D-Alanylcardiolipin, a major component of the unique lipid pattern of Vagococcus fluvialis

Abstract

Motile group N streptococci, classified as Vagococcus fluvialis, have been isolated from cows' udders, human and animal feces, river water, and seawater. They possess an unusual membrane lipid and fatty acid pattern. We isolated and characterized 13 polar lipids, 8 of them also found in other gram-positive bacteria: mono- and dihexosyldiacylglycerol, an acylated and a glycerophosphate-substituted derivative of the latter, cardiolipin, phosphatidylglycerol, D-alanylphosphatidylglycerol, and L-lysylphosphatidylglycerol. Besides them, we characterized two rare compounds, bis(acylglycero)phosphate and alpha-D-glucopyranosylcardiolipin, and two compounds so far not detected in nature, D-alanylbis(acylglycero)phosphate and D-alanylcardiolipin. The concomitant occurrence of four aminoacyl phospholipids in one organism is another unique finding. Substituted cardiolipins represent a novel lipid class: in vagococci, D-alanylcardiolipin is a major membrane lipid component, contributing 11 and 26 mol% of total lipids in the exponential and stationary phases of growth, respectively. The vagococcal lipids contain even-numbered straight-chain saturated and cis-monounsaturated fatty acids, but the cis-monoenic acids belong to the omega-9 series and not the omega-7 series, found in enterococci, lactococci, and streptococci.

FIG. 1

Polar-lipid pattern of V. fluvialis NCDO 2497 during exponential growth. For identification of the lipids, see Table 1. Lipids 14 and 15 reacted neither with the Dittmer-Lester reagent for lipid phosphorus nor with 1-naphthol–H₂SO₄ for carbohydrate and were not further studied. Two-dimensional TLC was performed on silica gel plates (Merck 60). The first dimension (upward) was developed with solvent A, and the second dimension was developed with solvent C. Visualization was done with iodine vapor. For quantitative determination, lipids 5 and 10 were separated in solvent E.

FIG. 2

Chromatographic behavior of the purified glyceroglycolipids 1 to 3, in comparison with the following reference lipids (from top to bottom): R₁, Glc(α1-3)acyl₂Gro, Glc(α1-2),acyl-6Glc(α1-3)acyl₂Gro, Glc(α1-2)Glc(α1-3)acyl₂Gro, and Glc(α1-2)Glc(α1-2)Glc(α1-3)acyl₂Gro; R₂, Glc(α1-3)acyl₂Gro, Gal(α1-2)Glc(α1-3)acyl₂Gro, and Glc(β1-6)Gal(α1-2)Glc(α1-3)acyl₂Gro. TLC was performed on silica gel plates (Merck 60) by using solvent D, and visualization was done with 1-naphthol–H₂SO₄.

FIG. 3

Chromatographic behavior of deacylated glyceroglycolipids 1 to 3 in comparison with the following reference glycosylglycerols: R₁, Glc(α1-3)Gro; R₂, Glc(α1-2)Gro; R₃, Gal(α1-2) Glc(α1-3)Gro; R₄, Glc(α1-2)Glc(α1-3)Gro. TLC was performed on silica gel plates (Merck 60) by using solvent F, and visualization was done with 1-naphthol–H₂SO₄.

FIG. 4

Hydrolysis of glycerophosphoglycolipid 4 with 98% (vol/vol) acetic acid (100°C, 30 min). Lanes 1 and 2, lipid 4 after and before treatment with acetic acid, respectively; lanes R₁ and R₂, Gal(α1-2)Glc(α1-3)acyl₂Gro before and after treatment with acetic acid, respectively; lane R₃, GroP-6Glc(α1-2)Glc(α1-3)acyl₂Gro. The two faster-moving bands in lane R₂ are partially acetylated derivatives; the more slowly moving band in lanes 1 and 2 is the monodeacyl derivative. TLC was performed on silica gel plates (Merck 60) by using solvent A, and visualization was done with 1-naphthol–H₂SO₄.

FIG. 5

Dealanylation of d-Ala(acylGro)₂P (lipid 9) by treatment with 0.1 M sodium borate, pH 9.5, containing Triton X-100. Lane R, (acylGro)₂P (lipid 8); lane 1, lipid 9, untreated; lanes 2 and 3, lipid 9 after treatment at pH 9.5 for 4 and 24 h, respectively. The heavy spot near the front in panel a and the ladderlike pattern in panel b are Triton X-100. TLC was performed on silica gel plates (Merck 60) by using solvents A (a) and D (b); visualization was done with iodine vapor.

FIG. 6

Hydrolysis of d-Ala-cardiolipin (lipid 11) and cardiolipin (lipid 10) with phospholipase A₂ (room temperature, 30 min). Lane R (from top to bottom), fatty acids, cardiolipin, and monodeacyl and dideacyl derivatives; lanes 1 and 2, lipid 11 before and after enzymic treatment, respectively; lanes 3 and 4, lipid 10 before and after enzymic treatment, respectively. The heavy band in lane 2 is the dideacyl derivative. TLC was performed on silica gel plates (Merck 60) by using solvent A, and visualization was done with iodine vapor.