The Segregation of Bacterial Chromosomes to Daughter Cel

细菌染色体向子细胞的分离

• 批准号: 6951348
• 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/6951348
• 关键词: DNA binding protein; DNA replication; DNA replication origin; Escherichia coli; bacterial DNA; bacterial genetics; bacterial proteins; cell cycle; chromosome movement; fluorescence microscopy; microorganism culture; protein localization

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 reletive 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. The overall mechanism for chromosome segregation in bacteria is clearly quite distinct from that of mitosis in higher organisms. However, there may be a strong resemblance between it and the mechanism that sgregates the newly replicated DNA in eucaryotes into distinct sister chromatids.

大肠杆菌有一个单一的环状染色体，在细胞分裂过程中，它被非常精确地复制和分离到子细胞。复制从一个起点开始双向进行，并终止于染色体的另一侧。该系统的简单性和其繁殖所需的有限数量的细胞组分使其成为一般DNA复制和分离的模型系统。 我们已经开发了一个P1 parS GFP-ParB系统，用于通过荧光显微镜定位E. coli染色体。该技术在活细胞中工作良好，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，我们使用的技术，结合流式细胞术，以确定在细胞周期中的细胞群体中的定义点的给定位点的空间分布。我们目前有数据的动态行为multipleloci分布在染色体周围的细胞生长与一个简单的细胞周期在中等的增长率。我们的主要结论如下： 1.染色体分离主要在复制仍在进行时完成。 2.末端在细胞中心复制，所有细胞的子末端保持附着在那里，直到细胞分裂，此时它们随着细胞分裂而迅速彼此分离。当细胞接近分裂时，终末病灶位于真正的细胞中心：病灶与FtsZ环的一段共定位。 3.在细胞中心的末端的捕获独立于xerCD位点特异性重组系统。它似乎也是独立的FtsK的C-末端结构域，涉及在细胞中心的DNA hadling的蛋白质，虽然它在许多ftsK突变细胞由于异常或缺乏细胞分裂事件被破坏。 4. 160 kb的末端结构域，以DIF重组位点为中心，在细胞分裂时作为一个单位分离。该区域侧翼的位置在细胞分裂之前分离。 5.复制的起源在细胞周期的相当早期分离。平均而言，在起源起始和分离之间存在约1/4代的延迟。然而，延迟在细胞与细胞之间变化很大，一些起源在启动后立即分离。 6.起源从细胞中心向两极分离，并在连接到新的细胞中心之前自由移动一段时间。女儿的起源有时会在最初的分离后重新结合并再次分离。起源的内聚虽然经常发生，但不是细胞周期的必要或不变的特征。 7.染色体周围位于末端结构域和原点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定时的标记有序分离所预测的要早。这些区域可能是由隔离机器以某种特殊方式处理的区域。 细菌染色体分离的总体机制显然与高等生物体中的有丝分裂完全不同。然而，它可能与真核生物中新复制的DNA分离成不同的姐妹染色单体的机制有很大的相似之处。