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EC-sponsored Research on Safety of Genetically Modified Organisms - A Review of Results
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image Studies of interspecific gene transfer, maintenance and expression in nature

Background and objectives

It is not known to what extent spontaneous gene transfer occurs in nature, nor the species limits to such transfer. This information is important for a realistic assessment of the effects of the release of genetically modified organisms (GMOs) into the environment. Given that plants and microbes share a microcosm, it is likely that transfer could occur between them. There are three principal questions which can provide information about this process. Firstly, is the gene(s) transferred? Secondly, is it expressed? And thirdly, is the expressed product, biologically active in its new environment?

The aim of this project was, to examine the potential transfer of DNA between rhizobial bacteria (common soil bacteria that form nitrogen-fixing root nodules in symbiotic association with compatible host legumes) which exist in close symbiotic association in the symbiotic plant organ designated root nodule.

Approach and methodology

We examined the transfer of the megaplasmid 2 between Rhizobium meliloti strains using the gentamicin acetyltransferase gene aacC1 as a marker. This gene was inserted in the megaplasmid 2 of R. meliloti via the transposon Tn5-Gmmob which additionally to the aacC1 gene provided the target for conjugation transfer factors. To examine gene transfer between two bacterial strains in the alfalfa root nodule, it is necessary to ensure that both strains are present inside this plant organ. Therefore, we employed complementary R. meliloti mutant strains which were either nodulation defective or infection defective, which ensured that functional nodules would only form if both mutant strains were present inside the nodule. In detail we analysed gene transfer in the infection threads of the root nodule during coinoculation with the two rhizobial mutants. To ensure optimal laboratory conditions for horizontal gene transfer we performed also filter matings with the two rhizobial strains.

Main findings and outcome

The inoculum containing both strains, filter matings and the inside of the nodules of coinoculated alfalfa were examined.

The lowest level of plasmid transfer occurred in the inoculum. Furthermore, it was noted that plasmid transfer induced by the broad host-range plasmid RP4-4 which provided the transfer factors for conjugation was highest in filter matings. Filter matings were used to provide optimal laboratory conditions for plasmid transfer. On the other hand, these experiments resulted in the observation that one of the two R. meliloti megaplasmids was able to self-transfer in filter matings without the help of RP4-4. The mobilisation of megaplasmid 2 induced by RP4-4 occurred at approximately the same frequency in filter matings and inside the nodules. RP4-4 independent self-mobilization of megaplasmid 2 was not detectable in the nodule.

These results indicate that the nodule creates conditions for plasmid transfer that are comparable to the optimal conditions created artificially in the laboratory.


This study showed that DNA can be transferred between rhizobial species within the root nodule where bacteria are in particularly close association. These findings highlight the need to closely monitor the potential biosafety risks associated with the transfer of foreign DNA between bacterial species and also between bacterial species and plants.


Major publication

Prätorius-Güth I.-M., Pühler A., Simon R., “Conjugal transfer of megaplasmid 2 between Rhizobium meliloti strains in alfalfa nodules”.
Appl Environ Microbiol, 56, 1990, pp. 2354-2359.
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Contract number

October 1988 – December 1990

J. Davies
Institut Pasteur
Paris (FR)

Follow-up of the project
This project was continued in EC project: BIOT-CT91-0282.



G. Freysinnet
Rhône-Poulenc Agrochimie
Lyon (FR)

A. Pühler
Universität Bielefeld (DE)

R. Guerrero
Universidad de Barcelona (ES)

A. Tsiftsoglou,
A. Tsaftsaris

Aristotle University of Thessaloniki (GR)

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