Comparative analysis of the amino acid sequence of the Cry protein of Bacillus thuringiensis codify the toxic protein to insects of the orders Lepidoptera, Diptera and Lepidoptera/ Diptera

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.

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

Insects ◽

10.3390/insects10090259 ◽

2019 ◽

Vol 10 (9) ◽

pp. 259 ◽

Cited By ~ 4

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.

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Nucleotide sequence and deduced amino acid sequence of a coleopteran-active delta-endotoxin gene from Bacillus thuringiensis subsp. san diego

Gene ◽

10.1016/0378-1119(87)90174-0 ◽

1987 ◽

Vol 57 (1) ◽

pp. 37-46 ◽

Cited By ~ 36

Author(s):

Corinna Herrnstadt ◽

Thomas E. Gilroy ◽

Donna A. Sobieski ◽

Brian D. Bennett ◽

Frank H. Gaertner

Keyword(s):

Amino Acid ◽

Bacillus Thuringiensis ◽

Nucleotide Sequence ◽

Amino Acid Sequence ◽

San Diego ◽

Bacillus Thuringiensis Subsp ◽

Delta Endotoxin

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Amino acid sequence and entomocidal activity of the P2 crystal protein. An insect toxin from Bacillus thuringiensis var. kurstaki.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)57428-2 ◽

1988 ◽

Vol 263 (1) ◽

pp. 561-567

Author(s):

W P Donovan ◽

C C Dankocsik ◽

M P Gilbert ◽

M C Gawron-Burke ◽

R G Groat ◽

...

Keyword(s):

Amino Acid ◽

Bacillus Thuringiensis ◽

Amino Acid Sequence ◽

Crystal Protein ◽

Insect Toxin

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

10.20944/preprints201905.0237.v1 ◽

2019 ◽

Author(s):

Mikel Dominguez- ◽

Maite Villanueva ◽

Ana Beatriz Fernandez ◽

Primitivo Caballero

Keyword(s):

Amino Acid ◽

Bacillus Thuringiensis ◽

Molecular Mass ◽

Leptinotarsa Decemlineata ◽

Insect Pests ◽

Amino Acid Residues ◽

Cry Protein ◽

Sds Page ◽

Neonate Larvae

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 respect to Cry7Aa1 protein and a predicted molecular mass of 129.4 kDa. The primary structure of this Cry7Aa2 protein, which revealed the presence of 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, it was degraded to a toxin of 62 kDa. However, when treated with digestive fluids from Leptinotarsa decemlineata larvae two proteinase-resistant fragments of 60 and 65 kDa were produced. Spore and crystal mixture produced by the wild-type BM311.1 strain against L. decemlineata neonate larvae resulted in a LC50 (18.8 μg/ml), which was statistically equal to the estimated LC50 (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 4 times approximately (LC50 = 4.9 μg/mL). The advantages of Cry7Aa2 protoxin compared to Cry7Aa1 protoxin when used in the control of insect pests are discussed.

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Proteolytic processing of a coleopteran-specific δ-endotoxin produced by Bacillus thuringiensis var. tenebrionis

Biochemical Journal ◽

10.1042/bj2610099 ◽

1989 ◽

Vol 261 (1) ◽

pp. 99-105 ◽

Cited By ~ 61

Author(s):

J Carroll ◽

J Li ◽

D J Ellar

Keyword(s):

Amino Acid ◽

Bacillus Thuringiensis ◽

Amino Acid Sequence ◽

Proteinase Inhibitors ◽

Cleavage Product ◽

Proteolytic Processing ◽

N Terminus ◽

Delta Endotoxin ◽

Insect Gut

Insecticidal protein delta-endotoxin crystals harvested from sporulated cultures of Bacillus thuringiensis var. tenebrionis contain a major polypeptide of 67 kDa and minor polypeptides of 73, 72, 55 and 46 kDa. During sporulation, only the 73 kDa polypeptide could be detected at stage I. The 67 kDa polypeptide was first detected at stage II and increased in concentration throughout the later stages of sporulation and after crystal release, with a concomitant decrease in the 73 kDa polypeptide. This change could be blocked by the addition of proteinase inhibitors. Trypsin or insect-gut-extract treatment of the delta-endotoxin crystals after solubilization resulted in a cleavage product of 55 kDa with asparagine-159 of the deduced amino acid sequence of the toxin [Höfte, Seurinck, van Houtven & Vaeck (1987) Nucleic Acids Res. 15, 71-83; Sekar, Thompson, Maroney, Bookland & Adang (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 7036-7040; McPherson, Perlak, Fuchs, Marrone, Lavrik & Fischhoff (1988) Biotechnology 6, 61-66] at the N-terminus. This polypeptide was found to be as toxic in vivo as native delta-endotoxin.

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