(p)ppGpp Measurements by Thin Layer Chromatography (TLC)

To determine the (p)ppGpp levels in cells we modified the method from Cashel (1994) and Tian et al. (2016). In brief, overnight cultures were diluted 100-fold in 5 ml of MOPS glucose minimal medium supplemented with all five nucleobases (10 μg/ml of each, Sigma-Alderich) and incubated at 37°C with shaking. All of the strains had similar growth rates in this medium. At an optical density at 600 nm (OD₆₀₀) of ≈0.5, cells were diluted 10-fold to an OD₆₀₀ of ≈0.05 and were left to grow with shaking at 37°C with H₃³²PO₄ (100 μCi/ml). When looking at the effect of RelA^CTD expressed from the low copy number plasmid (Figure 2C and Supplementary Figure S4), 1 mM IPTG was also added at this point. The cells were then grown for ≈2 generations (OD₆₀₀ ≈0.2) before amino acid starvation was induced by the addition of either 500 μg/ml valine (Sigma-Aldrich) or 400 μg/ml serine hydroxamate (SHX) (Sigma-Aldrich). Whereas, due to its inhibitory effect on growth, RelA^CTD was expressed from the high copy number plasmid for 30 min after the cells were grown for ≈2 generations (OD₆₀₀ ≈0.2). Subsequently, amino acid starvation was induced by the addition of valine (Figure 1C). Fifty-microliter samples were withdrawn before and at various times (see figures) after the induction of amino acid starvation. With the induction of RelA and RelA^ΔRRM (Figure 4D), after labeling of cells for ≈2 generations, 1 mM IPTG was added and 50 μl samples were taken at −2, 10, 30, and 60 min. The reactions were stopped by the addition of 10 μl of ice-cold 2 M formic acid. A 10 μl aliquot of each reaction mixture was loaded on polyethyleneimine (PEI) cellulose TLC plates (GE Healthcare) and separated by chromatography in 1.5 M potassium phosphate at pH 3.4. The TLC plates were revealed by phosphorimaging (GE Healthcare) and analyzed using ImageQuant software (GE Healthcare). The increase in the level of (p)ppGpp was normalized to the basal level (time zero) for each strain.

Low-level ectopic expression of RelA^CTD does not interfere with the functionality of endogenous RelA. (A,B) Overnight cultures of E. coli MG1655, transformed with low copy IPTG inducible vector pNDM220 (vector) or pNDM220:relA^CTD, were grown at 37°C overnight in M9 minimal medium with 30 μg/ml ampicillin. Ten-fold serial dilutions were made and spotted onto LB agar (LBA) and SMG plates, both supplemented with 30 μg/ml ampicillin and 1 mM IPTG. (C) Representative audioradiogram of a PEI Cellulose TLC plate showing (p)ppGpp accumulation of E. coli MG1655 carrying pNDM220 (vector) or pNDM220:relA^CTD upon valine-induced isoleucine starvation. The curves represent the average fold increase for three independent measurements, and the error bars represent standard errors. The levels of (p)ppGpp were normalized to the pre-starved level for each strain at –2 min. See experimental procedures for more details.

High-level ectopic expression of RelA^CTD impairs translation. Exponentially growing E. coli MG1655 cells harboring the CII-YFP plasmid were back-diluted to OD₆₀₀ 0.05, before the expression of relA, catalytically inactive G251E mutant relA^∗, or relA^CTD from high copy vector pMG25 was induced by addition of 1 mM IPTG (indicated by the arrow). (A) Growth, measured at 600 nm (OD₆₀₀) and (B) the fluorescence of YFP, measured at 520 nm was monitored every 15 min for 11 h. See section “Materials and Methods” for more details.