Irving et al 2021 Molecular Biology and Physiology

Bacteria face numerous environmental stresses throughout their life cycle, requiring prompt and efficient responses for survival. One key mechanism for bacterial adaptation involves stress signaling pathways, among which the stringent response is prominent, especially in scenarios of nutrient starvation. This review delves into the processes governing the stringent response, encapsulated by the alarmones guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp). These nucleotides are synthesized by the RelA/SpoT homologue (RSH) protein superfamily from guanosine diphosphate (GDP) and guanosine triphosphate (GTP) respectively, with a recently identified third member, guanosine 5′-monophosphate 3′-diphosphate (pGpp).

Alarmone Synthesis and Functions
The alarmones (collectively referred to as (pp)pGpp) have several intracellular targets, including proteins and RNA molecules that act as regulators of cellular processes. This broad range of interactions enables them to modulate various aspects of bacterial metabolism and physiology during stringent response activation. The target proteins include those involved in transcription and translation, as well as others that regulate nucleotide and amino acid synthesis pathways. Notably, some (pp)pGpp-binding targets are ubiquitous across bacteria, such as RNA polymerase and ribosomal proteins, while others are specific to certain lifestyles or ecological niches, illustrating the adaptability and evolutionary significance of this response mechanism.

Enzymatic Mechanism of Synthesis and Degradation
The synthesis of alarmones involves transferring a pyrophosphate group from ATP to the 3′-OH of GTP, GDP, or GMP, a process catalyzed by the synthetase domain of RSH enzymes. RSH enzymes with hydrolase domains can hydrolyze these alarmones, regulating their levels within the cell. Three categories of RSH enzymes exist: long RSH enzymes, which contain both synthetase and hydrolase domains; small alarmone synthetases (SASs), which synthesize (pp)pGpp but do not degrade it; and small alarmone hydrolases (SAHs), which specifically degrade the alarmones. Each type plays a unique role in managing the concentration of (pp)pGpp based on environmental cues, with long RSH enzymes typically acting under stress conditions while SASs and SAHs participate in more specific regulatory mechanisms.

Functional Roles of (pp)pGpp in Metabolic Pathways
The impact of (pp)pGpp on bacterial metabolism is extensive, influencing critical processes such as RNA polymerase activity, DNA replication, nucleotide synthesis, ribosome biogenesis, and lipid metabolism. For instance, in E. coli and Bacillus subtilis, the binding of these alarmones inhibits DnaG, a DNA primase essential for DNA replication, thereby controlling replication rates in response to nutrient deprivation. The inhibition extends to the synthesis of purine nucleotides, where ppGpp and pppGpp actively disrupt pathways involved in guanine and adenine synthesis, ensuring resource allocation aligns with cellular needs during stress conditions. This mechanism helps the bacteria conserve energy and materials, allowing them to prioritize survival and essential functions under duress.

The Role of (pp)pGpp in Virulence
The stringent response significantly impacts bacterial virulence during infection. Various bacterial pathogens exploit (pp)pGpp to regulate traits essential for adhesion, invasion, and immune evasion. For example, in enterohemorrhagic E. coli and Salmonella enterica, the accumulation of (pp)pGpp activates virulence factors necessary for colonization and invasion of host tissues. Additionally, it influences the expression of toxins and other secreted factors that facilitate disease progression. Furthermore, (pp)pGpp has been implicated in biofilm formation and modulation, highlighting its importance in persistent infections and chronic disease states. The regulation of genes associated with biofilm production enables bacteria to survive in hostile environments and evade host immune responses, contributing to their pathogenicity.

Future Directions in Research
Despite the advances in understanding the role of (pp)pGpp in bacterial physiology and pathogenesis, many aspects remain unexplored. Ongoing research aims to elucidate the complex regulatory networks involving (pp)pGpp, including its interactions with riboswitches and the identification of novel synthetases and hydrolases beyond the RSH superfamily. The impact of (pp)pGpp on chronic infections also warrants further investigation, particularly regarding its contributions to antibiotic tolerance and resistance mechanisms in pathogenic bacteria. There is a growing interest in how (pp)pGpp interacts with other signaling molecules in bacteria and how this could provide insights into new therapeutic targets that could enhance our ability to treat bacterial infections.

In summary, the stringent response mediated by (pp)pGpp is a pivotal component of bacterial adaptation to stress, influencing various metabolic processes and contributing significantly to virulence strategies in many pathogens. Understanding this regulatory network will enhance our insights into bacterial survival mechanisms and may inform strategies for combating antibiotic resistance and chronic infections in clinical settings.