Synthetic c di gmp

Nucleotide-enriched fractions were extracted from late-exponential growing cells from sulfur, thiosulfate or tetrathionate cultures. Sulfur cultures were separated in two independent cell populations: planktonic and biofilm cells [24 (link)]. The extraction was realized by hot lysis and HClO₄ treatments [53 (link)]. HPLC analysis was performed by a HPLC coupled to photodiode array detector (Waters 1525, 2996) (Waters, Milford, MA, USA) using a 15 cm × 3 mm SUPELCOSIL LC-18-DB C18, 3 μm particle size, Reverse Phase Column (SIGMA, Saint Louis, MO, USA). The liquid chromatography (LC) system consisted of degasser (Waters), binary pump (Waters) and oven (Waters). The mobile phase was methanol (A) and water pH 6.0 (6 mM KH₂PO₄) (B). Elution conditions were 5 min at 100% B, 15 min linear gradient from 100% B to 20% A and 80% B and finally 10 min with a gradient from 20% A and 80% B to 100% B with a constant flow of 0.4 mL/min. The temperature was set at 30 ± 3 °C. The injection volume was 20 µL. The calibration curve was performed with synthetic c-di-GMP (BIOLOG, Hayward, CA, USA) in a range of 6.9–552 ng (10–800 pmol) for injection. Signal signatures were identified by coincidence of retention times of 12.531 ± 0.295 min and comparison of absorption spectra at 252.4 nm. Data were expressed as pmol c-di-GMP and normalized against cellular wet weight.

Intracellular c-di-GMP levels in Y. pestis were detected as previously described (Ren et al., 2014 (link)). Overnight cultures of Y. pestis strains KIM6+ were diluted to an OD600 of 0.05 in LB broth supplemented with 4 mM CaCl₂, 4 mM MgCl₂, 2 mM FeCl₂, 100 μm DIP, 1 mM CuSO4, 0.01% SDS, 6% sucrose, 4 mM DTT, 10 mM H₂O₂, or 4% NaCl, or modified to pH 5 and grown at an appropriate temperature to OD₆₀₀ of 0.8. Cell pellets were collected and resuspended in 50 μL of extraction buffer (40% methanol and 40% acetonitrile prepared in 0.1 M formic acid) per 48 mg of wet cell weight. The slurries were incubated for 30 min at -20°C, and insoluble material was removed by centrifugation at 4°C. The supernatants were neutralized by adding 4 μL of 15% NH₄HCO₃ per 100 μL of sample. Ten microliters of each sample was analyzed using liquid chromatography tandem mass spectrometry. Synthetic c-di-GMP (BIOLOG Life Science Institute, Bremen, Germany) was used as a standard. Results from three independent experiments were analyzed using a one-way ANOVA with Dunnett’s test.

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.