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

ABSTRACT: 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 family (cry1Aa, cry1Ab, cry1C, and cry1D) as well as a gene each from the cry2 (cry2B) and the cry1I 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 cry1Aa 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.

SUBMITTER: Masson L

PROVIDER: S-EPMC90922 | biostudies-literature | 1998 Dec

REPOSITORIES: biostudies-literature

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

Masson L L Erlandson M M Puzstai-Carey M M Brousseau R R Juárez-Pérez V V Frutos R R

Applied and environmental microbiology 19981201 12

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 family (cry1Aa, cry1Ab, cry1C, and cry1D) as well as a gene each from the cry2 (cry2B) and the cry1I families. To directly determine which genes were expressed and crystallized in the purified parasporal inclusions, solubilized and trypsiniz ...[more]

PMID: 9835562

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Project description:For a surrogate bacterium to be used in outdoor studies, it is important to consider environmental and human safety and ease of detection. Recently, Bacillus thuringiensis, a popular bioinsecticide bacterium, has been gaining attention as a surrogate bacterium for use in biodefense. In this study, we constructed simulant strains of B. thuringiensis with enhanced characteristics for environmental studies. Through transposon mutagenesis, pigment genes were inserted into the chromosome, producing yellow-colored colonies for easy detection. To prevent persistence of spores in the environment, a genetic circuit was designed to produce a spore without sporulation capability. Two loxP sites were inserted, one on each side of the spo0A gene, which encodes a sporulation master regulator, and a sporulation-dependent Cre expression cassette was inserted into the chromosome. This genetic circuit successfully deleted spo0A during sporulation, producing spores that lacked the spo0A gene. In addition, two major α/β-type small acid-soluble spore protein (SASP) genes, predicted by synteny analysis, were deleted. The spores of the mutant strain showed increased UV-C sensitivity and quickly lost viability when tested in a solar simulator. When the spores of the mutant strain were administered to the lungs of BALB/c mice, cells were quickly removed from the body, suggesting enhanced in vivo safety. All strains constructed in this study contain no antibiotic resistance markers and all heterologous genes were inserted into the chromosome, which are useful features for simulants to be released into the environment.IMPORTANCEB. thuringiensis has recently been receiving increasing attention as a good spore simulant in biodefense research. However, few studies were done to properly address many important features of B. thuringiensis as a simulant in environmental studies. Since spores can persist in the environment for years after release, environmental contamination is a big problem, especially when genetically engineered strains are used. To solve these problems, we report here the development of B. thuringiensis simulant strains that are capable of forming yellow colonies for easy detection, incapable of forming spores more than once due to a genetic circuit, and lacking in two major SASP genes. The genetic circuit to produce a spore without sporulation capability, together with the deletion of SASP genes, ensures the environmental and human safety of the simulant strains developed in this study. All of these features will allow wider use of B. thuringiensis as a simulant for Bacillus anthracis in environmental release studies.

Project description:Bacillus thuringiensis (Bt) being an eco-friendly bioinsecticide is effectively used in pest management strategies and, therefore, isolation and identification of new strains effective against a broad range of target pests is important. In the present study, new indigenous B. thuringiensis strains were isolated and investigated so that these could be used as an alternative and/or support the current commercial strains/cry proteins in use. For this, 159 samples including soil, leaf and spider webs were collected from ten districts of Kashmir valley (India). Of 1447 bacterial strains screened, 68 Bt strains were identified with 4 types of crystalline inclusions. Crystal morphology ranking among the Bt strains was spherical (69.11%) > spore attached (8.82%) > rod (5.88%) = bipyramidal (5.88%) > spherical plus rod (4.41%) > spherical plus bipyramidal (2.94%) = cuboidal (2.94%). SDS-PAGE investigation of the spore-crystal mixture demonstrated Bt strains contained proteins of various molecular weights ranging from 150 to 28 kDa. Insecticidal activity of the 68 indigenous Bt strains against Spodoptera litura neonates showed that Bt strain SWK1 strain had the highest mortality. Lepidopteron active genes (cry1, cry2Ab, cry2Ab) were present in six Bt strains. Further, analysis of a full-length cry2A gene (~1.9 kb) by PCR-RFLP in strain SWK1 revealed that it was a new cry2A gene in Bt strain SWK1 and was named as cry2Al1 (GenBank Accession No. KJ149819.1) using the Bt toxin nomenclature ( http://www.btnomenclature.info ). Insect bioassays with neonate larvae of S. litura and H. armigera showed that the purified Cry2Al1 is toxic to S. litura with LC50 2.448 µg/ml and H. armigera with LC50 3.374 µg/ml, respectively. However, it did not produce any mortality in third instar larvae of Aedes aegypti, Culex quinquefasciatus and Anopheles stephensi larvae/pupae insects (100 µg/ml) at 28 ± 2 °C and 75 to 85% relative humidity under a photoperiod of 14L:10D.

Project description:Susceptibility of Tribolium castaneum (Tc) larvae was determined against spore-crystal mixtures of five coleopteran specific and one lepidopteran specific Bacillus thuringiensis Cry toxin producing strains and those containing the structurally unrelated Cry3Ba and Cry23Aa/Cry37Aa proteins were found toxic (LC(50) values 13.53 and 6.30 µg spore-crystal mixture/µL flour disc, respectively). Using iTRAQ combined with LC-MS/MS allowed the discovery of seven novel differentially expressed proteins in early response of Tc larvae to the two active spore-crystal mixtures. Proteins showing a statistically significant change in treated larvae compared to non-intoxicated larvae fell into two major categories; up-regulated proteins were involved in host defense (odorant binding protein C12, apolipophorin-III and chemosensory protein 18) and down-regulated proteins were linked to metabolic pathways affecting larval metabolism and development (pyruvate dehydrogenase Eα subunit, cuticular protein, ribosomal protein L13a and apolipoprotein LI-II). Among increased proteins, Odorant binding protein C12 showed the highest change, 4-fold increase in both toxin treatments. The protein displayed amino acid sequence and structural homology to Tenebrio molitor 12 kDa hemolymph protein b precursor, a non-olfactory odorant binding protein. Analysis of mRNA expression and mortality assays in Odorant binding protein C12 silenced larvae were consistent with a general immune defense function of non-olfactory odorant binding proteins. Regarding down-regulated proteins, at the transcriptional level, pyruvate dehydrogenase and cuticular genes were decreased in Tc larvae exposed to the Cry3Ba producing strain compared to the Cry23Aa/Cry37Aa producing strain, which may contribute to the developmental arrest that we observed with larvae fed the Cry3Ba producing strain. Results demonstrated a distinct host transcriptional regulation depending upon the Cry toxin treatment. Knowledge on how insects respond to Bt intoxication will allow designing more effective management strategies for pest control.

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

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

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