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ParE toxin encoded by the broad-host-range plasmid RK2 is an inhibitor of Escherichia coli gyrase
Yong Jiang1, Joe Pogliano, Donald R Helinski
1Department of Biology and Center for Molecular Genetics, University of California, San Diego, La Jolla, CA 92093-0322, USA.
Abstract:
Broad-host-range plasmid RK2 encodes a post-segregational killing system, parDE, which contributes to the stable maintenance of this plasmid in Escherichia coli and many distantly related bacteria. The ParE protein is a toxin that inhibits cell growth, causes cell filamentation and eventually cell death. The ParD protein is a specific ParE antitoxin. In this work, the in vitro activities of these two proteins were examined. The ParE protein was found to inhibit DNA synthesis using an E. coli oriC supercoiled template and a replication-proficient E. coli extract. Moreover, ParE inhibited the early stages of both chromosomal and plasmid DNA replication, as measured by the DnaB helicase- and gyrase-dependent formation of FI*, a highly unwound form of supercoiled DNA. The presence of ParD prevented these inhibitory activities of ParE. We also observed that the addition of ParE to supercoiled DNA plus gyrase alone resulted in the formation of a cleavable gyrase-DNA complex that was converted to a linear DNA form upon addition of sodium dodecyl sulphate (SDS). Adding ParD before or after the addition of ParE prevented the formation of this cleavable complex. These results demonstrate that the target of ParE toxin activity in vitro is E. coli gyrase.
Insights
The ParE toxin inhibits bacterial DNA replication by targeting E. coli gyrase. Its specific antitoxin, ParD, prevents this inhibition, highlighting a novel mechanism for plasmid stability.
Area of Science:
- Molecular Biology
- Bacteriology
Background:
- Broad-host-range plasmid RK2 utilizes a post-segregational killing system, parDE, for stable maintenance in bacteria.
- The ParE protein acts as a toxin, inhibiting cell growth and causing cell death, while ParD serves as its specific antitoxin.
Purpose of the Study:
- To investigate the in vitro activities of the ParE toxin and ParD antitoxin.
- To elucidate the molecular target of ParE's inhibitory action.
Main Methods:
- In vitro assays using Escherichia coli oriC supercoiled DNA templates and replication extracts.
- Measurement of DNA synthesis inhibition and DNA unwinding.
- Analysis of gyrase-DNA complex formation and cleavage.
Main Results:
- ParE protein inhibited DNA synthesis and early stages of DNA replication, including chromosomal and plasmid DNA replication.
- ParE's inhibitory effects were blocked by the presence of ParD.
- ParE induced the formation of a cleavable gyrase-DNA complex, which was prevented by ParD.
Conclusions:
- The molecular target of the ParE toxin's activity in vitro is identified as E. coli gyrase.
- ParD antitoxin neutralizes ParE's inhibitory effects on DNA replication and gyrase activity.
- These findings provide insights into the mechanism of plasmid stability conferred by the parDE system.