The Segregation of Bacterial Chromosomes to Daughter Cel

大肠杆菌有一个单一的圆形染色体，在细胞分裂过程中被复制和精确地分离到子细胞。复制从单一起点双向进行，并在染色体的另一侧终止。该系统的相对简单性及其繁殖所需的细胞成分数量有限，使其成为DNA复制和分离的典型系统。我们开发了一个P1 parS GFP-ParB系统，用于在荧光显微镜下定位大肠杆菌染色体上任何所需的位点。这项技术在活细胞中工作得很好，并允许我们通过延时显微镜跟踪几代染色体序列的命运。此外，我们还将该技术与流式细胞术结合使用，以确定细胞群体中细胞周期中特定点上给定位点的空间分布。我们目前有数据表明，在细胞以一个简单的细胞周期以中等生长速率生长时，分布在染色体周围的多位点的动态行为。我们的主要结论如下：1。染色体分离主要是在复制还在进行的时候完成的。终端在细胞中心复制，所有细胞的子终端都附着在那里，直到细胞分裂，这时它们随着细胞分裂而迅速分离。当细胞接近分裂时，终端病灶位于真正的细胞中心：病灶与FtsZ环的一段共定位。3. 细胞中心末端的捕获与xerCD位点特异性重组系统无关。它似乎也独立于FtsK的c端结构域，FtsK是一种涉及细胞中心DNA处理的蛋白质，尽管它在许多FtsK突变细胞中由于异常或缺失的细胞分裂事件而被破坏。4. 一个160kb的终端域。，集中在不同的重组位点，在细胞分裂时作为一个单位分离。这个区域两侧的位置在细胞分裂之前分离。5. 复制的起源在细胞周期中相当早地分离。在起源、起始和分离之间平均有1/4代的延迟。然而，细胞之间的延迟差异很大，有些起源在开始后立即分离。6. 起源从细胞中心向两极分离，在连接到新的细胞中心之前可以自由移动一段时间。子起源有时在最初的分离后重新结合，然后再次分离。起源内聚虽然经常发生，但并不是细胞周期的必要或不变特征。7. 染色体周围处于末端区域和起源区域之间的中间位置在细胞分裂前分离，有时大致对应于它们的图谱位置。有些位置的平均分离时间比按复制顺序计时的标记有序分离所预测的要早。这些可能是由隔离机器以某种特殊方式处理的区域。今年，我们特别强调改进我们对大肠杆菌染色体分离的分析。与弗莱明·汉森实验室合作。我们改进了用于DNA序列定位的GFP-ParB标记方法，并开发了一种用于细胞检测和荧光焦点测量的自动化方法。我们的结果清楚地表明，Bates和Kleckner最近发表的模型（Cell, 121, 899-911）认为姐妹染色体凝聚并作为一个单位分离是不正确的。相反，大多数染色体在复制过程中是平稳分离的。细菌中染色体分离的总体机制明显不同于高等生物的有丝分裂。

The bacterium Escherichia coli has a single, circular chromosome that is replicated and segregated with great precision to daughter cells during cell division. Replication proceeds bi-directionally from a single origin and terminates on the opposite side of the chromosome. The relative simplicity of this system and the limited number of cell components required for its propagation make it a model system for DNA replication and segregation in general. We have developed a P1 parS GFP-ParB system for localization by fluorescent microscopy of any desired locus on the E. coli chromosome. The technique works well in living cells and allows us to follow the fate of chromosomal sequences through several generations by time-lapse microscopy. In addition, we have used the technique, in combination with flow cytometry, to determine the spatial distributions of given loci at defined points in the cell cycle in a cell population. We currently have data for the dynamic behavior of multipleloci distributed around the chromosome in cells growing with a simple cell cycle at moderate growth rates. Our principle conclusions are as follows:1. Chromosome segregation is primarily accomplished while replication is still ongoing.2. The terminus is replicated at the cell center and the daughter termini of all cells remain attached there until cell division, at which time they rapidly segregate away from each other as the cell divides. The terminal foci are at the true cell center as the cell approaches division : the foci co-localize with a segment of the FtsZ ring. 3. The capture of the termini at the cell center is independent of the xerCD site-specific recombination system. It also appears to be independent of the C-terminal domain of FtsK, a protein implicated in DNA hadling at the cell center, although it is disrupted in many of the ftsK mutant cells due to aberrant or absent cell division events. 4. A terminus domain of 160kb., centered on the dif recombination site, segregates as a unit at cell division. Positions flanking this region segregate prior to cell division. 5. Origins of replication segregate fairly early in the cell cycle. On average, there is a delay of about 1/4 of a generation between origin initiation and segregation. However, the delay varies widely from cell to cell, with some origins segregating immediately after initiation. 6. Origins segregate from the cell center toward the poles and are free to move about for some time before becoming attached to the new cell centers. Daughter origins sometimes re-associate after initial segregation and dissociate again. Cohesion of origins, although it often occurs, is not a necessary or invariant feature of the cell cycle. 7. Positions around the chromosome that are intermediate between the terminal domain and the origin segregate before cell division, at times roughly corresponding to their map positions. There are some positions whose average segregation time appears earlier that predicted by an orderly segregation of markers timed by replication order. These may be regions that are handled in some special way by the segregation machinery. This year, we have put special emphasis on refining our analysis of E. coli chromosome segregation. In collaboration with the laboratory of Flemming Hansen., the technical University of Denmark, we have improved the GFP-ParB labelling method for the localization of DNA sequences, and have developed an automated method for the detection of cells and the measurement of the fluorescent foci. Our results clearly show that the recently published model of Bates and Kleckner (Cell, 121, 899-911) in which sister chromosomes cohere and are segregated as a unit, is incorrect. Rather, most of the chromosome is segregated smoothly as it is replicated.The overall mechanism for chromosome segregation in bacteria is clearly quite distinct from that of mitosis in higher organisms.