Chiral analysis of glycerol phosphates- can bacteria biosynthesize heterochiral phospholipid membranes?

Phosphatidylglycerol (1,2-diacyl- sn-glycero-3-phospho-glycerol) (PG) is one of the most abundant lipids in bacteria. However, the chirality of the carbon atom on glycerol phosphate is different between the three kingdoms, Archaea, Bacteria, and Eukarya. Archaea membranes consist of phospholipids with glycerol -1-phosphate (G1P) in the S configuration, whereas phospholipids of the other two kingdoms contain glycerol-3-phosphate (G3P) having R stereochemistry. In the present study, GC/MS and LC/MS methods sensitively detected G3P and G1P from four bacterial strains ( Bacillus amyloliquefaciens, B. subtilis, Clav-ibacter michiganensis, and Geobacillus stearothermophilus). Strain selection was carried out based on a GenBank search that revealed bacterial sequences associated with both enzymes involved in glycerol -phosphate synthesis, i.e., glycerol-3-phosphate dehydrogenase and glycerol-1-phosphate dehydrogenase. The detection of G1P and G3P was made by comparing the retention times of synthetic standards with those of analyzed samples. The structures of both glycerol phosphates were confirmed by selected ion monitoring (SIM) at m/z 171.006. The total concentration of G3P and G1P was around 30 mu M, with a ratio of G3P to G1P of 4:1. We showed that PG was the most abundant phospholipid in all four bac-teria by using the following analytical techniques and chromatographic modes: hydrophilic interaction liquid chromatography (HILIC), reversed-phase high-performance liquid chromatography high-resolution electrospray ionization tandem mass spectrometry (RP-HPLC/HR-ESI tandem MS) in negative and positive ionization modes, and an enzymatic cleavage by phospholipase C. By using chiral chromatography, the presence of both enantiomers in the glycerol backbone of some molecular species of PG was revealed. These results allow us to conclude that the bacteria examined here produce both enantiomer glycerol phosphates.(c) 2022 Elsevier B.V. All rights reserved.

Automated summary

In this study, G3P and G1P were detected in four bacterial strains using GC/MS and LC/MS methods. Different analytical techniques and chromatographic modes were employed to confirm that PG is the most abundant phospholipid in these bacteria.