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Profiling a single-stranded DNA region within an rDNA segment that affects the loading of bacterial condensin

Summary Bacterial condensin preferentially loads onto single-stranded DNA (ssDNA) in vitro and onto rDNA in vivo to support proper chromosome compaction. Thus, the actively transcribing rDNA would provide the ssDNA region for the loading of bacterial condensin. We attempted to detect the ssDNA region in the rrnI gene in situ. Non-denaturing sodium bisulfite treatment catalyzed the conversion of cytosines to thymines (CT-conversion) at the melted DNA of a genome. Using next-generation sequencing, we generated an average of 11,000 reads covering each cytosine on the rDNA segment. In principle, the CT-conversion rate is an accurate guide to detect ssDNA segment. We detected multiple ssDNA segments throughout the rDNA. The deletion mutations of the rDNA hindered the ssDNA formation at the 100-500 bp segment downstream of the promoter. These data support the idea that the ssDNA segment plays a crucial role as the condensin-loading site and suggest the mechanism of condensin loading onto rDNA.


INTRODUCTION
Proper organization of chromosomal DNA is crucial for the segregation of replicated chromosomes into daughter cells. The structural maintenance of chromosomes (SMC) proteins are major players in chromosomal DNA organization in prokaryotes and eukaryotes. [1][2][3][4][5] The SMC proteins form the core of the two distinct complexes, cohesin, and condensin, that manage chromosome cohesion and condensation. A two-armed structure of the SMC dimer forms a ring that holds DNA topologically. The topological DNA-binding activity is regulated by non-SMC subunits that interact with an ATP-binding cassette (ABC)-like domain at the distal end of each arm. In addition, the SMC proteins bind to not only doublestranded DNA (dsDNA), but also single-stranded DNA (ssDNA). The physiological significance of the ssDNA-binding activity is currently not clear. However, several reports indicate that the ssDNA binding of the SMC proteins contributes to topological DNA binding on genomic DNA. [6][7][8][9][10][11][12] The Bacillus subtilis Smc-ScpAB complex consists of the core of the SMC protein Smc and the non-SMC subunits ScpA/B and functions as a bacterial condensin. 13,14 The Smc protein specifically accumulates at parS which is the Spo0J binding site on the B. subtilis chromosome. 15 The Spo0J-parS facilitates the loading of the Smc-ScpAB complex on the chromosome to support efficient chromosome segregation by organizing the chromosome of B. subtilis. 15,16 Deficiency of Spo0J, deficiency of parS, or both cause moderate deterioration of chromosome segregation, whereas deficiency of Smc severely prevents chromosome partitioning. The contrast indicates that another loading pathway of the Smc-ScpAB complex must work independently. In addition to accumulating at the Spo0J binding sites, Smc is enriched at the highly transcribed genes containing rDNA. 15 Deficiencies of both rDNA and Spo0J cause aberrant nucleoid separation, which is similar to that of the smc deletion mutant. 17 Thus, rDNA may also be a loading site for the Smc-ScpAB complex to perfectly organize the chromosome of B. subtilis.
Moreover, a plasmid harboring a full-length rDNA segment is effectively co-immunoprecipitated with the Smc-ScpAB complex when rDNA is actively transcribed. 17 The highly transcribed rDNA would interact with the Smc-ScpAB complex. Although transcription is still highly activated, deletions of the rDNA segment on plasmids hinder efficiencies of co-immunoprecipitation of the plasmid DNA with the Smc-ScpAB complex. The full-length rDNA encodes 16, 23, 5S, and tRNA. These transcribed RNAs of the full-length rDNA RESULTS B. subtilis condensin binds ssDNA. 6 E. coli condensin preferentially loads onto ssDNA topologically in vitro. 10 Moreover, B. subtilis condensin interacts with rDNA in vivo, which would work to support proper chromosome compaction. 17 Based on those reports, we hypothesized that the rDNA provides the ssDNA region for the topological binding of the Smc-ScpAB complex. Therefore, we attempted to detect the ssDNA region on the rDNA segment in a living bacterial cell. Using next-generation sequencing (NGS), we generated 11,000 reads, on average, that cover each cytosine on the PCR-amplified rDNA segment to obtain the precise CT conversion rate. An outline of the non-denaturing sodium bisulfite sequencing is shown in Figure S1. The results showed that the CT conversion rates were higher than the frequency of error generation by NGS. Therefore, we expected that an increment in the CT conversion rate would reflect a trend toward ssDNA accumulation at a given site within the rDNA segment.
In situ detection of the single-stranded DNA region in rDNA on the chromosome The wild-type cell of B. subtilis has ten copies of rDNAs. By contrast, the "2 rrn strain" has only two copies of the rRNA gene due to deletions in the other eight rDNAs. The specific rDNA segment of the 2 rrn strain was easily amplified by PCR. The 2 rrn strain exhibited slight defects in cell growth 30 and had nucleoids that were separated and constricted in a dumbbell shape, as seen in the wild-type cell. And we carried out the co-immunoprecipitation of plasmids harboring a series of the rDNA segments with the Smc-ScpAB complex. 17 Then we used a series of the 2 rrn strains harboring the deletion mutation rDNA segments to detect the ssDNA region on the rDNA segment ( Figure S2).
First, we analyzed the CT conversion rates of the wild-type rrnI gene of the 2 rrn mutant without the sodium bisulfite treatment. We determined the CT conversion rates of each cytosine at the top and the bottom strand in the 5 0 half of the rrnI gene. The CT conversion rates of the top strand were similar to those of the bottom strand ( Figure 1A). We performed a sliding window analysis with the window size fixed at 64 bp and moving away from the promotor region of rrnI in one base step ( Figure 1B). The average CT conversion rate of each window was mapped on the rrnI gene to compensate for the irregular cytosine distributions in the gene. There were no characteristic profiles of the CT conversion rates at either strand. The average CT conversion rate was 0.16% (Table S1). When cells were not exposed to sodium bisulfite, the low CT conversion rates were well consistent with the error rate caused by the misreading of NGS (0.1-0.3%).
By contrast, the top and bottom strand CT conversion rates in the 5 0 half of the rrnI gene were about 40-fold increased when cells were exposed to sodium bisulfite (Table S1). The average CT conversion rate was 6.89%. The CT conversion rates fluctuated dramatically at a single-nucleotide resolution throughout the iScience Article rrnI gene; the lowest rate was 0.81%, and the highest rate was 21.5% ( Figure 1C). We further analyzed the CT conversion rates of the 3 0 half of the rrnI gene and then characterized the CT conversion rates throughout the rrnI gene. Similar results were obtained (Table S1 and Figure 1C). The CT conversion rates fluctuated dramatically throughout the rrnI gene. The average CT conversion rate of the rrnI gene was 6.77%, and the highest rate was 25.6%. The nucleotide positions corresponding to the higher CT conversion rates (>15%) on the top and the bottom strand are listed with the neighbor sequences in Tables S2 and S3. In addition, nucleotides with higher CT conversion rates were simultaneously found on both the top and the bottom strand of the DNA segment ( Figures S3 and S4). The nucleotides with higher CT conversion rates were positioned within DNA segments with relatively higher GC content, except for bp position 669 at the top strand,  iScience Article which was located at a DNA segment with relatively high AT content (Table S2). Thus, the higher CT conversion frequencies in the rrnI gene might not be caused by high AT content at the DNA segment.

Relationship between the CT conversion rates and the C content
The sliding window analysis showed that the CT conversion rates fluctuated dramatically throughout the whole rrnI gene. We conjectured that the cytosine content within the sliding window affected the fluctuation of the CT conversion rates because the cytosine content within the window size used (64 bp) is not constant in the rrnI gene ( Figure 1D). We analyzed the correlation coefficient between the CT conversion rates and the cytosine contents ( Figure 2A). Weak positive correlations were observed in the top and the bottom strand. The correlation coefficients were 0.41 and 0.53, respectively.
However, several segments were plotted apart from the main correlation lines. Indeed, some windows showed that the CT conversion rates were relatively high even though the cytosine content (C content) was low. Conversely, other windows showed that the CT conversion rates were relatively low even though the cytosine content was high. We selected the sliding windows with the higher and lower CT conversion (B) The graph points of the higher and the lower CT conversion rates are mapped on the rrnI segment, as shown in Figure 1D. The CT conversion rates on the top strand (five red hues) and bottom strand (four green hues), and the cytosine content (blue) are shown. The red and green hues correspond with those in (A). The rrnI map, including the transcription start sites at the promoter region, 16, 23, 5S rRNA genes, tRNA gene (trnI), and transcription terminator, are indicated above the graph. The asterisks indicate the points scattered above the +2SD lines of (A), whereas the arrow indicates the points scattered under the -2SD line of (A iScience Article rates (more and less than 1SD, respectively) from Figure 2A and plotted them on the rrnI gene map along with the CT conversion rates (Figures 2B, S5 and S6, Tables S4-S6). We confirmed that the CT conversion rates at the specific segments of the rrnI gene increased independently of the cytosine content. These results suggest that the rrnI gene contained the DNA segments that tend to form ssDNA.
Relation between CT conversion and transcription at the rDNA Next, we examined the effect of active transcription on the CT conversion rates. We analyzed the CT conversion rates of the rrnIDp gene in which the promoter region was deleted (YAN12675; Figure 3A). The transcriptional activity of the rrnIDp gene was reduced by 99% compared with that of the wild-type rrnI gene. 17 However, a remarkable CT conversion of rrnIDp was detected in the rrnIDp gene. Figure 3 shows the profiles of the CT conversion rates of the single nucleotides and the sliding window analysis. The whole profiles were strikingly similar in appearance to the profiles of the wild-type rrnI gene. However, we considered that the marked decline in transcription activity caused some differences in the CT conversion rates between the wild-type and the rrnIDp. We then subtracted the rrnIDp CT conversion rates from the wildtype CT conversion rates for further analysis ( Figure 3C). We found that, on the whole, the wild-type CT conversion rates were higher than the rrnIDp conversion rates. The transcription activity from the rrnI promoter does not directly affect DNA melting in the segment upstream of the rrnI promoter. Certainly, the CT conversion rate differences between the wild-type and the rrnIDp were slight at the segment upstream of the rrnI promoter. The average difference in the CT conversion rate at the promoter's upstream segment was 0.52% (Table S7). On the other hand, the average difference in the CT conversion rate was 2.06 percentage points in the promoter's downstream segment. Moreover, several segments showed differences of more than 4 percentage points in the CT conversion rate.
Notably, the differences in the CT conversion rates were particularly large in the top strand, i.e., the coding DNA strand. On the other hand, the differences in the CT conversion rates in the bottom strand, i.e., the template DNA strand, were mainly found at the former part of the rrnI gene but not at the latter part.
Next, we subtracted the CT conversion rates of the top strand from those of the bottom strand Figure 3D). The degree of influence caused by reducing the transcription activity was substantial in the top strand of the rrnI gene, and the influence was especially remarkable at the latter part of the top strand. The two peaks of the biased CT conversion rates at the segment 100 to 500 bp downstream of the promoter indicate that the ssDNA formation was affected by the reduction in transcription activity.

The CT conversion of the rrnID5S mutant
We carried out a further analysis to specify the portion of the ssDNA region within the rrnI gene that affects the interaction of Smc-ScpAB bacterial condensin. We analyzed the CT conversion rate of the rrnID5S gene (YAN12698), in which the 51 bp segment of the 5S rRNA coding region (116 bp) was deleted ( Figure 4A). Although the mutated rrnID5S gene results in a defect in the interaction of the Smc-ScpAB, it is actively transcribed. 17 The whole profiles of the CT conversion of rrnID5S shown by both the single nucleotides and the sliding window analysis were strikingly similar in appearance to those of the wild-type rrnI gene ( Figures 4A and 4B). As in the analysis of rrnIDp, the CT conversion rate of the rrnID5S gene was subtracted from that of the wild-type rrnI. As a result, we found marked differences in the CT conversion rates in the segment 100 to 500 bp downstream of the promoter but not in the other segments ( Figure 4C). The CT conversion rates within the segment 100 to 500 bp downstream of the promoter were higher than the average CT conversion rate of the rrnI segment. Similar results were obtained from the bottom strand.
We subtracted the CT conversion rates of the bottom strand from those of the top strand ( Figure 4D). There were two peaks in the CT conversion rates of the top strand in the segment 100 to 500 bp downstream of the promoter, as shown in Figure 4D. The deletion of the 51 bp segment at the 5S rRNA coding region specifically affected the CT conversion rates of the top strand. Thus, the efficiency of ssDNA formation within the rrnID5S gene was reduced only at the downstream segment of the promoter.
The CT conversion of an rDNA mutant defective in the interaction of the Smc-ScpAB complex iScience Article the remaining segments were similar in appearance to those of the wild-type rrnI gene ( Figure 1D). We found that the CT conversion rates were affected in the segment 100 to 500 bp downstream of the promoter when we subtracted the CT conversions of rrnID23 0 -5S from the wild-type or the CT conversions of rrnID23-5S from the wild-type. Other deletion mutants of the rrnI gene lost the region containing the segment 100 to 500 bp downstream of the promoter. The remaining segment was similar to that of the wild-type rrnI gene, and the differences were very small between the wild-type and the rrnI mutant genes. The results for both rrnID23 0 -5S and rrnID23-5S supported the idea that the segment 100 to 500 bp downstream of the promoter had the greatest effect on the CT conversion rates throughout the rrnI segment.
The CT conversion of rDNA in the Dsmc mutant cells We conjectured that the interaction of Smc with rDNA affected the structure of the transcribing rDNA segment, and it facilitated the formation of the ssDNA. To determine whether the loaded Smc affects iScience Article the rate of the ssDNA at the 100-500 bp segment downstream of the promoter, we analyzed the CT conversion rates in the Dsmc mutant cells. Our results showed that the CT conversion rates were drastically increased at the 100-500 bp segment of rDNA in the smc deletion mutant cells ( Figure S10). This suggested that the bound Smc proteins protect the cytosine residues from sodium bisulfite or suppress the formation of the ssDNA segments.

The CT conversion of inactive genes during vegetative growth
We evaluated the difference in the effect of the non-denaturing sodium bisulfite treatment between the wild-type and the mutant rrnI genes. Because the mutant rrnI cells did not exhibit the same growth as iScience Article the wild-type rrnI strains, and the mutant rrnI strains exhibited slow cell growth, we analyzed the CT conversion rates of another chromosomal segment as an internal standard to clarify the effect of cell growth on the CT conversion rates. The spsA and the spsB genes are inactive during vegetative growth and are activated by a sporulation-specific sigma factor, s K , in response to the deterioration of nutrients in a growing environment. 31,32 Thus, the spsAB segment is suitable for analysis of the effect of the non-denaturing sodium bisulfite treatment on cell activities other than transcription.
We analyzed the CT conversion rates of the DNA segment, including the spsAB genes in the wild-type rrnI strain. Figures 6A and 6B show the whole profile of the CT conversion of the spsAB segment by both the single nucleotides and the sliding window analysis. The top and bottom strand nucleotides showed markedly higher CT conversion rates (more than 10%). In particular, the CT conversion rates were high in the region upstream of the promoter on the top strand. The average CT conversion rates of the spsAB segment were 4.20% on the top strand and 3.65% on the bottom strand (Table S7). These rates were less than those for the rrnI and rrnIDp genes. Thus, the CT conversion of a chromosomal segment decreased due to the inactivation of transcription.
Next, we analyzed the CT conversion rates of the spsAB gene in the 2 rrn strains harboring a series of mutated rDNA segments. We obtained whole profiles of the CT conversion of the spsAB segment by sliding window analysis ( Figure S8). The results showed that the spsAB segment profiles in the mutated rDNA strains were similar. Moreover, the profiles were also very similar to that in the 2 rrn strain harboring the wild-type rrnI. In fact, we did not find any apparent differences when the CT conversion rates in the mutated rDNA strains were subtracted from those of the 2 rrn strain harboring the wild-type rrnI ( Figures 6C-6E and S9). The differences in the CT conversion rate between the wild-type and the mutated rrnI strains were less than 0.7 percentage points. Thus, the deletion within the rrnI gene did not affect the CT conversion of the other genes on the genome.

Interaction of smc with a full-length of rDNA under an RNase H-overproduction condition
We applied the co-immunoprecipitation assay without any chemical reaction for cross-linking between a protein and DNA to analyze the interaction of the Smc-ScpAB complex with a full-length of rDNA (rrnI) in a living cell. 17 The assay was carried out using a cell in which two additional genes, rnhB and rnhC, could be expressed under an inducible promoter. rnhB, and rnhC are genes of B. subtilis that independently encode RNase H. 33 RNase H catalyzes the cleavage of RNA in a hybrid of DNA and RNA-namely, the R-loop. If the R-loop is formed at the segment 100 to 500 bp downstream of the promoter of rDNA, increased production of RNase H will affect the formation of the ssDNA and result in a decrease in the interaction of the Smc-ScpAB with rDNA. We repeated the assay three times independently. When the gene expression of rnhB and rnhC was induced, the amount of recovered rDNA that participated with the Smc-ScpAB complex was clearly decreased in every experiment compared to non-inducible conditions, suggesting that overexpression of RNase H hindered the binding of the Smc-ScpAB complex on rDNA ( Figure S11).

DISCUSSION
We used a combination of biochemical and genomic approaches to detect where chromosome DNA segments melt into ssDNA in a bacterial cell. Non-denaturing sodium bisulfite treatment effectively catalyzed the conversion of unpaired cytosines to thymines via uracils without any DNA denaturation treatment. The CT conversion induced by non-denaturing sodium bisulfite treatment showed high performance for the detection of ssDNA segments in the genome by using NGS. Our analyses demonstrated that bacterial genomic DNA melts into ssDNA in living cells in a largely unpredictable manner.
We applied the CT conversion rates of the spsAB segment as an internal standard. The differences in the CT conversion rate at the spsAB segment between the wild-type and the mutated rrnI (0.67%) strains were less than those at the rrnI segment (1.26%) (Table S7). Judging from the above, the reduction of the cell growth rate did not reflect the CT conversion rates. Next, therefore, we carried out the non-denaturing sodium bisulfite treatment until the progression of the cytosine deamination was nearly saturated and then performed next-generation sequencing, with an average of 11,000 reads, covering each cytosine on the PCR-amplified rDNA segment. As a result, we were able to obtain the actual CT conversion rate to overcome perturbation by the cell growth rate. Therefore, we conclude that the CT conversion rate differences at the rrnI segment between the wild-type and the mutated iScience Article rrnI were directly affected by the efficiency of the ssDNA formation, which deteriorates due to the deletion mutations.
When we compared the CT conversion rates between the wild-type and the mutated rrnI gene, the difference in the rates led us to discover ssDNA segments in the bacterial rDNA under active transcription. Multiple ssDNA segments were formed throughout the rDNA segment under active transcription. Temporary hybridization between a new RNA transcript and the template DNA strand reduced the CT conversion rate The ssDNA segment was essential for loading the Smc-ScpAB complex. Biases in the CT conversion rates between the top and the bottom strand were obvious in this segment. It is conceivable that the ssDNA segment was exposed by the R-loop formation in a manner dependent on the rRNA transcription.
The results are consistent with previous reports that the bacterial SMC proteins bind to ssDNA and preferentially load onto the actively transcribing rDNA. 10,17 It is likely that the Smc-ScpAB complex is loaded onto the ssDNA segment located 100 to 500 bp downstream of the rrnI promoter, although further biochemical experiments will be needed to confirm the idea. The segment seems to stably maintain its unwound state during active transcription. Smc suppressed the formation of the ssDNA, partly because the binding of Smc blocked the sodium bisulfite and partly because of the reannealing activity of Smc from ssDNA to dsDNA. 6 Malig et al. 34 reported a sophisticated approach for identifying R-loops in the human genome by analyzing the CT conversion rates of consecutive cytosines. In addition, a nuclease-and bisulfite-based method has been applied to identify R-loops in a strand-specific manner in the mouse genome. 35 In contrast, in the present study we were able to detect R-loops of the bacterial genome in a straightforward manner. It is conceivable that the R-loop formation during transcription directly reflects differences in the CT conversion rates between the coding DNA strand and the template DNA strand. If so, this could be because the bacterial genome structure is simpler than the human genome structure. Modified cytosine bases in bacterial genomes are restricted to a specific motif of DNA. DNA methyltransferase of B. subtilis 168 (BsuM) modifies cytosine in the sequence 5 0 -YTCGAR-3 0 . 36 Nine cytosines in the sequence were located in the rrnI genes. In addition, bacterial chromatin does not form nucleosome or nucleosome-like structures. Thus, the properties of bacterial genomes make it easier to detect the R-loop formation during transcription by using the CT conversion rates. 29 Our procedure used in this experiment could be applied for analysis of the genome dynamics of prokaryotes.
The deletions in the latter half of the rrnI gene affected the ssDNA formation at the segment 100 to 500 bp downstream of the rrnI promoter. Even the 51 bp deletion in the 5S encoding segment was effective in reducing ssDNA formation at the downstream segment of the rrnI promoter. The transcription activity increases by 3.5-fold in the rrnID5S gene. 17 The ssDNA formation depended on the transcription from the rrnI promoter. Therefore, our results suggested that the latter half of the rrnI gene could somehow affect the transcription at the region upstream of the rrnI gene. The transcribed rRNA, including 5S rRNA, forms the secondary structure of RNA.
Moreover, the transcribed product develops a higher-order structure than the tertiary structure of transcribed products. It is a plausible explanation that the higher-order structure of the transcribed RNA, including 5S rRNA, would affect the R-loop formation at the ssDNA segment at 100 to 500 bp downstream of the rrnI promoter. Indeed, an R-loop was reported to be formed at the replication origin of the ColE1 plasmid and stabilized by the higher-order structure of a transcribed RNA. 37 We consider the ssDNA formation at the 100-500 bp segment downstream of the promoter to have promoted the interaction of the bacterial condensin to rDNA. Indeed, the 100-500 bp segment deletion mutations of rDNA hinder efficiencies of co-immunoprecipitation of the derivatives of pRRN, which are plasmids harboring the deleted rrnI segment, with the Smc-ScpAB complex, except for pRRN/D16-23 0 S 17 . The amount of retrieved DNA of that plasmid is relatively higher than those of other deletion plasmids, even though the 100-500 bp segment is lost. The reason why pRRN/D16-23 0 S is partially active in interaction with the Smc-ScpAB complex is because there are relatively higher CT conversion rates in the remaining segment. The CT conversion rates at the remained 23S segment of rrnID16-23 0 S were higher than that of the wild type. Such segments are shown as the minus CT conversion rates of the upper strand ( Figure 5D). The D16-23 0 S deletion mutation of rrnI could alter DNA structure or R-loop formation, thereby partially recovering the loss of the 100-500 bp segment. In any case, an effective ssDNA formation would be crucial for the interaction of rDNA with bacterial condensin.
Our results cannot distinguish whether the marked difference of the ssDNA formation at the 100-500 bp downstream of the promoter resulted from the direct binding of the Smc-ScpAB complex onto the iScience Article 100-500 bp segment or binding of the Smc-ScpAB complex somewhere on rDNA. It is possible that the Smc-ScpAB complex binds to sites other than the 100-500 bp segment, and the Smc binding causes an alteration of DNA structure at the 100 -500 bp segment. Further experiments with the combined investigations of biochemical analyses and Bisulfite sequencing will reveal more precise mechanisms.
High rates of CT conversion were detected in the B. subtilis genome, irrespective of transcription activity. The CT conversion rates by the single-nucleotide analysis fluctuated dramatically throughout the rDNA. The positions of the cytosines with the higher CT conversion rates were shared in common among the rDNA segments we analyzed. However, we could not find a prominent consensus motif in the flanking sequences of the cytosines with the higher CT conversion rates. In general, the AT-rich motifs tend to be locally melted with a complete disruption of base pairing. This was not the case in our present analysis. Instead, the flanking sequences of the cytosines with the higher CT conversion rates contained CC, CCC, CCCC, CCCCC, and CCCCCC motifs. The cytosines with the higher CT conversion rates were also detected in the spsB gene. Thus, melting of the rDNA was not necessarily dependent on AT content, even though the higher CT conversion was precisely reflected by the melting of dsDNA. In addition, transcription did not directly influence the higher CT conversion rates, based on similar results for the rrnIDp mutant. It may be that DNA replication was involved in the ssDNA formation of the cytosine-rich motifs.
Alternatively, these ssDNA segments may have been formed due to DNA breathing by torsion of a specific conformation rather than by thermal fluctuations. Genome-wide analysis of the CT conversion rate may clarify the correlation between a DNA sequence and a tendency to form ssDNA segments.
Members of the SMC protein family possess ssDNA-binding ability in addition to dsDNA-binding ability. For example, yeast cohesin can topologically load onto the ssDNA and thereby capture the secondary DNA to establish DNA-DNA interaction by the cohesin ring. 9 Bacterial RecN, which is closely related to the SMC family of proteins, topologically loads onto both ssDNA and dsDNA, and has a preference for ssDNA 8 . Yeast and human condensin recognize unwound DNA in highly transcribed genes, or ssDNA. 12 Indeed, the ssDNA binding ability is an important property for the loading of SMC proteins. It is not yet clear how the ssDNA-binding ability of the SMC proteins contributes to the maintenance of chromosomal DNA. Bacterial SMC proteins utilize the ssDNA-binding ability for topological loading to specific sites on the genome DNA, such as the stable ssDNA segment. A thorough analysis of the ssDNA formation in the genome would help elucidate the function of the ssDNA binding of the SMC proteins for topological loading.

Limitations of the study
In this study, we demonstrate that the ssDNA segment is formed at the 100-500 bp downstream of the rDNA promoter due to active transcription, and the ssDNA segment is crucial for the Smc-ScpAB loading onto rDNA. It seems that the Smc-ScpAB complex topologically loads onto the exposed ssDNA segment in the R-loop. However, it remains an open question whether Smc-ScpAB directly loads on the ssDNA segment or not. In addition, there is no direct evidence for the formation of the R-loop at the segment. Further experiments are needed to clarify the loading site of Smc-ScpAB and the R-loop formation at rDNA.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following: All the strains used in this study are listed in the key resources table. The 2rrn mutant, YAN13122, carries two copies of the rRNA gene (rrnA and rrnI). Various lengths of deletion in rDNA were introduced in the rrnI gene, as shown in Figure S2. A gene replacement technique was applied to substitute a gene of interest.
The smc gene was replaced with the erm gene according to the technique. A linear DNA fragment for the gene replacement technique was constructed by using the Splicing by Overlap Extension PCR (SOE PCR).
To overproduce RNaseH, we built the Pspac-rnhB-rnhC operon in which both the rnhB and the rnhC genes encoding RNaseH were placed under the control of the IPTG (isopropyl b-D-thiogalactopyranoside)-regulated promoter, Pspac. We constructed a linear DNA fragment containing tandemly arraigned genes of rnhB and rnhC by using SOE PCR. The linear DNA fragment was used to transform YAN16141, in which the Pspac promotor was located downstream of the aprE:erm gene, resulting in YAN17462. To test the expression of RnhB and RnhC, the gfp gene without a promoter was inserted downstream of the rnhB and rnhC genes, and the intensity of GFP was measured. An image of fluorescence microscopy showed that the intensity of GFP indeed increased in the presence of IPTG.

Plasmid construction
Plasmid DNA was constructed by using the in vivo ligation system, iVEC (in vivo E. coli cloning. 38 We had used the pGETS118-t0-Pr-SfiI-pBR322 plasmids in which the replication initiator Rep is regulated by gene expression under the control of the IPTG-inducible promoter, Pspac. 17 We substituted the Pspac promotor with the PspsA promoter to avert the IPTG induction of the Rep protein, resulting in pVEC2. The rrnI segment of pRRN 17 was cloned into pVEC2, resulting in pRRN2.

Cultivation of cells for sodium bisulfite treatment
The B. subtilis cells were cultured in L medium. When the smc deletion mutant was cultivated, the SMG medium was used for a growth medium with modification. 17 Cells were cultured overnight at 37 C with shaking, and the cells were diluted to be an optical density 600 (OD 600 ) of 0.04. When OD 600 reached 0.4, one mL of the culture was used for the sodium bisulfite treatment. After the centrifugation, the supernatant was discarded. The cell pellet was resuspended in 500 mL of an RNA stabilizing reagent (RNAlater; Thermo Fisher Scientific) and incubated at room temperature for 10 min. After centrifugation and discarding of the supernatant, the cell pellet was frozen in liquid nitrogen and stored at À80 C until use.

Sodium bisulfite treatment
Aqueous 80% methanol (methanol: water, 80:20 v/v) was directly added to the frozen cell pellet and left for 5 min, and then the cells were collected by centrifugation. After discarding the supernatant, the cells were lysed with lysozyme at concentrations of 0.4 mg/mL in a buffer (10 mM Tris-HCl, pH 8.0, 4 mM EDTA, 4% (w/v) sucrose) for 30 min at 37 C. Sodium bisulfite treatment was carried out using commercially available bisulfite conversion kits (EpiTect Fast Bisulfite Kit and EpiTect Fast LyseAll Bisulfite Kit; Qiagen) according to the manufacturer's instructions except for the DNA denaturation step and sodium bisulfite reaction time. We carried out the sodium bisulfite reaction at 45 C without DNA denaturation, and incubated it for 120 min. The incubation time was determined according to the CT conversion rates after a series of the sodium bisulfite reactions with incubations ranging from 0 to 180 min ( Figure S12). After the bisulfite-treated DNA was eluted from the DNA spin column, the DNA was precipitated with ethanol, rinsed with 70% ethanol, resuspended with Milli-Q water, and frozen at À80 C until use. Reagents were prepared just before using.

PCR amplification of the rDNA and the sps segment
To determine CT conversion rates in the whole region of the rrnI gene, the region divided into 5 0 -and -3 00 -half were separately amplified by PCR using the primers listed in Table S8. To amplify the 5 0 -half of the rrnI gene, specific primers that hybridize to the region upstream of the rrnI promoter (#1) and the inside of the gene encoding 23S rRNA (#2) were used. To amplify the 3 0 -half of the rrnI gene, specific primers that hybridize to the inside of the gene encoding 23S rRNA (#3) and the spectinomycin-resistance gene (spc), which was inserted at the region downstream of the trnI gene (#4), were used. To determine CT conversion rates at the spsAB genes, specific primers that hybridize to the upstream region of the spsA promoter (#5) and the inside of the spsB gene (#6) were used. The amplified segments are shown in Figure S13. To amplify the regions for sequencing, the target DNA fragments were amplified by PCR (TaKaRa EpiTaq HS; TAKARA). The PCR fragment was purified by precipitation with PEG 8000. The concentration of the DNA fragments was quantified by using a fluorescence dye and a fluorometer (Qubit dsDNA BR assay kit; Thermo Fisher Scientific). The amplified DNA fragments (100 ng) were sheared to the average length of 250 bp by using an ultrasonicator (Covaris focused-ultrasonicator S220; Covaris) and purified by using magnetic beads (Agencourt AMPure XP; Beckman Coulter). Adaptor and index primers were added to the sheared DNA fragments using a next-generation sequencing library preparation kit (NEBNext Ultra DNA Library Prep Kit for Illumina; NEB) according to the manufacturer's instructions. PCR enrichment was carried out with nine cycles. The index-ligated fragments were mixed to construct a sequencing library and sequenced using the MiSeq system and its reagent kit (MiSeq Reagent Kits v2 (500 cycles); Illumina) according to the manufacturer's instructions. Sequencing was carried out with 250 cycles and a pairedend mode.

Determination of the parameters for mapping of the sequenced reads
Sequenced reads were first trimmed using a trimming tool, Trimmomatic (v0.33). 39 To trim the reads, the following parameters were used: accepted mismatches, 2; palindrome clip threshold, 30; simple clip threshold, 10; leading, 20; trailing, 20; window size, 4; average quality, 15; minimal length, 30. The trimmed reads were mapped to the sequence of the 5 0 half of rrnI as a reference by using mapping software, Bismark (v0.20.0). 40 The version of Bowtie2, the software required to run Bismark, was v2.2.6. 41 In order to map as many reads as possible, we determined parameters for the minimum alignment function of Bismark (scoremin option). The option consists of three parameters: (a) a function type, (b) a constant term, X, and (c) a coefficient, Y. We used a linear equation (L) for the function type and ran Bismark with score-min L, -X, and -Y, where X and Y were changed from 0 to 150 and 0 to 4, respectively. While Y was being changed, X was fixed as 0. While X was being changed, Y was fixed as 2. The mapping efficiencies were plotted against the increasing values of X or Y ( Figure S14). The values of X and Y required to map as many reads as possible were determined as 100 and 2, respectively.

Quality control of the mapped reads
Mapped reads below MAPQ<= one were removed using SAMtools (v1.9), 42 and reads with insertions and deletions were removed using an in-house Perl script pid3.pl.

Calculation of the CT conversion rates
Depths of the coverage for each nucleotide position in the mapped reads were counted for four bases (A, T, G, C) by using SAMtools. CT conversion rates were calculated for each cytosine on both the top and bottom strands according to the following equations: CT conversion rates for top strand (%) = (Counts for T)/ (Counts for C + Counts for T) 3 100; CT conversion rates for bottom strand (%) = (Counts for A)/(Counts for G + Counts for A) 3 100.
Pull-down assay for Smc-ScpAB binding to DNA Cells were cultivated overnight in L medium containing tetracycline (10 mg/mL) with shaking at 37 C. The overnight culture was diluted to 100 mL of fresh L medium containing tetracycline until OD 600 of 0.005. In half an hour, IPTG was added to the culture (a final concentration of 0.5 mM). When OD 600 reached 0.6, the culture was diluted to 600 mL of fresh L medium containing tetracycline until OD 600 of 0.05. When the OD 600 reached to 0.6, the culture was centrifuged. The supernatant was removed, and the cell pellet was frozen with liquid nitrogen and stored at À80 C until use.
Pull-down assay was performed essentially as described previously 17 and the details are described below; The frozen cell pellet was resuspended in 6 mL of buffer A: 50 mM HEPES-KOH (pH 7.6), 100 mM KCl, 1 mM PMSF, 0.33 mg/mL lysozyme, 3.3% (w/v) sucrose, 8.3 mM Tris-HCl (pH 7.5), 0.05 mg/mL RNaseA, and 2 mM MgCl 2 . After incubation at 37 C for 10 min, the cell suspension was frozen and thawed three times using liquid nitrogen. To lyse cells, 6 mL of a cell lysis reagent (CelLytic B, Sigma-Aldrich) was added, and the solution was gently mixed and then incubated on ice for 10 min. The cell lysate was centrifuged at 9,100 3 g for 10 min at 4 C. The supernatant was mixed with 37.5 mL of magnetic beads coated by Protein G (Dynabeads, Thermo Fisher Scientific) to eliminate organic matter that nonspecifically binds to the magnetic beads. The Dynabeads magnetic beads had been previously equilibrated with buffer B (50 mM ll OPEN ACCESS iScience Article