A moonlighting enzyme imposes second messenger bistability to drive lifestyle decisions in E. coli.

Bacteria preferentially colonize surfaces and air-liquid interfaces as matrix embedded communities called biofilms. Biofilms exhibit specific physiological properties, including general stress tolerance, increased antibiotic recalcitrance and tolerance against phagocytic clearance. Together this largely accounts for increased biofilm persistence, chronic infections and infection relapses. One of the principle regulators of biofilm formation is c-di-GMP, a bacterial second messenger controlling various cellular processes. Cellular levels of c-di-GMP are controlled by two antagonistic enzyme families, diguanylate cyclases and phosphodiesterases. But despite the identification and characterization of an increasing number of components of the c-di-GMP network in different bacterial model organisms, details of c-di- GMP mediated decision-making have remained unclear. In particular, how cells shuttle between specific c-di-GMP regimes at the population and single cell level is largely unknown and moreover how these transitions are deterministically made in time and space, given that bacterial networks of diguanylate cyclases and phosphodiesterases show a high degree of complexity. Here we describe a novel mechanism regulating c-di-GMP mediated biofilm formation in E. coli. This mechanism relies on the bistable expression of a key phosphodiesterase that acts both as catalyst for c- di-GMP degradation and as a transcription factor promoting its own production. Bistability results from two interconnected positive feedback loops operating on the catalytic and gene expression level. Based on genetic, structural and biochemical analyses we postulate a simple substrate-induced switch mechanism through which this enzyme can sense changing concentration of c-di-GMP and convert this information into a bistable c-di-GMP response. This mechanism may explain how cellular heterogeneity of small signaling molecules is generated in bacteria and used as a bet hedging strategy for important lifestyle transitions