A Novel Sphingoglycolipid Containing Galacturonic Acid and 2-Hydroxy Fatty Acid in Cellular Lipids of Sphingomonas yanoikuyae

Abstract

A novel sphingoglycolipid was isolated from Sphingomonas yanoikuyae, and its structure was identified as a galacturonosyl-β (1→1)-ceramide. This was a characteristic sphingoglycolipid present in S. yanoikuyae and certain other species of Sphingomonas, such as Sphingomonas mali, Sphingomonas terrae, and Sphingomonas macrogoltabidus, but not in the type species of Sphingomonas, Sphingomonas paucimobilis.

Sphingolipids are one of the most ubiquitous components of eukaryotic cell membranes. In contrast, the occurrence of sphingolipids in bacteria is rare. They have been reported as sphingomyelin in Mycoplasma, sphingophospholipid in Bacteroides and Bdellovibrio, aminoglycosphingolipid in Chlorobium, capnoid in Capnocytophaga, Cytophaga, Flavobacterium, Flexibacter, and Sporocytophaga, sphingoglycolipid (SGL) in Sphingomonas and Zymomonas, and ceramide in Bacteroides and Sphingobacterium (6–8, 10, 15, 19). SGL (2-N-2′-hydroxymyristoyl dihydrosphingosine-1-glucuronic acid) containing glucuronic acid was isolated first from cellular lipids of the type strain of Flavobacterium devorans ATCC 10829 (14, 18). A similar SGL was found in a group of deep-yellow pigmented organisms, including the type strain of Pseudomonas paucimobilis (5). We proposed previously a new genus Sphingomonas with the type species Sphingomonas paucimobilis, based on the existence of a unique SGL, the major type of ubiquinone (Q-10), 16S rRNA sequence, and phenotypic features. Furthermore, three new species, Sphingomonas yanoikuyae, Sphingomonas parapaucimobilis, and Sphingomonas adhaesiva, and one new combination, Sphingomonas capsulata, were described from the homology of DNA-DNA hybridization and phenotypic characterization (15). SGLs containing di-, tri-, and tetrasaccharides have been found in the type strain of S. paucimobilis (7, 13). The major ubiquitous SGL in Sphingomonas is a glucuronosyl ceramide which is called SGL-1.

During research leading to the proposal of a new genus, Sphingomonas, we have noticed the existence of a novel alkali-stable sphingoglycolipid (SGL-1′) in S. yanoikuyae migrating close to but distinctive from an SGL-1 spot on a thin-layer chromatogram (TLC) of silica gel G (Uniplate; Analtech, Newark, Del.) developed with an acidic solvent system, chloroform-methanol-acetic acid-water (100:20:12:5, by volume) (15). The objective of this study was to isolate SGL-1′ and to define its chemical structure. We have demonstrated here an alkali-stable lipid that is identical to SGL-1′ in S. yanoikuyae and other type strains of Sphingomonas mali, Sphingomonas terrae, and Sphingomonas macrogoltabidus.

Isolation of SGL-1′.

S. yanoikuyae EY 4208^T, S. mali EY 4206^T, S. terrae EY 4207^T, and S. macrogoltabidus EY 4304^T were used in this study. The detailed history, corresponding strain number, and culture conditions were described previously (11, 12, 15). The crude lipids were extracted by the method of Folch et al. (4). In brief, harvested bacteria were sonicated and lipids were extracted with a chloroform-methanol (2:1 and 1:3, by volume) mixture. After condensation of the organic phase, extractable lipids were hydrolyzed with 0.5 N KOH (30°C, 3 h), which was followed by neutralization. Alkali-stable lipids were again extracted with chloroform-methanol (2:1, vol/vol). Alkali-stable lipids in S. yanoikuyae EY 4208^T revealed two major glycolipid spots on a TLC developed with an acidic solvent system (Fig. 1A). The upper spot, with an R_f value of 0.48, coincided with glucuronosyl ceramide (SGL-1) isolated from S. paucimobilis EY 2395^T; however, the lower spot (SGL-1′) did not correspond to any sphingo- (or glycero-) glycolipid reported previously. When the plate was developed with the neutral solvent system, chloroform-methanol-water (65:25:4, by volume) (Fig. 1B), these two spots were united to form a long tailing spot resembling anionic phospholipid, suggesting that both compounds (SGL-1 and SGL-1′) had an anionic charge. SGL-1′ was purified by using TLC developed with an acidic solvent system until a single spot was obtained. Both SGL-1 and SGL-1′ were reactive with anthrone reagent to yield a brownish-purple color, but they did not react with Dittmer's and ninhydrin reagents. Similarly, two spots (SGL-1 and SGL-1′) were found ubiquitously in S. mali EY 4206^T, S. terrae EY 4207^T, and S. macrogoltabidus EY 4304^T and were purified. They were analogous to those of S. yanoikuyae EY 4208^T, although the spot of SGL-1′ was not seen in S. paucimobilis EY 2395^T.

FIG. 1.

Thin-layer chromatograms of SGL-1 and SGL-1′. Shown are solvent systems chloroform-methanol-acetic acid-water (100:20:12:5, by volume) (A) and chloroform-methanol-water (65:25:4, by volume) (B). 2395, S. paucimobilis EY 2395^T; 4208, S. yanoikuyae EY 4208^T; 4206, S. mali EY 4206^T; 4207, S. terrae EY 4207^T; 4304, S. macrogoltabidus EY 4304^T. Lipids used were crude lipids (a), alkali-stable lipids (b), SGL-1 (c), and SGL-1′ (d).

Molecular weight of SGL-1′.

Molecular weight was determined by fast atom bombardment-mass spectrometry (FAB/MS) with triethanolamine as a matrix. FAB/MS analysis of SGL-1 and SGL-1′ showed the similar result of quasimolecular ions at m/z 742.6 and 728.6, respectively, due to [M-H]⁻ as the major ions and fragment ions at m/z 566.6 and 552.6, respectively, due to ceramide moiety corresponding to two molecular species (Fig. 2). The molecular weight and fragmentation of SGL-1′ were essentially identical to those of SGL-1.

FIG. 2.

Negative FAB/MS spectra of SGL-1 and SGL-1′ from S. yanoikuyae EY 4208^T. The major molecular ions were detected at m/z 742.6 and 728.6, and fragment ions of ceramide cleaved of carbohydrate moiety were detected at m/z 566.6 and 552.6.

Composition of fatty acids and long-chain bases in ceramide.

SGL-1′ was hydrolyzed with saturated Ba(OH)₂ solution (100°C, 16 h). Long-chain bases were extracted with diethyl ether. After acidification of the residues, fatty acids were extracted with n-hexane. Trimethylsilyl (TMS) derivatives of long-chain bases and fatty acid methyl esters were identified by gas chromatography (GC) and GC-mass spectrometry (GC-MS). C₂₀-sphingosine and C₂₁-monocyclopropanoyl dihydrosphingosine were identified as the long-chain bases from fragment ions (C2-C3 cleavage of TMS derivative, m/z 132; [M-132]⁺, m/z 339; [M-103]⁺, m/z 368; [M-15]⁺, m/z 456 for C₂₀-sphingosine; and C2-C3 cleavage of TMS derivative, m/z 132; [M-132]⁺, m/z 353; [M-103]⁺, m/z 382; [M-15]⁺, m/z 470 for C₂₁-monocyclopropanoyl dihydrosphingosine) by GC-MS analysis (20) and TLC (R_f value and ninhydrin-positive spot). On the other hand, 2-hydroxymyristic acid was identified as the major fatty acid, based on the quasimolecular ion ([M]⁺, m/z 258) and characteristic fragment ions of 2-hydroxy fatty acid methyl ester ([M-59]⁺, m/z 199 and m/z 90 and 103).

Identification of carbohydrate moiety and linkage analysis.

SGL-1′ was first reduced with LiAlD₄ (70°C, 4 h) (18). After that, the alditol acetate derivative was obtained by hydrolysis with trifluoroacetic acid (120°C, 1 h), reduction with NaBD₄ (room temperature, 2 h), and acetylation with acetic anhydride-pyridine (1:1, by volume). For partially methylated alditol acetate analysis, SGL-1′ was treated with dimethyl iodide after reduction with LiAlD₄ (3). Alditol acetate and partially methylated alditol acetate derivatives were analyzed and characterized by GC and GC-MS (1, 2). In GC analysis, the alditol acetate derivative of SGL-1′ exhibited the same retention time as alditol acetate of galacturonic acid monohydrate (Sigma Chemical Co., St. Louis, Mo.) (the reference standard) and a different retention time than that of the alditol acetate derivative of SGL-1, which contained glucuronic acid (Fig. 3A and B). The mass spectrum showed characteristic fragment ions at m/z 218, 219, 290, 291, 362, and 363 (Fig. 3C). These fragment ions showed a signal of two mass units higher than those of the alditol acetate derivative of d-galactose, due to reduction with LiAlD₄ at the C-6 position. These results suggest the presence of galacturonic acid as a carbohydrate moiety. Moreover, the GC-MS spectrum of the partially methylated alditol acetate derivative showed characteristic fragment ions of 2,3,4,6-tetra-O-methyl-1,5-di-O-acetyl galactitol at m/z 207, 147, 118, 163, 131, 162, and 102 (Fig. 3D). This result implies that the linkage of ceramide is at position C-1 of galacturonic acid. On the other hand, the coupling constant (J_1.2) of galacturonic acid of SGL-1′ was 6.3 Hz (chemical shift, d = 5.693 ppm) from the nuclear magnetic resonance (NMR) analysis (Table 1). Heteronuclear coupling constants (¹J_H.C) were 162.1 Hz for SGL-1′. These data suggest that galacturonic acid of SGL-1′ is linked β-glycosidically to the long-chain base of lipid.

FIG. 3.

Total ion gas chromatograms and mass spectra of alditol acetate and partially methylated alditol acetate derivatives of SGL-1′ from S. yanoikuyae EY 4208^T. Alditol acetate derivatives of SGL-1 (A), SGL-1′ (B and C), and a partially methylated alditol acetate derivative of SGL-1′ (D) are shown. The partially methylated alditol acetate derivative of SGL-1′ showed the fragment pattern of 2,3,4,6-tetra-O-methyl-1,5-di-O-acetyl galactitol. The fused silica capillary column SP-2380 was used, and the GC oven temperature was programmed to increase linearly at 5°C/min from 180 to 250°C.

TABLE 1.

NMR analysis of anomeric regions of SGL-1 and SGL-1′ derived from S. yanoikuyae EY 4208^T

Source of anomeric regions	Results of NMR analysis
	1H		13C
	Chemical shift (ppm)	Coupling constant (J1.2 [Hz])	Chemical shift (ppm)	Coupling constant (JC.H. [Hz])
SGL-1	4.853	1.0	98.975	172.2
SGL-1′	5.693	6.3	90.443	162.1

Based on above findings, the difference between SGL-1 and SGL-1′ may be only in their carbohydrate moiety. Taken together, the molar ratio of galacturonic acid-fatty acid-long-chain base of SGL-1′ is 1:1:1 by the results of FAB/MS and component and quantitative analyses. The alkali-stable sphingoglycolipid SGL-1′ obtained from S. yanoikuyae appears to be most likely ceramide galacturonic acid (2-N-2′-hydroxymyristoyl C₂₀-sphingosine-1-galacturonic acid and 2-N-2′-hydroxymyristoyl C₂₁-monocyclopropanoyl dihydrosphingosine-1-galacturonic acid). In gram-negative bacteria, the presence of diacylglycerol-type glycolipids containing hexuronic acid and sphingoglycolipids containing glucuronic acid has been demonstrated (7, 10, 18), although the existence of sphingoglycolipid containing galacturonic acid has not yet been reported for any prokaryotic or eukaryotic cells. To our knowledge, this is the first study to demonstrate this type of acidic sphingoglycolipid. The relationship between SGL-1′ and the biologic activity of acidic sphingoglycolipid derived from bacteria and mammalian sources (9, 16, 17) is particularly interesting and will be discussed taxonomically and biochemically in the near future.