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標題: 十字花科黑腐病菌胞外酵素分泌所必需的PefD是一外胞膜酯蛋白
PefD required for the secretion of extracellular enzymes by Xanthomons campestris pv. campestris is an outer membrane protein
作者: 林明慧
Lin, Ming-Huei
關鍵字: Xanthomonas campestris pv. campestris.
extracellular enzymes
outer membrane
出版社: 植物學系
摘要: pefD,是十字花科黑腐病菌分泌胞外酵素所必需的基因之一.將含有PefD基 因的DNA片段,選殖於表現載體,送入大腸桿菌或十字花科黑腐病菌pefD缺 失之變異株,利用[35S]-methionine放射性標定或西方墨點,皆可測得大量 表現之PefD蛋白質,分子量約為80kDa.[3H]-palmitate標定的結果,證明 PefD是經過酯質修飾的酯蛋白.以蔗糖梯度超高速離心進行PefD在胞內分 佈之研究,說明PefD蛋白位在外胞膜.合成PefD的完整細胞,分別以蛋白■ 處理或進行免疫螢光之標定,進一步顯示PefD至少有部分裸露於菌體表面. 此外,本研究發現PefD於外胞膜之定位無需其它pef基因之存在.定點突變 pefD基因,造成pefD之C半邊之刪除,分析僅含N半邊PefD之表現,發現其仍 具有嵌入外胞膜及裸露菌表之定位特性,唯已喪失協助十字花科黑腐病菌 輸送胞外酵素之功能. The pefD gene is required for the secretion of extracellular enzymes by Xanthomonas campestris pv. campestris. Expression of the pefD gene under the control of lacZ promoter in Escherichia coli revealed a protein of apparent molecular weight of 80 kilo- daltons. Immunoprecipitation analysis of [3H]-palmitate labaled proteins demonstrated that PefD is a lipoprotein. Examination of the cellular localization of PefD upon sucrose- density centri- fugation showed that the protein co- fractionated with the outer membrane vesicles. Protease digestion or immunofluorescence labeling of PefD in intact cells futher indicated that some regions of the protein may be exposed on the cell surface. It was also found that the transport and insertion of the PefD into the outer membrane of X. campestris pv. campestris is independent of other pef gene products. Site-directed mutagenesis was con- ducted to remove the C-terminal half(amino acids 414 to 759) of the PefD. the truncated PefD still possesses the localization characteristics of the intact PefD. However, the truncated PefD can no longer support the secretion of extracellular enzymes by X. campestris pv. campestris.
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