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EC-sponsored Research on Safety of Genetically Modified Organisms - A Review of Results
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image Transgenic releases, analysis of gene flow and fluctuations in indigenous bacterial communities

Background and objectives

To assess the risks of releasing genetically modified (GM) bacteria into the environment, baseline studies should include the population structure of related indigenous strains, genetic interactions within such populations and with other bacteria, the maintenance of genes and the fluctuations in the genetic composition of populations that arise due to normal agricultural practice. Rhizobia are bacteria that survive in soil, colonise the rhizosphere, and form nitrogen-fixing root nodules in symbiotic association with compatible legumes. Being of biotechnological importance, and essential for developing sustainable agricultural systems, they are an excellent model for environmental risk assessments. In earlier studies, GM rhizobia designed to detect gene transfer were released in the field and Polymerase Chain Reaction (PCR) primers were developed to study the distribution of genetic elements in native strains. These molecular tools can be refined for more detailed examination of population stability and gene flux within rhizobia, between rhizobia and other bacteria, and with the introduced inoculants. Crop rotation has a major influence on soil microbes and its impact on these populations needs to be considered in such studies.

This project explores these issues in rhizobia, providing a context for studies on the potential for gene transfer between inoculant and native strains.

Approach and methodology

Molecular tools developed in a previous European Union project include PCR primers to amplify plasmid replication genes (rep) and insertion elements (IS) in rhizobial populations and bacteriophage genomes. One property of plasmids is transfer ability, but little is known about the genes involved in this (tra, oriT) in rhizobia. The mercury resistance gene mer, is commonly found in plasmids isolated from the environment. The distribution of these genetic elements will be assessed in rhizobia, including inoculant strains and other microbes, from sites where GM rhizobia were released. The impact of different agricultural practices (crop rotation, herbicide use, etc.) on the microbial population structure and the GM inoculants will be investigated using both cell culture and molecular methods.

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Main findings and outcome

An examination of IS showed that one Sinorhizobium IS was similar to a previously unrecognised IS in Eschericia coli. Three newly identified rhizobial IS form a group that can be invaded by a group II intron. This is important because these are ribozymes that can confer gene instability and may encode reverse transcriptase. Additional group II introns identified in different rhizobia, appeared to be truncated.

PCR primers designed from the tra genes amplified the oriT region in four Rhizobium strains. Sequences from two were identical but only a minority of plasmids screened, hybridised to oriT probes although they did hybridise to a well-characterised self-transmissible plasmid. New repC primers could identify the replication genes in a range of rhizobial species but sequence analysis indicated considerable divergence, with no obvious relationships between chromosomal background and plasmid at this scale. New repC sequences were obtained from at least eight different S. meliloti native isolates and from other rhizobia. Amplification of repC sequences from DNA extracted from soil microbial communities demonstrated their ubiquity and diversity in microbial populations, reflecting other results showing oriT and repC diversity in different groups of rhizobia. Some mer plasmids at the release site could transfer to rhizobia, but no natural transconjugants were isolated.

A temperate phage from the release site could lysogenise one release strain but no evidence for transduction was found, in contrast to a virulent phage isolated previously. Primer sets were designed to identify sequences arising from related phages. Analysis of 40 field isolates showed sequences that are similar to the virulent phage were present in 14% of isolates, with sequences from the temperate phage present at the same frequency but in different isolates. This indicates that phage infection is common in the field and confirms the potential importance of phages in horizontal gene transfer.

No clear crop or herbicide effects on numbers in different groups of culturable soil bacteria were seen. However, a GM rhizobial inoculant lacking its symbiotic plasmid, but which survived relatively well in bulk soil, was undetectable in the rhizosphere of its original host, the pea. In contrast, a symbiotically competent inoculant showed a 100-fold increase, indicating that this property confers a selective advantage in the host rhizosphere, although no evidence of symbiotic gene transfer to the GM inoculant was found.


A large diversity of genetic elements in rhizobial populations and evidence of genetic interactions between different genera, and with less closely related bacteria, was clearly shown. Thus, there is considerable genetic flux within populations in field soil, although the time scale is unclear. The presence of a host plant offers a selective advantage for symbiotically competent rhizobia and could be a ‘hot spot’ for gene transfer but the frequency is too low to determine with the methods available.


Major publications

Hirsch P.R., Mendum T.A., Pühler A., Selbitschka W., “The field release and monitoring of GUS-marked rhizobial strain CT0370”, in J.K. Jansson, J.D. van Elsas, M. Bailey (eds.), Tracking Genetically Engineered Micro-organisms: Method Development from Microcosms to the Field, R.G. Landes Co., Austin, Texas, USA, 2000, p. 145.

Schneiker S., Kosier B., Puhler A., Selbitschka W., “The Sinorhizobium meliloti insertion sequence (IS) element ISRm14 is related to a previously unrecognized IS element located adjacent to the Escherichia coli locus of enterocyte effacement (LEE) pathogenicity island.”
Current Microbiol, 39, 1999, p. 274.

Selbitschka W., Zekri S., Schroder G., Puhler A., Toro N., “The Sinorhizobium meliloti insertion sequence (IS) elements ISRm102F34-1/ISRm7 and ISRm220-13-5 belong to a new family of insertion sequence elements”.
FEMS Microbiol Letts, 172, 1999, p. 1.

Villadas P.J., Burgos P., Rodriguez Navarro D.N., Temprano F., Toro N., “Characterization of rhizobia homologues of Sinorhizobium meliloti insertion sequences ISRm3 and ISRm4”.
FEMS Microbiol Ecol, 25, 1998, p. 341.

Rigottier-Gois L., Turner S.L., Young J.P.W., Amarger N., “Distribution of repC plasmid-replication sequences among plasmids and isolates of Rhizobium leguminosarum bv. viciae from field populations”.
Microbiol UK, 144, 1998, p. 771.
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Contract number

September 1998 - August 2000

P. Hirsch
Harpenden (UK)

Project website address



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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