supporting information - pnas · 10/8/2014 · gmf1061042970635f_1_839_1 diplosphaera colitermitum...

Supporting InformationSakowski et al. 10.1073/pnas.1401322111SI Materials and MethodsMetagenomic Libraries.ChesapeakeBay libraryCBBwassampled inSeptember 2002 and amplified using the linker-amplified shotgunlibrary (LASL)method before transformation and picking randomcolonies for Sanger sequencing (1). Chesapeake Bay library CBJwas sampled in October 2004 as part of the first Global OceanSurvey (2). DNA was inserted in a medium-copy plasmid andrandomly selected clones were Sanger-sequenced (2). Dry Tortu-gas libraries were sampled from surface seawater near the DryTortugas, Florida (24°29′N, 83°4′W) in January 2004. ChesapeakeBay libraries CFA through CFDwere collected over 24 h at stationCB 858 (38°58′N, 76°23′W) in July 2007. Water samples werecollected on July 30, 2007 at 0600 hours (CFA), 1130 hours (CFB),1630 hours (CFC), and July 31, 2007 at 0600 hours (CFD). Totalviral nucleic acids from the time series samples were separatedinto dsDNA, ssDNA, and RNA fractions using hydroxyapatitechromatography. The ssDNA and RNA fractions from each timepoint were pooled and transformed into dsDNA to provide li-braries CBS and CBR, respectively. Dry Tortugas and ChesapeakeBay libraries were amplified by the LASL method before trans-formation and sequencing (3). After the induction treatment virusparticles were concentrated by tangential-flow filtration (4). TheGulf of Maine Library GMF was sampled at station GOM04(44°07′5 ″N, 67°58′3 ″W) in January 2006.

Identification and Distribution of Putative Viroplankton RibonucleotideReductases. All sequences were screened by a Conserved DomainBLAST search (5) to confirm homology to ribonucleotide re-ductase (RNR). Each read was queried against the CDD database(v3.10) using Conserved Domain BLAST (5) and BLASTx (6) andsorted by the top results.

Interlibrary RNR Frequency Normalization.Differences in read lengthbetween libraries were corrected as follows to allow for interlibraryRNR frequency comparisons:

% RNR of library corrected =

0B@

rR×RNR

L

1CA× 100%;

where r is the mean read length of the individual library, R is themean read length of all libraries being compared, RNR is thenumber of reads with homology to RNR within the individuallibrary, and L is the number of reads in the individual library.

Intralibrary RNR Frequency Normalization. RNR frequencies werenormalized within a library by the mean read length of the libraryand gene length of the top BLAST hit reference phage for eachmetagenomic sequence. The corrected number of reads wascalculated as follows:

# Corrected readsðRNRcÞ =�rG

�×RNRn;

where r is the mean read length of the individual library, G is thegene length of the RNR from reference phage, and RNRn is the

number of reads with a top BLAST hit to a particular referencephage within the individual library.The proportion of each group/subunit combination per library

was calculated as follows:

% RNR group=subunit =�

RNRcPRNRc

�× 100%;

where RNRc is the corrected number of reads for a given RNRgroup/subunit.

Alignments and Phylogenetic Trees. Subsequent alignments and treeswere made by extracting sequences from the original alignment(Figs. S2, S3, and S4). Because of the high number of referencesequences within the class I Other, class II Other, and class IIRibonucleotide TriPhosphate Reductase (RTPR) groups (Fig. 1) itwas necessary to cluster these sequences at 80% identity using thefurthest neighbor algorithm in mothur (7). Representative se-quences from each cluster were aligned with the metagenomicsequences. Metagenomic sequences belonging to the class IIRTPR group were also clustered at 80% identity to reduce thenumber of sequences on the tree (Fig. S1).

Predicted RNR Group Abundances in the Chesapeake Bay. Theabundance (viruses per milliliter) of identified RNR groups waspredicted for libraries CFA–CFD using direct count values ob-tained from epifluorescence microscopy (8) and recruitment toRefSeq viral genomes. The abundance of each group was calcu-lated as (VA) × (BR/TB) × (RNR/G), where VA is the observedviral abundance (permilliliter) for a given library, BR is the numberof bases in the library that recruited to reference viral genomes, TBis the number of total bases in the library, RNR is the number ofpredicted RNR genes sampled in a given group in the library, andG is the number of total predicted genomes sampled in the library.

Identification of Redoxins. Thioredoxins and glutaredoxins fromRNR-encoding phages within the order Caudovirales were ob-tained from the GenBank nr database. These sequences werecompiled to create a reference database. Phage genomes werequeried against this reference database using BLASTx with ane-value cutoff of 1e-01 to identify hypothetical or unannotatedproteins that may be putative redoxins.

Contig Assembly and Annotation from the Rhode River. Fifty liters ofsurface water from theRhodeRiver was sampled at the SmithsonianEnvironmental Research Center in Edgewater, Maryland. The

4. Wommack KE, Sime-Ngando T, Winget DM, Jamindar S, Helton RR (2010) Filtration-based methods for the collection of viral concentrates from large water samples.Manual of Aquatic Viral Ecology, eds Wilhelm SW, Weinbaur MG, Suttle CA (Am. Soc.of Limnology and Oceanography, Waco, TX), pp 110–117.

5. Marchler-Bauer A, et al. (2011) CDD: A Conserved Domain Database for the functionalannotation of proteins. Nucleic Acids Res 39(Database issue):D225–D229.

6. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignmentsearch tool. J Mol Biol 215(3):403–410.

7. Schloss PD, et al. (2009) Introducing mothur: Open-source, platform-independent,community-supported software for describing and comparing microbial communities.Appl Environ Microbiol 75(23):7537–7541.

8. Chen F, Lu JR, Binder BJ, Liu YC, Hodson RE (2001) Application of digital imageanalysis and flow cytometry to enumerate marine viruses stained with SYBR gold.Appl Environ Microbiol 67(2):539–545.

9. John SG, et al. (2011) A simple and efficient method for concentration of ocean vi-ruses by chemical flocculation. Environ Microbiol Rep 3(2):195–202.

10. Noguchi H, Taniguchi T, Itoh T (2008) MetaGeneAnnotator: Detecting species-specific patterns of ribosomal binding site for precise gene prediction in anony-mous prokaryotic and phage genomes. DNA Res 15(6):387–396.

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www.pnas.org/cgi/content/short/1401322111

DTF1061043203475r_1_800_1; DT (1); GM (9)

Ectocarpus siliculosus (9)

GMF1061042942020f_103_880_1; CB (1); DT (1); GM (2)

CFA1061053817669f_1_691_1

Rhodococcus phage RGL3 (2)

Bacteriophage RM378

Alkaliphilus metalliredigens QYMF (2)

Candidatus Protochlamydia amoebophila UWE25

Mycobacterium phage Tiger (22)

Mycobacterium phage Gladiator (16)

CFA Contig 1 (6); CB (21); DT (12); GM (13)

DTF Contig 15 (2)

Thermus phage P23-45


CFA1061053822165r_5_739_1

Nitrosococcus halophilus Nc4

Roseiflexus castenholzii DSM 13941 (4)

CBJ1098127015314_915_1_1

Celeribacter phage P12053L

Roseophage SIO1

Acanthocystis turfacea Chlorella virus 1 (7)

GMF Contig 14 (3); DT (2); GM (9)

GMF1061042970635f_1_839_1

Diplosphaera colitermitum TAV2 (2)

Salpingoeca sp. ATCC 50818

Monosiga brevicollis MX1

GMF1061042661264f_1_935_1

GMF Contig 3 (8); DT (6); GM (21)

DTF Contig 5 (3); CB (1); DT (25); GM (2)

Puniceispirillum phage HMO-2011

Chlamydomonas reinhardtii

Volvox carteri f. nagariensis

0.4

Mycobacterium phage

Phage RM378

Clade II

ATCV-1Clade I

Clade III

Clade IV

Clade I

Clade II

Clade III

Clade IV

Fig. S1. Unrooted maximum likelihood tree with 100 bootstrap replicates of class II RNR reference and putative metagenomic RTPR sequences. Metagenomicsequences from the large tree (Inset) were clustered at 80% identity. Representative metagenomic sequences were placed on the tree, with the number ofreads from each environment within that cluster listed. Bacterial references from the large tree (Inset) were clustered at 80% identity. Representative se-quences were placed on each tree. Numbers in parentheses following bacterial references indicate the number of reference sequences within that cluster. Scalebar represents amino acid substitutions per site. Bacteria are shown in purple, eukaryotes and eukaryotic viruses in orange, myoviruses in red, siphoviruses inblue, podoviruses in green, and metagenomic sequences in black. Celeribacter phage P12053L was colored as a podovirus based on its T7-like DNA polymeraseeven though it is officially listed as an unclassified dsDNA phage. Black, gray, and white circles represent bootstrap support ≥100%, 75%, and 50%, re-spectively. CB, Chesapeake Bay; DT, Dry Tortugas; GM, Gulf of Maine.



Cyanophage Ma-LMM01 Cyanophage Ma-HPM05

Cyanophage Ma-LMM02 Cyanophage Ma-LMM03

Cyanothece sp. CCY 0110 NrdA2

P-SSM2

Synechococcus sp. PCC7002

S-CBS2

CBR1061057323945r_825_1_1

P60

Pelagibacter phage HTVC008M CBJ1098101650305_23_923_1

Arthrospira platensis NIES-39 Arthrospira maxima CS-328

P-SSP9 DTS Contig 2 (4)

DTS1061059628551r_1_780_1

CBJ1098101635164_885_1_1 CFB1061053832039f_822_1_1

P-SS2

S-ShM2

Synechocystis sp. PCC6803

Cyanobacterium UCYN-A

DTF1061043186908f_854_1_1

S-SSM7

P-HM2 P-HM1

Cyanothece sp. PCC 7424 Microcystis aeruginosa PCC 7806

Cyanothece sp. CCY 0110 NrdA1 Cyanothece sp. ATCC 51142

Syn5

GMF Contig 7 (6)

Syn9

Syn1 S-PM2

CBJ1098127013765_232_910_2

Synechococcus sp. PCC 7335

CBJ Contig 2 (2) S-SM2

CFD Contig 14 (3)

S-CBS4

DTR1061059708722r_64_828_2

Synechococcus sp. JA-3-3Ab Synechococcus sp. JA-2-3B'a(2-13) Nostoc sp. PCC 7120 Anabaena variabilis ATCC 29413

Syn33

P-SSM7 P-SSM4

DTF1061043225462r_796_1_1 P-RSP5

P-SSP10

CBB017C04.y01_102_812_1

CFA Contig 10 (2) CFB Contig 5 (4)

P-GSP1

DTF Contig 8 (3) DTF1061042876837_990_1_1

Synechococcus elongatus PCC 7942 Thermosynechococcus elongatus BP-1

S-SM1

P-HP1

DTF1061043197464f_808_1_1 DTF1061043192230f_735_1_1

CBB008H08.y01_1_669_1

GMF Contig 39 (2)

P-SSP7

Prochlorococcus marinus subsp. marinus str. CCMP1375 Prochlorococcus marinus str. NATL1A

Syn19

P-SSP11 P-SSP5

S-CBP4

GMF1061042926875f_1_685_1

P-SSP3 P-SSP2

P-RSM4 S-SSM5

CBJ1098214050374_886_1_1 S-CBP3

Synechococcus sp. RCC307 Synechococcus sp. WH 5701

Prochlorococcus marinus str. MIT 9515 Prochlorococcus marinus subsp. pastoris str. CCMP1986 Prochlorococcus marinus str. MIT 9312

GMF Contig 11 (4)

Prochlorococcus marinus str. MIT 9303 Prochlorococcus marinus str. MIT 9313

Prochlorococcus marinus str. MIT 9301 Prochlorococcus marinus str. MIT 9215

GMF1061042934309f_687_1_1 GMF1061042937625r_817_1_1

GMF Contig 23 (3)

Synechococcus sp. WH 8102

GMF1061042970087r_1_912_1 KBS-S-1A

S-RIP2 DTS Contig 4 (2)

Synechococcus sp. CC9605

Synechococcus sp. WH 7803 Synechococcus sp. WH 7805

Synechococcus sp. RS9917

GMF Contig 5 (7)

CBB019C09.y01_1_709_1 S-RIP1

Synechococcus sp. CC9902 Synechococcus sp. BL107

Synechococcus sp. RS9916 Synechococcus sp. CC9311

DTR1061059708668r_1_902_1 DTS Contig 3 (3)

0.3

T4-like cyanomyoviruses

Cyanosiphoviruses&

cyanopodoviruses

Cyanobacteria

CyanobacteriaClass I

Class II

Cyanosiphoviruses&

cyanopodoviruses

T4-like cyanomyoviruses

Fig. S2. Unrooted maximum likelihood tree with 100 bootstrap replicates of class I alpha and class II RNR reference and putative metagenomic Cyano se-quences. Numbers in parentheses indicate the number of reads assembled in each contig. Scale bar represents amino acid substitutions per site. Bacteria areshown in purple, myoviruses in red, siphoviruses in blue, podoviruses in green, and metagenomic sequences in black. Black, gray, and white circles representbootstrap support ≥100%, 75%, and 50%, respectively.



Thermus phage phiYS40 Thermus phage TMA

Aeromonas salmonicida subsp. salmonicida A449 (15)Thioalkalivibrio sp. K90mix

Roseovarius sp. 217 phage 1

Magnetospirillum magneticum AMB-1 (3) Roseophage DSS3P2 Roseophage EE36P1

Phenylobacterium zucineum HLK1 (3) Roseobacter sp. CCS2 (14)

Clostridium difficile QCD-32g58 (4)

Thermobaculum terrenum ATCC BAA-798

CFD Contig 11 (3) DTF Contig 26 (2)

DTF1061043181921_1782_938_2 Salinibacter ruber DSM 13855

Rhodospirillum rubrum ATCC 11170 Burkholderia multivorans ATCC 17616 (3)

Bacillus coagulans 36D1 (8)

Phage phiJL001

Zunongwangia profunda SM-A87 (10)

Oxalobacter formigenes HOxBLS

Paenibacillus sp. JDR-2 (2) Bacillus tusciae DSM 2912 (2)

Roseobacter phage RDJL Phi 1

GMF1061042906920f_834_1_1 GMF1061042906719f_1_672_1

GMF Contig 40 (2) GMF1061042963326f_1_823_1

Burkholderia pseudomallei DM98 (7)

Pseudomonas phage YuA Pseudomonas phage M6

Candidatus Puniceispirillum marinum IMCC1322 GMF1061042661109r_1_940_1

GMF1061042967907r_717_1_1

Ralstonia eutropha H16 (7) Methylococcus capsulatus str. Bath

GMF1061042934442r_760_1_1 GMF1061042970573r_630_1_1

GMF1061043206965f_804_1_1 GMF Contig 10 (5)

0.4

α-proteobacteria

Clade I Clade II

Clade III

Bacteroidetes

γ-proteobacteria

PhiJL-likeclade

α-proteobacteria

β-proteobacteriaFirmicutes

Class CI lass II

Clade III

Clade II

Clade I

PhiJL-like

Class I

Class IIAeromonas phage 44RR2.8t Aeromonas phage 31

Aeromonas phage phiAS4 Aeromonas salmonicida bacteriophage 25

Blattabacterium sp. (Blattella germanica) str. Bge (7) Vibrio phage pVp-1

CFC Contig 5 (2)

Thermus aquaticus Y51MC23 (7) Cyanophage S-TIM5

GMF1061042927759r_1_807_1

GMF Contig 56 (2)

Deftia phage phiW-14

GMF Contig 4 (7) Halophage AAJ-2005

Lymphocystis disease virus-isolate China Lymphocystis disease virus 1

DTF1061042915561r_791_1_1 GMF1061043206175r_1_828_1

GMF Contig 42 (2)

CFA1061053829314f_1_770_1 Francisella novicida FTG (6)

Grouper iridovirus Singapore grouper iridovirus

GMF1061043245027r_933_1_1

Rana tigrina ranavirus

Frog virus 3 Soft-shelled turtle iridovirus

GMF1061042968114f_929_1_1

GMF1061042925962f_784_1_1

GMF1061042971560f_1_840_1 CFA1061053812415f_675_1_1

Regina ranavirus Ambystoma tigrinum stebbensi virus

GMF Contig 30 (3) GMF1061042926741f_1_727_1

Pelagibacter phage HTVC019P GMF1061042949966r_830_1_1

CBS1061057326800r_735_1_1

GMF1061042926426f_648_1_1 GMF1061042943969r_1_765_1

Candidatus_Pelagibacter_ubique_HTCC1002 (27)

Caulobacter phage CcrColossus

Pseudomonas phage KPP10

Caulobacter phage CcrRogue

Pseudomonas phage PAK P3 Pseudomonas phage P3 CHA

GMF Contig 2 (8) CBJ1098101801525_1_814_1

DTF1061043183606r_811_1_1 GMF1061042924671r_733_1_1

GMF Contig 22 (3) GMF1061042968032r_1_866_1

Invertebrate iridescent virus 6

Caulobacter phage phiCbK

GMF Contig 25 (3) GMF1061043214682f_1_747_1

CFB Contig 1 (13) CBJ1098101648625_866_21_1

DTF1061043129216r_923_1_1

Invertebrate iridescent virus 3 Aedes taeniorhynchus iridescent virus

Caulobacter phage CcrKarma

GMF1061042943894f_1_624_1 GMF1061042957534r_1_794_1

DTF Contig 3 (4)

Caulobacter phage CcrSwift Caulobacter phage CcrMagneto

GMF Contig 9 (6)

GMF Contig 24 (3) DTF1061042874584r_837_1_1

DTF1061043202875r_1_816_1

GMF Contig 49 (2)

GMF1061039282610r_1_967_1 GMF1061042969816r_1_904_1

GMF1061042925690f_1_710_1 CFC Contig 1 (6) CFA Contig 12 (2) CFD Contig 3 (5)

0.3

Fig. S3. Unrooted maximum likelihood tree with 100 bootstrap replicates of class I alpha (Left) and class II (Right) RNR reference and putative metagenomicOther sequences. Numbers in parentheses following metagenomic contigs indicate the number of reads assembled in each contig. Bacterial references fromthe large tree (Inset) were clustered at 80% identity. Representative sequences were placed on each tree. Numbers in parentheses following bacterial ref-erences indicate the number of reference sequences within that cluster. Scale bar represents amino acid substitutions per site. Bacteria are shown in purple,eukaryotic viruses in orange, myoviruses in red, siphoviruses in blue, podoviruses in green, and metagenomic sequences in black. Black, gray, and white circlesrepresent bootstrap support ≥100%, 75%, and 50%, respectively.



Fig. S4. Distribution and dynamics of cyanophage populations. ORFs with a top BLASTx hit to a cyanophage were translated, aligned, and clustered at 98%identity. (A) Rank abundance of cyanophage-like RNR clusters in the Chesapeake Bay, Gulf of Maine, and Dry Tortugas. The morphology of the referencephage with the closest homology to sequences in each cluster is identified. Myoviruses are shown in red, siphoviruses in blue, and podoviruses in green. (B)Comparison of RNR distribution frequency in extracted region for peptide cluster analysis and those predicted by normalization of RNR sequences. Gulf ofMaine (GM), Chesapeake Bay (CB), and Dry Tortugas (DT). (C) Dynamics of phage populations by cluster in the Chesapeake Bay time series. Myoviruses areshown in red, siphoviruses in blue, and podoviruses in green.



Primase/Helicase DNA Polymerase A

Hypo

thet

ical

Prot

ein

HypotheticalProtein

Methyltransferase

DUF3

310

Exonuclease

Endo

nucle

ase

I

Hypo

thet

ical

Prot

ein

Ribonucleotide Reductase

Primase/Helicase

DNA Polymerase AExonuclease Domain DNA Polymerase A

Hypo

thet

ical

Prot

ein

ThymidylateSynthase

Hypo

thet

ical

Prot

ein

DUF3

310

Exonuclease

Hypo

thet

ical

Prot

ein

Hypo

thet

ical

Prot

ein

Nucle

otid

e

Pyro

phos

phoh

ydro

lase


DNA Primase DNA Helicase

Hypo

thet

ical

Prot

ein

Hypo

thet

ical

Prot

ein

ThymidylateSynthase

Hypo

thet

ical

Prot

ein

Hypo

thet

ical

Prot

ein

DNA Polymerase A Hypothetical Protein Exonuclease

Hypo

thet

ical

Prot

ein

Endo

nucle

ase

I

Hypo

thet

ical

Prot

ein

DUF3

310

Hypo

thet

ical

Prot

ein


500bp

Myoviridae

Podoviridae

Eukaryotic virus

Non-viral/ No hit

Contig 5585

Top BLAST Hit

ORF1 ORF2 ORF3 ORF4 ORF5 ORF6 ORF7 ORF8 ORF9 ORF10

ORF # Top BLAST hit Viral Sequence with Greatest Homolgy123456789

10

gamma proteobactgerium SCGC AAA160-D02 (1e-165) Vibrio phage CHOED (7e-142)gamma proteobactgerium SCGC AAA160-D02 (2e-157) Podovirus GOM (7e-133)

Puniceispirillum phage HMO-2011 (8e-80) Puniceispirillum phage HMO-2011 (8e-80)gamma proteobactgerium SCGC AAA160-D02 (2e-06) Vibrio phage CHOED (1e-03)

Rhizobium leguminosarum (43-31) Tetraselmis viridis virus S20 (1e-18)Puniceispirillum phage HMO-2011 (6e-23) Puniceispirillum phage HMO-2011 (6e-23)

Celeribacter phage P12053L (1e-73) Celeribacter phage P12053L (1e-73)Puniceispirillum phage HMO-2011 (9e-32) Puniceispirillum phage HMO-2011 (9e-32)

Rickettsia felis URRWXCal2 (2e-04) N/APuniceispirillum phage HMO-2011 (0) Puniceispirillum phage HMO-2011 (0)


10111213

Enterobacteria phage phi92 (2e-06) Enterobacteria phage phi92 (2e-06)gamma proteobactgerium SCGC AAA160-D02 (1e-27) Roseobacter phage SIO1 (6e-26)

Roseobacter phage SIO1 (7e-77) Roseobacter phage SIO1 (7e-77)Dialister micraerophilus (9e-2) N/A

Roseobacter phage SIO1 (2e-101) Roseobacter phage SIO1 (2e-101)N/A N/A

Vibrio cholerae (1e-19) Yersinia phage phiA1122 (9e-18)Celeribacter phage P12053L (3e-50) Celeribacter phage P12053L (3e-50)

Synechococcus phage S-CRM01 (1e-29) Synechococcus phage S-CRM01 (1e-29)Odoribacter laneus (6e-2) N/A

N/A N/ACeleribacter phage P12053L (8e-29) Celeribacter phage P12053L (8e-29)Puniceispirillum phage HMO-2011 (0) Puniceispirillum phage HMO-2011 (0)

ORF1 ORF2 ORF3 ORF4 ORF5 ORF6 ORF7 ORF8 ORF9 ORF10 ORF11 ORF12 ORF13

ORF1 ORF2 ORF3 ORF4 ORF5 ORF6 ORF7 ORF8 ORF9 ORF10 ORF11 ORF12 ORF13 ORF14 ORF15


101112131415

Puniceispirillum phage HMO-2011 (4e-103) Puniceispirillum phage HMO-2011 (4e-103)Puniceispirillum phage HMO-2011 (0) Puniceispirillum phage HMO-2011 (0)

Polymorphum gilvum SL003B-26A1 (2e-09) N/ARoseobacter phage SIO1 (6e-97) Roseobacter phage SIO1 (6e-97)

Celeribacter phage P12053L (1e-09) Celeribacter phage P12053L (1e-09)N/A N/A

Puniceispirillum phage HMO-2011 (0) Puniceispirillum phage HMO-2011 (0)Puniceispirillum phage HMO-2011 (1e-115) Puniceispirillum phage HMO-2011 (1e-115)Puniceispirillum phage HMO-2011 (1e-134) Puniceispirillum phage HMO-2011 (1e-134)

N/A N/APuniceispirillum phage HMO-2011 (3e-56) Puniceispirillum phage HMO-2011 (3e-56)Puniceispirillum phage HMO-2011 (1e-08) Puniceispirillum phage HMO-2011 (1e-08)

Olsenella profusa (1e-15) Salicola phage CGphi29 (2e-15)N/A N/A

Paramecium bursaria Chlorella Virus OR0704.2.2 (7e-95)Paramecium bursaria Chlorella Virus OR0704.2.2 (7e-95) Paramecium bursaria Chlorella Virus OR0704.2.2 (7e-95)

DNA Primase DNA Polymerase A Exonuclease

Hypo

thet

ical

Prot

ein

Glut

ared

oxin

Endo

nucle

ase

IHy

poth

etica

lPr

otei

nHy

poth

etica

lPr

otei

n

Ribonucleotide Reductase Class I alpha

Ribo

nucle

otid

e Re

duct

ase

Clas

s I b

eta

ORF1ORF2ORF3ORF4ORF5ORF6ORF7ORF8ORF9ORF10

500bp

Contig 12643

ORF # Annotation Homologous SequencesHomologous SequencesHomologous Sequences Top BLAST hit (E value)

12345678910

HTVC011P HTVC019P SIO1RNR beta Novosphingobium sp. PP1Y (3e-14)RNR alpha Pelagibacter phage HTVC019P (0)

Hypothetical protein Clostridium leptum (4e-16)Hypothetical protein NA

Endonuclease I Celeribacter phage P12053L (1e-27)Glutaredoxin alpha proteobacterium HIMB59 (1e-20)

Hypothetical protein NAExonuclease Celeribacter phage P12053L (1e-77)

DNA Polymerase A Roseobacter phage SIO1 (2e-125)DNA Primase Azorhizobium caulinodans ORS 571 (3e-151)

Class I ‘Other’

Class II ‘RTPR’

Contig 8066

500bp

Contig 12399

500bp

Fig. S5. Predicted ORFs on Rhode River contigs 12643, 5585, 8066, and 12399. These contigs contained class I Other, clade II (contig 12643) and RTPR (contigs5585, 8066, and 12399) RNR sequences. The contigs were assembled from ∼50 million Illumina 2 × 150 bp reads using MetaVelvet. ORFs were predicted usingMetageneAnnotator. Annotations were assigned by consensus BLASTx results. ORFs without hits less than 1e-3 or lacking hits to definitive genes were an-notated as hypothetical protein. ORFs with homology to phage sequences in the Caudovirales were colored by the viral family of the top BLASTx repre-sentatives. Scale bar represents 500 nucleotides.



100

33

24

31

33

60

58

56

65

90

57

78

97

100

88

88

58

98

58

100

100

89

61

82

100

100

100

95

100

DTF Contig 5 (3); CB (1); DT (25); GM (2)

Chlamydomonas reinhardtii

Volvox carteri f. nagariensis

Rhode River Contig 5585

GMF Contig 14 (3); DT (2); GM (9)

Acanthocystis turfacea Chlorella virus 1 (7)


CFA Contig 1 (6); CB (21); DT (12); GM (13)

DTF Contig 15 (2)

Nitrosococcus halophilus Nc4

Roseiflexus castenholzii DSM 13941 (4)

Bacteriophage RM378

CBJ1098127015314915_1_1

GMF1061042970635f_1_839_1

Diplosphaera colitermitum TAV2 (2)

Salpingoeca sp. ATCC 50818

Monosiga brevicollis MX1

GMF1061042661264f_1_935_1

GMF Contig 3 (8); DT (6); GM (21)

Roseobacter phage SIO1

CFA1061053822165r_5_739_1

Candidatus Protochlamydia amoebophila UWE25



Alkaliphilus metalliredigens QYMF (2)

GMF1061042942020f_103_880_1; CB (1); DT (1); GM (2)

CFA1061053817669f_1_691_1

Mycobacterium phage Tiger (22)

Mycobacterium phage Gladiator (16)

Rhodococcus phage RGL3 (2)

Ectocarpus siliculosus (9)

DTF1061043203475r_1_800_1; DT (1); GM (9)

0.4

Clade I

Clade II

Clade III

Clade IV

44 38

100 97

95 97

70 40

50 100

100 49

87

60

99

96 32

70

98

75

18

100 84

100

67 31

84 27

100

36

66

33

28 70

100 100

97

42

29 100

46

6

95 99

7

23

98

23

38

73 55

67

100

100

86

99

95

100

53 100

61

56

62 43

57

51

100

80

99

45

20

71

100

100

98

100


CFA1061053812415f_675_1_1 GMF1061042971560f_1_840_1

GMF1061043214682f_1_747_1 GMF Contig 25 (3)

GMF Contig 2 (8)

CFD Contig 3 (5) CFA Contig 12 (2) CFC Contig 1 (6) GMF1061042925690f_1_710_1 GMF Contig 49 (2)

GMF Contig 9 (6) DTF Contig 3 (4)

GMF1061042957534r_1_794_1 GMF1061042943894f_1_624_1

CBJ1098101801525_1_814_1

GMF1061042968032r_1_866_1 GMF Contig 22 (3)

GMF1061042924671r_733_1_1 DTF1061043183606r_811_1_1

GMF1061042943969r_1_765_1 GMF1061042926426f_648_1_1

Candidatus Pelagibacter ubique HTCC1002 (27)

Aedes taeniorhynchus iridescent virus Invertebrate iridescent virus 3

Invertebrate iridescent virus 6

CBJ1098101648625_866_21_1 CFB Contig 1 (13)

GMF1061042969816r_1_904_1 GMF1061039282610r_1_967_1DTF1061043202875r_1_816_1

DTF1061042874584r_837_1_1 GMF Contig 24 (3)

DTF1061043129216r_923_1_1

Caulobacter phage CcrMagneto Caulobacter phage CcrSwift Caulobacter phage CcrKarma

Caulobacter phage phiCbK Caulobacter phage CcrRogue

Caulobacter phage CcrColossus

Pseudomonas phage P3 CHAPseudomonas phage PAK P3Pseudomonas phage KPP10

CBS1061057326800r_735_1_1

GMF1061042949966r_830_1_1 Pelagibacter phage HTVC019P

GMF1061042925962f_784_1_1

Francisella novicida FTG (6) CFA1061053829314f_1_770_1

Deftia phage phiW-14

Rana tigrina ranavirus

Ambystoma tigrinum stebbensi virus Regina ranavirus

Soft-shelled turtle iridovirus Frog virus 3

Singapore grouper iridovirus Grouper iridovirus

Lymphocystis disease virus 1 Lymphocystis disease virus-isolate China

GMF Contig 4 (7) Halophage AAJ-2005

Thermus aquaticus Y51MC23 (7) Cyanophage S-TIM5

GMF1061042927759r_1_807_1

GMF1061043245027r_933_1_1

GMF1061042926741f_1_727_1 GMF Contig 30 (3) GMF1061042968114f_929_1_1

GMF Contig 42 (2)

GMF1061043206175r_1_828_1 DTF1061042915561r_791_1_1

GMF Contig 56 (2) Blattabacterium sp. (Blattella germanica) str. Bge (7)

CFC Contig 5 (2) Vibrio phage pVp-1

Aeromonas salmonicida bacteriophage 25 Aeromonas phage phiAS4

Aeromonas phage 31 Aeromonas phage 44RR2.8t

Clade I

Clade II

Clade III

0.2

A. B.Class II ‘RTPR’ Class I ‘Other’

Fig. S6. Unrooted maximum likelihood trees with 100 bootstrap replicates of Rhode River RNRs from contigs >5 kb. (A) RTPR RNRs from contigs 5585 and 8066(bold) with class II RNR reference and putative metagenomic RTPR sequences. Metagenomic sequences were clustered at 80% identity. Representative met-agenomic sequences were placed on the tree, with the number of reads from each environment within that cluster listed. Bacterial references were clustered at80% identity. Representative sequences were placed on each tree. (B) Rhode River contig 12643 (bold) on the class I alpha Other tree. Numbers in parenthesesfollowing metagenomic contigs indicate the number of reads assembled in each contig. Bacterial references were clustered at 80% identity. Representativesequences were placed on the tree. Numbers in parentheses following bacterial references indicate the number of reference sequences within that cluster. Scalebar represents amino acid substitutions per site. Bacteria are shown in purple, eukaryotic viruses in orange, myoviruses in red, siphoviruses in blue, podoviruses ingreen, and metagenomic sequences in black. Integer values are bootstrap support values. CB, Chesapeake Bay; DT, Dry Tortugas; GM, Gulf of Maine.



CB (dsDNA) CB (ssDNA) DT (dsDNA) DT (ssDNA)

18% 48% 16% 25%

37% 84% 31% 73%

38% 94% 42% 93%

Do

mai

nd

sDN

A v

iru

ses

Po

do

viri

dae

2000 4000 6000 8000 10000 12000 14000 16000 18000 20000 22000 24000 26000 28000 30000 32000 34000 36000 38000 40000 42000 44000

2000 4000 6000 8000 10000 12000 14000 16000 18000 20000 22000 24000 26000 28000 30000 32000 34000 36000 38000 40000 42000 44000

2000 4000 6000 8000 10000 12000 14000 16000 18000 20000 22000 24000 26000 28000 30000 32000 34000 36000 38000 40000

2000 4000 6000 8000 10000 12000 14000 16000 18000 20000 22000 24000 26000 28000 30000 32000 34000 36000 38000 40000 42000 44000

24 33

50 270

52 183

2547

26225

CB (dsDNA)CB (ssDNA)DT (dsDNA)DT (ssDNA)

RNR

DN

A P

ol

DN

A p

rimas

e/ h

elic

ase

Endo

nucl

ease

RNA

pol

ymer

ase

A.

B.

Fig. S7. Taxonomic distribution and alignment of ssDNA virome sequences. (A) Taxonomic distribution of translated ORFs in Chesapeake Bay and Dry Tor-tugas dsDNA and ssDNA virome libraries. Chesapeake Bay libraries CFA–CFD were combined for taxonomic composition analysis. Podoviral sequences witha top BLASTp hit to known cyanopodoviral sequences were categorized as cyanophage-like. (B) Recruitment of dsDNA and ssDNA virome library reads tocyanopodoviral genomes. Reads from Chesapeake Bay dsDNA libraries CFA–CFD were combined before mapping. Reads were mapped to each genome in-dependently. Maximal coverage values of reads from dsDNA libraries and ssDNA libraries against each genome are listed on the left and right sides of eachplot, respectively. Genomes were aligned with Mauve. Colors on horizontal axes are aligned regions.



Table S1. Frequency of sampled genomes and RNR alpha subunits in cyanophage and pelagiphage populations

Group No. of predicted RNR genes No. of predicted genomes Predicted RNR frequency, %

Cyano I 22 16 138Cyano II 63 85 74Pelagiphage

HTVC008M 4 6HTVC010P — 44HTVC011P — 15HTVC019P 36 11

Pelagiphage total 40 76 53

Table S2. Distribution frequencies of RNR alpha subunit sequences among designated groups

Library

Groups CBB CBJ CBR CBS CFA CFB CFC CFD CIA DTF DTR DTS GMF

Nucleic acid type dsDNA dsDNA RNA ssDNA dsDNA dsDNA dsDNA dsDNA dsDNA dsDNA RNA ssDNA dsDNACyanoClass I 25% (5) 16% (4) — — 4% (1) 5% (1) 3% (

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