## 4 Discussion
### Expression of Stress Response Genes  
Disruption of normal [ATP]/[ADP] ratio in _E. coli_ by uncoupling the cytoplasmic activity of the F\({}_{1}\) subunit affects the normal balance of ATP an NADH, significantly affecting the redod balance of the cell, resulting in the accumulation of reactive oxygen species and genes involved in respiration [52]. The transcriptional factors SoxR and SoxS control a superoxide response regulon. SoxR induces _soxS_ expression, and SoxS activates the transcription of at least 14 genes of the regulon, including genes involved in the elimination of oxidative stress, repair or compensate for damage, or the induction of metabolic functions that led the cell to contend with oxidative stress [53; 54].  
The disruption of the [ATP]/[ADP] ratio in the AGD\({}^{+}\) mutant triggered a strong response to oxidative stress. Among the analyzed genes in both PB12 mutants, _zwf_ and _fumC_ are reported to be induced by _soxS_ and _soxR_ in response to oxidative stress conditions. Induction of _zwf_ results in the regeneration of NADPH, and induction of _fumC_ replaces the \(\text{O}_{2}\text{ }^{\bullet}\) - sensitive fumarase [4; 54]; nevertheless, the expression of these genes was lower in the AGD\({}^{+}\) mutant than in PB12 (Table 3). The higher level of relative expression detected for _soxR_ and _soxS_ in the AGD\({}^{+}\) mutant suggests that this strain is under some stress condition probably associated with the higher oxygen consumption rate observed = 1.17 mmol/gDCW.h, a higher value than the observed for PB12 (an increase of 138X), as it was reported that reactive oxygen species are produced as a byproduct of aerobic respiration [55; 56].  
Finally, _spoT_ encoding the bifunctional (p)ppGpp synthase/hydrolase SpoT was the highest upregulated gene in the AGD\({}^{+}\) mutant (\(\sim\)257X), compared to the PB12. SpoT is a crucial enzyme involved in the stringent response in _E. coli_[4], which is a global regulatory system activated under conditions of nutrient or energy starvation or other environmental stress, resulting in the modification of gene expression, metabolism, and cellular processes under these conditions [4; 57]. In _E. coli_, the stringent response is mediated by the concentration of guanosine 3',5'-bis(diphosphate) and guanosine 3'-diphosphate,5'-triphosphate, known as (p)ppGpp that modulates the affinity of sigma factors for the RNAp core. Environmental stress results in variable concentrations of (p)ppGpp concentrations; higher levels of (p)ppGpp result in higher expression of rpoS by the direct stimulation of the transcription of other genes related to stress protection [4; 57].  
The higher transcription level of _spoT_ in the AGD\({}^{+}\) mutant could be considered (as stated above) an indicator that this mutant is under energy starvation. In this condition, the synthetase activity of SpoT (together with ReIA) synthesizes ppGpp [58], increasing its concentration and regulating several cellular processes to adapt and live under stress. This physiological condition suggests a relevant role of RpoS. Nevertheless, as shown in Table 3, the expression of this sigma factor showed a lesser value than PB12. Remarkably, the analysis of the correlation of the (p)ppGpp levels in diverse _E. coli_ K12 strains suggests that there is not necessarily a strong correlation with concentration with the concentration of \(\sigma^{38}\)[59].