## 3 Results
### [ATP]/[ADP] Ratio, ATPase Activity, and cAMP Concentration Determinations
The strains JM101 and PB12 showed practically the same ATPase activity = 1.46 and 1.47 (U/mg protein), respectively, whereas the derivative strain PB12 AGD\({}^{+}\) showed an increase of 34% compared to both strains. The cAMP concentration in PB12 increased by 20% compared to the wild-type JM101, whereas the derivative PB12 AGD\({}^{+}\) decreased by 14% and 28% compared to the wild-type JM101 and PB12, respectively. The previous selection of fast-growing PTS\({}^{-}\) mutants of _E. coli_ in glucose, selected from adaptive laboratory evolution (ALE) experiments, showed an increase in \(\mu\) in respect to their non-evolved PTS-mutants. Still, the observed \(\mu\) was always lower than the observed for the wild-type strains. This decrement in growth capabilities was explained by a reduced ATP availability due to the selection of alternative glucose transporters (ABC type) requiring one APT for transport and the further glucose phosphorylation with Glk from ATP, reviewed in [18]. The observed decrement in the [ATP]/[ADP] ratio in a PTS\({}^{-}\) mutant with a compromised ATP availability for glucose transport, and the lower intracellular level of cAMP due to the absence of EIIA\({}^{\mathrm{Clk}}\) in the derivative PB12 AGD\({}^{+}\), are determinants of its lower growing capabilities compared to the JM101 and the PB12 strains. Differentially Expressed Genes in Response to the Disruption of the [ATP]/[ADP] Ratio in the Derivative Strain PB12 AGD\({}^{+}\)  
When _E. coli_ transports glucose by the PTS system, the intracellular level of cAMP indicates the presence of glucose in the medium. As glucose is transported by the cell, it results in an inhibition of CyaA. A higher cAMP concentration correlates with a glucose starvation condition [4]. When glucose or other PTS sugars are consumed from the culture medium, the PTS proteins are found in phosphorylated form. P-IIA\({}^{\mathrm{Glc}}\) activates the adenylate cyclase (CyaA), converting ATP to cAMP, forming a complex with the catabolite repressor protein (CRP). The complex CRP\({}^{-}\)cAMP activates and modulates the expression of several genes repressed by glucose or other PTS sugars [4]. Nevertheless, in a PTS\({}^{-}\) mutant of _E. coli_, like PB12 (\(\Delta\)_ptsHlcrr_), the protein EIIA\({}^{\mathrm{Glc}}\) is absent, avoiding the activation of CyaA and the synthesis of cAMP, resulting in the abolition of the catabolite repression mechanism leading the capability to this mutant to consume glucose + acetate and mixtures of glucose-arabinose, glucose-gluconate, and glucose-glycerol simultaneously [11; 19]. To assess the impact of the energetic disruption in the mutant PB12 AGD\({}^{+}\) and the lower cAMP concentration with a possible affectation of its role as DNA-binding transcriptional dual regulator coupled to CRP (CRP-cAMP), we analyzed the expression of 96 genes encoding for ATP synthase, glucose and acetate transport, glycolysis and gluconeogenesis, PPP, TCA cycle and glyoxylate shunt, several anaplerotic, respiratory chain, and fermentative pathways genes, sigma factors, and several global regulators (Table 3 and Figures 2 and 3). As previously reported, the wild-type strain JM101 was used as the control to normalize the data using the wild-type RT-qPCR value for that gene. The results showed an increased relative transcription level in all analyzed genes in PB12 and the PB12 AGD\({}^{+}\) mutant compared to JM101 (Table 3). Still, only 55 genes were found upregulated (\(\geq\)1.7) in PB12 and 34 genes in the mutant PB12 AGD\({}^{+}\). \begin{table}
\begin{tabular}{l c c c} \hline \hline
**Pathway, Group of Genes** & **Encoded Protein 1** & \multicolumn{2}{c}{**Relative Transcription**} \\
**or Cellular Process** & **Encoded Protein 1** & \multicolumn{2}{c}{**Levels as 2\({}^{-\Delta\)Ct**}} \\ \hline \hline  & & **PB12** & **PB12 AGD\({}^{+}\)** \\ \hline ATP synthase & & & \\ _atpl_ & Subunit I & 1.61 \(\pm\) 0.18 & 0.69 \(\pm\) 0.05 \\ _atpA_ & Subunit \(\alpha\) & \(-\)2.0 \(\pm\) 0.03 & 8.10 \(\pm\) 2.82 \\ \hline \hline \end{tabular}
\end{table}