According to Bledig SA, 1996 deletion of FruR (Cra) resulted in an appreciable increase of pykF-encoded pyruvate kinase synthesis on gluconeogenic carbon sources as compared to glycolytic carbon sources (casamino-acids were added to the culture medium to allow gluconeogenic growth). In addition, overexpressed FruR repressed the transcription of the pykF gene significantly on gluconeogenic carbon sources but not on glycolytic carbon sources. Thus the FruR (Cra) effect on pykF transcription significantly takes place under conditions of gluconeogenic growth. Therefore using pykF as a reporter gene for analyzing, under glycolytic growth conditions, a transcriptional regulation by FBP/Cra appears inappropriate. In addition, the concentration of FBP is dependent on the concentration of PEP because phosphofructokinase is subject to allosteric control by PEP (Blangy D, 1968). Therefore decreasing pyruvate kinase synthesis below physiological level also decreases the FBP concentration which is in conflict with the present data (see Fig. S1 and Fig. 2B).
As shown in Fig. 1C, concentrations of FBP controlling the glycolytic flux are below 1 mM. This concentration range has been reported to be insufficient for FBP to displace FruR from its operator sites. In addition, cAMP-dependent carbon catabolite repression was reported by Nanchen A, 2008 to be the dominant control mechanism of metabolic fluxes under glucose limitation condition (used in Fig. 1B, black squares).
Nucleic Acids Res. 2013A ptsI mutant strain of Escherichia coli does not grow on minimal medium glucose after 48 hours. Therefore the ptsI mutant strain from the Keio collection, which is reported to grow on glucose with growth defect (Supplementary Information, S3 and S7), is not adequate. Therefore it is irrelevant to state, "…the cyaA and crr mutants should behave as the ptsI strain" (in Confirmation of known regulators of acs expression), because unlike ptsI strain, cyaA and crr mutant strains grow on glucose, even though they do not grow as well as wild type strains (it is also reported in S3 that gltA and icd mutant strains do not grow on glucose but they actually do when supplemented with glutamate). In addition, under the experimental conditions described, it is not clear to which extent glucose is used preferentially over acetate by certain mutant strains particularly in the presence of cAMP (absorbance should have been provided in Figure 4C, D, E and F). Also the authors seem to ignore that any mutant strains lacking any of the TCA cycle enzymes fail to grow on acetate as they wrote, "Interestingly we found that the above mutants [sucB and lpdA] fail to grow on acetate". Researchers are better off reading the paper by Oh MK, 2002 titled, Global expression profiling of acetate-grown Escherichia coli, which investigates the transcript profile of an E. coli strain grown on acetate as compared to the same profile on glucose.
Most importantly, the authors should re-consider analyzing "connections between the metabolic state of the cell and gene expression" by using luciferase as a reporter system considering luciferase strongly depletes the pool of cellular ATP. It is therefore no wonder mutant strains lacking genes involved in energy supply display the lowest luciferase activity. And yet, the authors used a second reporter system to detect "artifacts due to metabolic influences on luciferase activity", as stated in the Introduction.
Nature. 2013In an article by Doucette CD, 2011, it is clearly demonstrated that the effect of α-ketoglutarate on cAMP synthesis is related to an inhibition of Enzyme I. As a consequence phosphorylation of Enzyme IIAGlc is impaired, which relates to a decrease in cAMP synthesis, and yet You C, 2013 studied the effect of α-ketoglutarate in pts mutant strains. Because "a strong transient repression was still observed upon the addition of α-ketoacids in strains with deletion of various PTS proteins", the authors basically conclude the PTS proteins are no longer necessary for mediating the main effect by α-ketoglutarate, and α-ketoglutarate directly affects the activity of adenylate cyclase (AC). However, 10 mM α-ketoglutarate does not inhibit purified AC (Yang JK, 1983, in Table VII, caption a).
The feedback strategy established by Doucette CD, 2011 relates to data obtained under conditions of 'extreme' catabolite repression elicited by nitrogen limitation. A direct inhibitory effect of α-ketoglutarate on Enzyme I inhibits phosphorylation of the components of the phosphotransferase system (PTS), including phosphorylation of EnzymeIIAGlc which activates AC. Therefore, cAMP-mediated catabolite repression occurs in these conditions. Another not-yet propounded effect of an increase in α-ketoglutarate is an enhanced inducer exclusion, which further hinders uptake of carbon sources other than PTS substrates. Under nitrogen-limited conditions, β-galactosidase synthesis in a wild-type strain growing on glucose is most likely affected by inducer exclusion. Thus, nitrogen-limited growth may not 'rely completely' on cAMP-mediated gene regulation, as transcriptional regulation by cAMP and inducer exclusion both interfere with β-galactosidase synthesis (Crasnier-Mednansky M, 2008). By not taking into account inducer exclusion the authors undermine the power of 'quantitative physiology'. Within the same frame of thought, using pts strains grown on lactose to measure β-galactosidase activity is physiologically irrelevant.
In sharp contrast with the present data, Daniel J, 1986 reported α-ketoglutarate (or pyruvate indirectly by accumulation of α-ketoglutarate) caused repression of the lac operon - but not oxaloacetate. In addition repression did not occur in crr strains (lacking Enzyme IIAGlc), in support of Doucette CD, 2011.