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 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 Development of a Homologous Expression System for and Systematic Site-Directed Mutagenesis Analysis of Thurincin H, a Bacteriocin Produced by Bacillus thuringiensis SF361

2014 ◽  
Vol 80 (12) ◽  
pp. 3576-3584 ◽  
Author(s):  
Gaoyan Wang ◽  
David C. Manns ◽  
John J. Churey ◽  
Randy W. Worobo
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Bacillus Thuringiensis ◽  
Gene Cluster ◽  
Expression Vector ◽  
Expression System ◽  
Structural Genes ◽  
Cry Protein ◽  
Content Type ◽  
Thurincin H

ABSTRACTThurincin H is an antimicrobial peptide produced byBacillus thuringiensisSF361. With a helical back bone, the 31 amino acids of thurincin H form a hairpin structure maintained by four pairs of very unique sulfur-to-α-carbon thioether bonds. The production of thurincin H depends on a putative gene cluster containing 10 open reading frames. The gene cluster includes three tandem structural genes (thnA1,thnA2, andthnA3) encoding three identical 40-amino-acid thurincin H prepeptides and seven other genes putatively responsible for prepeptide processing, regulation, modification, exportation, and self-immunity. A homologous thurincin H expression system was developed by transforming a thurincin H-deficient host with a novel expression vector, pGW133. The host, designatedB. thuringiensisSF361 ΔthnA1ΔthnA2ΔthnA3, was constructed by deletion of the three tandem structural genes from the chromosome of the natural thurincin H producer. The thurincin H expression vector pGW133 was constructed by cloning the thurincin H native promoter,thnA1, and a Cry protein terminator into theEscherichia coli-B. thuringiensisshuttle vector pHT315. Thirty-three different pGW133 variants, each containing a different point mutation in thethnA1gene, were generated and separately transformed intoB. thuringiensisSF361 ΔthnA1ΔthnA2ΔthnA3. Those site-directed mutants contained either a single radical or conservative amino acid substitution on the thioether linkage-forming positions or a radical substitution on all other nonalanine amino acids. The bacteriocin activities ofB. thuringiensisSF361 ΔthnA1ΔthnA2ΔthnA3carrying different pGW133 variants against three different indicator strains were subsequently compared.

scholarly journals Efficient Bioconversion of Echinocandin B to Its Nucleus by Overexpression of Deacylase Genes in Different Host Strains

2012 ◽  
Vol 79 (4) ◽  
pp. 1126-1133 ◽  
Author(s):  
Lei Shao ◽  
Jian Li ◽  
Aijuan Liu ◽  
Qing Chang ◽  
Huimin Lin ◽  
...  
Keyword(s):  
Type Strain ◽  
Wild Type Strain ◽  
Wild Type ◽  
Transcription Level ◽  
Reaction Conditions ◽  
Fungal Cell ◽  
Content Type ◽  
Native Promoter ◽  
Echinocandin B ◽  
Host Strains

ABSTRACTAnidulafungin, which noncompetitively inhibits β-(1,3)-d-glucan synthase in fungal cell wall biosynthesis, is the newest antifungal drug to be developed. Echinocandin B deacylase fromActinoplanes utahensisNRRL 12052 catalyzes the cleavage of the linoleoyl group of echinocandin B, a key step in the process of manufacturing anidulafungin. Unfortunately, the natural yield of echinocandin B nucleus is low. In our study, the echinocandin B deacylase gene was systematically overexpressed by genetic engineering in its original producer,A. utahensis, and in the heterologous hostsStreptomyces lividansTK24 andStreptomyces albus. The introduction of additional copies of the gene, under the control ofPermE* or its native promoter, into hosts showed significant increases in its transcription level and in the efficiency of the bioconversion of echinocandin B to its nucleus. The conditions for the cultivation and bioconversion ofA. utahensishave been optimized further to improve production. As a result, while the wild-type strain initially produced 0.36 g/liter, a concentration of 4.21 g/liter was obtained after the generation of a strain with additional copies of the gene and further optimization of the reaction conditions. These results are useful for enhancing echinocandin B nucleus production inA. utahensis. Our study could enable the engineering of commercially useful echinocandin B nucleus-overproducing stains.

scholarly journals Crystal Structure of VapBC-1 from NontypeableHaemophilus influenzaeand the Effect of PIN Domain Mutations on Survival during Infection

2019 ◽  
Vol 201 (12) ◽  
Author(s):  
Ashley L. Molinaro ◽  
Maithri M. Kashipathy ◽  
Scott Lovell ◽  
Kevin P. Battaile ◽  
Nathan P. Coussens ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Haemophilus Influenzae ◽  
Protein Interactions ◽  
Ex Vivo ◽  
Single Copy ◽  
Human Tissues ◽  
Type Ii ◽  
Wild Type ◽  
Content Type ◽  
Native Promoter

ABSTRACTToxin-antitoxin (TA) gene pairs have been identified in nearly all bacterial genomes sequenced to date and are thought to facilitate persistence and antibiotic tolerance. TA loci are classified into various types based upon the characteristics of their antitoxins, with those in type II expressing proteic antitoxins. Many toxins from type II modules are ribonucleases that maintain a PilT N-terminal (PIN) domain containing conserved amino acids considered essential for activity. ThevapBC(virulence-associatedprotein) TA system is the largest subfamily in this class and has been linked to pathogenesis of nontypeableHaemophilus influenzae(NTHi). In this study, the crystal structure of the VapBC-1 complex from NTHi was determined to 2.20 Å resolution. Based on this structure, aspartate-to-asparagine and glutamate-to-glutamine mutations of four conserved residues in the PIN domain of the VapC-1 toxin were constructed and the effects of the mutations on protein-protein interactions, growth ofEscherichia coli, and pathogenesisex vivowere tested. Finally, a novel model system was designed and utilized that consists of an NTHi ΔvapBC-1strain complemented inciswith the TA module containing a mutated or wild-type toxin at an ectopic site on the chromosome. This enabled the analysis of the effect of PIN domain toxin mutants in tandem with their wild-type antitoxin under the control of thevapBC-1native promoter and in single copy. This is the first report of a system facilitating the study of TA mutant operons in the background of NTHi during infections of primary human tissuesex vivo.IMPORTANCEHerein the crystal structure of the VapBC-1 complex from nontypeableHaemophilus influenzae(NTHi) is described. Our results show that some of the mutations in the PIN domain of the VapC-1 toxin were associated with decreased toxicity inE. coli, but the mutants retained the ability to homodimerize and to heterodimerize with the wild-type cognate antitoxin, VapB-1. A new system was designed and constructed to quantify the effects of these mutations on NTHi survival during infections of primary human tissuesex vivo. Any mutation to a conserved amino acid in the PIN domain significantly decreased the number of survivors compared to that of the inciswild-type toxin under the same conditions.

scholarly journals Large Crystal Toxin Formation in Chromosomally Engineered Bacillus thuringiensis subsp. aizawai Due to σEAccumulation

2012 ◽  
Vol 78 (6) ◽  
pp. 1682-1691 ◽  
Author(s):  
Wasin Buasri ◽  
Watanalai Panbangred
Keyword(s):  
Bacillus Thuringiensis ◽  
Expression Profiles ◽  
Toxin Production ◽  
Wild Type ◽  
Large Crystal ◽  
Content Type ◽  
Crystal Toxin ◽  
Cat Expression ◽  
Toxin Formation

ABSTRACTSeven distinctBacillus thuringiensissubsp.aizawaiintegrants were constructed that carried the chitinase (chiBlA) gene fromB. licheniformisunder the control of thecry11Aapromoter and terminator with and withoutp19andp20genes. The toxicity ofB. thuringiensissubsp.aizawaiintegrants against second-instarSpodoptera lituralarvae was increased 1.8- to 4.6-fold compared to that of the wild-type strain (BTA1). Surprisingly, the enhanced toxicity in some strains ofB. thuringiensissubsp.aizawaiintegrants (BtaP19CS,BtaP19CSter, andBtaCAT) correlated with an increase in toxin formation. To investigate the role of these genes in toxin production, the expression profiles of the toxin genes,cry1AaandchiBlA, as well as their transcriptional regulators (sigKandsigE), were analyzed by quantitative real-time RT-PCR (qPCR) from BTA1,BtaP19CS, andBtaCAT. Expression levels ofcry1Aain these two integrants increased about 2- to 3-fold compared to those of BTA1. The expression of the transcription factorsigKalso was prolonged in the integrants compared to that of the wild type; however,sigEexpression was unchanged. Western blot analysis of σEand σKshowed the prolonged accumulation of σEin the integrants compared to that of BTA1, resulting in the increased synthesis of pro-σKup toT17after the onset of sporulation in bothBtaP19CS andBtaCAT compared to that ofT13in BTA1. The results from qPCR indicate clearly that thecry1Aapromoter activity was influenced most strongly by σE, whereascry11Aadepended mostly on σK. These results on large-crystal toxin formation with enhanced toxicity should provide useful information for the generation of strains with improved insecticidal activity.

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 Insect Hsp90 Chaperone Assists Bacillus thuringiensis Cry Toxicity by Enhancing Protoxin Binding to the Receptor and by Protecting Protoxin from Gut Protease Degradation

mBio ◽  
2019 ◽  
Vol 10 (6) ◽  
Author(s):  
Blanca I. García-Gómez ◽  
Sayra N. Cano ◽  
Erika E. Zagal ◽  
Edgar Dantán-Gonzalez ◽  
Alejandra Bravo ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Receptor Binding ◽  
Insecticidal Activity ◽  
Insect Pests ◽  
Biotechnological Applications ◽  
Cry Proteins ◽  
Cry Toxins ◽  
Content Type ◽  
Hsp90 Chaperone ◽  
Protease Degradation

ABSTRACT Bacillus thuringiensis Cry proteins are pore-forming insecticidal toxins with specificity against different crop pests and insect vectors of human diseases. Previous work suggested that the insect host Hsp90 chaperone could be involved in Cry toxin action. Here, we show that the interaction of Cry toxins with insect Hsp90 constitutes a positive loop to enhance the performance of these toxins. Plutella xylostella Hsp90 (PxHsp90) greatly enhanced Cry1Ab or Cry1Ac toxicity when fed together to P. xylostella larvae and also in the less susceptible Spodoptera frugiperda larvae. PxHsp90 bound Cry1Ab and Cry1Ac protoxins in an ATP- and chaperone activity-dependent interaction. The chaperone Hsp90 participates in the correct folding of proteins and may suppress mutations of some client proteins, and we show here that PxHsp90 recovered the toxicity of the Cry1AbG439D protoxin affected in receptor binding, in contrast to the Cry1AbR99E or Cry1AbE129K mutant, affected in oligomerization or membrane insertion, respectively, which showed a slight toxicity improvement. Specifically, PxHsp90 enhanced the binding of Cry1AbG439D protoxin to the cadherin receptor. Furthermore, PxHsp90 protected Cry1A protoxins from degradation by insect midgut proteases. Our data show that PxHsp90 assists Cry1A proteins by enhancing their binding to the receptor and by protecting Cry protoxin from gut protease degradation. Finally, we show that the insect cochaperone protein PxHsp70 also increases the toxicity of Cry1Ac in P. xylostella larvae, in contrast to a bacterial GroEL chaperone, which had a marginal effect, indicating that the use of insect chaperones along with Cry toxins could have important biotechnological applications for the improvement of Cry insecticidal activity, resulting in effective control of insect pests. IMPORTANCE Bacillus thuringiensis took advantage of important insect cellular proteins, such as chaperones, involved in maintaining protein homeostasis, to enhance its insecticidal activity. This constitutes a positive loop where the concentrations of Hsp90 and Hsp70 in the gut lumen are likely to increase as midgut cells burst due to Cry1A pore formation action. Hsp90 protects Cry1A protoxin from degradation and enhances receptor binding, resulting in increased toxicity. The effect of insect chaperones on Cry toxicity could have important biotechnological applications to enhance the toxicity of Cry proteins to insect pests, especially those that show low susceptibility to these toxins.

scholarly journals Bacillus thuringiensis DB27 Produces Two Novel Protoxins, Cry21Fa1 and Cry21Ha1, Which Act Synergistically against Nematodes

2014 ◽  
Vol 80 (10) ◽  
pp. 3266-3275 ◽  
Author(s):  
Igor Iatsenko ◽  
Iuliia Boichenko ◽  
Ralf J. Sommer
Keyword(s):  
Bacillus Thuringiensis ◽  
Virulence Factors ◽  
Insect Pests ◽  
Parasitic Nematodes ◽  
Intestinal Damage ◽  
High Similarity ◽  
Free Living ◽  
Cry Proteins ◽  
Content Type ◽  
C Elegans

ABSTRACTBacillus thuringiensishas been widely used as a biopesticide, primarily for the control of insect pests, but someB. thuringiensisstrains specifically target nematodes. However, nematicidal virulence factors ofB. thuringiensisare poorly investigated. Here, we describe virulence factors of nematicidalB. thuringiensisDB27 usingCaenorhabditis elegansas a model. We show thatB. thuringiensisDB27 kills a number of free-living and animal-parasitic nematodes via intestinal damage. Its virulence factors are plasmid-encoded Cry protoxins, since plasmid-cured derivatives do not produce Cry proteins and are not toxic to nematodes. Whole-genome sequencing ofB. thuringiensisDB27 revealed multiple potential nematicidal factors, including several Cry-like proteins encoded by different plasmids. Two of these proteins appear to be novel and show high similarity to Cry21Ba1. Named Cry21Fa1 and Cry21Ha1, they were expressed inEscherichia coliand fed toC. elegans, resulting in intoxication, intestinal damage, and death of nematodes. Interestingly, the effects of the two protoxins onC. elegansare synergistic (synergism factor, 1.8 to 2.5). Using purified proteins, we determined the 50% lethal concentrations (LC50s) for Cry21Fa1 and Cry21Ha1 to be 13.6 μg/ml and 23.9 μg/ml, respectively, which are comparable to the LC50of nematicidal Cry5B. Finally, we found that signaling pathways which protectC. elegansagainst Cry5B toxin are also required for protection against Cry21Fa1. Thus,B. thuringiensisDB27 produces novel nematicidal protoxins Cry21Fa1 and Cry21Ha1 with synergistic action, which highlights the importance of naturally isolated strains as a source of novel toxins.

scholarly journals Improvement of Crystal Solubility and Increasing Toxicity against Caenorhabditis elegans by Asparagine Substitution in Block 3 of Bacillus thuringiensis Crystal Protein Cry5Ba

2012 ◽  
Vol 78 (20) ◽  
pp. 7197-7204 ◽  
Author(s):  
Fenshan Wang ◽  
Yingying Liu ◽  
Fengjuan Zhang ◽  
Lujun Chai ◽  
Lifang Ruan ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Ph Value ◽  
Site Directed Mutagenesis ◽  
Cry Protein ◽  
Content Type ◽  
Mutant Proteins ◽  
Conserved Sequence ◽  
Wide Range ◽  
Ultrasonic Process ◽  
And Function

ABSTRACTThe crystal proteins fromBacillus thuringiensisare widely used for their specific toxicity against insects and nematodes. The highly conserved sequence blocks play an important role in Cry protein stability and flexibility, the basis of toxicity. The block 3 in Cry5Ba subfamily has a shorter sequence (only 12 residues) and more asparagine residues than that of others which harbor about 48 residues but only one asparagine. Based on the theoretical structure model of Cry5Ba, all three asparagines in block 3 are closely located in the interface of putative three domains, implying their probable importance in structure and function. In this study, all three asparagines in Cry5Ba2 block 3 were individually substituted with alanine by site-directed mutagenesis. The wild-type and mutant proteins were overexpressed and crystallized in acrystalliferousB. thuringiensisstrain BMB171. However, the crystals formed in one of the mutants, designated N586A, abnormally disappeared and dissolved into the culture supernatant once the sporulation cells lysed, whereas the Cry5Ba crystal and the other mutant crystals were stable. The mutant N586A crystal, isolated from sporulation cells by the ultrasonic process, was found to be easily dissolved at wide range of pH value (5.0 to 10.0). Moreover, the toxicity assays showed that the mutant N586A exhibited nearly 9-fold-higher activity against nematodes and damaged the host's intestine more efficiently than the native Cry5Ba2. These data support the presumption that the amide residue Asn586 at the interface of domains might adversely affect the protein flexibility, solubility and resultant toxicity of Cry5Ba.

scholarly journals Novel Bacillus thuringiensis δ-Endotoxin Active against Locusta migratoria manilensis

2011 ◽  
Vol 77 (10) ◽  
pp. 3227-3233 ◽  
Author(s):  
Yan Wu ◽  
Cheng-Feng Lei ◽  
Dan Yi ◽  
Peng-Ming Liu ◽  
Mei-Ying Gao
Keyword(s):  
Amino Acid ◽  
Bacillus Thuringiensis ◽  
Locusta Migratoria ◽  
Significant Activity ◽  
Amino Acid Residues ◽  
Cry Protein ◽  
Content Type ◽  
Amino Terminal ◽  
C Terminus ◽  
Locusta Migratoria Manilensis

ABSTRACTA novel δ-endotoxin gene was cloned from aBacillus thuringiensisstrain with activity againstLocusta migratoria manilensisby PCR-based genome walking. The sequence of thecrygene was 3,432 bp long, and it encoded a Cry protein of 1,144 amino acid residues with a molecular mass of 129,196.5 kDa, which exhibited 62% homology with Cry7Ba1 in the amino acid sequence. The δ-endotoxin with five conserved sequence blocks in the amino-terminal region was designated Cry7Ca1 (GenBank accession no.EF486523). Protein structure analysis suggested that the activated toxin of Cry7Ca1 has three domains: 227 residues forming 7 α-helices (domain I); 213 residues forming three antiparallel β-sheets (domain II); and 134 residues forming a β-sandwich (domain III). The three domains, respectively, exhibited 47, 44, and 34% sequence identity with corresponding domains of known Cry toxins. SDS-PAGE and Western blot analysis showed that Cry7Ca1, encoded by the full-length open reading frame of thecrygene, the activated toxin 1, which included three domains but without the N-terminal 54 amino acid residues and the C terminus, and the activated toxin 2, which included three domains and N-terminal 54 amino acid residues but without the C terminus, could be expressed inEscherichia coli. Bioassay results indicated that the expressed proteins of Cry7Ca1 and the activated toxins (toxins 1 and 2) showed significant activity against 2nd instar locusts, and after 7 days of infection, the estimated 50% lethal concentrations (LC50s) were 8.98 μg/ml for the expressed Cry7Ca1, 0.87 μg/ml for the activated toxin 1, and 4.43 μg/ml for the activated toxin 2. The δ-endotoxin also induced histopathological changes in midgut epithelial cells of adultL. migratoria manilensis.

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