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EC-sponsored Research on Safety of Genetically Modified Organisms - A Review of Results
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image Ecological and modelling study of gene-mobilising capacity of soils and related ecosystems

Background and objectives (1)

The impact of gene mobilising elements (GMEs; DNA plasmids with mobilising capacity) on the potential of microbes in the environment to mobilise 'foreign' DNA is poorly understood. This is of particular importance when considering the biosafety of DNA plasmids and conjugative DNA transposons of genetically modified organisms (GMOs). In particular, there is a lack of understanding of the types of GMEs prevalent in the environment, and their in situ functioning. This project aimed to provide a better understanding of the gene mobilising capacity of polluted (e.g. with heavy metals or xenobiotic organics) and unpolluted soils, the wheat and sugar beet rhizosphere (the environment surrounding the plant root), manure, and the phytosphere (the habitat influenced by the plant as a whole) of sugar beet.

The objectives of this project were firstly, to assess the prevalence of genetic elements conferring gene mobilising capacity in selected soil and soil-related habitats. Secondly, to assess the impact of stresses imposed on these habitats, on the gene mobilising capacity of their microbial communities. Finally, to assess the fate of genetic elements following their release into soil ecosystems.

image Manured field.

(1) This project was a direct follow-on from EC project
BIOT-CT91-0284.


Approach and methodology

Endogenous, and both biparental and triparental exogenous plasmid isolation methods were used in order to obtain a representative sample of plasmids prevalent in the selected ecosystems. These plasmids were then characterised with respect to plasmid type and gene mobilising capacity, using a suite of microbiological and molecular methods. In the final stage of the project, the in situ role of selected plasmids in mobilising incoming DNA was tested in specific ecosystems, i.e. in wheat and sugar beet rhizospheres and in manured fields.
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Main findings and outcome

A diverse range of different GMEs was obtained from each of the ecosystems under study. Both the direct exogenous isolation method using mercury or antibiotic resistance as the selective marker, and the triparental exogenous isolation method, were shown to work well for Gram negative bacteria, providing a variety of novel plasmids for these hosts. The plasmid types found were strongly dependent on the isolation methods used. Thus, the biparental exogenous method generally yielded different plasmids than the triparental method, even when the same sample was analysed. Plasmid identification methods, including replicon typing using available rep/inc probes, polymerase chain reaction (PCR)-assisted assessment of replicon type, restriction typing and probing, as well as the host range of self-transfer and mobilisation, enabled classification of the plasmids into separate groups. Many of the plasmids with gene mobilising capacity were novel, and several seemed to belong to hitherto unknown plasmid incompatibility groups. A number of plasmids were tested for their retromobilisation capacity, and some with extreme retromobilisation capability were found.

As a corollary of this study, a suite of new primers and probes for plasmid recognition were developed, enabling improved plasmid detection, notably of plasmids of the broad host range Inc groups (i.e. the IncP, IncQ, IncN and IncW groups). A range of such plasmids was subsequently found in all soil-related systems, notably in manure.

One GMO, the bacteria Pseudomonas fluorescens SBW25, previously used in test releases in the United Kingdom, was shown to acquire plasmids when it was present in the sugar beet phytosphere in the field, without the occurrence of any apparent selective pressure. Plasmid acquisition was dependent on the developmental stage of the plant, indicating that an ecological triggering event is involved in this process. Moreover, carriage of some of these plasmids affected host fitness, depending on plant growth. These observations point to a temporary plasmid transfer activity as well as a plasmid role in the sugar beet phytosphere.

Testing of gene mobilisation in the wheat rhizosphere as well as in manured soil in the field, indicated that transfer of IncQ elements occurred in both habitats. In particular, soil with recent manure input, was shown to be conducive to gene transfer activity. Finally, the imposition of stress (mercury) on soil in microcosms, resulted in the enhancement of the prevalence of plasmids with gene mobilising capacity, thus indicating that soils under stress may be more conducive to mobilisation processes than unstressed soils.

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Conclusions

Soil and soil-related habitats commonly contain bacterial populations that have gene mobilising capacities conferred by a diverse range of genetic elements. Some of the GMEs identified were apparently novel, and some had extreme mobilisation and retromobilisation capacities. The sugar beet and wheat rhizospheres, as well as recently manured soil, represent habitats in which gene transfer processes are facilitated. Stress imposed on soil systems may lead to an enhancement of the prevalence of GMEs. The results of these studies provided important information that will be useful for assessing the safety of GMOs in agricultural applications.


Major publications

Götz A., Smalla K., “Manure enhances plasmid mobilization and survival of Pseudomonas putida introduced into field soil”.
Appl Environ Microbiol, 63, 1997, pp. 1980-1986.

Lilley A.K., Bailey M.J., “The acquisition of indigenous plasmids by a genetically marked pseudomonad population colonizing the sugar beet phytosphere is related to local environmental conditions”.
Appl Environ Microbiol,
63, 1997, pp. 1577-1583.

Smit E., Wolters A., Van Elsas J.D., “Self-transmissible plasmids with gene-mobilizing capacity in soil bacterial populations: influence of wheat roots and mercury addition”.
Appl Environ Microbiol,
64, 1998, pp. 1210-1219.

Top E.M., De Rore H., Collard J.M., Gellens V., Slobodkina G., Verstraete W., Mergeay M., “Retromobilization of heavy metal resistance genes in unpolluted and heavy metal polluted soil”.
FEMS Microbiol Ecol,
18, 1995, pp. 191-203.

Van Elsas J.D., McSpadden-Gardener B.B., Wolter A.C., Smit E., “Isolation, characterization, and transfer of cryptic gene-mobilizing plasmids in the wheat rhizosphere”.
Appl Environ Microbiol,
64, 1998, pp. 880-889.
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imageResearch project
 

Contract number
BIO2-CT92-0491

Period
January 1993 - June 1996

Coordinator
J.D. van Elsas
Plant Research International (formerly IPO-DLO)
Wageningen (NL)

 
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Partners


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

E. Top
University of Ghent (BE)

K. Smalla
Federal Biological Research Centre for Agriculture and Forestry
Braunschweig (DE)

H. Tschäpe
Robert Koch Institut
Wernigerode (DE)

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

M.J. Bailey
Natural Environment Research Council (NERC)
Oxford (UK)

 
 
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