scholarly journals Comparative analysis of the individual protoxin components in P1 crystals of Bacillus thuringiensis subsp. kurstaki isolates NRD-12 and HD-1

1990 ◽  
Vol 269 (2) ◽  
pp. 507-512 ◽  
Author(s):  
L Masson ◽  
G Préfontaine ◽  
L Péloquin ◽  
P C K Lau ◽  
R Brousseau
Keyword(s):  
Bacillus Thuringiensis ◽  
Choristoneura Fumiferana ◽  
Insect Cell Line ◽  
Gene Products ◽  
Bacillus Thuringiensis Subsp ◽  
Independent Verification ◽  
Lepidopteran Larvae ◽  
Bacterium Bacillus ◽  
The Individual

Two commercially important strains (NRD-12 and HD-1) of the entomopathogenic bacterium Bacillus thuringiensis subsp. kurstaki each contain three genes of partially identical sequence coding for three classes of 130-135 kDa protoxins (termed the 4.5, 5.3 and 6.6 protoxins) that display toxicity towards various lepidopteran larvae. These gene products combine to form the intracellular bipyramidal P1 crystal. Each of the genes from both strains was cloned and expressed in Escherichia coli. Analysis of the cloned genes at the restriction-endonuclease level revealed no detectable differences among genes within a particular gene class. The composition of the P1 crystal from both strains was quantitatively analysed by CNBr cleavage of the purified P1 crystal, with the purified recombinant gene products as reference proteins. Independent verification of the presence of high 6.6-protoxin gene product in the P1 crystal was provided by a rapid in vitro lawn cell toxicity assay directed against a Choristoneura fumiferana (CF-1) insect cell line. The results indicate that, although all three gene products are represented within the P1 crystal of either NRD-12 or HD-1, only the contents of the 4.5 and 5.3 protoxins vary between the two crystals, whereas the 6.6 protoxin contents are similar and represent approximately one-third of the P1 crystal in either strain.

scholarly journals Toxicity of Bacillus thuringiensis-Derived Pesticidal Proteins Cry1Ab and Cry1Ba against Asian Citrus Psyllid, Diaphorina citri (Hemiptera)

Toxins ◽  
2019 ◽  
Vol 11 (3) ◽  
pp. 173 ◽  
Author(s):  
Maria Fernandez-Luna ◽  
Pavan Kumar ◽  
David Hall ◽  
Ashaki Mitchell ◽  
Michael Blackburn ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Asian Citrus Psyllid ◽  
Diaphorina Citri ◽  
Cry Toxins ◽  
Diverse Range ◽  
Additional Tool ◽  
Incurable Disease ◽  
Important Pest ◽  
Bacterium Bacillus ◽  
The Individual

The Asian citrus psyllid (ACP), Diaphorina citri Kuwayama (Hemiptera), is an important pest of citriculture. The ACP vectors a bacterium that causes huanglongbing (HLB), a devastating and incurable disease of citrus. The bacterium Bacillus thuringiensis (Bt) produces multiple toxins with activity against a diverse range of insects. In efforts to provide additional control methods for the ACP vector of HLB, we identified pesticidal proteins derived from Bt for toxicity against ACP. The trypsin proteolytic profiles of strain-derived toxins were characterized. Strain IBL-00200, one of six strains with toxins shown to have basal activity against ACP was selected for liquid chromatography-mass spectrometry (LC-MS/MS) identification of the individual Cry toxins expressed. Toxicity assays with individual toxins derived from IBL-00200 were then performed. The activated form of the Cry toxins Cry1Ab and Cry1Ba were toxic to ACP with LC50 values of approximately 120 µg/mL. Disruption of the midgut epithelium was associated with the toxicity of both the IBL-00200-derived toxin mixture, and with Cry1Ba. With further optimization of the efficacy of Cry1Ab and Cry1Ba, these toxins may have practical utility against ACP. Bt toxins with activity against ACP may provide an additional tool for management of ACP and the associated HLB disease, thereby providing a more sustainable and environmentally benign approach than repeated application of broad-spectrum insecticides.

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

2012 ◽  
Vol 443 (3) ◽  
pp. 711-717 ◽  
Author(s):  
Claudia Rodríguez-Almazán ◽  
Esmeralda Z. Reyes ◽  
Fernando Zúñiga-Navarrete ◽  
Carlos Muñoz-Garay ◽  
Isabel Gómez ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Aedes Aegypti ◽  
Lipid Bilayers ◽  
Insect Pests ◽  
Cry Toxins ◽  
Bacillus Thuringiensis Subsp ◽  
Limiting Step ◽  
Cry Toxin ◽  
Receptor Fragment

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

Bacillus thuringiensis var israelensis crystal delta-endotoxin: effects on insect and mammalian cells in vitro and in vivo

1983 ◽  
Vol 60 (1) ◽  
pp. 181-197 ◽  
Author(s):  
W.E. Thomas ◽  
D.J. Ellar
Keyword(s):  
Body Weight ◽  
Bacillus Thuringiensis ◽  
Mammalian Cells ◽  
Choristoneura Fumiferana ◽  
Similar Response ◽  
Parasporal Crystal ◽  
Discontinuous Sucrose Gradient ◽  
Delta Endotoxin

Bacillus thuringiensis var israelensis parasporal crystal delta-endotoxin was purified by ultracentrifugation on a discontinuous sucrose gradient. Native delta-endotoxin crystals showed no detectable toxicity in the vitro and in vivo systems that are described. By contrast alkali-solubilized crystal delta-endotoxin caused rapid cytological and cytopathological changes in Aedes albopictus, Choristoneura fumiferana 63 CF1, Spodoptera frugiperda and Trichoplusia ni cell lines as observed by phase-contrast microscopy and vital staining. Mouse fibroblasts, primary pig lymphocytes and three mouse epithelial carcinoma cell types showed a similar response to the alkali-soluble crystal delta-endotoxin. In addition the soluble crystal delta-endotoxin protein caused haemolysis of rat, mouse, sheep, horse and human erythrocytes. Intravenous administration of the alkali-soluble crystal delta-endotoxin to Balb. c mice at a dose rate of 15–30 micrograms of protein per gram body weight resulted in rapid paralysis followed by death within 12h. Subcutaneous inoculation of 15–30 micrograms of protein per gram body weight resulted in death of suckling mice in 2–3 h. The alkali-solubilized crystal delta-endotoxin was not toxic however, when administered per os. A comparison is made with a similar alkali-soluble fraction from the parasporal crystal delta-endotoxin of B. thuringiensis var kurstaki. With the exception of the Lepidopteran cell line, Choristoneura fumiferana 63 CF1, this soluble crystal delta-endotoxin protein showed no in vitro or in vivo toxicity, and no haemolytic activity.

Characterization and partial purification of a plasma membrane receptor for Bacillus thuringiensis var. kurstaki lepidopteran-specific delta-endotoxin

1986 ◽  
Vol 83 (1) ◽  
pp. 89-101
Author(s):  
B.H. Knowles ◽  
D.J. Ellar
Keyword(s):  
Plasma Membrane ◽  
Bacillus Thuringiensis ◽  
Choristoneura Fumiferana ◽  
Galactose Oxidase ◽  
Partial Purification ◽  
Wide Range ◽  
Delta Endotoxin ◽  
Plasma Membrane Receptor ◽  
Insect Gut

The lepidopteran-specific P1 delta-endotoxin of Bacillus thuringiensis var. kurstaki HD-1 was activated in vitro using insect gut proteases and found to be highly specific for the lepidopteran cell line Choristoneura fumiferana CF1 among a wide range of lepidopteran and dipteran cell lines tested. The toxicity of P1 against CF1 cells is inhibited by N-acetylgalactosamine (GalNAc), and the lectins soybean agglutinin (SBA) and wheat-germ agglutinin. Protein blotting was used to identify a glycoprotein of 146 X 10(3) Mr in the plasma membrane of CF1 cells, capable of binding both the toxin and SBA, which is specific for GalNAc. This glycoprotein was labelled using galactose oxidase and sodium boro-[3H]hydride and solubilized in Triton X-100 before partial purification by affinity chromatography on SBA-agarose. We propose that this glycoprotein is a good candidate for the cellular receptor of the lepidopteran-specific P1 delta-endotoxin of B. thuringiensis var. kurstaki HD-1.

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