Amplification of a chimeric plasmid carrying an erythromycin-resistance determinant introduced into the genome of Streptococcus pneumoniae

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 ( ery r) 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 observations: (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 ery r promoter could account for a strong selective pressure for the amplified state, which then allows for rapid growth in the presence of ery.