Figure 1. Genetic tools used for perturbing cellular ppGpp levels in this study. The wild type E. coli cell has two ... Figure 1. Genetic tools used for.

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Figure 1. Genetic tools used for perturbing cellular ppGpp levels in this study. The wild type E. coli cell has two ... Figure 1. Genetic tools used for perturbing cellular ppGpp levels in this study. The wild type E. coli cell has two ppGpp synthetases, SpoT and RelA. SpoT possesses both ppGpp hydrolase activity and synthetase activity while RelA only has synthetase activity. Wild type SpoT protein contains four functional domains, the N-terminal hydrolase domain (red), the adjacent synthetase domain (green) and two C-terminal regulatory domains (blue and orange). RelA shares a similar topology structure with SpoT except that its hydrolase domain is inactive. The E. coli RelA<sup>+</sup> variant contains the N-terminal 455 residues of wild type RelA protein, exhibiting constitutive ppGpp-synthesizing activity. The E. coli RelA' variant contains the N-terminal 331 residues of wild type RelA protein, exhibiting no ppGpp-synthesizing activity. The E. coli SpoT E319Q mutant has its ppGpp synthetase activity abolished due to an E319Q mutation in the synthetase domain. The Mesh1 protein is a metazoan single-function ppGpp hydrolase from Drosophila melanogaster. It shares a similar structure topology with the hydrolase domain of SpoT. The Mesh1 H62F point mutant has its ppGpp-hydrolyzing activity completely abolished. Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Nucleic Acids Res, gkz211, https://doi.org/10.1093/nar/gkz211 The content of this slide may be subject to copyright: please see the slide notes for details.

Figure 2. The effect of increased ppGpp levels on growth rate, ribosome content and constitutive gene expression in E. ... Figure 2. The effect of increased ppGpp levels on growth rate, ribosome content and constitutive gene expression in E. coli. (A) The relA<sup>+</sup> gene is driven by Ptac promoter under the control of PlacIq-lacI cassette. (B) Effect of RelA<sup>+</sup> overexpression (OE) on the growth rate of E. coli in LB medium and glucose minimal medium. (C) The cellular guanosine tetraphosphate (ppGpp) level of E. coli upon RelA<sup>+</sup> OE. The standard deviation of ppGpp measurement by UPLC-MS is within 25%. (D) Ribosome content (RNA/protein ratio) plotted against growth rate under different degrees of RelA<sup>+</sup> protein overexpression. Data of nutrient limitation denotes the data of wild type cells growing in six different nutrient conditions including LB medium, glucose casamino acid medium, glucose minimal medium, glycerol minimal medium, acetate minimal medium and mannose minimal medium. (E) The constitutive PLtetO-lacZ activity plotted against growth rate under different degrees of RelA<sup>+</sup> protein overexpression. Data are average of triplicates with standard deviations being within 10% (approximately the size of symbol). Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Nucleic Acids Res, gkz211, https://doi.org/10.1093/nar/gkz211 The content of this slide may be subject to copyright: please see the slide notes for details.

Figure 5. Growth control by ppGpp via governing global resource allocation in E. coli. (A) ppGpp mediates the proteome ... Figure 5. Growth control by ppGpp via governing global resource allocation in E. coli. (A) ppGpp mediates the proteome resource allocation among metabolic proteins and ribosome-affiliated proteins. In a recently proposed coarse-grained model, the E. coli proteome is partitioned into three sectors, R-sector including ribosome proteins and related affiliated protein (e.g. EF-Tu, EF-G), M-sector including metabolic proteins (for nutrient processing) and the constant growth-rate independent Q-sector. At higher ppGpp level, R-sector becomes smaller while M-sector becomes larger. In contrast, lower ppGpp level leads to higher R-sector but smaller M-sector. (B) Coarse-grained schematics from nutrients metabolism to protein synthesis. The external nutrients need to be assimilated and metabolized by metabolic proteins (belonging to M-sector, ϕ<sub>M</sub>) to generate amino acids, which is further polymerized into protein product by the ribosomes (belongs to R-sector, ϕ<sub>R</sub>). The flux from metabolism to protein synthesis is balanced in a steady-state growth. Compared with normal condition, higher ppGpp limits the protein synthesis flux (due to lower ϕ<sub>R</sub>), while lower ppGpp level limits the metabolic flux (due to lower ϕ<sub>M</sub>), both causing slower growth rate compared with normal condition. The gray color means that metabolic proteins (at higher ppGpp case) and ribosomes proteins (at lower ppGpp case) become unnecessarily high (like ‘useless’ protein overexpression). The flux (orange arrow) for biomass growth becomes smaller in altered ppGpp levels due to either smaller ϕ<sub>R</sub> or ϕ<sub>M</sub>, leading to slower overall protein synthesis rate (purple gears) and cell growth. Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Nucleic Acids Res, gkz211, https://doi.org/10.1093/nar/gkz211 The content of this slide may be subject to copyright: please see the slide notes for details.

Figure 3. Lowering ppGpp levels in E Figure 3. Lowering ppGpp levels in E. coli by overexpression (OE) of Mesh1 protein from Drosophila melanogaster. (A) ... Figure 3. Lowering ppGpp levels in E. coli by overexpression (OE) of Mesh1 protein from Drosophila melanogaster. (A) The mesh1 gene is driven by Ptac promoter under the control of PlacIq-lacI cassette. (B) Expression of Mesh1 or SpoT E319Q in E. coli can suppress the lethal effect of SpoT knockout. The LB plate was supplemented with 30 μM IPTG to induce the expression of Mesh1 or SpoT E319Q. (C) Effect of Mesh1 protein overexpression on the growth rate of wild type E. coli cells in LB medium, glucose medium, glycerol medium and sorbitol medium. (D) The relative change of growth rate of wild type E. coli cells upon Mesh1 protein overexpression in each growth condition. The growth rate data of no-IPTG in each condition in panel C is set as ‘1’. (E) Effect of overexpression of Mesh1 H62F mutant protein on the growth rate of wild type E. coli cells in LB medium and three minimal media. (F) The cellular guanosine tetraphosphate (ppGpp) level of E. coli growing in glycerol minimal medium upon Mesh1 OE. The standard deviation of ppGpp measurement by UPLC-MS is within 25%. (G) The cellular NADP(H) pool of E. coli cells under Mesh1 overexpression. Cells were growing in either glucose minimal medium or glycerol minimal medium. The growth rate data in glucose and glycerol medium is exactly the same as shown in panel C. (H) Ribosome content (RNA/protein ratio) plotted against growth rate under Mesh1 protein overexpression. The r<sub>max</sub> denotes the offset of Y-axis when growth rate becomes zero. Data are average of triplicates with standard deviations being within 10%. Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Nucleic Acids Res, gkz211, https://doi.org/10.1093/nar/gkz211 The content of this slide may be subject to copyright: please see the slide notes for details.

Figure 4. The effect of decreased ppGpp levels on gene expression of E Figure 4. The effect of decreased ppGpp levels on gene expression of E. coli. Experiments were preformed for cells ... Figure 4. The effect of decreased ppGpp levels on gene expression of E. coli. Experiments were preformed for cells growing in either glucose minimal medium (circle) or glycerol minimal medium (triangle). The activity of six promoters plotted against growth rate under different degrees of Mesh1 protein overexpression. (A) PtetO promoter; (B) PglpFK promoter; (C) PfucP promoter; (D) PsdhC promoter; (E) PthrA promoter; (F) PlysC promoter. Data are average of triplicates with standard deviations being within 10%. Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Nucleic Acids Res, gkz211, https://doi.org/10.1093/nar/gkz211 The content of this slide may be subject to copyright: please see the slide notes for details.