School of Biological Sciences, University of
East Anglia, Norwich NR4 7TJ, United Kingdom,1
and Department of Molecular Cell Physiology, Faculty of
Biology, BioCentrum Amsterdam, Vrije Universiteit, NL-1081 HV
Amsterdam, The Netherlands2
 |
INTRODUCTION |
Denitrification is the respiratory
reduction of nitrate to dinitrogen via the intermediates nitrite,
nitric oxide (NO), and nitrous oxide. Since NO is toxic, there is
a requirement in denitrifying bacteria to regulate the expression
and activity of the enzymes of the pathway in order to maintain a low
intracellular concentration of NO. There is increasing evidence to
indicate that NO itself acts as a signal molecule that interacts
(either directly or indirectly) with transcriptional regulators, which
coordinate the expression of the enzymes that make and consume NO. For
Paracoccus denitrificans, activation of the nitrite
reductase (nirS) and nitric oxide reductase (norCBQDEF) operons by NO requires a transcription
factor designated NNR that belongs to the FNR/CRP family (26,
27). An nnr mutant of P. denitrificans has
undetectable levels of nitrite reductase activity and reduced levels of
NO reductase activity when grown under anaerobic denitrifying
conditions (27). The activities of the nirS and
norC promoters are substantially reduced in the nnr mutant (28). The nirS and
norC promoters are activated by nitrate and nitrite under
anaerobic conditions in vivo, though the true signal might be NO,
generated by the reduction of nitrate and nitrite. This is supported by
the observation that sodium nitroprusside (SNP) (a source of
NO+) also efficiently activates the nirS and
norC promoters (27). The NNR-regulated
nirI gene encodes another protein that is required for
nirS expression (21). In a nitrite
reductase-deficient mutant, the nirI promoter can be
activated by coculturing with an NO reductase-deficient strain that
acts as the source of NO (28). Taken together, these observations indicate that for P. denitrificans, NO (or
perhaps a chemical species related to NO) acts as a signal to
activate expression of the nitrite and NO reductases and that
transcriptional activation requires NNR. A similar situation exists for
Rhodobacter sphaeroides, for which a transcriptional
regulator designated NnrR activates expression of the nitrite and NO
reductase genes in response to NO (12, 25). In
Pseudomonas aeruginosa, the nirS and
norC promoters are activated by another FNR/CRP family member, designated DNR (1), which is closely related to NNR in sequence. DNR is responsive to nitrite in vivo (8), which may reflect the fact that nitrite can be reduced to NO by the nitrite
reductase. Nevertheless, there is no direct evidence that DNR is an NO
sensor, as are NNR and NnrR. In Pseudomonas stutzeri, there
are at least four members of the FNR/CRP family, one of which, DnrD,
activates the expression of nitrite reductase and NO reductase
(29). DnrD is a close relative of NNR, though it is not
known whether it also is responsive to NO. The expression of the genes
encoding DNR and DnrD is activated in anaerobically growing cultures
(1, 29), whereas NNR is expressed constitutively (28).
The mechanism by which NO activates NNR is not known, nor is it known
whether NO interacts directly with the protein or whether there is a
signal transduction pathway with additional components. Alignment
of the NNR sequence with those of other known (NnrR) and possible (DNR,
DnrD) NO sensors provides few clues as to possible signaling
mechanisms. The primary structure of NNR and the sequence of its
probable binding sites in the promoters that it regulates suggest that
NNR has the same DNA binding specificity as the FNR protein of
Escherichia coli. This has recently been confirmed by
mutagenesis of the NNR binding site in the nirI-nirS
intergenic region of P. denitrificans (21) and of
the NNR binding site in the norC promoter (Hutchings and
Spiro, unpublished data). The common DNA binding specificity raises the
possibility that NNR might activate FNR-dependent promoters in
E. coli. This paper reports the successful development
of a system for studying NNR activity in E. coli and its use
to characterize seven NNR proteins with single amino acid
substitutions. Several important conclusions can be drawn about the
properties of NNR.
| |
MATERIALS AND METHODS |
Bacterial strains, plasmids, and growth media.
E. coli
strain JM83 [ara 
(lac-proAB) rpsL
80lacZM15] was used for all routine DNA
manipulations, and JRG1728 [lacX74 galU galK rpsL
(ara-leu) (tyrR-fnr-rac-trg)]
(23) was used as the host for the reporter system. To create
a mobAB mutant of JRG1728, a P1 lysate grown on TP1000
[araD (argF-lac)U169 rpsL relA flbB ptsF devC rbsR mobAB::kan] (17)
was used to transduce JRG1728 to kanamycin resistance. The
Mob
phenotype of JRG1728
mobAB::kan was confirmed by showing
that unlike its parent, it is unable to accumulate nitrite when grown in media containing nitrate, indicating a total absence of nitrate reductase activity. For 
-galactosidase assays, E. coli
strains were grown in Lennox (L) broth (tryptone [10 g
liter1], yeast extract [5 g liter1],
NaCl [5 g liter1]) supplemented with 0.5% glucose and
50 mM nitrate, 2 mM nitrite, or 100 µM sodium nitroprusside, as
indicated, or in M9 minimal medium (15) with similar
supplements. Aerobic cultures (10 ml in 250-ml flasks) were shaken at
250 rpm; anaerobic cultures were grown in standing bottles filled to
the top, in both cases at 37°C. The plasmids used were pRW2A/FF,
which contains an FNR-activated lacZ reporter
(13), and the glutathione S-transferase fusion vector pGEX-KG (7). To clone the nnr gene, the
coding region was amplified using PCR with a 5' primer
(5'-GGCATATGAACGCCCCCCTGCCCG-3') that introduced an
NdeI site around the start codon of the gene and a stop
codon into the reading frame of the lacZ alpha peptide. The
PCR product was phosphorylated with T4 polynucleotide kinase and
ligated into SmaI-digested and dephosphorylated pUC18. A
clone, designated pNNR, with the nnr gene in the same
orientation as the lac promoter was selected, and the
sequence of the insert was confirmed.
PCR mutagenesis.
To incorporate single or double point
mutations into the nnr gene, two appropriate complementary
primers, both containing the mutation(s), were used in an amplification
reaction with plasmid DNA as the template. The template DNA was then
removed by treatment with DpnI (which digests only
methylated DNA), and the remaining DNA was used to transform JM83. Each
reaction mixture contained 25 ng of template DNA, 5 µl of
Pwo buffer, 1.5 µl of deoxynucleoside triphosphate mix (50 µM), 4 µM (each) primer, and 0.5 µl of Pwo polymerase
(5 U/µl), in a total volume of 50 µl. Reaction conditions were
94°C for 5 min and then 25 cycles of 94°C for 30 s, 67°C for
30 s, and 72°C for 10 min, followed by 72°C for 15 min. The reaction mixtures were transferred to 1.5-ml tubes and treated with 10 U of DpnI for 30 min at 37°C and then 72°C for 30 min. After cooling on ice, each reaction mixture was treated with 2 U of T4
DNA ligase for 1 h and then used to transform competent JM83.
Control reaction mixtures contained no primers. Mutant DNAs were
sequenced on both strands using an ABI Prism automated sequencer. Other
general recombinant DNA techniques were performed as described by
Sambrook et al. (20).
Analytical methods.
-Galactosidase was assayed in
duplicate according to the method of Miller (15) on at least
three independently grown log-phase cultures. Experiments in which
reduced hemoglobin was added to cultures were performed by a
modification of the method of Kwiatkowski and Shapleigh
(12). Ten-milliliter cultures were grown aerobically to log
phase and then transferred to 15-ml bottles sealed with Suba seals. The
bottles were shaken for 30 min at 37°C to remove the residual oxygen.
Additions of hemoglobin and nitrate were then made as required, and the
bottles were incubated without shaking for a further 2.5 h before
-galactosidase was assayed. Absorption spectra were collected from
culture supernatants in an Aminco DW2000 dual beam spectrophotometer,
with a supernatant from a similar culture grown in the absence of
hemoglobin as the reference. Reduced human hemoglobin (Sigma) was
prepared in an anaerobic cabinet as a 0.5 mM solution in 50 mM
morpholinepropanesulfonic acid (pH 7.5), according to the method of
Bazylinski et al. (3). The concentration of nitrate in L
broth was measured with a Dionex DX-100 Ion Chromatograph using a
Dionex Ionpac AS4A column.
Preparation of anti-NNR antiserum and immunoblotting.
The
nnr gene was cloned into the glutathione
S-transferase fusion vector pGEX-KG, and the fusion protein
was purified on a glutathione affinity column. The column-bound fusion
protein was cleaved with thrombin, and NNR was eluted. Full details of
the cloning and purification will be published elsewhere. An antiserum against the purified NNR was raised in rabbit by Abcam Limited (Cambridge, United Kingdom). Cultures were grown under the same conditions used for -galactosidase assays; cells were harvested and
disrupted by sonication at 4°C. Protein was assayed (using the
bicinchoninic acid assay; Sigma) in the soluble fractions, and 50 µg
of protein from each sample was separated by sodium dodecyl sulfate
(SDS)-polyacrylamide gel electrophoresis. The gel was assembled into a
Novablot semi-dry blotter (Pharmacia) with a nitrocellulose membrane
and three layers of filter paper soaked in transfer buffer (glycine
[2.93 g liter1], Tris [5.81 g liter1],
SDS [0.38 g liter1], methanol [20%]), and proteins
were transferred at 150 mA for 30 min. The membrane was soaked in
blocking solution (10% dried milk-0.3% Tween 20 in
phosphate-buffered saline [PBS]) for 1 h and then incubated for
1 h at room temperature with the anti-NNR antiserum diluted
100-fold in blocking solution. The membrane was rinsed (twice for 10 min) in wash buffer (0.3% Tween 20 in PBS) and then incubated for
1 h with anti-rabbit antibody (alkaline phosphatase conjugate)
diluted 10,000-fold in blocking solution. The membrane was rinsed twice
in wash buffer and then in PBS, and then was incubated in 20-ml
reaction buffer (100 mM NaCl, 5 mM MgCl2, 100 mM Tris-HCl
[pH 9]), 15 µl of nitroblue tetrazolium (50 mg ml1),
and 60 µl of 5-bromo-4-chloro-3-indolylphosphate (50 mg
ml1) for 10 min.
| |
RESULTS |
Correction of the NNR sequence.
Nucleotide sequencing of the
nnr gene revealed an error in the previously reported
sequence (26); the CG dinucleotide at coordinates 683 to 684 (EMBL accession no. U17435) is GC in the correct sequence. In the
predicted protein sequence, this changes the reported sequence RLRNDGV
(residues 197 to 203) to RLRKHGV. Reexamination of the original
sequence data confirmed the error and this correction.
NNR activates an FNR-dependent promoter in response to reactive
nitrogen species.
The nnr gene of P. denitrificans was cloned into pUC18 under the control of the
lac promoter to generate a plasmid designated pNNR. This was
introduced into an fnr mutant of E. coli
containing a second plasmid (pRW2A/FF), which carries a derivative of
the melR promoter, from which transcription depends upon the
binding of FNR to a consensus FNR-binding site centered at 
41.5 with respect to the transcription start site (13). The promoter
is fused to lacZ such that any ability of NNR to activate
transcription would be manifested as -galactosidase activity. In
aerobically grown cultures, NNR did not activate transcription from the
FF-melR promoter (Table 1).
Under anaerobic growth conditions, there was a 14-fold stimulation of
the promoter by NNR and a further 9-fold stimulation in the presence of
100 µM SNP, a nitrosating agent that is a source of NO+
(this concentration of SNP has a negligible effect on anaerobic growth). The S-nitrosothiols S-nitrosoglutathione
and S-nitrosoacetylpenicillamine stimulated
NNR-dependent transcription to an extent similar to that stimulated by
SNP (data not shown). Thus, NNR responds to reactive nitrogen species,
and specifically to SNP, similarly in E. coli and in
P. denitrificans (28). This implies that the mechanism by which NNR is activated by NO, whether it is direct or
indirect, functions in the heterologous background. The activity of NNR
in the E. coli system further implies that it is able to activate transcription catalyzed by E. coli RNA polymerase
containing the major sigma factor (
70), since the
synthetic melR promoter is transcribed by
70-containing RNA polymerase (22). This is,
potentially, a significant finding, in light of speculation that in
P. denitrificans, NNR might activate RNA polymerase
containing an alternative sigma factor (2). In similar
experiments, NNR showed no ability to activate a class I promoter,
where the FNR binding site is centered at 61.5 with respect to the
transcription start site (data not shown).
View this table:
[in this window]
[in a new window]
|
TABLE 1.
-Galactosidase activities directed by the
FF-melR promoter in the presence of plasmids expressing NNR
and FNRa
|
|
It was surprising that NNR-dependent transcription from the
FF-melR promoter could be observed under anaerobic growth
conditions without additions to the medium or in the presence of either
nitrate or nitrite (Table 1). In defined minimal media, a similar
response to nitrate, nitrite, and SNP was observed, but there was no
activation under anaerobic conditions in the absence of these additions
(data not shown). This suggests that activation in rich medium in the absence of additions might be due to the presence of traces of nitrate
in L broth. Using ion chromatography, the concentration of nitrate in
the L broth used for these experiments was found to be approximately
1.5 mM (data not shown). One possible explanation for the apparent
activation of NNR by nitrate and nitrite is that utilization of these
electron acceptors in E. coli is accompanied by the
accumulation of traces of NO (11). E. coli has
three nitrate reductases capable of reducing nitrate to nitrite, all of
which are molybdoenzymes (16). A mutation
(mobAB::kan) in the molybdenum cofactor
biosynthesis pathway (17) was introduced into JRG1728 to
generate a strain devoid of nitrate reductase activity. In this strain,
there was no NNR-dependent expression in unamended L broth or in the
presence of added nitrate (Table 1), indicating that the effect of
nitrate in the parent strain requires nitrate reductase activity.
However, the ability of nitrite to activate NNR was not affected by the
mobAB mutation (Table 1). This excludes the possibility that
the activating effect of nitrite is due to the ability of the E. coli membrane-bound nitrate reductase to reduce nitrite to NO
(11).
The ability of hemoglobin to trap NO in growing cultures and so to
modulate the activity of NO-responsive promoters (6, 12, 28)
was used to explore further the nature of the signal(s) activating NNR.
The addition of 4 µM hemoglobin to cultures grown anaerobically in
minimal medium containing 2 mM nitrate reduced NNR-mediated activation
by a factor of 3 (Fig. 1). The spectra of
hemoglobin from supernatants of cultures grown in the presence of
nitrate showed absorption maxima at approximately 546 and 571 nm (Fig.
1), which is consistent with the formation of a hemoglobin-NO complex
(6, 12). Addition of hemoglobin to cultures grown in L broth
plus glucose (LG) reduced expression by a factor of 2.5 and resulted in
the formation of an NO-hemoglobin complex (Fig. 1), which is further
evidence that activation of NNR in L broth is due to the reduction of
traces of nitrate. Hemoglobin also reacts with nitrite, though much
more slowly than with NO, to produce a rather similar spectrum that
also has a broad absorption feature at around 630 nm (6).
This feature is just discernible in the spectrum of hemoglobin from the
supernatant of a culture grown in minimal medium supplemented with
nitrate (Fig. 1), suggesting that hemoglobin might be acting by
sequestering nitrite rather than, or as well as, NO. Thus, the data
presented herein do not rigorously exclude the possibility that nitrite
itself is able to activate NNR. An alternative possibility is that in
E. coli, nitrite reduction to ammonia by the multiheme and
siroheme nitrite reductases is accompanied by the release of traces of
NO, which results in activation of NNR. Hemoglobin had no significant
effect on NNR activity in anaerobic cultures growing on 2 mM nitrite (not shown). This may be because the large excess of nitrite reacts with hemoglobin, preventing the formation of the NO-hemoglobin complex,
but it is also consistent with the possibility that nitrite itself
activates NNR, so that sequestration of NO by hemoglobin in a culture
containing 2 mM nitrite has no effect on NNR activity.
View larger version (24K):
[in this window]
[in a new window]
|
FIG. 1.
Effect of hemoglobin on NNR-mediated gene expression.
(a) JRG1728 (pRW2A/FF; pNNR) was assayed for -galactosidase
following anaerobic growth on LG (with no added nitrate) or M9 minimal
medium supplemented with 2 mM nitrate. Cultures were supplemented with
2 µM (LG) or 4 µM (M9) prereduced hemoglobin, as indicated. (b)
Absorption spectra of supernatants from cultures grown in M9 in the
presence and absence of nitrate or in L broth plus glucose, as
indicated.
|
|
Derivatives of the FF-melR promoter have been
constructed in which the consensus FNR binding site is changed to a CRP
binding site (CC), or to a site bound by neither FNR nor CRP (NN), by two symmetrically related substitutions (13). Neither of
these promoters was significantly activated by NNR in the E. coli system (data not shown), confirming that the DNA binding
specificity of NNR is the same as, or very similar to, that of FNR.
It has been suggested that the activity of FNR might be sensitive to
NO, since the oxygen-labile Fe-S center of FNR is a potential target
for NO (30), and it has recently been reported that the CydR
(FNR) protein of Azotobacter vinelandii is inactivated by NO
in vitro (33). As expected, FNR (expressed from the
fnr promoter in pGS24) activated the FF-melR
promoter most efficiently under anaerobic growth conditions (Table 1).
Activation was not affected by the presence of SNP in growth media
(Table 1), suggesting that FNR activity is insensitive to SNP-derived
NO+ in vivo, at least under the conditions used in these experiments.
Characterization of altered NNR proteins.
The E. coli-based system provides a facile means to characterize NNR
proteins with substitutions in amino acids that may be important for
NNR activity. Potential targets for NO modification in NNR include
tyrosine residues (by nitrosylation or dityrosine bridge formation) and
cysteine residues (by S nitrosylation). Tyr-93 and Tyr-35 of NNR were
replaced with phenylalanine, and Cys-103 was replaced with serine. A
fourth residue, Phe-82, was altered to both alanine and tyrosine,
because Phe-82 is conserved in the NnrR protein of R. sphaeroides, the DNR protein of P. aeruginosa, and the
DnrD protein of P. stutzeri but is not conserved in other members of the FNR/CRP family. Serine-96 of NNR was also targeted for
mutagenesis, because the above-mentioned relatives of NNR all have
either serine or threonine at this position, while other members of the
FNR/CRP family do not. The altered genes were introduced into the
E. coli reporter system, and -galactosidase activity was
used as a measure of the ability of their products to activate transcription. Both NNR Y35F and NNR C103S showed a reduced ability to
activate transcription of the FF-melR promoter but remained significantly responsive to SNP, though less so than the wild-type protein (Table 2). This suggests that in
both cases, the residue is not essential for the NO activation of NNR
but may play a significant role. On the other hand, replacement of
Tyr-93 with phenylalanine abolished the activity of NNR, and
replacement of Phe-82 with either alanine or tyrosine substantially
reduced activity (Table 2). Replacement of Ser-96 with alanine almost
completely abolished the activity of NNR. On the other hand,
replacement with threonine had no significant effect on NNR activity in
the presence of SNP and significantly increased the basal level of
activity seen in aerobic cultures and the intermediate level of
activity in anaerobic LG-grown cultures (Table 2). The apparent
decrease in the activation of NNR S96T by SNP is a consequence of the
increased basal level of activity of this protein. Some of the altered
proteins showed different responses to anaerobic growth on LG and
on LG amended with SNP. For example, the F82Y and Y93F proteins
retained a significant response to anaerobic growth on LG but showed
little or no additional response to SNP (Table 2). Similarly, the C103S
protein appeared to be specifically impaired in the SNP response. One
possible explanation is that the nitrate-derived signal persists
throughout growth of the culture, whereas SNP is unstable and likely to
be depleted during early stages of growth. The activating effect of SNP
on proteins retaining low levels of activity may therefore be less
apparent. An alternative possibility is that the mutant proteins retain
some responsiveness to nitrite (assuming nitrite itself can activate
NNR) but not to SNP.
View this table:
[in this window]
[in a new window]
|
TABLE 2.
-Galactosidase activities directed by the
FF-melR promoter in the presence of plasmids expressing
NNR and its altered derivativesa
|
|
To test whether the consequences of mutagenizing nnr were
due to specific effects on the activity of NNR or to changes in the
protein's stability or expression level, the abundance of altered
proteins in E. coli cell extracts was evaluated by
immunoblotting (Fig. 2). Cultures of the
same strains used for assays of NNR activity were grown aerobically and
anaerobically, and equal amounts of cellular protein were separated by
SDS-polyacrylamide gel electrophoresis. Proteins were transferred to a
nylon membrane by Western blotting and probed with a polyclonal
antiserum raised against purified NNR protein. This revealed that the
cellular concentrations of mutant proteins were approximately equal,
with the exception of proteins with a substitution at Ser-96. The S96A
protein was consistently undetectable in cell extracts grown under a
variety of conditions. The same result was demonstrated for three
independently isolated mutants and was reproduced when the mutant
coding region was subcloned into another vector. Thus, the lack of
expression, or instability, of NNR S96A is a direct consequence of the
single amino acid substitution. The inactivity of NNR S96A in the in
vivo assay (Table 2) can therefore be ascribed to the absence of
the protein from the cell. The S96T protein was detectable in cell
extracts, to slightly higher concentrations than those of the other
proteins (Fig. 2). The increased basal-level activity of this protein
(Table 2) may therefore be a simple consequence of its increased
abundance in the cell. Taken together, these results point to
serine-96 being important for the stability of the NNR protein; an
additional functional role for this residue in NNR activity cannot be
excluded at this stage.
View larger version (24K):
[in this window]
[in a new window]
|
FIG. 2.
Western blot of NNR and its altered derivatives probed
with an anti-NNR antiserum. E. coli JRG1728(pRW2A/FF)
transformed with the appropriate NNR-expressing plasmid was grown
aerobically (upper panel) and anaerobically (lower panel) in LG plus
nitrate. Equal amounts of protein were run on an SDS-polyacrylamide
gel, which was then transferred to a nylon membrane, which was probed
with an anti-NNR antiserum. Lanes: 1, wild-type NNR; 2, NNR Y35F; 3, NNR F82A; 4, NNR F82Y; 5, NNR Y93F; 6, NNR S96A; 7, NNR S96T; 8, NNR
C103S; 9, pure NNR.
|
|
NNR represses an FNR-repressible promoter in response to reactive
nitrogen species.
A derivative of the gal promoter has
been constructed which is subject to simple repression by FNR, binding
to a site overlapping the 35 sequence (31). This promoter
(FF-gal4) was repressed only about 1.4-fold by NNR when
cultures were grown anaerobically in the presence of 100 µM SNP.
Repression by NNR was more efficient in cultures containing higher
concentrations of SNP, but the reagent became rather toxic at these
increased concentrations. It was found that the use of nitrate to
activate NNR led to a more efficient repression in this assay (Table
3), presumably because nitrate provides a
signal that persists throughout the growth period of the culture,
whereas SNP might become exhausted, leading to derepression of the
promoter. Even under these conditions, NNR is a much poorer repressor
of the FF-gal4 promoter than is FNR, for reasons that are
not clear. Nevertheless, repression of this promoter could be used to
test the ability of mutant NNR proteins to bind to DNA in vivo, and
these tests were done in media containing nitrate. The S96T protein was
a significantly better repressor of the FF-gal4 promoter
than the wild-type protein (Table 3). This may reflect the slightly
greater cellular abundance (Fig. 2) of this protein and its somewhat
enhanced activity (Table 2). No significant repression of the
NN-gal4 derivative was observed with NNR (results not
shown), confirming that the repressing effect of NNR involves interaction with the FF site.
View this table:
[in this window]
[in a new window]
|
TABLE 3.
-Galactosidase activities directed by the
FF-gal 4 promoter in the presence of plasmids
expressing NNR and its altered derivatives
and FNRa
|
|
The particularly severe effects of the Y93F, F82A, and F82Y mutations
(Table 2) may indicate that these residues are specifically required
for activation of NNR by NO, have a purely structural role, or are
involved in making activating contacts with RNA polymerase. These
possibilities were resolved by evaluating the ability of NNR
derivatives to repress transcription from the repressible FF-gal4 promoter (repression requires DNA binding only
and does not involve contacts with RNA polymerase
[31]). NNR Y93F repressed FF-gal4 at
least as efficiently as the wild-type NNR protein (Table 3),
implying that NNR Y93F binds to DNA as well as the wild-type protein
does. Therefore, Tyr-93 may have a role in making an activating contact
with RNA polymerase, rather than in the signal recognition mechanism of
NNR. In other words, Y93F may be a positive control mutation that
identifies an activating region of the NNR protein that is involved in
an interaction with RNA polymerase. The F82Y protein also retained a
significant ability to repress the FF-gal4 promoter
(Table 3), suggesting that Phe-82 may also have a role in interacting
with RNA polymerase. However, the F82A protein appeared not to function
as a repressor, for reasons that are not clear. The two proteins that
retained significant activity in the activation assay (Y35F and C103S)
were also functional in the repression assay (Table 3).
| |
DISCUSSION |
This work has demonstrated that the NNR protein of
P. denitrificans can be activated in E. coli
by exposure to SNP in anaerobic cultures or by anaerobic growth on
nitrate or nitrite. The reason that NNR can be activated only in
anaerobic cultures is not clear, but it may be related to the fact that
E. coli deals with reactive nitrogen species in different
ways in the presence and absence of oxygen (10). SNP is one
of several agents that can induce a nitrosative stress in E. coli, which, among other things, results in the derepression of a
flavohemoglobin that plays a role in protection against nitrosating
agents and NO-related species (14). The pattern of
expression of the flavohemoglobin gene hmp is rather similar
to the pattern of NNR-mediated gene expression in E. coli, in that hmp is activated under anaerobic growth conditions
by nitrate, nitrite, and SNP (14, 18). It has been suggested that nitrate and nitrite might activate hmp by acting as
substrates for the endogenous generation of NO, perhaps through the
ability of oxidases to reduce nitrite to NO or through nonenzymatic
reduction of nitrite (18). The ability of nitrate and
nitrite to activate NNR may be explained in a similar way. On the other
hand, the possibility that nitrite itself activates NNR cannot be
excluded by the data from experiments performed with E. coli. In a P. denitrificans nitrite reductase mutant,
which cannot reduce nitrite to NO, the activities of the NNR-regulated
norC promoter and of the NO reductase itself are at near
wild-type levels (27, 28). This also implies that either
nitrite itself or NO derived from nitrite (in a nitrite reductase-independent reaction) can activate NNR.
Nitrosative stress also results in activation of the transcriptional
regulator OxyR, which controls oxidative and nitrosative stress
response regulons (9). Nitrosative stress (but not NO itself) causes S nitrosylation and activation of OxyR (9), though it is not clear whether SNP in particular elicits this response. Nitrate respiration in E. coli also causes NO
release (10; this study) and nitrosative stress. The
activation of NNR by the presence of nitrate in cultures of E. coli requires nitrate reductase activity. Thus, it might be
tempting to speculate that the activation of NNR by SNP and by nitrate
respiration is a consequence of S nitrosylation of NNR (analogous to
the mechanism of activation of OxyR). However, the fact that the C103S
mutant of NNR retains a significant response to SNP argues against this
idea. NO also activates the SoxR regulatory protein of E. coli by direct nitrosylation of an iron sulfur center
(5). Given that NNR has only a single cysteine residue
(which is, at least partially, dispensable), this seems to be an
unlikely mechanism for the activation of NNR. Besides reaction with
thiol groups, formation of metal-NO adducts is the most common
mechanism by which proteins can be influenced by NO (24).
The sequence of NNR provides no indication that the protein might
contain a metal ion, and preliminary characterization of the purified
protein has also provided no evidence for the presence of metal ions
(unpublished observations). Thus, the mechanism of activation of NNR
remains obscure, and the possibility that there is a signal
transduction pathway involving additional proteins (conserved in
E. coli) cannot be excluded at this stage.
The ability to study NNR activity in E. coli will facilitate
the design of experiments aimed at resolving mechanistic questions. A
number of residues are herein shown to have important roles in NNR
activity. Preliminary indications are that Phe-82 and Tyr-93 may
identify an activating region (AR) of NNR involved in an interaction with RNA polymerase. Interestingly, Tyr-93 of NNR aligns closely with
Phe-112 of FNR, which has been shown to be part of the activating region, designated AR3, that is involved in transcription activation at
class II promoters (19). The failure of NNR to activate a class I promoter suggests that at least in E. coli, NNR
lacks a functional AR1 that is required for interaction with RNA
polymerase when the activator is bound at 61.5 (19). In
this respect, NNR is rather similar to FNR, which has a marked
preference for class II promoters (19, 32). Thus, by analogy
with the CRP/FNR system (4, 19), it appears as though NNR
has a functional AR3 but no AR1. The activity of AR3 in CRP requires
Glu-58 (4), which is conserved in NNR.
Tyrosine-35 and cysteine-103 may play important roles in NNR activity
but are not the sole determinants of the response to NO, since
mutations at these residues do not produce a null phenotype. Serine-96
is clearly an important residue, though properties of mutant proteins
with a substitution at this position can be explained solely in terms
of protein stability and abundance in the cell. Further
characterization of NNR activity in both E. coli and
P. denitrificans will shed additional light on the
mechanisms of signal perception and transcription activation.
This work was supported by research grants from the Biotechnology
and Biological Sciences Research Council.
We are grateful to Jeff Green, Steve Busby, and Tracy Palmer for
generous gifts of strains and plasmids and for helpful discussions and
to Michael Hill for help with ion chromatography.
| 1.
|
Arai, H.,
Y. Igarashi, and T. Kodama.
1995.
Expression of the nir and nor genes for denitrification of Pseudomonas aeruginosa requires a novel CRP/FNR-related transcriptional regulator, DNR, in addition to ANR.
FEBS Lett.
371:73-76[CrossRef][Medline].
|
| 2.
|
Baker, S. C.,
S. J. Ferguson,
B. Ludwig,
M. D. Page,
O.-M. H. Richter, and R. J. M. van Spanning.
1998.
Molecular genetics of the genus Paracoccus: metabolically versatile bacteria with bioenergetic flexibility.
Microbiol. Mol. Biol. Rev.
62:1046-1078[Abstract/Free Full Text].
|
| 3.
|
Bazylinski, D. A.,
J. Goretski, and T. C. Hollocher.
1985.
On the reaction of trioxodinitrate (II) with hemoglobin and myoglobin.
J. Am. Chem. Soc.
107:7986-7989[CrossRef].
|
| 4.
|
Busby, S., and R. H. Ebright.
1999.
Transcription activation by catabolite activator protein (CAP).
J. Mol. Biol.
293:199-213[CrossRef][Medline].
|
| 5.
|
Ding, H., and B. Demple.
2000.
Direct nitric oxide signal transduction via nitrosylation of iron-sulfur centres in the SoxR transcription activator.
Proc. Natl. Acad. Sci. USA
97:5146-5150[Abstract/Free Full Text].
|
| 6.
|
Goretski, J., and T. C. Hollocher.
1988.
Trapping of nitric oxide produced during denitrification by extracellular hemoglobin.
J. Biol. Chem.
263:2316-2323[Abstract/Free Full Text].
|
| 7.
|
Guan, K., and J. E. Dixon.
1991.
Eukaryotic proteins expressed in Escherichia coli: an improved thrombin cleavage and purification procedure of fusion proteins with glutathione S-transferase.
Anal. Biochem.
192:262-267[CrossRef][Medline].
|
| 8.
|
Hasegawa, N.,
H. Arai, and Y. Igarashi.
1998.
Activation of a consensus FNR-dependent promoter by DNR of Pseudomonas aeruginosa in response to nitrite.
FEMS Microbiol. Lett.
166:213-217[CrossRef][Medline].
|
| 9.
|
Hausladen, A.,
C. T. Privalle,
T. Keng,
J. DeAngelo, and J. S. Stamler.
1996.
Nitrosative stress: activation of the transcription factor OxyR.
Cell
86:719-729[CrossRef][Medline].
|
| 10.
|
Hausladen, A.,
A. J. Gow, and J. S. Stamler.
1998.
Nitrosative stress: metabolic pathways involving the flavohemoglobin.
Proc. Natl. Acad. Sci. USA
95:14100-14105[Abstract/Free Full Text].
|
| 11.
|
Ji, X.-B., and T. C. Hollocher.
1988.
Reduction of nitrite to nitric oxide by enteric bacteria.
Biochem. Biophys. Res. Commun.
157:106-108[CrossRef][Medline].
|
| 12.
|
Kwiatkowski, A. V., and J. P. Shapleigh.
1996.
Requirement of nitric oxide for induction of genes whose products are involved in nitric oxide metabolism in Rhodobacter sphaeroides 2.4.3.
J. Biol. Chem.
271:24382-24388[Abstract/Free Full Text].
|
| 13.
|
Lodge, J.,
R. Williams,
A. Bell,
B. Chan, and S. Busby.
1990.
Comparison of promoter activities in Escherichia coli and Pseudomonas aeruginosa: use of a new broad-host-range promoter-probe plasmid.
FEMS Microbiol. Lett.
67:221-226[CrossRef].
|
| 14.
|
Membrillo-Hernandez, J.,
M. D. Coopamah,
M. F. Anjum,
T. M. Stevanin,
A. Kelly,
M. N. Hughes, and R. K. Poole.
1999.
The flavohemoglobin of Escherichia coli confers resistance to a nitrosating agent, a "nitric oxide releaser," and paraquat and is essential for transcriptional responses to oxidative stress.
J. Biol. Chem.
274:748-754[Abstract/Free Full Text].
|
| 15.
|
Miller, J. H.
1992.
A short course in bacterial genetics.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
|
| 16.
|
Moreno-Vivián, C.,
P. Cabello,
M. Martínez-Luque,
R. Blasco, and F. Castillo.
1999.
Prokaryotic nitrate reduction: molecular properties and functional diversity among bacterial nitrate reductases.
J. Bacteriol.
181:6573-6584[Free Full Text].
|
| 17.
|
Palmer, T.,
C. L. Santini,
C. Iobbi-Nivol,
D. J. Eaves,
D. H. Boxer, and G. Giordano.
1996.
Involvement of the narJ and mob gene products in distinct steps in the biosynthesis of the molybdoenzyme nitrate reductase in Escherichia coli.
Mol. Microbiol.
20:875-884[Medline].
|
| 18.
|
Poole, R. K.,
M. F. Anjum,
J. Membrillo-Hernandez,
S. O. Kim,
M. N. Hughes, and V. Stewart.
1996.
Nitric oxide, nitrite, and Fnr regulation of hmp (flavohemoglobin) gene expression in Escherichia coli K-12.
J. Bacteriol.
178:5487-5492[Abstract/Free Full Text].
|
| 19.
|
Ralph, E. T.,
J. R. Guest, and J. Green.
1998.
Altering the anaerobic transcription factor FNR confers a hemolytic phenotype on Escherichia coli K12.
Proc. Natl. Acad. Sci. USA
95:10449-10452[Abstract/Free Full Text].
|
| 20.
|
Sambrook, J.,
E. F. Fritsch, and T. Maniatis.
1989.
Molecular cloning: a laboratory manual, 2nd ed.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
|
| 21.
|
Saunders, N. F. W.,
E. N. G. Houben,
S. Koefoed,
S. de Weert,
W. N. M. Reijnders,
H. V. Westerhoff,
A. P. N. de Boer, and R. J. M. van Spanning.
1999.
Transcription regulation of the nir gene cluster encoding nitrite reductase of Paracoccus denitrificans involves NNR and NirI, a novel type of membrane protein.
Mol. Microbiol.
34:24-36[CrossRef][Medline].
|
| 22.
|
Savery, N. J.,
G. S. Lloyd,
M. Kainz,
T. Gaal,
W. Ross,
R. H. Ebright,
R. L. Gourse, and S. J. W. Busby.
1998.
Transcription activation at Class II CRP-dependent promoters: identification of determinants in the C-terminal domain of the RNA polymerase  subunit.
EMBO J.
17:3439-3447[CrossRef][Medline].
|
| 23.
|
Spiro, S., and J. R. Guest.
1988.
Inactivation of the FNR protein of Escherichia coli by targeted mutagenesis in the N-terminal region.
Mol. Microbiol.
2:701-707[CrossRef][Medline].
|
| 24.
|
Stamler, J. S.
1994.
Redox signalling: nitrosylation and related target interactions of nitric oxide.
Cell
78:931-936[CrossRef][Medline].
|
| 25.
|
Tosques, I. E.,
J. Shi, and J. P. Shapleigh.
1996.
Cloning and characterization of nnrR, whose product is required for the expression of proteins involved in nitric oxide metabolism in Rhodobacter sphaeroides 2.4.3.
J. Bacteriol.
178:4958-4964[Abstract/Free Full Text].
|
| 26.
|
Van Spanning, R. J. M.,
A. P. N. De Boer,
W. N. M. Reijnders,
S. Spiro,
H. V. Westerhoff,
A. H. Stouthamer, and J. Van der Oost.
1995.
Nitrite and nitric oxide reduction in Paracoccus denitrificans is under the control of NNR, a regulatory protein that belongs to the FNR family of transcriptional activators.
FEBS Lett.
360:151-154[CrossRef][Medline].
|
| 27.
|
Van Spanning, R. J. M.,
A. P. N. de Boer,
W. N. M. Reijnders,
H. V. Westerhoff,
A. H. Stouthamer, and J. Van Der Oost.
1997.
FnrP and NNR of Paracoccus denitrificans are both members of the FNR family of transcriptional activators but have distinct roles in respiratory adaptation in response to oxygen limitation.
Mol. Microbiol.
23:893-907[CrossRef][Medline].
|
| 28.
|
Van Spanning, R. J. M.,
E. Houben,
W. N. M. Reijnders,
S. Spiro,
H. V. Westerhoff, and N. Saunders.
1999.
Nitric oxide is a signal for NNR-mediated transcription activation in Paracoccus denitrificans.
J. Bacteriol.
181:4129-4132[Abstract/Free Full Text].
|
| 29.
|
Vollack, K. U.,
E. Härtig,
H. Körner, and W. G. Zumft.
1999.
Multiple transcription factors of the FNR family in denitrifying Pseudomonas stutzeri: characterization of four fnr-like genes, regulatory responses and cognate metabolic processes.
Mol. Microbiol.
31:1681-1694[CrossRef][Medline].
|
| 30.
|
Watmough, N. J.,
G. Butland,
M. R. Cheesman,
J. W. B. Moir,
D. J. Richardson, and S. Spiro.
1999.
Nitric oxide in bacteria: synthesis and consumption.
Biochim. Biophys. Acta
1411:456-474[Medline].
|
| 31.
|
Williams, S. M.,
H. J. Wing, and S. J. W. Busby.
1998.
Repression of transcription initiation by Escherichia coli FNR protein: repression by FNR can be simple.
FEMS Microbiol. Lett.
163:203-208[CrossRef][Medline].
|
| 32.
|
Wing, H. J.,
S. M. Williams, and S. J. W. Busby.
1995.
Spacing requirements for transcription activation by Escherichia coli FNR protein.
J. Bacteriol.
177:6704-6710[Abstract/Free Full Text].
|
| 33.
|
Wu, G. H.,
H. Cruz-Ramos,
S. Hill,
J. Green,
G. Sawers, and R. K. Poole.
2000.
Regulation of cytochrome bd expression in the obligate aerobe Azotobacter vinelandii by CydR (Fnr) sensitivity to oxygen, reactive oxygen species, and nitric oxide.
J. Biol. Chem.
275:4679-4686[Abstract/Free Full Text].
|
![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
Top
Abstract
Introduction
