EC Slater Institute, University of Amsterdam,
1018 WS Amsterdam, The Netherlands,1 and
Department of Molecular Microbiology, John Innes Centre,
Norwich Research Park, Colney, Norwich NR4 7UH, United
Kingdom2
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INTRODUCTION |
Escherichia coli
possesses distinct catabolic routes that enable it to conserve energy
efficiently under wide ranges of redox conditions. In environments that
provide the cell with external electron acceptors such as oxygen,
nitrate, fumarate, and dimethyl sulfoxide, reoxidation of reducing
equivalents generated by the oxidation of the energy source occurs in
the respiratory chain. This process can be coupled to the formation of
a proton motive force (PMF) and thus constitutes an efficient pathway
for energy conservation. In the absence of oxygen or other external
electron acceptors, ATP synthesis occurs at the level of substrate
phosphorylation. Under such fermentative conditions E. coli,
when growing on glucose, excretes specific products such as ethanol,
acetate, lactate, succinate, and formate (or CO2 and
H2). The relative rates of formation of these products are
governed by the demand for redox neutrality (5, 21).
The actual in vivo fluxes of carbon and electrons through the various
pathways are determined largely by three major branch points (Fig.
1). The first of these involves the
cleavage of pyruvate, which serves as a common substrate for pyruvate
formate-lyase (PFL) and the pyruvate dehydrogenase complex (PDHc).
Entry into the fermentative pathway depends largely on the activity of
PFL, whereas entry into the respiratory pathway is largely governed by
the activity of the PDHc. At the second branch point, acetyl-coenzyme A, the product of both of the above reactions, can be converted to
either the major fermentation products acetate and ethanol or can
subsequently undergo further oxidation in the tricarboxylic acid (TCA)
cycle. Finally, E. coli also possesses a branched
respiratory chain. The electron flow into respiration can follow
alternative routes to oxygen, via a coupled or an uncoupled NADH
dehydrogenase (NDH-1 or NDH-2, respectively) to quinone (19,
42). Quinol is then oxidized by either the cytochrome
bd or the cytochrome bo terminal oxidase complex,
which in turn passes the electrons to oxygen with concomitant reduction
of the latter to water. The two terminal oxidases differ in their
affinities for oxygen as well as in their
H+-to-e
stoichiometries. Cytochrome
bd translocates one H+ per e
(44, 48) and has a high affinity for oxygen (10, 34, 49), whereas the low-affinity cytochrome bo oxidase is
thought to translocate two H+s per e (3,
48).
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FIG. 1.
Major routes of the anaerobic (left) and aerobic (right)
catabolic pathways in E. coli. Open circles, branch points
of the respiratory and fermentative catabolism. Glc, glucose.
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The regulation of expression of genes encoding catabolic enzymes in
enterobacteria has been the subject of many studies. In E. coli, two global regulatory systems which control aerobic
respiration and fermentation have been identified. These are the ArcAB
two-component regulatory system and the FNR protein (18, 27, 57,
58). FNR serves as an activator of a number of genes whose
products are involved in anaerobic respiratory metabolism,
whereas the ArcAB system plays an important role in transcriptional
regulation under both anaerobic and aerobic conditions. It has
been shown to repress a number of genes coding for TCA cycle enzymes,
cyoABCDE (coding for the low-affinity terminal oxidase),
and, to a lesser extent, transcription of pdhR-aceEF-lpd
encoding PDHc and its regulator during fermentative growth (4, 8,
9, 25, 53, 56). Conversely, ArcA acts as an activator of
transcription, under appropriate conditions, of two catabolic operons.
These are the focApfl operon encoding PFL and a formate
transport protein (51, 59) and the cydAB operon
coding for cytochrome bd oxidase (61).
Regulation of PFL synthesis and activity in vivo is very complex. Both
are controlled by the prevalent oxygen status, which consequently has a
major bearing on the control of catabolism (33). First, ArcA
and FNR function in combination as anaerobic activators of
focApfl gene expression. Second, interconversion of PFL
between inactive and active glycyl radical-bearing species occurs at
low oxygen tensions and is controlled by the activities of the
iron-sulfur protein PFL activase and the product of the adhE
gene, PFL deactivase (33, 52). Third, the active glycyl radical form of PFL is irreversibly destroyed by molecular oxygen and
hence must be either protected from oxygen damage or converted to the
inactive, oxygen-stable species during the transition between anaerobiosis and aerobiosis (63).
Aerobic respiratory activity in vivo is strictly dependent on the
presence of terminal oxidases. As the oxygen supply decreases, a
concomitant increase in cydAB expression and decrease in
cyoABCDE expression occur (61). Cytochrome
bd oxidase may therefore provide a means of affording
protection to the active PFL enzyme at low oxygen levels.
Transcriptional regulation of cyd is both ArcA and FNR
dependent, whereby FNR functions as an anaerobic repressor of
cyd expression (6, 7, 13).
Although a substantial amount of information regarding the mechanism of
signal transduction by the ArcAB system is now available (14, 15,
22, 26, 32, 39-41, 62), no unequivocal proof as to the nature of
the signal that stimulates the regulatory cascade in vivo has been
presented. Since molecular oxygen has been excluded as the
biochemical signal being sensed by ArcB (24, 28, 38), a
number of other potential signals have been proposed, including
intracellular metabolites (e.g., NADH, D-lactate, and pyruvate), the redox state of the respiratory chain, and the PMF (2, 22, 24, 30). In this study we used steady-state
chemostat cultures to investigate in detail the response of E. coli to varying oxygen availability. In particular, we provide
evidence to support the dual sensory mechanism of ArcB originally
proposed by Matsushika and Mizuno (39), whereby microaerobic
activation of the Arc system is mainly determined by a
respiration-dependent signal, while deactivation of the system is
governed by the redox state of the cell when oxygen levels become less
restrictive. Furthermore, we propose that in E. coli
respiratory protection of the active species of PFL by cytochrome
bd oxidase may occur during microaerobic growth.
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MATERIALS AND METHODS |
Strains and growth conditions.
E. coli strains
RM123
RM23 [MC4100 recA 
(pfl-lacZ)]
(50) and RM3133RM23 [MC4100
arcA::tet (pfl-lacZ)]
were used throughout this study. These strains are derivatives of
E. coli MC4100 containing a chromosomal pfl-lacZ
fusion in the attachment site. RM3133 arcA::tet was constructed by
transducing the arcA::tet allele from MG1655 arcA::tet (a gift from D. Touati) into MC4100. MC4100 was used in a number of experiments as a control.
Cells were grown in chemostat cultures under glucose-limited conditions
(Bioflo 3000 and III; New Brunswick) at a constant dilution rate of
0.15 ± 0.01 h1 at various oxygen supply rates.
Glucose (45 mM) was used as the single carbon and energy source. A
simple salts medium as described by Evans et al. (11) was
used, but instead of citrate, nitriloacetic acid (2 mM) was used as the
chelator. Selenite (30 µg/liter) and thiamine (15 mg/liter) were
added to the medium. The pH value was maintained at 7.0 ± 0.1 by
titrating with sterile 4 M NaOH, and the temperature was set to 35°C.
The cultures were regularly checked for kanamycin resistance by plating
on Luria-Bertani (LB) plates containing 20 µg of kanamycin/ml. The
arcA genotype was confirmed by Western blot analysis with
polyclonal ArcA antiserum each time the arcA strain was
used. The strains were maintained in vials in LB medium with 30%
(wt/vol) glycerol at 
70°C. The oxygen supply was varied by varying
the percentage of oxygen in a gas mixture of air and N2
while stirring the culture at a constant speed. In order to prevent
long-term adaptive responses the oxygen availability was varied
randomly. Residual dissolved oxygen was monitored by an INGOLD
polarographic (Ø [diameter of the electrode's tip], 12 mm)
O2 sensor with a detection limit of 100 nM and an accuracy
of approximately 30 nM as determined by examining O2 input
versus O2 output (electrode reading) under conditions
similar to those used for cultivation (medium, pH, temperature), but in the absence of cells. The O2 input/output ratio was found
to be linear in the range tested (0.05 to 100% of air saturation).
Analytical procedures.
Steady-state bacterial dry weight was
measured by the procedure of Herbert et al. (20). Levels of
glucose, pyruvate, lactate, formate, acetate, succinate, and ethanol
were determined by high-pressure liquid chromatography (LKB) with a
REZEX organic acid analysis column (Phenomenex) at a temperature of
65°C with 4 mM H2SO4 as the eluent, using a
2142 refractive index detector (LKB) and an SP 4270 integrator (Spectra
Physics). CO2 production and O2 consumption were measured by passing the eluent gas from the fermentor through a
Servomex CO2 analyzer and a Servomex O2
analyzer, respectively.
-Galactosidase activity was measured in permeabilized cells taken
from steady-state cultures as originally described by Miller (43) and modified by Giacomini et al. (16). The
cytochrome bd content of cells in French press (2.068 × 107 Pa) extracts in 50 mM Tris-HCl, pH 7, was estimated
by UV/visual difference spectrophotometry (46).
Dithionite-reduced versus oxidized difference spectra (absorption
maximum at 625 nm), recorded on an SLM AMINCO DW-2000 UV/visual
spectrophotometer, were analyzed to quantitate cytochrome d,
using the wavelength pair (628 and 649 nm) and extinction coefficient
(18.8 cm1 mM1) given by Kita et al.
(34).
Concentrations of NADH and NAD were determined in extracts obtained by
rapid sampling of chemostat cultures into 5 M KOH and 5 M HCl,
respectively, and were assayed after neutralization and filtration as
described previously (55).
Calculations of balances and fluxes.
The determination of
the steady-state concentrations of biomass and excreted end products
and CO2 production and O2 consumption rates
allowed the calculation of specific product and substrate consumption
rates (q values; millimoles per gram of dry weight per
hour). By converting these q values into amounts of carbon atoms produced (it was assumed that 50% of the dry weight consists of
carbon [20]) versus amounts consumed per gram of dry
weight per hour, the carbon recovery (Crecovery;
carbon balance) was determined according to the equation
Similarly, redox balances were constructed for fully aerobic and
fully anaerobic conditions. Here, net NADH and net NAD production rates
were calculated on the basis of the stoichiometric values known for
fermentation pathways, the TCA cycle, and succinate formation
(8). It was assumed that biomass formation from glucose as
the carbon source and ammonium ions as the nitrogen source is a
redox-neutral process (20):
where J is the catabolic flux. Redox balances were
defined as
qNAD/qNADH · 100.
All data present a carbon balance of 95% ± 3%. Redox balances,
calculated as indicated, were found to be 95 to 98%. Here, it was
assumed that under fully anaerobic conditions PDHc activity was
completely absent and that under fully aerobic conditions PFL activity
was absent (8).
Typical product formation patterns and carbon and redox balances for a
number of steady states are shown in Table
1. The q values were used to
calculate the carbon fluxes through PDHc, PFL, and TCA cycles
(J values; millimoles per gram of dry weight per hour). For
microaerobic conditions, these calculations are based on the scheme
depicted in Fig. 1 under the assumption that the prerequisite for a
complete redox balance was fulfilled (see also reference
8):
where qCO2(TCA) = 2/3 · (qCO2 + qformate qethanol qacetate). The calculations for
JPFL and JPDHc showed the
former flux to approach zero under anaerobic conditions and the latter
to approach zero under aerobic conditions, which is consistent with the
assumptions made for the calculations of redox balances under these
conditions.
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TABLE 1.
Typical steady-state product formation patterns and
carbon and redox balances under different oxygen
availability conditions
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RESULTS |
Experimental setup.
It is important to realize that it is
technically rather complicated to obtain data from cells grown under
steady-state conditions at very low oxygen tensions. It is not
surprising therefore that the bulk of data available deal with cells
grown under either fully aerobic or anaerobic conditions. Except for
the studies of Wimpenny and Necklen (64) and Rice and
Hempfling (49) only a few investigations have been performed
under conditions of low oxygen tension (1, 36, 61). From
these studies two conclusions can be drawn. First, for different
experimental setups (for example, alteration of the geometry of the
fermentor) a different oxygen supply may be required to achieve the
same physiological response of the cells (49). Second, a
certain oxygen supply condition at which further addition of oxygen has
no significant effect can be determined (1, 61).
E. coli was grown in chemostat cultures using glucose as the
sole carbon and energy source, and the experiments were carried out in
two different chemostats, with culture volumes of 1,250 (A) and 1,380 ml (B). Although cells responded qualitatively similarly upon changes
in the oxygen supply in the two chemostats, different actual
percentages of oxygen input were required to evoke the same response.
Consistently, a 2.2-fold-higher percentage of oxygen in the inflowing
gas was required for chemostat A than for chemostat B to obtain similar
responses of the cells. We assume that this factor reflects the
relative differences in Kl,a values (the Kl,a value of the system is the rate of oxygen transfer from the gaseous to
the liquid phase [12]) due to a difference in geometry
and stirring efficiency between the two vessels. It should be realized that the supply rate of O2 does not define the
O2 availability to a cell because the latter is also
dependent on the Kl,a values and the biomass. To eliminate
the effects of biomass, equal biomass concentrations for the same
percentage of aerobiosis in the two chemostats were obtained by using
the same glucose concentrations in all the experiments. Increased
oxygen contents of the inflowing gas mixture resulted in a new steady
state with cells displaying an increased oxygen consumption rate (Fig.
2a). As a consequence, up to an oxygen
input of 12 (A) or 5.6% (B), the steady-state dissolved oxygen
concentrations in experiments with the wild type did not change
significantly (Fig. 2b). Apparently, the organisms responded to these
changes in oxygen availability in a "homeostatic" manner, as indeed
is exemplified by the observed changes in the composition of the
respiratory chain (see below). The cell yield per mole of glucose
increased from 20.4 ± 1.5 g under anaerobic conditions to
69.33 ± 0.8 g under fully aerobic conditions. To be able to
compare the results obtained from the two fermentors, the data are
normalized to the minimal percentage of oxygen in the inflowing gas
(30% for chemostat A and 14% for chemostat B) required to reach
complete aerobic behavior (i.e., CO2 as the sole end
product). Thus, we refer to the minimum oxygen input resulting in
complete aerobiosis as 100% aerobiosis. We conclude that
dissolved-oxygen tension (DOT) and actual oxygen input are not
appropriate parameters to describe the responses of E. coli to variations in oxygen availability. The response of the cells depends
on the oxygen transfer rate of a particular setup, and this precludes
the possibility of comparing results if actual oxygen input is used. If
DOT is used as a variable, it is not possible to discriminate between
different responses of the cell within a low-oxygen-supply range.
Therefore, we propose to use the percentage of aerobiosis as a variable
to study responses of the cell to different oxygen availabilities as
described here.
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FIG. 2.
(a) Changes in the specific rate of respiration ( ,
chemostat A;
 ,
chemostat B) and intracellular redox state (NADH/NAD ratio) ( ,
chemostat A;  , chemostat B) in response to a change in the oxygen
supply in wild-type, glucose-limited E. coli. Upper
x axis, actual percentage of oxygen in the inflowing gas in
chemostat B. It took 2.2-fold more oxygen in the inflowing gas to evoke
the same effect in chemostat A. (b) Residual dissolved oxygen
concentrations ( ) in steady-state glucose-limited chemostat cultures
of E. coli (wild type) with increasing oxygen supply. Bars,
amplitude of fluctuations in DOT. (Inset) Control of O2
electrode sensitivity in the low (below 1 µM O2) and high
ranges. The control was performed in the absence of cells.
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We define the microaerobic range as the range between 0 and 100% aerobiosis.
Glucose catabolism at variable oxygen availability.
In Fig.
3a the specific production rates of the
major fermentation products are presented. For fully aerobic and fully
fermentative conditions the data are quantitatively virtually the same
as those found earlier (8).
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FIG. 3.
(a) Changes in specific rates of ethanol ( ,
chemostat B; , chemostat A), acetate ( , chemostat B;  ,
chemostat A), and formate (inset;  , chemostat A;
 , chemostat
B) formation in response to change in oxygen availability in
wild-type E. coli. For an explanation of x-axis
values, see the Fig. 2 legend. (b) Effect of oxygen availability on
distribution of in vivo flux (J) between PFL (, chemostat
A;  , chemostat B) and PDHc ( , chemostat A;  , chemostat B) in
wild-type E. coli. (c) Effect of oxygen availability on in
vivo TCA activity
( , chemostat A;
 , chemostat B) in
wild-type E. coli.
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Clearly, the cell switches gradually from completely fermentative to
completely respiratory metabolism as more oxygen becomes available,
formate being the first product to disappear.
The steady-state specific product formation rates and respiration rates
(Fig. 2a) allow for a calculation (based on a closed redox cycle; see
Materials and Methods) of the fluxes through TCA, PFL, and PDHc (Fig.
3b and c). From these calculations it follows that under fully
fermentative conditions the catabolic flux proceeds solely through PFL.
What is of particular interest here is how the flux distribution shifts
upon changes in the availability of the electron acceptor. The analysis
shows (Fig. 3b) that PFL and PDHc can be active simultaneously under
low microaerobic conditions (20 to 40% aerobiosis) and that their in
vivo activities change smoothly in response to oxygen availability. It
is noteworthy that there is a range where ethanol production is due to
PDHc activity. Moreover, as catabolism now proceeds more efficiently with respect to the conservation of energy, a decrease in the qglucose (the specific rate of glucose
consumption), and hence a decrease in the absolute value of
JPDHc, are observed. TCA cycle activity
initiates at 30% aerobiosis and gradually reaches its maximal activity
by 100% aerobiosis (Fig. 3c). At levels of aerobiosis greater than
70% acetate is the only product excreted (Fig. 3a).
Respiratory activity at variable oxygen availability.
Oxygen
consumption was already observed with the lowest oxygen supply rate
applied (Fig. 2a). Increasing the oxygen supply resulted in a new
steady state, with the cells respiring at a higher specific rate, but
it is remarkable that initially (up to an oxygen supply rate allowing
approximately 40% of full aerobiosis) no statistically significant
changes in dissolved oxygen concentration were detected (Fig. 2b). This
suggests that in this range the overall capacity of the respiratory
chain is not constant due to either quantitative (affecting the
capacity of the respiratory chain) or qualitative (affecting the
affinity for oxygen) changes in its composition. This explanation is in
accord with the observed changes in the cytochrome bd
oxidase content of the cell (Fig. 4a).
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FIG. 4.
(a) Effect of oxygen availability on cytochrome
d synthesis in wild-type () and arcA ( )
strains of E. coli. For an explanation of x-axis
values, see the Fig. 2 legend. (b) Effect of oxygen availability on
pfl expression (, chemostat A;
,
chemostat B) in wild-type and arcA () strains of
E. coli.
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At very low oxygen availability a flux of electrons through the
respiratory chain occurs, although the main carbon flux finds its way
to the end products through the fermentative pathway. As NADH is now
also reoxidized by respiration, there is no need for a 1:1 ratio of
acetate and ethanol production to maintain redox neutrality (Fig. 2a
and 3a).
Cellular cytochrome bd oxidase and PFL synthesis at
different oxygen availabilities.
In order to determine the
influence of the oxygen supply on the transcription of Arc-dependent
genes for all conditions, the expression of focApfl and
cydAB in wild-type and arcA strains was
studied. Expression of focApfl was monitored directly as a chromosomal lacZ fusion, while cydAB expression
was monitored indirectly by measuring the cytochrome bd
content of the cells. Both focApfl and cydAB
operons are positively regulated by the phosphorylated form of ArcA
(ArcA-P) under anaerobic conditions but are oppositely regulated by the
fnr gene product. It was assumed that the cytochrome
bd oxidase content correlated with cyd expression (6). From Fig. 4 it can be seen that expression of both
operons in the wild type displays a maximum at intermediate oxygen
supply rates, whereas this is not the case in the arcA
strain, suggesting a strong dependence of expression on ArcA. Increased
pfl expression, however, initiates at approximately 15%
aerobiosis only. This pattern of expression of both genes is in stark
contrast to the gradual changes seen in the overall catabolic fluxes
(Fig. 3b and c). Clearly, changes in the synthesis of PFL do not
coincide with its in vivo activity. Remarkably, maximal pfl
expression coincides with the microaerobic range where PDHc is
inactivated (25% aerobiosis). In addition, under fully anaerobic
conditions cyd expression is very low and comparable to that
under the fully aerated condition.
Changes in NADH/NAD ratio.
Significant differences between the
steady-state NADH/NAD ratios of aerobic and anaerobic cultures (0.02 and 0.75, respectively) have been observed previously, as well as a
decrease in this ratio whenever an increase in the DOT of a culture is
applied (8). We analyzed changes in intracellular
concentrations of the pyridine nucleotides over the complete range of
oxygen supply rates at small increments to be able to correlate them
with changes in Arc-dependent enzyme synthesis and metabolic fluxes in
vivo. The total amount of NADH plus NAD was found to be 4 ± 0.3 µmol (g of dry weight)1 under all conditions. When
trace amounts of oxygen were supplied to the culture the NADH/NAD ratio
decreased significantly to approximately 60% of the value found under
anaerobic conditions (Fig. 2a). Subsequent increases in oxygen supply
did not affect the ratio significantly up to the rate (between 30 and
60% aerobiosis) at which a detectable increase in the steady-state DOT
occurred (Fig. 2b). This range coincided with the range where major
shifts in both pfl and cyd were observed (Fig.
4). At higher oxygen supply rates, the NADH/NAD ratio decreased
gradually to the level under fully aerobic conditions.
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DISCUSSION |
The catabolism of E. coli is to a large extent
determined by the availability of an external electron acceptor. Not
surprisingly, elaborate sensing and regulatory systems have evolved to
monitor the presence and nature of these acceptors and to tune
catabolism accordingly. A major role in the regulation of the synthesis
of a number of key catabolic enzymes has been assigned to the ArcAB two-component phosphorelay system, which consists of the membrane sensor ArcB and the transcriptional regulator ArcA (22, 23, 25,
29).
To date, the nature of the biochemical signal(s) that triggers
phosphorylation of ArcB has not been identified. It is clear that
oxygen per se is not the direct signal for ArcB phosphorylation (28, 38). Rather, NADH, D-lactate,
acetate, and pyruvate, which can accumulate under microaerobic or
anaerobic conditions, have been proposed as potential signals
(22), based on the finding that in vitro high concentrations
of these intermediates inhibit the intrinsic phosphatase activity of
ArcB. Moreover, intracellular concentrations of at least one of the
proposed intermediates, NADH, has been found to vary significantly
under aerobic, microaerobic, and anaerobic conditions (8,
54).
The PMF (p) has been proposed as a signal being sensed by
the Arc system (2, 30) as ArcAB-dependent induction of
cytochrome bd synthesis has been observed in aerobically
growing cells by the addition of protonophores and the inducing potency
of the protonophores was found to be proportional to their uncoupling activity.
In this study we have analyzed the expression of two positively
regulated, ArcA-P-dependent operons as a function of increasing oxygen
availability. Changes in the intracellular redox state (as reflected by
the NADH/NAD ratio) and steady-state respiration rates were measured as
well. It has been established that cells growing aerobically have a
significantly higher value of the PMF than anaerobically grown cells
(31), and it seems therefore justified to assume that, with
increasing respiration rates, the magnitude of the PMF will increase.
In our experiments, the respiration rate reaches its maximum at 60 to
70% aerobiosis, the same condition where the PMF reaches its maximal
value (35).
In contrast to the respiration rate, which increased gradually with
increasing oxygen availability under stringent microaerobic conditions,
the intracellular NADH/NAD ratio was found to remain essentially
constant. Over this range, the expression level of the two
positively regulated ArcA-P-dependent genes, pfl and
cyd, increased. On the other hand, varying oxygen
availability under less-stringent microaerobic conditions (above
50% aerobiosis) did not result in significant changes in respiration
rate but did affect the NADH/NAD ratio dramatically. Now, derepression of ArcA-P-dependent genes (deduced from the increase of TCA cycle activity) and deactivation of focApfl and cydAB
operon expression were observed. So the ArcA-dependent increase in
cyd and pfl expression coincides with increasing
respiratory activity in the lower range of oxygen availability, whereas
the pattern of decreasing NADH concentrations correlates with the
decrease in the expression in the higher range of oxygen availability.
Whereas in our experiments ArcA-P-dependent pfl and
cyd induction coincided with increasing PMF, earlier work
(2) suggested an inverse correlation. In both experiments,
however, the induction coincided with increasing respiratory activity:
it was found earlier that the addition of uncouplers stimulates
respiration (45). This discrepancy indicates that the
overall electron flow rate per se may operate as a signal. The means by
which the electron flow rate can be sensed by ArcB remains to be
resolved, but it is tempting to speculate that the recently identified
PAS domain of ArcB (60) plays a role in this sensing.
In conclusion, our observations strongly suggest that both the redox
state and the rate of respiration rate play a role in ArcAB-dependent
regulation, albeit the former does so under conditions of higher oxygen
availability than the latter. In this context it is relevant to note
that recently Matsushika and Mizuno (39) have shown that
dual-signal sensing is possible for ArcB. Their study revealed that
ArcB is capable of propagating two types of stimuli through two
distinct phosphotransfer pathways (39).
It should be mentioned here that besides the ArcAB system, the
regulatory FNR protein is a strong effector for expression of genes
involved in the aerobic and anaerobic catabolic pathways. Indeed, the
observed differences in expression of pfl and cyd under anaerobic conditions can be explained by interference through FNR
regulation of these genes. In contrast to pfl
(51) and genes coding for TCA cycle enzymes, for
which ArcA-P and FNR exhibit similar effects (17, 25, 37,
38), cyd is subject to competitive ArcA-P induction
and FNR repression (7, 12, 61), the latter being dominant
under anaerobic conditions.
Our results suggest that the cytochrome bd content in
steady-state cultures of E. coli seems to be adjusted to
the oxygen availability in such a manner that the consumption of oxygen
is maximized. Up to a certain level, an increased input of oxygen results in an increased respiratory capacity, which as a
consequence will maintain a low residual oxygen concentration. This
explains why no statistical difference among dissolved oxygen
concentrations could be found in wild-type cultures in the
microaerobic range, where the most significant microaerobic
up-regulation of cyd and pfl occurs.
Maximal expression of cyd under microaerobic
conditions has been reported earlier (13, 49, 61).
Significantly, increased expression of cyd and
pfl under microaerobic conditions is coordinated. It is also
noteworthy that PFL activity is observed only under conditions where
dissolved oxygen concentrations remain low. PFL is, in its active form,
a radical-containing enzyme, known to be readily destroyed by oxygen
(63). Consequently, it has always been assumed that PFL
activity was exclusive to anaerobiosis. We now present proof that this
is not the case. Even under microaerobic conditions where respiration
reaches its half-maximal rate, as much as half of the total flux from
pyruvate is due to PFL activity. We propose that PFL activity can be
maintained under these conditions by a mechanism very similar to the
so-called respiratory protection known to occur in nitrogen-fixing
microorganisms, e.g., Azotobacter vinelandii, where
cytochrome bd plays a crucial role in creating an
intracellular environment that allows nitrogenase to function by
rapidly consuming oxygen via uncoupled respiration (for a review see
reference 47). Below 30% aerobiosis, the NADH/NAD
ratio is found to remain as high as 0.42, a ratio known to inhibit PDHc activity in vitro (54). Hence, the organism needs an
alternative route for pyruvate catabolism. In analogy to the concept of
respiratory protection, the physiological role of Arc-dependent
up-regulation of both cyd and pfl expression
under microaerobic conditions may reside in the rapid consumption of
oxygen by cytochrome bd. Thus, the resulting low
intracellular oxygen concentration allows for maintaining a high
catabolic flux through the oxygen-sensitive PFL.
| 1.
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D. Vlad,
S. Schuster,
P. Pfeiffer, and G. Unden.
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Regulatory O2 tensions for the synthesis of fermentation products in Escherichia coli and relation to aerobic respiration.
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R. Murtazina,
A. Shestopalov, and V. Skulachev.
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Abstract
Introduction
