Abstract
A procedure for the isolation of mutants affected in components containing glycerol derived from phospholipids yielded two mutant strains that contain membrane-derived oligosaccharides (MDO) devoid of glycerol (Rotering, H., Fiedler, W., Rollinger, W., and Braun, V. (1984) FEMS Microbiol. Lett. 22, 61-68). MDO are found in the periplasmic space of Escherichia coli and other Gram-negative bacteria, and they may comprise up to 7% of the cells dry weight. The biosynthesis of MDO is osmoregulated (Kennedy, E. P. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 1092-1095) and linked to the metabolism of phospholipids (van Golde, L. M. G., Schulman, H., and Kennedy, E. P. (1973) Proc. Natl. Acad. Sci. U. S. A. 70, 1368-1372). This leads to substitution of MDO with sn-1-phosphoglycerol and phosphoethanolamine (Kennedy, E. P., Rumley, M. K., Schulman, P., and van Golde, L. M. G. (1976) J. Biol. Chem. 251, 4208-4213). MDO also contain succinate in O-ester linkage. We now report that one mutant strain lacks phosphoglycerol transferase I activity and thus is unable to transfer sn-1-phosphoglycerol residues from phosphatidylglycerol to MDO. The mdoB gene affected in this mutant has been located at 99.2 min on the E. coli chromosome. The ethanolamine content of MDO isolated from the mutant strain is elevated, whereas the number of succinate residues is not affected. The only phenotype of mdoB mutants we found is a dramatic reduction of the diglyceride content observed in dgk mdoB double mutants when the beta-glucoside arbutin is present in the growth medium.
Highlights
The mdoB gene affected in thismutant hasbeen and UDP-glucose for the synthesis of branched &linked glulocated at 99.2 min on the E. coli chromosome
The only phenotype of mdoB mutants we found is a dramatic reduction of the diglyceride content observed in dgk mdoB double mutants when the &glucoside arbutin is present in the phosphoglycerol from phosphatidylglycerol to carrier bound pre-membrane-derived oligosaccharides (MDO) is catalyzed by a membrane-bound transferase, specified as mdoB, leading to membrane-boundpre-MDO-1-phosphoglyceroalnd 1,2-diglyceride (9)
Blocked in the salvage of diglyceride accumulate the neutral lipid resulting from the biosynthesis of MDO (10)
Summary
MM), M&lZ (0.08 mM),FeSO.(0.5 mg/hter), thiamine (20 pglml), Materials casamino acids (5 g/liter), and arbutin (10 mM). GlycerolDetermination-The glycerolcontent of the MDO samples 1-propranol This solution was passed over a Sephadex G-10 column was determined with the glycerol kinase reaction as described previously (12). Succinate Determination-2 ml of the desalted MDO samples were (22).Arbutin was used as standard for the calibration curve. The samples were concentrated by freeze drying To demonstrate the presence of phosphoglycerol in the derivative and dissolved in 500 plof 50 mM triethanolamine buffer, pH 8.3 Ethanolamine Determination-For amino acid analysis, 50-pl samples of the desalted MDO solutions were hydrolyzed in 6 N HCI for 18 h at 110"C. Under these conditions ethanolamine is liberated from the MDO oligosaccharide chains.
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