Abstract
Xanthan is an industrially important exopolysaccharide produced by the phytopathogenic, gram-negative bacterium Xanthomonas campestris pv. campestris. It is composed of polymerized pentasaccharide repeating units which are assembled by the sequential addition of glucose-1-phosphate, glucose, mannose, glucuronic acid, and mannose on a polyprenol phosphate carrier (L. Ielpi, R. O. Couso, and M. A. Dankert, J. Bacteriol. 175:2490-2500, 1993). A cluster of 12 genes in a region designated xpsI or gum has been suggested to encode proteins involved in the synthesis and polymerization of the lipid intermediate. However, no experimental evidence supporting this suggestion has been published. In this work, from the biochemical analysis of a defined set of X. campestris gum mutants, we report experimental data for assigning functions to the products of the gum genes. We also show that the first step in the assembly of the lipid-linked intermediate is severely affected by the combination of certain gum and non-gum mutations. In addition, we provide evidence that the C-terminal domain of the gumD gene product is sufficient for its glucosyl-1-phosphate transferase activity. Finally, we found that alterations in the later stages of xanthan biosynthesis reduce the aggressiveness of X. campestris against the plant.
Highlights
Xanthomonas campestris pv. campestris is a gram-negative bacterium which is a pathogen of cruciferous plants [15]
Since gum genes were shown to be encoded by a single transcriptional unit [34], nonpolar gum mutants were constructed by transcriptional fusion or plasmid integration (Table 1)
X. campestris strains with hybrid plasmids integrated into the genome by single crossover events were selected by use of the vector-encoded antibiotic resistance, and the changes were verified by Southern hybridization
Summary
Xanthomonas campestris pv. campestris is a gram-negative bacterium which is a pathogen of cruciferous plants [15]. The biosynthetic pathway of xanthan comprises five stages: (i) conversion of simple sugars to nucleotidyl derivative precursors, (ii) assembly of pentasaccharide subunits attached to the inner membrane polyprenol phosphate carrier, (iii) addition of acetyl and pyruvate groups, (iv) polymerization of pentasaccharide repeat units, and (v) secretion of the polymer [24, 29, 31, 32]. Chromosomal regions xpsIII, xpsIV, and xpsVI and a 35.3-kb gene cluster are required for the first stage of xanthan biosynthesis [25, 36]. These regions comprise gene functions involved in the biosynthesis of the sugar nucleotide precursors. The only gum gene characterized by genetic studies and biochemical analysis is the kpt or gumL gene encoding the ketal pyruvate transferase enzyme [37]
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