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Plasmids as Tools for Containment

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  • Authors: José L. GarcíA1, Eduardo Díaz2
  • Editors: Marcelo Tolmasky3, Juan Carlos Alonso4
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    Affiliations: 1: Department of Environmental Biology, Centro de Investigaciones Biológicas (CSIC), 28040 Madrid, Spain; 2: Department of Environmental Biology, Centro de Investigaciones Biológicas (CSIC), 28040 Madrid, Spain; 3: California State University, Fullerton, CA; 4: Centro Nacional de Biotecnología, Cantoblanco, Madrid, Spain
  • Source: microbiolspec October 2014 vol. 2 no. 5 doi:10.1128/microbiolspec.PLAS-0011-2013
  • Received 04 February 2014 Accepted 05 February 2014 Published 10 October 2014
  • Eduardo Díaz, ediaz@cib.csic.es
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  • Abstract:

    Active containment systems are a major tool for reducing the uncertainty associated with the introduction of monocultures, genetically engineered or not, into target habitats for a large number of biotechnological applications (e.g., bioremediation, bioleaching, biopesticides, biofuels, biotransformations, live vaccines, etc.). While biological containment reduces the survival of the introduced organism outside the target habitat and/or upon completion of the projected task, gene containment strategies reduce the lateral spread of the key genetic determinants to indigenous microorganisms. In fundamental research, suicide circuits become relevant tools to address the role of gene transfer, mainly plasmid transfer, in evolution and how this transfer contributes to genome plasticity and to the rapid adaptation of microbial communities to environmental changes. Many lethal functions and regulatory circuits have been used and combined to design efficient containment systems. As many new genomes are being sequenced, novel lethal genes and regulatory elements are available, e.g., new toxin-antitoxin modules, and they could be used to increase further the current containment efficiencies and to expand containment to other organisms. Although the current containment systems can increase the predictability of genetically modified organisms in the environment, containment will never be absolute, due to the existence of mutations that lead to the appearance of surviving subpopulations. In this sense, orthogonal systems (xenobiology) appear to be the solution for setting a functional genetic firewall that will allow absolute containment of recombinant organisms.

  • Citation: GarcíA J, Díaz E. 2014. Plasmids as Tools for Containment. Microbiol Spectrum 2(5):PLAS-0011-2013. doi:10.1128/microbiolspec.PLAS-0011-2013.

Key Concept Ranking

Microbial Ecology
0.5111254
Recombinant Vector Vaccines
0.43830776
Type 1 Fimbriae
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Bacterial Vaccines
0.4259529
0.5111254

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2014-10-10
2017-11-24

Abstract:

Active containment systems are a major tool for reducing the uncertainty associated with the introduction of monocultures, genetically engineered or not, into target habitats for a large number of biotechnological applications (e.g., bioremediation, bioleaching, biopesticides, biofuels, biotransformations, live vaccines, etc.). While biological containment reduces the survival of the introduced organism outside the target habitat and/or upon completion of the projected task, gene containment strategies reduce the lateral spread of the key genetic determinants to indigenous microorganisms. In fundamental research, suicide circuits become relevant tools to address the role of gene transfer, mainly plasmid transfer, in evolution and how this transfer contributes to genome plasticity and to the rapid adaptation of microbial communities to environmental changes. Many lethal functions and regulatory circuits have been used and combined to design efficient containment systems. As many new genomes are being sequenced, novel lethal genes and regulatory elements are available, e.g., new toxin-antitoxin modules, and they could be used to increase further the current containment efficiencies and to expand containment to other organisms. Although the current containment systems can increase the predictability of genetically modified organisms in the environment, containment will never be absolute, due to the existence of mutations that lead to the appearance of surviving subpopulations. In this sense, orthogonal systems (xenobiology) appear to be the solution for setting a functional genetic firewall that will allow absolute containment of recombinant organisms.

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FIGURE 1

Concept of biological and gene containment. In biological containment the GMO is restricted to the target habitat for a limited period of time. To accomplish this, the organism is engineered with a suicide circuit encoding a toxin (TOX) that usually is located in a plasmid and switched off (SURVIVAL), but it becomes activated in response to a specific environmental signal leading to cell death (DEATH). 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 toxic function (TOX) closely linked to the recombinant DNA needs to be engineered in such a way that the lethal function is inactive in the host GMO (e.g., formation of an antitoxin [ANT]-toxin [TOX] complex) but becomes activated (TOX) in the potential recipients of the contained DNA that lack the antitoxin function, leading to the death of such cells. doi:10.1128/microbiolspec.PLAS-0011-2013.f1

Source: microbiolspec October 2014 vol. 2 no. 5 doi:10.1128/microbiolspec.PLAS-0011-2013
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FIGURE 2

Schematic representations of molecular mechanisms for active containment systems. A control element responds to the appropriate environmental signal through a sensor protein and a cognate regulated promoter (), and regulates at the transcriptional level the expression of a lethal function. 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 (), which in turn drives transcription of a regulator that controls the expression of a second regulated promoter () running transcription of the lethal gene. The control element may involve 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 promoter under control of the sensor protein that responds to the environmental signal. doi:10.1128/microbiolspec.PLAS-0011-2013.f2

Source: microbiolspec October 2014 vol. 2 no. 5 doi:10.1128/microbiolspec.PLAS-0011-2013
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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 promoter and (ii) the gene, whose expression is driven by the promoter, coding for the LacI repressor protein that inhibits the promoter (*). The expression of the lethal gene is under control of the (*) promoter. In the presence of benzoate or benzoate analogues (+ Benzoate) the control element is switched on and the lethal function is not produced (SURVIVAL). Once bacteria complete the degradation of the aromatic compound or spread to a nonpolluted site (− Benzoate), the control system is switched off and, as a consequence, the lethal function is produced (DEATH). XylS, active conformation of XylS protein. XylS, inactive conformation of XylS protein. doi:10.1128/microbiolspec.PLAS-0011-2013.f3

Source: microbiolspec October 2014 vol. 2 no. 5 doi:10.1128/microbiolspec.PLAS-0011-2013
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FIGURE 4

Rationale of a model plasmid containment system. The scheme has been modified from Diaz et al. ( 38 ). Gene containment is achieved by a lethal donation of a toxic 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 (TOX) is neutralized by the product of an antitoxin gene () located at the chromosome, such that cotransfer of the and genes will be an extremely low-frequency event. Plasmid transfer to a nonimmune organism (recipient cell) will lead to the activation of the lethal function and to cell death, thus preventing the spread of the plasmid and the novel associated trait. doi:10.1128/microbiolspec.PLAS-0011-2013.f4

Source: microbiolspec October 2014 vol. 2 no. 5 doi:10.1128/microbiolspec.PLAS-0011-2013
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Tables

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TABLE 1

Lethal functions used in active containment systems

Source: microbiolspec October 2014 vol. 2 no. 5 doi:10.1128/microbiolspec.PLAS-0011-2013
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TABLE 2

Control elements used in active containment systems

Source: microbiolspec October 2014 vol. 2 no. 5 doi:10.1128/microbiolspec.PLAS-0011-2013
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TABLE 3

Applications of active containment systems

Source: microbiolspec October 2014 vol. 2 no. 5 doi:10.1128/microbiolspec.PLAS-0011-2013

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