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Testing the Role of Multicopy Plasmids in the Evolution of Antibiotic Resistance
Published on: May 2, 2018
Molecular cloning and functional characterization of a copy number control gene (copB) of plasmid R1
Abstract:
Deletions or insertions in the copB gene of plasmid R1 result in a copy mutant phenotype. The wild-type copB gene has been cloned on various plasmid vectors. The presence of such chimeric plasmids reduced the copy number of R1 copB mutant plasmids to normal or subnormal levels, indicating the expression of a trans-acting inhibitor activity from the copB chimeras. However, the cloned copB gene did not affect the copy number of wild-type R1, and no incompatibility was exerted by the cloned copB gene against wild-type R1 (or R100). Although the copB gene is not normally required for the incompatibility exerted by copA, it is shown that the CopB function is required for expression of incompatibility by the copA gene from some types of chimeric plasmids. Mutant plasmids that have lost both Cop functions replicate in an uncontrolled fashion.
Insights
Mutations in the copB gene of plasmid R1 cause copy number defects. Cloning copB revealed trans-acting inhibitor activity, affecting mutant plasmid replication and revealing CopB
Area of Science:
- Molecular biology
- Genetics
- Plasmid biology
Background:
- Plasmid R1 replication is regulated by copy number control genes, including copA and copB.
- Mutations in copB lead to altered plasmid copy numbers, termed copy mutants.
- Understanding these regulatory mechanisms is crucial for plasmid stability and genetic engineering.
Purpose of the Study:
- To investigate the function of the copB gene in plasmid R1 copy number regulation.
- To characterize the inhibitory activity associated with the copB gene.
- To explore the interplay between copB and copA in plasmid incompatibility.
Main Methods:
- Cloning of the wild-type copB gene into various plasmid vectors to create chimeric plasmids.
- Assessing the effect of chimeric plasmids on the copy number of R1 copB mutant plasmids.
- Evaluating the incompatibility of cloned copB against wild-type R1 and R100 plasmids.
- Analyzing the role of CopB function in copA-mediated incompatibility.
Main Results:
- Chimeric plasmids expressing copB reduced the copy number of R1 copB mutant plasmids.
- This reduction indicated a trans-acting inhibitor activity encoded by the cloned copB gene.
- The cloned copB gene did not affect wild-type R1 copy number or cause incompatibility.
- CopB function was found to be necessary for copA-mediated incompatibility in certain chimeric plasmid contexts.
- Mutant plasmids lacking both Cop functions exhibited uncontrolled replication.
Conclusions:
- The copB gene encodes a trans-acting inhibitor that regulates plasmid R1 copy number.
- CopB plays a role in plasmid incompatibility, particularly in conjunction with copA.
- Loss of both CopB and CopA functions leads to deregulation of plasmid replication.
