Zusammenfassung
Accurate segregation of genetic material is essential for the survival and proliferation of all organisms, whether eukaryotic or prokaryotic. In addition to the chromosome, bacteria are known to carry extrachromosomal DNA molecules called plasmids, which can provide growth advantages in certain situations and often encode antibiotic resistances. The copy numbers of plasmids can differ greatly based on the specific plasmid, ranging from hundreds of copies per cell for some to less than a handful for others. Plasmids with high copy numbers can rely on their abundance to ensure that at least one plasmid is distributed to each daughter cell during cell division. Low-copy number plasmids, on the other hand, require specialized partitioning systems to ensure their faithful segregation and prevent their loss during cell division. The ParABS system is the most common of these partitioning systems, consisting of three key components: parS, a centromere-like sequence located near the origin of replication on the plasmid; ParA, a walker-type ATPase that binds nonspecifically to the nucleoid in its dimeric ATP-bound state; ParB, is also a protein that forms dimers and binds specifically to a parS site. Once bound to parS, ParB spreads several kilobases in both directions and together parS and ParB form a nucleoprotein complex. This complex interacts with nucleoid-bound ParA, resulting in the equidistant positioning of plasmids along the long axis of the cell. This kind of positioning ensures that both daughter cells receive an equal number of plasmids upon cell division. The primary focus of this thesis is the F-plasmid, a low-copy number plasmid that harbors a ParABS system and maintains an average of three copies per cell. The placement of this plasmid and other ParABS carrying plasmids within the cell has been a topic of debate. Some suggest that plasmids within the cell are positioned through oscillatory motion, while others propose directed motion, placing the plasmids directly at their target positions. By utilizing high-throughput data acquisition and analysis, we captured thousands of cell cycles containing F-plasmids to reveal the true dynamics of plasmid positioning of this system. To track the motion of the F-plasmid, we used ParB fused to a fluorophore, which colocalizes with the plasmid and forms bright foci that are easily trackable. Furthermore, based on a previously developed molecular-scale model, we developed a unifying model that accurately reproduces the in vivo observations not only of the F-plasmid but also of another distantly related ParABS system (pB171). Our findings, based on both experimental data and simulations, prove that the F-plasmid exhibits true regular positioning: Plasmids move precisely to their target positions, ensuring they are equidistantly spaced throughout the cell. However, our model indicates that the F-plasmid operates just above the threshold of an oscillatory instability. As predicted by our model, large cells with a single plasmid crossed this threshold and exhibited low amplitude oscillations. In contrast, cells containing a different plasmid, the pB171 plasmids, exhibited clear pole-to-pole oscillations at low plasmid concentrations, but as the concentration increased, the plasmids became regularly positioned. Despite these significant differences in plasmid locomotion, our model was able to explain these differing plasmid dynamics using a single dimensionless parameter named lambda, which describes the average distance that ParA diffuses on the nucleoid. Further, our simulations yielded an interesting result: as the system approached the oscillatory regime, its energy consumption decreased. This finding provides a possible explanation for why these systems operate in such close proximity to the oscillatory instability. One challenge we encountered during the investigation of the ParABS system was the difficulty of tracking plasmids accurately at high numbers. To address this challenge, we developed an algorithm called *Track, which tracks persistent objects like plasmids with high accuracy. We tested this algorithm on both F-plasmid and chromosomal loci, revealing interesting observations and findings in both systems.
