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A dual lethal system to enhance containment of recombinant micro-organisms

Begoña Torres^1,

Susanne Jaenecke^2,

¹ Department of Molecular Microbiology, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Ramiro de Maeztu 9, 28040 Madrid, Spain
² Division of Microbiology, GBF-National Research Centre for Biotechnology, Mascheroder Weg 1, D-38124 Braunschweig, Germany

Correspondence
Eduardo Díaz
ediaz{at}cib.csic.es

Active containment systems based on the controlled expression of a lethal gene are designed to increase containment of recombinant micro-organisms used for environmental applications. A major drawback in containment is the existence of mutations that generate surviving cells that cease to respond to the toxic effect of the lethal function. In this work the authors have developed for the first time a strategy to reduce the problem of mutations and increase the efficiency of containment based on the combination of two lethal functions acting on different cellular targets of major concern in containment, DNA and RNA, and whose expression is under control of different regulatory signals. To engineer the dual gene containment circuit, two toxin–antitoxin pairs, i.e. the colicin E3–immunity E3 and the EcoRI restriction–modification systems, were combined. The genes encoding the immunity E3 and the EcoRI methyltransferase proteins (antitoxins) were stably inserted into the chromosome of the host cell, whereas the broad-host-range lethal genes encoding the colicin E3 RNase and the EcoRI restriction endonuclease (toxins) were flanking the contained trait in a plasmid. This dual lethal cassette decreased gene transfer frequencies, through killing of the recipient cells, by eight orders of magnitude, which provides experimental evidence that the anticipated containment level due to the combination of single containment systems is generally achieved. Survivors that escaped killing were analysed and the mutational events involved were characterized.

Present address: EuMeCom, Warburgstr. 4, 20354 Hamburg, Germany.

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