ppGpp extraction and measurement

ppGpp was extracted as described (30) with modifications. To this end, aliquots of ~100 mg of frozen cecal samples were transferred from −80°C storage to dry ice. Samples were extracted individually by the addition of 1 mL of ice-cold 2 M formic acid along with ~250 mg of acid-washed beads (BioSpec). Samples were then transferred to ice to thaw for 5 min before disruption by bead beating (Biospec) for 2 × 20 s with 1 min on ice between each 20-s treatment. Homogenized samples were then transferred to 15-mL conical tubes (Corning), and 2 mL of ice-cold 2 M formic acid was added to each sample. After 30 min incubation on ice, 50 mM NH₄OAc (pH 4.5) was added to each sample to bring the total volume to 6 mL. One sample per tissue group/time was split into two 3-mL aliquots in 15-mL conical tubes: 20 µL of 200 µm ppGpp or pppGpp standard (Jena Biosciences) was added to one of the aliquots to estimate extraction efficiency. Samples were then centrifuged at 2,400g for 5 min at 4°C or until the material was firmly pelleted at the bottom of the conical tube. Samples were then placed on ice prior to extraction using an Oasis WAX 1 cc Vac Cartridge, 30 mg Sorbent per Cartridge, 60 µm (Waters).

The Oasis WAX 1 cc Vac Cartridge was pre-treated with 1 mL of MeOH followed by 1 mL of 50 mM NH₄OAc (pH 4.5), and the supernatant of the sample solution was loaded 1 mL at a time onto the column; the column was washed with 1 mL of 50 mM NH₄OAc (pH 4.5), followed by 1 mL of MeOH. The nucleotide pool was eluted from the cartridge with 1 mL MeOH/H₂O/NH₄OH (20:70:10) solution into a 15-mL conical tube (Corning) and immediately frozen on dry ice before storing at −80°C for at least 2 h before preparing samples for lyophilization.

To lyophilize the extracted samples, tubes were transferred to dry ice, and caps were removed and replaced with a Kimwipe (KimTech) secured with a rubber band around the neck of the tube. Samples were returned to −80°C for 30 min to ensure the samples were frozen before lyophilization. Frozen samples were quickly transferred on dry ice to a lyophilizer (Labconco), and dry ice was packed around the glass holder to ensure samples remained frozen while reaching pressure. Lyophilized samples were stored at −80°C.

For measurement, lyophilized samples were dissolved in 100 µL of nuclease-free water, filtered through a Qiaquick spin column (Qiagen) by centrifugation for 1 min at 13,000g, and immediately used for ppGpp quantification. After measurement, samples were stored in HPLC vials at −80°C and retained similar ppGpp measurements for at least 3 months.

Chromatography was performed as described (31) with modifications. Specifically, 20 µL of extracted samples and standards were analyzed with an Agilent 1200 Infinity Series HPLC System (equipped with a Sphereclone SAX column, 4.6 × 150 mm, 5 µm) via DAD detection. The mobile phase employed a 2 mL/min isocratic delivery of 0.36 M NH₄H₂PO₄ (pH 3.4, 2.5% [vol/vol] acetonitrile) up to 1 h. The detection wavelength was set to 252 nm. The mobile phase was prepared with analytical grade NH₄H₂PO₄ (>99% pure), H₂O filtered through a Milli-Q system, and HPLC-grade acetonitrile. The pH of the solution was adjusted to 3.4 through the addition of H₃PO₄ (85% aq. solution). Output data were converted into concentrations using a standard curve of ppGpp (Jena Biosciences).