scholarly journals Construction of a Conditionally Asporogenous Bacillus thuringiensis Recombinant Strain Overproducing Cry Protein by Deletion of the leuB Gene

2020 ◽  
Vol 11 ◽  
Author(s):  
Meifang Quan ◽  
Jinli Peng ◽  
Zirong Zhu ◽  
Pengji Zhou ◽  
Sisi Luo ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Recombinant Strain ◽  
Cry Protein

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 A Strain of Bacillus thuringiensis Containing a Novel cry7Aa2 Gene that Is Toxic to Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae)

Insects ◽  
2019 ◽  
Vol 10 (9) ◽  
pp. 259 ◽  
Author(s):  
Mikel Domínguez-Arrizabalaga ◽  
Maite Villanueva ◽  
Ana Beatriz Fernandez ◽  
Primitivo Caballero
Keyword(s):  
Amino Acid ◽  
Bacillus Thuringiensis ◽  
Molecular Mass ◽  
Leptinotarsa Decemlineata ◽  
Insect Pests ◽  
Amino Acid Residues ◽  
Cry Protein ◽  
Lc50 Value ◽  
Leptinotarsa Decemlineata Say

The genome of the Bacillus thuringiensis BM311.1 strain was sequenced and assembled in 359 contigs containing a total of 6,390,221 bp. The plasmidic ORF of a putative cry gene from this strain was identified as a potential novel Cry protein of 1138 amino acid residues with a 98% identity compared to Cry7Aa1 and a predicted molecular mass of 129.4 kDa. The primary structure of Cry7Aa2, which had eight conserved blocks and the classical structure of three domains, differed in 28 amino acid residues from that of Cry7Aa1. The cry7Aa2 gene was amplified by PCR and then expressed in the acrystalliferous strain BMB171. SDS-PAGE analysis confirmed the predicted molecular mass for the Cry7Aa2 protein and revealed that after in vitro trypsin incubation, the protein was degraded to a toxin of 62 kDa. However, when treated with digestive fluids from Leptinotarsa decemlineata larvae, one major proteinase-resistant fragment of slightly smaller size was produced. The spore and crystal mixture produced by the wild-type BM311.1 strain against L. decemlineata neonate larvae resulted in a LC50 value of 18.8 μg/mL, which was statistically similar to the estimated LC50 of 20.8 μg/mL for the recombinant BMB17-Cry7Aa2 strain. In addition, when this novel toxin was activated in vitro with commercial trypsin, the LC50 value was reduced 3.8-fold to LC50 = 4.9 μg/mL. The potential advantages of Cry7Aa2 protoxin compared to Cry7Aa1 protoxin when used in the control of insect pests are discussed.

Insecticidal Activity of a Cry1Ca toxin of Bacillus thuringiensis Berliner (Firmicutes: Bacillaceae) and Its Synergism with the Cyt1Aa Toxin Against Aedes aegypti (Diptera: Culicidae)

2020 ◽  
Vol 57 (6) ◽  
pp. 1852-1856
Author(s):  
Sebastian E González-Villarreal ◽  
Mónica García-Montelongo ◽  
Jorge E Ibarra
Keyword(s):  
Bacillus Thuringiensis ◽  
Aedes Aegypti ◽  
Synergistic Effect ◽  
Joint Action ◽  
Recombinant Strain ◽  
Crystal Morphology ◽  
Graphical Method ◽  
Toxic Activity ◽  
Lepidopteran Species ◽  
Gene Coding

Abstract The Cry1C protein family of Bacillus thuringiensis form bipyramidal crystals, which are commonly associated with toxic activity against lepidopteran species; however, some members of this family may also be toxic to dipterans. In the present work, the Cry1Ca16 protein, synthesized by the B. thuringiensis LBIT-1217 strain, was analyzed. The gene coding for this protein was amplified, sequenced, and cloned into the pSTAB vector, which was electro-transferred into the acrystalliferous B. thuringiensis 4Q7 strain. The recombinant strain showed the expected bipyramidal crystal morphology, identical to the original LBIT-1217 strain and exhibited toxicity against larvae of Aedes aegypti (Diptera). Pure crystals from the recombinant strain were used in bioassays against Ae. aegypti larvae, estimating an LC50 of 4.61 μg/ml. Further studies on Cry1Ca16 mosquitocidal potential included joint-action tests with the Cyt1Aa protein crystals from B. thuringiensis israelensis. An LC50 using pure Cyt1Aa crystals was estimated at 0.73 μg/ml, whereas an LC50 of 0.61 μg/ml was estimated when both toxins were tested together. Data from these bioassays was analyzed using joint-action tests such as the Tammes-Bakuniak graphical method and the formula proposed by Tabashnik (1992). Both tests clearly showed a synergistic effect between these two toxins.

Exploration of insecticidal potential of Cry protein purified from Bacillus thuringiensis VIID1

2021 ◽  
Vol 174 ◽  
pp. 362-369
Author(s):  
Drishtant Singh ◽  
Samiksha ◽  
Seema Madhumal Thayil ◽  
Satwinder Kaur Sohal ◽  
Anup Kumar Kesavan
Keyword(s):  
Bacillus Thuringiensis ◽  
Cry Protein

Expression of a Bacillus thuringiensis crystal protein gene in Pseudomonas cepacia 526

1990 ◽  
Vol 36 (12) ◽  
pp. 879-884 ◽  
Author(s):  
Carolyn A. Stock ◽  
Thomas J. McLoughlin ◽  
Janet A. Klein ◽  
Michael J. Adang
Keyword(s):  
Bacillus Thuringiensis ◽  
Manduca Sexta ◽  
Protein Gene ◽  
Crystal Protein ◽  
Pseudomonas Cepacia ◽  
Broad Host Range ◽  
Cry Protein ◽  
Insecticidal Crystal Protein ◽  
Tobacco Plants ◽  
Range Vector

An insecticidal crystal protein gene, cryIA(C), from Bacillus thuringiensis HD-1 was cloned into a broad host range vector, pSUP204, and the resulting plasmid, pSUP89A, was conjugated into a plant-colonizing bacterium, Pseudomonas cepacia 526. Southern blot analysis detected the presence of the cry gene in Pseudomonas cepacia 526 transconjugants. Production of a truncated insecticidal crystal protein in Pseudomonas cepacia was detected by Western blotting and ELISA techniques and was found to have insecticidal activity against the tobacco hornworm, Manduca sexta, in an artificial diet assay. We also followed the establishment of the Pseudomonas cepacia 526/Bt transconjugant on axenically grown tobacco plants and found that approximately 1% of the Pseudomonas cells retained pSUP89A 3 days after application. In spite of this instability, treated tobacco plants were protected from Manduca sexta infestation. Key words: Bacillus thuringiensis, Pseudomonas cepacia, insecticidal crystal protein, Cry protein.

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