Cardiolipin synthases of Escherichia coli have phospholipid class specific phospholipase D activity dependent on endogenous and foreign phospholipids.

E. coli has three Cls-isoenzymes for cardiolipin (CL) synthesis but the differences between these three enzymes remain unresolved. All three Cls enzymes contain the phospholipase D (PLD) characteristic HKD motive and synthesize CL using PLD activity. Here, using LC-MS we show the effect of overexpressing or deletion of the three individual Cls enzymes on the lipidome, which included changes in lipid class distribution and CL species profiles. We demonstrate, for the first time, that overexpression of only ClsB resulted in the appreciable synthesis of a variety of phosphatidylalcohols, thereby establishing a 'classic' PLD activity for this enzyme: phospholipid headgroup exchange. Endogenous E. coli lipids and primary alcohols were substrates for this trans-phosphatidylation reaction. Furthermore, we show that endogenous levels of ClsA mediated a similar trans-phosphatidylation reaction to form phosphatidylalcohols, however this reaction was dependent on the presence of the foreign phospholipid class phosphatidylcholine (PC). This allows us to clarify the different specificities of the cardiolipin synthases.