Study of the mechanism of bacterial chromosome partitioning systems

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。因此，在细胞分裂发生之前，系统已经进化到主动将基因组的复制拷贝分割成细胞的两半。一种常见的这类系统包括三个组成部分；一个在分离染色体上的特定DNA序列，其功能相当于细菌的着丝粒，以及两个蛋白质因子，一个与着丝粒结合，另一个与atp依赖的非特异性DNA结合活性的atp酶结合。大肠杆菌p1质粒和f质粒都配备了这样的系统。p1质粒的着丝粒被称为parS， ParB蛋白与着丝粒结合，parS是分割atp酶。f质粒的着丝粒称为sopC， SopB蛋白与着丝粒结合，SopA是分切atp酶。类似的系统已被发现不仅涉及到各种细菌的染色体DNA分离，而且还涉及到细菌中大型蛋白质细胞器的分离，如蓝藻中的羧酶体。

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.