Exploring single-cell heterogeneity in a developing biofilm

Biofilms consist of surface-associated bacteria embedded in an extracellular polymeric matrix. This matrix provides protection and allows for resource exchange within the community. Although bacterial colonies are known to exhibit broad levels of heterogeneity, it is not well understood how this extends to biofilms. It is largely unknown whether individual cells display phenotypic heterogeneity and how such heterogeneities affect the biofilm’s life cycle. We use the model organism Vibrio cholerae to study spatiotemporal patterning within biofilms, as well as heterogeneities in gene expression and second-messenger molecule concentrations at the single-cell level. Combining high-resolution time-lapse imaging and fluorescent reporters, we find multiple heterogeneities on developing biofilms grown from single cells. We observe variable expression levels in relevant biofilm genes, including matrix production and quorum sensing. We also see differences in c-di-GMP concentrations (a second-messenger molecule that regulates the cell’s transition from motile to sessile) across cells in the biofilm. These heterogeneities are coupled to the cells’ mechanical environment and their structural organization, exemplifying how variations on biofilm regulation, cell organization and local mechanical environments are correlated in the biofilm development process. We also show progress towards understanding the role of inheritance by tracking single-cells in a growing biofilm.