Methylococcus capsulatus (Bath)為嗜甲烷菌的一種，隨著環境中銅離子濃度的高低可以調控其分別表現不同型態的微粒型甲烷單加氧酶(pMMO)及可溶型甲烷單加氧酶(sMMO)，但是關於嗜甲烷菌如何偵測環境中銅離子濃度的高低並分別表現不同型態的甲烷單加氧酶，一直沒有一個定論，過去的幾年來，由於Methylococcus capsulatus (Bath)的全基因圖譜被分析完成並發表出來，有一些關於微粒型甲烷單加氧酶及可溶型甲烷單加氧酶基因前端以及中間的啟動區序列(或是疑似啟動區序列)被發現並報導出來，也有人對整個調控機制提出了假設。本論文主要針對整個調控機制進行研究，將微粒型甲烷單加氧酶(pMMO)及可溶型甲烷單加氧酶(sMMO) 前端以及中間的啟動區序列(或是疑似啟動區序列)以PCR的方式複製出來，再將可能的轉錄調控因子sigma-70以及sigma-54的基因片段以PCR的方式複製出來在嵌入E. coli中進行表現，再將表現出來的蛋白質與啟動區序列作用，得知轉錄調控因子sigma-70在有銅一價離子的環境下會和pMMO前端的啟動區序列結合，可能為控制pMMO的轉錄調控因子，而對於sMMO不論是前端的啟動區序列或是中間的啟動區序列皆沒有明顯的結合，應該和sMMO的表現與否沒有直接的關係，sMMO的表現與否可能是由其它的轉錄調控因子所控制。

Methylococcus capsulatus (Bath) is one of methanotrophic bacteria. The differential expression of either sMMO or pMMO is regulated by the concentration of copper ions within the cells, but how this bacterium under varisus copper concentration to express the appropriate MMO is still unkown. In 2004, the total gene map of Methylococcus capsulatus (Bath) was analysed and published. The promoter region at upstream or middlestream of pMMO and sMMO were also elucidated. A regulational system which expressed either sMMO or pMMO is relied on copper ion concentration is proposed, however, not well understood yet. In this study, we use polymerase chain reaction (PCR) to obtain the gene fragments corresponded to the promoter regions. We also over-expressed two transcriptional factors sigma-70 (rpoD) and sigma-54 (rpoN) which are devied from Methylococcus capsulatus (Bath) within the E. coli. (RILP) system. We presumably assume these sMMO or pMMO transcriptional factors could interact with the promoter regions, and they will bind with each other. In electrophoresis mobility shift assay (EMSA), once the DNA binded with protein, they can be separeted from the protein free DNA, therefore the mobility will be different. From this thesis, we observed sigma-70 (rpoD) has the ability to bind with the prmoter region in upstream of pMMO sequence in the presence of copper (I) ion. The protein sigma-70 (rpoD) should be most like a transcriptional factor of pMMO. From the subseqent expriments, we did not find any of the promoter regions at upstream or middlestream of sMMO binded with sigma-70. We cosidered sigma-70 might not be a transcriptional factor of sMMO. The expression of sMMO could be regulated by other promoter(s).

Elliott, S. J., Zhu, M., Tso, L., Nguyen, H. H.T., Yip, J. H. K. and Chan, S. I., Regio- and Stereoselectivity of Particulate Methane Monooxygenase from Methylococcus capsulatus (Bath). J. Am. Chem. Soc. 1997, 119, 9949-9955.

Green, J., Prior S. D. and Dalton, H., Copper ions as inhibitors of protein C of soluble methane monooxygenase of Methylococcus capsulatus (Bath). Fur. J. Biochem. 1985, 153, 137-144.

Hanson, R. S. and Hanson, T. E., Methanotrophic Bacteria. Microbiol. Rev. 1996, 60, (2), 439–471.

Lieberman, R. L. and Rosenzweig, A. C., Crystal structure of a membrane-bound metalloenzyme that catalyses the biological oxidation of methane. Nature 2005, 434, 177-182

Madigan, M. T., Martnko, J. M. and Parker, J., Prokaryote diversity,Bacteria. Biology of Microorganisms. 8th ed. 1997, 16, 666-671.

Murrell, J. C., Gilbert, B. and McDonald, I. R., Molecular biology and regulation of methane monooxygenase. Arch Microbiol 2000, 173, 325–332.

Murrell, J. C., McDonald, I. R. and Gilbert, B., Regulation of expression of methane monooxygenases by copper ions. Trends in Microbiology 2000, 8, (5), 221-225.

Nielsen, A. K., Gerdes, K. and Murrell, J. C., copper-dependent reciprocal transcriptional regulation of methane monooxygenase genes in methylococcus capsulatus and methylosinus trichosporium. Mol. Microbiol. 1997, 25, (2), 399-409.

Ricke, P., Erkel, C., Kube, M., Reinhardt, R. and Liesack, W., Comparative Analysis of the Conventional and Novel pmo (ParticulateMethane Monooxygenase) Operons from Methylocystis Strain SC2. Appl. Environ. Microbiol. 2004, 70, (5), 3055-3063.

Stolyar, S., Costello, A. M., Peeples, T. L. and Lidstrom, M. E., Role of multiple gene copies in particulate methane monooxygenase activity in the methane-oxidizing bacterium Methylococcus capsulatus Bath. Microbiology 1999, 145, 1235–1244.

Stolyar, S., Franke, M. and Lidstrom, M. E., Expression of Individual Copies of Methylococcus capsulatus (Bath) Particulate MethaneMonooxygenase Genes. J. Bacteriol. 2001, 183, (5), 1810–1812.

Whittembury, R., Phillps,K. C., and Wilkinson, J. F., Enrichment, Isolation and Some Properties of Methane-utilizing Bacteria. Journal of General Microbiology 1970, 61, 205-218.

Yu, S. S. F., Chen, K. H. C.,Tseng, M. Y. H., Wang,Y. S., Tseng, C. F., Chen, Y. J., Huang, D. S. and Chan, S. I., Production of High-Quality Particulate Methane Monooxygenase in High Yields from Methylococcus capsulatus (Bath) with a Hollow-Fiber Membrane Bioreactor. J. Bacteriol. 2003, 185, (20), 5915–5924.

Yu, S. S. F., Wu, L. Y., Chen, K. H. C. Luo, W. I., Huang, D. S. and Chan, S. I., The Stereospecific Hydroxylation of [2,2-2H2]Butane and Chiral Dideuteriobutanes by the Particulate Methane Monooxygenase from Methylococcus capsulatus (Bath). J. Biol. Chem. 2003, 278, (42), 40658–40669.

ZAHN, J. A. and Dispirito, A. A., Membrane-Associated Methane Monooxygenase from Methylococcus capsulatus (Bath). J. Bacteriol. 1996, 178, (4), 1018–1029.