Separation of the cytolytic and mosquitocidal proteins of Bacillusthuringiensis subsp. israelensis

Hurley, J. Michael; Lee, Sung G.; Andrews, Robert E.; Klowden, Marc J.; Bulla, Lee A.

doi:10.1016/0006-291x(85)90279-7

Cited by 79 publications

(27 citation statements)

References 10 publications

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“…The latter investigators reported that mosquitocidal activity is associated with peptides of 130 and 230 kDa (31). Others (14,19) have attributed the mosquitocidal activity to a ca. 66-kDa peptide and the hemolytic activity to the 27-kDa peptide.…”

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Expression in Escherichia coli of a cloned crystal protein gene of Bacillus thuringiensis subsp. israelensis

McLEAN¹,

Whiteley²

1987

J Bacteriol

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A ca. 10-kilobase (kb) HindHII fragment of plasmid DNA from Bacillus thuringiensis subsp. israelensis was cloned into plasmid pUC9 and transformed into Escherichia coli. Extracts of the recombinant strain contained a 27-kilodalton (kDa) peptide that reacted with antibodies to a 27-kDa peptide isolated from crystals produced by B. thuringiensis subsp. israelensis. Extracts of the recombinant strain were hemolytic and toxic to Aedes aegypti larvae. Full expression of the 27-kDa peptide required the presence of a ca. 0.8-kb region of DNA located 4 kb upstream from the structural gene; the 0.8-kb region could be present in cis or trans relative to the gene and apparently acted post-transcriptionally. Analysis of maxicelis showed that the 10-kb insert also coded for peptides of 67, 20, and 16 kDa; data obtained with different subclones suggest that the 20-kDa peptide is encoded in the 0.8-kb DNA region.During sporulation, Bacillus thuringiensis subsp. israelensis (hereafter referred to as Bti) produces irregular, multicomponent parasporal crystals (6,15,29). The crystals are lethal to larvae of mosquitoes and black flies (11); solubilized preparations of crystals lyse erythrocytes and cultured mosquito cells (26). Electrophoretic analyses of solubilized, denatured crystals show several peptides ranging in size from 27 to 130 kilodaltons (kDa) (29). The relationship of these peptides to each other is not known, and there are conflicting reports concerning the identity of the toxic and cytolytic peptides. A number of investigators (1,9,15,16,35,38) have presented evidence that toxicity alone or that toxicity and hemolytic and cytolytic activities are associated with a 27-kDa peptide or its 25-to 26-kDa derivative. A gene coding for a peptide with a deduced molecular mass of 27,340 daltons has been cloned in Escherichia coli (32, 35) and sequenced (32, 34). reported that extracts of recombinant E. coli strains containing the 27-kDa gene are toxic and hemolytic, whereas Waalwijk et al. (32) found that extracts of recombinant strains are hemolytic but not mosquitocidal. The latter investigators reported that mosquitocidal activity is associated with peptides of 130 and 230 kDa (31). Others (14,19) have attributed the mosquitocidal activity to a ca. 66-kDa peptide and the hemolytic activity to the 27-kDa peptide. Walfield et al. (33) and Thorne et al. (27) have cloned and sequenced a gene coding for a 72-kDa peptide and have reported that the apparent processed product of this gene, a peptide of 58 kDa, is highly toxic but not hemolytic. Yet other investigators (7) propose that toxicity resides in a 31-to 35-kDa peptide or that toxicity involves the synergistic action of two peptides (15, 37).The present communication reports studies made with a recombinant E. coli strain containing the genes coding for the 27-kDa crystal protein peptide of Bti. Extracts of the recombinant strain are toxic to mosquito larvae and are hemolytic in vitro. We also report the unexpected observation that the presence of the 27-kDa peptide, bu...

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confidence: 98%

Expression in Escherichia coli of a cloned crystal protein gene of Bacillus thuringiensis subsp. israelensis

McLEAN¹,

Whiteley²

1987

J Bacteriol

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“…Several studies have provided strong evidence that the 28-kDa protein is responsible, after cleavage to a 25-kDa protein, for the hemolytic and cytolytic activities of the parasporal body, as well as for its toxicity for insect larvae in vivo (2,5,19,20,25). However, Hurley et al (9) and Lee et al. (15) recently reported that the 28-kDa protein accounts only for the cytolytic and hemolytic activities and that the 65-kDa protein is the actual mosquitocidal toxin.…”

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Isolation of a relatively nontoxic 65-kilodalton protein inclusion from the parasporal body of Bacillus thuringiensis subsp. israelensis

Ibarra¹,

Federici²

1986

J Bacteriol

153

108

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Ultrastructural studies of the mosquitocidal bacterium Bacillus thuringiensis subsp. israelensis revealed that the parasporal body contained three major inclusion types, designated types 1, 2, and 3, which could be differentiated on the basis of electron opacity and size and, to some extent, shape. The type-2 inclusion, which was of moderate electron density and often appeared as a bar-shaped polyhedral body, was isolated on NaBr gradients from purified parasporal bodies and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transmission electron microscopy, and bioassays agaikst neonate larvae of Aedes aegypti. Purified inclusions averaged 150 to 200 nm by 500 to 700 nm in transverse sections and consisted almost exclusively of a 65-kilodalton (kDa) protein contaminated with minor quantities of 38-and 28-kDa proteins.Lethal concentration values at the 50% level for preparations of the purified parasporal body and the type-2 inclusion were, respectively, 0.66 and 43 ng/ml, indicating that the 65-kDa protein is only slightly toxic to mosquitoes in comparison to the intact parasporal body. Analysis of the type-2 polyhedral inclusion by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and bioassays during different stages of purification demonstrated a positive correlation between the toxicity of the preparation and the degree of contamination with the 28-kDa protein. These results indicate that the 65-kDa protein is not the primary larvicidal toxin, although it may act in conjunction with other parasporal body proteins to produce the high mosquitocidal toxicity characteristic of this bacterium.

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“…For the last decade, naturally occurring mosquito-specific strains have been found occasionally in B. thuringiensis H serotypes 14 (1, 2),1 la: 1 lc (13, 14), 10 (15), and 8a: 8b (16). Several investigators have purified the 65-to 67-kD proteins from these strains as the mosquito larvicidal principles (5, 6,8,18,20). However, little is known about the serological relationship between purified toxic proteins except that Kim et al (8), using rabbit polyclonal antibodies, found no serological relationship between purified 67-kD mosquitocidal proteins from the strain 73-E-10-2 (H serotype 10) and the type strain of B. thuringiensis subsp.…”

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confidence: 99%

Monoclonal antibodies against the 65-kilodalton mosquitocidal protein of the Bacillus thuringiensis strain PG-14 (serotype 8A:8B).

Ohba

Aizawa

1989

J. Gen. Appl. Microbiol.

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Eight hybridoma cell clones, obtained by fusing murine myeloma cells (P3-NS 1 / 1-Ag-1) with the spleen cells of immunized BALB/c mice, secreted monoclonal antibodies against the 65-kD mosquitocidal protein of the Bacillus thuringiensis strain PG-14 (flagellar serotype 8a: 8b) . Of these monoclonal antibodies, six belonged to the IgM class and two belonged to the IgG 1 class. In binding tests, they were all specific for the 65-kD protein and did not react to the 25-kD hemolytic protein produced by the same bacterial strain. Indirect competitive enzyme-linked immunosorbent assays with two monoclonal antibodies (IgM), designated D8C 1 and D8B4, had a sensitivity of 1 ng/ml for the 65-kD protein. There was no competition by the 25-kD protein.Bacillus thuringiensis strain PG-14 [flagellar (H) serotype 8a: 8b] exhibits a highly preferential mosquitocidal activity similar to that of the type strain of B. thuringiensis subsp. israelensis (H serotype 14) (7,10,16). In preceding studies, we successfully purified the 25-and 65-kD proteins from the parasporal inclusion proteins of the strain PG-14 digested by silkworm (Bombyx mori) gut juice (18)(19)(20). Of these proteins, only the 65-kD protein was highly toxic to mosquito larvae. To analyze the highly mosquito-specific property of this protein and its relationship to the toxic proteins of the other mosquito-specific B. thuringiensis strains, it appeared that the application of monoclonal antibody-producing hybridomas (9) would be useful. In this study, we produced monoclonal antibodies against the 65-kD mosquitocidal protein of the strain PG-14 and developed the indirect competitive enzyme-linked immunosorbent assay (ELISA) to detect the 65-kD protein. We also conducted a comparative serology of the 65-and 25-kD proteins with monoclonal antibodies.

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Separation of the cytolytic and mosquitocidal proteins of Bacillusthuringiensis subsp. israelensis

Cited by 79 publications

References 10 publications

Expression in Escherichia coli of a cloned crystal protein gene of Bacillus thuringiensis subsp. israelensis

Expression in Escherichia coli of a cloned crystal protein gene of Bacillus thuringiensis subsp. israelensis

Isolation of a relatively nontoxic 65-kilodalton protein inclusion from the parasporal body of Bacillus thuringiensis subsp. israelensis

Monoclonal antibodies against the 65-kilodalton mosquitocidal protein of the Bacillus thuringiensis strain PG-14 (serotype 8A:8B).

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