Matela, A. M.; Siatkowski, C. W.; Yan, C.; Thiagarajan, S.; Cooper, V. S.

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 Pseudomonas fluorescens 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 (wsp, yfiBNR, morA, fuzY). 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 bmo (biofilm and motility optimizer). Competition experiments and microscopy demonstrated bmo mutants employ generalist strategies and coexist with the ancestor and specialist mutants through niche differentiation. Consequently, phenotypic diversity is maintained, with smooth (ancestral and bmo) 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 Pseudomonas infection biology and provide transformative STEM experiences. IMPORTANCE: Bacterial biofilms dominate microbial life, yet their evolutionary genetics remain incompletely understood. This science education project engages thousands of high school students in experimental evolution, yielding discoveries about biofilm adaptation while transforming their science education. Selected mutants included PFLU0185/bmo, a conserved phosphodiesterase that helps bacteria balance the competing demands of attachment and dispersal essential for biofilm life cycle success. The finding that smooth-colony generalists rather than conspicuous variants dominate biofilm adaptation adds to our understanding of the process of niche differentiation in biofilms. This work also demonstrates the power of distributed research networks for discovery of new genetic pathways of adaptation. Students gained authentic research experience, potentially inspiring them to join the next generation of scientists, while identifying mutants adapted to discrete conditions that maintain diversity within biofilms. This synergy between education and discovery offers a scalable model for addressing complex biological questions while developing scientific literacy in diverse classrooms.