d-Alanylcardiolipin,a Major Component of the Unique Lipid Pattern of Vagococcus fluvialis

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 α-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 ω-9 series and not the ω-7 series, found in enterococci, lactococci, and streptococci.Chemical and molecular systematic studies have recently been done to clarify the phylogenetic relationship of the group N streptococci (42, 43). Nucleic acid hybridization studies and immunological relationships of superoxide dismutases demonstrated that “Streptococcus lactis” and its subspecies are closely related to each other but not to other streptococci, which led to the formation of a new genus, Lactococcus (43). When during this study the membrane lipids were investigated, group N streptococcus strain Kiel 48809 displayed a pattern that differed greatly from the lipid pattern of the S. lactis group. This strain, which had been isolated from a cow’s udder in Germany (19a), was motile and formed a group with other motile group N streptococci (NCDO 2497, NCDO 2498, and NCDO 2499) (43) which had been isolated in Japan from feces of humans and animals and from river water and seawater (20, 21). Although the motile strains possess the group N antigen, they are not genetically related to Lactococcus or to other streptococci examined. The polar-lipid patterns and their long-chain fatty acid compositions reinforced their distinctiveness and, along with genetic data, suggested that these strains may represent the nucleus of a new taxon (43). This was confirmed by 16S RNA sequence analyses which located the motile group N streptococci on a phylogenetic tree and led to their classification in a new genus, Vagococcus, as Vagococcus fluvialis sp. nov. (7). By using seven isolates, molecular characterization was done and evidence of a possible connection between V. fluvialis and human infections was provided (46).In this report, we describe the polar-lipid pattern of V. fluvialis, which was the same for all the four strains investigated. We isolated and characterized 13 polar lipids and found, in addition to the ubiquitous membrane lipids of gram-positive bacteria, rare and so-far-unknown structures. Of particular interest is d-alanylcardiolipin, which is a major component among the membrane lipids of vagococci. It is a novel representative of the lipid class of substituted cardiolipins. Other examples are α-d-glucopyranosylcardiolipin, found in this study and earlier in group B streptococci (10), and l-lysylcardiolipin, isolated from species of the genus Listeria (16a). So far, substituted cardiolipins have been found only in gram-positive bacteria.