Genomic reconstruction of multiple lineages of uncultured benthic archaea suggests distinct

12 Pages·2017·1.99 MB·English

by  C S Lazar

TheISMEJournal(2017)11,1118–1129
©2017InternationalSocietyforMicrobialEcology Allrightsreserved 1751-7362/17
www.nature.com/ismej
ORIGINAL ARTICLE
Genomic reconstruction of multiple lineages of
uncultured benthic archaea suggests distinct
biogeochemical roles and ecological niches
CS Lazar1,2,3,5, BJ Baker4,5, KW Seitz4 and AP Teske1
1Department of Marine Sciences, University of North Carolina Chapel Hill, Chapel Hill, NC, USA;
2Department of Geosciences, Organic Geochemistry Group, MARUM Center for Marine Environmental
Sciences, University of Bremen, Bremen, Germany; 3Department of Aquatic Geomicrobiology, Institute of
Ecology, Friedrich SchillerUniversity Jena,Jena,Germanyand 4Department ofMarine Science, University of
Texas Austin, Marine Science Institute, Port Aransas, TX, USA
Genomicbinsbelongingtomultiplearchaeallineageswererecoveredfromdistinctredoxregimesin
sediments of the White Oak River estuary. The reconstructed archaeal genomes were identified as
belongingtothericeclustersubgroupsIIIandV(RC-III,RC-V),theMarineBenthicGroupD(MBG-D),
and a newly described archaeal class, the Theionarchaea. The metabolic capabilities of these
uncultured archaea were inferred and indicated a common capability for extracellular protein
degradation,supplementedbyotherpathways.ThemultiplegenomicbinswithintheMBG-Darchaea
shared a nearly complete reductive acetyl-CoA pathway suggesting acetogenic capabilities. In
contrast, the RC-III metabolism appeared centered on the degradation of detrital proteins and
productionofH , whereasthe RC-Varchaealackedcapabilities forproteindegradation and uptake,
2
and appeared to be specialized on carbohydrate fermentation. The Theionarchaea appeared as
complexmetabolic hybrids; encodingacomplete tricarboxylicacid cyclepermitting carbon (acetyl-
CoA) oxidation, together with a complete reductive acetyl-CoA pathway and sulfur reduction by a
sulfhydrogenase. The differentiated inferred capabilities of these uncultured archaeal lineages
indicated lineage-specific linkages with the nitrogen, carbon and sulfur cycles. The predicted
metabolisms of these archaea suggest preferences for distinct geochemical niches within the
estuarine sedimentary environment.
TheISME Journal (2017) 11,1118–1129; doi:10.1038/ismej.2016.189; publishedonline13January 2017
Introduction representatives (Teske and Sorensen, 2008). Hence,
the physiological capabilities and specific biogeo-
Particulate organic matter in the ocean is continu-
chemical roles of uncultured clades of Archaea in
ously sinking to the seafloor and accumulates in
marine sediment ecosystems remain poorly under-
subsurface sediments, where it is assimilated, trans-
stood and represent a major research challenge.
formedandoxidizedmainlybyresidentbacteriaand
Coastal estuaries, where sea water and riverine
archaea that constitute the global marine sedimen-
fresh water meet and mix, are particularly rich in
tary biosphere (Kallmeyer et al., 2012). Although
nutrients in both the water and sediment columns.
Bacteria are generally thought to dominate prokar-
In estuarine environments, reactive organic matter
yoticcommunitiesinsubsurfacesediments,Archaea
accumulating in sediments fuels intense microbial
were shown to contribute significantly to marine
activity (Poremba et al., 1999). Microbial processes
sediment microbial communities and their biogeo-
inestuarinesedimentshavebeenwell-studiedinthe
chemicalcycles(Biddleetal.,2006;Lippetal.,2008;
White Oak River (WOR) estuary, a drowned river
TeskeandSorensen,2008).Onthebasisof16SrRNA
valley that drains a swamp forest-dominated coastal
gene surveys, benthic archaeal communities consist
plainofNorthCarolina,USA(LandandPaull,2001).
of distinct lineages with few to no cultured
The WOR sediments have been thoroughly charac-
terized with regard to sulfate reduction, methano-
genesis and anaerobic methane oxidation (Martens
Correspondence:CSLazar,DepartmentofAquaticGeomicrobiol-
ogy, Institute of Ecology, Friedrich Schiller University Jena, and Goldhaber, 1978; Kelley et al., 1990; Kelley
DornburgerStraße159,07743Jena,Germany. et al., 1995; Lloyd et al., 2011). Recently, bacterial
E-mail:[email protected] genomes—including representatives of two novel
5Theseauthorscontributedequallytothiswork. bacterial phyla—were reconstructed from different
Received 29 April 2016; revised 10 November 2016; accepted
19November2016;publishedonline13January2017 sulfate-reducing and methane-producing sediment
Genomicreconstructionofunculturedarchaea
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1119
layers of the WOR estuary (Baker et al., 2015). The and prepared as detailed previously (Lazar et al.,
sulfate-reducing zone (SRZ) hosted Deltaproteobac- 2015). Briefly, a focused-ultrasonicator (Covaris,
teria and uncultured Gammaproteobacteria with Woburn, MA, USA) was used to shear 100ng of
a potential for sulfur oxidation and nitrate/nitrite DNA to 270bp. The sheared DNA fragments were
reduction; whereas the bacteria of the sulfate– size selected for a preferred size of 270bp using
methane transition zone (SMTZ) and the methane- SPRI beads (Beckman Coulter, Brea, CA, USA).
producingzone(MPZ)werepredominantlyinvolved The selected fragments were then end-repaired,
in fermentative organic carbon degradation. A-tailed and ligated with Illumina compatible
The WOR estuary also represents a preferred adapters (IDT, Inc., San Jose, CA, USA) using
habitat for uncultured benthic Archaea, as demon- KAPA-Illumina library creation kit (KAPA Biosys-
strated by archaeal 16S rRNA gene and metage- tems, Wilmington, USA).
nomics surveys. In particular, the WOR estuary
harbors a natural enrichment of the Miscellaneous
CrenarchaeotalGroup,nowrenamedBathyarchaeota Genomic assembly, binning and annotation
(Mengetal.,2014),thesearchaeadominatedthe16S Illumina (HiSeq 2500 PE 2x150) shotgun genomic
rRNA gene pool of all analyzed sediment depths readswerescreenedagainstIlluminaartifacts(adap-
(Lazar et al., 2015). Analysis of genomes belonging ters, DNA spike-ins) using a sliding window with a
to four different subgroups of the Bathyarchaeota kmer size of 28 and a step size of 1 using cutadapt
showed that they shared common pathways for (v1.1, https://pypi.python.org/pypi/cutadapt). Reads
protein degradation and acetogenesis, but suggested with three or more N’s or with average quality
distinctecologicalnichesforthedifferentsubgroups score of less than Q20 and a length o50bps were
(Lazaretal.,2016).Genomesoftwootheruncultured removed. Screened reads were trimmed from both
WOR archaeal groups, the former South African ends using a minimum quality cutoff of 5 using
Gold Mine Euryarchaeotal Group, now renamed Sickle (v1.33 https://github.com/najoshi/sickle).
Hadesarchaea (Baker et al., 2016), and a novel Trimmed, screened, paired-end Illumina reads were
archaeal phylum called Thorarchaeota (Seitz et al., assembledusingIDBA-UD(version1.0.9;Pengetal.,
2016),containedthegenomicblueprintofCOandH 2012) with the following parameters (pre_correction
2
oxidation, and of elemental sulfur and thiosulfate –mink 55 –maxk 95 –step 10 –seed_kmer 55). To
reduction, respectively. maximize assembly reads from different sites were
Inthisstudy,weuseddenovoassemblyofrandom co-assembled.TheSRZassemblywasacombination
shotgungenomiclibrariestoreconstructandidentify of high-quality reads (474179948 with an average
archaealgenomicbinsrecoveredfrommoderatelyto readlength 148bp) from sites2 (8–12cm),and3(8–
strongly reduced sediment layers, and their distinct 10cm). The SMTZ assembly was generated from
geochemical niches in the WOR sediments. We also a combination of high-quality reads (698574240,
analyzed the reconstructed genomes for insights average read length 143bp and average insert
into the metabolic capabilities of these uncultured 274bp) from sites 2 (30–32cm) and 3 (24–28cm).
archaea. These reconstructed archaeal genomes The MPZ assembly was generated from high-quality
were identified as belonging to the rice cluster reads (378027948, average read length 124bp and
subgroups III and V (RC-III, RC-V), the Marine average insert 284bp) of site 1 (52–54cm). The co-
BenthicGroupD(MBG-D),andonenewlydescribed assembledsampleshavebeengroupedpreviouslyby
archaealphylum-levelgrouppreviouslyidentifiedas LINKTREE analysis, a form of constrained cluster
Z7ME43, and renamed Theionarchaea. analysis involving a divisive partition of the sedi-
ment samples with their archaeal communities into
increasingly specific groups (Lazar et al., 2015).
Materials and methods As we were not able to co-assemble all three of the
samplesfromtheSMTZduetocomputationallimits
Sampling, DNA extraction and sequencing (computer memory maxed), an additional assembly
Six 1m sediment plunger cores, each containing ca. was generated from the third sample (site 1
60cm sediment, were collected at specific site 26–30cm)fromhigh-quality345710832reads(aver-
coordinates in three mid-estuary locations of the age length of 129bp and average insert 281bp).
WOR (North Carolina) in October 2010 (Lazar et al., The contigs from this SMTZ sample (site 1) were
2015).Thesesedimentcoreswerepreviouslyusedto co-binned with the combined assembly of the other
characterize the archaeal 16S rRNA gene diversity two, mutually closely related SMTZ samples ofsites
forallthreesitesatselecteddepths,representingthe 2and3(Lazaretal.,2015).Thissequenceofbinning
SRZ, SMTZ and MPZ, based on pore water concen- the most closely related sample pair before adding
trations of methane, sulfate and sulfide (Lazar et al., the more distinct third sample, resulted in differ-
2015). Subsequently, a wide range of archaeal and entiating some closely related but distinct bins,
bacterial genomes was obtained by metagenomic SM1-50 from site 1 and SM23-78 from the site 2/3
analyses of these estuarine sediments (Baker et al., co-assembly. Contigs with genes of particular inter-
2015;Lazaretal.,2016;Seitz etal.,2016).Sediment estwere checked forchimerasby lookingfordipsin
cores were processed and DNA was extracted coverage within read mappings.
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Figure1 Archaealphylogenetictreeinferredfrom16syntenousribosomalproteingenespresentwithingenomicbinsfromthesediment
metagenomicassemblies.RibosomalproteingeneslistcanbefoundinSupplementaryTableS5.Eachindividualsequenceinboldisfrom
asinglegenomicbin.PhylanamesinboldrepresentotherlineagesrecoveredfromtheWORandareincludedintheoverviewshownin
Figure3.Thetaxonomicidentityoftheselineageswasconfirmedby16SrRNAgenephylogeny(SupplementaryFigureS1).Largecircles
show 490 percent Bayesian posterior probabilities, and smaller circles show 470 percent. The scale bar indicates 10% estimated
phylogeneticdivergence.
Initial binning of the assembled fragments was BLASTN(bitscore475),whichwasthennormalized
done using tetra-nucleotide frequencies signatures to the number of reads from each library in order to
using5kbfragmentsofthecontigs(Dicketal.,2009). effectively compare sample coverages. Comparison
The emergent self-organizing maps (Ultsch and of the genomic bins, each of the 5kb sub-portions of
Moerchen, 2005) were manually delineated based the contigs, was used to assess the accuracy of the
on distinct clusters that were separated by visible binning.Ifthecontigwas415kbthenthecontigwas
valleys within the emergent self-organizing maps, assigned to the bin where the majority of the 5kb
seeBakeretal.,2015forthemapsusedinthisstudy. sub-portions were assigned. Genes were called and
Thecompletenessandcontaminationofthegenomic putative function was assigned using the JGI IMG/
bins was then estimated by counting universal MER system (Markowitz et al., 2012). The predicted
ribosomal proteins and single-copy genes using gene and protein sequence data supporting the
CheckM (v1.0.5; Parks et al., 2014). Genomic bins results of this article are being made available
that were found to contain contaminants were in NCBI Genbank under the accession numbers
re-examined with a second round of binning, using LSSB00000000 to LSSJ00000000.
differential coverage. Candidate contaminants were
identified by plotting the coverage of all the contigs
in the bin between different sites; the coverage Phylogenetic analyses
outliers that emerged in these plots were then The concatenated ribosomal protein tree was gener-
removed from the bins. Duplications of single-copy ated using 16 genes that have been shown to have
genes identified with checkM were also considered limited lateral gene transfer (rpL2, 3, 4, 5, 6, 14, 15,
duringthecleaningofthegenomicbins.Single-copy 16, 18, 22, 24 and rpS3, 8, 10, 17, 19; Sorek et al.,
geneswithextremelyloworhighcoveragewerealso 2007). The reference data sets were derived from
consideredlikelycontaminantcontigs,andremoved. the Phylosift database (Darling et al., 2014), which
Coverage was determined by recruiting reads (from additionalsets from the Joint GenomicInstitute IMG
each individual library/sample) to scaffolds by database (Castelle et al., 2013). Scaffolds, from bins
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Table1 GeneralbincharacteristicsandCheckMcompletenessanalysisofthegenomicbins
BinID SM1-50 SG8-52-1 SG8-52-2 SG8-52-3 SG8-5 SG8-52-4 DG-70 DG-70-1 SM23-78
Completeness 94.78 71.62 61.51 85.32 72.59 77.38 72.49 82.44 82.41
Contamination 3.24 7.20 2.38 2.78 0.79 8.33 7.47 7.55 0.93
GC(%) 39 31 31 31 56 31 41 41 37
Totallengthofbin(bp) 2091705 1914472 1355901 1909404 1247229 2246471 3559548 4794391 1480827
Longestscaffold(bp) 111154 93713 68198 131306 53836 104598 66889 144352 96136
Numberofscaffolds 94 79 68 84 111 91 177 194 47
Lineage MBG-D MBG-D MBG-D MBG-D RC-III MBG-D Z7ME43 Z7ME43 RC-V
CheckMcompletenesswasdeterminedusingmultiplearchaealgenemarkers.Highcontaminationisrepresentativeofmultiplemarkerhitswithin
agenomicbin.
in this study, containing o50% of the 16 selected genomic bins belong to the RC-III, RC-V MBG-D
ribosomal proteins were not included in the ana- and Z7ME43 archaealclades. Based onthe presence
lyses. Wesearched NCBI to include referenceamino of single-copy genes, these genomes range from
acid sequences for phylogenetic analyses. The trees partially to near complete (61–94% complete,
showninaminoacidalignmentsoftheconcatenated Table 1). Some of the bins contain fragments from
ribosomal proteins (Figure 1) were generated using duplication of single-copy genes, which may repre-
MAFFT (v1.3.3; Katoh et al., 2002) and trimmed to sent strain-level variants that could not be distin-
1904 residues using BMGE (v2.0; Criscuolo and guished using differential coverage. Therefore, these
Gribaldo, 2010; with the following settings: -m contigs were retained in the genomic bins. Binning
BLOSUM30 –g 0.5). The curated alignments were was manually curated by generating phylogenetic
then concatenated for phylogenetic analyses, and trees from markers on contigs and by accounting for
then phylogeny was generated by running the differences in coverage, to avoid contamination
RAxML (maximum likelihood) tool (version 2.2.1) between the subgroups. Four of the reconstructed
with PROTGAMMA rate distribution model and genomes (SG8-52-1, -2, -3 and -4) were initially
CAT substitution model. Bayesian posterior prob- binned together based on tetra-emergent self-orga-
abilities were generated using MRBAYES (v2.2.2; nizing maps clustering, due to their high similarity,
Huelsenbeck and Ronquist, 2001) for 120000 and then were separated based on differential
generations. coverage. All the genomic bins show considerable
The phylogenetic archaeal 16S rRNA tree was sizevariation,withRC-IIIbeing1.25Mb,RC-Vbeing
calculated with ARB (Ludwig et al.,2004);usingthe 1.48Mb, MBG-D averaging 1.9Mb and Z7ME43
neighbor-joining method based on Jukes–Cantor averaging 4.2Mb (Table 1).
distances. The 16S rRNA gene sequences were
aligned using the SINA webaligner, available online
at http://www.arb-silva.de/ (Pruesse et al., 2012). Archaea in the SRZ
Extremelyshort16SrRNAsequences(o130bp)that The shallow SRZ layers of the WOR sediments were
distorted the phylogeny by introducing predomi- characterizedbyhighsulfateconcentrations(ca.12–
nantly unknown positions into the alignment were 16mM)coincidingwithbarelydetectablesulfideand
assigned phylogenetically by closest match (499% methane concentrations (Lazar et al., 2015). Here,
similarity) obtained by BLASTn on the NCBI bins belonging to two Thermoplasmatales clades
database. The phylogenetic trees of the functional within the Euryarchaeota were identified. The
genes (hydA, hydB, hydD, hydG and cdhA) were RC-III archaea, which were first identified in anoxic
calculatedinMEGA4(Tamuraetal.,2007)usingthe incubations of flooded rice roots (Grosskopf et al.,
neighbor-joining method, and based on amino acid 1998),arerepresentedbythe72%completegenomic
sequence alignments. The amino acid sequences bin SG8-5 (Table 1). The MBG-D archaea, which
were aligned using ClustalW (v1.4). were initially detected in shallow sediments of the
Atlantic continental slope (Vetriani et al., 1999), are
represented by four closely related genomic bins
Results and discussion SG8-52-1, -2, -3 and -4 that are 73, 61, 85 and 77%
complete, respectively (Table 1).
Genome reconstruction of WOR archaea This metagenomic analysis suggests that the
De novo genomic assembly and binning of three metabolisms of the RC-III and MBG-D archaea are
sediment cores from three sites situated mid-estuary centered on the degradation of extracellular detrital
of the WOR, resulted in the reconstruction of nine proteins(SupplementaryFigures2and3),consistent
draft archaeal genomes (Table 1) from the distinct with previous studies (Kemnitz et al., 2005; Lloyd
redox regimes of the SRZ, SMTZ and MPZ (Lazar et al., 2013). The MBG-D bins have genes encoding
et al., 2015). Phylogenies based on concatenated secreted extracellular proteases such as clostripain
ribosomal proteins (Figure 1) and 16S rRNA genes (Merops family C11) and gingipain (Merops family
(Supplementary Figure S1) showed that these C25), interpain (Merops family C10) and legumain
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(Merops family C13) cysteine peptidases. No genes conversion of pyruvate, obtained by protein degra-
encoding extracellular proteases were detected in dation,toacetyl-CoAandformate,anditsexpression
theRC-IIIbin,withthecaveatthatthesegenescould is typically induced and enhanced under anaerobic
be located in the missing parts of this incomplete conditions(SawersandBock,1988).Theacetyl-CoA
genome. Both RC-III and MBG-D bins contain genes produced during protein degradation can be con-
encodingdi-andoligo-peptidesmembranetranspor- verted to acetate and ATP via an ATP-producing
ters, intracellular aminopeptidases and proteases acetyl-CoA synthetase. As no genes involved in
involved in the breakdown of amino acids, as fermentation were detected (for example, alcohol or
well as pyruvate:ferredoxin andindolpyruvate:ferre- lactate dehydrogenases), the originally suggested
doxin oxidoreductases converting 2-oxo acids fermentative metabolism for the RC-III archaea
to acyl-CoA and CO using ferredoxin as electron (Kemnitz et al., 2005) could not be confirmed.
2
carrier (Supplementary Figures 2 and 3; Supple- However,theMBG-Darchaeacouldfermentproteins
mentary Tables S1 and S2). In addition, the MBG-D to ethanol as their bins contain genes encoding an
bins encode an oligopeptide transporter system, ATP-producing acetyl-CoA synthetase, commonly
apossibleadaptationforutilizingsmallaminoacids, found in peptide-degrading archaea (Sapra et al.,
dependingonenvironmentalconditions.Indeedthis 2003), an aldehyde:ferredoxin oxidoreductase and
genewasshowntoberegulatedandenhancedbythe an alcohol dehydrogenase.
presence of small peptides in sulfur-depleted envir- Typically,organismsgrowingonaminoacidsrequire
onments (Wiles et al., 2006). The MBG-D genomes an Embden–Meyerhoff–Parnas gluconeogenesis path-
also contain genes encoding enzymes involved in way (EMP) to provide carbon sources for biosynthesis
assimilatory sulfate reduction to sulfite (Supple- of cellular building blocks (Ng et al., 2000). Both the
mentary Figure 3), the first steps of cysteine and RC-III and MBG-D bins include genes encoding a
methionine biosynthesis. In contrast to frequently partial EMP. Specifically, the fbp gene encodes the
found peptide transporters, genes involved in carbo- unidirectional gluconeogenetic fructose 1,6 bispho-
hydrateuptakeandassimilationwerenotdetectedin sphatase; which produces fructose 6-phosphate for
theMBG-Dbins,suggestingthatpeptidesarethesole further use in anabolic pathways (Supplementary
carbon sources for MBG-D. Figures 2 and 3; Supplementary Tables S1 and S2).
Both the RC-III and MBG-D bins contain genes An additional near-complete pathway providing
encoding subunits of a Ni,Fe hydrogenase type 3 and anaboliccarbonsourcesintheMBG-Darchaeaisthe
genes encoding an ATP synthase (Supplementary reductive acetyl-CoA (Wood–Ljungdahl) pathway
Figures2and3).Thus,theRC-IIIandMBG-Darchaea using tetrahydrofolate as a cofactor (Supplementary
might possess a simple energy-conserving respiratory Figure S3 and Supplementary Table S2). The
system similar to the one described in Pyrococcus gene cdhA, encoding the alpha subunit of the CO
furiosus (Sapra et al., 2003). In this system, reduced dehydrogenase/acetyl-CoA synthetase, was detected
ferredoxinisusedasanelectrondonorandprotonsare in the MBG-D bins (Supplementary Table S2).
usedasterminalelectronacceptors,andproductionof A phylogenetic tree based on amino acid sequences
H is directly coupled to ATP synthesis via proton placed the MBG-D CdhA sequences within a cluster
2
translocation. Consistent with this scenario, a com- containingtheheterotrophic,acidophilicandsulfur-
binedqPCRandculturingstudy(Kemnitzetal.,2005) reducing archaeon Aciduliprofundum (Supple-
demonstrated no growth stimulation in an RC-III mentary Figure S4), a sister lineage of the MBG-D
archaeal enrichment after addition of various electron archaea within the Thermoplasmatales. The Acid-
acceptors(NO3-,SO2-,Fe(III),S0,S O2-andfumarate).In uliprofundum sequences are annotated as [Fe4-S4]
4 2 3
addition,theRC-IIIbincontains8ofthe11subunitsof metalclusterswhichwereshowntobepresentinthe
a NADH dehydrogenase complex I (without a NADH- active sites of the CO dehydrogenase/acetyl-CoA
binding module), all subunits of a heterodisulfide synthetase of anaerobic bacteria (Dobbek et al.,
reductase HdrABC as well as subunits of a F420-non- 2001).Thus,thelowsequencesimilarityofthecdhA
reducing hydrogenase MvhADG (Supplementary MBG-D genes to other versions of the cdhA gene
Figure 2). It has been suggested that the 11 subunit suggests a modified CdhA protein in the MBG-D
NADH dehydrogenase complex could recycle archaea.
ferredoxin by accepting electrons from reduced So far, the existence of Wood–Ljungdahl genes in
ferredoxin (Battchikova et al., 2011), possibly MBG-D does not provide conclusive evidence for
provided by the HdrABC/MvhADG hydrogenase autotrophy. As the tricarboxylic acid (TCA) cycle is
complex (Castelle et al., 2013). In non-methanogenic completeintheMBG-Dbins,itwouldproducecitrate,
archaea, the HdrABC/MvhADG complex could oxoglutarate or oxaloacetate for biosynthesis of amino
couple the reduction of ferredoxin with H to the and/orfattyacids;thebyproductCO couldberecycled
2 2
reduction of an unknown disulfide (Mander et al., in the Wood–Ljungdahl pathway (Schuchmann and
2004; Castelle et al., 2013), hence yielding reduced Müller, 2014). To date no cultured anaerobic repre-
ferredoxin. sentativeoftheThermoplasmataleshasbeenshownto
TheRC-IIIbincontainsapyruvate-formatelyaseas use the reductive acetyl-CoA pathway for carbon
well as a pyruvate-formate lyase activating enzyme fixation. However, Ferroplasma, a cultured aerobic
(SupplementaryFigure2).Thisenzymecatalyzesthe Thermoplasmatales, has been predicted to use a
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modified Wood–Ljungdahl pathway with an aerobic transported into the cytoplasm, intracellular poly-
CO dehydrogenase (Cárdenas et al., 2009). saccharides may undergo further breakdown to
TheMBG-Dbinsharbornumerousgenesencoding glucose-1P or fructose-6P via the galactose and/or
one- and two-component systems (sensory protein mannose degradation pathway (Supplementary
andresponseregulators)thatareinvolvedinsensing Figure S5). The glucose-1P and/or fructose-6P can
and responding to a variety of changes in the then enter the glycolysis pathway, producing ATP
surrounding environment; for example, osmolarity, and NADH. The RC-V bin contains all genes
oxidative stress or nutrient availability (Supple- constituting the EMP pathway (Supplementary
mentary Figure S3 and Supplementary Table S2). FigureS5)includingthepfkandpykgenesencoding
Inadditiontoregulatorysystemsforchemotaxis,the a phosphofructokinase and a pyruvate kinase which
MBG-D genomes possess genes involved in archaeal operateexclusivelyinthecatabolicdirection(Bräsen
flagellin proteins and flagella assembly (Patenge etal.,2014).Theendproductofglycolysis,pyruvate,
et al., 2001; Chaban et al., 2007; Supplementary can then be converted to acetyl-CoA via a pyruvate:
Table S2). Hence, the MBG-D archaea are predicted ferredoxin oxidoreductase using ferredoxin as
to be motile, and capable of chemotactic response electron carrier (Supplementary Figure S5 and
towards different environmental factors. These Supplementary Table S3). Genes encoding proteins
genomes also contain a gene encoding a CRISPR involvedincellularrespirationandintheTCAcycle
exonucleasewhichmightconferresistancetophages were not detected; hence the WOR RC-V archaea
(Barrangouetal.,2007),andtheuspAgeneencoding might be strict fermenters relying on carbohydrates
a small cytoplasmic protein that is induced during as sole source of energy and carbon. The RC-V bin
starvation periods when substrate limitation inhibits also contains the pta and ackA genes encoding a
the growth rate (Nystrom and Neidhardt, 1994). phosphate acetyltransferase and an acetate kinase,
catalysingtheconversionofacetyl-CoAtoacetatevia
acetyl-P, and producing ATP. This pathway poten-
Archaea in the SMTZ tially used by the RC-V archaea leading to acetate
Bins belonging to two archaeal clades—the RC-V production, was thought to be unusual for Archaea
(bin SM23-78, 82.4% complete; Table 1) and an as the acetate-forming archaea are suggested to
additional MBG-D (bin SM1-50, 94.8% complete; mainly use the acetyl-CoA synthetase pathway
Table 1) distinct from those recovered from the SRZ (Schäfer et al., 1993). However, an acetate kinase
—were identified from the SMTZ layers of the WOR was detected in the methanogen Methanosarcina
sediments,whereoverlappingporewatersulfateand barkeri, in which cell-free enzyme extracts showed
methane gradients coincided with a sulfide peak activity in the direction of acetate formation (Smith
(Lazar et al., 2015). Phylogenetic analyses based on and Lequerica, 1985). Recently, the ack gene was
concatenated ribosomal proteins (Figure 1) and 16S detected in Bathyarchaeota, and the expressed and
rRNA genes (Supplementary Figure S1) place the purified protein showed reversible catalysing activ-
WOR RC-V genomic bin within the Woesearchaeota ity; suggesting that the Bathyarchaeota can consume
phylum (Castelle et al., 2015). The core metabolism and/or produce acetate through the pta-ack pathway
of the WOR RC-V archaea appears to be centered (He et al., 2016). Recycling of oxidized ferredoxin
on carbohydrate fermentation, whereas the SMTZ and NAD+ could occur via ethanol fermentation
MBG-D core metabolism appears to be centered on (Köpke et al., 2010), as the RC-V bin has genes
the degradation of extracellular detrital proteins. In encoding an aldehyde:ferredoxin oxidoreductase
comparison with the MBG-D bins from the SRZ, and an alcohol dehydrogenase (Supplementary
genes encoding interpain and legumain cysteine Figure S5).
peptidases were not detected in the SMTZ bin; and
both the gluconeogenetic EMP and the TCA cycle
appear to be incomplete (Supplementary Table S2), Theionarchaea in the MPZ
with the caveat that the pathway gaps could be Two bins (DG-70 and DG-70-1, 74.5 and 83%
artifacts resulting from incomplete genomic bins. complete; Table 1) belonging to an as-yet unde-
The RC-V bin contains two genes encoding scribed deeply branched class of the Euryarchaeota
potential extracellular enzymes involved in hydro- (Figure 1), were recovered from the MPZ layers (52–
lyzing plant-derived polymeric carbohydrates to 54cmbsf) of the WOR sediments, where methane
smaller oligosaccharides and glucose, that is, an concentrations near 0.5mM were measured, no
exo-beta-1,3-glucanase and a glucan-1,4-alpha- sulfatewasdetectedandsulfideconcentrationswere
glucosidase (Supplementary Figure S5 and Table S3). low(Lazaretal.,2015).Thiseuryarchaealgroupwas
The exo-beta-1,3-glucanase has been shown to use previouslyidentifiedasZ7ME43(Sahletal.,unpub-
laminarin, storage polysaccharides found in brown lished; NCBI GeneBank reference FJ902807); we
algae, as specific substrate (Bara et al., 2003). propose to call this new WOR archaeal class
Furthermore, the RC-V archaea have the potential Theionarchaea, as their genomes indicate strong
totransporttheseextracellularsugarsintothecellas capabilities for sulfur cycling (Figure 2). This new
the RC-V bin contains genes encoding an ABC-type archaeal lineage appears to comprise widespread
sugar transport system and a sugar permease. Once archaea,asphylogeneticanalysisbasedon16SrRNA
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Figure 2 Reconstructed metabolic pathways of genes detected in Theionarchaeota, based on the DG-70 and DG-70-1 bins. Predicted
proteinsindicatedbynumbersinthefigurecanbefoundintheSupplementaryTableS4.Alistofpredictedsignalpeptidescanbefound
in the Supplementary Table S6. DHAP, dihydroxyacetone phosphate; Fd, ferredoxin; Fru-6P/-1,6bP, Fructose-6-phopshate/-1,6-
bisphosphate;GAP,glyceraldehyde3-phosphate;Glu-1P/-6P,Glucose-1-phosphate/-6-phosphate;NAD,nicotinamideadeninedinucleo-
tide;PEP,phosphoenolpyruvate;THM,tetrahydromethanopterin.
genes show that sequences belonging to this new enzymes have been shown to preferentially reduce
phylum were recovered from coastal marine sedi- S0 rather than protons, H-II was shown to have a
ments, estuary sediments, lagoon carbonaceous higher affinity for S0 and polysulfides than H-I
sediments, a subsurface limestone sinkhole biomat, (Ma et al., 2000). Genes encoding all four subunits
lake sediment, deep subsurface marine sediments of the (sulf)hydrogenase in bins DG-70 and DG-70-1
from the Peru Margin and a terrestrial mud volcano were found to be similar to the genes belonging to
(Supplementary Figure S1). the H-II of P. furiosus (Supplementary Figure S6).
As their most distinctive feature, bins DG-70 and The Theionarchaea could also use thiosulfate as an
DG-70-1 contain genes encoding all four subunits of electron acceptor, as bin DG-70-1 contains a phsB
a sulfhydrogenase (or sulfur reductase; Figure 2; gene encoding a subunit of a thiosulfate reductase
Supplementary Figure S6). This enzyme has been (Heinzinger et al., 1995).
described in hyperthermophilic archaea such as Bins DG-70 and DG-70-1 have the potential for
P. furiosus, in which sulfur reduction was initially detrital protein uptake and degradation to pyruvate
assumed to function as sink for fermentatively and acetyl-CoA (Figure 2), similar to RC-III and
generated H (Fiala and Stetter, 1986). However, MBG-D archaea. Bins DG-70 and DG-70-1 contain
2
sulfur reduction catalyzed by the sulfhydrogenase genes encoding all subunits of a pyruvate dehydro-
was later suggested to be an energy-conserving genase complex, converting pyruvate derived from
mechanism allowing disposal of excess reductant amino acid degradation to acetyl-CoA. Like the RC-
usingpolysulfidesorS0(orH+)aselectronacceptors III archaea, bins DG-70 and DG-70-1 include genes
andH ororganiccompoundsaselectron donor (Ma encoding a pyruvate-formate lyase and pyruvate-
2
et al., 1993; Schicho et al., 1993). Two cytoplasmic formate lyase activating enzymes, probably induced
(sulf)hydrogenases (H-I and H-II) have been identi- by anoxic conditions asshownpreviously forE. coli
fied and purified in P. furiosus, and whereas both (Sawers and Bock, 1988). In addition, bins DG-70
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and DG-70-1 have genes encoding a formate dehy- constitute membrane proteins forming a large elec-
drogenase,whichwoulddisposeofthetoxicformate tron transfer complex (Tersteegen and Hedderich,
produced by conversion of pyruvate to acetyl-CoA 1999; Major et al., 2010) catalysing energy-driven
via the pyruvate-formate lyase (Figure 2). reduction of low-potential electron carriers, such as
The carbohydrate metabolism of the Theionarch- ferredoxin. The expression levels of hydrogenase B
aea appears to be limited. Bins DG-70 and DG-70-1 in Methanobacterium thermoautotrophicum cells
contain one gene encoding a sugar permease; and increased significantly in response to H limiting
2
their partial EMP pathways are lacking genes conditions (Tersteegen and Hedderich, 1999), sug-
involved in the first steps of glycolysis (Figure 2). gestingthatthegeneexpression levelsofferredoxin-
BinsDG-70andDG-70-1alsocontaingenesinvolved reducing enzyme systems in the Theionarchea are
inthemethylglyoxalpathway,alow-energyyielding environmentally modulated.
bypass of the lower EMPthat does not produce ATP The Theionarchaea can potentially fix nitrogen
and is typically activated under phosphate limiting to ammonium, as suggested by the detection of a nif
conditions (Weber et al., 2005). One possible fate of operon encoding subunits of a nitrogenase and
acetyl-CoA obtained by either protein or fructose/ accessory proteins (Figure 2; Supplementary Table
glucose degradation is conversion to acetate via an S4). Nitrogen fixation could allow Theionarchaea to
ATP-producing acetyl-CoA synthase (EC:6.2.1.13); use N as sole nitrogen source in nitrogen-limiting
2
the acetate produced this way, or taken up directly conditions. In addition, bins DG-70 and DG-70-1
via an cation/acetate symporter, can ultimately be have genes encoding proteins (NtrC, NtrY) involved
fermented to ethanol (Figure 2). The Theinarchaeoa in the Ntr two-component system which regulates
could also convert acetate to acetyl-CoA as bins nitrogen fixation (Gussin et al., 1986; Figure 2).
DG-70 and DG-70-1 have an acs gene encoding an SimilarlytotheMBG-Dgenomes,thetheionarchaeal
acetyl-CoA synthase (EC:6.2.1.1). As bins DG-70 and bins DG-70 and DG-70-1 possess numerous genes
DG-70-1havegenesencodingacompleteTCAcycle, encoding one- and two-component systems, six
acetyl-CoAcan enterthispathway, hence producing genes encoding CRISPR exonucleases, genes encod-
ATP and amino acid biosynthesis precursors. ing enzymes involved in DNA repair and the uspA
BinsDG-70andDG-70-1containgenesencodinga gene (Figure 2; Supplementary Table S4).
complete reductive acetyl-CoA (Wood–Ljungdahl)
pathway using the methanogenic C -carrier tetra-
1
methanopterin, as well as genes involved in coen-
Habitat specificity and predicted biogeochemical roles
zyme F420 biosynthesis and genes encoding a
of the WOR archaea
coenzyme F420-reducing hydrogenase (Figure 2,
Estuaries such as the WOR are known for harboring
Supplementary Table S4). The phylogenetic tree
highly active heterotrophic and autotrophic micro-
based on amino acid sequences of CdhA placed the
bial communities as they are continuously replen-
Theionarchaea CdhA sequences within a cluster
ishedinorganicmatterandnutrientsfrombothland
containing mainly methanogens and Archaeoglobus
and sea (Poremba et al., 1999). Thus, the roles of
(Supplementary Figure S4). In contrast, the MBG-D
archaea in these complex microbial ecosystems
genome encodes a pathway using tetrahydrofolate,
extendbeyondtheuptakeanddegradationofdetrital
the typical C -carrier in acetogenesis; and its CdhA
1 proteins (Ouverney and Fuhrman, 2000; Kemnitz
sequences form a distinct cluster lacking methano-
etal.,2005;Lloydetal.,2013).Althougheightofthe
gens(SupplementaryFigureS4).Thesecarriershave
nine reconstructed WOR archaeal genomes in this
been suggested to functionally distinct, based on study—belonging to the RC-III, MBG-D and Theio-
energy metabolism, thermodynamics of both reac- narchaea—encode the capability for extracellular
tions and biosynthetic pathways of the two carriers
protein degradation, they also show additional
(Maden,2000).Thetetrahydrofolatepathwayplaysa
capabilities that link them to carbohydrate degrada-
major role in biosynthesis of purines and regenera-
tion, acetogenesis and sulfur reduction.
tion of methionine, although in acetogenic bacteria
In contrast to the MBG-D and Theionarchaea
most of the C1 fluxwas shown to beusedforenergy
genomes, the RC-III genome recovered from the
generation. In the reductive direction, the tetrahy-
shallow SRZ layers did not contain any genes
drofolate pathway consumes ATP, whereas the
encoding extracellular peptidases. It is possible that
redox reactions involved in the methanopterin
the RC-III archaea depend on other archaea to pre-
pathway generate electrochemical ion gradients
digest detrital proteins into oligopeptides that are
(Deppenmeier et al., 1996) leading to chemiosmotic
small enough for transmembrane uptake (Figure 3).
synthesis of ATP (Maden, 2000).
Compatible with such a metabolic limitation, mole-
The Theionarchea potentially have two ways of
cularsurveyshavedetected RC-III archaeamostlyin
obtaining reduced ferredoxin that may substitute for
each other. In addition to containing a HdrABC- nutrient-rich rhizosphere habitats (Kemnitz et al.,
MvhADG complex, a likely source of reduced 2005), whereas the MBG-D are found in a wider
ferredoxin, bins DG-70 and DG-70-1 also have eight range of aquatic benthic environments with limited
subunits of an energy-converting hydrogenase B carbon substrates and energy sources (Teske and
(ehb). These subunits have been suggested to Sorensen, 2008; Lloyd et al., 2013).
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Figure3 PredictedgeneralmetabolismsoftheWORarchaeainrelationtothegeochemicallayerfromwhichtheywererecovered.In
addition, the genome-based metabolic predictions for the Bathyarchaeota subgroups −6, −1, −7/17 and −15 (Lazar et al., 2015), the
Thorarchaeota(Seitzetal.,2016)andtheHadesarchaea(Bakeretal.,2016)wereadded.TOC,totalorganiccarbon;MCG,miscellaneous
crenarchaeotal group (Bathyarchaeota); CYS, cysteine. WOR picture modified from http://www.learnnc.org/lp/editions/cede_black
waterriver/54.
Metabolic predictions for the RC-V archaea, based sediments (Meador et al., 2015). Thus, the avail-
on the genome reconstructed from the sulfate- ability of marine and terrestrial sources of organic
depleted sediment layers of the SMTZ in the WOR carbon could allow for the coexistence of many
estuary, indicate that these archaea seem to possess different types of fermenters in the WOR estuary
limited metabolic capacities and are probably obli- sediments, including the RC-V archaea.
gate fermenters of carbohydrates (Figure 3). In contrast to the carbohydrate-dependent RC-V
Although the RC-V archaea appear usually as a archaea, the MBG-D archaea lack genes involved in
non-dominant archaeal group in molecular surveys carbohydrate uptake and assimilation. On the basis
(Jurgens et al., 2000; Glissmann et al., 2004), they of the observed set of genes, cellular carbon would
were sufficiently abundant in the sampled WOR originate from protein degradation, complemented
sediments for genomic reconstruction. As no sedi- most likely by heterotrophic CO recycling through
2
mentary habitat has been shown so far to be the Wood–Ljungdahl pathway. The MBG-D genome
exclusively dominated by the RC-V archaea, it is recovered from the SMTZ might possess a more
likely that they are generalists sharing the fermenta- limited capacity for extracellular degradation of
tive ecological niche with a wide range of other proteins compared with the MBG-D genome recon-
microorganisms. Consistent with this interpretation, structed from the shallow SPZ layers, and its core
high concentrations of total organic carbon in the metabolism seems to be protein degradation either
rangeof4–6%were measuredintheWORsediment by respiration or fermentation.
samples used for genomic reconstruction (Lazar Genomic analysis of the Theionarchaea high-
et al., 2015). An average δ13C-total organic carbon lighted their repertoire of metabolic strategies to
of−25‰indicatedamajorcontributionofterrestrial dealwithsubstrate-andnutrient-limitingconditions.
C4plant-derivedorganicmatterinthesampledWOR The Theionarchaea genomes show potential for
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1127
using and converting sugar phosphates and amino Sequencing at the Joint Genome Institute is supported by
acids to acetyl-CoA and further to acetate with their theOfficeofScienceoftheU.S.DepartmentofEnergyunder
ATP-producing acetyl-CoA synthase; they also have Contract No. DE-AC02-05CH11231 given to B.J. Baker.
AdditionalfundingwasprovidedbytheCRC1706AquaDiva
the potential for taking up and oxidizing acetate by
funded by the Deutsche Forschungsgemeinschaft (DFG)
convertingittoacetyl-CoAandinsertingitintotheir
given to Kirsten Küsel. A. Teske was supported in part by
complete TCA cycle. Hence these archaea could
theCenterforDarkEnergyBiosphereInvestigations(C-DEBI).
scavenge extracellular acetate when acetate- Theassembliesthatwereusedinthisstudywererecovered
producing carbon sources become depleted, an fromthesameDNAsamplesthatwereusedfromtheauthors
adaptation strategy also known as ‘acetate switch’ in previous studies of the WOR estuary (Lazar et al., 2015,
(Wolfe, 2005). The Theionarchaea could also cope 2016;Bakeretal.,2015,2016;Seitzetal.,2016).
withphosphatelimitingconditionsbyactivatingthe
methylglyoxal pathway, a low-energy bypass of the
lower EMP pathway; they could adapt to nitrogen-
limiting conditions by fixing atmospheric N ; and
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Conflict of Interest of the fla genes in the flagella operon identify several
genes essential for proper assembly and function of
The authors declare no conflict of interest. flagella in the archaeon Methanococcus maripaludis.
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