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[SANET-MG] field testing GM microbes
http://www.i-sis.org.uk/fieldTest.phpISIS Press Release 29/08/05
University researchers have applied to the US Environment Protection
Agency to field test bacterial pathogens and fungus living inside plants
carrying antibiotic resistance marker genes. These should be strenuously
contested.
Organic growers should consider making an objection to the field testing
of GM microbes. Such microbes certainly pose a threat to organic
producers. There is still time to comment to USDA/APHIS and those public
comments may be anonymous.
Field-testing Bacterial Pathogens with Antibiotic Resistance Genes
Prof. Joe Cummins objects on behalf of Independent Science Panel, so
should you
This article has been submitted to the US EPA on behalf of the
Independent Science Pane (identifier: APHIS-2005-0069-0005). Please add
your support by registering your opposition in the docket and referring
to this article and identifer number.
The University of Wisconsin is preparing to release genetically modified
(GM) Erwinia carotovora bacteria containing antibiotic resistance marker
genes in environmental field tests. The United States department of
Agriculture (USDA) Animal and Plant Health Inspection Service (APHIS)
has prepared a docket with background information on the release and
there is a public comment period ending 15 September 2005 [1]. The tests
are to be conducted at the University of Wisconsin Experiment Station
near Hancock, Wisconsin. The environmental release of bacterial strains
each containing a different antibiotic should be a matter of grave
concern and a strong public response is in order.
The purpose of the field trial seems to be simply experimental with no
potential commercial application of the genetically modified bacteria.
Erwinia carotovora causes a rotting disease in potatoes. The experiment
is designed to test the fitness of bacteria that have genes known to be
associated with pathogenesis disrupted by inserting into those genes the
antibiotic resistance genes. The antibiotic resistance genes not only
eliminate the function of the pathogenesis genes but also serve as
selectable markers for the modified bacteria. The antibiotic resistance
genes are precisely inserted into the target pathogenesis gene by adding
short DNA tails to the resistance genes that are homologous to the
target pathogenesis gene. The pathogenesis gene targeted is hrp, which
controls secretion of virulence proteins into the host plant. Green
fluorescence protein was inserted as a visual marker to identify
potatoes with the modified bacteria [2].
The field test involves four GM mutant strains, WPP60,WPP198, WPP195 and
WPP40. WPP60 was created by inserting the streptomycin/spectomycin genes
into the hrpC, while WPP98 had chloramphenicol resistance gene inserted
into hrpL. WPP195 was created by deleting the hrpN gene and inserting
the choramphenicol resistance gene in its place. WPP40 has the kanamycin
resistance gene inserted into the outD gene required for protein
secretion, so it cannot secrete plant cell-wall degrading enzymes.
Inserting antibiotic resistance genes into the hrp genes interferes with
pathogenesis. However, only strain WPP40 that inactivates the outD gene
is reduced in virulence in a direct assay, nevertheless, the researchers
decided to carry out the field experiment [2].
They claim that the field tests are not expected to impact agricultural
practice because “the test is solely for research purposes” even though
Erwinia carotovora affects potato, cucumber, capsicum, turnip, brussel
sprouts, carrots and celery, all of which are grown in the general area
of the test site. Because the antibiotic resistance genes are not being
selected by antibiotic application, they are not deemed to be a cause
for concern, according to the applicants. The proposal believes that the
antibiotic resistance genes cannot be transferred to bacteria affecting
humans; justifying that position by referring to the US Food and Drug
Administration’s judgment that antibiotic resistance is already
prevalent in bacteria affecting humans and in soil-borne bacteria. Even
though the soil is sandy and porous in the test site, the applicants
claim that the spread of the GM bacteria is not expected, nor has it
been tested for [2]. In general, the application focuses on the benefits
of the experiments and ignores any evidence that might delay the
experiment. Apart from the release of antibiotic resistance genes, the
most obvious question is why release so many strains that are
genetically modified as well as still virulent? That procedure provides
high risk with little or no benefit.
The claim in the proposal that GM Erwinia carotovora would not transfer
antibiotic resistance genes to other soil bacteria, is inconsistent with
the scientific literature. Horizontal gene transfer is commonplace in
Erwinia and Klebsiella and transfer could extend to Salmonella, Shigella
and E. coli [3]. The GM bacteria contaminating surfaces of food crops
may also transfer antibiotic resistance gene to enteric bacteria when
ingested by humans or animals. Bacteria phage were found to transfer
genes between Erwinia species [4]. Transposons capable of mobilizing
antibiotic resistance genes have been identified in Erwinia carotovora
[5]. There is thus clear evidence that Erwinia is capable of moving
resistance genes among soil Erwinia species, between other soil
bacterial species and among enteric bacteria.
A number of GM microbes bearing antibiotic resistance markers have been
released commercially in the United States, these include Sinorhizobium,
Agrobacterium and Psuedomonas [6]. Little effort appears to have been
made to monitor the consequences of these releases. Mae-Wan Ho has
discussed the hazards of horizontal gene transfer in some detail [7, 8].
The main problem with the proposed field test of GM Erwinia carotovara
is the failure to monitor the spread of the modified bacteria and the
horizontal spread of transgenes. The release of virulent GM Erwinia does
not appear to provide any benefit that would justify the risks of
spreading the antibiotic genes.
References
1. University of Wisconsin-Madison Availability of Environmental
Assessment for Field Tests of Genetically Engineered Erwinia carotovora
Agency Document Number APHIS-2005-0069-0001 Comment Period End Date
(mm/dd/yyyy): 09-12-2005
http://docket.epa.gov/edkfed/do/EDKStaffCollectionDetailView?objectId=0b0007d48094780b
2. USDA/APHIS Environmental Assessment in response to permit application
(05-097-01r) received from University of Wisconsin for field testing of
genetically engineered strains of bacterium, Erwinia carotovora subsp.
Carotovora 2005
http://docket.epa.gov/edkfed/do/EDKStaffAttachDownloadPDF?objectId=090007d48094780e
3 Mulec J, Starcic M and Zgur-Bertok D. F-like plasmid sequences in
enteric bacteria of diverse origin, with implication of horizontal
transfer and plasmid host range. Curr Microbiol. 2002, 44, 231-5.
4. Chatterjee AK, Ross LM, McEvoy JL and Thurn KK. pULB113, an
RP4::mini-Mu plasmid, mediates chromosomal mobilization and R-prime
formation in Erwinia amylovora, Erwinia chrysanthemi, and subspecies of
Erwinia carotovora. Appl Environ Microbiol. 1985, 50(1),1-9.
5. Kotoujansky A, Lemattre M and Boistard P. Utilization of a
thermosensitive episome bearing transposon TN10 to isolate Hfr donor
strains of Erwinia carotovora subsp. Chrysanthemi. J Bacteriol. 1982,
150(1),122-31.
6. Cummins J. GM microbes invade North America. Science in Society 2003,
19, 39.
7. Ho MW Horizontal gene transfer – The hidden hazards of genetic
engineering. ISIS Report
http://www.i-sis.org.uk/full/HGTFull.php; also Biotechnology Series,
Third World Network, Penang 2001.
8. Ho MW Recent evidence confirms risks of horizontal gene transfer.
ISIS contribution to ACNFP/Food Standards Agency open meeting 2002
http://www.i-sis.org.uk/FSAopenmeeting.php
Field Testing GM fungus
Prof. Joe Cummins has objected to the field release of GM fungus
containing an antibiotic resistance gene on behalf of the Independent
Science Panel. Please do the same.
This article has been submitted to the US EPA on behalf of the
Independent Science Panel (identifier: APHIS-2005-0067-0005). Please add
your support by registering your opposition in the docket and referring
to this article and its identifier number.
The University of Kentucky has prepared Environmental Assessment for
Field Tests of Genetically Engineered Neotyphodium, an endophyte fungus
living inside ryegrass. USDA/APHIS has prepared a docket, which is
available for comment until12 September 2005 [1].
Fungal endophytes live symbiotically inside plants without harming them.
The fungus grows in or around plant cells in a cozy relationship whereby
the plant feeds the fungus, which takes what it is given and no more to
avoid becoming a parasite on the plant. The fungus Neotyphhodium
provides rye grass with alkaloids that protect the plant from animal
predators. The grass bearing the endophyte has a clear advantage over
grass lacking the fungus. However, alkaloids such as the ergot family
adversely affect grazing mammals. The fungus reproduces asexually in the
grass and does not produce sexual spores. However, fungi have active
mitotic recombination and somatic gene conversion in what is called a
“para sexual cycle”. The fungus is passed through the maternal tissue to
the seed, and inoculation of a plant lacking the fungus is difficult.
The proposed release involves two different strains modified in
different genes for alkaloid formation. One of the transgenic endophytes
has a gene for dimethylallytryptophan synthase (dmaW) disrupted, the
other has a gene for lysergyl peptide synthetase (IpsA) disrupted. The
disrupted dmaW eliminated production of the alkaloid ergovaline and its
precursors including lysergic acid. Disrupted lpsA eliminated
ergovaline, but not lysergic acid. In that strain, lysergic acid
increases markedly about twenty five times. Both transgenic strains were
disrupted by introducing the hygomycin B phosphotransferase (hph) gene
from a bacterium into the dmaW and lpsA genes. The hph confers
antibiotic resistance and is driven by a promoter from the fungus
Neurospora and a transcription terminator from the fungus Aspergillus.
The hph genes are targeted specifically to disrupt the dmaW and lpsA
genes by adding short DNA sequences from the target gene at both ends of
the hph insert [2- 4].
In the proposal, the inserted antibiotic resistance gene is assumed to
have no impact on the growth of the endophyte in the absence of
antibiotic treatment, but evidence in support of that assumption was not
provided. Furthermore, gene disruption leads to “pop out” in some fungi
– an intra-chromosomal homologous recombination that splices out the
inserted gene, mobilizing the excised insert as a circular DNA unit and
restoring the disrupted gene to full activity. This possibility seems
not to have been considered in the proposal.
The proposal claims that the fungal endophyte and its antibiotic
resistance gene will be stable and not be transmitted horizontally.
There was little or no discussion of the potential impact of the protein
produced by the antibiotic resistance gene, hygromycin B
phosphotransferase, other than to note the United States Environmental
Protection agency had granted it an exemption from tolerance. Exemption
from tolerance means that any level of the exempt protein is deemed to
be safe and acceptable and allowed in food and feed. The toxicity and
allergenicity of the protein does not appear to have been considered in
the proposal. The proposal argues that there will be little or no
horizontal transfer of the resistance marker but does not allow for the
fact that the grass carrying the transgenic endophyte is bound to
breakdown. In that eventuality, antibiotic resistance gene will be
released to the soil environment where it may transform soil bacteria.
Furthermore, transformation of gut bacteria may also occur during
digestion of the grass by the pasture animals.
GM endophytes have previously been tested. A bacterial endophyte of
yellow lupine was modified with genes for degrading an organic pollutant
along with genes for antibiotic and nickel resistance. The bacterial
enodphyte turned out to be a pathogen for humans! As in the present
proposal, little concern was given to the spread of antibiotic genes in
the environment [5].
The proposal to field test GM endophyte modified pasture grass should
have been preceded by feeding experiment with the modified grass. The
animals should be examined by a full necropsy to study the impact of the
grass. The fate of the introduced antibiotic resistance gene in decaying
and fed modified grass should be examined. All those experiments should
at least be undertaken before open field tests are contemplated.
References
1. University of Kentucky; Availability of Environmental Assessment for
Field Tests of Genetically Engineered Neotyphodium Docket ID:
APHIS-2005-0067 Comment Period End Date September 12,2005
http://docket.epa.gov/edkfed/do/EDKStaffCollectionDetailView?objectId=0b0007d4809475d5
2. USDA/APHIS Environmental Assessment in response to permit application
(05-152-01r) received from the University of Kentucky for field testing
of two genetically engineered fungal endophyte Neotyphodium sp. isolate
Lp1 strains introduced in perennial ryegrass (Lolium perenne). 2005
http://docket.epa.gov/edkfed/do/EDKStaffAttachDownloadPDF?objectId=090007d480947684
3. Panaccione DG, Johnson RD, Wang J, Young CA, Damrongkool P, Scott and
Schardl CL. Elimination of ergovaline from a grass-Neotyphodium
endophyte symbiosis by genetic modification of the endophyte. Proc Natl
Acad Sci U S A. 2001, 98(22), 12820-5.
4. Wang J, Machado C, Panaccione DG, Tsai HFand Schardl CL. The
determinant step in ergot alkaloid biosynthesis by an endophyte of
perennial ryegrass. Fungal Genet Biol. 2004, 41(2),189-98.
5. Cummins J and Ho MW. Bio-remediation without caution. Science in
Society 2004, 23, 40.
http://www.i-sis.org.uk/isisnews.php
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