03801nam a2200157 a 450000100080000000500110000800800410001910000250006024500590008526000160014430000110016050000880017152033460025965000270360565000110363211878942013-01-31       2006    bl uuuu  m   00u1 u    #d1 aSILVA-WERNECK, J. O.  aInvestigation of a novel Bacillus thuringiensis toxin.  a2006.c2006  a241 f.  aTese (Doutorado) - University of Cambridge, Cambridge, UK. Orientador: David Ellar.  aThe aim of the present work was to investigate the toxicity, biochemical characteristics, gene and protein contents, and the mechanism of action of toxins from the Brazilian Bacil/us thuringiensis (Bt) strain 8725. This strain has been reported as toxic against the cotton boll weevil (Anthonomus grandis), a severe pest of cotton crops in America, which is considered a recalcitrant target to most of the known Bt toxins. It was serotyped as Bt serovar japonensis and produced spherical crystals that were purified on discontinuous sucrose gradient. 8D8-P AGE analysis of the solubilised inclusions showed two proteins of about 130 kDa which were converted to fragments of between 50 and 70 kDa upon trypsin activation. N-terminal sequencing of a trypsin activated fragment revealed a high leveI of similarity to Cry9 õ-endotoxins. The protein comprising the crystals was found to react positively with a polyclonal antibody raised against a Cry9 toxin. The cry9-like gene was PCR amplified, cloned in Escherichia coli XLlO-Gold, and sequenced. A BLA8T search of the deduced amino acid sequence revealed it to be unique and to have 73% identity to Cry9Ba, 64% identity to Cry9Ea, 63% identity to Cry9Da, and 59% identity to Cry9Ca proteins. .The Cry9-like protein gene was transformed into the acrystalliferous strain Bti IP878/11 where it formed spherical cytoplasmic inclusions as viewed by ~canning electron microscopy. The novel Cry9-like õ-endotoxin was assigned to a new subclass (Cry9Bb) by the Bt Toxin Nomenclature Committee. Modelling of the novel protein sequence produced a 3D model containing three structural domains, similarly to the Cry proteins whose structures have been solved by X-ray crystallography. The similarities and phylogenetic distances of Cry9Bb to Cry9 holotype proteins were determined. The Cry9Bb protein was solubilised under different conditions and activated by A. grandis or Pieris brassicae gut extract or by trypsin at different trypsin:toxin ratios. The biotinylated toxin was used in overlay assays for detection of A. grandis BBMV -bound toxin. Bioassays with native crystals from the 8725 strain, Cry9Bb crystals from the recombinant strain and with solubilised or activated Cry9Bb protein were performed against coleopteran, lepidopteran and dipteran insects. The protein showed no toxicity against most of the insects tested, including the coleopteran A. grandis. Mortality was observed against the lepidopterans Manduca sexta and Anticarsia gemmatalis, although only at high toxin concentrations. The LCso for the recombinant Cry9Bb crystals against M sexta larvae was determined. Cytotoxicity assays of the activated Cry9Bb toxin were performed against Choristoneura fumiferana, M sexta and A. gemmatalis celI lines. The toxin showed no visible effects on the tested celIs. PCR screening revealed that in addition to cry9Bb, Bt strain S725 also contains crylI and vip3 genes. Transcription analysis, using RT-PCR, showed that the crylI gene was transcribed at low levels. Site directed mutagenesis was carried out to replace an alanine found in Cry9Bb with a proline conserved at that position in alI other Cry9 toxins. The effect of this change was investigated using bioassays against M sexta as welI as analysis of solubilisation, activation, and simulated gastric digestion of the toxin.  aBacillus Thuringiensis  aToxina