A SUBSTRATE-DEPENDENT BIOLOGICAL CONTAINMENT SYSTEM FOR PSEUDOMONAS-PUTIDA BASED ON THE ESCHERICHIA-COLI GEF GENE

A model substrate-dependent suicide system to biologically contain Pse udomonas putida KT2440 is reported. The system consists of two element s. One element carries a fusion between a synthetic lac promoter (P(A1 -04/03) and the gef gene, which encodes a killing function. This eleme nt is contained within a transposaseless mini-Tn5 transposon so that i t can be integrated at random locations on the Pseudomonas chromosome. The second element, harbored by plasmid pCC102, is designed to contro l the first and bears a fusion between the promoter of the P. putida T OL plasmid-encoded meta-cleavage pathway operon (Pm) and the lacI gene , encoding the Lac repressor, plus xylS2, coding for a positive regula tor of Pm. In liquid culture under optimal growth conditions and in st erile and nonsterile soil microcosms, P. putida KT2440(pWWO) bearing t he containment system behaves as designed. In the presence of a XylS e ffector, such as m-methylbenzoate, the LacI protein is synthesized, pr eventing the expression of the killing function. In the absence of eff ectors, expression of the P(A1-04/03)=gef cassette is no longer preven ted and a high rate of cell killing is observed. Fluctuation test anal yses revealed that mutants resistant to cell killing arise at a freque ncy of around 10(-5) to 10(-6) per cell per generation. Mutations are linked to the killing element rather than to the regulatory one. In ba cteria bearing two copies of the killing cassette, the rate of appeara nce of mutants resistant to killing decreased to as low as 10(-8) per cell per generation.