Metabolic traits of an uncultured archaeal lineage -MSBL1- from brine pools of the Red Sea

Abstract

The candidate Division MSBL1 (Mediterranean Sea Brine
Lakes 1) comprises a monophyletic group of uncultured archaea found in
different hypersaline environments. Previous studies propose methanogenesis as the
main metabolism. Here, we describe a metabolic reconstruction of MSBL1 based on 32
single-cell amplified genomes from Brine Pools of the Red Sea (Atlantis II,
Discovery, Nereus, Erba and Kebrit). Phylogeny based on rRNA genes as well as
conserved single copy genes delineates the group as a putative novel lineage of
archaea. Our analysis shows that MSBL1 may ferment glucose via the
Embden–Meyerhof–Parnas pathway. However, in the absence of
organic carbon, carbon dioxide may be fixed via the ribulose bisphosphate
carboxylase, Wood-Ljungdahl pathway or reductive TCA cycle. Therefore, based on the
occurrence of genes for glycolysis, absence of the core genes found in genomes of
all sequenced methanogens and the phylogenetic position, we hypothesize that the
MSBL1 are not methanogens, but probably sugar-fermenting organisms capable of
autotrophic growth. Such a mixotrophic lifestyle would confer survival advantage (or
possibly provide a unique narrow niche) when glucose and other fermentable sugars
are not available.

Full text

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Scientific RepoRts | 6:19181 | DOI: 10.1038/srep19181
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Metabolic traits of an uncultured
archaeal lineage -MSBL1- from
brine pools of the Red Sea
Romano Mwirichia1,†, Intikhab Alam2, Mamoon Rashid1, Manikandan Vinu1, Wail Ba-
Alawi2, Allan Anthony Kamau2, David Kamanda Ngugi1, Markus Göker3, Hans-Peter Klenk4,
Vladimir Bajic2 & Ulrich Stingl1
The candidate Division MSBL1 (Mediterranean Sea Brine Lakes 1) comprises a monophyletic group
of uncultured archaea found in dierent hypersaline environments. Previous studies propose
methanogenesis as the main metabolism. Here, we describe a metabolic reconstruction of MSBL1
based on 32 single-cell amplied genomes from Brine Pools of the Red Sea (Atlantis II, Discovery,
Nereus, Erba and Kebrit). Phylogeny based on rRNA genes as well as conserved single copy genes
delineates the group as a putative novel lineage of archaea. Our analysis shows that MSBL1 may
ferment glucose via the Embden–Meyerhof–Parnas pathway. However, in the absence of organic
carbon, carbon dioxide may be xed via the ribulose bisphosphate carboxylase, Wood-Ljungdahl
pathway or reductive TCA cycle. Therefore, based on the occurrence of genes for glycolysis, absence
of the core genes found in genomes of all sequenced methanogens and the phylogenetic position, we
hypothesize that the MSBL1 are not methanogens, but probably sugar-fermenting organisms capable
of autotrophic growth. Such a mixotrophic lifestyle would confer survival advantage (or possibly provide
a unique narrow niche) when glucose and other fermentable sugars are not available.
More than half of the 60 major lines of descent within the bacterial and archaeal domains that have been described
based on SSU rRNA phylogeny1 remain uncultured and make up the so-called “microbial dark matter”2, since
their metabolic capabilities and ecological role remain obscure. Members of the candidate division MSBL1
(Mediterranean Sea Brine Lakes 1) encompass an uncultured archaeal lineage that is abundant and widespread in
deep hyper-saline anoxic basins (DHABs) of the Mediterranean Sea, the Red Sea, and the Gulf of Mexico3–5. 16S
rRNA signature sequences of this group were also reported from the anoxic hypolimnion of a shallow hyper-saline
Solar Lake in Egypt6, sediments of hyper-saline Lake Chaka in China7, from a crystallizer in a multi-pond solar
saltern in the south of Mallorca Island8,9 and recently in metagenomic libraries from a hyper-saline lake in Kenya
(Mwirichia et al. unpublished data). MSBL1 have been postulated to be methanogenic based on their phyloge-
netic position and circumstantially because they are the numerically dominant archaeal group in DHABs, where
incidentally also high methane concentrations of presumably biogenic origin are present3,10,4,11. However, with
the exception of few sequences distantly related to Methanohalophilus4,12 no other homologs of the key methano-
genic enzyme, methyl coenzyme-M reductase (mcrA) have been recovered from the brine pools studied so far.
erefore, the exact metabolism of this group remains enigmatic in the absence of cultured representatives or
larger contigs of genomic sequences. In this study, we applied single-cell genomics using cells of the MSBL1 clade
from the Red Sea brine pools to reconstruct their metabolic potential. Our study provides the rst evidence of
their non-methanogenic metabolic capabilities that enable them to thrive in the anoxic DHABs.
1Red Sea Research Center, King Abdullah University of Science and Technology (KAUST), Thuwal, Saudi Arabia.
2Computational Bioscience Research Center, King Abdullah University of Science and Technology (KAUST), Thuwal,
Saudi Arabia. 3German Collection for Microorganisms and Cell Cultures GmbH (DSMZ), Inhoenstraße 7b, 38124
Braunschweig, Germany. 4School of Biology, Newcastle University, Newcastle upon Tyne, United Kingdom. †Present
address: Embu University College, Embu, Kenya. Correspondence and requests for materials should be
addre ssed to R .M. (email: mwirichia.romano@embuni.ac.ke) or U.S. (email: uli.stingl@kaust.edu.sa)
Received: 10 April 2015
Accepted: 04 December 2015
Published: 13 January 2016
OPEN

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Results
Genome analysis. e physico-chemistries of the dierent Red Sea brines have been described previ-
ously5,13. A total of thirty-two single-cell amplied genomes (SAGs) described in this study were recovered from
four brine pools (Atlantis II, Discovery, Erba, and Nereus) in the Red Sea (Fig.1).
e origins of the single cell genomes are as follows-Atlantis II (8), Discovery (13), Erba (3) and Nereus Deep
(9). e features of the assembled single cell genomes including size (137,797 – 1,424,127 bases) and estimated
genome completeness (0.0% – 56.7%), as evaluated using a set of 104 conserved single-copy archaeal genes as
described by AMPHORA214 are shown in Table1.
Genome completeness computed using a larger set of 191 genes described by Dodsworth et al.15 shows that best
result/completeness was 54.2% in the SAG AAA259I09, a slight variation from 56.7% shown in AMPHORA14.
The complete set of 191 genes (including those described in AMPHORA) is listed in Supplementary Table
S1. Phylogenetic inference using the full-length 16S rRNA gene sequences16 delineates the MSBL1 as a novel
deep-branching order, distinct from the described methanogens (Fig.2). e matrix for the 16S rRNA gene
sequences comprised 107 operational taxonomic units and 1714 characters, 1269 of which were variable and
1070 of which were parsimony-informative. ML analysis under the GTR model yielded a highest log likelihood of
-47688.24, whereas the estimated alpha parameter was 0.52. e bootstrapping converged aer 650 replicates; the
average support was 74.73%. MP analysis yielded a best score of 10431 (consistency index 0.24, retention index
0.69) and 2 best tree(s). e MP bootstrapping average support was 70.88%. On the basis of the Silva aligner17,
members of the MSBL1 are placed within the class ermoplasmata with identity scores between 83 to 86.9%.
A phylogenetic tree based on partial 16S rRNA genes and encompassing MSBL1 sequences from other environ-
ments is shown in Supplementary Figure S1. In this tree, the sequences from the Red Sea brine pools cluster with
those from brine pools in the Mediterranean Sea. Complementary phylogenetic analyses using a concatenated set
of ten conserved single-copy genes present in 8 MSBL1 SAGs and other sequenced archaeal genomes (Fig.3) also
conrms the placement of MSBL1 as a novel archaeal lineage distinct from methanogens. Unexpectedly, we found
that, although this novel archaeal group preferentially occurs in hyper saline environments, the largely unimodal
distribution in the isoelectric point (pI) of their overall protein-coding genes places their proteomes in the same
range as moderate halophiles (Supplementary Figure S2). An exception is the genome AAA385M11, whose pI is
slightly higher, which may be an artefact due to the small size (215 Kb) of the assembled genome.
Carbohydrate transport and metabolism. Genes encoding for transcriptional regulators involved in
carbohydrate transport and metabolism were identied in 18 of the SAGs (Supplementary Table S2), the most
important being the archaeal sugar-specic transcriptional regulator TrmB. In ermococcus litoralis, the respec-
tive protein is involved in the maltose-specic regulation of a gene cluster (malE, malF, malG, malK) that encodes
a trehalose/maltose-binding protein-dependent ABC transporter for trehalose and maltose18. e genes malE and
malG were identied in the genomes of AAA259A05 and AAA259E19, respectively. MalE is a maltose binding
Figure 1. Map of the Red Sea, showing ve dierent deep-sea brines along the N-S axis of the Red Sea. e
map was generated using ArcGIS v.10.1.

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protein whereas MalG is a maltose transport system permease. Sugar transporters include a putative catabolism
phosphotransferase system, putative sugar ABC transport system and a glucose import ATP-binding protein
TsgD13 (Supplementary Table S2). Potential substrates include glucose, galactose arabinose, maltodextrin, malt-
ose, xylose and ribose (Supplementary Table S2). Trehalose could play a signicant role both as a carbon source
and also compatible solute involved in osmoprotection. In this group, trehalose is synthesized from maltose,
starch or UDP glucose (Supplementary Table S2; Supplementary Figure S3). e ability to utilize trehalose as an
osmolyte would explain their rather normal pI as compared to that of other extreme halophiles. In the genome
of AAA259B11, α -D-glucanotransferase may be involved in conversion of starch to trehalose. Supplementary
Figure S3 summarizes the initial sugar metabolism to either α -D-glucose or trehalose.
Glycolysis/ Gluconeogenesis. Diversity in sugar metabolism pathways in archaea as well as the variabil-
ity in enzymes involved has been reviewed recently19. e MSBL1 group uses a fermentative sugar metabolism
that combines the classical and recently discovered (archaeal) enzymes of the Embden-Meyerhof (EM) pathway
(Fig.4; Supplementary Table S2). e absence of cytochromes, cytochrome oxidases and quinones in all the SAGs
reinforce our hypothesis that these Archaea are likely to ferment and also that they probably do not contain an
electron transport chain. Besides, presence of oxygen-sensitive enzymes (pyruvate-ferredoxin oxidoreductase)
and absence of catalase indicates a strictly anaerobic lifestyle as expected within the anoxic brine environment.
During sugar metabolism via the EM pathway, glucose is converted to two molecules of pyruvate and
yields two ATPs, reducing equivalents and intermediates that are precursors for cellular building blocks.
e products of sugar fermentation are acetate, carbon dioxide and H2. e two key genes for the alternative
SAG Contigs
Length
in bp Longest Smallest Average
Genome
Completeness (%)
104
Markers
191
Markers
AAA261C02 79 628,204 41,092 2,004 7,952 49.0 39.8
AAA261F17 62 527,268 47,307 2,094 8,504 32.7 21.5
AAA261G05 68 532,132 27,533 2,021 7,825 50.0 39.3
AAA261O19 99 731,948 41,274 2,022 7,393 41.3 35.6
AAA261D19 82 673,667 37,168 2,063 8,215 50.0 48.7
AAA261F19 82 655,981 44,778 2,016 7,999 49.0 45.5
AAA259A05 161 1,167,671 38,447 2,002 7,252 36.5 36.1
AAA259D18 56 387,594 17,623 2,039 6,921 14.4 6.8
AAA259E17 141 883,132 39,302 2,014 6,264 24.0 19.4
AAA259E19 211 1,424,127 37,031 2,003 6,749 42.3 37.2
AAA259O05 152 1,067,938 38,713 2,025 7,025 39.4 36.1
AAA259B11 118 828,207 32,654 2,022 7,018 19.2 23.0
AAA259D14 117 852,718 37,663 2,014 7,247 49.0 36.6
AAA259E22 100 825,701 37,800 2,026 8,257 14.4 16.8
AAA259I07 92 643,115 36,324 2,032 6,990 9.6 18.8
AAA259I09 215 1,350,271 42,722 2,011 6,280 56.7 54.5
AAA259I14 121 771,138 37,050 2,016 6,373 19.2 16.2
AAA259J03 148 896,033 45,149 2,003 6,054 29.8 31.4
AAA259M10 127 865,782 48,524 2,053 6,817 12.5 18.8
AAA382A03 88 428,827 17,372 2,040 4,866 14.4 16.8
AAA382A13 72 408,808 19,309 2,004 5,677 18.2 15.7
AAA382A20 111 925,397 41,226 2,010 8,336 36.5 27.7
AAA382C18 74 546,193 38,914 2,036 7,380 12.5 15.7
AAA382F02 34 336,176 29,731 2,226 9,887 25.9 34.0
AAA382K21 35 258,290 18,371 2,067 7,379 8.7 7.9
AAA382M17 63 378,040 25,329 2,007 6,000 32.7 22.5
AAA382N08 58 422,936 37,140 2,024 7,292 7.7 8.4
AAA385D11 35 306,358 26,504 2,012 8,753 8.7 12.6
AAA385M02 16 137,797 36,276 2,118 8,612 1.0 1.0
AAA385M11* 32 215,229 23,432 2,461 6,726 - 0.5
AAA833F18 24 269,215 63,868 2,373 11,246 10.6 9.9
AAA833K04 25 162,840 21,872 2,030 6,513 26.0 15.7
Table 1. Summary statistics of the clean contigs for SAGs described in this study. Contigs that were less
than 2 kb were agged as suspicious and omitted from the analysis. Genome completeness was computed using
104 and 191 conserved marker genes, respectively. *None of the AMPHORA marker genes were detected in this
genome.

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Entner-Doudoro pathway, gluconate dehydratase and KDG aldolase are missing in all the SAGs, which could
be related to the fact this pathway has one less ATP net yield compared to the EM pathway that yields two ATP
molecules. As illustrated in Fig.3, the transported glucose molecules or those emanating from the hydrolysis
of cellulose are probably converted to α -D-glucose 1-phosphate and s α -D-glucose-6-phosphate, eventually
entering the Embden-Meyerhof pathway. e genes involved in glycolysis are summarized in Supplementary
Table S2. Archaeal glyceraldehyde-3-phosphate ferredoxin oxidoreductase was only identied in the genome of
AAA259A05. Usually, the conversion of pyruvate to phosphoenolpyruvate is catalyzed by phosphoenolpyruvate
synthase (EC 2.7.9.2). However, during gluconeogenesis, phosphoenolpyruvate can also be synthesized from
oxaloacetate by phosphoenolpyruvate carboxykinase (EC 4.1.1.32) present in eleven of the SAGs. Similar to the
Figure 2. Maximum likelihood (ML) phylogenetic tree inferred using only nearly complete 16S rRNA
genes from SAGs of Brine Pools from the Red Sea (in red) and comparative sequences from the NCBI
database including MSBL1 sequences from Brine Pools in the Mediterranean (blue). e branches are
scaled in terms of the expected number of substitutions per site. Numbers above branches are support values
from ML (le) and maximum parsimony (MP; right) bootstrapping. e tree was rooted with selected bacterial
sequences.

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haloarchaea, the MSBL1 group does not encode archaeal-type pyruvate carboxylase which catalyses the irrevers-
ible carboxylation of pyruvate to oxaloacetate20.
Pentose metabolism. e oxidative pentose phosphate pathway is lacking in all the SAGs, consistent
with ndings in other archaea. Instead, pentoses are metabolized non-oxidatively by conversion of fructose
6-phosphate (C6) to ribulose 5-phosphate (C5). e four enzymes required in this archaeal pathway (fructose
1,6 bisphosphatase, fructose 16-bisphosphatase, 6-phospho-3-hexuloisomerase and 3-hexulose-6-phosphate
synthase) were identified in ten of the genomes (Supplementary Table S2). Another source of the ribulose
5-phosphate could be ribose sugars via the nucleotide salvage pathway. Ribulose bisphosphate carboxylase
(identied in 14 of the SAGs) is an enzyme known to convert ribulose 1,5-biphosphate to the highly unstable
six-carbon intermediate 3-keto-2-carboxyarabinitol 1,5-bisphosphate, which spontaneously decays to two mole-
cules of glycerate 3-phosphate. is end product is fed into the central metabolic pathway. e ribulose bisphos-
phate carboxylase proteins identied in nine of the SAGs are phylogenetically closely related to the archaeal form
III cluster of RuBisCo proteins, which are able to x CO2 to ribulose bisphosphate21. ese form III RuBisCo
proteins have also been shown to participate in the AMP salvage pathway22. In the genome of AAA259A05,
Glyceladehyde-3P is synthesized from deoxyribose sugars catalysed by ribokinase/phosphopentomutase and
Figure 3. Maximum likelihood (ML) phylogenetic tree inferred from the amino-acid matrix of 10
concatenated archaeal proteins present in eight of the MSBL1 SAGs (in blue) and other archaeal genomes.
e same set of proteins from selected Eukaryota was included as out-group. e branches are scaled in terms
of the expected number of substitutions per site. Numbers above branches are bootstrapping support values
from ML (le) and maximum parsimony (MP; right). e ‘prot’ amino-acid matrix comprised 94 operational
taxonomic units and 1305 characters, 1207 of which were variable and 1159 of which were parsimony-
informative. ML analysis under the LG model yielded a highest log likelihood of -104791.64, whereas the
estimated alpha parameter was 0.95. e bootstrapping converged aer 350 replicates; the average support was
78.52%. MP analysis yielded a best score of 20784 (consistency index 0.32, retention index 0.59) and 14 best
tree(s). e MP bootstrapping average support was 73.25%.

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Figure 4. A putative global metabolism of the MSBL1 based on 32 Single Amplied Genomes. e
gure summarizes glycolysis/gluconeogenesis, autotrophic carbon xation, one-carbon metabolism via the
tetrahydrofolate/tetrahydromethanopterin pathways, sulfur, nitrogen, amino acid degradation and aldehyde
metabolism. Membrane associated proteins, proteins involved in solute or ion transport are anchored in the
membrane and the arrows indicate the ow direction (import, export or symport). Encircled numbers represent
the various enzymes, whereas the color of the tiny balls on the periphery indicate in how many of the SAGs
was the enzyme identied: Grey color 1–5 SAGs, Blue 6–10; Yellow 11–16 SAGs. * denotes not detected. e
enzymes are: (1) phosphoglucomutase; (2) PTS system cellobiose-specic IIA component protein; (3) glucose-6-
phosphate isomerase; (4) 6-phosphofructokinase/Pyrophosphate—fructose 6-phosphate 1-phosphotransferase
protein; (5) fructose 16-bisphosphate aldolase; (6) fructose 16-bisphosphate aldolase-phosphatase protein; (7)
glyceraldehyde-3-phosphate dehydrogenase; (8) tungsten-containing aldehyde ferredoxin oxidoreductase (GAPOR)/
Aldehyde oxidoreductase protein; (9) phosphoglycerate kinase protein; (10) 2,3-bisphosphoglycerate-dependent
phosphoglycerate mutase/2,3-bisphosphoglycerate-dependent phosphoglycerate mutase; (11) enolase; (12)
pyruvate kinase protein; (13) ribulose bisphosphate carboxylase protein; (14*) Formate-tetrahydrofolate ligase
is missing; (15) bifunctional protein FolD; (16) putative thymidylate synthase protein/5-methyltetrahydrofolate-
homocysteine methyltransferase; (17*) methylenetetrahydrofolate reductase; (18) acetyl-CoA synthase (+ Ni,
Fe), carbon monoxide dehydrogenase; corrinoid protein (19) Acetyl-CoA decarbonylase-synthase complex/
Carbon monoxide dehydrogenase; (20) formate dehydrogenase; (21*) tungsten-containing hydrogen
dependent formate dehydrogenase); (22) formylmethanofuran dehydrogenase; (23) formylmethanofuran-
tetrahydromethanopterin formyltransferase; (24) methenyltetrahydromethanopterin cyclohydrolase; (25) coenzyme
F420-dependent N-methenyltetrahydromethanopterin dehydrogenase; (26) methylene-tetrahydromethanopterin
dehydrogenase; (27) 5,10-methylenetetrahydromethanopterin reductase; (28) coenzyme F420 hydrogenase; (29)
tetrahydromethanopterin S-methyltransferase; (30) CoB—CoM heterodisulde reductase; (31) coenzyme F420-
reducing hydrogenase; (32) thiosulfate sulfurtransferase GlpE protein; (33) sulfate adenylyltransferase protein; (34)
adenylylsulfate kinase protein/ Probable adenylyl-sulfate kinase protein; (35) sulfoxide reductase catalytic subunit
YedY protein; (36) sulte oxidase protein/ phosphoadenosine phosphosulfate reductase protein; (37) ferredoxin-
nitrite reductase protein/ sulte reductase ferredoxin 2 protein; (38) periplasmic nitrate reductase protein; (39)
NADH-quinone oxidoreductase. *Formate—tetrahydrofolate ligase is missing. Enzymes involved in amino acid
degradation are labelled as: (ADH) Alcohol dehydrogenase; (OFOR) 2-Oxoacid:ferredoxin oxidoreductase;
(AOR) tungsten-containing aldehyde ferredoxin oxidoreductase; (POR) Pyruvate ferredoxin oxidoreductase;
(VOR) 2-ketoisovalerate ferredoxin oxidoreductase; (IOR) indolepyruvate: ferredoxin oxidoreductase; (KGOR )
2-Oxoglutarate ferredoxin oxidoreductase subunit beta; (ACS) acetyl-CoA synthetase II (NDP forming); (SCS)
archaeal succinyl-CoA synthetase (NDP forming).

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deoxyribose-phosphate aldolase. e enzymes involved in the dierent reactions are listed in Supplementary
Table S2.
Carbon xation. ough the genomes of the individual SAGs are largely incomplete, the complete TCA
cycle was recovered in AAA259A05 and AAA259I09 and the genes involved are listed in Supplementary Table
S2. MSBL1 possesses genes that are typically involved in autotrophic and anaplerotic CO2 xation. e reductive
TCA cycle leads to the xation of two molecules of CO2 and the production of one molecule of acetyl-CoA cat-
alysed by the key enzymes 2-oxoglutarate-ferredoxin oxidoreductase and isocitrate dehydrogenase. ese two
genes were identied in seven SAGs indicating that MSBL1 may have a functional reductive citric acid cycle.
However, ATP-citrate lyase, which catalyses an ATP-dependent cleavage of citrate to oxaloacetate and acetyl-coA
was not detected in any of the SAGs. Instead, two homologs were identied in the genomes AAA261O19 (gene.
AAA261O19_00625C) and AAA261C02 (gene.AAA261C02_00763C) albeit with low similarity value of 32% to
the known enzyme. In the anaplerotic reaction acetyl-CoA is (reversibly) reductively carboxylated to pyruvate
by pyruvate:ferredoxin oxidoreductase (porA, porB, porC were identied in nine genomes) from which all other
central metabolites can be formed or used for gluconeogenesis via a reversal of the EMP pathway. Alternatively,
the enzyme phosphoenolpyruvate carboxylase (present in SAGs AAA382A03_00089C and AAA382N08) is able
to x CO2 by using phosphoenolpyruvate23. Neither pyruvate decarboxylase, which catalyses the decarboxylation
of pyruvic acid to acetaldehyde and carbon dioxide, nor lactate dehydrogenase were detected in any of the SAGs.
A glyoxylate bypass is also probably missing as the two key genes (isocitrate lyase and malate synthase) were
not detected. However, the organisms may import and degrade a variety of organic acids since beta-oxidation
enzymes such as ferredoxin-dependent oxidoreductases are present (Supplementary Table S2). Acquisition of
amino acids and proteins from the surrounding environment is evidenced by the occurrence of binding/transport
proteins for branched chain amino acids as well as oligopeptides (Supplementary Table S2). Beta-oxidation of
the branched chain amino acids uses enzymes that are also involved in the citric acid cycle (Fig.4). e MSBL1
SAGs lack the enzyme acetate kinase, which catalyses the transfer of phosphate from ATP to short chain ali-
phatic acids. However, genes for acetyl-CoA synthetase (which converts acetate to acetyl-CoA) were found in 16
of the SAGs. On the other hand, CO dehydrogenase/acetyl-CoA synthase (Table S2), which participates in the
Wood-Ljungdahl pathway, through which CO2 is xed under anaerobic conditions24, is present. e oxidative
and reductive branches25 of this pathway are present in the MSBL1 indicating that both, one-carbon metabolism
and carbon dioxide/carbon monoxide xation might be possible. e occurrence of the various carbon xation
pathways is summarized in Fig.4 and Supplementary Table S4. Among the autotrophic CO2 xation pathways,
the reductive acetyl-CoA pathway has the lowest energetic costs, requiring probably less than one ATP to pro-
duce pyruvate26. We cannot exclude the possibility that this pathway is used in the oxidative direction to oxidize
acetate as energy substrate. is pathway has a requirement for metals, cofactors, strict anaerobic environment
and substrates with low-reducing potential such as H2 or CO, which restricts the pathway to anoxic niches-such
as the deep-sea brine pools. Many facultative autotrophic archaea oen down-regulate the enzymes that are spe-
cically required for CO2 xation when organic substrates (such as acetate) are available26. Pyruvate formate
lyase (present AAA259B11, AAA259E22 and AAAA259I09) catalyses the reversible conversion of pyruvate and
coenzyme-A into formate and acetyl-CoA. Formate dehydrogenase detected in the SAGs may be involved in
the oxidation of formate to CO2 and donating the electrons to NAD+ (since no cytochromes were detected).
However, formate might also be reversibly incorporated into tetrahydrofolate by formyltetrahydrofolate ligase
(although this is missing in the SAGs) and goes through a series of rearrangements resulting in the formation
of 5-methyl-tetrahydrofolate (Fig.4). e transfer of the methyl group of methyl-THF to carbon monoxide is
mediated by a multi-enzyme complex catalysed by CO-methylating acetyl CoA synthase yielding acetyl-CoA26.
erefore, the acetyl-CoA decarboxylase/synthase complex (ACDS) bidirectionally links the tetrahydromethan-
opterin and tetrahydrofolate pathways with CO2 as the initial substrate (Fig.4). Notably, the pterin-containing
tetrahydromethanopterin and tetrahydrofolate serve as carriers of C1 fragments between formyl and methyl oxi-
dation levels in both anabolic/ catabolic reactions27. Tetrahydromethanopterin may be involved in autotrophy
(Wood-Ljungdhal pathway in some archaea) as well as purine biosynthesis whereas H4-folate could be used in
the biosynthesis of methionine, serine and acetyl-CoA28. None of the core genes usually found in methanogenic
archaea were detected in the MSBL1 SAGs (Supplementary Table S4).
Branched-chain amino acid transporters and permeases, transporters and genes for fatty acid beta-oxidation
pathway were identied (Fig.4, Supplementary Table S2). In the absence of fermentable sugars, these long-chain
fatty acids (LCFA) could serve as an alternative carbon source for the MSBL1 group. e end product of LCFA
biodegradation is acetyl-CoA, which can then be converted to pyruvate by 1.2.7.1, acetate by 2.8.3.1/6.2.1.13,
acetoacetyl-CoA by 2.3.1.9, homocitrate by 2.3.3.14 or 3-carboxy-3-hydroxy-4-methylpentanoate by
2-isopropylmalate synthase (2.3.3.13).
Sulphur metabolism. Sulphate, thiosulfate and sulfonates can be transported into the cell from the sur-
rounding environment via ABC transporters and/or molybdate-tungstate transport system permeases or cysA/
cysA2 proteins (Fig.4, Supplementary Table S2). Assimilatory sulphate reduction occurs through ATP sulfurylase
into adenylylsulfate (APS), which gets further reduced to either sulphite directly through the activity of adeny-
lylsulfate reductase (1.8.99.2), or to form 3-phosphoadenylyl sulfate (PAPS) due to the activity of sulfate adenylyl
transferase. Finally, PAPS is reduced to sulphite by PAPS reductase (Fig.4). erefore, in MSBL1, sulphate reduc-
tion is putatively assimilatory leading to synthesis of cysteine and homocysteine catalysed by cysteine synthase
and cystathionine gamma-synthase, respectively (Supplementary Table S2). In six of the genomes, we detected
a thiosulfate sulfurtransferase GlpE protein, which contains a rhodanese domain. eoretically, the role of this
protein is to transfer sulphur from thiosulfate to cyanide yielding sulphite and thiocyanate. e sulfoxide reduc-
tase catalytic subunit YedY protein, which was identied in AAA259E22 and AAA259I07, is an inner-membrane

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bound protein, which catalyses the reduction of sulfoxide to sulphite (Fig.4). e sulphate reduction mechanism
in MSBL1 probably proceeds in the same manner as in A. fulgidus and sulphate-reducing bacteria where by
the CoB-CoM heterodisulde reductase iron-sulphur subunit A protein transfers electrons via the adenosine
5′ -phosphosulfate reductase (AprAB 1.8.99.2 adenylylsulfate reductase subunit) from the reduced menaquinone
pool in the membrane to activated sulphate (APS, adenosine-5′ -phosphosulfate) forming sulphite. Localization
prediction on the TMHMM server (http://www.cbs.dtu.dk/services/TMHMM-2.0/) shows that these reductases
are located outside the membrane probably. e membrane-associated dsrMKJOP complex essential for sulphur
oxidation as well as dissimilatory sulphite reductase are absent in MSBL1. Finally, ve of the SAGs encode a gene
identied as ferredoxin-nitrite reductase which is a homolog of the F420-dependent sulphite reductase29,30. It has
been hypothesized that this enzyme may be involved in assimilatory nitrite/sulphite reduction31.
Nitrogen metabolism. e SAG AAA259D14 encodes two genes nrtA and nrtD that are essential for nitrate
uptake from the environment. Neither assimilatory nitrate reductases (Nas) nor respiratory nitrate reductase
(Nar) were identied in the genomes. However genes encoding for a periplasmic nitrate reductase (napA) were
identied in four genomes (Supplementary Table S2). It has been proposed that periplasmic nitrate reductase can
participate indirectly in respiration as part of the electron transport chain when coupled to a proton-translocating
enzyme, such as NADH dehydrogenase I (NuoA-N enzyme), reviewed in references32,33. In SAG AAA259E22,
the napA gene is located on the same contig with heterodisulde reductase (hdrABC), tetrathionate reductase
sub-unit B, Coenzyme F420 reducing hydrogenase and succinate dehydrogenase (iron sulfur and avoproteins
subunits). In genome AAA261O19, the gene is located on the same contig as NADH-quinone oxidoreductase
(subunits ACDHIK). e link between nitrate reduction and electron transport is also supported by the occur-
rence of genes such for ferredoxin-nitrite reductase protein, cytochrome c-type protein NrfB, 4Fe-4S ferredoxin
iron-sulfur binding domain protein, electron transfer avoprotein subunit alpha, electron transfer avoprotein
and periplasmic Fe-hydrogenase large subunit proteins (Supplementary Table S2). Other sources of nitrogen
could be nitrile that is converted to ammonia catalysed by a nitrilase (3.5.5.1) or nitroalkene (also called nitro
olen) that is oxidized to nitrite by nitronate monooxygenase (EC: 1.13.12.16), whose orthologues were found
in 15 genomes. Nitrilases act solely on carbon-nitrogen bonds to produce a carboxylate and ammonia. Eight
SAGs encode an anaerobic nitric oxide reductase avorubredoxin that can be used to detoxify nitric oxide using
NADH34.
Energy metabolism. Oxidative phosphorylation in MSBL1 consists of oxidoreductases, membrane bound
hydrogenases and dehydrogenases, NADH-quinone/ubiquinone oxidoreductases, fumarate reductase and an
ATPase complex (Supplementary Table S2). Based on the sub-unit composition of the NADH-quinone/ubiqui-
none oxidoreductase, the potential electron donor is NADH catalyzed by NADH dehydrogenase (found in 9
of the SAGs as shown in Table S2). e NADH dehydrogenase is a avoprotein that contains iron-sulfur cen-
tres. Iron-sulphur binding proteins, oxidoreductases and fumarate reductase possibly contribute to energiz-
ing the cell membrane as well as general intracellular ow of electrons. Several genes encoding for coenzyme
F420 hydrogenase and a putative hydrogenase maturation protease (EC 3.4.23.-) were identied in the SAGs
(Supplementary Table S2). e CoB-CoM heterodisulde reductase iron-sulfur protein (1.12.98.1) is similar to
that of Methanothermobacter fervidus and is involved in sulphate reduction as discussed above. e subunit FrhB
of F420-reducing hydrogenase carries the binding site for the prosthetic groups F420, FAD and a [4Fe-4S] cluster35.
Putative K+-stimulated pyrophosphate-energized sodium pumps are probably involved in oxidative phosphoryl-
ation in the MSBL1.
Transport. Transporters identied in the SAGs include ABC transport systems for branched-chain amino
acid, arginine, ornithine, dipeptide, spermidine/putrescine, sugars and acids as well as for uptake of metal ions
(Supplementary Table S2). ese compounds provide the necessary substrates for numerous biosynthetic and
degradation pathways. Additionally, ion transporters facilitate the ux of the dierent ions into, and also out
of the cells (Fig.4). For example, iron ions are essential for the synthesis of iron-sulphur clusters in the [NiFe]
hydrogenases, formylmethanofuran dehydrogenases, heterodisulde reductase, ferredoxins, and [Fe] hydroge-
nase. Phosphate is probably taken up by a PstABCS and PhoU system as described by Aguena and Spira36. is is
conrmed by the occurrence of the respective genes/proteins involved in regulation and uptake of phosphorous
from the environment. For example, in the SAG AAA259B11, genes for phosphate uptake regulation protein
(PhoU), phosphate binding (PstS), ABC transporter permease protein (YqgH), phosphate Import ATP binding
protein (PstB) and phosphate transport system permease protein PstA are all located on the same contig. Neither
anion permeases nor sodium dependent phosphate transporters were identied in any of the SAGs. In micro-
organisms, molybdate ions are required for the synthesis of the molybdenum-dependent formylmethanofuran
dehydrogenase, formate dehydrogenase and nitrogenases37,38. On the other hand, tungstate ions are required for
the synthesis of the tungsten-dependent formylmethanofuran dehydrogenase39 and their uptake from the envi-
ronment is mediated by a tungsten transport protein (WtpA).
Stress response. Carbon starvation genes (carbon starvation-induced protein A) were detected in the SAGs
AAA261C02 and AAA261O19, AAA833F18 and AAA833K04. is is a predicted membrane protein probably
involved in peptide utilization when carbon becomes limiting40,41. Stress response genes include the small heat
shock protein C4 and a universal stress protein YxiE (14 of the genomes). e universal stress protein UspA
identied in four of the genomes is a small cytoplasmic bacterial protein whose expression is enhanced when
the cell is exposed to stress agents42. Oxidative stress genes in MSBL1 include a putative oxidative stress-related
rubrerythrin protein, putative superoxide reductases, glutaredoxins and thioredoxins. Glutathione and glutare-
doxins are involved in disulphide reductions in the presence of NADPH and glutathione reductase43. Genes for

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resistance to heavy metals as well as antibiotics are listed in Supplementary Table S2. ese include genes for the
resistance to cadmium and arsenate as well as antibiotics such as danorubicin, methicillin, quinolones and tetra-
cycline. e complete archaeal gene cluster for motility is missing in all the genomes though twitching motility
protein PilT occurs in 21 SAGs. On the other hand, genes for pilus assembly are more widespread in the group
(Supplementary Table S1) and could be responsible for secretion and cell-to-cell signalling.
Discussion
MSBL1 have been presumed to be methanogens on the basis of phylogenetic placement or the presence of large
amount of methane in the environments where they have been detected3,9,10,44,45. Phylogeny based on the Silva
aligner places the MSBL1 within the class ermoplasmata16 with identity scores between 83 to 86.9%. Previous
phylogenetic placement of this group was summarized by Antunes et al.5. When shorter clone sequences are
included in the analysis, the MSBL1 lie in the radiation of the uncultured Euryarchaeota group-SAGMEG3,44,46
or other uncultured groups9. However, the low bootstrap values in the phylogenetic trees do not allow for a
clear placement at this point in time. In our analysis, we chose only full-length 16S sequences from the SAGs
and comparative genomes from the NCBI database in order to have consistency between both the phylogenetic
trees using 16S rRNA genes and core proteins. e MSBL1 group has been exclusively reported from hypersa-
line environments. e brine environment for example is one of the most extreme environments and therefore
specically adapted microorganisms probably have evolved mechanisms that enable them to adapt and thrive
under these conditions. e common adaptation mechanisms have been previously described11,47. Ability of the
MSBL1 archaea to import or synthesize osmolytes enables them to maintain intracellular osmotic balance and
hence cope with salt stress in hypersaline environments where they have been reported. is is evidenced by the
presence of transporters for glycine-betaine (Fig.4) and also genes for biosynthesis of trehalose (Supplementary
Figure S3). Furthermore, the slightly acidic proteome signature is associated with organisms employing the “salt
out” strategy in contrast to the extreme halophiles that have a highly acidic proteome (Supplementary Figure
S2) and use the “salt in” strategy47,48. MSBL1’s ability to operate between heterotrophy (sugar fermentation) and
potentially autotrophic CO2 xation (Fig.2) highlights a possibility of a exible mixotrophic lifestyle that might
explain why MSBL1 is the major group reported for example in Lake Medee brine4 as well as in the metagenomic
samples collected from the Atlantis II and Discovery brine pools12. Methanogenesis as we know it cannot occur in
the absence of methyl-coenzyme M reductase as well as the associated cofactor (F430). We were not able to detect
mcrA genes in the genomes nor were we able to amplify mcrA genes from the MDA-DNA that was used to gener-
ate the genome sequences. In addition, none of 15 core genes found in methanogenic archaea49 were detected in
the MSBL1 SAGs (Supplementary Table S4). Moreover, at high salinity methanogenesis from H2 + CO2 or from
acetate, dissimilatory sulphate reduction coupled to the oxidation of acetate, and autotrophic nitrication have
been mentioned as some of the energy-producing reactions that are bioenergetically unfavourable47,50. erefore,
methane encountered in the brines could be from other biochemical processes or is produced by MSBL1 through
a novel pathway independent of the canonical mcrA-associated pathway. For example, low amounts of methane
observed in Archaeoglobus51 and in sulphate-reducing bacteria52 result from transfers of methyl groups by CO
dehydrogenase. It is reported that the methyl group of N5-methyltetrahydromethanopterin can be reduced to
methane and tetrahydromethanopterin by carbon monoxide (CO) dehydrogenase53,54. Assimilatory sulphate /
nitrite reduction in the MSBL1 is catalysed by a ferredoxin-nitrite reductase31. Dissimilatory nitrate reduction
serves to oxidize excess reducing equivalents32 potentially catalysed by the periplasmic nitrate reductase with
electrons from formate dehydrogenase as the electron donor55. Stress response involves a repertoire of genes in
the dierent SAGs (Supplementary Table S2). ese include the universal stress protein UspA42, rubrerythrin (Rr)
also found in anaerobic sulphate-reducing bacteria56 and rubredoxin57. Glutaredoxins and thioredoxins are pro-
teins that act as antioxidants by facilitating the reduction of other proteins by cysteine thiol-disulphide exchange
and therefore play a role in alleviating oxidative stress in MSBL1. e data presented here provide a rst insight
into the metabolism of this enigmatic uncultured archaeal lineage encountered in hypersaline environments. We
are convinced that the metabolic reconstruction and genome sequences here will guide future isolation eorts.
Materials and Methods
Sampling sites and sample preparation. Samples used in this study were collected from the Atlantis II,
Nereus, Erba and Discovery brine pools in the Red Sea (Table2; Fig.1) between 16th and 29th of November 2011
during the 3rd KAUST Red Sea Expedition-Leg 2 on the vessel R/V Aegaeo.
Samples for the single-cell sorting were processed as follows: Small volumes of sample (ca. 30 ml) were col-
lected and divided into two parts. e rst aliquot of the sample was stored unxed whereas the second part
was xed by adding glycerol (nal concentration 10%) and immediately placed at –20 °C. Ten ml of the unxed
sample were transferred into a serum bottle and sent for cell sorting. Big volumes (ca. 480 L) of sample were col-
lected into 20 L carboys, bubbled with nitrogen gas. Concentration was done using a Tangential Flow Filtration
(TFF) system equipped with a 0.1μ m cassette lter and coupled with a 5.0-μ m pre-lter. A 10-ml portion of the
concentrates was transferred to a serum bottle and sent for cell sorting. Cell sorting, lysis, whole genome ampli-
cation and SSU rRNA PCR were performed as described58 at the Bigelow Laboratory Single Cell Genomics
Centre (http://www.bigelow.org). Amplication of the mcrA gene from the MDA reactions was done as published
previously59.
Sequencing, assembly, annotation. e SAG DNA was cleaned in preparation for sequencing using the
ethanol/sodium acetate precipitation method and re-suspended in 25 μ L of MilliQ water. Quantication of the
DNA was performed using Quant-iT dsDNA HS assay kit and a Qubit uorometer (Invitrogen GmbH, Karlsruhe,
Germany) as recommended by the manufacturer. Sequencing was done at the Bioscience core facility, King
Abdullah University of Science and Technology on an Illumina HiSeq 2000 platform. Assemblies of the single-cell

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amplied genomes (SAGs) were generated using a pipeline that employs assemblers designed for single-cell
sequencing data including VelvetSC60, Spades61, and IDBA-UD62, along with several pre and post assembly data
quality checks using Trimmomatic63. In our benchmarking tests, IDBA-UD showed better contig-level assemblies
and the assemble contigs were used in further analysis. Aer quality control (described below), genome anno-
tation for each of the SAGs was carried out as described in Alam et al.64. Briey, given a set of DNA sequences
from particular SAG, the Automatic Annotation of Microbial Genomes (AAMG) pipeline rst detects rRNA and
tRNA. To avoid prediction of Open Reading Frames (ORFs) in RNA detected regions, all DNA regions detected
with RNA are masked, followed by ORF predictions using Prodigal65 and MetaGeneAnnotator66. Aer ORFs pre-
diction is complete, a series of similarity searches are performed to select optimal gene annotations using UniProt,
NCBI’s NR, NCBI’s Conserved Domain Database (CDD), KEGG database and nally Interproscan. All annota-
tions, including DNA and ORF sequences are then stored in an integrative data-warehouse of microbial genomes
(INDIGO, see methods in reference 64)for easy look up. As none of the SAGs represent complete genomes, the
metabolic reconstruction in Fig.4 represents our current working hypothesis for the metabolism of MSBL1.
Quality control. Our approach towards decontaminating the dra assembly of SAGs was simple and some
of the genomic features like GC content, size, gene content and tetranucleotide frequency (TNF)67 of the contigs
were exploited. ese lters were kept independent and a contig had to pass all lters in order to be put into the
clean bin. A contig with %GC content lying outside + /− 10% range around the average was marked as potentially
contaminated68. e calculation of the average G + C% for any dra assembly might be spurious and mislead-
ing if the assembly contains a lot of contaminations. We observed some real contigs (passing size, gene content
and all lters) ending in the contamination bin just due to having slightly lower or higher G + C% around the
range. is problem was overcome in two dierent ways. Firstly, if the analysis was done on a single SAG, the
average GC content was calculated on the set of large contigs or the contigs constituting more than y per cent
SAGID Sampling Site Layer Temp. (°C)
Depth
(M) Salinity (%) pH
Coordinates (La.t, N/
Long., E)
AAA833F18 Atlantis II Deep Upper convective
layer 1 ≈ 54 2048 15.2 5.6 21°/38°
AAA833K04 Atlantis II Deep Upper convective
layer 1 ≈ 54 2048 15.2 5.6 21°/38°
AAA259A05 Discovery Brine 44.8 2141 26.2 6.2 21° 16.98′ /38° 03.18′
AAA259B11 Discovery Brine 44.8 2141 26.2 6.2 21° 16.98′ /38° 03.18′
AAA259D14 Discovery Brine 44.8 2141 26.2 6.2 21° 16.98′ /38° 03.18′
AAA259D18 Discovery Brine 44.8 2141 26.2 6.2 21° 16.98′ /38° 03.18′
AAA259E17 Discovery Brine 44.8 2141 26.2 6.2 21° 16.98′ /38° 03.18′
AAA259E19 Discovery Brine 44.8 2141 26.2 6.2 21° 16.98′ /38° 03.18′
AAA259E22 Discovery Brine 44.8 2141 26.2 6.2 21° 16.98′ /38° 03.18′
AAA259I07 Discovery Brine 44.8 2141 26.2 6.2 21° 16.98′ /38° 03.18′
AAA259I09 Discovery Brine 44.8 2141 26.2 6.2 21° 16.98′ /38° 03.18′
AAA259I14 Discovery Brine 44.8 2141 26.2 6.2 21° 16.98′ /38° 03.18′
AAA259J03 Discovery Brine 44.8 2141 26.2 6.2 21° 16.98′ /38° 03.18′
AAA259M10 Discovery Brine 44.8 2141 26.2 6.2 21° 16.98′ /38° 03.18′
AAA259O05 Discovery Brine 44.8 2141 26.2 6.2 21° 16.98′ /38° 03.18′
AAA261C02 Atlantis II Deep Brine -Interphase 57–63 2036 15.1–16.8 5.6 21°/38°
AAA261D19 Atlantis II Deep Brine -Interphase 57–63 2036 15.1–16.8 5.6 21°/38°
AAA261F17 Atlantis II Deep Brine -Interphase 57–63 2036 15.1–16.8 5.6 21°/38°
AAA261F19 Atlantis II Deep Brine -Interphase 57–63 2036 15.1–16.8 5.6 21°/38°
AAA261G05 Atlantis II Deep Brine -Interphase 57–63 2036 15.1–16.8 5.6 21°/38°
AAA261O19 Atlantis II Deep Brine -Interphase 57–63 2036 15.1–16.8 5.6 21°/38°
AAA382A03 Nereus Nereus Brine 30.1 2445 22.4 5.5 23° 11.53′ /37° 25.09′
AAA382A13 Nereus Nereus Brine 30.1 2445 22.4 5.5 23° 11.53′ /37° 25.09′
AAA382A20 Nereus Nereus Brine 30.1 2445 22.4 5.5 23° 11.53′ /37° 25.09′
AAA382C18 Nereus Nereus Brine 30.1 2445 22.4 5.5 23° 11.53′ /37° 25.09′
AAA382F02 Nereus Nereus Brine 30.1 2445 22.4 5.5 23° 11.53′ /37° 25.09′
AAA382K21 Nereus Nereus Brine 30.1 2445 22.4 5.5 23° 11.53′ /37° 25.09′
AAA382M17 Nereus Nereus Brine 30.1 2445 22.4 5.5 23° 11.53′ /37° 25.09′
AAA382N08 Nereus Nereus Brine 30.1 2445 22.4 5.5 23° 11.53′ /37° 25.09′
AAA385D11 Erba Erba Brine 28.2–28.5 2392 18.1 7.15 20° 43.80′ /38° 10.98′
AAA385M02 Erba Erba Brine 28.2–28.5 2392 18.1 7.15 20° 43.80′ /38° 10.98′
AAA385M11 Erba Erba Brine 28.2–28.5 2392 18.1 7.15 20° 43.80′ /38° 10.98′
Table 2. Metadata and geographical coordinates of sampled stations. Physicochemical parameters recorded
on site were depth, temperature, salinity and pH.

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of the dra assembly (i.e N50 contigs). e second solution was applied when the analysis contained a group of
SAGs belonging to same taxa and they needed to be cleaned in concert. In this case the single copy conserved
genes were identied using Bio-Hal pipeline (PMID:21327165) for each of them and pooling their corresponding
contigs to constitute a set (named “seed contigs”) on which G + C% was calculated. is single value of G + C
content calculated on “seed contigs” could be applied for cleaning all SAGs of this group. Alternatively, the seed
contigs could be separated for each SAG and G + C% calculated on dierent set. Size lter was relatively simple
and cut-o could be xed using the contig statistics of the dra assembly. For this cleaning the size threshold was
2000 bp i.e any contig below 2kb was discarded68. Nonetheless, a manual inspection of the contigs discarded just
due to size lter is always advised. e smaller contigs (500 bp < x < 2000bp) might also contain some impor-
tant gene. If majority (50% or more) of the genes in a scaold/contig hits to the non-target phylum, that contig
was discarded68. Binning of contigs was done at domain level either bacteria or archaea. Once the binning was
complete based on the above three lters the clean bin and all bin were subjected to Canonical Correspondence
Analysis (CCA) using TNF of the sequences and the contigs visualized on the plots67. Canonical Correspondence
Analysis was done in R using Vegan package (https://cran.r-project.org/web/packages/vegan/index.html), while
plotting was done in R using custom scripts developed in our group. e plot showing all contigs of the assem-
bled genome gave a clear idea of the level of contamination in terms of phylogeny and G + C content proles of
contigs. e subsequent plot using only the clean contigs was much clearer and helped in nding out the few false
positives, which were very dispersed but passed all the above three lters to be in clean bin. Manual inspection of
such contigs was done to decide whether keep or discard them. e number of multiple single-copy conserved
genes in the genome is a very important indicator of the contamination or might represent spurious assembly. To
check the distribution of conserved cluster of orthologous groups (COGs) in the genome (to have an idea about
“genome completeness”), we used dierent COG set for bacteria and archaea (adopted from Human Microbiome
Project; R package Vegan was used (https://cran.r-project.org/web/packages/vegan/index.html). We observed in
our single cell genomes data multiple copies of conserved genes could belong to multiple contigs. In most cases,
the contig with largest size and more genes content was retained as part of the genome. Altogether, the QC pipe-
line takes care of the contamination present in the dra single cell genome using various genomic features both
sequence-dependent and independent.
Evolutionary relationships. e SAGs and the representative genomes were scanned for common marker
genes (CMG) using the phylogenomic inference tool AMPHORA214 along with its set of predened marker
genes. Identied marker genes were concatenated in the same order across all the samples and saved in multi-fasta
format with headers being the sample names. e concatenated sequence in multi-fasta was then aligned using
Muscle69 (reference) with default settings. Simple gblocks tool70-with default settings – was used to remove any
ambiguous bases from the Muscle alignment. Phylogenetic trees were inferred from either trimmed alignments of
nucleotide sequences (16S tree) or the amino-acid (protein tree) alignments of ten concatenated proteins present
in all the genomes included in the analysis using the pthreads-parallelized RAxML71 version 7.2.8. e ten concat-
enated proteins are: translation initiation factor IF-2 (infB); 50S ribosomal proteins rpl18p, rpl19e, rpl32e, rpl5p,
rpl6p, rpl7ae, and 30S ribosomal proteins rps28e, rps6e and rps8p. Fast bootstrapping was applied with subse-
quent search for the best tree72, the autoMRE bootstopping criterion73 and the LG model of amino acid evolu-
tion74 (with which these data yielded the highest log likelihood among all empirical protein models implemented
in RAxML) in conjunction with gamma-distributed substitution rates75 and empirical amino acid frequencies.
Tree searches under the maximum parsimony (MP) criterion were conducted with PAUP* version 4b1076 using
100 rounds of random sequence addition MP bootstrap support was calculated with PAUP* using 1000 replicates
with ten rounds of heuristic search per replicate. e 16S rRNA gene datasets were analyzed in the same way, but
using GTR as substitution model.
pI estimation. In addition to the predicted protein-coding genes of our SAGs, we extracted the
proteins-coding coding genes from GenBank files from the NCBI of exemplary extreme halophiles
(Halobacterium sp. NRC1 and Salinibacter ruber), moderate halophiles (Chromohalobacter salexigens, Idomarina
loihiensis L2TR, and Nitrosococcus halophilus Nc4), as well those from typical marine bacterioplankton
(Pelagibacter ubique HTCC1062, Pelagibacter sp. HTCC 7211, Nitrosopumilus maritimus SCM1, and Nitrococcus
mobilis). e isoelectric points (pIs) of these proteomes were calculated using the “iep” script in the EMBOSS
soware package (v6.5.1; http://emboss.sourceforge.net/what/) using the following settings: -amino 1 -termini
YES –step 0.2.
Data deposition. This Whole Genome Shotgun project has been deposited at DDBJ/EMBL/
GenBank under the Bioproject PRJNA291812. The accession numbers for the Individual SAGS are
LHXJ00000000-LHYO00000000.
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Acknowledgements
is study was supported by King Abdullah University of Science and Technology (KAUST) though the baseline
grant to US as well as through the SEDCO Research Excellence Award and the SABIC Chair donation to US. We
thank the KAUST Core Facility for Bioscience for sequencing services and the KAUST Core Facility for Marine
Research for help in sampling. We also want to thank Dr. Andre Antunes for guidance in sampling and initiation
of the study.
Author Contributions
U.S. initiated the study and the S.A.G. sequencing. R.M. was responsible for the metabolic reconstruction
and wrote the rst dra the manuscript. I.A. led the bioinformatics team. M.R. was responsible for the de-
contamination of the SAGs. M.V. assisted in all bioinformatics approaches. W.B.A. was responsible for the
rst assemblies and annotation eorts. A.A.K. was responsible for Indigo-related issues. D.K.N. assisted with
phylogenetic analyses. M.G. was responsible for phylogenetic trees. H.P.K. had oversight over the phylogenetic
analyses. V.B. had oversight over most bioinformatics approaches, except phylogenies, and Indigo. All authors
reviewed and approved the manuscript.

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Scientific RepoRts | 6:19181 | DOI: 10.1038/srep19181
Additional Information
Supplementary information accompanies this paper at http://www.nature.com/srep
Competing nancial interests: e authors declare no competing nancial interests.
How to cite this article: Mwirichia, R. et al. Metabolic traits of an uncultured archaeal lineage -MSBL1- from
brine pools of the Red Sea. Sci. Rep. 6, 19181; doi: 10.1038/srep19181 (2016).
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