<i>B. subtilis</i> uses c-di-GMP accumulation as a long-term decision making factor during biofilm formation

Cells such as bacteria that make decisions about their fate often do so over timescales longer than a single cell cycle, which poses a design challenge. Here, we suggest that B. subtilis uses c-di-GMP accumulation as a long-term decision making factor for biofilm formation. C-di-GMP is implicated in motility switching and biofilm matrix production. Previous literature has shown that during biofilm formation, B. subtilis exhibit a bimodal distribution of c-di-GMP. This is thought to be driven by transcriptional repression of the enzyme that degrades c-di-GMP. We constructed a model that stochastically simulates formation and degradation of c-di-GMP and examined distributions of intracellular concentrations across a population. The model predicts that accumulation in the high subpopulation occurs over five or six cell generations. To experimentally confirm these predictions, we use a fluorescent c-di-GMP sensor. Bulk population measurements of c-di-GMP accumulation rates appear to take 150 minutes to saturate, roughly corresponding to the five cell generation prediction. Together, these results suggest that through c-di-GMP accumulation, B. subtilis uses intracellular signaling factors to integrate information beyond an individual cell cycle to coordinate biofilm formation.