Measurement of in vivo ppGpp levels by thin‐layer chromatography (TLC)

Cells were grown to OD₆₀₀ ~ 0.05 as described for CdiA‐CT expression from plasmid. Cells were labelled with ³²P (orthophosphate (H₃ ³²PO₄), NEX053H005MC, PerkinElmer) to a 100 μCi/ml final concentration. After three to four generations of growth, at OD₆₀₀ of 0.25–0.3, cells were centrifuged and resuspended in spent LB + 0.02% l‐arabinose. After 15 min, D‐glucose was added to 0.1% to terminate induction, and 0.5 ml of culture was harvested. Cells were resuspended in 1 ml 0.9% NaCl and washed by centrifugation three times. Pellets were resuspended in 200 μl 0.9% NaCl, and 200 μl of 20% formic acid was added to lyse the cells. Cell lysates were kept at −20°C. Cell debris was removed by centrifugation at 10,000 g for 5 min, and 20 μl of supernatant was loaded onto a TLC PEI Cellulose membrane (Merck, Germany). As a size marker, 0.2 μM of non‐radioactive ppGpp (TriLink Biotechnologies, San Diego, USA) was applied. Chromatography was performed in 1.5 M KH₂PO₄ (pH 3.0) (modified from Schneider et al, 2003). The unlabelled ppGpp marker was visualized under UV‐light. The membrane was dried, and labelled nucleotides were visualized on a PhosphorImager (Molecular Dynamics), quantified with ImageQuant software, version 4.2a (Molecular Dynamics). Ratios of ppGpp/GTP were calculated to normalize ppGpp content to the number of cells as described previously (Denapoli et al, 2013; Benoist et al, 2015).