RHIZOBIUM-MELILOTI MUTANTS DEFICIENT IN PHOSPHOLIPID N-METHYLTRANSFERASE STILL CONTAIN PHOSPHATIDYLCHOLINE

Phosphatidylcholine (PC) is the major membrane-forming phospholipid in eukaryotes. In addition to this structural function, PC is thought to play a major role in lipid turnover and signalling in eukaryotic syst ems, In prokaryotes, only some groups of bacteria, among them the memb ers of the family Rhizobiaceae, contain PC. To understand the role; of PC in bacteria, we have studied Rhizobium meliloti 1021, which is abl e to form nitrogen-fixing nodules on its legume host plants and theref ore has a very complex phenotype. R. meliloti was mutagenized with N-m ethyl-N'-nitro-N-nitrosoguanidine, and potential mutants defective in phospholipid N-methyltransferase were screened by using a colony autor adiography procedure. Filters carrying lysed replicas of mutagenized c olonies were incubated with S-adenosyl-L-[methyl-C-14] methionine. Enz ymatic transfer of methyl groups to phosphatidylethanolamine (PE) lead s to the formation of PC and therefore to the incorporation of radiola bel into lipid material, Screening of 24,000 colonies for reduced inco rporation of radiolabel into lipids led to the identification of seven mutants which have a much-reduced specific activity of phospholipid N -methyltransferase. In vivo labelling of mutant lipids with [C-14]acet ate showed that the methylated PC biosynthesis intermediates phosphati dylmonomethylethanolamine and phosphatidyldimethylethanolamine are no longer detectable. This loss is combined with a corresponding increase in the potential methyl acceptor PE. These results indicate that PC b iosynthesis via the methylation pathway is indeed blocked in the mutan ts isolated. However, mass spectrometric analysis of the lipids shows that PC was still present when the mutants had been grown on complex m edium and that it was present in the mutants in wild-type amounts. In vivo labelling with [methyl-C-14] methionine shows that in phospholipi d N-methyltransferase-deficient mutants, the choline moiety of PC is n ot formed by methylation. These findings suggest the existence Of a se cond pathway for PC biosynthesis in Rhizobium.