Publications

• Expression, purification and biochemical characterization of GumI, a monotopic membrane GDP-mannose:glycolipid 4-beta-D-mannosyltransferase from Xanthomonas campestris pv. campestris.

  Salinas S.R., Bianco M.I., Barreras M., Ielpi L.

  We describe the first biochemical characterization of the gumI gene product, an essential protein for xanthan polysaccharide synthesis. Cellular fractionation experiments reveal the presence of a protein associated with the membrane fraction, even in the absence of the other proteins responsible f ... >> More

  We describe the first biochemical characterization of the gumI gene product, an essential protein for xanthan polysaccharide synthesis. Cellular fractionation experiments reveal the presence of a protein associated with the membrane fraction, even in the absence of the other proteins responsible for the synthesis of glycolipid intermediates and the proteins involved in the polymerization and transport of the xanthan chains. By alkaline buffer extraction and detergent phase partitioning, GumI was categorized as a monotopic membrane protein. GumI was overexpressed in Escherichia coli, solubilized and purified in an active and stable form using a simple and reproducible two-step procedure. The purified recombinant GumI is a nonprocessive β-mannosyltransferase that uses GDP-Man as a donor substrate and glucuronic acid-β-1,2-mannose-α-1,3-glucose-β-1,4-glucose-PP-polyisoprenyl as an acceptor. We also established the optimal biochemical conditions for GumI enzymatic activity. Sequence analysis revealed the presence of a conserved domain for glycosyltransferases (GTs) of the GT-B superfamily and homologous proteins in several prokaryote organisms. On the basis of this biochemical characterization, GumI may represent the founding member of a new GT family in the Carbohydrate-Active EnZymes classification. << Less

  Glycobiology 21:903-913(2011) [PubMed] [EuropePMC]

• Sequential assembly and polymerization of the polyprenol-linked pentasaccharide repeating unit of the xanthan polysaccharide in Xanthomonas campestris.

  Ielpi L., Couso R.O., Dankert M.A.

  Lipid-linked intermediates are involved in the synthesis of the exopolysaccharide xanthan produced by the bacterium Xanthomonas campestris (L. Ielpi, R. O. Couso, and M. A. Dankert, FEBS Lett. 130:253-256, 1981). In this study, the stepwise assembly of the repeating pentasaccharide unit of xanthan ... >> More

  Lipid-linked intermediates are involved in the synthesis of the exopolysaccharide xanthan produced by the bacterium Xanthomonas campestris (L. Ielpi, R. O. Couso, and M. A. Dankert, FEBS Lett. 130:253-256, 1981). In this study, the stepwise assembly of the repeating pentasaccharide unit of xanthan is described. EDTA-treated X. campestris cells were used as both enzyme preparation and lipid-P acceptor, and UDP-Glc, GDP-Man, and UDP-glucuronic acid were used as sugar donors. A linear pentasaccharide unit is assembled on a polyprenol-P lipid carrier by the sequential addition of glucose-1-P, glucose, mannose, glucuronic acid, and mannose. The in vitro synthesis of pentasaccharide-P-P-polyprenol was also accompanied by the incorporation of radioactivity into a polymeric product, which was characterized as xanthan, on the basis of gel filtration and permethylation studies. Results from two-stage reactions showed that essentially pentasaccharide-P-P-polyprenol is polymerized. In addition, the direction of chain elongation has been studied by in vivo experiments. The polymerization of lipid-linked repeat units occurs by the successive transfer of the growing chain to a new pentasaccharide-P-P-polyprenol. The reaction involves C-1 of glucose at the reducing end of the polyprenol-linked growing chain and C-4 of glucose at the nonreducing position of the newly formed polyprenol-linked pentasaccharide, generating a branched polymer with a trisaccharide side chain. << Less

  J Bacteriol 175:2490-2500(1993) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Mutational analysis of the gum gene cluster required for xanthan biosynthesis in Xanthomonas oryzae pv oryzae.

  Kim S.Y., Kim J.G., Lee B.M., Cho J.Y.

  Genome sequence analysis of Xanthomonas oryzae pv. oryzae has revealed a cluster of 12 ORFs that are closely related to the gum gene cluster of Xanthomonas campestris pv. campestris. The gum gene cluster of X. oryzae encodes proteins involved in xanthan production; however, there is little experim ... >> More

  Genome sequence analysis of Xanthomonas oryzae pv. oryzae has revealed a cluster of 12 ORFs that are closely related to the gum gene cluster of Xanthomonas campestris pv. campestris. The gum gene cluster of X. oryzae encodes proteins involved in xanthan production; however, there is little experimental evidence supporting this. In this study, biochemical analyses of xanthan produced by a defined set of X. oryzae gum mutant strains allowed us to preliminarily assign functions to most of the gum gene products: biosynthesis of the pentasaccharide repeating unit for GumD, GumM, GumH, GumK, and GumI, xanthan polymerization and transport for GumB, GumC, GumE, and GumJ, and modification of the pentasaccharide repeating unit for GumF, GumG, and GumL. In addition, we found that the exopolysaccharides are essential but not specific for the virulence of X. oryzae. << Less

  Biotechnol. Lett. 31:265-270(2009) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Xanthomonas campestris pv. campestris gum mutants: effects on xanthan biosynthesis and plant virulence.

  Katzen F., Ferreiro D.U., Oddo C.G., Ielmini M.V., Becker A., Puhler A., Ielpi L.

  Xanthan is an industrially important exopolysaccharide produced by the phytopathogenic, gram-negative bacterium Xanthomonas campestris pv. campestris. It is composed of polymerized pentasaccharide repeating units which are assembled by the sequential addition of glucose-1-phosphate, glucose, manno ... >> More

  Xanthan is an industrially important exopolysaccharide produced by the phytopathogenic, gram-negative bacterium Xanthomonas campestris pv. campestris. It is composed of polymerized pentasaccharide repeating units which are assembled by the sequential addition of glucose-1-phosphate, glucose, mannose, glucuronic acid, and mannose on a polyprenol phosphate carrier (L. Ielpi, R. O. Couso, and M. A. Dankert, J. Bacteriol. 175:2490-2500, 1993). A cluster of 12 genes in a region designated xpsI or gum has been suggested to encode proteins involved in the synthesis and polymerization of the lipid intermediate. However, no experimental evidence supporting this suggestion has been published. In this work, from the biochemical analysis of a defined set of X. campestris gum mutants, we report experimental data for assigning functions to the products of the gum genes. We also show that the first step in the assembly of the lipid-linked intermediate is severely affected by the combination of certain gum and non-gum mutations. In addition, we provide evidence that the C-terminal domain of the gumD gene product is sufficient for its glucosyl-1-phosphate transferase activity. Finally, we found that alterations in the later stages of xanthan biosynthesis reduce the aggressiveness of X. campestris against the plant. << Less

  J. Bacteriol. 180:1607-1617(1998) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.