Sphaerophorus ridiculosis: Plasmalogen Composition

Glyceryl ethers are present in most mammalian tissues (17, 21) but have until recently been isolated from only a few anaerobic microorganisms (1, 4, 8, 9). Recently, Kamio et al. (12) have shown that these lipids are present in a number of anaerobic soil microorganisms, and Meyer and Meyer (14) have reported alk-1'-enyl glyceryl ethers (plasmalogens) in Treponema pallidum (Reiter) isolated from soil. It seems evident, therefore, that ether-containing lipids are widespread among the anaerobic bacteria. We report here the identification of alk-1'enyl glyceryl ethers and the possible presence of alkyl glyceryl ethers in a species of the gram-negative, anaerobic, highly pleomorphic bacteria Sphaerophorus most closely resembling Sphaerophorus ridiculosis. The organism (isolated 13 June 1968) was isolated and grown to late-exponential growth phase as previously described (15). The cells were harvested and washed, and the lipids were extracted as described (7). About 4.2% of the dry weight of the cells was lipid extractable, as determined by the Folch et al. (6) extraction procedure. The total lipids were separated by silicic acid column chromatography into neutral lipids (16.2%), glycolipids (1.4%), and polar lipids (82.4%) (10). The glycolipids were not investigated further. The neutral lipids were separated by thin-layer chromatography (TLC) on Silica Gel G in hexane-diethylether-acetic acid (80:20: 1 or 50:50:2, vol/vol/vol) or hexanechloroform-ethanol (135:60:5, vol/vol/vol) and were identified by co-chromatography with the appropriate standards (Superlco, Analabs, NuChek Prep). The major neutral lipids, free fatty acids (61.4%), triglycerides (11%), and diglycerides (2%), were quantitated by the method of Privett et al. (16). A compound (9%) which co-chromatographed with cholesterol and showed pink coloration after H2SO spray has not been further identified. Trace amounts of monoglycerides, wax esters, and long-chain fatty alcohols were also present. The total polar lipids were subjected to LiAlH4 reduction (24), and the reaction products were determined by TLC (24). Photodensitometric determinations (16) indicated approximately 18% of the total polar lipid to be the alk-1'-enyl glyceryl ether form. A lipid which yields a spot when sprayed with H2SO4 spray (16) co-chromatographed with alkyl glyceryl ethers and represented 1.4% of the total lipids. Whether this band is composed of 1,2or 1,3-alkane diols or both in addition to alkyl glyceryl ethers, as observed in Clostridium butyricum (9) lipids, is at present unknown. The alk-1'-enyl glycerols obtained were hydrogenated in the presence of Adams catalyst, and the resulting alkyl glycerol ethers were shown to co-chromatograph with 1-O-alkyl glycerol ether (gift from Randell Wood) on sodium arsenite-impregnated, silica gel thinlayer plates (23). This suggests the structure of 1-O-enyl-2-acyl-3 glyceryl phosphatide for these lipids. Table 1 shows the carbon number of the aldehydes liberated by acid from the 1-0-alk-1'enyl glycerols (2) and the acetate derivatives (19) of the fatty alcohols obtained by LiAlH4 reduction of the polar lipids. The aldehydogenic chain is relatively simple and predominantly saturated. The major polar lipids were shown by TLC on silica gel HRB (Brinkman), developed in chloroform-methanol-acetic acid-0.9% sodium chloride (85:25:8:4, vol/vol/vol/vol) or chloroformmethanol-ammonium hydroxide (65:35:5, vol/ vol/vol), to co-chromatograph with phosphatidylethanolamine, lyso-phosphatidylethanolamine, phosphatidylglycerol, and cardi-