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Characterization of a plasmid mutation affecting maintenance, transfer and elimination by novobiocin
Abstract:
We have isolated a mutant H group plasmid temperature-sensitive for plasmid maintenance. Unlike the wild type plasmid (pSD114), the mutant (pDT4) was eliminated at 37 degrees C and also at 30 degrees C after novobiocin treatment. The mutant plasmid interfered with host cell growth at the non-permissive temperature. Conjugative transfer of the mutant was reduced at 30 degrees C compared to the wild-type plasmid. Introduction of a coumermycin-novobiocin resistance DNA gyrase (cou) mutation into Escherichia coli prevented pDT4 elimination by novobiocin but did not affect the temperature-sensitive phenotype. The evidence indicates that the mutant plasmid used bacterial DNA gyrase for replication. Models to account for the behaviour of this unusual mutant are discussed.
Insights
A novel temperature-sensitive plasmid (pDT4) was identified, exhibiting instability at elevated temperatures and after novobiocin treatment. This mutant plasmid appears to utilize bacterial DNA gyrase for replication, impacting host cell growth and conjugative transfer.
Area of Science:
- Molecular Biology
- Microbiology
- Bacterial Genetics
Background:
- Plasmids are extrachromosomal DNA elements crucial for bacterial adaptation and evolution.
- Plasmid maintenance mechanisms are essential for stability and inheritance within bacterial populations.
- Understanding plasmid replication is key to controlling genetic exchange and bacterial physiology.
Purpose of the Study:
- To characterize a newly isolated temperature-sensitive mutant H group plasmid (pDT4).
- To investigate the replication mechanism and stability of the mutant plasmid.
- To elucidate the interaction between the mutant plasmid and bacterial DNA gyrase.
Main Methods:
- Isolation and characterization of a temperature-sensitive mutant plasmid (pDT4).
- Plasmid stability assays at different temperatures and in the presence of novobiocin.
- Assessment of conjugative transfer efficiency.
- Genetic analysis involving DNA gyrase mutations (cou) in Escherichia coli.
Main Results:
- The mutant plasmid pDT4 showed temperature-sensitive instability, being eliminated at 37°C and 30°C post-novobiocin treatment.
- pDT4 interfered with host cell growth at non-permissive temperatures.
- Conjugative transfer of pDT4 was reduced at 30°C compared to wild-type.
- A coumermycin-novobiocin resistance mutation prevented pDT4 elimination by novobiocin but not its temperature sensitivity.
Conclusions:
- The mutant plasmid pDT4 appears to depend on bacterial DNA gyrase for its replication.
- The study provides evidence for an unusual plasmid replication strategy involving host DNA gyrase.
- Further models are proposed to explain the observed behavior of this unique plasmid mutant.