嗜甲烷菌 Methylococcus capsulatus (Bath)之系統生物學研究

Systemsbiology Studies of the Methanotrophic Methylococcus capsulatus (Bath)

高偉鈞(Wei-Chun Kao)

指導教授 ： 陳長謙

國立臺灣大學/理學院/化學研究所/碩士(2004年)

https://doi.org/10.6342/NTU.2004.00185

摘要

在某些嗜甲烷菌之中，銅離子可以轉換氧化甲烷的催化機制從細胞質 (cytosol) 中的溶解性甲烷單氧化酶 (soluble methane momooxygenase, sMMO) 至細胞內膜 (intracellular membrane) 中的微粒體甲烷單氧化酶 (particulate mathane monooxygenase, pMMO) 上。銅離子除了具有啟動轉譯 (transcription) 的弁鄍~，在本菌 Methylococcus capsulatus (Bath) 中也可以刺激生化合成的進行。在高銅離子濃度的環境之下，微粒體甲烷單氧化酶會大量表現，同時脂質的生合成也會被刺激，開始大量製造細胞內膜以形成支持微粒體甲烷單氧化酶的網路架構。為闡明銅離子調控本菌生理狀態之分子機制，我們使用基因體定序 (genomic sequencing) 以及大規模的定量比較蛋白質體解析 (large scale quantitative comparative proteomic analysis) 來解決在不同銅離子濃度的環境之下，細胞質以及細胞內膜中所有蛋白質的表現變化 (differential expression) 之問題。我們分別將在不同銅離子濃度下的細胞質以及細胞內膜中所有蛋白質標識上酸裂性同位素親和性標識 (acid cleavable isotope-coded affinity tag, cICAT) 試劑，用二次元管柱層析將所有標識並酵素水解過的片段分離，並且用電灑式串連質譜 (electrospray ionization-tandem mass spectrometry, ESI-MS/MS) 以及基因體定序資料庫決定蛋白質序列。我們在本菌中一共鑑定出 682 個蛋白質，其中有 623 個為之前沒有發表過序列的新結果。定量比較蛋白質體解析顯示銅離子對於本菌的生化代謝有顯著之影響，在沒有銅離子以及 30 mM 銅離子的環境之下，分別有 68 及 60 個蛋白質的表現量超過兩倍。本菌中重要的代謝路徑亦被解明，和氧化以及處理甲烷的單碳相關代謝路徑均有被銅離子濃度調控的情形，包括 RuMP, RuBP, serine, 以及 H4MPT pathway。其中前三者的第一步反應酵素在高銅離子濃度下會被大量表現，而最後者在沒有銅離子的情況下才會被活化，本實驗結果亦顯示在高銅離子濃度下微粒體甲烷單氧化酶會被大量表現，而在沒有銅離子的情形本菌使用溶解性甲烷單氧化酶 來氧化甲烷，這一點和已知的事實完全相符，反應出銅離子對於本菌的甲烷氧化以及能量代謝的調控為一體之情形。
我們亦發現運送氧氣的 hemerythrin 在高銅離子濃度之環境下被大量表現，為了證實上述實驗結果，hemerythrin 之純化以及光譜特性必須完成。我們使用除鹽，離子交換法，以及膠體過濾法順序成左滲瞻ぁX hemerythrin，其紫外光-可見光 (UV-visible) 光譜亦與現有文獻資料相符。膠體過濾法的分子量計算亦指出本菌的 hemerythrin 為單體 (mo-nomer)，而在現有文獻中，大部分的 hemerythrin 則以四元體 (tetramer) 以上的形式存在。
本研究進行首次的嗜甲烷菌的基因體定序，完成第一個銅離子調控的定量比較蛋白質體解析，並純化出第一個細菌的原態 hemerythrin。系統生物學研究不僅解決基因體，蛋白質體，以及代謝上的問題，生物演化的課題亦已從單碳代謝以及氧氣運送機制的角度出發探討。

關鍵字

系統生物學 ； 微生物代謝 ； 蛋白質體學 ； 生物資訊學 ； 嗜甲烷菌

並列摘要

Copper ions are known to switch the methane oxidation from the soluble methane monooxygenase (sMMO) to the particulate methane monooxygenase (pMMO) in certain strains of methanotrophic bacteria. Aside from being a transcriptional switch, copper is also a metabolic activator, stimulating the production of high concentrations of pMMO and lipid biosynthesis to form the extensive networks of intracellular membranes in which the membrane-bound pMMO resides. To elucidate the molecular mechanism associated with this copper ion regulation, we have applied genomic sequencing and large-scale comparative quantitative proteome analysis to the cytosolic and membrane-associated proteins in Methylococcus capsulatus (Bath) grown under different copper-to-biomass ratios. Proteins associated with the membrane and the cytosolic portions of the proteome under different copper ion concentrations were differentially labeled with an acid cleavable isotope-coded affinity tag (cICAT) reagent, and the labeled peptides were separated by two-dimensional chromatography and characterized by electrospray ionization-tandem mass spectrometry (ESI-MS/MS). The study confirmed the expression of 682 unique proteins, of which 623 have not been sequenced previously. In 682 proteins identified, 68 and 60 proteins were overexpressed at least two fold in the absence and presence of Cu, respectively. These 68 proteins are mainly responsible for cellular signaling process while the 60 proteins cover methane and carbohydrate metabolic enzymes, indicating a role of copper in expression of the genes associated with metabolism of the organism downstream of methane oxidation.
The metabolism route of M. capsulatus (Bath) was also clarified. Levels of the enzymes involved in the C1 metabolism (RuMP, RuBP, serine, and H4MPT pathway) of the organism were dramatically affected, most notably, the soluble and particulate forms of the methane monooxygenase that mediate the methane oxidation under copper-deficient and copper-rich medium, respectively. In addition, high expression levels were observed for a number of key enzymes involved in the C1 metabolism pathway, suggesting a regulatory role of copper ions in regulating the expression of genes involved in the metabolism of the organism downstream of methane oxidation as well.
Hemerythrin was observed to be overexpressed about three fold in high copper environment. In order to prove the existence and expression status of M. capsulatus (Bath) hemerythrin, purification and spectroscopic studies were undertaken. The hemerythrin was purified successfully by desalting, ion-exchange, and gel-filtration chromatography techniques, and UV-visible spectrum revealed that the absorbance of M. capsulatus (Bath) hemerythrin was in accord with those previously reported for hemerythrins from higher organisms. A molecular weight estimation indicated that the native hemerythrin in M. capsulatus (Bath) is most likely a monomer, in contrast to hemerythrins from other organisms, where they usually exist as a tetramer or higher oligomers.
In summary, we have demonstrated in this work the first applications of the genomic sequencing of M. capsulatus (Bath): the quantitative proteomic analysis of copper regulation, and the discovery and purification of a native hemerythrin in bacteria. The present Systemsbiology study of M. capsulatus (Bath) has not only provided a genomic, proteomic and metabolic overview of this organism, but also clarified a number of long standing evolutionary issues, including elucidation of the C1 metabolic pathways and oxygen transporting mechanisms in this interesting methanotroph.

並列關鍵字

proteomics ； ICAT ； methanotrophic bacteria ； microbiol metabolism ； systemsbiology ； bioinformatics

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