# Toolbox of FRET-based c-di-GMP biosensors and its FRET-To-Sort application for genome-wide mapping of c-di-GMP regulation

## TL;DR

The paper introduces a set of FRET-based biosensors to study c-di-GMP regulation in bacteria and reveals a link between flagellar function and c-di-GMP levels.

## Contribution

A toolbox of FRET-based c-di-GMP biosensors and a novel FRET-To-Sort method for genome-wide regulatory mapping in bacteria.

## Key findings

- Mutations in flagellar genes are enriched in c-di-GMP regulation.
- Inhibited flagellar rotation reduces c-di-GMP biosynthesis via increased proton motive force.

## Abstract

C-di-GMP is a widespread second messenger that coordinates transitions between different lifestyles in bacteria. Levels of c-di-GMP are controlled by complex regulatory networks, and they can vary dynamically over a wide range of concentrations. To enable studies of c-di-GMP regulation under a variety of conditions, here we construct and characterize a large set of FRET-based c-di-GMP biosensors that undergo large FRET signal changes and display a stepwise coverage of diverse binding affinities, thus capable of sensitively detecting diverse cellular c-di-GMP concentrations. We subsequently apply different-affinity FRET biosensors from this toolbox to systematically investigate genome-wide network of c-di-GMP regulation in planktonic Escherichia coli cells by establishing FRET-To-Sort, which relies on FRET-based cell sorting of a barcoded transposon library. We observe prominent enrichment of mutations in two classes of flagellar genes among those affecting c-di-GMP levels, and demonstrate that inhibited flagellar rotation reduces biosynthesis of c-di-GMP due to increased proton motive force.

Cyclic di-GMP, a bacterial second messenger, governs many cellular processes including bacterial lifestyle transitions. Here, authors develop a set of FRET-based biosensors that enables them to monitor real-time cellular dynamics of c-di-GMP and to perform a genome-wide analysis that reveals complex interplay between motility and c-di-GMP regulation.