細菌基因表現之調控: HptB磷酸根轉移系統與三磷酸腺苷水解功能必須之轉錄蛋白AcoK之功能分析

Regulation of bacterial gene expression: elucidation of HptB-mediated phosphorelay signaling systems and an ATPase dependent transcriptional factor AcoK

徐婕琳(Jye-Lin Hsu)

指導教授 ： 張晃猷

國立清華大學/生命科學院/分子醫學研究所/博士(2009年)

摘要

本論文主要包含兩個主題，第一個為「鑑定磷酸根轉移分子HptB在綠膿桿菌PAO1中參與的多階層訊息傳遞系統」，第二個主題為「AcoK之三磷酸腺苷結合區域為此蛋白調控克雷白氏菌acetoin代謝之必要區域」。
綠膿桿菌為格蘭氏陰性致病菌，尤其對於住院治療的病人，可導致多種急慢性感染病症。此菌共有三個可表現具有磷酸根轉移區域，卻缺乏磷酸激脢功能區域的蛋白分子之基因。磷酸根轉移分子為感應蛋白(hybrid sensor)和接受子(response regulator)之間的訊息傳遞分子，被認為在細菌、真菌以及植物界都扮演關鍵性的訊息傳遞功能。在第一個主題中的研究顯示，綠膿桿菌HptB訊息傳遞系統包括了四個游離的感應激脢蛋白、HptB分子，及一個專一的接受子PA3346。我們也證實了PA3346具有磷酸化水解能力，並能將其隔壁基因PA3347的蛋白質產物去磷酸化。最後，在基因HptB、PA3346以及PA3347突變株，其swarming和生物膜形成之能力也被加以分析。
在本論文第二個主題，為探討Aco此蛋白調控克雷白氏菌參與acetoin代謝的功能區域。許多細菌種類都可利用acetoin為其碳源。在克雷白氏肺炎桿菌中，此種化合物可被acoABCD基因群轉譯的蛋白acetoin dehydrogenase 氧化。之前的研究顯示，此基因群的表現依賴位於其上游緊鄰之基因所產生的蛋白分子AcoK。AcoK在C端具有一個LuxR形式之helix-turn-helix去氧核醣核酸附著區域，在N端則有一個推測為Walker A和Walker B之核苷三磷酸結合區域。第二個研究目標就是去了解AcoK不同功能區域，尤其是在核苷三磷酸結合區域，對於它轉錄功能的重要性。在這研究中，我們建構了數個AcoK的截短及點突變蛋白並研究其生化及轉譯功能。其中一個在推測為Walker A區域之點突變，造成AcoK對於三磷酸腺苷水解能力以及其導致之轉錄活性的喪失。雖然此轉錄因子具有專一結合至acoABCD特定-66到-36啟動子核酸區域的性質，但Walker A區域點突變所造成之三磷酸腺苷水解能力喪失，並不影響其去氧核醣核酸結合能力。總結，本研究提供另一個多功能區域之訊息傳遞腺嘌呤三磷酸水解酵素 (Signal Transduction ATPases with Numerous Domains) 家族中，如何活化其標的基因表現的範例。

關鍵字

訊息調控 ； 轉錄蛋白

並列摘要

This thesis contains two major topics. The first one is “Characterization of the histidine-containing phosphotransfer (Hpt) protein Bmediated multi-step phosphorelay system in Pseudomonas aeruginosa PAO1” and the second one is “The ATP-binding motif in AcoK is required for regulation of acetoin catabolism in Klebsiella pneumoniae CG43”.
Pseudomonas aeruginosa is a gram-negative pathogen causing many acute and chronic infections, particularly in hospitalized individuals. It contains three genes that encode proteins with an Hpt domain but lack a kinase domain. Hpt proteins are signal mediators between hybrid sensors and response regulators. The proteins play a crucial role in directing signal transduction in bacteria, yeasts and plants. The study in first topic demonstrates that the Pseudomonas aeruginosa HptB-mediated signaling system consists of four orphan sensor kinases, HptB, and a specific response regulator, PA3346. We also present evidence of phosphatase activity of PA3346 on its neighboring gene product, PA3347. Finally, the swarming and biofilm forming activites of hptB, PA3346, and PA3347 knockout mutants are described.
The second part of this thesis is to characterize a transcriptional factor AcoK in the regulation of acetoin catabolism. Many bacterial species utilize acetoin as a carbon source. The compound is oxidized by acetoin dehydrogenase encoded by acoABCD operon in Klebsiella pneumoniae. Previously, we have shown the expression of this operon is induced by acetoin through AcoK, the product of a gene located immediately upstream of acoABCD. AcoK contains a helix-turn-helix DNA binding domain of the LuxR transcription activator family at the C-terminal region and putative Walker A and B nucleotide binding motifs at N-terminal portion. The goal of the second study is to understand the contribution of different domains, in particular the nucleotide binding motif, in AcoK on its transcriptional activity. A number of truncations and site-directed mutations were constructed on AcoK and the biochemical and trans-activation activities of the resulting proteins were determined and reported herein. A mutation in the putative Walker A motif resulted in a significant reduction of ATP hydrolysis and trans-activation activity of AcoK on acoABCD expression, presumably was due to the loss of ATP-binding ability. The transcription factor bound specifically to a region comprising nucleotide -66 to -36 of the acoABCD promoter, although the DNA binding ability was not affected by the Walker A motif mutation. Together, this study provides an additional example in how a member of the Signal Transduction ATPases with Numerous Domains family activates its target gene expression.

並列關鍵字

AcoK ； HptB

參考文獻

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