Cloning and characterization of a cytolytic and mosquito larvicidal delta-endotoxin from Bacillus thuringiensis subsp. darmstadiensis.
The cytolytic delta-endotoxin gene from Bacillus thuringiensis subsp. darmstadiensis was amplified from genomic DNA by PCR by using primers designed from the sequence of cyt2Aa1 gene of B. thuringiensis subsp. kyushuensis. DNA sequence analysis revealed an open reading frame translating to a 259-amino acid sequence. The cloned gene was designated cyt2Aa2. This gene was highly expressed in Escherichia coli as inclusion bodies that could be solubilized in 50 m M Na(2)CO(3), pH 10.5. Activation of the solubilized protoxin by proteinase K (1% wt/wt, proteinase K/protoxin) yielded the active fragment of about 23 kDa. Cyt2Aa2 showed high hemolytic activity against sheep erythrocytes (hemolytic end- point 0.25 microgram/ml) and was toxic to Culex quinquefasciatus and Aedes aegypti larvae (LC(50) 0.5-1.0 microgram/ml).
Promdonkoy
Boonhiang
B
National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Rama 6 Road, Bangkok 10400, Thailand. boonhiang@biotec.or.th
Chewawiwat
Namchai
N
Tanapongpipat
Sutipa
S
Luxananil
Plearnpis
P
Panyim
Sakol
S
D000330:Aedes / Q000187:drug effects* / Q000254:growth & development; D000595:Amino Acid Sequence; D000818:Animals; D001413:Bacillus thuringiensis / Q000378:metabolism; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism* / Q000633:toxicity*; D001427:Bacterial Toxins; D001483:Base Sequence; D003001:Cloning, Molecular; D003465:Culex / Q000187:drug effects* / Q000254:growth & development; D019286:Endopeptidase K / Q000235:genetics / Q000378:metabolism* / Q000633:toxicity; D004731:Endotoxins / Q000235:genetics / Q000378:metabolism* / Q000633:toxicity*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006460:Hemolysin Proteins; D006461:Hemolysis; D007814:Larva / Q000187:drug effects; D008969:Molecular Sequence Data; D017422:Sequence Analysis, DNA
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004731:Endotoxins; D006460:Hemolysin Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D019286:Endopeptidase K