Stable integration and expression of mosquito-larvicidal genes fromBacillus thuringiensissubsp.israelensisandBacillus sphaericusinto the chromosome ofEnterobacter amnigenus: a potential breakthrough in mosquito biocontrol

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Abstract
Previously, we have successfully integrated a spectinomycin/streptomycin resistance gene into Enterobacter amnigenus strain An11, a potential host for mosquito control, using in vivo recombination via homologous recombination (An11S4::Ω). We now report the successful transfer of two mosquito-larvicidal genes. cry4B from Bacillus thuringiensis subsp. israelensis and binary toxin genes from Bacillus sphaericus, into the host genome. To facilitate the screening procedure, the E. amnigenus derivative, An11S4::Ω, was used as a host. The integration of both toxin genes by two successive crossover events interrupted the Ω region yielding two integrants designated An11S4::cry4B and An11S4::Ω::bin, respectively. Differences in the integration efficiency of these toxin genes were observed. The presence of both genes in the target sites of the host genome was verified by PCR. Cry4B was expressed weakly from An11S4::cry4B, but no expression of the binary toxin gene could be detected from An11S4::Ω::bin. Nevertheless, these two integrants exhibited mosquito-larvicidal activity against Aedes and Culex, suggesting that both proteins were expressed, but at very low levels.

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