Role of the ATP-binding site of SopA protein in partition of the F plasmid

SopA belongs to a large family of bacterial partition protein ATPases. It h elps stabilize the F plasmid by acting as the primary repressor of transcri ption of the sopAB operon, preventing the destabilizing effects of Sop prot ein excess. It is also thought to act directly in the F partition mechanism . We have examined the role of SopA in partition and repression by observin g the consequences of replacing an invariant ATP-binding site lysine, K120, by glutamine or arginine. Circular dichroism studies of the purified mutan t proteins revealed no major differences from wild-type, but in the presenc e of ADP or ATP each protein showed a characteristic spectrum which suggest ed a distinct conformational change. The K120Q mutant retained most of the wild-type ATPase activity while the K120R mutant lost it. In neither case w as the residual activity stimulated by SopB, as occurs for wild-type SopA. The strength of sop promoter repression by the mutant SopA proteins alone w as comparable to that resulting from SopB-enhancement of wild-type SopA, bu t SopB enhanced repression by the mutant SopA proteins either slightly (K12 0R) or not at all (K120Q). Mini-Fs in which the sop operon was controlled b y a constitutive promoter were destabilized by the mutations, demonstrating the need for SopA and its ATP-binding site in the partition process. The K 120R mini-F was lost at the same rate as a mini-F lacking the sopC centrome re, the K120Q mutant was lost faster. SopAK120R at high levels was more eff ective than SopA I in disrupting the partition complex, whereas SopAK120Q d id not disrupt it at all. These results suggest that one function of SopA i n the partition mechanism is to break the paired plasmid structure to allow F molecules to segregate to daughter cells. (C) 2001 Academic Press.