Chapter 29 : Plasmids as Tools for Containment

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.

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.

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.

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.