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 P seudomonas strains potentially destined for environmental release. In this study, the activated Tel(r) determinants encoded by the cryptic t elAB genes of plasmid RK2 were produced, along with the associated kil A 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 fr om a variant of Pseudomonas plasmid pPS10, which is able to replicate in a variety of gram-negative hosts and is endowed with a modular coll ection of cloning and expression assets. Either in the plasmid or in t he 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 resistan ce-free but selectable P. putida strains with the ability to grow on t oluene as the sole carbon source through an ortho-cleavage catabolic p athway.