## 4 Discussion
### The TCA Cycle and the Glyoxylate Shunt  
The conversion of PEP to oxaloacetate in the reaction catalyzed by phosphoenolpyruvate carboxylase (Ppc), and the conversion of pyruvate to acetyl-CoA (ACoA) catalyzed by the pyruvate dehydrogenase complex (AceE, AceF, and Lpd) feed ACoA into the TCA cycle as substrates of the enzyme citrate synthase (GltA) (Figure 2). At each turn of the TCA cycle, one molecule of ACoA is converted into two molecules of CO2, reducing four NAD+, NADP+, or quinones to NADH, NADPH, and quinol, respectively. The TCA cycle phosphorylates one molecule of GDP to GTP and produces one molecule of fumarate from succinate. Additionally, the TCA cycle includes the glyoxylate shunt, bypassing the reactions of the TCA cycle and eliminating the loss of carbon as CO2 [4]. In a biosynthetic direction, carbon from the TCA cycle is withdrawn in two ways: by the decarboxylation of malate to from pyruvate by the malic enzymes MaeA (requiring NAD+) and MaeB (requiring NADP+), and from oxaloacetate to PEP by the phosphoenolpyruvate carboxylase (ATP) enzyme (Pck) [4,11]. Transcriptomic analysis of most of the genes encoding enzymes of the TCA cycle and the glyoxylate shunt in the PB12 and PB12 AGD+ mutants showed an increased relative transcription in these genes than in JM101 (1.7-fold or higher) (Table 3, Figure 2). These results indicate that both strains use the TCA cycle for energy and reducing power. As proposed, the glyoxylate shunt is used under aerobic growing conditions with glucose as the carbon source in the PB12 mutant [11]. Our results indicate that the _apt_ mutant maintains this metabolic trait despite the imposed energetic perturbation. Among the analyzed TCA cycle and the glyoxylate shunt genes in the mutant PB12 AGD+, _sdhC_, _funB_, and _funC_ showed a higher transcription than in PB12 (Table 3, Figure 2). _sdhC_ encodes a succinate:quinone oxidoreductase membrane protein, SdhC, which is a component of the four-subunit succinate dehydrogenase (SQR) enzyme [4]. SdhC is the large subunit of cytochrome b556, and the quinone binding (Qp) site resides in the interface between SdhB, SdhC, and SdhD [4,23,24]. The maximal expression of SQR was reported during aerobic growth, and the oxidation of succinate to fumarate concomitant was catalyzed with the reduction of ubiquinone to ubiquinol. SQR is essential in cellular metabolism, connecting the TCA cycle with the respiratory electron transport chain. As part of the TCA cycle, succinate is oxidized to fumarate by SQR, and electrons are transferred to the membrane quinone pool for entry into the electron transport chain, but do not contribute to the PMF [4; 25]. FumA (fumarase A) is one of the three fumarase isoenzymes in _E. coli_ (encoded by _fumA_, _fumB_, and _fumC_), participating in the TCA cycle catalyzing the reversible conversion of (\(S\))-malate to fumarate + H\({}_{2}\)O [4]. FumA was reported as the most abundant fumarase under aerobic growth conditions, and its expression is controlled by the transcriptional aerobic/anaerobic regulators ArcA, whereas Fnr represses _fumA_[26]. As our results showed that the expression of _fumA_ in the _atp_ mutant practically duplicated the observed in PB12, this difference could be considered an indicator of an active role in the TCA cycle in this mutant. The sensor histidine kinase ArcB represses genes encoding enzymes that catalyze the reaction of the TCA cycle. The DNA-binding transcriptional dual regulator ArcA is part of the ArcAB two-component system involved in response to respiratory conditions, and growth changes with a role in the anaerobic repression of genes associated with aerobic metabolism [4]. Previous results on the mutant PB12 showed that this strain possesses an ArcAB\({}^{-}\) phenotype due to a point mutation resulting in the mutation Tyr\(\tau_{\text{71}}\)Cys of ArcB [11].