A holistic approach for determining the entomopathogenic potential of Bacillus thuringiensis strains

The cry gene content of Bacillus thuringiensis subsp. aizawai HD-133 was an alyzed by a combination of high-pressure liquid chromatography (HPLC) and e xclusive PCR. A total of six ay genes were detected in genomic DNA purified from HD-133, four from the cry1 family (cry1Aa, cry1Ab, cry1C, and cry1D) as well as a gene each from the cry2 (cry2B) and the cry1I families. To dir ectly determine which genes were expressed and crystallized in the purified parasporal inclusions, solubilized and trypsinized HD-133 crystals were su bjected to chromatographic separation by HPLC. Only three proteins, Cry1Ab, Cry1C, and Cry1D, were found, in a 60/37/3 ratio, Dot blot analysis of tot al mRNA purified from HD-133 showed that both the cry2B and cry1I genes, bu t not the cry1Aa gene, were transcribed. Cloning and sequencing of the cry1 Aa gene revealed an inserted DNA sequence within the gy coding sequence, re sulting in a disrupted reading frame, Taken together, our results show that combining crystal protein analysts with a genetic approach is a highly com plementary and powerful way to assess the potential of B, thuringiensis iso lates for new insecticidal genes and specificities, Furthermore, based on t he number of cryptic genes found in HD-133, the total cry gene content of B . thuringiensis strains may be higher than previously thought.