Isolation and Classification of Methanogens and Investigation of Their Osmolyte Glycine Betaine Transport Systems

絕對厭氧的甲烷太古生物廣泛地存在在自然與人為的環境中。近幾年來，賴美津博士實驗室於台灣環境中採樣並純化數十株甲烷太古生物。其中從彰化王功附近的海水養殖池與河海口環境中分別得到菌株N2F9704T與P2F9704aT。形態上，這兩株甲烷太古生物皆為不規則球狀菌、屬中溫菌(最適生長溫度為37 °C)、且皆能夠利用甲酸(formate)或氫氣與二氧化碳作為碳源來進行甲烷化作用(methanogenesis)而獲得生長所需的能量。生長測試結果發現微量元素鎢可以促進菌株N2F9704T的生長並擴大其生長條件的上下限，但對菌株P2F9704aT只有輕微促進生長的效果。另外，16S rDNA序列分析的結果顯示菌株N2F9704T與P2F9704aT分別屬於Methanofollis與Methanocalculus這兩屬的甲烷太古生物，並分別命名為Methanofollis aquaemaris N2F9704T和Methanocalculus taiwanensis P2F9704aT。此外，菌株N2F9704T亦發現其具有直徑約200 nm的球狀病毒MMV，為少數從太古生物中發現的病毒之一。而這兩株甲烷太古生物為台灣第一次發表的新種甲烷太古生物。 本研究另以能自體生合成相容質betaine的高鹽甲烷太古生物Methanohalophilus portucalensis FDF1作為研究材料來探討其滲透壓調控機制，並期許能進一步了解太古生物與真細菌滲透壓調控機制的異同。首先分析目前已公佈於NCBI網站的25株太古生物的基因體序列，發現太古生物基因體中具有類似真細菌參與滲透壓調控之數種運輸系統的基因(proU、opuD和proP)。藉由這些資訊設計了一組引子並得到高鹽甲烷太古生物類似ABC型式betaine運輸系統ProU的基因，命名為bta (betaine transporter in archaea)。經由北方點漬法的分析，發現bta基因可以受到高鹽逆境而緩慢增加基因表現；若在高鹽逆境下，bta基因可以被環境中的betaine、choline、glycine、sarcosine和dimethylglycine誘導而快速地在15-30分鐘中達到最高峰的表現量。另外，高溫逆境下，bta基因表現量亦會因外界有betaine而增加；且發現bta基因上游序列與一些太古生物的熱逆境蛋白基因之上游序列相似，可推測bta基因於此高鹽甲烷太古生物當中扮演著對鹽與熱逆境的調控。而bta基因為第一個於能自體生合成相容質betaine菌株中發現的ABC型式betaine運輸系統。 為了分析Bta系統的特性，Bta系統藉由乳酸菌Lactococcus lactis的nisin A所控制的蛋白表現系統來表現並可用Ni2+-NTA管柱層析做進一步純化。螢光光譜儀分析結果，顯示betaine對於受質鍵結蛋白-BtaC的KD大約為15 μM，但是測試的條件仍然需要修正以期了解BtaC受質的種類。另外，重組Bta系統於人工膜上來進行運輸活性的測試，目前此系統運輸膜外的放射性betaine的活性仍然很低，需要進一步找到最佳的測試條件或材料，例如採用從此高鹽甲烷太古生物純化其本身的細胞膜質(archaeal lipids or archaeosomes)來建構此系統。Strictly anaerobic methanogenic archaea are ubiquitous. Dr. Lai's lab has isolated over twenty methanogens from Taiwan. Among them, strain N2F9704T was isolated from the seawater fish pond and strain P2F9704aT isolated from the estuarine near Wong-gong, Taiwan. Both strains are mesophilic, irregular cocci. Cells could metabolize formate and H2/CO2 as carbon source to obtain the energy through methanogenesis. Trace amounts of tungstate not only promoted growth of strain N2F9704T but also extended the range of growth conditions. However the trace element of tungstate only lightly stimulated the growth of strain P2F9704aT. Analyses of 16S rDNA sequences revealed that strain N2F9704T and strain P2F9704aT are members of genera Methanofollis and Methanocalculus respectively, and are named as Methanofollis aquaemaris N2F9704T and Methanocalculus taiwanensis P2F9704aT. Additionally, strain N2F9704T was infected with a novel coccus-shaped, enveloped virus MMV with a diameter of 200 nm. Both strains were the first published new methanogenic species from Taiwan. In addition to characterization of methanogens from Taiwan, the investigation of the osmoregulation of the betaine synthesizing halophilic methanogen, Methanohalophilus portucalensis FDF1, and the comparisons of osmoregulatory mechanisms between bacteria and archaea are our subject. Firstly, searching the bacterial osmoprotectants transporters through 25 archaeal genomes showed it may be the effect of the lateral gene transfer that archaea contain several homologues genes of bacterial osmoregulatory transporters, including proU, opuD and proP. Based on these informations, an ABC type transporter for betaine, the Bta (betaine transporter in Archaea) system, was identified in M. portucalensis FDF1. The gene structure of the bta gene was similar to proU of Escherichia coli. Transcriptional inducer tests revealed that betaine, choline and its de novo synthesized intermediates, glycine, sarcosine and dimethylglycine, could immediately increase the transcriptional level of the bta gene under the hyperosmotic stress. But proline and carnitine, the osmolytes occurred in bacteria, couldn't increase the bta expression. Upon the heat shock, the transcription of the bta gene was also activated immediately in the presence of external glycine betaine. Moreover, the upstream sequences of the bta gene have conserved sequences similar to other archaeal heat shock responsed genes, like dnaK and grpE. It suggests that in addition to play a role in osmotic stress, bta also responds to the heat stress. This ABC type glycine betaine transporter Bta system is first dicovered in the betaine-synthesizing organisms.Abstract-P1 中文摘要-P3 Chapter 1-Introduction-P5 Chapter 2-Methanofollis aquaemaris sp. nov., a methanogen isolated from an aquaculture fish pond-P37 Chapter 3-Methanocalculus taiwanensis sp. nov., isolated from an estuarine environment-P69 Chapter 4-Investigation of the osmolyte glycine betaine transport system in halophilic methanogen Methanohalophilus portucalensis FDF1-P100 Chapter 5-Determination of substrate specificity of Bta system and reconstitution of Bta-hybrid membranes for transport assays-P188 Chapter 6-Conclusions and Perspectives-P22