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Characterization of a replication mutant of the bacteriocinogenic plasmid Clo DF13
Abstract:
In a previous paper (Kool, A.J. and Nijkamp, H.J.J. (1974) J. Bacteriol. 120, 569--578) the isolation of a mutant of the bacteriocinogenic plasmid Clo DF13-Rep3, has been described. It was observed that cells harbouring the wild type plasmid synthesize more plasmid DNA cells harbouring the wild type plasmid. This paper deals with the characterization of the nature of this plasmid-specific mutation. The following properties of the Clo DF13-Rep3 mutant plasmid could be observed: 1. The plasmid-specific mutation did not lead to a significant change in the sedimentation value of Clo DF13 DNA. 2. The specific rate of Clo DF13-Rep3 DNA synthesis (expressed as the number of plasmid DNA molecules synthesized per min) is on average seven times the specific rate of wild type Clo DF13 DNA synthesis. 3. Also chromosomeless minicells, harbouring the Clo DF 13-Rep 3 plasmid, contain about seven times more plasmid DNA as wild type Clo DF13 harbouring minicells. 4. The replication time of the Clo DF13-Rep3 plasmid is approx. 90 s at 30 degrees C and does not differ significantly from the replication time of the wild type plasmid. 5. The Rep3 mutation did not alter the dependence of Clo DF13 plasmid replication on the dnaA and dnaC gene products. 6. The plasmid-specific mutation is cis-dominant over wild type. The data presented in this paper indicate that this mutant plasmid is not affected in the elongation but in the initiation of plasmid DNA replication.
Insights
A Clo DF13-Rep3 mutant plasmid shows increased DNA synthesis due to a mutation affecting initiation, not elongation. This mutation is cis-dominant, leading to higher plasmid DNA levels in cells and minicells.
Area of Science:
- Molecular Biology
- Bacteriology
- Genetics
Background:
- A previously isolated mutant bacteriocinogenic plasmid, Clo DF13-Rep3, was observed to have increased DNA synthesis compared to the wild type.
- This study aims to characterize the specific mutation responsible for the altered DNA synthesis in the Clo DF13-Rep3 mutant plasmid.
Purpose of the Study:
- To elucidate the nature of the plasmid-specific mutation in Clo DF13-Rep3.
- To determine whether the mutation affects plasmid DNA replication initiation or elongation.
- To investigate the cis-dominance and genetic dependencies of the mutation.
Main Methods:
- Characterization of Clo DF13-Rep3 DNA sedimentation value.
- Measurement of specific DNA synthesis rates in cells and minicells.
- Determination of plasmid replication time.
- Assessment of dependence on host dnaA and dnaC gene products.
Main Results:
- The Clo DF13-Rep3 mutation did not alter DNA sedimentation value or replication time.
- The specific rate of Clo DF13-Rep3 DNA synthesis was approximately seven times higher than wild type.
- Minicells harboring Clo DF13-Rep3 also showed a sevenfold increase in plasmid DNA content.
- The Rep3 mutation is cis-dominant and does not affect dependence on dnaA and dnaC gene products.
Conclusions:
- The Rep3 mutation in Clo DF13-Rep3 affects the initiation of plasmid DNA replication.
- The mutation leads to a significant increase in plasmid DNA synthesis and copy number.
- The findings provide insights into plasmid replication control mechanisms.