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Isolation and characterization of mutants of the RP4 plasmid coding for increased resistance to ampicillin
Abstract:
E. coli strain J53(RP4) was mutagenized with ethyl methanesulfonate and N-methyl-N'-nitro-N-nitrosoguanidine. Clones showing a two-to threefold increase in resistance to ampicillin were produced. This increase was not due to an increased number of RP4 copies per chromosome. The level of penicillinase activity was twice higher in comparison with the parental strain. No detectable changes were found in the region coding for the resistance to ampicillin on the plasmid by restriction analysis.
Insights
Mutagenesis of E. coli J53(RP4) yielded clones with increased ampicillin resistance, not from more plasmid copies. Penicillinase activity doubled, but plasmid restriction analysis showed no detectable changes in the resistance gene region.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Antibiotic resistance is a growing global health concern.
- Understanding the genetic basis of antibiotic resistance is crucial for developing new therapeutic strategies.
- Escherichia coli (E. coli) is a common bacterium that can harbor resistance plasmids.
Purpose of the Study:
- To investigate the genetic mechanisms behind increased ampicillin resistance in E. coli.
- To characterize mutations affecting ampicillin resistance and penicillinase activity in E. coli strain J53(RP4).
Main Methods:
- Ethyl methanesulfonate (EMS) and N-methyl-N'-nitro-N-nitrosoguanidine (NTG) were used for mutagenesis.
- Ampicillin resistance levels were measured in mutagenized clones.
- Plasmid copy number was assessed.
- Penicillinase activity was quantified.
- Restriction analysis was performed on the plasmid DNA.
Main Results:
- Mutagenesis resulted in E. coli clones with a two-to threefold increase in ampicillin resistance.
- The enhanced resistance was not attributed to an increased number of RP4 plasmid copies.
- Penicillinase activity was found to be twice as high in resistant clones compared to the parental strain.
- Restriction analysis did not reveal detectable alterations in the ampicillin resistance-coding region of the plasmid.
Conclusions:
- Mutagenesis can induce increased ampicillin resistance in E. coli through mechanisms independent of plasmid copy number.
- Elevated penicillinase activity correlates with increased ampicillin resistance.
- Further investigation is needed to identify the specific mutations responsible for the observed resistance phenotype.