Segregation of Bacterial Chromosomes to Daughter Cells

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，我们特别强调了染色体分离机制的一个组成部分，SeqA蛋白。SeqA是一种在腺嘌呤碱基半甲基化时特异性结合GATC序列的蛋白质。这种底物是在染色体位点被新复制时产生的。SeqA短暂地与新复制的DNA结合，随着复制叉的进展。我们有证据表明，SeqA结合以一种对正确的DNA分离很重要的方式组织和压缩新复制的DNA。与Wei Yang （NIDDK）和Alba Guarne （McMaster U.）合作，我们已经解决了大部分SeqA蛋白的结构，并利用结构信息来探测其功能。我们发现SeqA形成一个螺旋状的细丝，上面包裹着新复制的DNA。通过向新复制的GATC序列添加二聚体，丝从复制叉生长。在丝的另一端，二聚体随着DNA完全甲基化而消失。因此，一束DNA围绕着与SeqA丝结合的染色体前进。这种DNA组织似乎在复制过程中染色体的分离中起着核心作用。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。然而，它可能与真核生物中新复制的DNA分离成不同姐妹染色单体的机制有很强的相似性。

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. This year, we have put special emphasis on one component of the chromosome segregation machinery, the SeqA protein. SeqA is a protein that binds specifically to the sequence GATC when the adenine bases are hemi-methylated. This substrate is produced when chromosome loci are newly replicated. SeqA binds transiently to the newly replicated DNA that follows the progression of the replication forks. We have evidence that SeqA binding organizes and compacts the newly replicated DNA in a way that is important for proper DNA segregation. In collaboration with Wei Yang (NIDDK), and Alba Guarne (McMaster U.) we have solved the structure of much of the SeqA protein, and used the structural information to probe its function. We find that SeqA forms a helical filament on which the newly replicated DNA is wrapped. The filament grows from the replication fork by addition of dimers to the newly replicated GATC sequences. At the other end of the filament, dimers are lost as the DNA becomes fully methylated. Thus, a tract of the DNA progresses around the chromosome bound to a SeqA filament. This DNA organization appears to play a central role in segregation of the chromosome during replication. 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 segregates the newly replicated DNA in eucaryotes into distinct sister chromatids.