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Microbiology 152 (2006), 1089-1097; DOI  10.1099/mic.0.28575-0
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Microbiology 152 (2006), 1089-1097; DOI  10.1099/mic.0.28575-0
© 2006 Society for General Microbiology

Exploitation of a beta-lactamase reporter gene fusion in the carbapenem antibiotic production operon to study adaptive evolution in Erwinia carotovora

Steven D. Bowden and George P. C. Salmond

Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1QW, UK

Correspondence
George P. C. Salmond
gpcs{at}mole.bio.cam.ac.uk

Erwinia carotovora subsp. carotovora strain ATTn10 produces the beta-lactam antibiotic 1-carbapen-2-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.

Abbreviations: Ap, ampicillin; Tc, tetracycline; Car, 1-carbapen-2-em-3-carboxylic acid; Ecc, Erwinia carotovora subspecies carotovora






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