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ARTICLEReceived29Jul2013|Accepted5Feb2014|Published26Feb2014Electronuptakebyiron-oxidizingphototrophicbacteriaA.
Bose1,E.
J.
Gardel1,2,*,C.
Vidoudez1,*,E.
A.
Parra1&P.
R.
Girguis1Oxidation–reductionreactionsunderlieenergygenerationinnearlyalllifeforms.
Althoughmostorganismsusesolubleoxidantsandreductants,somemicrobescanaccesssolid-phasematerialsaselectron-acceptorsor-donorsviaextracellularelectrontransfer.
Manystudieshavefocusedonthereductionofsolid-phaseoxidants.
Farlessisknownaboutelectronuptakeviamicrobialextracellularelectrontransfer,andalmostnothingisknownabouttheassociatedmechanisms.
Hereweshowthattheiron-oxidizingphotoautotrophRhodopseu-domonaspalustrisTIE-1acceptselectronsfromapoisedelectrode,withcarbondioxideasthesolecarbonsource/electronacceptor.
BothelectronuptakeandruBisCoformIexpressionarestimulatedbylight.
Electronuptakealsooccursinthedark,uncoupledfromphotosynthesis.
Notably,thepioABCoperon,whichencodesaproteinsystemessentialforphotoautotrophicgrowthbyferrousironoxidation,inuenceselectronuptake.
Thesedatarevealapreviouslyunknownmetabolicversatilityofphotoferrotrophstouseextracellularelectrontransferforelectronuptake.
DOI:10.
1038/ncomms43911DepartmentofOrganismicandEvolutionaryBiology,HarvardUniversity,16DivinityAvenue,Cambridge,Massachusetts02138,USA.
2SchoolofEngineeringandAppliedSciences,HarvardUniversity,29OxfordStreet,Cambridge,Massachusetts02138,USA.
*Theseauthorscontributedequallytothiswork.
CorrespondenceandrequestsformaterialsshouldbeaddressedtoP.
R.
G.
(email:pgirguis@oeb.
harvard.
edu).
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Microbialmetabolicactivitysubstantiallyinuencesmatterandenergyowthroughthebiosphereanddrivesglobalbiogeochemicalcycles1.
Microorganismshavebroadmetaboliccapabilitiesandcanusechemicallydiverse,solublesubstratesforenergygeneration.
Somemicrobescanalsousesolid-phaseelectronacceptorsand-donorsviaaprocesscalledextracellularelectrontransfer(EET)2–5.
RecentyearshavebeenawatershedformicrobialEET,withmanystudiesfocusingontherelevanceofEETinbioremediationandbiotechnology6,7.
AlthoughstudiesoverthepastfewdecadeshaveexaminedtheroleofmicrobialEETindonatingelectronstometaloxidesandoxygen6,8–10,theinvolvementofmicrobialEETinfacilitatingelectronuptakehascometotheforeonlyrecently11.
Studiesshowthatmixedmicrobialcommunitiesfacilitatecathodicreactionsinbioelectrochemicalsystems(BESs),impli-catingmicrobesinelectronuptake11.
Recentstudiesusingpurecultureshaveshownthatatleastthreemicrobesarecapableoftakingupcurrentfromanelectrode:Sporomusaovata12,MariprofundusferrooxydansPV-1(ref.
13)andShewanellaoneidensisMR-1(ref.
14).
OnlythestudyperformedonShewanellaconsideredthegeneticlocitobelikelyinvolvedinelectronuptake14.
Assuch,themechanismsunderlyingelectronuptakebymicrobesincludingShewanellaremainpoorlyunderstood.
Characterizinghowmicrobestakeupelectronsfromsolid-phaseelectrondonorsiscriticaltoourunderstandingoftheecologicalandevolutionaryimplicationsofthisprocess,aswellastoanyfuturebiotechnologyeffortssuchaselectrosynthesis6,15.
Theestablishmentofgenetic,genomicandmetabolicstudiesinmicrobesthatnaturallytakeupelectronsviaEETwillleadtothefollowing:(1)identicationoftheassociatedgeneticdeterminants;(2)theunderlyingmolecularmechanisms;and(3)alsofacilitateexperimentsthatexaminetherelationshipbetweenelectronuptakeandcellularmetabolism.
HerewepresentdataonourstudiesofRhodopseudomonaspalustrisTIE-1(TIE-1),aphotoautotrophicmicrobecapableofacceptingelectronsfromavarietyofelectrondonors,includingiron16–18(SupplementaryFig.
1).
WechoseTIE-1asthemodelorganismbecauseitusesferrousiron,Fe(II),asanelectrondonorforphotosynthesis(photoferrotrophy)16.
Moreover,themetabolicversatilityandgenetictractabilityofTIE-1helpstoreadilyinterrogatethefundamentalphysiologicalrelevanceofelectronuptake,includingthedegreeandconditionsunderwhichTIE-1takesupelectrons,somegeneticlociencodingsystemsinvolvedinelectronuptake,andtherelationshipbetweenelectronuptakeandotherphysiologicalprocessessuchasphotosynthesisandcarbonxation.
WeobservethatTIE-1acceptselectronsfromapoisedelectrode,withcarbondioxideasthesolecarbonsource/electronacceptor.
BothelectronuptakeandruBisCo(ribulose-1,5-bisphosphatecarboxylase/oxygenase)formIexpressionarestimulatedbylight.
Electronuptakealsooccursinthedark,uncoupledfromphotosynthesis.
ThepioABCoperon,whichencodesaproteinsystemessentialforphotoautotrophicgrowthbyferrousironoxidation,inuenceselectronuptake.
ResultsTIE-1acceptselectronsfromapoisedelectrode.
TocharacterizeelectronuptakebyTIE-1,BESswereused.
BESsareexperimentalsystemswhereanelectrodeissubmergedinabioreactorandisusedtomimicthemidpointpotentialofsolid-phaseminerals3,6.
BESsprovideanattractivealternativetousingnaturalredoxactiveminerals,allowingonetostudymicrobialEETwithoutconfoundingissuessuchasmineralogicalchangesduringexperimentation6,9,13,19,20.
Theelectrodeswerepoisedat100mVversusStandardHydrogenElectrode(SHE)(SupplementaryFig.
2),asthispotentialisconsistentwithformsofFe(II)usedbyTIE-1(ref.
21).
TIE-1wassubjectedtothreetreatmentsasfollows:(1)illuminatedreactorswithpoisedelectrodespassingcurrent(illuminatedtreatment);(2)non-illuminatedreactorswithpoisedelectrodespassingcurrent(darktreatment);and(3)illuminatedreactorswithelectrodesatopencircuit,passingnocurrent(controltreatment).
ThehighestratesofcurrentuptakebytheTIE-1wild-type(WT)wereobservedinilluminatedtreatments,uptoB1.
5mAcm2(Fig.
1a).
Cyclicvoltammetry(CV)oftheelectrodesintheilluminatedtreatmentsrevealedtwomodestbutdiscernablecathodicpeaksat0.
27and0.
4V(versusSHE)intheWT,whichwereabsentintheabioticcontrol(Fig.
1b),suggestingthepresenceofredoxactivecomponentsintheilluminatedreactors.
Cathodiccurrentwasalsoobservedinthedarktreatments,suggestingthatcurrentuptakeoccurredundertheseconditions,althoughB70%lowerthanwhenilluminated(Fig.
1a).
Weobservedthatcellswereattachedtoelectrodesduringallbiotictreatments,withthehighestviablecelldensitiesoccurringinthelighttreatment(Fig.
2a,b,SupplementaryFig.
3,SupplementaryTables1and2).
Planktoniccellnumbersincreasedduringthecourseofthe1-dayincubations,althoughtheincreaseintheWTilluminatedandcontroltreatmentswerenotsignicantlydifferentattheendoftheseexperiments(SupplementaryTables3and4).
Currentdensity(μAcm–2)00.
5121.
5Wild-type–0.
200.
20.
40.
60.
811.
23020100102030Currentdensity(μAcm–2)Potential(VversusSHE)WTΔpioABCWT-ΔpioABCWTnormalWTdarkFigure1|CurrentuptakebyWTR.
palustrisTIE-1.
(a)CurrentdensitiesofR.
palustrisTIE-1WTunderilluminatedanddarkconditions.
Errorbarsindicates.
d.
oftheseaverages(n3),anddatareportedareconsistentwith10independentruns.
(b)CyclicvoltammogramsofWTandDpioABCmutantafter96hoftreatmentinbioelectrochemicalreactorswithelectrodespoisedat100mVversusSHE.
Twosetsofanodic–cathodicpeakpairswereidentiedat0.
27and0.
40V,respectively.
TheredtracedepictsthedifferenceinmagnitudebetweentheWTandtheDpioABCmutantstrain.
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TobestcapturethechangesingeneexpressionduringtheonsetofEET,thesetreatmentslastedB24htoavoidissuesthatcanariseduringprolongedexperiments(forexample,differencesingrowthphase).
Nevertheless,theapparentchangesinplanktoniccelldensitywouldsuggestthat(1)currentwasbeingusedtosupportplanktonicgrowth;or(2)anexogenouselectrondonorwasavailableforgrowth.
Notably,inseparate5-dayilluminatedtreatments,TIE-1exhibited10-foldhigherdensitiesthandarkandcontroltreatments.
However,intheseshorter-termilluminatedtreatments,massbalancecalculationssuggestthattheplanktoniccellincreaseinthebioreactorsistwoordersofmagnitudelowerthanthatpredictedifallcurrentwenttobiomass(SupplementaryNote1).
Moreover,thetraceconcentra-tionsofironpresentinthemedium(tosupportbiosynthesis)couldonlyaccountforupto4.
0104cellspermloftheobservedcellincrease(forcalculationsseeSupplementaryNote2).
Thus,thereisanelectronsinkotherthanbiomass,andnotablythegeneexpressiondatasuggestthatthiscouldbereductiveCO2assimilation(discussedindetailbelow).
Thesedataprovidetherstevidenceoflight-stimulatedelectronuptakebyaphotoferrotroph,withsomeelectronuptakealsooccurringinthedark,independentofphotosynthesis.
PhototrophicmicrobesrelatedtoTIE-1usephoticenergyforATPsynthesisthroughcyclicelectronow,withouttheneedforanelectrondonor22.
Anelectrondonorisonlyrequiredtoproducereducingequivalents(NADPH)forcellularmetabolismmostlikelybyreverseelectrontransfer23.
Inthedark,noATPcanbegeneratedviaphotosynthesisbutcellularmetabolismcontinues22,thusrequiringanelectrondonorthatislikelyrepresentedbytheobserveddarkcurrentinourexperiments.
Thedarkcurrentalsosuggeststhattheelectronuptakemachineryisindependent(orcanbeuncoupled)ofthecyclicphotosyntheticapparatus.
TheincreaseinelectronuptakeinthepresenceoflightsuggeststhattheATPgeneratedusingtheenergyoflightisusedbycellularprocesses,necessitatingahigherlevelofelectronuptake.
ThepioABCoperonhasaroleinelectronuptake.
BecausethesedatarevealthatTIE-1acceptselectronsfromasolid-phasecon-ductor,wereasonedthatitmightuseconservedstrategiestomediatethiselectronuptake.
PreviousstudieshaveshownthatpioABCisessentialforphotoferrotrophy,andhavespeculatedthatthePioproteinsmightbeinvolvedinelectrontransferfromFe(II)totheelectrontransportchain17,21.
ThepioABCoperonencodestheputativeproteinsPioA,aperiplasmicdecahemecytochrome,PioB,anoutermembraneporin,andPioC,aperiplasmichighpotentialiron–sulfurclusterprotein17,21(SupplementaryFig.
4).
Usingmutantstudiesandexpressionanalysis,wedirectlytestedwhetherthePioABCsystemhasaroleinelectronuptake.
WeobservedthatDpioABC-illuminatedbiolmsaccepted30%lesscurrentthantheWT(Fig.
3a),andthemutantilluminatedbiolmswereB8–10-foldlessdensethantheWT(SupplementaryTable2).
FewerDpioABCmutantscolonizedtheelectrodeintheilluminatedtreatments,whichmightresultfromanattachmentdefect.
However,thiswasnotobservedinthecontroltreatments,thatis,intheabsenceofcurrent,asthemutantcelldensitieswerecomparabletotheWT(DpioABC:9.
2106cellspercm2;WT:8.
1106cellspercm2).
Ifweassumethatonlyattachedcellscontributetoelectronuptake,thentheDpioABCmutantsseemtoacceptmorecurrentpercellthantheWT(SupplementaryTables5and6).
ThiswouldimplythattheDpioABCmutantcellscantakeupelectronsmoreactively,perhapsviacompensatorychanges.
Weposit,however,thatsuchanassumptionisinaccurate.
asitdisregardsthepotentialcontributionofplanktoniccellstoelectronuptake(SupplementaryFig.
2).
Regardless,thesedatacollectivelyshowthatthePiosysteminuenceselectronuptake,althoughothermechanismsofelectronuptakeclearlyexistinTIE-1,asthemutantmaintainsnearly70%ofthecurrentuptakeseenintheWT(Fig.
3a).
FuturestudiesshouldexaminethemeansbywhichthePiosysteminuencesbothphototrophicironoxidationandEET,anditspotentialroleingoverningattachmenttopoisedelectrodes.
WehypothesizedthatelectronuptakemightinuencephysiologicalsystemsthathavearoleinEETaswellasredoxbalance.
Accordingly,weassessedtheexpressionofthetargetgenes,includingthoseencodingthePioABCproteins,acrossalltreatments.
ExpressionofpioAintheWT-illuminatedbiolmwasupregulatedbyB48-fold,whereaspioBandpioCshowedmoremodestupregulationcomparedwiththecontroltreatment(11-and3-fold,respectively;Fig.
3b).
TheobservedlevelsofpioAintheWT-illuminatedbiolmwerewellabovethoseoftheinoculum(grownonH2:CO2;Fig.
3b).
Theywere,however,comparabletogeneexpressionobservedduringphotoferro-trophicgrowthonsolubleFe(II)inconventionalcultureapparatus.
ThedecreasedcurrentuptakeoftheDpioABCmutantaswellastheobservedupregulationofthePiogenesintheBESsystemtogethersuggestthatthePioABCproteinsmaybeinvolvedinelectronuptakebyTIE-1undertheseconditions.
ItshouldbenotedthatthePioABCmoduleoccursinanumberofanoxygenicphototrophicmicrobes,whichmightshowFigure2|R.
palustrisTIE-1cellsattachedtocathodes.
(a)FluorescencemicrographsofaR.
palustrisTIE-1WT-illuminatedbiolm.
Scalebar,10mm.
(b)ScanningelectronmicrographofaWT-illuminatedbiolm.
Scalebar,3mm.
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light-enhancedelectronuptakeasobservedinTIE-1(SupplementaryTable7),Moreover,non-phototrophicferrousiron-oxidizingbacteria(FeOB)alsopossessthePioABmodule,raisingthequestionastowhethertheseorganismsperformlight-independentelectronuptakesimilartotheobserveddarkcurrentinTIE-1.
TheinvolvementoftheMtrAB(relatedtoPioAB17)systemintheelectronacceptancebyS.
oneidensis(MR-1)fromapoisedelectrodealsosuggeststhatthismodulemighthaveadirectroleinelectronuptake14.
Electronuptakestimulatesexpressionofothergenes.
WeusedexpressionanalysesandmicroscopytofurtherexamineTIE-1'sresponsetoelectronuptake.
Exopolysaccharide(eps)geneswerehighlyupregulatedintheWT-illuminatedbiolmsand,insomecases,intheDpioABC-illuminatedbiolms.
ExpressionanalysisfurthershowedthatthepioChomologue(anotherhighpotentialiron–sulfurclusterproteinlocatedelsewhereonthechromosome)wasupregulated(fourfold)intheWT-illuminatedplanktoniccells(cellsnotattachedtotheelectrodepresentinthemedium)comparedwiththecontroltreatment(Fig.
4A(b)),suggestingthattheencodedproteinmighthavearoleinplanktoniccellincreaseundertheseconditions.
MicroscopyrevealedthatEPSproductionwasmostabundantinilluminatedbiolms(Fig.
4A(a)andB(a);SupplementaryFig.
5a,b).
Proteinstainingestablishedthepre-senceofextracellularproteinsinthecellsattachedtotheelec-trodes(SupplementaryFig.
5c,d).
FutureanalysisonbiolmandplanktoniccellsandtheproducedEPSwillhelpdeterminetheroleoftheseelementsinelectronuptake.
ruBisCoformIexpressionincreasesduringelectronuptake.
PreviousstudieshaveshownthatinorganismsrelatedtoTIE-1,electrondonorsarerequiredforgenerationofreducingequivalents,namelyNAD(P)H,whichservesasareductantforcellularprocessessuchascarbonxationviatheCalvincycle22,24–26.
RuBisCo,akeyenzymeintheCalvincycle,assimilatesCO2intoribulose-1,5-bisphosphateyieldingtwoPhotoferrotrophyInoculumDarkplanktonicControlplanktonicIlluminatedplanktonicIlluminatedbiofilmpioChomologue0100200300400500600700060120204080100140160–20020406080100abepsIepsIIepsIVepsVIPhotoferrotrophyInoculumDarkplanktonicControlplanktonicIlluminatedplanktonicIlluminatedbiofilmmRNAabundanceversusaerobicWTgrowthmRNAabundanceversusaerobicWTgrowthmRNAabundanceversusaerobicWTgrowthmRNAabundanceversusaerobicWTgrowthmRNAabundanceversusaerobicWTgrowthmRNAabundanceversusaerobicWTgrowth–20020406080100InoculumControlplanktonicIlluminatedplanktonicIlluminatedbiofilmWTIlluminatedbiofilmepsIepsIIepsIVepsVIWTΔpioABC010203040506070InoculumControlplanktonicIlluminatedplanktonicIlluminatedbiofilmWTIlluminatedbiofilmpioChomologueWTΔpioABCWTWT–40–30–20–10010203040PhotoferrotrophyInoculumDarkplanktonicControlplanktonicIlluminatedplanktonicIlluminatedbiofilmruBisCoformI–40–30–20–10010203040InoculumControlplanktonicIlluminatedplanktonicIlluminatedBiofilmWTIlluminatedbiofilmWTΔpioABCWTruBisCoformIIruBisCoformIIruBisCoformIABcFigure4|mRNAabundancedeterminedintheR.
palustrisTIE-1WT(A)andDpioABC(B).
(a)Exopolysaccharide(eps)genes;(b)pioChomologue;and(c)ruBisCoformIandIImRNAabundance.
Cellweregrownphotoautotrophicallyonhydrogenand5mMFeCl2.
qRT–PCRdataaretheaverages±s.
e.
forthreebiologicalreplicatesassayedintriplicate.
epsIRpal_3203,epsIIRpal_3763,epsIVRpal_3771,epsVIRpal_3777,pioChomologueRpal_4085.
WTnormalΔpioABCnormalΔpioABC02004006008001,0001,200050100150200250PhotoferrotrophyInoculumDarkplanktonicControlplanktonicpioApioBpioCIlluminatedplanktonicIlluminatedbiofilmmRNAabundanceversusaerobicWTgrowthCurrentdensity(μAcm–2)00.
5121.
5Figure3|CurrentuptakeandmRNAabundanceofthepioABCoperonbyWTR.
palustrisTIE-1andDpioABCmutantundervariousconditions.
(a)CurrentdensitiesofR.
palustrisTIE-1WTandDpioABCmutantunderilluminatedconditions.
Errorbarsindicates.
d.
oftheseaverages(n3),anddataareconsistentwith10independentruns.
(b)pioABCgenetranscriptabundancedeterminedintheWTgrownphotoautotrophicallyonhydrogenand5mMFeCl2.
qRT–PCRdataaretheaverages±s.
e.
forthreebiologicalreplicatesassayedintriplicate.
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moleculesof3-phosphoglycerate,whichareeventuallyreducedbyNAD(P)Htoglyceraldehyde3-phosphateinthereductivephaseofthecycle24–26.
TIE-1andrelatedmicrobesharbourgenesencodingtwoformsofruBisCo:formsIandII(refs24–26).
WeobservedthatruBisCOformIwasmosthighlyexpressedinWT-illuminatedbiolms(Fig.
4A(c)),andwastypicallyhigherthanruBisCoformIIduringconventional,photoautotrophicgrowthonhydrogenandFe(II)(Fig.
4A(c)).
Notably,ruBisCoformIexpressionwasnotinducedinthedarktreatment(Fig.
4A(c)).
PreviousstudiesontheregulationofruBisCoformIexpressioninCGA009/10(B99%identitytoTIE-1(ref.
16))showthatitisunderexquisitecontrolandispositivelyregulatedbyATPandNAD(P)H,metabolitesthatindicatetheenergystatusofthecell27.
Duringelectronuptakeinthepresenceoflight,TIE-1likelyproducesabundantATPandNAD(P)H,whichwepositleadstotheincreaseinruBisCoformIexpression.
Incontrast,boththesemetabolitesarelikelylowerinthedark,andthusruBisCoformIexpressiondecreases.
IthasalsobeensuggestedthatRuBisCoformIcanindirectlyactasanelectronsink(becauseincreaseinitsexpressionleadstohigherproductionof3-phosphoglycerate,whichservesasthesubstrateforthereductivepartoftheCalvincycle)tomaintainredoxbalanceinphotosyntheticbacteriarelatedtoTIE-1(refs25,26).
DiscussionOurdataprovidearstglimpseontheabilityofthephotoautotrophicbacteriumR.
palustrisTIE-1toacceptelectronsfromasolid-phaseelectrondonor.
Becausephotoautotrophsareexposedtodiurnalcyclesoflightanddarkconditions,wetestedtheeffectofilluminationontheabilityofTIE-1toacceptelectrons.
OurresultsshowthatTIE-1acceptselectronsunderbothlightanddarkconditions,althoughlightstronglystimulateselectronacceptance(Fig.
1a).
ThemassiveupregulationingenesthatencodeforthepioABCsystem(encodingproteinsthataresuggestedtohaveacriticalroleinphototrophicironoxidationbyTIE-1(ref.
17)),aswellasthedecreaseincurrentobservedinpioABCmutants,implythatthePioproteinsareengagedinelectronuptake.
Incontrast,thepioABCmutantsappeartohaveanattachmentdefecttopoisedelectrodes,thusexhibitinghighercell-specicelectronuptakeratescomparedwiththeWT(Fig.
3a,SupplementaryTables2and6).
ThisapparentpleiotropymakesitdifculttoascribeasimpleroletothepioABCsysteminelectronuptake.
Itshouldbenotedthatourexperimentaldesigndoesnotallowustoexcludethepossibilitythatboththebiolmandtheplanktoniccellswereengagedinelectronuptake,andthefreelivingandbiolmlifestylesmightbedynamic(SupplementaryFig.
2).
Theplanktoniccellsmayhavecontributedtocurrentuptakethroughdirectencounterswiththeelectrode(thesereactorswerewellstirred)orviasolublecompounds(wewereunable,however,todetectanyredoxactivecompoundsinthespentmedium;SupplementaryFigs2and6).
TranscriptomicanalysisshowedthatruBisCoformIexpressionwashighestinthepoisedilluminatedelectrodes(Fig.
4A(c)),suggestingthatthisenzymecouldbeanindirectelectronsinkashasbeenobservedinotherrelatedorganisms25,26.
BecauseRuBisCoispartoftheCalvinCycle,thecarbonxationpathwayinTIE-1,itisplausiblethatsomeoftheseelectronswouldgotobiomass.
Althoughwedidseeanincreaseinthetotalcelldensity,therewasnosignicantdifferenceamongtheseshort-termtreatments,andthemassbalanceanalysessuggestthatbiomassonlyaccountsforamodestamountofthetotalcurrentpassed(SupplementaryTables1–4).
Asmentioned,theseexperimentsweredesignedtobeshortindurationtoavoidconfoundingfactorsassociatedwithgrowthandchangesingrowthphase.
InlightofTIE-1'stypicallymodestgrowthrates,itislikelythatincreasesinbiomassattributabletoelectronuptakeduringtheseshort-termtreatmentsarebelowourlimitsofresolution.
Innature,electronuptakeviaEETcouldamelioratemetabolicdilemmasthatneutrophilicFeOBs,suchasTIE-1,areknowntoface.
FeOBsoftencontendwiththeprecipitationofinsolubleironoxidesoutsidethecellthatareabyproductoftheirmetabolicactivityandpotentiallylimitFe(II)availability21,28.
TIE-1producespoorlycrystallineFe(III)hydroxides,whichovertimeareabioticallytransformedtothe(semi)conductivemineralsgoethiteandmagnetite16,29.
ConductionofelectronsthroughthismatrixwouldallowTIE-1(andpotentiallyotherFeOBs)accesstoelectronsfromremoteelectrondonors,includingFe(II)(SupplementaryFig.
7),viaprocessessuchaselectronconductionandironatomexchange30–32.
Indeed,recentstudieshaveshownthatconductivemineralscanfacilitateelectrontransfertomicrobesfromremoteelectrondonors(includingothermicrobes)33.
Thesedataextendthisphenomenontophotoautotrophsthatishighlyrelevantbecausetheirrestrictiontothephoticzonemighthinderaccesstoreductantsindeeper,anaerobiclayers22,34.
InadditiontotheecologicaladvantagesofelectronuptakeviaEET,thereissubstantialinterestinexploitingphotoautotrophsforbothenergyandbiofuelgeneration11,andidentifyingageneticallytractablephotoautotrophthatcanuseelectriccurrentasanelectrondonorholdspromiseinfutureelectrosynthesisapplications11.
AlthoughtheecologicalsignicanceofEETisjustcomingtothefore,ourdataillustratethepotentialvalueofEETtomicroorganismsinnature,inparticularphotoautotrophs.
MethodsBacterialstrains,mediaandgrowthconditions.
R.
palustrisTIE-1wasgrownasdescribedpreviously18.
Forexperiments,cellswerepre-grownautotrophicallyon80%hydrogen:20%carbondioxide(H2:CO2)at200kPainfreshwatermedium(FW)with20mMbicarbonate.
TheDpioABCstrainusedhereinwasconstructedaspreviouslydescribed17.
Phototrophicpre-growthwasat30°Cusinga60-WincandescentlightsourceprovidingtotalirradianceofB40Wm2.
BioelectrochemicalreactorstudieswereconductedwithFWmedium(minimalsaltsmediumlackinganyaddedelectrondonors16–18)with20mMbicarbonate(solecarbonsource16–18),bufferedtopH6.
8andwithnoexogenouselectrondonor.
AllbacterialstrainswereroutinelytestedforpuritybystandardPCRusingprimersindicatedinSupplementaryTable8.
Owingtobiologicalvariationinthecultivationeffort,whichresultedindifferentcelldensitiesintheinoculumandprohibitscomparisonacrosstreatments,weranaWTcontrolinparallelwitheveryindividualtreatmenttoaccountforthesedifferences.
AllcomparisonsbetweenWTandtreatmentsaremadeusingthesepairedruns.
BESandconditions.
TheBESsconsistedofnew,acid-washed,combusted350mlborosilicateglassH-cellreactorsequippedwithtwobutylrubbersamplingportsinthecathodicchamber(AdamsandChittendenScienticGlass,Berkeley,CA,USA).
Avacuumclampheldtheanodicandcathodicchamberstogether,andelectrolyteswereseparatedusingacation-exchangemembrane(Naon117)withanactivecross-sectionof20cm2(FuelCellStore,Boulder,CO,USA).
Theworkingelectrodesconsistedofspectroscopicallypure1/800-diametergraphiteevaporationrods(SPI01685-FA,StructureProbeInc.
,WestChester,PA,USA)thatweremechanicallypolishedwith1200gritsandpaper,soakedin5%HClfor12handstoredinultrapuredeionizedwater.
Thegraphiterodswerethoroughlydriedbeforeusebyallowingthewatertoevaporate.
Eachreactorwasttedwiththreegraphiterodstoprovideatotalimmersedprojectedelectrodesurfaceareaof18cm2.
Therodsweresealedwithttingsandferrulesonthereactorcap(UpchurchScientic,OakHarbor,WA,USA).
Outsidethereactor,rodswereelectricallyconnectedtoonepotentiostatusingalligatorclips(describedbelow).
Thecounterelectrodeconsistedofcarboncloth(FuelCellStore),whichwasmechanicallyattachedtoatitaniumwirepiercedthrougharubberstopper(VWR)andsuspendedinthecounterchamber.
Electricalconditionsandcyclicvoltammograms.
Thereactorswerepoisedusingcustom-builtpotentiostatsengineeredformicrobialchronoamperometry(KarmaElectronicsInc.
,Somerville,MA,USA).
DatawerecollectedthroughaNationalInstrumentsDAQ(NI-6225)every10susingLabviewSignalExpresssoftware(NationalInstruments,Austin,TX,USA).
Basedonpreliminaryanalysesofelec-troactivityinWTR.
palustrisTIE-1,thereactorswerepoisedat100mVversusNATURECOMMUNICATIONS|DOI:10.
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SHE(100mVofthebiologicalEpcroughlyat200mVversusSHE)toassurecathodicconditionsduringtheexperiment.
Importantly,thispotentialalsoensuresthatareductiveFe(III)/Fe(II)cycleisnotestablishedduringtheseexperiments(theredoxpotentialatpH7.
0oftheFe(III)/Fe(II)coupleis14mV,andelectrontransferfromanelectrodepoisedat100mVwillbeanendergonicprocess)15.
Reportedcurrentdensities(mAcm2)wereobtainedbyaveragingregionsof48hofstablecurrentineachreactor.
CVwasconductedusingaGamryR600potentiostat(Gamry,Warminster,PA,USA).
BiolmCVswereobtainedwithascanrangeof100to900mVversusSHEatarateof20mVpersecond.
Supernatantvoltammogramswereobtainedusinga3-mmdiameterglassycarbonelectrode(partno.
A-002012,BioLogic,Claix,France),underaN2atmosphere,scannedbetween0and500mVversusSHEat20mVpersecond.
Wewereunabletodetectanyelectro-activesolublespeciesinthe0.
2-mMlteredspentmedium(SupplementaryFig.
7).
Toassesstheactivesurfaceareavariabilitybetweenelectrodes,CVswerecollectedabioticallyinFWmedium.
PotentialisreferencedtotheSHEunlessotherwisespecied.
Descriptionofbioelectrochemicalsetups.
ThedistancebetweentheworkingandcounterelectrodeswasB11cm.
AssembledBESreactorsweresterilizedbyautoclavinginsterilizationpouchesandplacedinsideananaerobicchamber(Coy,2%hydrogenandpalladiumcatalysts).
Ag/AgClreferenceelectrodeswerecustom-madeusingglasstubing(4mmKIMAX),silverwire(0.
5mmdiameter)andporousvycortips(1/80diameter,MF-2064,BASi).
Referenceelectrodeswerecalibratedbeforeeachexperiment,placedintheanaerobicchamber,sterilizedwith70%ethanolandplacedinthecounterchamberforthedurationoftheexperiments.
Althoughinsidetheanaerobicchamber,mediaandcounterbufferwereaddedtothecathodeandanodechambers,respectively.
InoculationoftheBESsoccurredinsidetheanaerobicchamberbeforetransferringthemoutsidetheanaerobicchambertoestablishelectricalconnections.
Thereactorsystemwaspurgedcon-tinuouslywitha1-cm3min1streamof0.
2mmlter-sterilized,deoxygenatedgasstreamof80%:20%N2:CO2and100%N2onthecathodicandanodicside,respectively,usingahypodermicneedleimmersed1cmbelowthemediasurface.
Thegasesweredeoxygenatedusingahighcapacityoxygentraploweringtheoxygenlevelstoo0.
01p.
p.
m.
(Restek,Bellefonte,PA,USA).
EachBESwasindividuallyhousedwithafreshincandescent60Wbulbprovidingatotalirra-dianceofB40Wm2.
DarkBESslackedabulbandwerecoveredthoroughlywithblackpapertopreventlightexposure.
Allworkingchamberswerestirredgentlywithamagneticbarandincubatedat30°C.
Allincubations,acrossalltreatments,lasted24h.
Sampling.
Thereactorswereinoculatedwith10mlofcellsinthemid-exponentialphaseofphotoautotrophicgrowthon80%H2:20%CO2.
Media(1ml)waswithdrawnfromthereactorsimmediatelyfollowinginoculationandusedforopticaldensity(OD660)determinationwitha4802spectrophotometer(ColePar-mer,VernonHills,IL,USA),andforpHmeasurements(InlabExpertPropHmetreandprobe;MettlerToledo,Schwerzback,Switzerland).
Culture(4ml)wasalsowithdrawnfromthereactorsforcellcounts.
Cellswerexedin4%paraf-ormaldehydeforcellcounting(ElectronMicroscopySciences,Hateld,PA,USA).
Attheendofeachexperiment,oneoftheelectrodeswasimmediatelydippedintoRNAlater(Qiagen,Valencia,CA,USA)forRNAextraction.
Also,5mlofplank-toniccellswereimmediatelypreservedinRNAlaterandlteredonapoly-ethersulfonemembraneforRNAextraction(Corning,Tewksbury,MA,USA).
AllRNAsampleswerestoredat80°C.
AsecondelectrodewascutintoB5mmpiecesandtransferredintoxativesorstainingsolutionsformicroscopicanalyses(describedbelow).
Postexperimentation,1mlofplanktoniccellswassampledforOD660determination,and2–4mlforpHmeasurements.
Theremainingculturevolumewasthenlteredona0.
2-mmcelluloseacetatelter(Corning).
Afterresuspensionin8mlofmedia,theseplanktoniccellswerepelletedintwo2mlmicrocentrifugetubes(18,000gfor10min)andkeptat80°Calongwiththelteredspentmedium.
Proteinanalysis.
SubsamplesfortotalproteinanalysiswereprocessedinProtloBind1.
5or2mlmicrocentrifugetubes(Eppendorf,Hauppauge,NY,USA).
Trichloroaceticacidprecipitationwasusedaspreviouslydescribed18.
Thepelletsweredriedundervacuumfor1htoremoveresidualacetoneandthenresuspendedin650mlof3MUrea(ACSgrade,Alfa-Aesar,WardHill,MA,USA).
Toensurecompleteresuspension,thesampleswereincubatedat80°Cfor3dayswithfrequentsonicationinasonicbath(FS30H;ThermoFisherScientic,Waltham,MA,USA).
ThePierceBCA(bichinchoninicacid)ProteinAssayKit(ThermoScientic,Rockford,IL,USA)wasusedusingthemicrotiterplatemethodforproteinestimationasspeciedbythemanufacturerwiththeprovidedbovineserumalbuminasthestandardprotein.
Eachsamplewasquantiedintriplicate.
Absorbanceat562nmwasmeasuredafter30sshakingat37°CusingaSpectramaxPlus384platereader(MolecularDevices,Sunnyvale,CA,USA).
Fluorescencemicroscopysamplepreparationandimaging.
Sectionsoftheelectrodewereplacedintooneofthreesolutionscontaining1mM40,6-diamidino-2-phenylindole(LifeTechnologies,GrandIsland,NY,USA)aswellas(1)LIVE/DEADstain(0.
5mMSYTO9and3mMpropidiumiodide,L7012,LifeTechnologies);(2)EPSstain(200mgl1ConcanavalinAandAlexa488,LifeTechnologies);and(3)Proteinstain(undilutedFilmTracerSYPRORubyBiolmMatrixStain,LifeTechnologies).
Tubeswerewrappedinaluminiumfoilandkeptatroomtemperatureforatleast30min.
Sampleswerethenplacedin1phos-phate-bufferedsaline(PBS)inaglass-bottomeddishandimagedwithaZeiss700invertedconfocalmicroscopewiththefollowingimaginglasersandZeisslters:(1)live/dead555and488nm,SP490;405nm,SP555;(2)EPS488and405nm,SP490andLP490;and(3)protein555nm,SP490;405nm,SP555.
ThisworkwasperformedattheHarvardCenterforBiologicalImaging.
Scanningelectronmicroscopy.
Sectionsoftheelectrodewerecutusingsteriletechniquesandimmediatelyplacedintoasterilemicrocentrifugetubecontainingoneofthreesolutions:(1)5%glutaraldehyde(ElectronMicroscopySciences)in1PBS;(2)2%paraformaldehyde(ElectronMicroscopySciences)in1PBS;and(3)2%glutaraldehydein1PBSwith0.
15%SafraninO(Sigma-Aldrich,StLouis,MO,USA),whichhaspreviouslybeenshowntoaidinEPSpreservation7.
Sampleswereheldat4°Cfor24hbeforebeingsubjectedtoethanoldehydrationbyplacingthemin35,50,70,95and100%ethanol(200proof)inPBSor0.
1MPBSsolutionsfor10mineach.
The100%ethanolsolutionwaschangedvetimes,andthesamplewasleftinethanolforcriticalpointdrying(Autosamdri815A;Tousimis,Inc.
)witha15-minpurgetime.
Thesampleswereadheredtoscanningelectronmicroscopy(SEM)postswithcarbonlmtapeandthenimagedwithaSEMat5kV(JEOL,Inc.
).
Cellcountsforelectrodesampleswereperformedbyanalysingmicroscopyeldstakenatthesameworkingdistance(4.
5mm)toimage,countingatleast500cellsorexamining12eldsofviewifcelldensitywaslowandnormalizedtototalarea.
ThisworkwasperformedattheHarvardCenterforNanoscaleSystems(CNS).
RNAisolation.
Forplanktonicassessments,preservedcellsweredislodgedfromthepolyethersulfonemembranebeforeRNAextractionbyvortexingfor3mininaTris-EDTAbuffer.
Forbiolmassessment,thecellsweredislodgedfromthegra-phitebyscrapingwithasterilerazor,thenvortexingvigorouslyinTris-EDTAbuffer.
RNAwasextractedasdescribedpreviously5.
TheRNAconcentrationwasquantiedusingaNanoDropND1000(ThermoScientic,Wilmington,DE,USA).
RNAamplication.
TheRNAobtainedfromthebiolmonthegraphitewascleanedwiththeMEGAclearKit(LifeTechnologies)asperthemanufacturer'sguidelines.
ThepuriedRNAwasprecipitatedusingammoniumacetate.
ThereconstitutedRNAwasusedastemplatefortheMessageAmpII-BacteriaKitasperthemanufacturer'sguidelines(LifeTechnologies).
QuantitativereversetranscriptionPCR.
Geneexpressionanalysiswasper-formedusingquantitativereversetranscriptionPCR(qRT–PCR).
ThecomparativeCtmethodwasusedasdescribedpreviouslytoassessexpressionofthepioABCoperonandotherrelevantgenes5.
Primerefcienciesweredeterminedusingthemanufacturer'smethod(AppliedBiosystems,Inc.
UserBulletinno.
2).
clpXandrecAwereusedasthetwointernalstandards,whichhavebeenpreviouslyusedandvalidatedasinternalstandards18.
TheprimersusedfortheassaysareindicatedinSupplementaryTable5.
TheiScriptcDNAsynthesiskitwasusedforreversetranscription(Bio-Rad,Hercules,CA,USA).
TheiTaqFASTSYBRGreenSupermixwithROX(Bio-Rad)andtheStratageneMx3005PQPCRSystem(Agilent,SantaClara,CA,USA)wereusedforallquantitativeassays.
Cellcounting.
Theparaformaldehyde-xedsamplesweretransferredintoAmiconcentrifugelters(AmiconUltrael100k,regeneratedcellulosemembrane;Millipore,Carrigtwohill,CO,Ireland)andcentrifugedfor10minat1,000g.
ThepelletwasresuspendedinPBSandwashedtwice.
ThecellswererecoveredbycentrifugationoftheAmiconinreversepositionfor15minat3,000g.
Theresultingsampleshado0.
04%paraformaldehyde.
Picogreenwasaddedtothecells(Quant-iTPicoGreendsDNA,LifeTechnologies),andthecellswerecountedin96-wellplatesalongwith50mlofSpheroAccuCountblankbeads(Spheroteck,LakeForest,IL,USA).
CelldensitywasestimatedwithaLSRIIowcytometer(BD,Sparks,MD,USA)usinga488-nmlaser.
Acalibrationcurverelatingtheratioofcelleventstobeadseventswithcelldensitywasconstructedbyanalysingadilutionseriesofacellsample,thedensityofwhichhasbeendeterminedbymicroscopy(withaHelberBacteriaCellcountingchamberwithThomaruling,Hawksley,Lancing,Sussex,UK).
Inductivelycoupledplasmamassspectrometry.
TomeasuretheconcentrationofironpresentinFWmediuminductivelycoupledplasmamassspectrometry(ICP-MS)wasperformedusinganAgilent7700xICP-MSwithanoctopoleMS(Agilent).
InternalstandardsusedwereGermaniumandManganese,whichwerewithinthedetectionlimitofoursystem.
Theamountofironinthebasalmediumwas4mMandrangedfrom2–4mMinthespentmedium.
Insilicomethods.
ForidentifyinghomologuesofthePioABCproteins,delta-blast35,FASTA36(http://www.
ebi.
ac.
uk/Tools/sss/fasta/),andtheIMGorthologneighbourhoodsearchwasused37(http://img.
jgi.
doe.
gov/cgi-bin/w/main.
cgi).
ARTICLENATURECOMMUNICATIONS|DOI:10.
1038/ncomms43916NATURECOMMUNICATIONS|5:3391|DOI:10.
1038/ncomms4391|www.
nature.
com/naturecommunications&2014MacmillanPublishersLimited.
Allrightsreserved.
SequencesimilaritywascalculatedusingEMBOSSmatcher38,39(http://www.
ebi.
ac.
uk/Tools/psa/emboss_matcher/).
ThedatareportedisaccurateasofOctober2nd,2012.
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AcknowledgementsThiseffortwassupportedbygrantsfromNASA(NNX09AB78G),NSF(OCE-1061934)andtheAdvancedResearchProjectsAgency—Energy(ARPA-E),U.
S.
DepartmentofEnergy(DoE)(DE-AR0000079)toP.
R.
G.
A.
B.
wasaHowardHughesMedicalInstitutefellowoftheLifeSciencesResearchFoundationandiscurrentlyaL'OrealUSAForWomeninScienceFellow.
E.
J.
G.
isaDoEfellow(DoESCGF,DE-AC05-06OR23100).
TheHarvardCenterforNanoscaleSystems(CNS)issupportedbytheNationalScienceFoundation(ECS-0335765).
WethankDianneNewmanforprovidingTIE-1strains,aswellasDanielRogers,ColleenHansel&EmilyFlemingforconstructivecomments,WealsothankWilliamDaleyBonicioforcollectingtheICP-MSdata.
AuthorcontributionsA.
B.
,E.
J.
G.
,C.
V.
,E.
A.
P.
andP.
R.
G.
designedtheresearch.
A.
B.
,C.
V.
,E.
A.
P.
,E.
J.
G.
andP.
R.
G.
analysedthedata.
E.
J.
G.
andC.
V.
contributedequallytothiswork.
A.
B.
andP.
R.
G.
wrotethemanuscriptwithinputfromallco-authors.
AdditionalinformationSupplementaryInformationaccompaniesthispaperathttp://www.
nature.
com/naturecommunicationsCompetingnancialinterests:Theauthorsdeclarenocompetingnancialinterests.
Reprintsandpermissioninformationisavailableonlineathttp://npg.
nature.
com/reprintsandpermissions/Howtocitethisarticle:Bose,A.
etal.
Electronuptakebyiron-oxidizingphototrophicbacteria.
Nat.
Commun.
5:3391doi:10.
1038/ncomms4391(2014).
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1038/ncomms4391ARTICLENATURECOMMUNICATIONS|5:3391|DOI:10.
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Allrightsreserved.
Bose1,E.
J.
Gardel1,2,*,C.
Vidoudez1,*,E.
A.
Parra1&P.
R.
Girguis1Oxidation–reductionreactionsunderlieenergygenerationinnearlyalllifeforms.
Althoughmostorganismsusesolubleoxidantsandreductants,somemicrobescanaccesssolid-phasematerialsaselectron-acceptorsor-donorsviaextracellularelectrontransfer.
Manystudieshavefocusedonthereductionofsolid-phaseoxidants.
Farlessisknownaboutelectronuptakeviamicrobialextracellularelectrontransfer,andalmostnothingisknownabouttheassociatedmechanisms.
Hereweshowthattheiron-oxidizingphotoautotrophRhodopseu-domonaspalustrisTIE-1acceptselectronsfromapoisedelectrode,withcarbondioxideasthesolecarbonsource/electronacceptor.
BothelectronuptakeandruBisCoformIexpressionarestimulatedbylight.
Electronuptakealsooccursinthedark,uncoupledfromphotosynthesis.
Notably,thepioABCoperon,whichencodesaproteinsystemessentialforphotoautotrophicgrowthbyferrousironoxidation,inuenceselectronuptake.
Thesedatarevealapreviouslyunknownmetabolicversatilityofphotoferrotrophstouseextracellularelectrontransferforelectronuptake.
DOI:10.
1038/ncomms43911DepartmentofOrganismicandEvolutionaryBiology,HarvardUniversity,16DivinityAvenue,Cambridge,Massachusetts02138,USA.
2SchoolofEngineeringandAppliedSciences,HarvardUniversity,29OxfordStreet,Cambridge,Massachusetts02138,USA.
*Theseauthorscontributedequallytothiswork.
CorrespondenceandrequestsformaterialsshouldbeaddressedtoP.
R.
G.
(email:pgirguis@oeb.
harvard.
edu).
NATURECOMMUNICATIONS|5:3391|DOI:10.
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Allrightsreserved.
Microbialmetabolicactivitysubstantiallyinuencesmatterandenergyowthroughthebiosphereanddrivesglobalbiogeochemicalcycles1.
Microorganismshavebroadmetaboliccapabilitiesandcanusechemicallydiverse,solublesubstratesforenergygeneration.
Somemicrobescanalsousesolid-phaseelectronacceptorsand-donorsviaaprocesscalledextracellularelectrontransfer(EET)2–5.
RecentyearshavebeenawatershedformicrobialEET,withmanystudiesfocusingontherelevanceofEETinbioremediationandbiotechnology6,7.
AlthoughstudiesoverthepastfewdecadeshaveexaminedtheroleofmicrobialEETindonatingelectronstometaloxidesandoxygen6,8–10,theinvolvementofmicrobialEETinfacilitatingelectronuptakehascometotheforeonlyrecently11.
Studiesshowthatmixedmicrobialcommunitiesfacilitatecathodicreactionsinbioelectrochemicalsystems(BESs),impli-catingmicrobesinelectronuptake11.
Recentstudiesusingpurecultureshaveshownthatatleastthreemicrobesarecapableoftakingupcurrentfromanelectrode:Sporomusaovata12,MariprofundusferrooxydansPV-1(ref.
13)andShewanellaoneidensisMR-1(ref.
14).
OnlythestudyperformedonShewanellaconsideredthegeneticlocitobelikelyinvolvedinelectronuptake14.
Assuch,themechanismsunderlyingelectronuptakebymicrobesincludingShewanellaremainpoorlyunderstood.
Characterizinghowmicrobestakeupelectronsfromsolid-phaseelectrondonorsiscriticaltoourunderstandingoftheecologicalandevolutionaryimplicationsofthisprocess,aswellastoanyfuturebiotechnologyeffortssuchaselectrosynthesis6,15.
Theestablishmentofgenetic,genomicandmetabolicstudiesinmicrobesthatnaturallytakeupelectronsviaEETwillleadtothefollowing:(1)identicationoftheassociatedgeneticdeterminants;(2)theunderlyingmolecularmechanisms;and(3)alsofacilitateexperimentsthatexaminetherelationshipbetweenelectronuptakeandcellularmetabolism.
HerewepresentdataonourstudiesofRhodopseudomonaspalustrisTIE-1(TIE-1),aphotoautotrophicmicrobecapableofacceptingelectronsfromavarietyofelectrondonors,includingiron16–18(SupplementaryFig.
1).
WechoseTIE-1asthemodelorganismbecauseitusesferrousiron,Fe(II),asanelectrondonorforphotosynthesis(photoferrotrophy)16.
Moreover,themetabolicversatilityandgenetictractabilityofTIE-1helpstoreadilyinterrogatethefundamentalphysiologicalrelevanceofelectronuptake,includingthedegreeandconditionsunderwhichTIE-1takesupelectrons,somegeneticlociencodingsystemsinvolvedinelectronuptake,andtherelationshipbetweenelectronuptakeandotherphysiologicalprocessessuchasphotosynthesisandcarbonxation.
WeobservethatTIE-1acceptselectronsfromapoisedelectrode,withcarbondioxideasthesolecarbonsource/electronacceptor.
BothelectronuptakeandruBisCo(ribulose-1,5-bisphosphatecarboxylase/oxygenase)formIexpressionarestimulatedbylight.
Electronuptakealsooccursinthedark,uncoupledfromphotosynthesis.
ThepioABCoperon,whichencodesaproteinsystemessentialforphotoautotrophicgrowthbyferrousironoxidation,inuenceselectronuptake.
ResultsTIE-1acceptselectronsfromapoisedelectrode.
TocharacterizeelectronuptakebyTIE-1,BESswereused.
BESsareexperimentalsystemswhereanelectrodeissubmergedinabioreactorandisusedtomimicthemidpointpotentialofsolid-phaseminerals3,6.
BESsprovideanattractivealternativetousingnaturalredoxactiveminerals,allowingonetostudymicrobialEETwithoutconfoundingissuessuchasmineralogicalchangesduringexperimentation6,9,13,19,20.
Theelectrodeswerepoisedat100mVversusStandardHydrogenElectrode(SHE)(SupplementaryFig.
2),asthispotentialisconsistentwithformsofFe(II)usedbyTIE-1(ref.
21).
TIE-1wassubjectedtothreetreatmentsasfollows:(1)illuminatedreactorswithpoisedelectrodespassingcurrent(illuminatedtreatment);(2)non-illuminatedreactorswithpoisedelectrodespassingcurrent(darktreatment);and(3)illuminatedreactorswithelectrodesatopencircuit,passingnocurrent(controltreatment).
ThehighestratesofcurrentuptakebytheTIE-1wild-type(WT)wereobservedinilluminatedtreatments,uptoB1.
5mAcm2(Fig.
1a).
Cyclicvoltammetry(CV)oftheelectrodesintheilluminatedtreatmentsrevealedtwomodestbutdiscernablecathodicpeaksat0.
27and0.
4V(versusSHE)intheWT,whichwereabsentintheabioticcontrol(Fig.
1b),suggestingthepresenceofredoxactivecomponentsintheilluminatedreactors.
Cathodiccurrentwasalsoobservedinthedarktreatments,suggestingthatcurrentuptakeoccurredundertheseconditions,althoughB70%lowerthanwhenilluminated(Fig.
1a).
Weobservedthatcellswereattachedtoelectrodesduringallbiotictreatments,withthehighestviablecelldensitiesoccurringinthelighttreatment(Fig.
2a,b,SupplementaryFig.
3,SupplementaryTables1and2).
Planktoniccellnumbersincreasedduringthecourseofthe1-dayincubations,althoughtheincreaseintheWTilluminatedandcontroltreatmentswerenotsignicantlydifferentattheendoftheseexperiments(SupplementaryTables3and4).
Currentdensity(μAcm–2)00.
5121.
5Wild-type–0.
200.
20.
40.
60.
811.
23020100102030Currentdensity(μAcm–2)Potential(VversusSHE)WTΔpioABCWT-ΔpioABCWTnormalWTdarkFigure1|CurrentuptakebyWTR.
palustrisTIE-1.
(a)CurrentdensitiesofR.
palustrisTIE-1WTunderilluminatedanddarkconditions.
Errorbarsindicates.
d.
oftheseaverages(n3),anddatareportedareconsistentwith10independentruns.
(b)CyclicvoltammogramsofWTandDpioABCmutantafter96hoftreatmentinbioelectrochemicalreactorswithelectrodespoisedat100mVversusSHE.
Twosetsofanodic–cathodicpeakpairswereidentiedat0.
27and0.
40V,respectively.
TheredtracedepictsthedifferenceinmagnitudebetweentheWTandtheDpioABCmutantstrain.
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TobestcapturethechangesingeneexpressionduringtheonsetofEET,thesetreatmentslastedB24htoavoidissuesthatcanariseduringprolongedexperiments(forexample,differencesingrowthphase).
Nevertheless,theapparentchangesinplanktoniccelldensitywouldsuggestthat(1)currentwasbeingusedtosupportplanktonicgrowth;or(2)anexogenouselectrondonorwasavailableforgrowth.
Notably,inseparate5-dayilluminatedtreatments,TIE-1exhibited10-foldhigherdensitiesthandarkandcontroltreatments.
However,intheseshorter-termilluminatedtreatments,massbalancecalculationssuggestthattheplanktoniccellincreaseinthebioreactorsistwoordersofmagnitudelowerthanthatpredictedifallcurrentwenttobiomass(SupplementaryNote1).
Moreover,thetraceconcentra-tionsofironpresentinthemedium(tosupportbiosynthesis)couldonlyaccountforupto4.
0104cellspermloftheobservedcellincrease(forcalculationsseeSupplementaryNote2).
Thus,thereisanelectronsinkotherthanbiomass,andnotablythegeneexpressiondatasuggestthatthiscouldbereductiveCO2assimilation(discussedindetailbelow).
Thesedataprovidetherstevidenceoflight-stimulatedelectronuptakebyaphotoferrotroph,withsomeelectronuptakealsooccurringinthedark,independentofphotosynthesis.
PhototrophicmicrobesrelatedtoTIE-1usephoticenergyforATPsynthesisthroughcyclicelectronow,withouttheneedforanelectrondonor22.
Anelectrondonorisonlyrequiredtoproducereducingequivalents(NADPH)forcellularmetabolismmostlikelybyreverseelectrontransfer23.
Inthedark,noATPcanbegeneratedviaphotosynthesisbutcellularmetabolismcontinues22,thusrequiringanelectrondonorthatislikelyrepresentedbytheobserveddarkcurrentinourexperiments.
Thedarkcurrentalsosuggeststhattheelectronuptakemachineryisindependent(orcanbeuncoupled)ofthecyclicphotosyntheticapparatus.
TheincreaseinelectronuptakeinthepresenceoflightsuggeststhattheATPgeneratedusingtheenergyoflightisusedbycellularprocesses,necessitatingahigherlevelofelectronuptake.
ThepioABCoperonhasaroleinelectronuptake.
BecausethesedatarevealthatTIE-1acceptselectronsfromasolid-phasecon-ductor,wereasonedthatitmightuseconservedstrategiestomediatethiselectronuptake.
PreviousstudieshaveshownthatpioABCisessentialforphotoferrotrophy,andhavespeculatedthatthePioproteinsmightbeinvolvedinelectrontransferfromFe(II)totheelectrontransportchain17,21.
ThepioABCoperonencodestheputativeproteinsPioA,aperiplasmicdecahemecytochrome,PioB,anoutermembraneporin,andPioC,aperiplasmichighpotentialiron–sulfurclusterprotein17,21(SupplementaryFig.
4).
Usingmutantstudiesandexpressionanalysis,wedirectlytestedwhetherthePioABCsystemhasaroleinelectronuptake.
WeobservedthatDpioABC-illuminatedbiolmsaccepted30%lesscurrentthantheWT(Fig.
3a),andthemutantilluminatedbiolmswereB8–10-foldlessdensethantheWT(SupplementaryTable2).
FewerDpioABCmutantscolonizedtheelectrodeintheilluminatedtreatments,whichmightresultfromanattachmentdefect.
However,thiswasnotobservedinthecontroltreatments,thatis,intheabsenceofcurrent,asthemutantcelldensitieswerecomparabletotheWT(DpioABC:9.
2106cellspercm2;WT:8.
1106cellspercm2).
Ifweassumethatonlyattachedcellscontributetoelectronuptake,thentheDpioABCmutantsseemtoacceptmorecurrentpercellthantheWT(SupplementaryTables5and6).
ThiswouldimplythattheDpioABCmutantcellscantakeupelectronsmoreactively,perhapsviacompensatorychanges.
Weposit,however,thatsuchanassumptionisinaccurate.
asitdisregardsthepotentialcontributionofplanktoniccellstoelectronuptake(SupplementaryFig.
2).
Regardless,thesedatacollectivelyshowthatthePiosysteminuenceselectronuptake,althoughothermechanismsofelectronuptakeclearlyexistinTIE-1,asthemutantmaintainsnearly70%ofthecurrentuptakeseenintheWT(Fig.
3a).
FuturestudiesshouldexaminethemeansbywhichthePiosysteminuencesbothphototrophicironoxidationandEET,anditspotentialroleingoverningattachmenttopoisedelectrodes.
WehypothesizedthatelectronuptakemightinuencephysiologicalsystemsthathavearoleinEETaswellasredoxbalance.
Accordingly,weassessedtheexpressionofthetargetgenes,includingthoseencodingthePioABCproteins,acrossalltreatments.
ExpressionofpioAintheWT-illuminatedbiolmwasupregulatedbyB48-fold,whereaspioBandpioCshowedmoremodestupregulationcomparedwiththecontroltreatment(11-and3-fold,respectively;Fig.
3b).
TheobservedlevelsofpioAintheWT-illuminatedbiolmwerewellabovethoseoftheinoculum(grownonH2:CO2;Fig.
3b).
Theywere,however,comparabletogeneexpressionobservedduringphotoferro-trophicgrowthonsolubleFe(II)inconventionalcultureapparatus.
ThedecreasedcurrentuptakeoftheDpioABCmutantaswellastheobservedupregulationofthePiogenesintheBESsystemtogethersuggestthatthePioABCproteinsmaybeinvolvedinelectronuptakebyTIE-1undertheseconditions.
ItshouldbenotedthatthePioABCmoduleoccursinanumberofanoxygenicphototrophicmicrobes,whichmightshowFigure2|R.
palustrisTIE-1cellsattachedtocathodes.
(a)FluorescencemicrographsofaR.
palustrisTIE-1WT-illuminatedbiolm.
Scalebar,10mm.
(b)ScanningelectronmicrographofaWT-illuminatedbiolm.
Scalebar,3mm.
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light-enhancedelectronuptakeasobservedinTIE-1(SupplementaryTable7),Moreover,non-phototrophicferrousiron-oxidizingbacteria(FeOB)alsopossessthePioABmodule,raisingthequestionastowhethertheseorganismsperformlight-independentelectronuptakesimilartotheobserveddarkcurrentinTIE-1.
TheinvolvementoftheMtrAB(relatedtoPioAB17)systemintheelectronacceptancebyS.
oneidensis(MR-1)fromapoisedelectrodealsosuggeststhatthismodulemighthaveadirectroleinelectronuptake14.
Electronuptakestimulatesexpressionofothergenes.
WeusedexpressionanalysesandmicroscopytofurtherexamineTIE-1'sresponsetoelectronuptake.
Exopolysaccharide(eps)geneswerehighlyupregulatedintheWT-illuminatedbiolmsand,insomecases,intheDpioABC-illuminatedbiolms.
ExpressionanalysisfurthershowedthatthepioChomologue(anotherhighpotentialiron–sulfurclusterproteinlocatedelsewhereonthechromosome)wasupregulated(fourfold)intheWT-illuminatedplanktoniccells(cellsnotattachedtotheelectrodepresentinthemedium)comparedwiththecontroltreatment(Fig.
4A(b)),suggestingthattheencodedproteinmighthavearoleinplanktoniccellincreaseundertheseconditions.
MicroscopyrevealedthatEPSproductionwasmostabundantinilluminatedbiolms(Fig.
4A(a)andB(a);SupplementaryFig.
5a,b).
Proteinstainingestablishedthepre-senceofextracellularproteinsinthecellsattachedtotheelec-trodes(SupplementaryFig.
5c,d).
FutureanalysisonbiolmandplanktoniccellsandtheproducedEPSwillhelpdeterminetheroleoftheseelementsinelectronuptake.
ruBisCoformIexpressionincreasesduringelectronuptake.
PreviousstudieshaveshownthatinorganismsrelatedtoTIE-1,electrondonorsarerequiredforgenerationofreducingequivalents,namelyNAD(P)H,whichservesasareductantforcellularprocessessuchascarbonxationviatheCalvincycle22,24–26.
RuBisCo,akeyenzymeintheCalvincycle,assimilatesCO2intoribulose-1,5-bisphosphateyieldingtwoPhotoferrotrophyInoculumDarkplanktonicControlplanktonicIlluminatedplanktonicIlluminatedbiofilmpioChomologue0100200300400500600700060120204080100140160–20020406080100abepsIepsIIepsIVepsVIPhotoferrotrophyInoculumDarkplanktonicControlplanktonicIlluminatedplanktonicIlluminatedbiofilmmRNAabundanceversusaerobicWTgrowthmRNAabundanceversusaerobicWTgrowthmRNAabundanceversusaerobicWTgrowthmRNAabundanceversusaerobicWTgrowthmRNAabundanceversusaerobicWTgrowthmRNAabundanceversusaerobicWTgrowth–20020406080100InoculumControlplanktonicIlluminatedplanktonicIlluminatedbiofilmWTIlluminatedbiofilmepsIepsIIepsIVepsVIWTΔpioABC010203040506070InoculumControlplanktonicIlluminatedplanktonicIlluminatedbiofilmWTIlluminatedbiofilmpioChomologueWTΔpioABCWTWT–40–30–20–10010203040PhotoferrotrophyInoculumDarkplanktonicControlplanktonicIlluminatedplanktonicIlluminatedbiofilmruBisCoformI–40–30–20–10010203040InoculumControlplanktonicIlluminatedplanktonicIlluminatedBiofilmWTIlluminatedbiofilmWTΔpioABCWTruBisCoformIIruBisCoformIIruBisCoformIABcFigure4|mRNAabundancedeterminedintheR.
palustrisTIE-1WT(A)andDpioABC(B).
(a)Exopolysaccharide(eps)genes;(b)pioChomologue;and(c)ruBisCoformIandIImRNAabundance.
Cellweregrownphotoautotrophicallyonhydrogenand5mMFeCl2.
qRT–PCRdataaretheaverages±s.
e.
forthreebiologicalreplicatesassayedintriplicate.
epsIRpal_3203,epsIIRpal_3763,epsIVRpal_3771,epsVIRpal_3777,pioChomologueRpal_4085.
WTnormalΔpioABCnormalΔpioABC02004006008001,0001,200050100150200250PhotoferrotrophyInoculumDarkplanktonicControlplanktonicpioApioBpioCIlluminatedplanktonicIlluminatedbiofilmmRNAabundanceversusaerobicWTgrowthCurrentdensity(μAcm–2)00.
5121.
5Figure3|CurrentuptakeandmRNAabundanceofthepioABCoperonbyWTR.
palustrisTIE-1andDpioABCmutantundervariousconditions.
(a)CurrentdensitiesofR.
palustrisTIE-1WTandDpioABCmutantunderilluminatedconditions.
Errorbarsindicates.
d.
oftheseaverages(n3),anddataareconsistentwith10independentruns.
(b)pioABCgenetranscriptabundancedeterminedintheWTgrownphotoautotrophicallyonhydrogenand5mMFeCl2.
qRT–PCRdataaretheaverages±s.
e.
forthreebiologicalreplicatesassayedintriplicate.
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moleculesof3-phosphoglycerate,whichareeventuallyreducedbyNAD(P)Htoglyceraldehyde3-phosphateinthereductivephaseofthecycle24–26.
TIE-1andrelatedmicrobesharbourgenesencodingtwoformsofruBisCo:formsIandII(refs24–26).
WeobservedthatruBisCOformIwasmosthighlyexpressedinWT-illuminatedbiolms(Fig.
4A(c)),andwastypicallyhigherthanruBisCoformIIduringconventional,photoautotrophicgrowthonhydrogenandFe(II)(Fig.
4A(c)).
Notably,ruBisCoformIexpressionwasnotinducedinthedarktreatment(Fig.
4A(c)).
PreviousstudiesontheregulationofruBisCoformIexpressioninCGA009/10(B99%identitytoTIE-1(ref.
16))showthatitisunderexquisitecontrolandispositivelyregulatedbyATPandNAD(P)H,metabolitesthatindicatetheenergystatusofthecell27.
Duringelectronuptakeinthepresenceoflight,TIE-1likelyproducesabundantATPandNAD(P)H,whichwepositleadstotheincreaseinruBisCoformIexpression.
Incontrast,boththesemetabolitesarelikelylowerinthedark,andthusruBisCoformIexpressiondecreases.
IthasalsobeensuggestedthatRuBisCoformIcanindirectlyactasanelectronsink(becauseincreaseinitsexpressionleadstohigherproductionof3-phosphoglycerate,whichservesasthesubstrateforthereductivepartoftheCalvincycle)tomaintainredoxbalanceinphotosyntheticbacteriarelatedtoTIE-1(refs25,26).
DiscussionOurdataprovidearstglimpseontheabilityofthephotoautotrophicbacteriumR.
palustrisTIE-1toacceptelectronsfromasolid-phaseelectrondonor.
Becausephotoautotrophsareexposedtodiurnalcyclesoflightanddarkconditions,wetestedtheeffectofilluminationontheabilityofTIE-1toacceptelectrons.
OurresultsshowthatTIE-1acceptselectronsunderbothlightanddarkconditions,althoughlightstronglystimulateselectronacceptance(Fig.
1a).
ThemassiveupregulationingenesthatencodeforthepioABCsystem(encodingproteinsthataresuggestedtohaveacriticalroleinphototrophicironoxidationbyTIE-1(ref.
17)),aswellasthedecreaseincurrentobservedinpioABCmutants,implythatthePioproteinsareengagedinelectronuptake.
Incontrast,thepioABCmutantsappeartohaveanattachmentdefecttopoisedelectrodes,thusexhibitinghighercell-specicelectronuptakeratescomparedwiththeWT(Fig.
3a,SupplementaryTables2and6).
ThisapparentpleiotropymakesitdifculttoascribeasimpleroletothepioABCsysteminelectronuptake.
Itshouldbenotedthatourexperimentaldesigndoesnotallowustoexcludethepossibilitythatboththebiolmandtheplanktoniccellswereengagedinelectronuptake,andthefreelivingandbiolmlifestylesmightbedynamic(SupplementaryFig.
2).
Theplanktoniccellsmayhavecontributedtocurrentuptakethroughdirectencounterswiththeelectrode(thesereactorswerewellstirred)orviasolublecompounds(wewereunable,however,todetectanyredoxactivecompoundsinthespentmedium;SupplementaryFigs2and6).
TranscriptomicanalysisshowedthatruBisCoformIexpressionwashighestinthepoisedilluminatedelectrodes(Fig.
4A(c)),suggestingthatthisenzymecouldbeanindirectelectronsinkashasbeenobservedinotherrelatedorganisms25,26.
BecauseRuBisCoispartoftheCalvinCycle,thecarbonxationpathwayinTIE-1,itisplausiblethatsomeoftheseelectronswouldgotobiomass.
Althoughwedidseeanincreaseinthetotalcelldensity,therewasnosignicantdifferenceamongtheseshort-termtreatments,andthemassbalanceanalysessuggestthatbiomassonlyaccountsforamodestamountofthetotalcurrentpassed(SupplementaryTables1–4).
Asmentioned,theseexperimentsweredesignedtobeshortindurationtoavoidconfoundingfactorsassociatedwithgrowthandchangesingrowthphase.
InlightofTIE-1'stypicallymodestgrowthrates,itislikelythatincreasesinbiomassattributabletoelectronuptakeduringtheseshort-termtreatmentsarebelowourlimitsofresolution.
Innature,electronuptakeviaEETcouldamelioratemetabolicdilemmasthatneutrophilicFeOBs,suchasTIE-1,areknowntoface.
FeOBsoftencontendwiththeprecipitationofinsolubleironoxidesoutsidethecellthatareabyproductoftheirmetabolicactivityandpotentiallylimitFe(II)availability21,28.
TIE-1producespoorlycrystallineFe(III)hydroxides,whichovertimeareabioticallytransformedtothe(semi)conductivemineralsgoethiteandmagnetite16,29.
ConductionofelectronsthroughthismatrixwouldallowTIE-1(andpotentiallyotherFeOBs)accesstoelectronsfromremoteelectrondonors,includingFe(II)(SupplementaryFig.
7),viaprocessessuchaselectronconductionandironatomexchange30–32.
Indeed,recentstudieshaveshownthatconductivemineralscanfacilitateelectrontransfertomicrobesfromremoteelectrondonors(includingothermicrobes)33.
Thesedataextendthisphenomenontophotoautotrophsthatishighlyrelevantbecausetheirrestrictiontothephoticzonemighthinderaccesstoreductantsindeeper,anaerobiclayers22,34.
InadditiontotheecologicaladvantagesofelectronuptakeviaEET,thereissubstantialinterestinexploitingphotoautotrophsforbothenergyandbiofuelgeneration11,andidentifyingageneticallytractablephotoautotrophthatcanuseelectriccurrentasanelectrondonorholdspromiseinfutureelectrosynthesisapplications11.
AlthoughtheecologicalsignicanceofEETisjustcomingtothefore,ourdataillustratethepotentialvalueofEETtomicroorganismsinnature,inparticularphotoautotrophs.
MethodsBacterialstrains,mediaandgrowthconditions.
R.
palustrisTIE-1wasgrownasdescribedpreviously18.
Forexperiments,cellswerepre-grownautotrophicallyon80%hydrogen:20%carbondioxide(H2:CO2)at200kPainfreshwatermedium(FW)with20mMbicarbonate.
TheDpioABCstrainusedhereinwasconstructedaspreviouslydescribed17.
Phototrophicpre-growthwasat30°Cusinga60-WincandescentlightsourceprovidingtotalirradianceofB40Wm2.
BioelectrochemicalreactorstudieswereconductedwithFWmedium(minimalsaltsmediumlackinganyaddedelectrondonors16–18)with20mMbicarbonate(solecarbonsource16–18),bufferedtopH6.
8andwithnoexogenouselectrondonor.
AllbacterialstrainswereroutinelytestedforpuritybystandardPCRusingprimersindicatedinSupplementaryTable8.
Owingtobiologicalvariationinthecultivationeffort,whichresultedindifferentcelldensitiesintheinoculumandprohibitscomparisonacrosstreatments,weranaWTcontrolinparallelwitheveryindividualtreatmenttoaccountforthesedifferences.
AllcomparisonsbetweenWTandtreatmentsaremadeusingthesepairedruns.
BESandconditions.
TheBESsconsistedofnew,acid-washed,combusted350mlborosilicateglassH-cellreactorsequippedwithtwobutylrubbersamplingportsinthecathodicchamber(AdamsandChittendenScienticGlass,Berkeley,CA,USA).
Avacuumclampheldtheanodicandcathodicchamberstogether,andelectrolyteswereseparatedusingacation-exchangemembrane(Naon117)withanactivecross-sectionof20cm2(FuelCellStore,Boulder,CO,USA).
Theworkingelectrodesconsistedofspectroscopicallypure1/800-diametergraphiteevaporationrods(SPI01685-FA,StructureProbeInc.
,WestChester,PA,USA)thatweremechanicallypolishedwith1200gritsandpaper,soakedin5%HClfor12handstoredinultrapuredeionizedwater.
Thegraphiterodswerethoroughlydriedbeforeusebyallowingthewatertoevaporate.
Eachreactorwasttedwiththreegraphiterodstoprovideatotalimmersedprojectedelectrodesurfaceareaof18cm2.
Therodsweresealedwithttingsandferrulesonthereactorcap(UpchurchScientic,OakHarbor,WA,USA).
Outsidethereactor,rodswereelectricallyconnectedtoonepotentiostatusingalligatorclips(describedbelow).
Thecounterelectrodeconsistedofcarboncloth(FuelCellStore),whichwasmechanicallyattachedtoatitaniumwirepiercedthrougharubberstopper(VWR)andsuspendedinthecounterchamber.
Electricalconditionsandcyclicvoltammograms.
Thereactorswerepoisedusingcustom-builtpotentiostatsengineeredformicrobialchronoamperometry(KarmaElectronicsInc.
,Somerville,MA,USA).
DatawerecollectedthroughaNationalInstrumentsDAQ(NI-6225)every10susingLabviewSignalExpresssoftware(NationalInstruments,Austin,TX,USA).
Basedonpreliminaryanalysesofelec-troactivityinWTR.
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SHE(100mVofthebiologicalEpcroughlyat200mVversusSHE)toassurecathodicconditionsduringtheexperiment.
Importantly,thispotentialalsoensuresthatareductiveFe(III)/Fe(II)cycleisnotestablishedduringtheseexperiments(theredoxpotentialatpH7.
0oftheFe(III)/Fe(II)coupleis14mV,andelectrontransferfromanelectrodepoisedat100mVwillbeanendergonicprocess)15.
Reportedcurrentdensities(mAcm2)wereobtainedbyaveragingregionsof48hofstablecurrentineachreactor.
CVwasconductedusingaGamryR600potentiostat(Gamry,Warminster,PA,USA).
BiolmCVswereobtainedwithascanrangeof100to900mVversusSHEatarateof20mVpersecond.
Supernatantvoltammogramswereobtainedusinga3-mmdiameterglassycarbonelectrode(partno.
A-002012,BioLogic,Claix,France),underaN2atmosphere,scannedbetween0and500mVversusSHEat20mVpersecond.
Wewereunabletodetectanyelectro-activesolublespeciesinthe0.
2-mMlteredspentmedium(SupplementaryFig.
7).
Toassesstheactivesurfaceareavariabilitybetweenelectrodes,CVswerecollectedabioticallyinFWmedium.
PotentialisreferencedtotheSHEunlessotherwisespecied.
Descriptionofbioelectrochemicalsetups.
ThedistancebetweentheworkingandcounterelectrodeswasB11cm.
AssembledBESreactorsweresterilizedbyautoclavinginsterilizationpouchesandplacedinsideananaerobicchamber(Coy,2%hydrogenandpalladiumcatalysts).
Ag/AgClreferenceelectrodeswerecustom-madeusingglasstubing(4mmKIMAX),silverwire(0.
5mmdiameter)andporousvycortips(1/80diameter,MF-2064,BASi).
Referenceelectrodeswerecalibratedbeforeeachexperiment,placedintheanaerobicchamber,sterilizedwith70%ethanolandplacedinthecounterchamberforthedurationoftheexperiments.
Althoughinsidetheanaerobicchamber,mediaandcounterbufferwereaddedtothecathodeandanodechambers,respectively.
InoculationoftheBESsoccurredinsidetheanaerobicchamberbeforetransferringthemoutsidetheanaerobicchambertoestablishelectricalconnections.
Thereactorsystemwaspurgedcon-tinuouslywitha1-cm3min1streamof0.
2mmlter-sterilized,deoxygenatedgasstreamof80%:20%N2:CO2and100%N2onthecathodicandanodicside,respectively,usingahypodermicneedleimmersed1cmbelowthemediasurface.
Thegasesweredeoxygenatedusingahighcapacityoxygentraploweringtheoxygenlevelstoo0.
01p.
p.
m.
(Restek,Bellefonte,PA,USA).
EachBESwasindividuallyhousedwithafreshincandescent60Wbulbprovidingatotalirra-dianceofB40Wm2.
DarkBESslackedabulbandwerecoveredthoroughlywithblackpapertopreventlightexposure.
Allworkingchamberswerestirredgentlywithamagneticbarandincubatedat30°C.
Allincubations,acrossalltreatments,lasted24h.
Sampling.
Thereactorswereinoculatedwith10mlofcellsinthemid-exponentialphaseofphotoautotrophicgrowthon80%H2:20%CO2.
Media(1ml)waswithdrawnfromthereactorsimmediatelyfollowinginoculationandusedforopticaldensity(OD660)determinationwitha4802spectrophotometer(ColePar-mer,VernonHills,IL,USA),andforpHmeasurements(InlabExpertPropHmetreandprobe;MettlerToledo,Schwerzback,Switzerland).
Culture(4ml)wasalsowithdrawnfromthereactorsforcellcounts.
Cellswerexedin4%paraf-ormaldehydeforcellcounting(ElectronMicroscopySciences,Hateld,PA,USA).
Attheendofeachexperiment,oneoftheelectrodeswasimmediatelydippedintoRNAlater(Qiagen,Valencia,CA,USA)forRNAextraction.
Also,5mlofplank-toniccellswereimmediatelypreservedinRNAlaterandlteredonapoly-ethersulfonemembraneforRNAextraction(Corning,Tewksbury,MA,USA).
AllRNAsampleswerestoredat80°C.
AsecondelectrodewascutintoB5mmpiecesandtransferredintoxativesorstainingsolutionsformicroscopicanalyses(describedbelow).
Postexperimentation,1mlofplanktoniccellswassampledforOD660determination,and2–4mlforpHmeasurements.
Theremainingculturevolumewasthenlteredona0.
2-mmcelluloseacetatelter(Corning).
Afterresuspensionin8mlofmedia,theseplanktoniccellswerepelletedintwo2mlmicrocentrifugetubes(18,000gfor10min)andkeptat80°Calongwiththelteredspentmedium.
Proteinanalysis.
SubsamplesfortotalproteinanalysiswereprocessedinProtloBind1.
5or2mlmicrocentrifugetubes(Eppendorf,Hauppauge,NY,USA).
Trichloroaceticacidprecipitationwasusedaspreviouslydescribed18.
Thepelletsweredriedundervacuumfor1htoremoveresidualacetoneandthenresuspendedin650mlof3MUrea(ACSgrade,Alfa-Aesar,WardHill,MA,USA).
Toensurecompleteresuspension,thesampleswereincubatedat80°Cfor3dayswithfrequentsonicationinasonicbath(FS30H;ThermoFisherScientic,Waltham,MA,USA).
ThePierceBCA(bichinchoninicacid)ProteinAssayKit(ThermoScientic,Rockford,IL,USA)wasusedusingthemicrotiterplatemethodforproteinestimationasspeciedbythemanufacturerwiththeprovidedbovineserumalbuminasthestandardprotein.
Eachsamplewasquantiedintriplicate.
Absorbanceat562nmwasmeasuredafter30sshakingat37°CusingaSpectramaxPlus384platereader(MolecularDevices,Sunnyvale,CA,USA).
Fluorescencemicroscopysamplepreparationandimaging.
Sectionsoftheelectrodewereplacedintooneofthreesolutionscontaining1mM40,6-diamidino-2-phenylindole(LifeTechnologies,GrandIsland,NY,USA)aswellas(1)LIVE/DEADstain(0.
5mMSYTO9and3mMpropidiumiodide,L7012,LifeTechnologies);(2)EPSstain(200mgl1ConcanavalinAandAlexa488,LifeTechnologies);and(3)Proteinstain(undilutedFilmTracerSYPRORubyBiolmMatrixStain,LifeTechnologies).
Tubeswerewrappedinaluminiumfoilandkeptatroomtemperatureforatleast30min.
Sampleswerethenplacedin1phos-phate-bufferedsaline(PBS)inaglass-bottomeddishandimagedwithaZeiss700invertedconfocalmicroscopewiththefollowingimaginglasersandZeisslters:(1)live/dead555and488nm,SP490;405nm,SP555;(2)EPS488and405nm,SP490andLP490;and(3)protein555nm,SP490;405nm,SP555.
ThisworkwasperformedattheHarvardCenterforBiologicalImaging.
Scanningelectronmicroscopy.
Sectionsoftheelectrodewerecutusingsteriletechniquesandimmediatelyplacedintoasterilemicrocentrifugetubecontainingoneofthreesolutions:(1)5%glutaraldehyde(ElectronMicroscopySciences)in1PBS;(2)2%paraformaldehyde(ElectronMicroscopySciences)in1PBS;and(3)2%glutaraldehydein1PBSwith0.
15%SafraninO(Sigma-Aldrich,StLouis,MO,USA),whichhaspreviouslybeenshowntoaidinEPSpreservation7.
Sampleswereheldat4°Cfor24hbeforebeingsubjectedtoethanoldehydrationbyplacingthemin35,50,70,95and100%ethanol(200proof)inPBSor0.
1MPBSsolutionsfor10mineach.
The100%ethanolsolutionwaschangedvetimes,andthesamplewasleftinethanolforcriticalpointdrying(Autosamdri815A;Tousimis,Inc.
)witha15-minpurgetime.
Thesampleswereadheredtoscanningelectronmicroscopy(SEM)postswithcarbonlmtapeandthenimagedwithaSEMat5kV(JEOL,Inc.
).
Cellcountsforelectrodesampleswereperformedbyanalysingmicroscopyeldstakenatthesameworkingdistance(4.
5mm)toimage,countingatleast500cellsorexamining12eldsofviewifcelldensitywaslowandnormalizedtototalarea.
ThisworkwasperformedattheHarvardCenterforNanoscaleSystems(CNS).
RNAisolation.
Forplanktonicassessments,preservedcellsweredislodgedfromthepolyethersulfonemembranebeforeRNAextractionbyvortexingfor3mininaTris-EDTAbuffer.
Forbiolmassessment,thecellsweredislodgedfromthegra-phitebyscrapingwithasterilerazor,thenvortexingvigorouslyinTris-EDTAbuffer.
RNAwasextractedasdescribedpreviously5.
TheRNAconcentrationwasquantiedusingaNanoDropND1000(ThermoScientic,Wilmington,DE,USA).
RNAamplication.
TheRNAobtainedfromthebiolmonthegraphitewascleanedwiththeMEGAclearKit(LifeTechnologies)asperthemanufacturer'sguidelines.
ThepuriedRNAwasprecipitatedusingammoniumacetate.
ThereconstitutedRNAwasusedastemplatefortheMessageAmpII-BacteriaKitasperthemanufacturer'sguidelines(LifeTechnologies).
QuantitativereversetranscriptionPCR.
Geneexpressionanalysiswasper-formedusingquantitativereversetranscriptionPCR(qRT–PCR).
ThecomparativeCtmethodwasusedasdescribedpreviouslytoassessexpressionofthepioABCoperonandotherrelevantgenes5.
Primerefcienciesweredeterminedusingthemanufacturer'smethod(AppliedBiosystems,Inc.
UserBulletinno.
2).
clpXandrecAwereusedasthetwointernalstandards,whichhavebeenpreviouslyusedandvalidatedasinternalstandards18.
TheprimersusedfortheassaysareindicatedinSupplementaryTable5.
TheiScriptcDNAsynthesiskitwasusedforreversetranscription(Bio-Rad,Hercules,CA,USA).
TheiTaqFASTSYBRGreenSupermixwithROX(Bio-Rad)andtheStratageneMx3005PQPCRSystem(Agilent,SantaClara,CA,USA)wereusedforallquantitativeassays.
Cellcounting.
Theparaformaldehyde-xedsamplesweretransferredintoAmiconcentrifugelters(AmiconUltrael100k,regeneratedcellulosemembrane;Millipore,Carrigtwohill,CO,Ireland)andcentrifugedfor10minat1,000g.
ThepelletwasresuspendedinPBSandwashedtwice.
ThecellswererecoveredbycentrifugationoftheAmiconinreversepositionfor15minat3,000g.
Theresultingsampleshado0.
04%paraformaldehyde.
Picogreenwasaddedtothecells(Quant-iTPicoGreendsDNA,LifeTechnologies),andthecellswerecountedin96-wellplatesalongwith50mlofSpheroAccuCountblankbeads(Spheroteck,LakeForest,IL,USA).
CelldensitywasestimatedwithaLSRIIowcytometer(BD,Sparks,MD,USA)usinga488-nmlaser.
Acalibrationcurverelatingtheratioofcelleventstobeadseventswithcelldensitywasconstructedbyanalysingadilutionseriesofacellsample,thedensityofwhichhasbeendeterminedbymicroscopy(withaHelberBacteriaCellcountingchamberwithThomaruling,Hawksley,Lancing,Sussex,UK).
Inductivelycoupledplasmamassspectrometry.
TomeasuretheconcentrationofironpresentinFWmediuminductivelycoupledplasmamassspectrometry(ICP-MS)wasperformedusinganAgilent7700xICP-MSwithanoctopoleMS(Agilent).
InternalstandardsusedwereGermaniumandManganese,whichwerewithinthedetectionlimitofoursystem.
Theamountofironinthebasalmediumwas4mMandrangedfrom2–4mMinthespentmedium.
Insilicomethods.
ForidentifyinghomologuesofthePioABCproteins,delta-blast35,FASTA36(http://www.
ebi.
ac.
uk/Tools/sss/fasta/),andtheIMGorthologneighbourhoodsearchwasused37(http://img.
jgi.
doe.
gov/cgi-bin/w/main.
cgi).
ARTICLENATURECOMMUNICATIONS|DOI:10.
1038/ncomms43916NATURECOMMUNICATIONS|5:3391|DOI:10.
1038/ncomms4391|www.
nature.
com/naturecommunications&2014MacmillanPublishersLimited.
Allrightsreserved.
SequencesimilaritywascalculatedusingEMBOSSmatcher38,39(http://www.
ebi.
ac.
uk/Tools/psa/emboss_matcher/).
ThedatareportedisaccurateasofOctober2nd,2012.
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AcknowledgementsThiseffortwassupportedbygrantsfromNASA(NNX09AB78G),NSF(OCE-1061934)andtheAdvancedResearchProjectsAgency—Energy(ARPA-E),U.
S.
DepartmentofEnergy(DoE)(DE-AR0000079)toP.
R.
G.
A.
B.
wasaHowardHughesMedicalInstitutefellowoftheLifeSciencesResearchFoundationandiscurrentlyaL'OrealUSAForWomeninScienceFellow.
E.
J.
G.
isaDoEfellow(DoESCGF,DE-AC05-06OR23100).
TheHarvardCenterforNanoscaleSystems(CNS)issupportedbytheNationalScienceFoundation(ECS-0335765).
WethankDianneNewmanforprovidingTIE-1strains,aswellasDanielRogers,ColleenHansel&EmilyFlemingforconstructivecomments,WealsothankWilliamDaleyBonicioforcollectingtheICP-MSdata.
AuthorcontributionsA.
B.
,E.
J.
G.
,C.
V.
,E.
A.
P.
andP.
R.
G.
designedtheresearch.
A.
B.
,C.
V.
,E.
A.
P.
,E.
J.
G.
andP.
R.
G.
analysedthedata.
E.
J.
G.
andC.
V.
contributedequallytothiswork.
A.
B.
andP.
R.
G.
wrotethemanuscriptwithinputfromallco-authors.
AdditionalinformationSupplementaryInformationaccompaniesthispaperathttp://www.
nature.
com/naturecommunicationsCompetingnancialinterests:Theauthorsdeclarenocompetingnancialinterests.
Reprintsandpermissioninformationisavailableonlineathttp://npg.
nature.
com/reprintsandpermissions/Howtocitethisarticle:Bose,A.
etal.
Electronuptakebyiron-oxidizingphototrophicbacteria.
Nat.
Commun.
5:3391doi:10.
1038/ncomms4391(2014).
NATURECOMMUNICATIONS|DOI:10.
1038/ncomms4391ARTICLENATURECOMMUNICATIONS|5:3391|DOI:10.
1038/ncomms4391|www.
nature.
com/naturecommunications7&2014MacmillanPublishersLimited.
Allrightsreserved.
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