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Journal of Bacteriology, June 2001, p. 3318-3327, Vol. 183, No. 11
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.11.3318-3327.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Functional Analysis of the Galactosyltransferases
Required for Biosynthesis of D-Galactan I, a Component of
the Lipopolysaccharide O1 Antigen of Klebsiella
pneumoniae
Shukui
Guan,
Anthony J.
Clarke, and
Chris
Whitfield*
Department of Microbiology, University of
Guelph, Guelph, Ontario N1G 2W1, Canada
Received 22 December 2000/Accepted 13 March 2001
D-Galactan I is an O-antigenic polymer with the repeat
unit structure
[
3)-
-D-Galf-(1
3)-
-D-Galp-(1
],
that is found in the lipopolysaccharide of Klebsiella
pneumoniae O1 and other gram-negative bacteria. A genetic locus
containing six genes is responsible for the synthesis and assembly of
D-galactan I via an ATP-binding cassette (ABC)
transporter-dependent pathway. The galactosyltransferase activities
that are required for the processive polymerization of
D-galactan I were identified by using in vitro reactions.
The activities were determined with endogenous lipid acceptors in membrane preparations from Escherichia coli K-12
expressing individual enzymes (or combinations of enzymes) or in
membranes reconstituted with specific lipid acceptors. The
D-galactan I polymer is built on a lipid acceptor,
undecaprenyl pyrophosphoryl-GlcpNAc, a product of the
WecA enzyme that participates in the biosynthesis of enterobacterial common antigen and O-antigenic polysaccharide (O-PS)
biosynthesis pathways. This intermediate is directed into
D-galactan I biosynthesis by the bifunctional
wbbO gene product, which sequentially adds one
Galp and one Galf residue from the
corresponding UDP-sugars to form a lipid-linked trisaccharide. The two
galactosyltransferase activities of WbbO are separable by limiting the
UDP-Galf precursor. Galactosyltransferase activity in
membranes reconstituted with exogenous lipid-linked trisaccharide
acceptor and the known structure of D-galactan I indicate
that WbbM catalyzes the subsequent transfer of a single
Galp residue to form a lipid-linked tetrasaccharide. Chain extension of the D-galactan I polymer requires WbbM
for Galp transferase, together with Galf
transferase activity provided by WbbO. Comparison of the biosynthetic
pathways for D-galactan I and the polymannose E.
coli O9a antigen reveals some interesting features that may
reflect a common theme in ABC transporter-dependent O-PS assembly systems.
*
Corresponding author. Mailing address: Department of
Microbiology, University of Guelph, Guelph, Ontario N1G 2W1, Canada. Phone: (519) 824-4120, ext. 3478. Fax: (519) 837-1802. E-mail: cwhitfie{at}uoguelph.ca.
Journal of Bacteriology, June 2001, p. 3318-3327, Vol. 183, No. 11
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.11.3318-3327.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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