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EC-sponsored Research on Safety of Genetically Modified Organisms - A Review of Results
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image Acquisition of genes from indigenous bacteria by inoculant strains at long-term release sites

Background and objectives

Bacteria used as agricultural inoculants can be improved by genetic manipulation. This includes modifications to minimise the transfer of novel genes to other microbes in the environment. Nevertheless, indigenous bacteria can transfer their DNA to inoculants, resulting in new, potentially undesirable gene combinations. Rhizobia, bacteria that form nitrogen-fixing root nodules in symbiotic association with compatible host legumes, are the most important plant inoculants in Europe and world-wide. They are potential targets for genetic modifications, and can be used as a model for modifying other soil bacteria. There are several mechanisms by which bacteria can exchange genetic material, and rhizobia are known to carry forms of DNA (conjugative plasmids and bacteriophages) that can transfer between strains, often conferring significant novel properties. For example, in many rhizobial genera, symbiotic genes are known to occur on large plasmids. Plasmids and bacteriophages may also mediate the transfer of other plasmids, genes and transposable insertion (IS) elements.

This project investigated gene acquisition by rhizobial inoculants from native bacteria in the field, to provide baseline information relevant to biosafety risk assessments.

Planting peas inoculated   Planting peas inoculated
with GM rhizobia.
Monitoring GUS- marked rhizobia.   Monitoring GUS-
marked rhizobia.

Approach and methodology

Genetic elements including plasmid replication origins, IS elements and bacteriophages, in naturally-occurring rhizobial populations were isolated and characterised. This facilitated the design of molecular tools for detection of genetic elements, collection of information on their distribution within populations, and the search for evidence of transfer to inoculant strains. In the course of the project, rhizobial inoculants designed to act as acceptors of indigenous genetic elements, were released. These strains were re-isolated from the field together with established, previously-introduced rhizobial populations, and screened for the acquisition of genetic elements present in the native rhizobial population.

Main findings and outcome

Plasmid replication origins from Rhizobium leguminosarum and Sinorhizobium meliloti were cloned and sequenced to design PCR primers to amplify repC from field isolates. Four repC families were found in R. leguminosarum isolates from Dijon, Bielefeld, Erlangen and Rothamsted. Two repC groups were identified in S. meliloti, one was widespread and was also found in R. tropici.

Primers were designed to amplify IS elements from field isolates, and the distribution of the various elements was assessed in inoculant strains and field populations. One R. leguminosarum IS isolate from Dijon was identical to an independently isolated IS from Bielefeld. Similar sequences were detected in IS from R. leguminosarum and S. meliloti, and using primers for two S. meliloti IS, amplified related elements were detected in R. leguminosarum. Five completely new S. meliloti IS were found, one with 90% sequence similarity to a Methylobacterium IS.

The majority of R. leguminosarum field isolates were lysogenic, and bacteriophages were isolated from soil. A virulent phage isolated from a field release site could transduce genes in one inoculant strain.

To measure gene acquisition in the field, an R. leguminosarum strain that had lost its symbiotic plasmid, had a gusA marker gene inserted in its chromosome. This strain, CT0370, could form nodules only after acquiring a new symbiotic plasmid, and the nodules could be readily identified because of their gusA expression. After field release at Rothamsted, > 20,000 nodules were screened for gusA but no expression was found. In addition, 1000 CT0370 re-isolates were screened after two years, for the acquisition of repC groups I and II using PCR, but all were negative. Similarly, 500 colonies of a previously-released inoculant strain re-isolated after 8 years, were screened for ISRm3 but it was not detected. In Dijon, an estimated 125,000 T2 inoculant bacteria released 7 years previously, were screened for IS and repC. One re-isolate was repCII-positive and appeared to have a small plasmid that was lost with subculture, so it could not be verified. In Granada, 27,000 nodules formed by an S. fredii inoculant were screened with primers for IS which are present only in the native population, but expression was not detected in the inoculant.

Thus, fewer than 4x10-5S. fredii acquired an IS element. The frequency of transfer of the symbiotic plasmid CT0370 was less than 5x10-5 per donor, and the Dijon results indicate a possible frequency of 8x10-6 per T2 recipient.


Genetic elements were found to be ubiquitous in rhizobial populations. Related elements were present in isolates from soils throughout Europe and in different genera, providing circumstantial evidence that transfer occurs in the field, although quantitative data obtained over the relatively short time periods studied was mostly negative. This has major ramifications for the safety of introducing genetically modified organisms (GMOs) into the environment. Another important outcome of this research was the development of a means of identifying various genetic elements, providing a molecular toolbox for future investigations.


Major publications

Villadas P.J., Burgos P., Jording D., Selbitschka W., Puhler A., Toro N., “Comparative analysis of the genetic structure of a Rhizobium meliloti field population before and after environmental release of the highly competitive R. meliloti strain GR4”.
FEMS Microbiol Ecol, 21, 1996, p. 37.

Turner S.L., Rigottier-Gois L., Power R.S., Amarger N., Young J.P.W., “Diversity of repC plasmid replication origins in Rhizobium leguminosarum”.
Microbiol UK, 142, 1996, p. 1705.

Mazurier S.I., Rigottier-Gois L., Amarger N., “Characterization, distribution, and localization of ISRl2, an insertion sequence element isolated from Rhizobium leguminosarum bv viciae”.
Appl Env Microbiol, 62, 1996, p. 685.

Hirsch P.R., “Population dynamics of indigenous and genetically modified rhizobia in the field”.
New Phytol, 133, 1996, p. 159.

Selbitschka W., Jording D., Nieman S., Schmidt R., Puhler A., Mendum T. & Hirsch P., “Construction and characterization of a Rhizobium leguminosarum biovar viciae strain designed to assess horizontal gene transfer in the environment”.
FEMS Microbiol Lett, 128, 1995, p. 255.
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imageResearch project
Contract number

January 1992 - March 1996

P. Hirsch
Harpenden (UK)


P. Young
University of York (UK)

N. Amarger
INRA - Dijon (FR)

N. Toro
Estación Experimental del Zaidín
Granada (ES)

W. Lotz
Universität Erlangen-Nürnberg
Erlangen (DE)

A. Pühler,
W. Selbitschka

Universität Bielefeld (DE)

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