CONDITIONAL-SUICIDE CONTAINMENT SYSTEM FOR BACTERIA WHICH MINERALIZE AROMATICS

A model conditional-suicide system to control genetically engineered microorganisms able to degrade substituted benzoates is reported. The system is based on two elements. One element consists of a fusion between the promoter of the Pseudomonas putida TOL plasmid-encoded meta-cleavage pathway operon (P(m)) and the lacI gene encoding Lac repressor plus xylS, coding for the positive regulator of P(m). The other element carries a fusion between the P(tac) promoter and the gef gene, which encodes a killing function. In the presence of XylS effectors, LacI protein is synthesized, preventing the expression of the killing function. In the absence of effectors, expression of the P(tac)::gef cassette is no longer prevented and a high rate of cell killing is observed. The substitution of XylS for XylSthr45, a mutant regulator with altered effector specificity and increased affinity for benzoates, allows the control of populations able to degrade a wider range of benzoates at micromolar substrate concentrations. Given the wide effector specificity of the key regulators, the wild-type and mutant XylS proteins, the system should allow the control of populations able to metabolize benzoate; methyl-, dimethyl-, chloro-, dichloro-, ethyl, and methoxybenzoates; salicylate; and methyl- and chlorosalicyclates. A small population of genetically engineered microorganisms became Gef resistant; however, the mechanism of such survival remains unknown.