Overproduction of the xylS gene product and activation of the xylDLEGF operon on the TOL plasmid

Abstract

The effect of high-level expression of the regulatory gene xylS of the Pseudomonas putida TOL plasmid on the activation of the xylDLEGF operon was investigated in Escherichia coli. The xylS gene was placed downstream from the tac promoter, and the resultant fusion was cloned in cis to the xylDLEGF operon. The expression of the operon was monitored by the level of catechol 2,3-dioxygenase, whose structural gene xylE was placed directly after the operator-promoter region of xylDLEGF. xylS transcription was also determined by reverse transcriptase mapping of mRNA. Overproduction of the xylS gene product elicited constitutive high expression of the xylDLEGF operon even in the absence of the inducer for the operon. The results were consistent with a cascade model for the positive control of the xylDLEGF operon by the xylR and xylS genes (S. Inouye, A. Nakazawa, and T. Nakazawa, Proc. Natl. Acad. Sci. USA, in press): m-xylene, a substrate of the degradative pathway, binds to the xylR gene product; the m-xylene-xylR product complex activates the xylS gene; and the xylS product thus synthesized de novo activates the xylDLEGF operon. Images