The Segregation of Bacterial Chromosomes to Daughter Cel

细菌染色体向子细胞的分离

• 批准号: 6763553
• 负责人: STUART AUSTIN
• 金额: --
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6763553
• 关键词: DNA binding protein; DNA replication; Escherichia coli; bacterial DNA; bacterial genetics; bacterial proteins; chromosome movement; microorganism culture

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.

噬菌体 P1 的质粒原噬菌体每个分裂细胞只有两个拷贝。它们通过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 分区机制由一个 2.1 kb 区域和一个下游顺式作用位点 parS 组成，其中操纵子编码 parA 和 parB 基因。 P1 ParA 和 ParB 是由不同组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。它们负责几种细菌物种的染色体分离。 ParB 与 parS 特异性结合，形成蛋白质-DNA 复合物。 ParA 是一种 ATP 酶，在分配过程中与核心复合物相互作用。今年，我们通过活细胞显微镜观察细胞周期中质粒 DNA 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 杂合蛋白的特性实现的，该蛋白在 parS 位点作为多个拷贝加载到 DNA 上，并在细胞内产生明亮的荧光焦点。获得的结果是令人惊讶的。两个或多个质粒拷贝在细胞中心聚集成一个焦点，在那里它们附着在某种结构上：可能是细胞分裂装置。分裂是在细胞分裂时，这些拷贝向外向细胞两极爆炸性扩散。由于拷贝总是在两个方向上喷射，细胞分裂总是产生两个子细胞，这两个子细胞都含有至少一个质粒拷贝。对分区有缺陷的突变质粒的初步研究表明，它们无法附着到中央结构上，或者在一个例子中，附着发生了，但没有爆炸性扩散。质粒分割是一个与染色体分离类似的过程，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性。因此，我们对阐明染色体分离过程的持续努力应该大大受益于我们关于 P1 质粒分割的新发现。此外，我们还表明，上述 DNA 位点的荧光标记可以适应染色体复制和分离的动态。 细菌染色体从一个独特的起点复制并双向前进，终止于末端区域，大约围绕圆形染色体的一半。我们之前发现复制叉锚定在细胞中心。新复制的 DNA 从这个中心复制“工厂”中产生，而模板染色体则逐渐被吸引到其中。关于姐妹染色体随后如何分离的两种截然不同的模型最近得到了文献的支持。其一，DNA 复制驱动分离。指定给每个姐妹染色体的序列以相反的方向从工厂位点挤出，最终在细胞分裂之前在每个细胞半部中形成两个基本上独立的团块。染色体上的个体标记逐渐与其姐妹标记分离。 它们的复制顺序。在第二种情况下，从工厂出来的姐妹染色体区域是配对的，形成配对的姐妹染色体，类似于姐妹染色单体 高等生物。在细胞周期后期，姐妹细胞通过类似于有丝分裂的过程作为完整的单位彼此分离。使用整合在染色体中的 P1 parS 位点及其同源荧光标记的 GFP-ParB 结合蛋白，我们已经能够跟踪活细胞中起点和末端序列的分离。在同一培养物中准确监测细胞周期 使用流式细胞术。我们发现原始序列在复制后不久就分离成两个独立的姐妹副本。相反，末端序列的分离要晚得多，就在细胞分裂之前。这一观察结果为复制过程中的渐进分离提供了强有力的支持，并且与姐妹染色体配对模型不一致。