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Partitioning of plasmid R1. The ParM protein exhibits ATPase activity and interacts with the centromere-like
1Department of Molecular Biology, Odense University, Denmark.
Abstract:
The parA system of plasmid R1 consists of two genes, parM and parR, and a cis-acting centromere-like site parC. The ParM protein exhibits similarity with a superfamily of ATPases that includes actin, hsp70 and hexokinase. ParM was purified to near-homogeneity and assayed for in vitro ATPase activity. The wild-type ParM protein was found to posses ATPase activity. Mutant ParM derivatives that exhibited decreased in vitro ATPase activity were non-functional in vivo, indicating that the ATP turnover by ParM is essential for correct plasmid partitioning. The mutant ParM proteins exhibited trans-dominance, suggesting that ParM participates as a structural component of the partitioning apparatus. The ATPase activity of ParM was activated slightly by the presence of ParR and activated to a much greater extent when ParR was bound to the centromere-like parC region. An analysis using the yeast two-hybrid system indicated that ParM and ParR interact, and demonstrated that ParR interacts with itself. Thus our results suggest a direct interaction of ParM and ParR at the natural partition site parC, and that the ATPase activity of ParM is specifically stimulated by this interaction.
Insights
The plasmid R1 parA system
Area of Science:
- Molecular Biology
- Microbiology
- Genetics
Background:
- The parA system of plasmid R1 is crucial for ensuring accurate plasmid segregation during cell division.
- This system comprises two genes, parM and parR, and a cis-acting site, parC.
- The ParM protein shares similarities with known ATPases, suggesting a role in energy-dependent processes.
Purpose of the Study:
- To investigate the ATPase activity of the ParM protein.
- To determine the functional significance of ParM's ATPase activity in plasmid partitioning.
- To elucidate the interaction between ParM, ParR, and the parC site.
Main Methods:
- Purification of wild-type and mutant ParM proteins.
- In vitro ATPase assays to measure enzymatic activity.
- Yeast two-hybrid system to analyze protein-protein interactions.
Main Results:
- Wild-type ParM protein exhibits ATPase activity, essential for plasmid partitioning in vivo.
- Mutant ParM proteins with reduced ATPase activity were non-functional and showed trans-dominance.
- ParM's ATPase activity is stimulated by ParR, particularly when ParR is bound to the parC site.
- ParM and ParR interact directly, and ParR self-interacts.
Conclusions:
- ATP turnover by ParM is essential for the correct partitioning of plasmid R1.
- ParM likely functions as a structural component of the plasmid partitioning apparatus.
- Specific interactions between ParM, ParR, and parC regulate ParM's ATPase activity, facilitating efficient plasmid segregation.