The extraction and quantification of c-di-GMP followed established methods (21). The light-responsive strain E. coli/pYYDT-RB was grown at 25°C in modified M9 minimal medium [33.7 mM Na2HPO4, 22.0 mM KH2PO4, 8.55 mM NaCl, and 9.35 mM NH4Cl supplemented with 1/10-strength LB (pH 7.0)] in the dark, NIR light (660 nm, 5.3 mW cm−2), and blue light (465 nm, 5.3 mW cm−2; pulses of 15 s light and 60 s dark). At late exponential growth phase, 15 ml of culture was collected by centrifugation at 10,000g for 5 min at 4°C for c-di-GMP extraction using organic solvents (22). The concentration of c-di-GMP was determined by using a Thermo Accela 1250 series liquid chromatography system with a Thermo Velos Pro Orbitrap mass spectrometer, as described earlier (23). The c-di-GMP concentration was normalized to total protein quantified by Qubit protein assay.

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