A
BS5
BS1
1m
B
&"#$%#
C
!"#$%#
Supplementary Figure 1: Spring fluid was collected at Barnes springs (BS) for the
microbial isolation.
A; BS complex, B; BS1 and C; BS5. The surface of springs is covered with calcite skin.
Due to the gas bubbling from the bottom, part of the calcite at the surface was broken
in both springs.
!
"
<?/@)
#
<?/@)
<?/@)
$%&&'()(*+,-./012%-(/3//45,6(/78*+-,6+/1),2(6/89/168',+(:/6+-,1*6/!;/!<=/";/"</,*:/#;/><
A
B
1 cm
1 cm
Supplementary Figure 3 Two-week cultivation of strain A1 in the liquid medium
A) no cell control, B) strain A1
"
!""#$%#
!
&'#$%#
()**+,%,-./01#234)0,#5#67-879/+#%3907:97*39#7;:,0</.37-:#78#.=,#9,++#/440,4/.,:#7-#9/+93)%#9/0;7-/.,#>3.=#
?388,0,-.#%/4-3839/.37-:@#(.0/3-#A!#>/:#407>-#/).7.07*=39/++1#870#.=0,,#?/1:@#6,++:#>,0,#:./3-,?#>3.=#(BCD#E0,,-#F#GE0,,-H@#
6/+93)%#9/0;7-/.,#*0,93*3./.,#>/:#/+:7#7;:,0<,?#GI=3.,H@
A
Mixotrophic growth!
7
100
Autotrophic growth!
6
80
5
4
60
3
40
Acetate conc. (mM)
Relative abundance of hydrogen (%)
Hydrogen!
Acetate!
Mixotrophic growth!
2
20
1
0
0
2
4
6
8
0
10 (days)
Time
B
Cell density!
Cell density (cells/mL)
Mixotrophic growth!
Autotrophic growth!
1.00E+07
1.00E+06
1.00E+05
0
2
4
6
8
10 (days)
Time
Supplementary Figure 5; Growth curves of strain A1 (n=3). A) Acetate (triangle) and
hydrogen (circle) consumption and B) cell densities (triangle). Samples were collected at
0, 3 and 8 days after cell innoculation. To count cell numbers, the samples were shaken
vigorously. Thus the cells were detouched from calcite and the autotrophic growth for
strain A1 was stopped. Bars indicate standard deviation"!!!!
A
B
Supplementary Figure 6 Thin-section TEM observations of strain A1. The cells were fixed after five-days
cultivations. A) Autotrophic growth, B) Heterotrophic growth. C; Carboxysome, N; Nucleoid, PHB;
Polyhydroxybutyrate
;
!"
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'"
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*"
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7589:4+;!+<643=6
01+2345645
13>689<6
?9:86@+@:A59426
7:4<B6+C9:86@+869@A
D34EAC62:F:2+=952G6A
H
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7589:4+H!+0643=6+
01+2345645
13>689<6
?9:86@+@:A59426
7:4<B6+C9:86@+869@A
D34EAC62:F:2+=952G6A
7ICCB6=64598J+K:<I86+(+L9M+869@A+=9CC:4<+53+5G6+<9CE2B3A6@+<643=6+A6NI6426A+3F+A589:4+;!+,;/+94@+H!+,H/
A
B
2600
(kbp)
2400
2600
(kbp)
200
2200
400
400
2200
Strain A1
Strain B1
2574013 bp
2606868 bp
2000
1800
200
2400
1st circle: Strain H1
2nd circle: Strain B1
3rd circle: Tablelands metagenome
4th circle: Hydrogenophaga sp. PBC
600
2000
800
1600
1800
1000
1400
600
1st circle: Strain H1
2nd circle: Strain A1
3rd circle: Tablelands metagenome
4th circle: Hydrogenophaga sp. PBC
800
1600
1000
1200
1400
1200
C
2400
(bp)
2200
200
400
2000
Strain H1
2475906 bp
600
1800
1st circle: Strain B1
2nd circle: Strain A1
3rd circle: Tablelands metagenome
4th circle: Hydrogenophaga sp. PBC
800
1600
1000
1400
1200
Supplementary Figure 8 Genome-wide comparisons of strain A1 (A), B1 (B) and H1 (C).
The inner scales designate the coordinates (in kbp). The first to fourth circles show the
blastn analysis of strains A1, B1 and H1 against each genome or metagenome as indicated
in the figure. The lower (gray) and upper (red) identity threshold in the blastn analysis is >90
or >95 respectively.
Tablelands contig 1
A1 Genome (2479529 -­ 2539338)
Tablelands contig 7
A1 Genome (976009 -­ 1005354)
Tablelands contig 2
A1 Genome (2363990 -­ 2558128)
Tablelands contig 6
A1 Genome (592637 -­ 613825)
Tablelands contig 8
A1 Genome (1317038 -­ 1340218)
Tablelands contig 5
A1 Genome (2377000 -­ 2545000)
Tablelands contig 17
A1 Genome (2233380 -­ 2252300)
Tablelands contig 9
A1 Genome (2435626 -­ 2465900)
Tablelands contig 15
10 kbp
A1 Genome (1198330 -­ 1219431)
Supplementary Figure 9 Blastn comparisons of the long contigs from the Tablelands1 (yellow) with the genome sequence of strain A1 (Blue)
COG clustering
Strain H1
(Contig 11)
Strain B1
Strain A1
Tableland
(Contig 1)
2 kbp
CO dehydro-­
genase Ni,Fe-­
hydro-­
genase
carboxy-­
some
shell
protein
Rubisco
C
D
E
G
H
J
K
M
N
O
P
Q
R
S
V
T
L
F
I
Supplementary Figure 10 Comparisons of predicted gene clusters encoding carbon fixation related proteins
Na+ - type
H+ - type
Strain A1
Strain B1
Strain H1
H+-type Comamonas testosteroni
Na+-type Alkaliphilus metalliredigens
Conserved region
Conserved region
Supplementary Figure 11 Comparison of cation binding site in F0F1-ATPase. The conserved regions were reported by Mulkidjanian et al.2
100
90
80
70
60
50
40
30
20
Mapped reads (%)
A
B
B1
H1
70
80
90
Mapped reads (%)
100
90
80
70
60
50
40
30
20
A1
100
90
80
70
60
50
40
30
20
70
80
90
Mapped reads (%)
C
70
80
90
Supplementary Figure 12 Mapped percent of raw reads to each genome. Raw reads from strain A1, B1 and H1 with average read length ~100 bp were mapped to the (draft) genome sequences of strains A1 (A), B1 (B) and H1 (C). The length cut off is 95 %. The numbers shown in the bottom of each graph
indicate the similarity cut-­off value (e.g. 70 means 70% similarity in 95% length.). USA
Cabeço
de Vide
Portugal
WHC2b
Table
lands
Canada
Finland
Mizunami
Japan
LC4
Chicago
USA
Present*
Outokumpu
MIU
(Sep, 2005)
Absent*
11.9
-
-
8.8
-
-
9-9.8
Mid-Atrantic
8.0
<dl
-656
Norway
-
USA
12.5
<dl
South Africa
-45
Lost City
8.2
<0.12
Lake
Ophiolite
-
-308
The
Cedars
-
30° N e
-
Gw3
-
GPS1
-
1500 m
deep
-
1000 m
deep
12.3
-
100 m
deep
Supplementary Table 1 Geochemical properties of the studied terrestrial serpentinizing sites and other alkaline soil or ground water
The Cedars
AC3
11.4
-
-552
BS5
11.6
<dl
-383
BS1
11.5
<dl
-550
Component
pH
<dl
-250
Kalahari
Shield
Evander,
EV818
DRLW
Eh (mV)
8-9
DO (mM)
-
-
0.25-0.43
-
-
-
-
479-485
0.13-0.28
-
-
7.4
-
-
-
-
0.54
-
-
-
-
36.6
-
-
14.69
21.0-23.3
50.9
-
-
0.012
-
-
-
18.90
0.031
-
-
-
0.13
-
48
-
2.2
0.94
-
-
-
-
-
49
-
22.7
13.70
-
58
-
-
27
1.2
-
9-19
38
-
57.85
17.2
0.041
0.42
0.02
-
-
1.64
0.00063
40
-
76.56
65.81
<dl
0.004
<0.003
-
-
0.59
-
33
1.04
57.42
55.55
-
0.024
5.3
-
-
0.02
0.76
-
<0.001
-
-
-
38.29
0.07
-
-
2.26
-
<0.105
-
-
-
1.12
-
3.79
-
-
1.98
0.12
-
0.71
34
N2 (aq) mM
-
0.58
-
0.77
53.6
H2 (aq) mM
2.0
0.03
-
0.03
49.6
CH2 (aq) mM
1.17
-
<dl
39.2
Na+ (mM)
-
0.038
<dl
0.036
by vol)
K+ (mM)
-
N2 (% by vol)
Ca2+ (mM)
<dl
6.5
(mM)
<dl
H2 (%
-
CH4 (% by vol)
PO4
<dl
-
<dl
-
546-549
40.00
-
-
0.54
2.1
8.73
-
-
52.6
5.9-12.9
-
0.019
-
198.57
<dl
0.038
-
-
<dl
0.792 (DIC)
~0.05
184.75
0.56
0.035
0.06
0.00135 (TOC)
-
3.08
0.17
128.34
-
0.275 (DIC)
0.06
-
0.055
-
9.59
-
0.72
-
0.014
<0.005
-
1.48
-
-
12.27
1.49
1.65
0.13
1.7
-
-
0.76
SO4
-
1.67
-
3-
Mg2+ (mM)
HCO3 (mM)
-
-
1.14
Cl (mM)
1.58§
-
<dl
-
0.73
<dl
-
-
<dl
0.03
0.37
0.67
-
0.11
NA
0.08
+
3, 4
+
5, 6
+
7
+
8
-
8
-
8
+
9
-
10
-
11
-
3,4
+
12
+
13
+
§
0.001
-
This study
§
<dl
0.07
0.26
-
-
0.59
0.02
(mM)
-
2-
TIC (mM)
NA
NO3 (mM)
DOC (mM)
Ca/Na ratio
occurrence of
serpentinization
Reference
-; not available, <dl; below the detection lmit, * Alkaline environments where strains A1, B1 and H1 related phylotypes are present or absent, §Total S concentration
Supplementary Table 2 Percent similarities of 16S rRNA gene sequence between strains A1,
B1 and H1, and type strains in the three closest genera
1
2
3
4
5
6
Strain A1 1
100
-
-
-
-
-
Strain B1 2
97.7
100
-
-
-
-
100
-
-
-
100
-
-
Strain H1 3 98.95 98.75
T
Malikia granosa DSM 15619
T
Hydrogenophaga flava DSM 619
4 95.56 96.51 96.58
5 94.96 96.12 95.98 96.64 100
Macromonas bipunctata IAM 14880T 6 94.07 94.72 94.65 96.5 95.61
100
Supplementary Table 3
Substrate utilization for strains A1, B1 and H1
A1
B1
H1
Autotrophic growth
Electron donor and Carbon source
H2 + CaCO3
H2 + CaCO3
H2 + CaCO3
Thiosulfate + CaCO3
Electron Acceptor
Oxygen
Thiosulfate
Nitrate
Oxygen
++
NT
++
NT
-
++
NT
++
NT
Heterotrophic growth
Electron donor and carbon source
Formate + CaCO3
Acetate + CaCO3
Propionate + CaCO3
Butyrate + CaCO3
Lactate + CaCO3
Pyruvate + CaCO3
Glucose + CaCO3
Glutamate + CaCO3
Glycerol + CaCO3
Cyclohexane + CaCO3
Electron Acceptor
Oxygen
Oxygen
Oxygen
Oxygen
Oxygen
Oxygen
Oxygen
Oxygen
Oxygen
Oxygen
+
+
+
+
+
+
+
+++
+++
+++
+++
+++
±
+
++
NT
+
++
NT
++
NT
NT
-
Electron donor and/or carbon source
Formate + H2 + CaCO3
Acetate + H2 + CaCO3
Butyrate + H2 + CaCO3
Lactate + H2 + CaCO3
Pyruvate + H2 + CaCO3
Glucose + H2 + CaCO3
Cyclohexane + H2 + CaCO3
Oxygen
Oxygen
Oxygen
Oxygen
Oxygen
Oxygen
Oxygen
++
+++
+++
+++
+++
+
+++
+
+++
+++
+++
+++
+++
±
++
+++
+
+++
NT
+++
-
Electron Acceptor
Acetate + H2 + CaCO3
Acetate + H2 + CaCO3
Acetate + H2 + CaCO3
Acetate + H2 + CaCO3
Acetate + H2 + CaCO3
Acetate + H2 + CaCO3
Oxygen
Nitrate
Sulfate
Thiosulfate
Fumarate
Iron
+++
+++
-
+++
+++
-
+++
+++
-
-
+++
-
+++
NT
Fermentation
Glucose
Cyclohexane
+; positive, - Negative, NT; not tested
Supplementary Table 4
Statstics for assembled genomes of strains A1, B1 and H1
Contig
Word Contig
Count
Sample Length Count* Over_1kbp
N50
Length
Max
Contig
Total
Contig
Length
Total
Reads
Reads
assembled
A1
53
36
25
226,682
471,724
2,561,383
3,857,324
3,829,752
B1
53
22
20
267,175
516,238
2,611,506
4,385,866
4,346,338
H1
53
96
70
87,939
198,940
2,511,299
4,927,302
4,836,043
*Length cut off > 300 bp
Supplementary Table 5 Primer sequences for the qPCR analyses
Targeted Genes
Forward (5' - 3')
Reverse (5' - 3')
SRAA-0853
CGTCGCCAACTGGTTCTT
CCGTACCACCACTGAATCA
SRAA-747
CCGAGCACAAGACCTACA
GAATGGGCACCGAGATGTT
SRAA-1136
CCACCACGCAGAACAATCAA
CAGCTCAAACAGGGGTCGTA
SRAA-2301
CGCCTTTTTGGAGCAGCA
GAGGAAAAAGCGCAGGTC
SRAA-2294
CCCGCATCAAGAAGATCAAC
GGCATCCAGTGCAGCTTTA
SRAA-2317
AGCGCCGGTGTCAAAGAATA
GTGTCGAGCGGGATGTAA
Supplementary References
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production by serpentinite-hosted subsurface microbial communities. Front Microbiol 2, 268,
(2012).
2. Mulkidjanian, A. Y., Galperin, M. Y., Makarova, K. S., Wolf, Y. I. & Koonin, E. V. Evolutionary
primacy of sodium bioenergetics. Biol Direct 3, 13 (2008).
3. Morrill, P. L. et al. Geochemistry and geobiology of a present-day serpentinization site in
California: The Cedars. Geochimica Et Cosmochimica Acta 109, 222-240 (2013).
4. Suzuki, S. et al. Microbial diversity in The Cedars, an ultrabasic, ultrareducing, and low salinity
serpentinizing ecosystem. Proc Natl Acad Sci U S A 110, 15336-15341 (2013).
5. Marques, J. M. et al. Origins of high pH mineral waters from ultramafic rocks, Central Portugal.
Applied Geochemistry 23, 3278-3289 (2008).
6. Tiago, I. & Verissimo, A. Microbial and functional diversity of a subterrestrial high pH
groundwater associated to serpentinization. Environmental Microbiology 15, 1687-1706 (2013).
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Gros Morne National Park: A Mars analogue. Icarus 224, 286-296 (2013).
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deep borehole, Fennoscandian Shield. FEMS Microbiology Ecology 77, 295-309 (2011).
9. Fukuda, A. et al. Geomicrobiological properties of ultra-deep granitic groundwater from the
Mizunami Underground Research Laboratory (MIU), central Japan. Microb Ecol 60, 214-225
(2010).
10. Roadcap, G. S., Sanford, R. A., Jin, Q. S., Pardinas, J. R. & Bethke, C. M. Extremely alkaline
(pH > 12) ground water hosts diverse microbial community. Ground Water 44, 511-517 (2006).
11. Davidson, M. M. et al. Capture of Planktonic Microbial Diversity in Fractures by Long-Term
Monitoring of Flowing Boreholes, Evander Basin, South Africa. Geomicrobiology Journal 28,
275-300 (2011).
12. Daae, F. L. et al. Microbial life associated with low-temperature alteration of ultramafic rocks in
the Leka ophiolite complex. Geobiology 11, 318-339 (2013).
13. Kelley, D. S. et al. An off-axis hydrothermal vent field near the Mid-Atlantic Ridge at 30 degrees
N. Nature 412, 145-149 (2001).