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Taxonomic group: bacteria / Proteobacteria (Phylum: Proteobacteria)
Associated disease: infection due to Escherichia coli [ICD11: XN6P4

]

NCBI PubMed ID: 20208062
Journal NLM ID: 9104124
Publisher: IRL Press at Oxford University Press
Correspondence: bgibson

buckinstitute.org
Institutions: Department of Pharmaceutical Chemistry and Pharmaceutical Sciences, University of California, San Francisco, CA, USA

In previous work, our laboratory generated novel chimeric lipopolysaccharides (LPS) in Escherichia coli transformed with a plasmid containing exogenous lipooligosaccharide synthesis genes (lsg) from Haemophilus influenzae. Analysis of these novel oligosaccharide-LPS chimeras allowed characterization of the carbohydrate structures generated by several putative glycosyltransferase genes within the lsg locus. Here, we adapted this strategy to construct a modular approach to study the synthetic properties of individual glycosyltransferases expressed alone and in combinations. To this end, a set of expression vectors containing one to four putative glycosyltransferase genes from the lsg locus, lsgC-F, were transformed into E. coli K12 (XL-1) which is defective in LPS O-antigen biosynthesis. This strategy relied on the inclusion of the H. influenzae gene product lsgG in every plasmid construct, which partially rescues the E. coli LPS biosynthesis defect by priming UDP-undecaprenyl in the WecA-dependent O-antigen synthetic pathway with N-acetyl-glucosamine (GlcNAc). This GlcNAc-undecaprenyl then served as an acceptor substrate for further carbohydrate extension by transformed glycosyltransferases. The resultant LPS-linked chimeric glycans were isolated from their E. coli constructs and characterized by mass spectrometry, methylation analysis and ELISA. These structural data allowed the specificity of various glycosyltransferases to be unambiguously assigned to individual genes. LsgF was found to transfer a galactose (Gal) to terminal GlcNAc. LsgE was found to transfer GlcNAc to Gal-GlcNAc, and both LsgF and LsgD were found to transfer Gal to GlcNAc-Gal-GlcNAc, but with differing linkage specificities. This method can be generalized and readily adapted to study the substrate specificity of other putative or uncharacterized glycosyltransferases.

Escherichia coli, mass spectrometry, glycosyltransferase, assay, E. coli, chimera

Structure type: oligomer
Location inside paper: p.764, table I, p.767, fig.2
Compound class: core oligosaccharide
Contained glycoepitopes: IEDB_130646,IEDB_130650,IEDB_130670,IEDB_130697,IEDB_135813,IEDB_136044,IEDB_136906,IEDB_137340,IEDB_137472,IEDB_137776,IEDB_1391962,IEDB_140088,IEDB_140108,IEDB_140122,IEDB_140529,IEDB_141794,IEDB_141807,IEDB_142078,IEDB_142488,IEDB_143794,IEDB_144998,IEDB_145003,IEDB_146664,IEDB_150899,IEDB_151528,IEDB_151531,IEDB_167070,IEDB_190606,IEDB_2189047,IEDB_226811,IEDB_232584,IEDB_983931,SB_137,SB_165,SB_166,SB_173,SB_187,SB_192,SB_195,SB_29,SB_30,SB_7,SB_88

Methods: HF solvolysis, de-O-acylation, SDS-PAGE, ELISA, MALDI-TOF MS, genetic methods
Biosynthesis and genetic data: genetic data
Comments, role: core 1, core 2, E1, E2, E3 and E4 glycoforms of the chimeric E.coli LPS structures from clones CG, DG, EG, FG, CFG, DFG, EFG, CEFG, DEFG and LOS-05.

NCBI Taxonomy refs (TaxIDs): 83333
Reference(s) to other database(s): GTC:G90168NC, GlycomeDB:37805
Show glycosyltransferases

There are 2 chemically distinct structures. Please, select:

CC(=O)N[C@H]1C(O[C@H]2[C@@H](O)[C@@H](CO)OC(O[C@H]3[C@H](O)[C@@H](CO)OC(OC[C@H](O)[C@H]4O[C@H](OC[C@H]5O[C@H](O[C@@H]6[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]6O[C@H]6[C@H](O)[C@@H](CO[C@H]7O[C@H](CO)[C@H](O)[C@H](O)[C@H]7O)O[C@H](O[C@@H]7[C@H](O)[C@@H](O[C@@H]8[C@H](O)[C@@H](O[C@@H]9[C@H](O)CC(O)(C(=O)O)O[C@@H]9[C@H](O)CO)O[C@H]([C@@H](O)CO)[C@H]8O)O[C@H]([C@@H](O)CO[C@H]8O[C@H]([C@@H](O)CO)[C@@H](O)[C@H](O)[C@@H]8O)[C@H]7O)[C@@H]6O)[C@H](O)[C@@H](O)[C@@H]5O)[C@@H](O)[C@@H](O)[C@@H]4O)[C@@H]3NC(C)=O)[C@@H]2O)O[C@H](CO)[C@@H](OC2O[C@H](CO)[C@H](O)[C@H](O)[C@H]2O)[C@@H]1O

SVG MW SMILES 3D mol Ionize