砷還原菌Enterobacter sp. AR-8之特性分析

Characterization of a novel arsenate-reducing bacterium Enterobacter sp. AR-8

蘇煜程(Yu-Chen Su)

指導教授 ： 廖秀娟

國立臺灣大學/生物資源暨農學院/生物環境系統工程學研究所/碩士(2009年)

https://doi.org/10.6342/NTU.2009.10643

摘要

砷是廣泛存在自然界中的物質。全世界有超過數百萬人長期處於環境中砷的影響而引發的毒性危害中。在過去十年間，能夠氧化或還原無機砷的微生物被從不同環境中分離出來並進行深入的研究。台灣地下水中的砷污染在1960年代就被發現但是直到目前為止卻沒有台灣地區砷污染與微生物相關的研究。在本篇研究中，一株能夠進行五價砷還原的細菌AR-8被從砷污染的地下水中分離出來並進行生理與分子生物的分析。利用聚合酶連鎖反應得到AR-8的16S rDNA序列後在資料庫中進行比對顯示AR-8是屬於Entertobacter 屬的細菌。在MSM液態培養基中進行的測試顯示AR-8能夠利用pyruvate，citrate、glucose或是sucrose作為生長所需的碳源。無論在好氧或厭氧的培養狀況下，AR-8都能夠在兩天以內將250 micromolar 的五價砷完全還原。利用聚合酶連鎖反應，成功得到位於細胞質中，在進行五價砷還原作用時需要Grx/GSH的五價砷還原酶基因arsC。除此之外，另一段與DMSO family reductase酵素有關的基因dmsA也利用聚合酶連鎖反應而得到。為了了解在AR-8中與五價砷還原有關的基因，本研究利用跳躍子進行隨機突變，得到許多喪失五價砷還原能力的突變株並進行下一步的分析。利用類似聚合酶連鎖反應的方法，受影響的基因可以被確認並定序。這些基因包含arsD、phoE、sbcC以及yqgE等。本研究在實驗室中利用現地取得的土樣及依照地下水成分設計的液態培養基模擬微生物原始生存環境，並加入具三價砷氧化能力的微生物L7506及具五價砷還原能力的微生物AR-8，來測試微生物在砷的循環扮演的角色及氧氣的影響。結果顯示當AR-8及L7506同時存在時能夠使無機砷循環加快，同時也顯示氧氣的有無是重要的因素之一。

關鍵字

五價砷還原 ； 地下水 ； 現地模擬砷循環試驗 ； 跳躍子隨機突變

並列摘要

Arsenic poisoning affects millions of people worldwide. Microbes able to oxidize or reduce arsenic have been isolated from various environments and extensively studied in past 10 years. Although Taiwan is noted for arsenic contamination in the groundwater, no reports was published about the relationship between microbes and arsenic yet. In this study we isolated an indigenous arsenate-reducing bacterium AR-8 from arsenic-contaminated groundwater. BLAST search of the 16S rDNA showed that AR-8 belongs to the genus of Enterobacter. AR-8 grows in basal medium amended with pyruvate, citrate, glucose, or sucrose. Under both aerobic and anaerobic condition, bacterial strain AR-8 can reduce 250 micromolar arsenate within 2 days. The PCR method successfully amplified the gene encodes the cytoplasmic arsenate reductase arsC that is a member of the Grx/GSH clade arsenate reductase. The gene dmsA, encodes the anaerobic DMSO reductase, was also cloned. In order to identify genes involved in arsenate reduction in AR-8, random mutagenesis was used to generate loss of arsenate reduction mutants. Interrupted genes that caused loss of arsenate reduction were identified by inverse PCR and sequenced. These genes include arsD, phoE, sbcC, and yqgE. A microcosm experiment was performed to mimic arsenic cycle in the environment. AR-8 was incubated with an arsenite oxidizer L7506 and sediments collected from arsenic-contaminated site to examine the response of microbes to arsenic and oxygen. The result of arsenic cycling experiment demonstrates that AR-8 and L7506 together can stimulate the cycling of arsenic and oxygen is an important factor.

並列關鍵字

arsenate reduction, ； groundwater ； microcosm arsenic cycle ； transposon mutagenesis

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