Resistance to Tellurite as a Selection Marker for Genetic Manipulations of Pseudomonas Strains

Resistance to the toxic compound potassium tellurite (Tel(r)) has been employed as a selection marker built into a set of transposon vectors and broad-host-range plasmids tailored for genetic manipulations of Pseudomonas strains potentially destined for environmental release. In this study, the activated Tel(r) determinants encoded by the cryptic telAB genes of plasmid RK2 were produced, along with the associated kilA gene, as DNA cassettes compatible with cognate vectors. In one case, the Tel(r) determinants were assembled between the I and O ends of a suicide delivery vector for mini-Tn5 transposons. In another case, the kilA and telAB genes were combined with a minimal replicon derived from a variant of Pseudomonas plasmid pPS10, which is able to replicate in a variety of gram-negative hosts and is endowed with a modular collection of cloning and expression assets. Either in the plasmid or in the transposon vector, the Tel(r) marker was combined with a 12-kb DNA segment of plasmid pWW0 of Pseudomonas putida mt-2 encoding the upper TOL pathway enzymes. This allowed construction of antibiotic resistance-free but selectable P. putida strains with the ability to grow on toluene as the sole carbon source through an ortho-cleavage catabolic pathway.