Bacillus thuringiensis entomocidal protoxin gene sequence and gene product analysis.

Area of Science:

• Molecular biology
• Insect toxicology
• Biochemistry

Background:

• Bacillus thuringiensis produces insecticidal crystal proteins (protoxins).
• The protoxin gene sequence and its protein product are crucial for understanding insecticidal activity.
• Variations in protoxins among subspecies contribute to differences in insect host range.

Purpose of the Study:

• To determine the DNA sequence of the insecticidal protoxin gene from Bacillus thuringiensis subspecies berliner 1715.
• To identify the specific DNA region encoding the toxic activity of the protoxin.
• To compare the protoxin sequence with related subspecies and propose a structural model.

Main Methods:

• DNA sequencing of the 3778-bp protoxin gene and flanking regions.
• Construction of sequential deletions from the 3' end of the coding region using Bal-31 nuclease.
• Insect bioassays using deletion mutants to assess toxicity.

Main Results:

• The protoxin gene codes for a 1155-amino acid protein with a molecular mass of 130,615 daltons.
• An amino-terminal peptide of 612 amino acids was found to be toxic, while a 603-amino acid peptide was not.
• The berliner 1715 protoxin showed 90% homology to kurstaki HD-1-Dipel and sotto protoxins, with clustered differences in both toxic and non-toxic regions.

Conclusions:

• The N-terminal portion of the Bacillus thuringiensis protoxin contains the toxic domain.
• Differences in protoxin structure, particularly in distinct domains, likely account for variations in insecticidal toxicity and host range.
• A structural and functional domain model for the protoxin molecule is proposed based on comparative analysis.