Phospholipids are an important component of bacterial membranes. Borrelia burgdorferidiffers from many other bacteria in that it contains only two major membrane phospholipids:phosphatidylglycerol (PG) and phosphatidylcholine (PC). B. burgdorferi appears to lack enzymesrequired for synthesis of PC through the well-described methylation pathway. However,B. burgdorferi does contain a gene (BB0249) with significant identity to a recently describedphosphatidylcholine synthase gene (pcs) of Sinorhizobiummeliloti.B. burgdorferi also contains a gene(BB0721) with significant identity to the gene (pgs) encoding phosphatidylglycerolphosphatesynthase, an enzyme in the synthetic pathway of PG. Activity of BB0249 was confirmed by cloningthe gene into Escherichia coli, which does not produce PC. Transformation with a plasmidcarrying BB0249 resulted in production of PC by E. coli, but only in the presence of exogenouslysupplied choline, as would be predicted for a Pcs. Because loss of Pgs activity is lethal toE. coli, activity of BB0721 was confirmed by the ability of BB0721 to complement an E. coliPgs "mutant. A plasmid containing BB0721 was transformed into a Pgs" mutant of E. coli containinga copy of the native gene on a temperature-regulated plasmid. The temperature-regulatedplasmid was exchanged for a plasmid containing BB0721 and it was shown that BB0721 wasable to replace the lost Pgs function and restore bacterial growth. This study has established theexistence and function of two critical enzymes in the synthesis of PC and PG in B. burgdorferi.Understanding of the biosynthetic pathways of PC and PG in B. burgdorferi is the first step indelineating the role of these phospholipids in the pathogenesis of Lyme disease.