BIOCHEMICAL, IMMUNOLOGICAL AND TOXICOLOGICAL CHARACTERISTICS OF THE CRYSTAL PROTEINS OF BACILLUS-THURINGIENSIS SUBSP MEDELLIN

Characterization of the insecticidal and hemolytic activity of solubil ized crystal proteins of Bacillus thuringiensis (Bt) subsp. medellin ( Btmed) was performed and compared to solubilized crystal proteins of i solates 1884 of B. thuringiensis subsp. israelensis (Bti) and isolate PG-14 of B. thuringiensis subsp. morrisoni (Btm). In general, al acid pH values solubilization of the Bt crystalline parasporal inclusions ( CPI) was lower than at alkaline pH. The larvicidal activity demonstrat ed by the CPI of Btmed indicated that optimal solubilization of CPI ta kes place at a pH value of 11.3, in Bti at pH values from 5.03 to 11.3 and in Btm at pH values from 9.05 to 11.3. Hemolytic activity against sheep red blood cells was mainly found following extraction at pH 11. 3 in all Bt strains tested Polyacrylamide gel electrophoresis under de naturing conditions revealed that optimal solubilization of the CPI in all Bt strains takes place at the alkaline pH values from 9.05 to 11. 3. An enriched preparation of Btmed crystals was obtained solubilized and crystal proteins were separated on a size exclusion column (Sephac ryl S-200). Three main protein peaks were observed on the chromatogram . The first peak had two main proteins that migrate between 90 to 100 kDa. These proteins are apparently not common to other Bt strains isol ated to date. The second and third peaks obtained from the size exclus ion column yielded polypeptides of 68 and 28-30 kDa, respectively. Eac h peak independently, showed toxicity against Ist instar Culex quinque fasciatus larvae. Interestingly, combinations of the fractions corresp onding to the 68 and 30 kDa protein showed an increased toxicity. Thes e results suggest that the 94 kDa protein is an important component of the Btmed toxins with the highest potency to kill mosquito larvae Whe n crystal proteins of Bti were probed with antisera raised independent ly against the three main protein fractions of Btmed, the only crystal protein that showed cross reaction was the 28 kDa protein. These data suggest that Btmed could be an alternative bacterium for mosquito con trol programs in case mosquito larval resistance emerges to Bti toxic proteins.