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EC-sponsored Research on Safety of Genetically Modified Organisms - A Review of Results
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Novel tools and techniques
to track GMOs


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

To exploit the full potential of genetically modified micro-organisms (GMOs) and to assess adequately the potential impacts on the environment of their applications, an improved base-line knowledge of microbial ecology is still required. In particular, the facts that only a small proportion of bacteria are readily accessible through standard cultivation techniques, and that bacterial cells can lose the ability to grow on solid media as a response to environmental stress, complicate assessment of the environmental fate and performance of GMOs. To overcome this cultivation bias, novel tools and techniques need to be developed. The EU-supported projects which are summarised in this chapter have contributed considerably to rapid progress in developing such tools and techniques in order to provide the necessary new insights into microbial diversity and the methodology for monitoring the fate of environmentally released GMOs and their potential effects on microbial communities.

Since clear phylogenetic identification and classification of bacterial or fungal hosts subjected to genetic modification is a crucial aspect of the information required for safety evaluation, according to the Directive 90/220/EEC, the projects BIOT-CT91-0294 and BIO2-CT94-3098 aimed at developing rapid, reliable and inexpensive techniques to identify micro-organisms. Many of the techniques which have been developed by these projects have now become routine and are widely applied for the characterisation and identification of environmental isolates. In particular, the use of the 16S rRNA, based on cloning of 16S rDNA fragments amplified from directly extracted nucleic acid genes as a molecular marker, is now an established method for determining phylogenetic relationships and for analysing ecosystems. The development of an automated and highly sensitive software programme for probe design and assignment of newly determined 16S sequences into the phylogenetic tree is also a major achievement. This publicly available database is of great use to microbial ecologists. In addition, DNA-based techniques have been developed for the detection and rapid identification of biotechnologically or medically important fungi (BIO-CT91-0301 and BIO2-CT94-3011).

The potential of marker and reporter genes, which enable sensitive and specific monitoring of GMOs and their DNA in different environmental conditions, has been demonstrated for examining the fate of GMOs released into the environment in a series of projects (BAP-0141/0369, BAP-0040/0361/0419 and BIO4-CT96-0434). In addition, combining advanced microscopic techniques with reporter genes or fluorescently labelled probes has facilitated in situ analysis of microbes. As a result, it is now possible to gain information on the population structure, compartmentalisation of bacterial communities, specific gene expression and transfer, without disturbing the complex interactions that exist, for example, within a biofilm. Reporter gene fusions have been extremely valuable for the analysis of gene expression in rhizobacteria in response to the presence of fungal pathogens (BIO2-CT94-3001). The concerted action MAREP (BIO4-CT96-0434) greatly facilitated information and technology exchange between laboratories having expertise with different marker and reporter genes. Marker genes were in general considered as safe for use in the laboratory or under field conditions. However, it was recommended that the use of genes encoding resistance to clinically relevant antibiotics in the environment should be avoided.

A better understanding of horizontal gene flux as a natural phenomenon and as a feature of microbial flexibility and adaptability in response to changing environmental conditions, and its implication for the biosafety evaluations of GMOs, was obtained through the projects (BAP 0141/0369, BAP 0383/0421/0485, BAP 0370/0378, BIOT-CT91-0285, BIOT-CT98-0287 and BIO2-CT92-0491). New approaches and molecular tools have opened up a new dimension in our ability to investigate the diversity and distribution of the horizontal gene pool. Thus it could be demonstrated that soils and soil-related habitats commonly contain bacteria that have a gene mobilising capacity (BIO2-CT92-0491). For the first time, in situ acquisition of mercury resistance plasmids by GMOs colonising the phytosphere of sugar beets and the mobilisation of non-self-transferable plasmids by indigenous bacteria in freshly manured soil was detected.

New approaches and molecular tools were developed and applied to survey the reservoirs of antibiotic resistance genes and mobile genetic elements in different environments (BIO2-CT92-0491, BIO4-CT98-0053, BIO4-CT98-0099 and BIO4-CT98-0424) and their distribution in response to selective pressure. The antibiotic resistance genes surveyed were found in all environments analysed and were often located on mobile genetic elements. Hot spots of antibiotic resistance genes and mobile genetic elements could be identified. The flow of antibiotic resistance genes, aided by mobile genetic elements, between different environmental compartments and the role of selective pressure is not only important for improving management of the serious problems raised by antibiotic resistant pathogens but also for predicting the fate of introduced transgenic DNA. In the concerted action MECBAD (BIO4-CT98-0099), groups working on the molecular biology and ecology of mobile genetic elements link their expertise to improve the basic knowledge of the environmental factors which stimulate or limit the maintenance and transfer of mobile genetic elements under environmental conditions, to understand better gene acquisition and spread of novel traits, and to provide new tools for the biotechnological exploitation of mobile genetic elements. The baseline data generated through these EU-supported projects has thus provided important information, particularly for biosafety evaluation of antibiotic resistance genes used as markers in transgenic plants, on the fate of transgenic DNA and on the role of selective pressure.

During the past decade the application of new methods to study microbial communities has greatly improved our ability to analyse microbial communities, and important base-line data has become available. At the same time it has become evident how little we know about the structural and functional diversity of microbial communities. More and more bacteria are recognised as multicellular organisms, and intercellular communication has become one of the fields in microbial ecology attracting a lot of attention and revolutionising our view of how bacteria operate. Thus, to study microbes in complex microbial communities adequately remains a challenge to microbial ecologists and will require further efforts and support.

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