From the Netherlands Institute for Sea Research
(NIOZ), Department of Marine Biogeochemistry and Toxicology,
P. O. Box 59, 1790 AB Den Burg, Texel, The Netherlands,
¶ Albert-Ludwigs-Universität Freiburg, Mikrobiologie,
Institut für Biologie II, Schaenzlestrasse 1, D 79104 Freiburg, Germany, and
Montana State University,
Department of Land Resources and Environmental Sciences,
Bozeman, Montana 59717
Received for publication, October 24, 2000, and in revised form, January 3, 2001
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ABSTRACT |
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To assess the effects related to known and
proposed biosynthetic pathways on the 13C content of
lipids and storage products of the photoautotrophic bacterium
Chloroflexus aurantiacus, the isotopic compositions of bulk
cell material, alkyl and isoprenoid lipids, and storage products such
as glycogen and polyhydroxyalkanoic acids have been investigated. The
bulk cell material was 13 Inorganic carbon fixation by many living organisms commonly
proceeds by the ribulose-bisphosphate carboxylase/oxygenase
(Rubisco)1-catalyzed
reaction, which feeds CO2 directly into the Calvin cycle,
the principal biochemical mechanism for reducing CO2 to carbohydrates (1-7). The Calvin cycle is used for carbon assimilation by all green plants, algae, and many autotrophic bacteria. Another well
known carbon fixation mechanism using phosphoenolpyruvate (PEP)
carboxylase is found in, for instance, C4 plants and many other
organisms in anaplerotic reactions compensating any loss of
intermediary components from the tricarboxylic acid cycle. Also, CAM
plants, which have a crassulacean acid metabolism, can fix carbon by
both Rubisco and PEP carboxylase reactions. The reversed tricarboxylic
acid cycle is used by green sulfur bacteria and various other bacteria
to fix CO2 (8-11). The acetyl-CoA pathway or variants
thereof are used by various anaerobic bacteria and archaea to fix
inorganic carbon (12-14). Finally, the 3-hydroxypropionate pathway
(Fig. 1) was proposed to function in
Chloroflexus aurantiacus, a green nonsulfur bacterium
(15-17). This pathway is a cyclic inorganic carbon fixation mechanism
in which acetyl-CoA is carboxylated and reductively converted via
3-hydroxypropionate to propionyl-CoA. Propionyl-CoA is carboxylated and
converted to malyl-CoA, which is cleaved yielding the first inorganic
carbon acceptor molecule acetyl-CoA and glyoxylate (16). The presence
of PEP carboxylase has been demonstrated in C. aurantiacus
(15) and may have an anaplerotic function (17). Recently, indications
have been found for the operation of a 3-hydroxypropionate-like pathway
in autotrophic Crenarchaeota (17).
depleted in 13C relative to
the dissolved inorganic carbon. Evidently, inorganic carbon fixation by
the main carboxylating enzymes used by C. aurantiacus, which are assumed to use bicarbonate rather than CO2,
results in a relatively small carbon isotopic fractionation compared
with CO2 fixation by the Calvin cycle. Even carbon numbered
fatty acids, odd carbon numbered fatty acids, and isoprenoid lipids
were 14, 15, and 17-18
depleted in 13C relative to the
carbon source, respectively. Based on the 13C contents of
alkyl and isoprenoid lipids, a 40
difference in 13C
content between the carboxyl and methyl carbon from acetyl-coenzyme A
has been calculated. Both sugars and polyhydroxyalkanoic acid were
enriched in 13C relative to the alkyl and isoprenoid
lipids. To the best of our knowledge this is the first report in which
the stable carbon isotopic composition of a large range of biosynthetic
products in a photoautotrophic organism has been investigated and
interpreted based on previously proposed inorganic carbon fixation and
biosynthetic pathways. Our results indicate that compound-specific
stable carbon isotope analysis may provide a rapid screening tool for
carbon fixation pathways.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
IMPLICATIONS
REFERENCES
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Fig. 1.
The 3-hydroxypropionate pathway as proposed
by Strauss and Fuchs (16).
Stable carbon isotope differences between organic carbon synthesized by
autotrophic organisms and that of the inorganic carbon source used can
assist in distinguishing between the different CO2 fixation
pathways. Because of the preference of Rubisco for 12CO2 relative to
13CO2, the Calvin cycle yields bulk cell
material that is ~20-25 depleted in 13C
(i.e. isotopically "lighter") relative to the
13C value (defined as
13C = (Rsample/RPDB standard
1)103;
R = 13C/12C) of the CO2 from
which it is formed (18-21). In other words, the stable carbon isotopic
fractionation relative to the carbon source of photoautotrophic
organisms
p (defined as
= (Rcarbon
source/Rfixed carbon
1)103; R = 13C/12C) using the Calvin cycle is in the range
of 20-25
. In contrast,
p of Chlorobium spp.,
green sulfur bacteria using the reversed tricarboxylic acid cycle, has
been reported to be only 2-12
(18, 22). Isotopic fractionation by
nonphototrophic bacteria using the reversed tricarboxylic acid cycle to
fix CO2 has been reported to be in the same range as for
photoautotrophic bacteria (~10
(23)). There are also indications
that the
p related to the 3-hydroxypropionate pathway is
small (~14
) relative to that of the Calvin cycle (15).
More recently, compound-specific isotope analyses (24) have been used
to ascribe certain sedimentary compounds from modern and ancient
ecosystems to groups of organisms with a specific CO2
fixation pathway (25, 26). For organisms using the Calvin cycle, like
micro-algae and cyanobacteria, it has been reported that lipids are
depleted in 13C relative to the bulk cell material and that
isoprenoid compounds are enriched in 13C relative to the
straight chain compounds (20, 27). For organisms using the reversed
tricarboxylic acid cycle, it has been reported that lipids are enriched
in 13C relative to the bulk cell material and that straight
chain lipids are enriched in 13C relative to isoprenoid
lipids (28). Hence, the isotopic composition of lipids may also reveal
the CO2 fixation pathway that organisms use. So far, no
studies have been reported on compound-specific stable carbon isotopic
fractionation effects of the 3-hydroxypropionate pathway. Thus, we
analyzed a C. aurantiacus culture for the isotopic composition of bulk cell material, both even and odd carbon numbered alkyl lipids, isoprenoid lipids, and storage products like polyglucose and polyhydroxyalkanoic acids (PHA). To the best of our knowledge this
is the first report in which the stable carbon isotopic compositions of
different compound classes, including storage products, are directly
linked to proposed and known biosynthetic pathways in an organism.
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MATERIALS AND METHODS |
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Cultures--
C. aurantiacus OK-70fl (DSM 636) was
grown under phototrophic anaerobic conditions on a mineral salt medium
supplemented with vitamins in a continuously stirred (200 rpm) 5-liter
fermenter. The culture was gassed with 250 ml/min of a mixture of
H2/CO2 (80:20) at 55 °C and pH 8.3 (16, 17).
The inorganic carbon source, supplied as CO2, was not
limiting. The carbon supply rate was 26 mg of carbon
supplied/min, whereas the culture, even at the end of cultivation (~5
g cell dry mass), consumed 1 mg of carbon/min. Cells were harvested
during the exponential growth phase by centrifugation and subsequently
frozen in liquid nitrogen and lyophilized before lipid extraction. The
supplied CO2 was trapped as carbonate by leading the
H2/CO2 mixture through an NaOH solution of
approximately pH 13; the carbonate was precipitated as
BaCO3 by addition of BaCl2 (29). The stable
carbon isotopic composition of the BaCO3 was determined by
automated on-line combustion followed by conventional isotope
ratio-mass spectrometry. From this the isotopic composition of the
dissolved inorganic carbon (DIC) in the culture medium, which is
present mainly as bicarbonate and carbonate at pH 8.3 and 55 °C, was
calculated using the temperature-dependent isotopic
equilibrium equation of Mook et al. (30).
Lipid Analysis--
Harvested cells were ultrasonically
extracted with methanol (MeOH) (3 times), dichloromethane (DCM)/MeOH
(1:1, v/v mixture) (3 times), and DCM (3 times) to obtain a total lipid
extract. To methylate the fatty acids, the extracts were heated with 2 ml of a 10% BF3 in MeOH solution at 60 °C (5 min).
Water was added, and the derivatized compounds were extracted with DCM
(3 times). For carbon isotopic correction of the added methyl group, a
hexadecanoic acid standard, with a known carbon isotopic composition
(27.6
), was derivatized in parallel using the same
BF3/MeOH mixture. The extracts were filtered over an
SiO2 column using ethyl acetate as eluent. The alcohols in
the fractions were subsequently silylated by adding 25 µl of
bis(trimethylsilyl)trifluoroacetamide (BSTFA) and pyridine and heating
the mixture at 60 °C (20 min). To correct for the isotopic change
due to the introduction of carbon derived from the trimethylsilyl
group, a hexadecanol standard with known isotopic composition
(
30.1
) was silylated in parallel with the same BSTFA. By
determining the carbon isotopic composition of the derivatized
standards, the carbon isotopic composition of the methyl and
trimethylsilyl groups were calculated. These values were used to
calculate the carbon isotopic compositions of the parent alcohols and
fatty acids present in the different fractions.
To analyze the isotopic composition of phytol, it was converted to phytane. To this end, part of the total extract of C. aurantiacus was hydrogenated in ethyl acetate with H2, a few drops of acetic acid, and PtO2 for 1 h. Hydrogenated apolar compounds were isolated using column chromatography with Al2O3 as stationary phase and a hexane/DCM 9:1 (v/v) mixture as eluent (for details see Schouten et al. (27)). Lipids were analyzed by gas chromatography (GC), gas chromatography-mass spectrometry (GC-MS), and isotope-ratio-monitoring GC-MS (GC-irMS).
Sugar Analysis-- Approximately 10 mg of Chloroflexus residue after lipid extraction was hydrolyzed in 12 M H2SO4 in a closed tube for 2 h at room temperature, followed by 4.5 h at 85 °C after dilution of the acid to 1 M. The hydrolyzed residue was neutralized with BaCO3, and after centrifugation the water fraction was removed, and the precipitate was repeatedly washed with double-distilled water. The water fractions were combined and subsequently freeze-dried. The sugar monomers were derivatized in 0.5 ml of a 10 mg/ml solution of methylboronic acid in pyridine for 30 min at 60 °C. Subsequently, 15 µl of BSTFA was added, and the solution was kept at 60 °C for 5 min.2 The carbohydrate fraction was analyzed by GC, GC/MS, and GC-irMS.
Polyhydroxyalkanoic Acid Analysis--
Approximately 10 mg of
Chloroflexus residue after lipid extraction was used to
determine the polyhydroxyalkanoic acid (PHA) composition of the cell
material. The PHA was transformed by methanolysis into its derivatized
monomers, the -hydroxycarboxylic acid methyl esters, by refluxing
for 2 h at 100 °C in a solution containing 2 ml of chloroform,
1.7 ml of methanol, and 0.3 ml of sulfuric acid (modified after Ref.
32). For carbon isotopic correction of the added methyl group,
polyhydroxybutyric acid (Aldrich) with a known carbon isotopic
composition (
10.3
) was derivatized in parallel using the same
procedure. The hydrolyzed PHA fraction was analyzed by GC, GC-MS, and
GC-irMS.
Instrumental-- Stable carbon isotopic compositions of the bulk cell material and BaCO3 were determined by automated on-line combustion (Carlo Erba CN analyzer 1502 series) followed by conventional isotope ratio-mass spectrometry (Fisons optima (33)).
The different component fractions were analyzed by gas chromatography
(GC), gas chromatography-mass spectrometry (GC-MS), and
isotope-ratio-monitoring GC-MS (GC-irMS) (see Schouten et al. (27) for details). The -hydroxycarboxylic acid methyl
esters were analyzed on a CP Sil-88 column (25-m length, 0.22-mm
internal diameter, 0.18-µm film thickness), with helium as carrier
gas, and a constant column pressure of 70 kPa. For GC analysis the detector temperature (flame ionization detector) was 280 °C.
The
-hydroxycarboxylic acid methyl esters were analyzed using a
programmed temperature increase from 40 (10 min) to 240 °C at a rate
of 4 °C/min. The temperature was held at 240 °C for 20 min.
Sugars were analyzed on a DB-1701 column (30-m length, 0.25-mm internal diameter, 0.25-µm film thickness), with helium as carrier gas, and a
constant gas flow of 1.15 ml/min. The sugars were analyzed using a
programmed temperature increase from 70 to 180 °C at 4 °C/min and
then to 280 °C at 10 °C/min where it was held isothermal for 10 min.2
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RESULTS |
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The stable carbon isotope compositions of the bulk cell material and inorganic carbon source of a C. aurantiacus OK-70fl culture grown under photoautotrophic conditions were determined. In addition the stable carbon isotopic compositions of individual compounds from different lipid fractions were analyzed as well. The residue left after lipid extraction was partly used for sugar analysis and for PHA analysis.
Lipid Analysis--
The total lipid extract of C. aurantiacus contained fatty acids ranging from C15 to
C20 including C16, C18,
C19 and C20, mono-unsaturated fatty acids and
was dominated by hexadecanoic acid and octadecanoic acid. Besides fatty
acids the total lipid extract contained verrucosanol (see
Fig. 2, structure I (34)),
C17-19 alkenols, long chain polyunsaturated alkenes, and
wax esters (35, 36). The long chain polyunsaturated alkenes ranged from
C29 to C32 with 1-3 double bonds and were
dominated by hentria-9,15,22-contatriene (Fig. 2, structure
II (36, 37)). The wax esters ranged from C31 to
C38, including mono-unsaturated C34-36 wax
esters, and were dominated by saturated C34-36 homologs
(36). The hydrogenated total extract fraction was dominated by phytane (Fig. 2, structure III), produced by the reduction of phytol (Fig. 2, structure IV (38)), the esterified alcohol of one
of the C. aurantiacus pigments, bacteriochlorophyll
c (39).
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Sugar and PHA Analysis--
The sugar fraction contained both
C5 and C6 sugars and was dominated by glucose
(see Fig. 2, structure V and Fig.
3). The hydrolyzed PHA fraction contained
mainly 3-hydroxyvaleric acid (Fig. 2, structure VI),
3-hydroxybutyric acid (structure VII), di- and trimers
consisting of 3-hydroxybutyric and/or valeric acid units (Fig. 2,
structures VIII and IX) and fatty acids ranging
from C15 to C20 (Fig.
4). The presence of dimers and trimers
could be due to a method artifact since the methanolysis method used is
an equilibrium reaction. Excess methanol will shift the equilibrium
toward the methylated monomers, but the conversion will not be
complete, and consequently some dimers and trimers may remain.
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13C Analysis--
The stable carbon isotopic
composition of the DIC in the medium was 35.9
, as calculated based
on the isotopic composition of the CO2 supplied to the
culture using the temperature-dependent isotopic
equilibrium equation of Mook et al. (30). The bulk cell
material of the Chloroflexus culture was 13
depleted in 13C relative to the DIC, whereas straight chain lipids,
like fatty acids, alcohols, and wax esters were consistently 13-15
depleted relative to the DIC (Table
I). Fatty acids with an even carbon number, such as the C16 and C18 fatty acids,
were ~14
depleted relative to the DIC, whereas the odd carbon
numbered fatty acids, such as the C17 and C19
homologs, were ~15
depleted in 13C relative to the DIC
(Table I). The isoprenoid lipids, i.e. phytane and
verrucosanol, were 17 and 18
depleted in 13C relative to
the DIC, respectively. Isotopic analysis of glucose (C6
sugar) showed a 6
depletion relative to the DIC, whereas xylose
(C5 sugar) was ~2
depleted in 13C relative
to the DIC (Table I). Finally, 3-hydroxyvaleric acid is ~12
depleted in 13C relative to the DIC (Table I).
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DISCUSSION |
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Overall Fractionation Effect--
In this study p of
the C. aurantiacus culture was 13.7
, calculated as
described by Hayes (40) and based on the isotopic composition of the
bulk cell material and the dissolved inorganic carbon in the culture
medium. Holo and Sirevåg (15) also reported C. aurantiacus
OK-70fl bulk cell material to be 13.7
depleted in 13C
relative to the carbon source. The results of both this study and the
work of Holo and Sirevåg (15) indicate that stable carbon isotope
fractionation by the 3-hydroxypropionate pathway is reduced compared
with the Calvin cycle operating in autotrophic organisms as well as the
isotopic fractionation of CO2 by Rubisco as determined in vitro (~27
(20, 21)). The explanation for the
reduced fractionation of the 3-hydroxypropionate pathway could be that both inorganic carbon-fixing enzymes proposed to be used by C. aurantiacus (acetyl-CoA and propionyl-CoA carboxylase) fix
bicarbonate instead of CO2 (16). It is known that
bicarbonate incorporation by PEP carboxylase, which is known to be
present in C. aurantiacus and may have an anaplerotic
function (17), results in none or hardly any stable carbon isotopic
fractionation (41). The fractionation effects of the carboxylating
enzymes in the 3-hydroxypropionate pathway, acetyl-CoA and
propionyl-CoA carboxylase (Fig. 1), are not known. However, the
relatively small
p indicates that they either have a
relatively small fractionation effect compared with CO2
fixation catalyzed by the enzyme Rubisco or, less likely, that a major
part of the cell material has been fixed by PEP carboxylase in C. aurantiacus.
Alkyl Versus Isoprenoid Lipids--
Even carbon numbered alkyl
lipids are ~14 depleted in 13C relative to the DIC,
while odd numbered alkyl and isoprenoid lipids were more depleted in
13C relative to the DIC, 15 and ~18
, respectively. The
carbon chain of even numbered fatty acids is most likely formed from
multiple acetyl-coenzyme A (CoA) units by chain elongation via
malonyl-CoA. For example, acetyl-CoA is carboxylated to form
malonyl-CoA, and one acetyl-CoA unit plus seven malonyl-CoA units form
a C16 fatty acid with the release of the seven carbon atoms
used to form malonyl-CoA (42). If it is assumed that this synthesis of
short chain alkyl lipids with an even carbon number does not result in
a large fractionation, then the isotopic composition of acetyl-CoA
should be rather similar to that of the C16 and
C18 fatty acids (43), i.e. approximately
49.5
. Labeling studies have shown that verrucosanol, a cyclic C20 isoprenoid (Fig. 2, structure I), contains 8 carboxyl carbon atoms and 12 methyl carbon atoms originating from
acetyl-CoA (44), indicating that isoprenoid lipids in C. aurantiacus are formed via the mevalonate pathway (44). In this
pathway the precursor for isoprenoid biosynthesis,
3-isopentenyl pyrophosphate, is formed from three
acetyl-CoA units via mevalonic acid, which is decarboxylated. In
comparison, the C16 fatty acid contains 8 carboxyl and 8 methyl carbon atoms originating from acetyl-CoA. Based on the stable
carbon isotope values of both verrucosanol and the C16
fatty acid, we can calculate the difference in 13C content
between the methyl and carboxyl carbon atoms in acetyl-CoA presuming
that they are formed from the same acetyl-CoA pool (40). Two simple
equations with two unknown parameters then yield a carbon isotope value
for the methyl carbon, i.e.
70 ± 4
, and for the
carboxyl carbon, i.e.
30 ± 4
. Surprisingly, this
difference in 13C content between the carboxyl and methyl
carbon of acetyl-CoA is relatively large, ~40
, compared with the
differences reported for acetyl-CoA in Escherichia coli,
although there are large differences between different reports (~6
(43) and ~25
(45)). Acetyl-CoA in E. coli is formed via
the Embden-Meyerhof pathway, i.e. the breakdown of sugars
via glyceraldehyde 3-phosphate and pyruvate to acetyl-CoA. Acetyl-CoA
is finally oxidized to CO2 in the tricarboxylic acid cycle.
The difference in stable carbon isotopic composition of the carboxyl
and methyl carbon in acetyl-CoA from E. coli is explained by
a fractionation effect occurring when pyruvate is oxidatively
decarboxylated to give acetyl-CoA (43, 46). The breaking of the
carbon-carbon bond in pyruvate results in a 13C depletion
of the carboxyl carbon in acetyl-CoA, whereas the methyl carbon retains
the original stable carbon isotopic signature of the sugar (43, 46).
However, Blair et al. (45) suggested that fractionation may
also occur downstream from acetyl-CoA and reported that the carboxyl
carbon can become more enriched in 13C relative to the
methyl carbon of acetyl-CoA in E. coli. The difference in
stable carbon isotopic composition of the carboxyl and methyl carbon in
acetyl-CoA from C. aurantiacus is not only larger than
reported by Blair et al. (45), but it is also the reverse of
what others have reported for E. coli (43, 46). A possible
explanation for the relative 13C depletion of the methyl
carbon in C. aurantiacus could be a fractionation effect
related to the breaking of the C-2
C-3 bond of malyl-CoA by the fully
reversible malyl-CoA lyase resulting in acetyl-CoA and glyoxylate (Fig.
1). If so, the residual malyl-CoA, and products formed thereof, would
be enriched in 13C. The carboxyl carbon of acetyl-CoA is
not only enriched in 13C relative to the methyl carbon but
is even somewhat enriched in 13C relative to the DIC,
although this might be within analytical error.
The 13C depletion of odd carbon numbered fatty acids
relative to even carbon numbered fatty acids can be explained by the
large difference in 13C content between methyl and carboxyl
carbon in acetyl-CoA. Although it is not clear how odd numbered fatty
acids are formed in C. aurantiacus, a possibility might be
an -oxidation reaction, i.e. the anaerobic
-hydroxylation of an even numbered fatty acid followed by
decarboxylation (42, 47, 48). The removal of an isotopically heavy
carboxyl carbon would result in a calculated isotope value of
50.7
and
50.6
for C17 and C19 fatty acids,
respectively, which compares favorably with the measured isotope value
for C17 and C19 fatty acids,
50.7
and
50.5
, respectively. Another possibility is chain elongation
starting from propionyl-CoA. However, the formation of propionyl-CoA
from acetyl-CoA would include a carboxylation step, and assuming that
the added carbon atom has a similar isotopic composition as the DIC,
this would result in odd carbon numbered fatty acids enriched in
13C relative to even carbon numbered fatty acids. Although
the exact mechanism is not known, the isotope data again seem to
suggest that the carboxyl carbon is enriched in 13C
relative to the methyl carbon of acetyl-CoA and that this enrichment is
relatively large. The even carbon numbered long chain
n-alkenes have isotopic compositions similar to the
C16 and C18 fatty acids, which is expected if
n-alkenes are formed from fatty acids by chain elongation
and reduction of carboxyl groups. The odd carbon numbered
n-alkene, hentriacontatriene, is enriched in 13C
relative to the C16 and C18 fatty acids. It may
either be formed by chain elongation from an odd carbon numbered fatty
acid or by chain elongation and decarboxylation from an even carbon
numbered fatty acid. Both biosynthetic pathways include a
decarboxylation step, which would result in a 13C depletion
of the n-alkene relative to the C16 and
C18 fatty acids. The 13C enrichment of
hentriacontatriene may possibly be explained by an isotope effect
related to its biosynthetic pathway, as indicated by the
13C enrichment of the unsaturated C18 fatty
acid relative to the saturated C18 fatty acid.
Sugars--
Both C6 and C5 sugars are
enriched in 13C relative to the bulk cell material and
lipids. However, glucose is 6 depleted in 13C relative
to the DIC, whereas xylose (Fig. 2, structure X) is only
~2
depleted. Sugars in C. aurantiacus are probably
synthesized via the reversed Embden-Meyerhof pathway (49), which forms
glucose from 2-phosphoenolpyruvate (PEP) molecules (Fig.
5). Labeling studies have indicated that
the C-1, C-2, C-5, and C-6 from glucose are most likely derived
from acetyl-CoA, whereas the C-3 and C-4 of glucose are
mainly derived from newly fixed inorganic carbon (49-51). Labeling
studies with 13C-labeled substrates other than acetate
suggest that glucose may be formed from malate via oxaloacetate and
pyruvate (50). If we assume that the newly fixed carbon atoms in
glucose are similar in isotopic composition to the DIC, the calculated
isotope value for glucose is approximately
45
. The difference
between the measured (approximately
42
) and the calculated isotope
value (
45
) indicates that the newly fixed carbon atoms are
possibly more enriched in 13C than the DIC. Assuming that
glucose (approximately
42
) consists of two acetyl-CoA units
(
49.5
, see above) and two newly fixed carbon atoms, the stable
carbon isotopic composition of the newly fixed carbon atoms is
calculated to be approximately
26
, which compares favorably with
the isotopic composition calculated for the carboxyl carbon of
acetyl-CoA.
|
The measured 13C enrichment of xylose relative to glucose
can be explained by the removal of an isotopically light carbon atom from glucose. Based on the measured isotope value of glucose and the
calculated isotope value of the methyl carbon (70
, see above), the
13C value for xylose can be estimated to be
approximately
36
, which is ~2
enriched in 13C
relative to the measured carbon isotope composition for xylose (
37.8
). The difference between the calculated and measured
isotopic composition of xylose could be attributed to scrambling of
carbon atoms (49-51). In contrast arabinose, also a C5
sugar, is as much depleted in 13C as xylose is enriched in
13C relative to glucose (4
(Table I)), which could be
explained by the removal of an isotopically heavy carbon atom from
glucose. Although the biosynthetic pathways for xylose and arabinose in C. aurantiacus are currently not known, their isotopic
compositions suggest that they are formed via different biosynthetic pathways.
Polyhydroxy Alkanoic Acid--
PHA in C. aurantiacus
consists mainly of 3-hydroxyvaleric acid, a C5 compound.
3-Hydroxyvaleric acid is enriched in 13C relative to the
alkyl lipids. Assuming that PHAs are formed by polymerization of
acetyl-CoA (31), the 13C value for 3-hydroxyvaleric acid
suggests that in the synthesis of this C5 compound an
isotopically "light" carbon atom is removed. The isotope value for
3-hydroxyvaleric acid calculated on the basis of 3 acetyl-CoA molecules
and the removal of a methyl carbon (
70
, see above) is
45.5
,
which is 2.5
different from that of the measured value (
48
).
Another possibility is the carboxylation of acetyl-CoA to form a
C3 compound and subsequent reaction with another acetyl-CoA
to give a C5 compound. Based on the calculated isotope
value for acetyl-CoA (see above) and the DIC isotopic composition,
assuming that the additional carbon atom is coming from the DIC, the
13C value for 3-hydroxyvaleric acid would be
46.8
,
which is close to the measured carbon isotope composition for
3-hydroxyvaleric acid (
48
). This indicates that 3-hydroxyvaleric
acid is probably synthesized from a C3 compound and
acetyl-CoA rather than from 3 acetyl-CoA units and subsequent
decarboxylation. Our results show that the carbon isotopic composition
of storage products such as PHA and polyglucose can have a large effect
on the carbon isotopic composition of the bulk cell material relative
to the carbon isotopic composition of lipids. Since the amount of
storage products formed by organisms depends largely on the growth
conditions, the difference in isotopic composition between the bulk
cell material and lipids can also vary with growth conditions. Many
organisms produce storage products, and this effect should be taken
into account when the isotopic composition of the bulk cell material is
used as reference value for the isotopic composition of lipids.
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IMPLICATIONS |
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To the best of our knowledge this is the first report on the stable carbon isotopic composition of storage products as well as bulk cell material and alkyl and isoprenoid lipids of a single organism. The stable carbon isotopic composition of the bulk cell material, different lipid classes, and storage products in C. aurantiacus can be rationalized on the basis of its proposed CO2 fixation and biosynthetic pathways and possibly indicates novel information about these pathways.
The pattern of 13C depletion for the 3-hydroxypropionate
pathway differs significantly from the patterns reported for organisms using the Calvin or tricarboxylic acid cycle (20, 27, 28). For
organisms using the Calvin cycle, it has been reported that lipids are
depleted in 13C relative to the bulk cell material and that
straight chain compounds are depleted in 13C relative to
isoprenoid compounds (20, 27). In contrast, for organisms using the
reversed tricarboxylic acid cycle, it has been reported that lipids are
enriched in 13C relative to the bulk cell material and that
straight chain lipids are enriched in 13C relative
isoprenoid lipids (28). We have shown for C. aurantiacus, which uses the 3-hydroxypropionate pathway, that lipids are depleted in
13C relative to the bulk cell material and that straight
chain lipids are enriched in 13C relative to the isoprenoid
lipids. This report indicates that compound-specific stable carbon
isotope analysis can be a useful rapid screening tool for inorganic
carbon fixation and biosynthetic pathways in autotrophic organisms.
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ACKNOWLEDGEMENTS |
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We thank Nasser Gadón and Dr. Castor Menendez, Freiburg University, for growing cells. We also thank Richard D. Pancost and Rikus Kloosterhuis for analytical assistance with isotope analysis. We thank Shell International Petroleum Maatschappij BV for financial support for the GC-irMS facility.
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FOOTNOTES |
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* This study was supported by Grants NAGW-2764 and NA65-3652 from the United States National Aeronautics and Space Administration. This is NIOZ Contribution 3504.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed. Tel.: 31-0-222-369584; Fax: 31-0-222-319674; E-mail: mmeer@nioz.nl.
Published, JBC Papers in Press, January 5, 2001, DOI 10.1074/jbc.M009701200
2 van Dongen, B. E., Schouten, S. & Sinninghe Damsté, J. S. (2001) Rapid Commun. Mass Spectrom., in press.
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ABBREVIATIONS |
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The abbreviations used are: Rubisco, ribulose-bisphosphate carboxylase/oxygenase; PEP, phosphoenolpyruvate; PHA, polyhydroxyalkanoic acids; DCM, dichloromethane; BSTFA, bis(trimethylsilyl)trifluoroacetamide; GC, gas chromatography; MS, mass spectrometry; irMS, isotope-ratio-monitoring; DIC, dissolved inorganic carbon.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Eichmann, R., and Schidlowski, M. (1975) Geochim. Cosmochim. Acta 39, 585-595 |
2. | Schidlowski, M., Matzigkeit, U., and Krumbein, W. E. (1984) Naturwissenschaften 71, 303-308 |
3. | Schidlowski, M. (1988) Nature 333, 313-318[CrossRef] |
4. | Des Marais, D. J., Cohen, Y., Nguyen, H., Cheatham, M., and Munoz, E. (1989) in Physiological Ecology of Benthic Microbial Communities (Cohen, Y. , and Rosenberg, E., eds) , pp. 191-203, American Society for Microbiology, Washington, D. C. |
5. | Des Marais, D. J., Strauss, H., Summons, R. E., and Hayes, J. M. (1992) Nature 359, 605-609[Medline] [Order article via Infotrieve] |
6. | Des Marais, D. J., and Canfield, D. E. (1994) in Microbial Mats: Structure, Development and Environmental Significance (Stal, L. J. , and Caumette, P., eds) , pp. 289-298, Springer-Verlag, Berlin, Germany |
7. | Tabita, F. R. (1995) in Anoxygenic Photosynthetic Bacteria (Blankenship, R. E. , Madigan, M. T. , and Bauer, C. E., eds), Vol. 2 , pp. 885-914, Kluwer Academic Publishers, Dordrecht, Netherlands |
8. | Evans, M. C. W., Buchanan, B. B., and Arnon, D. I. (1966) Biochemistry 55, 928-934 |
9. | Sirevåg, R. (1974) Arch. Microbiol. 98, 3-18 |
10. | Fuchs, G., Stupperich, E., and Jaenchen, R. (1980) Arch. Microbiol. 128, 56-63 |
11. | Fuchs, G., Stupperich, E., and Eden, G. (1980) Arch. Microbiol. 128, 64-71 |
12. | Fuchs, G. (1989) in Autotrophic Bacteria (Schlegel, H. G. , and Bowien, B., eds) , pp. 365-382, Science Tech Publishers, Madison, WI |
13. | Hafenbradl, D., Keller, M., Dirmeier, R., Rachel, R., Rossnagel, P., Burggraf, S., Huber, H., and Stetter, K. O. (1996) Arch. Microbiol. 166, 308-314[CrossRef][Medline] [Order article via Infotrieve] |
14. | Dai, Y. R., Reed, D. W., Millstein, J. H., Hartzell, P. L., Grahame, D. A., and DeMoll, E. (1998) Arch. Microbiol. 169, 525-529[CrossRef][Medline] [Order article via Infotrieve] |
15. | Holo, H., and Sirevåg, R. (1986) Arch. Microbiol. 145, 173-180 |
16. | Strauss, G., and Fuchs, G. (1993) Eur. J. Biochem. 215, 633-643[Abstract] |
17. |
Menendez, C.,
Bauer, Z.,
Huber, H.,
Gadón, N.,
Stetter, K. O.,
and Fuchs, G.
(1999)
J. Bacteriol.
181,
1088-1098 |
18. | Sirevåg, R., Buchanan, B. B., Berry, J. A., and Troughton, J. H. (1977) Arch. Microbiol. 112, 35-38[Medline] [Order article via Infotrieve] |
19. | Madigan, M. T., Takigiku, R., Lee, R. G., Gest, H., and Hayes, J. M. (1989) Appl. Environ. Microbiol. 55, 639-644[Medline] [Order article via Infotrieve] |
20. | Sakata, S., Hayes, J. M., McTaggart, A. R., Evans, R. A., Leckrone, K. J., and Togasaki, R. K. (1997) Geochim. Cosmochim. Acta 61, 5379-5389[CrossRef][Medline] [Order article via Infotrieve] |
21. | Popp, B. N., Laws, E. A., Bridigare, R. R., Dore, J. E., Hanson, K. L., and Wakeham, S. G. (1998) Geochim. Cosmochim. Acta 62, 69-77[CrossRef] |
22. | Quandt, L., Gottschalk, G., Ziegler, H., and Stichler, W. (1977) FEMS Microbiol. Lett. 1, 125-128 |
23. | Preuß, A., Schauder, R., Fuchs, G., and Stichler, W. (1989) Z. Naturforsch. 44, 397-402 |
24. | Hayes, J. M., Freeman, K. H., Popp, B. N., and Hoham, C. H. (1990) in Advances in Organic Geochemistry (Durand, B. , and Behar, F., eds) , pp. 1115-1128, Pergamon Press, Frankfurt |
25. | Freeman, K. H., Hayes, J. M., Trendel, J.-M., and Albrecht, P. (1990) Nature 353, 254-256 |
26. | Kohnen, M. E. L., Schouten, S., Sinninghe Damsté, J. S., de Leeuw, J. W., Merrit, D. A., and Hayes, J. M. (1992) Science 256, 358-362[Medline] [Order article via Infotrieve] |
27. | Schouten, S., Klein Breteler, W. C. M., Blokker, P., Schogt, N., Rijpstra, W. I. C., Grice, K., Baas, M., and Sinninghe Damsté, J. S. (1998) Geochim. Cosmochim. Acta 62, 1397-1406[CrossRef] |
28. | van der Meer, M. T. J., Schouten, S., and Sinninghe Damsté, J. S. (1998) Org. Geochem. 28, 527-533[CrossRef] |
29. | Simon, H., and Floss, H. G. (1967) Bestimmung der Isotopenverteilung in Markierten Verbindungen , Springer-Verlag, Berlin |
30. | Mook, W. G., Bommerson, J. C., and Staberman, W. H. (1974) Earth Planet. Sci. Lett. 22, 169-176[CrossRef] |
31. | Fuller, R. C. (1995) in Anoxygenic Photosynthetic Bacteria (Blankenship, R. E. , Madigan, M. T. , and Bauer, C. E., eds), Vol. 2 , pp. 1245-1256, Kluwer Academic Publishers Group, Dordrecht, Netherlands |
32. | Brandl, H., Gross, R. A., Lenz, R. W., and Fuller, R. C. (1988) Appl. Environ. Microbiol. 54, 1977-1982 |
33. | Fry, B., Brand, W., Mensch, F. J., Tholke, K., and Garritt, R. (1992) Anal. Chem. 64, 288-291 |
34. | Hefter, J., Richnow, H. H., Fischer, U., Trendel, J. M., and Michaelis, W. (1993) J. Gen. Microbiol. 139, 2757-2761 |
35. | Knudsen, E., Jantzen, E., Bryn, K., Ormerod, J. G., and Sirevåg, R. (1982) Arch. Microbiol. 132, 149-154 |
36. | Shiea, J., Brassell, S. C., and Ward, D. M. (1991) Org. Geochem. 17, 309-319[CrossRef] |
37. | van der Meer, M. T. J., Schouten, S., Ward, D. M., Geenevasen, J. A. J., and Sinninghe Damsté, J. S. (1999) Org. Geochem. 30, 1585-1587[CrossRef][Medline] [Order article via Infotrieve] |
38. | Hartgers, W. A., Lopez, J. F., de las Heras, F. X. C., and Grimalt, J. O. (1996) Org. Geochem. 25, 353-365[CrossRef] |
39. | Pierson, B. K., and Castenholz, R. W. (1974) Arch. Microbiol. 100, 283-305 |
40. | Hayes, J. M. (1993) Mar. Geol. 113, 111-125[CrossRef] |
41. | Goericke, R., Montya, J. B., and Fry, B. (1994) in Stable Isotopes in Ecology and Environmental Science (Lajtha, K. , and Michener, R. H., eds) , pp. 187-221, Blackwell Scientific Publications, Oxford |
42. | Schweizer, E. (1989) in Microbial Lipids (Ratledge, C. , and Wilkinson, S. G., eds), Vol. 2 , pp. 3-50, Academic Press Ltd., London |
43. | Monson, K. D., and Hayes, J. M. (1982) Geochim. Cosmochim. Acta 46, 139-149[CrossRef] |
44. |
Rieder, C.,
Strauss, G.,
Fuchs, G.,
Arigoni, D.,
Bacher, A.,
and Eisenreich, W.
(1998)
J. Biol. Chem.
273,
18099-18108 |
45. | Blair, N., Leu, A., Muñoz, E., Olsen, J., Kwong, E., and DesMarais, D. (1985) Appl. Environ. Microbiol. 50, 996-1001[Medline] [Order article via Infotrieve] |
46. |
Melzer, E.,
and Schmidt, H.-L.
(1987)
J. Biol. Chem.
262,
8159-8164 |
47. | Emmanuel, B. (1974) Biochim. Biophys. Acta 337, 404-413[Medline] [Order article via Infotrieve] |
48. | Yano, I., Furukawa, Y., and Kusunose, M. (1970) Biochim. Biophys. Acta 210, 105-115[Medline] [Order article via Infotrieve] |
49. | Holo, H., and Grace, D. (1987) Arch. Microbiol. 148, 292-297 |
50. | Strauss, G., Eisenreich, W., Bacher, A., and Fuchs, G. (1992) Eur. J. Biochem. 205, 853-866[Abstract] |
51. | Eisenreich, W., Strauss, G., Werz, U., Fuchs, G., and Bacher, A. (1993) Eur. J. Biochem. 215, 619-632[Abstract] |