## 2 Plurality of ppGpp Synthetic and Homeostatic Pathways  
Nutrient deprivation, commonly referred to as the'stringent response', is the originally discovered signal of (p)ppGpp synthesis [5; 19]. In the early experiments, the pathway was routinely induced by shifting _E. coli_ cells from rich media such as the customary Luria Broth (with glucose) to synthetic minimal media such as M9, devoid of amino acids. The (p)ppGpp synthesis is catalyzed by bifunctional proteins, RelA and SpoT, which use ATP and GTP/GDP as substrates; together, these two enzymes comprise the founding members of the eponymous RelA-SpoT homolog (RSH) family [17]. The members of this family share domain structures, notably a (p)ppGpp synthesis (SYNTH) and a (p)ppGpp hydrolysis (HD) domain; however, RelA is responsible for the bulk of synthesis, whereas SpoT is more involved in (p)ppGpp hydrolytic degradation (Figure 1). This is because the SYNTH domain of SpoT has very weak activity, and reciprocally, the HD domain of RelA has no degradative activity. In the predominant stringent response mechanism, when amino acids are in short supply, RelA detects the 3'-CCA terminus of non-acylated tRNA; the nucleoprotein complex then loads onto the ribosomal A-site of the translating ribosome, and the resultant structural changes preclude the binding of aminoacylated tRNA [20; 21; 22]. Simultaneously, the interaction changes the RelA structure, activating its (p)ppGpp synthesis activity [23; 24; 25; 26; 27]. In general, the RelA, SpoT, and several other players regulate the steady-state level of (p)ppGpp in the bacterial cell, maintaining strict homeostasis in response to changing nutritional conditions, i.e., not only raising (p)ppGpp levels in nutrient deficiency but also bringing it down to low basal levels when nutrients become available [28; 29; 30]. Precise tuning of the opposing synthetic and hydrolytic activities also allow the cell to achieve a continuum of (p)ppGpp concentrations ranging from micromolar to millimolar, which suits the exact degree of nutritional deficiency and the diverse affinity of the (p)ppGpp-binding proteins [31; 32]. Besides SpoT, two other notable degradative enzymes include guanosine  
Figure 1: Major pathways of metabolism of nucleoside phosphates relevant in stringent response. (**A**) The synthesis of the two guanosyl phosphates, ppGpp and pppGpp, in bacteria is catalyzed in steps of phosphorylation and dephosphorylation, as shown, using GDP and GTP as substrates. The phosphorylation reactions are reversed by the hydrolase activity of SpoT. Conversion of pppGpp to ppGpp is also catalyzed by various hydrolytic enzymes, such as guanosine pentaphosphohydrolase (GppA) and Elong Factor G. (**B**) In the ‘stringent-like’ response in the metazoo, NADPH is dephosphorylated by MESH1, a mammalian SpoT homolog, and can be resynthesized by phosphorylation with NAD kinase (not shown here) (details of MESH1 in Section 4). pentaphosphapthabydrolas (GppA) and several GTPase enzymes, such as the GTPase activity of the translation Elongation Factor EF-G (Figure 1) [33]. Intriguingly, a plethora of other deficiencies and stress conditions also activate (p)ppGpp synthesis; they include, but are not limited to, fatty acid limitation [34], temperature shift [35; 36], carbon [37; 38] and phosphate [39] starvation, and iron limitation [40]. While RelA is the canonical enzyme for (p)ppGpp synthesis in amino acid starvation, SpoT is capable of mediating (p)ppGpp synthesis in response to these other signals, namely carbon, iron, phosphate, and fatty acid deprivation [34; 40; 41; 42]. The mechanisms of SpoT activation are relatively unclear, but direct interactions with cytosolic accessory proteins appear to modulate the ratio of its synthetase (also termed'synthase') versus hydrolase activities. Fatty acid deficiency, for example, is sensed by the Acyl Carrier Protein (ACP), a central cofactor in fatty acid biosynthesis, which also physically binds to SpoT but not RelA.