Biofilm Initiation via Extracellular Matrix Production Driven by Cell Orientation Patterning in Growing Escherichia coli Populations

Abstract

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 is associated with the spatial initiation of Rcs stress-response activation, which is known to be involved in extracellular matrix production. Using an Escherichia coli strain reporting mechanically induced stress responses, we observed that Rcs activation was accompanied by mechanically regulated out-of-plane growth under agar-pad confinement. Notably, this activation 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 Rcs activation. These findings indicate an association between cell orientation patterning and the spatial activation of a stress-response pathway involved in extracellular matrix production, suggesting a possible physical link to biofilm initiation.