Transfer of the toxin protein genes of Bacillus sphaericus into Bacillus thuringiensis subsp. israelensis and their expression

Abstract

The genes encoding the toxic determinants of Bacillus sphaericus have been expressed in a nontoxic and a toxic strain of Bacillus thuringiensis subsp. israelensis. In both cases, the B. sphaericus toxin proteins were produced at a high level during sporulation of B. thuringiensis and accumulated as crystalline structures. B. thuringiensis transformants expressing B. sphaericus and B. thuringiensis subsp. israelensis toxins did not show a significant enhancement of toxicity against Aedes aegypti, Anopheles stephensi, and Culex pipiens larvae.

Identification and molecular structural prediction analysis of a toxicity determinant in the Bacillus sphaericus crystal larvicidal toxin

“…Similarly, the activated toxins were identical in size ( Fig. 2) and similar to the native Bin toxin of the B. sphaericus wild-type strain 1593 [26].…”

“…The wild-type B. sphaericus strain LP1-G (serotype H3) was originally isolated in Singapore [4]. Cloning and expression in B. thuringiensis of the binary toxin genes from strain 1593 have been described elsewhere [26]. The same B. thuringiensis crystal negative strain, 4Q2-81, was used as a recipient strain for transformation with the bin operon of LP1-G. Escherichia coli strains DH5-a and JM110 were used as host strains, DH5-a for general cloning and JM110 for the production of demethylated DNA.…”

“…The vector, pAlter-1 (Promega), was used for site-directed mutagenesis. The E. coli± B. thuringiensis shuttle vector, pBU4 [26], was used for expression of the cloned operon in B. thuringiensis 4Q2-81.…”

“…The native bin4 operon from strain LP1-G was inserted into the shuttle vector pBU4 to yield the recombinant plasmid, p1G. The bin2 operon from strain 1593 has previously been inserted into pBU4 and the resulting plasmid named pGSP10 [26]. Similarly, the mutated binA2 and binA4 genes were inserted into the pBU4 shuttle vector to yield the recombinant plasmids pGSP10 [BinA2(L93S)] named pGSP10* and p1G [BiA4(S93L)] named p1G*.…”

Resistance in a Laboratory Population of Culex Quinquefasciatus (Diptera: Culicidae) to Bacillus Sphaericus Binary Toxin is Due to a Change in the Receptor on Midgut Brush-Border Membranes

“…B. sphaericus toxin purification. Toxin of B. sphaericus strain 1593 (of which the binary toxin sequence is the same as that of the toxin from 2362; Berry et al, 1989) was purified from crystals isolated from a transformed crystal-minus strain of B. thuringiensis serovar isruelensis (Bourgouin et al, 1990). The crystals were then solubilized, activated by trypsin and radiolabelled with ' ' ,I as previously described (Nielsen-LeRoux and Charles, 1992), except that 0.02% NaN, was included in the sodium phosphate buffer for storage at 4"C, instead of using filtration through a 0.45-pm-pore-size membrane.…”