CHARACTERIZATION OF THE LIPID-CARRIER INVOLVED IN THE SYNTHESIS OF ENTEROBACTERIAL COMMON ANTIGEN (ECA) AND IDENTIFICATION OF A NOVEL PHOSPHOGLYCERIDE IN A MUTANT OF SALMONELLA-TYPHIMURIUM DEFECTIVE IN ECA SYNTHESIS

The polysaccharide chains of enterobacterial common antigen (ECA) cons ist of linear trisaccharide repeat units with the structure -->4)-beta -D-ManNAcA-(1-->4)-alpha-D-GlcNAc-(1-->, where Fuc4NAc is 4-acetamido- 4,6-di-deoxy-D-galactose, ManNAcA is N-acetyl-D- mannosaminuronic acid , and GlcNAc is N-acetyl-D-glucosamine. The major form of ECA (ECA(PG) ) consists of polysaccharide chains that are believed to be covalently linked to diacylglycerol through phosphodiester linkage; the phosphol ipid moiety functions to anchor molecules in the outer membrane. The E CA trisaccharide repeat unit has been tentatively identified as Fuc4NA c-ManNAcA-GlcNAc-pyrophosphorylundecaprenol (lipid III). Subsequent ch ain-elongation presumably occurs by a blocking-polymerization mechanis m. However, the identity of the polyisoprenoid carrier-lipid has not b een established. Accordingly, the current studies were conducted in an effort to structurally characterize the polyisoprenyl lipid-carrier i nvolved in ECA synthesis. Isolation and characterization of the lipid carrier was facilitated by the accumulation of a ManNA-cA-GlcNAc-pyrop hosphorylpolyisoprenyl lipid (lipid II) in mutants of Salmonella typhi murium defective in the synthesis of TDP-Fuc4NAc, the donor of Fuc4NAc residues for ECA synthesis. Analyses of lipid II preparations by fast atom bombardment tandem mass spectroscopy (FAB-MS/MS) resulted in the identification of a novel glycolipid which copurified with lipid II. FAB-MS/MS analyses of this glycolipid revealed its structure to be -di acyl-sn-glycero-3-pryophosphoryl-GlcNAc-ManNAcA (DGP-disaccharide). An examination of purified ECA(PG) by phosphorus-31 nuclear magnetic res onance spectroscopy confirmed that the polysaccharide chains are linke d to diacylglycerol through phosphodiester linkage. Thus, DGP-disaccha ride does not appear to be an intermediate in ECA(PG) synthesis. Never theless, although the available evidence clearly indicate that lipid I I is a precursor of DGP-disaccharide, the function of this novel glyco lipid is not yet known, and it may be an intermediate in the biosynthe sis of a molecule other than ECA(PG).