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Sample records for bacillus thuringiensis cry

  1. Evolution of Bacillus thuringiensis Cry toxins insecticidal activity

    OpenAIRE

    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 specificit...

  2. Regulation of cry Gene Expression in Bacillus thuringiensis

    OpenAIRE

    Chao Deng; Qi Peng; Fuping Song; Didier Lereclus

    2014-01-01

    Bacillus thuringiensis differs from the closely related Bacillus cereus group species by its ability to produce crystalline inclusions. The production of these crystals mainly results from the expression of the cry genes, from the stability of their transcripts and from the synthesis, accumulation and crystallization of large amounts of insecticidal Cry proteins. This process normally coincides with sporulation and is regulated by various factors operating at the transcriptional, post-transcr...

  3. Regulation of cry Gene Expression in Bacillus thuringiensis

    Directory of Open Access Journals (Sweden)

    Chao Deng

    2014-07-01

    Full Text Available Bacillus thuringiensis differs from the closely related Bacillus cereus group species by its ability to produce crystalline inclusions. The production of these crystals mainly results from the expression of the cry genes, from the stability of their transcripts and from the synthesis, accumulation and crystallization of large amounts of insecticidal Cry proteins. This process normally coincides with sporulation and is regulated by various factors operating at the transcriptional, post-transcriptional, metabolic and post-translational levels.

  4. Characterization of Cry34/Cry35 Binary Insecticidal Proteins from Diverse Bacillus thuringiensis Strain Collections

    OpenAIRE

    Schnepf, H. Ernest; Lee, Stacey; Dojillo, JoAnna; Burmeister, Paula; Fencil, Kristin; Morera, Lisa; Nygaard, Linda; Narva, Kenneth E.; Wolt, Jeff D.

    2005-01-01

    Bacillus thuringiensis crystal proteins of the Cry34 and Cry35 classes function as binary toxins showing activity on the western corn rootworm, Diabrotica virgifera virgifera LeConte. We surveyed 6,499 B. thuringiensis isolates by hybridization for sequences related to cry35A genes, identifying 78 strains. Proteins of the appropriate molecular mass (ca. 44 kDa) for Cry35 were observed in 42 of the strains. Full-length, or nearly full-length, sequences of 34 cry34 genes and 16 cry35 genes were...

  5. Complete Genome Sequence of Bacillus thuringiensis Strain 407 Cry-

    OpenAIRE

    Poehlein, Anja; Liesegang, Heiko

    2013-01-01

    Bacillus thuringiensis is an insect pathogen that has been used widely as a biopesticide. Here, we report the genome sequence of strain 407 Cry-, which is used to study the genetic determinants of pathogenicity. The genome consists of a 5.5-Mb chromosome and nine plasmids, including a novel 502-kb megaplasmid.

  6. Cross-Resistance to Bacillus thuringiensis Toxin CryIF in the Diamondback Moth (Plutella xylostella)

    OpenAIRE

    Tabashnik, Bruce E.; Finson, Naomi; Johnson, Marshall W.; David G Heckel

    1994-01-01

    Selection with Bacillus thuringiensis subsp. kurstaki, which contains CryIA and CryII toxins, caused a >200-fold cross-resistance to CryIF toxin from B. thuringiensis subsp. aizawai in the diamondback moth, Plutella xylostella. CryIE was not toxic, but CryIB was highly toxic to both selected and unselected larvae. The results show that extremely high levels of cross-resistance can be conferred across classes of CryI toxins of B. thuringiensis.

  7. Bacillus thuringiensis delta-endotoxin Cry1Ac domain III enhances activity against Heliothis virescens in some, but not all Cry1-Cry1Ac hybrids

    NARCIS (Netherlands)

    Karlova, R.B.; Weemen, W.M.J.; Naimov, S.; Ceron, J.; Dukiandjiev, S.; Maagd, de R.A.

    2005-01-01

    We investigated the role of domain III of Bacillus thuringiensis d-endotoxin Cry1Ac in determining toxicity against Heliothis virescens. Hybrid toxins, containing domain III of Cry1Ac with domains I and II of Cry1Ba, Cry1Ca, Cry1Da, Cry1Ea, and Cry1Fb, respectively, were created. In this way Cry1Ca,

  8. Evolution of Bacillus thuringiensis Cry toxins insecticidal activity.

    Science.gov (United States)

    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.

  9. Shared binding sites in Lepidoptera for Bacillus thuringiensis Cry1Ja and Cry1A toxins.

    Science.gov (United States)

    Herrero, S; González-Cabrera, J; Tabashnik, B E; Ferré, J

    2001-12-01

    Bacillus thuringiensis toxins act by binding to specific target sites in the insect midgut epithelial membrane. The best-known mechanism of resistance to B. thuringiensis toxins is reduced binding to target sites. Because alteration of a binding site shared by several toxins may cause resistance to all of them, knowledge of which toxins share binding sites is useful for predicting cross-resistance. Conversely, cross-resistance among toxins suggests that the toxins share a binding site. At least two strains of diamondback moth (Plutella xylostella) with resistance to Cry1A toxins and reduced binding of Cry1A toxins have strong cross-resistance to Cry1Ja. Thus, we hypothesized that Cry1Ja shares binding sites with Cry1A toxins. We tested this hypothesis in six moth and butterfly species, each from a different family: Cacyreus marshalli (Lycaenidae), Lobesia botrana (Tortricidae), Manduca sexta (Sphingidae), Pectinophora gossypiella (Gelechiidae), P. xylostella (Plutellidae), and Spodoptera exigua (Noctuidae). Although the extent of competition varied among species, experiments with biotinylated Cry1Ja and radiolabeled Cry1Ac showed that Cry1Ja and Cry1Ac competed for binding sites in all six species. A recent report also indicates shared binding sites for Cry1Ja and Cry1A toxins in Heliothis virescens (Noctuidae). Thus, shared binding sites for Cry1Ja and Cry1A occur in all lepidopteran species tested so far.

  10. 40 CFR 174.509 - Bacillus thuringiensis Cry3A protein; exemption from the requirement of a tolerance.

    Science.gov (United States)

    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...

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

    Science.gov (United States)

    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 174.529 - Bacillus thuringiensis modified Cry1Ab protein as identified under OECD Unique Identifier SYN...

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Bacillus thuringiensis modified Cry1Ab... Tolerance Exemptions § 174.529 Bacillus thuringiensis modified Cry1Ab protein as identified under OECD... Bacillus thuringiensis modified Cry1Ab protein as identified under OECD Unique Identifier SYN-IR67B-1...

  13. Flexibility Analysis of Bacillus thuringiensis Cry1Aa

    Institute of Scientific and Technical Information of China (English)

    ZHAO Xin Min; XIA Li Qiu; YANG Xiao Ping; PENG Xiao Yun

    2015-01-01

    Objective To investigate the flexibility and mobility of the Bacillus thuringiensis toxin Cry1Aa. Methods The graph theory-based program Constraint Network Analysis and normal mode-based program NMsim were used to analyze the global and local flexibility indices as well as the fluctuation of individual residues in detail. Results The decrease in Cry1Aa network rigidity with the increase of temperature was evident. Two phase transition points in which the Cry1Aa structure lost rigidity during the thermal simulation were identified. Two rigid clusters were found in domains I and II. Weak spots were found in C-terminal domain III. Several flexible regions were found in all three domains;the largest residue fluctuation was present in the apical loop2 of domain II. Conclusion Although several flexible regions could be found in all the three domains, the most flexible regions were in the apical loops of domain II.

  14. Distribution of cryV-type insecticidal protein genes in Bacillus thuringiensis and cloning of cryV-type genes from Bacillus thuringiensis subsp. kurstaki and Bacillus thuringiensis subsp. entomocidus.

    OpenAIRE

    Shin, B.S.; Park, S.H.; Choi, S. K.; Koo, B T; Lee, S. T.; Kim, J. I.

    1995-01-01

    DNA dot blot hybridizations with a cryV-specific probe and a cryI-specific probe were performed to screen 24 Bacillus thuringiensis strains for their cryV-type (lepidopteran- and coleopteran-specific) and cryI-type (lepidopteran-specific) insecticidal crystal protein gene contents, respectively. The cryV-specific probe hybridized to 12 of the B. thuringiensis strains examined. Most of the cryV-positive strains also hybridized to the cryI-specific probe, indicating that the cryV genes are clos...

  15. Dominant negative phenotype of Bacillus thuringiensis Cry1Ab, Cry11Aa and Cry4Ba mutants suggest hetero-oligomer formation among different Cry toxins.

    NARCIS (Netherlands)

    Carmona, D.; Rodriguez-Almazan, C.; Munoz-Garay, C.; Portugal, L.; Perez, C.; Maagd, de R.A.; Bakker, P.; Soberon, M.; Bravo, A.

    2011-01-01

    Background - Bacillus thuringiensis Cry toxins are used worldwide in the control of different insect pests important in agriculture or in human health. The Cry proteins are pore-forming toxins that affect the midgut cell of target insects. It was shown that non-toxic Cry1Ab helix a-4 mutants had a d

  16. Coleopteran-specific and putative novel cry genes in Iranian native Bacillus thuringiensis collection.

    Science.gov (United States)

    Nazarian, Amin; Jahangiri, Rosa; Jouzani, Gholamreza Salehi; Seifinejad, Ali; Soheilivand, Saeed; Bagheri, Omolbanin; Keshavarzi, Mansoureh; Alamisaeid, Khalil

    2009-10-01

    The characterization of the strains containing Coleopteran-specific and also putative novel cry genes in Iranian native Bacillus thuringiensis collection is presented. Characterization was based on PCR analysis using 31 general and specific primers for cry1B, cry1I, cry3A, cry3B, cry3C, cry7A, cry8A, cry8B, cry8C, cry14, cry18, cry26, cry28, cry34 and cry35 genes, protein band patterns as well as their insecticidal activity on Xanthogaleruca luteola Mull. larvae. Forty six isolates (65.7%) contained minimum one Coleopteran-active cry gene. Based on universal primers, strains containing cry18 and cry26 genes were the most abundant and represent 27.1% and 24% of the isolates, respectively, whereas cry14, cry3, cry28, cry34, cry35, cry7, cry8 genes were less abundant, found in 14.2, 12.5, 10, 7, 7 and 5.6% of the strains, respectively. Based on specific primers, isolates containing cry1I were the most abundant (48.5%). Two strains containing Coleopteran-active cry genes showed higher activity against X. luteola larvae than B. thuringiensis subsp. morrisoni pathovar tenebrionis. Thirty isolates, when assayed for cry1C, cry5, cry6, cry8b, cry9, cry10, cry11, cry18, cry24 and cry35 genes, showed unexpected size bands. Cloning and sequencing of the amplicons allowed both the identification of known cry genes and the detection of putative novel cry1C sequences.

  17. 40 CFR 174.517 - Bacillus thuringiensis Cry9C protein in corn; exemption from the requirement of a tolerance.

    Science.gov (United States)

    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...

  18. 40 CFR 174.505 - Bacillus thuringiensis modified Cry3A protein (mCry3A) in corn; exemption from the requirement of...

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Bacillus thuringiensis modified Cry3A... REQUIREMENTS FOR PLANT-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.505 Bacillus... 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...

    Science.gov (United States)

    2010-07-01

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

  20. Isolation, toxicity and detection of cry gene in Bacillus thuringiensis isolates in Krabi province, Thailand

    Directory of Open Access Journals (Sweden)

    Prakai Thaphan

    2008-06-01

    Full Text Available One hundred twenty one isolates of Bacillus thuringiensis were isolated from 91 soil samples collected in the national park and wildlife sanctuary in Krabi province. All isolates of B.thuringiensis were tested for their insecticidal activity against Spodoptera litura, S. exigua and Plutella xylostella larvae. Seven isolates of B. thuringiensis named JCPT7, JCPT16, JCPT18, JCPT64, JCPT68, JCPT74 and JCPT89 exhibited toxic activities against the insects, more than 90% mortality. The detection of cry gene of these isolates was done by a method based on polymerase chain reaction (PCR. The PCR result indicated that cry1Ab, cry1Ac, cry1C, cry1D, cry1I, cry9A, cry9B and cry2A were on chromosomal DNA and cry1Aa, cry1Ab, cry1Ac, cry1C, cry1D, cry1I and cry2A were on plasmid DNA. This study has introduced the promising B. thuringiensis isolates collected from soil samples which could be developed as an effective biocontrol agent for Lepidopterous pest.

  1. Biological characteristics of Bacillus thuringiensis strain Btll and identification of its cry-type genes

    Institute of Scientific and Technical Information of China (English)

    Tinghui LIU; Wei GUO; Weiming SUN; Yongxiang SUN

    2009-01-01

    A novel strain of Bacillus thuringiensis Bt11, isolated from soil samples in China, was classified and characterized in terms of its crystal proteins, cry genes content. The Bt11 strain showed high toxicity against Spodoptera exigua and Helicoverpa armigera neonates. Btll strain shares morphological and biochemical characteristics with the previously described Bacillus thuringiensis subsp. kurstaki. SDS-polyacrylamide gel electrophoresis revealed that crystals were composed of several polypeptides ranging from 20 to 130 kDa, of which the 35, 80, and 130 kDa proteins were the major components. PCR-RFLP with total DNA from strain Btll and specific primers for cryl, cry2, cry3, cry4/10, cry7, cry8, cry9, and cryll genes revealed that crylAa, crylAb, crylla, and cry9Ea genes were present.

  2. 40 CFR 174.530 - Bacillus thuringiensis Cry2Ae protein in cotton; temporary exemption from the requirement of a...

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Bacillus thuringiensis Cry2Ae protein... REQUIREMENTS FOR PLANT-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.530 Bacillus... Bacillus thuringiensis Cry2Ae protein in or on the food commodities of cotton, cotton; cotton,...

  3. 40 CFR 174.520 - Bacillus thuringiensis Cry1F protein in corn; exemption from the requirement of a tolerance.

    Science.gov (United States)

    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...

  4. 77 FR 6471 - Bacillus thuringiensis Cry2Ae Protein in Cotton; Exemption from the Requirement of a Tolerance

    Science.gov (United States)

    2012-02-08

    ... AGENCY 40 CFR Part 174 Bacillus thuringiensis Cry2Ae Protein in Cotton; Exemption from the Requirement of... regulation establishes an exemption from the requirement of a tolerance for residues of Bacillus... residues of Bacillus thuringiensis Cry2Ae protein in cotton under the FFDCA. DATES: This regulation...

  5. Structural Insights into Bacillus thuringiensis Cry, Cyt and Parasporin Toxins

    OpenAIRE

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

    2014-01-01

    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 grea...

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

    Science.gov (United States)

    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 ...

  7. Bacillus thuringiensis delta-endotoxin Cry1 hybrid proteins with increased activity against the Colorado potato beetle

    NARCIS (Netherlands)

    Naimov, S.; Weemen-Hendriks, M.; Dukiandjiev, S.; Maagd, de R.A.

    2001-01-01

    Cry1 delta-endotoxins of Bacillus thuringiensis are generally active against lepidopteran insects, but Cry1Ba and Cry1Ia have additional, though low, levels of activity against coleopterans such as the Colorado potato beetle. Here we report the construction of Cry1Ba/Cry1Ia hybrid toxins which have

  8. 77 FR 47287 - Bacillus thuringiensis eCry3.1Ab Protein in Corn; Exemption From the Requirement of a Tolerance

    Science.gov (United States)

    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...

  9. 40 CFR 174.504 - Bacillus thuringiensis Cry1F protein in cotton; exemption from the requirement of a tolerance.

    Science.gov (United States)

    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 § 174.504 Bacillus thuringiensis Cry1F protein in cotton; exemption from the requirement of a tolerance. Residues of...

  10. Abundance and diversity of Bacillus thuringiensis in Bangladesh and their cry genes profile

    Directory of Open Access Journals (Sweden)

    Md. MozammelHoq

    1900-01-01

    Full Text Available Bacillus thuringiensis (Bt biopesticides, a recognized eco-friendly pest control agent, can be used to reduce many problems associated with indiscriminate use of chemical pesticides such as environmental pollutions, public health problems, emergence of resistance among pests in many developing countries etc. Bt strains were, therefore, isolated from different ecosystems of Bangladesh and characterized based on biochemical typing, 16S rRNA gene analysis, plasmid and cry genes profiles. Bt index was calculated 0.86 in this study and variations in abundance and distribution pattern of 16 different biotypes were demonstrated within 316 indigenous Bt strains which was compared to the other parts of the world. Bt indiana (17.8%, Bt kurstaki (16.7% and Bt thuringiensis (12.7% were found to be the most prevalent in Bangladesh among other biotypes. Hemolytic activity was variable among the biotypes and it was maximum for Bt biotype 10 (100%. Plasmids in the biotypes indiana, kurstaki, thuringiensis and israelensis were observed to occupy a wider range than other biotypes. The screening for insecticidal genes viz. cry1, cry2, cry3, cry4A, cry8, cry9, cry10 and cry11 in the native Bt strains revealed their presence in varied proportion rendering cry1, cry2 and cry3 the most abundant. The abundance of Bt strains, their diversities and the cry genes profile were thus analyzed in this study which will be the basis for further research development with Bt biopesticide in Bangladesh.

  11. 40 CFR 180.1154 - CryIA(c) and CryIC derived delta-endotoxins of Bacillus thuringiensis var. kurstaki encapsulated...

    Science.gov (United States)

    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...

  12. Production and characterization of Bacillus thuringiensis Cry1Ac-resistant cotton bollworm Helicoverpa zea (Boddie).

    Science.gov (United States)

    Anilkumar, Konasale J; Rodrigo-Simón, Ana; Ferré, Juan; Pusztai-Carey, Marianne; Sivasupramaniam, Sakuntala; Moar, William J

    2008-01-01

    Laboratory-selected Bacillus thuringiensis-resistant colonies are important tools for elucidating B. thuringiensis resistance mechanisms. However, cotton bollworm, Helicoverpa zea, a target pest of transgenic corn and cotton expressing B. thuringiensis Cry1Ac (Bt corn and cotton), has proven difficult to select for stable resistance. Two populations of H. zea (AR and MR), resistant to the B. thuringiensis protein found in all commercial Bt cotton varieties (Cry1Ac), were established by selection with Cry1Ac activated toxin (AR) or MVP II (MR). Cry1Ac toxin reflects the form ingested by H. zea when feeding on Bt cotton, whereas MVP II is a Cry1Ac formulation used for resistance selection and monitoring. The resistance ratio (RR) for AR exceeded 100-fold after 11 generations and has been maintained at this level for nine generations. This is the first report of stable Cry1Ac resistance in H. zea. MR crashed after 11 generations, reaching only an RR of 12. AR was only partially cross-resistant to MVP II, suggesting that MVP II does not have the same Cry1Ac selection pressure as Cry1Ac toxin against H. zea and that proteases may be involved with resistance. AR was highly cross-resistant to Cry1Ab toxin but only slightly cross-resistant to Cry1Ab expressing corn leaf powder. AR was not cross-resistant to Cry2Aa2, Cry2Ab2-expressing corn leaf powder, Vip3A, and cypermethrin. Toxin-binding assays showed no significant differences, indicating that resistance was not linked to a reduction in binding. These results aid in understanding why this pest has not evolved B. thuringiensis resistance, and highlight the need to choose carefully the form of B. thuringiensis protein used in experiments.

  13. Study of Thermokinetic Properties of Sodium Selenite on Bacillus thuringiensis Cry B by Microcalorimetry

    Institute of Scientific and Technical Information of China (English)

    LI,Xi; LIU,Yi; ZHAO,Ru-Ming; YU,Zi-Niu; QU Song-Sheng

    2001-01-01

    By using an LKB2277 Bioactivity Monitor, the power-time curves of Bacillus thuringiensis Cry B at 28℃ effected by Na2SeO3 were determined. Some paarameters, such as growh rate constant k, inhibitory ratio I, the maximum heat production rate Pmax, heat output Q, were obtained. Considering both the growth rate constant k and heat output Q, it was found that a low concentration of Na2SeO3 had a promoting action on the growth of Bacillus thuringiensis Cry B, but a high concentration of Na2SeO3 had an inhibitory action.

  14. Structural Insights into Bacillus thuringiensis Cry, Cyt and Parasporin Toxins

    Science.gov (United States)

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

    2014-01-01

    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. PMID:25229189

  15. Structural Insights into Bacillus thuringiensis Cry, Cyt and Parasporin Toxins

    Directory of Open Access Journals (Sweden)

    Chengchen Xu

    2014-09-01

    Full Text Available 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.

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

    Science.gov (United States)

    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.

  17. Common, but complex, mode of resistance of Plutella xylostella to Bacillus thuringiensis toxins Cry1Ab and Cry1Ac.

    Science.gov (United States)

    Sayyed, Ali H; Gatsi, Roxani; Ibiza-Palacios, M Sales; Escriche, Baltasar; Wright, Denis J; Crickmore, Neil

    2005-11-01

    A field collected population of Plutella xylostella (SERD4) was selected in the laboratory with Bacillus thuringiensis endotoxins Cry1Ac (Cry1Ac-SEL) and Cry1Ab (Cry1Ab-SEL). Both subpopulations showed similar phenotypes: high resistance to the Cry1A toxins and little cross-resistance to Cry1Ca or Cry1D. A previous analysis of the Cry1Ac-SEL showed incompletely dominant resistance to Cry1Ac with more than one factor, at least one of which was sex influenced. In the present study reciprocal mass crosses between Cry1Ab-SEL and a laboratory susceptible population (ROTH) provided evidence that Cry1Ab resistance was also inherited as incompletely dominant trait with more than one factor, and at least one of the factors was sex influenced. Analysis of single pair mating indicated that Cry1Ab-SEL was still heterogeneous for Cry1Ab resistance genes, showing genes with different degrees of dominance. Binding studies showed a large reduction of specific binding of Cry1Ab and Cry1Ac to midgut membrane vesicles of the Cry1Ab-SEL subpopulation. Cry1Ab-SEL was found to be more susceptible to trypsin-activated Cry1Ab toxin than protoxin, although no defect in toxin activation was found. Present and previous results indicate a common basis of resistance to both Cry1Ab and Cry1Ac in selected subpopulations and suggest that a similar set of resistance genes are responsible for resistance to Cry1Ab and Cry1Ac and are selected whichever toxin was used. The possibility of an incompletely dominant trait of resistant to these toxins should be taken into account when considering refuge resistance management strategies.

  18. Susceptibility of northern corn rootworm Diabrotica barberi Smith & Lawrence (Coleoptera: Chrysomelidae) to mCry3A and eCry3.1Ab Bacillus thuringiensis proteins

    Science.gov (United States)

    Susceptibility of the northern corn rootworm (NCR), to mCry3A and eCry3.1Ab proteins derived from Bacillus thuringiensis (Bt) was determined using a diet bioassay. Northern corn rootworm neonates were exposed to different concentrations of mCry3A and eCry3.1Ab, incorporated into artificial diet. Lar...

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

    Science.gov (United States)

    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

  20. Dominant negative phenotype of Bacillus thuringiensis Cry1Ab, Cry11Aa and Cry4Ba mutants suggest hetero-oligomer formation among different Cry toxins.

    Directory of Open Access Journals (Sweden)

    Daniela Carmona

    Full Text Available BACKGROUND: Bacillus thuringiensis Cry toxins are used worldwide in the control of different insect pests important in agriculture or in human health. The Cry proteins are pore-forming toxins that affect the midgut cell of target insects. It was shown that non-toxic Cry1Ab helix α-4 mutants had a dominant negative (DN phenotype inhibiting the toxicity of wildtype Cry1Ab when used in equimolar or sub-stoichiometric ratios (1∶1, 0.5∶1, mutant∶wt indicating that oligomer formation is a key step in toxicity of Cry toxins. METHODOLOGY/PRINCIPAL FINDINGS: The DN Cry1Ab-D136N/T143D mutant that is able to block toxicity of Cry1Ab toxin, was used to analyze its capacity to block the activity against Manduca sexta larvae of other Cry1 toxins, such as Cry1Aa, Cry1Ac, Cry1Ca, Cry1Da, Cry1Ea and Cry1Fa. Cry1Ab-DN mutant inhibited toxicity of Cry1Aa, Cry1Ac and Cry1Fa. In addition, we isolated mutants in helix α-4 of Cry4Ba and Cry11Aa, and demonstrate that Cry4Ba-E159K and Cry11Aa-V142D are inactive and completely block the toxicity against Aedes aegypti of both wildtype toxins, when used at sub-stoichiometric ratios, confirming a DN phenotype. As controls we analyzed Cry1Ab-R99A or Cry11Aa-E97A mutants that are located in helix α-3 and are affected in toxin oligomerization. These mutants do not show a DN phenotype but were able to block toxicity when used in 10∶1 or 100∶1 ratios (mutant∶wt probably by competition of binding with toxin receptors. CONCLUSIONS/SIGNIFICANCE: We show that DN phenotype can be observed among different Cry toxins suggesting that may interact in vivo forming hetero-oligomers. The DN phenotype cannot be observed in mutants affected in oligomerization, suggesting that this step is important to inhibit toxicity of other toxins.

  1. Bacillus thuringiensis Cry34Ab1/Cry35Ab1 interactions with western corn rootworm midgut membrane binding sites.

    Directory of Open Access Journals (Sweden)

    Huarong Li

    Full Text Available BACKGROUND: Bacillus thuringiensis (Bt Cry34Ab1/Cry35Ab1 are binary insecticidal proteins that are co-expressed in transgenic corn hybrids for control of western corn rootworm, Diabrotica virgifera virgifera LeConte. Bt crystal (Cry proteins with limited potential for field-relevant cross-resistance are used in combination, along with non-transgenic corn refuges, as a strategy to delay development of resistant rootworm populations. Differences in insect midgut membrane binding site interactions are one line of evidence that Bt protein mechanisms of action differ and that the probability of receptor-mediated cross-resistance is low. METHODOLOGY/PRINCIPAL FINDINGS: Binding site interactions were investigated between Cry34Ab1/Cry35Ab1 and coleopteran active insecticidal proteins Cry3Aa, Cry6Aa, and Cry8Ba on western corn rootworm midgut brush border membrane vesicles (BBMV. Competitive binding of radio-labeled proteins to western corn rootworm BBMV was used as a measure of shared binding sites. Our work shows that (125I-Cry35Ab1 binds to rootworm BBMV, Cry34Ab1 enhances (125I-Cry35Ab1 specific binding, and that (125I-Cry35Ab1 with or without unlabeled Cry34Ab1 does not share binding sites with Cry3Aa, Cry6Aa, or Cry8Ba. Two primary lines of evidence presented here support the lack of shared binding sites between Cry34Ab1/Cry35Ab1 and the aforementioned proteins: 1 No competitive binding to rootworm BBMV was observed for competitor proteins when used in excess with (125I-Cry35Ab1 alone or combined with unlabeled Cry34Ab1, and 2 No competitive binding to rootworm BBMV was observed for unlabeled Cry34Ab1 and Cry35Ab1, or a combination of the two, when used in excess with (125I-Cry3Aa, or (125I-Cry8Ba. CONCLUSIONS/SIGNIFICANCE: Combining two or more insecticidal proteins active against the same target pest is one tactic to delay the onset of resistance to either protein. We conclude that Cry34Ab1/Cry35Ab1 are compatible with Cry3Aa, Cry6Aa, or Cry8Ba

  2. The pre-pore from Bacillus thuringiensis Cry1Ab toxin is necessary to induce insect death in Manduca sexta

    OpenAIRE

    Jiménez-Juárez, N.; Muñoz-Garay, C.; Gómez, I.; Gill, S. S.; Soberón, M; Bravo, A.

    2007-01-01

    The insecticidal Cry toxins from Bacillus thuringiensis bacteria are pore-forming toxins that lyse midgut epithelial cells in insects. We have previously proposed that they form pre-pore oligomeric intermediates before membrane insertion.

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

    Science.gov (United States)

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

    2009-07-03

    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.

  4. Structural and biophysical characterization of Bacillus thuringiensis insecticidal proteins Cry34Ab1 and Cry35Ab1.

    Directory of Open Access Journals (Sweden)

    Matthew S Kelker

    Full Text Available 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.

  5. 76 FR 57653 - Bacillus thuringiensis eCry3.1Ab Protein in Corn; Temporary Exemption From the Requirement of a...

    Science.gov (United States)

    2011-09-16

    ... AGENCY 40 CFR Part 174 Bacillus thuringiensis eCry3.1Ab Protein in Corn; Temporary Exemption From the... residues of Bacillus thuringiensis eCry3.1Ab protein in corn, in or on the food or feed commodities of corn... temporary tolerance exemption for Bacillus thuringiensis eCry3.1Ab protein in corn that was set to expire...

  6. Bacillus thuringiensis plants expressing Cry1Ac, Cry2Ab and Cry1F are not toxic to the assassin bug, Zelus renardii

    Science.gov (United States)

    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...

  7. 40 CFR 174.518 - Bacillus thuringiensis Cry3Bb1 protein in corn; exemption from the requirement of a tolerance.

    Science.gov (United States)

    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...

  8. 40 CFR 174.519 - Bacillus thuringiensis Cry2Ab2 protein in corn and cotton; exemption from the requirement of a...

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Bacillus thuringiensis Cry2Ab2 protein... REQUIREMENTS FOR PLANT-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.519 Bacillus... Bacillus thuringiensis Cry2Ab2 protein in or on corn or cotton are exempt from the requirement of...

  9. 40 CFR 174.532 - Bacillus thuringiensis eCry3.1Ab protein in corn; temporary exemption from the requirement of a...

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Bacillus thuringiensis eCry3.1Ab... REQUIREMENTS FOR PLANT-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.532 Bacillus... Bacillus thuringiensis eCry3.1Ab protein in corn, in or on the food and feed commodities of corn;...

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

    Science.gov (United States)

    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.

  11. High instability of a nematicidal Cry toxin plasmid in Bacillus thuringiensis.

    Science.gov (United States)

    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.

  12. Asymmetrical cross-resistance between Bacillus thuringiensis toxins Cry1Ac and Cry2Ab in pink bollworm.

    Science.gov (United States)

    Tabashnik, Bruce E; Unnithan, Gopalan C; Masson, Luke; Crowder, David W; Li, Xianchun; Carrière, Yves

    2009-07-21

    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.

  13. Expression of the Bacillus thuringiensis mosquitocidal toxin Cry11Aa in the aquatic bacterium Asticcacaulis excentricus.

    Science.gov (United States)

    Armengol, Gemma; Guevara, Oscar Enrique; Orduz, Sergio; Crickmore, Neil

    2005-12-01

    A mosquitocidal aquatic bacterium has been developed by introducing an operon containing the cry11Aa, and p20 genes from Bacillus thuringiensis subsp. israelensis (Bti) into the gram-negative aquatic bacterium Asticcacaulis excentricus. After transformation, the cry11Aa gene was successfully expressed in recombinant A. excentricus under the tac promoter, at the level of 0.04 pg/cell. The recombinant bacteria were toxic to Aedes aegypti larvae with an LC(50) of 6.83 x 10(5) cells/mL. We believe that these bacteria may have potential as genetically engineered microorganisms for the control of mosquito larvae.

  14. A coleopteran cadherin fragment synergizes toxicity of Bacillus thuringiensis toxins Cry3Aa, Cry3Bb, and Cry8Ca against lesser mealworm, Alphitobius diaperinus (Coleoptera: Tenebrionidae).

    Science.gov (United States)

    Park, Youngjin; Hua, Gang; Taylor, Milton D; Adang, Michael J

    2014-11-01

    The lesser mealworm, Alphitobius diaperinus, is a serious cosmopolitan pest of commercial poultry facilities because of its involvement in structural damage to poultry houses, reduction in feed conversion efficiency, and transfer of avian and human pathogens. Cry3Aa, Cry3Bb, and Cry8Ca insecticidal proteins of Bacillus thuringiensis are used to control coleopteran larvae. Cadherins localized in the midgut epithelium function as receptors for Cry toxins in lepidopteran, coleopteran, and dipteran insects. Previously, we demonstrated that the truncated cadherin (DvCad1) from Diabrotica virgifera virgifera, which consists of the C-terminal cadherin repeats (CR) 8-10 and expressed in Escherichia coli, enhanced Cry3Aa and Cry3Bb toxicity against several coleopteran species. Here we report that the DvCad1-CR8-10 enhances Cry3Aa, Cry3Bb, and Cry8Ca toxicity to lesser mealworm. Previously, by an enzyme linked immunosorbent microplate assay, we demonstrated that the DvCad1-CR8-10 binds activated-Cry3Aa (11.8 nM), -Cry3Bb (1.4nM), and now report that CR8-10 binds activated-Cry8Ca (5.7 nM) toxin. The extent of Cry toxins enhancement by DvCad1-CR8-10, which ranged from 3.30- to 5.93-fold, may have practical application for lesser mealworm control in preventing avian and human pathogen transfer in poultry facilities.

  15. Susceptibility of Cry1Ab-resistant and -susceptible Sugarcane Borer (Lepidoptera: crambidae) to Four Bacillus thuringiensis Toxins

    Science.gov (United States)

    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...

  16. Antagonism between Cry1Ac1 and Cyt1A1 toxins of bacillus thuringiensis

    Science.gov (United States)

    del Rincon-Castro MC; Barajas-Huerta; Ibarra

    1999-05-01

    Most strains of the insecticidal bacterium Bacillus thuringiensis have a combination of different protoxins in their parasporal crystals. Some of the combinations clearly interact synergistically, like the toxins present in B. thuringiensis subsp. israelensis. In this paper we describe a novel joint activity of toxins from different strains of B. thuringiensis. In vitro bioassays in which we used pure, trypsin-activated Cry1Ac1 proteins from B. thuringiensis subsp. kurstaki, Cyt1A1 from B. thuringiensis subsp. israelensis, and Trichoplusia ni BTI-Tn5B1-4 cells revealed contrasting susceptibility characteristics. The 50% lethal concentrations (LC50s) were estimated to be 4,967 of Cry1Ac1 per ml of medium and 11.69 ng of Cyt1A1 per ml of medium. When mixtures of these toxins in different proportions were assayed, eight different LC50s were obtained. All of these LC50s were significantly higher than the expected LC50s of the mixtures. In addition, a series of bioassays were performed with late first-instar larvae of the cabbage looper and pure Cry1Ac1 and Cyt1A1 crystals, as well as two different combinations of the two toxins. The estimated mean LC50 of Cry1Ac1 was 2.46 ng/cm2 of diet, while Cyt1A1 crystals exhibited no toxicity, even at very high concentrations. The estimated mean LC50s of Cry1Ac1 crystals were 15.69 and 19.05 ng per cm2 of diet when these crystals were mixed with 100 and 1,000 ng of Cyt1A1 crystals per cm2 of diet, respectively. These results indicate that there is clear antagonism between the two toxins both in vitro and in vivo. Other joint-action analyses corroborated these results. Although this is the second report of antagonism between B. thuringiensis toxins, our evidence is the first evidence of antagonism between toxins from different subspecies of B. thuringiensis (B. thuringiensis subsp. kurstaki and B. thuringiensis subsp. israelensis) detected both in vivo and in vitro. Some possible explanations for this relationship are discussed.

  17. The expression and crystallization of Cry65Aa require two C-termini, revealing a novel evolutionary strategy of Bacillus thuringiensis Cry proteins

    Science.gov (United States)

    Peng, Dong-hai; Pang, Cui-yun; Wu, Han; Huang, Qiong; Zheng, Jin-shui; Sun, Ming

    2015-01-01

    The insecticidal crystal protein (Cry) genes of Bacillus thuringiensis are a key gene resource for generating transgenic crops with pest resistance. However, many cry genes cannot be expressed or form crystals in mother cells. Here, we report a novel Cry protein gene, cry65Aa1, which exists in an operon that contains a downstream gene encoding a hypothetical protein ORF2. We demonstrated that ORF2 is required for Cry65Aa1 expression and crystallization by function as a C-terminal crystallization domain. The orf2 sequence is also required for Cry65Aa expression, because orf2 transcripts have a stabilizing effect on cry65Aa1 transcripts. Furthermore, we found that the crystallization of Cry65Aa1 required the Cry65Aa1 C-terminus in addition to ORF2 or a typical Cry protein C-terminal region. Finally, we showed that Cry65Aa1 has a selective cytotoxic effect on MDA-MB231 cancer cells. This report is the first description of a 130-kDa mass range Cry protein requiring two C-termini for crystallization. Our findings reveal a novel evolutionary strategy of Cry proteins and provide an explanation for the existence of Cry protein genes that cannot form crystals in B. thuringiensis. This study also provides a potential framework for isolating novel cry genes from “no crystal” B. thuringiensis strains. PMID:25656389

  18. New resistance mechanism in Helicoverpa armigera threatens transgenic crops expressing Bacillus thuringiensis Cry1Ac toxin.

    Science.gov (United States)

    Gunning, Robin V; Dang, Ho T; Kemp, Fred C; Nicholson, Ian C; Moores, Graham D

    2005-05-01

    In Australia, the cotton bollworm, Helicoverpa armigera, has a long history of resistance to conventional insecticides. Transgenic cotton (expressing the Bacillus thuringiensis toxin Cry1Ac) has been grown for H. armigera control since 1996. It is demonstrated here that a population of Australian H. armigera has developed resistance to Cry1Ac toxin (275-fold). Some 70% of resistant H. armigera larvae were able to survive on Cry1Ac transgenic cotton (Ingard) The resistance phenotype is inherited as an autosomal semidominant trait. Resistance was associated with elevated esterase levels, which cosegregated with resistance. In vitro studies employing surface plasmon resonance technology and other biochemical techniques demonstrated that resistant strain esterase could bind to Cry1Ac protoxin and activated toxin. In vivo studies showed that Cry1Ac-resistant larvae fed Cy1Ac transgenic cotton or Cry1Ac-treated artificial diet had lower esterase activity than non-Cry1Ac-fed larvae. A resistance mechanism in which esterase sequesters Cry1Ac is proposed.

  19. 76 FR 14289 - Bacillus thuringiensis

    Science.gov (United States)

    2011-03-16

    ... AGENCY 40 CFR Part 174 Bacillus thuringiensis eCry3.1Ab Protein in Corn; Temporary Exemption From the... regulation extends a temporary exemption from the requirement of a tolerance for residues of Bacillus... permissible level for residues of Bacillus thuringiensis eCry3.1Ab protein in corn. The temporary...

  20. 75 FR 34040 - Bacillus thuringiensis

    Science.gov (United States)

    2010-06-16

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

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

    Science.gov (United States)

    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.

  2. 40 CFR 174.511 - Bacillus thuringiensis Cry1Ab protein in all plants; exemption from the requirement of a tolerance.

    Science.gov (United States)

    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...

  3. 40 CFR 174.510 - Bacillus thuringiensis Cry1Ac protein in all plants; exemption from the requirement of a tolerance.

    Science.gov (United States)

    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...

  4. Shared Binding Sites for the Bacillus thuringiensis Proteins Cry3Bb, Cry3Ca, and Cry7Aa in the African Sweet Potato Pest Cylas puncticollis (Brentidae)

    Science.gov (United States)

    Hernández-Martínez, Patricia; Vera-Velasco, Natalia Mara; Martínez-Solís, María; Ghislain, Marc; Ferré, Juan

    2014-01-01

    Bacillus thuringiensis Cry3Bb, Cry3Ca, and Cry7Aa have been reported to be toxic against larvae of the genus Cylas, which are important pests of sweet potato worldwide and particularly in sub-Saharan Africa. However, relatively little is known about the processing and binding interactions of these coleopteran-specific Cry proteins. The aim of the present study was to determine whether Cry3Bb, Cry3Ca, and Cry7Aa proteins have shared binding sites in Cylas puncticollis to orient the pest resistance strategy by genetic transformation. Interestingly, processing of the 129-kDa Cry7Aa protoxin using commercial trypsin or chymotrypsin rendered two fragments of about 70 kDa and 65 kDa. N-terminal sequencing of the trypsin-activated Cry7Aa fragments revealed that processing occurs at Glu47 for the 70-kDa form or Ile88 for the 65-kDa form. Homologous binding assays showed specific binding of the two Cry3 proteins and the 65-kDa Cry7Aa fragment to brush border membrane vesicles (BBMV) from C. puncticollis larvae. The 70-kDa fragment did not bind to BBMV. Heterologous-competition assays showed that Cry3Bb, Cry3Ca, and Cry7Aa (65-kDa fragment) competed for the same binding sites. Hence, our results suggest that pest resistance mediated by the alteration of a shared Cry receptor binding site might render all three Cry toxins ineffective. PMID:25261517

  5. Proteolytic Activation of Bacillus thuringiensis Cry2Ab through a Belt-and-Braces Approach.

    Science.gov (United States)

    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.

  6. Mode of action of Bacillus thuringiensis Cry and Cyt toxins and their potential for insect control.

    Science.gov (United States)

    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.

  7. Structure and distribution of the Bacillus thuringiensis Cry4Ba toxin in lipid membranes

    Energy Technology Data Exchange (ETDEWEB)

    Puntheeranurak, Theeraporn [Institute for Biophysics, Johannes Kepler University of Linz, Altenbergerstr. 69, A-4040 Linz (Austria); Laboratory of Molecular Biophysics, Institute of Molecular Biology and Genetics, Mahidol University, Salaya Campus, Nakornpathom 73170 (Thailand); Stroh, Cordula [Institute for Biophysics, Johannes Kepler University of Linz, Altenbergerstr. 69, A-4040 Linz (Austria); Zhu Rong [Institute for Biophysics, Johannes Kepler University of Linz, Altenbergerstr. 69, A-4040 Linz (Austria); Angsuthanasombat, Chanan [Laboratory of Molecular Biophysics, Institute of Molecular Biology and Genetics, Mahidol University, Salaya Campus, Nakornpathom 73170 (Thailand); Hinterdorfer, Peter [Institute for Biophysics, Johannes Kepler University of Linz, Altenbergerstr. 69, A-4040 Linz (Austria)]. E-mail: peter.hinterdorfer@jku.at

    2005-11-15

    Bacillus thuringiensis Cry {delta}-endotoxins cause death of susceptible insect larvae by forming lytic pores in the midgut epithelial cell membranes. The 65 kDa trypsin activated Cry4Ba toxin was previously shown to be capable of permeabilizing liposomes and forming ionic channels in receptor-free planar lipid bilayers. Here, magnetic ACmode (MACmode) atomic force microscopy (AFM) was used to characterize the lateral distribution and the native molecular structure of the Cry4Ba toxin in the membrane. Liposome fusion and the Langmuir-Blodgett technique were employed for supported lipid bilayer preparations. The toxin preferentially inserted in a self-assembled structure, rather than as a single monomeric molecule. In addition, the spontaneous insertion into receptor-free lipid bilayers lead to formation of characteristic pore-like structures with four-fold symmetry, suggesting that tetramers are the preferred oligomerization state of this toxin.

  8. Tribolium castaneum Apolipophorin-III acts as an immune response protein against Bacillus thuringiensis Cry3Ba toxic activity.

    Science.gov (United States)

    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.

  9. Characterization of cry2-type genes of Bacillus thuringiensis strains from soil-isolated of Sichuan basin, China

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    Hongxia Liang

    2011-03-01

    Full Text Available Sichuan basin, situated in the west of China, is the fourth biggest basin in China. In order to describe a systematic study of the cry2-type genes resources from Bacillus thuringiensis strains of Sichuan basin, a total of 791 Bacillus thuringiensis strains have been screened from 2650 soil samples in different ecological regions. The method of PCR-restriction fragment length polymorphism (PCR-RFLP was used to identify the type of cry2 genes. The results showed that 322 Bacillus thuringiensis strains harbored cry2-type genes and four different RFLP patterns were found. The combination of cry2Aa/cry2Ab genes was the most frequent (90.4%, followed by cry2Aa (6.8% and cry2Ab alone (2.5%, and only one novel type of cry2 gene was cloned from one isolate (JF19-2. The full-length of this novel gene was obtained by the method of thermal asymmetric interlaced PCR (Tail-PCR, which was designated as cry2Ag1 (GenBank No. ACH91610 by the Bt Pesticide Crystal Protein Nomenclature Committee. In addition, the result of scanning electron microscopic (SEM observation showed that these strains had erose, spherical, bipyramidal, and square crystal. And the results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE demonstrated that these strains harbored about one to three major proteins. These strains exhibited a wide range of insecticidal spectrum toxic to Aedes aegypti (Diptera and Pieris rapae Linnaeus, 1758 (Lepidoptera. Particularly, JF19-2 contained cry2Ag gene had the highest insecticidal activity. All these researches mentioned above revealed the diversity and particularity of cry2-type gene resources from Bacillus thuringiensis strains in Sichuan basin.

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

    Science.gov (United States)

    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.

  11. Determinación de la presencia de genes cry en cepas nativas de Bacillus thuringiensis

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    Chaparro Giraldo Luis Alejandro

    2004-12-01

    Full Text Available A partir de dos tipos de muestras, suelo asociado a cultivos de papa (Solanum tuberosum y polvo asociado a sitios de almacenamiento de granos (cereales colectado en las centrales de Corabastos y Paloquemao en Bogotá, se aislaron 88 cepas nativas de Bacillus thuringiensis bacilo
    grampositivo patógeno de insectos plaga de cultivos agrícolas. De las 88 cepas obtenidas 57 de ellas se escogieron para separarlas posteriormente mediante subcultivos en virtud a sus características fenotípicas al interior de la colonia llevando esto a obtener un número de 145 cepas nuevas para un total de 176 cepas de B. thuringiensis caracterizadas  morfológicamente y por su patrón de formas de las ICPs mediante microscopía de contraste de fases, y de las proteínas Cry arrojado por los perfiles de SDS-PAGE; en el total de cepas hubo presencia predominante de ICPs con forma romboide acompañadas por al menos otras dos formas distintas, y mostraron perfiles electroforéticos de proteínas de peso molecular entre el rango de 130, 116 y 60 kDa. Del total de cepas B. thuringiensis nativas aisladas en este estudio, 59 (30 de suelos y 29 de polvo fueron analizadas mediante la técnica PCR, encontrando que 100% de ellas son portadoras de alguno de los seis tipos de genes cry1 estudiados cry1Aa5 (71,2%, cry1Ab9 (66,1%, cry1Ac5 (45,8%, cry1Ba1 (39%, cry1Ca3 (49,1%, y cry1Da1 (71,2%; adicionalmente se observó la presencia de productos génicos inespecíficos, posiblemente nuevos, amplificados por la PCR y relacionados con los genes cry1C (55,9%, cry1B (30,5% y otros genes cry1 no estudiados por este método en al menos el 50% de las cepas estudiadas. De acuerdo con estos resultados se estableció un total de 19 genotipos diferentes según los genes cry1 estudiados, presentados por las cepas nativas en estudio determinando que estos son diferentes en composición genética según la fuente de procedencia del aislamiento, permitiendo establecer estrecha correlaci

  12. Structural studies of {delta}-endotoxin Cry 1 C from Bacillus thuringiensis

    Energy Technology Data Exchange (ETDEWEB)

    Guimaraes, B.G.; Garratt, R.C.; Oliva, G. [Sao Paulo Univ., Sao Carlos, SP (Brazil). Inst. de Fisica; Lemos, M.V.F. [UNESP, Jaboticabal, SP (Brazil). Dept. de Biologia Aplicada Agropecuaria; Arantes, O.M.N. [Universidade Estadual de Londrina, PR (Brazil). Dept. de Biologia Geral

    1996-12-31

    Full text. The {delta}-endotoxins are a family of crystal protein by a soil bacterium, Bacillus thuringiensis. The study of these proteins has been of great interest due to their highly specific activity against insects of the orders Lepidoptera, Diptera and Coleoptera. Thus, the {delta}a-endotoxins have been used for more than two decades as biological insecticides to control agricultural pests and, more recently, insects vectors of some diseases. The knowledge of their three-dimensional structures is very important to understand their mechanism of action and their high specificity. To date, the structure of only three proteins of the {delta}-endotoxins family have been reported: Cry3A, a coleopteran-specific toxin (beetle toxin){sup 1}, Cry1Aa, a lepidopteran-specific toxin (butterfly toxin){sup 2} and CytB, a dipteran-specific toxin (mosquito toxin){sup 3} Our work is aimed at the determination of the crystallographic structure by X-ray diffraction of {delta}-endotoxin Cry1C, also toxic to insects of the Lepidoptera order but towards families other than those affected by Cry1Aa. A comparison between these structures may lead to important conclusions about the reasons for the specificity and would allow the planning of mutants with more efficient activity. The cry1C gene was cloned into an adequate vector and expressed in an acrystalliferous B. thuringiensis strain. After cell culture and sporulation the microcrystals of Cry1C were separated by ultra-centrifugation in sacharose. The protoxin inclusion bodies were activated by commercial trpsin and the protease-resistant core was purified by anion-exchange chromatography. Crystallization experiments are being conducted in order to obtain single crystals suitable for diffraction measurements. We intend to use the Protein Crystallograph Station of the LNLS to collect data as soon as it is available and we have suitable crystals. (author) 3 refs.

  13. Adaptive Evolution of cry Genes in Bacillus thuringiensis:Implications for Their Specificity Determination

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    The cry gene family, produced during the late exponential phase of growth in Bacillus thuringiensis, is a large, still-growing family of homologous genes, in which each gene encodes a protein with strong specific activity against only one or a few insect species. Extensive studies are mostly focusing on the structural and functional relationships of Cry proteins, and have revealed several residues or domains that are important for the target recognition and receptor attachment. In this study,we have employed a maximum likelihood method to detect evidence of adaptive evolution in Cry proteins, and have identified 24 positively selected residues, which are all located in Domain Ⅱ or Ⅲ. Combined with known data from mutagenesis studies, the majority of these residues, at the molecular level, contribute much to the insect specificity determination. We postulate that the potential pressures driving the diversification of Cry proteins may be in an attempt to adapt for the "arm race" between δ-endotoxins and the targeted insects, or to enlarge their target spectra, hence result in the functional divergence. The sites identified to be under positive selection would provide targets for further structural and functional analyses on Cry proteins.

  14. Monitoring resistance to Bacillus thuringiensis subsp. israelensis in the field by performing bioassays with each Cry toxin separately

    OpenAIRE

    Guillaume Tetreau; Renaud Stalinski; Jean-Philippe David; Laurence Despres

    2013-01-01

    Bacillus thuringiensis subsp. israelensis (Bti) is increasingly used worldwide for mosquito control and is the only larvicide used in the French Rhône-Alpes region since decades. The artificial selection of mosquitoes with field-persistent Bti collected in breeding sites from this region led to a moderate level of resistance to Bti, but to relatively high levels of resistance to individual Bti Cry toxins. Based on this observation, we developed a bioassay procedure using each Bti Cry toxin se...

  15. Crystalline protein profiling and cry gene detection in Bacillus thuringiensis strains isolated during epizootics in Cydia pomonella L.

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    Konecka Edyta

    2014-12-01

    Full Text Available The composition of Bacillus thuringiensis crystalline inclusions was characterized in 18 strains: 12 isolates were obtained from the intestinal tract of Cydia pomonella larvae during epizootics, 2 isolates were cultured from Leucoma salicis larvae taken from their natural populations, and 4 reference strains. The number and molecular mass of B. thuringiensis crystalline proteins (Cry and Cyt was estimated by the sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE. The crystals contained 1-8 proteins with molecular masses of 36-155 kDa. The toxin profiles differed both quantatively and qualitatively. The B. thuringiensis MPU B9 isolate had the highest number and diversity of Cry toxins. The analysis of crystal composition by SDS-PAGE was insufficient to detect groups and subgroups of Cry proteins. We identified 20 groups and 3 subgroups of Cry and Cyt crystalline toxins. Only one epizootic strain harboured cry25. In single reference strains, the cry1H, cry10 and cry25 genes were found. We did not find any correlation between the occurrence of cry genes and electrophoretic protein profiles of crystalline toxins.

  16. Cadherin is a functional receptor of bacillus thuringiensis toxin Cry2Aa in the beet armyworm, spodoptera exigua

    Science.gov (United States)

    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 ...

  17. Isolation of transcripts from Diabrotica virgifera virgifera LeConte responsive to the Bacillus thuringiensis toxin Cry3Bb1

    Science.gov (United States)

    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...

  18. Strategies to improve the insecticidal activity of Cry toxins from Bacillus thuringiensis.

    Science.gov (United States)

    Pardo-López, L; Muñoz-Garay, C; Porta, H; Rodríguez-Almazán, C; Soberón, M; Bravo, A

    2009-03-01

    Bacillus thuringiensis Cry toxins have been widely used in the control of insect pests either as spray products or expressed in transgenic crops. These proteins are pore-forming toxins with a complex mechanism of action that involves the sequential interaction with several toxin-receptors. Cry toxins are specific against susceptible larvae and although they are often highly effective, some insect pests are not affected by them or show low susceptibility. In addition, the development of resistance threatens their effectiveness, so strategies to cope with all these problems are necessary. In this review we will discuss and compare the different strategies that have been used to improve insecticidal activity of Cry toxins. The activity of Cry toxins can be enhanced by using additional proteins in the bioassay like serine protease inhibitors, chitinases, Cyt toxins, or a fragment of cadherin receptor containing a toxin-binding site. On the other hand, different modifications performed in the toxin gene such as site-directed mutagenesis, introduction of cleavage sites in specific regions of the protein, and deletion of small fragments from the amino-terminal region lead to improved toxicity or overcome resistance, representing interesting alternatives for insect pest control.

  19. A Western Corn Rootworm Cadherin-like Protein is not Involved in the Binding and Toxicity of Cry34/35Ab1 and Cry3Aa Bacillus Thuringiensis Proteins

    Science.gov (United States)

    The western corn rootworm (WCR) Diabrotica virgifera virgifera LeConte is an important insect pest of corn. Bacillus thuringiensis (Bt) insecticidal proteins Cry3Aa (as mCry3A) and Cry34Ab1/Cry35Ab1 have been expressed in transgenic corn and are used to control the insect in the U.S. To date, there ...

  20. Efficient production of Bacillus thuringiensis Cry1AMod toxins under regulation of cry3Aa promoter and single cysteine mutations in the protoxin region.

    Science.gov (United States)

    García-Gómez, Blanca I; Sánchez, Jorge; Martínez de Castro, Diana L; Ibarra, Jorge E; Bravo, Alejandra; Soberón, Mario

    2013-11-01

    Bacillus thuringiensis Cry1AbMod toxins are engineered versions of Cry1Ab that lack the amino-terminal end, including domain I helix α-1 and part of helix α-2. This deletion improves oligomerization of these toxins in solution in the absence of cadherin receptor and counters resistance to Cry1A toxins in different lepidopteran insects, suggesting that oligomerization plays a major role in their toxicity. However, Cry1AbMod toxins are toxic to Escherichia coli cells, since the cry1A promoter that drives its expression in B. thuringiensis has readthrough expression activity in E. coli, making difficult the construction of these CryMod toxins. In this work, we show that Cry1AbMod and Cry1AcMod toxins can be cloned efficiently under regulation of the cry3A promoter region to drive its expression in B. thuringiensis without expression in E. coli cells. However, p3A-Cry1Ab(c)Mod construction promotes the formation of Cry1AMod crystals in B. thuringiensis cells that were not soluble at pH 10.5 and showed no toxicity to Plutella xylostella larvae. Cysteine residues in the protoxin carboxyl-terminal end of Cry1A toxins have been shown to be involved in disulfide bond formation, which is important for crystallization. Six individual cysteine substitutions for serine residues were constructed in the carboxyl-terminal protoxin end of the p3A-Cry1AbMod construct and one in the carboxyl-terminal protoxin end of p3A-Cry1AcMod. Interestingly, p3A-Cry1AbMod C654S and C729S and p3A-Cry1AcMod C730S recover crystal solubility at pH 10.5 and toxicity to P. xylostella. These results show that combining the cry3A promoter expression system with single cysteine mutations is a useful system for efficient expression of Cry1AMod toxins in B. thuringiensis.

  1. A theoretical model of the tridimensional structure of Bacillus thuringiensis subsp. medellin Cry 11Bb toxin deduced by homology modelling

    Directory of Open Access Journals (Sweden)

    Pablo Gutierrez

    2001-04-01

    Full Text Available Cry11Bb is an insecticidal crystal protein produced by Bacillus thuringiensis subsp. medellin during its stationary phase; this ¶-endotoxin is active against dipteran insects and has great potential for mosquito borne disease control. Here, we report the first theoretical model of the tridimensional structure of a Cry11 toxin. The tridimensional structure of the Cry11Bb toxin was obtained by homology modelling on the structures of the Cry1Aa and Cry3Aa toxins. In this work we give a brief description of our model and hypothesize the residues of the Cry11Bb toxin that could be important in receptor recognition and pore formation. This model will serve as a starting point for the design of mutagenesis experiments aimed to the improvement of toxicity, and to provide a new tool for the elucidation of the mechanism of action of these mosquitocidal proteins.

  2. Lack of detrimental effects of Bacillus thuringiensis Cry toxins on the insect predator Chrysoperla carnea: a toxicological, histopathological, and biochemical analysis

    NARCIS (Netherlands)

    Rodrigo-Simón, A.; Maagd, de R.A.; Avilla, C.; Bakker, P.L.; Molthoff, J.W.; González-Zamora, J.; Ferré, J.

    2006-01-01

    The effect of Cry proteins of Bacillus thuringiensis on the green lacewing (Chrysoperla carnea) was studied by using a holistic approach which consisted of independent, complementary experimental strategies. Tritrophic experiments were performed, in which lacewing larvae were fed Helicoverpa armiger

  3. pH-controlled Bacillus thuringiensis Cry1Ac protoxin loading and release from polyelectrolyte microcapsules.

    Directory of Open Access Journals (Sweden)

    Wenhui Yang

    Full Text Available 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 CaCO(3 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.

  4. Bacillus thuringiensis-derived Cry5B has potent anthelmintic activity against Ascaris suum.

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    Joseph F Urban

    Full Text Available Ascaris suum and Ascaris lumbricoides are two closely related geo-helminth parasites that ubiquitously infect pigs and humans, respectively. Ascaris suum infection in pigs is considered a good model for A. lumbricoides infection in humans because of a similar biology and tissue migration to the intestines. Ascaris lumbricoides infections in children are associated with malnutrition, growth and cognitive stunting, immune defects, and, in extreme cases, life-threatening blockage of the digestive tract and aberrant migration into the bile duct and peritoneum. Similar effects can be seen with A. suum infections in pigs related to poor feed efficiency and performance. New strategies to control Ascaris infections are needed largely due to reduced treatment efficacies of current anthelmintics in the field, the threat of resistance development, and the general lack of new drug development for intestinal soil-transmitted helminths for humans and animals. Here we demonstrate for the first time that A. suum expresses the receptors for Bacillus thuringiensis crystal protein and novel anthelmintic Cry5B, which has been previously shown to intoxicate hookworms and which belongs to a class of proteins considered non-toxic to vertebrates. Cry5B is able to intoxicate A. suum larvae and adults and triggers the activation of the p38 mitogen-activated protein kinase pathway similar to that observed with other nematodes. Most importantly, two moderate doses of 20 mg/kg body weight (143 nM/kg of Cry5B resulted in a near complete cure of intestinal A. suum infections in pigs. Taken together, these results demonstrate the excellent potential of Cry5B to treat Ascaris infections in pigs and in humans and for Cry5B to work effectively in the human gastrointestinal tract.

  5. Luminal proteinases from Plodia interpunctella and the hydrolysis of Bacillus thuringiensis CryIA(c) protoxin.

    Science.gov (United States)

    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.

  6. Isolation and distribution of mosquito-larvicidal cry genes in Bacillus thuringiensis strains native to Saudi Arabia.

    Science.gov (United States)

    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.

  7. Binding of Bacillus thuringiensis Cry1A toxins with brush border membrane vesicles of maize stem borer (Chilo partellus Swinhoe).

    Science.gov (United States)

    Sharma, Priyanka; Nain, Vikrant; Lakhanpaul, Suman; Kumar, P A

    2011-02-01

    Maize stem borer (Chilo partellus) is a major insect pest of maize and sorghum in Asia and Africa. Bacillus thuringiensis (Bt) δ-endotoxins have been found effective against C. partellus, both in diet-overlay assay and in transgenic plants. Gene stacking as one of the resistance management strategies in Bt maize requires an understanding of receptor sharing and binding affinity of δ-endotoxins. In the present study, binding affinity of three fluorescein isothiocyanate labeled Cry1A toxins showed high correlation with the toxicity of respective δ-endotoxins. Competitive binding studies showed that Cry1Ab toxins share some of the binding sites with Cry1Aa and Cry1Ac with low affinity and that Cry1Ab may have additional binding sites that are unavailable to the other two toxins tested.

  8. An ABC transporter mutation is correlated with insect resistance to Bacillus thuringiensis Cry1Ac toxin.

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    Linda J Gahan

    2010-12-01

    Full Text Available 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.

  9. Cadherin is involved in the action of Bacillus thuringiensis toxins Cry1Ac and Cry2Aa in the beet armyworm, Spodoptera exigua.

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    Qiu, Lin; Hou, Leilei; Zhang, Boyao; Liu, Lang; Li, Bo; Deng, Pan; Ma, Weihua; Wang, Xiaoping; Fabrick, Jeffrey A; Chen, Lizhen; Lei, Chaoliang

    2015-05-01

    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 Bt proteins that bind to distinct receptor proteins within the insect midgut. The most common Bt pyramid in cotton (Gossypium hirsutum L.) employs Cry1Ac with Cry2Ab to target several key lepidopteran pests, including the beet armyworm, Spodoptera exigua (Hübner), which is a serious migratory pest of many vegetable crops and is increasingly important in cotton in China. While cadherin and aminopeptidase-N are key receptors of Cry1 toxins in many lepidopterans including S. exigua, the receptor for Cry2A toxins remains poorly characterized. Here, we show that a heterologous expressed peptide corresponding to cadherin repeat 7 to the membrane proximal extracellular domain (CR7-MPED) in the S. exigua cadherin 1b (SeCad1b) binds Cry1Ac and Cry2Aa. Moreover, SeCad1b transcription was suppressed in S. exigua larvae by oral RNA interference and susceptibility to Cry1Ac and Cry2Aa was significantly reduced. These results indicate that SeCad1b plays important functional roles of both Cry1Ac and Cry2Aa, having major implications for resistance management for S. exigua in Bt crops.

  10. Three toxins, two receptors, one mechanism: Mode of action of Cry1A toxins from Bacillus thuringiensis in Heliothis virescens.

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    Bretschneider, Anne; Heckel, David G; Pauchet, Yannick

    2016-09-01

    Insecticidal crystal (Cry) proteins from Bacillus thuringiensis (Bt) are highly active against Lepidoptera. However, field-evolved resistance to Bt toxins is on the rise. The 12-cadherin domain protein HevCaLP and the ABC transporter HevABCC2 are both genetically linked to Cry toxin resistance in Heliothis virescens. We investigated their interaction using stably expressing non-lytic clonal Sf9 cell lines expressing either protein or both together. Untransfected Sf9 cells are innately sensitive to Cry1Ca toxin, but not to Cry1A toxins; and quantitative PCR revealed negligible expression of genes involved in Cry1A toxicity such as cadherin, ABCC2, alkaline phosphatase (ALP) and aminopeptidase N (APN). Cry1Aa, Cry1Ab or Cry1Ac caused swelling of Sf9 cells expressing HevABCC2, and caused faster swelling, lysis and up to 86% mortality in cells expressing both proteins. No such effect was observed in control Sf9 cells or in cells expressing only HevCaLP. The results of a mixing experiment demonstrated that both proteins need to be expressed within the same cell for high cytotoxicity, and suggest a novel role for HevCaLP. Binding assays showed that the toxin-receptor interaction is specific. Our findings confirm that HevABCC2 is the central target in Cry1A toxin mode of action, and that HevCaLP plays a supporting role in increasing Cry1A toxicity.

  11. Bacillus thuringiensis insecticidal three-domain Cry toxins: mode of action, insect resistance and consequences for crop protection.

    Science.gov (United States)

    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.

  12. Resistance to Bacillus thuringiensis Toxin Cry2Ab in Trichoplusia ni Is Conferred by a Novel Genetic Mechanism

    Science.gov (United States)

    Song, Xiaozhao; Kain, Wendy; Cassidy, Douglas

    2015-01-01

    The resistance to the Bacillus thuringiensis (Bt) toxin Cry2Ab in a greenhouse-originated Trichoplusia ni strain resistant to both Bt toxins Cry1Ac and Cry2Ab was characterized. Biological assays determined that the Cry2Ab resistance in the T. ni strain was a monogenic recessive trait independent of Cry1Ac resistance, and there existed no significant cross-resistance between Cry1Ac and Cry2Ab in T. ni. From the dual-toxin-resistant T. ni strain, a strain resistant to Cry2Ab only was isolated, and the Cry2Ab resistance trait was introgressed into a susceptible laboratory strain to facilitate comparative analysis of the Cry2Ab resistance with the susceptible T. ni strain. Results from biochemical analysis showed no significant difference between the Cry2Ab-resistant and -susceptible T. ni larvae in midgut proteases, including caseinolytic proteolytic activity and zymogram profile and serine protease activities, in midgut aminopeptidase and alkaline phosphatase activity, and in midgut esterases and hemolymph plasma melanization activity. For analysis of genetic linkage of Cry2Ab resistance with potential Cry toxin receptor genes, molecular markers for the midgut cadherin, alkaline phosphatase (ALP), and aminopeptidase N (APN) genes were identified between the original greenhouse-derived dual-toxin-resistant and the susceptible laboratory T. ni strains. Genetic linkage analysis showed that the Cry2Ab resistance in T. ni was not genetically associated with the midgut genes coding for the cadherin, ALP, and 6 APNs (APN1 to APN6) nor associated with the ABC transporter gene ABCC2. Therefore, the Cry2Ab resistance in T. ni is conferred by a novel but unknown genetic mechanism. PMID:26025894

  13. Aedes aegypti cadherin serves as a putative receptor of the Cry11Aa toxin from Bacillus thuringiensis subsp. israelensis

    OpenAIRE

    Chen, Jianwu; Aimanova, Karlygash G.; Fernandez, Luisa E.; Bravo, Alejandra; Soberon, Mario; Gill, Sarjeet S.

    2009-01-01

    Cry11Aa of Bacillus thuringiensis subsp. israelensis is the most active toxin to Aedes aegypti in this strain. We previously reported that, in addition to a 65 kDa GPI (glycosylphosphatidylinositol)-anchored ALP (alkaline phosphatase), the toxin also binds a 250 kDa membrane protein. Since this protein is the same size as cadherin, which in lepidopteran insects is an important Cry toxin receptor, we developed an anti-AaeCad antibody. This antibody detects a 250 kDa protein in immunoblots of l...

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

    Science.gov (United States)

    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.

  15. Binding specificity of Bacillus thuringiensis Cry1Aa for purified, native Bombyx mori aminopeptidase N and cadherin-like receptors

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    Jenkins Jeremy L

    2001-10-01

    Full Text Available Abstract Background To better understand the molecular interactions of Bt toxins with non-target insects, we have examined the real-time binding specificity and affinity of Cry1 toxins to native silkworm (Bombyx mori midgut receptors. Previous studies on B. mori receptors utilized brush border membrane vesicles or purifed receptors in blot-type assays. Results The Bombyx mori (silkworm aminopeptidase N (APN and cadherin-like receptors for Bacillus thuringiensis insecticidal Cry1Aa toxin were purified and their real-time binding affinities for Cry toxins were examined by surface plasmon resonance. Cry1Ab and Cry1Ac toxins did not bind to the immobilized native receptors, correlating with their low toxicities. Cry1Aa displayed moderate affinity for B. mori APN (75 nM, and unusually tight binding to the cadherin-like receptor (2.6 nM, which results from slow dissociation rates. The binding of a hybrid toxin (Aa/Aa/Ac was identical to Cry1Aa. Conclusions These results indicate domain II of Cry1Aa is essential for binding to native B. mori receptors and for toxicity. Moreover, the high-affinity binding of Cry1Aa to native cadherin-like receptor emphasizes the importance of this receptor class for Bt toxin research.

  16. Histopathological effects and determination of the putative receptor of Bacillus thuringiensis Cry1Da toxin in Spodoptera littoralis midgut.

    Science.gov (United States)

    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.

  17. Interactions of Bacillus thuringiensis Cry1Ac toxin in genetically engineered cotton with predatory heteropterans.

    Science.gov (United States)

    Torres, Jorge B; Ruberson, John R

    2008-06-01

    A number of cotton varieties have been genetically transformed with genes from Bacillus thuringiensis (Bt) to continuously produce Bt endotoxins, offering whole plant and season-long protection against many lepidopteran larvae. Constant whole-plant toxin expression creates a significant opportunity for non-target herbivores to acquire and bio-accumulate the toxin for higher trophic levels. In the present study we investigated movement of Cry1Ac toxin from the transgenic cotton plant through specific predator-prey pairings, using omnivorous predators with common cotton pests as prey: (1) the beet armyworm, Spodoptera exigua (Lepidoptera: Noctuidae), with the predator Podisus maculiventris (Heteroptera: Pentatomidae); (2) the two-spotted spider mite, Tetranychus urticae (Acarina: Tetranychidae), with the predatory big-eyed bug Geocoris punctipes (Heteroptera: Geocoridae) and (3) with the predatory damsel bug Nabis roseipennis (Heteropera: Nabidae); and (4) the thrips Frankliniella occidentalis (Thysanoptera: Thripidae) with the predatory pirate bug Orius insidiosus (Heteroptera: Anthocoridae). We quantified Cry1Ac toxin in the cotton plants, and in the pests and predators, and the effects of continuous feeding on S. exigua larvae fed either Bt or non-Bt cotton on life history traits of P. maculiventris. All three herbivores were able to convey Cry1Ac toxin to their respective predators. Among the herbivores, T. urticae exhibited 16.8 times more toxin in their bodies than that expressed in Bt-cotton plant, followed by S. exigua (1.05 times), and F. occidentalis immatures and adults (0.63 and 0.73 times, respectively). Of the toxin in the respective herbivorous prey, 4, 40, 17 and 14% of that amount was measured in the predators G. punctipes, P. maculiventris, O. insidiosus, and N. roseipennis, respectively. The predator P. maculiventris exhibited similar life history characteristics (developmental time, survival, longevity, and fecundity) regardless of the prey's food

  18. Specificity and combinatorial effects of Bacillus thuringiensis Cry toxins in the context of GMO environmental risk assessment

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    Angelika eHilbeck

    2015-11-01

    Full Text Available Stacked GM crops expressing up to six Cry toxins from Bacillus thuringiensis are today replacing the formerly grown single- transgene GM crop varieties. Stacking of multiple Cry toxins not only increase the environmental load of toxins but also raise the question on how possible interactions of the toxins can be assessed for risk assessment, which is mandatory for GM crops. However, no operational guidelines for a testing strategy or testing procedures exist. From the developers point of view, little data testing for combinatorial effects of Cry toxins is necessary as the range of affected organisms is focused on pest species and no evidence is claimed to exists pointing to combinatorial effects on nontarget organisms. We have examined this rationale critically using information reported in the scientific literature. To do so we address the hypothesis of narrow specificity of Cry toxins subdivided into three underlying different conceptual conditions i 'efficacy' in target pests as indicator for 'narrow specificity', ii lack of reported adverse effects of Cry toxins on nontarget organisms, and iii proposed modes of action of Cry toxins (or the lack thereof as mechanisms underlying the reported activity/efficacy/specificity of Cry toxins. Complementary to this information we evaluate reports about outcomes of combinatorial effect testing of Cry toxins in the scientific literature and relate those findings to the practice of the environmental risk assessment of Bt-corps in general and of stacked Bt-events in particular.

  19. Bacillus thuringiensis Crystal Protein Cry6Aa Triggers Caenorhabditis elegans Necrosis Pathway Mediated by Aspartic Protease (ASP-1)

    Science.gov (United States)

    Zhang, Fengjuan; Peng, Donghai; Cheng, Chunsheng; Zhou, Wei; Ju, Shouyong; Wan, Danfeng; Yu, Ziquan; Shi, Jianwei; Deng, Yaoyao; Wang, Fenshan; Ye, Xiaobo; Hu, Zhenfei; Lin, Jian; Ruan, Lifang; Sun, Ming

    2016-01-01

    Cell death plays an important role in host-pathogen interactions. Crystal proteins (toxins) are essential components of Bacillus thuringiensis (Bt) biological pesticides because of their specific toxicity against insects and nematodes. However, the mode of action by which crystal toxins to induce cell death is not completely understood. Here we show that crystal toxin triggers cell death by necrosis signaling pathway using crystal toxin Cry6Aa-Caenorhabditis elegans toxin-host interaction system, which involves an increase in concentrations of cytoplasmic calcium, lysosomal lyses, uptake of propidium iodide, and burst of death fluorescence. We find that a deficiency in the necrosis pathway confers tolerance to Cry6Aa toxin. Intriguingly, the necrosis pathway is specifically triggered by Cry6Aa, not by Cry5Ba, whose amino acid sequence is different from that of Cry6Aa. Furthermore, Cry6Aa-induced necrosis pathway requires aspartic protease (ASP-1). In addition, ASP-1 protects Cry6Aa from over-degradation in C. elegans. This is the first demonstration that deficiency in necrosis pathway confers tolerance to Bt crystal protein, and that Cry6A triggers necrosis represents a newly added necrosis paradigm in the C. elegans. Understanding this model could lead to new strategies for nematode control. PMID:26795495

  20. Molecular and Insecticidal Characterization of a Novel Cry-Related Protein from Bacillus Thuringiensis Toxic against Myzus persicae

    Science.gov (United States)

    Palma, Leopoldo; Muñoz, Delia; Berry, Colin; Murillo, Jesús; Ruiz de Escudero, Iñigo; Caballero, Primitivo

    2014-01-01

    This study describes the insecticidal activity of a novel Bacillus thuringiensis Cry-related protein with a deduced 799 amino acid sequence (~89 kDa) and ~19% pairwise identity to the 95-kDa-aphidicidal protein (sequence number 204) from patent US 8318900 and ~40% pairwise identity to the cancer cell killing Cry proteins (parasporins Cry41Ab1 and Cry41Aa1), respectively. This novel Cry-related protein contained the five conserved amino acid blocks and the three conserved domains commonly found in 3-domain Cry proteins. The protein exhibited toxic activity against the green peach aphid, Myzus persicae (Sulzer) (Homoptera: Aphididae) with the lowest mean lethal concentration (LC50 = 32.7 μg/mL) reported to date for a given Cry protein and this insect species, whereas it had no lethal toxicity against the Lepidoptera of the family Noctuidae Helicoverpa armigera (Hübner), Mamestra brassicae (L.), Spodoptera exigua (Hübner), S. frugiperda (J.E. Smith) and S. littoralis (Boisduval), at concentrations as high as ~3.5 μg/cm2. This novel Cry-related protein may become a promising environmentally friendly tool for the biological control of M. persicae and possibly also for other sap sucking insect pests. PMID:25384108

  1. Bacillus thuringiensis Crystal Protein Cry6Aa Triggers Caenorhabditis elegans Necrosis Pathway Mediated by Aspartic Protease (ASP-1.

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    Fengjuan Zhang

    2016-01-01

    Full Text Available Cell death plays an important role in host-pathogen interactions. Crystal proteins (toxins are essential components of Bacillus thuringiensis (Bt biological pesticides because of their specific toxicity against insects and nematodes. However, the mode of action by which crystal toxins to induce cell death is not completely understood. Here we show that crystal toxin triggers cell death by necrosis signaling pathway using crystal toxin Cry6Aa-Caenorhabditis elegans toxin-host interaction system, which involves an increase in concentrations of cytoplasmic calcium, lysosomal lyses, uptake of propidium iodide, and burst of death fluorescence. We find that a deficiency in the necrosis pathway confers tolerance to Cry6Aa toxin. Intriguingly, the necrosis pathway is specifically triggered by Cry6Aa, not by Cry5Ba, whose amino acid sequence is different from that of Cry6Aa. Furthermore, Cry6Aa-induced necrosis pathway requires aspartic protease (ASP-1. In addition, ASP-1 protects Cry6Aa from over-degradation in C. elegans. This is the first demonstration that deficiency in necrosis pathway confers tolerance to Bt crystal protein, and that Cry6A triggers necrosis represents a newly added necrosis paradigm in the C. elegans. Understanding this model could lead to new strategies for nematode control.

  2. Molecular and Insecticidal Characterization of a Novel Cry-Related Protein from Bacillus Thuringiensis Toxic against Myzus persicae

    Directory of Open Access Journals (Sweden)

    Leopoldo Palma

    2014-11-01

    Full Text Available This study describes the insecticidal activity of a novel Bacillus thuringiensis Cry-related protein with a deduced 799 amino acid sequence (~89 kDa and ~19% pairwise identity to the 95-kDa-aphidicidal protein (sequence number 204 from patent US 8318900 and ~40% pairwise identity to the cancer cell killing Cry proteins (parasporins Cry41Ab1 and Cry41Aa1, respectively. This novel Cry-related protein contained the five conserved amino acid blocks and the three conserved domains commonly found in 3-domain Cry proteins. The protein exhibited toxic activity against the green peach aphid, Myzus persicae (Sulzer (Homoptera: Aphididae with the lowest mean lethal concentration (LC50 = 32.7 μg/mL reported to date for a given Cry protein and this insect species, whereas it had no lethal toxicity against the Lepidoptera of the family Noctuidae Helicoverpa armigera (Hübner, Mamestra brassicae (L., Spodoptera exigua (Hübner, S. frugiperda (J.E. Smith and S. littoralis (Boisduval, at concentrations as high as ~3.5 μg/cm2. This novel Cry-related protein may become a promising environmentally friendly tool for the biological control of M. persicae and possibly also for other sap sucking insect pests.

  3. Molecular Approaches to Improve the Insecticidal Activity of Bacillus thuringiensis Cry Toxins

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    Wagner A. Lucena

    2014-08-01

    Full Text Available 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.

  4. Molecular approaches to improve the insecticidal activity of Bacillus thuringiensis Cry toxins.

    Science.gov (United States)

    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.

  5. A P-Glycoprotein Is Linked to Resistance to the Bacillus thuringiensis Cry3Aa Toxin in a Leaf Beetle

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    Yannick Pauchet

    2016-12-01

    Full Text Available Chrysomela tremula is a polyvoltine oligophagous leaf beetle responsible for massive attacks on poplar trees. This beetle is an important model for understanding mechanisms of resistance to Bacillus thuringiensis (Bt insecticidal toxins, because a resistant C. tremula strain has been found that can survive and reproduce on transgenic poplar trees expressing high levels of the Cry3Aa Bt toxin. Resistance to Cry3Aa in this strain is recessive and is controlled by a single autosomal locus. We used a larval midgut transcriptome for C. tremula to search for candidate resistance genes. We discovered a mutation in an ABC protein, member of the B subfamily homologous to P-glycoprotein, which is genetically linked to Cry3Aa resistance in C. tremula. Cultured insect cells heterologously expressing this ABC protein swell and lyse when incubated with Cry3Aa toxin. In light of previous findings in Lepidoptera implicating A subfamily ABC proteins as receptors for Cry2A toxins and C subfamily proteins as receptors for Cry1A and Cry1C toxins, this result suggests that ABC proteins may be targets of insecticidal three-domain Bt toxins in Coleoptera as well.

  6. A P-Glycoprotein Is Linked to Resistance to the Bacillus thuringiensis Cry3Aa Toxin in a Leaf Beetle

    Science.gov (United States)

    Pauchet, Yannick; Bretschneider, Anne; Augustin, Sylvie; Heckel, David G.

    2016-01-01

    Chrysomela tremula is a polyvoltine oligophagous leaf beetle responsible for massive attacks on poplar trees. This beetle is an important model for understanding mechanisms of resistance to Bacillus thuringiensis (Bt) insecticidal toxins, because a resistant C. tremula strain has been found that can survive and reproduce on transgenic poplar trees expressing high levels of the Cry3Aa Bt toxin. Resistance to Cry3Aa in this strain is recessive and is controlled by a single autosomal locus. We used a larval midgut transcriptome for C. tremula to search for candidate resistance genes. We discovered a mutation in an ABC protein, member of the B subfamily homologous to P-glycoprotein, which is genetically linked to Cry3Aa resistance in C. tremula. Cultured insect cells heterologously expressing this ABC protein swell and lyse when incubated with Cry3Aa toxin. In light of previous findings in Lepidoptera implicating A subfamily ABC proteins as receptors for Cry2A toxins and C subfamily proteins as receptors for Cry1A and Cry1C toxins, this result suggests that ABC proteins may be targets of insecticidal three-domain Bt toxins in Coleoptera as well. PMID:27929397

  7. Expression of cry1Ab gene from a novel Bacillus thuringiensis strain SY49-1 active on pest insects.

    Science.gov (United States)

    Azizoglu, Ugur; Ayvaz, Abdurrahman; Yılmaz, Semih; Karabörklü, Salih; Temizgul, Rıdvan

    2016-01-01

    In this study, the cry1Ab gene of previously characterized and Lepidoptera-, Diptera-, and Coleoptera-active Bacillus thuringiensis SY49-1 strain was cloned, expressed and individually tested on Ephestia kuehniella (Lepidoptera: Pyralidae) and Plodia interpunctella (Lepidoptera: Pyralidae) larvae. pET-cry1Ab plasmids were constructed by ligating the cry1Ab into pET28a (+) expression vector. Constructed plasmids were transferred to an Escherichia coli BL21 (DE3) strain rendered competent with CaCl2. Isopropyl β-d-1-thiogalactopyranoside was used to induce the expression of cry1Ab in E. coli BL21(DE3), and consequently, ∼130kDa of Cry1Ab was obtained. Bioassay results indicated that recombinant Cry1Ab at a dose of 1000μgg(-1) caused 40% and 64% mortality on P. interpunctella and E. kuehniella larvae, respectively. However, the mortality rates of Bt SY49-1 strains' spore-crystal mixture at the same dose were observed to be 70% on P. interpunctella and 90% on E. kuehniella larvae. The results indicated that cry1Ab may be considered as a good candidate in transgenic crop production and as an alternative biocontrol agent in controlling stored product moths.

  8. Effects of Bacillus thuringiensis toxin Cry1Ac and Beauveria bassiana on Asiatic corn borer (Lepidoptera: Crambidae).

    Science.gov (United States)

    Ma, Xiao-Mu; Liu, Xiao-Xia; Ning, Xia; Zhang, Bo; Han, Fei; Guan, Xiu-Min; Tan, Yun-Feng; Zhang, Qing-Wen

    2008-10-01

    In this study, interactions between Cry1Ac, a toxic crystal protein produced by Bacillus thuringiensis (Berliner), and Beauveria bassiana on the mortality and survival of Ostrinia furnacalis was evaluated in the laboratory. The results showed that Cry1Ac is toxic to O. furnacalis. Not only were larval growth and development delayed, but pupation, pupal weight and adult emergency also decreased when larvae were fed on artificial diet containing purified Cry1Ac toxin. When third instars O. furnacalis were exposed to combination of B. bassiana (1.8x10(5), 1.8x10(6) or 1.8x10(7) conidia ml(-1)) and Cry1Ac, (0.2 or 0.8 microg g(-1)), the effect on mortality was additive, however, the combinations of sublethal concentrations showed antagonism between Cry1Ac (3.2 or 13 microg g(-1)) and B. bassiana (1.8x10(5) or 1.8x10(6) conidia ml(-1)). When neonates were reared on sublethal concentrations of Cry1AC until the third instar, and survivors exposed B. bassiana conidial suspension, such treatments showed additive effect on mortality of O. furnacalis except for the combination of Cry1Ac (0.2 microg g(-1)) and B. bassiana (1.8x10(6) conidia ml(-1)) that showed antagonism.

  9. Persistence of Bt Bacillus thuringiensis Cry1Aa toxin in various soils determined by physicochemical reactions

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    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

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

    Science.gov (United States)

    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.

  11. Toxicity of seven Bacillus thuringiensis Cry proteins against Cylas puncticollis and Cylas brunneus (Coleoptera: Brentidae) using a novel artificial diet.

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    Ekobu, Moses; Solera, Maureen; Kyamanywa, Samuel; Mwanga, Robert O M; Odongo, Benson; Ghislain, Marc; Moar, William J

    2010-08-01

    "Sweetpotato weevils" Cylas puncticollis (Boheman) and Cylas brunneus F. (Coleoptera: Brentidae) are the most important biological threat to sweetpotato, Ipomoea batatas L. (Lam), productivity in sub-Saharan Africa. Sweetpotato weevil control is difficult due to their cryptic feeding behavior. Expression of Cylas-active Bacillus thuringiensis (Bt) Cry proteins in sweetpotato could provide an effective control strategy. Unfortunately, Bt Cry proteins with relatively high toxicity against Cylas spp. have not been identified, partly because no published methodology for screening Bt Cry proteins against Cylas spp. in artificial diet exists. Therefore, the initial aim of this study was to develop an artificial diet for conducting bioassays with Cylas spp. and then to determine Bt Cry protein efficacy against C. puncticollis and C. brunneus by using this artificial diet. Five diets varying in their composition were evaluated. The highest survival rates for sweetpotato weevil larvae were observed for diet E that contained the highest amount of sweetpotato powder and supported weevil development from first instar to adulthood, similar to sweetpotato storage roots. Seven coleopteran-active Bt Cry proteins were incorporated into diet E and toxicity data were generated against neonate C. puncticollis and second-instar C. brunneus. All Bt Cry proteins tested had toxicity greater than the untreated control. Cry7Aa1, ET33/34, and Cry3Ca1 had LC50 values below 1 microg/g diet against both species. This study demonstrates the feasibility of using an artificial diet bioassay for screening Bt Cry proteins against sweetpotato weevil larvae and identifies candidate Bt Cry proteins for use in transforming sweetpotato varieties potentially conferring field resistance against these pests.

  12. Use of endophytic diazotrophic bacteria as a vector to express the cry3A gene from Bacillus thuringiensis

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    Salles Joana Falcão

    2000-01-01

    Full Text Available The goal of this study was to evaluate the potential of endophytic diazotrophic bacteria as a vector to express a cry gene from Bacillus thuringiensis, envisaging the control of pests that attack sugarcane plants. The endophytic nitrogen-fixing bacteria Gluconacetobacter diazotrophicus strain BR11281 and Herbaspirillum seropedicae strain BR11335 were used as models. The cry3A gene was transferred by conjugation using a suicide plasmid and the recombinant strains were selected by their ability to fix nitrogen in semi-solid N-free medium. The presence of the cry gene was detected by Southern-blot using an internal fragment of 1.0 kb as a probe. The production of delta-endotoxin by the recombinant H. seropedicae strain was detected by dot blot while for G. diazotrophicus the Western-blot technique was used. In both cases, a specific antibody raised against the B. thuringiensis toxin was applied. The delta-endotoxin production showed by the G. diazotrophicus recombinant strain was dependent on the nitrogen fixing conditions since the cry3A gene was fused to a nif promoter. In the case of H. seropedicae the delta-endotoxin expression was not affected by the promoter (rhi used. These results suggest that endophytic diazotrophic bacteria can be used as vectors to express entomopathogenic genes envisaging control of sugarcane pests.

  13. Monitoring Susceptibility of Western Bean Cutworm (Lepidoptera: Noctuidae) Field Populations to Bacillus thuringiensis Cry1F Protein.

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    Ostrem, Jared S; Pan, Zaiqi; Flexner, John Lindsey; Owens, Elizabeth; Binning, Rachel; Higgins, Laura S

    2016-04-01

    Zea mays L. (maize) hybrids producing the Cry1F protein from Bacillus thuringiensis were first commercialized in the United States in 2003. These products demonstrated varying levels of moderate control, but not immunity to Striacosta albicosta (Smith) (Lepidoptera: Noctuidae) (western bean cutworm). Susceptibility of western bean cutworm to Cry1F protein was assessed in field populations collected in the mid- and western United States in 2003, 2004, 2013, and 2014 using diet bioassay. A meta-analysis of 32 western bean cutworm field collections assessed for susceptibility to Cry1F was conducted to investigate changes in susceptibility over time. Based on meta-analysis results, these data suggest a 5.2-fold increase in median lethal concentration (LC50) response to Cry1F in the 2013–2014 populations compared with collections that were assessed 10 yr earlier. Widespread use of Cry1F-producing maize hybrids over the past 10 yr may have contributed to favoring western bean cutworm populations with tolerance to the Cry1F protein.

  14. Monitoring resistance to Bacillus thuringiensis subsp. israelensis in the field by performing bioassays with each Cry toxin separately

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    Guillaume Tetreau

    2013-11-01

    Full Text Available Bacillus thuringiensis subsp. israelensis (Bti is increasingly used worldwide for mosquito control and is the only larvicide used in the French Rhône-Alpes region since decades. The artificial selection of mosquitoes with field-persistent Bti collected in breeding sites from this region led to a moderate level of resistance to Bti, but to relatively high levels of resistance to individual Bti Cry toxins. Based on this observation, we developed a bioassay procedure using each Bti Cry toxin separately to detect cryptic Bti-resistance evolving in field mosquito populations. Although no resistance to Bti was detected in none of the three mosquito species tested (Aedes rusticus, Aedes sticticus and Aedes vexans, an increased tolerance to Cry4Aa (3.5-fold and Cry11Aa toxins (8-fold was found in one Ae. sticticus population compared to other populations of the same species, suggesting that resistance to Bti may be arising in this population. This study confirms previous works showing a lack of Bti resistance in field mosquito populations treated for decades with this bioinsecticide. It also provides a first panorama of their susceptibility status to individual Bti Cry toxins. In combination with bioassays with Bti, bioassays with separate Cry toxins allow a more sensitive monitoring of Bti-resistance in the field.

  15. Reduced levels of membrane-bound alkaline phosphatase are common to lepidopteran strains resistant to Cry toxins from Bacillus thuringiensis.

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    Juan Luis Jurat-Fuentes

    Full Text Available Development of insect resistance is one of the main concerns with the use of transgenic crops expressing Cry toxins from the bacterium Bacillus thuringiensis. Identification of biomarkers would assist in the development of sensitive DNA-based methods to monitor evolution of resistance to Bt toxins in natural populations. We report on the proteomic and genomic detection of reduced levels of midgut membrane-bound alkaline phosphatase (mALP as a common feature in strains of Cry-resistant Heliothis virescens, Helicoverpa armigera and Spodoptera frugiperda when compared to susceptible larvae. Reduced levels of H. virescens mALP protein (HvmALP were detected by two dimensional differential in-gel electrophoresis (2D-DIGE analysis in Cry-resistant compared to susceptible larvae, further supported by alkaline phosphatase activity assays and Western blotting. Through quantitative real-time polymerase chain reaction (qRT-PCR we demonstrate that the reduction in HvmALP protein levels in resistant larvae are the result of reduced transcript amounts. Similar reductions in ALP activity and mALP transcript levels were also detected for a Cry1Ac-resistant strain of H. armigera and field-derived strains of S. frugiperda resistant to Cry1Fa. Considering the unique resistance and cross-resistance phenotypes of the insect strains used in this work, our data suggest that reduced mALP expression should be targeted for development of effective biomarkers for resistance to Cry toxins in lepidopteran pests.

  16. Cry4Ba and Cyt1Aa proteins from Bacillus thuringiensis israelensis: Interactions and toxicity mechanism against Aedes aegypti.

    Science.gov (United States)

    Elleuch, Jihen; Jaoua, Samir; Darriet, Frédéric; Chandre, Fabrice; Tounsi, Slim; Zghal, Raida Zribi

    2015-09-15

    Individual crystal proteins from Bacillus thuringiensis israelensis exhibit variable levels of insecticidal activities against mosquito larvae. In all cases, they are much less active compared to the whole crystal proteins due to described complex synergistic interactions among them. In the present study we investigated the effects of Cyt1A98 (a Cyt1Aa type protein) on Cry4BLB (a Cry4Ba type toxin) insecticidal activity toward the dengue vector Aedes aegypti. The bioassay analyses demonstrated the ability of Cyt1A98 protein to enhance Cry4BLB toxin larvicidal activity even at a low proportion in the mixture (1%). In vitro interaction assays showed that Cyt1A98 provides supplementary binding sites for Cry4BLB in A. aegypti BBMVs. Moreover, it enhances the formation of Cry4BLB oligomeric structure. These results support that Cyt1A98 protein could act as a membrane-bound receptor fixing Cry4BLB δ-endotoxins and promoting its oligomerization.

  17. Bacillus thuringiensis Cry3Aa protoxin intoxication of Tenebrio molitor induces widespread changes in the expression of serine peptidase transcripts.

    Science.gov (United States)

    Oppert, Brenda; Martynov, Alexander G; Elpidina, Elena N

    2012-09-01

    The yellow mealworm, Tenebrio molitor, is a pest of stored grain products and is sensitive to the Bacillus thuringiensis (Bt) Cry3Aa toxin. As digestive peptidases are a determining factor in Cry toxicity and resistance, we evaluated the expression of peptidase transcripts in the midgut of T. molitor larvae fed either a control or Cry3Aa protoxin diet for 24 h (RNA-Seq), or in larvae exposed to the protoxin for 6, 12, or 24 h (microarrays). Cysteine peptidase transcripts (9) were similar to cathepsins B, L, and K, and their expression did not vary more than 2.5-fold in control and Cry3Aa-treated larvae. Serine peptidase transcripts (48) included trypsin, chymotrypsin and chymotrypsin-like, elastase 1-like, and unclassified serine peptidases, as well as homologs lacking functional amino acids. Highly expressed trypsin and chymotrypsin transcripts were severely repressed, and most serine peptidase transcripts were expressed 2- to 15-fold lower in Cry3Aa-treated larvae. Many serine peptidase and homolog transcripts were found only in control larvae. However, expression of a few serine peptidase transcripts was increased or found only in Cry3Aa-treated larvae. Therefore, Bt intoxication significantly impacted the expression of serine peptidases, potentially important in protoxin processing, while the insect maintained the production of critical digestive cysteine peptidases.

  18. Evaluation of Cytotoxicity, Genotoxicity and Hematotoxicity of the Recombinant Spore-Crystal Complexes Cry1Ia, Cry10Aa and Cry1Ba6 from Bacillus thuringiensis in Swiss Mice

    Science.gov (United States)

    de Souza Freire, Ingrid; Miranda-Vilela, Ana Luisa; Barbosa, Lilian Carla Pereira; Martins, Erica Soares; Monnerat, Rose Gomes; Grisolia, Cesar Koppe

    2014-01-01

    The insecticidal properties of Cry-endotoxins from Bacillus thuringiensis (Bt) have long been used as spore-crystals in commercial spray formulations for insect control. Recently, some Bt-endotoxin genes have been cloned in many different plants. Toxicological evaluations of three spore-crystal endotoxins, BtCry1Ia, BtCry10Aa and BtCry1Ba6 from B. thuringiensis, were carried out on mice to understand their adverse effects on hematological systems and on genetic material. These three spore-crystals have shown toxic activity to the boll weevil, which is one of the most aggressive pests of the cotton crop. Cry1Ia, Cry10Aa and Cry1Ba6 did not increase the micronucleus frequency in the peripheral erythrocytes of mice and did not cause changes in the frequency of polychromatic erythrocytes. However, some hematologic disburbances were observed, specifically related to Cry1Ia and Cry1Ba6, respectively, for the erythroid and lymphoid lineage. Thus, although the profile of such adverse side effects can be related to their high level of exposure, which is not commonly found in the environment, results showed that these Bt spore-crystals were not harmless to mice, indicating that each spore-crystal endotoxin presents a characteristic profile of toxicity and might be investigated individually. PMID:25268978

  19. Evaluation of cytotoxicity, genotoxicity and hematotoxicity of the recombinant spore-crystal complexes Cry1Ia, Cry10Aa and Cry1Ba6 from Bacillus thuringiensis in Swiss mice.

    Science.gov (United States)

    de Souza Freire, Ingrid; Miranda-Vilela, Ana Luisa; Barbosa, Lilian Carla Pereira; Martins, Erica Soares; Monnerat, Rose Gomes; Grisolia, Cesar Koppe

    2014-09-29

    The insecticidal properties of Cry-endotoxins from Bacillus thuringiensis (Bt) have long been used as spore-crystals in commercial spray formulations for insect control. Recently, some Bt-endotoxin genes have been cloned in many different plants. Toxicological evaluations of three spore-crystal endotoxins, BtCry1Ia, BtCry10Aa and BtCry1Ba6 from B. thuringiensis, were carried out on mice to understand their adverse effects on hematological systems and on genetic material. These three spore-crystals have shown toxic activity to the boll weevil, which is one of the most aggressive pests of the cotton crop. Cry1Ia, Cry10Aa and Cry1Ba6 did not increase the micronucleus frequency in the peripheral erythrocytes of mice and did not cause changes in the frequency of polychromatic erythrocytes. However, some hematologic disburbances were observed, specifically related to Cry1Ia and Cry1Ba6, respectively, for the erythroid and lymphoid lineage. Thus, although the profile of such adverse side effects can be related to their high level of exposure, which is not commonly found in the environment, results showed that these Bt spore-crystals were not harmless to mice, indicating that each spore-crystal endotoxin presents a characteristic profile of toxicity and might be investigated individually.

  20. Evaluation of Cytotoxicity, Genotoxicity and Hematotoxicity of the Recombinant Spore-Crystal Complexes Cry1Ia, Cry10Aa and Cry1Ba6 from Bacillus thuringiensis in Swiss Mice

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    Ingrid de Souza Freire

    2014-09-01

    Full Text Available The insecticidal properties of Cry-endotoxins from Bacillus thuringiensis (Bt have long been used as spore-crystals in commercial spray formulations for insect control. Recently, some Bt-endotoxin genes have been cloned in many different plants. Toxicological evaluations of three spore-crystal endotoxins, BtCry1Ia, BtCry10Aa and BtCry1Ba6 from B. thuringiensis, were carried out on mice to understand their adverse effects on hematological systems and on genetic material. These three spore-crystals have shown toxic activity to the boll weevil, which is one of the most aggressive pests of the cotton crop. Cry1Ia, Cry10Aa and Cry1Ba6 did not increase the micronucleus frequency in the peripheral erythrocytes of mice and did not cause changes in the frequency of polychromatic erythrocytes. However, some hematologic disburbances were observed, specifically related to Cry1Ia and Cry1Ba6, respectively, for the erythroid and lymphoid lineage. Thus, although the profile of such adverse side effects can be related to their high level of exposure, which is not commonly found in the environment, results showed that these Bt spore-crystals were not harmless to mice, indicating that each spore-crystal endotoxin presents a characteristic profile of toxicity and might be investigated individually.

  1. Competition of Bacillus thuringiensis Cry1 toxins for midgut binding sites: a basis for the development and management of transgenic tropical maize resistant to several stemborers.

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    Rang, Cécile; Bergvingson, David; Bohorova, Natasha; Hoisington, David; Frutos, Roger

    2004-07-01

    Binding and competition of five Bacillus thuringiensis toxins--Cry1Ab, Cry1Ac, Cry1Ba, Cry1Ca, and Cry1Ea--for midgut binding sites from three pests, Spodoptera frugiperda, Diatraea saccharalis, and Diatraea grandiosella, were investigated as part of a strategy to develop tropical transgenic maize resistant to several stemborers. On S. frugiperda, Cry1Ab and Cry1Ac compete for the same binding site; Cry1Ba and Cry1Ca compete for a second binding site. Cry1Ea recognizes a third specific binding site in S. frugiperda and does not compete with any of the other toxins. On D. grandiosella and D. saccharalis, Cry1Ac competes with Cry1Ab and not with Cry1Ba and Cry1Ca. Cry1Ba and Cry1Ca recognize each a specific binding site and do not compete with any of the other four toxins. Cry1Ea does not recognize any binding site on Diatraea species. Combinations of toxins are proposed to develop transgenic maize resistant to the three stemborers while allowing resistance management.

  2. Binding sites for Bacillus thuringiensis Cry2Ae toxin on heliothine brush border membrane vesicles are not shared with Cry1A, Cry1F, or Vip3A toxin.

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    Gouffon, C; Van Vliet, A; Van Rie, J; Jansens, S; Jurat-Fuentes, J L

    2011-05-01

    The use of combinations of Bacillus thuringiensis (Bt) toxins with diverse modes of action for insect pest control has been proposed as the most efficient strategy to increase target range and delay the onset of insect resistance. Considering that most cases of cross-resistance to Bt toxins in laboratory-selected insect colonies are due to alteration of common toxin binding sites, independent modes of action can be defined as toxins sharing limited or no binding sites in brush border membrane vesicles (BBMV) prepared from the target insect larvae. In this paper, we report on the specific binding of Cry2Ae toxin to binding sites on BBMV from larvae of the three most commercially relevant heliothine species, Heliothis virescens, Helicoverpa zea, and Helicoverpa armigera. Using chromatographic purification under reducing conditions before labeling, we detected specific binding of radiolabeled Cry2Ae, which allowed us to perform competition assays using Cry1Ab, Cry1Ac, Cry1Fa, Vip3A, Cry2Ae, and Cry2Ab toxins as competitors. In these assays, Cry2Ae binding sites were shared with Cry2Ab but not with the tested Cry1 or Vip3A toxins. Our data support the use of Cry2Ae toxin in combination with Cry1 or Vip3A toxins in strategies to increase target range and delay the onset of heliothine resistance.

  3. Binding Sites for Bacillus thuringiensis Cry2Ae Toxin on Heliothine Brush Border Membrane Vesicles Are Not Shared with Cry1A, Cry1F, or Vip3A Toxin ▿

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    Gouffon, C.; Van Vliet, A.; Van Rie, J.; Jansens, S.; Jurat-Fuentes, J. L.

    2011-01-01

    The use of combinations of Bacillus thuringiensis (Bt) toxins with diverse modes of action for insect pest control has been proposed as the most efficient strategy to increase target range and delay the onset of insect resistance. Considering that most cases of cross-resistance to Bt toxins in laboratory-selected insect colonies are due to alteration of common toxin binding sites, independent modes of action can be defined as toxins sharing limited or no binding sites in brush border membrane vesicles (BBMV) prepared from the target insect larvae. In this paper, we report on the specific binding of Cry2Ae toxin to binding sites on BBMV from larvae of the three most commercially relevant heliothine species, Heliothis virescens, Helicoverpa zea, and Helicoverpa armigera. Using chromatographic purification under reducing conditions before labeling, we detected specific binding of radiolabeled Cry2Ae, which allowed us to perform competition assays using Cry1Ab, Cry1Ac, Cry1Fa, Vip3A, Cry2Ae, and Cry2Ab toxins as competitors. In these assays, Cry2Ae binding sites were shared with Cry2Ab but not with the tested Cry1 or Vip3A toxins. Our data support the use of Cry2Ae toxin in combination with Cry1 or Vip3A toxins in strategies to increase target range and delay the onset of heliothine resistance. PMID:21441333

  4. Cry1A toxins of Bacillus thuringiensis bind specifically to a region adjacent to the membrane-proximal extracellular domain of BT-R(1) in Manduca sexta: involvement of a cadherin in the entomopathogenicity of Bacillus thuringiensis.

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    Dorsch, J A; Candas, M; Griko, N B; Maaty, W S A; Midboe, E G; Vadlamudi, R K; Bulla, L A

    2002-09-01

    Many subspecies of the soil bacterium Bacillus thuringiensis produce various parasporal crystal proteins, also known as Cry toxins, that exhibit insecticidal activity upon binding to specific receptors in the midgut of susceptible insects. One such receptor, BT-R(1) (210 kDa), is a cadherin located in the midgut epithelium of the tobacco hornworm, Manduca sexta. It has a high binding affinity (K(d) approximately 1nM) for the Cry1A toxins of B. thuringiensis. Truncation analysis of BT-R(1) revealed that the only fragment capable of binding the Cry1A toxins of B. thuringiensis was a contiguous 169-amino acid sequence adjacent to the membrane-proximal extracellular domain. The purified toxin-binding fragment acted as an antagonist to Cry1Ab toxin by blocking the binding of toxin to the tobacco hornworm midgut and inhibiting insecticidal action. Exogenous Cry1Ab toxin bound to intact COS-7 cells expressing BT-R(1) cDNA, subsequently killing the cells. Recruitment of BT-R(1) by B. thuringiensis indicates that the bacterium interacts with a specific cell adhesion molecule during its pathogenesis. Apparently, Cry toxins, like other bacterial toxins, attack epithelial barriers by targeting cell adhesion molecules within susceptible insect hosts.

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

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    N. Knaak

    Full Text Available 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 μg.cm-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.

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

    Science.gov (United States)

    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.

  7. Bacillus thuringiensis Cry1Ca-resistant Spodoptera exigua lacks expression of one of four Aminopeptidase N genes

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    Moar William J

    2005-06-01

    Full Text Available Abstract Background Insecticidal toxins from Bacillus thuringiensis bind to receptors on midgut epithelial cells of susceptible insect larvae. Aminopeptidases N (APNs from several insect species have been shown to be putative receptors for these toxins. Here we report the cloning and expression analysis of four APN cDNAs from Spodoptera exigua. Results Suppression Subtractive Hybridization (SSH was used to construct cDNA libraries of genes that are up-and down-regulated in the midgut of last instar larvae of beet armyworm, S. exigua exposed to B. thuringiensis Cry1Ca toxin. Among the clones from the SSH libraries, cDNA fragments coding for two different APNs were obtained (APN2 and APN4. A similar procedure was employed to compare mRNA differences between susceptible and Cry1Ca resistant S. exigua. Among the clones from this last comparison, cDNA fragments belonging to a third APN (APN1 were detected. Using sequences obtained from the three APN cDNA fragments and degenerate primers for a fourth APN (APN3, the full length sequences of four S. exigua APN cDNAs were obtained. Northern blot analysis of expression of the four APNs showed complete absence of APN1 expression in the resistant insects, while the other three APNs showed similar expression levels in the resistant and susceptible insects. Conclusion We have cloned and characterized four different midgut APN cDNAs from S. exigua. Expression analysis revealed the lack of expression of one of these APNs in the larvae of a Cry1Ca-resistant colony. Combined with previous evidence that shows the importance of APN in the mode of action of B. thuringiensis toxins, these results suggest that the lack of APN1 expression plays a role in the resistance to Cry1Ca in this S. exigua colony.

  8. Induction of Manduca sexta Larvae Caspases Expression in Midgut Cells by Bacillus thuringiensis Cry1Ab Toxin

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    Helena Porta

    2011-01-01

    Full Text Available Bacillus thuringiensis produces crystal toxins known as Cry that are highly selective against important agricultural and human health-related insect pests. Cry proteins are pore-forming toxins that interact with specific receptors in the midgut cell membrane of susceptible larvae making pores that cause osmotic shock, leading finally to insect death. In the case of pore-forming toxins that are specific to mammalian cells, death responses at low doses may induce apoptosis or pyroptosis, depending on the cell type. The death mechanism induced by Cry toxins in insect midgut cells is poorly understood. Here, we analyze the caspases expression by RT-PCR analysis, showing that the initial response of Manduca sexta midgut cells after low dose of Cry1Ab toxin administration involves a fast and transient accumulation of caspase-1 mRNA, suggesting that pyroptosis was activated by Cry1Ab toxin as an initial response but was repressed later. In contrast, caspase-3 mRNA requires a longer period of time of toxin exposure to be activated but presents a sustained activation, suggesting that apoptosis may be a cell death mechanism induced also at low dose of toxin.

  9. Role of UPR Pathway in Defense Response of Aedes aegypti against Cry11Aa Toxin from Bacillus thuringiensis

    Directory of Open Access Journals (Sweden)

    Alejandra Bravo

    2013-04-01

    Full Text Available The insecticidal Cry toxins are pore-forming toxins produced by the bacteria Bacillus thuringiensis that disrupt insect-midgut cells. Cells can trigger different survival mechanisms to counteract the effects of sub-lytic doses of pore forming toxins. Particularly, two signaling pathways have been demonstrated to play a role in the defense mechanism to other toxins in Caenorhabditis elegans and in mammalian cells. These are the unfolded protein response (UPR and the sterol regulatory element binding proteins (SREBP pathways, which are proposed to facilitate membrane repair responses. In this work we analyzed the role of these pathways in Aedes aegypti response to intoxication with Cry11Aa toxin. We show that UPR is activated upon toxin ingestion. The role of these two pathways was analyzed in vivo by using RNA interference. We silenced the expression of specific proteins in A. aegypti larvae. Gene silencing of Ire-1 and Xbp-1 proteins from UPR system, resulted in hypersensitive to Cry11Aa toxin action. In contrast, silencing of Cas-1, Scap and S2P from SREBP pathway had no affect on Cry11Aa toxicity in A. aegypti larvae. However, the role of SREBP pathway requires further studies to be conclusive. Our data indicate that the UPR pathway is involved in the insect defense against Cry toxins.

  10. The role of β18-β19 loop structure in insecticidal activity of Cry1Ac toxin from Bacillus thuringiensis

    Institute of Scientific and Technical Information of China (English)

    XIA LiQiu; WANG FaXiang; DING XueZhi; ZHAO XinMin; FU ZuJiao; QUAN MeiFang; YU ZiNiu

    2008-01-01

    The β18-β19 loop in domain Ⅲ of Cry1Ac toxin is unique among Bacillus thuringiensis Cry proteins. In this study, the role of the loop structure in insecticidal activity of Cry1Ac toxin was investigated. Alanine scanning mutations within the loop were initially generated and most mutants were over-expressed and reduced toxicity at different degrees, except mutant N546A that showed almost 2 times enhanced toxicity against Helicoverpa armigera larvae. Further mutagenic analysis of N546 re-vealed that a charged amino acid in this position would cause very unfavorable influence on insecti-cidal activity. In addition, the deletion of N546 led to protein instability because of destruction of the loop integrity. Besides, mutant W544F was much more toxic than W544Y, indicating that hydrophobic nature of the position was important for maintaining the stability and activity of Cry1Ac protein. These findings are the first biological evidence for a structural function of β18-β19 loop in insecticidal activity of the Cry1Ac toxin.

  11. Prevalence of cry2-type genes in Bacillus thuringiensis isolates recovered from diverse habitats in India and isolation of a novel cry2Af2 gene toxic to Helicoverpa armigera (cotton boll worm).

    Science.gov (United States)

    Katara, Jawahar Lal; Kaur, Sarvjeet; Kumari, Gouthami Krishna; Singh, Nagendra Kumar

    2016-12-01

    Insecticidal cry and vip genes from Bacillus thuringiensis (Bt) have been used for control of lepidopteran insects in transgenic crops. However, novel genes are required for gene pyramiding to delay evolution of resistance to the currently deployed genes. Two PCR-based techniques were employed for screening of cry2-type genes in 129 Bt isolates from diverse habitats in India and 27 known Bt strains. cry2Ab-type genes were more prevalent than cry2Aa- and cry2Ac-type genes. Correlation between source of isolates and abundance of cry2-type genes was not observed. Full-length cry2A-type genes were amplified by PCR from 9 Bt isolates and 4 Bt strains. The genes from Bt isolates SK-758 from Sorghum grain dust and SK-793 from Chilli seeds warehouse, Andhra Pradesh, were cloned and sequenced. The gene from SK-758 (NCBI GenBank accession No. GQ866915) was novel, while that from SK-793 (NCBI GenBank accession No. GQ866914) was identical to the cry2Ab1 gene. The Bacillus thuringiensis Nomenclature Committee ( http://www.lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/toxins2.html ) named these genes cry2Af2 and cry2Ab16, respectively. The cry2Af2 gene was expressed in Escherichia coli and found to be toxic towards Helicoverpa armigera. The cry2Af2 gene will be useful for pyramiding in transgenic crops.

  12. Synergism and Antagonism between Bacillus thuringiensis Vip3A and Cry1 Proteins in Heliothis virescens, Diatraea saccharalis and Spodoptera frugiperda

    OpenAIRE

    Ana Rita Nunes Lemes; Camila Chiaradia Davolos; Paula Cristina Brunini Crialesi Legori; Odair Aparecido Fernandes; Juan Ferré; Manoel Victor Franco Lemos; Janete Apparecida Desiderio

    2014-01-01

    Second generation Bt crops (insect resistant crops carrying Bacillus thuringiensis genes) combine more than one gene that codes for insecticidal proteins in the same plant to provide better control of agricultural pests. Some of the new combinations involve co-expression of cry and vip genes. Because Cry and Vip proteins have different midgut targets and possibly different mechanisms of toxicity, it is important to evaluate possible synergistic or antagonistic interactions between these two c...

  13. Linkage of an ABCC transporter to a single QTL that controls Ostrinia nubilalis larval resistance to the Bacillus thuringiensis Cry1Fa toxin

    Science.gov (United States)

    Field evolved resistance of insect populations to Bacillus thuringiensis (Bt) crystalline (Cry) toxins expressed by crop plants has resulted in reduced control of insect feeding damage to field crops, and threatens the sustainability of Bt transgenic technologies. A single quantitative trait locus ...

  14. A synthetic cryIC gene, encoding a Bacillus thuringiensis δ-endotoxin, confers Spodoptera resistance in alfalfa and tobacco

    NARCIS (Netherlands)

    Strizhov, N.; Keller, M.; Mathur, J.; Koncz-Kaiman, Z.; Bosch, D.; Prudovksy, E.; Schell, J.; Sneh, B.; Koncz, C.; Zilberstein, A.

    1996-01-01

    Spodoptera species, representing widespread polyphagous insect pests, are resistant to Bacillus thuringiensis δ-endotoxins used thus far as insecticides in transgenic plants. Here we describe the chemical synthesis of a cryIC gene by a novel template directed ligation–PCR method. This simple and eco

  15. Mutations in the Bacillus thuringiensis Cry1Ca toxin demonstrate the role of domain II and III in the specificity towards Spodoptera exigua larvae

    NARCIS (Netherlands)

    Herrero Sendra, S.; González-Cabrera, J.; Ferré, J.; Bakker, P.L.; Maagd, de R.A.

    2004-01-01

    Several mutants of the Bacillus thuringiensis Cry1Ca toxin affected with regard to specific activity towards Spodoptera exigua were studied. Alanine was used to replace single residues in loops 2 and 3 of domain II (mutant pPB19) and to replace residues 541-544 in domain III (mutant pPB20). Addition

  16. In vitro effect of Bacillus thuringiensis strains and Cry proteins in phytopathogenic fungi of paddy rice-field Efeito in vitro de cepas e proteínas Cry de Bacillus thuringiensis em fungos fitopatogênicos da cultura do arroz irrigado

    Directory of Open Access Journals (Sweden)

    Neiva Knaak

    2007-09-01

    Full Text Available Cry1Ab and Cry1Ac strains and proteins synthesized by Bacillus thuringiensis thuringiensis and B. thuringiensis kurstaki were assessed in the following phytopathogens: Rhizoctonia solani,Pyricularia grisea,Fusarium oxysporum and F. solani, which had their micelial growth decreased after incubation in the presence of the bacterial strains. As to Cry proteins, there were no inhibition halo development in the assessed concentrations.As cepas e proteínas Cry1Ab e Cry1Ac sintetizadas por Bacillus thuringiensis thuringiensis e B. thuringiensis kurstaki, foram avaliadas nos fitopatógenos: Rhizoctonia solani,Pyricularia grisea,Fusarium oxysporum e F. solani, os quais tiveram seu crescimento micelial reduzido após a incubação na presença das cepas bacterianas. Em relação às proteínas Cry, não houve formação de halo de inibição nas concentrações avaliadas.

  17. Preferential Protection of Domains II and III of Bacillus thuringiensis Cry1Aa Toxin by Brush Border Membrane Vesicles

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    Syed-Rehan A. Hussain

    2011-01-01

    Full Text Available Título español: Protección preferencial de los dominios II y III de la toxina Cry1Aa de Bacillus thuringiensis en Vesículas de Membrana de Borde de Cepillo Abstract The surface exposed Leucine 371 on loop 2 of domain II, in Cry1Aa toxin, was mutated to Lysine to generate the trypsin-sensitive mutant, L371K. Upon trypsin digestion L371K is cleaved into approximately 37 and 26 kDa fragments. These are separable on SDS-PAGE, but remain as a single molecule of 65 kDa upon purification by liquid chromatography. The larger fragment is domain I and a portion of domain II (amino acid residues 1 to 371. The smaller 26-kDa polypeptide is the remainder of domain II and domain III (amino acids 372 to 609. When the mutant toxin was treated with high dose of M. sexta gut juice both fragments were degraded. However, when incubated with M. sexta BBMV, the 26 kDa fragment (domains II and III was preferentially protected from gut juice proteases. As previously reported, wild type Cry1Aa toxin was also protected against degradation by gut juice proteases when incubated with M. sexta BBMV. On the contrary, when mouse BBMV was added to the reaction mixture neither Cry1Aa nor L371K toxins showed resistance to M. sexta gut juice proteases and were degraded. Since the whole Cry1Aa toxin and most of the domain II and domain III of L371K are protected from proteases in the presence of BBMV of the target insect, we suggest that the insertion of the toxin into the membrane is complex and involves all three domains. Key words: Bacillus thuringiensis, site directed mutagenesis,  -endotoxin. Resumen La superficie de la toxina Cry1Aa, en el asa 2 del dominio II contiene expuesta la leucina 371, la cual fue modificada a lisina produciendo una mutante sensible a la tripsina, L371K. Esta mutante produce dos fragmentos de 37 y 26 kDa por acción de la tripsina que son separables por SDS-PAGE, pero que a la purificación por cromatografía líquida se mantienen como una sola

  18. Fatores de Virulência de Bacillus thuringiensis Berlinier: O Que Existe Além das Proteínas Cry?

    OpenAIRE

    Laurival A Vilas-Boas; Rita C. Alvarez; dos Santos, Clelton A.; Gislayne Trindade Vilas-Bôas

    2012-01-01

    As proteínas Cry produzidas pela bactéria entomopatogênica Bacillus thuringiensis Berliner são bem conhecidas devido a alta citotoxicidade que exibem a uma variedade de insetos-alvo. O modo de ação destas proteínas é específico e torna os produtos à base de B. thuringiensis os mais amplamente utilizados em programas de controle biológico de pragas na agricultura e de importantes vetores de doenças humanas. Contudo, embora as proteínas Cry sejam os fatores de virulência inseto-específico mais ...

  19. Helicoverpa armigera baseline susceptibility to Bacillus thuringiensis Cry toxins and resistance management for Bt cotton in India.

    Science.gov (United States)

    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.

  20. Functional characterizations of residues Arg-158 and Tyr-170 of the mosquito-larvicidal Bacillus thuringiensis Cry4Ba

    Science.gov (United States)

    Leetachewa, Somphob; Moonsom, Saengduen; Chaisri, Urai; Khomkhum, Narumol; Yoonim, Nonglak; Wang, Ping; Angsuthanasombat, Chanan

    2014-01-01

    The insecticidal activity of Bacillus thuringiensis (Bt) Cry toxins involves toxin stabilization, oligomerization, passage across the peritrophic membrane (PM), binding to midgut receptors and pore-formation. The residues Arg-158 and Tyr-170 have been shown to be crucial for the toxicity of Bt Cry4Ba. We characterized the biological function of these residues. In mosquito larvae, the mutants R158A/E/Q (R158) could hardly penetrate the PM due to a significantly reduced ability to alter PM permeability; the mutant Y170A, however, could pass through the PM, but degraded in the space between the PM and the midgut epithelium. Further characterization by oligomerization demonstrated that Arg-158 mutants failed to form correctly sized high-molecular weight oligomers. This is the first report that Arg-158 plays a role in the formation of Cry4Ba oligomers, which are essential for toxin passage across the PM. Tyr-170, meanwhile, is involved in toxin stabilization in the toxic mechanism of Cry4Ba in mosquito larvae. [BMB Reports 2014; 47(10): 546-551] PMID:24286331

  1. Penetration of a Single Domain of Bacillus thuringiensis Cry1Ie-Domain I to a Lipid Membrane In vitro

    Institute of Scientific and Technical Information of China (English)

    GUO Shu-yuan; LI Jie; CHEN Zhen; HE Kang-lai

    2014-01-01

    Domain I of the activated Crystal protein from Bacillus thuringiensis has a sevenα-helix bundle structure, which is responsible for membrane channel formation in its insecticidal mechanism. Cry1Ie is toxic to Asian corn borer, Ostrinia furnacalis (Guenée), and plays important roles in insect biological control. The domain I from Cry1Ie has been expressed and puriifed in its normal conformation, as embedded in the full length homologous toxin structure. The membrane insertion ability of this single domain was compared with the full length homologous toxin using a monolayer insertion experiment. The results indicated that the Cry1Ie-domain I had the ability to insert into the lipid monolayer, and this ability is greater than that of the IE648 toxin. However, the state of insertion is not stable and remains for only a short period of time. The Cry1Ie-domain I plays no role in receptor binding as it had a nonspeciifc binding with the brush border membrane vesicles of the Asian corn borer.

  2. Mutagenic analysis of putative domain II and surface residues in mosquitocidal Bacillus thuringiensis Cry19Aa toxin.

    Science.gov (United States)

    Roh, Jong Yul; Nair, Manoj S; Liu, Xinyan Sylvia; Dean, Donald H

    2009-06-01

    The mosquitocidal crystal protein, Cry19Aa, from Bacillus thuringiensis ssp. jegathesan, has high toxicity to Anopheles stephensi and Culex pipiens but is less toxic to Aedes aegypti. To study the functional role of putative domain II and surface residues in mosquito toxicity, 16 alanine substitution mutations were introduced into Cry19Aa. All mutant constructs were expressed as 65-kDa protoxins and subsequently digested by trypsin to produce further fragmented polypeptides of 40 and 25 kDa. With chymotrypsin, however, most protoxins were digested to 60 kDa and minor bands. The circular dichroism spectra of the chymotrypsin-activated toxins of Cry19Aa and muteins, Y324A, W357A, Y412A, Y414A, W416A, D418A and F485A indicated that there was no significant variation in their structure. In mosquito bioassays, Y324A, W357A, Y410A, W416A, D418A and F485A muteins showed substantial reductions in mosquitocidal activity toward A. aegypti and C. pipiens. These muteins also showed reduced competition with wild-type fluorescein 5-isothiocyanate-labeled Cry19Aa for binding to C. pipiens brush border membrane vesicles. These data suggest that the reduction of toxicity was a result of the reduced binding affinity. From these studies we have identified loop residues of domain II that are important in toxicity and receptor binding to Culex larval midgut.

  3. Genetic Basis of Cry1F-Resistance in a Laboratory Selected Asian Corn Borer Strain and Its Cross-Resistance to Other Bacillus thuringiensis Toxins.

    Science.gov (United States)

    Wang, Yueqin; Wang, Yidong; Wang, Zhenying; Bravo, Alejandra; Soberón, Mario; He, Kanglai

    2016-01-01

    The Asian corn borer (ACB), Ostrinia furnacalis (Guenée) (Lepidoptera: Crambidae), is the most destructive insect pest of corn in China. Susceptibility to the Cry1F toxin derived from Bacillus thuringiensis has been demonstrated for ACB, suggesting the potential for Cry1F inclusion as part of an insect pest management program. Insects can develop resistance to Cry toxins, which threatens the development and use of Bt formulations and Bt crops in the field. To determine possible resistance mechanisms to Cry1F, a Cry1F-resistant colony of ACB (ACB-FR) that exhibited more than 1700-fold resistance was established through selection experiments after 49 generations of selection under laboratory conditions. The ACB-FR strain showed moderate cross-resistance to Cry1Ab and Cry1Ac of 22.8- and 26.9-fold, respectively, marginally cross-resistance to Cry1Ah (3.7-fold), and no cross-resistance to Cry1Ie (0.6-fold). The bioassay responses of progeny from reciprocal F1 crosses to different Cry1 toxin concentrations indicated that the resistance trait to Cry1Ab, Cry1Ac and Cry1F has autosomal inheritance with no maternal effect or sex linked. The effective dominance (h) of F1 offspring was calculated at different concentrations of Cry1F, showing that h decreased as concentration of Cry1F increased. Finally, the analysis of actual and expected mortality of the progeny from a backcross (F1 × resistant strain) indicated that the inheritance of the resistance to Cry1F in ACB-FR was due to more than one locus. The present study provides an understanding of the genetic basis of Cry1F resistance in ACB-FR and also shows that pyramiding Cry1F with Cry1Ah or Cry1Ie could be used as a strategy to delay the development of ACB resistance to Bt proteins.

  4. Cloning, characterization and expression of a novel haplotype cry2A-type gene from Bacillus thuringiensis strain SWK1, native to Himalayan valley Kashmir.

    Science.gov (United States)

    Reyaz, A L; Arulselvi, P Indra

    2016-05-01

    Bacillus thuringiensis (Bt) is a gram positive bacterium which is effectively being used in pest management strategies as an eco-friendly bioinsecticide. In the present study a new cry2A gene was cloned from a promising indigenous B. thuringiensis SWK1 strain previously characterized for its toxicity against Spodoptera litura and Helicoverpa armigera larvae. The nucleotide sequence of the cloned cry2A gene pointed out that the open reading frame has 1902 bases encoding a polypeptide of 634 amino acid residues with a probable molecular weight of 70kDa. Homology comparisons showed that the deduced amino acid sequence of Cry2A had a similarity of 94% compared to that of the known Cry2Aa protein in the NCBI database and this gene has been named as cry2Al1 by the B. thuringiensis δ-endotoxin Nomenclature Committee. cry2Al1 was ligated into pET 22b vector and expressed in Escherichia coli BL21 (DE3) pLysS under the control of T7 promoter induced by isopropyl-beta-d-thiogalactopyranoside (IPTG). SDS-PAGE analysis confirmed the expression of cry2Al1 as ∼65kDa protein. Insect pest bioassays with neonate larvae of S. litura and H. armigera showed that the purified Cry2Al1 are toxic to S. litura and H. armigera with LC50 2.448μg/ml and H. armigera with 3.374μg/ml respectively.

  5. Transcriptome profiling of the intoxication response of Tenebrio molitor larvae to Bacillus thuringiensis Cry3Aa protoxin.

    Directory of Open Access Journals (Sweden)

    Brenda Oppert

    Full Text Available Bacillus thuringiensis (Bt crystal (Cry proteins are effective against a select number of insect pests, but improvements are needed to increase efficacy and decrease time to mortality for coleopteran pests. To gain insight into the Bt intoxication process in Coleoptera, we performed RNA-Seq on cDNA generated from the guts of Tenebrio molitor larvae that consumed either a control diet or a diet containing Cry3Aa protoxin. Approximately 134,090 and 124,287 sequence reads from the control and Cry3Aa-treated groups were assembled into 1,318 and 1,140 contigs, respectively. Enrichment analyses indicated that functions associated with mitochondrial respiration, signalling, maintenance of cell structure, membrane integrity, protein recycling/synthesis, and glycosyl hydrolases were significantly increased in Cry3Aa-treated larvae, whereas functions associated with many metabolic processes were reduced, especially glycolysis, tricarboxylic acid cycle, and fatty acid synthesis. Microarray analysis was used to evaluate temporal changes in gene expression after 6, 12 or 24 h of Cry3Aa exposure. Overall, microarray analysis indicated that transcripts related to allergens, chitin-binding proteins, glycosyl hydrolases, and tubulins were induced, and those related to immunity and metabolism were repressed in Cry3Aa-intoxicated larvae. The 24 h microarray data validated most of the RNA-Seq data. Of the three intoxication intervals, larvae demonstrated more differential expression of transcripts after 12 h exposure to Cry3Aa. Gene expression examined by three different methods in control vs. Cry3Aa-treated larvae at the 24 h time point indicated that transcripts encoding proteins with chitin-binding domain 3 were the most differentially expressed in Cry3Aa-intoxicated larvae. Overall, the data suggest that T. molitor larvae mount a complex response to Cry3Aa during the initial 24 h of intoxication. Data from this study represent the largest genetic sequence

  6. Transcriptome profiling of the intoxication response of Tenebrio molitor larvae to Bacillus thuringiensis Cry3Aa protoxin.

    Science.gov (United States)

    Oppert, Brenda; Dowd, Scot E; Bouffard, Pascal; Li, Lewyn; Conesa, Ana; Lorenzen, Marcé D; Toutges, Michelle; Marshall, Jeremy; Huestis, Diana L; Fabrick, Jeff; Oppert, Cris; Jurat-Fuentes, Juan Luis

    2012-01-01

    Bacillus thuringiensis (Bt) crystal (Cry) proteins are effective against a select number of insect pests, but improvements are needed to increase efficacy and decrease time to mortality for coleopteran pests. To gain insight into the Bt intoxication process in Coleoptera, we performed RNA-Seq on cDNA generated from the guts of Tenebrio molitor larvae that consumed either a control diet or a diet containing Cry3Aa protoxin. Approximately 134,090 and 124,287 sequence reads from the control and Cry3Aa-treated groups were assembled into 1,318 and 1,140 contigs, respectively. Enrichment analyses indicated that functions associated with mitochondrial respiration, signalling, maintenance of cell structure, membrane integrity, protein recycling/synthesis, and glycosyl hydrolases were significantly increased in Cry3Aa-treated larvae, whereas functions associated with many metabolic processes were reduced, especially glycolysis, tricarboxylic acid cycle, and fatty acid synthesis. Microarray analysis was used to evaluate temporal changes in gene expression after 6, 12 or 24 h of Cry3Aa exposure. Overall, microarray analysis indicated that transcripts related to allergens, chitin-binding proteins, glycosyl hydrolases, and tubulins were induced, and those related to immunity and metabolism were repressed in Cry3Aa-intoxicated larvae. The 24 h microarray data validated most of the RNA-Seq data. Of the three intoxication intervals, larvae demonstrated more differential expression of transcripts after 12 h exposure to Cry3Aa. Gene expression examined by three different methods in control vs. Cry3Aa-treated larvae at the 24 h time point indicated that transcripts encoding proteins with chitin-binding domain 3 were the most differentially expressed in Cry3Aa-intoxicated larvae. Overall, the data suggest that T. molitor larvae mount a complex response to Cry3Aa during the initial 24 h of intoxication. Data from this study represent the largest genetic sequence dataset for T. molitor

  7. Isolation and characterization of a new Bacillus thuringiensis strain Lip harboring a new cry1Aa gene highly toxic to Ephestia kuehniella (Lepidoptera: Pyralidae) larvae.

    Science.gov (United States)

    El Khoury, Micheline; Azzouz, Hichem; Chavanieu, Alain; Abdelmalak, Nouha; Chopineau, Joël; Awad, Mireille Kallassy

    2014-06-01

    The aim of this study was to characterize new Bacillus thuringiensis strains that have a potent insecticidal activity against Ephestia kuehniella larvae. Strains harboring cry1A genes were tested for their toxicity, and the Lip strain showed a higher insecticidal activity compared to that of the reference strain HD1 (LC50 of Lip and HD1 were 33.27 and 128.61 μg toxin/g semolina, respectively). B. thuringiensis Lip harbors and expresses cry1Aa, cry1Ab, cry1Ac, cry1Ad and cry2A. DNA sequencing revealed several polymorphisms in Lip Cry1Aa and Cry1Ac compared to the corresponding proteins of HD1. The activation process using Ephestia kuehniella midgut juice showed that Lip Cry1A proteins were more stable in the presence of larval proteases. Moreover, LipCry1A proteins exhibited higher insecticidal activity against these larvae. These results indicate that Lip is an interesting strain that could be used as an alternative to the worldwide used strain HD1.

  8. SKPDT is a signaling peptide that stimulates sporulation and cry1Aa expression in Bacillus thuringiensis but not in Bacillus subtilis.

    Science.gov (United States)

    Aceves-Diez, Angel E; Robles-Burgueño, Refugio; de la Torre, Mayra

    2007-08-01

    We have identified and characterized in the supernatant of the transition phase of Bacillus thuringiensis var. kurstaki the peptide SKPDT. This peptide was previously identified by in silico analysis by Pottathil and Lazazzera (Front Biosci 8:32-45 2003) as a putative signaling peptide (NprRB) of the Phr family in B. thuringiensis. The chemically synthesized NprRB did not affect the growth kinetics of B. thuringiensis var. kurstaki but stimulated the sporulation, spore release, and transcription of cry1Aa when added to cultures during the transition phase. In fact, when the peptide (100 nM) was added to a culture in transition phase, the transcription of cry1Aa was stimulated almost threefold, mainly from the late promoter BtII, which requires the late-stage sporulation-specific transcription factor sigma (K). On the other hand, NprRB did not have any effect on B. subtilis. Thus, SKPDT seems to be a signaling peptide specific for B. thuringiensis.

  9. Effect of crop plants on fitness costs associated with resistance to Bacillus thuringiensis toxins Cry1Ac and Cry2Ab in cabbage loopers

    Science.gov (United States)

    Wang, Ran; Tetreau, Guillaume; Wang, Ping

    2016-01-01

    Fitness costs associated with resistance to Bacillus thuringiensis (Bt) toxins critically impact the development of resistance in insect populations. In this study, the fitness costs in Trichoplusia ni strains associated with two genetically independent resistance mechanisms to Bt toxins Cry1Ac and Cry2Ab, individually and in combination, on four crop plants (cabbage, cotton, tobacco and tomato) were analyzed, in comparison with their near-isogenic susceptible strain. The net reproductive rate (R0) and intrinsic rate of increase (r) of the T. ni strains, regardless of their resistance traits, were strongly affected by the host plants. The ABCC2 gene-linked mechanism of Cry1Ac resistance was associated with relatively low fitness costs, while the Cry2Ab resistance mechanism was associated with higher fitness costs. The fitness costs in the presence of both resistance mechanisms in T. ni appeared to be non-additive. The relative fitness of Bt-resistant T. ni depended on the specific resistance mechanisms as well as host plants. In addition to difference in survivorship and fecundity, an asynchrony of adult emergence was observed among T. ni with different resistance mechanisms and on different host plants. Therefore, mechanisms of resistance and host plants available in the field are both important factors affecting development of Bt resistance in insects. PMID:26868936

  10. Effect of crop plants on fitness costs associated with resistance to Bacillus thuringiensis toxins Cry1Ac and Cry2Ab in cabbage loopers.

    Science.gov (United States)

    Wang, Ran; Tetreau, Guillaume; Wang, Ping

    2016-02-12

    Fitness costs associated with resistance to Bacillus thuringiensis (Bt) toxins critically impact the development of resistance in insect populations. In this study, the fitness costs in Trichoplusia ni strains associated with two genetically independent resistance mechanisms to Bt toxins Cry1Ac and Cry2Ab, individually and in combination, on four crop plants (cabbage, cotton, tobacco and tomato) were analyzed, in comparison with their near-isogenic susceptible strain. The net reproductive rate (R0) and intrinsic rate of increase (r) of the T. ni strains, regardless of their resistance traits, were strongly affected by the host plants. The ABCC2 gene-linked mechanism of Cry1Ac resistance was associated with relatively low fitness costs, while the Cry2Ab resistance mechanism was associated with higher fitness costs. The fitness costs in the presence of both resistance mechanisms in T. ni appeared to be non-additive. The relative fitness of Bt-resistant T. ni depended on the specific resistance mechanisms as well as host plants. In addition to difference in survivorship and fecundity, an asynchrony of adult emergence was observed among T. ni with different resistance mechanisms and on different host plants. Therefore, mechanisms of resistance and host plants available in the field are both important factors affecting development of Bt resistance in insects.

  11. Effect of gamma irradiation on nutritional components and Cry1Ab protein in the transgenic rice with a synthetic cry1Ab gene from Bacillus thuringiensis[Transgenic rice; Gamma irradiation; Nutritional components; Cry1Ab protein

    Energy Technology Data Exchange (ETDEWEB)

    Wu Dianxing E-mail: dxwu@zju.edu.cn; Ye Qingfu; Wang Zhonghua; Xia Yingwu

    2004-01-01

    The effects of gamma irradiation on the transgenic rice containing a synthetic cry1Ab gene from Bacillus thuringiensis were investigated. There was almost no difference in the content of the major nutritional components, i.e. crude protein, crude lipid, eight essential amino acids and total ash between the irradiated grains and the non-irradiated transgenic rice. However, the amounts of Cry1Ab protein and apparent amylose in the irradiated transgenic rice were reduced significantly by the doses higher than 200 Gy. In vivo observation showed that Cry1Ab protein contents also decreased in the fresh leaf tissues of survival seedlings after irradiation with 200 Gy or higher doses and showed inhibition of seedling growth. The results indicate that gamma irradiation might improve the quality of transgenic rice due to removal of the toxic Cry1Ab protein.

  12. In Silico Modeling and Functional Interpretations of Cry1Ab15 Toxin from Bacillus thuringiensis BtB-Hm-16

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    Sudhanshu Kashyap

    2013-01-01

    Full Text Available 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.

  13. Resistance to Bacillus thuringiensis toxin Cry2Ab in a strain of Helicoverpa armigera (Lepidoptera: Noctuidae) in Australia.

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    Mahon, R J; Olsen, K M; Garsia, K A; Young, S R

    2007-06-01

    Transgenic cotton, Gossypium hirsutum L., expressing the crylAc and cry2Ab genes from Bacillus thuringiensis (Bt) Berliner variety kurstaki in a pyramid (Bollgard II) was widely planted for the first time in Australia during the 2004-2005 growing season. Before the first commercial Bollgard II crops, limited amounts of cotton expressing only the crylAc gene (Ingard) was grown for seven seasons. No field failures due to resistance to CrylAc toxin were observed during that period and a monitoring program indicated that the frequency of genes conferring high level resistance to the CrylAc toxin were rare in the major pest of cotton, Helicoverpa armigera (Htibner) (Lepidoptera: Noctuidae). Before the deployment of Bollgard II, an allele conferring resistance to Cry2Ab toxin was detected in field-collected H. armigera. We established a colony (designated SP15) consisting of homozygous resistant individuals and examined their characteristics through comparison with individuals from a Bt-susceptible laboratory colony (GR). Through specific crosses and bioassays, we established that the resistance present in SP15 was due to a single autosomal gene. The resistance was recessive. Homozygotes were highly resistant to Cry2Ab toxin, so much so, that we were unable to induce significant mortality at the maximum concentration of toxin available. Homozygotes also were unaffected when fed leaves of a cotton variety expressing the cry2Ab gene. Although cross-resistant to Cry2Aa toxin, SP15 was susceptible to CrylAc and to the Bt product DiPel.

  14. Bacillus thuringiensis Cry1Ia10 and Vip3Aa protein interactions and their toxicity in Spodoptera spp. (Lepidoptera).

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    Bergamasco, V B; Mendes, D R P; Fernandes, O A; Desidério, J A; Lemos, M V F

    2013-02-01

    The polyphagous pests belonging to the genus Spodoptera are considered to be among the most important causes of damage and are widely distributed throughout the Americas'. Due to the extensive use of genetically modified plants containing Bacillus thuringiensis genes that code for insecticidal proteins, resistant insects may arise. To prevent the development of resistance, pyramided plants, which express multiple insecticidal proteins that act through distinct mode of actions, can be used. This study analyzed the mechanisms of action for the proteins Cry1Ia10 and Vip3Aa on neonatal Spodoptera frugiperda, Spodoptera albula, Spodoptera eridania and Spodoptera cosmioides larvae. The interactions of these toxins with receptors on the intestinal epithelial membrane were also analyzed by binding biotinylated toxins to brush border membrane vesicles (BBMVs) from the intestines of these insects. A putative receptor of approximately 65 kDa was found by ligand blotting in all of these species. In vitro competition assays using biotinylated proteins have indicated that Vip3Aa and Cry1Ia10 do not compete for the same receptor for S. frugiperda, S. albula and S. cosmioides and that Vip3Aa was more efficient than Cry1Ia10 when tested individually, by bioassays. A synergistic effect of the toxins in S. frugiperda, S. albula and S. cosmioides was observed when they were combined. However, in S. eridania, Cry1Ia10 and Vip3Aa might compete for the same receptor and through bioassays Cry1Ia10 was more efficient than Vip3Aa and showed an antagonistic effect when the proteins were combined. These results suggest that using these genes to develop pyramided plants may not prove effective in preventing the development of resistance in S. eridiana.

  15. Fatores de virulência de Bacillus thuringiensis: o que existe além das proteínas Cry

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    Gislayne Vilas-Bôas

    2012-03-01

    Virulence Factors of Bacillus thuringiensis Berliner: Something Beyond of Cry Proteins? Abstract. The Cry proteins produced by the entomopathogenic bacterium Bacillus thuringiensis Berliner are widely known due to its high toxicity against a variety of insects. The mode of action of these proteins is specific and becomes B. thuringiensis-based products the most used in biological control programs of insect pests in agriculture and of important human disease vectors. However, while the Cry proteins are the best-known insect-specific virulence factor, strains of B. thuringiensis show also a wide range of other virulence factors, which allow the bacteria to achieve the hemolymph and colonize efficiently the insect host. Among these factors, we highlight the Vip proteins, Cyt, enterotoxins, hemolysins, phospholipases, proteases and enzymes of degradation, in addition to the recently described parasporin. This review explores the action of these virulence factors, as well as, the characterization and control of expression of their genes. Additionally, we discuss aspects related to the ecological niche of the bacteria with emphasis on the characteristics involved in the biosafety of the use of B. thuringiensis-based products for biological control of target insects.

  16. Resistance of Trichoplusia ni to Bacillus thuringiensis toxin Cry1Ac is independent of alteration of the cadherin-like receptor for Cry toxins.

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    Xin Zhang

    Full Text Available Alteration of binding sites for Bacillus thuringiensis (Bt toxins in insect midgut is the major mechanism of high-level resistance to Bt toxins in insects. The midgut cadherin is known to be a major binding protein for Bt Cry1A toxins and linkage of Bt-resistance to cadherin gene mutations has been identified in lepidopterans. The resistance to Bt toxin Cry1Ac evolved in greenhouse populations of Trichoplusia ni has been identified to be associated with the down-regulation of an aminopeptidase N (APN1 gene by a trans-regulatory mechanism and the resistance gene has been mapped to the locus of an ABC transporter (ABCC2 gene. However, whether cadherin is also involved with Cry1Ac-resistance in T. ni requires to be understood. Here we report that the Cry1Ac-resistance in T. ni is independent of alteration of the cadherin. The T. ni cadherin cDNA was cloned and the cadherin sequence showed characteristic features known to cadherins from Lepidoptera. Various T. ni cadherin gene alleles were identified and genetic linkage analysis of the cadherin alleles with Cry1Ac-resistance showed no association of the cadherin gene with the Cry1Ac-resistance in T. ni. Analysis of cadherin transcripts showed no quantitative difference between the susceptible and Cry1Ac-resistant T. ni larvae. Quantitative proteomic analysis of midgut BBMV proteins by iTRAQ-2D-LC-MS/MS determined that there was no quantitative difference in cadherin content between the susceptible and the resistant larvae and the cadherin only accounted for 0.0014% (mol% of the midgut BBMV proteins, which is 1/300 of APN1 in molar ratio. The cadherin from both the susceptible and resistant larvae showed as a 200-kDa Cry1Ac-binding protein by toxin overlay binding analysis, and nano-LC-MS/MS analysis of the 200-kDa cadherin determined that there is no quantitative difference between the susceptible and resistant larvae. Results from this study indicate that the Cry1Ac-resistance in T. ni is

  17. Resistance of Trichoplusia ni to Bacillus thuringiensis toxin Cry1Ac is independent of alteration of the cadherin-like receptor for Cry toxins.

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    Zhang, Xin; Tiewsiri, Kasorn; Kain, Wendy; Huang, Lihua; Wang, Ping

    2012-01-01

    Alteration of binding sites for Bacillus thuringiensis (Bt) toxins in insect midgut is the major mechanism of high-level resistance to Bt toxins in insects. The midgut cadherin is known to be a major binding protein for Bt Cry1A toxins and linkage of Bt-resistance to cadherin gene mutations has been identified in lepidopterans. The resistance to Bt toxin Cry1Ac evolved in greenhouse populations of Trichoplusia ni has been identified to be associated with the down-regulation of an aminopeptidase N (APN1) gene by a trans-regulatory mechanism and the resistance gene has been mapped to the locus of an ABC transporter (ABCC2) gene. However, whether cadherin is also involved with Cry1Ac-resistance in T. ni requires to be understood. Here we report that the Cry1Ac-resistance in T. ni is independent of alteration of the cadherin. The T. ni cadherin cDNA was cloned and the cadherin sequence showed characteristic features known to cadherins from Lepidoptera. Various T. ni cadherin gene alleles were identified and genetic linkage analysis of the cadherin alleles with Cry1Ac-resistance showed no association of the cadherin gene with the Cry1Ac-resistance in T. ni. Analysis of cadherin transcripts showed no quantitative difference between the susceptible and Cry1Ac-resistant T. ni larvae. Quantitative proteomic analysis of midgut BBMV proteins by iTRAQ-2D-LC-MS/MS determined that there was no quantitative difference in cadherin content between the susceptible and the resistant larvae and the cadherin only accounted for 0.0014% (mol%) of the midgut BBMV proteins, which is 1/300 of APN1 in molar ratio. The cadherin from both the susceptible and resistant larvae showed as a 200-kDa Cry1Ac-binding protein by toxin overlay binding analysis, and nano-LC-MS/MS analysis of the 200-kDa cadherin determined that there is no quantitative difference between the susceptible and resistant larvae. Results from this study indicate that the Cry1Ac-resistance in T. ni is independent of cadherin

  18. Risk assessment of Cry toxins of Bacillus thuringiensis on the predatory mites Euseius concordis and Neoseiulus californicus (Acari: Phytoseiidae).

    Science.gov (United States)

    de Castro, Thiago Rodrigues; Ausique, John Jairo Saldarriaga; Nunes, Daiane Heloisa; Ibanhes, Fernando Henrique; Delalibera Júnior, Italo

    2013-04-01

    Genetically modified plants carrying Cry toxins of Bacillus thuringiensis (Bt) are widely used for pest control. Possible adverse effects as a result of the use of this control technique to non-target organisms is still a concern; however, few studies have addressed the effects of Bt crops on phytoseiid predatory mites. Phytoseiids are important for the natural control of phytophagous mites, but they can also feed on pollen, plant exudates, etc. Thus, phytoseiids may ingest Bt toxins through several pathways. In this paper, we evaluate the direct effect of Bt-toxins by feeding the predators on Bt cell suspensions, on solution of a Bt toxin and the tri-trophic effect by Bt expressed in transgenic plants. We present a method of conducting toxicological tests with Phytoseiidae which can be useful in studies of risk analysis of toxins to be expressed by genetically engineered plants. This method was used to evaluate the potential effect of ingestion of suspensions of Bt (1.25 × 10(8) spores/ml) and of purified protein Cry1Ia12 (0.006 mg/ml and 0.018 mg/ml) on Euseius concordis, a predatory mite that develops and reproduces best on pollen. The effects of genetically modified Bollgard(®) cotton, which carries the Cry1Ac protein, on Neoseiulus californicus, a selective predator that feeds more on spider mites than on pollen or insects, was determined by feeding them with Tetranychus urticae reared in Bollgard(®) cotton and on the non-transgenic isoline. When E. concordis was fed with suspension of Bt isolate derived from product Dipel(®) PM, no significant effects were detected. Similarly, Cry1Ia12 Bt toxin, at a concentration of 0.006 mg/ml, did not affect E. concordis. At a concentration of 0.018 mg/ml, however, the intake of this protein reduced the reproduction of E. concordis. There were no effects of Bollgard(®) cotton on the biological traits and on the predatory capacity of N. californicus. Results indicate that the Cry toxins of B. thuringiensis

  19. Hematotoxicity and genotoxicity evaluations in Swiss mice intraperitoneally exposed to Bacillus thuringiensis (var kurstaki) spore crystals genetically modified to express individually Cry1Aa, Cry1Ab, Cry1Ac, or Cry2Aa.

    Science.gov (United States)

    Mezzomo, Bélin Poletto; Miranda-Vilela, Ana Luisa; Barbosa, Lilian Carla Pereira; Albernaz, Vanessa Lima; Grisolia, Cesar Koppe

    2016-08-01

    Bacillus thuringiensis (Bt) has been widely used in foliar sprays as part of integrated pest management strategies against insect pests of agricultural crops. Since the advent of genetically modified plants expressing Bt δ-endotoxins, the bioavailability of Cry proteins has increased, and therefore for biosafety reasons their adverse effects should be studied, mainly for nontarget organisms. We evaluated, in Swiss mice, the hematotoxicity and genotoxicity of the genetically modified strains of Bt spore crystals Cry1Aa, 1Ab, 1Ac, or 2Aa at 27 mg/kg, and Cry1Aa, 1Ab and 2Aa also at 136 and 270 mg/kg, administered with a single intraperitoneal injection 24 h before euthanasia. Controls received filtered water or cyclophosphamide. Blood samples collected by cardiac puncture were used to perform hemogram, and bone marrow was extracted for the micronucleus test. Bt spore crystals presented toxicity for lymphocytes when in higher doses, which varied according to the type of spore crystal studied, besides promoting cytotoxic and genotoxic effects for the erythroid lineage of bone marrow, mainly at highest doses. Although the profile of such adverse side effects can be related to their high level of exposure, which is not commonly found in the environment, results indicated that these Bt spore crystals were not harmless to mice. This suggests that a more specific approach should be taken to increase knowledge about their toxicological properties and to establish the toxicological risks to nontarget organisms. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 970-978, 2016.

  20. A Tenebrio molitor GPI-anchored alkaline phosphatase is involved in binding of Bacillus thuringiensis Cry3Aa to brush border membrane vesicles.

    Science.gov (United States)

    Zúñiga-Navarrete, Fernando; Gómez, Isabel; Peña, Guadalupe; Bravo, Alejandra; Soberón, Mario

    2013-03-01

    Bacillus thuringiensis Cry toxins recognizes their target cells in part by the binding to glycosyl-phosphatidyl-inositol (GPI) anchored proteins such as aminopeptidase-N (APN) or alkaline phosphatases (ALP). Treatment of Tenebrio molitor brush border membrane vesicles (BBMV) with phospholipase C that cleaves out GPI-anchored proteins from the membranes, showed that GPI-anchored proteins are involved in binding of Cry3Aa toxin to BBMV. A 68 kDa GPI-anchored ALP was shown to bind Cry3Aa by toxin overlay assays. The 68 kDa GPI-anchored ALP was preferentially expressed in early instar larvae in comparison to late instar larvae. Our work shows for the first time that GPI-anchored ALP is important for Cry3Aa binding to T. molitor BBMV suggesting that the mode of action of Cry toxins is conserved in different insect orders.

  1. Comparative proteomic analysis of Aedes aegypti larval midgut after intoxication with Cry11Aa toxin from Bacillus thuringiensis.

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    Angeles Cancino-Rodezno

    Full Text Available 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.

  2. The 60-kilodalton protein encoded by orf2 in the cry19A operon of Bacillus thuringiensis subsp. jegathesan functions like a C-terminal crystallization domain.

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    Barboza-Corona, J Eleazar; Park, Hyun-Woo; Bideshi, Dennis K; Federici, Brian A

    2012-03-01

    The cry19A operon of Bacillus thuringiensis subsp. jegathesan encodes two proteins, mosquitocidal Cry19A (ORF1; 75 kDa) and an ORF2 (60 kDa) of unknown function. Expression of the cry19A operon in an acrystalliferous strain of B. thuringiensis (4Q7) yielded one small crystal per cell, whereas no crystals were produced when cry19A or orf2 was expressed alone. To determine the function of the ORF2 protein, different combinations of Cry19A, ORF2, and the N- or C-terminal half of Cry1C were synthesized in strain 4Q7. Stable crystalline inclusions of these fusion proteins similar in shape to those in the strain harboring the wild-type operon were observed in sporulating cells. Comparative analysis showed that ORF2 shares considerable amino acid sequence identity with the C-terminal region of large Cry proteins. Together, these results suggest that ORF2 assists in synthesis and crystallization of Cry19A by functioning like the C-terminal domain characteristic of Cry protein in the 130-kDa mass range. In addition, to determine whether overexpression of the cry19A operon stabilized its shape and increased Cry19A yield, it was expressed under the control of the strong chimeric cyt1A-p/STAB-SD promoter. Interestingly, in contrast to the expression seen with the native promoter, overexpression of the operon yielded uniform bipyramidal crystals that were 4-fold larger on average than the wild-type crystal. In bioassays using the 4th instar larvae of Culex quinquefasciatus, the strain producing the larger Cry19A crystal showed moderate larvicidal activity that was 4-fold (95% lethal concentration [LC(95)] = 1.9 μg/ml) more toxic than the activity produced in the strain harboring the wild-type operon (LC(95) = 8.2 μg/ml).

  3. Construction of occluded recombinant baculoviruses containing the full-length cry1Ab and cry1Ac genes from Bacillus thuringiensis

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    B.M. Ribeiro

    1998-06-01

    Full Text Available The administration of baculoviruses to insects for bioassay purposes is carried out, in most cases, by contamination of food surfaces with a known amount of occlusion bodies (OBs. Since per os infection is the natural route of infection, occluded recombinant viruses containing crystal protein genes (cry1Ab and cry1Ac from Bacillus thuringiensis were constructed for comparison with the baculovirus prototype Autographa californica nucleopolyhedrovirus (AcNPV. The transfer vector pAcUW2B was used for construction of occluded recombinant viruses. The transfer vector containing the crystal protein genes was cotransfected with linearized DNA from a non-occluded recombinant virus. The isolation of recombinant viruses was greatly facilitated by the reduction of background "wild type" virus and the increased proportion of recombinant viruses. Since the recombinant viruses containing full-length and truncated forms of the crystal protein genes did not seem to improve the pathogenicity of the recombinant viruses when compared with the wild type AcNPV, and in order to compare expression levels of the full-length crystal proteins produced by non-occluded and occluded recombinant viruses the full-length cry1Ab and cry1Ac genes were chosen for construction of occluded recombinant viruses. The recombinant viruses containing full-length and truncated forms of the crystal protein genes did not seem to improve its pathogenicity but the size of the larvae infected with the recombinant viruses was significantly smaller than that of larvae infected with the wild type virus.

  4. Fatores de virulência de Bacillus thuringiensis: o que existe além das proteínas Cry

    OpenAIRE

    Gislayne Vilas-Bôas; Rita Alvarez; Clelton dos Santos; Laurival Vilas-Boas

    2012-01-01

    As proteínas Cry produzidas pela bactéria entomopatogênica Bacillus thuringiensis Berliner são bem conhecidas devido a alta citotoxicidade que exibem a uma variedade de insetos-alvo. O modo de ação destas proteínas é específico e torna os produtos à base de B. thuringiensis os mais amplamente utilizados em programas de controle biológico de pragas na agricultura e de importantes vetores de doenças humanas. Cont...

  5. Activity of spores and extracellular proteins from six Cry+ strains and a Cry- strain of Bacillus thuringiensis subsp. kurstaki against the western spruce budworm, Choristoneura occidentalis (Lepidoptera: Tortricidae).

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    Kalmykova, Galina; Burtseva, Ljudmila; Milne, Ross; van Frankenhuyzen, Kees

    2009-05-01

    We characterized insecticidal activity of previously untested strains of Bacillus thuringiensis kurstaki belonging to two crystal serovars (K-1 and K-73) against the western spruce budworm (Choristoneura occidentalis Freeman 1967). By testing various components, we demonstrated that spores play a critical role in the pathogenesis of each strain. Spore-free crystals caused low mortality and purified spores were generally not toxic. The addition of spores to purified protoxin increased toxicity several hundred-fold, regardless of the parental strain from which the spores or protoxins were derived. The crystal and spore components did not account for full insecticidal activity of whole sporulated cultures owing to the toxicity of soluble proteins that are secreted during cell growth. We observed a marked difference in toxicity of secreted proteins between the K-1 and K-73 type strains, with the K-1 preparations causing much higher mortality, mass reduction, and inhibition of pupation. There was a consistent correlation between relative toxicity of secreted protein preparations and the presence and quantity of the Vip3A protein, suggesting that this protein contributes to the virulence of B. thuringiensis subsp. kurstaki in western spruce budworm larvae. However, other virulence factors have to be invoked to explain the synergizing effect of spores from both K-1 and K-73 strains on Cry protein toxicity.

  6. Agrotis segetum midgut putative receptor of Bacillus thuringiensis vegetative insecticidal protein Vip3Aa16 differs from that of Cry1Ac toxin

    OpenAIRE

    Ben Hamadou-Charfi, Dorra; Boukedi, Hanen; Abdelkefi-Mesrati, Lobna; Tounsi, Slim; Jaoua, Samir

    2013-01-01

    Considering the fact that Agrotis segetum is one of the most pathogenic insects to vegetables and cereals in the world, particularly in Africa, the mode of action of Vip3Aa16 of Bacillus thuringiensis BUPM95 and Cry1Ac of the recombinant strain BNS3Cry-(pHTcry1Ac) has been examined in this crop pest. A. segetum proteases activated the Vip3Aa16 protoxin (90kDa) yielding three bands of about 62, 45, 22kDa and the activated form of the toxin was active against this pest with an LC50 of about 86n...

  7. Coexpression of the silent cry2Ab27 together with cry1 genes in Bacillus thuringiensis subsp. aizawai SP41 leads to formation of amorphous crystal toxin and enhanced toxicity against Helicoverpa armigera.

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    Somwatcharajit, Rasapirose; Tiantad, Itsares; Panbangred, Watanalai

    2014-02-01

    The unexpressed cry2Ab27 gene of Bacillus thuringiensis subsp. aizawai SP41 (SP41) consists of a single open reading frame (ORF) of 1902bp encoding for 634 amino acid residues. The cry2Ab27 gene appears to be silent due to the lack of promoter and terminator sequences. In this study we fused the cry2Ab27 ORF with the cry1Ab promoter (500bp) and the terminator (300bp) in vector pHT304-18Z in order to drive the expression of cry2Ab27 in both SP41 and an acrystaliferous, B. thuringiensis subsp. thuringiensis 407 (407). A protein with a molecular mass of 65kDa, consistent with the Cry2Ab protein, was detected in both transformants using SDS-PAGE and Western blot analysis. Bipyramidal crystals were observed in SP41 and its transformant containing the pHT304-18Z vector (SPHT) in contrast, cells expressing cry2Ab27 (SPC2) exhibited crystal proteins with irregular shapes. No inclusion protein was detected in the 407 transformant expressing the cry2Ab27 gene. Cry2Ab27 was found in the purified crystal toxin from strain SPC2. The solubilized crystal toxin proteins from SPC2 were 6.9-fold more toxic toward the larvae of Helicoverpa armigera compared to toxin proteins from SPHT. However SPC2 crystal toxin displayed only slightly higher toxicity against the larvae of Spodoptera litura and S. exigua compared to SPHT produced toxin. Our data support the use of Cry2Ab in combination with the Cry1 toxin for enhanced control of heliothine insect pests.

  8. 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.

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    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.

  9. Leucine transport is affected by Bacillus thuringiensis Cry1 toxins in brush border membrane vesicles from Ostrinia nubilalis Hb (Lepidoptera: Pyralidae) and Sesamia nonagrioides Lefebvre (Lepidoptera: Noctuidae) midgut.

    Science.gov (United States)

    Leonardi, M Giovanna; Caccia, Silvia; González-Cabrera, Joel; Ferré, Juan; Giordana, Barbara

    2006-01-01

    The pore-forming activity of Cry1Ab, Cry1Fa and Cry1Ca toxins and their interaction with leucine transport mediated by the K(+)/leucine cotransporter were studied in brush border membrane vesicles (BBMVs) isolated from the midgut of Ostrinia nubilalis and Sesamia nonagrioides. In both species, as in other Lepidoptera, leucine uptake by BBMVs can take place in the absence of cations, but it can also be driven by a K(+) gradient. Experiments with the voltage-sensitive fluorescent dye 3,3'-diethylthiacarbocyanine iodide proved that Cry1Ab, a Bacillus thuringiensis toxin active in vivo, enhanced the membrane permeability to potassium in O. nubilalis BBMVs. This result is in agreement with similar effects observed in S. nonagrioides BBMV incubated with various Cry1 toxins active in vivo. The effect of the above toxins was tested on the initial rate of 0.1 mM: leucine influx. Instead of an increase in leucine influx, a reduction was observed with the Cry1 toxins active in vivo. Cry1Ab and Cry1Fa, but not the inactive toxin Cry1Da, inhibited in a dose-dependent manner leucine uptake both in the absence and in the presence of a K(+) gradient, a clear indication that their effect is independent of the channel formed by the toxins and that this effect is exerted directly on the amino acid transport system.

  10. Binding site alteration is responsible for field-isolated resistance to Bacillus thuringiensis Cry2A insecticidal proteins in two Helicoverpa species.

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    Silvia Caccia

    Full Text Available BACKGROUND: Evolution of resistance by target pests is the main threat to the long-term efficacy of crops expressing Bacillus thuringiensis (Bt insecticidal proteins. Cry2 proteins play a pivotal role in current Bt spray formulations and transgenic crops and they complement Cry1A proteins because of their different mode of action. Their presence is critical in the control of those lepidopteran species, such as Helicoverpa spp., which are not highly susceptible to Cry1A proteins. In Australia, a transgenic variety of cotton expressing Cry1Ac and Cry2Ab (Bollgard II comprises at least 80% of the total cotton area. Prior to the widespread adoption of Bollgard II, the frequency of alleles conferring resistance to Cry2Ab in field populations of Helicoverpa armigera and Helicoverpa punctigera was significantly higher than anticipated. Colonies established from survivors of F(2 screens against Cry2Ab are highly resistant to this toxin, but susceptible to Cry1Ac. METHODOLOGY/PRINCIPAL FINDINGS: Bioassays performed with surface-treated artificial diet on neonates of H. armigera and H. punctigera showed that Cry2Ab resistant insects were cross-resistant to Cry2Ae while susceptible to Cry1Ab. Binding analyses with (125I-labeled Cry2Ab were performed with brush border membrane vesicles from midguts of Cry2Ab susceptible and resistant insects. The results of the binding analyses correlated with bioassay data and demonstrated that resistant insects exhibited greatly reduced binding of Cry2Ab toxin to midgut receptors, whereas no change in (125I-labeled-Cry1Ac binding was detected. As previously demonstrated for H. armigera, Cry2Ab binding sites in H. punctigera were shown to be shared by Cry2Ae, which explains why an alteration of the shared binding site would lead to cross-resistance between the two Cry2A toxins. CONCLUSION/SIGNIFICANCE: This is the first time that a mechanism of resistance to the Cry2 class of insecticidal proteins has been reported

  11. Proteínas Cry1 e Vip3A de Bacillus thuringiensis: sinergismo e efeito sub-letal no controle de Heliothis virescens

    OpenAIRE

    Lemes, Ana Rita Nunes [UNESP

    2012-01-01

    A bactéria Bacillus thuringiensis (Bt) possui a capacidade de produzir inclusões protéicas (proteína Cry) e proteínas vegetativas (Vip). Estas proteínas podem ser tóxicas para insetos e por meio de transgenia, a expressão em plantas, podem também proporcionar controle de importantes pragas agrícolas. Nesse sentido, esta pesquisa teve por objetivo avaliar o potencial de controle das proteínas Cry1Aa, Cry1Ac, Cry1Ca, Vip3A(1), Vip3A(2) e Vip3A(3) em uma população brasileira da lagarta-da-maçã, ...

  12. Lack of detrimental effects of Bacillus thuringiensis Cry toxins on the insect predator Chrysoperla carnea: a toxicological, histopathological, and biochemical analysis.

    Science.gov (United States)

    Rodrigo-Simón, Ana; de Maagd, Ruud A; Avilla, Carlos; Bakker, Petra L; Molthoff, Jos; González-Zamora, Jose E; Ferré, Juan

    2006-02-01

    The effect of Cry proteins of Bacillus thuringiensis on the green lacewing (Chrysoperla carnea) was studied by using a holistic approach which consisted of independent, complementary experimental strategies. Tritrophic experiments were performed, in which lacewing larvae were fed Helicoverpa armigera larvae reared on Cry1Ac, Cry1Ab, or Cry2Ab toxins. In complementary experiments, a predetermined amount of purified Cry1Ac was directly fed to lacewing larvae. In both experiments no effects on prey utilization or fitness parameters were found. Since binding to the midgut is an indispensable step for toxicity of Cry proteins to known target insects, we hypothesized that specific binding of the Cry1A proteins should be found if the proteins were toxic to the green lacewing. In control experiments, Cry1Ac was detected bound to the midgut epithelium of intoxicated H. armigera larvae, and cell damage was observed. However, no binding or histopathological effects of the toxin were found in tissue sections of lacewing larvae. Similarly, Cry1Ab or Cry1Ac bound in a specific manner to brush border membrane vesicles from Spodoptera exigua but not to similar fractions from green lacewing larvae. The in vivo and in vitro binding results strongly suggest that the lacewing larval midgut lacks specific receptors for Cry1Ab or Cry1Ac. These results agree with those obtained in bioassays, and we concluded that the Cry toxins tested, even at concentrations higher than those expected in real-life situations, do not have a detrimental effect on the green lacewing when they are ingested either directly or through the prey.

  13. Toxicity of Cry1A toxins from Bacillus thuringiensis to CF1 cells does not involve activation of adenylate cyclase/PKA signaling pathway.

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    Portugal, Leivi; Muñóz-Garay, Carlos; Martínez de Castro, Diana L; Soberón, Mario; Bravo, Alejandra

    2017-01-01

    Bacillus thuringiensis (Bt) bacteria produce Cry toxins that are able to kill insect pests. Different models explaining the mode of action of these toxins have been proposed. The pore formation model proposes that the toxin creates pores in the membrane of the larval midgut cells after interaction with different receptors such as cadherin, aminopeptidase N and alkaline phosphatase and that this pore formation activity is responsible for the toxicity of these proteins. The alternative model proposes that interaction with cadherin receptor triggers an intracellular cascade response involving protein G, adenylate cyclase (AC) and protein kinase A (PKA). In addition, it was shown that Cry toxins induce a defense response in the larvae involving the activation of mitogen-activated kinases such as MAPK p38 in different insect orders. Here we analyzed the mechanism of action of Cry1Ab and Cry1Ac toxins and a collection of mutants from these toxins in the insect cell line CF1 from Choristoneura fumiferana, that is naturally sensitive to these toxins. Our results show that both toxins induced permeability of K(+) ions into the cells. The initial response after intoxication with Cry1Ab and Cry1Ac toxins involves the activation of a defense response that involves the phosphorylation of MAPK p38. Analysis of activation of PKA and AC activities indicated that the signal transduction involving PKA, AC and cAMP was not activated during Cry1Ab or Cry1Ac intoxication. In contrast we show that Cry1Ab and Cry1Ac activate apoptosis. These data indicate that Cry toxins can induce an apoptotic death response not related with AC/PKA activation. Since Cry1Ab and Cry1Ac toxins affected K(+) ion permeability into the cells, and that mutant toxins affected in pore formation are not toxic to CF1, we propose that pore formation activity of the toxins is responsible of triggering cell death response in CF1cells.

  14. Genetic Basis of Cry1F-Resistance in a Laboratory Selected Asian Corn Borer Strain and Its Cross-Resistance to Other Bacillus thuringiensis Toxins

    Science.gov (United States)

    Wang, Yueqin; Wang, Yidong; Wang, Zhenying; Bravo, Alejandra; Soberón, Mario; He, Kanglai

    2016-01-01

    The Asian corn borer (ACB), Ostrinia furnacalis (Guenée) (Lepidoptera: Crambidae), is the most destructive insect pest of corn in China. Susceptibility to the Cry1F toxin derived from Bacillus thuringiensis has been demonstrated for ACB, suggesting the potential for Cry1F inclusion as part of an insect pest management program. Insects can develop resistance to Cry toxins, which threatens the development and use of Bt formulations and Bt crops in the field. To determine possible resistance mechanisms to Cry1F, a Cry1F-resistant colony of ACB (ACB-FR) that exhibited more than 1700-fold resistance was established through selection experiments after 49 generations of selection under laboratory conditions. The ACB-FR strain showed moderate cross-resistance to Cry1Ab and Cry1Ac of 22.8- and 26.9-fold, respectively, marginally cross-resistance to Cry1Ah (3.7-fold), and no cross-resistance to Cry1Ie (0.6-fold). The bioassay responses of progeny from reciprocal F1 crosses to different Cry1 toxin concentrations indicated that the resistance trait to Cry1Ab, Cry1Ac and Cry1F has autosomal inheritance with no maternal effect or sex linked. The effective dominance (h) of F1 offspring was calculated at different concentrations of Cry1F, showing that h decreased as concentration of Cry1F increased. Finally, the analysis of actual and expected mortality of the progeny from a backcross (F1 × resistant strain) indicated that the inheritance of the resistance to Cry1F in ACB-FR was due to more than one locus. The present study provides an understanding of the genetic basis of Cry1F resistance in ACB-FR and also shows that pyramiding Cry1F with Cry1Ah or Cry1Ie could be used as a strategy to delay the development of ACB resistance to Bt proteins. PMID:27518813

  15. Host-Pathogen Coevolution: The Selective Advantage of Bacillus thuringiensis Virulence and Its Cry Toxin Genes.

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    Leila Masri

    2015-06-01

    Full Text Available 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.

  16. Distribuição de genes cry de Bacillus thuringiensis isolados de solos do Estado do Rio Grande do Sul, Brasil Distribution of cry genes of Bacillus thuringiensis isolated from soils of the State of Rio Grande do Sul, Brazil

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    Laura Massochin Nunes Pinto

    2003-08-01

    Full Text Available A bactéria Bacillus thuringiensis (Bt caracteriza-se pela produção de proteínas tóxicas a representantes de diversas ordens de insetos, as quais são codificadas por genes cry. Devido a esta característica, mais de 40.000 cepas de Bt foram isoladas e cerca de 190 genes cry, caracterizados. Como os dados sobre Bt são limitados no Rio Grande do Sul, essa pesquisa objetivou avaliar a distribuição de seis famílias de genes cry de Bt, desse estado, que codificam proteínas ativas contra insetos-praga. O perfil dos 46 isolados de solos do Rio Grande do Sul foi avaliado, por PCR com os primers que detectam os genes cry1, cry2, cry3, cry7 cry8 e cry9 e suas respectivas proteínas foram analisadas por SDS-PAGE a 10%. A presença de genes cry9 foi detectada em 47,82% dos isolados, seguido de cry3 (15,21%, cry1 e cry7 (ambos com 6,52% e cry2 (2,17%. Oito perfis genéticos foram identificados, sendo o perfil cry9 (39,13% o mais freqüente. A análise protéica de Bt identificou 14 famílias de proteínas Cry possivelmente codificadas por genes presentes nos isolados, além de proteínas desconhecidas que podem caracterizar novos genes cry. Esses isolados revelam a presença de genes que codificam proteínas específicas contra lepidópteros e coleópteros, as quais poderão ser avaliadas quanto à toxicidade in vivo contra insetos-praga das plantas cultivadas.The Bacillus thuringiensis (Bt bacterium is characterized by the production of toxic protein to representatives of several insect orders, which are coded by cry genes. Due to this characteristic, more than 40.000 Bt strains were isolated and around 190 cry genes characterized. As the data on Bt are limited in Rio Grande do Sul, this research aimed the evaluation of the distribution of six Bt families of cry genes, in this state, that codify active proteins against insect-pest. The 46 isolates profiles of soil samples from Rio Grande do Sul were evaluated, by PCR with primers that detect cry1

  17. Identification of Bacillus thuringiensis Cry3Aa toxin domain II loop 1 as the binding site of Tenebrio molitor cadherin repeat CR12.

    Science.gov (United States)

    Zúñiga-Navarrete, Fernando; Gómez, Isabel; Peña, Guadalupe; Amaro, Itzel; Ortíz, Ernesto; Becerril, Baltazar; Ibarra, Jorge E; Bravo, Alejandra; Soberón, Mario

    2015-04-01

    Bacillus thuringiensis Cry toxins exert their toxic effect by specific recognition of larval midgut proteins leading to oligomerization of the toxin, membrane insertion and pore formation. The exposed domain II loop regions of Cry toxins have been shown to be involved in receptor binding. Insect cadherins have shown to be functionally involved in toxin binding facilitating toxin oligomerization. Here, we isolated a VHH (VHHA5) antibody by phage display that binds Cry3Aa loop 1 and competed with the binding of Cry3Aa to Tenebrio molitor brush border membranes. VHHA5 also competed with the binding of Cry3Aa to a cadherin fragment (CR12) that was previously shown to be involved in binding and toxicity of Cry3Aa, indicating that Cry3Aa binds CR12 through domain II loop 1. Moreover, we show that a loop 1 mutant, previously characterized to have increased toxicity to T. molitor, displayed a correlative enhanced binding affinity to T. molitor CR12 and to VHHA5. These results show that Cry3Aa domain II loop 1 is a binding site of CR12 T. molitor cadherin.

  18. Genetic Variability of Spodoptera frugiperda Smith (Lepidoptera: Noctuidae) Populations from Latin America Is Associated with Variations in Susceptibility to Bacillus thuringiensis Cry Toxins▿

    Science.gov (United States)

    Monnerat, Rose ; Martins, Erica; Queiroz, Paulo; Ordúz, Sergio; Jaramillo, Gabriela; Benintende, Graciela ; Cozzi, Jorge; Real, M. Dolores; Martinez-Ramirez, Amparo; Rausell, Carolina ; Cerón, Jairo; Ibarra, Jorge E.; Del Rincon-Castro, M. Cristina; Espinoza, Ana M. ; Meza-Basso, Luis; Cabrera, Lizbeth; Sánchez, Jorge; Soberon, Mario ; Bravo, Alejandra

    2006-01-01

    Bacillus thuringiensis strains isolated from Latin American soil samples that showed toxicity against three Spodoptera frugiperda populations from different geographical areas (Mexico, Colombia, and Brazil) were characterized on the basis of their insecticidal activity, crystal morphology, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of parasporal crystals, plasmid profiles, and cry gene content. We found that the different S. frugiperda populations display different susceptibilities to the selected B. thuringiensis strains and also to pure preparations of Cry1B, Cry1C, and Cry1D toxins. Binding assays performed with pure toxin demonstrated that the differences in the toxin binding capacities of these insect populations correlated with the observed differences in susceptibility to the three Cry toxins analyzed. Finally, the genetic variability of the three insect populations was analyzed by random amplification of polymorphic DNA-PCR, which showed significant genetic diversity among the three S. frugiperda populations analyzed. The data presented here show that the genetic variability of S. frugiperda populations should be carefully considered in the development of insect pest control strategies, including the deployment of genetically modified maize in different geographical regions. PMID:16936049

  19. Genetic variability of Spodoptera frugiperda Smith (Lepidoptera: Noctuidae) populations from Latin America is associated with variations in susceptibility to Bacillus thuringiensis cry toxins.

    Science.gov (United States)

    Monnerat, Rose; Martins, Erica; Queiroz, Paulo; Ordúz, Sergio; Jaramillo, Gabriela; Benintende, Graciela; Cozzi, Jorge; Real, M Dolores; Martinez-Ramirez, Amparo; Rausell, Carolina; Cerón, Jairo; Ibarra, Jorge E; Del Rincon-Castro, M Cristina; Espinoza, Ana M; Meza-Basso, Luis; Cabrera, Lizbeth; Sánchez, Jorge; Soberon, Mario; Bravo, Alejandra

    2006-11-01

    Bacillus thuringiensis strains isolated from Latin American soil samples that showed toxicity against three Spodoptera frugiperda populations from different geographical areas (Mexico, Colombia, and Brazil) were characterized on the basis of their insecticidal activity, crystal morphology, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of parasporal crystals, plasmid profiles, and cry gene content. We found that the different S. frugiperda populations display different susceptibilities to the selected B. thuringiensis strains and also to pure preparations of Cry1B, Cry1C, and Cry1D toxins. Binding assays performed with pure toxin demonstrated that the differences in the toxin binding capacities of these insect populations correlated with the observed differences in susceptibility to the three Cry toxins analyzed. Finally, the genetic variability of the three insect populations was analyzed by random amplification of polymorphic DNA-PCR, which showed significant genetic diversity among the three S. frugiperda populations analyzed. The data presented here show that the genetic variability of S. frugiperda populations should be carefully considered in the development of insect pest control strategies, including the deployment of genetically modified maize in different geographical regions.

  20. RNAi-mediated knockdown of a Spodoptera frugiperda trypsin-like serine-protease gene reduces susceptibility to a Bacillus thuringiensis Cry1Ca1 protoxin.

    Science.gov (United States)

    Rodríguez-Cabrera, Lianet; Trujillo-Bacallao, Damian; Borrás-Hidalgo, Orlando; Wright, Denis J; Ayra-Pardo, Camilo

    2010-11-01

    SfT6 has been identified in a subtracted cDNA library of Spodoptera frugiperda larval midgut transcripts as a serine-protease gene downregulated within 24 h of intoxication with Bacillus thuringiensis Cry1Ca1 protein. In the present study, the specific role of SfT6 during Cry1Ca1 intoxication was investigated by RT-PCR and in vivo RNA interference. Quantitative real-time RT-PCR analysis showed SfT6 mRNA levels in the midgut tissue were significantly reduced after injecting or feeding 4th-instar larvae with specific long-size dsRNA. Gut juice-mediated in vitro protoxin activation and susceptibility for Cry1Ca1 were investigated in Sft6-knockdown larvae and compared with control treated with nonspecific dsRNA. Our results demonstrate SfT6 plays a determinant role in Cry1Ca1 toxicity against S. frugiperda since a decreased expression caused a reduced protoxin activation by larval gut juice and reduced susceptibility of insects to toxin in bioassays. We propose SfT6 downregulation occurring at the early stages of Cry1Ca1 intoxication is part of a complex and multifaceted defensive mechanism triggered in the insect gut to withstand B. thuringiensis pathogenesis.

  1. The Correlation of the Presence and Expression Levels of cry Genes with the Insecticidal Activities against Plutella xylostella for Bacillus thuringiensis Strains

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    Ming-Lun Chen

    2014-08-01

    Full Text Available The use of Bacillus thuringiensis (Bt strains with high insecticidal activity is essential for the preparation of bioinsecticide. In this study, for 60 Bt strains isolated in Taiwan, their genotypes and the correlation of some cry genes as well as the expression levels of cry1 genes, with their insecticidal activities against Plutella xylostella, were investigated. Pulsed field gel electrophoresis (PFGE and random amplified polymorphic DNA (RAPD results revealed that the genotypes of these Bt strains are highly diversified. Also, a considerable number of the Bt strains isolated in Taiwan were found to have high insecticidal activities. Since strains that showed individual combined patterns of PFGE and RAPD exhibited distinct insecticidal activities against P. xylostella, thus, these genotypes may be useful for the identification of the new Bt strains and those which have been used in bioinsecticides. In addition, although the presence of cry2Aa1 may have a greater effect on the insecticidal activity of Bt strains in bioassay than other cry genes, only high expression level of cry1 genes plays a key role to determine the insecticidal activity of Bt strains. In conclusion, both RAPD and PFGE are effective in the differentiation of Bt strains. The presence of cry2Aa1 and, especially, the expression level of cry1 genes are useful for the prediction of the insecticidal activities of Bt strains against P. xylostella.

  2. Involvement of the processing step in the susceptibility/tolerance of two lepidopteran larvae to Bacillus thuringiensis Cry1Aa toxin.

    Science.gov (United States)

    Dammak, Mariam; Khedher, Saoussen Ben; Boukedi, Hanen; Chaib, Ikbel; Laarif, Asma; Tounsi, Slim

    2016-02-01

    Bacillus thuringiensis (Bt) Cry1A toxins are known for their effectiveness against lepidopteran insects. In this study, the entomopathogenic activity of Cry1Aa was investigated against two lepidopteran larvae causing serious threat to various crops, Spodoptera littoralis and Tuta absoluta. Contrarily to S. littoralis, which showed low susceptibility to Cry1Aa (40% mortality with 1μg/cm(2)), T. absoluta was very sensitive to this delta-endotoxin (LC50 of 95.8ng/cm(2)). The different steps in the mode of action of this toxin on the two larvae were studied with the aim to understand the origin of their difference of susceptibility. Activation of the 130kDa Cry1Aa protein by T. absoluta larvae juice generated a 65kDa active toxin, whereas S. littoralis gut juice led to a complete degradation of the protoxin. The study of the interaction of the brush border membrane vesicles (BBMV) with purified biotinylated Cry1Aa toxin revealed six and seven toxin binding proteins in T. absoluta and S. littoralis BBMV, respectively. Midgut histopathology of Cry1Aa fed larvae demonstrated approximately similar damage caused by the toxin in the two larvae midguts. These results suggest that the activation step was strongly involved in the difference of susceptibility of the two larvae to Cry1Aa.

  3. The correlation of the presence and expression levels of cry genes with the insecticidal activities against Plutella xylostella for Bacillus thuringiensis strains.

    Science.gov (United States)

    Chen, Ming-Lun; Chen, Pin-Hsin; Pang, Jen-Chieh; Lin, Chia-Wei; Hwang, Chin-Fa; Tsen, Hau-Yang

    2014-08-19

    The use of Bacillus thuringiensis (Bt) strains with high insecticidal activity is essential for the preparation of bioinsecticide. In this study, for 60 Bt strains isolated in Taiwan, their genotypes and the correlation of some cry genes as well as the expression levels of cry1 genes, with their insecticidal activities against Plutella xylostella, were investigated. Pulsed field gel electrophoresis (PFGE) and random amplified polymorphic DNA (RAPD) results revealed that the genotypes of these Bt strains are highly diversified. Also, a considerable number of the Bt strains isolated in Taiwan were found to have high insecticidal activities. Since strains that showed individual combined patterns of PFGE and RAPD exhibited distinct insecticidal activities against P. xylostella, thus, these genotypes may be useful for the identification of the new Bt strains and those which have been used in bioinsecticides. In addition, although the presence of cry2Aa1 may have a greater effect on the insecticidal activity of Bt strains in bioassay than other cry genes, only high expression level of cry1 genes plays a key role to determine the insecticidal activity of Bt strains. In conclusion, both RAPD and PFGE are effective in the differentiation of Bt strains. The presence of cry2Aa1 and, especially, the expression level of cry1 genes are useful for the prediction of the insecticidal activities of Bt strains against P. xylostella.

  4. Single-reversal charge in the β10-β11 receptor-binding loop of Bacillus thuringiensis Cry4Aa and Cry4Ba toxins reflects their different toxicity against Culex spp. larvae.

    Science.gov (United States)

    Visitsattapongse, Sarinporn; Sakdee, Somsri; Leetacheewa, Somphob; Angsuthanasombat, Chanan

    2014-07-25

    Bacillus thuringiensis Cry4Aa toxin was previously shown to be much more toxic to Culex mosquito-larvae than its closely related toxin - Cry4Ba, conceivably due to their sequence differences within the β10-β11 receptor-binding loop. Here, single-Ala substitutions of five residues (Pro(510), Thr(512), Tyr(513), Lys(514) and Thr(515)) within the Cry4Aa β10-β11 loop revealed that only Lys(514) corresponding to the relative position of Cry4Ba-Asp(454) is crucial for toxicity against Culex quinquefasciatus larvae. Interestingly, charge-reversal mutations at Cry4Ba-Asp(454) (D454R and D454K) revealed a marked increase in toxicity against such less-susceptible larvae. In situ binding analyses revealed that both Cry4Ba-D454R and D454K mutants exhibited a significant increase in binding to apical microvilli of Culex larval midguts, albeit at lower-binding activity when compared with Cry4Aa. Altogether, our present data suggest that a positively charged side-chain near the tip of the β10-β11 loop plays a critical role in determining target specificity of Cry4Aa against Culex spp., and hence a great increase in the Culex larval toxicity of Cry4Ba was obtained toward an opposite-charge conversion of the corresponding Asp(454).

  5. Composition of the Putative Prepore Complex of Bacillus thuringiensis Cry1Ab Toxin

    Science.gov (United States)

    Nair, Manoj S.; Dean, Donald H.

    2015-01-01

    Prepore formation is hypothesized to be an obligate step in the insertion of Cry1Ab toxin into insect brush border membrane vesicles. We examined the architecture of the putative prepore when isolated using the published protocols [1] [2]. Our results demonstrate that the putative prepore form of Cry1Ab is a combination of receptor proteins attached to the toxin, when purified. The results also suggest that this prepore form as prepared by the methods published is different from other membrane-extracted oligomeric forms of Cry toxins and prepore of other toxins in general. While most other known prepores are composed of multimers of a single protein, the Cry1Ab prepore, as generated, is a protein-receptor complex oligomer and monomers of Cry toxins. PMID:26702367

  6. Peptide isolated from Cry1Ab16 toxin present in Bacillus thuringiensis: Synthesis and morphology data for layer-by-layer films studied by atomic force microscopy

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    Alexandra Plácido

    2016-09-01

    Full Text Available The peptide PcL342-354C was obtained from the Cry1Ab16 toxin present in Bacillus thuringiensis (“Computational Modeling Deduced Three Dimensional Structure of Cry1Ab16 Toxin from B. thuringiensis AC11” (Kashyap, 2012 [1]. In this data article, we report the synthesis and characterization of the PcL342-354C peptide by MALDI-TOF/TOF mass spectrometry. In addition, the preparation of layer-by-layer films is shown based on interspersion of this peptide with both polyethylenimine (PEI and poly(sodium 4-styrenesulfonate (PSS, self-assembled on ITO (indium tin oxide electrodes. The morphology of the ITO/PEI/PSS/PcL342-354C film was analyzed using atomic force microscopy (AFM. We also evaluated the effect of the number of bilayers in ITO/PEI/(PSS/PcL342-354Cn on the morphology of the film using AFM amplitude images. Further details about this study were published elsewhere, “Layer-by-layer films containing peptides of the Cry1Ab16 toxin from B. thuringiensis for potential biotechnological applications,” (Plácido et al., 2016 [2].

  7. Peptide isolated from Cry1Ab16 toxin present in Bacillus thuringiensis: Synthesis and morphology data for layer-by-layer films studied by atomic force microscopy.

    Science.gov (United States)

    Plácido, Alexandra; de Oliveira Farias, Emanuel Airton; Marani, Mariela M; Gomes Vasconcelos, Andreanne; Leite, José R S A; Delerue-Matos, Cristina

    2016-09-01

    The peptide PcL342-354C was obtained from the Cry1Ab16 toxin present in Bacillus thuringiensis ("Computational Modeling Deduced Three Dimensional Structure of Cry1Ab16 Toxin from B. thuringiensis AC11" (Kashyap, 2012) [1]). In this data article, we report the synthesis and characterization of the PcL342-354C peptide by MALDI-TOF/TOF mass spectrometry. In addition, the preparation of layer-by-layer films is shown based on interspersion of this peptide with both polyethylenimine (PEI) and poly(sodium 4-styrenesulfonate) (PSS), self-assembled on ITO (indium tin oxide) electrodes. The morphology of the ITO/PEI/PSS/PcL342-354C film was analyzed using atomic force microscopy (AFM). We also evaluated the effect of the number of bilayers in ITO/PEI/(PSS/PcL342-354C) n on the morphology of the film using AFM amplitude images. Further details about this study were published elsewhere, "Layer-by-layer films containing peptides of the Cry1Ab16 toxin from B. thuringiensis for potential biotechnological applications," (Plácido et al., 2016) [2].

  8. Synergism and Antagonism between Bacillus thuringiensis Vip3A and Cry1 Proteins in Heliothis virescens, Diatraea saccharalis and Spodoptera frugiperda

    Science.gov (United States)

    Lemes, Ana Rita Nunes; Davolos, Camila Chiaradia; Legori, Paula Cristina Brunini Crialesi; Fernandes, Odair Aparecido; Ferré, Juan; Lemos, Manoel Victor Franco; Desiderio, Janete Apparecida

    2014-01-01

    Second generation Bt crops (insect resistant crops carrying Bacillus thuringiensis genes) combine more than one gene that codes for insecticidal proteins in the same plant to provide better control of agricultural pests. Some of the new combinations involve co-expression of cry and vip genes. Because Cry and Vip proteins have different midgut targets and possibly different mechanisms of toxicity, it is important to evaluate possible synergistic or antagonistic interactions between these two classes of toxins. Three members of the Cry1 class of proteins and three from the Vip3A class were tested against Heliothis virescens for possible interactions. At the level of LC50, Cry1Ac was the most active protein, whereas the rest of proteins tested were similarly active. However, at the level of LC90, Cry1Aa and Cry1Ca were the least active proteins, and Cry1Ac and Vip3A proteins were not significantly different. Under the experimental conditions used in this study, we found an antagonistic effect of Cry1Ca with the three Vip3A proteins. The interaction between Cry1Ca and Vip3Aa was also tested on two other species of Lepidoptera. Whereas antagonism was observed in Spodoptera frugiperda, synergism was found in Diatraea saccharalis. In all cases, the interaction between Vip3A and Cry1 proteins was more evident at the LC90 level than at the LC50 level. The fact that the same combination of proteins may result in a synergistic or an antagonistic interaction may be an indication that there are different types of interactions within the host, depending on the insect species tested. PMID:25275646

  9. Synergism and antagonism between Bacillus thuringiensis Vip3A and Cry1 proteins in Heliothis virescens, Diatraea saccharalis and Spodoptera frugiperda.

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    Ana Rita Nunes Lemes

    Full Text Available Second generation Bt crops (insect resistant crops carrying Bacillus thuringiensis genes combine more than one gene that codes for insecticidal proteins in the same plant to provide better control of agricultural pests. Some of the new combinations involve co-expression of cry and vip genes. Because Cry and Vip proteins have different midgut targets and possibly different mechanisms of toxicity, it is important to evaluate possible synergistic or antagonistic interactions between these two classes of toxins. Three members of the Cry1 class of proteins and three from the Vip3A class were tested against Heliothis virescens for possible interactions. At the level of LC50, Cry1Ac was the most active protein, whereas the rest of proteins tested were similarly active. However, at the level of LC90, Cry1Aa and Cry1Ca were the least active proteins, and Cry1Ac and Vip3A proteins were not significantly different. Under the experimental conditions used in this study, we found an antagonistic effect of Cry1Ca with the three Vip3A proteins. The interaction between Cry1Ca and Vip3Aa was also tested on two other species of Lepidoptera. Whereas antagonism was observed in Spodoptera frugiperda, synergism was found in Diatraea saccharalis. In all cases, the interaction between Vip3A and Cry1 proteins was more evident at the LC90 level than at the LC50 level. The fact that the same combination of proteins may result in a synergistic or an antagonistic interaction may be an indication that there are different types of interactions within the host, depending on the insect species tested.

  10. Synergism and antagonism between Bacillus thuringiensis Vip3A and Cry1 proteins in Heliothis virescens, Diatraea saccharalis and Spodoptera frugiperda.

    Science.gov (United States)

    Lemes, Ana Rita Nunes; Davolos, Camila Chiaradia; Legori, Paula Cristina Brunini Crialesi; Fernandes, Odair Aparecido; Ferré, Juan; Lemos, Manoel Victor Franco; Desiderio, Janete Apparecida

    2014-01-01

    Second generation Bt crops (insect resistant crops carrying Bacillus thuringiensis genes) combine more than one gene that codes for insecticidal proteins in the same plant to provide better control of agricultural pests. Some of the new combinations involve co-expression of cry and vip genes. Because Cry and Vip proteins have different midgut targets and possibly different mechanisms of toxicity, it is important to evaluate possible synergistic or antagonistic interactions between these two classes of toxins. Three members of the Cry1 class of proteins and three from the Vip3A class were tested against Heliothis virescens for possible interactions. At the level of LC50, Cry1Ac was the most active protein, whereas the rest of proteins tested were similarly active. However, at the level of LC90, Cry1Aa and Cry1Ca were the least active proteins, and Cry1Ac and Vip3A proteins were not significantly different. Under the experimental conditions used in this study, we found an antagonistic effect of Cry1Ca with the three Vip3A proteins. The interaction between Cry1Ca and Vip3Aa was also tested on two other species of Lepidoptera. Whereas antagonism was observed in Spodoptera frugiperda, synergism was found in Diatraea saccharalis. In all cases, the interaction between Vip3A and Cry1 proteins was more evident at the LC90 level than at the LC50 level. The fact that the same combination of proteins may result in a synergistic or an antagonistic interaction may be an indication that there are different types of interactions within the host, depending on the insect species tested.

  11. In vivo binding of the Cry11Bb toxin of Bacillus thuringiensis subsp. medellin to the midgut of mosquito larvae (Diptera: Culicidae

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    Ruiz Lina María

    2004-01-01

    Full Text Available Bacillus thuringiensis subsp. medellin produces numerous proteins among which 94 kDa known as Cry11Bb, has mosquitocidal activity. The mode of action of the Cry11 proteins has been described as similar to those of the Cry1 toxins, nevertheless, the mechanism of action is still not clear. In this study we investigated the in vivo binding of the Cry11Bb toxin to the midgut of the insect species Anopheles albimanus, Aedes aegypti, and Culex quinquefasciatus by immunohistochemical analysis. Spodoptera frugiperda was included as negative control. The Cry11Bb protein was detected on the apical microvilli of the midgut epithelial cells, mostly on the posterior midgut and gastric caeca of the three mosquito species. Additionally, the toxin was detected in the Malpighian tubules of An. albimanus, Ae. aegypti, Cx. quinquefasciatus, and in the basal membrane of the epithelial cells of Ae. aegypti midgut. No toxin accumulation was observed in the peritrophic membrane of any of the mosquito species studied. These results confirm that the primary site of action of the Cry11 toxins is the apical membrane of the midgut epithelial cells of mosquito larvae.

  12. Characterization of the mucosal and systemic immune response induced by Cry1Ac protein from Bacillus thuringiensis HD 73 in mice

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    Vázquez-Padrón R.I.

    2000-01-01

    Full Text Available The present paper describes important features of the immune response induced by the Cry1Ac protein from Bacillus thuringiensis in mice. The kinetics of induction of serum and mucosal antibodies showed an immediate production of anti-Cry1Ac IgM and IgG antibodies in serum after the first immunization with the protoxin by either the intraperitoneal or intragastric route. The antibody fraction in serum and intestinal fluids consisted mainly of IgG1. In addition, plasma cells producing anti-Cry1Ac IgG antibodies in Peyer's patches were observed using the solid-phase enzyme-linked immunospot (ELISPOT. Cry1Ac toxin administration induced a strong immune response in serum but in the small intestinal fluids only anti-Cry1Ac IgA antibodies were detected. The data obtained in the present study confirm that the Cry1Ac protoxin is a potent immunogen able to induce a specific immune response in the mucosal tissue, which has not been observed in response to most other proteins.

  13. Genomic sequencing identifies novel Bacillus thuringiensis Vip1/Vip2 binary and Cry8 toxins that have high toxicity to Scarabaeoidea larvae.

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    Bi, Yang; Zhang, Yanrui; Shu, Changlong; Crickmore, Neil; Wang, Qinglei; Du, Lixin; Song, Fuping; Zhang, Jie

    2015-01-01

    The Bacillus thuringiensis strain HBF-18 (CGMCC 2070), which has previously been shown to encode the cry8Ga toxin gene, is active against both Holotrichia oblita and Holotrichia parallela. Recombinant Cry8Ga however is only weakly toxic to these insect pests suggesting the involvement of additional toxins in the native strain. We report that through the use of Illumina sequencing three additional, and novel, genes, namely vip1Ad1, vip2Ag1, and cry8-like, were identified in this strain. Although no protein corresponding to these genes could be identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the HBF-18 proteome, reverse transcription (RT)-PCR indicated that all three genes were transcribed in the native strain. The two vip genes were cloned and expressed and, as with other Vip1/2 toxins, appeared to function as a binary toxin and showed strong activity against H. oblita, H. parallela and Anomala corpulenta. This is the first report to demonstrate that the Vip1/Vip2 binary toxin is active against these Scarabaeoidea larvae. The cry8-like gene appeared to be a C-terminally truncated form of a typical cry8 gene and was not expressed in our usual recombinant Bt expression system. When however the missing C-terminal region was replaced with the corresponding sequence from cry8Ea, the resulting hybrid expressed well and the toxin was active against the three test insects.

  14. Crystal structure of Cry6Aa: A novel nematicidal ClyA-type α-pore-forming toxin from Bacillus thuringiensis.

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    Huang, Jinbo; Guan, Zeyuan; Wan, Liting; Zou, Tingting; Sun, Ming

    2016-09-09

    Crystal (Cry) proteins from Bacillus thuringiensis (Bt) are globally used in agriculture as proteinaceous insecticides. Numerous crystal structures have been determined, and most exhibit conserved three-dimensional architectures. Recently, we have identified a novel nematicidal mechanism by which Cry6Aa triggers cell death through a necrosis-signaling pathway via an interaction with the host protease ASP-1. However, we found little sequence conservation of Cry6Aa in our functional study. Here, we report the 1.90 angstrom (Å) resolution structure of the proteolytic form of Cry6Aa (1-396), determined by X-ray crystallography. The structure of Cry6Aa is highly similar to those of the pathogenic toxin family of ClyA-type α-pore-forming toxins (α-PFTs), which are characterized by a bipartite structure comprising a head domain and a tail domain, thus suggesting that Cry6Aa exhibits a previously undescribed nematicidal mode of action. This structure also provides a framework for the functional study of other nematicidal toxins.

  15. Fatores de Virulência de Bacillus thuringiensis Berlinier: O Que Existe Além das Proteínas Cry?

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    Laurival A. Vilas-Boas

    2012-04-01

    Full Text Available As proteínas Cry produzidas pela bactéria entomopatogênica Bacillus thuringiensis Berliner são bem conhecidas devido a alta citotoxicidade que exibem a uma variedade de insetos-alvo. O modo de ação destas proteínas é específico e torna os produtos à base de B. thuringiensis os mais amplamente utilizados em programas de controle biológico de pragas na agricultura e de importantes vetores de doenças humanas. Contudo, embora as proteínas Cry sejam os fatores de virulência inseto-específico mais conhecidos, linhagens de B. thuringiensis apresentam também uma ampla gama de fatores de virulência, os quais permitem à bactéria atingir a hemolinfa e colonizar eficientemente o inseto hospedeiro. Dentre estes fatores, destacam-se as proteínas Vip, Cyt, enterotoxinas, hemolisinas, fosfolipases, proteases, enzimas de degradação, além das recentemente descritas parasporinas. Essa revisão aborda a ação desses fatores de virulência, bem como a caracterização e o controle da expressão de seus genes. Adicionalmente, são discutidos aspectos relacionados ao nicho ecológico da bactéria com ênfase nas características envolvidas com a biossegurança da utilização dos produtos à base de B. thuringiensis para o controle biológico de insetos-alvo.Virulence Factors of Bacillus thuringiensis Berliner: Something Beyond of Cry Proteins?Abstract. The Cry proteins produced by the entomopathogenic bacterium Bacillus thuringiensis Berliner are widely known due to its high toxicity against a variety of insects. The mode of action of these proteins is specific and becomes B. thuringiensis-based products the most used in biological control programs of insect pests in agriculture and of important human disease vectors. However, while the Cry proteins are the best-known insect-specific virulence factor, strains of B. thuringiensis show also a wide range of other virulence factors, which allow the bacteria to achieve the hemolymph and colonize

  16. Identificação de genes cry de Bacillus thuringiensis no controle de Sphenophorus levis, o bicudo da cana-de-açúcar Identification of cry genes from Bacillus thuringiensis effective against Sphenophorus levis, the sugar-cane borer

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    Elaine Aparecida Silva Cícero

    2009-01-01

    Full Text Available A bactéria B. thuringiensis caracteriza-se pela produção de proteínas tóxicas a representantes de diversas ordens de insetos, as quais são codificadas por genes cry. Este trabalho foi realizado com objetivo de selecionar isolados de B. thuringiensis, por meio da caracterização morfológica e molecular, identificando as diferentes subclasses dos genes cry3 e cry35 e determinar a patogenicidade contra Sphenophorus levis, uma das mais importantes pragas da cultura da cana-de-açúcar. Foram utilizados 1163 isolados de B. thuringiensis e com a observação em microscópio com contraste de fases foram confirmadas como pertencentes à espécie de B. thuringiensis. O material genético foi purificado pela matriz de troca iônica "Instagene Matrix" e submetido a PCR com iniciadores gerais cry3 e cry35 identificando-se 30 isolados contendo genes com potencial para o controle de coleópteros, os quais juntamente com as linhagens-padrão de B. thuringiensis var. tenebrionis, B. thuringiensis var. morrissone e B. thuringiensis var. tolworthi foram utilizados para a realização do bioensaio. Através de análise discriminante alocaram-se os isolados em quatro grupos quanto à toxicidade de B. thuringiensis. Os grupos ficaram assim definidos: um grupo que promovem até 10% de mortalidade contendo as testemunhas e duas linhagens;um grupo que causou 39% de mortalidade contendo três linhagens padrão e dez isolados; um grupo com 52% de mortalidade contendo treze isolados e um grupo com 70% de mortalidade contendo cinco isolados, os quais devem ser considerados promissores no controle biológico de S. levis.Bacillus thuringiensis is well known for its ability to produce toxic proteins to different insect orders which are encoded by the cry genes. This work was carried out aiming to select among different B. thuringiensis isolates, by morphological and molecular characterization, identifying different classes of cry3 and cry35 genes, and to determine the

  17. Research Progress of the cry Gene of Bacillus thuringiensis%苏云金芽胞杆菌cry基因研究进展

    Institute of Scientific and Technical Information of China (English)

    郑琦; 唐玉明; 关雄

    2012-01-01

    从cry基因的分类、鉴定、遗传特性以及应用概况等4个方面综述了苏云金芽胞杆菌cry基因的研究进展,并对cry基因的研究进行展望.%The research and progress of the cry genes of Bacillus thuringiensis were reviewed from the following aspects; gene classification, identification methods, genetic characteristics, application survey. And the prospect of researching was expected simultaneously.

  18. Characteristics of resistance to Bacillus thuringiensis toxin Cry2Ab in a strain of Helicoverpa punctigera (Lepidoptera: Noctuidae) isolated from a field population.

    Science.gov (United States)

    Downes, S; Parker, T L; Mahon, R J

    2010-12-01

    In 1996, the Australian cotton industry adopted Ingard that expresses the Bacillus thuringiensis (Bt) toxin gene cry1Ac and was planted at a cap of 30%. In 2004-2005, Bollgard II, which expresses cry1Ac and cry2Ab, replaced Ingard in Australia, and subsequently has made up >80% of the area planted to cotton, Gossypium hirsutum L. The Australian target species Helicoverpa armigera (Hübner) and Helicoverpa punctigera (Wallengren) are innately moderately tolerant to Bt toxins, but the absence of a history of insecticide resistance indicates that the latter species is less likely to develop resistance to Bt cotton. From 2002-2003 to 2006-2007, F2 screens were deployed to detect resistance to CrylAc or Cry2Ab in natural populations of H. punctigera. Alleles that conferred an advantage against CrylAc were not detected, but those that conferred resistance to Cry2Ab were present at a frequency of 0.0018 (n = 2,192 alleles). Importantly, the first isolation of Cry2Ab resistance in H. punctigera occurred before significant opportunities to develop resistance in response to Bollgard II. We established a colony (designated Hp4-13) consisting of homozygous resistant individuals and examined their characteristics through comparison with individuals from a Bt-susceptible laboratory colony. Through specific crosses and bioassays, we established that the resistance present in Hp4-13 is due to a single autosomal gene. The resistance is fully recessive. Homozygotes are able to survive a dose of Cry2Ab toxin that is 15 times the reported concentration in field grown Bollgard II in Australia (500 microg/ml) and are fully susceptible to Cry1Ac and to the Bt product DiPel. These characteristics are the same as those described for the first Cry2Ab resistant strain of H. armigera isolated from a field population in Australia.

  19. Modified Bacillus thuringiensis toxins and a hybrid B. thuringiensis strain counter greenhouse-selected resistance in Trichoplusia ni.

    Science.gov (United States)

    Franklin, Michelle T; Nieman, Christal L; Janmaat, Alida F; Soberón, Mario; Bravo, Alejandra; Tabashnik, Bruce E; Myers, Judith H

    2009-09-01

    Resistance of greenhouse-selected strains of the cabbage looper, Trichoplusia ni, to Bacillus thuringiensis subsp. kurstaki was countered by a hybrid strain of B. thuringiensis and genetically modified toxins Cry1AbMod and Cry1AcMod, which lack helix alpha-1. Resistance to Cry1AbMod and Cry1AcMod was >100-fold less than resistance to native toxins Cry1Ab and Cry1Ac.

  20. Bacillus thuringiensis Cry1Ca-resistant Spodoptera exigua lacks expression of one of four Aminopeptidase N genes

    NARCIS (Netherlands)

    Herrero, S.; Gechev, T.; Bakker, P.L.; Moar, W.; Maagd, de R.A.

    2005-01-01

    BACKGROUND: Insecticidal toxins from Bacillus thuringiensis bind to receptors on midgut epithelial cells of susceptible insect larvae. Aminopeptidases N (APNs) from several insect species have been shown to be putative receptors for these toxins. Here we report the cloning and expression analysis of

  1. Bacillus thuringiensis Cry5B protein is highly efficacious as a single-dose therapy against an intestinal roundworm infection in mice.

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    Yan Hu

    Full Text Available Intestinal parasitic nematode diseases are one of the great diseases of our time. Intestinal roundworm parasites, including hookworms, whipworms, and Ascaris, infect well over 1 billion people and cause significant morbidity, especially in children and pregnant women. To date, there is only one drug, albendazole, with adequate efficacy against these parasites to be used in mass drug administration, although tribendimidine may emerge as a second. Given the hundreds of millions of people to be treated, the threat of parasite resistance, and the inadequacy of current treatments, new anthelmintics are urgently needed. Bacillus thuringiensis (Bt crystal (Cry proteins are the most common used biologically produced insecticides in the world and are considered non-toxic to vertebrates.Here we study the ability of a nematicidal Cry protein, Cry5B, to effect a cure in mice of a chronic roundworm infection caused by the natural intestinal parasite, Heligmosomoides bakeri (formerly polygyrus. We show that Cry5B produced from either of two Bt strains can act as an anthelmintic in vivo when administered as a single dose, achieving a approximately 98% reduction in parasite egg production and approximately 70% reduction in worm burdens when delivered per os at approximately 700 nmoles/kg (90-100 mg/kg. Furthermore, our data, combined with the findings of others, suggest that the relative efficacy of Cry5B is either comparable or superior to current anthelmintics. We also demonstrate that Cry5B is likely to be degraded quite rapidly in the stomach, suggesting that the actual dose reaching the parasites is very small.This study indicates that Bt Cry proteins such as Cry5B have excellent anthelmintic properties in vivo and that proper formulation of the protein is likely to reveal a superior anthelmintic.

  2. Bacillus thuringiensis protein production, signal transduction, and insect control in chemically inducible PR-1a/cry1Ab broccoli plants.

    Science.gov (United States)

    Cao, Jun; Bates, Sarah L; Zhao, Jian-Zhou; Shelton, Anthony M; Earle, Elizabeth D

    2006-06-01

    In an effort to develop a chemically inducible system for insect management, we studied production of Cry1Ab Bacillus thuringiensis (Bt) protein and control of the diamondback moth (DBM), Plutella xylostella L., in inducer-treated and untreated tissues of a broccoli line transformed with a PR-1a/cry1Ab expression cassette. Spraying leaves of these plants with the inducer acibenzolar-S-methyl (= 1,2,3 benzothiadiazole-7-thiocarboxylic acid-S-methyl-ester) (ASM) triggered expression of the cry1Ab gene and produced a high level of Cry1Ab protein within 2-3 days. Cry1Ab protein persisted in leaves for at least 8 weeks, providing prolonged protection from P. xylostella attack. Signals generated in inducer-treated leaves were transferred to untreated newly emerged leaves or heads, as seen by production of Cry1Ab protein and/or protection from insect damage in these plant parts. Signal transduction proceeded in an attenuated manner up to the sixth newly emerged leaf. No Cry1Ab protein was detectable by ELISA in uninduced young leaves, but small amounts of the protein were present in uninduced leaves older than 3 weeks and caused some insect mortality. Such basal expression of Bt genes without induction may favor the evolution of resistant insect populations and therefore limits the application of the PR-1a/cry1Ab system for insect management. However, the rapid production and steady maintenance of a high level of transgenic protein upon induction, the signal transduction observed, and the fact that the chemical inducer can be used in field conditions make the PR-1a promoter attractive for chemical regulation of other agriculturally or pharmaceutically important genes for which low expression in the absence of induction is not a concern.

  3. Receptors are affected by selection with each Bacillus thuringiensis israelensis Cry toxin but not with the full Bti mixture in Aedes aegypti.

    Science.gov (United States)

    Stalinski, Renaud; Laporte, Frederic; Tetreau, Guillaume; Després, Laurence

    2016-10-01

    Bacillus thuringiensis israelensis (Bti) toxins are increasingly used for mosquito control, but little is known about the precise mode of action of each of these toxins, and how they interact to kill mosquito larvae. By using RNA sequencing, we investigated change in gene transcription level and polymorphism variations associated with resistance to each Bti Cry toxin and to the full Bti toxin mixture in the dengue vector Aedes aegypti. The up-regulation of genes related to chitin metabolism in all selected strain suggests a generalist, non-toxin-specific response to Bti selection in Aedes aegypti. Changes in the transcription level and/or protein sequences of several putative Cry toxin receptors (APNs, ALPs, α-amylases, glucoside hydrolases, ABC transporters) were specific to each Cry toxin. Selective sweeps associated with Cry4Aa resistance were detected in 2 ALP and 1 APN genes. The lack of selection of toxin-specific receptors in the Bti-selected strain supports the hypothesis that Cyt toxin acts as a receptor for Cry toxins in mosquitoes.

  4. Knockdown of the MAPK p38 pathway increases the susceptibility of Chilo suppressalis larvae to Bacillus thuringiensis Cry1Ca toxin

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    Qiu, Lin; Fan, Jinxing; Liu, Lang; Zhang, Boyao; Wang, Xiaoping; Lei, Chaoliang; Lin, Yongjun; Ma, Weihua

    2017-01-01

    The bacterium Bacillus thuringiensis (Bt) produces a wide range of toxins that are effective against a number of insect pests. Identifying the mechanisms responsible for resistance to Bt toxin will improve both our ability to control important insect pests and our understanding of bacterial toxicology. In this study, we investigated the role of MAPK pathways in resistance against Cry1Ca toxin in Chilo suppressalis, an important lepidopteran pest of rice crops. We first cloned the full-length of C. suppressalis mitogen-activated protein kinase (MAPK) p38, ERK1, and ERK2, and a partial sequence of JNK (hereafter Csp38, CsERK1, CsERK2 and CsJNK). We could then measure the up-regulation of these MAPK genes in larvae at different times after ingestion of Cry1Ca toxin. Using RNA interference to knockdown Csp38, CsJNK, CsERK1 and CsERK2 showed that only knockdown of Csp38 significantly increased the mortality of larvae to Cry1Ca toxin ingested in either an artificial diet, or after feeding on transgenic rice expressed Cry1Ca. These results suggest that MAPK p38 is responsible for the resistance of C. suppressalis larvae to Bt Cry1Ca toxin. PMID:28262736

  5. Dominant negative mutants of Bacillus thuringiensis Cry1Ab toxin function as anti-toxins: demonstration of the role of oligomerization in toxicity.

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    Claudia Rodríguez-Almazán

    Full Text Available BACKGROUND: Bacillus thuringiensis Cry toxins, that are used worldwide in insect control, kill insects by a mechanism that depends on their ability to form oligomeric pores that insert into the insect-midgut cells. These toxins are being used worldwide in transgenic plants or spray to control insect pests in agriculture. However, a major concern has been the possible effects of these insecticidal proteins on non-target organisms mainly in ecosystems adjacent to agricultural fields. METHODOLOGY/PRINCIPAL FINDINGS: We isolated and characterized 11 non-toxic mutants of Cry1Ab toxin affected in different steps of the mechanism of action namely binding to receptors, oligomerization and pore-formation. These mutant toxins were analyzed for their capacity to block wild type toxin activity, presenting a dominant negative phenotype. The dominant negative phenotype was analyzed at two levels, in vivo by toxicity bioassays against susceptible Manduca sexta larvae and in vitro by pore formation activity in black lipid bilayers. We demonstrate that some mutations located in helix alpha-4 completely block the wild type toxin activity at sub-stoichiometric level confirming a dominant negative phenotype, thereby functioning as potent antitoxins. CONCLUSIONS/SIGNIFICANCE: This is the first reported case of a Cry toxin dominant inhibitor. These data demonstrate that oligomerization is a fundamental step in Cry toxin action and represent a potential mechanism to protect special ecosystems from the possible effect of Cry toxins on non-target organisms.

  6. Alkaline phosphatases are involved in the response of Aedes aegypti larvae to intoxication with Bacillus thuringiensis subsp. israelensis Cry toxins.

    Science.gov (United States)

    Stalinski, Renaud; Laporte, Frédéric; Després, Laurence; Tetreau, Guillaume

    2016-03-01

    Bacillus thuringiensis subsp. israelensis (Bti) is a natural pathogen of dipterans widely used as a biological insecticide for mosquito control. To characterize the response of mosquitoes to intoxication with Bti, the transcriptome profile of Bti-exposed susceptible Aedes aegypti larvae was analysed using Illumina RNA-seq. Gene expression of 11 alkaline phosphatases (ALPs) was further investigated by real time quantitative polymerase chain reaction and ALP activity was measured in the susceptible strain and in four strains resistant to a single Bti Cry toxin or to Bti. These strains were unexposed or exposed to their toxin of selection. Although all resistant strains constitutively exhibited a higher level of transcription of ALP genes than the susceptible strain, they showed a lower total ALP activity. The intoxication with different individual Cry toxins triggered a global pattern of ALP gene under-transcription in all the one-toxin-resistant strains but involving different specific sets of ALPs in each resistant phenotype. Most of the ALPs involved are not known Cry-binding proteins. RNA interference experiment demonstrated that reducing ALP expression conferred increased the survival of larvae exposed to Cry4Aa, confirming the involvement of ALP in Cry4Aa toxicity.

  7. Altered binding of the Cry1Ac toxin to larval membranes but not to the toxin-binding protein in Plodia interpunctella selected for resistance to different Bacillus thuringiensis isolates.

    Science.gov (United States)

    Mohammed, S I; Johnson, D E; Aronson, A I

    1996-11-01

    Immunoblotting and cytochemical procedures were used to determine whether toxin binding was altered in strains of the Indianmeal moth, Plodia interpunctella, selected for resistance to various strains of Bacillus thuringiensis. Each of these B. thuringiensis subspecies produces a mixture of protoxins, primarily Cry1 types, and the greatest insect resistance is to the Cry1A protoxins. In several cases, however, there was also resistance to toxins not present in the B. thuringiensis strains used for selection. The Cry1Ab and Cry1Ac toxins bound equally well over a range of toxin concentrations and times of incubation to a single protein of ca. 80-kDa in immunoblots of larval membrane extracts from all of the colonies. This binding protein is essential for toxicity since a mutant Cry1Ac toxin known to be defective in binding and thus less toxic bound poorly to the 80-kDa protein. This binding protein differed in size from the major aminopeptidase N antigens implicated in toxin binding in other insects. Binding of fluorescently labeled Cry1Ac or Cry1Ab toxin to larval sections was found at the tips of the brush border membrane prepared from the susceptible but not from any of the resistant P. interpunctella. Accessibility of a major Cry1A-binding protein appears to be altered in resistant larvae and could account for their broad resistance to several B. thuringiensis toxins.

  8. Co-expression of the mosquitocidal toxins Cyt1Aa and Cry11Aa from Bacillus thuringiensis subsp. israelensis in Asticcacaulis excentricus.

    Science.gov (United States)

    Zheng, Dasheng; Valdez-Cruz, Norma Adriana; Armengol, Gemma; Sevrez, Chloe; Munoz-Olaya, Jose Maurilio; Yuan, Zhiming; Orduz, Sergio; Crickmore, Neil

    2007-01-01

    The cyt1Aa gene from Bacillus thuringiensis subsp. israelensis (Bti), whose product synergizes other mosquitocidal toxins, and functions as a repressor of resistance developed by mosquitoes against Bacilli insecticides, was introduced into the aquatic Gram-negative bacterium Asticcacaulis excentricus alongside the cry11Aa gene. The genes were introduced as an operon, but although mRNA was detected for both genes, no Cyt1Aa toxin was detected. Both proteins were expressed using a construct in which a promoter was inserted upstream of each gene. Recombinant A. excentricus expressing both toxins was found to be approximately twice as toxic to third instar larvae of Culex quinquefasciatus as transformants expressing just Cry11Aa.

  9. Crystal structure of Cry51Aa1: A potential novel insecticidal aerolysin-type β-pore-forming toxin from Bacillus thuringiensis.

    Science.gov (United States)

    Xu, Chengchen; Chinte, Unmesh; Chen, Lirong; Yao, Qingqing; Meng, Ying; Zhou, Dayong; Bi, Li-Jun; Rose, John; Adang, Michael J; Wang, Bi-Cheng; Yu, Ziniu; Sun, Ming

    2015-07-03

    The structures of several Bacillus thuringiensis (Bt) insecticidal crystal proteins have been determined by crystallographic methods and a close relationship has been explicated between specific toxicities and conserved three-dimensional architectures. In this study, as a representative of the coleopteran- and hemipteran-specific Cry51A group, the complete structure of Cry51Aa1 protoxin has been determined by X-ray crystallography at 1.65 Å resolution. This is the first report of a coleopteran-active Bt insecticidal toxin with high structural similarity to the aerolysin-type β-pore forming toxins (β-PFTs). Moreover, study of featured residues and structural elements reveal their possible roles in receptor binding and pore formation events. This study provides new insights into the action of aerolysin-type β-PFTs from a structural perspective, and could be useful for the control of coleopteran and hemipteran insect pests in agricultures.

  10. Using phage display to map the binding epitope of the Bacillus thuringiensis Cry2Ab toxin%利用噬菌体展示技术筛选Cry2Ab毒素受体表位的方法

    Institute of Scientific and Technical Information of China (English)

    齐佳; 刘晨曦; 吴孔明

    2012-01-01

    苏云金芽孢杆菌(Bacillus thuringiensis,Bt)产生的内毒素具有杀虫活性,Cry2Ab毒素作为Bt棉花的杀虫活性蛋白,其在靶标昆虫体内的结合受体及作用位点尚不清楚,本研究采用噬菌体展示( phage display)的方法,经4轮的“吸附-洗脱-扩繁”筛选,并对阳性克隆所携带的外源DNA片段进行序列测定后,得到2段能够与活化Cry2Ab毒素相互作用的多肽序列,通过酶联免疫结合试验(ELISA)进一步证明,这2段多肽序列与活化Cry2Ab毒素具有较高的亲和力和特异性,结果表明,利用该方法能够由噬菌体随机肽库中高效捕获亲和序列,筛选到与活化Cry2Ab毒素具有高亲和力的多肽,该序列可以模拟Cry2Ab毒素的受体表位,为进一步研究Cry2Ab毒素作用机制奠定了基础,并为今后田间抗性基因频率检测,以及毒素-受体作用机制研究工作提供更有力的技术支持.%The insecticidal Cry toxins produced by Bacillus thuringiensis are highly specific to different insects. Various proteins, such as cadherin, aminopeptidase-N ( APN) and alkaline phosphatase ( ALP) are characterized as potential Cry-receptors. However, little is known about the mode of Cry2Ab action, such as its receptors and binding sites. We used phage display to characterize the binding epitope of the Cry2Ab toxin in vitro. A two peptide sequence was identified after four-rounds of screening. ELISA analysis showed that activated Cry2Ab toxin could bind these two peptides with high affinity. The results indicate that employing this method can efficiently screen out target peptides with high affinity and specificity. The method also provides a valuable platform to discover the mode of other Bt toxins.

  11. Biological Characteristics of Bacillus thuringiensis FB Strain and Identification of Its cry and vip-type Genes%Bt菌株FB的生物学特性及其cry、vip基因型鉴定

    Institute of Scientific and Technical Information of China (English)

    王品舒; 刘晓垒; 席景会; 彭琦; 束长龙; 张杰; 宋福平

    2014-01-01

    菌株FB是1株对小菜蛾Plutella xylostella幼虫具有高毒力的苏云金芽胞杆菌Bacillus thuringiensis (Bt)。本研究通过扫描电子显微镜、大质粒电泳、总蛋白SDS-PAGE及菌株生长特征观察的方式研究了菌株FB特征,克隆得到了基因cry1Ia、cry1Ea、cry1Ab、cry2Ab和vip3Aa全长,依据全基因组测序结果得到了1个cry8基因部分片段,首次在Bt菌株中同时发现基因cry1类和cry8类,这五种基因推导的氨基酸序列与已知基因序列相比,最高相似性分别为99%、98%、99%、100%、99%,而cry8半长基因与已知基因仅为63%。生测结果表明,蛋白Cry1Ia、Cry1Ea和Cry1Ab对小菜蛾幼虫具有较高杀虫活性。%Bacillus thuringiensis FB strain was high toxicity against larvae of Plutella xylostella (L.). FB strain was characterized with scanning electron microscope, large plasmids electrophoresis, SDS-PAGE and growth characteristics observation. Five full length toxin genes, cry1Ia, cry1Ea, cry1Ab, cry2Ab and vip3Aa, were cloned. Furthermore, a half-length fragment of cry8 gene was found by whole-genome sequencing, this is the first time that cry1 and cry8-type genes existed in the same strain. The highest identity of the deduced amino acid sequences with known B. thuringiensis toxins were 99%, 98%, 99%, 100%, 99%respectively, but deduced amino acid sequence of this Cry8 fragment was 63%similarity with known Cry8 proteins. Bioassay results showed that Cry1Ab, Cry1Ia, Cry1Ea were high toxicity against larvae of P. xylostella.

  12. Shared midgut binding sites for Cry1A.105, Cry1Aa, Cry1Ab, Cry1Ac and Cry1Fa proteins from Bacillus thuringiensis in two important corn pests, Ostrinia nubilalis and Spodoptera frugiperda.

    Science.gov (United States)

    Hernández-Rodríguez, Carmen Sara; Hernández-Martínez, Patricia; Van Rie, Jeroen; Escriche, Baltasar; Ferré, Juan

    2013-01-01

    First generation of insect-protected transgenic corn (Bt-corn) was based on the expression of Cry1Ab or Cry1Fa proteins. Currently, the trend is the combination of two or more genes expressing proteins that bind to different targets. In addition to broadening the spectrum of action, this strategy helps to delay the evolution of resistance in exposed insect populations. One of such examples is the combination of Cry1A.105 with Cry1Fa and Cry2Ab to control O. nubilalis and S. frugiperda. Cry1A.105 is a chimeric protein with domains I and II and the C-terminal half of the protein from Cry1Ac, and domain III almost identical to Cry1Fa. The aim of the present study was to determine whether the chimeric Cry1A.105 has shared binding sites either with Cry1A proteins, with Cry1Fa, or with both, in O. nubilalis and in S. frugiperda. Brush-border membrane vesicles (BBMV) from last instar larval midguts were used in competition binding assays with (125)I-labeled Cry1A.105, Cry1Ab, and Cry1Fa, and unlabeled Cry1A.105, Cry1Aa, Cry1Ab, Cry1Ac, Cry1Fa, Cry2Ab and Cry2Ae. The results showed that Cry1A.105, Cry1Ab, Cry1Ac and Cry1Fa competed with high affinity for the same binding sites in both insect species. However, Cry2Ab and Cry2Ae did not compete for the binding sites of Cry1 proteins. Therefore, according to our results, the development of cross-resistance among Cry1Ab/Ac, Cry1A.105, and Cry1Fa proteins is possible in these two insect species if the alteration of shared binding sites occurs. Conversely, cross-resistance between these proteins and Cry2A proteins is very unlikely in such case.

  13. Prokaryotic Expression of the Soluble Cry2Ab Protein from Bacillus thuringiensis and Preparation of the Polyclonal Antibody Against Cry2Ab%苏云金杆菌Cry2Ab可溶蛋白的原核表达及多克隆抗体的制备

    Institute of Scientific and Technical Information of China (English)

    邵恩斯; 林莉; 关雄

    2013-01-01

    Cry2Ab toxin of Bacillus thuringiensis is a toxic protein, which is wildly used in controlling lepidopteran pest in agricultural production. In this research, the cry2A b gene (1 914 bp) was amplified from total DNA of B. Thuringiensis WB9 strain by a pair of primer designed by the full-length sequence of published crylAb gene. Then, cry2Ab was ligated with linearized pGEX-KG vector by restriction enzyme BamH Ⅰ and Xho Ⅰ for the construction of cry2Ab-pk expression vector. The soluble Cry2Ab-GST fusion protein (approximately 90 kD) was obtained after transferring Cry2Ab-PK expression vector into Escherichia coli BL21 (DE3) and then inducing by 0.8 mmol/L IPTG at 16℃ for 36 h. Total soluble protein was purified by batch purification and GST tag was removed by the use of prescission protease to obtain soluble Cry2Ab protein (approximately 65 kD). Polyclonal antibody against Cry2Ab was produced by immunizing the purified Cry2Ab to New Zealand white rabbit (Oryctolagus cuniculus) after three times of intramuscular injection and one time of intravenous injection. The titter of antibody was over 1:150 000, measured by indirect ELISA. Specificity of the prepared antibody was determined by Western blot, showing that the polyclonal antibody against the Cry2Aa or Cry2Ab protein was positive and the antibody against Cry1Ab or Cry3Aa protein was negative. These results indicated that antibody against Cry2Ab protein can specifically identify Cry2A protein but cannot identify other three domains Cry protein including CrylAb and Cry3Aa. These results will provide technical support for further study of Cry2A toxins mechanism and the interaction between Cry2A toxins and its receptors.%苏云金杆菌Cry2Ab蛋白是一类对鳞翅目昆虫有特异性毒性作用的毒素蛋白,已广泛应用于针对鳞翅目害虫的防治之中.依据苏云金杆菌cry2Ab基因序列设计一对全长引物,从苏云金杆菌(Bacillus thuringiensis WB9菌株总DNA中克隆出cry2Ab基因全序列,构建Cry

  14. The toxicity test and hypothetical model of Bacillus thuringiensis Cry1Aa helix4

    Institute of Scientific and Technical Information of China (English)

    SU; Yanhui(苏彦辉); QU; Hong(曲红); Vachon; Vincent; LUO; Jingchu(罗静初); ZHANG; Jie(张杰); Laprade; Raynald; ZHU; Yuxian(朱玉贤)

    2002-01-01

    Development of targeted biological agents against agricultural insect pests is of prime importance for the elaboration and implementation of integrated pest management strategies that are environment-friendly, respectful of bio-diversity and safer to human health through reduced use of chemical pesticides. A major goal to understand how Bt toxins work is to elucidate the functions of their three domains. Domains II and III are involved in binding specificity and structural integrity, but the function of Domain I remains poorly understood. Using a Manduca sexta BBMV (brush border membrane vesicles) system, we analyzed its responses to Cry1Aa 15 single-point mutations with altered Domain I helix 4 residues. Light scattering assay showed that toxicity was almost lost in 3 mutants, and we observed significantly reduced toxicity in other 7 mutants. However, 5 mutants retained wild-type toxicity. Using computer software, we simulated the three-dimensional structures of helix 4. Both experimental and bioinformatic analysis showed that residues in Cry1Aa Domain I helix 4 were involved in the formation of ion channels that is critical for its insect toxicity.

  15. Domains of Bacillus thuringiensis crystal proteins involved in insecticidal activity

    NARCIS (Netherlands)

    Bosch, H.J.; Schipper, B.; Kleij, van der H.; Maagd, de R.A.; Stiekema, W.J.

    1994-01-01

    The expected increase in application of Bacillus thuringiensis (Bt) in crop protection makes it necessary to anticipate the development of Bt-resistant insects. To safeguard the long-term use of Bt-based insecticides, we studied the mode of action of Bt crystal proteins. CryIA(b), CryIC and CryIE ar

  16. Expresión de la toxina Cry11Aa de Bacillus thuringiensis serovar. israelensis en Asticcacaulis excentricus, para el control de larvas acuáticas de dípteros de la familia Culicidae, vectores de enfermedades Expression of Bacillus thuringiensis serovar. israelensis toxins in Asticcacaulis excentricus to control dipteran larvae of vectors of diseases

    OpenAIRE

    Orduz Sergio; Guevara Óscar Enrique; Armengol Gemma; Crickmore Neil

    2004-01-01

    Los genes cry de Bacillus thuringiensis codifican para un diverso grupo de proteínas formadoras de cristales que exhiben actividad insecticida contra larvas de dípteros, lepidópteros y coleópteros, entre otros. La efectividad de los insecticidas basados en formulaciones de proteínas de B. thuringiensis puede ser mejorada usando bacterias prostecadas acuáticas como portadoras alternativas de los genes cry, ya que no se sedimentan rápidamente; las proteínas expresadas en el citoplasma están pro...

  17. High Sensitivity Bacillus thuringiensis Cry1Ac Protein Detections Using Fluorescein Diacetate Nanoparticles.

    Science.gov (United States)

    Liu, Cui; Zhou, Zhen; Zou, Linling; Cao, Yuan-Cheng; Liu, Jun'An; Lin, Yongjun

    2016-03-01

    A highly sensitive transgenic protein analysis method was proposed here based on fluorescein diacetate (FDA). First, FDA was prepared by the ball mill to harvest the nano-sized organic particles. Further examines showed that the FDA size can be controlled by the speed of centrifugation which can obtain FDA in well-distributed size. Cy3 antibody immobilization tests showed that the proteins can attach onto the FDA particles while keep bioactivities. FDA and Cry1Ac antibody immunoassay tests showed that when the FDA particle was in 150 nm, the linear range was 0.01 ng/L-30 μg/mL. And it has the lower detection limitation of 0.01 ng/L, which is 100 times more sensitive than the ELISA methods. These results indicate that the FDA related immunoassays are the promising approach in the transgenic analysis.

  18. Isolation of Bacillus thuringiensis Strains from Mangrove Sediment Samples and Identification for cry Genes%红树林沉积物Bt菌株的分离与cry基因型的鉴定

    Institute of Scientific and Technical Information of China (English)

    林毅; 方光伟

    2010-01-01

    从收集自泉州洛江红树林保护区的400个沉积物土壤样品中,分离到18株苏云金芽孢杆菌(Bacillus thuringiensis,Bt).利用PCR-RFLP体系对Bt菌株杀虫晶体蛋白的基因型进行了鉴定,发现15株Bt含有cry1基因,4株含有cry2基因,有3株分别含有cry7、cry8、cry9基因.克隆了一个新型的cry2Ab基因,其编码蛋白与现有Cry2Ab型杀虫晶体蛋白的最高同源性为95%.

  19. Different Effects of Bacillus thuringiensis Toxin Cry1Ab on Midgut Cell Transmembrane Potential of Mythimna separata and Agrotis ipsilon Larvae

    Science.gov (United States)

    Wang, Yingying; Hu, Zhaonong; Wu, Wenjun

    2015-01-01

    Bacillus thuringiensis (Bt) Cry toxins from the Cry1A family demonstrate significantly different toxicities against members of the family Noctuidae for unknown reasons. In this study, membrane potential was measured and analyzed in freshly isolated midgut samples from Mythimna separata and Agrotis ipsilon larvae under oral administration and in vitro incubation with Bt toxin Cry1Ab to elucidate the mechanism of action for further control of these pests. Bioassay results showed that the larvae of M. separata achieved a LD50 of 258.84 ng/larva at 24 h after ingestion; M. separata larvae were at least eightfold more sensitive than A. ipsilon larvae to Cry1Ab. Force-feeding showed that the observed midgut apical-membrane potential (Vam) of M. separata larvae was significantly depolarized from −82.9 ± 6.6 mV to −19.9 ± 7.2 mV at 8 h after ingestion of 1 μg activated Cry1Ab, whereas no obvious changes were detected in A. ipsilon larvae with dosage of 5 μg Cry1Ab. The activated Cry1Ab caused a distinct concentration-dependent depolarization of the apical membrane; Vam was reduced by 50% after 14.7 ± 0.2, 9.8 ± 0.4, and 7.6 ± 0.6 min of treatment with 1, 5, and 10 μg/mL Cry1Ab, respectively. Cry1Ab showed a minimal effect on A. ipsilon larvae even at 20 μg/mL, and Vam decreased by 26.3% ± 2.3% after 15 min. The concentrations of Cry1Ab displayed no significant effect on the basolateral side of the epithelium. The Vam of A. ipsilon (−33.19 ± 6.29 mV, n = 51) was only half that of M. separata (−80.94 ± 6.95 mV, n = 75). The different degrees of sensitivity to Cry1Ab were speculatively associated with various habits, as well as the diverse physiological or biochemical characteristics of the midgut cell membranes. PMID:26694463

  20. Different Effects of Bacillus thuringiensis Toxin Cry1Ab on Midgut Cell Transmembrane Potential of Mythimna separata and Agrotis ipsilon Larvae

    Directory of Open Access Journals (Sweden)

    Yingying Wang

    2015-12-01

    Full Text Available Bacillus thuringiensis (Bt Cry toxins from the Cry1A family demonstrate significantly different toxicities against members of the family Noctuidae for unknown reasons. In this study, membrane potential was measured and analyzed in freshly isolated midgut samples from Mythimna separata and Agrotis ipsilon larvae under oral administration and in vitro incubation with Bt toxin Cry1Ab to elucidate the mechanism of action for further control of these pests. Bioassay results showed that the larvae of M. separata achieved a LD50 of 258.84 ng/larva at 24 h after ingestion; M. separata larvae were at least eightfold more sensitive than A. ipsilon larvae to Cry1Ab. Force-feeding showed that the observed midgut apical-membrane potential (Vam of M. separata larvae was significantly depolarized from −82.9 ± 6.6 mV to −19.9 ± 7.2 mV at 8 h after ingestion of 1 μg activated Cry1Ab, whereas no obvious changes were detected in A. ipsilon larvae with dosage of 5 μg Cry1Ab. The activated Cry1Ab caused a distinct concentration-dependent depolarization of the apical membrane; Vam was reduced by 50% after 14.7 ± 0.2, 9.8 ± 0.4, and 7.6 ± 0.6 min of treatment with 1, 5, and 10 μg/mL Cry1Ab, respectively. Cry1Ab showed a minimal effect on A. ipsilon larvae even at 20 μg/mL, and Vam decreased by 26.3% ± 2.3% after 15 min. The concentrations of Cry1Ab displayed no significant effect on the basolateral side of the epithelium. The Vam of A. ipsilon (−33.19 ± 6.29 mV, n = 51 was only half that of M. separata (−80.94 ± 6.95 mV, n = 75. The different degrees of sensitivity to Cry1Ab were speculatively associated with various habits, as well as the diverse physiological or biochemical characteristics of the midgut cell membranes.

  1. Cry1Ac and Vip3Aa proteins from Bacillus thuringiensis targeting Cry toxin resistance in Diatraea flavipennella and Elasmopalpus lignosellus from sugarcane

    Science.gov (United States)

    2017-01-01

    The biological potential of Vip and Cry proteins from Bacillus is well known and widely established. Thus, it is important to look for new genes showing different modes of action, selecting those with differentiated entomotoxic activity against Diatraea flavipennella and Elasmopalpus lignosellus, which are secondary pests of sugarcane. Therefore, Cry1 and Vip3 proteins were expressed in Escherichia coli, and their toxicities were evaluated based on bioassays using neonate larvae. Of those, the most toxic were Cry1Ac and Vip3Aa considering the LC50 values. Toxins from E. coli were purified, solubilized, trypsinized, and biotinylated. Brush Border Membrane Vesicles (BBMVs) were prepared from intestines of the two species to perform homologous and heterologous competition assays. The binding assays demonstrated interactions between Cry1Aa, Cry1Ac, and Vip3Aa toxins and proteins from the BBMV of D. flavipennella and E. lignosellus. Homologous competition assays demonstrated that binding to one of the BBMV proteins was specific for each toxin. Heterologous competition assays indicated that Vip3Aa was unable to compete for Cry1Ac toxin binding. Our results suggest that Cry1Ac and Vip3Aa may have potential in future production of transgenic sugarcane for control of D. flavipennella and E. lignosellus, but more research is needed on the potential antagonism or synergism of the toxins in these pests. PMID:28123906

  2. Cry1Ac and Vip3Aa proteins from Bacillus thuringiensis targeting Cry toxin resistance in Diatraea flavipennella and Elasmopalpus lignosellus from sugarcane

    Directory of Open Access Journals (Sweden)

    Ana Rita Nunes Lemes

    2017-01-01

    Full Text Available The biological potential of Vip and Cry proteins from Bacillus is well known and widely established. Thus, it is important to look for new genes showing different modes of action, selecting those with differentiated entomotoxic activity against Diatraea flavipennella and Elasmopalpus lignosellus, which are secondary pests of sugarcane. Therefore, Cry1 and Vip3 proteins were expressed in Escherichia coli, and their toxicities were evaluated based on bioassays using neonate larvae. Of those, the most toxic were Cry1Ac and Vip3Aa considering the LC50 values. Toxins from E. coli were purified, solubilized, trypsinized, and biotinylated. Brush Border Membrane Vesicles (BBMVs were prepared from intestines of the two species to perform homologous and heterologous competition assays. The binding assays demonstrated interactions between Cry1Aa, Cry1Ac, and Vip3Aa toxins and proteins from the BBMV of D. flavipennella and E. lignosellus. Homologous competition assays demonstrated that binding to one of the BBMV proteins was specific for each toxin. Heterologous competition assays indicated that Vip3Aa was unable to compete for Cry1Ac toxin binding. Our results suggest that Cry1Ac and Vip3Aa may have potential in future production of transgenic sugarcane for control of D. flavipennella and E. lignosellus, but more research is needed on the potential antagonism or synergism of the toxins in these pests.

  3. Cry1Ac and Vip3Aa proteins from Bacillus thuringiensis targeting Cry toxin resistance in Diatraea flavipennella and Elasmopalpus lignosellus from sugarcane.

    Science.gov (United States)

    Lemes, Ana Rita Nunes; Figueiredo, Camila Soares; Sebastião, Isis; Marques da Silva, Liliane; da Costa Alves, Rebeka; de Siqueira, Herbert Álvaro Abreu; Lemos, Manoel Victor Franco; Fernandes, Odair Aparecido; Desidério, Janete Apparecida

    2017-01-01

    The biological potential of Vip and Cry proteins from Bacillus is well known and widely established. Thus, it is important to look for new genes showing different modes of action, selecting those with differentiated entomotoxic activity against Diatraea flavipennella and Elasmopalpus lignosellus, which are secondary pests of sugarcane. Therefore, Cry1 and Vip3 proteins were expressed in Escherichia coli, and their toxicities were evaluated based on bioassays using neonate larvae. Of those, the most toxic were Cry1Ac and Vip3Aa considering the LC50 values. Toxins from E. coli were purified, solubilized, trypsinized, and biotinylated. Brush Border Membrane Vesicles (BBMVs) were prepared from intestines of the two species to perform homologous and heterologous competition assays. The binding assays demonstrated interactions between Cry1Aa, Cry1Ac, and Vip3Aa toxins and proteins from the BBMV of D. flavipennella and E. lignosellus. Homologous competition assays demonstrated that binding to one of the BBMV proteins was specific for each toxin. Heterologous competition assays indicated that Vip3Aa was unable to compete for Cry1Ac toxin binding. Our results suggest that Cry1Ac and Vip3Aa may have potential in future production of transgenic sugarcane for control of D. flavipennella and E. lignosellus, but more research is needed on the potential antagonism or synergism of the toxins in these pests.

  4. Frequency of alleles conferring resistance to the Bacillus thuringiensis toxins Cry1Ac and Cry2Ab in Australian populations of Helicoverpa punctigera (Lepidoptera: Noctuidae) from 2002 to 2006.

    Science.gov (United States)

    Downes, S; Parker, T L; Mahon, R J

    2009-04-01

    Helicoverpa punctigera and Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) are important pests of field and horticultural crops in Australia. The former is endemic to the continent, whereas the latter is also distributed in Africa and Asia. Although H. armigera rapidly developed resistance to virtually every group of insecticide used against it, there is only one report of resistance to an insecticide in H. punctigera. In 1996 the Australian cotton industry adopted Ingard, which expresses the Bacillus thuringiensis (Bt) toxin gene cry1Ac. In 2004/2005, Bollgard II (which expresses Cry1Ac and Cry2Ab) replaced Ingard and has subsequently been grown on 80% of the area planted to cotton, Gossypium hirsutum L. From 2002/2003 to 2006/2007, F2 screens were used to detect resistance to Cry1Ac or Cry2Ab. We detected no alleles conferring resistance to Cry1Ac; the frequency was < 0.0005 (n = 2,180 alleles), with a 95% credibility interval between 0 and 0.0014. However, during the same period, we detected alleles that confer resistance to Cry2Ab at a frequency of 0.0018 (n = 2,192 alleles), with a 95% credibility interval between 0.0005 and 0.0040. For both toxins, the experiment-wise detection probability was 94%, i.e., if there actually was a resistance allele in any tested lines, we would have detected it 94% of the time. The first isolation of Cry2Ab resistance in H. punctigera was before the widespread deployment of Bollgard II. This finding supports our published notion for H. armigera that alleles conferring resistance to Cry2Ab may be present at detectable frequencies in populations before selection by transgenic crops.

  5. An Improved Method for Purifying Cry1Ac Protein from Bacillus thuringiensis%一种纯化苏云金杆菌Cry1Ac蛋白的方法

    Institute of Scientific and Technical Information of China (English)

    王发祥; 刘永乐; 丁学知; 夏立秋

    2011-01-01

    The research of structure and function of Cry proteins was greatly restricted by their purification method. Therefore, a method that purifying Bacillus thuringiensis CrylAc protoxin by using isoelectric precipitation and purifying Bacillus thuringiensis CrylAc activated toxin by using molecular sieve chromatography was proposed. The results showed that the CrylAc protoxin had good effects of precipitation and purification when Ph value was 5.05; activating the purified protoxin to conduct molecular sieve chromatography, high concentration of CrylAc activated toxin could be obtained from the maximum peak of chromatography. This improved method not only has good purification effect, but also contribute a renovation to the purification method of Cry protein, which is significant for promoting researches on structure and function of Cry protein.%Cry蛋白的纯化技术在很大程度上制约了其结构与功能的研究.为此,提出了一种以等电点沉淀方法纯化苏云金杆菌Cry1Ac的原毒素,用分子筛层析纯化其活性毒素的方法.结果表明:Cry1Ac的原毒素在pH值5.05条件下沉淀纯化效果好;以纯化的原毒素激活进行分子筛层析,收集其最大峰便可获得较高浓度的活性毒素;该方法不仅纯化效果好,而且省去层析后的浓缩步骤,是Cry蛋白的纯化方法研究中的一次革新,对推动Cry蛋白的结构和功能研究具有重要的意义.

  6. Expresión de la toxina cry11aa de bacillus thuringiensis serovar. israelensis en asticcacaulis excentricus, para el control de larvas acuáticas de dípteros de la familia culicidae, vectores de enfermedades

    OpenAIRE

    Guevara, Óscar Enrique; Armengol, Gemma; Crickmore, Neil; Orduz,Sergio

    2007-01-01

    Los genes cry de Bacillus thuringiensis codifican para un diverso grupo de proteínas formadoras de cristales que exhiben actividad insecticida contra larvas de dípteros, lepidópteros y coleópteros, entre otros. La efectividad de los insecticidas basados en formulaciones de proteínas de B. thuringiensis puede ser mejorada usando bacterias prostecadas acuáticas como portadoras alternativas de los genes cry, ya que no se sedimentan rápidamente; las proteínas expresadas en el citoplasma están pro...

  7. Short-Term Evaluation in Growing Rats of Diet Containing Bacillus thuringiensis Cry1Ia12 Entomotoxin: Nutritional Responses and Some Safety Aspects

    Directory of Open Access Journals (Sweden)

    Luciane Mourão Guimarães

    2010-01-01

    Full Text Available The Cry1Ia12 entomotoxin from a Brazilian Bacillus thuringiensis strain is currently being expressed in cotton cultivars to confer resistance to insect-pests. The present study aimed to assess the effects of a diet containing Cry1Ia12 protein on growing rats. A test diet containing egg white and Cry1Ia12 (0.1% of total protein as a protein source was offered to rats for ten days. In addition, an acute toxicity bioassay was performed in rats with a single oral dose of the entomotoxin (12 mg/animal. No adverse effects were observed in the animals receiving the test diet when compared to those receiving a control diet (egg white. The analysed parameters included relative dry weight of internal organs, duodenum histology, blood biochemistry, and nutritional parameters. The results of the acute toxicity test showed no mortality or behaviour alteration. Thus, Cry1Ia12 toxin at the tested concentration does not cause deleterious effects on growing rats when incorporated in the diet for 10 days.

  8. The protoxin Cry1Ac of Bacillus thuringiensis improves the protection conferred by intranasal immunization with Brucella abortus RB51 in a mouse model.

    Science.gov (United States)

    González-González, Edith; García-Hernández, Ana Lilia; Flores-Mejía, Raúl; López-Santiago, Rubén; Moreno-Fierros, Leticia

    2015-02-25

    Brucellosis is a zoonotic disease affecting many people and animals worldwide. Preventing this infection requires improving vaccination strategies. The protoxin Cry1Ac of Bacillus thuringiensis is an adjuvant that, in addition to increasing the immunogenicity of different antigens, has shown to be protective in different models of parasitic infections. The objective of the present study was to test whether the intranasal co-administration of pCry1Ac with the RB51 vaccine strain of Brucella abortus confers protection against an intranasal challenge with the virulent strain B. abortus 2308 in BALB/c mice. The results showed that co-administration of pCry1Ac and RB51, increased the immunoprotection conferred by the vaccine as evidenced by the following: (1) decrease of the splenic bacterial load when challenged intranasally with the virulent strain; (2) greater in vivo cytotoxic activity in response to the transference of previously infected cells; (3) further proliferation of cytotoxic TCD8+ cells in response to stimulation with heat-inactivated bacteria; (4) increased production of TNF-α and IFN-γ; and (5) significant IgG2a response. These results indicate that the use of the Cry1Ac protein as a mucosal adjuvant via the intranasal route can be a promising alternative for improving current RB51 vaccine against brucellosis.

  9. Activity of Bacillus thuringiensis D(delta)-endotoxins against codling moth (Cydia pomonella L.) larvae

    NARCIS (Netherlands)

    Boncheva, R.; Dukiandjiev, S.; Minkov, I.; Maagd, de R.A.; Naimov, S.

    2006-01-01

    Solubilized protoxins of nine Cry1 and one hybrid Cry1 ¿-endotoxin from Bacillus thuringiensis were tested for their activity against larvae of the codling moth (Cydia pomonella L). Cry1Da was the most toxic, followed by Cry1Ab, Cry1Ba, and Cry1Ac, while Cry1Aa, Cry1Fa, Cry1Ia, and SN19 were still l

  10. MAPK signaling pathway alters expression of midgut ALP and ABCC genes and causes resistance to Bacillus thuringiensis Cry1Ac toxin in diamondback moth.

    Directory of Open Access Journals (Sweden)

    Zhaojiang Guo

    2015-04-01

    Full Text Available Insecticidal crystal toxins derived from the soil bacterium Bacillus thuringiensis (Bt are widely used as biopesticide sprays or expressed in transgenic crops to control insect pests. However, large-scale use of Bt has led to field-evolved resistance in several lepidopteran pests. Resistance to Bt Cry1Ac toxin in the diamondback moth, Plutella xylostella (L., was previously mapped to a multigenic resistance locus (BtR-1. Here, we assembled the 3.15 Mb BtR-1 locus and found high-level resistance to Cry1Ac and Bt biopesticide in four independent P. xylostella strains were all associated with differential expression of a midgut membrane-bound alkaline phosphatase (ALP outside this locus and a suite of ATP-binding cassette transporter subfamily C (ABCC genes inside this locus. The interplay between these resistance genes is controlled by a previously uncharacterized trans-regulatory mechanism via the mitogen-activated protein kinase (MAPK signaling pathway. Molecular, biochemical, and functional analyses have established ALP as a functional Cry1Ac receptor. Phenotypic association experiments revealed that the recessive Cry1Ac resistance was tightly linked to down-regulation of ALP, ABCC2 and ABCC3, whereas it was not linked to up-regulation of ABCC1. Silencing of ABCC2 and ABCC3 in susceptible larvae reduced their susceptibility to Cry1Ac but did not affect the expression of ALP, whereas suppression of MAP4K4, a constitutively transcriptionally-activated MAPK upstream gene within the BtR-1 locus, led to a transient recovery of gene expression thereby restoring the susceptibility in resistant larvae. These results highlight a crucial role for ALP and ABCC genes in field-evolved resistance to Cry1Ac and reveal a novel trans-regulatory signaling mechanism responsible for modulating the expression of these pivotal genes in P. xylostella.

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

    OpenAIRE

    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 resembl...

  12. Shared midgut binding sites for Cry1A.105, Cry1Aa, Cry1Ab, Cry1Ac and Cry1Fa proteins from Bacillus thuringiensis in two important corn pests, Ostrinia nubilalis and Spodoptera frugiperda.

    Directory of Open Access Journals (Sweden)

    Carmen Sara Hernández-Rodríguez

    Full Text Available First generation of insect-protected transgenic corn (Bt-corn was based on the expression of Cry1Ab or Cry1Fa proteins. Currently, the trend is the combination of two or more genes expressing proteins that bind to different targets. In addition to broadening the spectrum of action, this strategy helps to delay the evolution of resistance in exposed insect populations. One of such examples is the combination of Cry1A.105 with Cry1Fa and Cry2Ab to control O. nubilalis and S. frugiperda. Cry1A.105 is a chimeric protein with domains I and II and the C-terminal half of the protein from Cry1Ac, and domain III almost identical to Cry1Fa. The aim of the present study was to determine whether the chimeric Cry1A.105 has shared binding sites either with Cry1A proteins, with Cry1Fa, or with both, in O. nubilalis and in S. frugiperda. Brush-border membrane vesicles (BBMV from last instar larval midguts were used in competition binding assays with (125I-labeled Cry1A.105, Cry1Ab, and Cry1Fa, and unlabeled Cry1A.105, Cry1Aa, Cry1Ab, Cry1Ac, Cry1Fa, Cry2Ab and Cry2Ae. The results showed that Cry1A.105, Cry1Ab, Cry1Ac and Cry1Fa competed with high affinity for the same binding sites in both insect species. However, Cry2Ab and Cry2Ae did not compete for the binding sites of Cry1 proteins. Therefore, according to our results, the development of cross-resistance among Cry1Ab/Ac, Cry1A.105, and Cry1Fa proteins is possible in these two insect species if the alteration of shared binding sites occurs. Conversely, cross-resistance between these proteins and Cry2A proteins is very unlikely in such case.

  13. Interação de proteínas Vip3A e Cry1la10 de Bacillus thuringiensis com atividade inseticida a lepidópteros-praga

    OpenAIRE

    Marucci, Suzana Cristina [UNESP

    2015-01-01

    Vip3Aa and Cry1Ia proteins have potential in control of Lepidopteran pest and emerge as a promising alternative in the pest resistance management the Cry1A proteins, which has been highly used in the formulation of commercial insecticides based on Bacillus thuringiensis (Bt) and in transgenic plants. Therefore, this study aimed to cloning and expression of Vip3Aa42, Vip3Aa43 and Cry1Ia10 proteins in Escherichia coli, in order to analyze the correlation between the binding to receptors through...

  14. F2 screen for resistance to Bacillus thuringiensis Cry2Ab2-maize in field populations of Spodoptera frugiperda (Lepidoptera: Noctuidae) from the southern United States.

    Science.gov (United States)

    Niu, Ying; Qureshi, Jawwad A; Ni, Xinzhi; Head, Graham P; Price, Paula A; Meagher, Robert L; Kerns, David; Levy, Ronnie; Yang, Xiangbing; Huang, Fangneng

    2016-07-01

    The fall armyworm, Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae), is a target pest of transgenic maize and cotton expressing Bacillus thuringiensis (Bt) proteins in both North and South America. In 2013 and 2014, a total of 215 F2 two-parent families of S. frugiperda were established using single-pair mating of field individuals collected from seven locations in four states of the southern U.S.: Texas, Louisiana, Georgia, and Florida. The objective of the investigation was to detect resistance alleles in field populations to Cry2Ab2, a common Bt protein produced in transgenic maize and cotton. For each F2 family, 128 F2 neonates were screened on leaf tissue of Cry2Ab2 maize plants in the laboratory. A conservative estimate of the frequency of major Cry2Ab2 resistance alleles in S. frugiperda from the four states was 0.0023 with a 95% credibility interval of 0.0003-0.0064. In addition, six families were considered to likely possess minor resistance alleles at a frequency of 0.0082 with a 95% credibility interval of 0.0033-0.0152. One F2 family from Georgia (GA-15) was confirmed to possess a major resistance allele to the Cry2Ab2 protein. Larvae from this family survived well on whole maize plants expressing Cry2Ab2 protein and demonstrated a significant level (>15-fold) of resistance when fed with the same protein incorporated in a meridic diet. The detection of the major resistance allele along with the relatively abundant minor resistance alleles revealed in this study may have important implications for resistance management.

  15. Shared midgut binding sites for Cry1A.105, Cry1Aa, Cry1Ab, Cry1Ac and Cry1Fa proteins from Bacillus thuringiensis in two important corn pests, Ostrinia nubilalis and Spodoptera frugiperda

    OpenAIRE

    Carmen Sara Hernández-Rodríguez; Patricia Hernández-Martínez; Jeroen Van Rie; Baltasar Escriche; Juan Ferré

    2013-01-01

    First generation of insect-protected transgenic corn (Bt-corn) was based on the expression of Cry1Ab or Cry1Fa proteins. Currently, the trend is the combination of two or more genes expressing proteins that bind to different targets. In addition to broadening the spectrum of action, this strategy helps to delay the evolution of resistance in exposed insect populations. One of such examples is the combination of Cry1A.105 with Cry1Fa and Cry2Ab to control O. nubilalis and S. frugiperda. Cry1A....

  16. Screen of Bacillus thuringiensis toxins for transgenic rice to control Sesamia inferens and Chilo suppressalis

    Science.gov (United States)

    Transgenic rice to control stem borer damage is under development in China. To assess the potential of Bacillus thuringiensis (Bt) transgenes in stem borer control, the toxicity of five Bt protoxins (Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ba and Cry1Ca) against two rice stem borers, Sesamia inferens (pink stem...

  17. Activity of wild-type and hybrid Bacillus thuringiensis delta-endotoxins against Agrotis ipsilon

    NARCIS (Netherlands)

    Maagd, de R.A.; Weemen-Hendriks, M.; Molthoff, J.W.; Naimov, S.

    2003-01-01

    Twelve Cry1 and two Cry9 ?-endotoxins fromBacillus thuringiensis were tested for their activity against black cutworm (Agrotis ipsilon).A. ipsilon was not susceptible to many toxins, but three toxins had significant activity. Cry9Ca was the most toxic, followed by Cry1Aa and Cry1Fb. Hybrids between

  18. Separation,Purification and Insecticidal Activity of Cry8Ea2 Protein from Bacillus thuringiensis%苏云金杆菌Cry8Ea2蛋白的分离纯化及生物活性测定

    Institute of Scientific and Technical Information of China (English)

    王伟; 郭巍; 徐大庆; 孙伟明; 刘大群; 李瑞军

    2012-01-01

    Cry8 Ea2蛋白是一种新型苏云金杆菌杀虫晶体蛋白.通过穿梭载体pSXY422b将cry8Ea2导入Bt无晶体突变菌株中,获得工程菌HD8Ea2.Cry8Ea2蛋白在工程菌中获得表达.通过分子筛层析,成功获得纯化的130 kDa活性毒素蛋白.用改进的生物活性测定方法进行Cry8Ea2蛋白对华北大黑鳃金龟和暗黑鳃金龟一龄幼虫的杀虫活性测定,LC50分别为0.267 9μg/mL和0.071 9μg/mL.研究首次发现Cry8Ea2蛋白对华北大黑鳃金龟具有较高毒力,拓展了该蛋白的杀虫谱,为转基因工程提供了新资源.%Cry8Ea2 protein is a kind of insecticidal crystal protein of Bacillus thuringiensis. In this study, cry8Ea2 was cloned into E. coli- B. thuringiensist shuttle vector pSXY422b,resulting from recombinant pScry8Ea2, and pScry8Ea2 was transformed into the Bt, acrystalliferous mutant generating HD8Ea2 engineering strain. Cry8Ea2 protein was successfully expressed in HD8Ea2 stain. By anion exchange chromatography on molecular sieve chroma-tography,130 kDa of toxin protein Cry8Ea2 was purified. The activities of the Cry8Ea2 toxin were evaluated by improved bioassays, and the LC50 value to neonates of Holotrichia oblita and Holotrichia paralella was 0. 267 9 μg/mL and 0.071 9 μg/mL, respectively. Insecticidal activity of Cry8Ea2 to Holotrichia oblita was firstly reported in this study ,which will broaden the protein insecticidal spectrum and provide new resources for genetically modified project.

  19. Novel cloning vectors for Bacillus thuringiensis.

    Science.gov (United States)

    Baum, J A; Coyle, D M; Gilbert, M P; Jany, C S; Gawron-Burke, C

    1990-11-01

    Seven replication origins from resident plasmids of Bacillus thuringienis subsp. kurstaki HD263 and HD73 were cloned in Escherichia coli. Three of these replication origins, originating from plasmids of 43, 44, and 60 MDa, were used to construct a set of compatible shuttle vectors that exhibit structural and segregational stability in the Cry- strain B. thuringiensis HD73-26. These shuttle vectors, pEG597, pEG853, and pEG854, were designed with rare restriction sites that permit various adaptations, including the construction of small recombinant plasmids lacking antibiotic resistance genes. The cryIA(c) and cryIIA insecticidal crystal protein genes were inserted into these vectors to demonstrate crystal protein production in B. thuringiensis. Introduction of a cloned cryIA(c) gene from strain HD263 into a B. thuringiensis subsp. aizawai strain exhibiting good insecticidal activity against Spodoptera exigua resulted in a recombinant strain with an improved spectrum of insecticidal activity. Shuttle vectors of this sort should be valuable in future genetic studies of B. thuringiensis as well as in the development of B. thuringiensis strains for use as microbial pesticides.

  20. Expresión de la toxina Cry11Aa de Bacillus thuringiensis serovar. israelensis en Asticcacaulis excentricus, para el control de larvas acuáticas de dípteros de la familia Culicidae, vectores de enfermedades Expression of Bacillus thuringiensis serovar. israelensis toxins in Asticcacaulis excentricus to control dipteran larvae of vectors of diseases

    Directory of Open Access Journals (Sweden)

    Orduz Sergio

    2004-07-01

    Full Text Available Los genes cry de Bacillus thuringiensis codifican para un diverso grupo de proteínas formadoras de cristales que exhiben actividad insecticida contra larvas de dípteros, lepidópteros y coleópteros, entre otros. La efectividad de los insecticidas basados en formulaciones de proteínas de B. thuringiensis puede ser mejorada usando bacterias prostecadas acuáticas como portadoras alternativas de los genes cry, ya que no se sedimentan rápidamente; las proteínas expresadas en el citoplasma están protegidas de los rayos ultra violeta y, lo más importante, las larvas de los mosquitos se alimentan de ellas. Una cepa de referencia de Asticcacaulis excentricus fue transformada con el plásmido pSOD3, el cual contiene el gen que codifica para la proteína Cry1 1 Aa de B. thuringiensis serovar. israelensis. La expresión de la proteína recombinante fue evaluada por electroforesis de proteínas y por Western blot. El Western blot revelado con un anticuerpo policlonal anti-Cry1 1 Aa mostró una banda de 72 kDa correspondiente a la proteína Cry 11 Aa. La toxicidad de las cepas de A. excentricus transformadas fue evaluada en bioensayos con larvas de primer estadío del mosquito Culex quinquefasciatus. Se alcanzó un promedio de mortalidad del 50% de las larvas de primer instar a concentraciones de 23 ng/mL de la toxina. Otros bioensayos indican que A. excentricus recombinante es tóxica para larvas de primer instar de las especies Aedes aegypti y Anopheles albimanus. Los ensayos de flotabilidad indican que A. excentricus no sedimenta hasta pasados 7 días, mientras que B. thuringiensis serovar. israelensis sedimenta al cabo de algunas horas. Palabras clave: Asticcacaulis excentricus, Bacillus thuringiensis, bacterias prostecadas, dengue, malaria.Bacillus thuringiensis cry genes encode for a diverse group of crystal-forming proteins that exhibit insecticidal activity towards dipteran, lepidopteran and coleopteran larvae. The effectiveness of

  1. Binding of Cyt1Aa and Cry11Aa toxins of Bacillus thuringiensis serovar israelensis to brush border membrane vesicles of Tipula paludosa (Diptera: Nematocera) and subsequent pore formation.

    Science.gov (United States)

    Oestergaard, Jesko; Ehlers, Ralf-Udo; Martínez-Ramírez, Amparo C; Real, Maria Dolores

    2007-06-01

    Bacillus thuringiensis serovar israelensis (B. thuringiensis subsp. israelensis) produces four insecticidal crystal proteins (ICPs) (Cry4A, Cry4B, Cry11A, and Cyt1A). Toxicity of recombinant B. thuringiensis subsp. israelensis strains expressing only one of the toxins was determined with first instars of Tipula paludosa (Diptera: Nematocera). Cyt1A was the most toxic protein, whereas Cry4A, Cry4B, and Cry11A were virtually nontoxic. Synergistic effects were recorded when Cry4A and/or Cry4B was combined with Cyt1A but not with Cry11A. The binding and pore formation are key steps in the mode of action of B. thuringiensis subsp. israelensis ICPs. Binding and pore-forming activity of Cry11Aa, which is the most toxic protein against mosquitoes, and Cyt1Aa to brush border membrane vesicles (BBMVs) of T. paludosa were analyzed. Solubilization of Cry11Aa resulted in two fragments, with apparent molecular masses of 32 and 36 kDa. No binding of the 36-kDa fragment to T. paludosa BBMVs was detected, whereas the 32-kDa fragment bound to T. paludosa BBMVs. Only a partial reduction of binding of this fragment was observed in competition experiments, indicating a low specificity of the binding. In contrast to results for mosquitoes, the Cyt1Aa protein bound specifically to the BBMVs of T. paludosa, suggesting an insecticidal mechanism based on a receptor-mediated action, as described for Cry proteins. Cry11Aa and Cyt1Aa toxins were both able to produce pores in T. paludosa BBMVs. Protease treatment with trypsin and proteinase K, previously reported to activate Cry11Aa and Cyt1Aa toxins, respectively, had the opposite effect. A higher efficiency in pore formation was observed when Cyt1A was proteinase K treated, while the activity of trypsin-treated Cry11Aa was reduced. Results on binding and pore formation are consistent with results on ICP toxicity and synergistic effect with Cyt1Aa in T. paludosa.

  2. LSSP-PCR para la identificación de polimorfismos en el gen cry1B en cepas nativas de Bacillus thuringiensis

    Directory of Open Access Journals (Sweden)

    Martha Ilce Orozco Mera

    2012-02-01

    Full Text Available Normal 0 21 false false false ES-CO X-NONE X-NONE MicrosoftInternetExplorer4 Título en ingles: LSSP-PCR to identify polymorphisms in the gene cry1B of Bacillus thuringiensis native strain Resumen: Se estandarizó la técnica LSSP-PCR (reacción en cadena de la polimerasa con un único oligonucleótido en condiciones de baja astringencia, para identificar polimorfismos del gen cry1B en aislamientos nativos de Bacillus thuringiensis (Bt. Se evaluaron 164 aislamientos nativos colombianos identificándose el gen cry1Ba en 11 de estos aislamientos. Los 11 fragmentos amplificados, junto con el de la cepa de referencia Bt subsp. aizawai HD137, se analizaron por LSSP-PCR y los patrones electroforéticos obtenidos se compararon cualitativamente. Con los productos amplificados mediante el oligonucleótido directo se construyó un dendrograma utilizando UPGMA que  mostró tres agrupamientos con similitud de 83, 79 y 68%. La agrupación con 68% de similaridad correspondió al aislamiento nativo BtGC120 que presentó el patrón de bandas más variable. Con el oligonucleótido reverso el aislamiento BtGC120 mostró una menor variabilidad (43%. La secuencia nucleotidica obtenida de este fragmento de 806 pares de bases mostró una identidad de 93% con la secuencia de los genes cry1Bc1 de Bt morrisoni y cry1Bb1 de la cepa BT-EG5847. Se predijo del marco de lectura +3 una proteína de 268 residuos aminoácidicos, con 88% de identidad con la proteína Cry1Bc. Esta  secuencia reveló dos dominios, una endotoxina N implicada en la formación del poro y otra endotoxina M relacionada en el reconocimiento del receptor. La evaluación biológica del aislamiento BtGC120 sobre larvas de primer instar del insecto plaga Spodoptera frugiperda, mostró una CL50 de 1,896 ng de proteína total por cm2. Este estudio muestra que la LSSP-PCR es una técnica que permite identificar de una manera específica variaciones en las secuencias de los genes cry de Bt, con potencialidad

  3. Cloning and Expression Analysis of the cry1Ac Gene from Bacillus thuringiensis Strain 4.0718%Bt4.0718 cry1Ac基因的克隆与表达分析

    Institute of Scientific and Technical Information of China (English)

    丁学知; 张何; 孙运军; 黄潢; 张春艳; 夏立秋

    2006-01-01

    在基因库中比对14种cry1Ac基因序列,发现了同源性很高的上游启动子区域和下游终止子区域.根据这一同源序列设计引物,从Bt4.0718中扩增出包含双启动子和终止子的4.2 kb片段,用PCR-RFLP检测确定其中含有cry1Ac基因.然后将此片段克隆到穿梭载体pHT304中,转化大肠杆菌DH5α和Bt无晶体突变株XZM-101.同时,利用原子力显微镜观察发现重组菌株BXZM34能够产生菱形晶体.%14 cry1Ac genes from GenBank were aligned and the consensus regions in the upstream of promoter and in the downstream of terminator were found. Based on the consensus sequences, a pair of primers was designed and a 4.2 kb element was amplified that includes the dual overlapping promoter and the whole termination-associated sequence from Bacillus thuringiensis strain 4. 0718, and the amplified 4.2 kb element was confirmed to contain the crylAc gene by cryI sub-genetype PCR-RFLP cry gene typing system. The 4.2 kb element was cloned into Bt-E. coli shuttle vector pHT30.4 and the cry1Ac gene was also expressed in E. coli DH5α and acrystalliferous mutant XZM-101. Meanwhile,rhombic crystal was observed from recombinant strain BXZM34 by atomic force microscope.

  4. Transgenic elite indica rice plants expressing CryIAc delta-endotoxin of Bacillus thuringiensis are resistant against yellow stem borer (Scirpophaga incertulas).

    Science.gov (United States)

    Nayak, P; Basu, D; Das, S; Basu, A; Ghosh, D; Ramakrishnan, N A; Ghosh, M; Sen, S K

    1997-03-18

    Generation of insect-resistant, transgenic crop plants by expression of the insecticidal crystal protein (ICP) gene of Bacillus thuringiensis (Bt) is a standard crop improvement approach. In such cases, adequate expression of the most appropriate ICP against the target insect pest of the crop species is desirable. It is also considered advantageous to generate Bt-transgenics with multiple toxin systems to control rapid development of pest resistance to the ICP. Larvae of yellow stem borer (YSB), Scirpophaga incertulas, a major lepidopteran insect pest of rice, cause massive losses of rice yield. Studies on insect feeding and on the binding properties of ICP to brush border membrane receptors in the midgut of YSB larvae revealed that cryIAb and cryIAc are two individually suitable candidate genes for developing YSB-resistant rice. Programs were undertaken to develop Bt-transgenic rice with these ICP genes independently in a single cultivar. A cryIAc gene was reconstructed and placed under control of the maize ubiquitin 1 promoter, along with the first intron of the maize ubiquitin 1 gene, and the nos terminator. The gene construct was delivered to embryogenic calli of IR64, an elite indica rice cultivar, using the particle bombardment method. Six highly expressive independent transgenic ICP lines were identified. Molecular analyses and insect-feeding assays of two such lines revealed that the transferred synthetic cryIAc gene was expressed stably in the T2 generation of these lines and that the transgenic rice plants were highly toxic to YSB larvae and lessened the damage caused by their feeding.

  5. Toxicity of Bacillus thuringiensis (L.) Cry proteins against summer fruit tortrix (Adoxophyes orana - Fischer von Rösslerstamm).

    Science.gov (United States)

    Radosavljevic, Jelena; Naimov, Samir

    2016-07-01

    The activity of seven Cry1, one Cry9 and one hybrid Cry1 protoxins against neonate larvae of summer fruit tortrix (Adoxophyes orana - Fischer von Rösslerstamm) has been investigated. Cry1Ia is identified as the most toxic protein, followed by Cry1Aa and Cry1Ac. Cry1Ca, Cry1Cb, Cry1Da and Cry1Fa were less active, while SN19 (Cry1 hybrid protein with domain composition 1Ba/1Ia/1Ba) and Cry9Aa exhibited negligible toxicity against A. orana. In vitro trypsin-activated Cry1Ac is still less active than Cry1Ia protoxin, suggesting that toxicity of Cry1Ia is most probably due to more complex differences in further downstream processing, toxin-receptor interactions and pore formation in A. orana's midgut epithelium.

  6. Isolation and Identification of Bacillus thuringiensis Strains Containing cry8 Genes%含cry8型基因苏云金芽孢杆菌的分离和鉴定

    Institute of Scientific and Technical Information of China (English)

    王志鑫; 束长龙; 申培立; 苏旭东; 李志辉; 于妍; 张先舟; 马雯; 檀建新

    2014-01-01

    It is very important to explore the source of both Bacillus thuringiensis and its cry gene for pest con-trol. In this study,using a method that the soil suspensions were incubated at 80 ℃ for 20 min in a water bath,forty two Bt strains were isolated from 254 soil samples which were collected from different counties of Hebei province. Ten strains of them are identified that they contain the different plasmids harboring cry8 gene. PCR-RFLP analysis revealed that:One strain may have 1 or more kinds of plasmids;PCR-RFLP results showed that there were multiple cry8-type genes in the strains and one strain usually contained two subtypes of cry8 gene,for example,cry8E and cry8 F which were often coexist in the same strain;The Cry8-type insecticidal crystal proteins has a molecular weight around 130 -140 kDa with spherical parasporal crystals in shape. The soil samples containing the cry8-type Bt strains were collected from Baoding, Hengshui, Handan, Langfang, Tangshan and Xingtai areas of Hebei province with various vegetation. These results implied that the cry8-type Bt strains extensively distribute in Hebei province and may provide abundant resources of Bt with insecticidal activity against the cockchafer pests.%为发掘对蛴螬有防治作用的苏云金芽孢杆菌及其cry基因,在河北省各县市共采集土壤样品254份,采用温度筛选法分离出Bt菌株42株,经PCR-RFLP方法鉴定,其中10株含有cry8型基因。这些菌株有如下特点:同一菌株含1种或多种质粒;PCR-RFLP鉴定证明有多种cry8基因存在,多数菌株中含2种cry8基因,如cry8E和cry8F常共存于同一菌株中;Cry8杀虫晶体蛋白分子量约为130~140 kDa,晶体形态均为球形。这10株菌采样地点分布于保定、衡水、邯郸、廊坊、唐山和邢台等地,植被类型多样化,说明cry8型Bt菌株在河北省分布广泛,可为蛴螬类害虫的防治提供丰富的Bt资源。

  7. Diversity in gut microflora of Helicoverpa armigera populations from different regions in relation to biological activity of Bacillus thuringiensis δ-endotoxin Cry1Ac.

    Science.gov (United States)

    Paramasiva, Inakarla; Shouche, Yogesh; Kulkarni, Girish Jayant; Krishnayya, Pulipaka Venkata; Akbar, Shaik Mohammed; Sharma, Hari Chand

    2014-12-01

    Transgenic crops expressing toxin proteins from Bacillus thuringiensis (Bt) have been deployed on a large scale for management of Helicoverpa armigera. Resistance to Bt toxins has been documented in several papers, and therefore, we examined the role of midgut microflora of H. armigera in its susceptibility to Bt toxins. The susceptibility of H. armigera to Bt toxin Cry1Ac was assessed using Log-dose-Probit analysis, and the microbial communities were identified by 16S rRNA sequencing. The H. armigera populations from nine locations harbored diverse microbial communities, and had some unique bacteria, suggesting a wide geographical variation in microbial community in the midgut of the pod borer larvae. Phylotypes belonging to 32 genera were identified in the H. armigera midgut in field populations from nine locations. Bacteria belonging to Enterobacteriaceae (Order Bacillales) were present in all the populations, and these may be the common members of the H. armigera larval midgut microflora. Presence and/or absence of certain species were linked to H. armigera susceptibility to Bt toxins, but there were no clear trends across locations. Variation in susceptibility of F1 neonates of H. armigera from different locations to the Bt toxin Cry1Ac was found to be 3.4-fold. These findings support the idea that insect migut microflora may influence the biological activity of Bt toxins.

  8. Detection of cry1 genes in Bacillus thuringiensis isolates from South of Brazil and activity against Aanticarsia gemmatalis (Lepidoptera:Noctuidae

    Directory of Open Access Journals (Sweden)

    Bobrowski Vera Lucia

    2001-01-01

    Full Text Available The bacterium Bacillus thuringiensis (Bt is characterized by its ability to produce proteic crystalline inclusions during sporulation. Cry1 protein has insecticidal activity and is highly specific to certain insects and not toxic to unrelated insects, plants or vertebrates. In this work, the patogenicity of twelve Bt isolates was tested against Anticarsia gemmatalis, one of the most important insect pests of soybeans. Spore-crystal complex was applied to the surface of artificial diets and the mortality of A. gemmatalis larvae was assessed seven days after each treatment. When compared to a control Bt isolate known by its high toxicity to A. gemmatalis larvae, four novel Bt isolates exhibited even higher toxic activities against the insect, resulting in more than 90% mortality. PCR was used to amplify DNA fragments related to known cry1 genes. Bt strains with high toxicity produced expected PCR products of around 280 bp, whereas non-toxic or low toxic strains did not produce any PCR product or showed amplified fragments of different sizes. Toxic Bt isolates also exhibited an expected protein profile when total protein extracts were evaluated by SDS-PAGE.

  9. Interaction of Bacillus thuringiensis Cry1 and Vip3A proteins with Spodoptera frugiperda midgut binding sites

    OpenAIRE

    Sena, J.A.D. [UNESP; Hernández Rodríguez, Carmen Sara; Ferré Manzanero, Juan

    2009-01-01

    Vip3Aa, Vip3Af, Cry1Ab, and Cry1Fa were tested for their toxicities and binding interactions. Vip3A proteins were more toxic than Cry1 proteins. Binding assays showed independent specific binding sites for Cry1 and Vip3A proteins. Cry1Ab and Cry1Fa competed for the same binding sites, whereas Vip3Aa competed for those of Vip3Af.

  10. Interaction of Bacillus thuringiensis Cry1 and Vip3A proteins with Spodoptera frugiperda midgut binding sites.

    Science.gov (United States)

    Sena, Janete A D; Hernández-Rodríguez, Carmen Sara; Ferré, Juan

    2009-04-01

    Vip3Aa, Vip3Af, Cry1Ab, and Cry1Fa were tested for their toxicities and binding interactions. Vip3A proteins were more toxic than Cry1 proteins. Binding assays showed independent specific binding sites for Cry1 and Vip3A proteins. Cry1Ab and Cry1Fa competed for the same binding sites, whereas Vip3Aa competed for those of Vip3Af.

  11. Interaction of Bacillus thuringiensis Cry1 and Vip3A Proteins with Spodoptera frugiperda Midgut Binding Sites▿

    Science.gov (United States)

    Sena, Janete A. D.; Hernández-Rodríguez, Carmen Sara; Ferré, Juan

    2009-01-01

    Vip3Aa, Vip3Af, Cry1Ab, and Cry1Fa were tested for their toxicities and binding interactions. Vip3A proteins were more toxic than Cry1 proteins. Binding assays showed independent specific binding sites for Cry1 and Vip3A proteins. Cry1Ab and Cry1Fa competed for the same binding sites, whereas Vip3Aa competed for those of Vip3Af. PMID:19181834

  12. Is the Cry1Ab protein from Bacillus thuringiensis (Bt) taken up by plants from soils previously planted with Bt corn and by carrot from hydroponic culture?

    Science.gov (United States)

    Icoz, I; Andow, D; Zwahlen, C; Stotzky, G

    2009-07-01

    The uptake of the insecticidal Cry1Ab protein from Bacillus thuringiensis (Bt) by various crops from soils on which Bt corn had previously grown was determined. In 2005, the Cry1Ab protein was detected by Western blot in tissues (leaves plus stems) of basil, carrot, kale, lettuce, okra, parsnip, radish, snap bean, and soybean but not in tissues of beet and spinach and was estimated by enzyme-linked immunosorbent assay (ELISA) to be 0.05 +/- 0.003 ng g(-1) of fresh plant tissue in basil, 0.02 +/- 0.014 ng g(-1) in okra, and 0.34 +/- 0.176 ng g(-1) in snap bean. However, the protein was not detected by ELISA in carrot, kale, lettuce, parsnip, radish, and soybean or in the soils by Western blot. In 2006, the Cry1Ab protein was detected by Western blot in tissues of basil, carrot, kale, radish, snap bean, and soybean from soils on which Bt corn had been grown the previous year and was estimated by ELISA to be 0.02 +/- 0.014 ng g(-1) of fresh plant tissue in basil, 0.19 +/- 0.060 ng g(-1) in carrot, 0.05 +/- 0.018 ng g(-1) in kale, 0.04 +/- 0.022 ng g(-1) in radish, 0.53 +/- 0.170 ng g(-1) in snap bean, and 0.15 +/- 0.071 ng g(-1) in soybean. The Cry1Ab protein was also detected by Western blot in tissues of basil, carrot, kale, radish, and snap bean but not of soybean grown in soil on which Bt corn had not been grown since 2002; the concentration was estimated by ELISA to be 0.03 +/- 0.021 ng g(-1) in basil, 0.02 +/- 0.008 ng g(-1) in carrot, 0.04 +/- 0.017 ng g(-1) in kale, 0.02 +/- 0.012 ng g(-1) in radish, 0.05 +/- 0.004 ng g(-1) in snap bean, and 0.09 +/- 0.015 ng g(-1) in soybean. The protein was detected by Western blot in 2006 in most soils on which Bt corn had or had not been grown since 2002. The Cry1Ab protein was detected by Western blot in leaves plus stems and in roots of carrot after 56 days of growth in sterile hydroponic culture to which purified Cry1Ab protein had been added and was estimated by ELISA to be 0.08 +/- 0.021 and 0.60 +/- 0.148 ng g(-1) of

  13. Rapid topology probing using fluorescence spectroscopy in planar lipid bilayer: the pore-forming mechanism of the toxin Cry1Aa of Bacillus thuringiensis.

    Science.gov (United States)

    Groulx, Nicolas; Juteau, Marc; Blunck, Rikard

    2010-11-01

    Pore-forming toxins, many of which are pathogenic to humans, are highly dynamic proteins that adopt a different conformation in aqueous solution than in the lipid environment of the host membrane. Consequently, their crystal structures obtained in aqueous environment do not reflect the active conformation in the membrane, making it difficult to deduce the molecular determinants responsible for pore formation. To obtain structural information directly in the membrane, we introduce a fluorescence technique to probe the native topology of pore-forming toxins in planar lipid bilayers and follow their movement during pore formation. Using a Förster resonance energy transfer (FRET) approach between site-directedly labeled proteins and an absorbing compound (dipicrylamine) in the membrane, we simultaneously recorded the electrical current and fluorescence emission in horizontal planar lipid bilayers formed in plastic chips. With this system, we mapped the topology of the pore-forming domain of Cry1Aa, a biological pesticide from Bacillus thuringiensis, by determining the location of the loops between its seven α helices. We found that the majority of the toxins initially traverse from the cis to the trans leaflet of the membrane. Comparing the topologies of Cry1Aa in the active and inactive state in order to identify the pore-forming mechanism, we established that only the α3-α4 hairpin translocates through the membrane from the trans to the cis leaflet, whereas all other positions remained constant. As toxins are highly dynamic proteins, populations that differ in conformation might be present simultaneously. To test the presence of different populations, we designed double-FRET experiments, where a single donor interacts with two acceptors with very different kinetics (dipicrylamine and oxonol). Due to the nonlinear response of FRET and the dynamic change of the acceptor distribution, we can deduce the distribution of the acceptors in the membrane from the time

  14. Immunotoxicological evaluation of corn genetically modified with Bacillus thuringiensis Cry1Ah gene by a 30-day feeding study in BALB/c mice.

    Directory of Open Access Journals (Sweden)

    Yan Song

    Full Text Available This study was to investigate the immunotoxicological potential of corn genetically modified (GM with Bacillus thuringiensis (Bt Cry1Ah gene in BALB/c mice. Female BALB/c mice were randomly assigned to one of the four groups: the negative control group, the parental corn group, the GM corn group and the positive control group with 10 mice per group. Mice in the GM corn group and the parental corn group were fed with diets containing 70% corresponding corn for 30 days. Mice in the negative control group and the positive control group were fed with AIN93G diet, administered with saline or 200 mg/kg of cyclophosphamide (CY via intraperitoneal injection 24 h before the termination of the study, respectively. At the end of the study, the immunotoxicological effects of the GM corn were evaluated through immunopathology parameters including body and organ weights, hematology and clinical chemistry parameters, histological examination, peripheral blood lymphocytes phenotype; humoral immunity including antibody plaque-forming cell, serum immunoglobulin, cytokine and half hemolysis value; cellular immunity such as mitogen-induced splenocyte proliferation, cytotoxic T-lymphocyte reaction, delayed-type hypersensitivity reaction; non-specific immunity including phagocytic activities of phagocytes, natural killer cell activity. A single dose of cyclophosphamide (200 mg/kg bw was found to have significant adverse effects on immunopathology, cellular immunity, and humoral immunity in mice. The corn genetically modified with Bt Cry1Ah gene is considered consistent with the parental corn in terms of immunopathology, humoral immunity, cellular immunity and non-specific immunity. No adverse immunotoxicological effects of GM corn with Bt Cry1Ah gene were found when feeding mice for 30 days.

  15. Susceptibility of Spodoptera exigua to 9 toxins from Bacillus thuringiensis.

    Science.gov (United States)

    Hernández-Martínez, Patricia; Ferré, Juan; Escriche, Baltasar

    2008-03-01

    Nine of the most common lepidopteran active Cry proteins from Bacillus thuringiensis have been tested for activity against Spodoptera exigua. Because of possible intraspecific variability, three laboratory strains (FRA, HOL, and MUR) have been used. Mortality assays were performed with the three strains. LC(50) values for the active toxins were determined to the FRA and the HOL strains, whereas susceptibility of the MUR strain was assessed using only two concentrations. The results showed that Cry1Ca, Cry1Da, and Cry1Fa were the most effective toxins with all strains. Cry1Ab was found effective for the HOL strain, but very little effective against FRA (6.5-fold) and MUR strains. Cry1Aa and Cry1Ac were marginally toxic to all strains, whereas the rest of the toxins tested (Cry1Ba, Cry2Aa, and Cry2Ab) were non toxic. Significant differences in susceptibility among strains were also found for Cry1Da, being the FRA strain 25-fold more susceptible than the HOL strain. Growth inhibition, as an additional susceptibility parameter, was determined in the FRA strain with the 9 toxins. The toxicity profile obtained differed from that observed in mortality assays. Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ca, Cry1Da, and Cry1Fa toxins produced a similar larval growth inhibition. Cry2Aa had a lower but clear effect on larval growth inhibition, whereas Cry1Ba and Cry2Ab did not have any effect.

  16. Estudo da toxicidade de proteínas (Cry) recombinantes de Bacillus thuringiensis, utilizando o sistema de expressão baseado em baculovírus e células de inseto

    OpenAIRE

    Aguiar, Raimundo Wagner de Souza

    2007-01-01

    Os genes cry1Ca, cry2Ab e cry10Aa de diferentes estirpes brasileiras de Bacillus thuringiensis foram amplificados por PCR, clonados em um vetor de clonagem e seqüenciados. As análises das seqüências mostraram alta identidade com outros genes cry já descritos. Os genes foram removidos dos vetores de clonagem e introduzidos em um plasmídeo vetor de transferência (pSynXIVVI+X3) para construção de baculovírus recombinantes por recombinação homóloga. Os vírus recombinantes foram purificados por di...

  17. Specific binding of Bacillus thuringiensis Cry2A insecticidal proteins to a common site in the midgut of Helicoverpa species.

    Science.gov (United States)

    Hernández-Rodríguez, Carmen Sara; Van Vliet, Adri; Bautsoens, Nadine; Van Rie, Jeroen; Ferré, Juan

    2008-12-01

    For a long time, it has been assumed that the mode of action of Cry2A toxins was unique and different from that of other three-domain Cry toxins due to their apparent nonspecific and unsaturable binding to an unlimited number of receptors. However, based on the homology of the tertiary structure among three-domain Cry toxins, similar modes of action for all of them are expected. To confirm this hypothesis, binding assays were carried out with (125)I-labeled Cry2Ab. Saturation assays showed that Cry2Ab binds in a specific and saturable manner to brush border membrane vesicles (BBMVs) of Helicoverpa armigera. Homologous-competition assays with (125)I-Cry2Ab demonstrated that this toxin binds with high affinity to binding sites in H. armigera and Helicoverpa zea midgut. Heterologous-competition assays showed a common binding site for three toxins belonging to the Cry2A family (Cry2Aa, Cry2Ab, and Cry2Ae), which is not shared by Cry1Ac. Estimation of K(d) (dissociation constant) values revealed that Cry2Ab had around 35-fold less affinity than Cry1Ac for BBMV binding sites in both insect species. Only minor differences were found regarding R(t) (concentration of binding sites) values. This study questions previous interpretations from other authors performing binding assays with Cry2A toxins and establishes the basis for the mode of action of Cry2A toxins.

  18. Specific Binding of Bacillus thuringiensis Cry2A Insecticidal Proteins to a Common Site in the Midgut of Helicoverpa Species▿

    Science.gov (United States)

    Hernández-Rodríguez, Carmen Sara; Van Vliet, Adri; Bautsoens, Nadine; Van Rie, Jeroen; Ferré, Juan

    2008-01-01

    For a long time, it has been assumed that the mode of action of Cry2A toxins was unique and different from that of other three-domain Cry toxins due to their apparent nonspecific and unsaturable binding to an unlimited number of receptors. However, based on the homology of the tertiary structure among three-domain Cry toxins, similar modes of action for all of them are expected. To confirm this hypothesis, binding assays were carried out with 125I-labeled Cry2Ab. Saturation assays showed that Cry2Ab binds in a specific and saturable manner to brush border membrane vesicles (BBMVs) of Helicoverpa armigera. Homologous-competition assays with 125I-Cry2Ab demonstrated that this toxin binds with high affinity to binding sites in H. armigera and Helicoverpa zea midgut. Heterologous-competition assays showed a common binding site for three toxins belonging to the Cry2A family (Cry2Aa, Cry2Ab, and Cry2Ae), which is not shared by Cry1Ac. Estimation of Kd (dissociation constant) values revealed that Cry2Ab had around 35-fold less affinity than Cry1Ac for BBMV binding sites in both insect species. Only minor differences were found regarding Rt (concentration of binding sites) values. This study questions previous interpretations from other authors performing binding assays with Cry2A toxins and establishes the basis for the mode of action of Cry2A toxins. PMID:18931285

  19. Complete Genome Sequence of Bacillus thuringiensis subsp. thuringiensis Strain IS5056, an Isolate Highly Toxic to Trichoplusia ni

    Science.gov (United States)

    Murawska, Emilia; Fiedoruk, Krzysztof; Bideshi, Dennis K.

    2013-01-01

    The genome sequence of the entomopathogen Bacillus thuringiensis subsp. thuringiensis strain IS5056 was determined. The chromosome is composed of 5,491,935 bp. In addition, IS5056 harbors 14 plasmids ranging from 6,880 to 328,151 bp, four of which contain nine insecticidal protein genes, cry1Aa3, cry1Ab21, cry1Ba1, cry1Ia14, cry2Aa9, cry2Ab1, vip1, vip2, and vip3Aa10. PMID:23516221

  20. Occurrence of natural Bacillus thuringiensis contaminants and residues of Bacillus thuringiensis-based insecticides on fresh fruits and vegetables

    DEFF Research Database (Denmark)

    Frederiksen, Kristine; Rosenquist, Hanne; Jørgensen, Kirsten

    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...

  1. Toxicity and binding analyses of Bacillus thuringiensis toxin Vip3A in Cry1Ac-resistant and-susceptible strains of Helicoverpa armigera (Hübner)

    Institute of Scientific and Technical Information of China (English)

    ZHANG Qian; CHEN Li-zhen; LU Qiong; ZHANG Yan; LIANG Ge-mei

    2015-01-01

    The Bacil us thuringiensis vegetative insecticidal protein, Vip3A, represents a new family of Bt toxin and is currently ap-plied to commercial transgenic cotton. To determine whether the Cry1Ac-resistant Helicoverpa armigera is cross-resistant to Vip3Aa protein, insecticidal activities, proteolytic activations and binding properties of Vip3Aa toxin were investigated using Cry1Ac-susceptible (96S) and Cry1Ac-resistant H. armigera strain (Cry1Ac-R). The toxicity of Vip3Aa in Cry1Ac-R slightly reduced compared with 96S, the resistance ratio was only 1.7-fold. The digestion rate of ful-length Vip3Aa by gut juice extracts from 96S was little faster than that from Cry1Ac-R. Surface plasmon resonance (SPR) showed there was no signiifcant difference between the binding afifnity of Vip3Aa and BBMVs between 96S and Cry1Ac-R strains, and there was no signiifcant competitive binding between Vip3Aa and Cry1Ac in susceptible or resistant strains. So there had little cross-resistance between Vip3Aa and Cry1Ac,Vip3A+Cry proteins maybe the suitable pyramid strategy to control H. armigera in China in the future.

  2. Layer-by-layer films containing peptides of the Cry1Ab16 toxin from Bacillus thuringiensis for potential biotechnological applications.

    Science.gov (United States)

    Plácido, Alexandra; de Oliveira Farias, Emanuel Airton; Marani, Mariela M; Vasconcelos, Andreanne G; Mafud, Ana C; Mascarenhas, Yvonne P; Eiras, Carla; Leite, José R S A; Delerue-Matos, Cristina

    2016-04-01

    Cry1Ab16 is a toxin of crystalline insecticidal proteins that has been widely used in genetically modified organisms (GMOs) to gain resistance to pests. For the first time, in this study, peptides derived from the immunogenic Cry1Ab16 toxin (from Bacillus thuringiensis) were immobilized as layer-by-layer (LbL) films. Given the concern about food and environmental safety, a peptide with immunogenic potential, PcL342-354C, was selected for characterization of the electrochemical, optical, and morphological properties. The results obtained by cyclic voltammetry (CV) showed that the peptide have an irreversible oxidation process in electrolyte of 0.1 mol · L(-1) potassium phosphate buffer (PBS) at pH7.2. It was also observed that the electrochemical response of the peptide is governed mainly by charge transfer. In an attempt to maximize the electrochemical signal of peptide, it was intercalated with natural (agar, alginate and chitosan) or synthetic polymers (polyethylenimine (PEI) and poly(sodium 4-styrenesulfonate (PSS)). The presence of synthetic polymers on the film increased the electrochemical signal of PcL342-354C up to 100 times. Images by Atomic Force Microscopy (AFM) showed that the immobilized PcL342-354C formed self-assembled nanofibers with diameters ranging from 100 to 200 nm on the polymeric film. By UV-Visible spectroscopy (UV-Vis) it was observed that the ITO/PEI/PSS/PcL342-354C film grows linearly up to the fifth layer, thereafter tending to saturation. X-ray diffraction confirmed the presence on the films of crystalline ITO and amorphous polypeptide phases. In general, the ITO/PEI/PSS/PcL342-354C film characterization proved that this system is an excellent candidate for applications in electrochemical sensors and other biotechnological applications for GMOs and environmental indicators.

  3. Studies about Bacillus thuringiensis Strain Containing New cry Gene %一株含cry新基因的苏云金杆菌菌株的研究

    Institute of Scientific and Technical Information of China (English)

    宋萍; 郭丽伟; 苏俊平; 王勤英

    2011-01-01

    A high toxicity Bacillus thuringiensis (Bt)strain XPZ-9 against Lepidoptera pests was isolated and studied further. The strain formed the bipyramidal crystal and spherical crystal observed by optical microscope. SDSPAGE analysis revealed molecular weight of insecticidal crystal protein in this strain was 70 kDa. By using of PCRRFLP identification method,new crylAh gene was found in this strain. The bioassay results indicated Bt XPZ-9 toxin protein had high insecticidal activity against several Lepidoptera pests,such as 2 instar larvae of Helicoverpa armigera,Plutella xylostella, Ostrinia furnacalis, Hyphantria cunea.%苏云金杆菌XPZ-9菌株是从土壤中分离的对鳞翅目幼虫有特异杀虫活性的新菌株.光学显微镜下观察到该菌株可产生菱形和圆形伴胞晶体,SDS-PAGE 检测表达的主要蛋白条带分子量约为70 kDa.PCR-RFLP分析结果表明该菌株含有新基因cry1Ah.生物活性测定表明该菌株对鳞翅目害虫棉铃虫、小菜蛾、亚洲玉米螟和美国白蛾的二龄幼虫都具有较强的杀虫活性.

  4. Identificação e caracterização de genes vip e cry coleóptero‑específicos em isolados de Bacillus thuringiensis

    Directory of Open Access Journals (Sweden)

    Meire de Cássia Alves

    2011-09-01

    Full Text Available O objetivo deste trabalho foi identificar e caracterizar os genes cry3, vip1, vip2 e vip1/vip2 em uma coleção de 1.078 isolados de Bacillus thuringiensis potencialmente tóxicos para larvas de coleópteros. Foram utilizados pares de oligonucleotídeos iniciadores gerais obtidos a partir de regiões conservadas dos genes e do alinhamento de sequências consenso. Posteriormente, os isolados positivos foram caracterizados por meio da técnica de PCR‑RFLP, tendo-se utilizado enzimas de restrição específicas, para identificar novas subclasses de genes nos isolados. Cento e cinquenta e um isolados foram positivos para os genes avaliados, com maior frequência para o gene vip1/vip2 (139 isolados. Pela técnica de PCR‑RFLP, foram observados 14 perfis polimórficos, o que indica a presença de diferentes alelos e, consequentemente, de distintas subclasses desses genes.

  5. Binding Sites for Bacillus thuringiensis Cry2Ae Toxin on Heliothine Brush Border Membrane Vesicles Are Not Shared with Cry1A, Cry1F, or Vip3A Toxin ▿

    OpenAIRE

    Gouffon, C.; van Vliet, A.; Van Rie, J; Jansens, S.; Jurat-Fuentes, J. L.

    2011-01-01

    The use of combinations of Bacillus thuringiensis (Bt) toxins with diverse modes of action for insect pest control has been proposed as the most efficient strategy to increase target range and delay the onset of insect resistance. Considering that most cases of cross-resistance to Bt toxins in laboratory-selected insect colonies are due to alteration of common toxin binding sites, independent modes of action can be defined as toxins sharing limited or no binding sites in brush border membrane...

  6. Complete Genome Sequence of Bacillus thuringiensis subsp. chinensis Strain CT-43▿

    Science.gov (United States)

    He, Jin; Wang, Jieping; Yin, Wen; Shao, Xiaohu; Zheng, Huajun; Li, Mingshun; Zhao, Youwen; Sun, Ming; Wang, Shengyue; Yu, Ziniu

    2011-01-01

    Bacillus thuringiensis has been widely used as an agricultural biopesticide for a long time. As a producing strain, B. thuringiensis subsp. chinensis strain CT-43 is highly toxic to lepidopterous and dipterous insects. It can form various parasporal crystals consisting of Cry1Aa3, Cry1Ba1, Cry1Ia14, Cry2Aa9, and Cry2Ab1. During fermentation, it simultaneously generates vegetative insecticidal protein Vip3Aa10 and the insecticidal nucleotide analogue thuringiensin. Here, we report the finished, annotated genome sequence of B. thuringiensis strain CT-43. PMID:21551307

  7. Complete genome sequence of Bacillus thuringiensis subsp. chinensis strain CT-43.

    Science.gov (United States)

    He, Jin; Wang, Jieping; Yin, Wen; Shao, Xiaohu; Zheng, Huajun; Li, Mingshun; Zhao, Youwen; Sun, Ming; Wang, Shengyue; Yu, Ziniu

    2011-07-01

    Bacillus thuringiensis has been widely used as an agricultural biopesticide for a long time. As a producing strain, B. thuringiensis subsp. chinensis strain CT-43 is highly toxic to lepidopterous and dipterous insects. It can form various parasporal crystals consisting of Cry1Aa3, Cry1Ba1, Cry1Ia14, Cry2Aa9, and Cry2Ab1. During fermentation, it simultaneously generates vegetative insecticidal protein Vip3Aa10 and the insecticidal nucleotide analogue thuringiensin. Here, we report the finished, annotated genome sequence of B. thuringiensis strain CT-43.

  8. A pangenomic study of Bacillus thuringiensis.

    Science.gov (United States)

    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. A pangenomic study of Bacillus thuringiensis

    Institute of Scientific and Technical Information of China (English)

    Yongjun Fang; Songnian Hu; Jie Zhang; Ibrahim A1-Mssallem; Jun Yu; Zhaolong Li; Jiucheng Liu; Changlong Shu; Xumin Wang; Xiaowei Zhang; Xiaoguang Yu; Duojun Zhao; Guiming Liu

    2011-01-01

    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 2.3-2.8 Mb and 5.0-5.6 Mb.We concluded that high-coverage sequence assemblies from multiple strains,before all the gaps are closed,are very useful for pangenomic studies.

  10. Bioinformatic analysis for allergenicity assessment of Bacillus thuringiensis Cry proteins expressed in insect-resistant food crops.

    Science.gov (United States)

    Randhawa, Gurinder Jit; Singh, Monika; Grover, Monendra

    2011-02-01

    The novel proteins introduced into the genetically modified (GM) crops need to be evaluated for the potential allergenicity before their introduction into the food chain to address the safety concerns of consumers. At present, there is no single definitive test that can be relied upon to predict allergic response in humans to a new protein; hence a composite approach to allergic response prediction is described in this study. The present study reports on the evaluation of the Cry proteins, encoded by cry1Ac, cry1Ab, cry2Ab, cry1Ca, cry1Fa/cry1Ca hybrid, being expressed in Bt food crops that are under field trials in India, for potential allergenic cross-reactivity using bioinformatics search tools. The sequence identity of amino acids was analyzed using FASTA3 of AllergenOnline version 10.0 and BLASTX of NCBI Entrez to identify any potential sequence matches to allergen proteins. As a step further in the detection of allergens, an independent database of domains in the allergens available in the AllergenOnline database was also developed. The results indicated no significant alignment and similarity of Cry proteins at domain level with any of the known allergens revealing that there is no potential risk of allergenic cross-reactivity.

  11. Layer-by-layer films containing peptides of the Cry1Ab16 toxin from Bacillus thuringiensis for potential biotechnological applications

    Energy Technology Data Exchange (ETDEWEB)

    Plácido, Alexandra [REQUIMTE/LAQV, Instituto Superior de Engenharia do Porto, Instituto Politécnico do Porto, Rua Dr. António Bernardino de Almeida, 431, 4200-072 Porto (Portugal); Oliveira Farias, Emanuel Airton de [Núcleo de Pesquisa em Biodiversidade e Biotecnologia, BIOTEC, Campus Ministro Reis Velloso, CMRV, Universidade Federal do Piauí, UFPI, 64202020 Parnaíba, Piaui (Brazil); Marani, Mariela M. [IPEEC-CENPAT-CONICET, Centro Nacional Patagónico, Consejo Nacional de Investigaciones Científicas y Técnicas, 9120 Puerto Madryn, Chubut (Argentina); Vasconcelos, Andreanne G. [Núcleo de Pesquisa em Biodiversidade e Biotecnologia, BIOTEC, Campus Ministro Reis Velloso, CMRV, Universidade Federal do Piauí, UFPI, 64202020 Parnaíba, Piaui (Brazil); Mafud, Ana C.; Mascarenhas, Yvonne P. [Instituto de Física de São Carlos, Universidade de São Paulo, USP, 13566-590 São Carlos, SP (Brazil); Eiras, Carla [Núcleo de Pesquisa em Biodiversidade e Biotecnologia, BIOTEC, Campus Ministro Reis Velloso, CMRV, Universidade Federal do Piauí, UFPI, 64202020 Parnaíba, Piaui (Brazil); Laboratório de Materiais Avançados, LIMAV, Engenharia de Materiais, Centro de Tecnologia, CT, Universidade Federal do Piauí, UFPI, 64049550 Teresina, Piaui (Brazil); and others

    2016-04-01

    Cry1Ab16 is a toxin of crystalline insecticidal proteins that has been widely used in genetically modified organisms (GMOs) to gain resistance to pests. For the first time, in this study, peptides derived from the immunogenic Cry1Ab16 toxin (from Bacillus thuringiensis) were immobilized as layer-by-layer (LbL) films. Given the concern about food and environmental safety, a peptide with immunogenic potential, PcL342–354C, was selected for characterization of the electrochemical, optical, and morphological properties. The results obtained by cyclic voltammetry (CV) showed that the peptide have an irreversible oxidation process in electrolyte of 0.1 mol·L{sup −1} potassium phosphate buffer (PBS) at pH 7.2. It was also observed that the electrochemical response of the peptide is governed mainly by charge transfer. In an attempt to maximize the electrochemical signal of peptide, it was intercalated with natural (agar, alginate and chitosan) or synthetic polymers (polyethylenimine (PEI) and poly(sodium 4-styrenesulfonate (PSS)). The presence of synthetic polymers on the film increased the electrochemical signal of PcL342–354C up to 100 times. Images by Atomic Force Microscopy (AFM) showed that the immobilized PcL342–354C formed self-assembled nanofibers with diameters ranging from 100 to 200 nm on the polymeric film. By UV–Visible spectroscopy (UV–Vis) it was observed that the ITO/PEI/PSS/PcL342–354C film grows linearly up to the fifth layer, thereafter tending to saturation. X-ray diffraction confirmed the presence on the films of crystalline ITO and amorphous polypeptide phases. In general, the ITO/PEI/PSS/PcL342–354C film characterization proved that this system is an excellent candidate for applications in electrochemical sensors and other biotechnological applications for GMOs and environmental indicators. - Highlights: • Peptides of the Cry1Ab16 toxin for potential biotechnological applications • Optimized LbL film deposition for synergic

  12. Bacillus thuringiensis (Bt)

    Science.gov (United States)

    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.

  13. 北京植物园苏云金芽胞杆菌菌株的分离鉴定%Isolation of Bacillus thuringiensis strains and identification of cry genes from the samples collected in Beijing Botanical Garden

    Institute of Scientific and Technical Information of China (English)

    刘东明; 束长龙; 宋福平; 高继国; 张杰

    2012-01-01

    [Objective] To isolate highly insecticidal Bacillus thuringiensis strains from soil samples collected in Beijing Botanical Garden. [Methods] B. Thuringiensis strains were isolated from soil samples using temperature method. Comparison of plasmid DNA pattern by agarose gel electrophoresis was conducted to discriminate the types of the isolates, cryl - cry40 type genes were identified via PCR-RFLP method. SDS-PAGE was used to analyze insecticidal crystal proteins. Insecticidal activity of these isolates against Colaphellus bowringi larvae and Plu-tella xylostella larvae was assayed. [Results] One hundred and forty-nine soil samples were collected and 147 B. Thuringiensis isolates were obtained. The types of the isolates were divided into 12 types, and these isolates contained bipyramidal, square, spherical and irregular crystal, respectively. crylAa, crylAb, cryl Ac, crylAh, crylBa, cryl Be, crylla, cryl La, cry2Ab and cry7Aa type genes were found in 6 isolates, but the other cry gene-types were unknown. SDS-PAGE results showed that 9 isolates could express Cry proteins of 130 ku, 1 of 70 ku, 2 of 150 ku, and 3 of both 130 ku and 60 ku. Bioassay results of 12 isolates indicated that the isolate ZWY-7 was toxic to C. Bowringi larvae, and the isolate ZWY-9 had highly insecticidal activity to P. Xylostella larvae, but their cry gene-types were unknown. [Conclusion] The B. Thuringiensis strains spread widely in Beijing Botanical Garden, belonging to different types. We found 2 Bt strains which were highly insecticidal, and it is possible to find more novel genes.%[目的]从北京植物园采集的土壤样品中分离高毒力的苏云金芽胞杆菌.[方法]采用温度法筛选Bt菌株,比对大质粒DNA图谱,区分不同类型的Bt菌株,PCR-RFLP方法对cry1~cry40类基因型进行鉴定,SDS-PAGE分析杀虫晶体蛋白,测定Bt分离株对大猿叶甲、小菜蛾幼虫的杀虫活性,筛选出高毒力的菌株.[结果]从149份土壤样品中分离出147

  14. Insect Resistance to Bacillus thuringiensis Toxin Cry2Ab Is Conferred by Mutations in an ABC Transporter Subfamily A Protein.

    Directory of Open Access Journals (Sweden)

    Wee Tek Tay

    2015-11-01

    Full Text Available The use of conventional chemical insecticides and bacterial toxins to control lepidopteran pests of global agriculture has imposed significant selection pressure leading to the rapid evolution of insecticide resistance. Transgenic crops (e.g., cotton expressing the Bt Cry toxins are now used world wide to control these pests, including the highly polyphagous and invasive cotton bollworm Helicoverpa armigera. Since 2004, the Cry2Ab toxin has become widely used for controlling H. armigera, often used in combination with Cry1Ac to delay resistance evolution. Isolation of H. armigera and H. punctigera individuals heterozygous for Cry2Ab resistance in 2002 and 2004, respectively, allowed aspects of Cry2Ab resistance (level, fitness costs, genetic dominance, complementation tests to be characterised in both species. However, the gene identity and genetic changes conferring this resistance were unknown, as was the detailed Cry2Ab mode of action. No cross-resistance to Cry1Ac was observed in mutant lines. Biphasic linkage analysis of a Cry2Ab-resistant H. armigera family followed by exon-primed intron-crossing (EPIC marker mapping and candidate gene sequencing identified three independent resistance-associated INDEL mutations in an ATP-Binding Cassette (ABC transporter gene we named HaABCA2. A deletion mutation was also identified in the H. punctigera homolog from the resistant line. All mutations truncate the ABCA2 protein. Isolation of further Cry2Ab resistance alleles in the same gene from field H. armigera populations indicates unequal resistance allele frequencies and the potential for Bt resistance evolution. Identification of the gene involved in resistance as an ABC transporter of the A subfamily adds to the body of evidence on the crucial role this gene family plays in the mode of action of the Bt Cry toxins. The structural differences between the ABCA2, and that of the C subfamily required for Cry1Ac toxicity, indicate differences in the

  15. Insect Resistance to Bacillus thuringiensis Toxin Cry2Ab Is Conferred by Mutations in an ABC Transporter Subfamily A Protein

    Science.gov (United States)

    Tay, Wee Tek; Mahon, Rod J.; Heckel, David G.; Walsh, Thomas K.; Downes, Sharon; James, William J.; Lee, Sui-Fai; Reineke, Annette; Williams, Adam K.; Gordon, Karl H. J.

    2015-01-01

    The use of conventional chemical insecticides and bacterial toxins to control lepidopteran pests of global agriculture has imposed significant selection pressure leading to the rapid evolution of insecticide resistance. Transgenic crops (e.g., cotton) expressing the Bt Cry toxins are now used world wide to control these pests, including the highly polyphagous and invasive cotton bollworm Helicoverpa armigera. Since 2004, the Cry2Ab toxin has become widely used for controlling H. armigera, often used in combination with Cry1Ac to delay resistance evolution. Isolation of H. armigera and H. punctigera individuals heterozygous for Cry2Ab resistance in 2002 and 2004, respectively, allowed aspects of Cry2Ab resistance (level, fitness costs, genetic dominance, complementation tests) to be characterised in both species. However, the gene identity and genetic changes conferring this resistance were unknown, as was the detailed Cry2Ab mode of action. No cross-resistance to Cry1Ac was observed in mutant lines. Biphasic linkage analysis of a Cry2Ab-resistant H. armigera family followed by exon-primed intron-crossing (EPIC) marker mapping and candidate gene sequencing identified three independent resistance-associated INDEL mutations in an ATP-Binding Cassette (ABC) transporter gene we named HaABCA2. A deletion mutation was also identified in the H. punctigera homolog from the resistant line. All mutations truncate the ABCA2 protein. Isolation of further Cry2Ab resistance alleles in the same gene from field H. armigera populations indicates unequal resistance allele frequencies and the potential for Bt resistance evolution. Identification of the gene involved in resistance as an ABC transporter of the A subfamily adds to the body of evidence on the crucial role this gene family plays in the mode of action of the Bt Cry toxins. The structural differences between the ABCA2, and that of the C subfamily required for Cry1Ac toxicity, indicate differences in the detailed mode

  16. Identification of cry Gene from a Novel Bacillus thuringiensis Isolate SHJ-5%苏云金芽孢杆菌SHJ-5菌株杀虫晶体蛋白基因分析

    Institute of Scientific and Technical Information of China (English)

    杜立新; 董明; 王容燕; 马铭泽; 王金耀; 曹伟平; 宋健; 冯书亮

    2011-01-01

    The novel Bacillus thuringiensis SHJ-5 was isolated from the soil in Heilongjiang province. This paper was a study on the strain SHJ-5, including that the shape of the crystal, the cry-type, protein expression and insecticidal activity. It produced blunt diamond-shaped crystals. It was identified that strain SHJ-5 contained cryIAa ,cryIEa ,cryIBe,cryI0Ah,cryIAi,cry2Ab ,cryII and vip3A gene by PCR-RFLP. SDS-PAGE analysis indicated that the strain produced a 130 -150 kDa protein and a 60 kDa protein. Bioassay showed that strain SHJ-5 had high insecticidal activity to Helicoverpa armigera and Plutella xylostella. The corrected mortality to the two target insects was 100% and 96. 7% , respectively.%苏云金芽孢杆菌SHJ-5菌株是从黑龙江采集的土样中分离获得的新菌株,对其伴孢晶体形状、基因型、蛋白表达以及杀虫活性进行了系统研究.菌株SHJ-5产生的伴孢晶体形状为钝菱形;利用PCR-RFLP技术对该菌株的基因型进行鉴定,结果表明,该菌株含有cry1Aa、cry1Ea、cry1Be、cry1Ah、cry1Ai、cry2Ab、cry1i和vip3A基因;SDS-PAGE晶体蛋白检测结果显示,该菌株表达130~150 kDa和60 kDa大小蛋白;生物活性测定表明,菌株SHJ-5对棉铃虫和小菜蛾有较高杀虫活性,校正死亡率分别为100%和96.7%.

  17. Interaction between Functional Domains of Bacillus thuringiensis Insecticidal Crystal Proteins

    Science.gov (United States)

    Rang, Cécile; Vachon, Vincent; de Maagd, Ruud A.; Villalon, Mario; Schwartz, Jean-Louis; Bosch, Dirk; Frutos, Roger; Laprade, Raynald

    1999-01-01

    Interactions among the three structural domains of Bacillus thuringiensis Cry1 toxins were investigated by functional analysis of chimeric proteins. Hybrid genes were prepared by exchanging the regions coding for either domain I or domain III among Cry1Ab, Cry1Ac, Cry1C, and Cry1E. The activity of the purified trypsin-activated chimeric toxins was evaluated by testing their effects on the viability and plasma membrane permeability of Sf9 cells. Among the parental toxins, only Cry1C was active against these cells and only chimeras possessing domain II from Cry1C were functional. Combination of domain I from Cry1E with domains II and III from Cry1C, however, resulted in an inactive toxin, indicating that domain II from an active toxin is necessary, but not sufficient, for activity. Pores formed by chimeric toxins in which domain I was from Cry1Ab or Cry1Ac were slightly smaller than those formed by toxins in which domain I was from Cry1C. The properties of the pores formed by the chimeras are therefore likely to result from an interaction between domain I and domain II or III. Domain III appears to modulate the activity of the chimeric toxins: combination of domain III from Cry1Ab with domains I and II of Cry1C gave a protein which was more strongly active than Cry1C. PMID:10388684

  18. cry8C/cry3A工程菌BIOT1853A对花生田主要节肢动物类群结构的影响%Effects of Engineered Bacillus thuringiensis with cry8C and cry3A on the Major Arthropod Groups Structure in Peanut Field

    Institute of Scientific and Technical Information of China (English)

    谢明惠; 张海珊; 陈浩梁; 徐德进; 章东方; 张杰; 苏卫华

    2012-01-01

    The effects of engineered Bacillus thuringiensis with cry8C and cry3A on the number of major arthropod groups in peanut field were studied, with receipt strain Bt185 and conventional in- secticide as the controls. The results showed that: the main arthropod groups in peanut field were lo- custs, crickets, whiteflies, leaf beetles, leafhoppers, ladybirds, bugs, lacewings and spiders. Using different concentrations of BIOT1853A had no significant effect on the composition, number and tem- poral dynamics of major arthropod groups. It was apparent that BIOT1853A has no significant nega- tive effect on the peanut field arthropod community.%以受体菌株Bt185和常规化学农药为对照,研究转cry8C/cry3A工程菌BIOT1853A对花生田主要节肢动物类群数量的影响。研究结果表明:花生田地上部分主要节肢动物分为以下9个类群:蝗虫类、蟋蟀类、粉虱类、叶甲类、叶蝉类、瓢虫类、蝽类、草蛉类和蜘蛛类;施加不同浓度工程菌BIOT1853A对主要节肢动物类群的组成、数量和时序动态均无显著性影响。综合分析认为,工程菌BIOT1853A对花生田节肢动物群落无明显的负面影响。

  19. Genome Sequence of the Mosquitocidal Bacillus thuringiensis Strain BR58, a Biopesticide Product Effective against the Coffee Berry Borer (Hypothenemus hampei)

    Science.gov (United States)

    Zorzetti, Janaina; Ricietto, Ana P. S.; da Silva, Carlos R. M.; Wolf, Ivan R.; Neves, Pedro M. O. J.; Meneguim, Ana M.; Vilas-Boas, Laurival A.

    2015-01-01

    Bacillus thuringiensis is an important microbial control agent against insect pests. The draft genome sequence of the Brazilian strain BR58 described here contains the insecticidal genes cry4A, cry4B, cry10A, cry11A, cry60A, cry60B, and cyt1A, which show toxicity to both Aedes aegypti and Hypothenemus hampei larvae. PMID:26659669

  20. 苏云金芽胞杆菌cry1Ib6基因的克隆、表达及活性的研究%Cloning, Expression and Activity of cry1Ib6 Gene from Bacillus thuringiensis

    Institute of Scientific and Technical Information of China (English)

    张庆丽; 李海涛; 赵世源; 刘荣梅; 赵勇; 高继国

    2011-01-01

    Bacillus thuringiensis could produce insecticide crystal proteins (ICPs), which had strong toxicity to sensitive insects, but no toxicity to higher animals and human. A full length of 2. 1 kb cry ll gene fragment was obtained by PCR amplification using primers designed according to crylI-type gene sequences with the plasmid DNA of Bt LBS2 strain as template. It could express 79.9 kDa protein in the Escherichia coli by the expression vector pEB. By AlginX analysis, its protein was composed of 712 amino acids. The molecular weight was 79. 9 kDa, and the isoelectric point was 6.54, a weak acid protein. By blasting on NCBI, the crylIb6's amino acid sequences had 98% similarity with Cryl Ib3, with 12 amino acids different. The gene has been registered in CenBank with accession number ADK38579 and was named as cryl Ib6 by International Nomenclature Committee. Cryllb6 protein products had high toxicity to Plutella xylostella, with Lethal concentration (LC50) 1.196 μg/mL, which provided a new gene for insect-resistant transgenic plants.%苏云金芽胞杆菌(Bacillus thuringiensis)能产生杀虫晶体蛋白(Insecticida Crystal Proteins,ICPs),对敏感昆虫有强烈毒性,而对高等动物和人无毒性.ICPs由cry或cyt基因编码,根据cry1I型基因设计引物,以BtLB52菌株的质粒DNA为模板,扩增出了全长为2.1kb的cry1I基因,其能通过表达载体pEB在大肠杆菌中高效表达为79.9 kDa的蛋白.经过AlginX软件分析该蛋白由712个氨基酸组成,分子量为79.9 kDa,等电点为6.54,为弱酸性蛋白质,NCBI Blast比对该蛋白的氨基酸序列与Cry1 Ib3的相似性最高为98%,有12个氨基酸的差异.该基因已在GenBank中注册,登录号为ADK38579,并被国际基因命名委员会正式命名为cry1 Ib6.它的表达产物对小菜蛾具有较高的毒力,LC50为1.196 μg/mL,为抗虫转基因植物研究提供了新的基因.

  1. Novel cloning vectors for Bacillus thuringiensis.

    OpenAIRE

    Baum, J A; Coyle, D M; Gilbert, M P; Jany, C S; Gawron-Burke, C

    1990-01-01

    Seven replication origins from resident plasmids of Bacillus thuringienis subsp. kurstaki HD263 and HD73 were cloned in Escherichia coli. Three of these replication origins, originating from plasmids of 43, 44, and 60 MDa, were used to construct a set of compatible shuttle vectors that exhibit structural and segregational stability in the Cry- strain B. thuringiensis HD73-26. These shuttle vectors, pEG597, pEG853, and pEG854, were designed with rare restriction sites that permit various adapt...

  2. Uniform Orientation of Biotinylated Nanobody as an Affinity Binder for Detection of Bacillus thuringiensis (Bt) Cry1Ac Toxin

    Science.gov (United States)

    Li, Min; Zhu, Min; Zhang, Cunzheng; Liu, Xianjin; Wan, Yakun

    2014-01-01

    Nanobodies are the smallest natural fragments with useful properties such as high affinity, distinct paratope and high stability, which make them an ideal tool for detecting target antigens. In this study, we generated and characterized nanobodies against the Cry1Ac toxin and applied them in a biotin-streptavidin based double antibodies (nanobodies) sandwich-ELISA (DAS-ELISA) assay. After immunizing a camel with soluble Cry1Ac toxin, a phage displayed library was constructed to generate Nbs against the Cry1Ac toxin. Through successive rounds of affinity bio-panning, four nanobodies with greatest diversity in CDR3 sequences were obtained. After affinity determination and conjugating to HRP, two nanobodies with high affinity which can recognize different epitopes of the same antigen (Cry1Ac) were selected as capture antibody (Nb61) and detection antibody (Nb44). The capture antibody (Nb61) was biotinylated in vivo for directional immobilization on wells coated with streptavidin matrix. Both results of specificity analysis and thermal stability determination add support for reliability of the following DAS-ELISA with a minimum detection limit of 0.005 μg·mL−1 and a working range 0.010–1.0 μg·mL−1. The linear curve displayed an acceptable correlation coefficient of 0.9976. These results indicated promising applications of nanobodies for detection of Cry1Ac toxin with biotin-streptavidin based DAS-ELISA system. PMID:25474492

  3. Uniform Orientation of Biotinylated Nanobody as an Affinity Binder for Detection of Bacillus thuringiensis (Bt Cry1Ac Toxin

    Directory of Open Access Journals (Sweden)

    Min Li

    2014-12-01

    Full Text Available Nanobodies are the smallest natural fragments with useful properties such as high affinity, distinct paratope and high stability, which make them an ideal tool for detecting target antigens. In this study, we generated and characterized nanobodies against the Cry1Ac toxin and applied them in a biotin-streptavidin based double antibodies (nanobodies sandwich-ELISA (DAS-ELISA assay. After immunizing a camel with soluble Cry1Ac toxin, a phage displayed library was constructed to generate Nbs against the Cry1Ac toxin. Through successive rounds of affinity bio-panning, four nanobodies with greatest diversity in CDR3 sequences were obtained. After affinity determination and conjugating to HRP, two nanobodies with high affinity which can recognize different epitopes of the same antigen (Cry1Ac were selected as capture antibody (Nb61 and detection antibody (Nb44. The capture antibody (Nb61 was biotinylated in vivo for directional immobilization on wells coated with streptavidin matrix. Both results of specificity analysis and thermal stability determination add support for reliability of the following DAS-ELISA with a minimum detection limit of 0.005 μg·mL−1 and a working range 0.010–1.0 μg·mL−1. The linear curve displayed an acceptable correlation coefficient of 0.9976. These results indicated promising applications of nanobodies for detection of Cry1Ac toxin with biotin-streptavidin based DAS-ELISA system.

  4. F2 screen for resistance to Bacillus thuringiensis Cry2Ab2-maize in field populations of Spodoptera frugiperda (Lepidoptera: Noctuidae) from the southern United States

    Science.gov (United States)

    The fall armyworm, Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae), is a target of transgenic maize and cotton expressing Bacillus thuringiensis (Bt) proteins in both North and South America. In 2013 and 2014, a total of 215 F2 two-parent families of S. frugiperda were established usin...

  5. Mode of action of mosquitocidal Bacillus thuringiensis toxins.

    Science.gov (United States)

    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.

  6. Efeitos da interação e toxicidade das proteínas Cry1 e Vip3Aa de Bacillus thuringiensis, Berliner em Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae)

    OpenAIRE

    Pavani, Claudio Damasceno [UNESP

    2013-01-01

    A bactéria Bacillus thuringiensis é conhecida como uma das melhores opções no controle biológico de pragas devido à ação entomopatogênica e especificidade de suas proteínas. As proteínas Vip3, que são secretadas durante o crescimento vegetativo de B. thuringiensis e Cry1, que são produzidas durante a fase de esporulação, atuam no controle de importantes pragas de lepidópteros. Um dos principais riscos ambientais associados à utilização de inseticidas é o potencial para o desenvolvimento de gr...

  7. Complete genome sequence of Bacillus thuringiensis YC-10, a novel active strain against plant-parasitic nematodes.

    Science.gov (United States)

    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.

  8. Degradation of Cry1Ac protein from Bacillus thuringiensis by soil bacteria from transgenic and conventional cotton (Gossypium hirsutum) culture.

    OpenAIRE

    João Paulo Leite Tozzi

    2009-01-01

    Bt é uma bactéria formadora da proteína Cry1Ac, tóxica a lepidópteros. Plantas geneticamente modificadas expressam essa toxina. O objetivo deste trabalho foi isolamento e identificação de bactérias do solo de algodão transgênico e convencional potencialmente biodegradadoras dessa proteína. Estudou-se a dinâmica de crescimento das bactérias em meios com a proteína Cry1Ac ou glucose, a biodegradação, os genes apr, npr e sub. Em solo de algodão convencional a contagem foi menor; para algodão tra...

  9. Cloning and Expression of cry2Ac4 Gene from Bacillus thuringiensis WB9%苏云金芽胞杆菌WB9菌株cry2Ac4基因的克隆及表达

    Institute of Scientific and Technical Information of China (English)

    黄天培; 潘洁茹; 黄张敏; 陈志; 庄浩瀚; 李今煜; 关雄

    2008-01-01

    苏云金芽胞杆菌(Bacillus thuringiensis,Bt)WB9是我国分离自武夷山的对多种重要农业害虫具有高毒力的菌株,经PCR-RFLP鉴定含有cry2Ac基因.根据cry2基因序列设计引物,以WB9质粒为模板扩增cry2Ac全长基因,与大肠杆菌(Es-chetichia cob)克隆载体pMD18-T连接获得含有cry2Ac全长基因的重组质粒pMD2Ac并测序.该基因在GenBank登录号为DQ361267.被Bt国际命名委员会正式命名为cry2Ac4.通过亚克隆方法将cry2Ac4基因插入穿梭表达载体pHT315获得重组表达质粒pHT2Ac,将其转化大肠杆菌SCS110和Bt无晶体突变株HD73 Cry,得到的工程菌能正常表达70 kD蛋白,形成方形晶体.生物测定结果表明,cry2Ae4基因表达产物对桔小实蝇(Bactrocera dorsalis Hendel)幼虫具有显著的毒杀作用,但对小菜蛾(Plutella xylostella)和致倦库蚊(Culex quinquefasciatus)幼虫基本没有效果.

  10. Expression of an engineered synthetic cry2Aa (D42/K63F/K64P) gene of Bacillus thuringiensis in marker free transgenic tobacco facilitated full-protection from cotton leaf worm (S. littoralis) at very low concentration.

    Science.gov (United States)

    Gayen, Srimonta; Mandal, Chandi Charan; Samanta, Milan Kumar; Dey, Avishek; Sen, Soumitra Kumar

    2016-04-01

    Emergence of resistant insects limits the sustainability of Bacillus thuringiensis (Bt) transgenic crop plants for insect management. Beside this, the presence of unwanted marker gene(s) in the transgenic crops is also a major environmental and health concern. Thus, development of marker free transgenic crop plants expressing a new class of toxin having a different mortality mechanism is necessary for resistance management. In a previous study, we generated an engineered Cry2Aa (D42/K63F/K64P) toxin which has a different mortality mechanism as compared to first generation Bt toxin Cry1A, and this engineered toxin was found to enhance 4.1-6.6-fold toxicity against major lepidopteran insect pests of crop plants. In the present study, we have tested the potency of this engineered synthetic Cry2Aa (D42/K63F/K64P) toxin as a candidate in the development of insect resistant transgenic tobacco plants. Simultaneously, we have eliminated the selectable marker gene from the Cry2Aa (D42/K63F/K64P) expressing tobacco plants by exploiting the Cre/lox mediated recombination methodology, and successfully developed marker free T2 transgenic tobacco plants expressing the engineered Cry2Aa toxin. Realtime and western blot analysis demonstrated the expression of engineered toxin gene in transgenic plants. Insect feeding assays revealed that the marker free T2 progeny of transgenic plants expressing Cry2Aa (D42/K63F/K64P) toxin showed 82-92 and 52-61 % mortality to cotton leaf worm (CLW) and cotton bollworm (CBW) respectively. Thus, this engineered Cry2Aa toxin could be useful for the generation of insect resistant transgenic Bt lines which will protect the crop damages caused by different insect pests such as CLW and CBW.

  11. Synergisms of cadherin fragments to Cry toxins from Bacillus thuringiensis%钙粘蛋白片段与苏云金杆菌晶体毒素协同作用研究进展

    Institute of Scientific and Technical Information of China (English)

    崔金杰; 李国清

    2011-01-01

    从钙粘蛋白介导苏云金杆菌晶体(Bt Cry)毒素对害虫的毒杀过程、钙粘蛋白重复区和近膜区与Bt Cry毒素的分子间作用涉及的结合位点及可能的互作机制等方面,综述了钙粘蛋白片段与Bt Cry毒素协同作用的最新研究进展.昆虫钙粘蛋白某些片段在非折叠状态时,可与Bt Cry毒素形成寡聚体,从而增加Bt Cry毒素对靶标害虫的毒杀活性.相关研究成果有助于提高Bt Cry毒素毒杀害虫的能力,克服害虫抗药性,具有一定的应用前景.%The recent advances in synergism of cadherin fragments to Bacillus thuringiensis (Bt) Cry toxins were reviewed. Cadherin receptors mediated virulence of the Cry toxins to insect pests. The molecular interaction between cadherin extracellular repeats or membrane proximal domain and Cry toxins involved in several active sites. Some unfolding fragments of cadherins could facilitate the formation of Bt Cry toxin oligomers, and consequently exhibited synergistic effects on Bt Cry toxins to target pests. This discovery provides novel strategies to enhance the insecticidal activity of Bt Cry toxins, and to overcome the resistance of insects to Bt toxin-based bio-pesticides or transgenic crops.

  12. Complete Genome Sequence of Bacillus thuringiensis subsp. chinensis Strain CT-43▿

    OpenAIRE

    He, Jin; Wang, Jieping; Yin, Wen; Shao, Xiaohu; Zheng, Huajun; Li, Mingshun; Zhao, Youwen; Sun, Ming; Wang, Shengyue; Yu, Ziniu

    2011-01-01

    Bacillus thuringiensis has been widely used as an agricultural biopesticide for a long time. As a producing strain, B. thuringiensis subsp. chinensis strain CT-43 is highly toxic to lepidopterous and dipterous insects. It can form various parasporal crystals consisting of Cry1Aa3, Cry1Ba1, Cry1Ia14, Cry2Aa9, and Cry2Ab1. During fermentation, it simultaneously generates vegetative insecticidal protein Vip3Aa10 and the insecticidal nucleotide analogue thuringiensin. Here, we report the finished...

  13. A 90-day safety study of genetically modified rice expressing Cry1Ab protein (Bacillus thuringiensis toxin) in Wistar rats

    DEFF Research Database (Denmark)

    Schrøder, Malene; Poulsen, Morten; Wilcks, Andrea

    2007-01-01

    An animal model for safety assessment of genetically modified foods was tested as part of the SAFOTEST project. In a 90-day feeding study on Wistar rats, the transgenic KMD1 rice expressing Cry1Ab protein was compared to its non-transgenic parental wild type, Xiushui 11. The KMD1 rice contained 15......, macroscopic and histopathological examinations were performed with only minor changes to report. The aim of the study was to use a known animal model in performance of safety assessment of a GM crop, in this case KMD1 rice. The results show no adverse or toxic effects of KMD1 rice when tested in the design...... used in this 90-day study. Nevertheless the experiences from this study lead to the overall conclusion that safety assessment for unintended effects of a GM crop cannot be done without additional test group(s)....

  14. Draft Genome Sequence of Bacillus thuringiensis Serovar Tolworthi Strain Na205-3, an Isolate Toxic for Helicoverpa armigera

    Science.gov (United States)

    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

  15. Toxicological Evaluation of a Potential Immunosensitizer for Use as a Mucosal Adjuvant—Bacillus thuringiensis Cry1Ac Spore-Crystals: A Possible Inverse Agonist that Deserves Further Investigation

    Directory of Open Access Journals (Sweden)

    Bélin Poletto Mezzomo

    2015-12-01

    Full Text Available In addition to their applicability as biopesticides, Bacillus thuringiensis (Bt Cry1Ac spore-crystals are being researched in the immunology field for their potential as adjuvants in mucosal and parenteral immunizations. We aimed to investigate the hematotoxicity and genotoxicity of Bt spore-crystals genetically modified to express Cry1Ac individually, administered orally (p.o. or with a single intraperitoneal (i.p. injection 24 h before euthanasia, to simulate the routes of mucosal and parenteral immunizations in Swiss mice. Blood samples were used to perform hemogram, and bone marrow was used for the micronucleus test. Cry1Ac presented cytotoxic effects on erythroid lineage in both routes, being more severe in the i.p. route, which also showed genotoxic effects. The greater severity noted in this route, mainly at 6.75 mg/kg, as well as the intermediate effects at 13.5 mg/kg, and the very low hematotoxicity at 27 mg/kg, suggested a possible inverse agonism. The higher immunogenicity for the p.o. route, particularly at 27 mg/kg, suggested that at this dose, Cry 1Ac could potentially be used as a mucosal adjuvant (but not in parenteral immunizations, due to the genotoxic effects observed. This potential should be investigated further, including making an evaluation of the proposed inverse agonism and carrying out cytokine profiling.

  16. 小菜蛾高效Bt菌株的分离、生化特性及基因型鉴定%Isolation, biochemical and cry-type gene characterization of Bacillus thuringiensis strains with high toxic to Plutella xylostella

    Institute of Scientific and Technical Information of China (English)

    朱勋; 吴青君; 张友军; 刘春光; 刘岩林; 余亚军; 景伟; 薛原; 杨峰山

    2011-01-01

    The insecticidal activities of ten strains of Bacillus thuringiensis isolated from diseased Plutella xylostella ( L. )larvae were characterized. All strains caused > 80% larval mortality and the strain DBW902 had the highest insecticidal activity with an LC50 of 13.99 mg/L at 48 h. The biochemical characteristics and cry/cyt genes of each strain were determined. High insecticidal activity coincided with the detection of at least one crylA or cry2A family gene. The biochemical characteristics of the strains, DBW904, DBW93 and DBW962 were homologous to B. thuringiensis subsp.and Shandongiensis, but not the other strains. The identification of these 10 B. thuringiensis isolates with moderate to high insecticidal activity against P. xylostella is promising for the control of this pest in northeastern China.%从啥尔滨田间采集死亡小菜蛾Plutella xylostella(L.)幼虫,从中分离出10株苏云金芽孢杆菌Bacillusthuringiensis.对小菜蛾幼虫室内生物测定结果表明,各菌株对小菜蛾幼虫的死亡率均在85%以上,其中DBW902毒力最强,48 h的LC为13.99 mg/L.菌株cry/cyt基因检测表明,所有菌株均含有cry1A或cry2A基因,这与其高毒力的特性基本吻合.生化检测与分析表明,菌株DBW904、DBW93、DBW962与已报道的苏云金芽孢杆菌山东亚种B.thuringiensis subsp.shandongiensis的生化特性一致,但是其它菌株的生理生化特性与已报道菌株有区别.

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

    Science.gov (United States)

    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.

  18. Screening of Bacillus thuringiensis strains effective against mosquitoes Prospecção de estirpes de Bacillus thuringiensis efetivas contra mosquitos

    Directory of Open Access Journals (Sweden)

    Rose Gomes Monnerat

    2005-02-01

    Full Text Available The objective of this work was to evaluate 210 Bacillus thuringiensis strains against Aedes aegypti and Culex quinquefasciatus larvae to select the most effective. These strains were isolated from different regions of Brazil and are stored in a Bacillus spp. collection at Embrapa Recursos Genéticos e Biotecnologia, Brasília, Brazil. The selected strains were characterized by morphological (microscopy, biochemical (SDS-PAGE 10% and molecular (PCR methods. Six B. thuringiensis strains were identified as mosquito-toxic after the selective bioassays. None of the strains produced the expected PCR products for detection of cry4, cry11 and cyt1A genes. These results indicate that the activity of mosquitocidal Brazilian strains are not related with Cry4, Cry11 or Cyt proteins, so they could be used as an alternative bioinsecticide against mosquitoes.Neste trabalho foram realizados testes de patogenicidade com 210 estirpes de Bacillus thuringiensis contra larvas de Aedes aegypti e Culex quinquefasciatus, a fim de se determinar as mais eficazes. Estas estirpes foram isoladas de diversas regiões do Brasil e estão armazenadas na coleção de Bacillus spp. da Embrapa Recursos Genéticos e Biotecnologia. As estirpes selecionadas foram caracterizadas por métodos morfológicos (microscopia, bioquímicos (SDS-PAGE 10% e moleculares (Reação em Cadeia da Polimerase. Foram selecionadas seis estirpes entomopatogênicas de Bacillus thuringiensis. Nenhuma das estirpes de Bacillus thuringiensis apresentou produtos de PCR esperados para a detecção dos genes cry4, cry11 e cyt1A. A patogenicidade das estirpes não está associada à presença das toxinas Cry4, Cry11 ou Cyt, assim, essas estirpes poderão ser utilizadas para a formatação de um bioinseticida alternativo contra mosquitos.

  19. 苏云金芽胞杆菌LSZ9408菌株基因型的鉴定%Identification of Cry-type Gene of Bacillus thuringiensis Strain LSZ 9408

    Institute of Scientific and Technical Information of China (English)

    林营志; 朱育菁; 刘波

    2012-01-01

    PCR and SDS-PAGE were used to identify the cry-type gene of Bacillus thuringiensis (Bt) strain LSZ 9408. PCR analysis showed that Bt LSZ 9408 strain contained cry1 and cry2 genes. Five pairs of general primers-Un1, Un2, Un3, Un4 and Un7/8 were used to amplify PCR, and then the amplification results were detected by SDS-PAGE. Primes Unl and Un2 formed about 270 bp and 700 bp DNA fragments on agarose gel electrophoresis, while none PCR products was found with the other three pairs of primers. The results of SDS-PAGE also indicated that Bt LSZ 9408 possessed cry1 and cry2 genes, which coded 130kD and 65kD ICPs.%利用聚合酶链式反应(PCR)和聚丙烯酰胺凝胶电泳(SDS-PAGE)技术鉴定苏云金芽胞杆菌LSZ9408菌株的杀虫晶体蛋白及其基因组成.PCR结果表明:苏云金芽胞杆菌LSZ 9408菌株中含有cry1和cry2两种基因型.采用5对通用引物-Un1、Un2、Un3、Un4、Un7/8进行PCR扩增,结果显示:Un1和Un2引物扩增的DNA片段大小分别为270 bp和700 bp左右,其他引物无PCR扩增产物.SDS-PAGE结果也表明:苏云金芽胞杆菌LSZ 9408菌株中含有cry1和cry2两种基因,它们编码的杀虫晶体蛋白分子量约为130 kD和65 kD.

  20. Análisis de la unión de la toxina Cry11Bb1 de bacillus thuringiensis subesp. Medellín a vesículas del epitelio en borde de cepillo del mosquito Aedes aegypti

    Directory of Open Access Journals (Sweden)

    Sergio Ordúz

    2000-02-01

    Full Text Available Las proteínas Cry11 son producidas por Bacillus thuringiensis, estas son potentes toxinas insecticidas contra larvas de mosquitos y su modo de acción se cree que es similar al de las toxinas del tipo Cry1, las cuales interactúan con el intestino medio de larvas de lepidópteros. En este trabajo, se estudió la interacción de la toxina de 94 kDa Cry11Bb de Bacillus thuringiensis subesp. medellin con el epitelio intestinal de larvas del mosquito Aedes aegypti utilizando vesículas de la membrana en borde de cepillo (VMBC. Un intermediario de la protoxina Cry11Bb de 68 kDa, y su forma activa 30/35 kDa se marcaron con Iodo-radioactivo e incubaron con las VMBC del mosquito. La protoxina marcada (125I-68 kDa interactuó específicamente con las vesículas y los experimentos de saturación en presencia de cantidades crecientes de vesículas mostraron que la interacción vesícula-125I-68 kDa fue saturable en el rango entre 10 a 80 ?g de VMBC. En contraste, la toxina marcada (125I-30/35 kDa no mostró ni unión específica, ni saturabilidad en la interacción. Adicionalmente, la toxina fue susceptible al ataque de las proteasas presentes en las vesículas y mediante la técnica de dynamic Light scattering, se evidenció que la toxina se presenta como un agregado proteico. Los resultados sugieren que la protoxina Cry11Bb de 68 kDa podría determinar la especificidad de la interacción y después de la unión ésta ser procesada hacia una forma funcional de 30/35 kDa capaz de interactuar con los lípidos de la membrana y generar, mediante la formación de poros, el desbalance osmótico que lleva a la muerte a las larvas de mosquitos.

  1. Genome Sequence of Bacillus thuringiensis Strain Btm27, an Egyptian Isolate Highly Toxic to Cotton Leafworm

    Science.gov (United States)

    Rusconi, Brigida; Chen, Yue; Koenig, Sara S. K.; El-Helow, Ehab R.

    2015-01-01

    Bacillus thuringiensis is a potent microbial control agent against insect pests. Here, we present the draft genome of the Egyptian strain Btm27 that shows high toxicity toward the cotton leafworm. The genome contains three insecticidal genes cry1Ac9, cry2Ab1, and vip3V that have been implicated in conferring toxicity toward lepidoptera. PMID:25977430

  2. Solubilization, Activation, and Insecticidal Activity of Bacillus thuringiensis Serovar thompsoni HD542 Crystal Proteins

    NARCIS (Netherlands)

    Naimov, S.; Boncheva, R.; Karlova, R.B.; Dukiandjiev, S.; Minkov, I.; Maagd, de R.A.

    2008-01-01

    Cry15Aa protein, produced by Bacillus thuringiensis serovar thompsoni HD542 in a crystal together with a 40 kDa accompanying protein is one of a small group of non-typical, less well-studied members of the Cry family of insecticidal proteins, and may provide an alternative for the more commonly used

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

    Directory of Open Access Journals (Sweden)

    Eitan Ben-Dov

    2014-03-01

    Full Text Available 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.

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

    Science.gov (United States)

    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.

  5. Complete Genome of Bacillus thuringiensis Myophage Spock

    OpenAIRE

    Maroun, Justin W.; Whitcher, Kelvin J.; Chamakura, Karthik R.; Kuty Everett, Gabriel F.

    2013-01-01

    Bacillus thuringiensis is a Gram-positive, sporulating soil microbe with valuable pesticide-producing properties. The study of bacteriophages of B. thuringiensis could provide new biotechnological tools for the use of this bacterium. Here, we present the complete annotated genome of Spock, a myophage of B. thuringiensis, and describe its features.

  6. Cytolytic Toxin and Related Genes in Bacillus thuringiensis

    Institute of Scientific and Technical Information of China (English)

    QI Dong-lai; LI Yi-dan; GAO Ji-guo

    2005-01-01

    Bacillus thuringiensis is a ubiquitous gram-positive, spore-forming bacterium that forms parasporal crystal during the stationary phase of its growth cycle. These crystal proteins, including Cry and Cyt protein, are toxic to certain insects. Lately, some problems about Cyt classification, structural characteristic, action mechanism and resistance to Cyt toxin are becoming new hotspots. We review the progress of above problems in several foreign labs.

  7. Single amino acid insertions in extracellular loop 2 of Bombyx mori ABCC2 disrupt its receptor function for Bacillus thuringiensis Cry1Ab and Cry1Ac but not Cry1Aa toxins.

    Science.gov (United States)

    Tanaka, Shiho; Miyamoto, Kazuhisa; Noda, Hiroaki; Endo, Haruka; Kikuta, Shingo; Sato, Ryoichi

    2016-04-01

    In a previous report, seven Cry1Ab-resistant strains were identified in the silkworm, Bombyx mori; these strains were shown to have a tyrosine insertion at position 234 in extracellular loop 2 of the ABC transporter C2 (BmABCC2). This insertion was confirmed to destroy the receptor function of BmABCC2 and confer the strains resistance against Cry1Ab and Cry1Ac. However, these strains were susceptible to Cry1Aa. In this report, we examined the mechanisms of the loss of receptor function of the transporter by expressing mutations in Sf9 cells. After replacement of one or two of the five amino acid residues in loop 2 of the susceptible BmABCC2 gene [BmABCC2_S] with alanine, cells still showed susceptibility, retaining the receptor function. Five mutants with single amino acid insertions at position 234 in BmABCC2 were also generated, resulting in loop 2 having six amino acids, which corresponds to replacing the tyrosine insertion in the resistant BmABCC2 gene [BmABCC2_R(+(234)Y)] with another amino acid. All five mutants exhibited loss of function against Cry1Ab and Cry1Ac. These results suggest that the amino acid sequence in loop 2 is less important than the loop size (five vs. six amino acids) or loop structure for Cry1Ab and Cry1Ac activity. Several domain-swapped mutant toxins were then generated among Cry1Aa, Cry1Ab, and Cry1Ac, which are composed of three domains. Swapped mutants containing domain II of Cry1Ab or Cry1Ac did not kill Sf9 cells expressing BmABCC2_R(+(234)Y), suggesting that domain II of the Cry toxin is related to the interaction with the receptor function of BmABCC2. This also suggests that different reactions against Bt-toxins in some B. mori strains, that is, Cry1Ab resistance or Cry1Aa susceptibility, are attributable to structural differences in domain II of Cry1A toxins.

  8. Identification and Distribution of Bacillus thuringiensis Isolates from Primeval Forests in Yunnan and Hainan Provinces and Northeast Region of China

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Ninety-two Bacillus thuringiensis isolates were screened from 683 soil samples collected from tropical and semitropical primeval forests in Yunnan and Hainan provinces of China. Several shapes of crystals, including bipyramidal, square,ovoid, spherical, and amorphous, were observed in the B. thuringiensis isolates. Twenty-six pairs of primers were used to identify 31 holotype cry genes at primary rank of the B. thuringiensis cry gene nomenclature system. The cry gene-types of 92 B. thuringiensis isolates and 33 B. thuringiensis isolates screened from Northeast region of China were identified by PCR-RFLP and SDS-PAGE methods. Fifty-eight isolates harbored cryl genes, 32 isolates cry2 genes, 12 isolates cry8 genes, 3 isolates cry9 genes, 12 isolates cry11 genes, and 13 isolates cry30 genes. Of the tested isolates, 42 produced no reaction product with 26 pairs of primers and also exhibited no toxicity against 8 insect species tested. The isolate Z2-34 harbored a novel cry30 gene, exhibited insecticidal activity against Aedes albopictus of Dipterans. The accession number of the novel genes in this study is AY916046. Isolation and identification of B. thuringiensis and cry gene are important for investigating the diversity of B. thuringiensis resources and cloning new cry gene.

  9. Gene expression patterns and sequence polymorphisms associated with mosquito resistance to Bacillus thuringiensis israelensis toxins

    OpenAIRE

    Després, Laurence; Stalinski, Renaud; Tetreau, Guillaume; Paris, Margot; Bonin, Aurélie; Navratil, Vincent; Reynaud, Stéphane; David, Jean-Philippe

    2014-01-01

    Background Despite the intensive use of Bacillus thuringiensis israelensis (Bti) toxins for mosquito control, little is known about the long term effect of exposure to this cocktail of toxins on target mosquito populations. In contrast to the many cases of resistance to Bacillus thuringiensis Cry toxins observed in other insects, there is no evidence so far for Bti resistance evolution in field mosquito populations. High fitness costs measured in a Bti selected mosquito laboratory strain sugg...

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

    Science.gov (United States)

    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.

  11. Toxicity of Bacillus thuringiensis crystal proteins against eri silkworm, Samia cynthia ricini (Lepidoptera: Saturniidae).

    Science.gov (United States)

    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.

  12. Use of Bacillus thuringiensis toxins for control of the cotton pest Earias insulana (Boisd.) (Lepidoptera: Noctuidae).

    Science.gov (United States)

    Ibargutxi, María A; Estela, Anna; Ferré, Juan; Caballero, Primitivo

    2006-01-01

    Thirteen of the most common lepidopteran-specific Cry proteins of Bacillus thuringiensis have been tested for their efficacy against newly hatched larvae of two populations of the spiny bollworm, Earias insulana. At a concentration of 100 microg of toxin per milliliter of artificial diet, six Cry toxins (Cry1Ca, Cry1Ea, Cry1Fa, Cry1Ja, Cry2Aa, and Cry2Ab) were not toxic at all. Cry1Aa, Cry1Ja, and Cry2Aa did not cause mortality but caused significant inhibition of growth. The other Cry toxins (Cry1Ab, Cry1Ac, Cry1Ba, Cry1Da, Cry1Ia, and Cry9Ca) were toxic to E. insulana larvae. The 50% lethal concentration values of these toxins ranged from 0.39 to 21.13 microg/ml (for Cry9Ca and Cry1Ia, respectively) for an E. insulana laboratory colony originating from Egypt and from 0.20 to 4.25 microg/ml (for Cry9Ca and Cry1Da, respectively) for a laboratory colony originating from Spain. The relative potencies of the toxins in the population from Egypt were highest for Cry9Ca and Cry1Ab, and they were both significantly more toxic than Cry1Ac and Cry1Ba, followed by Cry1Da and finally Cry1Ia. In the population from Spain, Cry9Ca was the most toxic, followed in decreasing order by Cry1Ac and Cry1Ba, and the least toxic was Cry1Da. Binding experiments were performed to test whether the toxic Cry proteins shared binding sites in this insect. 125I-labeled Cry1Ac and Cry1Ab and biotinylated Cry1Ba, Cry1Ia, and Cry9Ca showed specific binding to the brush border membrane vesicles from E. insulana. Competition binding experiments among these toxins showed that only Cry1Ab and Cry1Ac competed for the same binding sites, indicating a high possibility that this insect may develop cross-resistance to Cry1Ab upon exposure to Cry1Ac transgenic cotton but not to the other toxins tested.

  13. Screen of Bacillus thuringiensis toxins for transgenic rice to control Sesamia inferens and Chilo suppressalis.

    Science.gov (United States)

    Gao, Yulin; Hu, Yang; Fu, Qiang; Zhang, Jie; Oppert, Brenda; Lai, Fengxiang; Peng, Yufa; Zhang, Zhitao

    2010-09-01

    Transgenic rice to control stem borer damage is under development in China. To assess the potential of Bacillus thuringiensis (Bt) transgenes in stem borer control, the toxicity of five Bt protoxins (Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ba and Cry1Ca) against two rice stem borers, Sesamia inferens (pink stem borer) and Chilo suppressalis (striped stem borer), was evaluated in the laboratory by feeding neonate larvae on artificial diets containing Bt protoxins. The results indicated that Cry1Ca exhibited the highest level of toxicity to both stem borers, with an LC(50) of 0.24 and 0.30 microg/g for C. suppressalis and S. inferens, respectively. However, S. inferens was 4-fold lower in susceptibility to Cry1Aa, and 6- and 47-fold less susceptible to Cry1Ab and Cry1Ba, respectively, compared to C. suppressalis. To evaluate interactions among Bt protoxins in stem borer larvae, toxicity assays were performed with mixtures of Cry1Aa/Cry1Ab, Cry1Aa/Cry1Ca, Cry1Ac/Cry1Ca, Cry1Ac/Cry1Ba, Cry1Ab/Cry1Ac, Cry1Ab/Cry1Ba, and Cry1Ab/Cry1Ca at 1:1 (w/w) ratios. All protoxin mixtures demonstrated significant synergistic toxicity activity against C. suppressalis, with values of 1.6- to 11-fold higher toxicity than the theoretical additive effect. Surprisingly, all but one of the Bt protoxin mixtures were antagonistic in toxicity to S. inferens. In mortality-time response experiments, S. inferens demonstrated increased tolerance to Cry1Ab and Cry1Ac compared to C. suppressalis when treated with low or high protoxin concentrations. The data indicate the utility of Cry1Ca protoxin and a Cry1Ac/Cry1Ca mixture to control both stem borer populations.

  14. 基于cry1Ac表达调控元件的苏云金芽孢杆菌表达载体构建%Construction of Bacillus thuringiensis Expression Vector by Using Regulatory Elements from cry1Ac Gene

    Institute of Scientific and Technical Information of China (English)

    郑文; 叶伟星; 彭东海; 孙明

    2012-01-01

    通过克隆cry1Ac基因BtⅠ-BtⅡ启动子、SD序列以及终止子,并引入pET28a的His标签和多克隆位点,在穿梭载体pHT304的基础上构建了一个苏云金芽孢杆菌表达载体pBMB1A.将cry1Ac以及具有非典型BtⅠ-BtⅡ启动子的cry2Ab、cry5Ba、cry6Aa、cry7Ba、cry55Aa6个基因装载到该表达载体上,转入BMB171构建了重组菌株,通过复红简单染色后的显微镜镜检结果表明,重组菌株BMB1A-1Ac、BMB1A-5Ba、BMB1A-7Ba和BMB1A-55Aa均能形成正常晶体,SDS-PAGE结果也证实这4个重组菌株的重组质粒均能表达出目的蛋白.同时,选取重组菌株BMB1A-1Ac来考察His标签对Cry1Ac杀虫活性的影响,生物活性测定结果显示重组菌株的LC50值与对照菌株相比无明显差异.该载体可用于快速克隆表达不同类别的Cry蛋白.%A Bacillus thuringiensis expression vector named Pbmbia was constructed by cloning BtI-BtII promoter, SD-sequence, and transcription terminator of cry 1 Ac gene, and adding His-tag and the following multiple cloning sites (MCS) from pET28a into the shuttle vector Pht304. Cry 1 Ac and other five cry genes with atypical Btl-Btll promoter including cry2Ab, cry5Ba, cry6Aa, cry7Ba, cry55Aa were cloned into Pbmbia. Microscopic observation showed that the recombinant strains containing CrylAc, Cry5Ba, Cry7Ba and Cry55Aa could form parasporal inclusion bodies; And SDS-PAGE proved that all of them could produce the corresponding major insecticidal crystal proteins bands. Meanwhile, CrylAc was selected to determine the effect of His-tag on its insecticidal activity, and the bioassay result showed that there was no significant difference between the LC50 of the recombinant strain and the control strain. The expression vector constructed was suitable for rapid cloning and expression of different cry genes.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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.

  17. Elevated atmospheric ozone increases concentration of insecticidal Bacillus thuringiensis (Bt) Cry1Ac protein in Bt Brassica napus and reduces feeding of a Bt target herbivore on the non-transgenic parent

    Energy Technology Data Exchange (ETDEWEB)

    Himanen, Sari J. [University of Kuopio, Department of Environmental Science, P.O. Box 1627, FIN-70211 Kuopio (Finland)], E-mail: sari.himanen@uku.fi; Nerg, Anne-Marja [University of Kuopio, Department of Environmental Science, P.O. Box 1627, FIN-70211 Kuopio (Finland); Nissinen, Anne [University of Kuopio, Department of Environmental Science, P.O. Box 1627, FIN-70211 Kuopio (Finland); MTT Agrifood Research Finland, Plant Protection, FIN-31600 Jokioinen (Finland); Stewart, C. Neal [University of Tennessee, Department of Plant Sciences, Knoxville, TN 37996-4561 (United States); Poppy, Guy M. [University of Southampton, School of Biological Sciences, Southampton SO16 7PX (United Kingdom); Holopainen, Jarmo K. [University of Kuopio, Department of Environmental Science, P.O. Box 1627, FIN-70211 Kuopio (Finland)

    2009-01-15

    Sustained cultivation of Bacillus thuringiensis (Bt) transgenic crops requires stable transgene expression under variable abiotic conditions. We studied the interactions of Bt toxin production and chronic ozone exposure in Bt cry1Ac-transgenic oilseed rape and found that the insect resistance trait is robust under ozone elevations. Bt Cry1Ac concentrations were higher in the leaves of Bt oilseed rape grown under elevated ozone compared to control treatment, measured either per leaf fresh weight or per total soluble protein of leaves. The mean relative growth rate of a Bt target herbivore, Plutella xylostella L. larvae was negative on Bt plants in all ozone treatments. On the non-transgenic plants, larval feeding damage was reduced under elevated ozone. Our results indicate the need for monitoring fluctuations in Bt toxin concentrations to reveal the potential of ozone exposure for altering dosing of Bt proteins to target and non-target herbivores in field environments experiencing increasing ozone pollution. - Elevated atmospheric ozone can induce fluctuations in insecticidal protein concentrations in transgenic plants.

  18. 杀线虫苏云金芽胞杆菌晶体蛋白Cry5Aa与糖脂受体互作模式%Interaction mode of nematicidal Bacillus thuringiensis crystal protein Cry5Aa with glycolipid receptor

    Institute of Scientific and Technical Information of China (English)

    赵新民; 彭晓赟; 刘淑云; 周攀登; 徐玲; 夏莉

    2014-01-01

    采用分子对接方法研究了杀线虫苏云金芽胞杆菌晶体蛋白 Cry5Aa 与线虫糖脂受体寡糖片段的互作模式。结果表明:Cry5Aa与寡糖片段的结合位点在晶体蛋白结构域I和结构域II之间。Cry5Aa与寡糖片段的之间作用能主要为构象能,其次为氢键能,而静电作用能为零。二者之间能够形成10个氢键和多个构象能作用。其中晶体蛋白Cry5Aa结构域I氨基酸残基T298可与寡糖片段形成3个氢键,并且该氨基酸残基同样对构象能的贡献较大。Cry5Aa与寡糖片段的之间可以形成稳定的复合物。本研究结果对了解苏云金芽胞杆菌晶体蛋白Cry5Aa的毒理机制和开展定点突变提高Cry5Aa杀线虫活性具有指导意义。%Molecular docking was used to simulate the interaction between nematicidal Bacillus thuringiensis crystal protein Cry5Aa and its oligosaccharides fragment of the glycolipid receptor of target nematode. Results showed that the receptor binding site was located between domain I and domain II of Cry5Aa. The main interaction energy was the steric energy, next was the hydrogen bonding energy and the electrostatic interaction energy zero. Ten hydrogen bonds and several steric interactions were found in the binding site. Three hydrogen bonds were related to Thr298 in domain I that also contributed much to the steric energy. And as a result Cry5Aa and its receptor oligosaccharides formed a stable complex. The available results in this paper may help in understanding the action mode of Cry5Aa and designing mutagenesis experiments aimed to the improvement of nematicidal toxicity.

  19. The mode of action of the Bacillus thuringiensis vegetative insecticidal protein Vip3A differs from that of Cry1Ab delta-endotoxin.

    Science.gov (United States)

    Lee, Mi Kyong; Walters, Frederick S; Hart, Hope; Palekar, Narendra; Chen, Jeng-Shong

    2003-08-01

    The Vip3A protein, secreted by Bacillus spp. during the vegetative stage of growth, represents a new family of insecticidal proteins. In our investigation of the mode of action of Vip3A, the 88-kDa Vip3A full-length toxin (Vip3A-F) was proteolytically activated to an approximately 62-kDa core toxin either by trypsin (Vip3A-T) or lepidopteran gut juice extracts (Vip3A-G). Biotinylated Vip3A-G demonstrated competitive binding to lepidopteran midgut brush border membrane vesicles (BBMV). Furthermore, in ligand blotting experiments with BBMV from the tobacco hornworm, Manduca sexta (Linnaeus), activated Cry1Ab bound to 120-kDa aminopeptidase N (APN)-like and 250-kDa cadherin-like molecules, whereas Vip3A-G bound to 80-kDa and 100-kDa molecules which are distinct from the known Cry1Ab receptors. In addition, separate blotting experiments with Vip3A-G did not show binding to isolated Cry1A receptors, such as M. sexta APN protein, or a cadherin Cry1Ab ecto-binding domain. In voltage clamping assays with dissected midgut from the susceptible insect, M. sexta, Vip3A-G clearly formed pores, whereas Vip3A-F was incapable of pore formation. In the same assay, Vip3A-G was incapable of forming pores with larvae of the nonsusceptible insect, monarch butterfly, Danaus plexippus (Linnaeus). In planar lipid bilayers, both Vip3A-G and Vip3A-T formed stable ion channels in the absence of any receptors, supporting pore formation as an inherent property of Vip3A. Both Cry1Ab and Vip3A channels were voltage independent and highly cation selective; however, they differed considerably in their principal conductance state and cation specificity. The mode of action of Vip3A supports its use as a novel insecticidal agent.

  20. 一些化学物质对苏云金芽孢杆菌Cry1Ac毒素与昆虫离体细胞相互作用的影响%Influence of some chemicals on the action of Bacillus thuringiensis Cry1Ac toxin with cultured insect cells

    Institute of Scientific and Technical Information of China (English)

    刘凯于; 阎江洪; 康薇; 郑进; 洪华珠

    2005-01-01

    为探讨苏云金芽孢杆菌Bacillus thuringiensis(Bt)杀虫晶体蛋白与昆虫细胞的相互作用,以Bt Cry1Ac毒素和对该毒素敏感的粉纹夜蛾Trichoplusia ni离体细胞BTI-TN-5B1-4为材料,研究了一些化学物质对Cry1Ac毒素与昆虫离体细胞相互作用的影响.结果表明:N-糖基化抑制剂衣霉素、蛋白质合成抑制剂放线菌酮、胞吞作用抑制剂莫能菌素和胰蛋白酶预处理,都能不同程度地提高BTI-TN-5B1-4细胞对Cry1Ac毒素的敏感性,其中胰蛋白酶预处理的作用最明显;而N-乙酰半乳糖胺不能抑制Cry1Ac毒素对这种离体细胞的毒力.

  1. Efficient transformation of Bacillus thuringiensis requires nonmethylated plasmid DNA.

    OpenAIRE

    Macaluso, A; Mettus, A M

    1991-01-01

    The transformation efficiency of Bacillus thuringiensis depends upon the source of plasmid DNA. DNA isolated from B. thuringiensis, Bacillus megaterium, or a Dam- Dcm- Escherichia coli strain efficiently transformed several B. thuringiensis strains, B. thuringiensis strains were grouped according to which B. thuringiensis backgrounds were suitable sources of DNA for transformation of other B. thuringiensis strains, suggesting that B. thuringiensis strains differ in DNA modification and restri...

  2. Activity of four cry gene promoters in spoⅢD mutant of Bacillus thuringiensis%四种cry基因启动子在spoⅢD基因突变株中的活性比较

    Institute of Scientific and Technical Information of China (English)

    王新梅; 杜立新; 彭琦; 梁影屏; 李杰; 张杰; 宋福平

    2012-01-01

    [目的]分析spoⅢD基因突变对苏云金芽胞杆菌cry1、cry、cry4和cry8基因启动子Pcry1Ac、Pcry3A、Pcry4A和Pcry8E的影响,比较以上启动子在无芽胞spoⅢD基因突变体(HD-△SpoⅢD)中的转录活性.[方法]分别构建了Pcry1Ac、Pcry3A、Pcry4A和Pcry8E与lacZ基因融合的转录分析载体,并导入HD-73野生型菌株和HD-△SpoⅢD突变株中测定β-半乳糖苷酶活性;通过高温诱变方法在HD-△SpoⅢD基础上筛选出缺失cry1Ac基因的HD - -△SpoⅢD突变体;构建了4种启动子与cry1Ac基因融合表达载体,分别将它们转入HD-△SpoⅢD和HD - -△SpoⅢD中,分析Cry1Ac蛋白表达量及其生物活性.[结果]HD-73和HD-△SpoⅢD菌株中四个启动子转录活性由高到低分别为:Pcry8E>Pcry1Ac>Pcry4A>Pcry3A;spoⅢD基因的缺失未影响Pcry1Ac和Pcry8E转录活性,Pcry3A在HD-△SpoⅢD菌株中转录活性略有升高,Pcry4A在HD-△SpoⅢD菌株中转录活性在T5到T10略有降低.从翻译水平来看在HD-△SpoⅢD中cry8E启动子略低于cry1Ac启动子,并高于Pcry4Aa,Pcry3A指导的Cry1Ac蛋白产量,生物活性测定结果与蛋白表达量相符.[结论]cry8E基因启动子Pcry8E在spoⅢD突变体中在转录水平活性是最高的启动子,而cry1Ac启动子指导自身基因cry1Ac表达时,在翻译水平上略高于cry8E启动子指导的Cry1Ac产量.%[Objective] We studied the influence of spoIIID gene deletion on the activity of cry 1 Ac, cry3A, cry4A and crySE gene promoters in Bacillus thuringiensis and compared the activity among these promoters in spoIIID mutant ( HD-A SpoIIID). [Methods] We constructed 4 promoter fusions with lacZ gene and transformed them into wild-type strain HD-73 and HD-ASpoIIID to analyze their transcriptional activity. We constructed a spoIIID gene mutant ( HD -ASpoIIID ) with deletion of the crylAc-harboring native plasmid based on HD-ASpoIIID strain. We constructed four promoter fusions with cryl Ac gene and transformed them into

  3. Complete genome sequence of Bacillus thuringiensis serovar galleriae strain HD-29, a typical strain of commercial biopesticide.

    Science.gov (United States)

    Zhu, Lei; Tian, Long-Jun; Zheng, Jinshui; Gao, Qiu-Ling; Wang, Yue-Ying; Peng, Dong-Hai; Ruan, Li-Fang; Sun, Ming

    2015-02-10

    Bacillus thuringiensis serovar galleriae is highly toxic to Lepidoptera insect pests, and has been widely used as Bt biopesticide in many countries. Here we reported the complete genome of strain HD-29, a standard serotype strain in galleriae serovariety. More than previous work reported, it harbors ten plasmids, and three large ones carry eight insecticidal protein genes (cry1Aa, cry1Ac, cry1Ca, cry1Da, cry1Ia, cry2Ab, cry9Ea and vip3Aa) and an intact zwittermicin A biosynthetic gene cluster.

  4. Estirpes de Bacillus thuringiensis efetivas contra insetos das ordens Lepidoptera, Coleoptera e Diptera Bacillus thuringiensis strains effective against insects of Lepidoptera, Coleoptera and Diptera orders

    Directory of Open Access Journals (Sweden)

    Lílian Botelho Praça

    2004-01-01

    Full Text Available O objetivo deste trabalho foi selecionar entre 300 estirpes de Bacillus thuringiensis as efetivas simultaneamente contra larvas de Spodoptera frugiperda J.E. Smith e Anticarsia gemmatalis Hübner (Lepidoptera: Noctuidae, Anthonomus grandis Boheman (Coleoptera: Curculionidae, Aedes aegypti Linnaeus e Culex quinquefasciatus Say (Diptera: Culicidae. Foram selecionadas duas estirpes de B. thuringiensis, denominadas S234 e S997, que apresentaram atividade contra as três ordens de insetos. As estirpes foram caracterizadas por métodos morfológicos, bioquímicos e moleculares. As mesmas apresentaram duas proteínas principais de 130 e 65 kDa, produtos de reação em cadeia da polimerase de tamanho esperado para a detecção dos genes cry1Aa, cry1Ab, cry1Ac, cry1B e cry2 e cristais bipiramidais, cubóides e esféricos.The aim of this work was to select among 300 strains of Bacillus thuringiensis those which are simultaneously effective against larvae of Spodoptera frugiperda J.E. Smith and Anticarsia gemmatalis Hübner (Lepidoptera: Noctuidae, Anthonomus grandis Boheman (Coleoptera: Curculionidae, Aedes aegypti Linnaeus and Culex quinquefasciatus Say (Diptera: Culicidae. Two strains of B. thuringiensis were selected, S234 and S997, which presented activity against those three insect orders. Both strains were characterized by morphological, biochemical and molecular methods. They have presented two main proteins with 130 and 65 kDa, polimerase chain reaction products with expected sizes for detection of the genes cry1Aa, cry1Ab, cry1Ac, cry1B and cry2 and bipiramidal, cubical and spherical crystals.

  5. A genetically modified broad-spectrum strain of Bacillus thuringiensis toxic against Holotrichia parallela, Anomala corpulenta and Holotrichia oblita.

    Science.gov (United States)

    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.

  6. Characterization of Insecticidal Genes of Bacillus thuringiensis Strains Isolated from Arid Environments.

    Science.gov (United States)

    Abulreesh, Hussein H; Osman, Gamal E H; Assaeedi, Abdulrahman S A

    2012-09-01

    This study aimed at characterizing the insecticidal genes of eight Bacillus thuringiensis isolates that were recovered from the local environment of western Saudi Arabia. The screening for the presence of lepidopteran-specific cry1A family and vip3A genes, dipteran-specific cry4 family and coleopteran-specific cry3A, vip1A and vip2A genes, was carried out by PCR. All eight isolates produced PCR products that confirmed the presence of cry1Aa, cry1Ab, cry1Ac, cry4A, cry4B genes, but not cry3A, vip1A and vip2A genes. However, three isolates only were found to carry vip3A genes as revealed by PCR. The observation of cry1 and cry4 genes suggests that these eight isolates may have dual activity against Lepidoptera and Diptera species, while three isolates possessed vip3 genes in addition to cry1 and cry4 which suggests that these three isolates have toxic crystals and vegetative proteins. The results of this study are interesting in the sense that they may help developing new strategies for controlling insects of economic and medical importance in Saudi Arabia, using B. thuringiensis strains that naturally exist in the local environment instead of the current control strategies that are based solely on chemical insecticides.

  7. 苏云金芽孢杆菌新疆分离株cry3基因的克隆和序列分析%Cloning and Sequence Analysis on Bacillus thuringiensis cry 3 Gene

    Institute of Scientific and Technical Information of China (English)

    顾爱星; 张桦; 张翠芳; 张璐; 高微; 颜君; 艾山江·艾则孜

    2011-01-01

    These Bacillus thuringiensis (Bt) strains,used in this experiment and separated from soil and insects of north slope of Tianshan Mountains in Xinjiang,showed high virulence to Coleoptera pests. They contained total DNA about 15 kb as PCR template, specific primers were designed,800 bp and 550 bp DNA were amplified. Then these fragments were recovered and connected with vector pBS-T,and transformed the product into E. coli DH5α. The positive clone was obtained with the blue-white blot screening method, the sequensing was conducted on positive clone (1-5 about 700 bp, G4-3 about 500 bp,G4-4 about 720 bp) , then the known genes were compared with the sequences registered on the GenBank. The result indicated that the strains 1-5 had a higher sequence homology of 84% -100% with insecticidal toxin protein gene of the cry 3 gene family.%从新疆天山北坡土壤及昆虫上分离到的苏云金芽孢杆菌(Bacillus thuringiensis,Bt)菌株对鞘翅目害虫具有高毒力,其总DNA约为15kb,以此为模板,并设计特异引物,扩增出800bp和550 bp DNA片段,将回收的片段与pBS-T质粒连接,转化大肠杆菌(Escherichia coli),用蓝白斑法筛选,将阳性克隆子(1号-5,700 bp左右、G4号-3,500 bp左右、G4号-4,720 bp左右)测序,与GenBank中已知的序列进行比对,结果表明1号-5与cry 3基因家族的杀虫蛋白基因序列同源性可达84%~100%.

  8. Susceptibility of legume pod borer (LPB), Maruca vitrata to delta-endotoxins of Bacillus thuringiensis (Bt) in Taiwan.

    Science.gov (United States)

    Srinivasan, R

    2008-01-01

    Baseline susceptibility of legume pod borer (LPB) to the insecticidal crystal proteins (ICPs) from Bacillus thuringiensis, viz, Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ca and Cry2Aa was assessed in Taiwan. Insect bioassays were performed by incorporating the Bt delta-endotoxins into the LPB artificial diet. The efficacy of different Bt delta-endotoxins against second instar larvae of LPB showed that the toxin Cry1Ab was the most potent toxin (LC(50) 0.207ppm), followed by Cry1Ca, Cry1Aa, Cry2Aa and Cry1Ac in descending order, with LC(50)s 0.477ppm, 0.812ppm, 1.058ppm and 1.666ppm, respectively. Hence, Cry1Ab and/or Cry1Ca toxins would provide effective control of early larval stages of LPB.

  9. Comparison of susceptibility of Chilo suppressalis and Bombyx mori to five Bacillus thuringiensis proteins.

    Science.gov (United States)

    Jiao, Yaoyu; Yang, Yan; Meissle, Michael; Peng, Yufa; Li, Yunhe

    2016-05-01

    Transformation of rice with genes encoding insecticidal Cry proteins from Bacillus thuringiensis (Bt) should confer high resistance to target lepidopteran pests, such as Chilo suppressalis, and low toxicity to non-target organisms, such as silkworm Bombyx mori. Five purified Cry proteins that have been used for plant transformation were tested using dietary exposure assays. The susceptibility of C. suppressalis larvae to the five insecticidal proteins in the decreasing order was: Cry1Ca>Cry1Ab>Cry1Ac>Cry2Aa>Cry1Fa. However, the toxicities of the Cry proteins to B. mori were in the order: Cry1Fa>Cry1Ca>Cry2Aa>Cry1Ab>Cry1Ac. The Cry1Ca, Cry1Ab and Cry1Ac proteins exhibited relatively high toxicity to C. suppressalis larvae, with EC50 values of 16.4, 45.8 and 89.6ng/g, respectively. The toxicities of the three Cry proteins to B. mori larvae were 8, 14, and 22times lower, with EC50 values of 138.3, 628.4 and 1939.2ng/g, respectively. The Cry1Fa and Cry2Aa proteins showed high toxicity to B. mori larvae, with EC50 values of 135.7 and 373.9ng/g, respectively, but low toxicity to C. suppressalis larvae, with EC50 values of 6092.1 and 1208.5ng/g, respectively. We thus conclude that Cry1Ab, Cry1Ac and Cry1Ca are appropriate for transforming rice to control lepidopteran rice pests. In contrast, Cry1Fa and Cry2Aa are not appropriate due to their high toxicity to silkworm larvae and low activity against the target pest.

  10. Expression of Aminopeptidase N1(APN1),the Main Receptor Protein for Bacillus thuringiensis Cry1A Toxin from Helicoverpa armigera Larval Midgut in Trichoplusia ni cells

    Institute of Scientific and Technical Information of China (English)

    CHANG Hong-lei; LIANG Ge-mei; WANG Gui-rong; YU Hong-kun; GUO Yu-yuan; WU Kong-ming

    2008-01-01

    The aim of this article is to successfully express the Bt(Bacillus thuringiensis)toxin receptor protein located on the internal membrane of larval midgut of cotton bollworm(Helicoverpa armigera Hiibner)within eukaryotic expression system,which is one of the key links for clarifying the relationship between receptor and Bt resistance.The fragments of aminopeptidase N1(APN1)gene without signal peptide in the susceptible and the resistant H. armigera were cloned separately using PCR method,and were separately cloned into pUC 19 vector.After sequencing the gene,the fragments encoding for APN1 without signal peptide were cloned into the Bac-to-Bac baculovirus expression system with transfer vector pFastBacHTB under the polyhedron gene promoter.The recombinant transposing plasmid pFastBacHTB/APN1 was screened and then transformed into Escherichia coli DH10Bac.It was cultured in LB medium,which contained Te, Kan,Ge,X-gal,and IPTG.The resulting recombinant bacmid was transfected into cells of the insect Trichoplusia ni and recombinant baculoviruse was obtained.The lysate of cells infected with recombinant baculoviruse was analyzed by SDS-PAGE and blot analysis.The results showed that the recombinant baculoviruse was fully capable of expressing APN1.The APN1 gene successfully expressed in T. ni cell established the base for continuing the research on its function and relationship of resistance with Bt.

  11. Nano-Mechanical Properties of Heat Inactivated Bacillus anthracis and Bacillus thuringiensis Spores

    Science.gov (United States)

    2008-03-01

    NANO-MECHANICAL PROPERTIES OF HEAT INACTIVATED BACILLUS ANTHRACIS AND BACILLUS THURINGIENSIS ...GAP/ENP/08-M07 NANO-MECHANICAL PROPERTIES OF HEAT INACTIVATED BACILLUS ANTHRACIS AND BACILLUS THURINGIENSIS SPORES THESIS...AFIT/GAP/ENP/08-M07 NANO-MECHANICAL PROPERTIES OF HEAT INACTIVATED BACILLUS ANTHRACIS AND BACILLUS THURINGIENSIS SPORES Jessica

  12. Penicillin ingestion influences toxic effects of several Cry toxins from Bacillus thuringiensis on Spodoptera exigua larvae%青霉素影响甜菜夜蛾幼虫耐受几种苏云金杆菌晶体蛋白的能力

    Institute of Scientific and Technical Information of China (English)

    陈瑞瑞; 崔金杰; 李国清

    2011-01-01

    本文评估了Cry1Ab、Cry1Ba和Cry1Ca蛋白对甜菜夜蛾2龄幼虫的毒力,其LC50分别为0.442 5、0.675 7μg/cm2和0.150 2 μg/cm2,Cry1Ba毒力最低,其次是Cry1Ab,而Cry1Ca毒力最高.青霉素影响幼虫对晶体毒素的耐受能力;一次添加高剂量(500 μg/ cm2)青霉素于饲料中显著提高幼虫的耐受能力,以含低剂量(60 μg/cm2)青霉素的饲料饲养幼虫多代也能达到相似的效果.%Toxicities of Cry1Ab, Cry1Ba and CrylCa from Bacillus thuringiensis to the 2nd-instar larvae of Spo-doptera exigua were evaluated. The LC50 values were 0. 442 5 μg/cm2, 0. 675 7μg/cm2and 0.150 2 μg/cm2, respectively. Cry1Ba was least toxic, followed by Cry1Ab, and Cry1Ca was most toxic. Penicillin affected larval tolerance to the 3 Cry toxins. At the concentration of 500 μg/cm2, penicillin mixed directly with the toxins could significantly increase larval tolerance. At the concentration of 60 μg/cm2, however, penicillin should be first added alone to the diet for the larvae for several generations to obtain similar tolerance.

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

    Science.gov (United States)

    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.

  14. 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.

    Science.gov (United States)

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

    2015-09-01

    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.

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

    Science.gov (United States)

    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

    2016-05-05

    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.

  16. Potential of the Bacillus thuringiensis toxin reservoir for the control of Lobesia botrana (Lepidoptera: Tortricidae), a major pest of grape plants.

    Science.gov (United States)

    Ruiz de Escudero, Iñigo; Estela, Anna; Escriche, Baltasar; Caballero, Primitivo

    2007-01-01

    The potential of Bacillus thuringiensis Cry proteins to control the grape pest Lobesia botrana was explored by testing first-instar larvae with Cry proteins belonging to the Cry1, Cry2, and Cry9 groups selected for their documented activities against Lepidoptera. Cry9Ca, a toxin from B. thuringiensis, was the protein most toxic to L. botrana larvae, followed in decreasing order by Cry2Ab, Cry1Ab, Cry2Aa, and Cry1Ia7, with 50% lethal concentration values of 0.09, 0.1, 1.4, 3.2, and 8.5 microg/ml of diet, respectively. In contrast, Cry1Fa and Cry1JA were not active at the assayed concentration (100 microg/ml). In vitro binding and competition experiments showed that none of the toxins tested (Cry1Ia, Cry2Aa, Cry2Ab, and Cry9C) shared binding sites with Cry1Ab. We conclude that either Cry1Ia or Cry9C could be used in combination with Cry1Ab to control this pest, either as the active components of B. thuringiensis sprays or expressed together in transgenic plants.

  17. Draft Genome Sequence of Bacillus thuringiensis INTA Fr7-4

    Science.gov (United States)

    Berretta, Marcelo F.; Ortiz, Elio M.; Sauka, Diego H.; Benintende, Graciela B.; Zandomeni, Rubén O.

    2017-01-01

    ABSTRACT We report here the complete annotated 6,035,547-bp draft genome sequence of Bacillus thuringiensis INTA Fr7-4. This strain contains three cry8 and two vip1 and vip2 insecticidal toxin genes. PMID:28360155

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

    Science.gov (United States)

    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. Specific binding  of Bacillus thuringiensis Cry2A insecticidal proteins to a common site in the midgut of Helicoverpa species

    OpenAIRE

    Hernández Rodríguez, Carmen Sara; van Vliet, A.; Bautsoens, Nadine; Van Rie, Jeroen; Ferré Manzanero, Juan

    2008-01-01

    For a long time, it has been assumed that the mode of action of Cry2A toxins was unique and different from that of other three-domain Cry toxins due to their apparent nonspecific and unsaturable binding to an unlimited number of receptors. However, based on the homology of the tertiary structure among three-domain Cry toxins, similar modes of action for all of them are expected. To confirm this hypothesis, binding assays were carried out with 125 I-labeled Cry2Ab. Saturation assays showed tha...

  20. Effect of Quorum Sensing response regulator nprR deletion on expression of Cry protein in Bacillus thuringiensis%Bt群体信号应答因子nprR基因的缺失对cry1Ac基因表达的影响

    Institute of Scientific and Technical Information of China (English)

    王垚; 邓超; 彭琦; 陈榛; 张杰; 黄大昉; 宋福平

    2010-01-01

    [目的]研究群体信号应答蛋白编码基因nprR在苏云金芽胞杆菌(Bacillus thuringiensis,Bt)HD-73菌株晶体蛋白形成过程中的作用.[方法]通过同源重组,构建了HD-73 nprR基因缺失突变菌株HD73(AnprR).利用启动子-lacZ融合、SDS-PAGE方法,测定不同培养基中nprR基因转录活性及nprR基因缺失对cry1Ac转录及表达的影响.[结果]启动子转录活性分析表明,在LB和SSM培养基中nprR基因从对数期结束(T0)开始表达,稳定期持续表达.在LB培养基中,nprR基因的缺失使cry1Ac基因在生长过渡期和稳定期前期转录活性显著提高,同时HD73(ΔnprR)菌株Cry蛋白生成量也明显高于出发菌株HD-73,但是在芽胞形成释放后,Cry蛋白的表达没有明显的区别.[结论]在丰富培养基中苏云金芽胞杆菌nprR基因的缺失在生长过渡期和稳定期前期能够提高cry1Ac基因转录和表达,从而缩短了cry基因表达时间,并且Cry蛋白总产量与出发菌株相当.

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

    Science.gov (United States)

    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.

  2. Interação de proteínas Cry1 e Vip3A de Bacillus thuringiensis para controle de lepidópteros-praga

    Directory of Open Access Journals (Sweden)

    Paula Cristina Brunini Crialesi-Legori

    2014-02-01

    Full Text Available O objetivo deste trabalho foi avaliar a suscetibilidade das lagartas Anticarsia gemmatalis (Lepidoptera: Erebidae e Chrysodeixis includens (Lepidoptera: Noctuidae às proteínas Cry1 e Vip3A, bem como determinar se há a interação entre essas proteínas no controle das duas espécies. Bioensaios com as proteínas isoladas e em combinações foram realizados, e as concentrações letais CL50 e CL90 foram estimadas para cada condição. As proteínas Cry1Aa, Cry1Ac e Vip3Af foram as mais efetivas no controle de A. gemmatalis, enquanto Cry1Ac, Vip3Aa e Vip3Af foram mais efetivas no de C. includens. As proteínas Cry1Ac e Cry1Ca causaram maior inibição do desenvolvimento das larvas sobreviventes à CL50, em ambas as espécies. Combinações entre Vip3A e Cry1 apresentam efeito sinérgico no controle das espécies e a combinação Vip3Aa+Cry1Ea destaca-se no controle de A. gemmatalis e C. includens. Essas proteínas combinadas são promissoras na construção de plantas piramidadas, para o controle simultâneo das pragas.

  3. Importance of polarity of the α4-α5 loop residue-Asn(166) in the pore-forming domain of the Bacillus thuringiensis Cry4Ba toxin: implications for ion permeation and pore opening.

    Science.gov (United States)

    Juntadech, Thanate; Kanintronkul, Yodsoi; Kanchanawarin, Chalermpol; Katzenmeier, Gerd; Angsuthanasombat, Chanan

    2014-01-01

    Bacillus thuringiensis Cry4Ba toxin is lethal to mosquito-larvae by forming ion-permeable pores in the target midgut cell membrane. Previously, the polarity of Asn(166) located within the α4-α5 loop composing the Cry4Ba pore-forming domain was shown to be crucial for larvicidal activity. Here, structurally stable-mutant toxins of both larvicidal-active (N166D) and inactive (N166A and N166I) mutants were FPLC-purified and characterized for their relative activities in liposomal-membrane permeation and single-channel formation. Similar to the 65-kDa trypsin-activated wild-type toxin, the N166D bio-active mutant toxin was still capable of releasing entrapped calcein from lipid vesicles. Conversely, the two other bio-inactive mutants showed a dramatic decrease in causing membrane permeation. When the N166D mutant was incorporated into planar lipid bilayers (under symmetrical conditions at 150mM KCl, pH8.5), it produced single-channel currents with a maximum conductance of about 425pS comparable to the wild-type toxin. However, maximum conductances for single K(+)-channels formed by both bio-inactive mutants (N166I and N166A) were reduced to approximately 165-205pS. Structural dynamics of 60-ns simulations of a trimeric α4-α5 pore model in a fully hydrated-DMPC system revealed that an open-pore structure could be observed only for the simulated pores of the wild type and N166D. Additionally, the number of lipid molecules interacting with both wild-type and N166D pores is relatively higher than those of N166A and N166I pores. Altogether, our results further signify that the polarity at the α4-α5 loop residue-Asn(166) is directly involved in ion permeation through the Cry4Ba toxin-induced ionic pore and pore opening at the membrane-water interface.

  4. Bacillus thuringiensis and Its Pesticidal Crystal Proteins

    OpenAIRE

    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 o...

  5. BACILLUS THURINGIENSIS ELASTASES WITH INSECTICIDE ACTIVITY

    OpenAIRE

    E. V. Matseliukh; N. A. Nidialkova; V. V. Krout'; L. D. Varbanets; A. V. Kalinichenko; V. F. Patyka

    2015-01-01

    The purpose of the research was a screening of proteases with elastase activity among Bacillus thuringiensis strains, their isolation, partially purification, study of physicochemical properties and insecticide activity in relation to the larvae of the Colorado beetle. The objects of the investigation were 18 strains of B. thuringiensis, isolated from different sources: sea water, dry biological product "Bitoksibatsillin" and also from natural populations of Colorado beetles of the Crimea, Kh...

  6. Characterization of chimeric Bacillus thuringiensis Vip3 toxins.

    Science.gov (United States)

    Fang, Jun; Xu, Xiaoli; Wang, Ping; Zhao, Jian-Zhou; Shelton, Anthony M; Cheng, Jiaan; Feng, Ming-Guang; Shen, Zhicheng

    2007-02-01

    Bacillus thuringiensis vegetative insecticidal proteins (Vip) are potential alternatives for B. thuringiensis endotoxins that are currently utilized in commercial transgenic insect-resistant crops. Screening a large number of B. thuringiensis isolates resulted in the cloning of vip3Ac1. Vip3Ac1 showed high insecticidal activity against the fall armyworm Spodoptera frugiperda and the cotton bollworm Helicoverpa zea but very low activity against the silkworm Bombyx mori. The host specificity of this Vip3 toxin was altered by sequence swapping with a previously identified toxin, Vip3Aa1. While both Vip3Aa1 and Vip3Ac1 showed no detectable toxicity against the European corn borer Ostrinia nubilalis, the chimeric protein Vip3AcAa, consisting of the N-terminal region of Vip3Ac1 and the C-terminal region of Vip3Aa1, became insecticidal to the European corn borer. In addition, the chimeric Vip3AcAa had increased toxicity to the fall armyworm. Furthermore, both Vip3Ac1 and Vip3AcAa are highly insecticidal to a strain of cabbage looper (Trichoplusia ni) that is highly resistant to the B. thuringiensis endotoxin Cry1Ac, thus experimentally showing for the first time the lack of cross-resistance between B. thuringiensis Cry1A proteins and Vip3A toxins. The results in this study demonstrated that vip3Ac1 and its chimeric vip3 genes can be excellent candidates for engineering a new generation of transgenic plants for insect pest control.

  7. Decreased toxicity of Bacillus thuringiensis subsp. israelensis to mosquito larvae after contact with leaf litter.

    Science.gov (United States)

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

    2012-08-01

    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.

  8. Cytotoxicity Analysis of Three Bacillus thuringiensis Subsp. israelensis δ-Endotoxins towards Insect and Mammalian Cells

    Science.gov (United States)

    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. Cytotoxicity analysis of three Bacillus thuringiensis subsp. israelensis δ-endotoxins towards insect and mammalian cells.

    Directory of Open Access Journals (Sweden)

    Roberto Franco Teixeira Corrêa

    Full Text Available 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.

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

    Science.gov (United States)

    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...

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

    Science.gov (United States)

    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.

  12. Domain III of Bacillus thuringiensis Cry1Ie Toxin Plays an Important Role in Binding to Peritrophic Membrane of Asian Corn Borer

    Science.gov (United States)

    Feng, Dongmei; Chen, Zhen; Wang, Zhiwen; Zhang, Chunlu; He, Kanglai; Guo, Shuyuan

    2015-01-01

    The insecticidal IE648 toxin is a truncated Cry1Ie protein with increased toxicity against Asian corn borer (ACB). Cry toxins are pore-forming toxins that disrupt insect midgut cells to kill the larvae. However, the peritrophic membrane (PM) is an important barrier that Cry toxins must cross before binding to midgut cells. Previously, it was shown that Cry toxins are able to bind and accumulate in the PM of several lepidopteran insects. Binding of IE648 toxin to PM of ACB was previously reported and the goal of the current work was the identification of the binding region between Cry1Ie and the PM of ACB. Homologous competition binding assays showed that this interaction was specific. Heterologous competition binding assays performed with different fragments corresponding to domain I, domain II and domain III allowed us to identify that domain III participates in the interaction of IE648 with the PM. Specifically, peptide D3-L8 (corresponding to Cry1Ie toxin residues 607 to 616), located in an exposed loop region of domain III is probably involved in this interaction. Ligand blot assays show that IE648 interact with chitin and PM proteins with sizes of 30, 32 and 80 kDa. The fact that domain III interacts with proteins of similar molecular masses supports that this region of the toxin might be involved in PM interaction. These data provide for the first time the identification of domain III as a putative binding region between PM and 3D-Cry toxin. PMID:26295704

  13. Domain III of Bacillus thuringiensis Cry1Ie Toxin Plays an Important Role in Binding to Peritrophic Membrane of Asian Corn Borer.

    Directory of Open Access Journals (Sweden)

    Dongmei Feng

    Full Text Available The insecticidal IE648 toxin is a truncated Cry1Ie protein with increased toxicity against Asian corn borer (ACB. Cry toxins are pore-forming toxins that disrupt insect midgut cells to kill the larvae. However, the peritrophic membrane (PM is an important barrier that Cry toxins must cross before binding to midgut cells. Previously, it was shown that Cry toxins are able to bind and accumulate in the PM of several lepidopteran insects. Binding of IE648 toxin to PM of ACB was previously reported and the goal of the current work was the identification of the binding region between Cry1Ie and the PM of ACB. Homologous competition binding assays showed that this interaction was specific. Heterologous competition binding assays performed with different fragments corresponding to domain I, domain II and domain III allowed us to identify that domain III participates in the interaction of IE648 with the PM. Specifically, peptide D3-L8 (corresponding to Cry1Ie toxin residues 607 to 616, located in an exposed loop region of domain III is probably involved in this interaction. Ligand blot assays show that IE648 interact with chitin and PM proteins with sizes of 30, 32 and 80 kDa. The fact that domain III interacts with proteins of similar molecular masses supports that this region of the toxin might be involved in PM interaction. These data provide for the first time the identification of domain III as a putative binding region between PM and 3D-Cry toxin.

  14. Isolation of Bacillus thuringiensis from the state of Amazonas, in Brazil, and screening against Aedes aegypti (Diptera, Culicidae

    Directory of Open Access Journals (Sweden)

    Joelma Soares-da-Silva

    2015-03-01

    Full Text Available We investigated the use of Bacillus thuringiensis isolated in the state of Amazonas, in Brazil, for the biological control of the dengue vector Aedes aegypti. From 25 soil samples collected in nine municipalities, 484 bacterial colonies were obtained, 57 (11.78% of which were identified as B. thuringiensis. Six isolates, IBt-03, IBt-06, IBt-07, IBt-28, IBt-30, and BtAM-27 showed insecticidal activity, and only BtAM-27 presents the five genes investigated cry4Aa, cry4Ba, cry10Aa, cry11Aa, and cry11Ba. The IBt-07 and IBt- 28, with lower LC50 values, showed equal toxicity compared to the standards. The isolates of B. thuringiensis from Amazonas constitute potential new means of biological control for A. aegypti, because of their larvicidal activity and the possibility that they may also contain new combinations of toxins.

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

    Science.gov (United States)

    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.

  16. La biotecnología de Bacillus thuringiensis en la agricultura

    OpenAIRE

    Portela Dussán, Diana Daniela; Chaparro Giraldo, Alejandra; López Pazos, Silvio Alejandro

    2013-01-01

    Bacillus thuringiensis es un bacilo Gram positivo que durante su fase de esporulación produce una inclusión parasporal, conformada por proteínas Cry con actividad biológica contra insectos-plaga. Gracias a estas proteínas Bacillus thuringiensis presenta toxicidad contra larvas de insectos-plaga de los órdenes Lepidóptera, Coleóptera y Díptera, entre otros. Además es amigable con el medioambiente, razones por la cuales se ha hecho común el uso y desarrollo de productos comerciales y plantas tr...

  17. Interação de proteínas Cry1 e Vip3A de Bacillus thuringiensis para controle de lepidópteros-praga

    OpenAIRE

    Paula Cristina Brunini Crialesi-Legori; Camila Chiaradia Davolos; Ana Rita Nunes Lemes; Suzana Cristina Marucci; Manoel Victor Franco Lemos; Odair Aparecido Fernandes; Janete Apparecida Desidério

    2014-01-01

    O objetivo deste trabalho foi avaliar a suscetibilidade das lagartas Anticarsia gemmatalis (Lepidoptera: Erebidae) e Chrysodeixis includens (Lepidoptera: Noctuidae) às proteínas Cry1 e Vip3A, bem como determinar se há a interação entre essas proteínas no controle das duas espécies. Bioensaios com as proteínas isoladas e em combinações foram realizados, e as concentrações letais CL50 e CL90 foram estimadas para cada condição. As proteínas Cry1Aa, Cry1Ac e Vip3Af foram as mais efetivas no contr...

  18. Effects and mechanisms of Bacillus thuringiensis crystal toxins for mosquito larvae.

    Science.gov (United States)

    Zhang, Qi; Hua, Gang; Adang, Michael J

    2016-09-15

    Bacillus thuringiensis is a Gram-positive aerobic bacterium that produces insecticidal crystalline inclusions during sporulation phases of the mother cell. The virulence factor, known as parasporal crystals, is composed of Cry and Cyt toxins. Most Cry toxins display a common 3-domain topology. Cry toxins exert intoxication through toxin activation, receptor binding and pore formation in a suitable larval gut environment. The mosquitocidal toxins of Bt subsp. israelensis (Bti) were found to be highly active against mosquito larvae and are widely used for vector control. Bt subsp. jegathesan is another strain which possesses high potency against broad range of mosquito larvae. The present review summarizes characterized receptors for Cry toxins in mosquito larvae, and will also discuss the diversity and effects of 3-D mosquitocidal Cry toxin and the ongoing research for Cry toxin mechanisms generated from investigations of lepidopteran and dipteran larvae.

  19. Estructura tridimensional de las toxinas de Bacillus thuringiensis: revisión Three dimensional structure of Bacillus thuringiensis toxins: a review

    Directory of Open Access Journals (Sweden)

    Cerón Salamanca JA.

    2007-08-01

    Full Text Available La ingeniería de proteínas de las d-endotoxinas de Bacillus thuringiensis puede orientar la búsqueda de variantes con un espectro mayor de especies susceptibles, potencia optimizada, y estabilidad apropiada. Aquí, nosotros revisamos las características más importantes de la estructura tridimensional de las proteínas Cry y Cyt. Es posible concluir que existe un modelo general obvio con propiedades específicas de acuerdo a su función y organismo susceptible.Structure-based protein engineering of Bacillus thuringiensis d-endotoxins may direct the search for variants with broader susceptible species spectra, optimal potency, and stability properties. Here, we revised the more important characteristics of the Cry and Cyt proteins three-dimensional structure; it is possible to conclude that an obvious general model exists with specific properties according to its function and target organism.

  20. Estimation of resistance allele frequency to maize incorporated Bacillus thuringiensis Cry2Ab2 protein in field populations of the fall army Spodoptera frugiperda (Lepidoptera: Noctuidae) from south region of the United State

    Science.gov (United States)

    The fall armyworm, Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae), is a target of transgenic maize and cotton expressing Bacillus thuringiensis (Bt) proteins in both North and South Americas. In the falls of 2013 and 2014, a total of 215 F2 two-parent families of S. frugiperda were es...

  1. Narrow terahertz attenuation signatures in Bacillus thuringiensis.

    Science.gov (United States)

    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.

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

    Science.gov (United States)

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

    2016-06-14

    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.

  3. The pore-forming protein Cry5B elicits the pathogenicity of Bacillus sp. against Caenorhabditis elegans.

    Directory of Open Access Journals (Sweden)

    Melanie F Kho

    Full Text Available The soil bacterium Bacillus thuringiensis is a pathogen of insects and nematodes and is very closely related to, if not the same species as, Bacillus cereus and Bacillus anthracis. The defining characteristic of B. thuringiensis that sets it apart from B. cereus and B. anthracis is the production of crystal (Cry proteins, which are pore-forming toxins or pore-forming proteins (PFPs. Although it is known that PFPs are important virulence factors since their elimination results in reduced virulence of many pathogenic bacteria, the functions by which PFPs promote virulence are incompletely understood. Here we study the effect of Cry proteins in B. thuringiensis pathogenesis of the nematode Caenorhabditis elegans. We find that whereas B. thuringiensis on its own is not able to infect C. elegans, the addition of the PFP Cry protein, Cry5B, results in a robust lethal infection that consumes the nematode host in 1-2 days, leading to a "Bob" or bag-of-bacteria phenotype. Unlike other infections of C. elegans characterized to date, the infection by B. thuringiensis shows dose-dependency based on bacterial inoculum size and based on PFP concentration. Although the infection process takes 1-2 days, the PFP-instigated infection process is irreversibly established within 15 minutes of initial exposure. Remarkably, treatment of C. elegans with Cry5B PFP is able to instigate many other Bacillus species, including B. anthracis and even "non-pathogenic" Bacillus subtilis, to become lethal and infectious agents to C. elegans. Co-culturing of Cry5B-expressing B. thuringiensis with B. anthracis can result in lethal infection of C. elegans by B. anthracis. Our data demonstrate that one potential property of PFPs is to sensitize the host to bacterial infection and further that C. elegans and probably other roundworms can be common hosts for B. cereus-group bacteria, findings with important ecological and research implications.

  4. Molecular characterization of Bacillus thuringiensis strains from Argentina.

    Science.gov (United States)

    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.

  5. The Complete Genome Sequence of Bacillus thuringiensis AlHakam

    Energy Technology Data Exchange (ETDEWEB)

    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).

  6. Biological Characteristics of Bacillus thuringiensis Isolate GS8 and Identification of its cry-type Genes%苏云金芽胞杆菌GS8菌株的生物学特性及其cry基因型鉴定

    Institute of Scientific and Technical Information of China (English)

    刘廷辉; 郭巍; 申建茹

    2009-01-01

    采用桑叶浸渍法和饲料表面覆盖法测定发现,从土壤中分离的一株苏云金芽胞杆菌菌株GS8对家蚕Bombyx mori、甜菜夜蛾Spodoptera exigrua和棉铃虫Helicoverpa armigera初孵幼虫均表现出较高的杀虫活性,经形态学和生理生化反应鉴定该菌株为东北亚种(Bacillus thuringiensis subsp.tohokuensis).聚丙烯酰胺凝胶电泳(SDS-PAGE)分析结果表明,GS8菌株的杀虫蛋白晶体主要由分子质量为130、81和60 kDa的蛋白组成.聚合酶链反应-限制性片段长度多态性(PCR-RFLP)鉴定结果显示,GS8菌株含有cry1Aa、cry1Ab、cry1Ib、cry2Ab和cry9Ba等基因.

  7. Characterization of a novel Bacillus thuringiensis phenotype possessing multiple appendages attached to a parasporal body.

    Science.gov (United States)

    Ventura-Suárez, Antonio; Cruz-Camarillo, Ramón; Rampersad, Joanne; Ammons, David R; López-Villegas, Edgar O; Ibarra, Jorge E; Rojas-Avelizapa, Luz I

    2011-01-01

    Bacillus thuringiensis is a bacterium best known for its production of crystal-like bodies comprised of one or more Cry-proteins, which can be toxic to insects, nematodes or cancer cells. Although strains of B. thuringiensis have occasionally been observed with filamentous appendages attached to their spores, appendages in association with their parasporal bodies are extremely rare. Herein we report the characterization of Bt1-88, a bacterial strain isolated from the Caribbean that produces a spore-crystal complex containing six long appendages, each comprised of numerous thinner filaments approximately 10 nm in diameter and 2.5 μm in length. Each of the multi-filament appendages was attached to a single, small parasporal body located at one end of the bacterial spore. Biochemical tests, 16S rDNA gene sequencing, and the identification of two Cry proteins by partial protein sequencing (putatively Cry1A and Cry2A), unambiguously identified Bt1-88 as a strain of B. thuringiensis. Bt1-88 represents the second reported strain of B. thuringiensis possessing a parasporal body/appendage phenotype characterized by one or more long appendages, comprised of numerous filaments in association with a parasporal body. This finding suggests that Bt1-88 is a member of a new phenotypic class of B. thuringiensis, in which the parasporal body may perform a novel structural role through its association with multi-filament appendages.

  8. Research advances on the cry genes of Bacillus thuringiensis%苏云金芽孢杆菌cry基因研究进展

    Institute of Scientific and Technical Information of China (English)

    李雪雁; 李照会; 许维岸

    2003-01-01

    从cry基因的分类、cry 基因与转座因子的关系、cry基因的表达调控以及cry基因的作用机理等4个方面综述了cry基因的研究进展,并简要展望了其研究和应用前景.

  9. 表面等离子共振传感检测Cry2A蛋白%Using a Surface Plasmon Resonance Sensor for the Detection of Bacillus thuringiensis Cry2A Protein

    Institute of Scientific and Technical Information of China (English)

    蔡淼; 黄新; 岳喜庆

    2014-01-01

    目的:建立利用表面等离子共振(surface plasmon resonance,SPR)传感器检测转基因植物中苏云金芽孢杆菌Cry2A蛋白的方法.方法:利用SPR检测技术,根据生物分子间的相互作用,在金片表面修饰Cry2A蛋白的特异性单克隆抗体,对不同质量浓度梯度的Cry2A蛋白进行检测研究.结果:该方法可有效地检测到Cry2A蛋白,检测灵敏度可达10 ng/mL,与同为抗虫基因编码的Cry1Ac和Cry2Ab蛋白未出现交叉反应.结论:利用SPR方法检测Cry2A蛋白操作简单,省时且灵敏度高、特异性强,可用于对Cry2A蛋白的定性检测.

  10. 苏云金芽孢杆菌杀蚊蛋白基因cry11Aa的克隆与表达%Clone and Expression of Mosquitocidal Toxin Gene cry11Aa of Bacillus thuringiensis

    Institute of Scientific and Technical Information of China (English)

    孙建光; 高俊莲; Soberon Mario

    2006-01-01

    目的 克隆苏云金芽孢杆菌以色列亚种的杀蚊毒素蛋白基因cry11Aa,并使其在酵母双杂交系统受体菌Saccharomyces cerevisiae L40中获得表达.方法 应用聚合酶链反应扩增获得cry11Aa基因,构建表达载体pHybLex/Zeo-cry11Aa,转化到酵母菌Saccharomyces cerevisiae L40.结果 用Cry11Aa抗体和LexA抗体进行的Western blot免疫杂交表明cry11Aa基因在酵母菌L40中成功表达,生成了Cry11Aa-LexA融合蛋白.结论 成功地克隆、表达了cry11Aa基因,为进一步利用酵母双杂交系统寻找与毒素蛋白Cry11Aa特异性结合的蚊幼中肠受体蛋白,揭示苏云金芽孢杆菌毒杀蚊虫的分子生物学机制奠定了基础.

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

    Science.gov (United States)

    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.

  12. 苏云金杆菌cry7Ab8基因的克隆表达及其杀虫活性%Cloning,Expression and Insecticidal Activity of cry7Ab8 Gene from Bacillus thuringiensis

    Institute of Scientific and Technical Information of China (English)

    冯静静; 郭巍; 刘廷辉; 孙永祥; 孙伟明; 徐大庆

    2011-01-01

    A cry7-type gene from Bt GWS7 strain was identified by full-length PCR method. The cry7Ab8 gene was amplified and cloned into pET28b. The recombinant plasmid pET cry7Ab8 was transformed into E. coli BL21 (DE3), resulting BL21(pET cry7Ab8). The SDS-PAGE results showed that the 130 kDa Cry7Ab8 protein was expressed. An engineering strain BioHD7Ab8 was construced by transforming the shuttle vector pSXY422b containing cry7Ab8 into Bt acrystalliferous mutant HD73- (cry-). The bioassay results indicated that the Cry7Ab8 protein was highly toxic against Henosepilachnavigintioctomaculata larvae, and the LCs0 value was 548 μg/mL.%克隆对鞘翅目害虫有毒力的Bt新菌株GWS7的cry7Ab8基因,并对该基因进行表达和杀虫活性的研究.利用全长PCR方法克隆cry7Ab8基因,将cry7Ab8基因插入到表达载体pET28b,获得重组质粒pETcry7Ab8,转化E.coli BL21(DE3),诱导后表达130 kDa的蛋白.将cry7Ab8基因连接到大肠杆菌-苏云金杆菌的穿梭载体pSXY422b,获得重组穿梭质粒pScry7Ab8,电激转化到苏云金杆菌无晶体突变株HD73-(cry-),获得工程菌BioHD7Ab8,表达蛋白后进行生物活性测定.生物活性测定结果显示Cry7Ab8蛋白对马铃薯瓢虫二龄幼虫有较强的杀虫活性,LCso为548μg/mL.

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

    Science.gov (United States)

    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.

  14. 蛋白酶抑制剂对苏云金杆菌晶体毒素活性的影响%Influence of Protease Inhititors on Bacillus thuringiensis Cry Toxins' Activity

    Institute of Scientific and Technical Information of China (English)

    李国清; 崔金杰; 陈瑞瑞

    2011-01-01

    综述了蛋白酶抑制剂对苏云金杆菌晶体毒素(B.t.Cry)活性影响的最新研究进展.蛋白酶抑制剂降低中肠中蛋白酶活性,这不仅影响B.t.cry原毒素的活化,而且延缓B.t.Cry毒素的水解失活.蛋白酶抑制剂对B.t.Cry毒素协同作用取决于这2种作用的均衡.%The recent advances of the influence of protease inhibitors on Cry toxins from B. Thuringiensis was reviewed. Protease inhibitors showes strong inhibitory effects on proteases in insect gut. Consequently, some protease inhibitors affects cleavage of B.t.Cry protoxin to form activated toxin. At the same time, these inhibitors may prevent excessive degradation of B.t.Cry active toxins. The synergistic effects of protease inhibitors on B.t.Cry toxins depends on these two proteolytic metabolisms.

  15. Novel toxicity of Bacillus thuringiensis strains against the melon fruit fly, Bactrocera cucurbitae (Diptera: Tephritidae).

    Science.gov (United States)

    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.

  16. Transport of Bacillus thuringiensis var. Kurstaki Via Fomites

    Science.gov (United States)

    2011-01-01

    Special Feature: Remediation Transport of Bacillus Thuringiensis var. Kurstaki Via Fomites Sheila Van Cuyk, Lee Ann B. Veal, Beverley Simpson, and...evaluate biodefense concepts of operations using routine spraying of Bacillus thuringiensis var. kurstaki (Btk). Btk is dispersed in large quantities as...used is a water-based slurry containing Bacillus thuringiensis var. kurstaki (Btk). This bacterium produces a toxin that is lethal to gypsy moth

  17. Indica rice cultivar IRGA 424, transformed with cry genes of B. thuringiensis, provided high resistance against Spodoptera frugiperda (Lepidoptera: Noctuidae).

    Science.gov (United States)

    Pinto, Laura Massochin Nunes; Fiuza, Lidia Mariana; Ziegler, Denize; De Oliveira, Jaime Vargas; Menezes, Valmir Gaedke; Bourrié, Isabelle; Meynard, Donaldo; Guiderdoni, Emmanuel; Breitler, Jean-Christophe; Altosaar, Illimar; Gantet, Pascal

    2013-12-01

    Plant expression of the entomopathogenic bacteria Bacillus thuringiensis cry gene has reduced the damage created by insect pests in several economically important cultures. For this study, we have conducted genetic transformation of the indica rice "IRGA 424", via Agrobacterium tumefaciens, using the B. thuringiensis cry1Aa and cry1B genes, with the objective of obtaining rice plants resistant to the insect pests from this culture. The gene constructions harbor the promoters maize proteinase inhibitor and ubiquitin. The results showed that high concentration of the hormone 2,4-dichlorophenoxyacetic acid and agarose as the gelling agent helped the production of embryogenic calli for the analyzed cultivar. More than 80% of the obtained transformed plants revealed the integration, using polymerase chain reaction, of the cry1Aa and cry1B genes. Analysis of the expression of the heterologous protein by Western blotting revealed the expression of the Cry1B delta-endotoxin in IRGA 424 plants transformed with the ubiquitin promoter. Data showed the production and dissemination of a high number of embryogenic calli in addition to obtaining plants transformed with the cry1Aa and cry1B genes until the reproductive phase. The feed bioassays with the transformed plants and Spodoptera frugiperda (JE Smith) larvae indicated high rates of mortality to the insect target. The highest corrected mortality rate achieved under laboratory conditions with Bt-rice plants transformed with the cry1B and cry1Aa genes was 94 and 84%, respectively. Thus, our results demonstrated the great potential of transformed Bt-rice plants in controlling the damage caused by these insect pests in rice paddy fields.

  18. Mobility of adsorbed Cry1Aa insecticidal toxin from Bacillus thuringiensis (Bt) on montmorillonite measured by fluorescence recovery after photobleaching (FRAP)

    Science.gov (United States)

    Helassa, Nordine; Daudin, Gabrielle; Noinville, Sylvie; Janot, Jean-Marc; Déjardin, Philippe; Staunton, Siobhán; Quiquampoix, Hervé

    2010-06-01

    The insecticidal toxins produced by genetically modified Bt crops are introduced into soil through root exudates and tissue decomposition and adsorb readily on soil components, especially on clays. This immobilisation and the consequent concentration of the toxins in "hot spots" could increase the exposure of soil organisms. Whereas the effects on non-target organisms are well documented, few studies consider the migration of the toxin in soil. In this study, the residual mobility of Bt Cry1Aa insecticidal toxin adsorbed on montmorillonite was assessed using fluorescence recovery after photobleaching (FRAP). This technique, which is usually used to study dynamics of cytoplasmic and membrane molecules in live cells, was applied for the first time to a protein adsorbed on a finely divided swelling clay mineral, montmorillonite. No mobility of adsorbed toxin was observed at any pH and at different degrees of surface saturation.

  19. Single concentration tests show synergism among Bacillus thuringiensis subsp. israelensis toxins against the malaria vector mosquito Anopheles albimanus.

    Science.gov (United States)

    Fernández-Luna, María Teresa; Tabashnik, Bruce E; Lanz-Mendoza, Humberto; Bravo, Alejandra; Soberón, Mario; Miranda-Ríos, Juan

    2010-07-01

    Bioassays of insecticidal proteins from Bacillus thuringiensis subsp. israelensis with larvae of the malaria vector mosquito Anopheles albimanus showed that the cytolytic protein Cyt1Aa was not toxic alone, but it increased the toxicity of the crystalline proteins Cry4Ba and Cry11Aa. Synergism also occurred between Cry4Ba and Cry11Aa toxins. Whereas many previous analyses of synergism have been based on a series of toxin concentrations leading to comparisons between expected and observed values for the concentration killing 50% of insects tested (LC(50)), we describe and apply a method here that enables testing for synergism based on single concentrations of toxins.

  20. Bacillus thuringiensis crystal proteins that target nematodes

    OpenAIRE

    Wei, Jun-Zhi; Hale, Kristina; Carta, Lynn; Platzer, Edward; Wong, Cynthie; Fang, Su-Chiung; Aroian, Raffi V.

    2003-01-01

    Bacillus thuringiensis (Bt) crystal proteins are pore-forming toxins used as insecticides around the world. Previously, the extent to which these proteins might also target the invertebrate phylum Nematoda has been mostly ignored. We have expressed seven different crystal toxin proteins from two largely unstudied Bt crystal protein subfamilies. By assaying their toxicity on diverse free-living nematode species, we demonstrate that four of these crystal proteins are active against multiple nem...

  1. Extent of Variation of the Bacillus thuringiensis Toxin Reservoir: the Case of the Geranium Bronze, Cacyreus marshalli Butler (Lepidoptera: Lycaenidae)

    Science.gov (United States)

    Herrero, Salvador; Borja, Marisé; Ferré, Juan

    2002-01-01

    Despite the fact that around 200 cry genes from Bacillus thuringiensis have already been cloned, only a few Cry proteins are toxic towards a given pest. A crucial step in the mode of action of Cry proteins is binding to specific sites in the midgut of susceptible insects. Binding studies in insects that have developed cross-resistance discourage the combined use of Cry proteins sharing the same binding site. If resistance management strategies are to be implemented, the arsenal of Cry proteins suitable to control a given pest may be not so vast as it might seem at first. The present study evaluates the potential of B. thuringiensis for the control of a new pest, the geranium bronze (Cacyreus marshalli Butler), a butterfly that is threatening the popularity of geraniums in Spain. Eleven of the most common Cry proteins from the three lepidopteran-active Cry families (Cry1, Cry2, and Cry9) were tested against the geranium bronze for their toxicity and binding site relationships. Using 125I-labeled Cry1A proteins we found that, of the seven most active Cry proteins, six competed for binding to the same site. For the long-term control of the geranium bronze with B. thuringiensis-based insecticides it would be advisable to combine any of the Cry proteins sharing the binding site (preferably Cry1Ab, since it is the most toxic) with those not competing for the same site. Cry1Ba would be the best choice of these proteins, since it is significantly more toxic than the others not binding to the common site. PMID:12147511

  2. The Mode of Action of the Bacillus thuringiensis Vegetative Insecticidal Protein Vip3A Differs from That of Cry1Ab δ-Endotoxin

    OpenAIRE

    Lee, Mi Kyong; Walters, Frederick S.; Hart, Hope; Palekar, Narendra; Chen, Jeng-Shong

    2003-01-01

    The Vip3A protein, secreted by Bacillus spp. during the vegetative stage of growth, represents a new family of insecticidal proteins. In our investigation of the mode of action of Vip3A, the 88-kDa Vip3A full-length toxin (Vip3A-F) was proteolytically activated to an approximately 62-kDa core toxin either by trypsin (Vip3A-T) or lepidopteran gut juice extracts (Vip3A-G). Biotinylated Vip3A-G demonstrated competitive binding to lepidopteran midgut brush border membrane vesicles (BBMV). Further...

  3. Bacillus thuringiensis monogenic strains: screening and interactions with insecticides used against rice pests

    Directory of Open Access Journals (Sweden)

    Laura M.N. Pinto

    2012-06-01

    Full Text Available The screening of Bacillus thuringiensis (Bt Cry proteins with high potential to control insect pests has been the goal of numerous research groups. In this study, we evaluated six monogenic Bt strains (Bt dendrolimus HD-37, Bt kurstaki HD-1, Bt kurstaki HD-73, Bt thuringiensis 4412, Bt kurstaki NRD-12 and Bt entomocidus 60.5, which codify the cry1Aa, cry1Ab, cry1Ac, cry1Ba, cry1C, cry2A genes respectively as potential insecticides for the most important insect pests of irrigated rice: Spodoptera frugiperda, Diatraea saccharalis, Oryzophagus oryzae, Oebalus poecilus and Tibraca limbativentris. We also analyzed their compatibility with chemical insecticides (thiamethoxam, labdacyhalothrin, malathion and fipronil, which are extensively used in rice crops. The bioassay results showed that Bt thuringiensis 4412 and Bt entomocidus 60.5 were the most toxic for the lepidopterans, with a 93% and 82% mortality rate for S. frugiperda and D. saccharalis, respectively. For O. oryzae, the Bt kurstaki NRD-12 (64% and Bt dendrolimus HD-37 (62% strains were the most toxic. The Bt dendrolimus HD-37 strain also caused high mortality (82% to O. poecilus, however the strains assessed to T. limbativentris caused a maximum rate of 5%. The assays for the Bt strains interaction with insecticides revealed the compatibility of the six strains with the four insecticides tested. The results from this study showed the high potential of cry1Aa and cry1Ba genes for genetic engineering of rice plants or the strains to biopesticide formulations.

  4. Susceptibility of Grapholita molesta (Busck, 1916) to formulations of Bacillus thuringiensis, individual toxins and their mixtures.

    Science.gov (United States)

    Ricietto, Ana Paula Scaramal; Gomis-Cebolla, Joaquín; Vilas-Bôas, Gislayne Trindade; Ferré, Juan

    2016-11-01

    The Oriental fruit moth, Grapholita molesta (Lepidoptera: Tortricidae), is a major pest of fruit trees worldwide, such as peach and apple. Bacillus thuringiensis has been shown to be an efficient alternative to synthetic insecticides in the control of many agricultural pests. The objective of this study was to evaluate the effectiveness of B. thuringiensis individual toxins and their mixtures for the control of G. molesta. Bioassays were performed with Cry1Aa, Cry1Ac, Cry1Ca, Vip3Aa, Vip3Af and Vip3Ca, as well as with the commercial products DiPel® and XenTari®. The most active proteins were Vip3Aa and Cry1Aa, with LC50 values of 1.8 and 7.5ng/cm(2), respectively. Vip3Ca was nontoxic to this insect species. Among the commercial products, DiPel® was slightly, but significantly, more toxic than XenTari®, with LC50 values of 13 and 33ng commercial product/cm(2), respectively. Since Vip3A and Cry1 proteins are expressed together in some insect-resistant crops, we evaluated possible synergistic or antagonistic interactions among them. The results showed moderate to high antagonism in the combinations of Vip3Aa with Cry1Aa and Cry1Ca.

  5. INSECTICIDAL TOXIN FROM BACILLUS THURINGIENSIS IS RELEASED FROM ROOTS OF TRANSGENIC BT CORN IN VITRO AND IN SITU. (R826107)

    Science.gov (United States)

    AbstractThe 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...

  6. Screening and identification of single-chain antibodies (scFvs) against Bacillus thuringiensis Cry1B toxin%抗苏云金芽孢杆菌Cry1B毒蛋白质单链抗体的筛选与鉴定

    Institute of Scientific and Technical Information of China (English)

    徐重新; 张霄; 刘媛; 王耘; 张存政; 刘贤进

    2012-01-01

    A large human synthetic phage displayed human library (Tomlinson J) was employed to generate single-chain antibodies (scFvs) against Bacillus thuringiensis (Bt) CrylB toxin by affinity panning. Specific anti-CrylB toxin antibodies were isolated from amplified naive phage-displayed human single-fold scFv Tomlinson J library after four rounds of "adsorption-elution-amplification" by using CrylB toxin protein as immobilized antigen. Monoclonal phage enzyme-linked immunosorbent assay (ELISA) was used for the positive clones identification by picking single colonies randomly from the final round of panning. The positive clones were confirmed by PCR, DNA electrophoresis and sequencing. Totally 8 positive clones with distinct nucleotide sequences and intact scFv gene were confirmed to be specific for the Cry1B recognition. The positive clone, namely 1E2, which showed better binding ability than others, was employed to develop an indirect competitive ELISA for the detecting of Cryi B. The results indicated that the IC50 reached 1. 075 μg/ml, and ihe minimum detection limit was 0.013 4 μg/ml for the determination of CrylB. The linear range of detection was approximately 0. 5-4. 0 μg/ml.%利用人源化噬菌体抗体库筛选抗Bt Cry1B毒蛋白质的单链抗体(Single-chain antibodies,scFFv).将扩增后的噬菌体抗体库与固相化包被的Cry1B毒蛋白质特异性结合,经4轮“吸附-洗脱-扩增”后,富集特异性识别Cry1B毒蛋白质的噬菌体单链抗体.从最后一轮筛选中随机挑取单菌落进行单克隆ELISA鉴定,对阳性克隆进行PCR扩增、DNA电泳鉴定及测序,成功筛选获得8个阳性噬菌体scFvs,经鉴定均有完整外源基因片段插入.挑取阳性值最高的scFv(1E2)建立了基于单链抗体的Cry1B毒蛋白质间接竞争ELISA检测方法.结果表明,Cry1B毒蛋白质对噬菌体scFv(1E2)的抑制中浓度(IC50)为1.075 μg/ml,最低检测限(IC10)为0.013 4 μg,/ml,线性检测范围在0.5 ug/ml至4.0,μg/ml之间.

  7. Elite Indica transgenic rice plants expressing modified Cry1Ac endotoxin of Bacillus thuringiensis show enhanced resistance to yellow stem borer (Scirpophaga incertulas).

    Science.gov (United States)

    Khanna, H K; Raina, S K

    2002-08-01

    Bt-transgenics of elite indica rice breeding lines (IR-64, Pusa Basmati-1 and Karnal Local) were generated through biolistic or Agrobacterium-mediated approaches. A synthetic cry1Ac gene, codon optimised for rice and driven by the maize ubiquitin-1 promoter, was used. Over 200 putative transformants of IR-64 and Pusa Basmati-1 and 26 of the Karnal Local were regenerated following use of the hpt (hygromycin phosphotransferase) selection system. Initial transformation frequency was in the range of 1 to 2% for particle bombardment while it was comparatively higher (approximately 9%) for Agrobacterium. An improved selection procedure, involving longer selection on the antibiotic-supplemented medium, enhanced the frequency of Bt-transformants and reduced the number of escapes. Molecular evaluation revealed multiple transgene insertions in transformants, whether generated through biolistic or Agrobacterium. In the latter case, it was also observed that all genes on the T-DNA do not necessarily get transferred as an intact insert. Selected Bt-lines of IR-64 and Pusa Basmati-1, having Bt-titers of 0.1% (of total soluble protein) and above were evaluated for resistance against manual infestation of freshly hatched neonate larvae of yellow stem borers collected from a hot spot stem borer infested area in northern India. Several Bt-lines were identified showing 100% mortality of larvae, within 4-days of infestation, in cut-stem as well as vegetative stage whole plant assays. However, there was an occasional white head even among such plants when assayed at the reproductive stage. Results are discussed in the light of resistance management strategies for deployment of Bt-rice.

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

    Science.gov (United States)

    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

  9. Characterization of Tunisian Bacillus thuringiensis strains with abundance of kurstaki subspecies harbouring insecticidal activities against the lepidopteran insect Ephestia kuehniella.

    Science.gov (United States)

    Saadaoui, Imen; Al-Thani, Roda; Al-Saadi, Fatma; Belguith-Ben Hassan, Najeh; Abdelkefi-Mesrati, Lobna; Schultz, Patrick; Rouis, Souad; Jaoua, Samir

    2010-12-01

    The study of 257 crystal-producing Bacillus thuringiensis isolates from bioinsecticide free soil samples collected from different sites in Tunisia, was performed by PCR amplification, using six primer pairs specific for cry1, cry2, cry3, cry4, and vip3A genes, by the investigation of strain plasmid pattern, crystal morphology and delta-endotoxin content and by the assessment of insecticidal activities against the lepidopteran insect Ephestia kuehniella. Based on plasmid pattern study, 11 representative strains of the different classes were subjected to morphological and molecular analyses. The comparison of the PFGE fingerprints confirmed the heterogeneity of these strains. B. thuringiensis kurstaki strains, harbouring at the same time the genes cry1A, cry2, cry1Ia, and vip3A, were the most abundant (65.4%). 33.34% of the new isolates showed particular delta-endotoxin profiles but no PCR products with the used primer sets. B. thuringiensis israelensis was shown to be also very rare among the Tunisian B. thuringiensis isolates diversity. These findings could have considerable impacts for the set up of new pest control biological agents.

  10. Response of larval Ephestia kuehniella (Lepidoptera: Pyralidae) to individual Bacillus thuringiensis kurstaki toxins mixed with Xenorhabdus nematophila.

    Science.gov (United States)

    BenFarhat, Dalel; Dammak, Mariam; Khedher, Saoussen Ben; Mahfoudh, Salima; Kammoun, Schema; Tounsi, Slim

    2013-09-01

    Bacillus thuringiensis kurstaki strain BNS3 produces parasporal crystals formed by Cry1Aa, Cry1Ac and Cry2Aa delta-endotoxins. In a previous work, we showed that the latter exhibited individually, a weak insecticidal activity against Ephestia kuehniella. In order to improve their toxicities, we studied the combined effect of each delta-endotoxin with X. nematophila cells on E. kuehniella larvae growth. Xenorhabdus cells were used in combination with spore crystal mixture of the wild strain BNS3, known to be active against E. kuehniella, but no improvement in toxicity was observed. This could be due to the high efficiency of BNS3 crystals against this insect. However, when X. nematophila was combined with each of Cry1Aa, Cry1Ac and Cry2Aa, improvement of toxicity was noticed. The best improvements were obtained with Cry1Ac and Cry2Aa, which are more toxic to E. kuehniella than Cry1Aa. The difference in toxicity improvement was attributed to the low affinity of Cry1Aa to BBMV receptors, compared to those of Cry1Ac and Cry2Aa. This synergism between Cry toxins and Xenorhabdus cells could be exploited on control target insect, particularly in case of resistance to Cry toxins.

  11. Transferrin Impacts Bacillus thuringiensis Biofilm Levels

    Directory of Open Access Journals (Sweden)

    Bianca Garner

    2016-01-01

    Full Text Available The present study examined the impact of transferrin on Bacillus thuringiensis biofilms. Three commercial strains, an environmental strain (33679, the type strain (10792, and an isolate from a diseased insect (700872, were cultured in iron restricted minimal medium. All strains produced biofilm when grown in vinyl plates at 30°C. B. thuringiensis 33679 had a biofilm biomass more than twice the concentration exhibited by the other strains. The addition of transferrin resulted in slightly increased growth yields for 2 of the 3 strains tested, including 33679. In contrast, the addition of 50 μg/mL of transferrin resulted in an 80% decrease in biofilm levels for strain 33679. When the growth temperature was increased to 37°C, the addition of 50 μg/mL of transferrin increased culture turbidity for only strain 33679. Biofilm levels were again decreased in strain 33679 at 37°C. Growth of B. thuringiensis cultures in polystyrene resulted in a decrease in overall growth yields at 30°C, with biofilm levels significantly decreased for 33679 in the presence of transferrin. These findings demonstrate that transferrin impacts biofilm formation in select strains of B. thuringiensis. Identification of these differences in biofilm regulation may be beneficial in elucidating potential virulence mechanisms among the differing strains.

  12. CHARACTERIZATION OF RESISTANT CELL LINE OF TRICHOPLUSIA NI TO BACILLUS THURINGIENSIS TOXIN Cry1Ac%抗苏云金杆菌毒素Cry1Ac粉纹夜蛾细胞系的特性研究

    Institute of Scientific and Technical Information of China (English)

    刘凯于; 杨红; 蒋才富; 彭建新; 洪华珠

    2004-01-01

    为了从离体细胞水平探讨昆虫对苏云金芽孢杆菌杀虫晶体蛋白的部分抗性机制,本文采用活化的Cry1Ac毒素对粉纹夜蛾BTI-TN-5B1-4细胞连续筛选86代,获得了高水平抗性细胞,研究了其某些特性.它对Cry1C产生了低水平的交互抗性,对低渗溶液的耐受性显著增强,双向电泳图谱表明抗性细胞膜蛋白组分发生了明显的变化.膜蛋白组分的变化可能导致了筛选细胞的耐低渗透压和抗Cry1C.

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

    Directory of Open Access Journals (Sweden)

    Naresh Arora

    Full Text Available 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.

  14. In vivo fluorescence observation of parasporal inclusion formation in Bacillus thuringiensis

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    A recombinant gene expressing a Cry1Ac-GFP fusion protein with a molecular mass of approximately 160 kD was constructed to investigate the expression of cry1Ac,the localization of its gene product Cry1Ac,and its role in crystal development in Bacillus thuringiensis.The cry1Ac-gfp fusion gene under the control of the cry1Ac promoter was cloned into the plasmid pHT304,and this construct was designated pHTcry1Ac-gfp.pHTcry1Ac-gfp was transformed into the crystal-negative strain,HD-73 cry-,and the resulting strain was named HD-73-(pHTcry1Ac-gfp).The gfp gene was then inserted into the large HD-73 endogenous plasmid pHT73 and fused with the 3’ terminal of the cry1Ac gene by homologous recombination,yielding HD-73Φ(cry1Ac-gfp)3534.Laser confocal microscopy and Western blot analyses showed for the first time that the Cry1Ac-GFP fusion proteins in both HD-73-(pHTcry1Ac-gfp) and HD-73Φ(cry1Ac-gfp)3534 were produced during asymmetric septum formation.Surprisingly,the Cry1Ac-GFP fusion protein showed polarity and was located near the septa in both strains.There was no significant difference between Cry1Ac-GFP and Cry1Ac in their toxicity to Plutella xylostella larvae.

  15. BACILLUS THURINGIENSIS ELASTASES WITH INSECTICIDE ACTIVITY

    Directory of Open Access Journals (Sweden)

    E. V. Matseliukh

    2015-10-01

    Full Text Available The purpose of the research was a screening of proteases with elastase activity among Bacillus thuringiensis strains, their isolation, partially purification, study of physicochemical properties and insecticide activity in relation to the larvae of the Colorado beetle. The objects of the investigation were 18 strains of B. thuringiensis, isolated from different sources: sea water, dry biological product "Bitoksibatsillin" and also from natural populations of Colorado beetles of the Crimea, Kherson, Odesa, Mykolaiv and Zaporizhiia regions of Ukraine. Purification of enzymes with elastase activity isolated from above mentioned strains was performed by gel-chromatography and insecticide activity was studied on the 3–4 larvae instar of Colorado beetle. The ability of a number of B. thuringiensis strains to synthesize the proteases with elastase activity has been established. The most active were enzymes obtained from strains IMV B-7465, IMV B-7324 isolated from sea water, and strains 9, 902, Bt-H and 0-239 isolated from Colorado beetles. The study of the physicochemical properties of the partially purified proteases of these strains showed that they belonged to enzymes of the serine type. Peptidases of a number of B. thuringiensis strains (IMV B-7324, IMV B-7465, 902, 0-239, 9 are metal-dependent enzymes. Optimal conditions of action of all tested enzymes are the neutral and alkaline рН values and the temperatures of 30–40 °С. The studies of influence of the complex enzyme preparations and partially purified ones of B. thuringiensis strains on the larvae instar of Colorado beetles indicated that enzymes with elastase activity could be responsible for insecticide action of the tested strains.

  16. Evidence of two mechanisms involved in Bacillus thuringiensis israelensis decreased toxicity against mosquito larvae: Genome dynamic and toxins stability.

    Science.gov (United States)

    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.

  17. Evaluación de la bacteria transformada Paenibacillus polymixa, que expresa la toxina Cry1C de Bacillus thuringiensis, como insecticida y biofertilizante en algodón

    OpenAIRE

    Hussien, A. I.

    2012-01-01

    Spodoptera littoralis (Boisudval) es un lepidóptero de la familia Noctuidae cuyas larvas se desarrollan a expensas de numerosas plantas, causando daños importantes en cultivos del área mediterránea como algodón, alfalfa y hortícolas, entre otros. Con objeto de desarrollar nuevas materias activas que puedan sustituir a los actuales insecticidas químicos de síntesis, la bacteria esporígena Bacillus thuringiensis constituye un recurso natural que se comercializa como insecticida b...

  18. Expression andpurification of Bacillus thuringiensis Cry2A insecticidal protein in E.coli%Cry2A杀虫蛋白在大肠杆菌中的表达及纯化

    Institute of Scientific and Technical Information of China (English)

    杨奇; 韩芳婷; 刘启文; 蒋玲曦; 谭芙蓉; 吴潇; 赵凯; 王金斌; 唐雪明

    2013-01-01

    通过PCR方法从克隆载体上扩增转基因水稻中的抗虫基因cry2A,经限制性内切酶EcoRI和BamHI双酶切定向插入到原核表达载体pET-30a(+)中,成功构建了蛋白表达载体pET-30a(+)/Cry2A,并转入大肠杆菌BL21 (DE3)中进行诱导表达.通过对其表达条件进行优化,发现在IPTG浓度为0.4 mmol/L、诱导时间为6h、诱导温度为30℃的表达条件下,目的蛋白表达量最高,大部分以包涵体形式表达.将包涵体裂解并用Ni-NTA亲和柱纯化,所得纯化蛋白在复性缓冲液中复性,最终得到有活性的高纯度目的蛋白.

  19. Regulation of protoxin synthesis in Bacillus thuringiensis.

    OpenAIRE

    Minnich, S A; Aronson, A I

    1984-01-01

    A derivative of Bacillus thuringiensis subsp. kurstaki (HD-1) formed parasporal inclusions at 25 degrees C, but not at 32 degrees C. This strain differed from the parent only in the loss of a 110-megadalton (Md) plasmid, but plasmid and chromosomal copies of protoxin genes were present in both strains. On the basis of temperature shift experiments, the sensitive period appeared to be during midexponential growth, long before the time of protoxin synthesis at 3 to 4 h after the end of exponent...

  20. 苏云金芽孢杆菌的cry2A芽孢期启动子和分子伴侣ORF1-ORF2对Cry11Aa蛋白表达的影响%Influence of cry2A sporulation-dependent promoter and molecular chaperone ORF1-ORF2 from Bacillus thuringiensis on Cry11Aa protein

    Institute of Scientific and Technical Information of China (English)

    师永霞; 曾少灵; 袁美妗; 孙钒; 庞义

    2008-01-01

    [目的]分析苏云金芽孢杆菌的cry2A型芽孢期启动子对晶体蛋白Cry11Aa的协调作用和分子伴侣ORF1-ORF2对Cry11Aa表达的促进功能.[方法]3个包括cry11Aa编码区的重组质粒pHcy1、pHcy2和pHcy4被构建并电激转化到苏云金芽孢杆菌晶体缺陷株4Q7中,其中pHcy1质粒携带cry11Aa基因自身启动子和分子伴侣p19基因,pHcy2携带cry2A型芽孢期启动子和分子伴侣orf1-orf2基因,pHcy4质粒在pHcy1的上游插入了cry2A型芽孢期启动子和分子伴侣orf1-orf2基因.SDS-PAGE分析了Cry11Aa蛋白在各重组苏云金菌株中的表达情况,并通过生物测定确定了其对蚊虫的生物活性.[结果]SDS-PAGE结果表明,Cry11Aa蛋白在4Q7(pHcy1)和4QT(pHcy4)均获得了表达,在4Q7(pHcy2)中未检测到Cry11Aa蛋白,推测晶体蛋白Cry11A不能利用cry2A型启动子进行表达调控;Cry11Aa蛋白在等体积4Q7(pHcy4)培养液中的表达量是4Q7(pHcy1)菌株的1.25倍,暗示着分子伴侣ORF1-ORF2在某种程度上能提高Cry11Aa的蛋白表达量.4Q7(pHcy1)和4Q7(pHcy4)形成的Cry11Aa蛋白晶体的形状和大小相似,两者对致倦库蚊的生物活性没有明显差异,LC50s分别为59.33 ng/mL和66.21 ng/mL,.[结论]推测晶体蛋白Cry11A能否成功表达与其使用启动子的类型和两者的协调配合有关.分子伴侣ORF1-ORF2虽然在某种程度上能提高Cry11Aa的蛋白表达量,但对提高Cry11Aa蛋白的杀蚊毒力没有显著性帮助.

  1. Investigation of Cytocidal Activity of Bacillus Thuringiensis Parasporal Toxin on CCRF-CEM Cell Line

    Directory of Open Access Journals (Sweden)

    Elham Moazamian

    2013-03-01

    Full Text Available Background & Objective: Parasporin is a parasporal protein of Bacillus thuringiensis and exhibits special cytocidal activity against human cancer cells. Similar to other insecticidal Bacillus thuringiensis crystal toxins, parasporin shows target specificity and damages the cellular membrane. In this study, different strains of Bacillus thuringiensis isolated from various regions of Iran and their cytocidal activity against CCRF-CEM cell line and human erythrocyte were investigated.   Materials & Methods: Fifty soil samples were collected from different Iranian provinces, and characterization was performed based on protein crystal morphology by phase-contrast microscope and variations of Cry protein toxin using SDS-PAGE. After parasporin was processed with proteinase K, the active form was produced and protein activity on the cell line was evaluated. Results: Parasporal inclusion proteins showed different cytotoxicity against acute lymphoblastic leukemia cells (ALL, but not against normal lymphocyte. Isolated parasporin demonstrated no hemolytic activity against human erythrocyte. It appears that these proteins have the ability to differentiate between normal lymphocytes and leukemia cells and have specific receptors on specific cancer cell lines. Conclusion: Our results provide evidence that the parasporin-producing organism is a common member in Bacillus thuringiensis populations occurring in the natural environments of Iran.

  2. Enduring toxicity of transgenic Anabaena PCC 7120 expressing mosquito larvicidal genes from Bacillus thuringiensis ssp. israelensis.

    Science.gov (United States)

    Manasherob, Robert; Otieno-Ayayo, Zachariah Ngalo; Ben-Dov, Eitan; Miaskovsky, Rina; Boussiba, Sammy; Zaritsky, Arieh

    2003-10-01

    Persistence of biological control agents against mosquito larvae was tested under simulated field conditions. Mosquito larvicidal activity of transgenic Anabaena PCC 7120 expressing cry4Aa, cry11Aa and p20 from Bacillus thuringiensis ssp. israelensis was greater than B. thuringiensis ssp. israelensis primary powder (fun 89C06D) or wettable powder (WP) (Bactimos products) when either mixed with silt or exposed to sunlight outdoors. Reduction of Bactimos primary powder toxicity was at least 10-fold higher than Anabaena's after mixing with silt. In outdoors experiments, Bactimos WP remained toxic (over 30% mortality of 3rd instar Aedes aegypti larvae) for 2-4 days only, while transgenic Anabaena's toxicity endured 8-21 days.

  3. Chitinolitic activity in proteic extracts of Bacillus thuringiensis toxic to boll weevil (Anthonomus grandis)

    Energy Technology Data Exchange (ETDEWEB)

    Silva, T.S; Rocha, T.L. [EMBRAPA Recursos Geneticos e Biotecnologia, DF (Brazil); Vasconcelos, E.A.R [Universidade de Brasilia (UnB), DF (Brazil); Grossi-de-Sa, M.F. [Universidade Catolica de Brasilia, DF (Brazil)

    2008-07-01

    Full text: Bacillus thuringiensis (Bt) is a spore forming bacteria, which produces Cry proteins toxic towards several insect orders. Bt S 811 strain produces at least three Cry toxins: Cry1Ab, Cry1Ia12, and Cry8, and shown toxicity to insects from Coleoptera order. In order to characterize the production of theses toxins, and check its activity against Boll weevil larvae, proteic extracts from Bt cells and supernatant proteins from the bacterial culture, were obtained at different stages of cell cycle; 8, 16, 24, and 32 hours after inoculation (HAI). Proteins from 32 HAI of the supernatant, and 8 HAI of the cellular fractions, shown highest activity towards the Boll weevil larvae. Western blotting assays using anti-Cry8 and anti-Cry1I were carried out to analyse these toxins in the Bt proteic extracts. The existence of a Cry8 was detected at 8 HAI in the cellular fraction, what allow associate this molecule with the toxicity of this fraction. However, toxicity observed at 32 HAI in the supernatant fraction, was not possible to be associated with Cry8 or Cry1Ia toxins, indicating that there are another protein(s) responsible for the toxicity. A protein homo log to Cry1Ab was identified by 'Peptide Mass Fingerprint' at 32 HAI of the supernatant fraction and a chitin binding protein was identified by 2DE/MS/MS in this same stage and chitinolitic activity was also observed by enzymatic assay. All our data suggest a possible synergism between Cry toxins and a chitinase in the activity of this strain towards Boll weevil.

  4. 初始碳源对苏云金芽孢杆菌cry-lacZ融合基因表达的影响%Effects of Primzry Carbon Sources on the Expression of Cry-lac Z in Bacillus thuringiensis

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    分别采用携带cry1A上游区、突变或缺失上游区的重组穿棱载体pHT/pSGMU-P,电转化到苏云金芽孢杆菌不同菌株中,并经含不同初始碳源的Gtris培养基培养,检测BtI-lacZ融合基因的表达.结果表明,各种转化子的β-半乳糖苷酶活性随着培养基中初始碳源的不同而异,在以丙酮酸钠为初始碳源的培养基中,BtI-lacZ的表达量最高,以葡萄糖为初始碳源的次之,在琥珀酸钠中最低,说明cry基因的表达与初始碳源的种类和细胞的代谢有关.

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

    Science.gov (United States)

    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.

  6. Characterisation of novel Bacillus thuringiensis isolates against Aedes aegypti (Diptera: Culicidae) and Ceratitis capitata (Diptera: Tephridae).

    Science.gov (United States)

    Elleuch, Jihen; Tounsi, Slim; Ben Hassen, Najeh Belguith; Lacoix, Marie Noël; Chandre, Fabrice; Jaoua, Samir; Zghal, Raida Zribi

    2015-01-01

    Bacillus thuringiensis is successfully used in pest management strategies as an eco-friendly bioinsecticide. Isolation and identification of new strains with a wide variety of target pests is an ever growing field. In this paper, new B. thuringiensis isolates were investigated to search for original strains active against diptera and able to produce novel toxins that could be used as an alternative for the commercial H14 strain. Biochemical and molecular characterization revealed a remarkable diversity among the studied strains. Using the PCR method, cry4C/Da1, cry30Ea, cry39A, cry40 and cry54 genes were detected in four isolates. Three strains, BLB355, BLB196 and BUPM109, showed feeble activities against Aedes aegypti larvae. Interestingly, spore-crystal mixtures of BLB361, BLB30 and BLB237 were found to be active against Ceratitis capitata with an LC50 value of about 65.375, 51.735 and 42.972 μg cm(-2), respectively. All the studied strains exhibited important mortality levels using culture supernatants against C. capitata larvae. This suggests that these strains produce a wide range of soluble factors active against C. capitata larvae.

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

    Science.gov (United States)

    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.

  8. Characterization of cry Gene from a Novel Bacillus thuringiensis CY1%苏云金芽胞杆菌CY1菌株的cry基因分析

    Institute of Scientific and Technical Information of China (English)

    吴会贤; 宋萍; 王勤英; 苏旭东

    2007-01-01

    通过醋酸钠-抗生素筛选法从河北省土壤中筛选得到了苏云金芽孢杆菌新菌株CY1.镜检可观察到伴孢晶体呈小菱形.SDS-PAGE分析结果表明菌株CY1表达的晶体蛋白分子量为130 kDa.利用25对通用引物检测cry1,cry11, cry7,cry2,cry3,cry4/cry10, cry5,cry6,cry7,cry8,cry9, cry11,cry13/14,cry16/17,cry18,cry19,cry21,cry22, cry24/25,cry26/28,cry27/29, cry30,cry32, cry34/35, cry40基因,仅从CY1菌株中检测出cry7基因,该cry7基因N末端的1 215 bp片段所编码的氨基酸序列与已发表的Cry7Ab1(Acc No.:U04367)序列同源性达99%,仅有2个氨基酸的差异.用3对cry7Ab特异引物检测,进一步证实该菌株携带有cry7Ab基因.

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

    Science.gov (United States)

    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...

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

    Science.gov (United States)

    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.

  11. Ecological aspects of Bacillus thuringiensis in an Oxisol Ecologia do Bacillus thuringiensis num Latossolo

    Directory of Open Access Journals (Sweden)

    Lessandra Heck Paes Leme Ferreira

    2003-02-01

    Full Text Available Bacillus thuringiensis is a Gram positive, sporangial bacterium, known for its insecticidal habilities. Survival and conjugation ability of B. thuringiensis strains were investigated; vegetative cells were evaluated in non-sterile soil. Vegetative cells decreased rapidly in number, and after 48 hours the population was predominantly spores. No plasmid transfer was observed in non-sterile soil, probably because the cells died and the remaining cells sporulated quickly. Soil is not a favorable environment for B. thuringiensis multiplication and conjugation. The fate of purified B. thuringiensis toxin was analyzed by extractable toxin quantification using ELISA. The extractable toxin probably declined due to binding on surface-active particles in the soil.O comportamento de células vegetativas do Bacillus thuringiensis foi estudado em solo não esterilizado. Após o inóculo grande parte das células morrem e o restante esporula em 24 horas. Não foi observada conjugação provavelmente porque poucas células sobrevivem no solo e rapidamente esporulam, mostrando que este não é o ambiente propício para a multiplicação e conjugação desta bactéria. A toxina purificada, portanto livre de células, diminui rapidamente sua quantidade em solo não esterilizado. Provavelmente a ligação da toxina na fração argilosa do solo é a principal responsável por este fenômeno.

  12. Bacillus thuringiensis Conjugation in Simulated Microgravity

    Science.gov (United States)

    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.

  13. Sludge based Bacillus thuringiensis biopesticides: viscosity impacts.

    Science.gov (United States)

    Brar, S K; Verma, M; Tyagi, R D; Valéro, J R; Surampalli, R Y

    2005-08-01

    Viscosity studies were performed on raw, pre-treated (sterilised and thermal alkaline hydrolysed or both types of treatment) and Bacillus thuringiensis (Bt) fermented sludges at different solids concentration (10-40 g/L) for production of biopesticides. Correlations were established among rheological parameter (viscosity), solids (total and dissolved) concentration and entomotoxicity (Tx) of Bt fermented sludges. Exponential and power laws were preferentially followed by hydrolysed fermented compared to raw fermented sludge. Soluble chemical oxygen demand variation corroborated with increase in dissolved solids concentration on pre-treatments, contributing to changes in viscosity. Moreover, Tx was higher for hydrolysed fermented sludge in comparison to raw fermented sludge owing to increased availability of nutrients and lower viscosity that improved oxygen transfer. The shake flask results were reproducible in fermenter. This study will have major impact on selecting fermentation, harvesting and formulation techniques of Bt fermented sludges for biopesticide production.

  14. Antagonistic competition moderates virulence in Bacillus thuringiensis.

    Science.gov (United States)

    Garbutt, Jennie; Bonsall, Michael B; Wright, Denis J; Raymond, Ben

    2011-08-01

    Classical models of the evolution of virulence predict that multiple infections should select for elevated virulence, if increased competitiveness arises from faster growth. However, diverse modes of parasite competition (resource-based, antagonism, immunity manipulation) can lead to adaptations with different implications for virulence. Using an experimental evolution approach we investigated the hypothesis that selection in mixed-strain infections will lead to increased antagonism that trades off against investment in virulence. Selection in mixed infections led to improved suppression of competitors in the bacterial insect pathogen Bacillus thuringiensis. Increased antagonism was associated with decreased virulence in three out of four selected lines. Moreover, mixed infections were less virulent than single-strain infections, and between-strain competition tended to decrease pathogen growth in vivo and in vitro. Spiteful interactions among these bacteria may be favoured because of the high metabolic costs of virulence factors and the high risk of mixed infections.

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

    Science.gov (United States)

    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.

  16. Synergy between toxins of Bacillus thuringiensis subsp. israelensis and Bacillus sphaericus.

    Science.gov (United States)

    Wirth, Margaret C; Jiannino, Joshua A; Federici, Brian A; Walton, William E

    2004-09-01

    Synergistic interactions among the multiple endotoxins of Bacillus thuringiensis subsp. israelensis de Barjac play an important role in its high toxicity to mosquito larvae and the absence of insecticide resistance in populations treated with this bacterium. A lack of toxin complexity and synergism are the apparent causes of resistance to Bacillus sphaericus Neide in particular Culex field populations. To identify endotoxin combinations of the two Bacillus species that might improve insecticidal activity and manage mosquito resistance to B. sphaericus, we tested their toxins alone and in combination. Most combinations of B. sphaericus and B. t. subsp. israelensis toxins were synergistic and enhanced toxicity relative to B. sphaericus, particularly against Culex quinquefasciatus Say larvae resistant to B. sphaericus and Aedes aegypti (L.), a species poorly susceptible to B. sphaericus. Toxicity also improved against susceptible Cx. quinquefasciatus. For example, when the CytlAa toxin from B. t. subsp. israelensis was added to Bin and Cry toxins, or when native B. t. subsp. israelensis was combined with B. sphaericus, synergism values as high as 883-fold were observed and combinations were 4-59,000-fold more active than B. sphaericus. These data, and previous studies using cytolytic toxins, validate proposed strategies for improving bacterial larvicides by combining B. sphaericus with B. t. subsp. israelensis or by engineering recombinant bacteria that express endotoxins from both strains. These combinations increase both endotoxin complexity and synergistic interactions and thereby enhance activity and help avoid insecticide resistance.

  17. [Expression of mosquitocidal Cyt1Aa toxin from Bacillus thuringiensis subsp. israelensis in Asticcacaulis excentricus].

    Science.gov (United States)

    Zheng, Da-sheng; Crickmore, Neil; Cai, Ya-jun; Yan, Jian-ping; Yuan, Zhi-ming

    2007-04-01

    Asticcacaulis excentricus, who lives in upper-layer waters providing food resource to the mosquito larvae and has been proven to be a successful host to produce the mosquitocidal binary toxins or Cry11Aa toxin from Bacilli (Liu et al., 1996, Nat Biotech 14: 343; Armengol, et al. , 2005, Curr Microbiol 51: 430), was developed to express cyt1Aa gene from Bacillus thuringiensis subsp. israelensis (Bti). Two A. excentricus transformants were constructed with the attempt of producing CytlAa alone and alongside with Cry11Aa, repectively. Detection of expressed Cry11Aa and CytlAa proteins by immunoblot in the recombinant A. excentricus clones showed that either cry11Aa or cyt1Aa was expressed well solely but not simultaneously although both restriction analyses of plasmid DNA and DNA sequencing showed that the transformed plasmid was identical to scheme. To investigate the reason why the recombinant A. excentricus harboring both genes and their ribosome binding site (RBS) sequences expressed only Cry11Aa, the total RNA of A. excentricus cells was extracted and revealed three-band pattern in which all RNA molecule weights are not greater than 16S RNA of Escherichia coli by formamide agarose gel electrophoresis, indicating that different RNA systems within these two Gram-negative strains required distinguishingly organised constructs to express multiple foreign genes. It is hypothesized that an extra promoter upstream of RBS sequence is required to express cyt1Aa in the cry11Aa-cyt1Aa tandom plasmid.

  18. Construction of an environmental safe Bacillus thuringiensis engineered strain against Coleoptera.

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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.

  2. Bacillus thuringiensis: legado para el siglo XXI Bacillus thuringiensis: the legacy to the XXI century

    Directory of Open Access Journals (Sweden)

    Orduz S.

    1998-06-01

    Full Text Available

    Los insecticidas basados en la bacteria Bacillus thuringiensis son el principal renglón productivo del mercado mundial de biopesticidas. La investigación dedicada a esta área, promovida por la urgente necesidad de resolver problemas agrícolas y de salud pública, ha dado lugar a un conocimiento exhaustivo de su biología. La diversidad de cepas diferentes de B. thuringiensis ha permitido desarrollar productos principalmente, pero no exclusivamente, para el control de insectos. Con los nuevos desarrollos de la biología molecular, se ha logrado comprender su mecanismo de acción a nivel molecular y también se ha logrado extender sus capacidades entomopatógenas. Como producto de su amplio uso en muchos países, se han presentado casos de resistencia en poblaciones de insectos susceptibles. Con esta revisión se pretende elaborar un contexto teórico del estado actual de la investigación sobre B. thuringiensis, describiendo brevemente el conocimiento sobre esta bacteria, haciendo hincapié en los fenómenos biológicos que subyacen su actividad tóxica y la problemática que se avecina en el próximo siglo con los fenómenos de resistencia cada vez más comunes, todo esto analizado desde una perspectiva biotecnológica.

    Bacillus thuringiensis-based insecticides are the main production line of the biopesticides world market. The research devoted to this area, promoted by the necessity to solve problems in agriculture and public health has resulted in an exhaustive knowledge of its biology. The diversity of the B. thuringiensis strains has permitted to develop several products mainly, but not exclusively, for insect control. With the new developments in the field of molecular biology, it has been possible to understand the molecular basis of the mode of action and to increase the range of activity as well. As a result

  3. Complete Genome Sequence of Bacillus thuringiensis Bacteriophage Smudge.

    Science.gov (United States)

    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

    2016-08-18

    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.

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

    Science.gov (United States)

    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.

  5. Does distant homology with Evf reveal a lipid binding site in Bacillus thuringiensis cytolytic toxins?

    Science.gov (United States)

    Rigden, Daniel J

    2009-05-19

    The Cry and Cyt classes of insecticidal toxins derived from the sporulating bacterium Bacillus thuringiensis are valuable substitutes for synthetic pesticides in agricultural contexts. Crystal structures and many biochemical data have provided insights into their molecular mechanisms, generally thought to involve oligomerization and pore formation, but have not localised the site on Cyt toxins responsible for selective binding of phospholipids containing unsaturated fatty acids. Here, distant homology between the structure of Cyt toxins and Erwinia virulence factor (Evf) is demonstrated which, along with sequence conservation analysis, allows a putative lipid binding site to be localised in the toxins.

  6. Analysis of cry 1Ca gene from Bacillus thuringiensis strains with high insecticidal activity against Spodoptera exigua%对甜菜夜蛾具有杀虫活性的Bt 菌株cry1Ca 基因研究

    Institute of Scientific and Technical Information of China (English)

    王杨; 周子珊; 刘永磊; 韩榕; 张林静; 张杰

    2016-01-01

    One pair of degenerate primers,designed based upon the 14 known sequences of cry 1Ca genes,was used to identify cry 1Ca genes from 472 wild isolates of Bacillus thuringiensis screened in our laboratory,and there were 22 isolates containing cry 1Ca genes.Five patterns of restriction fragment were obtained by the analysis of cry gene diversity depended on PCR-RFLP,indicating that 22 B .thuringiensis isolates could be classified as five types.SDS-PAGE analysis results showed that a main protein band with approximate 130 ku molecular mass was detected in each strain.Western Blotting analysis results also demonstrated that Cry1Ca proteins were expressed normally in all of the 22 strains.The preliminary bioassay was conducted with trypsin-activated Cry proteins ex-tracted from 22 Bt isolates against the neonate larvae of Spodoptera exigua (Hübner).The results showed that all of these strains hade high insecticidal activity against target larvae.The LC50 values of the five selected Bt isolates representing five types against S .exigua were measured,and the results indicated that the toxicity of the five strains was very high;the insecticidal activity of the strains T1-E12 (0.087 μg/g),B16-C8 (0.103 μg/g)and T1-B8 (0.202 μg/g)was similar to that of the positive strain G03 (0.090 μg/g),and these strains have great potential for commercialization.Our findings will lay the foundation for discovery of novel B .thuringiensis isolates.%根据已报道的14个 cry1Ca 基因,设计能够扩增 cry1Ca 全长基因的简并引物。利用该引物对本实验室分离的472株 Bt 菌株进行筛选。PCR 扩增发现22株菌含有 cry1Ca 基因。对22株菌株进行 cry 基因多样性分析,结果获得5种类型酶切图谱,表明这些菌株分为5种类型。SDS-PAGE 结果显示22株菌均表达约130 ku 的蛋白。Western Blotting 结果证实这些株菌中 cry1Ca 基因均正常表达。提取菌株的晶体蛋白,经胰蛋白

  7. Bacillus thuringiensis toxin resistance mechanisms among Lepidoptera: progress on genomic approaches to uncover causal mutations in the European corn borer, Ostrinia nubilalis

    Science.gov (United States)

    Transgenic plants that expressed Bacillus thuringiensis (Bt) crystalline (Cry) protein toxins can suffer feeding damage from a small number of lepidopteran insect species under field conditions, which has heightened concerns about the durability of pest control tactics. Genomics research has provid...

  8. A Brazilian Bacillus thuringiensis strain highly active to sugarcane borer Diatraea saccharalis (Lepidoptera: Crambidae Uma Estirpe Brasileira de Bacillus thuringiensis com elevada atividade para a broca da cana-de-açúcar Diatraea saccharalis (Lepidoptera: Crambidae

    Directory of Open Access Journals (Sweden)

    Patrícia de Medeiros Gitahy

    2007-09-01

    Full Text Available The control of the major sugarcane pest, Diatraea saccharalis, is limited by the stem location of the caterpillar. As part of a long-term project towards the development of an alternative and efficient delivery system of Cry proteins to control the sugarcane borer, the current work describes the selection and characterization of a Brazilian B. thuringiensis strain with prominent activity towards D. saccharalis. Strain S76 was eleven-fold more active than the HD-1 Lepidoptera-standard strain, as estimated by the LC50 of 13.06 µg/L and 143.88 µg/L, respectively. We observed bipiramidal and cuboidal crystals similar to those found in other B. thuringiensis strains with entomopathogenic activity against Lepidoptera and Diptera. In addition, smaller and spherical crystalline inclusions were also observed. The plasmid profile of strain S76 is similar to that of HD-1. PCR amplifications of S76 DNA using cry specific primers confirmed the presence of cry1Aa,cry1Ab,cry1Ac,cry2Aa1, and cry2Ab2, but not cry1Ad, cry2Ac and cry9 type genes. No differences that could explain the superior activity of S76 when compared to HD-1, the Lepidoptera standard strain, were observed. Nevertheless, its higher entomopathogenic activity has pointed this strain S76 as a potential source of cry genes to control sugarcane borer, an important pest that affects sugarcane, a crop that occupies a planted area of about 6 million ha in Brazil.Diatraea saccharalis é o inseto-praga que provoca os maiores danos a cultura da cana-de-açúcar, e seu controle é limitado pela localização do ataque no interior do colmo das plantas. Como parte de um projeto a longo prazo com o objetivo de desenvolver uma alternativa eficiente para o controle da broca da cana utilizando as proteínas Cry de Bacillus thuringiensis, o presente trabalho descreve a seleção e caracterização de uma estirpe desta bactéria com atividade larvicida para D. saccharalis. A estirpe brasileira S76, foi

  9. 40 CFR 180.1011 - Viable spores of the microorganism Bacillus thuringiensis Berliner; exemption from the...

    Science.gov (United States)

    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...

  10. 我国土壤中苏云金芽孢杆菌的分离与基因型的鉴定%IDENTIFICATION OF CRY- TYPE GENE AND ISOLATION OF BACILLUS THURINGIENSIS FROM SOIL IN CHINA

    Institute of Scientific and Technical Information of China (English)

    罗兰; 谢丙炎; 袁忠林; 杨之为

    2005-01-01

    从我国20个省市采集176份土壤中分离出苏云金芽孢杆菌51株.以cry1,cry2,cry3,cry4,cry-n,cry7,cry8,cry11,cry13和cyt的引物作PCR分析.结果表明:4株含有cry4,12株含有cry7,4株含有cry8,4株含有cry1,2株同时含有cry1和cry2,27株无PCR产物.未检测到其它基因型.经电镜扫描伴胞晶体形态多种多样,主要有菱形、球形、方形、多边形和不规则形.图3表3参15

  11. [Effects of helper protein P20 from Bacillus thuringiensis on Vip3A expression].

    Science.gov (United States)

    Shi, Yong-xia; Yuan, Mei-jin; Chen, Jian-wu; Sun, Fan; Pang, Yi

    2006-02-01

    Insecticidal crystal proteins (ICPs) produced in Bacillus thuringiensis accumulate as crystalline inclusions that represent up to 30% of total dry weight the cell produces. The mechanisms of in vivo crystallization of these insecticidal proteins remain interests, yet unclear. A 20-kDa protein (P20), the product of the third open reading frame of cry11A operon in B. thuringiensis subsp. israelensis has been defined to be an important molecular chaperone (helper protein) for forming Cyt1A crystal and enhancing Cry11A expression. The novel vegetative insecticidal proteins (VIPs) are secreted outside the cell of B. thuringiensis during mid-logarithmic growth. VIP3A shows activity against many lepidopteran insect larvae in a different mechanism from that of ICPs. To investigate the influence of helper protein P20 on Vip3A production and its insecticidal activity, P20 was coexpressed with Vip3A protein in B. thuringiensis and the yields and insecticidal toxicity of Vip3A were also analyzed. The recombinant plasmid pHVP20 was constructed by inserting a 5.4kb foreign fragment containing both vip3A gene and p20 gene into the shuttle vector pHT3101. The plasmid pHPT3 only containing vip3A gene was used as control. pHVP20 and pHPT3 were transformed into the B. thuringiensis acrystalliferous strain CryB not containing vip3A gene by electroporation. The obtained B. thuringiensis transformants were CryB(pHVP20) and CryB(pHPT3) respectively. Western blot showed that Vip3A protein reached its maximum yield after 48h of CryB (pHVP20) growth and remained high expression level during the sporulation. The maximum yield of Vip3A protein in CryB (pHVP20) was about 1.5 fold as compared with that in CryB(pHPT3) by the mean of ImageMaster VDS software. It is considered that P20 might combine with the native Vip3A protein during the sporulation, stabilize Vip3A and protect Vip3A from unspecific full proteolysis. Bioassay showed that the cell pellets of CryB (pHVP20) and CryB(pHPT3

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

    Science.gov (United States)

    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.

  13. Ultra-violet-resistant mutants of Bacillus thuringiensis

    Energy Technology Data Exchange (ETDEWEB)

    Jones, D.R.; Karunakaran, V. (Polytechnic of Central London (UK). Faculty of Engineering and Science, School of Biological and Health Sciences); Burges, H.D. (Institute of Horticultural Research, Littlehampton (UK)); Hacking, A.J. (Reading Univ. (UK). Dextra Labs.Ltd.)

    1991-06-01

    One of the main disadvantages of using Bacillus thuringiensis as an insecticide is that the spore and crystal preparations applied to foliage are readily washed away by rain and are inactivated by sunlight. Spores from some strains of B. thuringiensis have been shown to be highly sensitive to u.v. light. This study has demonstrated how mutants with increased resistance to u.v., isolated by successive rounds of u.v. irradiation, and additionally with increased specific pathogenicity can be isolated. These techniques should be applied to strains that are frequently used in the industrial production of B.thuringiensis toxin. (author).

  14. Spore and crystal formation in Bacillus thuringiensis var thuringiensis during growth in cystine and cysteine.

    OpenAIRE

    Rajalakshmi, S.; Shethna, YI

    1980-01-01

    The effect of the addition of different concentratons of cystine and cysteine on sporulation and parasporal crystal formation in Bacillus thuringiensis var. thuringiensis was studied. The effect was well pronounced when the systine/cysteine additions were made after the stationary phase. Heat stable spores and crystals were formed when the culture was provided with a low concentration of cystine/cysteine (0.05 per cent w/v). At a moderate concentration of cystine or cysteine (0.15%), only ...

  15. Plasmid-associated sensitivity of Bacillus thuringiensis to UV light

    Energy Technology Data Exchange (ETDEWEB)

    Benoit, T.G.; Wilson, G.R.; Bull, D.L.; Aronson, A.I. (Department of Agriculture, College Station, TX (USA))

    1990-08-01

    Spores and vegetative cells of Bacillus thuringiensis were more sensitive to UV light than were spores or cells of plasmid-cured B. thuringiensis strains or of the closely related Bacillus cereus. Introduction of B. thuringiensis plasmids into B. cereus by cell mating increased the UV sensitivity of the cells and spores. Protoxins encoded by one or more B. thuringiensis plasmids were not involved in spore sensitivity, since a B. thuringiensis strain conditional for protoxin accumulation was equally sensitive at the permissive and nonpermissive temperatures. In addition, introduction of either a cloned protoxin gene, the cloning vector, or another plasmid not containing a protoxin gene into a plasmid-cured strain of B. thuringiensis all increased the UV sensitivity of the spores. Although the variety of small, acid-soluble proteins was the same in the spores of all strains examined, the quantity of dipicolinic acid was about twice as high in the plasmid-containing strains, and this may account for the differences in UV sensitivity of the spores. The cells of some strains harboring only B. thuringiensis plasmids were much more sensitive than cells of any of the other strains, and the differences were much greater than observed with spores.

  16. Biological Activity of Bacillus thuringiensis in Drosophila suzukii (Diptera: Drosophilidae).

    Science.gov (United States)

    Cossentine, J; Robertson, M; Xu, D

    2016-04-22

    Whole-culture extracts of Bacillus thuringiensis Berliner strains were assayed against larval and adult Drosophila suzukii (Matsumura), an important invasive pest of many thin-skinned soft fruit crops in North America. Of the 22 serovars tested versus larval D. suzukii, strains of Bacillus thuringiensis var. thuringiensis, kurstaki, thompsoni, bolivia, and pakistani caused high (75 to 100%) first-instar mortalities. Pupal mortality, measured as a failure of adults to emerge, varied with serovar. The first D. suzukii instar was the most susceptible of the three larval instars to B. thuringiensis var. kurstaki HD-1. Larval D. suzukii are shielded from crop treatments, as they develop under the skin of infested fruit, and adults would be a more vulnerable target for an efficacious strain of B. thuringiensis Only one of the 21 B. thuringiensis serovars, var. thuringiensis, prepared as oral suspensions in sucrose for adult D. suzukii ingestion resulted in significant, albeit low mortality within 7 d. It is not a candidate for use in pest management, as it produces β-exotoxin that is toxic to vertebrates.

  17. Pathogenicity of Bacillus thuringiensis isolated from two species of Acromyrmex (Hymenoptera, Formicidae

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    L. M. N. Pinto

    Full Text Available The control of Acromyrmex leaf-cutting ants is necessary due to the severe damage they cause to diverse crops. A possibility was to control them using the bacterium Bacillus thuringiensis (Bt that characteristically produces insecticidal crystal proteins (ICPs. The ICPs have been effective in controlling lepidopterans, dipterans, and coleopterans, but their action against hymenopterans is unknown. This paper describes an attempt to isolate Bt from ants of two Acromyrmex species, to evaluate its pathogenicity towards these ants, and to test isolates by PCR. Bacterial isolates of Bt obtained from A. crassispinus and A. lundi have been assayed against A. lundi in the laboratory. The bioassays were carried out in BOD at 25°C, with a 12-hour photoperiod, until the seventh day after treatment. The Bt isolates obtained were submitted to total DNA extraction and tested by PCR with primers specific to cry genes. The results showed Bt presence in 40% of the assessed samples. The data from the in vivo assays showed a mortality rate higher than 50% in the target population, with the Bt HA48 isolate causing 100% of corrected mortality. The PCR results of Bt isolates showed a magnification of DNA fragments relative to cry1 genes in 22% of the isolates, and cry9 in 67%. Cry2, cry3, cry7, and cry8 genes were not detected in the tested samples, and 22% had no magnified DNA fragments corresponding to the assessed cry genes. The results are promising not only regarding allele identification in new isolates, but also fort the assays aimed at determining the Bt HA48 LC50's, which can eventually be applied in controlling of Acromyrmex leaf-cutting ants.

  18. Complete genome sequence of Bacillus thuringiensis serovar alesti BGSC 4C1, a typical strain with toxicity to Lepidoptera insects.

    Science.gov (United States)

    Wang, Yueying; Fu, Jingjing; Zhu, Qian; Zhu, Lei; Zheng, Jinshui; Liu, Hualin; Peng, Donghai; Ruan, Lifang; Sun, Ming

    2016-12-10

    Bacillus thuringiensis serovar alesti was used to control caterpillars from 1970s. Here we reported the complete genome of BGSC 4C1, the type strain of this serovar. It has a circular chromosome and six plasmids. The largest plasmid pBMB267 contains five insecticidal crystal protein genes (two copies of cry1Ae, cry1Gb, cry2Ab, and a novel cry1M-type gene) and three vegetative insecticidal protein genes (a novel binary toxin gene operon vip1-vip2 and vip3Aa). Besides, the strain also has many genes encodeing virulence factors, and some secondary metabolite biosynthetic gene clusters involved in synthesis of antimicrobial peptides and bacteriocins. In addition, there is a poly γ-glutamate synthesis gene cluster, whose product is a candidate to control inflammasome-mediated disorders and potential in many other fields.

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

    Science.gov (United States)

    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.

  20. Parasporal Proteins from Bacillus thuringiensis and Their Cytotoxicity on Human Cancer Cells

    Institute of Scientific and Technical Information of China (English)

    WANG Lei; LV Yuan; YI Yin-sha; YI Shang-hui; LI Lin

    2015-01-01

    Parasporins(PSs) represent a novel functional category of crystal proteins (Cry) produced by non-insecticidal Bacillus thuringiensisA distinct feature for PSs is their specific cytotoxicity against human cancer cells from diverse origins, other than hemolytic or insecticidal activityAs structurally/functionally Cry proteins, parasporins are expressed as protoxins that require protease cleavage for activationCurrently, identified PSs is classified into 6 groups:PS1, PS2, PS3, PS4, PS5 and PS6, which are heterogeneous in cytotoxic spectrum and activity levelSome PSs have been explored for their mode of anticancer activities, reports mainly include pore formation induced by binding to putative receptors on cell membrane and apoptosis by intracellular Ca 2+concentrationFurther work should focus on the identification of new PS or PS homologs and better understanding of their anticancer mechanism before possible application in cancer therapy.

  1. Occurrence and linkage between secreted insecticidal toxins in natural isolates of Bacillus thuringiensis.

    Science.gov (United States)

    Espinasse, Sylvain; Chaufaux, Josette; Buisson, Christophe; Perchat, Stéphane; Gohar, Michel; Bourguet, Denis; Sanchis, Vincent

    2003-12-01

    Little is known about the occurrence and linkage between secreted insecticidal virulence factors in natural populations of Bacillus thuringiensis (Bt). We carried out a survey of 392 Bt strains isolated from various samples originating from 31 countries. The toxicity profile of the culture supernatants of these strains was determined individually against Anthonomus grandis (Coleoptera) and Spodoptera littoralis (Lepidoptera). We analyzed beta-exotoxin I production and searched for the genes encoding Vip1-2, Vip3, and Cry1I toxins in 125 of these strains. Our results showed that these insecticidal toxins were widespread in Bt but that their distribution was nonrandom, with significant linkage observed between vip3 and cry1I and between vip1-2 and beta-exotoxin I. Strains producing significant amounts of beta-exotoxin I were more frequently isolated from invertebrate samples than from dust, water, soil, or plant samples.

  2. Aerobic granulation of pure bacterial strain Bacillus thuringiensis

    Institute of Scientific and Technical Information of China (English)

    Sunil S ADAV; Duu-Jong LEE

    2008-01-01

    The objective of this study is to cultivate aer-obic granules by pure bacterial strain, Bacillus thuringien-sis, in a sequencing batch reactor. Stable granules sized 2.0-2.2 mm were formed in the reactor after a five-week cultivation. These granules exhibited excellent settling attributes, and degraded phenol at rates of 1.49 and concentration, respectively. Confocal laser scanning microscopic test results show that Bacillus thuringiensis was distributed over the initial small aggregates, and the outer edge of the granule was away from the core regime in the following stage.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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.

  5. Study of the Bacillus thuringiensis Vip3Aa16 histopathological effects and determination of its putative binding proteins in the midgut of Spodoptera littoralis.

    Science.gov (United States)

    Abdelkefi-Mesrati, Lobna; Boukedi, Hanen; Dammak-Karray, Mariam; Sellami-Boudawara, Tahya; Jaoua, Samir; Tounsi, Slim

    2011-02-01

    The bacterium Bacillus thuringiensis produces, at the vegetative stage of its growth, Vip3A proteins with activity against a broad spectrum of lepidopteran insects. The Egyptian cotton leaf worm (Spodoptera littoralis) is an important agricultural pest that is susceptible to the Vip3Aa16 protein of Bacillus thuringiensis kurstaki strain BUPM95. The midgut histopathology of Vip3Aa fed larvae showed vacuolization of the cytoplasm, brush border membrane destruction, vesicle formation in the apical region and cellular disintegration. Biotinylated Vip3Aa toxin bound proteins of 55- and 100-kDa on blots of S. littoralis brush border membrane preparations. These binding proteins differ in molecular size from those recognized by Cry1C, one of the very few Cry proteins active against the polyphagous S. littoralis. This result supports the use of Vip3Aa16 proteins as insecticidal agent, especially in case of Cry-resistance management.

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

    Science.gov (United States)

    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. Association of bioassays and molecular characterization to select new Bacillus thuringiensis isolates effective against Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae); Associacao de bioensaios e caracterizacao molecular para selecao de novos isolados de Bacillus thuringiensis efetivos contra Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae)

    Energy Technology Data Exchange (ETDEWEB)

    Fatoretto, Julio C.; Sena, Janete A.D.; Lemos, Manoel V.F. [UNESP, Jaboticabal, SP (Brazil). Faculdade de Ciencias Agrarias e Veterinarias. Dept. de Biologia Aplicada a Agropecuaria; Barreto, Marliton R. [Universidade Federal do Mato Grosso (UFMT), Cuiaba, MT (Brazil). Inst. Universitario do Norte Matogrossense (IUNMAT)]. E-mail: mrbarreto@pop.com.br; Junior Boica, Arlindo L. (UNESP), Jaboticabal, SP (Brazil). Faculdade de Ciencias Agrarias e Veterinarias. Dept. de Fitossanidade)

    2007-09-15

    The fall armyworm, Spodoptera frugiperda (J. E. Smith), is one of the main corn pests and Bacillus thuringiensis is important in its control because of its entomopathogenic property. The objective of this study was the molecular characterization of B. thuringiensis isolates for cry1 locus presence and the assessment of the efficiency of these isolates in controlling S. frugiperda caterpillars. Gral-cry1 was used in the PCR analyses to confirm the presence of the cry1 locus in 15 isolates. A 3 x 108 spore/ml suspension bathed the diet used to feed 30 caterpillars per isolate, with three replications. The cry1 locus type genes of the different isolates were identified for five gene subclasses; linear regression analyses were carried out to ascertain possible associations between the presence of an individual cry1 locus gene and high levels of toxicity. All the DNAs amplified with Gral-cry1 presented an amplification product with the expected size. Regarding the levels of insecticide efficiency against the cob worm, 41 isolates presented 100% mortality and 16 presented an index between 70% and 90%. The cry1Ab gene was present in 80 isolates, cryb in 69 isolates, cry1Ac in all the isolates and cryv and cry1E in 93 and 27 isolates, respectively. The values regarding the individual effect of each gene on caterpillar mortality were significant at 1% probability for the cry1Ac and cry1E genes. (author)

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

    Science.gov (United States)

    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. Transfer of the toxin protein genes of Bacillus sphaericus into Bacillus thuringiensis subsp. israelensis and their expression.

    OpenAIRE

    Bourgouin, C.; Delécluse, A; de la Torre, F; Szulmajster, J.

    1990-01-01

    The genes encoding the toxic determinants of Bacillus sphaericus have been expressed in a nontoxic and a toxic strain of Bacillus thuringiensis subsp. israelensis. In both cases, the B. sphaericus toxin proteins were produced at a high level during sporulation of B. thuringiensis and accumulated as crystalline structures. B. thuringiensis transformants expressing B. sphaericus and B. thuringiensis subsp. israelensis toxins did not show a significant enhancement of toxicity against Aedes aegyp...

  10. A new enrichment method for isolation of Bacillus thuringiensis from diverse sample types.

    Science.gov (United States)

    Patel, Ketan D; Bhanshali, Forum C; Chaudhary, Avani V; Ingle, Sanjay S

    2013-05-01

    New or more efficient methodologies having different principles are needed, as one method could not be suitable for isolation of organisms from samples of diverse types and from various environments. In present investigation, growth kinetics study revealed a higher germination rate, a higher growth rate, and maximum sporulation of Bacillus thuringiensis (Bt) compared to other Bacillus species. Considering these facts, a simple and efficient enrichment method was devised which allowed propagation of spores and vegetative cells of Bt and thereby increased Bt cell population proportionately. The new enrichment method yielded Bt from 44 out of 58 samples. Contrarily, Bt was isolated only from 16 and 18 samples by sodium acetate selection and dry heat pretreatment methods, respectively. Moreover, the percentages of Bt colonies isolated by the enrichment method were higher comparatively. Vegetative whole cell protein profile analysis indicated isolation of diverse population of Bt from various samples. Bt strains isolated by the enrichment method represented novel serovars and possibly new cry2 gene.

  11. Bacillus thuringiensis-based Products for Insect Pest Control

    NARCIS (Netherlands)

    Maagd, de R.A.

    2015-01-01

    Bacillus thuringiensis (or Bt, as it has become generally known) is one of the oldest and widely used biological control agents and has a long history of use. Bt and a number of related bacteria produce a variety of toxins, mostly—but not exclusively- localized in the parasporal crystals, which are,

  12. Bacillus thuringiensis insecticidal crystal proteins affect lifespan and reproductive performance of Helicoverpa armigera and Spodoptera exigua adults.

    Science.gov (United States)

    Zhang, Ying; Ma, Yan; Wan, Pin-Jun; Mu, Li-Li; Li, Guo-Qing

    2013-04-01

    Being delivered as sprays or expressed in plant, Bacillus thuringiensis (Bt) crystalline proteins (Cry toxins) display insecticidal activities against numerous Lepidopteran, Dipteran, and Coleopteran larvae. Comparative study of toxicities of Bt Cry toxins between larvae and adults may afford important new insights into the interactions of the toxins with receptor proteins in host insect, and represent intriguing targets for the control of insect pests. However, the effectiveness of Bt Cry toxins in insect adults has paid less attention. In the present article, the effectiveness of Cry1Ac and Cry1Ca on lifespans and reproductive performance of Helicoverpa armigera (Hübner) and Spodoptera exigua (Hübner) adults were evaluated by in vivo experiments. Considering transgenic plants express modified, truncated versions of cry genes yielding active toxin fragment, we used activated Bt toxins at the concentration of 500, 100, and 20 microg/ml in a 10% sucrose aquous solution. At the highest concentration, Cry1Ac and Cry1Ca shortened 48.1 and 48.9% of H. armigera female lifespan, and 43.5 and 38.5% of S. exigua female lifespan, and they reduced 37.8 and 40.3%, and 50.5 and 47.4% of H. armigera and S. exigua male lifespans respectively. Bt toxins negatively affected copulation. Exposure to 500 microg/ml of Cry1Ac and Cry1Ca greatly reduced 50.0 and 46.8%, and 58.7 and 57.3% spermatophore acceptance by H. armigera and S. exigua females, respectively. Similarly, Cry1Ac and Cry1Ca exposure decreased 40.0 and 50.3%, and 61.3 and 60.0% of spermatophore transfer by H. armigera and S. exigua males, respectively. Moreover, exposure females rather than males to 500 microg/ml of Cry1Ac and Cry1Ca significantly dropped 57.5 and 57.5% of the number of eggs laid by H. armigera, and 35.4 and 45.8% of the number of egg masses deposited by S. exigua. In contrast, both Cry1Ac and Cry1Ca did not negatively influence the egg hatchability. At the middle and the lowest concentrations, however

  13. Development of photoperiod- and thermo-sensitive male sterility rice expressing transgene Bacillus thuringiensis

    Science.gov (United States)

    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

  14. Poly-β-hydroxybutyrate Metabolism Is Unrelated to the Sporulation and Parasporal Crystal Protein Formation in Bacillus thuringiensis.

    Science.gov (United States)

    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.

  15. Poly-β-hydroxybutyrate Metabolism Is Unrelated to the Sporulation and Parasporal Crystal Protein Formation in Bacillus thuringiensis

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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.

  17. Toxicidad de δ-endotoxinas recombinantes de Bacillus thuringiensis sobre larvas de la polilla guatemalteca (Tecia solanivora (Lepidóptera: Gelechiidae

    Directory of Open Access Journals (Sweden)

    Javier Hernández-Fernández

    2009-01-01

    Full Text Available Con el objetivo de determinar la actividad tóxica específica de las proteínas recombinantes Cry1Aa, Cry1Ac, Cry1B y Cry1C de Bacillus thuringiensis (Bt, sobre larvas de primer instar de Tecia solanivora se estableció la CL50 para las toxinas. Para este fin se implementó la cría masiva de este insecto bajo condiciones de laboratorio, 58±5% de humedad relativa, 18±5ºC de temperatura y un fotoperiodo de 23 h oscuridad y 1 h luz. Se utilizó una dieta seminatural consistente en láminas de papa variedad parda pastusa autoclavada con solución preservante (ácido ascórbico 7 g/L y metilparabeno 5 g/L, ya que fue estable en el tiempo, garantizó la reproducibilidad de los resultados y fue de fácil evaluación. Las proteínas recombinantes se evaluaron a una concentración de 0,1 μg/cm2. Los resultados obtenidos de porcentaje de mortalidad indicaron que no había diferencias entre las delta-endotoxinas recombinantes Cry1Aa, Cry1Ac, Cry1B y Cry1C de Bt (PPalabras clave: Bacillus thuringiensis; proteínas recombinantes; Tecia solanivora; papa parda pastusa; Bacillus thuringiensis; bioassay; recombinant endotoxin; Tecia solanivora; pastusa potato variety.

  18. Identification of cry-type genes of Bacillus thuringiensis isolated from animal feces%源自动物粪便苏云金芽胞杆菌的cry基因型鉴定

    Institute of Scientific and Technical Information of China (English)

    吴昌标

    2010-01-01

    从5种草食性哺乳动物的粪便中筛选获得8株苏云金芽胞杆菌(Bt);并用聚合酶链式反应-限制性酶切片段长度多态性(PCR-RFLP)鉴定体系鉴定各菌株的cry基因型.结果表明:BRC-WCB1、BRC-WCB2、BRC-WCB3、BRC-WCB8同时含有cry1和cry2基因;BRC-WCB6仅含有cry1I基因;BRC-WCB5含有cry3基因,但未出现预知的酶切条带;而BRC-WCB4无任何PCR扩增产物.

  19. 40 CFR 180.1107 - Delta endotoxin of Bacillus thuringiensis variety kurstaki encapsulated into killed Pseudomonas...

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Delta endotoxin of Bacillus... 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...

  20. Occurrence and diversity of mosquitocidal strains of Bacillus thuringiensis

    Directory of Open Access Journals (Sweden)

    K. Balaraman

    2005-09-01

    Full Text Available Ever since the discovery of the first Bacillus thuringiensis strain capable of killing mosquito larvae,namely, B. thuringiensis var israelensis, there are several reports from different parts of the worldabout the occurrence of mosquitocidal strains belonging to different subspecies/serotypes numberingthirty-six. The main sources of these wild type strains are soils/sediments, plants, animal feces,sick/moribund insects and waters. The toxicity of the strains within a subspecies/serotype variedwidely. Some of the strains exhibited toxicity to mosquitoes as well as lepidopterans and dipterans(including mosquitoes as well as plant parasitic nematodes.

  1. Isolation and characterization of gut bacterial proteases involved in inducing pathogenicity of Bacillus thuringiensis toxin in cotton bollworm, Helicoverpa armigera

    Directory of Open Access Journals (Sweden)

    Visweshwar Regode

    2016-10-01

    Full Text Available Bacillus thuringiensis (Bt 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 towards pro-Cry1Ac. Among twelve 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 oC. Maximum inhibition of total proteolytic activity was exerted by PMSF 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 towards H. armigera. The gut bacterial isolates IVS1, IVS2 and IVS3 showed homology with Bacillus thuringiensis (CP003763.1, Vibrio fischeri (CP000020.2 and Escherichia coli (CP011342.1, respectively. Proteases produced by midgut bacteria are involved in proteolytic processing of Bt protoxin and play a major role in inducing pathogenicity of Bt toxins in H. armigera.

  2. Diversity of Cry Gene from Bacillus thuringiensis in Different Ecological Region of Hebei Province%河北省不同生态区的苏云金芽孢杆菌cry基因多样性研究

    Institute of Scientific and Technical Information of China (English)

    谢月霞; 杜立新; 李瑞军; 王客燕; 王金耀; 曹伟平; 宋健; 冯书亮

    2008-01-01

    为了明确苏云金芽孢杆菌在河北省不同生态区的分布特点和cry基因的多样性,研究从河北省不同生态地区(阜平天生桥、涞源白石山、安新白洋淀、保定郊区大田和安国中草药田)采集土样806份.采用温度.抗生素法分离获得Bt菌株46株,利用PCR.RFLP技术对46株Bt进行了基因型研究,结果表明从这些菌株中发现7种不同的基因型cry1、cry2、cry3、cry4、cry8、cry30和cry32,11株未鉴定出基因型:通过SDS-PAGE分析发现这些菌株主要表达130-150 kDa、70-80 kDa、60 kDa和30 kDa蛋白.实验结果说明了苏云金芽孢杆菌在河北省不同生态区均有分布,并且其cry基因类型是复杂多样的.

  3. Mutually exclusive distribution of the sap and eag S-layer genes and the lytB/lytA cell wall hydrolase genes in Bacillus thuringiensis.

    Science.gov (United States)

    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.

  4. A novel Cry9Aa with increased toxicity for Spodoptera exigua (Hübner)

    NARCIS (Netherlands)

    Naimov, S.; Nedyalkova, R.; Staykov, N.; Weemen-Hendriks, M.; Minkov, I.; Maagd, de R.A.

    2014-01-01

    Cry9Aa, produced by Bacillus thuringiensis is reported to be not active against Spodoptera exigua (beet armyworm). In this study we have cloned a new cry9Aa5 gene encoding a protoxin with increased activity against S. exigua as compared to Cry9Aa1. When aligned to Cry9Aa1, four amino acid substituti

  5. Gene knockout demonstrates that vip3A contributes to the pathogenesis of Bacillus thuringiensis toward Agrotis ipsilon and Spodoptera exigua.