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

Figure 1. NirD can limit the RelA-dependent accumulation of (p)ppGpp.

(A) Schematic representation of the genetic assay using ASKA library to identify genes that in multiple copies would suppress the growth defect associated with toxic over-accumulation of (p)ppGpp. Question marks denote a putative candidate that limits RelA-dependent accumulation of (p)ppGpp. (B) NirD suppresses the growth defect associated with RelA overproduction. Wild-type E. coli MG1655 cells were co-transformed with the plasmid derivatives pBbS2k-relA and pEG25-nirD inducible by anhydrotetracycline (aTc) and isopropyl β-D-1-thiogalactopyranoside (IPTG), respectively. Serial dilutions of stationary-phase cultures were spotted on NA plates containing the indicated concentrations of inducers. Additional controls and inducer concentrations are shown in Figure 1—figure supplement 1A. The results are representative of three independent experiments with similar results. (C, D) In vivo (p)ppGpp dynamic in E. coli MG1655 cells carrying pBbS2k-relA and pEG25-nirD after consecutive expression of the relA and nirD genes using 100 ng/mL aTc and 1 mM IPTG, respectively. Nucleotides extracted from samples collected at the indicated times were separated by thin layer chromatography. The autoradiogram (C) is representative of three independent experiments, and the curves of the relative levels of (p)ppGpp (D) are represented as the means of the three independent experiments, the error bars depicting the SDs.

Figure 1—source data 1. Raw autoradiogram.
Figure 1—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 1D, which are represented as the means and SDs of three independent experiments.

Figure 1.

Figure 1—figure supplement 1. NirD can inhibit RelA-dependent (p)ppGpp synthesis.

Figure 1—figure supplement 1.

(A) Growth assay of the E. coli MG1655 wild-type strain and its isogenic ΔrelA spoT mutant co-transformed with the plasmid derivatives pBbS2k-relA (sd4) and pEG25-nirD inducible by anhydrotetracycline (aTc) and isopropyl β-D-1-thiogalactopyranoside (IPTG), respectively. Serial dilutions of stationary-phase cultures were spotted on NA plates containing the indicated concentrations of inducers. The results are representative of three independent experiments. (B) Growth assay of the ΔrelA mutant carrying pEG25-nirD or pEG25-spoT. Serial dilutions of stationary-phase cultures were spotted on M9-glucose minimal medium plates containing the indicated concentrations of IPTG. The results are representative of three independent experiments.