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Sample records for bacillus thuringiensis cry2ab2

  1. 40 CFR 174.519 - Bacillus thuringiensis Cry2Ab2 protein in corn and cotton; exemption from the requirement of a...

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 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...

  2. 40 CFR 174.519 - Bacillus thuringiensis Cry2Ab2 protein in corn and cotton; exemption from the requirement of a...

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... in corn and cotton; exemption from the requirement of a tolerance. 174.519 Section 174.519 Protection... thuringiensis Cry2Ab2 protein in corn and cotton; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry2Ab2 protein in or on corn or cotton are exempt from the requirement of...

  3. 40 CFR 174.519 - Bacillus thuringiensis Cry2Ab2 protein in corn and cotton; exemption from the requirement of a...

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... in corn and cotton; exemption from the requirement of a tolerance. 174.519 Section 174.519 Protection... thuringiensis Cry2Ab2 protein in corn and cotton; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry2Ab2 protein in or on corn or cotton are exempt from the requirement of...

  4. 40 CFR 174.519 - Bacillus thuringiensis Cry2Ab2 protein in corn and cotton; exemption from the requirement of a...

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... in corn and cotton; exemption from the requirement of a tolerance. 174.519 Section 174.519 Protection... thuringiensis Cry2Ab2 protein in corn and cotton; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry2Ab2 protein in or on corn or cotton are exempt from the requirement of...

  5. 40 CFR 174.519 - Bacillus thuringiensis Cry2Ab2 protein in corn and cotton; exemption from the requirement of a...

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... in corn and cotton; exemption from the requirement of a tolerance. 174.519 Section 174.519 Protection... thuringiensis Cry2Ab2 protein in corn and cotton; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry2Ab2 protein in or on corn or cotton are exempt from the requirement of...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  7. 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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Sugarcane borer, Diatraea saccharalis (F.), is a primary corn stalk borer pest targeted by transgenic corn expressing Bacillus thuringiensis (Bt) proteins in many areas of the mid-southern region of the United States. Recently, genes encoding for Cry1A.105 and Cry2Ab2 Bt proteins were transferred in...

  9. Evaluation of corn hybrids expressing Cry1F, cry1A.105, Cry2Ab2, Cry34Ab1/Cry35Ab1, and Cry3Bb1 against southern United States insect pests.

    PubMed

    Siebert, M W; Nolting, S P; Hendrix, W; Dhavala, S; Craig, C; Leonard, B R; Stewart, S D; All, J; Musser, F R; Buntin, G D; Samuel, L

    2012-10-01

    Studies were conducted across the southern United States to characterize the efficacy of multiple Bacillus thuringiensis (Bt) events in a field corn, Zea mays L., hybrid for control of common lepidopteran and coleopteran pests. Cry1F protein in event TC1507 and Cry1A.105 + Cry2Ab2 proteins in event MON 89034 were evaluated against pests infesting corn on above-ground plant tissue including foliage, stalks, and ears. Cry34Ab1/Cry35Ab1 proteins in event DAS-59122-7 and Cry3Bb1 in event MON 88017 were evaluated against the larvae of Mexican corn rootworm, Diabrotica virgifera zeae Krysan and Smith, which occur below-ground. Field corn hybrids containing Cry1F, Cry1A.105 + Cry2Ab2, Cry34Ab1/Cry35Ab1, and Cry3Bb1 insecticidal proteins (SmartStax) consistently demonstrated reductions in plant injury and/or reduced larval survivorship as compared with a non-Bt field corn hybrid. Efficacy provided by a field corn hybrid with multiple Bt proteins was statistically equal to or significantly better than corn hybrids containing a single event active against target pests. Single event field corn hybrids provided very high levels of control of southwestern corn borer, Diatraea grandiosella (Dyar), lesser cornstalk borer, Elasmopalpus lignosellus (Zeller), and fall armyworm, Spodoptera frugiperda (J.E. Smith), and were not significantly different than field corn hybrids with multiple events. Significant increases in efficacy were observed for a field corn hybrid with multiple Bt events for sugarcane borer, Diatraea saccharalis (F.), beet armyworm, Spodoptera exigua (Hübner), corn earworm, Helicoverpa zea (Boddie), and Mexican corn rootworm. Utilization of field corn hybrids containing multiple Bt events provides a means for managing insect resistance to Bt proteins and reduces non-Bt corn refuge requirements.

  10. Bacillus thuringiensis

    PubMed Central

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

    2010-01-01

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

  11. Brush border membrane binding properties of Bacillus thuringiensis Vip3A toxin to Heliothis virescens and Helicoverpa zea midguts.

    PubMed

    Lee, Mi Kyong; Miles, Paul; Chen, Jeng-Shong

    2006-01-27

    The binding properties of Vip3A, a new family of Bacillus thuringiensis insecticidal toxins, have been examined in the major cotton pests, Heliothis virescens and Helicoverpa zea. Vip3A bound specifically to brush border membrane vesicles (BBMV) prepared from both insect larval midguts. In order to examine the cross-resistance potential of Vip3A to the commercially available Cry1Ac and Cry2Ab2 toxins, the membrane binding site relationship among these toxins was investigated. Competition binding assays demonstrated that Vip3A does not inhibit the binding of either Cry1Ac or Cry2Ab2 and vice versa. BBMV protein blotting experiments showed that Vip3A does not bind to the known Cry1Ac receptors. These distinct binding properties and the unique protein sequence of Vip3A support its use as a novel insecticidal agent. This study indicates a very low cross-resistance potential between Vip3A and currently deployed Cry toxins and hence supports its use in an effective resistance management strategy in cotton.

  12. 76 FR 14289 - Bacillus thuringiensis

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-03-16

    ... AGENCY 40 CFR Part 174 Bacillus thuringiensis eCry3.1Ab Protein in Corn; Temporary Exemption From the... 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...

  13. 75 FR 34040 - Bacillus thuringiensis

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-06-16

    ... AGENCY 40 CFR Part 174 Bacillus thuringiensis eCry3.1Ab Protein in Corn; Temporary Exemption from the... 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...

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

    PubMed

    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.

  15. Bacillus thuringiensis (Bt)

    NASA Technical Reports Server (NTRS)

    2004-01-01

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

  16. Development and validation of real-time PCR screening methods for detection of cry1A.105 and cry2Ab2 genes in genetically modified organisms.

    PubMed

    Dinon, Andréia Z; Prins, Theo W; van Dijk, Jeroen P; Arisi, Ana Carolina M; Scholtens, Ingrid M J; Kok, Esther J

    2011-05-01

    Primers and probes were developed for the element-specific detection of cry1A.105 and cry2Ab2 genes, based on their DNA sequence as present in GM maize MON89034. Cry genes are present in many genetically modified (GM) plants and they are important targets for developing GMO element-specific detection methods. Element-specific methods can be of use to screen for the presence of GMOs in food and feed supply chains. Moreover, a combination of GMO elements may indicate the potential presence of unapproved GMOs (UGMs). Primer-probe combinations were evaluated in terms of specificity, efficiency and limit of detection. Except for specificity, the complete experiment was performed in 9 PCR runs, on 9 different days and by testing 8 DNA concentrations. The results showed a high specificity and efficiency for cry1A.105 and cry2Ab2 detection. The limit of detection was between 0.05 and 0.01 ng DNA per PCR reaction for both assays. These data confirm the applicability of these new primer-probe combinations for element detection that can contribute to the screening for GM and UGM crops in food and feed samples.

  17. [Extracellular ribonuclease from Bacillus thuringiensis].

    PubMed

    Chepurnova, N K; Liakhov, D L; Rechinskiĭ, V O; Karpeĭskiĭ, M Ia

    1988-04-01

    The ability of the strain Bacillus thuringiensis var. subtoxicus to produce extracellular ribonuclease (ribonuclease Bt) was studied. It was found that the culture medium possesses a RNA-depolymerizing activity whose maximum is observed 4-5 hours after the beginning of the linear growth phase. A three-step chromatography of the culture extract on phosphocellulose resulted in a homogeneous enzyme with a molecular mass of 12000 Da. The enzyme showed the maximum activity towards RNA at pH 8.5, catalyzed the hydrolysis of polyribonucleotides and guanosine-2',3'-cyclophosphate. Hence, the enzyme can be related to base-nonspecific cyclizing ribonucleases showing the guanylic specificity towards nucleoside-2',3'-cyclophosphates.

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

    EPA Science Inventory

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

  19. The Complete Genome Sequence of Bacillus thuringiensis AlHakam

    SciTech Connect

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

    2007-04-01

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

  20. Novel cloning vectors for Bacillus thuringiensis.

    PubMed

    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.

  1. Novel cloning vectors for Bacillus thuringiensis.

    PubMed Central

    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 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. Images PMID:2268153

  2. [Spontaneous bacteriophage induction in Bacillus thuringiensis].

    PubMed

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

    1987-01-01

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

  3. 75 FR 35805 - Pesticide Product Registrations; Conditional Approvals

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-06-23

    ... products (EPA File Symbols 524-LTL and 524-LTA) containing the active ingredients, Bacillus thuringiensis... with the proposed use of the Bacillus thuringiensis Cry1A.105 and Cry2Ab2 proteins and the genetic... use of the Bacillus thuringiensis Cry1A.105 and Cry2Ab2 proteins and the genetic material...

  4. Insecticidal crystal proteins of Bacillus thuringiensis.

    PubMed Central

    Höfte, H; Whiteley, H R

    1989-01-01

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

  5. Transferrin Impacts Bacillus thuringiensis Biofilm Levels

    PubMed Central

    Brown, Elrica; Taplin, Martha; Garcia, Angel; Williams-Mapp, Baracka

    2016-01-01

    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. PMID:28025643

  6. Bacillus thuringiensis and Its Pesticidal Crystal Proteins

    PubMed Central

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

    1998-01-01

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

  7. The occurrence of disporous Bacillus thuringiensis cells.

    PubMed

    Chapman, G B; Slob-van Herk, A; Eguía, J M

    1992-05-01

    Ultrathin sections of sporulating Bacillus thuringiensis were examined in a transmission electron microscope. Less than 1% of the about 2,000 approximately sagittal sections of the bacterial cells examined contained two endospores per cell. This finding clarifies the majority of textbook and research reports (which tend to be ambiguous), contradicts several of the most recent textbook reports, and confirms three unillustrated textbook reports, in relation to the occurrence of disporous bacilli. Electron microscopic evidence of the observation is presented, apparently for the first time.

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

    PubMed

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

    2012-10-01

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

  9. Stability and antibacterial activity of bacteriocins produced by Bacillus thuringiensis and Bacillus thuringiensis ssp. kurstaki.

    PubMed

    Jung, Woo-Jin; Mabood, Fazli; Souleimanov, Alfred; Zhou, Xiaomin; Jaoua, Samir; Kamoun, Fakher; Smith, Donald L

    2008-11-01

    Bacteriocins are antimicrobial peptides that are produced by bacteria and toxic to bacterial strains closely related to the producer strain. It has previously been reported that Bacillus thuringiensis strain NEB17 and Bacillus thuringiensis subsp. kurstaki BUPM4 produce the bacteriocins thuricin 17 (3,162 Da) and bacthuricin F4 (3,160.05 Da), respectively. Here, we demonstrate that these bacteriocins have functional similarities and show a similar spectrum of antimicrobial activities against indicator strains. We also studied the effects of sterilization methods on the recovery and biological activities of these bacteriocins. They were completely degraded by autoclaving and the two were similarly affected by the tested filter membranes. Polyvinylidene fluoride (PVDF), polyestersulfone (PES), and cellulose acetate (CA) are suitable for filter sterilization of these bacteriocins. The two bacteriocins were stable across a range of storage conditions. These data will facilitate their utilization in food preservation or agricultural applications.

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

    EPA Science Inventory

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

  11. Distribution of phenotypes among Bacillus thuringiensis strains.

    PubMed

    Martin, Phyllis A W; Gundersen-Rindal, Dawn E; Blackburn, Michael B

    2010-06-01

    An extensive collection of Bacillus thuringiensis isolates from around the world were phenotypically profiled using standard biochemical tests. Six phenotypic traits occurred in 20-86% of the isolates and were useful in distinguishing isolates: production of urease (U; 20.5% of isolates), hydrolysis of esculin (E; 32.3% of isolates), acid production from salicin (A; 37.4% of isolates), acid production from sucrose (S; 34.0% of isolates), production of phospholipase C or lecithinase (L; 79.7% of isolates), and hydrolysis of starch (T; 85.8% of isolates). With the exception of acid production from salicin and hydrolysis of esculin, which were associated, the traits assorted independently. Of the 64 possible combinations of these six phenotypic characteristics, 15 combinations accounted for ca. 80% of all isolates, with the most common phenotype being TL (23.6% of isolates). Surprisingly, while the biochemical traits generally assorted independently, certain phenotypic traits associated with the parasporal crystal were correlated with certain combinations of biochemical traits. Crystals that remained attached to spores (which tended to be non-toxic to insects) were highly correlated with the phenotypes that included both L and S. Among the 15 most abundant phenotypes characterizing B. thuringiensis strains, amorphous crystals were associated with TLE, TL, T, and Ø (the absence of positive tested biochemical traits). Amorphous crystal types displayed a distinct bias toward toxicity to dipteran insects. Although all common phenotypes included B. thuringiensis isolates producing bipyramidal crystals toxic to lepidopteran insects, those with the highest abundance of these toxic crystals displayed phenotypes TLU, TLUA, TLUAE, and TLAE.

  12. Bacillus thuringiensis Conjugation in Simulated Microgravity

    NASA Astrophysics Data System (ADS)

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

    2009-10-01

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

  13. Bacillus thuringiensis conjugation in simulated microgravity.

    PubMed

    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.

  14. Sludge based Bacillus thuringiensis biopesticides: viscosity impacts.

    PubMed

    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.

  15. Complete Genome Sequence of Bacillus thuringiensis Bacteriophage Smudge

    PubMed Central

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

    2016-01-01

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

  16. 40 CFR 180.1011 - Viable spores of the microorganism Bacillus thuringiensis Berliner; exemption from the...

    Code of Federal Regulations, 2010 CFR

    2010-07-01

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

  17. Bollgard II cotton: compositional analysis and feeding studies of cottonseed from insect-protected cotton (Gossypium hirsutum L.) producing the Cry1Ac and Cry2Ab2 proteins.

    PubMed

    Hamilton, Kathryn A; Pyla, Paul D; Breeze, Matthew; Olson, Tammy; Li, Menghe; Robinson, Edwin; Gallagher, Sean P; Sorbet, Roy; Chen, Yin

    2004-11-17

    Bollgard II cotton event 15985 producing the Cry1Ac and Cry2Ab2 proteins has been developed by genetic modification to broaden the spectrum of insects to which the plant is tolerant and to provide an insect resistance management tool to impede the onset of resistance. The purpose of this study was to evaluate the composition and nutrition of Bollgard II cotton, relative to the use for food and animal feed, compared to that of conventional cotton varieties. Compositional analyses were conducted to measure proximate, fiber, amino acid, fatty acid, gossypol, and mineral contents of cottonseed from a total of 14 U.S. field sites over two years. Compositional analysis results showed that the cottonseed and cottonseed oil from Bollgard II cotton were comparable in their composition to those of the conventional control cotton line and other commercial varieties. The composition data are supported by nutritional safety studies conducted with dairy cows, catfish, and quail. Results from these studies showed that Bollgard II performed similarly to the conventional control cotton varieties. These data demonstrate that Bollgard II cotton is compositionally and nutritionally equivalent to conventional cotton varieties. These data support the conclusion that Bollgard II cotton is as safe and nutritious as conventional cotton for food and feed use.

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

    PubMed

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

    2002-10-01

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

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

    NASA Astrophysics Data System (ADS)

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

    1990-01-01

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

  20. Biological Activity of Bacillus thuringiensis in Drosophila suzukii (Diptera: Drosophilidae).

    PubMed

    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.

  1. Plasmid-associated sensitivity of Bacillus thuringiensis to UV light

    SciTech Connect

    Benoit, T.G.; Wilson, G.R.; Bull, D.L.; Aronson, A.I. )

    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.

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

    PubMed

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

    2006-05-01

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

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

    PubMed Central

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

    2016-01-01

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

  4. Regulation of cry Gene Expression in Bacillus thuringiensis

    PubMed Central

    Deng, Chao; Peng, Qi; Song, Fuping; Lereclus, Didier

    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-transcriptional, metabolic and post-translational levels. PMID:25055802

  5. Transformation of vegetative cells of Bacillus thuringiensis by plasmid DNA.

    PubMed

    Heierson, A; Landén, R; Lövgren, A; Dalhammar, G; Boman, H G

    1987-03-01

    Plasmid DNA-mediated transformation of vegetative cells of Bacillus thuringiensis was studied with the following two plasmids: pBC16 coding for tetracycline resistance and pC194 expressing chloramphenicol resistance. A key step was the induction of competence by treatment of the bacteria with 50 mM Tris hydrochloride buffer (pH 8.9) containing 30% sucrose. Transformation frequency was strongly influenced by culture density during the uptake of DNA and required the presence of polyethylene glycol. Growth in a minimal medium supplemented with Casamino Acids gave 35 times more transformants than growth in a rich medium. The highest frequencies were obtained with covalently closed circular DNA. With all parameters optimized, the frequency was 10(-3) transformants per viable cell or 10(4) transformants per microgram of DNA. Cells previously frozen were also used as recipients in transformation experiments; such cells gave frequencies similar to those obtained with freshly grown cells. The procedure was optimized for B. thuringiensis subsp. gelechiae, but B. thuringiensis subsp. kurstaki, B. thuringiensis subsp. galleriae, B. thuringiensis subsp. thuringiensis, and B. thuringiensis subsp. israelensis were also transformed. Compared with protoplast transformation, our method is much faster and 3 orders of magnitude more efficient per microgram of added DNA.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  7. Parallel evolution of Bacillus thuringiensis toxin resistance in lepidoptera.

    PubMed

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

    2011-10-01

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

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

    PubMed

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

    2006-08-01

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

  9. Molecular methods to evaluate biodiversity in Bacillus cereus and Bacillus thuringiensis strains from different origins.

    PubMed

    Manzano, Marisa; Giusto, Cristina; Iacumin, Lucilla; Cantoni, Carlo; Comi, Giuseppe

    2009-05-01

    The spore-forming genus Bacillus includes species of industrial, clinical and environmental significance. The possibility of differentiating between Bacillus cereus and Bacillus thuringiensis, toxin producers associated with illness, is a real need in monitoring potentially contaminated foods to understand the real distribution of B. cereus/B. thuringiensis in different outbreak cases. As the use of DNA comparison obtains clearer results than classical microbiological methods in distinguishing B. cereus from B. thuringiensis in this work PCR-TTGE (Temporal Temperature Gradient gel Electrophoresis), rep-PCR and RAPD-PCR methods have been compared to assess the intra- and inter-specific variability of B. cereus and B. thuringiensis. 80 strains of B. cereus and B. thuringiensis isolated from food, patients and pesticides were analyzed using a gyrB gene DNA sequence in TTGE; primer M13 in the RAPD-PCR and primers REP1DT and REP2DT in the rep-PCR methods. A widespread distribution of the electrophoretic profiles was obtained either for B. cereus or for B. thuringiensis using TTGE. rep-PCR and RAPD-PCR were not always able to group strains from the same origin or belonging to the same species. The fingerprints obtained with the rep- and RAPD-PCR methods confirm the high intraspecific variability present in B. cereus and B. thuringiensis indicating the difficulty to discriminate between these two species in outbreak cases.

  10. 40 CFR 180.1107 - Delta endotoxin of Bacillus thuringiensis variety kurstaki encapsulated into killed Pseudomonas...

    Code of Federal Regulations, 2010 CFR

    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...

  11. Flourescence Assay Based on Aptamer-Quantum Dot Binding to Bacillus thuringiensis Spores

    DTIC Science & Technology

    2007-01-01

    Binding to Bacillus thuringiensis 5a. CONTRACT NUMBER N/A Spores 5b. GRANT NUMBER N/A 5c. PROGRAM ELEMENT NUMBER 62202F 6. AUTHOR(S) Milada...assay was developed for the detection of Bacillus thuringiensis (BT) spores. The assay is based on the fluorescence observed after binding an aptamer...units/ml. 15. SUBJECT TERMS Bacillus thuringiensis , Aptamer, Quantum dots, SELEX, Fluorescence 16. SECURITY CLASSIFICATION OF: Unclassified U

  12. Fluorescence Assay Based on Aptamer-Quantum Dot Binding to Bacillus thuringiensis Spores

    DTIC Science & Technology

    2007-01-01

    Binding to Bacillus thuringiensis 5a. CONTRACT NUMBER N/A Spores 5b. GRANT NUMBER N/A 5c. PROGRAM ELEMENT NUMBER 62202F 6. AUTHOR(S) Milada...assay was developed for the detection of Bacillus thuringiensis (BT) spores. The assay is based on the fluorescence observed after binding an aptamer...units/ml. 15. SUBJECT TERMS Bacillus thuringiensis , Aptamer, Quantum dots, SELEX, Fluorescence 16. SECURITY CLASSIFICATION OF: Unclassified U

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

    PubMed Central

    Gillis, Annika; Mahillon, Jacques

    2014-01-01

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

  14. Characterization study of the sporulation kinetics of Bacillus thuringiensis.

    PubMed

    Liu, B L; Tzeng, Y M

    2000-04-05

    A wild-type and an rDNA strain of Bacillus thuringiensis were cultured in a net-draft-tube modified 20-L airlift bioreactor. A comparison of the sporulation patterns suggests that the early sporulation strain has a lower final spore count. Results from off-gas analysis suggests that the CO(2) profile could be an alternative indication to spore counts for the examination of fermentation performance or even the mortality in bioassay of the cultivation product. The difference in mortality tests exhibited by the microorganism was attributed to different patterns of sporulation as well as different levels of gene control inside the cell itself. The sporulation kinetics of B. thuringiensis was simulated by a simple modified Hill equation, where the initial glucose concentration could affect the timing of the onset of sporulation. The equation matches well with the experimental sporulation data for B. thuringiensis in both wild-type and rDNA strains.

  15. Genetic differentiation between sympatric populations of Bacillus cereus and Bacillus thuringiensis.

    PubMed

    Vilas-Boas, Gislayne; Sanchis, Vincent; Lereclus, Didier; Lemos, Manoel Victor F; Bourguet, Denis

    2002-03-01

    Little is known about genetic exchanges in natural populations of bacteria of the spore-forming Bacillus cereus group, because no population genetics studies have been performed with local sympatric populations. We isolated strains of Bacillus thuringiensis and B. cereus from small samples of soil collected at the same time from two separate geographical sites, one within the forest and the other at the edge of the forest. A total of 100 B. cereus and 98 B. thuringiensis strains were isolated and characterized by electrophoresis to determine allelic composition at nine enzymatic loci. We observed genetic differentiation between populations of B. cereus and B. thuringiensis. Populations of a given Bacillus species--B. thuringiensis or B. cereus--were genetically more similar to each other than to populations of the other Bacillus species. Hemolytic activity provided further evidence of this genetic divergence, which remained evident even if putative clones were removed from the data set. Our results suggest that the rate of gene flow was higher between strains of the same species, but that exchanges between B. cereus and B. thuringiensis were nonetheless possible. Linkage disequilibrium analysis revealed sufficient recombination for B. cereus populations to be considered panmictic units. In B. thuringiensis, the balance between clonal proliferation and recombination seemed to depend on location. Overall, our data indicate that it is not important for risk assessment purposes to determine whether B. cereus and B. thuringiensis belong to a single or two species. Assessment of the biosafety of pest control based on B. thuringiensis requires evaluation of the extent of genetic exchange between strains in realistic natural conditions.

  16. MICs of Selected Antibiotics for Bacillus anthracis, Bacillus cereus, Bacillus thuringiensis, and Bacillus mycoides From a Range of Clinical and Environmental Sources as Determined by the Etest

    DTIC Science & Technology

    2004-08-01

    1184–1187. 21. Kemmerly, S. A., and G. A. Pankey. 1993. Oral ciprofloxacin therapy for Bacillus cereus wound infection and bacteremia . Clin. Infect...Antibiotics for Bacillus anthracis, Bacillus cereus , Bacillus thuringiensis, and Bacillus mycoides from a Range of Clinical and Environmental Sources...76 isolates of Bacillus anthracis chosen for their diverse histories and 67, 12, and 4 cultures, respectively, of its close relatives B. cereus , B

  17. Photoprotection of Bacillus thuringiensis kurstaki from ultraviolet irradiation

    SciTech Connect

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

    1991-05-01

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

  18. Genome Sequences of Bacillus thuringiensis Serovar kurstaki Strain BP865 and B. thuringiensis Serovar aizawai Strain HD-133

    PubMed Central

    Jeong, Haeyoung

    2017-01-01

    ABSTRACT We report the draft genome sequences of two insecticidal strains against lepidopteran pests, Bacillus thuringiensis serovar kurstaki strain BP865, an isolate from the South Korean phylloplane, and strain HD-133, a reference strain of B. thuringiensis serovar aizawai. PMID:28153898

  19. [Bioconversion of sewage sludge to biopesticide by Bacillus thuringiensis].

    PubMed

    Chang, Ming; Zhou, Shun-gui; Lu, Na; Ni, Jin-ren

    2006-07-01

    Feasibility of bioconversion of sewage sludge to biopesticide by Bacillus thuringiensis was studied using sewage sludge as a raw material. The fermentation was also compared with conventional medium. Results showed that without any pretreatment, the nutrients contained in sewage sludge were almost sufficient for Bacillus thuringiensis growth, even with a rapid multiplicational rate. Higher viable cells and viable spores values were obtained earlier at 24 h, with 9.48 x 10(8) CFU x mL(-1) and 8.51 x 10(8) CFU x mL(-1) respectively, which was 12 hours earlier and nearly 20 percent higher than conventional medium. SEM of 36 h samples gave a clear phenomenon that the metabolizability in sludge was much faster with spores and crystals spreading around. The crystals in sludge seemed rather bigger and more regular. Also a better crystal protein yield of 2.80 mg x mL(-1) was observed in sludge medium compared to conventional medium at the end of fermentation. Sludge fermentation for Bacillus thuringiensis reduces the producing cost, and gives better fermentation capabilities. It's expected to be a new method for sludge disposal.

  20. Expression of a Bacillus thuringiensis delta-endotoxin gene by Bacillus pumilus.

    PubMed

    Selinger, L B; Khachatourians, G G; Byers, J R; Hynes, M F

    1998-03-01

    The delta-endotoxin genes from Bacillus thuringiensis were introduced into a rhizosphere-inhabiting Bacillus pumilus isolate to create a delta-endotoxin expression and delivery system for subterranean feeding insects such as the larvae of pale western cutworm (Agrotis orthogonia Morrison (Lepidoptera: Noctuidae)). Preliminary experiments indicated that Bacillus thuringiensis subsp. kurstaki cultures were toxic to pale western cutworm larvae. Three different cry genes from Bacillus thuringiensis subsp. kurstaki were cloned into high and low copy number vectors and mated into Bacillus pumilus RB8. When carried on high copy number vectors, cry genes appeared to inhibit sporulation and delta-endotoxin production in Bacillus pumilus RB8 cultures, since microscopic examination of these cultures revealed that < 0.1% of the cells of late stationary phase cultures had sporulated and produced parasporal inclusions. On low copy number vectors, the cry genes did not inhibit sporulation; however, production of delta-endotoxins was undetectable. Using a heat shock regime for enrichment of sporogenous crystalliferous variants, a Bacillus pumilus isolate, carrying cryIA(c) on a high copy number plasmid, was obtained in which high level delta-endotoxin production occurred concomitant with sporulation. Synthesis of functional delta-endotoxin by this strain was confirmed by Western blot analysis and bioassay with pale western cutworm larvae. These results show that rhizosphere-inhabiting bacilli are indeed a potential route for introduction of delta-endotoxins to the root environment for biocontrol purposes.

  1. 40 CFR 174.529 - Bacillus thuringiensis modified Cry1Ab protein as identified under OECD Unique Identifier SYN...

    Code of Federal Regulations, 2010 CFR

    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...

  2. 40 CFR 174.502 - Bacillus thuringiensis Cry1A.105 protein; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

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

  3. 40 CFR 174.517 - Bacillus thuringiensis Cry9C protein in corn; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

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

  4. 40 CFR 174.509 - Bacillus thuringiensis Cry3A protein; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

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

  5. 40 CFR 174.509 - Bacillus thuringiensis Cry3A protein; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

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

  6. 40 CFR 174.509 - Bacillus thuringiensis Cry3A protein; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

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

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

    Code of Federal Regulations, 2013 CFR

    2013-07-01

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

  8. 40 CFR 174.509 - Bacillus thuringiensis Cry3A protein; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  10. Transformation of Bacillus thuringiensis subsp. galleria protoplasts by plasmid pBC16.

    PubMed Central

    Alikhanian, S I; Ryabchenko, N F; Bukanov, N O; Sakanyan, V A

    1981-01-01

    Protoplasts of the entomopathogenic bacterium Bacillus thuringiensis subsp. galleria were transformed by plasmid pBC16. The frequency of transformation was much lower than that of Bacillus subtilis. All isolated B. thuringiensis transformants were characterized by increased sensitivity to lysozyme as compared with the original strain. Images PMID:7217007

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

    PubMed Central

    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; Pennacchio, Francesco

    2016-01-01

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

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

    PubMed

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

    2016-08-23

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

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

    PubMed

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

    2014-12-01

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

  14. Phage Display of a Biologically Active Bacillus thuringiensis Toxin

    PubMed Central

    Kasman, Laura M.; Lukowiak, Andrew A.; Garczynski, Stephen F.; McNall, Rebecca J.; Youngman, Phil; Adang, Michael J.

    1998-01-01

    Activated forms of Bacillus thuringiensis insecticidal toxins have consistently been found to form insoluble and inactive precipitates when they are expressed in Escherichia coli. Genetic engineering of these proteins to improve their effectiveness as biological pesticides would be greatly facilitated by the ability to express them in E. coli, since the molecular biology tools available for Bacillus are limited. To this end, we show that activated B. thuringiensis toxin (Cry1Ac) can be expressed in E. coli as a translational fusion with the minor phage coat protein of filamentous phage. Phage particles displaying this fusion protein were viable, infectious, and as lethal as pure toxin on a molar basis when the phage particles were fed to insects susceptible to native Cry1Ac. Enzyme-linked immunosorbent assay and Western blot analysis showed the fusion protein to be antigenically equivalent to native toxin, and micropanning with anti-Cry1Ac antibody was positive for the toxin-expressing phage. Phage display of B. thuringiensis toxins has many advantages over previous expression systems for these proteins and should make it possible to construct large libraries of toxin variants for screening or biopanning. PMID:9687463

  15. Multilocus sequence analysis of Bacillus thuringiensis serovars navarrensis, bolivia and vazensis and Bacillus weihenstephanensis reveals a common phylogeny.

    PubMed

    Soufiane, Brahim; Baizet, Mathilde; Côté, Jean-Charles

    2013-01-01

    The Bacillus cereus group sensu lato includes six closely-related bacterial species: Bacillus cereus, Bacillus anthracis, Bacillus thuringiensis, Bacillus mycoides, Bacillus pseudomycoides and Bacillus weihenstephanensis. B. thuringiensis is distinguished from the other species mainly by the appearance of an inclusion body upon sporulation. B. weihenstephanensis is distinguished based on its psychrotolerance and the presence of specific signature sequences in the 16S rRNA gene and cspA genes. A total of seven housekeeping genes (glpF, gmK, ilvD, pta, purH, pycA and tpi) from different B. thuringiensis serovars and B. weihenstephanensis strains were amplified and their nucleotide sequences determined. A maximum likelihood phylogenetic tree was inferred from comparisons of the concatenated sequences. B. thuringiensis serovars navarrensis, bolivia and vazensis clustered not with the other B. thuringiensis serovars but rather with the B. weihenstephanensis strains, indicative of a common phylogeny. In addition, specific signature sequences and single nucleotide polymorphisms common to B. thuringiensis serovars navarrensis, bolivia and vazensis and the B. weihenstephanensis strains, and absent in the other B. thuringiensis serovars, were identified.

  16. Characterization of chimeric Bacillus thuringiensis Vip3 toxins.

    PubMed

    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.

  17. Construction of Bacillus thuringiensis simulant strains suitable for environmental release.

    PubMed

    Park, Sangjin; Kim, Changhwan; Lee, Daesang; Song, Dong Hyun; Cheon, Ki Cheol; Lee, Hong Suk; Kim, Seong Joo; Kim, Jee Cheon; Lee, Sang Yup

    2017-03-03

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

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

    PubMed

    Gomaa, Eman Zakaria

    2012-02-01

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

  19. [Growth and development kinetics of Bacillus thuringiensis in batch culture].

    PubMed

    Sakharova, Z V; Ignatenko, Iu N; Schulz, F; Khovrychev, M P; Rabotnova, I L

    1985-01-01

    The kinetics of Bacillus thuringiensis growth and its assimilation of nutrient substances were studied under the conditions of batch cultivation in a complex medium containing yeast extract and in a chemically defined medium with amino acids. The growth of B. thuringiensis can be divided into five phases: exponential growth; decelerated growth; stationary phase when protein crystals are formed; stationary phase when spores are formed; lysis of sporangia releasing spores. The first phase may in turn be subdivided into three stages according to changes in the specific growth rate and substrate assimilation: a high specific growth rate and no glucose assimilation; an abrupt drop in mu and the beginning of intensive glucose assimilation from the medium; a new rise in the specific growth rate. As follows from the results of studying the kinetics of B. thuringiensis growth in a chemically defined medium, the above changes in the exponential growth phase are due to the fact that the culture assimilates yeast extract components in the complex medium or amino acids in the chemically defined medium during this phase, and then starts to assimilate glucose and ammonium in the following phases of growth.

  20. The Bacillus thuringiensis insecticidal toxin binds biotin-containing proteins.

    PubMed Central

    Du, C; Nickerson, K W

    1996-01-01

    Brush border membrane vesicles from larvae of the tobacco hornworm, Manduca sexta, contain protein bands of 85 and 120 kDa which react directly with streptavidin conjugated to alkaline phosphatase. The binding could be prevented either by including 10 microM biotin in the reaction mixture or by prior incubation of the brush border membrane vesicles with an activated 60- to 65-kDa toxin from Bacillus thuringiensis HD-73. The ability of B. thuringiensis toxins to recognize biotin-containing proteins was confirmed by their binding to pyruvate carboxylase, a biotin-containing enzyme, as well as to biotinylated ovalbumin and biotinylated bovine serum albumin but not to their nonbiotinylated counterparts. Activated HD-73 toxin also inhibited the enzymatic activity of pyruvate carboxylase. The biotin binding site is likely contained in domain III of the toxin. Two highly conserved regions within domain III are similar in sequence to the biotin binding sites of avidin, streptavidin, and a biotin-specific monoclonal antibody. In particular, block 4 of the B. thuringiensis toxin contains the YAS biotin-specific motif. On the basis of its N-terminal amino acid sequence, the 120-kDa biotin-containing protein is totally distinct from the 120-kDa aminopeptidase N reported to be a receptor for Cry1Ac toxin. PMID:8702286

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

    PubMed

    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.

  2. Molecular characterization of Bacillus thuringiensis strains from Argentina.

    PubMed

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

    2004-07-01

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

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

    PubMed Central

    Ben-Dov, Eitan

    2014-01-01

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

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

    PubMed

    Ben-Dov, Eitan

    2014-03-28

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

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

    SciTech Connect

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

    2005-02-18

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

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

    PubMed

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

    2015-06-01

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

  7. Bacillus thuringiensis and Bacillus weihenstephanensis Inhibit the Growth of Phytopathogenic Verticillium Species.

    PubMed

    Hollensteiner, Jacqueline; Wemheuer, Franziska; Harting, Rebekka; Kolarzyk, Anna M; Diaz Valerio, Stefani M; Poehlein, Anja; Brzuszkiewicz, Elzbieta B; Nesemann, Kai; Braus-Stromeyer, Susanna A; Braus, Gerhard H; Daniel, Rolf; Liesegang, Heiko

    2016-01-01

    Verticillium wilt causes severe yield losses in a broad range of economically important crops worldwide. As many soil fumigants have a severe environmental impact, new biocontrol strategies are needed. Members of the genus Bacillus are known as plant growth-promoting bacteria (PGPB) as well as biocontrol agents of pests and diseases. In this study, we isolated 267 Bacillus strains from root-associated soil of field-grown tomato plants. We evaluated the antifungal potential of 20 phenotypically diverse strains according to their antagonistic activity against the two phytopathogenic fungi Verticillium dahliae and Verticillium longisporum. In addition, the 20 strains were sequenced and phylogenetically characterized by multi-locus sequence typing (MLST) resulting in 7 different Bacillus thuringiensis and 13 Bacillus weihenstephanensis strains. All B. thuringiensis isolates inhibited in vitro the tomato pathogen V. dahliae JR2, but had only low efficacy against the tomato-foreign pathogen V. longisporum 43. All B. weihenstephanensis isolates exhibited no fungicidal activity whereas three B. weihenstephanensis isolates showed antagonistic effects on both phytopathogens. These strains had a rhizoid colony morphology, which has not been described for B. weihenstephanensis strains previously. Genome analysis of all isolates revealed putative genes encoding fungicidal substances and resulted in identification of 304 secondary metabolite gene clusters including 101 non-ribosomal polypeptide synthetases and 203 ribosomal-synthesized and post-translationally modified peptides. All genomes encoded genes for the synthesis of the antifungal siderophore bacillibactin. In the genome of one B. thuringiensis strain, a gene cluster for zwittermicin A was detected. Isolates which either exhibited an inhibitory or an interfering effect on the growth of the phytopathogens carried one or two genes encoding putative mycolitic chitinases, which might contribute to antifungal activities

  8. Bacillus thuringiensis and Bacillus weihenstephanensis Inhibit the Growth of Phytopathogenic Verticillium Species

    PubMed Central

    Hollensteiner, Jacqueline; Wemheuer, Franziska; Harting, Rebekka; Kolarzyk, Anna M.; Diaz Valerio, Stefani M.; Poehlein, Anja; Brzuszkiewicz, Elzbieta B.; Nesemann, Kai; Braus-Stromeyer, Susanna A.; Braus, Gerhard H.; Daniel, Rolf; Liesegang, Heiko

    2017-01-01

    Verticillium wilt causes severe yield losses in a broad range of economically important crops worldwide. As many soil fumigants have a severe environmental impact, new biocontrol strategies are needed. Members of the genus Bacillus are known as plant growth-promoting bacteria (PGPB) as well as biocontrol agents of pests and diseases. In this study, we isolated 267 Bacillus strains from root-associated soil of field-grown tomato plants. We evaluated the antifungal potential of 20 phenotypically diverse strains according to their antagonistic activity against the two phytopathogenic fungi Verticillium dahliae and Verticillium longisporum. In addition, the 20 strains were sequenced and phylogenetically characterized by multi-locus sequence typing (MLST) resulting in 7 different Bacillus thuringiensis and 13 Bacillus weihenstephanensis strains. All B. thuringiensis isolates inhibited in vitro the tomato pathogen V. dahliae JR2, but had only low efficacy against the tomato-foreign pathogen V. longisporum 43. All B. weihenstephanensis isolates exhibited no fungicidal activity whereas three B. weihenstephanensis isolates showed antagonistic effects on both phytopathogens. These strains had a rhizoid colony morphology, which has not been described for B. weihenstephanensis strains previously. Genome analysis of all isolates revealed putative genes encoding fungicidal substances and resulted in identification of 304 secondary metabolite gene clusters including 101 non-ribosomal polypeptide synthetases and 203 ribosomal-synthesized and post-translationally modified peptides. All genomes encoded genes for the synthesis of the antifungal siderophore bacillibactin. In the genome of one B. thuringiensis strain, a gene cluster for zwittermicin A was detected. Isolates which either exhibited an inhibitory or an interfering effect on the growth of the phytopathogens carried one or two genes encoding putative mycolitic chitinases, which might contribute to antifungal activities

  9. Persistence of Bacillus thuringiensis subsp. kurstaki in Urban Environments following Spraying▿†‡

    PubMed Central

    Van Cuyk, Sheila; Deshpande, Alina; Hollander, Attelia; Duval, Nathan; Ticknor, Lawrence; Layshock, Julie; Gallegos-Graves, LaVerne; Omberg, Kristin M.

    2011-01-01

    Bacillus thuringiensis subsp. kurstaki is applied extensively in North America to control the gypsy moth, Lymantria dispar. Since B. thuringiensis subsp. kurstaki shares many physical and biological properties with Bacillus anthracis, it is a reasonable surrogate for biodefense studies. A key question in biodefense is how long a biothreat agent will persist in the environment. There is some information in the literature on the persistence of Bacillus anthracis in laboratories and historical testing areas and for Bacillus thuringiensis in agricultural settings, but there is no information on the persistence of Bacillus spp. in the type of environment that would be encountered in a city or on a military installation. Since it is not feasible to release B. anthracis in a developed area, the controlled release of B. thuringiensis subsp. kurstaki for pest control was used to gain insight into the potential persistence of Bacillus spp. in outdoor urban environments. Persistence was evaluated in two locations: Fairfax County, VA, and Seattle, WA. Environmental samples were collected from multiple matrices and evaluated for the presence of viable B. thuringiensis subsp. kurstaki at times ranging from less than 1 day to 4 years after spraying. Real-time PCR and culture were used for analysis. B. thuringiensis subsp. kurstaki was found to persist in urban environments for at least 4 years. It was most frequently detected in soils and less frequently detected in wipes, grass, foliage, and water. The collective results indicate that certain species of Bacillus may persist for years following their dispersal in urban environments. PMID:21926205

  10. Activity of Bacillus thuringiensis against Pryeria sinica(Lepidoptera: Zygaenidae), an invasive pest of Euonymus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Pryeria sinica Moore (Lepidoptera: Zygaenidae), an invasive pest of Euonymus, is susceptible in the second instar to the Bacillus thuringiensis Berliner product Thuricide®, and to several strains isolated from other B. thuringiensis products. Third instars are also susceptible, while susceptibility...

  11. Draft genome sequence of Bacillus thuringiensis strain DAR 81934, which exhibits molluscicidal activity.

    PubMed

    Wang, Aisuo; Pattemore, Julie; Ash, Gavin; Williams, Angela; Hane, James

    2013-03-21

    Bacillus thuringiensis has been widely used as a biopesticide for a long time. Its molluscicidal activity, however, is rarely realized. Here, we report the genome sequence of B. thuringiensis strain DAR 81934, a strain with molluscicidal activity against the pest snail Cernuella virgata.

  12. Draft Genome Sequence of Bacillus thuringiensis NBIN-866 with High Nematocidal Activity

    PubMed Central

    Zhou, Ronghua; Fu, Guiping; Zhang, Wei; Min, Yong; Tian, Yuxi; Huang, Daye; Wang, Kaimei; Wan, Zhongyi; Yao, Jingwu; Yang, Ziwen

    2014-01-01

    Bacillus thuringiensis NBIN-866, a Gram-positive bacterium, was isolated from soil in China. We announce here the draft genome sequence of strain B. thuringiensis NBIN-866, which possesses highly nematocidal factors, such as proteins and small molecular peptides. PMID:24855295

  13. 40 CFR 180.1108 - Delta endotoxin of Bacillus thuringiensis variety San Diego encapsulated into killed Pseudomonas...

    Code of Federal Regulations, 2010 CFR

    2010-07-01

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

  14. 77 FR 6471 - Bacillus thuringiensis Cry2Ae Protein in Cotton; Exemption from the Requirement of a Tolerance

    Federal Register 2010, 2011, 2012, 2013, 2014

    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...

  15. 40 CFR 174.520 - Bacillus thuringiensis Cry1F protein in corn; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

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

  16. 40 CFR 174.530 - Bacillus thuringiensis Cry2Ae protein in cotton; temporary exemption from the requirement of a...

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 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,...

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

    USGS Publications Warehouse

    Zhioua, Elyes; Heyer, Klaus; Browning, M.; Ginsberg, Howard S.; LeBrun, Roger A.

    1999-01-01

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

  18. Bacillus thuringiensis HCB6 Amylase Immobilization by Chitosan Beads

    NASA Astrophysics Data System (ADS)

    Zusfahair; Ningsih, D. R.; Kartika, D.; Fatoni, A.; Zuliana, A. L.

    2017-02-01

    The purpose of this study was to optimize the amylase immobilization using a chitosan bead and to characterize immobilized amylase of Bacillus thuringiensis Bacteria HCB6. This study was started of amylase production, continued by immobilization optimization including ratio of chitosan:enzymes, enzyme-matrix contact time, substrate concentration, pH effect, incubation temperature effect, reaction time, and stability of immobilized enzyme. Amylase activity assay was dinitro salicylic (DNS) method. The results showed the optimum chitosan:enzyme ratio was 2.5: 1 (v/v), immobilization contact time of 18 hours and immobilization efficiency of 87.93%. Furthermore, immobilized amylase of B. thuringiensis HCB6 showed optimum substrate concentration of 1.5%, optimum pH of 6, optimum incubation temperature of 37 ° C, and the reaction time of 30 minutes. The Michaelis-Menten constant KM value for free and immobilized amylase were 5.30% and 1.33% respectively. Immobilized amylase can be used up to five times with the remaining activity of 43.3%.

  19. Bacillus thuringiensis: a specific gamma-cyclodextrin producer strain.

    PubMed

    Goo, Bon Geun; Hwang, You Jin; Park, Jae Kweon

    2014-03-11

    An anaerobic microbial isolate Bacillus species, designated B. thuringiensis GU-2, was isolated from soil as a specific γ-cyclodextrin (CD) producer strain in alkaline medium under anaerobic conditions. The optimum pH and temperature for bacterial growth and γ-CD production were estimated to be pH 8.5 and 37°C in the presence of 1.0% starch substrate, respectively. A high purity yield >95% of γ-CD from the total CD yield in the reaction mixture was obtained from starch that was supposed to be converted by gamma-cyclodextrin glycotransferase, tentatively named as γ-CGTase. The maximum γ-CGTase activity was estimated at 2.45U/mL under optimized condition. This is the first report demonstrating the generation of a specific γ-cyclodextrin (CD) producer strain by the action of a γ-CGTase under anaerobic conditions.

  20. Mode of action of mosquitocidal Bacillus thuringiensis toxins.

    PubMed

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

    2007-04-01

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

  1. Novel fermentation media for production of Bacillus thuringiensis subsp. israelensis.

    PubMed

    Poopathi, Subbiah; Kumar, K Anup

    2003-08-01

    The production of Bacillus thuringiensis subsp. israelensis (deBarjac) (Bti) as a biopesticide is not cost-effective using existing fermentation technology. In this study, we explored the use of several less expensive alternative culture media (potato, common sugar, and Bengal gram) for the growth and production of Bti. Growth was obtained in all tested media and was comparable to that obtained in conventional medium (Luria-Bertani). Toxicity assays showed that the toxin produced from the novel growth media were effective in killing larvae of Culex quinquefasciatus, Anopheles stephensi, and Aedes aegypti and toxicity was comparable to that produced from Luria-Bertani medium. These observations suggest that potato can be used as a cheap source of culture medium for the production of Bti toxin in mosquito control programs.

  2. Endospore degradation in an oligosporogenic, crystalliferous mutant of Bacillus thuringiensis.

    PubMed

    Sierra-Martínez, Pável; Ibarra, Jorge E; de la Torre, Mayra; Olmedo, Gabriela

    2004-02-01

    We isolated a new oligosporogenic mutant from Bacillus thuringiensis var. kurstaki HD73 that retains the ability to produce insecticidal crystal inclusions. Sporulation in this mutant initiates in a manner similar to the wild-type strain, and under the electron microscope endospores are seen, but these do not reach maturity (except for 0.2% of them). At a late stage, the coat surrounding the forespore seems to lack shape and to be empty. Most mutant cells exhibit a well-formed bipyramidal crystal but are completely devoid of the forespore. The mutant has a functional SigK holoenzyme, which is required for the expression of genes involved in the formation of spore coat and cortex and for cry1A transcription from the BtII promoter. Defective maturation of spores could be due to an inadequate forespore coat or cortex structure resulting in the arrest of sporulation at late stage III or early stage IV.

  3. Batch fermentation and optimization of media for Bacillus thuringiensis.

    PubMed

    Guan, X; Chen, J; Huang, Z; Tang, Y; Gao, R

    1998-01-01

    The composition of No. II medium obtained with shaking cultivation contained three factors: nitrogen source, carbon source, and inorganic salts. The relationship between component factors (x(i)) of the media and spore numbers (y) of Bacillus thuringiensis (Bt) was demonstrated by the orthogonal-rotation-combination test. A response surface equation was formed as follows: y = 384 - 7.245x1 + 11.705x2 + 15.475x3 + 14.039x1(2) + 41.831x2(2) - 79.49x3(2) - 35.375x1x2 - 3.375x1x3 - 106.625x2x3. The results showed that this method is simple, practical, and rapid enough for selecting fermentation media for Bt. In addition, the whole course of batch fermentation was also investigated.

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

    NASA Astrophysics Data System (ADS)

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

    2012-02-01

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

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

    PubMed

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

    2016-04-01

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

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

    PubMed

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

    2015-12-28

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

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

    PubMed

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

    2011-09-01

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

  8. 40 CFR 180.1107 - Delta endotoxin of Bacillus thuringiensis variety kurstaki encapsulated into killed Pseudomonas...

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 24 2011-07-01 2011-07-01 false Delta endotoxin of Bacillus... From Tolerances § 180.1107 Delta endotoxin of Bacillus thuringiensis variety kurstaki encapsulated into killed Pseudomonas fluorescens; exemption from the requirement of a tolerance. The delta endotoxin...

  9. 40 CFR 180.1107 - Delta endotoxin of Bacillus thuringiensis variety kurstaki encapsulated into killed Pseudomonas...

    Code of Federal Regulations, 2013 CFR

    2013-07-01

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

  10. 40 CFR 180.1107 - Delta endotoxin of Bacillus thuringiensis variety kurstaki encapsulated into killed Pseudomonas...

    Code of Federal Regulations, 2014 CFR

    2014-07-01

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

  11. 40 CFR 180.1107 - Delta endotoxin of Bacillus thuringiensis variety kurstaki encapsulated into killed Pseudomonas...

    Code of Federal Regulations, 2012 CFR

    2012-07-01

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

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

    PubMed Central

    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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  14. 77 FR 47287 - Bacillus thuringiensis eCry3.1Ab Protein in Corn; Exemption From the Requirement of a Tolerance

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-08-08

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

  15. Strategy for identification of Bacillus cereus and Bacillus thuringiensis strains closely related to Bacillus anthracis.

    PubMed

    Daffonchio, Daniele; Raddadi, Noura; Merabishvili, Maya; Cherif, Ameur; Carmagnola, Lorenzo; Brusetti, Lorenzo; Rizzi, Aurora; Chanishvili, Nina; Visca, Paolo; Sharp, Richard; Borin, Sara

    2006-02-01

    Bacillus cereus strains that are genetically closely related to B. anthracis can display anthrax-like virulence traits (A. R. Hoffmaster et al., Proc. Natl. Acad. Sci. USA 101:8449-8454, 2004). Hence, approaches that rapidly identify these "near neighbors" are of great interest for the study of B. anthracis virulence mechanisms, as well as to prevent the use of such strains for B. anthracis-based bioweapon development. Here, a strategy is proposed for the identification of near neighbors of B. anthracis based on single nucleotide polymorphisms (SNP) in the 16S-23S rRNA intergenic spacer (ITS) containing tRNA genes, characteristic of B. anthracis. By using restriction site insertion-PCR (RSI-PCR) the presence of two SNP typical of B. anthracis was screened in 126 B. cereus group strains of different origin. Two B. cereus strains and one B. thuringiensis strain showed RSI-PCR profiles identical to that of B. anthracis. The sequencing of the entire ITS containing tRNA genes revealed two of the strains to be identical to B. anthracis. The strict relationship with B. anthracis was confirmed by multilocus sequence typing (MLST) of four other independent loci: cerA, plcR, AC-390, and SG-749. The relationship to B. anthracis of the three strains described by MLST was comparable and even higher to that of four B. cereus strains associated with periodontitis in humans and previously reported as the closest known strains to B. anthracis. SNP in ITS containing tRNA genes combined with RSI-PCR provide a very efficient tool for the identification of strains closely related to B. anthracis.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  18. Discovery of crystalline inclusions in Bacillus licheniformis that resemble parasporal crystals of Bacillus thuringiensis.

    PubMed

    Yan, Ming; Roehrl, Michael H; Wang, Julia Y

    2007-09-01

    Crystalline inclusions were discovered in stationary and sporulating cells of the spore-forming bacterium Bacillus licheniformis ATCC 9945a. As detected by electron microscopy, dying or sporulating bacterial cells contain a single crystal of strikingly large size. The crystals in sporulating cells are located next to nascent spores and can be several times larger than the spores. Morphologically, most crystals are rhomboid with uniformly spaced grids. These newly discovered crystalline inclusions of B. licheniformis closely resemble parasporal crystals of Bacillus thuringiensis that are formed by insecticidal toxin proteins and used widely as biopesticides. The taxonomic identity of this strain was verified by its 16S rRNA gene sequence and its fatty acid profile. The finding of crystal proteins in B. licheniformis may lead to the discovery of new protein toxins and may expand our pool of biopesticides.

  19. 40 CFR 174.504 - Bacillus thuringiensis Cry1F protein in cotton; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 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...

  20. Draft Genome Sequence of Bacillus thuringiensis var. thuringiensis Strain T01-328, a Brazilian Isolate That Produces a Soluble Pesticide Protein, Cry1Ia

    PubMed Central

    Varani, Alessandro M.; Fernandes, Camila C.; Lemos, Eliana G. M.; Alves, Eliane C. C.; Desidério, Janete A.

    2013-01-01

    Bacillus thuringiensis var. thuringiensis strain T01-328, isolated from Cubatão county (São Paulo State, Brazil), produces a soluble pesticide protein, Cry1Ia, during vegetative growth. Here, we report the 7.089-Mbp draft genome sequence, composed of a 5.5-Mb chromosome and 14 plasmids, which is the largest B. thuringiensis genome sequenced to date. PMID:24115545

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2016-02-01

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

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

    PubMed

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

    2016-02-10

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

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

    PubMed

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

    2014-02-10

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

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

    PubMed

    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.

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

    PubMed Central

    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. Is the Insect World Overcoming the Efficacy of Bacillus thuringiensis?

    PubMed Central

    Peralta, Cecilia; Palma, Leopoldo

    2017-01-01

    The use of chemical pesticides revolutionized agriculture with the introduction of DDT (Dichlorodiphenyltrichloroethane) as the first modern chemical insecticide. However, the effectiveness of DDT and other synthetic pesticides, together with their low cost and ease of use, have led to the generation of undesirable side effects, such as pollution of water and food sources, harm to non-target organisms and the generation of insect resistance. The alternative comes from biological control agents, which have taken an expanding share in the pesticide market over the last decades mainly promoted by the necessity to move towards more sustainable agriculture. Among such biological control agents, the bacterium Bacillus thuringiensis (Bt) and its insecticidal toxins have been the most studied and commercially used biological control agents over the last 40 years. However, some insect pests have acquired field-evolved resistance to the most commonly used Bt-based pesticides, threatening their efficacy, which necessitates the immediate search for novel strains and toxins exhibiting different modes of action and specificities in order to perpetuate the insecticidal potential of this bacterium. PMID:28106770

  8. Evolution of Bacillus thuringiensis Cry toxins insecticidal activity.

    PubMed

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

    2013-01-01

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

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

    PubMed

    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.

  10. Is the Insect World Overcoming the Efficacy of Bacillus thuringiensis?

    PubMed

    Peralta, Cecilia; Palma, Leopoldo

    2017-01-18

    The use of chemical pesticides revolutionized agriculture with the introduction of DDT (Dichlorodiphenyltrichloroethane) as the first modern chemical insecticide. However, the effectiveness of DDT and other synthetic pesticides, together with their low cost and ease of use, have led to the generation of undesirable side effects, such as pollution of water and food sources, harm to non-target organisms and the generation of insect resistance. The alternative comes from biological control agents, which have taken an expanding share in the pesticide market over the last decades mainly promoted by the necessity to move towards more sustainable agriculture. Among such biological control agents, the bacterium Bacillus thuringiensis (Bt) and its insecticidal toxins have been the most studied and commercially used biological control agents over the last 40 years. However, some insect pests have acquired field-evolved resistance to the most commonly used Bt-based pesticides, threatening their efficacy, which necessitates the immediate search for novel strains and toxins exhibiting different modes of action and specificities in order to perpetuate the insecticidal potential of this bacterium.

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

    PubMed

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

    2014-12-11

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

  12. Role of Receptors in Bacillus thuringiensis Crystal Toxin Activity

    PubMed Central

    Pigott, Craig R.; Ellar, David J.

    2007-01-01

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

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

    PubMed

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

    2015-11-01

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

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

    PubMed

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

    2001-10-09

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

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

    PubMed Central

    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

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

    PubMed

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

    2014-09-01

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

  17. Bacillus thuringiensis: A story of a successful bioinsecticide.

    PubMed

    Bravo, Alejandra; Likitvivatanavong, Supaporn; Gill, Sarjeet S; Soberón, Mario

    2011-07-01

    Bacillus thuringiensis (Bt) bacteria are insect pathogens that rely on insecticidal pore forming proteins known as Cry and Cyt toxins to kill their insect larval hosts. At least four different non-structurally related families of proteins form the Cry toxin group of toxins. The expression of certain Cry toxins in transgenic crops has contributed to an efficient control of insect pests resulting in a significant reduction in chemical insecticide use. The mode of action of the three domain Cry toxin family involves sequential interaction of these toxins with several insect midgut proteins facilitating the formation of a pre-pore oligomer structure and subsequent membrane insertion that leads to the killing of midgut insect cells by osmotic shock. In this manuscript we review recent progress in understanding the mode of action of this family of proteins in lepidopteran, dipteran and coleopteran insects. Interestingly, similar Cry-binding proteins have been identified in the three insect orders, as cadherin, aminopeptidase-N and alkaline phosphatase suggesting a conserved mode of action. Also, recent data on insect responses to Cry toxin attack is discussed. Finally, we review the different Bt based products, including transgenic crops, that are currently used in agriculture.

  18. Raman spectroscopy of Bacillus thuringiensis physiology and inactivation

    NASA Astrophysics Data System (ADS)

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

    2012-12-01

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

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

    SciTech Connect

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

    2010-01-01

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

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

    PubMed

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

    2016-05-05

    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.

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

    PubMed Central

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

    2014-01-01

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

  2. Evolution of Bacillus thuringiensis Cry toxins insecticidal activity

    PubMed Central

    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. PMID:22463726

  3. Novel Vip3-related protein from Bacillus thuringiensis.

    PubMed

    Rang, Cécile; Gil, Patricia; Neisner, Nathalie; Van Rie, Jeroen; Frutos, Roger

    2005-10-01

    A novel vip3-related gene was identified in Bacillus thuringiensis. This novel gene is 2,406 bp long and codes for a 91-kDa protein (801 amino acids). This novel protein exhibits between 61 and 62% similarity with Vip3A proteins and is designated Vip3Ba1. Vip3Ba1 has several specific features. Differences between Vip3Ba1 and the Vip3A proteins are spread throughout the sequence but are more frequent in the C-terminal part from amino acid 456 onward. The regions containing the two proteolytic processing sites, which are highly conserved among the Vip3A toxins, are markedly different in Vip3Ba1. The pattern DCCEE (Asp Cys Cys Glu Glu) is repeated four times between position 463 and 483 in Vip3Ba1, generating the sequence 463-DCCEEDCCEEDCCEEDCCEE-483. This sequence, which is rich in negatively charged amino acids, also contains 73% of the cysteines present in Vip3Ba1. This repeated sequence is not present in Vip3A proteins. The Vip3Ba1protein was produced in Escherichia coli and tested against Ostrinia nubilalis and Plutella xylostella, and it generated significant growth delays but had no larvicidal effect, indicating that its host range might be different than that of Vip3A proteins.

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

    PubMed

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

    2015-06-01

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

  5. Bacillus thuringiensis: fermentation process and risk assessment. A short review.

    PubMed

    Capalbo, D M

    1995-01-01

    Several factors make the local production of Bacillus thuringiensis (Bt) highly appropriate for pest control in developing nations. Bt can be cheaply produced on a wide variety of low cost, organic substrates. Local production results in considerable savings in hard currency which otherwise would be spent on importation of chemical and biological insecticides. The use of Bt in Brazil has been limited in comparison with chemical insecticides. Although Bt is imported, some Brazilian researchers have been working on its development and production. Fermentation processes (submerged and semi-solid) were applied, using by-products from agro-industries. As the semi-solid fermentation process demonstrated to be interesting for Bt endotoxins production, it could be adopted for small scale local production. Although promising results had been achieved, national products have not been registered due to the absence of a specific legislation for biological products. Effective actions are being developed in order to solve this gap. Regardless of the biocontrol agents being considered atoxic and harmless to the environment, information related to direct and indirect effects of microbials are still insufficient in many cases. The risk analysis of the use of microbial control agents is of upmost importance nowadays, and is also discussed.

  6. Starch industry wastewater-based stable Bacillus thuringiensis liquid formulations.

    PubMed

    Brar, Satinder K; Verma, M; Tyagi, R D; Valéro, J R; Surampalli, R Y

    2005-12-01

    Liquid formulations were developed from Bacillus thuringiensis (Bt)-fermented broths of starch industry wastewater (SIW) and of soya medium. Stability studies were carried out for 1 yr. Storage stability was tested by studying various physical and chemical (e.g., viscosity, particle size, corrosion, and suspendibility) and biological (e.g., microbial contamination, viable spores, and entomotoxicity) parameters at different pH levels and temperatures. Three suspending agents, sorbitol, sodium monophosphate, and sodium metabisulfite, were added to fermented broth in different concentrations. Sorbitol and sodium monophosphate in the ratio 3:1 was the best suspending agent combination for both formulations. Starch industry wastewater fermentation yielded cell and viable spore counts 10- and 4-fold greater than those from soya medium, respectively, and a 1.7-fold increase in entomotoxicity. However, both formulations started deteriorating at pH 6 and 6.5 and 40 and 50 degrees C. There were no signs of corrosion and microbial contamination in both types of formulations.

  7. Bacteriocin-like inhibitor substances produced by Mexican strains of Bacillus thuringiensis.

    PubMed

    Barboza-Corona, J Eleazar; Vázquez-Acosta, Herminia; Bideshi, Dennis K; Salcedo-Hernández, Rubén

    2007-02-01

    Bacteriocins are antimicrobial peptides synthesized and secreted by bacteria and could potentially be used as natural food preservatives. Here, we report the production of bacteriocin-like inhibitor substances (Bt-BLIS) by five Mexican strains of Bacillus thuringiensis. Bacillus thuringiensis subsp. morrisoni (LBIT 269), B. thuringiensis subsp. kurstaki (LBIT 287), B. thuringiensis subsp kenyae (LBIT 404), B. thuringiensis subsp. entomocidus (LBIT 420) and B. thuringiensis subsp. tolworthi (LBIT 524) produced proteinaceous Bt-BLIS with high levels of activity against Bacillus cereus and other gram-positive bacteria. Although none was active against the gram-negative bacteria, Escherichia coli, Shigella species and Pseudomonas aeruginosa, the five Bt-BLIS demonstrated antimicrobial activity against Vibrio cholerae, the etiologic agent of cholera. Biochemical and biophysical studies demonstrated that the five Bt-BLIS could be categorized into two groups, those produced by LBIT 269 and 287 (Group A) and LBIT 404, 420, 524 (Group B), based on relative time of peptide synthesis, distinctive bacterial target specificity and stability in a wide range of temperatures and pH. Because of their stability and bactericidal activities against B. cereus and V. cholerae agents of emetic, diarrheal and lethal syndromes in humans, these Bt-BLIS could potentially be used as biodegradable preservatives in the food industry.

  8. The role of a purine-specific nucleoside hydrolase in spore germination of Bacillus thuringiensis.

    PubMed

    Liang, Liang; He, Xihong; Liu, Gang; Tan, Huarong

    2008-05-01

    A homologous gene (iunH) of a putative nucleoside hydrolase (NH), which had been identified from the exosporia of Bacillus cereus and Bacillus anthracis spores, was cloned from Bacillus thuringiensis subsp. kurstaki. Disruption of iunH did not affect the vegetative growth and sporulation of Bacillus thuringiensis, but promoted both inosine- and adenosine-induced spore germination. The inosine- or adenosine-induced germination rate decreased when the wild-type iunH gene was overexpressed in Bacillus thuringiensis. The iunH gene product was characterized as a purine-specific NH. The kinetic parameters of IunH with inosine as substrate were K(m)=399+/-115 microM, k(cat)=48.9+/-8.5 s(-1) and k(cat)/K(m)=1.23 x 10(5) M(-1) s(-1). The optimal pH and temperature for IunH were found to be pH 6 and 80 degrees C. Meanwhile, the specific activity of inosine hydrolase in intact spores of the wild-type strain with inosine as substrate was 2.89+/-0.23x10(-2) micromol min(-1) (mg dry wt)(-1). These results indicate that IunH is important in moderating inosine- or adenosine-induced germination of Bacillus thuringiensis spores.

  9. Synergy between toxins of Bacillus thuringiensis subsp. israelensis and Bacillus sphaericus.

    PubMed

    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.

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

    PubMed Central

    Luckevich, M D; Beveridge, T J

    1989-01-01

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

  11. Production of molybdenum-coordinating compound by Bacillus thuringiensis.

    PubMed

    Ketchum, P A; Owens, M S

    1975-05-01

    Bacillus thuringiensis (ATCC 10792) produces a molybdenum reactive compound (given the trivial name chelin) during growth on iron-deficient medium. This compound accumulates in the culture medium in direct relation to the amount of L-arginine added and reaches a maximum concentration 24 to 48 h after the stationary phase of growth. Chelin absorbs light in the ultraviolet region with absorption maxima at 315 and 248 nm and minima at 284 and 240 nm. Chelin reacts with Na2MoO4, but not with Mo2O4(H2O)6-2+, to form a bright yellow molybdo-chelin complex which absorbs light with an absorption maximum at 330 nm, a minimum at 288 nm, and shoulders at 255 and 400 nm. The differential absorption of molybdo-chelin versus chelin at 425 nm can be used to quantify chelin. This differential absorbance is linear with increasing concentrations of Na2MoO4 and was used to calculate the molar extinction coefficient of molybdochelin at 425 nm (epsilon similar to 6,200). Chelin binds MoO4-2 minus to form a complex (molybdochelin) which migrates as a single band and elutes as a single peak, during acrylamide gel electrophoresis and Sephadex G-15 gel filtration. Molecular weight determinations using Sephadex G-15 gel filtration resulted in an estimated molecular weight of 550 for chelin and an estimated molecular weight of 760 for molybdo-chelin. The peptide nature of chelin is indicated by its positive ninhydrin reaction on thin-layer chromatography plates and by the presence of amino acids in acid-hydrolyzed samples. The major amino acid residues detected were threonine, glycine, and alanine.

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

    PubMed Central

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

    2016-01-01

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

  13. The surface layer protein of Bacillus thuringiensis CTC forms unique intracellular parasporal inclusion body.

    PubMed

    Zhu, Chenguang; Yu, Ziniu

    2008-08-01

    Bacillus thuringiensis subsp. finitimus strain CTC forms round parasporal inclusion body. The inclusion body protein gene ctc has been cloned and characterized. Sequence homology analysis reveals that the amino acid sequence of CTC protein shows 87% identity with the surface layer (S-layer) protein Sap (GenBank Z36946) in B. anthracis. In this report, transmission electron microscope observation showed that CTC formed intracellular parasporal inclusion body and sheet structure of S-layer-like protein at the spore phase. Furthermore, the ctc gene was transformed into an acrystalliferous B. thuringiensis strain BMB171. The resulting transformant could form parasporal body which had the same shape and molecular weight of protein with that of B. thuringiensis CTC. These results, together with the sequence homology analysis of ctc gene, confirmed that the unique intracellular parasporal inclusion body of B. thuringiensis was comprised of S-layer protein.

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

    PubMed

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

    2012-04-01

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

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

    PubMed Central

    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

  16. The occurrence of Photorhabdus-like toxin complexes in Bacillus thuringiensis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Recently, genomic sequencing of a Bacillus thuringiensis (Bt) isolate from our collection revealed the presence of an apparent operon encoding an insecticidal toxin complex (Tca) similar to that first described from the entomopathogen Photorhabdus luminescens. To determine whether these genes are w...

  17. 40 CFR 180.1011 - Viable spores of the microorganism Bacillus thuringiensis Berliner; exemption from the...

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 24 2014-07-01 2014-07-01 false Viable spores of the microorganism... microorganism Bacillus thuringiensis Berliner; exemption from the requirement of a tolerance. (a) For the... is being established shall have the following specifications: (1) The microorganism shall be...

  18. 40 CFR 180.1011 - Viable spores of the microorganism Bacillus thuringiensis Berliner; exemption from the...

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 24 2011-07-01 2011-07-01 false Viable spores of the microorganism... microorganism Bacillus thuringiensis Berliner; exemption from the requirement of a tolerance. (a) For the... is being established shall have the following specifications: (1) The microorganism shall be...

  19. 40 CFR 180.1011 - Viable spores of the microorganism Bacillus thuringiensis Berliner; exemption from the...

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 25 2013-07-01 2013-07-01 false Viable spores of the microorganism... microorganism Bacillus thuringiensis Berliner; exemption from the requirement of a tolerance. (a) For the... is being established shall have the following specifications: (1) The microorganism shall be...

  20. 40 CFR 180.1011 - Viable spores of the microorganism Bacillus thuringiensis Berliner; exemption from the...

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 25 2012-07-01 2012-07-01 false Viable spores of the microorganism... microorganism Bacillus thuringiensis Berliner; exemption from the requirement of a tolerance. (a) For the... is being established shall have the following specifications: (1) The microorganism shall be...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  2. Regulation by gut bacertia of immune response, Bacillus thuringiensis susceptibility and hemolin expression in Plodia interpunctella

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plodia interpunctella (Hübner) is an important stored grain insect pest worldwide, and the first lepidopteran with reported resistance to Bacillus thuringiensis (Bt) toxins. Since gut bacteria may affect Bt insecticidal activity, we determined whether P. interpunctella lacking gut enterobacteria had...

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

    ERIC Educational Resources Information Center

    Stalter, Richard; Nadal, Gerard; Kincaid, Dwight

    2000-01-01

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

  4. Fulminant phlegmonitis of the esophagus, stomach, and duodenum due to Bacillus thuringiensis.

    PubMed

    Matsumoto, Hisatake; Ogura, Hiroshi; Seki, Masafumi; Ohnishi, Mitsuo; Shimazu, Takeshi

    2015-03-28

    We report a case of phlegmonitis of the esophagus, stomach, and duodenum in patient in an immunocompromised state. Culture of gastric juice and blood yielded Bacillus thuringiensis. This case showed that even low-virulence bacilli can cause lethal gastrointestinal phlegmonous gastritis in conditions of immunodeficiency.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  6. Vip3C, a Novel Class of Vegetative Insecticidal Proteins from Bacillus thuringiensis

    PubMed Central

    Palma, Leopoldo; Hernández-Rodríguez, Carmen Sara; Maeztu, Mireya; Hernández-Martínez, Patricia; Ruiz de Escudero, Iñigo; Escriche, Baltasar; Muñoz, Delia; Van Rie, Jeroen; Ferré, Juan

    2012-01-01

    Three vip3 genes were identified in two Bacillus thuringiensis Spanish collections. Sequence analysis revealed a novel Vip3 protein class (Vip3C). Preliminary bioassays of larvae from 10 different lepidopteran species indicated that Vip3Ca3 caused more than 70% mortality in four species after 10 days at 4 μg/cm2. PMID:22865065

  7. An improved method for rapid generation and screening of Bacillus thuringiensis phage-resistant mutants.

    PubMed

    Gillis, Annika; Mahillon, Jacques

    2014-11-01

    A simple method to isolate, screen and select phage-resistant mutants of Bacillus thuringiensis was developed. The traditional double-layer agar method was improved by a combination of the spotting assay using a lytic phage, to generate the bacterial-resistant mutants, with an inverted spotting assay (ISA), to rapidly screen the candidate-resistant mutants.

  8. Vip3C, a novel class of vegetative insecticidal proteins from Bacillus thuringiensis.

    PubMed

    Palma, Leopoldo; Hernández-Rodríguez, Carmen Sara; Maeztu, Mireya; Hernández-Martínez, Patricia; Ruiz de Escudero, Iñigo; Escriche, Baltasar; Muñoz, Delia; Van Rie, Jeroen; Ferré, Juan; Caballero, Primitivo

    2012-10-01

    Three vip3 genes were identified in two Bacillus thuringiensis Spanish collections. Sequence analysis revealed a novel Vip3 protein class (Vip3C). Preliminary bioassays of larvae from 10 different lepidopteran species indicated that Vip3Ca3 caused more than 70% mortality in four species after 10 days at 4 μg/cm(2).

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

    EPA Science Inventory

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

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

    Code of Federal Regulations, 2011 CFR

    2011-07-01

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

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

    PubMed

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

    2015-09-28

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

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

    PubMed

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

    2014-04-01

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

  13. An anionic defensin from Plutella xylostella with potential activity against Bacillus thuringiensis.

    PubMed

    Xu, X-X; Zhang, Y-Q; Freed, S; Yu, J; Gao, Y-F; Wang, S; Ouyang, L-N; Ju, W-Y; Jin, F-L

    2016-12-01

    Insect defensins, are cationic peptides that play an important role in immunity against microbial infection. In the present study, an anionic defensin from Plutella xylostella, (designated as PxDef) was first cloned and characterized. Amino acid sequence analysis showed that the mature peptide owned characteristic six-cysteine motifs with predicted isoelectric point of 5.57, indicating an anionic defensin. Quantitative real-time polymerase chain reaction analysis showed that PxDef was significantly induced in epidermis, fat body, midgut and hemocytes after injection of heat-inactivated Bacillus thuringiensis, while such an induction was delayed by the injection of live B. thuringiensis in the 4th instar larvae of P. xylostella. Knocking down the expression of nuclear transcription factor Dorsal in P. xylostella by RNA interference significantly decreased the mRNA level of PxDef, and increased the sensitivity of P. xylostella larvae to the infection by live B. thuringiensis. The purified recombinant mature peptide (PxDef) showed higher activity against Gram-positive bacteria, with the minimum inhibition concentrations of 1.6 and 2.6 µM against B. thuringiensis and Bacillus subtilis, respectively. To our knowledge, this is the first report about an anionic PxDef, which may play an important role in the immune system of P. xylostella against B. thuringiensis.

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

    PubMed Central

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

    2015-01-01

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

  15. Effects of four entomopathogenic nematode species on fitness costs of pink bollworm resistance to Bacillus thuringiensis toxin Cry1Ac

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Evolution of resistance by pests can reduce the efficacy of transgenic crops that produce insecticidal toxins from the bacterium Bacillus thuringiensis (Bt). However, fitness costs can slow the evolution of resistance. We tested whether four species of entomopathogenic nematodes (Steinernematidae ...

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

    PubMed

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

    2014-06-01

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

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

    PubMed

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

    2016-03-01

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

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

    PubMed

    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.

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

    PubMed Central

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

    2012-01-01

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

  20. 40 CFR 174.506 - Bacillus thuringiensis Cry34Ab1 and Cry35Ab1 proteins in corn; exemption from the requirement of...

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Bacillus thuringiensis Cry34Ab1 and... 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...

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

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Bacillus thuringiensis modified Cry3A... 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...

  2. 40 CFR 174.518 - Bacillus thuringiensis Cry3Bb1 protein in corn; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

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

  3. 40 CFR 174.501 - Bacillus thuringiensis Vip3Aa protein in corn and cotton; exemption from the requirement of a...

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Bacillus thuringiensis Vip3Aa protein... REQUIREMENTS FOR PLANT-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.501 Bacillus... Bacillus thuringiensis Vip3Aa proteins in or on corn or cotton are exempt from the requirement of...

  4. 40 CFR 174.532 - Bacillus thuringiensis eCry3.1Ab protein in corn; temporary exemption from the requirement of a...

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Bacillus thuringiensis eCry3.1Ab... 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;...

  5. cry1 genes from Bacillus thuringiensis: specificity determination and implications for primer design.

    PubMed

    Davolos, Camila C; Guidelli-Thuler, Ana M; de Abreu, Irlan L; Sena, Janete A D; Lemos, Manoel V F

    2009-12-01

    Some pest management programs employ PCR to identify cry1 genes from Bacillus thuringiensis to predict bacterial toxicity towards different insect pests. However, due to changes on the mode of action of the Cry proteins, new primers had to be designed to detect the new genes. Therefore, an 'in-silico' study of genetic sequences from five cry1 subclasses was carried out and characterized by molecular tools. The design of new primers allows for more precise selection of B. thuringiensis isolates, helping to better direct the programs employing biological control.

  6. [Toxicity of isolates of Bacillus thuringiensis from Wroclaw against larvae of Aedes aegypti].

    PubMed

    Lonc, E; Kucińska, J; Rydzanicz, K

    2001-01-01

    Seven field isolates of Bacillus thuringiensis from the Lower Silesia, region of Poland, the Osola plain and phylloplane niches and soil samples from the Karkonosze National Park were tested in vitro for insecticidal activity against mosquito larvae Aedes aegypti. Both the spore/crystal mixture and pured crystals from B. thuringienis strains KpC1, KpF3 and OpQ3 (belonging to the first physiological group including the subspecies japonensis, yoso, jinghongiensis ) proved to be the most active against insects (61-65% of corrected mortality). The lowest toxicity (7-28% mortality) was caused by B. thuringiensis wratislaviensis strains (PO12 and 13).

  7. 76 FR 57653 - Bacillus thuringiensis eCry3.1Ab Protein in Corn; Temporary Exemption From the Requirement of a...

    Federal Register 2010, 2011, 2012, 2013, 2014

    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...

  8. Structure of cloned ribosomal DNA cistrons from Bacillus thuringiensis.

    PubMed Central

    Klier, A F; Kunst, F; Rapoport, G

    1979-01-01

    A library of B. thuringiensis DNA has been prepared by using the plasmid pBR322 as a cloning vehicle and E. coli as a host cell. By screening this collection with specific probes, 17 clones were identified whose hybrid plasmids contain rRNA genes of B. thuringiensis. Several of these plasmids have been mapped with restriction endonucleases and by DNA-RNA hybridization. By using maps of overlapping fragments, we have been able to establish an overall map of the ribosomal gene cluster. Images PMID:388353

  9. Unusually high frequency of genes encoding vegetative insecticidal proteins in an Australian Bacillus thuringiensis collection.

    PubMed

    Beard, Cheryl E; Court, Leon; Boets, Annemie; Mourant, Roslyn; Van Rie, Jeroen; Akhurst, Raymond J

    2008-09-01

    Of 188 Australian Bacillus thuringiensis strains screened for genes encoding soluble insecticidal proteins by polymerase chain reaction/restriction-length fragment polymorphism (RFLP) analysis, 87% showed the presence of such genes. Although 135 isolates (72%) produced an RFLP pattern identical to that expected for vip3A genes, 29 isolates possessed a novel vip-like gene. The novel vip-like gene was cloned from B. thuringiensis isolate C81, and sequence analysis demonstrated that it was 94% identical to the vip3Ba1 gene. The new gene was designated vip3Bb2. Cell-free supernatants from both the B. thuringiensis strain C81 and from Escherichia coli expressing the Vip3Bb2 protein were toxic for the cotton bollworm, Helicoverpa armigera.

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

    NASA Astrophysics Data System (ADS)

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

    2007-01-01

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

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

    PubMed

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

    2014-09-16

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

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

    PubMed Central

    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

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

    PubMed

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

    2012-01-01

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

  14. Ecological consequences of ingestion of Bacillus cereus on Bacillus thuringiensis infections and on the gut flora of a lepidopteran host.

    PubMed

    Raymond, Ben; Lijek, Rebeccah S; Griffiths, Robert I; Bonsall, Michael B

    2008-09-01

    The Bacillus cereus group comprises a diverse array of non-pathogenic bacteria as well as pathogens such as Bacillus thuringiensis. Their spores are found together in soil and leaves and are therefore likely to commonly interact within hosts. Mixed infections of pathogenic B. thuringiensis and non-pathogenic strains have been little studied, despite their potential impact on biological control and the evolutionary ecology of virulence. Antibiotic secreting strains of B. cereus have been shown to be able to synergize B. thuringiensis (Bt) infections. We explored the ecology of these mixed infections more broadly in the diamondback moth (DBM). We tested whether antibiotic-expressing B. cereus can synergize Bt infections initiated with spores, investigated whether ingestion of antibiotic-expressing B. cereus had any consequences for the larval gut flora and whether synergistic interactions with B. cereus increase Bt reproduction. Ingestion of high-antibiotic secreting B. cereus synergized infections of B. thuringiensis in diamondback moth larvae, but at a lower level than previously reported. Coinfection also increased slightly the number of Bt spores found in cadavers. Culture independent analysis of gut homogenates indicated that ingestion of an antibiotic-expressing strain of B. cereus reduced the abundance of the gut flora and led to gut communities being dominated bacteria with DGGE profiles very similar to pure B. cereus cultures. Ingestion of B. cereus, regardless of genotype, reduced densities of an enteric isolate of Enterobacter sp. These findings support the hypothesis that antibiotic secretion in the gut synergizes B. thuringiensis infections by reducing the abundance of the commensal gut flora and facilitating invasion by bacteria in the B. cereus group.

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed

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

    2013-01-01

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

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

    SciTech Connect

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

    2005-10-12

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

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

    PubMed

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

    2013-03-01

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

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

    PubMed

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

    2013-12-01

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

    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.

  2. [Use of bentonite in production of granular bioinsecticide on the basis of Bacillus thuringiensis].

    PubMed

    Drehval', O A; Hordiienko, A S; Cherevach, N V; Kurdysh, I K; Vinnikov, A I

    2008-01-01

    Electrokinetic properties of Bacillus thuringiensis spores and peculiarities of their interaction with clayey minerals montmorillonite and paligorskite were investigated. Existence of contact interaction between montmorillonite particles and bacteria endospores was shown. Granular bioinsecticide was prepared on the basis of bentonite, montmorillonite being its main component. It was discovered that the preparation can be well preserved for 3 months. While using the preparation after the long-term preservation for 12 months, it is recommended to increase the bioinsecticide suspension concentration twice.

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed

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

    2015-12-01

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

  5. Genomic characterization and comparison of seven Myoviridae bacteriophage infecting Bacillus thuringiensis.

    PubMed

    Sauder, Amber Brooke; Quinn, McKenzie Rea; Brouillette, Alexis; Caruso, Steven; Cresawn, Steven; Erill, Ivan; Lewis, Lynn; Loesser-Casey, Kathryn; Pate, Morgan; Scott, Crystal; Stockwell, Stephanie; Temple, Louise

    2016-02-01

    Bacillus thuringiensis Kurstaki, a bacterium that is a source of biopesticides and a safe simulant for pathogenic Bacillus species, was used to isolate seven unique bacteriophages. The phage genomes were sequenced and ranged in size from 158,100 to 163,019 bp encoding 290-299 genes, and the GC content of ~38% was similar to that of the host bacterium. All phages had terminal repeats 2-3 kb long. Three of the phages encoded tRNAs and three contained a self-splicing intron in the DNA polymerase gene. They were categorized as a single cluster (>60% nucleotide conservation) containing three subclusters (>80% nucleotide conservation), supported by genomic synteny and phylogenetic analysis. Considering the published genomes of phages that infect the genus Bacillus and noting the ability of many of the Bacillus cereus group phages to infect multiple species, a clustering system based on gene content is proposed.

  6. 40 CFR 180.1108 - Delta endotoxin of Bacillus thuringiensis variety San Diego encapsulated into killed Pseudomonas...

    Code of Federal Regulations, 2013 CFR

    2013-07-01

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

  7. 40 CFR 180.1108 - Delta endotoxin of Bacillus thuringiensis variety San Diego encapsulated into killed Pseudomonas...

    Code of Federal Regulations, 2012 CFR

    2012-07-01

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

  8. 40 CFR 180.1108 - Delta endotoxin of Bacillus thuringiensis variety San Diego encapsulated into killed Pseudomonas...

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 24 2011-07-01 2011-07-01 false Delta endotoxin of Bacillus... From Tolerances § 180.1108 Delta endotoxin of Bacillus thuringiensis variety San Diego encapsulated into killed Pseudomonas fluorescens; exemption from the requirement of a tolerance. The delta...

  9. 40 CFR 180.1108 - Delta endotoxin of Bacillus thuringiensis variety San Diego encapsulated into killed Pseudomonas...

    Code of Federal Regulations, 2014 CFR

    2014-07-01

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

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

    PubMed

    Hariram, Upasana; Labbé, Ronald

    2015-03-01

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

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

    Code of Federal Regulations, 2010 CFR

    2010-07-01

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

  12. 40 CFR 174.511 - Bacillus thuringiensis Cry1Ab protein in all plants; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

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

  13. 40 CFR 180.1154 - CryIA(c) and CryIC derived delta-endotoxins of Bacillus thuringiensis var. kurstaki encapsulated...

    Code of Federal Regulations, 2010 CFR

    2010-07-01

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

  14. 40 CFR 174.511 - Bacillus thuringiensis Cry1Ab protein in all plants; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

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

  15. 40 CFR 174.510 - Bacillus thuringiensis Cry1Ac protein in all plants; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

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

  16. 40 CFR 174.511 - Bacillus thuringiensis Cry1Ab protein in all plants; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

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

  17. 40 CFR 174.510 - Bacillus thuringiensis Cry1Ac protein in all plants; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

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

  18. 40 CFR 174.511 - Bacillus thuringiensis Cry1Ab protein in all plants; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 24 2011-07-01 2011-07-01 false Bacillus thuringiensis Cry1Ab protein... Cry1Ab protein in all plants; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry1Ab protein in all plants are exempt from the requirement of a tolerance when used as...

  19. 40 CFR 174.510 - Bacillus thuringiensis Cry1Ac protein in all plants; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

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

  20. 40 CFR 174.511 - Bacillus thuringiensis Cry1Ab protein in all plants; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

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

  1. 40 CFR 174.510 - Bacillus thuringiensis Cry1Ac protein in all plants; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 24 2011-07-01 2011-07-01 false Bacillus thuringiensis Cry1Ac protein... Cry1Ac protein in all plants; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry1Ac protein in all plants are exempt from the requirement of a tolerance when used as...

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

    PubMed Central

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

    1991-01-01

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

  3. A facile analytical method for the identification of protease gene profiles from Bacillus thuringiensis strains.

    PubMed

    Chen, Fu-Chu; Shen, Li-Fen; Chak, Kin-Fu

    2004-01-01

    Five pairs of degenerate universal primers have been designed to identify the general protease gene profiles from some distinct Bacillus thuringiensis strains. Based on the PCR amplification patterns and DNA sequences of the cloned fragments, it was noted that the protease gene profiles of the three distinct strains of B. thuringiensis subsp. kurstaki HD73, tenebrionis and israelensis T14001 are varied. Seven protease genes, neutral protease B (nprB), intracellular serine protease A (ispA), extracellular serine protease (vpr), envelope-associated protease (prtH), neutral protease F (nprF), thermostable alkaline serine protease and alkaline serine protease (aprS), with known functions were identified from three distinct B. thuringiensis strains. In addition, five DNA sequences with unknown functions were also identified by this facile analytical method. However, based on the alignment of the derived protein sequences with the protein domain database, it suggested that at least one of these unknown genes, yunA, might be highly protease-related. Thus, the proposed PCR-mediated amplification design could be a facile method for identifying the protease gene profiles as well as for detecting novel protease genes of the B. thuringiensis strains.

  4. Bacillus thuringiensis Metalloproteinase Bmp1 Functions as a Nematicidal Virulence Factor

    PubMed Central

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

    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. PMID:23124228

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

    PubMed

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

    2016-10-01

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

  6. New variants of lepidoptericidal toxin genes encoding Bacillus thuringiensis Vip3Aa proteins.

    PubMed

    Sauka, Diego H; Rodriguez, Sonia E; Benintende, Graciela B

    2012-01-01

    Bacillus thuringiensis is an entomopathogenic bacterium characterized by producing parasporal proteinaceous insecticidal crystal inclusions during sporulation. Many strains are capable of also expressing other insecticidal proteins called Vip during the vegetative growing phase. Particularly, Vip3A proteins have activity against certain Lepidoptera species through a unique mechanism of action which emphasized their possible use in resistance management strategies against resistant pests. The aim of the work was to develop a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method that can distinguish between vip3A genes from B. thuringiensis strains. In addition, 4 novel vip3Aa genes were cloned and sequenced. The method was originally based on amplification of a single PCR amplicon and the use of 2 restriction enzymes with recognition sites that facilitate simultaneous detection. Subsequently, a third restriction enzyme was used to distinguish between vip3A variants. Thirteen vip3Aa genes were identified in strains belonging to 10 different B. thuringiensis serovars. Three intra-subclass variants of vip3Aa genes could be differentiated. The presented method can serve as an invaluable tool for the investigation of known and novel vip3A genes in B. thuringiensis strains. To the best of our knowledge, this is the first report where variants of a same subclass of insecticidal genes could be distinguished following PCR-RFLP.

  7. Dark fermentative bioconversion of glycerol to hydrogen by Bacillus thuringiensis.

    PubMed

    Kumar, Prasun; Sharma, Rishi; Ray, Subhasree; Mehariya, Sanjeet; Patel, Sanjay K S; Lee, Jung-Kul; Kalia, Vipin C

    2015-04-01

    Biodiesel manufacturing units discharge effluents rich in glycerol. The need is to convert crude glycerol (CG) into useful products such as hydrogen (H2). Under batch culture, Bacillusthuringiensis EGU45 adapted on pure glycerol (PG, 2% v/v) resulted in an H2 yield of 0.646 mol/mol glycerol consumed on minimal media (250 mL) supplemented with 1% ammonium nitrate at 37°C over 4 days. Here, H2 constituted 67% of the total biogas. Under continuous culture, at 2 days of hydraulic retention time, B. thuringiensis immobilized on ligno-cellulosic materials (banana leaves - BL, 10% v/v) resulted in a H2 yield of 0.386 mol/mol PG consumed. On CG, the maximal H2 yield of 0.393 mol/mol feed consumed was recorded. In brief, B. thuringiensis could transform CG, on limited resources - minimal medium with sodium nitrate, by immobilizing them on cheap and easily available biowaste, which makes it a suitable candidate for H2 production on a large scale.

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

    PubMed

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

    2013-08-01

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

  9. Behavior of transition state regulator AbrB in batch cultures of Bacillus thuringiensis.

    PubMed

    Lozano Goné, Astrid Magdalena; Dinorín Téllez Girón, Jabel; Jiménez Montejo, Fabiola Eloisa; Hidalgo-Lara, María Eugenia; López Y López, Víctor Eric

    2014-11-01

    The transition state regulator AbrB is involved in the regulation of various cellular functions such as exponential growth, transition state and sporulation onset, due to its ability to activate, suppress or prevent the inappropriate expression of various genes in Bacillus subtilis. In order to understand combined behavior in batch cultures of AbrB in Bacillus thuringiensis, we cloned and expressed the abrB gene of B. thuringiensis in Escherichia coli. The deduced sequence of abrB gene coded for a protein consisting of 94 amino acids with ~10.5 kDa protein that shares 100 and 85 % identity with those from Bacillus cereus and Bacillus subtilis. The recombinant AbrB protein was used as antigen for the production of rabbit polyclonal antibodies anti-AbrB. Two media cultures with carbon: nitrogen ratios of 7.0, but varying access to nutrients were tested in batch cultures. In the case of both media, AbrB accumulation occurred from the beginning of the process and was maximal during early exponential growth. Thereafter, the level of AbrB decreased when there were no nutrient limitations and coincided with a decreased value in specific growth rate, although growth continued exponentially. Nonetheless, sporulation onset was determined 3 h and 4 h later, in media with highly metabolizable nutrients clean medium and Farrera medium, respectively. Hence, the maximal level of AbrB accumulation in batch cultures of B. thuringiensis is not influenced by limiting nutrients; however, nutrient availability affects the required time lapse for transition state regulator accumulation.

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

    PubMed

    Murawska, Emilia; Fiedoruk, Krzysztof; Swiecicka, Izabela

    2014-01-01

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

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

    PubMed

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

    2015-06-01

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

  12. Transcriptional response of Choristoneura fumiferana to sublethal exposure of Cry1Ab protoxin from Bacillus thuringiensis.

    PubMed

    Meunier, L; Préfontaine, G; Van Munster, M; Brousseau, R; Masson, L

    2006-08-01

    Bacillus thuringiensis is a microbial control agent active against Choristoneura fumiferana, a lepidopteran defoliator of North American forests. Although the B. thuringiensis insecticidal crystal protoxins have a relatively narrow host range, there is concern about their impact on non-target species where intoxication effects may not be overt. Larval toxicity effects can be assessed at the molecular level by determining altered transcriptional profiles in response to sublethal protoxin exposure in sensitive insects. Subtraction hybridization libraries were created using two larval populations, control and protoxin-fed and were characterized by sequencing 1091 clones. Differential mRNA expression of selected clones, as measured by quantitative polymerase chain reaction, identified a number of metabolic and stress-related genes that were either transcriptionally enhanced or repressed after protoxin exposure.

  13. A purification and some properties of an insecticidal exotoxin from Bacillus thuringiensis Berliner

    PubMed Central

    Bond, R. P. M.; Boyce, C. B. C.; French, S. J.

    1969-01-01

    An insecticidal exotoxin from Bacillus thuringiensis var. thuringiensis (Berliner) has been purified. The efficiency of each stage of the purification has been ascertained and the yield of toxic material estimated by means of a quantitative bioassay. It is shown that the exotoxin is an adenine derivative substituted at position 9 and having a molecular weight of approximately 825. It can be dephosphorylated enzymically or chemically under conditions that define the exotoxin as a phosphomonoester. This results in loss of toxicity, both to insects and to mice. Spectroscopic and kinetic data are presented which suggest that a β-ribofuranosyl moiety may be attached to the adenine. Glucose and allomucic acid have been positively identified as hydrolysis fragments from the exotoxin. These results are discussed and compared with the results of others on similar (or possibly identical) compounds. PMID:5820635

  14. FlhA influences Bacillus thuringiensis PlcR-regulated gene transcription, protein production, and virulence.

    PubMed

    Bouillaut, Laurent; Ramarao, Nalini; Buisson, Christophe; Gilois, Nathalie; Gohar, Michel; Lereclus, Didier; Nielsen-Leroux, Christina

    2005-12-01

    Bacillus thuringiensis and Bacillus cereus are closely related. B. thuringiensis is well known for its entomopathogenic properties, principally due to the synthesis of plasmid-encoded crystal toxins. B. cereus appears to be an emerging opportunistic human pathogen. B. thuringiensis and B. cereus produce many putative virulence factors which are positively controlled by the pleiotropic transcriptional regulator PlcR. The inactivation of plcR decreases but does not abolish virulence, indicating that additional factors like flagella may contribute to pathogenicity. Therefore, we further analyzed a mutant (B. thuringiensis 407 Cry(-) DeltaflhA) previously described as being defective in flagellar apparatus assembly and in motility as well as in the production of hemolysin BL and phospholipases. A large picture of secreted proteins was obtained by two-dimensional electrophoresis analysis, which revealed that flagellar proteins are not secreted and that production of several virulence-associated factors is reduced in the flhA mutant. Moreover, we quantified the effect of FlhA on plcA and hblC gene transcription. The results show that the flhA mutation results in a significant reduction of plcA and hblC transcription. These results indicate that the transcription of several PlcR-regulated virulence factors is coordinated with the flagellar apparatus. Consistently, the flhA mutant also shows a strong decrease in cytotoxicity towards HeLa cells and in virulence against Galleria mellonella larvae following oral and intrahemocoelic inoculation. The decrease in virulence may be due to both a lack of flagella and a lower production of secreted factors. Hence, FlhA appears to be an essential virulence factor with a pleiotropic role.

  15. Molecular and toxigenic characterization of Bacillus cereus and Bacillus thuringiensis strains isolated from commercial ground roasted coffee.

    PubMed

    Chaves, Jeane Quintanilha; Cavados, Clara de Fátima Gomes; Vivoni, Adriana Marcos

    2012-03-01

    Thirty samples of roasted ground coffee beans from 10 different commercial brands were analyzed to investigate the occurrence and levels of Bacillus cereus and Bacillus thuringiensis strains. Strains were evaluated for their genetic diversity by repetitive element sequence polymorphism PCR (Rep-PCR) and for their toxigenic profiles, i.e., the presence of hblA, hblC, hblD, nheA, nheB, nheC, cytK, ces, and entFM. Survival and multiplication of B. cereus sensu lato in the ready-to-drink coffee was determined to evaluate this beverage as a possible vehicle for B. cereus infection. B. cereus was detected in 17 (56.7%) of the 30 samples, and B. thuringiensis was detected in 8 (26.7%) of the 30 samples. Five samples did not produce any characteristic growth. The most common gene, entFM, was detected in 23 strains (92%). The NHE complex (nheA, nheB, and nheC genes) was found in 19 strains (76%). The HBL complex (hblA, hblC, and hblD) was found in 16 strains (64%). All strains were negative for ces. The cytK gene was found in 16 strains (64%). The computer-assisted cluster analysis of Rep-PCR profiles using a clustering criterion of 80% similarity revealed four main clusters. Cluster 1 was the predominant and comprised three B. thuringiensis strains with 100% similarity, cluster 2 comprised two B. cereus strains (100% similarity), cluster 3 comprised two B. thuringiensis strains (90% similarity), and cluster 4 comprised one B. thuringiensis strain and one B. cereus strain (85% similarity). The cluster analysis of fingerprints generated by Rep-PCR revealed a high genetic diversity among the B. cereus strains, suggesting that the contamination could have originated from different sources. In our experiments, when sugar was added and the beverage was kept in thermic bottles there was a significant increase in B. cereus sensu lato levels, which may increase the risk of food poisoning. These results highlight the need for additional studies on this subject to better evaluate

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

    PubMed

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

    2014-01-01

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

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

    PubMed

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

    2014-07-14

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

  18. Identification of Distinct Bacillus thuringiensis 4A4 Nematicidal Factors Using the Model Nematodes Pristionchus pacificus and Caenorhabditis elegans

    PubMed Central

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

    2014-01-01

    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. PMID:25025708

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

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

    2013-01-01

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

  2. Changes in Morphology of Trichostrongylus colubriformis Eggs and Juveniles Caused by Bacillus thuringiensis israelensis

    PubMed Central

    Bottjer, Kurt P.; Bone, Leon W.

    1987-01-01

    Eggs and rhabditiform juveniles of the ruminant parasite Trichostrongylus colubriformis developed normally in Caenorhabditis briggsae Maintenance Medium. A toxin from a crystal-enriched preparation of the bacterium Bacillus thuringiensis israelensis was lethal to nematode eggs and juveniles within 24 hours and to eggs and juveniles after 24 hours of development. Treated eggs had refractive granules and development was arrested, whereas nontreated eggs developed normally. Eggs treated after 24 hours of development contained juveniles that were granulated, had esophageal derangements, and were moribund or dead. The ovicidal toxin from B. t. israelensis may facilitate microbial control of parasitic nematodes. PMID:19290144

  3. Cloning and expression of the lepidopteran toxin produced by Bacillus thuringiensis var Sotto in Escherichia coli.

    PubMed

    Rady, M H

    1991-01-01

    During sporulation, Bacillus thuringiensis var. Sotto produces a parasporal crystalline protein which is toxic for the silk-worm, Bombyx mori and the cotton leaf worm, Spodoptera littoralis. The gene coding this crystal protein is present in a single plasmid. The plasmid DNA was isolated, purified and physically mapped using restriction endonuclease enzymes (R.E.). The gene coding the delta-endotoxin was inserted into Escherichia coli-Jm103, using cloning vector pUC8. Transformed E. coli cells were found to synthesize the delta-endotoxin as demonstrated by the pathogenicity of the transformed cells against 4th instar larvae of S. littoralis.

  4. Transformation studies of Bacillus thuringiensis cryIC gene into a nitrogen-fixing Azospirillum lipoferum.

    PubMed

    Gounder, R; Rajendran, N

    2001-01-01

    A lepidopteran toxin gene, cryIC (pSB607) from entomopathogenic Bacillus thuringiensis subsp. aizawai was introduced into nitrogen-fixing Azospirillum lipoferum by transformation. Regeneration of spheroplasts was achieved at 99% with 39% frequency of regeneration. Transformants were screened on NB kanamycin with ampicillin plates and 4 transformants were selected after ten generations. SDS-PAGE and Western blot analysis confirmed the presence of a 68 kDa protein in the transformants. Studies on utilization of carbon sources indicate that glucose and sucrose are the most favorable carbon sources and 2% molasses is the cheap alternate carbon source for the better growth of parent A. lipoferum and transformants.

  5. Does distant homology with Evf reveal a lipid binding site in Bacillus thuringiensis cytolytic toxins?

    PubMed

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

    PubMed

    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.

  7. Cloning and localization of vip3A gene of Bacillus thuringiensis.

    PubMed

    Wu, Zeng Ling; Guo, Wen Yi; Qiu, Jun Zhi; Huang, Tian Pei; Li, Xun Bo; Guan, Xiong

    2004-09-01

    An insecticidal protein gene, vip3A, was cloned from Bacillus thuringiensis strain WB50. The nucleotide sequence of 2,460 bp (GenBank acc. No. AY295778) showed 99% homology with the known vip3A genes. Using specific primers for vip3A gene, PCR was performed to demonstrate that the gene was not located on the bacterial chromosome and this was confirmed by Southern blotting using an internal fragment (486 bp) from vip3A gene as a probe. The gene was carried on a plasmid of 31.8 kb.

  8. Fluorescence assay based on aptamer-quantum dot binding to Bacillus thuringiensis spores.

    PubMed

    Ikanovic, Milada; Rudzinski, Walter E; Bruno, John G; Allman, Amity; Carrillo, Maria P; Dwarakanath, Sulatha; Bhahdigadi, Suneetha; Rao, Poornima; Kiel, Johnathan L; Andrews, Carrie J

    2007-03-01

    A novel assay was developed for the detection of Bacillus thuringiensis (BT) spores. The assay is based on the fluorescence observed after binding an aptamer-quantum dot conjugate to BT spores. The in vitro selection and amplification technique called SELEX (Systematic Evolution of Ligands by EXponential enrichment) was used in order to identify the DNA aptamer sequence specific for BT. The 60 base aptamer was then coupled to fluorescent zinc sulfide-capped, cadmium selenide quantum dots (QD). The assay is semi-quantitative, specific and can detect BT at concentrations of about 1,000 colony forming units/ml.

  9. Relationship between poly-beta-hydroxybutyrate production and delta-endotoxin for Bacillus thuringiensis var. kurstaki.

    PubMed

    Navarro, A Karin; Farrera, Reynold R; López, Ruth; Pérez-Guevara, Fermín

    2006-05-01

    A linear relationship between total solid concentration (TSC), delta-endotoxin production [Cry = 0.2795(TSC)-0.2472, R2 = 0.8644] and poly-beta-hydroxybutyrate (PHB) accumulation [PHB = 0.1327(TSC) + 0.3974, R2 = 0.9877] in Bacillus thuringiensis var. kurstaki HD-73 was observed. A similar correlation between delta-endotoxin and PHB accumulation [Cry = 2.1573(PHB)-1.1248, R2 = 0.9181] was found. A minimum PHB accumulation of 0.52 mg l(-1) was required before the onset of delta-endotoxin production.

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

    PubMed

    Gillis, Annika; Mahillon, Jacques

    2014-12-01

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

  11. Influence of Lysogeny of Tectiviruses GIL01 and GIL16 on Bacillus thuringiensis Growth, Biofilm Formation, and Swarming Motility

    PubMed Central

    Gillis, Annika

    2014-01-01

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

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

    PubMed

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

    2015-01-01

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

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

    PubMed

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

    2015-03-05

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

  14. Location of the Bombyx mori specificity domain on a Bacillus thuringiensis delta-endotoxin protein.

    PubMed Central

    Ge, A Z; Shivarova, N I; Dean, D H

    1989-01-01

    Bacillus thuringiensis produces different types of insecticidal crystal proteins (ICPs) or delta-endotoxins. In an effort to identify the insect specificity of ICP toxins, two icp genes were cloned into the Escherichia coli expression vector pKK223-3, and bioassays were performed with purified crystals. The type A protein [from an icpA1, or 4.5-kilobase (kb) gene, from B. thuringiensis var. kurstaki HD-1] was found to be 400 times more active against Bombyx mori than type C protein (from an icpC73, or 6.6-kb gene, from B. thuringiensis var. kurstaki HD-244). The type C protein was 9 times more active against Trichoplusia ni than the type A protein, while both have similar activity against Manduca sexta. To locate the specificity domain of the type A protein for B. mori, site-directed mutagenesis was used to introduce or remove restriction enzyme sites, facilitating the exchange of regions of the two genes. The hybrid genes were overexpressed, and purified ICP was used in bioassays. The B. mori specificity domain for the ICP A toxin is located in the amino-terminal portion of the hypervariable region between amino acids 332 and 450. PMID:2542961

  15. Comparative biochemistry of entomocidal parasporal crystals of selected Bacillus thuringiensis strains.

    PubMed Central

    Tyrell, D J; Bulla, L A; Andrews, R E; Kramer, K J; Davidson, L I; Nordin, P

    1981-01-01

    Parasporal crystals of Bacillus thuringiensis subspp. kurstaki, tolworthi, alesti, berliner, and israelensis were compared by electron microscopy, polyacrylamide gel electrophoresis, amino acid analysis, tryptic peptide mapping, immunological analysis, and insecticidal activity. Spore coats also were compared by polyacrylamide gel electrophoresis. B. thuringiensis subsp. israelensis crystals were lethally toxic to mosquito larvae and nontoxic to tobacco hornworm larvae. Conversely, crystals from the other subspecies killed tobacco hornworm larvae but were ineffective against mosquitoes. Crystalline inclusion bodies of all subspecies contained a protoxic subunit that had an apparent molecular weight of approximately 1.34 X 10(5). However, polyacrylamide gel electrophoretic patterns of solubilized crystals revealed a small-molecular-weight component (apparent molecular weight, 26,000) in B. thuringiensis subsp. israelensis that was absent in the other subspecies. Also, differences were noted in amino acid composition and tryptic peptide fingerprints. Crystal proteins were found in spore coats of all subspecies. The results suggest that insecticidal specificity is due to unique polypeptide toxins. Images PMID:7462158

  16. Phase-specific optimization of multiple endotoxin-protein production with genetically engineered Bacillus thuringiensis.

    PubMed

    Yang, X M; Wang, S S

    2000-02-01

    An optimization approach was designed to specifically study the toxin-expression phase of the fermentation process of a genetically engineered Bacillus thuringiensis strain expressing dual toxin proteins (CryI and CryIII). The study has resulted in the discovery of important nutrient and process factors affecting toxin-protein yield. The results show that the existence of nitrogen sources in the medium during the toxin-expression phase is detrimental to the toxin-protein expression, while a high carbon-source level (40 g/l) encouraged protein expression. The study also suggests that the depletion of nitrogen source is the trigger for B. thuringiensis to initiate sporulation and toxin expression. A temperature setting of 28 degrees C for B. thuringiensis fermentation processes is optimal for protein yield, and reduces the oxygen requirement. It was found that the optimal conditions for spore yield and for toxin-protein yield were not the same, even though sporulation and toxin formation proceed simultaneously during the fermentation process. Scale-up studies were also conducted to confirm the optimal conditions obtained from a small-scale optimization study.

  17. Phylogenetic distribution of phenotypic traits in Bacillus thuringiensis determined by multilocus sequence analysis.

    PubMed

    Blackburn, Michael B; Martin, Phyllis A W; Kuhar, Daniel; Farrar, Robert R; Gundersen-Rindal, Dawn E

    2013-01-01

    Diverse isolates from a world-wide collection of Bacillus thuringiensis were classified based on phenotypic profiles resulting from six biochemical tests; production of amylase (T), lecithinase (L), urease (U), acid from sucrose (S) and salicin (A), and the hydrolysis of esculin (E). Eighty two isolates representing the 15 most common phenotypic profiles were subjected to phylogenetic analysis by multilocus sequence typing; these were found to be distributed among 19 sequence types, 8 of which were novel. Approximately 70% of the isolates belonged to sequence types corresponding to the classical B. thuringiensis varieties kurstaki (20 isolates), finitimus (15 isolates), morrisoni (11 isolates) and israelensis (11 isolates). Generally, there was little apparent correlation between phenotypic traits and phylogenetic position, and phenotypic variation was often substantial within a sequence type. Isolates of the sequence type corresponding to kurstaki displayed the greatest apparent phenotypic variation with 6 of the 15 phenotypic profiles represented. Despite the phenotypic variation often observed within a given sequence type, certain phenotypes appeared highly correlated with particular sequence types. Isolates with the phenotypic profiles TLUAE and LSAE were found to be exclusively associated with sequence types associated with varieties kurstaki and finitimus, respectively, and 7 of 8 TS isolates were found to be associated with the morrisoni sequence type. Our results suggest that the B. thuringiensis varieties israelensis and kurstaki represent the most abundant varieties of Bt in soil.

  18. A new enrichment method for isolation of Bacillus thuringiensis from diverse sample types.

    PubMed

    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.

  19. The aggregation-mediated conjugation system of Bacillus thuringiensis subsp. israelensis: host range and kinetics of transfer.

    PubMed

    Jensen, G B; Andrup, L; Wilcks, A; Smidt, L; Poulsen, O M

    1996-10-01

    The aggregation-mediated conjugation system in Bacillus thuringiensis subsp. israelensis encoded on the plasmid pXO16 is characterized by the formation of aggregates when Agr+ and Agr- cells are socialized in exponential growth. Using the aggregation phenotypes, we have identified potential recipients of the aggregation-plasmid pXO16 among Bacillus cereus, Bacillus subtilis, Bacillus megaterium, Bacillus sphaericus, and 24 subspecies of B. thuringiensis. We found 14 Agr- strains, i.e., potential recipients of the aggregation system encoded by plasmid pXO16. Five strains contained a conjugative apparatus of their own and were excluded from further examinations. To monitor the transfer of plasmid pXO16, we constructed a transposon insertion of the plasmid with Tn5401. The study of the plasmid transfer of pXO16::Tn5401 indicated the secretion of bacteriocins from both donor strain and recipient strains. Only one out of the nine strains examined was unable to receive the aggregation-plasmid pXO16 and express the aggregation phenotype and the conjugative abilities. It was found that the transfer of plasmid pXO16 to Bacillus thuringiensis subsp. israelensis Agr- strains was 100%. All recipients had acquired the aggregation-plasmid pXO16 and converted to the Agr+ phenotype.

  20. Haematological, biochemical and histopathological alterations induced by abamectin and Bacillus thuringiensis in male albino rats.

    PubMed

    Eissa, F I; Zidan, N A

    2010-03-01

    The renal- and hepato-toxicity induced by abamectin pesticide (Vertimec) and a commercial form of a bio-insecticide Bacillus thuringiensis (Agerin) in male albino rats were evaluated. Blood picture and blood glucose level were investigated. Male albino rats were administered dietary doses each equivalent to 1/10 or 1/100 of the LD50 values of each toxicant for 30 consecutive days. Abamectin was found to pose risks of renal- and hepato-toxicity in rats, since the biochemical parameters of liver function (i.e. aspartate aminotransferase activity, alanine aminotransferase activity, acid phosphatase activity, albumin, and total protein levels) and kidney function (uric acid and creatinine concentration) were severely affected. These effects were verified by histopathological examination of liver and kidney tissues. Likewise, some haematological indices (i.e. erythrocyte count, leukocyte count and haemoglobin concentration) were also influenced; in addition abamectin might cause hypoglycaemia. On the other hand, the above-mentioned lesions were less pronounced in the case of Bacillus thuringiensis -treated rats.

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

    DTIC Science & Technology

    2008-03-01

    possible simulant for B. anthracis in counter-proliferation studies because it is closely related to B. anthracis and is not harmful to humans. In order...to be a good simulant in counter-proliferation studies, B. thuringiensis spores must have similar properties to B. anthracis spores. In particular...Preparation ......................32 Reflection Amplitude Curves

  2. A novel Bacillus thuringiensis Cry-like protein from a rare filamentous strain promotes crystal localization within the exosporium.

    PubMed

    Ammons, David R; Reyna, Antonio; Granados, Jose C; Ventura-Suárez, Antonio; Rojas-Avelizapa, Luz I; Short, John D; Rampersad, Joanne N

    2013-09-01

    Mutation of a novel cry-like gene (cry256) from Bacillus thuringiensis resulted in a protein crystal, normally located within the spore's exosporium, being found predominately outside the exosporium. The cry256 gene codes for a 3-domain Cry-like protein that does not correspond to any of the known Cry protein holotypes.

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

    EPA Science Inventory

    Crystal proteins derived from Bacillus thuringiensis (Bt) have been widely used as a method of insect pest management for several decades. In recent years, a transgenic corn expressing the Cry3Bb1 toxin has been successfully used for protection against corn rootworm larvae (Genus...

  4. Expression of Bacillus thuringiensis cytolytic toxin (Cyt2Ca1) in citrus roots to control Diaprepes abbreviatus larvae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Diaprepes abbreviatus (L.) is an important pest of citrus in the USA. Currently, no effective management strategies of Diaprepes abbreviatus exist in citriculture. To protect citrus against Diaprepes abbreviatus a transgenic citrus rootstock expressing Bacillus thuringiensis Cyt2Ca1, an insect toxin...

  5. 40 CFR 174.517 - Bacillus thuringiensis Cry9C protein in corn; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... in corn; exemption from the requirement of a tolerance. 174.517 Section 174.517 Protection of... Cry9C protein in corn; exemption from the requirement of a tolerance. The plant-incorporated protectant Bacillus thuringiensis Cry9C protein in corn is exempted from the requirement of a tolerance for...

  6. 40 CFR 174.517 - Bacillus thuringiensis Cry9C protein in corn; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... in corn; exemption from the requirement of a tolerance. 174.517 Section 174.517 Protection of... Cry9C protein in corn; exemption from the requirement of a tolerance. The plant-incorporated protectant Bacillus thuringiensis Cry9C protein in corn is exempted from the requirement of a tolerance for...

  7. 40 CFR 174.520 - Bacillus thuringiensis Cry1F protein in corn; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... in corn; exemption from the requirement of a tolerance. 174.520 Section 174.520 Protection of... Cry1F protein in corn; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry1F protein in corn are exempt from the requirement of a tolerance when used as...

  8. 40 CFR 174.517 - Bacillus thuringiensis Cry9C protein in corn; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... in corn; exemption from the requirement of a tolerance. 174.517 Section 174.517 Protection of... Cry9C protein in corn; exemption from the requirement of a tolerance. The plant-incorporated protectant Bacillus thuringiensis Cry9C protein in corn is exempted from the requirement of a tolerance for...

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

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... in corn; exemption from the requirement of a tolerance. 174.517 Section 174.517 Protection of... Cry9C protein in corn; exemption from the requirement of a tolerance. The plant-incorporated protectant Bacillus thuringiensis Cry9C protein in corn is exempted from the requirement of a tolerance for...

  10. 40 CFR 174.520 - Bacillus thuringiensis Cry1F protein in corn; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... in corn; exemption from the requirement of a tolerance. 174.520 Section 174.520 Protection of... Cry1F protein in corn; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry1F protein in corn are exempt from the requirement of a tolerance when used as...

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

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... in corn; exemption from the requirement of a tolerance. 174.520 Section 174.520 Protection of... Cry1F protein in corn; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry1F protein in corn are exempt from the requirement of a tolerance when used as...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The yellow mealworm, Tenebrio molitor, is a pest of stored grain products and is sensitive to the coleopteran-specific Cry3Aa toxin from Bacillus thuringiensis (Bt). Larvae digest protein initially with cysteine peptidases in the anterior midgut and further with serine peptidases in middle and poste...

  13. 40 CFR 174.530 - Bacillus thuringiensis Cry2Ae protein in cotton; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... in cotton; exemption from the requirement of a tolerance. 174.530 Section 174.530 Protection of... Cry2Ae protein in cotton; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry2Ae protein in or on the food and feed commodities of cotton; cotton, undelinted seed;...

  14. 40 CFR 174.504 - Bacillus thuringiensis Cry1F protein in cotton; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... in cotton; exemption from the requirement of a tolerance. 174.504 Section 174.504 Protection of... Cry1F protein in cotton; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry1F protein in cotton are exempt from the requirement of a tolerance when used as a...

  15. 40 CFR 174.530 - Bacillus thuringiensis Cry2Ae protein in cotton; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... in cotton; exemption from the requirement of a tolerance. 174.530 Section 174.530 Protection of... Cry2Ae protein in cotton; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry2Ae protein in or on the food and feed commodities of cotton; cotton, undelinted seed;...

  16. 40 CFR 174.504 - Bacillus thuringiensis Cry1F protein in cotton; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... in cotton; exemption from the requirement of a tolerance. 174.504 Section 174.504 Protection of... Cry1F protein in cotton; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry1F protein in cotton are exempt from the requirement of a tolerance when used as a...

  17. 40 CFR 174.530 - Bacillus thuringiensis Cry2Ae protein in cotton; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... in cotton; exemption from the requirement of a tolerance. 174.530 Section 174.530 Protection of... Cry2Ae protein in cotton; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry2Ae protein in or on the food and feed commodities of cotton; cotton, undelinted seed;...

  18. 40 CFR 174.504 - Bacillus thuringiensis Cry1F protein in cotton; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... in cotton; exemption from the requirement of a tolerance. 174.504 Section 174.504 Protection of... Cry1F protein in cotton; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry1F protein in cotton are exempt from the requirement of a tolerance when used as a...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bacillus thuringiensis (Bt) crystal (Cry) proteins are effective against some coleopteran pests, but improvements are needed in both efficacy and “time to kill” for Cry toxins to become valuable tools for use in integrated pest management. To gain insight into Bt intoxication of Coleoptera, we perfo...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Development of insect resistance is one of the main concerns with the use of transgenic crops expressing Cry toxins from the bacterium Bacillus thuringiensis. Biomarkers for development of sensitive DNA-based methods to detect and monitor evolution of resistance to Bt toxins are currently needed. ...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bacillus thuringiensis (Bt) insecticidal crystal (Cry) proteins are effective against some insect pests in sprays and transgenic crops, although the evolution of resistance could threaten the long-term efficacy of such Bt use. One strategy to delay resistance to Bt crops is to “pyramid” two or more ...

  2. Effects of four nematodes species on fitness costs of pink bollworm resistance to Bacillus thuringiensis toxin Cry1Ac

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Evolution of resistance by pests can reduce efficacy of transgenic crops that produce insecticidal toxins from the bacterium Bacillus thuringiensis (Bt). In conjunction with refuges of non-Bt host plants, fitness costs can delay the evolution of resistance. Furthermore, fitness costs often vary wit...

  3. Cloning and localization of the lepidopteran protoxin gene of Bacillus thuringiensis subsp. kurstaki.

    PubMed Central

    Held, G A; Bulla, L A; Ferrari, E; Hoch, J; Aronson, A I; Minnich, S A

    1982-01-01

    Bacillus thuringiensis subsp. kurstaki produces a proteinaceous crystalline inclusion that is toxic for lepidopteran larvae. There are several size classes of plasmids in this organism and the presence of one or more has been correlated with production of this protein, defined as a protoxin. DNA fragments of B. thuringiensis subsp. kurstaki, obtained by EcoRI digestion, were cloned into the vector Charon 4A. Recombinant phage were screened immunologically for the production of protoxin. Cells infected with one phage, C4K6c, produced antigen that was the same size as the protoxin and was toxic to Manduca sexta larvae. A 4.6-kilobase-pair (kbp) EcoRI fragment from C4K6c was subcloned into pBR328 and in both orientations in pHV33. Both Escherichia coli and Bacillus subtilis containing these recombinant plasmids produced antigen that crossreacted with antibody directed against the protoxin. The various sized plasmids of B. thuringiensis were purified and only an EcoRI fragment from the 45-kbp plasmid hybridized to phage C4K6c. One of the pHV33 subclones, pSM36, hybridized to the same size EcoRI/HindIII restriction fragments from plasmid or chromosomal DNA. The cloned EcoRI fragment contained a 0.9-kbp Pvu II fragment that was also present in chromosomal but not in plasmid digests. The original clone was therefore of chromosomal origin, although very similar or identical protoxin genes were present in both the 45-kbp plasmid and the chromosome. Several acrystalliferous nontoxic mutants have been isolated that lacked the 45-kbp plasmid and in some cases all plasmids. All of the mutants contained the chromosomal gene but did not produce protoxin antigen. Images PMID:6310560

  4. Discovery of a significant optical chromatographic difference between spores of Bacillus anthracis and its close relative, Bacillus thuringiensis.

    PubMed

    Hart, Sean J; Terray, Alex; Leski, Tomasz A; Arnold, Jonathan; Stroud, Rhonda

    2006-05-01

    A significant difference between two closely related Bacillus spores has been discovered using optical chromatography. This difference can be harnessed for the separation of microscopic particles using opposing laser and fluid flow forces. Particles of different size, composition, and shape experience different optical and fluid forces and come to rest at unique equilibrium positions where the two forces balance. Separations in excess of 600 mum have been observed between Bacillus anthracis Sterne strain and its genetic relative, Bacillus thuringiensis. These findings open new possibilities for detection and characterization of the biological warfare agent, B. anthracis, the causative agent of anthrax, the deadly mammalian disease. The large optical separation between these species is surprising given their close genetic relationship but may be explained by differences in their shape and exosporium morphology, which may result in differences in fluid drag force. The observation of large differences due to less common variables indicates the complex nature of the force balance in optical chromatography, which may in the future be used to separate and characterize microbiological samples. In general, the discovery of such large differences between such closely related biological species suggests new possibilities for the separation and characterization of microorganisms using the full range of emerging techniques that employ radiation pressure (optical filtering, laser tweezers, optical chromatography, etc.).

  5. 40 CFR 174.505 - Bacillus thuringiensis modified Cry3A protein (mCry3A) in corn; exemption from the requirement of...

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... protein (mCry3A) in corn; exemption from the requirement of a tolerance. 174.505 Section 174.505... thuringiensis modified Cry3A protein (mCry3A) in corn; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis modified Cry3A protein (mCry3A) in corn are exempt from the requirement...

  6. 40 CFR 174.532 - Bacillus thuringiensis eCry3.1Ab protein in corn; temporary exemption from the requirement of a...

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... protein in corn; temporary exemption from the requirement of a tolerance. 174.532 Section 174.532... thuringiensis eCry3.1Ab protein in corn; temporary exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis eCry3.1Ab protein in corn, in or on the food and feed commodities of corn;...

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

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... Cry35Ab1 proteins in corn; exemption from the requirement of a tolerance. 174.506 Section 174.506... thuringiensis Cry34Ab1 and Cry35Ab1 proteins in corn; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry34Ab1 and Cry35Ab1 proteins in corn are exempted from the requirement of...

  8. 40 CFR 174.501 - Bacillus thuringiensis Vip3Aa protein in corn and cotton; exemption from the requirement of a...

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... in corn and cotton; exemption from the requirement of a tolerance. 174.501 Section 174.501 Protection... thuringiensis Vip3Aa protein in corn and cotton; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Vip3Aa proteins in or on corn or cotton are exempt from the requirement of...

  9. 40 CFR 174.505 - Bacillus thuringiensis modified Cry3A protein (mCry3A) in corn; exemption from the requirement of...

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... protein (mCry3A) in corn; exemption from the requirement of a tolerance. 174.505 Section 174.505... thuringiensis modified Cry3A protein (mCry3A) in corn; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis modified Cry3A protein (mCry3A) in corn are exempt from the requirement...

  10. 40 CFR 174.506 - Bacillus thuringiensis Cry34Ab1 and Cry35Ab1 proteins in corn; exemption from the requirement of...

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... Cry35Ab1 proteins in corn; exemption from the requirement of a tolerance. 174.506 Section 174.506... thuringiensis Cry34Ab1 and Cry35Ab1 proteins in corn; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry34Ab1 and Cry35Ab1 proteins in corn are exempted from the requirement of...

  11. 40 CFR 174.506 - Bacillus thuringiensis Cry34Ab1 and Cry35Ab1 proteins in corn; exemption from the requirement of...

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... Cry35Ab1 proteins in corn; exemption from the requirement of a tolerance. 174.506 Section 174.506... thuringiensis Cry34Ab1 and Cry35Ab1 proteins in corn; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry34Ab1 and Cry35Ab1 proteins in corn are exempted from the requirement of...

  12. 40 CFR 174.505 - Bacillus thuringiensis modified Cry3A protein (mCry3A) in corn; exemption from the requirement of...

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... protein (mCry3A) in corn; exemption from the requirement of a tolerance. 174.505 Section 174.505... thuringiensis modified Cry3A protein (mCry3A) in corn; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis modified Cry3A protein (mCry3A) in corn are exempt from the requirement...

  13. 40 CFR 174.501 - Bacillus thuringiensis Vip3Aa protein in corn and cotton; exemption from the requirement of a...

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... in corn and cotton; exemption from the requirement of a tolerance. 174.501 Section 174.501 Protection... thuringiensis Vip3Aa protein in corn and cotton; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Vip3Aa proteins in or on corn or cotton are exempt from the requirement of...

  14. 40 CFR 174.501 - Bacillus thuringiensis Vip3Aa protein in corn and cotton; exemption from the requirement of a...

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... in corn and cotton; exemption from the requirement of a tolerance. 174.501 Section 174.501 Protection... thuringiensis Vip3Aa protein in corn and cotton; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Vip3Aa proteins in or on corn or cotton are exempt from the requirement of...

  15. 40 CFR 174.506 - Bacillus thuringiensis Cry34Ab1 and Cry35Ab1 proteins in corn; exemption from the requirement of...

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... Cry35Ab1 proteins in corn; exemption from the requirement of a tolerance. 174.506 Section 174.506... thuringiensis Cry34Ab1 and Cry35Ab1 proteins in corn; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry34Ab1 and Cry35Ab1 proteins in corn are exempted from the requirement of...

  16. 40 CFR 174.501 - Bacillus thuringiensis Vip3Aa protein in corn and cotton; exemption from the requirement of a...

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... in corn and cotton; exemption from the requirement of a tolerance. 174.501 Section 174.501 Protection... thuringiensis Vip3Aa protein in corn and cotton; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Vip3Aa proteins in or on corn or cotton are exempt from the requirement of...

  17. 40 CFR 174.532 - Bacillus thuringiensis eCry3.1Ab protein in corn; temporary exemption from the requirement of a...

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... protein in corn; temporary exemption from the requirement of a tolerance. 174.532 Section 174.532... thuringiensis eCry3.1Ab protein in corn; temporary exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis eCry3.1Ab protein in corn, in or on the food and feed commodities of corn;...

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

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... protein (mCry3A) in corn; exemption from the requirement of a tolerance. 174.505 Section 174.505... thuringiensis modified Cry3A protein (mCry3A) in corn; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis modified Cry3A protein (mCry3A) in corn are exempt from the requirement...

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

    PubMed

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

    2014-12-01

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

  20. Distribution of genes encoding putative virulence factors and fragment length polymorphisms in the vrrA gene among Brazilian isolates of Bacillus cereus and Bacillus thuringiensis.

    PubMed

    Zahner, Viviane; Cabral, Diana Aparecida; Régua-Mangia, Adriana Hamond; Rabinovitch, Leon; Moreau, Gaétan; McIntosh, Douglas

    2005-12-01

    One hundred twenty-one strains of the Bacillus cereus complex, of which 80 were isolated from a variety of sources in Brazil, were screened by PCR for the presence of sequences (bceT, hblA, nheBC, plc, sph, and vip3A) encoding putative virulence factors and for polymorphisms in variable-number tandem repeats (VNTR), using a variable region of the vrrA open reading frame as the target. Amplicons were generated from isolates of B. cereus and Bacillus thuringiensis for each of the sequences encoding factors suggested to play a role in infections of mammals. Intriguingly, the majority of these sequences were detected more frequently in Bacillus thuringiensis than in B. cereus. The vip3A sequence, which encodes an insecticidal toxin, was detected exclusively in B. thuringiensis. VNTR analysis demonstrated the presence of five different fragment length categories in both species, with two of these being widely distributed throughout both taxa. In common with data generated from previous studies examining European, Asian, or North American populations, our investigation of Brazilian isolates supports the notion that B. cereus and B. thuringiensis should be considered to represent a single species.

  1. Gut Bacteria Are Not Required for the Insecticidal Activity of Bacillus thuringiensis toward the Tobacco Hornworm, Manduca sexta▿ †

    PubMed Central

    Johnston, Paul R.; Crickmore, Neil

    2009-01-01

    It was recently proposed that gut bacteria are required for the insecticidal activity of the Bacillus thuringiensis-based insecticide, DiPel, toward the lepidopterans Manduca sexta, Pieris rapae, Vanessa cardui, and Lymantria dispar. Using a similar methodology, it was found that gut bacteria were not required for the toxicity of DiPel or Cry1Ac or for the synergism of an otherwise sublethal concentration of Cry1Ac toward M. sexta. The toxicities of DiPel and of B. thuringiensis HD73 Cry− spore/Cry1Ac synergism were attenuated by continuously exposing larvae to antibiotics before bioassays. Attenuation could be eliminated by exposing larvae to antibiotics only during the first instar without altering larval sterility. Prior antibiotic exposure did not attenuate Cry1Ac toxicity. The presence of enterococci in larval guts slowed mortality resulting from DiPel exposure and halved Cry1Ac toxicity but had little effect on B. thuringiensis HD73 Cry− spore/Cry1Ac synergism. B. thuringiensis Cry− cells killed larvae after intrahemocoelic inoculation of M. sexta, Galleria mellonella, and Spodoptera litura and grew rapidly in plasma from M. sexta, S. litura, and Tenebrio molitor. These findings suggest that gut bacteria are not required for B. thuringiensis insecticidal activity toward M. sexta but that B. thuringiensis lethality is reduced in larvae that are continuously exposed to antibiotics before bioassay. PMID:19525273

  2. Viability of Bacillus licheniformis and Bacillus thuringiensis Spores as a Model for Predicting the Fate of Bacillus anthracis Spores during Composting of Dead Livestock▿

    PubMed Central

    Reuter, Tim; Alexander, Trevor W.; McAllister, Tim A.

    2011-01-01

    Safe disposal of dead livestock and contaminated manure is essential for the effective control of infectious disease outbreaks. Composting has been shown to be an effective method of disposal, but no information exists on its ability to contain diseases caused by spore-forming bacteria, such as Bacillus anthracis. Duplicate composters (east and west), each containing 16 dead cattle, were constructed (final capacity, 85,000 kg). Spores (107 CFU/g manure) of Bacillus licheniformis and Bacillus thuringiensis were mixed with autoclaved feedlot manure and placed in either sterile vials or porous nylon bags. Compost temperatures in the west composter were slightly higher than in the east composter. Viable B. thuringiensis spores were reduced to ≤102 CFU in all samples after 112 days but were isolated from bags (west composter) at ≤102 and at 105 CFU (east composter) after 230 days. In contrast, B. licheniformis was at ≤102 CFU in vials (west composter) after 112 days but remained at 106 CFU after 230 days (east composter). Similarly, B. licheniformis in bags was not detected after 230 days in the west composter but remained at 107 CFU in the east composter. Our study suggests that spore viability was reduced in the west composter by exposure to compost and elevated temperatures over time. Different temperature profiles may explain why spores remained viable in the east structure but were largely rendered nonviable in the west structure. Under practical conditions, variation in composting microclimates may preclude the complete inactivation of Bacillus spores, including those of B. anthracis, during composting. However, composting may still have merit as a method of biocontainment, reducing and diluting the transfer of infectious spores into the environment. PMID:21193674

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

    PubMed

    Cheng, Feixue; Wang, Jian; Song, Zhiqiang; Cheng, Ju'e; Zhang, Deyong; Liu, Yong

    2015-09-20

    Bacillus thuringiensis is an important microbial biopesticide for controlling agricultural pests by the production of toxic parasporal crystals proteins.Here,we report the finished annotated genome sequence of B. thuringiensis YC-10,which is highly toxic to nematodes.The complete genome sequence consists of a circular chromosome and nine circular plasmids,which the biggest plasmid harbors six parasporal crystals proteins genes consisting of cry1Aa, cry1Ac, cry1Ia, cry2Aa, cry2Ab and cryB1. The crystals proteins of Cry1Ia and Cry1Aa have high nematicidal activity against Meloidogyne incognita.

  4. An amoeba phagocytosis model reveals a novel developmental switch in the insect pathogen Bacillus thuringiensis.

    PubMed

    Beeton, M L; Atkinson, D J; Waterfield, N R

    2013-02-01

    The Bacillus cereus group bacteria contain pathogens of economic and medical importance. From security and health perspectives, the lethal mammalian pathogen Bacillus anthracis remains a serious threat. In addition the potent insect pathogen Bacillus thuringiensis is extensively used as a biological control agent for insect pests. This relies upon the industrial scale induction of bacterial spore formation with the associated production of orally toxic Cry-toxins. Understanding the ecology and potential alternative developmental fates of these bacteria is therefore important. Here we describe the use of an amoeba host model to investigate the influence of environmental bactivorous protists on both spores and vegetative cells of these pathogens. We demonstrate that the bacteria can respond to different densities of amoeba by adopting different behaviours and developmental fates. We show that spores will germinate in response to factors excreted by the amoeba, and that the bacteria can grow and reproduce on these factors. We show that in low densities of amoeba, that the bacteria will seek to colonise the surface of the amoeba as micro-colonies, resisting phagocytosis. At high amoeba densities, the bacteria change morphology into long filaments and macroscopic rope-like structures which cannot be ingested due to size exclusion. We suggest these developmental fates are likely to be important both in the ecology of these bacteria and also during animal host colonisation and immune evasion.

  5. Bacillus thuringiensis monogenic strains: screening and interactions with insecticides used against rice pests

    PubMed Central

    Pinto, Laura M.N.; Dörr, Natália C.; Ribeiro, Ana Paula A.; de Salles, Silvia M.; de Oliveira, Jaime V.; Menezes, Valmir G.; Fiuza, Lidia M.

    2012-01-01

    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. PMID:24031872

  6. Efficiency of Intergeneric Recombinants Between Bacillus Thuringiensis and Bacillus Subtilis for Increasing Mortality Rate in Cotten Leaf Worm

    NASA Astrophysics Data System (ADS)

    AlOtaibi, Saad Aied

    2012-12-01

    In this study , two strains of Bacillus belonging to two serotypes and four of their transconjugants were screened with respect to their toxicity against lepidopterous cotton pest. . Bacterial transconjugants isolated from conjugation between both strains were evaluated for their transconjugant efficiency caused mortality in Spodoptera littoralis larvae . Two groups of bioinsecticides ; crystals , crystals and spores have been isolated from Bacillusstrains and their transconjugants . Insecticidal crystal protein ( ICP ) was specific for lepidopteran insects because of the toxin sufficient both for insect specificity and toxicity . The toxicities of these two groups against larvae of Spodoptera littoralis was expressed as transconjugant efficiency , which related to the mean number of larvae died expressed as mortality percentage . The results showed transconjugant efficiency in reducing the mean number of Spodoptera littoralis larvae feeding on leaves of Ricinus communis sprayed with bioinsecticides of Bt transconjugants. Most values of positive transconjugant efficiency related to increasing mortality percentage are due to toxicological effects appeared in response to the treatments with crystals + endospores than that of crystals alone .This indicated that crystals + endospores was more effective for increasing mortality percentage than that resulted by crystals . Higher positive transconjugant efficiency in relation to the mid parents and better parent was appeared at 168 h of treatment . The results indicated that recombinant Bacillus thuringiensis are important control agents for lepidopteran pests , as well as , susceptibility decreased with larval development . The results also suggested a potential for the deployment of these recominant entomopathogens in the management of Spodoptera. littoralis larvae .

  7. Mosquito biolarvicide production by sequential fermentation with dual strains of Bacillus thuringiensis subsp. israelensis and Bacillus sphaericus using sewage sludge.

    PubMed

    Zhuang, Li; Zhou, Shungui; Wang, Yueqiang; Chang, Min

    2011-01-01

    This study demonstrated the bioconversion of sewage sludge into a composite biolarvicide for mosquito control based on sequential fermentation with dual strains of Bacillus thuringiensis subsp. israelensis (Bti) and Bacillus sphaericus (Bs). Results showed that sewage sludge was a suitable fermentation substrate for supporting growth, sporulation and mosquitocidal proteins synthesis by Bti and Bs. Through sequential fermentation with dual strains, a 10-L bench-scale fermentor was capable of producing Bti and Bs at a cell concentration of 2.1×10(9) and 6.8×10(8) CFU/mL, respectively. Such sequential fermentation can save half of raw materials and energy consumption comparing with the sludge fermentation with single strain. The toxic activity and persistence of the composite biolarvicide against mosquito larvae in the polluted waters were enhanced by the increased toxin complexity and synergistic interactions. This study, for the first time, validates the technical feasibility of using sewage sludge to produce a cost-effective composite biolarvicide based on Bti and Bs.

  8. Insecticidal Activity and Histopathological Effects of Vip3Aa Protein from Bacillus thuringiensis on Spodoptera litura.

    PubMed

    Song, Feifei; Lin, Yunfeng; Chen, Chen; Shao, Ensi; Guan, Xiong; Huang, Zhipeng

    2016-10-28

    Vegetative insecticidal proteins (Vips) are insecticidal proteins synthesized by Bacillus thuringiensis during the vegetative stage of growth. In this study, Vip3Aa protein, obtained by in vitro expression of the vip3Aa gene from B. thuringiensis WB5, displayed high insecticidal activity against Spodoptera litura aside from Spodoptera exigua and Helicoverpa armigera. Bioassay results showed that the toxicity of Vip3Aa protein against S. litura larvae statistically decreased along with the increase of the age of the larvae, with LC50 = 2.609 ng/cm(2) for neonatal larvae, LC50 = 28.778 ng/cm(2) for first instar larvae, LC50 = 70.460 ng/cm(2) for second instar larvae, and LC50 = 200.627 ng/cm(2) for third instar larvae. The accumulative mortality of 100% larvae appeared at 72 h for all instars of S. litura larvae, when feeding respectively with 83.22, 213.04, 341.40, and 613.20 ng/cm(2) of Vip3Aa toxin to the neonatal and first to third instar larvae. The histopathological effects of Vip3Aa toxin on the midgut epithelial cells of S. litura larvae was also investigated. The TEM observations showed wide damage of the epithelial cell in the midgut of S. litura larvae fed with Vip3Aa toxin.

  9. Microbial control of the cotton leafworm Spodoptera littoralis (Boisd.) by Egyptian Bacillus thuringiensis isolates.

    PubMed

    Alfazairy, Ahlam A; El-Ahwany, Amani M D; Mohamed, Eman A; Zaghloul, Heba A H; El-Helow, Ehab R

    2013-03-01

    Four local Bacillus thuringiensis (Bt) isolates that had been serologically identified as Bt var. kurstaki (Btk2, Btk3, and Btk66) and Bt var. mexicanensis (Btm27), in addition to two reference strains (4D20 and 4AC1), were laboratory assayed as microbial control agents against the Egyptian cotton leafworm Spodoptera littoralis (Boisd.). Polymerase chain reaction (PCR) amplification analysis revealed that each of the six experimental strains carries, at least, a cry1 type gene which expresses a protein toxin active against lepidopterous insects. Additionally, PCR amplification results demonstrated that 4D20 and Btk66 contain the Lepidoptera- and Diptera-active cry2 type gene and that Btk66 contains Coleoptera-active cry7 and cry8 genes. Among the six strains, Btk66 and Btm27 were the most promising microbial control agents against S. littoralis. The present findings were the first to report that Btm27 (classified as B. thuringiensis var. mexicanensis) is a very potent microbial control agent against S. littoralis-tested larvae. For more characterization of these two isolates, the sspO gene was investigated as a molecular chronometer. The DNA sequencing results proved that Btk66 and Btm27 carry sspO open reading frames with identical nucleotide sequences, suggesting a strong phylogenetic relationship between the two strains.

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

    PubMed

    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.

  11. Novel toxicity of Bacillus thuringiensis strains against the melon fruit fly, Bactrocera cucurbitae (Diptera: Tephritidae).

    PubMed

    Shishir, Md Asaduzzaman; Akter, Asma; Bodiuzzaman, Md; Hossain, M Aftab; Alam, Md Musfiqul; Khan, Shakil Ahmed; Khan, Shakila Nargis; Hoq, M Mozammel

    2015-01-01

    Bactrocera cucurbitae (melon fruit fly) is one of the most detrimental vegetable-damaging pests in Bangladesh. The toxicity of Bacillus thuringiensis (Bt) has been reported against a few genera of Bactrocera in addition to numerous other insect species. Bt strains, harbouring cry1A-type genes were, therefore, assayed in vivo against the 3(rd) instar larvae of B. cucurbitae in this study. The biotype-based prevalence of cry1 and cry1A genes was calculated to be 30.8% and 11.16%, respectively, of the test strains (n=224) while their prevalence was greatest in biotype kurstaki. Though three indigenous Bt strains from biotype kurstaki with close genetic relationship exhibited higher toxicity, maximum mortalities were recorded for Btk HD-73 (96%) and the indigenous Bt JSc1 (93%). LC50 and LC99 values were determined to be 6.81 and 8.32 for Bt JSc1, 7.30 and 7.92 for Bt SSc2, and 6.99 and 7.67 for Btk HD-73, respectively. The cause of toxicity and its variation among the strains was found to be correlated with the synergistic toxic effects of cry1, cry2, cry3 and cry9 gene products, i.e. relevant Cry proteins. The novel toxicity of the B. thuringiensis strains against B. cucurbitae revealed in the present study thus will help in developing efficient and eco-friendly control measures such as Bt biopesticides and transgenic Bt cucurbits.

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

    PubMed

    Rubio-Infante, Néstor; Moreno-Fierros, Leticia

    2016-05-01

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

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

    PubMed

    Sheppard, Anna E; Nakad, Rania; Saebelfeld, Manja; Masche, Anna C; Dierking, Katja; Schulenburg, Hinrich

    2016-01-01

    In bacterial pathogens, virulence factors are often carried on plasmids and other mobile genetic elements, and as such, plasmid evolution is central in understanding pathogenicity. Bacillus thuringiensis is an invertebrate pathogen that uses plasmid-encoded crystal (Cry) toxins to establish infections inside the host. Our study aimed to quantify stability of two Cry toxin-encoding plasmids, BTI_23p and BTI_16p, under standard laboratory culturing conditions. These two plasmids are part of the genome of the B. thuringiensis strain MYBT18679, which is of particular interest because of its high pathogenicity towards nematodes. One of the plasmids, BTI_23p, was found to be highly unstable, with substantial loss occurring within a single growth cycle. Nevertheless, longer term experimental evolution in the absence of a host revealed maintenance of the plasmid at low levels in the bacterial populations. BTI_23p encodes two nematicidal Cry toxins, Cry21Aa2 and Cry14Aa1. Consistent with previous findings, loss of the plasmid abolished pathogenicity towards the nematode Caenorhabditis elegans, which could be rescued by addition of Cry21Aa2-expressing Escherichia coli. These results implicate BTI_23p as a plasmid that is required for successful infection, yet unstable when present at high frequency in the population, consistent with the role of Cry toxins as public goods.

  14. Expression in Escherichia coli of a cloned crystal protein gene of Bacillus thuringiensis subsp. israelensis.

    PubMed Central

    McLean, K M; Whiteley, H R

    1987-01-01

    A ca. 10-kilobase (kb) HindIII fragment of plasmid DNA from Bacillus thuringiensis subsp. israelensis was cloned into plasmid pUC9 and transformed into Escherichia coli. Extracts of the recombinant strain contained a 27-kilodalton (kDa) peptide that reacted with antibodies to a 27-kDa peptide isolated from crystals produced by B. thuringiensis subsp. israelensis. Extracts of the recombinant strain were hemolytic and toxic to Aedes aegypti larvae. Full expression of the 27-kDa peptide required the presence of a ca. 0.8-kb region of DNA located 4 kb upstream from the structural gene; the 0.8-kb region could be present in cis or trans relative to the gene and apparently acted post-transcriptionally. Analysis of maxicells showed that the 10-kb insert also coded for peptides of 67, 20, and 16 kDa; data obtained with different subclones suggest that the 20-kDa peptide is encoded in the 0.8-kb DNA region. Images PMID:3546262

  15. Enhancement of heavy metal phytoremediation by Alnus firma with endophytic Bacillus thuringiensis GDB-1.

    PubMed

    Babu, A Giridhar; Kim, Jong-Dae; Oh, Byung-Taek

    2013-04-15

    Phytoremediation shows potential for remediating mine tailing sites contaminated with heavy metals. Our aim was to isolate, characterize, and assess the potential of endophytic bacteria to enhance growth and metal accumulation by the hyperaccumulator Alnus firma. A bacterial strain isolated from roots of Pinus sylvestris had the capacity to remove heavy metals from mine tailing and was identified as Bacillus thuringiensis GDB-1 based on 16S ribosomal DNA sequencing. GDB-1 exhibited plant growth-promoting traits, including 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase activity, indole acetic acid (IAA) and siderophore production, and P solubilization. The efficiency of GDB-1 to remove heavy metals was influenced by pH and initial metal concentration. Removal capacity (mg/l) was 77% for Pb (100), 64% for Zn (50), 34% for As (50), 9% for Cd (10), 8% for Cu (10), and 8% for Ni (10) during the active growth cycle in heavy metal-amended, mine tailing extract medium. Inoculating soil with GDB-1 significantly increased biomass, chlorophyll content, nodule number, and heavy metal (As, Cu, Pb, Ni, and Zn) accumulation in A. firma seedlings. Results indicate that inoculating the native plant A. firma with B. thuringiensis GDB-1 improves its efficiency for phytoremediation of soil containing mine tailings contaminated with heavy metals.

  16. Electron Microscope Study of Sporulation and Parasporal Crystal Formation in Bacillus thuringiensis

    PubMed Central

    Bechtel, Donald B.; Bulla, Lee A.

    1976-01-01

    A comprehensive ultrastructural analysis of sporulation and parasporal crystal development is described for Bacillus thuringiensis. The insecticidal crystal of B. thuringiensis is initiated at the start of engulfment and is nearly complete by the time the exosporium forms. The crystal and a heretofore unobserved ovoid inclusion develop without any clear association with the forespore septum, exosporium, or mesosomes. These observations contradict previous hypotheses that the crystal is synthesized on the forespore membrane, exosporium, or mesosomes. Formation of forespore septa involves densely staining, double-membrane-bound, vesicular mesosomes that have a bridged appearance. Forespore engulfment is subpolar and also involves mesosomes. Upon completion of engulfment the following cytoplasmic changes occur: decrease in electron density of the incipient forespore membrane; loss of bridged appearance of incipient forespore membrane; change in stainability of incipient forespore, forespore, and mother cell cytoplasms; and alteration in staining quality of plasma membrane. These changes are involved in the conversion of the incipient forespore into a forespore and reflect “commitment” to sporulation. Images PMID:182671

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

    PubMed Central

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

    2016-01-01

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

  18. Preparation of nanoscale Bacillus thuringiensis chitinases using silica nanoparticles for nematicide delivery.

    PubMed

    Qin, Xu; Xiang, Xuemei; Sun, Xiaowen; Ni, Hong; Li, Lin

    2016-01-01

    A series of amino, carboxylic, and aldehydic surface-grafted silica nanoparticles (SNPs) was prepared based on SiO2 NYSi40 nanoparticles to develop an efficient, biocompatible, and cost-effective biopesticide delivery system. Bacillus thuringiensis chitinase (Chi9602) was immobilized onto SNP surface to prepare nanoscale chitinases (SNPCs) through electrostatic adsorption and covalent binding. The specimens were characterized by Fourier transform infrared, scanning electron microscopy, and zeta-potential analyses. The delivery capacity of the SNPs in Caenorhabditis elegans N2 was observed by immunofluorescence. Results demonstrated that amino-grafted SiO2 nanoparticles with Chi9602 electrostatically adsorbed onto their surface (SNPC2) exhibited a relatively high enzyme immobilization rate (80.2%) and the highest (94.1%) residual enzyme activity among all SNPCs. SNPC2 also showed wider pH tolerance and relatively higher thermostability and ultraviolet radiation resistance capacity than Chi9602. Bioassays further showed that SNPC2 synergistically enhanced the nematicidal effect of B. thuringiensis YBT-020 preparation against C. elegans, with a reduced LC50 of 8.35mg/mL and a shortened LT50 of 12.04h. Immunofluorescence assays showed that SNPC2 had considerable delivery capacity to carry a large protein into C. elegans. Therefore, SNP2 can serve as an efficient nanocarrier for the delivery of macromolecular proteic biopesticides or drugs, indicating potential agricultural or biotechnological applications.

  19. Interaction between Calcium Ions and Bacillus thuringiensis Toxin Activity against Sf9 Cells (Spodoptera frugiperda, Lepidoptera)

    PubMed Central

    Monette, R.; Potvin, L.; Baines, D.; Laprade, R.; Schwartz, J. L.

    1997-01-01

    The effects of calcium ions and modulators of calcium movement on Bacillus thuringiensis insecticidal protein toxicity were investigated with Sf9 cells (Spodoptera frugiperda, fall armyworm) by a new B. thuringiensis toxicity assay based on measurement of fluorescence of ethidium homodimer, a high-affinity DNA stain. CryIC toxicity was substantially stimulated by extracellular calcium in a dose-dependent way (in the millimolar range), while toxicity enhancement could not be replicated when calcium was replaced by barium. This incremental toxicity was reduced by cobalt and lanthanum ions, two inorganic-calcium transport inhibitors. Methoxyverapamil, a voltage-dependent calcium channel blocker, and nifedipine, an inhibitor of dihydropyridine-sensitive L-type calcium channels, had no effect on CryIC toxin activity, but BAY K 8644, an L-type calcium channel activator, increased CryIC activity at high concentrations of extracellular calcium. While A23187, a calcium ionophore, and TMB-8, an inhibitor of intracellular-calcium mobilization, did not change CryIC-induced mortality, thapsigargin, an inhibitor of calcium uptake in intracellular stores, and more particularly trifluoperazine, which inhibits calcium-calmodulin-dependent processes, increased CryIC-mediated toxicity. The incremental effect of extracellular calcium on CryIC-induced toxicity was consistent with an increased concentration of intracellular calcium. PMID:16535509

  20. Production of an Insecticidal Crystal Protein from Bacillus thuringiensis by the Methylotroph Methylobacterium extorquens▿

    PubMed Central

    Choi, Young J.; Gringorten, J. Lawrence; Bélanger, Louise; Morel, Lyne; Bourque, Denis; Masson, Luke; Groleau, Denis; Míguez, Carlos B.

    2008-01-01

    The Cry1Aa protein from Bacillus thuringiensis is an insecticidal protein that is highly active against several species of Lepidoptera. We cloned and expressed the cry1Aa gene in a plant-colonizing methylotroph, Methylobacterium extorquens, under the control of the strong M. extorquens AM1 methanol dehydrogenase promoter, PmxaF. Transmission electron microscopy revealed characteristic bipyramidal intracellular δ-endotoxin crystals similar to the crystalline inclusions formed by B. thuringiensis. Both the protoxin protein and the activated toxin were visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western analysis. In single-dose assays of the recombinant against the silkworm, Bombyx mori, both whole cells and cell lysates caused rapid feeding inhibition followed by mortality. The biomass and growth rate of recombinant cells in shake flask culture were similar to those of the wild-type strain, indicating a lack of fitness cost to the recombinant under controlled culture conditions. Recombinant Cry1Aa was expressed at a level of 4.5% of total M. extorquens cell protein. The potential benefits of modifying M. extorquens to deliver insecticidal Cry proteins for crop and forest protection are discussed. PMID:18552184

  1. Effects of Bacillus thuringiensis strains virulent to Varroa destructor on larvae and adults of Apis mellifera.

    PubMed

    Alquisira-Ramírez, Eva Vianey; Peña-Chora, Guadalupe; Hernández-Velázquez, Víctor Manuel; Alvear-García, Andrés; Arenas-Sosa, Iván; Suarez-Rodríguez, Ramón

    2017-04-04

    The sublethal effects of two strains of Bacillus thuringiensis, which were virulent in vitro to Varroa destructor, were measured on Apis mellifera. The effects of five concentrations of total protein (1, 5, 25, 50 and 100μg/mL) from the EA3 and EA26.1 strains on larval and adult honey bees were evaluated for two and seven days under laboratory conditions. Based on the concentrations evaluated, total protein from the two strains did not affect the development of larvae, the syrup consumption, locomotor activity or proboscis extension response of adults. These same parameters were also tested for the effects of three concentrations (1, 10 and 15μg/kg) of cypermethrin as a positive control. Although no significant differences were observed after two days of treatment with cypermethrin, a dose-response relationship in syrup consumption and locomotor activity was observed. A significant reduction in the proboscis extension response of the bees treated with cypermethrin was also observed. Therefore, in contrast to cypermethrin, our results indicate that the EA3 and EA26.1 strains of B. thuringiensis can be used in beehives to control V. destructor and reduce the negative effects of this mite on colonies without adverse effects on the larvae and adults of A. mellifera. Additionally, the overuse of synthetic miticides, which produce both lethal and sublethal effects on bees, can be reduced.

  2. Characterisation of novel Bacillus thuringiensis isolates against Aedes aegypti (Diptera: Culicidae) and Ceratitis capitata (Diptera: Tephridae).

    PubMed

    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.

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

    PubMed

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

    2016-01-01

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

  4. Insecticidal Activity of Bacillus thuringiensis Strains Isolated from Soil and Water

    PubMed Central

    Konecka, Edyta; Baranek, Jakub; Hrycak, Anita; Kaznowski, Adam

    2012-01-01

    We attempted to search novel Bacillus thuringiensis strains that produce crystals with potential utility in plant protection and with higher activity than strains already used in biopesticide production. Seven B. thuringiensis soil and water isolates were used in the research. We predicted the toxicity of their crystals by cry gene identification employing PCR method. The isolate MPU B63 with interesting, according to us, genes content was used in evaluating its crystal toxicity against Cydia pomonella caterpillars. The strain MPU B63 was cultured from water sample and had cry1Ab, cry1B, and cry15 genes. The LC50 crystals of MPU B63 were compared to LC50 of commercial bioinsecticide Foray determined against C. pomonella (codling moth). The activity of MPU B63 inclusions against codling moth larvae was approximately 24-fold higher than that of Foray. The results are a promising introduction for further study evaluating the potential usefulness of isolate MPU B63 crystals in plant protection. PMID:22666145

  5. Susceptibility of Grapholita molesta (Busck, 1916) to formulations of Bacillus thuringiensis, individual toxins and their mixtures.

    PubMed

    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.

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

    PubMed

    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. Expression of Vitreoscilla hemoglobin in Bacillus thuringiensis improve the cell density and insecticidal crystal proteins yield.

    PubMed

    Liang, Feng; Shouwen, Chen; Ming, Sun; Ziniu, Yu

    2007-02-01

    The Vitreoscilla hemoglobin (VHb) gene (vgb) was integrated into the chromosome of Bacillus thuringiensis BMB171 using integrative vector pEG491. The production of VHb was confirmed by CO-difference spectra analysis. Fermentation experiments results showed that with the production of VHb, the critical oxygen concentration (COC) of the host strain was reduced from 18 to 12%. The maximum viable cell counts of the VHb+ strain in high, middle, and low aeration/agitation fermentations were 0.94-, 1.23-, and 1.59-fold of those of the VHb- strain, respectively. Under the same conditions, the yields of insecticidal crystal proteins (ICP) by VHb+ strain were 1.22-, 1.63-, and 3.13-fold of those of the VHb- strain. The production of VHb also accelerated the formation of ICP and spores. These results indicated that the production of VHb could improve the cell density and ICP yield of B. thuringiensis, especially under low aeration/agitation condition.

  8. Toxicological safety assessment of genetically modified Bacillus thuringiensis with additional N-acyl homoserine lactonase gene.

    PubMed

    Peng, Donghai; Zhou, Chenfei; Chen, Shouwen; Ruan, Lifang; Yu, Ziniu; Sun, Ming

    2008-01-01

    The aim of the present study is to evaluate the toxicology safety to mammals of a genetically modified (GM) Bacillus thuringiensis with an additional N-acyl homoserine lactones gene (aiiA), which possesses insecticidal activity together with restraint of bacterial pathogenicity and is intended for use as a multifunctional biopesticide. Safety assessments included an acute oral toxicity test and 28-d animal feeding study in Wistar rats, primary eye and dermal irritation in Zealand White rabbits, and delayed contact hypersensitivity in guinea pigs. Tests were conducted using spray-dried powder preparation. This GM product showed toxicity neither in oral acute toxicity test nor in 28-d animal feeding test at a dose of 5,000 mg/kg body weight. During the animal feeding test, there were no significant differences in growth, food and water consumption, hematology, blood biochemical indices, organ weights, and histopathology finding between rats in controls and tested groups. Tested animals in primary eye and dermal irritation and delayed contact hypersensitivity test were also devoid of any toxicity compared to controls. All the above results demonstrated that the GM based multifunctional B. thuringiensis has low toxicity and low eye and dermal irritation and would not cause hypersensitivity to laboratory mammals and therefore could be regarded as safe for use as a pesticide.

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

    PubMed

    Starzak, M; Bajpai, R K

    1991-01-01

    A mathematical model has been developed for the delta-endotoxin producing Bacillus thuringiensis. The structure of the model involves the processes taking place during vegetative growth, those leading to the initiation of sporulation under conditions of carbon and/or nitrogen limitation, and the sporulation events. The key features in the model are the pools of compounds, such as PRPP, IMP, ADP/ATP, GDP/GTP, pyrimidine nucleotides, NAD/NADH2, amino acids, nucleic acids, cell wall, and vegetative and sporulation proteins. These, along with sigma-factors that control the nature of RNA-polymerase during the different phases, effectively stimulate the vegetative growth and sporulation. The initiation of sporulation is controlled by the intracellular concentration of GTP. Results of simulation of vegetative growth, initiation of sporulation, spore protein formation, and production of delta-endotoxin under C- or N-limitation are presented.

  10. Mobilization of Bacillus thuringiensis plasmid pTX14-3.

    PubMed

    Andrup, L; Bendixen, H H; Jensen, G B

    1995-05-01

    The Bacillus thuringiensis subsp. israelensis (Bti) plasmid pTX14-3 has been reported to contain a gene, mob14-3, with considerable homology to genes encoding mobilization proteins from other gram-positive bacteria. We have used the aggregation-mediated conjugation system recently discovered in Bti to compare the mobilization kinetics of different derivatives of plasmid pTX14-3. Plasmid pTX14-3 has been found to replicate by the rolling-circle mechanism and to contain a locus suppressing the formation of high-molecular-weight DNA. We found that deleting a DNA fragment containing this locus increased the transfer frequency about twofold. The mobilization frequency of the plasmid containing the intact mob14-3 gene did not indicate a mobilization-enhancing activity of the encoded polypeptide. However, the presence of the mob14-3 gene seemed to increase the stability of the plasmid in exponential growth.

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

    PubMed

    Gryspeirt, Aiko; Grégoire, Jean-Claude

    2012-05-01

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

  12. Variations in the mosquito larvicidal activities of toxins from Bacillus thuringiensis ssp. israelensis.

    PubMed

    Otieno-Ayayo, Zachariah Ngalo; Zaritsky, Arieh; Wirth, Margaret C; Manasherob, Robert; Khasdan, Vadim; Cahan, Rivka; Ben-Dov, Eitan

    2008-09-01

    Comparing activities of purified toxins from Bacillus thuringiensis ssp. israelensis against larvae of seven mosquito species (vectors of tropical diseases) that belong to three genera, gleaned from the literature, disclosed highly significant variations in the levels of LC(50) as well as in the hierarchy of susceptibilities. Similar toxicity comparisons were performed between nine transgenic Gram-negative species, four of which are cyanobacterial, expressing various combinations of cry genes, cyt1Aa and p20, against larvae of four mosquito species as potential agents for biological control. Reasons for inconsistencies are listed and discussed. Standard conditions for toxin isolation and presentation to larvae are sought. A set of lyophilized powders prepared identically from six Escherichia coli clones expressing combinations of four genes displayed toxicities against larvae of three mosquito species, with levels that differed between them but with identical hierarchy.

  13. Occurrence and linkage between secreted insecticidal toxins in natural isolates of Bacillus thuringiensis.

    PubMed

    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.

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

    PubMed

    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.

  15. [Modification and expression of insecticidal protein structural gene of Bacillus thuringiensis var. aizawai 7-29].

    PubMed

    Guo, S; Hong, Z; Wang, J; Wang, M; Yu, M; Fan, Y

    1992-06-01

    The regulative region (181bp) and the fifth toxic active domain (217bp) were removed from the insecticidal protein gene of Bacillus thuringiensis var. aizawai 7-29. After the synthesis of the adaptor (15bp) that contains initiation codon (ATG) and the PCR synthesis of the fifth toxic active domain (229bp) that contains stop codon (TAA), were inserted into on 5' truncated and 3' truncated of the coding fod N-terminal peptid's DNA fragment, that to become a modified structural gene. The modified structural gene can be play initiatic translation-function and stop translation-function during translation of insecticidal protein. The insecticidal protein was determined by western blotting, showed the expression of modified structural gene in Escherichia coli JM 103. The bioassay of insecticidal proteins showed the 3' truncated and 5' truncated of insecticidal gene was higher toxic active than the 3' truncated of insecticidal gene in Escherichia coli JM 103.

  16. Development of Bacillus thuringiensis fermentation and process control from a practical perspective.

    PubMed

    Yang, X M; Wang, S S

    1998-10-01

    Bacillus thuringiensis (Bt) is the most widely used biopesticide producer in the biological control market. It is very critical for the Bt pesticide industry to be able to achieve a high yield in the Bt fermentation process in order to reduce its cost and compete with chemical pesticides in the market. We review the overall development of Bt fermentation process research and provide our point of view for the future research opportunities and potential improvements. This minireview covers the areas of fermentation physiology, growth dynamics and high-yield process control. It is pointed out that many studies aimed to improve spore count and process research focusing on toxin protein yield is lacking. In addition, significant development opportunities reside in the process development for the genetically engineered Bt strains expressing multiple toxin proteins.

  17. Novel strategy for protein production using a peptide tag derived from Bacillus thuringiensis Cry4Aa.

    PubMed

    Hayakawa, Tohru; Sato, Shinya; Iwamoto, Shigehisa; Sudo, Shigeo; Sakamoto, Yoshiki; Yamashita, Takaaki; Uchida, Motoaki; Matsushima, Kenji; Kashino, Yohko; Sakai, Hiroshi

    2010-07-01

    Numerous proteins cannot be sufficiently prepared by ordinary recombinant DNA techniques because they are unstable or have deleterious effects on the host cell. One idea to prepare such proteins is to produce them as protein inclusions. Here we developed a novel system to effectively prepare proteins by using peptide tags derived from the insecticidal Cry toxin of a soil bacterium, Bacillus thuringiensis. Fusion with this peptide tag, designated 4AaCter, facilitates the formation of protein inclusions of glutathione S-transferase in Escherichia coli without losing the enzyme activity. Application of 4AaCter to the production of syphilis antigens TpN15, TpN17 and TpN47 from Treponema pallidum yielded excellent results, including a dramatic increase in the production level, simplification of the product purification and high reactivity with syphilis antibody. The use of 4AaCter may provide an innovational strategy for the efficient production of proteins.

  18. Overexpression of polyphosphate kinase gene (ppk) increases bioinsecticide production by Bacillus thuringiensis.

    PubMed

    Doruk, Tugrul; Avican, Ummehan; Camci, Irem Yalim; Gedik, Sedef Tunca

    2013-05-06

    Polyphosphate (polyP), synthesized by polyP kinase (PPK) using the terminal phosphate of ATP as substrate, performs important functions in every living cell. The present work reports on the relationship between polyP metabolism and bioinsecticide production in Bacillus thuringiensis subsp. israelensis (Bti). The ppk gene of Bti was cloned into vector pHT315 and the effect of its overexpression on endotoxin production was determined. Endotoxin production by the recombinant strain was found to be consistently higher than that by the wild type strain and the strain that carried the empty plasmid. The toxicity of the recombinant mutant strain (LC50 5.8±0.6ngml(-1)) against late 2nd instar Culex quinquefasciatus was about 7.7 times higher than that of Bti (LC50 44.9±7ngml(-1)). To our knowledge this is the first reported study which relates polyP metabolism with bioinsecticide biosynthesis.

  19. Pretreatment of poultry litter improves Bacillus thuringiensis-based biopesticides production.

    PubMed

    Ozcan, Orhan; Icgen, Bulent; Ozcengiz, Gulay

    2010-04-01

    Pretreated poultry litter was used in batch cultures for the production of Bacillus thuringiensis (Bt)-based biopesticide of lepidoptera- and diptera-specific Cry1 and Cry2, diptera-specific Cry4Ba and Cry11Aa and coleoptera-specific Cry3Aa toxins by Bt subsp. kurstaki 81, subsp. israelensis HD500 and subsp. tenebrionis 3203, respectively. Bt kurstaki 81 showed improved growth and produced more toxin in this medium as compared to other subspecies. Base and acid hydrolysis were tested as the methods of substrate pretreatment. The use of poultry litter pretreated with 2N HCl yielded 94% more bioinsecticidal protein than 2N NaOH-pretreated poultry litter when Bt kurstaki 81 was cultured. With appropriate pretreatment, poultry litter demonstrated potential as a valuable raw material for a low-cost complex medium to produce Bt-based biopesticides.

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

    SciTech Connect

    Starzak, M.; Bajpai, R.K.

    1991-12-31

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

  1. Analysis of insecticidal proteins from Bacillus thuringiensis and recombinant Escherichia coli by capillary electrokinetic chromatography.

    PubMed

    Luong, John H T; Male, Keith B; Mazza, Alberto; Masson, Luke; Brousseau, Roland

    2004-10-01

    Bacillus thuringiensis and recombinant Escherichia coli proteinaceous protoxins were subject to proteolysis and analyzed by capillary electrokinetic chromatography. Three resulting toxins (65 kDa) were baseline-resolved within 22 min using a 10 mM borate, pH 11 separation buffer consisting of 25 mM sodium dodecyl sulfate (SDS) and 30 mM phytic acid. The toxins displayed differential interactions with the SDS and phytic acid phases to effect their separation. The ion-pairing interaction between the analyte and phytic acid was also useful in preventing adsorption to the capillary walls and thus enhanced separation resolution and efficiency. The use of electrokinetic chromatography allows achievement of the separation in a significantly shorter time than conventional high-performance liquid chromatography (HPLC) using a diethylaminoethyl (DEAE) weak-anion exchanger.

  2. Growth, sporulation, delta-endotoxins synthesis, and toxicity during culture of bacillus thuringiensis H14.

    PubMed

    Sarrafzadeh, Mohammad H; Guiraud, Joseph P; Lagneau, Christophe; Gaven, Bruno; Carron, Alexandre; Navarro, Jean-Marie

    2005-08-01

    Growth, sporulation, synthesis of delta-endotoxins, and toxicity against the larvae of Aedes aegypti and Culex pipiens were studied during fermentation of Bacillus thuringiensis H14 in a 20-L fermentor. Measurements of optical density and dielectric permittivity for biomass determination suggest a highly promising technique for on-line evaluation of sporulation. The synthesis of 65-, 25- and 130-kDa proteins started at 16, 18, and 23 h, respectively. These proteins were enriched in different ways until the end of culture (48 h). Toxicity in the course of sporulation was significantly different for the larvae of both mosquito species. Maximal activity against Ae. aegypti was obtained at the end of culture, whereas for Cx. pipiens, the sample at 38 h was the most active.

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

    PubMed Central

    Gryspeirt, Aiko; Grégoire, Jean-Claude

    2012-01-01

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

  4. Friction and Adhesion Forces of Bacillus thuringiensis Spores on Planar Surfaces in Atmospheric Systems

    SciTech Connect

    Kweon, Hyojin; Yiacoumi, Sotira; Tsouris, Costas

    2011-01-01

    The kinetic friction force and the adhesion force of Bacillus thuringiensis spores on planar surfaces in atmospheric systems were studied using atomic force microscopy. The influence of relative humidity (RH) on these forces varied for different surface properties including hydrophobicity, roughness, and surface charge. The friction force of the spore was greater on a rougher surface than on mica, which is atomically flat. As RH increases, the friction force of the spores decreases on mica whereas it increases on rough surfaces. The influence of RH on the interaction forces between hydrophobic surfaces is not as strong as for hydrophilic surfaces. The friction force of the spore is linear to the sum of the adhesion force and normal load on the hydrophobic surface. The poorly defined surface structure of the spore and the adsorption of contaminants from the surrounding atmosphere are believed to cause a discrepancy between the calculated and measured adhesion forces.

  5. Characterization of a highly pathogenic Bacillus thuringiensis strain isolated from common cockchafer, Melolontha melolontha.

    PubMed

    Kati, H; Sezen, K; Demirbağ, Z

    2007-01-01

    A bacterial isolate (Mm2) of Melolontha melolontha was identified and characterized. Based on various morphological, physiological, biochemical and molecular characteristics, it was identified as Bacillus thuringiensis subsp. tenebrionis. This isolate was compared to the reference strains by electron microscopy, SDS-PAGE analysis, plasmid pattern, cry gene content and insecticidal activity. Cells of the isolate harbored flat square inclusions containing a protein component of approximately equal to65 kDa. After trypsin digestion of solubilized crystals, SDS-PAGE resolved a unique proteinase-resistant peptide of approximately equal to 50 kDa. Plasmid pattern showed similar bands to those of the reference strain, PCR analysis showed that the isolate has cry3 gene. Toxicity tests (against 5 coleopteran species) showed 80 % insecticidal activity against the larvae of M. melolontha. The isolate Mm2 may be valuable as biological control agent for M. melolontha and other coleopteran insects.

  6. Bacillus thuringiensis as a specific, safe, and effective tool for insect pest control.

    PubMed

    Roh, Jong Yul; Choi, Jae Young; Li, Ming Shun; Jin, Byung Rae; Je, Yeon Ho

    2007-04-01

    Bacillus thuringiensis (Bt) was first described by Berliner [10] when he isolated a Bacillus species from the Mediterranean flour moth, Anagasta kuehniella, and named it after the province Thuringia in Germany where the infected moth was found. Although this was the first description under the name B. thuringiensis, it was not the first isolation. In 1901, a Japanese biologist, Ishiwata Shigetane, discovered a previously undescribed bacterium as the causative agent of a disease afflicting silkworms. Bt was originally considered a risk for silkworm rearing but it has become the heart of microbial insect control. The earliest commercial production began in France in, 1938, under the name Sporeine [72]. A resurgence of interest in Bt has been attributed to Edward Steinhaus [105], who obtained a culture in 1942 and attracted attention to the potential of Bt through his subsequent studies. In 1956, T. Angus [3] demonstrated that the crystalline protein inclusions formed in the course of sporulation were responsible for the insecticidal action of Bt. By the early 1980's, Gonzalez et al. [48] revealed that the genes coding for crystal proteins were localized on transmissible plasmids, using a plasmid curing technique, and Schnepf and Whiteley [103] first cloned and characterized the genes coding for crystal proteins that had toxicity to larvae of the tobacco hornworm, from plasmid DNA of Bt subsp. kurstaki HD-1. This first cloning was followed quickly by the cloning of many other cry genes and eventually led to the development of Bt transgenic plants. In the 1980s, several scientists successively demonstrated that plants can be genetically engineered, and finally, Bt cotton reached the market in 1996 [104].

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

    PubMed

    Prabhakar, A; Bishop, A H

    2011-06-01

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

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

    PubMed

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

    2014-09-01

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

  9. [Screening of Bacillus thuringiensis strains containing vip3A genes and analysis of gene conservation].

    PubMed

    Chen, Jian-Wu; Tang, Li-Xia; Song, Shao-Yun; Yuan, Mei-Jin; Pang, Yi

    2003-09-01

    Vip3A, a novel insecticidal protein, is secreted by Bacillus thuringiensis (Bt) during vegetative growth. Vip3A protein possesses insecticidal activity against a wild spectrum of lepidopteran insect larvae. Since the first cloning of vip3A gene from Bt, many other vip3A genes have been isolated. To investigate vip3A genes contribution to Bt and reflect the revolution relationships, the strains containing vip3A genes were screened and gene similarity was analyzed. 114 wild-type Bacillus thuringiensis (Bt) strains isolated from different regions and 41 standard Bt strains from the Institute of Pasteur were screened for the vip3A genes using PCR amplification. 39 strains including B. thuringiensis subsp. kurstaki (Btk) HD-1 were found to contain the vip3A genes. Because acrystallerous strain Cry- B derived from Btk HD-1 was proved not to contain vip3A gene, it suppose that the vip3A gene may be located at the plasmids. Vip3A proteins expressed in these strains were detected with polyclonal antibody by Western blot and 4 strains among them were shown not to express the Vip3A proteins. The vip3A genes amplified from wild-type Bacillus thuringiensis strains S101 and 611 with different levels of activity against lepidopteran insect larvae were cloned into pGEM-T Easy vector. Alignment of these 2 putative Vip3A proteins with 6 others (Vip3A (a), Vip3A(b), Vip3A-S, Vip3A-S184, Vip83 and Vip3V) in the GenBank data base and 2 reported Vip3A proteins (Vip14 and Vip15) showed that vip3A genes are highly conservative. The plasmids pOTP-S101 and pOTP-611 were constructed by in- serting 2 vip3A genes (vip3A-S101 and vip3A-611) into the expression vector pQE30 respectively and were transformed into E. coli M15. E. coli M15 cells harboring the pOTP plasmids were induced with 1 mmol/L IPTG to express 89 kDa protein. Experiments showed that the level of soluble proteins of Vip3A-S101 in E. coli M15[pOTP-S101] and Vip3A-611 in E. coli M15 [pOTP-611] were about 48% and 35% respectively

  10. [Effects of helper protein P20 from Bacillus thuringiensis on Vip3A expression].

    PubMed

    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

  11. Evidence of Bacillus thuringiensis intra-serovar diversity revealed by Bacillus cereus group-specific repetitive extragenic palindromic sequence-based PCR genomic fingerprinting.

    PubMed

    Sauka, Diego H; Basile, Juan I; Benintende, Graciela

    2011-01-01

    Bacillus thuringiensis is classified into serovars on the basis of H-flagellar antigens. Several alternative typing methods have been described. Among them, a B. cereus group-specific repetitive extragenic palindromic (Rep)-PCR fingerprinting technique was shown to be discriminative and able to identify B. thuringiensis serovars. The aim of this study was to investigate the genomic diversity and relationship among B. thuringiensis strains collected from different Argentinean ecosystems. Thirty-seven B. thuringiensis reference strains and 131 Argentinean isolates were analyzed using a B. cereus group-specific Rep-PCR. Fourteen different patterns were identified among the Argentinean isolates. Eight could not be associated to any pattern obtained from a reference strain. The pattern identical to the serovar kurstaki HD-1 strain was the most frequently identified in 68 native isolates. The profiles allowed tracing a single dendrogram with two groups and eight main lineages. Some strains showed distinctive patterns despite belonging to the same serovar. An intraspecific diversity resulted from this analysis that was highlighted by this technique since strains from a given serovar showed distinct profiles. This study may help to establish a system of B. thuringiensis classification with a higher discrimination level than established by the H antigen serotyping.

  12. The Pathogenomic Sequence Analysis of B. cereus and B.thuringiensis Isolates Closely Related to Bacillus anthracis

    SciTech Connect

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

    2005-08-18

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

  13. Identification of a Bacillus thuringiensis Cry11Ba toxin-binding aminopeptidase from the mosquito, Anopheles quadrimaculatus

    PubMed Central

    Abdullah, Mohd Amir F; Valaitis, Algimantas P; Dean, Donald H

    2006-01-01

    Background Aminopeptidase N (APN) type proteins isolated from several species of lepidopteran insects have been implicated as Bacillus thuringiensis (Bt) toxin-binding proteins (receptors) for Cry toxins. We examined brush border membrane vesicle (BBMV) proteins from the mosquito Anopheles quadrimaculatus to determine if APNs from this organism would bind mosquitocidal Cry toxins that are active to it. Results A 100-kDa protein with APN activity (APNAnq 100) was isolated from the brush border membrane of Anopheles quadrimaculatus. Native state binding analysis by surface plasmon resonance shows that APNAnq 100 forms tight binding to a mosquitocidal Bt toxin, Cry11Ba, but not to Cry2Aa, Cry4Ba or Cry11Aa. Conclusion An aminopeptidase from Anopheles quadrimaculatus mosquitoes is a specific binding protein for Bacillus thuringiensis Cry11Ba. PMID:16716213

  14. Efficacy of Bacillus thuringiensis, Paecilomyces marquandii,and Streptomyces costaricanus with and without Organic Amendments against Meloidogyne hapla Infecting Lettuce

    PubMed Central

    Chen, J.; Abawi, G. S.; Zuckerman, B. M.

    2000-01-01

    Chitin, wheat mash, or brewery compost were incorporated into unfumigated and methyl bromide-fumigated organic soils placed in microplots formed from cylindrical drainage tiles (0.25 m-diam. clay tile). After 3 weeks, Meloidogyne hapla and cell or spore suspensions of Bacillus thuringiensis, Paecilomyces marquandii, and Streptomyces costaricanus were individually added to the soils of designated microplots. A B. thuringiensis + S. costaricanus combination was also tested. Lettuce seedlings, cv. Montello, were transplanted into the soils 3 to 4 days later. All the bacterial and fungal antagonists applied without a soil amendment, except the B. thuringiensis + S. costaricanus treatment, reduced root galling and increased lettuce head weight in the unfumigated organic soil, but not in the fumigated soil. All three amendments were also effective against M. hapla and reduced root galling in fumigated and unfumigated soils. Wheat mash amendment increased lettuce head weight in the unfumigated soil. In general, no antagonist × amendment interaction was detected. Soil populations of B. thuringiensis were maintained at ≥4.0 log10 colony-forming units/g organic soil during the first 14 days after planting. However, viable cells of B. thuringiensis were not detected after 49 days. PMID:19270951

  15. In Vitro Ovicidal and Cestocidal Effects of Toxins from Bacillus thuringiensis on the Canine and Human Parasite Dipylidium caninum

    PubMed Central

    Peña, Guadalupe; Aguilar Jiménez, Fortino Agustín; Hallal-Calleros, Claudia; Morales-Montor, Jorge; Hernández-Velázquez, Víctor Manuel; Flores-Pérez, Fernando Iván

    2013-01-01

    Bacillus thuringiensis is a gram-positive soil-dwelling bacterium that is commonly used as a biological pesticide. This bacterium may also be used for biological control of helminth parasites in domestic animals. In this study, we evaluated the possible ovicidal and cestocidal effects of a total protein extract of B. thuringiensis native strains on the zoonotic cestode parasite of dogs, Dipylidium caninum (D. caninum). Dose and time response curves were determined by coincubating B. thuringiensis proteins at concentration ranging from 100 to 1000 μg/mL along with 4000 egg capsules of D. caninum. Egg viability was evaluated using the trypan blue exclusion test. The lethal concentration of toxins on eggs was 600 μg/ml, and the best incubation time to produce this effect was 3 h. In the adult stage, the motility and the thickness of the tegument were used as indicators of damage. The motility was inhibited by 100% after 8 hours of culture compared to the control group, while the thickness of the cestode was reduced by 34%. Conclusively, proteins of the strain GP526 of B. thuringiensis directly act upon D. caninum showing ovicidal and cestocidal effects. Thus, B. thuringiensis is proposed as a potential biological control agent against this zoonosis. PMID:23484087

  16. Evidence of two mechanisms involved in Bacillus thuringiensis israelensis decreased toxicity against mosquito larvae: Genome dynamic and toxins stability.

    PubMed

    Elleuch, Jihen; Zribi Zghal, Raida; Lacoix, Marie Noël; Chandre, Fabrice; Tounsi, Slim; Jaoua, Samir

    2015-07-01

    Biopesticides based on Bacillus thuringiensis israelensis are the most used and most successful around the world. This bacterium is characterized by a dynamic genome able to win or lose genetic materials which leads to a decrease in its effectiveness. The detection of such phenomena is of great importance to monitor the stability of B. thuringiensis strains in industrial production processes of biopesticides. New local B. thuringiensis israelensis isolates were investigated. They present variable levels of delta-endotoxins production and insecticidal activities against Aedes aegypti larvae. Searching on the origin of this variability, molecular and biochemical analyses were performed. The obtained results describe two main reasons of the decrease of B. thuringiensis israelensis insecticidal activity. The first reason was the deletion of cry4Aa and cry10Aa genes from the 128-kb pBtoxis plasmid as evidenced in three strains (BLB124, BLB199 and BLB506) among five. The second was the early degradation of Cry toxins by proteases in larvae midgut mainly due to some amino acids substitutions evidenced in Cry4Ba and Cry11Aa δ-endotoxins detected in BLB356. Before biological treatment based on B. thuringiensis israelensis, the studies of microflore in each ecosystem have a great importance to succeed pest management programs.

  17. In vitro ovicidal and cestocidal effects of toxins from Bacillus thuringiensis on the canine and human parasite Dipylidium caninum.

    PubMed

    Peña, Guadalupe; Aguilar Jiménez, Fortino Agustín; Hallal-Calleros, Claudia; Morales-Montor, Jorge; Hernández-Velázquez, Víctor Manuel; Flores-Pérez, Fernando Iván

    2013-01-01

    Bacillus thuringiensis is a gram-positive soil-dwelling bacterium that is commonly used as a biological pesticide. This bacterium may also be used for biological control of helminth parasites in domestic animals. In this study, we evaluated the possible ovicidal and cestocidal effects of a total protein extract of B. thuringiensis native strains on the zoonotic cestode parasite of dogs, Dipylidium caninum (D. caninum). Dose and time response curves were determined by coincubating B. thuringiensis proteins at concentration ranging from 100 to 1000 μ g/mL along with 4000 egg capsules of D. caninum. Egg viability was evaluated using the trypan blue exclusion test. The lethal concentration of toxins on eggs was 600 μ g/ml, and the best incubation time to produce this effect was 3 h. In the adult stage, the motility and the thickness of the tegument were used as indicators of damage. The motility was inhibited by 100% after 8 hours of culture compared to the control group, while the thickness of the cestode was reduced by 34%. Conclusively, proteins of the strain GP526 of B. thuringiensis directly act upon D. caninum showing ovicidal and cestocidal effects. Thus, B. thuringiensis is proposed as a potential biological control agent against this zoonosis.

  18. Effect of fermentation conditions on the enterotoxigenicity, cytotoxicity and pesticidal activity of Bacillus thuringiensis strains isolated in Taiwan.

    PubMed

    Pang, Jen-Chieh; Chen, Ming-Lun; Ho, Yi-Cheng; Yang, Chi-Yea; Tzeng, Ching-Chou; Kao, Suey-Sheng; Tsen, Hau-Yang

    2010-03-01

    A total of 75 Bacillus thuringiensis strains, among them 62 of Taiwan's microbiota, were screened for their enterotoxin genes, hemolysin BL activity and cytotoxicity. All the strains harbored enterotoxin genes and were cytotoxic to the cultivated Chinese hamster ovary (CHO) cells. The hemolysin BL and cytotoxicity titers of the B. thuringiensis culture in casitone yeast sucrose (CYS) broth were lower than those in brain heart infusion (BHI) broth, and when the B. thuringiensis strains were cultivated in CYS broth for 5 days, no cytotoxicity was detected. The spores and crystal toxins collected from 40 isolates showed high levels of insecticidal activity against Plutella xylostella. All strains exhibiting low cytotoxicity also had low pesticidal activity. Our study demonstrated that it is difficult to find B. thuringiensis strains that are both effective against insect targets and do not produce enterotoxins or cytotoxic effects in CHO cells. However, it is possible to avoid or reduce unwanted properties, but not the insecticidal activity, of some B. thuringiensis preparations by alteration of culture media and conditions.

  19. Complete genome sequence of Bacillus thuringiensis CTC-A typical strain with high production of S-layer proteins.

    PubMed

    Dong, Zhaoxia; Li, Junhua; Zheng, Jinshui; Geng, Ce; Peng, Donghai; Sun, Ming

    2016-02-20

    Bacillus thuringiensis CTC, which is identified as serotype H2, serovar. finitimus, is high production of S-layer protein. Due to the property of forming isoporous lattices on the whole cell surface, S-layer protein has been widely used in (nano) biotechnology, biomimetics, biomedicine, especially been employed for displaying many important active proteins. Here, we report the complete genome of strain CTC, which contains one circular chromosome and one linear plasmid.

  20. Efficacy of Bacillus thuringiensis var. Kurstaki in the control of two mosquito species (Anopheles stephensi and Culex quinquefasciatus).

    PubMed

    Kumar, A Senthil; Moorthi, M; Ramakrishnan, N; Dhanapakiam, P; Ramasamy, V K; Rajeswari, A Raja

    2009-05-01

    Bioinsecticide Bacillus thuringiensis var. kurstaki (Btk) was used for controlling the mosquito species (Anopheles stephensi and Culex quinquefasciatus) which gave a significant (p<0.05) mortality in both species. The higher concentration of Btk was highly effective compared to the control ones. The controlling effect was dose and time dependent. Among the studied mosquitoes the C. quinquefasciatus (LC5 0.154%) was more susceptible than A. stephensi (LC50 0.372%) towards the bioinsecticide Btk.

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

    PubMed Central

    Palma, Leopoldo; Muñoz, Delia; Murillo, Jesús

    2014-01-01

    We report here the complete annotated 6,510,053-bp draft genome sequence of Bacillus thuringiensis serovar tolworthi strain Na205-3, which is toxic for Helicoverpa armigera. This strain potentially contains nine insecticidal toxin genes homologous to cry1Aa12, cry1Ab1, cry1Ab8, cry1Ba1, cry1Af1, cry1Ia10, vip1Bb1, vip2Ba2, and vip3Aa6. PMID:24625875

  2. INSECTICIDAL TOXIN FROM BACILLUS THURINGIENSIS IS RELEASED FROM ROOTS OF TRANSGENIC BT CORN IN VITRO AND IN SITU. (R826107)

    EPA Science Inventory

    Abstract

    The insecticidal toxin encoded by the cry1Ab gene from Bacillus thuringiensis was released in root exudates from transgenic Bt corn during 40 days of growth in soil amended to 0, 3, 6, 9, or 12% (v/v) with montmorillonite or kaolinite in a...

  3. Chromosome-Directed PCR-Based Detection and Quantification of Bacillus cereus Group Members with Focus on B. thuringiensis Serovar israelensis Active against Nematoceran Larvae.

    PubMed

    Schneider, Salome; Hendriksen, Niels B; Melin, Petter; Lundström, Jan O; Sundh, Ingvar

    2015-08-01

    Bacillus thuringiensis serovar israelensis is a wide-spread soil bacterium affiliated with the B. cereus group (Bcg) and is widely used in biocontrol products applied against mosquito and black fly larvae. For monitoring and quantification of applied B. thuringiensis serovar israelensis and its effect on indigenous B. thuringiensis serovar israelensis and Bcg assemblages, efficient and reliable tools are essential. The abundance and properties of B. thuringiensis serovar israelensis strains in the environment traditionally have been investigated with cultivation-dependent techniques, which are hampered by low sensitivity and the morphological similarity between B. cereus and B. thuringiensis. Currently available PCR-based detection and quantification tools target markers located on plasmids. In this study, a new cultivation-independent PCR-based method for efficient and specific quantification of B. thuringiensis serovar israelensis and Bcg is presented, utilizing two sets of PCR primers targeting the bacterial chromosome. Sequence database searches and empirical tests performed on target and nontarget species, as well as on bulk soil DNA samples, demonstrated that this diagnostic tool is specific for B. thuringiensis serovar israelensis and Bcg. The method will be useful for comparisons of Bcg and B. thuringiensis serovar israelensis abundances in the same samples. Moreover, the effect of B. thuringiensis serovar israelensis-based insecticide application on the total Bcg assemblages, including indigenous populations, can be investigated. This type of information is valuable in risk assessment and policy making for use of B. thuringiensis serovar israelensis in the environment.

  4. Chromosome-Directed PCR-Based Detection and Quantification of Bacillus cereus Group Members with Focus on B. thuringiensis Serovar israelensis Active against Nematoceran Larvae

    PubMed Central

    Hendriksen, Niels B.; Melin, Petter; Lundström, Jan O.; Sundh, Ingvar

    2015-01-01

    Bacillus thuringiensis serovar israelensis is a wide-spread soil bacterium affiliated with the B. cereus group (Bcg) and is widely used in biocontrol products applied against mosquito and black fly larvae. For monitoring and quantification of applied B. thuringiensis serovar israelensis and its effect on indigenous B. thuringiensis serovar israelensis and Bcg assemblages, efficient and reliable tools are essential. The abundance and properties of B. thuringiensis serovar israelensis strains in the environment traditionally have been investigated with cultivation-dependent techniques, which are hampered by low sensitivity and the morphological similarity between B. cereus and B. thuringiensis. Currently available PCR-based detection and quantification tools target markers located on plasmids. In this study, a new cultivation-independent PCR-based method for efficient and specific quantification of B. thuringiensis serovar israelensis and Bcg is presented, utilizing two sets of PCR primers targeting the bacterial chromosome. Sequence database searches and empirical tests performed on target and nontarget species, as well as on bulk soil DNA samples, demonstrated that this diagnostic tool is specific for B. thuringiensis serovar israelensis and Bcg. The method will be useful for comparisons of Bcg and B. thuringiensis serovar israelensis abundances in the same samples. Moreover, the effect of B. thuringiensis serovar israelensis-based insecticide application on the total Bcg assemblages, including indigenous populations, can be investigated. This type of information is valuable in risk assessment and policy making for use of B. thuringiensis serovar israelensis in the environment. PMID:25979887

  5. A constitutively expressed 36 kDa exochitinase from Bacillus thuringiensis HD-1.

    PubMed

    Arora, Naresh; Ahmad, Tarannum; Rajagopal, R; Bhatnagar, Raj K

    2003-08-01

    A 36 kDa chitinase was purified by ion exchange and gel filtration chromatography from the culture supernatant of Bacillus thuringiensis HD-1. The chitinase production was independent of the presence of chitin in the growth medium and was produced even in the presence of glucose. The purified chitinase was active at acidic pH, had an optimal activity at pH 6.5, and showed maximum activity at 65 degrees C. Of the various substrates, the enzyme catalyzed the hydrolysis of the disaccharide 4-MU(GlnAc)(2) most efficiently and was therefore classified as an exochitinase. The sequence of the tryptic peptides showed extensive homology with Bacillus cereus 36 kDa exochitinase. The 1083 bp open reading frame encoding 36 kDa chitinase was amplified with primers based on the gene sequence of B. cereus 36 kDa exochitinase. The deduced amino-acid sequence showed that the protein contained an N-terminal signal peptide and consisted of a single catalytic domain. The two conserved signature sequences characteristic of family 18 chitinases were mapped at positions 105-109 and 138-145 of Chi36. The recombinant chitinase was expressed in a catalytically active form in Escherichia coli in the vector pQE-32. The expressed 36 kDa chitinase potentiated the insecticidal effect of the vegetative insecticidal protein (Vip) when used against neonate larvae of Spodoptera litura.

  6. Purification and identification of a novel leucine aminopeptidase from Bacillus thuringiensis israelensis.

    PubMed

    Cahan, Rivka; Hetzroni, Efrat; Nisnevitch, Marina; Nitzan, Yeshayahu

    2007-11-01

    A novel leucine aminopeptidase was purified from a Bacillus thuringiensis israelensis (Bti) culture. The purification stages included heating the concentrated supernatant to 65 degrees C for 90 min, anion-exchange chromatography by DEAE cellulose, and hydrophobic chromatography by phenyl Sepharose. The specific activity of leucine aminopeptidase after the hydrophobic chromatography increased by 215.5-fold and the yield was 16%. The molecular weight of the active enzyme was 59 kDa. Mass spectrometry analysis of the 59-kDa leucine aminopeptidase revealed that this protein has at least 41% homology with the cytosol leucine aminopeptidase produced by Bacillus cereus. Maximal leucine aminopeptidase activity occurred at 65 degrees C, pH 10 toward leucine as the amino acid terminus. The enzyme was strongly inhibited by bestatin, dithiothreitol, and 1,10-phenanthroline, indicating that the enzyme might be considered as a metallo-aminopeptidase that has disulfide bonds at the catalytic site or at a region that influences its configuration. Examination of the purified leucine aminopeptidase's effect on the activation of the protoxin Cyt1Aa from Bti revealed that when it acts synergistically with Bti endogenous proteases, it has only a minor role in the processing of Cyt1Aa into an active toxin.

  7. Re-aerosolization of Bacillus thuringiensis spores from concrete and turf.

    PubMed

    Bishop, A H; O'Sullivan, C M; Lane, A; Butler Ellis, M C; Sellors, W J

    2017-03-03

    Spores of Bacillus anthracis deposited on surfaces can become airborne again as a result of air currents and mechanical forces. As such they are a potential source of infection by inhalation. Spores of Bacillus thuringiensis were used to quantify this phenomenon in a simulation of outdoor conditions. Concrete and turf surfaces were inoculated by aerosol to produce high spore densities (greater than 1 x 10(9) CFU m(-2) ) which were then subjected to the passage of air at 10 ms(-1) with and without simulated walking. Re-aerosolized spores were sampled by wetted wall cyclone air samplers. The mean total re-aerosolization rate from concrete (m(-2) min(-1) ) was 1.16 x 10(-3) for wind alone and 3.2 x 10(-3) for wind and simulated walking while for turf the respective values were 2.7 x 10(-4) and 6.7 x 10(-4) . This article is protected by copyright. All rights reserved.

  8. Application of statistical experimental design for optimisation of bioinsecticides production by sporeless Bacillus thuringiensis strain on cheap medium.

    PubMed

    Ben Khedher, Saoussen; Jaoua, Samir; Zouari, Nabil

    2013-01-01

    In order to overproduce bioinsecticides production by a sporeless Bacillus thuringiensis strain, an optimal composition of a cheap medium was defined using a response surface methodology. In a first step, a Plackett-Burman design used to evaluate the effects of eight medium components on delta-endotoxin production showed that starch, soya bean and sodium chloride exhibited significant effects on bioinsecticides production. In a second step, these parameters were selected for further optimisation by central composite design. The obtained results revealed that the optimum culture medium for delta-endotoxin production consists of 30 g L(-1) starch, 30 g L(-1) soya bean and 9 g L(-1) sodium chloride. When compared to the basal production medium, an improvement in delta-endotoxin production up to 50% was noted. Moreover, relative toxin yield of sporeless Bacillus thuringiensis S22 was improved markedly by using optimised cheap medium (148.5 mg delta-endotoxins per g starch) when compared to the yield obtained in the basal medium (94.46 mg delta-endotoxins per g starch). Therefore, the use of optimised culture cheap medium appeared to be a good alternative for a low cost production of sporeless Bacillus thuringiensis bioinsecticides at industrial scale which is of great importance in practical point of view.

  9. Diagnostic properties of three conventional selective plating media for selection of Bacillus cereus, B. thuringiensis and B. weihenstephanensis.

    PubMed

    Hendriksen, Niels Bohse; Hansen, Bjarne Munk

    2011-11-01

    The aim of this study was to assess the diagnostic properties of the two selective plating media and a chromogenic medium for identification of Bacillus cereus. The 324 isolates were B. cereus (37%), Bacillus weihenstephanensis (45%) or Bacillus thuringiensis (18%), as identified by a new combination of techniques. All isolates were growing on mannitol-egg yolk-polymyxin agar (MYP), and they did not form acid from mannitol. However, a significant lower number of B. thuringiensis isolates did not show lecithinase activity. All isolates were also growing on polymyxin-egg yolk-mannitol-bromothymol blue agar (PEMBA); however, 11% isolates indicated that they did produce acid from mannitol, and 15% isolates did not show any lecithinase activity. Five of the isolates did not grow at all on the chromogenic agar, and 14 of the growing isolates were β-glucosidase negative. It is concluded that the two recommended selective plating media MYP and PEMBA for detection of B. cereus group bacteria both have their limitations for identification of some B. cereus, B. weihenstephanensis or B. thuringiensis. However, MYP is preferable compared to PEMBA. The chromogenic medium has its own advantages and limitations, and some of the limitations seem to be solved by incubation at 30°C instead of the recommended 37°C.

  10. Mutually exclusive distribution of the sap and eag S-layer genes and the lytB/lytA cell wall hydrolase genes in Bacillus thuringiensis.

    PubMed

    Soufiane, Brahim; Sirois, Marc; Côté, Jean-Charles

    2011-10-01

    Recently, two Bacillus thuringiensis strains were reported to synthesize parasporal inclusion bodies made not of the expected crystal (Cry) proteins but rather of the surface layer proteins (SLP) Sap (encoded by sap) and EA1 (encoded by eag), respectively. Whether the presence of the sap and eag genes is restricted to these two B. thuringiensis strains or ubiquitous in B. thuringiensis is unknown. We report here the distribution of the sap and eag genes in B. thuringiensis. Strains in the Bacillus cereus group were added for comparison purposes. We show that sap and eag are either present in tandem in 35% of the B. thuringiensis strains analysed and absent in 65% of the strains. When absent, a different tandem, the lytB/lytA cell wall hydrolase genes, is present. The distribution of the sap and eag S-layer and the lytB/lytA cell wall hydrolase genes is not species-specific in B. thuringiensis, B. cereus and Bacillus weihenstephanensis. Bacillus anthracis and Bacillus mycoides harbor sap and eag but not lytB/lytA. The sap, eag and lytB/lytA genes were absent in Bacillus pseudomycoides. Clearly, the distribution of the sap and eag S-layer and the lytB/lytA cell wall hydrolase genes in B. thuringiensis and in the Bacillus cereus group is mutually exclusive. We also showed that two genes involved in cell wall metabolism, csaA and csaB, are present not only upstream of the sap and eag S-layer genes, but also upstream of the lytB/lytA tandem in strains where sap and eag are absent. Bootstrapped neighbor-joining trees were inferred from the translated amino acid sequences of sap, eag and the tandem lytB/lytA, respectively.

  11. Coexpression of chitinase and the cry11Aa1 toxin genes in Bacillus thuringiensis serovar israelensis.

    PubMed

    Sirichotpakorn, N; Rongnoparut, P; Choosang, K; Panbangred, W

    2001-10-01

    At the spore stage, a cloned chitinase gene was coexpressed with the regulatory gene p19 and the toxin gene cry11Aa1 in the hosts Bacillus thuringiensis serovar israelensis strains 4Q2-72 and c4Q2-72. The chitinase gene was derived from a high-chitinase producer, Bacillus licheniformis TP-1. Two transcriptional fusion plasmids between the p19 or p19-cry11Aa1 genes and the promoterless chitinase gene were constructed. In transcription order, the p16-19CHI construct contained the p19 gene together with the chitinase gene only while the p16-1968CHI construct contained p19 together with the toxin gene cry11Aa1 and the chitinase gene. The inserted sequences were regulated by a spore-specific promoter located upstream of p19. The recombinant chitinase of all transformed B. thuringiensis serovar israelensis strains was initially synthesized at low level at about 9 h of growth when a portion of the cells started to sporulate. It increased thereafter and reached maximum levels of 5.5, 4.9, and 4.7 mU/ml at 48 h, for strain 4Q2-72 transformed with p16-19CHI and p16-1968CHI and strain c4Q2-72 transformed with p16-19CHI, respectively. This activity was approximately 2 times higher than the maximum activity (2.7 mU/ml) of the parental strain, B. licheniformis TP-1. Although crude chitinase alone from B. thuringiensis serovar israelensis c4Q2-72 (p16-19CHI) at 4.5 mU/ml caused 40% mortality in second instar Aedes aegypti larvae, transformants containing the chitinase alone or in combination with cry11Aa1 resulted in lower toxicity to A. aegypti larvae than the untransformed 4Q2-72 host. For example the LC(50) for the transformed 4Q2-72 harboring the chitinase gene only (p16-19CHI) was 5.6 x 10(4) +/- 0.7 x 10(4) cells, 40 times higher than that of the untransformed host at 1.4 x 10(3) +/- 0.19 x 10(3). The lower toxicity correlated with poor sporulation in the transformants (i.e., 35 times lower than that in the untransformed host). However, the transformed 4Q2-72 strain

  12. Potato flour mediated solid-state fermentation for the enhanced production of Bacillus thuringiensis-toxin.

    PubMed

    Smitha, Robinson Babysarojam; Jisha, Veloorvalappil Narayanan; Pradeep, Selvanesan; Josh, Moolakkariyil Sarath; Benjamin, Sailas

    2013-11-01

    In this study, we explored the efficacy of raw potato flour (PF) as supplement to the conventional LB medium (LB control, designated as M1) for enhancing the concomitant production of endospores and δ-endotoxin from Bacillus thuringiensis subsp. kurstaki by solid-state fermentation (SSF). Of different concentrations and combinations of media tested, 10% (w/v) PF supplemented LB medium (M2) was found as the best source for the maximum yield of toxin. After 12 h submerged fermentation (SmF) at 37°C and 125 rpm, M2 was made into a wet-solid matter for SSF by removing the supernatant (1000 ×g, 10 min); the resultant pellet subsequently incubated statically (37°C) for the production of B. thuringiensis subsp. kurstaki toxin (Btk-toxin). In comparison to M1, yield of δ-endotoxin purified by sucrose density gradient centrifugation method from M2 was about 6-fold higher (53% recovery). This maximum yield from M2 was obtained at 48 h (as against 72 h from M1), thus the gestation period of M2 was reduced by 24 h with higher yield. In addition to the quantitative data, qualitative photomicrographs taken by image analyzer, scanning electron and fluorescent microscopes and digital camera showed physical evidences for the upper hand of SSF over conventional SmF for the enhanced production of Btk-toxin. SDS-PAGE image of the purified δ-endotoxin showed three major fractions with apparent MWs 66, 45 and 30 kDa. Briefly, if low-cost agricultural products like PF is used as supplement to LB, by SSF strategy, production of Btk-toxin could be enhanced to 6-fold in short gestation time without losing its entomotoxicity efficiency.

  13. Long Lasting Persistence of Bacillus thuringiensis Subsp. israelensis (Bti) in Mosquito Natural Habitats

    PubMed Central

    Tilquin, Mathieu; Paris, Margot; Reynaud, Stéphane; Despres, Laurence; Ravanel, Patrick; Geremia, Roberto A.; Gury, Jérôme

    2008-01-01

    Background The detrimental effects of chemical insecticides on the environment and human health have lead to the call for biological alternatives. Today, one of the most promising solutions is the use of spray formulations based on Bacillus thuringiensis subsp. israelensis (Bti) in insect control programs. As a result, the amounts of Bti spread in the environment are expected to increase worldwide, whilst the common belief that commercial Bti is easily cleared from the ecosystem has not yet been clearly established. Methodology/Main Findings In this study, we aimed to determine the nature and origin of the high toxicity toward mosquito larvae found in decaying leaf litter collected in several natural mosquito breeding sites in the Rhône-Alpes region. From the toxic fraction of the leaf litter, we isolated B. cereus-like bacteria that were further characterized as B. thuringiensis subsp. israelensis using PCR amplification of specific toxin genes. Immunological analysis of these Bti strains showed that they belong to the H14 group. We finally used amplified length polymorphism (AFLP) markers to show that the strains isolated from the leaf litter were closely related to those present in the commercial insecticide used for field application, and differed from natural worldwide genotypes. Conclusions/Significance Our results raise the issue of the persistence, potential proliferation and environmental accumulation of human-spread Bti in natural mosquito habitats. Such Bti environmental persistence may lengthen the exposure time of insects to this bio-insecticide, thereby increasing the risk of resistance acquisition in target insects, and of a negative impact on non-target insects. PMID:18941501

  14. Long-term survival and germination of Bacillus thuringiensis var. kurstaki in a field trial.

    PubMed

    Hendriksen, Niels Bohse; Hansen, Bjarne Munk

    2002-03-01

    Long-term survival, dispersal, and germination of Bacillus thuringiensis var. kurstaki DMU67R has been investigated in a field trial. An experimental cabbage plot was sprayed with DMU67R in 1993 and allowed to lie fallow since. The investigations reported here were carried out from 1997 to 2000 in this plot. High persistence of DMU67R for 7 years in the bulk soil of the plot has been demonstrated. The numbers have not significantly reduced since 1994, stabilizing around 6.6 x 10(2) cfu/g from 1996 to 2000. Horizontal dispersal of DMU67R in the 1994-1999 period was limited. Vertical dispersal occurred from 1994 to 1999, as 77% of the population of DMU67R occurred in the 0-2 cm layer in 1994, while only 22% of the population was found there in 1999. Most of the population in 1999 was present homogeneously in the upper 6 cm of the soil profile. Germination, as evidenced by the ratio of DMU67R cfu before and after heat treatment, was not observed in the bulk soil. However, in the rhizospheres of dandelion (Taraxacum officinalis) and quackgrass (Agropyron repens), 40 and 50% of DMU67R was present as vegetative germinated cells, respectively. No germination occurred in the rhizosphere of red fescue (Festuca rubra). The material from the gut of the earthworm species Lumbricus rubellus, Lumbricus terrestris, and Apporrectodea caliginosa and from a tipulid larvae from the plot also contained vegetative cells of DMU67R. Further investigations of A. caliginosa showed that germination seemed to be restricted to the gut and that sporulation occurred after defecation. The germination of DMU67R in rhizospheres and in the gut of nontarget invertebrates suggests that survival in the soil of B. thuringiensis is a dynamic process involving germination, cell divisions, and sporulation in specific microhabitats.

  15. Molecular characterization and genetic diversity of insecticidal crystal protein genes in native Bacillus thuringiensis isolates.

    PubMed

    Mahadeva Swamy, H M; Asokan, R; Mahmood, Riaz; Nagesha, S N

    2013-04-01

    The Western Ghats of Karnataka natural ecosystem are among the most diverse and is one of the eight hottest hotspots of biological diversity in the world, that runs along the western part of India through four states including Karnataka. Bacillus thuringiensis (Bt) strains were isolated from soils of Western Ghats of Karnataka and characterized by molecular and analytical methods as a result of which 28 new Bt-like isolates were identified. Bt strains were isolated from soil samples using sodium acetate selection method. The morphology of crystals was studied using light and phase contrast microscopy. Isolates were further characterized for insecticidal cry gene by PCR, composition of toxins in bacterial crystals by SDS-PAGE cloning, sequencing and evaluation of toxicity was done. As a result 28 new Bt-like isolates were identified. Majority of the isolates showed the presence of a 55 kDa protein bands on SDS-PAGE while the rest showed 130, 73, 34, and 25 kDa bands. PCR analysis revealed predominance of Coleopteran-active cry genes in these isolates. The variations in the nucleotide sequences, crystal morphology, and mass of crystal protein(s) purified from the Bt isolates revealed genetic and molecular diversity. Three strains containing Coleopteran-active cry genes showed higher activity against larvae Myllocerus undecimpustulatus undatus Marshall (Coleoptera: Curculionidae) than B. thuringiensis subsp. Morrisoni. Results indicated that Bt isolates could be utilized for bioinsecticide production, aiming to reduce the use of chemical insecticide which could be useful to use in integrated pest management to control agriculturally important pests for sustainable crop production.

  16. 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.

    PubMed

    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.

  17. Formation of Crystalline δ-Endotoxin or Poly-β-Hydroxybutyric Acid Granules by Asporogenous Mutants of Bacillus thuringiensis

    PubMed Central

    Wakisaka, Yoshiharu; Masaki, Emiko; Nishimoto, Yoji

    1982-01-01

    Parental strains and asporogenous mutants of Bacillus thuringiensis subspp. kurstaki and aizawai produced high yields of δ-endotoxin on M medium, which contained 330 μg of potassium per ml, but not on ST and ST-a media, each of which contained only 11 μg of potassium per ml. On ST and ST-a media, refractile granules were formed instead. These granules had no insecticidal activity against silkworms and were isolated and identified as poly-β-hydroxybutyric acid. Supplementation of the potassium-deficient ST-a medium with 0.1% KH2PO4 (3.7 mM) led to the formation of crystalline δ-endotoxin. The replacement of KH2PO4 with equimolar amounts of KCl, KNO3, and potassium acetate or an equivalent amount of K2SO4 had a similar effect, whereas the addition of an equimolar amount of NaH2PO4 or NH4H2PO4 did not cause the endotoxin to form. An asporogenous mutant, B. thuringiensis subsp. kurstaki strain 290-1, produced δ-endotoxin on ST-a medium supplemented with 3 mM or more potassium but formed only poly-β-hydroxybutyric acid granules on the media containing ≤1 mM potassium. These results clearly indicate that a certain concentration of potassium is essential for the fermentative production of δ-endotoxin by these isolates of B. thuringiensis. Manganese could not be substituted for potassium. Phosphate ions stimulated poly-β-hydroxybutyric acid formation by strain 290-1. The sporulation of B. thuringiensis and several other Bacillus strains was suppressed on the potassium-deficient ST medium. This suggests that potassium plays an essential role not only in Bacillus cell growth and δ-endotoxin formation but also in sporulation. Images PMID:16346040

  18. The distribution pattern of DNA and protoxin in Bacillus thuringiensis as revealed by laser confocal microscopy analysis.

    PubMed

    Hu, Quanfang; Wang, Jingfang; Fu, Zujiao; Mo, Xiangtao; Ding, Xuezhi; Xia, Liqiu; Zhang, Youming; Sun, Yunjun

    2015-07-01

    It was reported that the parasporal crystal from Bacillus thuringiensis contained DNA fragments. To investigate the distribution of protoxin and DNA in B. thuringiensis cells at different growth stages, a cry1Ac-gfp fusion gene was constructed and expressed in an acrystalliferous B. thuringiensis strain, in which the localization of DNA and protoxin were indicated by DNA-specific dye and green fluorescent protein, respectively. When the recombinant cells were at the vegetative growth stage, the Cry1Ac-GFP fusion protein was not expressed and the DNA fluorescent signal was evenly distributed throughout the cell. At the initial stage of sporulation, the Cry1Ac-GFP fusion protein was expressed and accumulated as inclusion body, while two condensed DNA signals existed at each pole of the cell. With the extension of culture time, it seemed that the DNA fluorescence from the region of spore development gradually became faint or vanishing, while the DNA signal was still present in the other pole or the remaining area of the mother cell. Interestingly and unexpectedly, there was no DNA fluorescence signal in the region of the growing and mature inclusion body of Cry1Ac-GFP in B. thuringiensis cell, which might indicate that the DNA embodied in the inclusion body was not accessible to the DNA-specific dye. This was the first investigation devoted exclusively to the in vivo distribution of protoxin and DNA in B. thuringiensis at different growth stages. These data shed light on deeply understanding the process of sporulation and parasporal crystal formation as well as further exploring the interaction of DNA and protoxin in B. thuringiensis.

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

    PubMed

    Jia, Yanhua; Zhao, Can; Wang, Qinglei; Shu, Changlong; Feng, Xiaojie; Song, Fuping; Zhang, Jie

    2014-02-01

    Cry8Ea1 from Bacillus thuringiensis strain Bt185 has insecticidal activity against Holotrichia parallela. Cry8Ca2 from strain HBF-1 is effective against Anomala corpulenta. Cry8Ga1 from strain HBF-18 is toxic to H. oblita. Given the need to broaden the spectrum of B. thuringiensis, a broad-spectrum coleopteran active strain of B. thuringiensis, BIOT185, engineered to express multiple cry genes, including cry8Ea1, cry8Fa1 and cry8Ca2, was created by homologous recombination introducing the cry8Ca2 into the B. thuringiensis strain Bt185 by Liu et al. (Appl Microbiol Biotechnol 87:243-249, 2010). To further broaden the spectrum, an engineered B. thuringiensis strain BIOT1858G was constructed by introducing the recombinant plasmid pSTK-8G containing cry8Ga1 into the engineered B. thuringiensis strain BIOT185. PCR and Southern blotting demonstrated that the cry8Ga1 gene was transferred to the novel strain BIOT1858G. SDS-PAGE and RT-PCR confirmed the normal expression and transcription of the cry8Ga1 gene in addition to the cry8Ea1, cry8Fa1 and cry8Ca2 genes. Bioassays of BIOT1858G indicated that the recombinant strain broadened the spectrum against not only H. parallela susceptible to the Cry8E protein and A. corpulenta susceptible to the Cry8C protein but also H. oblita susceptible to the Cry8G protein. The pesticide could also decrease the cost of production and field labor.

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

    PubMed

    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.

  1. [Bacillus thuringiensis: general aspects. An approach to its use in the biological control of lepidopteran insects behaving as agricultural pests].

    PubMed

    Sauka, Diego H; Benintende, Graciela B

    2008-01-01

    Bacillus thuringiensis is the most widely applied biological pesticide used to control insects that affect agriculture and forestry and which transmit human and animal pathogens. During the past decades B. thuringiensis has been the subject of intensive research. These efforts have yielded considerable data about the relationships between the structure, mechanism of action, and genetics of their pesticidal crystal proteins. As a result, a coherent picture of these relationships has emerged. Other studies have focused on the ecological role of the B. thuringiensis crystal proteins and their performance in agricultural and other natural settings. With this knowledge as background and the help of biotechnological tools, researchers are now reporting promising results in the development of more useful toxins, recombinant bacteria, new formulations and transgenic plants that express pesticidal activity, in order to assure that these products are utilized with the best efficiency and benefit. This article is an attempt to integrate all these recent developments in the study of B. thuringiensis into a context of biological control of lepidopteran insect pest of agricultural importance.

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

    PubMed

    Baig, Deeba Noreen; Mehnaz, Samina

    2010-07-20

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

  3. Detection of the cryptic prophage-like molecule pBtic235 in Bacillus thuringiensis subsp. israelensis.

    PubMed

    Gillis, Annika; Guo, Suxia; Bolotin, Alexandre; Makart, Lionel; Sorokin, Alexei; Mahillon, Jacques

    2016-10-26

    Bacillus thuringiensis has long been recognized to carry numerous extrachromosomal molecules. Of particular interest are the strains belonging to the B. thuringiensis subsp. israelensis lineage, as they can harbor at least seven extrachromosomal molecules. One of these elements seems to be a cryptic molecule that may have been disregarded in strains considered plasmid-less. Therefore, this work focused on this cryptic molecule, named pBtic235. Using different approaches that included transposition-tagging, large plasmid gel electrophoresis and Southern blotting, conjugation and phage-induction experiments, in combination with bioinformatics analyses, it was found that pBtic235 is a hybrid molecule of 235,425 bp whose genome displays potential plasmid- and phage-like modules. The sequence of pBtic235 has been identified in all sequenced genomes of B. thuringiensis subsp. israelensis strains. Here, the pBtic235 sequence was considered identical to that of plasmid pBTHD789-2 from strain HD-789. Despite the fact that the pBtic235 genome possesses 240 putative CDSs, many of them have no homologs in the databases. However, CDSs coding for potential proteins involved in replication, genome packaging and virion structure, cell lysis, regulation of lytic-lysogenic cycles, metabolite transporters, stress and metal resistance, were identified. The candidate plasmidial prophage pBtic235 exemplifies the notable diversity of the extrachromosomal realm found in B. thuringiensis.

  4. Gene knockout demonstrates that vip3A contributes to the pathogenesis of Bacillus thuringiensis toward Agrotis ipsilon and Spodoptera exigua.

    PubMed

    Donovan, W P; Donovan, J C; Engleman, J T

    2001-07-01

    Vip3A is an 89-kDa protein secreted by Bacillus thuringiensis during vegetative growth. To determine the importance of Vip3A for the insect pathogenicity of B. thuringiensis the vip3A gene was deleted from strain HD1, yielding strain HD1Deltavip3A. Compared with HD1, strain HD1Deltavip3A was one-fourth as toxic to Agrotis ipsilon larvae and less than one-tenth as toxic to Spodoptera exigua larvae. When streptomycin was included in the S. exigua diet the toxicity of HD1Deltavip3A was approximately half that of HD1. Addition of HD1 spores increased the toxicity of purified Cry1 protein more than 600-fold against S. exigua, whereas addition of HD1Deltavip3A spores increased toxicity of Cry1 protein approximately 10-fold. These results demonstrate that an important component of B. thuringiensis insecticidal activity against S. exigua is the synthesis of Vip3A protein by B. thuringiensis cells after ingestion of spores and crystal proteins by insect larvae.

  5. Characterization of Tunisian Bacillus thuringiensis strains with abundance of kurstaki subspecies harbouring insecticidal activities against the lepidopteran insect Ephestia kuehniella.

    PubMed

    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.

  6. Transcriptome analysis of Bacillus thuringiensis spore life, germination and cell outgrowth in a vegetable-based food model.

    PubMed

    Bassi, Daniela; Colla, Francesca; Gazzola, Simona; Puglisi, Edoardo; Delledonne, Massimo; Cocconcelli, Pier Sandro

    2016-05-01

    Toxigenic species belonging to Bacillus cereus sensu lato, including Bacillus thuringiensis, cause foodborne outbreaks thanks to their capacity to survive as spores and to grow in food matrixes. The goal of this work was to assess by means of a genome-wide transcriptional assay, in the food isolate B. thuringiensis UC10070, the gene expression behind the process of spore germination and consequent outgrowth in a vegetable-based food model. Scanning electron microscopy and Energy Dispersive X-ray microanalysis were applied to select the key steps of B. thuringiensis UC10070 cell cycle to be analyzed with DNA-microarrays. At only 40 min from heat activation, germination started rapidly and in less than two hours spores transformed in active growing cells. A total of 1646 genes were found to be differentially expressed and modulated during the entire B. cereus life cycle in the food model, with most of the significant genes belonging to transport, transcriptional regulation and protein synthesis, cell wall and motility and DNA repair groups. Gene expression studies revealed that toxin-coding genes nheC, cytK and hblC were found to be expressed in vegetative cells growing in the food model.

  7. Vip3A, a novel Bacillus thuringiensis vegetative insecticidal protein with a wide spectrum of activities against lepidopteran insects.

    PubMed

    Estruch, J J; Warren, G W; Mullins, M A; Nye, G J; Craig, J A; Koziel, M G

    1996-05-28

    A novel vegetative insecticidal gene, vip3A(a), whose gene product shows activity against lepidopteran insect larvae including black cutworm (Agrotis ipsilon), fall armyworm (Spodoptera frugiperda), beet armyworm (Spodoptera exigua), tobacco budworm (Heliothis virescens), and corn earworm (Helicoverpa zea) has been isolated from Bacillus thuringiensis strain AB88. VIP3-insecticidal gene homologues have been detected in approximately 15% of Bacillus strains analyzed. The sequence of the vip3A(b) gene, a homologue of vip3A(a) isolated from B. thuringiensis strain AB424 is also reported. Vip3A(a) and (b) proteins confer upon Escherichia coli insecticidal activity against the lepidopteran insect larvae mentioned above. The sequence of the gene predicts a 791-amino acid (88.5 kDa) protein that contains no homology with known proteins. Vip3A insecticidal proteins are secreted without N-terminal processing. Unlike the B. thuringiensis 5-endotoxins, whose expression is restricted to sporulation, Vip3A insecticidal proteins are expressed in the vegetative stage of growth starting at mid-log phase as well as during sporulation. Vip3A represents a novel class of proteins insecticidal to lepidopteran insect larvae.

  8. Genetic analysis of cryIIIA gene expression in Bacillus thuringiensis.

    PubMed

    Salamitou, S; Agaisse, H; Bravo, A; Lereclus, D

    1996-08-01

    The Bacillus thuringiensis (Bt) cryIIIA gene is regulated by a different mechanism from that of most of the other cry genes. Its expression begins during late-exponential growth and not during sporulation as for the other classes of cry genes. Moreover, in Bacillus subtilis, cryIIIA expression is independent of the major sporulation-specific sigma factors and is increased in a spoOA genetic background. We used lacZ fusions and primer-extension analysis to follow the time-course of cryIIIA transcription in Bt wild-type and in various Spo- genetic backgrounds (spoOA, sigE and sigK). cryIIIA was activated from the end of vegetative growth to stage II of sporulation (t3) in the wild-type strain. Thereafter, transcription from the same promoter continued, at a decreasing rate, until the end of stage III. In the spoOA mutant strain, the same promoter was activated for at least 15 h during the stationary phase. cryIIIA activation in the sigK genetic background was similar to that in the wild-type but was extended in a sigma E mutant strain. Thus cryIIIA expression in Bt is not directly dependent on the major sporulation-specific sigma factors. Furthermore, an event linked with the thE-dependent period of sporulation ends cryIIIA activation, although transcription of this gene does not switch off before the end of stage III.

  9. No Adjuvant Effect of Bacillus thuringiensis-Maize on Allergic Responses in Mice

    PubMed Central

    Dekan, Gerhard; Epstein, Michelle M.

    2014-01-01

    Genetically modified (GM) foods are evaluated carefully for their ability to induce allergic disease. However, few studies have tested the capacity of a GM food to act as an adjuvant, i.e. influencing allergic responses to other unrelated allergens at acute onset and in individuals with pre-existing allergy. We sought to evaluate the effect of short-term feeding of GM Bacillus thuringiensis (Bt)-maize (MON810) on the initiation and relapse of allergic asthma in mice. BALB/c mice were provided a diet containing 33% GM or non-GM maize for up to 34 days either before ovalbumin (OVA)-induced experimental allergic asthma or disease relapse in mice with pre-existing allergy. We observed that GM-maize feeding did not affect OVA-induced eosinophilic airway and lung inflammation, mucus hypersecretion or OVA-specific antibody production at initiation or relapse of allergic asthma. There was no adjuvant effect upon GM-maize consumption on the onset or severity of allergic responses in a mouse model of allergic asthma. PMID:25084284

  10. Bacillus thuringiensis: a successful insecticide with new environmental features and tidings.

    PubMed

    Jouzani, Gholamreza Salehi; Valijanian, Elena; Sharafi, Reza

    2017-04-01

    Bacillus thuringiensis (Bt) is known as the most successful microbial insecticide against different orders of insect pests in agriculture and medicine. Moreover, Bt toxin genes also have been efficiently used to enhance resistance to insect pests in genetically modified crops. In light of the scientific advantages of new molecular biology technologies, recently, some other new potentials of Bt have been explored. These new environmental features include the toxicity against nematodes, mites, and ticks, antagonistic effects against plant and animal pathogenic bacteria and fungi, plant growth-promoting activities (PGPR), bioremediation of different heavy metals and other pollutants, biosynthesis of metal nanoparticles, production of polyhydroxyalkanoate biopolymer, and anticancer activities (due to parasporins). This review comprehensively describes recent advances in the Bt whole-genome studies, the last updated known Bt toxins and their functions, and application of cry genes in plant genetic engineering. Moreover, the review thoroughly describes the new features of Bt which make it a suitable cell factory that might be used for production of different novel valuable bioproducts.

  11. A new formulation of Bacillus thuringiensis: UV protection and sustained release mosquito larvae studies

    PubMed Central

    Zhang, Lingling; Zhang, Xiaojuan; Zhang, Yi; Wu, Songqin; Gelbič, Ivan; Xu, Lei; Guan, Xiong

    2016-01-01

    Persistence of Bacillus thuringiensis is an important factor in determining the success of this product as a pest control agent. In this report we present the development of a highly active mosquitocidal formulation with high resistance to UV. LLP29-M19 strain of Bt, selected by repeated exposure to UV was found to be highly resistant to UV. The product was optimized and the methods used were statistically analyzed. Using single-factor experiments it was determined that the optimal concentration of sodium alginate, CaCl2 and hollow glass beads in the formulation were 1.0%, 2.0% and 3.5%, respectively. Plackett-Burman design was used to screen the interaction of the three factors, CaCl2, sodium alginate and hollow glass beads in the sustained-release formulation. The best combined concentration and mutual effects of the three factors were optimized by response surface methodology. The results showed that the most favorable composition was sodium alginate 0.78%, CaCl2 4.52%, hollow glass bead 3.12%, bacterial powder 3.0%, melanin 0.015%, sodium benzoate 0.2%, and mouse feed 0.5%, resulting in the immobilization time of 4.5 h, at which time the corrected sustained-release virulence rose 2391.67 fold, which was 6.07-fold higher than the basic formulation and deviated only 5.0% from the value predicted by RSM. PMID:28004743

  12. A screening of five Bacillus thuringiensis Vip3A proteins for their activity against lepidopteran pests.

    PubMed

    Ruiz de Escudero, Iñigo; Banyuls, Núria; Bel, Yolanda; Maeztu, Mireya; Escriche, Baltasar; Muñoz, Delia; Caballero, Primitivo; Ferré, Juan

    2014-03-01

    Five Bacillus thuringiensis Vip3A proteins (Vip3Aa, Vip3Ab, Vip3Ad, Vip3Ae and Vip3Af) and their corresponding trypsin-activated toxins were tested for their toxicity against eight lepidopteran pests: Agrotis ipsilon, Helicoverpa armigera, Mamestra brassicae, Spodoptera exigua, Spodoptera frugiperda, Spodoptera littoralis, Ostrinia nubilalis and Lobesia botrana. Toxicity was first tested at a high dose at 7 and 10 days. No major differences were found when comparing protoxins vs. trypsin-activated toxins. The proteins that were active against most of the insect species were Vip3Aa, Vip3Ae and Vip3Af, followed by Vip3Ab. Vip3Ad was non-toxic to any of the species tested. Considering the results by insect species, A. ipsilon, S. frugiperda and S. littoralis were susceptible to Vip3Aa, Vip3Ab, Vip3Ae and Vip3Af; S. exigua was susceptible to Vip3Aa and Vip3Ae, and moderately susceptible to Vip3Ab; M. brassicae and L. botrana were susceptible to Vip3Aa, Vip3Ae and Vip3Af; H. armigera was moderately susceptible to Vip3Aa, Vip3Ae and Vip3Af, and O. nubilalis was tolerant to all Vip3 proteins tested, although it showed some susceptibility to Vip3Af. The results obtained will help to design new combinations of insecticidal protein genes in transgenic crops or in recombinant bacteria for the control of insect pests.

  13. Isolation, purification and physicochemical characterization of water-soluble Bacillus thuringiensis melanin.

    PubMed

    Aghajanyan, Armen E; Hambardzumyan, Artur A; Hovsepyan, Anichka S; Asaturian, Rafael A; Vardanyan, Andranik A; Saghiyan, Ashot A

    2005-04-01

    Melanins are widely used in medicine, pharmacology, cosmetics and other fields. Although several technologies for the purification of water-insoluble dioxyphenylalanine (DOPA) melanins have been described, a source of water-soluble melanin is highly desirable. Here we describe an effective procedure for the isolation and purification of water-soluble melanin using the culture medium of Bacillus thuringiensis subsp. galleriae strain K1. Water-soluble melanin from this organism has an isoelectric point (pI=3.0-3.2) and was purified optimally by adsorbtion using the IA-1r resin and elution as a concentrated solution. The purified melanin obtained exhibited a similar infra-red absorbtion spectrum to synthetic melanin and contained quinolic and phenolic structures and an amino acid content of around 20% after acid hydrolysis. The molecular weight of the purified melanin determined by SDS-PAGE was 4 kDa and the electromagnetic spin resonance spectrum of the purified microbial melanin was a slightly asymmetric singlet without hyperfine structure with about 7 Gauss width of the line between points of the maximum incline and g=2.006. The concentration of paramagnetic centers in melanin is 0.21x10(18) spin/g. The results obtained provide a rapid, simple and inexpensive method for the large scale purification of water soluble melanin that may have widespread applications.

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

    PubMed

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

    2015-02-01

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

  15. Quantitative spectral light scattering polarimetry for monitoring fractal growth pattern of Bacillus thuringiensis bacterial colonies

    NASA Astrophysics Data System (ADS)

    Banerjee, Paromita; Soni, Jalpa; Ghosh, Nirmalya; Sengupta, Tapas K.

    2013-02-01

    It is of considerable current interest to develop various methods which help to understand and quantify the cellular association in growing bacterial colonies and is also important in terms of detection and identification of a bacterial species. A novel approach is used here to probe the morphological structural changes occurring during the growth of the bacterial colony of Bacillus thuringiensis under different environmental conditions (in normal nutrient agar, in presence of glucose - acting as additional nutrient and additional 3mM arsenate as additional toxic material). This approach combines the quantitative Mueller matrix polarimetry to extract intrinsic polarization properties and inverse analysis of the polarization preserving part of the light scattering spectra to determine the fractal parameter H (Hurst exponent) using Born approximation. Interesting differences are observed in the intrinsic polarization parameters and also in the Hurst exponent, which is a measurement of the fractality of a pattern formed by bacteria while growing as a colony. These findings are further confirmed with optical microscopic studies of the same sample and the results indicate a very strong and distinct dependence on the environmental conditions during growth, which can be exploited to quantify different bacterial species and their growth patterns.

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

    PubMed Central

    Papkou, Andrei; Laehnemann, David; Guenther, Patrick S.; Prahl, Swantje; Saebelfeld, Manja; Hollensteiner, Jacqueline; Liesegang, Heiko; Brzuszkiewicz, Elzbieta; Daniel, Rolf; Michiels, Nicolaas K.; Schulte, Rebecca D.; Kurtz, Joachim; Rosenstiel, Philip; Telschow, Arndt; Bornberg-Bauer, Erich; Schulenburg, Hinrich

    2015-01-01

    Reciprocal coevolution between host and pathogen is widely seen as a major driver of evolution and biological innovation. Yet, to date, the underlying genetic mechanisms and associated trait functions that are unique to rapid coevolutionary change are generally unknown. We here combined experimental evolution of the bacterial biocontrol agent Bacillus thuringiensis and its nematode host Caenorhabditis elegans with large-scale phenotyping, whole genome analysis, and functional genetics to demonstrate the selective benefit of pathogen virulence and the underlying toxin genes during the adaptation process. We show that: (i) high virulence was specifically favoured during pathogen–host coevolution rather than pathogen one-sided adaptation to a nonchanging host or to an environment without host; (ii) the pathogen genotype BT-679 with known nematocidal toxin genes and high virulence specifically swept to fixation in all of the independent replicate populations under coevolution but only some under one-sided adaptation; (iii) high virulence in the BT-679-dominated populations correlated with elevated copy numbers of the plasmid containing the nematocidal toxin genes; (iv) loss of virulence in a toxin-plasmid lacking BT-679 isolate was reconstituted by genetic reintroduction or external addition of the toxins. We conclude that sustained coevolution is distinct from unidirectional selection in shaping the pathogen's genome and life history characteristics. To our knowledge, this study is the first to characterize the pathogen genes involved in coevolutionary adaptation in an animal host–pathogen interaction system. PMID:26042786

  17. Active enhancement of Bacillus thuringiensis subsp-israelensis toxins and protein-purification.

    PubMed

    Al-Zahrani, Hind A A

    2011-12-01

    Bacillus thuringiensis sub sp. ilsraelensis de Barjac, that produce insecticidal protein endotoxins are used for mosquito control. The bacterium produces several Cry and Cytoxins that individually show activity against mosquitoes. A CryllA protein IA848, which corresponds to the first 848 amino acids from N-terminal of CryllA-gene was purified from E. coli by Ni-NTA affinity isolation, Q-Sepharose Fast-Flow chromatography & Super-200 size exclusion chromatography. It was determined using laboratory bioassays that the purified-IA848 protein has good insecticidal competitive binding bioassays show that IA848 does not compete with CryIAb for binding to the brush border membrane vesicles (BBM) of the Aedes aegyptíborer and does not compete with CryIAb at concentrations below 400-fold excess of unlabeled CryIAb for binding to the peritrophic matrix (PM) of the insect. This IA848 proved good competitive in control strategies. CryllA protein purification demonstrate molecular mechanism by which CytIA synergizes or suppresses resistance to toxins by providing a binding site for CryIIAa that resulted in an efficient formation of CryIIAa pre-pore that inserts into membranes and forms ionic pores.

  18. Optimization of photobioreactor growth conditions for a cyanobacterium expressing mosquitocidal Bacillus thuringiensis Cry proteins.

    PubMed

    Ketseoglou, Irene; Bouwer, Gustav

    2013-08-10

    An Anabaena strain (PCC 7120#11) that was genetically engineered to express Bacillus thuringiensis subsp. israelensis cry genes has shown good larvicidal activity against Anopheles arabiensis, a major vector of malaria in Africa. Response surface methodology was used to evaluate the relationship between key growth factors and the volumetric productivity of PCC 7120#11 in an indoor, flat-plate photobioreactor. The interaction of input CO₂ concentration and airflow rate had a statistically significant effect on the volumetric productivity of PCC 7120#11, as did the interaction of airflow rate and photosynthetic photon flux density. Model-based numerical optimization indicated that the optimal factor level combination for maximizing PCC 7120#11 volumetric productivity was a photosynthetic photon flux density of 154 μmol m⁻² s⁻¹ and air enriched with 3.18% (v/v) CO₂ supplied at a flow rate of 1.02 vessel volumes per minute. At the levels evaluated in the study, none of the growth factors had a significant effect on the median lethal concentration of PCC 7120#11 against An. arabiensis larvae. This finding is important because loss of mosquitocidal activity under growth conditions that maximize volumetric productivity would impact on the feasibility of using PCC 7120#11 in malaria vector control programs. The study showed the usefulness of response surface methodology for determination of the optimal growth conditions for a cyanobacterium that is genetically engineered to have larvicidal activity against malaria vectors.

  19. Transgenic organisms expressing genes from Bacillus thuringiensis to combat insect pests.

    PubMed

    Zaritsky, Arieh; Ben-Dov, Eitan; Borovsky, Dov; Boussiba, Sammy; Einav, Monica; Gindin, Galina; Horowitz, A Rami; Kolot, Mikhail; Melnikov, Olga; Mendel, Zvi; Yagil, Ezra

    2010-01-01

    Various subspecies (ssp.) of Bacillus thuringiensis (Bt) are considered the best agents known so far to control insects, being highly specific and safe, easily mass produced and with long shelf life.1 The para-crystalline body that is produced during sporulation in the exosporium includes polypeptides named δ-endotoxins, each killing a specific set of insects. The different entomopathogenic toxins of various Bt ssp. can be manipulated genetically in an educated way to construct more efficient transgenic bacteria or plants that express combinations of toxin genes to control pests.2 Joint research projects in our respective laboratories during the last decade demonstrate what can be done by implementing certain ideas using molecular biology with Bt ssp. israelensis (Bti) as a model system. Here, we describe our progress achieved with Gram-negative bacterial species, including cyanobacteria, and some preliminary experiments to form transgenic plants, mainly to control mosquitoes (Diptera), but also a particular Lepidopteran and Coleopteran pest species. In addition, a system is described by which environment-damaging genes can be removed from the recombinants thus alleviating procedures for obtaining permits to release them in nature.

  20. Protease activation of the entomocidal protoxin of Bacillus thuringiensis subsp. kurstaki.

    PubMed

    Andrews, R E; Bibilos, M M; Bulla, L A

    1985-10-01

    Two isolates of Bacillus thuringiensis subsp. kurstaki were examined which produced different levels of intracellular proteases. Although the crystals from both strains had comparable toxicity, one of the strains, LB1, had a strong polypeptide band at 68,000 molecular weight in the protein from the crystal; in the other, HD251, no such band was evident. When the intracellular proteases in both strains were measured, strain HD251 produced less than 10% of the proteolytic activity found in LB1. These proteases were primarily neutral metalloproteases, although low levels of other proteases were detected. In LB1, the synthesis of protease increased as the cells began to sporulate; however, in HD251, protease activity appeared much later in the sporulation cycle. The protease activity in strain LB1 was very high when the cells were making crystal toxin, whereas in HD251 reduced proteolytic activity was present during crystal toxin synthesis. The insecticidal toxin (molecular weight, 68,000) from both strains could be prepared by cleaving the protoxin (molecular weight, 135,000) with trypsin, followed by ion-exchange chromatography. The procedure described gave quantitative recovery of toxic activity, and approximately half of the total protein was recovered. Calculations show that these results correspond to stoichiometric conversion of protoxin to insecticidal toxin. The toxicities of whole crystals, soluble crystal protein, and purified toxin from both strains were comparable.

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

    PubMed

    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.

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

    PubMed

    Lucena, Wagner A; Pelegrini, Patrícia B; Martins-de-Sa, Diogo; Fonseca, Fernando C A; Gomes, Jose E; de Macedo, Leonardo L P; da Silva, Maria Cristina M; Oliveira, Raquel S; Grossi-de-Sa, Maria F

    2014-08-13

    Bacillus thuringiensis (Bt) is a gram-positive spore-forming soil bacterium that is distributed worldwide. Originally recognized as a pathogen of the silkworm, several strains were found on epizootic events in insect pests. In the 1960s, Bt began to be successfully used to control insect pests in agriculture, particularly because of its specificity, which reflects directly on their lack of cytotoxicity to human health, non-target organisms and the environment. Since the introduction of transgenic plants expressing Bt genes in the mid-1980s, numerous methodologies have been used to search for and improve toxins derived from native Bt strains. These improvements directly influence the increase in productivity and the decreased use of chemical insecticides on Bt-crops. Recently, DNA shuffling and in silico evaluations are emerging as promising tools for the development and exploration of mutant Bt toxins with enhanced activity against target insect pests. In this report, we describe natural and in vitro evolution of Cry toxins, as well as their relevance in the mechanism of action for insect control. Moreover, the use of DNA shuffling to improve two Bt toxins will be discussed together with in silico analyses of the generated mutations to evaluate their potential effect on protein structure and cytotoxicity.

  3. A single type of cadherin is involved in Bacillus thuringiensis toxicity in Plutella xylostella.

    PubMed

    Park, Y; Herrero, S; Kim, Y

    2015-12-01

    Cadherins have been described as one the main functional receptors for the toxins of the entomopathogenic bacterium, Bacillus thuringiensis (Bt). With the availability of the whole genome of Plutella xylostella, different types of cadherins have been annotated. In this study we focused on determining those members of the cadherin-related proteins that potentially play a role in the mode of action of Bt toxins. For this, we mined the genome of P. xylostella to identify these putative cadherins. The genome screening revealed 52 genes that were annotated as cadherin or cadherin-like genes. Further analysis revealed that six of these putative cadherins had three motifs common to all Bt-related cadherins: a signal peptide, cadherin repeats and a transmembrane domain. From the six selected cadherins, only P. xylostella cadherin 1 (PxCad1) was expressed in the larval midgut and only the silencing of this gene by RNA interference (double-stranded RNA feeding) reduce toxicity and binding to the midgut of the Cry1Ac type toxin from Bt. These results indicate that from the whole set of cadherin-related genes identified in P. xylostella, only PxCad1 is associated with the Cry1Ac mode of action.

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

    PubMed

    Bravo, Alejandra; Gill, Sarjeet S; Soberón, Mario

    2007-03-15

    Bacillus thuringiensis Crystal (Cry) and Cytolitic (Cyt) protein families are a diverse group of proteins with activity against insects of different orders--Lepidoptera, Coleoptera, Diptera and also against other invertebrates such as nematodes. Their primary action is to lyse midgut epithelial cells by inserting into the target membrane and forming pores. Among this group of proteins, members of the 3-Domain Cry family are used worldwide for insect control, and their mode of action has been characterized in some detail. Phylogenetic analyses established that the diversity of the 3-Domain Cry family evolved by the independent evolution of the three domains and by swapping of domain III among toxins. Like other pore-forming toxins (PFT) that affect mammals, Cry toxins interact with specific receptors located on the host cell surface and are activated by host proteases following receptor binding resulting in the formation of a pre-pore oligomeric structure that is insertion competent. In contrast, Cyt toxins directly interact with membrane lipids and insert into the membrane. Recent evidence suggests that Cyt synergize or overcome resistance to mosquitocidal-Cry proteins by functioning as a Cry-membrane bound receptor. In this review we summarize recent findings on the mode of action of Cry and Cyt toxins, and compare them to the mode of action of other bacterial PFT. Also, we discuss their use in the control of agricultural insect pests and insect vectors of human diseases.

  5. Current models of the mode of action of Bacillus thuringiensis insecticidal crystal proteins: a critical review.

    PubMed

    Vachon, Vincent; Laprade, Raynald; Schwartz, Jean-Louis

    2012-09-15

    Bacillus thuringiensis (Bt) Cry toxins constitute the active ingredient in the most widely used biological insecticides and insect-resistant transgenic crops. A clear understanding of their mode of action is necessary for improving these products and ensuring their continued use. Accordingly, a long history of intensive research has established that their toxic effect is due primarily to their ability to form pores in the plasma membrane of the midgut epithelial cells of susceptible insects. In recent years, a rather elaborate model involving the sequential binding of the toxins to different membrane receptors has been developed to describe the events leading to membrane insertion and pore formation. However, it was also proposed recently that, in contradiction with this mechanism, Bt toxins function by activating certain intracellular signaling pathways which lead to the necrotic death of their target cells without the need for pore formation. Because work in this field has largely focused, for several years, on the elaboration and promotion of these two models, the present revue examines in detail the experimental evidence on which they are based. It is concluded that the presently available information still supports the notion that Bt Cry toxins act by forming pores, but most events leading to their formation, following binding of the activated toxins to their receptors, remain relatively poorly understood.

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

    PubMed Central

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

    2003-01-01

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

  7. Biopolymer microencapsulations of Bacillus thuringiensis crystal preparations for increased stability and resistance to environmental stress.

    PubMed

    He, Xiaolin; Sun, Zhongqin; He, Kanglai; Guo, Shuyuan

    2017-04-01

    Parasporal crystals synthesized by Bacillus thuringiensis (Bt) have been widely used as microbial pesticides because of their toxicity to the larval stages of specific insects. However, parasporal crystals can be damaged by environmental stresses, such as high temperature, ultraviolet radiation, and desiccation. To reduce environmental susceptibility of parasporal crystals and extend the duration of their activity, we developed a new type of protection by making microcapsules of crystals (MCs). The microcapsules were self-assembled by alternate deposition (layer by layer) of low-cost chitosan and sodium alginate (or sodium carboxymethyl cellulose) on the crystal surface. Crystal toxins (Cry1Ac) were released from microcapsules at pH values above 9.0. Bioassay results demonstrated that microencapsulated preparations had larvicidal toxicity equivalent to the non-encapsulated form. Microencapsuled crystals were protected from environmental stresses such as high temperature and desiccation. The results indicate that microcapsule protection can enhance the efficacy of Bt in pest control, especially to Lepidoptera larvae that have a alkaline midgut.

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

    PubMed Central

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

    2000-01-01

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

  9. Mutagenesis of specificity and toxicity regions of a Bacillus thuringiensis protoxin gene.

    PubMed Central

    Aronson, A I; Wu, D; Zhang, C

    1995-01-01

    Two different 30-nucleotide regions of the cryIAc insecticidal protoxin gene from Bacillus thuringiensis were randomly mutagenized. One region was within one of seven amphipathic helices believed to be important for the formation of ion channels. There was no loss of toxicity for three test insects by any of 27 mutants, a result similar to that obtained previously for mutations within another such helix. Only mutations within a region encoding the central helix have resulted in a substantial number of mutants with low or no toxicity. A second mutagenized region encodes amino acids which are unique to this toxin and are within one of the loops in a portion of the toxin important for specificity. Among 21 different mutations of these 10 residues, only changes of two adjacent serine residues resulted in decreased toxicity which was greater for Manduca sexta than for Heliothis virescens larvae. These mutant toxins bound poorly to the single M. sexta CryIAc vesicle-binding protein and to several of the multiple H. virescens-binding proteins. The loop containing these serines must be involved in the formation of a specific toxin recognition domain. PMID:7608080

  10. Differences in susceptibility and physiological fitness of Mexican field Trichoplusia ni strains exposed to Bacillus thuringiensis.

    PubMed

    Tamez-Guerra, Patricia; Damas, Gabriela; Iracheta, Maria M; Oppert, Brenda; Gomez-Flores, Ricardo; Rodríguez-Padilla, Cristina

    2006-06-01

    The use of different commercial Bacillus thuringiensis (Bt) products in the Bajio guanajuatense area in Mexico began 12 yr ago, and resistance to Bt in this area has been reported for Plutella xylostella (L.) The current study provides a baseline response and resistance potential to Bt in field and laboratory strains of Bajio Trichoplusia ni (Hübner). Differences in susceptibility to Bt among T. ni populations were observed. T. ni neonates collected in Romita, Guanajuato, were more susceptible to Bt than those collected in Salvatierra or San Luis de la Paz, Guanajuato. After five generations of exposure to XenTari in the laboratory, decreased susceptibility was found only in the Salvatierra insects, with an LC50 that was 2.1-fold greater than that of a Mexican laboratory strain. The XenTari-selected San Luis de la Paz strain was from 16- to 87-fold more resistant to CrylA protoxins than U.S. (US) and Mexican laboratory strains. Although CrylAb is not a component of XenTari, this strain also was significantly less susceptible to CrylAb toxin compared with a US strain, with a resistance ratio of 40.4. The larval weights and lengths, pupal lengths, and percentage of pupation were significantly lower for the Salvatierra strain than for all other strains. The relationship of T. ni susceptibilities to Bt Cry toxins and protoxins after several generations of exposure to XenTari and its similarity to P. xylostella behavior.

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

    PubMed Central

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

    2016-01-01

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

  12. [Expression of mosquitocidal Cyt1Aa toxin from Bacillus thuringiensis subsp. israelensis in Asticcacaulis excentricus].

    PubMed

    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.

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

    PubMed

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

    2011-09-01

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

  14. New Resistance Mechanism in Helicoverpa armigera Threatens Transgenic Crops Expressing Bacillus thuringiensis Cry1Ac Toxin

    PubMed Central

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

    2005-01-01

    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. PMID:15870346

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

    PubMed

    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.

  16. Construction of an environmental safe Bacillus thuringiensis engineered strain against Coleoptera.

    PubMed

    Yu, Yajun; Yuan, Yihui; Gao, Meiying

    2016-05-01

    Cloning of new toxic genes from Bacillus thuringiensis (Bt) and construction of Bt engineered strains are two key strategies for bio-control of coleopteran pests in agriculture and forestry. In this study, we cloned a new cry3Aa-type gene, cry3Aa8, from wild Bt strain YC-03 against coleopteran, and constructed a Bt engineered strain, ACE-38, containing insecticidal protein-encoding gene cry3Aa8. The engineered strain, with almost four times of Cry3Aa yield compared with strain YC-03, was an antibiotic marker-free strain. Though no selective pressure was presented in the medium, cry3Aa8 in the engineered strain ACE-38 remained stable. The yield of Cry3Aa by strain ACE-38 reached 2.09 mg/ml in the optimized fermentation medium. The activity of strain ACE-38 against Plagiodera versicolora was tested, and the LC50 of ACE-38 cultures in the optimized fermentation medium was 1.13 μl/ml. Strain ACE-38 is a non-antibiotic Bt engineered strain with high Chrysomelidae toxicity and exhibits good fermentation property. The modified indigenous site-specific recombination system constructed in this study might be useful for the construction of Bt engineered strains containing genes that cannot be expressed in the indigenous site-specific recombination system using plasmid pBMB1205R.

  17. Batch and fed-batch fermentation of Bacillus thuringiensis using starch industry wastewater as fermentation substrate.

    PubMed

    Vu, Khanh Dang; Tyagi, Rajeshwar Dayal; Valéro, José R; Surampalli, Rao Y

    2010-08-01

    Bacillus thuringiensis var. kurstaki biopesticide was produced in batch and fed-batch fermentation modes using starch industry wastewater as sole substrate. Fed-batch fermentation with two intermittent feeds (at 10 and 20 h) during the fermentation of 72 h gave the maximum delta-endotoxin concentration (1,672.6 mg/L) and entomotoxicity (Tx) (18.5 x 10(6) SBU/mL) in fermented broth which were significantly higher than maximum delta-endotoxin concentration (511.0 mg/L) and Tx (15.8 x 10(6) SBU/mL) obtained in batch process. However, fed-batch fermentation with three intermittent feeds (at 10, 20 and 34 h) of the fermentation resulted in the formation of asporogenous variant (Spo-) from 36 h to the end of fermentation (72 h) which resulted in a significant decrease in spore and delta-endotoxin concentration and finally the Tx value. Tx of suspended pellets (27.4 x 10(6) SBU/mL) obtained in fed-batch fermentation with two feeds was the highest value as compared to other cases.

  18. Bioconversion of industrial wastewater and wastewater sludge into Bacillus thuringiensis based biopesticides in pilot fermentor.

    PubMed

    Yezza, A; Tyagi, R D; Valéro, J R; Surampalli, R Y

    2006-10-01

    Starch industry wastewater (SWW), slaughterhouse wastewater (SHWW) and secondary sludges from three different wastewater treatment plants (Jonquière--JQS, Communauté Urbaine de Québec--CUQS and Black lake-BLS) were used as raw materials for the production of Bacillus thuringiensis (Bt) based biopesticides in a pilot scale fermentor (100 L working volume). The slaughterhouse wastewater exhibited the lowest Bt growth and entomotoxcity (Tx) potential (measured against spruce budworm) due to low availability of carbon, nitrogen and other nutrients. Performance variation (growth, sporulation, proteolytic activity and Tx potential) within the three types of sludges was directly related to the availability of nitrogen and carbohydrates, which could change with sludge origin and methods employed for its generation. The Tx potential of Bt obtained in different secondary sludges (JQS: 12 x 10(9) SBU/L; CUQS: 13 x 10(9) SBU/L and BLS: 16 x 10(9) SBU/L) and SWW (18 x 10(9) SBU/L) was higher than the soybean based synthetic medium (10 x 10(9) SBU/L). The maximum protease activity was obtained in CUQ secondary sludge (4.1 IU/mL) due to its high complex protein concentration. Nevertheless, high carbohydrate concentration in SWW repressed enzyme production. The secondary sludges and SWW were found to be suitable raw materials for high potency Bt biopesticide production.

  19. Risk assessment and ecological effects of transgenic Bacillus thuringiensis crops on non-target organisms.

    PubMed

    Yu, Hui-Lin; Li, Yun-He; Wu, Kong-Ming

    2011-07-01

    The application of recombinant DNA technology has resulted in many insect-resistant varieties by genetic engineering (GE). Crops expressing Cry toxins derived from Bacillus thuringiensis (Bt) have been planted worldwide, and are an effective tool for pest control. However, one ecological concern regarding the potential effects of insect-resistant GE plants on non-target organisms (NTOs) has been continually debated. In the present study, we briefly summarize the data regarding the development and commercial use of transgenic Bt varieties, elaborate on the procedure and methods for assessing the non-target effects of insect-resistant GE plants, and synthetically analyze the related research results, mostly those published between 2005 and 2010. A mass of laboratory and field studies have shown that the currently available Bt crops have no direct detrimental effects on NTOs due to their narrow spectrum of activity, and Bt crops are increasing the abundance of some beneficial insects and improving the natural control of specific pests. The use of Bt crops, such as Bt maize and Bt cotton, results in significant reductions of insecticide application and clear benefits on the environment and farmer health. Consequently, Bt crops can be a useful component of integrated pest management systems to protect the crop from targeted pests.

  20. Design and construction of a synthetic Bacillus thuringiensis Cry4Aa gene: hyperexpression in Escherichia coli.

    PubMed

    Hayakawa, Tohru; Howlader, Mohammad Tofazzal Hossain; Yamagiwa, Masashi; Sakai, Hiroshi

    2008-10-01

    Cry4Aa produced by Bacillus thuringiensis is a dipteran-specific toxin and is, therefore, of great interest for developing a bioinsecticide to control mosquitoes. However, the expression of Cry4Aa in Escherichia coli is relatively low, which is a major disadvantage in its development as a bioinsecticide. In this study, to establish an effective production system, a 1,914-bp modified gene (cry4Aa-S1) encoding Cry4Aa was designed and synthesized in accordance with the G + C content and codon preference of E. coli genes without altering the encoded amino acid sequence. The cry4Aa-S1 gene allowed a significant improvement in expression level, over five-fold, compared to that of the original cry4Aa gene. The product of the cry4Aa-S1 gene showed the same level of insecticidal activity against Culex pipiens larvae as that from cry4Aa. This suggested that unfavorable codon usage was one of the reasons for poor expression of cry4Aa in E. coli, and, therefore, changing the cry4Aa codons to accord with the codon usage in E. coli led to efficient production of Cry4Aa. Efficient production of Cry4Aa in E. coli can be a powerful measure to prepare a sufficient amount of Cry4Aa protein for both basic analytical and applied researches.

  1. Improvement of Bacillus thuringiensis bioinsecticide production by sporeless and sporulating strains using response surface methodology.

    PubMed

    Ben Khedher, Saoussen; Kamoun, Amel; Jaoua, Samir; Zouari, Nabil

    2011-10-01

    Statistical experimental designs, involving a Plackett-Burman design followed by a rotatable central composite design were used to optimize the culture medium constituents for Bacillus thuringiensis bioinsecticide production. This was carried out by using firstly an asporogenic strain and extrapolated to some sporeless and sporulating strains. Initial screening of production parameters was performed and the variables with statistically significant effects on delta-endotoxin production were identified: glucose, glycerol, yeast extract and MnSO(4). These variables were selected for further optimization by response surface methodology. The obtained results revealed that the optimum culture medium for delta-endotoxin production consists of 22.5 g/l of glucose, 4.8g/l of glycerol, 5.8 g/l of yeast extract and 0.008 g/l of MnSO(4). Under these conditions, delta-endotoxin production was 2,130 and 2,260 mg/l into 250 and 1,000 ml flask respectively, which represent more than 38% improvement in toxin production over the basal medium (1,636 mg/l). Such medium composition was shown to be suitable for overproducing delta-endotoxins by sporeless and sporulating strains.

  2. Activity of Bacillus thuringiensis hybrid protein against a lepidopteran and a coleopteran pest.

    PubMed

    López-Pazos, Silvio Alejandro; Rojas Arias, Adriana Carolina; Ospina, Sonia A; Cerón, Jairo

    2010-01-01

    The use of Cry proteins from Bacillus thuringiensis are an important strategy for biological control. Recently it has been demonstrated that Cry hybrid proteins (by domain swapping) resulted in improved toxicities in comparison with parental proteins. Here, an SN1917 hybrid toxin was constructed and tested against Colombian pest insects Tecia solanivora (Lepidoptera: Gelechiidae), a severe potato pest, and Hypothenemus hampei (Coleoptera: Scolytidae), which attacks coffee crops. The SN1917 protoxin had a concentration causing 50% mortality (LC(50)) of 392 ng cm(-2), and SN1917 toxin showed an LC(50) of 201 ng cm(-2) against T. solanivora first instar larvae. The two parental toxins (Cry1B and Cry1I) used to make this new hybrid protein caused a mortality of 60% and 52%, respectively. Unfortunately, H. hampei first instar larvae proved to be resistant to the toxin. We conclude that SN1917 is an option for biological control and resistance management of T. solanivora. Implications for H. hampei are discussed.

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

    PubMed Central

    Lucena, Wagner A.; Pelegrini, Patrícia B.; Martins-de-Sa, Diogo; Fonseca, Fernando C. A.; Gomes, Jose E.; de Macedo, Leonardo L. P.; da Silva, Maria Cristina M.; Oliveira, Raquel S.; Grossi-de-Sa, Maria F.

    2014-01-01

    Bacillus thuringiensis (Bt) is a gram-positive spore-forming soil bacterium that is distributed worldwide. Originally recognized as a pathogen of the silkworm, several strains were found on epizootic events in insect pests. In the 1960s, Bt began to be successfully used to control insect pests in agriculture, particularly because of its specificity, which reflects directly on their lack of cytotoxicity to human health, non-target organisms and the environment. Since the introduction of transgenic plants expressing Bt genes in the mid-1980s, numerous methodologies have been used to search for and improve toxins derived from native Bt strains. These improvements directly influence the increase in productivity and the decreased use of chemical insecticides on Bt-crops. Recently, DNA shuffling and in silico evaluations are emerging as promising tools for the development and exploration of mutant Bt toxins with enhanced activity against target insect pests. In this report, we describe natural and in vitro evolution of Cry toxins, as well as their relevance in the mechanism of action for insect control. Moreover, the use of DNA shuffling to improve two Bt toxins will be discussed together with in silico analyses of the generated mutations to evaluate their potential effect on protein structure and cytotoxicity. PMID:25123558

  4. The cost of resistance to Bacillus thuringiensis varies with the host plant of Trichoplusia ni

    PubMed Central

    Janmaat, Alida F; Myers, Judith H

    2005-01-01

    Selection for resistance to insecticides, diseases and parasitoids is assumed to be costly and often requires tradeoffs with reproductive fitness. The costs of resistance, however, are often difficult to measure. Cabbage looper, Trichoplusia ni, a generalist Lepidopteran herbivore, has become highly resistant following the extensive use of the microbial insecticide, Bacillus thuringiensis kurstaki (Bt) in vegetable greenhouses. We compared the growth rate, pupal size and survival of resistant, susceptible and hybrid T. ni larvae fed on tomato, bell pepper and cucumber. Performance was best on cucumber and worst on pepper, and the magnitude of fitness costs associated with Bt resistance increased with declining host plant suitability. This supports the hypothesis that in this system, resistance costs are condition dependent and are greatest in the most stressful environment. Management strategies that rely on the presence of fitness costs to reduce the frequency of resistance genes must consider this variation and should be more successful on crops that are less suitable food plants. In general, condition dependence should be considered in studies designed to measure the costs of resistance. PMID:16024361

  5. Wastewater treatment sludge as a raw material for the production of Bacillus thuringiensis based biopesticides.

    PubMed

    Montiel, M D; Tyagi, R D; Valero, J R

    2001-11-01

    Seven wastewater sludges of different origins and types were used as an alternate culture medium for producing Bacillus thuringiensis variety kurstaki HD-1. The sludge samples were used under three different preparations: without pre-treatment, with acid treatment (hydrolysed sludge) and the supernatant obtained after centrifugation of the hydrolysed sludge. The sludge composition varied widely with origin and the type of sludge. Growth and sporulation were evaluated by the total viable cell count and spore count of the preparations. Growth, sporulation and endotoxin production were affected by the sludge origin. Hydrolysed sludge gave the highest viable cell and spore counts while the liquid phase (supernatant) gave the lowest. Non-hydrolysed primary sludge from Valcartier was unable to sustain bacterial growth because of its low pH. Bioassays were conducted against larvae of spruce budworm to evaluate entomotoxic potential of the preparations obtained. In general, sludge hydrolysis increased the entomotoxicity yields. Similar entomotoxicity was observed in Black Lake secondary sludge (4100 IU/microL) as that obtained in the reference soya medium (3800 IU/microL). The use of the sludge supernatant (liquid phase) was not recommended due to the low entomotoxic potential obtained.

  6. Diversity of Bacillus thuringiensis isolated from Western Ghats of Tamil Nadu State, India.

    PubMed

    Ramalakshmi, A; Udayasuriyan, V

    2010-07-01

    The Western Ghats of India is the one of the world's 10 "Hottest biodiversity hotspots" that runs along the western part of India through four states including Tamil Nadu. The only biodiversity reserve in the Western Ghats is the Nilgiri biosphere located in the Tamil Nadu state. In the present study, 525 soil samples were collected from all the 14 different divisions of the Western Ghats in Tamil Nadu state, India. A total of 316 new isolates of Bacillus thuringiensis (Bt) that produce parasporal crystalline inclusions were isolated from 525 soil samples. Seven different types of crystalline inclusions were observed in the 316 new isolates of Bt. Cuboidal inclusion was predominantly present in 26.9% of the Bt isolates when compared to other shapes. Further characterization of 70 of the 316 Bt isolates for crystal protein profile through SDS-PAGE revealed six different types of crystal protein profile viz., 135 and 65, 135, 95, 65, 43, and 30 kDa crystal proteins. Variation in the mass of crystal protein(s) purified from the isolates of Bt revealed molecular diversity of this bacterium prevalent in the Western Ghats of Tamil Nadu, India.

  7. Mining rare and ubiquitous toxin genes from a large collection of Bacillus thuringiensis strains.

    PubMed

    Li, Ying; Shu, Changlong; Zhang, Xuewen; Crickmore, Neil; Liang, Gemei; Jiang, Xingfu; Liu, Rongmei; Song, Fuping; Zhang, Jie

    2014-10-01

    There has been considerable effort made in recent years for research groups and other organizations to build up large collections of strains of Bacillus thuringiensis in the search for genes encoding novel insecticidal toxins, or encoding novel metabolic pathways. Whilst next generation sequencing allows the detailed genetic characterization of a bacterial strain with relative ease it is still not practicable for large strain collections. In this work we assess the practicability of mining a mixture of genomic DNA from a two thousand strain collection for particular genes. Using PCR the collection was screened for both a rare (cry15) toxin gene as well as a more commonly found gene (vip3A). The method was successful in identifying both a cry15 gene and multiple examples of the vip3A gene family including a novel member of this family (vip3Aj). A number of variants of vip3Ag were cloned and expressed, and differences in toxicity observed despite extremely high sequence similarity.

  8. Characterization of vegetative insecticidal protein vip genes of Bacillus thuringiensis from Sichuan Basin in China.

    PubMed

    Yu, Xiumei; Zheng, Aiping; Zhu, Jun; Wang, Shiquan; Wang, Lingxia; Deng, Qiming; Li, Shuangcheng; Liu, Huainian; Li, Ping

    2011-03-01

    Vegetative insecticidal proteins (Vip), the second generation of insecticides, are produced during the vegetative growth stage of Bacillus thuringiensis (Bt). To perform a systematic study of vip genes in Bt strains from different ecological regions of Sichuan Basin, 1,789 soil samples were collected from this basin, which is situated in the western region of China. The basin has a complicated geomorphology and contains mountains, forests, highlands, hursts, and plains. A total of 2,134 Bt strains have been screened from the 1,789 soil samples. According to the results, three vip-type genes were found in this basin, namely the vip1, vip2, and vip3-type genes. Strains containing vip3-type genes were the most abundant in our collection (67.4%), followed by vip2-type genes (14.6%) and vip1-type genes (8.1%). The three types of vip genes were distributed in most of the regions, but E Mei Mountain and the Ba Lang Mountains only contained vip3 genes in environments with high elevation, low temperature, insufficient oxygen, and abundant snow. Moreover, five novel vip3 genes were found, and these Vip proteins were toxic for Chilo suppressalis. All the results mentioned above suggest that Sichuan Basin is a rich resource for vip genes.

  9. Susceptibility of Spodoptera frugiperda and S. exigua to Bacillus thuringiensis Vip3Aa insecticidal protein.

    PubMed

    Chakroun, Maissa; Bel, Yolanda; Caccia, Silvia; Abdelkefi-Mesrati, Lobna; Escriche, Baltasar; Ferré, Juan

    2012-07-01

    The Vip3Aa protein is an insecticidal protein secreted by Bacillus thuringiensis during the vegetative stage of growth. The activity of this protein has been tested after different steps/protocols of purification using Spodoptera frugiperda as a control insect. The results showed that the Vip3Aa protoxin was stable and retained full toxicity after being subjected to common biochemical steps used in protein purification. Bioassays with the protoxin in S. frugiperda and S. exigua showed pronounced differences in LC(50) values when mortality was measured at 7 vs. 10d. At 7d most live larvae were arrested in their development. LC(50) values of "functional mortality" (dead larvae plus larvae remaining in the first instar), measured at 7d, were similar or even lower than the LC(50) values of mortality at 10d. This strong growth inhibition was not observed when testing the trypsin-activated protein (62 kDa) in either species. S. exigua was less susceptible than S. frugiperda to the protoxin form, with LC(50) values around 10-fold higher. However, both species were equally susceptible to the trypsin-activated form. Processing of Vip3Aa protoxin to the activated form was faster with S. frugiperda midgut juice than with S. exigua midgut juice. The results strongly suggest that the differences in the rate of activation of the Vip3Aa protoxin between both species are the basis for the differences in susceptibility towards the protoxin form.

  10. Acute, Sublethal, and Combination Effects of Azadirachtin and Bacillus thuringiensis on the Cotton Bollworm, Helicoverpa armigera

    PubMed Central

    Abedi, Zahra; Saber, Moosa; Vojoudi, Samad; Mahdavi, Vahid; Parsaeyan, Ehsan

    2014-01-01

    The cotton bollworm, Helicoverpa armigera Hübner (Lepidoptera: Noctuidae) is a polyphagous and cosmopolitan insect pest that causes damage to various plants. In this study, the lethal and sublethal effects of azadirachtin and Bacillus thuringiensis Berliner sub sp. kurstaki (Bacillales: Bacillaceae) were evaluated on third instar H. armigera under laboratory conditions. The LC50 values of azadirachtin and Bt were 12.95 and 96.8 µg a.i./mL, respectively. A total mortality of 56.7% was caused on third instar larvae when LC20 values of the insecticides were applied in combination with each other. The LT50 values of azadirachtin and Bt were 4.8 and 3.6 days, respectively. The results of the sublethal study showed that the application of LC30 value of azadirachtin and Bt reduced the larval and pupal weight and increased larval and pupal duration of H. armigera. The longevity and fecundity of female adults were affected significantly by the insecticides. Female fecundity was reduced by the treatments, respectively. The lowest adult emergence ratio and pupation ratio were observed in the azadirachtin treatment. The results indicated that both insecticides have high potential for controlling of the pest. PMID:25373177

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

    PubMed

    Rajendran, Deepak; Venkatachalam, Ponnusami; Ramakrishnan, Jayapradha

    2014-01-01

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

  12. Structural and genetic organization of IS232, a new insertion sequence of Bacillus thuringiensis.

    PubMed Central

    Menou, G; Mahillon, J; Lecadet, M M; Lereclus, D

    1990-01-01

    In the Bacillus thuringiensis strains toxic for the lepidopteran larvae, the delta-endotoxin genes cryIA are frequently found within a composite transposonlike structure flanked by two inverted repeat sequences. We report that these elements are true insertion sequences and designate them IS232. IS232 is a 2,184-bp element and is delimited by two imperfect inverted repeats (28 of 37 bp are identical). Two adjacent open reading frames, overlapping for three codons, span almost the entire sequence of IS232. The potential encoded polypeptides of 50 and 30-kDa are homologous to the IstA and IstB proteins of the gram-negative insertion sequence IS21. The N-terminal part of the 50-kDa polypeptide contains a helix-turn-helix DNA-binding motif. The junctions at the insertion sites of three IS232 elements were analyzed. Each case was different, with 0, 4, or 6 bp of the target DNA being duplicated. Transposition of IS232 in Escherichia coli was demonstrated by using a genetic marker inserted upstream of the two open reading frames. PMID:2174857

  13. Biosynthesis of silver nanoparticles using Bacillus thuringiensis against dengue vector, Aedes aegypti (Diptera: Culicidae).

    PubMed

    Banu, A Najitha; Balasubramanian, C; Moorthi, P Vinayaga

    2014-01-01

    The present study reveals the larvicidal activity of silver nanoparticles (AgNPs) synthesized by Bacillus thuringiensis (Bt) against Aedes aegypti responsible for the diseases of public health importance. The Bt-AgNPs were characterized by using UV-visible spectrophotometer followed by scanning electron microscopy (SEM) and energy-dispersive X-ray (EDX) spectroscopy. A surface plasmon resonance spectrum of AgNps was obtained at 420 nm. The particle sizes were measured through SEM imaging ranging from 43.52 to 142.97 nm. The Bt-AgNPs has also given a characteristic peak at 3 keV in EDX image. Interestingly, the mortality rendered by Bt-AgNPs was comparatively high than that of the control against third-instar larvae of A. aegypti (LC50 0.10 ppm and LC90 0.39 ppm) in all the tested concentrations, viz. 0.03, 0.06, 0.09, 0.12, and 0.15 ppm. Hence, Bt-AgNPs would be significantly used as a potent mosquito larvicide against A. aegypti.

  14. Bacillus thuringiensis fermentation of hydrolyzed sludge--rheology and formulation studies.

    PubMed

    Brar, Satinder K; Verma, M; Tyagi, R D; Valéro, J R; Surampalli, R Y

    2007-03-01

    Rheology of Bacillus thuringiensis fermentation of hydrolyzed sludge was investigated in bench scale fermenter. Stable liquid formulations were developed and optimized for two-year based studies comprising various physical/chemical (viscosity, particle size, corrosion and suspendibility) and biological (microbial contamination, viable spores and entomotoxicity) parameters at different pHs and temperatures. The hydrolyzed sludge depicted non-Newtonian and pseudoplastic behaviour during fermentation with 90% to 96% confidence of fits into Casson, Power and IPC paste models. Higher values of consistency and flow index during exponential growth and stationary phase, respectively, affected downstream processing. The power law was also followed by stable formulations. Sorbitol, sodium monophosphate and sodium metabisulfite (2.2:1:1) as suspending agents produced suspendibility ranging from 69% to 94%. The stable formulation (FH-4) comprising sorbitol, sodium monophosphate and sodium metabisulfite deteriorated at pHs 6, 6.5 and temperatures, 40 and 50 degrees C, with no signs of corrosion and microbial contamination. The viscosity of FH-4 formulations decreased with shear rate which could improve handling and consequent spraying.

  15. Entomotoxicity, protease and chitinase activity of Bacillus thuringiensis fermented wastewater sludge with a high solids content.

    PubMed

    Brar, Satinder K; Verma, M; Tyagi, R D; Valéro, J R; Surampalli, R Y

    2009-10-01

    This study investigated the production of biopesticides, protease and chitinase activity by Bacillus thuringiensis grown in raw wastewater sludge at high solids concentration (30 g/L). The rheology of wastewater sludge was modified with addition of Tween-80 (0.2% v/v). This addition resulted in 1.6 and 1.3-fold increase in cell and spore count, respectively. The maximum specific growth rate (micro(max)) augmented from 0.17 to 0.22 h(-1) and entomotoxicity (Tx) increased by 29.7%. Meanwhile, volumetric mass transfer coefficient (k(L)a) showed marked variations during fermentation, and oxygen uptake rate (OUR) increased 2-fold. The proteolytic activity increased while chitinase decreased for Tween amended wastewater sludge, but the entomotoxicity increased. The specific entomotoxicity followed power law when plotted against spore concentration and the relation between Tx and protease activity was linear. The viscosity varied and volume percent of particles increased in Tween-80 amended wastewater sludge and particle size (D(50)) decreased at the end of fermentation. Thus, there was an increase in entomotoxicity at higher suspended solids (30 g/L) as Tween addition improved rheology (viscosity, particle size, surface tension); enhanced maximum growth rate and OUR.

  16. Determination of spore concentration in Bacillus thuringiensis through the analysis of dipicolinate by capillary zone electrophoresis.

    PubMed

    He, Jin; Luo, Xiaofeng; Chen, Shouwen; Cao, Lili; Sun, Ming; Yu, Ziniu

    2003-04-25

    A new capillary zone electrophoresis (CZE) method for the analysis of dipicolinic acid, a specific component found in spores but not in vegetative cells, was used to determine spore concentration in Bacillus thuringiensis according to the relationship between the spore concentration and the content of dipicolinate. The quantitative relationship was established by using purified spores. Electrolyte conditions that affected the separation efficiency of dipicolinate and the reproducibility were investigated. With 10 mM phosphate, 10 mM ethylenediaminetetraacetic acid and 0.25 mM tetradecyltrimethylammonium bromide at pH 6.2 as the carrier electrolyte, dipicolinate can be determined within 8 min at an applied voltage of -25 kV (anode at detector) and a capillary temperature of 25 degrees C. The method has a high separation efficiency with which the number of theoretical plates is above 300,000 plates m(-1). The relative standard deviations for migration time and peak area are less than 0.5% and 2.0%, respectively. The detection limit for dipicolinate was 10 ng ml(-1), which corresponds to 7.2 x 10(5) spores ml(-1). The method was used to determine spores in fermentation broths, and the results obtained agreed well with the values obtained by plate counting.

  17. Kinetics of Bacillus thuringiensis var. israelensis growth on high glucose concentrations.

    PubMed

    Berbert-Molina, M A; Prata, A M R; Pessanha, L G; Silveira, M M

    2008-11-01

    The kinetic and general growth features of Bacillus thuringiensis var. israelensis were evaluated. Initial glucose concentration (S0) in fermentation media varied from 10 to 152 g/l. The results afforded to characterize four morphologically and physiologically well-defined culture phases, independent of S0 values: Phase I, vegetative growth; Phase II, transition to sporulation; Phase III, sporulation; and Phase IV, spores maturation and cell lysis. Important process parameters were also determined. The maximum specific growth rates (microX,m) were not affected with S0 up to 75 g/l (1.0-1.1 per hour), but higher glucose concentrations resulted in growth inhibition by substrate, revealed by a reduction in microX,m values. These higher S0 values led to longer Phases III and IV and delayed sporulation. Similar biomass concentrations (Xm=15.2-15.9 g/l) were achieved with S0 over 30.8 g/l, with increasing residual substrate, suggesting a limitation in some other nutrients and the use of glucose to form other metabolites. In this case, with S0 from 30.8 to 152 g/l, cell yield (YX/S) decreased from 0.58 to 0.41 g/g. On the other hand, with S0=10 g/l growth was limited by substrate, and YX/S has shown its maximum value (0.83 g/g).

  18. Long-term regional suppression of pink bollworm by Bacillus thuringiensis cotton

    PubMed Central

    Carrière, Yves; Ellers-Kirk, Christa; Sisterson, Mark; Antilla, Larry; Whitlow, Mike; Dennehy, Timothy J.; Tabashnik, Bruce E.

    2003-01-01

    Despite the potentially profound impact of genetically modified crops on agriculture and the environment, we know little about their long-term effects. Transgenic crops that produce toxins from Bacillus thuringiensis (Bt) to control insects are grown widely, but rapid evolution of resistance by pests could nullify their benefits. Here, we present theoretical analyses showing that long-term suppression of pest populations is governed by interactions among reproductive rate, dispersal propensity, and regional abundance of a Bt crop. Supporting this theory, a 10-year study in 15 regions across Arizona shows that Bt cotton suppressed a major pest, pink bollworm (Pectinophora gossypiella), independent of demographic effects of weather and variation among regions. Pink bollworm population density declined only in regions where Bt cotton was abundant. Such long-term suppression has not been observed with insecticide sprays, showing that transgenic crops open new avenues for pest control. The debate about putative benefits of Bt crops has focused primarily on short-term decreases in insecticide use. The present findings suggest that long-term regional pest suppression after deployment of Bt crops may also contribute to reducing the need for insecticide sprays. PMID:12571355

  19. Transgenic organisms expressing genes from Bacillus thuringiensis to combat insect pests§

    PubMed Central

    Ben-Dov, Eitan; Borovsky, Dov; Boussiba, Sammy; Einav, Monica; Gindin, Galina; Horowitz, A Rami; Kolot, Mikhail; Melnikov, Olga; Mendel, Zvi; Yagil, Ezra

    2010-01-01

    Various subspecies (ssp.) of Bacillus thuringiensis (Bt) are considered the best agents known so far to control insects, being highly specific and safe, easily mass produced and with long shelf life.1 The para-crystalline body that is produced during sporulation in the exosporium includes polypeptides named δ-endotoxins, each killing a specific set of insects. The different entomopathogenic toxins of various Bt ssp. can be manipulated genetically in an educated way to construct more efficient transgenic bacteria or plants that express combinations of toxin genes to control pests.2 Joint research projects in our respective laboratories during the last decade demonstrate what can be done by implementing certain ideas using molecular biology with Bt ssp. israelensis (Bti) as a model system. Here, we describe our progress achieved with Gram-negative bacterial species, including cyanobacteria, and some preliminary experiments to form transgenic plants, mainly to control mosquitoes (Diptera), but also a particular Lepidopteran and Coleopteran pest species. In addition, a system is described by which environment-damaging genes can be removed from the recombinants thus alleviating procedures for obtaining permits to release them in nature. PMID:21326834

  20. [Enhancing effect of Tween 80 on degradation of triphenyltin by Bacillus thuringiensis].

    PubMed

    Huang, Jie; Ye, Jin-Shao; Yin, Hua; Peng, Hui; Ma, Jia-Wen; Tang, Li-Tao; Wang, Xi-Ruo

    2014-05-01

    So far, the information regarding enhanced degradation and biodegradation mechanisms of TPhT, an endocrine disruptor, is severely limited. Whether dearylation during TPhT degradation occurs successively or synchronously is not revealed clearly. To deal with these problems, this study focused on the biodegradation of TPhT and its metabolites by Bacillus thuringiensis through the acceleration of Tween 80. The results showed that Tween 80 obviously increased the TPhT solubility. After degradation by cells in the presence of 80 mg L-1 Tween 80 for 2 d, the residual TPhT at 1 mg L-1 initially was decreased to 48.4%. During the biodegradation process, Tween 80 significantly reduced intracellular Na+, NH+4: and Mg2+ release, and increased extracellular Cl- , PO(3-)4 and K+ utilization. Metabolites analysis revealed that phenyltin biodegradation initially proceeded by cleaving the aromatic ring, not by splitting the covalent bonds between the benzene rings and tin atom. Ring-cleavage reactions in the benzenes of TPhT occurred individually and synchronously, producing diphenyltin, monophenyltin and tin accordingly.

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

    PubMed

    Chilcutt, Charles F; Tabashnik, Bruce E

    2004-05-18

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

  2. The Efficacy of Bacillus thuringiensis spp. galleriae Against Rice Water Weevil (Coleoptera: Curculionidae) for Integrated Pest Management in California Rice.

    PubMed

    Aghaee, Mohammad-Amir; Godfrey, Larry D

    2015-02-01

    Rice water weevil (Lissorhoptrus oryzophilus Kushel) is the most damaging insect pest of rice in the United States. Larval feeding on the roots stunt growth and reduce yield. Current pest management against the weevil in California relies heavily on pyrethroids that can be damaging to aquatic food webs. Examination of an environmentally friendly alternative biopesticide based on Bacillus thuringiensis spp. galleriae chemistry against rice water weevil larvae showed moderate levels of activity in pilot studies. We further examined the performance of different formulations of Bt.galleriae against the leading insecticide used in California rice, λ-cyhalothrin. The granular formulation performed as well as the λ-cyhalothrin in use in California in some of our greenhouse and field studies. This is the first reported use of B. thuringiensis spp. galleriae against rice water weevil.

  3. Rapid evolution and the cost of resistance to Bacillus thuringiensis in greenhouse populations of cabbage loopers, Trichoplusia ni.

    PubMed Central

    Janmaat, Alida F; Myers, Judith

    2003-01-01

    The microbial insecticide Bacillus thuringiensis (Bt) has become the mainstay of non-chemical control of Lepidopteran pests, either as sprays or through the incorporation of Bt toxins into transgenic crops. Given the wide use of Bt, it is striking that currently only one pest species, Plutella xylostella, has been reported to have developed significant resistance to Bt outside the laboratory. By contrast, we report here the frequent and rapid development of resistance to B. thuringiensis kurstaki (Dipel, Abbott) in populations of cabbage loopers, Trichoplusia ni, in commercial greenhouses. Resistance to Bt appears to be costly and there is a rapid decline of resistance in populations collected from greenhouses and maintained in the laboratory without selection. Management of pests resistant to Bt in vegetable greenhouses will require sporadic use of Bt-based sprays or alternatively use of sprays containing other Bt toxins. PMID:14613613

  4. Genetic diversity among Bacillus anthracis, Bacillus cereus and Bacillus thuringiensis strains using repetitive element polymorphism-PCR.

    PubMed

    Brumlik, Michael J; Bielawska-Drózd, Agata; Zakowska, Dorota; Liang, Xudong; Spalletta, Ronald A; Patra, Guy; Delvecchio, Vito G

    2004-01-01

    Repetitive element polymorphism-PCR (REP-PCR) is one of the tools that has been used to elucidate genetic diversity of related microorganisms. Using the MB1 primer, REP-PCR fingerprints from 110 Bacillus strains within the "B. cereus group" have identified eighteen distinct categories, while other more distantly related bacterial species fell within six additional categories. All Bacillus anthracis strains tested were found to be monomorphic by fluorophore-enhanced REP-PCR (FERP) fingerprinting using the MB1 primer. In contrast, other non- B. anthracis isolates displayed a high degree of polymorphism. Dendrogramic analysis revealed that the non- B. anthracis strains possessing the Ba813 chromosomal marker were divided into two clusters. One of the clusters shared identity with the B. cereus strains examined.

  5. The response of Vaal River drift and benthos to Simulium (Diptera: Nematocera) control using Bacillus thuringiensis var. israelensis (H-14).

    PubMed

    Car, M; De Moor, F C

    1984-09-01

    Two trials to test the efficacy of Bacillus thuringiensis Berliner var. israelensis de Barjac (serotype H-14) against target simuliid and non-target aquatic invertebrates were undertaken in the Vaal River near Warrenton in South Africa. In the 1st trail an application of 1,6 ppm/10 min of B. thuringiensis resulted in a significant (P less than 0,05) reduction of simuliid larvae in rapids 70 m below the treatment point 40 hours after its application. Further downstream the larvicide was ineffectual because the low flow of the river (6 m3/s) allowed the Bacillus pores to settle out in calmer stretches. The 2nd trial was carried out upstream of small rapids with a calculated flow of 0,5 m3/s at a spore concentration of 2,3 ppm/7 min. The effect of B. thuringiensis on the benthic population density and drift activity of the benthos was recorded. A high mortality of simuliid larvae and Ephemeroptera was observed 35 m below the application point 9 hours after the application of the larvicide. The mortality in Ephemeroptera was partially due to the handling of these animals. Population densities of simuliid larvae in the treated rapids decreased 18 hours after application of the larvicide, but small simuliid larvae showed a numerical increase again after 72 hours, indicating rapid recolonization from drifting larvae. Tanytarsine Chironomidae decreased after the application of B. thuringiensis, but most other fauna either increased or did not decrease significantly (P greater than 0,05). Within 43 minutes after treatment of the rapids with the larvicide, simuliid drift increased more than sixtyfold, revealing the immediate irritating effect of the product on the target organisms.(ABSTRACT TRUNCATED AT 250 WORDS)

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

    PubMed

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

    2015-04-01

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

  7. Distribution of Bacillus thuringiensis subsp. israelensis in Soil of a Swiss Wetland Reserve after 22 Years of Mosquito Control▿†

    PubMed Central

    Guidi, Valeria; Patocchi, Nicola; Lüthy, Peter; Tonolla, Mauro

    2011-01-01

    Recurrent treatments with Bacillus thuringiensis subsp. israelensis are required to control the floodwater mosquito Aedes vexans that breeds in large numbers in the wetlands of the Bolle di Magadino Reserve in Canton Ticino, Switzerland. Interventions have been carried out since 1988. In the present study, the spatial distribution of resting B. thuringiensis subsp. israelensis spores in the soil was measured. The B. thuringiensis subsp. israelensis concentration was determined in soil samples collected along six transects covering different elevations within the periodically flooded zones. A total of 258 samples were processed and analyzed by quantitative PCR that targeted an identical fragment of 159 bp for the B. thuringiensis subsp. israelensis cry4Aa and cry4Ba genes. B. thuringiensis subsp. israelensis spores were found to persist in soils of the wetland reserve at concentrations of up to 6.8 log per gram of soil. Continuous accumulation due to regular treatments could be excluded, as the decrease in spores amounted to 95.8% (95% confidence interval, 93.9 to 97.7%). The distribution of spores was correlated to the number of B. thuringiensis subsp. israelensis treatments, the elevation of the sampling point, and the duration of the flooding periods. The number of B. thuringiensis subsp. israelensis treatments was the major factor influencing the distribution of spores in the different topographic zones (P < 0.0001). These findings indicated that B. thuringiensis subsp. israelensis spores are rather immobile after their introduction into the environment. PMID:21498758

  8. Response surface modeling for hot, humid air decontamination of materials contaminated with Bacillus anthracis ∆Sterne and Bacillus thuringiensis Al Hakam spores

    PubMed Central

    2014-01-01

    Response surface methodology using a face-centered cube design was used to describe and predict spore inactivation of Bacillus anthracis ∆Sterne and Bacillus thuringiensis Al Hakam spores after exposure of six spore-contaminated materials to hot, humid air. For each strain/material pair, an attempt was made to fit a first or second order model. All three independent predictor variables (temperature, relative humidity, and time) were significant in the models except that time was not significant for B. thuringiensis Al Hakam on nylon. Modeling was unsuccessful for wiring insulation and wet spores because there was complete spore inactivation in the majority of the experimental space. In cases where a predictive equation could be fit, response surface plots with time set to four days were generated. The survival of highly purified Bacillus spores can be predicted for most materials tested when given the settings for temperature, relative humidity, and time. These predictions were cross-checked with spore inactivation measurements. PMID:24949256

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

    PubMed

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

    2010-08-01

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

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

    NASA Technical Reports Server (NTRS)

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

    1973-01-01

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

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

    PubMed

    El-kersh, T A; Al-akeel, R A; Al-sheikh, Y A; Alharbi, S A

    2014-12-01

    A total of 157 environmental samples were collected from 11 ecological regions across Saudi Arabia to isolate native Bacillus thuringiensis (Bt) strains. Bt isolates (n=103) were recovered by the 50% (v/v) ethanol treatment method with Bt index range of 0.01 to 0.4. Most of Bt isolates showed spherical crystals (54%), while, irregular, bi-pyramidal, and spore-attached crystal constituted 27, 16 and 3% respectively. PCR analysis with eight general and specific dipteran primers of Cry and Cyt genes, revealed positive amplification for cry4 & cyt1, and cry4A, cry4B and cyt2, and cry 10 and cry 11 genes in 28%, 26%, 22%, and 25% of tested strains respectively; whereas cry2 gene was not detected except with the reference Bt kurstaki HD-1 strain. Bioassays against Aedes caspuis and Culex pipiens larvae indicated that 11 strains displayed better larvicidal activity compared with Bacillus thuringiensis H14 (Bti) reference (LC50 0.6 μg/ml) strain against Ae. caspuis, but only two strains (620A & 633R1, LC50 of 0.09 μg/ml & 0.064 μg/ml) that gave significant enhancement. Additionally, one strain (633R1) showed LC50 similar to that of Bti H14 (LC50 0.064 μg/ml) against Cx. pipiens. With the exception of cyt primers, sequenced DNA of all positive primers amplicons revealed 95 to 99% identity in GenBank with Bacillus thuringiensis subsp. israelensis plasmid pBtoxis and also correlated with its SDS-PAGE expressed protein profiles analysis. It is hoped that our wild bio-insecticide Bt strains can be explored in future in the control of mosquito-vector borne diseases in Saudi Arabia.

  12. Operational Evaluation Of Vectomax® WSP (Bacillus thuringiensis Subsp. israelensis+Bacillus sphaericus) Against Larval Culex pipiens in Septic Tanks (1).

    PubMed

    Cetin, Huseyin; Oz, Emre; Yanikoglu, Atila; Cilek, James E

    2015-06-01

    The residual effectiveness of VectoMax® WSP (a water-soluble pouch formulation containing a combination of Bacillus thuringiensis subsp. israelensis strain AM65-52 and B. sphaericus strain ABTS 1743) when applied to septic tanks against 3rd- and 4th-stage larvae of Culex pipiens L. was evaluated in this study. This formulation was evaluated at operational application rates of 1 pouch (10 g) and 2 pouches (20 g) per septic tank. Both application rates resulted in >96% control of larvae for 24 days. Operationally, VectoMax WSP has proven to be a useful tool for the nonchemical control of Culex species in septic tank environments.

  13. [Effect of delta-endotoxin of Bacillus thuringiensis israëlensis on biochemical functional relationships in Diptera Aedes aegypti].

    PubMed

    Bounias, M; Nizeyimana, B; Vago, C

    1986-01-01

    A study of the action of very low doses of the delta-endotoxin of Bacillus thuringiensis israëlensis on the larvae of Aedes aegypti (Diptera) gave evidence for peculiar properties of the dose/effect relations based on the variations of the regression slopes of functional relationships between the pairs cysteine/serine and fatty acids/histidine, by contrast with the pair fatty acids/glucose which exhibited a classical shaped relation. This indicates "crypto-toxicological" processes, not lethal by themselves and without pathological symptoms, but able to initiate "pathogen-chaining" mechanisms leading, at the end, to a lethal secondary syndrome.

  14. Single concentration tests show synergism among Bacillus thuringiensis subsp. israelensis toxins against the malaria vector mosquito Anopheles albimanus.

    PubMed

    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.

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

    PubMed

    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.

  16. Complete genome sequence of Bacillus thuringiensis tenebrionis 4AA1, a typical strain with toxicity to Coleopteran insects.

    PubMed

    Gao, Qiuling; Zheng, Jinshui; Zhu, Lei; Ruan, Lifang; Peng, Donghai; Sun, Ming

    2015-06-20

    Bacillus thuringiensis serovar morrisoni biovar tenebrionis has been developed as an bioinsecticide to control Coleopteran insects in agriculture and forestry for a few decades. Its major crystal protein Cry3Aa was also applied to transgenic crops. Here we report the complete genome sequence of strain tenebrionis 4AA1, which has one chromosome of 5,652,292bp and six plasmids. Two crystal protein genes, cry3Aa and cry15Aa, locate on one single plasmid named pBMB51. This strain also possesses plentiful virulence factors besides crystal proteins.

  17. Cry9Ca1 Toxin, a Bacillus thuringiensis Insecticidal Crystal Protein with High Activity against the Spruce Budworm (Choristoneura fumiferana).

    PubMed

    Van Frankenhuyzen, K; Gringorten, L; Gauthier, D

    1997-10-01

    The Cry9Ca1 toxin from Bacillus thuringiensis was significantly more toxic to spruce budworm (Choristoneura fumiferana) than the Cry1Ab6, Cry1Ba1, Cry1Ca2, Cry1Da1, Cry1Ea1, and Cry1Fa2 toxins. It displayed high activity against silkworm (Bombyx mori) but was not toxic to black army cutworm (Actebia fennica) or gypsy moth (Lymantria dispar). The Cry9Ca1 is the most effective spruce budworm toxin known to date and may offer promise for control and resistance management of that species.

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

    PubMed Central

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

    2006-01-01

    Crystal (Cry) proteins produced by the soil bacterium Bacillus thuringiensis (Bt) are harmless to vertebrates, but they are highly toxic to insects and nematodes. Their value in controlling insects that destroy crops and transmit human diseases is well established. Although it has recently been demonstrated that a few individual Bt Cry proteins, such as Cry5B, are toxic to a wide range of free-living nematodes, the potential activity of purified Cry proteins against parasitic nematodes remains largely unknown. We report here studies aimed at characterizing in vitro and in vivo anthelminthic activities of purified recombinant Cry5B against the hookworm parasite Ancylostoma ceylanicum, a bloodfeeding gastrointestinal nematode for which humans are permissive hosts. By using in vitro larval development assays, Cry5B was found to be highly toxic to early stage hookworm larvae. Exposure of adult A. ceylanicum to Cry5B was also associated with significant toxicity, including a substantial reduction in egg excretion by adult female worms. To demonstrate therapeutic efficacy in vivo, hamsters infected with A. ceylanicum were treated with three daily oral doses of purified Cry5B, the benzimidazole anthelminthic mebendazole, or buffer. Compared with control (buffer-treated) animals, infected hamsters that received Cry5B showed statistically significant improvements in growth and blood hemoglobin levels as well as reduced worm burdens that were comparable to the mebendazole-treated animals. These data demonstrate that Cry5B is highly active in vitro and in vivo against a globally significant nematode parasite and that Cry5B warrants further clinical development for human and veterinary use. PMID:17005719

  19. Transgenic Bacillus thuringiensis (Bt) rice is safer to aquatic ecosystems than its non-transgenic counterpart.

    PubMed

    Li, Guangsheng; Wang, Yongmo; Liu, Biao; Zhang, Guoan

    2014-01-01

    Rice lines genetically modified with the crystal toxin genes from Bacillus thuringiensis (Bt) have experienced rapid development, with biosafety certificates for two Bt rice lines issued in 2009. There has still been no commercial release of these lines yet due to public concerns about human health and environmental risks. Some studies confirmed that Bt rice was as safe as conventional rice to non-target organisms when pesticides were not applied, however, pesticides are still required in Bt rice to control non-lepidopteran pests. In this study, we assessed the environmental effects of two Bt rice lines expressing either the cry1Ab/1Ac or cry2A genes, respectively, by using zooplanktons as indicator species under normal field management practices using pesticides when required. In the whole rice growing season, non-Bt rice was sprayed 5 times while Bt rice was sprayed 2 times, which ensured both rice achieved a normal yield. Field investigations showed that rice type (Bt and non-Bt) significantly influenced zooplankton abundance and diversity, which were up to 95% and 80% lower in non-Bt rice fields than Bt rice fields. Laboratory rearing showed that water from non-Bt rice fields was significantly less suitable for the survival and reproduction of Daphnia magna and Paramecium caudatum in comparison with water from Bt rice fields. Higher pesticide residues were detected in the water from non-Bt than Bt rice fields, accounting for the bad performance of zooplankton in non-Bt field water. Our results demonstrate that Bt rice is safer to aquatic ecosystems than non-Bt rice, and its commercialization will be beneficial for biodiversity restoration in rice-based ecosystems.

  20. Bacillus thuringiensis: mechanism of action, resistance, and new applications: a review.

    PubMed

    Melo, André Luiz de Almeida; Soccol, Vanete Thomaz; Soccol, Carlos Ricardo

    2016-01-01

    Since the first report by Ishiwata in 1902 of a Bombyx mori infection, followed by the description by Berliner, Bacillus thuringiensis (Bt) has become the main microorganism used in biological control. The application of Bt to combat invertebrates of human interest gained momentum with the growing demand for food free of chemical pesticides and with the implementation of agriculture methods that were less damaging to the environment. However, the mechanisms of action of these products have not been fully elucidated. There are two proposed models: the first is that Bt causes an osmotic imbalance in response to the formation of pores in a cell membrane, and the second is that it causes an opening of ion channels that activate the process of cell death. There are various ways in which Bt resistance can develop: changes in the receptors that do not recognize the Cry toxin, the synthesis of membrane transporters that eliminate the peptides from the cytosol and the development of regulatory mechanisms that disrupt the production of toxin receptors. Besides the potential for formulation of biopesticides and the use in developing genetically modified cultivars, recent studies with Bt have discussed promising applications in other branches of science. Chitinase, an enzyme that degrades chitin, increases the efficiency of Bt insecticides, and there has been of increasing interest in the industry, given that its substrate is extremely abundant in nature. Another promising field is the potential for Bt proteins to act against cancer cells. Parasporins, toxins of Bt that do not have an entomopathogenic effect, have a cytotoxic effect on the cells changed by some cancers. This demonstrates the potential of the microorganism and new opportunities opening for future applications.

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

    PubMed

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

    2003-12-01

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

  2. Transgenic Bacillus thuringiensis (Bt) Rice Is Safer to Aquatic Ecosystems than Its Non-Transgenic Counterpart

    PubMed Central

    Li, Guangsheng; Wang, Yongmo; Liu, Biao; Zhang, Guoan

    2014-01-01

    Rice lines genetically modified with the crystal toxin genes from Bacillus thuringiensis (Bt) have experienced rapid development, with biosafety certificates for two Bt rice lines issued in 2009. There has still been no commercial release of these lines yet due to public concerns about human health and environmental risks. Some studies confirmed that Bt rice was as safe as conventional rice to non-target organisms when pesticides were not applied, however, pesticides are still required in Bt rice to control non-lepidopteran pests. In this study, we assessed the environmental effects of two Bt rice lines expressing either the cry1Ab/1Ac or cry2A genes, respectively, by using zooplanktons as indicator species under normal field management practices using pesticides when required. In the whole rice growing season, non-Bt rice was sprayed 5 times while Bt rice was sprayed 2 times, which ensured both rice achieved a normal yield. Field investigations showed that rice type (Bt and non-Bt) significantly influenced zooplankton abundance and diversity, which were up to 95% and 80% lower in non-Bt rice fields than Bt rice fields. Laboratory rearing showed that water from non-Bt rice fields was significantly less suitable for the survival and reproduction of Daphnia magna and Paramecium caudatum in comparison with water from Bt rice fields. Higher pesticide residues were detected in the water from non-Bt than Bt rice fields, accounting for the bad performance of zooplankton in non-Bt field water. Our results demonstrate that Bt rice is safer to aquatic ecosystems than non-Bt rice, and its commercialization will be beneficial for biodiversity restoration in rice-based ecosystems. PMID:25105299

  3. Synergism of Bacillus thuringiensis toxins by a fragment of a toxin-binding cadherin.

    PubMed

    Chen, Jiang; Hua, Gang; Jurat-Fuentes, Juan Luis; Abdullah, Mohd Amir; Adang, Michael J

    2007-08-28

    The insecticidal crystal proteins produced by Bacillus thuringiensis (Bt) are broadly used to control insect pests with agricultural importance. The cadherin Bt-R(1) is a binding protein for Bt Cry1A toxins in midgut epithelia of tobacco hornworm (Manduca sexta). We previously identified the Bt-R(1) region most proximal to the cell membrane (CR12-MPED) as the essential binding region required for Cry1Ab-mediated cytotoxicity. Here, we report that a peptide containing this region expressed in Escherichia coli functions as a synergist of Cry1A toxicity against lepidopteran larvae. Far-UV circular dichroism and (1)H-NMR spectroscopy confirmed that our purified CR12-MPED peptide mainly consisted of beta-strands and random coils with unfolded structure. CR12-MPED peptide bound brush border membrane vesicles with high affinity (K(d) = 32 nM) and insect midgut microvilli but did not alter Cry1Ab or Cry1Ac binding localization in the midgut. By BIAcore analysis we demonstrate that Cry1Ab binds CR12-MPED at high (9 nM)- and low (1 microM)-affinity sites. CR12-MPED-mediated Cry1A toxicity enhancement was significantly reduced when the high-affinity Cry1A-binding epitope ((1416)GVLTLNIQ(1423)) within the peptide was altered. Because the mixtures of low Bt toxin dose and CR12-MPED peptide effectively control target insect pests, our discovery has important implications related to the use of this peptide to enhance insecticidal activity of Bt toxin-based biopesticides and transgenic Bt crops.

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

    PubMed

    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.

  5. Insecticidal spectrum and mode of action of the Bacillus thuringiensis Vip3Ca insecticidal protein.

    PubMed

    Gomis-Cebolla, Joaquín; Ruiz de Escudero, Iñigo; Vera-Velasco, Natalia Mara; Hernández-Martínez, Patricia; Hernández-Rodríguez, Carmen Sara; Ceballos, Tomás; Palma, Leopoldo; Escriche, Baltasar; Caballero, Primitivo; Ferré, Juan

    2017-01-01

    The Vip3Ca protein, discovered in a screening of Spanish collections of Bacillus thuringiensis, was known to be toxic to Chrysodeixis chalcites, Mamestra brassicae and Trichoplusia ni. In the present study, its activity has been tested with additional insect species and we found that Cydia pomonella is moderately susceptible to this protein. Vip3Ca (of approximately 90kDa) was processed to an approximately 70kDa protein when incubated with midgut juice in all tested species. The kinetics of proteolysis correlated with the susceptibility of the insect species to Vip3Ca. The activation was faster to slower in the following order: M. brassicae (susceptible), Spodoptera littoralis (moderately susceptible), Agrotis ipsilon and Ostrinia nubilalis (slightly susceptible). Processing Vip3Ca by O. nubilalis or M. brassicae midgut juice did not significantly changed its toxicity to either insect species, indicating that the low susceptibility of O. nubilalis is not due to a problem in the midgut processing of the toxin. M. brassicae larvae fed with Vip3Ca showed binding of this toxin to the apical membrane of the midgut epithelial cells. Histopathological inspection showed sloughing of the epithelial cells with further disruption, which suggests that the mode of action of Vip3Ca is similar to that described for Vip3Aa. Biotin-labeled Vip3Ca and Vip3Aa bound specifically to M. brassicae brush border membrane vesicles and both toxins competed for binding sites. This result suggests that insects resistant to Vip3A may also be cross-resistant to Vip3C, which has implications for Insect Resistance Management (IRM).

  6. Aggregation of Bacillus thuringiensis Cry1A Toxins upon Binding to Target Insect Larval Midgut Vesicles

    PubMed Central

    Aronson, Arthur I.; Geng, Chaoxian; Wu, Lan

    1999-01-01

    During sporulation, Bacillus thuringiensis produces crystalline inclusions comprised of a mixture of δ-endotoxins. Following ingestion by insect larvae, these inclusion proteins are solubilized, and the protoxins are converted to toxins. These bind specifically to receptors on the surfaces of midgut apical cells and are then incorporated into the membrane to form ion channels. The steps required for toxin insertion into the membrane and possible oligomerization to form a channel have been examined. When bound to vesicles from the midguts of Manduca sexta larvae, the Cry1Ac toxin was largely resistant to digestion with protease K. Only about 60 amino acids were removed from the Cry1Ac amino terminus, which included primarily helix α1. Following incubation of the Cry1Ab or Cry1Ac toxins with vesicles, the preparations were solubilized by relatively mild conditions, and the toxin antigens were analyzed by immunoblotting. In both cases, most of the toxin formed a large, antigenic aggregate of ca. 200 kDa. These toxin aggregates did not include the toxin receptor aminopeptidase N, but interactions with other vesicle components were not excluded. No oligomerization occurred when inactive toxins with mutations in amphipathic helices (α5) and known to insert into the membrane were tested. Active toxins with other mutations in this helix did form oligomers. There was one exception; a very active helix α5 mutant toxin bound very well to membranes, but no oligomers were detected. Toxins with mutations in the loop connecting helices α2 and α3, which affected the irreversible binding to vesicles, also did not oligomerize. There was a greater extent of oligomerization of the Cry1Ac toxin with vesicles from the Heliothis virescens midgut than with those from the M. sexta midgut, which correlated with observed differences in toxicity. Tight binding of virtually the entire toxin molecule to the membrane and the subsequent oligomerization are both important steps in toxicity

  7. Synergism of Bacillus thuringiensis toxins by a fragment of a toxin-binding cadherin

    PubMed Central

    Chen, Jiang; Hua, Gang; Jurat-Fuentes, Juan Luis; Abdullah, Mohd Amir; Adang, Michael J.

    2007-01-01

    The insecticidal crystal proteins produced by Bacillus thuringiensis (Bt) are broadly used to control insect pests with agricultural importance. The cadherin Bt-R1 is a binding protein for Bt Cry1A toxins in midgut epithelia of tobacco hornworm (Manduca sexta). We previously identified the Bt-R1 region most proximal to the cell membrane (CR12-MPED) as the essential binding region required for Cry1Ab-mediated cytotoxicity. Here, we report that a peptide containing this region expressed in Escherichia coli functions as a synergist of Cry1A toxicity against lepidopteran larvae. Far-UV circular dichroism and 1H-NMR spectroscopy confirmed that our purified CR12-MPED peptide mainly consisted of β-strands and random coils with unfolded structure. CR12-MPED peptide bound brush border membrane vesicles with high affinity (Kd = 32 nM) and insect midgut microvilli but did not alter Cry1Ab or Cry1Ac binding localization in the midgut. By BIAcore analysis we demonstrate that Cry1Ab binds CR12-MPED at high (9 nM)- and low (1 μM)-affinity sites. CR12-MPED-mediated Cry1A toxicity enhancement was significantly reduced when the high-affinity Cry1A-binding epitope (1416GVLTLNIQ1423) within the peptide was altered. Because the mixtures of low Bt toxin dose and CR12-MPED peptide effectively control target insect pests, our discovery has important implications related to the use of this peptide to enhance insecticidal activity of Bt toxin-based biopesticides and transgenic Bt crops. PMID:17724346

  8. Pest management through Bacillus thuringiensis (Bt) in a tea-silkworm ecosystem: status and potential prospects.

    PubMed

    Dashora, Kavya; Roy, Somnath; Nagpal, Akanksha; Roy, Sudipta Mukhopadhyay; Flood, Julie; Prasad, Anjali Km; Khetarpal, Ravinder; Neave, Suzanne; Muraleedharan, N

    2017-03-01

    Bacillus thuringiensis (Bt) is a soil bacterium that forms spores containing crystals comprising one or more Cry or Cyt proteins having potential and specific insecticidal activity. Different strains of Bt produce different types of toxins, affecting a narrow taxonomic group of insects. Therefore, it is used in non-chemical pest management, including inherent pest resistance through GM crops. The specificity of action of Bt toxins reduces the concern of adverse effects on non-target species, a concern which remains with chemical insecticides as well. To make use of Bt more sustainable, new strains expressing novel toxins are actively being sought globally. Since Bt is successfully used against many pests including the lepidopteran pests in different crop groups, the insecticidal activity against Samia cynthia (Drury) (Eri silkworm) and Antheraea assamensis Helfer (Muga silkworm) becomes a concern in the state of Assam in India which is a predominantly tea- and silk-producing zone. Though Bt can be used as an effective non-chemical approach for pest management for tea pests in the same geographical region, yet, it may potentially affect the silk industry which depends on silkworm. There is a need to identify the potentially lethal impact (through evaluating their mortality potential) of local Bt strains on key silkworm species in North Eastern India. This will allow the use of existing Bt for which the silkworms have natural resistance. Through this review, the authors aim to highlight recent progress in the use of Bt and its insecticidal toxins in tea pest control and the potential sensitivity for tea- and silk-producing zone of Assam in India.

  9. Regulation by gut bacteria of immune response, Bacillus thuringiensis susceptibility and hemolin expression in Plodia interpunctella.

    PubMed

    Orozco-Flores, Alonso A; Valadez-Lira, Jose A; Oppert, Brenda; Gomez-Flores, Ricardo; Tamez-Guerra, Reyes; Rodríguez-Padilla, Cristina; Tamez-Guerra, Patricia

    2017-02-03

    Plodia interpunctella (Hübner) is an important stored grain insect pest worldwide, and the first lepidopteran with reported resistance to Bacillus thuringiensis (Bt) toxins. Since gut bacteria may affect Bt insecticidal activity, we evaluated whether P. interpunctella lacking gut bacteria had differences in immune responses and susceptibility to the Bt formulation, Bactospeine. In order to clear gut bacteria, third instar larvae were reared on artificial diet containing antibiotics, or were obtained from sterilized eggs and reared under sterile conditions, and larvae were fed diets with or without Bt. Mortality was significantly lower (p<0.05) in bacteria-free larvae treated with Bt, compared with Bt-treated larvae with unaffected gut bacteria. The number of hemocytes was lower in control and Bt-treated larvae, but was significantly higher (p<0.001) in larvae treated with antibiotics and Bt, and larvae from presterilized eggs and reared on sterile diet had the highest number of hemocytes. Phenoloxidase activity was significantly lower (p<0.05) in Bt-treated larvae from presterilized eggs reared on antibiotics for 24h or in larvae reared on antibiotic-treated diets prior to Bt introduction compared with those fed control diet. Hemolin gene expression was reduced in larvae fed Bt diets compared with control and was not detected in larvae treated with antibiotics. Larvae from sterilized eggs and fed sterile diet never reached the pupal stage. Therefore, the loss of gut bacteria in P. interpunctella larvae affected the host immune response and expression of the hemolin gene, and significantly reduced susceptibility to Bt.

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

    Urban, Joseph F; Hu, Yan; Miller, Melanie M; Scheib, Ulrike; Yiu, Ying Y; Aroian, Raffi V

    2013-01-01

    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.

  13. Immuno-physiological adaptations confer wax moth Galleria mellonella resistance to Bacillus thuringiensis

    PubMed Central

    Dubovskiy, Ivan M.; Grizanova, Ekaterina V.; Whitten, Miranda M. A.; Mukherjee, Krishnendu; Greig, Carolyn; Alikina, Tatiana; Kabilov, Marsel; Vilcinskas, Andreas; Glupov, Viktor V.; Butt, Tariq M.

    2016-01-01

    ABSTRACT Microevolutionary mechanisms of resistance to a bacterial pathogen were explored in a population of the Greater wax moth, Galleria mellonella, selected for an 8.8-fold increased resistance against the entomopathogenic bacterium Bacillus thuringiensis (Bt) compared with a non-selected (suspectible) line. Defense strategies of the resistant and susceptible insect lines were compared to uncover mechanisms underpinning resistance, and the possible cost of those survival strategies. In the uninfected state, resistant insects exhibited enhanced basal expression of genes related to regeneration and amelioration of Bt toxin activity in the midgut. In addition, these insects also exhibited elevated activity of genes linked to inflammation/stress management and immune defense in the fat body. Following oral infection with Bt, the expression of these genes was further elevated in the fat body and midgut of both lines and to a greater extent some of them in resistant line than the susceptible line. This gene expression analysis reveals a pattern of resistance mechanisms targeted to sites damaged by Bt with the insect placing greater emphasis on tissue repair as revealed by elevated expression of these genes in both the fat body and midgut epithelium. Unlike the susceptible insects, Bt infection significantly reduced the diversity and richness (abundance) of the gut microbiota in the resistant insects. These observations suggest that the resistant line not only has a more intact midgut but is secreting antimicrobial factors into the gut lumen which not only mitigate Bt activity but also affects the viability of other gut bacteria. Remarkably the resistant line employs multifactorial adaptations for resistance to Bt without any detected negative trade off since the insects exhibited higher fecundity. PMID:27029421

  14. Bacillus thuringiensis-derived Cry5B Has Potent Anthelmintic Activity against Ascaris suum

    PubMed Central

    Miller, Melanie M.; Scheib, Ulrike; Yiu, Ying Y.; Aroian, Raffi V.

    2013-01-01

    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. PMID:23818995

  15. Biphasic fermentation is an efficient strategy for the overproduction of δ-endotoxin from Bacillus thuringiensis.

    PubMed

    Jisha, Veloorvalappil Narayanan; Smitha, Robinson Babysarojam; Priji, Prakasan; Sajith, Sreedharan; Benjamin, Sailas

    2015-02-01

    This study illustrates a biphasic solid-state fermentation (SSF) strategy for the overproduction of δ-endotoxin from Bacillus thuringiensis subsp. kurstaki (Btk) and also purification of δ-endotoxin from the solid-fermented medium. The fermentation strategy had two phases (biphasic); i.e., the first short phase was semisolid state (12 h), and the remaining long phase was strict SSF. To achieve the biphasic SSF, after 12 h (150 rpm, 37 °C) fermentation of the medium [Luria-Bertani (LB) supplemented with 30 % (w/v) raw soybean flour (phase I)], the supernatant in it was completely centrifuged out (1,000 × g, 10 min) aseptically for harvesting the extracellular enzymes as by-product. The resultant wet solid matter without free-flowing liquid but with embedded Btk was incubated 60 h more (phase II) for enhancing δ-endotoxin production at static condition (37 °C). Coupled with this, δ-endotoxin was purified by the modified phase separation method, and its purity was physically confirmed by both staining and microscopic techniques. The maximum δ-endotoxin yield from solid medium (48 h) was 15.8 mg/mL (recovery was 55-59 %) LB-equivalent, while that of LB control (recovery was 95 %) was only 0.43 mg/mL (72 h), i.e., thus, in comparison, 36.74-fold more yield in solid medium obtained by 24 h less gestation period. The purified crystal proteins showed apparent molecular weights (MWs) of 45, 35, and 6 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Briefly, this unique study physically demonstrates how Btk δ-endotoxin is purified (95-99 % purity) from solid-fermented matter for the first time, coupled with its overproduction at the expense of only 21.5 % higher production cost.

  16. pH-Controlled Bacillus thuringiensis Cry1Ac Protoxin Loading and Release from Polyelectrolyte Microcapsules

    PubMed Central

    Yang, Wenhui; He, Kanglai; Zhang, Jie; Guo, Shuyuan

    2012-01-01

    Crystal proteins synthesized by Bacillus thuringiensis (Bt) have been used as biopesticides because of their toxicity to the insect larval hosts. To protect the proteins from environmental stress to extend their activity, we have developed a new microcapsule formulation. Poly (acrylic acid) (PAH) and poly (styrene sulfonate) (PSS) were fabricated through layer-by-layer self-assembly based on a CaCO3 core. Cry1Ac protoxins were loaded into microcapsules through layer-by-layer self-assembly at low pH, and the encapsulated product was stored in water at 4°C. Scanning electron microscopy (SEM) was used to observe the morphology of the capsules. To confirm the successful encapsulation, the loading results were observed with a confocal laser scattering microscope (CLSM), using fluorescein-labeled Cry1Ac protoxin (FITC-Cry1Ac). The protoxins were released from the capsule under the alkaline condition corresponding to the midgut of certain insects, a condition which seldom exists elsewhere in the environment. The following bioassay experiment demonstrated that the microcapsules with Cry1Ac protoxins displayed approximately equivalent insecticidal activity to the Asian corn borer compared with free Cry1Ac protoxins, and empty capsules proved to have no effect on insects. Further result also indicated that the formulation could keep stable under the condition of heat and desiccation. These results suggest that this formulation provides a promising methodology that protects protoxins from the environment and releases them specifically in the target insects’ midgut, which has shown potential as biopesticide in the field. PMID:23024810

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

    PubMed Central

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

    2016-01-01

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

  18. Bacillus thuringiensis Cyt2Aa2 toxin disrupts cell membranes by forming large protein aggregates

    PubMed Central

    Tharad, Sudarat; Toca-Herrera, José L.; Promdonkoy, Boonhiang; Krittanai, Chartchai

    2016-01-01

    Bacillus thuringiensis (Bt) Cyt2Aa2 showed toxicity against Dipteran insect larvae and in vitro lysis activity on several cells. It has potential applications in the biological control of insect larvae. Although pore-forming and/or detergent-like mechanisms were proposed, the mechanism underlying cytolytic activity remains unclear. Analysis of the haemolytic activity of Cyt2Aa2 with osmotic stabilizers revealed partial toxin inhibition, suggesting a distinctive mechanism from the putative pore formation model. Membrane permeability was studied using fluorescent dye entrapped in large unilamellar vesicles (LUVs) at various protein/lipid molar ratios. Binding of Cyt2Aa2 monomer to the lipid membrane did not disturb membrane integrity until the critical protein/lipid molar ratio was reached, when Cyt2Aa2 complexes and cytolytic activity were detected. The complexes are large aggregates that appeared as a ladder when separated by agarose gel electrophoresis. Interaction of Cyt2Aa2 with Aedes albopictus cells was investigated by confocal microscopy and total internal reflection fluorescent microscopy (TIRF). The results showed that Cyt2Aa2 binds on the cell membrane at an early stage without cell membrane disruption. Protein aggregation on the cell membrane was detected later which coincided with cell swelling. Cyt2Aa2 aggregations on supported lipid bilayers (SLBs) were visualized by AFM. The AFM topographic images revealed Cyt2Aa2 aggregates on the lipid bilayer at low protein concentration and subsequently disrupts the lipid bilayer by forming a lesion as the protein concentration increased. These results supported the mechanism whereby Cyt2Aa2 binds and aggregates on the lipid membrane leading to the formation of non-specific hole and disruption of the cell membrane. PMID:27612497

  19. Proteolytic processing of Bacillus thuringiensis Vip3A proteins by two Spodoptera species.

    PubMed

    Caccia, Silvia; Chakroun, Maissa; Vinokurov, Konstantin; Ferré, Juan

    2014-08-01

    Vip3 proteins have been described to be secreted by Bacillus thuringiensis during the vegetative growth phase and to display a broad insecticidal spectrum against lepidopteran larvae. Vip3Aa protoxin has been reported to be significantly more toxic to Spodoptera frugiperda than to Spodoptera exigua and differences in the midgut processing have been proposed to be responsible. In contrast, we have found that Vip3Ae is essentially equally toxic against these two species. Proteolysis experiments were performed to study the stability of Vip3A proteins to peptidase digestion and to see whether the differences found could explain differences in toxicity against these two Spodoptera species. It was found that activation of the protoxin form and degradation of the 62kDa band took place at lower concentrations of trypsin when using Vip3Aa than when using Vip3Ae. The opposite effect was observed for chymotrypsin. Vip3Aa and Vip3Ae protoxins were effectively processed by midgut content extracts from the two Spodoptera species and the proteolytic activation did not produce a peptidase resistant core under these in vitro conditions. Digestion experiments performed with S. frugiperda chromatography-purified digestive serine peptidases showed that the degradation of the Vip3A toxins active core is mainly due to the action of cationic chymotrypsin-like peptidase. Although the digestion patterns of Vip3A proteins do not always correlate with toxicity, the peptidase stability of the 62kDa core is in agreement with intraspecific differences of toxicity of the Vip3Aa protein.

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

    PubMed

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

    2015-12-01

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

  1. Effects of proportion and configuration of Bacillus thuringiensis cotton on pest abundance, damage, and yield.

    PubMed

    Chilcutt, Charles F

    2007-08-01

    Bacillus thuringiensis (Bt) transgenic cotton, Gossypium hirsutum L., kills several economically important pests, reducing injury and increasing yields. Refuges of non-Bt cotton are currently planted with Bt cotton in different designs to slow pest resistance evolution. To compare the effects of differences in Bt/non-Bt plant heterogeneity found in different refuge designs on square (flower bud) damage, abscissions, sap-feeding herbivore densities, and yield in cotton, four types of 24-row cotton plots were planted in 2001 and 2002: 1) seed mixtures of Bt and non-Bt varieties, 2) 12-row strips of Bt and non-Bt, 3) solid Bt, and 4) solid non-Bt. For both years cotton bollworm, Helicoverpa zea (Boddie), damage was less in solid Bt plots than strips and mixtures and all were less than solid non-Bt plots. Cotton fleahopper, Pseudatomoscelis seriatus (Reuter), damage was affected by refuge, but only in 2002 when damage was greater in solid Bt plots than all other plots and greater in strips than solid non-Bt plots. Abscissions were least in solid non-Bt plots, and less in mixtures and strips than solid Bt plots. In 2001, western flower thrips, Frankliniella occidentalis (Pergande), density was greatest in mixtures, whereas sweetpotato whitefly, Bemisia tabaci (Gennadius), was greatest in solid Bt plots, and greater in mixtures than solid non-Bt plots. Yield also was affected by refuge, it was greater for solid Bt plots than for solid non-Bt plots and mixtures in 2001, but the reverse was true in 2002.

  2. Cell Differentiation in a Bacillus thuringiensis Population during Planktonic Growth, Biofilm Formation, and Host Infection

    PubMed Central

    Verplaetse, Emilie; Slamti, Leyla; Gohar, Michel

    2015-01-01

    ABSTRACT Bacillus thuringiensis (Bt) is armed to complete a full cycle in its insect host. During infection, virulence factors are expressed under the control of the quorum sensor PlcR to kill the host. After the host’s death, the quorum sensor NprR controls a necrotrophic lifestyle, allowing the vegetative cells to use the insect cadaver as a bioincubator and to survive. Only a part of the Bt population sporulates in the insect cadaver, and the precise composition of the whole population and its evolution over time are unknown. Using fluorescent reporters to record gene expression at the single-cell level, we have determined the differentiation course of a Bt population and explored the lineage existing among virulent, necrotrophic, and sporulating cells. The dynamics of cell differentiation were monitored during growth in homogenized medium, biofilm formation, and colonization of insect larvae. We demonstrated that in the insect host and in planktonic culture in rich medium, the virulence, necrotrophism, and sporulation regulators are successively activated in the same cell. In contrast, in biofilms, activation of PlcR is dispensable for NprR activation and we observed a greater heterogeneity than under the other two growth conditions. We also showed that sporulating cells arise almost exclusively from necrotrophic cells. In biofilm and in the insect cadaver, we identified an as-yet-uncharacterized category of cells that do not express any of the reporters used. Overall, we showed that PlcR, NprR, and Spo0A act as interconnected integrators to allow finely tuned adaptation of the pathogen to its environment. PMID:25922389

  3. Biosterilant effects of Bacillus thuringiensis kurstaki HD-73 extract on male Wistar albino rats.

    PubMed

    Roy, Vikas Kumar; Peki, Vanlal; Devi, Maibam Sunita; Sanjeev, Sanasam; Khusboo, Maurya; Zothansanga, R; Ibrahim, Kalibulla Syed; Kumar, Nachimuthu Senthil; Gurusubramanian, Guruswami

    2017-01-15

    Chemosterilants have long been used in sterilization programs for managing pet, stray, and wild animals but adverse effects such as trauma, incomplete responses to treatment, and complete abolition of gonadal sources of testosterone often occurs. This study describes the biosterilant effects of administering three doses of Bacillus thuringiensis kurstaki HD-73 (Bt 5, 50, and 250 mg/kg; 20 rats/dose) extract in adult male Wistar albino rats on testicular parameters, function, histology, and a number of biochemical markers of overall health, free radical production, and cell proliferation. Intratesticular administration of Bt extract to rats induces testicular oxidative stress and damages and consequently, perturb spermatogenesis, degeneration of testis, reduction in testes size, and depletion of testosterone and antioxidant enzyme concentrations in a dose-dependent manner because of free radical-mediated lipid peroxidation. No morbidity or mortality adverse effects were observed in both the saline control and Bt extract-treated rats. Significant variation was noted in clinical manifestations, weight and volume of testes, and hormonal and biochemical profiles between Bt doses in comparison with the saline control. Aspermia/azoospermia (100%) resulted in Bt-treated rats without any adverse effects. Histopathological analysis showed degeneration, necrosis, vacuolation, fewer germ cells, formation of multinucleated giant cells, and a lack of elongated spermatids in atrophic seminiferous tubules in Bt extract-treated groups in the presence of low concentrations of testosterone, antioxidant enzymes, and suppression of germ cell proliferation. Dose-dependent effects were evident in most parameters that were measured. The vast array of tests that were undertaken also provides some important indicators of the physiological effects associated with the treatments that were applied. Intratesticular injection of Bt extract impairs spermatogenesis and induces permanent sterility

  4. A Single Point Mutation Resulting in Cadherin Mislocalization Underpins Resistance against Bacillus thuringiensis Toxin in Cotton Bollworm.

    PubMed

    Xiao, Yutao; Dai, Qing; Hu, Ruqin; Pacheco, Sabino; Yang, Yongbo; Liang, Gemei; Soberón, Mario; Bravo, Alejandra; Liu, Kaiyu; Wu, Kongming

    2017-02-17

    Transgenic plants that produce Bacillus thuringiensis (Bt) crystalline (Cry) toxins are cultivated worldwide to control insect pests. Resistance to B. thuringiensis toxins threatens this technology, and although different resistance mechanisms have been identified, some have not been completely elucidated. To gain new insights into these mechanisms, we performed multiple back-crossing from a 3000-fold Cry1Ac-resistant BtR strain from cotton bollworm (Helicoverpa armigera), isolating a 516-fold Cry1Ac-resistant strain (96CAD). Cry1Ac resistance in 96CAD was tightly linked to a mutant cadherin allele (mHaCad) that contained 35 amino acid substitutions compared with HaCad from a susceptible strain (96S). We observed significantly reduced levels of the mHaCad protein on the surface of the midgut epithelium in 96CAD as compared with 96S. Expression of both cadherin alleles from 96CAD and 96S in insect cells and immunofluorescence localization in insect midgut tissue sections showed that the HaCAD protein from 96S localizes on the cell membrane, whereas the mutant 96CAD-mHaCad was retained in the endoplasmic reticulum (ER). Mapping of the mutations identified a D172G substitution mainly responsible for cadherin mislocalization. Our finding of a mutation affecting membrane receptor trafficking represents an unusual and previously unrecognized B. thuringiensis resistance mechanism.

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

    PubMed

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

    2015-03-01

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

  6. Curing of plasmid pBMB28 from Bacillus thuringiensis YBT-020 using an unstable replication region.

    PubMed

    Wang, Pengxia; Zhu, Qian; Shang, Hui; Zhu, Yiguang; Sun, Ming

    2016-02-01

    Bacillus thuringiensis serovar finitimus strain YBT-020 is the well-studied spore-crystal association (SCA) phenotypic strain, whose parasporal crystals adhere to spore after lysis of the mother cell. Its endogenous plasmids pBMB26 and pBMB28 were proved essential for this SCA phenotype. In our previous study, using conventional methods, pBMB26 cured derivative and both pBMB26 and pBMB28 cured derivative of YBT-020 were obtained. However, YBT-020 solely cured of pBMB28 could not be obtained. In this study, an unstable replication region of pBMB28 was identified and was used to construct an incompatible plasmid pRep28B. This incompatible plasmid was successfully used to cure plasmid pBMB28 and was easily eliminated through segregational instability under the optimum growth temperature of YBT-020. Therefore, an endogenous plasmid was cured from the B. thuringiensis strain utilizing plasmid incompatibility. Moreover, using an unstable replication region instead of a temperature sensitive (Ts) replication region is better to cure the incompatible plasmid because it can avoid culturing at higher temperature. This method provides an efficient method for plasmid curing in B. thuringiensis and other bacteria.

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

    PubMed Central

    Muthukumar, G; Nickerson, K W

    1987-01-01

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

  8. Screening of Bacillus thuringiensis serotypes by polymerase chain reaction (PCR) for insecticidal crystal genes toxic against coffee berry borer.

    PubMed

    Naidu, M M; Rang, C; Frutos, R; Sreenivasan, C S; Naidu, R

    2001-02-01

    Using PCR,257 isolates of Bacillus thuringiensis(Bt) were screened for cry-type genes. Of 257 isolates/strains, 60 isolates were identified as cry7/8, 10 isolates as cry3 and 36 isolates as cry 1I. One specific strain of B. thuringiensis (sumiyoshiensis; T03B 001) was investigated for the presence of cry7 and cry8 genes. Genes Cry7 and cry8 were first detected in this strain using family primers prior to analysis by exclusion polymerase chain reaction (E-PCR) using specific type primers. E-PCR conducted with the above said primers led to the identification by agarose gel electrophoresis of a remaining 1.5 Kb family band indicating a potentially novel gene. This PCR product, (1.5 Kb), was purified from the gel and cloned in pGEM-T Easy vector. Twenty recombinant colonies bearing 1.5 Kb insert were identified and three randomly selected representatives of the group, clones 7, 8 and 10, were sequenced and compared to all cry7 and cry8 sequences available from Gene Bank. Alignments with available DNA and protein sequences showed that all these clones contained a gene related to cry8Aa1. Analysis using protein sequence alignment showed that the sequence from clone 7 differed from the closest relative, known under the new nomenclature as cry 8Aa1, by 44%. The crystal proteins from B. thuringiensis sumiyoshiensis (T03B 001) was toxic to coffee berry borer larvae.

  9. Multiple-locus sequence typing analysis of Bacillus thuringiensis recovered from the phylloplane of clover (Trifolium hybridum) in vegetative form.

    PubMed

    Bizzarri, M F; Prabhakar, A; Bishop, A H

    2008-05-01

    Abstract The chromosomal genotype, as judged by multi locus sequence typing, and the episomal genotype, as judged by plasmid profile and cry gene content, were analyzed for a collection of strains of Bacillus thuringiensis. These had been recovered in vegetative form over a period of several months from the leaves of a small plot of clover (Trifolium hybridum). A clonal population structure was indicated, although greater variation in sequence types (STs) was discovered than in previous collections of B. cereus/B. thuringiensis. Isolates taken at the same time had quite different genotypes, whereas those of identical genotypes were recovered at different times. The profiles of plasmid content and cry genes generally bore no relation to each other nor to the STs. Evidently, although relatively little recombination was occurring in the seven chromosomal genes analyzed, a great deal of conjugal transfer, and perhaps recombination, was occurring involving plasmids. A clinical diarrheal isolate of B. cereus and the commercial biopesticide strain HD-1 of B. thuringiensis, both included as out-groups, were found to have very similar STs. This further emphasizes the role of episomal elements in the characteristics and differentiation of these two species.

  10. Bacillus thuringiensis serovar israelensis is highly toxic to the coffee berry borer, Hypothenemus hampei Ferr. (Coleoptera: Scolytidae).

    PubMed

    Méndez-López, Ismael; Basurto-Ríos, Regina; Ibarra, Jorge E

    2003-09-12

    A native collection of Bacillus thuringiensis strains was screened, once a reliable bioassay technique to assess the toxicity against the coffee berry borer (CBB) first-instar larvae was developed. A first round of bioassays with 170 strains indicated that the great majority of them showed no or very little insecticidal activity and that very few showed significant levels of toxicity. Interestingly, only those strains that had previously been associated with mosquitocidal activity were also toxic to CBB. Qualitative bioassays (using one high dose) were carried out only with those native mosquitocidal strains, corroborating their significant toxicity towards the CBB first-instar larvae. Most of these strains belong to serovar israelensis. In a second approach, strains from the Institut Pasteur type collection, whose mosquitocidal activity had been previously demonstrated, were also subjected to bioassays. Only those strains that showed a comparable protein content in their parasporal crystals to the israelensis type strain also showed high levels of toxicity towards CBB. Finally, an accurate LC(50) was estimated, using purified parasporal crystals from B. thuringiensis serovar israelensis type strain, at 219.5 ng cm(-2) of diet. All the statistical requirements for a reliable estimator were fulfilled. This is the first report of B. thuringiensis serovar israelensis being active against a coleopteran species.

  11. Low persistence of Bacillus thuringiensis serovar israelensis spores in four mosquito biotopes of a salt marsh in southern France.

    PubMed

    Hajaij, Myriam; Carron, Alexandre; Deleuze, Julien; Gaven, Bruno; Setier-Rio, Marie-Laure; Vigo, Gerard; Thiéry, Isabelle; Nielsen-LeRoux, Christina; Lagneau, Christophe

    2005-11-01

    We studied the persistence of Bacillus thuringiensis serovar israelensis (Bti) in a typical breeding site of the mosquito Ochlerotatus caspius in a particularly sensitive salt marsh ecosystem following two Bti-based larvicidal applications (Vectobac 12AS, 1.95 L/ha). The treated area was composed of four larval biotopes that differed in terms of the most representative plant species (Sarcocornia fruticosa, Bolboschoenus maritimus, Phragmites australis, and Juncus maritimus) and the physical and chemical characteristics of the soil. We sampled water, soil, and plants at various times before and after the applications (from spring to autumn, 2001) and quantified the spores of B. thuringiensis (Bt) and Bacillus species. The B. cereus group accounted for between 0% and 20% of all Bacillus spp. before application depending on the larval biotope. No Bti were found before application. The variation in the quantity of bacilli during the mosquito breeding season depended more on the larval biotope than on the season or the larvicidal application. More bacilli were found in soil (10(4)-10(6) spores/g) than on plant samples (10(2)-10(4) spores/g). The abundance in water (10(5) to 10(7) spores/L) appeared to be correlated to the water level of the breeding site. The number of Bti spores increased just after application, after declining; no spores were detected in soil or water 3 months after application. However, low numbers of Bti spores were present on foliage from three of the four studied plant strata. In conclusion, the larvicidal application has very little impact on Bacillus spp. flora after one breeding season (two applications).

  12. Complete sequence of three plasmids from Bacillus thuringiensis INTA-FR7-4 environmental isolate and comparison with related plasmids from the Bacillus cereus group.

    PubMed

    Amadio, Ariel F; Benintende, Graciela B; Zandomeni, Rubén O

    2009-11-01

    Bacillus thuringiensis is an insect pathogen used worldwide as a bioinsecticide. It belongs to the Bacillus cereus sensu lato group as well as Bacillus anthracis and B. cereus. Plasmids from this group of organisms have been implicated in pathogenicity as they carry the genes responsible for different types of diseases that affect mammals and insects. Some plasmids, like pAW63 and pBT9727, encode a functional conjugation machinery allowing them to be transferred to a recipient cell. They also share extensive homology with the non-functional conjugation apparatus of pXO2 from B. anthracis. In this study we report the complete sequence of three plasmids from an environmental B. thuringiensis isolate from Argentina, obtained by a shotgun sequencing method. We obtained the complete nucleotide sequence of plasmids pFR12 (12,095bp), pFR12.5 (12,459bp) and pFR55 (55,712bp) from B. thuringiensis INTA-FR7-4. pFR12 and pFR12.5 were classified as cryptic as they do not code for any obvious functions besides replication and mobilization. Both small plasmids were classified as RCR plasmids due to similarities with the replicases they encode. Plasmid pFR55 showed a structural organization similar to that observed for plasmids pAW63, pBT9727 and pXO2. pFR55 also shares a tra region with these plasmids, containing genes related to T4SS and conjugation. A comparison between pFR55 and conjugative plasmids led to the postulation that pFR55 is a conjugative plasmid. Genes related to replication functions in pFR55 are different to those described for plasmids with known complete sequences. pFR55 is the first completely sequenced plasmid with a replication machinery related to that of ori44. The analysis of the complete sequence of plasmids from an environmental isolate of B. thuringiensis permitted the identification of a near complete conjugation apparatus in pFR55, resembling those of plasmids pAW63, pBT9727 and pXO2. The availability of this sequence is a step forward in the study

  13. The master transcription factor Spo0A is required for poly(3-hydroxybutyrate) (PHB) accumulation and expression of genes involved in PHB biosynthesis in Bacillus thuringiensis.

    PubMed

    Chen, Hui-Ju; Tsai, Teng-Kuan; Pan, Shih-Chuan; Lin, Jer-Sheng; Tseng, Chi-Ling; Shaw, Gwo-Chyuan

    2010-03-01

    Bacillus thuringiensis is a gram-positive spore-forming bacterium that can accumulate poly(3-hydroxybutyrate) (PHB) as a carbon and energy storage substance in response to nutritional stress. The regulatory mechanism for PHB biosynthesis in B. thuringiensis and diverse Bacillus species is still poorly understood. We now report that disruption of the sigH gene or the gene encoding the master sporulation transcription factor Spo0A severely impaired PHB accumulation in B. thuringiensis. Complementation of the spo0A mutation with the spo0A gene restored PHB accumulation. We have found that the requirement of Spo0A for PHB accumulation is independent of the transition state regulator AbrB and of loss of sporulation ability. We also show that Spo0A is required for the expression of three genes involved in PHB biosynthesis. These findings have uncovered a new role of Spo0A in the regulation of stationary-phase-associated cellular events.

  14. Recovery of commercially produced Bacillus thuringiensis var. israelensis and Bacillus sphaericus from tires and prevalence of bacilli in artificial and natural containers.

    PubMed

    Siegel, J P; Smith, A R; Novak, R J

    2001-03-01

    We conducted surveys to identify the species of spore-forming bacteria present in natural and artificial containers. Most of our samples came from Illinois. Identification was based on the cellular fatty acid composition of the bacterial cell wall. In addition, we utilized a custom database for commercially produced strains of Bacillus thuringiensis var. israelensis (Bti) and B. sphaericus, to differentiate between larvicidal isolates with commercial or native origin. Native Bti was present at low levels in almost all habitats but was not recovered from bromeliads and metal containers. In temporary woodland pools, 27.9% of the colonies recovered were native Bti. We did not recover larvicidal B. sphaericus in untreated habitats. VectoBac and VectoLex were applied to tires containing water and the tires were sampled 3 months and 9 months after treatment. Isolates of Bti and B. sphaericus with commercial origin were recovered as long as 9 months after application. We noticed numerous cadavers of Aedes triseriatus in several tires 9 months after treatment with VectoBac. We could not determine if this mortality resulted from recycling of Bti in these tires or whether insecticidal crystal proteins from the original treatment were resuspended. Bacillus thuringiensis var. israelensis isolates with commercial ancestry were recovered from untreated tires 9 months after application. Isolates of larvicidal B. sphaericus that differed from the bacteria in VectoLex were also recovered from untreated tires.

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

    PubMed

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

    2016-01-01

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

  16. Effects of physical culture parameters on bacteriocin production by Mexican strains of Bacillus thuringiensis after cellular induction.

    PubMed

    Martínez-Cardeñas, Janeth Adriana; de la Fuente-Salcido, Norma M; Salcedo-Hernández, Rubén; Bideshi, Dennis K; Barboza-Corona, J Eleazar

    2012-01-01

    We have shown previously that in the presence of inducer Bacillus cereus 183, significant increases in bacteriocin production and bactericidal activity of B. thuringiensis occur when the latter is cultivated at pH 7.2, 28°C, and 180 rpm. Here we show that this activity can be further improved when B. thuringiensis is induced with B. cereus 183 and then cultivated with modification of pH, temperature, and agitation. Five native strains of B. thuringiensis, LBIT 269, LBIT 287, LBIT 404, LBIT 420, and LBIT 524 which synthesize, respectively, morricin 269, kurstacin 287, kenyacin 404, entomocin 420, and tolworthcin 524, were cultivated in four different fermentation media. Of these, fermentation in tryptic soy broth (TSB) yielded the highest level of bacteriocin activity (~100-133 FU). Bacteria grown in TSB were induced with B. cereus 183 and cultivated at different pH (6.0, 7.2, 8.0), temperature (26, 28, 30°C), and agitation (150, 180, 210 rpm). Full factorial design was performed and results were analyzed with analysis of variance (ANOVA) and Tukey multiple comparison tests at significant level of α ≤ 0.05 to study the influence of the three variables on bacterial growth and bacteriocin production. Our data show that the highest bacteriocin activity was found with LBIT 269 and LBIT 404 with an increase of ~95-100% compared with induced B. thuringiensis strains cultivated under fixed conditions (pH 7.2, 28°C, 180 rpm), for which the data were set at 0%. The optimal conditions for morricin 269 and kenyacin 404 production were, respectively, pH 8, 30°C, 210 rpm and pH 7.2, 26°C, 210 rpm.

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

    PubMed Central

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

    2016-01-01

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

  18. Bacillus thuringiensis HD-73 Spores Have Surface-Localized Cry1Ac Toxin: Physiological and Pathogenic Consequences

    PubMed Central

    Du, C.; Nickerson, K. W.

    1996-01-01

    Spores from Cry(sup+) strains of Bacillus thuringiensis bound fluorescein isothiocyanate-labeled antibodies specific for the 65-kDa activated Cry 1Ac toxin, whereas spores from Bacillus cereus and Cry(sup-) strains of B. thuringiensis did not. The Cry(sup+) spores could be activated for germination by alkaline conditions (pH 10.3), whereas Cry(sup-) spores could not. Once the surrounding exosporia had been removed or permeabilized, Cry(sup+) spores were able to bind the toxin receptor(s) from insect gut brush border membrane vesicle preparations, and their germination rates were increased ca. threefold in the presence of brush border membrane vesicles. A model is presented whereby in the soil the Cry toxins on the spore surface are protected by the exosporium while in the gut they are exposed and available for binding to the insect receptors. This model explains why the disulfide-rich C terminus of the cry genes is so highly conserved even though it is removed during the processing of the protoxin to the activated toxin. It also highlights the trade-off resulting from having Cry toxins located on the spore surface, i.e., decreased spore resistance versus enhanced insect pathogenesis. PMID:16535421

  19. Use of Redundant Exclusion PCR To Identify a Novel Bacillus thuringiensis Cry8 Toxin Gene from Pooled Genomic DNA

    PubMed Central

    Zhang, Fengjiao; Shu, Changlong; Crickmore, Neil; Li, Yanqiu; Song, Fuping; Liu, Chunqin

    2016-01-01

    ABSTRACT With the aim of optimizing the cloning of novel genes from a genomic pool containing many previously identified homologous genes, we designed a redundant exclusion PCR (RE-PCR) technique. In RE-PCR, a pair of generic amplification primers are combined with additional primers that are designed to specifically bind to redundant, unwanted genes that are a subset of those copied by the amplification primers. During RE-PCR, the specific primer blocks amplification of the full-length redundant gene. Using this method, we managed to clone a number of cry8 or cry9 toxin genes from a pool of Bacillus thuringiensis genomic DNA while excluding amplicons for cry9Da, cry9Ea, and cry9Eb. The method proved to be very efficient at increasing the number of rare genes in the resulting library. One such rare (and novel) cry8-like gene was expressed, and the encoded toxin was shown to be toxic to Anomala corpulenta. IMPORTANCE Protein toxins from the bacterium Bacillus thuringiensis are being increasingly used as biopesticides against a wide range of insect pests, yet the search for new or improved toxins is becoming more difficult, as traditional methods for gene discovery routinely isolate previously identified clones. This paper describes an approach that we have developed to increase the success rate for novel toxin gene identification through reducing or eliminating the cloning of previously characterized genes. PMID:27084017

  20. Identification of a Bacillus thuringiensis Surface-Layer-Protein with Cytotoxic Activity against MDA-MB-231 Breast Cancer Cells.

    PubMed

    Rubio, Viviana P; Bravo, Alejandra; Olmos, Jorge

    2016-10-06

    In this work we isolated a Surface-Layer-Protein (SLP) from a Bacillus thuringiensis (Bt) strain to evaluate it cytotoxic effects against MDA-MB-231 human breast cancer cells. AP11 was selected from a group of Bt strains using SLP oligonucleotides developed from Bacillus conserved regions. AP11 strain was grown in Luria Bertani (LB) medium until late exponential phase; an 86 kDa protein was extracted using 5 M LiCl and identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). It corresponded to a multispecies S-layer protein highly similar to previously described SLP in B. thuringiensis. MDA-MB-231 breast cancer cells LC₅₀ was obtained using 0.25 µg/ml of the isolated SLP. HaCat non-cancerous cells presented 90% survival using the same protein concentration. Our data suggest that SLP cytotoxicity against MDA-MB-231 could be induced by an interaction with CDH11 cell membrane receptor.