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[SANET-MG] Field Test of Two Non-pathogenic Genetically Engineered Strains of Burkhoderia glumae



Comments on the APHIS EA for the field test release of GM bacteria are due by the 19 July 2007. I urge as many as can to make brief comment on the proposed EA. Burkholderia species are pathogens or endophytes to several crop plants. Burkholeria spedies are also modified to serve in remediation of polluted sites.Unfortunately, several Burholderia species are human pathogens that infect older people or others who have compromised immune systems such as AIDS victims, Cystic fibrosis victims, people taking anti transplant rejection drugs or people treated for arthritis or other autoimmune diseases. Burkhoderia glumae has been shown to infect a child but APHIS agrees that infection of just one child is not sufficient evidence to require careful handling and by-stander infection of people by the plant pathogen .
2 July 2007
Professor Joe Cummins
University of Western Ontario , London, Ontario. Canada and
The Institute in Science and Society, London, UK
Comments on a Field Test of Two Non-pathogenic Genetically Engineered Strains of Burkhoderia glumae The following comments deal with USDA APHIS Environmental Assessment (EA) In response to a permit application (06-111-01r) received from Dr. Milton Rush of Louisiana State University for a field test of two non-pathogenic, genetically engineered strains of Burkholderia glumae. APHIS-2007-0021 Availability of an Environmental Assessment for a Field Test of Two Non-pathogenic, Genetically Engineered Strains of Burkholderia glumae Comments Due by 07/19/2007 http://www.regulations.gov/fdmspublic/component/main The proposal summary is as follows: Burkholderia glumae Kurita et Tabei is a bacterial plant pathogen that causes bacterial panicle blight in rice , and is transmitted by infected seed . This bacterium was first described in Japan as the cause of grain rotting and seedling blight and is considered one of the most important rice pathogens in Japan Epidemics of panicle blight occurred in the southern rice producing area of the United States during the 1995 and 1998 growing seasons, with yield losses in some fields estimated to be as high as 40 percent.Currently, there is no control method for panicle blight in the United States , and most commercially grown rice varieties in the United States are susceptible to the disease. By field testing non-pathogenic, transgenic strains of B. glumae, the proposed field release will provide information on bacterial panicle blight infection of rice, and potential routes for control of the pathogen. The transgenic B. glumae strains have been modified to disrupt the disease-causing gene (gene that produces toxoflavin), producing avirulent, non-pathogenic strains. The transgenic B. glumae strains also express the genes for kanamycin and ampicillin resistance as selectable markers. The proposed field release will also involve the challenge of rice plants with wild-type B. glumae during the field test (1). One virulence factor in B. Glumae is a enzyme called Toxiflavin, a yellowish substance. Toxoflavin production results in significant rice damage to inoculated plants. Toxoflavin is produced in Burkholderia by an operon consisting of the tox gene cluster (toxABCDE) and the toxR gene. Disruption of the toxA gene (methyltransferase) results in mutants that do not produce toxoflavin. The cloning vector also contains two selectable markers, the gene (nptII) for neomycin phosphotransferase from Streptomyces kanamyceticus and the gene (bla) for beta-lactamase from Escherichia coli. The selectable markers provide resistance to kanamycin and ampicillin, respectively. The promoter for each of the genes is the Bacteriophage T7 promoter, and the terminator for each of the selectable markers is a synthetic TAA codon sequence. The donor DNA sequences are stably and irreversibly integrated into the bacterial genome, where they are maintained and inherited as any other genes of the bacteria cell (1). The avirulent non-pathogenic strain bears stable resistance to the antibiotics kanamycin and ampicllin.. The proposed field tests will include challenge of the rice plants with virulent wild type B. glumae. Two experiments will be conducted the first evaluates the toxoflavin as a disease causing agent the second experiment inoculates the rice with the mutant avirulent bacterium followed by challenge with the virulent strain to see whether or not the presence of the avirulent strain will protect rice from B. Glumae infection, Horizontal gene transfer of the antibiotic reistance gens to soil bacteria is acknowledged in the APHIS EA but is presumed to have insignificant consequences in the proposed experiment (1). The presumption that horizontal gene transfer will have insignificant consequences is not born out in a wealth of publication such as those covered in the review by Ho etal (2). An alternative approach to controlling B. glumae has been to regulate quorum sensing a regulatory network influencing virulence of the bacterium Quorum sensing coordinates behaviour or actions between bacteria, based on the local density of bacteria. Quorum sensing can occur within a single bacterial species as well as between disparate species, and can regulate a host of different processes, essentially serving as a simple communication network.Signaling between bactria is modulated by molecules specified by genes.Toxoflavin has been found to be regulated by a quorum sensing mechanism that uses N-acyl homoserine lactones as signal molecules.An endophyte , Burholderia sp., was selected from rice and found to be non-pathoogenic to rice and to inhibit pathogenic fungi. That endophyte was modified with a gene from Bacillus thuringiensis specifying N-acyl homoserine lactones and the transgenic endophyte was found to prevent Toxoflavin synthesis and virulence in B. glumae (3). Genetic modification of quorum sensing provides an alternative and to avoid use of the antibiotic résistance genes described above. Burkhoderia species such as B. Cepacia is a potent human pathogen (4). B. thailandensis caused pheumonia and septicemia in human(5) B. dolosa is pathogenic in people with cystic fibrosis (6). B. gladioli caused ocular keratitis in an indivual with diabetes and it also is found in other diseases as well (7). A number of other Burholderia species are associated with human infections. Based on those observations it is not at all surprising that a B. glumea infection was observed in an infant with chronic granulomatous disease (8). Further investigation of the clinically isolated strain of B. glumea showed that the bacterium caused severe disease symptoms in rice., The involvement of a quorum sensing regulated secreted lipase was implicated in the pathogenesis of the clinical strain (9). Surprisingly, the APHIS EA (1) did not consider human infection to be a serious matter based on a single human case nor did the EA consider precautions for those working with the pathogen who will likely bear the pathogen to their homes , families and neighbours. The EA argued that a single case was not a matter of concern, implying that a large “body count” was required to require careful handling of the pathogen. In other words, APHIS seemed to agree that the field tests experiments were more important than a few human infections! APHIS has shown a clear bias in favouring field test releases of GM crops but the present EA certainly cries out that APHIS should not be controlling release of experimental GM crops, instead , an unbiased , independent and humane body should regulate field tests and de-regulation of GM crops.
References
1. U.S. Department of Agriculture Animal and Plant Health Inspection Service Biotechnology Regulatory Services USDA APHIS Environmental Assessment In response to a permit application (06-111-01r) received from Dr. Milton Rush of Louisiana State University for a field test of two non-pathogenic, genetically engineered strains of Burkholderia glumae. http://www.regulations.gov/fdmspublic/component/main 2. Ho M-W , Cummins,J and Saunders,P. GM food nightmare unfolding in the regulatory sham Microbial Ecology in Health and Disease 2007,19, 2 , 66 – 77 3. . Cho HS, Park SY, Ryu CM, Kim JF, Kim JG and Park SH. Interference of quorum sensing and virulence of the rice pathogen Burkholderia glumae by an engineered endophytic bacterium FEMS Microbiol Ecol. 2007 Apr;60(1):14-23. 4. Cummins,J and Ho, M-W. Bio-remediation without caution Science in Society 2004,23,40 5. Glass MB, Gee JE, Steigerwalt AG, Cavuoti D, Barton T, Hardy RD, Godoy D, Spratt BG, Clark TA and Wilkins PP. Pneumonia and septicemia caused by Burkholderia thailandensis in the United States. J Clin Microbiol. 2006 Dec;44(12):4601-4. 6. Caraher E, Duff C, Mullen T, Mc Keon S, Murphy P, Callaghan M and McClean S. Invasion and biofilm formation of Burkholderia dolosa is comparable with Burkholderia cenocepacia and Burkholderia multivorans. J Cyst Fibros. 2007 Jan;6(1):49-56. 7. Ritterband D, Shah M, Cohen K, Lawrence J and Seedor J Burkholderia gladioli keratitis associated with consecutive recurrent endophthalmitis. Cornea. 2002 Aug;21(6):602-3. 8. Weinberg JB, Alexander BD, Majure JM, Williams LW, Kim JY, Vandamme P and LiPuma JJ. Burkholderia glumae infection in an infant with chronic granulomatous disease. J Clin Microbiol. 2007 Feb;45(2):662-5. 9. Devescovi G, Bigirimana J, Degrassi G, Cabrio L, Lipuma JJ, Kim J, Hwang I and Venturi V. A clinical isolate of Burkholderia glumae causes severe disease symptoms in rice; involvement of a quorum sensing regulated secreted lipase. Appl Environ Microbiol. 2007 Jun 8; [Epub ahead of print]
doi:10.1128/AEM.00105-07

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