LIPID A MODIFICATION SYSTEMS IN GRAM-NEGATIVE BACTERIA

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Lipopolysaccharides (LPSs) are remarkable glycolipids that comprise the outer surfaces of Gram-negative bacteria, including the symbiotic organism, Rhizobium leguminosamm. In Escherichia coli, the lipid A anchor of LPS is a hexa-acylated disaccharide of glucosamine, bearing phosphate moieties at positions 1 and 4'. The minimal LPS required for growth of E. coli consists of lipid A and two extra sugars. Emerging genomic sequences indicate that the enzymes that make lipid A in E. coli are present in most other Gram-negative bacteria. Lipid A (often termed endotoxin) is also the active component of LPS responsible for the clinical complications of Gram-negative sepsis. Minor modifications in the structure of lipid A can have profound effects on pathogenesis. Some lipid A analogs are actually potent endotoxin antagonists. Compared to E. coli, the chemical structures of the lipid A and core domains of/?, leguminosarum LPS are very unusual. R. leguminosarum lipid A lacks the 1 and 4' phosphates, but is modified with galacturonic acid at position 4'. It is acylated with a peculiar 28 carbon fatty acid, and contains 2-deoxy-2-aminogluconate in place of the proximal glucosamine. The structure of/?, leguminosarum LPS indicates the existence of novel enzymes for generating diverse lipid A and core species. It is now established that the first seven enzymes of lipid A I biosynthesis are in fact the same in E. coli and /?. leguminosarum. The differences arise in the later stages of the pathway. To date, enzymes identified as unique to /?. leguminosarum include a 4'-phosphatase that is also a phosphotransferase, a 1-phosphatase, a long chain acyltransferase with its own acyl carrier protein, and three distinct core glycosyltransferases. Characterization of the /?. leguminosarum system should provide insights into the functions of lipid A-like molecules, including special roles during symbiosis in plants, and affords the opportunity to create novel lipid A hybrids that may have interesting adjuvant or antagonist activities. Some structural features of/?, leguminosarum lipid A are seen in human pathogens. Legionella pneumophila lipid A i contains a C28 chain, while Porphyromonas gingivalis and Helicobacter pylori lipid A lack the 4' phosphate. | In the coming grant period, the specific aims are: I) cloning of the C28 acyltransferase of/?, leguminosarum; i II) analysis of the lipid A 4'-phosphatase/phosphotransferase, especially its ability to synthesize PtdIns-4-P; III) determination of the enzymatic basis for proximal unit diversity in /?. leguminosarum lipid A; and IV) i characterization of enzymes that incorporate the unique inner core sugars of/?, leguminosarum LPS.