Characterization of udgH and the Flanking Genes Involved in Carbohydrate Metabolism in Xanthomonas campestris pv. campestris

• Main Authors: Kai-Wei Chang, 張楷煒
• Other Authors: Yi—Hsiung Tseng
• Format: Others
• Language: zh-TW
• Published: 2001
• Online Access: http://ndltd.ncl.edu.tw/handle/62981894289427603228

碩士 === 國立中興大學 === 分子生物學研究所 === 89 === Xanthomonas campestris pv. campestris is a gram-negative, rod-shaped and monotrichously flagellated plant pathogenic bacterium. The chromosome of X. campestris pv. campestris has a G + C content of 64%. It produces copious amounts of xanthan gum which has been implicated to associate with the virulence of the bacterium. Biosynthesis of xanthan gum involves multiple steps and a multitude of enzymes. Rescently, our laboratory has localized the genes involved in xanthan synthesis at eight loci on the X. campestris pv. campestris 17 (Xc17) chromosome map. Thes loci are designated as eps1, eps2, eps3, eps4, eps5, eps6, eps7 and eps8, respectively. Among them, eps3 contains the gene encoding UDP-glucose dehydrogenase gene (udgH). In this study, I employed a chromosome walking strategy to clone the DNA fragments flanking the udgH gene from the (Xc17) chromosome. Two clones, pCKW-1 and pCKW-2, carrying the upstream and the downstream DNA fragment, respectively, were thus obtained. Sequence analysis revealed 10,200 bp containing nine ORFs : gntR gene, nt 441 to nt 79; glutathione peroxidase gene (gpxX)， nt 801 to nt 1,385； peptidyl-prolyl cis-trans isomerase (PPIase) gene (ppiA), nt 1,546 to nt 2,439； UDP-glucose dehydrogenase gene (udgH); nt 2,464 to nt 3,885; ORF225, nt 4,050 to nt 4,727; ORF278, nt 4,981 to nt 5,817; PckR gene (pckR), nt 6,134 to nt 7,171; glucose/galactose transporter gene (gluP), nt 7,185 to nt 8,495; fructokinase gene (pfkB), nt 8,492 to nt 9,475. Mutants of these genes were constructed by insertional mutagenesis. Mutation of udgH gene resulted in a non-mucoid phenotype and a loss of pathogenicity; the mutation of pfkB gene caused higher rates of growth and increased virulence; the mutation in ppiA, pckR and gluP gene did not affect the colony morphology, pathogenicity and growth rate. No differences were found in sugar utilization between the mutant and the wild-type strains. Sucrose can support better growth than dose glucose, and the oder of efficiency in sugar utilization by X campestris pv. campestris is fructose> maltose> xylose> galactose> lactose. The udgH gene promoter is stronger than that of the other four genes, and levels of promoter activity are always higher when cells are cultured rich medium which can support higher growth rates.