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Applied and Environmental Microbiology, November 2001, p. 5179-5189, Vol. 67, No. 11
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.11.5179-5189.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Signature Lipids and Stable Carbon Isotope Analyses of Octopus
Spring Hyperthermophilic Communities Compared with Those of
Aquificales Representatives
Linda L.
Jahnke,1,*
Wolfgang
Eder,2
Robert
Huber,2
Janet M.
Hope,3
Kai-Uwe
Hinrichs,4
John M.
Hayes,4
David J.
Des
Marais,1
Sherry L.
Cady,5 and
Roger E.
Summons3,
Exobiology Branch, NASA Ames Research Center,
Moffett Field, California 940351;
Lehrstuhl für Mikrobiologie and Archaeenzentrum,
Universität Regensburg, D-93053 Regensburg,
Germany2; Australian Geological Survey
Organisation, Canberra, ACT 2601, Australia3; Department of Geology and
Geophysics, Woods Hole Oceanographic Institution, Woods Hole,
Massachusetts 025434; and Department of
Geology, Portland State University, Portland, Oregon
972015
Received 10 May 2001/Accepted 20 August 2001
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ABSTRACT |
The molecular and isotopic compositions of lipid biomarkers of
cultured Aquificales genera have been used to study the
community and trophic structure of the hyperthermophilic pink streamers and vent biofilm from Octopus Spring. Thermocrinis
ruber, Thermocrinis sp. strain HI 11/12,
Hydrogenobacter thermophilus TK-6,
Aquifex pyrophilus, and Aquifex aeolicus
all contained glycerol-ether phospholipids as well as acyl glycerides.
The n-C20:1 and
cy-C21 fatty acids dominated all of the
Aquificales, while the alkyl glycerol ethers were mainly
C18:0. These Aquificales biomarkers were
major constituents of the lipid extracts of two Octopus Spring samples,
a biofilm associated with the siliceous vent walls, and the well-known
pink streamer community (PSC). Both the biofilm and the PSC contained
mono- and dialkyl glycerol ethers in which C18 and
C20 alkyl groups were prevalent. Phospholipid fatty acids included both the Aquificales n-C20:1 and
cy-C21, plus a series of
iso-branched fatty acids
(i-C15:0 to
i-C21:0), indicating an additional bacterial
component. Biomass and lipids from the PSC were depleted in
13C relative to source water CO2 by 10.9 and
17.2
, respectively. The C20-21 fatty acids of the PSC
were less depleted than the iso-branched fatty acids,
18.4 and 22.6, respectively. The biomass of T. ruber
grown on CO2 was depleted in 13C by only 3.3
relative to C source. In contrast, biomass was depleted by 19.7 when
formate was the C source. Independent of carbon source, T.
ruber lipids were heavier than biomass (+1.3). The depletion
in the C20-21 fatty acids from the PSC indicates that
Thermocrinis biomass must be similarly depleted and too
light to be explained by growth on CO2. Accordingly,
Thermocrinis in the PSC is likely to have utilized
formate, presumably generated in the spring source region.
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INTRODUCTION |
Based on phylogenetic
analysis of small-subunit rRNA sequences, hyperthermophilic organisms
proliferate in the deepest branches of the Bacterial and Archaeal
domains. The branch lengths of these hyperthermophilic lineages tend to
be short, which further suggests that such organisms are the closest
known extant descendants of the last common ancestor and retain many
ancestral phenotypic properties (49). The recent discovery
of filamentous microfossils preserved in a 3,235-million-year-old
submarine volcanogenic deposit lends considerable weight to the theory
that hydrothermal vent organisms have had a very long history on Earth
(41). Hyperthermophilic microbes are also attracting
astrobiological and biogeochemical interest because of their potential
role in the formation of many kinds of mineral deposits and the
generation of rock textures and mineral assemblages that may be
diagnostic for extant or extinct life beyond Earth (5).
A well-known example of a hyperthermophilic chemolithotrophic ecosystem
is the pink filamentous streamers found at Octopus Spring in
Yellowstone National Park (YNP), United States that were described by
Brock in 1965 (3, 4). Similar streamer communities were
first reported by Setchell in 1903 (45) and have
subsequently been identified in neutral to alkaline springs of
geothermal areas in Iceland, Japan, and Kamchatka, Russia (21, 48, 56) and, more recently, as distinct black streamers at Calcite Springs, YNP (42).
Molecular analysis of the small-subunit 16S rRNA sequences of the
filamentous pink streamer community (PSC) indicates dominance of the
domain Bacteria, in particular two deeply diverging phylotypes affiliated with the Aquificales and Thermotogales
(43). From the PCS, the first pink streamer isolate,
Thermocrinis ruber, was recently brought into culture
(18). T. ruber forms a separate lineage within
the order Aquificales and shares many features with the two
previously isolated genera, Aquifex and
Hydrogenobacter (22, 28, 29).
The use of lipid biomarkers for revealing microbial community structure
is well established. Branched-chain fatty acids are not uncommon in
thermophilic organisms (31) and have been identified in
pink streamer samples from Octopus Spring previously (1). Other more distinctive lipids are now recognized as valuable biomarkers in some thermophiles. Thermomicrobium roseum contains
internally methyl-branched C18 fatty acid and
long-chain 1,2-diols as major components (38). The core
lipids of members of the order Thermotogales are composed of
unusual dicarboxylic fatty acids and a recently discovered ether lipid,
15,16-dimethyl-30-glyceroloxytriacontanoic acid (20).
Other novel mono- and dialkyl glycerol ether (GME and GDE,
respectively) lipids have been described in
Thermodesulfobacterium commune (30) and
Aquifex pyrophilus (22).
In addition to carrying distinctive chemical structures, lipid
biomarkers also encode the stable isotopic signature that
provides information about the physiologies of the source
organisms (10, 26). However, interpretation of these
isotopic signatures requires specific knowledge about C isotopic
discriminations associated with the biochemical pathways involved in
carbon fixation and lipid synthesis. Only a limited amount of
information is available on the bulk isotopic fractionation factors for
cultured Aquificales (17), and to our
knowledge, nothing has been reported on the isotopic composition of lipids.
In this study, we initially set out to examine the microbial
composition of the Octopus Spring PSC and nearby vent biofilms through
a comprehensive lipid analysis. The resultant data revealed a more
complex situation than was apparent from genomic analysis alone and
also indicated a need for appropriate supporting data from pure-culture
studies. A comparison of the lipid profiles of several genera within
the Aquificales as well as measurements of the carbon
isotopic fractionation associated with autotrophic and heterotrophic
growth of T. ruber formed a framework for improved understanding of the population structure of the Octopus Spring PSC and
associated vent microbiota.
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MATERIALS AND METHODS |
Sample collection and preparation.
Biomass consisting
of the PSC was collected using forceps from an 87°C, pH 8.3 site in
the main outflow just below the source pool vent of Octopus Spring in
May 1997. The filaments were placed in glass tubes, sealed with
Teflon-lined caps, frozen on dry ice within 3 h, and maintained so
in transit to the National Aeronautics and Space Administration (NASA)
Ames Research Center. A vent wall geyserite sample, approximately 25 cm2, was removed in 1996 from the shallower main
pool that contains the main effluent of Octopus Spring (
92°C, pH
8.0). The geyserite sample was also kept frozen until it was prepared
for analysis.
Working in a glove box, the topmost 1 to 2 mm of the frozen geyserite
biofilm was carefully removed by scraping with a sterilized spatula.
The remaining material was then crushed in a sapphire mortar and pestle
that had been cleaned with methanol and transferred to sterilized glass
vials. PSC and vent geyserite samples were lyophilized and then ground
to a powder in a glass mortar previously cleaned with sequential
solvent washes of dichloromethane, methanol, and acetone. All glassware
and metal implements used in our procedures were baked at 450°C for a
minimum of 4 h. Only Teflon stoppers and/or Teflon-lined screw
caps were used in analyses.
Strains and culture conditions.
Thermocrinis
ruber OC 1/4 (DSM 12173), Aquifex pyrophilus Kol5a (DSM
6858), Hydrogenobacter thermophilus TK-6 (IAM 12695), Aquifex aeolicus VF5 (21), and
Thermocrinis sp. strain HI 11/12 (18) were
obtained from the culture collection of the Lehrstuhl für
Mikrobiologie, Universität Regensburg, Regensburg, Germany. Cell
masses of the Aquificales strains were grown at 85°C
(70°C for H. thermophilus and 80°C for T. ruber with formate) with stirring (up to 400 rpm) in a 300-liter
enamel-protected fermentor (Bioengineering, Wald, Switzerland) as
described in Table 1. For growth of
T. ruber in experiment 1 (isotope study), the cell titer was
monitored and the culture was gassed with increasing flow rates (2, 5, 7.5, and 10 liter min
1) to maintain the growth
rate.
Phylogenetic analyses.
For the analyses, an alignment of
about 11,000 homologous full primary sequences available in public
databases (ARB project [32, 33]) was used. The
Aquificales 16S rRNA gene sequences were fitted in the 16S
rRNA tree by using the automated tools of the ARB software package
(33). Distance matrix (Jukes and Cantor correction),
maximum parsimony, and maximum likelihood (fastDNAml) methods were
applied as implemented in the ARB software package (34).
Lipid extraction, separation, and analysis.
Lipids were
extracted from lyophilized ground sinter or Aquificales
biomass using a single-phase modification of the Bligh and Dyer
procedure, and water-soluble contaminants were removed as previously
reported (24). Elemental sulfur was removed by passing the
total lipid extract over activated copper powder. The total lipid
extract (TLE) was dried under nitrogen and then maintained in a vacuum
desiccator over Drierite until it reached a constant weight.
A portion of the PSC total lipid was used for an oxidation-reduction
procedure to convert bacteriohopanepolyol to its hopanol
derivative
(
44) and analyzed as previously reported
(
24).
Fatty acid methyl esters (FAME) and glycerol ethers (GME and GDE) were
prepared by two procedures. In procedure I, FAME were
prepared by
subjecting a portion of the TLE to mild alkaline methanolysis
(
36) with heating at 37°C for 1 h. FAME were
separated from
the remaining polar ether lipids (GME and GDE) by
thin-layer chromatography
(TLC) using a methylene chloride mobile phase
as previously reported
(
24). The FAME
(
Rf = 0.80) were recovered by
eluting the silica
gel with methylene chloride, and the ether-linked
components were
recovered from the origin of the TLC plate by Bligh and
Dyer extraction
of the silica gel zone. The polar ether components were
hydrolyzed
in 1 ml of chloroform-methanol-concentrated HCl (1:10:1) by
heating
to 100°C for 2 h (
36), and the glycerol
ethers were separated
by TLC using hexane-diethyl ether-acetic acid
(70:30:1) into GME
(
Rf = 0.04), GDE
(
Rf = 0.40), and diphytanylglycerol ether
(
Rf = 0.49) using reference compounds
1-
O-hexadecyl-glycerol and
1,2-di-
O-hexadecyl-glycerol
(Sigma, St. Louis, Mo.) and
diphytanylglycerol ether isolated
from a
Halobacterium sp.
(
52).
Procedure II was used in an attempt to analyze small samples such as
the Octopus Spring vent geyserite. In this approach,
the TLE was
directly hydrolyzed with acid as described above,
followed by
trimethylsilyl (TMS) derivatization of the resulting
free glycerol
ethers, and gas chromatography-mass spectrometry
(GC-MS) of the treated
TLE. Some TLE samples were also analyzed
for free fatty acids and
glycerides by preparation of TMS derivatives.
Abundance calculations
were based on comparison of peak areas
to internal standards, methyl
tricosanoate (C
23) for FAME and
cholestanol for
glycerol ethers. Weight percent of FAME (Table
2) was calculated based on flame
ionization detector (FID) response
of individual fatty acids
(C
14 to C
22) relative to
C
23; for GME
and GDE, values are based on
the areas of the total ion chromatographs
and should be
considered semiquantitative.
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TABLE 2.
Comparison of ester-linked fatty acid and glycerol ether
composition of Octopus Spring PSC and Aquificales
culturesa
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Some of the PSC and
T. ruber (experiment 1) TLE were also
preparatively separated into a polar lipid (phospholipids) and a
neutral lipid (glycolipids and glycerides) fraction by
precipitation
in cold acetone (
27). The components of the
polar and neutral
fractions were then separated by thin layer
chromatography on
Silica gel G plates (Merck) using
acetone-benzene-water (91:30:8)
(
37) or, in some cases,
chloroform-methanol-water (65:25:4)
(
27). Preliminary
characterization of TLC zones was made based
on migration of standard
diacyl compounds (phosphatidylcholine,
phosphatidylethanolamine,
and di- and monogalactosyl diglycerides)
and staining with specific
detection reagents, phosphomolybdic
acid, ninhydrin, and
-naphthol
(
27). TLC zones for lipid analysis
were detected by UV
fluorescence with rhodamine 6G and recovered
by the Bligh and Dyer
elution. FAME and glycerol ethers were prepared
from each fraction as
described above. The double bond positions
of the monounsaturated FAME
were determined by preparing the dimethyl
disulfide adducts
(
57). TMS derivatives of the glycerides were
prepared
using
N,
O-bis(trimethylsilyl)trifluoroacetamide
with
1% trimethylchlorosilane (1:1 in
pyridine).
Alkyl moieties were released from the glycerol ether compounds by
reaction with BBr
3 as reported previously
(
50).
Gas chromatographic analyses.
FAME were analyzed by using a
Perkin-Elmer Sigma 3B gas chromatograph equipped with an FID and 30-m
megabore columns (J & W Scientific), either a DB-5ms programmed to
increase at 4°C/min from 160 to 280°C, or a DB-27 programmed to
increase at 4°C/min from 120 to 220°C. Compound identification was
based on retention times on the nonpolar and the polar columns and on
mass spectral analysis (see below).
GC-MS analyses of fatty acids as FAME or TMS esters and glycerol ethers
as TMS derivatives, were performed using an HP 6890
gas chromatograph
equipped with a J&W DB-5 (60 m by 0.32 mm, 0.25-µm
film) capillary
column and an HP 5973 mass-selective detector
operated at 60°C for 10 min, then programmed at 10°C/min to 320°C,
and held for 60 min
(Fig.
1). The bond positions of the
monounsatured
FAME were determined by analyzing their dimethyl
disulfide adducts
as previously described (
25).
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FIG. 1.
Total ion chromatogram of biofilm-associated siliceous
sinter from within Octopus Spring vent pool (92°C) showing
predominance of C18 mono- and dialkyl glycerol ethers.
Structures are shown for 1,2-di-O-phytanylglycerol and
C18,18-diakylglycerol ether. Symbols designate FAME
(n- or i-18), isoprenoid lipid (I-),
monoalkyl glycerol (C18), and dialkyl glycerol
(C18,18) with accompanying carbon chain lengths.
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Isotopic measurements.
The dissolved inorganic carbon (DIC)
was measured by taking three 40-ml water samples from the outflow site
using a syringe and immediately filtering through Whatman GF/F filters
into preevacuated 130-ml serum bottles sealed with silicone stoppers
and containing a few drops of saturated HgCl2 to
inhibit bacterial growth. The bottles were kept chilled until analysis.
At Ames, samples were withdrawn and acidified, and the
CO2 gas was collected on a vacuum line for
isotopic analysis on a Nuclide 6-60RMS mass spectrometer modified for
small samples (9, 14). Analysis of the DIC composition of
the Thermocrinis culture medium was similar except that
samples were collected by filling three glass tubes with the gassed
culture medium prior to inoculation and immediately stoppering them
with crimp seals before shipment by air to Ames from Germany. Biomass and total lipid were determined at Ames using a Carlo Erba CHN EA1108
elemental analyzer interfaced to a Finnigan Delta Plus XL isotope ratio
mass spectrometer (EA-IRMS). Compound specific isotope analyses were
done at the Australian Geological Survey Organisation (AGSO) as
previously described using a Finnigan MAT 252 mass spectrometer
equipped with a CuO/Pt microvolume combustion furnace and a Varian 1400 gas chromatograph with DB-5 column (25). Reported 
values for FAME and GME were the averages of two runs which agreed
within ±1.2% and have been corrected for the presence of carbon added
during derivatization (
43 in the case of TMS-C and 50 in the
case of methanol carbon used to form FAME).
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RESULTS |
Comparison of PSC and vent biofilm lipids.
The total organic
carbon (TOC) recovered from the vent geyserite surface was only 0.24%,
compared with 7.2% for PSC and 36.7% for T. ruber biomass
(experiment 1). Procedure II, direct acid hydrolysis, was used to
minimize loss of material during preparation of the vent sample (Fig.
1) and to allow comparison with PSC and T. ruber samples.
Although this method does degrade the cyclopropyl FAME present in these
extracts (6), it provides valuable comparative information.
The fatty acids of vent communities and PSC were predominantly
C
17 to C
22 chain lengths,
distinguished by
iso-homologues of
the
C
16 to C
21 acids plus a
diminished (see above) amount of
cy-C
21.
The TMS derivatives of the
free fatty acids present in the vent
TLE confirmed that high amounts of
cy-C
21 (25% of total) were
characteristic of the Octopus Spring vent biofilm community. The
fatty
acid composition of vent and PSC extracts, while qualitatively
similar,
differed quantitatively. Streamers had more abundant
branched-chain
fatty acid (41% in PSC versus 13% in Octopus Spring
vent) with a
somewhat shorter chain distribution: the ratio of
i-C
17:0 to
i-C
19:0 was 1:2.1 in PSC but 1:3.7 in
the Octopus Spring
vent. The
anteiso analogues
ai-C
17:0 and
ai-C
19:0 were present
in PSC (1.3%)
but almost absent in the Octopus Spring vent. The
Octopus Spring vent
TLE also contained small amounts of even-carbon-numbered
chain fatty
acids,
n-22:0 to
n-30:0, and a mid-chain branched
octadecanoic acid (1%), possibly 10- or
12-methyl-C
18:0. Alkyl
glycerol ethers were
abundant in both vent and PSC, however, the
vent GDE represented a
higher proportion (43%) of total ether
lipids than in PSC (29%).
Significant amounts of archaeal biomarkers,
1,2-di-
O-phytanylglycerol (archaeol) and both
C
20- and C
25-isoprenoid
glycerol monoethers, were present in the vent biofilm and PSC
(
5%
of total ether lipids). BBr
3 cleavage of the
ether alkyl
chains showed that, apart from the isoprenoid moieties,
only straight-chain
compounds were present, with carbon numbers
consistent with the
GC-MS characterization of the intact glycerol
ethers. C
18 chains
dominated both GME and GDE
(Fig.
1).
PSC and Aquificales polar lipids.
The acid
hydrolysate of the T. ruber TLE (procedure II) was composed
primarily of saturated and monounsaturated C20
fatty acids and a C18-GME (Table 2). No
iso-branched fatty acids or GDE were detected. Additional
analyses of the lipid using an alkaline methanolysis procedure (I)
confirmed the high proportion of C20 fatty acids
with C20:1, which together comprised over 49% of
the total for T. ruber OC1/4 (Table 2, experiment 1). Large
amounts of cy-C21 were also recovered
in T. ruber and PSC by this procedure, and all subsequent
FAME analyses were so carried out (Tables 2 and
3).
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TABLE 3.
Stable carbon isotopic composition (13C)
and distribution of fatty acids in major lipid fractions isolated from
PSCa
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T. ruber biomass from a variety of growth conditions, as
shown in Table
1, was analyzed. In experiment 1,
T. ruber
was grown
using thiosulfate with a constant gassing of
H
2-CO
2-O
2
to maintain
high substrate levels for measurement of the carbon
isotopic discrimination
associated with CO
2
fixation (results below). These conditions
resulted in accumulation of
relatively large amounts of intracellular
sulfur (
15% of dry
weight). S
0 accumulation was not apparent in
T. ruber grown with thiosulfate
but without
H
2 (experiment 2) or in PSC extracts. The lipid
composition
of additional
Aquificales cultures (Table
1) was
also analyzed
to assess the potential use of
Thermocrinis-like lipids as group
biomarkers to characterize
the pink streamer community (Table
2). All
Aquificales
cultures contained GME, and no
iso-branched
fatty acids were
detected (Table
2). GDE were only present in
the lipids of the PSC and
the two marine
Aquifex cultures,
A. aeolicus VF5 and
A. pyrophilus Kol 5a.
BBr
3 cleavage of the
Aquificales glycerol ethers showed only straight-chain alkyls with chain length
distributions similar to those characterized for the intact
molecules.
Using the methods of Rohmer et al. (
44), no hopanoids were
detected in the PSC total lipid extract, either as the polar
bacteriohopanepolyol or as the free lipids, diplopterol or
diploptene.
A preliminary attempt was made to separate the complex lipids of the
PSC extract to aid in biomarker identification and as
a preparative
step to carbon isotope analysis. The diversity of
polar head groups and
the occurrence of both diacyl and dialkyl
moieties made separation by
one-dimensional TLC difficult. TLC
separation of the polar lipid (PL)
fraction using the chloroform-methanol-water
system recovered 92% of
PL-FAME in two zones, PL-2 (
Rf = 0.35
to
0.25) and PL-4 (
Rf = 0.60 to 0.56) that
roughly comigrated
with diacylphosphatidylcholine
(
Rf = 0.33) and
diacylphosphatidylethanolamine
(
Rf = 0.66)
(Table
3). All PL zones showed the presence of multiple
components, and
PL-4 was dominated by an aminolipid similar to
that observed from
A. pyrophilus (
27).
FAME present in the neutral lipid (NL) fraction were separated using
the acetone-benzene-water system. Both a glycolipid zone
that migrated
closely with a digalactosyl diglyceride standard
(
Rf = 0.43) and a rapidly migrating
component (
Rf = 0.95), a probable
free
glyceride designated NL-2, accounted for 12 and 82% of recovered
NL-FAME, respectively (Table
3). The GDE present in the PL fraction
were recovered primarily in PL-4 and those in the NL fraction
in NL-2.
Together these two zones accounted for 60 and 25% of
total GDE,
respectively. While the recoveries of FAME and GDE
were in good
agreement with analyses made using the TLE, the recovery
of GME was
poor (see
below).
Lipid fractions as described above were also prepared from the total
lipid of
T. ruber (experiment 1). In this case, most
of the
FAME were recovered from two fractions equivalent to PL-2
and PL-4 with
24 and 70%, respectively. No GDE were detected in
any of the isolated
fractions, and although most of the GME was
also recovered in PL-2 and
PL-4, the recovery was not significant
(0.2 µmol/g [dry weight])
relative to the amount measured by the
direct acid hydrolysis procedure
(Table
2). This discrepancy
appears to be associated with the use of
TLC to separate the polar
compounds and not procedure I, as relatively
large amounts of
GME were recovered during analysis of the additional
Aquificales cultures using procedure
I.
Carbon isotopic composition.
The Octopus Spring water had a
relatively high DIC content, 5.3 mM, with a
13C value of 1.5 ± 0.3
(n = 3). At the temperature (87°C) and pH (8.3) of
the pink streamer site, a dissolved CO2
concentration ([CO2]) of 71 µM and
13C of 4.7 can be calculated based on a
pKa for carbonic acid at 90°C of 6.42 and the
equation of Mook et al. (35) in which the fractionation
between dissolved CO2 (d) and
dissolved bicarbonate (b) is given by
Ed/b = 24.12 9866/T, where T is the absolute temperature. The
streamer biomass and TLE from this site are depleted in
13C relative to [CO2] by
10.9 and 17.2, respectively (Table 4).
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TABLE 4.
C isotopic composition of isolated components from PSC
and T. ruber grown as a lithoautotroph with
thiosulfate-H2-O2-CO2 and as a
chemoorganotroph with formate and
O2a
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In laboratory cultures (Table
1, experiment 1), a sample of medium
removed prior to inoculation measured 14.1 mM DIC with
a
13C of
25.5
± 0.9
(
n = 3). At the temperature and pH of the fluid,
the
[CO
2] was 6.2 mM, with a
13C of
27.4
. The carbon available from
the continuously flowing
gas mixture far exceeded that assimilated by
the culture. The
T. ruber biomass and lipids were depleted
in
13C relative to the
[CO
2] by 3.3 and 2.1
, respectively. As shown
in Table
4, this depletion is much smaller than that observed
for the
PSC. Moreover, the patterns of depletion differ, with
lipids depleted
relative to biomass in the PSC and enriched in
T. ruber.
In a second experiment where
T. ruber was grown with 0.1%
formate (Table
1, experiment 4), biomass was much more strongly
depleted in
13C relative to carbon source
(19.7
). The yield from 250 liters
of medium was 5 g (dry
weight) (39.2% carbon), which accounted
for 0.36% of available
carbon. As with growth on CO
2, lipids were
slightly enriched relative to
biomass.
The carbon isotopic compositions of several individual lipid biomarkers
were also determined (Table
5). In the
PSC fractions,
fatty acids clustered into two isotopically distinct
groups. Among
the peaks with sufficient material for isotopic analysis,
the
iso-branched fatty acids
(
i-C
17:0,
i-C
18:0, and
i-C
19:0) in PL-2,
PL-4, and NL-2 were
more depleted in
13C than the longer-chain
C
20 and
cy-C
21
fatty acids. Depletions
relative to CO
2 averaged
22.6
± 0.4
(
n = 8) for
iso-branched
and 18.4
± 1.4
(
n = 8) for the
C
20 and C
21 acids. Bulk
fractions
varied in parallel. PL-2 and NL-2 contained greater
proportions
of
iso acids relative to long-chain acids and
were depleted in
13C relative to PL-4, in which
longer-chain acids were more abundant.
Alkyl chains from glycerol
ethers analyzed after BBr
3 cleavage
yielded
values of
23.8,
21.8, and
23.8
for C
18,
C
19, and
C
20, respectively.
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TABLE 5.
Stable carbon isotopic composition (13C)
and distribution of fatty acids and glycerol ethers of T. ruber grown with CO2 or
formatea
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DISCUSSION |
Lipid composition and makeup of PSC and vent communities.
A
previous study of the pink filamentous streamers at Octopus Spring
identified three phylotypes, EM3, EM17, and EM19, from amplification of
the mixed-population DNA (43). A phylogenetic tree was
constructed to take advantage of a current, more extensive sequence
database (Fig. 2) and confirms that the
EM17 gene sequence clusters among the Aquificales and is
closely related (99% sequence identity) to the pink streamer isolate
T. ruber (18), whereas the EM3 sequence is
related to the Thermotogales. EM19, however, constitutes a
separate, more deeply diverging lineage, well outside the
Aquificales and Thermotogales. In Reysenbach's
study (43), the EM17 sequence represented the majority of
clones examined (26 of 35) and a fluorescently labeled oligonucleotide
probe complementary to EM17 hybridized in situ to the pink filaments.
No hybridization was noted for EM3 or EM19 probes even though all
morphotypes in the PSC did bind to universal and bacterial probes
(43).
View larger version (31K):
[in this window]
[in a new window]
|
FIG. 2.
16S rRNA gene-based phylogenetic tree of the
Aquificales based on the results of a maximum-likelihood
analysis showing C20-22 signature lipids for this group.
Reference sequences were chosen to represent the broadest diversity of
Bacteria. Only sequence positions that have identical
residues in 50% or more of all available bacterial 16S rRNA sequences
were included for tree reconstruction. Accession numbers for the
sequences are indicated. The scale bar represents 0.10 fixed mutations
per nucleotide position. Of the sequences previously identified in the
PSC (°), only EM17 is closely related to Aquificales
from this study (*).
|
|
The pink streamers of the Octopus Spring outflow channel were
characterized by high levels of
iso- and cyclopropane
ester-linked
fatty acids and straight-chain ether-linked alkyl lipids
(Table
2). Similar lipids were associated with the biofilm growing on
the siliceous sinter walls of the vent pool (Fig.
1). The presence
of
two fatty acid pools, the
C
15-C
19 iso-branched
fatty acids
and the
C
20-
cy-C
21 fatty
acids (Tables
2 and
3), together with
the absence of
iso-branched fatty acids in
Aquificales cultures
(Table
2), indicated that the PSC contained more than one distinct
bacterial
population.
To date,
T. ruber OC 1/4 is the only cultivated isolate from
the PSC (
18,
21). Its fatty acid composition is similar to
that reported previously for a number of
Hydrogenobacter
thermophilus strains (
28) and to those of the
additional
Aquificales cultures
analyzed here (Table
2). The
fatty acids of these
Aquificales were dominated by
n-C
18:0,
n-C
20:1, and
cy-C
21 (Table
2). Two
sets of
monounsaturated isomers, C
18:1
9 and
C
18:111,
and their chain elongation products,
C
20:111 and C
20:113,
were detected. Since cyclopropane fatty acids are formed by the
addition of a methylene group from
S-adenosylmethionine
across
the double bond of a monounsaturated fatty acid, the two
cy-C
21 isomers are probably
derivatives of the
n-C
20:1 isomers.
C
20 fatty
acids are rare in bacteria, and the
presence of large amounts
of the
n-C
20:1 and
cy-C
21, with lesser amounts of
n-C
20:0,
n-C
21:0,
n-C
22:0, and
n-C
22:1 in representatives of four
distinct subclusters
within the
Aquificales (Fig.
2, Table
2), demonstrates a phylogenetic
clustering for these membrane lipids
and suggests that these fatty
acids can serve as taxonomic marker
signatures for this order.
Notably, the pink streamers also contained
similar C
20, C
21, and
C
22 fatty
acids.
Nonisoprenoid alkyl glycerol ethers are being increasingly recognized
as bacterial membrane lipids. In addition to the GME
and GDE with the
n-C
16-18 alkyl chains previously
described
in
A. pyrophilus (
22), GME and GDE
with
iso- and
anteiso-branched
chains have also
been identified as major membrane lipids in two
anaerobic thermophiles,
Thermodesulfobacterium commune (
30)
and
Ammonifex degensii (
19). An unusual glycerol
monoether with
a dimethyltriacontanyl chain has been identified in
another thermophile,
Thermotoga maritima (
8).
Additionally, small amounts of glycerol
monoethers with normal or
methyl-branched chains have been detected
in mesophilic and
thermophilic clostridia. These presumably are
derived from
1-
O-alkylglycerols in which an ester-linked fatty
acid is
initially present at position 2 (
31). Environmental
analyses have identified small amounts of
n-C
18 and
br-C
17 1-
O-alkyl
ethers and
a C
15,C
15
1,2-
O-alkyl diether in hot spring cyanobacterial
mats
(
58,
59), and more recently, relatively abundant
n- and
br-C
14-18
1-
O-alkylglycerols have been found in association
with
anaerobic methane-oxidizing consortia in marine sediments
(
15).
All of the
Aquificales cultures in our study synthesize at
least some alkyl glycerol ether lipids (Table
2). The
A. pyrophilus ether lipids most closely approximated the distribution
observed
for the PSC and vent alkyl ether lipids. To date, however, the
only identified
Aquifex spp. are marine bacteria.
T. ruber does
not appear to be the source of the PSC ether lipids.
The relative
abundance of GME and GDE and the alkyl chain distribution
in the
PSC suggest that an additional
Aquificales-like
organism was present
in these thermophilic communities or that some
environmental condition
is responsible for the presence of GDE.
Although we attempted
to grow
T. ruber under a variety of
conditions in our study, we
cannot preclude this latter point.
Simulation of natural flow
conditions in the laboratory results in
growth of
T. ruber as
filaments rather than as the
individual cells characteristic of
our batch cultures
(
18). It is interesting to speculate that
the physical
environment of more natural growth conditions might
allow expression of
GDE
synthesis.
Although limited by the small amount of biomass available, our analyses
suggest that the community of organisms present in
the biofilm of the
Octopus Spring vent differs somewhat from the
PSC. Specifically, the
organisms producing the
iso-fatty acids
appear to be less
abundant than the
Aquificales.
Iso-C
17:0 and
iso-C
19:0 are abundant in the PSC,
and, while present in the vent
biofilm, the
iso-C
19:0 is now the only major
branched acid and
is present in much lower amounts relative to the
C
20-22 fatty acids and the glycerol ethers
representing the
Aquificales community members. The GDE also
make up a much higher proportion
of the glycerol ether lipids in the
vent biofilm than in the PSC,
which may reflect an environmental effect
(i.e., higher temperature
of vent water) or possibly a different
Aquificales population.
Carbon isotopic patterns.
Four CO2
fixation pathways have been described for bacteria (see reference
13 for a review). Carbon isotopic fractionation varies
widely, depending on CO2 assimilation pathway.
Generally, the enzymes of the reductive acetyl-coenzyme A (CoA) pathway
express the largest 13C discriminations, with
13C values for biomass relative to
CO2 ranging from 20 to 36 (11, 39). Organisms using the Calvin-Benson cycle and
ribulose-1,5-bisphosphate carboxylase (Rubisco) for
CO2 incorporation display somewhat less discrimination, with 
13C from 11 to 26
(12, 39, 40, 47). A much broader range of values is found
for the metabolically diverse organisms using the hydroxypropionate
cycle (13C values from 2 to 13 relative
to DIC [16, 53, 55]), and those using the reductive
citric acid cycle (3 to 13 [39, 40, 47]). A
particular hallmark of organisms of this last group, reductive
tricarboxylic acid (TCA), is enrichment of 13C in
lipids relative to biomass (54).
The enzymes of the reductive citric acid cycle are present in
A. pyrophilus,
A. aeolicus, and
H. thermophilus, whereas Rubisco
is absent (
2,
7,
46).
However, no information has been
available about the C isotopic
discrimination associated with
growth of these obligately autotrophic
bacteria. Moreover, the
enzymology and carbon fixation pathways of
T. ruber which can
grow either autotrophically or
organotrophically (
18), have
not been investigated. Our
results indicate that
T. ruber cells
grown autotrophically
at 85°C are depleted in
13C relative to
CO
2 by 3.3
and that the lipids are enriched in
13C relative to biomass by 1.2
. Although both
of these results
are consistent with fixation of inorganic carbon by
the reductive
citric acid cycle, a novel metabolism cannot be
precluded.
The pink streamer community overall is strongly depleted relative to
CO
2 by 10.9
(Table
4). Moreover, as shown in
Table
3, the individual fatty acids are even more strongly depleted
(by
15.7 to 23.2
). Neither the relatively large depletion in
biomass nor
the depletion in fatty acids relative to biomass is
consistent with
dominance of the PSC by
T. ruber or any other
organism
utilizing the reductive citric acid cycle for the assimilation
of
CO
2.
The isotopic composition of the
T. ruber-like biomass in the
PSC can be estimated from the
13C value of the
relevant biomarkers, namely the C
20 and
C
21 fatty
acids. As shown in Table
4, whether
grown on CO
2 or on formate,
the lipids in
T. ruber are enriched in
13C by 1.2 to
1.8
relative to biomass. Accordingly, the
23
value
of the
C
20-C
21 fatty acids (Table
3) corresponds to a
13C biomass of
24.5
(Fig.
3). This would be
consistent with a
CO
2 carbon source with
13C
21
or a formate carbon
source with
13C
5
(Fig.
3). The
former can be excluded. The isotope composition
of the dissolved
CO
2 in the pink streamer community is known to
be
4.7
(Table
4).
View larger version (29K):
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|
FIG. 3.
Proposed carbon isotopic profile for PSC showing
measured 13C values for isolated PSC components on the
left. In the center and to the right, an assumed value of 24 for
Thermocrinis biomass is based on the PSC biomarker
C20 fatty acids and the established relationship between
T. ruber biomass and fatty acids measured in laboratory
cultures. On the right, suggested 13C values for formate
and CO2 based on expressed discriminations measured in
laboratory culture experiments. Isotopic value for formate in Octopus
Spring vent (upper right) is unknown. In this scenario, mass balance
between PSC organic C (hatched box) and Thermocrinis
biomass requires a relatively heavy component (gray box) and leaves
unresolved the relationship of iso-fatty acids and their
associated biomass to the heavy organic carbon pool proposed.
|
|
It is not known whether formate was present in the waters around the
pink streamer community at the time these samples were
collected. In
subsequent investigations, Shock (E. Shock, personal
communication) has
found formate downstream but not at the location
of the pink streamers
and also measured H
2 and CO in the gas phase
of
the vent source. It is in any case possible that the organisms
within
the pink streamer communities both produced (from
CO
2 +
H
2 or from CO + O
2 + H
2O) and
quantitatively consumed HCO
2H. Given
the misfit
between isotopic composition of DIC and
T. ruber biomass,
this provides the explanation most consistent with the available
evidence.
T. ruber is the only member of the
Aquificales to grow
either autotrophically or as a
chemoorganotroph using formate
or formamide (
18). The
nature of this metabolic capacity is
unknown (
18).
The
13C value for the PSC organic carbon
overall is
15.6
(Table
4). The difference between this value and
the
24.5
estimated
for
T. ruber biomass requires that
the remaining organic carbon
be enriched in
13C
(Fig.
3). If its abundance is equal to
T. ruber, by mass
balance,
it must have
13C
6
in
order to produce an average
13C
15.6
.
The nature of this heavy portion of the bulk organic carbon is then
problematic. The
13C of two additional,
independently collected PSC samples (September
1994 and 1999) agreed
within ±0.5
(data not shown), indicating
that the isotopic
composition of this community is stable. It
does not appear to arise
from the
iso-fatty acids. These have
13C values near
27
. Based on our current
knowledge, the biomass
to which they are related must have a
13C value of
15
or lighter (the greatest
observed depletion of
lipids relative to biomass in microorganisms is
12
[
13]).
The total lipid fraction represents a
relatively small portion
(<10%) of total bulk organic carbon in the
PSC and is much too
light (
22.6
), in itself, to account for all of
the heavy carbon.
Some other nonlipid component, synthesized within the
community
or brought in from the surrounding environment, must be
present
to balance the isotopic
abundances.
Information is needed about the concentration and isotopic composition
of formate in Octopus Spring water, the potential nature
of an
isotopically heavy component associated with the PSC filaments,
the
apparent lack of GDE in
T. ruber, and the physiological
mechanisms
leading to the novel fractionations associated with its
growth
on CO
2 and formate in order to fully
assess the PSC on physiological,
structural, and ecosystem
levels.
Hydrogen-oxidizing members of the
Aquificales are widely
distributed in hot springs and thought to play a major role in the
biogeochemical processes in these ecosystems (
29). The
carbon
isotopic composition of the biomarker lipids in the PSC points
to the expression of a novel metabolic potential by the
Thermocrinis-like
organisms in this hyperthermophilic
community, but leaves unanswered
the role and/or relationship of the
population represented by
the
iso-fatty acids.
Aquificales are considered the prevalent
phylotype in
filamentous bacterial communities found in geothermal
springs
(
29,
42,
43,
51), but identification of
Aquificales signature lipids associated with this vent
geyserite suggests
a broader ecological role for this group. Electron
microscopic
images of Octopus Spring geyserite indicate that bacterial
communities
readily colonize the vent walls and contribute to geyserite
morphogenesis
(
5). Because of their phylogenetic position
and their potential
microfossil record, this group of organisms is
particularly important
to a better understanding of Earth's earliest
microbial
life.
| |
ACKNOWLEDGMENTS |
Tsege Embaye (ARC), Kendra Turk (ARC), Sean Sylva (WHOI), and
Thomas Hader (Regensburg) provided technical assistance. We thank Mitch
Schulte and Everett Shock for useful discussions on the geothermal
chemical mechanisms. We are grateful to Karl O. Stetter for stimulating
and critical discussions. We are also grateful for support from the
staff of the Research Division of YNP. L.J. thanks Jack Farmer for
logistic and field support in YNP.
The work of Linda Jahnke and David Des Marais was supported by grants
from NASA's Exobiology Program and the NASA Astrobiology Institute.
The work of Wolfgang Eder was supported by the Fonds der Chemischen
Industrie (to K.O.S.). Work by Sherry Cady was supported by the NASA
Exobiology and the NSF Life in Extreme Environments Programs. Roger
Summons and Janet Hope publish with the permission of the CEO, AGSO.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: M/S 239-4, NASA
Ames Research Center, Moffett Field, CA 94035. Phone: (650) 604-3221. Fax: (650) 604-1088. E-mail:
ljahnke{at}mail.arc.nasa.gov.
Present address: Department of Earth, Atmospheric and Planetary
Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139.
| |
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Applied and Environmental Microbiology, November 2001, p. 5179-5189, Vol. 67, No. 11
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.11.5179-5189.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
