摘要
陽光是地球上所有生命重要的直接或間接能量來源。光感受體是細胞內調控與反應光照環境十分重要的蛋白質。由於基因體學的發展，科學家發現光感受體廣泛的存在各式微生物中，除了有將光能轉變成化學能使用的功能之外，還可以影響微生物的趨性、毒性以及生長。嗜鹽古細菌中的視紫紅質，是目前研究中最具代表性的對象。 視紫紅質的功能可以分為兩大類，第一類是光驅動離子泵，包括菌視紫紅質和氯視紫紅質，功能分別是光驅動外向氫離子泵和內向氯離子泵。第二類是感光型視紫紅質，分別是可以同時控制正負光趨性的光趨性視紫紅質第一型與控制負光趨性的光趨性視紫紅質的第二型。 死海鹽古菌 (Haloarcula marismortui) 是死海中目前少數已知的嗜鹽古細菌。而死海鹽古菌利用何種獨特的機制，成為死海的倖存者，是個未知的主題。2004 年完成的死海鹽古菌基因體計畫中，發現其擁有六個視紅質的基因，是目前單一嗜鹽古細菌中最多的。其中包括兩個菌視紫紅質、一個氯視紫紅質、調控光趨性的光趨性視紫紅質第一型與光趨性視紫紅質的第二型和一個功能未知的視紫紅質。本論文主要針對其六個視紅質之獨特性，所浮現的兩大問題來尋求其生存之獨特性：(A) 為何同時擁有兩個菌視紫紅質，以及 (B) 死海鹽古菌是否具有對光譜更好的感受性。 本研究中發現，死海鹽古菌相對於只有一個菌視紫紅質 (HsBR) 的嗜鹽桿古菌 (Halobacterium salinarum)，可以同時利用兩個菌視紫紅質，不同卻為互補的最佳酸鹼值作用範圍，營造在較廣的 pH 範圍中，維持至少一個光驅動氫離子泵的活性。實驗分別針對死海鹽古菌中的兩個菌視紫紅質 (HmBRI 與 HmBRII) 進行不同 pH 值之下的活性測試，發現 HmBRI 和先前已知之 HsBR 相似，在 pH 值分別低於 5 和 6 時就會失去活性；而 HmBRII 在 pH 值高於 4 以上就具有活性。所以，在死海鹽古菌中同時具有兩個菌視紫紅質，可以針對不同的 pH 值之下維持持續的光驅動氫離子泵的活性；而此一互補後有活性的酸鹼範圍，恰可涵蓋死海 pH 5.5 的環境，是其它單菌視紫紅質的嗜鹽古細菌生物無法涵蓋的。據此推斷，這可能是死海鹽古菌能存活在死海的原因之一。 微生物光趨性視紫紅質扮演調控正負光趨性的功能時，需要專一地與一具有兩個穿膜區之觸發器蛋白質共同完成這個生理功能。在死海鹽古菌的基因組中，意外地沒有其他類同嗜鹽古生菌控制浮沈的氣胞 (gas vacuole)。過去研究顯示，氣胞可能同時扮演著折射或反射光線、和利用光線調控浮沉的角色。使得死海鹽古菌中，這些未被完整標註與鑑定的協同觸發器蛋白質變得可能更為重要。本研究中發現，於基因體定序中，標註為正趨光性的光趨性視紫紅質第一型基因，其上游被標註之可能的觸發器蛋白質，為一缺少兩個穿膜區之可溶蛋白質，而非膜蛋白質。綜合分析目前完成基因體解碼的嗜鹽古細菌之基因體資訊，比較兩型光趨性視紫紅質與其對應觸發器之生物資訊學資料，發現死海鹽古菌光趨性視紫紅質第一型基因上游的觸發器蛋白質基因上游 291 個核苷酸，可能利用替代起始密碼子 GTG，而此新的轉譯序列會形成一標準之雙穿膜蛋白質區，將符合所有其他已知協同觸發器之構形。經過實驗證明，其確實與光趨性視紫紅質第一型交互作用。此外綜合光趨性的實驗結果，證實死海鹽古菌是第一個能同時感應三原色之單細胞生物。 總結而言，本研究發現了死海鹽古菌以兩個最佳功能酸鹼值範圍互補的菌視紫紅質，組成了可以適應其他多數嗜鹽古生菌無法生存酸性水質的死海。並且，死海鹽古菌有個別對三原色有反應的光趨性視紫紅質，可能對缺乏調節浮力的氣胞，有更精確功能的取代作用。研究死海鹽古菌中六個視紫紅質的感光系統，揭露微生物視紫紅質在單細胞微生物中，相較於傳統過去四十年已知只有四個視紫紅質的感光系統，能夠在各式生理功能中扮演不同面貌的特性。
並列摘要
Solar radiation is the most important energy source for all life forms on earth. Photoreceptors play important roles in responding to the light from environment. Since the blooming proceeding of the genome projects, scientists find that the photoreceptors are ubiquitous in almost all the microbes. The photoreceptors relate to the solar energy conversion, phototaxis, virulence and life cycle. Halobacterium rhodopsins are the conventional targets for the photoreceptor studies. Two distinct functions have been identified. One is a light-driven ion translocator, including bacteriorhodopsin (BR) which is an outward proton pump and halorhodopsin (HR) which serves as an inward chloride pump. The other mediates phototaxis response, including the sensory rhodopsin I (SRI) shown to mediate both attractant and repellent signaling and sensory rhodopsin (SRII) that triggers repellent signaling against near-UV light. Haloarcula marismortui is one of the survivors in Dead Sea, but the primary reason behind such phenomenon is unknown. There were a total of six predicted opsins genes in H. marismortui genome, the most numbered opsins in a single archaeon, and all of them were cloned, over-expressed and confirmed to have two BRs, one HR, one SRI, one SRII and a new photosensory rhodopsin (SRM) in our previous study. This dissertation intends to answer two questions: (A) what is the physiological significance of this two-BR system? (B) Do the three-sensory rhodopsin system sense and response to red, green, and blue light and have spectrum sensitivity higher than the traditional two-SR system. The results showed that H. marismortui cells have a unique isochromatic dual-BR system, consisting of HmBRI and HmBRII and they were shown to translocate protons upon light illumination within wider pH range than H. salinarum which has a single BR (solo-BR). Series pH-dependent assays using purified BR proteins demonstrated that HsBR and HmBRI were not functional at pH < 5.0 and 6.0, respectively, but HmBRII remained functional at pH > 4.0. Therefore, our results conclude that the dual-HmBR system is composed of two BRs with different optimal functional pH ranges and together they maintain light-driven proton transport activity under pH > 4.0, which might contribute the survival of H. marismortui under the acidic pH 5.5 of the Dead Sea. Microbial sensory rhodopsins are known to mediate phototaxis with a specific cognate transducer that has two-transmembrane (2-TM) regions. In the genome of H. marismortui, there was no gene encoding gas vacuole. In other halophilies, such as Halobacterium salinarum, gas vacuole was shown to be related to the light shielding and light-driven buoyancy control. The lacking of the related genes in H. marismortui suggests other motile regulation of the whole H. marismortui cells other than gas vacuole is important. In this study, it is found that the translated amino acid sequnece of the candidate transducer gene for HmSRI was missing the 2-TM region - a cytosolic protein but not transmembrane protein. Combining the bioinformatics analysis of all SRs and cognate transducers from the known Halophile genome projects, it is shown that this transducer gene featured a preceding 2-TM region when the alternative start codon GTG located 291 nucleotides upstream of the original annotated open reading frame was introduced. The 2-TM-restored HmHtrI was shown to be a transmembrane protein and it interacted with HmSRI like other transducers of SRs. Together with the findings of phototaxis responses results, the H. marismortui appears as a microbe features a total of three sensory-transducer proteins that response to red, green and blue (RGB) light. In conclusion, the dual-BR system in H. marismortui with complementary optimal pH ranges accommodated itself to exist in the acidic aqua of Dead Sea. Besides, the trichromacy RGB sensory rhodopsins in H. marismortui may be a precision substitute for gas vesicles which may provide an even more precise phototaxis response than a gas vascuole system. Studying the six-rhodopsin photosensory system in H. marismortui unveiled the new faces of the physiological roles with microbial rhodopsins, comparing to the conventional four-rhodopsins system within the past fourty years, in an unicellular organisms.