ppGpp Measurements by HPLC and Estimation of Intracellular Concentrations.

Synechococcus cultures grown under the conditions specified in the text were harvested by filtration. For the light/dark shift experiment shown in Fig. 1A, a WT culture was grown to log phase (OD₇₅₀ ∼0.25) and split into seven flasks with 57 mL each (corresponding to ∼13 OD₇₅₀ units), and each culture was harvested at the appropriate time point. For the experiment shown in Fig. 1B, cultures of the appropriate strains were grown to log phase (OD₇₅₀ ∼0.4), and strains in the light (control and ppGpp⁺) were induced with IPTG for 17 h before harvesting 20 OD₇₅₀ units per culture.

Cells were isolated by filtration, resuspended, and lysed in 400 μL of 13 M formic acid, subsequently flash-frozen in liquid nitrogen, and stored at −80 °C until further processing. Lysates were thawed at room temperature, were subjected to three freeze/thaw cycles alternating between a dry ice/ethanol bath and room temperature, and were clarified by centrifugation (16,000 × g, 5 min, 4 °C). Extracts were filtered using PES syringe filters and were flash-frozen in liquid nitrogen and stored at −80 °C until HPLC analysis.

Levels of ppGpp were measured using anion-exchange chromatography and a Phenomenex Luna NH₂ column, 50 × 4.60 mm. Using a protocol modified from ref. 61, an isocratic method was developed to isolate the ppGpp peak on an Agilent Technologies 1200 series HPLC. The buffer consisted of 0.85 M ammonium phosphate, pH 2.1. Samples were thawed on ice before transfer to a prechilled HPLC vial; all samples were kept on ice until just before injection. Then, 25 μL was injected at time 0, and the method was run at 1 mL/min for 8–10 min. The column temperature was maintained at 30 °C, and absorbance was monitored at 254 nm. A small ppGpp peak was found to elute at ∼3.5 min. Concentrations were estimated by manually integrating peaks using Agilent Chemstation software and comparing with a ppGpp standard added to the sample. The ppGpp standard was obtained from TriLink Biotechnologies. Using this method, the limit of detection for ppGpp was ∼1 μM. We did not measure pppGpp by HPLC because we were unable to resolve any candidate peaks eluting after ppGpp.

To estimate intracellular ppGpp concentrations, we used the equation

where each parameter was determined as follows: mAU⋅s, value determined for each sample by integrating ppGpp peaks from HPLC traces; moles/(mAU⋅s) conversion, determined by running a standard curve with known molar amounts of the ppGpp standard vs. measured mAU⋅s and taking the slope of a linear regression (R² = 0.998) [value = 2.4 × 10⁻¹² moles/(mAU⋅s)]; fraction of total lysate measured, 25 μL was analyzed by HPLC from an original volume of 400 μL (value = 16); efficiency of ppGpp extraction, determined by spiking a known amount of ppGpp into the 400 μL of formic acid used to lyse cells and measuring how much was recovered compared with that measured after spiking in ppGpp at the end of the extract preparation (∼37% efficiency; value = 2.69); number of cells, determined by plating dilutions from cultures harvested for ppGpp extraction and averaging the number of colony-forming units across replicates (value = 4.69 × 10⁹ cells); and estimated cell volume, determined by analyzing microscopy images of WT Synechococcus using MicrobeTracker software (62) and converting pixels³ into μm³ into L (value = 3 fL = 3 × 10⁻¹⁵ L).