Fluorescence-activated cell sorting FACS analysis

All samples were analyzed using a Beckman CytoFLEX SRT flow cytometer equipped with a 100 μm nozzle, and normal saline served as the sheath fluid. The c-di-GMP sensor-labeled strain in the 24 hr static biofilm growth phase was washed and resuspended in sterile PBS. Bacterial cells were identified based on FSC (forward scatter) and SSC (side scatter) parameters. Cells were sorted into distinct groups according to their fluorescence intensity (PB450 for BFP, FITC for mVenus^NB, ECD for mScarlet-I). The obtained results were analyzed using FlowJo V10 software (Treestar, Inc).