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

verfasst von

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.

Organisationseinheit(en)

Institut für Mikrobiologie

Externe Organisation(en)

Humboldt-Universität zu Berlin
Max-Planck-Forschungsstelle für die Wissenschaft der Pathogene (MPUSP)

Typ

Artikel

Journal

Molecular microbiology

Band

117

Seiten

705-713

Anzahl der Seiten

9

ISSN

0950-382X

Publikationsdatum

18.03.2022

Publikationsstatus

Veröffentlicht

Peer-reviewed

Ja

ASJC Scopus Sachgebiete

Molekularbiologie, Mikrobiologie

Elektronische Version(en)

https://doi.org/10.1111/mmi.14876 (Zugang: Offen)