Anaerobic Toluene Metabolism in the T1 Denitrifying Strain

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; R o o t E n t r y F VxZ6 @ C o m p O b j b W o r d D o c u m e n t F O b j e c t P o o l 6 uZ6 6 uZ6 4 @ 6 7 8 9 : ; = ? @ F Microsoft Word 6.0 Document MSWordDoc Word.Document.6 ; { { { { Frazer, Anne C. MCB-9507132 The goal of this project is to characterize the biochemical features of the anaerobic degradation of toluene by strain T1. This is a novel isolate obtained in this laboratory and preliminary work has already been done on the anaerobic pathway for toluene utilization in the microbe. Toluene has two fates in T1, either productive metabolism and eventual conversion to carbon dioxide and biomass, or transformation to two dead-end metabolites, benzylsuccinate and benzylfumarate. The formation of the same dead-end products during anaerobic toluene metabolism has also recently been reported in certain sulfate-reducing environments, and in several other denitrifying toluene degraders. Thus, ths transformation may be environmentally significant. It is proposed to develop enzyme assays using anaerobic cell extracts with an emphasis on identifying the reactants and products of key enzyme steps in the toluene metabolism by T1. Emphasis includes, determining the initial enzyme step in the toluene utilization pathway, designated the TUT pathway, as well as defining the initi al step in the formation of the toluene dead-end metabolites. The research proposed is complementary to work currently underway by colleagues in the laboratory working on the genetic features of anaerobic toluene degradation by strain T1, and will make use of mutants unable to use toluene in order to resolve toluene degradation pathways. %%% Toluene is a major industrial chemical as well as a component of fuel. It is also a hazardous waste in soil and aquatic environments. A good deal has been learned about the degradation of toluene in air. However, oxygen in pollution sites is rapidly used up, which makes the study of anaerobic metabolism so important. The goal of this project is to characterize the beginning reactions performed by T1 anaerobically metabolize toluene. Toluene has two fates in T1, either mineralization or transformation into two dead-end chemical products that accumulate in the growth medium. Studies will first be undertaken to identify initial reactions of toluene metabolism in extracts from cells, with the intention of continuing on to identify key enzymatic reactants and products in the separate degradation pathways. This project will help provide a better understanding about the natural degradative processes in the environment and to use them effectively to remediate contaminated sites. *** ; Oh +' 0 $ H l D h S u m m a r y I n f o r m a t i o n ( 5 R:\WWUSER\TEMPLATE\NORMAL.DOT Frazer, Anne C. Robert Uffen Shelley A. Graves @ Sv 4 @ @ tfZ6 @ Ik Microsoft Word 6.0 2 ; ; e = e F j j j j j j j 1 # % % % C c T @ j j j j j # ~ j j j j # { Frazer, Anne C. MCB-9507132 The goal of this project is to ch