Effects of pH and Temperature on the Composition of Polar Lipids in Thermoplasma acidophilum HO-62

Abbreviations.EI-MS, electron impact mass spectrometry; ELSD, evaporative light-scattering detector; GC, gas chromatography; HPLC, high-performance liquid chromatography; liquid SIMS, liquid secondary ion mass spectrometry; MS, mass spectrometry; TMS, trimethylsilyl; the legend to Fig. 1 lists abbreviations of the names of polar lipids.

Chemicals.HPLC grade chloroform and methanol were obtained from Nacalai Tesque, Inc. (Kyoto, Japan). l-Gulose was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). All the other chemicals used were of analytical grade.

Organism and cultivation.T. acidophilum HO-62 was statically grown at the desired temperatures in 1 liter of the medium described by Yasuda et al. (32); the pH of the medium was adjusted to the desired pHs by using sulfuric acid. Cultivation was repeated three times under each condition. Lipids were extracted from cells from each culture independently.

Extraction of polar lipids.The polar lipid extraction process using the Bligh and Dyer method (4) was described previously (26). Lyophilized cells were suspended in chloroform-methanol-water (4:1:5 [vol/vol/vol]), and then chloroform-water (1:1 [vol/vol]) was added to the suspension. The lower chloroform phase was collected. The upper aqueous phase, including the fluff layer and the insoluble sediment, was extracted two times with chloroform. The chloroform extracts were pooled and evaporated. The residue was designated TPL1. The aqueous layer was lyophilized. Polar lipids that remained in the lyophilized aqueous layer were reextracted using chloroform-methanol (2:1 [vol/vol]). The reextraction was repeated five times. The pooled solution was evaporated and designated TPL2. The proportions of TPL1 and TPL2 in the total polar lipids were about 75% dry weight and 25% dry weight, respectively.

Separation of polar lipids by ELSD-HPLC.TPL1 and TPL2 were separately applied to a Capcellpak silica SG80 column (2.0 mm [inside diameter] by 250 mm [length]; 5-μm particle size; Shiseido, Tokyo, Japan) at 35°C, attached to an LC10A HPLC system (Shimadzu, Kyoto, Japan) and were monitored with an Alltech model 500 ELSD (Deerfield, IL). Nebulization was carried out with compressed air at a flow rate of 2.0 liters/min at 80°C. The eluents used were as follows: H1, chloroform; H2, chloroform-methanol-trifluoroacetic acid (50:50:1 [vol/vol/vol]); and H3, chloroform-methanol-trifluoroacetic acid (10:10:1 [vol/vol/vol]). The polar lipids were separated with gradients of H1 and H2, with the program H2 %(min) 0(0)-10(2)-50(42)-100(62), at a flow rate of 0.25 ml/min. After each analysis, the column was washed with H3.

Quantification of polar lipids.GuCGp, the main polar lipid of T. acidophilum, purified from the total amount of polar lipid from a 50-liter culture (26), was used as the polar lipid standard. GuCGp was diluted, and 10 μl of the appropriate dilution was subjected to ELSD-HPLC. The peak area of GuCGp in the HPLC chromatogram was plotted and used as the standard curve.

Analysis of core lipids.TPL1 and TPL2 were mixed, and polar lipids were decomposed to methylglycosides and caldarchaeol by acid methanolysis, according to the methods described by Yang and Haug (31). The caldarchaeol remaining in the hexane layer after acid methanolysis was applied directly to the ELSD-HPLC system, as described above. ELSD nebulization was performed with compressed air at a flow rate of 1.6 liters/min at 55°C. An Adsorbosphere NH₂ column (2.0 mm [inside diameter] by 250 mm [length]; 3-μm particle size; Alltech, Deerfield, IL) was used, and the column temperature was maintained at 30°C. The eluent used was J1 (n-hexane-1-propanol; 99:1 [vol/vol]). Caldarchaeol samples containing different numbers of cyclopentane rings were separated with J1 at a flow rate of 0.4 ml/min. To identify the HPLC peaks, caldarchaeols were collected by splitting the solvent line before the detector. The mass number of the component in each HPLC fraction was measured by liquid SIMS. Liquid SIMS was performed at an acceleration voltage of 7 keV, using an Autospec E mass spectrometer (Micromass Inc., Manchester, England) equipped with a SIMS probe. Dithiothreitol-dithioerythritol (3:1 [vol/vol]) was used as the matrix.

Biphytanyl chains in caldarchaeol were investigated by GC. Each caldarchaeol collected from the HPLC peaks was decomposed to two biphytanyl alcohols, which were converted to their TMS derivatives and analyzed by GC with a flame ionization detector as described previously (26). Each GC peak was identified by GC-MS using a Hewlett-Packard model HP5890 gas chromatograph (Avondale, PA) attached to an Autospec E mass spectrometer (Micromass Inc., Manchester, England) equipped with an EI-MS probe operated at a 70 eV ionization voltage. The GC procedure is described in the legend to Fig. 3.

Chemical stability of GuCGp.GuCGp was put into a screw-cap test tube and dried under a flow of nitrogen gas. The residue was overlaid with distilled water adjusted to pH 1.8 with sulfuric acid and incubated at 56°C for 3 days. Lipids were extracted with chloroform-methanol (2:1 [vol/vol]) and analyzed by ELSD-HPLC.

Liposome preparation.The polar lipid in the TPL2 fraction was applied to a silica gel (Wakogel C200, 100/200 mesh; Wako Pure Chemical Industries, Ltd., Osaka, Japan) column (10 mm [inside diameter] by 100 mm [length]) equilibrated with chloroform. After the elution of low-polarity lipids with chloroform, GuCGP was eluted with chloroform-methanol (3:2 [vol/vol]). GuCCp is a phosphoglycolipid that contains one sugar unit in the structure. The other phosphoglycolipids, named MGuCGp, DMGuCGp, and TMGuCGp, that contain two, three, and four sugar units, respectively, were eluted with methanol. The chloroform-methanol (3:2 [vol/vol]) and methanol fractions were dried with nitrogen gas, dissolved separately in chloroform-methanol (2:1 [vol/vol]), and designated chloroform-methanol fractions (Fr. CM) and methanol fractions (Fr. M), respectively.

Each fraction corresponding to 100 μg of lipid was put into a glass test tube, and valinomycin was added to the solution to make 1 nmol/mg lipid. The solutions were dried with nitrogen gas. A buffer solution containing 2 mM pyranine, 5 mM potassium phosphate (pH 7.0), and 0.1 M potassium chloride was then added to the residue, and the mixture was preheated at 70°C for 30 min to hydrate the lipids. A liposome suspension was prepared by sonication using an ultrasonicator VS-100 machine (Asone, Osaka, Japan) for 2.5 min at 70°C. The size of the liposomes was then adjusted to about 0.1 μm by using a Mini-Extruder (Avanti, Alabaster, AL). The resulting lysate was dialyzed against a pyranine-free buffer (5 mM potassium phosphate and 0.1 M potassium chloride [pH 7.0]). The internal pH of the liposomes was estimated by the method described by Kano and Feneler (18). Standard solutions containing 0.33 μM pyranine, 5 mM potassium phosphate, and 0.1 M potassium chloride at pHs of 5.0, 5.5, 6.0, 7.0, and 7.5 were prepared. The fluorescence of pyranine was measured with an RF-5300PC model (Shimadzu, Kyoto, Japan) spectrophotofluorometer at 50°C in a thermostatted holder. For the calibration curve, the logarithm of the ratios of the emission intensities (I) at 510 nm on excitation at 460 and 400 nm, log (I_{460 nm}/I_{400 nm}), was plotted against the pHs of the standard solutions. The internal pH of the liposomes was estimated from the calibration curve. The liposome suspension was diluted with the buffer (pH 7.0). The diluted liposome suspension (60 μl) was then mixed with the buffer (540 μl) containing 5 mM potassium phosphate and 0.1 M potassium chloride (pH 3.6). Fluorescence was measured for 30 min at 50°C.

Effects of pH and temperature on the number of cyclopentane rings in caldarchaeol of T. acidophilum HO-62.Caldarchaeol obtained from acid methanolysis of the total amount of polar lipid of T. acidophilum HO-62 was analyzed by ELSD-HPLC (Fig. 2). Caldarchaeols containing different numbers of cyclopentane rings were separated by normal phase HPLC (17). Each peak observed in the chromatogram (Fig. 2) was collected and analyzed by MS and GC. The relative masses of the protonated molecular ions ([M+H]⁺) of the HPLC peaks were measured by liquid SIMS (using a positive ion mode). Peaks 0, 1, 2, 3, 4, 5, and 6, shown in Fig. 2, showed [M+H]⁺ at m/z ratios of 1,302, 1,300, 1,298, 1,296, 1,294, 1,292, and 1290, respectively. These molecular ion masses agreed well with those of previously identified caldarchaeols that contained zero, one, two, three, four, five, and six cyclopentane rings, respectively (17). Although components with m/z ratios of 1,308, 1,306, and 1,304 were also observed in peaks 4, 5, and 6, respectively, they could not be identified.

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FIG. 2.

HPLC chromatograms of caldarchaeols with different numbers of cyclopentane rings, which are indicated beside their respective peaks. The caldarchaeols obtained from the cultures grown at different temperatures at pH 1.8 (A) and at 55°C at different pHs (B) are shown. The analytical conditions shown are described in Materials and Methods.

Liquid SIMS provides only the molecular mass of caldarchaeol; it does not clearly show the number of cyclopentane rings in each biphytanyl chain. To clarify the number of cyclopentane rings in each biphytanyl chain, the caldarchaeol in each HPLC peak was decomposed to biphytanyl alcohols, which were converted to their TMS derivatives. TMS-biphytanyl alcohols were analyzed by GC. The GC profiles and structures of caldarchaeols in the respective HPLC peaks are shown in Fig. 3. The GC peaks have been identified previously (26). Peak 0 contained only the biphytanyl alcohol without cyclopentane rings. Peaks 1, 2, 3, 4, and 5 contained biphytanyl alcohols with zero and one, one and one, one and two, two and two, and two and three cyclopentane rings, respectively. The EI-MS fragment pattern of each TMS-biphytanyl alcohol (data not shown) was similar to that reported previously (31) and consistent with the ring position shown in Fig. 3B. The amounts of caldarchaeols with high numbers of cyclopentane rings increased at high temperatures (Fig. 2A) and at high pHs (Fig. 2B).

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FIG. 3.

(A) GC chromatograms of TMS derivatives obtained from caldarchaeols corresponding to HPLC peaks shown in Fig. 2. (B) Structures corresponding to caldarchaeols shown in panel A. Caldarchaeols collected from the HPLC peaks in Fig. 2 were decomposed to biphytanyl alcohols and then converted to their TMS derivatives. TMS-biphytanyl alcohols were analyzed by GC using an HP-5ht capillary column (0.3 mm inside diameter by 30 m; thickness of film [df] = 0.1 μm) at a temperature gradient of 200°C to 350°C with an incremental step of 10 degrees/min and monitored with a flame ionization detector. The number of cyclopentane rings in the biphytanyl chains shown in the GC chart was identified previously (26).

Effects of pH and temperature on polar lipid composition in T. acidophilum HO-62.TPL1 and TPL2 extracted from T. acidophilum grown at different pHs and temperatures were analyzed by ELSD-HPLC. A typical chromatogram is shown in Fig. 4. The peaks observed with the chromatogram have been identified previously (26), and the structures of polar lipids are summarized in Fig. 1. The retention time of each component is also listed in Fig. 1. GuCGp, a main polar lipid of T. acidophilum, eluted at 22.5 min. MGuCGp and DMGuCGp, phosphoglycolipids in which one and two mannose residues, respectively are attached to gulose of GuCGp, eluted at 38.3 and 47.3 min, respectively. Polar lipids with more polar residues tended to elute at later times.

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FIG. 4.

HPLC chromatogram of TPL1, the chloroform extract obtained from T. acidophilum grown in medium at pH 1.2. Figure 1 shows the abbreviations and structures of the polar lipids. The asterisk (*) indicates the peak of an unknown component. HPLC was performed with a Capcellpak silica SG80 column (2.0 mm [inside diameter] by 250 mm [length]) and monitored by ELSD. The eluents used were H1, chloroform, and H2, chloroform-methanol-trifluoroacetic acid (50:50:1 [vol/vol/vol]). The gradient program was H2 %(min) 0(0)-10(2)-50(42)-100(62) at 35°C at a flow rate of 0.25 ml/min.

Polar lipids were quantified by using a calibration curve plotted with GuCGp as the standard (Fig. 5). Because the response of ELSD does not depend on the molecular structure but on the number and size of the analyte particles in a drift tube, the response is not expected to depend on the type of analyte (16). The relative deviation of the ELSD signal was 13% when eight different compounds of the same amount were tested, while that of the refractive index detector was 23% (16). The calibration curve obtained from ELSD is not always linear (5, 16), as was the calibration curve obtained in this study (Fig. 5). Glycolipids, phospholipids, and part of phosphoglycolipids were detected in TPL1 obtained from the first extraction using chloroform, methanol, and water. Phosphoglycolipids were found in the TPL2 obtained from the second extraction using chloroform-methanol (2:1 [vol/vol]). Since some lipids were found in both TPL1 and TPL2, the amounts of all the components in TPL1 and TPL2 were added. The weighted percentage of each polar lipid in the cells grown under different conditions is shown in Table 1.

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FIG. 5.

Calibration curves for GuCGp. Analytical conditions are described in Materials and Methods. The area under the peak of ELSD-HPLC was plotted against the weight of the standard GuCGp injected.

The amount of the main polar lipid GuCGp was low at low pHs and high temperatures. The amount of GuCGu showed a similar trend. However, the amounts of most of the glycolipids, namely, GuC, MGuC, MGuCGu, DMGuC, and TMGlC, and the sum of the amounts of the phospholipids CGp and AGp were high at low pHs and high temperatures. The other phosphoglycolipids, namely, MGuCGp, DMGuCGp, and TMGuCGp, showed a trend that was different from those of both types.

The general trends in the polar lipid compositions against pH and temperature can be seen in Table 2. The total amount of phosphoglycolipids was low at low pHs and high temperatures, whereas the total amounts of glycolipids and phospholipids were high at low pHs and high temperatures. The relative amounts of the polar lipids with zero to one sugar unit were low at low pHs and high temperatures, whereas those of the polar lipids with two to four sugar units were high under these conditions.

Chemical stability of GuCGp.GuCGp was exposed to water adjusted to pH 1.8 with sulfuric acid and incubated at 56°C for 3 days. These are the optimal growth conditions for T. acidophilum HO-62. Since GuCGp has a sugar moiety and a phosphate moiety on both ends of caldarchaeol, the differences in the chemical stabilities of these polar residues can be tested. The HPLC profiles before and after the incubation are shown in Fig. 6. GuCGp was decomposed mainly to GuC under these conditions. A phosphoester linkage was unstable under these conditions, whereas a sugar moiety was stable. The ether linkages in the caldarchaeol were also stable under these conditions.

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FIG. 6.

Hydrolysis of GuCGp. The chromatograms were obtained from the GuCGp that was exposed to distilled water adjusted to pH 1.8 and incubated at 56°C for 3 days. Panels A and B show the chromatograms before and after incubation, respectively.

Proton permeability of liposomes.Proton permeability was investigated by monitoring the internal pH of two liposomes made of Fr. CM (consisting of glycophospholipids containing one sugar unit) and Fr. M (consisting of glycophospholipids containing two to four sugar units). After the size of the liposome was adjusted to 0.1 μm, external pyranine of the liposomes was removed by dialysis, using a pyranine-free buffer. Pyranine is a pH-sensitive fluorescence compound. The lipid compositions of Fr. CM and Fr. M before and after the preparation of liposome were investigated using a thin layer chromatography (26). Lipid composition did not change significantly during the preparation of liposome for both liposomes made of Fr. CM and Fr. M (see Fig. S1 in the supplemental material). The calibration curve obtained from the pH standard solutions (pHs 5.0, 5.5, 6.0, 7.0, and 7.5) was linear (y = 0.8011 x = 5.5806, r² = 0.999). Fluorescence was measured after the liposome suspension was mixed with the buffer at pH 3.6. When the liposome suspension (60 μl; pH 7.0) was mixed with the bulk of the buffer (540 μl; pH 3.6), the solution pH was about 4.6. Figure 7 shows the internal pHs of the liposomes made of Fr. CM and the liposomes made of Fr. M. The internal pHs of the liposomes decreased after the suspension was mixed with the bulk of the buffer. The pH-decreasing speed of the liposomes made of Fr. M was lower than that of the liposomes made of Fr. CM. This result shows that the proton permeability of the liposomes made of phosphoglycolipids that contained two or more sugar units was lower than that of the liposomes made of phosphoglycolipids that contained one sugar unit.

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FIG. 7.

Internal pH of liposomes obtained from different phosphoglycolipid fractions. Fluorescence was measured after 60 μl of the liposome suspension (pH 7.0) was mixed with 540 μl of the buffer (pH 3.6). The ratio of emission intensities at 510 nm with excitation at 460 and 400 nm, I₄₆₀/I₄₀₀, was plotted. The internal pH of liposomes was estimated from the calibration curve (as described in Results). Closed circles indicate the liposomes obtained from Fr. CM, consisting of GuCGp. Open circles indicate the liposomes obtained from Fr. M, consisting of MGuCGp, DMGuCGp, and TMGuCGp. Each bar indicates the means ± standard deviations (n = 6). Unpaired Student's t test was used to determine the significance between the liposomes made of Fr. CM and Fr. M. Asterisks indicate a significant difference between corresponding data (P < 0.05).