Molecular cloning of gene xylS of the TOL plasmid: evidence for positive regulation of the xylDEGF operon by xylS

Abstract

The xylDEGF operon and the regulatory gene xylS of the TOL plasmid found in Pseudomonas putida mt-2 were cloned onto Escherichia coli vector plasmids. A 9.5-kilobase fragment, derived from the TOL segment of pTN2 deoxyribonucleic acid, carried the xyl genes D, E, G, and F, which encode toluate oxygenase, catechol 2,3-oxygenase, 2-hydroxymuconic semialdehyde dehydrogenase, and 2-hydroxymuconic semialdehyde hydrolase, respectively. The enzymes were noninducible unless a 3-kilobase PstI fragment, derived also from the TOL segment, was provided in either cis or trans. The PstI fragment appeared to contain the regulatory gene xylS, which produced a positive regulator. The regulator was activated by m-toluate or benzoate, but not by m-xylene or m-methylbenzyl alcohol. the map positions of xylG and xylF were also determined.