Isolation and characterization of a composite plasmid Rms201 mutant temperature sensitive for replication

Area of Science:

• Molecular Biology
• Microbiology
• Genetics

Background:

• R-plasmids are crucial for antibiotic resistance in bacteria.
• Understanding plasmid replication mechanisms is vital for controlling antibiotic resistance.
• Mutagenesis studies help identify genes essential for plasmid maintenance.

Purpose of the Study:

• To isolate and characterize a temperature-sensitive mutant of R-plasmid replication.
• To identify the genetic basis of temperature sensitivity in R-plasmid replication.
• To determine if the temperature-sensitive replication defect resides on the miniplasmid.

Main Methods:

• Chemical mutagenesis of P1 transducing lysate using hydroxylamine.
• Growth of Escherichia coli strains with R-plasmids at permissive and restrictive temperatures.
• Segregation analysis of antibiotic resistance markers.
• Quantification of plasmid covalently closed circular deoxyribonucleic acid (cccDNA) via incorporation of radioactive thymidine.
• Isolation of miniplasmids from parent and mutant strains.

Main Results:

• A temperature-sensitive mutant, Rms201ts14, was isolated from Rms201.
• At 42°C, Rms201ts14 exhibited temperature-sensitive replication, leading to segregation of antibiotic-sensitive cells.
• Plasmid cccDNA synthesis was significantly blocked in Rms201ts14 at 42°C compared to Rms201.
• A temperature-sensitive miniplasmid (pMSts214) encoding ampicillin resistance was isolated from Rms201ts14.

Conclusions:

• The gene(s) responsible for R-plasmid replication are affected in the Rms201ts14 mutant.
• The genetic determinant for temperature-sensitive replication of Rms201 resides on its miniplasmid.
• This finding contributes to understanding plasmid replication control mechanisms.