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EC-sponsored Research on Safety of Genetically Modified Organisms - A Review of Results
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image Fate of genetically engineered microorganisms (GEMs) and genetically engineered DNA Sequences (GEDs) in some environmental hot spots

Background and objectives (1)

Genetically modified micro-organisms (GMMOs) may be expected to be released in soil environments either via deliberate release (for agricultural or bioremediation purposes) or via accidental (undesired) release. Among the possible ways of gene dissemination, plasmid-mediated bacterial conjugation proved to be highly relevant, as the transfer of environmental conjugative plasmids into recipient strains that were introduced in river and other ecosystems was found to occur. Furthermore, special attention has to be given to Broad Host Range (BHR) plasmids and their potential for gene dissemination and gene rearrangements: some BHR plasmids display the phenotype of gene capture (retrotransfer), to the advantage of their hosts and may also carry degradative genes that allow their hosts to degrade man-made chlorinated chemicals.

We have studied the behaviour of GMMOs in some environmental “hot spots” (agricultural and polluted soils), with special emphasis on the presence of BHR plasmids in such environments and the role of these plasmids in gene dissemination.

(1) This project was a direct follow-on from EC project: Transfer, survival and spread of genetically manipulated organisms (GMOs) in river sediments, soils and agricultural environments (BAP-0043/0366/0367/0379).


Approach and methodology

The behaviour of GMMOs and their recombinant DNA (rDNA) was assessed in microcosms that simulate environmental hot spots: plant-soil ecosystems, river sediments, soils polluted with chemicals. The survival and mobility of GMMOs and the transfer of cloned rDNAs (metal resistance or degradative genes have been used here as engineered DNA) into appropriate introduced recipient strains and into the indigenous population were followed. The role of BHR plasmids in the dissemination of rDNA was assessed by introducing the plasmids into microcosms in either the donor or recipient strain, or in a helper strain. The presence of natural transfer potential in the environmental hot spots was assessed by the exogenous isolation of BHR plasmids from the environmental samples. The genetic and molecular composition of the samples was determined, with emphasis on their conjugation and gene dissemination properties. The mechanism of retrotransfer was also investigated.
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Main findings and outcome

The microcosm studies showed that recombinant DNA cloned in a variety of vectors could be disseminated from host strains into the recipients tested. This required the intervention of conjugative plasmids and occurred at variable frequencies, depending on the nature of the vector in which the recombinant DNA was cloned. It was shown that the presence of BHR plasmids in a soil recipient (especially those equipped with efficient transposons or other mobile genetic elements) may facilitate the dissemination of genes cloned in vectors that were supposed to ensure gene confinement. Since genes can be captured by BHR plasmids, (a manifestation of the retrotransfer phenotype of some BHR plasmids) this puts a limit on the safety of such vectors. However, this kind of conjugation-mediated release of recombinant DNA was almost only observed in sterile soils. Competition with indigenous microflora, predation by other (mostly eukaryotic) microbiota, lack of available nutrients and the absence of selection pressure linked to the recombinant DNA cumulatively compromised the survival of the tested GMMOs. However, if selection pressure acting on the relevant degradation or metal resistance genes is present, gene dissemination to the added recipient cells and even to the indigenous microflora may be detected as a consequence of the enrichment of rare transconjugants. These conditions of selection pressure are not at all representative for most recombinant DNA, especially those of eukaryotic origin, that is cloned in bacterial GMMOs, and for which there is usually no selective pressure in the environment.

Thus, the systematic exploitation of a catastrophe scenario that was specifically designed to easily detect very rare events and to optimise gene dissemination emphasises how limited the survival of released lab strains and their rDNA seems to be. These studies also emphasise the importance of BHR plasmids and other mobile genetic elements in the transfer of genes in all the tested environments. Furthermore, selection pressure may help the dissemination of genotypes with relevance to soil remediation.

Two strategies of exogenous isolation of plasmids from the indigenous communities (river stream and soil biotopes) successfully provided new BHR plasmids that were further studied in subsequent EC programmes. GMMOs (E. coli, Pseudomonas or Ralstonia strains) were shown to easily get new plasmids from these environments. It was also found for the first time that PCB (polychlorobiphenyl) degradative genes can be carried by large transposons which can transpose onto BHR plasmids, and a first approach to analyse the mechanism of such retrotransfer (plasmid-mediated gene capture to the benefit of the plasmid host) was carried out, emphasising the high frequency of the phenomenon and its ecological significance.
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Conclusions

BHR plasmid-mediated gene transfer plays a role in the capture and the subsequent dissemination of recombinant DNA cloned in current vectors carried by standard GMMOs. On plate matings or in sterile microcosms, the frequency of such events is measurable but generally low, and they virtually escape any detection under real environmental conditions that accompany the release of GMMOs. New BHR plasmids could easily be isolated from all the tested microbial communities. These plasmids have a measurable potential to disseminate genes across taxonomic barriers. These findings are of importance in the context of natural horizontal gene transfer as an evolutionary force and as a part of the “genetic landscape” in which long term biosafety issues must be considered.

 

Major publications

De Rore H., Top E., Houwen F., Mergeay M., Verstraete W., “Evolution of heavy metal resistant transconjugants in a soil environment with a concomitant selective pressure”.
FEMS Microbiol. Ecol.,
15, 1994, p. 71.

Dijkmans R., Jagers A., Kreps S., Collard J-M., Mergeay M., “Rapid method for purification of soil DNA for hybridization and PCR analysis”.
Microbial releases,
2, 1993, p. 29.

Springael D., Kreps S., Mergeay M., “Identification of a catabolic transposon Tn4371, carrying biphenyl and 4-chlorobiphenyl degradation genes in Alcaligenes eutrophus cells”.
J. Bacteriol.,
175, 1993, p. 1674.

Top E., Van Rolleghem P., Mergeay M., Verstraete W., “Determination of the mechanism of retrotransfer by mechanistic mathematical modelling”.
J. Bacteriol.,
174, 1992, p. 5953.

Top E., De Smet I., Verstraete W., Dijkmans R., Mergeay M., “Exogenous isolation of mobilizing plasmids from polluted soils and sludges”.
Appl. Environ. Microbiol, 60, 1994, p. 831.
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imageResearch project
 

Contract number
BIOT-CT91-0284

Period
October 1991- September 1993

Coordinator
M. Mergeay
Laboratory of Genetics & Biotechnology (SCK/VITO)
Mol (BE)

Follow-up of the project
This project was continued in EC project BIO2-CT92-0491.

 
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Partners


W. Verstraete
University of Ghent (BE)

J. Figuereido Marques
Instituto de Biologia Experimental e Tecnologica
Oeiras (PT)

J.C. Fry
University of Wales
College of Cardiff (UK)

 
 
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