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

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.