1
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
23
24
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 The copyright holder for this preprint (which was not peer-reviewed) is the author/funder..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
2
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
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
3
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
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
4
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
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
5
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
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
6
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
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
7
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
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
8
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
DNA
poly
mer
aseε
Topo
isom
eras
e IB
RNA
poly
mer
ase
subu
nit α
*RN
A po
lym
eras
e su
buni
t RPB
8Ri
boso
mal
pro
tein
L22
e
Cons
erve
d lo
kiac
tins
Prof
ilin
ARP
2/3
com
plex
sub
unit
4G
elso
lin
Actin
-rela
ted
prot
ein
Ribo
som
al p
rote
in L
28e
ESCR
T-I:
Vps2
8ES
CRT-
I: st
eadi
ness
box
dom
ain
ESCR
T-II:
Vps
22/3
6-lik
e (E
AP30
)ES
CRT-
II: V
ps25
ESCR
T-III
: Vps
2/24
/46
ESCR
T-III
: Vps
20/3
2/60
Long
in-d
omai
n pr
otei
nRo
adbl
ock/
RLC7
TRAP
P do
mai
n
RWD
dom
ain
E2-fi
ndin
g do
mai
n
Ub-a
ctiv
atin
g en
zym
e E1
-cat
alyt
icPu
tativ
e E2
-like
pro
tein
Ub-a
ctiv
atin
g en
zym
e E1
Tubu
linUb
iqui
tin-d
omai
n pr
otei
n
UFM
1-do
mai
n pr
otei
n
OST
3/O
ST6
Ribo
phor
in I
STT3
Puta
tive
deub
iqui
tinat
ing
enzy
me
Puta
tive
E3-li
ke p
rote
in
IPR029703
arCOG08649
arCOG04256-7
arCOG04271
IPR002671
IPR029004
IPR007143
IPR017916
IPR007286
IPR014041/008570
IPR005024
IPR005024
IPR011012
IPR004942
IPR007194
IPR004000/020902
IPR004000
IPR008384
IPR005455
IPR007122
IPR002453
IPR029071/000626
IPR006575
IPR000594
IPR019572
IPR014929
IPR000608
IPR013083
IPR018611
IPR000555
IPR007676
IPR021149
IPR003674
Sec2
3/Se
c24
IPR007194
Informationalproteins Trafficking machinery Cytoskeleton Ubiquitin system OST
complex
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
9
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
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
10
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
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
11
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
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
12
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
hdrB
hd
rC
Alkane activation
bcd
echA
hbdato
Bfix
Bfix
Afix
Xfix
CFe-S
OFe-S
ctr
Acyl-CoA oxidation
ACDSmermchmtd ftr fw
d
Wood-Ljungdahl
mta mtx mt-11mtr Misc
mt
Alkyryl-CoAConversion
FDHfrh
Bco
fEFqo
FNADH-Q
O
NADH-UQO
Energy transfer
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
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
13
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
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
14
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
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
15
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
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
16
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
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
17
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
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
18
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
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
19
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
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
20
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
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
21
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
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
22
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
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
23
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
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
24
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
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
25
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
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
26
Tables505
Table1.BinstatisticsforHelarchaeotaBins.Degreeofcompleteness,contaminationandheterogeneity506
wasdeterminedusingCheckM37.507
508
509
5101.GEClaypool&Kvenvolden,andK.A.MethaneandotherHydrocarbonGasesinMarine511
Sediment.Annu.Rev.EarthPlanet.Sci.11,299–327(1983).512
2.Thauer,R.K.,Kaster,A.-K.,Seedorf,H.,Buckel,W.&Hedderich,R.Methanogenicarchaea:513
ecologicallyrelevantdifferencesinenergyconservation.Nat.Rev.Microbiol.6,579–591514
(2008).515
3.Reeburgh,W.S.OceanicMethaneBiogeochemistry.Chem.Rev.107,486–513(2007).516
4.Spang,A.,Caceres,E.F.&Ettema,T.J.G.Genomicexplorationofthediversity,ecology,and517
evolutionofthearchaealdomainoflife.Science357,eaaf3883(2017).518
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
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
27
6.Vanwonterghem,I.etal.Methylotrophicmethanogenesisdiscoveredinthearchaealphylum521
Verstraetearchaeota.Nat.Microbiol.1,16170(2016).522
7.Laso-Pérez,R.etal.Thermophilicarchaeaactivatebutaneviaalkyl-coenzymeMformation.523
Nature539,396–401(2016).524
8.Dombrowski,N.,Seitz,K.W.,Teske,A.P.&Baker,B.J.Genomicinsightsintopotential525
interdependenciesinmicrobialhydrocarbonandnutrientcyclinginhydrothermalsediments.526
Microbiome5,106(2017).527
9.Bazylinski,D.A.,Farrington,J.W.&Jannasch,H.W.Hydrocarbonsinsurfacesedimentsfrom528
aGuaymasBasinhydrothermalventsite.Org.Geochem.12,547–558(1988).529
10. Teske,A.,Callaghan,A.V.&LaRowe,D.E.Biospherefrontiersofsubsurfacelifeinthe530
sedimentedhydrothermalsystemofGuaymasBasin.Front.Microbiol.5,(2014).531
11. VonDamm,K.L.,Edmond,J.M.,Measures,C.I.&Grant,B.Chemistryofsubmarine532
hydrothermalsolutionsatGuaymasBasin,GulfofCalifornia.Geochim.Cosmochim.Acta49,533
2221–2237(1985).534
12. Zaremba-Niedzwiedzka,K.etal.Asgardarchaeailluminatetheoriginofeukaryotic535
cellularcomplexity.Nature541,353–358(2017).536
13. Spang,A.etal.Complexarchaeathatbridgethegapbetweenprokaryotesand537
eukaryotes.Nature521,173–179(2015).538
14. Seitz,K.W.,Lazar,C.S.,Hinrichs,K.-U.,Teske,A.P.&Baker,B.J.Genomic539
reconstructionofanovel,deeplybranchedsedimentarchaealphylumwithpathwaysfor540
acetogenesisandsulfurreduction.ISMEJ10,1696–1705(2016).541
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
28
15. Jørgensen,S.L.,Thorseth,I.H.,Pedersen,R.B.,Baumberger,T.&Schleper,C.542
QuantitativeandphylogeneticstudyoftheDeepSeaArchaealGroupinsedimentsofthe543
Arcticmid-oceanspreadingridge.Front.Microbiol.4,(2013).544
16. Jorgensen,S.L.etal.Correlatingmicrobialcommunityprofileswithgeochemicaldatain545
highlystratifiedsedimentsfromtheArcticMid-OceanRidge.Proc.Natl.Acad.Sci.U.S.A.546
109,E2846-2855(2012).547
17. Hartman,H.&Fedorov,A.Theoriginoftheeukaryoticcell:agenomicinvestigation.548
Proc.Natl.Acad.Sci.99,1420–1425(2002).549
18. Eme,L.,Spang,A.,Lombard,J.,Stairs,C.&J.G.Ettema,T.Archaeaandtheoriginof550
eukaryotes.15,(2017).551
19. Spang,A.etal.Arenewedsyntrophyhypothesisfortheoriginoftheeukaryoticcell552
basedoncomparativeanalysisofAsgardarchaealmetabolism.Nat.Microbiol.Submitted,553
20. Dombrowski,N.,Teske,A.P.&Baker,B.J.Extensivemetabolicversatilityand554
redundancyinmicrobiallydiverse,dynamicGuaymasBasinhydrothermalsediments.Nat.555
Commun.9:4999,(2018).556
21. McKay,L.etal.Thermalandgeochemicalinfluencesonmicrobialbiogeographyinthe557
hydrothermalsedimentsofGuaymasBasin,GulfofCalifornia.Environ.Microbiol.Rep.8,558
150–161(2016).559
22. Yarza,P.etal.Unitingtheclassificationofculturedandunculturedbacteriaandarchaea560
using16SrRNAgenesequences.Nat.Rev.Microbiol.12,635–645(2014).561
23. Lazar,C.S.etal.Environmentalcontrolsonintragroupdiversityoftheuncultured562
benthicarchaeaofthemiscellaneousCrenarchaeotalgrouplineagenaturallyenrichedin563
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
29
anoxicsedimentsoftheWhiteOakRiverestuary(NorthCarolina,USA).Environ.Microbiol.564
17,2228–2238(2015).565
24. Tabita,F.R.,Satagopan,S.,Hanson,T.E.,Kreel,N.E.&Scott,S.S.DistinctformI,II,III,566
andIVRubiscoproteinsfromthethreekingdomsoflifeprovidecluesaboutRubisco567
evolutionandstructure/functionrelationships.J.Exp.Bot.59,1515–1524(2007).568
25. Dowell,F.etal.MicrobialCommunitiesinMethane-andShortChainAlkane-Rich569
HydrothermalSedimentsofGuaymasBasin.Front.Microbiol.7,(2016).570
26. Krukenberg,V.etal.CandidatusDesulfofervidusauxilii,ahydrogenotrophicsulfate-571
reducingbacteriuminvolvedinthethermophilicanaerobicoxidationofmethane.Environ.572
Microbiol.18,3073–3091(2016).573
27. Stams,A.J.M.&Plugge,C.M.Electrontransferinsyntrophiccommunitiesofanaerobic574
bacteriaandarchaea.Nat.Rev.Microbiol.7,568–577(2009).575
28. Meuer,J.,Kuettner,H.C.,Zhang,J.K.,Hedderich,R.&Metcalf,W.W.Geneticanalysis576
ofthearchaeonMethanosarcinabarkeriFusarorevealsacentralroleforEchhydrogenase577
andferredoxininmethanogenesisandcarbonfixation.Proc.Natl.Acad.Sci.99,5632–5637578
(2002).579
29. Kunow,J.,Linder,D.,Stetter,K.O.&Thauer,R.K.F420H2:quinoneoxidoreductase580
fromArchaeoglobusfulgidus.Eur.J.Biochem.223,503–511(1994).581
30. Wegener,G.,Krukenberg,V.,Riedel,D.,Tegetmeyer,H.E.&Boetius,A.Intercellular582
wiringenableselectrontransferbetweenmethanotrophicarchaeaandbacteria.Nature526,583
587–590(2015).584
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
30
31. McKay,L.J.etal.Spatialheterogeneityandunderlyinggeochemistryofphylogenetically585
diverseorangeandwhiteBeggiatoamatsinGuaymasBasinhydrothermalsediments.Deep586
SeaRes.PartOceanogr.Res.Pap.67,21–31(2012).587
32. Meyer,S.etal.Microbialhabitatconnectivityacrossspatialscalesandhydrothermal588
temperaturegradientsatGuaymasBasin.Front.Microbiol.4,(2013).589
33. Dick,G.J.etal.Community-wideanalysisofmicrobialgenomesequencesignatures.590
GenomeBiol.10,R85(2009).591
34. Kang,D.D.,Froula,J.,Egan,R.&Wang,Z.MetaBAT,anefficienttoolforaccurately592
reconstructingsinglegenomesfromcomplexmicrobialcommunities.PeerJ3,e1165(2015).593
35. Alneberg,J.etal.Binningmetagenomiccontigsbycoverageandcomposition.Nat.594
Methods11,nmeth.3103(2014).595
36. Sieber,C.M.K.etal.Recoveryofgenomesfrommetagenomesviaadereplication,596
aggregation,andscoringstrategy.bioRxiv107789(2017).doi:10.1101/107789597
37. Parks,D.H.,Imelfort,M.,Skennerton,C.T.,Hugenholtz,P.&Tyson,G.W.CheckM:598
assessingthequalityofmicrobialgenomesrecoveredfromisolates,singlecells,and599
metagenomes.GenomeRes.gr.186072.114(2015).doi:10.1101/gr.186072.114600
38. Darling,A.E.etal.PhyloSift:phylogeneticanalysisofgenomesandmetagenomes.PeerJ601
2,e243(2014).602
39. Altschul,S.F.,Gish,W.,Miller,W.,Myers,E.W.&Lipman,D.J.Basiclocalalignment603
searchtool.J.Mol.Biol.215,403–410(1990).604
40. Karst,S.M.,Kirkegaard,R.H.&Albertsen,M.mmgenome:atoolboxforreproducible605
genomeextractionfrommetagenomes.bioRxiv059121(2016).doi:10.1101/059121606
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
31
41. https://github.com/dparks1134/CompareM.607
42. Pruesse,E.,Peplies,J.&Glöckner,F.O.SINA:Accuratehigh-throughputmultiple608
sequencealignmentofribosomalRNAgenes.Bioinformatics28,1823–1829(2012).609
43. Kearse,M.etal.GeneiousBasic:anintegratedandextendabledesktopsoftware610
platformfortheorganizationandanalysisofsequencedata.Bioinforma.Oxf.Engl.28,1647–611
1649(2012).612
44. Altschul,S.F.etal.GappedBLASTandPSI-BLAST:anewgenerationofproteindatabase613
searchprograms.NucleicAcidsRes.25,3389–3402(1997).614
45. Katoh,K.&Standley,D.M.MAFFTmultiplesequencealignmentsoftwareversion7:615
Improvementsinperformanceandusability.Mol.Biol.Evol.30,772–780(2013).616
46. Criscuolo,A.&Gribaldo,S.BMGE(BlockMappingandGatheringwithEntropy):anew617
softwareforselectionofphylogeneticinformativeregionsfrommultiplesequence618
alignments.BMCEvol.Biol.10,210(2010).619
47. Nguyen,L.-T.,Schmidt,H.A.,vonHaeseler,A.&Minh,B.Q.Iq-tree:Afastandeffective620
stochasticalgorithmforestimatingmaximum-likelihoodphylogenies.Mol.Biol.Evol.32,621
268–274(2015).622
48. Wang,H.-C.,Minh,B.Q.,Susko,E.&Roger,A.J.ModelingSiteHeterogeneitywith623
PosteriorMeanSiteFrequencyProfilesAcceleratesAccuratePhylogenomicEstimation.Syst.624
Biol.syx068(2017).625
49. Jeffroy,O.,Brinkmann,H.,Delsuc,F.&Philippe,H.Phylogenomics:thebeginningof626
incongruence?TrendsGenet.22,225–231(2006).627
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
32
50. Lartillot,N.&Philippe,H.Improvementofmolecularphylogeneticinferenceandthe628
phylogenyofBilateria.Philos.Trans.R.Soc.Lond.B.Biol.Sci.363,1463–72(2008).629
51. Brown,M.W.M.etal.Phylogenomicsdemonstratesthatbreviateflagellatesarerelated630
toopisthokontsandapusomonads.Proc.R.Soc.BBiol.Sci.280,20131755(2013).631
52. Susko,E.&Roger,A.J.Onreducedaminoacidalphabetsforphylogeneticinference.632
Mol.Biol.Evol.24,2139–2150(2007).633
53. Capella-Gutiérrez,S.,Silla-Martínez,J.M.&Gabaldón,T.trimAl:atoolforautomated634
alignmenttrimminginlarge-scalephylogeneticanalyses.Bioinforma.Oxf.Engl.25,1972–635
1973(2009).636
54. Minh,B.Q.,Nguyen,M.A.T.&vonHaeseler,A.Ultrafastapproximationfor637
phylogeneticbootstrap.Mol.Biol.Evol.30,1188–1195(2013).638
55. Guindon,S.etal.Newalgorithmsandmethodstoestimatemaximum-likelihood639
phylogenies:assessingtheperformanceofPhyML3.0.Syst.Biol.59,307–321(2010).640
56. Lartillot,N.&Philippe,H.ABayesianmixturemodelforacross-siteheterogeneitiesin641
theamino-acidreplacementprocess.Mol.Biol.Evol.21,1095–1109(2004).642
57. Sukumaran,J.&Holder,M.T.DendroPy:aPythonlibraryforphylogeneticcomputing.643
Bioinforma.Oxf.Engl.26,1569–1571(2010).644
58. Hyatt,D.etal.Prodigal:prokaryoticgenerecognitionandtranslationinitiationsite645
identification.BMCBioinformatics11,119(2010).646
59. Seemann,T.Prokka:rapidprokaryoticgenomeannotation.Bioinforma.Oxf.Engl.30,647
2068–2069(2014).648
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
33
60. Moriya,Y.,Itoh,M.,Okuda,S.,Yoshizawa,A.C.&Kanehisa,M.KAAS:anautomatic649
genomeannotationandpathwayreconstructionserver.NucleicAcidsRes.35,W182–W185650
(2007).651
61. Markowitz,V.M.etal.IMG:theintegratedmicrobialgenomesdatabaseand652
comparativeanalysissystem.NucleicAcidsRes.40,D115–D122(2012).653
62. Yin,Y.etal.dbCAN:awebresourceforautomatedcarbohydrate-activeenzyme654
annotation.NucleicAcidsRes.40,W445–W451(2012).655
63. Rawlings,N.D.,Barrett,A.J.&Bateman,A.MEROPS:thepeptidasedatabase.Nucleic656
AcidsRes.38,D227–D233(2010).657
64. Jones,P.etal.InterProScan5:genome-scaleproteinfunctionclassification.Bioinforma.658
Oxf.Engl.30,1236–1240(2014).659
65. Johnson,L.S.,Eddy,S.R.&Portugaly,E.HiddenMarkovmodelspeedheuristicand660
iterativeHMMsearchprocedure.BMCBioinformatics11,431(2010).661
66. Anantharaman,K.etal.Thousandsofmicrobialgenomesshedlightoninterconnected662
biogeochemicalprocessesinanaquifersystem.Nat.Commun.7,13219(2016).663
67. Anantharaman,K.etal.Thousandsofmicrobialgenomesshedlightoninterconnected664
biogeochemicalprocessesinanaquifersystem.7,(2016).665
68. Søndergaard,D.,Pedersen,C.N.S.&Greening,C.HydDB:Awebtoolforhydrogenase666
classificationandanalysis.Sci.Rep.6,34212(2016).667
69. Vignais,P.M.&Billoud,B.Occurrence,classification,andbiologicalfunctionof668
hydrogenases:anoverview.Chem.Rev.107,4206–4272(2007).669
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
34
70. Vignais,P.M.,Billoud,B.&Meyer,J.Classificationandphylogenyofhydrogenases1.670
FEMSMicrobiol.Rev.25,455–501671
71. Makarova,K.S.,Wolf,Y.I.&Koonin,E.V.ArchaealClustersofOrthologousGenes672
(arCOGs):AnUpdateandApplicationforAnalysisofSharedFeaturesbetween673
Thermococcales,Methanococcales,andMethanobacteriales.Life5,818–840(2015).674
675
676
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
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
Top Related