Characterized full-length and truncated plasmid clones of the crystal protein of Bacillus thuringiensis subsp. kurstaki HD-73 and their toxicity to Manduca sexta

Adang, Michael J.; Staver, Michael J.; Rocheleau, Thomas A.; Leighton, Jacqui; Barker, Richard F.; Thompson, David V.

doi:10.1016/0378-1119(85)90184-2

Cited by 247 publications

(99 citation statements)

References 28 publications

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“…The amino-terminal domain is essential for toxicity, and roughly corresponds to the protease-activated toxin, whereas the carboxyterminal domain is not essential for toxicity (Adang et al, 1985;Hofte e t al., 1986;Wabiko e t al., 1986). The toxic domain is further subdivided into unique and conserved sequences.…”

Section: Introductionmentioning

confidence: 99%

Bacillus thuringiensis protoxin: location of toxic border and requirement of non-toxic domain for high-level in vivo production of active toxin

Wabiko¹,

Yasuda²

1995

Microbiology

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Insecticidal crystal proteins, or protoxins, of Bacillus thuringiensis are composed of two domains, an amino-terminal half essential for toxicity, and a carboxy-terminal half with an as yet unassigned function. To define the boundary of the two domains, sequential termination codons were introduced from the 3'-end of the DNA sequence encoding the toxic domain of the 1155-residue crylA(b) gene product. The mutated and the intact genes were placed under the control of the Escherichia coli inducible promoter PrecA, and toxicity of the cell extracts was determined using silkworm larvae. Under non-induced conditions, in which the gene products accumulated to a limited degree, mutations encoding 606 amino acid residues or more were toxic, whereas those encoding 605 residues or less were non-toxic. Comparison of the toxicities and the levels of the toxic proteins suggested that the mutant proteins had comparable activity to that of the intact protoxin. Furthermore, the non-toxic protein seemed to be unstable in the extracts. T o investigate the roles of the non-toxic domain, the mutant proteins were overproduced in both E. coli and B. thuringiensis. The intact and the mutated genes carrying natural promoters were introduced into acrystalliferous B. thuringiensis. Upon induction of PrecA in Em coli, and upon sporulation in B. thuringiensis, there was a large accumulation of gene products which formed inclusion bodies. The inclusion bodies of the intact protoxin were active, whereas those of the mutant proteins were inactive. Inclusion bodies of the intact protein could be solubilized in alkali, whereas the mutant inclusion bodies were insoluble. Since solubilization under alkaline conditions in the insect midgut is considered to be the first step of toxic action, the non-toxic domain is required to direct the synthesis of inclusion bodies as an active soluble form.

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Section: Introductionmentioning

confidence: 99%

Bacillus thuringiensis protoxin: location of toxic border and requirement of non-toxic domain for high-level in vivo production of active toxin

Wabiko¹,

Yasuda²

1995

Microbiology

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“…Particular attention was focused on the 5'-terminal region of the cryIF gene, since this region has been shown to encode the active toxin moiety of other Cryl ICPs (2,19,35).…”

Section: Resultsmentioning

confidence: 99%

Isolation and characterization of a novel insecticidal crystal protein gene from Bacillus thuringiensis subsp. aizawai

et al. 1991

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Bacillus thuringiensis subsp. aizawai EG6346, a novel grain dust isolate, was analyzed by Southern blot hybridization for its insecticidal crystal protein (ICP) gene profile. Strain EG6346 lacks previously characterized cryIA ICP genes yet does possess novel cryI-related gene sequences. A recombinant genomic plasmid library was constructed for strain EG6346 in Escherichia coli. One recombinant plasmid, pEG640, isolated from the library contained a novel ICP gene on a 5.7-kb Sau3A insert. The sequence of this gene, designated cryIF, was related to, but distinct from, the published sequences for other cryI genes. A second novel cryI-related sequence was also located on pEG640, approximately 500 bp downstream from cryIF. Introduction of cryIF into a Cry- B. thuringiensis recipient strain via electroporation enabled sufficient production of CryIF protein for quantitative bioassay analyses of insecticidal specificity. The CryIF crystal protein was selectively toxic to a subset of lepidopteran insects tested, including the larvae of Ostrinia nubilalis and Spodoptera exigua.

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“…Similarly, the Spel -Hind111 DNA fragment coding for the N-terminal portion of the CryIA(c) toxin from B. thuringiensis var. kurstaki HD73 [32] was cloned between the equivalent SpeI and the second Hind111 site, found in the CryIA(b) coding sequence of pX193. The resulting plasmid was called pX195 and encodes the cryIA (c) gene.…”

Section: Bucterial Strains and Plusmid D N Amentioning

confidence: 99%

Identification and characterization of Heliothis virescens midgut membrane proteins binding Bacillus thuringiensisδ‐endotoxins

Oddou

Hartmann

Geiser

1991

European Journal of Biochemistry

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To investigate the specificity of Bacillus thuringiensis var. kurstaki strain HD1 insecticidal crystal proteins (ICP), we used membrane preparations obtained from the midgut of Heliothis virescens larvae to perform separate ligand-blot experiments with the three activated CryIA toxins. The CrylA(a) and the CrylA(b) toxins bind the same 170-kDa protein, but most likely at two different binding sites. The CryIA(c) toxin binds two proteins of molecular masses 140 kDa and 120 kDa. We also demonstrate that the binding proteins for each of the B. thuringiensis toxins are not part of a covalent complex. Although the 170-kDa protein is a glycoprotein, endoglycosidase treatment does not prevent the binding of the CryIA

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Characterized full-length and truncated plasmid clones of the crystal protein of Bacillus thuringiensis subsp. kurstaki HD-73 and their toxicity to Manduca sexta

Cited by 247 publications

References 28 publications

Bacillus thuringiensis protoxin: location of toxic border and requirement of non-toxic domain for high-level in vivo production of active toxin

Bacillus thuringiensis protoxin: location of toxic border and requirement of non-toxic domain for high-level in vivo production of active toxin

Isolation and characterization of a novel insecticidal crystal protein gene from Bacillus thuringiensis subsp. aizawai

Identification and characterization of Heliothis virescens midgut membrane proteins binding Bacillus thuringiensisδ‐endotoxins

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