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Phil. Trans. R. Soc. B (2012) 367, 1213–1225
doi:10.1098/rstb.2011.0309
Review
Nitrous oxide production and consumption:
regulation of gene expression by gassensitive transcription factors
Stephen Spiro*
Department of Molecular and Cell Biology, University of Texas at Dallas, 800 W Campbell Road,
Richardson, TX 75080, USA
Several biochemical mechanisms contribute to the biological generation of nitrous oxide (N2O).
N2O generating enzymes include the respiratory nitric oxide (NO) reductase, an enzyme from
the flavo-diiron family, and flavohaemoglobin. On the other hand, there is only one enzyme that
is known to use N2O as a substrate, which is the respiratory N2O reductase typically found in bacteria capable of denitrification (the respiratory reduction of nitrate and nitrite to dinitrogen). This
article will briefly review the properties of the enzymes that make and consume N2O, together with
the accessory proteins that have roles in the assembly and maturation of those enzymes. The
expression of the genes encoding the enzymes that produce and consume N2O is regulated by
environmental signals (typically oxygen and NO) acting through regulatory proteins, which,
either directly or indirectly, control the frequency of transcription initiation. The roles and mechanisms of these proteins, and the structures of the regulatory networks in which they participate will
also be reviewed.
Keywords: denitrification; nitrous oxide; nitric oxide; nitrous oxide reductase;
nitric oxide reductase
1. INTRODUCTION
Nitrous oxide (N2O) is a water-soluble gas that attracts
current interest because of its contribution to the
atmospheric greenhouse effect. N2O has well known
and useful anaesthetic and analgesic properties, and
has also found applications as an oxidant in fuels.
N2O is relatively inert at ambient temperature, and
(unlike nitric oxide, NO) has a very low affinity for
metal centres in proteins. Thus, N2O is not toxic,
and micro-organisms can tolerate relatively high (millimolar) concentrations. The0 reduction potential of the
N2O/N2 couple is high (Eo ¼ þ1.35 V at pH 7), and
some bacteria exploit this property by using N2O as
the terminal electron acceptor in energy conserving
respiratory metabolism.
N2O is an intermediate (or, in some cases, endproduct) of the respiratory pathway denitrification,
so denitrification is a major contributor to global
N2O emissions [1]. The ammonia-oxidizing bacteria
are another significant source of N2O, although those
that have been studied are also capable of partial denitrification (the respiratory reduction of nitrite to N2O),
so the enzymes responsible for N2O production are
similar to those of the denitrifying bacteria [2]. The
respiratory reduction of nitrate to nitrite and ammonia
(sometimes called respiratory ammonification) can
*[email protected]
One contribution of 12 to a Theo Murphy Meeting Issue ‘Nitrous
oxide: the forgotten greenhouse gas’.
also be a source of N2O, since some nitric oxide
(NO) is made as a by-product of this pathway and is
subsequently reduced to N2O. In contrast to the multiplicity of mechanisms by which N2O can be
generated, only a single sink for N2O is known,
which is the respiratory N2O reductase typically
found in denitrifying bacteria.
The factors that contribute to the emission of
N2O from bacterial populations are numerous and
complex, clearly one important determinant being
the cellular abundance and activities of the enzymes
that produce and consume N2O. A key contributor
to enzyme abundance is the regulation of expression
of the corresponding genes by regulatory systems and
signal transduction pathways that respond to intraor extra-cellular signals. The goal of this review is to
present a brief overview of the enzymatic mechanisms
of N2O production and consumption, and then to
focus on the regulatory mechanisms that control the
expression of the genes that encode these enzymes.
The structures of the regulatory networks that integrate environmental signals and connect regulatory
proteins with their target genes will also be reviewed.
There is a surprising degree of diversity in the organization of regulatory networks, even among organisms
that are phylogenetically quite closely related. This
diversity presents a challenge when it comes to extrapolating from studies done on one organism to
another, and from pure culture experiments to studies
of complex microbial populations.
1213
This journal is q 2012 The Royal Society
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S. Spiro Review. N2O flux: regulation of gene expression
(a)
c551
Fe
CuA
MQ
P
Fe Fe
NorC
N
FeB
Fe
Fe
qNOR
cNOR
FMN
Rd
NorV
FAD
NorW
Escherichia coli
FeB
NorB
NorB
qCuANOR
Ralstonia eutropha
Pseudomonas aeruginosa
(b)
Fe Fe
Fe Fe
FeB
MQ
Fe
Fe
FMN
FprA
Rd
FMN
Hrb
Moorella thermoacetica
Bacillus azotoformans
Fe
Fe
FMN
Rd
FAD
FprA (ROO)
Rd
NRO
Desulfovibrio sp.
Figure 1. Organization of membrane-bound and soluble NO reductase complexes. (a) The cNOR has two membrane subunits
and accepts electrons from a small soluble c-type cytochrome (cytochrome c551 in Ps. aeruginosa) or from pseudoazurin. The
qNOR is a single-subunit enzyme that accepts electrons from the quinone pool. The qCuANOR accepts electrons either from a
membrane-associated c-type cytochrome (not shown) or from menaquinol. (b) The soluble enzymes have roles in NO detoxification. NO is reduced by a flavo-diiron protein (NorV, FprA or ROO), which accepts electrons from an NADH-dependent
flavo-enzyme: NorW, high molecular weight rubredoxin (Hrb) or NADH : rubredoxin oxidoreductase (NRO). P and N denote
periplasmic and cytoplasmic compartments, respectively.
2. ENZYMES OF NITROUS OXIDE SYNTHESIS
(a) Respiratory nitric oxide reductase
The major contributor to the biological production of
N2O is almost certainly the respiratory NO reductase
(NOR) found in denitrifying bacteria and in some
ammonia-oxidizing organisms. Three types of NOR
have been described [3], as will be discussed below.
All three catalyse the two-electron reduction of NO
to N2O:
2NO þ 2Hþ þ 2e ! N2 O þ H2 O
with the electrons derived from a small c-type cytochrome, the copper protein pseudoazurin or from the
quinone pool. In each case, the catalytic subunit contains a binuclear centre comprising a high-spin haem
b and a non-haem iron (FeB). A low spin haem b participates in electron transfer to the binuclear centre,
which is the site of NO reduction. The three types of
NOR differ in their subunit architectures and in the
entry routes for electrons (figure 1a).
In the c-type NOR (also called scNOR, for short
chain), the catalytic NorB subunt is in a complex
with NorC, a small c-type cytochrome with a single
membrane-spanning helix (figure 1a). Electrons
from a periplasmic c-type cytochrome or from pseudoazurin pass through the haem of NorC, to the low
spin haem of NorB, and thence to the binuclear
centre. The best-characterized cNORs are those
from Paracoccus denitrificans, Pseudomonas stutzeri and
Pseudomonas aeruginosa. The structure of the NorBC
complex from Ps. aeruginosa [4] confirmed the
Phil. Trans. R. Soc. B (2012)
predicted presence of 12 membrane-spanning ahelices in NorB. Biochemical experiments indicated
that the protons required for NO reduction are taken
from the periplasmic side of the membrane [5], and
that NorB does not function as a proton pump [6].
The latter is confirmed in the structure by the absence
of trans-membrane proton channels in NorB analogous to those found in the proton-translocating
haem-copper oxidases, which are otherwise structurally related to NorB [4].
The norCB genes are usually co-transcribed with
accessory genes designated norD, norE, norF and
norQ; the gene order norEFCBQD is typical though
not universal [3]. The norQ and norD genes are
always linked to norCB; the other accessory genes
may be distantly located or absent altogether in some
genomes [3]. The functions of the accessory genes
and their protein products are not well understood.
NorE is a predicted membrane protein with some
sequence similarity to part of subunit 3 of the cytochrome c oxidase. This led to speculation that NorE
might be a component of the NOR complex [7],
although purified and active preparations of the
cNOR only contain NorB and NorC. Mutation of
the accessory genes tends to lead to variable phenotypes in different organisms [3], but in no case is the
biochemical function of the accessory proteins understood; this is an area worthy of further investigation.
The q-type NOR is a single-subunit enzyme that is
similar to but larger than the B subunit of the c-type
NOR, hence this enzyme has also been called the
long chain NOR. The single subunit of the qNOR
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Review. N2O flux: regulation of gene expression S. Spiro 1215
has two additional trans-membrane helices (compared
with NorB) that flank a periplasmic domain of approximately 200 amino acids (figure 1a). The qNOR
receives electrons from the quinone pool, and the
additional N-terminal domain is the presumed location
of the quinol oxidase activity [8]. A qNOR has been
characterized in the hyperthermophilic Archaea Pyrobaculum aerophilum [9]. The archaeal enzyme is similar to
the bacterial qNORs, with the exception of covalent
modifications to its haem groups. Interestingly, the
genes encoding qNORs are not typically co-expressed
with accessory genes, implying that the accessory
proteins have functions that are specific to the activity
and/or assembly of the cNORs. In Ralstonia eutropha,
the norB gene that encodes the qNOR is transcribed
with a gene designated norA (also called ytfE, dnrN
and scdA in other organisms). The NorA/YtfE protein
contains a diiron centre, and has been implicated in
the repair of damaged iron–sulphur clusters [10] and/
or in the buffering of cytoplasmic NO [11].
An unusual hybrid NOR has been described in the
Gram-positive organism Bacillus azotoformans [12,13].
This is a two subunit enzyme that functions as a menaquinol : NO oxidoreductase, but can also accept
electrons from small membrane-bound c-type cytochromes [13]. The B. azotoformans NOR contains a
CuA centre that is similar to the CuA of cytochrome
oxidases, and is proposed to be bound to the small
subunit [12]. Hence, there appear to be two routes
of electron entry into this enzyme (designated qCuANOR), either from the quinone pool, or from small
c-type cytochromes (figure 1a).
(b) Flavo-diiron proteins
In several non-denitrifying bacteria, an enzyme from
the flavo-diiron family reduces NO to N2O. The physiological role of this enzyme seems to be NO
detoxification, a reaction which may be particularly
important in organisms (such as Escherichia coli)
which make low concentrations of NO as a by-product
of the respiratory reduction of nitrite to ammonia.
Although N2O is (probably) the end-product of this
pathway, the contribution that it makes to the global
N2O budget is likely to be very small. In this enzyme
system, NO is reduced by a flavo-diiron protein,
which receives electrons from a rubredoxin domain
or protein. The rubredoxin is itself reduced by an
NADH-dependent flavoenzyme (figure 1b). The
flavo-diiron protein of E. coli has a fused rubredoxin
domain, and so is called flavorubredoxin, FlRd (also
called NorV). In complex with the NADH-dependent
FlRd oxidoreductase (NorW), this enzyme functions
as an NO reductase in vitro [14] and in vivo [15]. In
Moorella thermoacetica, a similar complex (figure 1b)
functions as an NO reductase, though in this case
the flavo-diiron protein (FprA) is reduced by an
NADH-dependent high molecular weight rubredoxin,
Hrb [16]. In Desulfovibrio gigas, an equivalent
enzyme complex was initially characterized as an oxidase, comprising the flavo-diiron protein (ROO, for
rubredoxin oxygen oxido-reductase), a standalone
rubredoxin and an NADH-rubredoxin oxidoreductase. The recombinant D. gigas ROO and reduced
Phil. Trans. R. Soc. B (2012)
rubredoxin were found also to reduce NO to N2O,
and physiological data suggested that the enzyme functions as an NO reductase in vivo [17]. Similarly, the
D. vulgaris FprA functions as an NO reductase both
in vitro and in vivo [18]. The oxidase activities associated with these flavo-diiron proteins are probably
not physiologically relevant, since oxygen turnover
irreversibly inactivates the enzyme [16,18]. Thus, a
consensus has emerged in which the physiological
role of these enzymes is to scavenge low concentrations
of NO in cells growing under micro-oxic conditions.
(c) Flavohaemoglobin
The flavohaemoglobin Hmp is another NO detoxification enzyme, which is phylogenetically widespread,
being found in denitrifying bacteria (such as R. eutropha)
and non-denitrifiers, including E. coli. Hmp has a globin
like domain, and an FAD-containing domain that binds
NAD(P)H [19]. In the presence of oxygen, Hmp oxidizes NO to nitrate, an activity that has been described
as an NO dioxygenase or NO denitrosylase. In the
absence of oxygen, Hmp reduces NO to N2O [20].
However, the consumption of NO by Hmp under
anaerobic conditions is rather slow [21], so Hmp may
not be a significant source of N2O.
3. ENZYMES OF NITROUS OXIDE CONSUMPTION
(a) Respiratory nitrous oxide reductase
While N2O is generated by a diversity of enzymes, N2O
consumption appears to be exclusively due to the respiratory N2O reductase Nos (also called NosZ).
Genetic, biochemical and molecular aspects of N2O
reduction have been reviewed comprehensively [22]
and will be summarized only briefly here. Nos enzymes
are soluble periplasmic copper proteins that catalyse the
two-electron reduction of N2O to dinitrogen:
N2 O þ 2Hþ þ 2e ! N2 þ H2 O:
The electrons required for N2O reduction originate in
the quinone pool and are transferred to Nos either
via the cytochrome bc1 complex and small soluble
periplasmic proteins, or via Nos-specific membraneassociated electron transfer proteins (figure 2).
The well-characterized Nos (referred to as the Z-type
Nos) is a homodimeric protein in which each monomer
contains two copper centres designated CuA and CuZ.
Three-dimensional structures are available for the
enzymes from Marinobacter hydrocarbonclasticus (Pseudomonas nautica) and Pa. denitrificans [24,25]. The electron
transfer route to this enzyme is typically depicted as
involving the cytochrome bc1 complex and small soluble
periplasmic electron carriers, such as cytochrome c550
and pseudoazurin [1]. However, there are observations
that are consistent with Nos-specific proteins participating in electron transfer to the Z-type Nos. Specifically,
NosR is a membrane-bound iron–sulphur flavoprotein,
which has been suggested to mediate electron transfer
between the quinone pool and Nos [26].
The Z-type Nos is encoded by a gene called nosZ
that is typically linked to other nos genes, whose products have roles in the maturation of the active
enzyme. The functions of the accessory Nos proteins
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S. Spiro Review. N2O flux: regulation of gene expression
N2O + 2H+
N2O + 2H+
N2 + H2O
NosZ (Z-type)
CuZ
CuA
N2 + H2O
c
NosC1/C2
CuZ
CuA
c
NosZ (c-type)
c550
Cu
2 [4Fe–4S]
NosG
P
c1
2UQH2
2UQ
UQH2
UQ
N
bL
2Fe–2S
MKH2
MK
bH
cytochrome bc1
complex
2 [4Fe–4S]
Paracoccus
denitrificans
Wolinella
succinogenes
NosH
Figure 2. Electron transfer pathways to the Z-type and c-type nitrous oxide reductase, exemplified by those of Pa. denitrificans
and W. succinogenes, respectively. In Pa. denitrificans, electron transfer from the cytochrome bc1 complex is via cytochrome c550
or pseudoazurin. In the W. succinogenes system, the small mono-haem c-type cytochromes NosC1 and NosC2 may be periplasmic or attached to the membrane, and may or may not be on the electron transfer route from NosGH to NosZ [23]. P and N
denote periplasmic and cytoplasmic compartments, respectively.
are not well understood, but they include an ABC
transporter (NosFYD) that may export a sulphur
compound to the periplasm, an outer membrane
copper porin (NosA), an outer membrane anchored
copper protein (NosL), a periplasmic flavoprotein
(NosX) and NosR [22]. The catalytic Nos subunit is
exported as an apo protein to the periplasm via the
Tat transport system, which is unusual, since Tat typically secretes fully assembled and folded proteins.
Thus, the copper centres are inserted into Nos in the
periplasm, and it seems likely that some or all of the
periplasmic accessory Nos proteins have roles in
assembly of the copper clusters. As is also the case
for the c-type NOR, the maturation of Nos is an
area that demands further investigation.
An unusual variant of Nos is found in Wolinella
succinogenes and some of its relatives. This enzyme
(called the c-type Nos) is characterized by an additional
C-terminal mono-haem cytochrome c domain. Export
to the periplasm is by the Sec secretory system rather
than the Tat pathway used by the Z-type Nos. Electron
transfer to the c-type Nos involves membraneassociated iron–sulphur proteins (NosG and NosH)
which mediate electron transfer between the quinone
pool and NosZ, perhaps via small soluble periplasmic
c-type cytochromes [23]. The physiological role of the
Nos in W. succinogenes is not certain, given that the
Phil. Trans. R. Soc. B (2012)
organism reduces nitrite to ammonia. One possibility
is that the enzyme acts on the N2O produced by
detoxifying activities that remove the NO made as a
by-product of nitrite respiration [23].
4. REGULATORY PROTEINS
The genes and operons encoding the enzymes of N2O
production and consumption are controlled in different
organisms by a diverse array of transcriptional regulators (figure 3). The signals to which these regulatory
proteins respond are also diverse, and include oxygen,
NO, nitrate and the activity of the electron transport
chain. In most species that have been studied, multiple
environmental signals are integrated by regulatory
pathways to ensure that the NO and N2O reductases
are expressed optimally according to the prevailing
needs of the organism. In the following sections, the
properties of the various regulatory proteins will first
be reviewed briefly, with a focus on mechanistic
aspects. Then, the signalling networks that connect
regulatory proteins with their target genes in selected
model organisms are described. The intention is to
present only a thumbnail of each regulatory system.
Thus, references to the literature are not exhaustive,
with a focus on reviews, where available, and recent
developments. Regulators of genes of the entire
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Review. N2O flux: regulation of gene expression S. Spiro 1217
haem
FixL
FixJ
[4Fe – 4S]
FNR/FnrP/ANR/FnrN
1 2
3/4 5
6
RegB/PrrB/ActS
Cys265
RegB/PrrB/ActS
GGXXNPF
NNR/NnrR/DNR
haem?
Fe2+
NorR
P-Box
NarL
NarX
NarR
FixK
2
3
4
5
6
[4Fe–4S]
NirI
[4Fe–4S]
CX3CP
1
2
3
4
CX3CP
5
6
[4Fe–4S]
NosR
[4Fe–4S]
CX3CP
CX3CP
Figure 3. Domain organizations of proteins involved in regulating expression of genes encoding the respiratory nitrite and NO
reductases. Domain architectures were generated from primary structures of representative proteins with the simple modular
architecture research tool, SMART [27]. The proteins shown are those discussed in the text, with the exception of NsrR, for
which SMART predicts no conserved domains. Known or suspected cofactors are indicated, along with conserved sequence
motifs that are discussed in the text. The P-box in NarL is in a periplasmic loop (although SMART does not predict transmembrane helices for NarL) and is the site of nitrate binding [28]. For RegB and its orthologues, SMART predicts a
single trans-membrane helix in place of the experimentally verified helices 3 and 4 [29]. The GGXXNPF motif occurs between
helices 3 and 4, and is likely to be part of a quinine-binding site [30]. PAS, domain found in Per-Arnt-Sim proteins; PAC,
domain occurring C-terminal to a subset of PAS domains; HisKA, dimerization and phosphoacceptor domain of histidine
kinases; HATPase_c, histidine kinase-like ATPases; REC, cheY-homologous receiver domain; HTH LuxR, helix-turn-helix
domain in LuxR family of response regulators, cNMP, cyclic nucleotide-monophosphate-binding domain; HTH CRP,
helix-turn-helix domain in cAMP receptor protein family; GAF, domain present in phytochromes and cGMP-specific phosphodiesterases; AAA, ATPases associated with a variety of cellular activities; HAMP, domain found in histidine kinases,
adenylyl cyclases, methyl-binding proteins and phosphatases; FMN bind, flavin-binding site.
Phil. Trans. R. Soc. B (2012)
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S. Spiro Review. N2O flux: regulation of gene expression
denitrification pathway are considered, since the flux
through the early reactions of the pathway will be a
contributor to the emission of N2O.
(a) FixLJ
FixL is a multi-domain sensory histidine kinase which, in
its active state, phosphorylates a conserved aspartic acid
residue in the receiver domain of its cognate response
regulator FixJ (figure 3). Sensory input into this system
is via the PAS (Per-Arnt-Sim) domain of FixL, which
coordinates a single molecule of haem. There is some
structural diversity in FixL proteins from different
organisms. For example, the Bradyrhizobium japonicum
FixL is soluble and cytoplasmic, while the Sinorhizobium
meliloti protein is membrane-associated, such that its
PAS domain is anchored on the cytoplasmic face of the
membrane (figure 3). In all cases that have been studied,
FixL is in its ‘on’ state in the absence of oxygen, in which
it auto-phosphorylates prior to phospho-transfer to FixJ.
Phosphorylated FixJ is active for DNA binding and regulation of the transcription of target genes. Molecular
oxygen binds to FixL (with an affinity in the range 50–
150 mM), converting the ferrous iron of the haem from
high spin to low spin. The consequent movement of
the iron in the plane of the porphyrin ring triggers a
series of conformational changes that inhibit the kinase
activity of FixL. There is a great deal of structural and
biochemical information for FixL proteins, so the mechanism by which oxygen regulates the kinase activity is
quite well understood [31–33]. FixL can bind other
haem ligands (such as CO and NO) but these do not
inhibit the kinase activity, and so their interaction with
FixL is probably not physiologically significant.
(b) FNR
FNR from E. coli is a prototypical member of the FNR/
CRP superfamily of transcriptional activators. Four
conserved cysteines of FNR coordinate a [4Fe–4S]2þ
cluster, which is converted to a [2Fe–2S]2þ cluster on
exposure to oxygen. This transition is accompanied by
a reduced tendency of FNR to dimerize, and so a
reduced affinity for its DNA targets. Details of the
mechanism of the reaction of the cluster with oxygen
(which may proceed via a [3Fe–4S]1þ intermediate)
are beginning to emerge [32,34]. Prolonged reaction
with oxygen results in a cluster-free apo-protein,
which is the form isolated from aerobically grown
cells. Orthologues of FNR from other organisms
(such as FnrP, ANR and FnrN) are presumed to
work in similar ways, although the limited information
that is available suggests that the oxygen sensitivity
of the cluster can be fine-tuned by the protein
environment in different FNR relatives [35,36].
Iron– sulphur proteins are potential targets for NO,
and the reaction of FNR proteins with NO has been
documented for proteins from several sources
[35,37 –39]. The product of this reaction has not
been well characterized, but may include dinitrosyl
iron complexes [37], and/or, perhaps, a [2Fe –2S]
cluster [38]. However, FNR proteins do not provide
the dominant mechanism for NO sensing, and so
the reaction of FNR with NO may serve only to
Phil. Trans. R. Soc. B (2012)
fine-tune the expression of genes encoding the
enzymes of denitrification (see below).
(c) RegBA/PrrBA
RegB and its orthologues are membrane-associated
histidine kinases that phosphorylate their cognate
response regulators (figure 3). The RegBA system
was first described in the photosynthetic bacterium
Rhodobacter capsulatus, and its close relative Rhodobacter sphaeroides (in which it is called PrrBA). RegBA/
PrrBA two component systems have been well characterized in these organisms, and other members of the
alpha proteobacteria, where they are typically involved
in regulating energy generating or consuming processes, including denitrification [29]. RegB/PrrB
functions as a redox sensor, such that it is active as a
kinase under conditions that lead to a relatively
reduced state of the cell. Thus, in the facultatively
anaerobic Rhodobacter species, RegB/PrrB behaves as
if it is inactivated by oxygen, though this is not a
direct effect. Instead, the RegB/PrrB proteins appear
to measure the ‘redox state’ of the cell by multiple
mechanisms. In one mechanism, metal-dependent
oxidation of a key cysteine residue (Cys-265 in the
R. capsulatus RegB, figure 3) causes a dimer to tetramer transition, and inactivation of the kinase [40].
Purified RegB also contains bound ubiquinone, and
oxidation of the quinone inhibits kinase activity [30].
Quinone binding requires a conserved GGXXNPF
motif that is located in a short periplasmic loop
(figure 3, [40]). This mechanism presumably allows
RegB to monitor the redox state of the electron transport chain. The PrrB protein of R. sphaeroides appears
to be controlled by different mechanisms. In this case,
it has been suggested that the cytochrome cbb3 oxidase
serves as a redox sensor for PrrB, in a manner that
does not require oxidase activity [41]. Signal transfer
is independent of the quinone pool, implying a more
direct interaction between the cbb3 oxidase and PrrB.
Furthermore, while PrrB kinase activity is inhibited
by ubiquinone, this effect does not require the
GGXXNPF motif or the membrane domain [41].
Thus, the current evidence points to a multitude of
mechanisms in the RegB/PrrB sensor kinases.
(d) NNR/NnrR/DNR and NarR
The FNR/CRP superfamily includes several proteins
that regulate the expression of genes encoding the respiratory NO reductase. These proteins have been
variously designated NNR/NnrR and DNR, and evidence from in vivo experiments indicates that these
proteins activate transcription in response to NO. In
fact, the NNR/NnrR/DNR proteins do not form a
single coherent group, rather they fall into two of the
phylogenetically distinct branches of the wider FNR/
CRP superfamily [42]. The mechanism(s) of NO
sensing by these proteins has proved difficult to establish. NNR from Pa. denitrificans and DNR from
Ps. aeruginosa require haem for their NO-dependent
activity in heterologous reporter systems in E. coli
[43,44]. The structure of the sensory domain of
DNR reveals a hydrophobic pocket that might be a
haem-binding site, and purified apo-DNR can bind
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Review. N2O flux: regulation of gene expression S. Spiro 1219
one equivalent of haem [45]. The haem-reconstituted
DNR binds NO and CO, and also shows some DNAbinding activity that is neither stimulated nor inhibited
by NO [45]. The current model proposes that full activation of DNR requires haem and NO, though the
complete details of this mechanism remain to be established [46]. Results from in vivo experiments suggest
that NNR/NnrR/DNA activity is sensitive to oxygen
[43,47]; this would be consistent with the physiological roles of these proteins and with a haem-based
sensing mechanism.
NarR is another FNR/CRP family member, which
in Pa. denitrificans regulates expression of the respiratory nitrate reductase and a nitrate transport system in
response to nitrate and/or nitrite [48]. NarR can
also be activated by azide, which is suggestive of a
metal-based sensing mechanism [49].
(e) NorR
In the denitrifying bacterium R. eutropha, the respiratory NO reductase gene norB (of which there are two
copies, one on the chromosome and one on a megaplasmid) is activated in response to NO by a protein
designated NorR [50]. The NorR protein of E. coli
activates transcription of the norVW genes, which
encode the FlRd and its redox partner [51,52].
NorR-dependent transcription requires RNA polymerase containing the alternative sigma factor, s54,
so NorR belongs to the s54-dependent enhancer-binding protein (EBP) family of transcriptional activators.
NorR has a three-domain structure that is typical of
EBPs, with a C-terminal DNA-binding domain, a central domain from the AAAþ family that has ATPase
activity and interacts with RNA polymerase, and an
N-terminal signalling domain [50]. The N-terminal
GAF domain of NorR contains a mono-nuclear nonhaem iron, which is the binding site for NO. Formation of a mono-nitrosyl complex at this centre
disrupts an intra-molecular interaction, by which the
GAF domain inhibits the activity of the AAAþ
domain in the absence of NO [53,54]. The nonhaem iron is believed to be coordinated by the side
chains of three aspartate residues, an arginine and a
cysteine [55,56].
(f) NarXL
The NarXL proteins are a two-component sensor regulator system that responds to nitrate and/or nitrite.
NarXL and their orthologues NarQP have been well
characterized in E. coli [57]. In the denitrifying bacteria, these proteins are not known to regulate
expression of the genes encoding the enzymes of
N2O production and consumption. However, by regulating the expression of nitrate reductase genes,
NarXL do have a role to play in some organisms in
controlling the rate of flux through the denitrification
pathway. The sensor kinase NarX has two trans-membrane helices (not shown in the SMART prediction in
figure 3) that flank an approximately 100 residue periplasmic domain that contains the ‘P-box’, a region
implicated in nitrate and nitrite binding by genetic
and biochemical evidence [58,59]. In the three-dimensional structure of the periplasmic domain of NarX,
Phil. Trans. R. Soc. B (2012)
nitrate binds to a single site at the monomer–monomer
interface, where it makes contact with residues from
the P-box [28]. Comparison with the structure of the
apo-protein suggests that a piston-like displacement
between the trans-membrane helices might be involved
in coupling ligand binding to the kinase activity of
NarX [28]. This signal is propagated through the
cytoplasmic HAMP domain, and the signalling helix,
which is a C-terminal extension of the HAMP
domain [60].
(g) FixK
Rhizobial FixK proteins are FNR/CRP family members that occupy intermediate positions in the
regulatory hierarchies that control the expression of
genes involved in nitrogen fixation and denitrification.
In B. japonicum, expression of the gene encoding
FixK2 is upregulated under micro-oxic conditions by
FixLJ. Interestingly, this transcriptional regulation of
the fixK2 gene appears not to be reflected in changes
in the abundance of FixK2 protein, a paradox that
remains to be resolved [61]. Nevertheless, the fact
that FixK2 is regulated at the level of its expression
originally led to the view that FixK2 activity is not
itself sensitive to environmental signals. Recently,
however, it has been shown that the activity of the
B. japonicum FixK2 is sensitive to oxidative stress.
Specifically, oxidation of a cysteine residue (either to
a disulphide bridge, or to a sulphinic or sulphonic
acid derivative) reduces the activity of FixK2 and so
causes downregulation of its target genes [61]. This
regulatory mechanism may be peculiar to B. japonicum,
given that the cysteine residue involved is not
conserved in other FixK proteins.
(h) NosR and NirI
NirI and NosR are related proteins that are required
for transcription of the nitrite reductase and N2O
reductase genes, respectively, in some organisms.
Both are polytopic membrane proteins with a periplasmic flavin-containing domain, and two cytoplasmic
iron sulphur clusters (figure 3). The presence of a
covalently bound flavin and two [4Fe– 4S] clusters
has been confirmed biochemically for the NosR
protein of Ps. stutzeri [26]. Both NirI and NosR also
contain cytoplasmic CXXXCP motifs, which may be
metal ion-binding sites, or sites for thiol chemistry.
In Ps. stutzeri, the nosR gene is adjacent to and
upstream of the nosZ structural gene, and the nosD
operon, which encodes proteins involved in Nos maturation, but the three are in separate transcription units.
Transposon insertions in nosR abolished or severely
reduced production of the mono-cistronic nosZ
mRNA [62]. Similarly, transcription of the nosD
operon is abolished in a nosR mutant [63]. Although
nosR is upstream of nosZ and the nosD operon, these
results are probably not due to polarity, since both
nosR and nosZ are mono-cistonic. Thus, NosR behaves
as a factor that is required for the transcription of nosZ
and the nosD operon. The membrane location and
domain organization of NorR, and specifically the
absence of a predicted DNA-binding domain, argue
against a direct role in transcriptional control.
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1220
S. Spiro Review. N2O flux: regulation of gene expression
(i) NsrR
NsrR was originally identified as a negative regulator
of the respiratory nitrite reductase gene in the ammonia oxidizer Nitrosomonas europaea [66], an organism
that also expresses an NOR and so is capable of
N2O production. An orthologue of NsrR is a repressor
of the hmp gene of E. coli that encodes the NO detoxifying flavohaemoglobin [67]. In the denitrifying
pathogenic organisms Moraxella catarrhalis, Neisseria
meningitidis and Neisseria gonorrhoeae, NsrR is a repressor of the norB gene encoding the respiratory NO
reductase [68– 70]. NsrR is a negative regulator that
is inactivated by exposure to NO, the mechanism
probably involving NO-mediated modification of a
protein-bound iron– sulphur cluster [71].
Figure 4a displays a compendium of regulatory
interactions that impinge upon the nor and nos genes
in model denitrifiers (with some exceptions, see
below). It should be stressed that not all of these interactions are present in all denitrifiers, and no one
organism has all of the regulatory interactions shown
in the figure. Rather, the diagram depicts the totality
of interactions that have been described in those
organisms that have been studied. Clearly, the important environmental signals that control expression of
the nor and nos genes are the concentrations of
oxygen and NO, and the key regulatory proteins use
iron-based mechanisms to sense oxygen and NO.
Notably absent is a mechanism for regulating the nos
genes by the abundance of N2O. So, as far as is
known, the denitrifying bacteria do not induce
expression of the nos genes in response to elevated
N2O. Figure 4b shows an equivalent diagram for the
regulators of the genes encoding detoxification activities, that is Hmp and the flavo-diiron protein (FlRd,
in the case of E. coli ). Exceptions to this division
between denitrification and detoxification include
R. eutropha, M. catarrhalis and Neisseria species, in
which the respiratory NO reductase gene norB is regulated by NorR or NsrR [68– 70], rather than by the
regulators that generally regulate denitrification
genes, shown in figure 4a.
Clearly, the flux from nitrate to NO will also influence N2O production, and so it is appropriate to
consider the regulation of all steps of the denitrification
pathway. Figure 5 shows the mechanisms of regulation
of denitrification genes in six model organisms. Again,
it is evident that regulatory interactions can be diverse,
even in closely related organisms. For example, while
NosR is required for expression of the nosZ gene in
Ps. stutzeri, this is not the case in Ps. aeruginosa [64].
In three members of the alpha proteobacteria that
have been studied quite extensively, the mechanisms
of oxygen regulation of denitrification genes are rather
different. In B. japonicum, the haem protein FixL is
the key sensor of oxygen, while in Pa. denitrificans it is
the iron–sulphur protein FnrP, and in R. sphaeroides
oxygen is sensed indirectly by PrrB (figure 5). Thus,
there apparently are not universal mechanisms for the
regulation of denitrification genes, and so extrapolating
from studies done on one model organism to other
species is unlikely to be appropriate. Given the degree
of diversity that there is in the rather small number of
model organisms, it seems likely that novel regulatory
mechanism will be encountered as the set of model
species expands. Further, extrapolating from laboratory
studies done on a small number of model systems to
complex poly-species communities in the field would
seem to be especially problematic.
5. REGULATORY NETWORKS
The wiring diagrams that connect the regulators discussed above to their target genes are surprisingly
variable in different organisms [72,73]. In the context
of factors that directly influence N2O production and
consumption, mechanisms that regulate expression of
the nor and nos genes can be considered in isolation
from the remainder of the denitrification pathway.
6. CONCLUSIONS
The environmental signals that most commonly influence the expression of genes involved in denitrification
are the concentrations of oxygen and NO. Since denitrification is an anaerobic respiration, it makes good
physiological sense for denitrification genes to be upregulated by low oxygen concentrations. NO is an
intermediate of the pathway, and is somewhat toxic.
Moreover, deletion analysis of NosR showed that only
the periplasmic flavin-containing domain is required
for nosZ expression [26]. Thus, the mechanism by
which NosR controls expression of its target genes
remains mysterious, but is likely to be indirect. Interestingly, in Ps. aeruginosa, NosR is not required for
transcription of the nosZ gene [64]. NosR also has a
role to play in the activity of Nos, perhaps by acting
as an electron donor to the enzyme [26]. As is discussed
by Wunsch & Zumft [26], W. succinogenes expresses a
c-type Nos, and has no nosR gene. Interestingly, the
cytoplasmic domain of NosH of W. succinogenes
(figure 2) resembles NosR in that it coordinates two
[4Fe – 4S] clusters and has two CXXXP motifs. Perhaps the cytoplasmic domains of NorR and NosH
play similar roles in N2O reduction.
The NirI protein has been less extensively characterized, but shows some interesting similarities and
differences to NosR. In Pa. denitrificans, nirI is divergently transcribed from nirS, which is the structural
gene encoding nitrite reductase. Mutation of nirI abolished activity of the nirS promoter [65]. Thus, NirI
has a similar structure to NosR and seems also to be
required for the expression of its target gene. Careful
attempts to complement a nirI mutation with nirI
cloned on a plasmid were unsuccessful. Complementation could only be achieved by integrating nirI into the
chromosome, into a position directly upstream of nirS.
The basis for this unusual observation is not understood
[65]. Note that nosR cloned on a plasmid is capable of
complementing a Ps. stutzeri nosR mutant [26,62].
In summary, NosR and NirI are related multidomain membrane proteins, with redox centres in
both the periplasm and cytoplasm. Both behave as regulators of gene expression, although this is unlikely to
be a direct effect requiring DNA binding, particularly
in the case of NosR.
Phil. Trans. R. Soc. B (2012)
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Review. N2O flux: regulation of gene expression S. Spiro 1221
(a)
1
2 3 4
5 6
[4Fe–4S]
[4Fe–4S]
CX3CP CX3CP
NosR
?
nos
low
O2
NO
low
O2
ANR
haem
FixL
FixJ
nor
NnrR
FixK2
NO
low
O2
(b)
NO
norVW
Fe2+
NorR
norB
NO
NsrR
hmp
Figure 4. Regulatory proteins that control expression of the genes encoding enzymes that produce and consume N2O. (a) Summary of regulatory interactions that control expression of the nor (NO reductase) and nos (N2O reductase) genes in model
denitrifying bacteria. The domain organizations of membrane proteins NosR and FixL are shown as the SMART predictions
from figure 3. Filled arrow heads represent the control of protein activity, open arrow heads denote the control of gene
expression (so, for example, FixL activates its partner protein FixJ, which then activates expression of the gene-encoding
FixK2. NosR is required for transcription of the nosZ gene (in Ps. stutzeri but not in Ps. aeruginosa) but this effect is likely
to be indirect. (b) Summary of regulatory interactions that control expression of genes encoding NO detoxification activities
(norVW and hmp), and the norB gene encoding a respiratory NO reductase in R. eutropha, M. catarrhalis and Neisseria spp.
Negative regulatory interactions are denoted by lines with a ‘T’ end.
Regulation of denitrification gene expression by NO is
therefore presumed to be a mechanism to coordinate
NO production and consumption so as to avoid its
accumulation to toxic levels. There is no such requirement to maintain a low N2O concentration in
denitrifiers, and N2O does not appear to be a signal
Phil. Trans. R. Soc. B (2012)
that regulates the expression of any of the denitrification genes. From the point of view of mitigating N2O
releases from denitrification, the absence of regulation
by N2O is a significant observation, since denitrifying
populations do not (as far as is known) respond to
N2O accumulation by making more of the N2O
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1222
S. Spiro Review. N2O flux: regulation of gene expression
Rhodobacter sphaeroides
Paracoccus denitrificans
low
O2
nitrate
NarR
NO
O2
FnrP
narKGHJI
Nirl
nirSECF
NNR
low
O2
NosR
FnrP
norCBQDEF
low
O2
NO
PrrAB
nosZ
napKEFDABC
Bradyrhizobium japonicum
H2O2
NO
nitrate
low
O2
NO
FixLJ
FixK2
NnrR
NarXL
ANR
DNR
napEDAC
nirK
norCBQD
narK1K2GHJI
Pseudomonas stutzeri
NO
NarXL
DNR
narGHJI
nirSTBM
NO
NnrR
nirK
norCBQD
Pseudomonas aeruginosa
low
O2
nitrate
O2
nirSMCFDLGHJEN
nosRZDFYL
Agrobacterium tumifaciens
low
O2
NosR
norCB
norCBD
nosZ
ActRS
NO
FnrN
nirK
NnrR
norCBQD
Figure 5. Regulatory networks controlling expression of denitrification genes in a selection of model organisms. In each case,
the diagram is organized into three layers, these being the regulatory signals, regulatory proteins and the structural genes.
Thus, arrows between the upper and middle layers represent signalling events, while arrows within the middle layer, and
between the middle and lower layers represent gene regulation. Proteins boxed by double lines are two-component systems
(histidine kinase and response regulator). Genes and operons associated with denitrification include those designated nap
and nar (for periplasmic and membrane-bound nitrate reductase, respectively), nir (nitrite reductase), nor (NO reductase)
and nos (N2O reductase). The nos genes are not shown for B. japonicum since their regulation is not understood, while
these genes are absent from Agrobacterium tumifaciens. The ability to express N2O reductase in Rhodobacter strains is variable,
and nos gene expression has not been studied in this genus.
reductase. Nevertheless, understanding those signals
that do influence the expression of denitrification
genes, and those that influence the abundance and
activities of the corresponding enzymes, is very likely
to be helpful to any effort to intervene in the activities
of the denitrifying bacteria.
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