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Title: Structure-function analysis of a Bacillus thuringiensis entomocidal toxin
Author: Burton, S. L.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 1999
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This dissertation describes a mutagenic analysis of the structural determinants of toxicity, specificity and receptor binding of the Bacillus thuringiensis (Bt) insecticidal Cry 1 Ac δ-endotoxin. In a previous mutagenesis study of Cry1Ca δ-endotoxin, positively charged amino acids were replaced by uncharged residues. Some of the mutations gave increased crystal expression and/or toxicity against Spodoptera exigua larvae. In the present study, eight of the latter mutations were introduced by site-directed mutagenesis at the equivalent positions in Cry1Ac to investigate if the increase in potency is true for other δ-endotoxins. All but one of the mutants expressed normally in Bt. The other seven mutants were solubilised and activated and then screened for activity against Manduca sexta larvae, and for in vitro qualitative binding to brush border membrane vesicles (BBMV) prepared from Manduca sexta. One arginine residue was found to be important in toxicity. Residues found to be important in Ga1NAc-recognition were identified, and from this information, a triple mutant was constructed. In toxicity assays using neonate Manduca sexta larvae, there was no significant difference in toxicity between wild type Cry1Ac and any of the domain III mutants. In both a qualitative binding assay and the osmotic swelling assay, no inhibitory effect was observed by Ga1NAc on the action of the triple mutant on Manduca sexta BBMV. This indicates that Ga1NAc binding is located on domain III of Cry1Ac. The data presented in this dissertation provides strong evidence to support the hypothesis that Cry1Ac causes pore formation in Manduca sexta BBMV by two distinct receptor-mediated mechanisms. The triple mutant has lost the Ga1NAc-inhibitable aminopeptidase N-dependent mechanism of action and shows only Ga1NAc independent pore formation in BBMV permeability assays.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available