Streptococcus pyogenes pSM19035 requires dynamic assembly of ATP-bound ParA and ParB on parS DNA during plasmid segregation

The accurate partitioning of Firmicute plasmid pSM19035 at cell division depends on ATP binding and hydrolysis by homodimeric ATPase δ(2) (ParA) and binding of ω(2) (ParB) to its cognate parS DNA. The 1.83 Å resolution crystal structure of δ(2) in a complex with non-hydrolyzable ATPγS reveals a unique ParA dimer assembly that permits nucleotide exchange without requiring dissociation into monomers. In vitro, δ(2) had minimal ATPase activity in the absence of ω(2) and parS DNA. However, stoichiometric amounts of ω(2) and parS DNA stimulated the δ(2) ATPase activity and mediated plasmid pairing, whereas at high (4:1) ω(2) : δ(2) ratios, stimulation of the ATPase activity was reduced and δ(2) polymerized onto DNA. Stimulation of the δ(2) ATPase activity and its polymerization on DNA required ability of ω(2) to bind parS DNA and its N-terminus. In vivo experiments showed that δ(2) alone associated with the nucleoid, and in the presence of ω(2) and parS DNA, δ(2) oscillated between the nucleoid and the cell poles and formed spiral-like structures. Our studies indicate that the molar ω(2) : δ(2) ratio regulates the polymerization properties of (δ•ATP•Mg(2+))(2) on and depolymerization from parS DNA, thereby controlling the temporal and spatial segregation of pSM19035 before cell division.