Transcript Document

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.
CONCLUSIONS
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 glucoselimited 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.
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