A purification and some properties of an insecticidal exotoxin from Bacillus thuringiensis Berliner.
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ABSTRACT:
SUBMITTER: Bond RP
PROVIDER: S-EPMC1184919 | biostudies-other | 1969 Sep
REPOSITORIES: biostudies-other
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Project description: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.
| S-EPMC3815381 | biostudies-literature
Project description:Bacillus thuringiensis (Bt) has been used as sprayable pesticides for many decades. Bt strains utilized in these products produce multiple insecticidal proteins to complement a narrow insect specificity of each protein. In the late 1990s, genes encoding Bt insecticidal proteins were expressed in crop plants such as cotton and corn to protect these crops from insect damage. The first Bt protein used in transgenic cotton was Cry1Ac to control Heliothis virescens (tobacco budworm). Cry1Ab was applied to corn to control Ostrinia nubilalis (European corn borer). Since these insects have developed resistance to Cry1Ac and Cry1Ab, new Bt proteins are required to overcome the resistance. In order to protect corn furthermore, it is desired to control Diabrotica virgifera (Western corn rootworm), Helicoverpa zea (corn earworm) and Spodoptera frugiperda (fall armyworm). Recently, many new Bt insecticidal proteins have been discovered, but most of them require protein engineering to meet the high activity standard for commercialization. The engineering process for higher activity necessary for Bt crops is called optimization. The seed industry has been optimizing Bt insecticidal proteins to improve their insecticidal activity. In this review, several optimization projects, which have led to substantial activity increases of Bt insecticidal proteins, are described.
| S-EPMC9184247 | biostudies-literature
Project description:Bacillus thuringiensis is the most successful microbial insecticide agent and its proteins have been studied for many years due to its toxicity against insects mainly belonging to the orders Lepidoptera, Diptera and Coleoptera, which are pests of agro-forestry and medical-veterinary interest. However, studies on the interactions between this bacterium and the insect species classified in the order Coleoptera are more limited when compared to other insect orders. To date, 45 Cry proteins, 2 Cyt proteins, 11 Vip proteins, and 2 Sip proteins have been reported with activity against coleopteran species. A number of these proteins have been successfully used in some insecticidal formulations and in the construction of transgenic crops to provide protection against main beetle pests. In this review, we provide an update on the activity of Bt toxins against coleopteran insects, as well as specific information about the structure and mode of action of coleopteran Bt proteins.
| S-EPMC7404982 | biostudies-literature
Project description:Bacillus thuringiensis (Bt) Vip3A proteins are important insecticidal proteins used for control of lepidopteran insects. However, the mode of action of Vip3A toxin is still unclear. In this study, the amino acid residue S164 in Vip3Aa was identified to be critical for the toxicity in Spodoptera litura. Results from substitution mutations of the S164 indicate that the insecticidal activity of Vip3Aa correlated with the formation of a >240 kDa complex of the toxin upon proteolytic activation. The >240 kDa complex was found to be composed of the 19 kDa and the 65 kDa fragments of Vip3Aa. Substitution of the S164 in Vip3Aa protein with Ala or Pro resulted in loss of the >240 kDa complex and loss of toxicity in Spodoptera litura. In contrast, substitution of S164 with Thr did not affect the >240 kDa complex formation, and the toxicity of the mutant was only reduced by 35%. Therefore, the results from this study indicated that formation of the >240 kDa complex correlates with the toxicity of Vip3Aa in insects and the residue S164 is important for the formation of the complex.
| S-EPMC7232161 | biostudies-literature
Project description:A protoxin gene, localized to a high-molecular-weight plasmid from Bacillus thuringiensis subsp. kenyae, was cloned on a 19-kb BamHI DNA fragment into Escherichia coli. Characterization of the gene revealed it to be a member of the CryIE toxin subclass which has been reported to be as toxic as the CryIC subclass to larvae from Spodoptera exigua in assays with crude E. coli extracts. To directly test the purified recombinant gene product, the gene was subcloned as a 4.8-kb fragment into an expression vector resulting in the overexpression of a 134-kDa protein in the form of phase-bright inclusions in E. coli. Treatment of solubilized inclusion bodies with either trypsin or gut juice from the silkworm Bombyx mori resulted in the appearance of a protease-resistant 65-kDa protein. In force-feeding bioassays, the purified activated protein was highly toxic to larvae of B. mori but not to larvae of Choristoneura fumiferana. In diet bioassays with larvae from S. exigua, the purified protoxin was nontoxic. However, prior activation of the protoxin by tryptic digestion resulted in the appearance of some toxic activity. These results demonstrate that this new subclass of protein toxin may not be useful for the control of Spodoptera species as previously reported. Hierarchical clustering of the nine known lepidopteran-specific CryI toxin subclasses through multiple sequence alignment suggests that the toxins fall into four possible subgroups or clusters.
| S-EPMC195296 | biostudies-other
Project description:beta-Exotoxin I is a nonspecific insecticidal metabolite secreted by some Bacillus thuringiensis strains. Several studies of B. thuringiensis strains that have lost the capacity to produce beta-exotoxin I have suggested that there is a strong correlation between high levels of beta-exotoxin I production and the ability to synthesize crystal proteins. In this study, we showed that a mutant strain, B. thuringiensis 407-1(Cry(-))(Pig(+)), with no crystal gene, produced considerable amounts of beta-exotoxin I together with a soluble brown melanin pigment. Therefore, beta-exotoxin I production can take place after a strain has lost the plasmids bearing the cry genes, which suggests that these curable plasmids probably contain determinants involved in the regulation of beta-exotoxin I production. Using a mini-Tn10 transposon, we constructed a library of strain 407-1(Cry(-))(Pig(+)) mutants. We screened for nonpigmented mutants with impaired beta-exotoxin I production and identified a genetic locus harboring two genes (berA and berB) essential for beta-exotoxin I production. The deduced amino acid sequence of the berA gene displayed significant similarity to the ATP-binding domains of the DRI (drug resistance and immunity) family of ATP-binding cassette (ABC) proteins involved in drug resistance and immunity to bacteriocins and lantibiotics. The berB gene encodes a protein with six putative transmembrane helices, which probably constitutes the integral membrane component of the transporter. The demonstration that berAB is required for beta-exotoxin I production and/or resistance in B. thuringiensis adds an adenine nucleotide analog to the wide range of substrates of the superfamily of ABC proteins. We suggest that berAB confers beta-exotoxin I immunity in B. thuringiensis, through active efflux of the molecule.
| S-EPMC135382 | biostudies-literature
Project description: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 ?g/cm(2).
| S-EPMC3457495 | biostudies-literature
Project description:Nanotechnology is a promising way to enhance the stability of Bacillus thuringiensis (Bt) insecticidal proteins under environmental conditions. In this work, two emulsions were prepared through the Pickering emulsion technique, stabilized by Cu2+-SQDs/S-CN nanocomposites and by GO nanosheets. In addition, a pH-sensitive polymer was incorporated into these emulsions, allowing the Bt protein, Cry1Ab, to be released in an alkaline pH environment, as it occurs in the lepidopteran pests' gut. The effectiveness of these two nanomaterials in protecting Cry1Ab from degradation, and therefore enhancing its pesticidal activity, was assessed by exposing samples of the purified unprotected protein and encapsulated protein to high-intensity UV light and 40°C temperature treatments. The UV treatment results were evaluated using SDS-PAGE analysis and pointed out that Cry1Ab could be structurally protected by the emulsions. The bioassays with first instar larvae of the lepidopteran pest Ostrinia nubilalis confirm the nanomaterial protection to UV and temperature treatments, i.e., decreasing about half the degradation rate and increasing up to 12-fold the residual activity after UV treatment. Our results indicate that encapsulation could be an effective strategy to improve the effectiveness of Cry1Ab under environmental conditions. KEY POINTS: • Pickering emulsions are effective for solubilized Cry1Ab encapsulation. • Structural and toxicity Cry1Ab properties are enhanced by pH-sensitive encapsulation. • Cu2+-SQDs/S-CN and GO nanomaterials improve the efficacy of Bt insecticides.
| S-EPMC10560195 | biostudies-literature
Project description:Bacillus thuringiensis Vip3 (Vegetative Insecticidal Protein 3) toxins are widely used in biotech crops to control Lepidopteran pests. These proteins are produced as inactive protoxins that need to be activated by midgut proteases to trigger cell death. However, little is known about their three-dimensional organization and activation mechanism at the molecular level. Here, we have determined the structures of the protoxin and the protease-activated state of Vip3Aa at 2.9 Å using cryo-electron microscopy. The reconstructions show that the protoxin assembles into a pyramid-shaped tetramer with the C-terminal domains exposed to the solvent and the N-terminal region folded into a spring-loaded apex that, after protease activation, drastically remodels into an extended needle by a mechanism akin to that of influenza haemagglutinin. These results provide the molecular basis for Vip3 activation and function, and serves as a strong foundation for the development of more efficient insecticidal proteins.
| S-EPMC7414852 | biostudies-literature
Project description:BackgroundA new Bacillus thuringiensis X023 (BtX023) with high insecticidal activity was isolated in Hunan Province, China. The addition of metals (Cu, Fe, Mg and Mn) to the medium could influence the formation of spores and/or insecticidal crystal proteins (ICPs). In previous studies, Cu ions considerably increased the synthesis of ICPs by enhancing the synthesis of poly-β-hydroxy butyrate. However, the present study could provide new insights into the function of Cu ions in ICPs.ResultsBioassay results showed that wild strain BtX023 exhibited high insecticidal activity against Plutella xylostella. The addition of 1 × 10-5 M Cu2+ could considerably increase the expression of cry1Ac and vip3Aa, and the insecticidal activity was enhanced. Quantitative real-time polymerase chain reaction (qRT-PCR) and proteomic analyses revealed that the upregulated proteins included amino acid synthesis, the glyoxylate pathway, oxidative phosphorylation, and poly-β-hydroxy butyrate synthesis. The Cu ions enhanced energy metabolism and primary amino acid synthesis, will providing abundant raw material accumulation for ICP synthesis.ConclusionThe new strain BtX023 exerted a strong insecticidal effect on P. xylostella by producing ICPs. The addition of 1 × 10-5 M Cu2+ in the medium could considerably enhance the expression of the cry1Ac and vip3Aa genes, thereby further increasing the toxicity of BtX023 to Helicoverpa armigera and P. xylostella by enhancing energy synthesis, the glyoxylate cycle, and branched-chain amino acids synthesis, but not poly-β-hydroxy butyrate synthesis.
| S-EPMC7568400 | biostudies-literature