Bt Cry toxin is the mostly studied and widely used biological insect resistance protein, which plays a leading role in the green control of agricultural pests worldwide. However, with the wide application of its preparations and transgenic insecticidal crops, the resistance to target pests and potential ecological risks induced by the drive are increasingly prominent and attracting much attention. The researchers seek to explore new insecticidal protein materials that can simulate the insecticidal function of Bt Cry toxin. This will help to escort the sustainable and healthy production of crops, and relieve the pressure of target pests' resistance to Bt Cry toxin to a certain extent. In recent years, the author's team has proposed that Ab2β anti-idiotype antibody has the property of mimicking antigen structure and function based on the "Immune network theory" of antibody. With the help of phage display antibody library and specific antibody high-throughput screening and identification technology, Bt Cry toxin antibody was designed as the coating target antigen, and a series of Ab2β anti-idiotype antibodies (namely Bt Cry toxin insecticidal mimics) were screened from the phage antibody library. Among them, the lethality of Bt Cry toxin insecticidal mimics with the strongest activity was close to 80% of the corresponding original Bt Cry toxin, showing great promise for the targeted design of Bt Cry toxin insecticidal mimics. This paper systematically summarized the theoretical basis, technical conditions, research status, and discussed the development trend of relevant technologies and how to promote the application of existing achievements, aiming to facilitate the research and development of green insect-resistant materials.
Insecticides/metabolism* ; Bacillus thuringiensis ; Endotoxins/pharmacology* ; Bacillus thuringiensis Toxins/metabolism* ; Hemolysin Proteins/pharmacology* ; Bacterial Proteins/chemistry* ; Plants, Genetically Modified/genetics* ; Pest Control, Biological

Bacillus thuringiensis is widely used as an insecticide which is safe and environmentally friendly to humans and animals. One of the important insecticidal mechanisms is the binding of Bt toxins to specific toxin receptors in insect midgut and forming a toxin perforation which eventually leads to insect death. The resistance of target pests to Bt toxins is an important factor hampering the long-term effective cultivation of Bt crops and the continuous use of Bt toxins. This review summarizes the mechanism of insect resistance to Bt toxins from the perspective of important Bt toxin receptors in midgut cells of Lepidopteran insects, which may facilitate the in-depth study of Bt resistance mechanism and pest control.
Animals ; Bacillus thuringiensis/genetics* ; Bacillus thuringiensis Toxins ; Bacterial Proteins/metabolism* ; Endotoxins/metabolism* ; Hemolysin Proteins/metabolism* ; Insecta/metabolism* ; Insecticide Resistance/genetics* ; Insecticides/pharmacology* ; Pest Control, Biological

Aims: Potatoes are considered one of the most strategic vegetable crops all over the world. Alternaria alternata has recently contaminated certain potatoes farms in different regions in Egypt. Among thirteen samples from fifteen regions were studied in a precedent study. Our study was aimed to investigate the effect of Bacillus thuringiensis subsp. Kurosaki suspension on inhibiting the growth of the three tested isolates of A. alternata and minimizing their mycotoxins production in vitro using three isolates with three levels of highly, moderate and low pathogenicity with unequal amounts of dual mycotoxins production. Methodology and results: Three isolates of A. alternata from three regions, Kom Hamada (KH3), Alamin (Alam1) and Nobaria (NO3), which were determined as a producer of tenuazonic acid (TeA) and alternariol monomethyl ether (AME) toxins. Bacillus thuringiensis (Bt) use as commercial fungicide was applied with three suspension concentrations (75, 150 and 300 μg/mL) as inhibitor for the two mycotoxins. Our results illustrated that the three tested isolates recorded high TeA and AME inhibition efficacies by increasing the Bt suspension concentration. The highest inhibitory concentration of Bt was at concentration 75 μg/mL for isolated from Nobaria third region (NO3) and Alam1 it was (99.83 and 99.74%) for mycotoxin (AME) while, TeA mycotoxin had the most inhibition percentage (99.58%) at concentration 150 μg/mL for the isolate (NO3). Conclusion, significance and impact of study The preliminary results of the study suggest that B. thuringiensis spores’ suspension with different concentrations can be used as anti-mycotoxigenic agents to inhibit the (TeA) and (AME) mycotoxins produced by Alternaria alternata.
Bacillus thuringiensis ; Alternaria--isolation & ; purification ; Solanum tuberosum

Aims: Oryctes rhinoceros beetle is one of the most damaging pests of oil palm and cause high oil palm mortality. The empty fruit bunch mulch and rotten old trunk of oil palm in the field provide the organic matter for the breeding sites and increases the number of O. rhinoceros larvae. Bacillus thuringiensis as bioinsecticide can synthesize crystal proteins toxic to the larvae. The present study was aimed to find effective B. thuringiensis isolates as biopesticide against O. rhinoceros larvae. Methodology and results: Screening process was carried out through heating of soil sample suspension at 80 °C to eliminate the non-spore formers and plated onto T3 medium. Colony morphology was observed, followed by Gram and endospore staining. The crystal protein was observed by Coomassie Brilliant Blue (CBB) staining. Bioassay test was conducted by force-feed method followed by food contamination method. The results showed isolates SBB33 and SBB35 were able to infect and caused high mortality to the O. rhinoceros larvae. Isolates SBB33 and SBB35 showed the highest mortality against 1st instar larvae (94.44% and 75% respectively) and 3rd instar larvae (64.8% and 60% respectively) compared to control treatments. The 16S rRNA gene sequencing showed SBB33 has high similarity with B. thuringiensis strain 3S2-3, while SBB35 has high similarity with B. thuringiensis strain GCU_BTi10. Protein separation of the spore-crystal mixture by SDS-PAGE showed the prominence of 66 kDa protein band that was predicted to be Cry toxins which is specific to coleopterans insect. Conclusion, significance and impact of study Bacillus thuringiensis isolates SBB33 and SBB35 have high potential as biopesticides against O. rhinoceros larvae and could be used to control major pests in oil palm plantation.
Bacillus thuringiensis--isolation & ; purification ; Coleoptera

L-2-aminobutyric acid (L-ABA) is an important chemical raw material and chiral pharmaceutical intermediate. The aim of this study was to develop an efficient method for L-ABA production from L-threonine using a trienzyme cascade route with Threonine deaminase (TD) from Escherichia. coli, Leucine dehydrogenase (LDH) from Bacillus thuringiensis and Formate dehydrogenase (FDH) from Candida boidinii. In order to simplify the production process, the activity ratio of TD, LDH and FDH was 1:1:0.2 after combining different activity ratios in the system in vitro. The above ratio was achieved in the recombinant strain E. coli 3FT+L. Moreover, the transformation conditions were optimized. Finally, we achieved L-ABA production of 68.5 g/L with a conversion rate of 99.0% for 12 h in a 30-L bioreactor by whole-cell catalyst. The environmentally safe and efficient process route represents a promising strategy for large-scale L-ABA production in the future.
Aminobutyrates ; chemical synthesis ; Bacillus thuringiensis ; enzymology ; Candida ; enzymology ; Escherichia coli ; enzymology ; Formate Dehydrogenases ; metabolism ; Leucine Dehydrogenase ; metabolism ; Threonine ; metabolism ; Threonine Dehydratase ; metabolism

With the rapid development of transgenic technology, the safety of genetically modified products has received extensive attention. Certified reference materials for the detection of genetically modified organisms play important roles in ensuring comparability and traceability of the qualitative and quantitative detection of genetically modified products. However, the development of protein reference materials is relatively slow, and one of the difficulties is the preparation of protein candidates with high purity. The cry1Ah1 gene of Bacillus thuringiensis has been used for the development of transgenic insect-resistant crops because of its excellent insecticidal activity against lepidopteran pests such as Asian corn borer, and has obtained transgenic lines with good insect resistance traits. In order to develop Cry1Ah protein certified reference material, it is urgent to establish a preparation and purification system. In this study, a system for preparing Cry1Ah protein by Bt expression system was optimized, and a high-purity Cry1Ah protein (size exclusion chromatography purity: 99.6%) was obtained by ion-exchange chromatography and size exclusion chromatography stepwise purification. The results of biological activity assay showed that there was no significant difference in the insecticidal activity of purified Cry1Ah protein and protoxin against diamondback moths (Plutella xylostella). Finally, the amino acid sequence of the activated Cry1Ah protein was determined using Edman degradation and mass spectrometry. In summary, the obtained Cry1Ah pure protein can be used for the development of protein reference materials.
Animals ; Bacillus thuringiensis ; Bacterial Proteins ; Cryptochromes ; metabolism ; Endotoxins ; Hemolysin Proteins ; Moths ; Pest Control, Biological ; Plants, Genetically Modified

Bacillus thuringiensis (Bt) produces Cry toxins that are widely used as insecticides in agriculture and forestry. Receptors are important to elucidate the mode of interaction with Cry toxins and toxicity in lepidopteran insects. Here, we purified the Cry toxin from Bt and identified this toxin by flight mass spectrometry as Cry1Ac, and then recombinantly expressed aminopeptidase N (BmAPN6) and repeat domains of cadherin-like protein (CaLP) of B. mori. Using co-immunoprecipitation (co-IP), Far-Western blotting, and enzyme-linked immunosorbent assays (ELISAs), we identified the interaction between Cry1Ac and BmAPN6. Furthermore, analysis of the cytotoxic activity of Cry1Ac toxin in Sf9 cells showed that BmAPN6 directly interacted with Cry1Ac toxin to induce morphological aberrations and cell lysis. We also used co-IP, Far-Western blotting and ELISAs to analyze the interactions of Cry1Ac with three binding sites corresponding to cadherin repeat (CR) 7 CR11, and CR12 of CaLP. Notably, the three repeat domains were essential Cry1Ac binding components in CaLP. These results indicated that BmAPN6 and CaLP served as a functional receptor involved in Bt Cry1Ac toxin pathogenicity. These findings represent an important advancement in our understanding of the mechanisms of Cry1Ac toxicity and provide promising candidate targets for gene editing to enhance resistance to pathogens and increase the economic value of B. mori.
Animals ; Bacillus thuringiensis ; Bacterial Proteins ; metabolism ; Bombyx ; enzymology ; CD13 Antigens ; metabolism ; Cadherins ; metabolism ; Endotoxins ; metabolism ; Hemolysin Proteins ; metabolism ; Larva

A transgenic maize event ZD12-6 expressing a Bacillus thuringiensis (Bt) fusion protein Cry1Ab/Cry2Aj and a modified 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) protein G10 was characterized and evaluated. Southern blot analysis indicated that ZD12-6 is a single copy integration event. The insert site was determined to be at chromosome 1 by border sequence analysis. Expression analyses of Bt fusion protein Cry1Ab/Cry2Aj and the EPSPS protein G10 suggested that they are both expressed stably in different generations. Insect bioassays demonstrated that the transgenic plants are highly resistant to Asian corn borer (Ostrinia furnacalis), cotton boll worm (Helicoverpa armigera), and armyworm (Mythimna separata). This study suggested that ZD12-6 has the potential to be developed into a commercial transgenic line.
3-Phosphoshikimate 1-Carboxyvinyltransferase/metabolism* ; Animals ; Bacillus thuringiensis Toxins ; Bacterial Proteins/metabolism* ; China ; Disease Resistance/genetics* ; Drug Resistance/genetics* ; Endotoxins/metabolism* ; Gene Expression Profiling ; Glycine/chemistry* ; Hemolysin Proteins/metabolism* ; Insecta ; Plant Diseases/prevention & control* ; Plants, Genetically Modified/genetics* ; Zea mays/genetics* ; Glyphosate

ABSTRACT Aims: Dickeya dadantii is a pathogenic bacterium causing bacterial soft rot disease in plants. The bacterium uses a homoserine lactone signal in its quorum sensing process to express the virulence factor genes. Anti-quorum sensing is a new approach to control plant pathogenic bacteria. The aims of this study are to characterize AHL-lactonase enzyme produced by Bacillus thuringiensis SGT3g and to determine its effectiveness in inhibiting virulence of D. dadantii. Methodology and results: Activity of AHL-lactonase was determined using Chromobacterium violaceum as a bacterial biosensor. The crude extract enzymes of AHL-lactonase on both as extracellular and intracellular enzymes were analyzed their enzyme activity of protein precipitation and dialysis products. The optimum activity of AHL-lactonase was found at 30 °C and pH 5-8. Bacillus thuringiensis SGT3g was capable to reduce soft rot symptom disease caused by D. dadantii on Phalaenopsis orchid leaves after 24 h of incubation. Conclusion, significance and impact study: Bacillus thuringiensis SGT3g was capable to degrade AHL signal of C. violaceum and D. dadantii. The activity AHL-lactonase of B. thuringiensis SGT3g had a wide range of pH and temperature. The lactonase could reduce soft rot symptom disease caused by D. dadantii without any growth inhibition of D. dadantii on orchid leaves. Bacillus thuringiensis SGT3g can be used as an alternative biopesticide to control phytopathogenic bacteria due to its capability to suppress bacterial pathogenic virulence.
Bacillus thuringiensis

OBJECTIVETo investigate the flexibility and mobility of the Bacillus thuringiensis toxin Cry1Aa.

METHODSThe graph theory-based program Constraint Network Analysis and normal mode-based program NMsim were used to analyze the global and local flexibility indices as well as the fluctuation of individual residues in detail.

RESULTSThe decrease in Cry1Aa network rigidity with the increase of temperature was evident. Two phase transition points in which the Cry1Aa structure lost rigidity during the thermal simulation were identified. Two rigid clusters were found in domains I and II. Weak spots were found in C-terminal domain III. Several flexible regions were found in all three domains; the largest residue fluctuation was present in the apical loop2 of domain II.

CONCLUSIONAlthough several flexible regions could be found in all the three domains, the most flexible regions were in the apical loops of domain II.

Bacillus thuringiensis ; Bacterial Proteins ; chemistry ; genetics ; metabolism ; Cluster Analysis ; Computer Simulation ; Endotoxins ; chemistry ; genetics ; metabolism ; Entropy ; Hemolysin Proteins ; chemistry ; genetics ; metabolism ; Models, Structural ; Mutation ; Protein Conformation ; Protein Unfolding ; Software ; Temperature