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Eight Metagenome-Assembled Genomes Provide Evidence for
Microbial Adaptation in 20,000- to 1,000,000-Year-Old Siberian
Permafrost
Katie Sipes,
a
Abraham Almatari,
a
Alexander Eddie,
a
Daniel Williams,
a
Elena Spirina,
b
Elizaveta Rivkina,
b
Renxing Liang,
c
Tullis C. Onstott,
c
Tatiana A. Vishnivetskaya,
a,b
Karen G. Lloyd
a
a
University of Tennessee, Knoxville, Tennessee, USA
b
Institute of Physicochemical and Biological Problems of Soil Science, Pushchino, Russia
c
Princeton University, Department of Geosciences, Princeton, New Jersey, USA
ABSTRACT Permafrost microbes may be metabolically active in microscopic layers of liq-
uid brines, even in ancient soil. Metagenomics can help discern whether permafrost
microbes show adaptations to this environment. Thirty-three metagenome-assembled
genomes (MAGs) were obtained from six depths (3.5 m to 20 m) of freshly cored perma-
frost from the Siberian Kolyma-Indigirka Lowland region. These soils have been continu-
ously frozen for ;20,000 to 1,000,000 years. Eight of these MAGs were $80% complete
with ,10% contamination and were taxonomically identified as Aminicenantes,Atribacteria,
Chloroflexi,andActinobacteria within bacteria and Thermoprofundales within archaea. MAGs
from these taxa have been obtained previously from nonpermafrost environments and
have been suggested to show adaptations to long-term energy starvation, but they have
never been explored in ancient permafrost. The permafrost MAGs had greater proportions
in the Clusters of Orthologous Groups (COGs) categories of energy production and conver-
sion and carbohydrate transport and metabolism than did their nonpermafrost counter-
parts. They also contained genes for trehalose synthesis, thymine metabolism, mevalonate
biosynthesis, and cellulose degradation, which were less prevalent in nonpermafrost
genomes. Many of these genes are involved in membrane stabilization and osmotic stress
responses, consistent with adaptation to the anoxic, high-ionic-strength, cold environments
of permafrost brine films. Our results suggest that this ancient permafrost contains DNA of
high enough quality to assemble MAGs from microorganisms with adaptations to survive
long-term freezing in this extreme environment.
IMPORTANCE Permafrost around the world is thawing rapidly. Many scientists from a
variety of disciplines have shown the importance of understanding what will happen
to our ecosystem, commerce, and climate when permafrost thaws. The fate of perma-
frost microorganisms is connected to these predicted rapid environmental changes.
Studying ancient permafrost with culture-independent techniques can give a glimpse
into how these microorganisms function under these extreme low-temperature and
low-energy conditions. This will facilitate understanding how they will change with
the environment. This study presents genomic data from this unique environment
;20,000 to 1,000,000 years of age.
KEYWORDS permafrost, MAGs, exobiology, environmental, bioinformatics
Arctic soils are often studied by sampling the upper, seasonally thawed, active layer (1).
Climate change is rapidly increasing the depths of permafrost active layers worldwide
(2), making it necessary to learn more about the microbes living in the deeply buried per-
mafrost before it thaws completely. Evidence for microbes persisting in ancient permafrost
has come in the form of revivable cultured isolates, microscopic visualization of intact cells,
Citation Sipes K, Almatari A, Eddie A, Williams
D, Spirina E, Rivkina E, Liang R, Onstott TC,
Vishnivetskaya TA, Lloyd KG. 2021. Eight
metagenome-assembled genomes provide
evidence for microbial adaptation in 20,000- to
1,000,000-year-old Siberian permafrost. Appl
Environ Microbiol 87:e00972-21. https://doi
.org/10.1128/AEM.00972-21.
Editor Robert M. Kelly, North Carolina State
University
Copyright © 2021 American Society for
Microbiology. All Rights Reserved.
Address correspondence to Karen G. Lloyd,
klloyd@utk.edu.
Received 18 May 2021
Accepted 11 July 2021
Accepted manuscript posted online
21 July 2021
Published
October 2021 Volume 87 Issue 19 e00972-21 Applied and Environmental Microbiology aem.asm.org 1
ENVIRONMENTAL MICROBIOLOGY
10 September 2021
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live/dead cell staining, low D/L-amino acid ratios in cellular biomass, high-quality metage-
nomic reconstructions of microbial genomes, and the presence of high-quality RNA mole-
cules (3–8). Evidence from other studies of 33,000-year-old permafrost in Alaska suggests
that microbial communities are equipped to survive in this type of environment by using
unique energy acquisition mechanisms and stress responses such as scavenging detrital
biomass and environmental sensing (6). Variations in metagenomic functions of the in situ
microbial populations in 30,000-year-old permafrost in the Kolyma-Indigirka Lowland
region in northeastern Siberia corresponded to local geochemistry, suggesting that the
populations were alive (8). Additionally, a study of .500,000-year-old permafrost samples
from northeastern Russia contained microbial DNA with likely DNA repair mechanisms (9).
Thus, some microbes in ancient permafrost may not be dormant (7, 10, 11) but instead
maintain a continuous torpor-like state that allows them to subsist in the low-energy
environment.
The Yedoma geological suite in the Kolyma-Indigirka Lowland region in northeast-
ern Siberia originates from the late Pleistocene Era (12). Yedoma, or Ice Complex, per-
mafrost deposits were frozen syngenetically (deposited and frozen simultaneously)
;20,000 to ;60,000 years ago (13). Yedoma spans approximately 1 million km
2
(14)
and is found, on average, up to 10 m below the surface (13, 15). The next layer down,
called Olyor, is unlike Yedoma since sediments were frozen epigenetically (deposited
and then frozen) after being deposited ;0.6 million to 1.6 million years ago (15, 16)
and they contain few ice formations (17). Some of the perennially frozen soils of this
region have been dated as up to 3 million years old (18, 19). Although the permafrost
of the Kolyma area is of freshwater origin (12), other studies showed that, during freez-
ing, small solutes were occluded from the surrounding mineral material, creating small
brine films (5).
To live in the permanently frozen soils underneath the active layer without seasonal
nutrient replenishment, a microbe must be able to sustain cellular integrity with a lim-
ited source of liquid water (18). Liquid water exists in permafrost in the form of cryo-
pegs or small brine films (5), which are hypersaline liquid water films and lenses that
can occur throughout the permafrost at temperatures of 210°C and salt contents of
140 to 300 g/liter (18). These saline fluids can be in the shape of flattened films along
sediment grains or in small pockets, depending on depositional characteristics (20).
While it is thought that the presence of liquid water in these brine films is sufficient
to maintain live microbes within the thermostable deep permafrost, the interactions
between microbial cells and the brine film have not been visualized at a microscopic
level (21). However, plump cells with unique membrane adaptations have been visual-
ized with electron microscopy in 200,000-year-old permafrost samples (22). Additionally,
cells visualized from ice core and permafrost samples are notably very small (,1-
m
mdi-
ameter) (23, 24) but not small enough to be completely immersed in the brine vein (18).
Since these brine films are so small, ;1 nm wide (25), the cell membrane would have to
make contact with the brine in order to act as a conduit for small molecule transport.
Necessary microbial metabolic processes could occur in permafrost as long as physical
mass transfer can occur in the environment (26). Since the cell’s internal environment
remains unfrozen (27), the cell could transfer small metabolites (26) between the intra-
cellular matrix and the brine films.
Many organisms have been able to be cultured from Kolyma-Indigirka Lowland perma-
frost, including representatives from the Carnobacterium genus (28), Gammaproteobacteria,
Actinobacteria,andFirmicutes (29). Similar groups have been detected from 40,000-to
50,000-year-old permafrost (30). Microbes have also been isolated from Siberian permafrost
in high salt concentrations (0.5% to 15% NaCl) (18, 31, 32) and with various medium types
(33),suggestingthatthemicrobescouldsurviveinbrines.Culturework,however,doesnot
assess the entire microbial community, since many organisms have not been cultured, de-
spite many attempts (34).
To supplement culturing studies, we used metagenomes to study the microbial
populations in ;20,000- to 1,000,000-year-old permafrost formed near the freshwater
Sipes et al. Applied and Environmental Microbiology
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Alazeya River in the Kolyma-Indigirka Lowland region in northeastern Siberia. We
determined the geological age of the upper Yedoma suite by
14
C dating. The deeper
soil age was determined to be 650,000 to 1,000,000 years in previous studies (12, 14,
16, 18, 35). Microbial communities, largely Firmicutes and Proteobacteria, have been
identified in permafrost in this location by 16S rRNA gene analysis and are likely alive
since they have substantial amounts of intracellular DNA (4). In this study, we used
metagenomes to determine whether microbes in these ancient sediments (i) produce
metagenome-assembled genomes (MAGs), which suggest the presence of high-quality
nucleic matter, and (ii) contain genes that suggest adaptation to long-term subsistence
under permafrost environmental conditions. We identified 8 MAGs with $80% ge-
nome completeness and #10% contamination from five taxonomic groups. A unique
set of functional genes were present in permafrost MAGs but absent in publicly avail-
able genomes of the same taxonomic groups in nonpermafrost environments (referred
to as nonpermafrost genomes). These ancient permafrost MAGs give preliminary
insights into the survival capabilities of these microbes in 20,000- to 1,000,000-year-old
permafrost.
RESULTS
Site characteristics. A 20-m-deep permafrost core (AL3-15) from the Kolyma-Indigirka
Lowland region (69.339600, 154.996450) was subsectioned for microbiology and geology
immediately upon retrieval in the field (Fig. 1A). The permafrost geology enters a transition
zone at about 6.2 m, where it changes from the Yedoma geological suite to Olyor at
7.7 m. The temperature inside the borehole decreased from 22°C 1 m below the surface
to 28.7°C5mbelowthesurfaceandstayedinaconstantrangeof28°C to 28.6°C in
the lower 10 m of the borehole (see Fig. S1 in the supplemental material). The
d
13
Cofthe
inorganic carbon fraction (23.4 to 26%) was more
13
C enriched than the organic carbon
(228.8 to 224.3%) at 2.9, 3.5, and 5.6 m (see Table S1). The
14
C ages of the inorganic car-
bon increased from 21,760 6120 years to 33,900 6550 years with stratigraphic depth
over the upper 5.6 m of the Yedoma suite. However, the
14
C ages of the two organic car-
bon pools showed no stratigraphic trend, with great variability at different depths (see
Table S1). The large difference between 2.9 m (38,590 6980 to 41,700 61,400 years) and
3.5 m (18,228 678 to 20,158 699 years) suggested that the ice-rich layer at 3.5 m might
be an ice wedge that was much younger than the surrounding strata. The underlying sedi-
ments of the Olyor suite were too old for radiocarbon dating. With other methods such as
fossil and palynology records, however, these soils were previously dated as between
650,000 and 1,000,000 years old (12, 14, 16, 18, 35). The porewater extracted from thawed
FIG 1 Downcore permafrost borehole characteristics. (A) Photos of Yedoma and Olyor sediments and their relative depths, with
triangles indicating depths that produced MAGs and filled triangles indicating depths with MAGs that were .80% complete and
,10% contaminated. Concentrations of major ions are shown for each depth. Note that the depth intervals are discrete in panel B to
spread the data points evenly across the depths. (B) Concentrations of methane for each depth. Other geochemical measurements,
including temperature, pH, and total carbon, are available in Fig. S1 in the supplemental material. The small dashed line at 7.7 m
shows the depth of the transition between the Yedoma and Olyor layers.
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subsamples of permafrost showed fluctuations in dissolved ions downcore but they gener-
ally increased with depth (Fig. 1A). Chloride had the greatest increase down the core, rang-
ing from 1 mmol/100 g of soil at 3.5 m to 2.7 mmol/100 g of soil at 10.2 m. No chloride
measurements were made below this depth. Methane increased from 1.2 to 17.6 mmol/
100 g of soil and sulfate increased from 0.019 to 2.5mmol/100 g of soil at depths of 3 m
to 17.2 m, respectively (Fig. 1B). Methane levels were high only in the older Olyor deposits
below about 13 m. The pH stayed in a constant range of 8 to 8.5 throughout the core (see
Fig. S1). Cells were visualized with 49,6-diamidino-2-phenylindole (DAPI) and SYBR gold
staining (see Fig. S2A) but were below the quantification limit (36) for direct microscopic
cell counts. Further evidence of intact cells comes from flow cytometry, which showed a
population of similarly sized particles that stained with SYBR gold (see Fig. S2B and C).
None of the 10 sequenced single-cell amplified genomes (SAGs) had high enough quality
for use in this study (0% to 45% completeness). The only Genome Taxonomy Database-
Toolkit (GTDB-Tk)-classifiable SAG was a Firmicutes organism (45% complete, with 5% con-
tamination) from 7.2 m that matched the taxonomy of MAG_07_7.2m (which was not ana-
lyzed in this study due to its poor quality but can be found under BioProject accession
number PRJNA596250).
Metagenomes. Of the seven depths from which DNA was extracted (3.5, 7.2, 14.1,
14.8, 16.6, 17.2, and 20 m), six samples produced metagenomic assemblies (Table 1). The
metagenome from 17.2 m produced only ;1 MB of data that did not assemble; there-
fore, it was not included in the analysis. The numbers of reads, numbers of assembled
contigs, mean read lengths, N
50
values, and total numbers of MAGs did not trend with
the sample depth or the total amount of retrieved data (Table 1). Every assembled meta-
genome had at least 91% of the contigs incorporated into the MAGs, suggesting that
the MAGs represented most of the microbial community that was assembled (Table 1).
The 6 metagenomes produced a total of 33 MAGs (see Table S2), 8 of which were $80%
complete with ,10% contamination (see Fig. S2). GTDB-Tk and a maximum likelihood
tree with 139 conserved proteins placed these MAGs into the groups Atribacteria (n=3),
Chloroflexi (n=1), Aminicenantes (n=1), Actinobacteria (n= 1), and Thermoprofundales
(n= 2) among a total of 230 publicly available reference genomes (Fig. 2). A search
through the National Center for Biotechnology Information (NCBI) and Joint Genome
Institute (JGI) Integrated Microbial Genomes (IMG) public databases resulted in no MAGs
from these groups from other ancient permafrost studies. The remaining 25 Siberian
MAGs did not meet the quality standards for inclusion in our analysis; these were classified
as Acidobacteriota (n=1),Actinobacteriota (n=9),Aminicenantes (n=3),Atribacterota (n=2),
Chloroflexota (n=2),Crenarchaeota (n=1),Bathyarchaeota (n=1),Firmicutes (n=2),
Planctomycetota (n=3),andThermoplasmatota (n=1)(seeTableS2).
Description of major taxonomic groups. (i) Thermoprofundales.Two MAGs, 1
from 7.2 m (96% complete, with 6% contamination) and 1 from 14.8 m (94% complete,
with 2% contamination) grouped with Thermoprofundales genomes (Fig. 2; also see Fig. S2).
Thermoprofundales is an order (37) of archaea within the phylum Euryarchaeota and the
class Thermoplasmata (see Table S2). Thermoplasmata has been redesignated as the phy-
lum Thermoplasmatota,basedonGTDBtaxonomy(38),andtheclassIzemarchaea (39). It
was previously called DHVEG, which stands for deep-sea hydrothermal vent euryarchaeotal
TABLE 1 Metagenome details for the seven sampling depths
Sample depth (m)
Total no. of
unassembled reads
Total no. of
assembled reads
Total no. of
assembled contigs N
50
(bp)
Total no.
of MAGs
% of contigs
binned
3.5 14,187,214 13,195,548 115 2,842 1 91
7.2 9,621,340 9,045,678 7,556 4,323 8 94
14.1 11,513,548 10,956,796 4,513 4,527 7 93
14.8 13,544,854 12,728,740 7,098 4,625 11 93
16.6 5,698,300 5,115,016 900 2,871 2 99
17.2
a
420 NA NA NA 0 0
20 13,338,036 11,783,820 3,058 4,353 4 96
a
The sample depth of 17.2 m did not yield enough data to curate a metagenome. NA, not applicable.
Sipes et al. Applied and Environmental Microbiology
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FIG 2 Phylogenetic tree of Siberian MAGs. A maximum likelihood phylogenetic tree of 139 concatenated conserved genes for 8
Siberian MAGs and 230 total reference MAGs and genomes that were used in the comparative analysis is shown. No reference MAGs or
genomes from these groups were available from permafrost. The tree was visualized with iTOL. A full list of shared genes can be found
in Supplemental Data File S6 in the supplemental material.
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group (40), and marine benthic group D (41). Thermoprofundales are uncultured archaea
that are predominantly found in marine subseafloor seeps worldwide (37, 41–43) and were
recently detected in anoxic freshwater sediments (44). All 12,042 of the Thermoprofundales
16S rRNA gene sequences in the SILVA r138 database were from anoxic marine sediments.
To our knowledge, only 9 Thermoprofundales genomes are publicly available (see
Supplemental Data File S1), originating from sediment from the White Oak River estuary in
North Carolina (5 MAGs) and the Aarhus Bay in Denmark (4 SAGs).
(ii) Atribacteria.Atribacteria MAGs were retrieved from 7.2 m (90% complete, with
0% contamination), 14.1 m (81% complete, with 3% contamination), and 14.8 m (89%
complete, with 0% contamination) (Fig. 2; also see Fig. S2). The phylum Atribacteria
was previously called JS1/OP10 (45) and has been proposed to be renamed through
GTDB taxonomy as Caldatribacteriota (38). The 66 nonpermafrost Atribacteria MAGs
(see Supplemental Data File S2) originated across a variety of locations, including ma-
rine sediment from Aarhus Bay, Denmark; Great Boiling Springs, Nevada; soil from an
acetate-fed aquifer in Rifle, Colorado; Green River sediment in Utah; Sakinaw Lake in
Canada; a terephthalate-degrading reactor biofilm in Mexico; Etoliko Lagoon in Greece;
and sediment from Lake Baikal in Russia. The 455 Atribacteria 16S rRNA genes in the
SILVA r138 database are typically from temperate soil, deep marine sediment, and bio-
reactors. To our knowledge, neither 16S rRNA genes nor MAGs from this group from
permafrost environments have been reported previously.
(iii) Chloroflexi.One MAG from 20 m (81% complete, with 10% contamination)
grouped within the Chloroflexi order Anaerolineales (Fig. 2; also see Fig. S2). Anaerolineales
is abundant in many different types of environments; a meta-analysis of 1,504 metage-
nomes showed that Chloroflexi DNA sequences were in the top 10 most abundant groups
in metagenomes from marine sediment, host-associated environments, hypersaline envi-
ronments, freshwater, hot springs, and terrestrial subsurface (34). Forty-two nonpermafrost
Chloroflexi genomes (see Supplemental Data File S3) were chosen to represent the phylo-
genetic and environmental diversity available among the nonpermafrost genomes, from
deep Pacific Ocean basalt-hosted subsurface hydrothermal fluid; White Oak River estuary
sediment; Utah Grand County groundwater; a biological phosphorous bioreactor; marine
samples from the TARA04 Ocean Project; groundwater from an acetate-fed aquifer in Rifle,
Colorado; freshwater from Green River, Utah; sediment from Australia; and marine aquatic
samples from unreported locations.
(iv) Actinobacteria.One MAG (MAG_01_20m; 96% complete, with 8% contamina-
tion) was in the Actinobacteria phylum, with the family Dermatophilaceae and genus
Cutibacterium, which is commonly cultured from human skin (46). A complete nuclease
gene from this MAG had 100% similarity to Cutibacterium acnes in a BLASTN search, and
the entire MAG had 99.72% average nucleotide identity (ANI) (47) to Propionibacterium,
Cutibacterium sp. strain KPL2009, from the Human Genome Project (46). The other
Siberian permafrost Actinobacteria MAGs were not closely related to the skin microbe
(see Table S2) but, since they had lower completeness and higher contamination levels,
their phylogenetic relatedness could not be accurately determined.
(v) Aminicenantes.One MAG (MAG_02_14.8m) with 94% completeness and 3%
contamination came from the 14.8-m sample and grouped with Aminicenantes genomes
on a maximum likelihood tree based on concatenated conserved genes (Fig. 2; also see
Fig. S2). Aminicenantes was previously called OP8 and has been suggested to be in the
Acidobacteriota phylum based on the GTDB reclassification (45). The Aminicenantes MAG
was compared to 54 nonpermafrost Aminicenantes genomes (see Supplemental Data File
S5), chosen so that each available study site was represented, i.e., Etoliko Lagoon in
Greece; Sakinaw Lake in Canada; soil in Rifle, Colorado; hydrothermal fluid from the Juan
de Fuca Ridge flank in the Pacific Ocean; and marine sediment from Baltimore, Maryland.
Aminicenantes has been found in a variety of marine and terrestrial environments but
only up to 10.2% maximum relative abundance (48). Aminicenantes represented a
slightly higher percentage of mapped reads in most of our permafrost metagenomes
(3.5 m, 12.77%; 7.2 m, 15.77%; 14.1 m, 16.81%; 14.8 m, 14.7%; 16.6 m, 5.6%; 20 m, 0.14%)
(see Fig. 5; also see Supplemental Data File S7). This suggests that Aminicenantes may be
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at higher relative abundance in ancient permafrost, or the discrepancy may be due to
primer bias (49) during PCR amplification in the published studies.
Comparison of Siberian MAGs and nonpermafrost genomes. Genes that were
present in Siberian MAGs that were rare or absent in the nonpermafrost MAGs/SAGs
included 21 major Clusters of Orthologous Groups (COG) categories (Fig. 3A), excluding COG
categories chromatin structure and dynamics, RNA processing, secondary metabolites, and cy-
toskeleton. The COG categories with the largest numbers of genes unique to the Siberian
MAGs were carbohydrate metabolism and transport, energy production and conversion, func-
tion unknown, and general functional prediction only.
The Siberian Thermoprofundales MAGs had the most genes that were absent in non-
permafrost genomes (47 genes) (see Supplemental Data File S6). The greatest numbers
were carbohydrate metabolism and transport, with four components of an ABC-type
sugar transport system, ABC-type glycerol phosphate systems, cellobiose phosphoryl-
ase, and pyruvate kinase, energy production and conversion, with FoF1-type ATP syn-
thase and isocitrate dehydrogenase, and inorganic ion transport and metabolism, with
two components of a Ca
21
/H
1
antiporter and a Na
1
/H
1
antiporter-related arsenite per-
mease (Fig. 3A). The Siberian Thermoprofundales MAGs also had more genes per ge-
nome related to the carbohydrate metabolism and transport (2.5) and defense (3)
COG categories than the nonpermafrost genomes (see Fig. S3).
There were no genes that were present in all Siberian Atribacteria MAGs that were
also absent from all of the nonpermafrost MAGs. Therefore, we investigated the genes
in all Siberian Atribacteria MAGs found in only 10% to 33% of the nonpermafrost
Atribacteria MAGs (see Supplemental Data File S6). Of those 32 genes, 5 were in the
carbohydrate metabolism and transport COG category (Fig. 3; also see Supplemental
Data File S2). The 7 genes with the lowest representation (11% to 23%) in nonpermafrost
Atribacteria were pyruvate/oxaloacetate carboxyltransferase, trehalose-6-phosphate
synthase/hydroxylamine reductase (hybrid-cluster protein), cellobiose phosphorylase,
nucleoside diphosphate kinase, cation transport ATPase, and ribosomal protein L32 (see
Supplemental Data Files S2 and S6).
FIG 3 Genes unique to the Siberian MAGs. (A) Numbers of genes in each COG category that are present in the Siberian MAG groups but absent in the
reference genomes of the same groups (except for Atribacteria, for which the genes were in less than one-third of the reference genomes). Exact names of
the genes can be found in the main text and in Supplemental Data Files S1 to S5 in the supplemental material. (B) Functional categories encompassing
genes from various other COG categories that are known to have multiple functions. The full list of genes can be found in Supplemental Data File S6.
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Thirty-three genes were present in at least 1 of 3 Siberian Atribacteria MAGs but
were absent in all nonpermafrost Atribacteria. Six of these were from the energy pro-
duction and conversion COG category, i.e., four subunits of NADH:ubiquinone oxidore-
ductase and two subunits of a heme/copper type cytochrome/quinol oxidase. Three
Siberian Atribacteria MAG genes were in the carbohydrate metabolism and transport
COG category, including mevalonate-3-phosphate 5-kinase, aryl-phospho-
b
-D-glucosi-
dase, and a phosphoglycerate mutase. The three permafrost Atribacteria MAGs had
twice the number of genes per genome related to the energy production and conver-
sion, inorganic ion transport and metabolism, replication, recombination, and repair,
cell wall/membrane/envelope biogenesis, and lipid transport and metabolism COG cat-
egories as did nonpermafrost genomes (see Fig. S3).
The Siberian Chloroflexi MAG had 20 genes that were absent in all nonpermafrost
genomes (Fig. 3; also see Supplemental Data File S6), including a Na
1
/alanine sym-
porter, fumarate hydratase class II, and nitric oxide reductase large subunit. A complete
methyl-coenzyme M reductase contig (subunits
a
,
b
, and
g
and operon protein D;
3,483 amino acids long) binned in the Chloroflexi MAG, but this contig had no other
genes on it (see Supplemental Data File S3). The COG categories with the most genes
that were only found in the Siberian Chloroflexi MAG, relative to nonpermafrost MAGs,
were coenzyme transport and metabolism, extracellular structures, and cell motility
(Fig. 3; also see Supplemental Data File S3). The Siberian Chloroflexi MAG generally had
1 to 1.5genes per genome in each COG category, compared with the 42 nonperma-
frost Chloroflexi (see Fig. S3).
Eleven genes were unique to the Siberian Aminicenantes MAG, relative to nonper-
mafrost Aminicenantes MAGs, including 2 genes in the replication, recombination, and
repair COG category and 2 in the function unknown category (see Supplemental Data
File S6). The Aminicenantes MAG had only 1 gene (endo-1,4-
b
-mannosidase) unique to
the carbohydrate metabolism and transport COG category, but it had 2.7more genes
per genome than in the nonpermafrost genomes (see Fig. S3). Even though there
were few genes that were unique to the Aminicenantes Siberian MAG, it did have
more genes per genome in the COG categories than did the nonpermafrost genomes
(see Fig. S3).
Since many of the genes that were unique to the Siberian MAGs were involved in
transport, osmoregulation, and carbohydrate utilization but spanned multiple COG cat-
egories, we made custom groups for these three functions to examine them together
(Fig. 3B; also see Supplemental Data File S6 and Table S3). The transporters group had
the most genes that were unique to the Siberian MAGs, led by Thermoprofundales and
Atribacteria, with 9 and 8 genes, respectively. Atribacteria and Thermoprofundales also
had the most unique genes in the carbohydrate utilization group, with 5 and 4 genes,
respectively. Carbohydrate metabolism and transport had the greatest numbers of
genes, 4 of which were ABC-type sugar/transport system genes (Fig. 3B). The average
numbers of genes within each COG category in the Actinobacteria MAG were less than
or equal to the number of genes per genome in the nonpermafrost genomes (see Fig.
S3). Thermoprofundales had the most unique genes in the osmoregulation group too,
and all taxa except Aminicenantes had a Na
1
symporter for osmoregulation. The only
gene from Aminicenantes that fell within these three groups was a succinate-acetate
transporter.
Comparison of Siberian MAGs to each other. While no genes had identical anno-
tations among all 8 of the Siberian MAGs (Fig. 4), each MAG had some version of three
Na
1
/H
1
antiporters, NhaD, MnhC, and MnhG, as well as a biotin transporter. Also,
many genes had similar annotations in different unions (see Supplemental Data File
S6). Forty genes were present in 7 Siberian permafrost MAGs that were not in the
Actinobacteria MAG (Fig. 4; also see Supplemental Data File S6); these included pyru-
vate-formate lyase-activating enzyme, formate hydrogenlyase, carbamoyltransferase,
Na
1
/H
1
antiporter, M28 family peptidase, predicted nucleotidyltransferase component
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of viral defense system, protein-L-isoaspartate O-methyltransferase, and cellobiose
phosphorylase.
The 6 bacterial Siberian MAGs shared 145 gene annotations with each other, which
was more than they did with the Thermoprofundales, the only archaeal group (Fig. 4). The
Siberian Actinobacteria had the largest number of genes (243 genes) that were uniquely
annotated, compared with the other 7 Siberian MAGs (Fig. 4). Some of these genes
included H
1
/Cl
2
,Na
1
/H
1
,andK
1
/H
1
antiporters with C-terminal TrkAC and CorC domains
(see Supplemental Data File S6). The Siberian Thermoprofundales MAGs contained 206
genes that were uniquely annotated, relative to the other Siberian MAGs (Fig. 4; also see
Supplemental Data File S6); these included acetate kinase, isocitrate dehydrogenase, Ca
21
/
H
1
antiporter,HSP90ATPase,andaNa
1
/proline symporter (see Supplemental Data File
S4). Other genes specifictotheSiberianThermoprofundales MAGs included 21 different
archaea-type regulatory proteins and an uncharacterized conserved protein related to py-
ruvate-formate lyase-activating enzyme, many archaea-type synthetases, and many genes
with general or predicted functions (see Fig. S6A to C and Supplemental Data File S3).
Membrane stability genes were shared between two or more groups. Multiple genes
andenzymekinasesforthemevalonatepathwaywerefoundinthe2Thermoprofundales
MAGs, 1 Chloroflexi MAG, and 1 Atribacteria MAG (see Supplemental Data File S6). Apart
from Thermoprofundales, the other Siberian permafrost MAGs shared membrane-associated
genes, such as peptidoglycan biosynthesis protein MviN/MurJ, bacterial cell division protein
FtsW, and energy-coupling factor transporter ATP-binding protein EcfA2 (Fig. 4; also see
FIG 4 Numbers of unique genes shared by different Siberian MAG groups. The numbers in parentheses are total numbers of genes
present in all of the MAGs for that group. The complete list of genes in each MAG group and each union can be found in
Supplemental Data File S6 in the supplemental material.
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Supplemental Data File S6). The Aminicenantes MAG had 96 unique genes that were not
present in any other Siberian MAG (Fig. 4). This MAG also contained 111 genes similar to
genes annotated in Actinobacteria and Chloroflexi MAGs, which are two other widely found
soil and permafrost microbe groups (50, 51).
DISCUSSION
Comparison of the Siberian MAG communities to those found in other
permafrost studies. Apart from the 3.5-m depth, which was an ice wedge with few
sediment inclusions, and the 17.2-m depth, which did not produce a metagenome for
unknown reasons, DNA was present in sufficient quantities to produce MAGs throughout
the 20,000- to 1,000,000-year-old permafrost. The identities of the MAGs from ancient
Siberian permafrost differed greatly from those found in active layer permafrost environ-
ments. In the active layer of the Stordalen Mire, Woodcroft et al. found 1,434 MAGs
(.70% completeness and ,10% contamination), including 27% Actinobacteria,27%
Aminicenantes,14%Proteobacteria,4%Chloroflexi,and6%Euryarchaeota (52). Our an-
cient Siberian permafrost contained Chloroflexi,Actinobacteria,andAminicenantes mem-
bers but also had MAGs from groups that were absent in the Stordalen Mire active layer,
such as Atribacteria and Thermoprofundales. This suggests that there could be large dif-
ferences in microbial community compositions between seasonally thawed active layers
and ancient permafrost. Atribacteria and Thermoprofundales were absent in Alaskan an-
cient permafrost from 19,000 to 33,000years ago (6, 7). The ancient Alaskan permafrost
study showed an increase of Firmicutes 16S rRNA genes and a decrease of Actinobacteria
with permafrost age (6). A similar trend was observed in the borehole adjacent to this
study, AL3-15, called AL1-15 (4), where Firmicutes and Proteobacteria dominated 16S
rRNA gene amplicon libraries. These taxa, Firmicutes,Planctomycetota,Crenarchaeota,
and Bacteroidota, were found in Siberian MAGs but were not analyzed due to insufficient
quality. This discrepancy may be due to the differential biases of amplicon-based libra-
ries and metagenome-based libraries in low-biomass samples (34, 49). The presence of
groups such as Atribacteria,Thermoprofundales,andAminicenantes, which are not com-
monly found in ancient permafrost or freshwater environments but are more commonly
found in marine environments, suggest that these ancient freshwater Siberian deposits
may contain organisms adapted to saline conditions.
Genes common in Siberian permafrost MAGs indicate adaptation to a cold,
saline, low-energy environment. (i) Saline regulatory genes. Many of the genes
shared among two or more groups of Siberian MAGs were involved in the transport of
small molecules like sodium and carbohydrates. Having a variety of genes for ion and
salt transportation could mean that these MAGs resemble organisms that are adapted
to function in high-ionic-strength environments, like brines. All groups shared a similar
annotation for three Na
1
/H
1
antiporters and osmoregulators (NhaD, MnhC, and
MnhG), as well as a biotin transporter. All Siberian MAGs had unique genes in the trans-
porter category, compared to the nonpermafrost genomes (Fig. 3B).
(ii) Trehalose. Many of the genes that were specific to Siberian MAGs and were not
present in nonpermafrost outgroup genomes were involved in transport, osmoregulation,
and carbohydrate utilization. The prominence of these categories suggests the Siberian
MAGs had unique adaptations to interact with their environment, including dealing with
osmotic stress and using carbohydrates as energy sources. Trehalose-6-phosphate syn-
thase was found in the Siberian MAGs and lacked homologues in nonpermafrost
genomes. It was in both Thermoprofundales MAGs (22% of the nonpermafrost outgroup),
all 3 Atribacteria MAGs (13% of the nonpermafrost outgroup), the Aminicenantes MAG (2%
of the nonpermafrost outgroup), and the Actinobacteria MAG (59% of the nonpermafrost
outgroup). The Chloroflexi MAG had a gene annotated as trehalose and maltose hydrolase
(possible phosphorylase). Trehalose-6-phosphate synthase has been suggested to help
deep subsurface inhabitants maintain a low-energy state in marine sediments by produc-
ing trehalose, a disaccharide that prevents aggregation of degraded proteins, protects
against osmotic stress, and increases cellular longevity (53, 54). Trehalose is a cryoprotec-
tant and stabilizes cellular membranes and DNA at low temperatures and high osmolarity
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to increase cell longevity and to slow replication rates (55–57). Trehalose synthase has also
been found in metagenomes from Antarctic soil and sediment samples (58).
(iii) Mevalonate pathway. Multiple genes and enzyme kinases for the mevalonate
pathway were found in both Thermoprofundales MAGs, the Chloroflexi MAG, and the
Atribacteria MAG. The mevalonate pathway uses acetic acid for the biosynthesis of iso-
prenoids, which have been shown to stabilize membranes, increasing the organism’ssur-
vival at low temperatures (59–61), and have been found in bacteria (62, 63). A functional
mevalonate pathway has also been identified in a Methanosarcina MAG from a deep
Antarctic permafrost enrichment (64). The acetate ions for the mevalonate pathway
could be from fermentation of the surrounding organic matter from the cellobiose, ara-
binoxylans, and proteinaceous peptides. Genes to ferment these substrates were also
identified in the Siberian MAGs. Additionally, Psychrobacter arcticus 273-4, cultured from
Siberian permafrost, has phenotypic evidence of an acetate-based metabolism (65, 66),
and all 8 MAGs from this study had genes indicating mixed acid-acetate metabolism (see
Fig. S6 in the supplemental material).
(iv) Cellobiose phosphorylase and carbamate kinase. Metagenomes of thawed
permafrost have shown increases in degradation of cellulose (67). Genes for cellobiose
phosphorylase, which is an enzyme that aids in the degradation of cellulose as a carbon
source (68–70), were identified in all of our Siberian MAGs except the Actinobacteria MAG.
Cellobiose phosphorylase converts cellobiose into glucose and glucose-1-phosphate,
offering an energetic advantage in anoxic environments. Cellobiose phosphorylase sug-
gests that permafrost organisms may be able to slowly degrade available carbon sub-
strates (43, 44). The Siberian Atribacteria MAGs had other genes indicative of adaptation
to low-energy environments. Further, two Siberian Atribacteria MAGs contained aconitase
A,
a
-L-arabinofuranosidase, and
a
-amylase/
a
-mannosidase, which were present in only
6%, 15%, and 19% of nonpermafrost genomes, respectively. These are important in cellu-
lose degradation and carbohydrate metabolism (45). Carbon starvation protein (CstA)
was in two Siberian Atribacteria MAGs and 30% of nonpermafrost Atribacteria genomes.
This protein enhances peptide catabolism during carbon starvation (44). Carbamate ki-
nase was in two Siberian Atribacteria MAGs and 25% of nonpermafrost Atribacteria
genomes, suggesting that the permafrost Atribacteria can conserve energy by creating
ADP and carbamoyl phosphate from the combination of ATP, CO
2
,andNH
3
(46). This
would benefit organisms in a low-energy environment, because this enzyme has roles in
purine, glutamate, proline, and nitrogen metabolism (47).
(v) Biotic methane. Through their genome-centric analysis of functional genes in
active layer soils, Woodcroft et al. found genetic evidence for organic matter decomposi-
tion into CO
2
and CH
4
(52). Studies of 33,000-year-old permafrost (6) and recently thawed
permafrost (67) from Alaska also generated a variety of methanogens. A gene for methyl
coenzyme M reductase (mcr)wasidentified in another study’s metagenome from a
30,696-year-old Siberian permafrost sample from where methane was measured (8).
Additionally, biogenic methane was detected in one of two boreholes in another study
of Kolyma-Indigirka Lowland permafrost, while methanogens were distributed through-
out both boreholes (71). Other permafrosts up to 33,000years old, where methane was
absent, did not yield 16S rRNA genes from known methanogenic groups (7) or any mcr
genes (8). There was no evidence of methane metabolism in any of the Siberian
Thermoprofundales MAGs, unlike their nonpermafrost counterparts. We found four subu-
nits of the mcr gene on a single contig from the 20-m metagenome sample in the
Chloroflexi MAG, suggesting that the methane observed there could have been biotically
produced (Fig. 1B). The gene’s top BLASTP hit was an mcr gene found in the “Candidatus
Methanoperedens ferrireducens”archeaon from an Australian marine sediment incuba-
tion (72). Since this MAG did not contain a full methanogenic pathway and no bacteria
were previously shown to contain mcr,itislikelythatthismcr gene was part of the 10%
contamination.
(vi) DNA scavenging. Ureidoglycolate dehydrogenase was present in 2 Siberian
Atribacteria MAGs and 48% of nonpermafrost Atribacteria genomes. This is involved in the
degradation of allantoin, a DNA decomposition product, and allows the use of DNA as a
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nitrogen source. Other Atribacteria strains have been suggested to use an allantoin degra-
dation pathway to access detrital DNA as an energy source under extreme starvation con-
ditions in deep subsurface marine sediment (38). The presence of DNA-foraging enzymes
like those involved in allantoin degradation (54) and cellular debris recycling (30) may be
another key to survival in low-energy environments. The Siberian Aminicenantes MAG had
a protein-degrading metabolic pathway similar to that inferred for other Aminicenantes
(54), since they all had the same types of extracellular peptidases, such as dipeptidase,
dipeptidyl aminopeptidase, acylaminoacyl peptidase, RecA-mediated SOS response auto-
peptidase, D-alanyl-D-alanine carboxypeptidase, and cyanophycinase exopeptidases (see
Fig. S6 and Supplemental Data File S5). Atribacteria,Thermoprofundales, and Aminicenantes
from subseafloor sediments also have been found to have these extracellular enzymes
(73). Additionally, Thermoprofundales have been suggested to ferment proteinaceous or-
ganic matter, since they have a high genomic content of extracellular peptidases whose
activity could be measured in the bulk sediment (42). Therefore, the presence of genes for
osmotic, cold, and energetic stress tolerance in our ancient Siberian permafrost MAGs sug-
gests that they may be adapted to this environment.
Conclusions. The Siberian permafrost MAGs belong to taxonomic groups that are com-
monly found in low-energy, anoxic, saline habitats. Finding MAGs related to marine-associ-
ated microbes like Thermoprofundales,Aminicenantes,andAtribacteria further supports that
these MAGs are adapted to increased salinity (74) due to freezing of freshwater sediments,
since the Yedoma and Olyor deposits in this area of Siberia have never been inundated by
seawater (12, 13, 16). The concentrations of chloride increased with depth (Fig. 1) up to
879.8 ppm (248 mM), which is hypersaline, compared to accepted baseline tested agriculture
soils, where 50 ppm is considered excessive (75). The high prevalence of Na
1
/H
1
and Ca
21
/
alanine antiporters, mechanosensitive channels, osmoprotectants, and other ion transporters
(Fig. 3B), relative to the nonpermafrost genomes, may suggest adaptation to the saline con-
ditions observed in our geochemistry (Fig. 1), since these genes are common in halotolerant
organisms (18, 76). Additionally, microbial cultures from Siberian permafrost have osmosis-
specific adaptations and activity in 215°C permafrost (77, 78). Genes for osmotic stress
tolerance have also been found within Antarctic (30) and Canadian (77) permafrost
metagenomes.
We speculate that, upon burial and freezing, these permafrost microbes became the
dominant organisms because those not adapted to the high-salinity liquid water films in
permafrost died. If these organisms are adapted to surviving in saline brine films, then it
is unlikely that they will retain their dominance in these microbial communities upon
permafrost thawing due to climate change. As the permafrost thaws, these native brine-
adapted microbial communities will likely be replaced by freshwater-adapted organisms
when surface waters penetrate the newly thawed permafrost.
Further evidence to support extant life in this ancient Siberian permafrost is that the DNA
was intact enough to produce reads that assembled into MAGs, with less than 9% of the assem-
blies remaining unbinned (Table 1) and high read recruitment in the MAGs (Fig. 5). Each MAG
had some DNA read recruitment from other sample depths, which suggests that, with even
greater sequencing depth, a more diverse population of MAGs might have been observed.
Finding evidence for these organisms in ancient 1,000,000-year-old permafrost sam-
ples furthers the idea of a microbial community being able to persist in ancient perma-
frost within the high-salinity brine films. The Siberian permafrost MAGs analyzed in this
study demonstrate how individual organisms can be adapted to their environment, rel-
ative to members of the same taxonomic groups in nonpermafrost environments.
Although it is impossible to have absolute certainty that these organisms were alive at
the time of sampling, their genome functions and unique functionality, compared to
nonpermafrost genomes (Fig. 3 and 4), coupled with the geochemistry of the environ-
ment (Fig. 1), suggest adaptations to the liquid brine films in ancient permafrost.
MATERIALS AND METHODS
Field sampling details. Permafrost cores were taken from the northeastern Kolyma-Indigirka
Lowland region in Siberia, Russia (69°20.376N, 154°59.787E), at the end of July 2015. A 20-m-long core
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(AL3-15) was retrieved in a similar fashion as its sibling core (AL1-15), which was taken at the foothill in
the same location, as described previously (4). The temperature was measured inside the borehole at
the end of each sampling day, to allow the borehole to calibrate back to the in situ temperature, using
an Onset HOBO data logger. The geological suites changed from Yedoma to Olyor depositional charac-
teristics near 7.7 m below the surface.
Environmental characteristics. Permafrost samples were analyzed for pH, methane levels, and total
carbon concentrations. pH was measured with a benchtop pH meter (SevenEasy pH meter; Mettler Toledo).
Conductivity was measured using a conductometer (Ekspert-002; Soil Services, Russia). To measure ions in
the permafrost, pore water was extracted from 100 g of permafrost following procedures described by Van
Reeuwijk (79). Porewater analyses were performed following the standards of the Russian Federation GOST-R
Certificate. Methane was liberated from the frozen permafrost soon after sample retrieval in the field, as
described previously (11), using a headspace method (80). Concentrations of the gas were analyzed on a GC-
mini-2 gas chromatograph (Shimadzu, Tokyo, Japan) with a hydrogen flame ionization detector and argon
as a carrier gas. The
14
C age of inorganic and organic carbon from three subsamples of the upper layers (2.9
to 3, 3.5, and 5.6 m) was determined at the Arizona Accelerator Mass Spectrometry Laboratory according to
the following procedures. The inorganic carbon was released by acidifying the bulk material using phos-
phoric acid (under vacuum) and collecting the CO
2.
The residue was dried and combusted at 400°C to release
a relatively “younger”organic carbon fraction (81) (i.e., microbial cell material) from the permafrost sediment.
The resulting residue was further combusted at 800°C to oxidize any tightly bound carbon fraction that was
potentially associated with clay particles in the sediments. This presumptive “older”carbon fraction could be
inherited photosynthate or detrital carbon through geological time. An additional feature of this gas collec-
tion is the ability to determine the
d
13
Cofthesample,comparedtoPeeDeeBelemnite.
Metagenomic processing and analysis. Seven subsampled depths (3.5, 7.2, 14.1, 14.8, 16.6, 17.2,
and 20 m below the surface), spanning ;20,000 to 1,000,000 years of age, were used for metagenome
sequencing. DNA was extracted from approximately 5 g of permafrost using 10 technical replicate
extractions with the DNeasy PowerSoil kit (Qiagen, Germantown, MD, USA) with 0.5 g of sample in each
extraction. The 10 extractions per sample were pooled, and DNA was precipitated with ethanol and
resuspended in 50
m
l molecular-grade water. DNA concentrations ranged from 2.9 to 29.2 ng/
m
l.
Metagenomic libraries were prepared using the Nextera XT DNA library preparation kit (Illumina, USA),
following the manufacturer’s instructions, and sequenced at the University of Tennessee Genomics Core
on an Illumina MiSeq system with the 2 250-bp protocol.
A diagram describing the following bioinformatic methods can be found in Fig. S7 in the supple-
mental material. The raw metagenomic files were analyzed using KBase.us (82), and all links are provided
in “Data availability”(registration with https://www.kbase.us is free and is required to access the narra-
tive). The sample depths were kept separate for single-sample assembling and metagenomic binning, in
order to avoid combining contigs from different sample depths into the same MAG. Trimmomatic v0.36
(83) was used for poor-quality data trimming, removal of barcodes, and creation of a paired-end assem-
bly with the following parameters: leading_min_quality, 3; min_length, 36; sliding_window, 28; sliding_
FIG 5 Metagenomic read coverage for each MAG at each sample depth. Abundance is reported in RPKM.
MAGsinbluetextareMAGsanalyzedinthisstudy($80% completeness and ,10% contamination);
MAGs in black text came from these samples but were not analyzed extensively in this study (although
they are available under BioProject accession number PRJNA596250). Calculations can be found in
Supplemental Data File S7 in the supplemental material.
Eight MAGs from Million-Year-Old Siberian Permafrost Applied and Environmental Microbiology
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window_size, 10; trailing_min_quality, 3. MetaSPAdes v3.11.1 (84) was used for metagenomic assembly
with minimum contig length of 2,000 bp. MaxBin2 v2.2.4 (85) with default settings was used to place
contigs into metagenomic bins with the following parameters: prob_threshold, 0.8; min_contig_length,
2000. Prodigal (86) was used for gene finding and DIAMOND (87) for gene translation within anvi’ov6
esther (88) using the parameter anvi-run-ncbi-cogs in the anvi-gen-contigs-database command. GTDB-
Tk v1.4.0 was used for initial taxonomic classification of the created bins (38) with the classify_wf com-
mand with a minimum alignment percent (min_perc_aa) of 10. A maximum likelihood phylogenetic tree
was made in CLC Workbench v6 using 139 shared conserved genes gathered by using the anvi-get-
sequences-for-hmm-hits command with -return-best-hit, -get-aa-sequences, and -concatenate in anvi’o
(see Supplemental Data File S6). MAGs were quality assessed with CheckM v1.0.18 (89) for completeness
and contamination percentages. Bowtie2 v2.3.3 (90) with default settings was used to map the paired
metagenomes against each of the MAGs to determine the presence of MAGs at other depths. This map-
ping also helped to find similarities and difference of the MAGs in the metagenomic samples. Mapping
results were standardized by calculating reads per kilobase per million mapped reads (RPKM), which
accounts for the gene length and each sample’s library size (see Supplemental Data File S7).
Nonpermafrost genomes were downloaded from the JGI IMG database for comparison of genetic con-
tent (91). The five taxonomic groups to which the MAGs belonged were Actinobacteria (n=1, o= 57),
Aminicenantes (n=1, o= 54), Thermoprofundales (n=2, o= 9), Atribacteria (n=3, o=66), and Chloroflexi
(n=1, o=42), where nis the number of permafrost MAGs and ois the number of nonpermafrost
genomes downloaded from the JGI IMG database.
The program anvi’o (88) was used to compare the annotated genes for similarity between genomes
using DIAMOND (87) and the NCBI database with an MCL inflation score of 2 within the groups.
Comparisons were made between two experimental groups: (i) MAGs from this study against down-
loaded genomes (MAGs and SAGs) from the same phylogenetic groups and (ii) all of the Siberian MAG
groups against each other. These comparisons were made on the basis of gene annotations and, more
broadly, COG categories (92). To investigate the KEGG pathways and metabolic pathways of the anno-
tated Siberian MAGs, the KEGG Orthology (KO) numbers from protein fasta files were compiled in
GhostKoala and analyzed with KEGG-Decoder (93).
Cell visualization. Cells were visualized by filtering from sonicated bulk soil with Nanopore 0.2-
m
m
filters and staining with SYBR gold and DAPI (94) and were imaged under a Zeiss Imager M2 with 100
magnification (see Fig. S4). A Guava easyCyte 12HT benchtop cytometer was also used with SYBR green
cell dye to examine cells. Only cells frozen at 280°C were available, without cell preservation in the field;
therefore, many of the cells might have lysed. Cell counting and live/dead staining methods did not
yield quantifiable results. Additionally, SAGs were made from sample depths of 5.6 m and 7.2 m at the
Bigelow Single Cell Genomics Center (East Boothbay, ME, USA) (95). Ten single cells with the best whole-
genome amplification (see Fig. S5) with Bigelow’s whole-genome amplification and multiple displace-
ment amplification methods were sequenced using an Illumina MiSeq 2 250-bp protocol at the
University of Tennessee (Knoxville, TN).
Data availability. The 6 assembled metagenome files and resulting MAGs are available in the NCBI
database under BioProject accession number PRJNA596250. Sample methods are available to view, as
follows: 3.5 m, https://narrative.kbase.us/narrative/27695; 7.2 m, https://narrative.kbase.us/narrative/
27731; 14.1 m, https://narrative.kbase.us/narrative/27724; 14.8 m, https://narrative.kbase.us/narrative/
27725; 16.6 m, https://narrative.kbase.us/narrative/37690; 17.2 m, https://narrative.kbase.us/narrative/
37878;20m,https://narrative.kbase.us/narrative/37504.
SUPPLEMENTAL MATERIAL
Supplemental material is available online only.
SUPPLEMENTAL FILE 1, XLS file, 0.3 MB.
SUPPLEMENTAL FILE 2, XLS file, 0.6 MB.
SUPPLEMENTAL FILE 3, XLS file, 2.4 MB.
SUPPLEMENTAL FILE 4, XLS file, 0.9 MB.
SUPPLEMENTAL FILE 5, XLS file, 0.5 MB.
SUPPLEMENTAL FILE 6, XLSX file, 0.1 MB.
SUPPLEMENTAL FILE 7, XLSX file, 5.6 MB.
SUPPLEMENTAL FILE 8, PDF file, 7.8 MB.
ACKNOWLEDGMENTS
We thank Andrey Abramov, Nikita Demidov, Denis Shmelev, and Victor Sorokovikov
from the Institute of Physicochemical and Biological Problems of Soil Science (Pushchino,
Russia) for cooperation in the collection of permafrost samples. Peibo Li and Mackenzie
Thorton helped extensively with microscopy. Nicholas T. Sipes helped with Python
scripts; all in-house scripts are available at https://github.com/sipesk/SiberianMAGsPaper.
This study was supported by the National Science Foundation (grants DEB-1442262 and
DEB-1460058), the U.S. Department of Energy, Office of Science, Office of Biological and
Environmental Research, Genomic Science Program (grant DE-SC0020369), the Russian
Sipes et al. Applied and Environmental Microbiology
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Government (assignment AAAA-A18-118013190181-6), and the Russian Foundation for
Basic Research (grant 19-29-05003-mk).
We declare no conflicts of interests.
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