| Summary: | 碩士 === 國立中興大學 === 生物化學研究所 === 98 === Xanthomonas are Gram-negative aerobic pathogenic bacteria with yellow mucoid. X. campestris pv.campestris (Xcc) B24 has seven transcription units related to the formation of cell pigment (xanthomonadin). Mutation in one of the transcription units, pigB, concerned reduction of xanthomonadin in bactaria.Region contains two genes, encoding reductase/halogenase and pteridine-dependent dioxygenase like protein (named DogA), respectively. It has been known that DogA protein is involved in the synthesis of Xcc signal factor diffusible factor (DF).The aim of this study is to understand the formation of DF in Xc17, and the relationship between DF and DSF regulated pathways.Firstly, the dogA mutant, Xc17dogA::Gm, was constructed. Compared to that of Xc17, extracellular enzyme levels of protease, α-amylase, and cellulase in Xc17dogA::Gm had not significant differecens.However, lower levels of xanthomonadin, EPS, and pathogenicity. DF were found in the mutant strain. In addition DF secreted by Xc17 can restore the mutations to the wild type.Supernatants of Xc17, Xc17dogA::Gm, RM17F (rpfF mutant strain), and TC817 (clp mutnat) were collected, membrane filtered, and added to the culture of DF deficient strain Xc17dogA::Gm and DSF deficient strain RM17F. Compared the DF and DSF levels among wild type and mutant strain xanthomonadin, EPS, and engA promoter activity. Results showed that DSF increased in the DF deletion strain, production or activity, the supernatant of DSF deficient strain, also contained DF with lower levels than that of the wild type strain, the supernatant of clp mutant strain, contained DF and DSF with lower levels than that of the wild type strain. To understand proteins affected by DF, two-dimensional gel electrophoresis was used to analyze proteins of Xc17 and Xc17dogA::Gm.Six proteins may be affected by DF, two of them are emzymes involved in the TCA cycle.In addition Streptomyces A-factor is similar to DF in chemical structure, indicating that Xcc DF may be recognized by a corresponding receptor, and exercise it’s regulatory function on. The transcription factor then controls the transcription of enzymes that related in TCA cycle, xanthomonadin synthesis, and EPS synthesis. Therefor, loss of DF reduced the synthesis of xanthomonadin and EPS.
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