Related Experiment Video
Updated: Aug 12, 2026
Generation of Marked and Markerless Mutants in Model Cyanobacterial Species
Published on: May 29, 2016
A mutant defective in partitioning of composite plasmid Rms201
Abstract:
Escherichia coli harboring mutant plasmids defective in maintenance stability (from the conjugative plasmid Rms201) showed a wide distribution of ampicillin resistance levels, as well as increased frequency of plasmid loss from the cell. The amounts of covalently closed circular deoxyribonucleic acid of mutant plasmid Rms268 and parental plasmid Rms201 per chromosome were 5.3 and 6.1%, respectively. The beta-lactamase activities of strains W3630(Rms268) and W3630(Rms201) were 0.56 and 0.44 U/mg of protein, respectively. Frequency of plasmid loss from W3630(Rms268) was about 0.8 to 1.2% per cell generation, 100 times more than that of the wild-type strain. Ampicillin resistance levels of the colonies harboring the mutant plasmid showed a wide distribution, from low (100 micrograms/ml) to high (1,600 micrograms/ml). A miniplasmid (pMS268) with a mass of 7 X 10(6) daltons and encoding ampicillin resistance was isolated from Rms268. Frequency of pMS268 loss from W3630(pMS268) was about 0.8 to 1.9% per cell generation. W3630(pMS268) also showed a wide range of distribution in the levels of ampicillin resistance. These results indicated that the copies of Rms268 in E. coli did not segregate evenly between daughter cells at cell division and that the gene involved was located on the miniplasmid.
Insights
Mutant plasmids in Escherichia coli exhibit unstable maintenance, leading to increased ampicillin resistance and frequent plasmid loss. A miniplasmid, pMS268, responsible for ampicillin resistance, also shows unstable segregation.
Area of Science:
- Molecular Biology
- Microbiology
- Genetics
Background:
- Conjugative plasmids play crucial roles in bacterial adaptation and evolution.
- Plasmid stability is essential for maintaining genetic traits within bacterial populations.
- Mutations affecting plasmid maintenance can lead to altered bacterial phenotypes.
Purpose of the Study:
- To investigate the stability and ampicillin resistance characteristics of a mutant plasmid (Rms268) derived from Rms201 in Escherichia coli.
- To identify the genetic elements responsible for the observed instability and resistance phenotype.
Main Methods:
- Comparative analysis of plasmid copy number and beta-lactamase activity between mutant and parental plasmids.
- Quantification of plasmid loss frequency per cell generation.
- Isolation and characterization of a miniplasmid encoding ampicillin resistance.
Main Results:
- Mutant plasmid Rms268 showed a higher frequency of plasmid loss (0.8-1.2% per cell generation) compared to the wild-type Rms201.
- Strains harboring Rms268 exhibited a wide distribution of ampicillin resistance levels (100-1600 µg/ml).
- A miniplasmid, pMS268, isolated from Rms268, also demonstrated unstable segregation and conferred variable ampicillin resistance.
Conclusions:
- The Rms268 plasmid in E. coli exhibits unstable maintenance due to uneven segregation during cell division.
- The ampicillin resistance gene and the instability determinant are located on the miniplasmid pMS268.
- Plasmid instability can lead to significant variations in antibiotic resistance within bacterial populations.
