Mitra Nivedita, Mishra Dipika, Mudaliyar Manasi, Yadav Rinku, Zinjurte Suyog, Puthethu Irene Aniyan, Gayathri Pananghat, Ghosal Debnath, Srinivasan Ramanujam
School of Biological Sciences, National Institute of Science Education and Research, Bhubaneswar, 752050, India.
Homi Bhabha National Institutes (HBNI), Training School Complex, Anushakti Nagar, Mumbai, 400094, India.
Nucleic Acids Res. 2025 Jun 20;53(12). doi: 10.1093/nar/gkaf537.
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.
诸如质粒之类的可移动遗传元件在抗菌素耐药性的出现中起着至关重要的作用。因此，诸如沃克A型ATP酶/ParA超家族的ParA等质粒维持蛋白是新型抗生素的潜在靶点。ParA介导的质粒分配依赖于ATP依赖性二聚化以及ParA-ATP在细菌类核上形成趋化梯度。尽管在许多情况下都报道了ParA的聚合，但质粒维持中聚合的必要性仍不清楚。在这项研究中，我们深入了解了ParA的聚合以及聚合对质粒维持的影响。我们报告了来自F质粒（ParAF）的ParA中的两个突变，Q351H和W362E，它们形成独立于ParBSF分配复合体的细胞质细丝。这两种变体都无法分配质粒，不结合非特异性DNA，并作为超级阻遏物抑制来自ParAF启动子的转录。此外，我们表明ParAF的聚合需要ATP依赖性构象转换。我们在预测的ParAF细丝结构的界面处的螺旋12中的R320和螺旋14中的E375这两个残基，其突变消除了ParAF W362E的细丝组装并影响质粒分配。因此，我们的结果表明ParAF的C末端螺旋在质粒维持和组装成更高阶结构中起作用。
