Heterogeneity in a developing biofilm*

Biofilm is an important lifestyle of bacteria where individual cells form aggregates embedded in a secreted matrix. Biofilm development is known to consist of three stages: initial attachment, maturation, and dispersal. However, whether individual cells display phenotypic heterogeneity in biofilm formation and the consequence of such heterogeneity in biofilm development is still largely unknown. Using Vibrio cholerae as a model organism, we aim to reveal the spatiotemporal pattern and heterogeneity of gene expression and second-messenger molecule concentration at the single-cell level. By combining fluorescent reporters and high-resolution time-lapse imaging, we found various levels of heterogeneity in a developing biofilm in expression of biofilm relevant genes such as matrix production and quorum sensing, as well as heterogeneity in cyclic-di-GMP concentration, a critical second-messenger molecule that modulates sessile-to-motile transition in many bacterial species. The heterogeneity was also found to couple to the biofilm’s mechanical environment and structural organization. Our study elucidates how biofilm regulation and heterogeneity, cell organization, and mechanical environment are correlated in driving biofilm development.