Biological activity of nitrosubstituted compounds in complex mixtures

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Nitroaromatic, nitroheterocyclic and nitropolyaromatic compounds are examples of nitrosubstituted compounds (NSCs) that are important genotoxic components of complex matrices such as contaminated soil.  Our work over the next five years will examine the genomics of the microbial communities in contaminated soils that degrade NSCs, and will characterize the enzymes responsible for biotransformation of NSCs.1) CHARACTERIZATION OF NITROREDUCTASES.  Nitroreductses (NRs) are a group of enzymes that initiate the degradation of NSCs in bacteria.  They are important not only because of their role in biodegradation but also because the intermediates of metabolism are capable of interacting with biological macromolecules, and are responsible for the observed genotoxic and antimicrobial activity of NSCs.  Our studies will characterize NRs from a range of bacteria in order to  more completely understand a) the structural properties of NRs that are important for substrate specificity; and b) the relationships between NRs from different phylogenetic groups.2) GENOMICS OF CONTAMINATED SOILS.  Our objective in this part of the project will be to identify soil bacteria that contribute to NSC transformation, and also to identify novel pathways that may play a role in NSC transformation.  Since only a small proportion of the microbial population (~1%) is culturable by standard methods, we will focus on the soil metagenome that represents the DNA content of all microbes in the soil.  Microbial diversity of contaminated soils will be determined by molecular methods and this information will help us design PCR primers for the isolation of genes encoding NRs.  In addition we will attempt to identify novel genes associated with NSC degradation using a substrate induced gene expression screen (SIGEX), which is designed to isolate elements that are unregulated by specific NSC compounds or mixtures.