Molecular cloning of regulatory gene xylR and operator-promoter regions of the xylABC and xylDEGF operons of the TOL plasmid

Abstract

The regulatory gene xylR of the [Pseudomonas putida] TOL plasmid, which functions positively on xylABC and xylDEGF operons in the presence of m-xylene or m-methylbenzyl alcohol, was cloned onto an Escherichia coli vector, pACYC177. A fused operon consisting of the operator-promoter region of the xylABC operon and the xylE gene was cloned onto pBR322. The xylE product, catechol 2,3-dioxygenase, was induced by m-xylene or m-methylbenzyl alcohol in the cells containing the fused operon when a 2.8-kilobase segment of the TOL plasmid was provided in trans. The segment appeared to contain the regulatory gene xylR. The xylR gene was mapped very close to the other regulatory gene, xylS, determined previously. The xylR gene was not effective on activation of the xylDEGF operon unless an additional region containing xylS was provided together with the inducer. xylR and xylS are essential to the m-methylbenzyl alcohol-dependent induction of the xylDEGF operon. The map positions of xylR and xylS were precisely determined by subcloning or insertion inactivation. The operator-promoter regions of the xylABC and xylDEGF operons were mapped to the 0.6- and 0.4-kilobase regions of the TOL plasmid, respectively.