BldD-based bimolecular fluorescence complementation for in vivo detection of the second messenger cyclic di-GMP

The widespread bacterial second messenger bis-(3-5)-cyclic diguanosine monophosphate (c-di-GMP) is an important regulator of biofilm formation, virulence and cell differentiation. C-di-GMP-specific biosensors that allow detection and visualization of c-di-GMP levels in living cells are key to our understanding of how c-di-GMP fluctuations drive cellular responses. Here, we describe a novel c-di-GMP biosensor, CensYBL, that is based on c-di-GMP-induced dimerization of the effector protein BldD from Streptomyces resulting in bimolecular fluorescence complementation of split-YPet fusion proteins. As a proof-of-principle, we demonstrate that CensYBL is functional in detecting fluctuations in intracellular c-di-GMP levels in the Gram-negative model bacteria Escherichia coli and Salmonella enterica serovar Typhimurium. Using deletion mutants of c-di-GMP diguanylate cyclases and phosphodiesterases, we show that c-di-GMP dependent dimerization of CBldD-YPet results in fluorescence complementation reflecting intracellular c-di-GMP levels. Overall, we demonstrate that the CensYBL biosensor is a user-friendly and versatile tool that allows to investigate c-di-GMP variations using single-cell and population-wide experimental set-ups. ImportanceThe second messenger c-di-GMP controls various bacterial functions including development of resistant biofilm communities and transition into dormant spores. In vivo detection of c-di-GMP levels is therefore crucial for a better understanding of how intracellular c-di-GMP levels induce changes of bacterial physiology. Here, we describe the design of a novel c-di-GMP biosensor and demonstrate its effective application in investigating fluctuations in intracellular c-di-GMP levels in Escherichia coli and Salmonella enterica serovar Typhimurium on a population-based and single-cell level.