Haemophilus ducreyi produces a novel sialyltransferase - Identification ofthe sialyltransferase gene and construction of mutants deficient in the production of the sialic acid-containing glycoform of the lipooligosaccharide

Haemophilus ducreyi, the cause of the sexually transmitted disease chancroi d produces a lipooligosaccharide (LOS) containing a terminal sialyl N-acety llactosamine trisaccharide, Previously, we reported the identification and characterization of the N-acetylneuraminic acid cytidylsynthetase gene (neu A), Forty-nine base pairs downstream of the synthetase gene is an open read ing frame (ORF) encoding a protein with a predicted molecular weight of 34, 646, This protein has weak homology to the polysialyltransferase of Escheri chia colt K92, Downstream of this ORF is the gene encoding the H, ducreyi h omologue of the Salmonella typhimurium rmlB gene. Mutations were constructe d in the neuA gene and the gene encoding the second ORF by insertion of an Omega kanamycin cassette, and isogenic strains were constructed. LOS was is olated from each strain and characterized by SDS-polyacrylamide gel electro phoresis, carbohydrate, and mass spectrometric analysis, LOS isolated from strains containing a mutation in neuA or in the second ORF, designated Ist, lacked the sialic acid-containing glycoform, Complementation studies were performed, The neuA gene and the ist gene were each cloned into the shuttle vector pLS88 after polymerase chain reaction amplification. Complementatio n of the mutation in the ist gene was observed, but we were unable to compl ement the neuA mutation, Since it is possible that transcription of the neu A gene and the Ist gene were coupled, we constructed a nonpolar mutation in the neuA gene, in this construct, the neuA mutation was complemented, sugg esting transcriptional coupling of the neuA gene and the ist gene, Sialyltr ansferase activity was detected by incorporation of C-14-labeled NeuAc from CMP-NeuAc into trichloroacetic acid-precipitable material when the Ist gen e was overexpressed in the nonpolar neuA mutant. We conclude that the Ist g ene encodes the H, ducreyi sialyltransferase, Since the Ist gene product ha s little, if any, structural relationship to other sialyltransferases, this protein represents a new class of sialyltransferase.