<i>Erwinia carotovora</i> subspecies<i> carotovora (Ecc) </i>is a phytopathogenic member of the Enterobacteriaceae and causes soft rot in potato tubers. <i>Ecc</i> 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 <i>carH</i>, the last gene in the Car biosynthetic and auto-resistance operon. The <i>carH::blaM</i> reporter was shown to encode for a functional β-lactamase and by selecting in the presence of ampicillin (Ap), spontaneous <i>Ecc</i> mutants with increased Ap<sup>R</sup> were isolated. The mechanism of increased Ap<sup>R</sup> in the spontaneous mutants was shown to be due to IS<i>10</i> and Tn<i>10</i> mobile genetic elements that had been introduced during the previous construction of ATTn10 from its parent strain, ATCC 39048. Subsequently, <i>carH::blaM</i> was introduced <i>via</i> a series of generalised transductions into ATCC 39048 in order to employ the positive selection system in a strain that did not possess IS<i>10</i> or Tn<i>10</i> elements. By using a combined mini-transposon approach, the <i>carH::blaM</i> positive selection system enabled the isolation of a mutant disrupted in a porin gene with high homology to <i>ompF </i>from <i>Escherichia coli.</i> 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.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:596817 |
| Date | January 2006 |
| Creators | Bowden, S. D. |
| Publisher | University of Cambridge |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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