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Fate of Genetically Engineered Microorganisms (GEMs) and Genetically Engineered DNA Sequences (GEDs) in some environmental hot spots: polluted soils, river sediment, decaying and diseased plants; role of BHR plasmids and development of cont

Objective

The study of the behaviour and influence of GEM's in microbial environmental "hot spots" with special emphasis on the role of broad host range plasmids in gene dissemination and the development of bacterial containment systems.
Current objectives for the continuing project include;
monitoring of the fate genetically engineered microorganism (GEM's) and genetically engineered DS (GEDS) in potential environmental hot spots with special emphasis on the role of broad host range (BHR) plasmids;
study of the genetic transfer potential in the environmental hot spots using exogenous plasmid isolation;
development of a rapid method for total extraction of deoxyribonucleic acid (DNA) from soils;
development of bacterial containment systems.

Different GEM's and GEDS were constructed which will be used in the different microcosms to study gene escape. The GEM's contain marker genes based on heavy metal resistance and organic xenobiotioc degradation. Heavy metal resistance genes can be used as useful marker genes to study gene transfer form P putida and E coli to A eutrophus in microcosms as they allow the use of very selective media against donor and the natural microbial population. On the other hand in order to study the escape of genes from an introduced micro-organism to the indigenous micropopulation marker genes are needed which are expressed in a broad range of bacteria. Genes encoding resistance to mercury and encoding the meta cleavage of 2,3-dihydroxybiphenyl or catechol into an easily detectable yellow cleavage product were found to be expressed in a wide range of bacteria.

The transfer from E coli to A eutrophus of Mob{-}Tra{+} and Mob{-}Tra{-} plasmids carrying the czc genes encoding resistance to cobalt, cadmium, and zinc as marker genes was studied in unpolluted and heavy metal polluted soil microcosms as a model to study the risks associated with accidental release of GEM's. Special emphasis was put on the presence of broad host ranges (BHR) plasmids in the microcosms.

Several bacterial systems were evaulated for use as donor recipient pairs. A pair of E coli K12 strains, XL-2 and RB85 were chosen for further accidental release studies. Results suggest differences in spati al and temporal colonization patterns of the two strains. This may explain the low frequency of transfer over roots detected with this system. In future studies the E coli and P putida GEMS and gene escape system will be assessed in the plant-soil microcosms.

Experiments were done to isolate BHR plasmids from some of the proposed environmental hot spots such as River Taff epilithon and organic polluted soils and sludges by exogenous plasmid isolation. Plasmids were isolated from the river epilithon and from polluted soils and activated sludge.

To study the transfer of genes from released bacteria into the natural population and in particular to none-culturable bacteria. Total DNA extraction and the use of appropriate probes and polymerase chain reaction (PCR) may be a suitable method. A rapid method was developed to purify DNA from soils. The procedure takes no longer than 3 hours and the purified DNA can be used for DNA-DNA hybridization.

The presence of natural broad host range plasmids in the environment may represent an enhanced risk considering the escape of genes from introduced GEM's as they may be able to invade the GEM and pick up genes from it by means of retrotransfer. Therefore, the use of bacterial strains deficient in conjugation exchange may decrease the risk. A mutant of E coli ED8739 defective in conjugation with a P-type donor and a mutant of P aeruginosa impaired in survival were isolated and show promise for a possible containment system.

The presence of BHR plasmids and especially those provided with efficient transposons in a soil microflora influence the dissemination of genes cloned in pBR-vectors. This puts a limit on the safety of such vectors.
The behaviour of GEM's and GED's will be assessed in microcosms simulating environmental "hot spots". We define environmental "hot spots" as sites where gene transfer is likely to occur. The microcosms will simulate soils polluted with xenobiotics, river sediments, diseased plant material and decaying plant roots and plant-soil ecosystems. The survival, mobility of GEM's and the transfer of cloned GED's into an appropriate introduced recipient strain and into the indigenous population will be followed. The cloned GED's are genes which are only well expressed if they have been transferred into an appropriate host such as czc (coding for resistance to heavy metals). Special emphasis will be put on the role of broad host range plasmids (BHR) in gene transfer by introducing well known BHR plasmids to the microcosms.

Furthermore, we will assess the presence of natural transfer potential in the environmental hot spots by the exogenous isolation of plasmids from the environmental samples and their further characterization, by retrotransfer of non self transferable environmental plasmids and by DNA-DNA hybridization on total DNA extracted from the samples with appropriate probes, such as oriV and oriT of Incp1 and IncW plasmids.

The possible disturbance of the ecosystem after introduction of GEM's will be investigated by determining changes in different metabolic parameters in the samples, whereas the influence of indigenous organisms on the survival of the introduced GEM will be studied by selective elimination of populations of the natural microbial community.

Different bacterial containment systems will be constructed by construction of strains bearing mutations which affect physiological and genetic adaptation to chemical stress and toxic compounds and by construction of strains deficient in conjugation exchange.

Funding Scheme

CSC - Cost-sharing contracts

Coordinator

Vlaamse Instelling voor Technologisch Onderzoek
Address
200,Boeretang
2400 Mol
Belgium

Participants (3)

GENT UNIVERSITY
Belgium
Address
653,Coupure Links 653
9000 Gent
Instituto de Biologia Experimental e Tecnológica
Portugal
Address
12,Apartado
2780 Oeiras
University of Wales Cardiff
United Kingdom
Address

CF1 3TL Cardiff