Density and temperature controlled fluid extraction in a bacterial biofilm is determined by poly-γ-glutamic acid production

A hallmark of microbial biofilms is the self-production of an extracellular molecular matrix that encases the resident cells. The matrix provides protection from the environment, while spatial heterogeneity of gene expression influences the structural morphology and colony spreading dynamics. Bacillus subtilis is a model bacterial system used to uncover the regulatory pathways and key building blocks required for biofilm growth and development. In this work, we report on the emergence of a highly active population of bacteria during the early stages of biofilm formation, facilitated by the extraction of fluid from the underlying agar substrate. We trace the origin of this fluid extraction to the production of poly-gamma-glutamic acid (PGA). The flagella-dependent activity develops behind a moving front of fluid that propagates from the boundary of the biofilm towards the interior. The extent of fluid proliferation is controlled by the presence of extracellular polysaccharides (EPS). We also find that PGA production is positively correlated with higher temperatures, resulting in high-temperature mature biofilm morphologies that are distinct from the rugose colony biofilm architecture typically associated with B. subtilis. Although previous reports have suggested that PGA production does not play a major role in biofilm morphology in the undomesticated isolate NCIB 3610, our results suggest that this strain produces distinct biofilm matrices in response to environmental conditions.