Quantification of the intracellular c-di-GMP concentration.

c-di-GMP was extracted from whole cells as previously described (12). Briefly, the indicated strains were grown overnight in LB or LB^Ca with antibiotics when appropriate. The next day, cells were diluted 1:100 in fresh medium and grown to an OD₆₀₀ of 0.5. Cells were again diluted to an OD₆₀₀ of 0.025 into fresh LB supplemented with the indicated cations and l-Ara where indicated. Cells were grown for 12 h, when peak brp expression was observed in the presence of CaCl₂. Six OD units of each sample was centrifuged at 4°C for 10 min at 12,000 × g. The pellet was resuspended in 200 µl of 40% (vol/vol) acetonitrile–40% (vol/vol) methanol–20% 0.1 N formic acid. This suspension was incubated at −20°C for 30 min and then centrifuged at 4°C for 5 min. The supernatant was recovered, and 10 µl of each sample was analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) on a Quattro Premier XE mass spectrometer (Waters) coupled with an Acquity Ultra Performance LC system (Waters). c-di-GMP was detected by electrospray ionization using multiple reaction monitoring in negative-ion mode at m/z 689.16→344.31. The MS parameters were as follows: capillary voltage, 3.5 kV; cone voltage, 50 V; collision energy, 34 V; source temperature, 110°C; desolvation temperature, 350°C; cone gas flow (nitrogen), 50 liters/h; desolvation gas flow (nitrogen), 800 liters/h; collision gas flow (nitrogen), 0.15 ml/min; and multiplier voltage, 650 V. Chromatography separation was reverse phase using a Waters BEH C₁₈ 2.1- by 50-mm column at a flow rate of 0.3 ml/min with a gradient of 10 mM tributylamine plus 15 mM acetic acid in 97:3 water-methanol (solution A) to methanol (solution B) with the following parameters: t = 0 min; 99% A:1% B, t = 2.5 min; 80% A:20% B, t = 7.0 min; 35% A:65% B, t = 7.5 min; 5% A:95% B, t = 9.01 min; and 99% A:1% B, t = 10 min. A c-di-GMP standard curve for calculating the c-di-GMP concentration in each extract was generated by dissolving synthesized c-di-GMP (Axxora) in extraction buffer to 250 nM, followed by 2-fold serial dilutions to 0.975 nM. Data from three biological replicates were analyzed for each strain.