Construction and characterization of a recombinant Bacillus thuringiensis subsp israelensis strain that produces Cry11B

The mosquitocidal bacterium Bacillus thuringiensis subsp. israelensis (Bti) produces four major endotoxin proteins, Cry4A, Cry4B, Cry11k, and Cyt1A, a nd has toxicity in the range of many synthetic chemical insecticides. Cry11 B, which occurs naturally in B. thuringiensis subsp. jegathesan is a close relative of Cry11A, but is approximately 10-fold as toxic to Culex quinquef asciatus. To determine whether the addition of Cry11B to Bti would improve its toxicity, we produced this protein in Bti. High levels of Cry11B synthe sis were obtained by expression of the cry11B gene under the control of cyt 1A promoters and the STAB-SD sequence. This construct was cloned into the s huttle vector pHT3101, yielding the derivative plasmid pPFT11Bs, which was then transformed by electroporation. into acrystalliferous (4Q7) and crysta lliferous (IPS-82) strains of Bti. Synthesis of Cry11B in Bti 4Q7 produced crystals approximately 50% larger than those produced with its natural prom oters without STAB-SD. However, less Cry11B was produced per unit culture m edium with this construct than with the wild-type construct, apparently bec ause the latter construct produced more cells per unit medium. Nevertheless , the Bti IPS-82 strain that produced Cry11B with pPFT11Bs was twice as tox ic as the parental IPS-82 strain (LC50 = 1.4 ng/ml versus 3.3 ng/ml, respec tively) to fourth instars of C. quinquefasciatus. Against fourth instars of Aedes aegypti, no statistically significant difference between parental Bt i IPS-82 (LC50 = 4.7 ng/ml) and the Bti IPS-82 recombinant producing Cry11B (LC50 = 3.5 ng/ml) was found in toxicity. (C) 2001 Academic Press.