Risk analysis of the release of genetically modified, recombination-defective rhizobia

(1988 - 1995) Bielefeld University, Faculty of Biology, Chair of Genetics

Topic

The aim of the project was to test the suitability of recombination -defective Rhizobium strains (recA-) in the model ecosystem as a “biological safety measure” for releases.

The rhizobia under investigation (Rhizobium meliloti) are nodule bacteria that live in symbiosis with alfalfa and fix the nitrogen in the air by converting it to a form that the plants can utilise.

To what extent is the ability to perform homologous recombination necessary for the survival of bacteria?
Are foreign genes stable in recA- strains?
What effect does the modification have on the horizontal gene transfer between Rhizobium strains?
To what extent do the recA- strains spread in the soil?
How is the native Rhizobium population affected by genetically modified rhizobia?

The recombination-defective Rhizobium strains (recA-) are compared with "normal" strains (recA+).

Summary

Survival of the recA- strain. The recA- strain was less capable of surviving than the recA+ strain. The survival of both strains was positively influenced by the presence of the host plant, alfalfa.

Stability of the foreign genes. The foreign genes were equally stable in both strains.

Horizontal gene transfer. The recA- strain showed a clearly reduced transfer to other strains and was unable to take up DNA itself by homologous recombination.

Distribution in the soil. The luciferase gene used as a marker (luc; reporter gene ) was not detected either free or in possibly non-cultivable cells of the two strains.

Influence on the native Rhizobium population. The recA- strain was able to compete with individual wild type isolates in the microcosm despite being defective. However, the native Rhizobium population reduced the survival of both strains.

Since containment of recombination-defective strains is determined by the prevailing conditions, the recA- strain cannot be used as a standard biological safety measure.

Experiment description and Results

Two isogenic Rhizobium meliloti strains were genetically produced which carry a marker gene (luc) either in the recombinase gene (recA-) or next to it (recA+).

Survival of the recA- strain

The two strains were incubated in competition with their parent strain in microcosms (sealed containers with soil). The survival of the strains was studied in both sterile and non-sterile agricultural soils.

The influence of the host plant (alfalfa) and two non-host plants (clover and wheat) on the survival of the two strains was also investigated.

The effect of temperature was recorded at –20 °C and at 50 °C (16 hours).

The recA- strain was less competitive than its parent strain. The recA+ strain did not differ from the parent strain. In many of the non-sterile soils the recA- strain declined more quickly than the recA+ strain. By contrast, in the other soil types there was scarcely any difference in the survival of the two transgenic strains.

Alfalfa encouraged the growth of both strains, whereas clover and wheat had no effect on the survival of the strains.

Low temperatures (–20°C) had no effect on survival. Heat treatment, on the other hand, resulted in a clear reduction in both strains. The recA- strain declined more rapidly than the recA+ strain. Both strains recovered after ten days.

Stability of the foreign genes

To investigate the stability of the foreign genes, the strains were placed in the soil with alfalfa plants and the luciferase activity was then analysed by PCR .

Both foreign genes proved to be stable in the soil and also after passing into the nodules. There was no difference between the two strains.

Horizontal gene transfer

The transfer of an antibiotic resistance gene between R. meliloti strains that colonise the same root nodule was studied.

In addition, work was conducted on a strain pair that carries a blue marker gene (gusA), unlike the released strains that have a luminescent marker. The transfer of a plasmid with a broad host range from this strain pair to an antibiotic-resistant R. meliloti strain in the soil was examined.

The transfer of homologous DNA to the recA- strain was studied by inoculating the soil with this strain together with an E. coli strain which carries DNA from the Rhizobium wild type strain.

Transfer within the root nodules could only be detected when the resistance gene was located on a plasmid, but not when it was integrated in the chromosome .

Transfer in the soil occurred only when a culture medium was added. Then the transfer from the recA- strain was approximately ten times less than from the recA+ strain.

As anticipated, no integration took place in the recA- strain due to the defective homologous recombination. In the recA+ strain a very low rate of transfer occurred.

Distribution in the soil

Distribution in the soil was assessed according to the presence of the luc gene (PCR) and the cell count of the two strains.

The luc gene was not detected in the soil either as extra-cellular DNA or in non-cultivable cells.

Influence on the native (endogenous) Rhizobium population

Wild type strains of an endogenous Rhizobium population were initially isolated from alfalfa root nodules and also from soil samples. Twenty strains were characterised using various molecular methods.

The influence of the transgenic strains on a reconstructed Rhizobium population was studied using three of the wild type strains, which were cultivated with the two strains in microcosms together with alfalfa plants. After six weeks the strains were extracted from the root nodules and identified by PCR analysis. Microcosms that had only been inoculated with the wild type strains were used as controls.

The survival of both strains was impaired by the endogenous Rhizobium population in the soil.

With the artificial Rhizobium population the recA+ strain was dominant. The recA- strain was, however, clearly less competitive. Although individually it lost out to one of the wild type strains, it was surprisingly able to assert itself over the other two wild type strains.