ABSTRACT
Glucose-substituted cardiolipins account for about 4 mol% of total phospholipid extracted from exponentially grown cells of Geobacillus stearothermophilus NRS 2004/3a. Individual glucocardiolipin species exhibited differences in fatty acid substitution, with iso-C15:0 and anteiso-C17:0 prevailing. The compounds were purified to homogeneity by a novel protocol and precharacterized by matrix-assisted laser desorption ionization time-of-flight mass spectrometry.
The cell wall of Geobacillus (formerly Bacillus) (14) stearothermophilus NRS 2004/3a comprises an oblique S-layer glycoprotein lattice as the outermost surface layer (20), a rather thin peptidoglycan layer to which the S-layer is noncovalently bound via an anionic secondary cell wall polymer (18), and the cytoplasmic membrane. Since lipid-bound carbohydrates (which are expected to be involved in the biosynthesis of the S-layer glycoprotein glycans) have to cross the cytoplasmic membrane, they should be contained in membrane fractions and whole-cell lipid extracts, respectively. To unravel the nature of these lipid intermediates is one of our objectives in the course of determining S-layer glycobiology (for review, see reference 13).
In a crude lipid extract obtained from exponentially grown cells of Geobacillus stearothermophilus NRS 2004/3a, an unusual glycolipid was detected, the prevalence of which justified a more detailed investigation. In general, the ester phospholipid composition of G. stearothermophilus isolates is known to be similar to that of other Geobacillus or Bacillus species, except for the absence of amino acid derivatives of phosphatidylglycerol and for the presence of a higher concentration of cardiolipin (CL) (bisphosphatidylglycerol) (4). Thus, the major constituents include phosphatidylethanolamine (PE), accounting for 40 to 50% of the total phospholipids; and the interconvertible anionic lipids phosphatidylglycerol (PG) and cardiolipin (CL), the concentrations of which are growth phase dependent (5).
In this contribution, we report on the purification of substituted CL species from a whole-cell lipid extract of G. stearothermophilus NRS 2004/3a and its precharacterization by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS).
Extraction of lipids from G. stearothermophilus.
Strain NRS 2004/3a was grown on modified SVIII medium, supplemented with 0.2% (wt/vol) glucose, at pH 7.2 and 60°C with a dilution rate of 0.15 h−1 as described previously (20). Cells were harvested from the late-exponential growth phase (optical density at 600 nm of ∼0.7), and the biomass was stored at −20°C until use. Fractionated lipid extraction was performed with 50 g of biomass by using 1:1 (vol/vol) CHCl3-methanol (MeOH), 1:2 (vol/vol) CHCl3-MeOH, and 48:35:10 (vol/vol/vol) CHCl3-MeOH-H2O. Cells were suspended in 500 ml of the first solvent and disrupted by pulsed sonication for 10 min on ice, followed by extraction for 30 min at 25°C with vigorous stirring. After centrifugation at 31,400 × g, extraction was repeated with 350 ml of each of the other two solvents. The supernatants were combined, evaporated to dryness, and resuspended in 15 ml of 12:6:1 CHCl3-MeOH-H2O. Phases were separated by centrifugation at 4,200 × g. The lower phase, representing the crude lipid extract (CLE), contained a total of 1,075 μmol of lipid phosphorus (1). One corner of a 10- by 10-cm high-performance thin-layer chromatography (HPTLC) plate (Merck, Darmstadt, Germany) was spotted with 3 μl of CLE. The HPTLC plate was developed in the first dimension in 65:25:4 (vol/vol/vol) CHCl3-MeOH-H2O (solvent A) to 9 cm and in the second dimension in 8:1:1 (vol/vol/vol) 1-propanol-H2O-acetic acid (solvent B) to 9 cm. Compounds were analyzed with the following reagents: 0.5 ml of anisaldehyde (Merck) in 10 ml of acetic acid plus 85 ml of MeOH plus 5 ml of H2SO4 (for detection of lipids, as modified after reference 23); 5% primulin in 4:1 acetone-H2O (for detection of lipids) (22); iodine vapor (for detection of double bonds); molybdenum blue (for detection of phosphorus); 3.4% N-(1-naphthyl)-ethylenediamine in 97:3 MeOH-H2SO4 (for detection of carbohydrates; as modified from reference 3), and 0.2% ninhydrin in ethanol (for detection of amino groups). Unless indicated otherwise, reagents were purchased from Sigma (Vienna, Austria). Plates were developed at 110°C between 5 and 25 min, except for components reacting with primulin, which were visualized under UV light at 366 nm. The two-dimensional TLC pattern of the CLE from G. stearothermophilus NRS 2004/3a showed only one spot giving a triple-positive reaction when analyzed for lipid, phosphorus, and carbohydrate (Fig. 1a, b, and c). It was later identified as glucocardiolipin (GCL). In a separate run, the prominent component staining with anisaldehyde, molybdenum blue, and ninhydrin was identified as PE (Fig. 1a, b, and d). This result concurs with the preponderance of PE among the phospholipids of this bacterial species. To obtain sufficient amounts of GCL for detailed analysis and potential future applications, a purification protocol was elaborated.
Two-dimensional TLC pattern of a crude lipid extract from G. stearothermophilus NRS 2004/3a. Staining was performed with anisaldehyde (a), molybdenum blue (b), N-(1-naphthyl)-ethylenediamine (c), and ninhydrin (d). GCL was identified by MALDI-TOF MS from a homogeneous fraction, and PE was identified with a respective standard. Compounds were separated with solvent A in the first dimension and with solvent B in the second dimension.
Purification of GCLs.
The CLE was fractionated in portions containing ∼200 μmol of lipid phosphorus by column chromatography on Servacel DEAE 52 cellulose (1.0 by 48 cm; flow rate of 0.5 ml/min; Serva, Heidelberg, Germany) according to the method described by Fischer and Arneth-Seifert (7), with slight modifications. Stepwise gradient elution was done with CHCl3, 95:5 (vol/vol) CHCl3-MeOH, 9:1 (vol/vol) CHCl3-MeOH, 8:1 (vol/vol) CHCl3-MeOH, 2:1 (vol/vol) CHCl3-MeOH, and 2:1 (vol/vol) CHCl3-MeOH containing 6.5, 15, 50, 80, and 100 mM NH4OAc, respectively. Fractions of 80 ml were collected and evaporated to a final volume of 650 μl. Material from NH4OAc-containing fractions was extracted with 0.2% CaCl2 in saline, taken to dryness several times with benzene, dissolved in 650 μl of 2:1 (vol/vol) CHCl3-MeOH, and acidified with acetic acid. Fractions 1 to 10 were analyzed by one-dimensional TLC (solvent A) on Merck Silica 60 plates (data not shown). Fractions 5 and 9 each contained a single, prominent carbohydrate-staining component exhibiting Rf values of 0.34 and 0.25, respectively. Fractions 7, 8, and 9 revealed an intensive staining pattern when detected with iodine vapor and molybdenum blue, whereas the other fractions stained less effectively; pigments were recovered from fractions 1 and 2. The phosphorus contents were determined to be 0.7, 61, 55, 61, and 10 μmol in fractions 6, 7, 8, 9, and 10, respectively. In fractions 1 to 5, total phosphorus was <0.1 μmol.
Phosphoglycolipid-containing fraction 9, which accounted for 14.1% of the total extracted phospholipids, was subsequently applied to chromatography on an Iatrobeads 6RS-8060 column (1 cm by 50 cm; SES GmbH, Bechheim, Germany). The column was packed in 1:3 (vol/vol) CHCl3-MeOH, reequilibrated with 2:1 (vol/vol) CHCl3-MeOH, and eluted with the same solvent at a flow rate of 0.9 ml/min. The elution profile was recorded at 280 nm, and individual fractions (1 ml) were analyzed by one-dimensional TLC (solvent A) (data not shown). It was evident from the carbohydrate TLC pattern that the phosphoglycolipid was contained in fractions 23 to 36. However, minor amounts of the second, coeluting carbohydrate-staining component of the CLE were also contained in this pool. The pool was evaporated to dryness and dissolved in 100 μl of 2:1 (vol/vol) CHCl3-MeOH for final purification by TLC on HPTLC plates. The spot corresponding to phosphoglycolipid was scraped off from the plate after development in the first dimension (solvent A), extracted twice with 800 μl of 2:1 (vol/vol) CHCl3-MeOH, and 1:1 (vol/vol) CHCl3-MeOH for 15 min each, and run in the second dimension (solvent B). Extraction was repeated, and the dried extract was finally dissolved in 100 μl of 2:1 (vol/vol) CHCl3-MeOH for further characterization. Two-dimensional TLC of 2 μl of that extract verified that a pure GCL preparation had been obtained (Fig. 2). Based on the phosphorus and glucose (described below) contents of that preparation, the yield of GCL isolated from G. stearothermophilus NRS 2004/3a was ∼8 nmol per mg of cells (dry weight), which is 4.1 mol% of the total extracted phospholipids. Considering that in group N Streptococcus sp. strain NCDO 2497, the content of GCL varied from 0.5 mol% of membrane lipids in the exponential growth phase to 3.7 mol% in the stationary phase (7), this yield could obviously be increased when starting with G. stearothermophilus NRS 2004/3a biomass harvested from the stationary growth phase. Whether the overall content of GCLs in the membrane of that organism is dependent on the amount of glucose added to the growth medium has not been investigated.
Characterization of GCL from G. stearothermophilus NRS 2004/3a. (a) Glucose was identified as the sole carbohydrate constituent by HPAEC/PED. IS, internal standard (2-deoxygalactose). (b to d) The TLC staining pattern (silica 60 plates) of the crude lipid extract (lanes 1) is compared to those of the purified material (lanes 2) for anisaldehyde (b), N-(1-naphthyl)-ethylenediamine (c), and ninhydrin (d). (e and f) The homogeneity of the GCL preparation is demonstrated by two-dimensional TLC (HPTLC plates), involving a reaction with primulin (e) and staining with molybdenum blue (f). The first and second dimensions were developed with solvents A and B, respectively.
Precharacterization of GCL.
For carbohydrate analysis by high-performance anion-exchange chromatography with pulsed electrochemical detection (HPAEC/PED) (19), 5 μl of the GCL preparation was hydrolyzed with 200 μl of 25% trifluoroacetic acid for 4 h at 110°C. The acid was removed under a stream of nitrogen, lipid was extracted twice with 400 μl of 1:1 (vol/vol) dichloromethane-H2O, and the combined upper phases were backwashed twice with 400 μl of H2O. Glucose was identified as the sole carbohydrate component of the GCL preparation (Fig. 2a). Fatty acids were determined as methyl esters by gas-liquid chromatography as described elsewhere (12). The major fatty acids present were iso-C15:0 (26 mol%), anteiso-C15:0 (9.7 mol%), iso-C17:0 (18.6 mol%), and anteiso-C17:0 (31.8 mol%), in addition to minor amounts of iso-C16:0, C16:0, iso-C14:0, and C14:0. This fatty acid composition is in accordance with that recently described for G. stearothermophilus isolates (14). It is, however, different from the fatty acids contained in all other GCL species known thus far. The GCLs of several strains of Streptococcus group B (9) contain exclusively even-numbered fatty acids, with C16 and C18 saturated and monounsaturated fatty acids being predominant, in addition to cis-11,12-methylenoctadecanoic acid. These fatty acids are mostly linked to position 2 of the glycerol moieties (9). In Vagococcus fluvialis, the cis-dihydrooxylated monounsaturated fatty acids C14:1, C16:1, and C18:1 prevail (7).
MALDI-TOF MS.
MALDI-TOF MS mapping is a rapid and sensitive approach to obtain basic structural information about complex mixtures of biomolecules (16). Particularly the low sample consumption is advantageous for precharacterization. For the GCL species from G. stearothermophilus NRS 2004/3a, MALDI-TOF MS mapping was performed in the positive- and negative-ion modes. MALDI-TOF mass spectra were acquired on a TOFSpec 2E mass spectrometer (Micromass, Manchester, United Kingdom) equipped with a UV laser (λ = 337 nm) (this study). The GCL preparation was dissolved in 2:1 (vol/vol) CHCl3-MeOH. A 9:1 (vol/vol) mixture of 2,5-dihydroxybenzoic acid and 2-methoxy-5-hydroxybenzoic acid in 9:1 (vol/vol) H2O-ethanol was used as a matrix. An average of 100 laser shots were collected in one spectrum.
In Fig. 3, the positive (Fig. 3a)- and negative (Fig. 3b)-ion MALDI-TOF mass spectra of the GCL species are presented. In both spectra, besides lower-mass-range ions up to an m/z of 800, a wide variety of peaks attributed to the GCL basic structure (for details, see reference 2) were detected in the mass range of m/z = 1,500 to 1,700. In the positive-ion mode (Fig. 3a), a complex pattern of three overlapping series of molecular ions could be identified. The mass difference of m/z = 22 between the three series of peaks is due to the formation of sodium adduct ions. The mass difference of m/z = 14 between the peaks of one series corresponds to one −CH2 moiety originating from the different length of the fatty acid chain. These three series are represented by the molecular ions of the [M + Na]+ type (∗; at m/z = 1,510.25, 1,524.21, 1,538.23, 1,552.29, and 1,566.27), the [M − H + 2Na]+ type (#; at m/z = 1,532.10, 1,546.23, 1,560.25, 1,574.20, and 1,588.29), and the [M − 2H + 3Na]+ type (+; at m/z = 1,554.21, 1,568.27, 1,582.22, 1,596.31, and 1,610.28), as marked in the figure. At m/z = 1,576.17, the [M − H + Na + K]+ mixed adduct ion was detected, and at m/z = 1,598.22 and 1,626.22, respectively, the [M − 2H + 2Na + K]+ mixed adduct ions were detected. In the negative-ion-mode MALDI mass spectrum, two overlapping series of molecular ions were identified (Fig. 3b). The [M − H]− type series at m/z = 1,486.46, 1,500.51, 1,514.50, 1,528.55, and 1,524.56 is marked by an asterisk (∗), whereas the second series of molecular ions at m/z = 1,508.54, 1,522.57, 1,536.58, 1,550.60, 1,564.60, and 1,592.59, which belongs to the [M − 2H + Na]− type, are not marked. Due to the decreased alkali ion adduct formation, the negative-ion MALDI MS map of the complex GCL mixture provides a less complicated pattern than the positive-ion-mode map.
MALDI-TOF MS of GCL species. In the positive-ion mode (a), three overlapping series of sodiated GCL molecular ions are denoted as follows: ∗, [M+Na]+; #, [M − H + 2Na]+; and +, [M − 2H − 3Na]+. Peaks not marked originate from potassium adduct ions. In the negative-ion mode (b), two overlapping series of GCL molecular ions, [M − H]− (∗) and [M − 2H + Na]−, are detected.
The origins of single molecular species need to be traced by more detailed analysis, in particular by tandem MS (MS/MS) experiments. Mapping and sequencing of the GCL species from G. stearothermophilus NRS 2004/3a by positive- and negative-ion nano electrospray ionization quadrupole-time-of-flight MS (nanoESI-QTOF MS) and MS/MS were described in detail recently (2). Screening of lipid libraries by tandem MS methods, and precursor ion scans can provide a higher degree of data refinement by assigning particular functional elements in isomeric structures, by using a MALDI-CID (collision-induced damage) approach, as proposed recently (A. I. Beckedorf, M. Jovanovic, W. Fischer, C. Schäffer, P. Messner, and J. Peter-Katalinic, 50th Am. Soc. Mass Spectrom. Conf., poster no. 250, 2002).
In the MALDI-TOF analysis, in both ion modes, the most abundant peaks can be correlated to the theoretical fatty acid composition of four C16:0 fatty acid moieties or to compositions with different fatty acid moieties giving the same summation formula. Fragmentation analysis by MS/MS carried out by nanoESI tandem MS analysis in both the positive- and negative-ion modes (details are given in reference 2), as well the fatty acid composition derived from gas chromatography data (this study), indicated that the most abundant fatty acids are C15:0 and C17:0. The combination of two pairs, each in a single molecule, results in the same theoretical mass that of four C16:0 fatty acid moieties do. Furthermore, the MS/MS data (2) provide clear evidence that all molecular ion peaks detected by MALDI-TOF (this study) are in fact mixtures of isobaric structures containing different fatty acid compositions. The detected fatty acid chain lengths range from C12:0 to C22:0, with C15:0 and C17:0 as the most abundant species, and all of them are saturated. The ions appearing in the low-mass region could originate from the in-source fragmentation due to their high internal energy after laser desorption. Another option for their appearance in the spectra would be the formation of truncated GCL structures in vivo. This aspect needs to be investigated in further studies.
Conclusions.
G. stearothermophilus NRS 2004/3a is a source of the rare glycolipid GCL. Whereas CLs have been known as widespread negatively charged lipid components in the cytoplasmic membrane of gram-negative and -positive bacteria for a long time (15, 24), substituted CLs, such as the aminoacylcardiolipins d-alanylcardiolipin and l-lysylcardiolipin (7, 17), d-lysylcardiolipin (8), and GCLs represent a more recently discovered class of lipids present in gram-positive bacteria. So far, GCLs have been exclusively described in coccoid organisms, including group B streptococci, where they amount to approximately 18% of the lipid phosphorus (6), and vagococci, where they occur in only minor amounts (7). Furthermore, in Bacillus megaterium, a 1-amino-2-deoxy-β-d-glycopyranosyl derivative has been detected (11). G. stearothermophilus NRS 2004/3a is the first thermophilic organism for which GCL has been described (this study and reference 2). In analogy to previous studies of bacterial GCL species (17), it is conceivable that this GCL is an α-d-GCL (2). It is novel in so far that the fatty acid distribution pattern exhibits predominantly uneven numbered fatty acids, which are on average one C atom shorter than those in the other known GCLs, and all fatty acids are saturated. Even though the in vivo physiological relevance of GCL in bacterial membranes is generally poorly understood, it is conceivable that this phosphoglycolipid is involved in the regulation of the membrane lipid composition of G. stearothermophilus NRS 2004/3a to compensate for the destabilizing effect of high temperatures on the membrane organization or to provide an appropriate packaging of phospholipid molecules in a stable lipid bilayer (10).
We have elaborated a combined column chromatography-TLC protocol for the purification of GCL from G. stearothermophilus NRS 2004/3a in reasonable quantities. The combination of positive- and negative-ion-mode MALDI-TOF MS provided a full set of data concerning the molecular complexity of the phosphoglycolipid sample, which allowed the unambiguous identification of the GCL species as a basis for detailed characterization by more sensitive MS techniques (2). Concerning application aspects, this rare glycolipid might display several functions when incorporated into artificial membranes. These could include influencing the regulation of overall membrane stability and serving as an anchoring structure for the immobilization of macromolecules via carbohydrate coupling chemistry. For additional stabilization of these artificial composite membranes, S-layer technology offers a versatile tool (21).
ACKNOWLEDGMENTS
We thank A. Makristathis for performing GC analysis.
This work was supported by the Austrian Science Fund, project P14209 (to P.M.), the Hochschuljubiläumsstiftung der Stadt Wien, project H-148/2001 (to C.S.), and the Interdisciplinary Center for Clinical Research (IZKF) at the University of Münster, Germany, for use of the TofSpec2E instrument (to J.P.-K.). A.B. was supported by the Z2 project in the Sonderforschungsbereich 492, financed by Deutsche Forschungsgemeinschaft (to J.P.-K.)
FOOTNOTES
- Received 2 July 2002.
- Accepted 3 September 2002.
- Copyright © 2002 American Society for Microbiology
