larvae. The three indigenous strains were screened by PCR amplification for the presence of binary toxin genes. Among the strains, only ISPC-8
showed the presence of bin genes, whereas these genes were absent in ISPC-5 and ISPC-6 strains. The results agree with the medium larvicidal activity of ISPC-5 and ISPC-6 strains, as also noted earlier (de Barjac et al., 1985; Charles et al., 1996). Most of the highly toxic strains (1593, 2362) of B. sphaericus showed the presence of these genes (Yousten, 1984; Baumann et al., 1987). The binA (1.1 kb) and binB (1.3 kb) genes from ISPC-8 were PCR amplified (Fig. 1). The sequences of binA (GenBank accession no. EU3753086) and binB (GenBank accession no. EU3753089) from ISPC-8 were compared with other highly toxic strains 1593/2362. The BinA protein differed by one amino acid (R197M), whereas BinB differs by two amino acids (H99P,
P174S) as compared with standard 1593/2362 strains. find more The insecticidal activity of this organism is mainly due to the presence of Bin (41.9 and 51.4 kDa) proteins (Broadwell & Baumann, 1987). The Bin proteins from ISPC-8 were purified using ion-exchange and gel-filtration chromatography. selleck inhibitor These proteins coeluted as a single peak on a gel filtration column with an elution volume that corresponded to an ∼65-kDa protein. The eluted peak showed two distinct bands of BinA and BinB when resolved on 12% SDS-PAGE (Fig. 2). The apparent molecular mass of ∼65 kDa is much lower than the complex of the BinA/BinB monomer, which essentially should show an elution volume corresponding to ∼93 kDa. These results indicate that the BinA and BinB proteins did not interact under these elution conditions, but coeluted, Niclosamide most likely due to the resolution in the gel
filtration. The purified proteins were tested against third-instar larvae of C. quinquefasciatus. The results showed higher toxicity of purified proteins with an LC50 value 6.32 ng mL−1 (Fig. 3). These results are particularly significant as there are very few reports on the toxicity of purified binary proteins. Baumann et al. (1991) have shown that the purified crystal from strain 2362 showed an LC50 value of 7 ng protein mL−1, whereas N NaOH-solubilized crystal yielded an LC50 dose of 2700 ng mL−1. The purified 51- and 42-kDa proteins from strain 2362 showed an LC50 value of 12 ng mL−1 (Baumann et al., 1991). When these Bin protein genes were expressed in Bacillus subtilis, the purified inclusion bodies showed an LC50 dose of 16 ng mL−1 (Baumann et al., 1991). Thus, a large variation in the LC50 doses and in the preparation methods has been observed. Perhaps more accurate estimates of toxicity profiles can be obtained using in situ folded BinA and BinB proteins in the assay, which may reveal the effect of amino acid substitutions observed in BinA and BinB proteins in our indigenous high-activity strain, ISPC-8.