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. 2019 Dec 17;10:5763. doi: 10.1038/s41467-019-13764-4

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 1 — a Genetic setup based on the multicopy suppression (ASKA library) of the nongrowing phenotype of ΔrelA mutant on SMG plates. SMG plate induces isoleucine starvation and requires high (p)ppGpp level to de-repress isoleucine biosynthesis operon (see the Methods section). Question mark denotes putative candidate that promotes SpoT-dependent (p)ppGpp accumulation. b ytfK overexpression suppresses the nongrowing phenotype of the ΔrelA mutant on SMG plates. WT and ΔrelA mutant were transformed with pEG25 harboring either relA, ytfK, or spoT under an IPTG-inducible promoter. Cells were serial diluted and spotted both on nutrient agar (NA) and SMG plates with or without IPTG. This experiment was repeated five times with identical results. Additional controls and concentration of IPTG are provided in Supplementary Fig. 1. c In vivo (p)ppGpp accumulation following ectopic expression of ytfK. ΔrelA mutant carrying ytfK on pEG25 was grown exponentially in phosphate MOPS minimal medium (see the Methods section). Samples were collected before and after ytfK induction (1 mM IPTG) prior to nucleotide extraction and separation by TLC. Representative autoradiograph of the TLC plates is shown. This experiment was repeated three times with equivalent pattern of (p)ppGpp accumulation. Source data are provided as a Source Data file.