ParA systems play an important role in chromosome segregation and are responsible for the segregation of many bacterial plasmids. This proposal focuses on the ParA partitioning system of E. coli plasmid F, which consists of the SopA (ParA) ATPase, the SopB (ParB) DNA binding protein, and a DNA sequence, sopC, which contains the recognition sites for SopB binding. Our cell biological studies have demonstrated that SopA-GFP assembles into dynamic polymers that appear in the fluorescence microscope as a bright cloud surrounding the plasmid. This dynamic assembly of SopA is regulated by the SopB/sopC nucleoprotein complex in vivo and is essential for the ability of the Sop system to perform DNA segregation. The mechanism by which a ParA system performs DNA segregation remains unknown. We do not yet understand the mechanism of SopA polymerization or how polymerization contributes to each of the steps in plasmid segregation. We therefore propose to investigate the mechanism of ParA- mediated plasmid segregation in order to gain a better understanding of how this widespread family of proteins contributes to DNA segregation in bacteria. Specifically, we will characterize SopA polymerization in vivo using fluorescence recovery after photopbleaching (FRAP), total internal reflection fluorescence (TIRF) microscopy and speckle microscopy. Using time-lapse fluorescence microscopy and electron cryotomography, we will characterize the dynamic behavior of SopA during oscillation and plasmid separation. These studies will determine some of the basic features of SopA in vivo polymerization behavior and determine how polymerization is coupled to DNA segregation. The findings from this analysis of a representative ParAB plasmid partitioning system should have wide implications for our understanding of plasmid and chromosome segregation.
Many bacteria rely upon the ParA proteins to segregate chromosomal and plasmid DNA, but how they function within the cell is not understood. This proposal focuses on using the latest fluorescence and electron microscopy methods to determine how ParA proteins assemble into polymers within the cell and how these polymers contribute to DNA segregation in vivo. Since ParA proteins are essential for segregating many virulence plasmids as well as the chromosomes of important human pathogens (such as Vibrio cholerae) these studies may provide insight into novel strategies for combating many infectious diseases.
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