The long-term objective of the project is to understand the mechanisms by which the toxins of the bacterium, Bacillus thuringiensis Subsp. israelensis (BTI) exert their toxic effect. The toxins from this bacterium are increasingly being used for insect control. However, in spite of this increasing use there is little information on how the toxins exert their precise mode of action. In this renewal our principal focus will be to understand the structure and function relationships of the cytolytic 25KD protein toxin of BTI. While this toxin has been sequenced, the relationship of its structure to its mode of action is not known. Such studies will not only enable us to further understand the molecular basis of toxicity of this toxin both on mammalian and insect systems, but will also enable us to further understand cell membranes, both in mammals and insects. The specific objectives of this proposal are therefore to: I. Evaluate the structure an function relationships of the 25KD BTI toxin. Within this objective we will characterize the mechanism of action of the 25KD toxin, evaluate the interaction of the toxin on cell membranes, both mammalian and insect derived, and characterize the structural features of the 25KD toxin which cause the toxin to exert its toxin effect. II. Determine the similarities/differences in the action of toxins derived from Bacillus thuringiensis subsp. morrisoni (BTM) with those derived from BTI. III. Identify and characterize the nematocidal factor in BTI. Characterization of the structure-function relationships will be achieved by a variety of approaches including electrophsiological and biochemical techniques using purified toxins, antibodies, cell culture and liposomes. Purification of toxins from both BTI and BTM will be achieved by gel-filtration and ion-exchange chromatography, and toxin activity assay against insect and mammalian cels. The nematocidal factor from BTI will be purified by high-pressure liquid chromatography. Bioassays for this factor will be performed with nematode eggs.

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Research Project (R01)
Project #
2R01ES003298-04
Application #
3250473
Study Section
Tropical Medicine and Parasitology Study Section (TMP)
Project Start
1983-12-01
Project End
1990-03-31
Budget Start
1987-04-01
Budget End
1988-03-31
Support Year
4
Fiscal Year
1987
Total Cost
Indirect Cost
Name
University of California Riverside
Department
Type
Earth Sciences/Resources
DUNS #
City
Riverside
State
CA
Country
United States
Zip Code
92521
Cheong, H; Gill, S S (1997) Cloning and characterization of a cytolytic and mosquitocidal delta-endotoxin from Bacillus thuringiensis subsp. jegathesan. Appl Environ Microbiol 63:3254-60
Lee, H K; Gill, S S (1997) Molecular cloning and characterization of a novel mosquitocidal protein gene from Bacillus thuringiensis subsp. fukuokaensis. Appl Environ Microbiol 63:4664-70
Cheong, H; Dhesi, R K; Gill, S S (1997) Marginal cross-resistance to mosquitocidal Bacillus thuringiensis strains in Cry11A-resistant larvae: presence of Cry11A-like toxins in these strains. FEMS Microbiol Lett 153:419-24
Gill, S S; Cowles, E A; Francis, V (1995) Identification, isolation, and cloning of a Bacillus thuringiensis CryIAc toxin-binding protein from the midgut of the lepidopteran insect Heliothis virescens. J Biol Chem 270:27277-82
Kawalek, M D; Benjamin, S; Lee, H L et al. (1995) Isolation and Identification of novel toxins from a new mosquitocidal isolate from Malaysia, Bacillus thuringiensis subsp. jegathesan. Appl Environ Microbiol 61:2965-9
Gill, S S (1995) Mechanism of action of Bacillus thuringiensis toxins. Mem Inst Oswaldo Cruz 90:69-74
Cowles, E A; Yunovitz, H; Charles, J F et al. (1995) Comparison of toxin overlay and solid-phase binding assays to identify diverse CryIA(c) toxin-binding proteins in Heliothis virescens midgut. Appl Environ Microbiol 61:2738-44
Dai, S M; Gill, S S (1993) In vitro and in vivo proteolysis of the Bacillus thuringiensis subsp. israelensis CryIVD protein by Culex quinquefasciatus larval midgut proteases. Insect Biochem Mol Biol 23:273-83
Chang, C; Yu, Y M; Dai, S M et al. (1993) High-level cryIVD and cytA gene expression in Bacillus thuringiensis does not require the 20-kilodalton protein, and the coexpressed gene products are synergistic in their toxicity to mosquitoes. Appl Environ Microbiol 59:815-21
Gill, S S; Cowles, E A; Pietrantonio, P V (1992) The mode of action of Bacillus thuringiensis endotoxins. Annu Rev Entomol 37:615-36

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