, published 26 March 2012, doi: 10.1098/rstb.2011.0309367 2012 Phil. Trans. R. Soc. B Stephen Spiro expression by gas-sensitive transcription factorsNitrous oxide production and consumption: regulation of gene  

Supplementary data

ml http://rstb.royalsocietypublishing.org/content/suppl/2012/03/29/rstb.2011.0309.DC1.ht

"Audio supplement"

References

http://rstb.royalsocietypublishing.org/content/367/1593/1213.full.html#related-urls Article cited in:

 http://rstb.royalsocietypublishing.org/content/367/1593/1213.full.html#ref-list-1

This article cites 71 articles, 34 of which can be accessed free

Subject collections

(150 articles)molecular biology   � (49 articles)microbiology   � (125 articles)biochemistry   �

 Articles on similar topics can be found in the following collections

Email alerting service hereright-hand corner of the article or click Receive free email alerts when new articles cite this article - sign up in the box at the top

http://rstb.royalsocietypublishing.org/subscriptions go to: Phil. Trans. R. Soc. BTo subscribe to

on September 17, 2013rstb.royalsocietypublishing.orgDownloaded from

on September 17, 2013rstb.royalsocietypublishing.orgDownloaded from

Phil. Trans. R. Soc. B (2012) 367, 1213–1225

doi:10.1098/rstb.2011.0309

Review

*stephen

One conoxide: th

Nitrous oxide production and consumption:regulation of gene expression by gas-

sensitive transcription factorsStephen 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 fromthe flavo-diiron family, and flavohaemoglobin. On the other hand, there is only one enzyme thatis known to use N2O as a substrate, which is the respiratory N2O reductase typically found in bac-teria capable of denitrification (the respiratory reduction of nitrate and nitrite to dinitrogen). Thisarticle will briefly review the properties of the enzymes that make and consume N2O, together withthe accessory proteins that have roles in the assembly and maturation of those enzymes. Theexpression of the genes encoding the enzymes that produce and consume N2O is regulated byenvironmental signals (typically oxygen and NO) acting through regulatory proteins, which,either directly or indirectly, control the frequency of transcription initiation. The roles and mechan-isms of these proteins, and the structures of the regulatory networks in which they participate willalso be reviewed.

Keywords: denitrification; nitrous oxide; nitric oxide; nitrous oxide reductase;nitric oxide reductase

1. INTRODUCTIONNitrous oxide (N2O) is a water-soluble gas that attractscurrent interest because of its contribution to theatmospheric greenhouse effect. N2O has well knownand useful anaesthetic and analgesic properties, andhas 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 formetal centres in proteins. Thus, N2O is not toxic,and micro-organisms can tolerate relatively high (milli-molar) concentrations. The reduction potential of theN2O/N2 couple is high (Eo0 ¼ þ1.35 V at pH 7), andsome bacteria exploit this property by using N2O asthe terminal electron acceptor in energy conservingrespiratory metabolism.

N2O is an intermediate (or, in some cases, end-product) of the respiratory pathway denitrification,so denitrification is a major contributor to globalN2O emissions [1]. The ammonia-oxidizing bacteriaare another significant source of N2O, although thosethat have been studied are also capable of partial deni-trification (the respiratory reduction of nitrite to N2O),so the enzymes responsible for N2O production aresimilar to those of the denitrifying bacteria [2]. Therespiratory reduction of nitrate to nitrite and ammonia(sometimes called respiratory ammonification) can

.spiro@utdallas.edu

tribution of 12 to a Theo Murphy Meeting Issue ‘Nitrouse forgotten greenhouse gas’.

1213

also be a source of N2O, since some nitric oxide(NO) is made as a by-product of this pathway and issubsequently reduced to N2O. In contrast to the mul-tiplicity of mechanisms by which N2O can begenerated, only a single sink for N2O is known,which is the respiratory N2O reductase typicallyfound in denitrifying bacteria.

The factors that contribute to the emission ofN2O from bacterial populations are numerous andcomplex, clearly one important determinant beingthe cellular abundance and activities of the enzymesthat produce and consume N2O. A key contributorto enzyme abundance is the regulation of expressionof the corresponding genes by regulatory systems andsignal transduction pathways that respond to intra-or extra-cellular signals. The goal of this review is topresent a brief overview of the enzymatic mechanismsof N2O production and consumption, and then tofocus on the regulatory mechanisms that control theexpression of the genes that encode these enzymes.The structures of the regulatory networks that inte-grate environmental signals and connect regulatoryproteins with their target genes will also be reviewed.There is a surprising degree of diversity in the organiz-ation of regulatory networks, even among organismsthat are phylogenetically quite closely related. Thisdiversity presents a challenge when it comes to extra-polating from studies done on one organism toanother, and from pure culture experiments to studiesof complex microbial populations.

This journal is q 2012 The Royal Society

FeMQ MQ

Fe

FeFe FMN

NorV NorW FprA FprA NRO(ROO) RdHrb

FADRd FeFe FMN FMNRd Fe

Fe FMN FADRd

NorB

cNOR

(a)

(b)

Pseudomonas aeruginosa

Escherichia coli Moorella thermoacetica Desulfovibrio sp.

Ralstonia eutropha Bacillus azotoformans

qNOR qCuANOR

NorBNorC

Fe Fe Fe FeFe

FeB

c551

FeB FeB

CuAP

N

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. TheqNOR is a single-subunit enzyme that accepts electrons from the quinone pool. The qCuANOR accepts electrons either from amembrane-associated c-type cytochrome (not shown) or from menaquinol. (b) The soluble enzymes have roles in NO detox-ification. NO is reduced by a flavo-diiron protein (NorV, FprA or ROO), which accepts electrons from an NADH-dependentflavo-enzyme: NorW, high molecular weight rubredoxin (Hrb) or NADH : rubredoxin oxidoreductase (NRO). P and N denote

periplasmic and cytoplasmic compartments, respectively.

1214 S. Spiro Review. N2O flux: regulation of gene expression

on September 17, 2013rstb.royalsocietypublishing.orgDownloaded from

2. ENZYMES OF NITROUS OXIDE SYNTHESIS(a) Respiratory nitric oxide reductase

The major contributor to the biological production ofN2O is almost certainly the respiratory NO reductase(NOR) found in denitrifying bacteria and in someammonia-oxidizing organisms. Three types of NORhave been described [3], as will be discussed below.All three catalyse the two-electron reduction of NOto N2O:

2NOþ 2Hþ þ 2e� ! N2OþH2O

with the electrons derived from a small c-type cyto-chrome, the copper protein pseudoazurin or from thequinone pool. In each case, the catalytic subunit con-tains a binuclear centre comprising a high-spin haemb and a non-haem iron (FeB). A low spin haem b par-ticipates in electron transfer to the binuclear centre,which is the site of NO reduction. The three types ofNOR differ in their subunit architectures and in theentry routes for electrons (figure 1a).

In the c-type NOR (also called scNOR, for shortchain), the catalytic NorB subunt is in a complexwith NorC, a small c-type cytochrome with a singlemembrane-spanning helix (figure 1a). Electronsfrom a periplasmic c-type cytochrome or from pseu-doazurin pass through the haem of NorC, to the lowspin haem of NorB, and thence to the binuclearcentre. The best-characterized cNORs are thosefrom Paracoccus denitrificans, Pseudomonas stutzeri andPseudomonas aeruginosa. The structure of the NorBCcomplex from Ps. aeruginosa [4] confirmed the

Phil. Trans. R. Soc. B (2012)

predicted presence of 12 membrane-spanning a-helices in NorB. Biochemical experiments indicatedthat the protons required for NO reduction are takenfrom the periplasmic side of the membrane [5], andthat NorB does not function as a proton pump [6].The latter is confirmed in the structure by the absenceof trans-membrane proton channels in NorB analo-gous to those found in the proton-translocatinghaem-copper oxidases, which are otherwise structu-rally related to NorB [4].

The norCB genes are usually co-transcribed withaccessory genes designated norD, norE, norF andnorQ; the gene order norEFCBQD is typical thoughnot universal [3]. The norQ and norD genes arealways linked to norCB; the other accessory genesmay be distantly located or absent altogether in somegenomes [3]. The functions of the accessory genesand their protein products are not well understood.NorE is a predicted membrane protein with somesequence similarity to part of subunit 3 of the cyto-chrome c oxidase. This led to speculation that NorEmight be a component of the NOR complex [7],although purified and active preparations of thecNOR only contain NorB and NorC. Mutation ofthe accessory genes tends to lead to variable pheno-types in different organisms [3], but in no case is thebiochemical function of the accessory proteins under-stood; this is an area worthy of further investigation.

The q-type NOR is a single-subunit enzyme that issimilar to but larger than the B subunit of the c-typeNOR, hence this enzyme has also been called thelong chain NOR. The single subunit of the qNOR

Review. N2O flux: regulation of gene expression S. Spiro 1215

on September 17, 2013rstb.royalsocietypublishing.orgDownloaded from

has two additional trans-membrane helices (comparedwith NorB) that flank a periplasmic domain of approxi-mately 200 amino acids (figure 1a). The qNORreceives electrons from the quinone pool, and theadditional N-terminal domain is the presumed locationof the quinol oxidase activity [8]. A qNOR has beencharacterized in the hyperthermophilic Archaea Pyroba-culum aerophilum [9]. The archaeal enzyme is similar tothe bacterial qNORs, with the exception of covalentmodifications to its haem groups. Interestingly, thegenes encoding qNORs are not typically co-expressedwith accessory genes, implying that the accessoryproteins have functions that are specific to the activityand/or assembly of the cNORs. In Ralstonia eutropha,the norB gene that encodes the qNOR is transcribedwith a gene designated norA (also called ytfE, dnrNand scdA in other organisms). The NorA/YtfE proteincontains a diiron centre, and has been implicated inthe repair of damaged iron–sulphur clusters [10] and/or in the buffering of cytoplasmic NO [11].

An unusual hybrid NOR has been described in theGram-positive organism Bacillus azotoformans [12,13].This is a two subunit enzyme that functions as a mena-quinol : NO oxidoreductase, but can also acceptelectrons from small membrane-bound c-type cyto-chromes [13]. The B. azotoformans NOR contains aCuA centre that is similar to the CuA of cytochromeoxidases, and is proposed to be bound to the smallsubunit [12]. Hence, there appear to be two routesof electron entry into this enzyme (designated qCuA-

NOR), either from the quinone pool, or from smallc-type cytochromes (figure 1a).

(b) Flavo-diiron proteins

In several non-denitrifying bacteria, an enzyme fromthe flavo-diiron family reduces NO to N2O. The phys-iological role of this enzyme seems to be NOdetoxification, a reaction which may be particularlyimportant in organisms (such as Escherichia coli)which make low concentrations of NO as a by-productof the respiratory reduction of nitrite to ammonia.Although N2O is (probably) the end-product of thispathway, the contribution that it makes to the globalN2O budget is likely to be very small. In this enzymesystem, NO is reduced by a flavo-diiron protein,which receives electrons from a rubredoxin domainor protein. The rubredoxin is itself reduced by anNADH-dependent flavoenzyme (figure 1b). Theflavo-diiron protein of E. coli has a fused rubredoxindomain, and so is called flavorubredoxin, FlRd (alsocalled NorV). In complex with the NADH-dependentFlRd oxidoreductase (NorW), this enzyme functionsas an NO reductase in vitro [14] and in vivo [15]. InMoorella thermoacetica, a similar complex (figure 1b)functions as an NO reductase, though in this casethe flavo-diiron protein (FprA) is reduced by anNADH-dependent high molecular weight rubredoxin,Hrb [16]. In Desulfovibrio gigas, an equivalentenzyme complex was initially characterized as an oxi-dase, comprising the flavo-diiron protein (ROO, forrubredoxin oxygen oxido-reductase), a standalonerubredoxin and an NADH-rubredoxin oxidoreduc-tase. 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 func-tions as an NO reductase in vivo [17]. Similarly, theD. vulgaris FprA functions as an NO reductase bothin vitro and in vivo [18]. The oxidase activities associ-ated with these flavo-diiron proteins are probablynot physiologically relevant, since oxygen turnoverirreversibly inactivates the enzyme [16,18]. Thus, aconsensus has emerged in which the physiologicalrole of these enzymes is to scavenge low concentrationsof NO in cells growing under micro-oxic conditions.

(c) Flavohaemoglobin

The flavohaemoglobin Hmp is another NO detoxifica-tion enzyme, which is phylogenetically widespread,being found in denitrifying bacteria (such as R. eutropha)and non-denitrifiers, including E. coli. Hmp has a globinlike domain, and an FAD-containing domain that bindsNAD(P)H [19]. In the presence of oxygen, Hmp oxi-dizes NO to nitrate, an activity that has been describedas an NO dioxygenase or NO denitrosylase. In theabsence of oxygen, Hmp reduces NO to N2O [20].However, the consumption of NO by Hmp underanaerobic conditions is rather slow [21], so Hmp maynot 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, N2Oconsumption appears to be exclusively due to the res-piratory N2O reductase Nos (also called NosZ).Genetic, biochemical and molecular aspects of N2Oreduction have been reviewed comprehensively [22]and will be summarized only briefly here. Nos enzymesare soluble periplasmic copper proteins that catalyse thetwo-electron reduction of N2O to dinitrogen:

N2Oþ 2Hþ þ 2e� ! N2 þH2O:

The electrons required for N2O reduction originate inthe quinone pool and are transferred to Nos eithervia the cytochrome bc1 complex and small solubleperiplasmic proteins, or via Nos-specific membrane-associated electron transfer proteins (figure 2).

The well-characterized Nos (referred to as the Z-typeNos) is a homodimeric protein in which each monomercontains two copper centres designated CuA and CuZ.Three-dimensional structures are available for theenzymes from Marinobacter hydrocarbonclasticus (Pseudo-monas nautica) and Pa. denitrificans [24,25]. The electrontransfer route to this enzyme is typically depicted asinvolving the cytochrome bc1 complex and small solubleperiplasmic electron carriers, such as cytochrome c550

and pseudoazurin [1]. However, there are observationsthat are consistent with Nos-specific proteins participat-ing in electron transfer to the Z-type Nos. Specifically,NosR is a membrane-bound iron–sulphur flavoprotein,which has been suggested to mediate electron transferbetween the quinone pool and Nos [26].

The Z-type Nos is encoded by a gene called nosZthat is typically linked to other nos genes, whose pro-ducts have roles in the maturation of the activeenzyme. The functions of the accessory Nos proteins

N2O + 2H+ N2O + 2H+

NosZ (Z-type)

NosZ (c-type)

NosG2 [4Fe–4S]

2Fe–2S

2 [4Fe–4S]

NosC1/C2CuZ

CuZ

Cuc550

c

c

CuA

2UQH2

P

N

2UQ

UQH2

cytochrome bc1complex

UQbH

bL

Paracoccusdenitrificans

Wolinellasuccinogenes

c1

MKH2

MK

NosH

CuA

N2 + H2O N2 + H2O

Figure 2. Electron transfer pathways to the Z-type and c-type nitrous oxide reductase, exemplified by those of Pa. denitrificansand 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 periplas-mic or attached to the membrane, and may or may not be on the electron transfer route from NosGH to NosZ [23]. P and Ndenote periplasmic and cytoplasmic compartments, respectively.

1216 S. Spiro Review. N2O flux: regulation of gene expression

on September 17, 2013rstb.royalsocietypublishing.orgDownloaded from

are not well understood, but they include an ABCtransporter (NosFYD) that may export a sulphurcompound to the periplasm, an outer membranecopper porin (NosA), an outer membrane anchoredcopper protein (NosL), a periplasmic flavoprotein(NosX) and NosR [22]. The catalytic Nos subunit isexported as an apo protein to the periplasm via theTat transport system, which is unusual, since Tat typi-cally secretes fully assembled and folded proteins.Thus, the copper centres are inserted into Nos in theperiplasm, and it seems likely that some or all of theperiplasmic accessory Nos proteins have roles inassembly of the copper clusters. As is also the casefor the c-type NOR, the maturation of Nos is anarea that demands further investigation.

An unusual variant of Nos is found in Wolinellasuccinogenes and some of its relatives. This enzyme(called the c-type Nos) is characterized by an additionalC-terminal mono-haem cytochrome c domain. Exportto the periplasm is by the Sec secretory system ratherthan the Tat pathway used by the Z-type Nos. Electrontransfer to the c-type Nos involves membrane-associated iron–sulphur proteins (NosG and NosH)which mediate electron transfer between the quinonepool and NosZ, perhaps via small soluble periplasmicc-type cytochromes [23]. The physiological role of theNos in W. succinogenes is not certain, given that the

Phil. Trans. R. Soc. B (2012)

organism reduces nitrite to ammonia. One possibilityis that the enzyme acts on the N2O produced bydetoxifying activities that remove the NO made as aby-product of nitrite respiration [23].

4. REGULATORY PROTEINSThe genes and operons encoding the enzymes of N2Oproduction and consumption are controlled in differentorganisms by a diverse array of transcriptional regula-tors (figure 3). The signals to which these regulatoryproteins respond are also diverse, and include oxygen,NO, nitrate and the activity of the electron transportchain. In most species that have been studied, multipleenvironmental signals are integrated by regulatorypathways to ensure that the NO and N2O reductasesare expressed optimally according to the prevailingneeds of the organism. In the following sections, theproperties of the various regulatory proteins will firstbe reviewed briefly, with a focus on mechanisticaspects. Then, the signalling networks that connectregulatory proteins with their target genes in selectedmodel organisms are described. The intention is topresent only a thumbnail of each regulatory system.Thus, references to the literature are not exhaustive,with a focus on reviews, where available, and recentdevelopments. Regulators of genes of the entire

haem

[4Fe–4S]

GGXXNPF

Cys265

NorR

NarL

NarR

FixK

NarX

FNR/FnrP/ANR/FnrN

NNR/NnrR/DNR

haem?

P-Box

2 3 4 5 6

21 3 4 5 6

[4Fe–4S]

[4Fe–4S]

[4Fe–4S]

[4Fe–4S]

CX3CP CX3CP

CX3CP CX3CP

NirI

NosR

Fe2+

RegB/PrrB/ActS

RegB/PrrB/ActS

1 2 3/4 5 6

FixL

FixJ

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 modulararchitecture research tool, SMART [27]. The proteins shown are those discussed in the text, with the exception of NsrR, forwhich SMART predicts no conserved domains. Known or suspected cofactors are indicated, along with conserved sequencemotifs that are discussed in the text. The P-box in NarL is in a periplasmic loop (although SMART does not predict trans-membrane 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 betweenhelices 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 histidinekinases; HATPase_c, histidine kinase-like ATPases; REC, cheY-homologous receiver domain; HTH LuxR, helix-turn-helixdomain 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 phos-phodiesterases; 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.

Review. N2O flux: regulation of gene expression S. Spiro 1217

Phil. Trans. R. Soc. B (2012)

on September 17, 2013rstb.royalsocietypublishing.orgDownloaded from

1218 S. Spiro Review. N2O flux: regulation of gene expression

on September 17, 2013rstb.royalsocietypublishing.orgDownloaded from

denitrification pathway are considered, since the fluxthrough the early reactions of the pathway will be acontributor to the emission of N2O.

(a) FixLJ

FixL is a multi-domain sensory histidine kinase which, inits active state, phosphorylates a conserved aspartic acidresidue in the receiver domain of its cognate responseregulator FixJ (figure 3). Sensory input into this systemis via the PAS (Per-Arnt-Sim) domain of FixL, whichcoordinates a single molecule of haem. There is somestructural diversity in FixL proteins from differentorganisms. For example, the Bradyrhizobium japonicumFixL is soluble and cytoplasmic, while the Sinorhizobiummeliloti protein is membrane-associated, such that itsPAS domain is anchored on the cytoplasmic face of themembrane (figure 3). In all cases that have been studied,FixL is in its ‘on’ state in the absence of oxygen, in whichit auto-phosphorylates prior to phospho-transfer to FixJ.Phosphorylated FixJ is active for DNA binding and regu-lation of the transcription of target genes. Molecularoxygen binds to FixL (with an affinity in the range 50–150 mM), converting the ferrous iron of the haem fromhigh spin to low spin. The consequent movement ofthe iron in the plane of the porphyrin ring triggers aseries of conformational changes that inhibit the kinaseactivity of FixL. There is a great deal of structural andbiochemical information for FixL proteins, so the mech-anism by which oxygen regulates the kinase activity isquite well understood [31–33]. FixL can bind otherhaem ligands (such as CO and NO) but these do notinhibit the kinase activity, and so their interaction withFixL is probably not physiologically significant.

(b) FNR

FNR from E. coli is a prototypical member of the FNR/CRP superfamily of transcriptional activators. Fourconserved cysteines of FNR coordinate a [4Fe–4S]2þ

cluster, which is converted to a [2Fe–2S]2þ cluster onexposure to oxygen. This transition is accompanied bya reduced tendency of FNR to dimerize, and so areduced affinity for its DNA targets. Details of themechanism of the reaction of the cluster with oxygen(which may proceed via a [3Fe–4S]1þ intermediate)are beginning to emerge [32,34]. Prolonged reactionwith oxygen results in a cluster-free apo-protein,which is the form isolated from aerobically growncells. Orthologues of FNR from other organisms(such as FnrP, ANR and FnrN) are presumed towork in similar ways, although the limited informationthat is available suggests that the oxygen sensitivityof the cluster can be fine-tuned by the proteinenvironment in different FNR relatives [35,36].

Iron–sulphur proteins are potential targets for NO,and the reaction of FNR proteins with NO has beendocumented for proteins from several sources[35,37–39]. The product of this reaction has notbeen well characterized, but may include dinitrosyliron complexes [37], and/or, perhaps, a [2Fe–2S]cluster [38]. However, FNR proteins do not providethe dominant mechanism for NO sensing, and sothe reaction of FNR with NO may serve only to

Phil. Trans. R. Soc. B (2012)

fine-tune the expression of genes encoding theenzymes of denitrification (see below).

(c) RegBA/PrrBA

RegB and its orthologues are membrane-associatedhistidine kinases that phosphorylate their cognateresponse regulators (figure 3). The RegBA systemwas first described in the photosynthetic bacteriumRhodobacter capsulatus, and its close relative Rhodobac-ter sphaeroides (in which it is called PrrBA). RegBA/PrrBA two component systems have been well charac-terized in these organisms, and other members of thealpha proteobacteria, where they are typically involvedin regulating energy generating or consuming pro-cesses, including denitrification [29]. RegB/PrrBfunctions as a redox sensor, such that it is active as akinase under conditions that lead to a relativelyreduced state of the cell. Thus, in the facultativelyanaerobic Rhodobacter species, RegB/PrrB behaves asif it is inactivated by oxygen, though this is not adirect effect. Instead, the RegB/PrrB proteins appearto measure the ‘redox state’ of the cell by multiplemechanisms. In one mechanism, metal-dependentoxidation of a key cysteine residue (Cys-265 in theR. capsulatus RegB, figure 3) causes a dimer to tetra-mer transition, and inactivation of the kinase [40].Purified RegB also contains bound ubiquinone, andoxidation of the quinone inhibits kinase activity [30].Quinone binding requires a conserved GGXXNPFmotif that is located in a short periplasmic loop(figure 3, [40]). This mechanism presumably allowsRegB to monitor the redox state of the electron trans-port chain. The PrrB protein of R. sphaeroides appearsto be controlled by different mechanisms. In this case,it has been suggested that the cytochrome cbb3 oxidaseserves as a redox sensor for PrrB, in a manner thatdoes not require oxidase activity [41]. Signal transferis independent of the quinone pool, implying a moredirect interaction between the cbb3 oxidase and PrrB.Furthermore, while PrrB kinase activity is inhibitedby ubiquinone, this effect does not require theGGXXNPF motif or the membrane domain [41].Thus, the current evidence points to a multitude ofmechanisms in the RegB/PrrB sensor kinases.

(d) NNR/NnrR/DNR and NarR

The FNR/CRP superfamily includes several proteinsthat regulate the expression of genes encoding the res-piratory NO reductase. These proteins have beenvariously designated NNR/NnrR and DNR, and evi-dence from in vivo experiments indicates that theseproteins activate transcription in response to NO. Infact, the NNR/NnrR/DNR proteins do not form asingle coherent group, rather they fall into two of thephylogenetically distinct branches of the wider FNR/CRP superfamily [42]. The mechanism(s) of NOsensing by these proteins has proved difficult to estab-lish. NNR from Pa. denitrificans and DNR fromPs. aeruginosa require haem for their NO-dependentactivity in heterologous reporter systems in E. coli[43,44]. The structure of the sensory domain ofDNR reveals a hydrophobic pocket that might be ahaem-binding site, and purified apo-DNR can bind

Review. N2O flux: regulation of gene expression S. Spiro 1219

on September 17, 2013rstb.royalsocietypublishing.orgDownloaded from

one equivalent of haem [45]. The haem-reconstitutedDNR binds NO and CO, and also shows some DNA-binding activity that is neither stimulated nor inhibitedby NO [45]. The current model proposes that full acti-vation of DNR requires haem and NO, though thecomplete details of this mechanism remain to be estab-lished [46]. Results from in vivo experiments suggestthat NNR/NnrR/DNA activity is sensitive to oxygen[43,47]; this would be consistent with the physiologi-cal roles of these proteins and with a haem-basedsensing mechanism.

NarR is another FNR/CRP family member, whichin Pa. denitrificans regulates expression of the respirat-ory nitrate reductase and a nitrate transport system inresponse to nitrate and/or nitrite [48]. NarR canalso be activated by azide, which is suggestive of ametal-based sensing mechanism [49].

(e) NorR

In the denitrifying bacterium R. eutropha, the respirat-ory NO reductase gene norB (of which there are twocopies, one on the chromosome and one on a mega-plasmid) is activated in response to NO by a proteindesignated NorR [50]. The NorR protein of E. coliactivates transcription of the norVW genes, whichencode the FlRd and its redox partner [51,52].NorR-dependent transcription requires RNA poly-merase containing the alternative sigma factor, s54,so NorR belongs to the s54-dependent enhancer-bind-ing protein (EBP) family of transcriptional activators.NorR has a three-domain structure that is typical ofEBPs, with a C-terminal DNA-binding domain, a cen-tral domain from the AAAþ family that has ATPaseactivity and interacts with RNA polymerase, and anN-terminal signalling domain [50]. The N-terminalGAF domain of NorR contains a mono-nuclear non-haem iron, which is the binding site for NO. For-mation of a mono-nitrosyl complex at this centredisrupts an intra-molecular interaction, by which theGAF domain inhibits the activity of the AAAþ

domain in the absence of NO [53,54]. The non-haem iron is believed to be coordinated by the sidechains of three aspartate residues, an arginine and acysteine [55,56].

(f) NarXL

The NarXL proteins are a two-component sensor reg-ulator system that responds to nitrate and/or nitrite.NarXL and their orthologues NarQP have been wellcharacterized in E. coli [57]. In the denitrifying bac-teria, these proteins are not known to regulateexpression of the genes encoding the enzymes ofN2O production and consumption. However, by regu-lating the expression of nitrate reductase genes,NarXL do have a role to play in some organisms incontrolling the rate of flux through the denitrificationpathway. The sensor kinase NarX has two trans-mem-brane helices (not shown in the SMART prediction infigure 3) that flank an approximately 100 residue peri-plasmic domain that contains the ‘P-box’, a regionimplicated in nitrate and nitrite binding by geneticand biochemical evidence [58,59]. In the three-dimen-sional structure of the periplasmic domain of NarX,

Phil. Trans. R. Soc. B (2012)

nitrate binds to a single site at the monomer–monomerinterface, where it makes contact with residues fromthe P-box [28]. Comparison with the structure of theapo-protein suggests that a piston-like displacementbetween the trans-membrane helices might be involvedin coupling ligand binding to the kinase activity ofNarX [28]. This signal is propagated through thecytoplasmic HAMP domain, and the signalling helix,which is a C-terminal extension of the HAMPdomain [60].

(g) FixK

Rhizobial FixK proteins are FNR/CRP family mem-bers that occupy intermediate positions in theregulatory hierarchies that control the expression ofgenes involved in nitrogen fixation and denitrification.In B. japonicum, expression of the gene encodingFixK2 is upregulated under micro-oxic conditions byFixLJ. Interestingly, this transcriptional regulation ofthe fixK2 gene appears not to be reflected in changesin the abundance of FixK2 protein, a paradox thatremains to be resolved [61]. Nevertheless, the factthat FixK2 is regulated at the level of its expressionoriginally led to the view that FixK2 activity is notitself sensitive to environmental signals. Recently,however, it has been shown that the activity of theB. japonicum FixK2 is sensitive to oxidative stress.Specifically, oxidation of a cysteine residue (either toa disulphide bridge, or to a sulphinic or sulphonicacid derivative) reduces the activity of FixK2 and socauses downregulation of its target genes [61]. Thisregulatory mechanism may be peculiar to B. japonicum,given that the cysteine residue involved is notconserved in other FixK proteins.

(h) NosR and NirI

NirI and NosR are related proteins that are requiredfor transcription of the nitrite reductase and N2Oreductase genes, respectively, in some organisms.Both are polytopic membrane proteins with a periplas-mic flavin-containing domain, and two cytoplasmiciron sulphur clusters (figure 3). The presence of acovalently bound flavin and two [4Fe–4S] clustershas been confirmed biochemically for the NosRprotein of Ps. stutzeri [26]. Both NirI and NosR alsocontain cytoplasmic CXXXCP motifs, which may bemetal ion-binding sites, or sites for thiol chemistry.

In Ps. stutzeri, the nosR gene is adjacent to andupstream of the nosZ structural gene, and the nosDoperon, which encodes proteins involved in Nos matu-ration, but the three are in separate transcription units.Transposon insertions in nosR abolished or severelyreduced production of the mono-cistronic nosZmRNA [62]. Similarly, transcription of the nosDoperon is abolished in a nosR mutant [63]. AlthoughnosR is upstream of nosZ and the nosD operon, theseresults are probably not due to polarity, since bothnosR and nosZ are mono-cistonic. Thus, NosR behavesas a factor that is required for the transcription of nosZand the nosD operon. The membrane location anddomain organization of NorR, and specifically theabsence of a predicted DNA-binding domain, argueagainst a direct role in transcriptional control.

1220 S. Spiro Review. N2O flux: regulation of gene expression

on September 17, 2013rstb.royalsocietypublishing.orgDownloaded from

Moreover, deletion analysis of NosR showed that onlythe periplasmic flavin-containing domain is requiredfor nosZ expression [26]. Thus, the mechanism bywhich NosR controls expression of its target genesremains mysterious, but is likely to be indirect. Interest-ingly, in Ps. aeruginosa, NosR is not required fortranscription of the nosZ gene [64]. NosR also has arole to play in the activity of Nos, perhaps by actingas an electron donor to the enzyme [26]. As is discussedby Wunsch & Zumft [26], W. succinogenes expresses ac-type Nos, and has no nosR gene. Interestingly, thecytoplasmic domain of NosH of W. succinogenes(figure 2) resembles NosR in that it coordinates two[4Fe–4S] clusters and has two CXXXP motifs. Per-haps the cytoplasmic domains of NorR and NosHplay similar roles in N2O reduction.

The NirI protein has been less extensively character-ized, but shows some interesting similarities anddifferences to NosR. In Pa. denitrificans, nirI is diver-gently transcribed from nirS, which is the structuralgene encoding nitrite reductase. Mutation of nirI abol-ished activity of the nirS promoter [65]. Thus, NirIhas a similar structure to NosR and seems also to berequired for the expression of its target gene. Carefulattempts to complement a nirI mutation with nirIcloned on a plasmid were unsuccessful. Complementa-tion could only be achieved by integrating nirI into thechromosome, 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 ofcomplementing a Ps. stutzeri nosR mutant [26,62].

In summary, NosR and NirI are related multi-domain membrane proteins, with redox centres inboth the periplasm and cytoplasm. Both behave as reg-ulators of gene expression, although this is unlikely tobe a direct effect requiring DNA binding, particularlyin the case of NosR.

(i) NsrR

NsrR was originally identified as a negative regulatorof the respiratory nitrite reductase gene in the ammo-nia oxidizer Nitrosomonas europaea [66], an organismthat also expresses an NOR and so is capable ofN2O production. An orthologue of NsrR is a repressorof the hmp gene of E. coli that encodes the NO detox-ifying flavohaemoglobin [67]. In the denitrifyingpathogenic organisms Moraxella catarrhalis, Neisseriameningitidis and Neisseria gonorrhoeae, NsrR is a repres-sor of the norB gene encoding the respiratory NOreductase [68–70]. NsrR is a negative regulator thatis inactivated by exposure to NO, the mechanismprobably involving NO-mediated modification of aprotein-bound iron–sulphur cluster [71].

5. REGULATORY NETWORKSThe wiring diagrams that connect the regulators dis-cussed above to their target genes are surprisinglyvariable in different organisms [72,73]. In the contextof factors that directly influence N2O production andconsumption, mechanisms that regulate expression ofthe nor and nos genes can be considered in isolationfrom the remainder of the denitrification pathway.

Phil. Trans. R. Soc. B (2012)

Figure 4a displays a compendium of regulatoryinteractions that impinge upon the nor and nos genesin model denitrifiers (with some exceptions, seebelow). It should be stressed that not all of these inter-actions are present in all denitrifiers, and no oneorganism has all of the regulatory interactions shownin the figure. Rather, the diagram depicts the totalityof interactions that have been described in thoseorganisms that have been studied. Clearly, the impor-tant environmental signals that control expression ofthe nor and nos genes are the concentrations ofoxygen and NO, and the key regulatory proteins useiron-based mechanisms to sense oxygen and NO.Notably absent is a mechanism for regulating the nosgenes by the abundance of N2O. So, as far as isknown, the denitrifying bacteria do not induceexpression of the nos genes in response to elevatedN2O. Figure 4b shows an equivalent diagram for theregulators of the genes encoding detoxification activi-ties, that is Hmp and the flavo-diiron protein (FlRd,in the case of E. coli). Exceptions to this divisionbetween denitrification and detoxification includeR. eutropha, M. catarrhalis and Neisseria species, inwhich the respiratory NO reductase gene norB is regu-lated by NorR or NsrR [68–70], rather than by theregulators that generally regulate denitrificationgenes, shown in figure 4a.

Clearly, the flux from nitrate to NO will also influ-ence N2O production, and so it is appropriate toconsider the regulation of all steps of the denitrificationpathway. Figure 5 shows the mechanisms of regulationof denitrification genes in six model organisms. Again,it is evident that regulatory interactions can be diverse,even in closely related organisms. For example, whileNosR is required for expression of the nosZ gene inPs. stutzeri, this is not the case in Ps. aeruginosa [64].In three members of the alpha proteobacteria thathave been studied quite extensively, the mechanismsof oxygen regulation of denitrification genes are ratherdifferent. In B. japonicum, the haem protein FixL isthe key sensor of oxygen, while in Pa. denitrificans it isthe iron–sulphur protein FnrP, and in R. sphaeroidesoxygen is sensed indirectly by PrrB (figure 5). Thus,there apparently are not universal mechanisms for theregulation of denitrification genes, and so extrapolatingfrom studies done on one model organism to otherspecies is unlikely to be appropriate. Given the degreeof diversity that there is in the rather small number ofmodel organisms, it seems likely that novel regulatorymechanism will be encountered as the set of modelspecies expands. Further, extrapolating from laboratorystudies done on a small number of model systems tocomplex poly-species communities in the field wouldseem to be especially problematic.

6. CONCLUSIONSThe environmental signals that most commonly influ-ence the expression of genes involved in denitrificationare the concentrations of oxygen and NO. Since deni-trification is an anaerobic respiration, it makes goodphysiological sense for denitrification genes to be upre-gulated by low oxygen concentrations. NO is anintermediate of the pathway, and is somewhat toxic.

1 2

(a)

(b)

3 4 5 6[4Fe–4S] NosR

?[4Fe–4S]

Fe2+

CX3CP

lowO2

lowO2

lowO2

NO

NO

ANR

FixL

FixJ FixK2 NnrR

haem

nos

nor

norVW

norB

hmpNsrRNO

NO

NorR

CX3CP

Figure 4. Regulatory proteins that control expression of the genes encoding enzymes that produce and consume N2O. (a) Sum-mary of regulatory interactions that control expression of the nor (NO reductase) and nos (N2O reductase) genes in modeldenitrifying bacteria. The domain organizations of membrane proteins NosR and FixL are shown as the SMART predictionsfrom 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-encodingFixK2. NosR is required for transcription of the nosZ gene (in Ps. stutzeri but not in Ps. aeruginosa) but this effect is likelyto 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.

Review. N2O flux: regulation of gene expression S. Spiro 1221

on September 17, 2013rstb.royalsocietypublishing.orgDownloaded from

Regulation of denitrification gene expression by NO istherefore presumed to be a mechanism to coordinateNO production and consumption so as to avoid itsaccumulation to toxic levels. There is no such require-ment to maintain a low N2O concentration indenitrifiers, and N2O does not appear to be a signal

Phil. Trans. R. Soc. B (2012)

that regulates the expression of any of the denitrifica-tion genes. From the point of view of mitigating N2Oreleases from denitrification, the absence of regulationby N2O is a significant observation, since denitrifyingpopulations do not (as far as is known) respond toN2O accumulation by making more of the N2O

Paracoccus denitrificans

Bradyrhizobium japonicum

Pseudomonas stutzeri

narGHJI nirSTBM norCB nirKnosZ norCBQD

Agrobacterium tumifaciens

Pseudomonas aeruginosa

narKGHJI

napEDACnirK norCBQD norCBD nosRZDFYLnirSMCFDLGHJENnarK1K2GHJI

nirSECF norCBQDEF nosZ napKEFDABC norCBQDnirK

nitrate

NarR Nirl NNR NosRFnrP

NnrR NarXL ANR DNR

lowO2

lowO2

lowO2

lowO2

lowO2

lowO2O2 O2

H2O2

NO

NO

FixLJ

nitrate NO

DNR ActRS FnrN NnrRNarXL NosR

FixK2

NO

NO

nitrate

NO NO

FnrP NnrRPrrAB

Rhodobacter sphaeroides

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, andbetween 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 napand 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, whilethese 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.

1222 S. Spiro Review. N2O flux: regulation of gene expression

on September 17, 2013rstb.royalsocietypublishing.orgDownloaded from

reductase. Nevertheless, understanding those signalsthat do influence the expression of denitrificationgenes, and those that influence the abundance andactivities of the corresponding enzymes, is very likelyto be helpful to any effort to intervene in the activitiesof the denitrifying bacteria.

REFERENCES1 Richardson, D., Felgate, H., Watmough, N., Thomson,

A. & Baggs, E. 2009 Mitigating release of the potentgreenhouse gas N2O from the nitrogen cycle: could

Phil. Trans. R. Soc. B (2012)

enzymic regulation hold the key? Trends Biotechnol. 27,388–397. (doi:10.1016/j.tibtech.2009.03.009)

2 Ferguson, S. J., Richardson, D. J. & van Spanning,

R. J. M. 2007 Biochemistry and molecular biologyof nitrification. In Biology of the nitrogen cycle (edsH. Bothe, S. J. Ferguson & W. E. Newton), pp.209–222. Amsterdam, The Netherlands: Elsevier.

3 Zumft, W. G. 2005 Nitric oxide reductases of prokar-

yotes with emphasis on the respiratory, heme-copperoxidase type. J. Inorg. Biochem. 99, 194–215. (doi:10.1016/j.jinorgbio.2004.09.024)

4 Hino, T., Matsumoto, Y., Nagano, S., Sugimoto, H.,

Fukumori, Y., Murata, T., Iwata, S. & Shiro, Y. 2010Structural basis of biological N2O generation by bacterial

Review. N2O flux: regulation of gene expression S. Spiro 1223

on September 17, 2013rstb.royalsocietypublishing.orgDownloaded from

nitric oxide reductase. Science 330, 1666–1670. (doi:10.1126/science.1195591)

5 Shapleigh, J. P. & Payne, W. J. 1985 Nitric oxide-

dependent proton translocation in various denitrifiers.J. Bacteriol. 163, 837–840.

6 Bell, L. C., Richardson, D. J. & Ferguson, S. J. 1992Identification of nitric oxide reductase activity inRhodobacter capsulatus: the electron transport pathway

can either use or bypass both cytochrome c2 and the cyto-chrome bc1 complex. J. Gen. Microbiol. 138, 437–443.

7 de Boer, A. P., van der Oost, J., Reijnders, W. N.,Westerhoff, H. V., Stouthamer, A. H. & van Spanning,

R. J. 1996 Mutational analysis of the nor gene clusterwhich encodes nitric-oxide reductase from Paracoccusdenitrificans. Eur. J. Biochem. 242, 592–600. (doi:10.1111/j.1432-1033.1996.0592r.x)

8 Cramm, R., Pohlmann, A. & Friedrich, B. 1999 Purifi-

cation and characterization of the single-component nitricoxide reductase from Ralstonia eutropha H16. FEBS Lett.460, 6–10. (doi:10.1016/S0014-5793(99)01315-0)

9 de Vries, S., Strampraad, M. J. F., Lu, S., Moenne-Loccoz, P. & Schroder, I. 2003 Purification and

characterization of the MQH2 : NO oxidoreductase fromthe hyperthermophilic archaeon Pyrobaculum aerophilum.J. Biol. Chem. 278, 35 861–35 868. (doi:10.1074/jbc.M300857200)

10 Justino, M. C., Almeida, C. C., Teixeira, M. & Saraiva,

L. M. 2007 Escherichia coli di-iron YtfE protein is neces-sary for the repair of stress-damaged iron–sulfur clusters.J. Biol. Chem. 282, 10 352–10 359. (doi:10.1074/jbc.M610656200)

11 Strube, K., de Vries, S. & Cramm, R. 2007 Formation ofa dinitrosyl iron complex by NorA, a nitric oxide-bindingdi-iron protein from Ralstonia eutropha H16. J. Biol.Chem. 282, 20 292–20 300. (doi:10.1074/jbc.M702003200)

12 Suharti, Strampraad, M. J., Schroder, I. & de Vries, S.2001 A novel copper A containing menaquinol NOreductase from Bacillus azotoformans. Biochemistry 40,2632–2639. (doi:10.1021/bi0020067)

13 Suharti, Heering, H. A. & de Vries, S. 2004 NO

reductase from Bacillus azotoformans is a bifunctionalenzyme accepting electrons from menaquinol and aspecific endogenous membrane-bound cytochromec551. Biochemistry 43, 13 487–13 495. (doi:10.1021/bi0488101)

14 Gomes, C. M., Giuffre, A., Forte, E., Vicente, J. B.,Saraiva, L. M., Brunori, M. & Teixeira, M. 2002 Anovel type of nitric-oxide reductase. Escherichia coliflavorubredoxin. J. Biol. Chem. 277, 25 273–25 276.

(doi:10.1074/jbc.M203886200)15 Gardner, A. M., Helmick, R. A. & Gardner, P. R. 2002

Flavorubredoxin, an inducible catalyst for nitric oxidereduction and detoxification in Escherichia coli. J. Biol.Chem. 277, 8172–8177. (doi:10.1074/jbc.M110471200)

16 Silaghi-Dumitrescu, R., Coulter, E. D., Das, A., Ljungdahl,L. G., Jameson, G. N., Huynh, B. H. & Kurtz, D. M. 2003A flavodiiron protein and high molecular weight rubredoxinfrom Moorella thermoacetica with nitric oxide reductaseactivity. Biochemistry 42, 2806–2815. (doi:10.1021/

bi027253k)17 Rodrigues, R., Vicente, J. B., Felix, R., Oliveira, S.,

Teixeira, M. & Rodrigues-Pousada, C. 2006 Desulfovibriogigas flavodiiron protein affords protection against nitro-sative stress in vivo. J. Bacteriol. 188, 2745–2751.

(doi:10.1128/JB.188.8.2745-2751.2006)18 Silaghi-Dumitrescu, R., Ng, K. Y., Viswanathan, R. &

Kurtz, D. M. 2005 A flavo-diiron protein from Desulfovi-brio vulgaris with oxidase and nitric oxide reductaseactivities. Evidence for an in vivo nitric oxide scavenging

Phil. Trans. R. Soc. B (2012)

function. Biochemistry 44, 3572–3579. (doi:10.1021/bi0477337)

19 Poole, R. K. & Hughes, M. N. 2000 New functions for

the ancient globin family: bacterial responses to nitricoxide and nitrosative stress. Mol. Microbiol. 36, 775–783. (doi:10.1046/j.1365-2958.2000.01889.x)

20 Kim, S. O., Orii, Y., Lloyd, D., Hughes, M. N. & Poole,R. K. 1999 Anoxic function for the Escherichia coli flavo-

hemeoglobin (Hmp): reversible binding of nitric oxideand reduction to nitrous oxide. FEBS Lett. 445, 389–394. (doi:10.1016/S0014-5793(99)00157-X)

21 Mills, C. E., Sedelnikova, S., Soballe, B., Hughes, M. N. &

Poole, R. K. 2001 Escherichia coli flavohemeoglobin (Hmp)with equistoichiometric FAD and haem contents has a lowaffinity for dioxygen in the absence or presence of nitricoxide. Biochem. J. 353, 207–213. (doi:10.1042/0264-6021:3530207)

22 Zumft, W. G. & Kroneck, P. M. H. 2007 Respiratorytransformation of nitrous oxide (N2O) to dinitrogen byBacteria and Archaea. Adv. Microb. Physiol. 52, 107–227. (doi:10.1016/S0065-2911(06)52003-X)

23 Kern, M. & Simon, J. 2009 Electron transport chains

and bioenergetics of respiratory nitrogen metabolism inWolinella succinogenes and other Epsilonproteobacteria.Biochim. Biophys. Acta. 1787, 646–656. (doi:10.1016/j.bbabio.2008.12.010)

24 Brown, K., Tegoni, M., Prudencio, M., Pereira, A. S.,

Besson, S., Moura, J. J., Moura, I. & Cambillau, C.2000 A novel type of catalytic copper cluster in nitrousoxide reductase. Nat. Struct. Biol. 7, 191–195. (doi:10.1038/73288)

25 Haltia, T., Brown, K., Tegoni, M., Cambillau, C.,Saraste, M., Mattila, K. & Djinovic-Carugo, K. Crystalstructure of nitrous oxide reductase from Paracoccusdenitrificans at 1.6 A resolution. Biochem. J. 369,77–88. (doi:10.1042/BJ20020782)

26 Wunsch, P. & Zumft, W. G. 2005 Functional domains ofNosR, a novel transmembrane iron–sulfur flavoproteinnecessary for nitrous oxide respiration. J. Bacteriol. 187,1992–2001. (doi:10.1128/JB.187.6.1992-2001.2005)

27 Letunic, I., Doerks, T. & Bork, P. 2009 SMART 6:

recent updates and new developments. Nucleic AcidsRes. 37, D229–D232. (doi:10.1093/nar/gkn808)

28 Cheung, J. & Hendrickson, W. A. 2009 Structural analy-sis of ligand stimulation of the histidine kinase NarX.Structure 17, 190–201. (doi:10.1016/j.str.2008.12.013)

29 Elsen, S., Swem, L. R., Swem, D. L. & Bauer, C. E.2004 RegB/RegA, a highly conserved redox-respondingglobal two-component regulatory system. Microbiol.Mol. Biol. Rev. 68, 263–279. (doi:10.1128/MMBR.68.

2.263-279.2004)30 Wu, J. & Bauer, C. E. 2010 RegB kinase activity is con-

trolled in part by monitoring the ratio of oxidized toreduced ubiquinones in the ubiquinone pool. MBio 1,e00272-10. (doi:10.1128/mBio.00272-10)

31 Rodgers, K. R. & Lukat-Rodgers, G. S. 2005 Insightsinto heme-based O2 sensing from structure–functionrelationships in the FixL proteins. J. Inorg. Biochem. 99,963–977. (doi:10.1016/j.jinorgbio.2005.02.016)

32 Green, J., Crack, J. C., Thomson, A. J. & LeBrun, N. E.

2009 Bacterial sensors of oxygen. Curr. Opin. Microbiol.12, 145–151. (doi:10.1016/j.mib.2009.01.008)

33 Gilles-Gonzalez, M. A. & Gonzalez, G. 2005 Heme-based sensors: defining characteristics, recent develop-ments, and regulatory hypotheses. J. Inorg. Biochem. 99,

1–22. (doi:10.1016/j.jinorgbio.2004.11.006)34 Fleischhacker, A. S. & Kiley, P. J. 2011 Iron-containing

transcription factors and their roles as sensors. Curr.Opin. Chem. Biol. 15, 335–341. (doi:10.1016/j.cbpa.2011.01.006)

1224 S. Spiro Review. N2O flux: regulation of gene expression

on September 17, 2013rstb.royalsocietypublishing.orgDownloaded from

35 Wu, G., Cruz-Ramos, H., Hill, S., Green, J., Sawers, G. &Poole, R. K. 2000 Regulation of cytochrome bd expressionin the obligate aerobe Azotobacter vinelandii by CydR

(Fnr). Sensitivity to oxygen, reactive oxygen species, andnitric oxide. J. Biol. Chem. 275, 4679–4686. (doi:10.1074/jbc.275.7.4679)

36 Edwards, J., Cole, L. J., Green, J. B., Thomson, M. J.,Wood, A. J., Whittingham, J. L. & Moir, J. W. B. 2010

Binding to DNA protects Neisseria meningitidis fumarateand nitrate reductase regulator (FNR) from oxygen.J. Biol. Chem. 285, 1105–1112. (doi:10.1074/jbc.M109.057810)

37 Cruz-Ramos, H., Crack, J., Wu, G., Hughes, M. N.,Scott, C., Thomson, A. J., Green, J. & Poole, R. K.2002 NO sensing by FNR: regulation of the Escherichiacoli NO-detoxifying flavohemeoglobin, Hmp. EMBO J.21, 3235–3244. (doi:10.1093/emboj/cdf339)

38 Yoon, S. S. et al. 2007 Two-pronged survival strategy forthe major cystic fibrosis pathogen, Pseudomonasaeruginosa, lacking the capacity to degrade nitric oxideduring anaerobic respiration. EMBO J. 26, 3662–3672.(doi:10.1038/sj.emboj.7601787)

39 Hutchings, M. I., Crack, J. C., Shearer, N., Thompson,B. J., Thomson, A. J. & Spiro, S. 2002 Transcriptionfactor FnrP from Paracoccus denitrificans contains aniron–sulfur cluster and is activated by anoxia: identifi-cation of essential cysteine residues. J. Bacteriol. 184,

503–508. (doi:10.1128/JB.184.2.503-508.2002)40 Swem, L. R., Kraft, B. J., Swem, D. L., Setterdahl, A. T.,

Masuda, S., Knaff, D. B., Zalski, J. M. & Bauer, C. E.2003 Signal transduction by the global regulator RegB

is mediated by a redox-active cysteine. EMBO J. 22,4699–4708. (doi:10.1093/emboj/cdg461)

41 Kim, Y.-J., Ko, I.-J., Lee, J.-M., Kang, H.-Y., Kim,Y. M., Kaplan, S. & Oh, J.-I. 2007 Dominant role ofthe cbb3 oxidase in regulation of photosynthesis gene

expression through the PrrBA system in Rhodobactersphaeroides 2.4.1. J. Bacteriol. 189, 5617–5625. (doi:10.1128/JB.00443-07)

42 Korner, H., Sofia, H. J. & Zumft, W. G. 2003 Phylogenyof the bacterial superfamily of Crp-Fnr transcription reg-

ulators: exploiting the metabolic spectrum by controllingalternative gene programs. FEMS Microbiol. Rev. 27,559–592. (doi:10.1016/S0168-6445(03)00066-4)

43 Lee, Y.-Y., Shearer, N. & Spiro, S. 2006 Transcriptionfactor NNR from Paracoccus denitrificans is a sensor of

both nitric oxide and oxygen: isolation of nnr* allelesencoding effector-independent proteins and evidencefor a haem-based sensing mechanism. Microbiology 152,1461–1470. (doi:10.1099/mic.0.28796-0)

44 Castiglione, N., Rinaldo, S., Giardina, G. & Cutruzzola,F. 2009 The transcription factor DNR from Pseudomonasaeruginosa specifically requires nitric oxide and haem forthe activation of a target promoter in Escherichia coli.Microbiology 155, 2838–2844. (doi:10.1099/mic.0.

028027-0)45 Giardina, G., Rinaldo, S., Johnson, K. A., Di Matteo, A.,

Brunori, M. & Cutruzzola, F. 2008 NO sensing in Pseu-domonas aeruginosa: structure of the transcriptionalregulator DNR. J. Mol. Biol. 378, 1002–1015. (doi:10.

1016/j.jmb.2008.03.013)46 Giardina, G., Castiglione, N., Caruso, M., Cutruzzola,

F. & Rinaldo, S. 2011 The Pseudomonas aeruginosaDNR transcription factor: light and shade of nitricoxide-sensing mechanisms. Biochem. Soc. Trans. 39,

294–298. (doi:10.1042/BST0390294)47 Hartsock, A. & Shapleigh, J. P. 2010 Mechanisms of

oxygen inhibition of nirK expression in Rhodobactersphaeroides. Microbiology 156, 3158–3165. (doi:10.1099/mic.0.038703-0)

Phil. Trans. R. Soc. B (2012)

48 Wood, N. J., Alizadeh, T., Bennett, S., Pearce, J., Ferguson,S. J., Richardson, D. J. & Moir, J. W. 2001 Maximalexpression of membrane-bound nitrate reductase in Para-coccus is induced by nitrate via a third FNR-like regulatornamed NarR. J. Bacteriol. 183, 3606–3613. (doi:10.1128/JB.183.12.3606-3613.2001)

49 Wood, N. J., Alizadeh, T., Richardson, D. J., Ferguson,S. J. & Moir, J. W. 2002 Two domains of a dual-function

NarK protein are required for nitrate uptake, the firststep of denitrification in Paracoccus pantotrophus. Mol.Microbiol. 44, 157–170. (doi:10.1046/j.1365-2958.2002.02859.x)

50 Pohlmann, A., Cramm, R., Schmelz, K. & Friedrich, B.2000 A novel NO-responding regulator controls thereduction of nitric oxide in Ralstonia eutropha. Mol.Microbiol. 38, 626–638. (doi:10.1046/j.1365-2958.2000.02157.x)

51 Hutchings, M. I., Mandhana, N. & Spiro, S. 2002 TheNorR protein of Escherichia coli activates expression ofthe flavorubredoxin gene norV in response to reactivenitrogen species. J. Bacteriol. 184, 4640–4643. (doi:10.1128/JB.184.16.4640-4643.2002)

52 Gardner, A. M., Gessner, C. R. & Gardner, P. R. 2003Regulation of the nitric oxide reduction operon(norRVW ) in Escherichia coli. Role of NorR and s54 inthe nitric oxide stress response. J. Biol. Chem. 278,10 081–10 086.

53 Bush, M., Ghosh, T., Tucker, N., Zhang, X. & Dixon, R.2010 Nitric oxide-responsive interdomain regulation tar-gets the s54-interaction surface in the enhancer bindingprotein NorR. Mol. Microbiol. 77, 1278–1288. (doi:10.

1111/j.1365-2958.2010.07290.x)54 D’Autreaux, B., Tucker, N. P., Dixon, R. & Spiro, S.

2005 A non-heme iron centre in the transcriptionfactor NorR senses nitric oxide. Nature 437, 769–772.(doi:10.1038/nature03953)

55 Klink, A., Elsner, B., Strube, K. & Cramm, R. 2007Characterization of the signaling domain of the NO-responsive regulator NorR from Ralstonia eutrophaH16 by site-directed mutagenesis. J. Bacteriol. 189,2743–2749. (doi:10.1128/JB.01865-06)

56 Tucker, N. P., D’Autreaux, B., Yousafzai, F. K.,Fairhurst, S. A., Spiro, S. & Dixon, R. 2008 Analysisof the nitric oxide-sensing non-heme iron center in theNorR regulatory protein. J. Biol. Chem. 283, 908–918.(doi:10.1074/jbc.M705850200)

57 Stewart, V. 2003 Nitrate- and nitrite-responsive sensorsNarX and NarQ of proteobacteria. Biochem. Soc. Trans.31, 1–10. (doi:10.1042/BST0310001)

58 Cavicchioli, R., Chiang, R. C., Kalman, L. V. &

Gunsalus, R. P. 1996 Role of the periplasmic domainof the Escherichia coli NarX sensor-transmitter proteinin nitrate-dependent signal transduction and gene regu-lation. Mol. Microbiol. 21, 901–911. (doi:10.1046/j.1365-2958.1996.491422.x)

59 Williams, S. B. & Stewart, V. 1997 Discriminationbetween structurally related ligands nitrate and nitrite con-trols autokinase activity of the NarX transmembrane signaltransducer of Escherichia coli K-12. Mol. Microbiol. 26,911–925. (doi:10.1046/j.1365-2958.1997.6262002.x)

60 Stewart, V. & Chen, L.-L. 2010 The S helix mediatessignal transmission as a HAMP domain coiled-coilextension in the NarX nitrate sensor from Escherichiacoli K-12. J. Bacteriol. 192, 734–745. (doi:10.1128/JB.00172-09)

61 Mesa, S., Reutimann, L., Fischer, H.-M. & Hennecke,H. 2009 Posttranslational control of transcription factorFixK2, a key regulator for the Bradyrhizobium japoni-cum-soybean symbiosis. Proc. Natl Acad. Sci. USA 106,21 860–21 865. (doi:10.1073/pnas.0908097106)

Review. N2O flux: regulation of gene expression S. Spiro 1225

on September 17, 2013rstb.royalsocietypublishing.orgDownloaded from

62 Cuypers, H., Viebrock-Sambale, A. & Zumft, W. G.1992 NosR, a membrane-bound regulatory componentnecessary for expression of nitrous oxide reductase in

denitrifying Pseudomonas stutzeri. J Bacteriol. 174,5332–5339.

63 Honisch, U. & Zumft, W. G. 2003 Operon structure andregulation of the nos gene region of Pseudomonas stutzeri,encoding an ABC-type ATPase for maturation of nitrous

oxide reductase. J. Bacteriol. 185, 1895–1902. (doi:10.1128/JB.185.6.1895-1902.2003)

64 Arai, H., Mizutani, M. & Igarashi, Y. 2003 Transcrip-tional regulation of the nos genes for nitrous oxide

reductase in Pseudomonas aeruginosa. Microbiology 149,29–36. (doi:10.1099/mic.0.25936-0)

65 Saunders, N. F., Houben, E. N., Koefoed, S., de Weert,S., Reijnders, W. N., Westerhoff, H. V., de Boer, A. P. &van Spanning, R. J. 1999 Transcription regulation of the

nir gene cluster encoding nitrite reductase of Paracoccusdenitrificans involves NNR and NirI, a novel type ofmembrane protein. Mol. Microbiol. 34, 24–36. (doi:10.1046/j.1365-2958.1999.01563.x)

66 Beaumont, H. J. E., Lens, S. I., Reijnders, W. N. M.,

Westerhoff, H. V. & van Spanning, R. J. M. 2004Expression of nitrite reductase in Nitrosomonas europaeainvolves NsrR, a novel nitrite-sensitive transcriptionrepressor. Mol. Microbiol. 54, 148–158. (doi:10.1111/j.1365-2958.2004.04248.x)

67 Bodenmiller, D. M. & Spiro, S. 2006 The yjeB (nsrR)gene of Escherichia coli encodes a nitric oxide sensitivetranscriptional regulator. J. Bacteriol. 188, 874–881.(doi:10.1128/JB.188.3.874-881.2006)

Phil. Trans. R. Soc. B (2012)

68 Overton, T. W., Whitehead, R., Li, Y., Snyder, L. A. S.,Saunders, N. J., Smith, H. & Cole, J. A. 2006 Coordi-nated regulation of the Neisseria gonorrhoeae truncated

denitrification pathway by the nitric oxide-sensitiverepressor, NsrR, and nitrite-insensitive NarQ-NarP.J. Biol. Chem. 281, 33 115–33 126. (doi:10.1074/jbc.M607056200)

69 Rock, J. D., Thomson, M. J., Read, R. C. & Moir,

J. W. B. 2007 Regulation of denitrification genes inNeisseria meningitidis by nitric oxide and the repressorNsrR. J. Bacteriol. 189, 1138–1144. (doi:10.1128/JB.01368-06)

70 Wang, W., Richardson, A. R., Martens-Habbena, W.,Stahl, D. A., Fang, F. C. & Hansen, E. J. 2008 Identifi-cation of a repressor of a truncated denitrificationpathway in Moraxella catarrhalis. J. Bacteriol. 190,7762–7772. (doi:10.1128/JB.01032-08)

71 Tucker, N. P., Le Brun, N. E., Dixon, R. & Hutchings,M. I. 2010 There’s NO stopping NsrR, a global regulatorof the bacterial NO stress response. Trends Microbiol. 18,149–156. (doi:10.1016/j.tim.2009.12.009)

72 Rodionov, D. A., Dubchak, I. L., Arkin, A. P., Alm, E. J. &

Gelfand, M. S. 2005 Dissimilatory metabolim of nitrogenoxides in Bacteria: comparative reconstruction oftranscriptional networks. PLOS Comput. Biol. 1, e55.(doi:10.1371/journal.pcbi.0010055)

73 van Spanning, R. J. M., Richardson, D. J. & Ferguson, S.

J. 2007 Introduction to the biochemistry and molecularbiology of denitrification. In Biology of the nitrogen cycle(eds H. Bothe, S. J. Ferguson & W. E. Newton), pp.3–20. Amsterdam, The Netherlands: Elsevier.