Genomics and Ecology of Novel N 2 O-Reducing Microorganisms

2018·3.1 MB·English

by  Sara Hallin

Review
Genomics and Ecology of

Novel N O-Reducing
2

Microorganisms
Sara Hallin,1,* Laurent Philippot,2 Frank E. Löffler,3,4

Robert A. Sanford,5 and Christopher M. Jones1

Microorganismswiththecapacitytoreducethegreenhousegasnitrousoxide
Trends
(N O) to harmless dinitrogen gas are receiving increased attention due to
2
increa sin g N O em issions (an d ou r ne ed to mitig ate climat e change) and to N2O is produced in several microbial
2 processes, but N2O reduction is lim-
recentdiscoveriesofnovelN O-reducingbacteriaandarchaea.Thediversityof itedtoasingleprocesscatalyzedby
denitrif ying and no nd enitri fyi2ng microorg anisms w ith capacity for N 2O redu c- othneo N m2iOc a rl leyducdtiav seers pereseb natc atemrioang ta anxd-
tion was recently shown to be greater than previously expected. A formerly
archaea. This enzyme is used for

overlookedgroup(cladeII)intheenvironment includealargefractionofnon- energyconservationthroughanaero-
bic re spiration, d etoxificat ion, or
denitrifying N2O reducers, which could be N2O sinks without major contribution rem ovalofexces selectrons.
to N O formation. We review the recent advances about fundamental under-
2

standing of the genomics, physiology, and ecology of N2O reducers and the The N2O reductase has evolved into
importan ce of t hese findin gs for curbin g N O emiss ion s. two lineages, clade I and the recently
2 described clade II. Surveys indicate

that clade II N2O reductase (NosZ) is
abundantinmanybiomes.
Microbial Sources and Sinks of Nitrous Oxide: Introducing the Nitrous Oxide

Reducers Genome analyses and culture-based

Inorganic nitrogen compounds are primarily transformed through reduction–oxidation reac- studiessuggestthatthephysiologyof

NO reduction differs between the
tionsmediatedbymicrobialcommunitiesinthebiogeochemicalnitrogencycle(Figure1).Their 2
clades. Evidence from community
activityregulatestheretentionorlossofnitrogenfromecosystems,anddeterminesinwhich ecology supports niched ifferentiation

formnitrogenislost.Nitrousoxideisproducedinseveralnitrogenturnoverprocesses(Box1).It oforganismswitheithertypeofNO
isan importan tg reen houseg aswi th awarming p otential nearly30 0times thatofCO2 over 10 0 re ductase, an d wit hin the two cla de2s.

years, and the major cause of stratospheric ozone depletion [1,2]. Terrestrial ecosystems
accou nt for the majori ty of pla ne tary N2O emis sions, w hile aquat ic eco systems co ntribute one gIdiceanlt aifynidn ge cfaoclotogricsa tl hreast pdornivsee ps hoyf sNio2lOo--
third [1], of which the anthropogenic origins can be assigned to agricultural soils [3,4], and reducing communities could lead to
agric ultu ral run-of f an d animal wast e [5], r espe ctiv ely. Thes e e missions a re ex pecte d to effective and innovativ e strat egies to
increasedu etoinc rease duseo ffertiliz ers andmineraliz ationof soilorgani cma tterfollow ing curb N2O emissions.

land usechange [6,7].

AlthoughmultiplesourcesofN Oexist,thereisonlyoneknownsinkforN Ointhebiosphere–
2 2

themicrobialreductionofN2OtoN2,catalyzedbytheN2Oreductase(NosZ).Thisenzymehas 1SwedishUniversityofAgricultural
trad itionallyb eenassig ne dtob ac teri aandarch ae ape rform ingcomp leteden itrifi cation( see Sciences, Departme nt ofForest
Mycology andPlantPa th ology,75007
Glossary): a pathway ending with N2O reduction to N2 [8,9]. A few nondenitrifying bacteria, Uppsala,S wed en
such as Wolinella succinogenes and Campylobacter fetus, were reported early on for their 2Agroéco logie,AgroSupDijon,INRA,

capacitytogrowwithN Oasthesoleelectronacceptor[10,11].Duetotheincreasingnumber Univ.BourgogneFranche-Comté,
of seque nc ed mi crob ial2 ge no me s fro m a rang e of taxa , the div ersit y o f bo th denitrify ing and 3FC-2e1n0t e0r0 foDrijoEnn,v iFror annmceental
nondenitrifying microorganisms with the genetic capacity for N2O reduction was recently Biotech nolo gy,Departmentof
showntobegr eaterthanprevio uslye xpe cted[12 –14].Inco ntr astto denitrifying mic roorgan- Microbiology,D epartmento fCiviland
Environmenta lEngineering ,
isms, nondenitrifying N2O-reducing bacteria and archaea have the potential to be true N2O Departmentof Biosystems
sinks without contributing to N2O production (Box 1, Figure 1). Agricultural production and Engineering an dSoilScience,

TrendsinMicrobiology,January2018,Vol.26,No.1 http://dx.doi.org/10.1016/j.tim.2017.07.003 43

©2017ElsevierLtd.Allrightsreserved.
water treatment facilities are focal points for N O mitigation efforts, and the capacity for UniversityofTennessee,Knoxville,TN
2
37996,USA
smtricartoeogriegsanthisamtsc otmo baactt Na2sO Ne2mO isssinioknss isa ngdaminiintigg aitnecrceliamsaetde acthteanntgioen[ 1in5 –t1h7e] .development of I4nBsitoitsuctie enfocresB iDoilvoigsiicoanl, Sacnide nJcoeinst(JIBS),

OakRidgeNationalLaboratory,Oak

Ridge,TN37831,USA
An important discovery was the realization that NosZ protein phylogeny has two distinct 5Unive rsity ofIllino is,Departmentof
groups,cladeIandthenewlydescribedcladeII[13,14].Thesecladeshavealsobeenknownas Geology,C ha mpaign ,IL,USA

typical and atypical NosZ [13], but the authors of this review have agreed on using the

terminology clade I and II NosZ. It is known that denitrifiers within clade I reduce N O at
2
differentrate s[18] ,i mply ing thatd en itr ifiercom mun itycompos itionco uldin flu encein situn et *Correspondence:
[email protected](S.Hallin).
N Ofluxes.LessisknownaboutcladeIItypes,especiallythenondenitrifiers,althoughgenomic
2

and physiologicaldifferencesareemerging (e.g.,[19,20]).Wereviewthe recentliterature on

N O reducers and focus on genomic, physiological, and ecological information that would
2

explainnichedifferentiationbetweenandwithinthetwocladesofN O-reducingbacteriaand
2

archaea. We also discuss the importance of these findings for mitigation of N O emissions,
2

especiallyfromsoils.

Evolution and Genomics of N O-Reducing Microorganisms
2

TheabilitytoreduceN Oisataxonomicallywidespreadtrait.HomologsofthenosZgene,
2

whichcodeforthecatalyticsubunitinvolvedinN Oreduction,arefoundinapproximately12%
2

of sequenced microbial genomes, representing 12 archaeal and bacterial phyla (Figure 2)

isolatedfromvariousenvironments[19].Phylogeneticanalysisindicatesthatthediversification

ofNosZoccurredpredominantlythroughverticalinheritancewithsomeinstancesofhorizontal

genetransfer,asthenosZphylogenyislargelycongruentwithorganismalphylogenyathigher

taxonomiclevels[19,21].WhilethissuggeststhatN Oreductionisapotentiallyancienttrait,the
2

literatureoffersconflictingscenariosonwhetheranN Oreductaseappearedbeforeorafterthe
2

‘great oxygenation event (GOE)’ of the late Archaean, 2.8–2.5 Gyr before present. Isotopic

evidencesuggeststhatthedenitrificationpathwayappearedonlyshortlyaftertheGOE[22].

Copper, a key cofactor for several enzymes, including the N O reductase, was likely not
2

biologically available in the reduced conditions prior to the GOE, although recent isotopic

evidence suggests that copper concentrations may have been less limiting than previously

assumed[23].TheformationofNO,theprecursortoN O,isexpectedtohaveoccurredpriorto
2

theGOE[24],andiron-andheme-basedNOreductasescouldhavegeneratedN Opriortothe
2

build-upofoxygenlevels.Inaddition,N Ocouldhavebeenformedbychemodenitrification,
2

an abiotic reaction of nitrite with iron (II) [25,26]. Thus, N O reductase activity could have
2

evolved due to the accumulation of N O in parallel with nitrite reduction, but before nitrate
2

reduction becamesignificant.

Despite evidence for vertical inheritance, the distribution of nosZ genes across microbial

taxonomicgroupsatafinerscaleispatchier,asnosZcanbepresentorabsentinthegenomes

ofcloselyrelatedorganisms[19,21,27–30].ThepolyphyleticdistributionofN Oreductionasa
2

traitsuggeststhattheacquisitionorlossofnosgenescouldbeinfluencedbyenvironmental

conditions [27,31]. Gene loss through deletion is a common evolutionary phenomenon in

bacteria,wherelossoffunctionsmayresultinanincreaseinfitness[32].Indeed,organisms

capableofcompletedenitrificationmaydownregulatenosZexpressionwhennitrateisreplete,

but other resources are limiting [33]. This may explain why nearly 40% of all genomes that

possessgenesencodingthenitritereductasesassociatedwithdenitrificationlacknosZ[19].

Thispatternisnotrandomlydistributedacrosstaxa.Forexample,nosZhasnotbeendetected

in fungal genomes and is conspicuously absent from the genomes of Actinobacteria or

Acidobacteria [19,34]. These highly abundant and diverse phyla are commonly associated

withlowresourceavailabilityorlowpHconditionsinterrestrialecosystems[35–38],suggesting

a lowerfrequency ofnosZ insuchenvironments.

44 TrendsinMicrobiology,January2018,Vol.26,No.1
ThediversityofthenosZgenecanbedividedintotwodistinctgroupsofN2Oreducers,cladeI Glossary

andII[13,14](Figure3).ThisisbasednotonlyonthephylogenyoftheNosZprotein,butalsoon Chemodenitrification:achemical

differencesbetweenthetwocladesregardingthenosgenecluster(NGC)organization,NosZ reactionbetweenareduced

translocationpathway,andfrequenciesofco-occurrencewithothergenesassociatedwith chemical[e.g.,Fe(II)orreduced
denitrificationo rrespirator yam monification .C ladeIconsistsa lmos texcl usively ofAlpha-,B eta- humic su bstan ces] a nd NO2(cid:1)to
,andGamma pr oteobacter ia.ThenosZgen esfro m theha lophilic Euryarchae ot a(Haloa rcula pDreondiutrciefi cNaOti oann:d aNn2aOn.aerobic

andNatronomonas)arealsogroupedwithcladeIbasedonevolutionarydistance,butforma respiratorypathwayconsistingofthe
subc ladefromtheP rote obac teria.The sign alpep t idesre vea lthatcladeI NosZistr ansl ocate d sequential reduction ofsoluble nit rate
totheper iplasm us ingthetwin-arg inine translo cationpa thway ,inw hichp r oteins a refirstfolded (NO3(cid:1)) or nitrite (NO 2(cid:1)) to the
in the cytoplasm and then transported acrossthecy toplasmic m embr ane[39]. Th eNG Csof pgaasthewouasy cparondbuecttsr uNn2cOa teadnda nNd2. The

aonsorgssaeRnm,isebmnlycs, o awdniidnth gp coalasmdsieeb mlyI bncoroaspnZpe ehbra otvruean nads pnFooers-tSZ [ 4flg0ae]v n(oFepi gfrouolrtleoe win4e).lid kA eb ldyye iDnfinvFoinYlgvLe fgdeeainntueersel e inicsvt rtoohlvnee ptdrra einnss eppnrocorette toionf noteixtrrimdifiaeintria-odtnee inwsi ticrthiofi cuNap2tliOeodn. ,At oi n s NpwOehc2ici(cid:1)ahl cNaHse3 is
NosZ. Analysis o f theco-occu rrence of no sZ withothe rgen esinthe d enitrificat ionpathw ay rperdoudcutciotino,n wohficNh Oretshurlotsu ginh tbheioticor
indicat ethatorg an ism swithcladeIno sZ arem ore likelyt obeco m plet edenitrifiers,a s83%of acombinat ion o2fb ioticand abiot ic
re actions.
genomeswithcladeInosZalsopossessnirSornirKgenes,whichencodethetwodifferent
Fitness:ameasureofthe
nitrite reductases NirSor NirK,respectively [19](Figure3).
reproductivesuccessofaparticular

genotypeduringnaturalselection

AdiverserangeofarchaealandbacterialphylapossesscladeIInosZ.IncontrasttocladeI, comparedtoothergenotypesina
givenenvir on ment.
51% of the organisms with clade II nosZ appear to be nondenitrifying N2O reducers [19] Geno me:anorganism’scomplete
(Figure3).Interestingly,approximately27%ofgenomeswithcladeIInosZinanearlieranalysis
setofgeneticinformation,which

alsoharboredthenrfAgeneencodingtheformate-dependentnitritereductase[13],ahallmark includesallgenes.

for respiratory ammonification (Figure 1, Box 1). However, Photobacterium profundum and Homolog(gene):homologous

Sh ewanellaloi hicapossesscla deINos Z, whic hd emonstrate sthatrespiratory ammonifica tion genes have a common ancestor and
sharesequencesimilarities.
doesnotexclusivelyoccurwithcladeIINosZ.Shewanellaloihicaisabletoperformcomplete Meta genomics :DNA-based
denitr ifica tionandre spirato ryam moni fic ation[ 41],afeature share d bya few otherb acteria(e. analyseswhere‘a ll’genetic
g.,Opitituster raea ndsomeB acillusspp.).Inc ontr as ttoclad eI,nea rly al lclad eIIno sZ,exce pt informatio nina sam pleis

sequenced.Sequencedepthwill
thosefromarchaeaandthermophilicbacteria,possesssignalpeptidesassociatedwiththeSec
determinethecoverage.
translocationpathwaythatextrudesthenascentpolypeptidechainintotheperiplasm,where Niche:the ec ologicalroleofan

folding and insertion of cofactors then occurs [42]. Whether the difference in translocation organismwithinacommunitywith
pathwa ybe tweenthe tw ocladesh asan yfunctio nalsi gnificance bey ondthetra ns locationitself, respect to use o f resources.
Phylogeny:theinferredevolutionary
or is merely coincident with the distribution of taxa across the nosZ phylogeny is unclear.
Ho w ever,stu diesofsev eralc lade IIorganism s, includ ingW. succ inoge nes,Bacillu sa zotofor- rperloatteioinnss,hgipe noof ma essetb oafs egdenoens,the

mans and Solitalea canadensis, indicate that NosZ may be closely associated with the similaritiesanddifferencesintheir
characteris tics.
cytoplasmicmembrane[11,43].Thisisfurthersupportedbythepresenceofanopenreading
Trait:anorganism’sfunctional
frame(ORF)immediatelydownstreamofnosZ,referredtoasnosBandpredictedtoencodea
characteristicthatisaproductof
memb rane-s panningpro teininvolved in electro ntransp or t[4 4].Th eha lophilicarc ha eawith in geneticcomp ositio n a nd

cladeIareanexceptiontothispattern,astheirNGCsalsocontainnosB.Overall,thegenomic environmentalfactors.
Translocation pathway:proteins
signaturesindicateobviousdifferencesbetweenorganismswithcladeIandIInosZ,butfurther
thathaveevolvedtofunctionoutside
biochemicalandphysiologicalstudiesarerequiredtodeterminehowthesedifferencesinNGC
thecytoplasmicmembranecanbe

arrangementandtranslocationpathwaytranslateintophysiologicaldifferencesthatpotentially translocatedthroughthemembrane

drivenichedifferentiation withinandbetween eachclade. bydifferentmechanisms.

Physiology of N O-Reducing Bacterial Populations
2

ThedistributionofnosZgenesinbacterialgenomesandthemultiplesourcesofN Oformation
2

suggestthatN Oreductionisaffiliatedwitharangeofphysiologicalresponses.Reductionof
2

N Oisoftenassociatedwithenergyconservation,butthatdoesnotappeartobethecasefor
2

allmicroorganismspossessingnosZ.Instead,itisawaytocopewithpotentiallytoxiceffectsof

N O or functions as an electron sink for metabolism. Sullivan et al. [45] demonstrated that
2

copper limitation downregulates expression of nosZ in Paraccocus denitrificans, resulting in

cytotoxiceffectsofN OduetoinactivationofvitaminB .Gemmatimonasaurantiacastrain
2 12

T-27, which lacks a complete set of denitrification genes and cannot grow under anoxic

TrendsinMicrobiology,January2018,Vol.26,No.1 45
Figure1.MicrobialPathwaysintheNitrogen(N)Cycle.Backgroundcolorsdifferentiatebetweenprocessesoccurringunderoxicoranoxicconditions.The

compoundsarepositionedaccordingtotheoxidationstateofN.Solidcoloredlinesindicatemicrobialpathways,andthegenesencodingtherespectiveenzymesthat

catalyzeeachstepinthepathwayareindicatedasfollows:amo,ammoniamonooxygenase;hao,hydroxylamineoxidoreductase;hdh(alsoknownashzo),hydrazine

dehydrogenase;hzs,hydrazinesynthase,nar/nap,cytoplasmicandperiplasmicdissimilatorynitratereductases,respectively;nas,assimilatorynitratereductase;nif,
nitrogenase;nas /nirB ,assimilato rynitriter eductase ;nirS/nirK,cy toch rome-cd a ndcopper-ba seddi ssimilatoryn itritereductas es,r espectively; ‘nor’,a nyofthemu lti-
hemeorhem e–copper nitricoxider educt ases;nosZ, nitrousox idereductase;n1rf,for mate-depende ntdissimilato rynitr itereductase ;nxr,nitriteo xidore duc tas e.D otted

lines indicatethecontributingpathways toatmosphericloadingofgaseous nitrogencompounds,whereasdashedgray linesindicatebiotic/abioticprocesses

generatinggaseousNcompoundsthatalsofeedintotheoverallN-cycle.DNRA,dissimilatorynitratereductiontoammonium.

conditions, reduces N O following growth under oxic conditions [46]. Here, N O apparently
2 2

actsasanelectronsink,andNosZactivitysustainsviabilityduringtemporaryanoxia.Interest-

ingly,nosZgenesaffiliatedwiththephylumGemmatimonadetesareamongthemostfrequently

encountered nosZgenesinmanyterrestrial ecosystems[47,48].

46 TrendsinMicrobiology,January2018,Vol.26,No.1
Box1.MicrobialProcessesGeneratingNitrousOxide(N O)
2
Nitrousoxideisproducedbymanydifferentmicrobialguildsaspartofvariouspathways,inwhichinorganicnitrogenous

compoundsaretransformed(Figure1).TherelativecontributiontonetNOemissionsofthedifferentprocessesvaries
considerably and isinfluence dbythe m icrob ialcom munityande nv iron m2en talconditio n. How ever,th emajorityo fNO
emittedisprim aril ya ttributedto d enitr ificationa ndammonia oxi dationtoNO(cid:1) ,thefirsts tepinthen itrifi cationp roc es2s.
Denitrific a tionoften includes red uctionofNO to N,butde nitrifiersm a yno2ta lway sp erform com pletedenit rification,
resultinginthe form ationofN Oasterm in al2pro du ct2(cid:1) eit herbecaus ethe ylac ktheg eneticca pacityfor NOreduction
2 2
toN orbecauseenvironmentalfactorssuppressthereaction[4,19,73].Anexampleoftheformerarefungithatdenitrify
[81 –283]. Ammon iaoxidationis anene rgy-yieldin g,a erobicp rocessin wh ichNH is oxi dizedt oN O(cid:1)v iath einter-
3 2
mediateNHOH.WhileNOcanbeproducedfromabioticoxidationofNHOH[84],ithasbeenshownthatNH
oxidation cou2pled to enz ym2at ic NO 2(cid:1) and NO re ductio n (i.e., n itrifier-den itr ificat2ion) is the m ain r oute f or N2O e miss ions3
during ammonia oxidation [85]. Recent studies show that ammonia-oxidizing archaea generate N2O through hybrid
ofoxrimdiazitniogn bvaiac tae rsiapo[8n6ta–n8e8o].uDs urreinagctiroens poifr aNtoHr2yOaHm amnodn iNficOa,t iaolnth(oduisgshim thilaetyo lriykenlyit rpartoedruecdeu cletisosn Nto2Oa mthmano nthiuem am(DmNoRnAia))-,
crpNeorOpono3ds(cid:1)ruet/csrNv ecaOd ot i2onw(cid:1)nchl iue[s9dn 1re eN] .d t OhTuah citnee t dhtch eote onp tcrrNoeiblHclu ei4tss+io sdnw ec itootohfn x rtiterfihiesbepdu i tcr[ea9ost0 no m]cr.y oaS marogmimtinamean lotlD ynf NoitfiorRcm NaAat 2ibtoOioan nc et tmoeo rifosi avss emicoranaansllll aNcaloms2Oomo plubinanu krtde sNg do2e tOfto sN rdies2eO dnuu int(cercit fi.eigocr.nta,a ttii[noo8,n 9 Na ]a)2l.tn hwTdoh iu taehgm hNem n2meOoron gsiiasyt
oxidation[85,92].NitritealsoreactschemicallywithferrousirontoformNO,whichreactsfurtherwithferrousirontoform
N2O, and this ch emode nitrifi cation process c an m ake rel evan t contr ibutio ns to N2O fo rmatio n in environ men ts with
activeferric-iron-reducingmicroorganisms.Nitrousoxideproductionbymethane-oxidizingbacteria,nitriteoxidizing
bacter ia,andanaerobicm ethane-andanae robicam mon ium-oxidizin gb acteriahavealsob eenrepor ted[93 –96]but

theircontributionstoglobalNOemissionsareunclear.
2
WhenN Oisusedforrespirationbydenitrifiers,reductionmaynotoccurwiththesamekinetics
2

[18],suggestingadaptationstodifferentenvironmentalconditions.Recentworkonthekinetics

and regulation of N O reduction by Bacillus vireti highlights how environmental conditions
2

controlthepossibilityofanorganismwithcladeIInosZactingasapotentialN Osink[49,50].
2

ReductionofN Owasdependentonnitrateconcentrationsintheculturemedium,withhigher
2

concentrations(>30mM)inhibitingNosZactivity.nosZwashighlyexpressedatlowernitrate

levels(5mM),andreductionofexogenousN Owascoupledtogrowth,providedthatlowlevels
2

of nitrate or nitrite were present to induce expression. A recent comparison of five bacterial

species thatsustain growthwithN Oasthe onlyelectron acceptor showed thatthose with
2

cladeIInosZexhibitedlowerwhole-cellhalf-saturationconstants(K values)thanthosewith
s

cladeInosZ,althoughthelattertendedtoshowhighermaximumN Oreductionrates[20].The
2

cladeIIbacteriaproduced50–80%morebiomasspermolofN Oreduced,indicatinganN O
2 2

respiratorychainthatallowsformoreefficientfreeenergyconservationcomparedtothecladeI

systeminthestudiedorganisms.Whetherthisobservationcanbegeneralizedtoallorganisms

withcladeIInosZhasyettobeconfirmed.ShouldcladeIInosZorganismsindeedhavedifferent

N OreductionkineticsfromthosewithcladeI,thiscouldexplainnichedifferentiationandwould
2

haveecological andenvironmentalconsequences.

Ecology of N O-Reducing Communities
2

AfterthenewlineagesofN O-reducingmicroorganismsweredescribed,microbialecologists
2

studied these microorganisms in different environments. A number of studies assessed the

diversityandabundanceofN Oreducerstodeterminefactorsexplainingtheirdistribution.The
2

diversity is typically higher for nosZ clade II than for clade I (e.g., [47,51]), which would be

expectedduetothelargertaxonomicbreadthofnosZcladeIIamongsequencedgenomes

[19].ThemajorityofenvironmentalcladeInosZsequencesaremostsimilartonosZfromAlpha-

and Betaproteobacteria (Burkholderiales) in both marine and terrestrial habitats [47,51–54].

Within the Alphaproteobacteria, Rhodobacteralesdominate in marine sediments and Rhizo-

biaceae in soils. Clade II nosZ sequences are predominantly affiliated with Bacteroidetes,

Gemmatimonadetes, and Deltaproteobacteria in most environments, although Gammapro-

teobacteria and unclassified archaea are also commonly detected in marine and estuary

sediments [47,48,51–56]. Several environmental clade II nosZ reads are assigned to

TrendsinMicrobiology,January2018,Vol.26,No.1 47
rFeigduucreta s2e. sT(anxirKonaondmnicir SD,irsetsrpibeucttiiovenly o)ifn Dvoelvneitdriifinedre annitdrifi Nca2tOio nR,easdwuceellar sGtehenodimffeeresn. tGveanrioamntesso hfathrbeonroinsgZ ggeenneese ennccooddininggt htheeN coOppreedr-u acntads cey.tTohcehcroemntee rdt1r-eteypiseb naitsreitde
2
onNCBItaxonomicrankings,withcladesizeproportionaltothenumberofgenomesfoundwithineachphylumor,fortheProteobacteria,classgrouping.Venn

diagramsillustratethefrequencyofnirandnosZgeneco-occurrencesinthegenomesofeachtaxonomicgroup,wherecirclesizeisproportionaltothefrequencyof

geneoccurrencewithineachgroup,andoverlapindicatesdegreeofgeneco-occurrences.Color-codingforeachcombinationisshownatlowerright.Basedondata

reportedin[19].

nondenitrifying genomes of genera such as Anaeromyxobacter, Dyadobacter, Gemmatimo-

nas, Ignavibacterium, Melioribacter, and Pedobacter. These organisms could be using N O
2

originating from other organisms, chemodenitrification, or other sources [13,55,57] (Box 2).

TheirwidespreaddistributionindicatesthattheabilitytoreduceN Oisanecologicallyrelevant
2

trait.
PioneeringworkshowedthatcladeIInosZwasequallyormoreabundantthancladeIinarange

ofenvironments,forexample,soils,wastewater-treatmentplants,wetlands,andricepaddies

[14].Subsequentstudieshavemainlyfocusedonterrestrialenvironments,especiallyagricul-

tural soilsdueto theirlargecontributiontoN Oemissions.Numericaldominance ofcladeII
2

nosZovercladeI,withratiosof1.5–10,iscommonlyreportedwhenusingquantitativePCR

[14,58,59],althoughtherearesoilswithbothhigherandlowerratios[47,52,56].Studiesusing

metagenomics,whichcircumventsprimerbias,supportsthedominanceofcladeIInosZin

severalterrestrialhabitats[48,54].Nitrousoxidereducersinaquaticandengineeredsystems,

forexample,wastewater-treatmentplants,havesofarreceivedlessattention.Inasaltmarsh,

48 TrendsinMicrobiology,January2018,Vol.26,No.1
Figure3.Maximum-Likelihood-BasedPhylogenyofNosZAmino-AcidSequencesShowingtheTwoMajorCladesofNosZ(IandII).Majorlineagesof

monophyletictaxonomiccompositionattheclassorphylumlevelarecollapsed,withpolygonsizeproportionaltoamino-acidsequencediversitywithineachgrouping,

anddottedbranchesindicatingbootstrapsupportlessthan70%.PiechartsillustratethepercentageofgenomeswitheachnosZvariantthatlacksthenirgenes
asso ciated withdenitr ification(b lack),orpo ssesses eithe rnirS (light gre y)ornir K(darkg ray) .Percentage sa reshown forg enom eswit handw itho utthe norB /Z genes
that encode the heme–coppe r nitric o xid e reductase that r educ es N O to N 2O . Th e num ber o f genomes fo r ea ch grou p is shown be low each pie cha rt. B ased o n data
reportedin[19].

clade II nosZ was three times more abundant than clade I nosZ based on metagenomics

analysis[53].InPCR-basedstudiesofaquaticenvironments,thetwocladeswereeitherequally

abundantorcladeInosZdominated.Inwetlands,boreallakes,andaeutrophiclagoon,cladeI

nosZwasonlyslightlymoreabundantthancladeIInosZ[60–62],butupto1000timesmore

abundantinconstructedwetlandsandinabioreactortreatingnitrate-richwater[63,64].Alsoin

coastalsedimentsandsaltmarshes,cladeInosZoccurredata(cid:3)10-foldhigherabundance

than clade II nosZ [51,65]. Based on the abundance patterns, complete denitrifiers, being

overrepresentedincladeInosZ[19],couldhaveanimportantroleinN Oreductioninaquatic
2

systems,althoughactivityneedstobeconfirmed.Bycontrast,organismswithcladeIInosZ

would, in general, be more relevant in soils. The high abundance of clade II nosZ micro-

organisms,togetherwiththeirgreatdiversityintheenvironment,emphasizesN Oreductionas
2

anecologicallyfavorabletrait,whichpotentiallycontrolsnetN Oemissionsfromecosystems.
2

TrendsinMicrobiology,January2018,Vol.26,No.1 49
Figure4.ComparisonofnosGeneClustersfromGenomesHarboringCladeIorCladeIInosZGenes.Phylogenyatrightisbasedonthephylogenyshown

inFigure2,withphylumorclassleveltaxonomicdesignationundereachname,andthescalebarindicatingestimatedamino-acidsubstitutionspersite.Foreachgene

cluster,nosZanditsaccessorygenes(BDFGHLRXY)arelabeledandcoloredaccordingtohomologyacrossthedifferentgeneclusters.ForeachnosZreadingframe,

N-terminalregionsarecoloredaccordingtosignalpeptidestargetingtheSec(yellow)ortwin-argininetranslocation(TAT;green)pathwaysthatinsertthenosZproduct
intothepe riplasmi csp ace.Ad ditionalpro te ins,inc ludingiro n–sulfur- bind ing proteins (F eS),Rieskeir on–sulfurprot eins( S),and b-andc- type cytoc hrom es(C yband

Cyc,respectively)arealsolabeled.Noncoloredgenesdenoteopenreadingframeswithnoorthologsinothernosgeneclusters,andthescalebaratlower-right

indicatesgenesize.Adaptedfrom[13].

ContrastingpatternsintermsofabundanceanddiversityofthetwomainnosZcladeswere

typically observed within the studies referenced above, which suggest niche differentiation

betweenthetwoclades.Nevertheless,therearefewspecificfactorsthatcorrelatewithoneor

the other clade when comparing across individual studies. Soil textural components, for

example, clay or loam content [47,58,62], as well as nutrient status, for example, nitrogen,

carbon,organicmatter,orC:Nratio[47,53,58,61,65],wereshowntopromotethesizeofthe

nosZ clade Icommunity, yet no specific factors favoringnosZ clade IIhave emerged. Thus,

underlying drivers are only indirectly captured or context-dependent, and biotic drivers or

interactionscouldhavebeenoverlooked.Interestingly,microorganismsharboringeithergene

occupieddifferentecologicalnichesintheroot–soilinterface,withnosZcladeIItypesfavoredin

the soil compartment and type I associated with crop roots regardless of soil type [19]. In

agreement,theabundanceratioofcladeIandcladeIInosZwashigherintherhizosphereof

silverbirchcomparedtobulksoil[66].BothofthesestudiesindicateatighterlinkbetweenN O
2

productionandreductionviathedenitrificationpathwayintherhizospherecomparedtobulk

soil(Box2).GiventhephylogeneticdiversityoforganismswiththecapacitytoreduceN O,itis
2

notsurprisingthatdifferentnichesexistalsowithineachofthetwoclades.Forexample,niche

partitioningforbothcladeIandcladeIInosZwasevidencedinrelationtooxygenavailability,

50 TrendsinMicrobiology,January2018,Vol.26,No.1
Box2.HypothesizedInteractionsbetweenN OProducersandN OReducers
2 2
SinceNOisformedinanumberofprocesses,butonlyconsumedviathereductiontoN,interactionsbetweenthe
2 2
producersandreducerscanbeassumed.Indeed,consumptionofNOwasdemonstratedexperimentallyinsoil
2
microcosmsinoculatedwithanondenitrifyingbacteriumcapableofeliminatingNOproducedbythenativemicrobial
community[5 7].Thede nitrific a tionpathwayco uldpotent iallyalso be splitoverm u2ltip letaxaas the pat hwayis modular

[8,19],consistingofasuiteoffunctionsthatcaneitheractinconcertorbeperformedindividually,dependingon

genomicorenvironmentalconstraints.Thisraisesseveralquestions.Isthismodularityanexampleoflabordivisionin

communitiesassembledintoconsortiatocarryoutacooperativefunction?Ifso,whyisthepathwaytruncatedforsome
microorganis ms but not for others? I n other wo rd s, what are the fitnes s ad vant ag es of comp lete and t run cated
denitrificationpa thwa ys, and underw hic hcon ditionsa rethe yfa vore d?Itca nbehypoth esi zedthats ubstr atecross-

feeding eliminates interenzyme competition and accelerates substrate consumption. This was demonstrated in
experim entswithis ogenicmutan tsofthecom plete denitrifierP seudomon asstutzeri,asn itrate and nitritereductase s,

whenfunctionalinseparatepopulationsinthesameculture,nolongercompetedforelectrondonorsastheywouldif

functioninginthesamecell,therebyavoidingaccumulationofgrowth-inhibitinglevelsofnitrite[97].Cross-feedingof
Nsu2bOs taamntoianl gn u cmobme prleotfeg eonr opm aertsiawl dithe nnitorisfiZertsh aa tnddo Nn2oOt proesdsu ecses rsa mdeanyi traifilscoa tbioen a pdavthawnt aayg. eTohuiss,t yapse so ufgingteesrtaecdti obny wt haes

inferredaftergenomicreconstructionofshotgunsequencereadsfromanestuaryshowingthatgenesforatruncated
denitrific ation pathway – terminating in N 2O – wer e present i n popu lation s o f Gamm aproteob acte ria, wh ile th e ability to
rBreoodlexu fc1oera NNn2odOsGZ w loianss ns faoornuydn).edAn oitnrreilfiyce ienrsn G twesomuurmvldea ybtiemu stooinn gsacdsaetvateebsnle gaenN dN iMs2oOyt xogopecenosecrrcaeatveleedas lbethyda ott thphaoetsr csbehiosestmi cco laadsde enw iIteIr nlilfi oacssaZ tai o[b5ni5od]t.i rcAo npveorotahceesrsi gpsneosisfi sc(siabenleet

fractionoftheNOgenerationinestuarinesediments[26].SinceAnaeromyxobacterdehalogenans2CP-Cconstitu-
tivelyex pre sse s2Fe( III)-reducing a ctivityand canreduce NO (cid:1)toN O(cid:1)andNOtoN [98,99],onec ouldhyp othesize
thatt hisbacteriu mreducesNO gener ated viac hemod enitr3ificat ion.2Thisw ou2ld be an2example ofa nond enitrifierthat
com pete swithden itrifiersb y2co uplingbioti ca ndbioticreactions.

eutrophicationlevel,pH,C:Nratios,croppingsystems,andbiocharadditiontoagriculturalsoil

[51–53,56,67].
Ramifications for Ecosystem Functioning

ThediversityoftheN O-producingandN O-reducingcommunitiesultimatelydeterminesthe
2 2

netN Oemissions,sincecommunitieswithdifferentgeneticpotentials(e.g.,assemblagesof
2

variousfunctionalmodules)aresubjecttoecologicalprocessesthatinfluencetheirabundance,

diversity, and activity. The feedback between dynamic environmental conditions and evolu-

tionary processes will determine the structure and diversity of N O-producing and N O-
2 2

reducingcommunities.By‘structureanddiversity’,wemeannotonlytheassemblageoftaxa

thatconstitutethecommunities,butalsotheassociationofthevariousprocesseswithinthe

denitrification pathway or in association with other processes. Early work suggested that

differences in relative rates of N O production between an agricultural and a successional
2

field could only be explained by the denitrifier community composition when controlling all

known environmental factors regulating N O production and consumption by denitrification
2

[68].However,theinfluenceofthedenitrifiercommunitycompositionandthemodularityofthe

denitrificationpathwayareinterwovensinceco-occurrencepatternsofdenitrificationgenesare

notrandomlydistributedacrosstaxa[19].Forinstance,thehigherfrequencyofco-occurrence

ofnosZwithnirSthanwithnirK(Figure3)suggeststhatnirStypedenitrifiersaremorelikelyto

performcompletedenitrification,andtherebycontributelesstoN Oemissions,thanisthecase
2

fornirKtypedenitrifiers.AfieldstudyreportedapositivecorrelationbetweennirKabundance

and N O emissions, and qPCR analyses suggested that only 10–30% of the nirK type
2

denitrifiersalsocarriedthenosZgene[69].Theputativeimportanceofatruncationinthelast

step of the denitrification pathwaywascorroborated by a negativerelationshipbetween the

proportion of bacteria with nosZ and the N O-to-N denitrification end-product ratio in a
2 2

pasture[70].Similarly,interlakevariationinN Oaccumulationwasconnectedtotherelative
2

abundance of nir versus nosZ genes [60]. Other examples of such correlations have been

reported in different ecosystems, for example [71,72], but experimental testing of causal

relationships is needed. Addition of Agrobacterium tumefaciens C58, a denitrifying strain

lacking nosZ and therefore terminating with N O, to different soils resulted in an increase in
2

TrendsinMicrobiology,January2018,Vol.26,No.1 51
therelativeamountofN Oemitted[73].Thisdirectmanipulationexperimentprovidesaproofof Outstanding Questions
2
principlethattheinabilitytosynthesizeNosZcaninfluencetheratioofthedenitrificationend- Whatisthemainpurposeofreduction

products,andthattheextentofthereductionofN2OtoN2hasbothgeneticandenvironmental of N2O in the environment? Microor-
controls. ganisms can reduce N2O for different
purposes,thatis,energyconservation,
detoxificat ion, or remov al of excess
AfterthefindingthatnosZcouldbedelineatedintotwodistinctclades,oneofthequestions electrons. En viro nmental seq uences
raised wa swhet herd ifferen cesb etw eenmicroo rgan isms withcla deIand IIno sZ co uldimpact indicateth atsuchfunctio nalvariation

is present, if we can assume that
net N O emissions. In earlier studies considering only the clade I nosZ community, the link
2 sequence similarity equals functional
betweennosZcommunitymetricsandN2Oemissionsrangesfrombeingstrong,weak,oreven similarity.

absent[74–77].However,arecentlandmarkpaperhighlightedtheputativeroleofthecladeII

nosZratherthancladeInosZcommunityforthecapacityofsoilstoactasN Osinks[47].This Areresponsestoenvironmentalcon-
ehwmaalfsiso ss fuiocplnapsdo e[r5 tIe2Idn,5o b4s y]Z. laTmtheiecr s rs oeto uordgbieas sne irlsivnmekdsin ldgai cftfhkeern e icrnlgati aedlne re oI Is lnewosh saiZlre ecm oc omosn mtsoiusfntteiht nyet w cwliatihtdh er ethId neuo 2cfisneZdd ti ynppgoe ttesh napttoia asl sbNeo2suOst Sadtiogiitmnieo ,sinla wisnr lh yct,eho anenr seNce o2erOfvufi ep crdleiee ddwnucictwihetiaistnhs o edfe ipNfnfhee2yrOreglo nyrget edclinounynce?---

nirKornirS[19].TheproposedroleofcladeIInosZmicroorganismsinthesoilN O-reduction servation, conserved in the
capa ci tywa srec entl yverified[5 7].I no culatio n ofDy adobacterferme nt ans –a no2ndenitrifying phylogeny ? This type o f infor mation
is needed to understand if and how
bacterium possessing clade II nosZ and commonly detected in soil metagenomes [48] – to
evolution and niche differentiation
different s oils resulted in a de creas e in N2O emiss ion of up t o 18 9% in one-thi rd of t he among a nd w ithin t he two major
inoculated soils [57]. This demonstrates that clade II nosZ bacteria can consume N2O emitted icslmadseas reofl inNk2eOd .reducing microorgan-
insoilbyothermicroorganisms,andsuggeststhatatighterlinkbetweenN2Oproducersand

reducers could have relevance for mitigating N O emissions (Box 2). It has been known
2 IsnichedifferentiationbetweencladeI
anecdotallyforsomeyearsthatsoilscanconsumeN Oinsitu[78–80],butthemechanisms
2 andIIorganismsmainlyduetodiffer-

have not been investigated. Further work is needed to determine if N O emissions are encesrelatedtotheNOreductaseor
2 2
controlled by N O production or N O consumption, why and when soils increase N O areothertraitsthatvarybetweenthese
2 2 2
twogroupsmoreimportantinthiscon-
consumption, and whether microbial N2O reduction can be important, in quantitative terms, text. Thedi stribu tionofthe tw ot ypes
for curbing global N2O emissions. of N2 O re ductase is c orr elat ed to taxa,
andtherebyinterlinked.Thisneedsto

Concluding Remarks beteasedaparttobeabletomanipu-

The recent dis covery of clade II nosZ, and the realization that nondenitrifying N O-reducing late and manage the N2O reducers in
2 managedecosystems.
microorganismsarepresentinhighnumbersandgreatdiversityinessentiallyallecosystems,

has opened up a new field of research. Research conducted over the past 5 years has Are direct associations between N2O
souf bthseta fnutniacltlyio enxapl uannidtse dth oaut rd ruivned eNr2sOta nreddinugc toiof nt hteo oNr2gaanndis munadl, egrsecnoormesic t,h aen ads gseernteiotnic mdaivdeers bityy mtpargooends,u cafoenrrdst hifae nsoodr, g Nawn2hOisam t raser?deu tcheer sa dcvoamn--
ZumftandKroneck[40],thatN Oreduction‘representsarespiratoryprocessinitsownright’.
2

Both N O production and N O reduction are integral parts of the nitrogen cycle, yet most
ecos yst2em s emit N2O . Em iss2ion of N2O is of environm ental co nce rn, and e xperim ent s and Draote tsh eo fr aNte2sO o rf eNd2uOct ifoonrm caotniotnro ol rN t2hOe
surveyscomparingN OreducerswithincladeIandcladeIIsuggestthatcladeIIorganismsare emission,andisitecosystem-depen-
key det erminants o f 2a soil’s N2O sink capac ity. M ore i ns ight into the ecol og y of the N 2O sdieonnt?c oKnnto rowlsled cog ue ld of auidndinerldyeinvge loempinisg-
reducers is needed, especially mechanisms and factors explaining niche differentiation
andimprovingstrategiestodecrease
between org anisms w ith clade I and II nosZ, and betwee n organism s with in each of the NO emissions .
2
clades. Additional questions to further our understanding revolve around what the main

purpose of N O reduction is in the environment, whether close interactions between N O
2 2

producers and N O reducers are common, and if there are advantages of labor division in
2

consortia splittingthe denitrificationpathway (seeOutstanding Questions).Thisreview high-

lights advancements in the field, but further progress is essential for developing predictive

understandingofN Ofluxesandgeneratingimprovedmanagementstrategiesthatcancurb
2

N Oemissions.
2

Acknowledgments
ThisworkwassupportedbygrantsfromtheSwedishResearchCouncilVR(grant2016-03551toSH),theSwedish

Research Council FORMAS (grant 2013-656 to SH), and the US Department of Energy, Office of Biological and

EnvironmentalResearch.

52 TrendsinMicrobiology,January2018,Vol.26,No.1