## 2 Materials and Methods  
### Bacterial Strains, Plasmids, Culture Media, and Growing Conditions  
The bacterial strains JM101, PB12, and PB12 AGD\({}^{+}\) used in this contribution are described in Table 1. Plasmids pTrc99A [12] and pTcratp_AGD [6] were used to transform by electroporation of competent cells of _E. coli_ PB12 by standard procedures [13]. Cultures were performed using quadruplicate, as performed previously [6; 11], on 1 L Applikon ADI 1010 fermenters on M9 medium [13] with 2 g/L of glucose as the carbon source with a working volume of 750 mL, pH 7.0 controlled with NH\({}_{4}\)OH 2.9%, at 37 \({}^{\circ}\)C, 600 rpm, an airflow rate of 1 vvm. Cultures started with an OD\({}_{600\text{nm}}\) of 0.05. Samples containing 50 mL of the different strains growing logarithmically in the reactor were withdrawn at OD\({}_{600\text{nm}}\) = 1.0 corresponding to the mild-exponential growth phase [6; 11] for ATP and ADP concentrations ([ATP]/[ADP] ratio), ATPase activity, cAMP measurements, and for total RNA extraction for RT-qPCR analysis [6]. The energetic level was estimated as reported previously by using the [ATP]/[ADP] ratio based on the method reported by [6; 9]. Aliquots of 40 mL of cultures were withdrawn from bioreactors when the cellular growth was an OD\({}_{600\text{nm}}\) = 1.0, corresponding to the mild-exponential growth phase. Cell lysis was performed as described by [9]. According to the supplier specifications, the ATP concentration was determined using the ENLTEN(r) ATP Assay System (PROMEGA, Madison, WI, USA). For the [ADP] determinations, the ADP content in the same sample used for ATP determination was converted to ATP by adding 1 U of pyruvate kinase enzyme (PK) (Sigma-Aldrich, St. Louis, MO, USA) and 1 mM phosphoenolpyruvate (PEP) and recorded as the increase in luminescence. Finally, a standard ATP concentration (1 uM) was added to quantify [ATP] and [ADP]. The results were corrected for quenching of the ATP signal by the PK preparation. Soluble ATPase activities were measured in enzymatic assays using the above cellular extracts. The intracellular cAMP concentration determinations were measured in bioreactor cultures at OD\({}_{600\text{nm}}\) = 1.0 using Cyclic AMP Enzyme Immunoassay Kit (Enzo Life Sciences, Farmingdale, NY, USA) following the manufacturer's instructions. For ATPase activity, withdrawn cells were centrifuged as above and resuspended in 2.0 mL of ATP buffer (0.1 M Tris-acetate, pH 7.75, 10 mM potassium acetate, 2.0 mM EDTA) and supplemented with 10 uM MgCl\({}_{2}\) per mL. Resuspended cells were lysed  
\begin{table}
\begin{tabular}{c l l} \hline \hline
**Plasmids** & **Relevant Features** & **References** \\ \hline \hline \multirow{3}{*}{pTrc9A} & Cloning vector under the _trc_ promoter. & \\  & Carries _bla_ and _lac_\# genes. Replication origin & [12] \\  & from pBR322. & \\ \cline{1-1}  
and centrifuged as above, and incubated for 5 min at 37 \({}^{\circ}\)C. According to the supplier specifications, ATPase activity was determined using the ENLITEN(r)ATP Assay System (PROMEGA, Madison, WI, USA). The plasmid _atpAGD_ encodes the F1 ATP part of the membrane-bound (F\({}_{1}\)-F\({}_{0}\)) H+ ATP synthase. The expression of this operon in the PTS\({}^{-}\) mutant PB12 results in an uncoupled cytoplasmic ATPase activity. As enzymatic assays for determination of [ATP]/[ADP] ratio were performed in cellular extracts, results in the unique cellular enzymatic activities, and was considered as the total determined ATPase activity. Determination of Uncoupled ATPase Activity and Determination of [ATP]/[ADP] Ratio and cAMP Concentration  
The energetic level was estimated, as reported previously, by using the [ATP]/[ADP] ratio based on the method reported by [6; 9]. Aliquots of 40 mL of cultures were withdrawn from bioreactors when the cellular growth was an OD\({}_{600\mathrm{nm}}\) = 1.0, corresponding to the mild-exponential growth phase. Cell lysis was performed as described by [9].