CLONING OF THE LIPOOLIGOSACCHARIDE ALPHA-2,3-SIALYLTRANSFERASE FROM THE BACTERIAL PATHOGENS NEISSERIA-MENINGITIDIS AND NEISSERIA-GONORRHOEAE

The genes encoding the alpha-2,3-sialyltransferases involved in lipool igosaccharide biosynthesis from Neisseria meningitidis and Neisseria g onorrhoeae have been cloned and expressed in Escherichia coli, A high sensitivity enzyme assay using a synthetic fluorescent glycosyltransfe rase acceptor and capillary electrophoresis was used to screen a genom ic library of N. meningitidis MC58 L3 in a ''divide and conquer'' stra tegy, The gene, denoted 1st, was found on a 2.0 kilobase fragment of D NA, and its sequence was determined and then used to design probes to amplify and subsequently clone the corresponding Ist genes from N. men ingitidis 406Y L3, N. meningitidis M982B L7, and N. gonorrhoeae F62, F unctional sialyltransferase was produced from the genes derived from b oth L3 N. meningitidis strains and the N. gonorrhoeae F62, However, th e N. meningitidis M982B L7 gene contained a frameshift mutation that r enders it inactive, The expression of the 1st gene was easily detected using the enzyme assay, and the protein expression could be detected when an immunodetection tag was added to the COOH-terminal end of the protein, Using the synthetic acceptor inophenyl-(6-(5-(fluorescein-car boxamido)-hexanoic acid amide), the alpha-2,3 specificity of the enzym e was confirmed by NMR examination of the reaction product. The enzyme could also use synthetic accepters with lactose or galactose as the s accharide portion, This study is the first example of the cloning, exp ression, and examination of alpha-2,5-sialyltransferase activity from a bacterial source.