Upload
others
View
2
Download
0
Embed Size (px)
Citation preview
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)