PROJECT SUMMARY Bacterial biofilms are communities of bacteria stuck together by a self-produced polymer matrix. They exist in nearly every environment on earth and are the cause of most hospital-borne infections. These adherent communities are highly tolerant of antibiotics and their extreme resilience cannot merely be attributed to the inability of drug molecules to penetrate the biofilm matrix. It has become clear that emergent, multicellular behaviors in biofilms impart unique survival strategies. In recent years, we have learned detailed mechanisms of gene regulation and phenotypic heterogeneity of single bacterial cells, but we have neither explored these cellular phenomena in the context of crowded, multicellular biofilms, nor do we understand how multicellular behaviors emerge from these single-cell properties. The goal of my lab for the next five years will be to understand how single-cell properties and physics of the local environment give rise to beneficial emergent behaviors in biofilms. With this MIRA award, my group will explore these questions by focusing on two collective phenomena: 1) electrical cell-to-cell communication and 2) emergence of patterns of motile and matrix-producing cells during biofilm development. We will approach these problems using spatial gene expression measurements, single-cell-level time-lapse imaging of biofilms, and novel transistor devices for electrical perturbation of biofilms. To probe the emergence of group behaviors from single-cell properties, we will use the experimental biofilm model species Bacillus subtilis to perturb known cell-to-cell communication mechanisms and gene regulatory circuits. Our data will inform quantitative models of multicellular phenomena, which will give us a system-level understanding of multicellular behaviors in microbes, as well as suggesting new hypotheses and targets for disrupting the emergent behaviors that allow biofilms to elude treatment.