# The Link between Purine Metabolism and Production of Antibiotics in _Streptomyces_
## 2 Stringent Response and Morphological Differentiation  
Nutrient limitation or starvation induces the stringent response in the vast majority of bacteria. This response was first described in _Escherichia coli_ and shown to involve reduced accumulation of stable RNAs (rRNA and tRNA); this stringent response was'relaxed' by mutation in the gene accordingly named _relA_. Stringent response depends on a transient increase in the level of (p)ppGpp, also known as the magic spot [5]. Members of the RSH (RelA/SpoT Homolog) protein superfamily are responsible for (p)ppGpp synthesis from ATP and either GTP or GDP and for the degradation of (p)ppGpp to GTP or GDP and pyrophosphate, with some members possessing both enzymatic functions. The activity of different RSH family proteins is controlled by distinct mechanisms, including interaction with idling ribosomes in many cases, allowing responses to various stresses [1]. The stringent response may be initiated by limitation of nutrients such as amino acids, nitrogen, phosphorous, and carbon sources. Once (p)ppGpp accumulates, it exerts global changes designed to downregulate transcription of genes linked to growth (e.g., ribosome biogenesis) and upregulation of genes required for survival (e.g., nutrient acquisition and stress responses), and it also directly binds a number of proteins to regulate their activity [6]. While specific binding targets may differ between Gram-negative and Gram-positive species, (p)ppGpp has been shown to regulate replication, transcription, translation and GTP biosynthesis by binding to proteins that participate in these processes, including DNA primase, RNA polymerase, small GTPases, enzymes involved in purine biosynthesis, and transcriptional regulators [7; 8; 9; 10; 11]. Considerable differences exist between bacterial species with regard to both the mode of action and metabolism of (p)ppGpp. In Gram-negative species such as _E. coli_, ribosomes sense the uncharged tRNAs at the ribosomal A site during amino acid starvation, causing protein synthesis to stall and resulting in the activation of RelA [12]. This leads to synthesis of (p)ppGpp, which acts as an allosteric regulator of RNA polymerase. The (p)ppGpp binds together with the regulatory protein DksA; this leads to inhibition of transcription from stable RNA promoters and promoters driving expression of genes encoding, for example, ribosomal proteins, and it mediates an activation of genes critical for cell survival [13; 14]. This mechanism is likely to be conserved among other proteobacteria [14]. The stringent response in Gram-positive bacteria is not mediated by direct binding of (p)ppGpp to RNA polymerase [15; 16]. In _Bacillus subtilis_, (p)ppGpp regulates intracellular GTP levels, even during normal growth, and dysregulated GTP homeostasis leads to cell death [17; 18]. Two mechanisms contribute to the observed reduction in GTP levels during the stringent response, namely, consumption of GTP for the purpose of (p)ppGpp synthesis and inhibition of GTP biosynthesis. GTP can be synthesized by both expensive _de novo_ and less resource-demanding salvage pathways, and (p)ppGpp binds directly to several proteins that participate in these pathways to inhibit their activity [10; 18]. Figure 1 summarizes purine metabolism, identifying key regulated enzymes participating in synthesis of GTP; _de novo_ synthesis initiated by GuaB-catalyzed conversion of inosine monophosphate (IMP) to XMP, or formation of XMP by purine salvage, which requires xanthine dehydrogenase (Xdh). As a consequence of reduced cellular GTP levels, transcription from genes whose initiating nucleotide is GTP is decreased, thereby accounting for the reduced accumulation of stable RNAs [15]. Expression of other genes is affected indirectly by the reduced levels of GTP, for example, by favoring transcription of genes whose initiating nucleotide is ATP [19].