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J Biol Chem, Vol. 274, Issue 7, 4106-4114, February 12, 1999
Haemophilus ducreyi Produces a Novel
Sialyltransferase
IDENTIFICATION OF THE SIALYLTRANSFERASE GENE AND CONSTRUCTION OF
MUTANTS DEFICIENT IN THE PRODUCTION OF THE SIALIC ACID-CONTAINING
GLYCOFORM OF THE LIPOOLIGOSACCHARIDE
Joel A.
Bozue ,
Michael V.
Tullius¶,
Jing
Wang ,
Bradford W.
Gibson¶, and
Robert S.
Munson Jr.
From the Children's Hospital Research Foundation and Departments
of Pediatrics and Medical Microbiology, Ohio State
University, Columbus, Ohio 43205-2696, ¶ Department of
Pharmaceutical Chemistry, University of California,
San Francisco, California 94143-0446
Haemophilus ducreyi, the
cause of the sexually transmitted disease chancroid produces a
lipooligosaccharide (LOS) containing a terminal sialyl
N-acetyllactosamine trisaccharide. Previously, we reported
the identification and characterization of the
N-acetylneuraminic acid cytidylsynthetase gene
(neuA). Forty-nine base pairs downstream of the synthetase
gene is an open reading frame (ORF) encoding a protein with a predicted
molecular weight of 34,646. This protein has weak homology to the
polysialyltransferase of Escherichia coli K92. Downstream
of this ORF is the gene encoding the H. ducreyi homologue
of the Salmonella typhimurium rmlB gene. Mutations were constructed in the neuA gene and the gene encoding the
second ORF by insertion of an kanamycin cassette, and isogenic
strains were constructed. LOS was isolated from each strain and
characterized by SDS-polyacrylamide gel electrophoresis, carbohydrate,
and mass spectrometric analysis. LOS isolated from strains containing a mutation in neuA or in the second ORF, designated
lst, lacked the sialic acid-containing glycoform.
Complementation studies were performed. The neuA gene and
the lst gene were each cloned into the shuttle vector pLS88
after polymerase chain reaction amplification. Complementation of the
mutation in the lst gene was observed, but we were unable
to complement the neuA mutation. Since it is possible that
transcription of the neuA gene and the lst gene
were coupled, we constructed a nonpolar mutation in the neuA gene. In this construct, the neuA
mutation was complemented, suggesting transcriptional coupling of the
neuA gene and the lst gene. Sialyltransferase
activity was detected by incorporation of 14C-labeled NeuAc
from CMP-NeuAc into trichloroacetic acid-precipitable material when the
lst gene was overexpressed in the nonpolar neuA mutant. We conclude that the lst gene encodes the H. ducreyi sialyltransferase. Since the lst gene product
has little, if any, structural relationship to other
sialyltransferases, this protein represents a new class of sialyltransferase.
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.

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Copyright © 1999 by the American Society for Biochemistry and Molecular Biology.
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