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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 [right-arrow3)-beta -D-Galf-(1right-arrow3)-alpha -D-Galp-(1right-arrow], 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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