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Sample records for produce bacillus thuringiensis

  1. Bacillus thuringiensis

    PubMed Central

    Ibrahim, Mohamed A; Griko, Natalya; Junker, Matthew

    2010-01-01

    Bacillus thuringiensis (Bt) is a unique bacterium in that it shares a common place with a number of chemical compounds which are used commercially to control insects important to agriculture and public health. Although other bacteria, including B. popilliae and B. sphaericus, are used as microbial insecticides, their spectrum of insecticidal activity is quite limited compared to Bt. Importantly, Bt is safe for humans and is the most widely used environmentally compatible biopesticide worldwide. Furthermore, insecticidal Bt genes have been incorporated into several major crops, rendering them insect resistant, and thus providing a model for genetic engineering in agriculture. This review highlights what the authors consider the most relevant issues and topics pertaining to the genomics and proteomics of Bt. At least one of the authors (L.A.B.) has spent most of his professional life studying different aspects of this bacterium with the goal in mind of determining the mechanism(s) by which it kills insects. The other authors have a much shorter experience with Bt but their intellect and personal insight have greatly enriched our understanding of what makes Bt distinctive in the microbial world. Obviously, there is personal interest and bias reflected in this article notwithstanding oversight of a number of published studies. This review contains some material not published elsewhere although several ideas and concepts were developed from a broad base of scientific literature up to 2010. PMID:21327125

  2. An Ultra-Violet Tolerant Wild-Type Strain of Melanin-Producing Bacillus thuringiensis

    PubMed Central

    Sansinenea, Estibaliz; Salazar, Francisco; Ramirez, Melanie; Ortiz, Aurelio

    2015-01-01

    Background: Bacillus thuringiensis is the most successful biological control agent used in agriculture, forestry and mosquito control. However, the insecticidal activity of the B. thuringiensis formulation is not very stable and rapidly loses its biological activity under field conditions, due to the ultraviolet radiation in sunlight. Melanin is known to absorb radiation therefore photo protection of B. thuringiensis based on melanin has been extensively studied. Objectives: The aim of this study was to find a wild type strain of naturally melanin-producing B. thuringiensis to avoid any mutation or manipulation that can affect the Cry protein content. Materials and Methods: Bacillus thuringiensis strains were isolated from soils of different States of Mexico and pigment extraction was followed by lowering the pH to 2 using 1N HCl. Pigment was characterized by some chemical tests based on its solubility, bleaching by H2O2 and flocculation with FeCl3, and using an Infrared (IR) spectrum. Ultraviolet (UV) irradiation experiment was performed to probe the melanin efficacy. Results: ELI52 strain of B. thuringiensis was confirmed to naturally produce melanin. The Cry protein analysis suggested that ELI52 is probably a B. thuringiensis subsp. israelensis strain with toxic activity against the Diptera order of insects. Ultra Violet protection efficacy of melanin was probed counting total viable colonies after UV radiation and comparing the results with the non-producing melanin strain L-DOPA (L-3, 4-dihydroxyphenylalanine) was also detected in the culture. ELI52 strain showed an antagonistic effect over some common bacteria from the environment. Conclusions: ELI52 wild-type strain of B. thuringiensis is a good bio-insecticide that produces melanin with UV-resistance that is probably toxic against the Diptera order of insects and can inhibit the growth of other environmental bacteria. PMID:26421136

  3. Cyt toxins produced by Bacillus thuringiensis: a protein fold conserved in several pathogenic microorganisms.

    PubMed

    Soberón, Mario; López-Díaz, Jazmin A; Bravo, Alejandra

    2013-03-01

    Bacillus thuringiensis bacteria produce different insecticidal proteins known as Cry and Cyt toxins. Among them the Cyt toxins represent a special and interesting group of proteins. Cyt toxins are able to affect insect midgut cells but also are able to increase the insecticidal damage of certain Cry toxins. Furthermore, the Cyt toxins are able to overcome resistance to Cry toxins in mosquitoes. There is an increasing potential for the use of Cyt toxins in insect control. However, we still need to learn more about its mechanism of action in order to define it at the molecular level. In this review we summarize important aspects of Cyt toxins produced by Bacillus thuringiensis, including current knowledge of their mechanism of action against mosquitoes and also we will present a primary sequence and structural comparison with related proteins found in other pathogenic bacteria and fungus that may indicate that Cyt toxins have been selected by several pathogenic organisms to exert their virulence phenotypes.

  4. Ubiquity of parasporin-1 producers in Bacillus thuringiensis natural populations of Japan

    NASA Astrophysics Data System (ADS)

    Uemori, Akiko; Maeda, Minoru; Yasutake, Koichi; Ohgushi, Akira; Kagoshima, Kumiko; Mizuki, Eiichi; Ohba, Michio

    2007-01-01

    Parasporin, a Bacillus thuringiensis parasporal protein, is unique in having a strong cytocidal activity preferential for human cancer cells. In this study, we characterized parasporin activities associated with three novel geographical isolates of B. thuringiensis. Parasporal inclusion proteins of the three isolates were highly toxic to human uterus cervix cancer cells (HeLa), but not to non-cancer uterine smooth muscle cells (UtSMC). Inclusions of the isolates lacked insect toxicity and hemolytic activity against sheep erythrocytes. Ouchterlony immunodiffusion tests revealed that the proteins of the three isolates are immunologically closely related to parasporin-1 (Cry31A), but dissimilar to the three other existing parasporin groups. Our results provide evidence that the parasporin-1-producing organism is a common member in B. thuringiensis populations occurring in natural environments of Japan.

  5. 75 FR 34040 - Bacillus thuringiensis

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-06-16

    ... AGENCY 40 CFR Part 174 Bacillus thuringiensis eCry3.1Ab Protein in Corn; Temporary Exemption from the... Bacillus thuringiensis eCry3.1Ab protein in corn under the FFDCA. The temporary tolerance exemption expires... establishing an exemption from the requirement of a tolerance for residues of Bacillus thuringiensis...

  6. 76 FR 14289 - Bacillus thuringiensis

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-03-16

    ... AGENCY 40 CFR Part 174 Bacillus thuringiensis eCry3.1Ab Protein in Corn; Temporary Exemption From the... permissible level for residues of Bacillus thuringiensis eCry3.1Ab protein in corn. The temporary tolerance... for residues of Bacillus thuringiensis eCry3.1Ab protein in corn. This notice referenced a summary...

  7. Invertebrate pathogenicity and toxin-producing potential of strains of Bacillus thuringiensis endemic to Antarctica.

    PubMed

    Prabhakar, A; Bishop, A H

    2011-06-01

    Several strains of Bacillus thuringiensis were previously isolated from soil in Antarctica and appeared to have physiological adaptations to this cold, nutrient-poor environment. In spite of this they could produce abnormally large, parasporal crystals under laboratory conditions. Here, they have been further characterised for toxin genes and invertebrate pathogenicity. All of the strains were positive in PCR assays for the cry1Aa and cry2 genes. This was confirmed by sequence analysis and the parasporal crystals of all strains contained polypeptides of about 130kDa. This potential for lepidopteran toxicity was borne out in bioassays of purified δ-endotoxins against larvae of Pieris brassicae: the LD(50) values of B2408 (288μg) were comparable to that of the reference strain, HD-12 (201μg). There was no activity against the nematode Caenorhabditis elegans in spite of the fact that all strains appeared to possess the cry6 gene. PCR screening for genes encoding other nematode-toxic classes of toxins (Cry5, 4 and 21) was negative. B. thuringiensis has never previously been shown to be toxic to Collembola (springtails) but the purified δ-endotoxins of one of the Antarctic strains showed some activity against Folsomia candida and Seira domestica (224μg and 238μg, respectively). It seems unlikely that the level of toxicity demonstrated against springtails would support a pathogenic life-style in nature. All of the strains were positive for genes encoding Bacillus cereus-type enterotoxins. In the absence of higher insects and mammals the ecological value of retaining the toxic capability demonstrated here is uncertain. PMID:21457716

  8. Identification and characterization of a novel cytotoxic protein, parasporin-4, produced by Bacillus thuringiensis A1470 strain.

    PubMed

    Okumura, Shiro; Saitoh, Hiroyuki; Ishikawa, Tomoyuki; Mizuki, Eiichi; Inouye, Kuniyo

    2008-01-01

    In 1901, a unique bacterium was isolated as a pathogen of the sotto disease of the silkmoth larvae, and later in 1915, the organism was described as Bacillus thuringiensis. Since the discovery, this bacterium has widely attracted attention of not only insect pathologists but many other scientists who are interested in strong and specific insecticidal activity associated with inclusion bodies of B. thuringiensis. This has led to the recent worldwide development of B. thuringiensis-based microbial insecticides and insect-resistant transgenic plants, as well as the epoch-making discovery of parasporin, a cancer cell-specific cytotoxin. In the review, we introduce a detection study of interaction between inclusion proteins of B. thuringiensis and brush border membrane of insects using surface plasmon resonance-based biosensor, and then identification and cloning of parasporin-4, a latest cancer cell-killing protein produced by B. thuringiensis A1470 strain. Inclusion bodies of the parasporin-4 produced by recombinant Escherichia coli were solubilized and activated with a new method and purified by an anion-exchange chromatography. At last the characterization of the recombinant parasporin-4 was shown. PMID:18606366

  9. Bacillus thuringiensis (Bt)

    NASA Technical Reports Server (NTRS)

    2004-01-01

    Bacillus thuringiensis (Bt), a natural bacteria found all over the Earth, has a fairly novel way of getting rid of unwanted insects. Bt forms a protein substance (shown on the right) that is not harmful to humans, birds, fish or other vertebrates. When eaten by insect larvae the protein causes a fatal loss of appetite. For over 25 years agricultural chemical companies have relied heavily upon safe Bt pesticides. New space based research promises to give the insecticide a new dimension in effectiveness and applicability. Researchers from the Consortium for Materials Development in Space along with industrial affiliates such as Abott Labs and Pern State University flew Bt on a Space Shuttle mission in the fall of 1996. Researchers expect that the Shuttle's microgravity environment will reveal new information about the protein that will make it more effective against a wider variety of pests.

  10. Proteome Response of Tribolium castaneum Larvae to Bacillus thuringiensis Toxin Producing Strains

    PubMed Central

    Contreras, Estefanía; Rausell, Carolina; Real, M. Dolores

    2013-01-01

    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 (LC50 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

  11. Proteome response of Tribolium castaneum larvae to Bacillus thuringiensis toxin producing strains.

    PubMed

    Contreras, Estefanía; Rausell, Carolina; Real, M Dolores

    2013-01-01

    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

  12. Proteolytic processing of a coleopteran-specific delta-endotoxin produced by Bacillus thuringiensis var. tenebrionis.

    PubMed Central

    Carroll, J; Li, J; Ellar, D J

    1989-01-01

    Insecticidal protein delta-endotoxin crystals harvested from sporulated cultures of Bacillus thuringiensis var. tenebrionis contain a major polypeptide of 67 kDa and minor polypeptides of 73, 72, 55 and 46 kDa. During sporulation, only the 73 kDa polypeptide could be detected at stage I. The 67 kDa polypeptide was first detected at stage II and increased in concentration throughout the later stages of sporulation and after crystal release, with a concomitant decrease in the 73 kDa polypeptide. This change could be blocked by the addition of proteinase inhibitors. Trypsin or insect-gut-extract treatment of the delta-endotoxin crystals after solubilization resulted in a cleavage product of 55 kDa with asparagine-159 of the deduced amino acid sequence of the toxin [Höfte, Seurinck, van Houtven & Vaeck (1987) Nucleic Acids Res. 15, 71-83; Sekar, Thompson, Maroney, Bookland & Adang (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 7036-7040; McPherson, Perlak, Fuchs, Marrone, Lavrik & Fischhoff (1988) Biotechnology 6, 61-66] at the N-terminus. This polypeptide was found to be as toxic in vivo as native delta-endotoxin. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. PMID:2549968

  13. Characterization, N-terminal sequencing and classification of Tolworthcin 524: A bacteriocin produced by Bacillus thuringiensis subsp. tolworthi.

    PubMed

    Pacheco-Cano, Rubén D; de la Fuente-Salcido, Norma M; Salcedo-Hernández, Rubén; León-Galván, M Fabiola; Bideshi, Dennis K; Hernández-Guzmán, Gustavo; Barboza-Corona, J Eleazar

    2014-12-01

    Bacteriocins synthesized by entomopathogenic Bacillus thuringiensis are gaining attention owing to their inhibitory effects against a wide variety of pathogenic bacteria. In the present study, we purified and characterized Tolworthcin 524, a bacteriocin synthesized by B. thuringiensis subsp. tolworthi, and compared it with other bacteriocins synthesized by B. thuringiensis. Tolworthcin 524 was separated and purified from the secretome of B. thuringiensis by fast protein liquid chromatography with a gel filtration column to obtain yields of 17% and a specific activity of ∼3600U/mgprotein. The purified product showed two peptides of ∼9 and 6kDa with antimicrobial activity in a gel-screening assay. The purified product was analyzed by two-dimensional electrophoresis and the resolved peptides of ∼9 and 6kDa with isoelectric points of ∼8 were sequenced. Partial sequences (METPVVQPR and DWTCWSCLVCAACS) were obtained suggesting that the ∼9 and 6kDa correspond to the prebacteriocin and mature Tolworthcin 524, respectively. Sequences showed high identity with Thurincin H and Thuricin 17 and had a conserved motif with other bacteriocins of B. thuringiensis. Based on sequence data, Tolworthcin 524 was classified in subclass II.2 (Thuricin-like peptides) of the Bacillus bacteriocin classification scheme. The larger peptide did not harbor a sequence suggestive of a signal peptide neither did it contain the double-glycine (GG) motif characteristic of the secretion leader recognized by the ABC transport system. Implications of these properties in Tolworthcin 524 secretion are discussed.

  14. Bacillus thuringiensis DB27 produces two novel protoxins, Cry21Fa1 and Cry21Ha1, which act synergistically against nematodes.

    PubMed

    Iatsenko, Igor; Boichenko, Iuliia; Sommer, Ralf J

    2014-05-01

    Bacillus thuringiensis has been widely used as a biopesticide, primarily for the control of insect pests, but some B. thuringiensis strains specifically target nematodes. However, nematicidal virulence factors of B. thuringiensis are poorly investigated. Here, we describe virulence factors of nematicidal B. thuringiensis DB27 using Caenorhabditis elegans as a model. We show that B. thuringiensis DB27 kills a number of free-living and animal-parasitic nematodes via intestinal damage. Its virulence factors are plasmid-encoded Cry protoxins, since plasmid-cured derivatives do not produce Cry proteins and are not toxic to nematodes. Whole-genome sequencing of B. thuringiensis DB27 revealed multiple potential nematicidal factors, including several Cry-like proteins encoded by different plasmids. Two of these proteins appear to be novel and show high similarity to Cry21Ba1. Named Cry21Fa1 and Cry21Ha1, they were expressed in Escherichia coli and fed to C. elegans, resulting in intoxication, intestinal damage, and death of nematodes. Interestingly, the effects of the two protoxins on C. elegans are synergistic (synergism factor, 1.8 to 2.5). Using purified proteins, we determined the 50% lethal concentrations (LC50s) for Cry21Fa1 and Cry21Ha1 to be 13.6 μg/ml and 23.9 μg/ml, respectively, which are comparable to the LC50 of nematicidal Cry5B. Finally, we found that signaling pathways which protect C. elegans against Cry5B toxin are also required for protection against Cry21Fa1. Thus, B. thuringiensis DB27 produces novel nematicidal protoxins Cry21Fa1 and Cry21Ha1 with synergistic action, which highlights the importance of naturally isolated strains as a source of novel toxins.

  15. Short communication: Naturally sensitive Bacillus thuringiensis EG10368 produces thurincin H and acquires immunity after heterologous expression of the one-step-amplified thurincin H gene cluster.

    PubMed

    Wang, G; Manns, D C; Churey, J J; Worobo, R W

    2014-07-01

    Heterologous expression of bacteriocin genetic determinants (or operons) has long been a research interest for the functional analysis of genes involved in bacteriocin biosynthesis, regulation, modification, and immunity. Previously, construction of genomic libraries of the bacteriocin producer strains was usually required to identify new bacteriocin operons, a method that is tedious and time consuming. For the first time, we directly amplified an 8.14-kb bioinformatically identified thurincin H gene cluster using a one-step PCR method with 100% accuracy. This amplified gene cluster was cloned into plasmid pHT315, resulting in plasmid pGW139, and subsequently transformed to Bacillus thuringiensis EG10368, a strain naturally sensitive to thurincin H. Heterologous expression of the gene cluster makes the sensitive B. thuringiensis EG10368 produce thurincin H at a higher level compared with the wild-type producer, B. thuringiensis SF361. Moreover, B. thuringiensis EG10368pGW139 acquired complete immunity to thurincin H. The results indicated that one-step PCR is a promising tool to accurately amplify long bacteriocin gene clusters used in bacteriocin functional analysis studies and it is an effective way to produce bacteriocins at a higher level, without the need to clone large chromosomal fragments.

  16. Melanin: a photoprotection for Bacillus thuringiensis based biopesticides.

    PubMed

    Sansinenea, Estibaliz; Ortiz, Aurelio

    2015-03-01

    Melanins are negatively-charged, hydrophobic, dark high molecular weight irregular biopolymers, composed of polymerized phenolic and/or indolic compounds. They are produced by most organisms. Bacillus thuringiensis is a Gram-positive, spore-forming, soil bacterium and the most successful biological control agent that produces distinctly shaped crystals during sporulation that have insecticidal activity. However, one of the main disadvantages is that the insecticidal activity of B. thuringiensis formulation is unstable and rapidly loses its activity under field conditions due to UV radiation. Melanin absorbs radiation; therefore photoprotection of B. thuringiensis based on melanin has been studied and is herewith reviewed.

  17. Keratinolytic proteinase from Bacillus thuringiensis AD-12.

    PubMed

    Gegeckas, Audrius; Gudiukaitė, Renata; Citavicius, Donaldas

    2014-08-01

    A new isolated strain noted to produce a novel detergent-stable serine keratinolytic proteinase and identified as Bacillus thuringiensis AD-12. Native keratinolytic proteinase from B. thuringiensis (BtKER) was purified and characterized. The purified BtKER enzyme is a monomer with a molecular mass of 39kDa. Biochemical characterization assays revealed that the BtKER attained optimal activity at pH 7 and 30°C. Residual activity after 1h incubation at 50°C was higher than 80%. The enzyme was activated and stabilized by Mn(2+) and Li(+) metal ions but inactivated by organic solvents. Purified BtKER showed the highest substrate specificity toward keratin from wool>sodium caseinate>collagen>BSA>gelatin in descending order. BtKER is the first reported keratinolytic proteinase from B. thuringiensis and obtained results suggested that new characterized enzyme can be a powerful biocatalyst in peptide production associated to hydrolysis of keratinous and/or keratin-like waste.

  18. An in-depth characterization of the entomopathogenic strain Bacillus pumilus 15.1 reveals that it produces inclusion bodies similar to the parasporal crystals of Bacillus thuringiensis.

    PubMed

    Garcia-Ramon, Diana C; Molina, C Alfonso; Osuna, Antonio; Vílchez, Susana

    2016-04-01

    In the present work, the local isolate Bacillus pumilus 15.1 has been morphologically and biochemically characterized in order to gain a better understanding of this novel entomopathogenic strain active against Ceratitis capitata. This strain could represent an interesting biothechnological tool for the control of this pest. Here, we report on its nutrient preferences, extracellular enzyme production, motility mechanism, biofilm production, antibiotic suceptibility, natural resistance to chemical and physical insults, and morphology of the vegetative cells and spores. The pathogen was found to be β-hemolytic and susceptible to penicillin, ampicillin, chloramphenicol, gentamicin, kanamycin, rifampicin, tetracycline, and streptomycin. We also report a series of biocide, thermal, and UV treatments that reduce the viability of B. pumilus 15.1 by several orders of magnitude. Heat and chemical treatments kill at least 99.9 % of vegetative cells, but spores were much more resistant. Bleach was the only chemical that was able to completely eliminate B. pumilus 15.1 spores. Compared to the B. subtilis 168 spores, B. pumilus 15.1 spores were between 2.67 and 350 times more resistant to UV radiation while the vegetative cells of B. pumilus 15.1 were almost up to 3 orders of magnitude more resistant than the model strain. We performed electron microscopy for morphological characterization, and we observed geometric structures resembling the parasporal crystal inclusions synthesized by Bacillus thuringiensis. Some of the results obtained here such as the parasporal inclusion bodies produced by B. pumilus 15.1 could potentially represent virulence factors of this novel and potentially interesting strain.

  19. Development of a homologous expression system for and systematic site-directed mutagenesis analysis of thurincin H, a bacteriocin produced by Bacillus thuringiensis SF361.

    PubMed

    Wang, Gaoyan; Manns, David C; Churey, John J; Worobo, Randy W

    2014-06-01

    Thurincin H is an antimicrobial peptide produced by Bacillus thuringiensis SF361. With a helical back bone, the 31 amino acids of thurincin H form a hairpin structure maintained by four pairs of very unique sulfur-to-α-carbon thioether bonds. The production of thurincin H depends on a putative gene cluster containing 10 open reading frames. The gene cluster includes three tandem structural genes (thnA1, thnA2, and thnA3) encoding three identical 40-amino-acid thurincin H prepeptides and seven other genes putatively responsible for prepeptide processing, regulation, modification, exportation, and self-immunity. A homologous thurincin H expression system was developed by transforming a thurincin H-deficient host with a novel expression vector, pGW133. The host, designated B. thuringiensis SF361 ΔthnA1 ΔthnA2 ΔthnA3, was constructed by deletion of the three tandem structural genes from the chromosome of the natural thurincin H producer. The thurincin H expression vector pGW133 was constructed by cloning the thurincin H native promoter, thnA1, and a Cry protein terminator into the Escherichia coli-B. thuringiensis shuttle vector pHT315. Thirty-three different pGW133 variants, each containing a different point mutation in the thnA1 gene, were generated and separately transformed into B. thuringiensis SF361 ΔthnA1 ΔthnA2 ΔthnA3. Those site-directed mutants contained either a single radical or conservative amino acid substitution on the thioether linkage-forming positions or a radical substitution on all other nonalanine amino acids. The bacteriocin activities of B. thuringiensis SF361 ΔthnA1 ΔthnA2 ΔthnA3 carrying different pGW133 variants against three different indicator strains were subsequently compared.

  20. Development of a Homologous Expression System for and Systematic Site-Directed Mutagenesis Analysis of Thurincin H, a Bacteriocin Produced by Bacillus thuringiensis SF361

    PubMed Central

    Wang, Gaoyan; Manns, David C.; Churey, John J.

    2014-01-01

    Thurincin H is an antimicrobial peptide produced by Bacillus thuringiensis SF361. With a helical back bone, the 31 amino acids of thurincin H form a hairpin structure maintained by four pairs of very unique sulfur-to-α-carbon thioether bonds. The production of thurincin H depends on a putative gene cluster containing 10 open reading frames. The gene cluster includes three tandem structural genes (thnA1, thnA2, and thnA3) encoding three identical 40-amino-acid thurincin H prepeptides and seven other genes putatively responsible for prepeptide processing, regulation, modification, exportation, and self-immunity. A homologous thurincin H expression system was developed by transforming a thurincin H-deficient host with a novel expression vector, pGW133. The host, designated B. thuringiensis SF361 ΔthnA1 ΔthnA2 ΔthnA3, was constructed by deletion of the three tandem structural genes from the chromosome of the natural thurincin H producer. The thurincin H expression vector pGW133 was constructed by cloning the thurincin H native promoter, thnA1, and a Cry protein terminator into the Escherichia coli-B. thuringiensis shuttle vector pHT315. Thirty-three different pGW133 variants, each containing a different point mutation in the thnA1 gene, were generated and separately transformed into B. thuringiensis SF361 ΔthnA1 ΔthnA2 ΔthnA3. Those site-directed mutants contained either a single radical or conservative amino acid substitution on the thioether linkage-forming positions or a radical substitution on all other nonalanine amino acids. The bacteriocin activities of B. thuringiensis SF361 ΔthnA1 ΔthnA2 ΔthnA3 carrying different pGW133 variants against three different indicator strains were subsequently compared. PMID:24682301

  1. Bacteriocins synthesized by Bacillus thuringiensis: generalities and potential applications

    PubMed Central

    Salazar-Marroquín, Elma Laura; Galán-Wong, Luis J.; Moreno-Medina, Víctor Ricardo; Reyes-López, Miguel Ángel; Pereyra-Alférez, Benito

    2016-01-01

    The members of the Bacillus thuringiensis group, commonly known as Bt, produce a huge number of metabolites, which show biocidal and antagonistic activity. B. thuringiensis is widely known for synthesizing Cry, Vip and Cyt proteins, active against insects and other parasporins with biocidal activity against certain types of cancerous cells. Nevertheless, B. thuringiensis also synthesizes compounds with antimicrobial activity, especially bacteriocins. Some B. thuringiensis bacteriocins resemble lantibiotics and other small linear peptides (class IIa) from the lactic acid bacteria bacteriocins classification system. Although many bacteriocins produced by Bt have been reported, there is no proper classification for them. In this work, we have grouped these based on molecular weight and functionality. Bacteriocins are small peptides synthesized by bacteria, presenting inhibitory activity against Gram-positive and Gram-negative bacteria and to a lesser extent against fungi. These molecules represent a good study model in the search for microbial control alternatives. Lactic acid bacteria produces a huge number of these types of molecules with great potential. Nonetheless, members of the Bacillus, cereus group, especially B. thuringiensis, emerge as an attractive alternative for obtaining bacteriocins showing novel activities. This review describes the potential applications of B. thuringiensis bacteriocins in the control of foodborne pathogens, environment and medical area. PMID:27340340

  2. PERSISTENCE IN SOIL OF TRANSGENIC PLANT PRODUCED BACILLUS THURINGIENSIS VAR. KURSTAKI O-ENDOTOXIN1

    EPA Science Inventory

    Transgenic plants that produce pesticidal proteins will release these proteins into the soil when these plants are incorporated into the soil by tillage or as leaf litter. Little is known about the fate and persistence of transgenic plant pesticidal products in the soil. We used ...

  3. Role of receptor interaction in the mode of action of insecticidal Cry and Cyt toxins produced by Bacillus thuringiensis.

    PubMed

    Gómez, I; Pardo-López, L; Muñoz-Garay, C; Fernandez, L E; Pérez, C; Sánchez, J; Soberón, M; Bravo, A

    2007-01-01

    Cry toxins from Bacillus thuringiensis are used for insect control. Their primary action is to lyse midgut epithelial cells. In this review we will summarize recent findings on the Cry toxin-receptor interaction and the role of receptor recognition in their mode of action. Cry toxins interact sequentially with multiple receptors. In lepidopteran insects, Cry1A monomeric toxins interact with the first receptor and this interaction triggers oligomerization of the toxins. The oligomer then interacts with second receptor inducing insertion into membrane microdomains and larval death. In the case of mosquitocidal toxins, Cry and Cyt toxins play a part. These toxins have a synergistic effect and Cyt1Aa overcomes Cry toxin resistance. Recently, it was proposed that Cyt1Aa synergizes or suppresses resistance to Cry toxins by functioning as a membrane-bound receptor for Cry toxin. PMID:17145116

  4. BOOK REVIEW – BACILLUS THURINGIENSIS: A CORNERSTONE OF MODERN AGRICULTURE BACILLUS THURINGIENSIS

    EPA Science Inventory

    Are you interested in the technical issues surrounding the use of Bacillus thuringiensis pesticidal traits as sprays and as plant incorporated protectants (transgenic crops)? Should the dimensions of human health, ecology, entomology, risk assessment, resistance management, and d...

  5. Narrow terahertz attenuation signatures in Bacillus thuringiensis.

    PubMed

    Zhang, Weidong; Brown, Elliott R; Viveros, Leamon; Burris, Kellie P; Stewart, C Neal

    2014-10-01

    Terahertz absorption signatures from culture-cultivated Bacillus thuringiensis were measured with a THz photomixing spectrometer operating from 400 to 1200 GHz. We observe two distinct signatures centered at ∼955 and 1015 GHz, and attribute them to the optically coupled particle vibrational resonance (surface phonon-polariton) of Bacillus spores. This demonstrates the potential of the THz attenuation signatures as "fingerprints" for label-free biomolecular detection.

  6. The Complete Genome Sequence of Bacillus thuringiensis AlHakam

    SciTech Connect

    Challacombe, Jean F.; Altherr, Michael R.; Xie, Gary; Bhotika,Smriti S.; Brown, Nancy; Bruce, David; Campbell, Connie S.; Campbell,Mary L.; Chen, Jin; Chertkov, Olga; Cleland, Cathy; Dimitrijevic, Mira; Doggett, Norman A.; Fawcett, John J.; Glavina, Tijana; Goodwin, Lynne A.; Green, Lance D.; Han, Cliff S.; Hill, Karen K.; Hitchcock, Penny; Jackson, Paul J.; Keim, Paul; Kewalramani, Avinash Ramesh; Longmire, Jon; Lucas, Susan; Malfatti, Stephanie; Martinez, Diego; McMurry, Kim; Meincke, Linda J.; Misra, Monica; Moseman, Bernice L.; Mundt, Mark; Munk,A. Christine; Okinaka, Richard T.; Parson-Quintana, B.; Reilly, LeePhilip; Richardson, Paul; Robinson, Donna L.; Rubin, Eddy; Saunders,Elizabeth; Tapia, Roxanne; Tesmer, Judith G.; Thayer, Nina; Thompson,Linda S.; Tice, Hope; Ticknor, Lawrence O.; Wills, Patti L.; Gilna, Paul; Brettin, Thomas S.

    2007-04-01

    Bacillus thuringiensis is an insect pathogen that is widelyused as a biopesticide (3). Here we report the finished, annotated genomesequence of B. thuringiensis Al Hakam, which was collected in Iraq by theUnited Nations Special Commission (2).

  7. A pangenomic study of Bacillus thuringiensis.

    PubMed

    Fang, Yongjun; Li, Zhaolong; Liu, Jiucheng; Shu, Changlong; Wang, Xumin; Zhang, Xiaowei; Yu, Xiaoguang; Zhao, Duojun; Liu, Guiming; Hu, Songnian; Zhang, Jie; Al-Mssallem, Ibrahim; Yu, Jun

    2011-12-20

    Bacillus thuringiensis (B. thuringiensis) is a soil-dwelling Gram-positive bacterium and its plasmid-encoded toxins (Cry) are commonly used as biological alternatives to pesticides. In a pangenomic study, we sequenced seven B. thuringiensis isolates in both high coverage and base-quality using the next-generation sequencing platform. The B. thuringiensis pangenome was extrapolated to have 4196 core genes and an asymptotic value of 558 unique genes when a new genome is added. Compared to the pangenomes of its closely related species of the same genus, B. thuringiensis pangenome shows an open characteristic, similar to B. cereus but not to B. anthracis; the latter has a closed pangenome. We also found extensive divergence among the seven B. thuringiensis genome assemblies, which harbor ample repeats and single nucleotide polymorphisms (SNPs). The identities among orthologous genes are greater than 84.5% and the hotspots for the genome variations were discovered in genomic regions of 2.3-2.8Mb and 5.0-5.6Mb. We concluded that high-coverage sequence assemblies from multiple strains, before all the gaps are closed, are very useful for pangenomic studies. PMID:22196399

  8. A pangenomic study of Bacillus thuringiensis.

    PubMed

    Fang, Yongjun; Li, Zhaolong; Liu, Jiucheng; Shu, Changlong; Wang, Xumin; Zhang, Xiaowei; Yu, Xiaoguang; Zhao, Duojun; Liu, Guiming; Hu, Songnian; Zhang, Jie; Al-Mssallem, Ibrahim; Yu, Jun

    2011-12-20

    Bacillus thuringiensis (B. thuringiensis) is a soil-dwelling Gram-positive bacterium and its plasmid-encoded toxins (Cry) are commonly used as biological alternatives to pesticides. In a pangenomic study, we sequenced seven B. thuringiensis isolates in both high coverage and base-quality using the next-generation sequencing platform. The B. thuringiensis pangenome was extrapolated to have 4196 core genes and an asymptotic value of 558 unique genes when a new genome is added. Compared to the pangenomes of its closely related species of the same genus, B. thuringiensis pangenome shows an open characteristic, similar to B. cereus but not to B. anthracis; the latter has a closed pangenome. We also found extensive divergence among the seven B. thuringiensis genome assemblies, which harbor ample repeats and single nucleotide polymorphisms (SNPs). The identities among orthologous genes are greater than 84.5% and the hotspots for the genome variations were discovered in genomic regions of 2.3-2.8Mb and 5.0-5.6Mb. We concluded that high-coverage sequence assemblies from multiple strains, before all the gaps are closed, are very useful for pangenomic studies.

  9. The effect of seston on mortality of Simulium vittatum (Diptera: Simuliidae) from insecticidal proteins produced by Bacillus thuringiensis subsp. israelensis.

    PubMed

    Iburg, Joseph P; Gray, Elmer W; Wyatt, Roger D; Cox, Julia E; Fusco, Robert A; Noblet, Raymond

    2011-12-01

    Water was collected from a site on the Susquehanna River in eastern Pennsylvania, where less-than-optimal black fly larval mortality had been occasionally observed after treatment with Bacillus thuringiensis subsp. israelensis de Barjac insecticidal crystalline proteins (Bti ICPs). A series of experiments was conducted with Simulium vittatum Zetterstedt larvae to determine the water related factors responsible for the impaired response to Bti ICPs (Vectobac 12S, strain AM 65-52). Seston in the water impaired the effectiveness of the ICPs, whereas the dissolved substances had no impact on larval mortality. Individual components of the seston then were exposed to the larvae followed by exposure to Bti ICPs. Exposure of larvae to selected minerals and nutritive organic material before ICP exposure resulted in no significant decrease in mortality. Exposure of larvae to silicon dioxide, cellulose, viable diatoms, and purified diatom frustules before Bti ICP exposure resulted in significant reductions in mortality. Exposure of larvae to purified diatom frustules from Cyclotella meneghiniana Kützing resulted in the most severe impairment of mortality after Bti ICP exposure. It is postulated that frustule-induced impairment of feeding behavior is responsible for the impairment of larval mortality. PMID:22217757

  10. A selective chromogenic agar that distinguishes Bacillus anthracis from Bacillus cereus and Bacillus thuringiensis.

    PubMed

    Juergensmeyer, Margaret A; Gingras, Bruce A; Restaino, Lawrence; Frampton, Elon W

    2006-08-01

    A selective and differential plating medium, R & F anthracis chromogenic agar (ACA), has been developed for isolating and identifying presumptive colonies of Bacillus anthracis. ACA contains the chromogenic substrate 5-bromo-4-chloro-3-indoxyl-choline phosphate that upon hydrolysis yields teal (blue green) colonies indicating the presence of phosphatidylcholine-specific phospholipase C (PC-PLC) activity. Among seven Bacillus species tested on ACA, only members of the Bacillus cereus group (B. anthracis, B. cereus, and B. thuringiensis) produced teal colonies (PC-PLC positive) having cream rings. Examination of colony morphology in 18 pure culture strains of B. anthracis (15 ATCC strains plus AMES-1-RIID, ANR-1, and AMED-RIID), with one exception, required 48 h at 35 to 37 degrees C for significant color production, whereas only 24 h was required for B. cereus and B. thuringiensis. This differential rate of PC-PLC synthesis in B. anthracis (due to the truncated plcR gene and PlcR regulator in B. anthracis) allowed for the rapid differentiation on ACA of presumptive colonies of B. anthracis from B. cereus and B. thuringiensis in both pure and mixed cultures. Effective recovery of B. anthracis from a variety of matrices having both high (soil and sewage) and low microbial backgrounds (cloth, paper, and blood) spiked with B. anthracis ANR-1 spores suggests the probable utility of ACA plating for B. anthracis recovery in a diversity of applications.

  11. [Spontaneous bacteriophage induction in Bacillus thuringiensis].

    PubMed

    Besaeva, S G; Mikhaĭlov, A A; Petrova, T M; Tur, A I; Bystrova, E V

    1987-01-01

    The production of temperate bacteriophages was studied in the process of batch cultivation of three Bacillus thuringiensis lysogenic strains. Phage titres were determined using an indicator culture (IPM-1148). The growth of bacteriophages was induced when thermoactivated spores germinated. Some cells (1.10(-3)-2.10(-3)) underwent lysis without their division. The subsequent lytic cycles occurred in the actively growing culture. Phage titres ceased to rise before the exponential growth phase was over.

  12. Bacillus cereus and Bacillus thuringiensis isolated in a gastroenteritis outbreak investigation.

    PubMed

    Jackson, S G; Goodbrand, R B; Ahmed, R; Kasatiya, S

    1995-08-01

    During investigation of a gastroenteritis outbreak in a chronic care institution, Norwalk virus was found in stool specimens from two individuals and bacterial isolates presumptively identified as Bacillus cereus were isolated from four individuals (including one with Norwalk virus) and spice. Phage typing confirmed all Bacillus clinical isolates were phage type 2. All clinical isolates were subsequently identified as B. thuringiensis when tested as a result of a related study (L. Leroux, personal communication). Eight of 10 spice isolates were phage type 4. All B. cereus and B. thuringiensis isolates showed cytotoxic effects characteristic of enterotoxin-producing B. cereus. An additional 20 isolates each of B. cereus and B. thuringiensis from other sources were tested for cytotoxicity. With the exception of one B. cereus, all showed characteristic cytotoxic patterns.

  13. Insecticidal crystal proteins of Bacillus thuringiensis.

    PubMed Central

    Höfte, H; Whiteley, H R

    1989-01-01

    A classification for crystal protein genes of Bacillus thuringiensis is presented. Criteria used are the insecticidal spectra and the amino acid sequences of the encoded proteins. Fourteen genes are distinguished, encoding proteins active against either Lepidoptera (cryI), Lepidoptera and Diptera (cryII), Coleoptera (cryIII), or Diptera (cryIV). One gene, cytA, encodes a general cytolytic protein and shows no structural similarities with the other genes. Toxicity studies with single purified proteins demonstrated that every described crystal protein is characterized by a highly specific, and sometimes very restricted, insect host spectrum. Comparison of the deduced amino acid sequences reveals sequence elements which are conserved for Cry proteins. The expression of crystal protein genes is affected by a number of factors. Recently, two distinct sigma subunits regulating transcription during different stages of sporulation have been identified, as well as a protein regulating the expression of a crystal protein at a posttranslational level. Studies on the biochemical mechanisms of toxicity suggest that B. thuringiensis crystal proteins induce the formation of pores in membranes of susceptible cells. In vitro binding studies with radiolabeled toxins demonstrated a strong correlation between the specificity of B. thuringiensis toxins and the interaction with specific binding sites on the insect midgut epithelium. The expression of B. thuringiensis crystal proteins in plant-associated microorganisms and in transgenic plants has been reported. These approaches are potentially powerful strategies for the protection of agriculturally important crops against insect damage. Images PMID:2666844

  14. Bacillus thuringiensis and Its Pesticidal Crystal Proteins

    PubMed Central

    Schnepf, E.; Crickmore, N.; Van Rie, J.; Lereclus, D.; Baum, J.; Feitelson, J.; Zeigler, D. R.; Dean, D. H.

    1998-01-01

    During the past decade the pesticidal bacterium Bacillus thuringiensis has been the subject of intensive research. These efforts have yielded considerable data about the complex relationships between the structure, mechanism of action, and genetics of the organism’s pesticidal crystal proteins, and a coherent picture of these relationships is beginning to emerge. Other studies have focused on the ecological role of the B. thuringiensis crystal proteins, their performance in agricultural and other natural settings, and the evolution of resistance mechanisms in target pests. Armed with this knowledge base and with the tools of modern biotechnology, researchers are now reporting promising results in engineering more-useful toxins and formulations, in creating transgenic plants that express pesticidal activity, and in constructing integrated management strategies to insure that these products are utilized with maximum efficiency and benefit. PMID:9729609

  15. Bacillus thuringiensis membrane-damaging toxins acting on mammalian cells.

    PubMed

    Celandroni, Francesco; Salvetti, Sara; Senesi, Sonia; Ghelardi, Emilia

    2014-12-01

    Bacillus thuringiensis is widely used as a biopesticide in forestry and agriculture, being able to produce potent species-specific insecticidal toxins and considered nonpathogenic to other animals. More recently, however, repeated observations are documenting the association of this microorganism with various infectious diseases in humans, such as food-poisoning-associated diarrheas, periodontitis, bacteremia, as well as ocular, burn, and wound infections. Similar to B. cereus, B. thuringiensis produces an array of virulence factors acting against mammalian cells, such as phosphatidylcholine- and phosphatidylinositol-specific phospholipase C (PC-PLC and PI-PLC), hemolysins, in particular hemolysin BL (HBL), and various enterotoxins. The contribution of some of these toxins to B. thuringiensis pathogenicity has been studied in animal models of infection, following intravitreous, intranasal, or intratracheal inoculation. These studies lead to the speculation that the activities of PC-PLC, PI-PLC, and HBL are responsible for most of the pathogenic properties of B. thuringiensis in nongastrointestinal infections in mammals. This review summarizes data regarding the biological activity, the genetic basis, and the structural features of these membrane-damaging toxins.

  16. Cry-like genes, in an uncommon gene configuration, produce a crystal that localizes within the exosporium when expressed in an acrystalliferous strain of Bacillus thuringiensis.

    PubMed

    Ammons, David; Toal, Graham; Roman, Angel; Rojas-Avelizapa, Luz I; Ventura-Suárez, Antonio; Rampersad, Joanne

    2016-02-01

    Cry proteins are pesticidal toxins produced by the bacterium Bacillus thuringiensis (Bt), which aggregate in sporulating cells to form a crystal. Except in a relatively few cases, these crystals are located outside the exosporium that surrounds the spore. Bt2-56 is a strain of Bt that has the relatively uncommon characteristic of locating its Cry protein-containing crystal within the exosporium, and in association with a long, multifiber filament. With the ultimate goal of both understanding and manipulating the localization of Cry proteins within the exosporium, we sought to identify the genes coding for the exosporium-localized Cry proteins in Bt2-56. Herein we show (i) that five cry-like genes are present in the genome of Bt2-56, (ii) that two pairs of these genes show organizational similarity to a relatively uncommon gene configuration that coexpress a cry gene along with a gene whose product aids crystal formation and (iii) that when one of these two gene pairs (cry21A-cdA) is expressed in an acrystalliferous strain of Bt, crystals are formed that localize within the exosporium. In Bt ssp. finitimus, the only other strain in which crystal localization has been studied, a Cry protein needed expression of two non-cry ORFs in order to localize within the exosporium, indicating that there are some mechanistic differences for crystal localization between Bt ssp. finitimus and Bt2-56.

  17. Cry-like genes, in an uncommon gene configuration, produce a crystal that localizes within the exosporium when expressed in an acrystalliferous strain of Bacillus thuringiensis.

    PubMed

    Ammons, David; Toal, Graham; Roman, Angel; Rojas-Avelizapa, Luz I; Ventura-Suárez, Antonio; Rampersad, Joanne

    2016-02-01

    Cry proteins are pesticidal toxins produced by the bacterium Bacillus thuringiensis (Bt), which aggregate in sporulating cells to form a crystal. Except in a relatively few cases, these crystals are located outside the exosporium that surrounds the spore. Bt2-56 is a strain of Bt that has the relatively uncommon characteristic of locating its Cry protein-containing crystal within the exosporium, and in association with a long, multifiber filament. With the ultimate goal of both understanding and manipulating the localization of Cry proteins within the exosporium, we sought to identify the genes coding for the exosporium-localized Cry proteins in Bt2-56. Herein we show (i) that five cry-like genes are present in the genome of Bt2-56, (ii) that two pairs of these genes show organizational similarity to a relatively uncommon gene configuration that coexpress a cry gene along with a gene whose product aids crystal formation and (iii) that when one of these two gene pairs (cry21A-cdA) is expressed in an acrystalliferous strain of Bt, crystals are formed that localize within the exosporium. In Bt ssp. finitimus, the only other strain in which crystal localization has been studied, a Cry protein needed expression of two non-cry ORFs in order to localize within the exosporium, indicating that there are some mechanistic differences for crystal localization between Bt ssp. finitimus and Bt2-56. PMID:26781916

  18. Midgut microbiota and host immunocompetence underlie Bacillus thuringiensis killing mechanism.

    PubMed

    Caccia, Silvia; Di Lelio, Ilaria; La Storia, Antonietta; Marinelli, Adriana; Varricchio, Paola; Franzetti, Eleonora; Banyuls, Núria; Tettamanti, Gianluca; Casartelli, Morena; Giordana, Barbara; Ferré, Juan; Gigliotti, Silvia; Ercolini, Danilo; Pennacchio, Francesco

    2016-08-23

    Bacillus thuringiensis is a widely used bacterial entomopathogen producing insecticidal toxins, some of which are expressed in insect-resistant transgenic crops. Surprisingly, the killing mechanism of B. thuringiensis remains controversial. In particular, the importance of the septicemia induced by the host midgut microbiota is still debated as a result of the lack of experimental evidence obtained without drastic manipulation of the midgut and its content. Here this key issue is addressed by RNAi-mediated silencing of an immune gene in a lepidopteran host Spodoptera littoralis, leaving the midgut microbiota unaltered. The resulting cellular immunosuppression was characterized by a reduced nodulation response, which was associated with a significant enhancement of host larvae mortality triggered by B. thuringiensis and a Cry toxin. This was determined by an uncontrolled proliferation of midgut bacteria, after entering the body cavity through toxin-induced epithelial lesions. Consequently, the hemolymphatic microbiota dramatically changed upon treatment with Cry1Ca toxin, showing a remarkable predominance of Serratia and Clostridium species, which switched from asymptomatic gut symbionts to hemocoelic pathogens. These experimental results demonstrate the important contribution of host enteric flora in B. thuringiensis-killing activity and provide a sound foundation for developing new insect control strategies aimed at enhancing the impact of biocontrol agents by reducing the immunocompetence of the host. PMID:27506800

  19. Mortality of adult Stomoxys calcitrans fed isolates of Bacillus thuringiensis.

    PubMed

    Lysyk, T J; Kalischuk-Tymensen, L D; Selinger, L B

    2012-10-01

    We examined the ability of five isolates of Bacillus thuringiensis Berliner to cause mortality in adult stable flies, Stomoxys calcitrans (L.). Isolates Bacillus thuringiensis tolworthi 4L3 (serotype 9), Bacillus thuringiensis darmstadiensis 4M1 (serotype 10a10b), Bacillus thuringiensis thompsoni 401 (serotype 12), Bacillus thuringiensis thuringiensis HD2 (serotype 1), and Bacillus thuringiensis kurstaki HD945 (serotype 3a3b3c) were administered to adult flies in diets containing blood only, sugar only, and both sugar and blood combined. B. t. tolworthi 4L3 had no effect on adult mortality regardless of the feeding substrate. The remaining isolates tended to cause the greatest mortality when administered in blood alone. B. t. thompsoni 401 was the only isolate that consistently caused adult mortality when fed in blood at concentrations ranging from 0.21 to 50.0 microg of protein per ml of blood. This isolate also caused mortality when applied topically. The time to 50% mortality declined with dose and reached a lower asymptote at approximately equal to 1.3 d at an oral dose of 8.75 microg/ml and at a topical dose of 0.14 microg per fly.

  20. BOOK REVIEW: BACILLUS THURINGIENSIS: A CORNERSTONE OF MODERN AGRICULTURE

    EPA Science Inventory

    Are you interested in the technical issues surrounding the use of Bacillus thuringiensis pesticidal traits as sprays and as plant incorporated protectants (transgenic crops)? Should the dimensions of human health, ecology, entomology, risk assessment, resistance management, and d...

  1. Expression of Bacillus thuringiensis delta-endotoxin genes during vegetative growth.

    PubMed Central

    Mettus, A M; Macaluso, A

    1990-01-01

    Bacillus thuringiensis delta-endotoxin (crystal protein) genes are normally expressed only during sporulation. It is possible to produce crystal protein during vegetative growth by placing B. thuringiensis crystal protein genes downstream of a strong vegetative promoter. By removing a possible transcriptional terminator of the tetracycline resistance gene of pBC16 and inserting a multiple cloning site, delta-endotoxin genes can be cloned downstream from the tetracycline resistance gene promoter. This construct allows for readthrough transcription from the strong vegetative promoter. Crystal protein is then produced during vegetative growth as well as during sporulation in both B. thuringiensis and Bacillus megaterium. This construct also allows for production of delta-endotoxin in B. thuringiensis strains that do not normally produce delta-endotoxin because of a defect in sporulation. Images PMID:2160219

  2. Distribution of Toxin Genes and Enterotoxins in Bacillus thuringiensis Isolated from Microbial Insecticide Products.

    PubMed

    Cho, Seung-Hak; Kang, Suk-Ho; Lee, Yea-Eun; Kim, Sung-Jo; Yoo, Young-Bin; Bak, Yeong-Seok; Kim, Jung-Beom

    2015-12-28

    Bacillus thuringiensis microbial insecticide products have been applied worldwide. Although a few cases of B. thuringiensis foodborne illness have been reported, little is known about the toxigenic properties of B. thuringiensis isolates. The aims of this study were to estimate the pathogenic potential of B. thuringiensis selected from microbial insecticide products, based on its possession of toxin genes and production of enterotoxins. Fifty-two B. thuringiensis strains selected from four kinds of microbial insecticide products were analyzed. PCR assay for detection of toxin genes and immunoassay for detection of enterotoxins were performed. The hemolysin BL complex as a major enterotoxin was produced by 17 (32.7%), whereas the nonhemolytic enterotoxin complex was detected in 1 (1.9%) of 52 B. thuringiensis strains. However, cytK, entFM, and ces genes were not detected in any of the tested B. thuringiensis strains. The potential risk of food poisoning by B. thuringiensis along with concerns over B. thuringiensis microbial insecticide products has gained attention recently. Thus, microbial insecticide products based on B. thuringiensis should be carefully controlled.

  3. Bacillus thuringiensis Conjugation in Simulated Microgravity

    NASA Astrophysics Data System (ADS)

    Beuls, Elise; van Houdt, Rob; Leys, Natalie; Dijkstra, Camelia; Larkin, Oliver; Mahillon, Jacques

    2009-10-01

    Spaceflight experiments have suggested a possible effect of microgravity on the plasmid transfer among strains of the Gram-positive Bacillus thuringiensis, as opposed to no effect recorded for Gram-negative conjugation. To investigate these potential effects in a more affordable experimental setup, three ground-based microgravity simulators were tested: the Rotating Wall Vessel (RWV), the Random Positioning Machine (RPM), and a superconducting magnet. The bacterial conjugative system consisted in biparental matings between two B. thuringiensis strains, where the transfer frequencies of the conjugative plasmid pAW63 and its ability to mobilize the nonconjugative plasmid pUB110 were assessed. Specifically, potential plasmid transfers in a 0-g position (simulated microgravity) were compared to those obtained under 1-g (normal gravity) condition in each device. Statistical analyses revealed no significant difference in the conjugative and mobilizable transfer frequencies between the three different simulated microgravitational conditions and our standard laboratory condition. These important ground-based observations emphasize the fact that, though no stimulation of plasmid transfer was observed, no inhibition was observed either. In the case of Gram-positive bacteria, this ability to exchange plasmids in weightlessness, as occurs under Earth's conditions, should be seen as particularly relevant in the scope of spread of antibiotic resistances and bacterial virulence.

  4. Complete Genome Sequence of Bacillus thuringiensis Bacteriophage Smudge

    PubMed Central

    Cornell, Jessica L.; Breslin, Eileen; Schuhmacher, Zachary; Himelright, Madison; Berluti, Cassandra; Boyd, Charles; Carson, Rachel; Del Gallo, Elle; Giessler, Caris; Gilliam, Benjamin; Heatherly, Catherine; Nevin, Julius; Nguyen, Bryan; Nguyen, Justin; Parada, Jocelyn; Sutterfield, Blake; Tukruni, Muruj

    2016-01-01

    Smudge, a bacteriophage enriched from soil using Bacillus thuringiensis DSM-350 as the host, had its complete genome sequenced. Smudge is a myovirus with a genome consisting of 292 genes and was identified as belonging to the C1 cluster of Bacillus phages. PMID:27540049

  5. Complete Genome Sequence of Bacillus thuringiensis Bacteriophage Smudge.

    PubMed

    Cornell, Jessica L; Breslin, Eileen; Schuhmacher, Zachary; Himelright, Madison; Berluti, Cassandra; Boyd, Charles; Carson, Rachel; Del Gallo, Elle; Giessler, Caris; Gilliam, Benjamin; Heatherly, Catherine; Nevin, Julius; Nguyen, Bryan; Nguyen, Justin; Parada, Jocelyn; Sutterfield, Blake; Tukruni, Muruj; Temple, Louise

    2016-01-01

    Smudge, a bacteriophage enriched from soil using Bacillus thuringiensis DSM-350 as the host, had its complete genome sequenced. Smudge is a myovirus with a genome consisting of 292 genes and was identified as belonging to the C1 cluster of Bacillus phages. PMID:27540049

  6. 40 CFR 180.1011 - Viable spores of the microorganism Bacillus thuringiensis Berliner; exemption from the...

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... Bacillus thuringiensis Berliner; exemption from the requirement of a tolerance. 180.1011 Section 180.1011... microorganism Bacillus thuringiensis Berliner; exemption from the requirement of a tolerance. (a) For the... authentic strain of Bacillus thuringiensis Berliner conforming to the morphological and...

  7. 40 CFR 180.1011 - Viable spores of the microorganism Bacillus thuringiensis Berliner; exemption from the...

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... Bacillus thuringiensis Berliner; exemption from the requirement of a tolerance. 180.1011 Section 180.1011... microorganism Bacillus thuringiensis Berliner; exemption from the requirement of a tolerance. (a) For the... authentic strain of Bacillus thuringiensis Berliner conforming to the morphological and...

  8. Complete genome sequence of Bacillus thuringiensis strain HD521.

    PubMed

    Li, Qiao; Xu, Li Z; Zou, Ting; Ai, Peng; Huang, Gang H; Li, Ping; Zheng, Ai P

    2015-01-01

    Bacillus thuringiensis is the most widely used biological pesticide in the world. It belongs to the Bacillus cereus sensu lato group, which contains six species. Among these six species, B. thuringiensis, B. anthracis, and B. cereus have a low genetic diversity. B. thuringiensis strain HD521 shows maroon colony which is different from most of the B. thuringiensis strains. Strain HD521 also displays an ability to inhibit plant sheath blight disease pathogen (Rhizoctonia solani AG1 IB) growth and can form bipyramidal parasporal crystals consisting of three cry7 genes. These crystals have an insecticidal activity against Henosepilachna vigintioctomaculata larva (Coleoptera). Here we report the complete genome sequence of strain HD521, which has one chromosome and six circular plasmids.

  9. Bacillus thuringiensis in fecal samples from greenhouse workers after exposure to B. thuringiensis-based pesticides.

    PubMed

    Jensen, Gert B; Larsen, Preben; Jacobsen, Bodil L; Madsen, Bodil; Smidt, Lasse; Andrup, Lars

    2002-10-01

    In a study of occupational exposure to Bacillus thuringiensis, 20 exposed greenhouse workers were examined for Bacillus cereus-like bacteria in fecal samples and on biomonitoring filters. Bacteria with the following characteristics were isolated from eight individuals: intracellular crystalline inclusions characteristic of B. thuringiensis, genes for and production of B. cereus enterotoxins, and positivity for cry11 as determined by PCR. DNA fingerprints of the fecal isolates were identical to those of strains isolated from the commercial products used. Work processes (i.e., spraying) correlated with the presence of B. thuringiensis in the fecal samples (10(2) to 10(3) CFU/g of feces). However, no gastrointestinal symptoms correlated with the presence of B. thuringiensis in the fecal samples.

  10. Mechanism of Insect Resistance to the Microbial Insecticide Bacillus thuringiensis

    NASA Astrophysics Data System (ADS)

    van Rie, J.; McGaughey, W. H.; Johnson, D. E.; Barnett, B. D.; van Mellaert, H.

    1990-01-01

    Receptor binding studies show that resistance of a laboratory-selected Plodia interpunctella strain to a Bacillus thuringiensis insecticidal crystal protein (ICP) is correlated with a 50-fold reduction in affinity of the membrane receptor for this protein. The strain is sensitive to a second type of ICP that apparently recognizes a different receptor. Understanding the mechanism of resistance will provide strategies to prevent or delay resistance and hence prolong the usefulness of B. thuringiensis ICPs as environmentally safe insecticides.

  11. A Novel Tenebrio molitor Cadherin is a Functional Receptor for Bacillus thuringiensis Toxin Cry3Aa

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cry toxins produced by the bacterium Bacillus thuringiensis (Bt) are effective biological insecticides. Cadherin-like proteins have been reported as functional Cry1A toxin receptors in Lepidoptera. We present the first report demonstrating a functional interaction between the coleopteran-specific ...

  12. Occurrence of Natural Bacillus thuringiensis Contaminants and Residues of Bacillus thuringiensis-Based Insecticides on Fresh Fruits and Vegetables

    PubMed Central

    Frederiksen, Kristine; Rosenquist, Hanne; Jørgensen, Kirsten; Wilcks, Andrea

    2006-01-01

    A total of 128 Bacillus cereus-like strains isolated from fresh fruits and vegetables for sale in retail shops in Denmark were characterized. Of these strains, 39% (50/128) were classified as Bacillus thuringiensis on the basis of their content of cry genes determined by PCR or crystal proteins visualized by microscopy. Random amplified polymorphic DNA analysis and plasmid profiling indicated that 23 of the 50 B. thuringiensis strains were of the same subtype as B. thuringiensis strains used as commercial bioinsecticides. Fourteen isolates were indistinguishable from B. thuringiensis subsp. kurstaki HD1 present in the products Dipel, Biobit, and Foray, and nine isolates grouped with B. thuringiensis subsp. aizawai present in Turex. The commercial strains were primarily isolated from samples of tomatoes, cucumbers, and peppers. A multiplex PCR method was developed to simultaneously detect all three genes in the enterotoxin hemolysin BL (HBL) and the nonhemolytic enterotoxin (NHE), respectively. This revealed that the frequency of these enterotoxin genes was higher among the strains indistinguishable from the commercial strains than among the other B. thuringiensis and B. cereus-like strains isolated from fruits and vegetables. The same was seen for a third enterotoxin, CytK. In conclusion, the present study strongly indicates that residues of B. thuringiensis-based insecticides can be found on fresh fruits and vegetables and that these are potentially enterotoxigenic. PMID:16672488

  13. Bacillus thuringiensis: a century of research, development and commercial applications.

    PubMed

    Sanahuja, Georgina; Banakar, Raviraj; Twyman, Richard M; Capell, Teresa; Christou, Paul

    2011-04-01

    Bacillus thuringiensis (Bt) is a soil bacterium that forms spores during the stationary phase of its growth cycle. The spores contain crystals, predominantly comprising one or more Cry and/or Cyt proteins (also known as δ-endotoxins) that have potent and specific insecticidal activity. Different strains of Bt produce different types of toxin, each of which affects a narrow taxonomic group of insects. Therefore, Bt toxins have been used as topical pesticides to protect crops, and more recently the proteins have been expressed in transgenic plants to confer inherent pest resistance. Bt transgenic crops have been overwhelmingly successful and beneficial, leading to higher yields and reducing the use of chemical pesticides and fossil fuels. However, their deployment has attracted some criticism particularly with regard to the potential evolution of pest-resistant insect strains. Here, we review recent progress in the development of Bt technology and the countermeasures that have been introduced to prevent the evolution of resistant insect populations.

  14. A strategy for shuffling numerous Bacillus thuringiensis crystal protein domains.

    PubMed

    Knight, Jacqueline S; Broadwell, Andrew H; Grant, Warwick N; Shoemaker, Charles B

    2004-12-01

    Bacillus thuringiensis that produce Cry1Ba are toxic to Lucilia cuprina Wiedemann blow fly maggots in vivo, and when applied in quantity to sheep fleece, provide up to 6 wk protection against flystrike in the field. These strains also are toxic to Epiphyas postvittana (Walker) light brown apple moth caterpillars. B. thuringiensis expressing Cry1Db are toxic only to E. postvittana. When Cry1Ba and Cry1Db proteins are expressed within Escherichia coli, the recombinant bacteria have the same toxicity profile as the wild-type B. thuringiensis strain. In an effort to develop a Cry protein with improved blow fly toxicity, three different internal regions of Cry1Ba coding DNA, encoding all or part of domains I, II and III respectively were systematically exchanged with the corresponding region from a pool of other Cry protein coding DNAs. The chimeric products were then expressed in recombinant E. coli, and the resulting bacteria assayed for toxicity on L. cuprina and E. postvittana. Clones having insecticide bioactivity were characterized to identify the source of the replacement Cry domain. Despite successfully expressing a large number and variety of chimeric proteins within E. coli, many with measurable insecticidal activity, none of the chimeras had greater potency against L. cuprina than the wild-type Cry1Ba. Chimeric replacements involving domains I and II were rarely active, whereas a much higher proportion of domain III chimeras had some bioactivity. We conclude that shuffling of Cry coding regions through joining at the major conserved sequence motifs is an effective means for the production of a diverse number of chimeric Cry proteins but that such toxins with enhanced bioactive properties will be rare or nonexistent. PMID:15666731

  15. Effects of transgenic Bacillus thuringiensis maize grain on B. thuringiensis-susceptible Plodia interpunctella (Lepidoptera: Pyralidae).

    PubMed

    Giles, K L; Hellmich, R L; Iverson, C T; Lewis, L C

    2000-06-01

    Percentage survivorship, developmental time, adult body length, and sex ratio of Plodia interpunctella (Hübner) reared on field-produced grain from sixteen cultivars of maize, Zea mays L., including several transgenic Bacillus thuringiensis (Bt) Berliner hybrids and selected non-Bt isolines, were evaluated under laboratory conditions. Compared with isolines, development was delayed and survivorship reduced for P. interpunctella reared on grain from transgenic hybrids with the CaMV/35s promoter that express Cry1Ab protein. Similarly, compared with non-Bt hybrids, a transgenic hybrid with the CaMV/35s promoter that expresses Cry9C protein delayed development, decreased survivorship, and caused reductions in adult body length of P. interpunctella. In contrast, no significant differences in P. interpunctella developmental times or survivorship were observed between transgenic hybrids with the PEPC promoter expressing Cry1Ab and their isolines. Additionally, developmental time, survivorship, and adult body length were similar between P. interpunctella reared on a transgenic hybrid with the CaMV/35s promoter expressing Cry1Ac and non-Bt hybrids. Our data demonstrate that transgenic Bt maize grain, especially grain from hybrids with the CaMV/35s promoter expressing Cry1Ab or Cry9C, can significantly affect B. thuringiensis-susceptible P. interpunctella populations up to 4 or 5 mo after harvest.

  16. Parallel Evolution of Bacillus thuringiensis Toxin Resistance in Lepidoptera

    PubMed Central

    Baxter, Simon W.; Badenes-Pérez, Francisco R.; Morrison, Anna; Vogel, Heiko; Crickmore, Neil; Kain, Wendy; Wang, Ping; Heckel, David G.; Jiggins, Chris D.

    2011-01-01

    Despite the prominent and worldwide use of Bacillus thuringiensis (Bt) insecticidal toxins in agriculture, knowledge of the mechanism by which they kill pests remains incomplete. Here we report genetic mapping of a membrane transporter (ABCC2) to a locus controlling Bt Cry1Ac toxin resistance in two lepidopterans, implying that this protein plays a critical role in Bt function. PMID:21840855

  17. Interactions of transgenic Bacillus thuringiensis insecticidal crops with spiders (Araneae)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Genetically modified crops expressing insecticidal proteins from Bacillus thuringiensis (Bt) have dramatically increased in acreage since their introduction in the mid-1990’s. Although the insecticidal mechanisms of Bt target specific pests, concerns persist regarding direct and indirect effects on...

  18. Decreased Toxicity of Bacillus thuringiensis subsp. israelensis to Mosquito Larvae after Contact with Leaf Litter

    PubMed Central

    Stalinski, Renaud; Kersusan, Dylann; Veyrenc, Sylvie; David, Jean-Philippe; Reynaud, Stéphane; Després, Laurence

    2012-01-01

    Bacillus thuringiensis subsp. israelensis is a bacterium producing crystals containing Cry and Cyt proteins, which are toxic for mosquito larvae. Nothing is known about the interaction between crystal toxins and decaying leaf litter, which is a major component of several mosquito breeding sites and represents an important food source. In the present work, we investigated the behavior of B. thuringiensis subsp. israelensis toxic crystals sprayed on leaf litter. In the presence of leaf litter, a 60% decrease in the amount of Cyt toxin detectable by immunology (enzyme-linked immunosorbent assays [ELISAs]) was observed, while the respective proportions of Cry toxins were not affected. The toxicity of Cry toxins toward Aedes aegypti larvae was not affected by leaf litter, while the synergistic effect of Cyt toxins on all B. thuringiensis subsp. israelensis Cry toxins was decreased by about 20% when mixed with leaf litter. The toxicity of two commercial B. thuringiensis subsp. israelensis strains (VectoBac WG and VectoBac 12AS) and a laboratory-produced B. thuringiensis subsp. israelensis strain decreased by about 70% when mixed with leaf litter. Taken together, these results suggest that Cyt toxins interact with leaf litter, resulting in a decreased toxicity of B. thuringiensis subsp. israelensis in litter-rich environments and thereby dramatically reducing the efficiency of mosquitocidal treatments. PMID:22610426

  19. Phages preying on Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis: past, present and future.

    PubMed

    Gillis, Annika; Mahillon, Jacques

    2014-07-01

    Many bacteriophages (phages) have been widely studied due to their major role in virulence evolution of bacterial pathogens. However, less attention has been paid to phages preying on bacteria from the Bacillus cereus group and their contribution to the bacterial genetic pool has been disregarded. Therefore, this review brings together the main information for the B. cereus group phages, from their discovery to their modern biotechnological applications. A special focus is given to phages infecting Bacillus anthracis, B. cereus and Bacillus thuringiensis. These phages belong to the Myoviridae, Siphoviridae, Podoviridae and Tectiviridae families. For the sake of clarity, several phage categories have been made according to significant characteristics such as lifestyles and lysogenic states. The main categories comprise the transducing phages, phages with a chromosomal or plasmidial prophage state, γ-like phages and jumbo-phages. The current genomic characterization of some of these phages is also addressed throughout this work and some promising applications are discussed here.

  20. Phages preying on Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis: past, present and future.

    PubMed

    Gillis, Annika; Mahillon, Jacques

    2014-07-01

    Many bacteriophages (phages) have been widely studied due to their major role in virulence evolution of bacterial pathogens. However, less attention has been paid to phages preying on bacteria from the Bacillus cereus group and their contribution to the bacterial genetic pool has been disregarded. Therefore, this review brings together the main information for the B. cereus group phages, from their discovery to their modern biotechnological applications. A special focus is given to phages infecting Bacillus anthracis, B. cereus and Bacillus thuringiensis. These phages belong to the Myoviridae, Siphoviridae, Podoviridae and Tectiviridae families. For the sake of clarity, several phage categories have been made according to significant characteristics such as lifestyles and lysogenic states. The main categories comprise the transducing phages, phages with a chromosomal or plasmidial prophage state, γ-like phages and jumbo-phages. The current genomic characterization of some of these phages is also addressed throughout this work and some promising applications are discussed here. PMID:25010767

  1. Phages Preying on Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis: Past, Present and Future

    PubMed Central

    Gillis, Annika; Mahillon, Jacques

    2014-01-01

    Many bacteriophages (phages) have been widely studied due to their major role in virulence evolution of bacterial pathogens. However, less attention has been paid to phages preying on bacteria from the Bacillus cereus group and their contribution to the bacterial genetic pool has been disregarded. Therefore, this review brings together the main information for the B. cereus group phages, from their discovery to their modern biotechnological applications. A special focus is given to phages infecting Bacillus anthracis, B. cereus and Bacillus thuringiensis. These phages belong to the Myoviridae, Siphoviridae, Podoviridae and Tectiviridae families. For the sake of clarity, several phage categories have been made according to significant characteristics such as lifestyles and lysogenic states. The main categories comprise the transducing phages, phages with a chromosomal or plasmidial prophage state, γ-like phages and jumbo-phages. The current genomic characterization of some of these phages is also addressed throughout this work and some promising applications are discussed here. PMID:25010767

  2. Cultivable gut bacteria of scarabs (Coleoptera: Scarabaeidae) inhibit Bacillus thuringiensis multiplication.

    PubMed

    Shan, Yueming; Shu, Changlong; Crickmore, Neil; Liu, Chunqin; Xiang, Wensheng; Song, Fuping; Zhang, Jie

    2014-06-01

    The entomopathogen Bacillus thuringiensis is used to control various pest species of scarab beetle but is not particularly effective. Gut bacteria have diverse ecological and evolutionary effects on their hosts, but whether gut bacteria can protect scarabs from B. thuringiensis infection remains poorly understood. To investigate this, we isolated 32 cultivable gut bacteria from Holotrichia oblita Faldermann, Holotrichia parallela Motschulsky, and Anomala corpulenta Motschulsky, and analyzed their effect on B. thuringiensis multiplication and Cry toxin stability. 16S rDNA analysis indicated that these gut bacteria belong to the Proteobacteria, Actinobacteria, Firmicutes, and Bacteroidetes phyla. A confrontation culture analyses of the 32 isolates against three scarab-specific B. thuringiensis strains showed that the majority of the scarab gut bacteria had antibacterial activity against the B. thuringiensis strains. The Cry toxin stability analysis results showed that while several strains produced proteases capable of processing the scarab-specific toxin Cry8Ea, none were able to completely degrade it. These results suggest that gut bacteria can potentially affect the susceptibility of scarabs to B. thuringiensis and that this should be considered when considering future control measures.

  3. The genetic architecture of a complex trait: Resistance to multiple toxins produced by Bacillus thuringiensis israelensis in the dengue and yellow fever vector, the mosquito Aedes aegypti.

    PubMed

    Bonin, Aurélie; Paris, Margot; Frérot, Hélène; Bianco, Erica; Tetreau, Guillaume; Després, Laurence

    2015-10-01

    The bacterial insecticide Bacillus thuringiensis subsp. israelensis (Bti) is an increasingly popular alternative to chemical insecticides for controlling mosquito populations. Because Bti toxicity relies on the action of four main toxins, resistance to Bti is very likely a complex phenotype involving several genes simultaneously. Dissecting the underlying genetic basis thus requires associating a quantitative measure of resistance to genetic variation at many loci in a segregating population. Here, we undertake this task using the dengue and yellow fever vector, the mosquito Aedes aegypti, as a study model. We conducted QTL (Quantitative Trait Locus) and admixture mapping analyses on two controlled crosses and on an artificial admixed population, respectively, all obtained from resistant and susceptible lab strains. We detected 16 QTL regions, among which four QTLs were revealed by different analysis methods. These four robust QTLs explained altogether 29.2% and 62.2% of the total phenotypic variance in the two QTL crosses, respectively. They also all showed a dominant mode of action. In addition, we found six loci showing statistical association with Bti resistance in the admixed population. Five of the supercontigs highlighted in this study contained candidate genes as suggested by their function, or by prior evidence from expression and/or outlier analyses. These genomic regions are thus good starting points for fine mapping of resistance to Bti or functional analyses aiming at identifying the underlying genes and mutations. Moreover, for the purpose of this work, we built the first Ae. aegypti genetic map based on markers associated with genes expressed in larvae. This genetic map harbors 229 SNP markers mapped across the three chromosomes for a total length of 311.9cM. It brought to light several assembly discrepancies with the reference genome, suggesting a high level of genome plasticity in Ae. aegypti.

  4. fAFLP analysis of Brazilian Bacillus thuringiensis isolates.

    PubMed

    Valicente, Fernando Hercos; da Silva, Rosane Bezerra

    2014-01-01

    A total of 65 Bacillus thuringiensis (Bt) isolates were subjected to analysis of genetic relationship using fAFLP (fluorescent Fragment Length Polymorphism), in order to determine the genetic diversity within a group of Bt strains. 26 strains from different subspecies were identified as it follows: 9 kindly provided by the USDA (United States Department of Agriculture), 9 kindly provided by the Institute Pasteur and eight from Embrapa Maize and Sorghum Bt Collection, and 39 strains with no subspecies information also from Embrapa's Bt Collection. DNA sample was double digested with restriction enzymes EcoRI and MseI, and the fragments were linked to adapters. Selective amplification reactions were performed using five primer combinations and the amplified fragments were separated by gel electrophoresis on an ABI377 sequencer. Genetic distances were obtained by the complement of the Jaccard coefficient and the groups were performed by the UPGMA method. Five primer combinations generated 495 scorable fragments and 483 were found to be polymorphic. Out of 26 subspecies, strains 344 and T09 (B. thuringiensis subsp. tolworthi) showed the highest similarity (15%), while isolates HD3 B. thuringiensis subsp finitimus and T24 B. thuringiensis subsp neoleonensis were the most genetically distant (92%). B. thuringiensis isolates with no subspecies identification, found in samples from Goiás State showed higher similarity forming a group with an average distance of 6%, and the closest subspecies to this group was B. thuringiensis subsp thuringiensis (HD2) with 52% of similarity. This similarity may be due to the fact that these organism exchange genetic material by conjugation, and it is relatively common to have evolutionary characteristics of their ancestors.

  5. Photoprotection of Bacillus thuringiensis kurstaki from ultraviolet irradiation

    SciTech Connect

    Cohen, E.; Rozen, H.; Joseph, T.; Braun, S.; Margulies, L. )

    1991-05-01

    Irradiation of Bacillus thuringiensis var. kurstaki HD1 at 300-350 nm for up to 12 hr using a photochemical reactor results in a rapid loss of its toxicity to larvae of Heliothis armigera. Photoprotection of the toxic component was obtained by adsorption of cationic chromophores such as acriflavin (AF), methyl green, and rhodamine B to B. thuringiensis. AF gave the best photoprotection and a level of 0.42 mmol/g dye absorbed per gram of B. thuringiensis was highly toxic even after 12 hr of ultraviolet (uv) irradiation as compared to the control (77.5 and 5% of insect mortality, respectively). Ultraviolet and Fourier-transform infrared spectroscopic studies indicate molecular interactions between B. thuringiensis and AF. The nature of these interactions and energy or charge transfer as possible mechanisms of photoprotection are discussed. It is speculated that tryptophan residues are essential for the toxic effect of B. thuringiensis. It is suggested that photoprotection is attained as energy is transferred from the excited tryptophan moieties to the chromophore molecules.

  6. Synergistic activity of Bacillus thuringiensis toxins against Simulium spp. larvae.

    PubMed

    Monnerat, Rose; Pereira, Eleny; Teles, Beatriz; Martins, Erica; Praça, Lilian; Queiroz, Paulo; Soberon, Mario; Bravo, Alejandra; Ramos, Felipe; Soares, Carlos Marcelo

    2014-09-01

    Species of Simulium spread diseases in humans and animals such as onchocerciasis and mansonelosis, causing health problems and economic loses. One alternative for controlling these insects is the use of Bacillus thuringiensis serovar israelensis (Bti). This bacterium produces different dipteran-active Cry and Cyt toxins and has been widely used in blackfly biological control programs worldwide. Studies on other insect targets have revealed the role of individual Cry and Cyt proteins in toxicity and demonstrated a synergistic effect among them. However, the insecticidal activity and interactions of these proteins against Simulium larvae have not been reported. In this study we demonstrate that Cry4Ba is the most effective toxin followed by Cry4Aa and Cry11Aa. Cry10Aa and Cyt1Aa were not toxic when administered alone but both were able to synergise the activity of Cry4B and Cry11Aa toxins. Cyt1Aa is also able to synergise with Cry4Aa. The mixture of all toxin-producing strains showed the greatest level of synergism, but still lower than the Bti parental strain.

  7. 40 CFR 174.509 - Bacillus thuringiensis Cry3A protein; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 24 2011-07-01 2011-07-01 false Bacillus thuringiensis Cry3A protein...-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.509 Bacillus thuringiensis Cry3A protein; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry3A protein are...

  8. 40 CFR 174.502 - Bacillus thuringiensis Cry1A.105 protein; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 24 2011-07-01 2011-07-01 false Bacillus thuringiensis Cry1A.105...-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.502 Bacillus thuringiensis Cry1A.105 protein; exemption from the requirement of a tolerance. (a) Residues of Bacillus thuringiensis Cry1A.105 protein...

  9. 40 CFR 174.517 - Bacillus thuringiensis Cry9C protein in corn; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 24 2011-07-01 2011-07-01 false Bacillus thuringiensis Cry9C protein... PLANT-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.517 Bacillus thuringiensis... Bacillus thuringiensis Cry9C protein in corn is exempted from the requirement of a tolerance for...

  10. 40 CFR 174.517 - Bacillus thuringiensis Cry9C protein in corn; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Bacillus thuringiensis Cry9C protein... PLANT-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.517 Bacillus thuringiensis... Bacillus thuringiensis Cry9C protein in corn is exempted from the requirement of a tolerance for...

  11. 40 CFR 174.502 - Bacillus thuringiensis Cry1A.105 protein; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Bacillus thuringiensis Cry1A.105...-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.502 Bacillus thuringiensis Cry1A.105 protein; exemption from the requirement of a tolerance. (a) Residues of Bacillus thuringiensis Cry1A.105 protein...

  12. 40 CFR 174.509 - Bacillus thuringiensis Cry3A protein; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Bacillus thuringiensis Cry3A protein...-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.509 Bacillus thuringiensis Cry3A protein; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry3A protein are...

  13. 40 CFR 174.509 - Bacillus thuringiensis Cry3A protein; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 25 2013-07-01 2013-07-01 false Bacillus thuringiensis Cry3A protein...-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.509 Bacillus thuringiensis Cry3A protein; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry3A protein are...

  14. 40 CFR 174.509 - Bacillus thuringiensis Cry3A protein; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 25 2012-07-01 2012-07-01 false Bacillus thuringiensis Cry3A protein...-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.509 Bacillus thuringiensis Cry3A protein; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry3A protein are...

  15. 40 CFR 174.509 - Bacillus thuringiensis Cry3A protein; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 24 2014-07-01 2014-07-01 false Bacillus thuringiensis Cry3A protein...-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.509 Bacillus thuringiensis Cry3A protein; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry3A protein are...

  16. Bacillus thuringiensis Toxins: An Overview of Their Biocidal Activity

    PubMed Central

    Palma, Leopoldo; Muñoz, Delia; Berry, Colin; Murillo, Jesús; Caballero, Primitivo

    2014-01-01

    Bacillus thuringiensis (Bt) is a Gram positive, spore-forming bacterium that synthesizes parasporal crystalline inclusions containing Cry and Cyt proteins, some of which are toxic against a wide range of insect orders, nematodes and human-cancer cells. These toxins have been successfully used as bioinsecticides against caterpillars, beetles, and flies, including mosquitoes and blackflies. Bt also synthesizes insecticidal proteins during the vegetative growth phase, which are subsequently secreted into the growth medium. These proteins are commonly known as vegetative insecticidal proteins (Vips) and hold insecticidal activity against lepidopteran, coleopteran and some homopteran pests. A less well characterized secretory protein with no amino acid similarity to Vip proteins has shown insecticidal activity against coleopteran pests and is termed Sip (secreted insecticidal protein). Bin-like and ETX_MTX2-family proteins (Pfam PF03318), which share amino acid similarities with mosquitocidal binary (Bin) and Mtx2 toxins, respectively, from Lysinibacillus sphaericus, are also produced by some Bt strains. In addition, vast numbers of Bt isolates naturally present in the soil and the phylloplane also synthesize crystal proteins whose biological activity is still unknown. In this review, we provide an updated overview of the known active Bt toxins to date and discuss their activities. PMID:25514092

  17. Role of Receptors in Bacillus thuringiensis Crystal Toxin Activity

    PubMed Central

    Pigott, Craig R.; Ellar, David J.

    2007-01-01

    Bacillus thuringiensis produces crystalline protein inclusions with insecticidal or nematocidal properties. These crystal (Cry) proteins determine a particular strain's toxicity profile. Transgenic crops expressing one or more recombinant Cry toxins have become agriculturally important. Individual Cry toxins are usually toxic to only a few species within an order, and receptors on midgut epithelial cells have been shown to be critical determinants of Cry specificity. The best characterized of these receptors have been identified for lepidopterans, and two major receptor classes have emerged: the aminopeptidase N (APN) receptors and the cadherin-like receptors. Currently, 38 different APNs have been reported for 12 different lepidopterans. Each APN belongs to one of five groups that have unique structural features and Cry-binding properties. While 17 different APNs have been reported to bind to Cry toxins, only 2 have been shown to mediate toxin susceptibly in vivo. In contrast, several cadherin-like proteins bind to Cry toxins and confer toxin susceptibility in vitro, and disruption of the cadherin gene has been associated with toxin resistance. Nonetheless, only a small subset of the lepidopteran-specific Cry toxins has been shown to interact with cadherin-like proteins. This review analyzes the interactions between Cry toxins and their receptors, focusing on the identification and validation of receptors, the molecular basis for receptor recognition, the role of the receptor in resistant insects, and proposed models to explain the sequence of events at the cell surface by which receptor binding leads to cell death. PMID:17554045

  18. Evolution of Bacillus thuringiensis Cry toxins insecticidal activity.

    PubMed

    Bravo, Alejandra; Gómez, Isabel; Porta, Helena; García-Gómez, Blanca Ines; Rodriguez-Almazan, Claudia; Pardo, Liliana; Soberón, Mario

    2013-01-01

    Insecticidal Cry proteins produced by Bacillus thuringiensis are use worldwide in transgenic crops for efficient pest control. Among the family of Cry toxins, the three domain Cry family is the better characterized regarding their natural evolution leading to a large number of Cry proteins with similar structure, mode of action but different insect specificity. Also, this group is the better characterized regarding the study of their mode of action and the molecular basis of insect specificity. In this review we discuss how Cry toxins have evolved insect specificity in nature and analyse several cases of improvement of Cry toxin action by genetic engineering, some of these examples are currently used in transgenic crops. We believe that the success in the improvement of insecticidal activity by genetic evolution of Cry toxins will depend on the knowledge of the rate-limiting steps of Cry toxicity in different insect pests, the mapping of the specificity binding regions in the Cry toxins, as well as the improvement of mutagenesis strategies and selection procedures.

  19. Bacillus thuringiensis toxins: an overview of their biocidal activity.

    PubMed

    Palma, Leopoldo; Muñoz, Delia; Berry, Colin; Murillo, Jesús; Caballero, Primitivo

    2014-12-11

    Bacillus thuringiensis (Bt) is a Gram positive, spore-forming bacterium that synthesizes parasporal crystalline inclusions containing Cry and Cyt proteins, some of which are toxic against a wide range of insect orders, nematodes and human-cancer cells. These toxins have been successfully used as bioinsecticides against caterpillars, beetles, and flies, including mosquitoes and blackflies. Bt also synthesizes insecticidal proteins during the vegetative growth phase, which are subsequently secreted into the growth medium. These proteins are commonly known as vegetative insecticidal proteins (Vips) and hold insecticidal activity against lepidopteran, coleopteran and some homopteran pests. A less well characterized secretory protein with no amino acid similarity to Vip proteins has shown insecticidal activity against coleopteran pests and is termed Sip (secreted insecticidal protein). Bin-like and ETX_MTX2-family proteins (Pfam PF03318), which share amino acid similarities with mosquitocidal binary (Bin) and Mtx2 toxins, respectively, from Lysinibacillus sphaericus, are also produced by some Bt strains. In addition, vast numbers of Bt isolates naturally present in the soil and the phylloplane also synthesize crystal proteins whose biological activity is still unknown. In this review, we provide an updated overview of the known active Bt toxins to date and discuss their activities.

  20. Role of receptors in Bacillus thuringiensis crystal toxin activity.

    PubMed

    Pigott, Craig R; Ellar, David J

    2007-06-01

    Bacillus thuringiensis produces crystalline protein inclusions with insecticidal or nematocidal properties. These crystal (Cry) proteins determine a particular strain's toxicity profile. Transgenic crops expressing one or more recombinant Cry toxins have become agriculturally important. Individual Cry toxins are usually toxic to only a few species within an order, and receptors on midgut epithelial cells have been shown to be critical determinants of Cry specificity. The best characterized of these receptors have been identified for lepidopterans, and two major receptor classes have emerged: the aminopeptidase N (APN) receptors and the cadherin-like receptors. Currently, 38 different APNs have been reported for 12 different lepidopterans. Each APN belongs to one of five groups that have unique structural features and Cry-binding properties. While 17 different APNs have been reported to bind to Cry toxins, only 2 have been shown to mediate toxin susceptibly in vivo. In contrast, several cadherin-like proteins bind to Cry toxins and confer toxin susceptibility in vitro, and disruption of the cadherin gene has been associated with toxin resistance. Nonetheless, only a small subset of the lepidopteran-specific Cry toxins has been shown to interact with cadherin-like proteins. This review analyzes the interactions between Cry toxins and their receptors, focusing on the identification and validation of receptors, the molecular basis for receptor recognition, the role of the receptor in resistant insects, and proposed models to explain the sequence of events at the cell surface by which receptor binding leads to cell death. PMID:17554045

  1. Monarch larvae sensitivity to Bacillus thuringiensis- purified proteins and pollen

    PubMed Central

    Hellmich, Richard L.; Siegfried, Blair D.; Sears, Mark K.; Stanley-Horn, Diane E.; Daniels, Michael J.; Mattila, Heather R.; Spencer, Terrence; Bidne, Keith G.; Lewis, Leslie C.

    2001-01-01

    Laboratory tests were conducted to establish the relative toxicity of Bacillus thuringiensis (Bt) toxins and pollen from Bt corn to monarch larvae. Toxins tested included Cry1Ab, Cry1Ac, Cry9C, and Cry1F. Three methods were used: (i) purified toxins incorporated into artificial diet, (ii) pollen collected from Bt corn hybrids applied directly to milkweed leaf discs, and (iii) Bt pollen contaminated with corn tassel material applied directly to milkweed leaf discs. Bioassays of purified Bt toxins indicate that Cry9C and Cry1F proteins are relatively nontoxic to monarch first instars, whereas first instars are sensitive to Cry1Ab and Cry1Ac proteins. Older instars were 12 to 23 times less susceptible to Cry1Ab toxin compared with first instars. Pollen bioassays suggest that pollen contaminants, an artifact of pollen processing, can dramatically influence larval survival and weight gains and produce spurious results. The only transgenic corn pollen that consistently affected monarch larvae was from Cry1Ab event 176 hybrids, currently <2% corn planted and for which re-registration has not been applied. Results from the other types of Bt corn suggest that pollen from the Cry1Ab (events Bt11 and Mon810) and Cry1F, and experimental Cry9C hybrids, will have no acute effects on monarch butterfly larvae in field settings. PMID:11559841

  2. Monarch larvae sensitivity to Bacillus thuringiensis- purified proteins and pollen.

    PubMed

    Hellmich, R L; Siegfried, B D; Sears, M K; Stanley-Horn, D E; Daniels, M J; Mattila, H R; Spencer, T; Bidne, K G; Lewis, L C

    2001-10-01

    Laboratory tests were conducted to establish the relative toxicity of Bacillus thuringiensis (Bt) toxins and pollen from Bt corn to monarch larvae. Toxins tested included Cry1Ab, Cry1Ac, Cry9C, and Cry1F. Three methods were used: (i) purified toxins incorporated into artificial diet, (ii) pollen collected from Bt corn hybrids applied directly to milkweed leaf discs, and (iii) Bt pollen contaminated with corn tassel material applied directly to milkweed leaf discs. Bioassays of purified Bt toxins indicate that Cry9C and Cry1F proteins are relatively nontoxic to monarch first instars, whereas first instars are sensitive to Cry1Ab and Cry1Ac proteins. Older instars were 12 to 23 times less susceptible to Cry1Ab toxin compared with first instars. Pollen bioassays suggest that pollen contaminants, an artifact of pollen processing, can dramatically influence larval survival and weight gains and produce spurious results. The only transgenic corn pollen that consistently affected monarch larvae was from Cry1Ab event 176 hybrids, currently <2% corn planted and for which re-registration has not been applied. Results from the other types of Bt corn suggest that pollen from the Cry1Ab (events Bt11 and Mon810) and Cry1F, and experimental Cry9C hybrids, will have no acute effects on monarch butterfly larvae in field settings. PMID:11559841

  3. Proteomic profiling and identification of immunodominant spore antigens of Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis.

    PubMed

    Delvecchio, Vito G; Connolly, Joseph P; Alefantis, Timothy G; Walz, Alexander; Quan, Marian A; Patra, Guy; Ashton, John M; Whittington, Jessica T; Chafin, Ryan D; Liang, Xudong; Grewal, Paul; Khan, Akbar S; Mujer, Cesar V

    2006-09-01

    Differentially expressed and immunogenic spore proteins of the Bacillus cereus group of bacteria, which includes Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis, were identified. Comparative proteomic profiling of their spore proteins distinguished the three species from each other as well as the virulent from the avirulent strains. A total of 458 proteins encoded by 232 open reading frames were identified by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry analysis for all the species. A number of highly expressed proteins, including elongation factor Tu (EF-Tu), elongation factor G, 60-kDa chaperonin, enolase, pyruvate dehydrogenase complex, and others exist as charge variants on two-dimensional gels. These charge variants have similar masses but different isoelectric points. The majority of identified proteins have cellular roles associated with energy production, carbohydrate transport and metabolism, amino acid transport and metabolism, posttranslational modifications, and translation. Novel vaccine candidate proteins were identified using B. anthracis polyclonal antisera from humans postinfected with cutaneous anthrax. Fifteen immunoreactive proteins were identified in B. anthracis spores, whereas 7, 14, and 7 immunoreactive proteins were identified for B. cereus and in the virulent and avirulent strains of B. thuringiensis spores, respectively. Some of the immunodominant antigens include charge variants of EF-Tu, glyceraldehyde-3-phosphate dehydrogenase, dihydrolipoamide acetyltransferase, Delta-1-pyrroline-5-carboxylate dehydrogenase, and a dihydrolipoamide dehydrogenase. Alanine racemase and neutral protease were uniquely immunogenic to B. anthracis. Comparative analysis of the spore immunome will be of significance for further nucleic acid- and immuno-based detection systems as well as next-generation vaccine development.

  4. [Estimation of the spectrum of the aerial dispersion of Bacillus thuringiensis].

    PubMed

    Smirnoff, W A; Valéro, J R

    1983-10-01

    The present paper estimates the number of viable spores of Bacillus thuringiensis per droplet and reveals the importance of this data to improve B. thuringiensis treatments. For a given diameter, droplets from Futura formula contained two times more spores than droplets from the formulas used to date. It is preferable to have B. thuringiensis droplets larger than those of chemical insecticides. A Grumman AgCat aircraft calibrated to give the desired larger droplets resulted in successful B. thuringiensis treatments.

  5. SR450 and Superhawk XP applications of Bacillus thuringiensis israelensis de Barjac against Culex quinquefasciatus Say

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Sprayer comparisons and larval morality assays were conducted following SR450 backpack mist blower and Superhawk XP thermal fogger applications of Vectobac® WDG Bacillus thuringiensis israelensis (Bti) de Barjac against Culex quinquefasciatus Say. Bacillus thuringiensis israelensis was applied at m...

  6. 40 CFR 180.1107 - Delta endotoxin of Bacillus thuringiensis variety kurstaki encapsulated into killed Pseudomonas...

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... From Tolerances § 180.1107 Delta endotoxin of Bacillus thuringiensis variety kurstaki encapsulated into... Bacillus thuringiensis variety kurstaki encapsulated into killed Pseudomonas fluorescens is exempt from the... 40 Protection of Environment 24 2011-07-01 2011-07-01 false Delta endotoxin of...

  7. 40 CFR 180.1107 - Delta endotoxin of Bacillus thuringiensis variety kurstaki encapsulated into killed Pseudomonas...

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... From Tolerances § 180.1107 Delta endotoxin of Bacillus thuringiensis variety kurstaki encapsulated into... Bacillus thuringiensis variety kurstaki encapsulated into killed Pseudomonas fluorescens is exempt from the... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Delta endotoxin of...

  8. [Characterization of crystal-forming bacteria Bacillus thuringiensis subsp. tohokuensis toxic to mosquitos].

    PubMed

    Khodyrev, V P; Kalmykova, G V; Burtseva, L I; Glupov, V V

    2006-01-01

    Distribution study of Bacillus thuringiensis strains in Western Siberian soils allowed us to isolate crystal-forming bacteria assigned to a new pathovar of Bacillus thuringiensis ssp. tohokuensis with a toxic effect on mosquito larvae. A description of this bacterial pathovar is presented.

  9. Production of a Thermostable and Alkaline Chitinase by Bacillus thuringiensis subsp. kurstaki Strain HBK-51

    PubMed Central

    Kuzu, Secil Berna; Güvenmez, Hatice Korkmaz; Denizci, Aziz Akin

    2012-01-01

    This paper reports the isolation and identification of chitinase-producing Bacillus from chitin-containing wastes, production of a thermostable and alkaline chitinasese, and enzyme characterization. Bacillus thuringiensis subsp. kurstaki HBK-51 was isolated from soil and was identified. Chitinase was obtained from supernatant of B. thuringiensis HBK-51 strain and showed its optimum activity at 110°C and at pH 9.0. Following 3 hours of incubation period, the enzyme showed a high level of activity at 110°C (96% remaining activity) and between pH 9.0 and 12.0 (98% remaining activity). Considering these characteristics, the enzyme was described as hyperthermophile-thermostable and highly alkaline. Two bands of the enzyme weighing 50 and 125 kDa were obtained following 12% SDS-PAGE analyses. Among the metal ions and chemicals used, Ni2+ (32%), K+ (44%), and Cu2+ (56%) increased the enzyme activity while EDTA (7%), SDS (7%), Hg2+ (11%), and ethyl-acetimidate (20%) decreased the activity of the enzyme. Bacillus thuringiensis subsp. kurstaki HBK-51 is an important strain which can be used in several biotechnological applications as a chitinase producer. PMID:23304523

  10. Bacillus thuringiensis colonises plant roots in a phylogeny-dependent manner.

    PubMed

    Vidal-Quist, J Cristian; Rogers, Hilary J; Mahenthiralingam, Eshwar; Berry, Colin

    2013-12-01

    Although much is known about the pathology of Bacillus thuringiensis against invertebrates, current understanding of its natural ecology is limited. This study evaluated the biodiversity of B. thuringiensis in relation to its interaction with plants. Phylogenetic relationships between 44 reference and field-collected strains, determined using 16S rRNA and gyrB gene sequences, revealed a high degree of variability, similar to that found in databases. An Arabidopsis thaliana in vitro inoculation model was developed to screen the ability of B. thuringiensis to colonise roots. Significant colonisation differences up to 91-fold were observed between strains, and correlation between strain phylogeny and colonisation was found. The genetics and biochemistry of auxin production; presence of the gene encoding indole pyruvate decarboxylase; and the abilities of Bt strains to swarm, grow in rich/minimal media and affect root growth differed between the strains, but only auxin production correlated significantly with ability to colonise roots. Co-inoculation with Burkholderia phytofirmans PsJN or Pseudomonas fluorescens SBW25 produced no effect on B. thuringiensis colonisation levels, regardless of the co-inoculant. Similarly, root colonisation of A. thaliana mutants impaired in plant defences was not significantly higher compared with controls. This is the first systematic and phylogenetic evaluation of B. thuringiensis interaction with plants.

  11. Genome characteristics of a novel phage from Bacillus thuringiensis showing high similarity with phage from Bacillus cereus.

    PubMed

    Yuan, Yihui; Gao, Meiying; Wu, Dandan; Liu, Pengming; Wu, Yan

    2012-01-01

    Bacillus thuringiensis is an important entomopathogenic bacterium belongs to the Bacillus cereus group, which also includes B. anthracis and B. cereus. Several genomes of phages originating from this group had been sequenced, but no genome of Siphoviridae phage from B. thuringiensis has been reported. We recently sequenced and analyzed the genome of a novel phage, BtCS33, from a B. thuringiensis strain, subsp. kurstaki CS33, and compared the gneome of this phage to other phages of the B. cereus group. BtCS33 was the first Siphoviridae phage among the sequenced B. thuringiensis phages. It produced small, turbid plaques on bacterial plates and had a narrow host range. BtCS33 possessed a linear, double-stranded DNA genome of 41,992 bp with 57 putative open reading frames (ORFs). It had a typical genome structure consisting of three modules: the "late" region, the "lysogeny-lysis" region and the "early" region. BtCS33 exhibited high similarity with several phages, B. cereus phage Wβ and some variants of Wβ, in genome organization and the amino acid sequences of structural proteins. There were two ORFs, ORF22 and ORF35, in the genome of BtCS33 that were also found in the genomes of B. cereus phage Wβ and may be involved in regulating sporulation of the host cell. Based on these observations and analysis of phylogenetic trees, we deduced that B. thuringiensis phage BtCS33 and B. cereus phage Wβ may have a common distant ancestor.

  12. Genome Characteristics of a Novel Phage from Bacillus thuringiensis Showing High Similarity with Phage from Bacillus cereus

    PubMed Central

    Yuan, Yihui; Gao, Meiying; Wu, Dandan; Liu, Pengming; Wu, Yan

    2012-01-01

    Bacillus thuringiensis is an important entomopathogenic bacterium belongs to the Bacillus cereus group, which also includes B. anthracis and B. cereus. Several genomes of phages originating from this group had been sequenced, but no genome of Siphoviridae phage from B. thuringiensis has been reported. We recently sequenced and analyzed the genome of a novel phage, BtCS33, from a B. thuringiensis strain, subsp. kurstaki CS33, and compared the gneome of this phage to other phages of the B. cereus group. BtCS33 was the first Siphoviridae phage among the sequenced B. thuringiensis phages. It produced small, turbid plaques on bacterial plates and had a narrow host range. BtCS33 possessed a linear, double-stranded DNA genome of 41,992 bp with 57 putative open reading frames (ORFs). It had a typical genome structure consisting of three modules: the “late” region, the “lysogeny-lysis” region and the “early” region. BtCS33 exhibited high similarity with several phages, B. cereus phage Wβ and some variants of Wβ, in genome organization and the amino acid sequences of structural proteins. There were two ORFs, ORF22 and ORF35, in the genome of BtCS33 that were also found in the genomes of B. cereus phage Wβ and may be involved in regulating sporulation of the host cell. Based on these observations and analysis of phylogenetic trees, we deduced that B. thuringiensis phage BtCS33 and B. cereus phage Wβ may have a common distant ancestor. PMID:22649540

  13. Chitinase production by Bacillus thuringiensis and Bacillus licheniformis: their potential in antifungal biocontrol.

    PubMed

    Gomaa, Eman Zakaria

    2012-02-01

    Thirty bacterial strains were isolated from the rhizosphere of plants collected from Egypt and screened for production of chitinase enzymes. Bacillus thuringiensis NM101-19 and Bacillus licheniformis NM120-17 had the highest chitinolytic activities amongst those investigated. The production of chitinase by B. thuringiensis and B. licheniformis was optimized using colloidal chitin medium amended with 1.5% colloidal chitin, with casein as a nitrogen source, at 30°C after five days of incubation. An enhancement of chitinase production by the two species was observed by addition of sugar substances and dried fungal mats to the colloidal chitin media. The optimal conditions for chitinase activity by B. thuringiensis and B. licheniformis were at 40°C, pH 7.0 and pH 8.0, respectively. Na(+), Mg(2+), Cu(2+), and Ca(2+) caused enhancement of enzyme activities whereas they were markedly inhibited by Zn(2+), Hg(2+), and Ag(+). In vitro, B. thuringiensis and B. licheniformis chitinases had potential for cell wall lysis of many phytopathogenic fungi tested. The addition of B. thuringiensis chitinase was more effective than that of B. licheniformis in increasing the germination of soybean seeds infected with various phytopathogenic fungi.

  14. Activity of a Brazilian strain of Bacillus thuringiensis israelensis against the cotton Boll Weevil Anthonomus grandis Boheman (Coleoptera: Tenebrionidae).

    PubMed

    Monnerat, R; Martins, E; Praça, L; Dumas, V; Berry, C

    2012-02-01

    A Brazilian Bacillus thuringiensis subspecies israelensis, toxic to Diptera, including mosquitoes, was found also to show toxicity to the coleopteran boll weevil Anthonomus grandis Boheman at an equivalent level to that of the standard coleopteran-active B. thuringiensis subspecies tenebrionis T08017. Recombinant B. thuringiensis strains expressing the individual Cyt1Aa, Cry4Aa, Cry4Ba and Cry11Aa toxins from this strain were assessed to evaluate their potential contribution to the activity against A. grandis, either alone or in combination. Whilst individual toxins produced mortality, none was sufficiently potent to allow calculation of LC50 values. Combinations of toxins were unable to attain the same potency as the parental B. thuringiensis subsp. israelensis, suggesting a major role for other factors produced by this strain. PMID:23950011

  15. Existence of lysogenic bacteriophages in Bacillus thuringiensis type strains.

    PubMed

    Roh, Jong Yul; Park, Jong Bin; Liu, Qin; Kim, Song Eun; Tao, Xueying; Choi, Tae Woong; Choi, Jae Young; Kim, Woo Jin; Jin, Byung Rae; Je, Yeon Ho

    2013-07-01

    We screened the existence of bacteriophages in 67 Bacillus thuringiensis type strains by phage DNA extraction and PCR using phage terminase small subunit (TerS)-specific primers to the supernatants and the precipitated pellets of Bt cultures, and by transmission electron microscopy. The various bacteriophages were observed from the supernatants of 22 type strains. Ten type strains showed the extracted phage DNAs and the amplified fragment by TerS PCR but 12 type strains showed only the phage DNAs. Their morphological characteristic suggests that they belong to Family Siphoviridae which had a long tail and symmetrical head. PMID:23632013

  16. Bacillus thuringiensis resistance in Plutella - too many trees?

    PubMed

    Crickmore, Neil

    2016-06-01

    Plutella xylostella was the first insect for which resistance to Bacillus thuringiensis was reported in the field, yet despite many studies on the nature of this resistance phenotype its genetic and molecular basis remains elusive. Many different factors have been proposed as contributing to resistance, although in many cases it has not been possible to establish a causal link. Indeed, there are so many studies published that it has become very difficult to 'see the wood for the trees'. This article will attempt to clarify our current understanding of Bt resistance in P. xylostella and consider the criteria that are used when validating a particular model. PMID:27436736

  17. Modular genetic architecture of the toxigenic plasmid pIS56-63 harboring cry1Ab21 in Bacillus thuringiensis subsp. thuringiensis strain IS5056.

    PubMed

    Murawska, Emilia; Fiedoruk, Krzysztof; Swiecicka, Izabela

    2014-01-01

    Bacillus thuringiensis subsp. thuringiensis IS5056, a strain highly toxic to Trichoplusia ni larvae, produces the newly described Cry1Ab21 delta-endotoxin encoded by a gene located in the 63.8 kb pIS56-63 plasmid. In this report we present the structure and functional similarity of this plasmid to other B. thuringiensis large toxigenic plasmids with particular interest focused on its modular architecture. The 61 open reading frames (ORFs) of the plasmid made four functional modules: (i) M1-mic, the mobile insertion cassette harboring cry1Ab21; (ii) M2-tra, the putative conjugative element; (iii) M3-reg, regulation sequence; and (iv) M4-rep, the ori44 replicon. These modules display similarity to corresponding sequences in distinct B. thuringiensis plasmids, but, in general, not to plasmid of other Bacillus cereus sensu lato. The nucleotide sequence and organization of genes in pIS56-63 were highly similar (80-100%) to those in pHT73 of B. thuringiensis HT73, and in p03 of B. thuringiensis HD771, particularly within the M3-reg and M4-rep modules, and slightly less in M2-tra, the latter of which is composed of two segments exhibiting homology to sequences in pBMB28, pAH187_45, pCT83, and pIS56-85 or to pCT72, pBMB67, p04, and pIS56-68. The tetrapartite structure of the toxigenic pIS56-63 plasmid strongly suggests that its hybrid nature is a result of recombination of various genetic elements originating from different extrachromosomal and chromosomal sources in B. thuringiensis. The presence of cry1Ab21 in the mobile cassette suggests that its occurrence on pIS56-63 resulted from recombination and transposition events during the evolution of the plasmid.

  18. Spore prevalence and toxigenicity of Bacillus cereus and Bacillus thuringiensis isolates from U.S. retail spices.

    PubMed

    Hariram, Upasana; Labbé, Ronald

    2015-03-01

    Recent incidents of foodborne illness associated with spices as the vehicle of transmission prompted this examination of U.S. retail spices with regard to Bacillus cereus. This study focused on the levels of aerobic-mesophilic spore-forming bacteria and B cereus spores associated with 247 retail spices purchased from five states in the United States. Samples contained a wide range of aerobic-mesophilic bacterial spore counts (< 200 to 8.3 × 10(7) CFU/g), with 19.1% of samples at levels above 10(5) CFU/g. For examples, paprika, allspice, peppercorns, and mixed spices had high levels of aerobic spores (> 10(7) CFU/g). Using a novel chromogenic agar, B. cereus and B. thuringiensis spores were isolated from 77 (31%) and 11 (4%) samples, respectively. Levels of B. cereus were <3 to 1,600 MPN/g. Eighty-eight percent of B. cereus isolates and 91% of B. thuringiensis isolates possessed at least one type of enterotoxin gene: HBL (hemolysin BL) or nonhemolytic enterotoxin (NHE). None of the 88 isolates obtained in this study possessed the emetic toxin gene (ces). Using commercially available immunological toxin detection kits, the toxigenicity of the isolates was confirmed. The NHE enterotoxin was expressed in 98% of B. cereus and 91% of B. thuringiensis isolates that possessed the responsible gene. HBL enterotoxin was detected in 87% of B. cereus and 100% of B. thuringiensis PCR-positive isolates. Fifty-two percent of B. cereus and 54% of B. thuringiensis isolates produced both enterotoxins. Ninety-seven percent of B. cereus isolates grew at 12°C, although only two isolates grew well at 9°C. The ability of these spice isolates to form spores, produce diarrheal toxins, and grow at moderately abusive temperatures makes retail spices an important potential vehicle for foodborne illness caused by B. cereus strains, in particular those that produce diarrheal toxins.

  19. Spatio-Temporal Evolution of Sporulation in Bacillus thuringiensis Biofilm.

    PubMed

    El-Khoury, Nay; Majed, Racha; Perchat, Stéphane; Kallassy, Mireille; Lereclus, Didier; Gohar, Michel

    2016-01-01

    Bacillus thuringiensis can produce a floating biofilm which includes two parts: a ring and a pellicle. The ring is a thick structure which sticks to the culture container, while the pellicle extends over the whole liquid surface and joins the ring. We have followed over time, from 16 to 96 h, sporulation in the two biofilm parts. Sporulation was followed in situ in 48-wells polystyrene microtiterplates with a fluorescence binocular stereomicroscope and a spoIID-yfp transcriptional fusion. Sporulation took place much earlier in the ring than in the pellicle. In 20 h-aged biofilms, spoIID was expressed only in the ring, which could be seen as a green fluorescent circle surrounding the non-fluorescent pellicle. However, after 48 h of culture, the pellicle started to express spoIID in specific area corresponding to protrusions, and after 96 h both the ring and the whole pellicle expressed spoIID. Spore counts and microscopy observations of the ring and the pellicle harvested separately confirmed these results and revealed that sporulation occured 24 h-later in the pellicle comparatively to the ring, although both structures contained nearly 100% spores after 96 h of culture. We hypothesize that two mechanisms, due to microenvironments in the biofilm, can explain this difference. First, the ring experiences a decreased concentration of nutrients earlier than the pellicle, because of a lower exchange area with the culture medium. An second, the ring is exposed to partial dryness. Both reasons could speed up sporulation in this biofilm structure. Our results also suggest that spores in the biofilm display a phenotypic heterogeneity. These observations might be of particular significance for the food industry, since the biofilm part sticking to container walls - the ring - is likely to contain spores and will therefore resist both to washing and to cleaning procedures, and will be able to restart a new biofilm when food production has resumed. PMID:27536298

  20. Spatio-Temporal Evolution of Sporulation in Bacillus thuringiensis Biofilm.

    PubMed

    El-Khoury, Nay; Majed, Racha; Perchat, Stéphane; Kallassy, Mireille; Lereclus, Didier; Gohar, Michel

    2016-01-01

    Bacillus thuringiensis can produce a floating biofilm which includes two parts: a ring and a pellicle. The ring is a thick structure which sticks to the culture container, while the pellicle extends over the whole liquid surface and joins the ring. We have followed over time, from 16 to 96 h, sporulation in the two biofilm parts. Sporulation was followed in situ in 48-wells polystyrene microtiterplates with a fluorescence binocular stereomicroscope and a spoIID-yfp transcriptional fusion. Sporulation took place much earlier in the ring than in the pellicle. In 20 h-aged biofilms, spoIID was expressed only in the ring, which could be seen as a green fluorescent circle surrounding the non-fluorescent pellicle. However, after 48 h of culture, the pellicle started to express spoIID in specific area corresponding to protrusions, and after 96 h both the ring and the whole pellicle expressed spoIID. Spore counts and microscopy observations of the ring and the pellicle harvested separately confirmed these results and revealed that sporulation occured 24 h-later in the pellicle comparatively to the ring, although both structures contained nearly 100% spores after 96 h of culture. We hypothesize that two mechanisms, due to microenvironments in the biofilm, can explain this difference. First, the ring experiences a decreased concentration of nutrients earlier than the pellicle, because of a lower exchange area with the culture medium. An second, the ring is exposed to partial dryness. Both reasons could speed up sporulation in this biofilm structure. Our results also suggest that spores in the biofilm display a phenotypic heterogeneity. These observations might be of particular significance for the food industry, since the biofilm part sticking to container walls - the ring - is likely to contain spores and will therefore resist both to washing and to cleaning procedures, and will be able to restart a new biofilm when food production has resumed.

  1. Spatio-Temporal Evolution of Sporulation in Bacillus thuringiensis Biofilm

    PubMed Central

    El-Khoury, Nay; Majed, Racha; Perchat, Stéphane; Kallassy, Mireille; Lereclus, Didier; Gohar, Michel

    2016-01-01

    Bacillus thuringiensis can produce a floating biofilm which includes two parts: a ring and a pellicle. The ring is a thick structure which sticks to the culture container, while the pellicle extends over the whole liquid surface and joins the ring. We have followed over time, from 16 to 96 h, sporulation in the two biofilm parts. Sporulation was followed in situ in 48-wells polystyrene microtiterplates with a fluorescence binocular stereomicroscope and a spoIID-yfp transcriptional fusion. Sporulation took place much earlier in the ring than in the pellicle. In 20 h-aged biofilms, spoIID was expressed only in the ring, which could be seen as a green fluorescent circle surrounding the non-fluorescent pellicle. However, after 48 h of culture, the pellicle started to express spoIID in specific area corresponding to protrusions, and after 96 h both the ring and the whole pellicle expressed spoIID. Spore counts and microscopy observations of the ring and the pellicle harvested separately confirmed these results and revealed that sporulation occured 24 h-later in the pellicle comparatively to the ring, although both structures contained nearly 100% spores after 96 h of culture. We hypothesize that two mechanisms, due to microenvironments in the biofilm, can explain this difference. First, the ring experiences a decreased concentration of nutrients earlier than the pellicle, because of a lower exchange area with the culture medium. An second, the ring is exposed to partial dryness. Both reasons could speed up sporulation in this biofilm structure. Our results also suggest that spores in the biofilm display a phenotypic heterogeneity. These observations might be of particular significance for the food industry, since the biofilm part sticking to container walls – the ring – is likely to contain spores and will therefore resist both to washing and to cleaning procedures, and will be able to restart a new biofilm when food production has resumed. PMID:27536298

  2. Cloning of a novel cryIC-type gene from a strain of Bacillus thuringiensis subsp. galleriae.

    PubMed

    Kalman, S; Kiehne, K L; Libs, J L; Yamamoto, T

    1993-04-01

    A novel cryIC-type gene was isolated from a strain of Bacillus thuringiensis subsp. galleriae. A new polymerase chain reaction (PCR) technique with a set of several oligonucleotide primer pairs specific to the cryIC gene was used to screen a number of B. thuringiensis strains. PCR amplified several DNA fragments ranging from 100 bp to 1 kb for B. thuringiensis strains containing a cryIC gene. PCR fragments amplified from the Bacillus thuringiensis subsp. galleriae HD29 DNA differed from the fragments amplified from other cryIC-containing strains, indicating strain HD29 contained a novel cryIC-type gene. To isolate crystal genes homologous to cryIC, an HD29 gene library was probed with a 984-bp fragment of the amino-terminal coding region of the cryIC gene cloned from Bacillus thuringiensis subsp. aizawai HD229. A putative toxin gene was isolated from a phage that hybridized strongly to the cryIC probe. Translation of the putative toxin DNA sequence revealed an open reading frame of 1,176 amino acids whose predicted molecular mass was 132.8 kDa. Comparisons of the toxin gene sequence with sequences of other cry genes indicated that this gene is a subclass of cryIC. We propose to designate this gene cryIC(b). In Escherichia coli, the cryIC(b) gene produced a protein of approximately 130 kDa toxic to Spodoptera exigua and Trichoplusia ni.

  3. Molecular characterization of Bacillus thuringiensis strains from Argentina.

    PubMed

    Franco-Rivera, Alejandro; Benintende, Graciela; Cozzi, Jorge; Baizabal-Aguirre, Victor Manuel; Valdez-Alarcón, Juan José; López-Meza, Joel Edmundo

    2004-07-01

    Bacillus thuringiensis INTA 7-3, INTA 51-3, INTA Mo9-5 and INTA Mo14-4 strains were obtained from Argentina and characterized by determination of serotype, toxicity, plasmid composition, insecticidal gene content ( cry and vip ) and the cloning of the single- vip3A gene of the INTA Mo9-5 strain. The serotype analysis identified the serovars tohokuensis and darmstadiensis for the INTA 51-3 and INTA Mo14-4 strains, respectively, whereas the INTA Mo9-5 strain was classified as "autoagglutinated". In contrast to the plasmid patterns of INTA 7-3, INTA 51-3 and INTA Mo9-5 (which were similar to B. thuringiensis HD-1 strain), strain INTA Mo14-4 showed a unique plasmid array. PCR analysis of the four strains revealed the presence of cry genes and vip3A genes. Interestingly, it was found that B. thuringiensis 4Q7 strain, which is a plasmid cured strain, contained vip3A genes indicating the presence of these insecticidal genes in the chromosome. Bioassays towards various lepidopteran species revealed that B. thuringiensis INTA Mo9-5 and INTA 7-3 strains were highly active. In particular, the mean LC(50) obtained against A. gemmatalis larvae with the INTA Mo9-5 and INTA 7-3 strains were 7 (5.7-8.6) and 6.7 (5.6-8.0) ppm, respectively. The INTA Mo14-4 strain was non-toxic and strain INTA 51-3 showed only a weak larvicidal activity. PMID:15103240

  4. Bacillus thuringiensis subsp. israelensis and Its Dipteran-Specific Toxins

    PubMed Central

    Ben-Dov, Eitan

    2014-01-01

    Bacillus thuringiensis subsp. israelensis (Bti) is the first Bacillus thuringiensis to be found and used as an effective biological control agent against larvae of many mosquito and black fly species around the world. Its larvicidal activity resides in four major (of 134, 128, 72 and 27 kDa) and at least two minor (of 78 and 29 kDa) polypeptides encoded respectively by cry4Aa, cry4Ba, cry11Aa, cyt1Aa, cry10Aa and cyt2Ba, all mapped on the 128 kb plasmid known as pBtoxis. These six δ-endotoxins form a complex parasporal crystalline body with remarkably high, specific and different toxicities to Aedes, Culex and Anopheles larvae. Cry toxins are composed of three domains (perforating domain I and receptor binding II and III) and create cation-selective channels, whereas Cyts are composed of one domain that acts as well as a detergent-like membrane perforator. Despite the low toxicities of Cyt1Aa and Cyt2Ba alone against exposed larvae, they are highly synergistic with the Cry toxins and hence their combinations prevent emergence of resistance in the targets. The lack of significant levels of resistance in field mosquito populations treated for decades with Bti-bioinsecticide suggests that this bacterium will be an effective biocontrol agent for years to come. PMID:24686769

  5. Bacillus thuringiensis subsp. israelensis and its dipteran-specific toxins.

    PubMed

    Ben-Dov, Eitan

    2014-03-28

    Bacillus thuringiensis subsp. israelensis (Bti) is the first Bacillus thuringiensis to be found and used as an effective biological control agent against larvae of many mosquito and black fly species around the world. Its larvicidal activity resides in four major (of 134, 128, 72 and 27 kDa) and at least two minor (of 78 and 29 kDa) polypeptides encoded respectively by cry4Aa, cry4Ba, cry11Aa, cyt1Aa, cry10Aa and cyt2Ba, all mapped on the 128 kb plasmid known as pBtoxis. These six δ-endotoxins form a complex parasporal crystalline body with remarkably high, specific and different toxicities to Aedes, Culex and Anopheles larvae. Cry toxins are composed of three domains (perforating domain I and receptor binding II and III) and create cation-selective channels, whereas Cyts are composed of one domain that acts as well as a detergent-like membrane perforator. Despite the low toxicities of Cyt1Aa and Cyt2Ba alone against exposed larvae, they are highly synergistic with the Cry toxins and hence their combinations prevent emergence of resistance in the targets. The lack of significant levels of resistance in field mosquito populations treated for decades with Bti-bioinsecticide suggests that this bacterium will be an effective biocontrol agent for years to come.

  6. Altered protoxin activation by midgut enzymes from a Bacillus thuringiensis resistant strain of Plodia interpunctella.

    PubMed

    Oppert, B; Kramer, K J; Johnson, D E; MacIntosh, S C; McGaughey, W H

    1994-02-15

    Processing of Bacillus thuringiensis protoxins to toxins by midgut proteinases from a strain of the Indianmeal moth, Plodia interpunctella (Hubner), resistant to B. thuringiensis subspecies entomocidus (HD-198) was slower than that by midgut proteinases from the susceptible parent strain or a strain resistant to B. thuringiensis subspecies kurstaki (HD-1, Dipel). Midgut extracts from entomocidus-resistant insects exhibited five-fold lower activity toward the synthetic substrate alpha-N-benzoyl-DL-arginine rho-nitroanilide than extracts from susceptible or kurstaki-resistant insects. Midgut enzymes from susceptible or kurstaki-resistant insects converted the 133 kDa CryIA(c) protoxin to 61-63 kDa proteins, while incubations with entomocidus-resistant enzymes resulted in predominantly products of intermediate size, even with increased amounts of midgut extract. The 61-63 kDa proteins were only produced by entomocidus-resistant midgut extracts after long term incubations with the protoxin. The data suggest that altered protoxin activation by midgut proteinases is involved in some types of insect resistance to B. thuringiensis.

  7. Architecture and High-Resolution Structure of Bacillus thuringiensis and Bacillus cereus Spore Coat Surfaces

    SciTech Connect

    Plomp, M; Leighton, T; Wheeler, K; Malkin, A

    2005-02-18

    We have utilized atomic force microscopy (AFM) to visualize the native surface topology and ultrastructure of Bacillus thuringiensis and Bacillus cereus spores in water and in air. AFM was able to resolve the nanostructure of the exosporium and three distinctive classes of appendages. Removal of the exosporium exposed either a hexagonal honeycomb layer (B. thuringiensis) or a rodlet outer spore coat layer (B. cereus). Removal of the rodlet structure from B. cereus spores revealed an underlying honeycomb layer similar to that observed with B. thuringiensis spores. The periodicity of the rodlet structure on the outer spore coat of B. cereus was {approx}8 nm, and the length of the rodlets was limited to the cross-patched domain structure of this layer to {approx}200 nm. The lattice constant of the honeycomb structures was {approx}9 nm for both B. cereus and B. thuringiensis spores. Both honeycomb structures were composed of multiple, disoriented domains with distinct boundaries. Our results demonstrate that variations in storage and preparation procedures result in architectural changes in individual spore surfaces, which establish AFM as a useful tool for evaluation of preparation and processing ''fingerprints'' of bacterial spores. These results establish that high-resolution AFM has the capacity to reveal species-specific assembly and nanometer scale structure of spore surfaces. These species-specific spore surface structural variations are correlated with sequence divergences in a spore core structural protein SspE.

  8. Various Enterotoxin and Other Virulence Factor Genes Widespread Among Bacillus cereus and Bacillus thuringiensis Strains.

    PubMed

    Kim, Min-Ju; Han, Jae-Kwang; Park, Jong-Su; Lee, Jin-Sung; Lee, Soon-Ho; Cho, Joon-Il; Kim, Keun-Sung

    2015-06-01

    Many strains of Bacillus cereus cause gastrointestinal diseases, and the closely related insect pathogen Bacillus thuringiensis has also been involved in outbreaks of diarrhea. The diarrheal diseases are attributed to enterotoxins. Sixteen reference strains of B. cereus and nine commercial and 12 reference strains of B. thuringiensis were screened by PCR for the presence of 10 enterotoxigenic genes (hblA, hblC, hblD, nheA, nheB, nheC, cytK, bceT, entFM, and entS), one emetogenic gene (ces), seven hemolytic genes (hlyA, hlyII, hlyIII, plcA, cerA, cerB, and cerO), and a pleiotropic transcriptional activator gene (plcR). These genes encode various enterotoxins and other virulence factors thought to play a role in infections of mammals. Amplicons were successfully generated from the strains of B. cereus and B. thuringiensis for each of these sequences, except the ces gene. Intriguingly, the majority of these B. cereus enterotoxin genes and other virulence factor genes appeared to be widespread among B. thuringiensis strains as well as B. cereus strains.

  9. Susceptibility, mechanisms of response and resistance to Bacillus thuringiensis toxins in Spodoptera spp.

    PubMed

    Herrero, Salvador; Bel, Yolanda; Hernández-Martínez, Patricia; Ferré, Juan

    2016-06-01

    Bioinsecticides based on Bacillus thuringiensis have long been used as an alternative to synthetic insecticides to control insect pests. In this review, we focus on insects of the genus Spodoptera, including relevant polyphagous species that are primary and secondary pests of many crops, and how B. thuringiensis toxins can be used for Spodoptera spp. pest management. We summarize the main findings related to susceptibility, midgut binding specificity, mechanisms of response and resistance of this insect genus to B. thuringiensis toxins. PMID:27436737

  10. Pathogenicity of Bacillus thuringiensis variety kurstaki to Ixodes scapularis (Acari: Ixodidae)

    USGS Publications Warehouse

    Zhioua, E.; Heyer, K.; Browning, M.; Ginsberg, H.S.; LeBrun, R.A.

    1999-01-01

    Pathogenicity of the entomopathogenic bacterium Bacillus thuringiensis var. kurstaki de Barjac & Lemille was tested against the black-legged tick, Ixodes scapularis Say. Engorged larvae dipped in a solution of 108 spores per ml showed 96% mortality, 3 wk post-infection. The LC50 value for engorged larvae (concentration required to kill 50% of ticks) was 107 spores/ml. Bacillus thuringiensis shows considerable potential as a microbial control agent for the management of Ixodes scapularis.

  11. 40 CFR 174.520 - Bacillus thuringiensis Cry1F protein in corn; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Bacillus thuringiensis Cry1F protein... PLANT-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.520 Bacillus thuringiensis Cry1F protein in corn; exemption from the requirement of a tolerance. Residues of Bacillus...

  12. 40 CFR 174.520 - Bacillus thuringiensis Cry1F protein in corn; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 24 2011-07-01 2011-07-01 false Bacillus thuringiensis Cry1F protein... PLANT-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.520 Bacillus thuringiensis Cry1F protein in corn; exemption from the requirement of a tolerance. Residues of Bacillus...

  13. Glucose induced fractal colony pattern of Bacillus thuringiensis.

    PubMed

    Roy, Manas K; Banerjee, Paromita; Sengupta, Tapas K; Dattagupta, Sushanta

    2010-08-01

    Growing colonies of bacteria on the surface of thin agar plates exhibit fractal patterns as a result of nonlinear response to environmental conditions, such as nutrients, solidity of the agar medium and temperature. Here, we examine the effect of glucose on pattern formation by growing colonies of Bacillus thuringiensis isolate KPWP1. We also present the theoretical modeling of the colony growth of KPWP1 and the associated spatio-temporal patterns. Our experimental results are in excellent agreement with simulations based on a reaction-diffusion model that describes diffusion-limited aggregation and branching, in which individual cells move actively in the periphery, but become immotile in the inner regions of the growing colony. We obtain the Hausdorff fractal dimension of the colony patterns: D(H.Expt)=1.1969 and D(H, R.D.=)1.1965, for experiment and reaction-diffusion model, respectively. Results of our experiments and modeling clearly show how glucose at higher concentration can prove to be inhibitory for motility of growing colonies of B. thuringiensis cells on semisolid support and be responsible for changes in the growth pattern. PMID:20553734

  14. Biochemistry and genetics of insect resistance to Bacillus thuringiensis.

    PubMed

    Ferré, Juan; Van Rie, Jeroen

    2002-01-01

    Bacillus thuringiensis (Bt) is a valuable source of insecticidal proteins for use in conventional sprayable formulations and in transgenic crops, and it is the most promising alternative to synthetic insecticides. However, evolution of resistance in insect populations is a serious threat to this technology. So far, only one insect species has evolved significant levels of resistance in the field, but laboratory selection experiments have shown the high potential of other species to evolve resistance against Bt. We have reviewed the current knowledge on the biochemical mechanisms and genetics of resistance to Bt products and insecticidal crystal proteins. The understanding of the biochemical and genetic basis of resistance to Bt can help design appropriate management tactics to delay or reduce the evolution of resistance in insect populations.

  15. Mode of action of mosquitocidal Bacillus thuringiensis toxins.

    PubMed

    Soberón, Mario; Fernández, Luisa E; Pérez, Claudia; Gill, Sarjeet S; Bravo, Alejandra

    2007-04-01

    Cry toxins from Bacillus thuringiensis (Bt) are used for insect control. Their primary action is to lyse midgut epithelial cells. In lepidopteran insects, Cry1A monomeric toxins interact with a first receptor and this interaction triggers toxin oligomerization. The oligomeric structure interacts then with a second GPI-anchored receptor that induces insertion into membrane microdomains and larvae death. In the case of mosquitocidal Bt strains, two different toxins participate, Cry and Cyt. These toxins have a synergistic effect and Cyt1Aa overcomes Cry toxin-resistance. We will summarize recent findings on the identification of Cry receptors in mosquitoes and the mechanism of synergism: Cyt1Aa synergizes or suppresses resistance to Cry toxins by functioning as a Cry membrane-bound receptor. PMID:17145072

  16. Nanoscale imaging of Bacillus thuringiensis flagella using atomic force microscopy

    NASA Astrophysics Data System (ADS)

    Gillis, Annika; Dupres, Vincent; Delestrait, Guillaume; Mahillon, Jacques; Dufrêne, Yves F.

    2012-02-01

    Because bacterial flagella play essential roles in various processes (motility, adhesion, host interactions, secretion), studying their expression in relation to function is an important challenge. Here, we use atomic force microscopy (AFM) to gain insight into the nanoscale surface properties of two wild-type and four mutant strains of Bacillus thuringiensis exhibiting various levels of flagellation. We show that, unlike AFM in liquid, AFM in air is a simple and reliable approach to observe the morphological details of the bacteria, and to quantify the density and dimensions of their flagella. We found that the amount of flagella expressed by the six strains, as observed at the nanoscale, correlates with their microscopic swarming motility. These observations provide novel information on flagella expression in Gram-positive bacteria and demonstrate the power of AFM in genetic studies for the fast assessment of the phenotypic characteristics of bacterial strains altered in cell surface appendages.Because bacterial flagella play essential roles in various processes (motility, adhesion, host interactions, secretion), studying their expression in relation to function is an important challenge. Here, we use atomic force microscopy (AFM) to gain insight into the nanoscale surface properties of two wild-type and four mutant strains of Bacillus thuringiensis exhibiting various levels of flagellation. We show that, unlike AFM in liquid, AFM in air is a simple and reliable approach to observe the morphological details of the bacteria, and to quantify the density and dimensions of their flagella. We found that the amount of flagella expressed by the six strains, as observed at the nanoscale, correlates with their microscopic swarming motility. These observations provide novel information on flagella expression in Gram-positive bacteria and demonstrate the power of AFM in genetic studies for the fast assessment of the phenotypic characteristics of bacterial strains altered in

  17. Occurrence of Toxigenic Bacillus cereus and Bacillus thuringiensis in Doenjang, a Korean Fermented Soybean Paste.

    PubMed

    Park, Kyung Min; Kim, Hyun Jung; Jeong, Moon Cheol; Koo, Minseon

    2016-04-01

    This study determined the prevalence and toxin profile of Bacillus cereus and Bacillus thuringiensis in doenjang, a fermented soybean food, made using both traditional and commercial methods. The 51 doenjang samples tested were broadly contaminated with B. cereus; in contrast, only one sample was positive for B. thuringiensis. All B. cereus isolates from doenjang were positive for diarrheal toxin genes. The frequencies of nheABC and hblACD in traditional samples were 22.7 and 0%, respectively, whereas 5.1 and 5.1% of B. cereus isolates from commercial samples possessed nheABC and hblACD, respectively. The detection rate of ces gene was 10.8%. The predominant toxin profile among isolates from enterotoxigenic B. cereus in doenjang was profile 4 (entFM-bceT-cytK). The major enterotoxin genes in emetic B. cereus were cytK, entFM, and nheA genes. The B. thuringiensis isolate was of the diarrheagenic type. These results provide a better understanding of the epidemiology of the enterotoxigenic and emetic B. cereus groups in Korean fermented soybean products.

  18. Characteristics of a broad lytic spectrum endolysin from phage BtCS33 of Bacillus thuringiensis

    PubMed Central

    2012-01-01

    Background Endolysins produced by bacteriophages lyse bacteria, and are thus considered a novel type of antimicrobial agent. Several endolysins from Bacillus phages or prophages have previously been characterized and used to target Bacillus strains that cause disease in animals and humans. B. thuringiensis phage BtCS33 is a Siphoviridae family phage and its genome has been sequenced and analyzed. In the BtCS33 genome, orf18 was found to encode an endolysin protein (PlyBt33). Results Bioinformatic analyses showed that endolysin PlyBt33 was composed of two functional domains, the N-terminal catalytic domain and the C-terminal cell wall binding domain. In this study, the entire endolysin PlyBt33, and both the N- and C-termini,were expressed in Escherichia coli and then purified. The lytic activities of PlyBt33 and its N-terminus were tested on bacteria. Both regions exhibited lytic activity, although PlyBt33 showed a higher lytic activity than the N-terminus. PlyBt33 exhibited activity against all Bacillus strains tested from five different species, but was not active against Gram-negative bacteria. Optimal conditions for PlyBt33 reactivity were pH 9.0 and 50°C. PlyBt33 showed high thermostability, with 40% of initial activity remaining following 1 h of treatment at 60°C. The C-terminus of PlyBt33 bound to B. thuringiensis strain HD-73 and Bacillus subtilis strain 168. This cell wall binding domain might be novel, as its amino acid sequence showed little similarity to previously reported endolysins. Conclusions PlyBt33 showed potential as a novel antimicrobial agent at a relatively high temperature and had a broad lytic spectrum within the Bacillus genus. The C-terminus of PlyBt33 might be a novel kind of cell wall binding domain. PMID:23249212

  19. Transport of Bacillus thuringiensis var. kurstaki via fomites.

    PubMed

    Van Cuyk, Sheila; Veal, Lee Ann B; Simpson, Beverley; Omberg, Kristin M

    2011-09-01

    The intentional and controlled release of an aerosolized bacterium provides an opportunity to investigate the implications of a biological attack. Since 2006, Los Alamos National Laboratory has worked with several urban areas, including Fairfax County, VA, to design experiments to evaluate biodefense concepts of operations using routine spraying of Bacillus thuringiensis var. kurstaki (Btk). Btk is dispersed in large quantities as a slurry to control the gypsy moth, Lymantria dispar. Understanding whether personnel and equipment pick up residual contamination during sampling activities and transport it to other areas is critical for the formulation of appropriate response and recovery plans. While there is a growing body of literature surrounding the transmission of viral diseases via fomites, there is limited information on the transport of Bacillus species via this route. In 2008, LANL investigated whether field sampling activities conducted near sprayed areas, post-spray, resulted in measurable cross-contamination of sampling personnel, equipment, vehicles, and hotel rooms. Viable Btk was detected in all sample types, indicating transport of the agent occurred via fomites.

  20. Characterization of a Chitin-Binding Protein from Bacillus thuringiensis HD-1

    PubMed Central

    Gupta, Rani; Vimala, Y.; Bhatnagar, Raj K.

    2013-01-01

    Strains of Bacillus thuringiensis produce insecticidal proteins. These strains have been isolated from diverse ecological niches, such as soil, phylloplane, insect cadavers and grain dust. To effectively propagate, these strains produce a range of molecules that facilitate its multiplication in a competing environment. In this report, we have examined synthesis of a chitin-binding protein and evaluated its effect on fungi encountered in environment and its interaction with insecticidal proteins synthesized by B. thuringiensis. The gene encoding chitin-binding protein has been cloned and expressed. The purified protein has been demonstrated to interact with Cry insecticidal protein, Cry1Ac by Circular Dichrosim spectroscopy (CD) and in vitro pull down assays. The chitin-binding protein potentiates insecticidal activity of bacillar insecticidal protein, Cry1Ac. Further, chitin-binding protein was fungistatic against several soil fungi. The chitin binding protein is expressed in spore mother cell and deposited along with insecticidal protein, Cry1Ac. It interacts with Cry1Ac to potentiate its insecticidal activity and facilitate propagation of Bacillus strain in environment by inhibiting growth of certain fungi. PMID:23824872

  1. 40 CFR 180.1107 - Delta endotoxin of Bacillus thuringiensis variety kurstaki encapsulated into killed Pseudomonas...

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 25 2012-07-01 2012-07-01 false Delta endotoxin of Bacillus... From Tolerances § 180.1107 Delta endotoxin of Bacillus thuringiensis variety kurstaki encapsulated into killed Pseudomonas fluorescens; exemption from the requirement of a tolerance. The delta endotoxin...

  2. 40 CFR 180.1107 - Delta endotoxin of Bacillus thuringiensis variety kurstaki encapsulated into killed Pseudomonas...

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 25 2013-07-01 2013-07-01 false Delta endotoxin of Bacillus... From Tolerances § 180.1107 Delta endotoxin of Bacillus thuringiensis variety kurstaki encapsulated into killed Pseudomonas fluorescens; exemption from the requirement of a tolerance. The delta endotoxin...

  3. 40 CFR 180.1107 - Delta endotoxin of Bacillus thuringiensis variety kurstaki encapsulated into killed Pseudomonas...

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 24 2014-07-01 2014-07-01 false Delta endotoxin of Bacillus... From Tolerances § 180.1107 Delta endotoxin of Bacillus thuringiensis variety kurstaki encapsulated into killed Pseudomonas fluorescens; exemption from the requirement of a tolerance. The delta endotoxin...

  4. Bacillus cereus and Bacillus thuringiensis spores in Korean rice: prevalence and toxin production as affected by production area and degree of milling.

    PubMed

    Kim, Booyoung; Bang, Jihyun; Kim, Hoikyung; Kim, Yoonsook; Kim, Byeong-Sam; Beuchat, Larry R; Ryu, Jee-Hoon

    2014-09-01

    We determined the prevalence of and toxin production by Bacillus cereus and Bacillus thuringiensis in Korean rice as affected by production area and degree of milling. Rough rice was collected from 64 farms in 22 agricultural areas and polished to produce brown and white rice. In total, rice samples were broadly contaminated with B. cereus spores, with no effect of production area. The prevalence and counts of B. cereus spores declined as milling progressed. Frequencies of hemolysin BL (HBL) production by isolates were significantly (P ≤ 0.01) reduced as milling progressed. This pattern corresponded with the presence of genes encoding the diarrheal enterotoxins. The frequency of B. cereus isolates positive for hblC, hblD, or nheB genes decreased as milling progressed. Because most B. cereus isolates from rice samples contained six enterotoxin genes, we concluded that B. cereus in rice produced in Korea is predominantly of the diarrheagenic type. The prevalence of B. thuringiensis in rice was significantly lower than that of B. cereus and not correlated with production area. All B. thuringiensis isolates were of the diarrheagenic type. This study provides information useful for predicting safety risks associated with B. cereus and B. thuringiensis in rough and processed Korean rice.

  5. Cell membrane interaction of Bacillus thuringiensis subsp. israelensis cytolytic toxins.

    PubMed

    Gill, S S; Singh, G J; Hornung, J M

    1987-05-01

    Two toxic polypeptides of 24 and 25 kilodaltons (kDa) were purified from parasporal proteinaceous crystals of Bacillus thuringiensis subsp. israelensis. Both of these polypeptides, which are antigenically similar and have identical N terminals, lysed human erythrocytes and cultured mosquito cells. Although the 24-kDa peptide was more toxic than the 25-kDa peptide, both were less toxic than the crude alkali-solubilized crystal toxin. However, a 1:1 mixture of these 24- and 25-kDa proteins was more toxic than either of these polypeptides individually, indicating a possible interaction between these proteins at the cell membrane. Both the 24- and the 25-kDa proteins were inactivated by aqueous suspensions of dioleolylphosphatidylcholine, indicating the involvement of phospholipids in the cytotoxic action of these toxins. Thus the role of cell membrane phospholipids in mediating the toxin action was studied by using phospholipases as probes. Treatment of erythrocytes with high levels of phospholipase D increased their susceptibility to the toxin; however, phospholipase A2-treated erythrocytes were less susceptible to the toxin. These erythrocytes also bound less 125I-labeled 25-kDa toxin. These results support the role of fatty acyl residues at the syn-2 position of membrane phospholipids in toxin action. The cytolytic toxin of B. thuringiensis subsp. israelensis is thought to damage cell membranes in a detergentlike manner. However, there was a difference between the cytolytic action of this toxin and that of a nonionic detergent such as Triton X-100 because phospholipase A2-treated erythrocytes were more susceptible to Triton X-100, whereas such erythrocytes were less sensitive to the toxin. Thus, the cytolytic toxin apparently did not act as a nonspecific detergent, but rather interacted with phospholipid receptors on the cell membrane. Such an interaction of the toxin with phospholipid receptors probably results in the increased cell permeability, thereby causing

  6. Nematoda: susceptibility of the egg to Bacillus thuringiensis toxins.

    PubMed

    Bottjer, K P; Bone, L W; Gill, S S

    1985-10-01

    Crystalline toxins from Bacillus thuringiensis israelensis and B.t. kurstaki were lethal in vitro to eggs of the nematode Trichostrongylus colubriformis. The LD50 values for the two toxins were 0.38 ng and 37.5 micrograms total protein/ml, respectively. After 1 week at ambient temperature, the LD50 of B.t. kurstaki decreased to less than 4 micrograms/ml. Toxin from B.t. israelensis had no effect within 48 hr on survival of adult nematodes or on their feeding in vitro. Third-stage larvae of T. colubriformis were also unaffected by B.t. israelensis toxin. Exposure of third-stage larvae of Nippostrongylus brasiliensis to 1.1 micrograms total protein/ml of B.t. israelensis for 4 hr had no effect on their infectivity in mice, based on recovery of helminths at 7 days after infection. Similar exposure of 5-day-old N. brasiliensis and subsequent transfer into the intestine of mice gave recoveries that were similar to the untreated control. Thirty strains of B. thuringiensis caused mortality in nematode eggs, but over a 77,000-fold range of activity was found, based on the LD50 values. Toxin from B.t. israelensis was lethal to eggs of six zooparasitic and one free-living species of nematode, but the LD50 values varied 28-fold. Addition of B.t. israelensis to feces that contained eggs of T. colubriformis reduced subsequent recovery of larvae, with an LD50 of 260 ng/g of feces.

  7. How Quorum Sensing Connects Sporulation to Necrotrophism in Bacillus thuringiensis.

    PubMed

    Perchat, Stéphane; Talagas, Antoine; Poncet, Sandrine; Lazar, Noureddine; Li de la Sierra-Gallay, Inès; Gohar, Michel; Lereclus, Didier; Nessler, Sylvie

    2016-08-01

    Bacteria use quorum sensing to coordinate adaptation properties, cell fate or commitment to sporulation. The infectious cycle of Bacillus thuringiensis in the insect host is a powerful model to investigate the role of quorum sensing in natural conditions. It is tuned by communication systems regulators belonging to the RNPP family and directly regulated by re-internalized signaling peptides. One such RNPP regulator, NprR, acts in the presence of its cognate signaling peptide NprX as a transcription factor, regulating a set of genes involved in the survival of these bacteria in the insect cadaver. Here, we demonstrate that, in the absence of NprX and independently of its transcriptional activator function, NprR negatively controls sporulation. NprR inhibits expression of Spo0A-regulated genes by preventing the KinA-dependent phosphorylation of the phosphotransferase Spo0F, thus delaying initiation of the sporulation process. This NprR function displays striking similarities with the Rap proteins, which also belong to the RNPP family, but are devoid of DNA-binding domain and indirectly control gene expression via protein-protein interactions in Bacilli. Conservation of the Rap residues directly interacting with Spo0F further suggests a common inhibition of the sporulation phosphorelay. The crystal structure of apo NprR confirms that NprR displays a highly flexible Rap-like structure. We propose a molecular regulatory mechanism in which key residues of the bifunctional regulator NprR are directly and alternatively involved in its two functions. NprX binding switches NprR from a dimeric inhibitor of sporulation to a tetrameric transcriptional activator involved in the necrotrophic lifestyle of B. thuringiensis. NprR thus tightly coordinates sporulation and necrotrophism, ensuring survival and dissemination of the bacteria during host infection. PMID:27483473

  8. How Quorum Sensing Connects Sporulation to Necrotrophism in Bacillus thuringiensis

    PubMed Central

    Poncet, Sandrine; Lazar, Noureddine; Li de la Sierra-Gallay, Inès; Gohar, Michel; Lereclus, Didier; Nessler, Sylvie

    2016-01-01

    Bacteria use quorum sensing to coordinate adaptation properties, cell fate or commitment to sporulation. The infectious cycle of Bacillus thuringiensis in the insect host is a powerful model to investigate the role of quorum sensing in natural conditions. It is tuned by communication systems regulators belonging to the RNPP family and directly regulated by re-internalized signaling peptides. One such RNPP regulator, NprR, acts in the presence of its cognate signaling peptide NprX as a transcription factor, regulating a set of genes involved in the survival of these bacteria in the insect cadaver. Here, we demonstrate that, in the absence of NprX and independently of its transcriptional activator function, NprR negatively controls sporulation. NprR inhibits expression of Spo0A-regulated genes by preventing the KinA-dependent phosphorylation of the phosphotransferase Spo0F, thus delaying initiation of the sporulation process. This NprR function displays striking similarities with the Rap proteins, which also belong to the RNPP family, but are devoid of DNA-binding domain and indirectly control gene expression via protein-protein interactions in Bacilli. Conservation of the Rap residues directly interacting with Spo0F further suggests a common inhibition of the sporulation phosphorelay. The crystal structure of apo NprR confirms that NprR displays a highly flexible Rap-like structure. We propose a molecular regulatory mechanism in which key residues of the bifunctional regulator NprR are directly and alternatively involved in its two functions. NprX binding switches NprR from a dimeric inhibitor of sporulation to a tetrameric transcriptional activator involved in the necrotrophic lifestyle of B. thuringiensis. NprR thus tightly coordinates sporulation and necrotrophism, ensuring survival and dissemination of the bacteria during host infection. PMID:27483473

  9. Spider mite infestations reduce Bacillus thuringiensis toxin concentration in corn leaves and predators avoid spider mites that have fed on Bacillus thuringiensis corn

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Transgenic crops containing pyramid-stacked genes for Bacillus thuringiensis derived toxins for controlling coleopteran and lepidopteran pests are increasingly common. As part of environmental risk assessments, these crops are evaluated for toxicity against non-target organisms, and for their poten...

  10. 77 FR 47287 - Bacillus thuringiensis eCry3.1Ab Protein in Corn; Exemption From the Requirement of a Tolerance

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-08-08

    ... AGENCY 40 CFR Part 174 Bacillus thuringiensis eCry3.1Ab Protein in Corn; Exemption From the Requirement...-incorporated protectant (PIP), Bacillus thuringiensis eCry3.1Ab protein in corn, in or on the food and feed... permissible level for residues of Bacillus thuringiensis eCry3.1Ab protein in corn. DATES: This regulation...

  11. Cry Proteins from Bacillus thuringiensis Active against Diamondback Moth and Fall Armyworm.

    PubMed

    Silva, M C; Siqueira, H A A; Silva, L M; Marques, E J; Barros, R

    2015-08-01

    Biopesticides based on Bacillus thuringiensis and genetically modified plants with genes from this bacterium have been used to control Plutella xylostella (L.) and Spodoptera frugiperda (J.E. Smith). However, the selection pressure imposed by these technologies may undermine the efficiency of this important alternative to synthetic insecticides. Toxins with different modes of action allow a satisfactory control of these insects. The purpose of this study was to characterize the protein and gene contents of 20 B. thuringiensis isolates from soil and insect samples collected in several areas of Northeast Brazil which are active against P. xylostella and S. frugiperda. Protein profiles were obtained by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Polymerase chain reaction assays were used to determine toxin genes present within bacterial isolates. The protein profile of the majority of the isolates produced bands of approximately 130 kDa, suggesting the presence of Cry1, Cry8 and Cry9 proteins. The gene content of the isolates of B. thuringiensis investigated showed different gene profiles. Isolates LIIT-4306 and LIIT-4311 were the most actives against both species, with LC50 of 0.03 and 0.02 × 10(8) spores mL(-1), respectively, for P. xylostella, and LC50 of 0.001 × 10(8) spores mL(-1) for S. frugiperda. These isolates carried the cry1, cry1Aa, cry1Ab, cry1Ac, cry1B, cry1C, cry1D, cry1F, cry2, cry2A, cry8, and cry9C genes. The obtained gene profiles showed great potential for the control of P. xylostella and S. frugiperda, primarily because of the presence of several cry1A genes, which are found in isolates of B. thuringiensis active against these insects. PMID:26070631

  12. Comparative genomics analysis of the companion mechanisms of Bacillus thuringiensis Bc601 and Bacillus endophyticus Hbe603 in bacterial consortium.

    PubMed

    Jia, Nan; Ding, Ming-Zhu; Gao, Feng; Yuan, Ying-Jin

    2016-01-01

    Bacillus thuringiensis and Bacillus endophyticus both act as the companion bacteria, which cooperate with Ketogulonigenium vulgare in vitamin C two-step fermentation. Two Bacillus species have different morphologies, swarming motility and 2-keto-L-gulonic acid productivities when they co-culture with K. vulgare. Here, we report the complete genome sequencing of B. thuringiensis Bc601 and eight plasmids of B. endophyticus Hbe603, and carry out the comparative genomics analysis. Consequently, B. thuringiensis Bc601, with greater ability of response to the external environment, has been found more two-component system, sporulation coat and peptidoglycan biosynthesis related proteins than B. endophyticus Hbe603, and B. endophyticus Hbe603, with greater ability of nutrients biosynthesis, has been found more alpha-galactosidase, propanoate, glutathione and inositol phosphate metabolism, and amino acid degradation related proteins than B. thuringiensis Bc601. Different ability of swarming motility, response to the external environment and nutrients biosynthesis may reflect different companion mechanisms of two Bacillus species. Comparative genomic analysis of B. endophyticus and B. thuringiensis enables us to further understand the cooperative mechanism with K. vulgare, and facilitate the optimization of bacterial consortium. PMID:27353048

  13. Comparative genomics analysis of the companion mechanisms of Bacillus thuringiensis Bc601 and Bacillus endophyticus Hbe603 in bacterial consortium

    PubMed Central

    Jia, Nan; Ding, Ming-Zhu; Gao, Feng; Yuan, Ying-Jin

    2016-01-01

    Bacillus thuringiensis and Bacillus endophyticus both act as the companion bacteria, which cooperate with Ketogulonigenium vulgare in vitamin C two-step fermentation. Two Bacillus species have different morphologies, swarming motility and 2-keto-L-gulonic acid productivities when they co-culture with K. vulgare. Here, we report the complete genome sequencing of B. thuringiensis Bc601 and eight plasmids of B. endophyticus Hbe603, and carry out the comparative genomics analysis. Consequently, B. thuringiensis Bc601, with greater ability of response to the external environment, has been found more two-component system, sporulation coat and peptidoglycan biosynthesis related proteins than B. endophyticus Hbe603, and B. endophyticus Hbe603, with greater ability of nutrients biosynthesis, has been found more alpha-galactosidase, propanoate, glutathione and inositol phosphate metabolism, and amino acid degradation related proteins than B. thuringiensis Bc601. Different ability of swarming motility, response to the external environment and nutrients biosynthesis may reflect different companion mechanisms of two Bacillus species. Comparative genomic analysis of B. endophyticus and B. thuringiensis enables us to further understand the cooperative mechanism with K. vulgare, and facilitate the optimization of bacterial consortium. PMID:27353048

  14. Cyt1A from Bacillus thuringiensis Synergizes Activity of Bacillus sphaericus against Aedes aegypti (Diptera: Culicidae)

    PubMed Central

    Wirth, Margaret C.; Federici, Brian A.; Walton, William E.

    2000-01-01

    Bacillus sphaericus is a mosquitocidal bacterium recently developed as a commercial larvicide that is used worldwide to control pestiferous and vector mosquitoes. Whereas B. sphaericus is highly active against larvae of Culex and Anopheles mosquitoes, it is virtually nontoxic to Aedes aegypti, an important vector species. In the present study, we evaluated the capacity of the cytolytic protein Cyt1A from Bacillus thuringiensis subsp. israelensis to enhance the toxicity of B. sphaericus toward A. aegypti. Various combinations of these two materials were evaluated, and all were highly toxic. A ratio of 10:1 of B. sphaericus to Cyt1A was 3,600-fold more toxic to A. aegypti than B. sphaericus alone. Statistical analysis showed this high activity was due to synergism between the Cyt1A toxin and B. sphaericus. These results suggest that Cyt1A could be useful in expanding the host range of B. sphaericus. PMID:10698776

  15. ssp genes and spore osmotolerance in Bacillus thuringiensis israelensis and Bacillus sphaericus.

    PubMed

    Cucchi, A; Sanchez de Rivas, C

    1995-10-01

    It was shown previously that spores and vegetative cells of Bacillus sphaericus (Bf) and Bacillus thuringiensis israelensis (Bti) are very sensitive to osmotic variations. Since spore osmotolerance has been associated with their SASP (small acid soluble spore proteins) content coded by ssp genes, hybridization assays were performed with sspE and sspA genes from B. subtilis as probes and showed that Bti and Bf strains could lack an sspE-like gene. The B. subtilis sspE gene was then introduced into Bti 4Q2 strain; spores were obtained and showed a 65 to 650 times higher level of osmotolerance to NaCl, without affecting other important properties: hypoosmotic resistance in vegetative cells, spore UV resistance, and larvicidal activity against diptera larvae. PMID:7549769

  16. New role for DCR-1/dicer in Caenorhabditis elegans innate immunity against the highly virulent bacterium Bacillus thuringiensis DB27.

    PubMed

    Iatsenko, Igor; Sinha, Amit; Rödelsperger, Christian; Sommer, Ralf J

    2013-10-01

    Bacillus thuringiensis produces toxins that target invertebrates, including Caenorhabditis elegans. Virulence of Bacillus strains is often highly specific, such that B. thuringiensis strain DB27 is highly pathogenic to C. elegans but shows no virulence for another model nematode, Pristionchus pacificus. To uncover the underlying mechanisms of the differential responses of the two nematodes to B. thuringiensis DB27 and to reveal the C. elegans defense mechanisms against this pathogen, we conducted a genetic screen for C. elegans mutants resistant to B. thuringiensis DB27. Here, we describe a B. thuringiensis DB27-resistant C. elegans mutant that is identical to nasp-1, which encodes the C. elegans homolog of the nuclear-autoantigenic-sperm protein. Gene expression analysis indicated a substantial overlap between the genes downregulated in the nasp-1 mutant and targets of C. elegans dcr-1/Dicer, suggesting that dcr-1 is repressed in nasp-1 mutants, which was confirmed by quantitative PCR. Consistent with this, the nasp-1 mutant exhibits RNA interference (RNAi) deficiency and reduced longevity similar to those of a dcr-1 mutant. Building on these surprising findings, we further explored a potential role for dcr-1 in C. elegans innate immunity. We show that dcr-1 mutant alleles deficient in microRNA (miRNA) processing, but not those deficient only in RNAi, are resistant to B. thuringiensis DB27. Furthermore, dcr-1 overexpression rescues the nasp-1 mutant's resistance, suggesting that repression of dcr-1 determines the nasp-1 mutant's resistance. Additionally, we identified the collagen-encoding gene col-92 as one of the downstream effectors of nasp-1 that play an important role in resistance to DB27. Taken together, these results uncover a previously unknown role for DCR-1/Dicer in C. elegans antibacterial immunity that is largely associated with miRNA processing.

  17. 40 CFR 180.1108 - Delta endotoxin of Bacillus thuringiensis variety San Diego encapsulated into killed Pseudomonas...

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... From Tolerances § 180.1108 Delta endotoxin of Bacillus thuringiensis variety San Diego encapsulated... of Bacillus thuringiensis variety San Diego encapsulated into killed Pseudomonas fluorescens is... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Delta endotoxin of...

  18. 40 CFR 180.1108 - Delta endotoxin of Bacillus thuringiensis variety San Diego encapsulated into killed Pseudomonas...

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... From Tolerances § 180.1108 Delta endotoxin of Bacillus thuringiensis variety San Diego encapsulated... of Bacillus thuringiensis variety San Diego encapsulated into killed Pseudomonas fluorescens is... 40 Protection of Environment 24 2011-07-01 2011-07-01 false Delta endotoxin of...

  19. Performance of dairy cows fed silage and grain produced from second-generation insect-protected (Bacillus thuringiensis) corn (MON 89034), compared with parental line corn or reference corn.

    PubMed

    Castillo-Lopez, E; Clark, K J; Paz, H A; Ramirez Ramirez, H A; Klusmeyer, T H; Hartnell, G F; Kononoff, P J

    2014-01-01

    Corn grain and corn silage are major feed components in lactating dairy cow rations. Bacillus thuringiensis (B.t.) is a naturally occurring soil bacterium that produces a protein that is toxic to lepidopteran insects that may damage plant tissues and reduce corn quality and yields. During each of the four 28-d periods, cows were offered 1 of 4 rations in which the corn grain and silage originated from different corn hybrids: a nontransgenic corn control (from hybrid DKC63-78; Monsanto Co., St. Louis, MO), a B.t. test substance corn (MON 89034 in hybrid DKC63-78; Monsanto Co.), and 2 commercial nontransgenic reference (Ref) hybrids: DKC61-42 (Ref 1) and DKC62-30 (Ref 2; Monsanto Co.). Sixteen multiparous Holstein cows averaging 110 ± 21 d in milk and weighing 684 ± 62.3 kg were blocked by days in milk and milk yield and randomly assigned to one of four 4 × 4 Latin squares. Diets were formulated to contain 36.4% corn silage and 16.3% corn grain. Dry matter intake was greater for cows consuming B.t. corn (26.6 ± 0.59 kg/d) compared with the control, Ref 1, and Ref 2 corn diets (25.4, 25.0, and 25.6 ± 0.59 kg/d, respectively). Milk yield, fat yield, and percentage of fat (36.8 ± 0.98 kg/d, 1.22 ± 0.05 kg/d, and 3.3 ± 0.10%), milk protein yield and percentage of protein (1.11 ± 0.03 kg/d and 3.01 ± 0.05%), milk urea nitrogen concentration (14.01 ± 0.49 mg/dL), and 3.5% fat-corrected milk yield (35.7 ± 1.07 kg/d) were not different across treatments. The results from this study show that lactating dairy cows that consume B.t. corn (MON 89034) do not differ from lactating dairy cows that consume nontransgenic corn in milk yield, 3.5% fat-corrected milk per unit of dry matter intake, or milk components.

  20. Extended genetic analysis of Brazilian isolates of Bacillus cereus and Bacillus thuringiensis

    PubMed Central

    Zahner, Viviane; Silva, Ana Carolina Telles de Carvalho e; de Moraes, Gabriela Pinhel; McIntosh, Douglas; de Filippis, Ivano

    2013-01-01

    Multiple locus sequence typing (MLST) was undertaken to extend the genetic characterization of 29 isolates of Bacillus cereus and Bacillus thuringiensis previously characterized in terms of presence/absence of sequences encoding virulence factors and via variable number tandem repeat (VNTR). Additional analysis involved polymerase chain reaction for the presence of sequences (be, cytK, inA, pag, lef, cya and cap), encoding putative virulence factors, not investigated in the earlier study. MLST analysis ascribed novel and unique sequence types to each of the isolates. A phylogenetic tree was constructed from a single sequence of 2,838 bp of concatenated loci sequences. The strains were not monophyletic by analysis of any specific housekeeping gene or virulence characteristic. No clear association in relation to source of isolation or to genotypic profile based on the presence or absence of putative virulence genes could be identified. Comparison of VNTR profiling with MLST data suggested a correlation between these two methods of genetic analysis. In common with the majority of previous studies, MLST was unable to provide clarification of the basis for pathogenicity among members of the B. cereus complex. Nevertheless, our application of MLST served to reinforce the notion that B. cereus and B. thuringiensis should be considered as the same species. PMID:23440117

  1. Side effects of Bacillus thuringiensis var. kurstaki on the hymenopterous parasitic wasp Trichogramma chilonis.

    PubMed

    Amichot, Marcel; Curty, Christine; Benguettat-Magliano, Olivia; Gallet, Armel; Wajnberg, Eric

    2016-02-01

    Most of the detrimental effects of using conventional insecticides to control crop pests are now well identified and are nowadays major arguments for replacing such compounds by the use of biological control agents. In this respect, the bacterium Bacillus thuringiensis var. kurstaki and Trichogramma (Hymenoptera: Trichogrammatidae) parasitic wasp species are both effective against lepidopterous pests and can actually be used concomitantly. In this work, we studied the potential side effects of B. thuringiensis var. kurstaki on Trichogramma chilonis females. We first evidenced an acute toxicity of B. thuringiensis on T. chilonis. Then, after ingestion of B. thuringiensis at sublethal doses, we focused on life history traits of T. chilonis such as longevity, reproductive success and the time spent on host eggs patches. The reproductive success of T. chilonis was not modified by B. thuringiensis while a significant effect was observed on longevity and the time spent on host eggs patches. The physiological and ecological meanings of the results obtained are discussed.

  2. Raman spectroscopy of Bacillus thuringiensis physiology and inactivation

    NASA Astrophysics Data System (ADS)

    Morrow, J. B.; Almeida, J.; Cole, K. D.; Reipa, V.

    2012-12-01

    The ability to detect spore contamination and inactivation is relevant to developing and determining decontamination strategy success for food and water safety. This study was conducted to develop a systematic comparison of nondestructive vibrational spectroscopy techniques (Surface-Enhanced Raman Spectroscopy, SERS, and normal Raman) to determine indicators of Bacillus thuringiensis physiology (spore, vegetative, outgrown, germinated and inactivated spore forms). SERS was found to provide better resolution of commonly utilized signatures of spore physiology (dipicolinic acid at 1006 cm-1 and 1387 cm-1) compared to normal Raman and native fluorescence indigenous to vegetative and outgrown cell samples was quenched in SERS experiment. New features including carotenoid pigments (Raman features at 1142 cm-1, 1512 cm-1) were identified for spore cell forms. Pronounced changes in the low frequency region (300 cm-1 to 500 cm-1) in spore spectra occurred upon germination and inactivation (with both free chlorine and by autoclaving) which is relevant to guiding decontamination and detection strategies using Raman techniques.

  3. Continuous evolution of Bacillus thuringiensis toxins overcomes insect resistance.

    PubMed

    Badran, Ahmed H; Guzov, Victor M; Huai, Qing; Kemp, Melissa M; Vishwanath, Prashanth; Kain, Wendy; Nance, Autumn M; Evdokimov, Artem; Moshiri, Farhad; Turner, Keith H; Wang, Ping; Malvar, Thomas; Liu, David R

    2016-05-01

    The Bacillus thuringiensis δ-endotoxins (Bt toxins) are widely used insecticidal proteins in engineered crops that provide agricultural, economic, and environmental benefits. The development of insect resistance to Bt toxins endangers their long-term effectiveness. Here we have developed a phage-assisted continuous evolution selection that rapidly evolves high-affinity protein-protein interactions, and applied this system to evolve variants of the Bt toxin Cry1Ac that bind a cadherin-like receptor from the insect pest Trichoplusia ni (TnCAD) that is not natively bound by wild-type Cry1Ac. The resulting evolved Cry1Ac variants bind TnCAD with high affinity (dissociation constant Kd = 11-41 nM), kill TnCAD-expressing insect cells that are not susceptible to wild-type Cry1Ac, and kill Cry1Ac-resistant T. ni insects up to 335-fold more potently than wild-type Cry1Ac. Our findings establish that the evolution of Bt toxins with novel insect cell receptor affinity can overcome insect Bt toxin resistance and confer lethality approaching that of the wild-type Bt toxin against non-resistant insects.

  4. Adhesion of Spores of Bacillus thuringiensis on a Planar Surface

    SciTech Connect

    Chung, Eunhyea; Kweon, Hyojin; Yiacoumi, Sotira; Lee, Ida; Joy, David Charles; Palumbo, Anthony Vito; Tsouris, Costas

    2010-01-01

    Adhesion of spores of Bacillus thuringiensis (Bt) and spherical silica particles on surfaces was experimentally and theoretically investigated in this study. Topography analysis via atomic force microscopy (AFM) and electron microscopy indicates that Bt spores are rod shaped, {approx}1.3 {mu}m in length and {approx}0.8 {mu}m in diameter. The adhesion force of Bt spores and silica particles on gold-coated glass was measured at various relative humidity (RH) levels by AFM. It was expected that the adhesion force would vary with RH because the individual force components contributing to the adhesion force depend on RH. The adhesion force between a particle and a planar surface in atmospheric environments was modeled as the contribution of three major force components: capillary, van der Waals, and electrostatic interaction forces. Adhesion force measurements for Bt spore (silica particle) and the gold surface system were comparable with calculations. Modeling results show that there is a critical RH value, which depends on the hydrophobicity of the materials involved, below which the water meniscus does not form and the contribution of the capillary force is zero. As RH increases, the van der Waals force decreases while the capillary force increases to a maximum value.

  5. The ecological roles of Bacillus thuringiensis within phyllosphere environments.

    PubMed

    Wang, Xiaoxian; Xue, Yarong; Han, Meizhe; Bu, Yuanqing; Liu, Changhong

    2014-08-01

    Bacillus thuringiensis (Bt) is one of the most used bio-control agents to control plant insects, but little is known about its effect on the microbial population and communities on plant leaves. With the culture dependent method, it has been observed that the dynamics of Bt within the phyllosphere varied dependent on both the doses of Bt sprayed on the leaves and the plant species, however, Bt's population size kept stable at about 1000 cfu g(-1) after 15 d since inoculation. By comparing the bacterial abundances and community structures within the phyllosphere of three plant species, we confirmed that Bt at the doses of 1.5×10(7) and 1.5×10(9) cfu mL(-1) respectively did not significantly influence the natural bacterial population size on the leaf surfaces based on culture dependent assay. However, based on culture independent denaturing gradient gel electrophoresis (DGGE), Shannon-Wiener index (H') and Unweighted Pair Group Method with Arithmetic Mean (UPGMA) analysis, Bt has a significant influence on the bacterial communities within the phyllosphere of amaranth and cotton, but not rice. These results indicate that Bt exhibits different behaviors and ecological roles on the microbial diversity within the phyllosphere, and its environmental safety has to be concerned and evaluated in the future. PMID:24534157

  6. Investigation of lead(II) uptake by Bacillus thuringiensis 016.

    PubMed

    Chen, Zhi; Pan, Xiaohong; Chen, Hui; Lin, Zhang; Guan, Xiong

    2015-11-01

    In this work, we investigated the lead(II) biosorption mechanism of Bacillus thuringiensis (Bt) 016 through batch and microscopic experiments. We found that the maximum lead(II) biosorption capacity of Bt 016 was 164.77 mg/g (dry weight). The pH value could affect the biosorption of lead(II) in a large extent. Fourier transform infrared analyses and selective passivation experiments suggested that the carboxyl, amide and phosphate functional groups of Bt 016 played an important role in lead(II) biosorption. Scanning electron microscopy observation showed that noticeable lead(II) precipitates were accumulated on bacterial surfaces. Further transmission electron microscopy thin section analysis coupled with energy dispersive X-ray spectroscopy as well as selected area electron diffraction indicated that lead(II) immobilized on the bacteria could be transformated into random-shaped crystalline lead-containing minerals eventually. This work provided a new insight into lead(II) uptake of Bt, highlighting the potential of Bt in the restoration of lead(II) contaminated repositories. PMID:26271773

  7. Investigation of lead(II) uptake by Bacillus thuringiensis 016.

    PubMed

    Chen, Zhi; Pan, Xiaohong; Chen, Hui; Lin, Zhang; Guan, Xiong

    2015-11-01

    In this work, we investigated the lead(II) biosorption mechanism of Bacillus thuringiensis (Bt) 016 through batch and microscopic experiments. We found that the maximum lead(II) biosorption capacity of Bt 016 was 164.77 mg/g (dry weight). The pH value could affect the biosorption of lead(II) in a large extent. Fourier transform infrared analyses and selective passivation experiments suggested that the carboxyl, amide and phosphate functional groups of Bt 016 played an important role in lead(II) biosorption. Scanning electron microscopy observation showed that noticeable lead(II) precipitates were accumulated on bacterial surfaces. Further transmission electron microscopy thin section analysis coupled with energy dispersive X-ray spectroscopy as well as selected area electron diffraction indicated that lead(II) immobilized on the bacteria could be transformated into random-shaped crystalline lead-containing minerals eventually. This work provided a new insight into lead(II) uptake of Bt, highlighting the potential of Bt in the restoration of lead(II) contaminated repositories.

  8. Characterization of the chitinase gene in Bacillus thuringiensis Mexican isolates.

    PubMed

    Rosas-García, Ninfa M; Fortuna-González, Juan M; Barboza-Corona, J Eleazar

    2013-11-01

    The chitinase gene was molecularly characterized in five Bacillus thuringiensis Mexican isolates, MR10, MR11, MR21, MR33, and RN52. The proteins derived from these genes were tested for their chitinase activity using fluorogenic chitin derivatives. In order to verify if chitinase genes were functional, they were cloned, and enzymatic activity of recombinant chitinases was also tested. Results indicated that enzymes exhibited endochitinase activity. The highest hydrolytic activity shown against the chitin tetrameric derivative occurred at pH value of 6.5, and the optimum activity temperature was around 60 °C. The recombinant endochitinases showed a molecular mass of ∼77 kDa with isoelectric points from 6.5 to 7.0. Analysis of the nucleotide sequences showed highly conserved sequences among all isolates (97-99 %). Gene sequence analysis revealed a putative promoter (-35 TTGAGA and -10 TTAATA) and a Shine-Dalgarno sequence (5´-AGGAGA-3´) upstream from the open reading frame. The deduced amino acid sequence revealed that the proteins are modular enzymes composed by a family 18 glycosyl hydrolase domain located between amino acids 134 and 549, a fibronectin-binding domain (580 through 656), and a chitin-binding domain (664 through 771). The deduced amino acid sequences of our isolates showed a similarity close to 100 % respect to the sequences reported in the GenBank database.

  9. The impact of secondary pests on Bacillus thuringiensis (Bt) crops.

    PubMed

    Catarino, Rui; Ceddia, Graziano; Areal, Francisco J; Park, Julian

    2015-06-01

    The intensification of agriculture and the development of synthetic insecticides enabled worldwide grain production to more than double in the last third of the 20th century. However, the heavy dependence and, in some cases, overuse of insecticides has been responsible for negative environmental and ecological impacts across the globe, such as a reduction in biodiversity, insect resistance to insecticides, negative effects on nontarget species (e.g. natural enemies) and the development of secondary pests. The use of recombinant DNA technology to develop genetically engineered insect-resistant crops could mitigate many of the negative side effects of insecticides. One such genetic alteration enables crops to express toxic crystalline (Cry) proteins from the soil bacteria Bacillus thuringiensis (Bt). Despite the widespread adoption of Bt crops, there are still a range of unanswered questions concerning longer term agro-ecosystem interactions. For instance, insect species that are not susceptible to the expressed toxin can develop into secondary pests and cause significant damage to the crop. Here, we review the main causes surrounding secondary pest dynamics in Bt crops and the impact of such outbreaks. Regardless of the causes, if nonsusceptible secondary pest populations exceed economic thresholds, insecticide spraying could become the immediate solution at farmers' disposal, and the sustainable use of this genetic modification technology may be in jeopardy. Based on the literature, recommendations for future research are outlined that will help to improve the knowledge of the possible long-term ecological trophic interactions of employing this technology. PMID:25832330

  10. Continuous evolution of Bacillus thuringiensis toxins overcomes insect resistance.

    PubMed

    Badran, Ahmed H; Guzov, Victor M; Huai, Qing; Kemp, Melissa M; Vishwanath, Prashanth; Kain, Wendy; Nance, Autumn M; Evdokimov, Artem; Moshiri, Farhad; Turner, Keith H; Wang, Ping; Malvar, Thomas; Liu, David R

    2016-05-01

    The Bacillus thuringiensis δ-endotoxins (Bt toxins) are widely used insecticidal proteins in engineered crops that provide agricultural, economic, and environmental benefits. The development of insect resistance to Bt toxins endangers their long-term effectiveness. Here we have developed a phage-assisted continuous evolution selection that rapidly evolves high-affinity protein-protein interactions, and applied this system to evolve variants of the Bt toxin Cry1Ac that bind a cadherin-like receptor from the insect pest Trichoplusia ni (TnCAD) that is not natively bound by wild-type Cry1Ac. The resulting evolved Cry1Ac variants bind TnCAD with high affinity (dissociation constant Kd = 11-41 nM), kill TnCAD-expressing insect cells that are not susceptible to wild-type Cry1Ac, and kill Cry1Ac-resistant T. ni insects up to 335-fold more potently than wild-type Cry1Ac. Our findings establish that the evolution of Bt toxins with novel insect cell receptor affinity can overcome insect Bt toxin resistance and confer lethality approaching that of the wild-type Bt toxin against non-resistant insects. PMID:27120167

  11. Complete genome sequence of Bacillus thuringiensis YBT-1518, a typical strain with high toxicity to nematodes.

    PubMed

    Wang, Pengxia; Zhang, Chunyi; Guo, Mengmeng; Guo, Suxia; Zhu, Yiguang; Zheng, Jinshui; Zhu, Lei; Ruan, Lifang; Peng, Donghai; Sun, Ming

    2014-02-10

    Bacillus thuringiensis is a ubiquitous spore-forming bacterium and has been widely used as a biopesticide for controlling agricultural insects by the production of insecticidal crystal proteins (ICPs). B. thuringiensis YBT-1518 displays effective toxicity to nematodes. This strain harbors three nematicidal crystal protein genes, including cry55Aa1, cry6Aa2 and cry5Ba2, and also contains multiple potential virulence factors. Here, we report the complete genome sequence of B. thuringiensis YBT-1518, which consists of one circular chromosome and six circular plasmids.

  12. The complete genome sequence of Bacillus thuringiensis serovar Hailuosis YWC2-8.

    PubMed

    Zhu, Jun; Zhang, Qinbin; Cao, Ye; Li, Qiao; Zhu, Zizhong; Wang, Linxia; Li, Ping

    2016-02-10

    Bacillus thuringiensis, a typical aerobic, Gram-positive, spore-forming bacterium, is an important microbial insecticide widely used in the control of agricultural pests. B. thuringiensis serovar Hailuosis YWC2-8 with high insecticidal activity against Diptera and Lepidoptera insects has three insecticidal crystal protein genes, such as cry4Cb2, cry30Ea2, and cry56Aa1. In this study, the complete genome sequence of B. thuringiensis YWC2-8 was analyzed, which contains one circular gapless chromosome and six circular plasmids.

  13. Histopathology and the lethal effect of Cry proteins and strains of Bacillus thuringiensis Berliner in Spodoptera frugiperda J.E. Smith Caterpillars (Lepidoptera, Noctuidae).

    PubMed

    Knaak, N; Franz, A R; Santos, G F; Fiuza, L M

    2010-08-01

    Among the phytophagous insects which attack crops, the fall armyworm, Spodoptera frugiperda (J.E. Smith, 1797) (Lepidoptera, Noctuidae) is particularly harmful in the initial growth phase of rice plants. As a potential means of controlling this pest, and considering that the entomopathogen Bacillus thuringiensis Berliner demonstrates toxicity due to synthesis of the Cry protein, the present study was undertaken to evaluate this toxic effect of B. thuringiensis thuringiensis 407 (pH 408) and B. thuringiensis kurstaki HD-73 on S. frugiperda. The following method was used. Both bacterial strains were evaluated in vitro in 1st instar S. frugiperda caterpillars, by means of histopathological assays. The Cry1Ab and Cry1Ac proteins, codified by the respective strains of B. thuringiensis, were evaluated in vivo by bioassays of 1st instar S. frugiperda caterpillars in order to determine the Mean Lethal Concentration (LC50). The results of the histopathological analysis of the midget of S. frugiperda caterpillars demonstrate that treatment with the B. thuringiensis thuringiensis strain was more efficient, because the degradations of the microvilosities started 9 hours after treatment application (HAT), while in the B. thuringiensis kurstaki the same effect was noticed only after 12 HAT. Toxicity data of the Cry1Ab and Cry1Ac proteins presented for the target-species LC50 levels of 9.29 and 1.79 microgxcm-2 respectively. The strains and proteins synthesised by B. thuringiensis thuringiensis and B. thuringiensis kurstaki are effective in controlling S. frugiperda, and may be used to produce new biopesticides or the genes may be utilised in the genetic transformation of Oryza sativa L.

  14. Interaction between the predator Podisus nigrispinus (Hemiptera: Pentatomidae) and the entomopathogenic bacteria Bacillus thuringiensis.

    PubMed

    Carvalho, V F P; Vacari, A M; Pomari, A F; De Bortoli, C P; Ramalho, D G; De Bortoli, S A

    2012-12-01

    Plutella xylostella (L.) is susceptible to both the entomopathogen Bacillus thuringiensis and the predator, Brazilian spined soldier bug [Podisus nigrispinus (Dallas)]. The objective of this study was to measure the interaction between the bacterium B. thuringiensis and the predator P. nigrispinus. We also studied the behavior of P. nigrispinus in relation to its choice between B. thuringiensis-infected and healthy P. xylostellais larvae. In the first treatment, P. nigrispinus nymphs were fed daily with B. thuringiensis-infected P. xylostella larvae and distilled water. In the second treatment, nymphs were fed daily with healthy larvae and a suspension of B. thuringiensis as a source of water. The control nymphs were fed daily with healthy larvae and water. Adult P. nigrispinus were separated by sex, couples were formed, and they were fed daily with P. xylostella larvae derived from the treatments. We followed the development of P. nigrispinus and measured its biological characteristics. On the basis of these data, parameters were determined for the construction of life tables. A choice test was used to compare infected and healthy larvae. The HD1 strain of B. thuringiensis does not affect the biological characteristics of P. nigrispinus when fed infected larvae and water or healthy larvae and B. thuringiensis suspension. Our study shows that integrated management of P. xylostella, a pest of the Brassicaceae, is feasible by using the HD1 strain of B. thuringiensis and the predator P. nigrispinus, because the predator shows no preference for infected or healthy P. xylostella larvae.

  15. Genetic mapping of resistance to Bacillus thuringiensis toxins in diamondback moth using biphasic linkage analysis.

    PubMed

    Heckel, D G; Gahan, L J; Liu, Y B; Tabashnik, B E

    1999-07-20

    Transgenic plants producing environmentally benign Bacillus thuringiensis (Bt) toxins are deployed increasingly for insect control, but their efficacy will be short-lived if pests adapt quickly. The diamondback moth (Plutella xylostella), a worldwide pest of vegetables, is the first insect to evolve resistance to Bt toxins in open-field populations. A recessive autosomal gene confers resistance to at least four Bt toxins and enables survival without adverse effects on transgenic plants. Allelic variants of this gene confer resistance in strains from Hawaii, Pennsylvania, and the Philippines. Here we exploited the biphasic nature of Lepidopteran genetic linkage to map this gene in diamondback moth with 207 amplified fragment length polymorphisms as DNA markers. We also cloned and sequenced an amplified fragment length polymorphism marker for the chromosome containing the Bt resistance gene. The results provide a powerful tool for facilitating progress in understanding, monitoring, and managing resistance to Bt.

  16. Effects of Two Varieties of Bacillus thuringiensis Maize on the Biology of Plodia interpunctella

    PubMed Central

    Gryspeirt, Aiko; Grégoire, Jean-Claude

    2012-01-01

    On the market since 1996, genetically modified plants expressing an insecticidal toxin (Cry toxin stemmed from Bacillus thuringiensis) target several lepidopteran and coleopteran pests. In this study, we assessed the impact of two varieties of Bt maize producing different toxins (Cry1Ab or Cry1Fa, respectively) on the biology of a storage pest: Plodia interpunctella (Hübner) (Lepidoptera: Pyralidae). The Indianmeal moths were susceptible to both toxins but showed an escape behavior only from Cry1Fa. The weight of females issued from larvae reared on Cry1Ab increased with increasing toxin concentration, but adults of both sexes reared on Cry1Fa had decreased weight. Both toxins increased development time from egg to adult regardless of sex and had no impact on the male adult lifespan. Finally, we recorded a time lag between metamorphosis from the non-Bt and the Bt diets, which increased proportionally to Cry concentration in the Bt diet. PMID:22778907

  17. Diversity of Bacillus thuringiensis in the rice field soils of different ecologies in India.

    PubMed

    Das, J; Dangar, T K

    2007-12-01

    Diversity of the Bacillus thuringiensis (Bt) in the rice field soils of different ecologies viz. the island (Port Blair), the Himalayan (Srinagar), brackish water (Mahe) and coastal mesophilic (Mangalore) habitats was analyzed by phenotypic characterization of 5, 66, 14 and 54 Bt isolates, respectively. The Bt isolates produced either monotypic (bipyramidal or spherical) or heterotypic (polymorphic-bipyramidal or bipyramidal-rhomboidal) crystals. The organisms were generally resistant to the penicillin group of antibiotics, tolerated 5-12% NaCl and 0.5M Na-acetate. The Bt isolates contained 1-5 plasmids of 0.89-58.61 kbp sizes. The plasmid profiles had no correlation with crystal morphology or salt tolerance of different bacteria. Each soil was inhabited by different types of Bt. Two Bt strains of Mangalore and one strain each of the other places were phenotypically similar. One Bt strain each of Port Blair and Srinagar was different from all other strains. PMID:23100691

  18. Effects of two varieties of Bacillus thuringiensis maize on the biology of Plodia interpunctella.

    PubMed

    Gryspeirt, Aiko; Grégoire, Jean-Claude

    2012-05-01

    On the market since 1996, genetically modified plants expressing an insecticidal toxin (Cry toxin stemmed from Bacillus thuringiensis) target several lepidopteran and coleopteran pests. In this study, we assessed the impact of two varieties of Bt maize producing different toxins (Cry1Ab or Cry1Fa, respectively) on the biology of a storage pest: Plodia interpunctella (Hübner) (Lepidoptera: Pyralidae). The Indianmeal moths were susceptible to both toxins but showed an escape behavior only from Cry1Fa. The weight of females issued from larvae reared on Cry1Ab increased with increasing toxin concentration, but adults of both sexes reared on Cry1Fa had decreased weight. Both toxins increased development time from egg to adult regardless of sex and had no impact on the male adult lifespan. Finally, we recorded a time lag between metamorphosis from the non-Bt and the Bt diets, which increased proportionally to Cry concentration in the Bt diet.

  19. A structured model for vegetative growth and sporulation in Bacillus thuringiensis

    SciTech Connect

    Starzak, M.; Bajpai, R.K.

    1991-12-31

    A mathematical model has been developed for the 6-endotoxin producing Bacillus thuringiensis. The structure of the model involves the processes taking place during vegetative growth, those leading to the initiation of sporulation under conditions of carbon and/or nitrogen limitation, and the sporulation events. The key features in the model are the pools of compounds, such as PRPP, IMP, ADP/ATP, GDP/GTP, pyrimidine nucleotides, NAD/NADH{sub 2}, amino acids, nucleic acids, cell wall, and vegetative and sporulation proteins. These, along with a-factors that control the nature of RNA-polymerase during the different phases, effectively stimulate the vegetative growth and sporulation. The initiation of sporulation is controlled by the intracellular concentration of GTP. Results of simulation of vegetative growth, initiation of sporulation, spore protein formation, and production of {delta}-endotoxin under C- or N-limitation are presented.

  20. Characterization of a dynamic S layer on Bacillus thuringiensis.

    PubMed Central

    Luckevich, M D; Beveridge, T J

    1989-01-01

    The surfaces of three Bacillus thuringiensis strains possess an S layer composed of linear arrays of small particles arranged with p2 symmetry and with a = 8.5 nm, b = 7.2 nm, and gamma = 73 degrees. Platinum shadows of whole cells and S-layer fragments revealed the outer surface of the array to be smooth and the inner surface to be corrugated. Treatment with 2 M guanidine hydrochloride at pH 2.5 to 4 best removed the S layer for chemical characterization; it was a relatively hydrophilic 91.4-kilodalton protein with a pI of 5, no detectable carbohydrate, cysteine, methionine or tryptophan, and 21.2% nonpolar residues. No N-terminal homology with other S-layer proteins was evident. Antibody labeling experiments confirmed that the amount of S layer was proportional to the growth phase in broth cultures. Late-exponential- and stationary-growth-phase cells typically sloughed off fragments of S layer, and this may be the result of wall turnover. Indigenous autolytic activity in isolated walls rapidly digested the wall fabric, liberating soluble S-layer protein. At the same time, proteases frequently reduced the molecular weight of the 91.4-kilodalton protein, but these polypeptides could still be identified as S-layer components by immunoblotting. As cultures were serially subcultured, the frequency of appearance of the S layer diminished, and it was eventually lost. The dynamic nature of this S layer makes it atypical of most previously identified S layers and made it unusually difficult to characterize. Images PMID:2592346

  1. Postflight analyses of Bacillus thuringiensis organisms exposed to space flight conditions

    NASA Technical Reports Server (NTRS)

    Wrenn, R. T.; Simmonds, R. C.; Heimpel, A. M.

    1973-01-01

    Cultures of B. thuringiensis returned from space flight appeared to be normal to slightly affected adversely in their ability to produce three toxins that affect insects. In addition, it can be stated that B. thuringiensis spores are very resistant to ultraviolet irradiation at the individual wavelengths and energy levels previously described. Full sunlight, however, does have a detrimental effect on the viability of B. thuringiensis spores.

  2. Lack of Cross-Resistance to Cry19A from Bacillus thuringiensis subsp. jegathesan in Culex quinquefasciatus (Diptera: Culicidae) Resistant to Cry Toxins from Bacillus thuringiensis subsp. israelensis

    PubMed Central

    Wirth, Margaret C.; Delécluse, Armelle; Walton, William E.

    2001-01-01

    Culex quinquefasciatus mosquitoes with high levels of resistance to single or multiple toxins from Bacillus thuringiensis subsp. israelensis were tested for cross-resistance to the Bacillus thuringiensis subsp. jegathesan polypeptide Cry19A. No cross-resistance was detected in mosquitoes that had been selected with the Cry11A, Cry4A and Cry4B, or Cry4A, Cry4B, Cry11A, and CytA toxins. A low but statistically significant level of cross-resistance, three to fourfold, was detected in the colony selected with Cry4A, Cry4B, and Cry11A. This cross-resistance was similar to that previously detected with B. thuringiensis subsp. jegathesan in the same colony. These data help explain the toxicity of B. thuringiensis subsp. jegathesan against the resistant colonies and indicate that the Cry19A polypeptide might be useful in managing resistance and/or as a component of synthetic combinations of mosquitocidal toxins. PMID:11282656

  3. Combinatorial effect of Bacillus thuringiensis kurstaki and Photorhabdus luminescens against Spodoptera littoralis (Lepidoptera: Noctuidae).

    PubMed

    Benfarhat-Touzri, Dalel; Ben Amira, Amal; Ben khedher, Saoussen; Givaudan, Alain; Jaoua, Samir; Tounsi, Slim

    2014-11-01

    Spodoptera littoralis, one of the major pests of many important crop plants, is more susceptible to Bacillus thuringiensis aizawai delta-endotoxins than to those of Bacillus thuringiensis kurstaki. Within the framework of the development of efficient bioinsecticides and the prevention against insect resistance, we tested the effect of mixing B. thuringiensis kurstaki delta-endotoxins and Photorhabdus luminescens cells on S. littoralis growth. The obtained results showed that the growth inhibition of this insect was more effective when B. thuringiensis kurstaki spore-crystal mixture and Photorhabdus luminescens cells were used in combination. Furthermore, this synergism is mainly due to the presence of Cry1Ac, which is one of the three delta-endotoxins that form the crystal of B. thuringiensis kurstaki strain BNS3 in addition to Cry1Aa and Cry2Aa. This work shows a possibility to use B. thuringiensis as a delivery means for Photorhabdus bacteria in order to infect the insect hemocoel and to reduce the risk of developing resistance in the target organism.

  4. Combinatorial effect of Bacillus thuringiensis kurstaki and Photorhabdus luminescens against Spodoptera littoralis (Lepidoptera: Noctuidae).

    PubMed

    Benfarhat-Touzri, Dalel; Ben Amira, Amal; Ben khedher, Saoussen; Givaudan, Alain; Jaoua, Samir; Tounsi, Slim

    2014-11-01

    Spodoptera littoralis, one of the major pests of many important crop plants, is more susceptible to Bacillus thuringiensis aizawai delta-endotoxins than to those of Bacillus thuringiensis kurstaki. Within the framework of the development of efficient bioinsecticides and the prevention against insect resistance, we tested the effect of mixing B. thuringiensis kurstaki delta-endotoxins and Photorhabdus luminescens cells on S. littoralis growth. The obtained results showed that the growth inhibition of this insect was more effective when B. thuringiensis kurstaki spore-crystal mixture and Photorhabdus luminescens cells were used in combination. Furthermore, this synergism is mainly due to the presence of Cry1Ac, which is one of the three delta-endotoxins that form the crystal of B. thuringiensis kurstaki strain BNS3 in addition to Cry1Aa and Cry2Aa. This work shows a possibility to use B. thuringiensis as a delivery means for Photorhabdus bacteria in order to infect the insect hemocoel and to reduce the risk of developing resistance in the target organism. PMID:23908000

  5. Characterization of Bacillus thuringiensis l-Isoleucine Dioxygenase for Production of Useful Amino Acids▿†

    PubMed Central

    Hibi, Makoto; Kawashima, Takashi; Kodera, Tomohiro; Smirnov, Sergey V.; Sokolov, Pavel M.; Sugiyama, Masakazu; Shimizu, Sakayu; Yokozeki, Kenzo; Ogawa, Jun

    2011-01-01

    We determined the enzymatic characteristics of an industrially important biocatalyst, α-ketoglutarate-dependent l-isoleucine dioxygenase (IDO), which was found to be the enzyme responsible for the generation of (2S,3R,4S)-4-hydroxyisoleucine in Bacillus thuringiensis 2e2. Depending on the amino acid used as the substrate, IDO catalyzed three different types of oxidation reactions: hydroxylation, dehydrogenation, and sulfoxidation. IDO stereoselectively hydroxylated several hydrophobic aliphatic l-amino acids, as well as l-isoleucine, and produced (S)-3-hydroxy-l-allo-isoleucine, 4-hydroxy-l-leucine, (S)-4-hydroxy-l-norvaline, 4-hydroxy-l-norleucine, and 5-hydroxy-l-norleucine. The IDO reaction product of l-isoleucine, (2S,3R,4S)-4-hydroxyisoleucine, was again reacted with IDO and dehydrogenated into (2S,3R)-2-amino-3-methyl-4-ketopentanoate, which is also a metabolite found in B. thuringiensis 2e2. Interestingly, IDO catalyzed the sulfoxidation of some sulfur-containing l-amino acids and generated l-methionine sulfoxide and l-ethionine sulfoxide. Consequently, the effective production of various modified amino acids would be possible using IDO as the biocatalyst. PMID:21821743

  6. Characterization of Bacillus thuringiensis L-isoleucine dioxygenase for production of useful amino acids.

    PubMed

    Hibi, Makoto; Kawashima, Takashi; Kodera, Tomohiro; Smirnov, Sergey V; Sokolov, Pavel M; Sugiyama, Masakazu; Shimizu, Sakayu; Yokozeki, Kenzo; Ogawa, Jun

    2011-10-01

    We determined the enzymatic characteristics of an industrially important biocatalyst, α-ketoglutarate-dependent l-isoleucine dioxygenase (IDO), which was found to be the enzyme responsible for the generation of (2S,3R,4S)-4-hydroxyisoleucine in Bacillus thuringiensis 2e2. Depending on the amino acid used as the substrate, IDO catalyzed three different types of oxidation reactions: hydroxylation, dehydrogenation, and sulfoxidation. IDO stereoselectively hydroxylated several hydrophobic aliphatic l-amino acids, as well as l-isoleucine, and produced (S)-3-hydroxy-l-allo-isoleucine, 4-hydroxy-l-leucine, (S)-4-hydroxy-l-norvaline, 4-hydroxy-l-norleucine, and 5-hydroxy-l-norleucine. The IDO reaction product of l-isoleucine, (2S,3R,4S)-4-hydroxyisoleucine, was again reacted with IDO and dehydrogenated into (2S,3R)-2-amino-3-methyl-4-ketopentanoate, which is also a metabolite found in B. thuringiensis 2e2. Interestingly, IDO catalyzed the sulfoxidation of some sulfur-containing l-amino acids and generated l-methionine sulfoxide and l-ethionine sulfoxide. Consequently, the effective production of various modified amino acids would be possible using IDO as the biocatalyst. PMID:21821743

  7. An overview of the safety and biological effects of Bacillus thuringiensis Cry toxins in mammals.

    PubMed

    Rubio-Infante, Néstor; Moreno-Fierros, Leticia

    2016-05-01

    Crystal proteins (Cry) produced during the growth and sporulation phases of Bacillus thuringiensis (Bt) bacterium are known as delta endotoxins. These toxins are being used worldwide as bioinsecticides to control pests in agriculture, and some Cry toxins are used against mosquitoes to control vector transmission. This review summarizes the relevant information currently available regarding the biosafety and biological effects that Bt and its insecticidal Cry proteins elicit in mammals. This work was performed because of concerns regarding the possible health impact of Cry toxins on vertebrates, particularly because Bt toxins might be associated with immune-activating or allergic responses. The controversial data published to date are discussed in this review considering earlier toxicological studies of B. thuringiensis, spores, toxins and Bt crops. We discussed the experimental studies performed in humans, mice, rats and sheep as well as in diverse mammalian cell lines. Although the term 'toxic' is not appropriate for defining the effects these toxins have on mammals, they cannot be considered innocuous, as they have some physiological effects that may become pathological; thus, trials that are more comprehensive are necessary to determine their effects on mammals because knowledge in this field remains limited. PMID:26537666

  8. An overview of the safety and biological effects of Bacillus thuringiensis Cry toxins in mammals.

    PubMed

    Rubio-Infante, Néstor; Moreno-Fierros, Leticia

    2016-05-01

    Crystal proteins (Cry) produced during the growth and sporulation phases of Bacillus thuringiensis (Bt) bacterium are known as delta endotoxins. These toxins are being used worldwide as bioinsecticides to control pests in agriculture, and some Cry toxins are used against mosquitoes to control vector transmission. This review summarizes the relevant information currently available regarding the biosafety and biological effects that Bt and its insecticidal Cry proteins elicit in mammals. This work was performed because of concerns regarding the possible health impact of Cry toxins on vertebrates, particularly because Bt toxins might be associated with immune-activating or allergic responses. The controversial data published to date are discussed in this review considering earlier toxicological studies of B. thuringiensis, spores, toxins and Bt crops. We discussed the experimental studies performed in humans, mice, rats and sheep as well as in diverse mammalian cell lines. Although the term 'toxic' is not appropriate for defining the effects these toxins have on mammals, they cannot be considered innocuous, as they have some physiological effects that may become pathological; thus, trials that are more comprehensive are necessary to determine their effects on mammals because knowledge in this field remains limited.

  9. Anthelmintic Effect of Bacillus thuringiensis Strains against the Gill Fish Trematode Centrocestus formosanus

    PubMed Central

    Mendoza-Estrada, Luis Javier; Hernández-Velázquez, Víctor Manuel; Arenas-Sosa, Iván; Flores-Pérez, Fernando Iván; Morales-Montor, Jorge; Peña-Chora, Guadalupe

    2016-01-01

    Parasitic agents, such as helminths, are the most important biotic factors affecting aquaculture, and the fluke Centrocestus formosanus is considered to be highly pathogenic in various fish species. There have been efforts to control this parasite with chemical helminthicides, but these efforts have had unsuccessful results. We evaluated the anthelmintic effect of 37 strains of Bacillus thuringiensis against C. formosanus metacercariae in vitro using two concentrations of total protein, and only six strains produced high mortality. The virulence (CL50) on matacercariae of three strains was obtained: the GP308, GP526, and ME1 strains exhibited a LC50 of 146.2 μg/mL, 289.2 μg/mL, and 1721.9 μg/mL, respectively. Additionally, these six B. thuringiensis strains were evaluated against the cercariae of C. formosanus; the LC50 obtained from the GP526 strain with solubilized protein was 83.8 μg/mL, and it could be considered as an alternative control of the metacercariae and cercariae of this parasite in the productivity systems of ornamental fishes. PMID:27294137

  10. A novel Tenebrio molitor cadherin is a functional receptor for Bacillus thuringiensis Cry3Aa toxin.

    PubMed

    Fabrick, Jeff; Oppert, Cris; Lorenzen, Marcé D; Morris, Kaley; Oppert, Brenda; Jurat-Fuentes, Juan Luis

    2009-07-01

    Cry toxins produced by the bacterium Bacillus thuringiensis are effective biological insecticides. Cadherin-like proteins have been reported as functional Cry1A toxin receptors in Lepidoptera. Here we present data that demonstrate that a coleopteran cadherin is a functional Cry3Aa toxin receptor. The Cry3Aa receptor cadherin was cloned from Tenebrio molitor larval midgut mRNA, and the predicted protein, TmCad1, has domain structure and a putative toxin binding region similar to those in lepidopteran cadherin B. thuringiensis receptors. A peptide containing the putative toxin binding region from TmCad1 bound specifically to Cry3Aa and promoted the formation of Cry3Aa toxin oligomers, proposed to be mediators of toxicity in lepidopterans. Injection of TmCad1-specific double-stranded RNA into T. molitor larvae resulted in knockdown of the TmCad1 transcript and conferred resistance to Cry3Aa toxicity. These data demonstrate the functional role of TmCad1 as a Cry3Aa receptor in T. molitor and reveal similarities between the mode of action of Cry toxins in Lepidoptera and Coleoptera. PMID:19416969

  11. A novel Tenebrio molitor cadherin is a functional receptor for Bacillus thuringiensis Cry3Aa toxin.

    PubMed

    Fabrick, Jeff; Oppert, Cris; Lorenzen, Marcé D; Morris, Kaley; Oppert, Brenda; Jurat-Fuentes, Juan Luis

    2009-07-01

    Cry toxins produced by the bacterium Bacillus thuringiensis are effective biological insecticides. Cadherin-like proteins have been reported as functional Cry1A toxin receptors in Lepidoptera. Here we present data that demonstrate that a coleopteran cadherin is a functional Cry3Aa toxin receptor. The Cry3Aa receptor cadherin was cloned from Tenebrio molitor larval midgut mRNA, and the predicted protein, TmCad1, has domain structure and a putative toxin binding region similar to those in lepidopteran cadherin B. thuringiensis receptors. A peptide containing the putative toxin binding region from TmCad1 bound specifically to Cry3Aa and promoted the formation of Cry3Aa toxin oligomers, proposed to be mediators of toxicity in lepidopterans. Injection of TmCad1-specific double-stranded RNA into T. molitor larvae resulted in knockdown of the TmCad1 transcript and conferred resistance to Cry3Aa toxicity. These data demonstrate the functional role of TmCad1 as a Cry3Aa receptor in T. molitor and reveal similarities between the mode of action of Cry toxins in Lepidoptera and Coleoptera.

  12. Anthelmintic Effect of Bacillus thuringiensis Strains against the Gill Fish Trematode Centrocestus formosanus.

    PubMed

    Mendoza-Estrada, Luis Javier; Hernández-Velázquez, Víctor Manuel; Arenas-Sosa, Iván; Flores-Pérez, Fernando Iván; Morales-Montor, Jorge; Peña-Chora, Guadalupe

    2016-01-01

    Parasitic agents, such as helminths, are the most important biotic factors affecting aquaculture, and the fluke Centrocestus formosanus is considered to be highly pathogenic in various fish species. There have been efforts to control this parasite with chemical helminthicides, but these efforts have had unsuccessful results. We evaluated the anthelmintic effect of 37 strains of Bacillus thuringiensis against C. formosanus metacercariae in vitro using two concentrations of total protein, and only six strains produced high mortality. The virulence (CL50) on matacercariae of three strains was obtained: the GP308, GP526, and ME1 strains exhibited a LC50 of 146.2 μg/mL, 289.2 μg/mL, and 1721.9 μg/mL, respectively. Additionally, these six B. thuringiensis strains were evaluated against the cercariae of C. formosanus; the LC50 obtained from the GP526 strain with solubilized protein was 83.8 μg/mL, and it could be considered as an alternative control of the metacercariae and cercariae of this parasite in the productivity systems of ornamental fishes. PMID:27294137

  13. Translocation and insecticidal activity of Bacillus thuringiensis living inside of plants

    PubMed Central

    Monnerat, Rose Gomes; Soares, Carlos Marcelo; Capdeville, Guy; Jones, Gareth; Martins, Érica Soares; Praça, Lilian; Cordeiro, Bruno Arrivabene; Braz, Shélida Vasconcelos; Dos Santos, Roseane Cavalcante; Berry, Colin

    2009-01-01

    Summary The major biological pesticide for the control of insect infestations of crops, Bacillus thuringiensis was found to be present naturally within cotton plants from fields that had never been treated with commercial formulations of this bacterium. The ability of B. thuringiensis to colonize plants as an endophyte was further established by the introduction of a strain marked by production of green fluorescent protein (GFP). After inoculation of this preparation close to the roots of cotton and cabbage seedlings, GFP‐marked bacteria could be re‐isolated from all parts of the plant, having entered the roots and migrated through the xylem. Leaves taken from the treated plants were able to cause toxicity when fed to the Lepidoptera Spodoptera frugiperda (cotton) and Plutella xylostella (cabbage). These results open up new horizons for understanding the natural ecology and evolution of B. thuringiensis and use of B. thuringiensis in insect control. PMID:21255282

  14. Analysis of opportunities and challenges in patenting of Bacillus thuringiensis insecticidal crystal protein genes.

    PubMed

    Swamy, H M Mahadeva; Asokan, R; Rajasekaran, P E; Mahmood, Riaz; Nagesha, S N; Arora, D K

    2012-04-01

    Bacillus thuringiensis (Bt) is the most widely used microbial control agent. The broad spectrum of susceptible hosts, production on artificial media and ease of application has caused the widespread use of this bacterium against several pests in agriculture, forest and vectors of human diseases. B.thuringiensis toxins are highly species specific which provide economic, environmental benefits, potential for future control and spread of the technology worldwide. This makes the B. thuringiensis crystal proteins an interesting tool for the implementation in integrated pest management programs. It has gained importance over the last 100 years for its biocontrol properties which is used in this review as a case study and analysis of the patents granted on B. thuringiensis was carried out. This study categorizes a number of patents related to B.thuringiensis insecticidal crystal proteins, application of B.thuringiensis insecticidal crystal proteins and the development of patentable technologies. The analyses were done using various criteria like patenting trends over the years, assignees playing a major role, comparison of the technology used in different patents and the patenting activity across the insect orders. Patent documents related to bacterium B.thuringiensis contain a trove of technical and commercial information and thus, patent analysis is considered as a useful tool for R management and techno economical development. Patent analysis also helps identifying and evaluating new and alternate technologies, keeping abreast with latest technologies for business interests, finding solutions to technical problems and ideas for new innovative trends.

  15. Analysis of opportunities and challenges in patenting of Bacillus thuringiensis insecticidal crystal protein genes.

    PubMed

    Swamy, H M Mahadeva; Asokan, R; Rajasekaran, P E; Mahmood, Riaz; Nagesha, S N; Arora, D K

    2012-04-01

    Bacillus thuringiensis (Bt) is the most widely used microbial control agent. The broad spectrum of susceptible hosts, production on artificial media and ease of application has caused the widespread use of this bacterium against several pests in agriculture, forest and vectors of human diseases. B.thuringiensis toxins are highly species specific which provide economic, environmental benefits, potential for future control and spread of the technology worldwide. This makes the B. thuringiensis crystal proteins an interesting tool for the implementation in integrated pest management programs. It has gained importance over the last 100 years for its biocontrol properties which is used in this review as a case study and analysis of the patents granted on B. thuringiensis was carried out. This study categorizes a number of patents related to B.thuringiensis insecticidal crystal proteins, application of B.thuringiensis insecticidal crystal proteins and the development of patentable technologies. The analyses were done using various criteria like patenting trends over the years, assignees playing a major role, comparison of the technology used in different patents and the patenting activity across the insect orders. Patent documents related to bacterium B.thuringiensis contain a trove of technical and commercial information and thus, patent analysis is considered as a useful tool for R management and techno economical development. Patent analysis also helps identifying and evaluating new and alternate technologies, keeping abreast with latest technologies for business interests, finding solutions to technical problems and ideas for new innovative trends. PMID:22239684

  16. Molecular markers to determine ecological fate of Bacillus thuringiensis subsp. kurstaki

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bacillus thuringiensis (“Bt”) is a ubiquitous soil bacterium with entomopathogenic properties. One strain, Bt subsp. kurstaki (“Btk”), is highly toxic to lepidopteran larvae and used in many commercial products for biological pest control. We designed a set of DNA markers that successfully identifi...

  17. Screening Bacillus thuringiensis strains for toxicity against Manduca sexta and Plutella xylostella

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Screening Bacillus thuringiensis (Bt) isolates or strains for toxicity has traditionally been performed with one bacterial isolate at time versus a specific insect. By testing of Bt strains in groups, we identified 28 of 147 Bt isolates as toxic to either diamondback moth, Plutella xylostella (L.),...

  18. Resistance to Bacillus thuringiensis endotoxins in the European corn borer (Ostrinia nubilalis)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The European corn borer, Ostrinia nubilalis (Hübner), is the primary target of the widely adopted transgenic corn events MON810 and Bt11, expressing the Bacillus thuringiensis (Bt) insecticidal toxin, Cry1Ab. Resistant strains of O. nubilalis have been selected in the laboratory by exposure to Bt ...

  19. IMPACT OF BT ( BACILLUS THURINGIENSIS ) CROPS ON BAT ACTIVITY IN SOUTH TEXAS AGROECOSYSTEMS

    EPA Science Inventory

    The widespread adoption of transgenic insecticidal crops raises concerns that nontarget species may be harmed and food webs disrupted. The goal of this research is to determine how transgenic Bt (Bacillus thuringiensis) crops impact the activity of Brazilian freetailed bats (Tada...

  20. Bacillus thuringiensis Cry3Aa toxin increases the susceptibility of Crioceris quatuordecimpunctata to Beauveria bassiana infection

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The spotted asparagus beetle, Crioceris quatuordecimpunctata (Coleoptera: Chrysomelidae), is one of the most devastating pests of asparagus in China and elsewhere. In this study, we investigated the interaction of Bacillus thuringiensis (Bt) Cry3Aa toxin and the entomopathogenic fungus Beauveria bas...

  1. Toxicity of "Bacillus thuringiensis var. Kurstaki" to the Painted Lady Butterfly, Vanessa cardui.

    ERIC Educational Resources Information Center

    Stalter, Richard; Nadal, Gerard; Kincaid, Dwight

    2000-01-01

    Reports the effects of Bacillus thuringiensis var. Kurstaki (BT), which is highly toxic, to a non-target lepidopteran, the Painted Lady butterfly. Indicates that BT kills some Painted Lady butterfly larvae at the lowest dilution tested after 48 hours. (ASK)

  2. Transcriptome of the gypsy moth (Lymantria dispar) larval midgut in response to infection by Bacillus thuringiensis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Transcriptomic profiles of the lepidopteran insect pest Lymantria dispar (gypsy moth) were characterized in the larval midgut in response to infection by the biopesticide Bacillus thuringiensis kurstaki. RNA-Seq approaches were used to define a set of 49,613 assembled transcript sequences, of which...

  3. Determination and distribution of cry-type genes in halophilc Bacillus thuringiensis isolates of Arabian Sea sedimentary rocks.

    PubMed

    Baig, Deeba Noreen; Mehnaz, Samina

    2010-07-20

    Bacillus thuringiensis produces insecticidal crystal during its sporulation phase. In this study, marine sediments from Arabian Sea along coastal area of Pakistan were examined for the occurrence of B. thuringiensis. On the basis of morphological and biochemical properties, 31 out of 200 colonies were assigned to B. thuringiensis. Isolated strains were characterized on the basis of cry genes profile. PCR approach was used to analyze the presence of different crystal toxin encoding genes with six pairs of universal primers that could detect the cry1, cry4, cry7, cry8, cry9, and cry10 genes. Strains containing cry1 genes were the most abundant in our collection (49.5%). Seventeen different profiles of cry genes were identified, i.e., twelve harboring two cry genes while five profiles of more than two cry genes. The characterization of these strains provided useful information on the ecological patterns of distribution of B. thuringiensis and opportunities for the selection of new strains to develop novel bio-insecticidal products.

  4. Evolution of Resistance in Culex quinquefasciatus (Say) Selected With a Recombinant Bacillus thuringiensis Strain-Producing Cyt1Aa and Cry11Ba, and the Binary Toxin, Bin, From Lysinibacillus sphaericus

    PubMed Central

    Wirth, Margaret C.; Walton, William E.; Federici, Brian A.

    2015-01-01

    Fourth instars of Culex quinquefasciatus (Say) (Diptera: Culicidae) were selected with a recombinant bacterial strain synthesizing the mosquitocidal proteins from Lysinibacillus sphaericus (Bin) and Cry11Ba and Cyt1Aa from Bacillus thuringiensis. Selection was initiated in Generation 1 with a concentration of 0.04 μg/ml, which rose to a maximum selection concentration of 8.0 μg/ml in Generation 14, followed by an unexpected, rapid increase in mortality in Generation 15. Subsequently, a selection concentration of 0.8 μg/ml was determined to be survivable. During this same period, resistance rose to nearly 1,000-fold (by Generation 12) and declined to 18.8-fold in Generation 19. Resistance remained low and fluctuated between 5.3 and 7.3 up to Generation 66. The cross-resistance patterns and interactions among the component proteins were analyzed to identify possible causes of this unusual pattern of evolution. Poor activity in the mid-range concentrations and lower-than-expected synergistic interactions were identified as potential sources of the early resistance. These findings should be considered in the development of genetically engineered strains intended to control nuisance and vector mosquitoes. PMID:26336254

  5. Bacillus thuringiensis serovar mogi (flagellar serotype 3a3b3d), a novel serogroup with a mosquitocidal activity.

    PubMed

    Roh, Jong Yul; Liu, Qin; Lee, Dae Weon; Tao, Xueying; Wang, Yong; Shim, Hee Jin; Choi, Jae Young; Seo, Jong Bok; Ohba, Michio; Mizuki, Eiichi; Je, Yeon Ho

    2009-11-01

    The flagellated vegetative cells of the Bacillus thuringiensis strain K4 were agglutinated with the H3 reference antiserum and further, agglutinated with 3b and 3d monospecific factor sera but non-reactive for 3c and 3e factor sera. This creates a new serogroup with flagellar antigenic structure of 3a3b3d: B. thuringiensis serovar mogi. The strain K4 showed high activity against dipteran larvae, Anopheles sinensis and Culex pipienspallens while no lepidopteran toxicity. It produced ovoidal parasporal inclusions (crystals) whose SDS-PAGE protein profile consisted of several bands ranging from 75 to 30kDa. Through the protein identification by nano-LC-ESI-IT MS analysis, the putative peptides of Cry19Ba, Cry40ORF2, Cry27Aa and Cry20Aa were detected.

  6. New Bacillus thuringiensis toxin combinations for biological control of lepidopteran larvae.

    PubMed

    Elleuch, Jihen; Zghal, Raida Zribi; Jemaà, Mohamed; Azzouz, Hichem; Tounsi, Slim; Jaoua, Samir

    2014-04-01

    Cyt1Aa from Bacillus thuringiensis israelensis is known by its synergistical activity with B. thuringiensis and Bacillus sphaericus toxins. It is able to improve dipteran specific toxins activity and can prevent or overcome larval resistance to those proteins. The objective of the current study was to investigate the possible improvement of larvicidal activity of B. thuringiensis kurstaki expressing heterogeneous proteins Cyt1A and P20. cyt1A98 and p20 genes encoding the cytolytic protein (Cyt1A98) and the accessory protein (P20), respectively, were introduced individually and in combination into B. thuringiensis kurstaki strain BNS3. Immunoblot analysis evidenced the expression of these genes in the recombinant strains and hinted that P20 acts as molecular chaperone protecting Cyt1A98 from proteolytic attack in BNS3. The toxicities of recombinant strains were studied and revealed that BNS3pHTp20 exhibited higher activity than that of the negative control (BNS3pHTBlue) toward Ephestia kuehniella, but not toward Spodoptera littoralis. When expressed in combination with P20, Cyt1A98 enhanced BNS3 activity against E. kuehniella and S. littoralis. Thus, Cyt1Aa protein could enhance lepidopteran Cry insecticidal activity and would prevent larval resistance to the most commercialized B. thuringiensis kurstaki toxins.

  7. Genomic and transcriptomic insights into the efficient entomopathogenicity of Bacillus thuringiensis.

    PubMed

    Zhu, Lei; Peng, Donghai; Wang, Yueying; Ye, Weixing; Zheng, Jinshui; Zhao, Changming; Han, Dongmei; Geng, Ce; Ruan, Lifang; He, Jin; Yu, Ziniu; Sun, Ming

    2015-09-28

    Bacillus thuringiensis has been globally used as a microbial pesticide for over 70 years. However, information regarding its various adaptions and virulence factors and their roles in the entomopathogenic process remains limited. In this work, we present the complete genomes of two industrially patented Bacillus thuringiensis strains (HD-1 and YBT-1520). A comparative genomic analysis showed a larger and more complicated genome constitution that included novel insecticidal toxicity-related genes (ITRGs). All of the putative ITRGs were summarized according to the steps of infection. A comparative genomic analysis showed that highly toxic strains contained significantly more ITRGs, thereby providing additional strategies for infection, immune evasion, and cadaver utilization. Furthermore, a comparative transcriptomic analysis suggested that a high expression of these ITRGs was a key factor in efficient entomopathogenicity. We identified an active extra urease synthesis system in the highly toxic strains that may aid B. thuringiensis survival in insects (similar to previous results with well-known pathogens). Taken together, these results explain the efficient entomopathogenicity of B. thuringiensis. It provides novel insights into the strategies used by B. thuringiensis to resist and overcome host immune defenses and helps identify novel toxicity factors.

  8. Genomic and transcriptomic insights into the efficient entomopathogenicity of Bacillus thuringiensis

    PubMed Central

    Zhu, Lei; Peng, Donghai; Wang, Yueying; Ye, Weixing; Zheng, Jinshui; Zhao, Changming; Han, Dongmei; Geng, Ce; Ruan, Lifang; He, Jin; Yu, Ziniu; Sun, Ming

    2015-01-01

    Bacillus thuringiensis has been globally used as a microbial pesticide for over 70 years. However, information regarding its various adaptions and virulence factors and their roles in the entomopathogenic process remains limited. In this work, we present the complete genomes of two industrially patented Bacillus thuringiensis strains (HD-1 and YBT-1520). A comparative genomic analysis showed a larger and more complicated genome constitution that included novel insecticidal toxicity-related genes (ITRGs). All of the putative ITRGs were summarized according to the steps of infection. A comparative genomic analysis showed that highly toxic strains contained significantly more ITRGs, thereby providing additional strategies for infection, immune evasion, and cadaver utilization. Furthermore, a comparative transcriptomic analysis suggested that a high expression of these ITRGs was a key factor in efficient entomopathogenicity. We identified an active extra urease synthesis system in the highly toxic strains that may aid B. thuringiensis survival in insects (similar to previous results with well-known pathogens). Taken together, these results explain the efficient entomopathogenicity of B. thuringiensis. It provides novel insights into the strategies used by B. thuringiensis to resist and overcome host immune defenses and helps identify novel toxicity factors. PMID:26411888

  9. Detection of toxin proteins from Bacillus thuringiensis strain 4.0718 by strategy of 2D-LC-MS/MS.

    PubMed

    Yang, Qi; Tang, Sijia; Rang, Jie; Zuo, Mingxing; Ding, Xuezhi; Sun, Yunjun; Feng, Pinghui; Xia, Liqiu

    2015-04-01

    Bacillus thuringiensis is a kind of insecticidal microorganism which can produce a variety of toxin proteins, it is particularly important to find an effective strategy to identify novel toxin proteins rapidly and comprehensively with the discovery of the wild-type strains. Multi-dimensional high-performance liquid chromatography combined with mass spectrometry has become one of the main methods to detect and identify toxin proteins and proteome of B. thuringiensis. In this study, protein samples from B. thuringiensis strain 4.0718 were analyzed on the basis of two-dimensional liquid chromatography-tandem mass spectrometry (2D-LC-MS/MS), and tryptic peptides of whole cell from the late sporulation phase were eluted at different concentration gradients of ammonium chloride and followed by secondary mass spectrum identification. 831 and 894 proteins were identified from two biological replicates, respectively, while 1,770 and 1,859 peptides were detected correspondingly. Among the identified proteins and peptides, 606 proteins and 1,259 peptides were detected in both replicates, which mean that 1,119 proteins and 2,370 peptides were unique to the proteome of this strain. A total of 15 toxins have been identified successfully, and seven of them were firstly discovered in B. thuringiensis strain 4.0718 that were Crystal protein (A1E259), pesticidal protein (U5KS09), Cry2Af1 (A4GVF0), Cry2Ad (Q9RM89), Cry1 (K4HMB5), Cry1Bc (Q45774), and Cry1Ga (Q45746). The proteomic strategy employed in the present study has provided quick and exhaustive identification of toxins produced by B. thuringiensis.

  10. 40 CFR 174.530 - Bacillus thuringiensis Cry2Ae protein in cotton; temporary exemption from the requirement of a...

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... in cotton; temporary exemption from the requirement of a tolerance. 174.530 Section 174.530... thuringiensis Cry2Ae protein in cotton; temporary exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry2Ae protein in or on the food commodities of cotton, cotton; cotton,...

  11. 40 CFR 174.530 - Bacillus thuringiensis Cry2Ae protein in cotton; temporary exemption from the requirement of a...

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... in cotton; temporary exemption from the requirement of a tolerance. 174.530 Section 174.530... thuringiensis Cry2Ae protein in cotton; temporary exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry2Ae protein in or on the food commodities of cotton, cotton; cotton,...

  12. Analysis of Bacillus thuringiensis Population Dynamics and Its Interaction With Pseudomonas fluorescens in Soil

    PubMed Central

    Rojas-Ruiz, Norma Elena; Sansinenea-Royano, Estibaliz; Cedillo-Ramirez, Maria Lilia; Marsch-Moreno, Rodolfo; Sanchez-Alonso, Patricia; Vazquez-Cruz, Candelario

    2015-01-01

    Background: Bacillus thuringiensis is the most successful biological control agent, however, studies so far have shown that B. thuringiensis is very sensitive to environmental factors such as soil moisture and pH. Ultraviolet light from the sun had been considered as the main limiting factor for its persistence in soil and it has recently been shown that the antagonism exerted by other native soil organisms, such as Pseudomonas fluorescens, is a determining factor in the persistence of this bacterium under in vitro culture conditions. Objectives: The aim of the present investigation was to analyze the population dynamics of B. thuringiensis and its interaction with P. fluorescens using microbiological and molecular methods in soil, under different conditions, and to determinate the effect of nutrients and moisture on its interaction. Materials and Methods: The monitoring was performed by microbiological methods, such as viable count of bacteria, and molecular methods such as Polymerase Chain Reaction (PCR) and hybridization, using the direct extraction of DNA from populations of inoculated soil. Results: The analysis of the interaction between B. thuringiensis and P. fluorescens in soil indicated that the disappearance of B. thuringiensis IPS82 is not dependent on the moisture but the composition of nutrients that may be affecting the secretion of toxic compounds in the environment of P. fluorescens. The results showed that the recovered cells were mostly spores and not vegetative cells in all proved treatments. The molecular methods were effective for monitoring bacterial population inoculated in soil. Conclusions: Bacillus thuringiensis is very sensitive to the interaction of P. fluorescens, however is capable to survive in soil due to its capacity of sporulate. Some of the cells in the form of spores germinated and folded slightly and remained in a constant cycle of sporulation and germination. This confirms that B. thuringiensis IPS82 can germinate, grow and

  13. A novel metalloproteinase virulence factor is involved in Bacillus thuringiensis pathogenesis in nematodes and insects.

    PubMed

    Peng, Donghai; Lin, Jian; Huang, Qiong; Zheng, Wen; Liu, Guoqiang; Zheng, Jinshui; Zhu, Lei; Sun, Ming

    2016-03-01

    The Gram-positive soil bacterium Bacillus thuringiensis has been developed as the leading microbial insecticide for years. The pathogenesis of B. thuringiensis requires common extracellular factors that depend on the PlcR regulon, which regulates a large number of virulence factors; however, the precise role of many of these proteins is not known. In this study, we describe the complete lifecycle of a nematicidal B. thuringiensis strain in the free living nematode Caenorhabditis elegans using in vitro and in vivo molecular techniques to follow host and bacterial effectors during the infection process. We then focus on the metalloproteinase ColB, a collagenase, which was found highly important for destruction of the intestine thereby facilitates the adaptation and colonization of B. thuringiensis in C. elegans. In vivo green fluorescent protein (GFP) reporter-gene studies showed that ColB expression is highly induced and regulated by the global activator PlcR. Finally, we demonstrated that ColB also takes part in B. thuringiensis virulence in an insect model following injection and oral infection. Indeed, addition of purified ColB accelerates the action of Cry toxin proteins in insects, too. These results give novel insights into host adaptation for B. thuringiensis and other B. cereus group bacteria and highlight the role of collagenase metalloproteases to synergize infection process.

  14. A novel metalloproteinase virulence factor is involved in Bacillus thuringiensis pathogenesis in nematodes and insects.

    PubMed

    Peng, Donghai; Lin, Jian; Huang, Qiong; Zheng, Wen; Liu, Guoqiang; Zheng, Jinshui; Zhu, Lei; Sun, Ming

    2016-03-01

    The Gram-positive soil bacterium Bacillus thuringiensis has been developed as the leading microbial insecticide for years. The pathogenesis of B. thuringiensis requires common extracellular factors that depend on the PlcR regulon, which regulates a large number of virulence factors; however, the precise role of many of these proteins is not known. In this study, we describe the complete lifecycle of a nematicidal B. thuringiensis strain in the free living nematode Caenorhabditis elegans using in vitro and in vivo molecular techniques to follow host and bacterial effectors during the infection process. We then focus on the metalloproteinase ColB, a collagenase, which was found highly important for destruction of the intestine thereby facilitates the adaptation and colonization of B. thuringiensis in C. elegans. In vivo green fluorescent protein (GFP) reporter-gene studies showed that ColB expression is highly induced and regulated by the global activator PlcR. Finally, we demonstrated that ColB also takes part in B. thuringiensis virulence in an insect model following injection and oral infection. Indeed, addition of purified ColB accelerates the action of Cry toxin proteins in insects, too. These results give novel insights into host adaptation for B. thuringiensis and other B. cereus group bacteria and highlight the role of collagenase metalloproteases to synergize infection process. PMID:26995589

  15. New insights into the binding and catalytic mechanisms of Bacillus thuringiensis lactonase: insights into B. thuringiensis AiiA mechanism.

    PubMed

    Charendoff, Marc N; Shah, Halie P; Briggs, James M

    2013-01-01

    The lactonase enzyme (AiiA) produced by Bacillus thuringiensis serves to degrade autoinducer-1 (AI-1) signaling molecules in what is an evolved mechanism by which to compete with other bacteria. Bioassays have been previously performed to determine whether the AI-1 aliphatic tail lengths have any effect on AiiA's bioactivity, however, data to date are conflicting. Additionally, specific residue contributions to the catalytic activity of AiiA provide for some interesting questions. For example, it has been proposed that Y194 serves to provide an oxyanion hole to AI-1 which is curious given the fact the substrate spans two Zn(2+) ions. These ions might conceivably provide enough charge to promote both ligand stability and the carbonyl activation necessary to drive a nucleophilic attack. To investigate these questions, multiple molecular dynamics simulations were performed across a family of seven acylated homoserine lactones (AHL) along with their associated intermediate and product states. Distance analyses and interaction energy analyses were performed to investigate current bioassay data. Our simulations are consistent with experimental studies showing that AiiA degrades AHLs in a tail length independent manner. However, the presence of the tail is required for activity. Also, the putative oxyanion hole function of Y194 toward the substrate is not observed in any of the reactant or product state simulation trajectories, but does seem to show efficacy in stabilizing the intermediate state. Last, we argue through ionization state analyses, that the proton shuttling necessary for catalytic activity might be mediated by both water and substrate-based intra-molecular proton transfer. Based on this argument, an alternate catalytic mechanism is proposed.

  16. 40 CFR 174.518 - Bacillus thuringiensis Cry3Bb1 protein in corn; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 24 2011-07-01 2011-07-01 false Bacillus thuringiensis Cry3Bb1 protein... PLANT-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.518 Bacillus thuringiensis Cry3Bb1 protein in corn; exemption from the requirement of a tolerance. Residues of Bacillus...

  17. 40 CFR 174.518 - Bacillus thuringiensis Cry3Bb1 protein in corn; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Bacillus thuringiensis Cry3Bb1 protein... PLANT-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.518 Bacillus thuringiensis Cry3Bb1 protein in corn; exemption from the requirement of a tolerance. Residues of Bacillus...

  18. Production of polyclonal and monoclonal antibodies against the Bacillus thuringiensis vegetative insecticidal protein Vip3Aa16.

    PubMed

    Ben Hamadou-Charfi, Dorra; Sauer, Annette Juliane; Abdelkafi-Mesrati, Lobna; Jaoua, Samir; Stephan, Dietrich

    2015-03-01

    The aim of this study is to establish a quantitative determination of the vegetative insecticidal protein Vip3A from the culture supernatant of Bacillus thuringiensis either by ELISA or by the conventional quantification method of the Western blot band. The Vip3A protein was produced by fermentation of the B. thuringiensis reference strain BUPM95 in 3 L. By Western blot, the Vip3Aa16 toxin was detected in the culture supernatant during the exponential growth phase of B. thuringiensis BUPM95. However, the detection of Vip3Aa16 on Western blot showed in addition to the toxin two other strips (62 and 180 kDa) recognized by the anti-Vip3Aa16 polyclonal antibodies prepared at the Centre of Biotechnology of Sfax Tunisia. For that reason and in order to develop a technique for reliable quantification of the toxin, we have considered the production of polyclonal antibodies at the Julius Kühn Institute, Germany. These antibodies were the basis for the production of monoclonal antibodies directed against the protein produced by the Vip3Aa16 recombinant strain Escherichia coli BL21 (DE3). These monoclonal antibodies were tested by plate-trapped antigen (PTA) and triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA). The selection of hybridoma supernatants gave us four positive clones producing monoclonal antibodies.

  19. Cyt1A of Bacillus thuringiensis Delays Evolution of Resistance to Cry11A in the Mosquito Culex quinquefasciatus

    PubMed Central

    Wirth, Margaret C.; Park, Hyun-Woo; Walton, William E.; Federici, Brian A.

    2005-01-01

    Insecticides based on Bacillus thuringiensis subsp. israelensis have been used for mosquito and blackfly control for more than 20 years, yet no resistance to this bacterium has been reported. Moreover, in contrast to B. thuringiensis subspecies toxic to coleopteran or lepidopteran larvae, only low levels of resistance to B. thuringiensis subsp. israelensis have been obtained in laboratory experiments where mosquito larvae were placed under heavy selection pressure for more than 30 generations. Selection of Culex quinquefasciatus with mutants of B. thuringiensis subsp. israelensis that contained different combinations of its Cry proteins and Cyt1Aa suggested that the latter protein delayed resistance. This hypothesis, however, has not been tested experimentally. Here we report experiments in which separate C. quinquefasciatus populations were selected for 20 generations to recombinant strains of B. thuringiensis that produced either Cyt1Aa, Cry11Aa, or a 1:3 mixture of these strains. At the end of selection, the resistance ratio was 1,237 in the Cry11Aa-selected population and 242 in the Cyt1Aa-selected population. The resistance ratio, however, was only 8 in the population selected with the 1:3 ratio of Cyt1Aa and Cry11Aa strains. When the resistant mosquito strain developed by selection to the Cyt1Aa-Cry11Aa combination was assayed against Cry11Aa after 48 generations, resistance to this protein was 9.3-fold. This indicates that in the presence of Cyt1Aa, resistance to Cry11Aa evolved, but at a much lower rate than when Cyt1Aa was absent. These results indicate that Cyt1Aa is the principal factor responsible for delaying the evolution and expression of resistance to mosquitocidal Cry proteins. PMID:15640186

  20. Identification and Characterization of Three Previously Undescribed Crystal Proteins from Bacillus thuringiensis subsp. jegathesan

    PubMed Central

    Sun, Yunjun; Zhao, Qiang; Ding, Xuezhi; Hu, Quanfang; Federici, Brian A.

    2013-01-01

    The total protoxin complement in the parasporal body of mosquitocidal strain, Bacillus thuringiensis subsp. jegathesan 367, was determined by use of a polyacrylamide gel block coupled to mass spectrometry. A total of eight protoxins were identified from this strain, including five reported protoxins (Cry11Ba, Cry19Aa, Cry24Aa, Cry25Aa, and Cyt2Bb), as well as three previously undescribed (Cry30Ca, Cry60Aa, and Cry60Ba) in this isolate. It was interesting that the encoding genes of three new protoxins existed as cry30Ca-gap-orf2 and cry60Ba-gap-cry60Aa. The cry30Ca and a downstream orf2 gene were oriented in the same direction and separated by 114 bp, and cry60Ba was located 156 bp upstream from and in the same orientation to cry60Aa. The three new protoxin genes were cloned from B. thuringiensis subsp. jegathesan and expressed in an acrystalliferous strain under the control of cyt1A gene promoters and the STAB-SD stabilizer sequence. Recombinant strain containing only cry30Ca did not produce visible inclusion under microscope observation, while that containing both cry30Ca and orf2 could produce large inclusions. Cry60Aa and Cry60Ba synthesized either alone or together in the acrystalliferous host could yield large inclusions. In bioassays using the fourth-instar larvae of Culex quinquefasciatus, Cry60Aa and Cry60Ba alone or together had estimated 50% lethal concentrations of 2.9 to 7.9 μg/ml; however, Cry30Ca with or without ORF2 was not toxic to this mosquito. PMID:23524673

  1. 40 CFR 174.532 - Bacillus thuringiensis eCry3.1Ab protein in corn; temporary exemption from the requirement of a...

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 24 2011-07-01 2011-07-01 false Bacillus thuringiensis eCry3.1Ab... Bacillus thuringiensis eCry3.1Ab protein in corn, in or on the food and feed commodities of corn; corn... Bacillus thuringiensis eCry3.1Ab protein in corn is used as a plant-incorporated protectant in...

  2. 40 CFR 174.532 - Bacillus thuringiensis eCry3.1Ab protein in corn; temporary exemption from the requirement of a...

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Bacillus thuringiensis eCry3.1Ab... Bacillus thuringiensis eCry3.1Ab protein in corn, in or on the food and feed commodities of corn; corn... Bacillus thuringiensis eCry3.1Ab protein in corn is used as a plant-incorporated protectant in...

  3. Structural insights into Bacillus thuringiensis Cry, Cyt and parasporin toxins.

    PubMed

    Xu, Chengchen; Wang, Bi-Cheng; Yu, Ziniu; Sun, Ming

    2014-09-16

    Since the first X-ray structure of Cry3Aa was revealed in 1991, numerous structures of B. thuringiensis toxins have been determined and published. In recent years, functional studies on the mode of action and resistance mechanism have been proposed, which notably promoted the developments of biological insecticides and insect-resistant transgenic crops. With the exploration of known pore-forming toxins (PFTs) structures, similarities between PFTs and B. thuringiensis toxins have provided great insights into receptor binding interactions and conformational changes from water-soluble to membrane pore-forming state of B. thuringiensis toxins. This review mainly focuses on the latest discoveries of the toxin working mechanism, with the emphasis on structural related progress. Based on the structural features, B. thuringiensis Cry, Cyt and parasporin toxins could be divided into three categories: three-domain type α-PFTs, Cyt toxin type β-PFTs and aerolysin type β-PFTs. Structures from each group are elucidated and discussed in relation to the latest data, respectively.

  4. Method of measuring Bacillus anthracis spores in the presence of copious amounts of Bacillus thuringiensis and Bacillus cereus.

    PubMed

    Campbell, Gossett A; Mutharasan, Raj

    2007-02-01

    A sensitive and reliable method for the detection of Bacillus anthracis (BA; Sterne strain 7702) spores in presence of large amounts of Bacillus thuringiensis (BT) and Bacillus cereus (BC) is presented based on a novel PZT-anchored piezoelectric excited millimeter-sized cantilever (PAPEMC) sensor with a sensing area of 1.5 mm2. Antibody (anti-BA) specific to BA spores was immobilized on the sensing area and exposed to various samples of BA, BT, and BC containing the same concentration of BA at 333 spores/mL, and the concentration of BT + BC was varied in concentration ratios of (BA:BT + BC) 0:1, 1:0, 1:1, 1:10, 1:100, and 1:1000. In each case, the sensor responded with an exponential decrease in resonant frequency and the steady-state frequency changes reached were 14 +/- 31 (n = 11), 2742 +/- 38 (n = 3), 3053 +/- 19 (n = 2), 2777 +/- 26 (n = 2), 2953 +/- 24 (n = 2), and 3105 +/- 27 (n = 2) Hz, respectively, in 0, 27, 45, 63, 154, and 219 min. The bound BA spores were released in each experiment, and the sensor response was nearly identical to the frequency change during attachment. These results suggest that the transport of BA spores to the antibody immobilized surface was hindered by the presence of other Bacillus species. The observed binding rate constant, based on the Langmuir kinetic model, was determined to be 0.15 min-1. A hindrance factor (alpha) is defined to describe the reduced attachment rate in the presence of BT + BC and found to increase exponentially with BT and BC concentration. The hindrance factor increased from 3.52 at 333 BT + BC spores/mL to 11.04 at 3.33 x 105 BT + BC spores/mL, suggesting that alpha is a strong function of BT and BC concentration. The significance of these results is that anti-BA functionalized PEMC sensors are highly selective to Bacillus anthracis spores and the presence of other Bacillus species, in large amounts, does not prevent binding but impedes BA transport to the sensor.

  5. Division of labour and terminal differentiation in a novel Bacillus thuringiensis strain.

    PubMed

    Deng, Chao; Slamti, Leyla; Raymond, Ben; Liu, Guiming; Lemy, Christelle; Gominet, Myriam; Yang, Jingni; Wang, Hengliang; Peng, Qi; Zhang, Jie; Lereclus, Didier; Song, Fuping

    2015-02-01

    A major challenge in bacterial developmental biology has been to understand the mechanisms underlying cell fate decisions. Some differentiated cell types display cooperative behaviour. Cooperation is one of the greatest mysteries of evolutionary biology and microbes have been considered as an excellent system for experimentally testing evolution theories. Bacillus thuringiensis (Bt) is a spore-forming bacterium, which is genetically closely related to B. anthracis, the agent of anthrax, and to B. cereus, an opportunistic human pathogen. The defining feature that distinguishes Bt from its relatives is its ability to produce crystal inclusions in the sporulating cells. These toxins are solubilized after ingestion and are cooperative public goods in insect hosts. In this study, we describe a Bt strain LM1212 that presents the unique ability to terminally differentiate into crystal producers and spore formers. Transcriptional analysis based on lacZ and gfp reporter genes suggested that this phenotype is the consequence of a new type of cell differentiation associated with a novel regulation mode of cry gene expression. The differentiating crystal-producer phenotype has higher spore productivity than a typical Bt strain and is better able to compete with Cry toxin null 'cheaters'. Potentially, this division of labour provides additional fitness benefits in terms of spore viability or durability of Cry toxin.

  6. Microbial ecology of Bacillus thuringiensis: fecal populations recovered from wildlife in Korea.

    PubMed

    Lee, Dong-Hyun; Cha, In Hwan; Woo, Doo Sung; Ohba, Michio

    2003-07-01

    A total of 34 fecal samples, collected from 14 species of wild mammals in Korea, were examined for the occurrence of Bacillus thuringiensis. The organism was detected in 18 (53%) samples. Among the three food-habit groups, herbivorous animals yielded the highest frequency (69%) of samples positive for B. thuringiensis, followed by omnivorous animals (50%). Of the six fecal samples from carnivorous animals, only one sample contained B. thurin giensis. Among 527 isolates belonging to the Bacillus cereus - B. thuringiensis group, 43 (8%) were assigned to B. thurin giensis on the basis of the formation of parasporal inclusions. Of the 43 isolates, 13 were serologically allocated to the nine H-antigenic serotypes: H3ad (serovar sumiyoshiensis), H15 (dakota), H17/27 (tohokuensis/ mexicanensis), H19 (tochigiensis), H21 (colmeri), H29 (amagiensis), H31/49 (toguchini/muju), H42 (jinghongiensis), and H44 (higo). Other isolates were untestable or untypable by the 55 reference H antisera available. Insecticidal activity was associated with 23% of the fecal populations: three isolates killed larvae of the silkworm, Bombyx mori (Lepidoptera), and seven exhibited larvicidal activity against the mosquito, Aedes aegypti (Diptera). There was no larvicidal activity against the three lepidopterous insects: Plutella xylostella, Spodoptera exigua, and Spodoptera litura. The overall results suggest that wild animals in Korea are in contact with naturally occurring B. thuringiensis at high frequencies through the daily food intake of plants.

  7. Effect of vegetation on the presence and genetic diversity of Bacillus thuringiensis in soil.

    PubMed

    Ricieto, Ana Paula Scaramal; Fazion, Fernanda Aparecida Pires; Carvalho Filho, Celso Duarte; Vilas-Boas, Laurival Antonio; Vilas-Bôas, Gislayne Trindade

    2013-01-01

    Bacillus thuringiensis isolates were obtained from soil samples collected at different sites located in the same region but with different vegetation. The sites showed different frequencies of B. thuringiensis, depending on the type of vegetation. Strains of B. thuringiensis were found to be less common in samples of riparian forest soil than in soil of other types of vegetation. The rate of occurrence of B. thuringiensis in the samples also varied according to the vegetation. These results show that whenever this bacterium was found, it showed a high rate of occurrence, indicating that this species could be better adapted to using soil as a reservoir than other Bacillus species. The presence of cry genes was analyzed by polymerase chain reaction, and genes that exhibited activity against Diptera species were the most commonly found. The isolates obtained were characterized by random amplified polymorphic DNA, and 50% were clustered into clonal groups. These results demonstrated the possible occurrence of a high number of genetically similar strains when samples are collected from the same region, even if they are from locations with different vegetation.

  8. The Pathogenomic Sequence Analysis of B. cereus and B. Thuringiensis isolates closely related to Bacillus anthracis

    SciTech Connect

    Han, C S; Xie, G; Challacombe, J F; Altherr, M R; Bhotika, S S; Bruce, D; Campbell, C S; Campbell, M L; Chen, J; Chertkov, O; Cleland, C; Dimitrijevic-Bussod, M; Doggett, N A; Fawcett, J J; Glavina, T; Goodwin, L A; Hill, K K; Hitchcock, P; Jackson, P J; Keim, P; Kewalramani, A R; Longmire, J; Lucas, S; Malfatti, S; McMurry, K; Meincke, L J; Misra, M; Moseman, B L; Mundt, M; Munk, A C; Okinaka, R T; Parson-Quintana, B; Reilly, L P; Richardson, P; Robinson, D L; Rubin, E; Saunders, E; Tapia, R; Tesmer, J G; Thayer, N; Thompson, L S; Tice, H; Ticknor, L O; Wills, P L; Gilna, P; Brettin, T S

    2005-10-12

    The sequencing and analysis of two close relatives of Bacillus anthracis are reported. AFLP analysis of over 300 isolates of B. cereus, B. thuringiensis and B. anthracis identified two isolates as being very closely related to B. anthracis. One, a B. cereus, BcE33L, was isolated from a zebra carcass in Nambia; the second, a B. thuringiensis, 97-27, was isolated from a necrotic human wound. The B. cereus appears to be the closest anthracis relative sequenced to date. A core genome of over 3,900 genes was compiled for the Bacillus cereus group, including B anthracis. Comparative analysis of these two genomes with other members of the B. cereus group provides insight into the evolutionary relationships among these organisms. Evidence is presented that differential regulation modulates virulence, rather than simple acquisition of virulence factors. These genome sequences provide insight into the molecular mechanisms contributing to the host range and virulence of this group of organisms.

  9. Effect of chemical additives on Bacillus thuringiensis (Bacillales: Bacillaceae) against Plutella xylostella (Lepidoptera: Pyralidae).

    PubMed

    Zhang, L; Qiu, S; Huang, T; Huang, Z; Xu, L; Wu, C; Gelbic, I; Guan, X

    2013-06-01

    To examine the effect of chemical additives on Bacillus thuringiensis (Berliner) against Plutella xylostella (L.), inorganic salts, nitrogenous compounds, protein solubilizing agents, and organic acids were selected and tested. The chosen materials are low in cost and environmentally safe. Results show that many inorganic salts can increase the activity of B. thuringiensis in a range of 1.31- to 3.08-fold. These include calcium acetate, calcium chloride, calcium hydroxide, calcium sulfate, calcium carbonate, sodium carbonate, sodium acetate, potassium hydroxide, potassium carbonate, potassium acetate, magnesium chloride, magnesium sulfate, and zinc sulfate. Nitrogenous compounds, including peptone, sodium nitrate, and ammonium nitrate, can enhance the activity of B. thuringiensis 1.62-, 1.32-, and 1.37-fold, respectively. Among the protein solubilizing agents, EDTA, urea, mercaptoethanol and dipotassium hydrogen phosphate increased the activity of B. thuringiensis 1.62- to 2.34-fold. Among the organic acids, maleic and citric acids boosted the activity 1.45- and 1.55-fold, respectively. Meanwhile, sodium benzoate and resorcinol led to 1.74- and 1.44-fold activity gains, respectively. Use of appropriate additives could provide great benefit not only in reducing the costs for field applications of biological insecticides but also by boosting the efficacy of B. thuringiensis. PMID:23865169

  10. Multi-method approach for characterizing the interaction between Fusarium verticillioides and Bacillus thuringiensis subsp. Kurstaki.

    PubMed

    Rocha, Liliana O; Tralamazza, Sabina Moser; Reis, Gabriela M; Rabinovitch, Leon; Barbosa, Cynara B; Corrêa, Benedito

    2014-01-01

    Bacterial antagonists used as biocontrol agents represent part of an integrated management program to reduce pesticides in the environment. Bacillus thuringiensis is considered a good alternative as a biocontrol agent for suppressing plant pathogens such as Fusarium. In this study, we used microscopy, flow cytometry, indirect immunofluorescence, and high performance liquid chromatography to determine the interaction between B. thuringiensis subsp. kurstaki LFB-FIOCRUZ (CCGB) 257 and F. verticillioides MRC 826, an important plant pathogen frequently associated with maize. B. thuringiensis showed a strong in vitro suppressive effect on F. verticillioides growth and inhibited fumonisin production. Flow cytometry analysis was found to be adequate for characterizing the fungal cell oscillations and death during these interactions. Further studies of the antagonistic effect of this isolate against other fungi and in vivo testing are necessary to determine the efficacy of B. thuringiensis subsp. kurstaki in controlling plant pathogens. This is the first report on the use of flow cytometry for quantifying living and apoptotic F. verticillioides cells and the B. thuringiensis Cry 1Ab toxin.

  11. Multi-Method Approach for Characterizing the Interaction between Fusarium verticillioides and Bacillus thuringiensis Subsp. Kurstaki

    PubMed Central

    Rocha, Liliana O.; Tralamazza, Sabina Moser.; Reis, Gabriela M.; Rabinovitch, Leon; Barbosa, Cynara B.; Corrêa, Benedito

    2014-01-01

    Bacterial antagonists used as biocontrol agents represent part of an integrated management program to reduce pesticides in the environment. Bacillus thuringiensis is considered a good alternative as a biocontrol agent for suppressing plant pathogens such as Fusarium. In this study, we used microscopy, flow cytometry, indirect immunofluorescence, and high performance liquid chromatography to determine the interaction between B. thuringiensis subsp. kurstaki LFB-FIOCRUZ (CCGB) 257 and F. verticillioides MRC 826, an important plant pathogen frequently associated with maize. B. thuringiensis showed a strong in vitro suppressive effect on F. verticillioides growth and inhibited fumonisin production. Flow cytometry analysis was found to be adequate for characterizing the fungal cell oscillations and death during these interactions. Further studies of the antagonistic effect of this isolate against other fungi and in vivo testing are necessary to determine the efficacy of B. thuringiensis subsp. kurstaki in controlling plant pathogens. This is the first report on the use of flow cytometry for quantifying living and apoptotic F. verticillioides cells and the B. thuringiensis Cry 1Ab toxin. PMID:24739804

  12. Toxicity Analysis of N- and C-Terminus-Deleted Vegetative Insecticidal Protein from Bacillus thuringiensis

    PubMed Central

    Selvapandiyan, A.; Arora, N.; Rajagopal, R.; Jalali, S. K.; Venkatesan, T.; Singh, S. P.; Bhatnagar, Raj K.

    2001-01-01

    A vegetative insecticidal protein (VIP)-encoding gene from a local isolate of Bacillus thuringiensis has been cloned, sequenced, and expressed in Escherichia coli. The expressed protein shows insecticidal activity against several lepidopteran pests but is ineffective against Agrotis ipsilon. Comparison of the amino acid sequence with those of reported VIPs revealed a few differences. Analysis of insecticidal activity with N- and C-terminus deletion mutants suggests a differential mode of action of VIP against different pests. PMID:11722946

  13. Cyt1Ab1 and Cyt2Ba1 from Bacillus thuringiensis subsp. medellin and B. thuringiensis subsp. israelensis Synergize Bacillus sphaericus against Aedes aegypti and Resistant Culex quinquefasciatus (Diptera: Culicidae)

    PubMed Central

    Wirth, Margaret C.; Delécluse, Armelle; Walton, William E.

    2001-01-01

    The interaction of two cytolytic toxins, Cyt1Ab from Bacillus thuringiensis subsp. medellin and Cyt2Ba from Bacillus thuringiensis subsp. israelensis, with Bacillus sphaericus was evaluated against susceptible and resistant Culex quinquefasciatus and the nonsensitive species Aedes aegypti. Mixtures of B. sphaericus with either cytolytic toxin were synergistic, and B. sphaericus resistance in C. quinquefasciatus was suppressed from >17,000- to 2-fold with a 3:1 mixture of B. sphaericus and Cyt1Ab. This trait may prove useful for combating insecticide resistance and for improving the activity of microbial insecticides. PMID:11425753

  14. The glycoprotein toxin of Bacillus thuringiensis subsp. israelensis indicates a lectinlike receptor in the larval mosquito gut.

    PubMed Central

    Muthukumar, G; Nickerson, K W

    1987-01-01

    The mosquito-active protein crystals produced by Bacillus thuringiensis subsp. israelensis contain covalently attached aminosugars which are critical for their larvicidal activity. The 50% lethal concentrations toward Aedes aegypti larvae were increased up to 10-fold by mild periodate treatment, up to 40-fold by forming the protein crystals in the presence of tunicamycin, and up to 7-fold by the presence during the mosquito bioassays of N-acetylglucosamine or its trimer, triacetylchitotriose. Periodate-treated crystals and crystals formed in the presence of tunicamycin had greatly reduced binding capacities for wheat germ agglutinin, an N-acetylglucosamine-specific lectin. These results suggest that the B. thuringiensis subsp. israelensis glycoprotein toxin binds to a lectinlike receptor in the larval mosquito gut. Furthermore, the distinct lectin-binding patterns exhibited by diptera-active versus lepidoptera-active B. thuringiensis crystals suggest that host specificity for the microbial insecticides is determined, in part, by the carbohydrate portion of their glycoprotein crystals. Images PMID:2827571

  15. Mutated Cadherin Alleles from a Field Population of Helicoverpa armigera Confer Resistance to Bacillus thuringiensis Toxin Cry1Ac▿

    PubMed Central

    Yang, Yajun; Chen, Haiyan; Wu, Yidong; Yang, Yihua; Wu, Shuwen

    2007-01-01

    The cotton bollworm Helicoverpa armigera is the major insect pest targeted by cotton genetically engineered to produce the Bacillus thuringiensis toxin (transgenic Bt cotton) in the Old World. The evolution of this pest's resistance to B. thuringiensis toxins is the main threat to the long-term effectiveness of transgenic Bt cotton. A deletion mutation allele (r1) of a cadherin gene (Ha_BtR) was previously identified as genetically linked with Cry1Ac resistance in a laboratory-selected strain of H. armigera. Using a biphasic screen strategy, we successfully trapped two new cadherin alleles (r2 and r3) associated with Cry1Ac resistance from a field population of H. armigera collected from the Yellow River cotton area of China in 2005. The r2 and r3 alleles, respectively, were created by inserting the long terminal repeat of a retrotransposon (designated HaRT1) and the intact HaRT1 retrotransposon at the same position in exon 8 of Ha_BtR, which results in a truncated cadherin containing only two ectodomain repeats in the N terminus of Ha_BtR. This is the first time that the B. thuringiensis resistance alleles of a target insect of Bt crops have been successfully detected in the open field. This study also demonstrated that bollworm larvae carrying two resistance alleles can complete development on Bt cotton. The cadherin locus should be an important target for intensive DNA-based screening of field populations of H. armigera. PMID:17827322

  16. Enhancement of Bacillus thuringiensis insecticidal activity by combining Cry1Ac and bi-functional toxin HWTX-XI from spider.

    PubMed

    Sun, Yunjun; Fu, Zujiao; He, Xiaohong; Yuan, Chunhua; Ding, Xuezhi; Xia, Liqiu

    2016-03-01

    In order to assess the potency of bi-functional HWTX-XI toxin from spider Ornithoctonus huwena in improving the insecticidal activity of Bacillus thuringiensis, a fusion gene of cry1Ac and hwtx-XI was constructed and expressed in an acrystalliferous B. thuringiensis strain Cry(-)B. Western blot analysis and microscopic observation revealed that the recombinant strain could express 140-kDa Cry1Ac-HWTX-XI fusion protein and produce parasporal inclusions during sporulation. Bioassay using the larvae of Helicoverpa armigera and Spodoptera exigua showed that the Cry1Ac-HWTX-XI fusion was more toxic than the control Cry1Ac protoxin, as revealed by 95% lethal concentration. Our study indicated that the HWTX-XI from spider might be a candidate for enhancing the toxicity of B. thuringiensis products. PMID:25721170

  17. Experimental design and Bayesian networks for enhancement of delta-endotoxin production by Bacillus thuringiensis.

    PubMed

    Ennouri, Karim; Ayed, Rayda Ben; Hassen, Hanen Ben; Mazzarello, Maura; Ottaviani, Ennio

    2015-12-01

    Bacillus thuringiensis (Bt) is a Gram-positive bacterium. The entomopathogenic activity of Bt is related to the existence of the crystal consisting of protoxins, also called delta-endotoxins. In order to optimize and explain the production of delta-endotoxins of Bacillus thuringiensis kurstaki, we studied seven medium components: soybean meal, starch, KH₂PO₄, K₂HPO₄, FeSO₄, MnSO₄, and MgSO₄and their relationships with the concentration of delta-endotoxins using an experimental design (Plackett-Burman design) and Bayesian networks modelling. The effects of the ingredients of the culture medium on delta-endotoxins production were estimated. The developed model showed that different medium components are important for the Bacillus thuringiensis fermentation. The most important factors influenced the production of delta-endotoxins are FeSO₄, K2HPO₄, starch and soybean meal. Indeed, it was found that soybean meal, K₂HPO₄, KH₂PO₄and starch also showed positive effect on the delta-endotoxins production. However, FeSO4 and MnSO4 expressed opposite effect. The developed model, based on Bayesian techniques, can automatically learn emerging models in data to serve in the prediction of delta-endotoxins concentrations. The constructed model in the present study implies that experimental design (Plackett-Burman design) joined with Bayesian networks method could be used for identification of effect variables on delta-endotoxins variation.

  18. Experimental design and Bayesian networks for enhancement of delta-endotoxin production by Bacillus thuringiensis.

    PubMed

    Ennouri, Karim; Ayed, Rayda Ben; Hassen, Hanen Ben; Mazzarello, Maura; Ottaviani, Ennio

    2015-12-01

    Bacillus thuringiensis (Bt) is a Gram-positive bacterium. The entomopathogenic activity of Bt is related to the existence of the crystal consisting of protoxins, also called delta-endotoxins. In order to optimize and explain the production of delta-endotoxins of Bacillus thuringiensis kurstaki, we studied seven medium components: soybean meal, starch, KH₂PO₄, K₂HPO₄, FeSO₄, MnSO₄, and MgSO₄and their relationships with the concentration of delta-endotoxins using an experimental design (Plackett-Burman design) and Bayesian networks modelling. The effects of the ingredients of the culture medium on delta-endotoxins production were estimated. The developed model showed that different medium components are important for the Bacillus thuringiensis fermentation. The most important factors influenced the production of delta-endotoxins are FeSO₄, K2HPO₄, starch and soybean meal. Indeed, it was found that soybean meal, K₂HPO₄, KH₂PO₄and starch also showed positive effect on the delta-endotoxins production. However, FeSO4 and MnSO4 expressed opposite effect. The developed model, based on Bayesian techniques, can automatically learn emerging models in data to serve in the prediction of delta-endotoxins concentrations. The constructed model in the present study implies that experimental design (Plackett-Burman design) joined with Bayesian networks method could be used for identification of effect variables on delta-endotoxins variation. PMID:26689874

  19. Response surface methodology: optimisation of antifungal bioemulsifier from novel Bacillus thuringiensis.

    PubMed

    Rajendran, Deepak; Venkatachalam, Ponnusami; Ramakrishnan, Jayapradha

    2014-01-01

    An antifungal bioemulsifier compound was produced from a novel strain of Bacillus thuringiensis pak2310. To accentuate the production and as the first step to improve the yield, a central composite design (CCD) was used to study the effect of various factors like minimal salts (1X and 3X), glycerol concentration (2% and 4%), beef extract concentration (1% and 3%), and sunflower oil concentration (2% and 4%) on the production of bioemulsifier molecule and to optimize the conditions to increase the production. The E 24 emulsification index was used as the response variable as the increase in surfactant production was seen to be proportional to increased emulsification. A quadratic equation was employed to express the response variable in terms of the independent variables. Statistical tools like student's t-test, F-test, and ANOVA were employed to identify the important factors and to test the adequacy of the model. Under optimum conditions (1X concentration of minimal salts (MS), 2.6% glycerol (v/v), 1% beef extract (w/v), and 2% sunflower oil (v/v)) a 65% increase in yield was produced. PMID:25379529

  20. 40 CFR 180.1108 - Delta endotoxin of Bacillus thuringiensis variety San Diego encapsulated into killed Pseudomonas...

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 25 2012-07-01 2012-07-01 false Delta endotoxin of Bacillus... From Tolerances § 180.1108 Delta endotoxin of Bacillus thuringiensis variety San Diego encapsulated into killed Pseudomonas fluorescens; exemption from the requirement of a tolerance. The delta...

  1. 40 CFR 180.1108 - Delta endotoxin of Bacillus thuringiensis variety San Diego encapsulated into killed Pseudomonas...

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 25 2013-07-01 2013-07-01 false Delta endotoxin of Bacillus... From Tolerances § 180.1108 Delta endotoxin of Bacillus thuringiensis variety San Diego encapsulated into killed Pseudomonas fluorescens; exemption from the requirement of a tolerance. The delta...

  2. 40 CFR 180.1108 - Delta endotoxin of Bacillus thuringiensis variety San Diego encapsulated into killed Pseudomonas...

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 24 2014-07-01 2014-07-01 false Delta endotoxin of Bacillus... From Tolerances § 180.1108 Delta endotoxin of Bacillus thuringiensis variety San Diego encapsulated into killed Pseudomonas fluorescens; exemption from the requirement of a tolerance. The delta...

  3. Expression of the mosquitocidal toxins of Bacillus sphaericus and Bacillus thuringiensis subsp. israelensis by recombinant Caulobacter crescentus, a vehicle for biological control of aquatic insect larvae.

    PubMed Central

    Thanabalu, T; Hindley, J; Brenner, S; Oei, C; Berry, C

    1992-01-01

    In the quest for effective control of mosquitoes, attention has turned increasingly to strains of the bacteria Bacillus sphaericus and Bacillus thuringiensis subsp. israelensis, which produce potent toxins with specific mosquitocidal activities. However, sedimentation of the bacterial spores limits the duration of effective control after field application of these bacilli. We describe here the cloning of genes encoding the 51.4- and 41.9-kDa toxins from B. sphaericus 2297, the 100-kDa toxin from B. sphaericus SSII-1, and the 130-kDa toxin from B. thuringiensis subsp. israelensis into the broad-host-range plasmid pRK248 and the transfer of these genes for expression in Caulobacter crescentus CB15. The recombinant C. crescentus cells were shown to be toxic to mosquito larvae. Caulobacter species are ubiquitous microorganisms residing in the upper regions of aquatic environments and therefore provide the potential for prolonged control by maintaining mosquitocidal toxins in larval feeding zones. PMID:1575492

  4. Phosphatidylinositol-specific phospholipase C of Bacillus thuringiensis as a probe for the distribution of phosphatidylinositol in hepatocyte membranes.

    PubMed Central

    Higgins, J A; Hitchin, B W; Low, M G

    1989-01-01

    Phosphatidylinositol-specific phospholipase C (PI-PLC) produced by Bacillus thuringiensis has been used as a probe for the distribution of phosphatidylinositol in hepatocyte membranes. Approx. 50% of this phospholipid was hydrolysed in microsomal vesicles (endoplasmic reticulum) with no significant hydrolysis of the remaining membrane phospholipids. Latency of mannose-6-phosphatase was retained during treatment indicating that the vesicles remained impermeable. Stripping of the ribosomes did not increase hydrolysis of phosphatidylinositol; however, when the vesicles were opened using dilute sodium carbonate, hydrolysis increased to greater than 90%. Hydrolysis of phosphatidylinositol of Golgi membranes was 35% and of plasma membranes was 50%. After treatment with PI-PLC, radiolabelled secretory proteins were retained in Golgi membranes and trapped lactate dehydrogenase was retained in plasma-membrane preparations indicating that the vesicles remained closed. Hydrolysis of phosphatidylinositol increased to greater than 90% when the membranes were opened by treatment with dilute sodium carbonate. These observations indicate that PI-PLC of Bacillus thuringiensis is a suitable probe for the distribution of phosphatidylinositol in membranes, and that in liver membranes this phospholipid occurs on each side of the bilayer, a topography consistent with its diverse roles. PMID:2543374

  5. Isolation and Characterization of Gut Bacterial Proteases Involved in Inducing Pathogenicity of Bacillus thuringiensis Toxin in Cotton Bollworm, Helicoverpa armigera

    PubMed Central

    Regode, Visweshwar; Kuruba, Sreeramulu; Mohammad, Akbar S.; Sharma, Hari C.

    2016-01-01

    Bacillus thuringiensis toxin proteins are deployed in transgenic plants for pest management. The present studies were aimed at characterization of gut bacterial proteases involved in activation of inactive Cry1Ac protoxin (pro-Cry1Ac) to active toxin in Helicoverpa armigera. Bacterial strains were isolated from H. armigera midgut and screened for their proteolytic activation toward pro-Cry1Ac. Among 12 gut bacterial isolates seven isolates showed proteolytic activity, and proteases from three isolates (IVS1, IVS2, and IVS3) were found to be involved in the proteolytic conversion of pro-Cry1Ac into active toxin. The proteases from IVS1, IVS2, and IVS3 isolates were purified to 11.90-, 15.50-, and 17.20-fold, respectively. The optimum pH and temperature for gut bacterial protease activity was 8.0 and 40°C. Maximum inhibition of total proteolytic activity was exerted by phenylmethane sulfonyl fluoride followed by EDTA. Fluorescence zymography revealed that proteases from IVS1, IVS2, and IVS3 were chymotrypsin-like and showing protease band at ~15, 65, and 15 kDa, respectively. Active Cry1Ac formed from processing pro-Cry1Ac by gut bacterial proteases exhibited toxicity toward H. armigera. The gut bacterial isolates IVS1, IVS2, and IVS3 showed homology with B. thuringiensis (CP003763.1), Vibrio fischeri (CP000020.2), and Escherichia coli (CP011342.1), respectively. Proteases produced by midgut bacteria are involved in proteolytic processing of B. thuringiensis protoxin and play a major role in inducing pathogenicity of B. thuringiensis toxins in H. armigera. PMID:27766093

  6. Effect of Promoters and Plasmid Copy Number on Cyt1A Synthesis and Crystal Assembly in Bacillus thuringiensis.

    PubMed

    Park, Hyun-Woo; Hice, Robert H; Federici, Brian A

    2016-01-01

    Cyt1Aa is a major mosquitocidal protein synthesized during sporulation of Bacillus thuringiensis subsp. israelensis, composing more than 50% of its parasporal body. This high level of synthesis is due to several factors including three strong sporulation-dependent promoters, a strong transcription termination sequence, and an associated 20-kDa helper protein. Cyt1Aa's toxicity is low compared to the Cry proteins of this species, namely, Cry4Aa, Cry4Ba, and Cry11Aa, but it nevertheless plays an important role in the biology of B. thuringiensis subsp. israelensis in that it synergizes their mosquitocidal toxicity and suppresses the evolution of resistance. In the present study, the effects of using different cyt1Aa promoter combinations and plasmid copy number on synthesis of Cyt1Aa were evaluated. Using the 4Q7 (plasmid-cured) strain of B. thuringiensis subsp. israelensis as an experimental host, a plasmid copy number of two or three yielded no Cyt1Aa, whereas a copy number of four yielded only small crystals, even when expression was driven by one of the wild-type promoters. However, using all three wild-type promoters and a plasmid copy number of 20 yielded Cyt1A crystals tenfold larger than those produced by one promoter and a plasmid copy number of four. High levels of Cyt1Aa synthesis resulted in significantly fewer spores per unit medium and imperfectly formed crystals. Similar results were obtained when Cyt1Aa synthesis was evaluated using the same expression constructs in a mutant strain of B. thuringiensis subsp. israelensis that lacks the cyt1Aa gene.

  7. Improvement of Bacillus sphaericus toxicity against dipteran larvae by integration, via homologous recombination, of the Cry11A toxin gene from Bacillus thuringiensis subsp. israelensis.

    PubMed Central

    Poncet, S; Bernard, C; Dervyn, E; Cayley, J; Klier, A; Rapoport, G

    1997-01-01

    Integrative plasmids were constructed to enable integration of foreign DNA into the chromosome of Bacillus sphaericus 2297 by in vivo recombination. Integration of the aphA3 kanamycin resistance gene by a two-step procedure demonstrated that this strategy was applicable with antibiotic resistance selection. Hybridization experiments evidenced two copies of the operon encoding the binary toxin from B. sphaericus in the recipient strain. The Bacillus thuringiensis subsp. israelensis cry11Aal gene (referred to as cry11A), encoding a delta-endotoxin with toxicity against Culex, Aedes, and Anopheles larvae, was integrated either by a single crossover event [strain 2297 (::pHT5601), harboring the entire recombinant plasmid] or by two successive crossover events [strain 2297 (::cry11A)]. The level of the Cry11A production in B. sphaericus was high; two crystalline inclusions were produced in strain 2297 (::pHT5601). Synthesis of the Cry11A toxin conferred toxicity to the recombinant strains against Aedes aegypti larvae, for which the parental strain was not toxic. Interestingly, the level of larvicidal activity of strain 2297 (::pHT5601) against Anopheles stephensi was as high as that of B. thuringiensis subsp. israelensis and suggested synergy between the B. thuringiensis and B. sphaericus toxins. The toxicities of parental and recombinant B. sphaericus strains against Culex quinquefasciatus were similar, but the recombinant strains killed the larvae more rapidly. The production of the Cry11A toxin in B. sphaericus also partially restored toxicity for C. quinquefasciatus larvae from a population resistant to B. sphaericus 1593. In vivo recombination therefore appears to be a promising approach to the creation of new B. sphaericus strains for vector control. PMID:9361428

  8. Persistence of detectable insecticidal proteins from Bacillus thuringiensis (Cry) and toxicity after adsorption on contrasting soils.

    PubMed

    Hung, T P; Truong, L V; Binh, N D; Frutos, R; Quiquampoix, H; Staunton, S

    2016-01-01

    Insecticidal Cry, or Bt, proteins are produced by the soil-endemic bacterium, Bacillus thuringiensis and some genetically modified crops. Their environmental fate depends on interactions with soil. Little is known about the toxicity of adsorbed proteins and the change in toxicity over time. We incubated Cry1Ac and Cry2A in contrasting soils subjected to different treatments to inhibit microbial activity. The toxin was chemically extracted and immunoassayed. Manduca sexta was the target insect for biotests. Extractable toxin decreased during incubation for up to four weeks. Toxicity of Cry1Ac was maintained in the adsorbed state, but lost after 2 weeks incubation at 25 °C. The decline in extractable protein and toxicity were much slower at 4 °C with no significant effect of soil sterilization. The major driving force for decline may be time-dependent fixation of adsorbed protein, leading to a decrease in the extraction yield in vitro, paralleled by decreasing solubilisation in the larval gut. PMID:26549751

  9. One gene in diamondback moth confers resistance to four Bacillus thuringiensis toxins.

    PubMed

    Tabashnik, B E; Liu, Y B; Finson, N; Masson, L; Heckel, D G

    1997-03-01

    Environmentally benign insecticides derived from the soil bacterium Bacillus thuringiensis (Bt) are the most widely used biopesticides, but their success will be short-lived if pests quickly adapt to them. The risk of evolution of resistance by pests has increased, because transgenic crops producing insecticidal proteins from Bt are being grown commercially. Efforts to delay resistance with two or more Bt toxins assume that independent mutations are required to counter each toxin. Moreover, it generally is assumed that resistance alleles are rare in susceptible populations. We tested these assumptions by conducting single-pair crosses with diamondback moth (Plutella xylostella), the first insect known to have evolved resistance to Bt in open field populations. An autosomal recessive gene conferred extremely high resistance to four Bt toxins (Cry1Aa, Cry1Ab, Cry1Ac, and Cry1F). The finding that 21% of the individuals from a susceptible strain were heterozygous for the multiple-toxin resistance gene implies that the resistance allele frequency was 10 times higher than the most widely cited estimate of the upper limit for the initial frequency of resistance alleles in susceptible populations. These findings suggest that pests may evolve resistance to some groups of toxins much faster than previously expected.

  10. Novel actin filaments from Bacillus thuringiensis form nanotubules for plasmid DNA segregation

    PubMed Central

    Jiang, Shimin; Narita, Akihiro; Popp, David; Ghoshdastider, Umesh; Lee, Lin Jie; Srinivasan, Ramanujam; Balasubramanian, Mohan K.; Oda, Toshiro; Koh, Fujiet; Larsson, Mårten; Robinson, Robert C.

    2016-01-01

    Here we report the discovery of a bacterial DNA-segregating actin-like protein (BtParM) from Bacillus thuringiensis, which forms novel antiparallel, two-stranded, supercoiled, nonpolar helical filaments, as determined by electron microscopy. The BtParM filament features of supercoiling and forming antiparallel double-strands are unique within the actin fold superfamily, and entirely different to the straight, double-stranded, polar helical filaments of all other known ParMs and of eukaryotic F-actin. The BtParM polymers show dynamic assembly and subsequent disassembly in the presence of ATP. BtParR, the DNA-BtParM linking protein, stimulated ATP hydrolysis/phosphate release by BtParM and paired two supercoiled BtParM filaments to form a cylinder, comprised of four strands with inner and outer diameters of 57 Å and 145 Å, respectively. Thus, in this prokaryote, the actin fold has evolved to produce a filament system with comparable features to the eukaryotic chromosome-segregating microtubule. PMID:26873105

  11. Characterization of Bacillus thuringiensis soil isolates from Cuba, with insecticidal activity against mosquitoes.

    PubMed

    González, Aileen; Díaz, Raúl; Díaz, Manuel; Borrero, Yainais; Bruzón, Rosa Y; Carreras, Bertha; Gato, René

    2011-09-01

    Chemical insecticides may be toxic and cause environmental degradation. Consequently, biological control for insects represents an alternative with low ecological impact. In this work, three soil isolates (A21, A51 and C17) from different regions of the Cuban archipelago were identified, characterized and evaluated against Aedes aegypti and Culex quinquefasciatus. The new isolates were compared with reference IPS82 strain and two strains isolated from biolarvicides Bactivec and Bactoculicida, respectively. The differentiation was done by morphological, biochemical, bioassays activity and molecular methods (SDS-PAGE, plasmid profile and random amplified polymorphic analysis). All isolates were identified as Bacillus thuringiensis. The A21, A51 and C17 isolates showed higher larvicide activity than Bactivec's isolated reference strain, against both A. aegypti and C. quinquefasciatus. A21 isolate had a protein profile similar to IPS82 and Bactivec strain. A51 and C17 isolates produced a characteristic proteins pattern. A21 and A51 isolates had plasmid patterns similar to IPS82 standard strain, while C17 isolate had different both plasmid profile and protein bands. All the studied isolates showed a diverse RAPD patterns and were different from the strains previously used in biological control in Cuba. PMID:22017108

  12. Three cadherin alleles associated with resistance to Bacillus thuringiensis in pink bollworm

    PubMed Central

    Morin, Shai; Biggs, Robert W.; Sisterson, Mark S.; Shriver, Laura; Ellers-Kirk, Christa; Higginson, Dawn; Holley, Daniel; Gahan, Linda J.; Heckel, David G.; Carrière, Yves; Dennehy, Timothy J.; Brown, Judith K.; Tabashnik, Bruce E.

    2003-01-01

    Evolution of resistance by pests is the main threat to long-term insect control by transgenic crops that produce Bacillus thuringiensis (Bt) toxins. Because inheritance of resistance to the Bt toxins in transgenic crops is typically recessive, DNA-based screening for resistance alleles in heterozygotes is potentially much more efficient than detection of resistant homozygotes with bioassays. Such screening, however, requires knowledge of the resistance alleles in field populations of pests that are associated with survival on Bt crops. Here we report that field populations of pink bollworm (Pectinophora gossypiella), a major cotton pest, harbored three mutant alleles of a cadherin-encoding gene linked with resistance to Bt toxin Cry1Ac and survival on transgenic Bt cotton. Each of the three resistance alleles has a deletion expected to eliminate at least eight amino acids upstream of the putative toxin-binding region of the cadherin protein. Larvae with two resistance alleles in any combination were resistant, whereas those with one or none were susceptible to Cry1Ac. Together with previous evidence, the results reported here identify the cadherin gene as a leading target for DNA-based screening of resistance to Bt crops in lepidopteran pests. PMID:12695565

  13. Characterization of Bacillus thuringiensis soil isolates from Cuba, with insecticidal activity against mosquitoes.

    PubMed

    González, Aileen; Díaz, Raúl; Díaz, Manuel; Borrero, Yainais; Bruzón, Rosa Y; Carreras, Bertha; Gato, René

    2011-09-01

    Chemical insecticides may be toxic and cause environmental degradation. Consequently, biological control for insects represents an alternative with low ecological impact. In this work, three soil isolates (A21, A51 and C17) from different regions of the Cuban archipelago were identified, characterized and evaluated against Aedes aegypti and Culex quinquefasciatus. The new isolates were compared with reference IPS82 strain and two strains isolated from biolarvicides Bactivec and Bactoculicida, respectively. The differentiation was done by morphological, biochemical, bioassays activity and molecular methods (SDS-PAGE, plasmid profile and random amplified polymorphic analysis). All isolates were identified as Bacillus thuringiensis. The A21, A51 and C17 isolates showed higher larvicide activity than Bactivec's isolated reference strain, against both A. aegypti and C. quinquefasciatus. A21 isolate had a protein profile similar to IPS82 and Bactivec strain. A51 and C17 isolates produced a characteristic proteins pattern. A21 and A51 isolates had plasmid patterns similar to IPS82 standard strain, while C17 isolate had different both plasmid profile and protein bands. All the studied isolates showed a diverse RAPD patterns and were different from the strains previously used in biological control in Cuba.

  14. Contamination of refuges by Bacillus thuringiensis toxin genes from transgenic maize.

    PubMed

    Chilcutt, Charles F; Tabashnik, Bruce E

    2004-05-18

    Transgenic crops producing insecticidal toxins from Bacillus thuringiensis (Bt) are widely used to control pests, but their benefits will be lost if pests evolve resistance. The mandated high-dose/refuge strategy for delaying pest resistance requires planting refuges of toxin-free crops near Bt crops to promote survival of susceptible pests. We report that pollen-mediated gene flow up to 31 m from Bt maize caused low to moderate Bt toxin levels in kernels of non-Bt maize refuge plants. Immunoassays of non-Bt maize sampled from the field showed that the mean concentration of Bt toxin Cry1Ab in kernels and the percentage of kernels with Cry1Ab decreased with distance from Bt maize. The highest Bt toxin concentration in pooled kernels of non-Bt maize plants was 45% of the mean concentration in kernels from adjacent Bt maize plants. Most previous work on gene flow from transgenic crops has emphasized potential effects of transgene movement on wild relatives of crops, landraces, and organic plantings, whereas implications for pest resistance have been largely ignored. Variable Bt toxin production in seeds of refuge plants undermines the high-dose/refuge strategy and could accelerate pest resistance to Bt crops. Thus, guidelines should be revised to reduce gene flow between Bt crops and refuge plants.

  15. Bacillus thuringiensis (Bt) toxin susceptibility and isolation of resistance mutants in the nematode Caenorhabditis elegans.

    PubMed Central

    Marroquin, L D; Elyassnia, D; Griffitts, J S; Feitelson, J S; Aroian, R V

    2000-01-01

    The protein toxins produced by Bacillus thuringiensis (Bt) are the most widely used natural insecticides in agriculture. Despite successful and extensive use of these toxins in transgenic crops, little is known about toxicity and resistance pathways in target insects since these organisms are not ideal for molecular genetic studies. To address this limitation and to investigate the potential use of these toxins to control parasitic nematodes, we are studying Bt toxin action and resistance in Caenorhabditis elegans. We demonstrate for the first time that a single Bt toxin can target a nematode. When fed Bt toxin, C. elegans hermaphrodites undergo extensive damage to the gut, a decrease in fertility, and death, consistent with toxin effects in insects. We have screened for and isolated 10 recessive mutants that resist the toxin's effects on the intestine, on fertility, and on viability. These mutants define five genes, indicating that more components are required for Bt toxicity than previously known. We find that a second, unrelated nematicidal Bt toxin may utilize a different toxicity pathway. Our data indicate that C. elegans can be used to undertake detailed molecular genetic analysis of Bt toxin pathways and that Bt toxins hold promise as nematicides. PMID:10924467

  16. Persistence of detectable insecticidal proteins from Bacillus thuringiensis (Cry) and toxicity after adsorption on contrasting soils.

    PubMed

    Hung, T P; Truong, L V; Binh, N D; Frutos, R; Quiquampoix, H; Staunton, S

    2016-01-01

    Insecticidal Cry, or Bt, proteins are produced by the soil-endemic bacterium, Bacillus thuringiensis and some genetically modified crops. Their environmental fate depends on interactions with soil. Little is known about the toxicity of adsorbed proteins and the change in toxicity over time. We incubated Cry1Ac and Cry2A in contrasting soils subjected to different treatments to inhibit microbial activity. The toxin was chemically extracted and immunoassayed. Manduca sexta was the target insect for biotests. Extractable toxin decreased during incubation for up to four weeks. Toxicity of Cry1Ac was maintained in the adsorbed state, but lost after 2 weeks incubation at 25 °C. The decline in extractable protein and toxicity were much slower at 4 °C with no significant effect of soil sterilization. The major driving force for decline may be time-dependent fixation of adsorbed protein, leading to a decrease in the extraction yield in vitro, paralleled by decreasing solubilisation in the larval gut.

  17. The theoretical three-dimensional structure of Bacillus thuringiensis Cry5Aa and its biological implications.

    PubMed

    Xin-Min, Zhao; Li-Qiu, Xia; Xue-Zhi, Ding; Fa-Xiang, Wang

    2009-02-01

    Cry5Aa is a crystal protein produced by Bacillus thuringiensis serovar. damstadiensis during its stationary phase, this delta-endotoxin is active against nematodes and has great potential for nematodes control. The theoretical model of the three-dimensional structure of Cry5Aa was predicted by homology modeling on the structures of the Cry1Aa which is specific to Lepidopteran insects. The structure of the Cry5Aa resembles previously reported Cry toxin structures but shows the following distinctions. Cry5Aa has a long insertion in alpha2 of domain I. Some loops in the domain II and III of Cry5Aa are exposed to the solvent. In this work we give a brief description of our model and hypothesize the residues of the Cry5Aa that could be important in receptor recognition and pore formation. This model will be helpful for the design of mutagenesis experiments aimed to the improvement of toxicity, and lead to a deep understanding of the mechanism of action of nematicidal toxins.

  18. The theoretical 3D structure of Bacillus thuringiensis Cry5Ba.

    PubMed

    Xia, Li-Qiu; Zhao, Xin-Min; Ding, Xue-Zhi; Wang, Fa-Xiang; Sun, Yun-Jun

    2008-09-01

    Cry5Ba is a delta-endotoxin produced by Bacillus thuringiensis PS86A1 NRRL B-18900. It is active against nematodes and has great potential for nematode control. Here, we predict the first theoretical model of the three-dimensional (3D) structure of a Cry5Ba toxin by homology modeling on the structure of the Cry1Aa toxin, which is specific to Lepidopteran insects. Cry5Ba resembles the previously reported Cry1Aa toxin structure in that they share a common 3D structure with three domains, but there are some distinctions, with the main differences being located in the loops of domain I. Cry5Ba exhibits a changeable extending conformation structure, and this special structure may also be involved in pore-forming and specificity determination. A fuller understanding of the 3D structure will be helpful in the design of mutagenesis experiments aimed at improving toxicity, and lead to a deep understanding of the mechanism of action of nematicidal toxins.

  19. Effect of resistance to Bacillus thuringiensis cotton on pink bollworm (Lepidoptera: Gelechiidae) response to sex pheromone.

    PubMed

    Carrière, Yves; Nyboer, Megan E; Ellers-Kirk, Christa; Sollome, James; Colletto, Nick; Antilla, Larry; Dennehy, Timothy J; Staten, Robert T; Tabashnik, Bruce E

    2006-06-01

    Fitness costs associated with resistance to transgenic crops producing toxins from Bacillus thuringiensis (Bt) could reduce male response to pheromone traps. Such costs would cause underestimation of resistance frequency if monitoring was based on analysis of males caught in pheromone traps. To develop a DNA-based resistance monitoring program for pink bollworm, Pectinophora gossypiella (Saunders) (Lepidoptera: Gelechiidae), we compared the response to pheromone traps of males with and without cadherin alleles associated with resistance to Bt cotton (Gossypium hirsutum L.). When irradiated males from two hybrid laboratory strains with an intermediate frequency of resistance alleles were released in large field cages, the probability of capture in pheromone traps was not lower for males with resistance alleles than for males without resistance alleles. These results suggest that analysis of trapped males would not underestimate the frequency of resistance. As the time males spent in traps in the field increased from 3 to 15 d, the success of DNA amplification declined from 100 to 30%. Thus, the efficiency of a DNA-based resistance monitoring program would be improved by analyzing males remaining in traps for 3 d or less. PMID:16813335

  20. 40 CFR 180.1154 - CryIA(c) and CryIC derived delta-endotoxins of Bacillus thuringiensis var. kurstaki encapsulated...

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ...-endotoxins of Bacillus thuringiensis var. kurstaki encapsulated in killed Pseudomonas fluorescens, and the... Bacillus thuringiensis var. kurstaki encapsulated in killed Pseudomonas fluorescens, and the expression plasmid and cloning vector genetic constructs. CryIA(c) and CryIC derived delta-endotoxins of...

  1. 40 CFR 174.505 - Bacillus thuringiensis modified Cry3A protein (mCry3A) in corn; exemption from the requirement of...

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 24 2011-07-01 2011-07-01 false Bacillus thuringiensis modified Cry3A... of Bacillus thuringiensis modified Cry3A protein (mCry3A) in corn are exempt from the requirement of... REQUIREMENTS FOR PLANT-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.505...

  2. 40 CFR 174.510 - Bacillus thuringiensis Cry1Ac protein in all plants; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 24 2011-07-01 2011-07-01 false Bacillus thuringiensis Cry1Ac protein... PLANT-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.510 Bacillus thuringiensis Cry1Ac protein in all plants; exemption from the requirement of a tolerance. Residues of...

  3. 40 CFR 174.505 - Bacillus thuringiensis modified Cry3A protein (mCry3A) in corn; exemption from the requirement of...

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Bacillus thuringiensis modified Cry3A... of Bacillus thuringiensis modified Cry3A protein (mCry3A) in corn are exempt from the requirement of... REQUIREMENTS FOR PLANT-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.505...

  4. 40 CFR 174.506 - Bacillus thuringiensis Cry34Ab1 and Cry35Ab1 proteins in corn; exemption from the requirement of...

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 24 2011-07-01 2011-07-01 false Bacillus thuringiensis Cry34Ab1 and... Bacillus thuringiensis Cry34Ab1 and Cry35Ab1 proteins in corn are exempted from the requirement of a... REQUIREMENTS FOR PLANT-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.506...

  5. 40 CFR 174.511 - Bacillus thuringiensis Cry1Ab protein in all plants; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 24 2011-07-01 2011-07-01 false Bacillus thuringiensis Cry1Ab protein... PLANT-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.511 Bacillus thuringiensis Cry1Ab protein in all plants; exemption from the requirement of a tolerance. Residues of...

  6. 40 CFR 174.510 - Bacillus thuringiensis Cry1Ac protein in all plants; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Bacillus thuringiensis Cry1Ac protein... PLANT-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.510 Bacillus thuringiensis Cry1Ac protein in all plants; exemption from the requirement of a tolerance. Residues of...

  7. 40 CFR 174.506 - Bacillus thuringiensis Cry34Ab1 and Cry35Ab1 proteins in corn; exemption from the requirement of...

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Bacillus thuringiensis Cry34Ab1 and... Bacillus thuringiensis Cry34Ab1 and Cry35Ab1 proteins in corn are exempted from the requirement of a... REQUIREMENTS FOR PLANT-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.506...

  8. 40 CFR 174.511 - Bacillus thuringiensis Cry1Ab protein in all plants; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Bacillus thuringiensis Cry1Ab protein... PLANT-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.511 Bacillus thuringiensis Cry1Ab protein in all plants; exemption from the requirement of a tolerance. Residues of...

  9. 40 CFR 180.1154 - CryIA(c) and CryIC derived delta-endotoxins of Bacillus thuringiensis var. kurstaki encapsulated...

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ...-endotoxins of Bacillus thuringiensis var. kurstaki encapsulated in killed Pseudomonas fluorescens, and the... Bacillus thuringiensis var. kurstaki encapsulated in killed Pseudomonas fluorescens, and the expression plasmid and cloning vector genetic constructs. CryIA(c) and CryIC derived delta-endotoxins of...

  10. 40 CFR 174.511 - Bacillus thuringiensis Cry1Ab protein in all plants; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 25 2012-07-01 2012-07-01 false Bacillus thuringiensis Cry1Ab protein... Cry1Ab protein in all plants; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry1Ab protein in all plants are exempt from the requirement of a tolerance when used as...

  11. 40 CFR 174.510 - Bacillus thuringiensis Cry1Ac protein in all plants; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 25 2013-07-01 2013-07-01 false Bacillus thuringiensis Cry1Ac protein... Cry1Ac protein in all plants; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry1Ac protein in all plants are exempt from the requirement of a tolerance when used as...

  12. 40 CFR 174.510 - Bacillus thuringiensis Cry1Ac protein in all plants; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 24 2014-07-01 2014-07-01 false Bacillus thuringiensis Cry1Ac protein... Cry1Ac protein in all plants; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry1Ac protein in all plants are exempt from the requirement of a tolerance when used as...

  13. 40 CFR 174.511 - Bacillus thuringiensis Cry1Ab protein in all plants; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 24 2014-07-01 2014-07-01 false Bacillus thuringiensis Cry1Ab protein... Cry1Ab protein in all plants; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry1Ab protein in all plants are exempt from the requirement of a tolerance when used as...

  14. 40 CFR 174.510 - Bacillus thuringiensis Cry1Ac protein in all plants; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 25 2012-07-01 2012-07-01 false Bacillus thuringiensis Cry1Ac protein... Cry1Ac protein in all plants; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry1Ac protein in all plants are exempt from the requirement of a tolerance when used as...

  15. 40 CFR 174.511 - Bacillus thuringiensis Cry1Ab protein in all plants; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 25 2013-07-01 2013-07-01 false Bacillus thuringiensis Cry1Ab protein... Cry1Ab protein in all plants; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry1Ab protein in all plants are exempt from the requirement of a tolerance when used as...

  16. Larvicidal activity of Bacillus thuringiensis var. israelensis Cry11Aa toxin against Haemonchus contortus.

    PubMed

    DE Lara, Ana Paula DE Souza Stori; Lorenzon, Lucas Bigolin; Vianna, Ana Muñoz; Santos, Francisco Denis Souza; Pinto, Luciano Silva; Aires Berne, Maria Elisabeth; Leite, Fábio Pereira Leivas

    2016-10-01

    Effective control of gastrointestinal parasites is necessary in sheep production. The development of anthelmintics resistance is causing the available chemically based anthelmintics to become less effective. Biological control strategies present an alternative to this problem. In the current study, we tested the larvicidal effects of Bacillus thuringiensis var. israelensis Cry11Aa toxin against Haemonchus contortus larvae. Bacterial suspensions [2 × 108 colony-forming units (CFU) g-1 of the feces] of B. thuringiensis var. israelensis and recombinant Escherichia coli expressing Cry11Aa toxin were added to naturally H. contortus egg-contaminated feces. The larvae were quantified, and significant reductions of 62 and 81% (P < 0·001) were, respectively observed, compared with the control group. A 30 mL bacterial suspension (1 × 108 CFU mL-1) of B. thuringiensis var. israelensis and recombinant E. coli expressing Cry11Aa toxin were then orally administered to lambs naturally infected with H. contortus. Twelve hours after administration, feces were collected and submitted to coprocultures. Significant larvae reductions (P < 0·001) of 79 and 90% were observed respectively compared with the control group. The results suggest that the Cry11Aa toxin of B. thuringiensis var. israelensis is a promising new class of biological anthelmintics for treating sheep against H. contortus.

  17. Larvicidal activity of Bacillus thuringiensis var. israelensis Cry11Aa toxin against Haemonchus contortus.

    PubMed

    DE Lara, Ana Paula DE Souza Stori; Lorenzon, Lucas Bigolin; Vianna, Ana Muñoz; Santos, Francisco Denis Souza; Pinto, Luciano Silva; Aires Berne, Maria Elisabeth; Leite, Fábio Pereira Leivas

    2016-10-01

    Effective control of gastrointestinal parasites is necessary in sheep production. The development of anthelmintics resistance is causing the available chemically based anthelmintics to become less effective. Biological control strategies present an alternative to this problem. In the current study, we tested the larvicidal effects of Bacillus thuringiensis var. israelensis Cry11Aa toxin against Haemonchus contortus larvae. Bacterial suspensions [2 × 108 colony-forming units (CFU) g-1 of the feces] of B. thuringiensis var. israelensis and recombinant Escherichia coli expressing Cry11Aa toxin were added to naturally H. contortus egg-contaminated feces. The larvae were quantified, and significant reductions of 62 and 81% (P < 0·001) were, respectively observed, compared with the control group. A 30 mL bacterial suspension (1 × 108 CFU mL-1) of B. thuringiensis var. israelensis and recombinant E. coli expressing Cry11Aa toxin were then orally administered to lambs naturally infected with H. contortus. Twelve hours after administration, feces were collected and submitted to coprocultures. Significant larvae reductions (P < 0·001) of 79 and 90% were observed respectively compared with the control group. The results suggest that the Cry11Aa toxin of B. thuringiensis var. israelensis is a promising new class of biological anthelmintics for treating sheep against H. contortus. PMID:27573677

  18. Bacillus thuringiensis metalloproteinase Bmp1 functions as a nematicidal virulence factor.

    PubMed

    Luo, Xiaoxia; Chen, Ling; Huang, Qiong; Zheng, Jinshui; Zhou, Wei; Peng, Donghai; Ruan, Lifang; Sun, Ming

    2013-01-01

    Some Bacillus thuringiensis strains have high toxicity to nematodes. Nematicidal activity has been found in several families of crystal proteins, such as Cry5, Cry6, and Cry55. The B. thuringiensis strain YBT-1518 has three cry genes that have high nematicidal activity. The whole genome sequence of this strain contains multiple potential virulence factors. To evaluate the pathogenic potential of virulence factors, we focused on a metalloproteinase called Bmp1. It encompasses a consecutive N-terminal signal peptide, an FTP superfamily domain, an M4 neutral protease GluZincin superfamily, two Big-3 superfamily motifs, and a Gram-positive anchor superfamily motif as a C-terminal domain. Here, we showed that purified Bmp1 protein showed metalloproteinase activity and toxicity against Caenorhabditis elegans (the 50% lethal concentration is 610 ± 9.37 μg/ml). In addition, mixing Cry5Ba with Bmp1 protein enhanced the toxicity 7.9-fold (the expected toxicity of the two proteins calculated from their separate toxicities) against C. elegans. Confocal microscopic observation revealed that Bmp1 protein was detected from around the mouth and esophagus to the intestine. Striking microscopic images revealed that Bmp1 degrades intestine tissues, and the Cry5Ba causes intestinal shrinkage from the body wall. Thus, the B. thuringiensis Bmp1 metalloproteinase is a nematicidal virulence factor. These findings give a new insight into the relationship between B. thuringiensis and its host nematodes.

  19. Determination and Distribution of cry-Type Genes of Bacillus thuringiensis Isolates from Taiwan

    PubMed Central

    Chak, K.-F.; Chao, D.-C.; Tseng, M.-Y.; Kao, S.-S.; Tuan, S.-J.; Feng, T.-Y.

    1994-01-01

    Using PCR with a set of specific oligonucleotide primers to detect cryI-type genes, we were able to screen the cry-type genes of 225 Bacillus thuringiensis soil isolates from Taiwan without much cost in time or labor. Some combinations of cry genes (the cry-type profile) in a single isolate were unique. We identified five distinct profiles of crystal genes from the B. thuringiensis soil isolates from Taiwan. The cry genes included cryIA(a), cryIA(b), cryIA(c), cryIC, cryID, and cryIV. Interestingly, 501 B. thuringiensis isolates (93.5% of the total number that we identified) were isolated from areas at high altitudes. The profiles of cry-type genes were distinct in all isolation areas. The distribution of cry-type genes of our isolates therefore depended on geography. Using PCR footprinting to detect cryIC-type genes, we identified two distinct cryIC footprints from some of our isolates, indicating that these isolates may contain novel cryIC-type genes. B. thuringiensis isolates containing cryIA(a)-, cryIA(b)-, and cryIA(c)-type genes exhibited much greater activity against Plutella xylostella than did other isolates, indicating that multiple cry-type genes may be used as markers for the prediction of insecticidal activities. Images PMID:16349324

  20. Binding of Bacillus thuringiensis proteins to a laboratory-selected line of Heliothis virescens.

    PubMed

    MacIntosh, S C; Stone, T B; Jokerst, R S; Fuchs, R L

    1991-10-15

    A laboratory-selected colony of Heliothis virescens displaying a 20- to 70-fold level of resistance to Bacillus thuringiensis proteins was evaluated to identify mechanism(s) of resistance. Brush-border membrane vesicles were isolated from larval midgut epithelium from the susceptible and resistant strains of H. virescens. Two B. thuringiensis proteins, CryIA(b) and CryIA(c), were iodinated and shown to specifically bind to brush-border membrane vesicles of both insect strains. Multiple changes in the receptor-binding parameters were seen in the resistant strain as compared with the susceptible strain. A 2- to 4-fold reduction in binding affinity was accompanied by a 4- to 6-fold increase in binding-site concentration for both proteins. Although these two B. thuringiensis proteins competed for the same high-affinity binding site, competition experiments revealed different receptor specificity toward these proteins in the resistant H. virescens line. The H. virescens strains were not sensitive to a coleopteran-active protein, CryIIIA, nor did these proteins compete with the CryIA proteins for binding. Complexity of the mechanism of resistance is consistent with the complex mode of action of B. thuringiensis proteins. PMID:1924353

  1. Safety assessment of transgenic Bacillus thuringiensis with VIP insecticidal protein gene by feeding studies.

    PubMed

    Peng, Donghai; Chen, Shouwen; Ruan, Lifang; Li, Lin; Yu, Ziniu; Sun, Ming

    2007-07-01

    The aim of this study was to evaluate the toxicology safety of a genetically modified (GM) Bacillus thuringiensis with vegetative insecticidal protein (VIP) gene. Acute and subacute toxicity studies by using its powder preparation were conducted in Wistar rats. The result of the acute study showed the no-observable-adverse-effect level (NOAEL) of this GM B. thuringiensis powder preparation was greater than 5000 mg/kg body weight (BW). In the subacute study, the data analysis of body weight gain, food and water consumptions, clinical observations, haematology, serum biochemistry, organ weight ratios and histopathological findings did not show significant differences between control and treated groups. These results proved the NOAEL of this GM B. thuringiensis powder preparation in subacute test was greater than 5000 mg/kg BW. Since both the acute and subacute oral toxicity were not detected at the highest dose recommended by OECD guidelines, this GM B. thuringiensis could be generally regarded as safe for use in bio-pesticide industry.

  2. Diversity of thermal ecotypes and potential pathotypes of Bacillus thuringiensis soil isolates.

    PubMed

    Swiecicka, Izabela; Bartoszewicz, Marek; Kasulyte-Creasey, Daiva; Drewnowska, Justyna M; Murawska, Emilia; Yernazarova, Aliya; Lukaszuk, Edyta; Mahillon, Jacques

    2013-08-01

    Ecological diversification of Bacillus thuringiensis soil isolates was examined to determine whether bacteria adapted to grow at low temperature and/or potentially pathogenic correspond to genetically distinct lineages. Altogether, nine phylogenetic lineages were found among bacilli originating from North-Eastern Poland (n = 24) and Lithuania (n = 25) using multi-locus sequence typing. This clustering was chiefly confirmed by pulsed-field gel electrophoresis. One third of the bacilli were found to be psychrotolerant, which strongly supports the hypothesis of the existence of thermal ecotypes among B. thuringiensis. PCR screening was also performed to detect potential enterotoxin genes and Bacillus anthracis pXO1- and pXO2-like replicons. The cytK-positive isolates (22%) were significantly associated with two phylogenetic lineages (potential CytK pathotypes), whereas there was no correlation between phylogenetic grouping and the presence of the potential tripartite enterotoxin pathotypes (86% of strains). A statistically significant association between phylogenetic lineages and ecologic properties was found with regard to the cry1-positive Lithuanian isolates, while the cry genes in Polish isolates and the pXO1- and pXO2 replicon-like elements showed scattered distribution across phylogenetic lineages. Our results support the hypothesis that B. thuringiensis comprises strains belonging to different phylogenetic lineages, which exhibit specific ecological properties.

  3. Genes and environment interact to determine the fitness costs of resistance to Bacillus thuringiensis

    PubMed Central

    Raymond, Ben; Sayyed, Ali H; Wright, Denis J

    2005-01-01

    Genes which provide resistance to novel challenges such as pesticides, toxins or pathogens often impose fitness costs on individuals with a resistant phenotype. Studies of resistance to Bacillus thuringiensis and its insecticidal Cry toxins indicate that fitness costs may be variable and cryptic. Using two field populations (Karak and Serd4) of the diamondback moth, Plutella xylostella, we tested the hypothesis that the costs associated with resistance to the B. thuringiensis toxin Cry1Ac would be evident when insects were grown under poor environmental conditions, namely limited or poor quality resources. On a poor quality resource, a cultivar of Brassica oleracea var. capitata with varietal resistance to P. xylostella, only one resistant population, Karak, showed reduced fitness. Conversely, when we limited a high quality resource, Brassica pekinensis, by imposing larval competition, only resistant Serd4 insects had reduced survival at high larval densities. Furthermore, Cry1Ac resistance in Serd4 insects declined when reared at high larval densities while resistance at low densities fluctuated but did not decline significantly. These results confirm the hypothesis that resistance costs can appear under stressful conditions and demonstrate that the fitness cost of resistance to Bacillus thuringiensis can depend on the particular interaction between genes and the environment. PMID:16011928

  4. Protection from UV-B damage of mosquito larvicidal toxins from Bacillus thuringiensis subsp. israelensis expressed in Anabaena PCC 7120.

    PubMed

    Manasherob, Robert; Ben-Dov, Eitan; Xiaoqiang, Wu; Boussiba, Sammy; Zaritsky, Arieh

    2002-09-01

    A transgenic strain of the nitrogen-fixing filamentous cyanobacterium Anabaena PCC 7120 protected expressed delta-endotoxin proteins of Bacillus thuringiensis subsp. israelensis from damage inflicted by UV-B, a sunlight component that penetrates Earth's ozone layer. This organism, which serves as a food source to mosquito larvae and could multiply in their breeding sites, may solve the environment-imposed limitations of B. thuringiensis subsp. israelensis as a mosquito biological control agent.

  5. Sequencing and characterization of plasmid pUIBI-1 from Bacillus thuringiensis serovar entomocidus LBIT-113.

    PubMed

    López-Meza, Joel E; Barboza-Corona, J Eleazar; Del Rincón-Castro, M Cristina; Ibarra, Jorge E

    2003-11-01

    Plasmid pUIBI-1 from Bacillus thuringiensis svr. entomocidus was sequenced and its replication mechanism analyzed. Sequence analysis revealed that pUIBI-1 contains 4671 bp and a 32% GC content. Plasmid pUIBI-1 also includes at least seven putative open reading frames (ORFs) encoding for proteins ranging from 5 to 50 kDa. ORF-1 encodes for a putative 16-kDa Rep protein, which lacks homology with proteins of similar function. ORF2 encodes for a protein of 50 kDa and shows homology with Mob proteins of plasmids pLUB1000 from Lactobacillus hilgardii (32.2%) and pGI2 from B. thuringiensis (33.7%). Detection of single-stranded DNA (ssDNA) intermediates indicated that pUIBI-1 replicates by the rolling-circle replication mechanism, as demonstrated by S1 treatment and Southern hybridization under non-denaturing conditions. PMID:14669916

  6. Are nematodes a missing link in the confounded ecology of the entomopathogen Bacillus thuringiensis?

    PubMed

    Ruan, Lifang; Crickmore, Neil; Peng, Donghai; Sun, Ming

    2015-06-01

    Bacillus thuringiensis, which is well known as an entomopathogen, has been accepted by the public as a safe bioinsecticide. The natural ecology of this bacterium has never been particularly clear, with views ranging from it being an obligate pathogen to an opportunist pathogen that can otherwise exist as a soil saprophyte or a plant endophyte. This confusion has recently led to it being considered as an environmental pathogen that has evolved to occupy a diverse set of environmental niches in which it can thrive without needing a host. A significant driving force behind this classification is the fact that B. thuringiensis is found in high numbers in environments that are not occupied by the insect hosts to which it is pathogenic. It is our opinion that the ubiquitous presence of this bacterium in the environment is the result of a variety of vectoring systems, particularly those that include nematodes.

  7. Binary toxins from Bacillus thuringiensis active against the western corn rootworm, Diabrotica virgifera virgifera LeConte.

    PubMed

    Baum, James A; Chu, Chi-Rei; Rupar, Mark; Brown, Gregory R; Donovan, William P; Huesing, Joseph E; Ilagan, Oliver; Malvar, Thomas M; Pleau, Michael; Walters, Matthew; Vaughn, Ty

    2004-08-01

    The western corn rootworm, Diabrotica virgifera virgifera LeConte, is a significant pest of corn in the United States. The development of transgenic corn hybrids resistant to rootworm feeding damage depends on the identification of genes encoding insecticidal proteins toxic to rootworm larvae. In this study, a bioassay screen was used to identify several isolates of the bacterium Bacillus thuringiensis active against rootworm. These bacterial isolates each produce distinct crystal proteins with approximate molecular masses of 13 to 15 kDa and 44 kDa. Insect bioassays demonstrated that both protein classes are required for insecticidal activity against this rootworm species. The genes encoding these proteins are organized in apparent operons and are associated with other genes encoding crystal proteins of unknown function. The antirootworm proteins produced by B. thuringiensis strains EG5899 and EG9444 closely resemble previously described crystal proteins of the Cry34A and Cry35A classes. The antirootworm proteins produced by strain EG4851, designated Cry34Ba1 and Cry35Ba1, represent a new binary toxin. Genes encoding these proteins could become an important component of a sustainable resistance management strategy against this insect pest.

  8. Field-evolved resistance by western corn rootworm to multiple Bacillus thuringiensis toxins in transgenic maize.

    PubMed

    Gassmann, Aaron J; Petzold-Maxwell, Jennifer L; Clifton, Eric H; Dunbar, Mike W; Hoffmann, Amanda M; Ingber, David A; Keweshan, Ryan S

    2014-04-01

    The widespread planting of crops genetically engineered to produce insecticidal toxins derived from the bacterium Bacillus thuringiensis (Bt) places intense selective pressure on pest populations to evolve resistance. Western corn rootworm is a key pest of maize, and in continuous maize fields it is often managed through planting of Bt maize. During 2009 and 2010, fields were identified in Iowa in which western corn rootworm imposed severe injury to maize producing Bt toxin Cry3Bb1. Subsequent bioassays revealed Cry3Bb1 resistance in these populations. Here, we report that, during 2011, injury to Bt maize in the field expanded to include mCry3A maize in addition to Cry3Bb1 maize and that laboratory analysis of western corn rootworm from these fields found resistance to Cry3Bb1 and mCry3A and cross-resistance between these toxins. Resistance to Bt maize has persisted in Iowa, with both the number of Bt fields identified with severe root injury and the ability western corn rootworm populations to survive on Cry3Bb1 maize increasing between 2009 and 2011. Additionally, Bt maize targeting western corn rootworm does not produce a high dose of Bt toxin, and the magnitude of resistance associated with feeding injury was less than that seen in a high-dose Bt crop. These first cases of resistance by western corn rootworm highlight the vulnerability of Bt maize to further evolution of resistance from this pest and, more broadly, point to the potential of insects to develop resistance rapidly when Bt crops do not achieve a high dose of Bt toxin.

  9. Field-evolved resistance by western corn rootworm to multiple Bacillus thuringiensis toxins in transgenic maize

    PubMed Central

    Gassmann, Aaron J.; Petzold-Maxwell, Jennifer L.; Clifton, Eric H.; Dunbar, Mike W.; Hoffmann, Amanda M.; Ingber, David A.; Keweshan, Ryan S.

    2014-01-01

    The widespread planting of crops genetically engineered to produce insecticidal toxins derived from the bacterium Bacillus thuringiensis (Bt) places intense selective pressure on pest populations to evolve resistance. Western corn rootworm is a key pest of maize, and in continuous maize fields it is often managed through planting of Bt maize. During 2009 and 2010, fields were identified in Iowa in which western corn rootworm imposed severe injury to maize producing Bt toxin Cry3Bb1. Subsequent bioassays revealed Cry3Bb1 resistance in these populations. Here, we report that, during 2011, injury to Bt maize in the field expanded to include mCry3A maize in addition to Cry3Bb1 maize and that laboratory analysis of western corn rootworm from these fields found resistance to Cry3Bb1 and mCry3A and cross-resistance between these toxins. Resistance to Bt maize has persisted in Iowa, with both the number of Bt fields identified with severe root injury and the ability western corn rootworm populations to survive on Cry3Bb1 maize increasing between 2009 and 2011. Additionally, Bt maize targeting western corn rootworm does not produce a high dose of Bt toxin, and the magnitude of resistance associated with feeding injury was less than that seen in a high-dose Bt crop. These first cases of resistance by western corn rootworm highlight the vulnerability of Bt maize to further evolution of resistance from this pest and, more broadly, point to the potential of insects to develop resistance rapidly when Bt crops do not achieve a high dose of Bt toxin. PMID:24639498

  10. Online monitoring of Escherichia coli and Bacillus thuringiensis spore inactivation after advanced oxidation treatment.

    PubMed

    Sherchan, Samendra P; Snyder, Shane A; Gerba, Charles P; Pepper, Ian L

    2014-01-01

    Various studies have shown that advanced oxidation processes (AOPs) such as UV light in combination with hydrogen peroxide is an efficient process for the removal of a large variety of emerging contaminants including microorganisms. The mechanism of destruction in the presence of hydrogen peroxide (H2O2) is the enhanced formation of hydroxyl (·OH) radicals, which have a high oxidation potential. The goal of this study was to utilize in-line advanced oxidation to inactivate microbes, and document the inactivation via an in-line, real-time sensor. Escherichia coli cells and Bacillus thuringiensis spores were exposed to UV/H2O2 treatment in DI water, and the online sensor BioSentry(®) was evaluated for its potential to monitor inactivation in real-time. B. thuringiensis was selected as a non-pathogenic surrogate for B. anthracis, the causative agent of anthrax and a proven biological weapon. UV radiation and UV/H2O2 exposure resulted in a >6 log10 reduction of the viable culturable counts of E. coli vegetative cells, and a 3 log10 reduction of B. thuringiensis spores. Scanning electron microscopy of the treated samples revealed severe damage on the surface of most E. coli cells, yet there was no significant change observed in the morphology of the B. thuringiensis spores. Following AOP exposure, the BioSentry sensor showed an increase in the categories of unknown, rod and spores counts, but overall, did not correspond well with viable count assays. Data from this study show that advanced oxidation processes effectively inactivate E. coli vegetative cells, but not B. thuringiensis spores, which were more resistant to AOP. Further, the BioSentry in-line sensor was not successful in documenting destruction of the microbial cells in real-time.

  11. Identification of distinct Bacillus thuringiensis 4A4 nematicidal factors using the model nematodes Pristionchus pacificus and Caenorhabditis elegans.

    PubMed

    Iatsenko, Igor; Nikolov, Angel; Sommer, Ralf J

    2014-07-14

    Bacillus thuringiensis has been extensively used for the biological control of insect pests. Nematicidal B. thuringiensis strains have also been identified; however, virulence factors of such strains are poorly investigated. Here, we describe virulence factors of the nematicidal B. thuringiensis 4A4 strain, using the model nematodes Pristionchus pacificus and Caenorhabditis elegans. We show that B. thuringiensis 4A4 kills both nematodes via intestinal damage. Whole genome sequencing of B. thuringiensis 4A4 identified Cry21Ha, Cry1Ba, Vip1/Vip2 and β-exotoxin as potential nematicidal factors. Only Cry21Ha showed toxicity to C. elegans, while neither Cry nor Vip toxins were active against P. pacificus, when expressed in E. coli. Purified crystals also failed to intoxicate P. pacificus, while autoclaved spore-crystal mixture of B. thuringiensis 4A4 retained toxicity, suggesting that primary β-exotoxin is responsible for P. pacificus killing. In support of this, we found that a β-exotoxin-deficient variant of B. thuringiensis 4A4, generated by plasmid curing lost virulence to the nematodes. Thus, using two model nematodes we revealed virulence factors of the nematicidal strain B. thuringiensis 4A4 and showed the multifactorial nature of its virulence.

  12. Functional analysis of the sporulation-specific diadenylate cyclase CdaS in Bacillus thuringiensis

    PubMed Central

    Zheng, Cao; Ma, Yang; Wang, Xun; Xie, Yuqun; Ali, Maria K.; He, Jin

    2015-01-01

    Cyclic di-AMP (c-di-AMP) is a recently discovered bacterial secondary messenger molecule, which is associated with various physiological functions. In the genus Bacillus, the intracellular level and turnover of c-di-AMP are mainly regulated by three diadenylate cyclases (DACs), including DisA, CdaA and CdaS, and two c-di-AMP-specific phosphodiesterases (GdpP and PgpH). In this study, we demonstrated that CdaS protein from B. thuringiensis is a hexameric DAC protein that can convert ATP or ADP to c-di-AMP in vitro and the N-terminal YojJ domain is essential for the DAC activity. Based on the markerless gene knock-out method, we demonstrated that the transcription of cdaS was initiated by the sporulation-specific sigma factor σH and the deletion of cdaS significantly delayed sporulation and parasporal crystal formation. These findings contrast with similar experiments conducted using B. subtilis, wherein transcription of its cdaS was initiated by the sigma factor σG. Deletion of all the three DAC genes from a single strain was unsuccessful, suggesting that c-di-AMP is an indispensable molecule in B. thuringiensis. Phylogenetic analysis indicated increased diversity of CdaS in the B. cereus and B. subtilis Bacillus subgroups. In summary, this study identifies important aspects in the regulation of c-di-AMP in the genus Bacillus. PMID:26441857

  13. Resistance of Trichoplusia ni Populations Selected by Bacillus thuringiensis Sprays to Cotton Plants Expressing Pyramided Bacillus thuringiensis Toxins Cry1Ac and Cry2Ab

    PubMed Central

    Kain, Wendy; Song, Xiaozhao; Janmaat, Alida F.; Zhao, Jian-Zhou; Myers, Judith; Shelton, Anthony M.

    2014-01-01

    Two populations of Trichoplusia ni that had developed resistance to Bacillus thuringiensis sprays (Bt sprays) in commercial greenhouse vegetable production were tested for resistance to Bt cotton (BollGard II) plants expressing pyramided Cry1Ac and Cry2Ab. The T. ni colonies resistant to Bacillus thuringiensis serovar kurstaki formulations were not only resistant to the Bt toxin Cry1Ac, as previously reported, but also had a high frequency of Cry2Ab-resistant alleles, exhibiting ca. 20% survival on BollGard II foliage. BollGard II-resistant T. ni strains were established by selection with BollGard II foliage to further remove Cry2Ab-sensitive alleles in the T. ni populations. The BollGard II-resistant strains showed incomplete resistance to BollGard II, with adjusted survival values of 0.50 to 0.78 after 7 days. The resistance to the dual-toxin cotton plants was conferred by two genetically independent resistance mechanisms: one to Cry1Ac and one to Cry2Ab. The 50% lethal concentration of Cry2Ab for the resistant strain was at least 1,467-fold that for the susceptible T. ni strain. The resistance to Cry2Ab in resistant T. ni was an autosomally inherited, incompletely recessive monogenic trait. Results from this study indicate that insect populations under selection by Bt sprays in agriculture can be resistant to multiple Bt toxins and may potentially confer resistance to multitoxin Bt crops. PMID:25480752

  14. Bio-insecticide Bacillus thuringiensis spores encapsulated with amaranth derivatized starches: studies on the propagation "in vitro".

    PubMed

    Rodríguez, Ana Priscila García; Martínez, Marcela Gaytán; Barrera-Cortés, Josefina; Ibarra, Jorge E; Bustos, Fernando Martínez

    2015-02-01

    Bacillus thuringiensis (Bt) is one of the bioinsecticides used worldwide due to its specific toxicity against target pests in their larval stage. Despite this advantage, its use is limited because of their short persistence in field when exposed to ultra violet light and changing environmental conditions. In this work, microencapsulation has been evaluated as a promising method to improve Bt activity. The objective of this study was to develop and characterize native and modified amaranth starch granules and evaluate their potential application as wall materials in the microcapsulation of B thuringiensis serovar kurstaki HD-1 (Bt- HD1), produced by spray drying. Native amaranth starch granules were treated by hydrolyzation, high energy milling (HEM) and were chemically modified by phosphorylation and succinylation. The size of the Bt microcapsules varied from 12.99 to 17.14 μm adequate to protect the spores of Bt from ultraviolet radiation. The aw coefficient of the microcapsules produced by the modified starches after drying was low (0.14-1.88), which prevent microbial growth. Microcapsules prepared with phosphorylated amaranth starch presented the highest bacterial count and active material yield. Different concentrations of the encapsulated Bt formulation in phosphorylated amaranth starch showed a high level of insecticidal activity when tested on M. sexta larvae and has great potential to be developed as a bioinsecticide formulation, also, the level of toxicity is much higher than that found in some of the products commercially available. PMID:25168123

  15. The crystal proteins from Bacillus thuringiensis subsp. thompsoni display a synergistic activity against the codling moth, Cydia pomonella.

    PubMed

    Rang, C; Lacey, L A; Frutos, R

    2000-03-01

    Crystal proteins from Bacillus thuringiensis subsp. thompsoni strain HnC are active against the codling moth, Cydia pomonella, a major pest of orchards. Inclusion bodies purified from strain HnC displayed an LC(50) of 3.34 x 10(-3) microgram/microliter. HnC-purified crystals were tenfold more active than Cry2Aa and Cry1Aa toxins, and 100-fold more toxic than Cry1Ab. The 34-kDa and 40-kDa proteins contained in HnC inclusion bodies were shown to act synergistically. The toxicity of crystal proteins produced by the recombinant B. thuringiensis strain BT-OP expressing the full-length native operon was about tenfold higher than that of the 34-kDa protein. When the gene encoding the non-insecticidal 40-kDa protein, which is not active, was introduced into the recombinant strain producing only the 34-kDa protein, the toxicity was raised tenfold and was similar to that of the strain BT-OP.

  16. Bio-insecticide Bacillus thuringiensis spores encapsulated with amaranth derivatized starches: studies on the propagation "in vitro".

    PubMed

    Rodríguez, Ana Priscila García; Martínez, Marcela Gaytán; Barrera-Cortés, Josefina; Ibarra, Jorge E; Bustos, Fernando Martínez

    2015-02-01

    Bacillus thuringiensis (Bt) is one of the bioinsecticides used worldwide due to its specific toxicity against target pests in their larval stage. Despite this advantage, its use is limited because of their short persistence in field when exposed to ultra violet light and changing environmental conditions. In this work, microencapsulation has been evaluated as a promising method to improve Bt activity. The objective of this study was to develop and characterize native and modified amaranth starch granules and evaluate their potential application as wall materials in the microcapsulation of B thuringiensis serovar kurstaki HD-1 (Bt- HD1), produced by spray drying. Native amaranth starch granules were treated by hydrolyzation, high energy milling (HEM) and were chemically modified by phosphorylation and succinylation. The size of the Bt microcapsules varied from 12.99 to 17.14 μm adequate to protect the spores of Bt from ultraviolet radiation. The aw coefficient of the microcapsules produced by the modified starches after drying was low (0.14-1.88), which prevent microbial growth. Microcapsules prepared with phosphorylated amaranth starch presented the highest bacterial count and active material yield. Different concentrations of the encapsulated Bt formulation in phosphorylated amaranth starch showed a high level of insecticidal activity when tested on M. sexta larvae and has great potential to be developed as a bioinsecticide formulation, also, the level of toxicity is much higher than that found in some of the products commercially available.

  17. Toxicity studies for indigenous Bacillus thuringiensis isolates from Malang city, East Java on Aedes aegypti larvae

    PubMed Central

    Gama, Zulfaidah Penata; Nakagoshi, Nobukazu; Suharjono; Setyowati, Faridah

    2013-01-01

    Objective To investigate the toxicity of indigenous Bacillus thuringiensis (B. thuringiensis)isolates from Malang City for controlling Aedes aegypti (Ae. aegypti) larvae. Methods Soil samples were taken from Purwantoro and Sawojajar sub-districts. Bacterial isolation was performed using B. thuringiensis selective media. Phenotypic characteristics of the isolates were obtained with the simple matching method. The growth and prevalence of spores were determined by the Total Plate Count method, and toxicity tests were also performed on the third instar larval stage of Ae. aegypti. The percentage of larval mortality was analysed using probit regression. The LC50 was analysed by ANOVA, and the Tukey HSD interval was 95%. Results Among the 33 selected bacterial isolates, six were obtained (PWR4-31, PWR4-32, SWJ4-2b, SWJ4-4b, SWJ-4k and SWJ5-1) that had a similar phenotype to reference B. thuringiensis. Based on the dendrogram, all of the bacterial isolates were 71% similar. Three isolates that had a higher prevalence of reference B. thuringiensis were PWR4-32, SWJ4-4b and SW5-1, of which the spore prevalence was 52.44%, 23.59%, 34.46%, respectively. These three indigenous isolates from Malang City successfully killed Ae. aegypti larvae. The PWR4-32 isolates were the most effective at killing the larvae. Conclusions Six indigenous B. thuringiensis isolates among the 33 bacterial isolates found in the Sawojajar and Purwantoro sub-districts were toxic to the third instar larvae of Ae. aegypti. The PWR4-32 isolates were identical to the reference B. thuringiensis and had 88% phenotype similarity. The PWR4-32 isolates had the highest spore prevalence (52.44%), and the early stationary phase occurred at 36 h. The PWR4-32 isolates were the most effective at killing Ae. aegypti larvae (LC50-72 h=2.3×108 cells/mL). PMID:23593589

  18. Effects of pink bollworm resistance to Bacillus thuringiensis on phenoloxidase activity and susceptibility to entomopathogenic nematodes.

    PubMed

    Gassmann, Aaron J; Fabrick, Jeffrey A; Sisterson, Mark S; Hannon, Eugene R; Stock, S Patricia; Carrière, Yves; Tabashnik, Bruce E

    2009-06-01

    Widespread planting of crops genetically engineered to produce insecticidal toxins from the bacterium Bacillus thuringiensis (Bt) imposes selection on many key agricultural pests to evolve resistance to Bt. Fitness costs can slow the evolution of Bt resistance. We examined effects of entomopathogenic nematodes on fitness costs of Bt resistance in the pink bollworm, Pectinophora gossypiella (Saunders) (Lepidoptera: Gelechiidae), a major pest of cotton, Gossypium hirsutum L., in the southwestern United States that is currently controlled by transgenic cotton that produces Bt toxin Cry1Ac. We tested whether the entomopathogenic nematodes Steinernema riobrave Cabanillas, Poinar, and Raulston (Rhabditida: Steinernematidae) and Heterorhabditis bacteriophora Poinar (Rhabditida: Heterorhabditidae) affected fitness costs of resistance to Cry1Ac in two laboratory-selected hybrid strains of pink bollworm reared on non-Bt cotton bolls. The nematode S. riobrave imposed a recessive fitness cost for one strain, and H. bacteriophora imposed a fitness cost affecting heterozygous resistant individuals for the other strain. Activity of phenoloxidase, an important component of insects' immune response, did not differ between Bt-resistant and Bt-susceptible families. This suggests phenoloxidase does not affect susceptibility to entomopathogenic nematodes in Bt-resistant pink bollworm. Additionally, phenoloxidase activity does not contribute to Bt resistance, as has been found in some species. We conclude that other mechanisms cause higher nematode-imposed mortality for pink bollworm with Bt resistance genes. Incorporation of nematode-imposed fitness costs into a spatially explicit simulation model suggests that entomopathogenic nematodes in non-Bt refuges could delay resistance by pink bollworm to Bt cotton.

  19. Purification and Characterization of a Novel Cold Shock Protein-Like Bacteriocin Synthesized by Bacillus thuringiensis

    PubMed Central

    Huang, Tianpei; Zhang, Xiaojuan; Pan, Jieru; Su, Xiaoyu; Jin, Xin; Guan, Xiong

    2016-01-01

    Bacillus thuringiensis (Bt), one of the most successful biopesticides, may expand its potential by producing bacteriocins (thuricins). The aim of this study was to investigate the antimicrobial potential of a novel Bt bacteriocin, thuricin BtCspB, produced by Bt BRC-ZYR2. The results showed that this bacteriocin has a high similarity with cold-shock protein B (CspB). BtCspB lost its activity after proteinase K treatment; however it was active at 60 °C for 30 min and was stable in the pH range 5–7. The partial loss of activity after the treatments of lipase II and catalase were likely due to the change in BtCspB structure and the partial degradation of BtCspB, respectively. The loss of activity at high temperatures and the activity variation at different pHs were not due to degradation or large conformational change. BtCspB did not inhibit four probiotics. It was only active against B. cereus strains 0938 and ATCC 10987 with MIC values of 3.125 μg/mL and 0.781 μg/mL, and MBC values of 12.5 μg/mL and 6.25 μg/mL, respectively. Taken together, these results provide new insights into a novel cold shock protein-like bacteriocin, BtCspB, which displayed promise for its use in food preservation and treatment of B. cereus-associated diseases. PMID:27762322

  20. Broad-spectrum resistance to Bacillus thuringiensis toxins by western corn rootworm (Diabrotica virgifera virgifera)

    PubMed Central

    Jakka, Siva R. K.; Shrestha, Ram B.; Gassmann, Aaron J.

    2016-01-01

    The evolution of resistance and cross-resistance threaten the sustainability of genetically engineered crops that produce insecticidal toxins derived from the bacterium Bacillus thuringiensis (Bt). Western corn rootworm, Diabrotica virgifera virgifera LeConte, is a serious pest of maize and has been managed with Bt maize since 2003. We conducted laboratory bioassays with maize hybrids producing Bt toxins Cry3Bb1, mCry3A, eCry3.1Ab, and Cry34/35Ab1, which represent all commercialized Bt toxins for management of western corn rootworm. We tested populations from fields where severe injury to Cry3Bb1 maize was observed, and populations that had never been exposed to Bt maize. Consistent with past studies, bioassays indicated that field populations were resistant to Cry3Bb1 maize and mCry3A maize, and that cross-resistance was present between these two types of Bt maize. Additionally, bioassays revealed resistance to eCry3.1Ab maize and cross-resistance among Cry3Bb1, mCry3A and eCry3.1Ab. However, no resistance or cross-resistance was detected for Cry34/35Ab1 maize. This broad-spectrum resistance illustrates the potential for insect pests to develop resistance rapidly to multiple Bt toxins when structural similarities are present among toxins, and raises concerns about the long-term durability of Bt crops for management of some insect pests. PMID:27297953

  1. Broad-spectrum resistance to Bacillus thuringiensis toxins by western corn rootworm (Diabrotica virgifera virgifera).

    PubMed

    Jakka, Siva R K; Shrestha, Ram B; Gassmann, Aaron J

    2016-01-01

    The evolution of resistance and cross-resistance threaten the sustainability of genetically engineered crops that produce insecticidal toxins derived from the bacterium Bacillus thuringiensis (Bt). Western corn rootworm, Diabrotica virgifera virgifera LeConte, is a serious pest of maize and has been managed with Bt maize since 2003. We conducted laboratory bioassays with maize hybrids producing Bt toxins Cry3Bb1, mCry3A, eCry3.1Ab, and Cry34/35Ab1, which represent all commercialized Bt toxins for management of western corn rootworm. We tested populations from fields where severe injury to Cry3Bb1 maize was observed, and populations that had never been exposed to Bt maize. Consistent with past studies, bioassays indicated that field populations were resistant to Cry3Bb1 maize and mCry3A maize, and that cross-resistance was present between these two types of Bt maize. Additionally, bioassays revealed resistance to eCry3.1Ab maize and cross-resistance among Cry3Bb1, mCry3A and eCry3.1Ab. However, no resistance or cross-resistance was detected for Cry34/35Ab1 maize. This broad-spectrum resistance illustrates the potential for insect pests to develop resistance rapidly to multiple Bt toxins when structural similarities are present among toxins, and raises concerns about the long-term durability of Bt crops for management of some insect pests. PMID:27297953

  2. gyrB as a phylogenetic discriminator for members of the Bacillus anthracis-cereus-thuringiensis group

    NASA Technical Reports Server (NTRS)

    La Duc, Myron T.; Satomi, Masataka; Agata, Norio; Venkateswaran, Kasthuri

    2004-01-01

    Bacillus anthracis, the causative agent of the human disease anthrax, Bacillus cereus, a food-borne pathogen capable of causing human illness, and Bacillus thuringiensis, a well-characterized insecticidal toxin producer, all cluster together within a very tight clade (B. cereus group) phylogenetically and are indistinguishable from one another via 16S rDNA sequence analysis. As new pathogens are continually emerging, it is imperative to devise a system capable of rapidly and accurately differentiating closely related, yet phenotypically distinct species. Although the gyrB gene has proven useful in discriminating closely related species, its sequence analysis has not yet been validated by DNA:DNA hybridization, the taxonomically accepted "gold standard". We phylogenetically characterized the gyrB sequences of various species and serotypes encompassed in the "B. cereus group," including lab strains and environmental isolates. Results were compared to those obtained from analyses of phenotypic characteristics, 16S rDNA sequence, DNA:DNA hybridization, and virulence factors. The gyrB gene proved more highly differential than 16S, while, at the same time, as analytical as costly and laborious DNA:DNA hybridization techniques in differentiating species within the B. cereus group.

  3. Influence of lysogeny of Tectiviruses GIL01 and GIL16 on Bacillus thuringiensis growth, biofilm formation, and swarming motility.

    PubMed

    Gillis, Annika; Mahillon, Jacques

    2014-12-01

    Bacillus thuringiensis is an entomopathogenic bacterium that has been used as an efficient biopesticide worldwide. Despite the fact that this bacterium is usually described as an insect pathogen, its life cycle in the environment is still largely unknown. B. thuringiensis belongs to the Bacillus cereus group of bacteria, which has been associated with many mobile genetic elements, such as species-specific temperate or virulent bacteriophages (phages). Temperate (lysogenic) phages are able to establish a long-term relationship with their host, providing, in some cases, novel ecological traits to the bacterial lysogens. Therefore, this work focuses on evaluating the potential influence of temperate tectiviruses GIL01 and GIL16 on the development of different life traits of B. thuringiensis. For this purpose, a B. thuringiensis serovar israelensis plasmid-cured (nonlysogenic) strain was used to establish bacterial lysogens for phages GIL01 and GIL16, and, subsequently, the following life traits were compared among the strains: kinetics of growth, metabolic profiles, antibiotics susceptibility, biofilm formation, swarming motility, and sporulation. The results revealed that GIL01 and GIL16 lysogeny has a significant influence on the bacterial growth, sporulation rate, biofilm formation, and swarming motility of B. thuringiensis. No changes in metabolic profiles or antibiotic susceptibilities were detected. These findings provide evidence that tectiviruses have a putative role in the B. thuringiensis life cycle as adapters of life traits with ecological advantages.

  4. Influence of lysogeny of Tectiviruses GIL01 and GIL16 on Bacillus thuringiensis growth, biofilm formation, and swarming motility.

    PubMed

    Gillis, Annika; Mahillon, Jacques

    2014-12-01

    Bacillus thuringiensis is an entomopathogenic bacterium that has been used as an efficient biopesticide worldwide. Despite the fact that this bacterium is usually described as an insect pathogen, its life cycle in the environment is still largely unknown. B. thuringiensis belongs to the Bacillus cereus group of bacteria, which has been associated with many mobile genetic elements, such as species-specific temperate or virulent bacteriophages (phages). Temperate (lysogenic) phages are able to establish a long-term relationship with their host, providing, in some cases, novel ecological traits to the bacterial lysogens. Therefore, this work focuses on evaluating the potential influence of temperate tectiviruses GIL01 and GIL16 on the development of different life traits of B. thuringiensis. For this purpose, a B. thuringiensis serovar israelensis plasmid-cured (nonlysogenic) strain was used to establish bacterial lysogens for phages GIL01 and GIL16, and, subsequently, the following life traits were compared among the strains: kinetics of growth, metabolic profiles, antibiotics susceptibility, biofilm formation, swarming motility, and sporulation. The results revealed that GIL01 and GIL16 lysogeny has a significant influence on the bacterial growth, sporulation rate, biofilm formation, and swarming motility of B. thuringiensis. No changes in metabolic profiles or antibiotic susceptibilities were detected. These findings provide evidence that tectiviruses have a putative role in the B. thuringiensis life cycle as adapters of life traits with ecological advantages. PMID:25261525

  5. Bacillus thuringiensis subsp. israelensis Cyt1Aa synergizes Cry11Aa toxin by functioning as a membrane-bound receptor.

    PubMed

    Pérez, Claudia; Fernandez, Luisa E; Sun, Jianguang; Folch, Jorge Luis; Gill, Sarjeet S; Soberón, Mario; Bravo, Alejandra

    2005-12-20

    Bacillus thuringiensis subsp. israelensis produces crystal proteins, Cry (4Aa, 4Ba, 10Aa, and 11Aa) and Cyt (1Aa and 2Ba) proteins, toxic to mosquito vectors of human diseases. Cyt1Aa overcomes insect resistance to Cry11Aa and Cry4 toxins and synergizes the toxicity of these toxins. However, the molecular mechanism of synergism remains unsolved. Here, we provide evidence that Cyt1Aa functions as a receptor of Cry11Aa. Sequential-binding analysis of Cyt1Aa and Cry11Aa revealed that Cyt1Aa binding to Aedes aegypti brush border membrane vesicles enhanced the binding of biotinylated-Cry11Aa. The Cyt1Aa- and Cry11Aa-binding epitopes were mapped by means of the yeast two-hybrid system, peptide arrays, and heterologous competition assays with synthetic peptides. Two exposed regions in Cyt1Aa, loop beta6-alphaE and part of beta7, bind Cry11Aa. On the other side, Cry11Aa binds Cyt1Aa proteins by means of domain II-loop alpha8 and beta-4, which are also involved in midgut receptor interaction. Characterization of single-point mutations in Cry11Aa and Cyt1Aa revealed key Cry11Aa (S259 and E266) and Cyt1Aa (K198, E204 and K225) residues involved in the interaction of both proteins and in synergism. Additionally, a Cyt1Aa loop beta6-alphaE mutant (K198A) with enhanced synergism to Cry11Aa was isolated. Data provided here strongly indicates that Cyt1Aa synergizes or suppresses resistance to Cry11Aa toxin by functioning as a membrane-bound receptor. Bacillus thuringiensis subsp. israelensis is a highly effective pathogenic bacterium because it produces a toxin and also its functional receptor, promoting toxin binding to the target membrane and causing toxicity. PMID:16339907

  6. Pathway and kinetics of cyhalothrin biodegradation by Bacillus thuringiensis strain ZS-19.

    PubMed

    Chen, Shaohua; Deng, Yinyue; Chang, Changqing; Lee, Jasmine; Cheng, Yingying; Cui, Zining; Zhou, Jianuan; He, Fei; Hu, Meiying; Zhang, Lian-Hui

    2015-03-05

    Cyhalothrin is a common environmental pollutant which poses increased risks to non-target organisms including human beings. This study reported for the first time a newly isolated strain, Bacillus thuringiensis ZS-19 completely degraded cyhalothrin in minimal medium within 72 h. The bacterium transformed cyhalothrin by cleavage of both the ester linkage and diaryl bond to yield six intermediate products. Moreover, a novel degradation pathway of cyhalothrin in strain ZS-19 was proposed on the basis of the identified metabolites. In addition to degradation of cyhalothrin, this strain was found to be capable of degrading 3-phenoxybenzoic acid, a common metabolite of pyrethroids. Furthermore, strain ZS-19 participated in efficient degradation of a wide range of pyrethroids including cyhalothrin, fenpropathrinn, deltamethrin, beta-cypermethrin, cyfluthrin and bifenthrin. Taken together, our results provide insights into the mechanism of cyhalothrin degradation and also highlight the promising potentials of B.thuringiensis ZS-19 in bioremediation of pyrethroid-contaminated environment. This is the first report of (i) degradation of cyhalothrin and other pyrethroids by B.thuringiensis, (ii) identification of 3-phenoxyphenyl acetonitrile and N-(2-isoproxy-phenyl)-4-phenoxy-benzamide as the metabolites in the degradation pathway of pyrethroids, and (iii) a pathway of degradation of cyhalothrin by cleavage of both the ester linkage and diaryl bond in a microorganism.

  7. pXO16 from Bacillus thuringiensis serovar israelensis: Almost 350 kb of terra incognita.

    PubMed

    Makart, Lionel; Gillis, Annika; Mahillon, Jacques

    2015-07-01

    Bacillus thuringiensis strains usually harbor large sets of plasmids, some of which carrying the entomopathogenic δ-endotoxins. B. thuringiensis serovar israelensis, active on Dipteran larvae, carries the very large conjugative plasmid pXO16 (350 kb). pXO16 displays a macroscopic aggregation phenotype when liquid cultures of conjugative partners are mixed. Its conjugative apparatus is able of transferring itself and other non-conjugative and non-mobilizable plasmids in a fast and very efficient manner. Even though its conjugative kinetics and capabilities have been extensively studied, the genetic bases for this unique transfer system remain largely unknown. In this work, the sequence of pXO16 has been identified in the existing sequenced genome of B. thuringiensis sv. israelensis HD-789 as corresponding to the p01 plasmid. Despite pXO16 sequence being highly coding, few CDS possess homologs in the databases. However, potential regions responsible for the aggregation phenotype and the plasmid replication have been highlighted. The common orientation of all CDS and the presence of a high number of potential paralogs suggested a phage-like nature. Concerning conjugative functions, no significant type IV secretion system homologs have been found, indicating that pXO16 encodes an unforeseen conjugative system.

  8. Susceptibility of Agrotis segetum (noctuidae) to Bacillus thuringiensis and analysis of midgut proteinases.

    PubMed

    Ben Hamadou-Charfi, Dorra; Sauer, Annette Juliane; Abdelkefi-Mesrati, Lobna; Tounsi, Slim; Jaoua, Samir; Stephan, Dietrich

    2015-01-01

    Seventy-eight Bacillus thuringiensis isolates were selected for a screening against the Lepidoptera species Agrotis segetum to search the higher insecticidal activity. In a preliminary bioassay, the spore-crystal mixture of 78 B. thuringiensis isolates was tested against L1 larvae of A. segetum. Fifty-two isolates had more than 60% corrected mortality after 3 days. Seven isolates caused a corrected mortality of 100% on A. segetum. Twelve isolates were selected for a second bioassay investigating the effect of the vegetative insecticidal protein (Vip) against third-instar larvae. After 7 days, the weight gain and the larval stage of each larva were recorded. This bioassay showed an aberration in larval growth increases, morphology, and weight gain. After plasmid pattern analysis, the most active strains are most likely B. thuringiensis kurstaki strains expressing the Vip3A toxin. The absence of two proteinase activities observed in the case of Cry1Ac would be the consequence of the difference in susceptibility of A. segetum to the toxins used.

  9. Pathway and kinetics of cyhalothrin biodegradation by Bacillus thuringiensis strain ZS-19

    PubMed Central

    Chen, Shaohua; Deng, Yinyue; Chang, Changqing; Lee, Jasmine; Cheng, Yingying; Cui, Zining; Zhou, Jianuan; He, Fei; Hu, Meiying; Zhang, Lian-Hui

    2015-01-01

    Cyhalothrin is a common environmental pollutant which poses increased risks to non-target organisms including human beings. This study reported for the first time a newly isolated strain, Bacillus thuringiensis ZS-19 completely degraded cyhalothrin in minimal medium within 72 h. The bacterium transformed cyhalothrin by cleavage of both the ester linkage and diaryl bond to yield six intermediate products. Moreover, a novel degradation pathway of cyhalothrin in strain ZS-19 was proposed on the basis of the identified metabolites. In addition to degradation of cyhalothrin, this strain was found to be capable of degrading 3-phenoxybenzoic acid, a common metabolite of pyrethroids. Furthermore, strain ZS-19 participated in efficient degradation of a wide range of pyrethroids including cyhalothrin, fenpropathrinn, deltamethrin, beta-cypermethrin, cyfluthrin and bifenthrin. Taken together, our results provide insights into the mechanism of cyhalothrin degradation and also highlight the promising potentials of B.thuringiensis ZS-19 in bioremediation of pyrethroid-contaminated environment. This is the first report of (i) degradation of cyhalothrin and other pyrethroids by B.thuringiensis, (ii) identification of 3-phenoxyphenyl acetonitrile and N-(2-isoproxy-phenyl)-4-phenoxy-benzamide as the metabolites in the degradation pathway of pyrethroids, and (iii) a pathway of degradation of cyhalothrin by cleavage of both the ester linkage and diaryl bond in a microorganism. PMID:25740758

  10. Efficacy of VectoBac (Bacillus thuringiensis variety israelensis) formulations for mosquito control in Australia.

    PubMed

    Russell, Tanya L; Brown, Michael D; Purdie, David M; Ryan, Peter A; Kay, Brian H

    2003-12-01

    Laboratory bioassays were conducted on the efficacy of a water-dispersible granule (WG) formulation of Bacillus thuringiensis variety israelensis (VectoBac WG; active ingredient [AI]: 3,000 Bti international toxic units [ITU]/mg) against third instars of six common Australian mosquito species, Aedes aegypti (L.), Ochlerotatus vigilax (Skuse), Ochlerotatus notoscriptus (Skuse), Culex sitiens Wiedemann, Culex annulirostris Skuse, and Culex quinquefasciatus Say. The normal model for log-linear mortality data was used to determine laboratory 48-h LC50 and LC95 values. The target mosquito species tested were extremely sensitive to the VectoBac WG formulation, with the most sensitive species (Cx. annulirostris and Cx. quinquefasciatus, LC95 value of 0.019 ppm) being twice as susceptible as the most tolerant (Oc. notoscriptus, LC95 value of 0.037 ppm). Cx. annulirostris was selected as a target species for a small-plot evaluation of VectoBac WG and VectoBac 12 aqueous solution (AS) ([AI]: 1,200 Bti ITU/mg) efficacy over time, in freshwater in southeastern Queensland, Australia. Replicated cohorts of caged third instars were exposed weekly to six concentrations of WG formulation (0.004-0.13 ppm) and three concentrations of the 12AS formulation (0.04-0.13 ppm). In water with high organic content, treatment concentrations of 0.008 ppm WG and 0.04 ppm 12AS and above produced significant larval control (> or = 96%) at 48 h posttreatment, with no residual control at week 1. Water quality was not affected by treatment with either formulation.

  11. An ABC Transporter Mutation Is Correlated with Insect Resistance to Bacillus thuringiensis Cry1Ac Toxin

    PubMed Central

    Gahan, Linda J.; Pauchet, Yannick; Vogel, Heiko; Heckel, David G.

    2010-01-01

    Transgenic crops producing insecticidal toxins from Bacillus thuringiensis (Bt) are commercially successful in reducing pest damage, yet knowledge of resistance mechanisms that threaten their sustainability is incomplete. Insect resistance to the pore-forming Cry1Ac toxin is correlated with the loss of high-affinity, irreversible binding to the mid-gut membrane, but the genetic factors responsible for this change have been elusive. Mutations in a 12-cadherin-domain protein confer some Cry1Ac resistance but do not block this toxin binding in in vitro assays. We sought to identify mutations in other genes that might be responsible for the loss of binding. We employed a map-based cloning approach using a series of backcrosses with 1,060 progeny to identify a resistance gene in the cotton pest Heliothis virescens that segregated independently from the cadherin mutation. We found an inactivating mutation of the ABC transporter ABCC2 that is genetically linked to Cry1Ac resistance and is correlated with loss of Cry1Ac binding to membrane vesicles. ABC proteins are integral membrane proteins with many functions, including export of toxic molecules from the cell, but have not been implicated in the mode of action of Bt toxins before. The reduction in toxin binding due to the inactivating mutation suggests that ABCC2 is involved in membrane integration of the toxin pore. Our findings suggest that ABC proteins may play a key role in the mode of action of Bt toxins and that ABC protein mutations can confer high levels of resistance that could threaten the continued utilization of Bt–expressing crops. However, such mutations may impose a physiological cost on resistant insects, by reducing export of other toxins such as plant secondary compounds from the cell. This weakness could be exploited to manage this mechanism of Bt resistance in the field. PMID:21187898

  12. Comparative Analysis of Genomics and Proteomics in Bacillus thuringiensis 4.0718

    PubMed Central

    Rang, Jie; He, Hao; Wang, Ting; Ding, Xuezhi; Zuo, Mingxing; Quan, Meifang; Sun, Yunjun; Yu, Ziquan; Hu, Shengbiao; Xia, Liqiu

    2015-01-01

    Bacillus thuringiensis is a widely used biopesticide that produced various insecticidal active substances during its life cycle. Separation and purification of numerous insecticide active substances have been difficult because of the relatively short half-life of such substances. On the other hand, substances can be synthetized at different times during development, so samples at different stages have to be studied, further complicating the analysis. A dual genomic and proteomic approach would enhance our ability to identify such substances, and particularily using mass spectrometry-based proteomic methods. The comparative analysis for genomic and proteomic data have showed that not all of the products deduced from the annotated genome could be identified among the proteomic data. For instance, genome annotation results showed that 39 coding sequences in the whole genome were related to insect pathogenicity, including five cry genes. However, Cry2Ab, Cry1Ia, Cytotoxin K, Bacteriocin, Exoenzyme C3 and Alveolysin could not be detected in the proteomic data obtained. The sporulation-related proteins were also compared analysis, results showed that the great majority sporulation-related proteins can be detected by mass spectrometry. This analysis revealed Spo0A~P, SigF, SigE(+), SigK(+) and SigG(+), all known to play an important role in the process of spore formation regulatory network, also were displayed in the proteomic data. Through the comparison of the two data sets, it was possible to infer that some genes were silenced or were expressed at very low levels. For instance, found that cry2Ab seems to lack a functional promoter while cry1Ia may not be expressed due to the presence of transposons. With this comparative study a relatively complete database can be constructed and used to transform hereditary material, thereby prompting the high expression of toxic proteins. A theoretical basis is provided for constructing highly virulent engineered bacteria and for

  13. A purified Bacillus thuringiensis crystal protein with therapeutic activity against the hookworm parasite Ancylostoma ceylanicum.

    PubMed

    Cappello, Michael; Bungiro, Richard D; Harrison, Lisa M; Bischof, Larry J; Griffitts, Joel S; Barrows, Brad D; Aroian, Raffi V

    2006-10-10

    Crystal (Cry) proteins produced by the soil bacterium Bacillus thuringiensis (Bt) are harmless to vertebrates, but they are highly toxic to insects and nematodes. Their value in controlling insects that destroy crops and transmit human diseases is well established. Although it has recently been demonstrated that a few individual Bt Cry proteins, such as Cry5B, are toxic to a wide range of free-living nematodes, the potential activity of purified Cry proteins against parasitic nematodes remains largely unknown. We report here studies aimed at characterizing in vitro and in vivo anthelminthic activities of purified recombinant Cry5B against the hookworm parasite Ancylostoma ceylanicum, a bloodfeeding gastrointestinal nematode for which humans are permissive hosts. By using in vitro larval development assays, Cry5B was found to be highly toxic to early stage hookworm larvae. Exposure of adult A. ceylanicum to Cry5B was also associated with significant toxicity, including a substantial reduction in egg excretion by adult female worms. To demonstrate therapeutic efficacy in vivo, hamsters infected with A. ceylanicum were treated with three daily oral doses of purified Cry5B, the benzimidazole anthelminthic mebendazole, or buffer. Compared with control (buffer-treated) animals, infected hamsters that received Cry5B showed statistically significant improvements in growth and blood hemoglobin levels as well as reduced worm burdens that were comparable to the mebendazole-treated animals. These data demonstrate that Cry5B is highly active in vitro and in vivo against a globally significant nematode parasite and that Cry5B warrants further clinical development for human and veterinary use.

  14. Diversity and Toxicity of Bacillus thuringiensis from Shifting Cultivation (Jhum) Habitat.

    PubMed

    Zothansanga, Ralte; Senthilkumar, Nachimuthu; Gurusubramanian, Guruswami

    2016-01-01

    Bacillus thuringiensis (Bt) strains were isolated from jhum-agriculture, jhum-forest, aquatic and fallow soil samples from Mizoram by acetate selection method. Isolates were characterized for biochemical typing, cry gene and protein profiling, growth curve study and toxicity against Culex tritaeniorhynchus. Bt frequency was high in jhum-agriculture land (69.56%) whereas low in jhum-forest soils (31.57%). Bt was found to be abundant in jhum shifting cultivation soil with an index ranging between 0.010 and 0.015. Majority of the isolates from jhum soils produced oval and spherical crystals and showed eleven types of crystal proteins groups. PCR analysis revealed predominance of dipteran-active cry genes (cry4 and cry9). The variations in crystal morphology, cry genes and Cry protein (s) from the isolates of Bt revealed molecular diversity. Higher mortality, lower lethal dose, and lesser time to kill were observed in Bt isolates from jhum soils than aquatic and fallow habitats. Based on the toxicity test, SC1 and HP7 isolates containing cry 4 and cry 9 genes showed higher activity. Growth curve analysis showed significant variations among Bt isolates to reach the sporulating stage. Higher growth index and lower mean generation time were observed in SC1 and HP7 Bt isolates. Bt strains express different endotoxin genes and crystal proteins and their harvesting time also varied from strain to strain. Significant variation was found in Bt isolates from jhum habitats in relation to the cry gene composition, protein profiling and toxicity. Results from this study suggest that novel Bt entomopathogens may complement for regulating mosquito vectors. PMID:27350428

  15. Improvement and efficient display of Bacillus thuringiensis toxins on M13 phages and ribosomes.

    PubMed

    Pacheco, Sabino; Cantón, Emiliano; Zuñiga-Navarrete, Fernando; Pecorari, Frédéric; Bravo, Alejandra; Soberón, Mario

    2015-12-01

    Bacillus thuringiensis (Bt) produces insecticidal proteins that have been used worldwide in the control of insect-pests in crops and vectors of human diseases. However, different insect species are poorly controlled by the available Bt toxins or have evolved resistance to these toxins. Evolution of Bt toxicity could provide novel toxins to control insect pests. To this aim, efficient display systems to select toxins with increased binding to insect membranes or midgut proteins involved in toxicity are likely to be helpful. Here we describe two display systems, phage display and ribosome display, that allow the efficient display of two non-structurally related Bt toxins, Cry1Ac and Cyt1Aa. Improved display of Cry1Ac and Cyt1Aa on M13 phages was achieved by changing the commonly used peptide leader sequence of the coat pIII-fusion protein, that relies on the Sec translocation pathway, for a peptide leader sequence that relies on the signal recognition particle pathway (SRP) and by using a modified M13 helper phage (Phaberge) that has an amber mutation in its pIII genomic sequence and preferentially assembles using the pIII-fusion protein. Also, both Cry1Ac and Cyt1Aa were efficiently displayed on ribosomes, which could allow the construction of large libraries of variants. Furthermore, Cry1Ac or Cyt1Aa displayed on M13 phages or ribosomes were specifically selected from a mixture of both toxins depending on which antigen was immobilized for binding selection. These improved systems may allow the selection of Cry toxin variants with improved insecticidal activities that could counter insect resistances. PMID:26606918

  16. Optimization of spray-drying conditions for the large-scale preparation of Bacillus thuringiensis var. israelensis after downstream processing.

    PubMed

    Prabakaran, G; Hoti, S L

    2008-05-01

    Reduction of water activity in the formulations of mosquito biocontrol agent, Bacillus thuringiensis var. israelensis is very important for long term and successful storage. A protocol for spray drying of B. thuringiensis var. israelensis was developed through optimizing parameters such as inlet temperature and atomization type. A indigenous isolate of B. thuringiensis var. israelensis (VCRC B-17) was dried by freeze and spray drying methods and the moisture content and mosquito larvicidal activity of materials produced by the two methods were compared. The larvicidal activity was checked against early fourth instars Aedes aegypti larvae. Results showed that the freeze-dried powders retained the larvicidal activity fairly well. The spray-dried powder moderately lost its larvicidal activity at different inlet temperatures. Between the two types of atomization, centrifugal atomization retained more activity than the nozzle type atomization. Optimum inlet temperature for both centrifugal and nozzle atomization was 160 degrees C. Keeping the outlet temperature constant at 70 degrees C the moisture contents for the spray-dried powders through centrifugal atomization and freeze-dried powders were 10.23% and 11.80%, respectively. The LC(50) values for the spray-dried and freeze-dried powders were 17.42 and 16.18 ng/mL, respectively. Spore count of materials before drying was 3 x 10(10) cfu/mL and after spray drying through nozzle and centrifugal atomization at inlet and outlet temperature of 160 degrees C/70 degrees C were 2.6 x 10(9) and 5.0 x 10(9) cfu/mL, respectively.

  17. Southern analysis of BT-R1, the Manduca sexta gene encoding the receptor for the Cry1Ab toxin of Bacillus thuringiensis.

    PubMed

    Franklin, S E; Young, L; Watson, D; Cigan, A; Meyer, T; Bulla, L A

    1997-11-01

    Various subspecies of the gram-positive bacterium Bacillus thuringiensis are known to produce a wide array of insecticidal crystal proteins (ICPs) upon sporulation. These ICPs act primarily on the brush border of midgut epithelial cells of susceptible larvae. Recently, a protein of 210 kDa, isolated from the midgut of Manduca sexta, has been demonstrated to bind the Cry1Ab toxin produced by B. thuringiensis subsp, berliner and is therefore postulated to be involved in mediating the toxicity of Cry1Ab. The cDNA encoding the 210 kDa protein, termed BT-R1 (Bacillus thuringiensis receptor-1), was recently cloned, and shows limited homology to the cadherin superfamily of proteins. Quite naturally, there is a great deal of interest in the characterization of BT-R1, the gene encoding the 210 kDa Cry1Ab binding protein. The studies presented here involve the use of various restriction fragments prepared from the cDNA encoding BT-R1 as probes of Southern blots bearing M. sexta genomic DNA cleaved with a variety of restriction endonucleases. These Southern blot data reveal that there are two discrete regions within the M. sexta genome which encode sequences homologous to BT-R1. On the basis of the signal intensities seen on Southern blots, it appears that only one of these genes encodes BT-R1, whereas the other is a closely related homologue. PMID:9413435

  18. Persistence and recycling of bioinsecticidal Bacillus thuringiensis subsp. israelensis spores in contrasting environments: evidence from field monitoring and laboratory experiments.

    PubMed

    Duchet, Claire; Tetreau, Guillaume; Marie, Albane; Rey, Delphine; Besnard, Gilles; Perrin, Yvon; Paris, Margot; David, Jean-Philippe; Lagneau, Christophe; Després, Laurence

    2014-04-01

    Sprays of commercial preparations of the bacterium Bacillus thuringiensis subsp. israelensis are widely used for the control of mosquito larvae. Despite an abundant literature on B. thuringiensis subsp. israelensis field efficiency on mosquito control, few studies have evaluated the fate of spores in the environment after treatments. In the present article, two complementary experiments were conducted to study the effect of different parameters on B. thuringiensis subsp. israelensis persistence and recycling, in field conditions and in the laboratory. First, we monitored B. thuringiensis subsp. israelensis persistence in the field in two contrasting regions in France: the Rhône-Alpes region, where mosquito breeding sites are temporary ponds under forest cover with large amounts of decaying leaf matter on the ground and the Mediterranean region characterized by open breeding sites such as brackish marshes. Viable B. thuringiensis subsp. israelensis spores can persist for months after a treatment, and their quantity is explained both by the vegetation type and by the number of local treatments. We found no evidence of B. thuringiensis subsp. israelensis recycling in the field. Then, we tested the effect of water level, substrate type, salinity and presence of mosquito larvae on the persistence/recycling of B. thuringiensis subsp. israelensis spores in controlled laboratory conditions (microcosms). We found no effect of change in water level or salinity on B. thuringiensis subsp. israelensis persistence over time (75 days). B. thuringiensis subsp. israelensis spores tended to persist longer in substrates containing organic matter compared to sand-only substrates. B. thuringiensis subsp. israelensis recycling only occurred in presence of mosquito larvae but was unrelated to the presence of organic matter.

  19. Variable cross-resistance to Cry11B from Bacillus thuringiensis subsp. jegathesan in Culex quinquefasciatus (Diptera: Culicidae) resistant to single or multiple toxins of Bacillus thuringiensis subsp. israelensis.

    PubMed

    Wirth, M C; Delécluse, A; Federici, B A; Walton, W E

    1998-11-01

    A novel mosquitocidal bacterium, Bacillus thuringiensis subsp. jegathesan, and one of its toxins, Cry11B, in a recombinant B. thuringiensis strain were evaluated for cross-resistance with strains of the mosquito Culex quinquefasciatus that are resistant to single and multiple toxins of Bacillus thuringiensis subsp. israelensis. The levels of cross-resistance (resistance ratios [RR]) at concentrations which caused 95% mortality (LC95) between B. thuringiensis subsp. jegathesan and the different B. thuringiensis subsp. israelensis-resistant mosquito strains were low, ranging from 2.3 to 5.1. However, the levels of cross-resistance to Cry11B were much higher and were directly related to the complexity of the B. thuringiensis subsp. israelensis Cry toxin mixtures used to select the resistant mosquito strains. The LC95 RR obtained with the mosquito strains were as follows: 53.1 against Cq4D, which was resistant to Cry11A; 80.7 against Cq4AB, which was resistant to Cry4A plus Cry4B; and 347 against Cq4ABD, which was resistant to Cry4A plus Cry4B plus Cry11A. Combining Cyt1A with Cry11B at a 1:3 ratio had little effect on suppressing Cry11A resistance in Cq4D but resulted in synergism factors of 4.8 and 11.2 against strains Cq4AB and Cq4ABD, respectively; this procedure eliminated cross-resistance in the former mosquito strain and reduced it markedly in the latter strain. The high levels of activity of B. thuringiensis subsp. jegathesan and B. thuringiensis subsp. israelensis, both of which contain a complex mixture of Cry and Cyt proteins, against Cry4- and Cry11-resistant mosquitoes suggest that novel bacterial strains with multiple Cry and Cyt proteins may be useful in managing resistance to bacterial insecticides in mosquito populations.

  20. Toxicity, activation process, and histopathological effect of Bacillus thuringiensis vegetative insecticidal protein Vip3Aa16 on Tuta absoluta.

    PubMed

    Sellami, Sameh; Cherif, Maroua; Abdelkefi-Mesrati, Lobna; Tounsi, Slim; Jamoussi, Kaïs

    2015-02-01

    Tuta absoluta is a destructive moth of Solanaceae plants and especially tomatoes. Here, we considered the entomopathogenic activity of the Bacillus thuringiensis Vip3Aa16 protein heterologously produced by Escherichia coli against T. absoluta. Purified Vip3Aa16 showed lower lethal concentration 50 % against third instar larvae (Toxin/tomato leaf) (335 ± 17 ng/cm(2)) compared to that of B. thuringiensis kurstaki HD1 δ-endotoxins (955 ± 4 ng/cm(2)) (P < 0.05). Action mode examination showed that Vip3Aa16 (88 kDa) was more sensitive to proteolysis activation by the chymotrypsin than the trypsin or the larvae gut soluble proteases, yielding derivative proteins essentially of about 62 and 33 kDa. The gut-soluble proteases could contain trypsin-like enzymes implicated in Vip3Aa16 activation since the proteolysis was inhibited using specific protease inhibitors. Additionally, we showed that the histopathological effect of Vip3Aa16 on T. absoluta larva midguts consisted on a microvillus damage and an epithelial cell rupture.

  1. Fermentation of a Malaysian Bacillus thuringiensis serotype H-14 isolate, a mosquito microbial control agent utilizing local wastes.

    PubMed

    Lee, H L; Seleena, P

    1991-03-01

    A screening program searching for indigenous microbial control agents of mosquitos in Malaysia is initiated since 1987 and to date at least 20 isolates of mosquitocidal Bacillus thuringiensis serotypes have been obtained. Preliminary field evaluation of several isolates indicated that they are highly effective in the control of medically important mosquito species. For operational purposes, there is an urgent need to produce this agent utilizing cheap and locally available wastes through fermentation biotechnology. Fermentation studies in shake-flasks containing standard nutrient broth and soya bean waste, respectively, indicate that it takes about 37 hours for a Malaysian isolate of B. thuringiensis serotype H-14 to mature. In the grated coconut waste, fishmeal and rice bran, the bacteria took 28 hours, 26 hours and 126 hours respectively to mature. The endotoxin was harvested from the standard nutrient broth at 55 hours and at 50 hours from soybean, grated coconut waste and fishmeal. The endotoxin could only be harvested 150 hours after inoculation from rice bran medium. However, no bacterial growth was detected in palm oil effluent. In terms of endotoxin and biomass production, fishmeal appears to be a suitable medium. Variations in the pH of the fermenting media were also noted.

  2. Cadherin binding is not a limiting step for Bacillus thuringiensis subsp. israelensis Cry4Ba toxicity to Aedes aegypti larvae.

    PubMed

    Rodríguez-Almazán, Claudia; Reyes, Esmeralda Z; Zúñiga-Navarrete, Fernando; Muñoz-Garay, Carlos; Gómez, Isabel; Evans, Amy M; Likitvivatanavong, Supaporn; Bravo, Alejandra; Gill, Sarjeet S; Soberón, Mario

    2012-05-01

    Bacillus thuringiensis subsp. israelensis produces three Cry toxins (Cry4Aa, Cry4Ba and Cry11Aa) that are active against Aedes aegypti larvae. The identification of the rate-limiting binding steps of Cry toxins that are used for insect control in the field, such as those of B. thuringiensis subsp. israelensis, should provide targets for improving insecticides against important insect pests. Previous studies showed that Cry11Aa binds to cadherin receptor fragment CR7-11 (cadherin repeats 7-11) with high affinity. Binding to cadherin has been proposed to facilitate Cry toxin oligomer formation. In the present study, we show that Cry4Ba binds to CR7-11 with 9-fold lower binding affinity compared with Cry11Aa. Oligomerization assays showed that Cry4Ba is capable of forming oligomers when proteolytically activated in vitro in the absence of the CR7-11 fragment in contrast with Cry11Aa that formed oligomers only in the presence of CR7-11. Pore-formation assays in planar lipid bilayers showed that Cry4Ba oligomers were proficient in opening ion channels. Finally, silencing the cadherin gene by dsRNA (double-stranded RNA) showed that silenced larvae were more tolerant to Cry11Aa in contrast with Cry4Ba, which showed similar toxic levels to those of control larvae. These findings show that cadherin binding is not a limiting step for Cry4Ba toxicity to A. aegypti larvae. PMID:22329749

  3. Potential use of Bacillus thuringiensis bacteriocins to control antibiotic-resistant bacteria associated with mastitis in dairy goats.

    PubMed

    Gutiérrez-Chávez, A J; Martínez-Ortega, E A; Valencia-Posadas, M; León-Galván, M F; de la Fuente-Salcido, N M; Bideshi, D K; Barboza-Corona, J E

    2016-01-01

    Mastitis caused by microbial infections in dairy goats reduces milk yield, modifies milk composition, and potentially contributes to morbidity in herds and consumers of dairy products. Microorganisms associated with mastitis in dairy goats are commonly controlled with antibiotics, but it is known that continued use of these chemical agents promotes antibiotic resistance among bacterial populations. Recently, it has been shown that bacteriocins of Bacillus thuringiensis inhibit growth of food-borne pathogens and also bacteria associated with bovine mastitis. However, there is no report on their ability to inhibit microorganisms linked to mastitis in dairy goats. In this study, using 16S rDNA and ITS regions of rDNA, we identified nine bacterial isolates and an encapsulated yeast associated with mastitis in dairy goats. Enterococcus durans, Brevibacillus sp., and Staphylococcus epidermidis 2 were resistant to, respectively, 75, ~67, ~42, and ~42 % of the antibiotics screened. In addition, 60 % of the bacterial isolates were resistant to penicillin, ampicillin, vancomycin, and dicloxacillin. Importantly, 60 % of the isolates were inhibited by the bacteriocins, but S. epidermidis 1, Enterobacter sp., Escherichia vulneris, and Cryptococcus neoformans were not susceptible to these antimicrobial peptides. Using Brevibacillus sp. and Staphylococcus chromogenes as indicator bacteria, we show that peptides of ~10 kDa that correspond to the molecular mass of bacteriocins used in this study are responsible for the inhibitory activity. Our results demonstrate that multiple antibiotic-resistant bacteria associated with subclinical mastitis in dairy goats from Guanajuato, Mexico, are susceptible to bacteriocins produced by B. thuringiensis.

  4. Potential use of Bacillus thuringiensis bacteriocins to control antibiotic-resistant bacteria associated with mastitis in dairy goats.

    PubMed

    Gutiérrez-Chávez, A J; Martínez-Ortega, E A; Valencia-Posadas, M; León-Galván, M F; de la Fuente-Salcido, N M; Bideshi, D K; Barboza-Corona, J E

    2016-01-01

    Mastitis caused by microbial infections in dairy goats reduces milk yield, modifies milk composition, and potentially contributes to morbidity in herds and consumers of dairy products. Microorganisms associated with mastitis in dairy goats are commonly controlled with antibiotics, but it is known that continued use of these chemical agents promotes antibiotic resistance among bacterial populations. Recently, it has been shown that bacteriocins of Bacillus thuringiensis inhibit growth of food-borne pathogens and also bacteria associated with bovine mastitis. However, there is no report on their ability to inhibit microorganisms linked to mastitis in dairy goats. In this study, using 16S rDNA and ITS regions of rDNA, we identified nine bacterial isolates and an encapsulated yeast associated with mastitis in dairy goats. Enterococcus durans, Brevibacillus sp., and Staphylococcus epidermidis 2 were resistant to, respectively, 75, ~67, ~42, and ~42 % of the antibiotics screened. In addition, 60 % of the bacterial isolates were resistant to penicillin, ampicillin, vancomycin, and dicloxacillin. Importantly, 60 % of the isolates were inhibited by the bacteriocins, but S. epidermidis 1, Enterobacter sp., Escherichia vulneris, and Cryptococcus neoformans were not susceptible to these antimicrobial peptides. Using Brevibacillus sp. and Staphylococcus chromogenes as indicator bacteria, we show that peptides of ~10 kDa that correspond to the molecular mass of bacteriocins used in this study are responsible for the inhibitory activity. Our results demonstrate that multiple antibiotic-resistant bacteria associated with subclinical mastitis in dairy goats from Guanajuato, Mexico, are susceptible to bacteriocins produced by B. thuringiensis. PMID:26022411

  5. Prays oleae midgut putative receptor of Bacillus thuringiensis vegetative insecticidal protein Vip3LB differs from that of Cry1Ac toxin.

    PubMed

    Abdelkefi-Mesrati, Lobna; Rouis, Souad; Sellami, Sameh; Jaoua, Samir

    2009-09-01

    Vegetative insecticidal protein (Vip) is a class of insecticidal proteins produced by many Bacillus thuringiensis strains during their vegetative growth stage. The vip3LB gene of B. thuringiensis strain BUPM95, which encodes a protein active against the Lepidoptera olive tree pathogenic insect Prays oleae, was cloned into pET-14b vector and overexpressed in Escherichia coli. The expressed Vip3LB protein, found in the E. coli cytoplasmic fraction, was purified and used to produce anti-Vip3LB antibodies. Using the midgut extract of P. oleae, the purified Vip3LB bound to a 65-kDa protein, whereas Cry1Ac toxin bound to a 210-kDa midgut putative receptor. This result justifies the importance of the biological pest control agent Vip3LB that could be used as another alternative particularly in case of resistance to Cry toxins. PMID:19434523

  6. Extraction of antibiotic zwittermicin A from Bacillus thuringiensis by macroporous resin and silica gel column chromatography.

    PubMed

    Hao, Zaibin; Yan, Li; Liu, Jianguo; Song, Fuping; Zhang, Jie; Li, Xia

    2015-01-01

    To establish a production process capable of providing refined zwittermicin A (ZwA) on a large scale, the macroporous resin and silica gel column chromatography were used to separate and purify the antibiotic ZwA from the fermentation broth of Bacillus thuringiensis HD-1. The result of high-performance liquid chromatography-mass spectrometry after purification suggests that the samples of ZwA were of high purity, 89%, and the average yield was 20 mg L(-1). Erwinia herbicola LS005, Escherichia coli, Staphylococcus aureus, and Bacillus subtilis were used to assess the toxicity of ZwA. The antibiotic had strong antibacterial activity against E. herbicola LS005 and a color reaction with ninhydrin. PMID:25099664

  7. Generalized transduction in Bacillus thuringiensis var. berliner 1715 using bacteriophage CP-54Ber.

    PubMed

    Lecadet, M M; Blondel, M O; Ribier, J

    1980-11-01

    A phage isolated from lysates of phage CP-54 grown on Bacillus cereus 569 and selected on the basis of its ability to infect Bacillus thuringiensis var. berliner 1715 (serotype I) was designated CP-54Ber. Phages CP-54Ber and CP-54 were similar in size, morphology, cryosensitivity and stabilization by dimethyl sulphoxide. They showed significant differences with regard to inactivation by specific antiserum, adsorption to the berliner strains and host range. Phage CP-54Ber was able to mediate generalized transduction in the host strain berliner 1715 with frequencies ranging between 1 x 10(-5) and 1 x 10(-6). Cotransduction of markers was demonstrated. Cross-transduction occurred between strains belonging to serotype I whereas it was more difficult to observe when lysates were prepared on strains from other serotypes.

  8. Function of global regulator CodY in Bacillus thuringiensis BMB171 by comparative proteomic analysis.

    PubMed

    Qi, Mingxia; Mei, Fei; Wang, Hui; Sun, Ming; Wang, Gejiao; Yu, Ziniu; Je, Yeonho; Li, Mingshun

    2015-02-01

    CodY is a highly conserved protein in low G+C gram-positive bacteria that regulates genes involved in sporulation and stationary-phase adaptation. Bacillus thuringiensis is a grampositive bacterium that forms spores and parasporal crystals during the stationary phase. To our knowledge, the regulatory mechanism of CodY in B. thuringiensis is unknown. To study the function of CodY protein in B. thuringiensis, BMB171codY(-) was constructed in a BMB171 strain. A shuttle vector containing the ORF of cry1Ac10 was transformed into BMB171 and BMB171codY(-), named BMB171cry1Ac and BMB171codY(-)cry1Ac, respectively. Some morphological and physiological changes of codY mutant BMB171codY(-)cry1Ac were observed. A comparative proteomic analysis was conducted for both BMB171codY(-)cry1Ac and BMB171cry1Ac through two-dimensional gel electrophoresis and MALDI-TOF-MS/MS analysis. The results showed that the proteins regulated by CodY are involved in microbial metabolism, including branched-chain amino acid metabolism, carbohydrate metabolism, fatty acid metabolism, and energy metabolism. Furthermore, we found CodY to be involved in sporulation, biosynthesis of poly-β-hydroxybutyrate, growth, genetic competence, and translation. According to the analysis of differentially expressed proteins, and physiological characterization of the codY mutant, we performed bacterial one-hybrid and electrophoretic mobility shift assay experiments and confirmed the direct regulation of genes by CodY, specifically those involved in metabolism of branched-chain amino acids, ribosomal recycling factor FRR, and the late competence protein ComER. Our data establish the foundation for in-depth study of the regulation of CodY in B. thuringiensis, and also offer a potential biocatalyst for functions of CodY in other bacteria.

  9. SR450 And Superhawk XP Applications Of Bacillus thuringiensis israelensis Against Culex quinquefasciatus.

    PubMed

    Dunford, James C; Stoops, Craig A; Estep, Alden S; Britch, Seth C; Richardson, Alec G; Walker, Todd W; Farooq, Muhammad; Hoel, David F; Platt, Raymond R; Smith, Vincent L; Wirtz, Robert A; Kerce, Jerry D

    2014-09-01

    Sprayer comparisons and larval morality assays were conducted following SR450 backpack mist blower and Superhawk XP thermal fogger applications of Vectobac® WDG Bacillus thuringiensis israelensis (Bti) against Culex quinquefasciatus. Bacillus thuringiensis israelensis was applied at maximum label rate in a 232.26-m(2) field plot located in north-central Florida with containers placed at 2 heights (ground level and 1.52 m above ground) on stakes positioned 3.04, 6.09, 9.14, 12.19, and 15.24 m from the spray line. Results indicated that there was no significant (P > 0.05) difference in 24- and 48-h larval mortality between the 2 sprayers or between the 2 heights. There was significant difference (P < 0.05) among the 5 rows, with mortality continuously decreasing with increasing distance from sprayer. Both sprayers provided on average >70% larval mortality 3.04-9.14 m from the spray line, and <60% mortality at 12.19 and 15.24 m. The data suggest that the SR450 and Superhawk XP may be comparable sprayers for use with Bti to control mosquito larvae. PMID:25843094

  10. Cellular fatty-acid analysis of Bacillus thuringiensis var. kurstaki commercial preparations.

    PubMed

    Adams, D Jack; Gurr, Susan; Hogge, Jason

    2005-02-01

    Cellular fatty acid (CFA) composition of Bacillus thuringiensis var. kurstaki (Btk) preparations was determined by use of the MIDI Sherlock microbial identification system on a Hewlett-Packard 5890 gas chromatograph. Four commercial preparations--one Btk sample obtained from the U.S. Forest Service, one Btk sample obtained from Dugway Proving Ground, and Btk and Bacillus thuringiensis var. israelensis (Bti) preparations obtained from American Type Culture Collection (ATCC)--were analyzed and evaluated. This study demonstrated the capability to detect the strain variation in the bacterial species Btk and to clearly differentiate strain variants on the basis of qualitative and quantitative differences in hydrolyzable whole CFA compositions in the preparations examined. We conclude that CFA analysis may be used to identify commercial products but that a more intensive study would be required to evaluate the potential of CFA to provide an inexpensive screening tool applicable to several levels of isolate or product evaluation, including how applied preparations might interact with natural populations over time.

  11. Revision of the nomenclature for the Bacillus thuringiensis pesticidal crystal proteins.

    PubMed

    Crickmore, N; Zeigler, D R; Feitelson, J; Schnepf, E; Van Rie, J; Lereclus, D; Baum, J; Dean, D H

    1998-09-01

    The crystal proteins of Bacillus thuringiensis have been extensively studied because of their pesticidal properties and their high natural levels of production. The increasingly rapid characterization of new crystal protein genes, triggered by an effort to discover proteins with new pesticidal properties, has resulted in a variety of sequences and activities that no longer fit the original nomenclature system proposed in 1989. Bacillus thuringiensis pesticidal crystal protein (Cry and Cyt) nomenclature was initially based on insecticidal activity for the primary ranking criterion. Many exceptions to this systematic arrangement have become apparent, however, making the nomenclature system inconsistent. Additionally, the original nomenclature, with four activity-based primary ranks for 13 genes, did not anticipate the current 73 holotype sequences that form many more than the original four subgroups. A new nomenclature, based on hierarchical clustering using amino acid sequence identity, is proposed. Roman numerals have been exchanged for Arabic numerals in the primary rank (e.g., Cry1Aa) to better accommodate the large number of expected new sequences. In this proposal, 133 crystal proteins comprising 24 primary ranks are systematically arranged. PMID:9729610

  12. Revision of the Nomenclature for the Bacillus thuringiensis Pesticidal Crystal Proteins

    PubMed Central

    Crickmore, N.; Zeigler, D. R.; Feitelson, J.; Schnepf, E.; Van Rie, J.; Lereclus, D.; Baum, J.; Dean, D. H.

    1998-01-01

    The crystal proteins of Bacillus thuringiensis have been extensively studied because of their pesticidal properties and their high natural levels of production. The increasingly rapid characterization of new crystal protein genes, triggered by an effort to discover proteins with new pesticidal properties, has resulted in a variety of sequences and activities that no longer fit the original nomenclature system proposed in 1989. Bacillus thuringiensis pesticidal crystal protein (Cry and Cyt) nomenclature was initially based on insecticidal activity for the primary ranking criterion. Many exceptions to this systematic arrangement have become apparent, however, making the nomenclature system inconsistent. Additionally, the original nomenclature, with four activity-based primary ranks for 13 genes, did not anticipate the current 73 holotype sequences that form many more than the original four subgroups. A new nomenclature, based on hierarchical clustering using amino acid sequence identity, is proposed. Roman numerals have been exchanged for Arabic numerals in the primary rank (e.g., Cry1Aa) to better accommodate the large number of expected new sequences. In this proposal, 133 crystal proteins comprising 24 primary ranks are systematically arranged. PMID:9729610

  13. Isolation of transcripts from Diabrotica virgifera virgifera LeConte responsive to the Bacillus thuringiensis toxin Cry3Bb1

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Crystal (Cry) proteins derived from Bacillus thuringiensis (Bt) have been widely used as a method of insect pest management for several decades. In recent years, a transgenic corn expressing the Cry3Bb1 toxin has been successfully used for protection against corn rootworm larvae (Genus Diabrotica). ...

  14. Susceptibility of Cry1Ab-resistant and -susceptible Sugarcane Borer (Lepidoptera: crambidae) to Four Bacillus thuringiensis Toxins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Sugarcane borer, Diatraea saccharalis (F.), is a primary corn stalk borer pest targeted by transgenic corn expressing Bacillus thuringiensis (Bt) proteins in many areas of the mid-southern region of the United States. Recently, genes encoding for Cry1A.105 and Cry2Ab2 Bt proteins were transferred in...

  15. Cadherin is a functional receptor of bacillus thuringiensis toxin Cry2Aa in the beet armyworm, spodoptera exigua

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bacillus thuringiensis (Bt) insecticidal crystal (Cry) proteins are effective against some insect pests in sprays and transgenic crops, although the evolution of resistance could threaten the long-term efficacy of such Bt use. One strategy to delay resistance to Bt crops is to “pyramid” two or more ...

  16. Isolation of transcripts from Diabrotica virgifera virgifera LeConte responsive to the Bacillus thuringiensis toxin Cry3Bb1

    EPA Science Inventory

    Crystal proteins derived from Bacillus thuringiensis (Bt) have been widely used as a method of insect pest management for several decades. In recent years, a transgenic corn expressing the Cry3Bb1 toxin has been successfully used for protection against corn rootworm larvae (Genus...

  17. Expression of Bacillus thuringiensis Cytolytic Toxin (Cyt2Ca1) in citrus roots to control Diaprepes abbreviatus larvae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Diaprepes abbreviatus (L.) is an important pest of citrus in the USA. Currently, no effective management strategies of Diaprepes abbreviatus exist in citriculture. To protect citrus against Diaprepes abbreviatus a transgenic citrus rootstock expressing Bacillus thuringiensis Cyt2Ca1, an insect toxin...

  18. 40 CFR 174.520 - Bacillus thuringiensis Cry1F protein in corn; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... in corn; exemption from the requirement of a tolerance. 174.520 Section 174.520 Protection of... Cry1F protein in corn; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry1F protein in corn are exempt from the requirement of a tolerance when used as...

  19. 40 CFR 174.517 - Bacillus thuringiensis Cry9C protein in corn; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... in corn; exemption from the requirement of a tolerance. 174.517 Section 174.517 Protection of... Cry9C protein in corn; exemption from the requirement of a tolerance. The plant-incorporated protectant Bacillus thuringiensis Cry9C protein in corn is exempted from the requirement of a tolerance for...

  20. 40 CFR 174.520 - Bacillus thuringiensis Cry1F protein in corn; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... in corn; exemption from the requirement of a tolerance. 174.520 Section 174.520 Protection of... Cry1F protein in corn; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry1F protein in corn are exempt from the requirement of a tolerance when used as...

  1. 40 CFR 174.517 - Bacillus thuringiensis Cry9C protein in corn; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... in corn; exemption from the requirement of a tolerance. 174.517 Section 174.517 Protection of... Cry9C protein in corn; exemption from the requirement of a tolerance. The plant-incorporated protectant Bacillus thuringiensis Cry9C protein in corn is exempted from the requirement of a tolerance for...

  2. 40 CFR 174.517 - Bacillus thuringiensis Cry9C protein in corn; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... in corn; exemption from the requirement of a tolerance. 174.517 Section 174.517 Protection of... Cry9C protein in corn; exemption from the requirement of a tolerance. The plant-incorporated protectant Bacillus thuringiensis Cry9C protein in corn is exempted from the requirement of a tolerance for...

  3. Transcriptome profiling of the intoxication response of Tenebrio molitor larvae to Bacillus thuringiensis Cry3Aa protoxin

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bacillus thuringiensis (Bt) crystal (Cry) proteins are effective against some coleopteran pests, but improvements are needed in both efficacy and “time to kill” for Cry toxins to become valuable tools for use in integrated pest management. To gain insight into Bt intoxication of Coleoptera, we perfo...

  4. Bacillus thuringiensis Cry3Aa protoxin intoxication of Tenebrio molitor induces widespread changes in the expression of serine peptidase transcripts

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The yellow mealworm, Tenebrio molitor, is a pest of stored grain products and is sensitive to the coleopteran-specific Cry3Aa toxin from Bacillus thuringiensis (Bt). Larvae digest protein initially with cysteine peptidases in the anterior midgut and further with serine peptidases in middle and poste...

  5. 40 CFR 174.504 - Bacillus thuringiensis Cry1F protein in cotton; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... in cotton; exemption from the requirement of a tolerance. 174.504 Section 174.504 Protection of... Cry1F protein in cotton; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry1F protein in cotton are exempt from the requirement of a tolerance when used as a...

  6. 40 CFR 174.530 - Bacillus thuringiensis Cry2Ae protein in cotton; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... in cotton; exemption from the requirement of a tolerance. 174.530 Section 174.530 Protection of... Cry2Ae protein in cotton; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry2Ae protein in or on the food and feed commodities of cotton; cotton, undelinted seed;...

  7. 40 CFR 174.530 - Bacillus thuringiensis Cry2Ae protein in cotton; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... in cotton; exemption from the requirement of a tolerance. 174.530 Section 174.530 Protection of... Cry2Ae protein in cotton; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry2Ae protein in or on the food and feed commodities of cotton; cotton, undelinted seed;...

  8. 40 CFR 174.530 - Bacillus thuringiensis Cry2Ae protein in cotton; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... in cotton; exemption from the requirement of a tolerance. 174.530 Section 174.530 Protection of... Cry2Ae protein in cotton; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry2Ae protein in or on the food and feed commodities of cotton; cotton, undelinted seed;...

  9. 40 CFR 174.504 - Bacillus thuringiensis Cry1F protein in cotton; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... in cotton; exemption from the requirement of a tolerance. 174.504 Section 174.504 Protection of... Cry1F protein in cotton; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry1F protein in cotton are exempt from the requirement of a tolerance when used as a...

  10. 40 CFR 174.504 - Bacillus thuringiensis Cry1F protein in cotton; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... in cotton; exemption from the requirement of a tolerance. 174.504 Section 174.504 Protection of... Cry1F protein in cotton; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry1F protein in cotton are exempt from the requirement of a tolerance when used as a...

  11. 40 CFR 174.504 - Bacillus thuringiensis Cry1F protein in cotton; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... in cotton; exemption from the requirement of a tolerance. 174.504 Section 174.504 Protection of... Cry1F protein in cotton; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry1F protein in cotton are exempt from the requirement of a tolerance when used as a...

  12. Reduced levels of membrane-bound alkaline phosphatase are common to lepidopteran strains resistant to Cry toxins from Bacillus thuringiensis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Development of insect resistance is one of the main concerns with the use of transgenic crops expressing Cry toxins from the bacterium Bacillus thuringiensis. Biomarkers for development of sensitive DNA-based methods to detect and monitor evolution of resistance to Bt toxins are currently needed. ...

  13. Transcriptome of the lymantria dispar (gypsy moth) larval midgut and its response to infection by bacillus thuringiensis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Transcriptomic profiles of the serious lepidopteran insect pest Lymantria dispar (gypsy moth) were characterized in the larval midgut in response to infection by Bacillus thuringiensis kurstaki, a biopesticide commonly used for its control in nature. RNA-Seq approaches were used to define a set of ...

  14. 40 CFR 174.532 - Bacillus thuringiensis eCry3.1Ab protein in corn; temporary exemption from the requirement of a...

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... protein in corn; temporary exemption from the requirement of a tolerance. 174.532 Section 174.532... thuringiensis eCry3.1Ab protein in corn; temporary exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis eCry3.1Ab protein in corn, in or on the food and feed commodities of corn;...

  15. 40 CFR 174.506 - Bacillus thuringiensis Cry34Ab1 and Cry35Ab1 proteins in corn; exemption from the requirement of...

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... Cry35Ab1 proteins in corn; exemption from the requirement of a tolerance. 174.506 Section 174.506... thuringiensis Cry34Ab1 and Cry35Ab1 proteins in corn; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry34Ab1 and Cry35Ab1 proteins in corn are exempted from the requirement of...

  16. 40 CFR 174.505 - Bacillus thuringiensis modified Cry3A protein (mCry3A) in corn; exemption from the requirement of...

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... protein (mCry3A) in corn; exemption from the requirement of a tolerance. 174.505 Section 174.505... thuringiensis modified Cry3A protein (mCry3A) in corn; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis modified Cry3A protein (mCry3A) in corn are exempt from the requirement...

  17. 40 CFR 174.505 - Bacillus thuringiensis modified Cry3A protein (mCry3A) in corn; exemption from the requirement of...

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... protein (mCry3A) in corn; exemption from the requirement of a tolerance. 174.505 Section 174.505... thuringiensis modified Cry3A protein (mCry3A) in corn; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis modified Cry3A protein (mCry3A) in corn are exempt from the requirement...

  18. 40 CFR 174.505 - Bacillus thuringiensis modified Cry3A protein (mCry3A) in corn; exemption from the requirement of...

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... protein (mCry3A) in corn; exemption from the requirement of a tolerance. 174.505 Section 174.505... thuringiensis modified Cry3A protein (mCry3A) in corn; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis modified Cry3A protein (mCry3A) in corn are exempt from the requirement...

  19. 40 CFR 174.506 - Bacillus thuringiensis Cry34Ab1 and Cry35Ab1 proteins in corn; exemption from the requirement of...

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... Cry35Ab1 proteins in corn; exemption from the requirement of a tolerance. 174.506 Section 174.506... thuringiensis Cry34Ab1 and Cry35Ab1 proteins in corn; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry34Ab1 and Cry35Ab1 proteins in corn are exempted from the requirement of...

  20. 40 CFR 174.506 - Bacillus thuringiensis Cry34Ab1 and Cry35Ab1 proteins in corn; exemption from the requirement of...

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... Cry35Ab1 proteins in corn; exemption from the requirement of a tolerance. 174.506 Section 174.506... thuringiensis Cry34Ab1 and Cry35Ab1 proteins in corn; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry34Ab1 and Cry35Ab1 proteins in corn are exempted from the requirement of...

  1. 40 CFR 174.519 - Bacillus thuringiensis Cry2Ab2 protein in corn and cotton; exemption from the requirement of a...

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... in corn and cotton; exemption from the requirement of a tolerance. 174.519 Section 174.519 Protection... thuringiensis Cry2Ab2 protein in corn and cotton; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry2Ab2 protein in or on corn or cotton are exempt from the requirement of...

  2. 40 CFR 174.501 - Bacillus thuringiensis Vip3Aa protein in corn and cotton; exemption from the requirement of a...

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... in corn and cotton; exemption from the requirement of a tolerance. 174.501 Section 174.501 Protection... thuringiensis Vip3Aa protein in corn and cotton; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Vip3Aa proteins in or on corn or cotton are exempt from the requirement of...

  3. 40 CFR 174.501 - Bacillus thuringiensis Vip3Aa protein in corn and cotton; exemption from the requirement of a...

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... in corn and cotton; exemption from the requirement of a tolerance. 174.501 Section 174.501 Protection... thuringiensis Vip3Aa protein in corn and cotton; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Vip3Aa proteins in or on corn or cotton are exempt from the requirement of...

  4. 40 CFR 174.519 - Bacillus thuringiensis Cry2Ab2 protein in corn and cotton; exemption from the requirement of a...

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... in corn and cotton; exemption from the requirement of a tolerance. 174.519 Section 174.519 Protection... thuringiensis Cry2Ab2 protein in corn and cotton; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry2Ab2 protein in or on corn or cotton are exempt from the requirement of...

  5. 40 CFR 174.501 - Bacillus thuringiensis Vip3Aa protein in corn and cotton; exemption from the requirement of a...

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... in corn and cotton; exemption from the requirement of a tolerance. 174.501 Section 174.501 Protection... thuringiensis Vip3Aa protein in corn and cotton; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Vip3Aa proteins in or on corn or cotton are exempt from the requirement of...

  6. 40 CFR 174.501 - Bacillus thuringiensis Vip3Aa protein in corn and cotton; exemption from the requirement of a...

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... in corn and cotton; exemption from the requirement of a tolerance. 174.501 Section 174.501 Protection... thuringiensis Vip3Aa protein in corn and cotton; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Vip3Aa proteins in or on corn or cotton are exempt from the requirement of...

  7. 40 CFR 174.519 - Bacillus thuringiensis Cry2Ab2 protein in corn and cotton; exemption from the requirement of a...

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... in corn and cotton; exemption from the requirement of a tolerance. 174.519 Section 174.519 Protection... thuringiensis Cry2Ab2 protein in corn and cotton; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry2Ab2 protein in or on corn or cotton are exempt from the requirement of...

  8. 40 CFR 174.519 - Bacillus thuringiensis Cry2Ab2 protein in corn and cotton; exemption from the requirement of a...

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... in corn and cotton; exemption from the requirement of a tolerance. 174.519 Section 174.519 Protection... thuringiensis Cry2Ab2 protein in corn and cotton; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry2Ab2 protein in or on corn or cotton are exempt from the requirement of...

  9. 40 CFR 174.519 - Bacillus thuringiensis Cry2Ab2 protein in corn and cotton; exemption from the requirement of a...

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... in corn and cotton; exemption from the requirement of a tolerance. 174.519 Section 174.519 Protection... thuringiensis Cry2Ab2 protein in corn and cotton; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry2Ab2 protein in or on corn or cotton are exempt from the requirement of...

  10. 40 CFR 174.501 - Bacillus thuringiensis Vip3Aa protein in corn and cotton; exemption from the requirement of a...

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... in corn and cotton; exemption from the requirement of a tolerance. 174.501 Section 174.501 Protection... thuringiensis Vip3Aa protein in corn and cotton; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Vip3Aa proteins in or on corn or cotton are exempt from the requirement of...

  11. Gut bacteria are not required for the insecticidal activity of Bacillus thuringiensis toward the tobacco hornworm, Manduca sexta.

    PubMed

    Johnston, Paul R; Crickmore, Neil

    2009-08-01

    It was recently proposed that gut bacteria are required for the insecticidal activity of the Bacillus thuringiensis-based insecticide, DiPel, toward the lepidopterans Manduca sexta, Pieris rapae, Vanessa cardui, and Lymantria dispar. Using a similar methodology, it was found that gut bacteria were not required for the toxicity of DiPel or Cry1Ac or for the synergism of an otherwise sublethal concentration of Cry1Ac toward M. sexta. The toxicities of DiPel and of B. thuringiensis HD73 Cry(-) spore/Cry1Ac synergism were attenuated by continuously exposing larvae to antibiotics before bioassays. Attenuation could be eliminated by exposing larvae to antibiotics only during the first instar without altering larval sterility. Prior antibiotic exposure did not attenuate Cry1Ac toxicity. The presence of enterococci in larval guts slowed mortality resulting from DiPel exposure and halved Cry1Ac toxicity but had little effect on B. thuringiensis HD73 Cry(-) spore/Cry1Ac synergism. B. thuringiensis Cry(-) cells killed larvae after intrahemocoelic inoculation of M. sexta, Galleria mellonella, and Spodoptera litura and grew rapidly in plasma from M. sexta, S. litura, and Tenebrio molitor. These findings suggest that gut bacteria are not required for B. thuringiensis insecticidal activity toward M. sexta but that B. thuringiensis lethality is reduced in larvae that are continuously exposed to antibiotics before bioassay. PMID:19525273

  12. A high-throughput, in-vitro assay for Bacillus thuringiensis insecticidal proteins.

    PubMed

    Izumi Willcoxon, Michi; Dennis, Jaclyn R; Lau, Sabina I; Xie, Weiping; You, You; Leng, Song; Fong, Ryan C; Yamamoto, Takashi

    2016-01-10

    A high-throughput, in-vitro assay for Bacillus thuringiensis (Bt) insecticidal proteins designated as Cry was developed and evaluated for screening a large number of Cry protein variants produced by DNA shuffling. This automation-amenable assay exploits an insect cell line expressing a single receptor of Bt Cry proteins. The Cry toxin used to develop this assay is a variant of the Cry1Ab protein called IP1-88, which was produced previously by DNA shuffling. Cell mortality caused by the activated Bt Cry toxin was determined by chemical cell viability assay in 96/384-well microtiter plates utilizing CellTiter 96(®) obtained from Promega. A widely-accepted mode-of-action theory of certain Bt Cry proteins suggests that the activated toxin binds to one or more receptors and forms a pore through the insect gut epithelial cell apical membrane. A number of insect proteins such as cadherin-like protein (Cad), aminopeptidase-N (APN), alkaline phosphatase (ALP) and ABC transporter (ABCC) have been identified as the receptors of Bt Cry toxins. In this study, Bt Cry toxin receptors Ostrinia nubilalis (European corn borer) cadherin-like protein (On-Cad) and aminopeptidase-N 1 and 3 (On-APN1, On-APN3) and Spodoptera frugiperda (fall armyworm) cadherin-like protein (Sf-Cad) were cloned in an insect cell line, Sf21, and a mammalian cell line, Expi293F. It was observed by ligand blotting and immunofluorescence microscopy that trypsin-activated IP1-88 bound to On-Cad and On-APN1, but not Sf-Cad or On-APN3. In contrast, IP1-88 bound only to APN1 in BBMV (Brush Border Membrane Vesicles) prepared from the third and fourth-instar O. nubilalis larval midgut. The sensitivity of the recombinant cells to the toxin was then tested. IP1-88 showed no toxicity to non-recombinant Sf21 and Expi293F. Toxicity was observed only when the On-Cad gene was cloned and expressed. Sf-Cad and On-APN1 were not able to make those cells sensitive to the toxin. Since the expression of On-Cad alone was

  13. A high-throughput, in-vitro assay for Bacillus thuringiensis insecticidal proteins.

    PubMed

    Izumi Willcoxon, Michi; Dennis, Jaclyn R; Lau, Sabina I; Xie, Weiping; You, You; Leng, Song; Fong, Ryan C; Yamamoto, Takashi

    2016-01-10

    A high-throughput, in-vitro assay for Bacillus thuringiensis (Bt) insecticidal proteins designated as Cry was developed and evaluated for screening a large number of Cry protein variants produced by DNA shuffling. This automation-amenable assay exploits an insect cell line expressing a single receptor of Bt Cry proteins. The Cry toxin used to develop this assay is a variant of the Cry1Ab protein called IP1-88, which was produced previously by DNA shuffling. Cell mortality caused by the activated Bt Cry toxin was determined by chemical cell viability assay in 96/384-well microtiter plates utilizing CellTiter 96(®) obtained from Promega. A widely-accepted mode-of-action theory of certain Bt Cry proteins suggests that the activated toxin binds to one or more receptors and forms a pore through the insect gut epithelial cell apical membrane. A number of insect proteins such as cadherin-like protein (Cad), aminopeptidase-N (APN), alkaline phosphatase (ALP) and ABC transporter (ABCC) have been identified as the receptors of Bt Cry toxins. In this study, Bt Cry toxin receptors Ostrinia nubilalis (European corn borer) cadherin-like protein (On-Cad) and aminopeptidase-N 1 and 3 (On-APN1, On-APN3) and Spodoptera frugiperda (fall armyworm) cadherin-like protein (Sf-Cad) were cloned in an insect cell line, Sf21, and a mammalian cell line, Expi293F. It was observed by ligand blotting and immunofluorescence microscopy that trypsin-activated IP1-88 bound to On-Cad and On-APN1, but not Sf-Cad or On-APN3. In contrast, IP1-88 bound only to APN1 in BBMV (Brush Border Membrane Vesicles) prepared from the third and fourth-instar O. nubilalis larval midgut. The sensitivity of the recombinant cells to the toxin was then tested. IP1-88 showed no toxicity to non-recombinant Sf21 and Expi293F. Toxicity was observed only when the On-Cad gene was cloned and expressed. Sf-Cad and On-APN1 were not able to make those cells sensitive to the toxin. Since the expression of On-Cad alone was

  14. Complete genome sequence of Bacillus thuringiensis YC-10, a novel active strain against plant-parasitic nematodes.

    PubMed

    Cheng, Feixue; Wang, Jian; Song, Zhiqiang; Cheng, Ju'e; Zhang, Deyong; Liu, Yong

    2015-09-20

    Bacillus thuringiensis is an important microbial biopesticide for controlling agricultural pests by the production of toxic parasporal crystals proteins.Here,we report the finished annotated genome sequence of B. thuringiensis YC-10,which is highly toxic to nematodes.The complete genome sequence consists of a circular chromosome and nine circular plasmids,which the biggest plasmid harbors six parasporal crystals proteins genes consisting of cry1Aa, cry1Ac, cry1Ia, cry2Aa, cry2Ab and cryB1. The crystals proteins of Cry1Ia and Cry1Aa have high nematicidal activity against Meloidogyne incognita.

  15. Complete genome sequence of Bacillus thuringiensis YC-10, a novel active strain against plant-parasitic nematodes.

    PubMed

    Cheng, Feixue; Wang, Jian; Song, Zhiqiang; Cheng, Ju'e; Zhang, Deyong; Liu, Yong

    2015-09-20

    Bacillus thuringiensis is an important microbial biopesticide for controlling agricultural pests by the production of toxic parasporal crystals proteins.Here,we report the finished annotated genome sequence of B. thuringiensis YC-10,which is highly toxic to nematodes.The complete genome sequence consists of a circular chromosome and nine circular plasmids,which the biggest plasmid harbors six parasporal crystals proteins genes consisting of cry1Aa, cry1Ac, cry1Ia, cry2Aa, cry2Ab and cryB1. The crystals proteins of Cry1Ia and Cry1Aa have high nematicidal activity against Meloidogyne incognita. PMID:26100235

  16. An amoeba phagocytosis model reveals a novel developmental switch in the insect pathogen Bacillus thuringiensis.

    PubMed

    Beeton, M L; Atkinson, D J; Waterfield, N R

    2013-02-01

    The Bacillus cereus group bacteria contain pathogens of economic and medical importance. From security and health perspectives, the lethal mammalian pathogen Bacillus anthracis remains a serious threat. In addition the potent insect pathogen Bacillus thuringiensis is extensively used as a biological control agent for insect pests. This relies upon the industrial scale induction of bacterial spore formation with the associated production of orally toxic Cry-toxins. Understanding the ecology and potential alternative developmental fates of these bacteria is therefore important. Here we describe the use of an amoeba host model to investigate the influence of environmental bactivorous protists on both spores and vegetative cells of these pathogens. We demonstrate that the bacteria can respond to different densities of amoeba by adopting different behaviours and developmental fates. We show that spores will germinate in response to factors excreted by the amoeba, and that the bacteria can grow and reproduce on these factors. We show that in low densities of amoeba, that the bacteria will seek to colonise the surface of the amoeba as micro-colonies, resisting phagocytosis. At high amoeba densities, the bacteria change morphology into long filaments and macroscopic rope-like structures which cannot be ingested due to size exclusion. We suggest these developmental fates are likely to be important both in the ecology of these bacteria and also during animal host colonisation and immune evasion. PMID:22750551

  17. An amoeba phagocytosis model reveals a novel developmental switch in the insect pathogen Bacillus thuringiensis.

    PubMed

    Beeton, M L; Atkinson, D J; Waterfield, N R

    2013-02-01

    The Bacillus cereus group bacteria contain pathogens of economic and medical importance. From security and health perspectives, the lethal mammalian pathogen Bacillus anthracis remains a serious threat. In addition the potent insect pathogen Bacillus thuringiensis is extensively used as a biological control agent for insect pests. This relies upon the industrial scale induction of bacterial spore formation with the associated production of orally toxic Cry-toxins. Understanding the ecology and potential alternative developmental fates of these bacteria is therefore important. Here we describe the use of an amoeba host model to investigate the influence of environmental bactivorous protists on both spores and vegetative cells of these pathogens. We demonstrate that the bacteria can respond to different densities of amoeba by adopting different behaviours and developmental fates. We show that spores will germinate in response to factors excreted by the amoeba, and that the bacteria can grow and reproduce on these factors. We show that in low densities of amoeba, that the bacteria will seek to colonise the surface of the amoeba as micro-colonies, resisting phagocytosis. At high amoeba densities, the bacteria change morphology into long filaments and macroscopic rope-like structures which cannot be ingested due to size exclusion. We suggest these developmental fates are likely to be important both in the ecology of these bacteria and also during animal host colonisation and immune evasion.

  18. Characterization of Bacillus thuringiensis isolates with potential for control of Aedes aegypti (Linnaeus, 1762) (Diptera: Culicidae).

    PubMed

    Santos, F P; Lopes, J; Vilas-Bôas, G T; Zequi, J A C

    2012-04-01

    Aedes aegypti (Linnaeus) is the vector of dengue virus in Brazil. Bioinsecticides based on Bacillus thuringiensis have shown satisfactory results in the control of Diptera, due to the production of proteins called Cry and Cyt. The aim of the present study was to select B. thuringiensis isolates carrying the cry and cyt genes, which are efficient in the control of Ae. aegypti. A collection of 27 isolates of B. thuringiensis, derived from various regions in Brazil, was evaluated using selective bioassays against A. aegypti larvae. Of the 27 isolates, five showed 100% larval mortality at a concentration of 0.05 ppm and the toxicity of these isolates in quantitative bioassays did not differ significantly at temperatures of 15, 25 and 35 °C. In addition, these isolates showed statistical differences for the LC50 values only above pH 9, which indicates a maintenance in insecticide potential in a wide range of alkaline pH values. This result is promising, considering that waste treatment reservoirs generally show an acid pH and higher temperatures. These isolates were also evaluated by PCR using specific primers for the genes cry4A, cry4B, cry10A, cry11, cyt1 and cyt2. The analyses demonstrated that all the five isolates showed amplification products for all the studied genes showing four different Cry proteins, besides Cyt proteins. The obtained results of bioassays and PCR demonstrates the great potential for the use of these isolates in controlling populations of Ae. Aegypti and perhaps other species of mosquitoes in a wide range of environments.

  19. Isolation and characterization of a new Bacillus thuringiensis strain with a promising toxicity against Lepidopteran pests.

    PubMed

    Boukedi, Hanen; Sellami, Sameh; Ktari, Sonia; Belguith-Ben Hassan, Najeh; Sellami-Boudawara, Tahya; Tounsi, Slim; Abdelkefi-Mesrati, Lobna

    2016-01-01

    Insecticides derived from Bacillus thuringiensis are gaining worldwide importance as environmentally desirable alternatives to chemicals for the control of pests in public health and agriculture. Isolation and characterization of new strains with higher and broader spectrum of activity is an ever growing field. In the present work, a novel Tunisian B. thuringiensis isolate named BLB459 was characterized and electrophoresis assay showed that among a collection of 200 B. thuringiensis strains, the plasmid profile of BLB459 was distinctive. SmaI-PFGE typing confirmed the uniqueness of the DNA pattern of this strain, compared with BUPM95 and HD1 reference strains. PCR and sequencing assays revealed that BLB459 harbored three cry genes (cry30, cry40 and cry54) corresponding to the obtained molecular sizes in the protein pattern. Interestingly, PCR-RFLP assay demonstrated the originality of the BLB459 cry30-type gene compared to the other published cry30 genes. Insecticidal bioassays showed that BLB459 spore-crystal suspension was highly toxic to both Ephestia kuehniella and Spodoptera littoralis with LC50 values of about 64 (53-75) and 80 (69-91) μg of toxin cm(-2), respectively, comparing with that of the commercial strain HD1 used as reference. Important histopathological effects of BLB459 δ-endotoxins on the two tested larvae midguts were detected, traduced by the vacuolization of the apical cells, the damage of microvilli, and the disruption of epithelial cells. These results proved that BLB459 strain could be of a great interest for lepidopteran biocontrol. PMID:27242138

  20. Purification and characterization of a new Bacillus thuringiensis bacteriocin active against Listeria monocytogenes, Bacillus cereus and Agrobacterium tumefaciens.

    PubMed

    Kamoun, Fakher; Fguira, Ines Ben; Hassen, Najeh Belguith Ben; Mejdoub, Hafedh; Lereclus, Didier; Jaoua, Samir

    2011-09-01

    This study reports on the identification, characterization and purification of a new bacteriocin, named Bacthuricin F103, from a Bacillus thuringiensis strain BUPM103. Bacthuricin F103 production began in the early exponential phase and reached a maximum in the middle of the same phase. Two chromatographic methods based on high performance liquid chromatography and fast protein liquid chromatography systems were used to purify Bacthuricin F103. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis revealed that this bacteriocin had a molecular weight of approximately 11 kDa. It also showed a wide range of thermostability of up to 80 °C for 60 min and a broad spectrum of antimicrobial activity over a pH range of 3.0-10.0. This bacteriocin was noted, and for the first time, to exhibit potent antimicrobial activity against Agrobacterium subsp. strains, the major causal agents of crown gall disease in tomato and vineyard crops, and against several challenging organisms in food, such as Listeria monocytogenes and Bacillus cereus. Complete killing with immediate impact on cells was observed within a short period of time. The sequence obtained for Bacthuricin F103 by direct N-terminal sequencing shared considerable homology with hemolysin. Bacthuricin F103 was noted to act through the depletion of intracellular ions, which suggest that the cell membrane was a possible target to Bacthuricin F103. PMID:21487734

  1. Efficiency of Intergeneric Recombinants Between Bacillus Thuringiensis and Bacillus Subtilis for Increasing Mortality Rate in Cotten Leaf Worm

    NASA Astrophysics Data System (ADS)

    AlOtaibi, Saad Aied

    2012-12-01

    In this study , two strains of Bacillus belonging to two serotypes and four of their transconjugants were screened with respect to their toxicity against lepidopterous cotton pest. . Bacterial transconjugants isolated from conjugation between both strains were evaluated for their transconjugant efficiency caused mortality in Spodoptera littoralis larvae . Two groups of bioinsecticides ; crystals , crystals and spores have been isolated from Bacillusstrains and their transconjugants . Insecticidal crystal protein ( ICP ) was specific for lepidopteran insects because of the toxin sufficient both for insect specificity and toxicity . The toxicities of these two groups against larvae of Spodoptera littoralis was expressed as transconjugant efficiency , which related to the mean number of larvae died expressed as mortality percentage . The results showed transconjugant efficiency in reducing the mean number of Spodoptera littoralis larvae feeding on leaves of Ricinus communis sprayed with bioinsecticides of Bt transconjugants. Most values of positive transconjugant efficiency related to increasing mortality percentage are due to toxicological effects appeared in response to the treatments with crystals + endospores than that of crystals alone .This indicated that crystals + endospores was more effective for increasing mortality percentage than that resulted by crystals . Higher positive transconjugant efficiency in relation to the mid parents and better parent was appeared at 168 h of treatment . The results indicated that recombinant Bacillus thuringiensis are important control agents for lepidopteran pests , as well as , susceptibility decreased with larval development . The results also suggested a potential for the deployment of these recominant entomopathogens in the management of Spodoptera. littoralis larvae .

  2. Persistence of Bt Bacillus thuringiensis Cry1Aa toxin in various soils determined by physicochemical reactions

    NASA Astrophysics Data System (ADS)

    Helassa, N.; Noinville, S.; Déjardin, P.; Janot, J. M.; Quiquampoix, H.; Staunton, S.

    2009-04-01

    Insecticidal Cry proteins from the soil bacterium, Bacillus thuringiensis (Bt) are produced by a class of genetically modified (GM) crops, and released into soils through root exudates and upon decomposition of residues. In contrast to the protoxin produced by the Bacillus, the protein produced in GM crops does not require activation in insect midguts and thereby potentially looses some of its species specificity. Although gene transfer and resistance emergence phenomena are well documented, the fate of these toxins in soil has not yet been clearly elucidated. Cry proteins, in common with other proteins, are adsorbed on soils and soil components. Adsorption on soil, and the reversibility of this adsorption is an important aspect of the environmental behaviour of these toxins. The orientation of the molecule and conformational changes on surfaces may modify the toxicity and confer some protection against microbial degradation. Adsorption will have important consequences for both the risk of exposition of non target species and the acquisition of resistance by target species. We have adopted different approaches to investigate the fate of Cry1Aa in soils and model minerals. In each series of experiments we endeavoured to maintain the protein in a monomeric form (pH above 6.5 and a high ionic strength imposed with 150 mM NaCl). The adsorption and the desorbability of the Cry1Aa Bt insecticidal protein were measured on two different homoionic clays: montmorillonite and kaolinite. Adsorption isotherms obtained followed a low affinity interaction for both clays and could be fitted using the Langmuir equation. Binding of the toxin decreased as the pH increased from 6.5 (close to the isoelectric point) to 9. Maximum adsorption was about 40 times greater on montmorillonite (1.71 g g-1) than on kaolinite (0.04 g g-1) in line with the contrasting respective specific surface areas of the minerals. Finally, some of the adsorbed toxin was desorbed by water and more, about 36

  3. Decolorization of dyehouse effluent and biodegradation of Congo red by Bacillus thuringiensis RUN1.

    PubMed

    Olukanni, Olumide David; Osuntoki, Akinniyi A; Awotula, Ayodeji Olushola; Kalyani, Dayanand C; Gbenle, George Olabode; Govindwar, Sanjay P

    2013-06-28

    A dye-decolorizing bacterium was isolated from a soil sample and identified as Bacillus thuringiensis using 16S rRNA sequencing. The bacterium was able to decolorize three different textile dyes, namely, Reactive blue 13, Reactive red 58, and Reactive yellow 42, and a real dyehouse effluent up to 80-95% within 6 h. Some non-textile industrially important dyes were also decolorized to different extents. Fourier transform infrared spectroscopy and gas chromatography-mass spectrometer analysis of the ethyl acetate extract of Congo red dye and its metabolites showed that the bacterium could degrade it by the asymmetric cleavage of the azo bonds to yield sodium (4- amino-3-diazenylnaphthalene-1-sulfonate) and phenylbenzene. Sodium (4-amino-3-diazenylnaphthalene-1-sulfonate) was further oxidized by the ortho-cleavage pathway to yield 2- (1-amino-2-diazenyl-2-formylvinyl) benzoic acid. There was induction of the activities of laccase and azoreductase during the decolorization of Congo red, which suggests their probable role in the biodegradation. B. thuringiensis was found to be versatile and could be used for industrial effluent biodegradation.

  4. Combining hexanoic acid plant priming with Bacillus thuringiensis insecticidal activity against Colorado potato beetle.

    PubMed

    García-Robles, Inmaculada; Ochoa-Campuzano, Camila; Fernández-Crespo, Emma; Camañes, Gemma; Martínez-Ramírez, Amparo C; González-Bosch, Carmen; García-Agustín, Pilar; Rausell, Carolina; Real, María Dolores

    2013-01-01

    Interaction between insect herbivores and host plants can be modulated by endogenous and exogenous compounds present in the source of food and might be successfully exploited in Colorado potato beetle (CPB) pest management. Feeding tests with CPB larvae reared on three solanaceous plants (potato, eggplant and tomato) resulted in variable larval growth rates and differential susceptibility to Bacillus thuringiensis Cry3Aa toxin as a function of the host plant. An inverse correlation with toxicity was observed in Cry3Aa proteolytic patterns generated by CPB midgut brush-border membrane vesicles (BBMV) from Solanaceae-fed larvae, being the toxin most extensively proteolyzed on potato, followed by eggplant and tomato. We found that CPB cysteine proteases intestains may interact with Cry3Aa toxin and, in CPB BBMV from larvae fed all three Solanaceae, the toxin was able to compete for the hydrolysis of a papain substrate. In response to treatment with the JA-dependent plant inducer Hexanoic acid (Hx), we showed that eggplant reduced OPDA basal levels and both, potato and eggplant induced JA-Ile. CPB larvae feeding on Hx-induced plants exhibited enhanced Cry3Aa toxicity, which correlated with altered papain activity. Results indicated host-mediated effects on B. thuringiensis efficacy against CPB that can be enhanced in combination with Hx plant induction. PMID:23743826

  5. Novel toxicity of Bacillus thuringiensis strains against the melon fruit fly, Bactrocera cucurbitae (Diptera: Tephritidae).

    PubMed

    Shishir, Md Asaduzzaman; Akter, Asma; Bodiuzzaman, Md; Hossain, M Aftab; Alam, Md Musfiqul; Khan, Shakil Ahmed; Khan, Shakila Nargis; Hoq, M Mozammel

    2015-01-01

    Bactrocera cucurbitae (melon fruit fly) is one of the most detrimental vegetable-damaging pests in Bangladesh. The toxicity of Bacillus thuringiensis (Bt) has been reported against a few genera of Bactrocera in addition to numerous other insect species. Bt strains, harbouring cry1A-type genes were, therefore, assayed in vivo against the 3(rd) instar larvae of B. cucurbitae in this study. The biotype-based prevalence of cry1 and cry1A genes was calculated to be 30.8% and 11.16%, respectively, of the test strains (n=224) while their prevalence was greatest in biotype kurstaki. Though three indigenous Bt strains from biotype kurstaki with close genetic relationship exhibited higher toxicity, maximum mortalities were recorded for Btk HD-73 (96%) and the indigenous Bt JSc1 (93%). LC50 and LC99 values were determined to be 6.81 and 8.32 for Bt JSc1, 7.30 and 7.92 for Bt SSc2, and 6.99 and 7.67 for Btk HD-73, respectively. The cause of toxicity and its variation among the strains was found to be correlated with the synergistic toxic effects of cry1, cry2, cry3 and cry9 gene products, i.e. relevant Cry proteins. The novel toxicity of the B. thuringiensis strains against B. cucurbitae revealed in the present study thus will help in developing efficient and eco-friendly control measures such as Bt biopesticides and transgenic Bt cucurbits.

  6. Bacillus thuringiensis strain 199 can induce systemic resistance in tomato against Fusarium wilt

    PubMed Central

    Mahboob, Asrar; Javed, Asmat Ali

    2013-01-01

    The research work was performed to investigate the potential of Bacillus thuringiensis strain 199 to induce systemic resistance in tomato against Fusarium wilt. Roots of two-week-old seedlings of tomato plants were primed with bacterial strain. After 10 days of transplantation, some pots of tomato seedlings were provided with inoculum of Fusarium oxysporum lycopersici according to experimental design to induce disease. After 15 days of incubation period, plants challenged with F. oxysporum lycopersici alone were having obvious symptoms of Fusarium wilt. Plants that were treated with B. thuringiensis 199 + F. oxysporum lycopersici were having significant reduction of disease severity. Quantity of total phenolics increased 1.7-fold in bacterial-treated plants as compared to nontreated. Likewise, in case of defense-related enzymes, a significant increase of 1.3-, 1.8-, and 1.4-fold in polyphenol oxidase (PPO), phenyl ammonia lyase (PAL), and peroxidase (PO) was observed in comparison with untreated control. These results, hence, prove the potential of this bacterial strain for use as plant protection agent. PMID:24294498

  7. Beta-glucosidase enzymatic activity of crystal polypeptide of the Bacillus thuringiensis strain 1.1.

    PubMed

    Papalazaridou, A; Charitidou, L; Sivropoulou, A

    2003-01-01

    The crystals of Bacillus thuringiensis strain 1.1 consist of the 140 kDa delta-endotoxin, which exhibits beta-glucosidase enzymatic activity, based on the following data. (i) Purified crystals exhibit beta-glucosidase enzymatic activity. When the crystals are reacted with specific antibodies directed either against the commercial (almond purified) beta-glucosidase or against the 140 kDa polypeptide, then considerable reduction of enzymatic activity is observed almost at the same level with both antibodies. (ii) Commercial beta-glucosidase and the 140 kDa crystal polypeptide share antigenic similarities; in Western immunoblots, the 140 kDa crystal polypeptide is recognized by anti-beta-glucosidase antibodies, and commercial beta-glucosidase is recognized by anti-140-kDa antibodies. (iii) The enzymatic properties of commercial beta-glucosidase and that resident in the crystals of B. thuringiensis strain 1.1 are very similar. Thus, both enzymes hydrolyze a wide range of substrates (aryl-beta-glucosides, disaccharides with alpha- or beta-linkage polysaccharides) and have an optimum activity at 40 degrees C and pH 5. Both enzymes are relatively thermostable and are resistant to end-product inhibition by glucose. Additionally, they show the same pattern of inhibition or activation by several chemical compounds. (iv) The crystals and commercial beta-glucosidase show almost equivalent levels of insecticidal activity against Drosophila melanogaster larvae and, furthermore, cause reduction in adult flies that emerge from larvae surviving treatment.

  8. Cytotoxicity Analysis of Three Bacillus thuringiensis Subsp. israelensis δ-Endotoxins towards Insect and Mammalian Cells

    PubMed Central

    Teixeira Corrêa, Roberto Franco; Ardisson-Araújo, Daniel Mendes Pereira; Monnerat, Rose Gomes; Ribeiro, Bergmann Morais

    2012-01-01

    Three members of the δ-endotoxin group of toxins expressed by Bacillus thuringiensis subsp. israelensis, Cyt2Ba, Cry4Aa and Cry11A, were individually expressed in recombinant acrystalliferous B. thuringiensis strains for in vitro evaluation of their toxic activities against insect and mammalian cell lines. Both Cry4Aa and Cry11A toxins, activated with either trypsin or Spodoptera frugiperda gastric juice (GJ), resulted in different cleavage patterns for the activated toxins as seen by SDS-PAGE. The GJ-processed proteins were not cytotoxic to insect cell cultures. On the other hand, the combination of the trypsin-activated Cry4Aa and Cry11A toxins yielded the highest levels of cytotoxicity to all insect cells tested. The combination of activated Cyt2Ba and Cry11A also showed higher toxic activity than that of toxins activated individually. When activated Cry4Aa, Cry11A and Cyt2Ba were used simultaneously in the same assay a decrease in toxic activity was observed in all insect cells tested. No toxic effect was observed for the trypsin-activated Cry toxins in mammalian cells, but activated Cyt2Ba was toxic to human breast cancer cells (MCF-7) when tested at 20 µg/mL. PMID:23029407

  9. Intravital imaging of Bacillus thuringiensis Cry1A toxin binding sites in the midgut of silkworm.

    PubMed

    Li, Na; Wang, Jing; Han, Heyou; Huang, Liang; Shao, Feng; Li, Xuepu

    2014-02-15

    Identification of the resistance mechanism of insects against Bacillus thuringiensis Cry1A toxin is becoming an increasingly challenging task. This fact highlights the need for establishing new methods to further explore the molecular interactions of Cry1A toxin with insects and the receptor-binding region of Cry1A toxins for their wider application as biopesticides and a gene source for gene-modified crops. In this contribution, a quantum dot-based near-infrared fluorescence imaging method has been applied for direct dynamic tracking of the specific binding of Cry1A toxins, CrylAa and CrylAc, to the midgut tissue of silkworm. The in vitro fluorescence imaging displayed the higher binding specificity of CrylAa-QD probes compared to CrylAc-QD to the brush border membrane vesicles of midgut from silkworm. The in vivo imaging demonstrated that more CrylAa-QDs binding to silkworm midgut could be effectively and distinctly monitored in living silkworms. Furthermore, frozen section analysis clearly indicated the broader receptor-binding region of Cry1Aa compared to that of Cry1Ac in the midgut part. These observations suggest that the insecticidal activity of Cry toxins may depend on the receptor-binding sites, and this scatheless and visual near-infrared fluorescence imaging could provide a new avenue to study the resistance mechanism to maintain the insecticidal activity of B. thuringiensis toxins. PMID:24252542

  10. Cytotoxicity analysis of three Bacillus thuringiensis subsp. israelensis δ-endotoxins towards insect and mammalian cells.

    PubMed

    Teixeira Corrêa, Roberto Franco; Ardisson-Araújo, Daniel Mendes Pereira; Monnerat, Rose Gomes; Ribeiro, Bergmann Morais

    2012-01-01

    Three members of the δ-endotoxin group of toxins expressed by Bacillus thuringiensis subsp. israelensis, Cyt2Ba, Cry4Aa and Cry11A, were individually expressed in recombinant acrystalliferous B. thuringiensis strains for in vitro evaluation of their toxic activities against insect and mammalian cell lines. Both Cry4Aa and Cry11A toxins, activated with either trypsin or Spodoptera frugiperda gastric juice (GJ), resulted in different cleavage patterns for the activated toxins as seen by SDS-PAGE. The GJ-processed proteins were not cytotoxic to insect cell cultures. On the other hand, the combination of the trypsin-activated Cry4Aa and Cry11A toxins yielded the highest levels of cytotoxicity to all insect cells tested. The combination of activated Cyt2Ba and Cry11A also showed higher toxic activity than that of toxins activated individually. When activated Cry4Aa, Cry11A and Cyt2Ba were used simultaneously in the same assay a decrease in toxic activity was observed in all insect cells tested. No toxic effect was observed for the trypsin-activated Cry toxins in mammalian cells, but activated Cyt2Ba was toxic to human breast cancer cells (MCF-7) when tested at 20 µg/mL. PMID:23029407

  11. Combining Hexanoic Acid Plant Priming with Bacillus thuringiensis Insecticidal Activity against Colorado Potato Beetle

    PubMed Central

    García-Robles, Inmaculada; Ochoa-Campuzano, Camila; Fernández-Crespo, Emma; Camañes, Gemma; Martínez-Ramírez, Amparo C.; González-Bosch, Carmen; García-Agustín, Pilar; Rausell, Carolina; Real, María Dolores

    2013-01-01

    Interaction between insect herbivores and host plants can be modulated by endogenous and exogenous compounds present in the source of food and might be successfully exploited in Colorado potato beetle (CPB) pest management. Feeding tests with CPB larvae reared on three solanaceous plants (potato, eggplant and tomato) resulted in variable larval growth rates and differential susceptibility to Bacillus thuringiensis Cry3Aa toxin as a function of the host plant. An inverse correlation with toxicity was observed in Cry3Aa proteolytic patterns generated by CPB midgut brush-border membrane vesicles (BBMV) from Solanaceae-fed larvae, being the toxin most extensively proteolyzed on potato, followed by eggplant and tomato. We found that CPB cysteine proteases intestains may interact with Cry3Aa toxin and, in CPB BBMV from larvae fed all three Solanaceae, the toxin was able to compete for the hydrolysis of a papain substrate. In response to treatment with the JA-dependent plant inducer Hexanoic acid (Hx), we showed that eggplant reduced OPDA basal levels and both, potato and eggplant induced JA-Ile. CPB larvae feeding on Hx-induced plants exhibited enhanced Cry3Aa toxicity, which correlated with altered papain activity. Results indicated host-mediated effects on B. thuringiensis efficacy against CPB that can be enhanced in combination with Hx plant induction. PMID:23743826

  12. Combining hexanoic acid plant priming with Bacillus thuringiensis insecticidal activity against Colorado potato beetle.

    PubMed

    García-Robles, Inmaculada; Ochoa-Campuzano, Camila; Fernández-Crespo, Emma; Camañes, Gemma; Martínez-Ramírez, Amparo C; González-Bosch, Carmen; García-Agustín, Pilar; Rausell, Carolina; Real, María Dolores

    2013-06-06

    Interaction between insect herbivores and host plants can be modulated by endogenous and exogenous compounds present in the source of food and might be successfully exploited in Colorado potato beetle (CPB) pest management. Feeding tests with CPB larvae reared on three solanaceous plants (potato, eggplant and tomato) resulted in variable larval growth rates and differential susceptibility to Bacillus thuringiensis Cry3Aa toxin as a function of the host plant. An inverse correlation with toxicity was observed in Cry3Aa proteolytic patterns generated by CPB midgut brush-border membrane vesicles (BBMV) from Solanaceae-fed larvae, being the toxin most extensively proteolyzed on potato, followed by eggplant and tomato. We found that CPB cysteine proteases intestains may interact with Cry3Aa toxin and, in CPB BBMV from larvae fed all three Solanaceae, the toxin was able to compete for the hydrolysis of a papain substrate. In response to treatment with the JA-dependent plant inducer Hexanoic acid (Hx), we showed that eggplant reduced OPDA basal levels and both, potato and eggplant induced JA-Ile. CPB larvae feeding on Hx-induced plants exhibited enhanced Cry3Aa toxicity, which correlated with altered papain activity. Results indicated host-mediated effects on B. thuringiensis efficacy against CPB that can be enhanced in combination with Hx plant induction.

  13. Electron Microscope Study of Sporulation and Parasporal Crystal Formation in Bacillus thuringiensis

    PubMed Central

    Bechtel, Donald B.; Bulla, Lee A.

    1976-01-01

    A comprehensive ultrastructural analysis of sporulation and parasporal crystal development is described for Bacillus thuringiensis. The insecticidal crystal of B. thuringiensis is initiated at the start of engulfment and is nearly complete by the time the exosporium forms. The crystal and a heretofore unobserved ovoid inclusion develop without any clear association with the forespore septum, exosporium, or mesosomes. These observations contradict previous hypotheses that the crystal is synthesized on the forespore membrane, exosporium, or mesosomes. Formation of forespore septa involves densely staining, double-membrane-bound, vesicular mesosomes that have a bridged appearance. Forespore engulfment is subpolar and also involves mesosomes. Upon completion of engulfment the following cytoplasmic changes occur: decrease in electron density of the incipient forespore membrane; loss of bridged appearance of incipient forespore membrane; change in stainability of incipient forespore, forespore, and mother cell cytoplasms; and alteration in staining quality of plasma membrane. These changes are involved in the conversion of the incipient forespore into a forespore and reflect “commitment” to sporulation. Images PMID:182671

  14. High instability of a nematicidal Cry toxin plasmid in Bacillus thuringiensis.

    PubMed

    Sheppard, Anna E; Nakad, Rania; Saebelfeld, Manja; Masche, Anna C; Dierking, Katja; Schulenburg, Hinrich

    2016-01-01

    In bacterial pathogens, virulence factors are often carried on plasmids and other mobile genetic elements, and as such, plasmid evolution is central in understanding pathogenicity. Bacillus thuringiensis is an invertebrate pathogen that uses plasmid-encoded crystal (Cry) toxins to establish infections inside the host. Our study aimed to quantify stability of two Cry toxin-encoding plasmids, BTI_23p and BTI_16p, under standard laboratory culturing conditions. These two plasmids are part of the genome of the B. thuringiensis strain MYBT18679, which is of particular interest because of its high pathogenicity towards nematodes. One of the plasmids, BTI_23p, was found to be highly unstable, with substantial loss occurring within a single growth cycle. Nevertheless, longer term experimental evolution in the absence of a host revealed maintenance of the plasmid at low levels in the bacterial populations. BTI_23p encodes two nematicidal Cry toxins, Cry21Aa2 and Cry14Aa1. Consistent with previous findings, loss of the plasmid abolished pathogenicity towards the nematode Caenorhabditis elegans, which could be rescued by addition of Cry21Aa2-expressing Escherichia coli. These results implicate BTI_23p as a plasmid that is required for successful infection, yet unstable when present at high frequency in the population, consistent with the role of Cry toxins as public goods. PMID:26592941

  15. Novel toxicity of Bacillus thuringiensis strains against the melon fruit fly, Bactrocera cucurbitae (Diptera: Tephritidae).

    PubMed

    Shishir, Md Asaduzzaman; Akter, Asma; Bodiuzzaman, Md; Hossain, M Aftab; Alam, Md Musfiqul; Khan, Shakil Ahmed; Khan, Shakila Nargis; Hoq, M Mozammel

    2015-01-01

    Bactrocera cucurbitae (melon fruit fly) is one of the most detrimental vegetable-damaging pests in Bangladesh. The toxicity of Bacillus thuringiensis (Bt) has been reported against a few genera of Bactrocera in addition to numerous other insect species. Bt strains, harbouring cry1A-type genes were, therefore, assayed in vivo against the 3(rd) instar larvae of B. cucurbitae in this study. The biotype-based prevalence of cry1 and cry1A genes was calculated to be 30.8% and 11.16%, respectively, of the test strains (n=224) while their prevalence was greatest in biotype kurstaki. Though three indigenous Bt strains from biotype kurstaki with close genetic relationship exhibited higher toxicity, maximum mortalities were recorded for Btk HD-73 (96%) and the indigenous Bt JSc1 (93%). LC50 and LC99 values were determined to be 6.81 and 8.32 for Bt JSc1, 7.30 and 7.92 for Bt SSc2, and 6.99 and 7.67 for Btk HD-73, respectively. The cause of toxicity and its variation among the strains was found to be correlated with the synergistic toxic effects of cry1, cry2, cry3 and cry9 gene products, i.e. relevant Cry proteins. The novel toxicity of the B. thuringiensis strains against B. cucurbitae revealed in the present study thus will help in developing efficient and eco-friendly control measures such as Bt biopesticides and transgenic Bt cucurbits. PMID:26133509

  16. Immobilization of alginate-encapsulated Bacillus thuringiensis var. israelensis containing different multivalent counterions for mosquito control.

    PubMed

    Prabakaran, G; Hoti, S L

    2008-08-01

    Immobilized techniques have been used widely for the controlled release formulation of mosquitoes. Among the microbial formulations, polymeric matrices play an important role in the controlled release of microbial pesticide at rates sufficiently effective to kill mosquitoes in the field. The advantage of these matrices is that they enhance the stability of both spores and toxin against pH, temperature variations, and UV irradiation. The disadvantage of using calcium alginate beads is that they are unstable upon contact with phosphate of potassium or sodium ions rich in the mosquito habitats. To overcome these problems, attempts were made to encapsulate Bacillus thuringiensis var. israelensis within alginate by using different multivalent counterions, namely, calcium chloride, zinc sulfate, copper sulfate, cobalt chloride, and ferric chloride, and the beads formed were tested for its mosquito larvicidal activity. Among all the beads tested, zinc alginate beads resulted in maximum larvicidal activity of 98% (+/-1.40 SE) against Culex quinquefasciatus IIIrd instar larvae and maximum spore count of 3.36 x 10(5) (+/-5291.50 SE) CFU/ml. Zinc alginate beads maintained their structure for up to 48 h when shaken vigorously on a rotary shaker at 180 rpm in the presence of 10 mM potassium phosphate buffer (pH 6.8 +/- 0.1). In conclusion, our results suggest that the use of zinc sulfate as counterions to encapsulate B. thuringiensis var. israelensis within alginate may be a potent mosquito control program in the habitats where more phosphate ions are present.

  17. Protist-type lysozymes of the nematode Caenorhabditis elegans contribute to resistance against pathogenic Bacillus thuringiensis.

    PubMed

    Boehnisch, Claudia; Wong, Daniel; Habig, Michael; Isermann, Kerstin; Michiels, Nicolaas K; Roeder, Thomas; May, Robin C; Schulenburg, Hinrich

    2011-01-01

    Pathogens represent a universal threat to other living organisms. Most organisms express antimicrobial proteins and peptides, such as lysozymes, as a protection against these challenges. The nematode Caenorhabditis elegans harbours 15 phylogenetically diverse lysozyme genes, belonging to two distinct types, the protist- or Entamoeba-type (lys genes) and the invertebrate-type (ilys genes) lysozymes. In the present study we characterized the role of several protist-type lysozyme genes in defence against a nematocidal strain of the Gram-positive bacterium Bacillus thuringiensis. Based on microarray and subsequent qRT-PCR gene expression analysis, we identified protist-type lysozyme genes as one of the differentially transcribed gene classes after infection. A functional genetic analysis was performed for three of these genes, each belonging to a distinct evolutionary lineage within the protist-type lysozymes (lys-2, lys-5, and lys-7). Their knock-out led to decreased pathogen resistance in all three cases, while an increase in resistance was observed when two out of three tested genes were overexpressed in transgenic lines (lys-5, lys-7, but not lys-2). We conclude that the lysozyme genes lys-5, lys-7, and possibly lys-2 contribute to resistance against B. thuringiensis, thus highlighting the particular role of lysozymes in the nematode's defence against pathogens.

  18. Isolation and characterization of a novel insecticidal crystal protein gene from Bacillus thuringiensis subsp. aizawai.

    PubMed

    Chambers, J A; Jelen, A; Gilbert, M P; Jany, C S; Johnson, T B; Gawron-Burke, C

    1991-07-01

    Bacillus thuringiensis subsp. aizawai EG6346, a novel grain dust isolate, was analyzed by Southern blot hybridization for its insecticidal crystal protein (ICP) gene profile. Strain EG6346 lacks previously characterized cryIA ICP genes yet does possess novel cryI-related gene sequences. A recombinant genomic plasmid library was constructed for strain EG6346 in Escherichia coli. One recombinant plasmid, pEG640, isolated from the library contained a novel ICP gene on a 5.7-kb Sau3A insert. The sequence of this gene, designated cryIF, was related to, but distinct from, the published sequences for other cryI genes. A second novel cryI-related sequence was also located on pEG640, approximately 500 bp downstream from cryIF. Introduction of cryIF into a Cry- B. thuringiensis recipient strain via electroporation enabled sufficient production of CryIF protein for quantitative bioassay analyses of insecticidal specificity. The CryIF crystal protein was selectively toxic to a subset of lepidopteran insects tested, including the larvae of Ostrinia nubilalis and Spodoptera exigua. PMID:2061280

  19. Toxicological safety assessment of genetically modified Bacillus thuringiensis with additional N-acyl homoserine lactonase gene.

    PubMed

    Peng, Donghai; Zhou, Chenfei; Chen, Shouwen; Ruan, Lifang; Yu, Ziniu; Sun, Ming

    2008-01-01

    The aim of the present study is to evaluate the toxicology safety to mammals of a genetically modified (GM) Bacillus thuringiensis with an additional N-acyl homoserine lactones gene (aiiA), which possesses insecticidal activity together with restraint of bacterial pathogenicity and is intended for use as a multifunctional biopesticide. Safety assessments included an acute oral toxicity test and 28-d animal feeding study in Wistar rats, primary eye and dermal irritation in Zealand White rabbits, and delayed contact hypersensitivity in guinea pigs. Tests were conducted using spray-dried powder preparation. This GM product showed toxicity neither in oral acute toxicity test nor in 28-d animal feeding test at a dose of 5,000 mg/kg body weight. During the animal feeding test, there were no significant differences in growth, food and water consumption, hematology, blood biochemical indices, organ weights, and histopathology finding between rats in controls and tested groups. Tested animals in primary eye and dermal irritation and delayed contact hypersensitivity test were also devoid of any toxicity compared to controls. All the above results demonstrated that the GM based multifunctional B. thuringiensis has low toxicity and low eye and dermal irritation and would not cause hypersensitivity to laboratory mammals and therefore could be regarded as safe for use as a pesticide.

  20. Analysis of insecticidal proteins from Bacillus thuringiensis and recombinant Escherichia coli by capillary electrokinetic chromatography.

    PubMed

    Luong, John H T; Male, Keith B; Mazza, Alberto; Masson, Luke; Brousseau, Roland

    2004-10-01

    Bacillus thuringiensis and recombinant Escherichia coli proteinaceous protoxins were subject to proteolysis and analyzed by capillary electrokinetic chromatography. Three resulting toxins (65 kDa) were baseline-resolved within 22 min using a 10 mM borate, pH 11 separation buffer consisting of 25 mM sodium dodecyl sulfate (SDS) and 30 mM phytic acid. The toxins displayed differential interactions with the SDS and phytic acid phases to effect their separation. The ion-pairing interaction between the analyte and phytic acid was also useful in preventing adsorption to the capillary walls and thus enhanced separation resolution and efficiency. The use of electrokinetic chromatography allows achievement of the separation in a significantly shorter time than conventional high-performance liquid chromatography (HPLC) using a diethylaminoethyl (DEAE) weak-anion exchanger.

  1. Global variation in the genetic and biochemical basis of diamondback moth resistance to Bacillus thuringiensis.

    PubMed

    Tabashnik, B E; Liu, Y B; Malvar, T; Heckel, D G; Masson, L; Ballester, V; Granero, F; Ménsua, J L; Ferré, J

    1997-11-25

    Insecticidal proteins from the soil bacterium Bacillus thuringiensis (Bt) are becoming a cornerstone of ecologically sound pest management. However, if pests quickly adapt, the benefits of environmentally benign Bt toxins in sprays and genetically engineered crops will be short-lived. The diamondback moth (Plutella xylostella) is the first insect to evolve resistance to Bt in open-field populations. Here we report that populations from Hawaii and Pennsylvania share a genetic locus at which a recessive mutation associated with reduced toxin binding confers extremely high resistance to four Bt toxins. In contrast, resistance in a population from the Philippines shows multilocus control, a narrower spectrum, and for some Bt toxins, inheritance that is not recessive and not associated with reduced binding. The observed variation in the genetic and biochemical basis of resistance to Bt, which is unlike patterns documented for some synthetic insecticides, profoundly affects the choice of strategies for combating resistance.

  2. Retargeting of the Bacillus thuringiensis toxin Cyt2Aa against hemipteran insect pests

    PubMed Central

    Chougule, Nanasaheb P.; Li, Huarong; Liu, Sijun; Linz, Lucas B.; Narva, Kenneth E.; Meade, Thomas; Bonning, Bryony C.

    2013-01-01

    Although transgenic crops expressing Bacillus thuringiensis (Bt) toxins have been used successfully for management of lepidopteran and coleopteran pest species, the sap-sucking insects (Hemiptera) are not particularly susceptible to Bt toxins. To overcome this limitation, we demonstrate that addition of a short peptide sequence selected for binding to the gut of the targeted pest species serves to increase toxicity against said pest. Insertion of a 12-aa pea aphid gut-binding peptide by adding to or replacing amino acids in one of three loops of the Bt cytolytic toxin, Cyt2Aa, resulted in enhanced binding and toxicity against both the pea aphid, Acyrthosiphon pisum, and the green peach aphid, Myzus persicae. This strategy may allow for transgenic plant-mediated suppression of other hemipteran pests, which include some of the most important pests of global agriculture. PMID:23650347

  3. Production of concentrates of bacterial bio-insecticide Bacillus thuringiensis var. israelensis by flocculation/sedimentation.

    PubMed

    Luna-Finkler, Christine Lamenha; Finkler, Leandro

    2008-08-01

    Flocculation/sedimentation of Bacillus thuringiensis var. israelensis (Bti) using flocculating agents has been studied. Batch cell production was performed in an agitated tank, and the flocculation assays were carried out in jar tests. Flocculent suspensions were characterized based on diameter of flocs and density. The best results were obtained with CaCl(2).2H(2)O, FeCl(3).6H(2)O, Al(2)(SO(4))(3) and tannin, with optimal flocculation concentrations of 2500, 2500, 3500 and 1000 mg l(-1), respectively. Thickening of the flocculent suspensions was investigated, leading to determination of the capacity curves of the settler. Bioassays against Aedes aegypti larvae demonstrated excellent results in insect control.

  4. Luminal proteinases from Plodia interpunctella and the hydrolysis of Bacillus thuringiensis CryIA(c) protoxin.

    PubMed

    Oppert, B; Kramer, K J; Johnson, D; Upton, S J; Mcgaughey, W H

    1996-06-01

    The ability of proteinases in gut extracts of the Indianmeal moth, Plodia interpunctella, to hydrolyze Bacillus thuringiensis (Bt) protoxin, casein, and rho-nitroanilide substrates was investigated. A polyclonal antiserum to protoxin CryIA(c) was used in Western blots to demonstrate slower protoxin processing by gut enzymes from Bt subspecies entomocidus-resistant larvae than enzymes from susceptible or kurstaki-resistant strains. Enzymes from all three strains hydrolyzed N-alpha-benzoyl-L-arginine rho-nitroanilide, N-succinyl-ala-ala-pro-phenylalanine rho-nitroanilide, and N-succinyl-ala-ala-pro-leucine rho-nitroanilide. Zymograms and activity blots were used to estimate the apparent molecular masses, number of enzymes, and relative activities in each strain. Several serine proteinase inhibitors reduced gut enzyme activities, with two soybean trypsin inhibitors, two potato inhibitors, and chymostatin the most effective in preventing protoxin hydrolysis.

  5. Expression and characterization of a lytic polysaccharide monooxygenase from Bacillus thuringiensis.

    PubMed

    Zhang, Huiyan; Zhao, Yong; Cao, Hailong; Mou, Guangqing; Yin, Heng

    2015-08-01

    Lytic polysaccharide monooxygenases (LPMOs) are recently discovered oxidative enzymes that are capable of oxidative cleavage of recalcitrant polysaccharides such as chitin or cellulose. Despite the importance of LPMOs in biomass conversion and the large number of lpmo genes in microorganisms, only a few LPMOs have been well studied, and further characterization of these proteins is thus of interest. In this study, a chitin-active AA10 family LPMO from Bacillus thuringiensis subsp. kurstaki, BtLPMO10A, is described. This enzyme generates even-numbered oxidized oligosaccharides as the dominated products from crystalline chitin, however, interestingly, when colloidal chitin is used as the substrate, a ladder of oxidized oligosaccharides is observed. These results provide new insights into the action mode of LPMOs that may be affected by the substrates. PMID:25936286

  6. Toxicity of Bacillus thuringiensis crystal proteins against eri silkworm, Samia cynthia ricini (Lepidoptera: Saturniidae).

    PubMed

    Sandeep Kumar, Donthula; Tarakeswari, Muddanuru; Lakshminarayana, Maddukuri; Sujatha, Mulpuri

    2016-07-01

    Ten purified crystal proteins of Bacillus thuringiensis (Bt) were tested at concentrations ranging from 2.93 to 3000ng/cm(2) for their toxicity to eri silkworm through protein paint bioassays using castor leaves. Based on LC50 values, Cry1Aa (2.6ng/cm(2)) was highly toxic followed by Cry1Ac (29.3ng/cm(2)) and Cry1Ab (68.7ng/cm(2)). The Cry1Ca and Cry1Ea proteins were moderately toxic to eri silkworm larvae and resulted in 23% and 28% mortality, respectively at the highest concentration tested (3000ng/cm(2)). Only reduction in larval weight was observed with Cry2Aa, Cry1Da and Cry9Aa proteins while Cry3Aa and Cry1Ba proteins were found to be nontoxic. PMID:27377590

  7. Biomimetic extraction of Bacillus thuringiensis insecticidal crystal proteins from soil based on invertebrate gut fluid chemistry.

    PubMed

    Shan, Guomin; Embrey, Shawna K; Herman, Rod A; Wolt, Jeffrey D; Weston, Donald; Mayer, Larry M

    2005-08-24

    Quantitative monitoring of Bacillus thuringiensis (Bt) insecticidal crystal proteins in soil has been hampered by the lack of efficient extraction/detection methods. A novel approach for simple and effective Bt protein extraction was explored by evaluating extraction solutions from invertebrate gut fluids. Marine worm gut fluids were identified as promising for extracting Bt protein from soil. An artificial gut fluid based on these marine worm gut fluids was developed using commercially available chemicals and was evaluated for its ability to extract Bt proteins from soil. On the basis of experiments with Cry1 proteins, the artificial gut fluid in combination with ELISA was highly effective for protein extraction and analysis in a variety of soil types and was well-correlated with bioassay results. Coupling of immunoassay with this extraction method provides, for the first time, an efficient, accurate, and quantitative assay for routine measurement of Bt protein residues in soil.

  8. Friction and Adhesion Forces of Bacillus thuringiensis Spores on Planar Surfaces in Atmospheric Systems

    SciTech Connect

    Kweon, Hyojin; Yiacoumi, Sotira; Tsouris, Costas

    2011-01-01

    The kinetic friction force and the adhesion force of Bacillus thuringiensis spores on planar surfaces in atmospheric systems were studied using atomic force microscopy. The influence of relative humidity (RH) on these forces varied for different surface properties including hydrophobicity, roughness, and surface charge. The friction force of the spore was greater on a rougher surface than on mica, which is atomically flat. As RH increases, the friction force of the spores decreases on mica whereas it increases on rough surfaces. The influence of RH on the interaction forces between hydrophobic surfaces is not as strong as for hydrophilic surfaces. The friction force of the spore is linear to the sum of the adhesion force and normal load on the hydrophobic surface. The poorly defined surface structure of the spore and the adsorption of contaminants from the surrounding atmosphere are believed to cause a discrepancy between the calculated and measured adhesion forces.

  9. Commercial formulations of Bacillus thuringiensis for control of Indian meal moth.

    PubMed Central

    Schesser, J H

    1976-01-01

    Doses of four commercial formulations and one experimental formulation of Bacillus thuringiensis Berliner were mixed with the diet used to rear colonies of the Indian meal moth Plodia interpunctella (Hübner). Indian meal moth eggs were introduced to the treated diet, and the resultant adult emergence was tabulated. The experimental formulations ranked as follows in efficacy in controlling the Indian meal moth: Dipel (50% lethal concentration [LC50], 25 mg/kg) greater than Bactospeine WP (LC50, 100 mg/kg) greater than Thuricide (LC50, 150 mg/kg) greater than IMC 90007 (LC30, 180 mg/kg) greater than Bactospeine Flowable (LC50, 440 mg/kg). PMID:984828

  10. Bacillus thuringiensis Cry1Aa toxin-binding region of Bombyx mori aminopeptidase N.

    PubMed

    Yaoi, K; Nakanishi, K; Kadotani, T; Imamura, M; Koizumi, N; Iwahana, H; Sato, R

    1999-12-17

    The Bacillus thuringiensis Cry1Aa toxin-binding region of Bombyx mori aminopeptidase N (APN) was analyzed, to better understand the molecular mechanism of susceptibility to the toxin and the development of resistance in insects. APN was digested with lysylendopeptidase and the ability of the resulting fragments to bind to Cry1Aa and 1Ac toxins was examined. The binding abilities of the two toxins to these fragments were different. The Cry1Aa toxin bound to the fragment containing 40-Asp to 313-Lys, suggesting that the Cry1Aa toxin-binding site is located in the region between 40-Asp and 313-Lys, while Cry1Ac toxin bound exclusively to mature APN. Next, recombinant APN of various lengths was expressed in Escherichia coli cells and its ability to bind to Cry1Aa toxin was examined. The results localized the Cry1Aa toxin binding to the region between 135-Ile and 198-Pro. PMID:10606725

  11. Efficient transformation of Cellulomonas flavigena by electroporation and conjugation with Bacillus thuringiensis.

    PubMed

    Montes-Horcasitas, Carmen; Ruiz-Medrano, Roberto; Magaña-Plaza, Ignacio; Silva, Lidia Gómez; Herrera-Martínez, Aseneth; Hernández-Montalvo, Lourdes; Xoconostle-Cázares, Beatriz

    2004-12-01

    The conjugative self-transmissible plasmid pHT73, harbored in Bacillus thuringiensis var. kurstaki, was demonstrated to be transferred to Cellulomonas flavigena, a cellulolytic bacterium. Both conjugation and transformation procedures yielded resistant colonies; however, chromosomal integration was observed only when bacterial conjugation occurred. The efficiency of conjugation was 10% of recipient strain, which is considered a very efficient process. When the plasmid pHT73 was introduced by transformation, erythromycin-resistant cells contained the plasmid as an episome with no arrangements, as assayed by Southern blot analysis. In contrast, conjugated-resistant cells harbor the plasmid integrated into the chromosome. These data suggest a common mechanism of cell communication between nonrelated bacterial species with similar ecological habitats, and also that both electroporation and conjugation can be used to transform C. flavigena efficiently.

  12. Tribolium castaneum Apolipophorin-III acts as an immune response protein against Bacillus thuringiensis Cry3Ba toxic activity.

    PubMed

    Contreras, Estefanía; Rausell, Carolina; Real, M Dolores

    2013-07-01

    In this study, a 2.1-fold Apolipophorin-III mRNA up-regulation was found in Tribolium castaneum larvae challenged with Bacillus thuringiensis Cry3Ba spore-crystal mixture. Knockdown of Apolipophorin-III by RNAi resulted in increased T. castaneum larvae susceptibility following Cry3Ba spore-crystal treatment, demonstrating Apolipophorin-III involvement in insect defense against B. thuringiensis. We showed that Apolipophorin-III participates in T. castaneum immune response to B. thuringiensis activating the prophenoloxidase cascade since: (i) phenoloxidase activity significantly increased after Cry3Ba spore-crystal treatment compared to untreated or Cry1Ac spore-crystal treated larvae and (ii) phenoloxidase activity in Cry3Ba spore-crystal treated Apolipophorin-III silenced larvae was 71±14% lower than that of non-silenced intoxicated larvae.

  13. Structural and Biophysical Characterization of Bacillus thuringiensis Insecticidal Proteins Cry34Ab1 and Cry35Ab1

    PubMed Central

    Kelker, Matthew S.; Berry, Colin; Evans, Steven L.; Pai, Reetal; McCaskill, David G.; Wang, Nick X.; Russell, Joshua C.; Baker, Matthew D.; Yang, Cheng; Pflugrath, J. W.; Wade, Matthew; Wess, Tim J.; Narva, Kenneth E.

    2014-01-01

    Bacillus thuringiensis strains are well known for the production of insecticidal proteins upon sporulation and these proteins are deposited in parasporal crystalline inclusions. The majority of these insect-specific toxins exhibit three domains in the mature toxin sequence. However, other Cry toxins are structurally and evolutionarily unrelated to this three-domain family and little is known of their three dimensional structures, limiting our understanding of their mechanisms of action and our ability to engineer the proteins to enhance their function. Among the non-three domain Cry toxins, the Cry34Ab1 and Cry35Ab1 proteins from B. thuringiensis strain PS149B1 are required to act together to produce toxicity to the western corn rootworm (WCR) Diabrotica virgifera virgifera Le Conte via a pore forming mechanism of action. Cry34Ab1 is a protein of ∼14 kDa with features of the aegerolysin family (Pfam06355) of proteins that have known membrane disrupting activity, while Cry35Ab1 is a ∼44 kDa member of the toxin_10 family (Pfam05431) that includes other insecticidal proteins such as the binary toxin BinA/BinB. The Cry34Ab1/Cry35Ab1 proteins represent an important seed trait technology having been developed as insect resistance traits in commercialized corn hybrids for control of WCR. The structures of Cry34Ab1 and Cry35Ab1 have been elucidated to 2.15 Å and 1.80 Å resolution, respectively. The solution structures of the toxins were further studied by small angle X-ray scattering and native electrospray ion mobility mass spectrometry. We present here the first published structure from the aegerolysin protein domain family and the structural comparisons of Cry34Ab1 and Cry35Ab1 with other pore forming toxins. PMID:25390338

  14. A Novel Formulation of Bacillus thuringiensis for the Control of Brassica Leaf Beetle, Phaedon brassicae (Coleoptera: Chrysomelidae).

    PubMed

    Kim, Eunseong; Jeoung, Sujin; Park, Youngjin; Kim, Kunwoo; Kim, Yonggyun

    2015-12-01

    Cabbage is a major vegetable crop over the world. Various insect pests can affect cabbage production. Excessive spray of chemical insecticides can lead to the development of insecticide resistance with various adverse effects on the environment and humans. Brassica leaf beetle, Phaedon brassicae Baly, is a coleopteran pest. Both larvae and adults cause damages to cabbage. The objective of this study was to develop an effective microbial insecticide against P. brassicae by adding an immunosuppressive agent to Bacillus thuringiensis (Bt). The immunosuppressive agent was chosen from bacterial cultured broth of Photorhabdus temperata subsp. temperata (Ptt). Reverse phase HPLC revealed that Ptt-cultured broth possessed at least two eicosanoid biosynthesis inhibitors (oxindole and indole) in its hexane extract. The bacterial cultured broth exhibited potent immunosuppressive activity against P. brassicae. Based on toxicity results, B. thuringiensis subsp. tenebrionis (BtT) was selected from four strains of Bts. When Ptt-cultured broth was added to spore-producing BtT cells, the insecticidal activities of BtT against both larvae and adults of P. brassicae were significantly increased. This bacterial mixture applied to develop a "Bt-Plus," which was formulated by mixing BtT cells (10(11) spores per ml) and 48-h Ptt-cultured broth along with additives (surfactant and preservative). When Bt-Plus was sprayed to cabbage infested by P. brassicae at 1,000-fold dilution, the mixture exhibited much higher control efficacy than BtT treatment alone, suggesting it could be used as a novel Bt insecticide for the control of P. brassicae. PMID:26470390

  15. Structural and biophysical characterization of Bacillus thuringiensis insecticidal proteins Cry34Ab1 and Cry35Ab1.

    PubMed

    Kelker, Matthew S; Berry, Colin; Evans, Steven L; Pai, Reetal; McCaskill, David G; Wang, Nick X; Russell, Joshua C; Baker, Matthew D; Yang, Cheng; Pflugrath, J W; Wade, Matthew; Wess, Tim J; Narva, Kenneth E

    2014-01-01

    Bacillus thuringiensis strains are well known for the production of insecticidal proteins upon sporulation and these proteins are deposited in parasporal crystalline inclusions. The majority of these insect-specific toxins exhibit three domains in the mature toxin sequence. However, other Cry toxins are structurally and evolutionarily unrelated to this three-domain family and little is known of their three dimensional structures, limiting our understanding of their mechanisms of action and our ability to engineer the proteins to enhance their function. Among the non-three domain Cry toxins, the Cry34Ab1 and Cry35Ab1 proteins from B. thuringiensis strain PS149B1 are required to act together to produce toxicity to the western corn rootworm (WCR) Diabrotica virgifera virgifera Le Conte via a pore forming mechanism of action. Cry34Ab1 is a protein of ∼14 kDa with features of the aegerolysin family (Pfam06355) of proteins that have known membrane disrupting activity, while Cry35Ab1 is a ∼44 kDa member of the toxin_10 family (Pfam05431) that includes other insecticidal proteins such as the binary toxin BinA/BinB. The Cry34Ab1/Cry35Ab1 proteins represent an important seed trait technology having been developed as insect resistance traits in commercialized corn hybrids for control of WCR. The structures of Cry34Ab1 and Cry35Ab1 have been elucidated to 2.15 Å and 1.80 Å resolution, respectively. The solution structures of the toxins were further studied by small angle X-ray scattering and native electrospray ion mobility mass spectrometry. We present here the first published structure from the aegerolysin protein domain family and the structural comparisons of Cry34Ab1 and Cry35Ab1 with other pore forming toxins.

  16. Initial frequency of alleles conferring resistance to Bacillus thuringiensis poplar in a field population of Chrysomela tremulae.

    PubMed Central

    Génissel, Anne; Augustin, Sylvie; Courtin, Claudine; Pilate, Gilles; Lorme, Philippe; Bourguet, Denis

    2003-01-01

    Globally, the estimated total area planted with transgenic plants producing Bacillus thuringiensis (Bt) toxins was 12 million hectares in 2001. The risk of target pests becoming resistant to these toxins has led to the implementation of resistance-management strategies. The efficiency and sustainability of these strategies, including the high-dose plus refuge strategy currently recommended for North American maize, depend on the initial frequency of resistance alleles. In this study, we estimated the initial frequencies of alleles conferring resistance to transgenic Bt poplars producing Cry3A in a natural population of the poplar pest Chrysomela tremulae (Coleoptera: Chrysomelidae). We used the F(2) screen method developed for detecting resistance alleles in natural pest populations. At least three parents of the 270 lines tested were heterozygous for a major Bt resistance allele. We estimated mean resistance-allele frequency for the period 1999-2001 at 0.0037 (95% confidence interval = 0.00045-0.0080) with a detection probability of 90%. These results demonstrate that (i) the F(2) screen method can be used to detect major alleles conferring resistance to Bt-producing plants in insects and (ii) the initial frequency of alleles conferring resistance to Bt toxin can be close to the highest theoretical values that are expected prior to the use of Bt plants if considering fitness costs and typical mutation rates. PMID:12737656

  17. The Pathogenomic Sequence Analysis of B. cereus and B.thuringiensis Isolates Closely Related to Bacillus anthracis

    SciTech Connect

    Han, Cliff S.; Xie, Gary; Challacombe, Jean F.; Altherr, MichaelR.; Smriti, B.; Bruce, David; Campbell, Connie S.; Campbell, Mary L.; Chen, Jin; Chertkov, Olga; Cleland, Cathy; Dimitrijevic-Bussod, M.; Doggett, Norman A.; Fawcett, John J.; Glavina, Tijana; Goodwin, Lynne A.; Hill, Karen K.; Hitchcock, Penny; Jackson, Paul J.; Keim, Paul; Kewalramani, Avinash Ramesh; Longmire, Jon; Lucas, Susan; Malfatti,Stephanie; McMurry, Kim; Meincke, Linda J.; Misra, Monica; Moseman,Bernice L.; Mundt, Mark; Munk, A. Christine; Okinaka, Richard T.; Parson-Quintana, B.; Reilly, Lee P.; Richardson, Paul; Robinson, DonnaL.; Rubin, Eddy; Saunders, Elizabeth; Tapia, Roxanne; Tesmer, Judith G.; Thayer, Nina; Thompson, Linda S.; Tice, Hope; Ticknor, Lawrence O.; Wills, Patti L.; Gilna, Payl; Brettin, Thomas S.

    2005-08-18

    The sequencing and analysis of two close relatives of Bacillus anthracis are reported. AFLP analysis of over 300 isolates of B.cereus, B. thuringiensis and B. anthracis identified two isolates as being very closely related to B. anthracis. One, a B. cereus, BcE33L, was isolated from a zebra carcass in Nambia; the second, a B. thuringiensis, 97-27, was isolated from a necrotic human wound. The B. cereus appears to be the closest anthracis relative sequenced to date. A core genome of over 3,900 genes was compiled for the Bacillus cereus group, including Banthracis. Comparative analysis of these two genomes with other members of the B. cereus group provides insight into the evolutionary relationships among these organisms. Evidence is presented that differential regulation modulates virulence, rather than simple acquisition of virulence factors. These genome sequences provide insight into the molecular mechanisms contributing to the host range and virulence of this group of organisms.

  18. Identification of a Bacillus thuringiensis Cry11Ba toxin-binding aminopeptidase from the mosquito, Anopheles quadrimaculatus

    PubMed Central

    Abdullah, Mohd Amir F; Valaitis, Algimantas P; Dean, Donald H

    2006-01-01

    Background Aminopeptidase N (APN) type proteins isolated from several species of lepidopteran insects have been implicated as Bacillus thuringiensis (Bt) toxin-binding proteins (receptors) for Cry toxins. We examined brush border membrane vesicle (BBMV) proteins from the mosquito Anopheles quadrimaculatus to determine if APNs from this organism would bind mosquitocidal Cry toxins that are active to it. Results A 100-kDa protein with APN activity (APNAnq 100) was isolated from the brush border membrane of Anopheles quadrimaculatus. Native state binding analysis by surface plasmon resonance shows that APNAnq 100 forms tight binding to a mosquitocidal Bt toxin, Cry11Ba, but not to Cry2Aa, Cry4Ba or Cry11Aa. Conclusion An aminopeptidase from Anopheles quadrimaculatus mosquitoes is a specific binding protein for Bacillus thuringiensis Cry11Ba. PMID:16716213

  19. Evidence of two mechanisms involved in Bacillus thuringiensis israelensis decreased toxicity against mosquito larvae: Genome dynamic and toxins stability.

    PubMed

    Elleuch, Jihen; Zribi Zghal, Raida; Lacoix, Marie Noël; Chandre, Fabrice; Tounsi, Slim; Jaoua, Samir

    2015-07-01

    Biopesticides based on Bacillus thuringiensis israelensis are the most used and most successful around the world. This bacterium is characterized by a dynamic genome able to win or lose genetic materials which leads to a decrease in its effectiveness. The detection of such phenomena is of great importance to monitor the stability of B. thuringiensis strains in industrial production processes of biopesticides. New local B. thuringiensis israelensis isolates were investigated. They present variable levels of delta-endotoxins production and insecticidal activities against Aedes aegypti larvae. Searching on the origin of this variability, molecular and biochemical analyses were performed. The obtained results describe two main reasons of the decrease of B. thuringiensis israelensis insecticidal activity. The first reason was the deletion of cry4Aa and cry10Aa genes from the 128-kb pBtoxis plasmid as evidenced in three strains (BLB124, BLB199 and BLB506) among five. The second was the early degradation of Cry toxins by proteases in larvae midgut mainly due to some amino acids substitutions evidenced in Cry4Ba and Cry11Aa δ-endotoxins detected in BLB356. Before biological treatment based on B. thuringiensis israelensis, the studies of microflore in each ecosystem have a great importance to succeed pest management programs.

  20. Effect of clonal variation among hybrid poplars on susceptibility of gypsy moth (Lepidoptera: Lymantriidae) to Bacillus thuringiensis subsp. kurstaki.

    PubMed

    Broderick, Nichole A; Vasquez, Eric; Handelsman, Jo; Raffa, Kenneth F

    2010-06-01

    Trees in the genus Populus can provide substantial commercial and ecological benefits, including sustainable alternatives to traditional forestry. Realization of this potential requires intensive management, but damage by defoliating insects can severely limit productivity in such systems. Two approaches to limiting these losses include cultivation of poplar varieties with inherent resistance to pests and application of microbial pesticides. Little is known about the interaction between host resistance and the ability of poplars to support the efficacy of biocontrol agents. The research described here was conducted to survey the effect of hybrid poplar clones on gypsy moth, Lymantria dispar (L.) (Lepidoptera: Lymantriidae), a pest on these trees. We assessed the effect of various poplar clones on larval performance and susceptibility to Bacillus thuringiensis subsp. kurstaki. Larvae were reared from hatching on the foliage of 25 hybrid poplar clones and we monitored larval survival, development time, and weight at fourth instar. Eight of these clones showed high resistance against gypsy moth. The remaining clones showed high variation in their effect on larval performance. We evaluated the susceptibility of third-instar larvae to B. thuringiensis subsp. kurstaki when reared on the 17 remaining clones. There was a significant effect of poplar clone on time to death after ingestion of B. thuringiensis subsp. kurstaki. The susceptibility of gypsy moth larvae to B. thuringiensis on various clones was not correlated with the effects of these clones on larval performance in the absence of B. thuringiensis, suggesting this interaction is more complex than merely reflecting higher mortality to previously stressed larvae.

  1. Evidence of two mechanisms involved in Bacillus thuringiensis israelensis decreased toxicity against mosquito larvae: Genome dynamic and toxins stability.

    PubMed

    Elleuch, Jihen; Zribi Zghal, Raida; Lacoix, Marie Noël; Chandre, Fabrice; Tounsi, Slim; Jaoua, Samir

    2015-07-01

    Biopesticides based on Bacillus thuringiensis israelensis are the most used and most successful around the world. This bacterium is characterized by a dynamic genome able to win or lose genetic materials which leads to a decrease in its effectiveness. The detection of such phenomena is of great importance to monitor the stability of B. thuringiensis strains in industrial production processes of biopesticides. New local B. thuringiensis israelensis isolates were investigated. They present variable levels of delta-endotoxins production and insecticidal activities against Aedes aegypti larvae. Searching on the origin of this variability, molecular and biochemical analyses were performed. The obtained results describe two main reasons of the decrease of B. thuringiensis israelensis insecticidal activity. The first reason was the deletion of cry4Aa and cry10Aa genes from the 128-kb pBtoxis plasmid as evidenced in three strains (BLB124, BLB199 and BLB506) among five. The second was the early degradation of Cry toxins by proteases in larvae midgut mainly due to some amino acids substitutions evidenced in Cry4Ba and Cry11Aa δ-endotoxins detected in BLB356. Before biological treatment based on B. thuringiensis israelensis, the studies of microflore in each ecosystem have a great importance to succeed pest management programs. PMID:26070692

  2. Draft Genome Sequence of Bacillus thuringiensis Serovar Tolworthi Strain Na205-3, an Isolate Toxic for Helicoverpa armigera

    PubMed Central

    Palma, Leopoldo; Muñoz, Delia; Murillo, Jesús

    2014-01-01

    We report here the complete annotated 6,510,053-bp draft genome sequence of Bacillus thuringiensis serovar tolworthi strain Na205-3, which is toxic for Helicoverpa armigera. This strain potentially contains nine insecticidal toxin genes homologous to cry1Aa12, cry1Ab1, cry1Ab8, cry1Ba1, cry1Af1, cry1Ia10, vip1Bb1, vip2Ba2, and vip3Aa6. PMID:24625875

  3. INSECTICIDAL TOXIN FROM BACILLUS THURINGIENSIS IS RELEASED FROM ROOTS OF TRANSGENIC BT CORN IN VITRO AND IN SITU. (R826107)

    EPA Science Inventory

    Abstract

    The insecticidal toxin encoded by the cry1Ab gene from Bacillus thuringiensis was released in root exudates from transgenic Bt corn during 40 days of growth in soil amended to 0, 3, 6, 9, or 12% (v/v) with montmorillonite or kaolinite in a...

  4. Isolation of Bacillus thuringiensis strains that contain Dipteran-specific cry genes from Ilha Bela (São Paulo, Brazil) soil samples.

    PubMed

    Campanini, E B; Davolos, C C; Alves, E C C; Lemos, M V F

    2012-05-01

    The entomophatogenic bacterium Bacillus thuringiensis produces crystal proteins, named Cry proteins which are encoded by the cry genes. This bacterium is used on biological control of important economical pests, as well as in the control of disease´s vectors, such as Aedes aegypti, a mosquito that transmits the dengue viruses. Isolates of this bacterium can be characterized by the content of cry genes and this prediction helps target different insect orders. In this research, we isolated 76 colonies of B. thuringiensis from 30 soil samples that were taken from Ilha Bela (SP, Brazil), a place where simulids are already biologically controlled by B. thuringiensis, to find bacterial isolates that were capable of controlling A. aegypti. The 16S ribosomal subunit genes of the selected isolates were sequenced, and the isolates were molecularly characterized based on their Dipteran-specific cry gene contents. Eight of the 76 isolates (10.52%) contained the cry4Aa, cry4Ba or cry10Aa genes, these isolates were carried out against A. aegypti larvae on bioassay. The presence or absence of specific cry genes was associated with the observed average larval mortalities. From the 76 isolates, seven (9.2%) were potentially able to control A. aegypti larvae. Therefore these are promising isolates for the biological control of A. aegypti larvae.

  5. Chromosome-Directed PCR-Based Detection and Quantification of Bacillus cereus Group Members with Focus on B. thuringiensis Serovar israelensis Active against Nematoceran Larvae.

    PubMed

    Schneider, Salome; Hendriksen, Niels B; Melin, Petter; Lundström, Jan O; Sundh, Ingvar

    2015-08-01

    Bacillus thuringiensis serovar israelensis is a wide-spread soil bacterium affiliated with the B. cereus group (Bcg) and is widely used in biocontrol products applied against mosquito and black fly larvae. For monitoring and quantification of applied B. thuringiensis serovar israelensis and its effect on indigenous B. thuringiensis serovar israelensis and Bcg assemblages, efficient and reliable tools are essential. The abundance and properties of B. thuringiensis serovar israelensis strains in the environment traditionally have been investigated with cultivation-dependent techniques, which are hampered by low sensitivity and the morphological similarity between B. cereus and B. thuringiensis. Currently available PCR-based detection and quantification tools target markers located on plasmids. In this study, a new cultivation-independent PCR-based method for efficient and specific quantification of B. thuringiensis serovar israelensis and Bcg is presented, utilizing two sets of PCR primers targeting the bacterial chromosome. Sequence database searches and empirical tests performed on target and nontarget species, as well as on bulk soil DNA samples, demonstrated that this diagnostic tool is specific for B. thuringiensis serovar israelensis and Bcg. The method will be useful for comparisons of Bcg and B. thuringiensis serovar israelensis abundances in the same samples. Moreover, the effect of B. thuringiensis serovar israelensis-based insecticide application on the total Bcg assemblages, including indigenous populations, can be investigated. This type of information is valuable in risk assessment and policy making for use of B. thuringiensis serovar israelensis in the environment.

  6. Chromosome-Directed PCR-Based Detection and Quantification of Bacillus cereus Group Members with Focus on B. thuringiensis Serovar israelensis Active against Nematoceran Larvae

    PubMed Central

    Hendriksen, Niels B.; Melin, Petter; Lundström, Jan O.; Sundh, Ingvar

    2015-01-01

    Bacillus thuringiensis serovar israelensis is a wide-spread soil bacterium affiliated with the B. cereus group (Bcg) and is widely used in biocontrol products applied against mosquito and black fly larvae. For monitoring and quantification of applied B. thuringiensis serovar israelensis and its effect on indigenous B. thuringiensis serovar israelensis and Bcg assemblages, efficient and reliable tools are essential. The abundance and properties of B. thuringiensis serovar israelensis strains in the environment traditionally have been investigated with cultivation-dependent techniques, which are hampered by low sensitivity and the morphological similarity between B. cereus and B. thuringiensis. Currently available PCR-based detection and quantification tools target markers located on plasmids. In this study, a new cultivation-independent PCR-based method for efficient and specific quantification of B. thuringiensis serovar israelensis and Bcg is presented, utilizing two sets of PCR primers targeting the bacterial chromosome. Sequence database searches and empirical tests performed on target and nontarget species, as well as on bulk soil DNA samples, demonstrated that this diagnostic tool is specific for B. thuringiensis serovar israelensis and Bcg. The method will be useful for comparisons of Bcg and B. thuringiensis serovar israelensis abundances in the same samples. Moreover, the effect of B. thuringiensis serovar israelensis-based insecticide application on the total Bcg assemblages, including indigenous populations, can be investigated. This type of information is valuable in risk assessment and policy making for use of B. thuringiensis serovar israelensis in the environment. PMID:25979887

  7. Application of statistical experimental design for optimisation of bioinsecticides production by sporeless Bacillus thuringiensis strain on cheap medium.

    PubMed

    Ben Khedher, Saoussen; Jaoua, Samir; Zouari, Nabil

    2013-01-01

    In order to overproduce bioinsecticides production by a sporeless Bacillus thuringiensis strain, an optimal composition of a cheap medium was defined using a response surface methodology. In a first step, a Plackett-Burman design used to evaluate the effects of eight medium components on delta-endotoxin production showed that starch, soya bean and sodium chloride exhibited significant effects on bioinsecticides production. In a second step, these parameters were selected for further optimisation by central composite design. The obtained results revealed that the optimum culture medium for delta-endotoxin production consists of 30 g L(-1) starch, 30 g L(-1) soya bean and 9 g L(-1) sodium chloride. When compared to the basal production medium, an improvement in delta-endotoxin production up to 50% was noted. Moreover, relative toxin yield of sporeless Bacillus thuringiensis S22 was improved markedly by using optimised cheap medium (148.5 mg delta-endotoxins per g starch) when compared to the yield obtained in the basal medium (94.46 mg delta-endotoxins per g starch). Therefore, the use of optimised culture cheap medium appeared to be a good alternative for a low cost production of sporeless Bacillus thuringiensis bioinsecticides at industrial scale which is of great importance in practical point of view.

  8. Nitric oxide participates in the toxicity of Bacillus thuringiensis Cry1Ab toxin to kill Manduca sexta larvae.

    PubMed

    Chavez, Carolina; Recio-Tótoro, Benito; Flores-Escobar, Biviana; Lanz-Mendoza, Humberto; Sanchez, Jorge; Soberón, Mario; Bravo, Alejandra

    2015-06-01

    Nitric oxide (NO) produced by the nitric oxide synthase (NOS) enzyme is a reactive oxygen molecule widely considered as important participant in the immune system of different organisms to confront microbial infections. In insects the NO molecule has also been implicated in immune response against microbial pathogens. Bacillus thuringiensis (Bt) is an insect-pathogenic bacterium that produces insecticidal proteins such as Cry toxins. These proteins kill insects because they form pores in the larval-midgut cells. Here we show that intoxication of Manduca sexta larvae with Cry1Ab activates expression of NOS with a corresponding increase in NO. This effect is not observed with a non-toxic mutant toxin Cry1Ab-E129K that is affected in pore formation. The increased production of NO triggered by intoxication with LC50 dose of Cry1Ab toxin is not associated with higher expression of antimicrobial peptides. NO participates in Cry1Ab toxicity since inhibition of NOS by selective l-NAME inhibitor prevented NO production and resulted in reduced mortality of the larvae. The fact that mortality was not completely abolished by L-NAME indicates that other processes participate in toxin action and induction of NO production upon Cry1Ab toxin administration accounts only for a part of the toxicity of this protein to M. sexta larvae.

  9. Specific PCR primers directed to identify cryI and cryIII genes within a Bacillus thuringiensis strain collection.

    PubMed Central

    Cerón, J; Ortíz, A; Quintero, R; Güereca, L; Bravo, A

    1995-01-01

    In this paper we describe a PCR strategy that can be used to rapidly identify Bacillus thuringiensis strains that harbor any of the known cryI or cryIII genes. Four general PCR primers which amplify DNA fragments from the known cryI or cryIII genes were selected from conserved regions. Once a strain was identified as an organism that contains a particular type of cry gene, it could be easily characterized by performing additional PCR with specific cryI and cryIII primers selected from variable regions. The method described in this paper can be used to identify the 10 different cryI genes and the five different cryIII genes. One feature of this screening method is that each cry gene is expected to produce a PCR product having a precise molecular weight. The genes which produce PCR products having different sizes probably represent strains that harbor a potentially novel cry gene. Finally, we present evidence that novel crystal genes can be identified by the method described in this paper. PMID:8526493

  10. Mutually exclusive distribution of the sap and eag S-layer genes and the lytB/lytA cell wall hydrolase genes in Bacillus thuringiensis.

    PubMed

    Soufiane, Brahim; Sirois, Marc; Côté, Jean-Charles

    2011-10-01

    Recently, two Bacillus thuringiensis strains were reported to synthesize parasporal inclusion bodies made not of the expected crystal (Cry) proteins but rather of the surface layer proteins (SLP) Sap (encoded by sap) and EA1 (encoded by eag), respectively. Whether the presence of the sap and eag genes is restricted to these two B. thuringiensis strains or ubiquitous in B. thuringiensis is unknown. We report here the distribution of the sap and eag genes in B. thuringiensis. Strains in the Bacillus cereus group were added for comparison purposes. We show that sap and eag are either present in tandem in 35% of the B. thuringiensis strains analysed and absent in 65% of the strains. When absent, a different tandem, the lytB/lytA cell wall hydrolase genes, is present. The distribution of the sap and eag S-layer and the lytB/lytA cell wall hydrolase genes is not species-specific in B. thuringiensis, B. cereus and Bacillus weihenstephanensis. Bacillus anthracis and Bacillus mycoides harbor sap and eag but not lytB/lytA. The sap, eag and lytB/lytA genes were absent in Bacillus pseudomycoides. Clearly, the distribution of the sap and eag S-layer and the lytB/lytA cell wall hydrolase genes in B. thuringiensis and in the Bacillus cereus group is mutually exclusive. We also showed that two genes involved in cell wall metabolism, csaA and csaB, are present not only upstream of the sap and eag S-layer genes, but also upstream of the lytB/lytA tandem in strains where sap and eag are absent. Bootstrapped neighbor-joining trees were inferred from the translated amino acid sequences of sap, eag and the tandem lytB/lytA, respectively.

  11. Potato flour mediated solid-state fermentation for the enhanced production of Bacillus thuringiensis-toxin.

    PubMed

    Smitha, Robinson Babysarojam; Jisha, Veloorvalappil Narayanan; Pradeep, Selvanesan; Josh, Moolakkariyil Sarath; Benjamin, Sailas

    2013-11-01

    In this study, we explored the efficacy of raw potato flour (PF) as supplement to the conventional LB medium (LB control, designated as M1) for enhancing the concomitant production of endospores and δ-endotoxin from Bacillus thuringiensis subsp. kurstaki by solid-state fermentation (SSF). Of different concentrations and combinations of media tested, 10% (w/v) PF supplemented LB medium (M2) was found as the best source for the maximum yield of toxin. After 12 h submerged fermentation (SmF) at 37°C and 125 rpm, M2 was made into a wet-solid matter for SSF by removing the supernatant (1000 ×g, 10 min); the resultant pellet subsequently incubated statically (37°C) for the production of B. thuringiensis subsp. kurstaki toxin (Btk-toxin). In comparison to M1, yield of δ-endotoxin purified by sucrose density gradient centrifugation method from M2 was about 6-fold higher (53% recovery). This maximum yield from M2 was obtained at 48 h (as against 72 h from M1), thus the gestation period of M2 was reduced by 24 h with higher yield. In addition to the quantitative data, qualitative photomicrographs taken by image analyzer, scanning electron and fluorescent microscopes and digital camera showed physical evidences for the upper hand of SSF over conventional SmF for the enhanced production of Btk-toxin. SDS-PAGE image of the purified δ-endotoxin showed three major fractions with apparent MWs 66, 45 and 30 kDa. Briefly, if low-cost agricultural products like PF is used as supplement to LB, by SSF strategy, production of Btk-toxin could be enhanced to 6-fold in short gestation time without losing its entomotoxicity efficiency.

  12. Molecular characterization and genetic diversity of insecticidal crystal protein genes in native Bacillus thuringiensis isolates.

    PubMed

    Mahadeva Swamy, H M; Asokan, R; Mahmood, Riaz; Nagesha, S N

    2013-04-01

    The Western Ghats of Karnataka natural ecosystem are among the most diverse and is one of the eight hottest hotspots of biological diversity in the world, that runs along the western part of India through four states including Karnataka. Bacillus thuringiensis (Bt) strains were isolated from soils of Western Ghats of Karnataka and characterized by molecular and analytical methods as a result of which 28 new Bt-like isolates were identified. Bt strains were isolated from soil samples using sodium acetate selection method. The morphology of crystals was studied using light and phase contrast microscopy. Isolates were further characterized for insecticidal cry gene by PCR, composition of toxins in bacterial crystals by SDS-PAGE cloning, sequencing and evaluation of toxicity was done. As a result 28 new Bt-like isolates were identified. Majority of the isolates showed the presence of a 55 kDa protein bands on SDS-PAGE while the rest showed 130, 73, 34, and 25 kDa bands. PCR analysis revealed predominance of Coleopteran-active cry genes in these isolates. The variations in the nucleotide sequences, crystal morphology, and mass of crystal protein(s) purified from the Bt isolates revealed genetic and molecular diversity. Three strains containing Coleopteran-active cry genes showed higher activity against larvae Myllocerus undecimpustulatus undatus Marshall (Coleoptera: Curculionidae) than B. thuringiensis subsp. Morrisoni. Results indicated that Bt isolates could be utilized for bioinsecticide production, aiming to reduce the use of chemical insecticide which could be useful to use in integrated pest management to control agriculturally important pests for sustainable crop production.

  13. The distribution pattern of DNA and protoxin in Bacillus thuringiensis as revealed by laser confocal microscopy analysis.

    PubMed

    Hu, Quanfang; Wang, Jingfang; Fu, Zujiao; Mo, Xiangtao; Ding, Xuezhi; Xia, Liqiu; Zhang, Youming; Sun, Yunjun

    2015-07-01

    It was reported that the parasporal crystal from Bacillus thuringiensis contained DNA fragments. To investigate the distribution of protoxin and DNA in B. thuringiensis cells at different growth stages, a cry1Ac-gfp fusion gene was constructed and expressed in an acrystalliferous B. thuringiensis strain, in which the localization of DNA and protoxin were indicated by DNA-specific dye and green fluorescent protein, respectively. When the recombinant cells were at the vegetative growth stage, the Cry1Ac-GFP fusion protein was not expressed and the DNA fluorescent signal was evenly distributed throughout the cell. At the initial stage of sporulation, the Cry1Ac-GFP fusion protein was expressed and accumulated as inclusion body, while two condensed DNA signals existed at each pole of the cell. With the extension of culture time, it seemed that the DNA fluorescence from the region of spore development gradually became faint or vanishing, while the DNA signal was still present in the other pole or the remaining area of the mother cell. Interestingly and unexpectedly, there was no DNA fluorescence signal in the region of the growing and mature inclusion body of Cry1Ac-GFP in B. thuringiensis cell, which might indicate that the DNA embodied in the inclusion body was not accessible to the DNA-specific dye. This was the first investigation devoted exclusively to the in vivo distribution of protoxin and DNA in B. thuringiensis at different growth stages. These data shed light on deeply understanding the process of sporulation and parasporal crystal formation as well as further exploring the interaction of DNA and protoxin in B. thuringiensis.

  14. A genetically modified broad-spectrum strain of Bacillus thuringiensis toxic against Holotrichia parallela, Anomala corpulenta and Holotrichia oblita.

    PubMed

    Jia, Yanhua; Zhao, Can; Wang, Qinglei; Shu, Changlong; Feng, Xiaojie; Song, Fuping; Zhang, Jie

    2014-02-01

    Cry8Ea1 from Bacillus thuringiensis strain Bt185 has insecticidal activity against Holotrichia parallela. Cry8Ca2 from strain HBF-1 is effective against Anomala corpulenta. Cry8Ga1 from strain HBF-18 is toxic to H. oblita. Given the need to broaden the spectrum of B. thuringiensis, a broad-spectrum coleopteran active strain of B. thuringiensis, BIOT185, engineered to express multiple cry genes, including cry8Ea1, cry8Fa1 and cry8Ca2, was created by homologous recombination introducing the cry8Ca2 into the B. thuringiensis strain Bt185 by Liu et al. (Appl Microbiol Biotechnol 87:243-249, 2010). To further broaden the spectrum, an engineered B. thuringiensis strain BIOT1858G was constructed by introducing the recombinant plasmid pSTK-8G containing cry8Ga1 into the engineered B. thuringiensis strain BIOT185. PCR and Southern blotting demonstrated that the cry8Ga1 gene was transferred to the novel strain BIOT1858G. SDS-PAGE and RT-PCR confirmed the normal expression and transcription of the cry8Ga1 gene in addition to the cry8Ea1, cry8Fa1 and cry8Ca2 genes. Bioassays of BIOT1858G indicated that the recombinant strain broadened the spectrum against not only H. parallela susceptible to the Cry8E protein and A. corpulenta susceptible to the Cry8C protein but also H. oblita susceptible to the Cry8G protein. The pesticide could also decrease the cost of production and field labor.

  15. Insecticidal activity of Bacillus thuringiensis Cry1Bh1 against Ostrinia nubilalis (Hubner) (Lepidoptera: Crambidae) and other lepidopteran pests.

    PubMed

    Lira, Justin; Beringer, Jeff; Burton, Stephanie; Griffin, Samantha; Sheets, Joel; Tan, Sek Yee; Woosley, Aaron; Worden, Sarah; Narva, Kenneth E

    2013-12-01

    Bacillus thuringiensis is an important source of insect resistance traits in commercial crops. In an effort to prolong B. thuringiensis trait durability, insect resistance management programs often include combinations of insecticidal proteins that are not cross resistant or have demonstrable differences in their site of action as a means to mitigate the development of resistant insect populations. In this report, we describe the activity spectrum of a novel B. thuringiensis Cry protein, Cry1Bh1, against several lepidopteran pests, including laboratory-selected B. thuringiensis-resistant strains of Ostrinia nubilalis and Heliothis virescens and progeny of field-evolved B. thuringiensis-resistant strains of Plutella xylostella and Spodoptera frugiperda. Cry1Bh1 is active against susceptible and B. thuringiensis-resistant colonies of O. nubilalis, P. xylostella, and H. virescens in laboratory diet-based assays, implying a lack of cross-resistance in these insects. However, Cry1Bh1 is not active against susceptible or Cry1F-resistant S. frugiperda. Further, Cry1Bh1 does not compete with Cry1Fa or Cry1Ab for O. nubilalis midgut brush border membrane binding sites. Cry1Bh1-expressing corn, while not completely resistant to insect damage, provided significantly better leaf protection against Cry1Fa-resistant O. nubilalis than did Cry1Fa-expressing hybrid corn. The lack of cross-resistance with Cry1Ab and Cry1Fa along with independent membrane binding sites in O. nubilalis makes Cry1Bh1 a candidate to further optimize for in-plant resistance to this pest.

  16. Helicoverpa armigera baseline susceptibility to Bacillus thuringiensis Cry toxins and resistance management for Bt cotton in India.

    PubMed

    Gujar, G T; Kalia, V; Kumari, A; Singh, B P; Mittal, A; Nair, R; Mohan, M

    2007-07-01

    Transgenic cotton that produces insecticidal proteins from Bacillus thuringiensis (Bt), often referred to as Bt cotton, is widely grown in many countries. Bt cotton with a single cry1A gene and stacked also with cry2A gene has provided satisfactory protection against the damage by the lepidopteran bollworms, especially the cotton bollworm, Helicoverpa armigera (Hübner) which is considered as a key pest. The baseline susceptibility of the larvae of H. armigera to Cry1Ac and other toxins carried out in many countries has provided a basis for monitoring resistance. There is no evidence of development of field-level resistance in H. armigera leading to the failure of Bt cotton crop anywhere in the world, despite the fact that Bt cotton was grown on the largest ever area of 12.1 million hectares in 2006 and its cumulative cultivation over the last 11 years has surpassed the annual cotton area in the world. Nevertheless, the Bt resistance management has become a necessity to sustain Bt cotton and other transgenic crops in view of potential of the target insects to evolve Cry toxin resistance.

  17. Effect of Larvae Treated with Mixed Biopesticide Bacillus thuringiensis - Abamectin on Sex Pheromone Communication System in Cotton Bollworm, Helicoverpa armigera

    PubMed Central

    Shen, Li-Ze; Chen, Peng-Zhou; Xu, Zhi-Hong; Deng, Jian-Yu; Harris, Marvin-K; Wanna, Ruchuon; Wang, Fu-Min; Zhou, Guo-Xin; Yao, Zhang-Liang

    2013-01-01

    Third instar larvae of the cotton bollworm (Helicoverpa armigera) were reared with artificial diet containing a Bacillus thuringiensis - abamectin (BtA) biopesticide mixture that resulted in 20% mortality (LD20). The adult male survivors from larvae treated with BtA exhibited a higher percentage of “orientation” than control males but lower percentages of “approaching” and “landing” in wind tunnel bioassays. Adult female survivors from larvae treated with BtA produced higher sex pheromone titers and displayed a lower calling percentage than control females. The ratio of Z-11-hexadecenal (Z11–16:Ald) and Z-9-hexadecenal (Z9–16:Ald) in BtA-treated females changed and coefficients of variation (CV) of Z11–16:Ald and Z9–16:Ald were expanded compared to control females. The peak circadian calling time of BtA-treated females occurred later than that of control females. In mating choice experiment, both control males and BtA-treated males preferred to mate with control females and a portion of the Bt-A treated males did not mate whereas all control males did. Our Data support that treatment of larvae with BtA had an effect on the sex pheromone communication system in surviving H.armigera moths that may contribute to assortative mating. PMID:23874751

  18. Ostrinia nubilalis parasitism and the field abundance of non-target insects in transgenic Bacillus thuringiensis corn (Zea mays).

    PubMed

    Bourguet, Denis; Chaufaux, Josette; Micoud, Annie; Delos, Marc; Naibo, Bernard; Bombarde, Fany; Marque, Gilles; Eychenne, Nathalie; Pagliari, Carine

    2002-10-01

    In this study, we evaluated in field trials the effects on non-target species, of transgenic corn producing the Cry1Ab toxin of Bacillus thuringiensis (Bt). In 1998, we collected Ostrinia nubilalis (Hübner) larvae from transgenic Bt corn (Novartis Hybrid 176) and non-Bt corn at four geographical sites. We found a significant variation in parasitism by the tachinids Lydella thompsoni (Herting) and Pseudoperichaeta nigrolineata (Walker) among sites, and more parasitism in non-Bt than in Bt fields. The Bt effect did not vary significantly among fields. In 1999, we performed a field experiment at two sites, comparing the temporal abundance of non-target arthropods in Bt corn (Monsanto Hybrid MON810) and non-Bt corn. The non-target insects studied included the aphids Metopolophium dirhodum (Walker), Rhopalosiphum padi (L.) and Sitobion avenae (F.), the bug Orius insidiosus (Say), the syrphid Syrphus corollae (Meigen), the ladybird Coccinella septempunctata (L.), the lacewing Chrysoperla carnea (Stephens), thrips and hymenopteran parasitoids. For all species but one, the number of individuals varied greatly over the season but did not differ between the types of corn. The only exception was thrips which, at one site, was significantly more abundant in Bt corn than in non-Bt corn. However this difference did not remain significant when we took the multiple tests into account. Implications for pest resistance management, population dynamics and risk assessment are discussed.

  19. Initial frequency of alleles for resistance to Bacillus thuringiensis toxins in field populations of Heliothis virescens

    PubMed Central

    Gould, F.; Anderson, A.; Jones, A.; Sumerford, D.; Heckel, D. G.; Lopez, J.; Micinski, S.; Leonard, R.; Laster, M.

    1997-01-01

    The risk of rapid pest adaptation to an insecticide is highly dependent on the initial frequency of resistance alleles in field populations. Because we have lacked empirical estimates of these frequencies, population–genetic models of resistance evolution have relied on a wide range of theoretical estimates. The recent commercialization of genetically engineered cotton that constitutively produces an insecticidal protein derived from the biocontrol agent, Bacillus thuringiensis (Bt) has raised concern that we lack data needed to quantify the risk of insect pests such as Heliothis virescens rapidly adapting to this ecologically valuable class of toxins. By individually mating over 2,000 male H. virescens moths collected in four states to females of a Bt toxin-resistant laboratory strain, and screening F1 and F2 offspring for tolerance of the toxic protein, we were able to directly estimate the field frequency of alleles for resistance as 1.5 × 10−3. This high initial frequency underscores the need for caution in deploying transgenic cotton to control insect pests. Our single-pair mating technique greatly increases the efficiency of detecting recessive resistance alleles. Because alleles that decrease target site sensitivity to Bt toxins and other insecticides are often recessive, this technique could be useful in estimating resistance allele frequencies in other insects exposed to transgenic insecticidal crops or conventional insecticides. PMID:11038613

  20. Bacillus thuringiensis insecticidal three-domain Cry toxins: mode of action, insect resistance and consequences for crop protection.

    PubMed

    Pardo-López, Liliana; Soberón, Mario; Bravo, Alejandra

    2013-01-01

    Bacillus thuringiensis bacteria are insect pathogens that produce different Cry and Cyt toxins to kill their hosts. Here we review the group of three-domain Cry (3d-Cry) toxins. Expression of these 3d-Cry toxins in transgenic crops has contributed to efficient control of insect pests and a reduction in the use of chemical insecticides. The mode of action of 3d-Cry toxins involves sequential interactions with several insect midgut proteins that facilitate the formation of an oligomeric structure and induce its insertion into the membrane, forming a pore that kills midgut cells. We review recent progress in our understanding of the mechanism of action of these Cry toxins and focus our attention on the different mechanisms of resistance that insects have evolved to counter their action, such as mutations in cadherin, APN and ABC transporter genes. Activity of Cry1AMod toxins, which are able to form toxin oligomers in the absence of receptors, against different resistant populations, including those affected in the ABC transporter and the role of dominant negative mutants as antitoxins, supports the hypothesis that toxin oligomerization is a limiting step in the Cry insecticidal activity. Knowledge of the action of 3d-Cry toxin and the resistance mechanisms to these toxins will set the basis for a rational design of novel toxins to overcome insect resistance, extending the useful lifespan of Cry toxins in insect control programs.

  1. Histopathological effects and determination of the putative receptor of Bacillus thuringiensis Cry1Da toxin in Spodoptera littoralis midgut.

    PubMed

    BenFarhat-Touzri, Dalel; Saadaoui, Marwa; Abdelkefi-Mesrati, Lobna; Saadaoui, Imen; Azzouz, Hichem; Tounsi, Slim

    2013-02-01

    Bacillus thuringiensis subsp. aizawai strain HD133, known by its effectiveness against Spodoptera species, produces many insecticidal proteins including Cry1Ab, Cry1Ca and Cry1Da. In the present study, the insecticidal activity of Cry1Da against Spodoptera littoralis was investigated. It showed toxicity with an LC(50) of 224.4 ng/cm(2) with 95% confidence limits of (178.61-270.19) and an LC(90) of 467.77 ng/cm(2) with 95% confidence limits of (392.89-542.65). The midgut histopathology of Cry1Da fed larvae showed vesicle formation in the apical region, vacuolization and destruction of epithelial cells. Biotinylated-activated Cry1Da toxin bound protein of about 65 kDa on blots of S. littoralis brush border membrane preparations. This putative receptor differs in molecular size from those recognized by Cry1C and Vip3A which are active against this polyphagous insect. This difference in midgut receptors strongly supports the use of Cry1Da as insecticidal agent, particularly in case of Cry and/or Vip-resistance management. PMID:23220238

  2. In silico modeling and functional interpretations of Cry1Ab15 toxin from Bacillus thuringiensis BtB-Hm-16.

    PubMed

    Kashyap, Sudhanshu

    2013-01-01

    The theoretical homology based structural model of Cry1Ab15 δ-endotoxin produced by Bacillus thuringiensis BtB-Hm-16 was predicted using the Cry1Aa template (resolution 2.25 Å). The Cry1Ab15 resembles the template structure by sharing a common three-domain extending conformation structure responsible for pore-forming and specificity determination. The novel structural differences found are the presence of β0 and α3, and the absence of α7b, β1a, α10a, α10b, β12, and α11a while α9 is located spatially downstream. Validation by SUPERPOSE and with the use of PROCHECK program showed folding of 98% of modeled residues in a favourable and stable orientation with a total energy Z-score of -6.56; the constructed model has an RMSD of only 1.15 Å. These increments of 3D structure information will be helpful in the design of domain swapping experiments aimed at improving toxicity and will help in elucidating the common mechanism of toxin action.

  3. Mode of Action and Specificity of Bacillus thuringiensis Toxins in the Control of Caterpillars and Stink Bugs in Soybean Culture.

    PubMed

    Schünemann, Rogério; Knaak, Neiva; Fiuza, Lidia Mariana

    2014-01-01

    The bacterium Bacillus thuringiensis (Bt) produces delta-endotoxins that possess toxic properties and can be used as biopesticides, as well as a source of genes for the construction of transgenic plants resistant to insects. In Brazil, the introduction of Bt soybean with insecticidal properties to the velvetbean caterpillar, the main insect pest of soybean, has been seen a promising tool in the management of these agroecosystems. However, the increase in stink bug populations in this culture, in various regions of the country, which are not susceptible to the existing genetically modified plants, requires application of chemicals that damage the environment. Little is known about the actual toxicity of Bt to Hemiptera, since these insects present sucking mouthparts, which hamper toxicity assays with artificial diets containing toxins of this bacterium. In recent studies of cytotoxicity with the gut of different hemipterans, susceptibility in the mechanism of action of delta-endotoxins has been demonstrated, which can generate promising subsidies for the control of these insect pests in soybean. This paper aims to review the studies related to the selection, application and mode of action of Bt in the biological control of the major pest of soybean, Anticarsia gemmatalis, and an analysis of advances in research on the use of Bt for control hemipterans.

  4. Mode of Action and Specificity of Bacillus thuringiensis Toxins in the Control of Caterpillars and Stink Bugs in Soybean Culture

    PubMed Central

    Fiuza, Lidia Mariana

    2014-01-01

    The bacterium Bacillus thuringiensis (Bt) produces delta-endotoxins that possess toxic properties and can be used as biopesticides, as well as a source of genes for the construction of transgenic plants resistant to insects. In Brazil, the introduction of Bt soybean with insecticidal properties to the velvetbean caterpillar, the main insect pest of soybean, has been seen a promising tool in the management of these agroecosystems. However, the increase in stink bug populations in this culture, in various regions of the country, which are not susceptible to the existing genetically modified plants, requires application of chemicals that damage the environment. Little is known about the actual toxicity of Bt to Hemiptera, since these insects present sucking mouthparts, which hamper toxicity assays with artificial diets containing toxins of this bacterium. In recent studies of cytotoxicity with the gut of different hemipterans, susceptibility in the mechanism of action of delta-endotoxins has been demonstrated, which can generate promising subsidies for the control of these insect pests in soybean. This paper aims to review the studies related to the selection, application and mode of action of Bt in the biological control of the major pest of soybean, Anticarsia gemmatalis, and an analysis of advances in research on the use of Bt for control hemipterans. PMID:24575310

  5. Identification and characterization of Aedes aegypti aminopeptidase N as a putative receptor of Bacillus thuringiensis Cry11A toxin

    PubMed Central

    Chen, Jianwu; Aimanova, Karlygash G.; Pan, Songqin; Gill, Sarjeet S.

    2009-01-01

    Bacillus thuringiensis subsp. israelensis, which is used worldwide to control Aedes aegypti larvae, produces Cry11Aa and other toxins during sporulation. In this study, pull-down assays were performed using biotinylated Cry11Aa toxin and solubilized brush border membrane vesicles prepared from midguts of Aedes larvae. Three of the eluted proteins were identified as aminopeptidease N (APN), one of which was a 140 kDa protein, named AaeAPN1 (AAEL 012778 in VectorBase). This protein localizes to the apical side of posterior midgut epithelial cells of larva. The full-length AaeAPN1 was cloned and expressed in E. coli and in Sf21 cells. AaeAPN1 protein expressed in Sf21 cells was enzymatically active, had a GPI-anchor but did not bind Cry11Aa. A truncated AaeAPN1, however, binds Cry11Aa with high affinity, and also Cry11Ba but with lower affinity. BBMV but not Sf21 expressed AaeAPN1 can be detected by wheat germ agglutinin suggesting the native but Sf21 cell expressed APN1 contains N-acetylglucosamine moieties. PMID:19698787

  6. Asymmetrical cross-resistance between Bacillus thuringiensis toxins Cry1Ac and Cry2Ab in pink bollworm

    PubMed Central

    Tabashnik, Bruce E.; Unnithan, Gopalan C.; Masson, Luke; Crowder, David W.; Li, Xianchun; Carrière, Yves

    2009-01-01

    Transgenic crops producing Bacillus thuringiensis (Bt) toxins kill some key insect pests and can reduce reliance on insecticide sprays. Sustainable use of such crops requires methods for delaying evolution of resistance by pests. To thwart pest resistance, some transgenic crops produce 2 different Bt toxins targeting the same pest. This “pyramid” strategy is expected to work best when selection for resistance to 1 toxin does not cause cross-resistance to the other toxin. The most widely used pyramid is transgenic cotton producing Bt toxins Cry1Ac and Cry2Ab. Cross-resistance between these toxins was presumed unlikely because they bind to different larval midgut target sites. Previous results showed that laboratory selection with Cry1Ac caused little or no cross-resistance to Cry2A toxins in pink bollworm (Pectinophora gossypiella), a major cotton pest. We show here, however, that laboratory selection of pink bollworm with Cry2Ab caused up to 420-fold cross-resistance to Cry1Ac as well as 240-fold resistance to Cry2Ab. Inheritance of resistance to high concentrations of Cry2Ab was recessive. Larvae from a laboratory strain resistant to Cry1Ac and Cry2Ab in diet bioassays survived on cotton bolls producing only Cry1Ac, but not on cotton bolls producing both toxins. Thus, the asymmetrical cross-resistance seen here does not threaten the efficacy of pyramided Bt cotton against pink bollworm. Nonetheless, the results here and previous evidence indicate that cross-resistance occurs between Cry1Ac and Cry2Ab in some key cotton pests. Incorporating the potential effects of such cross-resistance in resistance management plans may help to sustain the efficacy of pyramided Bt crops. PMID:19581574

  7. Bacillus thuringiensis associated with faeces of the Kerama-jika, Cervus nippon keramae, a wild deer indigenous to the Ryukyus, Japan.

    PubMed

    Ohba, Michio; Lee, Dong-Hyun

    2003-01-01

    Faeces of the Kerama-jika (Cervus nippon keramae), a wild shika deer indigenous to the Ryukyus, Japan, were examined for the natural occurrence of Bacillus thuringiensis. Of the ten faecal samples tested, seven contained this organism. The frequency of B. thuringiensis was 8.5% among 387 colonies of spore-forming bacteria belonging to the Bacillus cereus/B. thuringiensis group. Of 33 B. thuringiensis isolates recovered, only one isolate, assigned to the serotype H3abc (serovar kurstaki), exhibited dual toxicity against larvae of the silkworm, Bombyx mori, and the mosquito, Aedes aegypti. All of 32 other isolates were serologically untypable or untestable, and were non-toxic to larvae of the two insect species.

  8. Histopathological and combinatorial effects of the metalloprotease InhA1 and Cry proteins of Bacillus thuringiensis against Spodoptera littoralis.

    PubMed

    Dammak, Ines; Dammak, Mariam; Tounsi, Slim

    2015-11-01

    The zinc metalloprotease (InhA) of Bacillus thuringiensis specifically hydrolyzes cecropins and attacins, two antibacterial peptides in the immune hemolymph of insects, leading to a high resistance of the bacteria to the humoral defense system of its host. In the present study, the inhA gene of B. thuringiensis strain BUPM28 was cloned and the nucleotide sequence analysis revealed that it was identical to that of B. thuringiensis 8010. The expressed InhA1 protein in Escherichia coli showed toxicity to neonate Spodoptera littoralis larvae with a LC50 of 2.07±0.72μg/cm(2). Study of the effect of combining Cry proteins with InhA1 showed that one improves the toxicity of the other one against S. littoralis. Investigation of the histopathological effect of this metalloprotease showed an extensive damage of S. littoralis epithelium tissue. These results provide an insight to the use of InhA as supplement to Cry toxins to improve the efficacy of B. thuringiensis formulations and to overcome possible resistance problems.

  9. Resistance to Bacillus thuringiensis by the Indian meal moth, Plodia interpunctella: comparison of midgut proteinases from susceptible and resistant larvae.

    PubMed

    Johnson, D E; Brookhart, G L; Kramer, K J; Barnett, B D; McGaughey, W H

    1990-03-01

    Midgut homogenates from susceptible and resistant strains of the Indian meal moth, Plodia interpunctella, were compared for their ability to activate the entomocidal parasporal crystal protein from Bacillus thuringiensis. The properties of midgut proteinases from both types of larvae were also examined. Electrophoretic patterns of crystal protein from B. thuringiensis subspecies kurstaki (HD-1) and aizawai (HD-133 and HD-144) were virtually unchanged following digestion by either type of midgut homogenate. Changes in pH (9.5 to 11.5) or midgut homogenate concentration during digestion failed to substantially alter protein electrophoretic patterns of B. thuringiensis HD-1 crystal toxin. In vitro toxicity of crystal protein activated by either type of midgut preparation was equal toward cultured insect cells from either Manduca sexta or Choristoneura fumiferana. Electrophoresis of midgut extracts in polyacrylamide gels containing gelatin as substrate also yielded matching mobility patterns of proteinases from both types of midguts. Quantitation of midgut proteolytic activity using tritiated casein as a substrate revealed variation between midgut preparations, but no statistically significant differences between proteolytic activities from susceptible and resistant Indian meal moth larvae. Inhibition studies indicated that a trypsin-like proteinase with maximal activity at pH 10 is a major constituent of Indian meal moth midguts. The results demonstrated that midguts from susceptible and resistant strains of P. interpunctella are similar both in their ability to activate B. thuringiensis protoxin and in their proteolytic activity.

  10. Transcriptome analysis of Bacillus thuringiensis spore life, germination and cell outgrowth in a vegetable-based food model.

    PubMed

    Bassi, Daniela; Colla, Francesca; Gazzola, Simona; Puglisi, Edoardo; Delledonne, Massimo; Cocconcelli, Pier Sandro

    2016-05-01

    Toxigenic species belonging to Bacillus cereus sensu lato, including Bacillus thuringiensis, cause foodborne outbreaks thanks to their capacity to survive as spores and to grow in food matrixes. The goal of this work was to assess by means of a genome-wide transcriptional assay, in the food isolate B. thuringiensis UC10070, the gene expression behind the process of spore germination and consequent outgrowth in a vegetable-based food model. Scanning electron microscopy and Energy Dispersive X-ray microanalysis were applied to select the key steps of B. thuringiensis UC10070 cell cycle to be analyzed with DNA-microarrays. At only 40 min from heat activation, germination started rapidly and in less than two hours spores transformed in active growing cells. A total of 1646 genes were found to be differentially expressed and modulated during the entire B. cereus life cycle in the food model, with most of the significant genes belonging to transport, transcriptional regulation and protein synthesis, cell wall and motility and DNA repair groups. Gene expression studies revealed that toxin-coding genes nheC, cytK and hblC were found to be expressed in vegetative cells growing in the food model.

  11. Molecular Approaches to Improve the Insecticidal Activity of Bacillus thuringiensis Cry Toxins

    PubMed Central

    Lucena, Wagner A.; Pelegrini, Patrícia B.; Martins-de-Sa, Diogo; Fonseca, Fernando C. A.; Gomes, Jose E.; de Macedo, Leonardo L. P.; da Silva, Maria Cristina M.; Oliveira, Raquel S.; Grossi-de-Sa, Maria F.

    2014-01-01

    Bacillus thuringiensis (Bt) is a gram-positive spore-forming soil bacterium that is distributed worldwide. Originally recognized as a pathogen of the silkworm, several strains were found on epizootic events in insect pests. In the 1960s, Bt began to be successfully used to control insect pests in agriculture, particularly because of its specificity, which reflects directly on their lack of cytotoxicity to human health, non-target organisms and the environment. Since the introduction of transgenic plants expressing Bt genes in the mid-1980s, numerous methodologies have been used to search for and improve toxins derived from native Bt strains. These improvements directly influence the increase in productivity and the decreased use of chemical insecticides on Bt-crops. Recently, DNA shuffling and in silico evaluations are emerging as promising tools for the development and exploration of mutant Bt toxins with enhanced activity against target insect pests. In this report, we describe natural and in vitro evolution of Cry toxins, as well as their relevance in the mechanism of action for insect control. Moreover, the use of DNA shuffling to improve two Bt toxins will be discussed together with in silico analyses of the generated mutations to evaluate their potential effect on protein structure and cytotoxicity. PMID:25123558

  12. Development of photoperiod- and thermo-sensitive male sterility rice expressing transgene Bacillus thuringiensis

    PubMed Central

    Liu, Xin; Zhang, Jiwen; Zhang, Cuicui; Wang, Liangchao; Chen, Hao; Zhu, Zengrong; Tu, Jumin

    2015-01-01

    Stem borers and leaffolders are the main pests that cause severe damage in rice (Oryza sativa L.) production worldwide. We developed the first photoperiod- and thermo-sensitive male sterility (PTSMS) rice 208S with the cry1Ab/1Ac Bacillus thuringiensis (Bt) gene, through sexual crossing with Huahui 1 (elite line with the cry1Ab/1Ac gene). The novel 208S and its hybrids presented high and stable resistance to stem borers and leaffolders, and the content of Cry1Ab/1Ac protein in chlorophyllous tissues achieved the identical level as donor and showed little accumulation in non-chlorophyllous tissue. No dominant dosage effect in the Bt gene was observed in 208S and its derived hybrids. An analysis of fertility transition traits indicated that 208S was completely sterile under long day length/high temperature, but partially fertile under short day length/low temperature. With fine grain quality and favorable combining ability, 208S had no observed negative effects on fertility and agronomic traits from Bt (cry1Ab/1Ac). Additionally, 208S as a male sterile line showed no fertility decrease caused by Bt transgenic process, as it is the case in Huahui 1. Thus, 208S has great application value in two-line hybrid production for insect resistance, and can also be used as a bridge material in rice Bt transgenic breeding. PMID:26366116

  13. The impact of strain diversity and mixed infections on the evolution of resistance to Bacillus thuringiensis.

    PubMed

    Raymond, Ben; Wright, Denis J; Crickmore, Neil; Bonsall, Michael B

    2013-10-22

    Pesticide mixtures can reduce the rate at which insects evolve pesticide resistance. However, with live biopesticides such as the naturally abundant pathogen Bacillus thuringiensis (Bt), a range of additional biological considerations might affect the evolution of resistance. These can include ecological interactions in mixed infections, the different rates of transmission post-application and the impact of the native biodiversity on the frequency of mixed infections. Using multi-generation selection experiments, we tested how applications of single and mixed strains of Bt from diverse sources (natural isolates and biopesticides) affected the evolution of resistance in the diamondback moth, Plutella xylostella, to a focal strain. There was no significant difference in the rate of evolution of resistance between single and mixed-strain applications although the latter did result in lower insect populations. The relative survivorship of Bt-resistant genotypes was higher in the mixed-strain treatment, in part owing to elevated mortality of susceptible larvae in mixtures. Resistance evolved more quickly with treatments that contained natural isolates, and biological differences in transmission rate may have contributed to this. Our data indicate that the use of mixtures can have unexpected consequences on the fitness of resistant and susceptible insects.

  14. Stable Bacillus thuringiensis transgene introgression from Brassica napus to wild mustard B. juncea.

    PubMed

    Cao, Di; Stewart, C Neal; Zheng, Min; Guan, Zhengjun; Tang, Zhi-Xi; Wei, Wei; Ma, Ke-Ping

    2014-10-01

    Transgenic canola (Brassica napus) with a Bacillus thuringiensis cry1Ac gene and a green fluorescent protein (GFP) marker gene was used in hybridization experiments with wild Brassica juncea. Hybrid F1 and successive five backcross generations were obtained. The pod-set frequency on backcrossed B. juncea plants was over 66%, which suggested relatively high crossing compatibility between the hybrids and wild species. The seed setting in BC1 was the least of all generations tested, and then increased at the BC2 generation for which the thousand-seed weight was the highest of all generations. Seed size in backcrossed generations eventually approached that of the wild parent. The plants in all backcrossed generations were consistent with the expected 1:1 segregation ratio of the transgenes. The Bt Cry1Ac protein concentrations at bolting and flowering stages was higher compared to the 4-5-leaf and pod-formation stages. Nonetheless, the Bt toxin in the fifth backcrossing generation (BC5) was sufficient to kill both polyphagous (Helicoverpa armigera) and oligophagous (Plutella xylostella) Lepidoptera. As a consequence, the subsequent generations harboring the transgene from F1 to BC5 could have selection advantage against insect pests. The result is useful in understanding gene flow from transgenic crops and the followed transgene introgression into wild. PMID:25219305

  15. Acute, sublethal, and combination effects of azadirachtin and Bacillus thuringiensis on the cotton bollworm, Helicoverpa armigera.

    PubMed

    Abedi, Zahra; Saber, Moosa; Vojoudi, Samad; Mahdavi, Vahid; Parsaeyan, Ehsan

    2014-01-01

    The cotton bollworm, Helicoverpa armigera Hübner (Lepidoptera: Noctuidae) is a polyphagous and cosmopolitan insect pest that causes damage to various plants. In this study, the lethal and sublethal effects of azadirachtin and Bacillus thuringiensis Berliner sub sp . kurstaki (Bacillales: Bacillaceae) were evaluated on third instar H. armigera under laboratory conditions. The LC50 values of azadirachtin and Bt were 12.95 and 96.8 µg a.i./mL, respectively. A total mortality of 56.7% was caused on third instar larvae when LC20 values of the insecticides were applied in combination with each other. The LT50 values of azadirachtin and Bt were 4.8 and 3.6 days, respectively. The results of the sublethal study showed that the application of LC30 value of azadirachtin and Bt reduced the larval and pupal weight and increased larval and pupal duration of H. armigera. The longevity and fecundity of female adults were affected significantly by the insecticides. Female fecundity was reduced by the treatments, respectively. The lowest adult emergence ratio and pupation ratio were observed in the azadirachtin treatment. The results indicated that both insecticides have high potential for controlling of the pest. PMID:25373177

  16. Toxicity and affecting factors of Bacillus thuringiensis var. israelensis on Chironomus kiiensis larvae.

    PubMed

    Cao, Chuan-Wang; Sun, Li-Li; Wen, Rong-Rong; Li, Xiao-Peng; Wu, Hong-Qu; Wang, Zhi-Ying

    2012-01-01

    Abstract Bacillus thuringiensis var. israelensis (Bti) is a suitable agent for controlling Chironomus kiiensis, a major pest polluting water. In this study, laboratory bioassays were used to study toxicity and affecting factors of Bti on C. kiiensis larvae. Tests were conducted using three commercial Bti formulations (oil miscible suspension, 1,200 ITU/mL; wettable power, 1,200 ITU/mg; technical material, 5,000 ITU/mg) of Bti. The toxicity of Bti formulations to third and fourth instar C. kiiensis larvae was in decreasing order of technical material, oil miscible suspension, and wettable powder, based on the 12 and 24 hour LC50 values. Increasing larval densities (from 10 to 30 per bioassay cup) increased the LC50 values for fourth instar C. kiiensis larvae. The LC50 values for fourth instar larvae reared in sand substrate were higher than those from soil substrate, and autoclaved substrates significantly increased the LC50 values. The technical material of Bti at 12 and 24 hours responded similarly to changes in temperature between 30° C and 15° C, but the LC50 values at a range of tested temperatures showed distinct differences in time points.

  17. Aedes albopictus control with spray application of Bacillus thuringiensis israelensis, strain AM 65-52.

    PubMed

    Lam, Patrick H Y; Boon, Chia S; Yng, Ng Y; Benjamin, Seleena

    2010-09-01

    A Bacillus thuringiensis israelensis (Bti) formulation, VectoBac WG (strain AM 65-52), was evaluated for mosquito control in a training area with dense vegetation. Bti was spray applied to target Aedes albopictus larval habitats of 130 ha once every 2 weeks using a motorized back pack mist blower, Stihl SR420, and a vehicle mounted ultra low volume generator (ULV), IGEBA U40. Ovitrap index (OI) and larval density (LD) were used to measure the efficacy of larviciding. In the Bti treated area the OI and LD significantly decreased with time (p < 0.05); OI decreased from 84.3 +/- 1.7 to 27.5 +/- 2.5 (%) and LD decreased from 27.9 +/- 1.5 to 3.2 +/- 1.8 larvae per ovitrap by 3 months from the start of treatment. During the same period of time there was no significant reduction in OI and LD at the untreated site which was under a conventional mosquito control program. This large scale study indicates larvicidal spraying with Bti of natural breeding sites, was able to reduce Ae. albopictus adult density. This significant reduction was not achieved with conventional manual application methods. PMID:21073027

  18. Quantitative spectral light scattering polarimetry for monitoring fractal growth pattern of Bacillus thuringiensis bacterial colonies

    NASA Astrophysics Data System (ADS)

    Banerjee, Paromita; Soni, Jalpa; Ghosh, Nirmalya; Sengupta, Tapas K.

    2013-02-01

    It is of considerable current interest to develop various methods which help to understand and quantify the cellular association in growing bacterial colonies and is also important in terms of detection and identification of a bacterial species. A novel approach is used here to probe the morphological structural changes occurring during the growth of the bacterial colony of Bacillus thuringiensis under different environmental conditions (in normal nutrient agar, in presence of glucose - acting as additional nutrient and additional 3mM arsenate as additional toxic material). This approach combines the quantitative Mueller matrix polarimetry to extract intrinsic polarization properties and inverse analysis of the polarization preserving part of the light scattering spectra to determine the fractal parameter H (Hurst exponent) using Born approximation. Interesting differences are observed in the intrinsic polarization parameters and also in the Hurst exponent, which is a measurement of the fractality of a pattern formed by bacteria while growing as a colony. These findings are further confirmed with optical microscopic studies of the same sample and the results indicate a very strong and distinct dependence on the environmental conditions during growth, which can be exploited to quantify different bacterial species and their growth patterns.

  19. Susceptibility of field-collected populations of the southwestern corn borer, Diatraea grandiosella, to Bacillus thuringiensis.

    PubMed

    Trisyono, Y Andi; Chippendale, G Michael

    2002-10-01

    The susceptibilitity of newly hatched larvae of laboratory-adapted and field-collected populations of the Southwestern corn borer, Diatraea grandiosella (Dyar) (Lepidoptera: Crambidae), to a Bacillus thuringiensis protein (Cry1Ab) was examined using a larval feeding bioassay. D grandiosella populations were collected from five states: Missouri, Kansas, Texas, Tennessee and Kentucky. Using larval mortality as the end-point of the bioassay, the magnitude of differences in the susceptibility of the laboratory-adapted and the field-collected populations to Cry1Ab protein varied from one to 46 times depending on the time of observation and the standard of comparison (LC50 or LC95). However, significant differences in susceptibility to Cry1Ab protein among these populations were not detected when the comparisons were based on growth inhibition (EC50 or EC95); the magnitude of differences was less than fourfold. Either using larval mortality or larval growth inhibition, the results indicated that the field-collected populations of D grandiosella were susceptible to Cry1Ac, and differences in susceptibility may reflect natural variation among populations. The bioassay using larval growth inhibition offers advantages over that using larval mortality, including giving more accurate representation of the toxicological effects of the toxin.

  20. Transcriptome of the Lymantria dispar (Gypsy Moth) Larval Midgut in Response to Infection by Bacillus thuringiensis

    PubMed Central

    Sparks, Michael E.; Blackburn, Michael B.; Kuhar, Daniel; Gundersen-Rindal, Dawn E.

    2013-01-01

    Transcriptomic profiles of the serious lepidopteran insect pest Lymantria dispar (gypsy moth) were characterized in the larval midgut in response to infection by Bacillus thuringiensis kurstaki, a biopesticide commonly used for its control. RNA-Seq approaches were used to define a set of 49,613 assembled transcript sequences, of which 838, 1,248 and 3,305 were respectively partitioned into high-, mid- and low-quality tiers on the basis of homology information. Digital gene expression profiles suggested genes differentially expressed at 24 hours post infection, and qRT-PCR analyses were performed for verification. The differentially expressed genes primarily associated with digestive function, including α-amylase, lipase and carboxypeptidase; immune response, including C-type lectin 4; developmental genes such as arylphorin; as well as a variety of binding proteins: cellular retinoic acid binding protein (lipid-binding), insulin-related peptide binding protein (protein-binding) and ovary C/EBPg transcription factor (nucleic acid-binding). This is the first study conducted to specifically investigate gypsy moth response to a bacterial infection challenge using large-scale sequencing technologies, and the results highlight important genes that could be involved in biopesticide resistance development or could serve as targets for biologically-based control mechanisms of this insect pest. PMID:23658687

  1. Optimization of photobioreactor growth conditions for a cyanobacterium expressing mosquitocidal Bacillus thuringiensis Cry proteins.

    PubMed

    Ketseoglou, Irene; Bouwer, Gustav

    2013-08-10

    An Anabaena strain (PCC 7120#11) that was genetically engineered to express Bacillus thuringiensis subsp. israelensis cry genes has shown good larvicidal activity against Anopheles arabiensis, a major vector of malaria in Africa. Response surface methodology was used to evaluate the relationship between key growth factors and the volumetric productivity of PCC 7120#11 in an indoor, flat-plate photobioreactor. The interaction of input CO₂ concentration and airflow rate had a statistically significant effect on the volumetric productivity of PCC 7120#11, as did the interaction of airflow rate and photosynthetic photon flux density. Model-based numerical optimization indicated that the optimal factor level combination for maximizing PCC 7120#11 volumetric productivity was a photosynthetic photon flux density of 154 μmol m⁻² s⁻¹ and air enriched with 3.18% (v/v) CO₂ supplied at a flow rate of 1.02 vessel volumes per minute. At the levels evaluated in the study, none of the growth factors had a significant effect on the median lethal concentration of PCC 7120#11 against An. arabiensis larvae. This finding is important because loss of mosquitocidal activity under growth conditions that maximize volumetric productivity would impact on the feasibility of using PCC 7120#11 in malaria vector control programs. The study showed the usefulness of response surface methodology for determination of the optimal growth conditions for a cyanobacterium that is genetically engineered to have larvicidal activity against malaria vectors. PMID:23732832

  2. Isolation, purification and physicochemical characterization of water-soluble Bacillus thuringiensis melanin.

    PubMed

    Aghajanyan, Armen E; Hambardzumyan, Artur A; Hovsepyan, Anichka S; Asaturian, Rafael A; Vardanyan, Andranik A; Saghiyan, Ashot A

    2005-04-01

    Melanins are widely used in medicine, pharmacology, cosmetics and other fields. Although several technologies for the purification of water-insoluble dioxyphenylalanine (DOPA) melanins have been described, a source of water-soluble melanin is highly desirable. Here we describe an effective procedure for the isolation and purification of water-soluble melanin using the culture medium of Bacillus thuringiensis subsp. galleriae strain K1. Water-soluble melanin from this organism has an isoelectric point (pI=3.0-3.2) and was purified optimally by adsorbtion using the IA-1r resin and elution as a concentrated solution. The purified melanin obtained exhibited a similar infra-red absorbtion spectrum to synthetic melanin and contained quinolic and phenolic structures and an amino acid content of around 20% after acid hydrolysis. The molecular weight of the purified melanin determined by SDS-PAGE was 4 kDa and the electromagnetic spin resonance spectrum of the purified microbial melanin was a slightly asymmetric singlet without hyperfine structure with about 7 Gauss width of the line between points of the maximum incline and g=2.006. The concentration of paramagnetic centers in melanin is 0.21x10(18) spin/g. The results obtained provide a rapid, simple and inexpensive method for the large scale purification of water soluble melanin that may have widespread applications.

  3. Biosynthesis of silver nanoparticles using Bacillus thuringiensis against dengue vector, Aedes aegypti (Diptera: Culicidae).

    PubMed

    Banu, A Najitha; Balasubramanian, C; Moorthi, P Vinayaga

    2014-01-01

    The present study reveals the larvicidal activity of silver nanoparticles (AgNPs) synthesized by Bacillus thuringiensis (Bt) against Aedes aegypti responsible for the diseases of public health importance. The Bt-AgNPs were characterized by using UV-visible spectrophotometer followed by scanning electron microscopy (SEM) and energy-dispersive X-ray (EDX) spectroscopy. A surface plasmon resonance spectrum of AgNps was obtained at 420 nm. The particle sizes were measured through SEM imaging ranging from 43.52 to 142.97 nm. The Bt-AgNPs has also given a characteristic peak at 3 keV in EDX image. Interestingly, the mortality rendered by Bt-AgNPs was comparatively high than that of the control against third-instar larvae of A. aegypti (LC50 0.10 ppm and LC90 0.39 ppm) in all the tested concentrations, viz. 0.03, 0.06, 0.09, 0.12, and 0.15 ppm. Hence, Bt-AgNPs would be significantly used as a potent mosquito larvicide against A. aegypti.

  4. Mode of action of Bacillus thuringiensis Cry and Cyt toxins and their potential for insect control.

    PubMed

    Bravo, Alejandra; Gill, Sarjeet S; Soberón, Mario

    2007-03-15

    Bacillus thuringiensis Crystal (Cry) and Cytolitic (Cyt) protein families are a diverse group of proteins with activity against insects of different orders--Lepidoptera, Coleoptera, Diptera and also against other invertebrates such as nematodes. Their primary action is to lyse midgut epithelial cells by inserting into the target membrane and forming pores. Among this group of proteins, members of the 3-Domain Cry family are used worldwide for insect control, and their mode of action has been characterized in some detail. Phylogenetic analyses established that the diversity of the 3-Domain Cry family evolved by the independent evolution of the three domains and by swapping of domain III among toxins. Like other pore-forming toxins (PFT) that affect mammals, Cry toxins interact with specific receptors located on the host cell surface and are activated by host proteases following receptor binding resulting in the formation of a pre-pore oligomeric structure that is insertion competent. In contrast, Cyt toxins directly interact with membrane lipids and insert into the membrane. Recent evidence suggests that Cyt synergize or overcome resistance to mosquitocidal-Cry proteins by functioning as a Cry-membrane bound receptor. In this review we summarize recent findings on the mode of action of Cry and Cyt toxins, and compare them to the mode of action of other bacterial PFT. Also, we discuss their use in the control of agricultural insect pests and insect vectors of human diseases. PMID:17198720

  5. Risk assessment and ecological effects of transgenic Bacillus thuringiensis crops on non-target organisms.

    PubMed

    Yu, Hui-Lin; Li, Yun-He; Wu, Kong-Ming

    2011-07-01

    The application of recombinant DNA technology has resulted in many insect-resistant varieties by genetic engineering (GE). Crops expressing Cry toxins derived from Bacillus thuringiensis (Bt) have been planted worldwide, and are an effective tool for pest control. However, one ecological concern regarding the potential effects of insect-resistant GE plants on non-target organisms (NTOs) has been continually debated. In the present study, we briefly summarize the data regarding the development and commercial use of transgenic Bt varieties, elaborate on the procedure and methods for assessing the non-target effects of insect-resistant GE plants, and synthetically analyze the related research results, mostly those published between 2005 and 2010. A mass of laboratory and field studies have shown that the currently available Bt crops have no direct detrimental effects on NTOs due to their narrow spectrum of activity, and Bt crops are increasing the abundance of some beneficial insects and improving the natural control of specific pests. The use of Bt crops, such as Bt maize and Bt cotton, results in significant reductions of insecticide application and clear benefits on the environment and farmer health. Consequently, Bt crops can be a useful component of integrated pest management systems to protect the crop from targeted pests.

  6. Optimization of photobioreactor growth conditions for a cyanobacterium expressing mosquitocidal Bacillus thuringiensis Cry proteins.

    PubMed

    Ketseoglou, Irene; Bouwer, Gustav

    2013-08-10

    An Anabaena strain (PCC 7120#11) that was genetically engineered to express Bacillus thuringiensis subsp. israelensis cry genes has shown good larvicidal activity against Anopheles arabiensis, a major vector of malaria in Africa. Response surface methodology was used to evaluate the relationship between key growth factors and the volumetric productivity of PCC 7120#11 in an indoor, flat-plate photobioreactor. The interaction of input CO₂ concentration and airflow rate had a statistically significant effect on the volumetric productivity of PCC 7120#11, as did the interaction of airflow rate and photosynthetic photon flux density. Model-based numerical optimization indicated that the optimal factor level combination for maximizing PCC 7120#11 volumetric productivity was a photosynthetic photon flux density of 154 μmol m⁻² s⁻¹ and air enriched with 3.18% (v/v) CO₂ supplied at a flow rate of 1.02 vessel volumes per minute. At the levels evaluated in the study, none of the growth factors had a significant effect on the median lethal concentration of PCC 7120#11 against An. arabiensis larvae. This finding is important because loss of mosquitocidal activity under growth conditions that maximize volumetric productivity would impact on the feasibility of using PCC 7120#11 in malaria vector control programs. The study showed the usefulness of response surface methodology for determination of the optimal growth conditions for a cyanobacterium that is genetically engineered to have larvicidal activity against malaria vectors.

  7. The occurrence of photorhabdus-like toxin complexes in Bacillus thuringiensis.

    PubMed

    Blackburn, Michael B; Martin, Phyllis A W; Kuhar, Daniel; Farrar, Robert R; Gundersen-Rindal, Dawn E

    2011-01-01

    Recently, genomic sequencing of a Bacillus thuringiensis (Bt) isolate from our collection revealed the presence of an apparent operon encoding an insecticidal toxin complex (Tca) similar to that first described from the entomopathogen Photorhabdus luminescens. To determine whether these genes are widespread among Bt strains, we screened isolates from the collection for the presence of tccC, one of the genes needed for the expression of fully functional toxin complexes. Among 81 isolates chosen to represent commonly encountered biochemical phenotypes, 17 were found to possess a tccC. Phylogenetic analysis of the 81 isolates by multilocus sequence typing revealed that all the isolates possessing a tccC gene were restricted to two sequence types related to Bt varieties morrisoni, tenebrionis, israelensis and toumanoffi. Sequencing of the ∼17 kb tca operon from two isolates representing each of the two sequence types revealed >99% sequence identity. Optical mapping of DNA from Bt isolates representing each of the sequence types revealed nearly identical plasmids of ca. 333 and 338 kbp, respectively. Selected isolates were found to be toxic to gypsy moth larvae, but were not as effective as a commercial strain of Bt kurstaki. Some isolates were found to inhibit growth of Colorado potato beetle. Custom Taqman® relative quantitative real-time PCR assays for Tc-encoding Bt revealed both tcaA and tcaB genes were expressed within infected gypsy moth larvae. PMID:21464948

  8. Acute, Sublethal, and Combination Effects of Azadirachtin and Bacillus thuringiensis on the Cotton Bollworm, Helicoverpa armigera

    PubMed Central

    Abedi, Zahra; Saber, Moosa; Vojoudi, Samad; Mahdavi, Vahid; Parsaeyan, Ehsan

    2014-01-01

    The cotton bollworm, Helicoverpa armigera Hübner (Lepidoptera: Noctuidae) is a polyphagous and cosmopolitan insect pest that causes damage to various plants. In this study, the lethal and sublethal effects of azadirachtin and Bacillus thuringiensis Berliner sub sp. kurstaki (Bacillales: Bacillaceae) were evaluated on third instar H. armigera under laboratory conditions. The LC50 values of azadirachtin and Bt were 12.95 and 96.8 µg a.i./mL, respectively. A total mortality of 56.7% was caused on third instar larvae when LC20 values of the insecticides were applied in combination with each other. The LT50 values of azadirachtin and Bt were 4.8 and 3.6 days, respectively. The results of the sublethal study showed that the application of LC30 value of azadirachtin and Bt reduced the larval and pupal weight and increased larval and pupal duration of H. armigera. The longevity and fecundity of female adults were affected significantly by the insecticides. Female fecundity was reduced by the treatments, respectively. The lowest adult emergence ratio and pupation ratio were observed in the azadirachtin treatment. The results indicated that both insecticides have high potential for controlling of the pest. PMID:25373177

  9. Role of helix 3 in pore formation by the Bacillus thuringiensis insecticidal toxin Cry1Aa.

    PubMed

    Vachon, Vincent; Préfontaine, Gabrielle; Coux, Florence; Rang, Cécile; Marceau, Lucie; Masson, Luke; Brousseau, Roland; Frutos, Roger; Schwartz, Jean-Louis; Laprade, Raynald

    2002-05-14

    Helix 3 of the Cry1Aa toxin from Bacillus thuringiensis possesses eight charged amino acids. These residues, with the exception of those involved in intramolecular salt bridges (E90, R93, E112, and R115), were mutated individually either to a neutral or to an oppositely charged amino acid. The mutated genes were expressed, and the resultant, trypsin-activated toxins were assessed for their toxicity to Manduca sexta larvae and their ability to permeabilize M. sexta larval midgut brush border membrane vesicles to KCl, sucrose, raffinose, potassium gluconate, and N-methyl-D-glucamine hydrochloride with a light-scattering assay based on osmotic swelling. Most mutants were considerably less toxic than Cry1Aa. Replacing either E101, E116, E118, or D120 by cysteine, glutamine, or lysine residues had only minor effects on the properties of the pores formed by the modified toxins. However, half of these mutants (E101C, E101Q, E101K, E116K, E118C, and D120K) had a significantly slower rate of pore formation than Cry1Aa. Mutations at R99 (R99C, R99E, and R99Y) resulted in an almost complete loss of pore-forming ability. These results are consistent with a model in which alpha-helix 3 plays an important role in the mechanism of pore formation without being directly involved in determining the properties of the pores. PMID:11994014

  10. Kinetics of size changes of individual Bacillus thuringiensis spores in response to changes in relative humidity

    PubMed Central

    Westphal, Andrew J.; Price, P. Buford; Leighton, Terrance J.; Wheeler, Katherine E.

    2003-01-01

    Using an automated scanning microscope, we report the surprising result that individual dormant spores of Bacillus thuringiensis grow and shrink in response to increasing and decreasing relative humidity. We simultaneously monitored the size of inorganic calibration particles. We found that the spores consistently swell in response to increased relative humidity, and shrink to near their original size on reexposure to dry air. Although the dispersion of swelling amplitudes within an ensemble of spores is wide (≈30% of the average amplitude), amplitudes for individual spores are highly correlated between different swelling episodes, suggesting that individual spores respond consistently to changes in humidity. We find evidence for two distinct time scales for swelling: one with a time scale of no more than ≈50 s, and another with a time scale of ≈8 min. We speculate that these two mechanisms may be due to rapid diffusion of water into the spore coat + cortex, followed by slower diffusion of water into the spore core, respectively. Humidity-dependent swelling may account for the greater kill effectiveness of spores by gas-phase chlorine dioxide, formaldehyde, and ethylene oxide at very high relative humidity. PMID:12584363

  11. Current models of the mode of action of Bacillus thuringiensis insecticidal crystal proteins: a critical review.

    PubMed

    Vachon, Vincent; Laprade, Raynald; Schwartz, Jean-Louis

    2012-09-15

    Bacillus thuringiensis (Bt) Cry toxins constitute the active ingredient in the most widely used biological insecticides and insect-resistant transgenic crops. A clear understanding of their mode of action is necessary for improving these products and ensuring their continued use. Accordingly, a long history of intensive research has established that their toxic effect is due primarily to their ability to form pores in the plasma membrane of the midgut epithelial cells of susceptible insects. In recent years, a rather elaborate model involving the sequential binding of the toxins to different membrane receptors has been developed to describe the events leading to membrane insertion and pore formation. However, it was also proposed recently that, in contradiction with this mechanism, Bt toxins function by activating certain intracellular signaling pathways which lead to the necrotic death of their target cells without the need for pore formation. Because work in this field has largely focused, for several years, on the elaboration and promotion of these two models, the present revue examines in detail the experimental evidence on which they are based. It is concluded that the presently available information still supports the notion that Bt Cry toxins act by forming pores, but most events leading to their formation, following binding of the activated toxins to their receptors, remain relatively poorly understood.

  12. Molecular approaches to improve the insecticidal activity of Bacillus thuringiensis Cry toxins.

    PubMed

    Lucena, Wagner A; Pelegrini, Patrícia B; Martins-de-Sa, Diogo; Fonseca, Fernando C A; Gomes, Jose E; de Macedo, Leonardo L P; da Silva, Maria Cristina M; Oliveira, Raquel S; Grossi-de-Sa, Maria F

    2014-08-13

    Bacillus thuringiensis (Bt) is a gram-positive spore-forming soil bacterium that is distributed worldwide. Originally recognized as a pathogen of the silkworm, several strains were found on epizootic events in insect pests. In the 1960s, Bt began to be successfully used to control insect pests in agriculture, particularly because of its specificity, which reflects directly on their lack of cytotoxicity to human health, non-target organisms and the environment. Since the introduction of transgenic plants expressing Bt genes in the mid-1980s, numerous methodologies have been used to search for and improve toxins derived from native Bt strains. These improvements directly influence the increase in productivity and the decreased use of chemical insecticides on Bt-crops. Recently, DNA shuffling and in silico evaluations are emerging as promising tools for the development and exploration of mutant Bt toxins with enhanced activity against target insect pests. In this report, we describe natural and in vitro evolution of Cry toxins, as well as their relevance in the mechanism of action for insect control. Moreover, the use of DNA shuffling to improve two Bt toxins will be discussed together with in silico analyses of the generated mutations to evaluate their potential effect on protein structure and cytotoxicity.

  13. Alteration in Bacillus thuringiensis toxicity by curing gut flora: novel approach for mosquito resistance management.

    PubMed

    Patil, Chandrashekhar D; Borase, Hemant P; Salunke, Bipinchandra K; Patil, Satish V

    2013-09-01

    Mosquitoes are known for acquiring resistance against insecticides in many ways, namely target side mutation, enzyme modification, sequestration, quick elimination, etc. But, the role of microflora present in abundance in the larval midgut is less explored with respect to their role in insecticide resistance. During the course of their development, mosquitoes are continuously exposed to microbes and have naturally acquired midgut microbial flora. This midgut flora can modulate the mosquito's susceptibility to Bacillus thuringiensis (Bt) infection by degrading toxic Bt protein forms through an unknown mechanism. In this study, we show that microbe-free aseptic mosquito larvae displayed an increased susceptibility to Bt toxicity compared to larvae harboring natural microbial flora. Fourth instar larvae of Anopheles stephensi were treated separately with penicillin, streptomycin, erythromycin (100 μg/ml), and mixtures of all three antibiotics and then analyzed for Bt toxicity. We have also examined the influence of the mosquito's midgut microbial flora under microaerophilic condition on the Bt protein degradation through plate, broth, TLC, and UV-vis spectrophotometric assay. A better understanding of the roles of microbiota in preventing Bt toxicity to mosquitoes could potentially lead to the development of new sustainable mosquito control strategies.

  14. Transcriptome of the Lymantria dispar (gypsy moth) larval midgut in response to infection by Bacillus thuringiensis.

    PubMed

    Sparks, Michael E; Blackburn, Michael B; Kuhar, Daniel; Gundersen-Rindal, Dawn E

    2013-01-01

    Transcriptomic profiles of the serious lepidopteran insect pest Lymantria dispar (gypsy moth) were characterized in the larval midgut in response to infection by Bacillus thuringiensis kurstaki, a biopesticide commonly used for its control. RNA-Seq approaches were used to define a set of 49,613 assembled transcript sequences, of which 838, 1,248 and 3,305 were respectively partitioned into high-, mid- and low-quality tiers on the basis of homology information. Digital gene expression profiles suggested genes differentially expressed at 24 hours post infection, and qRT-PCR analyses were performed for verification. The differentially expressed genes primarily associated with digestive function, including α-amylase, lipase and carboxypeptidase; immune response, including C-type lectin 4; developmental genes such as arylphorin; as well as a variety of binding proteins: cellular retinoic acid binding protein (lipid-binding), insulin-related peptide binding protein (protein-binding) and ovary C/EBPg transcription factor (nucleic acid-binding). This is the first study conducted to specifically investigate gypsy moth response to a bacterial infection challenge using large-scale sequencing technologies, and the results highlight important genes that could be involved in biopesticide resistance development or could serve as targets for biologically-based control mechanisms of this insect pest. PMID:23658687

  15. Construction of an environmental safe Bacillus thuringiensis engineered strain against Coleoptera.

    PubMed

    Yu, Yajun; Yuan, Yihui; Gao, Meiying

    2016-05-01

    Cloning of new toxic genes from Bacillus thuringiensis (Bt) and construction of Bt engineered strains are two key strategies for bio-control of coleopteran pests in agriculture and forestry. In this study, we cloned a new cry3Aa-type gene, cry3Aa8, from wild Bt strain YC-03 against coleopteran, and constructed a Bt engineered strain, ACE-38, containing insecticidal protein-encoding gene cry3Aa8. The engineered strain, with almost four times of Cry3Aa yield compared with strain YC-03, was an antibiotic marker-free strain. Though no selective pressure was presented in the medium, cry3Aa8 in the engineered strain ACE-38 remained stable. The yield of Cry3Aa by strain ACE-38 reached 2.09 mg/ml in the optimized fermentation medium. The activity of strain ACE-38 against Plagiodera versicolora was tested, and the LC50 of ACE-38 cultures in the optimized fermentation medium was 1.13 μl/ml. Strain ACE-38 is a non-antibiotic Bt engineered strain with high Chrysomelidae toxicity and exhibits good fermentation property. The modified indigenous site-specific recombination system constructed in this study might be useful for the construction of Bt engineered strains containing genes that cannot be expressed in the indigenous site-specific recombination system using plasmid pBMB1205R. PMID:26767987

  16. The impact of strain diversity and mixed infections on the evolution of resistance to Bacillus thuringiensis.

    PubMed

    Raymond, Ben; Wright, Denis J; Crickmore, Neil; Bonsall, Michael B

    2013-10-22

    Pesticide mixtures can reduce the rate at which insects evolve pesticide resistance. However, with live biopesticides such as the naturally abundant pathogen Bacillus thuringiensis (Bt), a range of additional biological considerations might affect the evolution of resistance. These can include ecological interactions in mixed infections, the different rates of transmission post-application and the impact of the native biodiversity on the frequency of mixed infections. Using multi-generation selection experiments, we tested how applications of single and mixed strains of Bt from diverse sources (natural isolates and biopesticides) affected the evolution of resistance in the diamondback moth, Plutella xylostella, to a focal strain. There was no significant difference in the rate of evolution of resistance between single and mixed-strain applications although the latter did result in lower insect populations. The relative survivorship of Bt-resistant genotypes was higher in the mixed-strain treatment, in part owing to elevated mortality of susceptible larvae in mixtures. Resistance evolved more quickly with treatments that contained natural isolates, and biological differences in transmission rate may have contributed to this. Our data indicate that the use of mixtures can have unexpected consequences on the fitness of resistant and susceptible insects. PMID:24004937

  17. Stable Bacillus thuringiensis transgene introgression from Brassica napus to wild mustard B. juncea.

    PubMed

    Cao, Di; Stewart, C Neal; Zheng, Min; Guan, Zhengjun; Tang, Zhi-Xi; Wei, Wei; Ma, Ke-Ping

    2014-10-01

    Transgenic canola (Brassica napus) with a Bacillus thuringiensis cry1Ac gene and a green fluorescent protein (GFP) marker gene was used in hybridization experiments with wild Brassica juncea. Hybrid F1 and successive five backcross generations were obtained. The pod-set frequency on backcrossed B. juncea plants was over 66%, which suggested relatively high crossing compatibility between the hybrids and wild species. The seed setting in BC1 was the least of all generations tested, and then increased at the BC2 generation for which the thousand-seed weight was the highest of all generations. Seed size in backcrossed generations eventually approached that of the wild parent. The plants in all backcrossed generations were consistent with the expected 1:1 segregation ratio of the transgenes. The Bt Cry1Ac protein concentrations at bolting and flowering stages was higher compared to the 4-5-leaf and pod-formation stages. Nonetheless, the Bt toxin in the fifth backcrossing generation (BC5) was sufficient to kill both polyphagous (Helicoverpa armigera) and oligophagous (Plutella xylostella) Lepidoptera. As a consequence, the subsequent generations harboring the transgene from F1 to BC5 could have selection advantage against insect pests. The result is useful in understanding gene flow from transgenic crops and the followed transgene introgression into wild.

  18. Transcriptome of the Lymantria dispar (gypsy moth) larval midgut in response to infection by Bacillus thuringiensis.

    PubMed

    Sparks, Michael E; Blackburn, Michael B; Kuhar, Daniel; Gundersen-Rindal, Dawn E

    2013-01-01

    Transcriptomic profiles of the serious lepidopteran insect pest Lymantria dispar (gypsy moth) were characterized in the larval midgut in response to infection by Bacillus thuringiensis kurstaki, a biopesticide commonly used for its control. RNA-Seq approaches were used to define a set of 49,613 assembled transcript sequences, of which 838, 1,248 and 3,305 were respectively partitioned into high-, mid- and low-quality tiers on the basis of homology information. Digital gene expression profiles suggested genes differentially expressed at 24 hours post infection, and qRT-PCR analyses were performed for verification. The differentially expressed genes primarily associated with digestive function, including α-amylase, lipase and carboxypeptidase; immune response, including C-type lectin 4; developmental genes such as arylphorin; as well as a variety of binding proteins: cellular retinoic acid binding protein (lipid-binding), insulin-related peptide binding protein (protein-binding) and ovary C/EBPg transcription factor (nucleic acid-binding). This is the first study conducted to specifically investigate gypsy moth response to a bacterial infection challenge using large-scale sequencing technologies, and the results highlight important genes that could be involved in biopesticide resistance development or could serve as targets for biologically-based control mechanisms of this insect pest.

  19. No Adjuvant Effect of Bacillus thuringiensis-Maize on Allergic Responses in Mice

    PubMed Central

    Dekan, Gerhard; Epstein, Michelle M.

    2014-01-01

    Genetically modified (GM) foods are evaluated carefully for their ability to induce allergic disease. However, few studies have tested the capacity of a GM food to act as an adjuvant, i.e. influencing allergic responses to other unrelated allergens at acute onset and in individuals with pre-existing allergy. We sought to evaluate the effect of short-term feeding of GM Bacillus thuringiensis (Bt)-maize (MON810) on the initiation and relapse of allergic asthma in mice. BALB/c mice were provided a diet containing 33% GM or non-GM maize for up to 34 days either before ovalbumin (OVA)-induced experimental allergic asthma or disease relapse in mice with pre-existing allergy. We observed that GM-maize feeding did not affect OVA-induced eosinophilic airway and lung inflammation, mucus hypersecretion or OVA-specific antibody production at initiation or relapse of allergic asthma. There was no adjuvant effect upon GM-maize consumption on the onset or severity of allergic responses in a mouse model of allergic asthma. PMID:25084284

  20. Host-Pathogen Coevolution: The Selective Advantage of Bacillus thuringiensis Virulence and Its Cry Toxin Genes.

    PubMed

    Masri, Leila; Branca, Antoine; Sheppard, Anna E; Papkou, Andrei; Laehnemann, David; Guenther, Patrick S; Prahl, Swantje; Saebelfeld, Manja; Hollensteiner, Jacqueline; Liesegang, Heiko; Brzuszkiewicz, Elzbieta; Daniel, Rolf; Michiels, Nicolaas K; Schulte, Rebecca D; Kurtz, Joachim; Rosenstiel, Philip; Telschow, Arndt; Bornberg-Bauer, Erich; Schulenburg, Hinrich

    2015-06-01

    Reciprocal coevolution between host and pathogen is widely seen as a major driver of evolution and biological innovation. Yet, to date, the underlying genetic mechanisms and associated trait functions that are unique to rapid coevolutionary change are generally unknown. We here combined experimental evolution of the bacterial biocontrol agent Bacillus thuringiensis and its nematode host Caenorhabditis elegans with large-scale phenotyping, whole genome analysis, and functional genetics to demonstrate the selective benefit of pathogen virulence and the underlying toxin genes during the adaptation process. We show that: (i) high virulence was specifically favoured during pathogen-host coevolution rather than pathogen one-sided adaptation to a nonchanging host or to an environment without host; (ii) the pathogen genotype BT-679 with known nematocidal toxin genes and high virulence specifically swept to fixation in all of the independent replicate populations under coevolution but only some under one-sided adaptation; (iii) high virulence in the BT-679-dominated populations correlated with elevated copy numbers of the plasmid containing the nematocidal toxin genes; (iv) loss of virulence in a toxin-plasmid lacking BT-679 isolate was reconstituted by genetic reintroduction or external addition of the toxins. We conclude that sustained coevolution is distinct from unidirectional selection in shaping the pathogen's genome and life history characteristics. To our knowledge, this study is the first to characterize the pathogen genes involved in coevolutionary adaptation in an animal host-pathogen interaction system. PMID:26042786

  1. Evaluation of Bt (Bacillus thuringiensis) corn on mouse testicular development by dual parameter flow cytometry.

    PubMed

    Brake, Denise G; Thaler, Robert; Evenson, Donald P

    2004-04-01

    The health safety of Bt (Bacillus thuringiensis) corn (Zea mays L.) was studied using mouse testes as a sensitive biomonitor of potential toxic effects. Pregnant mice were fed a Bt corn or a nontransgenic (conventional) diet during gestation and lactation. After they were weaned, young male mice were maintained on the respective diets. At 8, 16, 26, 32, 63, and 87 days after birth, three male mice and an adult reference mouse were killed, the testes were surgically removed, and the percentage of germ cell populations was measured by flow cytometry. Multigenerational studies were conducted in the same manner. There were no apparent differences in percentages of testicular cell populations (haploid, diploid, and tetraploid) between the mice fed the Bt corn diet and those fed the conventional diet. Because of the high rate of cell proliferation and extensive differentiation that makes testicular germ cells highly susceptible to some toxic agents, it was concluded that the Bt corn diet had no measurable or observable effect on fetal, postnatal, pubertal, or adult testicular development. If data from this study were extrapolated to humans, Bt corn is not harmful to human reproductive development.

  2. The Efficacy of Bacillus thuringiensis spp. galleriae Against Rice Water Weevil (Coleoptera: Curculionidae) for Integrated Pest Management in California Rice.

    PubMed

    Aghaee, Mohammad-Amir; Godfrey, Larry D

    2015-02-01

    Rice water weevil (Lissorhoptrus oryzophilus Kushel) is the most damaging insect pest of rice in the United States. Larval feeding on the roots stunt growth and reduce yield. Current pest management against the weevil in California relies heavily on pyrethroids that can be damaging to aquatic food webs. Examination of an environmentally friendly alternative biopesticide based on Bacillus thuringiensis spp. galleriae chemistry against rice water weevil larvae showed moderate levels of activity in pilot studies. We further examined the performance of different formulations of Bt.galleriae against the leading insecticide used in California rice, λ-cyhalothrin. The granular formulation performed as well as the λ-cyhalothrin in use in California in some of our greenhouse and field studies. This is the first reported use of B. thuringiensis spp. galleriae against rice water weevil.

  3. Response surface modeling for hot, humid air decontamination of materials contaminated with Bacillus anthracis ∆Sterne and Bacillus thuringiensis Al Hakam spores

    PubMed Central

    2014-01-01

    Response surface methodology using a face-centered cube design was used to describe and predict spore inactivation of Bacillus anthracis ∆Sterne and Bacillus thuringiensis Al Hakam spores after exposure of six spore-contaminated materials to hot, humid air. For each strain/material pair, an attempt was made to fit a first or second order model. All three independent predictor variables (temperature, relative humidity, and time) were significant in the models except that time was not significant for B. thuringiensis Al Hakam on nylon. Modeling was unsuccessful for wiring insulation and wet spores because there was complete spore inactivation in the majority of the experimental space. In cases where a predictive equation could be fit, response surface plots with time set to four days were generated. The survival of highly purified Bacillus spores can be predicted for most materials tested when given the settings for temperature, relative humidity, and time. These predictions were cross-checked with spore inactivation measurements. PMID:24949256

  4. Isolation and distribution of mosquito-larvicidal cry genes in Bacillus thuringiensis strains native to Saudi Arabia.

    PubMed

    El-kersh, T A; Al-akeel, R A; Al-sheikh, Y A; Alharbi, S A

    2014-12-01

    A total of 157 environmental samples were collected from 11 ecological regions across Saudi Arabia to isolate native Bacillus thuringiensis (Bt) strains. Bt isolates (n=103) were recovered by the 50% (v/v) ethanol treatment method with Bt index range of 0.01 to 0.4. Most of Bt isolates showed spherical crystals (54%), while, irregular, bi-pyramidal, and spore-attached crystal constituted 27, 16 and 3% respectively. PCR analysis with eight general and specific dipteran primers of Cry and Cyt genes, revealed positive amplification for cry4 & cyt1, and cry4A, cry4B and cyt2, and cry 10 and cry 11 genes in 28%, 26%, 22%, and 25% of tested strains respectively; whereas cry2 gene was not detected except with the reference Bt kurstaki HD-1 strain. Bioassays against Aedes caspuis and Culex pipiens larvae indicated that 11 strains displayed better larvicidal activity compared with Bacillus thuringiensis H14 (Bti) reference (LC50 0.6 μg/ml) strain against Ae. caspuis, but only two strains (620A & 633R1, LC50 of 0.09 μg/ml & 0.064 μg/ml) that gave significant enhancement. Additionally, one strain (633R1) showed LC50 similar to that of Bti H14 (LC50 0.064 μg/ml) against Cx. pipiens. With the exception of cyt primers, sequenced DNA of all positive primers amplicons revealed 95 to 99% identity in GenBank with Bacillus thuringiensis subsp. israelensis plasmid pBtoxis and also correlated with its SDS-PAGE expressed protein profiles analysis. It is hoped that our wild bio-insecticide Bt strains can be explored in future in the control of mosquito-vector borne diseases in Saudi Arabia.

  5. Operational Evaluation Of Vectomax® WSP (Bacillus thuringiensis Subsp. israelensis+Bacillus sphaericus) Against Larval Culex pipiens in Septic Tanks (1).

    PubMed

    Cetin, Huseyin; Oz, Emre; Yanikoglu, Atila; Cilek, James E

    2015-06-01

    The residual effectiveness of VectoMax® WSP (a water-soluble pouch formulation containing a combination of Bacillus thuringiensis subsp. israelensis strain AM65-52 and B. sphaericus strain ABTS 1743) when applied to septic tanks against 3rd- and 4th-stage larvae of Culex pipiens L. was evaluated in this study. This formulation was evaluated at operational application rates of 1 pouch (10 g) and 2 pouches (20 g) per septic tank. Both application rates resulted in >96% control of larvae for 24 days. Operationally, VectoMax WSP has proven to be a useful tool for the nonchemical control of Culex species in septic tank environments.

  6. Transgenic Bacillus thuringiensis (Bt) Rice Is Safer to Aquatic Ecosystems than Its Non-Transgenic Counterpart

    PubMed Central

    Li, Guangsheng; Wang, Yongmo; Liu, Biao; Zhang, Guoan

    2014-01-01

    Rice lines genetically modified with the crystal toxin genes from Bacillus thuringiensis (Bt) have experienced rapid development, with biosafety certificates for two Bt rice lines issued in 2009. There has still been no commercial release of these lines yet due to public concerns about human health and environmental risks. Some studies confirmed that Bt rice was as safe as conventional rice to non-target organisms when pesticides were not applied, however, pesticides are still required in Bt rice to control non-lepidopteran pests. In this study, we assessed the environmental effects of two Bt rice lines expressing either the cry1Ab/1Ac or cry2A genes, respectively, by using zooplanktons as indicator species under normal field management practices using pesticides when required. In the whole rice growing season, non-Bt rice was sprayed 5 times while Bt rice was sprayed 2 times, which ensured both rice achieved a normal yield. Field investigations showed that rice type (Bt and non-Bt) significantly influenced zooplankton abundance and diversity, which were up to 95% and 80% lower in non-Bt rice fields than Bt rice fields. Laboratory rearing showed that water from non-Bt rice fields was significantly less suitable for the survival and reproduction of Daphnia magna and Paramecium caudatum in comparison with water from Bt rice fields. Higher pesticide residues were detected in the water from non-Bt than Bt rice fields, accounting for the bad performance of zooplankton in non-Bt field water. Our results demonstrate that Bt rice is safer to aquatic ecosystems than non-Bt rice, and its commercialization will be beneficial for biodiversity restoration in rice-based ecosystems. PMID:25105299

  7. Effects of bacillus thuringiensis transgenic corn on corn earworm and fall armyworm (Lepidoptera: Noctuidae) densities.

    PubMed

    Chilcutt, Charles F; Odvody, Gary N; Correa, J Carlos; Remmers, Jeff

    2007-04-01

    We examined 17 pairs of near-isogenic hybrids of Bacillus thuringiensis (Bt) (176, Mon810, and Bt11) and non-Bt corn, Zea mays L., to examine the effects of Bt on larval densities of Helicoverpa zea (Boddie) and Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae) during 2 yr. During ear formation, instar densities of H. zea and S. frugiperda were recorded for each hybrid. We found that H. zea first, second, and fifth instar densities were each affected by Mon810 and Bt11 Bt corn but not by 176 corn. Surprisingly, first and second instars were found in higher numbers on ears of Mon810 and Bt11 corn than on non-Bt corn. Densities of third and fourth instars were equal on Bt and non-Bt hybrids, whereas densities of fifth instars were lower on Bt plants. S. frugiperda larval densities were only affected during 1 yr when second, and fourth to sixth instars were lower on ears of Mon810 and Bt11 hybrids compared with their non-Bt counterparts. Two likely explanations for early instar H. zea densities being higher on Bt corn than non-Bt corn are that (1) Bt toxins delay development, creating a greater abundance of early instars that eventually die, and (2) reduced survival of H. zea to later instars on Bt corn decreased the normal asymmetric cannibalism or H. zea-S. frugiperda intraguild predation of late instars on early instars. Either explanation could explain why differences between Bt and non-Bt plants were greater for H. zea than S. frugiperda, because H. zea is more strongly affected by Bt toxins and more cannibalistic.

  8. Transgenic Bacillus thuringiensis (Bt) rice is safer to aquatic ecosystems than its non-transgenic counterpart.

    PubMed

    Li, Guangsheng; Wang, Yongmo; Liu, Biao; Zhang, Guoan

    2014-01-01

    Rice lines genetically modified with the crystal toxin genes from Bacillus thuringiensis (Bt) have experienced rapid development, with biosafety certificates for two Bt rice lines issued in 2009. There has still been no commercial release of these lines yet due to public concerns about human health and environmental risks. Some studies confirmed that Bt rice was as safe as conventional rice to non-target organisms when pesticides were not applied, however, pesticides are still required in Bt rice to control non-lepidopteran pests. In this study, we assessed the environmental effects of two Bt rice lines expressing either the cry1Ab/1Ac or cry2A genes, respectively, by using zooplanktons as indicator species under normal field management practices using pesticides when required. In the whole rice growing season, non-Bt rice was sprayed 5 times while Bt rice was sprayed 2 times, which ensured both rice achieved a normal yield. Field investigations showed that rice type (Bt and non-Bt) significantly influenced zooplankton abundance and diversity, which were up to 95% and 80% lower in non-Bt rice fields than Bt rice fields. Laboratory rearing showed that water from non-Bt rice fields was significantly less suitable for the survival and reproduction of Daphnia magna and Paramecium caudatum in comparison with water from Bt rice fields. Higher pesticide residues were detected in the water from non-Bt than Bt rice fields, accounting for the bad performance of zooplankton in non-Bt field water. Our results demonstrate that Bt rice is safer to aquatic ecosystems than non-Bt rice, and its commercialization will be beneficial for biodiversity restoration in rice-based ecosystems.

  9. Absence of toxicity of Bacillus thuringiensis pollen to black swallowtails under field conditions

    PubMed Central

    Wraight, C. L.; Zangerl, A. R.; Carroll, M. J.; Berenbaum, M. R.

    2000-01-01

    A single laboratory study on monarch butterflies has prompted widespread concern that corn pollen, engineered to express Bacillus thuringiensis (Bt) endotoxin, might travel beyond corn fields and cause mortality in nontarget lepidopterans. Among the lepidopterans at high potential risk from this technology is the black swallowtail butterfly, Papilio polyxenes, whose host plants in the midwestern U.S. are located principally in narrow strips between roads and crop fields. A field study was performed to assess whether mortality of early instar black swallowtails was associated either with proximity to a field of Bt corn or by levels of Bt pollen deposition on host plants. Potted host plants were infested with first instar black swallowtails and placed at intervals from the edge of a field of Bt corn (Pioneer 34R07 containing Monsanto event 810) at the beginning of anthesis. We confirmed by ELISA that pollen from these plants contained Cry1Ab endotoxin (2.125 ± 0.289 ng/g). Although many of the larvae died during the 7 days that the experiments were run, there was no relationship between mortality and proximity to the field or pollen deposition on host plants. Moreover, pollen from these same plants failed to cause mortality in the laboratory at the highest pollen dose tested (10,000 grains/cm2), a level that far exceeded the highest pollen density observed in the field (200 grains/cm2). We conclude that Bt pollen of the variety tested is unlikely to affect wild populations of black swallowtails. Thus, our results suggest that at least some potential nontarget effects of the use of transgenic plants may be manageable. PMID:10840067

  10. Absence of toxicity of Bacillus thuringiensis pollen to black swallowtails under field conditions.

    PubMed

    Wraight, C L; Zangerl, A R; Carroll, M J; Berenbaum, M R

    2000-07-01

    A single laboratory study on monarch butterflies has prompted widespread concern that corn pollen, engineered to express Bacillus thuringiensis (Bt) endotoxin, might travel beyond corn fields and cause mortality in nontarget lepidopterans. Among the lepidopterans at high potential risk from this technology is the black swallowtail butterfly, Papilio polyxenes, whose host plants in the midwestern U. S. are located principally in narrow strips between roads and crop fields. A field study was performed to assess whether mortality of early instar black swallowtails was associated either with proximity to a field of Bt corn or by levels of Bt pollen deposition on host plants. Potted host plants were infested with first instar black swallowtails and placed at intervals from the edge of a field of Bt corn (Pioneer 34R07 containing Monsanto event 810) at the beginning of anthesis. We confirmed by ELISA that pollen from these plants contained Cry1Ab endotoxin (2.125 +/- 0.289 ng/g). Although many of the larvae died during the 7 days that the experiments were run, there was no relationship between mortality and proximity to the field or pollen deposition on host plants. Moreover, pollen from these same plants failed to cause mortality in the laboratory at the highest pollen dose tested (10,000 grains/cm(2)), a level that far exceeded the highest pollen density observed in the field (200 grains/cm(2)). We conclude that Bt pollen of the variety tested is unlikely to affect wild populations of black swallowtails. Thus, our results suggest that at least some potential nontarget effects of the use of transgenic plants may be manageable. PMID:10840067

  11. Susceptibility of field-collected Aedes aegypti (L.) (Diptera: Culicidae) to Bacillus thuringiensis israelensis and temephos.

    PubMed

    Loke, S R; Andy-Tan, W A; Benjamin, S; Lee, H L; Sofian-Azirun, M

    2010-12-01

    The susceptibility status of field-collected Aedes aegypti (L.) from a dengue endemic area to Bacillus thuringiensis israelensis (Bti) and temephos was determined. Since August 2007, biweekly ovitrap surveillance (OS) was conducted for 12 mo in 2 sites, A & B, in Shah Alam, Selangor. Site A was treated with a Bti formulation, VectoBac® WG at 500 g/ha, from December 2007 - June 2008 while Site B was subjected to routine dengue vector control activities conducted by the local municipality. Aedes aegypti larvae collected from OS in both sites were bred until F3 and evaluated for their susceptibility. The larvae were pooled according to 3 time periods, which corresponded to Bti treatment phases in site A: August - November 2007 (Bti pre-treatment phase); December 2007 - June 2008 (Bti treatment phase); and July - September 2008 (Bti post-treatment phase). Larvae were bioassayed against Bti or temephos in accordance with WHO standard methods. Larvae collected from Site A was resistant to temephos, while incipient temephos resistant was detected in Site B throughout the study using WHO diagnostic dosage of 0.02 mg/L. The LC50 of temephos ranged between 0.007040 - 0.03799 mg/L throughout the year in both sites. Resistance ratios (LC50) indicated that temephos resistance increased with time, from 1.2 - 6.7 folds. The LC50 of Ae. aegypti larvae to Bti ranged between 0.08890 - 0.1814 mg/L throughout the year in both sites, showing uniform susceptibility of field larvae to Bti, in spite of Site A receiving 18 Bti treatments over a period of 7 mo. No cross-resistance of Ae. aegypti larvae from temephos to Bti was detected. PMID:21399591

  12. Bacillus thuringiensis Cyt2Aa2 toxin disrupts cell membranes by forming large protein aggregates

    PubMed Central

    Tharad, Sudarat; Toca-Herrera, José L.; Promdonkoy, Boonhiang; Krittanai, Chartchai

    2016-01-01

    Bacillus thuringiensis (Bt) Cyt2Aa2 showed toxicity against Dipteran insect larvae and in vitro lysis activity on several cells. It has potential applications in the biological control of insect larvae. Although pore-forming and/or detergent-like mechanisms were proposed, the mechanism underlying cytolytic activity remains unclear. Analysis of the haemolytic activity of Cyt2Aa2 with osmotic stabilizers revealed partial toxin inhibition, suggesting a distinctive mechanism from the putative pore formation model. Membrane permeability was studied using fluorescent dye entrapped in large unilamellar vesicles (LUVs) at various protein/lipid molar ratios. Binding of Cyt2Aa2 monomer to the lipid membrane did not disturb membrane integrity until the critical protein/lipid molar ratio was reached, when Cyt2Aa2 complexes and cytolytic activity were detected. The complexes are large aggregates that appeared as a ladder when separated by agarose gel electrophoresis. Interaction of Cyt2Aa2 with Aedes albopictus cells was investigated by confocal microscopy and total internal reflection fluorescent microscopy (TIRF). The results showed that Cyt2Aa2 binds on the cell membrane at an early stage without cell membrane disruption. Protein aggregation on the cell membrane was detected later which coincided with cell swelling. Cyt2Aa2 aggregations on supported lipid bilayers (SLBs) were visualized by AFM. The AFM topographic images revealed Cyt2Aa2 aggregates on the lipid bilayer at low protein concentration and subsequently disrupts the lipid bilayer by forming a lesion as the protein concentration increased. These results supported the mechanism whereby Cyt2Aa2 binds and aggregates on the lipid membrane leading to the formation of non-specific hole and disruption of the cell membrane. PMID:27612497

  13. pH-Controlled Bacillus thuringiensis Cry1Ac Protoxin Loading and Release from Polyelectrolyte Microcapsules

    PubMed Central

    Yang, Wenhui; He, Kanglai; Zhang, Jie; Guo, Shuyuan

    2012-01-01

    Crystal proteins synthesized by Bacillus thuringiensis (Bt) have been used as biopesticides because of their toxicity to the insect larval hosts. To protect the proteins from environmental stress to extend their activity, we have developed a new microcapsule formulation. Poly (acrylic acid) (PAH) and poly (styrene sulfonate) (PSS) were fabricated through layer-by-layer self-assembly based on a CaCO3 core. Cry1Ac protoxins were loaded into microcapsules through layer-by-layer self-assembly at low pH, and the encapsulated product was stored in water at 4°C. Scanning electron microscopy (SEM) was used to observe the morphology of the capsules. To confirm the successful encapsulation, the loading results were observed with a confocal laser scattering microscope (CLSM), using fluorescein-labeled Cry1Ac protoxin (FITC-Cry1Ac). The protoxins were released from the capsule under the alkaline condition corresponding to the midgut of certain insects, a condition which seldom exists elsewhere in the environment. The following bioassay experiment demonstrated that the microcapsules with Cry1Ac protoxins displayed approximately equivalent insecticidal activity to the Asian corn borer compared with free Cry1Ac protoxins, and empty capsules proved to have no effect on insects. Further result also indicated that the formulation could keep stable under the condition of heat and desiccation. These results suggest that this formulation provides a promising methodology that protects protoxins from the environment and releases them specifically in the target insects’ midgut, which has shown potential as biopesticide in the field. PMID:23024810

  14. Transgenic Bacillus thuringiensis (Bt) rice is safer to aquatic ecosystems than its non-transgenic counterpart.

    PubMed

    Li, Guangsheng; Wang, Yongmo; Liu, Biao; Zhang, Guoan

    2014-01-01

    Rice lines genetically modified with the crystal toxin genes from Bacillus thuringiensis (Bt) have experienced rapid development, with biosafety certificates for two Bt rice lines issued in 2009. There has still been no commercial release of these lines yet due to public concerns about human health and environmental risks. Some studies confirmed that Bt rice was as safe as conventional rice to non-target organisms when pesticides were not applied, however, pesticides are still required in Bt rice to control non-lepidopteran pests. In this study, we assessed the environmental effects of two Bt rice lines expressing either the cry1Ab/1Ac or cry2A genes, respectively, by using zooplanktons as indicator species under normal field management practices using pesticides when required. In the whole rice growing season, non-Bt rice was sprayed 5 times while Bt rice was sprayed 2 times, which ensured both rice achieved a normal yield. Field investigations showed that rice type (Bt and non-Bt) significantly influenced zooplankton abundance and diversity, which were up to 95% and 80% lower in non-Bt rice fields than Bt rice fields. Laboratory rearing showed that water from non-Bt rice fields was significantly less suitable for the survival and reproduction of Daphnia magna and Paramecium caudatum in comparison with water from Bt rice fields. Higher pesticide residues were detected in the water from non-Bt than Bt rice fields, accounting for the bad performance of zooplankton in non-Bt field water. Our results demonstrate that Bt rice is safer to aquatic ecosystems than non-Bt rice, and its commercialization will be beneficial for biodiversity restoration in rice-based ecosystems. PMID:25105299

  15. Bacillus thuringiensis delta-endotoxin binding to brush border membrane vesicles of rice stem borers.

    PubMed

    Alcantara, Edwin P; Aguda, Remedios M; Curtiss, April; Dean, Donald H; Cohen, Michael B

    2004-04-01

    The receptor binding step in the molecular mode of action of five delta-endotoxins (Cry1Ab, Cry1Ac, Cry1C, Cry2A, and Cry9C) from Bacillus thuringiensis was examined to find toxins with different receptor sites in the midgut of the striped stem borer (SSB) Chilo suppressalis (Walker) and yellow stem borer (YSB) Scirpophaga incertulas (Walker) (Lepidoptera: Pyralidae). Homologous competition assays were used to estimate binding affinities (K(com)) of (125)I-labelled toxins to brush border membrane vesicles (BBMV). The SSB BBMV affinities in decreasing order was: Cry1Ab = Cry1Ac > Cry9C > Cry2A > Cry1C. In YSB, the order of decreasing affinities was: Cry1Ac > Cry1Ab > Cry9C = Cry2A > Cry1C. The number of binding sites (B(max)) estimated by homologous competition binding among the Cry toxins did not affect toxin binding affinity (K(com)) to both insect midgut BBMVs. Results of the heterologous competition binding assays suggest that Cry1Ab and Cry1Ac compete for the same binding sites in SSB and YSB. Other toxins bind with weak (Cry1C, Cry2A) or no affinity (Cry9C) to Cry1Ab and Cry1Ac binding sites in both species. Cry2A had the lowest toxicity to 10-day-old SSB and Cry1Ab and Cry1Ac were the most toxic. Taken together, the results of this study show that Cry1Ab or Cry1Ac could be combined with either Cry1C, Cry2A, or Cry9C for more durable resistance in transgenic rice. Cry1Ab should not be used together with Cry1Ac because a mutation in one receptor site could diminish binding of both toxins. PMID:15027071

  16. Effects of bacillus thuringiensis transgenic corn on corn earworm and fall armyworm (Lepidoptera: Noctuidae) densities.

    PubMed

    Chilcutt, Charles F; Odvody, Gary N; Correa, J Carlos; Remmers, Jeff

    2007-04-01

    We examined 17 pairs of near-isogenic hybrids of Bacillus thuringiensis (Bt) (176, Mon810, and Bt11) and non-Bt corn, Zea mays L., to examine the effects of Bt on larval densities of Helicoverpa zea (Boddie) and Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae) during 2 yr. During ear formation, instar densities of H. zea and S. frugiperda were recorded for each hybrid. We found that H. zea first, second, and fifth instar densities were each affected by Mon810 and Bt11 Bt corn but not by 176 corn. Surprisingly, first and second instars were found in higher numbers on ears of Mon810 and Bt11 corn than on non-Bt corn. Densities of third and fourth instars were equal on Bt and non-Bt hybrids, whereas densities of fifth instars were lower on Bt plants. S. frugiperda larval densities were only affected during 1 yr when second, and fourth to sixth instars were lower on ears of Mon810 and Bt11 hybrids compared with their non-Bt counterparts. Two likely explanations for early instar H. zea densities being higher on Bt corn than non-Bt corn are that (1) Bt toxins delay development, creating a greater abundance of early instars that eventually die, and (2) reduced survival of H. zea to later instars on Bt corn decreased the normal asymmetric cannibalism or H. zea-S. frugiperda intraguild predation of late instars on early instars. Either explanation could explain why differences between Bt and non-Bt plants were greater for H. zea than S. frugiperda, because H. zea is more strongly affected by Bt toxins and more cannibalistic. PMID:17461054

  17. Biphasic fermentation is an efficient strategy for the overproduction of δ-endotoxin from Bacillus thuringiensis.

    PubMed

    Jisha, Veloorvalappil Narayanan; Smitha, Robinson Babysarojam; Priji, Prakasan; Sajith, Sreedharan; Benjamin, Sailas

    2015-02-01

    This study illustrates a biphasic solid-state fermentation (SSF) strategy for the overproduction of δ-endotoxin from Bacillus thuringiensis subsp. kurstaki (Btk) and also purification of δ-endotoxin from the solid-fermented medium. The fermentation strategy had two phases (biphasic); i.e., the first short phase was semisolid state (12 h), and the remaining long phase was strict SSF. To achieve the biphasic SSF, after 12 h (150 rpm, 37 °C) fermentation of the medium [Luria-Bertani (LB) supplemented with 30 % (w/v) raw soybean flour (phase I)], the supernatant in it was completely centrifuged out (1,000 × g, 10 min) aseptically for harvesting the extracellular enzymes as by-product. The resultant wet solid matter without free-flowing liquid but with embedded Btk was incubated 60 h more (phase II) for enhancing δ-endotoxin production at static condition (37 °C). Coupled with this, δ-endotoxin was purified by the modified phase separation method, and its purity was physically confirmed by both staining and microscopic techniques. The maximum δ-endotoxin yield from solid medium (48 h) was 15.8 mg/mL (recovery was 55-59 %) LB-equivalent, while that of LB control (recovery was 95 %) was only 0.43 mg/mL (72 h), i.e., thus, in comparison, 36.74-fold more yield in solid medium obtained by 24 h less gestation period. The purified crystal proteins showed apparent molecular weights (MWs) of 45, 35, and 6 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Briefly, this unique study physically demonstrates how Btk δ-endotoxin is purified (95-99 % purity) from solid-fermented matter for the first time, coupled with its overproduction at the expense of only 21.5 % higher production cost.

  18. Bacillus thuringiensis: mechanism of action, resistance, and new applications: a review.

    PubMed

    Melo, André Luiz de Almeida; Soccol, Vanete Thomaz; Soccol, Carlos Ricardo

    2016-01-01

    Since the first report by Ishiwata in 1902 of a Bombyx mori infection, followed by the description by Berliner, Bacillus thuringiensis (Bt) has become the main microorganism used in biological control. The application of Bt to combat invertebrates of human interest gained momentum with the growing demand for food free of chemical pesticides and with the implementation of agriculture methods that were less damaging to the environment. However, the mechanisms of action of these products have not been fully elucidated. There are two proposed models: the first is that Bt causes an osmotic imbalance in response to the formation of pores in a cell membrane, and the second is that it causes an opening of ion channels that activate the process of cell death. There are various ways in which Bt resistance can develop: changes in the receptors that do not recognize the Cry toxin, the synthesis of membrane transporters that eliminate the peptides from the cytosol and the development of regulatory mechanisms that disrupt the production of toxin receptors. Besides the potential for formulation of biopesticides and the use in developing genetically modified cultivars, recent studies with Bt have discussed promising applications in other branches of science. Chitinase, an enzyme that degrades chitin, increases the efficiency of Bt insecticides, and there has been of increasing interest in the industry, given that its substrate is extremely abundant in nature. Another promising field is the potential for Bt proteins to act against cancer cells. Parasporins, toxins of Bt that do not have an entomopathogenic effect, have a cytotoxic effect on the cells changed by some cancers. This demonstrates the potential of the microorganism and new opportunities opening for future applications.

  19. Proteolytic Activation of Bacillus thuringiensis Cry2Ab through a Belt-and-Braces Approach.

    PubMed

    Xu, Lian; Pan, Zhi-Zhen; Zhang, Jing; Liu, Bo; Zhu, Yu-Jing; Chen, Qing-Xi

    2016-09-28

    Proteolytic processing of Bacillus thuringiensis (Bt) crystal toxins by insect midgut proteases plays an essential role in their insecticidal toxicities against target insects. In the present study, proteolysis of Bt crystal toxin Cry2Ab by Plutella xylostella L. midgut proteases (PxMJ) was evaluated. Both trypsin and chymotrypsin were identified involving the proteolytic activation of Cry2Ab and cleaving Cry2Ab at Arg(139) and Leu(144), respectively. Three Cry2Ab mutants (R139A, L144A, and R139A-L144A) were constructed by replacing residues Arg(139), Leu(144), and Arg(139)-Leu(144) with alanine. Proteolysis assays revealed that mutants R139A and L144A but not R139A-L144A could be cleaved into 50 kDa activated toxins by PxMJ. Bioassays showed that mutants R139A and L144A were highly toxic against P. xylostella larvae, while mutant R139A-L144A was almost non-insecticidal. Those results demonstrated that proteolysis by PxMJ was associated with the toxicity of Cry2Ab against P. xylostella. It also revealed that either trypsin or chymotrypsin was enough to activate Cry2Ab protoxin. This characteristic was regarded as a belt-and-braces approach and might contribute to the control of resistance development in target insects. Our studies characterized the proteolytic processing of Cry2Ab and provided new insight into the activation of this Bt toxin. PMID:27598769

  20. Curing of plasmid pBMB28 from Bacillus thuringiensis YBT-020 using an unstable replication region.

    PubMed

    Wang, Pengxia; Zhu, Qian; Shang, Hui; Zhu, Yiguang; Sun, Ming

    2016-02-01

    Bacillus thuringiensis serovar finitimus strain YBT-020 is the well-studied spore-crystal association (SCA) phenotypic strain, whose parasporal crystals adhere to spore after lysis of the mother cell. Its endogenous plasmids pBMB26 and pBMB28 were proved essential for this SCA phenotype. In our previous study, using conventional methods, pBMB26 cured derivative and both pBMB26 and pBMB28 cured derivative of YBT-020 were obtained. However, YBT-020 solely cured of pBMB28 could not be obtained. In this study, an unstable replication region of pBMB28 was identified and was used to construct an incompatible plasmid pRep28B. This incompatible plasmid was successfully used to cure plasmid pBMB28 and was easily eliminated through segregational instability under the optimum growth temperature of YBT-020. Therefore, an endogenous plasmid was cured from the B. thuringiensis strain utilizing plasmid incompatibility. Moreover, using an unstable replication region instead of a temperature sensitive (Ts) replication region is better to cure the incompatible plasmid because it can avoid culturing at higher temperature. This method provides an efficient method for plasmid curing in B. thuringiensis and other bacteria.

  1. Cross-Resistance and Stability of Resistance to Bacillus thuringiensis Toxin Cry1C in Diamondback Moth

    PubMed Central

    Liu, Yong-Biao; Tabashnik, Bruce E.; Meyer, Susan K.; Crickmore, Neil

    2001-01-01

    We tested toxins of Bacillus thuringiensis against larvae from susceptible, Cry1C-resistant, and Cry1A-resistant strains of diamondback moth (Plutella xylostella). The Cry1C-resistant strain, which was derived from a field population that had evolved resistance to B. thuringiensis subsp. kurstaki and B. thuringiensis subsp. aizawai, was selected repeatedly with Cry1C in the laboratory. The Cry1C-resistant strain had strong cross-resistance to Cry1Ab, Cry1Ac, and Cry1F, low to moderate cross-resistance to Cry1Aa and Cry9Ca, and no cross-resistance to Cry1Bb, Cry1Ja, and Cry2A. Resistance to Cry1C declined when selection was relaxed. Together with previously reported data, the new data on the cross-resistance of a Cry1C-resistant strain reported here suggest that resistance to Cry1A and Cry1C toxins confers little or no cross-resistance to Cry1Bb, Cry2Aa, or Cry9Ca. Therefore, these toxins might be useful in rotations or combinations with Cry1A and Cry1C toxins. Cry9Ca was much more potent than Cry1Bb or Cry2Aa and thus might be especially useful against diamondback moth. PMID:11425744

  2. The Metabolic Regulation of Sporulation and Parasporal Crystal Formation in Bacillus thuringiensis Revealed by Transcriptomics and Proteomics*

    PubMed Central

    Wang, Jieping; Mei, Han; Zheng, Cao; Qian, Hongliang; Cui, Cui; Fu, Yang; Su, Jianmei; Liu, Ziduo; Yu, Ziniu; He, Jin

    2013-01-01

    Bacillus thuringiensis is a well-known entomopathogenic bacterium used worldwide as an environmentally compatible biopesticide. During sporulation, B. thuringiensis accumulates a large number of parasporal crystals consisting of insecticidal crystal proteins (ICPs) that can account for nearly 20–30% of the cell's dry weight. However, the metabolic regulation mechanisms of ICP synthesis remain to be elucidated. In this study, the combined efforts in transcriptomics and proteomics mainly uncovered the following 6 metabolic regulation mechanisms: (1) proteases and the amino acid metabolism (particularly, the branched-chain amino acids) became more active during sporulation; (2) stored poly-β-hydroxybutyrate and acetoin, together with some low-quality substances provided considerable carbon and energy sources for sporulation and parasporal crystal formation; (3) the pentose phosphate shunt demonstrated an interesting regulation mechanism involving gluconate when CT-43 cells were grown in GYS medium; (4) the tricarboxylic acid cycle was significantly modified during sporulation; (5) an obvious increase in the quantitative levels of enzymes and cytochromes involved in energy production via the electron transport system was observed; (6) most F0F1-ATPase subunits were remarkably up-regulated during sporulation. This study, for the first time, systematically reveals the metabolic regulation mechanisms involved in the supply of amino acids, carbon substances, and energy for B. thuringiensis spore and parasporal crystal formation at both the transcriptional and translational levels. PMID:23408684

  3. Comparative proteomic analysis of Aedes aegypti larval midgut after intoxication with Cry11Aa toxin from Bacillus thuringiensis.

    PubMed

    Cancino-Rodezno, Angeles; Lozano, Luis; Oppert, Cris; Castro, Julieta I; Lanz-Mendoza, Humberto; Encarnación, Sergio; Evans, Amy E; Gill, Sarjeet S; Soberón, Mario; Jurat-Fuentes, Juan L; Bravo, Alejandra

    2012-01-01

    Cry toxins produced by Bacillus thuringiensis bacteria are environmentally safe alternatives to control insect pests. They are pore-forming toxins that specifically affect cell permeability and cellular integrity of insect-midgut cells. In this work we analyzed the defensive response of Aedes aegypti larva to Cry11Aa toxin intoxication by proteomic and functional genomic analyses. Two dimensional differential in-gel electrophoresis (2D-DIGE) was utilized to analyze proteomic differences among A. aegypti larvae intoxicated with different doses of Cry11Aa toxin compared to a buffer treatment. Spots with significant differential expression (p<0.05) were then identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS), revealing 18 up-regulated and seven down-regulated proteins. The most abundant subcategories of differentially expressed proteins were proteins involved in protein turnover and folding, energy production, and cytoskeleton maintenance. We selected three candidate proteins based on their differential expression as representatives of the different functional categories to perform gene silencing by RNA interference and analyze their functional role. The heat shock protein HSP90 was selected from the proteins involved in protein turnover and chaperones; actin, was selected as representative of the cytoskeleton protein group, and ATP synthase subunit beta was selected from the group of proteins involved in energy production. When we affected the expression of ATP synthase subunit beta and actin by silencing with RNAi the larvae became hypersensitive to toxin action. In addition, we found that mosquito larvae displayed a resistant phenotype when the heat shock protein was silenced. These results provide insight into the molecular components influencing the defense to Cry toxin intoxication and facilitate further studies on the roles of identified genes. PMID:22615881

  4. Field Evaluation of Arbuscular Mycorrhizal Fungal Colonization in Bacillus thuringiensis Toxin-Expressing (Bt) and Non-Bt Maize

    PubMed Central

    Cruzan, Mitchell B.; Rosenstiel, Todd N.

    2013-01-01

    The cultivation of genetically engineered Bacillus thuringiensis toxin-expressing (Bt) maize continues to increase worldwide, yet the effects of Bt crops on arbuscular mycorrhizal fungi (AMF) in soil are poorly understood. In this field experiment, we investigated the impact of seven different genotypes of Bt maize and five corresponding non-Bt parental cultivars on AMF and evaluated plant growth responses at three different physiological time points. Plants were harvested 60 days (active growth), 90 days (tasseling and starting to produce ears), and 130 days (maturity) after sowing, and data on plant growth responses and percent AMF colonization of roots at each harvest were collected. Spore abundance and diversity were also evaluated at the beginning and end of the field season to determine whether the cultivation of Bt maize had a negative effect on AMF propagules in the soil. Plant growth and AMF colonization did not differ between Bt and non-Bt maize at any harvest period, but AMF colonization was positively correlated with leaf chlorophyll content at the 130-day harvest. Cultivation of Bt maize had no effect on spore abundance and diversity in Bt versus non-Bt plots over one field season. Plot had the most significant effect on total spore counts, indicating spatial heterogeneity in the field. Although previous greenhouse studies demonstrated that AMF colonization was lower in some Bt maize lines, our field study did not yield the same results, suggesting that the cultivation of Bt maize may not have an impact on AMF in the soil ecosystem under field conditions. PMID:23624473

  5. Field evaluation of arbuscular mycorrhizal fungal colonization in Bacillus thuringiensis toxin-expressing (Bt) and non-Bt maize.

    PubMed

    Cheeke, Tanya E; Cruzan, Mitchell B; Rosenstiel, Todd N

    2013-07-01

    The cultivation of genetically engineered Bacillus thuringiensis toxin-expressing (Bt) maize continues to increase worldwide, yet the effects of Bt crops on arbuscular mycorrhizal fungi (AMF) in soil are poorly understood. In this field experiment, we investigated the impact of seven different genotypes of Bt maize and five corresponding non-Bt parental cultivars on AMF and evaluated plant growth responses at three different physiological time points. Plants were harvested 60 days (active growth), 90 days (tasseling and starting to produce ears), and 130 days (maturity) after sowing, and data on plant growth responses and percent AMF colonization of roots at each harvest were collected. Spore abundance and diversity were also evaluated at the beginning and end of the field season to determine whether the cultivation of Bt maize had a negative effect on AMF propagules in the soil. Plant growth and AMF colonization did not differ between Bt and non-Bt maize at any harvest period, but AMF colonization was positively correlated with leaf chlorophyll content at the 130-day harvest. Cultivation of Bt maize had no effect on spore abundance and diversity in Bt versus non-Bt plots over one field season. Plot had the most significant effect on total spore counts, indicating spatial heterogeneity in the field. Although previous greenhouse studies demonstrated that AMF colonization was lower in some Bt maize lines, our field study did not yield the same results, suggesting that the cultivation of Bt maize may not have an impact on AMF in the soil ecosystem under field conditions.

  6. Laboratory and field evaluation of Teknar HP-D, a biolarvicidal formulation of Bacillus thuringiensis ssp. israelensis, against mosquito vectors.

    PubMed

    Gunasekaran, K; Doss, P S Boopathi; Vaidyanathan, K

    2004-10-01

    Larvicidal efficacy of Teknar HP-D, an improved biolarvicidal formulation of Bacillus thuringiensis ssp. israelensis (Bti), against Anopheles stephensi, Culex quinquefasciatus and Aedes aegypti was determined in the laboratory, and in field the efficacy of the formulation was tested against Cx. quinquefasciatus breeding in cesspits, unused wells and drains. The toxicity of the formulation to Gambusia affinis (larvivorous fish), Notonecta sp. and Diplonychus indicus (water bugs) was also evaluated in the laboratory. Teknar HP-D was field tested at three recommended dosages, 1, 1.5 and 2l/ha, selecting five habitats for each dosage. Another five habitats were kept untreated as controls. Ae. aegypti showed greatest susceptibility to the Bti toxin in the laboratory. In cesspits, all the three dosages caused >80% reduction of pupal recruitment up to day 6 post-treatment, indicating that a weekly application at the lowest would be necessary for sustained control. The residual activity of the formulation was longer in unused wells, causing >80% reduction of pupal recruitment for 17 days from the day of treatment. In controlling pupal recruitment the three dosages produced equal effect. Application of Teknar HP-D at 1 l/ha once in three weeks is therefore recommended to control Cx. quinquefasciatus in unused wells. However, in drains, >80% reduction of pupal recruitment was observed for only 3 days and hence, application of Teknar HP-D at 2 l/ha that caused significantly higher level of reduction twice in a week at 3-day interval is necessary. At dosages from 0.032 to 3.2 mg/l (ppm), Teknar HP-D was non-toxic to Gambusia fish. The two predatory water bugs, Notonecta sp. and Diplonychus indicus that fed on the surviving larvae of Cx. quinquefasciatus exposed to the sub-lethal doses (LC(50) and LC(80)) of Teknar HP-D were safe with out having any mortality.

  7. A cadherin-like protein influences Bacillus thuringiensis Cry1Ab toxicity in the oriental armyworm, Mythimna separata.

    PubMed

    Wang, Ling; Jiang, Xingfu; Luo, Lizhi; Stanley, David; Sappington, Thomas W; Zhang, Lei

    2013-06-01

    Cadherins comprise a family of calcium-dependent cell adhesion proteins that act in cell-cell interactions. Cadherin-like proteins (CADs) in midguts of some insects act as receptors that bind some of the toxins produced by the Bacillus thuringiensis (Bt). We cloned a CAD gene associated with larval midguts prepared from Mythimna separata. The full-length cDNA (MsCAD1, GenBank Accession No. JF951432) is 5642 bp, with an open reading frame encoding a 1757 amino acid and characteristics typical of insect CADs. Expression of MsCAD1 is predominantly in midgut tissue, with highest expression in the 3rd- to 6th-instars and lowest in newly hatched larvae. Knocking-down MsCAD1 decreased Cry1Ab susceptibility, indicated by reduced developmental time, increased larval weight and reduced larval mortality. We expressed MsCAD1 in E. coli and recovered the recombinant protein, rMsCAD1, which binds Cry1Ab toxin. Truncation analysis and binding experiments revealed that a contiguous 209-aa, located in CR11 and CR12, is the minimal Cry1Ab binding region. These results demonstrate that MsCAD1 is associated with Cry1Ab toxicity and is one of the Cry1Ab receptors in this insect. The significance of this work lies in identifying MsCAD1 as a Cry1Ab receptor, which helps understand the mechanism of Cry1Ab toxicity and of potential resistance to Bt in M. separata. PMID:23754724

  8. Genetically engineered vegetables expressing proteins from Bacillus thuringiensis for insect resistance: successes, disappointments, challenges and ways to move forward.

    PubMed

    Shelton, Anthony M

    2012-01-01

    Genetically engineered (GE) insect-resistant crops that express proteins from Bacillus thuringiensis (Bt) have been widely adopted in the two field crops currently commercially available, Bt cotton and Bt corn. However, the development and commercialization of Bt vegetables has lagged in comparison, which is unfortunate since vegetables tend to be insecticide-intensive crops due to high pest pressure and cosmetic standards required for the market. While it is often stated that consumer choice has played a major role in companies avoiding development of Bt vegetables, this concept requires re-evaluation. In market studies in North America when consumers have been provided basic information about Bt genetic engineering, then given a choice between Bt and conventional sweet corn, they have often preferred the former. Likewise, 77% of consumers in a US survey said they would likely purchase foods produced through biotechnology for their ability to reduce pesticide use. Presently, however, the only commercialized Bt vegetable is sweet corn. Perhaps more critical obstacles to Bt vegetables are their relatively smaller acreages and the cost of government biosafety regulations that inadvertently favor large acreage of field crops because companies can obtain a better return on investment. In developing countries, private-public partnerships may provide the vehicle to bring Bt vegetables to market. However, these can be subverted by misinformation from anti-biotech campaigns, as is the case with Bt eggplant in India. Without the use of Bt vegetables as a tool for integrated pest management, farmers and the general public will not be able to realize the substantial environmental and economic benefits that have been well documented with Bt cotton and Bt corn.

  9. Effect of entomocidal proteins from Bacillus thuringiensis on ion permeability of apical membranes of Tenebrio molitor larvae gut epithelium.

    PubMed

    Andreev, I M; Bulushova, N V; Zalunin, I A; Chestukhina, G G

    2009-10-01

    Effects of entomocidal Cry-type proteins, delta-endotoxins Cry3A and Cry11A produced by Bacillus thuringiensis, on ion permeability of the apical membranes of intestinal epithelium from Tenebrio molitor larvae midgut were studied. Using potential-sensitive dyes safranine O and oxonol VI and DeltapH indicator acridine orange, it was shown that placing brush border membrane vesicles (BBMV) (loaded with Mg2+ during their preparation) into a salt-free buffer medium resulted in spontaneous generation of transmembrane electric potential on the vesicular membrane (negative inside the vesicles) accompanied by acidification of the aqueous phase inside the vesicles. The generation of transmembrane ion gradients on the vesicular membrane was a result of an electrogenic efflux of Mg2+ from the vesicles as shown by abolishing of the membrane potential by such agents as MgSO4 or CaCl2 in centimolar concentrations, a highly lipophilic cation tetraphenylphosphonium, and some blockers of cell membrane Ca2+-channels in submillimolar concentrations. A passive generation of membrane potential on the vesicular membrane (but positive inside the vesicles) was also observed upon addition of centimolar concentrations of K2SO4. Addition of delta-endotoxins Cry3A and Cry11A to the vesicle suspension in a salt-free buffer medium or in the same medium supplemented with centimolar concentrations of K2SO4 exerted a pronounced hyperpolarization of the vesicular membrane. This hyperpolarization was sensitive to the same agents, which abolished the membrane potential generation in the absence of delta-endotoxin. It is concluded that Cry proteins induced in BBMV from T. molitor opening pores or ion channels, which were considerably more permeable for alkaline- and alkaline-earth metal cations than for the accompanying anions.

  10. Effect of entomocidal proteins from Bacillus thuringiensis on ion permeability of apical membranes of Tenebrio molitor larvae gut epithelium.

    PubMed

    Andreev, I M; Bulushova, N V; Zalunin, I A; Chestukhina, G G

    2009-10-01

    Effects of entomocidal Cry-type proteins, delta-endotoxins Cry3A and Cry11A produced by Bacillus thuringiensis, on ion permeability of the apical membranes of intestinal epithelium from Tenebrio molitor larvae midgut were studied. Using potential-sensitive dyes safranine O and oxonol VI and DeltapH indicator acridine orange, it was shown that placing brush border membrane vesicles (BBMV) (loaded with Mg2+ during their preparation) into a salt-free buffer medium resulted in spontaneous generation of transmembrane electric potential on the vesicular membrane (negative inside the vesicles) accompanied by acidification of the aqueous phase inside the vesicles. The generation of transmembrane ion gradients on the vesicular membrane was a result of an electrogenic efflux of Mg2+ from the vesicles as shown by abolishing of the membrane potential by such agents as MgSO4 or CaCl2 in centimolar concentrations, a highly lipophilic cation tetraphenylphosphonium, and some blockers of cell membrane Ca2+-channels in submillimolar concentrations. A passive generation of membrane potential on the vesicular membrane (but positive inside the vesicles) was also observed upon addition of centimolar concentrations of K2SO4. Addition of delta-endotoxins Cry3A and Cry11A to the vesicle suspension in a salt-free buffer medium or in the same medium supplemented with centimolar concentrations of K2SO4 exerted a pronounced hyperpolarization of the vesicular membrane. This hyperpolarization was sensitive to the same agents, which abolished the membrane potential generation in the absence of delta-endotoxin. It is concluded that Cry proteins induced in BBMV from T. molitor opening pores or ion channels, which were considerably more permeable for alkaline- and alkaline-earth metal cations than for the accompanying anions. PMID:19916922

  11. Resistance to the Cry1Ac delta-endotoxin of Bacillus thuringiensis in the cotton bollworm, Helicoverpa armigera (Lepidoptera: Noctuidae).

    PubMed

    Akhurst, Raymond Joseph; James, William; Bird, Lisa Jane; Beard, Cheryl

    2003-08-01

    Three laboratory strains of Helicoverpa armigera (Hübner) were established by mating of field-collected insects with an existing insecticide-susceptible laboratory strain. These strains were cultured on artificial diet containing the Cry1Ac protoxin of Bacillus thuringiensis using three different protocols. When no response to selection was detected after 7-11 generations of selection, the three strains were combined by controlled mating to preserve genetic diversity. The composite strain (BX) was selected on the basis of growth rate on artificial diet containing Cry1Ac crystals. Resistance to Cry1Ac was first detected after 16 generations of continuous selection. The resistance ratio (RR) peaked approximately 300-fold at generation 21, after which it declined to oscillate between 57- and 111-fold. First-instar H. armigera from generation 25 (RR = 63) were able to complete their larval development on transgenic cotton expressing Cry1Ac and produce fertile adults. There appeared to be a fitness cost associated with resistance on cotton and on artificial diet. The BX strain was not resistant to the commercial Bt spray formulations DiPel and XenTari, which contain multiple insecticidal crystal proteins, but was resistant to the MVP formulation, which only contains Cry1Ac. The strain was also resistant to Cry1Ab but not to Cry2Aa or Cry2Ab. Toxin binding assays showed that the resistant insects lacked the high affinity binding site that was detected in early generations of the strain. Genetic analysis confirmed that resistance in the BX strain of H. armigera is incompletely recessive.

  12. Bacillus thuringiensis pore-forming toxins trigger massive shedding of GPI-anchored aminopeptidase N from gypsy moth midgut epithelial cells.

    PubMed

    Valaitis, Algimantas P

    2008-06-01

    The insecticidal Cry proteins produced by Bacillus thuringiensis strains are pore-forming toxins (PFTs) that bind to the midgut brush border membrane and cause extensive damage to the midgut epithelial cells of susceptible insect larvae. Force-feeding B. thuringiensis PFTs to Lymantria dispar larvae elicited rapid and massive shedding of a glycosylphosphatidylinositol (GPI)-anchored aminopeptidase N (APN) from midgut epithelial cells into the luminal fluid, and depletion of the membrane-anchored enzyme on the midgut epithelial cells. The amount of APN released into the luminal fluid of intoxicated larvae was dose- and time-dependent, and directly related to insecticidal potency of the PFTs. The induction of toxin-induced shedding of APN was inhibited by cyclic AMP and MAPK kinase (MEK) inhibitors PD98059 and U0126, indicating that signal transduction in the MEK/ERK pathway is involved in the regulation of the shedding process. APN released from epithelial cells appears to be generated by the action of a phosphatidylinositol-specific phospholipase C (PI-PLC) cleavage of the GPI anchor based upon detection of a cross-reacting determinant (CRD) on the protein shed into the luminal fluid. Alkaline phosphatase was also released from the gut epithelial cells, supporting the conclusion that other GPI-anchored proteins are released as a consequence of the activation PI-PLC. These observations are the basis of a novel and highly sensitive tool for evaluating the insecticidal activity of new Cry proteins obtained though discovery or protein engineering.

  13. Isolation and characterization of EG2158, a new strain of Bacillus thuringiensis toxic to coleopteran larvae, and nucleotide sequence of the toxin gene.

    PubMed

    Donovan, W P; Gonzalez, J M; Gilbert, M P; Dankocsik, C

    1988-11-01

    A novel strain of Bacillus thuringiensis was isolated from soybean grain dust from Kansas and found to be toxic to larvae of Leptinotarsa decemlineata (Colorado potato beetle). The strain (EG2158) synthesized two parasporal crystals: a rhomboid crystal composed of a 73116 dalton protein of approximately 30 kDa. Plasmid transfer and gene cloning experiments demonstrated that the 73 kDa protein was encoded on an 88 MDa plasmid and that the protein was toxic to the larvae of Colorado potato beetle (CPB). The sequence of the 73 kDa protein, as deduced from the sequence of its gene (cryC), was found to have regions of similarity with several B. thuringiensis crystal proteins: the lepidopteran-toxic P1 proteins of var. kurstaki and berliner, the lepidopteran- and dipteran-toxic P2 (or CRYB1) protein of var. kurstaki, and the dipteran-toxic 130 kDa protein of var. israelensis. While B. megaterium cells harboring the cryC gene from EG2158 synthesized significant amounts of the 73 kDa CRYC protein, Escherichia coli cells did not. The cryC-containing B. megaterium cells produced rhomboid crystals that were toxic to CPB larvae. PMID:3146015

  14. Bacillus subtilis as potential producer for polyhydroxyalkanoates

    PubMed Central

    Singh, Mamtesh; Patel, Sanjay KS; Kalia, Vipin C

    2009-01-01

    Polyhydroxyalkanoates (PHAs) are biodegradable polymers produced by microbes to overcome environmental stress. Commercial production of PHAs is limited by the high cost of production compared to conventional plastics. Another hindrance is the brittle nature and low strength of polyhydroxybutyrate (PHB), the most widely studied PHA. The needs are to produce PHAs, which have better elastomeric properties suitable for biomedical applications, preferably from inexpensive renewable sources to reduce cost. Certain unique properties of Bacillus subtilis such as lack of the toxic lipo-polysaccharides, expression of self-lysing genes on completion of PHA biosynthetic process – for easy and timely recovery, usage of biowastes as feed enable it to compete as potential candidate for commercial production of PHA. PMID:19619289

  15. Complete sequence of three plasmids from Bacillus thuringiensis INTA-FR7-4 environmental isolate and comparison with related plasmids from the Bacillus cereus group.

    PubMed

    Amadio, Ariel F; Benintende, Graciela B; Zandomeni, Rubén O

    2009-11-01

    Bacillus thuringiensis is an insect pathogen used worldwide as a bioinsecticide. It belongs to the Bacillus cereus sensu lato group as well as Bacillus anthracis and B. cereus. Plasmids from this group of organisms have been implicated in pathogenicity as they carry the genes responsible for different types of diseases that affect mammals and insects. Some plasmids, like pAW63 and pBT9727, encode a functional conjugation machinery allowing them to be transferred to a recipient cell. They also share extensive homology with the non-functional conjugation apparatus of pXO2 from B. anthracis. In this study we report the complete sequence of three plasmids from an environmental B. thuringiensis isolate from Argentina, obtained by a shotgun sequencing method. We obtained the complete nucleotide sequence of plasmids pFR12 (12,095bp), pFR12.5 (12,459bp) and pFR55 (55,712bp) from B. thuringiensis INTA-FR7-4. pFR12 and pFR12.5 were classified as cryptic as they do not code for any obvious functions besides replication and mobilization. Both small plasmids were classified as RCR plasmids due to similarities with the replicases they encode. Plasmid pFR55 showed a structural organization similar to that observed for plasmids pAW63, pBT9727 and pXO2. pFR55 also shares a tra region with these plasmids, containing genes related to T4SS and conjugation. A comparison between pFR55 and conjugative plasmids led to the postulation that pFR55 is a conjugative plasmid. Genes related to replication functions in pFR55 are different to those described for plasmids with known complete sequences. pFR55 is the first completely sequenced plasmid with a replication machinery related to that of ori44. The analysis of the complete sequence of plasmids from an environmental isolate of B. thuringiensis permitted the identification of a near complete conjugation apparatus in pFR55, resembling those of plasmids pAW63, pBT9727 and pXO2. The availability of this sequence is a step forward in the study

  16. Poly-β-hydroxybutyrate Metabolism Is Unrelated to the Sporulation and Parasporal Crystal Protein Formation in Bacillus thuringiensis.

    PubMed

    Wang, Xun; Li, Zhou; Li, Xin; Qian, Hongliang; Cai, Xia; Li, Xinfeng; He, Jin

    2016-01-01

    Poly-3-hydroxybutyrate (PHB) is a natural polymer synthesized by many bacteria as a carbon-energy storage material. It was accumulated maximally prior to the spore formation but was degraded during the process of sporulation in Bacillus thuringiensis. Intriguingly, B. thuringiensis also accumulates large amounts of insecticidal crystal proteins (ICPs) during sporulation, which requires considerable input of carbon and energy sources. How PHB accumulation affects sporulation and ICP formation remains unclear to date. Intuitively, one would imagine that accumulated PHB provides the energy required for ICP formation. Yet our current data indicate that this is not the case. First, growth curves of the deletion mutants of phaC (encoding the PHB synthase) and phaZ (encoding the PHB depolymerase) were found to be similar to the parent strain BMB171; no difference in growth rate could be observed. In addition we further constructed the cry1Ac10 ICP gene overexpression strains of BMB171 (BMB171-cry), as well as its phaC and phaZ deletion mutants ΔphaC-cry and ΔphaZ-cry to compare their spore and ICP production rates. Again, not much change of ICP production was observed among these strains either. In fact, PHB was still degraded in most ΔphaZ-cry cells as observed by transmission electron microscopy. Together these results indicated that there is no direct association between the PHB accumulation and the sporulation and ICP formation in B. thuringiensis. Some other enzymes for PHB degradation or other energy source may be responsible for the sporulation and/or ICP formation in B. thuringiensis.

  17. Biosorption of heavy metals by Bacillus thuringiensis strain OSM29 originating from industrial effluent contaminated north Indian soil

    PubMed Central

    Oves, Mohammad; Khan, Mohammad Saghir; Zaidi, Almas

    2012-01-01

    The study was navigated to examine the metal biosorbing ability of bacterial strain OSM29 recovered from rhizosphere of cauliflower grown in soil irrigated consistently with industrial effluents. The metal tolerant bacterial strain OSM29 was identified as Bacillus thuringiensis following 16S rRNA gene sequence analysis. In the presence of the varying concentrations (25–150 mgl−1) of heavy metals, such as cadmium, chromium, copper, lead and nickel, the B. thuringiensis strain OSM29 showed an obvious metal removing potential. The effect of certain physico-chemical factors such as pH, initial metal concentration, and contact time on biosorption was also assessed. The optimum pH for nickel and chromium removal was 7, while for cadmium, copper and lead, it was 6. The optimal contact time was 30 min. for each metal at 32 ± 2 °C by strain OSM29. The biosorption capacity of the strain OSM29 for the metallic ions was highest for Ni (94%) which was followed by Cu (91.8%), while the lowest sorption by bacterial biomass was recorded for Cd (87%) at 25 mgl−1 initial metal ion concentration. The regression coefficients obtained for heavy metals from the Freundlich and Langmuir models were significant. The surface chemical functional groups of B. thuringiensis biomass identified by Fourier transform infrared (FTIR) were amino, carboxyl, hydroxyl, and carbonyl groups, which may be involved in the biosorption of heavy metals. The biosorption ability of B. thuringiensis OSM29 varied with metals and was pH and metal concentration dependent. The biosorption of each metal was fairly rapid which could be an advantage for large scale treatment of contaminated sites. PMID:24115905

  18. Poly-β-hydroxybutyrate Metabolism Is Unrelated to the Sporulation and Parasporal Crystal Protein Formation in Bacillus thuringiensis.

    PubMed

    Wang, Xun; Li, Zhou; Li, Xin; Qian, Hongliang; Cai, Xia; Li, Xinfeng; He, Jin

    2016-01-01

    Poly-3-hydroxybutyrate (PHB) is a natural polymer synthesized by many bacteria as a carbon-energy storage material. It was accumulated maximally prior to the spore formation but was degraded during the process of sporulation in Bacillus thuringiensis. Intriguingly, B. thuringiensis also accumulates large amounts of insecticidal crystal proteins (ICPs) during sporulation, which requires considerable input of carbon and energy sources. How PHB accumulation affects sporulation and ICP formation remains unclear to date. Intuitively, one would imagine that accumulated PHB provides the energy required for ICP formation. Yet our current data indicate that this is not the case. First, growth curves of the deletion mutants of phaC (encoding the PHB synthase) and phaZ (encoding the PHB depolymerase) were found to be similar to the parent strain BMB171; no difference in growth rate could be observed. In addition we further constructed the cry1Ac10 ICP gene overexpression strains of BMB171 (BMB171-cry), as well as its phaC and phaZ deletion mutants ΔphaC-cry and ΔphaZ-cry to compare their spore and ICP production rates. Again, not much change of ICP production was observed among these strains either. In fact, PHB was still degraded in most ΔphaZ-cry cells as observed by transmission electron microscopy. Together these results indicated that there is no direct association between the PHB accumulation and the sporulation and ICP formation in B. thuringiensis. Some other enzymes for PHB degradation or other energy source may be responsible for the sporulation and/or ICP formation in B. thuringiensis. PMID:27379025

  19. Poly-β-hydroxybutyrate Metabolism Is Unrelated to the Sporulation and Parasporal Crystal Protein Formation in Bacillus thuringiensis

    PubMed Central

    Wang, Xun; Li, Zhou; Li, Xin; Qian, Hongliang; Cai, Xia; Li, Xinfeng; He, Jin

    2016-01-01

    Poly-3-hydroxybutyrate (PHB) is a natural polymer synthesized by many bacteria as a carbon-energy storage material. It was accumulated maximally prior to the spore formation but was degraded during the process of sporulation in Bacillus thuringiensis. Intriguingly, B. thuringiensis also accumulates large amounts of insecticidal crystal proteins (ICPs) during sporulation, which requires considerable input of carbon and energy sources. How PHB accumulation affects sporulation and ICP formation remains unclear to date. Intuitively, one would imagine that accumulated PHB provides the energy required for ICP formation. Yet our current data indicate that this is not the case. First, growth curves of the deletion mutants of phaC (encoding the PHB synthase) and phaZ (encoding the PHB depolymerase) were found to be similar to the parent strain BMB171; no difference in growth rate could be observed. In addition we further constructed the cry1Ac10 ICP gene overexpression strains of BMB171 (BMB171-cry), as well as its phaC and phaZ deletion mutants ΔphaC-cry and ΔphaZ-cry to compare their spore and ICP production rates. Again, not much change of ICP production was observed among these strains either. In fact, PHB was still degraded in most ΔphaZ-cry cells as observed by transmission electron microscopy. Together these results indicated that there is no direct association between the PHB accumulation and the sporulation and ICP formation in B. thuringiensis. Some other enzymes for PHB degradation or other energy source may be responsible for the sporulation and/or ICP formation in B. thuringiensis. PMID:27379025

  20. Bacillus thuringiensis plants expressing Cry1Ac, Cry2Ab and Cry1F are not toxic to the assassin bug, Zelus renardii

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cotton and maize delivering insecticidal crystal (Cry) proteins from the bacterium, Bacillus thuringiensis (Bt), have been commercialized since 1996. Bt plants are subjected to environmental risk assessments for non-target organisms, especially natural enemies that suppress pest populations. In th...