(263d) Phenotypic Segregation in a Developing Biofilm

Biofilm is an important bacterial lifestyle in which individual bacterial cells form surface-associated aggregates embedded in a polymeric matrix they secrete. Biofilms are important in biomedical settings where they cause chronic infections resistant to antibiotic treatments. They are also relevant in industrial and engineering settings where they can lead to biofouling or be leveraged for bioremediation. Therefore, understanding the regulation and development of biofilms is of essential importance. The regulation of biofilm formation involves cell-cell communication that enables cells to make synchronous decisions. However, phenotypic heterogeneity is widely observed in clonal communities and can play a significant role in biofilm development. In this work, we use fluorescent biosensors and reporters to study the intracellular cyclic diguanylate (c-di-GMP) concentration, a key signaling molecule in biofilm regulation, and expression of biofilm matrix genes in a 3D biofilm. We found both c-di-GMP concentration and matrix production exhibit high levels of heterogeneity in a correlated fashion. Moreover, cells with different phenotypes spatially segregate during biofilm formation, a process that parallels morphogenesis in tissue and embryo development in multicellular organisms. We show that such spatial segregation arises from physical interactions medicated by the biofilm matrix and is modulated by the presence and expression level of specific matrix components. Combining bacterial genetics, single-cell tracking, and agent-based modeling, we reveal the physical principles behind phenotypic segregation in a developing biofilm. Our results further elucidate the associated fitness advantage of phenotypic heterogeneity in microbial communities, as well as provide a renewed understanding of the developmental cycle of biofilms.