Controlled-expression shuttle vector for pseudomonads based on the trpIBA genes of Pseudomonas putida

A cloning vector pPS7 (8.5 kb) for Pseudomonas was constructed from pBR322 and the Pseudomonas cryptic low-copy-number pMK1 plasmid. The vector confers resistance to kanamycin (Km) and tetracycline (Tc), contains the par locus of Pseudomonas plasmid pMT2 and a mob site. The new vector was used for construction of controlled-expression vector pPS10 (10.4 kb) based on the trpIBA genes of Pseudomonas putida. This KmR vector contains the trpI gene, encoding activator protein and promoter of trpBA genes (Pba), which are inducible by TrpI and indoleglycerol phosphate (InGP). InGP is an unstable compound, but it accumulates in trpE mutants grown in anthranilate (Anth)-supplemented medium. We show that expression of the Escherichia coli pheA gene, inserted into the pPS10 vector downstream from Pba, increases about 70-fold upon InGP accumulation.