Biosynthetic Controls on the 13C Contents of Organic Components in the Photoautotrophic Bacterium Chloroflexus aurantiacus*

Marcel T. J. van der MeerDagger §, Stefan SchoutenDagger , Bart E. van DongenDagger , W. Irene C. RijpstraDagger , Georg Fuchs, Jaap S. Sinninghe DamstéDagger , Jan W. de LeeuwDagger , and David M. Ward||

From the Dagger  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



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
IMPLICATIONS
REFERENCES

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 13per thousand 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 17-18per thousand depleted in 13C relative to the carbon source, respectively. Based on the 13C contents of alkyl and isoprenoid lipids, a 40per thousand 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

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).



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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 ~20-25per thousand depleted in 13C (i.e. isotopically "lighter") relative to the delta 13C value (defined as delta 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 epsilon p (defined as epsilon  = (Rcarbon source/Rfixed carbon -1)103; R = 13C/12C) using the Calvin cycle is in the range of 20-25per thousand . In contrast, epsilon p of Chlorobium spp., green sulfur bacteria using the reversed tricarboxylic acid cycle, has been reported to be only 2-12per thousand (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 (~10per thousand (23)). There are also indications that the epsilon p related to the 3-hydroxypropionate pathway is small (~14per thousand ) 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.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
IMPLICATIONS
REFERENCES

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 (-27.6per thousand ), 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 (-30.1per thousand ) 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 beta -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 (-10.3per thousand ) 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 beta -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 beta -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


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
IMPLICATIONS
REFERENCES

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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Fig. 2.   Structures of compounds.

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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Fig. 3.   Partial gas chromatogram of the sugar fraction of a C. aurantiacus OK-70fl culture.



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Fig. 4.   Partial gas chromatogram of the PHA fraction of a C. aurantiacus OK-70fl culture. Roman numbers correspond with structures shown in Fig. 2.

13C Analysis-- The stable carbon isotopic composition of the DIC in the medium was -35.9per thousand , 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 13per thousand depleted in 13C relative to the DIC, whereas straight chain lipids, like fatty acids, alcohols, and wax esters were consistently 13-15per thousand depleted relative to the DIC (Table I). Fatty acids with an even carbon number, such as the C16 and C18 fatty acids, were ~14per thousand depleted relative to the DIC, whereas the odd carbon numbered fatty acids, such as the C17 and C19 homologs, were ~15per thousand depleted in 13C relative to the DIC (Table I). The isoprenoid lipids, i.e. phytane and verrucosanol, were 17 and 18per thousand depleted in 13C relative to the DIC, respectively. Isotopic analysis of glucose (C6 sugar) showed a 6per thousand depletion relative to the DIC, whereas xylose (C5 sugar) was ~2per thousand depleted in 13C relative to the DIC (Table I). Finally, 3-hydroxyvaleric acid is ~12per thousand depleted in 13C relative to the DIC (Table I).


                              
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Table I
Stable carbon isotopic compositions of DIC, bulk cell material, alkyl, and isoprenoid lipids and storage products such as sugars and PHA, and their 13C content relative to the carbon source (DIC) of a C. aurantiacus OK-70fl culture in <FR><NU><UP>0</UP></NU><DE><UP>00</UP></DE></FR> relative to the PDB standard are shown.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
IMPLICATIONS
REFERENCES

Overall Fractionation Effect-- In this study epsilon p of the C. aurantiacus culture was 13.7per thousand , 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 13.7per thousand 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 (~27per thousand (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 epsilon 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 ~14per thousand 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 ~18per thousand , 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 -49.5per thousand . 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, Delta 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 ± 4per thousand , and for the carboxyl carbon, i.e. -30 ± 4per thousand . Surprisingly, this difference in 13C content between the carboxyl and methyl carbon of acetyl-CoA is relatively large, ~40per thousand , compared with the differences reported for acetyl-CoA in Escherichia coli, although there are large differences between different reports (~6per thousand (43) and ~25per thousand (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 alpha -oxidation reaction, i.e. the anaerobic alpha -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 -50.7per thousand and -50.6per thousand for C17 and C19 fatty acids, respectively, which compares favorably with the measured isotope value for C17 and C19 fatty acids, -50.7per thousand and -50.5per thousand , 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 6per thousand depleted in 13C relative to the DIC, whereas xylose (Fig. 2, structure X) is only ~2per thousand 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 -45per thousand . The difference between the measured (approximately -42per thousand ) and the calculated isotope value (-45per thousand ) indicates that the newly fixed carbon atoms are possibly more enriched in 13C than the DIC. Assuming that glucose (approximately -42per thousand ) consists of two acetyl-CoA units (-49.5per thousand , see above) and two newly fixed carbon atoms, the stable carbon isotopic composition of the newly fixed carbon atoms is calculated to be approximately -26per thousand , which compares favorably with the isotopic composition calculated for the carboxyl carbon of acetyl-CoA.



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Fig. 5.   Simplified pathway for glucose biosynthesis based on Strauss et al. (16).

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 (-70per thousand , see above), the delta 13C value for xylose can be estimated to be approximately -36per thousand , which is ~2per thousand enriched in 13C relative to the measured carbon isotope composition for xylose (-37.8per thousand ). 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 (4per thousand (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 delta 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 (-70per thousand , see above) is -45.5per thousand , which is 2.5per thousand different from that of the measured value (-48per thousand ). 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 delta 13C value for 3-hydroxyvaleric acid would be -46.8per thousand , which is close to the measured carbon isotope composition for 3-hydroxyvaleric acid (-48per thousand ). 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.


    IMPLICATIONS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
IMPLICATIONS
REFERENCES

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.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

* 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.


    ABBREVIATIONS

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
IMPLICATIONS
REFERENCES


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