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Trimethoprim-induced DNA polymerase I deficiency in Escherichia coli K-12
Abstract:
Curing of the mini-ColE1 plasmid pML21 was observed among cells of Escherichia coli K-12 strain C600(pML21) grown under subinhibitory conditions in the presence of trimethoprim, a specific inhibitor of dihydrofolate reductase. Some of the cured colonies showed (i) a reduction in frequency of transformation with pML21 compared with those of isogenic strains not treated with trimethoprim, (ii) loss of viability after acquisition of a recA mutation, and (iii) UV sensitivity greater than that of the original isogenic strain. These colonies therefore had PolA- phenotypes. Moreover, they were found to be deficient in DNA polymerase I activity in the in vitro assays, indicating the occurrence of a polA mutation in them. Many of the colonies with PolA- phenotypes were also thyA deoC mutants, and these mutations, in addition to the polA mutations, appeared to be involved in the expression of the PolA- phenotypes.
Insights
Trimethoprim treatment induced curing of the mini-ColE1 plasmid in Escherichia coli. This resulted in PolA- phenotypes, indicating mutations in DNA polymerase I, affecting cell viability and UV sensitivity.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Mini-ColE1 plasmid curing was observed in Escherichia coli K-12.
- Trimethoprim, a dihydrofolate reductase inhibitor, was used under subinhibitory conditions.
Purpose of the Study:
- To investigate the effects of trimethoprim on plasmid stability and bacterial phenotypes.
- To characterize the genetic basis of observed phenotypic changes.
Main Methods:
- Culturing of Escherichia coli K-12 strain C600(pML21) with trimethoprim.
- Phenotypic analysis of plasmid-cured colonies, including transformation frequency, viability assays, and UV sensitivity.
- In vitro DNA polymerase I activity assays.
- Genetic analysis to identify mutations (polA, thyA, deoC).
Main Results:
- Trimethoprim induced curing of the pML21 plasmid.
- Cured colonies exhibited PolA- phenotypes, characterized by reduced transformation, loss of viability with recA mutation, and increased UV sensitivity.
- Deficiency in DNA polymerase I activity confirmed polA mutations.
- Co-occurrence of thyA deoC mutations in many PolA- colonies suggested their involvement in phenotype expression.
Conclusions:
- Subinhibitory trimethoprim can lead to plasmid curing and polA mutations in E. coli.
- PolA mutations, potentially influenced by thyA and deoC mutations, alter DNA repair, viability, and plasmid maintenance.
- This study reveals a complex interplay between drug-induced stress, plasmid loss, and bacterial genetic instability.