Study of the mechanism of bacterial chromosome partitioning systems

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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. However, prokaryotes do not have the mitotic machinery of eukaryotes. One commonly found class of bacterial 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 centromeres of these systems are generally called parS, to which ParB centromere-binding protein binds, and ParA is the partition ATPase. Closely related systems involving homologous proteins 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. In some of the systems, one replicated copy of the ParB-bound parS locus 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 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, association/dissociation dynamics of these proteins with DNA molecules densely immobilized on a slide glass surface (DNA-carpet) mimicking bacterial nucleoid were monitored under a variety of reaction conditions. By using P1- and F-plasmid ParA/B/S as model systems, we learned that ParA, 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 in the presence of ParA and ParB 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 complexes that form in the presence of both non-specific and parS DNA. Latest advances in this project include progress in our understanding of the mechanism of the F-plasmid partition complex assembly. For more than two decades, it has been known that ParB protein of the F-plasmid and its homologues not only bind to their cognate parS DNA sequence, DNA-binding spread to nearby non-specific DNA areas, forming condensed partition protein-DNA complexes containing large numbers of ParB molecules. However, how such complexes are assembled remained a mystery. Recently, ParB proteins of this family have been found to bind CTP and have CTPase activity. We found that the way ParB protein of the F-plasmid interacts with ParA protein and induce ATP hydrolysis by ParA to dissociate it from non-specific (chromosomal) DNA is coordinately controlled by CTP and parS DNA to tune the system for efficient motive force generation by the diffusion-ratchet mechanism we proposed. We have also expanded this project to include Streptococcus pyogenes plasmid pSM19035 partition system, which belongs to a separate class within the same ParABS family of partition systems. The group of ParABS systems to which this system belongs have ParB centromere-binding proteins structurally unrelated to those of F- and P1 plasmid systems and chromosomal Par systems. Most notably, they do not have the CTPase activity and lack the ParB spreading capability. We are finding the pSM19035 Par system functions via partition complex architecture and ParA ATPase activation mechanism quite distinct from those systems involving ParB-CTPase proteins. 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.