| Summary: | 碩士 === 國立中興大學 === 生物科技學研究所 === 100 === Extracytoplasmic function (ECF) sigma factors are widely found in prokaryotic cells. These ECF-sigma factors can recognize specific promoter sequences, and thus induce the expression of certain genes, such as with drug resistance, ion transportation, cell envelope integrity and environmental stress related genes. Pseudomonas syringae also contains some of the ECF sigma factors, among which RpoE is not currently found in other saprophytic Pseudomonas spp and Pseudomonas aeruginosa. In order to study the features of rpoE gene product in P. syringae pv. averhoi (Pav), this gene is cloned from Pav HL1 and transformed into some Pseudomonas spp. strains by the tri-parental mating. The transconjugants were cultured on KA, NA and TTC agar plates. Compared with their wild-type strains, the colony morphology of the Pav PA5 and PA13 with a plasmid-borne rpoE is more mucoid; but in P. aeruginosa PAO1, Pav HL1 and P. syringae pv. tomato DC3000, those conjugants still showed the same colony morphology as their wild types did. In addition, the motility of PA5 overexpressing rpoE is better than the wild-type on nutrient agar, but is more sensitive to the beta-lactam antibiotics and peroxides such as hydrogen peroxide. Results of the tests for minimum inhibitory concentrations (MIC) of antibiotics showed that the overexpressed-RpoE PA5 is more resistant to tetracycline and Streptomycin. The cause of mucus-like morphology may be associated with the lipopolysaccharide (LPS) production, whereas the product of galU plays an important role in the biosynthesis of LPS. Overexpressing RpoE in Pav PA5-ΔgalU also causes more sticky colonies, but still cannot restore its ability to cause hypersensitive response on leaves of tobacco. A previous study in our laboratory showed that promoter of yedY gene is positively controlled by RpoE, a yedY mutant of Pav PA5 strain was constructed by unmarked-mutagenesis. The colony phenotype of Pav PA5-ΔyedY overexpressing RpoE remained viscous, indicating that YedY protein may not be involved in the mucous colony morphology. In order to explore the special colony phenotype and the interactions with plants, PA5 and its derivative strains were inoculated in non-host plant tobacco. The results showed that PA5 strain with the rpoE plasmid caused the delayed hypersensitive reaction on leaves of tobacco. Random mutagenesis of PA5 containing rpoE plasmid via transposon Tn5’lacZY showed that the transposon inserting in gacA and homologous PSPPH_0136 genes in PA5/RpoE resulted in loss of the mucus-like colony phenotypes. Moreover, in order to evaluate the RpoE-regulating gene products, two-dimensional protein gel electrophoresis analysis of protein profiles of PA5 and PA5 overexpressing RpoE were carried out. Some differential protein spots were displayed, suggesting that it may result from genes regulated by RpoE.
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