns (Qiagen). Sequencing and primer synthesis was performed at the Mobix Central Facility (McMaster University, Hamilton, Ontario, Canada) using the ABIPRISM 3100 Genetic Analyser using the BigDye terminator chemistry.
Copy number determination.
Total DNA was prepared from cultures grown to an OD600 of 0·5–0·6, according to a standard protocol. Genomic DNA (10 µg) was digested in a 30 µl reaction volume for 4 h. Digested genomic DNA was loaded onto a 0·8 % agarose gel and electrophoresed at 15 V overnight. Southern hybridization was done using either linearized plasmid DNA or purified PCR products as probes which were radioactively labelled with [
-32P]dATP using the Roche Random Primed DNA Labelling Kit. Following hybridization, the membrane was exposed to a Storage Phosphor Screen (Amersham Biosciences) for 1 h. After exposure, the screen was scanned on a Storm 820 Phosphoimager (Molecular Dynamics) at a pixel size of 50 µm. Band intensities were calculated using the Image Quant 5.2 program (Molecular Dynamics). Plasmid copy number was calculated as a ratio of plasmid signal to chromosome signal. Copy number was determined for triplicate samples and a mean copy number is reported.
Protein purification.
The dnaA gene was PCR-amplified from genomic DNA using the AB26340 and AB26341 primers (Table 2) and cloned via engineered BamHI and EcoRI restriction sites into the pET43a NusAHis6 tag expression vector (Novagen) to create pTH1081. The pTH1081 plasmid was transformed into E. coli BL21 STAR (Stratagene) to create the protein overexpression strain J1027. This strain was grown at 37 °C in 5 ml LB broth containing 50 µg ampicillin ml–1 and subcultured into 100 ml LB broth with antibiotic. The culture was grown to an OD600 of 0·6 at 37 °C and then IPTG was added to a final concentration of 0·3 mM. Following incubation for an additional 2·5 h at 30 °C, the cells were collected and resuspended in 10 ml ice-cold resuspension buffer (50 mM NaH2PO4, pH 8·0, 500 mM NaCl, 10 mM imidazole). Lysozyme was added to a 150 µg ml–1 final concentration and incubated on ice for approximately 30 min. The cell suspension was then sonicated twice with a Branson Sonifier Cell Disruptor with a 1/8'' (2·7 mm) tapered probe for 20 s at a power level of 3.5. Lysates were kept on ice between sonications. The crude lysate was centrifuged at 15 000 r.p.m. to remove insoluble material.
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Electrophoretic mobility shift assay.
Probe DNA (
100–200 bp purified PCR product) was quantified visually on a 1·8 % agarose gel. One picomole of 5' termini was then end-labelled with [
-32P]ATP using T4 polynucleotide kinase (New England Biolabs). Following the labelling reaction, probes were purified with a QIAquick PCR Purification Kit (Qiagen). Specific activity of the labelled probe was calculated using a liquid scintillation counter.
The binding reaction was set up as described by Schaper & Messer (1995). In a microfuge tube on ice the following were mixed (in this order): 4 µl 5x binding buffer (100 mM HEPES-KOH, pH 8·0, 25 mM magnesium acetate, 5 mM Na2EDTA, 20 mM DTT, 25 mg BSA ml–1, 1 % Triton X-100, 25 % glycerol), ddH2O, 0·4 µl ATP (50 mM), probe DNA (50 nM), 1 µl poly dI : dC (100 ng µl–1) and purified DnaA (100–500 nM) in a total reaction volume of 20 µl. The reaction was incubated on ice for 10 min and then at room temperature for 20 min. The reactions were loaded onto a 4 % polyacrylamide gel and electrophoresed at 14 V cm–1 (252 V) for 10 min and then 9 V cm–1 (162 V) for 2·5 h at room temperature. The gel was dried and exposed to Kodak Scientific Imaging Film and a Storage Phosphor Screen for quantification.
Environmental scanning electron microscopy.
For glutaraldehyde fixation, several colonies were used to inoculate 2 ml LBmc with 30 µg gentamicin ml–1 and the culture was grown to an OD600 of 0·5. The culture was pelleted in a microfuge tube and the supernatant was removed. The pellet was then resuspended in 1 ml 0·2 M sodium cacodylate buffer (pH 7·4). One millilitre of 0·2 M sodium cacodylate buffer containing 5 % glutaraldehyde (pH 7·4) was added, thus changing the effective concentration of glutaraldehyde to 2·5 %, and the tubes were inverted a couple of times and left for 1 h at room temperature.
For slide preparation, a cover glass was mounted on an aluminium ESEM stub with conductive glue (equal parts white Elmers glue and colloidal graphite) making sure that a line of conductive glue was made from the edge of the glue spot to the edge of the cover glass and just around to the sample side of the glass. The mounted cover glass was air-dried for 30 min and then coated with a 5 nm layer of gold using a Sputter Coater. The glutaraldehyde fixed sample was washed six times in 2 ml ddH2O to remove all traces of salt. Then, 1 µl resuspended sample was spotted onto the gold-coated glass slide and the spot was allowed time to air dry. For visualization and image capturing the stub was placed into an Electroscan 2020 Environmental Scanning Electron Microscope and set to Wet mode. Samples were viewed at 2·4–4·0 Torr with an accelerating voltage of 20–30 keV. Various magnifications were used and images were saved as TIFF files.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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) and this corresponds to the same location as the well studied chromosome origin from C. crescentus (Marczynski & Shapiro, 1992; Marczynski et al., 1995; Brassinga & Marczynski, 2001). The C. crescentus chromosome origin is located 5 kb from the dnaA gene region, a sequence with a similar genetic arrangement to the S. meliloti dnaA region (Brassinga et al., 2001). This fadB1-rpsT-dnaA region is unusual in that it deviates from the rnpA-rmpH-dnaA-dnaN-recF-gyrB-rnpA structure linked to many bacterial chromosome origins (Yoshikawa & Ogasawara, 1991; Ogasawara & Yoshikawa, 1992). Interestingly, the parB-parA-gidA-gidB-thdF gene cluster located 70 kb from the C. crescentus oriC is within 8 kb of the predicted S. meliloti oriC. We examined the predicted S. meliloti oriC region for features characteristic of bacterial chromosome origins. The most obvious feature found was an exceptionally AT-rich (30 mol% G+C over 80 nt) region contrasting with the flanking DNA and the rest of the chromosome which has a G+C content of 62 mol% (Fig. 1a). In E. coli, Bramhill & Kornberg (1988b) showed that duplex opening occurred in the AT-rich region of oriC and presumably the lower double-stranded thermodynamic stability in this region promotes strand dissociation.
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). As shown in Fig. 1(b)
, five putative DnaA boxes were identified in the 3 kb oriC region. An additional box was also found in the dnaA gene region. This DnaA box (box 5, Fig. 1b) is an 8/9 bp match to the E. coli consensus sequence and is an exact match to an experimentally identified DnaA box found in the C. crescentus chromosome origin (Marczynski & Shapiro, 1992). DnaA box 3 (also an 8/9 bp match to the consensus) is identical to the sequences of DnaA boxes found in chromosome origins from Streptomyces spp. (Majka et al., 2001) and Micrococcus luteus (Fujita et al., 1990).
Since the initiation of replication of the megaplasmid origins may also be subject to regulation by DnaA, the repABC replicator regions for the pSymA and pSymB megaplasmids were also scanned for DnaA-binding sites. As shown in Fig. 1(c), five putative DnaA boxes were identified in the pSymA repABC region. Interestingly, DnaA box 2, found in the hemE–Y02793 intergenic region, is found twice in the pSymA replicator region, once 158 bp upstream of the translational start site of repA2 and again inside the repA2 ORF. Eleven DnaA-binding sites were predicted in the pSymB repABC locus (Fig. 1d). It has been documented that only the repC ORF and sequence downstream of repC is necessary for repABC plasmid replication (Ramirez-Romero et al., 2000) and thus the origin of replication must be encoded either within repC or just downstream of the gene. This work predicts a single DnaA box in the sequence downstream of repC in both megaplasmid replicator regions and this box may therefore serve a role in recruiting the cellular replication machinery to the megaplasmid origins. However, the distribution of these putative DnaA boxes appears to be biased to upstream of or within the repA1 gene (8 of 11 predicted sites in the region). DnaA may possibly be involved in regulating repABC gene expression. There is no experimental evidence for the involvement of DnaA in repABC plasmid replication, but this work might point to such a role.
Autonomous replication of the DNA region encompassing the predicted oriC
Exploiting the ability of a cloned sequence to support autonomous replication of a normally non-replicating plasmid in the host cell has been a successful strategy for isolating the chromosome origins from Enterobacter aerogenes, Klebsiella pneumoniae (Harding et al., 1982), Erwinia carotovora (Takeda et al., 1982), Vibrio harveyi (Zyskind et al., 1983), Pseudomonas aeruginosa, Pseudomonas putida (Yee & Smith, 1990) and Streptomyces lividans (Zakrzewska-Czerwinska & Schrempf, 1992). We used this strategy to examine whether the previously predicted S. meliloti oriC region could support autonomous replication. For this purpose a 3 kb region encompassing the AT-rich region, the hemE and Y02793 genes, and the five putative DnaA boxes was amplified by PCR from S. meliloti genomic DNA using primers AB24853 and AB24854. This DNA was cloned into pUCP30T (a plasmid that cannot replicate in S. meliloti) to form plasmid pTH838 (see Fig. 3b). pTH838 was transferred from E. coli donor cells into a recA– derivative of S. meliloti (Rm5004) via conjugation. The ability of pTH838 to promote the formation of transconjugant colonies on medium containing an antibiotic (60 µg gentamicin ml–1) to select for its presence is indicative of autonomous replication. The pTH838 plasmid transferred into both Rm5004 and wild-type Rm1021 cells and transconjugants were obtained at a frequency of 10–1 per recipient cell. The high transfer frequency into both recipient strains reflects an ability of pTH838 to autonomously replicate because the recA mutation in Rm5004 prevents homologous recombination of the plasmid with the hemE locus on the chromosome. To our surprise, every time this mating experiment was performed, both small and large S. meliloti transconjugant colonies arose on selective medium after incubation. Both small and large Rm5004(pTH838) transconjugants colonies were purified three times on selective medium, then tested for the presence of an autonomously replicating plasmid by Southern blot hybridization. A single small colony and a single large colony (done in triplicate) was used to inoculate LB and total DNA was prepared following growth for 36 h. As a control, total DNA was prepared from a pUCP30T cointegrant strain RmK569 (a wild-type Rm1021 derivative in which a single copy of pUCP30T has integrated at the pstS locus). Total transconjugant genomic DNA was restricted and probed in a Southern blot with labelled pUCP30T. pTH838 DNA was detected as a single restriction fragment and this is indicative of the plasmid being maintained as a closed circular molecule in the transconjugant cells. Two restriction fragments hybridized with the pUCP30T probe in the control strain RmK569. Similarly, integration of pTH838 into the chromosomal hemE locus would result in two restriction fragments, whereas only a single fragment was observed (data not shown), revealing that pTH838 is an autonomously replicating plasmid.
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). The intensities of the plasmid signal (5·8 kb) and the chromosome signal (3·5 kb) were quantified with a phosphoimager. A ratio of plasmid signal : chromosome signal was determined for each transconjugant sample and then the three ratios for each of the small and large strains were used to calculate a mean number of plasmid molecules per chromosome. The mean copy number of the three small transconjugants was determined to be 0·053 plasmids per chromosome and the mean copy number of the three large transconjugants was 0·135 plasmids per chromosome. Thus the large colonies contained approximately twice as much autonomously replicating pTH838 plasmid DNA as the small colonies.
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). Mutations that impair the replication of C. crescentus minichromosomes do so in a way that leads to a reduction of the copy number of the autonomously replicating plasmid and results in a delayed growth phenotype on selective medium. This parallels our observation that Rm5004(pTH8383) transconjugant colony size directly correlates with the copy number of the pTH838 minichromosomes.
To validate the calculated pTH838 copy number of less than one, K1012 and K1013 and the control strain RmK569 (Rm1021 with a single copy of pUCP30T that has integrated at the pstS locus) were grown in LB with antibiotic selection to mid-exponential phase and plated onto LB plates without added antibiotic. Two hundred colonies were then patched back onto selective medium and incubated at 30 °C for 4 days. A total of 20 of the 200 (0·1) patches and 13 of the 200 (0·065) patches were gentamicin-resistant from the K1013 and K1012 cultures, respectively. As expected, all 200 of the RmK569 patches maintained gentamicin resistance because of the stability of the chromosomally integrated copy of pUCP30T. These values are very close to the mean copy number calculated from the Southern blot (Fig. 2), 0·053 vs 0·065 for the small transconjugants and 0·135 vs 0·1 for the large transconjugants. These ratios represent the fraction of cells in the cell population growing in the presence of gentamicin that actually contain a pTH838 plasmid molecule or the gentamicin 3'-acetyltransferase protein encoded by the pTH838 plasmid.
We employed a second indirect method to establish that pTH838 was autonomously replicating in Rm5004. The method is based on the premise that autonomously replicating plasmids, but not integrated plasmids, can be readily transferred from S. meliloti to E. coli. Thus we tested strains K1012 [Rm5004(pTH838) small transconjugant], K1013 [Rm5004(pTH838) large transconjugant] and strain RmK569 (as a control) for their ability to transfer pTH838 (as monitored by gentamicin resistance) to E. coli. The results from these experiments (Table 3) clearly showed a high frequency of transfer of gentamicin resistance from the Rm5004(pTH838) transconjugants K1012 and K1013 to E. coli (>10–1 per donor). In contrast no transfer of the integrated copy of pUCP30T in RmK569 to E. coli was detected (<10–8 per donor). Restriction analysis of plasmid DNA prepared from gentamicin-resistant E. coli transconjugant cells confirmed that the transferred plasmid was pTH838 (data not shown). These data demonstrate that pTH838 is capable of autonomous replication and therefore represents a mini-derivative of the chromosomal basic replicon.
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). The sequence encompassing the AT-rich region and DnaA boxes 2, 3 and 4 (pTH1245) was not sufficient for autonomous replication (transconjugant colony formation). The 5' limit of the sequence required for autonomous replication was mapped to within 19 bp (the difference between pTH1451 and pTH1452, and the difference between pTH1453 and pTH1454) and within this essential 19 bp is the predicted DnaA box 5. The 3' limits required for transconjugant colony formation (pTH1454) extends 250 bp into the Y02793 ORF and thus the plasmid copy of Y02793 is not required for autonomous replication. The minimal size of the S. meliloti oriC is 1802 bp and this is much larger than the 437 bp C. crescentus minimal chromosome origin (Marczynski & Shapiro, 1992). It may be that the hemE ORF itself is not required for replication, but only the DnaA box located downstream of hemE and thus deletion of the intervening 956 bp hemE gene would reduce the size of the S. meliloti oriC to 846 bp. The minimal nucleotide sequence required to support autonomous replication of the suicide plasmid pUCP30T suggests that the functional S. meliloti oriC sequence may be defined by the distribution of DnaA boxes that flank the AT-rich region.
We therefore examined the importance of the predicted DnaA boxes to replication by using a mutagenesis approach. The five DnaA boxes identified in pTH838 were mutated by oligonucleotide site-directed mutagenesis. The mutations in DnaA boxes 5, 2, 3 and 4 were 4 bp deletions and the mutation in DnaA box 1 was a 3 bp deletion (Fig. 3c). Plasmids carrying mutations in boxes 2, 3 and 4 lost the ability to autonomously replicate in S. meliloti, suggesting that these sequences are required for replication of plasmid-borne oriC (Fig. 3c). As expected, transconjugant colonies formed at a low frequency when mutant oriC plasmids were transferred into Rm1021, probably due to homologous recombination at the oriC genomic locus. Rm1021 was used as a control recipient in the replication assay to ensure that the oriC plasmids were still mobilizable after mutagenesis. The mutation in DnaA box 1 did not influence pTH838 replication and, surprisingly, the mutation carried on pTH1518 (mutation in DnaA box 5) also did not abolish replication even though our deletion analysis (Fig. 3b) demonstrated that this sequence was essential for replication. It is possible that the removal of the 4 bp in DnaA box 5 generated a sequence still capable of interacting with the replication initiator. DnaA box 5 (5'-TGATCCACA-3') is an 8/9 bp match to the expanded E. coli DnaA box consensus (Fig. 1.); the site-directed mutation (deletion of GATC) in this predicted binding site generates 5'-TCACAGATA-3' and this sequence only deviates from the consensus sequence at positions 4 and 6. In other words, the 4 bp deletion in box 5 may result in the reconstitution of a sequence that can still bind DnaA.
DnaA binds to predicted binding sites in the hemE–Y02793 intergenic region
The ability of DNA fragments carrying the various putative DnaA-binding sites to bind purified DnaA protein was examined in electrophoretic mobility shift assays. We tested mutations in putative DnaA-binding sites that abolish replication of plasmid-borne oriC for their influence on DnaA binding. The six DNA sequences that were used in DnaA-binding experiments included a 190 bp probe (ML700/ML701) that contained all three predicted DnaA boxes in the hemE–Y02793 intergenic region (DnaA boxes 2, 3 and 4), a 178 bp probe (ML1182/ML1183) with the same sequence as the 190 bp probe except with 4 bp deletions in all three predicted DnaA boxes, a 117 bp probe (ML2796/ML2797) containing DnaA box 5 downstream of hemE, a 197 bp fragment (ML3257/ML3258) from the dnaA promoter that contains a predicted DnaA box overlapping the translational start site of the dnaA gene, a 233 bp repA2 promoter probe (ML702/ML703) which includes a DnaA box with an exact match to DnaA box 2 in oriC and a 125 bp repA1 promoter probe (AB27527/AB27526) containing two DnaA boxes.
Two complexes were resolved with the 190 bp oriC probe as seen in Fig. 4; however, in some cases using this probe resulted in the formation of three complexes (data not shown), consistent with the number of binding sites in the probe. Three rounds of site-directed mutagenesis of pTH838 were required to introduce the three mutations needed to generate a template that could be used for PCR amplification of the 178 bp probe. DnaA did not interact with this target as it did with the wild-type sequence and thus it appears that the mutations created in the hemE–Y02793 intergenic region represent bona fide deletions in DnaA boxes (Fig. 3c). Only a weak interaction was observed with the 117 bp DnaA box 5 probe. Complexes were not detected with the dnaA promoter probe or the repA1 promoter probe. DnaA did complex with the repA2 promoter probe, probably at the predicted DnaA box 158 bp upstream of the repA2 translational start site. DnaA has been implicated as a transcription factor as it autoregulates dnaA gene expression (Atlung et al., 1985) and either represses or activates expression from several other genes (Messer & Weigel, 1997). Thus the DnaA-binding site upstream of the repA2B2C2 operon may be biologically relevant such that expression of the pSymA genes encoded in the replicator region are transcriptionally coordinated with chromosome replication.
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; Skarstad et al., 1989). This results in a loss of the correct timing in the cycle of replication initiation and leads to a block in cell division, thus generating filamentous cells (Pierucci et al., 1989). This block in cell division as a result of increased DnaA levels has also been reported for Mycobacterium smegmatis (Greendyke et al., 2002). We examined whether DnaA would have similar consequences on S. meliloti cell morphology if overexpressed. To this end the dnaA gene and 20 bp upstream of the predicted translational start (including a predicted ribosome-binding site) was PCR-amplified using primers AB29744 and AB29675. Primer AB29744 was designed with three engineered stop codons in all three reading frames to prevent a fusion to the Lac peptide encoded in pBBR1MCS-5. The PCR product was cloned into pBBR1MCS-5 such that the dnaA gene was under the transcriptional control of the E. coli lac promoter, generating plasmid pTH1091. The pTH1091 DnaA expression plasmid was transferred into wild-type S. meliloti Rm1021 and E. coli DH5 and transconjugant cells were visualized with an environmental scanning electron microscope. Many more of the E. coli cells expressing S. meliloti DnaA appeared filamentous compared to the parent DH5 (compare Fig. 5a and b), suggesting that S. meliloti DnaA has some form of cross-functionality and can perturb cell division in other Proteobacteria. S. meliloti cells expressing DnaA from the plasmid promoter also displayed an apparent block in cell division, with many of the cells growing up to 15 µm in length compared to the 1 µm length of Rm1021 wild-type cells (compare Fig. 5c and d). The S. meliloti filaments are complex and in most cases the cells display many branches with swollen areas flanked by regions that appear partially septated, as shown under higher magnification (Fig. 5e and f).
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). It would seem that the default pathway for a block in cell division that arises from perturbations in chromosome replication in this endosymbiont is a complex change in cell shape. Interestingly, many of the cells assume branched Y-shaped architectures that look very similar to the fully differentiated bacteroids found inside plant cells in root nodules.
Here we report the identification of a replication origin from the S. meliloti circular chromosome which represents the first such origin to be localized in the Rhizobiaceae and the second chromosome origin to be experimentally defined in the Alphaproteobacteria. This is the third autonomously replicating sequence to be identified in the S. meliloti genome. The other two sequences were isolated from the pSymB megaplasmid (Margolin & Long, 1993; Chain et al., 2000). The S. meliloti oriC possesses the hallmark features of a bacterial chromosome origin, such as sequences that interact specifically with the replication initiator DnaA (DnaA boxes) flanking an exceptionally AT-rich region. Interestingly, the S. meliloti chromosome origin is located greater than 400 kb from the dnaA gene which, in other bacteria, is often closely linked to the replication origin. This explains why an autonomously replicating sequence was not detected in the vicinity of dnaA in previous attempts to localize this replication origin (Margolin et al., 1995). S. meliloti transconjugant cells harbouring oriC plasmids display a delayed growth phenotype, perhaps explaining why a screen for autonomously replicating sequences encoded in the S. meliloti genome conducted by Margolin & Long (1993) did not detect the chromosome origin.
Previous reports have demonstrated that requirements for minichromosome replication may be very different than those for the chromosomal origin. The necessity of certain DnaA-binding sites and DNA-bending proteins HU and IHF for oriC plasmid replication, but not for chromosome replication (Weigel et al., 2001; Asai et al., 1998), suggests that the plasmid-borne oriC may adopt a very different DNA topology than the chromosomal origin. It is possible that constraints on DNA topology in S. meliloti minichromosomes may cause a severe reduction in the number of replication initiation events occurring at oriC in pTH838 and thus result in copy numbers of much less than one per chromosome.
The copy numbers of plasmids encoding bacterial oriC sequences is quite diverse. The best characterized bacterial minichromosome from E. coli has a copy number of approximately 38 per cell (Lobner-Olesen et al., 1987), but this does not represent the typical situation. Plasmids replicating from the cloned Pseudomonas chromosome origin have been reported to be present at as low as 0·7 copies per cell (Yee & Smith, 1990). The ColE1 replication origin has also been shown to exert a strong effect on the copy number of Mycobacterium tuberculosis minichromosomes and can reduce the copy number from approximately 17 to 0·6 plasmids per chromosome (Qin et al., 1999). The cause of this instability is unknown, but we do not rule out the possibility that the ColE1 replication origin present in pUCP30T may be the cause of the low S. meliloti minichromosome copy number.
While we refer to the region identified in this report as the oriC, we note that direct experimental proof that replication of the S. meliloti chromosome initiates from this region has yet to be obtained. Similarly, the presumed replication origins for the pSymA and pSymB megaplasmids have yet to be experimentally verified. Such verification would seem to be very worthwhile and since this would require synchronization of cell division, these experiments could also address whether replication of the chromosome and two megaplasmids is coordinately regulated.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Atlung, T., Clausen, E. S. & Hansen, F. G. (1985). Autoregulation of the dnaA gene of Escherichia coli K12. Mol Gen Genet 200, 442–450.[CrossRef][Medline]
Atlung, T., Lobner-Olesen, A. & Hansen, F. G. (1987). Overproduction of DnaA protein stimulates initiation of chromosome and minichromosome replication in Escherichia coli. Mol Gen Genet 206, 51–59.[CrossRef][Medline]
Bramhill, D. & Kornberg, A. (1988a). A model for initiation at origins of DNA replication. Cell 54, 915–918.[CrossRef][Medline]
Bramhill, D. & Kornberg, A. (1988b). Duplex opening by DnaA protein at novel sequences in initiation of replication at the origin of the E. coli chromosome. Cell 52, 743–755.[CrossRef][Medline]
Brassinga, A. K. & Marczynski, G. T. (2001). Replication intermediate analysis confirms that chromosomal replication origin initiates from an unusual intergenic region in Caulobacter crescentus. Nucleic Acids Res 29, 4441–4451.
Brassinga, A. K., Siam, R. & Marczynski, G. T. (2001). Conserved gene cluster at replication origins of the -Proteobacteria Caulobacter crescentus and Rickettsia prowazekii. J Bacteriol 183, 1824–1829.
Calcutt, M. J. & Schmidt, F. J. (1992). Conserved gene arrangement in the origin region of the Streptomyces coelicolor chromosome. J Bacteriol 174, 3220–3226.
Capela, D., Barloy-Hubler, F., Gouzy, J. & 25 other authors (2001). Analysis of the chromosome sequence of the legume symbiont Sinorhizobium meliloti strain 1021. Proc Natl Acad Sci U S A 98, 9877–9882.
Chain, P. S., Hernandez-Lucas, I., Golding, B. & Finan, T. M. (2000). oriT-directed cloning of defined large regions from bacterial genomes: identification of the Sinorhizobium meliloti pExo megaplasmid replicator region. J Bacteriol 182, 5486–5494.
Finan, T. M., Kunkel, B., De Vos, G. F. & Signer, E. R. (1986). Second symbiotic megaplasmid in Rhizobium meliloti carrying exopolysaccharide and thiamine synthesis genes. J Bacteriol 167, 66–72.
Fujita, M. Q., Yoshikawa, H. & Ogasawara, N. (1990). Structure of the dnaA region of Micrococcus luteus: conservation and variations among eubacteria. Gene 93, 73–78.[CrossRef][Medline]
Fujita, M. Q., Yoshikawa, H. & Ogasawara, N. (1992). Structure of the dnaA and DnaA-box region in the Mycoplasma capricolum chromosome: conservation and variations in the course of evolution. Gene 110, 17–23.[CrossRef][Medline]
Fuller, R. S., Kaguni, J. M. & Kornberg, A. (1981). Enzymatic replication of the origin of the Escherichia coli chromosome. Proc Natl Acad Sci U S A 78, 7370–7374.
Fuller, R. S., Funnell, B. E. & Kornberg, A. (1984). The DnaA protein complex with the E. coli chromosomal replication origin (oriC) and other DNA sites. Cell 38, 889–900.[CrossRef][Medline]
Galibert, F., Finan, T. M., Long, S. R. & 53 other authors (2001). The composite genome of the legume symbiont Sinorhizobium meliloti. Science 293, 668–672.
Greendyke, R., Rajagopalan, M., Parish, T. & Madiraju, M. V. (2002). Conditional expression of Mycobacterium smegmatis dnaA, an essential DNA replication gene. Microbiology 148, 3887–3900.
Harding, N. E., Cleary, J. M., Smith, D. W., Michon, J. J., Brusilow, W. S. & Zyskind, J. W. (1982). Chromosomal replication origins (oriC) of Enterobacter aerogenes and Klebsiella pneumoniae are functional in Escherichia coli. J Bacteriol 152, 983–993.
Jakimowicz, D., Majka, J., Messer, W. & 8 other authors (1998). Structural elements of the Streptomyces oriC region and their interactions with the DnaA protein. Microbiology 144, 1281–1290.[Abstract]
Kovach, M. E., Elzer, P. H., Hill, D. S., Robertson, G. T., Farris, M. A., Roop, R. M. & Peterson, K. M. (1995). Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes. Gene 166, 175–176.[CrossRef][Medline]
Latch, J. N. & Margolin, W. (1997). Generation of buds, swellings, and branches instead of filaments after blocking the cell cycle of Rhizobium meliloti. J Bacteriol 179, 2373–2381.
Lobner-Olesen, A., Atlung, T. & Rasmussen, K. V. (1987). Stability and replication control of Escherichia coli minichromosomes. J Bacteriol 169, 2835–2842.
Lobry, J. R. (1996). Asymmetric substitution patterns in the two DNA strands of bacteria. Mol Biol Evol 13, 660–665.[Abstract]
Mackiewicz, P., Zakrzewska-Czerwinska, J., Zawilak, A., Dudek, M. R. & Cebrat, S. (2004). Where does bacterial replication start? Rules for predicting the oriC region. Nucleic Acids Res 32, 3781–3791.
MacLellan, S. R., Smallbone, L. A., Sibley, C. D. & Finan, T. M. (2005). The expression of a novel antisense gene mediates incompatibility within the large repABC family of alpha-proteobacterial plasmids. Mol Microbiol 55, 611–623.[CrossRef][Medline]
Majka, J., Zakrzewska-Czerwinska, J. & Messer, W. (2001). Sequence recognition, cooperative interaction, and dimerization of the initiator protein DnaA of Streptomyces. J Biol Chem 276, 6243–6252.
Marczynski, G. T. & Shapiro, L. (1992). Cell-cycle control of a cloned chromosomal origin of replication from Caulobacter crescentus. J Mol Biol 226, 959–977.[CrossRef][Medline]
Marczynski, G. T., Lentine, K. & Shapiro, L. (1995). A developmentally regulated chromosomal origin of replication uses essential transcription elements. Genes Dev 9, 1543–1557.
Margolin, W. & Long, S. R. (1993). Isolation and characterization of a DNA replication origin from the 1,700-kilobase-pair symbiotic megaplasmid pSym-b of Rhizobium meliloti. J Bacteriol 175, 6553–6561.
Margolin, W., Bramhill, D. & Long, S. R. (1995). The dnaA gene of Rhizobium meliloti lies within an unusual gene arrangement. J Bacteriol 177, 2892–2900.
Meade, H. M., Long, S. R., Ruvkun, G. B., Brown, S. E. & Ausubel, F. M. (1982). Physical and genetic characterization of symbiotic and auxotrophic mutants of Rhizobium meliloti induced by transposon Tn5 mutagenesis. J Bacteriol 149, 114–122.
Messer, W. & Weigel, C. (1997). DnaA initiator – also a transcription factor. Mol Microbiol 24, 1–6.[CrossRef][Medline]
Moriya, S., Atlung, T., Hansen, F. G., Yoshikawa, H. & Ogasawara, N. (1992). Cloning of an autonomously replicating sequence (ars) from the Bacillus subtilis chromosome. Mol Microbiol 6, 309–315.[CrossRef][Medline]
Ogasawara, N. & Yoshikawa, H. (1992). Genes and their organization in the replication origin region of the bacterial chromosome. Mol Microbiol 6, 629–634.[Medline]
Paau, A. S., Lee, D. & Cowles, J. R. (1977). Comparison of nucleic acid content in populations of free-living and symbiotic Rhizobium meliloti by flow microfluorometry. J Bacteriol 129, 1156–1158.
Pierucci, O., Rickert, M. & Helmstetter, C. E. (1989). DnaA protein overproduction abolishes cell cycle specificity of DNA replication from oriC in Escherichia coli. J Bacteriol 171, 3760–3766.
Qin, M. H., Madiraju, M. V. & Rajagopalan, M. (1999). Characterization of the functional replication origin of Mycobacterium tuberculosis. Gene 233, 121–130.[CrossRef][Medline]
Ramirez-Romero, M. R., Soberon, N., Perez-Oseguera, A., Tellez-Sosa, J. & Cevallos, M. A. (2000). Structural elements required for replication and incompatibility of the Rhizobium etli symbiotic plasmid. J Bacteriol 182, 3117–3124.
Richter, S. & Messer, W. (1995). Genetic structure of the dnaA region of the cyanobacterium Synechocystis sp. strain PCC6803. J Bacteriol 177, 4245–4251.
Richter, S., Hess, W. R., Krause, M. & Messer, W. (1998). Unique organization of the dnaA region from Prochlorococcus marinus CCMP1375, a marine cyanobacterium. Mol Gen Genet 257, 534–541.[CrossRef][Medline]
Salazar, L., Fsihi, H., De Rossi, E., Riccardi, G., Rios, C., Cole, S. T. & Takiff, H. E. (1996). Organization of the origins of replication of the chromosomes of Mycobacterium smegmatis, Mycobacterium leprae and Mycobacterium tuberculosis and isolation of a functional origin from M. smegmatis. Mol Microbiol 20, 283–293.[CrossRef][Medline]
Schaefer, C. & Messer, W. (1991). DnaA protein/DNA interaction. Modulation of the recognition sequence. Mol Gen Genet 226, 34–40.[CrossRef][Medline]
Schaper, S. & Messer, W. (1995). Interaction of the initiator protein DnaA of Escherichia coli with its DNA target. J Biol Chem 270, 17622–17626.
Schaper, S., Nardmann, J., Luder, G., Lurz, R., Speck, C. & Messer, W. (2000). Identification of the chromosomal replication origin from Thermus thermophilus and its interaction with the replication initiator DnaA. J Mol Biol 299, 655–665.[CrossRef][Medline]
Schweizer, H. P., Klassen, T. R. & Hoang, T. (1996). Improved methods for gene analysis and expression in Pseudomonas. In Biology of Pseudomonas, pp. 229–237. Edited by T. Nakazawa, K. Furukawa, D. Haas & S. Silver. Washington, DC: American Society for Microbiology.
Skarstad, K., Lobner-Olesen, A., Atlung, T., von Meyenburg, K. & Boye, E. (1989). Initiation of DNA replication in Escherichia coli after overproduction of the DnaA protein. Mol Gen Genet 218, 50–56.[CrossRef][Medline]
Smith, D. W., Yee, T. W., Baird, C. & Krishnapillai, V. (1991). Pseudomonad replication origins: a paradigm for bacterial origins? Mol Microbiol 5, 2581–2587.[CrossRef][Medline]
Takeda, Y., Harding, N. E., Smith, D. W. & Zyskind, J. W. (1982). The chromosomal origin of replication (oriC) of Erwinia carotovora. Nucleic Acids Res 10, 2639–2650.
Weigel, C., Messer, W., Preiss, S., Welzeck, M., Morigen & Boye, E. (2001). The sequence requirements for a functional Escherichia coli replication origin are different for the chromosome and a minichromosome. Mol Microbiol 40, 498–507.[CrossRef][Medline]
Yee, T. W. & Smith, D. W. (1990). Pseudomonas chromosomal replication origins: a bacterial class distinct from Escherichia coli-type origins. Proc Natl Acad Sci U S A 87, 1278–1282.
Yoshikawa, H. & Ogasawara, N. (1991). Structure and function of DnaA and the DnaA-box in eubacteria: evolutionary relationships of bacterial replication origins. Mol Microbiol 5, 2589–2597.[CrossRef][Medline]
Zakrzewska-Czerwinska, J. & Schrempf, H. (1992). Characterization of an autonomously replicating region from the Streptomyces lividans chromosome. J Bacteriol 174, 2688–2693.
Zyskind, J. W., Cleary, J. M., Brusilow, W. S., Harding, N. E. & Smith, D. W. (1983). Chromosomal replication origin from the marine bacterium Vibrio harveyi functions in Escherichia coli: oriC consensus sequence. Proc Natl Acad Sci U S A 80, 1164–1168.
Received 18 August 2005;
revised 17 October 2005;
accepted 31 October 2005.
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pUCP30T pstS); 8,Rm1021 (wild-type); 9, Rm5004; 10, HindIII-restricted pTH838 plasmid DNA. A PCR product of the 477 bp hemE–Y02793 intergenic region (using primers AB32323 and AB32324) was randomly prime-labelled and used as a probe. The 5·8 kb band is probe-hybridized with plasmid DNA and the 3·4 kb band corresponds to the chromosomal signal. Intensities of the bands were determined and relative copy number was calculated as a ratio of plasmid to chromosome signal. The mean copy number was calculated from the three independent colonies tested.
