Identification of the nahR gene product and nucleotide sequences required for its activation of the sal operon

Abstract

The product of the nahR gene, a salicylate-dependent activator of transcription of the nah and sal hydrocarbon degradation operons of the NAH7 plasmid, was identified and characterized after synthesis in Escherichia coli maxicells. The nahR gene product had a subunit molecular weight of 36,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, whereas gel filtration analysis of the nondenatured nahR protein indicated a molecular weight in excess of 250,000. However, DNase I treatment of this high-molecular-weight complex shifted the apparent molecular weight of the nahR protein to 40,000. Various upstream portions of the sal operon promoter were transcriptionally fused to the E. coli galactokinase gene. Fusion plasmids containing the sal promoter sequence from --83 to 27 (relative to the transcription start site) showed salicylate-inducible expression of galactokinase in the presence of the cloned nahR gene, while expression of galactokinase from a fusion plasmid containing the sal promoter sequence from --45 to 27 was not induced by the nahR gene and salicylate. Results suggest that the nahR gene product is a 36-kilodalton polypeptide which exerts its salicylate-dependent activation of transcription of the sal operon by interacting with the promoter sequence in the region of --83 to --45 base pairs before the transcription start site.