Copper Bioavailability on Microbial Degradation of Trichloroethylene

9708557本提案是应环境地球化学和生物地球化学征集NSF96-152号文件的要求提交的，由分子和细胞生物科学处、化学和地球科学处以及海洋和大气管理局多学科活动办公室共同资助。这项研究将确定土壤表面如何限制甲烷氧化菌对铜的生物有效性，以及由此对两种形式的甲烷单加氧酶(MMO)的表达和活性的影响。铜/生物量比是调节甲烷氧化菌液体培养中可溶性和颗粒性甲烷单加氧酶(sMMO和pMMO)表达和活性的关键因素。为了确定不同的土壤表面如何限制铜的生物有效性以及由此对MMO表达和活性的影响，两种代表性的甲烷氧化菌--天花粉分枝杆菌OB3b和白色分枝杆菌Bg8将在不同的溶液条件下，在氧化铝(代表常见的金属氧化物)和蒙脱石(代表重要的离子交换部位的矿物)的纯培养条件下生长。之所以选择这些菌种进行初步研究，是因为丝孢霉OB3b可以表达两种形式的MMO，而白色丝孢霉Bg8只能表达pMMO。实验测量包括：使用原子吸附光谱或铜离子电极对水中、吸附的和与细胞相关的铜的形态和数量；通过X射线吸收光谱进行铜的吸附；通过萘氧化试验和Western blotts检测sMMO和pMMO的表达；通过环氧丙烷对可溶性和颗粒组分进行定位和测量MMO的活性；GC-MS分析以确定这些细胞氧化甲烷和三氯乙烯的能力。在随后的实验中，将在类似的土壤/溶液条件下培养这两种生物的人工混合培养物，以了解表面是如何通过充当铜的汇来引起种群波动的。实验方法包括：sMMO和pMMO的表达和活性；Western印迹、丫啶橙直接计数和16S rRNA、sMMO和pMMO基因的PCR扩增的种群组成；混合培养物对甲烷和三氯乙烯的氧化能力。这项多学科的研究结合了分子生物学、生物化学和地球化学的方法，是检验其他金属的生物有效性对微生物降解芳香族和脂肪族卤代烃的影响的模型系统。***

9708557 Semrau This proposal was submitted in response to the Environmental Geochemistry and Biogeochemistry solicitation NSF 96-152, and is being funded jointly by the Divisions of Molecular and Cellular Biosciences, Chemistry and Earth Sciences, and by the MPS Office of Multidisciplinary Activities. This research will determine how soil surfaces limit the bioavailability of copper to methanotrophs and the resulting effect on the expression and activity of both forms of methane monooxygenase (MMO). Copper to biomass ratio is a key factor in regulating expression and activity of soluble and particulate methane monooxygenases (sMMO and pMMO, respectively) in liquid cultures of methanotrophs. To determine how different soil surfaces can limit the bioavailability of copper and the resulting effect on MMO expression and activity, two representative methanotrophs, M. trichosporium OB3b and M. albus BG8 will be grown in pure cultures in the presence of aluminum oxide (a representative of commonly occurring metal oxides) and montmorillonlte (representative of an important ion-exchange site bearing mineral) under a variety of solution conditions. These organisms were chosen for initial study because M trichosporium OB3b can express both forms of MMO while M. albus BG8 can express only pMMO. Experimental measurements include: speciation and amount of aqueous, adsorbed and cell-associated copper using atomic adsorption spectroscopy or a copper ion electrode; copper sorption by X-ray absorption spectroscopy; sMMO and pMMO expression by naphthalene oxidation assays and Western blots; propylene oxide assays of soluble and particulate fractions to localize and measure MMO activity; GC-MS analysis to determine the ability of these cells to oxidize methane and trichloroethylene. In subsequent experiments, artificial mixed cultures of both organisms will be grown in similar soil/solution conditions to understand how surfaces can cause population fluctuations by acting as a sink for copper. Experimental mea surements include: expression and activity of sMMO and pMMO; population composition by Western blot, acridine orange direct count, and PCR amplification of the genes for 16s rRNA, sMMO and pMMO; and the ability of mixed cultures to oxidize methane and trichloroethylene. This multidisciplinary research incorporates approaches from molecular biology, biochemistry and geochemistry, and is a model system for examining the effect of bioavailability of other metals on microbial degradation of both aromatic and aliphatic halogenated hydrocarbons. ***