Biochemical, immunological and toxicological characteristics of the crystal proteins of Bacillus thuringiensis subsp. medellin

Abstract

Characterization of the insecticidal and hemo­lytic activity of solubilized crystal proteins of Bacillus thuringiensis subsp. medellin (Btmed) was performed and compared to solu­bilized crystal proteins of isolate 1884 of B. thuringiensis subsp. israelensis (Bti) and iso­late PG-14 of B. thuringiensis subsp. morri­soni (Btm). In general, at acid pH values, solu­bilization of the Bt crystalline parasporal in­clusions (CPI) was lower than at alkaline pH. The larvicidal activity demonstrated by the CPI of Btmed indicated that optimal solubili­zation of CPI takes 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. Hemoly­tical activity against sheep red blood cells was mainly found following extraction at pH 11.3 in all Bt strains tested. Polyacrylamide gel electrophoresis under denaturing conditions revealed that optima' solubilization of the CPI in all Bt strains takes place at alkaline pH val­ues from 9.05 to 11.3. An enriched prepara­tion of Btmed crystals was obtained, solubi­lized crystal proteins were separated on a size exclusion column (Sephacryl 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 isolated to date. The second and third peaks obtained from the size exclu­sion column yielded polypeptides of 68 and 28-30 kDa, respectively. Each peak indepen­dently showed toxicity against first instar Culex quinquefasciatus larvae. These results suggest that the 94 kDa protein is an impor­tant component of the Btmed toxins with the highest potency to kill mosquito larvae. Com­binations of the fractions corresponding to the 68 and 30 kDa protein showed increased toxicity. When crystal proteins of Bti were tested with antisera raised independently 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 control programs in case that mosquito larval resistance emerges to Bti toxic proteins develops.