從台灣分離之嗜鹽古細菌光趨性分析與其感受型視紫蛋白質之特性

科學界對古生菌的感光蛋白質研究，已有四十年以上之歷史，但其研究對象尚未包含台灣之菌種。本實驗室在2010年發表死海唯一倖存的嗜鹽古生菌，不僅有目前同類古生菌中，單一菌種最多的六個感光蛋白質，其中 HmBRII 性質和已知同類蛋白質具有差異。因此，不同地區感光蛋白質的性質，是值得研究和比較的。本研究要從台灣的嗜鹽古生菌著手，瞭解本地之感光蛋白質之性質。為達到此目的，有三個目標要完成，一是發現台灣本土菌種，二是選殖其感光蛋白質並加以大量表達，三是對其生化及生物物理性質做研究。首先，本實驗室已於井仔腳鹽田分離出一株暫命名為Ht之嗜鹽古細菌，初步利用16S rRNA 定序結果得知，Ht為一株屬於 Haloarcula 屬的嗜鹽古細菌，並與Haloarcula vallismortis 和 Haloarcula marismortui 具有高度16S rRNA序列相似度。因就選殖出來之感光蛋白質數量和生長酸鹼值之不同，知其非 H. marismortui。先前也已利用 FTIR，發現 Ht 與 H. vallismortis 具有不同的細胞膜成分，因此，將Ht視為一株台灣的特殊菌株。在本研究中，進一步利用不同鹽濃度下之生長曲線，發現 Ht 與 H. vallismortis 具有不同的鹽耐受性，因此，我們提出Ht為一新種，並暫名為 Haloarcula taiwanensis。第二目標上，我們選殖並比較其四個感光蛋白質，並進行感光蛋白質之大量表達。就感知環境因子及對外界環境反應對生物而言，感光是十分重要的生理功能，本研究先進一步針對H. taiwanensis之光趨性 (phototaxis response) 進行分析，觀察菌體被不同波長的光照射下之泳動現象。針對第三目標，先在分子層面，利用H. marismortui 之六個視紫蛋白質引子，以 H. taiwanensis 全基因體為模板，進行聚合酶連鎖反應，得到微生物感光型視紫蛋白質的擴增產物，包括 sensory rhodpsin I (SRI) 和 sensory rhodopsin II (SRII) 與其觸發器蛋白質。同時，建構其感光型蛋白質，利用大腸桿菌進行異源表現，進行其蛋白質的功能性分析。其中，實驗發現感光型視紫蛋白質I (Sensory rhodopsin I, SRI) 十分穩定，和現有 SRI 皆不同，目前發現嗜鹽古細菌中的 SRI 都十分不穩定，不利於蛋白質養晶過程，這也是目前為止，尚未解析出SRI蛋白質結構之原因。另外，在本研究中發現HtSRI-HtrI fusion protein 可以偵測650 nm的微弱光源，行使光週期，且其最大吸收峰為602 nm。此現象尚未在其他菌種之感光型視紫蛋白質發現。有別於其他嗜鹽古細菌，再度提高H. taiwanensis為一新種之可能性外，並對 HtSRI 進行養晶條件的篩選，如能解出結構，可望將過去四個主要的嗜鹽古細菌中的微生物視紫蛋白質結構研究中，唯一未能解出的結構補齊，啟發同類蛋白質研究之新可能性。鑑於所有證據都支持此一臺灣井仔腳古生菌為新種，本實驗對此菌做全基因定序，並進行基因註解 (gene annotation)，已確定所有菌視紫蛋白質之存在及胺基酸順序，並初步得知H. taiwanensis之基因圖譜。

關鍵字

光趨視紫蛋白質；光趨性；光週期

並列摘要

Studies in proteins from local species offer valuable information in biological diversity. The main aim of this study was to investigate whether microbial rhodopsins in haloarchaea from Taiwan exhibit a region-dependent adaptation. To achieve this goal, we first tried to isolate local haloarchaea and proved them to be a new species, then microbial rhodopsins from it were cloned for bioinformatic, biochemical and biophysical studies before we attempted to compare their differences with other known microbial rhodopsins. In a previous study, we have isolated a haloarchaea from Beiman saltern in southern Taiwan and and identified it as a new strain; it was temporarily named as Ht. In this study, further growth condition experiments suggested Ht as a new strain; we therefor name it Haloarcula taiann. A total of four microbial rhodopsins were successfully cloned and sequenced from Ht. Protein sequence alignment showed only 87-93% similarity to any known microbial rhodopsins; it further supported H. taiann as a new species. Among those four rhodopsins, we found sensory rhodopsin I, HtSRI, to be unusually more stable and was surprisingly responsive to light of > 650 nm, a feature not being observed in any other know SRI proteins. In addition to biological, biochemical and biophysical studies, we also tried to grow protein crystal, a procedure failed in all other SRI due to stability issue. Since SRI is the only microbial rhodopsin that still lacks atomic structure, we have list it as a goal in this study. We expected that solving protein structure for HtSRI can shine light on protein science. We also cloned its transducer, a cognate partner protein when SRI exerting positive phototaxis, and engineered them together as another strategy to further stabilize HtSRI for crystallographic study. Hence all the evidences supported H. taiwanenesis a new species from Taiwan, we further conducted whole genomic sequencing and gene annotation. Now we have obtained all the rhodopsins exiting in H. taiwanenesis and full length gene sequences.

並列關鍵字

haloarchaea ； Haloarcula ； senosory rhodopsin ； phototaxis ； photocycle

參考文獻

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從台灣分離之嗜鹽古細菌光趨性分析與其感受型視紫蛋白質之特性

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