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. 2021 Aug 4;10:e65467. doi: 10.7554/eLife.65467

Figure 4. Increasing the cellular pool of Smc hampers chromosome organization.

(A) Immunoblotting using α-Smc (top panel) and α-ScpB serum (bottom panel). SMChigh denotes strains with extra genes for Smc-ScpAB. Protein extracts of wild-type or SMChigh strains (harboring all parS sites or single parS site) were serially diluted with extracts from Δsmc or ΔscpB strains as indicated (see Materials and methods). * indicates unspecific bands generated by the α-ScpB serum. (B) Normalized 3C-seq contact map for SMChigh strain with all parS sites present. Inset shows 3C-seq contact map of a strain with wild-type protein levels (also displayed in Figure 1C) for direct comparison. (C) Normalized 3C-seq contact maps for SMChigh strains with parSopt at −9 kb only or at −304 kb only. (D) Normalized 3C-seq contact map for SMChigh strain with parSopt at positions: −9 kb and −304 kb. As in (B), with inset displaying respective control strain with normal Smc expression levels (also shown in Figure 3B). (E) Ratio plots for ChIP-seq read counts comparing SMChigh strains with all parS sites and a single parS site (–9kbparSopt). Representation as in Figure 2B (top panel). Read count for α-ParB ChIP-seq in SMChigh strain (bottom panel). (F) As in (E) involving a SMChigh strain with two parS sites (–304kbparSopt and –9kbparSopt) instead of all parS sites. (G) Normalized 3C-seq contact maps for time point t25 after IPTG-induced ParB expression with a single parSopt site (top panel) or two parSopt sites (at −9 kb and −304 kb) (bottom panel). Ellipsoids (in yellow colors) mark the position of contacts stemming from loop extrusion originating at –9kbparSopt.

Figure 4.

Figure 4—figure supplement 1. Synchronized loading of SMC hampers chromosome organization.

Figure 4—figure supplement 1.

(A) Spotting assay comparing strains with extra Smc-ScpAB genes to wild-type strains with normal or altered parS site distribution. Prepared as described in Figure 1B. (B) RNAP inhibition experiment using rifampicin. Normalized 3C-seq contact maps for SMChigh strains with all, single –9kbparSopt, or two parSopt sites spaced by ~300 kb (mock treatment controls ‘rif-’, top panels) (rif treatment, ‘rif+’, bottom panels). (C) Ratio plots for ChIP-seq read counts comparing SMChigh strain and wild-type levels of Smc with a single parS site (–9kbparSopt). Representation as in Figure 2B (top panel). Read count for α-ParB ChIP-seq in the SMChigh and wild-type Smc strains (middle and bottom panels, respectively). (D) α-ScpB ChIP-seq read counts for SMChigh strains with all parS sites present (top panel), single parSopt at position −9 kb (middle panel), and two parS sites at positions −304 kb and −9 kb (bottom panel). Represented as in Figure 3—figure supplement 1C. (E) Normalized 3C-seq contact maps for time-course experiments for strains with a single loading site parSopt (at −9 kb, top panels) or two parSopt (at −304 kb and −9 kb, bottom panels) and IPTG-inducible ParB.