JOURNAL OF BACTERIOLOGY, Dec. 1972, p. 1193-1200Copyright O 1972 American Society for Microbiology

Vol. 112, No. 3Printed in U.S.A.

Lipids of Thermoplasma acidophilumTHOMAS A. LANGWORTHY, PAUL F. SMITH, AND WILLIAM R. MAYBERRY

Department of Microbiology, School ofMedicine, University ofSouth Dakota, Vermillion, South Dakota 57069

Received for publication 22 August 1972

Cells of Thermoplasma acidophilum contain about 3% total lipid on a dryweight basis. Total lipid was found to contain 17.5% neutral lipid, 25.1% glyco-lipid, and 56.6% phospholipid by chromatography on silicic acid. The lipidscontain almost no fatty acid ester groups but appear to have long-chain alkylgroups in ether linkages to glycerol. The phospholipid fraction includes a majorcomponent which represents about 80% of the lipid phosphorus and 46% ofthe total lipids. We believe this component to be a long-chain isopranol glyceroldiether analogue of glycerolphosphoryl monoglycosyl diglyceride. The glycolip-ids appear to contain isopranol diether analogues. Several components of thecomplex, neutral lipid fraction have been identified as hydrocarbons, vitaminK2-7, and isopranol glycerol diether analogues. Sterols are present in the neu-tral lipids but do not appear to be synthesized by the organism.

Recently, Darland et al. (5) have reported onthe isolation and identification of Thermo-plasma acidophilum, an obligate thermophilic,acidophilic mycoplasma. The organism growsaerobically at a temperature optimum of 59 C.It grows only very slowly at 45 C, and does notgrow at 70 C. The organism grows over a pHrange of 1 to 4, with an optimum of pH 2. Wepresent here some initial observations on theunusual nature of the lipids of this organism.

MATERIALS AND METHODSGrowth of cells. T. acidophilum was provided by

M. Norman through the American Type CultureCollection. The organism was grown in a medium (5)containing 0.02% (NH4)2S04, 0.05% MgSO4, 0.025%CaCl2 2H2O, 0.3% KH2PO4, and 0.1% yeast extract(Difco). The pH was adjusted to 2 with 10 N H2SO4.After the medium was autoclaved, glucose was addedto a final concentration of 1%. The inoculum con-sisted of 10% (v/v) of a 24-hr culture in the samemedium. Incubation was carried out with shaking at59 C in 3-liter flasks, each containing 2 liters ofmedium. After 24 hr, cultures were brought to roomtemperature in the cold room and concentrated in aSharples centrifuge. Cells were sedimented andwashed two times with distilled water at 20,000 x gfor 15 min in a Sorvall RC-2B centrifuge and finallylyophilized. Under these conditions, the organismnormally yielded approximately 50 mg (dry weight) ofcells per liter of medium. Yields varied dependingupon the lot of yeast extract (Difco) used.

For labeling studies, 50 ml of medium in 125-mlflasks containing either 50 ACi of [2-'4C]acetate or 5uCi of [2-14C]mevalonic acid dibenzylethylendia-mine salt was inoculated with 5 ml of a 24-hr culture.When "5S-sulfate or 32P-orthophosphate was used, themedium was modified to avoid dilution of the isotope.

For 35S, 50 ml of medium containing chloride in placeof sulfate salts was adjusted to pH 2 with HCl. Thismedium containing 800 uCi of 3"S-sulfate was inocu-lated with 3 ml of a 24-hr culture grown in thestandard medium. For 32P, 50 ml of medium withoutKH2PO4 but containing 1 mCi of 32P-orthophosphatewas inoculated with 3 ml of a 24-hr culture. Radio-isotopes were a product of New England NuclearCorp.

Extraction of lipid. Lipids were extracted fromlyophilized cells or from washed, labeled pellets bystirring with chloroform-methanol (2: 1, v/v) at roomtemperature for 60 min. After evaporation to dryness,the lipid was passed through Sephadex G25 for theremoval of nonlipid contaminants (24).Chromatography. Silicic acid columns (2 by 8

cm) were prepared from Unisil (100-200 mesh; Clark-son Chemical Co., Inc., Williamsport, Pa.). Lipidswere eluted with 100 ml of chloroform (neutral lipids),100 ml of acetone (glycolipids), and 150 ml of meth-anol (phospholipids). Labeled lipids were frac-tionated on columns (1 by 2 cm) by using one-tenthsolvent volumes.

General purpose thin-layer chromatography (TLC)was carried out on 0.25-mm layers of Silica Gel Hactivated by heating at 105 C for 30 min unlessotherwise stated. Solvents for phospholipids includedchloroform-methanol-water (65:25:4, v/v) (18) andchloroform-methanol-acetic acid-water (100:20:12:5, v/v) (10). Glycolipids were examined inchloroform-methanol (9:1, v/v) (18). Neutral lipidswere examined in the two-step solvent system isopro-pyl ether-acetic acid (96:4, v/v) followed by n-hex-ane-diethyl ether-acetic acid (90:10:1, v/v) (19).Chloroform-diethyl ether (9:1, v/v) was used forglycerol ethers (11). For preparative TLC, bands werescraped and eluted through sintered-glass filters withchloroform-methanol (2:1, v/v) for lipids or diethylether for glycerol ethers.

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Lipid components were located by iodine vapor,Rhodamine 6G, and 50% methanolic-sulfuric acid.The latter also was used to observe the characteristicsequence of colors given by sterols after short periodsof heating. Specific sprays included 0.2% ninhydrin inwater-saturated n-butanol for amines; the reagent ofVaskovsky and Kostetsky (23) or Hanes and Isher-wood (9) for phosphorus; periodate-Schiff for vicinalglycols (3); phenol-sulfuric (8) or diphenylamine (19)for glycolipids; and sodium borohydride-neotet-razolium spray for quinones (25). Radioactive com-pounds were located by radioautography by usingKodak Royal Blue X-ray film.

Paper chromatography of water-soluble hydrolysisproducts was carried out on Whatman no. 1 paper byusing the solvent n-propanol-concentrated aqueousammonia-water (6:3: 1, v/v).Gas chromatography. A Biomedical gas chroma-

tograph (Hewlett-Packard, F and M model 402)equipped with a flame ionization detector and a3370A digital electronic integrator was used. Allqualitative and quantitative analyses were done oneither a glass column (1.83 m by 0.6 mm) packed with5.5T SE-30 on 80- to 100-mesh Gas Chrom Q (Ap-plied Science Laboratories, State College, Pa.) or on astainless-steel column (1.83 m by 0.6 mm) packedwith 7(' OV-17 (Ohio Valley Specialty Chemical Co.,Marietta. Ohio) on Gas Chrom Q. Long-chain al-kanes. alcohols. and alkyl chlorides were run at anoven temperature of 325 C. In some instances, sam-ples were run isothermally at 170 C for 45 minfollowed by temperature programming at 5 C/min to325 C. Carbohydrate derivatives were run at an oventemperature ot 175 C. Samples were injected directlyon column. Detector temperatures were 50 C higherthan oven temperature. Carrier gas (Helium) flow ratewas 60 ml/min. Corrections for molar response factorswere determined empirically. For the preparation oftrimethvlsilvl (TMS) derivatives, the silylation mix-ture of Carter and Gaver was used (4). Treatmentwith 10%' (v/v-) perchloric acid in methanol at 55 C for15 min was used to esterify free fattv acids.

Hydrolyses. All hvdrolyses were carried out insmall Teflon-lined screw-cap tubes with samplesevaporated under nitrogen. Strong base hydrolysiswas carried out in 1 N NaOH at 100 C for 1 hr. Mildalkaline methanolvsis was carried out in either so-dium methoxide (15) or by the method of Dittmer andWells (7). When it was found that the lipid wasresistant to mild alkaline methanolysis, the followingacid methanolvsis was used (12). Samples in 2.5%7methanolic-hydrochloride were heated at 100 C for 5hr. A 10%, volume of water was added, and theunsaponitiable material was extracted three timeswith an equal volume of n-hexane. For chromatogra-phy of phosphate ester, the methanol-water phase wastaken to dryness over KOH pellets, 0.2 N NaOH wasadded to the residue and hydrolyzed for 2 hr at 100 C.The base was neutralized with Dowex-50 (Hi), andsamples were taken for chromatography. Glycolipidwas hydrolyzed in 2.5% methanolic-hydrochloride at100 C tor 83 hr. taken to dryness under a stream ofnitrogen, and hydrolyzed for another 2 hr in 2 N HCIat 100 C. The HCI was removed under nitrogen, andthe samples were dried over KOH pellets.

Degradation of glycerol ethers. Hydrolysis ofglycerol ethers was conducted in 57% hydriodic acid(12, 14). Samples were heated under reflux for 24 hr,cooled, and extracted three times with three volumesof n-hexane. Alkyl iodides contained in the n-hexaneextract were washed once with one-third volume of10% NaCl, a saturated solution of K2COS, and finally50% Na2S2O3. Alkyl iodides were converted to thealkanes by refluxing with zinc in acetic acid for 3 hr(14). Alkyl iodides were converted to the acetates byrefluxing with silver acetate in acetic acid for 24 hr(12). Alcohols were prepared from the acetates byhydrolysis in 0.2 N NaOH at 100 C for 2 hr (12). Forthe estimation of glycerol, glycerol ethers in screw-cap tubes with Teflon liners were treated with liqui-fied boron-trichloride in chloroform (1: 1, v/v) at roomtemperature for 24 hr to release glycerol and alkylchlorides (12). After evaporation of the boron-trichlo-ride-chloroform mixture, the sample was partitionedbetween n-hexane and water, and the two phases wereexamined.

Analytical methods. Phosphorus was determinedby the method of Ames (1) or following fractionationby TLC by the method of Rouser et al. (17). Glycerolpresent in the aqueous phase from acid methanolysiswas assayed after an additional hydrolysis in 6 N HClat 100 C for 72 hr. Glycerol was determined eitherenzymatically (26) or colorimetrically after periodateoxidation (22). Carbohydrate was estimated by theanthrone (6) or phenol-sulfuric acid (2) procedures.Alkanes and alkyl chlorides were quantitativelv es-timated by gas-liquid chromatography (GLC) withn-dotriacontane or 1-chloro-octacosane as internalstandards. Lipid content was determined gravimetri-cally after drying to a constant weight at roomtemperature under a stream of nitrogen. Radioactivesamples were counted in a Packard Tri-Carb liquidscintillation spectrometer. Infrared spectra were ob-tained on a Beckman model IR 18A instrument withsamples spread as films between sodium chlorideplates. Ultraviolet spectra were obtained from sam-ples in n-hexane on a Beckman DK-2A scanningspectrophotometer.

Materials. All materials and standards were thebest grade commercially available.

RESULTSLipid composition. Total lipid extracted

from T. acidophilum accounted for 3.1% of thecells on a drv weight basis. The lipid was furtherfractionated on columns of silicic acid (Table1). Neutral lipids eluted with chloroform ac-counted for 17.5%. of the total lipids; glyco-lipids eluted with acetone, 25.1%; and phos-pholipids eluted with methanol, 56.6'%. Asecond extraction of the chloroform-methanol(2: 1, v/v)-extracted cell residue with chloro-form-methanol (2:1, v/v), adjusted to 0.1 Nwith respect to HCI, released another 0.5% oflipid containing 19.3% neutral lipids, 59.4%glycolipids, and 21.1% phospholipids. Each ofthe lipid classes obtained by the acidic extrac-tion contained the same components as the

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TABLE 1. Lipid composition of T. acidophiluma

F Per cent cell Per centFraction j dry weight total lipid

Total lipid .......... 3.1 100Neutral lipid ........ 0.54 17.5Glycolipid .......... 0.79 25.1Phospholipid ........ 1.75 56.6

a Average of three determinations. Lipid was frac-tionated on columns (2 by 8 cm) of silicic acid with100 ml of chloroform (neutral lipid), 100 ml of ace-tone (glycolipid), and 150 ml of methanol (phospho-lipid). Lipid percentages were determined gravi-metrically.

original neutral extraction when examined byTLC.Phospholipids. TLC of the phospholipid

fraction developed in chloroform-methanol-water (65:25:-4. v/v; Fig. 1) or in the two-dimensional system chloroform-methanol-water (65:25:4, v/v) followed by chloroform-methanol-acetic acid-water (100:20:12:5, v/v)(Fig. 2) indicated the presence of at least eightcomponents by charring with sulfuric acidspray. Phospholipids were examined by usingvarious labeled precursors. Distribution of thelabeled precursors between the neutral lipid,glycolipid, and phospholipid classes is illus-trated in Table 2. Phospholipids were examinedby autoradiography after one-dimensional andtwo-dimensional TLC. All of the components,except A, incorporated 32P, whereas none incor-porated 35S. All of the components incor-porated [2-'4C]acetate, and all except compo-nent B incorporated [2-'4C]mevalonate, sug-gesting that the phospholipids contain iso-prenoid-like components. Treatment of thephospholipid fraction with sodium methoxideat room temperature for 1 hr (15) or methanolic-potassium hydroxide at 37 C for 30 min (7) didnot release any water-soluble phosphate estersor fatty acids. The phospholipids remainedunaltered after saponification when examinedby TLC. Resistance to saponification suggestedthe presence of ether linkages. The stainingcharacteristics and distribution of phospholipidphosphorus are illustrated in Table 3.Characterization of component D. The

major phospholipid, component D, accountedfor 80.5% of the phospholipid phosphorus and45.9% of the total lipid from the cells. It wasrapid periodate-Schiff and diphenylamine posi-tive, and ninhydrin negative. Component Dwas isolated by preparative TLC after develop-ment in chloroform-methanol-acetic acid-water(100:20:12:5, v/v) (RF 0.29).The absence of fatty acid ester groups was

confirmed by the infrared absorption spectrumof the isolated phospholipid. No ester absorp-tion could be detected in the 1,740 to 1,750cm-' range. The infrared showed OH absorp-tion at 3,340 cm-- '; typical alkyl group absorp-

||||SF

A

BC

D

E

FIG. 1. Radioautograph of the phospholipids fromT. acidophilum. Organisms were grown in the pre-sence of 1 mCi of 32P-orthophosphate/50 ml for 24 hr.The thin-layer chromatogram was developed in chlo-roform-methanol-water (65:25:4, v/v). Letter A indi-cates the RF of material that does not incorporate 32pbut is detected in the phospholipid fraction bycharring with 50% methanolic-sulfuric acid.

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TABLE 3. Staining reactions of phospholipids onthin-layer platesa

SF

t

FIG. 2. Radioautograph of the phospholipids fromT. acidophilum. Organisms were grown in the pre-sence of 1 mCi of 32P-orthophosphate/50 ml for 24 hr.The thin-layer chromatogram was developed in thefirst dimension with the solvent chloroform-methanol-water (65:25:4, v/v). The solvent in thesecond dimension was chloroform-methanol-aceticacid-water (100:20:12:5, v/v). LetterA indicates theRF of material that does not incorporate 32P but isdetected in the phospholipid fraction by charringwith 50% methanolic-sulfuric acid.

TABLE 2. Distribution of various precursorsincorporated into lipid classes of T. acidophilum

Per cent distributionbPrecursor supplieda Neutral Glyco- Phospho-

lipid lipid lipid

[2-l4C]Acetate ........ 13 7 79[2-14C]Mevalonate ..... 18 5 763201pOrthophosphatec.0.. 1035S-Sulfatec ........... 0 0 0

a Fifty-milliliter cultures containing 50 AsCi of [2-"4Cjlacetate, 5 jCi of [2-"4C]mevalonate, 1 mCi of32P-orthophosphate, or 800 ACi of 3"S-sulfate wereextracted after 24 hr.

I Lipid was fractionated on silicic acid columns (1by 2 cm) with 10 ml of chloroform (neutral lipid), 10ml of acetone (glycolipid), and 15 ml of methanol(phospholipid).

c The medium was modified to avoid isotope dilu-tion, as described in Materials and Methods.

tion at 2,960, 2,925, 2,860, 1,460, and 1,375cm-'; P-O absorption at 1,235 cm-'; etherabsorption at 1,105 cm-'; and P-O-C absorp-tion at 1,055 cm- '.

Hydrolysis of component D in 2.5% meth-anolic-hydrochloride at 100 C for 5 hr re-leased an unsaponifiable n-hexane-soluble ma-

Staining reactions

Component ~~~Perio- P(%ofComponent Phos- Ninhy- Diphen- ttdatetoalphate drin ylaine d recov -Scif erv)'

A - - + Schif

B + - + 1.8C + 8.6D + + + 80.5E + + + + 4.4F + +1+ + 3.6c

H + + I + + 1.1

aThin-layer chromatography on Silica Gel Hplates, developed in chloroform-methanol-water (65:25:4, v/v).

I Lipids separated by two-dimensional TLC devel-oped in chloroform-methanol-water (65:25:4, v/v)followed by chloroform-methanol-acetic acid-water(100:20:12:5, v/v). Phosphorus was determined asdescribed by Rouser et al. (17). Total recovery oflipid phosphorus was 96.7%.

c Component F and G determined together.

terial and a methanol-water-soluble materialwith a phosphorus: glycerol: carbohydrate ratioof 1:1:1.01.TLC of the n-hexane-soluble unsaponifiable

material developed in chloroform-diethyl ether(9: 1, v/v) gave one major spot (RF 0.49) whichmigrated faster than the glycerol monoethers,batyl (RF 0.19), chimyl (RF 0.15), or selachyl (RF0.22) alcohols. The material was periodate-Schiff negative, whereas the glycerol monoeth-ers were positive, indicating the absence ofvicinal hydroxyl groups.The infrared spectrum of the material was

examined after preparative TLC in chloroform-diethyl ether (9: 1, v/v). The infrared spectrumshowed OH absorption at 3,450 cm '; alkylabsorption at 2,960, 2,929, 2,860, 1,460, and1,375 cm-'; C-O-C absorption at 1,115 cm-';and alcohol C-O absorption at 1,045 cm- ' in therange of a primary alcohol.A portion of the unsaponifiable ether was

treated with liquified boron trichloride in chlo-roform (1:1, v/v) to release polyol and alkylchlorides (12). After 24 hr at room temperature,boron trichloride was evaporated under nitro-gen, and n-hexane was added, followed by anequal volume of water. TLC of a sample of thewater phase on plates developed in chloroform-methanol-formic acid (65:25: 10, v/v) gave onerapid periodate-Schiff-positive spot (RF 0.55)identical to glycerol. Enzymatic quantitativeestimation of glycerol in the water phase and

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estimation of the n-hexane-soluble alkyl chlo-rides by GLC with 1-chlorooctacosane as theinternal standard gave a glycerol to alkyl chlo-ride ratio of 1: 2.1. The unsaponifiable materialappears to be a 1, 2-substituted glycerol dietheranalogue.

Hydrolysis of the diether by refluxing the 57%hydriodic acid for 24 hr released the alkyliodides. The alkyl iodides were either reducedto the alkanes by refluxing with zinc in aceticacid for 2 hr (14) or converted to the acetates byrefluxing with silver acetate in acetic acid for 24hr. The acetates were finally converted to thealcohols by hydrolysis in 0.2 N NaOH at 100 Cfor 2 hr (12). The alkanes and TMS derivativesof the alcohols from the diether were examinedby GLC on columns of SE-30 or 7% OV-17 at325 C. Both derivatives indicated two majorlong-chain alkyl components in the ratio of 2:1,representing 80% of the alkyl side chains. Theequivalent chain lengths of the two majoralkane derivatives were 35.2 and 36.7 on SE-30,and 34.6 and 36.6 on OV-17 columns. Theremaining 20% of the alkyl derivatives ap-peared to consist of a complete series of comnpo-nents between C,8 and C,9, differing by onecarbon. Analysis of the mass spectrum of thetwo major alkane derivatives showed molecularions with m/e 560 and 562 corresponding tomolecular formulas of C,4oH80 and C40H8, in theratio of 2: 1.The methanol-water phase from the acid

methanolysis of component D was evaporatedto dryness under a stream of nitrogen and driedin vacuo over KOH pellets. The material wasthen hydrolyzed in 0.2 N NaOH at 100 C for 2hr, neutralized with Dowex-50 (H+), and ex-amined by paper chromatography developed inn-propanol-concentrated ammonia-water(6:3:1, v/v). One major spot (RF 0.35) waspresent identical with glycerolphosphatetreated under the same conditions. Hydrolysisof the phosphate ester in 6 N HCl at 100 C for 72hr gave a glycerol to phosphorus ratio of 1: 1.01.

Hydrolysis of the dried methanol-water phasein 2 N HCI at 100 C for 3 hr followed bypreparation of the TMS derivatives showedglucose as the major carbohydrate when ex-amined by GLC.

Hydrolysis of component D in 1 N NaOH at100 C for 1 hr released a chloroform-solublematerial which migrated as glycolipid compo-nents I and J when examined by TLC deve-loped in chloroform-methanol (9: 1, v/v). The.major phospholipid component D appears to bean isopranol glycerol diether analogue ofglycerolphosphoryl monoglycosyl diglyceride.Minor phospholipids. The minor compo-

nents present in the phospholipid fraction re-main essentially unidentified due to the smallamounts of material available for assay. Com-ponent A was diphenylamine positive and slowperiodate-Schiff positive and ninhydrin andphosphorus negative, probably a glycolipid.Component B, containing 1.8% of the lipidphosphorus, was rapid periodate-Schiff posi-tive, diphenylamine negative, and ninhydrinnegative. It incorporated [2-'4C]acetate, butnot [2-'4C]mevalonate. Component C, contain-ing 8.6% of the lipid phosphorus, was rapidperiodate-Schiff positive. diphenylamine nega-tive, and ninhydrin negative. Components E, Fand G, and H representing 4.4, 3.6, and 1.1%,respectively, of the lipid phosphorus, were allrapid periodate-Schiff positive, diphenylaminepositive, and ninhydrin positive. Hydrolysis ofthe minor phospholipids in 2.5% methanolic-hydrochloride at 80 C for 24 hr did not releaseany long-chain base. All of the componentsreleased n-hexane-soluble ethers which mi-grated as the glycerol diether found in compo-nent D when examined by TLC. The amountsof methanol-water-soluble material released byacid methanolysis were too small for analysis.

Glycolipids. TLC of the glycolipid frac-tion developed in chloroform-methanol (9:1,v/v; Fig. 3) revealed six components. Com-ponents I (RF 0.72) and J (RF 0.65) migratedfaster than authentic monoglycosyl diglyc-eride (RF 0.55). Component K (RF 0.52) mi-grated similar to monoglucosyl diglyceride,whereas components L, M, and N (RF 0.27, 0.22,0.18, respectively) migrated similar to digluco-syl diglyceride (RF 0.21). Each of the compo-nents I through N were diphenylamine, phenol-sulfuric, and slow periodate-Schiff positive andninhydrin and phosphorus negative. All com-ponents incorporated [2-' 4C ]acetate and[2- 4C]mevalonate, but not 32P or 35S. Meth-anolysis of the glycolipid fraction in 0.2 Nsodium methoxide at room temperature for 1 hrdid not release any fatty acid esters. Thechromatographic mobility after methanolysisremained unaltered when examined by TLC.Components I, J, and K were separated, andcomponents L, M, and N were taken togetherby preparative TLC after development in chlo-roform-methanol (9:1, v/v). Each of the glyco-lipid fractions was hydrolyzed in 2.5% meth-anolic-hydrochloride at 100 C for 3 hr. Each ofthe component fractions released a n-hexane-soluble unsaponifiable material. When ex-amined by TLC in chloroform-diethyl ether(9:1, v/v), the unsaponifiable material fromeach glycolipid component migrated (RF 0.49)identical to the glycerol diether identified in

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SF.::..

I

Jv

K

L

M

N

FIG. 3. Radioautograph of the glycolipids from T.acidophilum. Organisms were grown in the presenceof 50 ,Ci of [2-"4C]acetate/50 ml for 24 hr. Thethin-layer chromatogram was developed in chloro-form-methanol (9:1, vlv).

phospholipid component D.The methanol phase of the acid hydrolysis

from each fraction was evaporated under nitro-gen and further hydrolyzed in 2 N HCl at 100 C

for 2 hr. Carbohydrates were examined as theTMS derivatives by GLC. All of the glycolipidfractions contain glucose, except component Iwhich contains an unidentified sugar. The gly-colipid appears to consist of isopranol glyceroldiether analogues of glycosyl diglycerides.Neutral lipids. Neutral lipids were ex-

amined in the two-step solvent isopropyl ether-acetic acid (96:4, v/v) followed by n-hexane-diethyl ether-acetic acid (90: 10: 1, v/v; Fig. 4).All of the components except component Uincorporated [2-'lC ]acetate and [2- '4C ]mevalo-nate. Treatment of the neutral lipids with 0.2N sodium methoxide at room temperaturefor 1 hr did not alter the chromatographicmobility of any of the components. The com-ponents 0, P, U, and V have been identifiedafter preparative TLC in the two-step solventsystem. Components Q, R, S, T, and W re-main unidentified.Component 0 contains a series of 3% to 5%

each of C,4 through C,2 hydrocarbons as es-timated by GLC.Component P appeared as a yellow band on

TLC plates and gave a rapid pink color indica-tive of naphthoquinones when examined withthe sodium borohydride-neo-tetrazolium spraydescribed by White (25). The ultraviolet absorp-tion spectrum of component P in n-hexane gaveabsorption maxima at 325, 269, 260, 248. and243 nm which was identical with that of vita-min K,-7. Upon reduction with sodium borohy-dride in methanol (13) both gave maxima at 330and 246 nm. Reverse-phase TLC was run onplates of Silica Gel H impregnated with 5%paraffin oil (16). Plates developed in acetone-water (9: 1, v/v) gave only one spot (RF 0.37)identical to vitamin K,-7.

After the TLC plates were heated briefly andsprayed with 50% methanolic-sulfuric acid,component U gave a red spot (RF 0.61) thatmigrated identical to cholesterol. Examinationof the isolated component U as the TMSderivative by GLC showed cholesterol and a C,,sterol in the ratio of 1: 2. When cells werelabeled with [2-'4C]acetate or [2-"C]mevalo-nate, however, radioactivity was not incor-porated into the sterol component, as deter-mined by autoradiography. There is doubt,therefore, that the sterols are synthesized bythe organism.Component V migrated as the glycerol

diether released from the glycolipids or phos-pholipids when chromatographed by TLC inthe neutral lipid solvent (RF 0.55) or chloro-form-diethyl ether (9: 1, v/v) (RF 0.49). Compo-nent V appears to be 1, 2-substituted isopranolglycerol diether analogue.

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DISCUSSIONThe lipids of T. acidophilum appear to con-

tain ether linkages and an almost completeabsence of ester-bound fatty acids. Of the totallipids almost 46% is accounted for by the majorphosphoglycolipid, component D. Upon acidmethanolysis, component D yielded glycerol-phosphate and carbohydrate in the ratio of 1:1and a 1,2-substituted long-chain diether ofglycerol. Analysis of the major glyceryl dietherside chains as the alkane derivatives by massspectrometry indicated hydrocarbons with amolecular weight of 560 and 562 accounted forby the formulas C4,0H80 and C4oH82. Compo-nent D incorporated [2- '4C]mevalonate, in-dicating isoprenoid branching. No double bondabsorption was apparent in the infrared spec-trum. These 0-alkyl side chains appear toresemble some type of isopranol derivatives.Since acid methanolysis of component D re-leased glycerolphosphate and carbohydrate inthe ratio of 1:1, and strong base hydrolysisreleased a glycolipid, it is reasonable to assumethe major phospholipid of T. acidophilum is anisopranol glycerol diether analogue of glycerol-phosphoryl monoglycosyl diglyceride.The other small amounts of phospholipids

and glycolipid components also appear to con-tain similar isopranol diether analogues. Sul-folipids do not appear to be present since 3Swas not incorporated into any components.Cholesterol and C29 sterol were found in theorganism as in other Mycoplasma (20, 21).However, since the sterols did not incorporate[2-'4C]acetate or [2-l4C]mevalonate, it is un-likely these sterols are synthesized by theorganism.The lipids of T. acidophilum resemble the

glycerol glycolipids and phosphoglycolipidsfound in other Mycopklsma (20, 21). The glyce-rol lipids contain ether linkages instead of esterlinkages which may be a response of the organ-ism to the acidic environment, whereas thepresence of long-chain isopranols may be re-lated to the thermostability of the organism. Itis interesting that these lipids resemble thosefound in the extreme halophile, Halobactercutirubrum, which has responded to its extremeenvironment by the formation of phosphatidescontaining dihydrophytyl glycerol diether ana-logues (12).A more detailed study is in progress to

confirm the structure of the major phospho-lipid, as well as elucidation of the other lipidcomponents of T. acidoohilum.

ACKNOWLEDGMENTSThis work was supported by Public Health Service grant

AI-04410-11 from the National Institute of Allergy andInfectious Diseases.

_____SFa 0L

w

FIG. 4. Radioautograph of the neutral lipids fromT. acidophilum. Organisms were grown in the pre-sence of 50,gCi of [2-'4C]acetate/50 ml for 24 hr. Thethin-layer chromatogram was developed a distance of10 cm with the solvent isopropyl ether-acetic acid(96:4, v/v) followed by the solvent n-hexane-diethylether-acetic acid (90:10: 1, v/v) in the same dimen-sion and developed a distance of 15 cm.

The authors gratefully acknowledge the help of Ralph T.Holman of the Hormel Institute, Austin. Minn., who per-formed the mass spectral analysis.

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LANGWORTHY, SMITH, AND MAYBERRY

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