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bioRxiv · 10.1101/2024.03.17.585406

Mutational analysis of the F plasmid partitioning protein ParA reveals novel residues required for oligomerisation and plasmid maintenance

Mitra, N.·Mishra, D.·Puthethu, I. A.·SRINIVASAN, R.
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Abstract

Mobile genetic elements such as plasmids play a crucial role in the emergence of antimicrobial resistance. Hence, plasmid maintenance proteins like ParA of the Walker A type cytoskeletal ATPases/ ParA superfamily are potential targets for novel antibiotics. Plasmid partitioning by ParA relies upon ATP-dependent dimerisation and formation of chemophoretic gradients of ParA-ATP on bacterial nucleoids. Though polymerisation of ParA has been reported in many instances, the need for polymerisation in plasmid maintenance remains unclear. In this study, we provide novel insights into the polymerisation of ParA and the effect of polymerisation on plasmid maintenance. We first characterise two mutations, Q351H and W362E, in ParA from F plasmid (ParAF) that form cytoplasmic filaments independent of the ParBSF partitioning complex. Both mutants fail to partition plasmids, do not bind non-specific DNA and act as super-repressors to suppress transcription from the ParA promoter. Further, we show that the polymerisation of ParAF requires the conformational switch to the ParA-ATP* state. We identify two mutations, R320A in the C-terminal helix-14 and E375A helix-16 of ParAF, that abolish filament assembly and affect plasmid partitioning. Our results thus suggest a role for higher-order structures or polymerisation of ParA in plasmid maintenance.

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Mitra, N., Mishra, D., Puthethu, I. A., SRINIVASAN, R.. 2024-03-18. Mutational analysis of the F plasmid partitioning protein ParA reveals novel residues required for oligomerisation and plasmid maintenance. https://doi.org/10.1101/2024.03.17.585406

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