and modelling study of gene-mobilising capacity of soils and related ecosystems
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
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.
This project was a direct follow-on from EC project
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.
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.
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.
Götz A., Smalla K., Manure enhances plasmid mobilization
and survival of Pseudomonas putida introduced into field
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.
January 1993 - June 1996
J.D. van Elsas
Plant Research International (formerly IPO-DLO)
Laboratory of Genetics and Biotechnology (SCK/VITO)
University of Ghent (BE)
Federal Biological Research Centre for Agriculture and Forestry
Robert Koch Institut
J.C. Fry, M. Day
University of Wales
College of Cardiff (UK)
Natural Environment Research Council (NERC)