Role of Rfe and RfbF in the initiation of biosynthesis of D-galactan I, the lipopolysaccharide O antigen from Klebsiella pneumoniae serotype O1

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Abstract
The 6.6-kb rfb gene cluster from Klebsiella pneumoniae serotype O1 (rfbKpO1) contains six genes whose products are required for the biosynthesis of a lipopolysaccharide O antigen with the following repeating unit structure: -->3-beta-D-Galf-1-->3-alpha-D-Galp-1-->(D-galactan I). rfbFKpO1 is the last gene in the cluster, and its gene product is required for the initiation of D-galactan I synthesis. Escherichia coli K-12 strains expressing the RfbFKpO1 polypeptide contain dual galactopyranosyl and galactofuranosyl transferase activity. This activity modifies the host lipopolysaccharide core by adding the disaccharide beta-D-Galf-1-->3-alpha-D-Galp, representing a single repeating unit of D-galactan I. The formation of the lipopolysaccharide substituted either with the disaccharide or with authentic polymeric D-galactan I is dependent on the activity of the Rfe enzyme. Rfe (UDP-GlcpNAc::undecaprenylphosphate GlcpNAc-1-phosphate transferase) catalyzes the formation of the lipid-linked biosynthetic intermediate to which galactosyl residues are transferred during the initial steps of D-galactan I synthesis. The rfbFKpO1 gene comprises 1,131 nucleotides, and the predicted polypeptide consists of 373 amino acid residues with a predicted M(r) of 42,600. A polypeptide with an M(r) of 42,000 was evident in sodium dodecyl sulfate-polyacrylamide gels when rfbKpO1 was expressed behind the T7 promoter. The carboxy-terminal region of RfbFKpO1 shares similarity with the carboxy terminus of RfpB, a galactopyranosyl transferase which is involved in the synthesis of the type 1 O antigen of Shigella dysenteriae.

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