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

Mol Microbiol. 2022 Mar;117(3):705-713. doi: 10.1111/mmi.14876. Epub 2022 Jan 10.


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.

Keywords: BldD; EAL; GGDEF; bimolecular fluorescence complementation; biosensor; c-di-GMP; diguanylate cyclase; phosphodiesterase.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cyclic GMP* / analogs & derivatives
  • Cyclic GMP* / metabolism
  • Escherichia coli / metabolism
  • Fluorescence
  • Salmonella typhimurium / metabolism
  • Second Messenger Systems*


  • bis(3',5')-cyclic diguanylic acid
  • Cyclic GMP