Identification and molecular structural prediction analysis of a toxicity determinant in the Bacillus sphaericus crystal larvicidal toxin.
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 amino-acid 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 site-directed 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 alpha 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.
Yuan
Z
Z
CIRAD, Montpellier, France.
Rang
C
C
Maroun
R C
RC
Juárez-Pérez
V
V
Frutos
R
R
Pasteur
N
N
Vendrely
C
C
Charles
J F
JF
Nielsen-Leroux
C
C
D000595:Amino Acid Sequence; D000818:Animals; D001407:Bacillus / Q000737:chemistry* / Q000235:genetics; D001427:Bacterial Toxins / Q000737:chemistry* / Q000235:genetics / Q000633:toxicity; D001483:Base Sequence; D001667:Binding, Competitive; D003465:Culex / Q000187:drug effects / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D004064:Digestive System / Q000378:metabolism; D007814:Larva / Q000187:drug effects / Q000378:metabolism; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D010572:Pest Control, Biological; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000633:toxicity; D017386:Sequence Homology, Amino Acid
D001427:Bacterial Toxins; D017931:DNA Primers; D011994:Recombinant Proteins; C046134:binB protein, Bacillus sphaericus