Roles of Predicted Glycosyltransferases in the Biosynthesis of the Rhizobium etli CE3 O

TheRhizobium etliCE3O antigen is a fixed-length heteropolymer. The genetic regions required for its synthesis have been identified, and the nucleotide sequences are known. The structure of theO antigen has been determined, but the roles of specific genes in synthe-sizing this structure are relatively unclear.Within the knownO-antigen genetic clusters of this strain, nine open reading frames (ORFs) were found to contain a conserved glycosyltransferase domain. EachORFwasmutated, and the resultingmutant lipopolysaccharide (LPS)was analyzed. Tricine SDS-PAGE revealed stepwise truncations of theO antigen thatwere consistentwith differences inmutant LPS sugar compositions and reactivitywithO-antigen-specificmonoclonal antibodies. Based on these results and current theories of O-antigen synthesis, specific roles were deduced for each of the nine glycosyltransferases, and amodel for biosynthesis of theR. etli CE3Oantigenwas proposed. In thismodel, O-antigen biosynthesis is initiatedwith the addition ofN-acetyl-quinovosamine-phos-phate (QuiNAc-P) to bactoprenol-phosphate by glycosyltransferaseWreU.GlycosyltransferasesWreG,WreE,WreS, andWreTwould each act once to attachmannose, fucose, a second fucose, and 3-O-methyl-6-deoxytalose (3OMe6dTal), respectively.WreHwould then catalyze the addition ofmethyl glucuronate (MeGlcA) to complete the first instance of theO-antigen repeat unit. Four subsequent re-peats of this unit composed of fucose, 3OMe6dTal, andMeGlcAwould be assembled by a cycle of reactions catalyzed by two additional glycosyltransferases,WreMandWreL, alongwithWreH. Finally, theO antigenwould be capped by attachment of di- or tri-O-methyl-ated fucose as catalyzed by glycosyltransferaseWreB. Lipopolysaccharide (LPS) is the major surface component ofGram-negative bacteria and consists of lipid A, a core oligo