Optimizing Conditions for Bacillus subtilis Ectopic Gene Expression and Delivery via Seed Treatment

Area of Science:

• Microbiology
• Biotechnology
• Agricultural Science

Background:

• Bacillus subtilis is a versatile bacterium used in agriculture as a biofertilizer, biocontrol agent, and probiotic.
• It also serves as a host for industrial protein and peptide synthesis.
• Combining these applications allows for in-situ production and delivery of plant health-promoting peptides.

Purpose of the Study:

• To optimize the ectopic expression of a soybean Plant Elicitor Peptide (GmPEP3) in Bacillus subtilis for enhanced seedling growth.
• To improve the formulation and viability of Bacillus subtilis for seed treatments.
• To assess the potential of Bacillus subtilis as a cell factory for delivering bioactive peptides.

Main Methods:

• Optimized Bacillus subtilis growth conditions (media, temperature, growth phase) for GmPEP3 ectopic expression.
• Compared retention and viability of vegetative cells versus endospores on seeds, with and without carboxymethylcellulose (CMC).
• Assessed the plant-growth-promoting activity of GmPEP3-expressing endospores.

Main Results:

• Ectopic expression of GmPEP3 was enhanced at 30°C in 2x YT media during the log phase.
• Tradeoffs between cell growth and expression per cell were observed between log and stationary phases.
• Carboxymethylcellulose (CMC) increased bacterial retention on seeds by ~40%, and endospores extended shelf life to 3 months.
• GmPEP3-expressing endospores demonstrated comparable plant-growth-promoting activity to vegetative cells.

Conclusions:

• Optimized conditions (30°C, 2x YT media, log phase) enhance GmPEP3 expression per Bacillus subtilis cell.
• Endospore formulation with CMC significantly improves Bacillus subtilis seed treatment viability and shelf life.
• Bacillus subtilis endospores are a viable delivery vehicle for bioactive peptides, offering a promising strategy for enhancing crop health.