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[SANET-MG] field testing GM bacteria in Wisconsin



August 12, 2005

Prof. Joe Cummins

“Field testing Erwinia carotovora bacteria containing antibiotic resistance genes”

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 has been a public comment period ending September 15,2005.. The tests are to be conducted at the University of Wisconsin Experiment Station near Hancock, Wisconsin. The docket is located as described in reference (1). The release to the environment of a 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 rot disease in potatoes. The experiment is designed to test the fitness of bacteria that have had genes known to be associated with pathogenesis which have been disrupted by the insertion in those genes of the antibiotic resistance genes. The antibiotic resistance genes not only eliminate the function of the pathogenesis gene but they also serve as selectable markers to identify 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 target is Hrp which controls secretion of virulence proteins into the host plant. The green fluorescence protein was inserted as a visual marker to identify potatoes bearing the modified bacteria (2).

The field test involves four GM mutant strains (WPP60,WPP198,WPP195 and WPP40. Strains WP60 was created by inserting the streptomycin/spectomycin genes into the hrcC gene while WPP98 had chloramphenicol resistance gene inserted into hrpL gene.WPP195 was created by deleting the hrpN gene and inserting the choramphenicol resistance gene in its place. Strain WPP was created by inserting the kanamycin resistance gene into the OutD gene that is required for a functioning protein secretion system in the bacterium.; The mutant cannot secrete plant cell wall degrading enzymes. Inserting antibiotic resistance genes into the hrp genes interferes with pathogenisis. However only strain WPP40 that eliminates the OutD gene is reduced in virulence in a direct assay while the other three strains are not reduced in virulence, but in spite of that they are carried in the field experiment (2).

The field tests were not expected to impact agricultural practice because “the test is solely for research purposes” even though Erwinia carotovora effects 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 believed to be a concern according to the applicants. The proposal believes that the antibiotic resistance genes cannot be transferred to bacteria effecting humans (as pointed out below that belief is not reasonable). The applicants justify that position by judgments from the US Food and Drug Administration that antibiotic resistance is already prevalent in bacteria effecting humans and in soil born bacteria. Even though the soil is sandy and porous in the area of the test site spread of the GM bacteria is not expected nor tested for(2). In general the application focuses on the benefits of the experiments and ignores any evidence that might delay the experiment.. The most obvious question is why release so many strains that are genetically modified but still virulent? That procedure provides high risk with little or no benefit.

The belief expressed in the proposal, that GM Erwinia carotovora would not transfer antibiotic resistance genes to other soil bacteria seems inconsistent with the scientific literature. Horizontal gene transfer was commonplace in Erwinia and Klebsiella and further that transfer could extend to Salmonella, Shigella and E. coli (3).Presumabley the GM bacteria on surfaces of food crops could transfer antibiotic resistance gene to enteric bacteria during ingestion 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 clear evidence that Erwinia is capapble 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 Psueomonas (6). Little effort seems to have been put forth to monitor the consequences of these releases. The hazards of horizontal gene transfer have been discussed in depth by Dr. MaeWan Ho (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 the genes with which they have been modified. The release of virulent GM Erwinia does not appear to provide any benefit in the face of the risk of spreading the antibiotic genes.

Reference

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 Apr;44(4):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 Jul;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 Apr;150(1):122-31

6. Cummins,J. GM Microbes invade North America Sience in Society 2003, 19.39

7. Ho,MW Horizontal gene transfer happens-II ISIS Report 2001

http://www.i-sis.org.uk/

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/

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