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Plasmid copy number control: isolation and characterization of high-copy-number mutants of plasmid pE194
Abstract:
A plasmid, pE194, obtained from Staphylococcus aureus confers resistance to macrolide, lincosamide, and streptogramin type B ("MLS") antibiotics. For full expression, the resistance phenotype requires a period of induction by subinhibitory concentrations of erythromycin. A copy number in the range of 10 to 25 copies per cell is maintained during cultivation at 32 degrees C. It is possible to transfer pE194 to Bacillus subtilis by transformation. In B. subtilis, the plasmid is maintained at a copy number of approximately 10 per cell at 37 degrees C, and resistance is inducible. Tylosin, a macrolide antibiotic which resembles erythromycin structurally and to which erythromycin induces resistance, lacks inducing activity. Two types of plasmid mutants were obtained and characterized after selection on medium containing 10 microgram of tylosin per ml. One mutant class appeared to express resistance constitutively and maintained a copy number indistinguishable from that of the parent plasmid. The other mutant type had a 5- to 10-fold-elevated plasmid copy number (i.e., 50 to 100 copies per cell) and expressed resistance inducibly. Both classes of tylosin-resistant mutants were shown to be due to alterations in the plasmid and not to modifications of the host genome.
Insights
The Staphylococcus aureus plasmid pE194 confers inducible resistance to macrolide, lincosamide, and streptogramin B antibiotics. Mutants selected for tylosin resistance exhibited either constitutive resistance or increased copy numbers, indicating plasmid-based alterations.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- The pE194 plasmid from Staphylococcus aureus confers resistance to macrolide, lincosamide, and streptogramin B (MLS) antibiotics.
- Expression of this resistance phenotype requires induction by subinhibitory concentrations of erythromycin.
- pE194 is maintained at 10-25 copies per cell in S. aureus and is transferable to Bacillus subtilis, where it maintains a similar copy number and inducible resistance.
Purpose of the Study:
- To investigate the genetic basis of pE194-mediated MLS resistance.
- To characterize plasmid mutants exhibiting altered resistance patterns or copy numbers.
- To understand the role of plasmid-borne genes in antibiotic resistance regulation.
Main Methods:
- Plasmid transfer via transformation into Bacillus subtilis.
- Cultivation of bacteria at specific temperatures (32°C and 37°C).
- Selection and characterization of tylosin-resistant mutants.
- Determination of plasmid copy number.
- Analysis of resistance inducibility.
Main Results:
- Tylosin, structurally similar to erythromycin, failed to induce resistance in wild-type pE194.
- Two classes of tylosin-resistant mutants were identified: one with constitutive resistance and normal copy number, and another with inducible resistance and a 5- to 10-fold increased copy number (50-100 copies/cell).
- Both mutant classes resulted from alterations within the plasmid, not the host genome.
Conclusions:
- Plasmid pE194 encodes resistance to MLS antibiotics, requiring induction for full expression.
- Mutations in pE194 can lead to constitutive resistance or altered plasmid copy number, affecting antibiotic resistance.
- The study highlights the plasmid's role in mediating antibiotic resistance and provides insights into resistance mechanisms and regulation.