# Nucleotides as Bacterial Second Messengers
## 6 The pppGpp or ppGpp Nucleotide  
Nutrients become limiting during bacterial growth, mainly in the stationary phase; in this condition, the bacterium resorts to other processes to reuse possible nutritional sources within the cell. The processes of synthesis of membrane components, ribosomal proteins, RNA, and DNA are arrested, and in response, the cell rapidly produces factors crucial for stress resistance [94]. In general, these events are known as nutritional stress responses. Under nutritional stress conditions, a signaling process is triggered by the nucleotide's guanosine tetraphosphate and guanosine pentaphosphate (ppGpp and ppPGPp, respectively), called alarmones. Two classes of enzymes carry out the synthesis of alarmones. One is the monofunctional synthetase enzyme, and the other is the bifunctional synthetase-hydrolase enzyme. The monofunctional synthetase corresponds to the ReLA protein; this enzyme uses GTP and ATP as phosphate donors to generate ppGpp and then convert it into pppGpp. The bifunctional synthetase-hydrolase enzymes are represented by SpoT, Rel, or RSH (Rel-Spo homologous) proteins. These enzymes synthesize ppGpp or ppGpp and hydrolyze these nucleotides to produce GDP and pyrophosphate (PPi) or GTP and PPi, respectively. These two classes of enzymes participate in alarmone synthesis by different metabolic states. ReLA functions in alarmone synthesis when there is a shortage of amino acids and this causes uncharged tRNAs, which stimulate this enzyme's activity. In contrast, the bifunctional synthetase-hydrolase enzyme is involved when bacteria are under nutritional stress from carbon, fatty acids, iron, or phosphate starvation. The synthesis of ppGpp under nutritional stress conditions causes a controlling effect on RNA polymerase (RNAP) activity in cooperation with the suppressor DnaK (DksA). The binding of ppGpp and DksA regulates transcription positive or negative by RNAP, as determined by the intrinsic properties of the promoter in question and causes numerous physiological effects [95]. Notably, DksA and ppGpp also regulate RNAP transcription through an event called \(\sigma\)-factor competition. This event occurs as follows: in the logarithmic growth phase, vegetative \(\sigma\)-factor 70 (RpoD) binds to RNAP to initiate DNA replication and lipid synthesis as well as transcription of essential proteins. In stringent conditions, bacteria produce high concentrations of ppGpp to inhibit RNAP-e70 binding; consequently, RNAP is free to bind to other types of sigma factors, such as sigma E and sigma S, which are present when the bacterium is under stress conditions [96]. When metabolic precursors are restored to high concentrations, SpoT degrades ppGpp. Thus, the \(\sigma\)-vegetative factor, \(\sigma\)70, controls RNAP to transcribe genes crucial for DNA replication and biomolecule synthesis. A mechanism is now known in _E. coli_ to stabilize and activate sigma S function under stress conditions. In the logarithmic growth phase, the sigma S protein is bound to the RssB protein (an adaptor protein), and this sigma S-RssB binding drives the proteolytic degradation of sigma S by the ClpXP proteasome [97]. However, during phosphate stress, the hydrolase activity of SpoT is inhibited. Therefore, ppGpp synthesis is elevated, then ppGpp binds to RNAP to transcribe and produce IraP and IraD, which are anti-adaptor proteins that sequester RssB and prevent sigma S delivery to the ClpXP proteasome [98]. When the concentration of amino acids is low, the bacterium uses the ppGpp message to recover amino acids from conventional proteins, such as ribosomal proteins. In _E. coli_, the ppGpp alarmone inhibits the transcription of ribosomal protein-coding genes, and the alarmones inhibit exopolynophatase activity, causing accumulation of polyphosphate in the cytosol. Accumulated polyphosphate binds to free ribosomal proteins which are subsequently degraded by Lon protease, producing free amino acids for enzymatic biosynthesis [99].