scholarly journals Structural, functional, and evolutionary analysis of Cry toxins of Bacillus thuringiensis: an in silico study

2021 ◽  
Vol 31 (1) ◽  
Author(s):  
Sujit Kumar Das ◽  
Sukanta Kumar Pradhan ◽  
Kailash Chandra Samal ◽  
Nihar Ranjan Singh
Keyword(s):  
Bacillus Thuringiensis ◽  
3D Structure ◽  
Evolutionary Relationship ◽  
Functional Domain ◽  
Main Body ◽  
Evolutionary Analysis ◽  
Structural Level ◽  
Cry Protein ◽  
Cry Proteins ◽  
Insect Order

Abstract Background Bacillus thuringiensis (Bt) is a gram-positive spore-forming soil bacterium that synthesizes crystalline (Cry) protein, which is toxic and causing pathogenicity against mainly three insect orders: Coleoptera, Diptera, and Lepidoptera. These crystalline protein inclusions, i.e., δ-endotoxins are successfully used as a bio-control agent against insect pests. Main body A total of 58 various Cry proteins belonging to these 3 insect orders were retrieved from SwissProt database and are categorized into different groups. Structural and functional analysis were performed to understand the functional domain arrangements at sequence level as well as at structural level involving both experimental and predicted 3-dimensional models. Besides, the analysis of evolutionary relationship involving all 58 observed Cry proteins at the sequence, domain, and structural levels were done using different bioinformatics tools. Evolutionary analysis revealed that some Cry proteins having toxicity for a specific insect order are found to be clustered for another different insect order, which concludes that they might have toxicity for more than one insect order. Three-dimensional (3D) structure analysis of both experimental and predicted models revealed that proteins might have toxicity for a specific insect order differ in their structural arrangements and was observed in Cry proteins belonging to 3 different insect orders. Conclusions It could be hypothesized that an inner-molecular domain shift or domain insertion/deletion might have taken place during the evolutionary process, which consequently causes structural and functional divergence of Bt. The study output may be helpful for understanding the diversity as well as specificity of the analyzed insecticidal proteins and their application as a biopesticide in the field of agriculture.

scholarly journals Engineering of Bacillus thuringiensis Cry Proteins to Enhance the Activity against Western Corn Rootworm

Toxins ◽  
2019 ◽  
Vol 11 (3) ◽  
pp. 162 ◽  
Author(s):  
Jingtong Hou ◽  
Ruth Cong ◽  
Michi Izumi-Willcoxon ◽  
Hana Ali ◽  
Yi Zheng ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
3D Structure ◽  
Diabrotica Virgifera Virgifera ◽  
Western Corn Rootworm ◽  
Corn Rootworm ◽  
Diabrotica Virgifera ◽  
Cry Protein ◽  
Neutral Ph ◽  
Highly Active ◽  
Cry Toxin

A novel Bacillus thuringiensis Cry protein, Cry8Hb, active against Diabrotica virgifera virgifera (Western corn rootworm, WCRW) was discovered. Unexpectedly, the anti-rootworm activity of the Cry8Hb toxin was enhanced significantly by fusing Escherichia coli maltose binding protein (MBP) to this Cry toxin. While the exact mechanism of the activity enhancement remains indefinite, it is probable that the enhancement is a result of increased solubility of the MBP-Cry8Hb fusion in the rootworm midgut. This hypothesis was examined using a synthetic Cry3 protein called IP3-1, which was not soluble at a neutral pH like Cry8Hb and marginally active to WCRW. When IP3-1 was fused to MBP, its anti-WCRW activity was enhanced 13-fold. To further test the hypothesis, DNA shuffling was performed on IP3-1 to increase the solubility without MBP. Screening of shuffled libraries found six new IP3 variants showing very high anti-WCRW activity without MBP. Sequence and 3D structure analysis of those highly active, shuffled IP3 variants revealed several charge-altering mutations such as Lys to Glu on the putative MBP-attaching side of the IP3 molecule. It is likely that those mutations make the protein acidic to substitute the functions of MBP including enhancing the solubility of IP3 at a neutral pH.

scholarly journals Bacillus thuringiensis as microbial biopesticide: uses and application for sustainable agriculture

2021 ◽  
Vol 31 (1) ◽  
Author(s):  
Pradeep Kumar ◽  
Madhu Kamle ◽  
Rituraj Borah ◽  
Dipendra Kumar Mahato ◽  
Bharti Sharma
Keyword(s):  
Bacillus Thuringiensis ◽  
Plant Growth ◽  
Sustainable Agriculture ◽  
Insect Pest ◽  
High Specificity ◽  
Environmental Safety ◽  
Main Body ◽  
Cry Protein ◽  
Insecticidal Proteins ◽  
Toxic Activity

Abstract Background Bacillus thuringiensis (Bt) has been used in agriculture for a long time because of its insecticidal proteins which make it a valuable environment-friendly biopesticide. However, its use is not only limited to insecticidal properties. Current and previous studies indicate its potential as a biofertilizer for promoting plant growth, the development of transgenic plants, and others. It is the presence of δ-endotoxins, especially cry protein, which attributes the insecticidal property to the bacteria. Besides, there are some vegetative and secreted insecticidal proteins that exert their toxic activity towards specific species. Main body of abstract The present review briefly provides an overview of the Bt uses and application as a biocontrol agent against insect pest for sustainable agriculture. Historical development of Bt as biocontrol, classification of various cry proteins, their mechanisms of actions against different insect-pest, and incorporation of cry genes in the plant for developing transgenic Bt plants such as Bt cotton, potato, and maize. Applications of Bt as biofertilizer and the various bioformulations as biopesticide are also described. Short conclusion Uses of harmful pesticides and chemical cause various health issues and environmental problem; therefore, the Bt served as the best alternative to overcome the above issue. Also, we aim to explore the potential as plant growth-promoting potential and solubilization of minerals and the uses as a biofertilizer, keeping the high specificity and environmental safety of Bt. Its various formulations are commercially available and considered an efficient alternative to chemical pesticides.

scholarly journals Study of the Bacillus thuringiensis Cry1Ia Protein Oligomerization Promoted by Midgut Brush Border Membrane Vesicles of Lepidopteran and Coleopteran Insects, or Cultured Insect Cells

Toxins ◽  
2020 ◽  
Vol 12 (2) ◽  
pp. 133 ◽  
Author(s):  
Ayda Khorramnejad ◽  
Mikel Domínguez-Arrizabalaga ◽  
Primitivo Caballero ◽  
Baltasar Escriche ◽  
Yolanda Bel
Keyword(s):  
Bacillus Thuringiensis ◽  
Growth Phase ◽  
Brush Border ◽  
Brush Border Membrane ◽  
Insect Cells ◽  
3D Structure ◽  
Membrane Vesicles ◽  
Brush Border Membrane Vesicles ◽  
Host Susceptibility ◽  
Cry Proteins

Bacillus thuringiensis (Bt) produces insecticidal proteins that are either secreted during the vegetative growth phase or accumulated in the crystal inclusions (Cry proteins) in the stationary phase. Cry1I proteins share the three domain (3D) structure typical of crystal proteins but are secreted to the media early in the stationary growth phase. In the generally accepted mode of action of 3D Cry proteins (sequential binding model), the formation of an oligomer (tetramer) has been described as a major step, necessary for pore formation and subsequent toxicity. To know if this could be extended to Cry1I proteins, the formation of Cry1Ia oligomers was studied by Western blot, after the incubation of trypsin activated Cry1Ia with insect brush border membrane vesicles (BBMV) or insect cultured cells, using Cry1Ab as control. Our results showed that Cry1Ia oligomers were observed only after incubation with susceptible coleopteran BBMV, but not following incubation with susceptible lepidopteran BBMV or non-susceptible Sf21 insect cells, while Cry1Ab oligomers were persistently detected after incubation with all insect tissues tested, regardless of its host susceptibility. The data suggested oligomerization may not necessarily be a requirement for the toxicity of Cry1I proteins.

scholarly journals The 60-Kilodalton Protein Encoded byorf2in thecry19AOperon of Bacillus thuringiensis subsp. jegathesan Functions Like a C-Terminal Crystallization Domain

2012 ◽  
Vol 78 (6) ◽  
pp. 2005-2012 ◽  
Author(s):  
J. Eleazar Barboza-Corona ◽  
Hyun-Woo Park ◽  
Dennis K. Bideshi ◽  
Brian A. Federici
Keyword(s):  
Bacillus Thuringiensis ◽  
Mass Range ◽  
Wild Type ◽  
Cry Protein ◽  
Cry Proteins ◽  
Content Type ◽  
Native Promoter ◽  
Orf2 Protein ◽  
Function Expression ◽  
Type Crystal

ABSTRACTThecry19Aoperon ofBacillus thuringiensissubsp.jegathesanencodes two proteins, mosquitocidal Cry19A (ORF1; 75 kDa) and an ORF2 (60 kDa) of unknown function. Expression of thecry19Aoperon in an acrystalliferous strain ofB. thuringiensis(4Q7) yielded one small crystal per cell, whereas no crystals were produced whencry19Aororf2was expressed alone. To determine the function of the ORF2 protein, different combinations of Cry19A, ORF2, and the N- or C-terminal half of Cry1C were synthesized in strain 4Q7. Stable crystalline inclusions of these fusion proteins similar in shape to those in the strain harboring the wild-type operon were observed in sporulating cells. Comparative analysis showed that ORF2 shares considerable amino acid sequence identity with the C-terminal region of large Cry proteins. Together, these results suggest that ORF2 assists in synthesis and crystallization of Cry19A by functioning like the C-terminal domain characteristic of Cry protein in the 130-kDa mass range. In addition, to determine whether overexpression of thecry19Aoperon stabilized its shape and increased Cry19A yield, it was expressed under the control of the strong chimericcyt1A-p/STAB-SD promoter. Interestingly, in contrast to the expression seen with the native promoter, overexpression of the operon yielded uniform bipyramidal crystals that were 4-fold larger on average than the wild-type crystal. In bioassays using the 4th instar larvae ofCulex quinquefasciatus, the strain producing the larger Cry19A crystal showed moderate larvicidal activity that was 4-fold (95% lethal concentration [LC95] = 1.9 μg/ml) more toxic than the activity produced in the strain harboring the wild-type operon (LC95= 8.2 μg/ml).

scholarly journals Importance of Cry Proteins in Biotechnology: Initially a Bioinsecticide, Now a Vaccine Adjuvant

Life ◽  
2021 ◽  
Vol 11 (10) ◽  
pp. 999
Author(s):  
Maria Cristina Gonzalez-Vazquez ◽  
Ruth Abril Vela-Sanchez ◽  
Norma Elena Rojas-Ruiz ◽  
Alejandro Carabarin-Lima
Keyword(s):  
Immune Response ◽  
Bacillus Thuringiensis ◽  
Specific Antigen ◽  
Structural Homology ◽  
Insect Larvae ◽  
Cry Protein ◽  
Parasporal Crystal ◽  
Cry Proteins ◽  
Experimental Animal Models ◽  
Specific Antigens

A hallmark of Bacillus thuringiensis bacteria is the formation of one or more parasporal crystal (Cry) proteins during sporulation. The toxicity of these proteins is highly specific to insect larvae, exerting lethal effects in different insect species but not in humans or other mammals. The aim of this review is to summarize previous findings on Bacillus thuringiensis, including the characteristics of the bacterium, its subsequent contribution to biotechnology as a bioinsecticide due to the presence of Cry proteins, and its potential application as an adjuvant. In several studies, Cry proteins have been administered together with specific antigens to immunize experimental animal models. The results have shown that these proteins can enhance immunogenicity by generating an adequate immune response capable of protecting the model against an experimental infectious challenge, whereas protection is decreased when the specific antigen is administered without the Cry protein. Therefore, based on previous results and the structural homology between Cry proteins, these molecules have arisen as potential adjuvants in the development of vaccines for both animals and humans. Finally, a model of the interaction of Cry proteins with different components of the immune response is proposed.

scholarly journals Toxicity and Mode of Action of Bacillus thuringiensis Cry Proteins in the Mediterranean Corn Borer, Sesamia nonagrioides (Lefebvre)

2006 ◽  
Vol 72 (4) ◽  
pp. 2594-2600 ◽  
Author(s):  
Joel González-Cabrera ◽  
Gema P. Farinós ◽  
Silvia Caccia ◽  
Mercedes Díaz-Mendoza ◽  
Pedro Castañera ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Binding Site ◽  
Mode Of Action ◽  
Membrane Vesicles ◽  
Bt Corn ◽  
Sesamia Nonagrioides ◽  
Cry Protein ◽  
Cry Proteins ◽  
Corn Borer ◽  
Mediterranean Countries

ABSTRACT Sesamia nonagrioides is one of the most damaging pests of corn in Spain and other Mediterranean countries. Bt corn expressing the Bacillus thuringiensis Cry1Ab toxin is being grown on about 58,000 ha in Spain. Here we studied the mode of action of this Cry protein on S. nonagrioides (binding to specific receptors, stability of binding, and pore formation) and the modes of action of other Cry proteins that were found to be active in this work (Cry1Ac, Cry1Ca, and Cry1Fa). Binding assays were performed with 125I- or biotin-labeled toxins and larval brush border membrane vesicles (BBMV). Competition experiments indicated that these toxins bind specifically and that Cry1Aa, Cry1Ab, and Cry1Ac share a binding site. Cry1Ca and Cry1Fa bind to different sites. In addition, Cry1Fa binds to Cry1A's binding site with very low affinity and vice versa. Binding of Cry1Ab and Cry1Ac was found to be stable over time, which indicates that the observed binding is irreversible. The pore-forming activity of Cry proteins on BBMV was determined using the voltage-sensitive fluorescent dye DiSC3(5). Membrane permeability increased in the presence of the active toxins Cry1Ab and Cry1Fa but not in the presence of the nonactive toxin Cry1Da. In terms of resistance management, based on our results and the fact that Cry1Ca is not toxic to Ostrinia nubilalis, we recommend pyramiding of Cry1Ab with Cry1Fa in the same Bt corn plant for better long-term control of corn borers.

scholarly journals Intracellular and Extracellular Expression of Bacillus thuringiensis Crystal Protein Cry5B in Lactococcus lactis for Use as an Anthelminthic

2015 ◽  
Vol 82 (4) ◽  
pp. 1286-1294 ◽  
Author(s):  
Evelyn Durmaz ◽  
Yan Hu ◽  
Raffi V. Aroian ◽  
Todd R. Klaenhammer
Keyword(s):  
Bacillus Thuringiensis ◽  
Lactococcus Lactis ◽  
Copy Number ◽  
Oral Delivery ◽  
Membrane Integrity ◽  
Biologically Active ◽  
High Copy Number ◽  
Cry Protein ◽  
Western Blots ◽  
Content Type

ABSTRACTTheBacillus thuringiensiscrystal (Cry) protein Cry5B (140 kDa) and a truncated version of the protein, tCry5B (79 kDa), are lethal to nematodes. Genes encoding the two proteins were separately cloned into a high-copy-number vector with a strong constitutive promoter (pTRK593) inLactococcus lactisfor potential oral delivery against parasitic nematode infections. Western blots using a Cry5B-specific antibody revealed that constitutively expressed Cry5B and tCry5B were present in both cells and supernatants. To increase production,cry5Bwas cloned into the high-copy-number plasmid pMSP3535H3, carrying a nisin-inducible promoter. Immunoblotting revealed that 3 h after nisin induction, intracellular Cry5B was strongly induced at 200 ng/ml nisin, without adversely affecting cell viability or cell membrane integrity. Both Cry5B genes were also cloned into plasmid pTRK1061, carrying a promoter and encoding a transcriptional activator that invoke low-level expression of prophage holin and lysin genes inLactococcuslysogens, resulting in a leaky phenotype. Cry5B and tCry5B were actively expressed in the lysogenic strainL. lactisKP1 and released into cell supernatants without affecting culture growth. Lactate dehydrogenase (LDH) assays indicated that Cry5B, but not LDH, leaked from the bacteria. Lastly, using intracellular lysates fromL. lactiscultures expressing both Cry5B and tCry5B,in vivochallenges ofCaenorhabditis elegansworms demonstrated that the Cry proteins were biologically active. Taken together, these results indicate that active Cry5B proteins can be expressed intracellularly in and released extracellularly fromL. lactis, showing potential for future use as an anthelminthic that could be delivered orally in a food-grade microbe.

scholarly journals New Strategy for Identification of Novel cry-Type Genes from Bacillus thuringiensis Strains

2005 ◽  
Vol 71 (2) ◽  
pp. 761-765 ◽  
Author(s):  
Corina M. Berón ◽  
Leonardo Curatti ◽  
Graciela L. Salerno
Keyword(s):  
Bacillus Thuringiensis ◽  
Phylogenetic Distance ◽  
Cloning And Sequencing ◽  
Degenerate Primers ◽  
Dna Templates ◽  
Cry Proteins ◽  
New Strategy ◽  
Genes Encoding ◽  
Encoding Protein

ABSTRACT We designed five degenerate primers for detection of novel cry genes from Bacillus thuringiensis strains. An efficient strategy was developed based on a two-step PCR approach with these primers in five pair combinations. In the first step, only one of the primer pairs is used in the PCR, which allows amplification of DNA fragments encoding protein regions that include consensus domains of representative proteins belonging to different Cry groups. A second PCR is performed by using the first-step amplification products as DNA templates and the set of five primer combinations. Cloning and sequencing of the last-step amplicons allow both the identification of known cry genes encoding Cry proteins covering a wide phylogenetic distance and the detection and characterization of cry-related sequences from novel B. thuringiensis isolates.

Sign in / Sign up

Export Citation Format

Share Document