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J Bacteriol, February 1998, p. 586-593, Vol. 180, No. 3
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Assignment of Biochemical Functions to Glycosyl Transferase Genes Which Are Essential for Biosynthesis of Exopolysaccharides in Sphingomonas Strain S88 and Rhizobium leguminosarum

Thomas J. Pollock,1,* Wilbert A. T. van Workum,2 Linda Thorne,1 Marcia J. Mikolajczak,1 Motohide Yamazaki,1 Jan W. Kijne,2 and Richard W. Armentrout1

Shin-Etsu Bio, Inc., San Diego, California 92121,1 and Institute of Molecular Plant Sciences, University of Leiden, Leiden, The Netherlands2

Received 15 September 1997/Accepted 19 November 1997

Glycosyl transferases which recognize identical substrates (nucleotide-sugars and lipid-linked carbohydrates) can substitute for one another in bacterial polysaccharide biosynthesis, even if the enzymes originate in different genera of bacteria. This substitution can be used to identify the substrate specificities of uncharacterized transferase genes. The spsK gene of Sphingomonas strain S88 and the pssDE genes of Rhizobium leguminosarum were identified as encoding glucuronosyl-(beta 1right-arrow4)-glucosyl transferases based on reciprocal genetic complementation of mutations in the spsK gene and the pssDE genes by segments of cloned DNA and by the SpsK-dependent incorporation of radioactive glucose (Glc) and glucuronic acid (GlcA) into lipid-linked disaccharides in EDTA-permeabilized cells. By contrast, glycosyl transferases which form alternative sugar linkages to the same substrate caused inhibition of polysaccharide synthesis or were deleterious or lethal in a foreign host. The negative effects also suggested specific substrate requirements: we propose that spsL codes for a glucosyl-(beta 1right-arrow4)-glucuronosyl transferase in Sphingomonas and that pssC codes for a glucuronosyl-(beta 1right-arrow4)-glucuronosyl transferase in R. leguminosarum. Finally, the complementation results indicate the order of attachment of sphingan main-chain sugars to the C55-isoprenylphosphate carrier as -Glc-GlcA-Glc-isoprenylpyrophosphate.

* Corresponding author. Mailing address: Shin-Etsu Bio, Inc., 6650 Lusk Blvd., Suite B106, San Diego, CA 92121. Phone: (619) 455-8500. Fax: (619) 587-2716. E-mail: 76554.3460{at}compuserve.com.




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