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Bacillus thuringiensis var. israelensis δ-endotoxin. Cloning and expression of the toxin in sporogenic and asporogenic strains of Bacillus subtilis

Bacillus thuringiensis var. israelensis δ-endotoxin. Cloning and expression of the toxin in sporogenic and asporogenic strains of Bacillus subtilis
Bacillus thuringiensis var. israelensis δ-endotoxin. Cloning and expression of the toxin in sporogenic and asporogenic strains of Bacillus subtilis

A plasmid-borne gene from Bacillus thuringiensis var. israelensis encoding a 27,340 Mr insecticidal δ-endotoxin has been cloned on a bifunctional multicopy plasmid in a wild-type sporogenic strain and two asporogenic mutants of Bacillus subtilis. The δ-endotoxin gene is expressed at a low level during vegetative growth in all three strains, but the synthesis of the toxin increases markedly during the third hour of stationary phase for both the sporogenic strain and an asporogenic mutant containing the OJ lesion. However, in a stage OA mutant, this increase in δ-endotoxin synthesis is not observed. In both the wild-type sporogenic B. subtilis and the asporogenic OJ strain, phase-bright inclusions, resembling the israelensis crystal in appearance, are visible during late stationary phase. The insoluble inclusions from the B. subtilis transformants, consisting solely of the 27,340 Mr polypeptide, were purified by density gradient centrifugation and found to be extremely toxic to Aedes aegypti larvae. After solubilization in alkaline buffer, this polypeptide was also shown to be haemolytic for human erythrocytes and to lyse Aedes albopictus cells with the same LC50 value as native israelensis δ-endotoxin crystals. During stationary phase, novel mRNA species appear in both the wild-type strain and the OJ mutant, but not in the OA mutant, and these appear to be the major gene-specific transcripts. Transcriptional mapping of δ-endotoxin-specific mRNA has shown that the same region of initiation is used at a relatively low level in all three strains during vegetative growth.

0022-2836
13-22
Ward, E. S.
b31c0877-8abe-485f-b800-244a9d3cd6cc
Ridley, A. R.
929870b2-1303-4464-87fc-8ea750169d86
Ellar, D. J.
92e52b64-6600-401e-a3d0-b15cfff77199
Todd, J. A.
d56a9d14-2502-480b-913c-5a4731b9a074
Ward, E. S.
b31c0877-8abe-485f-b800-244a9d3cd6cc
Ridley, A. R.
929870b2-1303-4464-87fc-8ea750169d86
Ellar, D. J.
92e52b64-6600-401e-a3d0-b15cfff77199
Todd, J. A.
d56a9d14-2502-480b-913c-5a4731b9a074

Ward, E. S., Ridley, A. R., Ellar, D. J. and Todd, J. A. (1986) Bacillus thuringiensis var. israelensis δ-endotoxin. Cloning and expression of the toxin in sporogenic and asporogenic strains of Bacillus subtilis. Journal of Molecular Biology, 191 (1), 13-22. (doi:10.1016/0022-2836(86)90418-3).

Record type: Article

Abstract

A plasmid-borne gene from Bacillus thuringiensis var. israelensis encoding a 27,340 Mr insecticidal δ-endotoxin has been cloned on a bifunctional multicopy plasmid in a wild-type sporogenic strain and two asporogenic mutants of Bacillus subtilis. The δ-endotoxin gene is expressed at a low level during vegetative growth in all three strains, but the synthesis of the toxin increases markedly during the third hour of stationary phase for both the sporogenic strain and an asporogenic mutant containing the OJ lesion. However, in a stage OA mutant, this increase in δ-endotoxin synthesis is not observed. In both the wild-type sporogenic B. subtilis and the asporogenic OJ strain, phase-bright inclusions, resembling the israelensis crystal in appearance, are visible during late stationary phase. The insoluble inclusions from the B. subtilis transformants, consisting solely of the 27,340 Mr polypeptide, were purified by density gradient centrifugation and found to be extremely toxic to Aedes aegypti larvae. After solubilization in alkaline buffer, this polypeptide was also shown to be haemolytic for human erythrocytes and to lyse Aedes albopictus cells with the same LC50 value as native israelensis δ-endotoxin crystals. During stationary phase, novel mRNA species appear in both the wild-type strain and the OJ mutant, but not in the OA mutant, and these appear to be the major gene-specific transcripts. Transcriptional mapping of δ-endotoxin-specific mRNA has shown that the same region of initiation is used at a relatively low level in all three strains during vegetative growth.

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Published date: 5 September 1986

Identifiers

Local EPrints ID: 425246
URI: https://eprints.soton.ac.uk/id/eprint/425246
ISSN: 0022-2836
PURE UUID: cb7f9b31-97d9-459a-acc2-ae9bb8016944
ORCID for E. S. Ward: ORCID iD orcid.org/0000-0003-3232-7238

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Date deposited: 12 Oct 2018 16:30
Last modified: 14 Mar 2019 01:21

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Contributors

Author: E. S. Ward ORCID iD
Author: A. R. Ridley
Author: D. J. Ellar
Author: J. A. Todd

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