BIOSYNTHESIS OF THE ESCHERICHIA-COLI K4 CAPSULE POLYSACCHARIDE - A PARALLEL SYSTEM FOR STUDIES OF GLYCOSYLTRANSFERASES IN CHONDROITIN FORMATION

Escherichia coli K4 bacteria synthesize a capsule polysaccharide (GalN Ac-GlcA(fructose))(n) with the carbohydrate backbone identical to chon droitin. GlcA- and GalNAc-transferase activities from the bacterial me m brane were assayed with accepters derived from the capsule polysacch aride and radiolabeled UDP-[C-14]GlcA and UDP-[H-3]GalNAc, respectivel y. It was shown that defructosylated oligosaccharides (chondroitin) co uld serve as substrates for both the GlcA- and the GalNAc-transferases . The radiolabeled products were completely degraded with chondroitina se AC; the [C-14]GlcA unit could be removed by beta-D-glucuronidase, a nd the [H-3]GalNAc could be removed by beta-N-acetylhexosaminidase. A fructosylated oligosaccharide acceptor tested for GlcA-transferase act ivity was found to be inactive. These results indicate that the chain elongation reaction of the K4 polysaccharide proceeds in the same way as the polymerization of the chondroitin chain, by the addition of the monosaccharide units one by one to the nonreducing end of the polymer . This makes the biosynthesis of the K4 polysaccharide an interesting parallel system for studies of chondroitin sulfate biosynthesis. In th e biosynthesis of capsule polysaccharides from E. coli, a similar mech anism has earlier been demonstrated for polysialic acid (NeuNAc)(n) (R ohr, T. E., and Troy, F. A. (1980) J. Biol. Chem. 255, 2332-2342) and for the K5 polysaccharide (GlcA beta 1-4GlcNAc alpha 1-4)(n) (Lidholt, K., Fjelstad, M., Jann, K., and Lindahl, U. (1994) Carbohydr. Res. 25 5, 87-101). In contrast, chain elongation of hyaluronan (GlcA beta 1-3 GlcNAc beta 1-4)(n) is claimed to occur at the reducing end (Prehm, P. (1983) Biochem. J. 211, 181-189).