ISIS Report - May 4, 2001
Horizontal Gene Transfer Happens - II
The evidence for horizontal gene transfer is accumulating.
Mae-Wan Ho reviews a selection of recent scientific papers.
Survival of free DNA encoding antibiotic resistance from transgenic
maize and the transformation activity of DNA in ovine saliva, ovine rumen fluid
and silage effluent. Duggan PS, Chambers PA, Heritage J and Forbes JM.
FEMS Microbiology Letters 2000, 191, 71-7.
Insect-resistant maize line CG00526-176 contains three bacterial genes:
the cry1A(b) specific to lepidopterans, the bar gene conferring
tolerance to glufosinate, and a bla gene encoding TEM-1
b-lactamase (ampicillin resistance). The
bla gene originates from the cloning vector PUC18 and is not expressed
in maize, but has bacterial regulatory sequences that would allow it to become
functional were it to be transferred back into bacteria. There are at least two
copies of crylA(b) and bla genes integrated into the DNA of maize
line CG00526-176.
The researchers investigated the survival of DNA from transgenic maize
and the transfer of the antibiotic resistance bla gene to bacteria in
the presence of saliva, rumen fluid and silage effluent, which are relevant to
horizontal gene transfer in the oral cavity, the rumen, and in silage.
E. coli strain DH5a was the
test micro-organism for horizontal gene transfer. Degradation of DNA was
followed by gel electrophoresis as well as by polymerase chain reaction (PCR).
Both pUC18 plasmid and transgenic maize DNA were used in the experiments.
On gel electrophoresis, plasmid DNA and maize DNA were shown to be
degraded rapidly by rumen fluid or silage effluent within one minute, but both
were incompletely degraded after at least l h exposure to saliva.
On PCR analysis, large fragments of the bla gene (>350bp) were
still found in rumen fluid up to 30 mins for the plasmid and up to1 min for
maize DNA. Even larger fragments (>350 and >684 bp) from plasmid and
maize DNA were found up to 30 min incubation in silage effluent, and up to 24h
and 2 h respectively in saliva.
PCR analysis also showed that fragments of the cryl1A(b)
(>1914bp) in maize DNA could be found up to 1 min with rumen fluid, 5 min
with silage effluent, and 60 min with saliva.
Plasmid DNA exposed to saliva for 24h was still capable of transforming
E. coli to ampicillin resistance, but at low efficiency: 20 cfu (colony
forming units) per ml compared with 1.6 x103 cfu per ml after 24h in
sterile water. Previous exposure to rumen fluid for 30s reduced transformation
5-fold. No transformants were obtained after the plasmid DNA was exposed to
silage eflluent or rumen fluid for longer than 1 min.
However, when E. coli and plasmid were simultaneously added to
filter-sterilized silage effluent or rumen fluid, 4.75x103 cfu per
ml transformants were recovered after 4.5h in rumen fluid and 11cfu per ml were
recovered after 3h in silage effluent.
In summary, horizontal gene transfer can occur before the DNA is
completely broken down, even when the breakdown is rapid, as in the rumen or in
silage. DNA breakdown is extremely slow in saliva, and hence the oral cavity
will be a very important site for horizontal gene transfer.
The natural transformation of the soil bacteria Pseudomonas
stutzeri and Acinetobacter sp. by transgenic plant DNA strictly
depends on homologous sequences in the recipient cells. DeVries J, Meier P
and Wackernagel W. FEMS Microbiology Letters 2001, 195, 211-5.
The nptII gene in transgenic potato plants coding for kanamycin
resistance, transforms naturally competent cells of the soil bacteria
Pseudomonas stutzeri and Acinetobacter BD413 (both harboring a
plasmid with an nptII gene containing a small deletion (hence
nonfunctional) with the same high efficiency as nptII genes on plasmid
DNA ( 3x10-5 -1x10-4) despite the presence of a more than
106 fold excess of plant DNA. However, in the absence of homologous
sequences in the recipient cells the transformation dropped by at least about
108 fold in P. stutzeri and 109 fold in
Acinetobacter, below the detection limit.
More than 60 bacterial species have been shown to take up and
incorporate DNA (undergo transformation). Many bacteria like Bacillus
subtilis and Acinetobacter sp. strain BD413, apparently take up DNA
of any source into the cytoplasm. Stable maintenance and expression depends on
integration into the genome by genetic recombination.
The authors state, "This indicates a very low probability of
non-homologous DNA fragments to be integrated by illegitimate recombination
events during transformation". Should we be reassured? Not at all.
The high frequencies of homologous recombination obtained are
relevant to GM constructs released in large concentrations into the environment
in GM crops and transgenic wastes. GM constructs contain homologies to many
different species of bacteria and viruses, and are therefore capable of
engaging in high frequencies of recombination with a wide variety of bacteria
and viruses.
Illegitimate recombination events may occur at lower frequencies, but
they become substantial as GM constructs are released on massive scales. In
particular, recombination hotspots associated with many GM constructs (such as
those containing CaMV 35S promoter) may increase the frequency of illegitimate
recombination.
Effect of genomic location on horizontal transfer of a recombinant
gene cassette between Pseudomonas strains in the rhizosphere and
spermosphere of barley seedlings. Sengelov G, Kristensen KJ, Sorensen AH,
Kroer N, and Sorensen SJ. Current Microbiology 2001, 42, 160-7.
The rhizosphere - surfaces around the plant roots - and the
spermosphere - surfaces around the germinated seeds – are recognised
hotspots for horizontal gene transfer between bacteria. But the frequency of
horizontal transfer also depends on the location of the genes; whether in the
bacterial chromosome, or in a mobilizable plasmid, ie, a plasmid that can be
transferred with helper functions supplied by other plasmids, or in a
conjugative plasmid, ie, a plasmid that has its own functions for transfer
during conjugation (mating between bacterial cells). Not surprisingly,
researchers found the highest frequencies of horizontal gene transfer in both
the rhizosphere and spermosphere when the GM cassette was in a conjugative
plasmid, somewhat lower when it was in a mobilizable plasmid, but could not be
detected when it was inserted into the bacterial chromosome.
However, that does not mean GM constructs located on bacterial
chromosomes do not transfer. The authors were careful to point out that the
main mode of horizontal gene transfer in both the rhizosphere and the
spermosphere is conjugation. Elsewhere, transformation (by direct uptake of
DNA) will be more important, and there is evidence that chromosomal constructs
are more efficient in transformation.
The authors warn: "On the basis of these experiments, we cannot
rule out the possibility that horizontal gene transfer by transformation occurs
at low frequencies in soil and that this process might have significant effect
at field scale, which is an especially important point as regards risk
assessment. Such rare events cannot be studied in microcosm experiments, but
must be addressed in retrospective field studies."
The only retrospective study carried out has indeed found evidence of
horizontal gene transfer from transgenic plants to soil bacteria (see "Horizontal gene transfer
happens", ISIS News 5). An investigation yet to be done is horizontal
transfer from GM plants to bacteria in the rhizosphere.
Evidence for recent invasion of the medaka fish genome by the
Tol2 transposable element. Koga A, Shimada A, Shima, A, Sakaizumi, M,
Tachida H and Hori H. Genetics 2000, 155, 273-81.
Transposable elements are genetic units that can move from one
chromosome to another, with or without multiplying themselves. Tol2 is a
4.7 kbp element found in the genome of the medaka fish Oryzias latipes.
It has terminal inverted repeats and contains four genes similar to a group of
transposable elements found in fruitflly, maize and snapdragon. There are some
10 to 30 copies of Tol2 in the medaka genome that are highly homogeneous
in structure, and no variation in base sequence was found when 5 random clones
were examined. This is unusual, as transposable elements are typically
heterogeneous, with many defective copies being present in the same genome.
The genus Oryzias contains more than 10 species. The authors examined
10 species but Tol2 was found in only 2 of them: O. curvinotus
and O. latipes. The structure of the Tol2 is homogenous and
identical both within each species and between the two species, which are not
closely related and do not crossbreed in nature.
These results suggest very recent horizontal gene transfer. The
two species overlap in distribution probably somewhere in Southern China. Tol2
could have transferred from one species to another, or both species could have
acquired it from the same source. They also illustrate the dangers of using
transposable elements as gene transfer vectors.
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