The capsular polysaccharide of Escherichia coli K92 contains alternating -8-NeuAcalpha2- and -9-NeuAcalpha2- linkages. The enzyme catalyzing this polymerizing reaction has been cloned from the genomic DNA of E. coli K92. The 1.2-kilobase polymerase chain reaction fragment was subcloned in pRSET vector and the protein was expressed in the BL21(DE3) strain of E. coli with a hexameric histidine at its N-terminal end. The enzyme was isolated in the supernatant after lysis of the cells and fractionated by ultracentrifugation. Western blotting using anti-histidine antibody showed the presence of a band that migrated at about 47.5 kDa on both reducing and nonreducing SDS-polyacrylamide gel electrophoresis, indicating a monomeric enzyme. Among the carbohydrate acceptors tested, N-acetylneuraminic acid and the gangliosides G(D3) and G(Q1b) were preferred substrates. The cell-free enzyme reaction products obtained were characterized by NMR and mass spectrometry, which indicated the presence of both alpha2,9- and alpha2,8-linked polysialyl structure. The K92 neuS gene was used to transform the K1 strain of E. coli, the capsule of which contains only -8-NeuAcalpha2- linkages. Analysis of the polysaccharides isolated from these transformed cells is consistent with the presence of both -8-NeuAcalpha2- and -9-NeuAcalpha2- linkages. Our results suggest that the neuS gene product of E. coli K92 catalyzes the synthesis of polysialic acid with alpha2,9- and alpha2,8-linkages in vitro and in vivo.