Supplementary Information Metagenomics uncovers a new ... · Tel: +34 965 919451 Email: [email protected] Keywords: ... with the scaffold identifier, sampling location, sampling depth

Supplementary Information

Metagenomics uncovers a new group of low GC and ultra-small marine

Actinobacteria

Short Title: Novel, ultra-small, low-GC marine Actinobacteria

Rohit Ghai1, Carolina Megumi Mizuno1, Antonio Picazo2, Antonio Camacho2,

Francisco Rodriguez-Valera1

1Evolutionary Genomics Group, Departamento de Producción Vegetal y Microbiología,

Universidad Miguel Hernández, San Juan de Alicante, 03550, Alicante, Spain 2Cavanilles Institute of Biodiversity and Evolutionary Biology – University of Valencia E-46100

Burjassot, Spain.

Corresponding Author: Francisco Rodriguez-Valera

Evolutionary Genomics Group, Departamento de Producción Vegetal y Microbiología, Universidad Miguel Hernández, San Juan de Alicante, 03550, Alicante, Spain Tel: +34 965 919451 Email: [email protected]

Keywords: Low-GC Actinobacteria, metagenomics, deep chlorophyll maximum, rhodopsins

Fig. S1. Alignment of ITS regions of Low GC Actinobacterial Contigs from the Mediterranean DCM and GOS scaffolds. The Mediterranean DCM contig ITS sequences are prefixed by MedDCM-OCT and details of the GOS sample, including the GOS scaffold identifier are shown.

1) Coastal upwelling Galapagos Islands Upwelling Fernandina Island JCVI_SCAF_1097263062467_GS031_its2) Estuary_North American East Coast_Delaware Bay NJ JCVI_SCAF_1097179020611_GS011_its3) Coastal_Galapagos Islands_Cabo Marshall Isabella Island JCVI_SCAF_1097263401211_GS036_its4) Coral Reef Atoll_Polynesia Archipelagos_Rangirora Atoll JCVI_SCAF_1097263566470_GS051_its5) Coastal_Eastern Tropical Pacific_Gulf of Panama_JCVI_SCAF 1097207858586_GS021_its6) Coastal_Galapagos Islands_Cabo Marshall Isabella Island JCVI_SCAF_1097263399889_GS036_its7) Coastal_Galapagos Islands_Coastal Floreana_JCVI_SCAF 1097263718363_GS028_its8) Coastal_Galapagos Islands_Devils Crown Floreana Island JCVI_SCAF_1097208922928_GS027_its9) Open Ocean_Eastern Tropical Pacific_250 miles from Panama City JCVI_SCAF_1097208170248_GS022_its10) Open Ocean_Eastern Tropical Pacific_30 miles from Cocos Island JCVI_SCAF_1097156482264_GS023_its11) Open Ocean_Eastern Tropical Pacific_Equatorial Pacific TAO Buoy JCVI_SCAF_1097263465119_GS037_its12) Warm Seep_Galapagos Islands_Warm seep Roca Redonda JCVI_SCAF_1097262600301_GS030_its13) MedDCM OCT S38 C68_its14) Coastal_Galapagos Islands_Wolf Island JCVI_SCAF_1097263366991_GS035_its15) Coastal_North American East Coast_South of Charleston SC JCVI_SCAF_1097205148428_GS014_its16) MedDCM OCT S40 C95_its

Fig.S2: Phylogenetic tree of 23S Ribosomal RNA. A maximum likelihood tree created from the alignments of complete 23s rRNA genes from several organisms belonging to all orders within the phylum Actinobacteria is shown. Along with the rRNA sequences from the fosmids from the Mediterranean DCM, we have also included several rRNA sequences from GOS scaffolds where we could identify complete 23S genes. The oceanic habitat (CRA-Coral Reef, Atoll C-Coastal), sampling location (PA-Polynesia Archipelagos, ETP-Eastern Tropical Pacific, GI-Galapagos Islands) and the GOS dataset identifier are all shown next to each GOS scaffold. Bootstrap values are indicated by circles on the nodes (Black circles > 90 %, gray circles > 65% white circles > 50%). Several sequences from Firmicutes were used as outgroup. The new suggested names of the subclass, Actinomarinidae and the order Actinomarinales are indicated. Names of Actinobacterial subclasses are in bold uppercase capitals, names of orders are in bold and italicized and families are shown in capitals. The tree was constructed using a total of 88 sequences.

0.06

FIRMICUTES

MICROMONOSPORINEAE

MedDCM-OCT-S38-C68

MedDCM-OCT-S40-C95

FRANKINEAE

CORYNEBACTERINEAE

STREPTOMYCINEAE

PROPIONIBACTERINEAE

STREPTOSPORANGINEAE

PSEUDOCARDINEAE

MICROCOCCINEAE

CRA-PA Rangirora Atoll JCVI_SCAF_1097263566470GS051

C-ETP Gulf of Panama GS021 JCVI_SCAF_1097207858586

C-GI Devils Crown Floreana Island JCVI_SCAF_1097208922928GS027

C-GI Coastal Floreana JCVI_SCAF_1097263718363GS028

C-GI Upwelling Fernandina Island JCVI_SCAF_1097263062467GS031

Uncultured actinobacterium MB11A03

Uncultured actinobacterium MB11C06

Uncultured actinobacterium HF0130 15N16

Acidimicrobium ferrooxidans DSM 10331

Acidithiomicrobium sp. P1

Catenulispora acidiphila DSM 44928 | CATENULISPORINEAE

Stackebrandtia nassauensis DSM 44728 | GLYCOMYCINEAE

Thermobispora bispora DSM 43833 | PSEUDONOCARDINEAE

CORIOBACTERIDAE Coriobacteriales

RUBROBACTERIDAE Rubrobacterales, Solirubrobacterales

C. ACTINOMARINIDAEC. Actinomarinales

ACIDIMICROBIDAEAcidimicrobiales

ACTINOBACTERIDAEActinomycetales

Fig. S3: Phylogenetic tree of concatenated 16S and 23S ribosomal RNA sequences. A maximum likelihood tree created from the concatenated alignments of 16s and 23s genes from several organisms belonging to all orders within the phylum Actinobacteria is shown. Along with the rRNA sequences from the fosmids from the Mediterranean DCM, we have also included several rRNA sequences from GOS scaffolds where we could identify complete 16S and 23S genes. The oceanic habitat (C-Coastal, CRA-Coral Reef Atoll), sampling location (ETP-Eastern Tropical Pacific, PA-Polynesia Archipelagos, GI-Galapagos Islands), and the GOS dataset identifier are all shown next to each GOS scaffold. Bootstrap values are indicated by circles on the nodes (Black circles > 90 %, gray circles > 65% white circles > 50%). Several sequences from Firmicutes were used as outgroup. The new suggested names of the subclass, Actinomarinidae and the order Actinomarinales are indicated. Names of Actinobacterial subclasses are in bold uppercase capitals, names of orders are in bold and italicized and families are shown in capitals. The tree was constructed using a total of 91 sequences.

0.06

C-GI Upwelling Fernandina Island JCVI_SCAF_1097263062467GS031

STREPTOSPORANGINEAE

Acidimicrobium ferrooxidans DSM 10331

CRA-PA Rangirora Atoll JCVI_SCAF_1097263566470GS051


Uncultured actinobacterium Hf0130 15N16

Thermobispora bispora DSM 43833 | PSEUDONOCARDINEAE

C-GI Devils Crown Floreana Island JCVI_SCAF_1097208922928GS027

C-GI Coastal Floreana JCVI_SCAF_1097263718363GS028

STREPTOMYCINEAE

Stackebrandtia nassauensis DSM 44728 | GLYCOMYCINEAE

MedDCM-OCT-S40-C95 | MedDCM-OCT-S38-C68

PSEUDOCARDINEAE

PROPIONIBACTERINEAE

MICROMONOSPORINEAE

C-ETP Gulf of Panama GS021 JCVI_SCAF_1097207858586

Uncultured actinobacterium MB11A03

Catenulispora acidiphila DSM 44928 | CATENULISPORINEAE

Uncultured actinobacterium MB11C06

MICROCOCCINEAE


FRANKINEAE

CORYNEBACTERINEAE

RUBROBACTERIDAE

FIRMICUTES

CORIOBACTERIDAE

C. ACTINOMARINIDAEC Actinomarinales

ACIDIMICROBIDAE

ACTINOBACTERIDAE

Rubrobacterales, Solirubrobacterales

Coriobacteriales

Acidimicrobiales

Actinomycetales

Fig. S4: Principal components analysis of tetranucleotide frequency of assembled contigs belonging to Marine Low GC Actinobacteria. Assembled scaffolds of Low GC Actinobacteria from two GOS datasets (Lake Gatun, Freshwater and Punta Cormoran, Hypersaline Lagoon), and fosmids from Lake Kinneret (freshwater), complete genomes of two low GC Actinobacteria (Candidatus Aquiluna and actinobacterium acIBI), along with assembled contigs of Low GC Marine Actinobacteria. Clusters are labeled and the total number of sequences, total length and mean length of all sequences assigned to a cluster and the GC% range for each are indicated. Complete genomes are shown in larger circles and labeled by name. Marine Low GC Actinobacterial contigs cluster is shown in red. Only contigs that were > 5kb in length and had a consistent taxonomic profile were included in this analysis.

10

5

-5

-10

0

-15

-300-20 -10

PC1

PC

3

PC1

PC

2

Lake Kinneret Lake Gatun

MedDCM Punta Cormorant

Candidatus Aquiluna1.35 Gb, GC% 51.6

AcIBI1.15 Gb, GC% 41.4

106 kb, mean 6.6 kbGC range 58-74

cluster 1 (n=16)

cluster 2 (n=135)1510 kb, mean 11.1 kbGC% range 61-68






cluster 7 (n=43)1317 kb, mean 30.6 kbGC% 29-36

GC range 58-74cluster 1 (n=16)

cluster 2 (n=135)GC% range 61-68





cluster 7 (n=43)GC% 29-36 cluster 3 (n=55)

GC% range 53-61

Candidatus Aquiluna1.35 Gb, GC% 51.6

AcIBI1.15 Gb, GC% 41.4

A B

Fig. S5: Median size of intergenic spacers for selected microbial genomes with the smallest intergenic spacers. The freshwater actinobacterial genome, (acI-BI actinobacterium) is also shown.

C. Actinomarina

C. Pelagibacter

Mycoplasma genitalium

Thermotaga maritima

Nanoarchaeum equitans

Campylobacter jejuni

Aquifex aeolicus

Thermus thermophilus

Pyrococcus horikoshii

acI-BI actinobacterium

Pyrococcus abyssi

Tropheryma whipplei

median size (in bp) of intergenic spacers

MedDCM-OCT-S44-C50(34.7 kb, GC%33.47)

MedDCM-OCT-S36-C22(39 kb, GC%33.49)

FeS assemblyATPase SufCFeS assembly

protein SufD

FeS assemblyprotein SufB

DNA modificationmethylase

fumarase

RpiB/LacA/LacBfamily sugar-phosphate

isomerase

carbamoylphosphatesynthase

large subunit

aspartatecarbamoyltransferase

catalytic subunitcarbamoylphosphatesynthase

small subunit

cysteine desulfuraseSufS subfamily

FeS assemblyATPase SufC

lipoylsynthaseABC transporter

related proteinFeS assemblyATPase SufC

FeS assemblyprotein SufB

ubiquinone/menaquinonebiosynthesis methyltransferase

ABC transporter

pyridoxamine 5-Poxidase-related

binding-protein-dependent transport system inner

membrane protein

antibiotic ABC transporterATP-binding protein

propionyl-CoAcarboxylase alcohol

dehydrogenase

tRNA Leu

ABCtransporter

alpha-glucosides-bindingperiplasmic protein AglE

ROK family protein

sugar ABCtransporterpermease

alpha-glucosides-bindingperiplasmic protein

AglE precursor

MazG family protein

50S ribosomalprotein L50

bioY protein

carbamoyl phosphatesynthase L chain ATP

binding proteintRNA Arg

MACrhodopsin

JCVI_SCAF_1097156482175_Eastern_Tropical_Pacific_Cocos_Island_2_m_GS023

JCVI_SCAF_1097205335775_Caribbean_Sea_Gulf_of_Mexico_2_m_GS016

JCVI_SCAF_1097205453281_Caribbean_Sea_Yucatan_Channel_2_m_GS017

JCVI_SCAF_1097205816890_Caribbean_Sea_Northeast_of_Colon_1.7_m_GS019

JCVI_SCAF_1097207869285_Eastern_Tropical_Pacific_Gulf_of_Panama_1.6_m_GS021

JCVI_SCAF_1097208168413_Eastern_Tropical_Pacific_250_miles_from_Panama_City_2_m_GS022

JCVI_SCAF_1097262602060_Galapagos_Islands_Warm_seep,_Roca_Redonda_19_m_GS030

JCVI_SCAF_1097263498021_Tropical_South_Pacific_201_miles_from_F._Polynesia_30_m_GS047

JCVI_SCAF_1099266104707_Sargasso_Sea_Station_5_m_GS000b

JCVI_SCAF_1099266274449_Sargasso_Sea_Sargasso_Station_5_m_GS000a42.7 kb, GC% 26.55

JCVI_SCAF_1099266666974_Sargasso_Sea_Station_5_m_GS000d

JCVI_SCAF_1099266920828_Sargasso_Sea_Hydrostation_5_m_GS001c



Color Scale (%identity)

30-50 50-70 70-80 80-90 90-95 95-100

photolyase

thiol disulfide reductase

Fig. S6: Comparison of C. Actinomarina rhodopsin contigs with GOS scaffolds containing similar rhodopsins. The rhodopsin genes are shown in red, and the flanking genes, a photolyase, and a thio-disulfide reductase are also colored.GOS scaffolds are labeled with the scaffold identifier, sampling location, sampling depth and the GOS dataset identifier. The alignments (performed using TBLASX, minimum length 50aa and minimum evalue 1e-5), are colored to show the varying degrees of similarity (see color key at bottom left)

Arc

tic M

eta

genom

e

Anta

rctic

a-S

tatio

n_347-0

p1-S

outh

Zone

Anta

rctic

a-S

tatio

n_349-0

p1-S

outh

Zone

Anta

rctic

a-S

tatio

n_351-0

p1-S

outh

Zone

Anta

rctic

a-S

tatio

n_352-0

p1-S

outh

Zone

Anta

rctic

a-S

tatio

n_353-0

p1-S

outh

Zone

Anta

rctic

a-S

tatio

n_357-0

p1-S

outh

Zone

Anta

rctic

a-S

tatio

n_358-0

p1-S

outh

Zone

Anta

rctic

a-S

tatio

n_359-0

p1-S

outh

Zone

Anta

rctic

a-S

tatio

n_360-0

p1-S

outh

Zone

Anta

rctic

a-S

tatio

n_362-0

p1-S

outh

Zone

Anta

rctic

a-S

tatio

n_346-0

p1-N

ort

hZ

one

Anta

rctic

a-S

tatio

n_363-0

p1-N

ort

hZ

one

Anta

rctic

a-S

tatio

n_364-0

p1-N

ort

hZ

one

Anta

rctic

a-S

tatio

n_366-0

p1-N

ort

hZ

one

Anta

rctic

a-S

tatio

n_366-0

p8-N

ort

hZ

one

Anta

rctic

a-S

tatio

n_367-0

p1-N

ort

hZ

one

Anta

rctic

a-S

tatio

n_367-0

p8-N

ort

hZ

one

Anta

rctic

a-S

tatio

n_368-0

p1-N

ort

hZ

one

BA

TS

20m

BA

TS

50m

BA

TS

100m

Medite

rranean D

CM

HO

Ts25m

HO

Ts75m

HO

Ts11

0m

HO

Ts500m

GS

000a-O

pen_O

cean-5

_m

-Sarg

ass

o_S

ea

GS

000b-O

pen_O

cean-5

_m

-Sarg

ass

o_S

ea

GS

000c-

Open_O

cea

n-5

_m

-Sarg

ass

o_S

ea

GS

000d-O

pen_O

cean-5

_m

-Sarg

ass

o_S

ea

GS

001c-

Open_O

cea

n-5

_m

-Sarg

ass

o_S

ea

GS

017-O

pen_O

cean

-2_m

-Caribbean_S

ea

GS

018-O

pen_O

cean-1

.7_m

-Caribbean_S

ea

GS

022-O

pen_O

cean-2

_m

-East

ern

_T

ropic

al_

Paci

fic

GS

023-O

pen_O

cean-2

_m

-East

ern

_T

ropic

al_

Paci

fic

GS

026-O

pen_O

cean-2

_m

-Gala

pagos_

Isla

nds

GS

047-O

pen_O

cean-3

0_m

-Tro

pic

al_

South

_P

aci

fic

GS

110a-O

pen_O

cean-1

.5_m

-India

n_O

cean

GS

112a-O

pen_O

cean-1

.8_m

-India

n_O

cean

GS

113-O

pen_O

cean-1

.8_m

-India

n_O

cean

GS

114-O

pen_O

cean-1

.5_m

-India

n_O

cean

GS

116-O

pen_O

cean-1

.5_m

-India

n_O

cean

GS

119-O

pen_O

cean-2

.0_m

-India

n_O

cean

GS

121-O

pen_O

cean-1

.5_m

-India

n_O

cean

GS

122a-O

pen_O

cean-1

.9_m

-India

n_O

cean

GS

123-O

pen_O

cean-2

.2_m

-India

n_O

cean

GS

002-C

oast

al-1_m

-Nort

h_A

merica

n_E

ast

_C

oast

GS

008-C

oast

al-1_m

-Nort

h_A

merica

n_E

ast

_C

oast

GS

009-C

oast

al-1_m

-Nort

h_A

merica

n_E

ast

_C

oast

GS

010-C

oast

al-1_m

-Nort

h_A

merica

n_E

ast

_C

oast

GS

013-C

oast

al-2.1

_m

-Nort

h_A

merica

n_E

ast

_C

oast

GS

014-C

oast

al-1_m

-Nort

h_A

merica

n_E

ast

_C

oast

GS

015-C

oast

al-1

.7_m

-Caribbean_S

ea

GS

016-C

oast

al_

Sea

-2_m

-Caribbean_S

ea

GS

019-C

oast

al-1

.7_m

-Caribbean_S

ea

GS

02

1-C

oast

al-1.6

_m

-East

ern

_T

ropic

al_

Paci

fic

GS

027-C

oast

al-2.2

_m

-Gala

pagos_

Isla

nds

GS

028-C

oast

al-2_

m-G

ala

pagos_

Isla

nds

GS

029-C

oast

al-2.1

_m

-Gala

pagos_

Isla

nds

GS

031-C

oast

al_

upw

elli

ng-1

2_

m-G

ala

pagos_

Isla

nds

GS

034-C

oast

al-2.1

_m

-Gala

pagos_

Isla

nds

GS

035-C

oast

al-1.7

_m

-Gala

pagos_

Isla

nds

GS

036-C

oast

al-2.1

_m

-Gala

pagos_

Isla

nds

GS

049-C

oast

al-1.4

_m

-Poly

nesi

a_A

rchip

ela

gos

GS

117a-C

oast

al_

sam

ple

-1.8

_m

-India

n_O

cean

GS

051-C

ora

l_R

eef_

Ato

ll-1_m

-Poly

nesi

a_A

rchip

ela

gos

GS

048a-C

ora

l_R

eef-

1.4

_m

-Poly

nesi

a_A

rchip

ela

gos

GS

011

-Est

uary

-1_m

-Nort

h_A

merica

n_E

ast

_C

oast

GS

012-E

stu

ary

-13.2

_m

-Nort

h_A

merica

n_E

ast

_C

oast

GS

020-F

resh

_W

ate

r-2_m

-Panam

a_C

anal

GS

030-W

arm

_S

eep-1

9_

m-G

ala

pagos_

Isla

nds

GS

032-M

angro

ve-0

.1_m

-Gala

pagos_

Isla

nds

GS

033-H

ypers

alin

e-0

.2_m

-Gala

pagos_

Isla

nds

Puert

o R

ico

Tre

nch

Meta

genom

e

% o

f to

tal 1

6S

rR

NA

0

10

20

30

40

Candidatus Actinomarina

Synechococcus

Prochlorococcus

Candidatus Pelagibacter

Polar High Latitude BATS/HOTS/MedDCM GOS Open Ocean GOS Coastal GOS Assorted Deep Sea

Fig. S7: Percentages of total rRNA reads (%identity >98% and coverage 98% of metagenomic read) of 16S ribosomal rRNA of Candidatus Actinomarina, Synechococcus, Prochlorococcus and Candidatus Pelagibacter detected in several metagenomes.

Supplementary Tables

Table S1: Oligonucleotide probes considered in this study and conditions used for hybridization.

Probe Specificity Sequence (5' to 3') Target site

(rRNA, 5' position)

Match

(%) % FAa Reference

LGC722 Actinobacteriab CAGCGTCGATAGCGGCCCAG 16S, 722 100 30-40 This Study

HGC236 Actinobacteria AACAAGCTGATAGGCCGC 16S, 236 83.3 10

Glöckner et al.,

2000((1))

Consensusc AACTAGCTGATAGAACGC 16S, 236

HGC664 Actinobacteria AGGAATTCCAGTCTCCCC 16S, 664 77.7 30

Glöckner et al.,

2000((1))

Consensus CGGAATTCCACTCACCTC 16S, 236

HGC840 Actinobacteria HG1 Cluster TCGCASAAACCGTGGAAG 16S, 840 70.0 10

Glöckner et al.,

2000((1))

Consensus ACACAGAAACCGTC-AAT- 16S, 236

aPercent formamide (FA) in hybridization buffer for hybridizations at 48°C

bSpecific for unclassified Actinobacteria, no hybridization with any known subclass of Actinobacteria (Ribosomal Database Project)

cSequence of the Low GC Actinobacteria of this study

Table S2: Summary of bacterial cell biovolumes (μm3), average and range, found in different aquatic environments

Biovolume ( m3) Environment Applied methods Study Reference

Candidatus Actinomarina

0.013 (0.006-0.024)

M FISH-Cy3-DAPI EFPM This study

Pelagibacter ubique (0.019-0.039) M SEM-images Steindler et al., 2011 (2)

Pelagibacter ubique (0.025-0.045) M Cryo-electron tomography

Nicastro et al., 2006 (3)

SAR11 0.045 (0.033-0.060)

M FISH-Cy3-DAPI EFPM Malmstrom et al., 2005 (4)

Bacterioplankton (0.015-0.303) M AO EFPM La Ferla et al., 2005 (5)

Bacterioplankton (0.036-0.073) M AO EFPM Lee and Fuhrman, 1987 (6)

Bacterioplankton (0.003-14.8) M,F,E TEM-images Fagerbakke et al., 1996 (7)

Bacterioplankton (0.026-0.400) M,F AO EFPM Norland et al., 1993 (8)

Bacterioplankton (0.011-7.1) M,F TEM-images Norland et al, 1987 (9)

Bacterioplankton (0.003-2.624) F,E DAPI EFPM Theil-Nielsen and Sondergaard, 1998

(10)

LD12-121 0.017 (0.005-0.041)

F CARD-FISH Salcher et al., 2011 (11)

EUB I-III 0.038 (0.002-0.569)

F CARD-FISH Salcher et al., 2011 (11)

Actinobacteria (0.015-0.16) F CARD-FISH Salcher et al., 2010 (12)

Bacterioplankton 0.16 (0.09-0.45) F DAPI EFPM Felip et al., 2007 (13)

Bacterioplankton (0.003-0.08) F DAPI EFPM Posch, 2001 (14)

Bacterioplankton (0.003-3.5) F TEM-DAPI Loferer-Krobacher et al., 1998 (15)

(M: Marine, F: Freshwater and, E: Estuarine). Biovolume estimations in the listed studies were made by applying different methodologies (SEM:

Scanning Electron Microscopy; TEM: Transmission Electron Microscopy; DAPI: 4',6-diamidino-2-phenylindole; AO: Acridine Orange, EFPM:

Epifluorescence photomicrography, FISH: Fluorescence in situ hybridization, Cy3: Cyanine 3).

Supplementary References

1. Glockner FO, et al. (2000) Comparative 16S rRNA analysis of lake bacterioplankton reveals globally distributed phylogenetic clusters including an abundant group of actinobacteria. Appl Environ Microbiol 66(11):5053-5065.

2. Steindler L, Schwalbach MS, Smith DP, Chan F, & Giovannoni SJ (2011) Energy starved Candidatus Pelagibacter ubique substitutes light -mediated ATP production for endogenous carbon respiration. PLoS One 6(5):e19725.

3. Nicastro D, et al. (2006) Three-dimensional structure of the tiny bacterium Pelagibacter ubique studied by cryo-electron tomography. Microscopy and Microanalysis 12(S02):180-181.

4. Malmstrom RR, Cottrell MT, Elifantz H, & Kirchman DL (2005) Biomass production and assimilation of dissolved organic matter b y SAR11 bacteria in the Northwest Atlantic Ocean. Applied and Environmental Microbiology 71(6):2979-2986.

5. La Ferla R & Leonardi M (2005) Ecological implications of biomass and morphotype variations of bacterioplankton: an example i n a coastal zone of the Northern Adriatic Sea (Mediterranean). Marine Ecology 26(2):82-88.

6. Lee S & Fuhrman JA (1987) Relationships between biovolume and biomass of naturally derived marine bacterioplankton. Applied and Environmental Microbiology 53(6):1298-1303.

7. Fagerbakke KM, Heldal M, & Norland S (1996) Content of carbon, nitrogen, oxygen, sulfur and phosphorus in native aquatic and cultured bacteria. Aquat Microb Ecol 10(1):15-27.

8. Norland S (1993) The relationship between biomass and volume of bacteria. Aquat Microb Ecol, eds Kemp PF, Sherr BF, Cole JJ, & Sherr EB (Lewis Publishers, Boca Raton), pp 303–307.

9. Norland S, Heldal M, & Tumyr O (1987) On the relation between dry matter and volume of bacteria. Microbial Ecology 13(2):95-101. 10. Theil-Nielsen J & Søndergaard M (1998) Bacterial carbon biomass calculated from biovolumes. Arch Hydrobiol 141:195–207. 11. Salcher MM, Pernthaler J, & Posch T (2011) Seasonal bloom dynamics and ecophysiology of the freshwater sister clade of SAR11 bacteria ‘that rule

the waves’(LD12). The ISME Journal 5(8):1242-1252. 12. Salcher MM, Pernthaler J, & Posch T (2010) Spatiotemporal distribution and activity patterns of bacteria from three phylogene tic groups in an

oligomesotrophic lake. Limnology and Oceanography 55(2):846. 13. Felip M, Andreatta S, Sommaruga R, Straskrábová V, & Catalan J (2007) Suitability of flow cytometry for estimating bacterial biovolume in natural

plankton samples: comparison with microscopy data. Applied and Environmental Microbiology 73(14):4508-4514. 14. Posch T, et al. (2001) Precision of bacterioplankton biomass determination: a comparison of two fluorescent dyes, and of allometric and linear

volume-to-carbon conversion factors. Aquat Microb Ecol 25(1):55-63. 15. Loferer-Krößbacher M, Klima J, & Psenner R (1998) Determination of bacterial cell dry mass by transmission electron microscopy and densitometric

image analysis. Applied and Environmental Microbiology 64(2):688-694.

Documents

Supplementary Information Metagenomics uncovers a new ... · Tel: +34 965 919451 Email: [email protected] Keywords: ... with the scaffold identifier, sampling location, sampling depth