分析高鹽甲烷太古生物鹽逆境反應基因與分子伴護蛋白系統

Abstract

高鹽甲烷太古生物Methanohalophilus portucalensis FDF1能生長在胞外鹽濃度1.2 M-2.9 M的環境中，並以methanol及trimethylamine做為甲烷反應的基質。為了克服高鹽環境滲透壓力的波動，細胞能運送betaine或自體生合成betaine、β-glutamine、以及Nε-acetyl-β-lysine做為相容質。以55組引子進行差異性表現逆轉錄聚合酶連鎖反應，用以比較細胞在高鹽逆境與低鹽逆境下基因表現的差異性。結果共偵測到14個在高鹽或低鹽逆境表現具差異的基因並完成選殖與定序。其中幾個基因與甲烷菌特有的甲烷化反應基因相關，分別是膜蛋白MttP (SU11312)，cobalamin生合成蛋白 (SU34312)，methenyl-H4MPT cyclohydrolase (SD44094) 以及monomethylamine 甲基轉移酶 (SU26311)。在高鹽逆境下有鑑別出一個與訊息傳遞有關的histidine kinase (SU37311)。另外有三個已知的逆境反應基因也在此實驗中被偵測到，包括DNA 修復蛋白 Muts (SU25311)，universal stress protein UspA (SD38092) 以及分子伴護蛋白系統的ClpB (SU12312)。從轉錄分析發現，能量代謝基因會受瞬間高鹽逆境而降低轉錄量。最值得一提的是，核糖體蛋白基因 (SU37311)、lysyl tRNA合成酶 (SD44093)、nitrite及sulfite還原酶以及translation elongation factor 1 alpha (SD17092) 則是第一次發現與鹽逆境反應有關。 ClpB和GroES及GroEL都屬於分子伴護蛋白系統，能促使蛋白進行去凝集化以及重新摺疊。此論文中我們提出了Methanohalophilus portucalensis的ClpB、GroES及GroEL基因。Methanohalophilus portucalensis ClpB基因序列全長2610 bp及其上游的GroEL (1611 bp) 與GroES (276 bp) 基因是利用inverse PCR及南方墨點法所得並完成定序。ClpB預測的多肽鏈包含869個胺基酸並含有一個會形成coiled coil的中間區域及兩個ATP結合區 (NBD1和NBD2)。NBD1有一個Walker A及兩個Walker B motif而NBD1則只有各一個Walker motif。在ClpB的N端則有兩個重複的Clp amino-terminal domain motifs (ClpN)。GroES和GroEL的多肽鏈分別為91及536個胺基酸。根據初步的系統演化分析則可發現MpClpB與革蘭氏陽性菌的ClpB演化距離較為接近而太古生物的GroES與GroEL則是自成一群且與細菌的該蛋白演化距離較遠。以北方墨點法分析發現clpB在瞬間高鹽逆境 (NaCL從2.1 M提高至3.1 M NaCl) 及低鹽逆境 (NaCl從2.1 M降至0.9 M) 的轉錄量均會提升約1.5倍。ClpB與GroEL/ES基因的轉錄量都會隨溫度上升 (37°C-55°C)而提升。在熱逆境下添加1 mM的betaine則會降低clpB的轉錄量。總結來說，本論文為第一次鑑別並分析高鹽甲烷太古生物與溫度、鹽濃度及相容質betaine波動反應相關的AAA+ chaperone ClpB基因。

Methanohalophilus portucalensis FDF1, a member of the halophilic genus of methanoarchaea, can grow over a range of external NaCl concentrations from 1.2 to 2.9 M and utilize methanol and trimethylamine as substrates for methanogenesis. To encounter the changing osmotic stress in hypersaline environment, cell could transport betaine or de novo synthesize betaine, β-glutamine, and Nε-acetyl-β-lysine as compatible solutes. Differential gene expression in response to long term hyper-salt stress (3.1 M NaCl) and hypo-salt stress (0.9 M NaCl) were analyzed and compared by differential display reverse transcription-PCR with fifty five primer sets. Fourteen differentially expressed genes respond to long term hyper-salt stress or hypo-salt stress were detected, cloned and sequenced. Several of differentially expressed genes were related to the unique energy-acquiring methanogenesis of methanogen as transmembrane protein MttP (SU11312), cobalamin biosynthesis protein (SU24312), methenyl-H4MPT cyclohydrolase (SD44094) and monomethylamine methyltransferase (SU26311). One signal transduction histidine kinase (SU12317) was identified from hyper-salt stress culture. Moreover, three known stress responsive gene homologues: the DNA mismatch repair protein Muts (SU25311), the universal stress protein UspA (SD38092), and the member of protein-disaggregating multichaperone system ClpB (SU12312) were also detected. The transcript analysis of the immediately expression indicated the energy metabolism was arrested during hyper-salt shock while the chaperone clpB gene was stimulated in both hypo and hyper salt shock. Notably, genes for ribosomal proteins (SU37311), lysyl tRNA synthetase (SD44093), nitrite and sulfite reductase (SD18092) and translation elongation factor 1 alpha (SD17092) were the first time to identified associated with salt stress. ClpB, GrpEL and GroES promote protein disaggregation and refolding and belong to the molecular chaperone system. Here, we describe a new ClpB gene and the group I chaperonin GroEL/ES genes from the halophilic methanoarchaeon Methanohalophilus portucalensis (Mp). The complete clpB sequence (2610 nt) and its up stream genes encoding the type I chaperonin GroEL/ES (1611 bp/276 bp) were obtained through the inverse PCR, southern hybridization, and sequencing. The predicted polypeptide of ClpB contains 869 amino acids and posses a long central domain, a predicted distinctly discontinuous coiled-coil motif separating two ATP-binding sites (NBD1 and NBD2). NBD1 has a single Walker A and two Walker B motifs and NBD2 has only one of each Walker motifs. Two repeated Clp amino-terminal domain motifs (ClpN) were identified in ClpB. The predicted polypeptide of GroES and GroEL contain 91 and 536 amino acids, respectively. The predicted mobile loop in GroES and the putative ATP-Mg2+ binding domains, GxxLE region in GroEL were identified. Preliminary phylogenetic analysis clustered MpClpB with the low G+C gram-positive bacteria while the archaeal GroES and GroEL formed a distinguish group distant from bacterial ones. The stress response analysis of clpB by Northern blot showed up to 1.5 fold increased transcriptional level in response to both salt upshock (from 2.1 M NaCl to 3.1 M NaCl) and downshock (from 2.1 M NaCl to 0.9 M NaCl). Both clpB and groEL/ES transcript levels increased while temperature shifted from 37°C to 55°C. Under heat stress clpB transcription was repressed with the addition of osmolyte betaine (1 mM). In conclusion, a novel AAA+ chaperone ClpB gene that responded to the fluctuation of temperature, salt concentrations, and osmolyte betaine from a halophilic methanogen was identified and analyzed for the first time.