Chromosome segregation in B. subtilis is highly heterogeneous.

OBJECTIVE: The bacterial cell cycle comprises initiation of replication and ensuing elongation, concomitant chromosome segregation (in some organisms with a delay termed cohesion), and finally cell division. By quantifying the number of origin and terminus regions in exponentially growing Bacillus subtilis cells, and after induction of DNA damage, we aimed at determining cell cycle parameters at different growth rates at a single cell level. RESULTS: B. subtilis cells are mostly mero-oligoploid during fast growth and diploid during slow growth. However, we found that the number of replication origins and of termini is highly heterogeneous within the cell population at two different growth rates, and that even at slow growth, a majority of cells attempts to maintain more than a single chromosome at all times of the cell cycle. Heterogeneity of chromosome copy numbers may reflect different subpopulations having diverging growth rates even during exponential growth conditions. Cells continued to initiate replication and segregate chromosomes after induction of DNA damage, as judged by an increase in origin numbers per cell, showing that replication and segregation are relatively robust against cell cycle perturbation.

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

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.

Bacterial cell growth is arrested by violet and blue, but not yellow light excitation during fluorescence microscopy.

BACKGROUND: Fluorescence microscopy is a powerful tool in cell biology, especially for the study of dynamic processes. Intensive irradiation of bacteria with UV, blue and violet light has been shown to be able to kill cells, but very little information is available on the effect of blue or violet light during live-cell imaging. RESULTS: We show here that in the model bacterium Bacillus subtilis chromosome segregation and cell growth are rapidly halted by standard violet (405 nm) and blue light (CFP) (445-457 nm) excitation, whereas they are largely unaffected by green light (YFP). The stress sigma factor σB and the blue-light receptor YtvA are not involved in growth arrest. Using synchronized B. subtilis cells, we show that the use of blue light for fluorescence microscopy likely induces non-specific toxic effects, rather than a specific cell cycle arrest. Escherichia coli and Caulobacter crescentus cells also stop to grow after 15 one-second exposures to blue light (CFP), but continue growth when imaged under similar conditions in the YFP channel. In the case of E. coli, YFP excitation slows growth relative to white light excitation, whereas CFP excitation leads to cell death in a majority of cells. Thus, even mild violet/blue light excitation interferes with bacterial growth. Analyzing the dose-dependent effects of violet light in B. subtilis, we show that short exposures to low-intensity violet light allow for continued cell growth, while longer exposures do not. CONCLUSIONS: Our experiments show that care must be taken in the design of live-cell imaging experiments in that violet or blue excitation effects must be closely controlled during and after imaging. Violet excitation during sptPALM or other imaging studies involving photoactivation has a threshold, below which little effects can be seen, but above which a sharp transition into cell death occurs. YFP imaging proves to be better suited for time-lapse studies, especially when cell cycle or cell growth parameters are to be examined.

Single-Molecule Tracking of DNA Translocases in Bacillus subtilis Reveals Strikingly Different Dynamics of SftA, SpoIIIE, and FtsA.

Like many bacteria, Bacillus subtilis possesses two DNA translocases that affect chromosome segregation at different steps. Prior to septum closure, nonsegregated DNA is moved into opposite cell halves by SftA, while septum-entrapped DNA is rescued by SpoIIIE. We have used single-molecule fluorescence microscopy and tracking (SMT) experiments to describe the dynamics of the two different DNA translocases, the cell division protein FtsA and the glycolytic enzyme phosphofructokinase (PfkA), in real time. SMT revealed that about 30% of SftA molecules move through the cytosol, while a fraction of 70% is septum bound and static. In contrast, only 35% of FtsA molecules are static at midcell, while SpoIIIE molecules diffuse within the membrane and show no enrichment at the septum. Several lines of evidence suggest that FtsA plays a role in septal recruitment of SftA: an ftsA deletion results in a significant reduction in septal SftA recruitment and a decrease in the average dwell time of SftA molecules. FtsA can recruit SftA to the membrane in a heterologous eukaryotic system, suggesting that SftA may be partially recruited via FtsA. Therefore, SftA is a component of the division machinery, while SpoIIIE is not, and it is otherwise a freely diffusive cytosolic enzyme in vivo Our developed SMT script is a powerful technique to determine if low-abundance proteins are membrane bound or cytosolic, to detect differences in populations of complex-bound and unbound/diffusive proteins, and to visualize the subcellular localization of slow- and fast-moving molecules in live cells.IMPORTANCE DNA translocases couple the late events of chromosome segregation to cell division and thereby play an important role in the bacterial cell cycle. The proteins fall into one of two categories, integral membrane translocases or nonintegral translocases. We show that the membrane-bound translocase SpoIIIE moves slowly throughout the cell membrane in B. subtilis and does not show a clear association with the division septum, in agreement with the idea that it binds membrane-bound DNA, which can occur through cell division across nonsegregated chromosomes. In contrast, SftA behaves like a soluble protein and is recruited to the division septum as a component of the division machinery. We show that FtsA contributes to the recruitment of SftA, revealing a dual role of FtsA at the division machinery, but it is not the only factor that binds SftA. Our work represents a detailed in vivo study of DNA translocases at the single-molecule level.

Requirements for Septal Localization and Chromosome Segregation Activity of the DNA Translocase SftA from Bacillus subtilis.

Bacillus subtilis possesses 2 DNA translocases that affect late stages of chromosome segregation: SftA separates nonsegregated DNA prior to septum closure, while SpoIIIE rescues septum-entrapped DNA. We provide evidence that SftA is associated with the division machinery via a stretch of 47 amino acids within its N-terminus, suggesting that SftA is recruited by protein-protein interactions with a component of the division machinery. SftA was also recruited to mid-cell in the absence of its first 20 amino acids, which are proposed to contain a membrane-binding motif. Cell fractionation experiments showed that SftA can be found in the cytosolic fraction, and to a minor degree in the membrane fraction, showing that it is a soluble protein in vivo. The expression of truncated SftA constructs led to a dominant sftA deletion phenotype, even at very low induction rates of the truncated proteins, indicating that the incorporation of nonfunctional monomers into SftA hexamers abolishes functionality. Mobility shift experiments and surface plasmon binding studies showed that SftA binds to DNA in a cooperative manner, and demonstrated low ATPase activity when binding to short nucleotides rather than to long stretches of DNA.