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

authored by

Manuel Halte, Mirka E Wörmann, Maxim Bogisch, Marc Erhardt, Natalia Tschowri

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.

Organisation(s)

Institute of Microbiology

External Organisation(s)

Humboldt-Universität zu Berlin
Max Planck Unit for the Science of Pathogens (MPUSP)

Type

Article

Journal

Molecular microbiology

Volume

117

Pages

705-713

No. of pages

9

ISSN

0950-382X

Publication date

18.03.2022

Publication status

Published

Peer reviewed

Yes

ASJC Scopus subject areas

Molecular Biology, Microbiology

Electronic version(s)

https://doi.org/10.1111/mmi.14876 (Access: Open)