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* Royal Netherlands Institute for Sea Research (NIOZ), Department of Marine Biogeochemistry and Toxicology, PO Box 59, 1790 AB Den Burg, The Netherlands
Newcastle University, Department of Fossil Fuels and Environmental Geochemistry, Drummond Building, Newcastle upon Tyne NE1 7RU, UK
University of Amsterdam, Faculty of Chemistry, Department of Organic Chemistry, Nieuwe Achtergracht 129, 1018 WS Amsterdam, The Netherlands
DOI 10.1194/jlr.M200148-JLR200
2 A number after GDGT indicates the total number of cyclopentane rings. 
1 To whom correspondence should be addressed. e-mail: damste{at}nioz.nl
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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This prevents the dense packing characteristic for the cyclopentane ring-containing GDGTs membrane lipids used by hyperthermophilic crenarchaeota to adjust their membrane fluidity to high temperatures.
Abbreviations: APCI, atmospheric pressure chemical ionization; APT, attached proton test; COSY, correlated spectroscopy; DCM, dichloromethane; DEPT, distorsionless enhancement by polarization transfer; HMBC, heteronuclear multiple bond correlation; HMQC, heteronuclear multiple quantum correlation; GDGT, glycerol dibiphytanyl glycerol tetraether; MS, mass spectrometry; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser effect spectroscopy; 2D, two-dimensional; TOCSY, total correlation spectroscopy
Supplementary key words archaea • ether-bound biphytanes • ocean • ether lipids
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Apart from their characteristic rRNA sequences, archaea are also biochemically distinct from bacteria and eukaryotes since they use biphytanyl glycerol diethers or (glycerol dibiphytanyl glycerol tetraethers) GDGTs instead of diacyl membrane lipids. This has been interpreted to be an adaptation to the extreme environments in which archaea thrive, as ether linkages are more stable than ester linkages (5). The use of membrane-spanning GDGTs by (hyper)thermophilic crenarchaeota is thought to be a further adaptation of their membranes to cope with high temperatures (>60°C). In addition, (hyper) thermophilic archaea form cyclopentane rings by internal cyclization of the dibiphytane moieties, resulting in a more densely packed and consequently thermally more stable membrane (6). The number of cyclopentane rings in GDGTs of (hyper)thermophilic crenarchaeota indeed increases with growth temperature (7). Hence, GDGTs seem to be specifically designed to cope with extremely high temperatures.
Remarkably, however, cyclopentane ring-containing GDGTs have recently also been identified in non-thermophilic crenarchaeota and in the marine environment. Ether cleavage studies on water column particulate matter (8) and Cenarchaeum symbiosum (9), the only uni-archaeal culture available from the group of pelagic crenarchaeota (10), and studies of intact GDGTs in marine sediments (3) have all indicated that the GDGTs of pelagic crenarchaeota comprise dibiphytanes with predominantly no, two, or three cyclopentane rings. Detailed mass spectrometry (MS) studies indicated that the dibiphytane comprising three cyclopentane rings is different from the isomer found in hyperthermophilic crenarchaeota since the position of one of the cyclopentane rings is different (11). These studies confirmed the phylogenetically close relationship between the thermophilic and pelagic crenarchaeota; both biosynthesize cyclopentane-containing GDGTs. It remains, however, unclear how pelagic crenarchaeotea can thrive in the relatively cold ocean waters with a set of membrane lipids specifically designed to cope with high temperatures.
Here we report a detailed account on the structural identification of the core GDGT membrane lipid of pelagic crenarchaeota using high-field two-dimensional (2D)-NMR techniques. Our results reveal that these organisms have adjusted their membrane lipids to cope with the much colder conditions in the ocean by the formation of an internal cyclohexane moiety, an unprecedented biochemical reaction for the archaea.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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GDGT-4 was isolated from a GDGT fraction of the thermophilic archaeon Sulfolobus solfataricus, prepared as previously described by Nicolaus et al. (12) and a kind gift of Dr. A. Gambacorta, Instituto di Chimica di Molecole di Interesse Biologico, Napoli, Italy. The GDGT fraction was dried, re-dissolved, and filtered as described above before injection.
GDGTs were isolated using an HP (Palo Alto, CA) 1100 series LC equipped with an auto-injector, and a fraction collector (Foxy Jr., Isco, Inc., Lincoln, NE). A first isolation was achieved on a semi-preparative Econosphere NH2 column (10 x 250 mm, 10 µm; Alltech, Deerfield, IL), maintained at 30°C. Typical injection volume was 100 µl containing up to 10 mg material. GDGTs were eluted isocratically with 99% hexane and 1% propanol for 5 min, followed by a linear gradient to 1.8% propanol in 45 min. The flow rate was set at 2.5 ml/min. After each run the column was cleaned by back-flushing hexane-propanol (9:1; v/v) at 2.5 ml/min for 10 min. Column effluent was collected in 1 min fractions, which were screened for the presence of the target GDGT by HPLC/atmospheric pressure chemical ionization (APCI)-MS as described by Hopmans et al. (13). Fractions containing the target GDGT were pooled and further purified on an Econosphere NH2 column (4.6 x 250 mm, 5 µm; Alltech, Deerfield, IL). Typical injection volumes were 50 µl containing up to 0.5 mg of material. GDGTs were eluted using an identical gradient and conditions as described above, but at a flow rate of 1 ml/min. After each run the column was cleaned by back-flushing hexane-propanol (95:5, v/v) at 1 ml/min for 10 min. Column effluent was collected in 0.5 min fractions and screened as described above.
GDGT extraction and analyses
In case of the sponge Axinella mexicana containing Cenarchaeum symbiosum (kind gift of Dr. E. DeLong, Monterey Bay Aquarium Research Institute, CA) direct solvent extraction did not yield significant amounts of GDGTs. The sponge was therefore extracted by refluxing with 2 N HCl in methanol for 8 h, followed by liquid/liquid extraction with DCM. Released GDGTs were subsequently analyzed by HPLC-APCI/MS as described previously (13).
Nuclear magnetic resonance
All GDGTs were solved in CDCl3 at a concentration of 3–6 µmol/ml. NMR spectroscopy was performed on a Varian Unity Inova 500, a Bruker DRX600, and a Bruker AV-750 spectrometer equipped with an SWBB probe, an inverse TBI-Z probe with a pulsed field gradient (PFG) accessory, and a BBI-zGRAD probe, respectively. All experiments were recorded at 300 K in CDCl3. Proton and carbon chemical shifts were referenced to internal CDCl3 (7.24/77.0 ppm). In the 2D 1H-13C correlated spectroscopy (COSY), the number of complex points and sweep widths were 2K points/6 ppm for 1H and 512 points/150 ppm for 13C. In the 2D 1H-1H COSY the number of complex points and sweep widths were 2 K points/5.5 ppm. Quadrature detection in the indirect dimension was achieved with the time-proportional-phase-incrementation method. The data were processed with Varian or NMRSuite software packages. After apodization with a 90 shifted sinebell, zero filling to 512 real points were applied for the indirect dimensions. For the direct dimensions zero filling to 4 K real points, Lorentz transformations were used.
Torsion angles in GDGT-4
To determine the average torsion angles in the cyclopentane rings of GDGT-4, a constant volume, constant temperature molecular dynamics (MD) simulation (14, 15) was performed at a system temperature of 298 K on a single GDGT-4 lipid. After equilibration at this temperature, a 25,000-iteration MD-simulation was performed. Atom positions were saved every 50 iterations, thus generating 500 conformational snapshots. Subsequently, average torsion angles in the five-membered rings and associated standard deviations were obtained from the analysis of these snapshots. The ReaxFF-force field (16) was used in these simulations.
Membrane volume determination
To determine the influence of molecular structure on membrane volume we performed constant pressure, constant temperature MD simulations (14, 15) on systems containing 3 x 3 GDGT-4 or crenarchaeol-lipid monomers. In accordance with work by Gabriel and Chong (6) we used ß-D-glucopyranose and myo-inositolphosphate as the polar groups in both the GDGT-4 and crenarchaeol-lipids. Periodic images of the 3 x 3-lipid system were used in the y- and z-direction (parallel to the membrane), no periodicity was used in the x-direction (perpendicular to the membrane). The system was allowed to expand and contract in the y- and z-directions according to the intermolecular forces. By this means the initial membrane structure, containing the 3 x 3 lipid monomers evenly and symmetrically distributed in a periodic box of dimensions 21 x 30 Å in y- and z-directions, was allowed to relax until it reached a steady-state configuration, after which the periodic box dimensions (the yz-surface area) could be used as a direct measure for the membrane volume. The system temperature (298 K) and pressure (10 bar) were controlled using the algorithm described by Berendsen et al. (17), with temperature and pressure damping constants of 1,000 femtoseconds and a MD time-step of 1 femtosecond.
A modified version of the AMBER-protein force field (18), as described by van Duin and Larter (19), was used to evaluate the interatomic forces in these simulations. To test for potential biases associated with this choice of force field all simulations were repeated with the ReaxFF-potential (16).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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60% of total GDGTs (Fig. 2A)
. This latter component showed a mass spectrum typical for a GDGT (i.e., loss of water and glycerol) (13) but with a protonated molecular ion 10 daltons lower than that of GDGT-0, establishing the molecular formula as C86H162O6 and indicating that this GDGT contained five rings. These results are in good agreement with ether cleavage studies of GDGTs of C. symbiosum (9), which revealed biphytanes with two and three cyclopentane rings (structures II and III) as major components.
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Isolation of the unknown GDGT
To fully elucidate the structure of this major unknown GDGT, it was isolated and its structure was determined by high-field 2D-NMR studies. As the source for isolation, surface sediments of the Arabian Sea were chosen. These sediments have a GDGT composition very similar to that of C. symbiosum (Fig. 2), contain high amounts of this GDGT, and are thus a good source for isolation. By solvent extraction, column chromatography over silica, and preparative HPLC a fraction significantly enriched in the unknown GDGT core membrane lipid was obtained. From this fraction, the unknown GDGT was isolated by repetitive analytical HPLC, resulting in 4 mg of isolate. HPLC-APCI/MS of this fraction indicated that the unknown GDGT was the only GDGT present in this fraction and did not reveal any other impurities. Consequently, this fraction was used for NMR studies.
For comparison of NMR data, GDGT-0 (I) and GDGT-4 (IV) were also isolated in high purity by HPLC from Arabian Sea sediments and cells of the hyperthermophilic crenarchaeon Sulfolobus acidocaldarius, respectively. Both GDGTs (but especially GDGT-4) share structural similarities with the unknown GDGT of pelagic crenarchaeota and 13C-NMR data have been reported for the biphytane carbon skeletons of these components (21–23), thus assisting in the identification of the unknown GDGT. However, no high-resolution 1H, 13C-NMR, and 2D-NMR correlation techniques have been applied to the intact GDGTs.
Basic skeleton
The 1H-NMR spectrum is extremely complex even if measured at 750 MHz. In the 3.4–3.7 ppm region multiplets representing 18 protons are observed (Table 1). These represent the protons of the two glycerol units and the first and ultimate methylene units of the biphytane moieties bound via the ether linkages. The same signals are observed in the 1H-NMR spectra of GDGT-0 and GDGT-4 (Table 2). At 2.2 ppm a broad singlet representing the two hydroxy groups is found. Between 0.82 and 0.88 ppm a complicated pattern of signals (mainly doublets) occurs in total representing eleven methyl groups. At 750 MHz, the resolution is high enough to separate a singlet at 
= 0.836 ppm, representing one methyl group, from a doublet at = 0.844 ppm, representing three methyl groups. In the 0.7–0.8 ppm region two "high-field" protons are observed; the remaining protons are all found in the 1.0–1.8 ppm region.
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= 0.836 ppm as a singlet in the 1H-NMR spectrum, which is absent in the 13C-NMR spectrum of GDGT-4 (Fig. 3)
. There are two possible structures (Va and Vb) for the second moiety through ring closure of the biphytanyl chain. Both are consistent with mass spectrometry data (11). Inverse long-range heteronuclear multiple bond correlation (HMBC) experiments enabled discrimination between these two possibilities since the singlet at = 0.836 ppm did not show correlation with the neighboring carbon atom (i.e., A10') of the cyclopentane ring, as would be expected for skeleton Vb, but instead with carbon atom A16' (Fig. 3). The remaining NMR data (Table 1) are also in agreement with this assignment. Furthermore, this structure is also in better agreement with published MS data of the biphytane moiety with three rings released after ether bond cleavage (11) because cleavage of the C-C bond between A15' and A16' explains why the fragment at m/z 263 is relatively abundant. This established that the abundant unknown GDGT membrane lipid in pelagic crenarchaeota is VI. We propose to call this component crenarchaeol, in analogy to the nomenclature of other archaeal ether lipids (24). Of the 86 carbon atoms of crenarchaeol, 23 are chiral and below we will explore literature and our NMR data to determine their stereochemistry.
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Heathcock et al. (26) have established the full stereostructure of GDGT-0 [2,3,2',3'-tetra-O-di-(3R,7R,11S,15S, 18S,22S,26R,30R-3,7,11,15,18,22,26,30-octamethyldotriacontanyl)-di-sn-glycerol; I]. Since all cyclopentane ring-containing GDGTs are biosynthesized by internal cyclization reactions of GDGT-0 (I), it is assumed that the remaining acyclic stereocentres in cyclopentane-containing GDGTs are also as in I (23). This assumption is also likely for crenarchaeol. This establishes the stereocentres of A3, A11, A3', A15', B3, B11, B3', B11' as (R) and A15, B15, and B15' as (S). Note that due to changes in the priorities of the groups on chiral carbon atoms according to the Cahn-Ingold-Prelog convention, the naming of the configuration may be different although the absolute stereochemistry remains the same.
Stereochemistry of the cyclopentane rings
The absolute stereochemistry of the cyclopentane-ring has not yet been established. De Rosa et al. (22) reported, on basis of the chemical shifts of the carbon atoms of the cyclopentane rings in comparison with 13C-NMR data of dimethylcyclopentanes (27), that the 1,3-substitution pattern of the cyclopentane ring in archaeal GDGTs is probably trans.
To determine the full stereochemistry of the cyclopentane rings, it was decided to first concentrate on the symmetrical GDGT-4 (IV), where no interference of signals from the cyclohexane ring occurs. HMQC, HMBC, COSY, and total correlation spectroscopy (TOCSY) experiments resulted in the assignment of all protons of the cyclopentane rings (Table 2). The two protons of both the A8 and A9 methylene groups showed a large difference (0.6–0.7 ppm) in chemical shift, whereas this difference for the two protons of the A18 methylene unit was only small (0.1 ppm) (Table 2). This suggested for the protons at A8 and A9 a situation comparable to that of cyclohexane rings, where the chemical shifts of protons strongly depend on their axial or equatorial position: the axial protons often resonate at much higher field than their equatorial counterparts. On the other hand, the protons at A18 seem to be more in eclipsed than in staggered positions. This assignment is, however, complicated by the fact that much conformational freedom exists in the cyclopentane compared with the cyclohexane ring. Therefore, we simulated the conformation of GDGT-4 using molecular dynamics and determined the average torsion angle of the protons and alkyl substituents of the cyclopentane ring. The results show that i) there is indeed a significant degree of conformational freedom in the cyclopentane ring and ii) the protons at A8 and A9 have a pronounced axial/equatorial character, whereas the torsion angles of the two protons at A18 with the ring are similar (Fig. 4A)
, consistent with our assignments of the NMR signals. The shifts of protons at the more substituted, tertiary carbon atoms A7 and A10 are at 1.79 and 1.68 ppm, respectively, not allowing any conclusion on whether they are axially or equatorially substituted.
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Based on these assignments, the stereochemistry of the cyclopentane rings of crenarchaeol was assessed to be identical to those in GDGT-4. All the protons and carbon atoms resonate at identical field strength (Tables 1 and 2), except those of the cyclopentane ring attached to the cyclohexane ring. In this cyclopentane ring, most chemical shifts are also identical except for the shift of the proton at carbon atom A10', which is at slightly higher field (1.47 ppm vs. 1.68 ppm in GDGT-4). This is attributed to the attached cyclohexane ring, which forces the proton in a slightly more "axial" position.
Stereochemistry of the cyclohexane ring
A prominent feature in the 1H-NMR data of crenarchaeol are the two high-field protons, which are absent in the spectrum of GDGT-4, and obviously related to the presence of the cyclohexyl moiety. The assignment of these high-field protons is based on COSY, TOCSY, HMQC, and HMBC correlations. The proton at = 0.70 represents a quasi triplet with a coupling constant of J = 12.5 Hz. This signal must be assigned to the axial proton at A19'; in addition to the relatively large geminal coupling there must be an equally large, axial-axial coupling with the proton at carbon atom A11'. The other high field proton absorbs at = 0.72 and forms a quasi-double quartet with coupling constants J = 13.0 and 4.5 Hz. This is the axial proton at carbon atom A12', which couples with the axial protons at A11' and A13', the geminal proton (all with large coupling constants of 13 Hz), and with the equatorial proton at carbon atom A13' with a much smaller coupling constant. The proton at carbon atom A11' absorbs at = 1.17 and is not well resolved from other signals. However, in 2D-NMR spectra it shows up as a double doublet with two relatively large coupling constants, in agreement with this assignment. Coupling with the proton at carbon atom A10' is only weak, indicating that the dihedral angle is probably close to 90°C. These results indicate that the cyclopentane ring is equatorially substituted at carbon atom A11' and that the stereochemistry at position A11' is thus (S).
The stereochemistry at position A15' follows from two observations. First, the chemical shift of the methyl group at A15' (A20') in the 13C-NMR spectrum is at relatively low field (22.39 ppm), indicative for equatorially substituted methyl groups of cyclohexane rings (28). Second, remarkably, both axial protons at carbon atoms A14' and A19' show a strong long-range (four bonds) correlation with methyl group A20' in the COSY spectrum. This established the (R) stereochemistry at A15'.
The stereochemistry of the cyclohexane ring is consistent with its presumed biosynthetic formation through ring closure via A15' and A19'; the resulting stereochemistry is "inherited" from the stereochemistry of the GDGT-0 (I) precursor. This also determines the equatorial/axial positions of the alkyl substituents of the cyclohexane ring. If the cyclohexane ring is in the more stable chair configuration, this fits with the stereochemical configuration of the cyclohexane ring in crenarchael as determined by NMR.
Regioisomerism
It has been assumed for a long time that archaeal GDGTs were characterized by an antiparallel arrangement of glycerol units as in I (29). However, Gräther and Arigoni (30) showed by selective chemical degradation for three archaeal species that GDGT-0 is in fact a 1:1 mixture of the regioisomeric components I and VII.
During isolation of GDGT-4 from S. solfataricus, a fraction enriched in a less abundant, slightly later eluting (13) (35% of GDGT-4) isomer (GDGT-4') was also isolated. This isomer had virtually identical 1H- and 13C-NMR spectra, indicating that the four cyclopentane rings must be in the same position and have the same stereochemistry. Detailed comparison of the 13C data indicated, however, a subtle difference; the carbon atoms A3, A4, A5, and A6 showed two signals in case of GDGT-4 but only one in case of GDGT-4' (Table 3). This observation led us to the conclusion that GDGT-4 is the antiparallel isomer whereas GDGT-4' is the parallel isomer. This latter isomer has a plane of symmetry and only one stereoisomer exists. For GDGT-4 there is no plane of symmetry and its mirror image is therefore different. This explains why for some carbon atoms two close but not identical signals are observed.
For crenarchaeol an even more complicated situation exists since for some carbon atoms even four different signals are observed (Table 3). This indicates that the isolated isomer probably has the antiparallel configuration of glycerol units like in GDGT-4. Indeed, a minor isomer of crenarchaeol (presumably the parallel regioisomer) elutes later on the HPLC column, just as with GDGT-4 and GDGT-4'. In case of crenarchaeol there are, however, two additional regioisomers (VI and VIII) both with the antiparallel configuration of glycerol units, since the two biphytanyl chains in crenarchaeol are not the same, resulting in four stereoisomers. This explains the even more complex 13C-NMR spectrum.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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This inferred evolutionary adaptation of membrane fluidity has not only resulted in the development of a dominant group of archaea but of microorganisms in general. Marine pelagic crenarchaeota probably represent one of the most abundant clades of microorganisms on earth. Their estimated total cell number in the oceans (1.3·1028) is 40% of the estimated total number of all bacteria in the ocean (4). This indicates that the oceans also contain a massive amount of crenarchaeol. We have recently estimated that one crenarchaeal cell contains 1·10-3pg GDGT (20). This indicates that the oceans contain 13 Mt GDGT, of which 50% (6.5 Mt) is comprised of crenarchaeol. Together with GDGT-0, crenarchaeol is by far the most abundant GDGT in the biosphere, much more abundant than the cyclopentane-containing GDGT's derived from (hyper) thermophilic archaea.
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ACKNOWLEDGMENTS |
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Manuscript received April 3, 2002 and in revised form June 5, 2002.
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