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Functional Characterization of Annotated Trehalose Anabolic Genes in Xanthomonas Campestris PV. Campestris

Author: YaoYuQun
Tutor: TangJiLiang
School: Guangxi University
Course: Biochemistry and Molecular Biology
Keywords: Xanthomonas Trehalose TPS TPP sole carbon source virulence high osmolarity
CLC: S432.42
Type: Master's thesis
Year: 2006
Downloads: 12
Quote: 0
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Abstract


Trehalose is a nonreducing disaccharide that consists of the two subunits of glucose bond by anα,α-1, 1-glycosidic linkage. In addition to being nonreducing, it possesses several unique physical properties, which include high hydrophilicity and chemical stability and the absence of internal hydrogen bond formation. In the Escherichia coli, trehalose biosynthesis is catalysed by trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate Phosphatase (TPP). TPS is encoded by the otsA gene and TPP is encoded by the otsB gene. TPS catalyses the transfer of glucose from UDP-glucose to glucose 6-phosphate and form trehalose-6-phosphate. TPP then converts trehalose-6-phosphate to free trehalose. According the blast of amino acids sequence, we found in Xcc 8004 that a putative protein which is encoded by the ORF designated as XC1076, shows 64% amino acids similarity with the TPS of Escherichia coli, a putative protein which is encoded by the ORF designated as XC1078, shows 53% amino acids similarity with the TPP of Escherichia coli, and a putative protein which is encoded by the ORF designated as XC1077, shows 45% amino acids similarity with the TPP of Nocardia farcinica. We had got separate three Tn5gusA5 insertional mutants of XC1076, XC1077 and XC1078, which are 006B12, 110C01 and 237E11 respectively in the Xcc 8004 mutant library constructed in our lab. Throught the analysis of transcription of 237E11, we consider that XC1076, XC1077 and XC 1078 are not transcripted together.Base on a series of phenotypic analysis, 006B12 and C006B12 grow not well than the wild type strain 8004 on NCM medium with different carbon sources. 006B12 and the wild type strain 8004 show the same virulence. 006B12 grows not well than C006B12 in rich medium supplemented with NaCl at the concentration of 3%. This finding suggests that the XC1076 plays a role in resisting high osmolarity. XC1076 is not related with extracellular enzymes synthesis and may be related to extracellular polysaccharide.Base on a series of phenotypic analysis, 110C01 grow well in minimal medium, rich medium and medium with different carbon sources, the same as the wild type strain 8004. 110C01and the wild type strain 8004 have the same phenotype in the examinations of pathogenic phenotype, hypersensitive response and extracellular enzymes. EPS production is reduced in 110C01. This result suggested that XC1077 might play a role in EPS production.237E11 grows poorly on NCM medium with glucose, or other simple sugars including sucrose, maltose and trehalose as sole carbon source, but not C237E11. In addition, 237E11 also grows poorly on NCM medium with sodium acetate, glycerol, malate, pyruvate or succinate as the sole carbon source, but not C237E11. These indicate that XC1078 is required for regulation of glycolysis and gluconeogenesis. Virulence assay was performed and disease length of 237E11 is average 6.2 mm, compared with 12.8 mm of 8004 and 13 mm of C237E11. These results show that XC1078 is essential for the full virulence of Xcc. Experiments show that there is no difference in the yield of extracellular enzymes and extracellular polysaccharide between 237E11 and 8004. There is no difference in the hypersensitive response between 237E11 and 8004. There is no difference in rich medium between 237E11 and 8004. In minimal medium, 237E11 grows poorly, while C237E11 and 8004 grow very well. And in the host, 237E11 grows not well than C237E11 and 8004. These suggest that the low virulence of 237E11 is related with the impaired viability. C237E11 and 8004 grow well in rich medium supplemented with NaCl at the concentration of 2%, but not 237E11. This finding indicates that the XC1078 plays a role in resisting high osmolarity.

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CLC: > Agricultural Sciences > Plant Protection > Pest and Disease Control > Plant diseases and their prevention > Transgression ( pass ) an infectious agent harmful > Bacterial
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