## 4 Discussion  
In the following sections, we discuss the physiological relevance of the differential upregulation on analyzed genes in response to ATP disruption in the PTS\({}^{-}\) mutants.  
Expression of the _atpAGD_ Operon, ATPase Activities, [ATP]/[ADP] Ratio, and Cyclic AMP (cAMP) Concentrations  
The total ATPase activity in _E. coli_ measures the bacterium's ability to hydrolyze ATP to ADP and inorganic phosphate. The activity of the F\({}_{1}\)F\({}_{0}\) ATP synthase is responsible for the synthesis of ATP during oxidative phosphorylation and for hydrolyzing ATP under certain conditions [9]. Uncoupled ATPase activity in the PB12 AGD\({}^{+}\) mutant results from the cloning and expression of the _atpAGD_ operon encoding for the F\({}_{1}\) component of the F\({}_{1}\)F\({}_{0}\) ATP synthase, increased the overall ATPase activity (U/mgprotein) by -135% than JM101 and PB12, while these strains showed practically the same activity (Table 2); nevertheless, as expected, the [ATP]/[ADP] ratio in the AGD\({}^{+}\) mutant was the lower one observed in the analyzed strains. A decreased [ATP]/[ADP] ratio in _E. coli_ results from a reduction in the rate of the synthesis of ATP and is considered an indicator of oxidative stress, resulting in a reduction in growth and protein synthesis [20,21].  
The relative transcription level of genes encoding the F\({}_{1}\)-ATPase in the plasmid-cloned operon _atpAGD_ encoding the \(\alpha\), \(\beta\), and \(\gamma\) components of the F\({}_{1}\)-ATP synthase showed, as expected, an increased expression by 8-10 X in PB12 ADG\({}^{+}\) than that observed in the strain PB12 (Table 3, Figure 3). The relative transcription level of the _atpAGD_ operon is related  
Figure 3: Relative transcript levels of genes involved in the respiratory chain and oxidative phosphorylation in PB12 and PB12 AGD\({}^{+}\) derivatives. RT-PCR values of those upregulated genes (1.7-fold or higher) are shown beside the gene’s name in parenthesis: The first value for PB12 and the second for PB12 AGD\({}^{+}\). The relative gene transcription value for JM101 is always equal to 1. NADH-I, NADH-quinone oxidoreductase kinase; FUM, fumarate, SUC, succinate. The corresponding names of the genes and the complete RT-qPCR values for all analyzed genes are shown in Table 3. The reaction mechanisms were adapted from the EcoCyc database ([[https://ecocy.org](https://ecocy.org)]([https://ecocy.org](https://ecocy.org)), accessed on 8 January 2024) [4].  
to the increment of the total ATPase activity detected in the AGD\({}^{+}\) mutant. As a control of the transcriptional level of the chromosomal _atp_ operon, the _atpl_ gene encoding for the ATP synthase accessory factor [4] showed a lower expression level than the observed in the PB12 strain (Table 3).  
The observed decrement in the [ATP]/[ADP] ratio in the AGD\({}^{+}\) mutant is associated with the reduction in the intracellular level of cAMP compared to the mutant PB12 (~28%). These results suggest that the decrement in the available ATP resulting from the uncoupled ATPase activity of the F1 component of the F1F0 ATP synthase has a negative impact on the availability of intracellular cAMP synthesized by adenylate cyclase enzyme CyaA in PB12 and the AGD\({}^{+}\) mutant. The analysis of the genes with expression regulated by cAMP-CRP, shown in Table 2 (32 genes flagged with *), reports a reduction of 15 genes (~47%), the upregulation of 1 gene, and no change in the expression of 16 genes (50%). These results suggest that despite the reduction in the concentration of cAMP in the _apt_ mutant, they do not have a severe effect on the expression of the target genes.