Separation and analysis of free ceramides containing 2-hydroxy fatty acids in Sphingobacterium species

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1 FEMS Microbiology Letters 20 (1983) Published by Elsevier Separation and analysis of free ceramides containing 2-hydroxy fatty acids in Sphingobacterium species (Bacterial sphingolipid; ceramide; d-iso 17 : 0 sphinganine; 2-hydroxy isopentadecanoic acid) Ikuya Yano, Sadao Imaizumi, Ikuko Tomiyasu * and Eiko Yabuuchi ** Department of Bacteriology, Niigata University School of Medicine, Niigata - shl Asahimachidori- 1, Niigata 951; * Tezukayama Junior College, Nara- shi, Gakuen , and ** Department of Bacteriology, Gifu University School of Medicine, Gifu- shi, Tsukasa- chyo, Gifu 500, Japan Received 12 August 1983 Accepted 31 August SUMMARY The occurrence of free ceramides was shown in the chloroform-methanol extractable lipids of 16 strains of Sphingobacterium including three species: S. versatilis, S. muhivorum and S. mizutae. The predominant long-chain base was identified as a branched-chain, saturated dihydroxy base with a carbon chain consisting of 17 carbon atoms, while the most abundant fatty acid was 2-hydroxy-13- methyltetradecanoic acid. The major molecular species of the intact ceramides were identified as LCB-d-iso-17 : O-2-OHiso-15 : 0FA, LCB-d-iso- 17 : O-iso-15 : 0FA and LCB-d-nl6 : O-iso- 15 : 0FA. 2. INTRODUCTION Sphingolipids are already established to be important components in membrane lipids of higher organisms. However, they are extremely rare in bacterial cells and reported to occur only in limited genera [1-6]. Previously, we have reported that strains of Pseudomonas such as group II-K type 2, a Gram-negative, aerobic, opportunistic pathogen, contained sphingophospholipids [7,8] and there- fore, Yabuuchi et al. [9] placed the organism in a new genus. Since the presence of sphingophospholipids in bacteria was very characteristic, we proposed new names, Sphingobacterium gen. nov., composing three separate species; biovar 1, S. versatilis (including KM2138), biovar 2, S. multivorum (including KM926) and biovar 3, S. mizutae (including KM1203) [10]. The present paper describes the ceramide molecular species composition of cellular lipids of 16 representative strains including three Sphingobacterium species. 3. MATERIALS AND METHODS Seven strains of S. versatilis, five strains of S. multivorum and four strains of S. mizutae were grown aerobically in a medium containing 1% polypeptone, 0.5% yeast extract and 1% glucose with ph 7.0. After 30 h incubation with shaking at 30 C, the cells were harvested and the lipids were extracted with 20 vols. of chloroform-methanol (2:1, by vol.). The lipids were separated on a thin-layer plate (0.25 mm thick) of Silica gel (Analtek, U.S.A.) with solvent systems of (1) chloroform-methanol-acetic acid (100 : 10 : 5, by vol.) or (2) chloroform-methanol-acetic acid-water /83/$ Federation of European Microbiological Societies

450 (100 : 20 : 12 : 5, by vol.). The phospholipids were visualized by spraying with Dittmer's reagent and the long-chain bases with ninhydrin reagent, respectively.

2 450 (100 : 20 : 12 : 5, by vol.). The phospholipids were visualized by spraying with Dittmer's reagent and the long-chain bases with ninhydrin reagent, respectively. Mild alkaline hydrolysis of the extractable lipids was performed with 0.5 M KOH in chloroform-methanol (1:1, by vol.) for 2 h at room temperature with occasional shaking. Acid methanolysis was carried out with methanol-12 M HCI (5:1, by vol.) at 100 C for 3 h [11]. After cooling, an equal volume of water was added and the fatty acid methyl esters were extracted twice with n-hexane. The aqueous phase was then made to ph 12 with KOH and the long-chain bases were extracted twice with hexane-diethyl ether (1 : 1, by vol.). The ceramides were also prepared by HF hydrolysis from alkali-stable phospholipids, essentially according to the method of Shaw and Stead [12]. Infrared spectra were recorded with Hitachi Grating IR Spectrophotometer Model EPI-G3 (Hitachi Co. Tokyo) as KBr discs. Trimethylsilyl (TMS) derivatives of the long-chain bases (or intact ceramides) were prepared with pyridine-bistrimethylsilyltrifluoroacetamide (BSTFA) (1 : 2, by vol.) by heating at 70 C for 20 min. Gas-liquid chromatography of fatty acid methyl esters or TMS derivatives of long-chain bases was performed on a Hitachi 063 apparatus with a column of 5% SE-30 or 10% DEGS (3 mm l.0 m), essentially according to the method described in [18]. Gas-chromatographic and mass-spectrometric analyses of TMS-ceramides were carried out on a Hitachi M-60 GC/MS system equipped with 2% OV-1 glass column (3 mm 0.5 m) at a temperature ranging from 200 to 300 C. The mass spectra were recorded at an electron energy of 20 ev and accelerating voltage 3.2 ev. 4. RESULTS AND DISCUSSION Thin-layer chromatograms of the extractable lipids obtained from 16 strains of Sphingobacterium showed five or more spots with solvent system (1) (Fig. 1). The upper three spots (cer, X l and X2) were alkali-stable and not stained with Dittmer's reagent, while the Rf value of the lowest alkali-labile spot (PE), showing phosphorus positive, coincided with authentic phosphatidyl- eqeeet6., eeeteeeeeeeeotte ee.oooe6e:;;;:eeoo Ib '' $$ t,a,o,ttttttttttl!l!t-tt Z ~t Cmr. 2t ~f103 g L~ ~89~H 2i20 Fig. 1. Thin-layer chromatograms of extractable lipids from 16 strains of Sphingobacterium. Cer, ceramide; X 1 and X 2, unknown; PE, phosphatidylethanolamine; PLP, other phospholipids. Numbers of each lane indicate strain numbers of Sphingobacterium. 2134, 2138, 2148, 3101, 3102, 3103 and 3104 for S. versatilis; 926, 2087, 2532, 2812 and 2813 for S. multivorum and 1203, 2055, 2274 and 2289 for S. mizutae. Cer-OH, bovine ceramide containing 2:hydroxy fatty acid (standard). Plates were developed with a solvent system of chloroform-methanol-acetic acid (100 : 10 : 5, by vol.). ethanolamine from egg yolk. The 'cer' spot migrated essentially with authentic bovine brain ceramide containing 2-hydroxy fatty acids. Since we have already reported on the occurrence and structural analyis of long-chain bases in the genera, we tried to characterize the molecular species of the ceramides. The IR spectra of free ceramides isolated from thin-layer plates showed a very similar profile, possessing absorptions at 1650 and 1560/cm, indicating the presence of an amide linkage. After methanolysis of free ceramides, the long-chain bases and fatty acid methyl esters were obtained. They were identified as 15-methylhexadecasphinganine and 2-hydroxy-13-methyltetradecanoic acid, as major components. The gas chromatogram of TMS derivatives of ceramide showed the presence of 2 major and several smaller peaks, as demonstrated in Fig. 2. All 16 strains of Sphingobaeterium showed similar results on gas chromatograms. The mass spectra of the two major ceramides are reproduced in Fig. 3. In the case of the mass spectrum of peak No. 3, the M r is indi-

3 451 q~ u} CL u} 4J J,J Ib C'q O 2b r~, 3b TMS ceramide Sphingobacterium KM b (mini Fig. 2. Gas chromatograms of TMS derivatives of ceramides from S. versatilis (KM2138). 1, LCB-d-nl6:0-isol5 : 0FA, 2, LCB-d-nl6 : 0-2-OH isol5 : 0FA, 3, LCBd-isol7 : O- isol5 : 0FA, 4, LCB-d-isol 7 : O-2-OHisol5 : 0FA, 5, LCB-d-nl8:0-isol5 : 0FA and 6, LCB-d-nl8:0-2- OH isol5:0fa. X l and X z, unknown. Conditions for gas chromatography are described in the text. cated by mass ions at m/z 640 (M-15) and m/z 552 (M-103) formed by the elimination of a methyl group and the terminal-ch2osi(ch3) 3. 'LCB fragments' are shown at m/z 299 (a), being the base peak of the spectrum and indicating that the long-chain base is a heptadecasphinganine. The presence of a prominent ion at m/z 217 is also indicative of a sphinganine ceramide, but not the sphingosine ceramide series [14]. This was also indicated by the absence of an absorption band due to a trans double bond in the IR spectra (970/cm) of intact sphingophospholipids. The occurrence of a methyl branch in the alkyl chain of the sphinganine base (iso type) was confirmed from the relationship between log retention time and carbon number of TMS long-chain bases on gas chromatographic separation. On the other hand, 'fatty acid fragments' are observed at m/z 356 (M-a), m/z 429 (M-(a-73)) and m/z 267 (M-(a + 89)), formed by the cleavage between C 2 and C 3 with or without elimination of a methyl or TMS group. Therefore, the fatty acid linked to the long-chain base by amide linkage was identified as a pentadecanoic acid. From the gas chromatographic analysis, the C~5 fatty acid was identified as 13-methyltetradecanoic acid, exclusively. From these results, peak No. 3 ceramide was identified as N-13-methyltetradecanoyl 15-methylhexadecasphinganine. In the case of a mass spectrum of ceramide No. 4, M r fragments (M and M-103) and fatty acid fragments (Ma, M- (a-73) and M-(a + 89)) were shifted by + 88, although LCB fragment (a) was not changed. Furthermore, the occurrence of mass ion peak at m/z 285 indicated Ce~tilbil7LCEI) M-a ~29 M-103 M ,~ M ( IOO Cer (2-aqi15- b -89 M-103.E l iltlcb) 158 M -(0"7"5)/ L--i~,21~ M-IO] M-90 M-If 728 M I.I. I t b ioo ~ o0 m/z Fig. 3. Mass spectra of TMS derivatives of ceramides from Sphingobacterium versatilis (KM 2138). Upper, LCB-d-isol7 : O-iso15 : 0FA and lower, LCB-d-isol7:O-2-OHisol5:OFA ceramides. Conditions for mass spectrometry are described in the text.

4 452 z, 5 0 r, r~ ',I~P "~- t~',,~r"- e,-,

5 453 the presence of a hydroxyl group at position 2 of a saturated C15 fatty acyl moiety. Therefore, ceramide No. 4, the most abundant species of ceramide was identified as N-2'-hydroxy-13-methyltetradecanoyl-15-methylhexadecasphinganine. All ceramide molecular species are identified and summarized in Table 1. Similar species were obtained in the cases of free ceramides from all 16 strains of Sphingobacterium. The similar results were also demonstrated in the cases of ceramides obtained after HF hydrolysis of isolated sphingophospholipids. It was noted that the ceramide molecular species of bacterial sphingolipids were rather simple and composed of shorter-chain fatty acids and bases than animal ceramides [15,16]. However, in animal tissues, sphingolipids are usually associated with non-hydroxy or 2-hydroxy fatty acids. Since the fatty acids from bacterial ceramide were also shown to by hydroxylated, 3-hydroxy in Bacteroides and 2-hydroxy in Sphingobacterium [18], it seems likley there is common specificity for the biosynthesis of ceramide in both the animals and bacteria, especially in the selection of hydroxy fatty acids. The detailed structure and the physiological role of sphingophospholipids will be described in a subsequent paper. REFERENCES [2] Rizza, V., Tucker, A.N. and White, D.C. (1970) J. Bacteriol. 101, [3] Kunsman, J.E. (1973) J. Bacteriol. 113, [4] Tourtellotte, M.E., Jensen, R.G., Gander, G.W. and Morowitz, H.J. (1963) L Bacteriol. 86, [5] Plackett, P., Smith, P.F. and Mayberry, W.R. (1970) J. Bacteriol. 104, [6] Steiner, S., Conti, S.F. and Lester, R.L. (1973) J. Bacteriol. 116, [7] Yamamoto, A., Yano, I., Masui, M. and Yabuuchi, E. (1978) J. Biochem. 83, [8] Yano, I., Yamamoto, A., Ohno, Y., Masui, M. and Yabuuchi, E. (1977) Proceedings of Japanese Conference on the Biochemistry of Lipids (in Japanese) 19, [9] Yabuuchi, E., Yano, I., Kaneko, T. and Ohyama, A. (1981) in The Flavobacterium cytophaga Group (Reichenbach, H. and Weeks, O.B., Eds.), pp , Verlag Chemie, Weinheim. [10] Yabuuchi, E., Kaneko, T., Yano, I., Wayne Moss, C. and Miyoshi, N. (1983) Int. J. Syst. Bacteriol. (in press). [11] Sweeley, C.C. and Moscatelli, E.A. (1959) J. Lipid Res. 1, [12] Shaw, N. and Stead, A. (1974) Biochem. J. 143, [13] Gaver, R.C. and Sweeley, C.C. (1965) J. Am. Oil. Chem. Soc. 42, [14] Samuelsson, B. and Samuelsson, K. (1969) J. Lipid Res. 10, [15] White, D.C., Tucker, A.N. and Sweeley, C.C. (1969) Biochim. Biophys. Acta 187, [16] Miyagawa, E., Azuma, R., Suto, T. and Yano, I. (1979) J. Biochem. 86, [17] Yabuuchi, E. and Moss, C.W. (1982) FEMS Microbiol. Lett. 13, [18] Yano, I., Tomiyasu, I. and Yabuuchi, E. (1982) FEMS Microbiol. Lett. 15, [1] LaBach, L.P. and White, D.C. (1969) J. Lipid Res. 10,

Distribution of molecular species of sphingomyelins in different parts of bovine digestive tract

Distribution of molecular species of sphingomyelins in different parts of bovine digestive tract M. E. Breimer Membrane Biochemistry Group, Department of Medical Biochemistry, University of Giiteborg,