Abstract
Phosphatidylcholine is a major lipid of eukaryotic membranes, but found in only few prokaryotes. Enzymatic methylation of phosphatidylethanolamine by phospholipid N-methyltransferase was thought to be the only biosynthetic pathway to yield phosphatidylcholine in bacteria. However, mutants of the microsymbiotic soil bacterium Sinorhizobium (Rhizobium) meliloti, defective in phospholipid N-methyltransferase, form phosphatidylcholine in wild type amounts when choline is provided in the growth medium. Here we describe a second bacterial pathway for phosphatidylcholine biosynthesis involving the novel enzymatic activity, phosphatidylcholine synthase, that forms phosphatidylcholine directly from choline and CDP-diacylglycerol in cell-free extracts of S. meliloti. We further demonstrate that roots of host plants of S. meliloti exude choline and that the amounts of exuded choline are sufficient to allow for maximal phosphatidylcholine biosynthesis in S. meliloti via the novel pathway.
Highlights
Eukaryotes can synthesize the membrane lipid phosphatidylcholine (PC)1 by two alternative biosynthetic pathways [1]
Choline-dependent PC Biosynthesis in S. meliloti—For the determination of lipid compositions, S. meliloti strains were grown on complex medium or on a MOPS minimal medium in the presence of radiolabeled acetate, and lipid extracts were subsequently separated by thin layer chromatography (TLC)
The methylated intermediates of the methylation pathway, monomethylphosphatidylethanolamine (MMPE) and dimethylphosphatidylethanolamine (DMPE), are not formed in the mutant, and these results suggest the existence of a second, methylation-independent pathway for PC biosynthesis in S. meliloti
Summary
Strains and Growth Conditions—S. (Rhizobium) meliloti 1021 (wild type) and derived mutants KDR309 (pmt-deficient) [6], KDR500 (betCBA-deficient), and KDR508 (pmt- and betCBA-deficient) were grown at 29 °C in tryptone/yeast extract (TY) medium [7] or in MOPS minimal medium containing 40 mM MOPS, 20 mM KOH, 20 mM NH4Cl, 100 mM NaCl, 2 mM MgSO4, 1.2 mM CaCl2, 0.3 mg biotin/l, 15 mM succinate, 10 mM potassium phosphate buffer, pH 7. Recombinant betCBA-deficient strains derived from S. meliloti 1021 wild type and pmt-deficient mutant KDR309 were named KDR500 and KDR508, respectively. The strains were labeled in MOPS minimal medium cultures (1 ml) inoculated from fresh overnight cultures. TY components (2.5% tryptone, 1.5% yeast extract; w/v), choline (1 mM), or root exudate of Medicago sativa after 7 days incubation (75 l of root exudate containing 14.7 M choline to 1 ml of minimal medium) were added as sterile filtrates to the final concentrations indicated. At a cell density of 2 ϫ 108/ml in MOPS minimal medium, [methyl-14C]choline was added to a final concentration of 100 M. Determination of Specific Phosphatidylcholine Synthase Activity— The in vitro incorporation of radiolabeled choline into PC was determined in cell-free extracts of the betCBA- and pmt-deficient double mutant S. meliloti KDR508. Final concentrations of additional compounds when applied were 10 mM MgCl2, 10 mM MnCl2, 343 M CDP-
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