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TWN Biotechnology & Biosafety Series
SURVIVAL,
PERSISTENCE,
TRANSFER
An update on
current knowledge
on GMOs and
the fate of their
recombinant DNA
by Beatrix Tappeser, Manuela Jager,
Claudia Eckelkamp
TWN
Third World Network
Survival, Persistence, Transfer — An update on current
knowledge on GMOs and the fate of their recombinant DNA
is published by
Third World Network
228 Macalister Rond
10400 Penang, Malaysia.
copyright © Third World Network 1999
This is part of a series of papers on biotechnology and biosafety that the
Third World Network is publishing with a view to deepening public
understanding on the ecological and safety aspects of the new
biotechnologies, especially genetic engineering.
Printed byjutaprint
2 Solok Sungei Pinang 3, Sg. Pinang
11600 Penang, Malaysia.
First Printing: 1999
Second Printing: 2002
ISBN: 983-9747-37-1
Contents
Chapter 1.
Introduction
1
Chapter 2.
Survival and Spread of GMOs
3
Survival in the digestive tract
Survival in wastewater and sludge
Survival in aquatic ecosystems
Survival in soil
Survival on plants
3
4
5
7
11
Chapter 3.
Further Points to Consider: VNC state and
13
adaptation ability
Chapter 4.
Spread of Cloned Sequences
16
Persistence of "naked" DNA
Persistence in wastewater treatment plants
(water/sludge)
Persistence in aquatic systems
Persistence in soils
Persistence in the digestive system
“Avenues and barriers of genetic transmission"
(Istok, 1991)
17
Discussion
24
Chapter 5.
References
17
17
18
19
19
27
Chapter 1
Introduction
During the last years the release, whether tolerated or permitted,
of genetically modified microorganisms (GMOs) and their nucleic
acids into various environments has increased worldwide. Legis
lation concerning the containment of GMOs has been deregulated
and safety measures have been relaxed throughout the industrial
world, as genetic engineering has not occasioned any obvious ac
cident or visible negative impact during the two decades of its
rapid development and constantly increasing use. Another deve
lopment of the last 10 to 15 years, according to the 1996 World
Health Organisation (WHO) Report, has been an increase in fre
quency of outbreaks of new and reemerging infectious diseases.
Moreover, current pathogen strains are often resistant to known
treatments, some even to nearly all commonly used antibiotics.
Horizontal gene transfer is now recognised to be the main avenue
of exchange of genetic material in the microbial world, and hence
also of the exchange and spread of antibiotic resistance genes.
These developments give rise to two questions. Does the exten
sive use of antibiotic resistance genes in genetic engineering con
tribute to the increase in frequency of antibiotic resistance in bac
terial pathogens? And what will be the outcome of a spread of
recombinant genes analogous to the spread of antibiotic resistance
genes?
Do we have risk assessment procedures available for monitoring
the fate of GMOs and their recombinant DNA? Are we capable of
1
detecting ecological impacts or health impacts in early warning
systems? The answer has to be no, and this for various reasons. We
are only beginning to understand microbial ecology. There is a lack
of basic knowledge by which to judge the possible impacts of a
given GMO or its recombinant DNA on different environments. In
particular, we do not know enough of the special conditions under
which gene transfer takes place. What are the selective conditions
which facilitate the transfer of specific genes and may, for exam
ple, promote the transfer of recombinant gene constructs?
A prerequisite for the possibility of GMOs and recombinant DNA
contributing to the spread of newly cloned genes is the viability
and/or persistence of GMOs and their recombinant DNA in cer
tain environments. Another point requiring analysis is the extent
to which artificial vectors facilitate and/or enhance the probability
of horizontal gene transfer.
In this review we present the latest data on survival of GMOs in
different environments, persistence of recombinant DNA, and what
is currently known of the different gene transfer mechanisms in
volved.
2
Chapter 2
Survival and Spread of GMOs
Survival in the digestive tract
The digestive system of vertebrates and invertebrates alike offers
possibilities for bacteria ingested with the food to get in close con
tact with each other and with the microflora of the animal. Its envi
ronment may prolong bacterial survival as compared with other
environments and, due to its high density of microorganisms, in
crease the probability of gene transfer (Adamo and Gealt, 1996).
Moreover, bacteria can be disseminated by their new host animals,
e.g. earthworms (Clegg et al., 1995). Some bacteria may even change
their metabolic activities and capacity for survival during their pas
sage through the digestive tract. (Clegg et al., 1995). Earthworm
activities may also influence the rate of transfer of conjugative
plasmids from Pseudomonas fluorescens to autochthonous soil mi
croorganisms (Daane et al., 1996). It is also possible for transfer
mechanisms other than conjugation to contribute to the dissemi
nation of DNA from GMOs to the endogenous microflora of ani
mals (Duval-Iflah, 1992).
Aside from these studies on bacterial interaction with the inverte
brate digestive system, Brockmann et al. (1996) investigated the
possibility of different strains of Lactococcus lactis colonising the rat
intestine and the transfer of plasmids between these bacteria. While
the digestive tract of germfree animals was found to be rapidly
colonisable by all kinds of bacteria, this only succeeded temporar
3
ily (for 2-3 days) in non-germfree controls. Studies with Lactococcus
lactis in the human intestine yielded similar results (Klijn et al.,
1995a). Moreover, the conjugative plasmid pAMBl, which has a
broad host-range, proved transferable both to a Lactococcus recipi
ent strain in the intestine of germ-free rats and an endogenous strain
of Enterococcus faecalis in that of non-germfree animals (Brockmann
et al., 1996). These authors conclude that even those microorgan
isms only temporarily detectable in the digestive tract are able to
transfer their conjugative plasmids to the endogenous microflora.
The rate of such events mainly depends on the structure of the
plasmids themselves.
Survival in wastewater and sludge
The most important factor influencing bacterial survival in
wastewater has to do with competitors. Because predator
populations fluctuate seasonally, the time of year is an important
factor in evaluating the risk of GMOs' survival (Inamori et al., 1992).
If they are not eliminated by their competitors, E. coli KI2 and
Pseudomonas putida species have the best chances of survival when
associated with snow particles containing sludge material (McClure
et al., 1989; Overbeck, 1991). Whereas viable cell counts in
supernatant were found to decrease quickly after introduction of
recombinant E. coli K12 into the aeration basin of a model
wastewater treatment plant, this was not usually the case in the
remaining bacteria attached to sludge particles (Heitkamp et al.,
1993).
Most studies on survival and gene transfer in wastewater and
sludge use in vitro systems or models of settlement tanks or acti
vated sludge for investigation. Ashelford et al. (1995) concentrated
on percolating filter beds, which are layered with living cells (a socalled biofilm) similar to the river epilithon. Two inoculated
Pseudomonas strains survived the whole investigation time of 145
days. The authors demonstrated the transfer of a conjugative
plasmid (pQKH6) harboured by one of these strains to another,
4
initially plasmid-less, strain (Ashelford et al., 1995).
Similar results were also published by Feldmann and Sahm (1994),
who investigated the survival of four different recombinant micro
organisms in laboratory wastewater systems.1 While none of the
GMOs persisted in the settlement tank, two yeasts tested in the
aeration basin remained detectable for more than 25 days.
Survival in aquatic ecosystems
A lot of the above data apply in a like manner to aquatic ecosys
tems. The chance of survival of, e.g., E. coli and Campylobacter jejuni
increases in filtered water taken from lakes, since filtering elimi
nates predators and competitors (Korhonen and Martikainen, 1991).
Sterile tapwater also allowed the persistence of four transgenic
microorganisms tested within the scope of the "Verbundprojekt
Sicherheitsforschung Gentechnik"2: in this medium survival lasted
the whole testing period of 440 days (Tebbe et al., 1994a).
Brettar et al. (1994) collected data on the survival of Pseudomonas
putida DSM3931 in lakewater mesocosms. Strains were not geneti
cally modified and survived the whole testing period of 10 weeks.
Mesocosms additionally inoculated with culture media even in
duced an increase of bacterial numbers during the first 10 days.
The most important factor responsible for the reduction of bacte
rial populations was predation, though many bacteria escaped by
associating with particles and/or sediments during the first two
days (Brettar et al., 1994).
Predation through protozoa also eliminated most bacteria
' These experiments were part of the "Verbundprojekt Sicherheitsforschung
Gentechnik", which aims at investigating the safety of GMOs released from pro
duction facilities, and were sponsored by Bayer AG, Germany.
2 Saccharomyces cerevisiae, Zyinomonns iiiobilis, Corynebacteriiini glutamicum, and
Hniiscmiln polymorphic
5
{Pseudomonas fluorescens AG1) inoculated in seawater mesocosms,
though the extent of this was found to depend on the state of bac
terial growth (Christoffersen et al., 1995). Elimination of bacteria
in seawater is also achieved by lysis through bacteriophages (bac
terial viruses), depending on seasonal changes (Weinbauer et al.,
1995).
Survival of bacteria in aquatic ecosystems also depends on abiotic
factors like temperature, nutrient availability and osmotic condi
tions (Ahl et al., 1995). These authors studied the survival of
Pseudomonas fluorescens Agl in experiments with seawater micro
cosms (0.51 water) and seawater mesocosms (5,300 1 water). Ahl et
al. (1995) demonstrated a less pronounced reduction of bacterial
cells inoculated in mesocosms as compared with earlier microcosmic experiments. Of the inoculated Pseudomonads 25% survived the
whole testing period of 14 days.
Intensity and seasonal changes of light are further important abiotic
factors influencing the survival of bacteria in natural environments
(Canteras et al., 1995). Five different strains of E. coli kept in com
plete darkness persisted for 96 hours in sterile riverwater with only
a minor reduction of cfu3. Differences in persistence were only ap
parent upon illumination. However, sterile conditions cannot be
taken to represent natural aquatic ecosystems. In the comparative
study of Alvarez et al. (1996b) on the survival of E. coli strains with
or without plasmids (DH1 and JM103) in tropical riverwater sys
tems, differences in survival only showed in unsterile test-cham
bers.
Bacteria taken from their natural environment, transformed with a
plasmid and then released again as GMOs, have a very high chance
of establishing themselves in their original ecosystem. For exam
ple, Sobecky et al. (1996) demonstrated that a marine bacterium
(Achromobacter sp.) is able to form stable populations in seawater
3 cfu = colony-forming units
6
microcosms within 2-3 weeks after being transformed with a
plasmid bearing a gene encoding an alkaline phosphatase. Even
the enzyme was proved to have retained its activity.
Bacteria not adapted to environmental conditions, like typical
enteropathogenic microorganisms (e.g. E. coli ETEC, Yersinia
enterocolitica and Campylobacter jejuni) are not only able to survive
within their hosts at body temperature but may also persist in a
cultivable stage for weeks in aquatic environments at temperatures
between 6°C and 16°C (Terzieva and McFeters, 1991). Davies et al.
(1995) demonstrated that the time of survival of enteropathogenic
bacteria (faecal Streptococci and Coliforme) in sediments exceeds
that in open water. While gram-negative bacteria inoculated in open
seawater soon entered a stage of non-culturability, E. coli cells re
siding in sediments remained cultivable during the whole testing
time of 68 days (Davies et al., 1995).
The same seems to be true of groundwater aquifers and freshwa
ter environments, where sediments provide a better chance of sur
vival than open water. This was demonstrated by Winkler et al.
(1995) with Burkholderia cepacia in a groundwater microcosm ex
periment and by Fish and Pettibone (1995) with E. coli. The latter
species remained cultivable in sediments for 56 days, i.e., for the
entire duration of the test.
Survival in soil
Since soil environments are especially complex and heterogene
ous systems, it is rather difficult to predict the chance of survival
they offer to newly introduced GMOs. The density of soil bacte
ria is estimated at about 108 to 109 cfu/g soil. However, less than
10% of these are cultivable using current techniques, and only
around 1% have been characterised to date (Hugenholz and Pace,
1996; Pace, 1997). In clay sediments bacteria are detectable at depths
down to 224 m (Boivin-Jahns et al., 1996). Microbial populations
from the laboratory decrease drastically during the first days after
7
their introduction into natural soil, but then often remain at a con
stant level (Schmidt, 1991).
It is most important in assessing and comparing published data on
the survival of GMOs in soil systems to thoroughly analyse the
design of the microcosm or mesocosm and detection method be
ing used. The variety of microcosms studied makes it is rather dif
ficult to directly compare the results collected (Angle et al., 1995).
An undisturbed soil texture is an important prerequisite for opti
mal imitation of natural environmental conditions, as Angle et al.
(1995) demonstrated. These authors compared survival of
Pseudomonas aureofaciens in microcosms and deliberate release ex
periments in naked soil for two years. Validation of the microcosms
used is also expedient with aquatic systems. Leser (1995) found
63%-76% agreement between data obtained from a lakewater mi
crocosm and a natural lake. After inoculation of Alcaligenes eutrophus
in both systems the degree of coincidence was reduced to 2-27%,
but increased again with decreasing cell counts (Leser, 1995).
As with aquatic and wastewater systems, in soils, too, predation
through protozoa is one of the most important factors influencing
the persistence of inoculated GMOs. In these environments
protozoal activity requires a certain degree of soil moisture and
free water in soil pores of suitable size (Van Elsas 1992; Wright et
al., 1995). Experiments in soil microcosms with Pseudomonas
fluorescens and the ciliate Colopoda steinii demonstrated that small
pores (< 6pm) may offer effective protection zones against preda
tors (Wright et al., 1995). While bacterial populations in larger pores
(< 30pm) diminish more rapidly, bacteria that do survive show a
higher level of metabolic activity in response to the richer supply
of nutrients there (Wright et al., 1995). Van Der Hoeven et al. (1996)
used a computer simulation model to evaluate the chance of sur
vival of GMOs in soil pores of different sizes. Bacteria introduced
into the soil were eliminated very slowly, in some cases taking years
for total elimination (Van Der Hoevan et al., 1996).
8
The number of bacteria inoculated into the soil is a very important
determinant of the time of possible survival, as differences as small
as 100 E. coli K12 cells/g soil suffice to change the results of an
assay (Recorbet et al., 1992). This strong correlation between in
oculation number and survival is also found in aquatic ecosystems
(Karapinar and Sahika, 1991). Another criterion is the depth of in
oculation. In the range from 10 to 50 cm the chance of survival of
the phytopathogenic bacterium Erwinia carotovora increases with
growing inoculation depth. This correlation largely reflects differ
ences in competitive pressure (Armon et al., 1995). Recorbet et al.
(1995) demonstrated uneven distribution of genetically modified
E. coli which had been inoculated in soil systems. Furthermore,
GMOs were concentrated in different soil layers than
autochthonous bacteria.
Spore-forming bacteria like Bacillus subtilis are particularly likely
to persist for prolonged time periods. Spores of Bacillus subtilis of
both native and genetically modified parent strains remained
culturable in sterile soil for at least 50 days (Tokuda et al., 1995).
GMOs' ability to adapt to natural environments makes it more dif
ficult to estimate their chance of survival after release. Cells of
Pseudomonas fluorescens showed improved stress resistance against
temperature or osmotic pressure one day after inoculation into two
different types of soil (Van Overbeek et al., 1995). When cultivated
under starving conditions in the laboratory these bacteria may stand
stress-factors like "hunger" for 70 days by reducing their cell size.
These physiological changes are soon reverted again when condi
tions are made to improve (Clegg et al., 1996).
There is still no evidence of plasmid-bearing microorganisms be
ing less fit than their native parents: e.g. Fujimura et al. (1994) tested
genetically modified Saccharomyces cerevisiae and their parent strains
under different environmental conditions but were unable to prove
any negative impact in terms of survival probability. Under selec
tive pressure, exerted, for example, by antibiotic substances, GMOs
9
may even profit from possessing the corresponding resistance genes
(Van Elsas, 1992). This was also demonstrated by Ramos et al. (1994)
with recombinant Pseudomonas putida (pWW0-EB62) in soil systems.
In unsterile soil and under assumed selective pressure cells num
bers even increased after inoculation (Ramos et al., 1994). How
ever, application of selective pressure is not a necessary prerequi
site for survival of GMOs.
Sjogren (1995) initiated a very profound project on survival of E.
coli using a multiresistant native strain isolated from a lake. The
bacteria were inoculated into different field plots seeded with rye.
There were no more bacteria detectable on the rye grass 41 days
later. After two months bacteria were reisolated from soil at a depth
of 20 to 50 cm. At 60-cm depth they reached groundwater and per
sisted there at the water-soil interface for two more years. Six years
later 1.5 cfu/ml were still detectable, and after eight years cell counts
had increased to 8 cfu/ml. After 13 days, at the end of the experi
ment, population density was still at 0.25 cfu/ml (Sjogren, 1995).
The author calculated the speed of distribution as about 2 cm/day.
In analogous experiments performed under laboratory conditions
with microcosms of the same soil the maximum survival time had
been 2.5 years (Sjogren, 1995).
GMOs intended for use in natural environments are designed to
persist and show at least some competitive ability versus
autochthonous microorganisms. Experiments with Rhizobium
meliloti, which had been genetically modified for biological con
tainment by disrupting a gene for recombination, only demon
strated reduced survival of the genetically modified as compared
with the native strain when performed in model ecosystems, but
not after deliberate release in agricultural soils (Dresing et al., 1995;
Selbitschka et al., 1994).
Beside surviving, GMOs inoculated in soil may also be distributed
further, thus increasing the chances of gene transfer. Gillespie et al.
(1995) simulated rainfall on wheat plants or naked soil in green
10
house experiments after inoculation with genetically modified
Pseudomonas aureofaciens. The number of cells detectable in the soil
was found to correlate with the number of cells in water running
from the plots. One exception to this was that 245 days after inocu
lation 108 cfu were measured in the water probes, whereas cell
counts in the soil were below the detection limit. The authors con
cluded that standard techniques for detection of GMOs in soil may
not be suitable for registering bacteria in all soil layers and that
short and heavy rainfalls can cause quite high numbers of GMOs
(about 1,014 cfu/ha) to be washed away from the place of release
(Gillespie et al., 1995). Similar testing was done by Hekman et al.
(1995) with Pseudomonas fluorescens and Burkholderia cepacia. In the
case of planted microcosms, these authors demonstrated enhanced
distribution of inoculated bacteria in deeper soil layers after mod
erate rainfall (Hekman et al., 1995).
Survival on plants
It is evident from the last paragraph that any analysis of survival
of GMOs in soil has to include the plants growing there. A
recombinant strain of Bacillus amploliquefaciens, used to produce (amylase) was no longer detectable in the soil of planted microcosms
six days after inoculation, though it persisted on the grass for at
least 70 days (Wendt-Potthoff et al., 1994). Genetically modified
strains of the phytopathogenic bacteria Xanthomonas campestris in
oculated on cabbage host plants survived for six months and were
distributed to other cruciferous plants and even unrelated plants
(Dane and Shaw, 1996).
Persistence of bacteria inoculated on plants may depend on the
plant species used. The population density of two strains of
Pseudomonas fluorescens remained at a constant level on leaf sur
faces of capsicum and eggplants during a 30-day glasshouse ex
periment, but decreased on leaves of strawberry and tomato
(Cirvilleri et al., 1996). Genetically modified Pseudomonas fluorescens
whose parent strain had been isolated from sugar beets persisted
11
Chapter 3
Further Points to Consider: VNC state and
adaptation ability
clear distinction must be made between cultivable and dor
mant cells, regardless of the environment under study (Dott et
al., 1991; Turpin et al., 1993). The dormancy problem is currently
receiving more and more attention, since results have been found
to differ tremendously according to whether all cells or only cul
tivable cells (Kragelund and Nybroe, 1996; Pickup, 1991; Stotzky
et al., 1991) are counted.
These VNC, i.e., viable non-culturable, bacteria are predominant
in marine environments with high salt concentration (Byrd and
Colwell, 1990; Garcia-Lara et al., 1993). There are various especially
adapted methods of detecting VNC cells and cultivable cells. How
ever, counts of VNC bacteria in environmental probes are usually
rather variable (Bianchi and Giuliano, 1996; Byrd and Colwell, 1990;
Jensen et al., 1996; Zweifel and Hagstrom, 1995). The problem of
poor culturability has also to be addressed in the case of bacteria in
activated sludge or soil probes (Binnerup et al., 1993; Wagner et al.,
1993).
VNC cells can be distinguished from dead cells by some metabo
lism and protein synthesis parameters. Moreover, rather than lose
their plasmids, they may even enrich their intracellular plasmid
content (Arturo-Schaan et al., 1996; Nwoguh et al., 1995; Nybroe et
al., 1996). These so-called dormant cells can be activated again when
environmental conditions change. This is not to say that an im
13
proved nutritional state is the only factor responsible for reactiva
tion; temperature fluctuations in natural environments may also
play a role (Colwell et al., 1996; Ferguson et al., 1995; Jiang and
Chai, 1996; Oliver et al., 1995a; Rahman et al., 1996). The presence
of plasmids can further contribute to induction of the VNC stage
(Oliver et al., 1995b). Bacteria detection methods which rely on the
expression of marker genes and are thus blind to VNC cells should
therefore at least be combined with techniques that record VNC
cells (Lee et al., 1996; Leff and Leff, 1996; Oliver et al., 1995b).
Obviously, the choice of a detection method has a great influence
on the record of bacteria released in natural environments. The
ability of microorganisms to adsorb to particles and sediments
renders detection more difficult, though application of PCR has
certainly lowered detection limits (Bej et al., 1990; Hodson et al.,
1995; Lindahl 1996; Lindahl and Bakken, 1995; Tsushima et al., 1995;
Vahjen and Tebbe, 1994). One disadvantage of this technique is the
remaining difficulty in distinguishing between isolated DNA and
DNA associated with living or VNC organisms (Brockmann et al.,
1996; England et al., 1995; Tamanai-Shacoori et al., 1996).
A further point to consider in the context of survival of GMOs in
natural environments is their capability to adapt to unfavourable
environmental conditions. When given a chance to adapt slowly,
bacterial populations in aquatic microcosms imitating natural sur
roundings show prolonged persistence as compared to non-adapted
populations of the same strain (Awong et al., 1990; Mezrioui et al.,
1994; Sobecky et al., 1992). Bacteria cultivated in the laboratory while
exposed to various primary stress factors like unfavourable pH or
nutrient deficiency are able to adapt, and also show higher resist
ance against different secondary stress factors (Ferianc et al., 1995;
Lou and Yousef, 1996). Consequently, any design of microcosm or
mesocosm experiments should include at least some environmen
tal stress factors in order to check GMOs' responsiveness to them.
In determining the time-scale of such experiments one has to con
sider that adaptation may be a slow process and that populations
14
of GMOs no longer detectable after a short time may multiply and
rise again (Clegg et al., 1995; Gillespie et al., 1995; Sjorgen, 1995;
Ramos et al., 1994; Thompson et al., 1995b). Stress factors may even
act as a positive selection pressure.
15
Chapter 4
Spread of Cloned Sequences
Ixisk assessment should not be satisfied with the assumption or
proof that a given GMO will not survive. It must rather extend to
the fate of the DNA, which may be stably integrated, eventually
expressed and may by chance provide advantages for indigenous
microorganisms (Henschke and Schmidt, 1990; Istock, 1991; Schmidt
et al., 1994). GMOs may only require a short time of survival in
order to be transported to other ecological niches or be able to trans
fer some of their nucleic acids to other members of a given ecosys
tem (Byzov et al., 1993; Heijnen and Marinissen, 1995; Weiskel et
al., 1996). While conjugation and transduction are restricted to liv
ing cells, transformation may also take place after the death of do
nor cells. The probability of gene transfer rises with the stability
and quantity of isolated DNA (Fulthorpe and Wyndham, 1991),
though selective forces have also to be taken into account and may
play a crucial role.
For example, Pukall et al. (1996) isolated plasmids with mobilising
capability from soil probes and pig manure in numbers substan
tially higher than estimated. Such plasmids can transform compe
tent cells living in soil or manure environments also under
suboptimal conditions and may even be able to mobilise
recombinant plasmids in these cells (Lorenz and Wackernagel, 1987;
Pukall et al., 1996).
16
Persistence of "naked" DNA
Isolated DNA does not only originate from dead and lysed cells
but can also be actively secreted (Paget and Simonet, 1994). Quan
tifying extracellular DNA in environmental media seems to pose
difficulties, judging by the great variability of published data
(Lorenz and Wackernagel, 1994). Some authors assume that bacte
ria in natural environments release large quantities of high mo
lecular DNA and plasmids into the extracellular gene pool (Lorenz
and Wackernagel, 1994; Romanowski et al., 1990; Wackernagel et
al. 1992). Secretion of DNA and competence for nucleic acid up
take are induced in particular in unfavourable environmental con
ditions like starvation (Lorenz and Wackernagel, 1994; Wackernagel
et al., 1992).
Persistence in wastewater treatment plants (water/sludge)
Wastewater treatment plants guarantee rapid and almost complete
inactivation and degradation of isolated DNA when it is suspended
in open water. Nucleic acids adsorbed to sludge particles, how
ever, find some degree of protection against degradation and also
against detection (Gross et al., 1994; Aardema et al., 1983; Bauda et
al., 1995). As efficiency of inactivation of naked DNA depends on
different factors like temperature, it varies with the season and type
of treatment plant under study (Bergemann et al., 1994; Lorenz and
Wackernagel, 1994).
Persistence in aquatic systems
Some recent studies have dealt with the origin and distribution of
naked DNA in aquatic systems (Beebee 1993; Hermansson and
Linberg, 1994; Jiang and Paul, 1995; Weinbauber et al., 1995). As in
wastewater environments, isolated and dissolved nucleic acids are
17
rapidly degraded in aquatic systems by enzymatic activity (Alvarez
et al., 1996a; Lorenz and Wackernagel, 1994). Adsorption to sand
or clay minerals protects DNA against nucleases even if these are
bound to the same particles (Aardema et al., 1983; Romanowski et
al., 1993a).
Prolonged protection of nucleic acids against inactivation is also
provided by VNC cells (Byrd et al., 1992; Lorenz and Wackernagel,
1994). If naked DNA remains biologically active, this can give rise
to the transformation of competent cells. Therefore, transforma
tion experiments are a suitable method of detecting intact molecules,
regardless of whether they were adsorbed to particles or not
(Alvarez et al., 1996a; Chamier et al., 1993; Mieschendahl and
Danneberg, 1994; Romanowski et al., 1993a).
Persistence in soils
Isolated DNA in non-sterile soils can persist for some time, de
pending on various factors like temperature, pH, salt concentra
tion, type of soil and nucleic acid material, all of which may influ
ence the binding of DNA on mineral or quartz particles (Lorenz
and Wackernagel, 1992). As in aquatic systems, when introduced
into non-sterile soils, particle-adsorbed nucleic acids are protected
against degradation by nucleases, even if these are bound to the
same particle samples (Khanna and Stotzky, 1992; Lorenz and
Wackernagel, 1987). Nucleic acids can bind to minerals quite rap
idly and relatively independently of pH (Lorenz and Wackernagel,
1994). One factor determining the speed of association in aquatic
systems is the structure of the DNA (Khanna and Stotzky 1992;
Lorenz and Wackernagel, 1992; Lorenz et al., 1988; Paget et al., 1993).
Factors relevant to soil environment include the humidity and
granular structure of the soil (Hobom, 1995).
Data on the persistence of isolated DNA in soils show great varia
tion. Smalla (1995) detected recombinant DNA from sugar beets in
soil 18 months after inoculation of plant material. Romanowski et
18
al. (1993b) found that particle-associated plasmids could persist in
different soils for at least 60 days. Plasmid conformation remained
intact during the first two days in this study. According to
Wackernagel et al. (1992) and Romanowski et al. (1992), the time
limit for detecting plasmids in unsterile soil probes without PCR
application is five to ten days. Intact genes or plasmids can fre
quently be detected for as long as the originating bacteria.
Persistence in the digestive system
Consequently, if isolated DNA is able to persist in different envi
ronmental media, then it is quite possible for the digestive system
of animals or humans to take up these nucleic acids with the food
or drinking water. Moreover, as Schubbert et al. (1996) demon
strated in feeding experiments on mice, nucleic acids reaching the
gastrointestinal tract must not necessarily be completely frag
mented, and hence inactivated, but may just as well reach the blood
stream and temporarily even be detected in leukocytes and liver
cells. The transferability of these results from mice experiments to
other mammalian digestive systems, including the human one, is
an obvious possibility. Investigating the persistence of bacterial cells
(Lactococcus lactis) and their DNA in the human intestine Klijn et
al. (1995b) were able to isolate the bacterial DNA from faeces four
days after administering the inoculated milk drinks.
"Avenues and barriers of genetic transmission" (Istock,
1991)
To date gene transfer has mostly been found by means of specially
designed plasmids, as in a number of studies carried out in micro
cosms (Collard et al., 1993; Del Solar et al., 1993; Mazodier and
Davies 1991; Miller et al., 1992; Paget et al., 1992; Paul et al., 1991;
Paul, 1992). However, the spread of antibiotic resistance markers
throughout bacterial communities shows that gene transfer is likely
to happen not only in more or less artificial settings but also under
natural conditions (Saye and Miller 1989; Gotz et al., 1996; Kruse
19
and Sorum, 1994). Moreover, some plasmids originating from gram
positive bacteria have also been isolated in gram-negative bacte
ria, stressing the possibility of a wide distribution of genetic infor
mation. It was not possible to ascertain in this case whether gene
transfer took place via conjugation, transformation or transduction
(Del Solar et al., 1993).
Conjugation, i.e., the transfer of nucleic acids by direct contact
between bacterial donor and recipient cells, is assumed to be the
most effective mechanism of genetic transmission under natural
conditions. Wastewater treatment plants offer perfect conditions
for this kind of transmission because of their abundance of cell
populations (Schneider et al., 1994). Analogously, conjugation in
the gastrointestinal tract is promoted by high population densities
of potential bacterial donor and receptor cells (Morrison, 1996).
Several recent studies have dealt with conjugation in digestive sys
tems (Adamo and Gealt, 1996; Brockmann et al., 1996; Klijn et al.,
1995a,b; Rang et al., 1996; Rybachenko et al., 1996). Microcosm ex
periments or in situ testing with soil or aquatic systems also dem
onstrate the transfer of plasmids by conjugation in other environ
ments (Lebaron et al., 1994; Sandaa and Enger, 1994; Bale et al.,
1988; Pukall et al., 1996; Lukin and Prozorov, 1992). Barkay et al.
(1995) have pointed out the importance of using autochthonous
bacteria as donors or recipients for efficient detection of transfer
through conjugation in natural environments. Other authors have
discussed the influence of different soil systems and supplements
of plant material, temperature and soil humidity (Ashelford et al.,
1995; Hermansson and Linberg, 1994; Klingmiiller, 1993; Temann
et al., 1992; Van Elsas et al., 1990). The main factors inhibiting or
promoting conjugation in natural environments are thought to be
selective forces from, e.g., heavy metals, herbicides, or antibiotics
(De Rore et al., 1994a,b; Gadkari, 1991; Klingmuller and Rieder,
1994; Kozdroj and Pietrowska-Seget, 1995). Lafuente et al. (1996)
have pointed out that conjugation in sterile soil microcosms is most
efficient in "natural" abiotic conditions. As conjugation is an en
ergy-consuming process, it can be promoted by any of a variety of
20
nutrients (Gotz and Smalla, 1997). Any environmental compart
ment fulfilling this requirement like sediments, biofilms or water
air interfaces, and additionally characterised by high numbers of
bacterial cells, will provide optimal conditions for conjugation
(Amabile-Cuevas and Chicurel, 1996; Barkay et al., 1995;
Hermansson and Linberg, 1994).
Transduction, a gene transmission process mediated by
bacteriophages, requires living cells. It is more likely to occur un
der conditions of high metabolic activity (Ripp and Miller, 1995).
Transduction may contribute to the spread of plasmids which are
not transferable by conjugation for lack of essential sequences
(Replicon et al., 1995). It is not restricted to plasmids, as chromo
somal DNA can be transferred as well (Lorenz and Wackernagel,
1993). In this case the host range of the phages determines the pos
sible radius of distribution of genetic material. Empirical evidence
of gene transfer by transduction also taking place in natural envi
ronments (Ogunseitan et al., 1992; Ripp et al., 1994; Schicklmaier
and Schmieger, 1995; Stotzky and Babich, 1994) has been further
substantiated by experiments carried out under natural conditions
(Kidambi et al., 1994; Miller et al., 1992) and by the detection of
high numbers of phage particles in some environmental media
(Kokjohn and Miller, 1992; Lorenz and Wackernagel, 1993; Schafer,
1996). As transduction requires a certain minimum concentration
of phage particles and corresponding bacterial host cells in order
to be efficient, it is more likely to occur in environmental compart
ments characterised by high cell density and plenteous nutrients
(Ogunseitan et al., 1992; Replicon et al., 1995). This is not an uncon
ditional prerequisite for transduction, however, since also
oligothrophic conditions and natural cell densities allow
transduction (Goodman, 1994; Ripp et al., 1994). Adsorption of
phages and bacteria to particles further increases the chances of
survival and interaction and, consequently, also of transduction
(Pickup et al., 1993; Ripp and Miller, 1995).
Transformation, the third mechanism of gene transfer, does not
depend on living cells as donors of nucleic acids. It requires the
active uptake of isolated DNA by recipient cells which are in a
physiological stage of competence (Lorenz and Wackernagel, 1994).
According to present knowledge, induction of competence is de
termined by growth stage, impact of stress factors and/or pres
ence of diffusible competence factors secreted by bacteria, and, not
least, by the species in question (Baur et al., 1996; Cheng et al., 1997;
Goodman, 1994; Lorenz and Wackernagel, 1994; Prozorov, 1997;
Schluter and Potrykus, 1996). The next step towards establishment
of the foreign nucleic acids involves recombination as a means of
integrating the new sequences into the recipient genome. Homolo
gous sequences have better chances of transformation, though up
take does not usually depend on the presence of similar DNA
(Lorenz and Wackernagel, 1994; Prozorov, 1997).
Establishment of plasmids does not depend on homology, but only
on suitable origins of replication (Lorenz and Wackernagel, 1994).
Experimental and empirical data both suggest that transformation
is common in natural environments (Boyle-Vavra and Seifert, 1996;
Hermansson and Linberg, 1994; Lorenz and Wackernagel, 1994;
Schafer, 1996). Microorganisms in aquatic and soil systems can also
be transformed by isolated DNA, as has been demonstrated by
Frischer et al. (1994), Lorenz and Wackernagel (1994), Paget and
Simonet (1993) and others. Transformation of endogenous bacteria
in the gastrointestinal tract was shown in the intestine of Folsomia
Candida after feeding with GMOs (Hobom 1995;Tebbe et al., 1994b).
Baur et al. (1996) postulate that natural environments can provide
optimal conditions for transformation which are not properly imitable in the laboratory. Environmental factors influencing transfor
mation include lack or oversupply of nutrients and certain miner
als, ionic strength of water and temperature (Baur et al., 1996;
Frischer et al., 1993; Hermansson and Linberg, 1994; Lunsford and
London, 1996; Williams et al., 1996). As in the case of conjugation
and transduction, enhanced rates of transformation are found on
surfaces and biofilms (Baur et al., 1996; Hermansson and Linberg,
1994).
22
Reports of intense natural gene transfer may appear to stand in
contradiction to the ability of bacterial strains and varieties to main
tain their genetic make-up. However, there are restriction systems
whose action consists much rather in degrading or "silencing" for
eign DN A than in preventing uptake of foreign DNA (Heinemann,
1991). Plasmid incompatibility is another means of hindering ge
netic transfer (Naik et al., 1994). Stressful conditions seem to re
duce the activity of restriction systems in bacteria and to induce
genes mediating recombination (Saunders and Saunders, 1993;
Schafer et al., 1994).
On the one hand, special plasmids are being designed to reinforce
the constraints of biological containment (safety vectors without
transfer genes), while on the other, progress in cloning relies on
shuttle vectors capable of transgressing existing borders between
bacterial classes and even kingdoms (Doucet-Populaire, 1992;
Henchke and Schmidt 1990; Schafer et al., 1994; Trieu-Cuot et al.,
1987). Plasmids are being constructed to undermine the different
levels of restriction they encounter under normal conditions in a
natural environment and so ensure their persistence and integra
tion (Dunn et al., 1993; Heinemann, 1991).
23
Chapter 5
Discussion
C-urrent security measures for research and production are based
on earlier assumptions about the survival and transfer abilities of
microorganisms. In our opinion, the data in this article expose the
concept of biological containment, on which legal regulation is
based, as unsound, and this for several reasons:
• GMOs can survive or transfer their transgenes to indigenous
organisms;
• DNA is more stable than has been hitherto imagined;
• and DNA taken up with the food is not completely degraded
in the gastrointestinal tract, but has rather been found to enter white
blood cells and spleen and liver cells.
• DNA can even be transferred to the cells of foetuses, as has
been shown in newborn mice. Here transfer probably took place
via the placenta (Doerfler and Schubbert, 1997).
Moreover, there are now nucleic acid constructs containing se
quences which contribute not only to effective replication in differ
ent cellular backgrounds but also to stability and integration via
recombination, transfer and extraordinary expression. They are —
this is the essence of the art — especially designed to fulfil these
jobs.
24
Moreover, neither laboratory nor in situ studies on survival, DNA
persistence and gene transfer provide a realistic measure of what
can happen in complex terrestrial, aquatic and gut environments
(Spielman et al., 1996; Stotzky et al., 1991). Crude as they may be,
validated microcosm and mesocosm studies imitating natural con
ditions as accurately as possible are nevertheless an indispensable
means of testing and prognosticating the influence of some of the
biotic and abiotic factors operating on GMOs in natural environ
ments (Pickup et al., 1993; Spielman et al., 1996).
What has been neglected until now in designing such studies are
the selective forces which may act on the spread of recombinant
genes. Especially in disturbed or polluted environments, contami
nants like heavy metals or high salt concentrations may possibly
facilitate gene transfer.
In a recent seminar organised by the Norwegian Biotechnology
Advisory Board on Antibiotic Resistance, Marker Genes and
Transgenic Plants one of the invited speakers summarised:
"The extent and consequences of horizontal gene transfer are ap
parent in the evolution of antibiotic resistant microorganisms and
evidence suggests that horizontal gene transfer may be equally fre
quent among multicellular eukaryotic organisms. But the actual
and potential frequencies of gene transfer are poor indicators of
risk; very common genes are not maintained in nature if unselected
and rare genes become common extremely quickly if they are the
subject of selection. What remains essential to assessing risk is iden
tifying all potential selective pressures a recombinant gene might
be suited to neutralise. New evidence suggests that current knowl
edge of evolutionary theory is inadequate to predict the fate of
recombinant organisms or recombinant genes." (Heinemann, 1997).
To this we would add that until there is sufficient scientific evi
dence that a given GMO or its recombinant genes will not pose
25
any environmental stress or health impact, we should abide by the
precautionary approach in regulating the contained use and delib
erate release of GMOs. This implies putting a stop to deregulation
in favour of contained use and to tolerated releases from produc
tion plants where environmental impacts are not routinely assessed.
We should bear in mind that soil ecology, for example, has much to
do with mineralisation and nutrient flow, which again is depend
ent on the enzymatic properties of the organismal network. Genes
currently perceived as harmless like many enzyme-coding genes
(or the organisms containing them) may alter soil chemistry and
so create selective pressure or conditions favourable to the survival
of GMOs (see Holmes, 1995 and for a review Doyle et al., 1995).
Moreover, there is no way to extrapolate from one region or envi
ronment to another, differing, environment. This is especially true
when GMOs are transferred to ecosystems and climates which dif
fer greatly from those where they were first developed and used.
This is evident and acknowledged for deliberate release but also
holds true for contained use in production plants with their multi
ple pathways for escape. Therefore, the scope of the biosafety pro
tocol should cover the transfer of GMOs intended for contained
use and have these included in the Advanced Informed Agreement
procedures.
26
any environmental stress or health impact, we should abide by the
precautionary approach in regulating the contained use and delib
erate release of GMOs. This implies putting a stop to deregulation
in favour of contained use and to tolerated releases from produc
tion plants where environmental impacts are not routinely assessed.
We should bear in mind that soil ecology, for example, has much to
do with mineralisation and nutrient flow, which again is depend
ent on the enzymatic properties of the organismal network. Genes
currently perceived as harmless like many enzyme-coding genes
(or the organisms containing them) may alter soil chemistry and
so create selective pressure or conditions favourable to the survival
of GMOs (see Holmes, 1995 and for a review Doyle et al., 1995).
Moreover, there is no way to extrapolate from one region or envi
ronment to another, differing, environment. This is especially true
when GMOs are transferred to ecosystems and climates which dif
fer greatly from those where they were first developed and used.
This is evident and acknowledged for deliberate release but also
holds true for contained use in production plants with their multi
ple pathways for escape. Therefore, the scope of the biosafety pro
tocol should cover the transfer of GMOs intended for contained
use and have these included in the Advanced Informed Agreement
procedures.
26
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44
ABOUT THE AUTHORS
Dr Beatrix Tappeser is a biologist and coordinator of
the Department of Risk Assessment in Genetic Engi
neering at the Institute for Applied Ecology, Freiburg,
Germany. She has been working for fifteen years on
questions concerning the possible social, ecological and
health-related impacts of genetic engineering in differ
ent application fields. The Institute for Applied Ecology
is a non-profit, citizen-founded research institution.
Dr Manuela Jager studied biology at the University of
Ulm, Germany, from 1983 to 1992, with microbiology,
molecular genetics and virology as her main subjects.
From 1992 to 1998, she was working at the Department
of Risk Assessment in Genetic Engineering at the Insti
tute for Applied Ecology, focusing on genetically engi
neered and novel foods and their impacts on human
health.
Dr Claudia Eckelkamp is a biologist and has been a
scientific collaborator at the Department of Risk Assess
ment in Genetic Engineering at the Institute for Applied
Ecology since 1995. Her field of work covers risk assess
ment and evaluation of the exploitation of genetic
engineering in different areas of application, particu
larly risk assessment of genetically modified microor
ganisms, environmental risks of the cultivation of
genetically engineered plants in agriculture, and evalu
ation of the safety of genetically engineered foodstuffs.
Position: 1779 (3 views)