2.4 Extraction and quantification of intracellular c-di-GMP
c-di-GMP was extracted as previously reported, with some modifications (Xu et al., 2013). Briefly, 15 mL of Z. mobilis culture was grown to the exponential stage and centrifuged at 5, 000 g for 3 min. The pellet was immediately suspended in 1000 μL buffer (40% methanol: 40% acetonitrile: 20% dH2O) by vigorous vortexing at −20°C for 30 min, which was by centrifuged at 12, 000 g and 4 °C for 5 min, and the supernatant was collected into a tube cooled with ice. Cell debris was extracted twice and the supernatant was collected. All supernatants were combined for vacuum evaporation to condense c-di-GMP, which was dissolved in 80 µL of buffer (50% acetonitrile and 50% dH2O) for analysis.
c-Di-GMP quantification was performed using a Waters I-Class Acquity UPLC (Waters, UK) coupled with a Vion IMS QToF mass spectrometer (Waters, UK). The separation of c-di-GMP was performed using a SeQuant ZIC-HILIC column (100 mm × 2.1 mm) packed with 3.5 µm polyetheretherketone and operated at 45°C; and the mobile phase composed of 50 mM ammonium formate in water (A), and acetonitrile (B) was pumped at 0.4 mL/min under the following gradient elution conditions: 0–10 min, 90–50% B; 10–12 min, 50–90% B; 12–15 min, 90% B. c-di-GMP was detected through electrospray ionization operated in the negative-ion mode. The software UNIFI 1.8.1 was used for data processing.
c-Di-GMP with a purity of 98% (Biolog, Germany) was used as the standard to calibrate the analysis.