Molecular Insight into Polyaromatic Toxicant Degradation

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The microbial world is diverse owing to its 3.7 billion years of evolution, which provides for both the opportunity of undiscovered metabolic capacity, including that for pollutant degradation, and the challenge of detecting and recovering this activity. It is well known that more than 99% of the microbial world has not been cultured and hence remains undiscovered. We propose to explore and recover genes for two key biodegradative steps in the detoxification of chlorinated polyaromatic compounds from the DNA of this uncultured microbial diversity, and then to use the molecular markers from this study to aid in site assessment and quantitative predictions of biodegradation at contaminated sites. We are targeting the reductive dehalogenases and the aromatic oxygenases as the key functions to recover since they are most often the rate limiting steps in the degradation of the polychlorinated dioxins and dibenzofurans, PCBs and polynuclear aromatic hydrocarbons (PAHs), the Superfund chemicals of focus in our study. Our specific aims are to: (1) explore and recover nature's catalytic diversity with the goal of developing a comprehensive profile of microbial metabolic capabilities for these polyaromatic compounds, (2) use the genome sequence information of Burkholderia xenovorans LB400, the most effective PCB degrader, to study the metabolic features important to the degradation of these chemicals and (3), develop quantitative diagnostic tools based on Bayesian probabilistic networks to predict biodegradation at contaminated sites. We propose to use enrichments to help identify the functionally active populations and hence genes, to use stable isotope probing to recover DNA from the active degrading populations, to use metagenomic libraries to recover the full genes and operons, and to use high throughput PCR screening for identifying clones with the targeted gene families. This project combines the expertise of the Rutgers University scientists in aromatic oxygenases and high throughput screening and metagenomics with the expertise at Michigan State University in reductive dechlorination and genomics and microarray technology.