A substrate-dependent biological containment system for Pseudomonas putida based on the Escherichia coli gef gene

Abstract

A model substrate-dependent suicide system to biologically contain Pseudomonas putida KT2440 is reported. The system consists of two elements. One element carries a fusion between a synthetic bc promoter (PA-4w03) and the gefgene, which encodes a killing function. This element is contained within a transposaseless mini-TnS transposon so that it can be integrated at random locations on the Pseudomonas chromosome. The second element, harbored by plasmid pCC102, is designed to control the first and bears a fusion between the promoter of the P. putida TOL plasmid-encoded meta-cleavage pathway operon (Pm) and the lacI gene, encoding the Lac repressor, plus xyLS2, coding for a positive regulator of Pm. In liquid culture under optimal growth conditions and in sterile and nonsterile soil microcosms, P. putida KT2440(pWWO) bearing the containment system behaves as designed. In the presence of a XylS effector, such as m-methylbenzoate, the LacI protein is synthesized, preventing the expression of the killing function. In the absence of effectors, expression of the PA,4w03::gef cassette is no longer prevented and a high rate of cell killing is observed. Fluctuation test analyses revealed that mutants resistant to cell killing arise at a frequency of around 10'S to 10-6 per cell per

Development of a conditional lethal system for a Streptomyces lividans strain and its use to investigate conjugative transfer in soil

“…For instance, various plasmidbearing killing genes including hok, gef and rel cloned under inducible promoters lacked e¤ciency due to the loss of the plasmid, to the insu¤cient production of the lethal protein, to chromosomal mutations leading to resistance to its action, or to mutational inactivation of the lethal gene itself. Only duplication of the suicide functions arranged on a plasmid in a way to prevent inactivation by deletion and recombination permitted a decrease in the mutation rate to about 10 38 [27]. The e¤ciency of the gylB-based system developed for the S. lividans strain in this study does not depend on stable maintenance of plasmids or expression of foreign genes.…”

The Behavior of Bacteria Designed for Biodegradation

“…It should be emphasized that effective containment systems can only be constructed if the performance of the organism in the proper environmental context is at least partially characterized. In fact, this biological containment system was shown to function in soils 55 (Fig. 4) .…”

Suicidal genetically engineered microorganisms for bioremediation: Need and perspectives

“…This substitution resulted in an expansion of the range of benzoate analogues allowing survival of the 'controlled' population. Following this study, Jensen et al (88) tried to construct a substrate-dependent biological containment system for Pseudomonas putida based on the gef killing gene and attempted to integrate the fused promoter and gef gene into the chromosome of a Pseudomonas strain by using a mini Tn-5 transposons. The second element was incorporated into the pCC102 wherein the promoter Pm, lacI and xylS2 were fused.…”

“…It was seen that, in bacteria with two copies of the 'killing' cassette, the rate of appearance of mutants resistant to 'gef' were as low as 10 À8 per cell per generation. (88) Ronchel et al (89) used a similar system for bioremediation of alkylbenzoate contamination. In their system, the killer gef gene was placed under the control of the P tac promoter, which could be switched on/off by the absence/presence of alkylbenzoates, respectively.…”