==== Front Microb Cell Microb Cell Microb Cell Microbial Cell 2311-2638 Shared Science Publishers OG 10.15698/mic2023.07.799 MIC0273E111 Viewpoint (p)ppGpp stringent response nucleotide amino acid ribose 5'-phosphate Ribose 5-phosphate: the key metabolite bridging the metabolisms of nucleotides and amino acids during stringent response in Escherichia coli? R5P links nucleotide and amino acid metabolisms Grucela Paulina Katarzyna 1 Fuhrer Tobias 2 Sauer Uwe 2 Chao Yanjie 3* Zhang Yong Everett 1* 1 Department of Biology, University of Copenhagen, DK-2200 Copenhagen, Denmark. 2 Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland. 3 The Center for Microbes, Development and Health (CMDH), Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai 200031, China. * Corresponding Author: Yanjie Chao, The Center for Microbes, Development and Health (CMDH), Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai 200031, China; E-mail: yjchao@ips.ac.cn * Corresponding Author: Yong Everett Zhang, Department of Biology, University of Copenhagen, DK-2200 Copenhagen, Denmark; E-mail: yong.zhang@bio.ku.dk Conflict of Interest: The authors have no conflict of interest to disclose. Please cite this article as: Paulina Katarzyna Grucela, Tobias Fuhrer, Uwe Sauer, Yanjie Chao and Yong Everett Zhang (2023). Ribose 5-phosphate: the key metabolite bridging the metabolisms of nucleotides and amino acids during stringent response in Escherichia coli? Microbial Cell 10(7): 141-144. doi: 10.15698/mic2023.07.799 01 6 2023 03 7 2023 10 7 141144 01 5 2023 18 5 2023 18 5 2023 Copyright: © 2023 Grucela et al. 2023 https://creativecommons.org/licenses/by/4.0/ This is an open-access article released under the terms of the Creative Commons Attribution (CC BY) license, which allows the unrestricted use, distribution, and reproduction in any medium, provided the original author and source are acknowledged. The bacterial stringent response and its effector alarmone guanosine penta- or tetra – phosphates (p)ppGpp are vital for bacterial tolerance and survival of various stresses in environments (including antibiotics) and host cells (virulence). (p)ppGpp does so by binding to its numerous target proteins and reprograming bacterial transcriptome to tune down the synthesis of nucleotides and rRNA/tRNA, and up-regulate amino acid biosynthesis genes. Recent identification of more novel (p)ppGpp direct binding proteins in Escherichia coli and their deep studies have unveiled unprecedented details of how (p)ppGpp coordinates the nucleotide and amino acid metabolic pathways upon stringent response; however, the mechanistic link between nucleotide and amino acid metabolisms remains still incompletely understood. Here we propose the metabolite ribose 5'-phosphate as the key link between nucleotide and amino acid metabolisms and a working model integrating both the transcriptional and metabolic effects of (p)ppGpp on E. coli physiological adaptation during the stringent response. (p)ppGpp stringent response nucleotide amino acid ribose 5'-phosphate This work is supported by National Key R&D Program of China (2022YFE0111800 to Y.C.), Novo Nordisk Foundation Project Grant (NNF19OC0058331 to Y.E.Z), Chinese Academy of Sciences (176002GJHZ2022022MI to Y.C.), Shanghai Municipal Science and Technology Commission (21ZR1471300 to Y.C.). ==== Body pmcUpon amino acid starvation, either artificially induced (e.g., by valine) or during nutrient downshift to amino acid free condition, the wild type (wt) Escherichia coli MG1655 strain elicits the stringent response by producing the small alarmone (p)ppGpp, globally reprogramming bacterial metabolism to tune down the synthesis of nucleotides and rRNA, and up-regulate amino acids biosynthesis [1–5] (Figure 1A). Despite the regulatory scheme being known for many years and studied at the levels of global transcription [1–3] and translation [6], the underlying mechanistic details and the relationship of nucleotide and amino acid metabolisms upon amino acid starvation started to unfold recently. Two different techniques, i.e., DRaCALA [7] and photocrosslinkable ppGpp-analogs based capture compound mass spectrometry [8], systematically identified novel direct binding proteins of (p)ppGpp in E. coli. Besides the large overlap, some novel (p)ppGpp targets were identified separately, e.g., PpnN [7], Gsk and PurF [8]. Subsequent more detailed analyses of these proteins [9, 10] have revealed unprecedented details of how (p)ppGpp regulates their molecular functions and thus reprogram global metabolism upon amino acid starvation. Here we discuss these two studies, emphasizing on the connection and potential role of ribose 5-phosphate (R5P) between nucleotide and amino acid metabolisms and propose a working model to integrate the effects of (p)ppGpp on both nucleotides and amino acids metabolism in E. coli upon amino acid starvation. Figure 1 FIGURE 1: A comprehensive model integrating both the metabolic and transcriptional coordination of nucleotide and amino acid metabolisms upon stringent response in E. coli. (A) Amino acid starvation in E. coli leads to the activation of RelA to produce (p)ppGpp, which binds to the RNA polymerase (RNAP) to downregulate stable RNA (rRNA, tRNA) and upregulate amino acid biosynthesis genes at the transcriptional level. (B) At the metabolic level, (p)ppGpp directly inhibits (in red broken lines) the de novo and the salvage pathways (highlighted in red shadow) of purine nucleotides synthesis by targeting the PurF and Gpt/Hpt/Gsk proteins, respectively (in red font). Meanwhile, (p)ppGpp binds to PpnN (light blue font) and stimulates its activity (in blue broken lines) to degrade excess nucleotides, from both the redundant nucleotides and the degraded rRNAs/tRNAs. Degraded nucleotides lead to increased nucleobases (Gua, Xan, Hyp) (in blue up arrows) and R5P. The former ones bind to PurR to repress expression of genes (in red dotted lines) in the de novo purine and pyrimidine biosynthesis pathways. R5P, used to produce pRpp, enters the pentose phosphate pathway and culminates with the synthesis of (aromatic) amino acids and the intermediate metabolites. Metabolites highlighted with light blue up arrows are the ones that increased their concentrations upon amino acid starvation in the wild type E. coli, but not in the ΔppnN mutant strain [10]. The list of abbreviated metabolites is included in the inset. The E. coli purine nucleotide biosynthesis pathways consist of both the de novo and salvage branches (Figure 1B, left). The de novo pathway starts from the formation of phosphoribosylamine from phosphoribosyl pyrophosphate (pRpp) and glutamine catalyzed by the glutamine aminophosphoribosyltransferase PurF. Subsequent nine reactions culminate with the synthesis of inosine 5'-monophosphate (IMP). From here, IMP is converted by two enzymes PurA and GuaB for the eventual biosynthesis of ATP and GTP, respectively. The salvage pathway catalyzes the one-step synthesis of purine nucleotides by transferring the R5P of pRpp to the respective nucleobases. This is catalyzed by three enzymes in E. coli, Gpt, Hpt, and Apt, which synthesize IMP, XMP, GMP and AMP by using the nucleobases hypoxanthine, xanthine, guanine and adenine, respectively. Additionally, the salvage pathway can be undertaken in a two-step reaction. Firstly, DeoD converts nucleobases to nucleosides, which are converted into nucleotides by Gsk. There is also a newly characterized nucleosidase PpnN which degrades nucleotides into nucleobases and R5P in a one-step reaction [11]. We recently found that during amino acid starvation (p)ppGpp allosterically stimulates the catalytic activity of PpnN to accelerate the degradation of nucleotides to nucleobases and R5P [10]. One reason underlying the stimulated PpnN activity by (p)ppGpp lies on the fact that during amino acid starvation (p)ppGpp inhibits the bulk RNA synthesis (mainly rRNA, tRNA) which consumes most of the nucleotide pool. Furthermore, stable RNA (rRNA, tRNA) is degraded during amino acid starvation [12, 13] to release more nucleotides inside cells. These two processes lead to high levels of nucleotides inside cells; however, nucleotides must be depleted to prevent resynthesis of NTPs (and ADP, see below) when cells do not need so much of them. To achieve this, besides the stimulation of PpnN, (p)ppGpp inhibits the activities of Gpt and Hpt [14] (and also Gsk [9], see below), preventing the salvage syntheses of purine nucleotides. Thus, the two opposite effects of (p)ppGpp synergistically reduce and hold the nucleotide level low upon amino acid starvation. Additionally, PpnN is ideal for this task because of its two other properties. First, PpnN is constitutively expressed inside cells and ready to be stimulated by (p)ppGpp upon amino acid starvation; but without (p)ppGpp its catalytic activity is held low [10]. Second, a sigmoidal activity curve of PpnN with the GMP substrate [10] showed that PpnN is a cooperative enzyme and more active at higher GMP levels. These properties make PpnN the ideal driver to rapidly deplete and drain the increased pool of nucleotides upon amino acid starvation. As the reaction products of PpnN, nucleobases are secreted outside cells and can be taken up and re-used for nucleotides synthesis to promote fast cell regrowth [15, 16]. However, the fate of the other reaction product R5P was not confirmed in our previous study [10], despite the obvious anticipation that R5P enters the carbon metabolic pathways. On the other hand, the study of another (p)ppGpp target protein Gsk [9] revealed another reason why the nucleotide levels, especially ADP, must be kept low. A (p)ppGpp non-binding Gsk mutant Gsk(K383A) converts exogenous supplied nucleosides, i.e., inosine and guanosine, into nucleotides by depriving the gamma phosphate of ATP during amino acid starvation even in the presence of (p)ppGpp [9]. This reaction caused higher intracellular levels of nucleotides, especially ADP. Notably, ADP is instantly produced from the Gsk reaction and ADP binds to the essential protein PrsA to inhibit the synthesis of the crucial biosynthetic intermediate pRpp [17, 18]. pRpp is essential for the biosynthesis of pyrimidine nucleotides, and the amino acids histidine and tryptophan, and NAD etc. Consistently, supplementation with uridine, histidine and tryptophan completely suppressed the growth defects of the Gsk(K383A) mutant strain in presence of nucleosides [9]. These data suggest a key role of (p)ppGpp in balancing the metabolic pathways of purine, pyrimidine and amino acid biosynthesis pathways by lowering the nucleotides concentration and thus maintaining the production of pRpp and amino acids during amino acid starvation. However, the synthesis of pRpp in wt E. coli cells requires both the substrates ATP and R5P. ATP levels did not change much during amino acid starvation [9]; however, the R5P level was not shown. Given the above analyses, we hypothesize that R5P produced from nucleotide degradation is used for the syntheses of pRpp and amino acids. We thus re-analyzed our previously published metabolomic data generated via untargeted mass spectrometry (MS) of the wt MG1655 and the ΔppnN mutant strains upon valine induced amino acid starvation [10]. We found that a series of metabolites increased five minutes after the starvation in wt E. coli cells, but not in the ΔppnN mutant. These include the pentose phosphate pathway metabolites sedoheptulose 7-phosphate (S7P), erythrose 4-phosphate (E4P), and the glycolysis metabolites phosphoenolpyruvate (PEP), pyruvate, which together contribute to the synthesis of aromatic amino acids and their biosynthesis intermediates (shikimate, chorismate; Figure 1B, right, in light blue up arrows). The abolished increase of these metabolites in the ΔppnN mutant indicates that R5P derived from degraded nucleotides as catalyzed by PpnN substantially contributes to the synthesis of aromatic amino acids. Consistently, aromatic (and other) amino acids were higher in the wt E. coli than in the Gsk(K383A) mutant strain (Figure 5E, S5C in [9]) wherein the salvage pathway is uninhibited. Altogether, these data indicate that the degraded nucleotides provide (at least one source of) R5P for the synthesis of pRpp, pyrimidine nucleotides and amino acids upon stringent response. With these, we propose a working model (Figure 1) integrating both transcriptional and metabolic effects of (p)ppGpp on E. coli stringent physiology. Upon amino acid starvation, RelA senses the uncharged tRNA at the ribosome A-site and synthesizes (p)ppGpp from GTP/GDP and ATP within several minutes. The abruptly increased (p)ppGpp on one hand binds to the RNA polymerase on two sites [19, 20] and reprogram the binding preference of RNAP [3, 21], to reduce expression of rRNA/tRNA and increase expression of amino acid biosynthesis genes. However, these amino acid synthesizing proteins need the respective precursors such as E4P which are derived from glucose catabolism but also from the intracellularly degraded rRNA/tRNA and nucleotides. Here, (p)ppGpp stimulates PpnN to degrade and deplete nucleotides, yielding nucleobases and R5P; on the other hand, (p)ppGpp inhibits further the futile cycle of re-synthesizing nucleotides via the salvage pathway enzymes Gsk, Gpt, Hpt. R5P is used to synthesize pRpp and enters the carbon metabolic pathways, which together lead to the synthesis of pyrimidine nucleotides and amino acids that are used to sustain the adapted cell growth of E. coli. The nucleobases can be secreted outside cells and conserved for further use during regrowth. Of note, nucleobases bind to a transcriptional repressor PurR and co-repress the expression of de novo synthesis genes (including PurF) [22] and in synergy (p)ppGpp directly inhibits the PurF activity [8], blocking the de novo pathway. All in all, (p)ppGpp coordinates the dynamic metabolic conversions of nucleotides to amino acids via affecting both the metabolic enzyme activities and the transcriptional expression of relevant enzymes. This timely synchronization of molecular events at different levels via various target proteins of (p)ppGp ensures a prompt reprograming of cell physiology and thus a competitive ecological fitness of E. coli and probably many other bacteria. Abbreviations: wt – wild type. ==== Refs REFERENCES 1 Traxler MF Summers SM Nguyen HT Zacharia VM Hightower GA Smith JT Conway T The global, ppGpp-mediated stringent response to amino acid starvation in Escherichia coli. Mol Microbiol 2008 68 5 1128 1148 10.1111/j.1365-2958.2008.06229.x 18430135 2 Durfee T Hansen AM Zhi H Blattner FR Jin DJ Transcription profiling of the stringent response in Escherichia coli. J Bacteriol 2008 190 3 1084 1096 10.1128/JB.01092-07 18039766 3 Sanchez-Vazquez P Dewey CN Kitten N Ross W Gourse RL Genome-wide effects on Escherichia coli transcription from ppGpp binding to its two sites on RNA polymerase. Proc Natl Acad Sci USA 2019 116 17 8310 8319 10.1073/pnas.1819682116 30971496 4 Hauryliuk V Atkinson GC Murakami KS Tenson T Gerdes K Recent functional insights into the role of (p)ppGpp in bacterial physiology. Nat Rev Microbiol 2015 13 5 298 309 10.1038/nrmicro3448 25853779 5 Gallant JA Stringent control in E. coli. Annu Rev Genet 1979 13 393 415 10.1146/annurev.ge.13.120179.002141 94252 6 Zhu M Dai X Stringent response ensures the timely adaptation of bacterial growth to nutrient downshift. Nat Commun 2023 14 1 467 10.1038/s41467-023-36254-0 36709335 7 Zhang Y Zbornikova E Rejman D Gerdes K Novel (p)ppGpp Binding and Metabolizing Proteins of Escherichia coli. mBio 2018 9 2 e02188 17 10.1128/mBio.02188-17 29511080 8 Wang B Dai P Ding D Del Rosario A Grant RA Pentelute BL Laub MT Affinity-based capture and identification of protein effectors of the growth regulator ppGpp. Nat Chem Biol 2019 15 2 141 150 10.1038/s41589-018-0183-4 30559427 9 Wang B Grant RA Laub MT ppGpp Coordinates Nucleotide and Amino-Acid Synthesis in E. coli During Starvation. Mol Cell 2020 80 1 29-42 e10 10.1016/j.molcel.2020.08.005 32857952 10 Zhang YE Baerentsen RL Fuhrer T Sauer U Gerdes K Brodersen DE (p)ppGpp Regulates a Bacterial Nucleosidase by an Allosteric Two-Domain Switch. Mol Cell 2019 74 6 1239-1249 e1234 10.1016/j.molcel.2019.03.035 31023582 11 Sevin DC Fuhrer T Zamboni N Sauer U Nontargeted in vitro metabolomics for high-throughput identification of novel enzymes in Escherichia coli. Nat Methods 2017 14 2 187 194 10.1038/nmeth.4103 27941785 12 Fessler M Gummesson B Charbon G Svenningsen SL Sorensen MA Short-term kinetics of rRNA degradation in Escherichia coli upon starvation for carbon, amino acid or phosphate. Mol Microbiol 2020 113 5 951 963 10.1111/mmi.14462 31960524 13 Svenningsen SL Kongstad M Stenum TS Munoz-Gomez AJ Sorensen MA Transfer RNA is highly unstable during early amino acid starvation in Escherichia coli. Nucleic Acids Res 2017 45 2 793 804 10.1093/nar/gkw1169 27903898 14 Hochstadt-Ozer J Cashel M The regulation of purine utilization in bacteria. V. Inhibition of purine phosphoribosyltransferase activities and purine uptake in isolated membrane vesicles by guanosine tetraphosphate. J Biol Chem 1972 247 21 7067 7072 doi: 4343167 15 Link H Fuhrer T Gerosa L Zamboni N Sauer U Real-time metabolome profiling of the metabolic switch between starvation and growth. Nat Methods 2015 12 11 1091 1097 10.1038/nmeth.3584 26366986 16 Rinas U Hellmuth K Kang R Seeger A Schlieker H Entry of Escherichia coli into stationary phase is indicated by endogenous and exogenous accumulation of nucleobases. Appl Environ Microbiol 1995 61 12 4147 4151 10.1128/aem.61.12.4147-4151.1995 8534082 17 Hove-Jensen B Andersen KR Kilstrup M Martinussen J Switzer RL Willemoes M Phosphoribosyl Diphosphate (PRPP): Biosynthesis, Enzymology, Utilization, and Metabolic Significance. Microbiol Mol Biol Rev 2017 81 1 e00040 16 10.1128/MMBR.00040-16 28031352 18 Hove-Jensen B Harlow KW King CJ Switzer RL Phosphoribosylpyrophosphate synthetase of Escherichia coli. Properties of the purified enzyme and primary structure of the prs gene. J Biol Chem 1986 261 15 6765 6771 10.1016/s0021-9258(19)62682-7 3009477 19 Ross W Vrentas CE Sanchez-Vazquez P Gaal T Gourse RL The magic spot: a ppGpp binding site on E. coli RNA polymerase responsible for regulation of transcription initiation. Mol Cell 2013 50 3 420 429 10.1016/j.molcel.2013.03.021 23623682 20 Ross W Sanchez-Vazquez P Chen AY Lee JH Burgos HL Gourse RL ppGpp Binding to a Site at the RNAP-DksA Interface Accounts for Its Dramatic Effects on Transcription Initiation during the Stringent Response. Mol Cell 2016 62 6 811 823 10.1016/j.molcel.2016.04.029 27237053 21 Gourse RL Chen AY Gopalkrishnan S Sanchez-Vazquez P Myers A Ross W Transcriptional Responses to ppGpp and DksA. Annu Rev Microbiol 2018 72 163 184 10.1146/annurev-micro-090817-062444 30200857 22 Cho BK Federowicz SA Embree M Park YS Kim D Palsson BO The PurR regulon in Escherichia coli K-12 MG1655. Nucleic Acids Res 2011 39 15 6456 6464 10.1093/nar/gkr307 21572102