www.checkbiotech.org ; www.raupp.info ; www.czu.cz

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; February 2006
by Gijs A. Kleter, Ad A.C.M. Peijnenburg, & Henk J.M. Aarts.

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 te 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
modification;


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?s 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?s 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.

References

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.
[www.isaaa.org]

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, 326-352.
[www.hindawi.com]

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.
[www.fao.org]

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.
[www.efsa.eu.int]

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.
[www.efsa.eu.int]

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.
[europa.eu.int]
1.pdf

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.
[www.entransfood.nl]
arked.pdf

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
gijs.kleter@wur.nl

[www.isb.vt.edu]

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