Intracellular c-di-GMP quantification

Intracellular c-di-GMP levels were measured as previously described (Wu et al., 2022). Briefly, the second round of bacterial culture was diluted 1000-fold into 5 mL of HI broth containing 0, 0.2 and 0.4 μg/mL chloramphenicol, respectively, and incubated at 37°C with shaking to an OD₆₀₀ value of 1.5. Bacterial cells were harvested and resuspended in 2 mL ice-cold phosphate buffered saline (PBS), and then incubated at 100°C for 5 min, followed by sonicated for 15 min (power 100%, frequency 37 kHz) in an ice-water bath. The supernatant containing c-di-GMP was collected, and the pellet was resuspended in 2 mL ice-cold PBS and re-extracted for another two times. The intracellular c-di-GMP levels were determined with a c-di-GMP Enzyme-linked Immunosorbent Assay (ELISA) Kit (Mskbio, Beijing, China). In addition, total protein in the supernatant was also determined by a Pierce BCA Protein Assay kit (ThermoFisher Scientific, USA). Intracellular c-di-GMP levels were expressed as pmol/g protein.