Mutational analysis of the F plasmid partitioning protein ParA reveals residues required for oligomerization and plasmid maintenance.

Mobile genetic elements such as plasmids play a crucial role in the emergence of antimicrobial resistance. Hence, plasmid maintenance proteins such as ParA of the Walker A-type ATPases/ParA superfamily are potential targets for novel antibiotics. Plasmid partitioning by ParA relies on ATP-dependent dimerization and formation of chemophoretic gradients of ParA-ATP on bacterial nucleoids. Though polymerization of ParA has been reported in many instances, the need for polymerization in plasmid maintenance remains unclear. In this study, we provide insights into the polymerization of ParA and the effect of polymerization on plasmid maintenance. We report two mutations, Q351H and W362E, in ParA from the F plasmid (ParAF) that form cytoplasmic filaments independent of the ParBSF partitioning complex. Both variants fail to partition plasmids, do not bind non-specific DNA, and act as super-repressors to suppress transcription from the ParAF promoter. Further, we show that the polymerization of ParAF requires an ATP-dependent conformational switch. We identify two residues, R320 in helix 12 and E375 in helix 14 at the interface of the predicted ParAF filament structure, whose mutations abolish filament assembly of ParAF W362E and affect plasmid partitioning. Our results thus suggest a role for the C-terminal helix of ParAF in plasmid maintenance and assembly into higher order structures.