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Biological properties of loop-replaced mutants of Bacillus thuringiensis mosquitocidal Cry4Aa
Mohammad Tofazzal Hossain Howlader1, Yasuhiro Kagawa, Hiroshi Sakai
1Graduate School of Natural Science and Technology, Okayama University, 3-1-1 Tsushima-Naka, Okayama-Shi, Okayama 700-8530, Japan.
Abstract:
Cry4Aa produced by Bacillus thuringiensis subsp. israelensis (Bti) exhibits a specific toxicity to Anopheles, Aedes, and Culex larvae, which are vectors of serious diseases, and formulations of Bti are used worldwide for mosquito control. In general, domain II of the Cry toxin is believed to be important for target specificity, and three loops (loops 1, 2, and 3) in domain II have been studied extensively. In this report, to analyze the biological functions of loops 1, 2, and 3 of Cry4Aa, mutants were constructed in which one of the loops was replaced with either of the other two loops. A bioassay using Culex pipiens larvae revealed that the mosquitocidal activity was virtually lost upon replacement of loop2. The mutants in which loops 1 and/or 3 were replaced also showed decreased activity, but they still maintained some activities. This suggested that loop2, but not loops 1 and 3, was essential for the mosquitocidal activity of Cry4Aa. Proteolytic digestion revealed the involvement of loops in the stability of the Cry4Aa structure. No significant differences were observed in the amount of wild-type and mutant Cry4Aa bound to the BBMVs prepared from the C. pipiens larvae.
Insights
Loop 2 of Bacillus thuringiensis subsp. israelensis (Bti) Cry4Aa is essential for mosquitocidal activity against Culex pipiens larvae. Replacing loop 2 virtually eliminated toxicity, while other loop modifications reduced but did not abolish activity.
Area of Science:
- Molecular Biology
- Entomology
- Biochemistry
Background:
- Bacillus thuringiensis subsp. israelensis (Bti) produces Cry4Aa, a toxin crucial for mosquito control.
- Domain II of Cry toxins, particularly loops 1, 2, and 3, is implicated in target specificity.
- Understanding these loops is vital for developing effective mosquito control strategies.
Purpose of the Study:
- To elucidate the biological functions of loops 1, 2, and 3 in the Cry4Aa toxin.
- To determine the role of individual loops in mosquitocidal activity and structural stability.
Main Methods:
- Construction of Cry4Aa mutants by loop replacement (loop 1, 2, or 3 swapped with others).
- Bioassays using Culex pipiens larvae to assess mosquitocidal activity.
- Proteolytic digestion to evaluate structural stability of wild-type and mutant Cry4Aa.
- Binding assays with brush border membrane vesicles (BBMVs) from C. pipiens.
Main Results:
- Replacement of loop 2 in Cry4Aa resulted in a near-complete loss of mosquitocidal activity.
- Mutants with replaced loops 1 and/or 3 exhibited reduced, but retained, insecticidal activity.
- Proteolytic digestion indicated loops are involved in Cry4Aa structural stability.
- No significant differences in Cry4Aa binding to C. pipiens BBMVs were observed between wild-type and mutants.
Conclusions:
- Loop 2 is essential for the mosquitocidal activity of Cry4Aa against Culex pipiens.
- Loops 1 and 3 contribute to, but are not solely responsible for, the toxin's activity.
- The loops play a role in maintaining the structural integrity of the Cry4Aa toxin.
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