Supplementary Figures for Photosynthesis is not a Universal

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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