Gene transfer of antibiotic resistance genes from genetically modified rhizobia to other bacteria

Topic

Using antibiotic resistance genes, the aim of the project was to investigate whether gene transfer is possible from transgenic micro-organisms to other bacteria.

The focus was on the following questions:

• Are antibiotic resistance genes transferred from transgenic rhizobia (Rhizobium meliloti) to other bacteria?

• Does the transfer take place directly (conjugation) or with the help of helper plasmids?

It is possible to produce new plasmids and transfer them to rhizobia using non-genetic methods (in-vivo recombination ). Another area to be investigated was the possible transfer of this kind of plasmid from rhizobia to other bacteria. (This is not, however, covered by the provisions of the genetic engineering law.)

Summary

When the plasmids were produced using genetic engineering methods, the resistance gene inserted into them was not transferred to other bacteria.

By contrast, it was possible to transfer a resistance plasmid that had been produced using in-vivo recombination to a range of bacteria.

Experiment description

Various plasmids were transferred to the rhizobia strains under investigation:

• Genetically engineered plasmids that did not themselves possess any genes for the gene transfer and contained an antibiotic resistance gene;

• An autotransferable, non-genetically modified plasmid with two antibiotic resistance genes.

Under laboratory conditions, the conjugation ability of the plasmids and plasmid transfer was investigated using helper plasmids. Transfer was analysed both within one bacterial species (E. coli ) and from rhizobia to other bacteria (Pseudomonas, Enterobacter and E. coli).

Results

Genetically engineered plasmids:

• The antibiotic resistance gene used in one of these plasmids (tetracyclin resistance) was not transferable either within the same species or to other bacteria. This plasmid therefore appeared suitable for release. However, the rhizobia strain with this plasmid did present altered characteristics (growth pattern, resistance) compared with the isogenic strain .

• The other genetically engineered plasmid could be transferred within the same species (E. coli) both with and without helper plasmids. It was not however, transferable to other types of bacteria. The plasmid was extremely unstable in the rhizobia strain used.

Non-genetically modified plasmid:

• By contrast, the non-GM plasmid was transferred from rhizobium to other groups of bacteria. With this plasmid too, transfer within a species was more frequent than between species.

• The probability of a dispersal of antibiotic resistance genes from the release of strains with this plasmid is higher than for the other two, genetically engineered plasmids.

Gene transfer was demonstrated under optimised laboratory conditions (bacteria density, nutrient supply) and can be applied to the environment only to a certain degree. The conditions for rhizobia in the root nodules are, however, similar to the laboratory conditions because of the high density of bacteria.