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Chapter 29 : Plasmids as Tools for Containment

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Abstract:

This chapter reviews and discusses different aspects of containment, highlighting those systems devoted to contain organisms that remove toxic pollutants. There are two main strategies to diminish the potential risks associated with the deliberate or unintentional release of genetically modified organisms (GMOs) into the open environment. The lethal functions most extensively used for developing active containment circuits are those that disrupt the membrane potential, specially the two-component toxin-antidote systems involved in post-segregational killing of plasmid-free cells and their chromosomal counterparts. Biological containment systems have been engineered on plasmids using the Plac promoter and the Lacl repressor from , and they are triggered by addition of isopropyl-β-D-thiogalactopyranoside (IPTG). The most advanced containment systems are those developed for bacteria that degrade pollutants. Although the biological containment systems increase the predictability of GMOs, one of the main concerns about the release of such GMOs to the environment is how recombinant DNA can spread among indigenous bacterial populations. Lethal donation circuits, such as those described for gene containment, constitute interesting tools to explore the ecological and evolutionary consequences of shifting the natural equilibrium between genetic change and genetic constancy toward the latter. Many lethal functions and regulatory circuits have been used and combined to design efficient containment systems. Active containment systems are a major tool to reduce the uncertainty associated with the introduction of monocultures, genetically engineered or not, into target habitats.

Citation: Torres B, García J, Diaz E. 2004. Plasmids as Tools for Containment, p 589-602. In Funnell B, Phillips G (ed), Plasmid Biology. ASM Press, Washington, DC. doi: 10.1128/9781555817732.ch29

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Figures

Image of Figure 1
Figure 1

Concept of biological and gene containment. (A) In biological containment the GMO is restricted to the target habitat for a limited time. To accomplish this, the organism is engineered with a suicide circuit (●) that usually is switched off (survival), but it becomes activated ( ) in response to a specific environmental signal, leading to cell death. (B) In gene containment, it is the recombinant DNA rather than the organism itself that is the subject of containment. To create a barrier that restricts dispersal of such novel DNA from the GMO to the indigenous microbiota, a genetic circuit based on a lethal function closely linked to the recombinant DNA (●) needs to be engineered in a way that the lethal function becomes activated ( ) in the potential recipients of the contained DNA, leading to the death of such cells.

Citation: Torres B, García J, Diaz E. 2004. Plasmids as Tools for Containment, p 589-602. In Funnell B, Phillips G (ed), Plasmid Biology. ASM Press, Washington, DC. doi: 10.1128/9781555817732.ch29
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Image of Figure 2
Figure 2

Schematic representations of molecular mechanisms for active containment systems. (A) A control element responds to the appropriate environmental signal through a sensor protein ( ) and a cognate regulated promoter (Preg) and regulates at the transcriptional level the expression of a lethal function (■). (B) The control element can be engineered as a double transcriptional regulatory circuit. The sensor protein ( ) recognizes an environmental signal and interacts with the cognate regulated promoter (Preg1), which, in turn, drives transcription of a second regulator ( ) that controls the expression of the Preg2 promoter running transcription of the lethal gene (■). (C) The control element may involve a posttranslational regulation through an immunity protein ( ) that specifically neutralizes the killing effect of the constitutively expressed lethal function ( ). To this end, the expression of the immunity gene is driven by the Preg promoter under control of the sensor protein ( ) that responds to the environmental signal.

Citation: Torres B, García J, Diaz E. 2004. Plasmids as Tools for Containment, p 589-602. In Funnell B, Phillips G (ed), Plasmid Biology. ASM Press, Washington, DC. doi: 10.1128/9781555817732.ch29
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Image of Figure 3
Figure 3

Rationale of a model biological containment system for biodegraders. The control element consists of a double regulatory circuit based on (i) the XylS sensor protein that recognizes benzoate or benzoate analogues (alkyl- and halo-benzoates) and stimulates gene expression from the Pm promoter; and (ii) the lael gene, whose expression is driven by the Pm promoter, coding for the Lael repressor protein that inhibits the PA1/04/03 promoter (P*). The expression of the lethal gene is under control of the PA1/04/03(P*) promoter. (A) In the presence of benzoate or benzoate analogues the control element is switched on and the lethal function is not produced (survival). XylSa, active conformation of XylS protein. (B) Once bacteria complete the degradation of the aromatic compound or they spread to a noil polluted site, the control system is switched off and, as a consequence, the lethal function is produced (cell death). XylS1, inactive conformation of XylS protein.

Citation: Torres B, García J, Diaz E. 2004. Plasmids as Tools for Containment, p 589-602. In Funnell B, Phillips G (ed), Plasmid Biology. ASM Press, Washington, DC. doi: 10.1128/9781555817732.ch29
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Figure 4

Rationale of a model plasmid containment system. The scheme has been modified from reference 10. Gene containment is achieved by a lethal donation of a killing function. In the GMO (donor cell), the lethal gene is closely linked to the novel trait in a plasmid, and the toxic effect of the lethal function is neutralized (Lethal1) by the product of an immunity gene (1mm) located at the chromosome, such that cotransfer of the lethal and immunity functions will be an extremely low-frequency event. Plasmid transfer to a nonimmune organism will lead to the activation of the lethal function (LethalA) and to the rapid killing of the recipient cells, thus preventing the spread of the plasmid and the associated novel trait.

Citation: Torres B, García J, Diaz E. 2004. Plasmids as Tools for Containment, p 589-602. In Funnell B, Phillips G (ed), Plasmid Biology. ASM Press, Washington, DC. doi: 10.1128/9781555817732.ch29
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Tables

Generic image for table
Table 1

Lethal functions used in active containment systems

Citation: Torres B, García J, Diaz E. 2004. Plasmids as Tools for Containment, p 589-602. In Funnell B, Phillips G (ed), Plasmid Biology. ASM Press, Washington, DC. doi: 10.1128/9781555817732.ch29
Generic image for table
Table 2

Control elements used in active containment systems

Citation: Torres B, García J, Diaz E. 2004. Plasmids as Tools for Containment, p 589-602. In Funnell B, Phillips G (ed), Plasmid Biology. ASM Press, Washington, DC. doi: 10.1128/9781555817732.ch29
Generic image for table
Table 3

Applications of active containment systems

Citation: Torres B, García J, Diaz E. 2004. Plasmids as Tools for Containment, p 589-602. In Funnell B, Phillips G (ed), Plasmid Biology. ASM Press, Washington, DC. doi: 10.1128/9781555817732.ch29

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