Effect of oxygen on the Escherichia coli ArcA and FNR regulation systems and metabolic responses

1
Effect of oxygen on the Escherichia coli ArcA and
FNR regulation systems and metabolic responses

• Chao Wang
• Jan 23, 2006

2
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.).
3
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.
4
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.
5
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.
6
Strains and Plasmids
7
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.
8
(No Transcript)
9
(No Transcript)
10
(No Transcript)
11
(No Transcript)
12
The internal redox state, reflected by the
NADH/NAD ratio
13
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 2.521, 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
110
14
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 110. Moreover,
since FNR is inactive at OCH of 2.521, the same
PFL fluxes were expected in cultures of the arcA
mutant strain and in cultures of the strain with
the arcAfnr double mutation under these
microaerobic conditions. Yet significantly lower
fluxes were obtained in cultures of the arcAfnr
double mutant strain.
15
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 arcAfnr double mutant strain
lower than those of cultures of the arcA mutant
strain at OCH of 2.521, while FNR is supposedly
inactive under these conditions?
16
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 110 compared to
the wildtype, it is possible that the cells
contain a higher level of the FNR protein in an
activated form.
17
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 arcAfnr
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 lt10), and the
active FNR influences the fluxes through the PFL.
18
(No Transcript)
19
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.
20
CONCLUSIONS
In this work the metabolic activity of wildtype
E. coli, an arcA mutant, an fnr mutant, and a
double arcAfnr 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.
21
The End