It is estimated that up to 20 percent of the 1.4 billion lbs. of Polychlorinated biphenyls (PCBs) produced from 1929 to 1978 have entered the environment. These pollutants pose a serious threat to human health. It appears that the most reasonable and cost efficient method of removing these compounds is via biological means. The ubiquitous nature and vast biochemical capabilities of bacteria make them the organisms of choice for bioremediation of PCBs. A group of bacteria composed of many different species contains a nearly identical suite of genes (called the bph cluster) encoding enzymes for PCB degradation. The organization and structure of the bph cluster is understood at a rudimentary level, but it is essential that the regulation of these genes be investigated. It is reasonable to assume that an understanding of bph cluster gene regulation will significantly enhance the usefulness of these organisms for solving the environmental PCB problem. The first specific aim of this proposal will be to characterize and clone the entire bph cluster. Although portion of the bph cluster from a Burkholderia sp. called LB400 has been cloned, it is not complete. We will complete the cloning of the bph_cluster and identify all the genes present. The second specific aim is to identify promoters for all of these genes. Previous work has shown that multiple promoters are present and some may be up-regulated when biphenyl is present. However, this study only investigated promoters from one region of the bph cluster. We will propose to complete this promoter study for all of the genes present. The final specific aim is to identify regulatory mechanisms that control the expression of bph cluster genes. This information is absolutely essential for a complete understanding of how PCBs are removed from the environment, and may be useful as a means of enhancing the biodegradative rates of bacteria. The persistence of PCBs in the ecosystem as well as the known health risks to humans clearly justify the proposed research project.
