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Chapter 31 : Plasmids as Tools for Containment

Authors: José L. García1, Eduardo Díaz2
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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
Content Type: Monograph
Publication Year: 2015

Category: Clinical Microbiology

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Abstract:

Biotechnology provides a large number of environmental applications (e.g., bioremediation, biofilters, bioleaching, biopesticides, biofuels, biotransformations [green chemistry], live vaccines, etc.) that support the development of the bioeconomy ( ). Nevertheless, the biotechnological processes planned to work in the open field are not easy to implement since they have to cope with a wide range of chemical, physical, and biological variations that could lead to a low level of predictability, making such processes difficult to control ( ). In addition, the biotechnological processes that introduce large quantities of microorganisms into the ecosystem have raised concerns about their potential impact on the environment. Such concerns have prompted the creation of risk assessment research programs oriented to developing new strategies in order to increase the safety and predictability of the microorganisms released into the environment and especially those that have been genetically modified ( ).

Citation: García J, Díaz E. 2015. Plasmids as Tools for Containment, p 615-631. In Tolmasky M, Alonso J (ed), Plasmids: Biology and Impact in Biotechnology and Discovery. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.PLAS-0011-2013
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Plasmids as Tools for Containment
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Citation: García J, Díaz E. 2015. Plasmids as Tools for Containment, p 615-631. In Tolmasky M, Alonso J (ed), Plasmids: Biology and Impact in Biotechnology and Discovery. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.PLAS-0011-2013
/content/10.1128/9781555818982.chap31.fig31-1
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.
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10.1128/9781555818982/fig31-1.gif
Image of Figure 1
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.

Citation: García J, Díaz E. 2015. Plasmids as Tools for Containment, p 615-631. In Tolmasky M, Alonso J (ed), Plasmids: Biology and Impact in Biotechnology and Discovery. ASM Press, Washington, DC. 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.
10.1128/9781555818982/fig31-2_thmb.gif
10.1128/9781555818982/fig31-2.gif
Image of Figure 2
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.

Citation: García J, Díaz E. 2015. Plasmids as Tools for Containment, p 615-631. In Tolmasky M, Alonso J (ed), Plasmids: Biology and Impact in Biotechnology and Discovery. ASM Press, Washington, DC. 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.
10.1128/9781555818982/fig31-3_thmb.gif
10.1128/9781555818982/fig31-3.gif
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 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.

Citation: García J, Díaz E. 2015. Plasmids as Tools for Containment, p 615-631. In Tolmasky M, Alonso J (ed), Plasmids: Biology and Impact in Biotechnology and Discovery. ASM Press, Washington, DC. 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. ( ). 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.
10.1128/9781555818982/fig31-4_thmb.gif
10.1128/9781555818982/fig31-4.gif
Image of Figure 4
Figure 4

Rationale of a model plasmid containment system. The scheme has been modified from Diaz et al. ( ). 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.

Citation: García J, Díaz E. 2015. Plasmids as Tools for Containment, p 615-631. In Tolmasky M, Alonso J (ed), Plasmids: Biology and Impact in Biotechnology and Discovery. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.PLAS-0011-2013
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Tables

Plasmids as Tools for Containment
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Citation: García J, Díaz E. 2015. Plasmids as Tools for Containment, p 615-631. In Tolmasky M, Alonso J (ed), Plasmids: Biology and Impact in Biotechnology and Discovery. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.PLAS-0011-2013
/content/10.1128/9781555818982.chap31.tab31-1
Table 1
Lethal functions used in active containment systems
Generic image for table
Table 1

Lethal functions used in active containment systems

Citation: García J, Díaz E. 2015. Plasmids as Tools for Containment, p 615-631. In Tolmasky M, Alonso J (ed), Plasmids: Biology and Impact in Biotechnology and Discovery. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.PLAS-0011-2013
/content/10.1128/9781555818982.chap31.tab31-2
Table 2
Control elements used in active containment systems
Generic image for table
Table 2

Control elements used in active containment systems

Citation: García J, Díaz E. 2015. Plasmids as Tools for Containment, p 615-631. In Tolmasky M, Alonso J (ed), Plasmids: Biology and Impact in Biotechnology and Discovery. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.PLAS-0011-2013
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Table 3
Applications of active containment systems
Generic image for table
Table 3

Applications of active containment systems

Citation: García J, Díaz E. 2015. Plasmids as Tools for Containment, p 615-631. In Tolmasky M, Alonso J (ed), Plasmids: Biology and Impact in Biotechnology and Discovery. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.PLAS-0011-2013

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