Abstract
The mosquitocidal crystal of Bacillus thuringiensis subsp. darmstadiensis 73-E10-2 was purified, bioassayed against third-instar Aedes aegypti larvae (50% lethal concentration, 7.5 micrograms/ml), and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, revealing polypeptides of 125, 50, 47, and 28 kilodaltons (kDa). When solubilized and proteolytically activated by insect gut proteases or proteinase K, the crystal was cytotoxic to insect and mammalian cells in vitro and was hemolytic. By using nondenaturing polyacrylamide gel electrophoresis, a polypeptide of 23 kDa, derived from the 28-kDa protoxin, was identified which was hemolytic and cytotoxic to Aedes albopictus, A. aegypti, and Choristoneura fumiferana CF1 insect cell lines. The 23-kDa polypeptide was purified by ion-exchange chromatography and gave 50% lethal dose values of 3.8, 3.3, and 6.9 micrograms/ml against A. albopictus, A. aegypti, and C. fumiferana CF1 cells lines, respectively. Cytotoxicity in vitro was both dose and temperature dependent, with a sigmoidal dose-response curve. The cytotoxicity of the 23-kDa toxin and the solubilized and proteolytically activated delta-endotoxin was inhibited by a range of phospholipids containing unsaturated fatty acids and by triglyceride and diglyceride dispersions. An interaction with membrane phospholipids appears important for toxicity. Polyclonal antisera prepared against the 23-kDa polypeptide did not cross-react with polypeptides in the native crystals of four other mosquitocidal strains.
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- Ansorge W. Fast and sensitive detection of protein and DNA bands by treatment with potassium permanganate. J Biochem Biophys Methods. 1985 May;11(1):13–20. doi: 10.1016/0165-022x(85)90037-5. [DOI] [PubMed] [Google Scholar]
- Arbuthnott J. P., Freer J. H., Billcliffe B. Lipid-induced polymerization of staphylococcal -toxin. J Gen Microbiol. 1973 Apr;75(2):309–319. doi: 10.1099/00221287-75-2-309. [DOI] [PubMed] [Google Scholar]
- Armstrong J. L., Rohrmann G. F., Beaudreau G. S. Delta endotoxin of Bacillus thuringiensis subsp. israelensis. J Bacteriol. 1985 Jan;161(1):39–46. doi: 10.1128/jb.161.1.39-46.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
- FOLCH J., LEES M., SLOANE STANLEY G. H. A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem. 1957 May;226(1):497–509. [PubMed] [Google Scholar]
- Faust R. M., Travers R. S., Hallam G. M. Preliminary investigations on the molecular mode of action of the delta-endotoxin produced by Bacillus thuringiensis var. alesti. J Invertebr Pathol. 1974 Mar;23(2):259–261. doi: 10.1016/0022-2011(74)90196-7. [DOI] [PubMed] [Google Scholar]
- Gill S. S., Singh G. J., Hornung J. M. Cell membrane interaction of Bacillus thuringiensis subsp. israelensis cytolytic toxins. Infect Immun. 1987 May;55(5):1300–1308. doi: 10.1128/iai.55.5.1300-1308.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
- HEIMPEL A. M., ANGUS T. A. Bacterial insecticides. Bacteriol Rev. 1960 Sep;24(3):266–288. doi: 10.1128/br.24.3.266-288.1960. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hedrick J. L., Smith A. J. Size and charge isomer separation and estimation of molecular weights of proteins by disc gel electrophoresis. Arch Biochem Biophys. 1968 Jul;126(1):155–164. doi: 10.1016/0003-9861(68)90569-9. [DOI] [PubMed] [Google Scholar]
- Hurley J. M., Lee S. G., Andrews R. E., Jr, Klowden M. J., Bulla L. A., Jr Separation of the cytolytic and mosquitocidal proteins of Bacillus thuringiensis subsp. israelensis. Biochem Biophys Res Commun. 1985 Jan 31;126(2):961–965. doi: 10.1016/0006-291x(85)90279-7. [DOI] [PubMed] [Google Scholar]
- Kim K. H., Ohba M., Aizawa K. Purification of the toxic protein from Bacillus thuringiensis serotype 10 isolate demonstrating a preferential larvicidal activity to the mosquito. J Invertebr Pathol. 1984 Sep;44(2):214–219. doi: 10.1016/0022-2011(84)90015-6. [DOI] [PubMed] [Google Scholar]
- Knowles B. H., Ellar D. J. Characterization and partial purification of a plasma membrane receptor for Bacillus thuringiensis var. kurstaki lepidopteran-specific delta-endotoxin. J Cell Sci. 1986 Jul;83:89–101. doi: 10.1242/jcs.83.1.89. [DOI] [PubMed] [Google Scholar]
- LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
- Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
- Lyman G. H., Preisler H. D., Papahadjopoulos D. Membrane action of DMSO and other chemical inducers of Friend leukaemic cell differentiation. Nature. 1976 Jul 29;262(5567):361–363. doi: 10.1038/262360a0. [DOI] [PubMed] [Google Scholar]
- Padua L. E., Ohba M., Aizawa K. The isolates of Bacillus thuringiensis serotype 10 with a highly preferential toxicity to mosquito larvae. J Invertebr Pathol. 1980 Sep;36(2):180–186. doi: 10.1016/0022-2011(80)90022-1. [DOI] [PubMed] [Google Scholar]
- Sriram R., Kamdar H., Jayaraman K. Identification of the peptides of the crystals of Bacillus thuringiensis var israelensis involved in the mosquito larvicidal activity. Biochem Biophys Res Commun. 1985 Oct 15;132(1):19–27. doi: 10.1016/0006-291x(85)90982-9. [DOI] [PubMed] [Google Scholar]
- Stewart G. S., Ellar D. J. Precursor processing during the maturation of a spore-coat protein in Bacillus megaterium KM. Biochem J. 1983 Feb 15;210(2):411–417. doi: 10.1042/bj2100411. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Stewart G. S., Johnstone K., Hagelberg E., Ellar D. J. Commitment of bacterial spores to germinate. A measure of the trigger reaction. Biochem J. 1981 Jul 15;198(1):101–106. doi: 10.1042/bj1980101. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Thomas W. E., Ellar D. J. Bacillus thuringiensis var israelensis crystal delta-endotoxin: effects on insect and mammalian cells in vitro and in vivo. J Cell Sci. 1983 Mar;60:181–197. doi: 10.1242/jcs.60.1.181. [DOI] [PubMed] [Google Scholar]
- Thomas W. E., Ellar D. J. Mechanism of action of Bacillus thuringiensis var israelensis insecticidal delta-endotoxin. FEBS Lett. 1983 Apr 18;154(2):362–368. doi: 10.1016/0014-5793(83)80183-5. [DOI] [PubMed] [Google Scholar]
- Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ward E. S., Ellar D. J. Bacillus thuringiensis var. israelensis delta-endotoxin. Nucleotide sequence and characterization of the transcripts in Bacillus thuringiensis and Escherichia coli. J Mol Biol. 1986 Sep 5;191(1):1–11. doi: 10.1016/0022-2836(86)90417-1. [DOI] [PubMed] [Google Scholar]
- Ward E. S., Ridley A. R., Ellar D. J., Todd J. A. Bacillus thuringiensis var. israelensis delta-endotoxin. Cloning and expression of the toxin in sporogenic and asporogenic strains of Bacillus subtilis. J Mol Biol. 1986 Sep 5;191(1):13–22. doi: 10.1016/0022-2836(86)90418-3. [DOI] [PubMed] [Google Scholar]
- Yamamoto T., McLaughlin R. E. Isolation of a protein from the parasporal crystal of Bacillus thuringiensis var. Kurstaki toxic to the mosquito larva, Aedes taeniorhynchus. Biochem Biophys Res Commun. 1981 Nov 30;103(2):414–421. doi: 10.1016/0006-291x(81)90468-x. [DOI] [PubMed] [Google Scholar]