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Title: The molecular and genetic characterization of the secretory apparatus of Erwinia carotovora subsp. carotovora
Author: Wharam, Susan Deborah
ISNI:       0000 0001 3566 4454
Awarding Body: University of Warwick
Current Institution: University of Warwick
Date of Award: 1992
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Erwinia carotovora subsp. carotovora (Ecc) is a Gram negative phytopathogen, which causes soft-rot of various crops. The major pathogenicity determinants are macerating enzymes: including pectinases (Pel) and Cellulases (Cel). These are thought to be secreted from the cell by a two-step mechanism, via a periplasmic intermediate. The proteins required for the second step of secretion (transport across the outer membrane) are encoded by a cluster of (out) genes. This thesis describes an analysis of the structure of the out gene cluster and secretion of Pel by Ecc. The synthesis and secretion of Pel by the wild-type Ecc strain HC131 was characterized, to enable accurate studies to be made of secretion-deficient (Out') mutants. The out gene cluster was analysed using restriction mapping and Southern hybridizations. Mutagenesis (using TnlacZ and TnphoA) was performed, in an attempt to identify a putative regulatory region of the out cluster. The expression of individual out genes under various conditions was investigated using gene fusions. DNA sequence analysis of the upstream region of the out cluster identified two genes: outC and outX. These were expressed in vivo, and their protein products were visualized using SDS-PAGE. OutX was found to encode exo-Peh: predicted to be a lipoprotein, bound to the Ecc outer membrane. This protein is proposed to have a role in the regulation of pel (and possibly out) gene expression. A putative promoter region was identified upstream of outX, suggesting that the start of the out gene cluster had been found. While this project was in progress, DNA sequence analysis and data bank searches revealed homologies between the Out proteins of Ecc (including OutC), and proteins involved in the trafficking of various macromolecules across the Gram negative cell envelope. It is thought that the Ecc Out system may represent the major pathway out of Gram negative cells.
Supervisor: Not available Sponsor: Science and Engineering Research Council ; Scottish Crop Research Institute
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: QR Microbiology