of interspecific gene transfer, maintenance and expression in nature
It is not known to what extent spontaneous gene transfer occurs in nature,
nor the species limits to such transfer. This information is important
for a realistic assessment of the effects of the release of genetically
modified organisms (GMOs) into the environment. Given that plants and
microbes share a microcosm, it is likely that transfer could occur between
them. There are three principal questions which can provide information
about this process. Firstly, is the gene(s) transferred? Secondly, is
it expressed? And thirdly, is the expressed product, biologically active
in its new environment?
The aim of this project was, to examine the potential transfer of DNA
between rhizobial bacteria (common soil bacteria that form nitrogen-fixing
root nodules in symbiotic association with compatible host legumes) which
exist in close symbiotic association in the symbiotic plant organ designated
Approach and methodology
We examined the transfer of the megaplasmid 2 between Rhizobium meliloti
strains using the gentamicin acetyltransferase gene aacC1 as a
marker. This gene was inserted in the megaplasmid 2 of R. meliloti
via the transposon Tn5-Gmmob which additionally to the aacC1
gene provided the target for conjugation transfer factors. To examine
gene transfer between two bacterial strains in the alfalfa root nodule,
it is necessary to ensure that both strains are present inside this plant
organ. Therefore, we employed complementary R. meliloti mutant
strains which were either nodulation defective or infection defective,
which ensured that functional nodules would only form if both mutant strains
were present inside the nodule. In detail we analysed gene transfer in
the infection threads of the root nodule during coinoculation with the
two rhizobial mutants. To ensure optimal laboratory conditions for horizontal
gene transfer we performed also filter matings with the two rhizobial
Main findings and outcome
The inoculum containing both strains, filter matings and the inside of
the nodules of coinoculated alfalfa were examined.
The lowest level of plasmid transfer occurred in the inoculum. Furthermore,
it was noted that plasmid transfer induced by the broad host-range plasmid
RP4-4 which provided the transfer factors for conjugation was highest
in filter matings. Filter matings were used to provide optimal laboratory
conditions for plasmid transfer. On the other hand, these experiments
resulted in the observation that one of the two R. meliloti megaplasmids
was able to self-transfer in filter matings without the help of RP4-4.
The mobilisation of megaplasmid 2 induced by RP4-4 occurred at approximately
the same frequency in filter matings and inside the nodules. RP4-4 independent
self-mobilization of megaplasmid 2 was not detectable in the nodule.
These results indicate that the nodule creates conditions for plasmid
transfer that are comparable to the optimal conditions created artificially
in the laboratory.
This study showed that DNA can be transferred between rhizobial species
within the root nodule where bacteria are in particularly close association.
These findings highlight the need to closely monitor the potential biosafety
risks associated with the transfer of foreign DNA between bacterial species
and also between bacterial species and plants.
Prätorius-Güth I.-M., Pühler A., Simon R., Conjugal
transfer of megaplasmid 2 between Rhizobium meliloti strains
in alfalfa nodules.
Appl Environ Microbiol, 56, 1990, pp. 2354-2359.
October 1988 December 1990
of the project
This project was continued in EC project: BIOT-CT91-0282.
Universität Bielefeld (DE)
Universidad de Barcelona (ES)
Aristotle University of Thessaloniki (GR)