The combined genomes of nature's thousands of thus far uncultivated species of microorganisms provide a vast resource of uncharacterized functional genetic information that will be examined toward the goal of discovery of novel genes involved in contaminant transformation. The approach to be employed involves the cloning of large fragments of soil community DNA in an E. coli Bacterial Artificial Chromosome (BAC) expression vector, followed by screening of BAC clone libraries for expression of novel genes involved in the biotransformation of environmental contaminants associated with Superfund sites. Libraries will be screened for single genes or gene clusters involved in the biodegradation of polycyclic aromatic hydrocarbons, nitrated munitions compounds, chlorinated solvents, and synthetic azo dyes. Libraries also will be screened for genes involved in sequestration of heavy metals. Our primary objectives are to (a) produce five BAC expression libraries from community DNAs of different pollutant-contaminated soils, (b) screen the libraries for expression of genes involved in degradation or sequestration of environmental contaminants, (c) sequence inserts from positive clones to identify putative novel genes or gene clusters involved in contaminant transformation or sequestration, (d) examine the biotransformation chemistry mediated by select clones containing unique DNA inserts, and (e) make the BAC libraries available at minimal cost to other investigators interested in screening for novel gene products of value to American biomedical science.Each library will be comprised of a total of approximately 10s clones and contain >10/10 bp of community DNA. This effort will provide tools for obtaining novel functional genes from the vast store of unexplored genetic information within natural microbial communities of contaminated soils. It should lead to discovery of multiple new gene products with promising environmental restoration application potential that will directly contribute to NIH's goal of protecting human health from adverse effects of industrial contaminants. This application is consistent with the NIH R21 mechanism of support in that the use of soil metagenomic expression libraries to discover novel biodegradation genes represents a new approach that is largely untested and has never before been applied to contaminant biotransformation research in a systematic manner as detailed here.
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