Chromosome Segregation in Bacillus subtilis Follows an Overall Pattern of Linear Movement and Is Highly Robust against Cell Cycle Perturbations

Abstract

We have followed the segregation of origin regions on the Bacillus subtilis chromosome in the fastest practically achievable temporal manner, for a large fraction of the cell cycle. We show that segregation occurred in highly variable patterns but overall in an almost linear manner throughout the cell cycle. Segregation was slowed down, but not arrested, by treatment of cells that led to transient blocks in DNA replication, showing that segregation is highly robust against cell cycle perturbation. Computer simulations based on entropy-driven separation of newly synthesized DNA polymers can recapitulate sudden bursts of movement and segregation patterns compatible with the observed in vivo patterns, indicating that for Bacillus , segregation patterns may include entropic forces helping to separate chromosomes during the cell cycle. Although several proteins have been identified that facilitate chromosome segregation in bacteria, no clear analogue of the mitotic machinery in eukaryotic cells has been identified. In order to investigate if recognizable patterns of segregation exist during the cell cycle, we tracked the segregation of duplicated origin regions in Bacillus subtilis for 60 min in the fastest practically achievable resolution, achieving 10-s intervals. We found that while separation occurred in random patterns, often including backwards movement, overall, segregation of loci near the origins of replication was linear for the entire cell cycle. Thus, the process of partitioning can be best described as directed motion. Simulations with entropy-driven separation of polymers synthesized by two polymerases show sudden bursts of movement and segregation patterns compatible with the observed in vivo patterns, showing that for Bacillus , segregation patterns can be modeled based on entropic forces. To test if obstacles for replication forks lead to an alteration of the partitioning pattern, we challenged cells with chemicals inducing DNA damage or blocking of topoisomerase activity. Both treatments led to a moderate slowing down of separation, but linear segregation was retained, showing that chromosome segregation is highly robust against cell cycle perturbation. IMPORTANCE We have followed the segregation of origin regions on the Bacillus subtilis chromosome in the fastest practically achievable temporal manner, for a large fraction of the cell cycle. We show that segregation occurred in highly variable patterns but overall in an almost linear manner throughout the cell cycle. Segregation was slowed down, but not arrested, by treatment of cells that led to transient blocks in DNA replication, showing that segregation is highly robust against cell cycle perturbation. Computer simulations based on entropy-driven separation of newly synthesized DNA polymers can recapitulate sudden bursts of movement and segregation patterns compatible with the observed in vivo patterns, indicating that for Bacillus , segregation patterns may include entropic forces helping to separate chromosomes during the cell cycle.