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Title: Novel Bacillus sphaericus binary toxin active against bin-resistant culex mosquito larvae
Author: Jones, Gareth Wyn
ISNI:       0000 0004 2749 5912
Awarding Body: Cardiff University
Current Institution: Cardiff University
Date of Award: 2006
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Some Bacillus sphaericus strains (e.g. IAB59, LP1G and 47-6B) can overcome resistance in Culex mosquito larvae, raised against the well characterised binary toxin from this bacterium. A common spore protein (P49), of approximately 49 kDa, produced by these strains has previously been proposed to be responsible for this toxicity. Protein fingerprint analysis of sporulated cultures of these strains identified a number of candidate toxins. Their N-terminal sequences were determined and used to design degenerate oligonucleotide probes. Southern blotting, cloning and colony hybridisation allowed the identification of clones containing genes encoding the putative toxins Cry49Aal (P49) and Cry48Aal (P135) from IAB59. The 1,395 bp cry49Aal gene encodes a protein of 53.3 kDa, showing homology to BinA and BinB from B. sphaericus as well as Cry36Aal and the Cry35 binary toxins from Bacillus thuringiensis. The 3,534 bp cry48Aal gene encodes a 135.6 kDa protein showing homology to the three-domain Cry toxins from B. thuringiensis, including the mosquitocidal Cry4Aa and Cry4Ba from B. thuringiensis subsp. israelensis. Individual expression of these proteins in an acrystalliferous B. thuringiensis subsp. israelensis strain, followed by bioassays against mosquito larvae revealed no toxicity. However, a Cry48Aal/Cry49Aal combination was toxic to both Bin-susceptible and Bin-resistant Culex quinquefasciatus larvae. Aedes aegypti and Anopheles gambiae mosquito larvae were insensitive to the combination, as were a range of other dipteran, coleopteran and lepidopteran insects. The components of this novel binary toxin from B. sphaericus are highly conserved among strains able to overcome resistance. Differential processing of Cry48Aal by C. quinquefasciatus and A. aegypti larval gut proteinases is not responsible for the non-toxicity towards the latter mosquito. Cry49Aal and Cry48Aal form bipyramidal and amorphous crystals respectively at sporulation and their expression involves RNA polymerase factor cr6 in B. subtilis. Discovery of Cry49Aal and Cry48Aal may prove central in the development of strategies to avoid resistance development against B. sphaericus in Culex populations.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available