The Segregation of Bacterial Chromosomes to Daughter Cel

噬菌体P1的质粒前噬菌体在每个分裂细胞中只有两个拷贝。它们通过一种类似于有丝分裂的精确机制分配给子细胞。在过去的二十年里，我们一直在研究这个系统作为染色体复制和分离的模型。P1分化机制由一个2.1 kb的区域和一个下游的顺式作用位点parS组成，该区域有两个编码parA和parB基因的操纵子。P1 ParA和ParB是由多种质粒和宿主染色体编码的蛋白质对家族中研究最多的成员。它们在一些细菌种类中负责染色体分离。ParB特异性地与parS结合，形成蛋白质- dna复合物。ParA是一种atp酶，在分割期间与核心复合体相互作用。今年，我们通过显微镜观察活细胞中质粒DNA在细胞周期中的位置和运动，在理解这一过程方面取得了相当大的进展。这是通过GFP-ParB杂交蛋白的特性实现的，该蛋白在parS位点以多个拷贝的形式加载到DNA上，并在细胞内产生明亮的荧光焦点。得到的结果令人惊讶。质粒的两个或多个拷贝聚集在细胞中心作为一个焦点，在那里它们附着在一些结构上：可能是细胞分裂装置。分裂包括细胞分裂时这些拷贝向外向细胞两极的爆炸性扩散。由于拷贝总是向两个方向喷射，细胞分裂总是产生两个子细胞，这两个子细胞都包含至少一个质粒拷贝。对有分裂缺陷的突变质粒的初步研究表明，它们不能附着在中心结构上，或者在一个例子中，附着发生了，但没有爆炸性的扩散。质粒分裂是一个类似于染色体分离的过程，ParA和ParB蛋白的细菌类似物的存在表明这两个过程之间存在机制上的相似之处。因此，我们继续努力阐明染色体分离的过程，应该从我们关于P1质粒分裂的新发现中受益匪浅。此外，我们已经表明，上述DNA位点的相同荧光标记可以适应于跟踪染色体复制和分离的动态。

The plasmid prophage of bacteriophage P1 can have as few as two copies per dividing cell. These are distributed to daughter cells by a precise mechanism, analogous to mitosis. We have been studying this system as a model for chromosome replication and segregation for the past twenty years. The P1 partition mechanism consists of a 2.1-kb region with and operon encoding parA and parB genes and a down-stream cis-acting site, parS. P1 ParA and ParB are the most studied members of a family of protein pairs encoded by a diverse group of plasmids and host chromosomes. They are responsible for chromosome segregation in several bacterial species. ParB binds specifically to parS, forming a protein-DNA complex. ParA is an ATPase that interacts with the core complex during partition. This year, we have made considerable progress in understanding the process by following the position and movement of the plasmid DNA during the cell cycle by microscopy in living cells. This is made possible by the properties of a GFP-ParB hybrid protein which loads onto the DNA as mutiple copies at the parS site, and produces a bright fluorescent focus within the cell. The results obtained are surprising. Two or more copies of the plasmid gather as a single focus at the cell center where they attached to some structure: probably the cell division apparatus. Partition consists of an explosive dispersal of these copies outward toward the cell poles that occurs just as the cell is dividing. As copies are always ejected in both directions cell division always results in two daughter cells, both of which contain at least one plasmid copy. Preliminary studies on mutant plasmids that are defective for partition suggest that they are unable to attach to the central structure, or in one example, that attachment occurs, but there is no explosive dispersal. Plasmid partition is an analogous process to chromosome segregation, and the existence of bacterial analogs of the ParA and ParB proteins suggests that there are mechanistic parallels between the two processes. Our continuing efforts to illuminate the process of chromosome segregation should therefore benefit greatly from our new findings concerning P1 plasmid partition. In addition, we have shown that the same fuorescence labeling of DNA sites described above can be adapted to following the dynamics of chromosome replication and segregation. The bacterial chromosome replicates from a unique origin and progresses bi-directionally, ending at a terminus region,approximately halfway around the circular chromosome. We have previously contributed to the finding that the replication forks are anchored to the cell center. Newly replicated DNA emerges from this central replication "factory" while the template chromosome is progressively drawn into it. Two very different models for how the sister chromosomes subsequently segregate have recently received support in the literature. In one, DNA replication drives segregation. The sequences destined for each sister chromosome are extruded away from the factory site in opposite directions, eventually forming two substantially separate masses in each cell half prior to cell division. The individual markers on the chromosome segregate away from their sisters progressively in the order in which they are replicated. In the second, the sister chromosome regions that emerge from the factory are paired, forming paired sister chromosomes that are analogous to sister chromatids in higher organisms. Late in the cell cycle, the sisters segregate away from each other as complete units by a process akin to mitosis. Using P1 parS sites integrated in the chromosome, and their cognate fluorescently labeled GFP-ParB binding protein, we have been able to follow the segregation of the origin and terminus sequences in living cells. The cell cycle was accurately monitored in the same culture using flow cytometry. We found that the origin sequence segregated into two separate sister copies soon after it was replicated. In contrast, the terminus sequences segregate much later, just before the cell divides. This observation provides strong support for progressive segregation during replication, and is not consistent with sister chromosome pairing models.