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標題: 噬菌體 P1201 的 Ant4 蛋白與 Corynebacterium glutamicum NCHU87078 蛋白之間的交互作用
Interaction of corynephage P1201 Ant4 to the proteins from Corynebacterium glutamicum NCHU 87078
作者: 黃炳綬
Huang, Bing-Shou
關鍵字: corynephage P1201;噬菌體P1201;Ant4;Corynebacterium glutamicum;protein-protein interaction;transcriptional factor;Ant4;Corynebacterium glutamicum;蛋白質交互作用;轉錄調控因子
出版社: 分子生物學研究所
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本實驗室從味精生產菌 Corynebacterium glutamicum NCHU 87078 醱酵液中分離出之corynephage P1201,屬於線性雙股 DNA 溶裂性噬菌體。其基因體具有 ant4 基因,此基因在溶裂性噬菌體中的生理角色,仍無文獻報導。實驗室前人曾發現 Ant4 蛋白會抑制C. glutamicum NCHU 87078 之生長。以 RT-PCR 及 Western blotting 偵測 ant4 在 corynephage P1201 感染時的表現情形,發現其屬於病毒感染的早期基因。由於 Ant4 蛋白之 C 端 KilAC domain 可能與其他蛋白產生交互作用,本研究利用固定化金屬離子親和性層析法從 C. glutamicum NCHU 87078分離出與 Ant4 蛋白交互作用的蛋白,並利用 pull down assay 及免疫沉澱法進行蛋白質交互作用的驗證。發現 Ant4 會與 C. glutamicum NCHU 87078 的轉錄調控因子: SugR 、 RamA 、 RamB 及 LexA 產生交互作用,並確認 Ant4 蛋白 C 端的 KilC domain 為參與蛋白質交互作用的主要區域。 gel shift assay 的結果顯示,高濃度的 Ant4會抑制RamA 與含有C/TGGGG(G)(G)T/C 及 ACCCC(C)A/T/G 序列之 DNA 片段的結合,此 DNA 片段位於 aceA 及 aceB 基因間的 promoter 區域內。 real-time PCR 分析的結果顯示,由 RamA 及 RamB所調控之酒精代謝以及 acetate 代謝相關的基因表現量皆下降,而由 LexA 所調控的 divS 基因則有 derepression 的現象。 divS 的基因產物為 cell division suppressor ,可能與 Ant4 抑制 C. glutamicum NCHU 87078 生長的機制有關。以上結果顯示Ant4蛋白能夠與宿主細胞的轉錄調控因子交互作用,並調控其與 promoter/operator 的結合能力。

Lytic phage P1201 was isolated from Corynebacterium glutamicum NCHU 87078 during an industrial fermentation for glutamic acid production. P1201 genome contains an ant4 gene encoding a putative antirepressor, while the biological roles of this gene in the lytic phage P1201 remain to be elucidated. Growth of C. glutamicum NCHU 87078 is inhibited by the expression of ant4 gene. Reverse transcription-PCR and Western Blotting analysis indicated that the expression of ant4 gene occurs at the early satge of infection. Since the C-terminal KilAC domain of Ant4 may involve in protein-protein interaction, immobilized metal affinity chromatography was performed to isolate Ant4-interacting proteins from C. glutamicum. Pull-down experiments and immunoprecipitation were also performed to identify the proteins interacting with Ant4. We found that Ant4 interacted with transcriptional regulators, SugR, RamA, RamB, and LexA of C. glutamicum NCHU 87078, and the C-terminal KilC domain is required for protein-protein interaction. Gel shift assay revealed that high concerntration of Ant4 could inhibit the binding of RamA to the sequence of C/TGGGG(G)(G)T/C and ACCCC(C)A/T/G in the promoter region between aceA and aceB genes. The data from real-time PCR assay indicated that Ant4 inhibited the expression of the genes regulated by RamA and RamB, including the genes involved in acetate and ethanol metabolisms, but up-regulated the divS gene, which is regulated by LexA. The expression of divS gene encoding a cell division suppressor may lead to cell growth inhibition of C. glutamicum. Our data suggested that Ant4 interacted with the transcriptional regulators of C. glutamicum NCHU 87078 and subsequently modulated their binding to promoter/operator regions.
其他識別: U0005-1208201013012500
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