How Do Biofilms Evolve to Endure Metal-rich Environments?

While bacteria can adapt to harsh environments, understanding of which pathways are preferred for modification and how they evolve remains limited. Directed evolution provides an opportunity to understand bacterial response to toxic environmental contaminants such as heavy metals, as development of a metal-tolerant bacterial strain can be paired with sequence analysis to gain insight into which mechanisms drive metal stress response and survival. Because its biofilms are already known to bind metals, we will leverage UV mutagenesis and selection to evolve a strain of the biofilm-forming bacteria B. subtilis that can tolerate metal-rich environments. Metal identity dictates baseline tolerance in wild-type B. subtilis; while it can survive in 50 mM copper, no growth is observed in 10-fold lower concentrations of zinc. UV irradiation yields survivors at higher zinc concentrations, suggesting that iterative rounds of mutagenesis and selection can produce zinc-tolerant mutants. Genome sequencing of mutants in parallel will allow us to identify and follow the trajectory of evolving pathways. Combined with physical characterization and comparison to predicted mutations, we will build a comprehensive understanding of acquired zinc tolerance mechanisms and viable routes for evolution.