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. ***
