ROLE OF RFE AND RFBF IN THE INITIATION OF BIOSYNTHESIS OF D-GALACTAN-I, THE LIPOPOLYSACCHARIDE O-ANTIGEN FROM KLEBSIELLA-PNEUMONIAE SEROTYPE-O1

The 6.6-kb rfb gene cluster from Klebsiella pneumoniae serotype O1 (rfb(KpO1)) 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). rfbF(KpO1) 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 RfbF(KpO1) 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 rfbF(KpO1) gene comprises 1,131 nucleotides, and the predicted polypeptide consists of 373 amino acid residues,vith 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 rfb(KpO1) was expressed behind the T7 promoter. The carboxyterminal region of RfbF(KpO1) 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.