Bacillus thuringiensis is characterized by the production of a large, intracellular inclusion during sporulation. Each of these inclusions is comprised of one or a few classes of large polypeptides (protoxins) that are toxic for specific species of insect larvae. There is a spectrum of these toxins produced by subspecies of B. thuringiensis each with a unique pattern of toxicity for certain insect larvae. Included among these are many that cause major damage to crops and trees (Lepidoptera) as well as others that serve as carriers of human diseases, i.e. mosquitoes and flies. In some subspecies, more than one inclusion is formed and each contains a unique toxin. These polypeptides are produced during a portion of the sporulation process and comprise a substantial part of the protein synthetic activity during the period of formation. Several of these protoxin genes have been cloned from B. thuringiensis plasmids into B. subtilis E. coli shuttle vector and express sufficiently well in both organisms to permit determination of lethal doses for select neonate larvae. The basis for toxin specificity will be determined by comparing relative LC50's for parental B. thuringiensis strains, cloned protoxin genes (alone or in combination) and of B. cereus transcipients containing selected arrays of B. thuringiensis plasmids. B. thuringiensis subspecies and cloned genes will be chosen to provide a comparison of those with overlapping as well as non-overlapping specificities. Various regions of the cloned protoxin genes will be subcloned to examine toxicity levels as well as alterations in specificity. The regulation of protoxin synthesis in at least one subspecies is dependent on the activities of two cryptic plasmids. One plasmid affects the temperature of protoxin synthesis, the other the amount produced. Bioassay systems will be developed employing cloned protoxin genes plus cloned fragments of these regulatory plasmids in order to define the portion of each plasmid involved in regulation, whether there is a gene product and whether the regulatory function is at the level of transcription or elsewhere.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM034035-02
Application #
3284440
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1985-09-05
Project End
1990-08-31
Budget Start
1986-09-01
Budget End
1987-08-31
Support Year
2
Fiscal Year
1986
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

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