Geoarchaeota: a new candidate phylum in the Archaea from high-temperature acidic iron mats in ...

13 Pages·2013·2.2 MB·English

by  Mark A Kozubal

TheISMEJournal(2013)7,622–634
&2013InternationalSocietyforMicrobialEcology Allrightsreserved 1751-7362/13
www.nature.com/ismej
ORIGINAL ARTICLE
Geoarchaeota: a new candidate phylum in the
Archaea from high-temperature acidic iron mats in
Yellowstone National Park
Mark AKozubal1,Margaret Romine2,Ryan deMJennings1,ZackJJay1,Susannah GTringe3,
Doug B Rusch4, Jacob P Beam1, Lee Ann McCue2 and William P Inskeep1
1Department of Land Resources and Environmental Sciences and Thermal Biology Institute, Montana State
University,Bozeman,MT,USA;2EnvironmentalMicrobiologyGroup,PacificNorthwestNationalLaboratory,
Richland, WA, USA; 3DOE Joint Genome Institute, Walnut Creek, CA, USA and 4Department of Biology,
Indiana University, Bloomington, IN, USA
Geothermal systems in Yellowstone National Park (YNP) provide an outstanding opportunity to
understand the origin and evolution of metabolic processes necessary for life in extreme
environments including low pH, high temperature, low oxygen and elevated concentrations of
reduced iron. Previous phylogenetic studies of acidic ferric iron mats from YNP have revealed
considerablediversityofuncultivatedandundescribedarchaea.Thegoalofthisstudywastoobtain
replicate de novo genome assemblies for a dominant archaeal population inhabiting acidic iron-
oxide mats in YNP. Detailed analysis of conserved ribosomal and informational processing genes
indicates that the replicate assemblies represent a new candidate phylum within the domain
Archaeareferredtohereas‘Geoarchaeota’or‘novelarchaealgroup1(NAG1)’.TheNAG1organisms
containpathwaysnecessaryforthecatabolismofpeptidesandcomplexcarbohydratesaswellasa
bacterial-likeFormIcarbonmonoxidedehydrogenasecomplexlikelyusedforenergyconservation.
Moreover, this novel population contains genes involved in the metabolism of oxygen including a
Type A heme copper oxidase, a bd-type terminal oxidase and a putative oxygen-sensing
protoglobin.NAG1hasavarietyofuniquebacterial-likecofactorbiosynthesisandtransportgenes
and a Type3-like CRISPR system. Discovery of NAG1 is critical to our understanding of microbial
community structure and function in extant thermophilic iron-oxide mats of YNP, and will provide
insight regarding the evolution of Archaea in early Earth environments that may have important
analogs activeinYNP today.
The ISME Journal(2013)7, 622–634;doi:10.1038/ismej.2012.132;published online15November2012
Subject Category: microbial ecology and functional diversity of natural habitats
Keywords: extremophiles; geothermal; Yellowstone National Park; heme copper oxidase; carbon
monoxide; iron-oxides
Introduction methanogensandhalophilestogetherinthephylum
Euryarchaeota, and a second major phylum (Cre-
Although members of the domain Archaea were
narchaeota) that included the first cultured thermo-
actually discovered in the late 18th century (Wolfe,
philes in the order Sulfolobales (Brock et al., 1972).
2006) and the first isolates were obtained in the
Sincethen,considerableprogresshasresultedinthe
early 20th century (Harrison and Kennedy, 1922;
isolation and subsequent genome sequencing of
Barker, 1936), these organisms were not recognized
numerous thermophiles and hyperthermophiles
as a separate lineage from the Bacteria and Eukarya
belonging to the Crenarchaeota (Wolfe, 2006).
until the groundbreaking work focused on the
Characterization of 16S rRNA gene sequences
phylogenetics of the 16S rRNA gene (Woese et al.,
from uncultivated organisms across a myriad of
1976; Woese and Fox, 1977). The newly recognized
natural environments as well as isolation of key
domain Archaea (Woese et al., 1990) included the
referenceorganismshasexpandedourknowledgeof
the phylogenetic diversity and evolutionary rela-
tionships among members of the domain Archaea.
Correspondence:WPInskeep,DepartmentofLandResourcesand In fact, phylogenetic placement of deeply rooted
Environmental Sciences, Thermal Biology Institute, Montana sequences from Obsidian Pool in Yellowstone
StateUniversity,POBox173120,Bozeman,MT59717,USA.
National Park (YNP) led to the proposal of a third
E-mail:[email protected]
candidate phylum in the mid-1990s, the Korarch-
Received 16 March 2012; revised 10 October 2012; accepted 10
October2012;publishedonline15November2012 aeota(Barnsetal.,1994).EnrichmentofCandidatus
Geoarchaeota:anewcandidatephylum
MAKozubaletal
623
Korarchaeum cryptofilum and subsequent full-gen- lineage (Geoarchaeota) within the domain Archaea.
ome analysis provided further evidence that the AnalysisofmetagenomesequencefromreplicateFe-
Korarchaeota represent a separate phylum within oxide mat samples (60–781C) reveals a dominant
the Archaea (Elkins et al., 2008). A fourth phylum archaeal population with novelstrategies forenergy
(Nanoarchaeota) was described after the discovery conservation and oxygen respiration. Evidence is
and genome analysis of the symbiont species provided here that these organisms are members of
Candidatus Nanoarchaeum equitans (Huber et al., one the earliest currently known lineages of the
2002),althoughplacementoftheNanoarchaeotaasa domain Archaea,andthat membersofthis phylum-
new phylum is still debated and is based primarily level group are limited primarily to thermophilic
on a single genome sequence and 16S rRNA habitats, especially those containing ferrous Fe and
sequences (Brochier et al., 2005). More recently, low levels of oxygen.
the candidate phylum Thaumarchaeota was pro-
posed (Brochier-Armanet et al., 2008), and detailed
analysisofgenomesequencefrommultiplerepresen- Materials and methods
tatives of this lineage provides convincing evidence
that this group of organisms is significantly different Sample collection and metagenome sequencing
from other recognized phyla within the Archaea DNA for metagenome sequencing was extracted
(Spang et al., 2010). Specifically, phylogenetic analy- from B10g of iron-oxide microbial mat from 100
sis of housekeeping genes (that is, ribosomal proteins Spring Plain (Norris Geyser Basin; YNP; (Easting
and enzymes involved in translation, replication, 523042/Northing 4953337) sampled at two different
cell division and repair) from Nitrosopumilus time points using a FastDNA Spin Kit (MO BIO
maritimus, Nitrososphaera gargensis and Candidatus Laboratories, Carlsbad, CA, USA). Environmental
Cenarchaeum symbiosum provides strong evidence DNAwasprecipitatedwith3.0Msodiumacetateand
fortheuniqueanddistinguishinggeneticfeaturesof 100% ethanol. Sanger sequencing of 3-kb random
members of this phylum. The first isolate from this insert pUC18 libraries was used to obtain random
groupwastheammonia-oxidizing,marineorganism shotgun metagenome sequence (average read length
N. maritimus (Ko¨nneke et al., 2005), and this B800bp) of the first sample obtained in Fall 2007
discovery led to numerous enrichment cultures (OSPB-1). Further replicates sampled 2.4 year after
and molecular studies demonstrating the ubiquity OSPB-1(OSPB-2,OSPCandOSPD)weresequenced
of these organisms in various marine settings using 454 titanium pyrosequencing (average read
(Hatzenpichler et al., 2008; Walker et al., 2010; length B380bp; Table 1).
Mulleretal.,2010;Blaineyetal.,2011).Isolatesand
enrichment cultures from this phylum now include
diverse members from marine, soil and hydrother- MetagenomesequenceanalysisandassemblyofNAG1
mal systems (de la Torre et al., 2008; Tourna et al., Random shotgun sequence reads were assembled
2011), and include organisms with novel metabo- using Celera (JCVI) and Newbler (Roche/454, Basel,
lisms and/or morphologies, such as the ‘giant’ Switzerland)assemblers.Assemblystatistics(coverage,
Thaumarchaeota(Mulleretal.,2010).Thediscovery qualityscoresandGCcontent)werecompiledbythe
of Thaumarchaeota has resulted in major contribu- JointGenomeInstitute(JGI;WalnutCreek,CA,USA).
tionstoourunderstandingofglobalCandNcycling All aspects of library construction and sequencing
and the potential importance of CO fixation and performed at the JGI can be found at http://www.jgi.
2
nitrification in the open ocean (Ingalls et al., 2006). doe.gov/includingassemblymethods,baseerrorand
High-temperature environments in YNP have possiblemis-assemblycorrections.Contigswerefurther
yielded a wealth of knowledge regarding the extant analyzed using nucleotide word frequency (Teeling
diversity, distribution and metabolism of archaea etal.,2004)principalcomponentanalysis(NWF-PCA)
(Barns et al., 1996; Inskeep et al., 2005; Meyer- to separate novel archaeal group 1 (NAG1) contigs
Dombardetal.,2005;Inskeep etal.,2010).Previous from other species (performed at http://gos.jcvi.org/
studiesofacidicferriciron mats,inparticular,have openAccess/scatterPlotViewer2.html with the follow-
shown a remarkable diversity of archaea, which ingparameters:wordsize¼four,minimumcontigsize
corresponds with the combination of unique geo- at 2000 bases, and the chop sequence size at 4000
chemistry and high temperature of these systems basesasdescribedpreviouslyforYNPferricironmat
(Inskeep et al., 2005; Kozubal et al., 2008; Inskeep systems;Inskeepetal.,2010).Furtherverificationof
et al., 2010; Kozubal et al., 2012). Prior 16S rRNA NAG1 contigs was obtained by plotting coverage vs
gene sequencing of high-temperature (65–801C) GþC content for all metagenome contigs, which
acidic Fe-oxide mats has revealed the presence of clearly verified the NAG1 sequences as separate
novel archaea, but cultivation of representative(s) from other populations. A group of contigs exhi-
from this group has yet to be successful. Here, we biting similar sequence character in NWF-PCA
report for the first time a thorough analysis and space were further screened using GþC%, contig
description of four replicated de novo assemblies of coverageandblastpidentitytoimprovethedenovo
a deeply rooted microorganism, which we propose assemblies for each replicate sample. For example,
as a representative of a candidate phylum-level the largest NAG1 scaffold (1.36Mb) was obtained
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Table1 SitegeochemistryandgenomestatisticsoffourreplicatesequenceassembliesoftheNAG1population(s)observedinacidic
high-temperatureferricironmatsofNorrisGeyserBasin(YNP)
OSPB-1 OSPB-2 OSPC OSPD
Sitegeochemistry
Sampledate September2007 January2010 January2010 January2010
Samplingdistancefromsource(m) 1.2 1.2 2 4
Temperature(1C) 76 78 73 60
O2(mM) 22 25 41 59
H2S(mM) 3 3 0.2 0.2
TotalFe(mM) 37 55 55 48
DIC(mM) 0.1 0.09 0.09 0.1
DOC(mM) 51 52 49 ND
Assemblystatistics
Assemblysize(Mb) 1.81 1.31 1.52 1.27
Sequencingmethod Sanger 454Titanium 454Titanium 454Titanium
Averagecontigcoverage 6 50 43 18
Numberofcontigs/scaffolds 30a 68 104 52
%GþCcontent 32.2 32.4 32.3 32.1
%Proteinsw/100%a.a.identitytoOSPB-1 NA 93.2 91.5 89.4
Numberofcodinggenes 1932 1442 1708 1407
Uniquecodinggenes(comparedwithOSPB-1) NA 25 32 35
Abbreviations:a.a.,aminoacid;DIC,dissolvedinorganiccarbon;DOC,dissolvedorganiccarbon;NA,notapplicable;ND,notdetermined.
aNonredundantsequenceof1.7Mbwasassembledfromthelargesteightscaffolds.
from replicate OSPB-1 and contained the 16S and analyzed from function prediction by alignment
23S ribosomal gene sequences as well as the with sequences with known biochemical functions.
majorityofribosomalproteinsfortheNAG1popula- The replicate OSPB-1 de novo assembly is located
tion. Nearly identical clustering occurred for the ontheJointGenomeInstituteIMG/Mwebsiteunder
replicate assemblies (OSPB-2, OSPC and OSPD) the IMG submission ID number 2423.
resulting in 68, 104 and 52 scaffolds, respectively.
Assembled sequence was compared with reference
databases (blastp) and top sequence hits were Phylogenetic analysis
assigned to their nearest phylum; however, as All phylogenies were constructed with the MEGA
indicated by the phylogenetic placement of this 4.0softwarepackage(Tamuraetal.,2007).Atotalof
candidatephylum,theNAG1assembliesdemonstrate 20 concatenated protein phylogenetic trees were
a lack of identity to currently described reference constructed around 32 full-length ribosomal pro-
genomes (470% of the NAG1 sequence is o50% teins(Figure 3)aswellassingle-copy proteinsRNA
identical to known references). NAG1 assemblies polymerase, DNA polymerase and transcription
were compared with four reference genomes from factors (Supplementary Figure S1). Two different
other related archaeal phyla including Ferroplasma alignmentmethods(ClustalWandMuscle)andthree
acidarmanus fer1, Thermofilum pendens HRK5, substitution models (Dayhoff and Jones–Taylor–
N. maritimus SCM1 and Candidatus Korarchaeum Thornton, no. of differences and p-distance) were
cryptofilum OPF8 using NWF-PCA analysis. used for both the neighbor-joining and minimum
evolution distance methods (Nei and Kumar, 2000).
In addition, three substitution models (Poisson,
Annotation of NAG1 de novo assemblies Dayhoff and Jones–Taylor–Thornton) were utilized
Gene models, predicted functions and pathway/ to construct three maximum likelihood trees for
subsystem assignments derived from IMG/ER and each of the two alignments (Jones et al., 1992; Nei
the rapid annotation using subsystems technology andKumar,2000).Finally,theClustalWandMuscle
were compared and used to annotate the NAG1 alignments were utilized to obtain corresponding
sequence clusters screened using NWF-PCA (Aziz maximum parsimony trees. Reliability of inferred
et al., 2008). Cofactor utilization and central meta- trees was conducted with the bootstrap test of
bolic subsystems were manually curated using the phylogeny using 1000 bootstraps. Alignments were
SEED subsystem editor (Overbeek et al., 2005) to obtained with default gap penalty parameters and
facilitate mapping genes to known subsystem var- were edited by manual inspection and deletion of
iants and the Artemis genome browser (Rutherford largegaps.Aconsensustreewasconstructedforthe
et al., 2000) to identify gene fragments that corre- concatenated protein tree of 32 ribosomal proteins
sponded to missed genes belonging to these sub- (Figure3a).Maximumlikelihoodtrees(obtainedfrom
systems. Individual protein sequences were further Muscle alignments and the Jones–Taylor–Thornton
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substitution model) are shown for single protein Results
trees and are similar to topologies of consensus
Metagenome assemblies
trees.
De novo ‘genome’ assemblies of a ‘novel archaeal
A total of 16 nucleotide 16S/23S phylogenetic
Group 1’ population were generated from random
trees(Figure3b)wereconstructedwithtwodifferent
shotgunsequenceofcommunityDNAobtainedfrom
alignment methods (ClustalW and Muscle), three
four separate sampling points (60–751C) within the
substitution models (no. of differences, p-distance
outflow channel of an acidic (pH 3.5) Fe-oxide
and maximum composite likelihood) for both
geothermal spring in YNP (Figure 1a; Table 1). Two
neighbor-joining and minimum evolution distance
replicateswereobtainedfromthesamelocationand
methods. Maximum likelihood (Jukes–Cantor substi-
tution model) and maximum parsimony trees were
constructedforbothClustalWandMusclealignments
(other details are as described above). Reliability of
inferred treeswasconductedwiththebootstraptest
ofphylogenyusing1000bootstraps.Alignmentswere
obtained with default gap penalty parameters and
were edited by manual inspection and deletion of
largegapsinthealignments.Resultsfromalltreeing
methods were utilized to construct a consensus
16S/23S phylogenetic tree.
Fluorescence in situ hybridization
Approximately 10g of dispersed OSP_B mat was
incubatedat751Cin160mlserumbottlesfor3days
with50mlofOSPsourcespringwater.An0.5-galiquot
of culture slurry was washed with Nycodenz buffer
(Bertin et al., 2011) then fixed with 4% paraformal-
dehyde at 41C for 16h (Fuchs et al., 2007). Fixed
cells were further separated from the solid ferric
iron with the nonionic density gradient medium
Nycodenz(60%w/v;Sigma-AldrichD2158;St.Louis,
MO, USA) and pelleted at 10000g for 30min (Bertin
et al., 2011). The cells were resuspended in 500ml
of 1:1 phosphate-buffered saline:ethanol. 50ml of
this suspension was fixed onto a glass microscope
slide and fluorescence in situ hybridization (FISH)
performed with Cy5-labeled probe (50-GAG TTC
TTA CCT ATC CGG G-30; Integrated DNA Techno-
logies, Coralville, IA, USA) specific for the NAG1
16S ribosomal RNA sequence. The Cy5 probe was
designed around 16S rRNA class I and II sites and
had no homology to 16S rRNA gene sequences of
otherorganismsfromOSPSpringsorotherYNPiron
mats(Behrensetal.,2003).Theprobesequencewas
further analyzed using IDT ScitTools OligoAnalyzer
3.0 (Integrated DNA Technologies) to determine
possiblehairpins,dimersandthemeltingtemperature.
Formamide stringency optimization was conducted at
concentrations of 0%, 5%, 20% and 35% according
to Fuchs et al. (2007), and highest specificity was Figure 1 (a) One Hundred Springs Plain Spring (OSP) located
determined to be 5% based on comparison of withinNorrisGeyserBasin(YNP).Arrowsindicatespringsource
fluorescence signal with appropriate Cy5 excitation and sampling points with corresponding temperature and
dissolved oxygen values obtained along primary flow path. (b)
laser. In addition, fresh OSPB mat was stained with
NWF-PCAoffourreplicatedenovoassembliesforNAG1(circled,
SYBR gold to show cell distribution in a natural four shades of brown and red) and genomes from T. pendens,
sample. Cy5 fluorescence staining was not success- Candidatus Korarchaeum cryptofilum, N. maritimus and F.
ful on natural mat samples because of high mineral acidarmanus. Axes x, y, z correspond to PC1, PC2 and PC3. (c)
Average GþC content (%) of NAG1 scaffolds from replicate
content. Both Nycodenz preparations and SYBR
OSPB-1(redcircles)plottedasafunctionofdecreasingscaffold
gold-labeledmatwereviewedonaLeicaSP5CSLM
length (solid black line, cumulative total sequence length; solid
(Leica Microsystems GmbH; Wetzlar, Germany) red line, average GþC content; dotted line, indicates first eight
inverted scanning confocal laser microscope. scaffoldswhereconsensussequenceis490%).
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temperature (76–781C), but sampled over 2 years reveal or contribute any additional sequence con-
apart (OSPB-1 and OSPB-2). Additional replicates tent. Furthermore, each replicate assembly contains
were sampled downstream at temperatures of 731C onecompleteandidenticalcopyof5S,16Sand23S
(OSP-C) and 601C (OSP-D) at the same time as rRNA genes. Definitive identification of NAG1 cells
OSPB-2 (Figure 1a). Contigs assigned to each using FISH probes specific for these 16S rRNA
replicate de novo assembly have similar GþC sequences show that NAG1 cells are B1mm dia-
content(%)andsequencereadcoveragethatclearly meter cocci and are easily located in Nycodenz cell
distinguish them from other population clusters in preparations (Figure 2a). The NAG1 organisms,
this community (example shown in Supplementary along with Metallosphaera yellowstonensis, are
Figure S1). Comparison of NAG1 sequence to among the dominant cocci in the Fe-mat samples.
reference databases (via blastp or blastn) revealed a Based on metagenome replicates, 480% of the
consistent pattern (that is, poor sequence similarity archaeal sequence in OSP_B belongs to NAG1
to current reference genomes), and is clearly (B50–55%), M. yellowstonensis (12–21%) and
different than the other three to four predominant Acidolobus spp. (10–15%) as determined by three
populationspresentinthesesystems(Kozubaletal., separate phylogenetic methods. All three of these
2012). The assemblies were evaluated using NWF- organisms are B1mm diameter cocci (Boyd et al.,
PCA (Teeling et al., 2004), which showed that the 2007; Kozubal et al., 2008; this study). Most of the
sequence content and character (that is, GþC remaining sequences belong to Vulcanisaeta- or
content, codon usage) are nearly identical among Hydrogenobaculum-like spp. (rods and filaments,
the four NAG1 replicates, and that these assemblies
differ significantly compared with four representa-
tive phyla within the domain Archaea, including
those from the Crenarchaeota, Euryarchaeota and
Thaumarchaeota (Figure 1b).
The largest assembly of the NAG1 population
(OSPB-1) was obtained using Sanger sequencing
(3kbshort-insertlibrary,pUC18vector)andresulted
in 1.7Mb of nonredundant sequence on eight
scaffolds (Figure 1c, Table 1). Blastp matches of
1932 protein-coding open reading frames identified
in the OSPB-1 assembly reveal a distribution of top
hits scattered across nearby phyla within the
domain Archaea, as well as a significant number
of protein-coding genes that have best matches to
references within the domain Bacteria (15%). How-
ever, the majority (490%) of these matches are
o60% amino acid (aa) identity to reference gen-
omes. Whole-genome nucleotide alignments of the
replicate NAG1 assemblies (OSPB-2, OSPC and
OSPD) obtained from 454 titanium pyrosequencing
show nearly identical sequence content (499%
nucleotide sequence identity) and synteny with
one another (Table 1).
The assembled NAG1 sequence from OSPB-1
(1.7Mb) likely corresponds to a near-complete
consensus genome, based on the finding that this
assembly contains the expected number of archaeal
RNA polymerase subunits, transcription factors,
ribosomalproteinsandallgenesnecessaryfortRNA
synthesis (Supplementary Table S1). Addition of
NAG1-like contigs or scaffolds less than B5kb in
size (that is, scaffolds 9–30) did not result in
significant increases in nonredundant sequence or
functional information; consequently, our analysis
is focused on the largest 8 scaffolds from OSPB-1
(Figure 1c) as well as a conservative group of
NAG1 scaffolds from replicate assemblies (those Figure 2 Confocal microscopy of cell preparations and undis-
contigs that could be clearly defined using NWF- turbedFe-oxidemicrobialmat.(a)NAG1-specific16SrRNAFISH
of Nycodenz cell preparations (to remove mineral phases) from
PCA, GþC content and blastp analysis; Table 1).
OSP_B iron mat samples. Cells were identified as B1mm cocci
ReplicateNAG1assembliesobtainedusingtitanium
(green). (b). Undisturbed Fe-oxide mat from site OSPB stained
pyrosequencing (OSPB-2, OSPC and OSPD) did not withSYBRgold(scalebar¼20mm).
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respectively). SYBR gold staining shows the dis- among archaeal species show consistently that the
tribution of these cell morphologies in an undis- four replicate NAG1 assemblies cluster together
turbed OSP_B ferric iron mat. Given the successful as a new lineage between the current phylum
identification of NAG1 organisms using a FISH Crenarchaeota and candidate phylum Korarchaeota
probeoncellseparationsfromthesamesample,itis (Figures 3a and b, Supplementary Figure S2).
likely that a fraction of the cocci identified using a Both the concatenated ribosomal protein and 16S/
global (SYBR gold) stain of intact iron mat are 23S trees are built as consensus trees using 18 and
indeed NAG1 organisms. FISH probes proved to be 16 phylogenetic analyses, respectively, and both
highly problematic on iron mat samples without approaches yield consistent phylogenetic place-
sample dispersion and Nycodenz preparation to ment of the NAG1 lineage. Several universally
remove mineral material. conserved housekeeping genes have been utilized
previously for phylum-level patterning in the
Archaea(Spangetal.,2010),andthissameanalysis
Phylogenetic analysis of NAG1: representative of a of de novo NAG1 assemblies supports the conclu-
candidate phylum in the Archaea sion that NAG1 organisms are representatives of a
Consensus concatenated trees of 32 ribosomal phylum-level lineage (Supplementary Figure S2;
proteins and 16S/23S nucleotide sequences shared SupplementaryTableS2).Specifically,topoisomerase
Figure3 (a)Consensusconcatenatedphylogeneticproteintreeof32ribosomalproteinssharedbythethreedomainsoflife(Giardia
intestinaliswasusedastheoutgroup).Treeisaconsensusof18differentphylogeneticmethods.Consensusbootstrapvaluesfornodes
4900/1000arenotedbysolidcirclesandvalues4700/1000arenotedbyhollowcircles.(b)Consensusconcatenated16S/23SrRNA
nucleotidetree(Hydrogenobaculumsp.Y04AAS1wasusedastheoutgroup).Treeisaconsensusof16differentphylogeneticmethods.
Consensusbootstrapvaluesfornodes4800/1000arenotedbysolidcirclesandvalues4600/1000arenotedbyhollowcircles.
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and translation proteins show a distinct pattern neutral pH thermophilic systems including Obsi-
differenceinNAG1populationscomparedwithother dian Pool (YNP). These 16S rRNA sequences are
Archaea. Topoisomerase IA, IB and IIA were not only81–85%similar(nt)toNAG1sequenceswithin
found in NAG1 consensus assemblies, but the clade A (Supplementary Figure S4).
genome(s) do contain a reverse gyrase most similar
to Thermococcus onnurineu, a member of the
Euryarchaeota. NAG1 organisms contain cdvABC Functional analysis of NAG1 genome sequence
genes for cell division (Bernander and Ettema, 2010),
which are phylogenetically distinct and fall between Energy conservation and central C metabolism.
the Crenarchaeota and the Thaumarchaeota. Further- The de novo NAG1 assemblies provide sequence
more,unliketheCrenarchaeota,theNAG1organisms data necessary for understanding metabolic attri-
contain sequences for small and large subunits of butes of these organisms in acidic high-temperature
DNA polymerase D; the small subunit represents the Fe-mats of YNP (Figure 4). Genes coding for
mostdeeplyrooteddeducedPolDsequencecurrently chemolithotrophicpathwaysknowntobeassociated
available in public databases, branching between with extreme environments (Inskeep et al., 2010;
recognized members of Archaea and Eukarya Kozubal et al., 2011), including the oxidation of
(Supplementary Figure S1). Phylogenetic analysis of ferrous iron, hydrogen, arsenic, sulfur, ammonium
other RNA polymerases places the NAG1 popula- ormethane,werenotfoundintheNAG1assemblies.
tion(s) between the Crenarchaeota and either the However, the NAG1 populations have two separate
Thaumarchaeota, Korarchaeota or Euryarchaeota, loci that encode aerobic carbon monoxide (CO)
depending on the specific subunit used dehydrogenases and associated maturases (Dobbek
(Supplementary Figure S2). Moreover, maximum et al., 2002; King and Weber, 2007). One locus
parsimony trees suggest that the NAG1 lineage is encodes a ‘Form I’ CO dehydrogenase along with
moredeeplyrootedthanothermethodsandbranches coxFSM and the other contains three coxL ‘Form II’
between the Thaumarchaeota and Euryarchaeota CO dehydrogenase sequences along with coxDEFG
(Supplementary Figure S3). (SupplementaryFiguresS5,S6).TheNAG1‘FormI’
Numerous full-length 16S rRNA gene sequences CO dehydrogenase contains the active binding site
from acidic Fe-oxide mats in YNP have been (amino-acid sequence¼IAYRCSFR) implicated in
characterized during the last decade, and many of aerobic CO oxidation and has similar gene arrange-
these belong to the proposed candidate phylum ment and sequence similarity to the known CO
Geoarchaeota. For example, 60% of B250 clones utilizer Thermomicrobium roseum (Wu et al., 2009;
from site OSPB are highly similar to (499.5%) and 68%, 73% and 47% amino-acid identity for CoxS,
clade with the 16S rRNA gene sequences obtained CoxM and CoxL subunits, respectively). Although
fromthedenovoassemblies(SupplementaryFigure the ‘Form II’ CO oxidases may be involved in CO
S4). This agrees favorably with analysis of random oxidation, they have been shown to have a broader
metagenome sequence where B55% of the total substrate range (King and Weber, 2007).
reads belong to the NAG1 assembly (true for both The NAG1 assemblies contain a number of
OSPB-1 Sanger and OSPB-2 pyrosequencing runs). protein-coding genes important in the external
Moreover, 16S rRNA sequences from previous acquisition and subsequent degradation of amino
studies of Fe mats in YNP and other extreme acids and peptides. For example, genes involved in
environmentsshowatleasttwomajorcladeswithin branched-chain amino acid, dipeptide and oligo-
thisnovellineage(SupplementaryFigureS4).Clade peptide uptake ABC transport systems, a CstA
A is represented by over 600 clones from low-pH peptide transporter, aminotransferases, peptidases
ferricironmatsfromYNP(withtheexceptionofone and proteases all support the hypothesis that NAG1
sequence from GreatBoilingSprings,NV,USA)and populationsacquireanddegradeproteinasacarbon
includes the 16S rRNA sequences of the four andenergysource.Theassembliescontaingenesfor
replicate de novo assemblies from OSP Spring, a variety of related transporters that include
which are all 100% identical. Moreover, the major- branchedchainaminoacids(livFGKHM),dipeptides
ity of environmental clones in clade A are 499.5% (dppABCDF), oligopeptides (appABCDF) and pep-
similar to the 16S rRNA sequences obtained using tides(cstA;Figure4).Inaddition,NAG1assemblies
independent metagenome sequencing. The absence contain a glycoside hydrolase (EC 3.2.1.23) for
of sequence entries from other locations indicates hydrolysis of glycosidic bonds as well as other
that the collective attributes of these Fe(III)-oxide genes necessary forglycogen and starch metabolism
mats are unique, or that similar habitats have not (Figure 4). It is unclear whether the polysaccharide
been characterized elsewhere on the planet. The degradation potential observed in the genome
combination of high temperature, ferrous iron, sequence of NAG1 assemblies relates to storage
hypoxic to oxic conditions and moderately acidic and utilization of glycogen and/or hydrolysis of
conditions appears to define the niche of these complex carbohydrates found in the environment.
deeply rooted organisms. The second major group The absence of a Type 1 4-hydroxybutyryl-CoA
within this candidate phylum (clade B) is repre- dehydratase and bifunctional acetyl/propionyl-CoA
sented by B25 sequences from a limited number of carboxylase suggests that NAG1 populations do not
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Figure4 SummaryofNAG1metabolicattributesbasedonreplicatedenovoassemblies.(bd,bd-typeoxygenreductase;CO,aerobicCO
dehydrogenase CoxSML; ED, Entner–Doudoroff; EM, Embden–Meyerhof; HCO, heme copper oxygen reductase complex IV of the
respiratory chain (NAG1 has subunits I, II and III of a Type A HCO); I, NADH dehydrogenase complexI of the respiratory chain; II,
succinatedehydrogenaseComplexII).
fixCO throughthe3-hydroxypropionate/4-hydroxy- member of the glucose-phosphorylating enzymes
2
butyrate pathway observed in the Crenarchaeota initially characterized in Sulfolobus tokodaii
and Thaumarchaeota (Berg et al., 2007). No other (Nishimasu et al., 2006). NAG1 contains all genes
obvious modes of CO or CO fixation were noted required for both the phosphorylating and non-
2
in replicate NAG1 assemblies, based on genes phosphorylating branches of the archaeal Entner–
coding for key marker enzymes of currently recog- Doudoroffpathway.Inaddition,NAG1hasallgenes
nized autotrophic pathways (Berg et al., 2010). necessaryfortheoxidativepentose phosphatepath-
Although NAG1 has a gene coding for a Type II way, which includes an F420-dependent (FGD),
4-hydroxybutyryl-CoA dehydratase, it is not known glucose-6-phosphate (G6P) dehydrogenase (that
whether this protein is involved in CO fixation. mayalsobelinkedwithoxidativestress—seebelow)
2
Consequently, the organism may either obtain and a bacterial-like 6-phospho-gluconate dehydro-
carbon from autotrophic organisms in the spring genase. Furthermore, NAG1 has the archaeal-
(for example, M. yellowstonensis, Hydrogenobacu- specific ribulose monophosphate pathway and
lum spp.) or from exogenous sources. The gene componentsofthenon-oxidativepentosephosphate
content of NAG1 suggests metabolism of simple pathway including a ribose-5-phosphate isomerase
carbon compounds such as peptides and fatty acids and a transketolase.
and/or degradation of more complex organic sub-
strates. Other NAG1 genes involved in central C
metabolism were annotated manually including Respiratory complexes, oxygen dependence and
those required for the Embden–Meyerhof pathway oxidative stress
and gluconeogenesis. Aunique feature ofthe NAG1 NAG1 sequence contains both a Type A heme
Embden–Meyerhof pathway is the presence of a copper oxidase (HCO) and a bd ubiquinol oxidase
glucokinase gene that is similar to an atypical complex.TheNAG1HCO(subunitI)isdistinctfrom
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bacterial Type A HCOs and forms a separate branch than tyrosine and has thiO rather than thiH, which
near the base of a subunit I protein tree would make NAG1 the only known archaeon with
(Supplementary Figure S7) between members of this ‘bacterial-like’ pathway. NAG1 also has a
theThaumarchaeotaandallotherarchaeawithType ‘bacterial-like’ SUF biosynthesis system as the sole
A HCOs. In addition, the terminal oxidase complex pathwayforFeSsynthesis,includingsufAandsufS,
contains three subunits and exhibits operon struc- which have been found exclusively in bacteria.
ture and gene content considerably similar to that NAG1containsallgenesnecessaryforthebiosynth-
found in mitochondrial terminal oxidases (along esis of coenzyme F420 and has 10 putative FGD
with thaumarchaeal sequences). The operon coding enzymes in addition to the (G6P) dehydrogenase
for the Rieske and cytochrome b components of mentioned above including four luciferase mono-
electron transport chain complex III was found in oxygenase-like proteins, four FGD N(5),N(10)-
NAG1 assemblies, but neither cytochrome C1 nor methylenetetrahydro-methanopterin reductases, a
CbsA are located on this operon, and are found F420 hydrogenase/dehydrogenase beta subunit and
elsewhere in the genome. This structure is more an nicotinamide adenine dinucleotide phosphate
typical of a euryarchaeotal-like complex III. Genes oxidase-dependent F420 reductase.
coding for proteins involved in anaerobic respira- NAG1 does not contain any biotin synthesis or
tion were not found in NAG1 assemblies (for salvage genes, and contains no genes coding for
example, respiration on ferric Fe, arsenate, nitrate, enzymes that require this cofactor. Biotin is con-
sulfate and/or sulfur). Although NAG1 contains an sidered an essential vitamin throughout the
alcohol dehydrogenase and certain components of domains Bacteria and Eukarya as it is utilized as a
the subsystem resulting in acetyl-CoA fermentation carboxyl carrier in pathways that mediate fatty acid
to butyrate, it does not appear to contain all the synthesis, branched-chain amino-acid catabolism
proteins necessary for mixed-acid fermentation. and gluconeogenesis. Moreover, the Sulfolobales,
NAG1 contains an FGD G6P dehydrogenase, which someDesulfurococcalesandpossiblyonememberof
is most similar to a functionally characterized the Thermoproteales (T. pendens) utilize biotin-
enzyme in Mycobacterium smegmatis (Hasan et al., dependent carboxylases for the 3-hydroxypropio-
2010)andisanotherexampleofspecificNAG1gene nate/4-hydroxybutyrate CO fixation pathway, but
2
content that is more ‘bacterial-like’. G6P is believed these enzymes are not required for central carbon
tobeasourceofreducingpowerinM.smegmatisfor metabolism and lipid biosynthesis (Berg et al.,
reduction of reactive oxygen species with FGD 2010). Interestingly, genes encoding biotin-dependent
having an active role in ensuring high amounts of enzymes are not found in the genomes of other
G6P. The NAG1 FGD homolog is part of a two gene archaea including the Korarchaeota, Thermo-
operon with a superoxide dismutase (sodA), which plasmatales and Acidilobus spp. Consequently,
further suggests a role for the FGD in oxidative including the candidate phylum Geoarchaeota,
stress. representatives of four archaeal phyla do not
The NAG1 assemblies also contain a globin contain biotin-dependent enzymes.
protein (169 aa) with a clear protoglobin loop and Heme is predicted to be required by NAG1
heme domain (pfam 11563). Moreover, the NAG1 organisms (for example, subunit I HCOs), but
sequence is the most deeply rooted entry of all identifiable heme uptake genes were not identified.
known globins/protoglobins (Supplementary Figure However, NAG1 ‘genomes’ have the early
S8).Theproteinsequenceisrelatedtocharacterized hemALBCD heme synthesis genes found in most
protoglobins in Aeropyrum pernix, which suggests archaea (missing in the Thermococcales, Nanoarch-
possible involvement in binding O , CO and NO aeota, Korarchaeota and some Crenarchaeota;
2
(Freitas et al., 2004). Other genes encoding proteins Storbeck et al., 2010). Interestingly, NAG1 also
for heavy metal detoxification and reduction of contains hemEH (only found in the bacteria), but
oxidative stress are found in the NAG1 assembly is missing hemFG necessary for a complete bacter-
and include mercuric reductase (merA), arsenic ial-likehemesynthesispathway.Furthermore,genes
resistance (arsABC), a superoxide dismutase (sodA) for a proposed alternative archaeal heme synthesis
and multiple peroxiredoxins. However, the ars pathway(SUMT,PC2-DH,nirD,nirHandnirJ12)are
genes were found distributed throughout the gen- not found in the NAG1 assemblies suggesting heme
ome(s) and are not part of a single operon. biosynthesis via an unknown alternative pathway
(Storbeck et al., 2010).
Cofactor biosynthesis
The NAG1 population has necessary pathways for Evidence for viral defense
synthesis of thioredoxin, pyridoxine, riboflavin, TheNAG1populationfromOSPB-1containsaunique
flavine adenine dinucleotide, cobalamin, siroheme, CRISPR(ClusteredRegularlyInterspacedShortPalin-
lipoic acid and salvage of cobalamin and niacin dromic Repeats) region with several putative CRISPR
(Figure 4), several of which differ from those protein-coding genes and a CRISPR array containing
commonly found in archaea. Specifically, NAG1 19 direct repeats (DR¼50-CTTAAACTCAGAAG
appears to synthesize thiozole from glycine rather AGGATTGAAAG)-30 with associated spacer regions
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(1217 total nucleotides). The putative Csm3-like Phylogenetic analysis (Figure 3; Supplementary
(RAMP)proteinsanda896aminoacidhypothetical Figure S2) and phylum-level patterning of specific
Cas10polymerasearefoundupstreamoftheCRISPR markergenes(SupplementaryTableS2;Spangetal.,
array with similar gene arrangement to T. pendens, 2010) provide strong evidence that the NAG1
MetallosphaerasedulaandS.tokodaii(Supplementary population is a representative of a new phylum,
FigureS9).However,unliketheseorganisms,homo- withphylogeneticplacementbetweentheEuryarch-
logstoCas1,Cas2andCas4proteinswerenotfound aeota,CrenarchaeotaandThaumarchaeotaaswellas
in NAG1 assemblies. Additional hypothetical the Eukarya. However, given that the de novo
CRISPR-associated genes were found on a separate assemblies of NAG1 may not constitute a complete
locus of NAG1 including Cas6, a Cas3 helix DNA- genome, missing genes may be located in gaps
binding region, a herA helicase and a nuclease. between contigs, although this is unlikely in cases
Although the herA helicase and nuclease are not where genes are missing from conserved operons.
similar to any known cas sequences, they may have Additional examples of genes that are not shared
similar function to Cas3 proteins. The CRISPR- with the Crenarchaeota include the prevalence of
associated proteins in NAG1 suggest a Type III- coenzyme FGD enzymes. The assembly proteins
polymerasesystemwithoutCas1andCas2(Makarova (cofHG) for coenzyme F-420 are not found in any
et al., 2011). crenarchaeal genomes, but are prevalent in metha-
Definitive CRISPR/Cas regions were not found in nogens and thaumarchaea.
the replicate assemblies from OSPB-2, OSPC and The discovery of the NAG1 lineage contributes to
OSPD, sampled 2.4 year after OSPB-1. Although our understanding of the evolution of archaea and
four homologous sequences to the OSPB-1 direct their metabolic interactions with oxygen. Specifi-
repeats were found in the replicate assemblies cally, the NAG1 contains the most deeply rooted
(OSPB-2, OSPC and OSPD) obtained using pyro- protoglobin known to date (Supplementary Figure
sequencing, they were not identified as complete S8), and appears to provide an evolutionary linkage
CRISPRs,andthismaybeduetopoorerassemblyof to higher globins such as hemoglobin that are
shorter read lengths from pyro-sequencing. Further thought to have evolved from an ancestral proto-
studies may clarify the temporal nature and identi- globin (Moens et al., 1996; Freitas et al., 2005;
fication of CRISPR direct repeats, variable spacer Nardini et al., 2008). Protoglobins from the obli-
regions and/or other CRISPR/Cas proteins. The gately aerobic hyperthermophile A. pernix and the
presence of a unique CRISPR/Cas region within strictlyanaerobicmethanogenMethanosarcinaacet-
the largest NAG1 assembly (OSPB-1) is strong ivorans have been found to bind molecular oxygen,
evidence that this population is a host to viruses nitric oxide and CO, potentially protecting the
not yet characterized. Moreover, variable spacer organisms from nitrosative and oxidative stress
regions found within the NAG1 assemblies did not (Freitas et al., 2004). Consequently, protoglobins
match any gene content from publically available have been found in both aerobic and anaerobic
viral sequences or gene content found within organisms. Possible functions of the NAG1 proto-
metagenome assemblies. globin include CO transfer to the CoxSML complex,
asensorforCOintheenvironment,orO transferto
2
a terminal oxidase in low O environments. The
2
Discussion NAG1 organisms also contain two terminal oxidase
complexes including an advanced Type AHCO and
Here, we describe for the first time de novo a bd-ubiquinol oxidase, which suggests that these
assemblies of a novel archaea, which we propose organisms can potentially metabolize O under
2
belongs in a candidate phylum Geoarchaeota (from different conditions (that is, oxic/hypoxic).
‘Geo’, Gr., meaning ‘Earth’). The large majority of The divergence of the Euryarchaeota from the
currently described 16S rRNA gene sequences from Crenarchaeota is thought to have occurred B3.2–
thislineagewereobtainedfromacidicferricFemats 3.6Ga, whereas the split between the Thermopro-
in YNP geothermal springs. These moderately teales and Sulfolobales/Desulfurococcales was
acidic, high-temperature, high-ferrous Fe and hypothesized to occur B2.3–2.7Ga (Blank, 2009).
hypoxic to oxic conditions appear to define the The divergence of the NAG1 lineage would have
niche of NAG1 organisms. The NAG1 population is occurred between these two periods, likely before
one of four to five predominant community mem- ‘the Great Oxidation Event’ B2.45Ga (Bekker et al.,
bers and represents B20–55% of the total sequence 2004) and perhaps before the evolution of oxygenic
readsinOSPSpring,dependingontheexactsample photosynthesis at B2.7Ga (Barley et al., 2005).
location and temperature (60–781C). Other commu- Phylogenetic analysis of the NAG1 HCO shows the
nity members present in the acidic Fe mats of OSP deducedproteintobeamongthemostdeeplyrooted
spring include organisms most closely related to M. TypeAHCOsintheArchaea(SupplementaryFigure
yellowstonensis Acidilobus spp., Vulcanisaeta spp. S7),andmayhavebeenimportantinthedivergence
and Hydrogenobaculum spp. (Aquificales), as well ofotherarchaealTypeAHCOsinhightemperature,
asotherlessabundant,uncharacterizedarchaeaand slightly acidic ferrous iron systems, before
bacteria (Kozubal et al., 2012). the proposed rise in oxygen (410(cid:2)5 present
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