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Amplification of a chimeric plasmid carrying an erythromycin-resistance determinant introduced into the genome of
Abstract:
Hybrid plasmid molecules carrying an insert of pneumococcal DNA can integrate into the pneumococcal genome by homologous recombination. The resulting structure is a duplication of the pneumococcal DNA insert bracketing a vector genome. To select for plasmid integration, the vector plasmid was marked with an erythromycin (ery) resistance determinant (eryr) originating from pVA736, a streptococcal plasmid. Experiments with pR27 and pR28, two plasmids carrying the same insert of pneumococcal DNA but in opposite orientations, led to the following observation: (i) In one orientation of the ery region with respect to the amiA locus, cells exhibited a low-level resistance to ery; when these cells were grown in the presence of ery, amplification of the integrated plasmid occurred and cells became resistant to a high level of antibiotic. (ii) In the opposite orientation, a high level of resistance was observed, without need for amplification. These results indicate that, in the orientation conferring a high-level resistance without amplification, the ery region is transcribed both from its own promoter and from the promoter of the amiA locus. In the opposite orientation, a low level of transcription from the eryr promoter could account for a strong selective pressure for the amplified state, which then allows for rapid growth in the presence of ery.
Insights
Plasmid integration into the pneumococcal genome depends on DNA insert orientation. Specific orientations enhance erythromycin resistance, influencing gene amplification and antibiotic selection strategies for genetic engineering.
Area of Science:
- Microbiology
- Molecular Biology
- Genetic Engineering
Background:
- Hybrid plasmid molecules can integrate into the pneumococcal genome via homologous recombination.
- The integration results in a duplication of the pneumococcal DNA insert flanking the vector genome.
- Plasmid integration selection is facilitated by an erythromycin resistance determinant (eryr) from a streptococcal plasmid.
Purpose of the Study:
- To investigate the effect of DNA insert orientation on plasmid integration and erythromycin resistance in Streptococcus pneumoniae.
- To understand the transcriptional regulation of the erythromycin resistance gene in relation to the pneumococcal amiA locus.
Main Methods:
- Utilized two plasmids (pR27 and pR28) with identical pneumococcal DNA inserts in opposite orientations.
- Introduced these plasmids into Streptococcus pneumoniae and selected for integration using erythromycin resistance.
- Analyzed erythromycin resistance levels and the necessity of gene amplification for high-level resistance.
Main Results:
- One orientation of the erythromycin resistance region resulted in low-level resistance, requiring gene amplification for high-level resistance in the presence of erythromycin.
- The opposite orientation conferred high-level erythromycin resistance without the need for amplification.
- The orientation yielding high-level resistance without amplification showed dual transcription of the eryr region from its own promoter and the amiA locus promoter.
Conclusions:
- The orientation of the DNA insert significantly impacts plasmid integration and antibiotic resistance.
- Dual transcription from the amiA locus promoter and the eryr promoter enhances antibiotic resistance.
- Understanding these orientation-dependent transcriptional effects is crucial for optimizing genetic manipulation strategies in Streptococcus pneumoniae.