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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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