<I>Erwinia carotovora </I>subspecies <I>caratovora</I> (<I>Ecc</I>) is a Gram-negative bacterial phytopathogen, belonging to the Enterbaceriaceae family. <I>Ecc </I>has a broad host range, including potato, causing soft rotting of tubers while in storage. This effect is due to the secretion of an arsenal of tissue-macerating extracellular enzymes, which includes multiple cellulases (Cel), pectate lyases (Pel) and proteases (Prt). The overall aim of this project was to learn more about the global regulatory mechanisms controlling the co-ordinate production of exoenzymes in <I>Ecc</I>. Global regulatory mutants were generated by λTn<I>phoA'-</I>2 mutagenesis. Both Rex (regulation of exoenzymes) and Hex (hyperproduction of exoenzymes) mutants were isolated. Rex and Hex mutants are down and up-regulated, respectively, for the synthesis of all the exoenzymes. This project concentrated on the analysis of the Hex mutants thereby identifying 'repressor' genes affecting exoenzyme synthesis. λTn<I>phoA</I>'-2 consists of a promoterless <I>lacZ</I> gene, such that an insertion of the transposon into a gene, in the correct orientation results in a transcriptional gene fusion. Of the 2500 gene fusions screened, 10 Hex and 22 Rex mutants were isolated. Southern hybridisation with a transposon-specific probe showed that the 10 Hex mutants carried a single transposon insertion in each case. Two of these Hex mutants were identified as defective in HexL. The other eight mutations from the Hex mutants were cloned and the DNA sequences flanking the transposons were sequenced. Only one of the disrupted genes encoded a product that showed significant sequence similarity with a known protein, HtrB (high temperature requirement) from <I>E. coli</I>.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:597423 |
| Date | January 2000 |
| Creators | Chan, M. |
| Publisher | University of Cambridge |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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