耐鹽菌 Halomonas beimenensis 與 Virgibacillus chiguensis 之耐鹽機制研究

Study of high saline adaptation mechanisms for Halomonas beimenensis and Virgibacillus chiguensis

陳彥惠(Yan-Huey Chen)

指導教授 ： 徐源泰

共同指導教授 ： 林詩舜

國立臺灣大學/生物資源暨農學院/園藝暨景觀學系/博士(2017年)

https://doi.org/10.6342/NTU201800287

摘要

近年來，因為耐鹽菌的極為耐鹽的特性，使其成為生技產業上的新星。由於耐鹽菌的極端逆境耐性，使其成為學術與產業所高度重視，期能藉由對極端逆境抗性的瞭解，以開發包括生技醫藥、與食品工業的應用；在蔬果加工領域，微生物的耐高鹽更可創新產品、加速製程、並減少污染等。然而，耐鹽菌完整的基因體資訊仍舊不足。本研究利用高通量體學分析 Halomonas beimenensis 與 Virgibacillus chiguensis 這兩株可生長於 20% 鹽濃度環境的耐鹽菌並探討其之可能的耐鹽機制。H. beimenensis 完整的基因體長度為 4.05 Mbp，其內含 3,807 個基因，而 Virgibacillus chiguensis 的基因體草圖總長度為4.15 Mbp，且預測有 4,347 個基因。在高鹽的環境下，我們發現許多參與在細胞移動以及無機鹽離子代謝通透的基因可被大量表現；而與能量轉換、轉錄、脂肪及胺基酸代謝通透相關的基因表現量則降低。此外，透過 Tn5 轉位子所得到失去耐鹽性的 H. beimenensis 突變株中，發現有十六個基因可能與耐鹽機制有關。nqrA、trkA、atpC、nadA 及 gdhB 這幾個和一般所知的耐鹽機制如鈉鉀離子通透、氫離子通透以及相容性溶質生合成有關。而其他的基因，如 spoT、prkA、mtnN、rsbV、lon、smpB、rfbC、rfbP、tatB、acrR及lacA 則是與細胞訊息傳遞、群體感應、基因轉錄與轉譯，以及細胞移動相關聯，且影響了細菌的耐鹽。而其中八個基因，如 trkA2、smpB、nadA、mtnN2、rfbP、spoT、lon 及 atpC，於 V. chiguensis 中亦為保守基因，且於高鹽環境下也和在 H. beimenensis 一樣，有相似的基因表現模式，這也說明了，此八個基因於不同門的分類下的細菌也可能是和耐鹽能力有關。另外，額外添加氯化鉀，則發現能夠增加H. beimenensis的耐鹽性與鉀離子依賴型的細胞移動。我們的結果顯示結合基因體與轉錄體，或者結合其他組學資料能有效地幫助探討細菌高鹽適應機制。以更長遠的角度來看，足夠的生物資訊提供了未來發現更多具耐鹽特性酵素重要的訊息，且也具備了開發遺傳工程工具的潛力。此外，這八個重要基因也可作為未來篩選耐鹽菌的分子標誌。

關鍵字

耐鹽菌 ； 次世代定序 ； 高通量體學 ； 全基因體 ； 轉錄體 ； Tn5突變 ； 耐鹽機制

並列摘要

According to the characterization of high saline adaptation, halotolerant bacteria become a coming star for industrial biotechnology in recent years. Because property of tolerance in extreme environment, halotolerant bacteria become a highly regarded biology in academia and industry. Through the comprehension of halotolerance mechanism, halotolerant bacteria might help the development of the biotechnology, medical industry, and food processing industry. In fruit and vegetable processing, the halotolerance ability of microorganism might lead new product creating, processing acceleration and contamination reduction. However, there is lack of sufficient genomic information for halotolerance study in bacteria. Here, we describe the possible molecular mechanisms of saline adaptation based on high-throughput omics in two halotolerant bacteria, Halomonas beimenensis and Virgibacillus chiguensis which could grow in the environment with 20% NaCl. The complete genome of H. beimenensis genome is 4.05 Mbp with 3,807 genes, while the draft genome of V. chiguensis genome is approximately 4.15 Mbp in length with 4,347 genes. In general, many genes involved in the function of “cell motility” and “inorganic ion transport and metabolism” are up-regulated; and many genes involved in the function of “energy production and conversion”, “amino acid transport and metabolism”, “lipid transport and metabolism”, and “transcription” are down-regulated in H. beimenensis and V. chiguensis under high-salinity environment. Moreover, sixteen genes are identified as halotolerance related genes with the loss of halotolerance ability in H. beimenensis Tn5 mutants. Orthologs of genes such as nqrA, trkA, atpC, nadA, and gdhB have significant biological functions in the well know halotolerance control of sodium efflux, potassium uptake, hydrogen ion transport for energy conversion, and compatible solute synthesis. Other genes, such as spoT, prkA, mtnN, rsbV, lon, smpB, rfbC, rfbP, tatB, acrR, and lacA, also shown critical functions for promoting a halotolerance in cellular signaling, quorum sensing, transcription/translation, and cell motility. Eight out of these sixteen orthologous halotolerance related genes, such as trkA2, smpB, nadA, mtnN2, rfbP, spoT, lon, and atpC, were conserved in V. chiguensis and shown the similar expression pattern with H. beimenensis under high saline condition. This result pointed out that these 8 genes might have conserved functions in different phyla. In addition, the additional KCl could increase halotolerance and potassium-dependent cell motility of H. beimenensis under high saline environment. Our results demonstrated that through the combination of genomic and transcriptomic profiles or the combination of omics data could be facilitated the high saline adaptation mechanism exploitation. Furthermore, the sufficient bioinformation also provide an important information for potential halotolerance enzyme mining and plays a forward-looking role in genetic tools development. Finally, the 8 conserved genes can be used as molecular markers for halotolerant bacteria identification.

並列關鍵字

lotolerant bacteria ； next generation sequence ； high-throughput omics ； whole genome ； transcriptome ； Tn5 mutagenesis ； halotolerance mechanism

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