The amino acid sequences of all Bacillus thuringiensis (B.t.) delta- endotoxins deduced from gene sequences share certain conserved regions which very likely reflect a similar mode of action. Inclusions containing the delta-endotoxins (or protoxins) are solubilized in insect larval guts and converted to toxins which bind to specific cell membrane receptors. The toxins then insert into the membrane to form or alter cation pores. The sequenced toxins are active on larvae from different insect orders including Lepidoptera, Coleoptera and Diptera. Among the target insects are those which damage trees and crops as well as vectors for pathogens. In fact, WHO is employing a B.t. isolate in Africa for vector control. Since there is likely to be a common mode of action, one protoxin gene has been selected for extensive mutagenic analysis in order to assess the contribution of various portions of the protoxin molecule to processing, specificity and toxicity. Both site-directed and mutagenic oligonucleotide techniques will be used to alter specific residues or regions of the gene. The mutated genes will be cloned back into B.t. so that pure inclusions may be isolated for bioassays and for in vitro vesicle binding and transport experiments. An understanding of the contribution of various conserved regions to these processes should be helpful in the design of new toxins, for elucidating the mode of action and for understanding the basis for resistance. Most B.t. isolates contain multiple protoxin genes which are expressed to different extents. In general, protoxin genes are on large plasmids so differential expression could be influenced by gene copy number, plasmid stability and the availability of transcription factors. These particular parameters will be examined in at least two isolates employing gene-specific probes to measure protoxin gene content., steady state mRNA levels and the presence or absence of a particular protoxin-encoding plasmid. Understanding the regulation of protoxin synthesis would be important for the evaluation of the efficacy of new isolates, for constructing new strains by mating and/or transformation and for determining the stability of certain genes in a particular isolate.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM034035-08
Application #
3284445
Study Section
Tropical Medicine and Parasitology Study Section (TMP)
Project Start
1985-09-05
Project End
1994-06-30
Budget Start
1993-07-01
Budget End
1994-06-30
Support Year
8
Fiscal Year
1993
Total Cost
Indirect Cost
Name
Purdue University
Department
Type
Schools of Arts and Sciences
DUNS #
072051394
City
West Lafayette
State
IN
Country
United States
Zip Code
47907
Sedlak, M; Walter, T; Aronson, A (2000) Regulation by overlapping promoters of the rate of synthesis and deposition into crystalline inclusions of Bacillus thuringiensis delta-endotoxins. J Bacteriol 182:734-41
Walter, T; Aronson, A (1999) Specific binding of the E2 subunit of pyruvate dehydrogenase to the upstream region of Bacillus thuringiensis protoxin genes. J Biol Chem 274:7901-6
Aronson, A (1995) The protoxin composition of Bacillus thuringiensis insecticidal inclusions affects solubility and toxicity. Appl Environ Microbiol 61:4057-60
Aronson, A I; Wu, D; Zhang, C (1995) Mutagenesis of specificity and toxicity regions of a Bacillus thuringiensis protoxin gene. J Bacteriol 177:4059-65
Aronson, A I (1993) The two faces of Bacillus thuringiensis: insecticidal proteins and post-exponential survival. Mol Microbiol 7:489-96
Wu, D; Aronson, A I (1992) Localized mutagenesis defines regions of the Bacillus thuringiensis delta-endotoxin involved in toxicity and specificity. J Biol Chem 267:2311-7
Walter, T M; Aronson, A I (1991) Transduction of certain genes by an autonomously replicating Bacillus thuringiensis phage. Appl Environ Microbiol 57:1000-5
Lee, C S; Aronson, A I (1991) Cloning and analysis of delta-endotoxin genes from Bacillus thuringiensis subsp. alesti. J Bacteriol 173:6635-8
Wu, D; Cao, X L; Bai, Y Y et al. (1991) Sequence of an operon containing a novel delta-endotoxin gene from Bacillus thuringiensis. FEMS Microbiol Lett 65:31-5
Benoit, T G; Wilson, G R; Bull, D L et al. (1990) Plasmid-associated sensitivity of Bacillus thuringiensis to UV light. Appl Environ Microbiol 56:2282-6

Showing the most recent 10 out of 12 publications