Iron assimilation and utilization in anaerobic ammonium oxidizing bacteria

Delft University of Technology

Iron assimilation and utilization in anaerobic ammonium oxidizing bacteria

Ferousi, Christina; Lindhoud, Simon; Baymann, Frauke; Kartal, Boran; Jetten, Mike SM; Reimann, Joachim DOI

10.1016/j.cbpa.2017.03.009 Publication date

2017

Document Version Final published version Published in

Current Opinion in Chemical Biology

Citation (APA)

Ferousi, C., Lindhoud, S., Baymann, F., Kartal, B., Jetten, M. SM., & Reimann, J. (2017). Iron assimilation and utilization in anaerobic ammonium oxidizing bacteria. Current Opinion in Chemical Biology, 37, 129-136. https://doi.org/10.1016/j.cbpa.2017.03.009

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Iron

assimilation

and

utilization

in

anaerobic

ammonium

oxidizing

bacteria

Christina

Ferousi

,

Simon

Lindhoud

,

Frauke

Baymann

,

Boran

Kartal

,

Mike

SM

Jetten

and

Joachim

Reimann

Themost abundanttransition metal in biological systems is iron. It

isincorporatedintoproteincofactorsandserveseithercatalytic,

redoxorregulatorypurposes.Anaerobicammoniumoxidizing

(anammox)bacteriarelyheavilyoniron-containingproteins–

especiallycytochromes–fortheirenergyconservation,which

occurswithinauniqueorganelle,theanammoxosome.Boththeir

anaerobiclifestyleandthepresenceofanadditionalcellular

compartmentchallengeourunderstandingofironprocessing.

Here,wecombineexistingconceptsofironuptake,utilization

andmetabolism,andcellularfatewithgenomicandstilllimited

biochemicalandphysiologicaldataonanammoxbacteriato

proposepathwaysthesebacteriamayemploy.

Addresses

1

DepartmentofMicrobiology,InstituteforWaterandWetlandResearch, RadboudUniversity,Heyendaalseweg135,6525AJNijmegen,The Netherlands

2_{LaboratoiredeBioe´nerge´tiqueetInge´nieriedesProte´inesUMR}

7281CNRS/AMU,FR3479,MarseilleCedex2013402,France

3

MicrobialPhysiologyGroup,MaxPlanckInstituteforMarine Microbiology,Celsiusstrasse1,28359Bremen,Germany

4_{DepartmentofBiotechnology,DelftUniversityofTechnology,Vander}

Maasweg9,2629HZDelft,TheNetherlands

5_{SoehngenInstituteofAnaerobicMicrobiology,Heyendaalseweg135,}

6525AJNijmegen,TheNetherlands

Correspondingauthor:Reimann,Joachim(j.reimann@science.ru.nl)

CurrentOpinioninChemicalBiology2017,37:129–136

ThisreviewcomesfromathemedissueonBioinorganicChemistry EditedbyMaartenMerkxandAntonioJPierik

ForacompleteoverviewseetheIssueandtheEditorial

Availableonline30thMarch2017

http://dx.doi.org/10.1016/j.cbpa.2017.03.009

1367-5931/ã2017TheAuthors.PublishedbyElsevierLtd.Thisisan openaccessarticleundertheCCBY-NC-NDlicense( http://creative-commons.org/licenses/by-nc-nd/4.0/).

Introduction

Iron is themostabundanttransition metalin biological systems,whereitisemployedasanessentialcofactorin redox chemistry, electrontransfer reactions and regula-tory processes. The capacity of iron to formcomplexes withcarbon,oxygen,sulfur,andnitrogen,andthe impres-sivelywiderangeofredoxpotentialsironmetalloproteins exhibit (
700mV to +350mV) [1] contributes to its involvementin allbiogeochemicalcycles.

The majority of iron-dependent redox proteins harbor ironwithinhemes andin theformofiron–sulfur(Fe–S) clusters [1]. Bacteria and Archaea show an enormous diversity and abundance of iron–sulfur and heme-con-taining proteins, which reflects their broad metabolic capacities [1,2]. Some of these organisms feature large amountsofiron-containingproteins.Thisisthecasefor anammoxbacteria,whichoxidizeammoniumwithnitrite astheterminalelectronacceptorintheabsenceofoxygen [3,4].Thegenomesofthesemicroorganismsencodefor about60c-typecytochromes[5–9],amongwhicharekey catabolicenzymesthatconstituteatleast50%ofthetotal protein mass. Compared to an Escherichia coli cell that contains 105–106 atoms of protein-bound iron [10,11], each anammox cell canbe estimated to manage apool ofabout107ironatoms.Thebrightredcolorofanammox enrichment culturesreflectsthis highcontent of heme-bound iron(Figure1a).

Anammox bacteriaareanaerobicGram-negative micro-organismswithinthephylumofPlanctomycetes,andhave acompartmentalizedcellplan.Inadditiontothe pepti-doglycan-containingcellwall[12]and thecytoplasmic membrane, they possess a third and innermost mem-branethatdefinestheanammoxosome,aunique energy-converting organelle that occupies a large volume of the cell (70%) [13,14] (Figure 1b). This additional compartmentaddsalayerofcomplexitytointracellular trafficking of nutrients and co-substrates, as well as protein sorting.

Theanammoxosomecontainsthevastmajorityofcellular iron in the form of cofactors withinFe–S proteins and multi-hemecytochromes[4],whichareinvolvedinthe oxidationofammoniumtodinitrogengas.Inthecurrent model(Figure2)[3,4],nitriteisreducedtonitricoxide, and subsequentlyhydrazine synthasecatalyzesthe con-densation of nitric oxide and ammonium to produce hydrazine[15,16],themostpowerfulchemicalreductant in nature(E00=
700mV).Thisisfollowedbythe oxi-dationofhydrazinetodinitrogengasbyhydrazine dehy-drogenase[17].Thefourlow-potentialelectronsreleased inthisreactionshouldpassthroughelectrogenic respira-torycomplexeswithintheanammoxosomemembranevia a sequence of electron transfer events to build up the membrane potential. Then, the electrons return to the anammoxosometofuelthefirsttwostepsoftheanammox reaction,thusclosingtheelectrontransfercycle.Electron withdrawalfromthecyclicanammoxpathwayduringCO2

fixation is compensated through nitrite oxidation to nitratecatalyzed bynitrite oxidoreductase(NXR). Despitethecentralroleofironintheanammoxprocess, mostaspectsofironinanammoxbacteriahaveremained unexploredtodate.Inthisreviewwewillbrieflydiscuss iron metabolism and protein systems that these micro-organisms may use to assimilate and utilize iron. We thereby follow the ions from outside the cell through

thecytoplasmtotheanammoxosomeinterior(Figure3), witha special emphasisonthe peculiarcellplan and a final discussion of iron-rich nanoparticles inside the anammoxosome.

Iron

metabolism

Thenaturalabundanceof ironcombinedwithitsredox propertiesrenders it aprevalentsubstrate for both het-erotrophic respiration and autotrophic growth. In the 130 BioinorganicChemistry Figure1 (a) (b) _S-Layer Outer membrane Periplasm Cytoplasm Cytoplasmic membrane Anammoxosome Anammoxosome membrane 200 nm

Current Opinion in Chemical Biology

Anammoxenrichmentcultureandcellplan.(a)Laboratory-scalemembranebioreactorenrichedwith95%oftheanammoxbacteriumKuenenia stuttgartiensis.(b)Thecellplanofananammoxbacterium,illustratedwithatransmissionelectronmicrographofK.stuttgartiensis.Cell compartmentsandmembranesareindicated.Arrowsindicateiron-richnanoparticles.Courtesy,L.vanNiftrik.

Figure2 Anammoxosome Cytoplasm NO₂ -NO₃ -N₂H₄ N₂ NO NH₄+ NXR Nir HZS HDH ? R/b ETM ? nH+ 3H+ _4H+ H+ nH+ nH+ nH+ 3-5H+ 2H+ ATP ADP 2e 1e 3e 4e Ψ Ψ+ Ψ

-Current Opinion in Chemical Biology

Currentmodeloftheanammoxpathway.Anaerobicoxidationofammonium(NH4+)todinitrogengas(N2)withnitrite(NO2
)asterminalelectron

acceptorproceedsvianitricoxide(NO)andhydrazine(N2H4)asfreeintermediates.Theavailablefreeenergyoftheanammoxprocessisutilized

tomaintainanelectrochemicalpotentialgradientacrosstheanammoxosomemembrane,whichdrivesATPsynthesis.Electronwithdrawalfrom thecyclicanammoxpathwayduringcarbondioxide(CO2)fixation(notshown)iscompensatedbynitriteoxidationtonitrate(NO3
)catalyzedby

nitriteoxidoreductase(NXR).Diamondsrepresentputativeelectroncarriersandindicatethenumberofelectronsthataretransferredineach reaction.Questionmarksrepresentthehypotheticalcytochrome:quinoneoxidoreductasesthatfeedthequinonepool(Q/QH2)withelectrons

yieldedfromtheoxidationofhydrazineandnitrite.Nir:putativenitritereductase;HZS:hydrazinesynthase;HDH:hydrazinedehydrogenase;R/b: Rieske/cytochromebcomplex,ETM:electrontransfermoduleforhydrazinesynthesis;_C+_{,C
:positiveandnegativesidesofthemembrane,}

formercase,oxidationoforganiccompoundsiscoupledto reductionofextracellularferriciron,whereasinthelatter caseoxidationofirondonateselectronstooxygen, bicar-bonate or nitrate. Theseprocesses involve c-type cyto-chromes andare performed byphylogeneticallydiverse microorganisms.Anammoxbacteriahavebeenobserved to couple formate oxidation to iron reduction [5,18,19] and performnitrate-dependent ironoxidation[5,20]. Fe(III)reduction

ShewanellaandGeobacterarethebest-studied iron-reduc-ingorganismsandprovideuswithgeneraltemplatesfor theidentificationandunderstandingofextracellular elec-tron transfer [21–23]. They employ different, but

functionally similar, systemsto enable electrontransfer fromthequinone(Q)poolatthecytoplasmicmembrane throughtheoutermembranetotheironmineraloutside thecell.Thefirstcomponentisamembranequinol(QH2) dehydrogenase thatislocatedonthecytoplasmic mem-brane and extends into the periplasm with its cyto-chrome-richdomain (CymA in Shewanellaand ImcH or CbcLinGeobacter)[21].Electrontransfertowarda multi-hemeproteinthat ischanneledthroughan outer mem-brane beta barrel occurs either via soluble periplasmic cytochromes [24] or direct interaction [25]. Ultimately, electronsreachtheinsolubleextracellularmineraleither viasolubleshuttlessuchas flavins[26],appendageslike nanowires[27]or directcontact[21].

Figure3

FeoB

Fe(II)

iron-sulfur

proteins _cytochromesc-type

c-type cytochromes iron-sulfur proteins OM OM SL SL PG PG CM CM AM AM IP IP Ψ+ Ψ+ Ψ -Ψ -TAT NifSU TAT Ahb FeoB ? Sec Sec R R S-IIP S-IIA Periplasm Cytoplasm Anammoxosome Current Opinion in Chemical Biology

Proposedpathwaysforironassimilationandutilizationinanammoxbacteria.Solubleferrousiron(Fe(II))freelydiffusesintotheperiplasmthrough poresintheS-Layer(SL)andporesintheoutermembrane(OM)(green).Transportacrossthecytoplasmicmembrane(CM)occursviathe Fe(II)-specificFeoBsystem.Thealternativehemebiosynthesis(Ahb)pathwaysynthesizesb-typehemes.BothaSec-translocon(Sec)andcytochromec maturationsystemsS-IIAandS-IIParerequiredformaturationofc-typecytochromesintheanammoxosomeandperiplasm,respectively.The NifSUmachineryassemblesmatureFe–Sproteins,whicharetranslocatedintotheperiplasmandanammoxosomeviatheTATpathway.The yellowboxinsidetheanammoxosomedenotesunknownpathwaysforthedisassemblyofiron–sulfurproteinsandcytochromesandreleaseof ironions.FeoBattheanammoxosomemembrane(AM)representsapossiblerouteforfreeironbackintothecytoplasmforrecycling.Iron-rich nanoparticles(IP)werehypothesizedtobeironstoragesites,butmayalternativelybecytochrome-containingencapsulinnanocompartments.Red spheresrepresentironions.Dottedlinesrepresentuncertainpathways.R:ribosome;SL:S-layer;PG:peptidoglycan;C+,C
:positiveand negativesidesofthemembrane,respectively.

Reduction of Fe(III) coupled to formate oxidation has beenreportedforanammoxbacteria[5,19],andseemsto adheretothesamedesignfor electrontransferto extra-cellulariron.TheyexpressamembraneQH2 dehydroge-nasehomologoustoCymAbutwitheleveninsteadoffour hemebindingsites,twoperiplasmicmultihemeproteins homologous to Shewanella-MtrA and at least one outer membranebetabarrelporin-likeprotein[28].Although theelectrontransfer throughtheperiplasm couldoccur via direct contact between anammox-CymA and ana-mmox-MtrA, electron transfer via soluble c-type cyto-chromes cannot be excluded. Once outside the cell, it isunclearhowthereductionofparticulateironproceeds. Furthermore, how the oxidation of organicacids inside the cell and the reduction of iron outside the cell are coupledto energyconservationremainselusive. Fe(II)oxidation

Incontrasttoironreduction,themolecularmechanismof bioticironoxidationislesswellunderstood.Inadditionto oxygen in microaerophilic and bicarbonate in photo-trophic iron oxidation, nitrate is identified as another possible terminal electron acceptor of iron oxidation [29,30]. Formationof insoluble ferric oxides in the cell andpossibleinterferenceofabioticandbioticironredox transitionsare potentialcomplications [31].

Anammoxbacteriawereshowntoperform nitrate-depen-dentironoxidation[5,20].Theonly candidatefor cata-lyzingnitratereduction(NXR)isexclusivelylocalizedin theanammoxosome[32],whichrequireseitherFe(II)to beimported intotheanammoxosome or electrons from Fe(II)tocrossthecytoplasmicmembrane,thecytoplasm and the anammoxosome membrane. In the first case, export of Fe(III) might be problematic (see Section ‘Degradation of iron-containing proteins’), whereas in thesecondcasethefreeenergyavailablefromthe reac-tionseemsinsufficienttodriveelectrontransferovertwo membranes.

Iron

uptake

Utilization of iron as metal cofactor of redox enzymes requirestranslocationofironacrossmembranesintothe cellinterior/cytoplasm,where cofactor assemblyoccurs. Because the availability of reduced and oxidized iron differsdramaticallybetweenecosystems,microorganisms employ different strategies to assimilate iron in their respectivehabitats.ProkaryotesthatthriveinpH-neutral aerobic environments where iron mainly exists in the poorlysolubleferricformuseawiderangeofmechanisms toassimilateironfromminerals[33].Aprominent exam-ple is secretion of siderophores: organic iron-chelators thatsolubilizeironand facilitateitsuptake[33,34]. Anammox bacteria live in oxygen-limited and anoxic habitats where soluble ferrous iron is the predominant iron species and do not possess genes required for

siderophore synthesis [5–9]. Soluble ferrous iron is believed to freely diffuse into the periplasm through outer membrane porins [28]. Although several non-specific divalent metal ion transporters exist, only two machineries are known to exclusively transport Fe(II) intothecytoplasm(i.e.,EfeUOBandFeoABC)[35,36]. TheFeoABCsystemexhibitsbroadphylogenetic distri-butionanditistheonlysystemforironuptakefoundin anammoxgenomes(Figure3).AlthoughFeoAandFeoC have been speculated to enhance iron uptake [35], anammoxbacteriaonlycarrythegenefortheiron trans-porter FeoB. The universal transcriptional regulator related to anaerobic metabolism Fnr (fumarate and nitratereductase)[37],aswellasthemetal-specific regu-latorFur(ferricuptakeregulator)[38]canbeassumedto participateinregulationofironuptakeandhomeostasisin anammox bacteria, but a detailed analysis is currently missing.

Biosynthesis

of

iron

cofactors

Themostcommonwayfor biologicalsystemstoexploit theredoxpropertiesofironisitsassemblyinto metallo-prostheticgroupsthatareincorporatedintoprotein com-plexes, whichserve either catalytic, redoxor regulatory purposes.Amongiron-containingcofactors,c-typehemes and Fe–S clusters are omnipresent throughout all domains of life,and anammox bacteria rely heavily on thesetwoclassesofcofactorsfortheirenergymetabolism [3].Belowwediscusshemebbiosynthesis,cytochromec maturation and Fe–S cluster biogenesis in anammox bacteria.

Hemebbiosynthesis

Hemesbelongto abroadclass of organiccofactorsthat useatetrapyrrolemacrocyclictemplateto accommodate thechelation of a metalcenter. Tetrapyrroleformation proceedsviafouror sixuniversallyconservedsteps,the C-4andC-5pathway,respectively,andleadstoformation ofuroporphyrinogenIII[39].Fromthisintermediateon, three different pathways for heme b biosynthesis have been described. The ‘classic pathway’ is conserved among Eukaryotes and Proteobacteria [40] and the ‘HemQ-based route’ is only present in Gram-positive bacteria[41].Remarkably,unliketheotherknown mem-bers of the phylum Planctomycetes, which employ the ‘classic pathway’, anammox genomes encode for the complete machinery of the so-called ‘alternative heme biosynthesis’ (Ahb) pathway [42,43] that is possibly phylogeneticallyolderthanitsclassiccounterpart[44,45]. Cytochromecmaturation

Enzymatic modification of heme b yields chemically distinct hemecofactors(a, b,c, d ando-type).In c-type cytochromes,whicharethemostabundanthemeproteins inanammox, hemebmolecules are covalentlyattached viathioetherbondsoftheirvinylgroupstothesulfhydryls of two, or in rare cases one, cysteine residue(s) [46]. 132 BioinorganicChemistry

Regardless of the heme b biosynthesis pathway, Sec-based protein translocation machinery and cytochrome cmaturationsystemsarerequiredfortheproductionof c-type cytochromes [47]. All three studied cytochrome c maturation systems (I–III) comprise membrane com-plexes that transport bheme across the membrane and catalyzeitscovalentattachmenttotheapoproteinatthe extra-cytoplasmic side [47]. Although, c-type cyto-chromes are likely to be present in the periplasm (see Section‘Ironmetabolism’),themainsiteforcytochromes is the anammoxosome [48]. Since anammox bacteria express two highly similar copies of maturation system II[49],weproposethatbothcytoplasmicand anammoxo-some membranes possess their individual maturation machinery (Figure 3). How proteins destined for the periplasm oranammoxosomearetargeted tothe respec-tive translocation and maturation systems remains an intriguing openquestion.

Iron–sulfurclusterbiosynthesis

Iron–sulfur clusters in anammox bacteria are found not only in ferredoxins, Rieske/cytochrome b complexes, complexI, andhydrogenases,butalsoas electron-trans-ferring cofactors of the highly abundant NXR protein complexresidinginsidetheanammoxosome[3,32]. Bio-synthesis of iron–sulfur clusters in prokaryotes is cata-lyzed by cytoplasmic proteins and involves donationof ironandsulfurtotheassemblyscaffold,maturationofthe cluster,anddeliverytotheapoprotein[50].Threeiron– sulfursynthesissystemsareknown:Isc,SufandNif[51]. TheIscsystemappearstobethegenericpathwayforFe– S cluster assembly whereas the Suf pathway has been associatedwithironlimitationandoxygenstress.TheNif system, on the contrary, was initially associated exclu-sively with the assembly of thenitrogenase enzyme in nitrogen-fixingbacteria[52].However,itsrecent identi-ficationasthesoleFe–Smaturationsystemintwodiverse non-nitrogen fixing organisms has changed this view [53,54]. Interestingly, also in anammox bacteria the NifsystemisseeminglytheonlyFe–Sassembly machin-ery, supporting the notionthat the Nif systemis more versatile than previously assumed. Matured iron–sulfur proteins destined for either the anammoxosome or the periplasm aretransportedacross themembranesviathe twin-argininetranslocationsystem(Figure3).

Degradation

of

iron-containing

proteins

Controlleddegradationofanammoxosomal iron-contain-ingproteinsasamechanismofmetabolicregulationorin thecontextofproteinqualitycontrolhasnotbeen stud-ied,butislikelytooccur.Whiletheanammoxgenomes providecandidateproteasesthatcouldcleavetheprotein backbone,theprocessingofironcofactors,andespecially hememoieties,isintriguinglymoreelusive.Heme deg-radation,forexample,requirestheoxidativecleavageof theporphyrinringbytheactionofhemeoxygenases[55]; a processthat isoxygen-dependentand notcompatible

with the anaerobic lifestyle of anammox bacteria. An alternative oxygen-independent system, similar to the recently discovered radical S-adenosyl-L-methionine

(SAM)-based pathway [56], is conceivable but difficult to identifyonthegenomiclevelandrequiresdedicated geneticandbiochemicalstudies.Furthermore,thefateof ironreleasedfromdisassemblediron–sulfurproteinsand cytochromes is unknown.Whether theanammoxosome membraneprovidesarouteforironintothecytoplasmis unclear,butwemighthypothesizethat,ifpresentinthe anammoxosome membrane, FeoB could play this role (Figure3).However,intheabsenceofsuchamechanism ironwouldaccumulateandcouldonlybedilutedthrough cell–and anammoxosome–division.

Iron-rich

nanoparticles

in

anammox

Anammoxbacteria culturedunderlaboratory conditions possessnanosized(diameter16–25nm)iron-richparticles inside theanammoxosome [13](Figure1).These parti-cles were speculated to be iron storage sites, possibly formed by bacterioferritins; spherical, hollow protein complexes that contain large amounts of iron oxides (2000Featomspercomplex)[57].However,anammox bacterioferritins lack signal peptides that would target themintotheanammoxosome.Therecentlydiscovered encapsulins[58,59]mayprovide analternative explana-tion for the observed particles.Encapsulins form nano-compartments that store cargo proteins with different functions [60]. Indeed, one encapsulin homologue that ispotentiallytargetedtotheanammoxosomeviaasignal sequence,anditsheme-richcargoproteinwere hypothe-sizedforanammoxbacteria[61].Thenatureofthe iron-rich particles in anammox bacteria and the proposed existence offunctional encapsulinsshould bethefocus of futureinvestigations.

Conclusions

Althoughwe havegained considerableinsightsintothe physiology, cell architecture and energy metabolism of anammox bacteria,ourknowledge ontheir iron uptake and incorporation is rather limited. In this review we presentourcurrentviewsonthevariousproteinsystems that anammox bacteria likely employ to support their iron-based lifestyle.

Inadditiontonitrite-dependentammoniumoxidation,a limited numberof studiesshow thatanammox bacteria utilizeextracellulariron(Fe(II)andFe(III))asrespiratory substrates.The pathwaysand bioenergeticsinvolvedin ironreductionandoxidationarepoorlyunderstood,and thisinterestingtopicclearlydeservesmoreattentionand experimentalefforts.

Eventhoughsubstantialamountsofironarepresentin c-typecytochromesandFe–Sproteins,anammoxbacteria relyoncommonassimilationsystems.Interestingly,they seemtobedependentonthepresenceofFe(II),whichis

taken up by the core component of the Feo system (FeoB).Assembly of Fe–Sclusters isperformed bythe compactNifSUsystem,andthealternativeheme biosyn-thesis(Ahb)pathwayproducesb-typehemes.Maturation systemIIcompletestheassemblyofc-typecytochromes intheanammoxosome,andweproposethatacopyofthis maturationsystemalsoprovidestheperiplasmwithc-type cytochromes.

Theoverviewpresentedinthisreviewisbasedon anno-tatedsystemsandinferredgenetichomology.We identi-fiedpathwaysforiron,aspartofmetalloproteins,intothe anammoxosome, but mechanisms of heme degradation andironexportfromanammoxosomeremainelusive.In thiscontext,theoriginandroleoftheobservediron-rich particlesareparticularlyintriguing.Slowgrowthandthe lackofmoleculartoolsmakebiochemicalstudiesonthese fascinatingorganismsverychallenging,butinvestigation of iron-related effects on anammox proteomes and transcriptomes certainly promises to yield valuable insights.

Acknowledgements

WethankLauravanNiftrikforprovidingtheEMimageshownin

Figure1b,andmembersoftheDepartmentofMicrobiologyatRadboud Universityforinsightfuldiscussions.C.FandM.S.M.Jaresupportedbya SpinozaPrizeawardedtoM.S.M.J.bytheNetherlandsOrganizationfor ScientificResearch[NWO62001581,2012],S.L.by[NWO824.15.011, 2015],B.K.bytheEuropeanResearchCouncil[ERC640422,2014],M.S. M.Jisfurthersupportedby[ERC232937,2009],[ERC339880,2014]and [NWO024002002,2014],andJ.R.by[ERC339880,2014].

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