Construction of a cassette enabling regulated gene expression in the presence of aromatic hydrocarbons.

A high-level expression cassette has been constructed from a TOL plasmid derived from Pseudomonas putida carrying all cis- and trans-acting regulatory elements necessary for transcriptional gene activation in the presence of aromatic hydrocarbons such as toluene. Foreign DNA can be inserted at unique KpnI, SacI, and EcoRI sites 7, 13, and 15 nucleotides downstream of a ribosome binding site. The cassette, flanked by BamHI and EcoRI restriction sites, was inserted into a broad-host-range vector and its efficacy demonstrated in various purple bacteria by monitoring the expression of a reporter gene spectrophotometrically and by SDS-PAGE. High-level induction (80- to 600-fold) was detected in Enterobacteriaceae and in Pseudomonas but was absent or low in Agrobacterium tumefaciens and Rhizobium leguminosarum.