Effect of oxygen on the Escherichia coli ArcA and FNR regulation systems and metabolic responses Chao Wang Jan 23, 2006

Metabolic products formed by E. coli change according to the growth condition with factors such as the availability of oxygen, pH, and nutrient source. With the increased use of genetically modified strains it is useful to be able to predict culture conditions that would be best suited for a particular strain to produce the desired product. A major factor is the aerobic vs. anaerobic nature of the culture; aerobic culture favors faster growth but anaerobic conditions are needed for the formation of certain desired products (e.g., ethanol, lactic acid, etc.).

Basically, in the aerobic state the TCA cycle operates to oxidize pyruvate with the reductants formed coupling with the electron transport chain to generate the proton gradient, which in turn is used for ATP production. In anaerobic conditions, succinate dehydrogenase is replaced by fumarate reductase and 2-ketoglutarate dehydrogenase is repressed blocking the TCA cycle at 2-oxoglutarate. Pyruvate can then be metabolized to lactate by lactate dehydrogenase, or converted to acetyl-CoA by pyruvate formate lyase instead of the pyruvate dehydrogenase, which acts under aerobic conditions. Under anaerobic conditions the acetyl-CoA forms either ethanol or acetate instead of combining with oxaloacetate to form citrate, as it does under aerobic conditions.

Escherichia coli possesses a large number of sensing/regulation systems for the rapid response to availability of oxygen and the presence of other electron acceptors The adaptive responses are coordinated by a group of global regulators, which includes the one component FNR (fumarate, nitrate reduction) protein, and the two-component Arc (aerobic respiration control) system.

A thorough quantitative characterization of the effect of ArcA, FNR, and their combination on the physiological behavior of cells under uniform culture conditions To quantitate the contribution of Arc and Fnr-dependent regulation in catabolism.

Strains and Plasmids

The cells were grown under a glucose-limiting condition at a dilution rate, and variable oxygen concentrations. The oxygen supply was varied by varying the percentage of oxygen in the gas mixture of oxygen and nitrogen. Glucose, succinate, lactate, formate, acetate, ethanol, and pyruvate were determined using HPLC. NADH and NAD+ were determined using HPLC. Oxygen and carbon dioxide concentrations in the headspace were measured using a CO2/O2 analyzer. The carbon dioxide concentration was also measured using CO2 detector tubes. Hydrogen was measured using H2 detector tubes. Fluxes were calculated using the metabolite concentrations.

The internal redox state, reflected by the NADH/NAD+ ratio

The metabolic activity is important, as deletion of one regulatory gene may affect the metabolite pattern, which in turn can affect the activity of various other enzymes. In particular, it can result in the activation of another regulatory system. Indeed, deletion of fnr did not affect the metabolite pattern at OCH of 2.5–21%, while deletion of arcA results in an increase in formate, the NADH/NAD+ ratio, lactate, and ethanol and a decrease in succinate at OCH of 1–10%

Active ArcA protein should induce the transcription of pfl. However, deletion of ArcA results in strain with higher PFL fluxes compared to the wildtype at OCH of 1–10%. Moreover, since FNR is inactive at OCH of 2.5–21%, the same PFL fluxes were expected in cultures of the arcA mutant strain and in cultures of the strain with the arcA–fnr double mutation under these microaerobic conditions. Yet significantly lower fluxes were obtained in cultures of the arcA–fnr double mutant strain.

Two questions: 1) Why are the PFL fluxes of the arcA mutant strain higher than the fluxes of the wildtype, while ArcA has a positive effect on pfl transcription? 2) Why are the PFL fluxes of cultures of the arcA–fnr double mutant strain lower than those of cultures of the arcA mutant strain at OCH of 2.5–21%, while FNR is supposedly inactive under these conditions?

To answer these questions it is important to examine the NADH/NAD+ ratios obtained in the various cultures. This ratio reflects the steady-state internal redox state, which may affect the activity of many enzymes. It has been shown that the FNR protein responds to redox potential. Moreover, it was suggested that high NADH/NAD+ ratios can activate the FNR protein in cell extracts. Thus, it seems reasonable that the NADH/NAD+ ratio, which affects the redox potential of the cells, can alter directly or indirectly the activation state of the FNR protein in the cells as well as affecting the activity of many enzymes. As the NADH/NAD+ ratios are significantly higher in cultures of the arcA mutant strain at OCH 1–10% compared to the wildtype, it is possible that the cells contain a higher level of the FNR protein in an activated form.

To investigate the hypothesis that FNR is more functionally active in cultures of the arcA mutant strain, we transferred a plasmid that expresses a mutated FNR protein under the control of the lac promoter (pRZ7382) into the arcA–fnr double mutant strain. A higher level of active FNR protein is present in cultures of the arcA mutant strain compared to the wildtype under microaerobic conditions (OCH <10%), and the active FNR influences the fluxes through the PFL.

The FNR protein may activate either the transcription of the pfl gene or could indirectly affect the activity of the PFL enzyme. It is more likely that the active FNR protein indirectly affects the activity of the PFL enzyme. One possible mechanism by which the FNR protein could affect the activity of the PFL enzyme is by the activation of yfiD. It is possible that in the arcA mutant cultures the active FNR protein induces the expression of YFiD protein, which in turn reactivates the PFL protein in the presence of oxygen.

In this work the metabolic activity of wildtype E. coli, an arcA mutant, an fnr mutant, and a double arcA–fnr mutant, via the fermentative pathways, in glucose- limited cultures and different oxygen concentrations was studied in chemostat cultures at steady state. It was found that the most significant role of ArcA is under microaerobic conditions, while that of FNR is under more strictly anaerobic conditions. The FNR protein is normally inactive during microaerobic conditions. However, our results indicate that in the arcA mutant strain the cells behave as if a higher level of the FNR regulator is in the activated form compared to the wildtype strain during the transition from aerobic to microanaerobic growth. The results show a significant increase in the flux through pyruvate formate lyase (PFL) in the presence of oxygen. The activity of FNR-regulated pathways in the arcA mutant strain is correlated with the high redox potential obtained under microaerobic growth. CONCLUSIONS

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