Multiple Phospholipid N-Methyltransferases with Distinct Substrate Specificities Are Encoded in Bradyrhizobium japonicum

Lipid formation after heterologous expression of pmtA from B. japonicum and four genes encoding putative Pmt enzymes from B. japonicum in E. coli. E. coli BL21(DE3)/pLysS cells expressing the indicated enzymes were labeled with [¹⁴C]acetate or [methyl-¹⁴C]methionine during growth on LB medium. Panel A shows the resulting phospholipid profile after single expression of (putative) Pmts in E. coli: panels B and C show the resulting phospholipid profile after coexpression of the phospholipid methyltransferase PmtA from B. japonicum with ORFs encoding putative Pmts in E. coli. After extraction, lipids were separated by one-dimensional TLC. Lane M, marker (S. meliloti lipids labeled with [¹⁴C]acetate); lane C, control with BL21(DE3)/pLysS/pET9a (+pRK404 for panels B and C); lane A, pmtA; lane X1, pmtX1; lane X2, pmtX2; lane X3, pmtX3; and lane X4, pmtX4 (genes encoded in the indicated vector backbone [see also Materials and Methods and Table 1]); lane Bj, B. japonicum lipids labeled with [¹⁴C]acetate. The lipids PG, cardiolipin (CL), PE, MMPE, DMPE, and PC are indicated.

Physical and genetic maps of the B. japonicum pmtA (A), pmtX1-4 (B to E), and pcs (F) gene regions. Physical maps are given for SalI, StuI, Ecl136II, and PstI (indicated by S, St, E, and P, respectively). Black arrows indicate the position of chromosomally integrated translational lacZ fusions and point to the corresponding strain designation. Below each map, the insertion sites for antibiotic resistance cassettes (Km in the case of pmtA and pmtX2-4; ΩSm/Sp in the case of pcs) or the plasmid integration site are depicted. Antibiotic resistance cassettes are not drawn to scale. Km, kanamycin; Sm, streptomycin; Sp, spectinomycin; dnaJ, chaperone protein DnaJ; pmtA, Pmt; blr0682, putative oxidoreductase; pyrF, orotidine-5′-monophosphat-decarboxylase; blr0684, blr6996, bll6993, bll6633, blr8167, bll4801, and bll4584, hypothetical ORFs; trmU, tRNA (5-methylaminomethyl-2-thiouridylate)-methyltransferase; pmtX1, phosphatidylethanolamine N-methyltransferase (EC 2.1.1.17); blr6636, ATP synthase subunit; pmtX2, phosphatidylethanolamine N-methyltransferase (EC 2.1.1.17); bll6635, probable glycosyl transferase; bll8168, Na⁺/H⁺ antiporter; pmtX3, Pmt; bll8165, putative alkaline phosphatase; blr4802, two-component response regulator; blr4803, two-component hybrid sensor and regulator; pmtX4, Pmt; pyrG, CTP synthetase; blr4586, putative oxidoreductase protein; pcs, Pcs; qor, quinone oxidoreductase.

Expression of B. japonicum PC biosynthesis genes. The expression of chromosomally integrated, translational lacZ reporter gene fusions (see Fig. 3) was measured as described previously (see Materials and Methods). Samples were collected after 24, 48, and 72 h. The results of a typical experiment, in which two parallel cultures of each strain were grown and assayed in duplicate, are shown. Four independent experiments were performed with individual strains.

Phospholipid profile of PC biosynthesis mutants after in vivo labeling with [¹⁴C]acetic acid. B. japonicum strains were labeled with [¹⁴C]acetate during growth in PSY medium. After extraction, lipids were separated by one-dimensional TLC. The lipids PC, DMPE, MMPE, and PE are indicated. The relative PC content (as the percentage of total phospholipids) is given below each PC spot.

Expression of the PC biosynthesis genes in the pmtA mutant compared to the wild type. The expression of chromosomally integrated, translational lacZ reporter gene fusions (see Fig. 3) was measured as described previously (see Materials and Methods). Samples were collected after 24, 48, and 72 h. The results of a typical experiment, in which two parallel cultures of each strain were grown and assayed in duplicate, are shown. Four independent experiments were performed with individual strains.

Symbiotic phenotype of B. japonicum pmt and pcs mutants. The nitrogen fixation activity was measured as the amount of C₂H₂ reduced per hour per gram (dry weight) of nodule. A 100% wild-type (WT) activity corresponds to 93.7 μmol of C₂H₂/h/g. Mean values and standard deviations derive from at least eight individual plants, except for the pcs mutant with six individual plants.