Download FS406-22

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FS406-22
µ (hr-1)
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Temperature ( oC)
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Axial volcano- intersection
theA34Juan de Fuca Ridge and Cobbhydrothermal of
vent clone
A
deep-sea clone B33
Eikelberg
hotspot
deep-sea clone B49
B
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1 2 3 4 5 6
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deep-sea clone B28
deep-sea clone B25
Hydrothermal venthydrothermal
fluid was
into diazotrophic medium at
vent cloneinoculated
E7
FS406-22 nifH1 (expressed at 90 C)
70 and 90 degrees C Methanothermococcus thermolithotrophicus nifH1
hydrothermal vent clone A12
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o
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hydrothermal vent clone A11
hydrothermal vent clone A16
hydrothermal vent clone E59
Isolated FS406-22
archaeal
culture capable of growth from 58-92
hydrothermal
vent clone A2
Azotobacter vinelandii nifH3
Clostridium pasteurianum
nifH3
degrees C (N2, H2, CO2 marine
medium)
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Rhodobacter capsulatus anfH
Methanosarcina acetivorans C2A anfH !"# vnfH
Methanosarcina barkeri
First microorganismMethanothermobacter
from the thermautotrophicus
deep sea $!%&'%(
to exhibit diazotrophy
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Methanothermobacter thermautotrophicus ∆H
Methanobacterium ivanovii
Methanococcus maripaludis
hydrothermal vent clone A8
Implications of a hyperthermophilic
methanogen
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hydrothermal vent clone A21
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Methanosarcina acetivorans C2A
hydrothermal vent clone C83
FS406-22 nifH2
Methanocaldococcus jannaschii nifH
Methanococcus voltae
Methanobacterium ivanovii nifH homolog
hydrothermal vent clone C61
NifH cluster 4
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Phylogenetic analyses of nitrogenase and chlorophyll iron protein
suggests that an ancestral iron protein diverged into nifH/anfH before
separation of bacteria
and methanogenic archaea (Burke etl al. 1993)
Methanothermococcus thermolithotrophicus nifH2
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Methanopyrus kandleri AV19
Plectonema boryanum frxC
hydrothermal
vent clone➔
A37 nitrogenase predates
nifH gave rise to chlorophyll iron
protein
photosynthesis
Fig. 2. (A) NifH amino acid phylogenetic tree constructed by quartet puzzling maximum likelihood
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(12). Cluster 2 includes molybdenum dinitrogenase reductases from methanogens, as well as
alternative vanadium and iron-only dinitrogenase reductases (VnfH, AnfH) from Methanosarcinales
and bacteria. Cluster 4 includes paralogous dinitrogenase reductases that are probably not
involved in nitrogen fixation. The scale bar indicates the number of amino acid substitutions per
site. The tree is outgroup rooted with Plectonema boryanum frxC, a dinitrogenase reductase–like
protein involved in the light-independent reduction of protochlorophyllide. GenBank ID numbers
for tree sequences are listed in table S2, and the alignment is shown in fig. S2. (B) Lanes 1 and 2:
the product of RT-PCR with nifH primers and 2 and 3 ml of RNA extracted from FS406-22 growing
Tuesday,
25,3:2011
on October
N2 at 90°C; lane
RT-PCR without RNA; lane 4: Hi-Lo DNA ladder; lane 5: nifH PCR with 2 ml of
Nitrogenase part of last common ancestor?
NifH cluster 4
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NifH cluster 2
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µ (hr-1)
NifH cluster 2
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archaea, are diazot
fixation (6). The su
bathed in reduced,
fluid and thus p
methanogens, strict
as a by-product. Th
coccus thermolithot
thermophilic microo
gen, at up to 64°C (7
microbial mats also
Here we describe the
deep-sea hydrotherm
at up to 92°C, whic
limit of biological n
Axial volcano is
at the intersection of
the Juan de Fuca Rid
crust beneath Axial
high porosity (9,
community associat
thermophilic and h
with maximal grow
above 80°C, respect
Millennium Observ
fluid exiting the su
diffuse vent named
which was measur
sampling, was inocu
select for diazotroph
70° and 90°C. The e
at both temperatu
antibiotic-containing
of bacteria. The am
medium was reduc
completely (12).
The archaeal c
capable of growth f
sole source of nitro
marine salts and H
occurred at 90°C, an
and 95°C (Fig. 1). A
essary for growth on
cocci were visible
during exponential g
of FS406-22, we seq
(rRNA) gene and fo
16S rRNA gene fr
caldococcus jannas
isolated from a deep
on the East Pacific
and grows in a tem
with an optimum ne
vent isolate FS40
termined by means
The specific growth
nitrate and ammon
which is lower th
jannaschii at 85°C,
The nifH gene
reductase, is highly
mostly consistent w
The nifD and nifK
containing dinitrogen
nase reductase cons
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range of diverse bacteria, and the methanogenic
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archaea, are diazotrophic, or capable of nitrogen
Temperature ( oC)
fixation (6). The subseafloor at mid-ocean ridges is
bathed in reduced, H2- and CO2-rich hydrothermal
fluid and thus provides an ideal habitat for
hydrothermal vent clone A34
A
methanogens, strict anaerobes that produce methane
deep-sea clone B33
as a by-product. The methanogen MethanothermoB
97 deep-sea clone B49
coccus thermolithotrophicus is currently the most
deep-sea clone B28
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thermophilic microorganism known that fixes nitrodeep-sea clone B25
gen, at up to 64°C (7). Synechococcus ecotypes from
84 69 hydrothermal vent clone A12
microbial mats also fix nitrogen at up to 63.4°C (8).
hydrothermal vent clone E7
Here we describe the isolation of a methanogen from
FS406-22 nifH1 (expressed at 90oC)
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deep-sea hydrothermal vent fluid that fixes nitrogen
Methanothermococcus thermolithotrophicus nifH1
at up to 92°C, which extends the upper temperature
hydrothermal vent clone A11
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limit of biological nitrogen fixation by 28°C.
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Axial volcano is located in the northeast Pacific,
hydrothermal vent clone E59
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at the intersection of the Cobb-Eikelberg hotspot and
hydrothermal vent clone A2
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the Juan de Fuca Ridge. The upper 100 m of oceanic
Azotobacter vinelandii nifH3
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crust beneath Axial seamount is estimated to have
Clostridium pasteurianum nifH3
high porosity (9, 10), and the diverse archaeal
Rhodobacter capsulatus anfH
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community associated with its subseafloor includes
97 Methanosarcina acetivorans C2A anfH !"# vnfH
thermophilic and hyperthermophilic methanogens,
Methanosarcina barkeri
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with maximal growth rates from 45° to 80°C and
Methanothermobacter thermautotrophicus $!%&'%(
95
70
above 80°C, respectively (11). During the 2004 New
Methanothermobacter thermautotrophicus ∆H
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Millennium Observatory cruise to Axial volcano,
Methanobacterium ivanovii
Methanococcus maripaludis
fluid exiting the subseafloor was sampled from a
diffuse vent named marker 113 (12). The fluid,
hydrothermal
vent clone A8
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which was measured to be 23°C at the point of
hydrothermal vent clone A21
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sampling, was inoculated into a medium designed to
Methanosarcina acetivorans C2A
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hydrothermal vent clone C83
select for diazotrophs and incubated anaerobically at
70° and 90°C. The enrichment cultures were positive
FS406-22 nifH2
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Methanocaldococcus jannaschii nifH
at both temperatures and transferred into an
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Methanococcus voltae
antibiotic-containing medium to prevent the growth
Methanobacterium ivanovii nifH homolog
of bacteria. The amount of fixed nitrogen in the
hydrothermal vent clone C61
medium was reduced over time and then omitted
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Methanothermococcus thermolithotrophicus nifH2
completely (12).
Methanopyrus kandleri AV19
The archaeal culture, named FS406-22, was
Plectonema boryanum frxC
capable of growth from 58° to 92°C with N2 as the
hydrothermal vent clone A37
sole source of nitrogen, in a medium containing
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marine salts and H2 plus CO2. Maximal growth
occurred at 90°C, and no growth was detected at 55°
Fig. 2. (A) NifH amino acid phylogenetic tree constructed by quartet puzzling maximum likelihood
and 95°C (Fig. 1). Agitation of the culture was nec(12). Cluster 2 includes molybdenum dinitrogenase reductases from methanogens, as well as
essary for growth on N2, and clusters of two or more
alternative vanadium and iron-only dinitrogenase reductases (VnfH, AnfH) from Methanosarcinales
cocci were visible by phase contrast microscopy
and bacteria. Cluster 4 includes paralogous dinitrogenase reductases that are probably not
during exponential growth. To determine the identity
involved in nitrogen fixation. The scale bar indicates the number of amino acid substitutions per
of FS406-22, we sequenced its 16S ribosomal RNA
site. The tree is outgroup rooted with Plectonema boryanum frxC, a dinitrogenase reductase–like
(rRNA) gene and found it to be 99% identical to the
protein involved in the light-independent reduction of protochlorophyllide. GenBank ID numbers
for tree sequences are listed in table S2, and the alignment is shown in fig. S2. (B) Lanes 1 and 2:
16S rRNA gene from the methanogen Methanothe product of RT-PCR with nifH primers and 2 and 3 ml of RNA extracted from FS406-22 growing
caldococcus jannaschii (fig. S1). M. jannaschii was
on N2 at 90°C; lane 3: RT-PCR without RNA; lane 4: Hi-Lo DNA ladder; lane 5: nifH PCR with 2 ml of
isolated from a deep-sea hydrothermal vent chimney
RNA; lane 6: nifH PCR without RNA. The product in lanes 1 and 2 lies between the 400- and 500-bp
on the East Pacific Rise in a nitrogen-rich medium,
bands of the ladder.
and grows in a temperature range of 50° to 86°C
with an optimum near 85°C (13). Our hydrothermal
vent isolate FS406-22 produced methane, de15 DECEMBER 2006 VOL 314 SCIENCE www.sciencemag.org
1784
termined by means of gas chromatography (GC).
The specific growth rate of FS406-22 grown with
nitrate and ammonium was 0.37 hour−1 at 90°C,
which is lower than the rate reported for M.
jannaschii at 85°C, which is ~1.5 hour−1 (13).
The nifH gene, which encodes dinitrogenase
reductase, is highly conserved, and its phylogeny is
mostly consistent with that of the 16S rRNA gene.
The nifD and nifK genes encode a molybdenum-
Downloaded from www.sciencemag.org on October 24, 2011
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Mehta and Baross 2006 (Science)
Fig. 1. Growth rate of FS406-22
grown with N2 as the sole source of
nitrogen, as a function of temperature. The specific growth rate (m) is
the average of three determinations
of growth rate during exponential
phase. Growth was monitored by
epifluorescence microscopy (12).
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A hyperthermophilic archaea that can fix N2 at 92 degrees C!
REPORTS
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grown with N2 as the sole source of
nitrogen, as a function of temperature. The specific growth rate (m) is
the average of three determinations
of growth rate during exponential
phase. Growth was monitored by
epifluorescence microscopy (12).