## 4 Discussion
Thus, the increasing levels of fatty acids at lower dilution rates are potentially associated with nutrient starvation responses, and in \(\Delta\)_rel_A mutant cells, these cellular responses are evidently suppressed or simply not triggered. Interestingly, in the succinate (_succ_) profile, metabolite levels were higher in \(\Delta\)_rel_A cultures, except at a dilution rate of 0.1 h\({}^{-1}\). This suggests that in the \(\Delta\)_rel_A culture, the metabolic activities involving this metabolite may be augmented, indicating a less stringent control of TCA enzyme activities or the activation of the glyoxylate cycle. However, the lack of information regarding other intermediaries of the TCA cycle does not support any further assumptions. Overall, the stringent control of _E. coli_ metabolism can be perturbed by the _rel_A mutation, in particular under slow growth steady states (0.05 and 0.1 h\({}^{-1}\)). Alterations in amino and fatty acids levels were significant, as was the poor correlation between several fatty acids profiles produced by the two _E. coli_ cultures. In particular, fatty acids profiles were strongly divergent when decreasing the dilution rate (from 0.1 to 0.05 h\({}^{-1}\)), _i.e._, while in the _E. coli_ W3110 culture fatty acid levels increased, they decreased in the _E. coli_\(\Delta\)_rel_A mutant culture. This supports the idea that the RelA enzyme is involved in the control of metabolic activities manipulating metabolite levels and thus, the metabolic state of cells. Many authors have stated that cells lacking the RelA-dependent stringent control have a relaxed phenotype [7, 42, 43, 44], which is often characterized by a limitation of certain cellular processes, including central metabolic activities (e.g., fatty acids biosynthesis). Therefore, alterations observed in metabolite profiles might be explained by the lack of this enzyme and most likely a deregulation of certain metabolic functions. Also, the effect of other regulators that play a role in the control of metabolism under nutrient-limited conditions cannot be disregarded. The CRP-cAMP transcriptional regulator is chiefly responsible for controlling metabolic fluxes under glucose limitation in _E. coli_ cells [24]. This regulator responds to alterations in the intracellular cAMP levels resulting from glucose availability, which are higher at dilution rates below 0.1 h\({}^{-1}\), and through the functional conversion of CRP into the active form CRP-cAMP that regulates the expression of various gene-encoding transporters and catabolic enzymes of sugars other than glucose [37]. At these conditions, we observed large differences between the \(\Delta\)_rel_A mutant and wild-type cultures, which suggests that the single gene mutation influences the CRP-cAMP metabolic control. This phenomenon has been previously associated with the stringent response [8, 45, 46], indicating that ppGpp potentiates the expression of several stress response genes, namely the transcriptional regulator CRP that governs the catabolite repression. Thus, it was expected that \(\Delta\)_rel_A mutants would be less effective in inducing anaplerotic reactions at a dilution rate of 0.1 h\({}^{-1}\).