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Title: A study of carbapenem production in Erwinia carotovora
Author: Bowden, S. D.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2006
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Erwinia carotovora subspecies carotovora (Ecc) is a phytopathogenic member of the Enterobacteriaceae and causes soft rot in potato tubers. Ecc strain ATTn10 also produces the β-lactam antibiotic, 1-carbapen-2-em-3-carboxylic acid (Car). In order to select for mutants with increased production of Car, a β-lactamase translational fusion was engineered into carH, the last gene in the Car biosynthetic and auto-resistance operon. The carH::blaM reporter was shown to encode for a functional β-lactamase and by selecting in the presence of ampicillin (Ap), spontaneous Ecc mutants with increased ApR were isolated. The mechanism of increased ApR in the spontaneous mutants was shown to be due to IS10 and Tn10 mobile genetic elements that had been introduced during the previous construction of ATTn10 from its parent strain, ATCC 39048. Subsequently, carH::blaM was introduced via a series of generalised transductions into ATCC 39048 in order to employ the positive selection system in a strain that did not possess IS10 or Tn10 elements. By using a combined mini-transposon approach, the carH::blaM positive selection system enabled the isolation of a mutant disrupted in a porin gene with high homology to ompF from Escherichia coli. An alternative approach to isolate mutants that were affected in Car production relied on the observation that many ATTn10 spontaneous caseinase mutants were also influenced in Car production. A mini-transposon mutagenesis screen of mutants with altered caseinase activity identified five mutants that were also reduced in Car production.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available