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

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Abstract
The accurate partitioning of Firmicute plasmid pSM19035 at cell division depends on ATP binding and hydrolysis by homodimeric ATPase δ2 (ParA) and binding of ω 2 (ParB) to its cognate parS DNA. The 1.83 Å resolution crystal structure of δ2 in a complex with non-hydrolyzable ATPγS reveals a unique ParA dimer assembly that permits nucleotide exchange without requiring dissociation into monomers. In vitro , δ2 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 : δ2 ratios, stimulation of the ATPase activity was reduced and δ2 polymerized onto DNA. Stimulation of the δ2 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 δ2 alone associated with the nucleoid, and in the presence of ω 2 and parS DNA, δ2 oscillated between the nucleoid and the cell poles and formed spiral-like structures. Our studies indicate that the molar ω 2 : δ2 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.