assessment of releasing recombinant streptomycetes into the environment
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 5412,
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 5412
by linking it to the amplified sequence.
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 5412
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.
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.
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
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.
October 1989 September 1991
University of Warwick
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.
Universität Kaiserslautern (DE)