Details
| Original language | English |
|---|---|
| Pages (from-to) | 705-713 |
| Number of pages | 9 |
| Journal | Molecular microbiology |
| Volume | 117 |
| Issue number | 3 |
| Early online date | 27 Dec 2021 |
| Publication status | Published - 18 Mar 2022 |
Abstract
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
- bimolecular fluorescence complementation, biosensor, BldD, c-di-GMP, diguanylate cyclase, EAL, GGDEF, phosphodiesterase
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology(all)
- Molecular Biology
- Immunology and Microbiology(all)
- Microbiology
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In: Molecular microbiology, Vol. 117, No. 3, 18.03.2022, p. 705-713.
Research output: Contribution to journal › Article › Research › peer review
}
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 -