advances.sciencemag.org/cgi/content/full/1/9/e1500675/DC1 Supplementary Materials for Global prevalence and distribution of genes and microorganisms involved in mercury methylation Mircea Podar, Cynthia C. Gilmour, Craig C. Brandt, Allyson Soren, Steven D. Brown, Bryan R. Crable, Anthony V. Palumbo, Anil C. Somenahally, Dwayne A. Elias Published 9 October 2015, Sci. Adv. 1, e1500675 (2015) DOI: 10.1126/sciadv.1500675 This PDF file includes: Fig. S1. Whisker plot distribution of genomic and metagenomic PFam3599 protein hits to various hidden Markov profiles. Fig. S2. Distribution of HgcA and WL pathway–associated CFeSP encoding genes in methanogenic Archaea. Fig. S3. Distribution of HgcA, HgcB, and WL pathway–associated CFeSP in genomes of Deltaproteobacteria and Firmicutes. Fig. S4. Hg methylation assays for M. luminyensis B10. Fig. S5. Repeat methylation assay for M. luminyensis B10. Fig. S6. Schematic representation of the domain architectures of HgcA, HgcB, and the fusion HgcAB. Fig. S7. Hg methylation assays for P. furiosus. Fig. S8. MEGAN-based co-occurrence profiles of the closest genera-assigned HgcAs based on all metagenomes. Other Supplementary Material for this manuscript includes the following: (available at advances.sciencemag.org/cgi/content/full/1/9/e1500675/DC1) Table S1 (Microsoft Excel format). List of metagenomic projects with hgcA counts. Table S2 (Microsoft Excel format). Identity matrix of sequence similarities. HgcA 400 350 300 250 200 HgcA-B fusion 150 HgcB HgcB HgcA 100 HgcA HgcA 50 Other ferredoxins WL CFeSP 0 PF3599 Uniprot PF3599 PF3599 Meta PF3599 PF13237 Uniprot HgcA HgcA Trans HgcA-Cap HgcA-Cap HgcB HgcB Figure S1. Whisker plot distribution of genomic and metagenomic PFam3599 protein hits to various Hidden Markov profiles (HgcA, HgcA Transmembrane domain, HgcA cap helix domain and HgcB) based on hmmsearch. The Uniprot database was also used as a reference. HgcA and B proteins are grouped in the shaded boxes and have distinct high scores (Y-axis). The other members of the superfamilies (HgcA-B fusion, Wood Lungdahl CFeSP and ferredoxins) have lower scores. Pyrococcus furiosus Pyrococcus sp ST04 Pyrococcus sp NA2 Pyrococcus abyssi Thermococcales Pyrococcus yayanosii Methanocaldococcus vulcanius Methanocaldococcus jannaschii Me Methanocaldococcus fervens Methanocaldococcus sp FS406-22 t Methanocaldococcus infernus hano Methanocaldococcus villosus* Methanotorris formicicus* Methanotorris igneus c o Methanococcus aeolicus cc Methanothermococcus okinawensis ale Methanothermococcus thermolithotrophicus* Methanococcus voltae s Methanococcus vannielii Methanococcus maripaludis Me Methanothermus fervidus t Methanothermobacter thermoautotrophicum hanoba Methanothermobacter marburgensis Methanobrevibacter ruminantium Methanobrevibacter smithii Methanosphaera stadtmanae ct Methanobacterium formicicum* Methanobacterium sp Maddingley* eriale Methanobacterium sp SWAN-1 Methanobacterium sp AL-21 Methanobacterium arcticum* s Methanopyrus kandleri Methanopyrales Cand Methanomethylophilus alvus Cand Methanomassillicoccus intestinalis “Methanomassillicoccales” Methanomassillicoccus luminyensis* Ferroglobus placidus Archaeoglobus sulfaticallidus Archaeoglobus veneficus Archaeoglobales Archaeoglobus fulgidus Archaeoglobus profundus Methanoflorens stordalenmirensis Methanocella arvoryzae Methanocella paludicola Methanocellales Methanocella conradii Cand Methanoperedens nitroreducens ANME-2D* Methanosaeta harundinacea Methanosaeta thermophila Methanosaeta concilii M e uncultured ANME2a2* t Methermicoccus shengliensis* h a n Methanosarcina barkeri o Me Methanosarcina acetivorans s Methanosarcina sp SMA-21* a r t Methanosarcina mazei c hanomicrobi i Methanohalobium evestigatum nal Methanohalophilus mahii Methanococcoides burtonii e Methanosalsum zhilinae s Methanomethylovorans hollandica Methanolobus psychrophilus Methanolobus tindarius* a Me Methanospirillum hungatei Methanosphaerula palustris t Methanoculleus bourgensis hanomicrobiale Methanoculleus sp MH98A* Methanoculleus marisnigri Methanoregula formicicum Methanoregula boonei Methanofollis liminatans Methanoplanus petrolearius Methanoplanus limicola* Methanomicrobium mobile* Methanocorpusculum bavaricum* Methanocorpusculum labreanum s Methanolinea tarda 0.2 WL CFeSP HgcA Figure S2. Distribution of HgcA and Wood-Lungdahl CFeSP encoding genes in methanogenic Archaea, relative to their phylogenetic classification based on SSU rRNA gene. The Pyrococcus genus (Thermococcales, Euryarchaeota) was used as outgroup as one of its species encodes an hgcA-B fusion gene. Syntrophorhabdus aromaticivorans Bacteria ferrireducans Syntrophobacterales Bdellovibrio bacteriovorus Stigmatella aurantiaca Deltaproteobacteria Corallococcus coralloides Myxococcus stipitatus Myxococcus xanthus Myxococcus fulvus Anaeromyxobacter sp Myxococcales Anaeromyxobacter dehalogenans Plesiocystis pacifica Haliangium ochraceum Sorangium cellulosum Geopsychrobacter electrodiphilus Pelobacter carbinolicus Desulfuromonas acetoxidans Geobacter lovleyi Desulfuromonadales Pelobacter propionicus Geobacter metallireducens Geobacter sulfurreducens KN400 Geobacter sp M18 Geobacter bemidjiensis Geobacter sp M21 Geobacter sp FRC32 Geobacter uraniumreducens Syntrophus aciditrophicus Desulfomonile tiedjei Syntrophobacterales Syntrophobacter fumaroxidans Desulfarculus baarsii Desulfarculales Desulfobacca acetoxidans Delta proteobacterium NaphS2 Desulfatibacillum alkenivorans Desulfococcus oleovorans Desulfobacterium sp.N47 Desulfobacter postgatei Desulfobacter curvatus Desulfotignum phosphitoidans Desulfobacterales Desulfotignum balticum Desulfobulbus sp. oral 41 Desulfosarcina sp Desulfobulbus propionicus Desulfobulbus japonicus Desulfobulbus mediterraneus Desulfotalea psychrophila Desulfurivibrio alkaliphilus delta proteobacterium MLMS1 Desulfonatronum lacustre Desulfovibrio alaskensis Desulfovibrio acrylicus Desulfovibrio sp A2 Desulfovibrio termitidis Desulfovibrio cuneatus Desulfovibrionales Desulfovibrio desulfuricans Desulfovibrio piger Desulfovibrio sp 3 1 Desulfovibrio sp. oral taxon 40 Lawsonia intracellularis Bilophila sp. Desulfovibrio vulgaris Hildenborough Desulfovibrio gigas Desulfovibrio africanus Desulfovibrio inopinatus Desulfovibrio putealis Desulfovibrio magneticus Desulfovibrio fructosovorans Desulfovibrio sp FW1012B Desulfovibrio sp U5L Desulfovibrio piezophilus Desulfovibrio aespoeensis Desulfovibrio desulfuricans ND132 Desulfovibrio oxyclinae Desulfovibrio longus Desulfovibrio aminophilus Desulfovibrio bastinii Desulfovibrio hydrothermalis Desulfovibrio salexigens Desulfovibrio zosterae Desulfomicrobium baculatum Desulfomicrobium escambiense Desulfonatronospira thiodismutans Desulfohalobium retbaense Caldicellulosiruptor obsidiansis Acetohalobium arabaticum Halanaerobium sp sapolanicus Halanaerobium praevalens Halanaeorobiales Dethiobacter alkaliphilus Firmicutes Natranaerobius thermophilus Natranaerobiales Moorella thermoacetica Desulfonispora thiosulfatigenes Thermoanaerobacterales Desulfitobacterium dichloroeliminans Desulfitobacterium dehalogenans Desulfitobacterium sp PCE1 Desulfitobacterium hafniense (5 strains) Desulfitobacterium sp LBE Syntrophobotulus glycolicus Dehalobacter restrictus; D.sp. FTH1 Dehalobacter sp 11DCA Desulfosporosinus acidophilus Desulfosporosinus youngiae Desulfosporosinus orientis Desulfosporosinus meridiei Clostridiales Desulfosporosinus sp OT Desulfitobacterium metallireducens Thermincola sp JR Desulfotomaculum kuznetsovii Desulfotomaculum acetoxidans Desulfotomaculum ruminis Desulfotomaculum carboxydivorans Desulfotomaculum reducens Desulfotomaculum gibsoniae Pelotomaculum thermopropionicum Desulfurispora thermophila Candidatus Desulforudis audaxviator Ammonifex degensii Carboxydothermus hydrogenoformans Acidaminococcus fermentans Dialister invisus Veillonella parvula Megasphaera elsdenii Pelosinus sp HCF1 Selenomonas sputigena Selenomonadales Zymophilus raffinosivorans Anaeromusa acidaminophila Thermosinus carboxydivorans Acetonema longum Sporomusa sp KB1 Syntrophothermus lipocalidus Syntrophomonas wolfei Clostridiales Thermaerobacter subterraneus Tepidanaerobacter acetatoxydans Thermosediminibacter oceani Thermoanaerobacterales Thermoanaerobacter tengcongensis Thermoanaerobacter ethanolicus Thermoanaerobacter brockii Blautia hydrogenotrophica Butyrivibrio fibrisolvens Bryantella formatexigens Blautia hansenii Ruminococcus torques Oscillibacter valericigenes Ethanoligenens harbinense Anaerotruncus colihominis Ruminococcus albus Faecalibacterium prausnitzii Subdoligranulum sp Anaerofustis stercorihominis Clostridiales Acetobacterium woodii Anaerococcus prevotii Anaerococcus hydrogenalis Anaerococcus vaginalis Clostridium difficile Anaerococcus senegalensis Alkaliphilus metalliredigens Acetivibrio cellulolyticus Clostridium termitidis CFeSP Caloramator sp ALD01 Clostridium acetobutylicum Clostridium ragsdalei HgcA,B Clostridium ljungdahlii Clostridium carboxidivorans 0.2 Figure S3. Distribution of HgcA, HgcB and WL CFeSP in genomes of Deltaproteobacteria and Firmicutes relative to other sequenced members of those taxa. The RNA polymerase subunits B-B’ sequences were used to construct’ a maximum likelihood phylogenetic tree (circles at major nodes denote bootstrap supports >50, nodes <50 were collapsed). 1,000 100.0% T0 T0 Tend Tend 100 10.0% M p g , H g e H 10 1.0% 1 M 0 2 % e M 1 0.1% 0 0.0% culture spent medium sterile medium culture spent medium sterile medium 700 10 T0 600 n Tend i 8 e t 500 o r M p d p g e , 6 c 400 μ g / u H d 1 o Hg 20 300 e pr 4 M