Exploitation of a beta-lactamase reporter gene fusion in the carbapenem antibiotic production operon to study adaptive evolution in Erwinia carotovora.
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.
Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1QW, UK.
Steven D
SD
Bowden
Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1QW, UK.
George P C
GPC
Salmond
D000220:Adaptation, Biological; D000668:Ampicillin Resistance / Q000235:genetics; D000900:Anti-Bacterial Agents / Q000096:biosynthesis / Q000378:metabolism / Q000494:pharmacology; D019677:Artificial Gene Fusion; D001483:Base Sequence; D015139:Blotting, Southern; D015780:Carbapenems / Q000096:biosynthesis* / Q000378:metabolism / Q000494:pharmacology; D015169:Colony Count, Microbial; D004251:DNA Transposable Elements / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D019143:Evolution, Molecular; D017930:Genes, Reporter; D008969:Molecular Sequence Data; D009154:Mutation; D009876:Operon; D016973:Pectobacterium carotovorum / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011995:Recombination, Genetic; D012641:Selection, Genetic; D014158:Transcription, Genetic; D001618:beta-Lactamases / Q000032:analysis* / Q000235:genetics
D000900:Anti-Bacterial Agents; D015780:Carbapenems; D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D001618:beta-Lactamases