EFFICIENT EXPRESSION OF A 100-KILODALTON MOSQUITOCIDAL TOXIN IN PROTEASE-DEFICIENT RECOMBINANT BACILLUS-SPHAERICUS

The expression of the 100-kDa mosquitocidal toxin (Mtx) during vegetat ive growth and sporulation in nine different mosquito-larvicidal strai ns of Bacillus sphaericus has been analyzed. In five out of the nine s trains the 100-kDa toxin was found to be expressed predominantly in th e vegetative phase of growth, and in all nine strains the level of the toxin in sporulated cells was very low or undetectable. Strains in fo ur out of the six DNA homology groups of B. sphaericus produced intrac ellular and extracellular proteases, which degraded the 100-kDa toxin, during sporulation. The 100-kDa toxin gene was expressed by using its native promoter on a multicopy number plasmid in B. sphaericus 1693 ( protease negative) and B. sphaericus 13052 (protease positive), High l evels of the 100-kDa toxin were produced in vegetative cells of both s trains as well as in sporulated cells of protease-negative strain 1693 , which is in contrast to the low levels of the 100-kDa toxin produced in sporulated cells of protease-positive strain 13052, Thus, the smal l amount of the 100-kDa toxin in sporulated cells of the nine mosquito -larvicidal strains is probably due to degradation of the 100-kDa toxi n synthesized during vegetative growth by a protease(s) produced durin g sporulation, B. sphaericus 1693 transformed with the 100-kDa toxin g ene was as toxic to mosquito larvae during both vegetative growth and sporulation as the natural high-toxicity strains of sporulated B. spha ericus. Therefore, it is conceivable that protease-negative strains of B. sphaericus expressing Mtx and other toxins may form the basis of a n alternative to the natural high-toxicity strains for mosquito contro l.