Study of the mechanism of bacterial chromosome partitioning systems

National Institute Of Diabetes And Digestive And Kidney Diseases

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Abstract

After DNA replication, two daughter copies of the bacterial chromosome and low copy number plasmids must be segregated into two daughter cells to ensure 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 ATPase. The centromere of F-plasmid is called sopC, to which SopB protein binds, and SopA is the 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 proteinus organelles in bacteria. 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 exploitation of 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/CCD camera system. By using fluorescence-labeled ParA and ParB proteins, or SopA and SopB proteins, association/dissociation dynamics of these proteins with DNA molecules immobilized on a slide glass surface 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 non-specific DNA. 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 distribution gradient 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. 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 has a much larger length-scale than the individual protein molecules involved, without assembling a polymeric protein assembly that spans the distance