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Characterization and complementation of pMB1 copy number mutant: effect of RNA I gene dosage on plasmid copy number
Abstract:
A 16-base-pair insertion has been identified as the mutation responsible for the high-copy-number phenotype of the plasmid copy number mutant pFH118. The mutation is located near the plasmid origin of replication in a region of the genome that encodes two overlapping RNA transcripts. One of these transcripts, RNA I, acts as a negative regulator of plasmid replication. The second transcript is the precursor to the primer for the initiation of DNA synthesis. We demonstrate through complementation that the pFH118 DNA overproduction phenotype is a consequence of the reduced effectiveness of the mutant RNA I at inhibiting plasmid replication and not a consequence of an altered target site on the primer precursor. In addition, a series of plasmids containing multiple RNA I-coding genes was constructed for investigating the effects of RNA I gene dosage on plasmid copy number and incompatibility. The results of this study strongly support the inhibitor dilution model of plasmid copy control with RNA I as the plasmid-specified inhibitor responsible for both copy number control and incompatibility.
Insights
A mutation in plasmid pFH118 causes high copy numbers by reducing RNA I's ability to inhibit replication. This supports the inhibitor dilution model for plasmid copy control.
Area of Science:
- Molecular biology
- Genetics
- Biochemistry
Background:
- Plasmid copy number is tightly regulated.
- RNA I is a known negative regulator of plasmid replication.
- The pFH118 mutant exhibits a high-copy-number phenotype.
Purpose of the Study:
- Identify the mutation responsible for the high-copy-number phenotype of pFH118.
- Investigate the mechanism by which this mutation affects plasmid replication.
- Determine the role of RNA I in plasmid copy number control and incompatibility.
Main Methods:
- DNA sequencing to identify the mutation.
- Complementation assays to assess functional effects.
- Construction of plasmids with varying RNA I gene dosage.
Main Results:
- A 16-base-pair insertion near the origin of replication was identified as the causative mutation.
- The mutation reduces the inhibitory effectiveness of RNA I on plasmid replication.
- Increased RNA I gene dosage correlates with changes in plasmid copy number and incompatibility.
Conclusions:
- The pFH118 high-copy-number phenotype results from impaired RNA I-mediated inhibition.
- The findings strongly support the inhibitor dilution model for plasmid copy number regulation.
- RNA I is confirmed as the key inhibitor for both copy number control and incompatibility.