Mosquito Larvicidal Activity ofEscherichia coliwith Combinations of Genes fromBacillus thuringiensis subsp.israelensis

The genescryIVAandcryIVD, encoding 134- and 72-kDa proteins, respectively, and the gene for a regulatory 20-kDa polypeptide ofBacillus thuringiensissubsp.israelensis(serovar H14) were cloned in all seven possible combinations by the Escherichia coli expression vectors pT7 and pUHE. The four combinations containing cryIVA (cryIVA alone, with cryIVD, with the 20-kDa-protein gene, and with both) displayed high levels of mosquitolarvicidalactivityinpUHE.ThetoxicityofthecombinationofcryIVAandcryIVD,withorwithoutthe 20-kDa-protein gene, was higher than has ever been achieved with d-endotoxin genes in recombinantE. coli. Fifty percent lethal concentrations against third-instar Aedes aegypti larvae for these clones decreased (i.e., toxicity increased) continuously to about 3 3 105 cells ml21 afte r4ho finduction. Larvicidal activities, obtained after 30 min of induction, were lower for clones in pT7 and decreased for an additional 3.5 h. Induction of eithercryIVDor the 20-kDa-protein gene alone resulted in no larvicidal activity in either pT7 or pUHE20. Cloned together, these genes were slightly toxic in pT7 but not in pUHE20. Five minutes of induction of this combination (cryIVDwith the 20-kDa-protein gene) in pT7 yielded a maximal mortality of about 40%, which decreased rapidly and disappeared completely after 50 min. CryIVD is thus apparently degraded inE. coliand partially stabilized by the 20-kDa regulatory protein. Larvicidal activity of the combination ofcryIVA andcryIVDwas sevenfold higher than that ofcryIVAalone, probably because of the cross-stabilization of the polypeptides or the synergism between their activities.