Dynamic Self-Organization of Bacterial DNA Segregation Machinery in a Cell-Free Reaction

Abstract

Bacterial plasmids have evolved segregation machineries to partition replicated DNA to the daughter cells at cell division. P1 phage lysogenizes as a low-copy-number plasmid in Escherichia coli. Its partition system consists of three components, a centromere-like region, parS, an adaptor protein that binds to the centromere, ParB and a partition ATPase, ParA. In general, it is known that a ParB/parS partition complex is formed when ParB oligomerize onto the centromere. This large nucleoprotein complex interacts with ParA and is thought to couple ATP hydrolysis to drive the movement and segregation of plasmids to opposite cell-halves. To understand ATP-driven DNA segregation, we reconstituted the P1 plasmid partition system in a cell-free reaction and visualized the spatiotemporal dynamics using TIRF microscopy. We coated a flow cell surface with non-specific DNA to mimic the bacterial nucleoid surface and flowed in the three-component reaction system. We found that ParA coats the artificial nucleoid creating a reference scaffold for plasmid movement. ParA assembles onto the ParB/parS complexes and anchors them onto the ParA-coated nucleoid surface. ParB stimulates ParA disassembly leading to vigorous Brownian motion of the plasmid as the complex loses bridging interactions with the nucleoid. The plasmid detaches from the nucleoid surface leaving a hole devoid of ParA, which is refilled rapidly with ParA rebinding onto the nucleoid. FRAP experiments demonstrate the dynamic exchange of proteins on the nucleoid surface and the partition complex. We present a Par partition model of ParB-stimulated ParA assembly/disassembly triggering dynamic instability leading to plasmid segregation and movement.