Stereochemistry of phosphatidylglycerols from thermotolerant bacteria isolated thermal springs

•Identification of phosphatidylglycerols (PG) from thermophilic bacteria is described.•A total of 4 isomers were separated by hydrophilic interaction liquid chromatography.•Many dozens molecular species of PG were identified by shotgun ESI.•PG were analyzed by HILIC/ESI-MS and chiral HPLC.•A total 4 isomers (2 enantiomers and 2 regioisomers) of PG were identified.•The ratio of isomers depends on the cultivation temperature. Phosphatidylglycerol (1,2-diacyl-sn-glycero-3-phospho-glycerol) (PG) is one of the most abundant lipids in biological membranes. However, the chirality of the carbon atom in glycerol phosphate differs among the three kingdoms: bacteria, archaea, and eukaryotes. It is commonly assumed that archaea, as well as bacteria and eukaryotes, produce only one isomer of PG. Archaeal membranes consist of phospholipids with glycerol-1-phosphate in the S configuration, while the phospholipids of the other two kingdoms contain glycerol-3-phosphate with (R) stereochemistry. Another chiral atom is found in glycerol with non-esterified hydroxy groups. Considering the high temperatures that accompanied the origin of life on Earth, it becomes obvious that it is necessary to clarify the importance of membrane lipids in early evolutionary times. To reconstruct the effect of high temperatures on membrane lipids, it is ideal to use microorganisms originating from a thermophilic environment analogous to the early Earth, such as the thermal groundwater of the famous spa town of Karlovy Vary. Here, we prepared all four isomers of PG, i.e., (R,S, R,R, S,R), and (S,S), by organic synthesis and analyzed the representation of individual molecular species in seven bacteria isolated from the Karlovy Vary thermal springs using chiral chromatography - mass spectrometry. Our results provide evidence that five of these strains produce all four isomers of PG and that this production is highly dependent on the cultivation temperature. Subsequent analysis by chiral chromatography revealed that the ratio of isomers, enantiomers, and diastereoisomers depends on the cultivation temperature of individual strains.