Biosynthesis of the Nod Factor Chito-oligosaccharide Backbone in Rhizobium fredii is Controlled by the Concentration of UDP-A/-acetyl-D-glucosamine. Nora Inon de lannino. Mnstituto de Investigaciones Bioqui'micas, Fundacion Campomar, CONICET, Universidad de Buenos Aires.1405, Buenos Aires, Argentina. Steven G. Pueppke(2), and Rodolfo A. Ugalde(1) (1)Mnstituto de Investigaciones Bioqui'micas, Fundacion Campomar, CONICET, Universidad de Buenos Aires.1405, Buenos Aires, Argentina; (2)Department of Plant Pathology, University of Missouri, Columbia, 65211, U.S.A. MPMI 8:292-301. Accepted 14 December 1994. Copyright 1995 The American Phytopathological Society.

A cell-free system was used to study the biochemical properties of the ( (l-4) A/-acetyl-D-glucosaminyl transferase that forms the Nod factor chito-oligosaccharide backbone in Rhizobium fredii. Total membrane fractions prepared from a genistein-induced culture of wild-type R. fredii USDA257 incorporated AZ-acetyl-D-glucosamine at the rate of 6.0 nmoles • h-1 * mg-1 protein into a ( (l-4) N-acetyl-D-glucosamine pentasaccharide. The Km for the substrate UDP-A/-acetyI-D-glucosamine was 42 (M. For maximal activity, the reaction required free jV-acetyl-D-glucosamine that could not be substituted by glucosamine, N-acetyl-D-galactosamine, or galactosamine. Chain elongation of the oligosaccharide proceeded toward the nonreducing end. Pulse-chase experiments demonstrated that the reaction follows a single chain mechanism; free chitobiose, chi-lotriose or chitotelraose were not substrates for elongation and/or initiation. The concentration of UDP-N-acetyl-D-glucosamine controlled the degree of polymerization of the chito-oligosaccharide formed in vitro. At low concentration (0.1 (M) the main product was chitobiose and chi-totriose, and at concentrations higher than 10 (M, chi-topentaose was the main product. Polyoxln D (200 (M) had no effect on the synthesis and or the degree of polymerization of the chito-oligosaccharide formed.