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

Abstract

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.

Chromosome segregation in B. subtilis is highly heterogeneous

“…These ndings show that although MMC induces events of replication arrests due to interstrand crosslinks, and leads to atypical nucleoid morphology, most MMC-treated cells were still able to complete a replication cycle, or even reinitiated another cycle, giving rise to a double set of chromosomes after induction of DNA damage. The ndings are in accordance with the report that MMC slows down the cell cycle considerably but does not stop it completely [6], and with ndings from Goranov et al showing that double strand breaks induced by MMC slow down the replication elongation beyond the origin proximal region [10].…”

Chromosome segregation in B. subtilis is highly heterogeneous

“…These ndings show that despite frequent replication arrests due to interstrand crosslinking by MMC and the atypical nucleoid morphology, many cells were either still able to complete a replication cycle and had a double set of chromosomes after induction of DNA damage, or that the cells simply retained the number of nucleoids and termini they had before damage induction. The latter is in accordance with the idea that MMC slows down the cell cycle considerably but does not stop it completely [6]. Goranov et al showed that double strand breaks induced by MMC inhibit the replication elongation beyond the origin proximal region [10].…”

“…As stated in reference [6], a small fraction of cells containing the FROS system showed irregularities in segregation. Cells were taken from exponential growth conditions and were imaged within 10 minutes, minimally in uencing the actual number of origins and termini.…”

Single molecule dynamics at a bacterial replication fork after nutritional downshift

“…We propose that this exchange is based on exchange of subunits from the hexameric helicase, which thus appear to exchange within a time frame of few seconds. We have recently shown that chromosome segregation and thus also DNA replication, which occur concomitantly, are relatively robust against DNA damageinduction via MMC or inhibition of DNA gyrase, continuing to follow a general pattern that resembles that of directed diffusion (48). Here, we show that replication forks can be seen…”

“…S3). Because chromosome segregation and thus replication continue after addition of MMC, albeit more slowly (48), likely based on frequent restart processes, it follows that DnaC molecules are more frequently exchanged during replication restart than polymerases. Unexpectedly, blocking of PolC by HPUra lead to an increase of fork-bound primase molecules (Fig.…”