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Regular segregation of composite plasmid Rms201
Abstract:
Copy number mutants Rms201ts15 and Rms201ts16 were isolated at 30 degrees C from a temperature-sensitive replication mutant (Rms201ts14) of the conjugative plasmid Rms201. The numbers of plasmids per chromosome of ML1410(Rms201ts14), ML1410(Rms201ts15), and ML1410(Rms201ts16) grown at 30 degrees C were 2.2, 7.4, and 20, respectively. The synthesis of covalently closed circular plasmid deoxyribonucleic acid stopped in Rms201ts14, Rms201ts15, and Rms201ts16 immediately after a "shift-up" in temperature (42 degrees C). At 42 degrees C, antibiotic-sensitive derivatives appeared after a certain lag time: the lag times of ML1410(Rms201ts14), ML1410(Rms201ts15), and ML1410(Rms201ts16) were 2.5, 5, and 6.8 generations, respectively. After these times, plasmid-positive cells in the populations decreased at a rate of about 50% per generation in all of the mutants. From these results we conclude that plasmid segregation (partition) of Rms201 occurs by regular segregation (partition).
Insights
New copy number mutants of the Rms201 plasmid were identified. These mutants demonstrate that Rms201 plasmid segregation occurs through a regular partitioning mechanism, impacting cell populations under temperature stress.
Area of Science:
- Molecular Biology
- Microbiology
- Genetics
Background:
- The conjugative plasmid Rms201 is essential for bacterial conjugation.
- Understanding plasmid replication and segregation is crucial for genetic stability.
- Temperature-sensitive mutants provide tools to study essential genetic processes.
Purpose of the Study:
- To isolate and characterize new copy number mutants of the Rms201 plasmid.
- To investigate the mechanism of Rms201 plasmid segregation.
- To determine the impact of temperature shifts on plasmid replication and stability.
Main Methods:
- Isolation of temperature-sensitive copy number mutants (Rms201ts15, Rms201ts16) from a parent mutant (Rms201ts14).
- Culturing bacterial strains (ML1410) with different Rms201 mutants at permissive (30°C) and restrictive (42°C) temperatures.
- Quantifying plasmid copy number per chromosome and monitoring the appearance of antibiotic-sensitive segregants over generations.
Main Results:
- Mutants Rms201ts15 and Rms201ts16 exhibited increased plasmid copy numbers (7.4 and 20, respectively) compared to the parent mutant (2.2) at 30°C.
- Plasmid DNA synthesis ceased in all mutants upon temperature shift to 42°C.
- Antibiotic-sensitive segregants appeared after lag times ranging from 2.5 to 6.8 generations, with plasmid-positive cells decreasing by ~50% per generation post-lag.
Conclusions:
- The Rms201 plasmid utilizes a regular segregation (partition) mechanism for distribution into daughter cells.
- The identified mutants provide insights into the regulation of plasmid copy number and segregation.
- Temperature-sensitive mutations disrupt Rms201 replication and lead to loss of plasmid-bearing cells through regular segregation.