Supplementary Figures for Photosynthesis is not a Universal Feature of the Phylum Cyanobacteria Rochelle M. Soo, Connor T. Skennerton, Yuji Sekiguchi, Michael Imelfort, Samuel J. Paech, Paul G. Dennis, Jason A. Steen, Donovan H. Parks, Gene W. Tyson, and Philip Hugenholtz† † Correspondence to: Philip Hugenholtz, [email protected] Sample collection Zagget feces (3 samples), EBPR (9 samples) and UASB (4 samples) DNA extraction MP-BIO FASTSPIN spin kit and bead-beating Paired end sequencing Community Profiling Upload data from MetaHIT database Illumina HiSeq and MiSeq 16SrRNA gene amplified and clustered to identify taxonomy (7 samples) CLC Assembly Samples co-assembled for GroopM GroopM Population genome binning based on coverage patterns CheckM Check for completion and contamination of standard draft population genome bins Phlylogenetic placement Phylogenetic placement of standard draft population genome bins using complete bacterial genomes from IMG and Melainabacteria Mate pair sequencing Illumina HiSeq and MiSeq SSpace scaffolding Improvement of draft population genome bins CheckM Check for completion and contamination of standard draft population genome bins IMG-ER submission 16S rRNA gene reconstruction Phylogenetic placement Automated annotation - HMM models of the 16S rRNA - Mapped to Greengenes using BWA-MEM Phylogenetic placement of standard draft population genome bins Metabolic reconstruction Kegg maps 16S rRNA gene trees - ML, MP and NJ Concatenated gene trees - 83 and 38 single copy marker genes -ML, MP and NJ Fig. S1. Flow diagram of the Methods used in this paper ML (RAxML, JTT+Gamma, 100x bootstraping) rooted with Archaea 100 c__Gammaproteobacteria c__Betaproteobacteria Mariprofundus ferrooxydans c__Alphaproteobacteria 100 98 68 62 100 100 100 95 100 p__Acidobacteria p__Nitrospirae 100 100 100 p__Bacteroidetes 100 100 74 75 100 68 98 Ca. Cloacamonas acidaminovorans p__Fibrobacteres Gemmatimonas aurantiaca p__Spirochaetes 100 100 p__Verrucomicrobia Lentisphaera araneosa HTCC2155 p__Planctomycetes 100 89 p__Elusimicrobia 100 p__Fusobacteria 100 P__Tenericutes 100 c__Oxyphotobacteria 100 MEL_C1 100 MH_37 Zag_1 100 MEL_A1 94 Zag_111 91 100 Ca. Gastranaerophilus phascolarctosicola 100 MEL_B1 100 MEL_B2 ACD20 Ca. Obscuribacter phosphatis Ca. Caenarcanum bioreactoricola p__Armatimonadetes 99 79 100 100 86 MP (PAUP*, 100x bootstraping, >50% consensus tree) 100 87 p__Thermi Ca. Saccharimonas aalborgensis 59 64 100 p__Firmicutes 100 100 97 100 79 100 0.10 p__Synergistetes Caldatribacterium p__Thermotogae Coprothermobacter proteolyticus DSM5265 Caldisericum exile AZM16c01 p__Dictyoglomus Thermodesulfobium narugense Na82 p__Aquificae c__Oxyphotobacteria MEL_C1 100 MH_37 Zag_1 100 97 MEL_A1 Zag_111 64 100 Ca. Gastranaerophilus phascolarctosicola 100 MEL_B1 MEL_B2 ACD20 80 Ca. Obscuribacter phosphatis Ca. Caenarcanum bioreactoricola 100 p__Chloroflexi 100 85 c__Oxyphotobacteria 100 MEL_C1 100 MH_37 Zag_1 100 MEL_A1 98 Zag_111 95 100 Ca. Gastranaerophilus phascolarctosicola 100 MEL_B1 100 MEL_B2 ACD20 Ca. Obscuribacter phosphatis Ca. Caenarcanum bioreactoricola p__Cyanobacteria p__Actinobacteria 100 96 80 79 c__Oxyphotobacteria 100 MEL_C1 100 MH_37 Zag_1 100 94 MEL_A1 91 Zag_111 100 Ca. Gastranaerophilus phascolarctosicola 100 MEL_B1 100 MEL_B2 ACD20 Ca. Obscuribacter phosphatis Ca. Caenarcanum bioreactoricola ML (FastTree, JTT+CAT, 100x bootstraping) on RAxML tree p__Chlamydiae 100 99 99 99 p__Chlorobi 100 68 99 p__Chrysiogenetes 100 100 100 c__Epsilonproteobacteria p__Deferribacteres 100 63 100 100 c__Deltaproteobacteria 100 87 65 100 73 ML (RAxML, JTT+Gamma, 100x bootstraping) 89 Fig. S2. Phylogeny of Oxyphotobacteria and Melainabacteria genomes among the bacterial phyla based on up to 38 marker genes Phylogenetic maximum likelihood (RAxML, JTT, G) and maximum parsimony (PAUP*) trees based on a concatenated alignment of up to 38 marker genes, showing the phylogenetic robustness of the phylum Cyanobacteria and the classes Oxyphotobacteria and Melainabacteria. 422 OTUs (operational taxonomic units) from Bacteria and Archaea were used to produce the phylogenetic tree (Table S4). Bootstrap analyses (100 times) were performed for the data set with maximum likelihood (RAxML, JTT, G; FastTree, JTT, CAT) and maximum parsimony (PAUP*) methods, and the values obtained are shown in respective trees, except for the values from FastTree, which are shown on the tree generated with RAxML bootstrapping. Genomes in green are representatives from Di Rienzi et al., 2013 and genomes in red are Melainabacteria from this study. Bootstrap values >60% are shown. p_ represents phylum and c_ represents class. Candidatus has been abbreviated to Ca. for the most complete genomes. ML (RAxML, JTT+Gamma, 100x bootstraping) 100 c__Oxyphotobacteria ML (RAxML, JTT+Gamma, 100x bootstraping) unrooted 100 c__Gammaproteobacteria c__Alphaproteobacteria 100 100 98 Mariprofundus ferrooxydans PV-1 c__Betaproteobacteria c__Deltaproteobacteria o__Acidithiobacillales ML (FastTree, JTT+CAT, 100x bootstraping) on RAxML tree p__Elusimicrobia p__Acidobacteria 100 p__Spirochaetes p__Deferribacteres c__Epsilonproteobacteria 100 MEL_C1 100 MH_37 Zag_1 100 MEL_A1 100 Zag_111 69 100 Ca. Gastroanaerophilus phascolarctosicola 100 MEL_B1 MEL_B2 ACD20 Ca. Obscuribacter phosphatis Ca. Caenarcanum bioreactoricola p__Chrysiogenetes p__Nitrospirae p__Planctomycetes c__Oxyphotobacteria 100 Lentisphaera araneosa p__Aquificae p__Verrucomicrobia p__Cyanobacteria 100 100 p__Chlamydiae c__Oxyphotobacteria 86 Gemmatimonas aurantiaca 100 MEL_C1 100 MH_37 100 Zag_1 100 MEL_A1 Zag_111 100 Ca. Gastroanaerophilus phascolarctosicola 100 MEL_B1 MEL_B2 ACD20 Ca. Obscuribacter phosphatis Ca. Caenarcanum bioreactoricola Ca. Cloacamonas acidami c__Melainabacteria p__Chlorobi p__Actinobacteria p__Thermi p__Chloroflexi p__Dictyoglomus p__Fusobacteria MP (PAUP*, 100x bootstraping, >50% consensus tree) p__Bacteroidetes 100 c__Oxyphotobacteria p__Synergistetes 100 100 p__Thermotogae Coprothermobacter proteolyticus 100 100 100 63 100 100 p__Firmicutes 0.10 100 68 MEL_C1 MH_37 Zag_1 MEL_A1 Zag_111 Ca. Gastroanaerphilus phascolarctosicola MEL_B1 MEL_B2 ACD20 Ca. Obscurbacter phosphatis Ca. Caenarcanum bioreactoricola Fig. S3. Phylogeny of Oxyphotobacteria and Melainabacteria among the bacterial phyla based on up to 83 marker genes Phylogenetic maximum likelihood (RAxML, JTT, G) and maximum parsimony (PAUP*) trees based on a concatenated alignment of up to 83 marker genes, showing the phylogenetic robustness of the phylum Cyanobacteria and the classes Oxyphotobacteria and Melainabacteria. 322 OTUs from Bacteria were used to produce the phylogenetic tree (Table S4). Bootstrap analyses (100 times) were performed for the data set with maximum likelihood (RAxML, JTT, G; FastTree, JTT, CAT) and maximum parsimony (PAUP*) methods, and the values obtained are shown in respective trees, except for the values from FastTree, which are shown on the tree generated with RAxML. Genomes in green are representatives from Di Rienzi et al., 2013 and genomes in red are Melainabacteria from this study. Bootstrap values >60% are shown. p_ represents phylum, c_ represents class and o_ represents order. Candidatus has been abbreviated to Ca. for the most complete genomes. ML (FastTree, JTT+CAT, 100x bootstraping) on RAxML tree ML (RAxML, JTT+Gamma, 100x bootstraping) 100 100 70 Group A 99 71 100 71 100 100 100 100 100 100 100 100 100 100 100 Group B Group D Geitlerinema sp. PCC 7407 Group E 100 100 Group C Group F 100 99 100 99 100 100 92 100 100 100 100 100 100 Group G 100 Gloeobacter violaceus PCC 7421 100 MEL_C1 100 MH_37 100 Zag_1 MEL_A1 Zag_111 100 100 Ca. Gastranaerophilus phascolarctosicola 100 MEL_B1 MEL_B2 ACD20 Ca. Obscuribacter phosphatis Ca. Caenarcanum bioreactoricola p__Chloroflexi 100 100 100 99 100 0.10 Group B Group A Group D Geitlerinema sp. PCC 7407 Group E Group C Group F Group G Gloeobacter violaceus PCC 7421 100 MEL_C1 100 MH_37 100 Zag_1 MEL_A1 100 Zag_111 100 Ca. Gastranaerophilus phascolarctosicola 100 MEL_B1 MEL_B2 ACD20 Ca. Obscuribacter phosphatis Ca. Caenarcanum bioreactoricola p__Chloroflexi 0.10 MP (PAUP*, 100x bootstraping, >50% consensus tree) 100 100 Group A 73 99 100 97 100 100 100 100 100 100 0.10 100 85 100 100 Group B Group D Geitlerinema sp. PCC 7407 Group E 100 Group C Group F 100 Group G Gloeobacter violaceus PCC 7421 100 MEL_C1 100 MH_37 100 Zag_1 MEL_A1 100 Zag_111 Ca. Gastranaerophilus phascolartosicola 100 100 MEL_B1 MEL_B2 ACD20 Ca. Obscuribacter phosphatis 67 Ca. Caenarcanum bioreactoricola p__Chloroflexi Fig. S4. Phylogeny of Oxyphotobacteria and Melainabacteria in the Cyanobacteria phylum based on up to 83 marker genes Phylogenetic maximum likelihood (RAxML, JTT, G) and maximum parsimony (PAUP*) trees based on a concatenated alignment of up to 83 marker genes, showing the phylogenetic robustness of the phylum Cyanobacteria and the classes with Chloroflexi used as the outgroup. The Oxyphotobacteria were grouped as according to Shih et al., 2013 (Table S3). Bootstrap analyses (100 times) were performed for the data set with maximum likelihood (RAxML, JTT, G; FastTree, JTT, CAT) and maximum parsimony (PAUP*) methods, and the values obtained are shown in respective trees, except for the values from FastTree, which are shown on the tree generated with RAxML. Genomes in green are representatives from Di Rienzi et al., 2013 and genomes in red are Melainabacteria from this study. Bootstrap values >60% are shown. p_ represents phylum, c_ represents class and o_ represents order. Candidatus has been abbreviated to Ca. for the most complete genomes. 100 78 100 o__Vampirovibrioniales 100 92 o__SHAS531 100 80 o__Obscuribacteriales 100 c__Oxyphotobacteria 81 99 100 p__Microgenomates 100 p__Microgenomates 100 80 73 p__Tenericutes p__Fusobacterium 100 93 77 p__Gracilibacteria 100 p__Saccharibacteria (TM7) 98 p__Chloroflexi p__Caldithrixae 100 p__Chlorobi 95 100 p__Bacteroidetes 100 85 p__Fibrobacteres 98 100 85 p__Cyanobacteria o__Gastroanaerophilales 100 87 o__Caenarcaophilales 99 p__Gemmatimonadetes 99 c__Gammaproteobacteria 100 c__Betaproteobacteria 100 c__Alphaproteobacteria 100 c__Epsilonproteobacteria c__Deltaproteobacteria 97 p__Nitrospirae 100 p__Deferribacteres 100 p__Chrysiogenetes 100 89 p__Verrucomicrobia 100 88 p__Lentisphaerae (vadinBE97) 100 p__Chlamydiae 93 p__Omnitrophica (OP3) 100 p__Poribacteria 83 p__Planctomycetes 100 p__Aminicenantes (OP8) 100 98 p__Elusimicrobia 100 p__Acidobacteria 89 p__Aminicenantes (OP8) 100 p__Caldiserica (OP5) 100 100 p__Aerophobetes (CD12) p__Nitrospirae 100 p__Synergistetes 100 p__Acetothermia (OP1) 100 p__Armatimonadetes (OP10) 100 p__Hydrogenedentes (NKB19) 100 100 70 89 100 100 0.10 78 71 100 100 c__Spirochaetes p__Actinobacteria p__Firmicutes p__Thermi p__Thermodesulfobacteria 100 p__Calescamantes (EM19) 100 p__Aquificales 100 p__Cloacimonetes (WWE1) p__Latescibacteria (WS3) p__Thermotogae k__Archaea Fig. S5. 16S rRNA gene tree showing the phylogenetic robustness of the Cyanobacteria and its class level lineages in the bacterial domain Maximum likelihood (RAxML, GTR, G, I; FastTree, JTT, CAT) trees based on the 16S rRNA gene, showing the phylogenetic robustness of the classes Oxyphotobacteria and Melainabacteria. 418 OTUs (operational taxonomic units) from Bacteria and Archaea were used to construct the trees. Bootstrap analyses (100 times) were performed for the data set with maximum likelihood (RAxML, GTR, G, I; FastTree, JTT, CAT). Bootstrap values >60% are shown. 97 AGI38755 HydG [Clostridium stercorarium subsp. stercorarium DSM 8532] HydG [Melainabacteria] HydG 95 92 99 97 96 85 HydE Clostridium stercorarium subsp. stercorarium DSM 8532] Shewanella oneidensis] Anabaena variabilis Methanoplanus petrolearius Methanoculleus bourgensis] Treponema brennaborense Spirochaeta thermophila DSM 6578] 84 92 Clostridium ljungdahlii Ca. 95 NAD(P)-dependent iron-only dehydrogenase catalytic subunit 93 76 Rhodobacter sphaeroides] Olsenella profusa] 74 [FeFe] hydrogenase, group B1/B3 Clostridium Methanoplanus petrolearius Escherichia coli Ca. Anabaena variabilis] Ca. 99 84 Escherichia coli 99 82 85 Thermodesulfatator atlanticus] Ca. 93 HypE Klebsiella pneumoniae] Synechococcus Ca. 69 Ca. Escherichia coli Lyngbya majuscula 85 HypD 83 84 Ca. Lyngbya majuscula Nostoc Clostridium Cyanobium 66 86 Ca. 82 Ca. Olsenella profusa 96 99 93 HydF Clostridium kluyveri DSM 555] 87 72 Escherichia coli Ca. 99 76 HypF Klebsiella pneumoniae] Microcystis aeruginosa Methanoplanus petrolearius Thermococcus onnurineus 84 Ca. Hydrogenobaculum sp. HO] 97 Burkholderia pseudomallei Clostridium termitidis] Enterobacter cloacae 97 85 97 93 Obscuribacter phosphatis] 69 Escherichia coli 66 Ca. 87 95 Treponema brennaborense Clostridium lentocellum DSM 5427] 98 Microcystis aeruginosa Ca. Ca hydrogenase 4 Ni,Fe-hydrogenase III large subunit Fig. S6. Gene tree showing the hydrogenase genes found in the Melainabacteria representatives. A representative collection of hydrogenase genes publicly available from NCBI was combined with genes identified from this study and from Di Rienzi et al., 2013. Hydrogenase genes common to genomes from both studies are colored blue and grouped together. Hydrogenases found only in this study are colored red and genes only identified in the genomes of Di Rienzi et al., 2013 are colored green. A 100 100 100 MEL_C1 MH_37 Zag_1 100 MEL_A1 69 Zag_111 100 100 100 Ca. G. phascolarctosicola MEL_B1 MEL_B2 ACD20 100 Ca. O. phosphatis 98 Flg FlgA FlgB Flg C FlgD FlgE Flg F FlgG FlgH Flg I Flg J FlgK Flg L FlhN FlhA FliB Fli C FliD FliE Fli F FliG H Fli Fli I Fli J FK Fli FlliL N.S iM po FliA FliO Fli P FliQ FliR Fli S Mo T M tA OmotB pA Ca. C. bioreactoricola Gain of flagella Loss of flagella Color Key 0 1 2 3 4 5 6 B 100 97 100 71 MEL_C1 98 Zag_1 99 MH_37 100 MEL_A1 92 96 Zag_111 Ca. Gastranaerophilus phascolarctosicola 98 MEL_B1 100 MEL_B2 ACD20 Ca. Caenarcanum bioreactoricola 100 p_Chlamydiae, type III secretory flagellar biosynthesis 89 p_Proteobacteria, type III secretion protein SctV 100 0.10 C p_Chlamydia flagellar secretion protein Opitutus terrae PB90 100 Thermotoga petrophila Thermotoga maritima MSB8, DSM 3109 76 Thermovirga lienii Cas60314, DSM 17291 88 84 Thermanaerovibrio acidaminovorans Su883, DSM 6589 ACD20 100 100 MEL_B2 MEL_B1 Halanaerobium praevalens GSL, DSM 2228 Gemmata obscuriglobus UQM 2246 80 Thermodesulfatator indicus CIR29812, DSM 15286 95 88 Rhodospirillum rubrum S1, ATCC 11170 Thermocrinis albus HI 11/12, DSM 14484 99 100 Thermovibrio ammonificans Sulfurihydrogenibium azorense Az-Fu1 Desulfurispirillum indicum S5, DSM 22839 Leptospirillum ferrooxidans C2-3 Borrelia duttonii Ly 79 94 68 Planctomyces brasiliensis IFAM, flagella 80 Rhodopirellula baltica SH 1, fla 1448, DSM 5305 Leptonema illini 3055, DSM 21528 Gemmatimonas aurantiaca Alicycliphilus denitrificans BC 100 Photorhabdus luminescens laumondii TTO1 100 100 Dickeya zeae Ech1591 95 Dickeya dadantii 3937 Pusillimonas Azotobacter vinelandii DJ, ATCC BAA-1303 Terriglobus saanensis SP1PR4 100 Acidobacterium capsulatum ATCC 51196 0.10 65 Fig. S7. Heat map showing the presence and absence of flagella genes from the Melainabacteria representatives (A) A maximum parsimony tree based on a concatenated alignment of up to 83 marker genes. Purple arrows indicate the gain of a functional flagella and blue arrows indicate the loss of a functional flagella. The heat map indicates the square root of the number of flagella genes found in each of the Melainabacteria representatives. (B) Flagella gene trees for FlhA and (C) flagella gene tree for FliM. Genomes used to make the trees are found in Table S6. Genomes in green are Melainabacteria representatives from Di Rienzi et al., 2013, genomes in red are Melaianabacteria representatives from this study. Candidatus has been abbreviated to Ca. for the most complete genomes. A. MH_37 total = 2358 MH_37 516 33 220 MEL_C1 1589 Zag_1 Zag_1 total = 2156 MEL_C1 total = 2114 230 75 459 Zag_1 MH_37 MH_37 MEL_C1 B. Zag_1 MEL_C1 Fig. S8. Venn diagram of the three Gastranaerophilales genomes from the same species (A) Shared orthologs between MH_37, MEL_C1 and Zag_1 showing the number of genes that are core to the three genomes, and those that are found between two genomes and individual genomes. The total number for each genome does not include paralogs. (B) Average nucleotide identity between MH_37, MEL_C1 and Zag_1, where ≥95% ANI is used to define a species. Melainabacteria 95% confidence intervals [COG0600] ABC-type nitrate/sulfonate/bicarbonate transport system, permease < 1e-15 [COG0715] ABC-type nitrate/sulfonate/bicarbonate transport systems, periplasmic components < 1e-15 < 1e-15 [COG1116] ABC-type nitrate/sulfonate/bicarbonate transport system, ATPase component [COG1122] ABC-type cobalt transport system, ATPase component 4.60e-14 [COG3221] ABC-type phosphate/phosphonate transport system, periplasmic component [COG0025] NhaP-type Na+/H+ and K+/H+ antiporters 1.22e-12 2.44e-11 5.94e-10 2.05e-9 3.44e-8 [COG1226] Kef-type K+ transport systems, predicted NAD-binding component [COG0226] ABC-type phosphate transport system, periplasmic component [COG0609] ABC-type Fe3+-siderophore transport system, permease component [COG0474] Cation transport ATPase 1.99e-7 2.11e-7 4.43e-7 3.72e-6 [COG1117] ABC-type phosphate transport system, ATPase component [COG1629] Outer membrane receptor proteins, mostly Fe transport [COG0614] ABC-type Fe3+-hydroxamate transport system, periplasmic component [COG0475] Kef-type K+ transport systems, membrane components [COG2217] Cation transport ATPase [COG0607] Rhodanese-related sulfurtransferase q-value (corrected) Oxyphotobacteria 1.20e-5 6.81e-4 1.28e-3 5.94e-3 [COG0735] Fe2+/Zn2+ uptake regulation proteins 0.0 0.1 Mean proportion (%) 0.00 0.02 0.04 0.06 0.08 0.10 Difference in mean proportions (%) Fig. S9. COG category P (inorganic ion transport and metabolism) for Oxyphotobacteria and Melainabacteria Shown are all COGs from category P with a difference in mean proportions >= 0.02% between Oxyphotobacteria and Melainabacteria and a Storey's q-value of <= 0.05. Fig. S10. COG category Q (secondary metabolites and biosynthesis, transport and catabolism) for Oxyphotobacteria and Melainabacteria Shown are all COGs from category Q with a difference in mean proportions >= 0.02% between Oxyphotobacteria and Melainabacteria and a Storey's q-value of <= 0.05
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