## 8 Stress Responses and Persistence: The Stringent Response  
Nutrient limitation, metabolic flux, drug efflux, and growth rate are not the only mechanisms by which persisters can arise. As the PASH model states, active transcriptional responses can trigger persisters, and major transcriptional responses are undoubtedly coupled with metabolic shifts. Recently, links between CCR and antibiotic tolerance have implicated the involvement of the stringent response [96]. The stringent response is a stress response pathway that activates during amino acid deprivation, fatty acid limitation, and other stressors [97; 98]. Stringent response pathways are activated via (p)ppGpp alarmone concentrations, which modulate subsequent cellular responses such as transcription, replication, and gyrase-mediated negative DNA supercoiling [28]. (p)ppGpp is synthesized and maintained by members of the RelA/SpoT Homolog (RSH) enzyme superfamily [99; 100]. When cellular concentrations of (p)ppGpp are high enough, the alarmone interacts with RNA polymerase and the DskA ribosome binding protein, ultimately reducing translational fidelity due to a reduction in the pool of filled aminoacyl-tRNAs [28]. Because the stringent response can modulate so many processes involved in direct targets of antibiotics, it has been implicated in tolerance development and biofilm recalcitrance, which likely share mechanistic triggers with persister development [28]. Bacterial mutants lacking the stringent response, such as a _relQ-rsh_ double knockout in _Enterococcus faecalis_, exhibit divergence in various metabolic operons, glycerol uptake, and glycerol metabolism [96]. These processes have been demonstrated to be under the control of CcpA in _E. faecalis_. This double knockout strain has significantly perturbed levels of _ccpA_ transcription, indicating an inability to accurately sense metabolic cues and properly adapt. In _S. aureus_, double knockouts of _rel-rsh_ have aberrant intracellular pools of (p)ppGpp, suggesting an inability to control the pace or directionality of carbon flux [96]. This inability to properly adapt to nutrient availability changes likely leads to nutrient starvation and limitation. This leads to dysbiosis of NAD+/NADH ratios, increased ROS generation, and unbalanced cellular homeostasis [96]. [PERSON] et al. found that when _Mycobacterium smegmatis_ populations were challenged with nutrient depletion, stringent response pathways were activated, representing a form of adaptive switching that generates persisters [101]. In vitro deletion of _relA_ in _E. coli_ ablates (p)ppGpp synthesis, and experimental nutrient starvation fails to elicit penicillin tolerance in this mutant [12]. Furthermore, the stringent response appears to be critical to persistence development in _E. coli_, as deletion of several pathway components inhibits persister formation in vitro [102]. In _P. aeruginosa_, antibiotic tolerance in nutrient-limited and biofilm contexts is mediated by active responses to starvation rather than by passive effects of growth and arrest, which closely resembles the PASH model of persistence [103]. In Pseudomonas, the stringent response can be linked to tolerance via reduction of oxidative stress in cells. By inactivating protective mechanisms, biofilms become sensitized to multiple antibiotic classes by several orders of magnitude. In experimental knockouts of _relA_ and _spoT_, cells were unable to produce (p)ppGpp during serine starvation [103]. When challenged with ofloxacin during starvation, wild type cells had a 2300-fold reduced killing, while knockout strains exhibited only a 34-fold reduction in antibiotic killing [103]. Ultimately, stringent response inactivation appears to modulate antibiotic tolerance via relief of oxidant stress, and this stress response has likely conserved functionality in persister cell formation. The stringent response has also been linked to indole-mediated antibiotic tolerance. [PERSON] et al.