A C

B

MQ-?

CH4C2H6C3H8C4H10CnHn

1/2 O2 +

CH3-OHC2H5-OHC3H7-OHC4H9-OHCnHn-OH

}

NAD+H+NADH2

H+

CH2=O

THF

Periplasm

Cytoplasm

Serine Cycle

HCOOH

CO2 + 2H+

Acetyl-CoA

TCA

Propionyl-CoAMethylmalonyl-CoA Butyryl-CoA

Acetyl-CoAȕ-oxidation

11

7,8,9,10

2

2

5

pHMO

4

6

CnHnOn-CoA

Form

alde

hyde

Deh

ydro

gena

se

Uni

dent

ified

Per

mea

se

11 Hexulose-6PRuMP

Formate

12

Purine Biosynthesis3 Ferrodoxin ox

Ferrodoxin red

}A.

phmoC

phmoA

phmoB

1 2 3 4 5 6 7a 7b 7c 8a 8b 9 10 11 12

Average Plume

Average Background

Log 10

Nor

mal

ized

Tra

nscr

ipt A

bund

ance

02

46

8

02

46

8

Methan

ol de

hydro

gena

se

5,10 Meth

ylene

-THF dehy

droge

nase

1,2

5,10-M

ethyle

ne-T

HF redu

ctase

1

Formate

-THF lig

ase1

Formate

dehy

droge

nase

1

Serine

hydro

xymeth

yltran

sferas

e

Alcoho

l deh

ydrog

enas

e Clas

s IV3

Zinc de

pend

ent a

lcoho

l deh

ydrog

enas

e

Short c

hain

alcoh

ol de

hydro

gena

se3

NAD depe

nden

t alde

hyde

dehy

droge

nase

3

Aldehy

de:fe

rredo

xin ox

idored

uctas

e3

Acetyl

-CoA

Syn

thase

3

Acyl-C

oA S

yntha

se3

Hexulo

se-6-

P synth

ase

Formald

ehyd

e deh

ydrog

enas

e Gene No. Fig Above

GB-SAR324 + + + + + + + + + + + + +

- --

++

AAA240-J09 + NR + + + + + + + + + +

- -- NR++

AAA001-C10 + + + + + + NR --- -- -- - NR--

Presence/Absence of genes in SAR324 B.

Mirr

or Im

ages

Bin

Stru

ctur

eC

ontig

s H

ighl

ight

ed

Closeup of Highlighted Bins

Emergent Self Organizing Map (ESOM)of assembled contigs from Mid-Cayman Rise

Bins are 3-DConceptually similar to a topographic Map

Each color represents a ‘bin’ of contigs with similar tetramer frequencies

Contigs within bins are identified taxonomically or functionally

with gene annotations

Contigs

Post-assembly binning using Tetramer Frequency

M H PV H ....ATGCACGTGCCCCAT...

ProteinCds

Tetramer ATGC ACGTCACGGCACTGCA

Extract

gDNA

Shotgun

Sequence

Assemble

Sequences

Variable length ContigsFilter microbes from seawater 100-300 bp fragmentsMicrobial Genomes

Metagenomic Hurdle

How to associate contigswith little to no taxonomic

resolution due to database limitations?

Year Environment# Reads (millions)

Genomes or Bins

2007 Acid mine1 0.3 102010 Cyano Mat2 1.2 22010 Guaymas Basin2 4 82012 Cyano Mat3 255 552013 Guaymas Basin3 343 902013 Mid-Cayman Rise3 1,959 1002013 Lau Basin3 2,053 3652013 Coastal Sediment3 1850 75+

1Sanger, 2454 pyrosequencing, 3Illumina HiSeq

Plumes as deep-sea, in situ laboratoriesHow do background microbes respond to nutrient pulses?

Neutrally Buoyant Plume

Ris

ing

Plum

e

BackgroundMicrobial

Communities

NH4 Ox.

H2 Ox.H2S Ox.

Mn Ox.

CH4 Ox.

ChemosyntheticBiomass

HeterotrophicBiomass

+ DOCFe (II) Ox.

AnimalCommunities

ChimneyCommunities

Vent derived DOC

Near BottomCommunities

BackgroundMicrobial

Communities

Partical associatedMicrobial

Communities

Adapted from Dick et al. 2013

Non-buoyant Plume

Seafloor

Orifice

Buoyant Plume

ChimneyDeepBackground

Above Plume Background

ROV Jason

B. Plume sampling schematic A. SUPR filtering bouyant plumes

Rapid in situ sampling/preservation

Cultivation independent approaches have revolutionized our understanding of the diversity, dispersion and metabolic capability of microbial communities. However the dearth of environmentally-relavent genome sequences in publically available databases hinders our ability to ascribe metabolic function and taxonomic LGHQWL¿FDWLRQ� WR� HQLJPDWLF� PLFUREHV� IURP� QH[W�gen shotgun sequencing libraries. Here we show that by leveraging de novo assembly of shotgun sequenced metagenomes and metatranscriptomes followed by advanced nucleotide composition based genomic binning, we are able to infer functional characteristics of novel, ubiquitous, deep-ocean microorganisms. At present over 200 genomic bins containing partial and near complete genome cohorts (species and subspecies) have been recovered from a handful of metagenomes of hydrothermal plumes at Guaymas Basin, Eastern Lau Spreading Center and the Mid-Cayman Rise. A majority of genomes recovered stem from dark biosphere microorganisms, e.g. microbes heretofore represented only by ribosomal RNA sequences, thus revealing putative ecological roles of these novel microorganisms. Furthermore, coupled metatranscriptomic data shows many of these dark biosphere microorganisms are highly responsive to the plume environment and likely contribute to the biogeochemical cycling of hydrothermally GHULYHG�SOXPH�ÀXLGV�LQ�WKH�GHHS�RFHDQV�����

AbstractLeveraging de novo assembly and post assembly

clustering to discover novel environmental genomes

A C

B

MQ-?

CH4C2H6C3H8C4H10CnHn

1/2 O2 +

CH3-OHC2H5-OHC3H7-OHC4H9-OHCnHn-OH

}

NAD+H+NADH2

H+

CH2=O

THF

Periplasm

Cytoplasm

Serine Cycle

HCOOH

CO2 + 2H+

Acetyl-CoA

TCA

Propionyl-CoAMethylmalonyl-CoA Butyryl-CoA

Acetyl-CoAȕ-oxidation

11

7,8,9,10

2

2

5

pHMO

4

6

CnHnOn-CoA

Form

alde

hyde

Deh

ydro

gena

se

Uni

dent

ified

Per

mea

se

11 Hexulose-6PRuMP

Formate

12

Purine Biosynthesis3 Ferrodoxin ox

Ferrodoxin red

}A.

phmoC

phmoA

phmoB

1 2 3 4 5 6 7a 7b 7c 8a 8b 9 10 11 12

Average Plume

Average Background

Log 10

Nor

mal

ized

Tra

nscr

ipt A

bund

ance

02

46

8

02

46

8

Methan

ol de

hydro

gena

se

5,10 Meth

ylene

-THF dehy

droge

nase

1,2

5,10-M

ethyle

ne-T

HF redu

ctase

1

Formate

-THF lig

ase1

Formate

dehy

droge

nase

1

Serine

hydro

xymeth

yltran

sferas

e

Alcoho

l deh

ydrog

enas

e Clas

s IV3

Zinc de

pend

ent a

lcoho

l deh

ydrog

enas

e

Short c

hain

alcoh

ol de

hydro

gena

se3

NAD depe

nden

t alde

hyde

dehy

droge

nase

3

Aldehy

de:fe

rredo

xin ox

idored

uctas

e3

Acetyl

-CoA

Syn

thase

3

Acyl-C

oA S

yntha

se3

Hexulo

se-6-

P synth

ase

Formald

ehyd

e deh

ydrog

enas

e Gene No. Fig Above

GB-SAR324 + + + + + + + + + + + + +

- --

++

AAA240-J09 + NR + + + + + + + + + +

- -- NR++

AAA001-C10 + + + + + + NR --- -- -- - NR--

Presence/Absence of genes in SAR324 B.

Unraveling the function of enigmatic microbes and viruses with metagenomics and metatranscriptomics in deep ocean hydrothermal plumes

Project supported by GBMF 2609: Unveiling the microbial

communities that underpin deep-sea biogeochemistry

Cody S. Sheik1, Jain, S.1, Anantharaman, K.1, Baker, B.J.1, Li, M.1, German, C.R. 3, Toner, B.M.2, Breier, J.A.3, Dick, G.J.1

1Dept. Earth and Environmental Sciences, University of Michigan 2Dept. Soil, Water and Climate, University of Minnesota

3Applied Ocean Physics and Engineering, Woods Hole Oceanographic Institution

M. japanense

100

M. v

adi

72

Uncu

lture

d ba

cter

ium

OPU

3

99

NC10

M. capsulatus str. Bath

63 93 89

M. trichosporium OB3b

90

M. fumari

olicu

m

100

63

N. e

urop

aea

100

78

Group

X M

etha

notro

phs

100

SAR324

N. bacterium Broad-1100

M. rhodesiae NBB3Nocardioides sp. CF8

100

100

0.05

Met

hylo

cocc

acea

e ET

-HIR

O

Ethane

C2- C4 Hydrocarbons

Ammonia

Methane

Unknown

Methylocaldus sp. T-025

GB-SAR324 contain a novel hydrocarbon monooxygenaseHydrothermal Plume WaterEnriched in Methane, Alkanes, Reduced Sulfur and Ammonia

Increasing O2

H +

qmoC

A B

Comp I

dsr AB

dsrC

Rhodanese

HS-SO3- + HCN 2 H+ + SO3

2-

S--CN

HCO2- CO2

+ H+

/SO42-

SO42-

NA

DH

+ H

+

NA

D+

B

nrfD

APS

B aprASat

ATP

e-

fdhAH+HS-SO3

-

K

J

dsrMP

O

CtyC

SO3-2

2H+

OM

CM

TCA

Beta oxidation

X e-

HCO3-

SO3-

NADPHNADP + +H +

dsrLe

dsr

hf

?S -

MQ

H 2

MQ

MQ

H 2

MQ

MQ

H 2

MQ Com

p III

CtyC

Cbb

3

1/2 O2

NirK

H2O

NO2- NO-

NO2-

TauE

Organic Sulfur

Alcohols

S0

01

23

4Lo

g 10 T

rans

crip

t Abu

ndan

ce

fdhAB

IGKïPHP SAT

aprAB

dsrA

dsrB

dsrC

dsrD ds

rE dsrF

dsrH ds

rLds

rM dsrK ds

rJds

rO dsrP

dsrN

TPR$%&

TauE

/SafE

Rhoda

nese

Cbb3

NirK

SKPR$%&

AOA

NH3

? ?

dsrL

0 1 2 3 4 5

01

23

45

Log10 Background cDNA Abundance

Log 10

Plu

me

cDN

A A

bund

ance

Trap C4/Chlorinated PermeaseABC Carbohydrate TransportersABC Amino Acid TransportersAlcohol Dehydrogenases

0 1 2 3 4 5

01

23

45

Log10 Background cDNA Abundance

Log 10

Plu

me

cDN

A A

bund

ance

Sulfur OxidationFormate DehydrogenaseHydrocarbon MonooxygenaseNitrite OxidaseCO Dehydrogenase

A) B)

Genes indicative of sulfur oxidation present in the GB-SAR324 bin are also highly transcribed in the plume

Sheik et al. Environ Micro (2014)

Transcriptional response of pHMO and key degradation genes suggest GB-SAR324 is responding to the hydrocarbons in the plume

Using plumes to disentangle the eco-physiology of enigmatic microorganisms: Example SAR324 from Guaymas Basin, Gulf of California

Metabolic genes more abundant in the plume

Genome recovery scales with sequencing platform

Learn more on ESOM, Dick et al. Genome Biology (2009)