The primary goal of the proposed project will be to elucidate at the molecular level the control mechanism of the expression of the nah and sal operons of the NAH plasmid in Pseudomonas putida. This will be accomplished by: (1) Using molecular hybridization of salicylate-induced and uninduced RNA from P. putida cells containing various mutant NAH plasmids to evaluate transcriptional control. (2) Purification and characterization of the nahR gene product which mediates induction of the nah and sal operons. (3) Cloning and sequence analysis of the regulatory regions from wild-type and constitutive mutants of the nah and sal operons. (4) Physical analysis of the interaction of the nahR protein with the regulatory sequences of the nah and sal operons by binding studies, in vitro transcription experiments and analysis of DNA-protein crosslinking. In addition, the molecular relationship of the NAH plasmid genes to very similar genes in other hydrocarbon oxidizing bacteria will be analyzed by Southern hybridization analysis. These studies should result in a better understanding of the evolution of gene control mechanisms and metabolic pathways responsible for biodegradation of toxic, carcinogenic pollutants. The studies should also provide basic information about molecular mechanisms of positive control of gene expression in the important genus Pseudomonas.
