The goal of the project is to further characterize the molecular basis of the regulation of the nah and sal operons of the NAH7 napthalene degradation plasmid from Pseudomonas putids. These two operons are coordinately controlled by a regulatory gene, nahR, which encodes an inducer-dependent activator of transcription. Specific experiments will involve analysis of the protein-DNA interactions between the nahR gene product and the promoters of the nah and sal operons. This will be accomplished by: (1) In vitro mutagenesis of sal promoter - galK fusion plasmids and subsequent analysis to determine critical nucleotides involved in transcription activation by the nahR protein and the inducer, salicylate. (2) In vitro mutagenesis and analysis of nahR promoter - galK fusion plasmids to determine promoter structure and possible regulation. (3) Purification of the nahR gene product from E. coli cells which overexpress the cloned nahR gene utilizing a radiochemical tracer method involving the radiochemically pure nahR protein as an assay. (4) Analysis and characterization of the biological properties of the nahR gene product and its molecular interactions with NAH7 DNA (e.g., specific binding to the nah and sal promoters, stimulation of transcription in vitro, salicylate binding, etc.). (5) Analysis of the DNA sequence of the nahR gene to determine homologies with known functional domains in other positive regulatory proteins. (6) Analysis of nah promoter - galK fusion plasmids to confirm that the nah and sal operons are controlled in an identical manner at the molecular level. The results of these experiments will provide a detailed understanding of the molecular interactions involved in this non-E. coli, positively regulated gene system. This information will be important to an understanding of bacterial disease processes and treatments and will demonstrate that many gram negative pathogenic bacteria are amenable to detailed molecular genetic analysis. In addition basic knowledge of unique genetic regulatory systems and mechanisms will help in understanding analogous genetic systems and situations involved in human disease states.
