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

Abstract

The operon containing the genes encoding the subunits of the binary crystal toxin of Bacillus sphaericus strain LP1-G, BinA and BinB (41.9 kDa and 51.4 kDa, respectively), was cloned and sequenced. Purified crystals were not toxic to Culex pipiens larvae. Comparison of the aminoacid sequences of this strain (Bin4) with those of the three other known toxin types (Bin1, Bin2 and Bin3) revealed mutations at six positions, including a serine at position 93 of BinA4, whereas all other types of BinA toxin from B. sphaericus had a leucine at this position. Reciprocal sitedirected mutagenesis was performed to replace this serine in BinA4 from LP1-G with a leucine and the leucine in the BinA2 protein from strain 1593 with a serine. Native and mutated genes were cloned and overexpressed. Inclusion bodies were tested on C. pipiens larvae. Unlike the native Bin4 toxin, the mutated protein was toxic, and the reciprocal mutation in Bin2 led to a significant loss of toxicity. In vitro receptor-binding studies showed similar binding behaviour for native and mutated toxins. In the absence of any experimental data on the 3D structure of these proteins, sequence analysis and secondary-structure predictions were performed. Amino acid 93 of the BinA polypeptide probably belongs to an a helix that is sensitive to amino-acid modifications. Position 93 may be a key element in the formation of the BinA±BinB complex responsible for the toxicity and stability of B. sphaericus Bin toxins.

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).…”

Phylogenetic Analysis and Heterologous Expression of Surface Layer Protein SlpC ofBacillus sphaericusC3-41

“…12,13) Although S-layers have been identified in some B. sphaericus and their structures and re-assembly characteristics have been described, 3,6,14) the occurrence of S-layers is not well documented in insect pathogenic B. sphaericus strains, and there is no report on the relationship between S-layer of pathogenic B. sphaericus and its toxicity against mosquito larvae.…”

Role of cysteine at positions 67, 161 and 241 of a Bacillus sphaericus binary toxin BinB

“…It is proposed that in solution binary toxin exists as tetramer composing of 2 molecules of BinA and 2 molecules of BinB, but the oligomer is not held together by disulfide bonds (13). However, dimers of both BinA and BinB withstood SDS-PAGE sample preparation to some degree so that they could be observed as bands of molecular masses of 110 kDa and 125 kDa, respectively, but these dimer bands disappeared after trypsin digestion (13,15). In agreement with those findings, our results indicate that the active form of BinB does not contain disulfide bond.…”

“…Interaction between BinA and BinB is crucial for activity of binary toxin and studies of binary toxin with lipid bilayers suggest that BinA and BinB may interact in solution prior to binding to the receptor (24). Significance of this interaction is emphasized in the work showing that amino acid at position 93 of BinA is important for biological activity of the toxin by playing a major role in the formation of BinA-BinB complex (15). Also, amino acids centered around position 100 in BinA which has been proved to be important for host ranges of binary toxin may play a part in BinA-BinB interaction (14).…”

“…In the same report, it was suggested that both Nand C-terminal regions of BinA are important for BinA-BinB interaction (8). Site-directed mutagenesis of amino acid positions 93, 99, and 104 of BinA revealed that these residues are crucial for toxicity and it was proposed that residues in this region might play a role in the formation of BinA-BinB complex (14,15). Interestingly, mutagenesis of charged residues (R97, E98, R101, and E114) in the same region showed that these charged residues are also essential for biological activity of binary toxin, but not involved in BinA-BinB interaction (16).…”