IMPROVEMENT OF BACILLUS-SPHAERICUS TOXICITY AGAINST DIPTERAN LARVAE BY INTEGRATION, VIA HOMOLOGOUS RECOMBINATION, OF THE CRY11A TOXIN GENE FROM BACILLUS-THURINGIENSIS SUBSP ISRAELENSIS

Integrative plasmids were constructed to enable integration of foreign DNA into the chromosome of Bacillus sphaericus 2297 by in vivo recomb ination. Integration of the aphA3 kanamycin resistance gene by a two-s tep procedure demonstrated that this strategy was applicable with anti biotic resistance selection. Hybridization experiments evidenced two c opies of the operon encoding the binary toxin from B. sphaericus in th e recipient strain. The Bacillus thuringiensis subsp. israelensis cry1 1Aa1 gene (referred to as cry11A), encoding a delta-endotoxin with tox icity against Culex, Aedes, and Anopheles larvae, was integrated eithe r by a single crossover event [strain 2297(::pHT5601), harboring the e ntire recombinant plasmid] or by two successive crossover events [stra in 2297(::cry11A)]. The level of the Cry11A production in B. sphaericu s was high; two crystalline inclusions were produced in strain 2297(:: pHT5601). Synthesis of the Cry11A toxin conferred toxicity to the reco mbinant strains against Aedes aegypti larvae, for which the parental s train was not toxic. Interestingly, the level of larvicidal activity o f strain 2297(::pHT5601) against Anopheles stephensi was as high as th at of B. thuringiensis subsp. israelensis and suggested synergy betwee n the B. thuringiensis and B. sphaericus toxins. The toxicities of par ental and recombinant B. sphaericus strains against Culex quinquefasci atus were similar, but the recombinant strains killed the larvae more rapidly. The production of the Cry11A toxin in B. sphaericus also part ially restored toxicity for C. quinquefasciatus larvae from a populati on resistant to B. sphaericus 1593. In vivo recombination therefore ap pears to be a promising approach to the creation of new B. sphaericus strains for vector control.