Figure 1.
The metabolic modes of Escherichia coli.
The main pathways of carbon and energy metabolism are shown for (A) aerobic respiration, (B) anaerobic (nitrate) respiration, and (C) fermentation. Under aerobic conditions the citric acid cycle (CAC) is functional, whereas under anaerobic conditions the non-cyclic form operates. Under aerobic conditions glucose can be completely oxidized to CO2 and water. Under anaerobic conditions in the presence of an alternative electron acceptor such as nitrate, glucose is partially oxidized to CO2 and acetate. Under anaerobic fermentative conditions glucose is converted to acetate, ethanol and formate and energy is conserved by substrate-level phosphorylation rather than oxidative phosphorylation. The enzyme activities responsible for each step are indicated: AckA, acetate kinase; Acn, aconitase B; AdhE, alcohol dehydrogenase; Cyd, cytochrome bd oxidase; Cyo, cytochrome bo oxidase; Fdn, formate dehydrogenase-N; Fum, fumarase (A, B and C); GltA, citrate synthase; IcdA, isocitrate dehydrogenase; Mdh, malate dehydrogenase; Nar, nitrate reductase; Nuo, NADH dehydrogenase I; Ndh, NADH dehydrogenase II; PCK, phosphoenolpyruvate carboxykinase; PDHC, pyruvate dehydrogenase complex; PFL, pyruvate formate-lyase; Pta, phosphotransacetylase; Sdh, succinate dehydrogenase; Suc, 2-oxoglutarate dehydrogenase/succinyl-CoA synthetase.
Table 1.
Transcripts encoding proteins of central metabolism that are present in altered abundance after shifting anaerobic cultures of E. coli MG1655 to micro-aerobic (10 µM O2) conditions.
Figure 2.
Western blot showing the induction of selected central metabolic proteins after transfer of anaerobic cultures to micro-aerobic conditions.
Anaerobic steady-state fermentative cultures were perturbed by introduction of air to maintain a dissolved O2 tension of 10 µM. Samples were taken before and after perturbation at the indicated times. The samples were analyzed by Western blotting using polyclonal antibodies to the PDHC components E1 and E3, aconitase B (AcnB), pyruvate formate-lyase (PFL), and PFL repair protein (YfiD). Total protein loading in each track is shown as the Coomassie blue-stained gel in the bottom panel.
Table 2.
Pyruvate dehydrogenase complex activity during transition from anaerobic to micro-aerobic conditions.
Figure 3.
Carbon balance for the anaerobic steady-state cultures.
The carbon input into the system comes from glucose and carbon dioxide supplied to the culture (Cin). Carbon output consists of overflow metabolite production and biomass (Cout). At steady-state Cin = Cout (i.e. Cbalance = 100%). The numbers in parentheses are calculated from the values in Table 3 and are the average (n = 4) concentrations of C (mM) in each of the indicated species as measured by NMR.
Table 3.
Measurements of extra-cellular metabolites and biomass production during transition of anaerobic cultures of E. coli MG1655 to micro-aerobic conditions.
Figure 4.
Inferred activity of FNR and PdhR.
The inferred activities of the transcription factors FNR (A) and PdhR (B) are shown as natural log (ln) values. The error bars (mostly within the size of the symbols) represent the standard deviation provided by the posterior distribution.
Figure 5.
Model illustrating the role of pyruvate as a secondary messenger in managing the transition of E. coli K-12 from anaerobic to micro-aerobic conditions.
In the absence of O2 (A) carbon (pyruvate) is incorporated into biomass or excreted as overflow metabolites (mostly formate, acetate and ethanol) in a process initiated by pyruvate formate-lyase (PFL). Upon transfer to micro-aerobic conditions (B) O2 inactivates PFL, directly by oxygenolytic cleavage, indirectly through the action of PFL deactivase (AdhE) [23]. Inactivation of the O2-sensing transcription factor FNR inhibits expression from promoter 6 of focA-pflB [24]. Inhibition of PFL activity at transcriptional and protein levels and a delay in induction of pyruvate dehydrogenase complex (PDHC) activity (Tables 1 and 2) results in a deficit in the capacity to metabolize the pyruvate generated by glycolysis. This hiatus in pyruvate metabolism is sufficient to inactivate the transcription factor PdhR. Inactivation of PdhR derepresses expression of genes encoding the PDHC and the PFL repair protein (YfiD). This response acts to restore the capacity to metabolize pyruvate and as a result extra-cellular pyruvate concentration begins to decline 60 min into the transition. In addition, pyruvate-mediated derepression of the PdhR-repressed genes encoding the primary dehydrogenase NdhII and the terminal oxidase cytochrome bo' oxidase (Cyo) [8] increases the consumption of O2 at the cell membrane, potentially stabilizing undamaged/newly activated cytoplasmic PFL activity and restoring some activity to the O2-sensing transcription factor, FNR, promoting limited anaerobic metabolism (e.g. acetate excretion, Table 3).