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

• Published in: Applied and environmental microbiology Vol. 64; no. 12; pp. 4782 - 4788
• Main Authors: Masson, L. (Biotechnology Research Institute, Montreal, Quebec.), Erlandson, M, Puzstai-Carey, M, Brousseau, R, Juarez-Perez, V, Frutos, R
• Format: Journal Article
• Language: English
• Published: Washington, DC American Society for Microbiology 01.12.1998
• Subjects: Animals; ARN MENSAJERO; ARN MESSAGER; Bacillus thuringiensis; Bacillus thuringiensis - genetics; Bacillus thuringiensis - pathogenicity; Bacillus thuringiensis Toxins; Bacteria; Bacterial Proteins - genetics; Bacterial Proteins - isolation & purification; Bacterial Proteins - toxicity; Bacterial Toxins; Bacteriological methods and techniques used in bacteriology; Bacteriology; Base Sequence; Biological and medical sciences; Biological Assay; Biotechnology; Cloning, Molecular; Crystallography; DNA Primers; Endotoxins - genetics; Endotoxins - isolation & purification; Endotoxins - toxicity; EXPRESION GENICA; EXPRESSION DES GENES; Fundamental and applied biological sciences. Psychology; GENBANK/AF093626; GENE EXPRESSION; Genes; Genes, Bacterial; Hemolysin Proteins; Insecta - microbiology; Insecticides; Invertebrate Microbiology; Larva; MAMESTRA CONFIGURATA; MESSENGER RNA; Microbiology; MOLECULAR SEQUENCE DATA; Moths; NUCLEOTIDE SEQUENCE; Pest Control, Biological; Polymerase Chain Reaction; Proteins; Repetitive Sequences, Nucleic Acid; SECUENCIA NUCLEOTIDICA; SEQUENCE NUCLEOTIDIQUE; Polymerase chain reaction; Toxin; Entomopathogen; Analysis method; Bacillales; Bacillus thuringiensis; Biological control; HPLC chromatography; Bacteria; Multigene family; Parasporal body; Bacillaceae
• ISSN: 0099-2240; 1098-5336
• DOI: 10.1128/aem.64.12.4782-4788.1998

The cry gene content of Bacillus thuringiensis subsp. aizawai HD-133 was analyzed by a combination of high-pressure liquid chromatography (HPLC) and exclusive PCR. A total of six cry genes were detected in genomic DNA purified from HD-133, four from the cry1 families (cry1Aa, cry1Ab, cry1C, and cry1D) as well as a gene each from the cry2 (cry2B) and the cryII families. To directly determine which genes were expressed and crystallized in the purified parasporal inclusions, solubilized and trypsinized HD-133 crystals were subjected to chromatographic separation by HPLC. Only three proteins, Cry1Ab, Cry1C, and Cry1D, were found, in a 60/37/3 ratio. Dot blot analysis of total mRNA purified from HD-133 showed that both the cry2B and cry1I genes, but not the cry1Aa gene, were transcribed. Cloning and sequencing of the cryAa gene revealed an inserted DNA sequence within the cry coding sequence, resulting in a disrupted reading frame. Taken together, our results show that combining crystal protein analysis with a genetic approach is a highly complementary and powerful way to assess the potential of B. thuringiensis isolates for new insecticidal genes and specificities. Furthermore, based on the number of cryptic genes found in HD-133, the total cry gene content of B. thuringiensis strains may be higher than previously thought