Sinorhizobium meliloti mutants deficient in phosphatidylserine decarboxylase accumulate phosphatidylserine and are strongly affected during symbiosis with alfalfa

Abstract

Sinorhizobium meliloti contains phosphatidylglycerol, cardiolipin, phosphatidylcholine, and phosphatidylethanolamine (PE) as major membrane lipids. PE is formed in two steps. In the first step, phosphatidylserine synthase (Pss) condenses serine with CDP-diglyceride to form phosphatidylserine (PS), and in the second step, PS is decarboxylated by phosphatidylserine decarboxylase (Psd) to form PE. In this study we identified the sinorhizobial psd gene coding for Psd. A sinorhizobial mutant deficient in psd is unable to form PE but accumulates the anionic phospholipid PS. Properties of PE-deficient mutants lacking either Pss or Psd were compared with those of the S. meliloti wild type. Whereas both PE-deficient mutants grew in a wild-type-like manner on many complex media, they were unable to grow on minimal medium containing high phosphate concentrations. Surprisingly, the psd-deficient mutant could grow on minimal medium containing low concentrations of inorganic phosphate, while the pss-deficient mutant could not. Addition of choline to the minimal medium rescued growth of the pss-deficient mutant, CS111, to some extent but inhibited growth of the psd-deficient mutant, MAV01. When the two distinct PE-deficient mutants were analyzed for their ability to form a nitrogen-fixing root nodule symbiosis with their alfalfa host plant, they behaved strikingly differently. The Pss-deficient mutant, CS111, initiated nodule formation at about the same time point as the wild type but did form about 30% fewer nodules than the wild type. In contrast, the PS-accumulating mutant, MAV01, initiated nodule formation much later than the wild type and formed 90% fewer nodules than the wild type. The few nodules formed by MAV01 seemed to be almost devoid of bacteria and were unable to fix nitrogen. Leaves of alfalfa plants inoculated with the mutant MAV01 were yellowish, indicating that the plants were starved for nitrogen. Therefore, changes in lipid composition, including the accumulation of bacterial PS, prevent the establishment of a nitrogen-fixing root nodule symbiosis.

FIG. 1.

Biosynthesis of phospholipids in Sinorhizobium meliloti. SAM, S-adenosylmethionine; SAHC, S-adenosylhomocysteine; PgsA, phosphatidylglycerolphosphate synthase; Pgp, phosphatidylglycerolphosphate phosphatase; Cls, cardiolipin synthase; Pss, phosphatidylserine synthase; Psd, phosphatidylserine decarboxylase; PmtA, phospholipid N-methyltransferase; Pcs, phosphatidylcholine synthase.

FIG. 2.

Separation of [¹⁴C]acetate-labeled lipids from the S. meliloti 1021 wild type (A), the psd-deficient mutant, MAV01 (B), the mutant MAV01 complemented with psd-expressing pTB2086 (C), or the pss-deficient mutant, CS111 (D), after growth in complex TY medium. The lipids PC, PE, MMPE, DMPE, OL, SL, PG, CL, PS, and an unknown lipid (U) are indicated.

FIG. 3.

Mutant MAV01 is defective in Psd activity. Lipid products obtained by in vitro activity tests for Pss/Psd using CDP-diacylglycerol and [¹⁴C]serine as substrates were separated by one-dimensional TLC. Activity tests were performed with crude cell extracts from the S. meliloti wild type, strain 1021 (lane 1), and the psd-deficient mutant, MAV01 (lane 2). [¹⁴C]acetate-labeled lipids from S. meliloti 1021 (lane 3) and MAV01 (lane 4) serve as lipid standards. The lipids PC, PE, PG, CL, and PS are indicated.

FIG. 4.

Growth of Sinorhizobium meliloti strains in Sherwood minimal medium with or without choline supplementation. S. meliloti wild-type strain 1021 (diamonds), S. meliloti Pss-deficient mutant CS111 (squares), and S. meliloti Psd-deficient mutant MAV01 (triangles) were grown in Sherwood minimal medium containing 1.3 mM phosphate (A and B) or in Sherwood minimal medium containing 20 μM of phosphate (C and D). Cultures analyzed in panels B and D were supplemented with 10 μM choline chloride. Results of a typical experiment are shown.

FIG. 5.

Symbiotic phenotypes of Sinorhizobium meliloti wild-type and mutant strains on alfalfa plants. Results for S. meliloti wild-type strain 1021 (filled squares), S. meliloti Pss-deficient mutant CS111 (crosses), S. meliloti Psd-deficient mutant MAV01 (filled triangles), water-inoculated control (open circles), S. meliloti complemented mutant MAV01.pTB2086 (open squares), and S. meliloti mutant MAV01 harboring the empty plasmid (open triangles) are shown. Nodules were counted every third day after inoculation. The experiment was continued until no more changes in nodule number were observed. The experiment was repeated three times. The result of a typical experiment is shown.

FIG. 6.

Symbiotic phenotypes of Sinorhizobium meliloti wild-type and mutant strains on alfalfa plants. Alfalfa plant inoculated with S. meliloti wild-type strain 1021 (A), with S. meliloti mutant MAV01 (B), with S. meliloti mutant CS111 (C), or with water (D).