Field-testing Bacterial Pathogens and Fungus

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30 Aug 2005 13:38:29 -0000

Field-testing Bacterial Pathogens and Fungus

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ISIS 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.

 

 

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?ob

jectId=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?ob

jectId=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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http://www.i-sis.org.uk/fieldTest.php

 

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