Student-led experimental evolution reveals novel biofilm regulatory networks underlying adaptations to multiple niches.

We established a research-education partnership known as EvolvingSTEM that provides secondary school students the opportunity to conduct authentic research experiments centered on microbial evolution. These experiments are currently conducted by thousands of high school students and can offer an unprecedented window into biofilm adaptation while building a community of young researchers. Providing high school students access to research experiences improves learning and can have positive and long-lasting impacts on their attitudes towards science. Moreover, student research can make impactful scientific contributions. Through EvolvingSTEM, students evolve populations of in a biofilm bead model and observe heritable changes in colony morphology. Genome sequencing of 70 mutants that they picked identified parallel mutations in genes known to regulate biofilm growth (, , ). We also uncovered novel adaptations: loss-of-function mutations in phosphodiesterase PFLU0185 that did not alter colony morphology, and mutations affecting periplasmic disulfide bond formation producing small colonies. PFLU0185 mutations consistently reached high frequencies and phenotyping revealed roles in cyclic di-GMP regulation, biofilm formation, and motility, prompting us to name this gene (biofilm and motility optimizer). Competition experiments and microscopy demonstrated mutants employ generalist strategies and coexist with the ancestor and specialist mutants through niche differentiation. Consequently, phenotypic diversity is maintained, with smooth (ancestral and ) colonies consistently outnumbering wrinkly and fuzzy variants. The study advances understanding of biofilm genetic architecture while demonstrating that student-led research can uncover mechanisms of microbial adaptation relevant to infection biology and provide transformative STEM experiences.