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Construction of Bacillus thuringiensis Simulant Strains Suitable for Environmental Release
Sangjin Park1,2, Changhwan Kim2, Daesang Lee2
1Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 Plus Program), Center for Systems and Synthetic Biotechnology, Institute for the BioCentury, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Republic of Korea.
Engineered Bacillus thuringiensis strains offer enhanced safety and detectability for biodefense research. These modified bacteria produce yellow colonies and spores with limited viability, improving environmental and human safety in outdoor studies.
Area of Science:
- Microbiology and Biotechnology
- Biodefense and Environmental Safety
Background:
- Bacillus thuringiensis (Bt) is increasingly recognized as a surrogate bacterium for biodefense research.
- Existing Bt strains require modifications for safe and effective use in environmental studies, particularly concerning spore persistence and detectability.
- Environmental release of genetically modified organisms necessitates robust safety features to prevent long-term contamination.
Purpose of the Study:
- To construct simulant strains of Bacillus thuringiensis with improved characteristics for environmental studies.
- To enhance detectability, control spore persistence, and ensure environmental and human safety of Bt simulants.
- To develop a safer surrogate bacterium for biodefense research, particularly for studies mimicking Bacillus anthracis.
Main Methods:
- Transposon mutagenesis was employed to insert pigment genes for yellow colony formation, aiding detection.
- A genetic circuit involving loxP sites and a Cre expression cassette was designed to delete the spo0A gene during sporulation, preventing recurrent sporulation.
- Two major small acid-soluble spore protein (SASP) genes were deleted, and antibiotic resistance markers were excluded from all constructed strains.
Main Results:
- Constructed Bt strains produced easily detectable yellow colonies.
- Engineered spores exhibited increased UV-C sensitivity and rapid viability loss under solar simulation, indicating reduced environmental persistence.
- Mutant spores showed enhanced in vivo safety, being quickly cleared from mouse lungs.
Conclusions:
- The developed Bt simulant strains possess enhanced detectability and controlled environmental persistence, addressing key safety concerns for biodefense research.
- The genetic circuit for non-recurrent sporulation and SASP gene deletion significantly improve the environmental and human safety profile of the simulants.
- These modified Bt strains are suitable for wider application as simulants in environmental release studies, particularly for Bacillus anthracis.
