Construction and behavior of biologically contained bacteria for environmental applications in bioremediation

The survival of microorganisms can be predicted through the use of active biological containment systems. We have constructed contained Pseudomonas putida strains that degrade alkylbenzoates. The modified strain carries a fusion of the Plac promoter to the gef gene, which encodes a killing protein. Expression from Plac is controlled through a regulatory cascade, so that Plac is switched on or off by the absence or presence of alkylbenzoates, respectively. Similar uncontained strains were also constructed and tested as a control. Contained and uncontained strains were genetically stable, and their survival and functionality in soil microcosms were as expected. Both contained and uncontained strains survived well in soils supplemented with alkylaromatics, whereas survival of the contained strain in soil microcosms without methylbenzoates was markedly reduced, in contrast to the control strain, which survived in these soils in the absence of alkylbenzoates. The TOL plasmid was transferred in soils between Pseudomonas strains but was not able to mobilize the elements of the containment system.