| Summary: | 碩士 === 中臺科技大學 === 醫學生物科技研究所 === 98 === Abstract
Xanthomonas campestris pv. campestris (Xcc) is a Gram-negative plant-pathogenic bacterium that causes black rot in crucifers, resulting in tremendous losses in agriculture. This organism is capable of producing large amounts of exopolysaccharides and secreting several extracellular enzymes (such as protease, endoglucanase, mannanase, and pectinase), which have been considered to be important virulence determinants. The production of these extracellular products is coordinated by a cell-cell communication mechanism through diffusible signal factor (DSF), which is dependent on RpfF, a putative enoyl-CoA hydratase, for synthesis and the RpfC/RpfG two component system for perception and signal transduction. A recent study also indicated that global transcription factor cAMP receptor protein-like protein (Clp) is essential for DSF regulation of virulence factor production.
The aim of this study was to characterize the pmeA gene encoding one of the pectinases, pectin methyl esterase (PME), in Xcc. First, the pmeA mutant YL17 and its complementary strain YL17(pRKpmeA) were constructed and the extracellular PME activity was compared with different Xcc strains. The result showed that the PME activity of AU56E (clp mutant), RM17F (rpfF mutant), and YL17 were slightly reduced, whereas the activity was restored in YL17(pRKpmeA). It was suggested that Clp and RpfF are involved in PME expression and pmeA gene might code for a secondary PME in Xcc. In pathogenicity test, the rate of symptoms development in YL17 was slightly slower than Xc17. These findings deduced that PmeA of Xcc plays a minor role in pathogenesis. Second, nucleotide G at 54 nt upstream of the pmeA start codon was mapped as the pmeA transcriptional initiation site by using the 5’-RACE (rapid amplication of cDNA ends) technique. Third, seven PpmeA-lacZ transcriptional fusion constructs were generated by cloning PCR fragments into the broad-host-range-promoter-probing vector pFY13-9, which uses lacZ as the reporter. The resultant PpmeA-lacZ transcriptional fusion constructs were introduced into Xc17, AU56E and RM17F by electroporation, respectively. Reporter assay indicated that the -359/+46 region contains the complete promoter and is capable of maximal-level expression. Transcriptional fusion analysis also revealed that the promoter activity of pmeA was reduced in either AU56E or RM17F, suggested that Clp and RpfF positively regulate transcription of the pmeA gene. In addition, the pmeA expression was affected by catabolite repression, osmolarity, oxygen limitation, and nitrogen starvation.
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