1 Metabolic
flexibility
of
enigmatic
Sar324
revealed
through
metagenomics
and
metatranscriptomics
Short Title: Disentangling the ecophysiological role of Sar324
Cody S. Sheik1, Sunit Jain1 and Gregory J. Dick1,2,3
1
Department of Earth and Environmental Sciences
2
Center for Computational Medicine and Bioinformatics
3
Department of Ecology and Evolutionary Biology
University of Michigan, Ann Arbor, MI, 48109, USA
Correspondence: Greg Dick, Department of Earth & Environmental Sciences, The University of
Michigan, 1100 N. University Ave., 2534 CC Little Bldg, Ann Arbor MI, 48109-1005, USA. E-mail:
[email protected]
2 SI Figure 1. Emergent Self Organizing Map based on tetranucleotide frequencies from metagenomic
contigs greater than 2500 bp. Each data point represents a genomic fragment (2500-5000 bp) and
the background topology represents the Euclidean distance between tetranucleotide frequency
profiles. Thus genomic bins are delineated by ridges. The GB-Sar324 bin is identified, and other
major genomic bins are colored: Blue =MG1 Archaea, Dark Red= SUP05 and black = methylotrophs.
Sar 324
3 SI Figure 2. Recruitment of genes from published single cell Sar324 genomes to GB-Sar324
Metagenome Bin. Recruitment was performed using BLASTn with high stringency cutoffs (e-value
cutoff 10-10, minimum length= 400 bases). Blue dots represent AFIB (AFIB00000000) and red dots
represent AFIA (AFIA00000000) single cell genomes. Blue and red lines represent the average
90
85
80
% ID
95
100
80
85
90
95
% ID
100
nucleotide similarity to GB-Sar324.
0
500000
1000000
GB-Sar324 Genome
1500000
2000000
SI Figure 3. Recruitment of Guaymas Basin 454 metagenomic reads to the dark ocean SAR324
single amplified genomes. Reads were recruited with BLASTn using a similarity of >90% and bit
scores over 70. Coverage was calculated with a 100 base pair sliding window.
4 SI Figure 4. Representative CTD depth profile from Guaymas Basin. A) Oxygen concentration, B)
Beam transmission, C) Temperature and D) Sar324 abundance from clone libraries (Dick and Tebo,
2010) and high throughput tagged ribosomal sequencing libraries (Anantharaman, 2012). Note that
the clone library data is as a percentage of Bacteria, whereas the tag sequence data is as a
percentage of Bacteria plus Archaea.
5 6 G
aI
ua
as
aym
as
v
ati
ym
Gu
Ba
sin
Ba
sin
(2
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(20
621
0.5
Bootstrap Support (%)
>95
90-95
85-89
80-84
70-79
50-69
eo
ha
oco
ccu
s ho
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riko
shii
co
(NP
cc
_14
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286
ko
1.1)
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ka
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is
(Y
P_
18
47
03
.1)
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_0
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idu
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5.1)
5804
(EAY
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itrosp
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(CBK
us
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.1)
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.1)
42
38
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is (
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Form IV
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b
lum ru
.1)
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spiril
P_
(N
6.
m
Lepto
Th
io
Pyr
tep
SC
(A
er
ium
ob
lor
Ch
84)
Archaeoglobus fulgidus (NP_070416.1)
973
2
062
GC
)
7.1
)
59
3.1
05
559
X
0
AD (ADX
(
G
06
03
07
-D 3-F
A0 007 A00
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X055
AA AA AA
0 (AD
1
C C ACGC
-C
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G G S
A00
A
A
C
S SC SCGC
SCGC AAA240-J17 (ADX05610.1)
SCGC
SCG
AAA24
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AA0
DX0560
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9.1)
20 (
ADX
055
95.1
)
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c
Ar
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um
AY
(E
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52
1)
4.
Form II
)
.1
99
55
0
DX
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ar32
GBS
SC
GC
AA
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us
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ic
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(ADX05
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628.1) .1) 163 .1)
96
.1)
05627.1 )
SCGC AAA240-N18 (ADX
red SU
P05 (A
Form I
bu
62
17
2
436 505
CX3051 18) )
3.1)
in (2062325545).1)
Guaymas Bas
7
748
_42
1)
(YP
0.
)
brum
0.1 478
u
r
9
2
m
5
2
lu
il
3
2
pir
41
dos
P_
00
Rho
(Y
P_
(Y
es
id
sis
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o
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e
oe
ae
sp
ph
ae
io
rs
ibr
te
v
c
a
lfo
su
ob
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od
Rh
uncultu
M
Ni ethyloco
ccus
tro
capsu
so
latus
mo
(YP_
na
1151
se
43.1)
uro
pa
ea
(N
P_
84
19
43
.1)
)
40.1
708
P_1
s (Y
s
gatu
n
a
lo
yz
se
Or ococcu
ech
Syn coccus mobilis (ZP_01125685.1)
Nitro
nd
ica
(Y
6
P_
54
22
1.1
)
SI Figure 5. Phylogenetic inference of large subunit 1,5-ribulose bisphosphate genes (RuBisCO).
Sar324 related sequences are highlighted in black and the GB-Sar324 RuBisCO gene is bolded.
Form III
SI Figure 6. Recruitment of 454 metagenome reads to the phmoABC operon from SAR324 single
amplified genome (AAA240-J09). Reads were recruited with BLASTn using a similarity of >90% and
bit scores over 70.
7 SI Figure 7. Recruitment of paired end illumina metatranscriptome reads mapped to the phmoABC
operon from SAR324 single amplified genome (AAA240-J09). Reads were mapped with BWA and
alignments were visualized with IGV (See methods section). The order of genes for the Phmo are
CAB, which can clearly be seen through mapping.
8 9 SI Figure 8. Depiction of contig synteny in GB-Sar324 and single cell genome isolates. Contigs all
contain sulfur cycling genes, the expression profiles of representative genes are shown in Figure 3.
These five contigs from GB-SAR324 are present in as single contig in the AFIA SAG (AAA240-J09),
thus is used as a reference. Colors represent contigs from GB-SAR324, while inside contig boxes
lines represent similarity between GB-SAR324 and SAGs
AFIA0000000 SAG
2000
4000
6000
AFIB0000000
SAG
2000
4000
6000
8000
10000
12000
14000
16000
18000
20000
22000
8000
10000
12000
14000
16000
18000
20000
22000
16000
18000
20000
22000
24000
26000
28000
30000
24000
26000
28000
30000
Guaymas Basin SAR324 Metagenome
2000
C1212
4000
6000
8000
10000
12000
14000
C352
32000
C41
34000
36000
38000
40000
42000
C669
44000
10 SI Figure 9. Phylogeny of the dsrA gene from SAR324 in relation to sulfur oxidizing and sulfur
reducing microorganisms.
Sulfur Reducing Organisms
77
Sulfur Oxidizing Organisms
92
60
Chlorobiacea
SAR324 SAG AAA240-C23
81
80 SAR324 SAG AAA003-G02
98 SAR324 SAG AAA240-N20
51
82
Guaymas Basin SAR324
Magnetococcus sp. MC-1 (NZ_AAAN02000009)
Moorella thermoacetica (NC_007644)
82
10 %
Pyrobaculum
11 SI Figure 10. Phylogeny of the aprA gene from SAR324 to sulfur oxidizing and sulfur reducing
microorganisms.
92
Sulfur Reducing Deltaproteobacteria
59
Thiobacillus denitrificans (637709551)
62
97 Thiobacillus thioparus (2515447707)
Thiobacillus denitrificans (2515457036)
100
Thiothrix nivea (2506752823)
Sedimenticola selenatireducens (2513982171)
Lamprocystis purpurea (2515666939)
72
Thiorhodococcus drewsii (2510114581)
Beggiatoa sp. PS (641100078)
Thiocystis violascens (2508739308)
Thermodesulfobium narugense (2504785014)
Desulfobacca acetoxidans (2504154265)
68
64
66
99
100
Chlorobium tepidum (637115811)
(Chlorobium chlorochromatii 637774125)
Pelodictyon phaeoclathratiforme (642726341)
Chlorobium phaeobacteroides (642684333)
63
Thermodesulfovibrio yellowstonii (643376069)
40
100
83
65
Guaymas Basin SAR324
SAR324 SAG AAA003-C18 (ADX05636.1)
SAR324 SAG AAA240-N20 (ADX05658.1)
Thermodesulfobacterium sp. OPB45 (2506601900)
81
100
Thermodesulfatator indicus (2505283574)
Sulfur Reducing Clostridia
100
100
98
75
Archaeoglobus
100
100
63
100
97
Succinate/fumarate Dehydrogenase
0.2
Thermophillic Crenarchaeota
Caldivirga
99
Alphaproteobacteria
Gammaproteobacteria
12 SI Figure 11. Phylogeny of the SAT gene from GB-SAR324 to sulfur oxidizing and sulfur reducing
microorganisms.
74
Sulfur Oxidizing Gammaproteobacteria
61
Alcanivoraceae
Gammaproteobacterium IMCC2047 ctg 507 (651416985)
92
85
68
33
100
Gammaproteobacterium NOR51-B
Marine Gammaproteobacteria NOR5
Marine Gammaproteobacteria OM60
Desulfarculus baarsii (648111637)
57
Deltaproteobacterium NaphS2 (648675202)
Guaymas Basin SAR324
100
61
Sulfur Reducing Deltaproteobacteria
64
Syntrophobacter fumaroxidans (639701810)
48
Desulfobacca acetoxidans (2504154267)
70
100
Chlorobi
90
61
47
45
46
44
Sideroxydans lithotrophicus (646689383)
96 Thioalkalivibrio
98
Thiobacillus
Ca. Ruthia magnifica (639757769)
Gammaproteobacterium HTCC5015 (647606004)
100
Ca. Desulforudis audaxviator (641593890)
Desulfotomaculum gibsoniae (2508514668)
100
100
0.1
Desulfotomaculum
Staphylococcus
13 SI Figure 12. Phylogeny of the SoxZ-like gene from SAR324 to sulfur oxidizing microorganisms.
Sulfurihydrogenibium sp. YO3AOP1 (YP_001930709.1)
97
Sulfurihydrogenibium yellowstonens (ZP_04584674.1)
96
88
Sulfurihydrogenibium azorense (YP_002729641.1)
Persephonella marina (YP_002731652.1)
Hydrogenobaculum sp. Y04AAS1 (YP_002122130.1)
41
Thermocrinis albus (YP_003473526.1)
81
Hydrogenobacter thermophilus (YP_003433166.1)
56
Hydrogenivirga sp. 128-5-R1-1 (ZP_02176716.1)
72
Aquifex aeolicus (NP_214240.1)
78
Acidithiobacillus caldus (ZP_05291811.1)
84
Acidithiobacillus caldus (YP_004749566.1)
91
Acidithiobacillus thiooxidans (ZP_09995370.1)
99
Acidithiobacillus ferrivorans (YP_004784910.1)
Thiocapsa marina (ZP_08771913.1)
44
marine gamma proteobacterium HTCC2080 (ZP_01627098.1)
68
73
78
gamma proteobacterium NOR51-B (ZP_04957680.1)
Thiorhodovibrio sp. 970 (ZP_09810551.1)
93
Thiorhodococcus drewsii (ZP_08823721.1)
49
Magnetococcus marinus (YP_866895.1)
GB-SAR324 (2062278661)
99
SAR324 cluster bacterium SCGC AAA001-C10 (ZP_10757415.1)
Ca. Methylomirabilis oxyfera (YP_003206554.1)
Anaeromyxobacter sp. Fw109-5 (YP_001380040.1)
Paracoccus denitrificans (CAA55824.2)
1
14 SI Figure 13. Phylogeny of the SoxY-like gene from GB-SAR324 to sulfur oxidizing microorganisms
and phylogenetically similar Desulfoferrodoxin genes.
65
Polymorphum gilvum (SoxY, YP_004304422.1)
Roseibium sp. TrichSKD4 (SoxY, ZP_07661876.1)
Aurantimonas manganoxydans (SoxY, ZP_01225768.1)
Fulvimarina pelagi (SoxY, ZP_01439479.1 )
49
Thalassospira profundimaris (SoxY, ZP_11122281.1)
Methylobacterium populi (SoxY, YP_001926807.1)
Hyphomicrobium denitrificans (SoxY, YP_003754845.1)
Rhodopseudomonas palustris (SoxY, YP_001993924.1)
Methylosinus trichosporium (SoxY, ZP_06890155.1)
Magnetospirillum gryphiswaldense (SoxY, CAM76245.1)
75
Magnetospirillum magneticum (Hypothetical protein, YP_420219.1)
Rhodopseudomonas palustris (SoxY, P_001990583.1)
Halomonas sp. KM-1 (Hypothetical protein, ZP_10777717.1)
Bradyrhizobium sp. STM 3843 (soxY, ZP_09432158.1)
53
Methylobacterium sp. 4-46 (SoxY, YP_001773624.1)
Hyphomonas neptunium (SoxY, YP_761173.1)
43
Oceanibaculum indicum (SoxY, ZP_11128108.1)
Sinorhizobium fredii ( SoxY, YP_005191851.1)
99
Hyphomicrobium denitrificans (SoxY, YP_003755533.1)
Hyphomicrobium denitrificans (SoxY, EHB75152.1)
Methylobacillus flagellatus (Hypothetical protein, YP_546685.1)
Sulfurimonas denitrificans (SoxY, YP_392777.1)
44
Sulfurimonas gotlandica (Tat pathway signal protein, ZP_05071877.1)
Sulfurimonas autotrophica (SoxY, YP_003892053.1)
83
Sulfuricurvum kujiense (SoxY, YP_004059313.1)
Sulfurihydrogenibium azorense (Tat signal sequence domain protein, YP_002729640.1)
50
79
Sulfurovum sp. AR (SoxY, ZP_10062574.1)
Sulfurovum sp. NBC37-1 (SoxY, YP_001357815.1)
Magnetococcus marinus (SoxY, YP_867607.1)
46
Magnetococcus marinus (SoxY, YP_866894.1)
52
82
Acidithiobacillus thiooxidans (SoxY, ZP_09995124.1)
Acidithiobacillus caldus (SoxY, YP_004749567.1)
77
Acidithiobacillus caldus (SoxY, ZP_05291810.1)
80
45
Acidithiobacillus ferrivorans (Hypothetical protein, YP_004784909.1)
Acidithiobacillus thiooxidans (Hypothetical protein, ZP_09995371.1)
89
71
42
Anaeromyxobacter sp. Fw109-5 (Hypothetical protein, YP_001380040.1|)
Ca. Methylomirabilis oxyfera (hypothetical protein, YP_003206554.1)
GB-SAR324 (Desulfoferrodoxin, 2062278663)
Magnetococcus marinus (Desulfoferrodoxin, YP_866847.1)
58
67
Methanocella conradii ( Rubredoxin, YP_005381203.1)
Ferroglobus placidus (Desulfoferrodoxin, YP_003436391.1)
Paracoccus denitrificans (soxB, CAA55824.2)
0.5
15 SI Figure 14. Phylogeny of the SoxB-like gene from GB-SAR324 to sulfur oxidizing microorganisms.
Sulfitobacter sp. EE-36 (ZP00956139.1)
100
64
Sulfitobacter sp. NAS-14.1 (ZP00963375.1)
Roseobacter denitrificans OCh 114 (YP681834.1)
Rhodobacterales bacterium HTCC2150 (ZP01743245.1)
Roseovarius nubinhibens ISM (ZP00961294.1)
Sulfitobacter sp. NAS-14.1 (ZP00963529.1)
54
Sagittula stellata E-37 (ZP01748359.1)
100
Roseobacter sp. MED193 (ZP01055912.1)
Ruegeria pomeroyi DSS-3 (YP166249.1)
100
Maritimibacter alkaliphilus HTCC2654 (ZP01014859.1)
Paracoccus denitrificans (CAA55824.2)
52
Rhodovulum sulfidophilum (AAF99435.1)
Pseudaminobacter salicylatoxidans KCT001 (CAC39170.2)
62
Stappia aggregata IAM 12614 (ZP01549055.1)
81
Aurantimonas manganoxydans SI85-9A1 (ZP01225765.1)
80
Fulvimarina pelagi HTCC2506 (ZP01439476.1)
100
Bradyrhizobium japonicum USDA 110 (NP770155.1)
99
Magnetospirillum magnetotacticum MS-1 (ZP00051299.2)
97
Starkeya novella DSM 506 (AAF61448.2)
51
Rhodopseudomonas palustris CGA009 (NP949801.1)
100
Rhodopseudomonas palustris HaA2 (YP487967.1)
100
Rhodopseudomonas palustris BisB5 (YP571371.1)
90
Magnetococcus marinus MC-1 (YP865819.1)
56
Halorhodospira halophila SL1-9 (YP001003505.1)
99
100
Chlorobium limicola (AAL68888.1)
Chlorobium tepidum TLS (NP661913.1)
Chlorobium chlorochromatii CaD3 (YP380218.1)
59
Thiomicrospira crunogena XCL-2 (YP391815.1)
72
Oceanospirillum sp. MED92 (ZP01167148.1)
58
Bradyrhizobium japonicum USDA 110 (NP767649.1)
98
100
Nitrobacter hamburgensis X14 (YP578859.1)
Allochromatium vinosum (ABE01359.1)
81
Marine Gammaproteobacterium HTCC2080 (ZP01627095.1)
Polaromonas sp. JS666 (YP549439.1)
54
Ralstonia solanacearum GMI1000 (NP521372.1)
48
Ralstonia eutropha JMP134 (YP297452.1)
57
Anaeromyxobacter dehalogenans 2CP-C (YP465486.1)
Methylibium petroleiphilum PM1 (YP001021622.1)
Herminiimonas arsenicoxydans (CAL61372.1)
Thiobacillus denitrificans ATCC 25259 (YP314321.1)
Dechloromonas aromatica RCB (YP286328.1)
59
96
Hydrogenophilus thermoluteolus (BAF34125.1)
Aquifex aeolicus VF5 (NP214237.1)
Sulfurimonas denitrificans DSM 1251 (YP392780.1)
Thermus thermophilus HB27 (YP005021.1)
75
100
Thermus thermophilus HB8 (YP144683.1)
GB-SAR324
0.05
16 SI Figure 15. Genes detected only in the background cDNA metatranscriptome. Percentages are
calculated as fraction of genes found only in the background cDNA libraries.
Protein Metabolism
es
at
dr
hy
bo
Fatty Acids, Lipids, and Isoprenoids
r
Ca
ed
bas
ingster
Clu
all
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ins
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tors
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an
ids
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, Pr
ino
osth
Am
etic
Gro
ups
, Pig
me
nts
2m
ore
SI Figure 16. Genes detected only in the plume cDNA metatranscriptome. Percentages are
calculated as fraction of genes found only in the plume cDNA libraries. The four unmarked categories
at the bottom are related to (from left to right) quorum sensing and biofilm formation, macromolecule
formation and synthesis, hormone synthesis, and proteorhodopsin synthesis.
s
ipid
, an
d Is
opr
ids
eno
Clus
terin
s, L
y
Fatt
Acid
DNA Metabolism
Plume only
Cell W
all and
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Carb
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Potassiu
ism
2m
or
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re
2 mo
2 more
18 SI Figure 17. Order of genes related to SAR324’s putative soxZ and comparison to closest
organisms in IMG determined by BLASTp. Genes that could potentially be related to the SOX
system were translated by IMG and searched individually to find closest homolog proteins. Numbers
between genes represent the percent similarity score from BLAST. Genes are color coded for
reference in closest relative genomes. * Denotes that both the soxZ (30%) and the desulfoferrodoxin
(35%) were both the top hits to the Anaeromyxobacter genome. SCGC AAA001-C10 is a Dark ocean
SAR324 single amplified genome. Lines behind genes denotes whether genes were found on a
single (no breaks) or multiple contigs and the number at the beginning and end denote region of the
genome.
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IMG Annotation
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Genome Name
15000
GB-SAR324 5000
97
96
96
98
ND
94/96
99 95
93 94 94
97
94 91
SCGC AAA001-C10
36
35
Persephonella marina EX-H1 1795420
1807941
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6394474
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BLASTp Similarity
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