of genetically engineered microorganisms (GEMs) and genetically engineered
DNA Sequences (GEDs) in some environmental hot spots
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
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
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.
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.
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.
October 1991- September 1993
Laboratory of Genetics & Biotechnology (SCK/VITO)
of the project
This project was continued in EC project BIO2-CT92-0491.
University of Ghent (BE)
J. Figuereido Marques
Instituto de Biologia Experimental e Tecnologica
University of Wales
College of Cardiff (UK)