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Journal of Bacteriology, October 2000, p. 5813-5822, Vol. 182, No. 20
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
The nrfA and nirB Nitrite
Reductase Operons in Escherichia coli Are Expressed
Differently in Response to Nitrate than to Nitrite
Henian
Wang and
Robert P.
Gunsalus*
Department of Microbiology and Molecular
Genetics, University of California, Los Angeles, California 90095-1489
Received 10 March 2000/Accepted 27 July 2000
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ABSTRACT |
Escherichia coli possesses two distinct nitrite
reductase enzymes encoded by the nrfA and nirB
operons. The expression of each operon is induced during anaerobic cell
growth conditions and is further modulated by the presence of either
nitrite or nitrate in the cells' environment. To examine how each
operon is expressed at low, intermediate, and high levels of either
nitrate or nitrite, anaerobic chemostat culture techniques were
employed using nrfA-lacZ and nirB-lacZ reporter
fusions. Steady-state gene expression studies revealed a differential
pattern of nitrite reductase gene expression where optimal
nrfA-lacZ expression occurred only at low to intermediate
levels of nitrate and where nirB-lacZ expression was
induced only by high nitrate conditions. Under these conditions, the
presence of high levels of nitrate suppressed nrfA gene
expression. While either NarL or NarP was able to induce nrfA-lacZ expression in response to low levels of nitrate,
only NarL could repress at high nitrate levels. The different
expression profile for the alternative nitrite reductase operon encoded
by nirBDC under high-nitrate conditions was due to
transcriptional activation by either NarL or NarP. Neither response
regulator could repress nirB expression. Nitrite was also
an inducer of nirB and nrfA gene expression,
but nitrate was always the more potent inducer by >100-fold. Lastly,
since nrfA operon expression is only induced under
low-nitrate concentrations, the NrfA enzyme is predicted to have a
physiological role only where nitrate (or nitrite) is limiting in the
cell environment. In contrast, the nirB nitrite reductase
is optimally synthesized only when nitrate or nitrite is in excess of
the cell's capacity to consume it. Revised regulatory schemes are
presented for NarL and NarP in control of the two operons.
 |
INTRODUCTION |
Escherichia coli
possesses two biochemically distinct nitrite reductase enzymes encoded
by the nrfABCDEFG and nirBDC operons (4). The NirB nitrite reductase is a soluble
siroheme-containing enzyme that uses NADH as an electron donor to
reduce nitrite in the cytoplasm. The NrfA nitrite reductase is a
membrane-associated respiratory enzyme that couples to the
membrane-associated formate-oxidizing enzymes via quinones in order to
generate membrane potential. The abundance of each enzyme is elevated
during anaerobic cell growth conditions when either nitrate and/or
nitrite is present (14). Nitrite, the substrate for the two
enzymes, must either be encountered environmentally or generated by the
cell from nitrate reduction by one of the three E. coli
nitrate reductases.
Expression of the nrfABCDEFG operon (previously described as
aeg-93 [3]) and the nirBDC
operon is elevated during anaerobic cell growth by the Fnr regulatory
protein (1, 10, 14). The addition of nitrite, but not
nitrate, is reported to further elevate nrfA expression via
either the NarL or NarP response regulators. In contrast, NarL is
reported to repress nrfA expression in response to nitrate,
whereas NarP cannot (14-16, 23, 24). Expression of the
nirB operon differs from nrfA in that NarL and
NarP are reported to activate nirB expression in response to
nitrate. In contrast, only NarL is reported to activate nirB
in response to nitrite (23, 24). The locations of sites for
NarL and NarP binding have been proposed for each operon (described below).
It is unknown how the nrfA and nirB operons are
expressed in response to either low or intermediate levels of either
nitrate or nitrite since the prior gene regulation studies were
performed in batch cultures using high levels of each anion. Thus, we
examined the steady-state expression of nrfA-lacZ and
nirB-lacZ reporter fusions using anaerobic chemostat culture
methods under limiting nitrite or nitrate conditions. The findings
reveal a differential pattern of nitrite reductase gene expression
whereby the nrfA operon is preferentially expressed only at
low nitrate concentrations. Maximal nirB expression occurs
only at high levels of nitrate. Nitrite, the substrate for each enzyme,
was shown to be a less potent regulatory signal for either operon
relative to nitrate in contrast to prior reports. The chemostat studies
also revealed new roles for the NarL and NarP proteins in the
activation and/or repression of the two operons in contrast to prior
conclusions derived from batch culture studies. Thus, revised models
for the control of the nrfA and nirB nitrite
reductase operons are proposed.
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MATERIALS AND METHODS |
Bacteria, plasmids, and phages.
E. coli MC4100
[F
(argF-lac)U169 araD139 rpsL150
deoC1 relA1 flbB5301 rbsR ptsF25] was used for all chemostat and
batch culture experiments unless indicated otherwise (19).
The lacZ reporter fusions used to monitor expression of the
two-nitrite reductase operons were
HW1 (nrfA-lacZ) and
HW3 (nirB-lacZ). As the fusions were integrated at the
lambda att site on the chromosome, each strain is wild type
for the nrfABCDEFGH and nirBDC operons. The nrfA-lacZ fusion was constructed by the generation of a DNA
fragment containing the nrfABCDEFG control region from
position
297 to position +120 relative to the start site of
nrfA transcription (17). The resulting DNA
fragment was then inserted into plasmid pRS415 (20) to give
the nrfA-lacZ operon fusion plasmid designated pHW1. The DNA
insert in pHW1 was DNA sequenced to confirm the intended construction
(18). The nrfA-lacZ fusion was then transferred onto
RS45 to generate
HW1. A high-titer lysate was then used to
introduce the phage into MC4100 in single copy as previously described
(20). The nirB-lacZ fusion was constructed by the generation of a DNA fragment containing the nirBDC control
region from position
342 to position +222 relative to the start site of transcription using standard PCR protocols (17). The
subsequent cloning steps were as described for nrfA-lacZ
above. The nrfA-lacZ and nirB-lacZ reporter
fusions were introduced into the isogenic wild-type, narL,
narP, and narL narP double mutant strains (Table 1) by P1 transduction methods as
previously described (13). The narL narP strain
HL101 was constructed by P1 transduction of
narP::kan Tn10 tet from strain HL101
into RCC70, followed by selection for tetracycline resistance. Strain
HL101 was constructed by inserting Tn10 nearby the
narP::kan allele of RCC71 by standard methods
(19).
Cell growth.
For routine cell growth and plasmid
construction, cells were grown in Luria-Bertani liquid or solid medium.
For batch cell culture, cells were grown in a glucose (40 mM) minimal
medium (5). Where indicated, sodium nitrate (20 mM) or
sodium nitrite (2.5 mM) was added to the growth medium after
sterilization. Anaerobic cell growth was performed at 37°C in 10-ml
anaerobic culture tubes fitted with butyl rubber stoppers
(11). Cells grown overnight under identical conditions in
the same medium were used for inoculation.
Continuous culture experiments were performed in a Bioflo 3000 Bioreactor (New Brunswick Scientific) fitted with a 2-liter
glass
vessel and operated at a 1-liter liquid working volume as
previously
described (
25). A modified Vogel-Bonner medium supplemented
with glucose (2.25 mM) was used to limit cell growth (i.e.,
carbon-limiting
conditions) (
21). During the experiments,
the chemostat was
maintained at a medium flow rate of 10 ml/min, which
corresponds
to a cell doubling time of 70 min. Anaerobic culture
conditions
were maintained by continuously sparging the vessel with
oxygen-free
nitrogen at a flow rate of 200 ml/min (
21). To
vary the concentration
of nitrate or nitrite in the medium,
NaNO
3 or NaNO
2 was added
at the amount
indicated after medium
sterilization.
When the chemostat was shifted to a new nitrate (or nitrite)
concentration, new steady-state levels of gene expression were
verified
as described earlier (
25). The values obtained for
each
culture condition were independently determined at least
twice and
there was <10% variation in the

-galactosidase activity.
Strain
stability and purity was monitored as described elsewhere
(
21).

-Galactosidase assays were performed as described
previously
(
5), where one unit of

-galactosidase is
defined as the hydrolysis
of 1 nmol of
o-nitrophenyl-

-
D-galactopyranoside (ONPG) per
min
per mg of
protein.
Determination of nitrate and nitrite levels.
The
concentration of nitrate and nitrite in the culture medium was
determined as previously described (25), where the
sensitivities were 0.03 and 0.04 µM, respectively.
Materials.
ONPG was purchased from Sigma Chemical Co., St.
Louis, Mo.; X-Gal
(5-bromo-4-chloro-3-indolyl-
-D-galactopyranoside) was
obtained from International Biotechnologies, Inc., New Haven, Conn.;
and the Casamino Acids were from Difco Co., Detroit, Mich. Nitrogen gas
was supplied by Arco, Inc. All other chemicals used in this study were
of reagent grade.
 |
RESULTS |
Effect of nitrate concentration on nrfA-lacZ
expression.
To determine how the nrfABCDEFG operon is
expressed in response to different levels of nitrate, a
nrfA-lacZ reporter fusion was examined using steady-state
chemostat cell culture methods (Fig. 1).
In the absence of any added nitrate, a basal level of nrfA-lacZ gene expression (ca. 100 U) was observed. When the
steady-state nitrate addition level was increased to 0.5 mM, expression
was elevated by 8-fold, while the maximal expression of 25-fold
induction was seen at 1 mM nitrate addition (ca. 2,500 U). As the level of nitrate was further elevated, nrfA-lacZ expression was
gradually decreased until nitrate additions reached 6 mM (Fig. 1).
Under these conditions, gene expression was still fivefold above the basal level seen when no nitrate anion was added. Between the 6 and 40 mM nitrate additions, nrfA-lacZ expression remained
unchanged (Fig. 1 and data not shown).

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FIG. 1.
Effect of nitrate on nrfA-lacZ and
nirB-lacZ expression during anaerobic cell growth. The
amount of nitrate present in the medium added to the chemostat vessel
was adjusted prior to sterilization (see Materials and Methods). Upon
the shift to a new condition, steady state was generally achieved
within five residence times. Expression of the nrfA-lacZ
fusion ( ) and the nirB-lacZ fusion ( ) is relative to
the maximum level achieved for each fusion. Maximal
nrfA-lacZ expression was 2,500 U versus 18,000 U for
nirB-lacZ expression.
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Since the
E. coli cells were continually consuming nitrate
by reducing it to nitrite via the various nitrate reductase enzymes,
the nitrate additions shown in Fig.
1 do not indicate the actual
level
of nitrate remaining in the culture vessel (
25). The
chemostat
was therefore sampled at each steady-state condition shown in
Fig.
1, and the residual level of nitrate or nitrite in the vessel
was
determined. These values were essentially identical to those
previously
reported (
25; Fig.
2
reproduced here). Nitrite accumulation,
first observed when nitrate was
added at a level of 1 mM, increased
to 5 mM as the nitrate additions
were raised from 1 to 6 mM. Maximum
nrfA-lacZ expression was
achieved only when submicromolar amounts
of nitrate were present in the
chemostat vessel (ca. 0.2 µM).
Therefore,
nrfA gene
expression is exquisitely sensitive to nitrate
as a regulatory signal
in contrast to prior reports that nitrite
was the primary regulatory
signal (
1,
10,
14).

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FIG. 2.
Levels of nitrate and nitrite accumulated in the
chemostat vessel at different concentrations of nitrate addition. Upon
the shift to each new condition, steady state was generally achieved
within five residence times. The vessel was then sampled, and the
concentration of nitrate and nitrite remaining in the growth medium was
determined by high-pressure liquid chromatography (HPLC) (Materials and
Methods). Symbols: , nitrate; , nitrite.
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Effect of nitrate concentration on nirB-lacZ gene
expression.
The pattern of nirB-lacZ expression in
response to steady-state nitrate additions was different than that for
nrfA-lacZ expression (Fig. 1). While nirB-lacZ
expression was also lowest (850 U) in the absence of added nitrate, it
was then increased by 21-fold as the level of nitrate addition was
incrementally raised to 2 mM. The level of nirB-lacZ
expression then remained high as the nitrate additions were raised up
to 40 mM (Fig. 1 and data not shown). In contrast, maximal
nrfA-lacZ expression occurred at 1 mM added nitrate but was
reduced to near basal activity at the higher levels of the oxyanion
signal, where nirB gene expression was optimal. Therefore,
the nirB and nrfA nitrite reductase operons are
differentially expressed in response to nitrate.
Effect of narL and narP mutations on
nitrate-dependent nirB-lacZ expression.
From prior
batch culture experiments, both NarL and NarP were reported to activate
nirB-lacZ in response to nitrate (23, 24). The
chemostat experiments (Fig. 3A) also
demonstrate that NarL or NarP can function independently of each other
to induce nirB expression in response to nitrate. However,
NarL appears to be the superior activator of nirB-lacZ
expression (Fig. 3A; compare the NarL+ NarP
strain and the NarL
NarP+ strain). At the
highest levels of nitrate tested, NarL-dependent induction was
approximately 34-fold, while NarP induction was about 26-fold.
Significantly, NarP also appears to somehow antagonize the ability of
NarL to activate nirB-lacZ expression at low nitrate levels
since, in a NarP
NarL+ strain,
nirB-lacZ expression was higher relative to the wild-type strain during low nitrate additions. Similar results were observed for
the nrfA operon (see below). Finally, the induction of
nirB-lacZ expression was abolished in a strain deleted for
both narL and narP.

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FIG. 3.
Effect of nitrate (A) and nitrite (B) concentration on
nirB-lacZ expression in narL and narP
strains. The chemostat was sampled after each steady-state condition
was achieved, and the -galactosidase activity was determined. The
fermentor conditions were identical to that used in Fig. 1. Expression
levels in the wild-type strain ( ), the narL ( ),
narP ( ), and the narL narP strain ( ) are
indicated.
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|
Effect of nitrite additions on nirB-lacZ
expression.
A nearly identical pattern of nirB-lacZ
gene expression was seen when nitrite was used in place of nitrate
(Fig. 3B). However, nitrate was a significantly more effective anion
signal compared to nitrite (compare the maximal induction of
nirB expression in each panel of Fig. 3). Maximal
nirB gene expression occurred when the nitrate level in the
vessel was still <50 µM, whereas an equivalent induction by nitrite
required anion additions of >2 mM (Fig. 2). In addition, the maximal
response to nitrite was only 70% of that achieved when nitrate was the
inducer (Fig. 3). Finally, the rate that nirB-lacZ
expression was increased in response to increasing levels of anion was
lower for nitrite than for nitrate.
Either NarL or NarP was able to induce
nirB-lacZ expression
in response to nitrite (compare the NarL

NarP
+ and the NarL
+ NarP

strains
to the wild-type strain [Fig.
3B]). In contrast, only
NarL was
reported to respond to the nitrite signal (
22). NarL
is
clearly the more effective activator of
nirB-lacZ expression
across the entire range of nitrite anion additions (compare the
NarL
+ NarP

and the NarL

NarP
+ strains). NarL also caused a higher final level of
gene expression
than NarP. Interestingly, when nitrite was present,
NarP did not
antagonize NarL activation of
nirB-lacZ
expression to the extent
that it did when nitrate was present (Fig.
3).
Finally, the induction
of gene expression was abolished in a
narL
narP double mutant.
In summary, NarP was the less-effective
response regulator for
inducing
nirB-lacZ expression
relative to NarL when either nitrate
or nitrite was present as the
inducer
signal.
Effect of narL and narP mutations on
nitrate-dependent nrfA-lacZ expression.
During batch
cell culture conditions, nitrite was reported to induce nrfA
expression via either the NarL or NarP response regulator proteins,
while only nitrate was reported to cause repression of nrfA
expression and only by NarL (14, 16, 24). However, when we
examined the effect of narL and narP mutations on
nrfA-lacZ expression in continuous culture (Fig.
4A), four points were immediately clear.
First, either NarL or NarP can activate nrfA-lacZ expression in response to added nitrate. Second, NarL can repress
nrfA-lacZ expression when nitrate is present, whereas NarP
cannot (compare the NarL+ NarP
strain to the
NarL
NarP+ strain and to the wild-type
strain). Third, NarL is the more effective response regulator in
activating nrfA-lacZ expression at low nitrate levels (as
revealed by comparing the NarL+ NarP
and the
NarL
NarP+ strains). Lastly, NarP appears to
weakly antagonize the ability of NarL to activate nrfA-lacZ
expression at low nitrate levels (compare the NarL+
NarP
strain to the wild-type strain). These findings
therefore require a reexamination of the regulatory models based on
batch culture studies wherein NarL or NarP can activate nrfA
expression in response to nitrite only (24). Rather, NarL
and nitrate clearly provide the major inputs in controlling of
nrfA gene expression.

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FIG. 4.
Effect of nitrate (A) and nitrite (B) concentration on
nrfA-lacZ expression in narL and narP
strains. The chemostat was sampled after each steady-state condition
was achieved, and the -galactosidase activity was determined. The
fermentor conditions were identical to that used in Fig. 1. Expression
levels in the wild-type strain ( ), the narL ( ),
narP ( ), and the narL narP strain
( ) are
indicated.
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Effect of nitrite additions on nrfA-lacZ
expression.
We next examined how nitrite additions modulate the
steady-state expression of the nrfA-lacZ reporter fusion.
Nitrite induced nrfA-lacZ expression in a pattern similar to
that shown for nitrate (Fig. 4B). However, a twofold-higher level of
nitrite addition was required to elicit the same level of gene
expression than nitrate did. The actual level of nitrate present in the
growth vessel at maximal nrfA-lacZ expression was over
1,000-fold lower than for nitrite (ca. 0.2 µM versus 1 mM). Thus,
nitrate is also the more potent regulatory signal for inducing
nrfA gene expression. This finding is in contrast to
previous reports in which nitrite was concluded to be the primary
signal (14). At saturating levels of nitrite (ca. 6 mM),
nrfA-lacZ expression was reduced to an intermediate level
that was about 25% of the maximal level seen with nitrite. Finally,
either NarL or NarP was able to activate nrfA-lacZ
expression independently of the other, while only NarL could repress expression.
Comparison of nrfA-lacZ and nirB-lacZ gene
expression in batch culture and in continuous culture.
To directly
compare the steady-state chemostat and batch culture methods, the above
nrfA-lacZ and nirB-lacZ reporter strains were
analyzed in batch culture (Table 2). The
batch culture results are in good agreement with previous studies
(22). Therefore, any differences between the chemostat and
batch culture data cannot be due to medium composition or strain
differences between the two methods. It is therefore evident that
considerable regulatory information was not revealed by the batch
culture studies. Similar conclusions resulted from chemostat studies
with the narG and napF operons that encode the
two nitrate-regulated nitrate reductase enzymes in E. coli
(25).
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TABLE 2.
Anaerobic expression of nrfA-lacZ and
nirB-lacZ reporter fusions in batch culture expression in
wild-type and narL and narP mutant strains
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For the
narL or the
narP strains growing in the
chemostat, it is unknown if the amount of nitrate or nitrite remaining
in
the chemostat differs significantly from the wild-type strain.
This
is unlikely under high-anion-addition conditions, but it
is less clear
when very low to intermediate levels of anion additions
are made. For
the batch culture experiments, the levels of nitrate
remaining and the
levels of nitrite accumulated in the vessel
at the time of cell
sampling for gene expression measurements
are unknown, and it is
unlikely that steady-state gene expression
was
achieved.
Roles of the
22,
50, and
70 NarL heptamer recognition sites
in the activation and repression of nrfA-lacZ
expression.
Prior DNA footprinting studies demonstrate that NarL
and NarP bind to and protect several NarL heptamer recognition sites located in the nrfA promoter region (8). The
nrfA operon has a promoter element containing an activation
site consisting of the
79 and
70 heptamers that can be recognized
by either NarL or NarP. Additional binding sites for NarL occur at
positions
50 and
22. To evaluate how the sites contribute to the
activation versus the repression of nrfA gene expression at
low, intermediate, and high levels of nitrate, single- and
double-base-pair mutations were introduced into the NarL heptamer sites
designated
22,
50, and
70, (Fig.
5). The intended mutations were confirmed
by DNA sequence analysis, and each altered DNA fragment was then used to construct a nrfA-lacZ reporter fusion for subsequent in
vivo chemostat analysis.

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FIG. 5.
Location of NarL heptamer recognition sites located at
positions 22, 50, 70, and 79 in the nrfA promoter
region. The sequence of the nrfA promoter region with the
associated NarL heptamer sites (8) is shown relative to the
start of nrfA transcription. The location of the Fnr
recognition site is indicated by the inverted arrows below the DNA
sequence. The mutations introduced into the 22, 50, and 70
heptamer sites are indicated below or above the sequence. The mutated
nrfA promoter designated m5 was made by combining the m1 and
the m4 mutations.
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Introduction of a single base change in the

70 NarL heptamer,
designated the
nrfA m6 promoter mutation, completely
abolished
the nitrate-dependent induction of
nrfA-lacZ
expression (Fig.
6D). Interestingly, in
the NarL

NarP
+ strain,
nrfA-lacZ
expression remained at a basal level (400 U),
whereas in the
NarL
+ NarP

strain, expression was gradually
reduced below the initial basal
level as the level of nitrate addition
was increased from 0 to
7.5 mM. This indicates that (i) the

70 NarL
heptamer site is
essential for NarL to function as an activator of
nrfA expression
and that (ii) other sites are used for NarL
to repress expression
independently of the

70 site, in contrast to
prior interpretations
(see Discussion).

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FIG. 6.
Effect of 22, 50, and 70 NarL heptamer site
mutations on nitrate-dependent nrfA-lacZ expression. The
expression of each nrfA-lacZ reporter fusion was evaluated
at different levels of nitrate addition as described in Fig. 1. (A)
Expression of the nrfA-lacZ m1 and m2 fusions. (B)
Expression of the nrfA-lacZ m3 and m4 fusions. (C)
Expression of the m5 nrfA-lacZ fusion. (D) Expression of the
m6 nrfA-lacZ fusion in wild-type and narL
strains.
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|
To further explore the regulatory process, single and double mutations
were introduced into the

50 NarL heptamer site to
give the altered
nrfA promoter fusions designated m3 and m4 (Fig.
5). While
NarL was still able to activate and to repress
nrfA gene
expression, the pattern differed from the wild-type
nrfA-lacZ reporter fusion (Fig.
6B). The maximal level of
gene expression
was equivalent, but the induction occurred at a lower
nitrate
level in the m3 and m4 reporter strains. Repression of
nrfA-lacZ expression was also seen, but it was delayed until
higher levels
of nitrate additions were made. Thus, the

50 NarL
heptamer site
appears to fine-tune the activation and repression of
nrfA gene
expression by narrowing the window of maximal gene
expression.
When a single- or double-base change was introduced into the

22 NarL
heptamer site to give the m1 or the m2
nrfA-lacZ fusions,
a
distinct pattern of
nrfA-lacZ expression was seen relative
to
either the

50 or the

70 mutant (Fig.
6A). The onset of
activation
seen for the m1 and m2
nrfA-lacZ reporter fusions
was like the
wild-type
nrfA-lacZ reporter fusion. However,
the maximum level
of gene expression was 40% higher, and it occurred
only at higher
levels of nitrate addition. Third, the subsequent
repression of
nrfA-lacZ expression by NarL was also delayed
until higher nitrate
additions. Fourth, the degree of repression was
less severe at
the highest level of nitrate tested. Therefore, the

22
heptamer
site is a critical site for mediating NarL repression of
nrfA gene expression. When mutations were introduced into
both the

22 and the

50 NarL heptamer sites (designated as the
nrfA-lacZ m5 reporter fusion), repression of
nrfA-lacZ expression was further
impaired relative to when
mutations were introduced only in the

22 site (Fig.
6C versus A).
Gene expression was nearly derepressed
relative to the wild-type
strain. These results suggest that both
the

22 and the

50 sites are
needed for optimal repression of
nrfA gene expression. This
repression also occurs in the absence
of an intact

70 activator site
(Fig.
6D).
When the m1, m2, m3, m4, m5, and m6
nrfA-lacZ reporter
fusions were tested in response to nitrite additions (Fig.
7), similar
patterns of
nrfA
gene expression were seen relative to the nitrate
addition experiments
(compare these results to those in Fig.
6).
It is therefore evident
that NarL and NarP regulate
nrfA-lacZ expression in response
to nitrite addition in the same qualitative
way that the nitrate anion
signal does in contrast to prior proposals.
However, a significantly
higher level of nitrite is required to
elicit a somewhat inferior
response relative to nitrate. These
findings also imply that the NarX
and/or NarQ sensor transmitters
detect each anion in the same general
way but where nitrate is
the more potent signal.

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FIG. 7.
Effect of 22, 50, and 70 NarL heptamer site
mutations on nitrite-dependent nrfA-lacZ expression. Effect
of 22, 50, and 70 NarL heptamer site mutations on nitrite
dependent nrfA-lacZ expression. The expression of each
nrfA-lacZ reporter fusion was evaluated at different levels
of nitrate addition as described in Fig. 1. (A) Expression of the
nrfA-lacZ m1 and m2 fusions. (B) Expression of the
nrfA-lacZ m3 and m4 fusions. (C) Expression of the m5
nrfA-lacZ fusion. (D) Expression of the m6
nrfA-lacZ fusion in wild-type and narL strains.
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 |
DISCUSSION |
The nrfA and nirB operons are expressed in
a complementary style.
The chemostat gene expression studies
clearly establish a complementary pattern of nitrite reductase gene
expression in E. coli (Fig. 1, 3, and 4). When the nitrate
level is very low, the cell induces the "first response" nitrite
reductase encoded by the nrfA operon to consume nitrite in
the environment. It is interesting to speculate that the NrfA nitrite
reductase may therefore have a higher (e.g., stronger) affinity for
nitrite than does the nirB encoded nitrite reductase. Tests
of this prediction must await additional biochemical characterization
of the NrfA enzyme.
When the level of nitrate in the environment becomes elevated, the
second nitrite reductase encoded by the
nirB operon is
preferentially synthesized due to transcriptional controls imposed
by
the Nar regulatory circuit. The cytoplasmic reductase is then
the
predominant nitrite reductase in the cell since the NrfA enzyme
is
nearly absent under high-nitrate conditions (Fig.
1)! Apparently,
the
two-nitrite reductase enzymes and their transcriptional regulatory
elements are evolved to function in a complementary way to provide
for
nitrite reduction under these different conditions (discussed
below).
The 25-fold induction of
nrf operon expression seen at low
concentrations of nitrate was not revealed by previous batch culture
studies since those experiments were performed only at saturating
levels of the anion (
1,
14,
22). Therefore, the steady-state
chemostat studies indicate that the
nrfA operon must serve a
far
more important role in cell metabolism than was previously
indicated
by the modest three- to fourfold change seen in batch culture
experiments (Table
2; discussed below). Second, the roles of
the two
anion inducer molecules on
nrfA gene expression (i.e.,
nitrate and nitrite), as well as the roles for the two response
regulatory proteins, NarL and NarP, are far more intricate than
previously envisioned. Likewise,
nirB operon expression was
examined
under identical chemostat conditions at low and intermediate
levels
of nitrate or nitrite (Fig.
3). With low nitrate additions of
1 mM where
nrfA-lacZ expression was maximal,
nirB
expression was
only 30% of its final maximal level. With nitrate
additions ranging
from 1 to 2 mM, both nitrite reductase operons are
simultaneously
expressed at significant
levels.
The conclusions derived from batch culture and chemostat experiments
for NarL- and NarP-dependent control of
nrfA and
nirB gene regulation in response to nitrate and nitrite are
summarized
in Table
3. From the chemostat
studies, new roles for NarL and
nitrate in the control of each operon
are evident. Also, since
NarL and NarP clearly function in more
versatile ways in response
to either nitrate or nitrite as a regulatory
signal, the prior
models based on the batch culture studies must be
reconsidered
(
6,
24). New regulatory schemes for the NarL
and NarP control
of
nrfA and
nirB operon
expression based on the chemostat data
shown in Fig.
1 to
7 are
depicted in Fig.
8 and
9. One of the
more striking points in
each model is that the NarP protein is
nonessential (i.e., not a major
player) and that it can function
as an antagonist of NarL in its
ability to activate
nirB or
nrfA operon
expression. Rather, NarP appears to fine-tune gene expression
under
low-nitrate conditions by delaying the regulatory response
to nitrate.
This antagonizing effect is relatively weak when nitrite
is present in
place of nitrate. The simplest explanation for these
findings is that
the cognate sensor transmitter proteins, NarX
and/or NarQ, are unable
to respond to nitrite in the same way
they do for nitrate. The latter
anion is a more potent stimulator
of NarX kinase activity by 2 to 3 orders of magnitude in concentration
relative to nitrite
(
12). Nitrite also fails to elicit the same
steady-state
level of NarX-phosphate that nitrate does.
View this table:
[in this window]
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|
TABLE 3.
Functioning of NarL and NarP in the control of
nrfA and nirB operon expression in response to
nitrate and nitrite
|
|

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|
FIG. 8.
Model for NarL- and NarP-dependent control of
nrfA operon expression. (A) Nitrate control. The Fnr protein
(F) induces nrfA operon expression under anaerobic
conditions. In the presence of low levels of nitrate, either NarL (L)
or NarP (P) bind at the 70 and 79 heptamer sites (hatched boxes) to
activate nrfA gene expression. NarL is the more proficient
activator under low-nitrate conditions, where NarP antagonizes NarL
binding (Fig. 4A). At elevated-nitrate conditions, additional NarL
molecules bind to the flanking NarL heptamer sites at 50 and 22 to
repress nrfA operon expression (Fig. 5). NarP is not
essential for either the activation or repression process. (B) Nitrite
control. A similar regulatory response is observed in response to
nitrite except that higher anion levels are required relative to the
nitrate (Fig. 4B).
|
|

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|
FIG. 9.
Model for NarL- and NarP-dependent control of
nirB operon expression. (A) Nitrate control. During
anaerobiosis, the Fnr protein (F) binds at its consensus recognition
site, centered at 42, to activate nirB operon expression.
When low-nitrate conditions are encountered, NarL protein (L) binds at
the two heptamer sites at 74 and 65 to activate nirB
expression (8). NarP interaction at the heptamer sites
somehow antagonizes NarL function (Fig. 3A). (B) Nitrite control. A
similar regulatory response is observed in response to nitrite except
that higher anion levels are required relative to nitrate (Fig. 3B).
|
|
Revised models for NarL and NarP control of nrfA and
nirB gene expression.
As noted above, the
nrfA operon has a promoter element containing an activation
site consisting of the
79 and
70 heptamers that can be recognized
by either NarL or NarP (7). Additional binding sites for
NarL are at positions
50 and
22 (8). NarL-phosphate binding at the
50 site presumably interferes with Fnr binding at its
site adjacent to the NarL heptamer. Additional NarL binding at the
22
site is envisioned to interfere with RNA polymerase interactions at the
nrfA promoter.
A revised regulatory model for the
nrfA operon is shown in
Fig.
8 based on the chemostat studies with strains defective in
narL or
narP (Fig.
4). Overall,
nrfA
gene expression can be modulated
by 25-fold by adjusting the balance
between the activation and
repression events. It is evident that NarL
can serve as an activator
and as a repressor of
nrfA
expression in vivo in response to either
nitrate or nitrite (Table
3).
In the absence of either inducer
anion, gene expression is minimal.
When sufficient amounts of
either anion are detected by the NarX and/or
NarQ sensors, both
NarL and NarP are phosphorylated where either
response regulator
can then activate
nrfA gene expression.
However, the chemostat
experiments demonstrate that NarP-phosphate
cannot repress
nrfA gene expression under high nitrate or
nitrite conditions. This
is consistent with the in vitro experiments
that both NarP-phosphate
and NarL-phosphate bind to the

70 and

79
heptamer sites, while
only NarL-phosphate bind at the

50 and

22
sites (
8). In fact,
NarL has the highest affinity for the
activation sites at

70
and

79 as previously shown by Darwin et al.
(
8), whereas NarP
exhibits a weaker affinity for these
sites. When high-nitrate
or -nitrite conditions are encountered,
additional NarL-phosphate
molecules are made that then bind at the
nearby NarL heptamers
(Fig.
5) to inhibit
nrfA gene
expression. As described above,
much higher levels of nitrite are
required to give the same result
that submicromolar amounts of nitrate
does (Fig.
4B). Batch culture
experiments clearly do not permit the
quality of the nitrate or
nitrite signals to be deduced
(
25). Interestingly, the
nrfA operon expression
pattern is similar to that of the
napF operon
(
25). However, the latter operon has a strikingly different
regulatory element where the locations of the NarL and NarP heptamer
sequences and the Fnr sites are reversed in relative position
(
7). Thus, positions of the binding sites alone cannot be
used
to predict the differential expression patterns for these two
operons.
A revised model for NarL-dependent activation of
nirB gene
expression is shown in Fig.
9. Under low-nitrate conditions,
NarP-phosphate
binding at its two heptamer recognition sites
(
8) weakly induces
nirB expression (Fig.
3A).
Under these conditions NarP also antagonizes
the superior activator
response by NarL-phosphate. However, when
elevated nitrate conditions
are encountered, NarL-phosphate overcomes
the antagonistic effect of
NarP to give optimal induction of
nirB expression.
NarP-phosphate can clearly also elicit activation
of
nirB
operon expression in response to nitrite (Figure
3B) in
contrast to
prior conclusions based on batch culture methods (
23,
24).
Different environmental conditions for the two-nitrite reductase
enzymes?
Since nrf operon expression is optimal only at
low-nitrate conditions, while nirB expression is optimal
during high-nitrate conditions (Fig. 1), the two nitrite reductase
enzymes must serve distinct roles within the cell. The high-nitrite
conditions needed for nirB induction are in keeping with the
proposed role of the NirB enzyme in detoxification (4). The
chemostat experiments also support a second plausible role for the NirB
enzyme whereby it recycles NADH by oxidizing it under conditions when
excess reducing equivalents are present inside the cell. Such
conditions would occur when sufficient energy is generated by
nitrate-dependent respiration via the NarG nitrate reductase complex.
Use of the NirB enzyme would thus allow the cell to effectively
decouple carbon dissimilation from the nitrite respiratory pathways by using a futile cycle for NADH-NAD recycling. It is apparent that the
respective regulatory elements for the two nitrite reductase operons
are evolved to ensure that each enzyme is synthesized under different
environmental conditions.
The differential and complementary patterns for
nrfA and
nirB expression in response to nitrate or nitrite (Fig.
1,
3, and
4) are similar to the expression patterns for the two nitrate
reductase operons encoded by the
narGHJI and
napAB genes (
25).
The
nirB operon is
induced primarily in response to intermediate
to high levels of nitrate
like the
narGHJI nitrate reductase operon
(
25).
The pattern of
nrfA operon expression is similar to that
seen for the
napAB operon where both operons are optimally
expressed
only during low-nitrate growth conditions
(
25; this study).
This supports a physiological role
of the NrfA respiratory enzyme
in providing additional membrane
potential needed for cellular
ATP synthesis in concert with the NapAB
respiratory nitrate reductase.
Since the NapAB enzyme is periplasmic
and apparently of lower
proton pumping efficiency due to its inability
to form vectoral
coupling, NapAB would therefore have a lower
efficiency in energy
generation relative to the NarG enzyme
(
4). The NrfA enzyme
could thus effectively assist in the
NapAB enzyme in energy harvesting
under low-nitrate growth conditions.
In high-nitrate conditions
when the expression of both the
nrfA and
napA operons is switched
off
(
25; this study) and where the expression of the
narG and
nirB operons are switched on, the NarG
respiratory nitrate reductase
appears to be proficient in generating an
appropriate proton gradient
for the cell. Under these conditions, most
of the newly formed
nitrite is excreted from the cell and accumulates
in the medium
(Fig.
2).
Is nitrite a significant physiological inducer of nrfA
or nirB gene expression?
From the chemostat studies,
it is evident that nitrate is the more potent inducer of
nrfA and nirB gene expression than nitrite by at
least 2 orders of magnitude in concentration. Submicromolar levels of
nitrate (i.e., <0.03 µM nitrate) are sufficient to induce both
nrfA and nirB gene expression, as also seen for
narG and napF gene expression (25).
Additionally, nitrite was not a coinducer nor an antagonist of the
cells' ability to recognize nitrate as a signal based on
chemostat
studies with the
napF and
narG operons
(
25). Therefore,
nitrite does not antagonize the cell
ability to detect nitrate
via NarX and NarQ. The cell exhibits a
considerable ability to
discriminate between the two structurally
related oxyanion molecules
(
12). Nitrate is also the
superior regulatory signal for control
of the
narG and
napF operons in vivo (
25).
Differentially expressed genes in E. coli.
These studies
provide the third example of differentially expressed pairs of
respiratory pathway genes in E. coli. These include two
nitrite reductase operons (nrfA and nirB; this
study), two nitrate reductase operons (napFDAGHBC and
narGHJI) (25), and two cytochrome oxidase operons
(cydAB and cyoABCDE) (21). The first
two sets of operons are regulated primarily by nitrate and to a lesser
extent by nitrite, while the latter set of operons are controlled in
response to oxygen availability. The differential and complementary
expression of these pairs of operons in response to their environmental
signals allows the cell to more effectively compete for respiratory
substrates at low concentrations versus when they are in excess. The
cell would thus conserve energy by not synthesizing unneeded
respiratory enzymes. Finally, the cell does not use an abrupt
"switch" to turn on or turn off respiratory gene expression.
Rather, the regulatory response can be adjusted over a range of signal
concentrations (Fig. 1 and 2).
Since many other enteric and soil bacteria also possess dual sets of
nitrate reductase, nitrite reductase, and cytochrome
oxidase enzymes
such as those present in
E. coli (
9,
26),
it is
conceivable that the operons in these organisms are also
regulated in
similar ways. From the above studies, it is evident
that the chemostat
approach provides a powerful tool for better
understanding the
physiological basis of gene expression in response
to environmental
signals. This is especially relevant when the
inducer or repressor
molecule is present in low
amounts.
 |
ACKNOWLEDGMENT |
This study was supported in part by the Public Health Service
grant AI21678 from the National Institutes of Health.
 |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology and Molecular Genetics, University of California, Los
Angeles, CA 90095-1489. Phone: (310) 206-8201. Fax: (310) 206-5231. E-mail: robg{at}microbiol.ucla.edu.
 |
REFERENCES |
| 1.
|
Bell, A.,
J. Cole, and S. Busby.
1990.
Molecular genetic analysis of an FNR-dependent anaerobically inducible Escherichia coli promoter.
Mol. Microbiol.
4:1753-1763[Medline].
|
| 2.
|
Chiang, R. C.,
R. Cavicchioli, and R. P. Gunsalus.
1997.
`Locked-on' and `locked-off' signal transduction mutations in the periplasmic domain of the Escherichia coli NarQ and NarX sensors affect nitrate- and nitrite-dependent regulation by NarL and NarP.
Mol. Microbiol.
24:1049-1060[CrossRef][Medline].
|
| 3.
|
Choe, M., and W. S. Reznikoff.
1993.
Identification of the regulatory sequence of anaerobically expressed locus aeg-46.5.
J. Bacteriol.
175:1165-1172[Abstract/Free Full Text].
|
| 4.
|
Cole, J.
1996.
Nitrate reduction to ammonia by enteric bacteria: redundancy, or a strategy for survival during oxygen starvation?
FEMS Microbiol. Lett.
136:1-11[CrossRef][Medline].
|
| 5.
|
Cotter, P. A., and R. P. Gunsalus.
1989.
Oxygen, nitrate, and molybdenum regulation of dmsABC gene expression in Escherichia coli.
J. Bacteriol.
171:3817-3823[Abstract/Free Full Text].
|
| 6.
|
Darwin, A. J., and V. Stewart.
1996.
The Nar modulon systems: nitrate and nitrite regulation of anaerobic gene expression, p. 333-359.
In
E. C. C. Lin, and A. S. Lynch (ed.), Regulation of gene expression in Escherichia coli. R. G. Landes Company, Austin, Tex.
|
| 7.
|
Darwin, A. J., and V. Stewart.
1995.
Nitrate and nitrite regulation of the Fnr-dependent aeg-46.5 promoter of Escherichia coli K-12 is mediated by competition between homologous response regulators (NarL and NarP) for a common DNA-binding site.
J. Mol. Biol.
251:15-29[CrossRef][Medline].
|
| 8.
|
Darwin, A. J.,
K. L. Tyson,
S. J. Busby, and V. Stewart.
1997.
Differential regulation by the homologous response regulators NarL and NarP of Escherichia coli K-12 depends on DNA binding site arrangement.
Mol. Microbiol.
25:583-595[CrossRef][Medline].
|
| 9.
|
Garcia-Horsmann, J. A.,
B. Barquera,
J. Rumbley,
J. Ma, and R. B. Gennis.
1994.
The superfamily of heme-copper respiratory oxidases.
J. Bacteriol.
176:5587-5600[Free Full Text].
|
| 10.
|
Jayaraman, P. S.,
J. Cole, and S. Busby.
1989.
Mutational analysis of the nucleotide sequence at the FNR-dependent nirB promoter in Escherichia coli.
Nucleic Acids Res.
17:135-145[Abstract/Free Full Text].
|
| 11.
|
Jones, H. M., and R. P. Gunsalus.
1987.
Regulation of Escherichia coli fumarate reductase (frdABCD) operon expression by respiratory electron acceptors and the fnr gene product.
J. Bacteriol.
169:3340-3349[Abstract/Free Full Text].
|
| 12.
|
Lee, A. I.,
A. Delgado, and R. P. Gunsalus.
1999.
Signal-dependent phosphorylation of the membrane bound NarX two-component sensor transmitter protein of Escherichia coli: nitrate elicits a superior anion ligand response compared to nitrite.
J. Bacteriol.
181:5309-5316[Abstract/Free Full Text].
|
| 13.
|
Miller, J. H.
1972.
Experiments in molecular genetics.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
|
| 14.
|
Page, L.,
L. Griffiths, and J. A. Cole.
1990.
Different physiological roles for two independent pathways for nitrite reduction to ammonia by enteric bacteria.
Arch. Microbiol.
154:349-354[Medline].
|
| 15.
|
Peakman, T.,
J. Crouzet,
J. F. Mayaux,
S. Busby,
S. Mohan,
N. Harborne,
J. Wootton,
R. Nicolson, and J. Cole.
1990.
Nucleotide sequence, organisation and structural analysis of the products of genes in the nirB-cysG region of the Escherichia coli K-12 chromosome.
Eur. J. Biochem.
191:315-323[Medline].
|
| 16.
|
Rabin, R. S., and V. Stewart.
1993.
Dual response regulators (NarL and NarP) interact with dual sensors (NarX and NarQ) to control nitrate- and nitrite-regulated gene expression in Escherichia coli K-12.
J. Bacteriol.
175:3259-3268[Abstract/Free Full Text].
|
| 17.
|
Saiki, R. K.,
S. Scharf,
F. Faloona,
K. B. Mullis,
G. T. Horn,
H. A. Erlich, and N. Arnheim.
1985.
Enzymatic amplification of b-globin genomic sequences for diagnosis of sickle cell anemia.
Science
230:1350-1354[Abstract/Free Full Text].
|
| 18.
|
Sanger, F.,
B. J. Nicklen, and A. R. Coulson.
1977.
DNA sequencing with chain terminating inhibitors.
Proc. Natl. Acad. Sci. USA
74:5463-5467[Abstract/Free Full Text].
|
| 19.
|
Silhavy, T. J.,
M. L. Berman, and L. W. Enquist.
1984.
Experiments with gene fusions.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
|
| 20.
|
Simons, R. W.,
F. Houman, and N. Kleckner.
1987.
Improved single and multicopy lac-based cloning vectors for protein and operon fusions.
Gene
53:85-96[CrossRef][Medline].
|
| 21.
|
Tseng, C. P.,
A. K. Hansen,
P. Cotter, and R. P. Gunsalus.
1994.
Effect of cell growth rate on expression of the anaerobic respiratory pathway operons frdABCD, dmsABC, and narGHJI of Escherichia coli.
J. Bacteriol.
176:6599-6605[Abstract/Free Full Text].
|
| 22.
|
Tyson, K.,
S. Busby, and J. Cole.
1997.
Catabolite regulation of two Escherichia coli operons encoding nitrite reductases: role of the Cra protein.
Arch. Microbiol.
168:240-244[CrossRef][Medline].
|
| 23.
|
Tyson, K. L.,
A. I. Bell,
J. A. Cole, and S. J. W. Busby.
1993.
Definition of nitrate and nitrite response elements at the anaerobically inducible Escherichia coli nirB promoter: interactions between FNR and NarL.
Mol. Microbiol.
7:151-157[CrossRef][Medline].
|
| 24.
|
Tyson, K. L.,
A. I. Bell,
J. A. Cole, and S. J. W. Busby.
1994.
Nitrite and nitrate regulation at the promoters of two Escherichia coli operons encoding nitrite reductase: identification of common target heptamers for both NarP- and NarL-dependent regulation.
Mol. Microbiol.
5:353-360[CrossRef].
|
| 25.
|
Wang, H.,
C.-P. Tseng, and R. P. Gunsalus.
1999.
The napF and narG nitrate reductase operons in Escherichia coli are differentially expressed in response to submicromolar concentrations of nitrate but not nitrite.
J. Bacteriol.
181:5303-5308[Abstract/Free Full Text].
|
| 26.
|
Zumft, W. G.
1997.
Cell biology and molecular basis of denitrification.
Microbiol. Mol. Biol. Rev.
61:533-616[Abstract].
|
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