ISIS Report 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.

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

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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