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

<i>Bacillus subtilis</i> is applied as a biofertilizer, biocontrol agent, and probiotic in agriculture, and is also used for industrial synthesis of proteins and peptides. These applications can be combined by using <i>B. subtilis</i> to synthesize plant health-promoting peptides in culture or to deliver them to roots via seed treatments. To facilitate the use of <i>B. subtilis</i> as a cell factory, we tested different media, temperatures, and growth phases to optimize ectopic expression of a Plant Elicitor Peptide from soybean (GmPEP3) that enhances seedling growth. Our results indicate that temperature, culture media, and growth phase have interactive effects, and that 30 °C and 2x YT media can enhance ectopic expression per cell compared to 37 °C or LB media in log phase bacteria. We also identified tradeoffs between cell growth and ectopic expression levels per cell, with the log phase favoring high expression per cell but the stationary phase yielding higher cell numbers and consequently higher expression levels per unit of growth media. In addition, to facilitate <i>B. subtilis</i> seed treatments, we compared retention of spores versus vegetative cells with and without carboxymethylcellulose (CMC) to improve the viability of <i>B. subtilis</i> seed treatments. Our results indicated that retention of viable bacteria on <i>B. subtilis</i>-treated seeds could be increased by ~40% by using the adhesive polymer CMC, and shelf life could be extended from 24 h to at least 3 months by using endospores rather than vegetative cells. For <i>B. subtilis</i> expressing GmPEP3, endospores also had comparable plant-growth-promoting activity as vegetative cells. This establishes the bioactivity of spores and illustrates the potential benefits of using <i>B. subtilis</i> to deliver heterologous peptides. These results provide valuable insights for deploying <i>B. subtilis</i> for crop health.