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. 2021 Jul 29;10:e64092. doi: 10.7554/eLife.64092

Figure 2. NirD inhibits accumulation of (p)ppGpp upon amino acid starvation.

Figure 2.

(A) NirD inhibits cell growth on M9-glucose minimal medium supplemented with serine, methionine, and glycine (SMG). Stationary-phase cultures of wild-type E. coli MG1655 strain harboring pEG25-nirD or pEG25-spoT were spotted on NA and SMG plates containing the indicated concentrations of isopropyl β-D-1-thiogalactopyranoside (IPTG). The results are representative of three independent experiments. (B, C) Exponentially growing cells of wild-type E. coli MG1655 harboring pEG25 or pEG25-nirD were challenged for amino acid starvation by addition of 500 µg/mL of L-valine. nirD is induced by the addition of 1 mM of IPTG. Nucleotides were extracted and separated by thin layer chromatography. The autoradiogram (B) is representative of three independent experiments, and the curves of the relative levels of (p)ppGpp (C) are represented as the means of the three independent experiments, the error bars depicting the SDs.

Figure 2—source data 1. Raw autoradiogram.
Figure 2—source data 2. Quantification of (p)ppGpp.
The amount of (p)ppGpp was normalized to total amount of G nucleotides observed in each sample. Total G is the sum of GTP and (p)ppGpp detected. The source data are provided for the relative levels of (p)ppGpp in Figure 2C, which are represented as the means and SDs of three independent experiments.