Revealing Acyl Chain Selectivity of Cis-to-Trans Isomerase through Profiling of C═C Geometry and Location Isomers of Bacterial Lipids

Bacteria adapt to environmental stress by modifying their membrane lipid structures, including the C═C geometry. Profiling of bacterial lipids with accurate C═C geometry assignment is challenging due to the lack of standards and interference from C═C location isomers. By leveraging two radical reactions: thiyl radical-catalyzed C═C isomerization and the Paternò-Büchi (PB) reaction, we developed an analytical workflow to profile C═C geometric and location isomers in bacterial lipidomes. The high yield (∼80%) of cis (Z)-to-trans (E) C═C isomerization catalyzed by thiyl radicals allows for on-demand synthesis of commercially unavailable lipid C═C geometric isomers. By comparing the retention behavior of Z vs E isomers from reversed-phase liquid chromatography-mass spectrometry (RPLC-MS), we can determine C═C geometry at sub-nM levels. The location of C═C can be further obtained by conducting an online acetone PB reaction after RPLC separation. Applying this workflow to Pseudomonas putida, we profiled 60 lipid species across six subclasses, including the rarely reported glucosaminyl phosphatidylglycerol. We found that both Z and E isomers were present in bacterial lipids, however, with an increase in E isomers after toluene exposure, which correlated with an upregulation of cis-to-trans isomerase (Cti). Our workflow further revealed the chain selectivity of Cti, with a preference for C16:1(n-7Z) > C18:1(n-7Z) > C18:1(n-9Z). This finding provides valuable insights into the dynamics of lipid metabolism during bacterial stress responses.