B. subtilis is a Gram-positive, soil-dwelling bacterium that has long served as a model organism for studying cellular differentiation in bacteria. As B. subtilis cells reach stationary phase, complex, interdependent regulatory networks govern differentiation into a variety of cell fates including matrix formation, sporulation, and competency. While B. subtilis is currently utilized in industrial production of recombinant proteins, antibiotics, and chemicals, engineering in this bacterium is hindered by comparatively low transformation efficiencies compared to other common biotechnology chassis organisms. Natural competence—the active ability to uptake and recombine exogenous DNA into the genome—can be a simple, efficient method of genome engineering. However, the native regulatory network limits the ease and efficiency of transformation in B. subtilis. Even under optimized conditions only 5-20/% of cells will enter the competent state. Therefore, the goal of this work is to enhance the transformation efficiency of this bacterium. We utilized inducible promoters to control expression of the global competence regulator, comK, to increase the fraction of the population that enters competency and, thereby, transformation frequency. We compare and characterize five inducible competence systems by exploring the relationship between the activation of comG promoter—a commonly used reporter for competence—and transformability. Through characterization and method optimization, we increase the number of transformants 2800-fold and the transformation frequency 1700-fold. Further, we demonstrate that the complex relationship between PcomG activation and transformation is dependent on growth conditions and genetic background, impacting how the reporter is used to model natural competence.
