Bioassay of Solubilized
            <i>Bacillus thuringiensis</i>
            var.
            <i>Israelensis</i>
            Crystals by Attachment to Latex Beads

Schnell, Danny J.; Pfannenstiel, Mary Ann; Nickerson, Kenneth W.

doi:10.1126/science.6701520

Cited by 56 publications

(30 citation statements)

References 16 publications

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“…Although we also observed mortality of C. quinquefascistus larvae challanged with recombinant Cyt1Ab1 crystal inclusions (LC 50 1.3 µg/ml), no toxicity was observed when soluble and protease treated Cyt1Ab1 protein was used. Similar results were found by Schnell et al (1984) where solubilized crystal proteins of B. thuringiensis subsp. israelensis were 7,000 times less active than crystals.…”

Section: Discussionsupporting

confidence: 87%

Proteolytic processing of the Cyt1Ab1 toxin produced by Bacillus thuringiensis subsp. medellin

Escobar

Segura

Vanegas³

et al. 2000

Mem. Inst. Oswaldo Cruz

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Bacillus thuringiensis produces δ-endotoxins that require proteolytic processing to become active. The activation of the B. thuringiensis subsp. medellin 28 kDa (Cyt1Ab1) cytolytic toxin by trypsin, chymotrypsin and gut extract from Culex quinquefasciatus larvae was analyzed. The Cyt1Ab1 toxin of B. thuringiensis subsp. medellin was processed by all proteases tested to fragments between 23 and 25 kDa, while processing of the Cyt1Aa1 toxin produce fragments between 22.5 and 24.5 kDa. The Cyt1Ab1 toxin was preferentially processed at the alkaline pH of 12. The in vitro proteolytic processing of the Cyt1Ab1 toxin by C. quinquefasciatus larvae midgut extract showed a 25 kDa fragment; a similar result was observed when the activation was performed in the in vivo experiments. The solubilized Cyt1Ab1 toxin and the protease resistant cores generated by in vitro processing showed hemolytic activity but not mosquitocidal activity. Amino terminal sequence of the C. quinquefasciatus gut extract resistant fragment indicated that the cutting site was located between Lys 31 and Asp 32 , with a sequence DDPNEKNNHNS; while for the trypsin-resistant fragment the cutting site was determined between Leu 29 and Arg 30 , and for the chymotrypsin-resistant fragment between Arg 30 and Lys 31 .

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Section: Discussionsupporting

confidence: 87%

Proteolytic processing of the Cyt1Ab1 toxin produced by Bacillus thuringiensis subsp. medellin

Escobar

Segura

Vanegas³

et al. 2000

Mem. Inst. Oswaldo Cruz

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“…Our 3.5-kb IlindIII subclone in pGEM-3-blue was expressed in the exponential as well as the stationary phase of growth (Table 1; results of bioassays and Western immunoblots not shown). Ultrasonic disruption of E. coli cells containing the toxin greatly reduced toxicity, as would be expected from the fact that mosquito larvae ingest particulate matter better than soluble material (Table 1) (25). The converse was true for the clone of Ganesan et al (13); cells disrupted in the presence of Triton X-100 exhibited greatly enhanced toxicity.…”

mentioning

confidence: 82%

Cloning of the gene for the larvicidal toxin of Bacillus sphaericus 2362: evidence for a family of related sequences

Baumann¹,

Baumann²,

Bowditch³

et al. 1987

J Bacteriol

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During sporulation, Bacillus sphaericus 2362 produces a parasporal crystalline protein which is toxic for the larvae of a number of mosquito species. Using the Eschertichia coli cloning vector Agtll, in which gene products of the inserts may be fused to I8-galactosidase, we isolated 29 bacteriophages which produced peptides reacting with antiserum to crystal protein. On the basis of restriction enzyme analyses of the recombinants and Ouchterlony immunodiffusion experiments with induced lysogens as a source of antigens, the recombinants were assigned to three groups, designated A, B, and C. Group A consisted of three clones which appeared to express all or part of the B. sphaericus toxin gene from their own promoters and one clone producing a ,-galactosidase-toxin fusion protein. The host cells of two induced recombinant lysogens of this group were toxic to larvae of Culex pipiens. A cell suspension containing 174 ng (dry weight) of the more toxic recombinant per ml killed 50% of the larvae. Both recombinants formed peptides with molecular sizes of 27, 43, and 63 kilodaltons (kDa). The antigenically related 27-and 43-kDa peptides were distinct from the 63-kDa peptide, which resembled crystals from sporulating cells of B. sphaericus in which antigenically distinct 43-and 63-kDa proteins are derived from a 125-kDa precursor. A 3.5-kilobase HindIII fragment from recombinants having toxic activity against larvae was subcloned into pGEM-3-blue. E. coli cells harboring this fragment were toxic to mosquito larvae and produced peptides of 27, 43, and 63 kDa. The distribution of the A gene among strains of B. sphaericus of different toxicities suggested that it is the sole or principal gene encoding the larvicidalcrystal protein. The two recombinants of group B and the 23 of group C were all ,-galactosidase fusion proteins, suggesting that in E. coli these genes were not readily expressed from their own promoters. The distribution of these two genes in different strains of B. sphaericus suggested that they do not have a role in the toxicity of this species to mosquito larvae.

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“…The bioassay of Cry11A protein was done essentially by the methods of Schnnel et al 25) The proteins were added to 1 ml of 0.1 M Tris (pH7.5), 0.1z latex beads (Sigma) of 0.8 mm in diameter, to give 0.1 mg W ml of aˆnal protein concentration. After a brief vortexing, the samples were rotated for 1 h at room temperature.…”

Section: Methodsmentioning

confidence: 99%

“…5). Because both the solubilized and the digested toxin were adsorbed to the latex beads for the bioassay against C. pipiens larvae, 25) the insecticidal activity of these toxins was lower than that of the Cry11A crystal. Especially, the toxicity was almost undetectable at the toxin concentrations lower than 0.01 mg W ml.…”

Section: Association Of the Two Processed Fragments Of Cry11amentioning

confidence: 99%

Active Form of Dipteran-Specific Insecticidal Protein Cry11A Produced byBacillus thuringiensissubsp.israelensis

Yamagiwa

Ogawa

Yasuda

et al. 2002

Bioscience, Biotechnology, and Biochemistry

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Bioassay of Solubilized Bacillus thuringiensis var. Israelensis Crystals by Attachment to Latex Beads

Cited by 56 publications

References 16 publications

Proteolytic processing of the Cyt1Ab1 toxin produced by Bacillus thuringiensis subsp. medellin

Proteolytic processing of the Cyt1Ab1 toxin produced by Bacillus thuringiensis subsp. medellin

Cloning of the gene for the larvicidal toxin of Bacillus sphaericus 2362: evidence for a family of related sequences

Active Form of Dipteran-Specific Insecticidal Protein Cry11A Produced byBacillus thuringiensissubsp.israelensis

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