Study of the mechanism of bacterial chromosome partitioning systems

细菌染色体分配系统机制研究

• 批准号: 10250240
• 负责人: KIYOSHI MIZUUCHI
• 金额: $ 148.48万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 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/10250240
• 关键词: ATP phosphohydrolase; Anti-Bacterial Agents; Bacteria; Bacterial Chromosomes; Behavior; Binding; Binding Proteins; Biochemical; Biophysical Process; Carpet; Cell division; Cell-Free System; Cells; Centromere; Chromosomes; Complex; Coupling; Cyanobacterium; DNA; DNA Binding; DNA Sequence; DNA biosynthesis; Daughter; Development; Diffusion; Dissociation; Ensure; Escherichia coli; Event; F Factor; Family; Fibrinogen; Filament; Fluorescence; Foundations; Generations; Genetic; Genome; Glass; Glycocalyx; Goals; Immobilization; Individual; Inherited; Kinetics; Knowledge; Label; Length; Modeling; Molecular; Molecular Conformation; Monitor; Motion; Organelles; Plasmids; Polymers; Process; Protein Dynamics; Proteins; Reaction; Set protein; Slide; Streptococcus; Surface; System; Techniques; Time; Transport Reaction; Transportation; biophysical techniques; daughter cell; fluorescence microscope; imaging study; in vivo; in vivo imaging; instrument; mathematical methods; novel; plasmid DNA; protein distribution; reconstitution; segregation

After DNA replication, daughter copies of the bacterial chromosome and low copy number plasmids must be segregated into two daughter cells to ensure genetic inheritance. Therefore, systems have evolved to actively partition the replicated copies of the genome to two halves of the cell before cell division takes place. One commonly found class of such systems involve three components; a specific DNA sequence on the segregating chromosome that functions as the bacterial equivalent of a centromere, and two protein factors, one binds to the centromere and the other an ATPase with ATP-dependent non-specific DNA binding activity. E. coli P1-plasmid and F-plasmid are both equipped with such systems. The centromere of P1-plasmid is called parS, to which ParB protein binds, and ParA is the partition ATPase. The centromere of F-plasmid is called sopC, to which SopB protein binds, and SopA is the partition ATPase. Analogous systems have been found to be involved not only in the chromosomal DNA segregation in a variety of bacterial species, but also in the segregation of large proteinous organelles in bacteria, such as carboxysomes in cyanobacteria. In vivo imaging studies on some of these systems have demonstrated oscillating focus formation of the ATPase protein and accompanied oscillation of the plasmid DNA within the cell prior to DNA replication. After replication, one DNA copy stays near one end of the cell and the other copy moves toward the other end prior to cell division. However, the detailed molecular mechanism of these bio-molecular transport reaction systems is still poorly understood. This project aims to investigate the biochemical and biophysical mechanism of the dynamic aspects of these reaction systems by combining a variety of techniques, including a reconstituted cell-free reaction systems we have established that recapitulates aspects of the in vivo system dynamics. Techniques and instruments have been developed to study these dynamic reaction systems by using a sensitive fluorescence microscope system. By using fluorescence-labeled ParA and ParB proteins, or SopA and SopB proteins, association/dissociation dynamics of these proteins with DNA molecules densely immobilized on a slide glass surface (DNA-carpet) were monitored under a variety of reaction conditions. We learned that ParA, or SopA, in the presence of ATP, associates with non-specific DNA with rapid on- and off-rates. A pre-steady state kinetic analysis of the ParA ATPase reaction and the ATP-induced conformational change of ParA have also been studied. The ParA conformational change necessary for DNA binding has been observed to take place with a time delay following ATP binding, leading to a mechanistic model of plasmid DNA motion. We have successfully reconstituted cell-free systems to observe ATP-driven dynamic behaviors of the fluorescence-labeled plasmid DNA carrying parS, or sopC, in the presence of ParA and ParB proteins, or SopA and SopB proteins, within a flow cell coated by DNA-carpet. This study led us to propose a new class of mechanistic model for bio-cargo transport systems we call diffusion-ratchet mechanism. This model comprises a reaction-diffusion process that generates a local protein distribution gradient and a chemophoretic principle of motive force generation that converts the protein distribution gradient on the DNA-carpet, or bacterial nucleoid in vivo, to the motive force via a mechano-coupling mechanism. Further mechanistic details of the ATP-driven plasmid DNA dynamics are currently studied combining biochemical, biophysical and mathematical approaches. Our efforts are currently focused on the characterization of ParA-ParB (SopA-SopB) complexes that form in the presence of both non-specific and parS (sopC) DNA. We have also extended this project to include Streptococcus plasmid pSM19035 partition system, which belongs to a separate class within the same ParABS family of partition systems. This study is in part aimed at advancement of our general knowledge on how a set of protein molecules could orchestrate a spatial control of cellular events that occur with a much larger length-scale than the individual protein molecules involved, without assembling polymeric protein filaments that spans the distance.

在DNA复制后，细菌染色体的子拷贝和低拷贝数质粒必须分离到两个子细胞中以确保遗传遗传。因此，系统已经进化到在细胞分裂发生之前主动地将基因组的复制拷贝分配到细胞的两半。一种常见的此类系统包括三种成分：分离染色体上的特异性DNA序列，其功能相当于细菌的着丝粒，以及两种蛋白质因子，一种与着丝粒结合，另一种是具有ATP依赖性非特异性DNA结合活性的ATP酶。E.大肠杆菌P1质粒和F质粒都配备了这样的系统。P1质粒的着丝粒称为parS，ParB蛋白与之结合，而帕拉是分区ATP酶。F质粒的着丝粒称为sopC，SopB蛋白与之结合，SopA是分配ATP酶。已经发现类似系统不仅参与多种细菌物种中的染色体DNA分离，而且还参与细菌中的大蛋白质细胞器（例如蓝藻中的羧基体）的分离。 对这些系统中的一些系统的体内成像研究已经证明了ATP酶蛋白的振荡焦点形成和伴随的DNA复制前细胞内质粒DNA的振荡。复制后，一个DNA拷贝留在细胞的一端附近，另一个拷贝在细胞分裂前向另一端移动。然而，这些生物分子运输反应系统的详细分子机制仍然知之甚少。该项目旨在通过结合各种技术，包括我们建立的重现体内系统动力学方面的重建无细胞反应系统，研究这些反应系统动态方面的生物化学和生物物理机制。 通过使用灵敏的荧光显微镜系统，已经开发了研究这些动态反应体系的技术和仪器。通过使用荧光标记的帕拉和ParB蛋白，或SopA和SopB蛋白，在各种反应条件下监测这些蛋白与密集固定在载玻片表面（DNA地毯）上的DNA分子的缔合/解离动力学。我们了解到，帕拉，或SopA，在ATP存在的情况下，与非特异性DNA以快速的结合和解离速率结合。对帕拉ATP酶反应的预稳态动力学分析和ATP诱导的帕拉构象变化也进行了研究。已观察到DNA结合所需的帕拉构象变化在ATP结合后延迟一段时间发生，导致质粒DNA运动的机械模型。我们已经成功地重建无细胞系统，观察ATP驱动的动力学行为的荧光标记的质粒DNA携带parS，或sopC，在帕拉和ParB蛋白质，或SopA和SopB蛋白质的存在下，在一个流动池内由DNA地毯包被。这项研究使我们提出了一类新的机制模型的生物货物运输系统，我们称之为扩散棘轮机制。该模型包括产生局部蛋白质分布梯度的反应扩散过程和动力产生的化学泳原理，该动力产生通过机械耦合机制将DNA地毯或体内细菌类核上的蛋白质分布梯度转化为动力。目前结合生物化学、生物物理和数学方法研究ATP驱动的质粒DNA动力学的进一步机制细节。我们的努力目前集中在ParA-ParB（SopA-SopB）复合物的特性，在非特异性和parS（sopC）DNA的存在下形成。我们还将该项目扩展到包括链球菌质粒pSM 19035分配系统，其属于同一ParABS分配系统家族中的单独类别。 这项研究的部分目的是为了提高我们的一般知识，即一组蛋白质分子如何能够协调对细胞事件的空间控制，这些事件发生的长度比所涉及的单个蛋白质分子大得多，而不组装跨越距离的聚合蛋白质丝。