Inactivation of the gene for phospholipid N-methyltransferase in Sinorhizobium meliloti: phosphatidylcholine is required for normal growth.

Abstract

In phosphatidylcholine (PC)-containing prokaryotes, only the methylation pathway of PC biosynthesis was thought to occur. However, a second choline-dependent pathway for PC formation, the PC synthase (Pcs) pathway, exists in Sinorhizobium (Rhizobium) meliloti in which choline is condensed with CDP-diacylglyceride. Here, we characterize the methylation pathway of PC biosynthesis in S. meliloti. A mutant deficient in phospholipid N-methyltransferase (Pmt) was complemented with a S. meliloti gene bank and the complementing DNA was sequenced. A gene coding for a S-adenosylmethionine-dependent N-methyltransferase was identified as the sinorhizobial Pmt, which showed little similarity to the corresponding enzyme from Rhodobacter sphaeroides. Upon expression of the sinorhizobial Pmt, besides phosphatidylcholine, the methylated intermediates of the methylation pathway, monomethylphosphatidylethanolamine and dimethylphosphatidylethanolamine, are also formed. When Pmt-deficient mutants of S. meliloti are grown on minimal medium, they cannot form PC, and they grow significantly more slowly than the wild type. Growth of the Pmt-deficient mutant in the presence of choline allows for PC formation via the Pcs pathway and restores wild-type-like growth. Double knock-out mutants, deficient in Pmt and in Pcs, are unable to form PC and show reduced growth even in the presence of choline. These results suggest that PC is required for normal growth of S. meliloti.