Physics of 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. The regulation of biofilm formation involves cell-cell communication that enables cells to make synchronous decisions. However, phenotypic heterogeneity can play a significant role in biofilm development. Using fluorescent biosensors and reporters, we reveal that a growing Vibrio cholerae biofilm exhibits high levels of heterogeneity in the intracellular cyclic diguanylate (c-di-GMP) concentration and expression of biofilm matrix genes. The intracellular c-di-GMP concentration correlates with biofilm matrix production, and cells with different c-di-GMP levels spatially segregate during biofilm development. We show that such spatial segregation arises from physical interactions associated with matrix production and is affected by the presence or absence of specific matrix components. Combining high-temporal-resolution tracking and single-cell gene expression, we reveal the physical principles behind phenotypic segregation in a developing biofilm and its implications on fitness.