The SMC condensin complex is required for origin segregation in Bacillus subtilis

Abstract

SMC condensin complexes play a central role in organizing and compacting chromosomes in all domains of life [1, 2]. In the bacterium Bacillus subtilis, cells lacking SMC are viable only during slow growth and display decondensed chromosomes, suggesting that SMC complexes function throughout the genome [3, 4]. Here, we show that rapid inactivation of SMC or its partner protein ScpB during fast growth leads to a failure to resolve newly replicated origins and a complete block to chromosome segregation. Importantly, the loss of origin segregation is not due to an inability to unlink precatenated sister chromosomes by Topoisomerase IV. In support of the idea that ParB-mediated recruitment of SMC complexes to the origin is important for their segregation, cells with reduced levels of SMC that lack ParB are severely impaired in origin resolution. Finally, we demonstrate that origin segregation is a task shared by the condensin complex and the parABS partitioning system. We propose that origin-localized SMC constrains adjacent DNA segments along their lengths, drawing replicated origins in on themselves and away from each other. This SMC-mediated lengthwise condensation, bolstered by the parABS system, drives origin segregation.

Figure 1. SMC complexes are required for origin segregation

(A) Heterogeneous nucleoid morphologies and cell sizes in the SMC null mutant grown under permissive conditions. Representative images of Δsmc (strain BWX2208) grown at 22°C in LB, casein hydrolysates (CH), minimal medium (S750) supplemented with glucose or sorbitol. The nucleoids (red) and the origins (green) were visualized with HbsU-GFP and TetR-mCherry bound to a tetO array inserted adjacent to the origin. (B) Spot-dilutions of indicated temperature-sensitive mutants grown on LB-agar plates at permissive (30°C) and restrictive (42°C) temperatures. Representative images of DAPI-stained nucleoids (red) and origin foci (green) in wild-type cells (BWX811) grown in CH medium at 37°C (C); smc^ts (BWX2090) grown in CH medium at 30°C (D) and after shift to 42°C (E–G); scpB^ts (BWX2092) grown in CH medium for 1.5 h at 42°C (H); smc-ssrA (BWX1497) 1.5 h after induction of SspB grown in CH medium at 37°C (I). 85% (n=1273) of the smc^ts cells; 81% (n=1336) of the scpB^ts cells; and 85% (n=1392) of smc-ssrA cells had a single origin focus or cluster of foci at hour 1.5. Membranes (false-colored blue) were stained with FM4-64. Yellow carets highlight septum formation on top of the nucleoid. (J) Origin (green), replisome foci (red, DnaX-YFP), and DAPI-stained nucleoids (blue) in the smc-ssrA strain (BWX1771) 1.5 h after induction of SspB in CH medium at 37°C. Scale bars are 4 μm. Strains harboring wild-type copies of smc, scpB, or parC with a linked antibiotic resistance gene displayed normal chromosome organization and segregation when grown at 42°C (Figure S1F). See also Figure S1.

Figure 2. ParB-mediated recruitment of SMC promotes efficient chromosome segregation

(A) Bulk chromosome segregation is blocked upon SMC inactivation. Representative images of DAPI-stained nucleoids (blue) and indicated chromosomal loci (green and red) in the smc^ts mutant (BWX2378, BWX2116, BWX2110) grown in CH medium for 1.5 h after shifting to 42°C. Schematic representations of the loci analyzed and their subcellular locations are shown above the micrographs. 72–76% (n>1320 per strain) had nucleoids and foci similar to those shown in (A). (B) Origin segregation is impaired in ParB mutants when SMC levels are reduced. Representative micrographs of DAPI-stained nucleoids (false-colored red) and origins (green) in cells (BWX1497, left panel) harboring an smc-ssrA fusion that results in a 2.5-fold reduction in SMC levels. The cells in the middle panel (BWX2551) harbor an in-frame deletion of parA and those in the right panel (BWX1569) lack parB. 2% (n=1165) of smc-ssrA cells; 8% (n=1038) of ΔparA, smc-ssrA cells; and 34% (n=1214) of ΔparB, smc-ssrA cells had a single bright origin focus. 5%, 13%, and 80%, respectively had an unsegregated nucleoid. Representative images of ParA and ParB mutants that have wild-type levels of SMC and quantitative analysis of nucleoid size in all strains can be found in Figure S2D. Scale bars are 4 μm. (C) Immunoblot analysis of SMC, ScpB, and a loading control (σ^A) in wild-type and the smc-ssrA mutant under conditions in which the adaptor protein is not induced. See also Figure S2.

Figure 3. Topoisomerase IV is essential for bulk chromosome segregation but is not necessary to resolve replicated origins

(A) Representative images of DAPI-stained nucleoids (blue) and indicated chromosomal loci (green and red) in a parC^ts mutant (BWX2112 and BWX2106) grown in CH medium for 1.5 h after shifting to 42°C. Clusters of green and red foci reflect a failure to segregate the replicated chromosomes. 70–75% (n>1190 per strain) had nucleoids and foci similar to those shown in (A). (B) Representative images of nucleoids (red) and origin loci (green) in a parC^ts mutant (BWX2082) grown at 30°C and after shift to 42°C. (C) The same time course as in (B) with a parC^ts mutant that also lacks ParA (BWX2574). Origin foci are more often clustered in the absence of ParA although this is not as pronounced as upon SMC inactivation (Figure 1). 70% (n=1143) of parC^ts and 19% (n=1238) of ΔparA, parC^ts had nucleoids with 3 or more well-resolved foci or clusters of foci at hour 1.5. Scale bars are 4 μm.

Figure 4. The parABS partitioning system and the SMC complex contribute to origin segregation

(A) Chromosome resolution and segregation occur in the absence of SMC if new rounds of replication are blocked. Representative micrographs of DAPI-stained nucleoids (false-colored red) and origin foci (green) in a strain (BWX1527) harboring the smc-ssrA degradable allele and a temperature-sensitive replication initiation mutant (dnaB^ts). After induction of SMC-SsrA degradation for 1 h most cells had unsegregated nucleoids with unresolved origin foci. Inhibition of replication leads to resolution and segregation of the chromosomes if the parABS system is intact (61%; n=1284) (A) but not if ParA is absent (strain BWX2558) (29%; n=1262) (B). (C) Cells lacking SMC and ParA grown under permissive conditions have severe defects in origin segregation. Cells with intact parABS (BWX1497) and lacking parA (BWX2551) were induced to degraded SMC-SsrA under permissive growth conditions (minimal S750 medium supplemented with sorbitol at 22°C). Representative images of nucleoids (false-colored red) and origin foci (green) are shown. 11% (n=1595) of smc-ssrA cells and 51% (n=1287) of ΔparA, smc-ssrA cells had unsegregated nucleoids. 2% and 30%, respectively had a single bright origin focus. Scale bars are 4 μm. (D) Schematic model depicting origin segregation in B. subtilis. The SMC condensin complex (green) is recruited to the origin by ParB (purple) bound to origin-proximal parS sites and is enriched at highly transcribed genes including ribosomal RNA genes (rrn) (blue) that reside adjacent to the origin [10, 11]. Compaction of contiguous DNA segments leads to individualization of the sister origins that are resolved and segregated, in part, through the action of ParA acting on ParB/parS complexes. See also Figure S3.