Downloaded from www.microbiologyresearch.org by IP: 54.70.40.11 On: Tue, 06 Nov 2018 23:53:31 A dual lethal system to enhance containment of recombinant micro-organisms Begon ˜ a Torres, 1 3 Susanne Jaenecke, 2 4 Kenneth N. Timmis, 2 Jose ´ L. Garcı ´a 1 and Eduardo Dı ´az 1 Correspondence Eduardo Dı ´az ediaz@cib.csic.es 1 Department of Molecular Microbiology, Centro de Investigaciones Biolo ´ gicas, Consejo Superior de Investigaciones Cientı ´ficas, Ramiro de Maeztu 9, 28040 Madrid, Spain 2 Division of Microbiology, GBF-National Research Centre for Biotechnology, Mascheroder Weg 1, D-38124 Braunschweig, Germany Received 1 July 2003 Revised 5 September 2003 Accepted 9 September 2003 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. INTRODUCTION Horizontal gene transfer is a source of concern when genetically engineered micro-organisms are intended to be released in large quantities to the environment for bio- technological applications (Ramos et al., 1995; Wackett, 2000). Gene spread could be also undesirable in contained environments, e.g. a bacterial consortium in a fermentation tank, for process protection and process optimization reasons (Dı´az et al., 1999). To make more predictable the behaviour of a genetically modified organism (GMO) introduced into a target habitat, its ability to spread new genetic information to potential recipients has to be reduced to avoid the appearance of undesired novel genetic combinations (gene containment). Moreover, the survival of the GMO has to be limited in time and space, i.e. engineering a controlled life cycle to reduce its dissemination and impact on the indigenous population of organisms (biological containment) (Molin et al., 1993; Ramos et al., 1995). Several gene and biological containment systems have been designed and they are based on a lethal function that, through a transcriptional and/or post-translational control element, becomes active in response to specific environmental signals (Molin et al., 1993; Dı ´az et al., 1999; Torres et al., 2000; Ronchel & Ramos, 2001; Davison, 2002). A general feature of the containment systems so far described is that a surviving subpopulation of cells, in the range of 10 27 to 10 23 per cell and generation depending on the particular system under study, ceases to respond to the toxic effect of the lethal function, which may be significant in environmental applications that use large quantities of cells (Molin et al., 1993; Szafranski et al., 1997). Analysis of the survivors has revealed that mutations are the main drawback in containment. Mutations that inacti- vate the containment system have been located either in the lethal function (Jensen et al., 1993; Munthali et al., 1996a; Torres et al., 2000) or in the control element (Bej et al., 1988; Knudsen et al., 1995). A way to reduce the problem of mutations and increase the efficiency of containment is to engineer genetic circuits 3Present address: Department Molecular and Cell Biology, Centro Nacional de Biotecnologı ´a, Consejo Superior de Investigaciones Cientı ´- ficas, Campus Universidad Auto ´ noma, Cantoblanco, 28049 Madrid, Spain. 4Present address: EuMeCom, Warburgstr. 4, 20354 Hamburg, Germany. 0002-6618 G 2003 SGM Printed in Great Britain 3595 Microbiology (2003), 149, 3595–3601 DOI 10.1099/mic.0.26618-0