細菌常利用雙分子調控系統來適應外界的刺激。雙分子系統通常包括了一個具有組胺酸激酶和組胺酸磷酸根轉移分子結構域的感應子，以及一個反應調控子。在Pseudomonas aeruginosa PAO1這株菌中，已發現有12個不具有組胺酸磷酸根轉移分子結構域的雜合感應子，以及3個只具有組胺酸磷酸根轉移分子結構域的Hpt蛋白質。本實驗室在先前的研究中，已發現其中五個雜合感應子具有自我磷酸化的能力。而且其中3個(PA2824，PA1611，PA1976)可將磷酸根傳遞給HptB蛋白質。為了能找尋其他可磷酸化HptB蛋白質的感應子，本研究純化了11個雜合感應子以及HptB蛋白質。磷酸化的實驗利用[γ-32P]ATP進行反應之後，以SDS-PAGE和自動放射顯影分析。在時間點分析的實驗中發現，5個具有自我磷酸化能力的感應子都可在30分鐘內表現磷酸化訊息。另外，依據反向磷酸化的結果也發現，RtsM感應子可從HptB獲得磷酸根。這個結果確認了共有四個雜合感應子(PA2824, PA1611, PA1976, and RtsM)可專一性將磷酸根經由傳遞給HptB，再傳給反應調控子。在先前的研究中，本實驗室已發現磷酸化的HptB可將磷酸根傳給反應調控子PA3346。而PA3346則可以將PA3347在Ser56的位置上去磷酸根。本實驗比較PA3346在Asp61以及PA3347在Ser56磷酸化位置突變而成的類磷酸化型和無法磷酸化型互補株，在細菌游動、swarming和生物膜形成能力的表現情況。結果顯示，不同型互補株之間的表現並無明顯差異。為了進一步尋找PA3345、PA3346以及PA3347調控的下游基因，本研究也進行了細菌群體感應分析，發現MPA45、MJL46以及MJL47所分泌的 3OC12-HSL訊息分子量下降。另外，我們也針對MPA45、MJL46和MJL47在swarming狀態下的全細胞蛋白質進行二維膠體電泳以及質譜技術的分析，找尋PA3347的下游基因。依據表現型分析以及蛋白質體的研究，我們認為，PA3345□PA3346□PA3347這條訊息傳遞路徑在Pseudomonas aeruginosa中可能扮演廣泛性的調控功能。

Bacterial two-component systems (2CS) are usually involved in adaptation to external stimuli. Most 2CS consists of a histidine kinase sensor containing the Hpt domain, and a response regulator. In Pseudomonas aeruginosa PAO1, there are twelve hybrid sensors which do not have the Hpt domain. In addition, there are three genes encoding a protein with only the Hpt domain. Autophosphorylation of five of the hybrid sensors in vitro were detected in our laboratory previously. Three of them (PA2824, PA1611, PA1976) displayed an ability to transfer the phosphoryl group to HptB. In order to identify additional sensors that may phosphorylate HptB protein, cytoplasmic region of eleven hybrid sensors and the HptB protein were purified and characterized by phosphorelay assays with [γ-32P]ATP followed by SDS-PAGE and autoradiography. Five sensors showed rapid self-phosphorylation within 30 min in the time course assay. Using the reverse phosphorelay assay, a hybrid sensor RtsM, which can receive phosphoryl group from HptB was identified. The results demonstrate that these four hybrid sensors (PA2824, PA1611, PA1976, and RtsM) perform specific signal transduction through HptB to response regulators. Phosphorylated HptB can transfer the phosphoryl group to response regulator PA3346. Our laboratory also found PA3346 can dephosphorylate PA3347 on Ser56. In this study, swimming, swarming, and biofilm assays were performed to compare PA3346 complementary strains harboring PA3346 with different mutations at Asp61 and PA3347 complementary strains harboring PA3347 with different mutations at Ser56. Our results show little difference among these strains in these aspects. In order to search the downstream genes which were regulated by PA3345, PA3346 and PA3347, we performed quorum sensing assay. Secretion of signal molecular 3OC12-HSL was found to be decreased in MPA45, MJL46 and MJL47. In addition, we analyzed proteome of MPA45, MJL46 and MJL47 swarming cells by 2D gel electrophoresis and mass spectrometry to search for the genes downstream of PA3347. According to the results of the phenotype assay and proteomic analysis, we propose that PA3345□PA3346□PA3347 signal pathway plays a global regulation role in Pseudomonas aeruginosa.

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