GENET archive


9-Misc: Possible health aspects of horizontal transfer of microbial trangenes in GM crops

------------------------------- GENET-news -------------------------------

SOURCE: ISB News Report, USA
        by Gijs A. Kleter, Ad A.C.M. Peijnenburg, & Henk J.M. Aarts
DATE:   Feb 2006

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Since the first large-scale introduction of genetically modified (GM)
crops a decade ago, the global area cultivated with these crops has
undergone a continuous increase, amounting to a total of 90 million
hectares in 2005.1 For comparison, this area equals the national sizes of
Portugal, Spain, and Italy together. Many of the "foreign" genes that
have been introduced into these crops, i.e., the transgenes, are derived
from microbial sources. As explained below, the issue of their potential
transfer to other organisms was addressed in a recent article published
by our group.2

Long before the first introduction of GM crops, international
organizations like the Food and Agriculture Organization (FAO), World
Health Organization (WHO), and Organization for Economic Co-operation and
Development (OECD), had been promoting international consensus on how to
assess the safety of such crops. An internationally harmonized approach
of comparative safety assessment was thus formulated in which the GM crop
is compared to a conventional counterpart with a known history of safe
use (reviewed in 3).

Usually, this comparison entails a description of the genetic
modification, such as the nature of the DNA used and the function of the
transgenes and encoded proteins, as well as of agronomic and phenotypic
traits and composition. Based upon the differences thus identified, a
strategy for further safety assessment can be chosen. Given the wide
variety in characteristics of both the host crops and the transgenes,
this approach entails decisions on a case-by-case basis, rather than a
"cook book" with standard recipes.

Issues that are commonly addressed during the regulatory safety
assessment of GM crops include:

- Molecular characteristics, such as the introduced DNA, its integration
site (e.g., flanking DNA), and its expression;

- Comparison of agronomic and/or phenotypic characteristics and
composition of key macro- and micro-nutrients, anti-nutrients, and toxins;

- Unintended effects that might have arisen from the genetic modification;

- Potential toxicity of newly introduced proteins and of possible changes
in the host crop itself, which may have been caused by the genetic

- Potential allergenicity of newly introduced proteins, i.e., the
likelihood that they may cause allergies in consumers of food containing
GM crops, and possible changes in the intrinsic allergenicity, if any, of
the host crop that may have been caused by the genetic modification;

- Nutritional characteristics of the GM crop, which have been already
partially addressed by the compositional analyses, and which may also
entail animal feeding studies;

- Horizontal gene transfer, i.e., the "natural" genetic modification of
organisms other than the crop itself with the newly introduced DNA, for
example after the transgene has been released from the crop during
processing or digestion. This would require, among others, the uptake of
the released DNA by cells of the other organism and also the successful
incorporation of this DNA into the new host's genetic material and its
expression. Consideration is given to the likelihood of such a transfer
to pathogenic microbes in the human intestines, and if it occurred, which
consequences it would entail for consumers' health.

In 2003, the activities on international consensus building culminated
into the establishment of Codex Alimentarius' guidelines on the conduct
of safety assessment of foods derived from genetically modified plants
and micro-organisms.4 Codex Alimentarius standards, guidelines, and other
documents are important because they serve as reference for international
trade disputes over the safety of internationally traded foods under the
international agreement on sanitary and phytosanitary standards (SPS).

Horizontal gene transfer is one of the important issues addressed during
the safety assessment of GM crops. In the Codex Alimentarius guidelines,
the focus of the assessment of this topic is restricted to the potential
transfer of antibiotic resistance marker genes and the consequences
thereof. These marker genes are used to facilitate the process of genetic
modification. This is done by co-introducing the gene of interest with an
antibiotic resistance gene into the DNA of a crop cell. Those cells that
have been successfully modified can be selected based upon their ability
to sustain on culture media containing the pertinent antibiotic, to which
non-modified cells are sensitive. Antibiotic resistance marker genes
therefore do not serve a purpose in the GM crop itself.

Antibiotic resistance currently is a matter of great priority to health
care, as evidenced, for example, by the attention devoted to this issue
by organizations like the WHO. For example, popular media give accounts
of the dissemination in hospitals of antibiotic-resistant pathogens, such
as methicillin-resistant Staphylococcus aureus (MRSA). In general, the
spread of antibiotic resistance is considered to be linked to the way
that antibiotics are used, among other factors.

During the safety assessment of GM crops, the possibility of the transfer
of antibiotic resistance genes that have been introduced into GM crops is
considered. The European Food Safety Authority°Øs Scientific Panel on
Genetically Modified Organisms recently issued an opinion on antibiotic
resistance genes.5 This opinion, among others, proposed a categorization
of the antibiotic resistance genes into three categories based on the
clinical importance of the antibiotic, the natural prevalence of
resistance to the same antibiotic in nature, and the likelihood of
transfer. Only antibiotic resistance genes that fall into the first
category of this scheme, such as the kanamycin resistance gene nptII, are
recommended to be allowed for use in GM crops that are to enter the market.

In practice, however, regulatory safety assessments do not limit the
scope of potential transfer of transgenes from GM crops only to
antibiotic resistance. These assessments also address other potential
effects of transgenes, including pathogenicity. The potential impacts of
gene transfer on health and the environment in a broad sense are
considered by European Union guidelines.6,7

Similar to antibiotic resistance, literature reports indicate that
characteristics associated with pathogenicity have been exchanged between
microorganisms like Escherichia coli and Salmonella enterica, such as
through transfer of DNA fragments containing "pathogenicity islands." A
wide array of biochemical characteristics are known to be involved in the
pathogenicity of microorganisms, such as the formation of adhesion
molecules that bind to host cells, enzymes that facilitate entrance into
host cells, self-sufficiency for some nutritional compounds, and "quorum
sensing" within groups of micro-organisms.

Various mechanisms by which DNA is horizontally transferred between
microorganisms are known to exist in nature, including transfer after
conjugation between bacteria, transduction by bacteriophages, and
transformation by free DNA. Potential transfer of transgenes from GM
crops to microbes in the gastro-intestinal tract likely proceeds through
a process in which competent cells are transformed with free DNA. As
stated above, this can occur after the DNA of the GM crops has been
released from its host cells, for example during digestion.

Various factors influence the likelihood that transfer of DNA from a GM
crop to a recipient bacterium will occur and become productive. One of
these factors is the level of the bacterium°Øs competence, i.e., the
physiological state of a bacterial cell during which it can bind, take
up, and recombine DNA molecules. The outcomes of a number of studies
indicate that the most likely mechanism by which DNA is transferred from
GM crops to microorganisms is by homologous recombination. This means
that the recipient microorganisms should already contain sequences that
are sufficiently similar ("homologous") to the incoming foreign DNA, such
that they can align with each other and allow for integration of the latter.

Finally, plant genes and microbial genes differ with respect to preferred
base composition of the codons. Plant genes also have other features that
differ from microbial genes, such as introns, which do not occur in
bacterial sequences, and different types of regulatory sequences.

On the one hand, based on these considerations, which have been reviewed
in more detail elsewhere,8 it appears that transgenes of microbial origin
carry an enhanced likelihood of being transferred from GM crops to
microorganisms. Genetic modification allows for the introduction of
foreign genes from one organism into another, unrelated organism. As a
result of this, many of the GM crops currently on the market contain
transgenes of microbial origin, such as enzymes metabolizing herbicides
obtained from soil microorganisms or insecticidal proteins obtained from
Bacillus thuringiensis.

In our review,2 we focused on transgenes of microbial origin other than
antibiotic resistance genes that are present within GM crops approved by
the regulatory authorities of the European Union, United States of
America, Canada, Australia, and New Zealand. A number of factors that
influence the transfer of these transgenes, as well as the potential
impact of such a transfer on the health of consumers, were considered.
For each gene studied, these factors, if applicable and information
available, included:

- Occurrence and pathogenicity of the microorganism from which a given
gene has been obtained;

- Natural function of the gene;

- Prevalence of the gene in other microorganisms;

- Geographical distribution of the gene;

- Similarity of the original gene and codon-modified transgene to genes
in other microorganisms. For this purpose, DNA sequences were compared
using the FASTA algorithm. A stringent threshold for similarity was used.
In addition, we checked whether the aligned sequences would have two
identical stretches of DNA of at least 20 contiguous base pairs each,
which is considered the minimum required for homologous recombination.
For many transgenes, the actual sequences introduced into GM crops are
treated as confidential information and are thus not publicly available.
A high degree of similarity may be indicative both for the background
presence of the gene in nature, and for the likelihood of transfer by
homologous recombination;

- Known horizontal gene transfer activity of the gene. Has this gene
previously been transferred in nature?

- Selective conditions and environments, e.g., does the gene confer a
selective advantage to its host? If yes, persistence of the transferred
gene may be more likely.

- Possible effect of the transgene on the pathogenicity or virulence of
its host.

None of these single items can be considered completely predictive for
adverse effects and therefore a combination of factors has to be
considered in a "weight of evidence"-based approach. Based upon these
considerations, a conclusion was formulated for each gene as to whether
its transfer from GM crops would be likely to have any adverse health
effects in consumers. In total, 20 microbial transgenes were considered,
including five that are linked with herbicide resistance, three with
hybrid breeding through male sterility, two with prolonged fruit
ripening, two linked with markers for genetic modification, and eight
with insecticidal properties. The genes with insecticidal properties all
encoded Cry proteins from B. thuringiensis.2

It was concluded that none of these cases raises safety concerns.
However, a number of conspicuous findings were made. For example, the
native forms of a number of genes appeared to have been transferred
horizontally in nature. In some cases, this transfer was postulated by
other authors based on sequence similarities between genes from different
species, or the ability to transfer plasmids between them under
laboratory conditions.2 This pertained, for example, to the uidA
transgene from E. coli encoding ¶¬-glucuronidase, which is used as a
marker enzyme in GM crops based on its ability to form a blue color under
test conditions. Similar genes with bacterial rather than fungal sequence
characteristics were found to occur in moulds residing in soils. The
authors of this particular study9 concluded that the transferred gene
would allow the recipient microorganisms to utilize glucuronide
compounds, which are formed, for example, in the liver of animals and
excreted through feces and urine. The transferred gene would thus have
conferred a selective advantage to its recipient in soil.

Another case of selective advantage in soil conditions was that of the 1-
aminocyclopropane-1-carboxylate (ACC) deaminase gene, which has been
isolated from a soil isolate of Pseudomonas and introduced into GM
tomatoes to suppress ethylene synthesis and thereby delay ripening. It
has been observed that this gene is expressed in soil microorganisms
colonizing plant roots and that its activity is associated with increased
root formation.10 We therefore postulated that the transfer of this gene
may confer a selective advantage to recipient microorganisms in the
vicinity of plants producing ACC.

It should be noted that the data on the original sequences from the
native hosts may represent a "worst case" situation. This is because in
GM crops the transgene sequences may have been optimized for expression
in plants. As stated above, plant genes have a number of features that
are different from bacterial genes, which decrease the likelihood of
effective transfer and expression of plant genes to bacteria.

In conclusion, it was recommended to include the abovementioned
considerations in safety assessments of GM crops carrying transgenes
other than the ones already reviewed in the current survey2.


1. James C (2005) Executive Summary, Global Status of Commercialized
Biotech/GM Crops: 2005. ISAAA Brief 34. International Service for the
Acquisition of Agri-biotech Applications: Ithaca.
2. Kleter GA, Peijnenburg AACM, Aarts HJM (2005) Health considerations
regarding horizontal transfer of microbial transgenes present in
genetically modified crops. Journal of Biomedicine and Biotechnology 4,
3. Kok EJ, Kuiper HA (2003) Comparative safety assessment for biotech
crops. Trends in Biotechnology 21, 439-444
4. Codex alimentarius (2003) Codex Principles and Guidelines on Foods
Derived from Biotechnology. Codex Alimentarius Commission, Joint FAO/WHO
Food Standards Programme, Food and Agriculture Organisation: Rome.
5. EFSA (2004) Opinion of the Scientific Panel on Genetically Modified
Organisms on the Use of antibiotic resistance genes as marker genes in
genetically modified plants. (Question N°? EFSA-Q-2003-109). EFSA Journal
48, 1-18.
6. EFSA (2005) Guidance Document of The Scientific Panel on Genetically
Modified Organisms for the Risk Assessment of Genetically Modified Plants
and Derived Food and Feed. European Food Safety Authority: Parma.
7. EU (2002) Council Decision of 3 October 2002 establishing pursuant to
Directive 2001/18/EC of the European Parliament and of the Council the
summary information format relating to the placing on the market of
genetically modified organisms as or in products (2002/812/EC). Official
Journal of the European Communities L 280:37-61
8 Van den Eede G, Aarts H, Buhk HJ, Corthier G, Flint HJ, Hammes W,
Jacobsen B, Midtvedt T, Van der Vossen J, Von Wright A, Wackernagel W,
Wilcks A (2004) The relevance of gene transfer to the safety of food and
feed derived from genetically modified (GM) plants. Food and Chemical
Toxicology 42, 1127-1156.
9 Wenzl P, Wong L, Kwang-won K, Jefferson RA (2005) A functional screen
identifies lateral transfer of ¶¬-glucuronidase (gus) from bacteria to
fungi. Molecular Biology and Evolution 22, 308-316
10 Belimov AA, Safronova VI, Sergeyava TA, Egorova TN, Matveyeva VA,
Tsyganov VE, Borisov AY, Tikhonovich IA, Kluge C, Preisfeld A, Dietz K-J,
Stepanok VV (2001) Characterization of plant growth promoting
rhizobacteria isolated from polluted soils and containing 1-
aminocyclopropane-1-carboxylate deaminase. Canadian Journal of
Microbiology 47, 642-652

Gijs A. Kleter, Ad A.C.M. Peijnenburg, Henk J.M. Aarts
RIKILT - Institute of Food Safety
Wageningen University and Research Center, The Netherlands


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