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National Chung Hsing University Institutional Repository - NCHUIR > 生命科學院 > 生命科學系所 > 依資料類型分類 > 碩博士論文 >  高鹽甲烷太古生物Methanohalophilus portucalensis FDF1T的一級甜菜鹼運輸系統Bta的分析

Please use this identifier to cite or link to this item: http://nchuir.lib.nchu.edu.tw/handle/309270000/152368

標題: 高鹽甲烷太古生物Methanohalophilus portucalensis FDF1T的一級甜菜鹼運輸系統Bta的分析
Osmolyte glycine betaine ABC transport system Bta in Methanohalophilus portucalensis FDF1T
作者: 吳舒堯
Wu, Sue-Yao
Contributors: 賴美津
生命科學系所
關鍵字: 太古生物;一級運輸系統;甜菜鹼
Archaea;ABC type transporter;glycine betaine
日期: 2012
Issue Date: 2013-11-07 13:23:13 (UTC+8)
Publisher: 生命科學系所
摘要: 高鹽甲烷太古生物Methanohalophilus portucalensis FDF1T於高滲透壓下的適應機制之研究,目前已證實M. portucalensis FDF1T能攝取鉀離子,或是自體生合成glycine betaine、β-glutamine及Nε-acetyl-β-lysine做為相容質於細胞內累積;或是利用高專一性、高親和性及需要消耗能量的甜菜鹼一級運輸系統,從胞外攝取glycine betaine至細胞內累積,以平衡胞內外滲透壓差。且由M. portucalensis FDF1T基因體中搜尋並獲得相容質一級運輸系統之三個基因btaABC1,命名為Bta (betaine transportor in Archaea)系統;在高鹽與高溫逆境下添加相容質glycine betaine,bta基因皆會被大量轉錄,顯示Bta系統是受高鹽、高溫及glycine betaine的誘導。
藉由高通量測定系統定序的資料、南方墨漬法及聚合酶連鎖反應重新檢視M. portucalensis FDF1T基因體中的Bta運輸系統基因群,發現btaC1基因下游有另兩套受質結合蛋白 (SBPs, Substrate-binding proteins)的基因,分別為距離btaC1基因72 bp的btaC1’基因,及距離1546 bp單獨的btaC2基因,Bta運輸系統的基因排列為btaA-btaB-btaC1-btaC1’---btaC2,是甲烷太古生物中第一個發現具有三個SBP基因的一級運輸系統。當處於高鹽及含有glycine betaine的條件下,btaA mRNA經後轉錄修飾而截去2 bp,使得能轉譯成有功能的BtaA蛋白,並經序列及蛋白結構分析發現,其C端延伸區域具有兩個CBS domains,及朝向細胞膜的面上有四個帶正電的胺基酸,做為胞內離子強度的感應因子,可能扮演活化並啟動Bta運輸系統的關鍵角色。三個SBP基因序列及蛋白結構分析顯示,BtaC1分別與BtaC1’及BtaC2的胺基酸序列相似度為39 %及80 %,且皆有與相容質glycine betaine結合的三個保留性Trp胺基酸。由於BtaC1與BtaC2胺基酸相似度高,經由基因轉錄的分析顯示,btaC1及btaC2基因亦會受到低溫及高鹽逆境下的誘導而增加,而btaC1基因更受到外界glycine betaine的誘導表現,且其基因轉錄量較btaC2高3-10倍。受質結合活性的結果顯示,BtaC1SPD及BtaC1’SPD對glycine betaine結合皆為高親和性,KD值在μM範圍,且分別可與dimethylglycine及sarcosine結合;BtaC2SPD為廣效型結合蛋白,可與glycine betaine、dimethylglycine、sarcosine、choline及carnitine結合,但親和性低。綜合以上結果顯示BtaC1在基因轉錄調控及高親和性結合glycine betaine的特性,可能為M. portucalensis FDF1T在溫度及鹽度逆境下主要攝取受質的重要結合蛋白。互補實驗亦證實BtaAB運輸中心可接受由BtaC1或BtaC2所攝取的glycine betaine,並運送至胞內累積,推測Bta運輸中心可以接收不同的受質結合蛋白,且三種受質結合蛋白具有不同glycine betaine親和性與glycine betaine相關相容質專一性,顯示M. portucalensis FDF1T擁有自體生合成及運輸glycine betaine的精緻網絡之滲透壓調節機制。
The osmotic adaptation of halophilic methanogen, Methanohalophilus portucalensis FDF1T could uptake potassium or de novo synthesized glycine betaine, β-glutamine and Nε-acetyl-β-lysine as compatible solutes to encounter the increasing external osmotic and salt stress. Most importantly, it also posses an energy-required, high affinity and highly specific glycine betaine transport system. An ABC type transporter for glycine betaine, Bta (betaine transporter in Archaea) system, in M. portucalensis FDF1T has revealed and the transcription of bta expression was activated immediately in presence of external glycine betaine upon the salt stress or heat stress. In this study, the bta gene cluster was confirmed as btaA-btaB-btaC1-btaC1’---btaC2 by metagenomic pyrosequencing data of M. portucalensis FDF1T, which is the first ABC transporter containing three substrate binding proteins (SBP) genes from methanogic archaea. RT-PCR analysis revealed the possible post-transcriptional modification through two base splicing in btaA while cells under salt upshock and the betaine is available in the environment. The sequence analysis and protein homology modeling of BtaA revealed two CBS (cystathionine β-synthase) domains were located at C-terminal extended region, and surface-exposed four cationic residues in the CBS domains are critical for ion sensing. It suggested BtaA play the major role of osmotic regulation in Bta system. All three substrate binding proteins, BtaC1, BtaC1’ and BtaC2, contained a tryptophan prism for betaine binding as other known substrate betaine-binding proteins. The transcription of btaC1 and btaC2 were both activated upon the hyper-salt or cold stress, whereas the expression of btaC1 are 3-10 fold higher than btaC2. BtaC1SPD and BtaC1’SPD are a high affinity betaine specific binding protein, and could also bind dimethylglycine or sarcosine, respectively, and carnitine. The glycine betaine binding affinity of BtaC2SPD is low, but it could bind multiple substrates, such as dimethylglycine, sarcosine, choline and carnitine. The functional complementation analysis indicated BtaAB core transporter could recruited BtaC1 and BtaC2 to translocate osmolyes into cell. This investigation revealed the occurrence of multiple substrate-binding proteins for betaine transport system Bta to response the salt and temperature stresses and the broad substrate spectrum and substrate betaine affinity range suggested the delicate network between osmolyte betaine transport and biosynthesis in osmotic regulatory system.
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