綠膿桿菌中磷酸根傳遞分子HptB在訊息傳遞中的功能研究

Abstract

綠膿桿菌(Pseudomonas aeruginosa)，為一臨床常見的伺機性病原，對環境有很強的適應性。在已解碼的基因體序列中，發現綠膿桿菌具有123個雙分子訊息傳遞系統，藉以偵測環境的變化與刺激、調控適當的基因表現，使細菌可以適應環境、躲避免疫系統及抗生素的攻擊。目前已建立的雙分子訊息傳遞系統模式分為三類：典型系統(classic system)、非典型系統(unorthodox system)、混成系統(hybrid system)。混成系統的訊息由感應蛋白質激脢透過游離的接受模組分子(Hpt domain)傳遞給調控蛋白質。為了確認這些Hpts在綠膿桿菌中傳遞磷酸根的功能，我利用聚合脢鏈鎖反應選殖出這三個Hpt基因序列，以pET30表現系統在大腸桿菌中表現並進而純化蛋白質，再以放射性元素標定磷酸根來偵測磷酸根在雜合感應子PA1611、Hpt分子及感應蛋白質間的轉移。結果證實專一的磷酸根傳遞路徑由感應子PA1611到HptB再到調控蛋白質PA3346。然而，以酵母菌雜合系統分析卻無法證實這些蛋白質間的交互作用。經由蛋白質序列的比對，在多種單胞桿菌中，均可發現HptB基因與鞭毛生合成的基因組座落在一起。為了探討HptB的功能，以同源互換方式構築HptB基因缺損株。此HptB缺損株在營養缺乏的狀態下生長速率降低；在添加碳源及氮源的情況下，其生物膜的生成量是野生株的兩倍；缺損株的游移能力在半液態環境下較差，而在固態界面中則較佳；趨化性的分析結果顯示缺損株朝趨化物移動的能力降低。由這些特性分析及基因體序列比較結果，我們推斷HptB分子可能與鞭毛的生合成及調控有關。

Pseudomonas aeruginosa PAO1 is an opportunistic pathogen, and owns great capability to adapt versatile environment. There are 123 genes encoding two-component system components in the bacteria, which serve as a stimulus-response coupling machinery allowing the organism sense and respond to the changes in environment. The system has been classified into three types: the classical system, the unorthodox system, and the hybrid system. We focus on the hybrid system which consists of a histidine kinase (HK) without output domain, a separated Histidine-containing phosphotransfer (Hpt) molecule, and a response regulator (RR). In order to demonstrate the role of Hpt modules in signaling phosphorelay, the gene fragments corresponding to each of the Hpt domain and one hybrid sensor (PA1611) and two RRs (PA3346 and PA0034) were amplified by PCR and subcloned respectively into pET30 expression vector. The recombinant proteins were expressed in E. coli and the proteins purified by His-Bind nickel column for the phosphorylation assays. The assay demonstrated a specific phosphoryl transfer from sensor kinase PA1611 to HptB and then to PA3346. However, using yeast two-hybrid screening failed to identify the protein interactions. Sequence comparisons of several Pseudomonas species revealed that the genome DNA contains the HptB homolog flanking by a flagella biosynthesis gene cluster. To identify the functional role of HptB, hptB mutant was constructed by homologous recombination. The hptB mutant showed a decrease of growth rate decreased in a nutrient limiting condition. The hptB mutation also affected the biofilm formation with a 2-fold higher activity than that of the wild type strain in a minimal medium supplemented with carbon and nitrogen source. A reducing swimming capability and an increase of twitching motility were also found in hptB mutant. Taken together, the HptB is likely mediated the signaling involved in flagella biosynthesis and regulation.