Characterization of cell lysis in Pseudomonas putida induced upon expression of heterologous killing genes

Active biological containment systems are based on the controlled expressio n of killing genes. These systems are of interest for the Pseudomonadaceae because of the potential applications of these microbes as bioremediation a gents and biopesticides, The physiological effects that lead to cell death upon the induction of expression of two different heterologous killing gene s in nonpathogenic Pseudomonas putida KT2440 derivatives have been analyzed , P. putida CMC4 and CMC12 carry in their chromosomes a fusion of the PAl-0 4/03 promoter to the Escherichia coli gef gene and the phi X174 lysis gene E, respectively. Expression of the killing genes is controlled by the LacI protein, whose expression is initiated from the XylS-dependent Pm promoter. Under induced conditions, killing of P. putida CMC12 cells mediated by phi X174 lysis protein E was faster than that observed for P. putida CMC4, for which the Gef protein was the killing agent. In both cases, cell death occ urred as a result of impaired respiration, altered membrane permeability, a nd the release of some cytoplasmic contents to the extracellular medium.