The 42- and 51-kilodalton mosquitocidal proteins of Bacillus sphaericus 2362: construction of recombinants with enhanced expression and in vivo studies of processing and toxicity

Abstract

After site-directed mutagenesis, the genes coding for the 42-and 51-kilodalton (kDa) mosquitocidal proteins of Bacillus sphaericus 2362 were placed under the regulation of the aprE (subtilisin) promoter of the BaciUus subtilis vector pUE (a derivative of pUB18). The levels of expression of the gene products in B. subtilis DB104 and B. sphaericus 718 were assessed by bioassays with larvae of Culex pipiens and by Western immunoblots. The results indicated that a higher amount of protein was produced in B. subtlis DB104. Electron microscopic examination of B. subilis DB104 and B. sphaericus 718 containing the 42-and 51-kDa proteins indicated that amorphous inclusions accumulated in the former species and that crystals identical in appearance to that found in B. sphaericus 2362 were produced in the latter. Strains producing only the 42-or the 51-kDa protein were not toxic to larvae of C. pipiens. A mixture of both strains, a single strain producing both proteins, or a fusion of the 51-and the 42-kDa proteins was toxic. The amount ofB. subtlis DB104 containing the 42-and the 51-kDa proteins necessary to kill 50% of the larvae of C. pipiens was 5.6 ng (dry weight) of cells per ml. This value was significantly lower than that for B. sphaericus 2362 (14 ng [dry weight] per ml). Larvae consuming purified amorphous inclusions containing the 42-kDa protein degraded this protein to primarily 39-and 24-kDa peptides, whereas inclusions with the 51-kDa protein were primarily degraded to a protein of 44 kDa. Past studies involving purified proteins from B. sphaericus 2362 indicate an association of toxicity with the 39-kDa peptide. The results presented here suggest that the 44-kDa degradation product of the 51-kDa protein may also be required for toxicity.

Proteolytic processing of the mosquitocidal toxin from Bacillus sphaericus SSII-1

“…The presence of trypsinlike proteases in mosquito larvae has been demonstrated (18). Toxin processing by trypsin and chymotrypsinlike enzymes in mosquito gut extracts has been demonstrated for the 51.4-and 41.9-kDa toxins from the highly toxic B. sphaericus strains (1,8,9,12,13) and for the mosquitocidal toxins of B. thuringiensis (28a). Given the highly specific cleavage of the 97-kDa toxin by gut extracts, we suggest a similar processing of the Mtx2l protein by a trypsinlike enzyme.…”

Essential role of tryptophan residues in toxicity of binary toxin from Bacillus sphaericus

“…Since the interaction of the binary toxin with target lipid membrane is one of the key steps in eliciting cytopathological effects on mosquito larvae, molecular insights into the interaction of the binary toxin with lipid membranes is required to gain a better understanding of the mechanism of toxicity. Due to the lack of three dimensional structure of the binary toxin, functional characterization has been based mainly on its amino acid sequence analysis and secondary structure prediction (10)(11)(12)(13)(14)(15). Aromatic residues, especially tyrosine and tryptophan, conceivably play a key role in membrane anchoring of many membrane proteins and are mainly found at the membrane-water interface (16).…”

Deletion analysis of the 51-kilodalton protein of the Bacillus sphaericus 2362 binary mosquitocidal toxin: construction of derivatives equivalent to the larva-processed toxin

“…thuringiensis toxins which consist of one protein, the B. sphaericus larvicide is a binary toxin requiring the presence of both the 51-and 42-kDa proteins for activity (7,8).…”

“…Following ingestion, the 51-and 42-kDa proteins are degraded to proteins of about 43 and 39 kDa, respectively (1,7). Evidence has been presented indicating that this processing leads to an activation of the toxin (6,9).…”