Determination of intracellular c-di-GMP levels.

c-di-GMP extraction was performed as previously described (58). Briefly, V. cholerae wild-type, ΔvxrB, ΔvxrC, and ΔvxrB Tn7::vxrB-complemented strains were grown in LB broth to an OD₆₀₀ of 0.4 before 40 ml of culture was harvested at 4,000 × g for 30 min. Cell pellets were allowed to dry briefly and then resuspended in 1 ml extraction solution (40% acetonitrile, 40% methanol, 0.1% formic acid, 19.9% high-pressure liquid chromatography [HPLC]-grade water), and incubated on ice for 15 min. Samples were then centrifuged at 16,000 × g for 5 min and 800 μl of supernatant was dried under vacuum and then lyophilized. Samples were resuspended in 50 μl of 184 mM NaCl and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) on a Thermo-Electron Finnigan LTQ mass spectrometer coupled to a surveyor HPLC. The amount of c-di-GMP in samples was calculated with a standard curve generated from pure c-di-GMP suspended in 184 mM NaCl (Biolog Life Science Institute, Bremen, Germany). The concentrations used for the standard curve generation were 50 nM, 100 nM, 500 nM, 2 μM, 3.5 μM, 5 μM, 7.5 μM, and 10 μM. The assay is linear from 50 nM to 10 μM, with an R² of 0.999. The c-di-GMP levels were normalized to total protein per ml of culture.

To determine protein concentration, 4 ml from each culture was harvested, the supernatant was removed, and cells were lysed in 1 ml of 2% sodium dodecyl sulfate (SDS). Total protein in the samples was determined with a bicinchoninic acid (BCA) assay (Thermo Fisher, Waltham, MA) using bovine serum albumin (BSA) as the standard. Each c-di-GMP quantification experiment was performed with four biological replicates. Statistical analysis was performed using ANOVA and Bonferroni's multiple-comparison test.