TY - JOUR

T1 - BldD-based bimolecular fluorescence complementation for in vivo detection of the second messenger cyclic di-GMP

AU - Halte, Manuel

AU - Wörmann, Mirka E

AU - Bogisch, Maxim

AU - Erhardt, Marc

AU - Tschowri, Natalia

N1 - Funding information: This work was supported in part by the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program (grant to Marc Erhardt; agreement no. 864971). Research in Natalia Tschowri's lab is funded by the DFG Emmy Noether?Program (TS 325/1?1) and the DFG Priority Program SPP 1879 (TS 325/2?2). We thank Heidi Landmesser and Kristin Funke for expert technical assistance. Open access funding enabled and organized by ProjektDEAL.

PY - 2022/3/18

Y1 - 2022/3/18

N2 - 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.

AB - 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.

KW - bimolecular fluorescence complementation

KW - biosensor

KW - BldD

KW - c-di-GMP

KW - diguanylate cyclase

KW - EAL

KW - GGDEF

KW - phosphodiesterase

UR - http://www.scopus.com/inward/record.url?scp=85122705115&partnerID=8YFLogxK

U2 - 10.1111/mmi.14876

DO - 10.1111/mmi.14876

M3 - Article

C2 - 34961989

VL - 117

SP - 705

EP - 713

JO - Molecular microbiology

JF - Molecular microbiology

SN - 0950-382X

IS - 3

ER -