Analysis of an upstream regulatory sequence required for activation of the regulatory gene xylS in xylene metabolism directed by the TOL plasmid of Pseudomonas putida

Abstract

Transcription from the promoter of a positive regulatory gene, xylS, on the TOL plasmid of Pseudomonas putida is activated by another positive regulator, XylR, in the presence of m-xylene and is dependent on RNA polymerase containing the NtrA protein (sigma 54). Deletion analysis of the upstream region of the xylS gene revealed an upstream regulatory sequence (URS), located between 145 and 188 bp upstream from the transcription start site. The URS is active in either orientation and can be placed 3.9 kb further upstream without loss of activity. Dependence of activation on helical periodicity was observed in the region between the URS and the promoter of the xylS gene, suggesting DNA loop formation between these two sites, which are located about 100 bp apart. The expression of xylR was autogenously repressed by XylR protein. This autogenous repression is decreased in an NtrA- background, irrespective of the presence of the xylS promoter in cis, indicating that NtrA protein, or NtrA-containing RNA polymerase that is not bound to the xylS promoter, is involved in the binding of XylR protein to the URS.