Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/35838
標題: 楊桃細菌性斑點病菌gacA基因突變株的特性分析
Characterization of Pseudomonas syringae pv. averrhoi gacA mutant
作者: 邱銘鋒
Chiou, Ming-Fong
關鍵字: Pseudomonas syringae pv. averrhoi;楊桃細菌性斑點病菌;two-compotent system;gacA;response regulator;雙組分調控系統;反應調節子
出版社: 生物科技學研究所
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摘要: 
Pseudomonas syringae pv. averrhoi (Pav) causes bacterial spot on carambola. The typical symptoms on leaves are purple spots surrounded by yellow haloes, and the infected leaves turn yellow and fall easily. RpoE is an ECF sigma factor which can recognize specific promoter sequences. A previous study revealed that the colony morphology of Pav PA5 carrying a plasmid-borne rpoE was more mucoid than wild type. By using transposon (Tn5’lacZY) induced mutagenesis, the RpoE-overexpressed PA5 carrying transposon-inserted gacA gene lost the mucus-like colony phenotype. GacA is a regulator which belongs to GacS/GacA two component system. In previous studies, this system is present in a wide variety of bacteria, and has been reported to regulate an array of phenotypes including virulence, motility, secondary metabolites production such as quorum sensing molecules, pigments, toxins, biofilm formation, and secretion systems. In this study, a gacA mutant was constructed by unmarked-mutagenesis, and also transformed with a plasmid-borne rpoE gene. The result verified that the GacA is involved in mucus-like phenotype. In this study, multiple phenotypes of gacA mutant and its derivative carrying a plasmid-borne rpoE gene were analyzed. In motility assays, results shows that gacA mutant lost its“swarming” motility, but not the “swimming”motility. Nevertheless, the motility of the strains carrying a plasmid-borne rpoE was suppressed in both swarming and swimming assays. Moreover, a decreased fluorescent pigment production caused by gacA deletion was observed when cultured in a KA medium. Furthermore, the level of siderophore production in gacA mutant was lower than wild type according to CAS assay. PA5 gacA mutant retained the ability to induce an HR on non-host tobacco. For bacterial multiplication assays, PA5 and its derivative strains were inoculated into the leaves of carambola. The data showed that the gacA deletion remarkably reduced bacterial growth in the carambola leaves. There was no symptom development on the carambola leaves infected with gacA mutant using spray inoculation. According to western blot assays of type III secretion system (T3SS) expression, results showed that the expression of HrpZ and HopAK1 were increased in gacA mutant compared to wild type. Moreover, using gfp as a reporter, the hrpC promoter is also enhanced in gacA mutant. However, the hrpL promoter activity was not affected by gacA mutation. In analyzing the expression of EPS related gene, the promoter activity of algD was lower in gacA mutant, indicating that the alginate biosynthesis is positively regulated by GacA. In addition, the rpoE promoter activity was lower in gacA mutant, but the gacA promoter activity was higher in rpoE mutant. The results reveal that that GacA and RpoE may regulate each other with an unknown mechanism.

Pseudomonas syringae pv. averrhoi (Pav) PA5為革蘭氏陰性之螢光假單胞菌,可引起楊桃細菌性斑點病害,在楊桃植株上產生的病徵為葉片上有紫紅色斑點,周圍有明顯黃暈,且葉片易黃化脫落。RpoE是一種ECF sigma factor,可以辨認特定的啟動子 (promoter)序列,先前研究發現,Pav PA5菌株過表現RpoE蛋白時,會產生明顯的黏液狀菌落型態,但利用跳躍子隨機誘變後,gacA基因被跳躍子插入的菌株,則失去其黏液狀的表現型。GacA屬於GacS/GacA雙組分系統 (two component system)中的反應調節子 (response regulator),這組系統廣泛存在於各種細菌,又前人研究已證實,此調控系統控制病原菌一系列的表現型,包括致病性、移動性、二級代謝物如群落感知 (quorum sensing)分子、色素和毒素的生合成、生物膜生成以及蛋白質分泌系統等。本研究利用非標記置換法構築gacA基因的突變株,同時送入帶rpoE基因的質體進行觀察,證實黏液狀菌落型態的性狀表現確實與gacA基因有關,針對gacA突變株與攜帶rpoE質體進行表型特性、病原性及其基因表現等能力進行分析。在swarming試驗分析顯示gacA突變株失去“表面移行”的移動能力,而swimming實驗中gacA突變株的“泳動”能力並未受到影響;然而帶有rpoE基因質體的菌株 (野生菌株及突變菌株)其移動能力皆會受到抑制。gacA突變株培養於KB固態培養基產生較少的螢光色素,在紫外燈照射下的螢光表現較弱,以CAS定量分析結果亦顯示gacA突變株嵌鐵化合物含量較低。於病原性分析的結果顯示,gacA基因的突變對於其在非寄主菸草引發過敏性反應的能力並無影響,但以注射接種法測定gacA突變株在寄主楊桃葉片內的族群生長則有較弱趨勢;而用噴灑接種後觀察楊桃葉片的發病情況,接種gacA突變株的葉片無任何病徵出現,推測gacA基因突變可能影響毒質的產生而降低此病菌對寄主的毒力。利用西方墨點法與啟動子活性分析gacA基因缺失株之第三型分泌系統的相應基因或蛋白的表現情形,結果顯示gacA基因缺失株之第三型分泌系統HrpZ及HopAK1蛋白表現量及hrpC operon啟動子活性皆高於野生株PA5的表現量,證實GacA於楊桃細菌性斑點病菌中為負調控第三型分泌系統,但是第三型分泌系統的調控蛋白HrpL其啟動子活性卻不受GacA影響。在分析合成胞外多醣體的相關基因algD的表現情形,可發現algD的啟動子活性於gacA突變株表現較低,顯示GacA正調控alginate生合成。另外也分析rpoE於gacA突變株中的表現時,可發現rpoE的啟動子活性於gacA突變株較低,而相反地,gacA的啟動子活性於rpoE突變株則表現較高,顯示GacA與RpoE相互影響彼此的表現,但其機制未明。
URI: http://hdl.handle.net/11455/35838
其他識別: U0005-2208201318513600
Appears in Collections:生物科技學研究所

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