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標題: 以枯草桿菌表現革蘭氏陽性菌汞離子結合蛋白之研究
Fundamental study of a Gram-positive bacterial mercury-binding protein (MerP) expressed in Bacillus subtilis
作者: 許淑瓊
Hsu, Shu-Chiung
關鍵字: Bacillus subtilis;枯草桿菌;MerP;MerR;remediation;汞離子結合蛋白;汞離子調控蛋白;生物復育.
出版社: 生命科學院碩士在職專班
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Metal sequestering biochemicals. P. 200-248. In B. Volesky(ed.), Biosorption of heavy metals. CRC Press, Boca Raton, Fla. Misra, T. K. 1992. Bacterial resistance to inorganic mercury salts and organomercurials. Plasmid. 27:4-16. Morby, A. P., J. L. Hobman, and N. L. Brown. 1995. The role of cysteine residues in the transport of mercuric ions by the Tn501 MerT and MerP mercury-resistance proteins. Mol. Microbiol. 17:25-35. Morel, F. M. M., A. M. L. Kraepiel., and M. Amyot. 1998. The chemical cycle and bioaccumulation of mercury. Annu. Rev. Ecol. Syst. 29: 543-566. Pilon S. 2000. Breeding mercury-breathing plants for environmental cleanup. Trends in plant Science. 5:235-236. Qian, H., L. Sahlman, P. Eriksson, C. Hambraeus, U. Edlund and I. Sethson. 1998. NMR Solution structure of the oxidized form of MerP, a mercury ion binding protein involved in bacterial mercuric ion resistance. Biochemistry. 37:9316-22. Rensing, C., B. Fan, R. Sharma, B. Mitra, and B. P. Rosen. 2000. CopA: An Escherichia coli Cu(I)-translocating P-type ATPase. Proc. Natl. Acad. Sci. U S A. 97:652-6 Robinson, J. B. and O. H. Tuovinen. 1984. Mechanisms of microbial resistance and detoxification of mercury and organomercury compounds: Physiological, Bio-chemical, and genetic analysis. Microbial. Rev. 48:95-124. Robinson, N. J., A. M. Tommey, C. Kuske, and P. J. Jackson. 1993. Plant metallothioneins. J. Biochem. 295:1-10. Ronald E. Yasbin, Vernon C. Maino, and frank E. Young. 1975. Bacteriophage Resistance in Bacillus subtilis 168, W23, and Interstrain Tranfomants. Journal of Bacteriology. Mar. 1120-1126. Sahlman, L., and B.-H. Jonsson. 1992. Purification and properties of the mercuric-ion-binding protein MerP. Eur. J. Biochem. 205:375-381. Sahlman, L., and E. G. Skärfstad. 1993. Mercuric ion binding abilities of MerP variants containing only one cysteine.Biochem. Biophys. Res. Commun. 196:583-588. Sahlman, L., Wong, W., and Powlowski, J. 1997. 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本研究以汞為模式重金屬汙染物,利用分生技術將已知對汞金屬具有抗性的Bacillus megaterium MB1之汞抗性基因組(mer operon)中汞離子結合蛋白merP與調控蛋白merR1基因,選殖在pHY300PLK質體上,所構築的重組質體包含帶有完整merR1與merP之pHYR1P,以及帶有刪除的merR1與一份(copy)完整merP之pHY△R1P,以及帶有刪除的merR1與兩份完整merP之pHY△R1PP。再將此三種重組質體與質體pHY300PLK分別轉形入革蘭氏陽性菌Bacillus subtilis 168(BS168)中,並將這些轉型株命名為BS168/R1P、BS168/△R1P、BS168/△R1PP以及BS168/pHY。接著測試不同重組菌株及無質體的原菌株BS168在不同濃度汞離子溶液中之耐受能力,在固態培養之汞抗性測試結果,重組菌株BS168/R1P、BS168/△R1P、BS168/△R1PP,皆可在含5 ppm汞的LB固態培養基下正常生長,其中BS168/R1P的抗性最佳,在含6 ppm汞的LB固態培養基下亦可正常生長;而對照組BS168/pHY和BS168在含3 ppm汞的LB固態培養基即生長受抑制。在液態培養之汞抗性測試結果可分兩方面,菌株直接在含3 ppm汞的LB培養下,全部生長皆受抑制;另外如果先利用低濃度0.2 ppm汞誘導後再置於含3 ppm汞的LB培養,重組菌株BS168/pHYR1P、BS168/△R1P、BS168/△R1PP皆可正常生長,而對照組BS168/pHY和BS168則生長受抑制;在1O.D菌液及汞離子濃度10μM的條件下,溶液中的汞離子幾乎可完全吸附,而無論在10μM或20μM下,BS168/R1P對汞離子吸附效果均較佳。本實驗的研究結果顯示,將含有merR1與merP基因的重組質體轉形進入目標菌株可提供其對汞的抗性及累積能力。我們預期此方法將可應用於不同的革蘭氏陽性或陰性菌株當中,並增加微生物在生物復育方面的應用價值。

In this study, mercury (Hg) was selected as a model heavy metal pollutant. At first, two full length gene fragments of merP and merR1, and the truncated merR1 fragment from known Hg-resistant Bacillus megaterium MB1 mer operon were amplified by PCR. Three recombinant plasmids which include pHYR1P with one copy of complete merR1 and merP gene, pHY△R1P with one truncated merR1 and one copy of complete merP gene, and pHY△R1PP with one truncated merR1 and two copies of merP gene, were constructed using pHY300PLK as vector. The recombinant plasmids were then transformed into Bacillus subtilis 168 (BS168) to conduct the Hg resistance tests under different conditions of Hg2+ concentrations. The results show that all the transformants carrying pHYR1P, pHY△R1P, pHY△R1PP can survive on LB agar plates containing 5 ppm of Hg2+, especially for the transformants carrying pHYR1P which can tolerate up to 6 ppm of Hg2+, conversely, the growth of control strians, BS168 and BS168 with pHY300PLK, were inhibited under 3ppm of Hg2+. In liquid medium with 3 ppm of Hg2+, the growth of all experimental strains were inhibited, however, under the induction with 0.2 ppm of Hg2+, all the transformants can grow normally except the control strains. The results from the adsorption test have shown that the Hg2+ in 10 uM almost can be absorbed completely by all transformants, particularly the transformants with pHYR1P, which have higher absorption ability, no matter under 10 uM or 20 uM of Hg2+.
These results demonstrate that the gene products of merP and merR1 provide the host cells the resistance to Hg. We anticipate that these result can apply to a variety of gram negative or positive bacteria, and enhance the value of application in environmental remediation.
其他識別: U0005-2901200713560200
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