Exploitation of a β-lactarnase reporter gene fusion in the carbapenern antibiotic production operon to study adaptive evolution in Erwinia carotovora

Erwinia carotovora subsp. carotovora strain ATTn10 produces the beta-lactam antibiotic 1-carbapen2-em-3-carboxylic acid (carbapenem) by expressing the carABCDEFGH operon. Mutants exhibiting increased carbapenem gene transcription were positively selected using an engineered strain with a functional beta-lactamase translational fusion in carH, the last gene of the operon. However, spontaneous ampicillin-resistant mutants were isolated even when transcription of carH:: blaM was blocked by a strongly polar mutation in carE The mechanism of resistance was shown to be due to cryptic IS10 elements transposing upstream of carH: : blaM, thereby providing new promoters enabling carH:: blaM transcription. Southern blots showed that IS10 was present in multicopy in ATTn10. In addition, a Tn10 genetic remnant was discovered. The results offer insights into the genetic archaeology of strain ATTn10 and highlight the powerful impacts of cryptic IS elements in bacterial adaptive evolution.