Multiple cis-Acting rDNAs Contribute to Nucleoid Separation and Recruit the Bacterial Condensin Smc-ScpAB  Koichi Yano, Hironori Niki  Cell Reports  Volume 21, Issue 5, Pages 1347-1360 (October 2017) DOI: 10.1016/j.celrep.2017.10.014 Copyright © 2017 The Authors Terms and Conditions

Cell Reports 2017 21, 1347-1360DOI: (10.1016/j.celrep.2017.10.014) Copyright © 2017 The Authors Terms and Conditions

Figure 1 Pull-Down Assay of Plasmid DNA Loaded by Smc-ScpAB (A) A schematic for isolation of the plasmid DNA onto which Smc-ScpAB loads. (B) Recovered plasmid DNA from cell extract (YAN13683 and YAN13684) after washing with buffer (100 mM KCl). Bar charts indicate the average values from three independent experiments. (C) Recovered plasmid DNA from cell extract (YAN13683 and YAN13684) after washing with buffer (500 mM KCl). Bar charts indicate the average values from six independent experiments. (D) Recovery of various plasmids harboring deletions or insertions in the rrnI operon (YAN13683, YAN13684, YAN13904, YAN13911, YAN13913, YAN13914, YAN14632, YAN14691, and YAN13981). The schematic of the rrn operon indicates deleted DNA segments (orange) and a replaced DNA segment (blue). The bar charts for the vector and pRRN show the average values as described in (C). Other bar charts show the average values from three independent experiments. (E) Recovered plasmid DNA from the Δspo0J mutant (YAN13939 and YAN13940) and the Δsmc mutant (YAN13982 and YAN13983) after washing with buffer (500 mM KCl). Bar charts indicate the average of three independent experiments. (F) Recovered plasmid DNA harboring parS (YAN14055 and YAN14405) after washing with buffer (500 mM KCl). Bar charts indicate the average of three independent experiments. (G) Recovered plasmid DNA from the 1 rrn mutant (YAN14288 and YAN14289) after washing with buffer (500 mM KCl). Bar charts indicate the average of five independent experiments. (B–G) Diamonds indicate the measured values. Diamonds with the same colors between vector and pRRN indicate that the measured values are derived from a pair of experiments. Error bars indicate SE. Calculations of the amounts of retrieved plasmid DNA are shown in Tables S4A–S4F. Cell Reports 2017 21, 1347-1360DOI: (10.1016/j.celrep.2017.10.014) Copyright © 2017 The Authors Terms and Conditions

Figure 2 Analysis of Nucleoid Separation in rrn Deletion Mutants (A) Images of wild-type (168), 2 rrn mutant (RIK1754), and 1 rrn mutant (RIK539) cells. (B) Images of cells of the wild-type and the smc deletion mutant (YAN12061). (C) A series of images of a growing wild-type cell. According to separation of nucleoids, each image was extracted from images of cells in unsynchronized culture. (A–C) Phase contrast images are merged into fluorescent images of DAPI staining (pseudocolor in magenta). Scale bars indicate 1 μm. (D) Histogram of nucleoid sizes of wild-type cells and Gaussian distribution by curve fitting. (E–H) Histograms of normalized nucleoid sizes for (E) the wild-type, (F) the 2 rrn mutants (RIK1754, RIK2226, and RIK2237), (G) the 1 rrn mutant (RIK539), and (H) the smc deletion mutant (YAN12061). The number of samples (n) is indicated in each histogram. Cell Reports 2017 21, 1347-1360DOI: (10.1016/j.celrep.2017.10.014) Copyright © 2017 The Authors Terms and Conditions

Figure 3 The cis-trans Test for Defects in Nucleoid Separation of the 1 rrn Mutant (A) Images of growing cells: wild-type cells harboring the vector pGETS118-t0-Pr-pBR322 (YAN12643) or pRRN (YAN12644) and 1 rrn mutant cells harboring the vector pGETS118-t0-Pr-pBR322 (YAN12641) or pRRN (YAN12642). Phase contrast images are merged with fluorescent images of DAPI staining (pseudocolor in magenta). The scale bar indicates 1 μm. (B) Histograms of normalized nucleoid sizes of wild-type (YAN12643, YAN12644) and 1 rrn mutant cells (YAN12641 and YAN12642). The number of samples is indicated in each histogram. (C) A schematic of the cis-trans test for the defect in nucleoid separation. The genotype indicates rrn operons in the strains, and the phenotype indicates complementation of the defect in nucleoid separation: Figure 2F and (B). Cell Reports 2017 21, 1347-1360DOI: (10.1016/j.celrep.2017.10.014) Copyright © 2017 The Authors Terms and Conditions

Figure 4 Translocation of the Second rrn Operon in the 2 rrn Mutant (A) Positions of the rrn operon on the B. subtilis chromosomal map. The position of each rrn operon is represented in degrees, where oriC is assigned a position of 0°. Native rrn operons are indicated as short bars on the map. rrn operons in the 2 rrn mutants (Figure 2F) are indicated in bold. Translocations of the rrnI operon are indicated as triangles. (B) Histograms of normalized nucleoid sizes of a series of 2 rrn mutants. The position of the second rrn operon is shown as a schematic of the B. subtilis chromosome. The bars represent the rrnA and the triangles represent the second rrn operon. The colors of bars and triangles indicate whether the second rrn located at the position can suppress the defect in nucleoid separation with rrnA; a positive effect is indicated in blue and a negative effect in orange. (C) Histograms of normalized nucleoid sizes of the 2 rrn mutants harboring the vector (YAN12672) or pRRN (YAN12673). The position of the rrn operon is shown as a schematic of the B. subtilis chromosome and plasmids. The bar and triangles represent rrn operons. Cell Reports 2017 21, 1347-1360DOI: (10.1016/j.celrep.2017.10.014) Copyright © 2017 The Authors Terms and Conditions

Figure 5 Translocation of rrn Operons at the Replication Terminus Region and Dissection of the Structure of the Second rrn Operon at the Native Location in the 2 rrn Mutant (A) A schematic indicating the position of two rrnI operons at the replication terminus region of the B. subtilis chromosome. Phase contrast images are merged into fluorescent images of DAPI staining (pseudocolor in magenta). The bar indicates 1 μm. Histograms of normalized nucleoid sizes of the 2 rrn@171°&175° mutant are shown (YAN14396). (B) A schematic of deletions or an insertion in the rrnI operon of the 2 rrn mutants. Deleted DNA segments (orange) and an inserted DNA segment (blue) are indicated. (C) Histograms of normalized nucleoid sizes of the 2 rrn mutants in which the rrnI operons at the native position are deleted or replaced by the lac genes of E. coli. Cell Reports 2017 21, 1347-1360DOI: (10.1016/j.celrep.2017.10.014) Copyright © 2017 The Authors Terms and Conditions

Figure 6 Analysis of Nucleoid Separation in the Double Deletion Mutant of spo0J and rrn Phase contrast images are merged into fluorescent images of DAPI staining (pseudocolor in magenta). (A) Images of growing cells of the spo0J mutant (YAN12106) in L medium at 37°C. The scale bar indicates 1 μm. (B) Images of growing cells of the double deletion mutant of spo0J and rrn (YAN13492) in L medium at 37°C. (C) Histogram of normalized nucleoid sizes of the spo0J mutant (YAN12106). (D) Histogram of normalized nucleoid sizes of the double mutant of the spo0J and rrn deletion (YAN13492). (E) Merged images of nucleoids (green) and oriC foci (yellow): wild-type (YAN14176), the rrn deletion mutant (YAN14595), the spo0J mutant (YAN14622), the smc mutant (YAN14625), and the double deletion mutant of spo0J rrn (YAN14606). Fluorescent images of LacI-GFP (false-colored in yellow) and Hoechst 33342-stained DNA (pseudocolor in green) were merged. The scale bars indicates 1 μm. See also Figure S4 for grayscale images for LacI-GFP and superimposed images of LacI-GFP and the membrane. Cell Reports 2017 21, 1347-1360DOI: (10.1016/j.celrep.2017.10.014) Copyright © 2017 The Authors Terms and Conditions

Figure 7 Models of rDNA-Dependent Smc-ScpAB Loading and Chromosome Compaction (A) A genetic map of parS (magenta arrowheads), rrn operons (blue arrowheads), and tRNA genes (black circles) on half of the Bacillus subtilis chromosome. rrn operons in bold are located at positions where the second rrn could suppress the defect in nucleoid separation with rrnA. Asterisks indicate rrn operons that do not contain tRNA genes. The yellow circle indicates oriC. (B) A model of loading of Smc-ScpAB at highly transcribed rrn genes. Formation of the R-loop provides stable ssDNA for loading of Smc-ScpAB. (C) Schematics of compaction of newly replicated DNA strands in spo0J, rrn deletion mutants, and double mutants of both spo0J and rrn. The yellow circle indicates oriC, black circles indicate replication forks, and magenta circles indicate rrn operons. The blue rings indicate Smc-ScpAB topologically loaded on rrn operons, and magenta rings represent Smc-ScpAB topologically loaded by ParB bound to parS, as shown in Wang et al. (2017). In addition to separation of oriC, chromosome compaction would always be accompanied by proper chromosome segregation. The chromosomal region adjacent to oriC is packed by Smc in the WT so that the nucleoids are clearly separated, but it is not packed by Smc in either 1 rrn, Δspo0J, or both, and thus the nucleoids are merged irrespective of the separation of oriC. Cell Reports 2017 21, 1347-1360DOI: (10.1016/j.celrep.2017.10.014) Copyright © 2017 The Authors Terms and Conditions