Expression of the p20 gene from Bacillus thuringiensis H-14 increases Cry11A toxin production and enhances mosquito-larvicidal activity in recombinant gram-negative bacteria.
Experimental analyses with recombinant Escherichia coli and Pseudomonas putida transformed with plasmids bearing genes coding for the Cry11A toxin and P20 protein from Bacillus thuringiensis H-14 showed that cells producing both proteins were more toxic when fed to third-instar Aedes aegypti larvae than were cells expressing cry11A alone; the 50% lethal concentrations were in the range of 10(4) to 10(5) cells/ml. Western blots revealed a higher production of Cry11A when the p20 gene was coexpressed. Cry11A was detected primarily in insoluble form in recombinant cells. Cry11A was not detected in P. putida when P20 was not coproduced, and these recombinants were not toxic to larvae, whereas P. putida recombinants producing both proteins were toxic at concentrations similar to those for E. coli. A coelution experiment was conducted, in which a p20 gene construct producing the P20 protein with an extension of six histidines on the C terminus was mixed with the Cry11A protein. The results showed that Cry11A bound to the P20(His(6)) on a nickel chelating column, whereas Cry11A produced without the P20(His(6)) protein was washed through the column, thus indicating that Cry11A and P20 physically interact. Thus, P20 protein either stabilizes Cry11A or helps it attain the folding important for its toxic activity.
Xu
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Department of Entomology, Michigan State University, East Lansing, Michigan 48824, USA.
Nagai
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M
Bagdasarian
M
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Smith
T W
TW
Walker
E D
ED
D000330:Aedes / Q000187:drug effects* / Q000254:growth & development; D000818:Animals; D001413:Bacillus thuringiensis / Q000235:genetics / Q000378:metabolism*; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D001427:Bacterial Toxins / Q000096:biosynthesis; D003227:Conjugation, Genetic; D004274:DNA, Recombinant; D004731:Endotoxins / Q000096:biosynthesis*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005798:Genes, Bacterial; D006460:Hemolysin Proteins; D007814:Larva / Q000187:drug effects; D010572:Pest Control, Biological / Q000379:methods*; D016958:Pseudomonas putida / Q000235:genetics / Q000378:metabolism
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004274:DNA, Recombinant; D004731:Endotoxins; D006460:Hemolysin Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis