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EC-sponsored Research on Safety of Genetically Modified Organisms - A Review of Results
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image Risk assessment of releasing recombinant streptomycetes into the environment

Background and objectives

Genetically modified organisms (GMOs) are increasingly being studied in economically important crops. Bacteria are easily modified genetically and are also easy to produce in large quantities and are a major target of ongoing research into GMOs. However, the release of GM bacteria into the environment requires much knowledge about the possible spread of GM DNA into other bacteria found in situ. The major aim of this project was therefore to investigate the survival of a crippled bacterium, Streptomyces lividans TK64 541image2, in soil and determine if it was capable of transferring genes to other bacteria in situ.


Approach and methodology

In order to study gene transfer events, it was first necessary to develop methods for the extraction of DNA from soil and to test the performance of different types of soil microcosm for gene transfer. Exploiting a naturally occurring amplified DNA fragment on the chromosome allowed development of a novel method for transfer detection using a variety of marker genes. An unusual feature of Streptomyces species is their ability to spontaneously amplify specific chromosomal DNA sequences in the absence of selection, with a corresponding deletion. This property was exploited for transfer detection in soil using cloned resistance genes by linking them to the amplified sequence. Amplified DNA containing marker genes was introduced by transformation into S. lividans 66. This property of spontaneous amplification was exploited to detect the agarase gene dag, originally from S. coelicolor, which had been introduced into S. lividans TK64 541image2 by linking it to the amplified sequence.
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Main findings and outcome

Amplification facilitated detection of the dag gene in soil and was useful in other soil experiments as a biomarker as dag has so far not been detected occurring naturally in any soil. The dag phenotype resulted in degradation of agar, producing characteristic depressions around the colonies. Such depressions were observed even before the colonies were seen by the naked eye. This marker gene thus allowed both genotypic and phenotypic detection of an inoculant actinomycete. Detection limits were investigated for the dag marker and found to be in the region of 104 c.f.u. g-1 without PCR amplification of the DNA. In nonsterile soil the disabled strain TK64 541delta2 became difficult to specifically culture using the selective media containing thiostrepton and neomycin. Under these conditions, however, it was still possible to monitor growth and survival using the dag or tsr (thiostrepton resistance) genotypes. Under conditions of thiostrepton selection in soil, the crippled strain was able to survive but no transfer of the dag or thiostrepton resistance genes was detected.

Differential DNA extractions were used to locate where the gene was present in the soil community. For example, extractions omitting the cell lysis steps extracted extracellular DNA, while marked propagules such as spores, recalcitrant to SDS-heat lysis, were detected in DNA extracted by bead-beating only (Cresswell et al., 1991). The figure illustrates the detection of an amplified sequence in Streptomyces lividans which codes for 5.7 kb and has been used to amplify marker genes on the chromosome for commercial application as a heterologous host for enzyme production and other biological products. Uninoculated soil did not give a signal when probed with a plasmid pJOE777 containing the 5.7 kb of DNA, whilst the marker was detected in soil inoculated with S. lividans both in the spores and mycelium growing or surviving in the soil. Extracellular marker was only detected at day 0, immediately after inoculation, when some lysis would have occurred. Free DNA was probably rapidly degraded after this time. The figure also shows that cleaner DNA was recovered by bead-beating compared to the SDS-lysis procedure which tends to extract more phenolic components from the soil.

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Conclusions

The use of molecular monitoring has greatly facilitated ecological studies of actinomycetes in soil and allowed detection of populations containing specific genes. The monitoring directly of gene expression in soil is still problematic although some limited studies have been done using transcriptional fusions to reporter genes. In situ hybridisations using 16S rRNA probes targeting ribosomes did indicate active mycelium of Streptomyces species in soil as the fluorescence was related to ribosome number. It is clear that under appropriate conditions of selection crippled strains can survive in soil.


Major publications

Wellington E.M.H., Cresswell N. and Saunders V.A., “Growth and survival of streptomycete inoculants and the extent of plasmid transfer in sterile and non-sterile soil”.
Appl. Environ. Microbiol.,
56, 1990, pp. 1413-1419.

Clewlow L.J., Cresswell N. and Wellington E.M.H., “A mathematical model of plasmid transfer between strains of streptomycetes in soil microcosms”.
Appl. Environ. Microbiol.,
56, 1990, pp. 3139-3145.

Wellington E.M.H., “Gene transfer in the natural environment?”.
Biotechnology Educ.,
1, 1990, pp. 133-135.

Cresswell N., Saunders V.A. and Wellington E.M.H., “Detection and quantification of Streptomyces violaceolatus plasmid DNA in soil”.
Lett. Appl. Microbiol., 13, 1991, pp. 193-197.
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imageResearch project
 

Contract number
BAP-0370/0378

Period
October 1989 – September 1991

Coordinator
E.M.H. Wellington
University of Warwick
Coventry (UK)

Follow-up of the project
This project was continued in EC project: BIOT-CT91-0285: The effects of selection on gene stability and transfer in populations of bacteria in soil.

 
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Partner


J. Cullum
LB Genetik
Universität Kaiserslautern (DE)

 
 
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