Rhizobium meliloti mutants deficient in phospholipid N-methyltransferase still contain phosphatidylcholine

Abstract

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 systems. In prokaryotes, only some groups of bacteria, among them the members of the family Rhizobiaceae, contain PC. To understand the role of PC in bacteria, we have studied Rhizobium meliloti 1021, which is able to form nitrogen-fixing nodules on its legume host plants and therefore has a very complex phenotype. R. meliloti was mutagenized with N-methyl-N-nitro-N-nitrosoguanidine, and potential mutants defective in phospholipid N-methyltransferase were screened by using a colony autoradiography procedure. Filters carrying lysed replicas of mutagenized colonies were incubated with S-adenosyl-L-[methyl-14 C]methionine. Enzymatic transfer of methyl groups to phosphatidylethanolamine (PE) leads to the formation of PC and therefore to the incorporation of radiolabel into lipid material. Screening of 24,000 colonies for reduced incorporation 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 [ 14 C]acetate showed that the methylated PC biosynthesis intermediates phosphatidylmonomethylethanolamine 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 biosynthesis via the methylation pathway is indeed blocked in the mutants isolated. However, mass spectrometric analysis of the lipids shows that PC was still present when the mutants had been grown on complex medium and that it was present in the mutants in wild-type amounts. In vivo labelling with [methyl-14 C]methionine shows that in phospholipid N-methyltransferasedeficient mutants, the choline moiety of PC is not formed by methylation. These findings suggest the existence of a second pathway for PC biosynthesis in Rhizobium.

Pathways for phosphatidylcholine biosynthesis in bacteria

“…The lipids were extracted according to Bligh & Dyer (1959). The chloroform phase was used for lipid analysis on TLC plates and, after one-dimensional separation (de Rudder et al, 1997), visualization of labelled compounds was obtained by autoradiography.…”

“…In this latter reaction, labelled methyl groups can be incorporated into phospholipids, especially into PE and to some extent into PC (de Rudder et al, 1997). However, for formation of MMPE and DMPE, methylation of the ethanolamine head group of PE is required, as this constitutes the only pathway of MMPE and DMPE formation, at least in S. meliloti (de Rudder et al, 1997). Therefore, the formation of radiolabelled MMPE and DMPE is indicative of a functional PMT pathway.…”

“…Only the methylation pathway of PC biosynthesis was thought to occur in prokaryotes (Rock et al, 1996); however, a second pathway for PC biosynthesis exists, the phosphatidylcholine synthase (PCS) pathway (Fig. 1), which is distinct from the CDPcholine pathway and seems to be exclusive for bacteria (de Rudder et al, 1997(de Rudder et al, , 1999Sohlenkamp et al, 2000). In this second pathway, choline is condensed directly with CDP-diacylglycerol to form PC and CMP in a reaction catalysed by PCS.…”

Phosphatidylcholine levels in Bradyrhizobium japonicum membranes are critical for an efficient symbiosis with the soybean host plant

“…In Sinorhizobium meliloti, a single gene coding for Pmt activity constitutes the methylation pathway . Apart from the methylation pathway, a second bacterial pathway for PC biosynthesis exists in this organism (de Rudder et al, 1997) involving the novel enzymatic activity, phosphatidylcholine synthase (Pcs), which forms PC directly from choline and CDP-diacylglycerol (de Rudder et al, 1999;Sohlenkamp et al, 2000). We have also shown that PC is required for normal growth of S. meliloti .…”

“…The lipids were extracted according to the method of Bligh and Dyer (1959). The chloroform phase was used for lipid analysis on TLC plates and, after one-dimensional or two-dimensional separation, the individual lipids were quantified as described previously (de Rudder et al, 1997).…”