Biofilm initiation regulated by cell orientation patterning in Escherichia coli

Unicellular microorganisms can transition to multicellular states that enhance survival under environmental fluctuations. In bacteria, one such state is the biofilm, defined by the production of an extracellular matrix. Although many aspects of biofilm formation have been extensively studied, how matrix production is spatially initiated within a growing population remains largely unknown. Here we show that cell orientation patterning governs the spatial initiation of matrix production in bacterial monolayers. Using an Escherichia coli strain reporting mechanically induced production of colanic acid, a major matrix component, we observed that matrix production was accompanied by mechanically regulated out-of-plane growth under agar-pad confinement. Notably, colanic acid production preferentially emerged near dense topological defects, where cell orientation mismatches and growth-induced pressure builds up. By controlling confinement geometry with microfluidic devices, we regulated cell orientation patterns and defect distributions, thereby biasing the spatial pattern of matrix production. These findings establish cell orientation patterning as a physical mechanism that spatially organizes mechanical cues to regulate extracellular matrix production, providing a general framework for the initiation of bacterial biofilms.