Quantifying Stringent Response Alarmones

Cellular levels of the stringent response alarmones, i.e., ppGpp and pppGpp, were measured basically as described previously [24 (link)], with modifications. The overnight bacterial culture was grown in the MOPS (4-morpholinepropanesulfonic acid) minimal medium and then was diluted in the same medium, but with a low phosphate concentration (0.4 mM) and then cultivated to A₆₀₀ of 0.2. Then, the cultures were diluted in a 1:10 ratio in the same medium, but with addition of [³²P]orthophosphoric acid (150 μCi/mL) and grown for two generations. Next, at time zero, ITCs at 1 × MIC, or serine hydroxamate (SHX), at 1 mM was added. At specific times, samples were collected and lysed with formic acid (13 M) in three cycles of the freeze-thaw procedure. Following centrifugation, nucleotides were separated by thin-layer chromatography on PEI cellulose plates (Sigma-Aldrich, Darmstadt, Germany) in 1.5 M potassium phosphate buffer (pH 3.4). The chromatograms were analyzed using the Typhoon 9200 Phosphorimager (GE Healthcare, Uppsala, Sweden). QuantityOne (BioRad, Hercules, CA, USA) software was used for densitometry analysis.