SUPPLEMENTAL INFORMATION
Detection of a key Hg methylation gene, hgcA, in wetland soils.
J. K. Schaefer1*, R-M. Kronberg2, F. M. M. Morel1, and U. Skyllberg2
1
Dept of Geosciences, Princeton University, Princeton, NJ 08544
2
Department of Forest Ecology and Management, Swedish University of Agricultural Sciences, SE-901 83
Umeå, Sweden
*Corresponding author
Running Title: Detection of the Hg methylation gene, hgcA, in soils
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SUPPLEMENTAL INFORMATION
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Growth of bacterial cultures for DNA extraction:
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Geobacter sulfurreducens PCA was grown under fumarate-reducing conditions as described in Schaefer
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et al 2011. Desulfovibrio sp. ND132 was grown under sulfate-reducing conditions as described in
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Gilmour et al. 2011 (Gilmour et al., 2011). Strains Desulfovibrio africanus, Desulfobulbus propionicus,
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and Desulfococcus multivorans were grown on sulfate as described in Ekstrom et al 2003 (Ekstrom et al.,
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2003). All other strains were grown in medium as recommended by ATCC (D. desulfuricans ATCC 27774)
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or DSMZ (remaining strains). DNA was extracted from cells using the UltraClean Microbial DNA Isolation
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kit (MoBio, Carlsbad, CA).
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Testing Hg methylation capacity in bacterial cultures:
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Desulfobacterium autotrophicum and Desulfosarcina variabilis were grown under sulfate-reducing
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conditions in DSMZ medium 383 and 193, respectively, and tested for the capacity to methylate Hg(II).
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Desulfovibrio sp. ND132 grown under sulfate-reducing conditions (Gilmour et al., 2011) was used as a
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positive control. Cells and sterile medium were exposed to 50 nM Hg(II) for 24 hours during mid-
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exponential growth and stored frozen until analysis. Methylmercury was analyzed by distillation (Tekran
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2750 Distillation System), followed by ethylation with tetraethylborate, and GC-CVAFS detection on a
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Tekran 2700 Methyl Mercury System (Schaefer et al., 2011). Results are shown in Fig. SI2.
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PCR amplification, cloning and sequencing of hgcA amplicons:
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Approximately 1 g soil was preserved with 3 ml LifeGuard solution (MoBio, Carlsbad, CA) from each site
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immediately after collection and stored at -80oC upon returning to the lab. Genomic DNA was extracted
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from soil following manufacturer’s protocol (MoBio, Carlsbad) using the PowerSoil Isolation and
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PowerClean DNA Clean-Up kits. Purified DNA from all samples (D. multivorans, native soil, and
2
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enrichments) was quantitated using Quant-iT Picogreen dsDNA kit (Life Technologies, USA). hgcA was
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PCR amplified in either 50 or 100 µl volumes with 1x Phusion buffer, 0.2 mM dNTPs, 3% DMSO, 0.5 µM
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each primer, 50 ng genomic DNA, and 1 U Phusion (NEB, Ipswich, MA) for an initial denaturation of 2
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min at 98oC followed by 35 cycles (10 s at 96oC, 30 s 60oC, and 45 s at 72oC). Amplification products were
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verified by gel electrophoresis on a 1% agarose gel stained with ethidium bromide. While multiple bands
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were observed from cultured isolates, typically only a single band (~650 bp) was observed from
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environmental DNA. This 650 bp band was excised from the gel and gel-purified using QiaQuick Gel
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Extraction kit (Qiagen, Valencia, CA). The isolated PCR product ends were adenylated by incubation with
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Taq polymerase (1U Taq , 1x Taq buffer, 0.2 mM dATP) for 15 min at 72oC and ligated into pGEM T-Easy
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vector (Promega, Madison, WI) overnight at 4oC according to manufacturer’s protocol. The ligated
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vector was subsequently cloned into chemically competent TOP10 cells (Life Technologies, USA), and
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transformants selected for on ampicillin-LB plates using blue-white screen. Plasmids were extracted
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from individual clones grown in broth overnight using QIAprep Spin MiniPrep Kit (Qiagen, Valencia, CA).
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The individual hgcA amplicons were sequenced from purified plasmids by Genewiz (South Plainfield, NJ)
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using M13F(-47) sequencing primer, and trimmed of the pGEM vector sequence. Only high quality
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sequences were kept for further study. Any sequences which contained ambiguous nucleotides or other
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problems were sequenced on the complementary strand using the M13R primer. Sequencing problems
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which could not be resolved by resequencing were discarded from further analysis. Sequences obtained
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from each library were analyzed by a TBLASTN search (http://blast.ncbi.nlm.nih.gov/) of translated
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nucleotide in the database using a protein query and evaluated by sequence alignment to other known
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hgcA sequences.
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Preparation of soil enrichments:
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Soil enrichments were prepared in triplicate in an anaerobic glove box (Coy) using freshly collected soil
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from sites B2 and B7 in southern Sweden (August 2012). For each enrichment, 8 g soil and 8 ml site
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water (0.2 µm filtered and N2-bubbled) was added to 50 ml serum bottles and sealed with butyl rubber
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stoppers. The enrichments varied by amendment: Control (no addition), +SO4 (+0.5 mM each sulfate
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and organic substrate), and +Fe (+0.5 mM each colloidal Fe(III) and organic substrate). The organic
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substrate was a mixture of equimolar sodium salts of propionate, butyrate, acetate, lactate, and
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ethanol. Each enrichment was then flushed with N2 gas and incubated at room temperature for 14 days
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in the dark. After two weeks, the enrichments were spiked with 30 and 45 ppb 198Hg(NO3)2 and
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incubated for an additional 48 h to allow for Hg methylation, after which the enrichments were analyzed
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for methane concentration and subsampled for DNA extraction (0.5 g) and CH3198Hg extraction (4 g) and
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analyzed by GC-ICPMS (Tjerngren et al., 2012).
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Methane analysis
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Methane samples were taken from the headspace of each serum bottle while in the glove box, and
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transferred into evacuated 22 mL glass GC-vials using a syringe with a three-way stop valve to keep the
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sample in the syringe during the transfer. The samples were analyzed on a gas chromatograph (Clarus
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580, Perkin Elmer Autosystem, Waltham, MA, USA) equipped with a TurboMatrix 110 headspace
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autosampler, a HeySep Q column and a flame ionization detector. Temperatures for the detector, oven
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and the injector were 100, 35 and 50oC, respectively. Nitrogen gas was used as the carrier gas at a rate
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of 40 mL min-1.
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REFERENCES
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Ekstrom, E.B., Morel, F.M.M., Benoit, J.M. (2003) Mercury methylation independent of the acetylcoenzyme A pathway in sulfate-reducing bacteria. Appl Environ Microbiol, 69: 5414-5422.
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70
71
72
73
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75
76
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Gilmour, C.C., Elias, D.A., Kucken, A.M., Brown, S.D., Palumbo, A.V., Schadt, C.W., Wall, J.D. (2011)
Sulfate-reducing bacterium Desulfovibrio desulfuricans ND132 as a model for understanding
bacterial mercury methylation. Appl Environ Microbiol, 77: 3938-3951.
Schaefer, J.K., Rocks, S.S., Zheng, W., Liang, L., Gu, B., Morel, F.M.M. (2011) Active transport, substrate
specificity, and methylation of Hg(II) in anaerobic bacteria. Proceedings of the National Academy
of Sciences, 108: 8714-8719.
Tjerngren, I., Karlsson, T., Björn, E., Skyllberg, U. (2012) Potential Hg methylation and MeHg
demethylation rates related to the nutrient status of different boreal wetlands.
Biogeochemistry, 108: 335-350.
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80
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Fig. SI1: A nucleotide sequence alignment of each forward and reverse hgcA-targeted PCR primers to select genomic sequences. Mismatched
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base pairs are indicated in red.
Species
Desulfovibrio sp. ND132
Desulfovibrio aespoeensis
Desulfobulbus propionicus
Desulfomicrobium baculatum
Desulfovibrio africanus
Geobacter sulfurreducens
Geobacter bemidjiensis
Geobacter metallireducens
Geobacter uraniireducens
Geobacter daltonii
Desulfomonile tiedjei
Syntrophus aciditrophicus
Gene Locus
Dnd132_1056
Daes_2662
Despr_0439
Dbac_0376
Desaf_0117
GSU1440
Gbem_1183
Gmet_1240
Gura_0480
Geob_2483
Desti_1022
SYN_00351
Binding
position
261
249
261
297
237
228
282
228
219
228
249
411
hgcA_261F forward primer
CGGCATCAAYGTCTGGTGYGC
CGGCATCAACGTCTGGTGCGC
CGGCATCAACATCTGGTGCGC
AGGGATCAATGTCTGGTGTGC
CGGCATCAACATCTGGTGCGC
GGGCGTCAACGTCTGGTGCGC
CGGCATTAATGTCTGGTGCGC
CGGCATCAACGTCTGGTGTGC
CGGGATCAATGTCTGGTGCGC
CGGCATTAACGTCTGGTGTGC
CGGCATCAATGTCTGGTGCGC
GGGAATAAATGTCTGGTGTGC
CGGGATCAATGTCTGGTGTGC
Binding
position
912
903
915
939
891
870
918
870
855
864
915
1080
hgcA_912R reverse primer
GGTGTAGGGGGTGCAGCCSGTRWARKT
GGTGTAGGGGGTCGAGCCCGTGAAATT
GGTGTAGGGCGTCGAGCCGGTGAAGTT
GGTGAAGGGGGTGGAGCCGGTGAAATT
CGTGTACGGCGTGGACCCGGTGAAGTT
AGTGAAGGTGCTCGATCCGGTGTAGTT
GGTAAAGGGCGTACTTCCGGTAAACGT
GGTGTAGGTGGTGCAGCCGGTGAAATT
GGTGAAGGGGGTGCTGCCGGTGAAATT
GGTAAACGTGGTGCACCCCGTGAAATT
GGTATAGGTGGAACAGCCGGTAAAATT
CGTGTACGTGGAACAACCGGTAAAATT
AGTGATCGGCGTGGAACCGGTGAATTT
Methanosphaerula palustris
Methanoregula boonei
Methanocella paludicola
Methanolobus psychrophilus
Methanospirillum hungatei
Mpal_1034
Mboo_0422
MCP_0718
Mpsy_0587
Mhun_0876
228
288
255
327
285
TGGAGTCAATGTCTGGTGTGC
AGGCGTAAATGTCTGGTGCGC
GGGCATCAACGTCTGGTGTGC
GGGAGTTAATGTGTGGTGTGC
AGGGATTAATGTCTGGTGTGC
849
912
876
951
909
GGTGAACGGGGTCGCACCAGTGAAGAG
GGTAAACGTGGTCGAGCCCGTGAAGTT
CGTGAAGGTCGAACATCCGGTAAAGTT
AGTGAATGTTGTACAACCTGTAAAGTT
AGTATATGGCGTGGACCCGGTAAAATT
Desulfosporosinus acidiphilus
Desulfosporosinus orientis
Desulfitobacterium
dehalogenans
Dehalobacter sp. CF
Syntrophobotulus glycolicus
Ethanoligenens harbinense
Desaci_1621
Desor_2652
261
261
AGGAATTAACGTTTGGTGTGC
GGGAGTCAATGTTTGGTGCGC
903
903
AGTATATGTTGAAGAGCCTGTGAAATT
GGTATAGGTTGAAGAGCCTGTAAAATT
Desde_2772
DCF50_p1170
Sgly_2352
Ethha_0975
261
408
318
348
GGGAATAAATGTATGGTGTGC
AGGCATTAATGTCTGGTGCGC
AGGTGTTAACGTCTGGTGCGC
TGGTGTGAATGTCTGGTGCGC
903
1050
954
984
AGTGTACGTTGAAGAACCAGTAAAATT
AGTATATGTTGAAGATCCGGTGAAGTT
CGTGTAGGTCGACGAGCCTGTGAAATC
CGTATAGGTTGAGCTTCCGGTAAAGTT
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Table SI1: The number of hgcA-like and non-hgcA sequences detected in each clonal library.
Library
ENP1
ENP3
B7
B2
B2_C
B2_SO4
B2_Fe
85
86
a
87
88
89
90
b
Description
Native soil
Native soil
Native soil
Native soil
Control Incubation
Sulfate Enrichment
Fe Enrichment
Total Sequences
9
8
29
34
27
17
18
hgcAa
9
8
29
25
18
9
17
Other (not hgcA)b
0
0
0
9
9
8
1
Eight of these reported hgcA sequences were dropped from further analysis due to the presence of
ambiguous bases not resolved by re-sequencing.
Sequences found not to be hgcA but detected in libraries included (no. of clones): benzoylCoA
reductase (6), Rieske 2Fe-2S domain containing protein (2), a glycosidase in the Type II secretory
pathway (7), L-aspartate oxidase (1), a Signal peptide protein (1), and the remaining were <400 bp and
unknown.
91
8
92
93
Table SI2: A description of the different OTUs and their similarity to other known hgcA-containing
microorganisms.
OTUa
No of clones
Sweden (Boreal)
ENP (Tropical)
B7
B2
B2 ENRb
ENP1
ENP3
1
28
2
3
11
3
1
3
6
4
B7-02, -03, -06, -15, -17, -26
1
B2-31
ENP1-01, -03, -04, -06, -09
5
5
4
6
hgcA clone IDc
B2_C-01, -02, -04, -07, -08, -10,
-15, -16, -17, -18, -20, -23, -27,
-28, B2_Fe-05, -07, -08, 09, -12, -15, -16, -19, B2_SO402, -03, -05, -08, -09, -10
ENP3-09
B2-06, -08, -13, -14, -17, 18, -21, -24, -27, -33, -34
B2_C-03, B2_Fe-03,
B2_SO4, -07, ENP1-02, -05, -08
1
7
ENP3-01, -02, -03, -05
2
B2-28, B2_Fe-02, -06
2
B2_C-13, B2_Fe-17
8
2
B7-18, B7-21
9
2
B7-04, -09
10
2
B7-14, -16
11
2
12
1
B2-15, -29
1
B2-26, ENP1-07
13
2
B7-12, -20
14
2
B7-01, -07
15
2
16
1
B2_Fe-10, B2_SO4-11
B2-10
17
1
B7-33
18
1
B7-34
19
1
B7-11
20
1
B2_C-14
21
1
B2_C-25
22
1
B2-25
23
1
B7-10
24
1
B7-25
25
1
B2-30
9
Closest match to GenBank by
TBLASTN search, accession
no., locus tag (% aa identity)
Inferred
Phylogeny
Methanocella arvoryzae,
AM114193, RCIX2342 (61%)
Methanomicrobia
Desulfobulbus propionicus
CP002364, Despr_0439 (52%)
-Proteobacteria
SRB
Geobacter uraniireducens
CP000698, Gura_0480 (64%)
Only distantly related to
sequenced genomes
Methanolobus psychrophilus,
CP003083, Mpsy_0587 (70%)
Geobacter uraniireducens
CP000698, Gura_0480 (74%)
Geobacter uraniireducens
CP000698, Gura_0480 (67%)
Geobacter uraniireducens
CP000698, Gura_0480 (69%)
Geobacter uraniireducens
CP000698, Gura_0480 (67%)
Geobacter uraniireducens
CP000698, Gura_0480 (71%)
Desulfobulbus propionicus
CP002364, Despr_0439 (54%)
Desulfomonile tiedjei,
CP003360, Desti_1022 (57%)
Only distantly related to
sequenced genomes
Only distantly related to
sequenced genomes
Only distantly related to
sequenced genomes
Geobacter uraniireducens
CP000698, Gura_0480 (66%)
Geobacter uraniireducens
CP000698, Gura_0480 (65%)
Geobacter uraniireducens
CP000698, Gura_0480 (66%)
Geobacter uraniireducens
CP000698, Gura_0480 (69%)
Geobacter uraniireducens
CP000698, Gura_0480 (73%)
Geobacter uraniireducens
CP000698, Gura_0480 (68%)
Geobacter uraniireducens
CP000698, Gura_0480 (73%)
Geobacter uraniireducens
CP000698, Gura_0480 (71%)
Geobacter uraniireducens
CP000698, Gura_0480 (68%)
Geobacter uraniireducens
CP000698, Gura_0480 (70%)
-Proteobacteria
FeRB
Unknown
Methanomicrobia
-Proteobacteria
FeRB
-Proteobacteria
FeRB
-Proteobacteria
FeRB
-Proteobacteria
FeRB
-Proteobacteria
FeRB
-Proteobacteria
SRB
-Proteobacteria
SRB
Unknown
Unknown
Unknown
-Proteobacteria
FeRB
-Proteobacteria
FeRB
-Proteobacteria
FeRB
-Proteobacteria
FeRB
-Proteobacteria
FeRB
-Proteobacteria
FeRB
-Proteobacteria
FeRB
-Proteobacteria
FeRB
-Proteobacteria
FeRB
-Proteobacteria
FeRB
26
1
B7-19
27
1
B7-08
28
1
B2-07
29
1
B2-03
30
1
B7-32
31
1
ENP3-04
32
1
ENP3-07
33
34
1
1
35
36
37
B2_Fe-13
B7-28
1
B2-19
1
B7-31
1
38
B2-23
1
ENP3-06
39
1
B2-35
40
1
B2-32
94
a
95
bENR
96
97
c
Geobacter uraniireducens
CP000698, Gura_0480 (66%)
Geobacter uraniireducens
CP000698, Gura_0480 (%)
Dehalococcoides mccartyi,
CP004079, dcmb_330 (54%)
Dehalococcoides mccartyi,
CP004079, dcmb_330 (55%)
Dehalococcoides mccartyi,
CP004079, dcmb_330 (55%)
Methanosphaerula palustris,
CP001338, Mpal_1034 (63%)
Methanoregula formicicum,
CP003167, Metfor_0951 (75%)
Methanolobus psychrophilus,
CP003083, Mpsy_0587 (59%)
Only distantly related to
sequenced genomes
Only distantly related to
sequenced genomes
Only distantly related to
sequenced genomes
Only distantly related to
sequenced genomes
Only distantly related to
sequenced genomes
Only distantly related to
sequenced genomes
Only distantly related to
sequenced genomes
-Proteobacteria
FeRB
-Proteobacteria
FeRB
Chloroflexi
Chloroflexi
Chloroflexi
Methanomicrobia
Methanomicrobia
Methanomicrobia
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
An OTU is defined as all hgcA clones exhibiting > 90% deduced amino acid identity.
= enrichments
Clone ID names are prefaced with the site location. Clones from B2 enrichments are further denoted as “_C”, “_Fe”, and “_SO4” for
unamended, +Fe(III), and +SO4 enrichments.
98
99
10