Quantification of c-di-GMP using HPLC

c-di-GMP was quantified using HPLC as described previously (Ueda and Wood, 2009 (link)) with slight modifications. Strains were grown in 1 L LB medium for 16 h at 250 rpm. HPLC was conducted using C18 reverse-phase column (150^*3.9 mm, 4 μm, Nova-Pak, Waters) at a flow rate of 1 ml/min. Solvent A was 0.15 M TEAA buffer (pH 5.0). Solvent B was acetonitrile. The gradient was as follows: t = 0, 0% solvent B; t = 35 min, 12% solvent B; t = 36 min, 80% solvent B; t = 41 min, 80% solvent B; t = 42 min, 0% solvent B; t = 55 min, 0% solvent B. Each sample had a running time of 55 min. A photodiode array detector (Waters, Milford, MA) was used to detect nucleotides at 254 nm after the HPLC separation step. Synthetic c-di-GMP (BIOLOG Life Science Institute, Bremen, Germany) was used as a standard. The peak corresponding to c-di-GMP from the extract of the shrA mutant was verified by co-elution with standard c-di-GMP. This experiment was performed with two independent cultures.