new asgard archaea capable of anaerobic hydrocarbon cycling1 1 new asgard archaea capable of...

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NewAsgardarchaeacapableofanaerobichydrocarboncycling1

KileyW.Seitz1,NinaDombrowski1,3,LauraEme2,AnjaSpang2,3,JonathanLombard2,JessicaR.Sieber4,2

AndreasP.Teske5,ThijsJ.G.Ettema2,andBrettJ.Baker1*3

1.DepartmentofMarineScience,UniversityofTexasAustin,PortAransas,TX783732.UppsalaUniversity,4Uppsala Sweden 3. NIOZ, Royal Netherlands Institute for Sea Research, and Utrecht University, The5Netherlands4.UniversityMinnesotaDuluth,MN5.DepartmentofMarineSciences,UniversityofNorth6Carolina,ChapelHill,NC*Correspondingauthor789Large reservoirs of natural gas in the oceanic subsurface sustain a complex biosphere of10

anaerobic microbes, including recently characterized archaeal lineages that extend the11

potential to mediate hydrocarbon oxidation (methane and butane) beyond the12

Methanomicrobia.Herewedescribeanewarchaealphylum,Helarchaeota,belongingtothe13

Asgard superphylum with the potential for hydrocarbon oxidation. We reconstructed14

Helarchaeotagenomesfromhydrothermaldeep-seasedimentmetagenomesinhydrocarbon-15

richGuaymasBasin,andshowthattheseencodenovelmethyl-CoMreductase-likeenzymes16

thataresimilartothosefoundinbutane-oxidizingarchaea.Basedontheseresultsaswellas17

the presence of several alkyl-CoA oxidation and Wood-Ljungdahl pathway genes in the18

Helarchaeotagenomes,wesuggestthatmembersoftheHelarchaeotahavethepotentialto19

activateandsubsequentlyanaerobicallyoxidizeshort-chainhydrocarbons.Thesefindingslink20

a new phylum of Asgard archaea to themicrobial utilization of hydrothermally generated21

hydrocarbons,andextendthisgenomicblueprintfurtherthroughthearchaealdomain.22

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All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder.. https://doi.org/10.1101/527697doi: bioRxiv preprint

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Short-chainalkanes,suchasmethaneandbutane,areabundantinmarinesedimentsandplay25

an important role in carbon cycling with methane concentrations of ~1 Gt being processed26

globallythroughanoxicmicrobialcommunities1–3.Untilrecently,archaealmethanecyclingwas27

thought to be limited to Euryarchaeota4. However, additional archaeal phyla, including28

Bathyarchaeota5andVerstraetarchaeota6,havebeenshowntocontainproteinswithhomology29

totheactivatingenzymemethyl-coenzymeMreductase(Mcr)andcorrespondingpathwaysfor30

methane utilization. Furthermore, new lineages within the Euryarchaeota belonging to31

Candidatus Syntrophoarchaeum spp., have been shown to use methyl-CoM reductase-like32

enzymesforanaerobicbutaneoxidation7.SimilartomethaneoxidationinmanyANME-1archaea,33

butane oxidation in Syntrophoarchaeum is proposed to be enabled through a syntrophic34

interactionwithsulfurreducingbacteria7.Metagenomicreconstructionsofgenomesrecovered35

fromdeep-seasedimentsfromnear2000mdepthinGuaymasBasin(GB)intheGulfofCalifornia36

have revealed the presence of additional uncharacterized alkyl methyl-CoM reductase-like37

enzymesinmetagenome-assembledgenomeswithintheMethanosarcinales(Gom-Arc1)8.GBis38

characterizedbyhydrothermalalterationsthattransformlargeamountsoforganiccarboninto39

methane,polycyclicaromatichydrocarbons(PAHs), low-molecularweightalkanesandorganic40

acidsallowingfordiversemicrobialcommunitiestothrive(SupplementaryTable1)8–11.41

Recently, genomes of novel clade of uncultured archaea, referred to as the Asgard42

superphylum that includes the closest archaeal relativesof eukaryotes, havebeen recovered43

fromanoxicenvironmentsaroundtheworld12–14.Diversitysurveysinanoxicmarinesediments44

show that Asgard archaea appear to be globally distributed9,11,12,13. Based on phylogenomic45

analyses, Asgard archaea have been divided into four distinct phyla: Lokiarchaeota,46


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Thorarchaeota,OdinarchaeotaandHeimdallarchaeota,withthelatterpossiblyrepresentingthe47

closest relatives of eukaryotes12. Supporting their close relationship to eukaryotes, Asgard48

archaeapossessawide repertoireofproteinspreviously thought tobeunique toeukaryotes49

known as eukaryotic signature proteins (ESPs)17. These ESPs include homologs of eukaryotic50

proteins, which in eukaryotes are involved in ubiquitin-mediated protein recycling, vesicle51

formationandtrafficking,endosomalsortingcomplexesrequiredfortransport(ESCRT)-mediated52

multivesicular body formation aswell as cytokinetic abscission and cytoskeleton formation18.53

Asgardarchaeahavebeensuggestedtopossessheterotrophiclifestylesandareproposedtoplay54

aroleincarbondegradationinsediments;however,severalmembersoftheAsgardarchaeaalso55

havegenesthatcodeforacompleteWood-Ljungdahlpathwayandarethereforeinterestingwith56

regardtocarboncyclinginsediments14,19.57

Here we present the first evidence of metagenome assembled genomes (MAGs),58

recovered from Guaymas Basin deep-sea hydrothermal sediments, which represent a new59

Asgard phylumwith themetabolic potential to perform anaerobic hydrocarbon degradation60

usingamethyl-CoMreductase-likehomolog.61

62

Results63

IdentificationofHelarchaeotagenomesfromGuaymasBasinsediments.Werecentlyobtained64

over~280gigabasesofsequencingdatafrom11samplestakenfromvarioussitesanddepthsat65

GuaymasBasinhydrothermalventsediments20.Denovoassemblyandbinningofmetagenomic66

contigsresulted in thereconstructionofover550genomes(>50%complete)20. Withinthese67

genomeswedetecteda surprisingdiversityofarchaea, including>20phyla,whichappear to68


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representupto50%ofthetotalmicrobialcommunityinsomeofthesesamples20.Preliminary69

phylogenyofthedatasetusing37concatenatedribosomalproteinsrevealedtwodraftgenomic70

bins representing a new lineage in the Asgard archaea. These draft genomes, referred to as71

Hel_GB_AandHel_GB_B,werere-assembledandre-binnedresultinginfinalbinsthatwere8272

and87%completeandhadabinsizeof3.54and3.84Mbp,respectively(Table1).Anin-depth73

phylogeneticanalysisconsistingof56concatenatedribosomalproteinswasusedtoconfirmthe74

placement of these final bins form a distant sister-groupwith the Lokiarchaeota (Figure 1a).75

Hel_GB_Apercentabundancerangedfrom3.41x10-3%to8.59x10-5%andrelativeabundancefrom8.4376

to0.212.Hel_GB_Bpercentabundancerangedfrom1.20x10-3%to7.99x10-5%andrelativeabundance77

from3.41 to0.22.ForbothHel_GB_AandHel_GB_B thehighestabundancewas seenat the site the78

genomesbinswere recovered from.Thesenumbersarecomparable tootherAsgardarchaea isolated79

formthesesites20.Hel_GB_AandHel_GB_BhadameanGCcontentof35.4%and28%,respectively,80

andwere recovered from twodistinct environmental samples,which share similarmethane-81

supersaturated and strongly reducing geochemical conditions (concentrations of methane82

rangingfrom2.3-3mM,dissolvedinorganiccarbonrangingfrom10.2-16.6mM,sulfatenear2183

mMandsulfidenear2mM;SupplementaryTable1)butdifferedintemperature(28oCand10oC,84

respectively,SupplementaryTable1)19.85

Phylogenetic analyses of a 16S rRNA gene sequence (1058bp in length) belonging to86

Hel_GB_A confirmed that they are related to Lokiarchaeota and Thorarchaeota, but are87

phylogeneticallydistinct fromeitherof these lineages (Figure1b).Acomparisontopublished88

Asgard archaeal 16S rRNA gene sequences indicate a phylum level division between the89

Hel_GB_AsequenceandotherAsgardarchaea22(SupplementaryTable2).AsearchforESPsin90


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bothbinsrevealedthattheycontainedasimilarsuitecomparedtothosepreviouslyidentifiedin91

Lokiarchaeota,whichisconsistentwiththeirdistantphylogenomicrelationship(Figure2).These92

lineages are relatively distantly related as evidenced by their difference in GC content and93

relatively low pairwise sequence identity of proteins. An analysis of the average amino acid94

identity(AAI)showedthatHel_GB_AandHel_GB_Bshared1477geneswithandAAIof51.96%.95

WhencomparedtoLokiarcheota_CR4,Hel_GB_Ashare634outoforthologousgenes3595and96

Hel_GB_Bhad624orthologousgenesoutof3157.HelarchaeotabinsshowedthehighestAAI97

similaritytoOdinarchaeotaLCB_4(45.9%);however,itcontainedfewerorthologousgenes(57498

outof3595and555outof3157forHel_GB_AandHel_GB_B,respectively).Additionally,the99

Hel_GB bins differed from Lokiarchaeota in their total gene number, for example Hel_GB_A100

possessed3595genesandCR_4possessed4218; thisdifference is consistentwith the larger101

estimatedgenomesizeforLokiarchaeumCR_4comparedtoHel_GB_A(~5.2Mbpto~4.6Mbp)102

(SupplementaryTable3,SupplementaryMethods).Theseresultsaddsupporttothephylumlevel103

distinctionobservedforHel_GB_AandHel_GB_Binboththeribosomalproteinand16srRNA104

phylogenetic trees.We propose the nameHelarchaeota after Hel, theNorse goddess of the105

underworldandLoki’sdaughterforthislineage.106


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107

Figure 1. Phylogenomic position of Helarchaeota within the Asgard archaea superphylum (A)108

Phylogenomicanalysisof56concatenatedribosomalproteinsidentifiedinHelarchaeotabins.Blackcircles109

indicateBootstrapvaluesgreaterthan95(LG+C60+F+G+PMSF);PosteriorProbability>=0.95(SR4).(B)110

Maximum-likelihoodphylogenetictreeof16SrRNAgenesequencesthoughttobelongtoHelarchaeota.111


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ThephylogenywasgeneratedusingRAxML(GTRGAMMAmodelandnumberofbootstrapsdetermined112

usingtheextendedmajority-ruleconsensustreecriterion).ThepurpleboxshowspossibleHelarchaeota113

sequences from GB data, as well as closely related published sequences and sequences form newly114

identifiedHelarchaeotabins(identifiedasMegxx_xxxx_Bin_xxx_scaffold_xxxxx).Numberofsequencesis115

depictedintheclosedbranches.116

117

MetabolicanalysisofHelarchaeota.Toreconstructthemetabolicpotentialofthesearchaea,118

theHelarchaeotaproteomeswerecomparedtoseveralfunctionalproteindatabases20(Figure119

3a).Likemanyarchaeainmarinesediments23,Helarchaeotamaybeabletoutilizeorganiccarbon120

astheypossessavarietyofextracellularpeptidasesandcarbohydratedegradationenzymesthat121

include theβ-glucosidase,α-L-arabinofuranosidaseandputative rhamnosidase,amongothers122

(Supplementary Table 4 and 5). Degraded organic substrates can then be metabolized via123

glycolysisandanincompleteTCAcyclefromcitratetomalateandapartialgamma-aminobutyric124

acidshunt(Figure3a,SupplementaryTable4).BothHelarchaeotabinsaremissingfructose-1,6-125

bisphosphataseandhavefewgenescodingforthepentosephosphatepathway.Genesencoding126

for the bifunctional enzyme 3-hexulose-6-phosphate synthase/6-phospho-3-hexuloisomerase127

(hps-phi)wereidentifiedinHel_GB_Bsuggestingtheymaybeusingtheribulosemonophosphate128

(RuMP)pathwayforformaldehydeanabolism.Genescodingforacetate-CoAligase(bothAPM129

and ADP-forming) and an alcohol dehydrogenase (adhE) were identified in both genomes130

suggestingthattheorganismsmaybecapableofbothfermentationandproductionofacetyl-131

CoA using acetate and alcohols (Supplementary Table 4). Like in Thorarchaeota and132

Lokiarchaeota, these genomes possess the large subunit of type IV Ribulose bisphosphate133

carboxylase19,24.Additionally,theHelarchaeotagenomesencodeforthecatalyticsubunitofthe134


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methanogenictypeIIIribulosebisphosphatecarboxylaseusedforC-fixation24.Helarchaeotaare135

metabolically distinct from Lokiarchaeota as both Hel_GB draft genomes appear to lack a136

completeTCAcycleasgenescodingforcitratesynthaseandmalate/lactatedehydrogenaseare137

absent. Both genomes also likely produce acetyl-CoA using glyceraldehyde 3-phosphate138

dehydrogenase which is absent in Lokiarchaeota19 (Supplementary Table 4). Helarchaeota139

genomes lack genes that code for enzymes involved in dissimilatory nitrogen and sulfur140

metabolism.Assimilatorygenesincludingsat,cysNandcysCwerefoundinHel_GB_Bhowever141

thesegeneswerenotidentifiedinHel_GB_A.Thisabsencemaybeindicativeofspecies-specific142

characteristics of their genomes or could be a results of genome incompleteness. Additional143

genomesofmembersof theHelarchaeotawillhelp to fullyunderstandthediversityof these144

pathwaysacrossthewholephylum.145

146

Helarchaeote GB_AHelarchaeote GB_B

Lokiarchaeote CR_4Lokiarchaeum GC14_75

Odinarchaeote LCB_4

Thorarchaeote SMTZ-83Thorarchaeote AB_25

Thorarchaeote SMTZ-45

Heimdallarchaeote AB_125Heimdallarchaeote LC_2Heimdallarchaeote LC_3Heimdallarchaeote CS_D

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Informationalproteins Trafficking machinery Cytoskeleton Ubiquitin system OST

complex


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Figure2.Distributionofeukaryoticsignatureproteins(ESPs)inHelarchaeotaandotherAsgardarchaea.147

NumbersundereachcolumncorrespondtotheInterProaccessionnumber(IPR)andArchaealClustersof148

OrthologousGenes(arcCOG)IDsthatweresearchedfor.Fullcirclesrefertocasesinwhichahomologue149

wasfoundintherespectivegenomes.EmptycircleswithblackoutlinesrepresenttheabsenceoftheESP.150

ThecheckeredpatternintheRNApolymerasesubunitalpharepresentsthefactthattheproteinswere151

split,whilethefusedproteinsarerepresentedbythefullcircles.Greycircleswithbordersinanyother152

colorrepresentcaseswherethestandardprofileswerenotfoundbutpotentialhomologswheredetected.153

IntheRoadblockproteins,potentialhomologsweredetectedbutthephylogenycouldnotsupportthe154

closerelationshipofanyofthesecopiestotheAsgardarchaeagroupclosesttoeukaryotes.IntheUb-155

activatingenzymeE1representshomologsfoundclusteredappropriatelywithitspotentialorthologsin156

the phylogeny but the synteny of this gene with other ubiquitin-related proteins in the genome is157

uncertain.158

159

Interestingly, both Helarchaeota genomes have mcrABG-containing gene clusters160

encodingputativemethyl-CoMreductase-likeenzymes(Figure3b,SupplementaryFigure2)4,5,7.161

Phylogenetic analyses of both the A subunit of methyl-CoM reductase-like enzymes162

(SupplementaryFigure2)aswellastheconcatenatedAandBsubunits(Figure3b)revealedthat163

theHelarchaeotasequencesaredistinctfromthoseinvolvedinmethanogenesisandmethane164

oxidation but cluster with homologs from butane oxidizing Syntrophoarchaea7 and165

Bathyarchaeota with high statistical support (rapid bootstrap support/single branch test166

bootstrapsupport/posteriorprobabilityof99.8/100/1;Figure3b)excludingthedistanthomolog167

ofCa.Syntrophoarchaeumcaldarius(OFV68676).AnalysisoftheHelarchaeotamcrAalignment168

confirmed that amino acids present at their active sites are similar to those identified on169


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Bathyarchaeota and Syntrophoarchaeummethyl-CoM reductase-like enzymes (Supplementary170

Figure3).InSyntrophoarchaeum,themethyl-CoMreductase-likeenzymeshavebeensuggested171

to activate butane to butyl-CoM7. It is proposed that this process is then followed by the172

conversion of butyl-CoM to butyryl-CoA; however, the mechanism of this reaction is still173

unknown.Butyryl-CoAcanthenbeoxidizedtoacetyl-CoAthatcanbefurtherfeedintotheWood-174

Ljungdahl pathway to produce CO27. While some n-butane is detected in Guaymas Basin175

sediments (usually below 10 micromolar), methane is the most abundant hydrocarbon176

(SupplementaryTable1)followedbyethaneandpropane(oftenreachingthe100micromolar177

range); thus, a spectrum of short-chain alkanes could potentially be metabolized by178

Helarchaeota26.179

180

Figure 3. Metabolic inference of Helarchaeota and phylogenetic analyses of concatenated McrAB181

proteins.(A)Enzymesshownindarkpurplearepresentinbothgenomes,thoseshowninlightpurpleare182

presentinasinglegenomeandonesingreyareabsent.(B)ThetreewasgeneratedusingIQ-treewith183

1000ultrafastbootstraps,singlebranchtestbootstrapsandposteriorpredictivevaluesfromtheBayesian184

phylogeny. Whitecircles indicatebootstrapvaluesof90-99.9/90-99.9/0.9-0.99andblack filledcircles185


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indicatevaluesof100/100/1.Thetreewasrootedarbitrarilybetweentheclustercomprisingcanonical186

McrABhomologsanddivergentMcrABhomologs,respectively.Scalebarsindicatetheaveragenumber187

ofsubstitutionspersite.188

189

Proposedhydrocarbondegradationpathway forHelarchaeota.Next,we searched forgenes190

encodingenzymespotentially involved inhydrocarbonutilizationpathways includingpropane191

and butane oxidation. Alongwith themethyl-CoM reductase-like enzyme that could convert192

alkane to alkyl-CoM, Helarchaeota possess heterodisulfide reductase subunits ABC (hdrABC)193

whichisneededtorecycletheCoMandCoBheterodisulfidesafterthisreactionoccurs(Figure3194

and4)7,8.Theconversionofalkyl-CoMtoacyl-CoAiscurrentlynotunderstoodinarchaeacapable195

ofbutaneoxidation.Novelalkyl-bindingversionsofmethyltransferaseswouldbe required to196

convertalkyl-CoMtobutyl-CoAorotheracyl-CoAs,asdiscussedforCa.S.butanivorans7.Genes197

codingformethyltransferaseswereidentifiedinbothHelarchaeotagenomes,includingalikely198

tetrahydromethanopterin S-methyltransferase subunit H (MtrH) homolog (Figure 4;199

SupplementaryTable4).Short-chainacyl-CoAcouldbeoxidizedtoacetyl-CoAusingthebeta-200

oxidation pathway via a short-chain acyl-CoA dehydrogenase, enoyl-CoA hydratase, 3-201

hydroxyacyl-CoAdehydrogenaseandacetyl-CoAacetyltransferase,candidateenzymesforallof202

whicharepresentintheHelarchaeotagenomesandarealsofoundingenomesofotherAsgard203

archaea(Figure4)19.Alongwiththeseenzymes,genescodingfortheassociatedelectrontransfer204

systems,includinganFe-Soxidoreductaseandallsubunitsoftheelectrontransferflavoprotein205

(ETF)complexwereidentifiedinHelarchaeota(Figure4).Acetyl-CoAproducedbybeta-oxidation206

might be further oxidized to CO2 via theWood-Ljungdahl pathway, using among others the207


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classical5,10-methylene-tetrahydromethanopterinreductase(Figure3aand4).208

209

210

Figure4.ComparisonofHelarchaeotaalkanemetabolismtootheralkaneoxidizingandmethanogenic211

archaea.AlkanemetabolismofHelarchaeotacomparedtoBathyarchaeotaandCa.Syntrophoarchaeum212

sp.,Verstraetearchaeota,GoM-Arc1sp.,ANME-1sp.andANME-2sp.Alistofgenesandcorresponding213

contigidentifierscanbefoundinSupplementaryTable4.214

215

Threepossibleenergy-transferringmechanisms forHelarchaeota.Tomakeanaerobicalkane216

oxidationenergetically favorable, itmustbe coupled to the reductionofan internalelectron217

acceptorortransferredtoasyntrophicpartnerthatcanperformthisreaction7,26,27.Wecouldnot218

identifyaninternalelectronsinkoranycanonicalterminalreductasesusedbyANMEarchaea219

(suchasiron,sulfurornitrogen),leadingtotheconclusionthatasyntrophicpartnerorganism220

would be necessary to enable growth on short-chain hydrocarbons. However, we could not221

identify any obvious syntrophic partner organisms based on co-occurrence analyses of222

abundanceprofilesofmetagenomicdatasetsgeneratedinthisstudy20.223

mcrAmcrBmcrC

hdrA

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bcd

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Hel_GB_A Hel_GB_B

Bathyarchaeota BA1Bathyarchaeota BA2

Syntrophoarchaeum butanivoransSyntrophoarchaeum caldarius

Methanomethylicus mesodigestumMethanosuratus petracarbonis

Methanomethylicus oleusabulum

Gom-Arc1 archaeon ex4484_138 Gom-Arc1 archaeon ex4572_44

Methanoperedens nitroreducensMethanoperedens sp. BLZ1

ANME-1 archaeon ex4572_4

ANME-2 archaeon HR1

Helarchaeota

Bathyarchaeota

Euryarchaeota

Verstraetearchaeota

COG4022

COG1571

COG4008

COG4073

COG4855

COG1810

COG4800

Methanogen-specific proteins


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An evaluation of traditional energy transferring mechanisms showed that our224

Helarchaeota bins lack genes coding for NADH:ubiquinone oxidoreductase, F420-dependent225

oxidoreductase, F420H2:quinone oxidoreductase andNADH:quinone oxidoreductase thatwere226

identifiedinCa.S.butanivorans(Figure4)7.Theseelectron-carryingproteinsareimportantfor227

energytransferacrossthecellmembraneandarecommonamongsyntrophicorganisms2,28,29.228

Helarchaeotaalsolackgenescodingforpiliorcytochromesthataregenerallyassociatedwith229

electron transfer to a bacterial partner, as demonstrated for different ANME archaea26,30.230

Therefore,Helarchaeotamayuseathusfarunknownapproachforenergyconservation.Below231

we analyzed potential energy-transferring mechanisms that might be involved in syntrophic232

interactionsbetweenHelarchaeotaandpotentialpartnerorganisms.233

Apossible candidate for energy transfer to apartnermaybe formatedehydrogenase234

becausesubstrateexchangeinformofformatehaspreviouslybeendescribedtooccurbetween235

methanogensandsulfur-reducingbacteria27.Helarchaeotagenomescodeforthealphaandbeta236

subunits ofamembrane-bound formatedehydrogenase (EC.1.2.1.2) thatcould facilitate this237

transfer(Figure2,SupplementaryTable4).However,toourknowledgeformatetransferhasnot238

been shown tomediatemethaneoxidation.Alternatively,Helarchaeotamaypossess a novel239

redox-active complex. In both Helarchaeota bins, a gene cluster was found encoding three240

proteins that were identified as members of the HydB/Nqo4-like superfamily, Oxidored_q6241

superfamilyandaFe-SdisulfidereductasewithaFlpDdomain(mvhD)(Figure5a).Ananalysisof242

these three proteins showed that each possessed transmembrane motifs (Figure 5b, and243

Supplementary Methods). While the membrane association of the disulfide reductase/FlpD244


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needs to be confirmed, interactionswith the other twomembrane-associated subunitsmay245

allowforthebifurcatedelectronstobetransferredacrossthemembrane.246

Finally,hydrogenproductionandreleasewasalsoconsideredaspossibleelectronsinkfor247

Helarchaeota. We identified several hydrogenases and putative Fe-S disulfide reductase-248

encodinggenesintheHelarchaeotagenomes.Subsequentphylogeneticanalysesrevealedthat249

themajorityofthesehydrogenasesrepresentsmallandlargesubunitsofgroupIIIChydrogenases250

(methanogenic F420-non-reducing hydrogenase (mvh)) that are usually involved in bifurcating251

electronsfromhydrogen(SupplementaryFigure4,SupplementaryTable4). Incontrast,while252

homologsbelongingtotheabovementionedOxidored_q6superfamilyproteinfamilyareoften253

found to be associated with group IV hydrogenases, canonical membrane-bound group IV-254

hydrogenases could not be identified in the genomes of the Helarchaeota. Altogether, this255

indicatesthathydrogencouldplayacentralroleinenergymetabolismofHelarcharota,butthe256

absence of a classicalmembrane-bound hydrogenasemakes it unlikely that hydrogen is the257

majorsyntrophicelectroncarrier.258


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259

Figure5.DepictionofageneclusterfoundinbothHelarchaeotagenomesthatconsistsofgenesthat260

encode for a possible energy-transferring complex. (A) In Hel_GB_A the complex was found on the261

reversestrandbuthasbeenorientedintheforwarddirectionforclarity(asterisk).Arrowsindicatethe262

lengthofthereadingframe.Genenameswerepredictedbyvariousdatabases(SupplementaryMethods).263

Smallnumberslocatedabovethearrowsrefertothenucleotidepositionforthefullcontig.Boldnumbers264

onHel_GB_Brefertotheaminoacidnumberofthewholecomplex. (B)Figuredepictsthemembrane265

motifs identified on NODE_147_length_7209_cov_4.62199_5, 6 and 7 using various programs266

(Supplementarymethods).Eachcirclerepresentsasingleaminoacid.Boldcirclesrepresentaminoacids267

atthestartoftheprotein,thestartandendofthetransmembranesites,andtheendofthecomplex.268

NumberingcorrespondstotheaminoacidnumbersofHel_GB_Binpanel(A).Afulllooprepresents50269

aminoacidsanddoesnotreflectthesecondarystructureofthecomplex.270

271

hydB_Nqo4-likeoxidored_q6Hel_GB_B

Hel_GB_A*

24,47329,170 26,47228,472

hydB_Nqo4-likeoxidored_q6Disulfide reductase-FlpD

hydB_Nqo4-likeDisulfide reductase-FlpD

2,595 7,2095,4043,404

235

253

864

1395

1413

1533

1 840 1088 1533

A.

B.

Hel_GB_B

cytoplasmic

non-cytoplasmic

1

917

934

840

845

1088

Disulfide reductase-FlpD

Disulfide reductase-FlpD oxidored_q6 hydB_Nqo4-like

NODE_1033_length_35804_cov_14.1912_31, 30 and 29

NODE_147_length_7209_cov_4.62199_5, 6 and 7


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Discussion272

Historically methanogenesis and anaerobic methane oxidation were regarded as the only273

examples of anaerobic archaeal short-chain alkanemetabolism. The enzymes acting in these274

pathways were considered to be biochemically and phylogenetically unique and limited to275

lineageswithintheEuryarchaeota4.Thisstudyrepresentsthediscoveryofanovelphylumand276

thefirstindicationsforanaerobicshort-chainalkaneoxidationusingaMCR-likehomologinthe277

Asgardarchaea.SincethepresenceofthesemcrgenesisrestrictedtoHelarchaeotaamongthe278

known Asgard archaea19, these genes were likely transferred to Helarchaeota and do not279

constitute an ancestral trait within the Asgard superphylum. Based on current phylogenetic280

analysis, theHelarchaeotamcr geneclustermayhavebeenhorizontallyacquired fromeither281

BathyarchaeotaorCa.Syntrophoarchaeum(Fig.1b,SupplementaryFigure3).Duetothisclose282

relationship,webasedouranalysisofHelarchaeota’sabilitytoperformanaerobicshort-chain283

hydrocarbon oxidation on the pathway proposed for Ca. Syntrophoarchaeum. Helarchaeota284

probablyutilizeasimilarshort-chainalkaneasasubstrateinlieuofmethane,butgiventhelow285

butaneconcentrationsatoursiteitmaynotbeanexclusivesubstrate.286

OurcomparisontoCa.S.butanivoransshowsaconsistentpresenceingenesnecessary287

forthismetabolismincludingacompleteWood-Ljungdahlpathway,acyloxidationpathwayand288

internal electron transferring systems. These electron-transferring systems are essential289

housekeepingcomponentsthatactaselectroncarriersforoxidationreactions.Interestingly,in290

theWood-Ljungdahl pathway identified inCa. S.butanivorans, the bacterial enzyme is5,10-291

methylene-tetrahydrofolatereductase(met)isthoughttobesubstitutingforthemissing5,10-292

methylene-tetrahydromethanopterin reductase (mer)7. In contrast, Helarchaeota encode the293


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canonicalarchaeal-typemer.To renderanaerobicbutaneoxidationenergetically favorable, it294

mustbecoupledtothereductionofanelectronacceptorsuchasnitrate,sulfateoriron7,26,27.In295

ANMEarchaea that lack genes for internal electron acceptors,methaneoxidation is enabled296

through the transfer of electrons to a syntrophic partner organism. In Syntrophoarchaeum,297

syntrophicbutaneoxidationisthoughttooccurthroughtheexchangeofelectronsviapiliand/or298

cytochromeswithsulfate-reducingbacteria7.Helarchaeotadonotappeartoencodeanyofthe299

systemstraditionallyassociatedwithsyntrophyandnopartnerwasidentifiedinthisstudy.Thus,300

furtherresearchisneededtoidentifypossiblebacterialpartners.301

Furthermore,thehypothesisforHelarchaeotagrowththroughtheanaerobicoxidationof302

short-chainalkanesremainstobeconfirmedasthegenomesofmembersofthisgroupdonot303

encodecanonicalroutesforelectrontransfertoapartnerbacterium.However,weidentifiedthe304

genetic potential for potential enzymes thatmay be involved in transfer of electrons. Some305

methanogenic archaea use formate for syntrophic energy transfer to a syntrophic partner;306

therefore, the reverse reactionhasbeen speculated tobeenergetically feasible formethane307

oxidation27. If this is true, thepresenceofamembrane-boundformatedehydrogenase inthe308

Helarchaeota genomes may support this electron-transferring mechanism, however to our309

knowledgethishasneverbeenshownforanANMEarchaeasofar.Alternatively,thetype3NiFe-310

hydrogenasesencodedbyHelarchaeotamaybe involved intransferofhydrogentoapartner311

organism. For example, we identified a protein complex distantly related to themvh-hdr of312

methanogens for electron transfer (Supplementary material).Mvh-hdr structures have been313

proposed to be potentially used by non-obligate hydrogenotrophicmethanogens for energy314

transfer, but the directionality of hydrogen exchange could easily be reversed2. These315


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methanogens form syntrophic associations with fermenting, H2-producing bacteria, lack316

dedicatedcytochromesorpiliandusethemvh-hdrforelectronbifurcation2.Thedetectionofa317

hydrophobic region in themvh-hdr complex led to thesuggestion that thiscomplexcouldbe318

membraneboundandactasmechanismforelectrontransferacrossthemembrane;however,a319

transmembrane association has never been successfully shown2. While the membrane320

associationofthedisulfidereductase/FlpDneedstobeconfirmed,wewereabletodetectseveral321

other transmembranemotifs in the associated proteins that could potentially allow electron322

transferinformofhydrogentoanexternalpartner.Thus,whileweproposethatthemostlikely323

explanation for anaerobic short-chain alkane oxidation in Helarchaeota is via a syntrophic324

interactionwithapartner,additionalexperimentsareneededtoconfirmthisworkinghypothesis.325

Thediscoveryofalkane-oxidizingpathwaysandpossiblesyntrophicinteractionsinanew326

phylumofAsgardarchaeaindicatesamuchwiderphylogeneticrangeforhydrocarbonutilization.327

Based on their phylogenetic distribution, the Helarchaeota mcr operon may have been328

horizontally transferred from either Bathyarchaeota or Syntrophoarchaeum. However, the329

preservation of a horizontally transferred pathway indicative of a competitive advantage; it330

followsthatgenetransfersamongdifferentarchaealphylareflectalkaneoxidationasadesirable331

metabolictrait.Thediscoveryofthealkyl-CoM reductasesandalkane-oxidizingpathwaysamong332

theAsgardarchaea indicatesecologicalrolesforthesestillcrypticorganisms,andopensupa333

wider perspective on the evolution and expansion of hydrocarbon-oxidizing pathways334

throughoutthearchaealdomain.335

336

337


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Methods338

Samplecollectionandprocessing.Samplesanalyzedherearepartofastudythataimstocharacterize339

the geochemical conditions and microbial community of Guaymas Basin (GB) hydrothermal vent340

sediments(GulfofCalifornia,Mexico)31,32.Thetwogenomicbinsdiscussedinthispaper,Hel_GB_Aand341

Hel_GB_B,wereobtainedfromsedimentcoresamplescollectedinDecember2009onAlvindives4569_2342

and4571_4respectively21.Immediatelyafterthedive,freshlyrecoveredsedimentcoreswereseparated343

intoshallow(0-3cm),intermediate(12-15cm)anddeep(21-24cm)sectionsforfurthermolecularand344

geochemicalanalysis,andfrozenat-80oContheshipuntilshore-basedDNAextraction.Hel_GB_Awas345

recoveredfromtheintermediatesediment(~28oC)andHel_GB_Bwasrecoveredfromshallowsediment346

(~10oC)fromanearbycore(SupplementaryTable1);thesamplingcontextandgeochemicalgradientsof347

thesehydrothermallyinfluencedsedimentsarepublishedanddescribedindetail21,31.348

DNAwasextractedfromsedimentsamplesusingtheMOBIO–PowerMaxSoilDNAIsolationkit349

andsenttotheJointGenomeInstitute(JGI)forsequencing.AlaneofIlluminareads(HiSeq–25001TB,350

readlengthof2x151bp)wasgeneratedforbothsamples.Atotalof226,647,966and241,605,888reads351

weregeneratedforsamplesfromdivesfor4569-2and4571-4,respectively.Trimmed,screened,paired-352

end Illumina reads were assembled using the megahit assembler using a range of Kmers (See353

SupplementaryMethods).354

355

Genomereconstruction.ThecontigsfromtheJGIassembleddatawerebinnedusingESOM33,MetaBAT34356

andCONCOCT35 and resultingbinswere combinedusingDASTool (version1.0)36 (See Supplementary357

Methods).CheckMlineage_wf(v1.0.5)wasrunonbinsgeneratedfromDAS_Tooland577binsshowed358

ancompleteness>50%andwerecharacterizedfurther37.37Phylosift38identifiedmarkergeneswereused359

for preliminary phylogenetic identification of individual bins (Supplementary Table 6). Thereby, we360

identifiedtwogenomes,belongingtoapreviouslyuncharacterizedphylumwithintheAsgardarchaea,361


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whichwe namedHelarchaeota. To improve the quality of these twoHelarchaeota bins (increase the362

lengthoftheDNAfragmentsandlowertotalnumber),weusedMetaspadestoreassemblethecontigsin363

each individual bin producing scaffolds. Additionally, we tried to improve the overall assemblies by364

reassemblingthetrimmed,screened,paired-endIlluminareadsprovidedbyJGIusingbothIDBA-UDand365

Metaspades (Supplementary Methods). Binning procedures (using scaffolds longer than 2000 bp) as366

previously described in SupplementaryMethods for the original bins were repeated with these new367

assembles. All binswere compared to the original Helarchaeota bins using blastn39 for identification.368

Mmgenome40andCheckM37wereusedtocalculategenomestatistics (i.e.contig length,genomesize,369

contaminationandcompleteness).ThehighestqualityHelarchaeotabinfromeachsamplewaschosen370

forfurtheranalyses.Forthe4572-4dataset,thebestbinwasgeneratedusingtheMetaspadesreassembly371

on the trimmed data and for the 4569-2 dataset the best bin was recovered using theMetaspades372

reassemblyontheoriginalHelbincontigs.ThefinalgenomeswerefurthercleanedbyGCcontent,paired-373

endconnections,sequencedepthandcoverageusingMmgenome40.CheckMwasrerunoncleanedbins374

toestimatetheHel_GB_Atobe82%andHel_GB_Btobe87%completeandbothbinswerecharacterized375

byalowdegreeofcontamination(between1.4-2.8%withnoredundancy)(Table1)37.Genomesizewas376

estimated to be 4.6 Mbp for Hel_GB_A and 4.1 for Hel_GB_B and was calculated using percent377

completenessandbinsizetoextrapolatethelikelysizeofthecompletegenome.CompareM41wasused378

toanalysisdifferencesbetweenHelarchaeotabinsandpublishedAsgardbinsusingthecommandpython379

comparemaai_wf--tmp_dirtmp/--file_extfa-c8aai_compair_lokiaai_compair_loki_output.380

381

16SrRNAgeneanalysis.Neitherbinpossesseda16SrRNAgenesequence38,andtouncoverpotentially382

unbinned 16S rRNA gene sequences fromHelarchaeota, all 16S rRNA gene sequences obtained from383

samples4569_2and4571_4wereidentifiedusingJGI-IMGannotations,regardlessofwhetherornotthe384

contigwassuccessfullybinned.These16SrRNAgenesequenceswerecomparedusingblastn39(blastn-385


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outfmt6-queryHel_possible_16s.fasta–dbNew_Hel_16s-outHel_possible_16s_blast.txt-evalue1E-20)386

tonewlyacquired16SrRNAgenesequencesfromMAGsrecoveredfrompreliminarydatafromnewGB387

sites.A37Phylosift38markergenestreewasusedtoassigntaxonomytotheseMAGs.Wewereableto388

identify fiveMAGs that possessed 16s and that formed amonophyletic groupwith our Hel_GB bins389

(SupplementaryTable2;MegxxinFigure2).Oftheunbinned16SrRNAgenesequencesonewasidentified390

as likely Helarchaeota sequence. The contig was retrieved from the 4572_4 assembly (designated391

Ga0180301_10078946)andwas2090bplongandencodedforan16SrRNAgenesequencethatwas1058392

bplong.Giventhesmallsizeofthiscontigrelativetothelengthofthe16SrRNAgenenoneoftheother393

genes on the contig could be annotated. Blastn39 comparison to published Asgard 16S rRNA gene394

sequenceswasperformedusingthefollowingcommand:blastn-outfmt6-queryHel_possible_16s.fasta395

–dbAsgrad_16s-outHel_possible_16s_blast.txt-evalue1E-20(SupplementaryTable2).TheGCcontent396

of each 16S rRNA gene sequence was calculated using the Geo-omics script length+GC.pl397

(https://github.com/Geo-omics/scripts/blob/master/AssemblyTools/length%2BGC.pl). For a further398

phylogeneticplacement,the16SrRNAgenesequenceswerealignedtotheSILVAdatabase(SINAv1.2.11)399

using the SILVA online server42 and Geneious (v10.1.3)43 was used to manually trim sequences. The400

alignmentalsocontained16SrRNAgenesequencesfromthenew,preliminaryHelarchaeotabins.The401

cleanedalignmentwasusedtogeneratedamaximum-likelihoodtreewithRAxMLasfollows:“/raxmlHPC-402

PTHREADS-AVX-T20-fa-mGTRGAMMA-NautoMRE-p12345-x12345-sNucleotide_alignment.phy-n403

output”(Figure1b).404

405

Phylogeneticanalysisofribosomalproteins.Foramoredetailedphylogeneticplacement,weused406

BLASTp44toidentifyorthologsof56ribosomalproteinsinthetwoHelarchaeotabins,aswellasfroma407

selectionof130representativetaxaofarchaealdiversityand14eukaryotes.Thefulllistofmarkergenes408

selectedforphylogenomicanalysesisshowninSupplementaryTable7.Individualproteindatasetswere409


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alignedusingmafft-linsi45andambiguouslyalignedpositionsweretrimmedusingBMGE(-mBLOSUM30)46.410

Maximum likelihood (ML) individualphylogenieswere reconstructedusing IQtree v. 1.5.547under the411

LG+C20+G substitutionmodelwith 1000 ultrafast bootstraps thatweremanually inspected. Trimmed412

alignments were concatenated into a supermatrix, and two additional datasets were generated by413

removingeukaryoticand/orDPANNhomologuestotesttheimpactoftaxonsamplingonphylogenetic414

reconstruction. For each of these concatenated datasets, phylogenies were inferred using ML and415

Bayesian approaches.ML phylogenieswere reconstructed using IQtree under the LG+C60+F+G+PMSF416

model48.Statisticalsupportforbrancheswascalculatedusing100bootstrapsreplicatedunderthesame417

model.Totestrobustnessofthephylogenies,thedatasetwassubjectedtoseveraltreatments.Forthe418

‘fulldataset’(i.e.,withall146taxa),wetestedtheimpactofremovingthe25%fastest-evolvingsites,as419

withinadeepphylogeneticanalysis,thesesitesareoftensaturatedwithmultiplesubstitutionsand,asa420

resultofmodel-misspecificationcanmanifestinanartifactualsignal50–52.ThecorrespondingMLtreewas421

inferredasdescribedabove.BayesianphylogenieswerereconstructedwithPhylobayesforthedataset422

“withoutDPANN”undertheLG+GTRmodel.FourindependentMarkovchainMonteCarlochainswere423

runfor~38,000generations.Afteraburn-inof20%,convergencewasachievedforthreeofthechains424

(maxdiff<0.29).Theinitialsupermatrixwasalsorecodedinto4categories,inordertoameliorateeffects425

ofmodelmisspecification and saturation52 and the corresponding phylogeny was reconstructed with426

Phylobayes,undertheCAT+GTRmodel.FourindependentMarkovchainMonteCarlochainswererun427

for~49,000generations.Afteraburn-inof20convergencewasachievedforallfourthechains(maxdiff428

<0.19).AllphylogeneticanalysesperformedaresummarizedinSupplementaryTable8,includingmaxdiff429

valuesandstatisticalsupportfortheplacementofHelarchaeota,andofeukaryotes.430

431

PhylogeneticanalysisofMcrAandconcatenatedMcrAandMcrBproteins.McrAhomologswerealigned432

usingmafft-linsi45,trimmedwithtrimAL53andthefinalalignmentconsistingof528siteswassubjectedto433


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phylogeneticanalysesusingv.1.5.547withtheLG+C60+R+Fmodel.Supportvalueswereestimatedusing434

1000 ultrafast boostraps54 and SH-like approximate likelihood ratio test55, respectively. Sequences for435

McrAandBwerealignedseparatelywithmafft-linsi45andtrimmedusingtrimALSubsequently,McrAand436

McrB encoded in the same gene cluster, were concatenated yielding a total alignment of 972 sites.437

BayesianandMaximum likelihoodphylogenieswere inferredusing IQtreev.1.5.5 47with themixture438

modelLG+C60+R+FandPhyloBayesv.3.256usingtheCAT-GTRmodel.ForMaximumlikelihoodinference,439

supportvalueswereestimatedusing1000ultrafastboostraps54andSH-likeapproximatelikelihoodratio440

test55,respectively.ForBayesiananalyses,fourchainswereruninparallel,samplingevery50pointsuntil441

convergencewasreached(maximumdifference<0.07;meandifference<0.002).Thefirst25%orthe442

respectivegenerationswereselectedasburn-in.Phylobayesposteriorpredictivevaluesweremapped443

onto the IQtreeusing sumlabels from theDendroPypackage57. The final treeswere rootedartificially444

betweenthecanonicalMcranddivergentMcr-likeproteins,respectively.445

446

Metabolic Analyses. Gene prediction for the two Helarchaeota bins was performed using prodigal58447

(V2.6.2)withdefaultsettingsandProkka59(v1.12)withtheextension‘–kingdomarchaea’.Resultsforboth448

methodswerecomparableandyieldedatotalof3,574-3,769and3,164-3,287genesforHel_GB_Aand449

Hel_GB_B, respectively, with Prokka consistently identifying fewer genes. Genes were annotated by450

uploading theprotein fasta files frombothmethods toKAAS (KEGGAutomaticAnnotationServer) for451

completeordraftgenomestoassignorthologs60.Fileswererunusingthefollowingsettings:prokaryotic452

option,GhostXandbi-directionalbesthit (BBH)60.Additionally,geneswereannotatedby JGI-IMG61 to453

confirmhitsusingtwoindependentdatabases.HitsofinterestwereconfirmedusingblastpontheNCBI454

webserver44.ThedbCAN62andMEROPS63webserverwererunusingdefaultconditionsforidentification455

ofcarbohydratedegradingenzymesandpeptidasesrespectively.Hitswithe-valueslowerthane^-20were456


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discarded. Inadditionto thesemethodsanextendedsearchwasusedtocategorizegenes involved in457

butanemetabolism,syntrophyandenergytransfer.458

Identifiedgenespredictedtocodeforputativealkaneoxidationproteinsweresimilartothose459

describedfromCandidatusSyntrophoarchaeumspp..Therefore,ablastp44databaseconsistingofproteins460

predictedtobeinvolvedinthealkaneoxidationpathwayofCa.Syntrophoarchaeumwascreatedinorder461

toidentifyadditionalproteinsinHelarchaeota,whichmayfunctioninalkaneoxidation.Positivehitswere462

confirmed with blastp44 on the NCBI webserver and compared to the annotations from JGI-IMG61,463

Interpro64,PROKKA59andKAAS60annotation.GenesformcrABGwerefurtherconfirmedbyaHMMER65464

search to a published database using the designated threshold values66 andmultipleMCR trees (see465

Methods).ToconfirmthatthecontigswiththemcrAgeneclusterwerenotmissbined,allothergeneson466

thesecontigswereanalyzed for theirphylogeneticplacementandgenecontent.Theprodigalprotein467

predictionsforgenesonthecontigswithmcrAoperonswereusedtodeterminedirectionalityandlength468

ofthepotentialoperon.469

To identifygenesthatare involved inelectronandhydrogentransferacrossthemembrane,a470

databasewascreatedofknowngenesrelevantinsyntrophythatweredownloadfromNCBI.Theprotein471

sequencesofthetwoHelarchaeotagenomeswereblastedagainstthedatabasetodetectrelevanthits472

(E-value ≥ e ^-10). All hits were confirmed using the NCBI webserver, Interpro, JGI-IMG and KEGG.473

HydrogenaseswereidentifiedbyaHMMERsearchtopublisheddatabaseusingthedesignatedthreshold474

values67. Hits were confirmed with comparisons against JGI annotations and NCBI blasts, the HydDB475

database68andamanualdatabasemadefrompublishedsequences69,70.Alldetectedhydrogenaseswere476

usedtogeneratetwophylogenetictrees,oneforproteinsidentifiedassmallsubunitsandoneforlarge477

subunitsinordertoproperlyidentifythedifferenthydrogenasesubgroups.Hydrogenasesthatarepart478

oftheproposedcomplexwerethenfurtheranalyzedtoevaluateifthiswasapossibleoperonbylooking479

forpossibletranscriptionfactorsandbindingmotifs(SupplementaryMethods).480


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481

ESPIdentification.GenepredictionforthetwoHelarchaeotabinswasperformedusingprodigal58(V2.6.2)482

withdefault settings.All thehypotheticalproteins inferred inbothHelarchaeaotawereusedasseeds483

againstInterPro64,arCOG71andnrusingBLAST44.TheannotationtablefromZaremba-Niedzwiedzka,etal.484

2017.wasusedasabasis for thecomparison12. The IPRs (or in somecases, thearCOGs) listed in the485

Zaremba-Niedzwiedzka,etal.2017weresearchedforintheHelarchaeotagenomes12andtheresulting486

informationwasusedtocompletethepresence/absencetable.Whensomethingthathadpreviouslybeen487

detected inanAsgardbinwasnot found inaHelarchaeotabinusingthe InterPro/arCOGannotations,488

BLASTswere carriedoutusing the closestAsgard seeds to verify theabsence. In somecases, specific489

analyseswereused to verify thehomologyor relevanceofparticular sequences. Thedetails for each490

individualESParedepictedinsupplementarymaterials.491

492

DataAvailability.TherawreadsfromthemetagenomesdescribedinthisstudyareavailableatJGIunder493

theIMGgenomeID3300014911and3300013103forsamples4569-2and4571-4,respectively.Genome494

sequencesareavailableatNCBIundertheaccessionnumbersSAMN09406154andSAMN09406174for495

Hel_GB_AandHel_GB_Brespectively.BothareassociatedwithBioProjectPRJNA362212.496

497

498

499

500

501

502

503

504


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Tables505

Table1.BinstatisticsforHelarchaeotaBins.Degreeofcompleteness,contaminationandheterogeneity506

wasdeterminedusingCheckM37.507

508

509

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5.Evans,P.N.etal.MethanemetabolisminthearchaealphylumBathyarchaeotarevealedby519

genome-centricmetagenomics.Science350,434–438(2015).520

Table1

SeqID Hel_GB_A Hel_GB_BCompleteness(%) 82.4 86.92Contamination(%) 2.8 1.40Strainheterogeneity(%) 0 0Scaffoldnumber 333 182GCcontent(%) 35.40 28.00N50(bp) 15,161 28,908Lengthtotal(Mbp) 3.84 3.54EstimatedGenomesize(Mbp) 4.6 4.1Longestcontig(bp) 52,512 72,379Meancontig(bp) 11,531 19,467


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Acknowledgements677

ThisstudywassupportedinpartbyanAlfredP.SloanFoundationfellowship(FG-2016-6301)and678NationalScienceFoundationDirectorateofBiologicalSciences(award1737298)toBJB.Sampling679inGuaymasBasinandpost-cruiseworkwassupportedbyNSFAwardsOCE-0647633andOCE-6801357238 to APT, respectively. The work conducted by the U.S. Department of Energy Joint681GenomeInstitute,aDOEOfficeofScienceUserFacility,issupportedbytheOfficeofScienceof682theU.S.DepartmentofEnergyunderContractNo.DE-AC02-05CH11231providedtoND.683684Authorcontributions685KWS,TJGE,NDandBJBconceivedthestudy.KWS,ND,andBJBanalyzedthegenomicdata.APT686collectedandprocessedsamples.KWS,AS,andLEperformedphylogeneticanalyses.JLanalyzed687ESPs. KWS, AS, JRS, APT, BJB handled the metabolic inferences. BJB and KWS wrote the688manuscriptwithinputsfromallauthors.689


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