Extraction, Purification, and Detection of ppGpp

To determine the intercellular level of ppGpp in M. aeruginosa under different stress conditions, 2.4-g fresh cell weight (FCW) of M. aeruginosa cells was collected, frozen in liquid nitrogen, crushed, and resuspended in 10 ml of 1 M cool formic acid. The broken cells were vigorously mixed, incubated on ice for 30 min, and then centrifuged at 10,000 rpm for 10 min at 4°C. The upper layer was transferred to a new tube and centrifuged to remove cell debris at 10,000 rpm for 10 min at 4°C. The aqueous layer was then collected and purified by solid phase extract (SPE). SPE purification was performed by using the OASIS^® WAX 3-cc Vac Cartridges (Waters, United States) according to the manufacturer’s instructions. Chromatographic separation was performed on a Waters BEH amide column (2.1 × 150 mm, 1.7 μm) according to our published method (Jin et al., 2018).