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EC-sponsored Research on Safety of Genetically Modified Organisms - A Review of Results
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image Genetic tools for constructing genetically-modified micro-organisms (GEMs) with high predictability in performance and behaviour in ecological microcosms, soils, rhizospheres and river sediments

Background and objectives

Many organic compounds including numerous pollutants are broken down by microbes. However, polychlorinated biphenyls (PCBs) are recalcitrant to microbial degradation. They persist in the environment, are toxic to micro-organisms, may inhibit degradative pathways and can enter the food chain where they present health hazards. Therefore, the removal of PCBs from the environment is a high priority task. Since PCBs are found in large volumes in soils and sediments, in situ degradation may be an effective approach. The aim was to transfer the PCB degradation pathway from PCB-degrading bacteria, which survive poorly in soil, into bacteria found associated with plant roots and which can survive in soils. The result would be to create recombinants with an improved ability to eliminate organic solvents. Pseudomonas sp. LB400, the best known PCB degrader, was used as a model system.




Approach and methodology

The catabolic bph genes were transferred into the chromosome of indigenous bacteria, particularly those of the sugar beet rhizosphere. The resultant recombinants were screened for their ability to degrade PCBs.

The behaviour of GEMs released into the environment has not been extensively documented, therefore it is useful to restrict their survival to increase their predictability in the environment. The Pseudomonas model was used to construct contained GEMs. The gef killing gene was coupled to the PCB regulatory system. The system was based on two elements. The control element consisted of a fusion between the TOL meta-cleavage degradation pathway promoter (Pm) and the LacI repressor protein, plus the xylS gene encoding the XylS protein that respond to chlorobenzoate effectors. The killing cassette element consisted of a fusion between the Plac promoter and gef. Thus in the presence of PCBs the bacteria produce the LacI protein, which prevents the expression of the killing gene. In the absence of PCBs the expression of the killing cassette is no longer repressed and the bacteria die.

To limit the rate of lateral transfer of recombinant DNA (rDNA) a gene containment system was developed. This consisted of a killing element (the colE3 gene which encodes an RNase that cleaves all prokaryotic 16S rRNA) and a control element (the immE3 gene which encodes a specific repressor of the lethal function). In the GEM the killing gene is closely linked to the rDNA determining the new trait, whereas the control element is not. Thus transfer of the rDNA would be accompanied by transfer of the lethal gene but not the control gene and so if gene transfer occurred the recipient organism would be killed.

Main findings and outcome

One major drawback of PCB-degraders is the responsiveness of the regulatory circuit to the substrate. Therefore we examined the regulation of the system in vivo, and particularly its potential to express the bph gene under different conditions and its inducibility by PCBs. Bph genes were constitutively expressed at high levels even in the absence of PCBs. Exposure of the cells to PCBs resulted in a further increase of bph expression. Thus bph genes can be expressed in diverse environmental conditions.

The bph operon was transferred to Pseudomonas F113 and seventeen Gram-negative bacteria isolated from river sediment. The functionality of the operon in the modified organisms was confirmed by their ability to grow on PCBs. The novel genetic trait was stably maintained for over 200 generations in the recombinant bacteria. The bph operon was not transferred to related bacteria. The survival, colonisation and competitive abilities of the recombinants were unaffected.

The TOL catalytic pathway was exploited to control gene expression through artificial cascades to develop biologically contained strains.

A suicide containment system was incorporated randomly into the P. putida chromosome. In both liquid cultures and sterile and non-sterile soil microcosms the recombinant bearing the containment system behaved as predicted. Mutants resistant to cell killing arose at a frequency of around 10-5 to 10-6 per cell per generation. In bacteria containing two copies of the killing cassette the frequency of such mutants decreased to around 10-8 per cell per generation. Mutations were therefore linked to the killing element and not to the regulatory element.

The introduction into the soil of the contained bacteria did not have significant effects on natural PCB-degraders already present.

A system was designed using colicin E3 and immunity E3 as containment genes and to decrease undesirable horizontal gene transfer. Colicin E3 kills all prokaryotes by inhibiting protein synthesis. This makes it a powerful tool for decreasing dispersal of recombinant genes among indigenous micro-organisms in ecosystems into which a GEM is deliberately or accidentally introduced.


We dissected the regulatory circuits involved in the control of a bacterial pathway for degrading chemical pollutants. The genes for this pathway were introduced into the host chromosome of indigenous bacteria isolated from various ecological niches in which in situ bioremediation treatment may be required. Ecologically, the recombinant bacteria behave like the wild types. Thus, the first GEMs specifically designed to degrade pollutants, equipped with either circuits for biological containment or barriers to limit lateral transfer of rDNA, were developed. We showed that the survival and behaviour of GEMs, and rDNA transfer, can be rendered predictable.


Major publications

González-Pérez M.M., Ramos J.L., Gallegos M.T. and Marqués S., “Critical nucleotides in the upstream region of the XylS-dependent TOL meta-cleavage pathway operon promoter as deduced from analysis of mutants”.
J. Biol. Chem., 274, 1999, pp. 2286-2290.

Espinosa-Urgel M., Salido A. and Ramos J.L., “Genetic analysis of functions involved in adhesion of Pseudomonas putida to seeds”.
J. Bacteriol.,
182, 2000, pp. 2363-2369.

Torres B., Jaenecke S., Timmis K.N., García J.L. and Díaz E., “A gene containment strategy based on a restriction-modification system”.
Environ. Microbiol.,
2, 2000 (in press).

Díaz E. and Prieto M.A., “Bacterial promoters triggering biodegradation of aromatic pollutants”.
Curr. Opin. Biotechnol., 2000 (in press).

Ronchel M.C., Ramos C., Jensen L.B., Molin S., Ramos J.L., “Construction and behavior of biologically contained bacteria for environmental applications in bioremediation”.
Appl. Environ. Microbiol,
61, 1995, pp. 2990-2994.

Ronchel M.C., Molina L., Witte A., Lutbiz W., Molin S., Ramos J.L., Ramos C., “Characterization of cell lysis in Pseudomonas putida induced upon expression of heterologous killing genes”.
Appl. Environ. Microbiol.,
64, 1998, pp. 4904-4911.

Ronchel M.C., Ramos-Díaz M.A., Ramos J.L., “Retrotransfer of DNA in the rhizosphere”.
Env. Microbiol.,
2, 2000, pp. 319-323.

Molina L., Ramos C., Ronchel M.C, Molin S., Ramos J.L., “Construction of an efficient biologically contained Pseudomonas putida strain and its survival in outdoor assays”.
Appl. Environ. Microbiol.,
64, 1998, pp. 2072-2078.

Molina L., Ramos C., Duque E., Ronchel M.C, García J.M., Wyke L., Ramos J.L., “Survival of Pseudomonas putida KT2440 in soil and in the rhizosphere of plants under greenhouse and environmental conditions”.
Soil Biol. Biochem.,
32, 2000, pp. 315-321.
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imageResearch project

Contract number

October 1991 – September 1993

J.L. Ramos
Estación Experimental del Zaidín
Granada (ES)

Follow-up of the project
This project was continued in EC project: BIO2-CT92-0084.



S. Molin
Technical University of Denmark
Lyngby (DK)

K.N. Timmis, D. Dwyer
National Research Centre for Biotechnology (GBF)
Braunschweig (DE)

V. de Lorenzo
Centro Nacional de Biotecnología
Madrid (ES)

D. Dowling
Institute of Technology
Carlow (IE)

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