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bioRxiv · 10.1101/2024.08.21.609041

A toolbox of FRET-based c-di-GMP biosensors and its FRET-To-Sort application for genome-wide mapping of the second messenger regulatory network

Wang, L.·Malengo, G.·Medeiros, A. S.·Chen, X.·Teteneva, N.·Gonzalez, S. S.·Hammond, M.·Sourjik, V.
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Abstract

C-di-GMP is a widespread second messenger, coordinating various cellular functions in bacteria. Levels of c-di-GMP can be highly dynamic and vary over a wide range of concentrations. Here we constructed a large set of FRET-based c-di-GMP biosensors, using homologues of c-di-GMP-binding effector YcgR from different bacterial species. This biosensor library was characterized using a newly established protocol to quantify FRET efficiency using flow cytometry. The resulting toolbox of 18 selected biosensors that undergo large FRET signal change upon c-di-GMP binding displays a [~]100-fold range of c-di-GMP binding affinities. We combined this toolbox with a barcoded Tn5 transposon library and cytometry-based cell sorting to develop FRET-To-Sort, a new application for systematic characterization of gene networks regulating levels of FRET-detected small molecules. Applied to planktonic E. coli cells, FRET-To-Sort identified both known and novel regulatory modules controlling c-di-GMP levels, including flagellum and fimbria biogenesis, lipid metabolism and stress response genes.

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Wang, L., Malengo, G., Medeiros, A. S., Chen, X., Teteneva, N., Gonzalez, S. S., Hammond, M., Sourjik, V.. 2024-08-21. A toolbox of FRET-based c-di-GMP biosensors and its FRET-To-Sort application for genome-wide mapping of the second messenger regulatory network. https://doi.org/10.1101/2024.08.21.609041

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