The sialyl-T antigen sialyl␣2-3Gal␤1-3GalNAc is a common O-glycan structure in human glycoproteins and is synthesized by sialyltransferase ST3Gal1. The enterohemorrhagic Escherichia coli serotype O104 has the rare ability to synthesize a sialyl-T antigen mimic. We showed here that the wbwA gene of the E. coli O104 antigen synthesis gene cluster encodes an ␣2,3-sialyltransferase WbwA that transfers sialic acid from CMP-sialic acid to Gal␤1-3GalNAc␣-diphosphate-lipid acceptor. Nuclear magnetic resonance (NMR) analysis of purified WbwA enzyme reaction product indicated that the sialyl-T antigen sialyl␣2-3Gal␤1-3GalNAc␣-diphosphate-lipid was synthesized. We showed that the conserved His-Pro (HP) motif and Glu/Asp residues of two EDG motifs in WbwA are important for the activity. The characterization studies showed that WbwA from E. coli O104 is a monofunctional ␣2,3-sialyltransferase and is distinct from human ST3Gal1 as well as all other known sialyltransferases due to its unique acceptor specificity. This work contributes to knowledge of the biosynthesis of bacterial virulence factors. IMPORTANCEThis is the first characterization of a sialyltransferase involved in the synthesis of an O antigen in E. coli. The enzyme contributes to the mimicry of human sialyl-T antigen and has unique substrate specificity but very little sequence identity to other sialyltransferases. Thus, the bacterial sialyltransferase is related to the human counterpart only by the similarity of biochemical activity. Sialic acids are ubiquitous in nature and contribute to the acidity and hydration of a glycoprotein, metal ion binding, and epitope exposure. Sialylation affects the adhesive properties of cells and has been implicated in the functions of cell surface receptors (1). Sialic acids are also found in O antigens of bacterial lipopolysaccharides (LPS) or lipooligosaccharides, for example, in Neisseria, Campylobacter, Pasteurella, or Photobacterium spp. (2-5). In Escherichia coli, O antigens containing sialic acid linkages are rare and have only been found in serotypes O24, O55, O104, O145, and O171 (ECODAB). Specific proteins in the mammalian nervous system as well as a number of bacterial capsules carry polysialic acids that regulate adhesive properties of the cell. Masking their own antigens with human-like structures derived from glycolipids and glycoproteins may help bacteria to escape the host immune system. For example, Campylobacter jejuni has lipooligosaccharides resembling human gangliosides (2). However, cross-reactivity in infected humans can lead to autoimmune disease affecting the nervous system, for example the Guillain-Barré syndrome (3). O-acetylation of sialic acids controls the exposure of sialyl epitopes (1).The enterohemorrhagic, Shiga toxin-producing E. coli serotype O104 is the causative agent of outbreaks of bloody diarrhea and hemolytic-uremic syndrome with significant mortality (6, 7). The E. coli O104 antigen has the unusual repeating unit structure (DGal␣1-4Neu5,7,9Ac 3 ␣2-3-D-Gal␤1-3-D-GalNAc␤1) n (ECOD...