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

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.