nature

ARTICLENitricoxide-dependentanaerobicammoniumoxidationZiyeHu1,3,HansJ.C.T.Wessels2,TheovanAlen1,MikeS.M.Jetten1&BoranKartal1,4Nitricoxide(NO)hasimportantfunctionsinbiologyandatmosphericchemistryasatoxin,signalingmolecule,ozonedepletingagentandtheprecursorofthegreenhousegasnitrousoxide(N2O).AlthoughNOisapotentoxidant,andwasavailableonEarthearlierthanoxygen,itisunclearwhetherNOcanbeusedbymicroorganismsforgrowth.Anaerobicammonium-oxidizing(anammox)bacteriacouplenitritereductiontoammoniumoxidationwithNOandhydrazineasintermediates,andproduceN2andnitrate.Here,weshowthattheanammoxbacteriumKueneniastuttgartiensisisabletogrowintheabsenceofnitritebycouplingammoniumoxidationtoNOreduction,andproduceonlyN2.Underthesegrowthconditions,thetranscriptionofproteinsnecessaryforNOgenerationisdownregulated.OurworkhaspotentialimplicationsinthecontrolofN2OandNOemissionsfromnaturalandmanmadeecosystems,whereanammoxbacteriacontributesignificantlytoN2releasetotheatmosphere.WehypothesizethatmicrobialNO-dependentammoniumoxidationmayhaveexistedonearlyEarth.:Sanquin,Plesmanlaan125,1066CXAmsterdam,TheNetherlands.4Presentaddress:MicrobialPhysiologyGroup,MaxPlanckInstituteforMarineMicrobiology,Celsiusstraße1,28359Bremen,Germany.CorrespondenceandrequestsformaterialsshouldbeaddressedtoB.K.(email:[e-mail])NATURECOMMUNICATIONS|(2019)10:1244||():,;Nitricoxide(NO)isahighlyreactivemoleculethatplaysakeyroleinatmosphericchemistryasanozonedepletingagentandastheprecursorofthegreenhousegasnitrousoxide(N2O)1–3.InbiologyNOhasseveraldistinctfunctions:itisaverypotenttoxin,butalsoasignalingmoleculeandanintermediateinthemicrobialnitrogencycle4.ToremovetoxicNO,microorganismsuseamultitudeofenzymes,andintricatemechanismsthatsense,scavenge,andconvertNOtolessreactivecompoundssuchasN2O5.Duetoitshighreactivity,NOexistsonlyatverylowconcentrationsincells,andisrapidlyturnedover,whichiswhyinbothdenitrificationandaerobicammoniaoxidationitwasthelastrecognizedintermediate6–8.Inmicrobialprocesses,NOiseithergeneratedvianitritereductioncatalyzedbyCu-containing(Cu-NIR)orcytochromecd1-containing(cd1-NIR)nitritereductases7,9,oralternativelybyhydroxylamineoxidationcatalyzedbyoctahemehydroxylamineoxidoreductases(HAO)6,10.Intriguingly,microbialgrowthwithNOastheterminalelectronacceptorhavenotbeenshownbefore.However,beforemolecularoxygenstartedtoaccumulateintheatmosphere,NOwasthestrongestoxidantavailableonearth11–14,whichsuggeststhatmicroorganismscapableofusingexternalNOastheterminalelectronacceptorcouldhaveevolvedearlyinthehistoryoflife.Anaerobicammonium-oxidizing(anammox)bacteriausetheoxidativepowerofNOtoactivateammoniumintheabsenceofoxygen.Thesemicroorganismsnormallyusenitriteastheirterminalelectronacceptor,producingnitrateandN215.Theircatabolismcanbedescribedinthreemainreactions:First,NO2−isreducedtoNO(Eq.(1));thenNOandNH4+arecondensedintohydrazine(N2H4)byhydrazinesynthase(Eq.(2)),whichisfollowedbytheoxidationofN2H4toN2byhydrazinedehy-drogenase(Eq.(3)).Thefourelectronsreleasedfromhydrazineoxidationareusedfornitritereduction(1electron)andhydrazinesynthesis(3electrons)completingtheanammoxcataboliccycle.Theelectronsthatarenecessaryforcellcarbonfixationaresuggestedtobedeliveredbytheoxidationofnitritetonitrate(Eq.(4))basedontheobservationthatthegrowthofanammoxbacteriaappearstobealwaysassociatedwithnitrateproduction15,16.NO
2þ2Hþþe
!NOþH2OðE′0¼þ0:38VÞð1ÞNOþNHþ4þ2Hþþ3e
!N2H4þH2OðE′0¼þ0:06VÞð2ÞN2H4!N2þ4Hþþ4e
ðE′0¼
0:75VÞð3ÞNO
2!NO
3þ2Hþþ2e
ðE′0¼þ0:42VÞð4ÞWhiletheenzymesresponsibleforreactions(2)–(4)arecon-servedinallknownanammoxgenera17,nitritereductiontoNOiscatalyzedbydistinctenzymesthatarealsofoundinothernitrogen-transformingmicroorganisms7,9.TheanammoxspeciesKueneniastuttgartiensisandScalinduaprofundaencodecd1-NIR18–20,Jetteniaspp.encodeCu-NIR21,whereasBrocadiaspp.donotencodeanyknownnitritereductases22.Moreover,allanammoxbacteriaencodeanoctahemeHAOthatcatalyzestheoxidationofhydroxylaminetoNO10,17.ItisevidentthatdifferentanammoxspecieshavedifferentNO-formingpathways,whichsuggeststhatnitritereductiontoNOmightbeatraitthatwasacquiredafterthecoreanammoxcatabolismwasalreadyinplace.Indeed,reactions(2)and(3)wouldbesufficientbothtoconserveenergyandsupplynecessaryelectronsforcellcarbonfixation(CO2)forbiomass.Inthisscenario,threeofthefourelectronsreleasedfromhydrazineoxidationwouldbeusedforhydrazinesynthesis,andtheremainingelectroncouldbeusedforbiomassproduction,withouttheneedfornitriteoxidationtonitrate.Totestthishypothesis,afree-livingplanktonicK.stuttgar-tiensisculturecontinuouslysuppliedwithammoniumandNOastheonlysubstratesinacontinuousmembranebioreactorisemployed.WeshowthatK.stuttgartien