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. The second messenger c‐di‐GMP controls various bacterial functions including development of resistant biofilm communities and transition into dormant spores. 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 on both population and single‐cell levels.