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Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

While bacteria such as Escherichia coli and Bacillus subtilis harbour a single circular chromosome, some freshwater cyanobacteria have multiple chromosomes per cell. The detailed mechanism(s) of cyanobacterial replication remains unclear. To elucidate the replication origin (ori), form and synchrony of the multi-copy genome in freshwater cyanobacteria Synechococcus elongatus PCC 7942 we constructed strain S. 7942TK that can incorporate 5-bromo-2'-deoxyuridine (BrdU) into genomic DNA and analysed its de novo DNA synthesis. The uptake of BrdU was blocked under dark and resumed after transfer of the culture to light conditions. Mapping analysis of nascent DNA fragments using a next-generation sequencer indicated that replication starts bidirectionally from a single ori, which locates in the upstream region of the dnaN gene. Quantitative analysis of BrdU-labelled DNA and whole-genome sequence analysis indicated that the peak timing of replication precedes that of cell division and that replication is initiated asynchronously not only among cell populations but also among the multi-copy chromosomes. Our findings suggest that replication initiation is regulated less stringently in S. 7942 than in E. coli and B. subtilis.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Replication is the most fundamental and essential process in the cell cycle of all organisms. In Escherichia coli and Bacillus subtilis, DNA replication starts from a single origin (oriC) in their single circular chromosome (Bramhill and Kornberg, 1988). The oriC region consists of multiple repeated sequences that contain consensus elements termed the DnaA box (Fuller et al., 1984). Replication proceeds bidirectionally around the chromosome and terminates at a region on the opposite side of oriC (terC). These replication processes are thought to be tightly coupled to cell division in the bacterial cell cycle (Cooper and Helmstetter, 1968; Wang and Levin, 2009).

Cyanobacteria are prokaryotic microorganisms; they manifest an oxygen-producing photosynthetic system similar to that of chloroplasts of higher plants. There is increased interest in cyanobacteria because these photosynthetic organisms convert solar energy to biomass and thus they may be useful for the production of biofuels. However, the exploitation of cyanobacteria for bioengineering requires a thorough understanding of their proliferation mechanism(s).

The freshwater cyanobacteria Synechococcus elongatus PCC 7942 and Synechocystis sp. PCC 6803 (S. 7942 and S. 6803 respectively) have been used as model organisms for phototrophs because their transformation efficiency and growth rate are superior to those of marine cyanobacteria and their complete genome sequences are published. Several species of freshwater cyanobacteria are oligo- and polyploid organisms harbouring multiple genomic copies per cell (Mann and Carr, 1974; Binder and Chisholm, 1995; Mori et al., 1996; Griese et al., 2011). Among them, it has been reported that S. 7942 is oligoploid, because it carries three to four genome copies per cell (Mori et al., 1996; Griese et al., 2011). Chloroplasts of plants, which are thought to derive from cyanobacteria, also contain multi-copy chromosomes (Bendich, 1987; Kuroiwa, 1991). Similar polyploidy are observed in other bacterial species such as Deinococcus radiodurans (Minton, 1994) and Thermus thermophilus (Ohtani et al., 2010).

The genome sequence of freshwater cyanobacteria is also unique in terms of its GC skew, a plot of the normalized excess of the guanine (G) over the cytosine (C) content in a subgenomic region with sliding windows along the entire genome sequence (Lobry, 1996). In many eubacteria including E. coli and B. subtilis, the GC skew plot divides the genome into a region with an excess of G over C and a region with an excess of C over G (the leading and lagging strand respectively). The shift points of the GC skew plot have been reported to correlate with the loci of ori and ter (Frank and Lobry, 1999). On the other hand, the GC skews of freshwater cyanobacteria (S. 6301, S. 7942 and S. 6803), chloroplasts and D. radiodurans, all of which carry multi-copy chromosomes, are distinct from those of other bacteria including the marine cyanobacterium Synechococcus sp. WH 8102 (Fig. S1). Since these asymmetrical genomes have many shift points between high-G and high-C regions, the location of oriC and terC cannot be predicted from GC skew information alone. In S. 7942, a cluster of dnaA boxes has been identified in the upstream region of dnaN (Liu and Tsinoremas, 1996), located on the border between the high- and low-GC regions in the GC skew analysis (Fig. S1), and therefore, this region has been predicted as a replication origin of the S. 7942 genome. Although there is an informatics method to identify the oriC regions (Zhang and Zhang, 2005), these predictions have not been confirmed experimentally.

Here we document the location of the replication origin, the replication form, and the replication synchrony of S. 7942. Our data provide significant information for a thorough understanding of not only the cyanobacterial cell cycle but also the replication mechanism(s) of organisms containing multi-copy and asymmetrical genomes.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Growth conditions for investigating DNA replication in S. 7942

Since S. 7942 cells grow photoautotrophically, their DNA replication is thought to be activated under light- and arrested under dark conditions (Binder and Chisholm, 1990). In the stationary phase, the level of DNA synthesis and the genome copy number are lower than in other growth phases (Asato, 1979; Binder and Chisholm, 1990).

To investigate the replication initiation process of S. 7942 we applied the following culture conditions. The stationary-phase culture grown under continuous light for 10 days was diluted and incubated in the dark for 18 h and then transferred to the light condition to restart cell growth (Fig. S2A). The growth curve and the cell number after transfer are shown in Fig. S2B and C. The cell mass, but not the cell number, was increased 9 h post transfer. As the S. 7942 cells showed exponential growth 24 h after transfer and both their cell mass and cell number increased significantly, we defined the periods around these time points as the pre-log (up to approximately 15 h post transfer) and log phases (15 h and longer post transfer) under this culture condition. During the log phase the cells multiplied almost synchronously with a doubling time of about 9 h (Fig. S2C).

Next we investigated the DNA content per cell in each growth phase. Compared with dark- and briefly light-exposed (1 h) cultures, about double the amount of DNA was recovered from pre-log- (9 h) and log-phase (24 h) cultures (Fig. S2D). When the dark culture was subjected to flow cytometry (FACS), the cellular DNA profile exhibited a spike-shaped pattern, suggesting that a full round of DNA replication was completed under the dark condition. On the other hand, the FACS profile of pre-log-phase cultures manifested a broader shape and the genome copy number was significantly higher than in the dark cultures (Fig. S2E). This suggests that replication of the multi-copy chromosomes is initiated under the light condition during the pre-log phase.

Quantitative analysis of sequencing reads using a next-generation sequencer

In rapidly growing bacterial cultures, e.g. E. coli and B. subtilis, most cells contain two or more replication forks; consequently they harbour two or more copies of the oriC region and one copy of the terC region (Yoshikawa et al., 1964). Sequencing of the whole genome on a next-generation sequencer showed that the read depth of oriC is significantly greater than of terC in cells with multiple replication forks (Srivatsan et al., 2008). To confirm this finding we ascertained the location of the replication origin in the B. subtilis genome (Yoshikawa and Ogasawara, 1991). We prepared genome libraries from log- and stationary-phase B. subtilis cells. Using a next-generation sequencer, we sequenced the libraries and mapped them onto the B. subtilis 168 genome as a reference. Since the replication frequency of log-phase is higher than of stationary-phase B. subtilis cells, in log-phase cells the ratio of sequence reads around the origin region was approximately twofold higher than of the terC region, resulting in a V-shaped distribution (Fig. 1A and B). Using this technique we next tried to identify the replication origin in the S. 7942 genome. Sequencing detected a circa 50 kb deletion in the genome of our laboratory strain (indicated by asterisks in Fig. 1C and D). This 50 kb deletion is from 711 254 to 759 931 of the S. 7942 genome (GenBank: CP000100) and it is specific to our laboratory strain, because the S. 7942 strains of Prof. Kondo's laboratory (Nagoya strain) do not contain the deletion. Since the growth rates of our strain and the Nagoya strains containing the deleted region were equivalent, we concluded that these genotypes have little effect on replication and used our strain in our experiments. Compared with the distribution of sequence reads in B. subtilis, in S. 7942 there was no dynamic change under dark and light (log phase) conditions (Fig. 1C and D, left). However, expanded plots revealed that the read depth ratios around the dnaN gene (Synpcc7942_0001) were slightly higher than the chromosome region opposite to dnaN in log-phase cells (Fig. 1C and D, right). We mapped the sequence reads of S. 7942 onto the S. 6301 genome as a reference; S. 6301 and S. 7942 are closely related except for the presence of a 188.6 kb inversion, and the location of the dnaN gene in the S. 6301 genome is distinct from that in S. 7942 (Sugita et al., 2007). A peak in the read depth ratio was observed around the dnaN gene (syc1496_c, which locates at 1.62 Mbp position in the S. 6301 genome) (Fig. S3). Nevertheless, there was only approximately 10% difference in the read depth ratio around the dnaN gene and the chromosome region opposite to dnaN (Figs 1D and S3B).

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Figure 1. Identification of the replication origin by sequencing. B. subtilis and S. 7942 genomes were quantitatively sequenced on a next-generation sequencer (GAII, Illumina) and the average read numbers per base in a 1 kb window (read depth) were mapped at the respective genomic positions. The ratio of the read depth at each position to the sum of the read depths throughout the whole genome is shown. B. subtilis genomic DNA prepared from stationary-phase cells (A), B. subtilis DNA from mid-log-phase cells (B), S. 7942 DNA from cells grown under dark conditions for 18 h (C), S. 7942 DNA from cells grown under dark conditions for 18 h and under light conditions for 22 h (D). Expanded plots of (C) and (D) are shown on the right. The locations of oriC and terC in the B. subtilis genome and the dnaN gene in S. 7942 are indicated by arrows. Asterisks: A sequence gap derived from the circa 50 kb genomic deletion in our S. 7942 strain.

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Analysis of de novo DNA synthesis in S. 7942

De novo DNA synthesis can be monitored by detecting the incorporation of BrdU, an analogue of thymidine (Dolbeare, 1995; Lewis and Errington, 1997; Hodson et al., 2003). Since the S. 7942 wild-type strain lacks thymidine kinase (TK) and therefore cannot incorporate BrdU into its genomic DNA, we introduced the TK gene from herpes simplex virus 2 into the S. 7942 genome (named S. 7942TK). We introduced the TK gene, HA-tagged and under the control of the Ptrc promoter, into the neutral site of the S. 7942 genome with a spectinomycin resistance gene (Fig. S4A). The growth of S. 7942TK was equivalent to that of the wild-type strain. In S. 7942TK the TK protein was expressed by a leaky activity of the Ptrc promoter without IPTG; its expression was increased by transferring the culture to the light condition (Fig. S4B). We monitored BrdU uptake in a dark-synchronized S. 7942TK culture. Under light- but not dark conditions, BrdU was incorporated into the S. 7942TK genome. Since BrdU incorporation was sufficient without IPTG, we cultured this strain without IPTG in subsequent experiments. After transfer of the culture to the light condition BrdU uptake increased and reached a plateau 9 h post transfer (Fig. 2A); it was clearly inhibited by the addition of the replication inhibitor nalidixic acid (Fig. 2B). To investigate the involvement of de novo protein synthesis in replication initiation, we added chloramphenicol, a translation inhibitor, at the time of transfer to the light condition. The uptake of BrdU was clearly inhibited by chloramphenicol (Fig. 2C), although the TK protein exists even after chloramphenicol addition (Fig. 2D). These results suggest that replication initiation depends on de novo protein synthesis.

image

Figure 2. Detection of de novo DNA synthesis. A–C. Immunoblot analyses of BrdU-labelled DNA. The growth phase of S. 7942TK was synchronized by dark–light conditioning and nascent DNA was labelled with BrdU during the indicated periods. DNA samples (50 ng for A, 100 ng each for B and C) extracted from the cells were blotted and analysed using anti-BrdU antibody. Nalidixic acid (B, NDX, 3 µg ml−1) and chloramphenicol (C, Cm, 100 µg ml−1) were added at the time of transfer to the light condition. Ethanol was the solvent control. D. Expression of the TK protein without (control) or with inhibitors (NDX, Cm). RpoD1 was analysed as an internal control. Samples (40 µg) were analysed by Western blotting using anti-HA and anti-RpoD1 antibodies.

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Identification of the replication origin and form

The location of the replication origin can be identified by quantitative sequencing of BrdU-labelled DNA (replication sequencing, hereafter referred to as Repli-seq) (Hansen et al., 2010). To identify the replication origin of S. 7942, BrdU-labelled DNA, labelled for 1 h before (dark) and 0.5, 1 and 2 h after light exposure, was purified by immunoprecipitation with an anti-BrdU antibody (Fig. S5), and genomic libraries prepared from the purified DNA were analysed using a next-generation sequencer. The read depth of each sample was normalized with a control prepared from immunoprecipitates using control mouse IgG. Although there were no marked changes in distribution between the dark cultures and cultures exposed for 30 min to light (Fig. 3A and B), we detected a clear peak around the dnaN gene locus in the culture exposed to light for 1 h (Fig. 3C). This peak was broadened in the 2 h light culture (Fig. 3D). When we mapped the sequence reads onto the S. 6301 genome as a reference (Fig. S6), we observed a clear peak around the dnaN gene locus. This finding was confirmed by quantitative real-time PCR (qPCR) analysis using BrdU immunoprecipitated DNA. We performed qPCR assays using primer sets specific for several genomic regions (see Table S1). The qPCR signal was first detected upstream of the dnaN gene; it spread bidirectionally with time after transfer to the light condition (Fig. S7). These findings were consistent with our Repli-seq analysis.

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Figure 3. Ratio of read depth at each genomic position analysed by Repli-seq. Libraries made from purified BrdU-labelled DNA were sequenced on a next-generation sequencer (GAII, Illumina). The sequence reads were mapped onto the S. 7942 genome as a reference. Each of the mapping data was normalized by control data using the library prepared from DNA precipitated with control mouse IgG. Cells were labelled with BrdU for 1 h under the dark condition (A), or 0.5 (B), 1 (C) and 2 h (D) after transfer to the light condition. The location of the dnaN gene in the S. 7942 genome is shown by arrows. Asterisks indicate the 50 kb deletions.

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To study the progression of DNA replication newly synthesized DNA was sequentially BrdU-labelled at 30 min intervals before and after cell transfer to the light condition and analysed by qPCR. The qPCR signal was first detected in the dnaN upstream region, indicating that the replication origin was near the dnaN gene (Fig. 4). In addition, later on the peak was detected in equidistant regions opposite the dnaN locus (Synpcc7942_2328 and Synpcc7942_0302). In the regions opposite to dnaN (Synpcc7942_1001, Synpcc7942_1294 and Synpcc7942_1595), the peak signal was observed 3.5 h post transfer to the light condition (Fig. 4). These results indicate that DNA replication is initiated as early as 0.5–1 h after the transfer of cells to the light condition and proceeds bidirectionally from around the dnaN gene.

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Figure 4. Quantitative real-time PCR analysis of purified BrdU-labelled DNA. Before and after transfer to the light condition, S. 7942TK cells were labelled with BrdU for 0.5 h before being harvested at the indicated times. BrdU-labelled DNA was purified by immunoprecipitation with anti-BrdU antibody and used as a template for qPCR analysis with primer sets specific for the genomic regions shown in the centre. In each diagram the actual number of amplified DNA molecules was calculated by subtracting the background number in the dark-labelled samples (−0.5 to 0). The BrdU labelling times are: −0.5 to 0 h (Dark), 0 to 0.5 h (0.5), 0.5 to 1 h (1), 1 to 1.5 h (1.5), 2 to 2.5 h (2.5), 3 to 3.5 h (3.5), 4 to 4.5 h (4.5), 5 to 5.5 h (5.5), 6 to 6.5 h (6.5) and 7 to 7.5 h (7.5).

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Asynchronous DNA replication initiation in S. 7942

To investigate the synchrony of DNA replication among the S. 7942 cells we examined BrdU-labelled cells using immunofluorescence microscopy. As in E. coli and B. subtilis (Lewis and Errington, 1997; Adachi et al., 2005) we observed one or two BrdU foci per S. 7942 cell (Fig. 5A). Their formation was dependent on the duration of light exposure and was clearly inhibited by nalidixic acid (Fig. 5C). These findings indicate that the replication machinery exists in a specific subcellular location although multi-copy chromosomes are widely distributed inside the S. 7942 cells (Fig. 5B), and that the formation of BrdU foci is dependent on ongoing chromosomal replication. The BrdU foci were randomly dispersed in the cells (Fig. S8) although in E. coli they were located in the cell centre (single focus) or at one-fourth and three-fourths points along the long axis of the cells (two foci) that correspond with potential division sites (Adachi et al., 2005).

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Figure 5. Asynchronous DNA replication in S. 7942 cells. A. Localization of BrdU-labelled DNA in S. 7942TK cells. The cells were cultured in the dark for 18 h and 6 h after their transfer to the light condition they were BrdU-labelled for 0.5 h and harvested. Fixed cells were examined by immunofluorescence microscopy using anti-BrdU antibody. The bright-field and immunofluorescence images of BrdU are merged. Scale bar: 2 µm. B. Localization of genomic DNA in S. 7942TK cells. After fixation as described in Experimental procedures S. 7942TK cells were stained with 1 µg ml−1 DAPI. A bright-field image (left) and DAPI-stained cells (right) are shown. Scale bar: 2 µm. C. The proportion of BrdU-positive cells and the number of BrdU foci before and after transfer to the light condition. S. 7942TK cells were labelled with BrdU for 0.5 h before being harvested at the indicated times and examined by anti-BrdU immunofluorescence microscopy. 8.5+NDX: 3 µg ml−1 nalidixic acid was added at the time of transfer to the light condition and BrdU-labelling was started from 8 h post transfer. The proportion of BrdU-positive cells among 300 examined cells is shown in different colours based on the focus number per cell: blue = one focus, red = 2 foci, green = 3 or more foci per cell. D. Plots of the sequencing read depth (presented as in Fig. 1) prepared from S. 7942TK cells grown in the dark for 18 h and under the light condition for 9 h. The location of the dnaN gene is shown by an arrow. Asterisk: the 50 kb deletion.

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The number of BrdU-positive cells gradually increased with the duration of light exposure (Fig. 5C) and reached a maximum (c. 70%) 6.5 h after transfer to the light condition, suggesting that replication started asynchronously among the cell populations in culture. The point at which the proportion of BrdU-positive cells reached its peak (i.e. the peak replication time, 6–9 h post transfer) preceded the point of cell division onset (15 h, Fig. S2C); this is consistent with our findings presented in Fig. 2A and Fig. S2D and E. We performed whole-genome sequencing at the peak replication time. Different from the V-shaped distribution of the sequence reads in B. subtilis log-phase cultures (Fig. 1B), the sequence reads in S. 7942 at the peak replication time (9 h post transfer) exhibited an almost flat-shaped distribution (Fig. 5D), as in log-phase cultures (Fig. 1D). This observation indicates that only a small number of the replication origins in the multi-copy chromosomes were fired even when most of the cells were undergoing DNA replication. Taken together, these results suggest that replication is initiated asynchronously not only among cell populations but also among multi-copy chromosomes. Cell division, on the other hand, is relatively synchronized (Fig. S2C).

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Our genome-wide analysis indicated that the DNA replication of S. 7942 is initiated from a single origin located proximate to the dnaN gene (Figs 1B and C and 3). Since a cluster of dnaA boxes has been found in the upstream region of dnaN (Liu and Tsinoremas, 1996) on the border between the high- and low-GC regions in the GC skew plot (Fig. S1), the dnaN upstream region was predicted as the replication origin of the S. 7942 genome. The evidence we provided here strongly supports this prediction. In the dnaN upstream region, the first qPCR signal appeared 1 h after the transfer of cultures to the light condition. No signal was observed after 30 min light exposure (Fig. 4), a period during which the cells are thought to prepare for growth (e.g. accumulation of energy, gene expression). In fact, the expression of RpoD1 and TK protein increased post transfer (Fig. S4B). Furthermore, DNA synthesis was clearly blocked by chloramphenicol (Fig. 2B), suggesting that de novo protein synthesis is essential for the initiation of DNA replication.

Consistent with an earlier report (Yoshikawa and Ogasawara, 1991) and the GC skew analysis (Fig. S1) (Lobry, 1996), the replication origin of B. subtilis was readily identified by sequencing the log-phase genome library on a next-generation sequencer (Fig. 1A and B). On the other hand, although the distribution of sequence reads in S. 7942 did not manifest an obvious peak, the reads ratio was slightly but significantly higher around the dnaN gene than in regions on the opposite side of dnaN in log-phase and peak replication-time cultures (Figs 1D and 5D). The qPCR signal in the dnaN upstream region did not disappear even when the first signal arrived at the site directly opposite of dnaN (Fig. 4). These results suggest that DNA replication was initiated asynchronously. In addition, the formation of BrdU foci exhibited asynchrony among cell populations in the culture (Fig. 5C). We posit that these asynchronies are reflected in the nearly flat-shaped distribution of the read depth ratios shown in Figs 1D and 5D. Since the cell number increased stepwise rather than linearly after transfer of the cultures to the light condition, the timing of cell division was apparently synchronized under this condition (Fig. S2C). We suggest that the replication initiation of multi-copy chromosomes occurs independently and asynchronously. We also found that DNA replication progressed bidirectionally (Figs 3 and 4) as in other eubacteria with a circular genome. On the side directly opposite of dnaN (Synpcc7942_1294), the qPCR peak appeared 3.5 h after transfer to the light condition (Fig. 4), suggesting that under our culture condition about 3 h are required for S. 7942 cells to complete one round of genome replication. This time is significantly shorter than the doubling time during the log phase (c. 9 h), suggesting that there is a gap between replication and cell division or that multiple rounds of replication occur within a cell division cycle. Indeed, the point of peak replication (6–9 h post transfer, Figs 2A, 5C and S2D and E) was significantly earlier than the onset of cell division (15 h, Fig. S2C), an observation consistent with an earlier report (Asato, 1979). Based on our findings we hypothesize that cell division is initiated when the multi-copy chromosomes reach a threshold number that is regulated by checkpoint(s) responding to environmental and/or internal conditions (see below).

Immunofluorescence microscopy revealed that like E. coli and B. subtilis, S. 7942 cells harboured one or two BrdU foci (Fig. 5A). In the former, subunits of DNA polymerase III colocalized with the BrdU foci (Lemon and Grossman, 1998; Kongsuwan et al., 2002; Onogi et al., 2002). We posit that the replication machinery is locoregional and colocalizes with newly synthesized DNA in the S. 7942 cell, although its multi-copy chromosomes are widely distributed inside the cell (Fig. 5B). Since the position of BrdU foci corresponds to potential division sites (Adachi et al., 2005) that colocalize with the FtsZ division rings, the replication process in E. coli is thought to be highly co-ordinated with cell division (Wang and Levin, 2009). On the other hand, in S. 7942 cells, BrdU foci were randomly dispersed (Fig. S8) although the FtsZ ring is located in the centre of the cell as in E. coli (Miyagishima et al., 2005). In S. 6803, multiple nucleoids segregate just before the complete closing of the division septum, leading to the suggestion that the co-ordination between chromosome segregation and cell division in S. 6803 is much less stringent than in B. subtilis (Schneider et al., 2007). This is consistent with our observation that the replication process of freshwater cyanobacteria containing multi-copy chromosomes is not tightly coupled to cell division. Others (Dong and Golden, 2008; Mori, 2009; Yang et al., 2010) proposed that cell division is under the control of the circadian rhythm, which measures daily time and adjusts to the predictable light–dark alteration. Its involvement in DNA replication remains unclear, although the rate of DNA synthesis was constant even when the circadian rhythm of S. 7942 cells was synchronized (Mori et al., 1996).

Why do freshwater cyanobacteria harbour multiple chromosomes and an asynchronous replication system? We posit a correlation with the genome structure represented as an asymmetrical GC skew. We suspect that this phenomenon is present in a range of organisms with multi-copy and asymmetrical genomes. To elucidate the process by which freshwater cyanobacteria replicate, the replication machinery and replication regulators in cyanobacteria require further study.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Culture conditions and synchronization of growth

Unless otherwise indicated, wild-type S. 7942 and its derivatives were grown photoautotrophically at 30°C under continuous illumination (40 µE m−2 s−1) in BG-11 medium with 2% CO2 bubbling. When appropriate, spectinomycin was added at a final concentration of 40 µg ml−1. To synchronize cell growth we cultured the cells in BG-11 medium until they reached the stationary phase. They were then diluted to OD750 = 0.2 with fresh BG-11 medium. After 18 h in the dark the cultures were transferred to the light condition (time 0) to restart cell growth.

Flow cytometry

For flow cytometry, 1 ml of cell culture was fixed in 0.005% Tween 20 and 1% glutaraldehyde for 30 min at 4°C, washed with 1 ml of PBS and stored at −30°C. After freeze–thawing with liquid N2 the cells were treated with 100 µg ml−1 RNase A in PBS at 37°C for 1 h, washed once with PBS and resuspended in 50 mM trisodium citrate (pH 8.0). Chromosomal DNA was stained with 10 µM Sytox Green (Invitrogen, Carlsbad, CA, USA) for 3 h and flow cytometry was performed on a FACS Calibur instrument (Becton-Dickinson, Palo Alto, CA, USA). The genome copy number of S. 7942 was estimated using the chloramphenicol-treated E. coli culture as a standard.

Construction of the strain carrying the HA-tagged thymidine kinase gene

To introduce the HA-tagged thymidine kinase (TK) gene into the chromosome, we constructed plasmid pNSHA based on plasmid pNSE1 that harbours a spectinomycin-resistant gene in the fragment derived from the neutral site (Kato et al., 2008). The SacI–BamHI fragment containing the HA-tag sequence from pCS2+HA (Turner and Weintraub, 1994) was cloned between the SacI and BamHI sites of pNSE1. A TK gene fragment of herpes simplex virus 2 was amplified by PCR from plasmid pMH6 (Hayashi et al., 2007) using the primers TK-Bam-f and TK-Bgl-r (Table S1). After digestion with BamHI and BglII, the fragment was cloned into pNSHA digested with BamHI. The resulting plasmid was used to transform the wild-type S. 7942 to spectinomycin resistance. After PCR confirmation that the TK gene was correctly introduced into the chromosomal neutral site, the resulting strain was named S. 7942TK.

Immunoblot analysis of BrdU-labelled DNA

S. 7942TK cells were cultured in BG-11 medium in the presence of 1 mM BrdU and harvested at the appropriate times. When appropriate, nalidixic acid (NDX) and chloramphenicol (Cm) were added at the time of transfer to the light condition at a final concentration of 3 µg ml−1 and 100 µg ml−1 respectively. DNA was extracted from each sample using the DNeasy Plant Kit (QIAGEN GmbH, Hilden, Germany) and spotted onto Hybond-N+ membranes (GE Healthcare). After blocking with 5% skim milk in TNT buffer (blocking buffer), the membranes were incubated with antibodies. For the detection of BrdU, an anti-BrdU monoclonal antibody (Invitrogen) was used as the primary antibody; horseradish peroxidase-conjugated anti-mouse IgG antibody (GE) was the secondary antibody. Signals were detected and visualized using the ChemiDoc XRS+ system (Bio-Rad Laboratories, Hercules, California, USA).

Immunoprecipitation of BrdU-labelled DNA

BrdU-labelled DNA was purified by immunoprecipitation as described previously (Cimbora et al., 2000) with minor modifications. BrdU-labelled DNA (20 µg) was sheared into 500 bp pieces using a Covaris S-2 sonicator (Covaris, Woburn, MA, USA) and boiled in 500 µl of immunoprecipitation (IP) buffer [0.1 M sodium phosphate (pH 7.0), 0.14 M NaCl, 0.05% Triton X-100]. After 10 min chilling on ice, biotin-conjugated mouse anti-BrdU monoclonal antibody (2 µg, Invitrogen) or control mouse IgG (Sigma) was added to each sample. After 30 min incubation at room temperature (RT), 100 µl of streptavidin-agarose (GE Healthcare) was added and the samples were incubated overnight at RT. DNA–protein complexes with agarose beads were pelleted by 1 min microcentrifugation at 4°C (the supernatant is indicated as [Sup.] in Fig. S5), the pellets were washed with 750 µl of IP buffer, resuspended in 200 µl of digestion buffer [50 mM Tris (pH 8.0), 10 mM EDTA, 0.5% SDS, 250 mg ml−1 proteinase K] and digested for 3 h at 37°C. After phenol/chloroform treatment, the DNA was ethanol-precipitated, briefly dried and resuspended in TE buffer (precipitate [Ppt.] in Fig. S5). Purified BrdU-labelled DNA was analysed by immunoblotting (Fig. S5) and qPCR, and sequenced.

Quantitative real-time PCR of BrdU-labelled DNA

BrdU-labelled DNA isolated by immunoprecipitation was analysed using the 7500 Real-Time PCR System (Applied Biosystems) with a KAPA SYBR FAST qPCR kit (KAPA Biosystems, Woburn, MA, USA) and a primer set for each locus (Table S1, target-f and target-r). Standard DNA (500 bp) was prepared using the corresponding standard-f and standard-r primer set (Table S1). The actual number of amplified DNA molecules for each locus was calculated by the amplification rate of the corresponding standard DNA fragment.

Immunofluorescence microscopy of BrdU-labelled cells

S. 7942TK cells were fixed in chilled methanol containing 1% (w/v) paraformaldehyde and 10% (v/v) dimethyl sulphoxide for 5 min at −80°C and washed two times with PBS. After 15 min treatment with 0.05% Triton X-100 in PBS the cells were permeabilized for 30 min at 37°C with 0.2 mg ml−1 lysozyme dissolved in 25 mM Tris-HCl (pH 7.5) and 10 mM EDTA, and then washed twice with PBS. For observation of the BrdU signals, cells were treated with 4 M HCl for 1 h at 37°C and then washed with PBS. After blocking with 5% bovine serum albumin in PBS (blocking buffer), the cells were immunostained for 2 h with mouse monoclonal anti-BrdU antibody (Invitrogen) diluted 1:20 in the blocking buffer, washed twice with the blocking buffer and incubated for 1 h with Alexa Fluor 488-conjugated goat anti-mouse antibody (Invitrogen) at a 1:200 dilution. After washing twice with the blocking buffer the cells were examined under a fluorescence microscope equipped with an Olympus DP71 digital camera (Olympus, Tokyo, Japan) and analysed with MetaMorph image analysis software (Molecular Devices, Downingtown, PA).

Library preparation for genome sequencing

Genomic DNA of B. subtilis and S. 7942 was extracted with the DNeasy Blood and Tissue kit (Qiagen) and the DNeasy Plant kit (Qiagen) respectively. For sequencing each sample was prepared according to Illumina protocols. Briefly, genomic DNA (5 µg) was fragmented to an average length of 200 bp using the Covaris S2 system (Covaris). After repair of the fragmented DNA and the ligation of ‘A’ to the 3′ end, Illumina Index PE adapters were ligated to the fragments and the samples were size-selected for a length of 300 bp using E-Gel SizeSelect 2% (Invitrogen). The size-selected products were PCR-amplified for 18 cycles with the primers InPE1.0, InPE2.0, and an index primer containing 6 nt barcodes (Illumina). The final products were validated using an Agilent Bioanalyser 2100 (Agilent, CA, USA). For Repli-seq analysis we used 20 µg of purified BrdU-labelled DNA for library preparation in the same manner, except that PCR amplification was with 30 cycles.

Sequencing and data analysis

The barcoded libraries were used for cluster generation in a multiplexed flow cell lane in the Illumina Genome Analyser II system. After the sequencing reactions were completed, the Illumina analysis pipeline (CASAVA 1.6.0) was used for image analysis, base calling and quality score calibration. Reads were sorted by the barcodes and exported in the FASTQ format. The reads from each sample were aligned to the published complete genomes of the B. subtilis 168 (GenBank: AL009126), S. 7942 (GenBank: CP000100) and S. 6301 strain (GenBank: AP008231) by MAQ software (Ver. 0.7.1) (Li et al., 2008). The read depth was obtained by calculating the average number of reads per base in each 1 kb window using the MAQ cns2win command and the ratio of each read depth to the total read depth was calculated. For Repli-seq analysis each read depth was normalized with control data using the library prepared from DNA precipitated with mouse IgG.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

We thank H. Masukata (Osaka University) for providing plasmid pMH6 which contains the thymidine kinase gene of herpes simplex virus 2 and T. Kondo (Nagoya University) for providing the S. 7942 Nagoya strains. All authors have no conflict of interest to declare. This work was supported by the Ministry of Education, Culture, Sports, Science and Technology [Grants-in-Aid for Scientific Research (S0801025)].

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information
FilenameFormatSizeDescription
MMI_7971_sm_Data.doc3345KSupporting info item

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