具有排出系統（czc操縱子）耐鎘機制之Cupriavidus metallidurans (Ralstonia eutrophus)和C. taiwanensis (R. taiwanensis)

Efflux Pump (czc Operon)-Mediated Cadmium-Resistance Mechanism of Cupriavidus metallidurans (Ralstonia eutrophu) and C. taiwanensis (R. taiwanensis)

陳心佩(Xin-Pei Chen) ； 朱紀實(Chi-Shih Chu) ； 蔡竹固(Jwu-Guh Tsay)

《環境保護》 28卷2期 (2005/12) Pp. 185-201

https://doi.org/10.30017/EP.200512.0006

摘要

本試驗目的在於分離具有鎘抗性的細菌，並探討其抗性的可能機制。由雲林色料工廠污染的土壤中分離出20個菌株，其中有15株對鎘的耐受濃度在650~900 mg/kg之間，其中菌株rcd 12、14及21可忍受鎘達900 mg/kg。利用Biolog微生物鑑定系統進行菌株rcd 1, 6, 7, 17, 18和20菌種鑑定，其生理生化特性皆類似於Ralstonia eutrophus（現已更名為Cupriavidus metallidurans）。經由16S rDNA序列比對結果亦屬Ralstonia spp.，並與NCBI(National Center for Biotechnology Information)資料庫已登錄的Ralstonia spp.進行序列比對與種系發生樹分析，菌株編號rcd 12、14及21之16S rDNA序列與Ralstonia taiwanensis（現已更名為Cupriavidus taiwanensis）之類緣關係較為接近，區分在同一分支；菌株rcd 2、3、4、8及9的類緣關係與Cupriavidus metallidurans (=Ralstonia eutrophus)較為接近。而其餘rcd 5、11、13、15及16等5個菌株對鎘的耐受能力小於400 mg/kg，利用16S rDNA序列與NCBI資料庫序列比對顯示屬於Streptomyces spp.，具有97-99%一致性。利用排出系統(Efflux pump system)的鎘抗性基因czc操縱子設計的專一性引子進行聚合酶連鎖反應，所有Cupriavidus屬的菌株都可增幅出czcA及czcC預期的產物片段，但只有C. taiwanensis的rcd 12、14及21菌株可以增幅出預期czcB及czcD的產物片段。分析czc操縱子存在於質體上的情形，利用Kado和Lui鹼性方法抽取質體，發現在Cupriavidus屬的15個菌株中，只有rcd 2、3、4、7、17、18、19及20共8個菌株含有一個大於150 kb的質體。利用czcA作為探針進行南方墨點雜合法後，發現czc操縱子可同時存在於質體及染色體上。

關鍵字

鎘 ； 聚合酶鏈鎖反應 ； 耐鎘基因 ； Ralstonia spp. ； Cupriavidus spp. ； 排出系統

並列摘要

The purpose of this study was to discover the cadmium-resistant microorganisms and then to determine their resistance mechanism. 20 cadmium-resistant organisms were isolated from cadmium-polluted and tainted soil by a waterink factory in Yunlin, Taiwan. According to cadmium tolerance these organism were then separated into two groups: lower than 400 mg/kg and ranges from 650 to 900 mg/kg, respectively. Some organisms were tested by BIOLOG bacterial identification system. Isolates rcd 1, 6, 7, 17, 18 and 20 appeared to be Ralstonia eutrophus. These organisms were further confirmed by nucleotide sequence of 16S rDNA. The results suggested that strains rcd 12, 14, and 21 were Cupriavidus taiwanensis (=Ralstonia taiwanensis)-like organisms; strains rcd 2, 3, 4, 8, and 9 were similar to Cupriavidus metallidurans (=Ralstonia eutrophus); the remaining strains rcd 5, 11, 13, 15, and 16 were related to Streptomyces spp. To determine the genes responsible cadmium resistance, one of efflux pump, czc operon was used for as PCR target, all Cupriavidus strains consisted of this operon with some variations. Whether czc operon was located on chromosme or plasmid, plasmid profile was first constructed by alkaline method and showed that 8 of Cupriavidus strains, rcd 2, 3, 4, 7, 17, 18, 19, and 20, harbored a large plasmid with the size over 150 kb. When czcA used as probe in a Southern blot analysis, the results revealed that this operon was located on plasmid and chromosome as well. Here we first reported that cadmium-resistant Cupriavidus metallidurans and C. taiwanensis strains use czc operon, which is located on plasmid or chromosome, release cadmium off the organism.

並列關鍵字

cadmium ； polymerase chain reaction PCR ； cadmium-resistant genes ； Ralstonia spp. ； Cupriavidus spp. ； efflux system