Antagonistic Effect of Truncated Fragments of Bacillus thuringiensis Vip3Aa on the Larvicidal Activity of its Full-length Protein

2020 ◽  
Vol 27 ◽  
Author(s):  
Patcharaporn Boonyos ◽  
Chutchanun Trakulnalueamsai ◽  
Amporn Rungrod ◽  
Sukumal Chongthammakun ◽  
Boonhiang Promdonkoy
Keyword(s):  
Bacillus Thuringiensis ◽  
Spodoptera Exigua ◽  
Antagonistic Effect ◽  
Full Length ◽  
Carbohydrate Binding ◽  
Specific Receptor ◽  
Terminal Part ◽  
Insect Larvae ◽  
Lethal Time ◽  
Insect Gut

Background: Vip3Aa is a vegetative insecticidal protein produced by Bacillus thuringiensis. The protein is produced as an 88- kDa protoxin that could be processed by insect gut proteases into a 22-kDa N-terminal and a 66-kDa Cterminal fragments. The C-terminal part could bind to a specific receptor while the N-terminal part is required for toxicity and structural stability. Objective: To demonstrate the antagonistic effect of truncated fragments on the insecticidal activity of the full-length Vip3Aa. Methods: The full-length protein (Vip3Aa), a 66-kDa C-terminal fragment (Vip3Aa-D199) and a predicted carbohydrate binding module (CBM) were produced in Escherichia coli. Purified proteins were mixed at different ratios and fed to Spodoptera litura and Spodoptera exigua larvae. Mortality was recorded and compared between larvae fed with individual toxin and mixtures of the full-length and truncated toxins. Results: Production level of the Vip3Aa-D199 was significantly decreased comparing to that of the full-length protein. Vip3Aa-D199 and CBM fragment were not toxic to insect larvae whereas Vip3Aa showed high toxicity with LC50 about 200 ng/cm2 . Feeding the larvae with mixtures of the Vip3Aa and Vip3Aa-D199 at different ratios revealed antagonistic effect of the Vip3Aa-D199 on the toxicity of Vip3Aa. Results showed that the lethal time ( LT50 and LT95) of larvae fed the mixture toxins was longer than those fed the Vip3Aa alone. In addition, a CBM fragment could inhibit toxicity of the full-length Vip3Aa. Conclusion: Our results demonstrated that the Vip3Aa-D199 and a CBM fragment could complete for the membrane binding thus rendering activity of the full-length Vip3Aa.

scholarly journals Regulation of the Packaging of Bacillus thuringiensis δ-Endotoxins into Inclusions

2001 ◽  
Vol 67 (11) ◽  
pp. 5032-5036 ◽  
Author(s):  
Lily Chang ◽  
Rian Grant ◽  
Arthur Aronson
Keyword(s):  
Bacillus Thuringiensis ◽  
Consensus Sequence ◽  
Luria Bertani ◽  
Insect Larvae ◽  
Inclusion Formation ◽  
Reducing Conditions ◽  
Mrna Ratio ◽  
Yeast Extract Medium ◽  
Relative Deficiency ◽  
Binding Sequence

ABSTRACT During sporulation, many Bacillus thuringiensis subspecies synthesize several related δ-endotoxins which are packaged into bipyramidal intracellular inclusions. These inclusions are solubilized in the alkaline, reducing conditions of the midguts of susceptible insect larvae and are converted by proteolysis to active toxins. The toxins insert into the membranes of cells lining the midgut and form cation-selective channels, which results in lethality. There are three δ-endotoxins, Cry1Ab3, Cry1Ca1, and Cry1Da1, present in the inclusions produced by a B. thuringiensis subsp.aizawai cell. While the ratio of the steady-state mRNAs for these three protoxins has been shown to differ (cry1Ab3/cry1Ca1/cry1Da1 mRNA ratio, 4:2:1), the half-lives of the cry1Da1 and cry1Ab3 mRNAs were found to be similar, indicating that there were differences in the transcription rates. The relative contents of these δ-endotoxins in purified inclusions from B. thuringiensissubsp. aizawai have been measured previously, and an even greater relative deficiency of the Cry1Da1 protoxin (ratio, 20:12:1) was found. In order to account for this deficiency, other steps which could be involved in inclusion formation, such as translation and packaging, were examined. The three cry genes have the same dual overlapping promoters, but the ribosome binding sequence for the cry1Da1 gene was not the consensus sequence. Translation was enhanced about fourfold by changing to the consensus sequence. In addition, the relative amount of Cry1Da1 protoxin in inclusions was twofold lower when cells were sporulated in Luria-Bertani (LB) medium than when cells were sporulated in a glucose-yeast extract medium. This difference was attributable to packaging since the relative amounts of Cry1Da1 antigen in cells sporulating in the two media were the same. Some factor(s) required for packaging of the Cry1Da1 protoxin in inclusions is apparently limiting in LB medium. Differences in the initial transcription rates, translation efficiencies, and packaging all contribute to the δ-endotoxin composition of an inclusion.

Technology of production of biopesticides based on Bacillus thuringiensis

2021 ◽  
Vol 9 (16) ◽  
pp. 28-38
Author(s):  
Anatolii Bezusov ◽  
◽  
Valentyna Krutiakova ◽  
Olena Myroshnichenko ◽  
Nataliia Dotsenko ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Corn Oil ◽  
Raw Materials ◽  
Culture Fluid ◽  
Scientific Work ◽  
Insect Larvae ◽  
Effective Prevention ◽  
Research Results ◽  
Wide Range ◽  
Vegetable Raw Materials

Subject of research. Biopesticides are based on live cultures of specially selected beneficial microorganisms with controlled properties. They have a pronounced phytoprotective and stimulating effect, thus providing effective prevention and protection of plants from diseases. The obtained biological product increases productivity, improves the quality and structure of the crop, does not accumulate in plants, which allows to obtain environmentally friendly agricultural products and does not harm the environment. Among the large number of bacteria, as a source of microbiological insecticide is Bacillus thuringiensis, which infects lepidopteran pests and leads to their death. Preparations based on this strain are effective when used in low concentrations of solutions. The Bacillus thuringiensis strain produces several toxins with insecticidal action, including β-exotoxin and δ-endotoxin. Toxic effect is manifested and leads to paralysis of the intestinal tract of parasites. Preparations β-exotoxin and δ-endotoxin are obtained by culturing Bacillus thuringiensis bacteria in a liquid medium. The scientific work proposes a method of industrial production using by-products of vegetable raw materials, which makes it economically feasible to use such substrates. The purpose of the study is the development of technology, formulation of nutrient medium, process parameters for the cultivation of bacteria of the genus Bacillus thuringiensis and obtaining a culture fluid containing substances of the class of biopesticides. Methods. Standard and generally accepted methods of research of bioproducts in biotechnology. The formation of bioinsecticides was established by hydrolysis methods, followed by determination of the component of β-exotoxin – ribose, the formation of octagonal crystals of exotoxin – by microscopic method. Research results. Three variants of nutrient media, which include yeast-polysaccharide complex: corn flour, corn oil, yeast autolysate were developed. The parameters of the bacterial cultivation process were studied. The final product is a paste or powder with a titer of 35 .109 spores in 1 g of the bioproduct. Scope of research results. Microbiological preparations based on Bacillus thuringiensis are highly specific and act only on insect larvae from the classes Lepidoptera and Diptera. The resulting biopesticide can be used against pests of a wide range of vegetable and fruit crops.

scholarly journals Mutations in the Bacillus thuringiensis Cry1Ca toxin demonstrate the role of domains II and III in specificity towards Spodoptera exigua larvae

2004 ◽  
Vol 384 (3) ◽  
pp. 507-513 ◽  
Author(s):  
Salvador HERRERO ◽  
Joel GONZÁLEZ-CABRERA ◽  
Juan FERRÉ ◽  
Petra L. BAKKER ◽  
Ruud A. de MAAGD
Keyword(s):  
Bacillus Thuringiensis ◽  
Spodoptera Exigua ◽  
Brush Border Membrane ◽  
Specific Activity ◽  
Membrane Vesicles ◽  
Wild Type ◽  
Oligomeric Form ◽  
Specific Receptors ◽  
Domain Iii

Several mutants of the Bacillus thuringiensis Cry1Ca toxin affected with regard to specific activity towards Spodoptera exigua were studied. Alanine was used to replace single residues in loops 2 and 3 of domain II (mutant pPB19) and to replace residues 541–544 in domain III (mutant pPB20). Additionally, a Cry1Ca mutant combining all mutations was constructed (mutant pPB21). Toxicity assays showed a marked decrease in toxicity against S. exigua for all mutants, while they retained their activity against Manduca sexta, confirming the importance of these residues in determining insect specificity. Parameters for binding to the specific receptors in BBMV (brush border membrane vesicles) of S. exigua were determined for all toxins. Compared with Cry1Ca, the affinity of mutant pPB19 was slightly affected (2-fold lower), whereas the affinity of the mutants with an altered domain III (pPB20 and pPB21) was approx. 8-fold lower. Activation of Cry1Ca protoxin by incubation with S. exigua or M. sexta BBMV revealed the transient formation of an oligomeric form of Cry1Ca. The presence of this oligomeric form was tested in the activation of the different Cry1Ca mutants, and we found that those mutated in domain II (pPB19 and pPB21) could not generate the oligomeric form when activated by S. exigua BBMV. In contrast, when oligomerization was tested using BBMV prepared from M. sexta, all of the Cry1Ca mutants showed the formation of a similar oligomeric form as did the wild-type toxin. Our results show how modification of insect specificity can be achieved by manipulation of different parts of the toxin structure involved in different steps of the mode of action of B. thuringiensis toxins.

scholarly journals Characterization of Cry34/Cry35 Binary Insecticidal Proteins from Diverse Bacillus thuringiensis Strain Collections

2005 ◽  
Vol 71 (4) ◽  
pp. 1765-1774 ◽  
Author(s):  
H. Ernest Schnepf ◽  
Stacey Lee ◽  
JoAnna Dojillo ◽  
Paula Burmeister ◽  
Kristin Fencil ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Sequence Similarity ◽  
Diabrotica Virgifera Virgifera ◽  
Full Length ◽  
Pcr Analysis ◽  
Binding Motif ◽  
Diabrotica Virgifera ◽  
Crystal Proteins ◽  
Corn Rootworms ◽  
Geographic Origins

ABSTRACT Bacillus thuringiensis crystal proteins of the Cry34 and Cry35 classes function as binary toxins showing activity on the western corn rootworm, Diabrotica virgifera virgifera LeConte. We surveyed 6,499 B. thuringiensis isolates by hybridization for sequences related to cry35A genes, identifying 78 strains. Proteins of the appropriate molecular mass (ca. 44 kDa) for Cry35 were observed in 42 of the strains. Full-length, or nearly full-length, sequences of 34 cry34 genes and 16 cry35 genes were also obtained from cloning, PCR analysis, and DNA sequencing. These included representatives of all known Cry34A, Cry34B, Cry35A, and Cry35B classes, as well as a novel Cry34A/Cry35A-like pair. Bioassay analysis indicated that cry35-hybridizing strains not producing a ca. 14-kDa protein, indicative of Cry34, were not active on corn rootworms, and that the previously identified Cry34A/Cry35A pairs were more active than the Cry34B/Cry35B pairs. The cry35-hybridizing B. thuringiensis strains were found in locales and materials typical for other B. thuringiensis strains. Comparison of the sequences with the geographic origins of the strains showed that identical, or nearly identical, sequences were found in strains from both Australasia and the Americas. Sequence similarity searches revealed that Cry34 proteins are similar to predicted proteins in Photorhabdus luminescens and Dictyostelium discoidium, and that Cry35Ab1 contains a segment similar to beta-trefoil domains that may be a binding motif. The binary Cry34/Cry35 B. thuringiensis crystal proteins thus appear closely related to each other, are environmentally ubiquitous, and share sequence similarities consistent with activity through membrane disruption in target organisms.

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